Synthesis, in vitro evaluation, and SAR studies of a potential antichagasic 1H-pyrazolo[3,4-b]pyridine series

Article abstract of DOI:10.1016/j.bmc.2006.09.067

The development of new drugs against Trypanosoma cruzi is still required since the only two drugs currently used cause severe side effects. In this work we described the synthesis, the in vitro biological evaluation, and the SAR results of 1H-pyrazolo[3,4

Full text of DOI:10.1016/j.bmc.2006.09.067

Bioorganic & Medicinal Chemistry 15 (2007) 211–219
Synthesis, in vitro evaluation, and SAR studies of a potential
antichagasic 1H-pyrazolo[3,4-b]pyridine series
a,b
c
Luiza R. S. Dias,a, Marcelo B. Santos, Sergio de Albuquerque,
*
´
Helena C. Castro,d Alessandra M. T. de Souza,b,d Antonio C. C. Freitas,
a
ˆ
Maria A. V. DiVaio,a Lucio M. Cabrale and Carlos R. Rodriguese
aUniversidade Federal Fluminense (UFF), Laborato´rio de Quı´mica Medicinal (LQMed), Faculdade de Farma´cia,
Rua Ma´rio Viana, 523, Santa Rosa, 24241-000, Nitero´i, RJ, Brazil
b
ˆ
Universidade Federal Fluminense, Instituto de Quı´mica, Departamento de Quı´mica Organica,
ˆ
´
Programa de Po´s-Graduac¸a˜o em Quı´mica Organica, Campus do Valonguinho, 24020-150, Niteroi, RJ, Brazil
cUniversidade de Sa˜o Paulo, Departamento de Ana´lises Clı´nicas, Toxicolo´gicas e Bromatolo´gicas, FCFRP/USP,
ZIP 29020-159, Sa˜o Paulo, SP, Brazil
dUniversidade Federal Fluminense (UFF), Laborato´rio de Antibio´ticos, Bioquı´mica e Modelagem Molecular (LABioMol),
Instituto de Biologia—Outeiro de Sa˜o Joa˜o Baptista, 24020-150, Nitero´i, RJ, Brazil
eUniversidade Federal do Rio de Janeiro, Laborato´rio de Modelagem Molecular e QSAR (ModMolQSAR),
Faculdade de Farma´cia, 21941-590, Rio de Janeiro, RJ, Brazil
Received 26 June 2006; revised 29 August 2006; accepted 29 September 2006
Available online 1 October 2006
Abstract—The development of new drugs against Trypanosoma cruzi is still required since the only two drugs currently used cause
severe side effects. In this work we described the synthesis, the in vitro biological evaluation, and the SAR results of 1H-pyrazol-
o[3,4-b]pyridine derivatives, a new antichagasic agent series. The presence of fluorine, hydroxyl or nitro group at Y position resulted
in at least one or two promising compounds in each set of derivatives (6f, 6g, 6i, 6l, and 6m). The SAR study showed that trypan-
ocidal activity observed depends on both geometric and stereoelectronic parameters (MEP and frontier molecular orbitals HOMO
and LUMO). We also used the Osiris program for calculating and comparing the fragment based druglikeness of the most active
derivative (6g) (IC50 = 1.9 lg/mL), the inactive compound (6o), and the current toxic antichagasic drugs (nifurtimox and benznidaz-
ole). Interestingly 6g presented a potential druglikeness higher than nifurtimox and benznidazole while 6o presented the lowest value
among them.
ꢀ 2006 Published by Elsevier Ltd.
1. Introduction
dazole] (2) (Fig. 1). However they are often poorly toler-
ated and may produce serious toxic effects in the host.4
In the acute phase of the disease, these compounds are
frequently ineffective due to differences in T. cruzi
strains’ drug susceptibility, and they are useless for the
chronic stage.5–8 Thus, effective and safe drugs are still
needed for Chagas disease.
Chagas’ disease is caused by Trypanosoma cruzi, a par-
asite with a large zoonotic reservoir in Central and
South America.1–3 Its endemy keeps 100 million people
at risk and about 20 million people chronically infected
with T. cruzi.4
Current treatment of Chagas’ disease involves the use of
nitrofurans [nifurtimox] (1) and nitroimidazoles [benzni-
N
O
N
N
O
N
N
H
S
O
O2N
Keywords: Structure–activity relationship (SAR); 1H-pyrazolo[3,4-b]-
pyridine; Antiparasite agent; Chagas.
O2N
O
(1)
(2)
*
Corresponding author. Tel.: +55 21 2629 9588; fax: +55 21 2629
9578; e-mail: ldias@vm.uff.br
Figure 1. Nifurtimox (1) and benznidazole (2).
0968-0896/$ - see front matter ꢀ 2006 Published by Elsevier Ltd.
doi:10.1016/j.bmc.2006.09.067

212
L. R. S. Dias et al. / Bioorg. Med. Chem. 15 (2007) 211–219
2. Results and discussion
N
H C
3
O
C
NH
H C
3
2.1. Chemistry
Y
N
NH
2
N
N
The synthetic pathway is depicted in Scheme 1. The
intermediate compound 4 may be obtained in ꢀ50%
yield by a sequence of five reactions as described for
ethyl-3-methyl-1-phenyl-1H-pyrazolo[3,4-b]pyridine-4-
carboxylate.10 This new method explores the commer-
cially available ethyl pyruvate in a modified Quiroga
reaction (Scheme 1).16
N
N
B
(3)
(6)
X
A
a
c
CO C H
2 5
CONHNH
N
2
2
H C
3
H C
3
Briefly the 5-amino-3-methyl-1-phenyl-1H-pyrazole 3
was reacted with aromatic aldehydes and ethyl pyruvate
N
N
b
N
N
N
1
to originate ester 4, which was confirmed by H NMR
X
X
(C-5 hydrogen atom = d8.20 ppm, ester function = d4.6
and d1.5 ppm), GC–MS (one peak at 31.93 min) and
the fragment m/z = 357 (100%) in MS spectrometry.
Ester 4 was converted to the hydrazine 5 using a func-
tional group conversion and the treatment with hydra-
zine hydrate (80%) in ethanol. Derivative 5 showed a
characteristic band of –NH group in m3210–3190 cmꢁ1
in the IR spectra. We performed the conversion of 5
to the benzylidene-carbohydrazide 6 by condensation
using the appropriate aromatic aldehydes in acidic medi-
um.17 The IR spectra also showed the carbonyl group of
(4)
(5)
X= H, CN, NO2 , Cl, OCH3 , OC2H5
Y=H,FF,, OH, NO2
a) 1b-enazroymlidaetnicealdaelhdyedheysd,eEst,OHH,CHl,C2l,hrefluaxn,d2h;ethyl
2- ethyl pyruvate, reflux, 2h (4 yield = 30-50%)
4
pyruvate , HCl, 2h ( yie d = 38-48%)
5
b) NH22NNHH2 .H2OO, reflux, 2h ( yield = 75-95% )
2
2
6
c) abreonmzyaltidiceanledehayldeh, yHdCels, E, tHOCHl,,reEfltuOxH; (,6ryeifeluldx=; (
60-99%)
yield = 60-99%)
Scheme 1. The synthetic pathway of N0-benzylidene-3-methyl-1,6-
diphenyl-1H-pyrazolo[3,4-b]pyridine-4-carbohydrazide.
the compounds 6a–6p in 1650 cmꢁ1
.
The presence of a tautomeric enidrazine form was consid-
ered18 but the IR analysis revealed only the hydrazine
form with a strong absorption band assigned to group
–NH in m3200 cmꢁ1. The 1H and 13C NMR spectra
showed signals in a range of d 8.3–9.1 ppm and d 147.1–
149.5 ppm corresponding to the methylenic groups (–
CH@phenyl).19 The benzylidene-carbohydrazide unit re-
Since 1990’s several phenylpyrazole derivatives have
been described as antiparasites, and the nucleus
1H- pyrazolo[3,4-b]pyridine has been evaluated for its
antiparasitic profile.9–12 Literature also described struc-
tural subunits such as carbohydrazides [R2C = N–NH–
CO–] and their importance for the antiparasitic
activity.13,14
vealed no characteristic doublet signal in the 1H and 13
C
NMR spectra, which suggest the existence of a single dia-
stereoisomer. These results suggested that the thermody-
namic control favored the formation of a unique
diastereoisomeric product in the condensation step
among hydrazide (5) and different aromatic aldehydes.
In order to identify a leading compound against T.
cruzi, in this work we designed and synthesized six-
teen new 4-benzylidene-hydrazide-1H-pyrazolo[3,4-
b]pyridine de-rivatives (6) (Scheme 1). We designed
this new series of 1H-pyrazolo[3,4-b]pyridine deriva-
tives presenting substituent groups with different re-
dox properties in the benzylidene hydrazide moiety
possibly analogous to the redox properties of the
conventional drugs (benznidazole, allopurinol, and
nifurtimox) (Scheme 1). Stereoelectronic properties
were also evaluated in the phenyl at 6 position of
the pyrazolopyridine heterocycle. Aromatic aldehydes
were chosen in a way to explore the contribution
of lipophilic and electrophilic properties of these
substituents to the biological activity.15 In addition
we tested the precursor of these compounds (5),
which does not present the benzylidene hydrazide
moiety, to analyze its influence in the anti-parasitic
profile. The structure–activity relationship study of
these compounds was also conducted by comparing
the biological effects with the theoretical parameters
of the compounds such as HOMO and LUMO
(energy, coefficient orbital, and density), GAP and
clogP calculated by using a molecular modeling
approach.
2.2. Biology
All compounds in the 1H-pyrazolo[3,4-b]pyridine series
(6a–6p) were tested and showed some level of antipara-
site activity, except for 6o derivative (Fig. 2). These data
reinforced pyrazolopyridine as a potential system for
antiparasite activity as suggested by literature.9–12
Compared to crystal violet (IC50 = 71 lg/mL), and the
non-substituted derivative (6a) (IC50 = 205.3 lg/mL),
the presence of fluorine atom or hydroxyl group at Y
position resulted in at least two promising compounds
(IC50 < 150 lg/mL) in each set of derivatives (6b and
6f, 6g and 6i, respectively) such as nitro group that re-
vealed three compounds (6l–n) (Fig. 2 and Table 1). In-
deed, among the three substituents used at Y position
(F, OH, and NO2), the better profile was observed with
OH (6g) (IC50 = 1.9 lg/mL) that exhibited approximate-
ly a 35- and 100-fold higher activity than crystal violet
and the non-substituted derivative 6a, respectively
(Fig. 2 and Table 1). Despite the chemical features of

L. R. S. Dias et al. / Bioorg. Med. Chem. 15 (2007) 211–219
213
Figure 2. The N0-benzylidene-3-methyl-1,6-diphenyl-1H-pyrazolo[3,4-b]pyridine-4-carbohydrazide (6a–6p) and its precursors 3-methyl-1,6-diphenyl-
1H-pyrazolo[3,4-b]pyridine-4-carbohydrazide (5a–5f) substituents (A), the in vitro trypanocidal activity (%) (B), and the inhibitory effect
(concentration that inhibits 50% of parasite growth—IC50) expressed as 1/IC50 (C). The non-substituted derivative (6a) is represented in all graphics
as filled square in (B) and control (Cont) is crystal violet in (C).
Y substituents, the methoxy in X decreased the activity
of 6e, 6j, and 6o, compared to the non-substituted com-
pound (6a) (Fig. 2 and Table 1). In fact, substitution in
X position seems to be critical for the antichagasic pro-
file as suggested by the biological results of 6b, 6g and 6l
(the non-substituted derivatives), which were higher

214
L. R. S. Dias et al. / Bioorg. Med. Chem. 15 (2007) 211–219
Table 1. Comparison of in vitro trypanocidal activity and the molecular electronic properties (HOMO and LUMO energy, GAP, and clogP) of the
N0-benzylidene-3-methyl-1,6-diphenyl-1H-pyrazolo[3,4-b]pyridine-4-carbohydrazide (6a–6p), and its precursors (5a–5f)
Compound
Y
X
IC50 (lg/mL)
HOMO (eV)
LUMO (eV)
GAPa (eV)
clogP
5a
5b
5c
5d
5e
5f
—
—
H
781.8
533.3
341.2
211.0
781.2
439.4
205.3
88.9
ꢁ8.15
ꢁ8.12
ꢁ8.08
ꢁ7.93
ꢁ8.00
ꢁ7.98
ꢁ7.87
ꢁ7.92
ꢁ8.15
ꢁ8.01
ꢁ7.86
ꢁ7.84
ꢁ7.87
ꢁ8.11
ꢁ7.96
ꢁ7.82
ꢁ7.81
ꢁ8.04
ꢁ8.28
ꢁ8.13
ꢁ7.97
ꢁ7.96
1.58
0.98
1.27
1.71
1.71
1.73
1.71
1.65
1.12
1.51
1.72
1.75
1.72
1.17
1.56
1.80
1.81
0.77
0.65
0.73
0.78
0.78
9.73
9.10
9.35
9.64
9.71
9.71
9.58
9.57
9.27
9.52
9.58
9.59
9.59
9.28
9.52
9.62
9.62
8.81
8.93
8.86
8.75
8.74
3.09
3.12
3.12
3.65
2.96
3.30
6.24
6.40
6.43
6.96
6.27
6.61
5.37
5.88
6.41
5.73
6.06
6.28
6.31
6.83
6.15
6.48
NO2
CN
—
—
Cl
—
—
OCH3
OC2H5
H
6a
6b
6c
6d
6e
6f
H
F
H
CN
F
378.2
197.2
1461.0
39.3
F
Cl
F
OCH3
OC2H5
H
F
6g
6h
6i
OH
OH
OH
OH
OH
NO2
NO2
NO2
NO2
NO2
1.9
260.3
71.8
CN
Cl
6j
OCH3
OC2H5
H
331.5
169.6
95.3
6k
6l
6m
6n
6o
6p
CN
Cl
37.3
108.5
>1500
1179.0
OCH3
OC2H5
a GAP = ELUMO ꢁ EHOMO
.
active than those of 6e, 6j, and 6o, the substituted com-
pounds in this position (Fig. 2 and Table 1). The analy-
sis of Table 1 suggested that increments in the atomic
size of the substituent in that position could induce a ste-
ric effect that may prevent the perfect interaction of the
target/receptor.
In order to evaluate the importance of the benzylidene-
carbohydrazide, we tested the derivative precursors,
which did not present this moiety (5a–5f) (Fig. 2). Our
experimental data showed that most of these com-
pounds were less active (IC50 = 341–781.8 lg/mL) than
6a (IC50 = 205.3 lg/mL), except for 5d, the chlorine
substituted derivative (IC50 = 211 lg/mL) (Fig. 2/Table
1). These data pointed out this region with a significant
role for the improvement of the antiparasitic profile.
Figure 3. Comparison of molecular electrostatic potential energy
isosurfaces (MEP), HOMO and LUMO orbital coefficients of 6g, 6f,
6i, 6m, the most active compounds, and 6o and 6p, the less active
derivatives of 1H-pyrazolo[3,4-b]pyridine series.
2.3. SAR studies
ed between B ring and the pyrazole-pyridine moiety,
except when nitro is in Y position (6m).
Three-dimensional isosurfaces of MEP superimposed
onto total electron density of the most representative
members (1000 lg/mL < IC50 < 50 lg/mL) of the 1H-
pyrazolo[3,4-b]pyridine series are presented in Figure
3. The MEP revealed that the negative potential (red re-
gion) is present in all three aromatic rings (A, B, and C,
see Scheme 1) of the most active compounds (6g, 6m, 6i,
and 6f) in contrast to the less active (6o and 6p), which
presented only two negative regions (Fig. 3).
Interestingly, the derivative precursors that do not pres-
ent the benzylidene-carbohydrazide moiety (5a–5f) were
poorly active, which infers the requirement of this moi-
ety for a good biological profile. These compounds pre-
sented higher GAP values (Table 1), which is the
difference between the energies of the frontier orbitals.
This feature may have contributed to their smaller activ-
ity. In addition the 5a–5f lowest clogP values may be
deleterious for their biological activity as it may com-
promise reaching the target inside the cell by these mol-
ecules. This is inferred by 5d that presents the highest
clogP and the lowest IC50 of this set of compounds.
To gain insight into the atomic contribution on the fron-
tier orbital, we analyzed the three-dimensional HOMO
and LUMO coefficient contribution. The study of these
compounds showed the HOMO orbital concentrated at
A ring in 6g, 6m, and 6i. Differently 6o and 6p showed
HOMO coefficient orbital distributed along both A
and B rings, which may affect their interaction with
the target receptor (Fig. 3). Meanwhile LUMO is locat-
The individual analysis of the most active compound
6g suggested that the hydroxyl group might act as a

L. R. S. Dias et al. / Bioorg. Med. Chem. 15 (2007) 211–219
215
hydrogen bond donor in the interaction with the
receptor. The HOMO density is concentrated on the
topside-left of 6g structure in a higher degree while A
ring presents a different orientation compared to 6j, 6k
and 6o, some of the less active compounds. These 6g
structural features seem to be important for displaying
a significant antiparasitic activity (Fig. 4 and Table 1).
toxicity risks in one value and that may be used to judge
the compound’s overall potential to qualify for a drug.18
Our theoretical data showed that 6g was comparable to
benznidazole but better than nifurtimox. Once more 6j
and 6k, the intermediate compounds and 6o, the inactive
compound, presented lower values in agreement to their
biological activity. These results suggested that the
structural features of 6g might be useful in designing
new antichagasic agents.
Currently there are many approaches that assess a com-
pound’s druglikeness partially based on topological
descriptors, fingerprints of molecular drug likeness
structure keys or other properties as clogP and molecu-
lar weights.20 In the Osiris program (http://www.organ-
ic-chemistry.org/prog/peo/), the occurrence frequency of
each fragment is determined within the collection of
traded drugs and within the supposedly non-drug-like
collection of Fluka compounds. On that case, positive
values (0.1–10) point out that the molecule contains pre-
dominantly the better fragments, which are frequently
present in commercial drugs. In this work we used the
Osiris program for calculating the fragment based drug-
likeness of the most active (6g), the inactive compound
(6o), and some intermediate compounds (6j and 6k)
along with nifurtimox and benznidazole, the current
3. Conclusion
In conclusion, we have developed an easy one-step syn-
thesis to obtain the N0-benzylidene-3-methyl-1,6-diphen-
yl-1H-pyrazolo[3,4-b]pyridine-4-carbohydrazide using a
modified Quiroga reaction. The benzylidene-carbohyd-
razide derivatives (6) could be obtained just by using
functional group conversions. The 6g derivative showed
the highest trypanocidal profile according to the higher
HOMO coefficient orbital and density, which is localized
in the A ring, and to its higher druglikeness value com-
pared to other trypanocidal drugs. The biological and
SAR results pointed out 6g as a potential leading com-
pound for the development of new drugs for Chagas
treatment.
toxic antichagasic drugs. Interestingly 6g (IC50
=
1.9 lg/mL) presented a potential druglikeness value
higher than those of the molecules studied including nif-
urtimox and benznidazole while 6o presented the lowest
value among them (Fig. 4).
4. Experimental
4.1. Chemistry
In this study we also verified the drug score, which com-
bines druglikeness, clogP, logS, molecular weight, and
Melting points were determined in a capillary Thomas
Hoover apparatus and are given uncorrected. Infrared
(IR) spectra were recorded on a Perkin-Elmer 1420 spec-
1
trometer in potassium bromide pellets. The H and 13C
NMR spectra were recorded using a Varian UNITY-
plus-300 at 300 MHz spectrophotometer for solutions
in DMSO. GC–MS spectra were recorded using a GC
6850N Network, GS system (Agilent), injector 7683 ser-
ies, detector 5973N, mass selective detector, column:
DB-5 ms, 30 m, ID: 0.25 · 0.25 lm. The progress of all
reactions was monitored by t.l.c. performed on Kilsegel
60F 0.2 mm (Merck-Darmstadt). The developed chro-
matograms were viewed under ultraviolet light (254–
365 mm).
4.2. General procedure for the preparation of ethyl 3-
methyl-1-phenyl-6-substituted-phenyl-1H-pyrazolo[3,4-
b]pyridine-4-carboxylate (4a–4f)
To a solution of aromatic aldehydes (5.78 mmol) in
EtOH (15 mL) and 5 drops of hydrochloric acid was
added 5-amino-3-methyl-1-phenyl-1H-pyrazole (1 g,
5.78 mmol). The reaction mixture was stirred at reflux
temperature for 2 h. Then ethyl pyruvate (5.78 mmol)
was added and the mixture was kept, at these condi-
tions, for additional 2 h. After cooling to room temper-
ature, the reaction mixture was poured into cold water
and 20% aqueous NaOH solution was added to obtain
the solid precipitated. The product was filtered off,
washed with cold water and dried. Recrystallization
from ethanol/water led to ethyl carboxylate derivatives.
Figure 4. Comparison of 6g, 6j, 6k, and 6o that vary from the highest
to the inactive profile. (A) HOMO density encoded onto a van der
Waals surface (isodensity 0.002 e/au3) with the HOMO’s absolute
density coefficient mapped from deepest red (0.00) to deepest blue
(0.03), (B) druglikeness and (C) drugscore values of the 6g, 6j, 6k, and
6o compared to those of nifurtimox and benznidazole using Osiris
program. (http://www.organic-chemistry.org/prog/peo/druglikeness.
html).

216
L. R. S. Dias et al. / Bioorg. Med. Chem. 15 (2007) 211–219
4.2.1. Ethyl3-methyl-1,6-diphenyl-1H-pyrazolo[3,4-b]pyr-
idine-4-carboxylate (4a). 50%, mp = 123 ꢁC. IR
(m-cmꢁ1): 3040 (C–H); 1760 (C@O); 1590–1570 (C@C).
GC/MS: rt 31.93 min, area 100%, 357 (100%); 328
ture was refluxed for 3 h under stirring. After cooling
to room temperature, the mixture was added to cold
water. The solid precipitated was filtered, washed with
water, and dried.
1
(15.78%); 77 (24.45%). HNMR: d 1.50 (t, 3H, H-11,
J = 7 Hz); 4.50 (q, 2H, H-10, J = 7 Hz); 2.80 (s, 3H,
H-8); 8.13 (s, 1H, H-5); 8.35 (dd, 2H, H-20, H-60,
J = 7.1 Hz, 1.4 Hz); 8.20 (m, 3H, H-30, H-40, H50);
7.50 (m, 5H, H200–H500).
4.3.1. 3-Methyl-1,6-diphenyl-1H-pyrazolo[3,4-b]pyridine-
4-carbohydrazide (5a). 92%, mp = 200 ꢁC. IR (m-cmꢁ1):
3300 (N–H); 3200 (N–H); 1650 (C@O); 1500 (C@C).
1HNMR: d 2.80 (s, 3H, H-8); 8.13 (s, 1H, H-5); 8.30
(m, 3H, H-300–H-500); 7.40 (m, 5H, H-20–H-60); 8.00 (m,
2H, H-200, H-600); 10.20(s, 1H, NH); 5.00 (s, 2H, NH2).
4.2.2. Ethyl 3-methyl-1-phenyl-6-p-nitrophenyl-1H-pyraz-
olo[3,4-b]pyridine-4-carboxylate (4b). 50%, mp = 138 ꢁC.
IR (m-cmꢁ1): 2980–2920 (C–H); 1760 (C@O); 1600–1570
(C@C); 1540, 1340 (NO2). 1HNMR: d 1.50 (t, 3H, H-11,
J = 7.2 Hz); 4.60 (q, 2H, H-10, J = 7.2 Hz); 2.00 (s, 3H,
H-8); 8.00 (s, 1H, H-5); 8.20 (m, 2H, H-300, H-500); 7.90
(m, 2H, H-200, H-600); 7.20 (m, 3H, H-30–H-50); 7.70
(m, 2H, H-20, H-60).
4.3.2. 3-Methyl-1-phenyl-6-p-nitrophenyl-1H-pyrazolo[3,4-
b]pyridine-4-carbohydrazide (5b). 75%, mp = 282 ꢁC. IR
(m-cmꢁ1): 3290 (N–H); 1640 (C@O); 1560, 1340 (NO2).
1HNMR: d 2.80 (s, 3H, H-8); 8.00 (s, 1H, H-5); 8.20
(m, 2H, H-300, H-500); 7.90 (m, 2H, H-200, H-600); 7.30 (m,
3H, H-30–H-50); 7.70 (m, 2H, H-20, H-60).
4.2.3. Ethyl 3-methyl-1-phenyl-6-p-cyanophenyl-1H-pyr-
azolo[3,4-b]pyridine-4-carboxylate (4c). 35%, mp =
144 ꢁC. IR (m-cmꢁ1): 3040–2920 (C–H); 2210 (CN);
1730 (C@O); 1600–1590 (C@C). 1HNMR: d 1.50 (t,
3H, H-11, J = 7.2 Hz); 4.60 (q, 2H, H-10, J = 7.2 Hz);
2.00 (s, 3H, H-8); 8.30 (s, 1H, H-5); 8.46 (d, 2H, H300,
H-500, J = 8.1 Hz); 8.15 (d, 2H, H-200, H-600, J = 8.1 Hz);
8.34 (d, 2H, H-20, H-60, J = 7.8 Hz); 7.48 (t, 1H, H-40);
7.75 (t, 2H, H-30, H-50, J = 7.8 Hz).
4.3.3. 3-Methyl-1-phenyl-6-p-cyanophenyl-1H-pyrazolo[3,4-
b]pyridine-4-carbohydrazide (5c). 95%, mp = 258 ꢁC. IR
(m-cmꢁ1): 3290 (N–H); 2210 (CN); 1680 (C@O). 1HNMR:
d 2.74 (s, 3H, H-8); 4.88 (sl, 2H, NH2); 10.55 (sl, 1H, NH);
7.72 (dd, 2H, H-30, J = 7.80 Hz); 7.48 (dd, 1H, H-40,
J = 7.2 Hz); 8.12 (s, 1H, H-5); 8.14 (d, 2H, H-20,
J = 8.1 Hz); 8.40 (d, 2H, H-300, J = 8.7 Hz); 8.55 (dd, 2H,
13
H-200, J = 8.4 Hz). CNMR: d 15.1 (C-8); 113.0 (C-400);
114.20 (C-3a); 119.3 (CN); 121.4 (C-5 e C-20); 126.6
(C-40); 128.8 (C-30e C-200); 129.9 (C-3); 133.6 (C-300);
139.5 (C-4); 140.2 (C-10); 151.6 (C-7a); 154.7 (C-6); 165.0
(C@O).
4.2.4. Ethyl 3-methyl-1-phenyl-6-p-chlorophenyl-1H-pyraz-
olo[3,4-b]pyridine-4-carboxylate (4d). 44%, mp = 123 ꢁC.
IR (m-cmꢁ1): 3040–2920 (C–H); 1730 (C@O); 1590–1570
1
(C@C). HNMR: d 1.50 (t, 3H, H-11, J = 7 Hz); 4.60
4.3.4. 3-Methyl-1-phenyl-6-p-chlorophenyl-1H-pyrazolo-
[3,4-b]pyridine-4-carbohydrazide(5d). 75%, mp = 238 ꢁC.
IR (m-cmꢁ1): 3280 (N–H); 1630 (C@O). 1HNMR: d
2.74 (s, 3H, H-8); 4.87 (sl, 2H, NH2); 10.19 (sl, 1H,
N–H); 7.47 (dd, 1H, H-40, J = 7.2 Hz); 7.70 (d, 2H, H-
300, J = 8.4 Hz); 7.75 (d, 2H, H-200, J = 8.7 Hz); 8.03 (s,
1H, H-5); 8.39 (dd, 2H, H-30, J = 5.4 Hz); 8.42 (d, 2H,
H-20, J = 5.4 Hz). 13CNMR: d 14.4 (C-8); 112.8 (C-3a);
120.5 (C-5 e C-20); 120.6 (C-40); 129.0 (C-30, C-200 e C-
300); 129.1 (C-3); 134.9 (C-400); 136.7 (C-4); 139.3 (C-
100); 142.4 (C-10); 150.9 (C-7a); 154.7 (C-6); 164.4 (C@O).
(q, 2H, H-10, J = 7 Hz); 2.00 (s, 3H, H-8); 8.26 (s, 1H,
H-5); 7.48 (m, 2H, H-200, H-600); 7.72 (m, 2H, H-300,
H-500); 8.35 (m, 5H, H-20–H-60).
4.2.5. Ethyl 3-methyl-1-phenyl-6-p-methoxyphenyl-1H-
pyrazolo[3,4-b]pyridine-4-carboxylate (4e). 30%, mp =
122 ꢁC. IR (m-cmꢁ1): 3040–2920 (C–H); 1760 (C@O);
1610–1570 (C@C). 1HNMR: d 1.55 (t, 3H, H-11,
J = 6.9 Hz); 4.60 (q, 2H, H-10, J = 6.9 Hz); 2.00 (s,
3H, H-8); 7.90 (s, 1H, H-5); 4.00 (s, 3H, OCH3); 7.25
(m, 2H, H-300, H-500); 7.48 (m, 2H, H-200, H-600); 7.75
(m, 3H, H-30–H-50); 8.40 (m, 2H, H-20, H-60).
4.3.5. 3-Methyl-1-phenyl-6-p-methoxyphenyl-1H-pyrazolo-
[3,4-b]pyridine-4-carbohydrazide (5e). 86%, mp = 205 ꢁC.
IR (m-cmꢁ1): 3280 (N–H); 1630 (C@O). 1HNMR: d
2.70 (s, 3H, H-8); 3.97 (s, 3H, OCH3); 4.93 (sl, 2H,
NH2); 10.17 (s, 1H, N–H); 7.24 (d, 2H, H-300,
J = 9 Hz); 7.45 (dd, 1H, H-40, J = 7.8 Hz); 7.71 (dd,
2H, H-30, J = 7.8 Hz); 7.96 (s, 1H, H-5); 8.34 (d, 2H,
H-200, J = 9 Hz); 8.44 (d, 2H, H-2, J = 7.8 Hz).
13CNMR: d 14.2 (C-8); 55.4 (OCH3); 112.3 (C-3a);
114.5 (C-300); 120.5 (C-5 e C-20); 125.5 (C-40); 128. 9
(C-200); 129.2 (C-30); 130.4 (C-100); 139.1 (C-4); 141.4
(C-10); 155.9 (C-400); 151.2 (C-7a); 161.0 (C-6); 164.8
(C@O).
4.2.6. Ethyl 3-methyl-1-phenyl-6-p-ethoxyphenyl-1H-pyr-
azolo[3,4-b]pyridine-4-carboxylate (4f). 30%, mp =
112 ꢁC. IR (m-cmꢁ1): 2980–2920 (C–H); 1740 (C@O);
1620–1570 (C@C). 1HNMR: d 1.45 (t, 3H, H-11,
J = 6.9 Hz); 4.60 (q, 2H, H-10, J = 6.9 Hz); d 1.60 (t,
3H, OCH2CH3, J = 7.0 Hz); 4.60 (q, 2H, OCH2CH3,
J = 7.0 Hz); 8.25 (s, 1H, H-5); 7.20 (d, 2H, H-300, H-500,
J = 9 Hz); 8.30 (d, 2H, H-200, H-600, J = 9 Hz); 8.40 (dd,
2H, H-20, H-60, J = 7.5 and 1.2 Hz); 7.70 (m, 2H, H-30,
H-50); 7.50 (m, 1H, H-40).
4.3. General procedure for the preparation of 3-methyl-1-
phenyl-6-substituted-phenyl-1H-pyrazolo[3,4-b]pyridine-
4-carbohydrazide (5a–5f)
4.3.6. 3-Methyl-1-phenyl-6-p-ethoxyphenyl-1H-pyrazolo-
[3,4-b]pyridine-4-carbohydrazide (5f). 70%, mp = 201 ꢁC.
IR (m-cmꢁ1) 3260 (N–H); 1650 (C@O). 1HNMR: d
1.49 (t, 3H, OCH2CH3); 2.71 (s, 3H, H-8); 4.68 (q,
2H, OCH2CH3); 4.86 (sl, 2H, NH2); 10.14 (sl, 1H,
Hydrazine monohydrate (1 mL) was added to a solution
of 6 (0.56 mmol) in ethanol (5 mL). The reaction mix-

L. R. S. Dias et al. / Bioorg. Med. Chem. 15 (2007) 211–219
217
N–H); 7.22 (d, 2H, H-300, J = 9 Hz); 7.45 (dd, 1H, H-
40, J = 7.5 Hz); 7.71 (dd, 2H, H-30, J = 7.5 Hz); 7.95 (s,
1H, H-5); 8.32 (d, 2H, H-200, J = 9 Hz); 8.44 (d, 2H,
4.4.4. N0-(4-Fluorobenzylidene)-3-Methyl-1-phenyl-6-p-
chlorophenyl-1H-pyrazolo[3,4-b]pyridine-4-carbohydraz-
ide (6d). 93%, mp = 230 ꢁC. IR (m-cmꢁ1): 3260 (N–H);
1
H-200, J = 7.5 Hz). 13CNMR:
d
14.3 (C-8); 14.5
1650 (C@O). HNMR: d 2.73 (s, 3H, H-8); 7.18–7.24
(OCH2CH3); 63.3 (OCH2CH3); 112.2 (C-3a); 114.8
(C-300); 120.3 (C-20); 125.4 (C-4000); 128.8 (C-200); 129.0
(C-30); 130.2 (C-3); 138.9 (C-1 ); 139.1 (C-4); 142.3
(C-10); 151.1 (C-7a); 155.9 (C-400); 160.2 (C-6); 164.7
(C@O).
(m, 1H, H-40); 7.41–7.51 (m, 2H, H-30); 7.69 (d, 2H, H-
300, J = 8.4 Hz); 7.73–7.77 (m, 2H, H-3000); 7.96–7.99 (m,
2H, H-2000); 8.20 (s, 1H, H-5); 8.36–8.44 (m, 4H, H-20 e
H-200); 8.45 (s, 1H, H-12); 12.31 (sl, 1H, H-10). 13CNMR:
d 14.2 (C-8); 111.9 (C-3a); 112.7 (C-3000); 120.4 (C-5 e C-
20); 125.5 (C-40); 128.9 (C-200 e C-2000); 129.0 (C-300); 129.4
(C-30); 130.5 (C-3); 134.8 (C-1000); 136.7 (C-400); 138.9 (C-
4); 139.2 (C-100); 142.3 (C-10); 148.0 (C-12); 150.9 (C-7a);
154.7 (C-6); 161.5 (C-4000); 164.4 (C@O).
4.4. General procedure for the preparation of N0-benzyl-
idene-3-methyl-1-phenyl-6-substituted-phenyl-1H-pyraz-
olo[3,4-b]pyridine-4-carbohydrazide (6a–6p)
Aromatic aldehyde (0.3 mmol) and five drops of
hydrochloric acid were added to a solution of (5)
(0.3 mmol) in absolute ethanol until complete dissolu-
tion. The mixture was refluxed under stirring and the
end of reaction monitored by t.l.c. The resulting solu-
tion was concentrated under reduced pressure and
added cold water. The precipitated was filtered off,
washed with cold water, and dried. Recrystallization
from ethanol/water led to the expected compounds
as colored solids.
4.4.5. N0-(4-Fluorobenzylidene)-3-methyl-1-phenyl-6-p-
methoxyphenyl-1H-pyrazolo[3,4-b]pyridine-4-carbohyd-
razide (6e). 94%, mp = 264 ꢁC. IR (m-cmꢁ1): 3560 (N–
1
H); 1650 (C@O); 1350 (C–O–C). HNMR: d 2.71 (s,
2H, H-8); 3.97 (s, 3H, OCH3); 7.25 (dd, 1H, H-40,
J = 8.7 Hz); 7.43–7.48 (m, 2H, H-3000); 7.69–7.75 (m,
4H, H-30 e H-200); 7.95–8.00 (m, 2H, H-300); 8.18 (s,
1H, H-5); 8.34 (d, 2H, H-200, J = 9 Hz); 8.44 (d, 2H,
H-20, J = 8.7 Hz); 8.54 (s, 1H, H-12); 12.38 (sl, 1H, H-
10). 13CNMR: d 14.2 (C-8); 55.2 (OCH3); 111.1 (C-
3a); 112.1 (C-3000); 114.2 (C-300); 120.5 (C-5 e C-20);
125.3 (C-40); 128.7 (C-200 e C-30); 128.9 (C-2000); 130.3
(C-3); 138.9 (C-4 e C-100); 142.2 (C-10); 152.2 (C-7a);
155.8 (C-6 e C-400); 160.8 (C-4000); 164.5 (C@O).
4.4.1. N0-(Benzylidene)-3-methyl-1,6-diphenyl-1H-pyraz-
olo[3,4-b]pyridine-4-carbohydrazide (6a). 98%, mp =
148–150 ꢁC. IR (m-cmꢁ1): 1650 (C@O); 3190 (N–H).
1HNMR: d 2.70 (s, 3H, H-8); 8.20 (s, 1H, H-5); 12.40
(s, 1H, H-10); 7.60–7.80 (m, 5H, H-2000–H-6000); 8.10 (s,
1H, H-12); 7.40–7.50 (m, 5H, H-20–H-60); 8.40–8.50
(m, 2H, H-200, H-600); 7.90 (m, 3H, H-300–H-500).
13CNMR:0 d 14.4 (C-8); 111 (C-3a); 120.3 (C-5); 120.5
(C-20, C-3 ); 129.0 (C-2000, C-3000, C-300, C-500, C-30, C-50);
127.0 (C-200, C-600); 130.0 (C-4000); 133 (C-1000); 134.0 (C-
10); 143 (C-3); 111.0 (C-3a); 161 (C@O); 155.8 (C-6);
151.0 (C-7a); 145.0 (CH-Ar); 141(C-10); 130.0 (C-100).
4.4.6. N0-(4-Fluorobenzylidene)-3-methyl-1-phenyl-6-p-
ethoxyphenyl-1H-pyrazolo[3,4-b]pyridine-4-carbohydraz-
ide (6f). 83%, mp = 190 ꢁC. IR (m-cmꢁ1): 3240–3200 (N–
1
H); 1650 (C@O). HNMR: d 1.50 (t, 3H, OCH2CH3);
2.72 (s, 3H, H-8); 4.24 (q, 2H, OCH2CH3); 7.21 (d,
2H, H-3000, J = 8.7 Hz); 7.46 (d, 1H, H-40, J = 6.3 Hz);
7.68–7.73 (m, 6H, H-30, H-300, H-200); 7.94 (s, 1H, H-5);
8.31 (d, 2H, H-200, J = 8.7 Hz); 8.44 (d, 2H, H-20,
J = 7.8 Hz); 12.30 (sl, 1H, H-10). 13CNMR: d 14.3 (C-
8); 14.5 (OCH2CH3); 63.2 (OCH2CH3); 111.2 (C-3a);
112.2 (C-3000); 114.8 (C-300); 120.3 (C-5 e C-20); 125.4
(C-40); 128.7 (C-200); 129.0 (C-30); 130.0 (C-3); 138.4
(C-1000); 138.9 (C-100); 139.1 (C-4); 142.3 (C-10); 147.9
(C-12); 151.1 (C-7a); 155.8 (C-400); 160.2 (C-4000); 161.8
(C-6); 164.8 (C@O).
4.4.2. N0-(4-Fluorobenzylidene)-3-methyl-1,6-diphenyl-
1H-pyrazolo[3,4-b]pyridine-4-carbohydrazide (6b). 90%,
mp = 284 ꢁC. IR (m-cmꢁ1): 3200 (N–H); 1650 (C@O).
1HNMR: d 2.70 (s, 3H, H-8); 8.30 (s, 1H, H-5); 12.30
(s, 1H, H-10); 8.10 (s, 1H, H-12); 7.50 (m, 2H, H-3000,
H-5000); 8.40 (m, 5H, H-200-H-600); 7.70 (m, 5H, H-20–H-
60); 8.00 (2H, H-2000, H-6000). 13CNMR: d 14.2 (C-8);
143.0 (C-3); 156.0 (C-6); 161.5 (C@O); 143.3 (C-10);
164.5 (C-4000); 115.6 (C-3a); 138.5 (C-4); 120.0 (C-5);
151.0 (C-7a); 147.9 (CH-Ar).
4.4.7. N0-(4-Hydroxybenzylidene)-3-methyl-1,6-diphenyl-
1H-pyrazolo[3,4-b]pyridine-4-carbohydrazide (6g). 97%,
mp = 278–280 ꢁC. IR (m-cmꢁ1): 3300–3100 (OH); 1660
1
(C@O). HNMR: d 2.73 (s, 3H, H-8); 8.20 (s, 1H, H-
4.4.3. N0-(4-Fluorobenzylidene)-3-methyl-1-phenyl-6-p-
cyanophenyl-1H-pyrazolo[3,4-b]pyridine-4-carbohydraz-
ide (6c). 92%, mp = 276–278 ꢁC. IR (m-cmꢁ1): 3290 (N–
5); 12.10 (s, 1H, H-10); 10.00 (s, 1H, H-12); 6.80 (d,
2H, H-3000, H-5000, J = 9 Hz); 7.35 (d, 2H, H-2000, H-6000,
J = 9 Hz); 7.50 (m, 3H, H-300–H-500); 7.60–7.80 (m, 7H,
H-20–H-60, H-200, H-600); 8.10 (s, 1H, OH). 13CNMR: d
15.6 (C-8); 144.0 (C-3); 156 (C-6); 163.0 (C@O); 140
(C-10); 151.0 (C-7a); 143.0 (CH-Ar); 160.0 (C-4000);
120.0 (C-5); 111.0 (C-3a).
1
H); 2220 (CN); 1650 (C@O). HNMR: d 2.74 (s, 3H,
H-8); 7.22 (dd, 1H, H-40, J = 8.7 Hz); 7.45–7.52 (m,
2H, H-30); 7.73 (dd, 2H, H-3000, J = 8.4 Hz); 7.96–8.01
(m, 2H, H300); 8.17 (d, 2H, H-2000, J = 8.7 Hz); 8.35 (s,
1H, H-5); 8.41 (d, 2H, H-200, J = 9 Hz); 8.54 (s, 1H, H-
13
12); 12.43 (sl, 1H, H-10). CNMR: d 14.4 (C-8); 112.3
4.4.8. N0-(4-Hydroxybenzylidene)-3-methyl-1-phenyl-6-p-
cyanophenyl-1H-pyrazolo[3,4-b]pyridine-4-carbohydraz-
ide (6h). 65%, mp = 278 ꢁC. IR (m-cmꢁ1): 3500–3200
(OH); 2220 (CN); 1650 (C@O). 1HNMR: d 2.72 (s,
3H, H-8); 6.99 (d, 2H, H-3000, J = 7.5 Hz); 7.46 (dd,
1H, H-40, J = 7.2 Hz); 7.69 (d, 2H, H-30, J = 7.2 Hz);
(C-3a e C-400); 113.2 (C-3000); 118.6 (CN); 120.5 (C-5 e
C-20); 125.8 (C-40); 128.0 (C-2000); 129.3 (C-30); 130.5
(C-3); 138.7 (C-1000); 140.0 (C-4); 141.8 (C-100); 142.0
(C-10); 148.0 (C-12); 150.9 (C-7a); 153.8 (C-6); 161.4
(C-4000); 165.0 (C@O).

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L. R. S. Dias et al. / Bioorg. Med. Chem. 15 (2007) 211–219
1
7.74 (d, 2H, H-300, J = 8.7 Hz); 8.11 (d, 2H, H-2000,
J = 8.1 Hz); 8.28 (s, 1H, H-5); 8.38 (d, 2H, H-200,
J = 8.4 Hz); 8.43 (s, 1H, H-12); 8.54 (d, 2H, H-20,
J = 7.2 Hz); 10.13 (s, 1H, OH); 12.17 (sl, 1H, H-10).
13CNMR: d 14.4 (C-8); 112.3 (C-3a e C-400); 118.6
(CN); 120.7 (C-5 e C-20); 124.9 (C-3); 125.7 (C-40);
128.4 (C-30); 129.2 (C-200); 132.8 (C-300); 138.8 (C-1000);
139.1 (C-4); 140.2 (C-10); 141.9 (C-100); 149.5 (C-12);
150.8 (C-7a); 153.7 (C-4000); 159.8 (C-6); 161.1 (C@O).
1540 (NO2). HNMR: d 2.80 (s, 3H, H-8); 8.30 (s, 1H,
H-5); 8.60 (s, 1H, H-12); 12.60 (s, 1H, H-10); 8.40–
8.50 (m, 4H, H-2000, H-6000, H-200, H-600); 8.20 (m, 2H,
H-300, H-500); 7.60–7.80 (m, 8H, H-20–H-60, H-300–H-500).
13CNMR: d 14.2 (C-8); 146.0 (C-3); 156.2 (C-6); 151.1
(c-7a); 161.9 (C@O); 143 (CH-Ar); 141.7 (C-10); 141.0
(C-100); 146.0 (C-4000).
4.4.13. N0-(4-Nitrobenzylidene)-3-methyl-1-phenyl-6-p-
cyanophenyl-1H-pyrazolo[3,4-b]pyridine-4-carbohydraz-
ide (6m). 95%, mp = 305 ꢁC. IR (m-cmꢁ1): 3220–3190
4.4.9. N0-(4-Hydroxybenzylidene)-3-methyl-1-phenyl-6-p-
chlorophenyl-1H-pyrazolo[3,4-b]pyridine-4-carbohydraz-
ide (6i). 73%, mp = >310 ꢁC. IR (m-cmꢁ1): 3400–3220
1
(N–H); 2220 (CN); 1660 (C@O); 1540 (NO2). HNMR:
d 3.23 (s, 3H, H-8); 7.96–8.02 (m, 1H, H-40); 8.14–8.17
(m, 2H, H-3000); 8.14–8.26 (m, 2H, H-30); 8.62–8.69 (M,
2H, H-300); 8.73–8.78 (m, 2H, H-2000); 8.89 (s, 1H, H-5);
8.90–8.96 (m, 2H, H-200); 9.06–9.11 (m, 2H, H-20); 9.12
(s,1H, H-12); 13.23 (sl, 1H, H-10). 13CNMR: d 14.3
(C-8); 112.3 (C-400); 113.7 (C-3a); 120.5 (C-5 e C-20);
124.1 (C-3000); 126.1 (C-3); 127.7 (C-40); 128.2 (C-200);
128.4 (C-2000); 129.3 (C-30); 133.0 (C-300); 138.8 (C-1000);
139.5 (C-4); 141.8 (C-100); 143.4 (C-10); 146.7 (C-12);
148.2 (C-4000); 150.9 (C-7a); 153.8 (C-6); 161.0 (C@O).
1
(OH); 1650 (C@O). HNMR: d2.72 (s, 3H, H-8); 6.76
(d, 2H, H-3000, J = 8.7 Hz); 7.25 (d, 2H, H-2000,
J = 8.4 Hz); 7.46 (dd, 1H, H-40, J = 7.5 Hz); 7.69–7.76
(m, 2H, H-30); 7.76–7.89 (m, 2H, H-300); 8.20 (s, 1H,
H-5); 8.31–8.37 (m, 2H, H-200); 8.40 (d, 2H, H-20,
J = 8.7 Hz); 8.46 (s, 1H, H-12); 10.08 (s, 1H, OH);
12.12 (sl, 1H, H-10). 13CNMR: d 14.4 (C-8); 11.9 (C-
3a); 115.8 (C-3000); 120.4 (C-5); 120.6 (C-200); 124.9 (C-
3); 125.8 (C-200); 129.0 (C-300); 129.2 (C-3 ); 135.0 (C-
10000); 136.6 (C-400); 139.0 (C-4); 140.1 (C-100); 142.3 (C-
1 ); 149.4 (C-12); 151.0 (C-7a); 154.9 (C-6); 159.8 (C-
4000); 161.3 (C@O).
4.4.14. N0-(4-Nitrobenzylidene)-3-methyl-1-phenyl-6-p-
chlorophenyl-1H-pyrazolo[3,4-b]pyridine-4-carbohydraz-
ide (6n). 97%, mp = 296–298 ꢁC. IR (m-cmꢁ1): 3220–3190
1
4.4.10. N0-(4-Hydroxybenzylidene)-3-methyl-1-phenyl-6-
p-methoxyphenyl-1H-pyrazolo[3,4-b] pyridine-4-carbo-
hydrazide (6j). 88%, mp = 270 ꢁC. IR (m-cmꢁ1): 3300–
(N–H); 1660 (C@O). HNMR: d 3.23 (s, 3H, H-8); 7.98
(dd, 1H, H-40, J = 7.5 Hz); 8.16 (d, 2H, H-3000,
J = 8.7 Hz); 8.22 (dd, 2H, H-30, J = 8.1 Hz); 8.27 (d,
2H, H-2000, J = 8.7 Hz); 8.66–8.70 (m, 2H, H-300); 8.80
(s, 1H, H-5); 8.90–8.96 (m, 4H, H-20 e H-200); 9.12 (s,
1H, H-12). 13CNMR: d 14.2 (C-8); 112.2 (C-3a); 120.6
(C-5 e C-20); 123.8 (C-40); 127.5 (C-2000); 128.9 (C-30);
129.0 (C-3); 132.7 (C-400); 138.4 (C-4); 138.6 (C-1000);
140.0 (C-100); 142.1 (C-10); 146.7 (C-12); 148.1 (C-4000);
150.1 (C-7a); 154.0 (C-6); 161.6 (C@O).
1
3220 (OH); 1650 (C@O). HNMR: d 2.71 (s, 3H, H-8);
3.77 (s, 3H OCH3); 6.99 (d, 2H, H-3000, J = 8.4 Hz);
7.20–7.27 (m, 2H, H-30); 7.46 (dd, 1H, H-40,
J = 8.7 Hz); 7.70 (d, 2H, H-2000, J = 8.4 Hz); 7,75 (d,
2H, H-300, J = 8.7 Hz); 8.14 (s, 1H, H-5); 8.38 (d, 2H,
H-200, J = 9 Hz); 8.43 (s, 1H, H-12); 8.45 (d, 2H, H-20,
J = 7.5 Hz); 10.12 (s, 1H, OH); 12.14 (sl, 1H H-10).
13CNMR: d 14.4 (C-8); 55.4 (OCH3); 111.2 (C-3a);
114.5 (C-300); 115.7 (C-3000); 120.5 (C-5 e C-20); 124.9
(C-3); 125.6 (C-40); 128.5 (C-2000); 129.0 (C-200); 129.2
(C-300); 130.2 (C-30); 138.8 (C-4); 139.1 (C-100); 142.2
(C-1 ); 149.3 (C-12); 151.1 (C-7a); 156.0 (C-4000); 159.8
(C-400); 161.0 (C-6); 161.5 (C@O).
4.4.15. N0-(4-Nitrobenzylidene)-3-methyl-1-phenyl-6-p-
methoxyphenyl-1H-pyrazolo[3,4-b]pyridine-4-carbohyd-
razide (6o). 98%, mp = 284 ꢁC. IR (m-cmꢁ1): 3220 (N–
H); 1660 (C@O); 1540 (NO2). 1HNMR: d 2.72 (s,
3H, H-8); 3.98 (s, 3H, OCH3); 7.21–7.27 (m, 2H, H-
30); 7.45–7.50 (m, 1H, H-40); 7.57–7.65 (m, 4H, H-30
e H-2000); 8.06–8.21 (m, 2H, H-300); 8.16 (s, 1H, H-5);
8.36–8.46 (m, 4H, H-20 e H-200); 8.64 (s, 1H, H-12);
12.66 (sl, 1H, H-10). 13CNMR: d 14.3 (C-8); 55.3
(OCH3); 112.6 (C-3a); 114.4 (C-300); 120.4 (C-5 e C-
20); 123.9 (C-3000); 125.5 (C-40); 127.5 (C-2000); 128.2
(C-200); 128.9 (C-30); 130.2 (C-3); 138.0 (C-4); 139.0
(C-100); 139.8 (C-1000); 143.1 (C-10); 146.6 (C-12); 148.1
(C-4000); 151.1 (C-7a); 155.7 (C-400); 161.0 (C-6); 162.1
(C@O) .
4.4.11. N0-(4-Hydroxybenzylidene)-3-methyl-1-phenyl-6-
p-ethoxyphenyl-1H-pyrazolo[3,4-b] pyridine-4-carbohyd-
razide (6k). 92%, mp = 288–290 ꢁC. IR (m-cmꢁ1): 3400–
3200 (OH); 1660 (C@O). 1HNMR: d 1.47 (t, 3H,
OCH2CH3); 2.87 (s, 3H, H-8); 4.66 (q, 2H, OCH2CH3);
7.51 (d, 2H, H-3000, J = 8.4 Hz); 7.73 (d, 2H, H-2000,
J = 8.4 Hz); 7.97 (d, 1H, H-40, J = 7.5 Hz); 8.21 (d, 2H,
H-30, J = 8.1 Hz); 8.26 (d, 2H, H-3000, J = 8.4 Hz); 8.65
(s, 1H, H-5); 8.87–8.96 (m, 2H, H-20); 8.89 (d, 2H, H-
200, J = 8.7 Hz); 8.99 (s, 1H, H-12); 10.62 (s, 1H, OH);
12.35 (sl, 1H, H-10). 13CNMR: d 14.4 (C-8); 14.5
(OCH2CH3); 63.3 (OCH2CH3); 11.2 (C-3a); 115.6 (C-
3000); 120.1 (C-20); 120.4 (C-5); 124.9 (C-3); 125.5 (C-40);
127.7 (C-2000); 128.7 (C-200); 129.1 (C-30); 130.0 (C-1000);
138.7 (C-1000); 139.1 (C-4); 141.7 (C-10); 149.2 (C-12);
151.1 (C-7a); 156.0 (C-4000); 160.3 (C-6); 161.4 (C@O).
4.4.16. N0-(4-Nitrobenzylidene)-3-methyl-1-phenyl-6-p-
ethoxyphenyl-1H-pyrazolo[3,4-b]pyridine-4-carbohydraz-
ide (6p). 94%, mp = 280–282 ꢁC. IR (m-cmꢁ1): 3240–3200
1
(N–H); 1650 (C@O); 1540 (NO2). HNMR: d 1.49 (t,
3H, OCH2CH3); 2.72 (s, 3H, H-8); 4.26 (q, 2H,
OCH2CH3); 7.24 (d, 2H, H-3000, J = 8.7 Hz); 7.49 (d,
1H, H-40, J = 7.5 Hz); 7.71 (d, 2H H-2000); 7.74 (d, 2H,
H-30, J = 7.5 Hz); 8.18 (d, 2H, H-300, J = 9 Hz); 8.20 (s,
1H, H-5); 8.38 (d, 2H, H-200, J = 8.7 Hz); 8.45 (d, 2H,
H-20, J = 8.7 Hz); 8.64 (s, 1H, H-12); 12.63 (sl, 1H, H-
4.4.12. N0-(4-Nitrobenzylidene)-3-methyl-1,6-diphenyl-
1H-pyrazolo[3,4-b]pyridine-4-carbohydrazide (6l). 78%,
mp = 300 ꢁC. IR (m-cmꢁ1): 3200 (N–H); 1650 (C@O);

L. R. S. Dias et al. / Bioorg. Med. Chem. 15 (2007) 211–219
219
10). 13CNMR: d 14.2 (C-8); 14.3 (OCH2CH3); 63.2
Acknowledgments
(OCH2CH3); 110.9 (C-3a); 114.8 (C-300); 120.5 (C-5 e
C-20); 125.5 (C-40); 128.1 (C-2‘00); 128.9 (C-200); 129.3
(C-30); 129.9 (C-3); 137.9 (C-100); 138.9 (C-4); 140.1 (C-
1000); 141.9 (C-10); 146.4 (C-12); 148.1 (C-1000 e C-4000);
151.1 (C-7a); 155.9 (C-400); 160.2 (C-6); 162.0 (C@O).
`
We thank Fundac¸a˜o de Amparo a Pesquisa do Estado
do Rio de Janeiro (FAPERJ—Brazil) for supporting
this research and to Conselho Nacional de Desenvolvi-
´
´
mento Cientıfico e Tecnologico (CNPq—Brazil) for fel-
lowships (M.B.S. and A.C.C.F.). We thank Edson F. da
Silva for technical support. We would like to extend spe-
cial thanks to Dr. Mishbaul A. Khan for helpful advice.
4.5. Pharmacology
Bloodstream forms were obtained at the peak of par-
asitaemia (7th day of infection) from albino mice
infected with Y strain of T. cruzi by cardiac puncture.
The infected blood was diluted with blood from
healthy mice and fetal bovine serum, in a way to
obtain a final parasite concentration of about 106 try-
pomastigotes forms/mL. Stock solution of the sub-
stances was prepared in dimethylsulfoxide (DMSO).
Parts of these stock solutions were added to the
infected blood in order to obtain final concentrations
of 10, 100, 250, 500, and 1000 lg/mL using micro-
plates (96 wheels). The solutions were incubated at
4 ꢁC for 24 h under constant shaking. Untreated and
crystal violet treated parasites were used as controls.
All assays were performed in triplicate. The activity
was determined by counting the trypomastigote forms
and determination of parasitical lyses (%) compared
to the non-treated control group.21 The compound
concentration corresponding to 50% activity was ex-
pressed as the IC50.
References and notes
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4.6. Molecular modeling
All computations were performed using SPARTAN004
(Wavefunction Inc., Irvine, CA, 2000) as described
elsewhere.22 The conformational analysis of the unsub-
stituted compound revealed the Syn conformation as
the most stable due to an antiparallel dipole of the
carbonyl and the NH of the amide moiety. Thus this
conformation was used in all theoretical derivatives
analysis.
In order to evaluate the electronic properties of all
compounds, we submitted them to a Single-Point
ab initio calculation with a 6-31G* basis set available
on SPARTAN004 program (Wavefunction Inc., Irvine,
CA, 2000). Molecular electrostatic potential maps
(MEPs), HOMO and LUMO eigenvalues and orbital
coefficients, GAP, and the clogP were calculated. In
this work, we generated the MEPs isoenergy contours
in a range from ꢁ25.000 to +40.000 kcal/mol and
superimposed onto a surface of constant electron den-
sity of 0.002 e/au3. HOMO density encoded onto a van
der Waals surface (isodensity 0.002 e/au3) with the
HOMO’s absolute density coefficient mapped from
deepest red (0.00) to deepest blue (0.03). The constants
employed in these isosurface calculations are default
parameters in the program used. Theoretical logP
(clogP) was calculated using Villar method, at AM1
semiempirical level.