Synthesis and antiviral activity of new 4-(phenylamino)/4-[(methylpyridin- 2-yl)amino]-1-phenyl-1H-pyrazolo[3,4-b]pyridine-4-carboxylic acids derivatives

Article abstract of DOI:10.1007/s00044-007-9035-6

The synthesis of new 4-(phenylamino)-1-phenyl-1H-pyrazolo[3,4-b]pyridine-4- carboxylic acid (3a-l) derivatives and the new 4-[(methylpyridin-2-yl)amino]-1- phenyl-1H-pyrazolo[3,4-b]pyridine-4-carboxylic acid (5a-c) derivatives was achieved with an efficient synthetic route. Ethyl 4-chloro-1-phenyl-1H- pyrazolo[3,4-b]pyridine-5-carboxylate (1) on fusion with appropriate substituted anilines or aminopicolines gave the required new ethyl 4-(phenylamino)-1- phenyl-1H-pyrazolo[3,4-b]pyridine-5-carboxylates (2a-l) (52-82%) or new ethyl 4-[(methylpyridin-2-yl)amino]-1-phenyl-1H-pyrazolo[3,4-b]pyridine-5- carboxylates (4a-c) (50-60%), respectively. Subsequent hydrolysis of the esters afforded the corresponding carboxylic acids (3a-l) (86-93%) and (5a-c) in high yield (80-93%). Inhibitory effects of 4-(phenylamino)/4-[(methylpyridin-2-yl) amino]-1-phenyl-1H-pyrazolo[3,4-b]pyridine-4-carboxylic acids. Derivatives on Herpes simplex virus type 1 (HSV-1), Mayaro virus (MAY) and vesicular stomatitis virus (VSV) were investigated. Compounds 2d, 3f, 3a, and 3c exhibited antiviral activity against HSV-1, MAY, and VSV virus with EC₅₀ values of 6.8, 2.2, 4.8, 0.52, 2.5, and 1.0. None of these compounds showed toxicity for Vero cells.

Full text of DOI:10.1007/s00044-007-9035-6

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2007) 16:352–369
DOI 10.1007/s00044-007-9035-6
ORIGINAL RESEARCH
Synthesis and antiviral activity of new 4-(phenylamino)/
4-[(methylpyridin-2-yl)amino]-1-phenyl-1H-
pyrazolo[3,4-b]pyridine-4-carboxylic acids derivatives
Alice Maria Rolim Bernardino Æ Alexandre Reis de Azevedo Æ
´
Luiz Carlos da Silva Pinheiro Æ Julio Cesar Borges Æ
´
Vinıcius Lucio Carvalho Æ Milene Dias Miranda Æ
Marcelo Damia˜o Ferreira de Meneses Æ Marcelo Nascimento Æ
Davis Ferreira Æ Moacyr Alcoforado Rebello Æ
Viveca Antonia Giongo Galva˜o da Silva Æ
Izabel Christina Palmer Paixa˜o de Frugulhetti
Received: 7 August 2007 / Accepted: 24 October 2007 / Published online: 13 December 2007
¨
Ó Birkhauser Boston 2007
Abstract The synthesis of new 4-(phenylamino)-1-phenyl-1H-pyrazolo[3,4-
b]pyridine-4-carboxylic acid (3a-l) derivatives and the new 4-[(methylpyridin-2-
yl)amino]-1-phenyl-1H-pyrazolo[3,4-b]pyridine-4-carboxylic acid (5a–c) deriva-
tives was achieved with an efficient synthetic route. Ethyl 4-chloro-1-phenyl-1H-
pyrazolo[3,4-b]pyridine-5-carboxylate (1) on fusion with appropriate substituted
anilines or aminopicolines gave the required new ethyl 4-(phenylamino)-1-phenyl-
1H-pyrazolo[3,4-b]pyridine-5-carboxylates (2a–l) (52–82%) or new ethyl 4-
[(methylpyridin-2-yl)amino]-1-phenyl-1H-pyrazolo[3,4-b]pyridine-5-carboxylates
(4a–c) (50–60%), respectively. Subsequent hydrolysis of the esters afforded the
corresponding carboxylic acids (3a–l) (86–93%) and (5a–c) in high yield (80–93%).
Inhibitory effects of 4-(phenylamino)/4-[(methylpyridin-2-yl)amino]-1-phenyl-1H-
pyrazolo[3,4-b]pyridine-4-carboxylic acids. Derivatives on Herpes simplex virus
type 1 (HSV-1), Mayaro virus (MAY) and vesicular stomatitis virus (VSV) were
investigated. Compounds 2d, 3f, 3a, and 3c exhibited antiviral activity against
A. M. R. Bernardino (&) ꢀ A. R. de Azevedo ꢀ L. C. d. S. Pinheiro ꢀ J. C. Borges ꢀ V. L. Carvalho
´
´
ˆ
´
´
Instituto de Quımica, Departamento de Quımica Organica, Programa de Pos-Graduac¸a˜o em Quımica
Organica, Universidade Federal Fluminense, Outeiro de Sa˜o Joa˜o Batista, s/n, Centro, Niteroi CEP
ˆ
´
24020-141, RJ, Brazil
e-mail: alicerolim@globo.com
M. D. Miranda ꢀ M. Nascimento ꢀ I. C. P. P. de Frugulhetti
Instituto de Biologia, Departamento de Biologia Celular e Molecular, Universidade Federal
´
Fluminense, Outeiro de Sa˜o Joa˜o Batista s/n, Niteroi CEP 24020-150, RJ, Brazil
M. D. F. de Meneses ꢀ D. Ferreira ꢀ M. A. Rebello ꢀ V. A. G. G. d. Silva
´
Instituto de Microbiologia, Universidade Federal do Rio de Janeiro Prof. Paulo de Goes. CCS -
Bloco I, Av. Carlos Chagas Filho, 373, Rio de Janeiro CEP 21941-902, RJ, Brazil

Med Chem Res (2007) 16:352–369
353
HSV-1, MAY, and VSV virus with EC50 values of 6.8, 2.2, 4.8, 0.52, 2.5, and 1.0.
None of these compounds showed toxicity for Vero cells.
Keywords 1H-pyrazolo[3,4-b]pyridine ꢀ antiviral ꢀ HSV-1 ꢀ MAY ꢀ
VSV
Introduction
Among the various important parent ring systems of pyrazolopyridine, substituted
1H-pyrazolo[3,4-b]pyridine is known to have several biological activities such as
anxyolitic, anticonvulsant, anti-inflammatory, analgesic, hypoglycemic, antipyretic,
vasodilators, and antileishmanial (Hohn et al., 1972; Lynch et al., 1988; Bernardino
et al., 1996a; Ahluwalia and Goyal, 1996; Quiroga et al., 1998; Bernardino et al.,
1999; Mello et al., 2004). Recently our research group described the synthesis and
antiviral activity of derivatives of the 1H-pyrazolo[3,4-b]pyridine and thieno[2,3-
b]pyridine systems with promising results (Bernardino et al., 1996b; Azevedo et al.,
2002a; Pinheiro et al., 2004; Bernardino et al., 2007).
In a attempt to explore the antiviral potential of 1H-pyrazolo[3,4-b]pyridine
derivatives, we have studied their effects of these compounds on Herpes simplex
virus type 1 (HSV-1), Mayaro virus (MAY), and vesicular stomatitis virus (VSV).
Herpes simplex type 1 (HSV-1) infects mucocutaneous epithelial cells and
establishes latency in sensory ganglions (Whitley and Roizman, 2001). HSV-1
causes gingivostomatitis, cold sores, keratoconjunctivitis, and encephalitis.
Although any viral protein essential for viral replication is a potential target, nearly
all currently available drugs for herpes viruses are primarily inhibitors of viral DNA
polymerase (Eizuru, 2003). Among the anti-herpes-virus agents, acyclovir, valac-
iclovir, penciclovir, famciclovir, idoxuridine, and trifluridine (applied topically) as
well as brivudin are used in the treatment of herpes simplex virus (De Clercq, 2005).
Mayaro virus is a member of the Alphavirus genus, Togaviridae family and is
antigenically closely related to Semiliki Forest virus (Casals and Whitman, 1957).
Mayaro virus is an arbovirus, isolated for the first time in Trinidad, in 1954 and has
since been reported as the cause of several epidemic outbreaks in Brazil, Bolivia,
and other regions, mainly at colonial borders in the Amazon region (Causey and
Maroja, 1957). Clinical manifestations of human infection have been described as
feverish illness accompanied by headache, chills, nausea, photophobia, myalgia, and
arthralgia. In some cases there is true arthritis that persists for months (Strauss and
Strauss, 1994). Vesicular stomatitis virus (VSV) is a single-strand RNA virus of the
Rhabdoviridae family. VSV has been isolated from a variety of animals and causes
nonfatal disease of significant economic importance in cattle and swine (Rose and
Whitt, 2007).
In order to identify the 1H-pyrazolo[3,4-b]pyridine system as promising
compounds for the development of new antiviral agents, we synthesized a new
set of derivatives 4-(phenylamino)/4-[(methylpyridin-2-yl)amino]-1-phenyl-1H-
pyrazolo[3,4-b]pyridines (2–5). The aim of this study was to assess their
cytotoxicity and the potential antiviral activity.

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Results and discussion
Chemistry
The synthesis of new 4-(phenylamino)-1-phenyl-1H-pyrazolo[3,4-b]pyridine-4-car-
boxylic acid (3a–l) derivatives and the new 4-[(methylpyridin-2-yl)amino]-1-phenyl-
1H-pyrazolo[3,4-b]pyridine-4-carboxylic acid (5a–c) derivatives was achieved with
an efficient synthetic route outlined in Scheme 1. A halogen in the C-4 position and an
ester group in the C-5 position of 1H-pyrazolo[3,4-b]pyridine react readily with
nucleophilic compounds to form a good precursor for the synthesis of these
derivatives. Ethyl 4-chloro-1-phenyl-1H-pyrazolo[3,4-b]pyridine-5-carboxylate (1)
was available in our laboratory and could be easily prepared from 5-aminopyrazoles
through condensation with diethyl ethoxymethylenemalonate followed by chloro-
cyclization with phosphorus oxychloride (Hohn et al., 1971; Azevedo et al., 2002a;
Azevedo et al., 2002b). Ethyl 4-chloro-1-phenyl-1H-pyrazolo[3,4-b]pyridine-5-car-
boxylate (1) on fusion with appropriate substituted anilines or aminopicolines gave the
required new ethyl 4-(phenylamino)-1-phenyl-1H-pyrazolo[3,4-b]pyridine-5-car-
boxylates (2a–l) (52–82%) or new ethyl 4-[(methylpyridin-2-yl)amino]-1-phenyl-
1H-pyrazolo[3,4-b]pyridine-5-carboxylates (4a–c) (50–60%), respectively. Subse-
quent hydrolysis of the ester afforded the corresponding 4-(phenylamino)-1-phenyl-
1H-pyrazolo[3,4-b]pyridine-4-carboxylic acids (3a–l) in high yields (86–93%) and 4-
[(methylpyridin-2-yl)amino]-1-phenyl-1H-pyrazolo[3,4-b]pyridine-4-carboxylic
acids (5a–c) in high yields (80–93%) (Azevedo et al., 2002b).
R
R
H
H
N
N
CO2Et
CO2H
(ii)
N
N
N
N
N
N
(i)
(2 a-l)
(3 a-l)
Cl
CO2Et
N
N
N
H3C
H3C
(iii)
(1)
H
H
N
N
N
N
CO2Et
CO2H
(ii)
N
N
N
N
N
N
(4 a-c)
(5 a-c)
(i) suitable aniline, DMF, 4h, reflux; (ii) NaOH 20%, ethanol, reflux, 1-3 h; (iii) suitable aminopicoline, DMF, 4h, reflux.
Scheme 1 2a, 3a R = H, 2b, 3b R = p-CH3, 2c, 3c R = m-Cl, 2d, 3d R = m-NO2, 2e, 3e R = p-NO2,
2f, 3f R = m-Br, 2g, 3g R = p-Br, 2h, 3h R = m-CH3, 2i, 3i R = p-Cl, 2f, 3f R = m-OCH3, 2g, 3g
R = p-OCH3

Med Chem Res (2007) 16:352–369
355
1
The structures of esters and acids were determined by infrared (IR), H and
13C nuclear magnetic resonance (NMR), and mass spectroscopy. In the IR
spectra, the carbonyl group absorptions were observed in the acids and esters
derivatives at 1649–1678 cm-1 and 1672–1696 cm-1, respectively. In the 3435–
3125 cm-1 region, NH bands were observed. The OH bands for the acid
compounds were observed in the 3491–2500 cm-1 region. 1H NMR spectra
indicated a chemical shift of the NH in the range of d = 10.98–9.46 ppm as
singlet signals. The aromatic protons signals in aniline and aminopicoline
moieties appeared as a multiplet in orto derivatives or a double doublet in para
derivatives. The quartet and triplet signals of the esters derivatives (2a–l and 4 a-
c) appeared in the ranges d = 4.62–4.42 ppm and 1.58–1.50 ppm, respectively.
The singlet signals of the acids (3a–l and 5a–c) appeared in the range d = 13.00–
11.36 ppm.
Biological evaluation
Initially, the anti-herpetic effect of all compounds investigate was evaluated in a
primary screening using the 50% end-point titration method described by Reed and
Muench (1938).
As shown in Table 1, compounds 2a, 2d, 2h, 2i, 2j, 4b, and 4c exhibited the
highest anti-HSV-1 activity, with the esters being, in general, more effective
inhibitors than the corresponding acids. In conclusion, the introduction of ester
group at C-5 in the 1H-pyrazolo[3,4-b]pyridine system increased activity against
HSV-1 virus.
To calculate the selective or therapeutic index of each compound for antiviral
activity we infected Vero cells with HSV-1, Mayaro, and VSV to determine the
EC50 value by plaque-reducing assay. The cytotoxicity of the compounds was
determined in parallel by the MTT 3-(4,5-dimethylthiazol-2-yl)-2-5-diphenyltet-
razolium bromide colorimetric method (Mossmann, 1983) (Table 2). As shown in
the Table 3, the effects of the compounds 3a and 3c, selected on the basis of
preliminary screening (data not shown), exhibit the best EC50 values against VSV
and Mayaro virus replication cycle.
Conclusion
In summary, 15 new derivatives of the 1H-pyrazolo[3,4-b]pyridine system 2a–l, 3a–
l, 4a–c, and 5a–c with different substituents were synthesized and exhibited a range
of significant anti-herpes, anti-Mayaro, and anti-VSV activities, suggesting that
these compounds are potential antivirus agents. Compounds 2d, 3f, 3a, and 3c
exhibited antiviral activity against HSV-1, MAY, and VSV virus, with EC50 values
of 6.8, 2.2, 4.8, 0.52, 2.5, and 1.0. None of the compounds showed toxicity for Vero
cells. The mechanism of antiviral activity is unknown and requires further study,
which is in progress in our laboratory.

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Med Chem Res (2007) 16:352–369
HSV-1 virus yield inhibition (%)
Table 1 Anti-HSV-1 virus
activity of 1H-pyrazolo[3,4-
b]pyridine derivatives 2a–l, 3a–
l, 4a–c, and 5a–c
Compounds
R
2a
2b
2c
2d
2e
2f
H
90.0 3.3
69.2 2.2
60.0 5.2
90.0 1.8
0
p-CH3
m-Cl
m-NO2
p-NO2
m-Br
70.0 2.7
75.0 3.4
90.0 5.7
92.0 5.5
98.0 2.3
75.5 5.5
0
2g
2h
2i
p-Br
m-CH3
p-Cl
2j
m-OCH3
p-OCH3
m-F
2k
2l
3a
3b
3c
3d
3e
3f
H
0
p-CH3
m-Cl
58.4 4.2
68.0 7.4
20.6 3.6
80.0 6.3
84.0 2.4
37.0 5.8
38.5 4.3
68.0 6.0
75.5 5.5
80.6 3.4
68.0 3.7
60.0 2.0
90.0 3.2
90.0 3.7
75.0 4.0
60.0 1.5
21.0 2.1
96.0 1.0
m-NO2
p-NO2
m-Br
3g
3h
3i
p-Br
m-CH3
p-Cl
3j
m-OCH3
p-OCH3
m-F
3k
3l
The experimental concentration
of 1H-pyrazolo[3,4-b]pyridine
derivatives 2a–l, 3a–l, 4a–c, and
5a–c was 50 lM and that for
ACV was 10 lM
4a
4b
4c
5a
5b
5c
ACV
4-CH3
5-CH3
6-CH3
4-CH3
5-CH3
6-CH3
–
Results are presented as the
mean of triplicate experiments
ACV: acyclovir has been
included for comparison
purposes
Experimental section
1H and 13C NMR spectra were obtained at 300 MHz and 75 MHz, respectively,
using a Varian Unity Plus instrument with tetramethylsilane as an internal standard.
The chemical shifts (d) are reported in ppm and the coupling constants (J) in Hertz.
Fourier-transform infrared (FT-IR) spectra were recorded in a Perkin–Elmer
Spectrum One instrument. The solid samples were determined in potassium bromide
(KBr) pellets. Melting points (m.p.) were determined with a Fisher–Johns apparatus.
Thin-layer chromatography (TLC) was carried out using silica gel F-254 glass

Med Chem Res (2007) 16:352–369
357
Table 2 Anti-HSV-1 virus activity, cytotoxicity, and selectivity index in Vero cells for 1H-pyrazolo[3,4-
b]pyridine derivatives
Compounds
R
ECa50(lM)
CCb50(lM)
S.I.c
2d
m-NO2
m-Br
6.8 0.1
2.2 0.05
1.09 0.25
1000 100
600 50
141.4
272.4
880
3f
ACV
960 156
ACV: acyclovir has been included for comparison purposes
a
50% effective concentration or concentration required to inhibit HSV-1 virus yield
b
c
50% cytotoxic concentration or concentration required to reduce the viability of host cells by 50%
Selective index (CC50/EC50)
Table 3 Anti-Mayaro and VSV virus activity, cytotoxicity, and selectivity index in Vero cells for 1H-
pyrazolo[3,4-b]pyridine derivatives
Compounds
R
ECa50(lM)
May
VSV
3a
m-H
m-Cl
R
4.80 0.05
0.52 0.01
CCb50(lM)
2.5 0.05
1.00 0.07
S.I.c
3c
Compounds
May
VSV
54
3a
m-H
m-Cl
–
135 0.9
37.4 0.5
960 156
28
3c
71
37
ACV
880
ACV: acyclovir has been included for comparison purposes
a
50% effective concentration or concentration required to inhibit HSV-1 virus yield
b
c
50% cytotoxic concentration or concentration required to reduce the viability of host cells by 50%
Selective index (CC50/EC50)
plate (20 9 20 cm). All reagents and solvents used were analytical grade.
The electron-ionization mass spectrometry (EI-MS) spectra were recorded using a
Finingan MAT 711 A instrument. The ionization energy was 70 eV with a source
temperature of 200°C and an accelerative voltage of 8 kV. Samples were introduced
by using a standard direct-insertion probe. High-resolution data were obtained with
the instrument using 10000 resolution).
Ethyl 4-chloro-1-phenyl-1H-pyrazolo[3,4-b]pyridine-5-carboxylate (1)
The procedure developed for the synthesis of 1 started from the previously prepared
5-amino-1-phenyl-1H-pyrazole. The condensation of aminopyrazole with diethyl
ethoxymethylenemalonate, based on the Gould–Jacobs method, produced ethyl
a-carboetoxi-b-(N-5-pyrazolylamino)acrylate, which was recrystallized from

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Med Chem Res (2007) 16:352–369
anhydrous ethanol. The resulting acrylate was mixed with phosphorus oxychloride
and refluxed for 5 hours. The excess solvent was removed under reduced pressure
and the resulting material was poured onto crushed ice and the product collected by
filtration. The solid material was recrystallized from ethanol to yield ethyl 4-chloro-
1-phenyl-1H-pyrazolo[3,4-b]pyridine-5-carboxylate (1) (Azevedo et al., 2002b).
General procedure for the preparation of the ethyl 4-(arylamino)-1-phenyl-1H-
pyrazolo[3,4-b]pyridine-5-carboxylates (2a–l)
An equimolar mixture of 1 (1,2 g, 4 mmol) and a slight excess of the appropriate
aniline (5 mmol), in 10 ml N,N-dimethylformamide (DMF) was heated in a silicone
oil bath under reflux for 4 hours. The reaction mixture, after cooling, was poured
into 50 ml of ice-cold water. The precipitate was filtered, dried, and recrystallized
from a mixture of ethanol and water. The compounds were isolated in good yields
1
(52–82%). The structures of the compounds were elucidated by IR, H and 13C
NMR, and mass spectrometry.
2a. Ethyl 4-(phenylamino)-1-phenyl-1H-pyrazolo[3,4-b]pyridine-5-carboxylate
Yield: 1.08g (76%), m.p.: 197°C; IR (KBr, cm-1): (m NH 3420; m C = O 1678); 1H
NMR (DMSO-d6, J in Hz, d in ppm) 6,81(s, H-3); 9,04(s, H-6); 8,08(d, 8.7, H-20,60),
7,51-7,26(10H, m), 4,42(q, 7.1, CH2), 1,42(t, 7.1, CH3), 10,61(s, N-H);13C NMR
(DMSO-d6, d in ppm) 135.7(C-3), 99.7(C-3a), 137.4(C-4), 105.1(C-5), 149.5(C-6),
150.3(C-7a), 131.9(C-10), 121.5(C-20, 60), 128.3(C-30, 50), 126.8(C-40), 158.1(C-100),
120.4(C-200), 128.5(C-300), 140.1(C-400), 128.5(C-500), 120.4(C-600), 164.9(CO2Et),
60.5(CH2), 13.7(CH3);
2b. Ethyl 4-(40-methylphenylamino)-1-phenyl-1H-pyrazolo[3,4-b]pyridine-5-
carboxylate
Yield: 0.93 g (63%), m.p.: 155°C; IR (KBr, cm-1): (m NH 3435; m C = O 1678); 1H
NMR (DMSO-d6, J in Hz, d in ppm) 6.73(s, H-3), 9.04(s, H-6), 8.23(d, 8.7, H-20,60),
7.67(dd, 7.5, H-30,50), 7.54–7.46(5H, m), 4.53(q, 7.2, CH2), 1.53(t, 7.2, CH3),
10.52(s, N-H), 3.38(s, Ar-CH3); 13C NMR (DMSO-d6, d in ppm) 135.5(C-3),
101.7(C-3a), 139.6(C-4), 105.4(C-5), 153.6(C-6), 151.7(C-7a), 137.3(C-10),
122.4(C-20, 60), 131.3(C-30, 50), 130.0(C-40), 158.6(C-100), 122.4(C-200), 128.1(C-
300), 138.6(C-400), 128.1(C-500), 122.4(C-600), 168.6(CO2Et), 21.7(Ar-CH3),
61.6(CH2), 15.1(CH3).
2c. Ethyl 4-(30-chlorophenylamino)-1-phenyl-1H-pyrazolo[3,4-b]pyridine-5-
carboxylate
Yield: 1.06 g (68%), m.p.: 182°C; IR (KBr, cm-1): (m NH 3208; m C = O 1681); 1H
NMR (DMSO-d6, J in Hz, d in ppm) 6.92(s, H-3), 9.03(s, H-6), 8.20(d, 8.7, H-20,60),

Med Chem Res (2007) 16:352–369
359
7.68–7.56(5H, m), 7.47(dd, 7.5, H-40), 7.73(dd, 8.7, H-500), 4.50(q, 7.2, CH2), 1.47(t,
7.2, CH3),10.57(s, N-H); 13C NMR (DMSO-d6, d in ppm) 133.9(C-3), 101.(C-3a),
138.3(C-4), 104.4(C-5), 152.2(C-6), 152.0(C-7a), 133.5(C-10), 121.1(C-20, 60),
130.4(30, 50), 128.6(C-40), 158.8(C-100), 124.8(C-200), 149.1(C-300), 140.4(C-400),
127.0(C-500), 126.1(C-600), 167.0(CO2Et), 60.3(CH2), 13.7(CH3); EI (70 eV) m/z
(%): M+. 392.10435 (95), 346.06091 (100), 311.09595 (25), 283.09918 (15),
173.03200 (7), 110.99878 (4), 77.03763 (10)
2d. Ethyl 4-(30-nitrophenylamino)-1-phenyl-1H-pyrazolo[3,4-b]pyridine-5-
carboxylate
Yield: 0.96 g (60%), m.p.: 173°C; IR (KBr, cm-1): (m NH 3207; m C = O 1681); 1H
NMR (DMSO-d6, J in Hz, d in ppm) 7.25(s, H-3), 9.13(s, H-6), 8.30(d, 8.7, H-20,60),
7.74(dd, 7.5, H-30,50), 7.55(dd, 7.5, H-40), 8.50(s, H-200), 8.44(d, 8.1, H-400), 7.97(dd,
8.7, H-500), 8.10(d, 8.7, H-600), 4.55(q, 7.2, CH2), 1.55(t, 7.2, CH3), 10.75(s, N-H);
13C NMR (DMSO-d6, d in ppm) 134.8(C-3), 102.7(C-3a), 138.8(C-4), 105.2(C-5),
153.0(C-6), 152.6(C-7a), 133.2(C-10), 122.1(C-20, 60), 129.4(C-30, 50), 126.9(C-40),
158.9(C-100), 120.9(C-200), 148.9(C-300), 140.9(C-400), 121.9(C-500), 120.9(C-600),
167.6(CO2Et), 61.2(CH2), 14.5(CH3); EI (70 eV) m/z (%): M+. 403.12863 (100),
357.09045 (70), 311.09466 (30), 283.10117 (30), 256.08533 (7), 77.03626 (11).
2e. Ethyl 4-(40-nitrophenylamino)-1-phenyl-1H-pyrazolo[3,4-b]pyridine-5-
carboxylate
Yield: 1.11 g (69%), m.p.: 176°C; IR (KBr, cm-1): (m NH 3208; m C = O 1681); 1H
NMR (DMSO-d6, J in Hz, d in ppm) 7.18(s, H-6), 9.09(s, H-6), 8.25(d, 8.7, H-20,60),
7.68(dd, 7.5, H-30,50), 7.50(dd, 7.5, H-40), 7.74(d, 8.7, H-200), 8.43(d, 8.7, H-300),
8.43(d, 8.7, H-500), 7.74(d, 8.7, H-600), 4.45(q, 7.2, CH2), 1.46(t, 7.2, CH3), 10.67(s, N-
H); 13C NMR (DMSO-d6, d in ppm) 134.4(C-3), 104.1(C-3a), 138.3(C-4), 105.6(C-
5), 152.1(C-6), 152.0(C-7a), 133.6(C-10), 121.1(C-20, 60), 128.8(C-30, 50), 126.3(C-
40), 158.7(C-100), 123.3(C-200), 124.9(C-300), 144.0(C-400), 124.9(C-500), 124.9(C-600),
166.7(CO2Et), 60.7(CH2), 13.8(CH3); EI (70 eV) m/z (%): M+. 403.12461 (100),
357.07820 (45), 311.08910 (80), 283.08977 (30), 256.08447 (7), 77.03500 (11).
2f. Ethyl 4-(30-bromophenylamino)-1-phenyl-1H-pyrazolo[3,4-b]pyridine-5-
carboxylate
Yield: 1.31 g (75%), m.p.: 180°C; IR (KBr, cm-1): (m NH 3224; m C = O 1679); 1H
NMR (DMSO-d6, J in Hz, d in ppm) 6.88(s, H-3), 8.99(s, H-6), 8.17(d, 8.7, H-20,60),
7.64–7.57(3H, m), 7.43(dd, 7.5, H-40), 7.82(s, H-200), 7.75(dd, 7.5, H-500), 7.75(d,
7.5, H-600), 4.44(q, 7.5, CH2), 1.44(t, 7.5, CH3),10.53(s, N-H); 13C NMR (DMSO-d6,
d in ppm) 134.7(C-3), 102.0(C-3a), 138.9(C-4), 104.9(C-5), 153.0(C-6), 153.2(C-
7a), 133.0(C-10), 121.8(C-20, 60), 129.1(C-30, 50), 126.8(C-40), 158.0(C-10000),

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120.0(C-200), 150.1(C-300), 139.0(C-400), 121.0(C-500), 120.0(C-600), 167.8(CO2Et),
61.0(CH2), 14.4(CH3); EI (70 eV) m/z (%): M+. 436.05399 (85), 392.00998 (55),
358.13943 (10), 311.09562 (100), 283.09590 (30), 256.08710 (8), 222.57337 (15),
155.54822 (18), 95.07887 (23).
2g. Ethyl 4-(40-bromophenylamino)-1-phenyl-1H-pyrazolo[3,4-b]pyridine-5-
carboxylate
Yield: 1.31 g (75%), m.p.: 187°C; IR (KBr, cm-1): (m NH 3219; m C = O 1683); 1H
NMR (DMSO-d6, J in Hz, d in ppm) 6.88(s, H-3), 8.99(s, H-6), 8.15(d, 8.7, H-20,60),
7.60–7.50(7H, m), 4.45(q, 7.5, CH2), 1.40(t, 7.5, CH3), 10.50(s, N-H); 13C NMR
(DMSO-d6, d in ppm) 134.1(C-3), 101.4(C-3a), 138.4(C-4), 104.5(C-5), 153.3(C-6),
152.1(C-7a), 132.6(C-10), 121.4(C-20, 60), 129.0(C-30, 50), 128.8(C-40), 158.2(C-100),
120.1(C-200), 126.4(C-300), 138.5(C-400), 126.4(C-500), 120.1(C-600), 167.4(CO2Et),
60.5(CH2), 13.5(CH3); EI (70 eV) m/z (%): M+. 436.05358 (65), 390.01168 (15),
311.09221 (100), 283.09354 (20), 256.08226 (4), 155.54525 (12), 77.17351(10).
2h. Ethyl 4-(30-methylphenylamino)-1-phenyl-1H-pyrazolo[3,4-b]pyridine-5-
carboxylate
Yield: 0.99 g (67%), m.p.: 187°C; IR (KBr, cm-1): (m NH 3210; m C = O 1680); 1H
NMR (DMSO-d6, J in Hz, d in ppm) 6.75(s, H-3), 9.03(s, H-6), 8.21(d, 8.7, H-20,60),
7.68–7.41(7H, m), 4.51(q, 7.2, CH2), 1.51(t, 7.2, CH3), 10.53(s, N-H), 3.37(s, Ar-
CH3); 13C NMR (DMSO-d6, d in ppm) 135.5(C-3), 101.1(C-3a), 138.4(C-4),
105.1(C-5), 153.0(C-6), 152.9(C-7a), 133.0(C-10), 122.0(C-20, 60), 128.7(C-30, 50),
126.1(C-40), 158.1(C-100), 120.5(C-200), 129.0(C-300), 138.5(C-400), 124.0(C-500),
120.5(C-600), 166.9(CO2Et), 1.7(Ar-CH3), 61.6(CH2), 15.1(CH3).
2i. Ethyl 4-(40-chlorophenylamino)-1-phenyl-1H-pyrazolo[3,4-b]pyridine-5-
carboxylate
Yield: 1.00 g (64%), m.p.: 159°C; IR (KBr, cm-1): (m NH 3218; m C = O 1681); 1H
NMR (DMSO-d6, J in Hz, d in ppm) 6.94(s, H-3), 9.05(s, H-6), 8.08(d, 8.7, H-20,60),
7.51–7.25(7H, m), 4.42(q, 7.1, CH2), 1.42(t, 7.1, CH3), 10.51(s, N-H); 13C NMR
(DMSO-d6, d in ppm) 134.4(C-3), 104.1(C-3a), 138.3(C-4), 105.6(C-5), 152.3(C-6),
152.6(C-7a), 133.0(C-10), 121.2(C-20, 60), 128.8(30, 50), 126.3(C-40), 158.4(C-100),
123.3(C-200), 126.6(C-300), 145.7(C-400), 126.6(C-500), 123.3(C-600), 166.7(CO2Et),
60.7(CH2), 13.8(CH3); EI (70 eV) m/z (%): M+. 392.10854 (95), 346.06119 (100),
311.08943 (25), 283.08789 (15), 173.03286 (7), 110.99432 (4), 77.03839 (10)
2j. Ethyl 4-(30-methoxyphenylamino)-1-phenyl-1H-pyrazolo[3,4-b]pyridine-5-
carboxylate
Yield: 0.97 g (63%), m.p.: 135°C; IR (KBr, cm-1): (m NH 3435; m C = O 1678); 1H
NMR (DMSO-d6, J in Hz, d in ppm) 6.77(s, H-3), 8.95(s, H-6), 8.16(d, 8.7, H-20,60),

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7.13(dd, 7.5, H-30,50), 7.41(dd, 7.5, H-40), 7.51(s, H-200), 7.55(d, 7.8, H-400), 7.60(dd,
7.5, H-500), 7.53(d, 7.8, H-600),4.43(q, 7.0, CH2), 1.43(t, 7.0, CH3), 10.51(s, N-H),
3.86(s, Ar-OCH3); 13C NMR (DMSO-d6, d in ppm) 135.8(C-3), 98.1(C-3a),
137.3(C-4), 101.6(C-5), 147.4(C-6), 149.9(C-7a), 131.8(C-10), 118.6(C-20, 60),
127.8(C-30, 50), 126.3(C-40), 157.6(C-100), 120.2(C-200), 148.5(C-300), 109.8(C-400),
123.5(C-500), 110.9(C-600), 164.6(CO2Et), 57.9(Ar-OCH3), 69.4(CH2), 11.4(CH3); EI
(70 eV) m/z (%): M+. 388.15321 (100), 342.11180 (100), 271.10042 (5),
244.08777(2), 171.06055 (7), 77.04092 (6).
2k. Ethyl 4-(40-methoxyphenylamino)-1-phenyl-1H-pyrazolo[3,4-b]pyridine-5-
carboxylate
Yield: 0.80 g (52%), m.p.: 148°C; IR (KBr, cm-1): (m NH 3435; m C = O 1672); 1H
NMR (DMSO-d6, J in Hz, d in ppm) 6.60(s, H-3), 8.98(s, H-6), 8.19(d, 8.7, H-20,60),
7.63(dd, 7.5, H-30,50), 7.45(dd, 7.5, H-40), 7.22(d, 8.7, H-200), 7.50(d, 8.7, H-300), 7.22
(d, 8.7, H-500), 7.22(d, 8.7, H-600), 4.49(q, 7.2, CH2), 1.48(t, 7.2, CH3), 10.46(s, N-H),
3.96(s, Ar-OCH3); 13C NMR (DMSO-d6, d in ppm) 134.0(C-3), 99.9(C-3a),
138.1(C-4), 103.9(C-5), 152.2(C-6), 150.8(C-7a), 131.0(C-10), 120.9(C-20, 60),
128.6(C-30, 50), 125.9(C-40), 158.5(C-100), 114.6(C-200), 128.5(C-300), 151.8(C-400),
128.5(C-500), 114.6(C-600), 167.1(CO2Et), 55.0(Ar-OCH3), 60.1(CH2), 13.7(CH3); EI
(70 eV) m/z (%): M+. 372.15870 (85), 326.12064 (100), 300.13490 (9), 163.06130
(7), 77.04092 (4).
2l. Ethyl 4-(30-fluorophenylamino)-1-phenyl-1H-pyrazolo[3,4-b]pyridine-5-
carboxylate
Yield: 1.23 g (82%), m.p.: 206°C; IR (KBr, cm-1): (m NH 3249; m C = O 1676); 1H
NMR (DMSO-d6, J in Hz, d in ppm) 7.03(s, H-3), 9.13(s, H-6), 8.30(d, 8.7, H-20,60),
7.77–7.53(7H, m), 4.57(q, 7.5, CH2), 1.55(t, 7.0, CH3), 10.67(s, N-H); 13C NMR
(DMSO-d6, d in ppm) 134.2(C-3), 104.0(C-3a), 138.4(C-4), 105.7(C-5), 153.0(C-6),
152.1(C-7a), 135.0(C-10), 121.0(C-20, 60), 128.0(C-30, 50), 127.0(C-40), 158.7(C-100),
123.2(C-200), 148.0(C-300), 142.1(C-400), 120.3(C-500), 112.6(C-60000), 167.0(CO2Et),
60.5(CH2), 13.5(CH3); EI (70 eV) m/z (%): M+. 376.12275 (90), 330.08058 (100),
303.07368 (10), 236.20421 (7), 165.03244 (11), 83.07803 (14).
General procedure for the preparation of the 4-(arylamino)-1-phenyl-1H-
pyrazolo[3,4-b]pyridine-4-carboxylic acids (3a–l)
A mixture of 3 mmol of (2a–g), 10 ml of 20% sodium hydroxide solution, and
10 ml of ethanol was heated under reflux for 1–3 hours. On cooling the mixture was
acidified with diluted hydrochloric acid (1:3), and the precipitate was filtered and
recrystallized from a mixture of DMF and water. The compounds were isolated in

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excellent yields (86–93%). The structures of the compounds were elucidated by IR,
1H and 13C NMR, and mass spectrometry.
3a. 4-Phenylamino-l-phenyl-1H-pyrazolo[3,4-b]pyridine-5-carboxylic acid
Yield: 0.85 g (86%), m.p.: 229°C; IR (KBr, cm-1): (m OH 3435-2598; m C = O
1
1654); H NMR (DMSO-d6, J in Hz, d in ppm) 7.01(s, H-3), 9.02(s, H-6), 8.23(d,
8.1, H-20,60), 7.86-7.45(8H, m), 10.78(s, N-H); 13C NMR (DMSO-d6, d in ppm)
135.4(C-3), 100.0(C-3a), 151.7(C-4), 105.5(C-5), 153.1(C-6), 153.5(C-7a),
114.2(C-10), 122.2(C-20, 60), 131.5(C-30, 50), 129.1(C-40), 140.2(C-100), 126.4(C-
200), 139.5(C-300), 126.9(C-400), 139.5(C-500), 126.4(C-600), 170.2(CO2H).
3b. 4-(40-Methylphenylamino)-1-phenyl-1H-pyrazolo[3,4-b]pyridine-5-carboxylic
acid
Yield: 0.94 g (92%), m.p.: 258°C; IR (KBr, cm-1): (m OH 3430-2603; m C = O
1
1654); H NMR (DMSO-d6, J in Hz, d in ppm) 6.67(s, H-3), 8.95(s, H-6), 8.18(d,
7.5, H-20,60), 7.61-7.40(7H, m), 10.73(s, N-H), 2.47(s, Ar-CH3); 13C NMR
(DMSO-d6, d in ppm) 134.8(C-3), 100.1(C-3a), 151.3(C-4), 104.5(C-5), 153.5(C-6),
152.8(C-7a), 139.1(C-10), 121.7(C-20, 60), 130.7(C-30, 50), 129.3(C-40), 139.1(C-100),
126.6(C-200), 137.8(C-300), 136.7(C-400), 137.8(C-500), 126.6(C-600), 170.3(CO2H),
21.0(Ar-CH3); EI (70 eV) m/z (%): M+. 344.13367 (95), 326.12041 (100),
297.10439 (5), 270.11045 (3), 199.93153 (4), 73.36787 (4).
3c. 4-(30-Chlorophenylamino)-1-phenyl-1H-pyrazolo[3,4-b]pyridine-5-carboxylic
acid
Yield: 1.01 g (93%), m.p.: 236°C; IR (KBr, cm-1): (m OH 3488-2766; m C = O
1
1649); H NMR (DMSO-d6, J in Hz, d in ppm) 7.01(s, H-3), 9.07(s, H-6), 8.27(d,
7.5, H-20,60), 7.74-7.62 (5H, m), 7.52(dd, 7.5, H-40), 7.79(s, H-200), 10.92(s, N-H);
13C NMR (DMSO-d6, d in ppm) 132.9(C-3), 100.5(C-3a), 148.6(C-4), 103.2(C-5),
151.6(C-6), 150.8(C-7a), 120.1(C-10), 125.2(C-20, 60), 130.0(C-30, 50), 127.7(C-40),
139.3(C-100), 124.0(C-200), 137.2(C-300), 126.1(C-400), 132.3(C-500), 120.1(C-600),
168.4(CO2H); EI (70 eV) m/z (%): M+. 364.07926 (100), 311.10332 (20),
283.10468 (9), 236.22030 (3), 155.55160 (7), 77.17758 (20).
3d. 4-(30-Nitrophenylamino)-1-phenyl-1H-pyrazolo[3,4-b]pyridine-5-carboxylic
acid
Yield: 1.01 g (90%), m.p.: 255 °C, IR (KBr, cm-1): (m OH 3401-2603; m C = O
1
l674); H NMR (DMSO-d6, J in Hz, d in ppm) 7.12(s, H-3), 9.04(s, H-6), 8.22(d,
7.5, H-20,60), 7.65(dd, 7.5, H-30,50), 7.46(dd, 7.5, H-40), 8.42(s, H-200), 8.35(d, 8.7, H-

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363
400), 7.88(d, 8.7, H-500), 8.03(d, 8.7, H-600), 10.99(s, N-H); 13C NMR (DMSO-d6, d in
ppm) 138.2(C-3), 102.1(C-3a), 148.8(C-4), 104.3(C-5), 152.7(C-6), 152.0(C-7a),
120.2(C-10), 126.1(C-20, 60), 132.0(C-30, 50), 130.5(C-40), 148.1(C-100), 121.3(C-200),
140.0(C-300), 128.7(C-400), 134.0(C-500), 120.9(C-600), 168.2(CO2H); EI (70 eV) m/z
(%): M+. 375.09687 (100); 310.09034 (27); 211.20865 (8); 178.57242 (23);
145.62161 (9); 121.49006 (10); 77.17135 (9).
3e. 4-(40-Nitrophenylamino)-1-phenyl-1H-pyrazolo[3,4-b]pyridine-5-carboxylic
acid
Yield: 1.04 g (93%), m.p.: 256°C; IR (KBr, cm-1): (m OH 3409-2603; m C = O
1
1678); H NMR (DMSO-d6, J in Hz, d in ppm) 7.16(s, H-3), 9.06(s, H-6), 8.23(d,
7.5, H-20,60), 7.65(dd, 7.5, H-30,50), 7.46(dd, 7.5, H-40), 7.74(d, 8.7, H-200), 8.40(d,
8.7, H-300), 8.40(d, 8.7, H-500), 7.74(d, 8.7, H-600), 10.98(s, N-H); 13C NMR (DMSO-
d6, d in ppm) 139.5(C-3), 130.3(C-3a), 148.2(C-4), 104.9(C-5), 153.7(C-6),
153.1(C-7a), 120.3(C-10), 126.0(C-20, 60), 133.2(C-30, 50), 129.9(C-40), 148.1(C-
100), 121.3(C-200), 139.4(C-300), 127.3(C-400), 131.7(C-500), 120.3(C-600),
170.4(CO2H); EI (70 eV) m/z (%): M+. 375.09575 (100), 311.09014 (45),
283.09583 (26), 204.83632 (23), 138.51953 (12), 116.02373 (20), 83.21144 (14).
3f. 4-(30-Bromophenylamino)-1-phenyl-1H-pyrazolo[3,4-b]pyridine-5-carboxylic
acid
Yield: 1.10 g (90%), m.p.: 246°C; IR (KBr, cm-1): (m OH 3490-2580; m C = O
1
1650); H NMR (DMSO-d6, J in Hz, d in ppm) 6.65(s, H-3), 8.72(s, H-6), 7.93(d,
7.5, H-20,60), 7.50-7.31(5H, m), 7.17(dd, 7.5, H-40), 7.57(s, H-200), 12.90(s, CO2H),
10.57(s, N-H); 13C NMR (DMSO-d6, d in ppm) 134.4(C-3), 102.0(C-3a), 150.0(C-
4), 104.7(C-5), 153.3(C-6), 152.6(C-7a), 120.5(C-10), 125.9(C-20, 60), 130.5(C-30,
50), 129.6(C-40), 141.0(C-100), 121.5(C-200), 138.8(C-300), 126.6(C-400), 131.7(C-500),
120.5(C-600), 170.0(CO2H); EI (70 eV) m/z (%): M+. 410.00421 (100), 339.37886
(8), 311.07759 (100), 210.05296 (7), 76.04006 (5).
3g. 4-(40-Bromophenylamino)-1-phenyl-1H-pyrazolo[3,4-b]pyridine-5-carboxylic
acid
Yield: 1.10 g (90%), m.p.: 265°C; IR (KBr, cm-1): (m OH 3431-2580; m C = O
1
1651); H NMR (DMSO-d6, J in Hz, d in ppm) 6.87(s, H-3), 8.95(s, H-6), 8.16(d,
7.5, H-20,60), 7.59(dd, 7.5, H-30,50), 7.40(dd, 7.5, H-40), 7.50(d, 8.7, H-200), 7.78(d,
8.7, H-300), 7.78(d, 8.7, H-500), 7.50(d, 8.7, H-600), 13.00(s, CO2H), 10.73(s, N-H);
13C NMR (DMSO-d6, d in ppm) 132.5(C-3), 101.5(C-3a), 149.9(C-4), 104.5(C-5),
153.0(C-6), 152.2(C-7a), 115.0(C-10), 120.0(C-20, 60), 128.7(C-30, 50), 126.3(C-40),
138.4(C-100), 120.0(C-200), 134.2(C-300), 121.2(C-400), 128.9(C-500), 115.0(C-600),

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169.7(CO2H); EI (70 eV) m/z (%): M+. 410.00732 (13), 386.21067 (4), 311.08624
(100), 283.04687 (13), 82.30021 (5).
3h. 4-(30-Methylphenylamino)-l-phenyl-1H-pyrazolo[3,4-b]pyridine-5-carboxylic
acid
Yield: 0.92 g (90%), m.p.: 237°C; IR (KBr, cm-1): (m OH 3482-2592; m C = O
1650); 1H NMR (DMSO-d6, J in Hz, d in ppm) 6.71(s, H-3), 8.95(s, H-6), 8.16(d, 7.5,
H-20,60), 7.60(dd, 7.5, H-30,50), 7.52(dd, 7.5, H-40), 7.44-7.32(4H, m), 10.81(s, N-H),
2.21(s, Ar-CH3); 13C NMR (DMSO-d6, d in ppm) 138.9(C-3), 101.4(C-3a), 151.1(C-
4), 104.7(C-5), 153.3(C-6), 152.6(C-7a), 124.4(C-10), 127.8(C-20, 60), 130.0(C-30,
50), 129.4(C-40), 140.0(C-100), 126.7(C-200), 139.1(C-300), 128.9(C-400), 134.8(C-500),
121.7(C-600), 170.3(CO2H), 21.3(Ar-CH3); EI (70 eV) m/z (%): M+. 344.13178 (95),
326.11951 (100), 297.11572 (5), 270.10950 (3), 199.93871 (4), 73.35493 (4).
3i. 4-(40-Chlorophenylamino)-l-phenyl-1H-pyrazolo[3,4-b]pyridine-5-carboxylic
acid
Yield: 0.97 g (89%), m.p.: 268°C; IR (KBr, cm-1): (m OH 3430-2603; m C = O
1
1654); H NMR (DMSO-d6, J in Hz, d in ppm) 6.87(s, H-3), 8.97(s, H-6), 8.17(d,
7.5, H-20,60), 7.61(dd, 7.5, H-30,50), 7.42(dd, 7.5, H-40), 7.58(d, 8.7, H-200), 7.67(d,
8.7, H-300), 7.67(d, 8.7, H-500), 7.58(d, 8.7, H-600), 10.77(s, N-H); 13C NMR (DMSO-
d6, d in ppm) 131.7(C-3), 101.4(C-3a), 150.0(C-4), 104.3(C-5), 153.0(C-6),
152.2(C-7a), 121.2(C-10), 128.5(C-20, 60), 128.9(C-30, 50), 128.9(C-40), 138.4(C-
100), 126.3(C-200), 134.2(C-300), 137.9(C-400), 129.6(C-500), 121.2(C-600),
169.6(CO2H); EI (70 eV) m/z (%): M+. 364.07828 (100), 311.10296 (20),
283.11744 (9), 236.22865 (3), 155.54781 (7), 77.17234 (20).
3j. 4-(30-Methoxyphenylamino)-l-phenyl-1H-pyrazolo[3,4-b]pyridine-5-carboxylic
acid
Yield: 0.97 g (90%), m.p.: 237°C; IR (KBr, cm-1): (m OH 3330-2590; m C = O
1
1654); H NMR (DMSO-d6, J in Hz, d in ppm) 6.91(s, H-3), 9.05(s, H-6), 8.27(d,
7.5, H-20,60), 7.69(dd, 7.5, H-30,50), 7.51(dd, 7.5, H-40), 7.66-7.61(4H, m), 10.88(s,
N-H), 3.95(s, Ar-OCH3); 13C NMR (DMSO-d6, d in ppm) 139.3(C-3), 101.8(C-3a),
151.1(C-4), 105.0(C-5), 153.7(C-6), 153.0(C-7a), 114.3(C-10), 122.0(C-20, 60),
131.3(C-30, 50), 129.7(C-40), 140.7(C-100), 119.6(C-200), 161.0(C-300), 127.0(C-400),
135.2(C-500), 113.1(C-600), 170.6(CO2H), 56.1(Ar-OCH3); EI (70 eV) m/z (%): M+.
360.12379 (100), 298.08825 (10), 230.57425 (4), 171.06160 (8), 77.17826 (13).
3k. 4-(40-Methoxyphenylamino)-l-phenyl-1H-pyrazolo[3,4-b]pyridine-5-carboxylic
acid
Yield: 0.98 g (91%), m.p.: 261°C; IR (KBr, cm-1): (m OH 3430-2588; m C = O
1
1654); H NMR (DMSO-d6, J in Hz, d in ppm) 6.70(s, H-3), 9.03(s, H-6), 8.26(d,

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7.5, H-20,60), 7.69(dd, 7.5, H-30,50), 7.51(dd, 7.5, H-40), 7.28(d, 8.7, H-200), 7.56(s, H-
300), 7.56(d, 8.7, H-50000), 7.28(d, 8.7, H-600), 10.77(s, N-H), 4.02(s, Ar-OCH3); 13C
NMR (DMSO-d6, d in ppm) 131.3(C-3), 100.5(C-3a), 151.3(C-4), 104.1(C-5),
152.9(C-6), 152.2(C-7a), 114.1(C-10), 121.1(C-20, 60), 128.7(C-30, 50), 126.1(C-40),
138.5(C-100), 120.0(C-200), 134.2(C-300), 158.7(C-400), 128.8(C-500), 114.0(C-600),
169.8(CO2H), 55.2(Ar-OCH3); EI (70 eV) m/z (%): M+. 360.12451 (100),
298.08978 (10), 230.58327 (4), 171.07122 (8), 77.17399 (13).
3l. 4-(30-Fluorophenylamino)-l-phenyl-1H-pyrazolo[3,4-b]pyridine-5-carboxylic
acid
Yield: 0.97 g (93%), m.p.: 258°C; IR (KBr, cm-1): (m OH 3412-2586; m C = O
1675); 1H NMR (DMSO-d6, J in Hz, d in ppm) 7.04(s, H-3), 9.08(s, H-6),
8.28(d,7.5, H-20,60), 7.79-7.54(6H, m), 7.49(dd,7.5, H-40), 10.98(s, N-H); 13C NMR
(DMSO-d6, d in ppm) 134.8(C-3), 102.3(C-3a), 150.4(C-4), 105.0(C-5), 153.6(C-6),
152.9(C-7a), 115.0(C-10), 121.9(C-20, 60), 129.6(C-30, 50), 126.9(C-40), 141.4(C-100),
121.9(C-200), 139.2(C-300), 123.3(C-400), 131.9(C-500), 114.8(C-600), 170.4(CO2H); EI
(70 eV) m/z (%): M+. 348.10136 (100), 304.11069 (7), 274.07411 (4), 198.04128
(4), 165.04218 (10), 77.17166 (12).
General procedure for the preparation of the ethyl 4-[(methylpyridin-2-
yl)amino]-1-phenyl-1H-pyrazolo[3,4-b]pyridine-5-carboxylates (4a–c)
An equimolar mixture of 1 (1.08 g, 4 mmol) and a slightly excess of the appropriate
aminopicoline (5 mmoles) in 10 ml DMF was heated in a silicone oil bath under
reflux for 4 hours. The reaction mixture, after cooling, was poured into 50 ml of ice-
cold water. The precipitate was filtered, dried, and recrystallized from a mixture of
ethanol and water. The compounds were isolated in good yields (50–60%). The
1
structures of the compounds were elucidated by IR, H and 13C NMR, and mass
spectrometry.
4a. Ethyl 4-[(40-methylpyridin-2-yl)amino]-1-phenyl-1H-pyrazolo[3,4-b]pyridine-5-
carboxylate
Yield: 0.72 g (60%), m.p.: 180°C; IR (KBr, cm-1): (m NH 3110; m C = O 1696); 1H
NMR (DMSO-d6, J in Hz, d in ppm) 8.12(d, 7.5, H-20,60), 7.52(dd, 7.5, H-30,50),
7.33(dd, 7.5, H-40), 7.33(dd, 7.5, H-300), 7.52(dd, 7.5, H-400), 8.13(d, 7.5, H-600),
4.38(q, 7.0, CH2), 1.40(t, 7.0, CH3), 9.56 (s, N-H), 3.30 (s, Ar-CH3); 13C NMR
(DMSO-d6, d in ppm) 136.7(C-3), 107.0(C-3a), 145.5(C-4), 108.0(C-5), 152.7(C-6),
152.5(C-7a), 134.0(C-10), 121.7(C-20, 60), 129.6(C-30, 50), 126.5(C-40), 157.1(C-200),
121.6(C-300), 139.4(C-400), 119.4(C-500), 152.0(C-600), 167.1(CO2Et), 26.1(Ar-CH3),
60.9(CH2), 14.6(CH3); EI (70 eV) m/z (%): M+. 373.4102 (100)

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4b. Ethyl 4-[(50-methylpyridin-2-yl)amino]-1-phenyl-1H-pyrazolo[3,4-b]pyridine-5-
carboxylate
Yield: 0.70 g (50%), m.p.: 193°C; IR (KBr, cm-1): (m NH 3110; m C = O 1695); 1H
NMR (DMSO-d6, J in Hz, d in ppm) RMN 8.90 (s, H-3), 9.26 (s, H-6), 8.38 (d, 7.5,
H-20,60), 7.82(dd, 7.5, H-30,50), 7.63(dd, 7.5, H-40), 8.38(d, 7.5, H-600), 4.62(q, 7.0,
CH2), 1.58(t, 7.0, CH3), 3.50(s, Ar-CH3); 13C NMR (DMSO-d6, d in ppm) 135.1(C-
3), 101.2(C-3a), 145.4(C-4), 103.4(C-5), 152.4(C-6), 152.3(C-7a), 139.0(C-10),
122.2(C-20, 60), 130.0(C-30, 50), 127.8(C-40), 157.5(C-200), 117.8(C-300), 139.1(C-400),
120.3(C-500), 152.1(C-600), 164.2(CO2Et), 21.9(Ar-CH3), 62.5(CH2), 14.8(CH3); EI
(70 eV) m/z (%): M+. 373.4139 (100)
4c. Ethyl 4-[(60-methylpyridin-2-yl)amino]-1-phenyl-1H-pyrazolo[3,4-b]pyridine-5-
carboxylate
Yield: 0.70 g (50%), m.p.: 165°C; IR (KBr, cm-1): (m NH 3125; m C = O 1693); 1H
NMR (DMSO-d6, J in Hz, d in ppm) 9.40 (s, H-3), 9.50 (s, H-6), 9.10 (d, 7.5, H-
20,60), 8.30-7.20(6H, m), 4.45(q, 7.0, CH2), 1.45(t, 7.0, CH3), 2.20 (s, Ar-CH3); 13C
NMR (DMSO-d6, d in ppm) 136.2(C-3), 104.0(C-3a), 145.9(C-4), 102.8(C-5),
152.1(C-6), 152.9(C-7a), 136.7(C-10), 122.4(C-20, 60), 129.1(C-30, 50), 126.9(C-40),
156.8(C-200), 120.2(C-300), 139.7(C-400), 119.6(C-500), 152.9(C-600), 164.9(CO2Et),
25.2(Ar-CH3), 60.5(CH2), 14.1(CH3); EI (70 eV) m/z (%): M+. 373.4186 (100)
General procedure for the preparation of the 4-[(methylpyridin-2-yl)amino]-1-
phenyl-1H-pyrazolo[3,4-b]pyridine-4-carboxylic acids (5a–c)
A mixture of 3 mmol of (4a–c), 10 ml of 20% sodium hydroxide solution, and
10 ml of ethanol was heated under reflux for 1–3 hours. On cooling mixture was
acidified with diluted hydrochloric acid (1:3), and the precipitated was filtered and
recrystallized from a mixture of DMF and water. The compounds were isolated in
excellent yields (80–93%). The structures of the compounds were elucidated by IR,
1H and 13C NMR, and mass spectrometry.
5a. 4-[(40-Methylpyridin-2-yl)amino]-l-phenyl-1H-pyrazolo[3,4-b]pyridine-5-
carboxylic acid
Yield: 0.73 g (80%), m.p.:[300°C; IR (KBr, cm-1): (m OH 3491-2580; m C = O
1
1674); H NMR (DMSO-d6, J in Hz, d in ppm) 8.42(s, H-3), 9.07(s, H-6), 8.22(d,
8.0, H-20,60), 7.69(dd, 7.5, H-30,50), 7.51(dd, 7.5, H-40), 7.90(d, 7.5,H-300), 7.42(d,
7.5, H-400), 7.73(s, H-600), 11.37(s, CO2H), 3.56(s, Ar-CH3); 13C NMR (DMSO-d6, d
in ppm) 135.8(C-3), 102.4(C-3a), 145.3(C-4), 106.1(C-5), 152.5(C-6), 151.1(C-7a),
137.0(C-10), 121.9(C-20, 60), 129.9(C-30, 50), 126.8(C-40), 157.3(C-200), 118.6(C-300),

Med Chem Res (2007) 16:352–369
367
139.7(C-400), 119.8(C-500), 152.6(C-600), 21.7(Ar-CH3), 170.9(CO2H); EI (70 eV) m/
z (%): M+. 345.3629 (100)
5b. 4-[(50-Methylpyridin-2-yl)amino]-l-phenyl-1H-pyrazolo[3,4-b]pyridine-5-
carboxylic acid
Yield: 0.85 g (93%), m.p.: 225°C; IR (KBr, cm-1): (m OH 3426-2600; m C = O
1
1678); H NMR (DMSO-d6, J in Hz, d in ppm) 8.81(s, H-3), 9.08(s, H-6), 8.23(d,
8.0, H-20,60), 7.61(dd, 7.5, H-30,50), 7.44(dd, 7.5, H-40), 7.95(s, H-300), 7.70(d, 7.5, H-
500), 8.43(d, 7.5, H-600), 11.36(s, CO2H), 9.46(s, N-H), 3.27(s, Ar-CH3); 13C NMR
(DMSO-d6, d in ppm) 135.6(C-3), 102.8(C-3a), 145.7(C-4), 104.2(C-5), 152.1(C-6),
152.5(C-7a), 138.5(C-10), 121.6(C-20, 60), 129.2(C-30, 50), 127.2(C-40), 157.8(C-200),
117.6(C-300), 139.0(C-400), 119.6(C-500), 152.2(C-600), 21.2(Ar-CH3), 170.2(CO2H);
EI (70 eV) m/z (%): M+. 345.3678 (100)
5c. 4-[(60-Methylpyridin-2-yl)amino]-l-phenyl-1H-pyrazolo[3,4-b]pyridine-5-
carboxylic acid
Yield: 0.78 g (85%), m.p.: 252°C; IR (KBr, cm-1): (m OH 3400-2500; m C = O
1
1678); H NMR (DMSO-d6, J in Hz, d in ppm) 8.86(s, H-3), 9.10(s, H-6), 8.25(d,
8.0, H-20,60), 7.65(dd, 7.5, H-30,50), 7.46(dd, 7.5, H-40), 7.36(d, 7.5, H-400), 7.77(dd,
7.5, H-500), 8.35(d, 7.5, H-600), 11.45(s, CO2H), 9.51(s, NH), 3.31(s, Ar-CH3); 13C
NMR (DMSO-d6, d in ppm) 135.4(C-3), 102.2(C-3a), 145.1(C-4), 103.7(C-5),
152.8(C-6), 152.0(C-7a), 138.9(C-10), 121.0(C-20, 60), 129.8(C-30, 50), 127.6(C-40),
157.1(C-200), 116.8(C-300), 139.7(C-400), 119.2(C-500), 152.8(C-600), 21.7(Ar-CH3),
171.2(CO2H); EI (70 eV) m/z (%): M+. 345.3634 (100)
Cell culture and virus
Vero cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM)
supplemented with 5% fetal bovine serum (FBS; HyClone, Logan, Utah), 100 U/
mL penicillin and 100 ug/mL streptomycin, at 37°C in 5% CO2.
Cytotoxicity assay
A monolayer of about 104 Vero cells in 96-multiwell plates was treated with
various concentrations of the compounds for the 72 h. Then, 50 ll of a 1 mg/mL
solution of 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyl tetrazolium bromide (MTT;
Sigma) was added to evaluate cell viability according to procedures described
elsewhere (Mossmann, 1983). The 50% cytotoxic concentration (CC50) was
calculated by linear regression analysis of the dose–response curves.

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Plaque-reduction assay
The assay followed procedures described previously (Lucero et al., 2006).
Acyclovir was used as a positive control. Vero cells in six-well plates were
exposed to different dilutions of the supernatant from the assay described above for
1 h at 37°C. Residual viruses were then rinsed with PBS, and DMEM containing
5% FBS and 1% methylcellulose (Fluka) (overlay medium) was added to cells.
After 72 h at 37°C, the monolayers were fixed with 10% formaldehyde in PBS and
stained with a 0.1% solution of crystal violet in 70% methanol, and the virus titers
were calculated by scoring the plaque-forming units (PFU). The 50% antiviral
concentration (EC50) was calculated by linear regression analysis of dose–response
curves.
´
Acknowledgments We thank the Conselho Nacional de Desenvolvimento Cientıfico e Tecnologico
(CNPq), Coordenac¸a˜o de Aperfeic¸oamento de Pessoal Docente (CAPES), and Fundac¸a˜o de Amparo a
´
`
Pesquisa do Estado do Rio de Janeiro (FAPERJ) for financial support. The technical assistance of Samara
and Hania is gratefully acknowledged.
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