Synthesis, reactions, and antiviral activity of 1-(1H-pyrazolo[3,4-b] pyridin-5-yl)ethanone and pyrido[2′,3′:3,4]pyrazolo[5,1-c][1,2,4] triazine derivatives

Article abstract of DOI:10.1080/10426500500326404

1-(3-Amino-6-methyl-4-pyridin-3-yl-1H-pyrazolo[3,4-b]pyridin-5-yl)ethano ne (3) was obtained in very pure state and used as a good starting material for the present study. It diazotized to give the corresponding diazonium salt 9 and also reacted with phenyl isothiocyanate to give the corresponding thiourea derivative 4. Compound 4 was used for the preparation of thiazole derivatives 5-8 via the reaction with active halogen-containing compounds. On the other hand, compound 9 coupled with several active -CH₂- containing compounds to afford the corresponding triazine derivatives 10-17. Considering the data from IR, ¹H NMR, mass spectra, and elemental analyses, the chemical structures of the newly synthesized heterocyclic compounds were elucidated. Cytotoxicity, anti-HSV1, and anti-HAV-MBB activity were evaluated for the newly synthesized heterocyclic compounds. Copyright Taylor & Francis Group, LLC.

Full text of DOI:10.1080/10426500500326404

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Phosphorus, Sulfur, and Silicon
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Synthesis, Reactions,
and Antiviral Activity
of 1-(1H-Pyrazolo[3,4-
b]pyridin-5-yl)ethanone and
Pyrido[2′,3′:3,4]pyrazolo[5,1-
c][1,2,4]triazine Derivatives
Fawzy A. Attaby a , A. H. H. Elghandour a , M. A. Ali
b & Yasser M. Ibrahem c
a Chemistry Department , Cairo University , Giza,
Egypt
b Virology Lab., National Research Center , Dokki,
Egypt
c Chemistry Department, Cairo University , Cairo,
Egypt
Published online: 01 Feb 2007.
To cite this article: Fawzy A. Attaby , A. H. H. Elghandour , M. A. Ali & Yasser M.
Ibrahem (2006) Synthesis, Reactions, and Antiviral Activity of 1-(1H-Pyrazolo[3,4-
b]pyridin-5-yl)ethanone and Pyrido[2′,3′:3,4]pyrazolo[5,1-c][1,2,4]triazine
Derivatives, Phosphorus, Sulfur, and Silicon and the Related Elements, 181:5,
1087-1102, DOI: 10.1080/10426500500326404
To link to this article: http://dx.doi.org/10.1080/10426500500326404
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Phosphorus, Sulfur, and Silicon, 181:1087–1102, 2006
Copyright © Taylor & Francis Group, LLC
ISSN: 1042-6507 print / 1563-5325 online
DOI: 10.1080/10426500500326404
Synthesis, Reactions, and Antiviral Activity of
1-(1H-Pyrazolo[3,4-b]pyridin-5-yl)ethanone and
Pyrido[2ꢀ,3ꢀ:3,4]pyrazolo[5,1-c][1,2,4]triazine Derivatives
Fawzy A. Attaby
A. H. H. Elghandour
Chemistry Department, Cairo University, Giza, Egypt
M. A. Ali
Virology Lab., National Research Center, Dokki, Egypt
Yasser M. Ibrahem
Chemistry Department, Cairo University, Cairo, Egypt
1-(3-Amino-6-methyl-4-pyridin-3-yl-1H-pyrazolo[3,4-b]pyridin-5-yl)ethanone (3)
was obtained in very pure state and used as a good starting material for the
present study. It diazotized to give the corresponding diazonium salt 9 and also
reacted with phenyl isothiocyanate to give the corresponding thiourea derivative
4. Compound 4 was used for the preparation of thiazole derivatives 5–8 via
the reaction with active halogen-containing compounds. On the other hand,
compound 9 coupled with several active –CH - containing compounds to afford
2
the corresponding triazine derivatives 10–17. Considering the data from IR, 1 H
NMR, mass spectra, and elemental analyses, the chemical structures of the newly
synthesized heterocyclic compounds were elucidated. Cytotoxicity, anti-HSV1,
and anti-HAV-MBB activity were evaluated for the newly synthesized heterocyclic
compounds.
Keywords N-phenylthiourea; 1H-pyrazolo[3,4-b]pyridin-5-yl)ethanone; 1,3-thiazolidin-
4-one; 2-cyanoethanethioamide; pyrido[2ꢀ,3ꢀ:3,4]pyrazolo[5,1-c][1,2,4]triazine
INTRODUCTION
In previous papers,1−17 the synthesis of pyrazolopyridine and pyridopy-
razolotriazine derivatives were described. As a continuation of our re-
search on the synthesis of these ring systems and owing to the re-
ported biological activities of pyrazolopyridine18,19 and thiazoles,20−22
Received May 1, 2005; accepted June 25, 2005.
Address correspondence to Fawzy A. Attaby, Cairo University, Chemistry Department,
Faculty of Science, Giza, Egypt. E-mail: fattaby@hotmail.com
1087

1088
F. A. Attaby et al.
as well as that of triazines,23−26 we were interested to synthesize several
derivatives of these ring systems that are required for several chemical
transformations as well as for our medicinal chemistry program.
RESULTS AND DISCUSSION
It was found that 5ꢀ-acetyl-6ꢀ-methyl-2ꢀ-(methylthio)-3,4ꢀ-bipyridine-
3ꢀ-carbonitrile (2) reacted with hydrazine hydrate in ethanol to give
the sulfur-free 1-(3-Amino-6-methyl-4-pyridin-3-yl-1H-pyrazolo-[3,4-
b]pyridin-5-yl)ethanone (3). The chemical structure of 3 was elucidated
1
based on the IR, H NMR, and elemental analysis (cf. Experimental
section). Moreover, its mass spectrum gave m/z = 267, which corre-
sponded to the molecular weight of a molecular formula C14H13N5O of
the assigned structure. A further elucidation of structure 3 arose from
its preparation from the reaction of 5ꢀ-acetyl-6ꢀ-methyl-2ꢀ-thioxo-1ꢀ,2ꢀ-
dihydro-3,4ꢀbipyridine-3ꢀ-carbonitrile (1) with hydrazine hydrate. It is
remarkable that the product of the two pathways was identical in all
physical and chemical properties (cf. Exp. section and Scheme 1).
The synthetic potentiality of 3 was investigated through its reaction
with phenyl isothiocyanate in pyridine to give the corresponding
N-(5-acetyl-6-methyl-4-pyridin-3-yl-1H-pyrazolo[3,4-b]pyridin-3-yl)-
N-phenyl-thiourea (4). The IR, 1H NMR, and elemental analysis of
this reaction product were found to be in a good agreement with the
assigned structure (cf. Exp. section and Scheme 1). Moreover, the mass
spectrum of 4 gave m/z = 402, which corresponded to the molecular
weight of a molecular formula C21H18N6OS of the assigned structure.
Compound 4 reacted with ethyl 2-chloro-3-oxobutanoate, 3-chlo-
ropentan-2,4-dione, 2-bromo-1-phenylethanone, and 2-bromo-1-(4ꢀ-
chlorophenyl)-ethanone in pyridine to give 1,3-thiazolidin-4-one
derivative 5, which tautomerized to the more stable isomer1-[(5-
acetyl-3-phenyl-4-hydroxy-1,3-thiazol-2(3H)-ylidene)amino]-6-methyl-
4-pyridin-3-yl-1 H-pyrazolo[3,4-b]pyridin-5-yl)ethanone (6), 4-(3-pyridyl)-
5-acetyl-6-methyl-N-[4-methyl-3-phenyl-5-acetyl-1,3-thiazol-2(2H)yli-
dine]-1H-pyrazolo-[3,4-b]pyridin-3-amine (7), and 1-([3,4-diaryl-1,3-
thiazol-2-ylidine]amino-6-methyl-4-pyridin-3-yl-1H-pyrazolo-[3,4-b]-
pyridin-5-yl)ethanone 8a,b, respectively. The chemical structures of
compounds 6–8 were elucidated by considering the data of IR, 1H
NMR, and elemental analyses (cf. Experimental section). Moreover,
the mass spectra of 6, 7, and 8a,b gave m/z = 442, 482, 502, and 537,
which corresponded to the molecular weights of the molecular formulas
C23H18N8O2S, C26H22N6O2S, C29H22N6O2S, and C29H21N6OS, of the
assigned structures, respectively (cf. Exp. section and Scheme 2).

1-(1H-Pyrazolo[3,4-b]pyridin-5-yl)ethanone
1089
SCHEME 1
The chemical reactivity of 3 was further investigated via its
reaction with nitrous acid to give the corresponding 3-diazotized
aminopyrazolo[3,4-b]pyridine derivative 9, which was used for the
preparation of triazine derivatives 10–17 via its coupling with several
active hydrogen-containing compounds. It is remarkable to report that
all trials to isolate 9 as a solid product failed. Thus, the treatment of 9

1090
F. A. Attaby et al.
SCHEME 2
with malononitrile, ethyl cyanoacetate, and 2-cyanoethanethioamide
in cold ethanolic sodium acetate gave reaction products, which corre-
sponded to addition of equimolecular amounts of the reactants with
dehydrochlorination. The IR spectra of these reaction products showed
the bands of NH2 that were detected also in the 1H NMR spectra.
Moreover, the IR spectra of these reaction products showed the CN
function, CO ester, and CSNH2 respectively (cf. Experimental section).
By considering the data of IR, 1H NMR, and elemental analyses,
these reaction products could be formulated as 9-acetyl-4-amino-
8-methyl-10-pyridin-3-ylpyrido[2ꢀ,3ꢀ:3,4]pyrazolo[5,1-c]-[1,2,4]triazine-
3-carbonitrile (10), ethyl9-acetyl-4-amino-8-methyl-10-pyridin-3-ylpyr-
ido[2ꢀ,3ꢀ:3,4]pyrazolo[5,1-c][1,2,4]-triazine-3-carboxylate (11), and 1-(4-
amino-3-thiocarboxamido-8-methyl-10-pyridin-3-yl pyrido-[2’,3’:3,4]-
pyrazolo[5,1-c][1,2,4]triazin-9-yl)ethanone (12), respectively. The

1-(1H-Pyrazolo[3,4-b]pyridin-5-yl)ethanone
1091
SCHEME 3
mass spectra of 10, 11, and 12 gave m/z = 346, 393, and 389, which
corresponded to the molecular weights of the molecular formulas
C17H14N8O, C19H19N7O3, and C17H16N8OS of the assigned structure,
respectively (Scheme 3).
In a similar reaction, 9 reacted with ethyl 3-oxobutanoate and/or
pentan-2,4-dione in cold ethanolic sodium acetate to afford the corre-
sponding triazine derivatives 13 and 14 whose structures were elu-
cidated from IR, 1H NMR, and elemental analyses (cf. Experimen-
tal section). Moreover, their mass spectra gave m/z = 392 and 362,
which corresponded to the molecular weights of the molecular formu-
lae C20H20N6O3 and C19H18N6O2 of the assigned structures. A fur-
ther confirmation of the 10, 11, 12, 13, and 14 structures obtained
from the reactions between 9 and malononitrile, ethyl cyanoacetate, 2-
cyanoethanethioamide, ethyl 3-oxobutanoate, and/or pentan-2,4-dione
in boiling ethanol containing catalytic amounts of triethylamine. It is
important to refer here that the reaction products obtained by the two
pathways were identical in all physical and chemical properties (cf. Exp.
section and Scheme 4).
Compound 9 reacted with diethyl malonate in cold ethanolic sodium
acetate to give a reaction product 15 via dehydrochlorination, followed
by cyclization through ethanol elimination. The structure of 15 was es-
tablished from analysis of IR, 1H NMR, and elemental analyses (cf. Ex-
perimental section) Moreover, its mass spectrum gave m/z = 394, which
corresponded to the molecular weight of a molecular formula of the as-
signed structure. In contrast to the behavior of 9 in the previously men-
tioned reactions, 9 reacted with ethyl 3-oxo-3-phenylpropanoate under
the same experimental conditions to give the open-chain structure 16,
which was obtained through the dehydrochlorination. Compound 16 un-
derwent cyclization by treatment with triethylamine in boiling ethanol
to give the corresponded triazine derivative 17 whose structure was

1092
F. A. Attaby et al.
SCHEME 4
SCHEME 5
1
elucidated from analysis of IR, H NMR, and elemental analyses (cf.
Exp. section and Scheme 5).
Further confirmation of both 15 and 17 structures was given through
their preparation authentically by boiling 9 with diethyl malonate and
ethyl 3-oxo-3-phenylpropanoate, respectively, in ethanol containing a
catalytic amount of triethylamine (cf. Experimental section).
Biological Evaluation
Cytotoxicity Assay
As shown in Table (I), compounds 3, 10, 11, 12, 13, 14, 15, and 17
were safe for cell culture inoculation than were the other compounds,
which show CPE (cytopathic effect) at 15 µg. Accordingly, the safe doses
were selected and used for an antivirus bioassay.

1-(1H-Pyrazolo[3,4-b]pyridin-5-yl)ethanone
1093
TABLE I Cytotoxicity Assay for the Synthetic Compound in a Vero
Cell Line
Cytotoxicity grade of the tested materials
Ser. no.
5 µg
10 µg
15 µg
20 µg
25 µg
30 µg
35 µg
40 µg
3
4
6
7
—
+2
+1
+2
+4
+1
—
—
—
—
—
—
+4
+4
+4
+4
+3
—
—
—
—
—
—
+4
+4
+4
+4
+4
—
—
+4
+4
+4
+4
+4
—
—
+4
+4
+4
+4
+4
—
—
+4
+4
+4
+4
+4
—
—
—
+4
+4
+4
+4
+4
—
+1
+3
+3
+4
+2
+4
+1
+4
+4
+4
+4
+4
—
+1
+4
+4
+4
+2
+4
+1
8a
8b
10
11
12
13
14
15
16
17
—
—
—
—
+1
+1
—
+1
+1
+1
+1
+3
—
+1
+1
+2
+1
+4
—
+2
+2
+3
+2
+4
+1
—
—
—
—
—
—
—
+1
—
Cytotoxicity grades are divided into four grads: +1 = 25% of the cell monolyer showed
CPE; +2 = 50% of the cell monolayer showed CPE; +3 = 75% of the cell monolayer showed
CPE; and +4 = 100% of the cell monolayer showed CPE.
Antivirus Bioassay
Screening for anti-HSV1 activity. Plaque reduction assay showed
that compounds 3, 4, 6, and 7 were promising as anti-HSV1 compounds
at a dose of 15 µg, and the percentage of virus reduction increased at
25 µg. Compound 12 only gave virus reduction by 55% at a dose of 15 µg,
and compound 8b only exhibited virus reduction at a dose of 25 µg. All
other compounds either had very week activity or no activity at all (cf.
Figure 1).
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 4, 6, and 7
exhibited good percentages of virus reduction at both 15 µg and 25 µg
doses, while compound 8a exhibited a 53% virus reduction at a dose of
25 µg. Compound 8b showed moderate activity at both 15 µg and 25 µg
doses. The other newly synthesized heterocyclic compounds exhibited
no activity against HAV (cf. Figure 2).

1094
F. A. Attaby et al.
FIGURE 1 Anti-HSV1 bioassay of synthetic compounds in a Vero cell line.
EXPERIMENTAL
All melting points were uncorrected. IR (KBr discs) spectra were
recorded on a Shimadzu FTIR-8201PC spectrophotometer. 1H-NMR
spectra were recorded on a Varian Mercury 300 MHz and Varian Gemini
200 MHz. spectrometers using TMS as an internal standard and CDCl3,
DMSO-d6, and (CD3)2CO as solvents. Chemical shifts were expressed
FIGURE 2 Anti-HAV bioassay of synthetic compounds in HepG2 cells.

1-(1H-Pyrazolo[3,4-b]pyridin-5-yl)ethanone
1095
as δ (ppm) units. Mass spectra were recorded on Shimadzu GCMS-
QP1000EX using an inlet type at 70 eV. The Microanalytical Center of
Cairo University performed the microanalyses.
Synthesis of 3
A mixture of 2 or 1 (0.01 mole of each) and hydrazine hydrate
(20–25 mL) was heated under reflux for 8–10 h. The product formed
after cooling was filtered off, washed with cold ethanol, and crystal-
lized from ethanol to give 3.
1-(3-Amino-6-methyl-4-pyridin-3-yl-1H-pyrazolo[3,4-b]pyridin-
5-yl)-ethanone (3)
Crystallized from ethanol as yellow crystals (81%); m.p. 284–286◦C;
IR (cm−1): 3450, 3267, 3179 (NH2, NH), 3043 (pyridine-CH), 2982 (sat.
1
CH) and 1705 (acetyl-CO); H-NMR (δ): 1.3 (s, 3H, CH3), 2.5 (s, 3H,
CH3CO), 3.9 (s, br., 2H, NH2), 5.8(s, br., 1H, NH) and 7.3–8.3 (m, 4H,
Pyridine H’s); Anal. for C14H13N5O (267): Calcd./Found: C, 62.91/63.1%;
H, 4.90/5.0%; N, 26.20/26.4%.
Synthesis of 4
A solution of 3 (0.01 mole) in pyridine (35 mL) was treated with phenyl
isothiocyanate (0.01 mole). The reaction mixture was heated under re-
flux for 5 h, then cooled, poured onto ice-cold water, and acidified with
dilute HCl. The solid product so formed was filtered off, washed with
water, and crystallized from ethanol to give 4.
1-(5-Acetyl-6-methyl-4-(pyridin-3-yl)-1H-pyrazolo[3,4-b]-
pyridin-3-yl)-3-phenylthiourea (4)
Crystallized from ethanol as white crystals, (77%), m.p. 206–
208◦C; IR (cm−1): 3382, 3277, 3205 (three NH), 3049 (pyridine-
CH), 2986 (sat. CH) and 1701 (acetyl-CO); 1H-NMR (δ): 1.2 (s,
3H, CH3), 2.4 (s, 3H, CH3CO), 5.8–6.3 (m, 3H, three NH) and
7.5–8.7 (m, 9H, ArH’s and Pyridine H’s); Anal. for C21H18N6OS
(402): Calcd./Found: C, 62.67/62.4%; H, 4.51/4.5%; N, 20.88/21.1%; S,
7.97/8.1%.
Synthesis of 6
A solution of 4 (0.01 mole) in ethanol (50 mL) in the presence of
sodium acetate (1 gm) was heated under reflux with ethyl 2-chloro-
3-oxobutanoate (0.01 mole) for 5 h. After cooling, the solid so formed

1096
F. A. Attaby et al.
was filtered off, washed with water, and crystallized with ethanol to
give 6.
1-[(5-Acetyl-3-phenyl-4-hydroxy-1,3-thiazol-2(3H)-
ylidene)amino]-6-methyl-4-pyridin-3-yl-1H-pyrazolo[3,4-
b]pyridin-5-yl)ethanone(6)
Crystallized from ethanol as pale yellow crystals (69%) m.p. 248–
250◦C; IR (cm−1): 3452, (OH), 3175 (NH), 3050 (aromatic-CH), 2973
(sat. CH) and 1698 (acetyl-CO); 1H-NMR (δ): 1.1 (s, 3H, CH3), 2.5
(s, 6H, two CH3CO), 5.1 (s, 1H, OH) 6.3 (s, br., 1H, NH) and
7.2–8.4 (m, 9H, ArH’s and Pyridine H’s); Anal. for C25H20N6O3S
(484): Calcd./Found: C, 61.97/62.1%; H, 4.16/4.2%; N, 17.34/17.1%; S,
6.62/6.7%.
Synthesis of 7
A solution of 4 (0.01 mole) in ethanol (50 mL) in the presence of sodium
acetate (1 gm) was heated under reflux with 3-chloropentan-2,4-dione
(0.01 mole) for 5 h. After cooling, the solid so formed was filtered off,
washed with water, and crystallized with ethanol to give 7.
4-(3-Pyridyl)-5-acetyl-6-methyl-N-[4-methyl-3-phenyl-5-acetyl-
1,3-thiazol-2(2H)-ylidine]-1H-pyrazolo-[3,4-b]pyridin-3-amine
(7)
Crystallized from ethanol as yellow crystals (72%) m.p. 264–266◦C;
IR (cm−1): 3135 (NH), 3061 (aromatic-CH), 2879 (sat. CH) and 1707
(acetyl-CO); 1H-NMR (δ): 1.3 (s, 6H, two CH3), 2.5 (s, 6H, two CH3CO),
6.2 (s, 1H, NH) and 7.3–8.5 (m, 9H, ArH’s and Pyridine H’s); Anal.
for C26H22N6O2S (482): Calcd./Found: C, 64.71/64.5%; H, 4.6/4.5%; N,
17.42/17.5%; S, 6.65/6.7%.
Synthesis of 8a,b
A solution of 4 (0.01 mole) in ethanol (50 mL) in the presence of sodium
acetate (1 gm) was heated under reflux with 2-bromo-1-phenylethanone
or 2-bromo-1-(4ꢀ-chlorophenyl)ethanone (0.01 mole) for 5 h. After cool-
ing, the solids so formed were filtered off, washed with water, and crys-
tallized with ethanol to give 8ab, respectively.
1-([3,4-Diphenyl-1,3-thiazol-2-ylidine]amino-6-methyl-4-
pyridin-3-yl-1H-pyrazolo[3,4-b]pyridin-5-yl)ethanone (8a)
Crystallized from ethanol asyellow crystals (61%) m.p. 146–148◦C;
IR (cm−1): 3125 (NH), 3055 (aromatic-CH), 2883 (sat. CH) and 1689

1-(1H-Pyrazolo[3,4-b]pyridin-5-yl)ethanone
1097
1
(acetyl-CO); H-NMR (δ): 1.2 (s, 3H, CH3), 2.3 (s, 3H, CH3CO), 5.1 (s,
1H, thiazole H-5), 5.9 (s, 1H, NH) and 7.2–8.4 (m, 14H, ArH’s and Pyri-
dine H’s); Anal. for C29H22N6OS (502): Calcd./Found: C, 69.30/69.5%;
H, 4.41/4.5%; N, 16.72/16.8%; S, 6.38/6.4%.
1-([3-Phenyl-4-(4-chlorophenyl)-1,3-thiazol-2-ylidine] amino-
6-methyl-4-pyridin-3-yl-1H-pyrazolo-[3,4-b]pyridin-5-
yl)ethanone (8b)
Crystallized from ethanol as yellowish-white crystals (71%) m.p.
172–174◦C; IR (cm−1): 3139 (NH), 3063 (aromatic-CH), 2878 (sat.
1
CH) and 1695 (ketone-CO); H-NMR (δ): 1.1 (s, 3H, CH3), 2.5 (s, 3H,
CH3CO), 5.4 (s, 1H, thiazole H-5, 6.1 (s, 1H, NH) and 7.2–8.3 (m, 13H,
ArH’s and Pyridine H’s); Anal. for C29H21N6OSCl (502): Calcd./Found:
C, 64.86/65.0%; H, 3.94/4.1%; N, 15.65/15.7%; S, 5.97/6.1%; Cl,
6.60/6.5%.
Synthesis of 10–16: General Procedure
A solution of 9 (0.01 mole) in ethanol (50 mL) in the presence of sodium
acetate (1 g) was treated with either malonitrile, ethyl cyanoacetate, 2-
cyanoethanethioamide, ethyl 3-oxobutanoate, pentan-2,4-dione, ethyl
3-oxo-3-phenylpropanoate, or diethyl malonate (0.01 mole of each) re-
spectively. The reaction mixture was stirred in an ice-cold bath for 1 h.
The solid product obtained in each case was filtered off, washed with
water, and crystallized from ethanol as 10–16, respectively.
9-Acetyl-4-amino-8-methyl-10-pyridin-3-ylpyrido-
[2’,3’:3,4]pyrazolo[5,1-c][1,2,4]triazine-3-carbonitrile (10)
Crystallized from ethanol as pale brown crystals (61%) m.p. > 300◦C;
IR (cm−1): 3339, 3211 (NH2), 3063 (pyridine-CH), 2872 (sat. CH) and
1703 (acetyl-CO); 1H-NMR (δ): 1.1 (s, 3H, CH3), 2.3 (s, 3H, CH3CO), 5.4
(s, br., 2H, NH2) and 7.0–8.1 (m, 4H PyridineH’s); Anal. for C17H12N8O
(344): Calcd./Found: C, 59.5/59.6%; H, 3.51/3.5%; N, 32.54/32.5%.
9-Acetyl-4-amino-8-methyl-10-pyridin-3-ylpyrido-
[2’,3’:3,4]pyrazolo[5,1-c][1,2,4]triazine-3-carboxylate (11)
Crystallized from ethanol as yellow crystals (69%) m.p. 250◦C; IR
(cm−1): 3359, 3231 (NH2), 3058 (pyridine-CH), 2878 (sat. CH) and 1711
1
(ester-CO), 1702 (acetyl-CO); H-NMR (δ): 1.1 (s, 3H, CH3), 1.5 (t, 3H,
CH3CH2), 2.4 (s, 3H, CH3CO), 4.2 (q, 2H, CH3CH2), 5.5 (s, br., 2H,
NH2) and 7.1–8.5 (m, 4H, PyridineH’s); Anal. for C19H17N7O3 (391):
Calcd./Found: C, 58.31/58.4%; H, 4.38/4.2%; N, 25.05/25.0%.

1098
F. A. Attaby et al.
1-(4-Amino-3-thiocarboxamido-8-methyl-10-pyridin-3-yl
pyrido[2’,3’:-3,4]pyrazolo[5,1-c][1,2,4]triazin-9-yl)ethanone (12)
Crystallized from ethanol as brown crystals (65%) m.p. > 300◦C; IR
(cm−1): 3452, 3298, 3152 (two NH2), 3038 (pyridine-CH), 2867 (sat. CH)
and 1697 (acetyl-CO), 1H-NMR (δ): 1.1 (s, 3H, CH3), 2.2 (s, 3H, CH3CO),
5.0–5.5 (m, 4H, two NH2) and 7.0–8.2 (m, 4H, Pyridine H’s); Anal.
for C17H14N8OS (378): Calcd./Found: C, 53.96/54.0%; H, 3.73/3.8%; N,
29.61/29.5%; S, 8.47/8.5%.
Ethyl 9-Acetyl-4,8-dimethyl-10-pyridin-3-ylpyrido-
[2’,3’:3,4]pyrazolo[5,1- c][1,2,4]triazine-3-carboxylate (13)
Crystallized from ethanol as yellowish-white crystals (65%) m.p.
180◦C; IR (cm−1): 3058 (pyridine-CH), 2864 (sat. CH) and 1701 (acetyl-
CO), 1H-NMR (δ): 1.0 (s, 6H, two CH3), 1.5 (t, 3H, CH3CH2), 2.3 (s, 3H,
CH3CO), 4.2 (q, 2H, CH3CH2) and 7.0–8.4 (m, 4H, Pyridine H’s); Anal.
for C20H18N6O3 (390): Calcd./Found: C, 61.53/61.4%; H, 4.65/4.5%; N,
21.53/21.5%.
1-(3-Acetyl-4,8-dimethyl-10-pyridin-3-ylpyrido-
[2’,3’:3,4]pyrazolo[5,1-c][1,2,4]triazin-9-yl)ethanone (14)
Crystallized from ethanol as yellowish-white crystals (61%) m.p.
195◦C; IR (cm−1): 3071 (pyridine-CH), 2868 (sat. CH) and 1705 (acetyl-
CO), 1H-NMR (δ): 1.1 (s, 6H, two CH3), 2.4 (s, 6H, two CH3CO) and 7.2–
8.3 (m, 4H, Pyridine H’s); Anal. for C19H16N6O2 (360): Calcd./Found: C,
63.32/63.4%; H, 4.48/4.5%; N, 23.32/23.5%.
Ethyl 9-Acetyl-8-methyl-4-oxo-10-pyridin-3-yl-3,4-
dihydropyrido[2’,3’:3,4]pyrazolo[5,1-c][1,2,4]triazine-
3-carboxylate (15)
As pale-yellow crystals (62%) m.p. > 300◦C; IR (cm−1): 3198 (NH),
3063 (pyridine-CH), 2851 (sat. CH) and 1713 (ester-CO), 1697 (acetyl-
CO), 1662 (ring-CO); 1H-NMR (δ): 1.1 (s, 3H, CH3), 1.5 (t, 3H, CH3CH2),
2.4 (s, 3H, CH3CO), 4.3 (q, 2H, CH3CH2) 5.4 (s, br., 1H, NH) and 6.9–
8.2 (m, 4H, PyridineH’s); Anal. for C19H16N6O4 (392): Calcd./Found: C,
58.16/58.4%; H, 4.11/4.2%; N, 21.42/21.5%.
Ethyl [(5-Acetyl-6-methyl-4-pyridin-3-yl-1H-pyrazolo-
[3,4-b]pyri-din-3-yl)hydrazono]phenylacetate (16)
As white crystals (69%) m.p. 120◦C; IR (cm−1): 3188 (NH), 3059
(aromatic-CH), 2859 (sat. CH) and 1710 (ester-CO), 1698 (acetyl-CO);
1H-NMR (δ): 1.0 (s, 3H, CH3), 1.4 (t, 3H, CH3CH2), 2.5 (s, 3H, CH3CO),
4.2 (q, 2H, CH3CH2) 5.6 (s, br., 2H, two NH) and 7.0–8.4 (m, 9H,

1-(1H-Pyrazolo[3,4-b]pyridin-5-yl)ethanone
1099
ArH’s and Pyridine H’s); Anal. for C25H22N6O4 (470): Calcd./Found: C,
63.82/63.9%; H, 4.71/4.8%; N, 17.86/18.0%.
Synthesis of 17
A solution of 15 (0.01 mole) in ethanol (50 mL) in the presence of tri-
ethylamine (0.5 mL) was heated under reflux for 3 h. The solid obtained
was filtered off and crystallized from ethanol as 17.
Ethyl 9-Acetyl-8-methyl-3-pheny-10-pyridin-3-ylpyrido
[2’,3’:3,4]pyrazolo[5,1-c][1,2,4]triazine-4-carboxylate(17)
As pale-yellow crystals (71%) m.p. 224–226◦C; IR (cm−1): 3057
(aromatic-CH), 2862 (sat. CH) and 1715 (ester-CO), 1702 (acetyl-CO);
1H-NMR (δ): 1.1 (s, 3H, CH3), 1.5 (t, 3H, CH3CH2), 2.3 (s, 3H, CH3CO),
4.3 (q, 2H, CH3CH2) and 7.1–8.5 (m, 9H, ArH’s and Pyridine H’s); Anal.
for C25H20N6O3 (452): Calcd./Found: C, 66.36/66.2%; H, 4.46/4.6%; N,
18.5718.6%.
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 geen monkey kidney cells (Vero) and human hepatoma
cell line (Hep G2).
Viruses
Two models of DNA and RNA viruses were used for the bioassay.
These viruses were Herpes simplex virus type 1 (HSV-1) and Hepatitis
A virus, MBB strain (HAV-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 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 used at a 10%
final concentration for growth medium and at 2% for maintenance
medium.

1100
F. A. Attaby et al.
Antibiotic-Antimycotic Mixture (GIBCO-BRL)
100 X antibiotic-antimycotic mixtures consisted of 10,000 U peni-
cillin G sodium 10,000 µg streptomycin sulfate and 25 µg of 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); 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 a 0.22 µm nitrocellulose
membrane. The solution was used to wash the cell monolayer sheets
and to prepare a cell dissociation solution as follows.
Trypsin 1:250 (Sigma)
1.5 gm 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 a 2 mM solution
(0.04 gm), which was mixed with an equal volume of trypsin solution.
The pH value of the trypsin-versene mixture was adjusted to 8.4 by
7.5% sodium bicarbonate solution and sterilized 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 antibiotic-
antimycotic mixture: 10,000 U penicillin G sodium or Gentamicin
(50 µg/mL), 10,000 µg streptomycin sulfate, and 250 µg of 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’ Minimum essential medium (MEM)

1-(1H-Pyrazolo[3,4-b]pyridin-5-yl)ethanone
1101
and supplemented with 10% Foetal bovine serum and 1% antibiotic-
antimycotic mixture. The pH was adjusted to 7.2–7.4 by 7.5% sodium
bicarbonate solution. The mixture was sterilized by filtration through
a 0.2-µm pore-size nitrocellulose membrane.
III: Viruses
Herpes Simplex virus type 1 was obtained from Environmental Vi-
rology Lab. Department of Water Pollution Res., National Research
Center, Dokki, 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 (105cell/mL) and incubated for 2 days at 37◦C. HSV-1 was
diluted to give 104PFU/mL as a final concentration, mixed with the
plant extract at the previous concentration, incubated overnight at 4◦C.
Growth medium was removed from the multiwell plate, and the virus-
compound mixture was inoculated (100 µL/well). After 1 h of a contact
time, the inoculum was aspirated, and 3 mL of MEM with 1% agar
rose was 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. 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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