Synthesis, reactions, and antiviral activity of 5′-acetyl-6′- methyl-2′-thioxo-1′,2′-dihydro-3,4′-bipyridine-3′- carbonitrile

Article abstract of DOI:10.1080/104265090968398

Bipyridine-3′0-carbonitrile derivatives 5 reacted with several halogen containing reagents, e.g., 1-chloroacetone, 3-chloropentan-2,4-dione, ethyl chloroacetate, ethyl 2-chloro-3-oxobutanoate, 2-chloroacetamide, chloroacetonitrile, and iodomethane to afford the corresponding thieno[2,3-b]-pyridine derivatives. Considering the data of IR, ¹H NMR, mass spectra and elemental analysis the chemical structures of the newly synthesized heterocyclic compounds elucidated. Cytotoxicity, anti-HSV1, (anti-Herpes Simplex virus type 1) anti-HAV (Hepatitis A virus), and MBB activity were evaluated for the newly synthesized heterocyclic compounds. Copyright Taylor & Francis LLC.

Full text of DOI:10.1080/104265090968398

This article was downloaded by: [University of Aberdeen]
On: 14 February 2015, At: 12:29
Publisher: Taylor & Francis
Informa Ltd Registered in England and Wales Registered Number: 1072954
Registered office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH,
UK
Phosphorus, Sulfur, and Silicon
and the Related Elements
Publication details, including instructions for
authors and subscription information:
http://www.tandfonline.com/loi/gpss20
Synthesis, Reactions,
and Antiviral Activity of
5′-Acetyl-6′-methyl-2′-
thioxo-1′,2′-dihydro-3,4′-
bipyridine-3′-carbonitrile
Fawzy A. Attaby a , M. A. Ali b , A. H. H. Elghandour
c & Yasser M. Ibrahem a
a Chemistry Department , Faculty of Science, Cairo
University , Giza, Egypt
b Virology Laboratory, National Research Center ,
Dokki, Egypt
c Chemistry Department , Cairo University , Giza,
Egypt
Published online: 01 Feb 2007.
To cite this article: Fawzy A. Attaby , M. A. Ali , A. H. H. Elghandour & Yasser M.
Ibrahem (2006) Synthesis, Reactions, and Antiviral Activity of 5′-Acetyl-6′-methyl-2′-
thioxo-1′,2′-dihydro-3,4′-bipyridine-3′-carbonitrile, Phosphorus, Sulfur, and Silicon
and the Related Elements, 181:1, 1-14, DOI: 10.1080/104265090968398
To link to this article: http://dx.doi.org/10.1080/104265090968398
PLEASE SCROLL DOWN FOR ARTICLE
Taylor & Francis makes every effort to ensure the accuracy of all the
information (the “Content”) contained in the publications on our platform.
However, Taylor & Francis, our agents, and our licensors make no
representations or warranties whatsoever as to the accuracy, completeness,

or suitability for any purpose of the Content. Any opinions and views
expressed in this publication are the opinions and views of the authors, and
are not the views of or endorsed by Taylor & Francis. The accuracy of the
Content should not be relied upon and should be independently verified with
primary sources of information. Taylor and Francis shall not be liable for any
losses, actions, claims, proceedings, demands, costs, expenses, damages,
and other liabilities whatsoever or howsoever caused arising directly or
indirectly in connection with, in relation to or arising out of the use of the
Content.
This article may be used for research, teaching, and private study purposes.
Any substantial or systematic reproduction, redistribution, reselling, loan,
sub-licensing, systematic supply, or distribution in any form to anyone is
expressly forbidden. Terms & Conditions of access and use can be found at
http://www.tandfonline.com/page/terms-and-conditions

Phosphorus, Sulfur, and Silicon, 181:1–14, 2006
Copyright © Taylor & Francis LLC
ISSN: 1042-6507 print / 1563-5325 online
DOI: 10.1080/104265090968398
Synthesis, Reactions, and Antiviral Activity
of 5ꢀ-Acetyl-6ꢀ-methyl-2ꢀ-thioxo-1ꢀ,2ꢀ-dihydro-
3,4ꢀ-bipyridine-3ꢀ-carbonitrile
Fawzy A. Attaby
Chemistry Department, Faculty of Science, Cairo University,
Giza, Egypt
M. A. Ali
Virology Laboratory, National Research Center, Dokki, Egypt
A. H. H. Elghandour
Chemistry Department, Cairo University, Giza, Egypt
Yasser M. Ibrahem
Chemistry Department, Faculty of Science, Cairo University,
Giza, Egypt
Bipyridine-3ꢀ0-carbonitrile derivatives 5 reacted with several halogen containing
reagents, e.g., 1-chloroacetone, 3-chloropentan-2,4-dione, ethyl chloroacetate, ethyl
2-chloro-3-oxobutanoate, 2-chloroacetamide, chloroacetonitrile, and iodomethane to
afford the corresponding thieno[2,3-b]-pyridine derivatives. Considering the data
of IR, 1 H NMR, mass spectra and elemental analysis the chemical structures of
the newly synthesized heterocyclic compounds elucidated. Cytotoxicity, anti-HSV1,
(anti-Herpes Simplex virus type 1) anti-HAV (Hepatitis A virus), and MBB activity
were evaluated for the newly synthesized heterocyclic compounds.
Keywords
2-Cyanoethanethioamide;
2-methyl-thiopyridine-3ꢀ-carbonitrile;
2-
thioxopyridine; bipyridine-3ꢀ-carbonitrile; thieno[2,3-b]pyridine
INTRODUCTION
In continuation to our previous work1−16 and the reported biological
activity of 2-thioxopyridine17−19 as well as that of thieno[2,3-
b]pyridine,20−21 we were interested in the synthesis of 5ꢀ-acetyl-6ꢀ-
methyl-2ꢀ-thioxo-1ꢀ,2ꢀ-dihydro-3,4ꢀ-bipyridine-3ꢀ-carbonitrile (5) using
Received February 7, 2004; in final form March 16, 2005.
Address correspondence to Fawzy A. Attaby, Cairo University, Chemistry Department,
Faculty of Science, Giza, Egypt. E-mail: fattaby@hotmail.com
1

2
F. A. Attaby et al.
each of 2-cyano-ethanthioamide, pyridine-3-carbaldehyde, and pentan-
2,4-dione. The synthetic potentiality of 5 also was investigated through
its reactions with several reagents, to synthesize several heterocyclic
derivatives, which was required for several chemical transformations
as well as for a medicinal chemistry program.
RESULTS AND DISCUSSION
It has been found that 2-cyanoethanethioamide 1 reacted with pyridine-
3-carbaldehyde 2 in ethanol containing a catalytic amount of triethyl
amine, which gave 2-cyano-3-pyridin-3-ylprop-2-enethioamide 3 that
cyclized in methanolic sodium methoxide with pentan-2,4-dione 4 to af-
ford the corresponding 5ꢀ-acetyl-6ꢀ-methyl-2ꢀ-thioxo-1ꢀ,2ꢀ-dihydro-3,4ꢀ-
bipyridine-3ꢀ-carbonitrile (5) according to Scheme 1.
SCHEME 1
The chemical structure of 5 was elucidated based on its IR, 1H NMR,
and its elemental analysis (cf. Experimental section). Moreover, its
mass spectrum gave m/z = 269, which corresponds to the molecular
weight of the molecular formula C14H11 NOS of the assigned structure.
5ꢀ-acetyl-6ꢀ-methyl-2ꢀ-thioxo-1ꢀ,2ꢀ-dihydro-3,4ꢀ-bipyridine-3ꢀ-carbon-
itrile (5) reacted with 1-chloroacetone (1:1) in methanolic sodium
methoxide solution via dehydrochlorination to afford a reaction prod-
uct, which formulated as 5ꢀ-acetyl-6ꢀ-methyl-2ꢀ-[(2-oxopropyl)thio]-3,4ꢀ-
biphenyl-3ꢀ-carbonitrile 6 by considering the data of IR, 1H NMR,
and elemental analysis (cf. Experimental section). Moreover, its mass
spectrum gave m/z = 325, which corresponds to the molecular weight
of the molecular formula C17H15 N3O2S of the assigned structure.
Compound 6 underwent ring closure through its boiling with 10%
ethanolic KOH for 3–5 h to give the corresponding 1-(2-acetyl-3-amino-
6-methyl-4-pyridin-3-ylthieno[2,3-b]pyridin-5-yl)ethanone (7). The IR
spectrum of compound 7 showed no bands corresponding to the CN
function, while the newly formed NH2 was detected in both IR and
1H NMR (cf. Experimental section). The previously-mentioned result

Carbonitriles
3
SCHEME 2
confirms that both CN and CH2- in 6 are involved in the cyclization
step to give NH2 in 7 (Scheme 2).
Compound 5 reacted with 3-chloropentan-2,4-dione via the dehy-
drochlorination to give 8, and this was followed by addition to the
CN function to give 9, which then adds one water molecule to yield
the corresponding imino derivatives 10 by the elimination of an acetic
SCHEME 3

4
F. A. Attaby et al.
acid molecule. The imino derivative 10 will be converted to more stable
tautomer 7.
It is interesting to note that compound 7 obtained by the two path-
ways is identical in all physical and chemical properties (cf. Experimen-
tal section) (Scheme 3).
In a similar reaction, compound 5 reacted with both ethyl chloroac-
etate and ethyl 2-chloro-3-oxobutanoate to give a reaction prod-
uct which could be formulated as ethyl 5-acetyl-3-amino-6-methyl-4-
pyridin-3-ylthieno[2,3-b]pyridine-2-carboxylate (11), whose structure
could be elucidated by considering the data of elemental analysis, and
1
IR and H NMR. Moreover, the mass spectrum of 11 gave m/z = 355,
which corresponds to the molecular weight of the molecular formula
C18H17N3O3S of the assigned structure.
It is important to report here that compound
5 reacted
with ethyl chloroacetate to give a reaction product 11 via the
SCHEME 4

Carbonitriles
5
isolated
intermediate
ethyl
[(5ꢀ-acetyl-3ꢀ-cyano-6ꢀ-methyl-3,4ꢀ-
bipyridin-2ꢀ-yl)thio]acetate (12), while all trials to isolate the in-
termediate 13 (that 5 reacted with) ethyl 2-chloro-3-oxobutanoate
failed (Scheme 4).
Synthon 5 also reacted with 2-chloroacetamide in ethanol containing
a catalytic amount of triethyl amine through dehydro-chlorination to
give 2-[(5ꢀ-acetyl-3ꢀ-cyano-6ꢀ-methyl-3,4ꢀ-bipyridin-2ꢀ-yl)thio]acetamide
(14), which cyclized in 10% ethanolic potassium hydroxide solution to
give 5-acetyl-3-amino-6-methyl-4-pyridin-3-ylthieno[2,3- b]pyridine-2-
carboxamide (15). The IR of 15 showed no bands of a CN function and
its 1H NMR did not reveal the signals of CH2 protons, while the newly
formed NH2 in 15 was detected by both IR and 1H NMR; this con-
firmed that both CN and CH2 are involved in cyclization of 14 to give
15 (Scheme 5).
SCHEME 5
In a similar investigation, 5 reacted with chloroacetonitrile
and iodomethane in methanolic sodium methoxide solution to
give 5-acetyl-3-amino-6-methyl-4-pyridin-3-ylthieno[2,3-b]pyridine-2-
carbonitrile and 5ꢀ-acetyl-6ꢀ-methyl-2ꢀ-(methylthio)-3,4ꢀ-bipyridine-3ꢀ-
carbonitrile 16 and 17, respectively. The structures (Scheme 6) of 16
SCHEME 6

6
F. A. Attaby et al.
and 17 were elucidated depending on the data given by elemental anal-
1
ysis, IR, and H NMR spectra. Moreover, their mass spectra gave m/z
= 308 and 283, respectively, and these correspond to the molecular for-
mulas C16H12N4 OS and C15H13N3OS of the assigned structures.
It is important to note that the reaction of 5 with chloroacetonitrile
in methoxide solution proceeded through the non-isolable intermedi-
ate 18, and all trials to isolate it failed. Authentic samples of 7, 11,
and 15 were prepared through the reaction of 5 with chloroacetone,
ethyl chloroacetate, and chloroacetamide, respectively, in 10% ethano-
lic KOH solution under reflux for 5–7 h. It is important to report here
that the isolated compounds 7, 11, and 15 are identical in all physical
and chemical properties with that given by the cyclization of 6, 12, and
14.
Biological Evaluation
Cytotoxicity assay
As shown in Table (I), compounds 5, 7, 9, 10, and 12 were much
safer for cell culture inoculation than the other compounds, which show
CPE at 15 µg. Accordingly, the safe doses were selected and used for
antivirus bioassay.
Antivirus bioassay
Screening for Anti-HSV1 Activity. Plaque reduction assay showed
that only compound 11 was promising to be an anti-HSV1 (anti-Herpes
Simplex Virus Type 1) compound. The percentage of virus reduction
TABLE I Cytotoxicity Assay for the Synthetic Compound in a Vero
Cell Line
Cytotoxicity grade of the tested materials
Comp. no.
5 µg
10 µg
15 µg
20 µg
25 µg
30 µg
35 µg
40 µg
5
6
7
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
+2
—
—
—
—
—
—
+3
—
—
+1
—
—
—
—
+2
—
—
—
—
+3
—
—
—
9
10
11
12
+4
+4
+4
—
+2
+2
Note: Cytotoxicity grades are divided into four grads: +1 = 25%, +2 = 50%, +3 =
75%, and +4 = 100% of the cell monolayer showed CPE

Carbonitriles
7
FIGURE 1 Anti-HSV1 bioassay of synthetic compounds in Vero cell line.
was 55% at a dose of 10 µg and increased to 83% with a dose of 15 µg
(Figure 1). The compounds 5, 9, and 12 exhibited a moderate percentage
of virus reduction while all the other compounds had either very weak
activity or no activity at all.
Screening for Anti-HAV, MBB Strain Activity. All compounds were
tested for anti-HAV activity in HepG2 cells by plaque reduction assay.
The results showed that compounds 7, 10, and 12 exhibited a moderate
percentage of virus reduction while the other newly synthesized hetero-
cyclic compounds either exhibited weak or no activity against Hepatitis
a virus (HAV) (Figure 2).
FIGURE 2 Anti-HAV bioassay of synthetic compounds in HepG2 cells.

8
F. A. Attaby et al.
EXPERIMENTAL
All melting points are uncorrected. IR spectra were recorded as KBr
discs on a Shimadzu FTIR-8201PC Spectrophotometer. 1H-NMR spec-
tra were recorded on Varian Mercury 300 MHz and Varian Gemini 200
MHz spectrometers using TMS as an internal standard and CDCl3,
DMSO-d6, and (CD3)2 CO as solvents. Chemical shifts are expressed as δ
ppm units. Mass spectra were recorded on Shimadzu GCMS-QP1000EX
using an inlet type at 70 eV. The Microanalytical Center of Cairo Uni-
versity performed microanalyses.
Synthesis of 5ꢀ-Acetyl-6ꢀ-methyl-2ꢀ-thioxo-1ꢀ,2ꢀ-dihydro-3,4ꢀ-
bipyridine-3ꢀ-carbonitrile (5)
A solution of each of 1 (5 g) and 2 (5.35 g) in absolute ethanol (50 mL)
containing a catalytic amount of piperidine (0.4 mL) was stirred at
room temperature for 15–20 min. The product formed was filtered off
and dried well. The isolated product 3 was obtained as yellow crystals
(8.2 g). Mixture of 3 (9.4 g) and 4 (5 g) reacted in absolute ethanol
(50 mL) containing a catalytic amount of piperidine under reflux for
5 h. The reaction mixture then was evaporated until it was dry and
then cooled. The product formed was collected by filtration, washed
with cold ethanol, and then crystallized from ethanol as yellow crys-
tals (9.8 g), m.p. 246–248◦C; IR (ν cm−1): 3348 (NH), 2897–2932 (CH-
aliphatic); 3051 (pyridine-CH), 2217(CN); 1732 (C Oester) and 1547
1
(C S); H-NMR (δppm): 1.5 (s, 3H, CH3), 2.3 (s, 3H, CH3CO), 4.3 (s,
1H, NH) and 7.3–8.6 (m, 4H, pyridine H’s); Anal. for C14H11N3OS
(269.32): Calcd./Found (%): C, 62.43/62.4; H, 4.12/4.0; N, 15.60/15.5; S,
11.91/11.3.
Synthesis of 5ꢀ-Acetyl-6ꢀ-methyl-2ꢀ-[(2-oxopropyl)thio]-3,4ꢀ-
biphenyl-3ꢀ-carbonitrile 60
A solution of 5 (6.7 g) and 1-chloroacetone (2.3 g) in methanol containing
sodium methoxide (prepared by 0.6 g of sodium in 40 mL methanol) was
stirred for 1 h. The product formed was collected by filtration, washed
with cold ethanol, and then recrystallized from ethanol to give 6 as yel-
low crystals (6.3 g), m.p. 150◦C; IR (ν cm−1): 2887–2922 (CH-aliphatic);
3055 (pyridine-CH), 2219(CN) and 1719 (C O acetyl); 1H-NMR (δppm):
1.8 (s, 3H, CH3 COCH2), 2.1 (s, 3H, CH3), 2.5 (s, 3H, CH3 CO), 4.1(s, 2H,
CH3CO CH2) and 7.3–8.6 (m, 4H, pyridine H’s); Anal. for C17 H15N3 O2
S (325): Calcd./Found (%): C, 62.75/62.4; H, 4.65/4.2; N, 12.91/12.5; S,
9.85/9.7.

Carbonitriles
9
Synthesis of Ethyl [(5ꢀ-Acetyl-3ꢀ-cyano-6ꢀ-methyl-3,4ꢀ-
bipyridin-2ꢀ-yl)thio]acetate 12
A solution of 5 (6.7 g) and ethyl chloroacetate (3.3 g) in methanol
containing sodium methoxide (prepared by 0.6 g of sodium in 40 mL
methanol) was stirred for 1 h. The product formed was collected by fil-
tration, washed with cold ethanol, and then recrystallized from ethanol
to give 12 as pale orange crystals (6.6 g), m.p. 204◦C; IR (ν cm−1): 2889–
2931 (CH-aliphatic); 3062 (pyridine-CH), 2222(CN) 1735 (C O ester)
and 1713 (C O acetyl); 1H-NMR (δppm): 1.2 (t, 3H, CH3 CH2-), 2.1 (s,
3H, CH3), 2.6 (s, 3H, CH3 CO), 3.7 (s, 2H, -CH2 COOC2 H5), 4.5 (q, 2H,
CH3 CH2-) and 7.1–8.7 (m, 4H, pyridine H’s); Anal. for C18 H17 N3 O3
S (355): Calcd./Found (%): C, 60.83/60.7; H, 4.82/4.7; N, 11.82/11.9; S,
9.02/9.1.
Synthesis of 2-[(5ꢀ-Acetyl-3ꢀ-cyano-6ꢀ-methyl-3,4ꢀ-bipyridin-
2ꢀ-yl)thio]-acetamide 14
A solution of 5 (6.7 g) and chloroacetamide (2.3 g) in methanol contain-
ing sodium methoxide (prepared by 0.6 g of sodium in 40 mL methanol)
was stirred for 1 h. The product formed was collected by filtration,
washed with cold ethanol, and then recrystallized from ethanol to give
14 as pale brown crystals (6.4 g), m.p. 165◦C; IR (ν cm−1): 3432, 3378,
3344 (NH2), 2882–2921 (CH-aliphatic); 3052 (pyridine-CH), 2220(CN)
1715 (C O acetyl) and 1693 (C O amide); 1H-NMR (δ ppm): 2.0 (s, 3H,
CH3), 2.5 (s, 3H, CH3CO), 3.8 (s, 2H, CH2 CONH2), 6.1 (s, br., 2H,
NH2) and 6.9–8.6 (m, 4H, pyridine H’s); Anal. for C16H14N4O2S (326):
Calcd./Found (%): C, 58.55/58.4; H, 4.32/4.2; N, 17.17/17.2; S, 9.82/9.8.
Synthesis of 5ꢀ-acetyl-6ꢀ-methyl-2ꢀ-(methylthio)-3,4ꢀ-
bipyridine-3ꢀ-carbonitrile 17
A solution of 5 (6.7 g) and iodomethane (4.7 g) in methanol containing
sodium methoxide (prepared by 0.6 g of sodium in 40 mL methanol) was
stirred for 1 h. The product formed was collected by filtration, washed
with cold ethanol, and then recrystallized from ethanol to give 17 as
pale yellow crystals (4.9 g), m.p. 188◦C; IR (ν cm−1): 2885–2933 (CH-
1
aliphatic); 3059 (pyridine-CH), 2219(CN) and 1718 (C O acetyl); H-
NMR (δppm): 1.9 (s, 6H, two CH3), 2.5 (s, 3H, CH3CO) and 7.1–8.8 (m,
4H, pyridine H’s); Anal. for C15H13N3OS (283): Calcd./Found (%): C,
63.58/63.4; H, 4.62/4.7; N, 14.83/14.9; S, 11.32/11.4.

10
F. A. Attaby et al.
Synthesis of 1-(2-Acetyl-3-amino-6-methyl-4-pyridin-3-
ylthieno[2,3-b]pyridin-5-yl)ethanone 7
First Method
A solution of 5 (6.7 g) and 1-chloroacetone (2.3 g) in ethanol (30 mL)
and KOH 10% solution (10 mL) was heated under reflux for 3 h. The
reaction mixture was cooled, poured onto ice-cold water, and acidified
with hydrochloric acid; the product formed was collected by filtration,
washed with water followed by cold ethanol, and recrystallized from
ethanol, to give 7.
Second Method: (Cyclization of 6)
A solution of 6 (3.6 g) in ethanol (30 mL) and KOH 10% solution
(10 mL) was heated under reflux for 3 h. The reaction mixture was
cooled, poured into ice-cold water, and acidified with hydrochloric acid;
the product formed was collected by filtration, washed with water fol-
lowed by cold ethanol, and recrystallized from ethanol to give 7 as
yellowish white crystals (2.1 g), m.p. 190◦C; IR (ν cm−1): 3432, 3389,
3332 (NH2), 2894–2938 (CH-aliphatic); 3073 (pyridine-CH), and 1715
1
(C O acetyl); H-NMR (δppm): 2.6 (s, 9H, three CH3), 4.3 (s, br., 2H,
NH2) and 7.2–8.7 (m, 4H, pyridine H’s); Anal. for C17 H15N3O2S (325):
Calcd./Found (%): C, 62.75/62.8; H, 4.65/4.7; N, 12.91/12.7; S, 9.83/9.9.
Synthesis of Ethyl 5-Acetyl-3-amino-6-methyl-4-pyridin-3-
ylthieno-[2,3-b]pyridine-2-carboxylate 11
First Method
A solution of 5 (6.7 g) and ethyl chloroacetate (3.1 g) or ethyl 2-
chloro-3-oxobutanoate (4.1 g) in ethanol (30 mL) and KOH of a 10%
solution (10 mL) was heated under reflux for 3 h. The reaction mixture
was cooled, poured onto ice-cold water, and acidified with hydrochloric
acid; the product formed was collected by filtration, washed with water
followed by cold ethanol, and recrystallized from ethanol to give 11.
Second Method: (Cyclization of 12)
A solution of 12 (3.9 g) in ethanol (30 mL) and KOH 10% solution
(10 mL) was heated under reflux for 3 h. The reaction mixture was
cooled, poured into ice-cold water, and acidified with hydrochloric acid,
the product formed was collected by filtration, washed with water fol-
lowed by cold ethanol, and recrystallized from ethanol to give 11 as pale
brown crystals (2.5 g), m.p. 187◦C; IR (ν cm−1): 3443, 3388, 3342 (NH2),
2887–2925 (CH-aliphatic); 3062 (pyridine-CH), 1732 (C O ester) and
1712 (C O acetyl); 1H-NMR (δppm): 1.2 (t, 3H, CH3 CH2 ), 2.5 (s, 6H,

Carbonitriles
11
two CH3), 3.9 (s, br., 2H, NH2), 4.5 (q, 2H, CH3 CH2 ) and 7.0–8.6 (m,
4H, pyridine H’s); Anal. for C18H17N3O3 S (355): Calcd./Found (%): C,
60.83/60.8; H, 4.82/4.7; N, 11.82/11.7; S, 9.02/9.1.
Synthesis of 5-Acetyl-3-amino-6-methyl-4-pyridin-3-
ylthieno[2,3-b-pyridine-2-carboxamide 15
First Method
A solution of 5 (6.7 g) and chloroacetamide (2.3 g) in ethanol (30 mL)
and KOH 10% solution (10 mL) was heated under reflux for 3 h. The
reaction mixture was cooled, poured onto ice-cold water, and acidified
with hydrochloric acid; the product formed was collected by filtration,
washed with water followed by cold ethanol, and recrystallized from
ethanol, to give 15.
Second Method: (Cyclization of 14)
A solution of 14 (3.6 g) in ethanol (30 mL) and KOH 10% solution
(10 mL) was heated under reflux for 3 h. The reaction mixture was
cooled, poured into ice-cold water, and acidified with hydrochloric acid;
the product formed was collected by filtration, washed with water fol-
lowed by cold ethanol, and recrystallized from ethanol to give 15 as
white crystals (2.6 g), m.p. 248◦C; IR (ν cm−1): 3454, 3394, 3364 (NH2),
2889–2932 (CH-aliphatic); 3056 (pyridine-CH), 1713 (C O acetyl) and
1697 (C O amide); 1H-NMR (δppm): 2.0 (t, 3H, CH3), 2.5 (s, 3H, CH3),
4.0 (s, br., 2H, NH2), 6.2 (s, br., 2H, CO NH2) and 7.3–8.9 (m, 4H, pyri-
dine H’s); Anal. for C18H17N3O3S (326): Calcd./Found (%): C, 58.88/58.7;
H, 4.32/4.5; N, 17.17/17.2; S, 9.82/9.9.
Synthesis of 5-Acetyl-3-amino-6-methyl-4-pyridin-3-
ylthieno[2,3-b-pyridine-2-carbonitrile 16
A solution of 5 (2.9 g) and chloroacetonitrile (0.8 g) in methanolic sodium
methoxide (0.3 g of sodium in 30 mL methanol) was stirred for 1–2 h
or was heated under reflux for 20–30 min. The product was formed,
filtrated off, washed with cold ethanol, and recrystallized from ethanol
to give 16 as dark brown crystals (2.7 g), m.p. 110◦C; IR (ν cm−1): 3433,
3389, 3367 (NH2), 2884–2927 (CH-aliphatic); 3054 (pyridine-CH), 2220
1
(CN) and 1717 (C O acetyl); H-NMR (δppm): 2.0 (t, 3H, CH3), 2.5 (s,
3H, CH3), 4.3 (s, br., 2H, NH2) and 7.1–8.7 (m, 4H, pyridine H’s); Anal.
for C16 H12N4OS (308): Calcd./Found (%): C, 62.32/62.4; H, 3.92/4.1; N,
18.17/18.0; S, 10.40/10.5.

12
F. A. Attaby et al.
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 were: African green monkey kidney-derived cells (VERO), cell
line obtained from American type culture collection (ATCC), and human
hepatoma cell line (Hep G2).
Viruses
Two models of DNA & RNA viruses were used for bioassay. These
viruses were HSV-1 and HAV, MBB strain (HAN-MBB).
II-Media and Supplements
Cell Culture Medium
Minimum Essential Media (MEM with Hank’s balanced salt solu-
tion, GIBCO-BRL) was prepared and sterilized by filtration through
0.22 µm of pore-size nitrocellulose membrane; the pH value was ad-
justed at 7.4 by sodium bicarbonate.
Foetal Bovine Serum (FBS): (Sigma)
FBS was inactivated at 56◦C for 30 min and used at 10% final con-
centration for a growth medium and at 2% for a maintenance medium.
Antibiotic–Antimycotic Mixture (GIBCO-BRL)
100 × antibiotic–antimycotic mixtures consisted of 10,000 U peni-
cillin G sodium, 10,000 µg streptomycin sulfate, and 25 µg ampho-
tericin 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 con-
centrations: NaCl (8.9 g/L), KCl (0.2 g/L), KH2 PO4 (0.12 g/L), Na2 HPO4
(0.91 g/L), and deionized H2O (up to 1 L). Ingredients were mixed gen-
tly in this order and the pH value was adjusted at 7.5. The buffer was
sterilized by filtration through a 0.22-µm nitrocellulose membrane. The
solution was used in the washing of cell monolayer sheets and in prepa-
ration of cell dissociation solution as follows.
Trypsin 1:250 (Sigma). 1.5 gm of trypsin powder (1:250) was dis-
solved in 500 ml PBS and digested at 4◦C overnight with stirring.

Carbonitriles
13
Versene Solution (0.04%). Tetrasodium salt of ethylenediamine
tetraacetic acid was dissolved in 500 mL of 0.15 M PBS pH 7.5 to pre-
pare 2 mM solution (0.04 gm) and mixed with equal volume of trypsin
solution. The pH value of the trypsin–versene mixture was adjusted
at 8.4 by a 7.5% sodium bicarbonate solution and sterilized by filtra-
tion through a 0.22-µm pore-size nitrocellulose membrane. 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% Dimethyl
sulfoxide (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 am-
photericin B. Sterility test were carried out in nutrient agar.
II: Cell Culture
VERO were used. The cells were Propagated in Hanks’ MEM, and
supplemented with 10% FBS, and 1% antibiotic-antimycotic mixture.
The pH was adjusted at 7.2–7.4 by a 7.5% sodium bicarbonate solution.
The mixture was sterilized by filtration through a 0.22 µm pore-size
nitrocellulose membrane.
III: Viruses
HSV-1, obtained from Environmental Virology lab Department of
Water Pollution Res., National Research Centre, Dokki, Egypt.
IV: Antiviral Assay
Cytotoxicity Assay. Cytotoxicity was assayed for both 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 it with trypan
blue dye.
Plaque Reduction Assay. A 6-well plate was cultivated with a VERO
cell culture (105 cell/mL) and incubated for 2 days at 37◦C. HSV-1 was
diluted to give a 104 PFU/mL final concentration and mixed with the
plant extract at the previous concentration and incubated overnight
at 4◦C. Growth medium was removed from the multiwell plate and a
virus–compound mixture was inoculated (100 µl/well). After 1 h contact

14
F. A. Attaby et al.
time, the inoculum was aspirated and 3 mL of MEM with 1% agar rose
overlaid 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 reduc-
tion was calculated.
REFERENCES
[1] F. A. Attaby, Arch. Pharmcol. Res., 13, 342 (1990).
[2] F. A. Attaby, L. I. Ibrahim, S. M. Eldin, and A. K. K El-louh, Phosphorous, Sulfur,
and Silicon, 73, 127 (1992).
[3] F. A. Attaby, S. M. Eldin, and M. Abdel Razik, Phosphorous, Sulfur, and Silicon,
106, 21 (1994).
[4] F. A. Attaby and A. M. Abdel-Fattah, Phosphorous, Sulfur, and Silicon, 119, 257
(1996).
[5] F. A. Attaby, Phosphorous, Sulfur, and Silicon, 126, 27 (1997).
[6] F. A. Attaby, Phosphorous, Sulfur, and Silicon, 139, 1 (1998).
[7] F. A. Attaby, S. M. Edin, and M. A. A. Elneairy, Heteroatom Chemistry, 9, 571 (1998).
[8] F. A. Attaby, S. M. Edin, and M. A. A. Elneairy, J. Chem. Res. (M), 10, 2754, (S)
(1998).
[9] F. A. Attaby, M. A. A. Elneairy, and M. S. Elsayed, Phosphorous, Sulfur, and Silicon,
149, 230 (1999).
[10] F. A. Attaby and A. M. Abdel-Fattah, Phosphorous, Sulfur, and Silicon, 155, 253
(1999).
[11] M. A. A. Elneairy, Sanaa M. Eldin, Fawzy A. Attaby, and A.K. K. El-Louh, Phospho-
rous, Sulfur, and Silicon, 167, 289 (2000).
[12] M. A. A. Elneairy, F. A. Attaby, and M. S. Elsayed, Phosphorus, Sulfur, and Silicon,
167, 161 (2000).
[13] F. A. Attaby, M. A. A. Elneairy, S. M. Eldin, and A. K. K. El-Louh, Journal of the
Chinese Chemical Society (China), 48, 893 (2001).
[14] F. A. Attaby, H. M. Mostafa, A. H. H. Elghandour, and Y. M. Ibrahem, Phosphorus,
Sulfur, and Silicon, 177, 2753 (2002).
[15] F. A. Attaby, A. H. H. Elghandour, H. M. Mustafa, and Y. M. Ibrahem, Journal of
the Chinese Chemical Society, 49, 561 (2002).
[16] F. A. Attaby, S. M. Eldin, M. A. A. Elneairy, and A. K. K. Elouh, Phosphorus, Sulfur,
and Silicon, 179, 2205 (2004).
[17] N. Borg and H. Tjaelve, Arch. Toxicol., 68, 450 (1994).
[18] J. Gottofrey and H. Tjaelve, Water, Air, Soil Pollut., 56, 521 (1991).
[19] A. Rumler, V. Hagen, and A. Hagen, Pharmazie, 45, 657 (1990).
[20] K. Umemura, H. Kawai, H. Tshihara, and M. Nakashima, Jpn. J. Pharmacol., 67,
253 (1995).
[21] S. C. Klein, J. P. Cazenave, J. M. Herbert, and J. P. Maffrand, J. Cell-Physiol., 160,
316 (1994).