Synthesis, reactions, and antiviral activity of 6′-amino-2′- thioxo-1′,2′-dihydro-3,4′-bipyridine-3′, 5′-dicarbonitrile

Article abstract of DOI:10.1080/10426500601087277

Nicotinaldehyde 1 reacted with 2-cyanoethanethioamide 2 to give 2-cyano-3-pyridin-3-ylprop-2-enethioamide 3, which reacted with a second mole of 2to give the corresponding 6′-amino-2′-thioxo-1′,2′- dihydro-3,4′-bipyridine-3′,5′-dicarbonitrile 6. The synthetic potentiality of compound 6 was investigated via its reaction with active halogen-containing reagents, e.g., chloroacetone, 2-chloro-3-oxobutanoate, 2-chloroacetamide, chloroaceto-nitrile, and ethyl chloroacetate, to afford the corresponding thieno[2,3-b]-pyridine derivatives. Structure elucidation of all newly synthesized heterocyclic compounds was based on the data of elemental analyses, and IR, ¹H NMR, as well as mass spectra. Cytotoxicity, anti-HSV1, anti-HAV, and MBB activities were evaluated for all newly synthesized heterocyclic compounds. Copyright Taylor & Francis Group, LLC.

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Synthesis, Reactions, and
Antiviral Activity of 6′-
Amino-2′-thioxo-1′,2′-
dihydro-3,4′-bipyridine-3′,5′-
dicarbonitrile
Fawzy A. Attaby a , A. H. H. Elghandour a , M. A. Ali
b & Yasser M. Ibrahem a
a Chemistry Department , Cairo University , Giza,
Egypt
b Virology Laboratory , National Research Center ,
Dokki, Egypt
Published online: 24 Feb 2007.
To cite this article: Fawzy A. Attaby , A. H. H. Elghandour , M. A. Ali & Yasser
M. Ibrahem (2007) Synthesis, Reactions, and Antiviral Activity of 6′-Amino-2′-
thioxo-1′,2′-dihydro-3,4′-bipyridine-3′,5′-dicarbonitrile, Phosphorus, Sulfur, and
Silicon and the Related Elements, 182:4, 695-709, DOI: 10.1080/10426500601087277
To link to this article: http://dx.doi.org/10.1080/10426500601087277
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Phosphorus, Sulfur, and Silicon, 182:695–709, 2007
Copyright © Taylor & Francis Group, LLC
ISSN: 1042-6507 print / 1563-5325 online
DOI: 10.1080/10426500601087277
Synthesis, Reactions, and Antiviral Activity of 6ꢀ-Amino-
2ꢀ-thioxo-1ꢀ,2ꢀ-dihydro-3,4ꢀ-bipyridine-3ꢀ,5ꢀ-dicarbonitrile
Fawzy A. Attaby
A. H. H. Elghandour
Chemistry Department, Cairo University, Giza, Egypt
M. A. Ali
Virology Laboratory, National Research Center, Dokki, Egypt
Yasser M. Ibrahem
Chemistry Department, Cairo University, Giza, Egypt
Nicotinaldehyde 1 reacted with 2-cyanoethanethioamide 2 to give 2-cyano-3-
pyridin-3-ylprop-2-enethioamide 3, which reacted with a second mole of 2 to give the
corresponding 6ꢀ-amino-2ꢀ-thioxo-1ꢀ,2ꢀ-dihydro-3,4ꢀ-bipyridine-3ꢀ,5ꢀ-dicarbonitrile
6. The synthetic potentiality of compound 6 was investigated via its reaction with
active halogen-containing reagents, e.g., chloroacetone, 2-chloro-3-oxobutanoate,
2-chloroacetamide, chloroaceto-nitrile, and ethyl chloroacetate, to afford the cor-
responding thieno[2,3-b]-pyridine derivatives. Structure elucidation of all newly
synthesized heterocyclic compounds was based on the data of elemental analyses,
1
and IR, H NMR, as well as mass spectra. Cytotoxicity, anti–HSV1, anti–HAV, and
MBB activities were evaluated for all newly synthesized heterocyclic compounds.
Keywords
2-Cyano-3-pyridin-3-yl-prop-2-enethioamide; 2-cyanoethanethioamide;
bipyridine-3ꢀ, 5ꢀ-dicarbonitrile; nicotinaldehyde; thieno[2,3-b]pyridine
INTRODUCTION
The reported biological activity of 2-thioxopyridine,1−3 and thieno-
pyridine,4,5 and the conjunction of our previous work5−23 stimulate our
interest to synthesize several derivatives of these ring systems required
for chemical transformations and a medicinal chemistry program. We
also are interested in investigating the chemical reactivity and syn-
thetic potentiality of both CH C in 3 and CH2 in 2.
Received January 4, 2006; accepted March 14, 2006.
Address correspondence to Fawzy A. Attaby, Chemistry Department, Faculty of Sci-
ence, Cairo University, Giza, Egypt. E-mail: fattaby@hotmail.com
695

696
F. A. Attaby et al.
RESULTS AND DISCUSSION
It has been found that nicotinaldehyde 1 reacted with 2-cyanoethane-
thioamide 2 to afford 2-cyano-3-pyridin-3-ylprop-2-enethioamide 3 that
reacted in turn with a second mole of 2 to give 6 via the non-isolable
structures 4 and 5. The IR (cm−1) of this reaction product showed
1
the bands of NH2, NH, and CN functions, and H NMR (δ ppm) re-
vealed the signals of NH2, NH, and pyridine protons. Moreover, its
mass spectrum gave m/z = 253, which corresponded to the molecular
weight of the molecular formula C12H7N5S of the assigned structure
(cf. Experimental section and Scheme 1). By considering the previously
mentioned data in addition to the data of elemental analyses, we rolled
out structure 7 and conclude that this reaction product underwent
auto-oxidation under the experimental conditions to afford 6, which
was formulated as 6ꢀ-amino-2ꢀ-thioxo-1ꢀ,2ꢀ-dihydro-3,4ꢀ-bipyridine-3ꢀ,5ꢀ-
dicarbo-nitrile 6 (cf. Scheme 1) and Experimental section). Authenti-
cally 6 was obtained also by a reaction of both 1 and 2 in the ratio 1:2
as a ternary mixture.
The synthetic potential of 6 was investigated through its reaction
with haloketones, haloesters, halonitriles, and haloalkanes. Thus, it
has been found that 6 reacted with chloroacetone (8a) in methanolic
sodium methoxide to afford a reaction product formed via dehydrochlo-
rination. The IR of this reaction product showed bands of NH2, CN, and
acetyl CO functions; its 1H NMR spectrum revealed signals of CH3CO,
NH2, and pyridine protons; moreover, its mass spectrum gave a peak
at m/z = 309, which corresponded to the molecular weight of the molec-
ular formula C15H11N5SO of the assigned structure (cf. Experimental
section). Considering the previously mentioned data in addition to
that of elemental analyses, this reaction product was obtained through
the non-isolable products 9a and 10a and was formulated as 2-acetyl-
3,6-diamino-4-pyridin-3-ylthieno[2,3-b]pyrid-ine-5-carbonitrile
(11)
(cf. Scheme 2).
The reaction product 11 was also obtained through different path-
ways. Thus, it has been found that 6 reacted with each of 3-chloro-
pentan-2,4-dione and ethyl 2-chloro-3-oxobutanoate 8b, c under the
same previously mentioned experimental conditions through the non-
isolable products 9b, c and 10b, c, respectively (cf. Scheme 2).
Structure 12 was not formed via the reaction of 6 with 8c depend-
ing on the data of 1H NMR, which revealed signals of COCH3 pro-
tons and did not reveal signals of COOCH2CH3 protons. This also con-
firmed using the data of mass spectra, which did not give m/z = 339,
which corresponded to the molecular weight of the molecular formula
C16H13N5O2S of the assigned structure 12, while it gave a peak at

6ꢀ-Amino-2ꢀ-thioxo-1ꢀ,2ꢀ-dihydro-3,4ꢀ-bipyridine-3ꢀ,5ꢀ-dicarbonitrile
697
SCHEME 1
m/z = 309, which corresponded to the molecular weight of the molecular
formula C15H11N5OS of the assigned structure 11. It is important to re-
port here a that compound 11 obtained via 8b formed through addition
of H2O molecule followed by the removal of the acetic acid molecule.
On the other hand, 11 formed via 8c through hydrolysis with ethyl

698
F. A. Attaby et al.
SCHEME 2
alcohol molecule removal, and this was followed by decarboxylation (cf.
Experimental section).
In further investigation, compound 6 reacted with each of chloroace-
tonitrile and 2-chloroacetamide 13a, b in a ratio of 1:1 in methanolic
sodium methoxide through dehydrochlorination to afford reaction prod-
ucts with the molecular formulas C14H8N6S and C14H10N6SO, respec-
tively. The IR (cm−1) of these reaction products showed bands of NH2
1
and CN functions; their H NMR spectra revealed signals of NH2 and
pyridine protons. Moreover, their mass spectra gave m/z = 292 and 310,
which corresponded to the molecular weights of the molecular formu-
las C14H8N6S and C14H10N6SO of the assigned structures 16a,b. The
formation of 16a,b proceeded via the non-isolable products 14a,b and
15a, b, respectively (cf. Scheme 3 and Experimental section).
The chemical reactivity and synthetic potentiality of 6 were further
investigated through its reaction with ethyl chloroacetate (17) to afford
a reaction product formed by dehydrochlorination. The IR of this reac-
tion product showed the bands of NH2, CN, and CO ester functions; its

6ꢀ-Amino-2ꢀ-thioxo-1ꢀ,2ꢀ-dihydro-3,4ꢀ-bipyridine-3ꢀ,5ꢀ-dicarbonitrile
699
SCHEME 3
1H NMR revealed the signals of CH3CH2, CH3CH2, NH2, and pyridine
protons. Moreover, its mass spectrum gave the parent peak at m/z = 339
(27.2%), which corresponded to the molecular weight of the molecular
formula C16H13N5O2S in addition to the base peak at m/z = 266 (100%),
which corresponded to M-73 (elimination of the COOEt fragment from
the parent peak). Considering the previously mentioned data and the
data of elemental analyses, we can conclude that the reaction prod-
uct and it represented as 12 formed through the non-isolable products
18 and 19, and it could be formulated as ethyl 3,6-diamino-5-cyano-4-
pyridin-3-ylthieno[2,3-b]pyridine-2-carboxylate (cf. Scheme 4).
The synthetic potentiality of 6 could be established through its reac-
tion with iodomethane followed by its reaction with hydrazine hydrate.
Thus, it has been found that 6 reacted with iodomethane in methanolic
sodium methoxide to afford a reaction product formed through dehydro-
iodonation. The IR spectrum of this reaction product showed bands of
1
NH2, CN, and aliphatic CH (no bands for NH function); its H NMR
spectrum revealed the signals of CH3, NH2, and pyridine protons. Fur-
thermore, its mass spectrum gave m/z = 267, which corresponded to the
molecular weight of the molecular formula C13H9N5S of the assigned

700
F. A. Attaby et al.
SCHEME 4
structure 20. Compound 20 reacted with hydrazine hydrate to give a
sulfur-free reaction product whose structure was established by consid-
1
ering the data of IR, H NMR, mass spectra, and elemental analyses
(cf. Experimental section). The element test of this reaction product
gave no S, and this proves the removal of the SCH3 group during the
reaction with hydrazine hydrate; this also confirms, through the sig-
1
nal absence of SCH3, protons from the H NMR spectrum. Moreover,
the mass spectrum of this reaction product gave m/z = 267, and this
confirms, that NH2 at the 6-position reacted with hydrazine hydrate
to give –NHNH2 instead of NH2 so that the reaction product could
be formulated as 3-amino-6-hydrazino-4-pyridin-3-yl-7,7a-dihydro-1H-
pyrazolo[3,4-b]pyridine-5-carbonitrile 21, not 22. Compound 21 could
be prepared authentically through the reaction of 6 with hydrazine hy-
drate (cf. Scheme 5).
Biological Evaluation
Cytotoxicity Assay
As shown in Table I, compounds 12, 16a, b, and 21 were much safer
for cell culture inoculation than the other compounds. Compound 11
showed high cytotoxicity at doses 15–45 μg, while all other compounds

6ꢀ-Amino-2ꢀ-thioxo-1ꢀ,2ꢀ-dihydro-3,4ꢀ-bipyridine-3ꢀ,5ꢀ-dicarbonitrile
701
SCHEME 5
showed CPE at 20 μg. Accordingly, the safe doses were selected and
used for anti-virus bioassay.
Antivirus Bioassay
Screening for Anti-HSV1 Activity
Plaque reduction assay showed that compounds 6, 11, 16a, 20, and
21 were promising to be anti-HSV1 compounds. The percentage of virus

702
F. A. Attaby et al.
TABLE I Cytotoxicity Assay for the Synthetic Compound in Vero
Cell Line
Cytotoxicity Grade of the Tested Materials (μg)
Compound
No.
5
10
15
20
25
30
35
40
6
—
+2
—
—
—
—
—
—
+3
—
—
—
—
—
—
+4
—
—
—
—
—
—
+4
—
—
—
—
—
—
+4
—
—
—
+1
+4
—
—
—
+2
+4
—
—
—
+3
+4
—
—
—
11
12
16a
16b
20
21
+1
+1
−1
+1
—
—
—
—
Note: Cytotoxicity grades are divided into four grades: +1 = 25%, +2 = 50%, +3 =
75%, and +4 = 100%, of the cell monolayer showed CPE.
reduction was 35% and increased to 57% at dose 25 μg, and the same
compounds showed a moderate anti-HSV1 activity at dose 15 μg. Com-
pound 16b showed no activity at dose 25 μg and showed 35% of virus
reduction at dose 15 μg, while compound 12 exhibited no activity at all
(Figure 1).
Screening for Anti-HAV and MBB Strain Activity
All compounds were tested for anti–Hepatitis A virus activity in
HepG2 cells by plaque reduction assay. The results shown that com-
pounds 6 and 21 exhibited a moderate percentage of virus reduction at
FIGURE 1 Anti-HSV1 bioassay of synthetic compounds in Vero cell line.

6ꢀ-Amino-2ꢀ-thioxo-1ꢀ,2ꢀ-dihydro-3,4ꢀ-bipyridine-3ꢀ,5ꢀ-dicarbonitrile
703
FIGURE 2 Anti-HAV bioassay of synthetic compounds in HepG2 cells.
dose 15 μg and high activity at dose 25 μg, while the other newly syn-
thesized heterocyclic compounds either exhibited weak or no activity
against HAV (Figures 1 and 2).
EXPERIMENTAL
All melting points are uncorrected. IR spectra were recorded as KBr
discs on a Shimadzu FTIR-8201PC spectrophotometer. 1H NMR spectra
were recorded on Varian Mercury 300 MHz and Varian Gemini 200-
MHz spectrometers using TMS as an internal standard and CDCl3,
DMSO-d6, and DMSO as solvents. Chemical shifts are expressed as
δ ppm units.
Mass spectra were recorded on Shimadzu GCMS-QP1000EX using
inlet type at 70 eV. The Microanalytical Center of Cairo University
(Giza, Egypt) performed microanalyses.
Synthesis of 6ꢀ-Amino-2ꢀ-thioxo-1ꢀ,2ꢀ-dihydro-3,4ꢀ-bipyridine-
3ꢀ,5ꢀ-dicarbonitrile (6)
A solution of each of 1 (3 mmole, 3.21 g) and 2 (3 mmole, 3 g) in absolute
ethanol (50 mL) containing a catalytic amount of piperidine (0.4 mL)
was stirred at r.t. for 15–20 min. The product that formed was filtered
off and dried well. The isolated product 3 was obtained as yellow crys-
tals. A mixture of 3 (3 mmole, 5.67 g) and 2 (3 mmole, 3 g) reacted in
absolute ethanol (50 mL) containing a catalytic amount of piperidine
under reflux for 5 h. The reaction mixture was then evaporated until
dryness and then cooled. The product formed was collected by filtra-
tion, washed with cold ethanol, and then crystallized from ethanol as

704
F. A. Attaby et al.
yellow crystals, m.p. 246–248◦C; IR (ν cm−1): 3455, 3348, 3155 (NH2
and NH), 3071 (pyridine-CH), 2213(CN at C-3), 2205(CN at C-5) and
1
1550 (C S); H NMR (δ ppm): 4.2 (s, 2H, NH2), 5.3 (s, br., 1H, NH)
and 7.3–8.7 (m, 4H, pyridineH’s); mass spectra (m/z, intensity %):
parent peak (m/z = 253, 54%), base peak (m/z = 84, 100%, due to the
fragment NC CH C SH), peak at m/z = 209, 9.8% due to the loss of
CS from the parent), the latter peak lost NH to afford peak at m/z =
194 (9.0%) that lost CH2N fragment to give peak at m/z = 166 (19.3%)
that lost a C2NH fragment to give peak at m/z = 131 (14.3%); anal.
for C12H7N5S (253.28): calcd./found (%): C, 56.90/57.0; H, 2.79/2.8; N,
27.65/27.5; S,12.66/12.7.
Synthesis of 11
A solution of 6 (0.5 mmole, 1.27 g) and 8a (0.5 mmole, 0.46 g), 8b
(0.5 mmole, 0.67 g), or 8c (0.5 mmole, 0.82 g) in methanol contain-
ing sodium methoxide (prepared by 0.6 g of sodium in 40 mL methanol)
was stirred for 5 h. The product that formed was collected by filtra-
tion, washed with cold ethanol, and then recrystallized from ethanol
to give 2-acetyl-3,6-diamino-4-pyridin-3-ylthieno[2,3-b]pyridine-
5-carbonitrile (11) as yellow crystals, m.p. >300◦C; IR (ν cm−1):
3456, 3344, 3188 (NH2); 2873–2950 (CH-aliphatic); 3048 (pyridine-
1
CH), 2215(CN) and 1724 (C=O acetyl); H-NMR (δ ppm): 2.3 (s, 3H,
CH3CO), 5.1(s, br., 2H, NH2) and 7.1–8.5 (m, 4H, pyridine H’s); mass
spectra (m/z, intensity %) parent peak and also the base peak (inten-
sity 100%) at m/z = 309, peak at m/z = 266 (36.9%), which corresponded
to M-43 (elimination of CH3CO from the parent peak), the latter peak
lost a CS fragment to give peak at m/z = 222 (5.9%) that lost a fragment
of NH2-CN to afford peak at m/z = 180 (17.3%) that also underwent
fragmentation to give peaks at m/z = 142 (8.4%) and 102 (7.3%). Anal.
for C15H11N5OS (309): calcd./found (%): C, 58.24/58.2; H, 3.58/3.6; N,
22.64/22.5; S, 10.37/10.4.
Synthesis of 12
A solution of 6 (0.5 mmole, 1.27 g) and ethyl chloroacetate 17 (0.5 mmole,
0.61 g) in methanol containing sodium methoxide (prepared by 0.6
g of sodium in 40 mL methanol) was stirred for 5 h. The product
that formed was collected by filtration, washed with cold ethanol, and
then recrystallized from ethanol to give ethyl 3,6-diamino-5-cyano-
4-pyridin-3-ylthieno[2,3-b]pyridine-2-carboxylate (12) as yellow
crystals, m.p. 306–308◦C; IR (ν cm−1): 3451, 3340, 3190 (NH2); 3050
1
(pyridine-CH); 2218 (CN) and 1735 (CO ester); H NMR (δ ppm): 1.5

6ꢀ-Amino-2ꢀ-thioxo-1ꢀ,2ꢀ-dihydro-3,4ꢀ-bipyridine-3ꢀ,5ꢀ-dicarbonitrile
705
(t, 3H, COOCH2CH3); 4.3 (q, 2H, COOCH2CH3); 5.2(s, br., 4H, two NH2)
and 7.3–8.6 (m, 4H, pyridine H’s); mass spectra (m/z, intensity %)
parent peak at m/z = 338 (47.6%), base peak at m/z = 266 (100%) in ad-
dition to several peaks of low intensity; anal. for C16H13N5SO2 (339.3):
calcd./found (%): C, 56.63/56.7; H, 3.86/3.9; N, 20.64/21.0; S, 9.45/9.4.
Synthesis of 16a,b
A solution of 6 (0.5 mmole, 1.27 g) and chloroacetonitrile 13a (0.5 mmole,
0.38 g) and 2-chloroacetamide 13b (0.5 mmole, 0.47 g) in methanol
containing sodium methoxide (prepared by 0.6 g of sodium in 40 mL
methanol) was stirred for 5 h. The products that formed were collected
by filtration, washed with cold ethanol, and then recrystallized from
ethanol to give 16a,b, respectively.
3,6-diamino-4-pyridin-3-ylthieno[2,3-b]pyridine-2,5-dicarbo-
nitrile (16a) as brown crystals, m.p. >300◦C; IR (ν cm−1): 3474,
3354, 3194 (NH2); 3051 (pyridine-CH) and 2220(CN); 1H-NMR (δ
ppm): 5.2(s, br., 4H, two NH2) and 7.3–8.6 (m, 4H, pyridine H’s); mass
spectra (m/z, intensity %) parent peak at m/z = 292 (70.1%) that
lost hydrogen to afford the base peak at m/z = 291 (100%) that lost CN
to give peak at m/z = 266 (3.3%) and peak at m/z = 194 (3.8%) due to
the loss of NH2 C=C S CN. Anal. for C14H8N6S (292.3): calcd./found
(%): C, 57.52/57.6; H, 2.76/2.8; N,28.75/28.5; S;10.97/10.1.
3,6-diamino-5-cyano-4-pyridin-3-ylthieno[2,3-b]pyridine-2-
carboxa-mide (16b) as yellow crystals, m.p. >300◦C; IR (ν cm−1):
3466, 3342, 3189 (NH2); 3055 (pyridine-CH); 2222 (CN) and 1675
1
(CO amidic); H NMR (δ ppm): 4.8 (s, br., 4H, two NH2); 5.7 (s, br.,
2H, CONH2) and 7.2–8.4 (m, 4H, pyridine H’s); mass spectra (m/z,
intensity %) parent peak at m/z = 310 (56.4%) that lost H2O to give
the base peak at m/z = 292 (100%), the latter peak lost HCN, and CN,
to give peaks at m/z = 265 (13.6%) and at m/z = 266 (8.5%); anal. for
C14H10N6SO (310.3): calcd./found (%): C, 54.18/54.2; H, 3.25/3.3; N,
27.08/27.1; S, 10.33/10.4.
Synthesis of 2ꢀ-Amino-6ꢀ-(methylthio)-3,4ꢀ-bipyridine-3ꢀ,5ꢀ-
dicarbnitrile (20)
A solution of 6 (0.5 mmole, 1.27 g) and iodomethane (1 mmole, 1.42 g)
in methanol containing sodium methoxide (prepared by 0.6 g of sodium
in 40 mL methanol) was stirred for 1 h. The product that formed was
collected by filtration, washed with cold ethanol, and then recrystal-
lized from ethanol to give 20 as pale yellow crystals, m.p. 280–282◦C;
IR (ν cm−1): 3453, 3442, 3188 (NH2); 2881–2923 (CH-aliphatic); 3066

706
F. A. Attaby et al.
1
(pyridine-CH) and 2217 (CN); H-NMR (δ ppm): 1.8 (s, 3H, CH3), 4.8
(s, br., 2H, NH2) and 7.3–8.6 (m, 4H, pyridine H’s); mass spectra (m/z,
intensity %) parent and base at m/z = 266 (100%) that lost SCH2 frag-
ment to give peak at m/z = 220 (7.1%) that lost a CN fragment to give
peak at m/z = 194 (14.8%); Anal. for C13H9N5S (267.3): calcd./found (%):
C, 58.41/58.4; H, 3.39/3.4; N, 26.20/26.3 S, 12.00/11.8.
Synthesis of 3-Amino-6-hydrazino-4-pyridin-3-yl-1H-pyrazolo-
[3,4-b]-pyridine-5-carbonitrile (21)
A mixture of 6 (0.5 mmole, 1.27 g) or 20 (0.5 mmole, 1.34 g) and hy-
drazine hydrate (20–25 mL) was heated under reflux for 8–10 h until
the odor of either H2S or CH3SH ceased. The product that formed af-
ter cooling was filtered off, washed with cold ethanol, and crystallized
from ethanol to give 21, as yellow crystals, m.p. 290–292◦C; IR (cm−1):
3462, 3287, 3187 (NH2, NH), 3062 (pyridine-CH), 2987 (sat. CH) and
2213 (CN); 1H-NMR (δ ppm): 1.5 (s, 3H, CH3), 3.7 (s, br., 4H, two NH2),
5.4(s, br., 2H, two NH) and 7.5–8.6 (m, 4H, Pyridine H’s); mass spectra
(m/z, intensity %) parent and base peak at m/z = 266 (100%) that lost
NH2 to give peak at m/z = 250 (46.2%), which fragmented to give sev-
eral peaks at low intensities; anal. for C12H10N8 (266.2): calcd./found:
C, 54.13/54.1, H, 3.79/3.8, N, 42.08/42.2.
Biological Evaluation
I
Two types of cell lines were used for propagation of Herpes simplex
virus type 1 (HSV-1) and hepatitis A virus (HAV-MBB strain). These
cell lines, African green monkey kidney cells (Vero), a cell line obtained
from American type culture collection (ATCC), and a human hepatoma
cell line (Hep G2).
Viruses
Two models of DNA and RNA viruses were used for bioassay. These
viruses were HSV-1 and Hepatitis A virus, MBB strain (HAN-MBB).
II: Media and Supplements
Cell Culture Medium. Minimum Essential Media (MEM with
Hank’s balanced salt solution, GIBCO-BRL) was prepared and steril-
ized by filtration through a 0.22-μm pore-size nitrocellulose membrane;
the pH value was adjusted at 7.4 by sodium bicarbonate.
Foetal Bovine Serum (FBS). (Sigma) FBS was inactivated at 56◦C
for 30 min and was used at 10% final concentration for the growth
medium and at 2% for the maintenance medium.

6ꢀ-Amino-2ꢀ-thioxo-1ꢀ,2ꢀ-dihydro-3,4ꢀ-bipyridine-3ꢀ,5ꢀ-dicarbonitrile
707
Antibiotic-Antimycotic
Mixture
(GIBCO-BRL). Hundered
X
antibiotic-antimycotic mixtures consisted of 10,000 U penicillin G
sodium, 10,000 μg streptomycin sulfate and 25 μg amphotericin B.
III: Cell Dissociation Solution (Trypsin-Versene Mixture)
Phosphate Buffered Saline. (PBS, pH 7.5, 0.15 M). The buffer was
prepared at the following concentrations: NaCl (8.9 g/L) KCl (0.2 g/L),
KH2PO4 (0.12 g/L), Na2HPO4 (0.91 g/L), deionized H2O (up to 1 L).
Ingredients were mixed gently in the order previously shown, and the
pH value was adjusted at 7.5. The buffer was sterilized by filtration
through 0.22 μm nitrocellulose membrane. The solution was used in
washing cell monolayer sheets and in preparation of cell dissociation
solution as follows.
Trypsin 1:250 (Sigma). Fifteen gram of trypsin powder (1:250) was
dissolved in 500 mL PBS and digested at 4◦C overnight with stirring.
Versene Solution (0.04%). Tetrasodium salt of Ethylenediamine
Tetraacetic Acid (EDTA) was dissolved in 500 mL of 0.15 M PBS pH
7.5 to prepare 2 mM solution (0.04 g) and was mixed with an equal vol-
ume of trypsin solution. The pH value of the trypsin-versene mixture
was adjusted at 8.4 by 7.5% sodium bicarbonate solution and was steril-
ized by filtration through a 0.22-μm pore size nitrocellulose membrane.
The mixture was aliquoted and stored at −20◦C until used.
Methods
I: Preparation of Synthetic Compounds for Bioassay
Compounds were dissolved as 100 mg each in 1 mL of 10% DMSO
in water. The final concentration was 100 μg/μL (stock solution). The
dissolved stock solutions were sterilized by the addition of an antibiotic-
antimycotic mixture: 10,000 U penicillin G sodium or Gentamicin
50 μg/mL, 10,000 μg streptomycin sulfate, and 250 μg amphotericin
B. Sterility tests were carried out in nutrient agar.
II: Cell Culture
African green monkey kidney-derived cells (VERO) were used. The
cells were propagated in Hanks’ MEM. The MEM was supplemented
with 10% FOBS and a 1% antibiotic-antimycotic mixture. The pH was
adjusted at 7.2–7.4 by 7.5% sodium bicarbonate solution. The mixture
was sterilized by filtration through a 0.22-μm pore-size nitrocellulose
membrane.

708
F. A. Attaby et al.
III: Viruses
HSV-1 was obtained from the Environmental Virology Labortories,
Department of Water Pollution Res., National Research Center (Dokki,
Giza, Egypt).
IV: Antiviral Assay
Cytotoxicity Assay. Cytotoxicity was assayed for both dimethyl sul-
foxide (DMSO) and the test compounds. Serial dilutions were prepared
and inoculated on Vero and HepG2 cells grown in 96 well tissue culture
plates. The maximum tolerated concentration for each compound was
determined by both cell morphology and cell viability by straining with
trypan blue dye.
Plaque Reduction Assay. A 6-well plate was cultivated with Vero cell
culture (105 cell/mL) and was incubated for 2 days at 37◦C. HSV-1 was
diluted to give 104 PFU/mL final concentration, and mixed with the
plant extract at the previous concentration, and incubated overnight
at 4◦C. The growth medium was removed from the multiwell plate,
and the virus-compound mixture was inoculated (100 μL/well). After
1 h of contact time, the inoculum was aspirated, and 3 mL of MEM
with 1% agar rose was overlaid on the cell sheets. The plates were
left to solidify and incubated at 37◦C until the development of virus
plaques. Cell sheets were fixed in 10% formaline solution for 2 h, and
stained with crystal violet stain. The control virus and cells were treated
identically without chemical compounds. Virus plaques were counted,
and the percentage of reduction was calculated.
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