Design, synthesis and anti-P. falciparum activity of pyrazolopyridine–sulfonamide derivatives

Article abstract of DOI:10.1016/j.bmc.2016.07.049

Ten 1-phenyl-1H-pyrazolo[3,4-b]pyridine derivatives connected by a linker group to benzenesulfonamide moieties with different substituents in the 4-position were synthesized and assayed against Plasmodium falciparum. These ten compounds exhibited activity in vitro against the chloroquine-resistant clone W2 with IC₅₀values ranging from 3.46 to 9.30 μM. The most active derivatives with substituent R₂= Cl or CH₃at the benzenesulfonamide moiety exhibited the lowest IC₅₀. Compounds with an R₁= CO₂Et substituent at the 5-position of the 1H-pyrazolo[3,4-b]pyridine ring presented lower activity than those with a CN substituent. The 1H-pyrazolo[3,4-b]pyridine system appears to be promising for further studies as an antimalarial for overcoming the burden of resistance in P. falciparum.

Full text of DOI:10.1016/j.bmc.2016.07.049

Accepted Manuscript
Design, synthesis and anti-P. falciparum activity of pyrazolopyridine-sulfona-
mide derivatives
Thais B. Silva, Alice M.R. Bernardino, Maria de Lourdes G. Ferreira, Kamilla
R. Rogerio, Leonardo J.M. Carvalho, Nubia Boechat, Luiz C.S. Pinheiro
PII:
S0968-0896(16)30566-1
DOI:
http://dx.doi.org/10.1016/j.bmc.2016.07.049
BMC 13162
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
24 June 2016
20 July 2016
22 July 2016
Please cite this article as: Silva, T.B., Bernardino, A.M.R., Ferreira, d.L.G., Rogerio, K.R., Carvalho, L.J.M.,
Boechat, N., Pinheiro, L.C.S., Design, synthesis and anti-P. falciparum activity of pyrazolopyridine-sulfonamide
derivatives, Bioorganic & Medicinal Chemistry (2016), doi: http://dx.doi.org/10.1016/j.bmc.2016.07.049
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Bioorganic & Medicinal Chemistry
Design, synthesis and anti-P. falciparum activity of pyrazolopyridine-sulfonamide
derivatives
Thais B. Silvaa, b, Alice M. R. Bernardinob, Maria de Lourdes G. Ferreiraa, Kamilla R. Rogerioa,c,d
Leonardo J. M. Carvalhoc, Nubia Boechata and Luiz C. S. Pinheiroa
,
a Departamento de Síntese de Fármacos, Instituto de Tecnologia em Fármacos, Farmanguinhos - FIOCRUZ, Fundação Oswaldo Cruz. Rua Sizenando Nabuco
100, Manguinhos, Rio de Janeiro, RJ, 21041-250, Brazil.
bPrograma de Pós-Graduação em Química, Departamento de Química Orgânica. Universidade Federal Fluminense, Niterói-RJ, Brazil
c Laboratório de Pesquisa em Malária, Instituto Oswaldo Cruz, Fundação Oswaldo Cruz, Rio de Janeiro, RJ, Brazil.
d Programa de Pós Graduação em Química, PGQu Instituto de Química, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
Ten 1-phenyl-1H-pyrazolo[3,4-b]pyridine derivatives connected by a linker group to
benzenesulfonamide moieties with different substituents in the 4-position were synthesized and
assayed against Plasmodium falciparum. These ten compounds exhibited activity in vitro against
the chloroquine-resistant clone W2 with IC50 values ranging from 3.46 to 9.30 µM. The most
active derivatives with substituent R2 = Cl or CH3 at the benzenesulfonamide moiety exhibited
the lowest IC50. Compounds with an R1 = CO2Et substituent at the 5-position of the 1H-
pyrazolo[3,4-b]pyridine ring presented lower activity than those with a CN substituent. The 1H-
pyrazolo[3,4-b]pyridine system appears to be promising for further studies as an antimalarial for
overcoming the burden of resistance in P. falciparum.
Available online
Keywords:
malaria
pyrazolopyridine
sulfonamide
2009 Elsevier Ltd. All rights reserved.
Plasmodium falciparum
1. Introduction
Due to the high parasitic resistance exhibited by Plasmodium
falciparum to most available drugs, monotherapy is no longer
Over the past fifteen years (2000 to 2015), there has been a
significant reduction in the number of cases and deaths caused by
malaria according to data from the World Health Organization. In
2000, it was estimated that there were 262 million cases of
malaria worldwide, leading to 839,000 deaths. In 2015, the
number of malaria cases decreased to 214 million, and the
number of deaths decreased to 438,000. These figures represent
an 18% decline in cases of malaria and a 48% decrease in the
number of deaths during this period1. The progress made in
reducing the number of deaths in children under 5 years of age
has been substantial; however, it is still high, and malaria remains
a major cause of death in children corresponding to one death
every 2 minutes1.
used to treat malaria. The WHO recommends the use of first-line
drugs against malaria, including a fixed dose of artemisinin-
combined therapies (ACTs) to treat simple and severe falciparum
malaria, using artemether plus lumefantrine, artesunate plus
amodiaquine or mefloquine, dihydroartemisinin plus piperaquine
or artesunate plus sulfadoxine-pyrimethamine, among other
ACTs3. Combination therapy is intended to prevent or delay the
onset of resistance with the simultaneous use of drugs that have
different modes of action. An artemisinin derivative with a quick
and effective action is used, however, if a parasite-resistant
mutant of the drugs arises during the course of infection is
rapidly cleared, allowing a longer lasting drug to kill the parasite
that survived the initial application of the artemisinin derivative3.
In addition to efforts to control the mosquito vector and the
search for an effective vaccine, developing malaria drugs is still
the main disease control strategy due to the spread of parasites
that are resistant to antimalarial drugs2.
There is an urgent need for novel antimalarials with better
safety profiles than current medicines in addition to new drugs
that prevent transmission and relapse due to the resistance against
quinoline derivatives and artemisinins4,5. In the recent literature, a
———
Corresponding authors. Tel.: +55-21-3977-2456; fax: +55-21-2560-2518
e-mail: lpinheiro@far.fiocruz.br

number of new antimalarial compounds in different states of
preclinical and clinical development have been described6-8
In an effort to enhance the anti-P. falciparum activity of the
precursor quinoline-sulfonamide hybrid V, new derivatives N-(4-
((1-phenyl-1H-pyrazolo[3,4-b]pyridin-4-
.
Quinoline derivatives are still the dominate class of
antimalarial compounds9,10; however, due to resistance to this
class of drugs, searching for other analogous compounds is
extremely important.
yl)amino)butyl)benzenesulfonamides (1-10) were designed.
The 7-chloroquinoline moiety was replaced by the system 1-
phenyl-1H-pyrazolo[3,4-b]pyridine by ring isosterism. An N-(4-
aminobutyl)benzenesulfonamide group was attached at the 4
position of the heterocyclic ring. The 1H-pyrazolo[3,4-b]pyridine
ring remains separated from the benzenesulfonamide moiety by
the linker containing four methylene groups, similar to that found
in the individual molecular framework of the precursor V.
The pyrazolopyridine ring is an isostere of quinoline, and the
literature has demonstrated that this system possesses anti-
inflammatory11, anti-cancer12, antibacterial13,14 and antiviral15-18
activities. The literature suggests that pyrazolopyridine is a
potential system with antiparasitic activity against T. cruzi19, as
well as antileishmanial20 and antimalarial activities21,22
.
In our previous search for new drugs against malaria, we
demonstrated the importance of MEFAS (I), a hybrid salt active
against the P. falciparum W2 clone that presents an IC50 value of
0.001 µM23.
The
derivative
2-(trifluoromethyl)[1,2,4]triazolo[1,5-
a]pyrimidine II was active against P. falciparum W2 with an
IC50= 0.023 µM and showed no toxicity. In this work, the
importance of the CF3 group at position 2 of the nucleus
triazolo[1,5-a]pyrimidine
for
increased
activity
was
demonstrated24.
A series of quinoline derivatives containing a 1,2,3-triazole
ring at position 4 were synthesized and tested against strains of P.
falciparum W2. Compound III was the most active, with an
IC50= 1.4 µM25.
We have recently synthesized a new class of hybrids of
atorvastatin (AVA) and aminoquinolines (primaquine and
chloroquine derivatives). The quinolinic moiety was connected to
the pentasubstituted pyrrole from AVA by a linker group. The
activity of the compounds increased with the size of the carbon
chain. Compound IV with four methylene groups and a 7-
chloroquinolinyl moiety exhibited better activity than CQ with an
IC50 of 0.40 µM26.
Figure 2. Rational approach to the design of compounds 1-10.
2. Results and discussion
2.1 Chemistry
New derivatives of quinoline-sulfadoxine hybrids were
planned by molecular hybridization between the quinoline ring
and the benzenesulfonamide moiety present in chloroquine and
sulfadoxine, which are drugs used in the treatment of malaria. All
15 compounds in this series were active in vitro against the P.
falciparum W2 clone, and none of these compounds were toxic
to BGM cells. Ten compounds presented IC50 values lower than
that of CQ, with values ranging from 0.05 to 0.40 µM. The most
active hybrids have a structural relationship between the four
methylene groups used as linkers between the quinoline ring and
the benzenesulfonamide moiety. Four compounds exhibited SI
values (3386.0 - 1031.3) higher than that of chloroquine
(834.74). When evaluated against P. berghei malaria, compound
V was the most active and inhibited parasitemia by 49% on day 5
after inoculation, contributing to the discovery of new prototypes
with antimalarial activity27.
The synthetic route for preparing the N-(4-((1-phenyl-1H-
pyrazolo[3,4-b]pyridin-4-yl)amino)butyl)benzenesulfonamides
(1-10) is shown in Scheme 1.
Ethyl 5-amino-1-phenyl-1H-pyrazole-4-carboxylate (13)
could easily be prepared in 80% yield from the reaction of
phenylhydrazine (11) (0.01 mol) and ethyl 2-cyano-3-
ethoxyacrylate (12) (0.01 mol), in ethanol under reflux. The
derivative 5-amino-1-phenyl-1H-pyrazol (14) was obtained in
85% yield via a hydrolysis reaction followed by decarboxylation
of the derivative ethyl 5-amino-1-phenyl-1H-pyrazole-4-
carboxylate (13) with phosphoric acid (85%).
The Michael addition of 5-aminopyrazole (14) with ethyl 2-
cyano-3-ethoxyacrylate
(12)
or
diethyl
2-
(ethoxymethylene)malonate (15) in ethanol under reflux afforded
the derivatives 16 and 17 in 86% and 88% yield, respectively.
Ethyl
4-chloro-1-phenyl-1H-pyrazolo[3,4-b]pyridine-5-
carboxylate (18) was prepared from diethyl 2-(((1-phenyl-1H-
pyrazol-5-yl)amino)methylene)malonate (16) through a reaction
with phosphorous oxychloride under reflux in 84% yield as
described in the literature16,20. However, the derivative 4-chloro-
1-phenyl-1H-pyrazolo[3,4-b]pyridine-5-carbonitrile (19) cannot
be obtained using this methodology.
The cyclization of pyrazole (17) was performed by refluxing
in dowtherm (250 °C) for 40 min and isolating 4-oxo-1-phenyl-
4,7-dihydro-1H-pyrazolo[3,4-b]pyridine-5-carbonitrile (22) by
Figure 1. Structures of compounds I-V.

precipitating from hexane with a yield of 84%28. The derivative
(22) was refluxed with phosphorous oxychloride to produce (19)
in 90% yield.
chloro-1-phenyl-1H-pyrazolo[3,4-b]pyridines (18, 19) (1.0
mmol) and butane-1,4-diamine (1.0 mmol)26,27
.
The addition-elimination reaction between the appropriate
sulfonyl chloride (1.2 mmol) and amines (20, 21) (1.0 mmol) was
performed in MeOH and TEA (1.0 mmol) at 25 °C to obtain the
target compounds N-(4-((1-phenyl-1H-pyrazolo[3,4-b]pyridin-4-
yl)amino)butyl)benzenesulfonamides (1-10) in 70-59% yield27.
The compounds ethyl 4-((4-aminobutyl)amino)-1-phenyl-1H-
pyrazolo[3,4-b]pyridine-5-carboxylate
(20)
and
4-((4-
aminobutyl)amino)-1-phenyl-1H-pyrazolo[3,4-b]pyridine-5-
carbonitrile (21) were synthesized in 79% and 66% yield,
respectively, by the nucleophilic substitution reaction between 4-
Reagents and conditions: (i) EtOH, reflux, 1 h, 70%; (ii) H3PO4, 170 °C, 6 h, 86%; (iii) EtOH, reflux, 2 h, 86-88%; (iv) POCl3, reflux, 6 h, 84%; (v) butane-1,4-
diamine, 1,4-dioxane, 25-80 °C, 1-4 h, 66-79%; (vi) appropriate sulfonyl chloride, MeOH, TEA, 25 °C, 24 h, 59-70%; (vii) dowtherm, 250 °C, 40 min, 71%;
(viii) POCl3, reflux, 6 h, 90%.
Scheme 1. Synthetic route used to prepare compounds 1-10.
The 10 derivatives 1-phenyl-1H-pyrazolo[3,4-b]pyridine (1-
10) that were synthesized with different substituents in the 4-
position of the benzenesulfonamide group were tested against
the W2 chloroquine and sulfadoxine-resistant P. falciparum
clone and for cytotoxicity (Table 1).
3. Conclusions
Among the 1-phenyl-1H-pyrazolo[3,4-b]pyridine derivatives
(1-10) synthesized with different substituents at the 4-position of
the benzenesulfonamide moiety, all exhibited anti-P. falciparum
activity in vitro against W2 chloroquine-resistant parasites and
low toxicity to BGM cells.
The values are summarized in Table 1. All the compounds
that presented activity showed IC50 values ranging from 3.46 to
9.30 µM in the anti-HPR2 assay, lower than that of sulfadoxine
(IC50 = 15.0 M); however, these compounds were not more
active than chloroquine (IC50 = 0.55 M).
The ten compounds (1-10) presented IC50 values lower than
that of the control drug sulfadoxine, with IC50 values ranging
from 3.46 to 9.30 µM. However, the observed activity was lower
than that of chloroquine and the quinolone-sulfonamide hybrid
V.
Compounds 2, 7 with substituent (R2 = CH3) and 4 (R2 = Cl)
at the 4-position of the benzenesulfonamide moiety were the
most active against P. falciparum, presenting IC50 values of 3.46
M (2) and 3.60 M (4 and 7). Compound 4 also showed the
highest SI values: >277.77.
Derivatives 2, 4 and 7 exhibited the lowest activity against P.
falciparum, with IC50 values of 3.46 M (2) and 3.60 M (4 and
7). Compounds with an R1 = CO2Et substituent at the 5-position
of the 1H-pyrazolo[3,4-b]pyridine ring showed higher activity
than those with a CN substituent. The amine 20 showed higher
activity than the sulfonamides (1-10) with an IC50 = 2.10 M and
the highest SI value (442.85).
Comparing the series in which substituents R1 = CO2Et and
CN at the 5-position of the 1H-pyrazolo[3,4-b]pyridine ring, it
can be observed that the 3 compounds with R1 = CO2Et showed
higher activity than those with CN. Also is observed in the
activity values of the intermediates 20 and 21 with IC50values of
2.10 and 5.00, respectively. In fact, the amine 20 exhibited
greater activity than the sulfonamides (1-10) and the highest SI
value (442.85).
The 1H-pyrazolo[3,4-b]pyridine system appears to be
promising for further studies as an antimalarial with the objective
of overcoming the burden of resistance in P. falciparum.

Table 1. Evaluation of activity against P. falciparum parasites (W2 clone) resistant to chloroquine, cytotoxicity tests in normal
monkey kidney cell line (BGM) and the selectivity index (SI) of compounds 1-10, 20 and 21, chloroquine and sulfadoxine.
Anti-P. falciparum activity
Cytotoxicity to BGM cells
Compounds
SI[MLD50 / IC50]
MLD50**(µM)
822.00
596.00
993.00
>1000.00
995.00
248.00
682.00
508.00
418.00
498.00
930.00
503.00
385.00
310.00
IC50*(µM)
1
2
R1 = CO2Et/ R2 = H
R1 = CO2Et/ R2 = CH3
R1 = CO2Et / R2 = F
R1 = CO2Et/ R2 = Cl
R1 = CO2Et/ R2 = OCH3
R1 = CN/ R2 = H
R1 = CN/ R2= CH3
R1 = CN / R2 = F
R1 = CN/ R2 = Cl
R1 = CN/ R2 = OCH3
R1 = CO2Et
6.52 ± 0.8
126.07
172.25
241.01
>277.77
106.98
59.04
3.46 ± 0.25
4.12 ± 0.48
3.60 ± 2.04
9.30 ± 1.25
4.20 ± 1.07
3.60 ± 0.09
9.30 ± 2.95
7.50 ± 0.09
5.00 ± 1.15
2.10 ± 0.08
5.00 ± 1.90
0.55
3
4
5
6
7
189.44
54.62
8
9
55.73
10
20
21
99.60
442.85
100.60
700.00
20.70
R1 = CN
chloroquine
sulfadoxine
15.00
*IC50 = inhibitory concentration for 50% of parasite growth, evaluated in three different experiments for each test (± standard deviation (SD)).
** Minimal lethal dose for 50% of cells (MLD50), used to calculate the selectivity index.
ethyl(ethoxymethylene)cyanoacetate (9 mmol) dissolved in 10
mL of ethanol was slowly added. The reaction mixture was
refluxed for 20 minutes. The reaction mixture was poured into 50
mL of ice-cold water. The precipitate was collected by filtration
and washed with water to afford 13 in 70% yield.
4. Experimental
4.1. Chemistry
The 1H, 13C and 19F nuclear magnetic resonance (NMR)
spectra were obtained at 400.00, 100.00 and 376.00 MHz,
respectively, using a BRUKER Avance instrument equipped with
a 5 mm probe. Tetramethylsilane was used as an internal
standard. The chemical shifts (δ) are reported in ppm, and the
coupling constants (J) are reported in Hertz. Electron-ionization
mass spectra (EI-MS, scan ES+ capillary (3.0 kV)/cone (30
V)/extractor (1 V)/RF lens (1.0 V)/source temperature (150
°C)/desolvation temperature (300 °C)) were recorded using a
Micromass/Waters Spectrometer (model: ZQ-4000). HRMS data
were obtained using LC-MS Bruker Daltonics MicroTOF (time
of flight analyzer). Fourier transform infrared (FT-IR) absorption
spectra were recorded on a Shimadzu mode IR Prestige-21
spectrophotometer through KBr reflectance. The melting points
(m.p.) were determined using a Büchi model B-545 apparatus.
TLC (thin layer chromatography) was performed using a silica
gel F-254 glass plate (20 x 20 cm). Column chromatography was
performed using silica gel 60 (0.040-0.063 mm). The remaining
reagents and solvents that were used were of analytical grade.
Yield: 70%. MP: 98°C. IR (KBr. cm-1): 3400-3140; 2995;
1
2912; 1677; 1621; 1552; 1528; 1280; 781-697. H NMR (400
MHz, CDCl3, TMS,  in ppm): 1.36 (t, 3H, J = 7.1 Hz; CH2CH3);
4.30 (q, 2H, J = 7.1 Hz CH2CH3); 5.31 (s, 2H, NH2); 7.41-7.37
(m, 1H, H4’); 7.55-7.48 (m, 4H, H2’, H3’, H5’, H6’); 7.78 (s,
1H, H3). 13C NMR (100 MHz, CDCl3, TMS,  in ppm): 14.5;
59.6; 96.2; 123.8; 128.1; 129.7; 137.6; 140.6; 149.0; 164.6. EI
[M-1]- 230.2.
4.2.2. Procedure
for
preparing
1-phenyl-1H-
pyrazol-5-amnine (14)
A mixture of 1.50 g (6 mmol) of ethyl 5-amine-1-phenyl-1H-
pyrazole-4-carboxylate (13) and 20 mL of 85% phosphoric acid
was maintained under reflux and stirring for 6 hours at 170 °C.
After completion of the reaction, 50 mL of crushed ice was
added, and then the mixture was basified to pH 7 with NaOH (6
M aq.). The mixture was extracted with dichloromethane (3 × 30
mL). The combined organic solution was washed with water (3 ×
50 mL), dried (anhydrous sodium sulfate), filtered, and
concentrated under vacuum to afford 14 as a brown oil.
4.2. Synthesis
4.2.1. Procedure for preparing ethyl 5-amino-1-
phenyl-1H-pyrazole-4-carboxylate (13)
A mixture of phenylhydrazine (9 mmol) and 10 mL of ethanol
was stirred and allowed to reflux for
Yield: 86%. IR (KBr. cm-1): 3418-3182; 1618; 1597; 1550;
1
760-694. H NMR (400 MHz, CDCl3, TMS,  in ppm): 3.84 (s,
1
h. Then,
2H, NH2); 5.61 (d, 1H, J = 2.0 Hz, H4); 7.42 (d, 1H, J = 1.8 Hz,

H3); 7.57-7.32 (m, 5H, H2’, H3’, H4’, H5’, H6’). 13C NMR (100
MHz, CDCl3, TMS,  in ppm): 90.6; 123.9; 127.4; 129.4; 138.6;
140.3; 144.7. EI [M-1]- 157.8.
The cyclization of 1.0 g (3.5 mmol) of 17 was performed by
refluxing in 15 mL of dowtherm at 250 °C for 40 min. After
completion of the reaction, the reaction mixture was cooled and
then poured into 50 mL of hexane. The precipitate was filtered
and dried to afford 22 in 71% yield.
4.2.3. General procedure for preparing diethyl 2-
(((1-phenyl-1H-pyrazol-5-
yl)amino)methylene)malonate (16), and (E)-ethyl 2-
cyano-3-((1-phenyl-1H-pyrazol-5-yl)amino)acrylate
(17).
A mixture of 11.13 g (7 mmol) of 1-phenyl-1H-pyrazol-5-
amine 14 and 7 mmol of diethyl 2-(ethoxymethylene)malonate or
ethyl 2-cyano-3-ethoxyacrylate was maintained under reflux and
stirring for 2 hours. After completion of the reaction, the reaction
mixture was poured into 50 mL of ice-cold water. The precipitate
was collected by filtration and washed with water to afford 16
and 17 in yields of 88% and 86%, respectively.
Yield: 71%. MP: 223°C. IR (KBr. cm-1): 3124; 2229; 1661;
1
1583; 1497. H NMR (400 MHz, CDCl3,TMS,  in ppm): 7.45-
7.41 (m, 1H, H4’); 7.61-7.57 (m, 2H, H3’, H5’); 8.06 (d, 1H, J =
7.9 Hz, H2’, H6’); 8.53 (s, 1H, H3); 8.64 (s, 1H, H6). 13C NMR
(100 MHz, CDCl3, TMS,  in ppm): 91.5; 108.4; 116.1; 121.9;
127.1; 129.2; 134.2; 138.0; 151.8; 164.4. EI [M-1]- 234.9.
4.2.6. Procedure for preparing 4-chloro-1-phenyl-
1H-pyrazolo[3,4-b]pyridine-5-carbonitrile (19)
To 1 g (4.2 mmol) of derivative 22 was used the same
procedure described in 4.2.1.
Yield: 90%. MP: 146°C. IR (KBr. cm-1): 3110; 2229; 1593;
1463. 1H NMR (400 MHz, DMSO-d6, TMS,  in ppm): 7.47-7.43
(m, 1H, H4’); 7.63-7.59 (m, 2H, H3’, H5’); 8.14-8.11 (m, 2H,
H2’, H6’); 8.81 (s, 1H, H3); 9.08 (s, 1H, H6). 13C NMR (100
MHz, DMSO-d6, TMS,  in ppm): 103.4; 114.9; 115.8; 121.5;
127.4; 129.3; 134.1; 137.7; 149.9; 141.3; 152.5. EI [M+23]+
276.9.
Diethyl
2-(((1-phenyl-1H-pyrazol-5-
yl)amino)methylene)malonate (16).
Yield: 88%. MP: 86°C. IR (KBr. cm-1): 3135; 3105; 2976;
1
2904; 1683; 1638; 1594; 1556; 1513; 1272. H NMR (400 MHz,
CDCl3, TMS,  in ppm): 1.30 (t, J = 7.1 Hz, 3H, CH2CH3); 4.23
(q, J = 7.1 Hz, 2H, CH2CH3); 6.19 (d, 1H, J = 1.9 Hz, H4); 7.58-
7.45 (m, 5H, H2’, H3’, H4’, H5’, H6’); 7.61 (d, 1H, J = 1.9 Hz,
H3); 7.64 (d, 1H, J = 12.7 Hz, Hβ); 10.89 (d, J = 12.4 Hz, NH).
13C NMR (100 MHz, CDCl3, TMS,  in ppm): 14.1; 61.6; 78.2;
94.0; 124.3; 128.9; 129.9; 137.1; 138.6; 140.6; 152.7; 166.7. EI
[M-1]- 328.2.
4.2.7. General
procedure
for
preparing4-[(4-
aminobutyl)amino]-1-phenyl-1H-pyrazolo[3,4-
b]pyridines (20, 21)
A solution of 1 g of derivative 18 or 19 (3.3 mmol or 3.9
mmol, respectively) in 20 mL of 1,4-dioxane was added
dropwise to 0.8 mL (8.2 mmol) of butane-1,4-diamine and stirred
at 80 °C for 4 h. The reaction mixture was poured into 50 mL of
ice-cold water. The precipitate was collected by filtration and
washed with ethyl ether to afford 20 and 21 in yields of 79 and
66%.
Ethyl 2-cyano-3-((1-phenyl-1H-pyrazol-5-yl)amino)acrylate (17).
Yield: 86%. MP: 169°C. IR (KBr. cm-1): 3175-3140; 3068;
1
2992; 2939; 2212; 1667; 1621; 1559; 1511; 1238. H NMR (400
MHz, CDCl3, TMS,  in ppm): 1.33-1.27 (m, 3H, CH2CH3);
4.26-4.19 (m, 2H, CH2CH3); 6.21 (d, 1H, J = 1.9 Hz, H4); 7.57-
7.43 (m, 5H, H2’, H3’, H4’, H5’, H6’); 7.61 (d, 1H, J = 1.9 Hz,
H3); 8.22 (d, 1H, J = 12.9 Hz, Hβ); 11.03 (d, J = 12.8 Hz, NH).
13C NMR (100 MHz, CDCl3, TMS,  in ppm): 14.2; 14.3; 60.4;
60.7; 94.0; 96.0; 124.3; 128.6; 130.0; 137.5; 140.0; 140.6; 152.5;
165.0; 168.2. EI [M-1]- 280.8.
Ethyl
4-[(4-aminobutyl)amino]-1-phenyl-1H-
pyrazolo[3,4-b]pyridine-5-carboxylate (20).
Yield: 79%. MP: 149°C. IR (KBr. cm-1): 3271-3239; 2979;
1
2867; 1667; 1578-1473; 1266. H NMR (400 MHz, DMSO-d6,
TMS,  in ppm): 1.34 (t, 3H, J = 7.1 Hz, CH2CH3); 1.80-1.72 (m;
4H, H9, H10); 2.84 (m, 2H, H11); 3.73 (m, 2H, H8); 4.31 (q, 2H,
J = 7.1 Hz, CH2CH3); 7.40-7.34 (m, 1H, H4’); 7.58-7.53 (m, 2H,
H3’-H5’); 8.19-8.16 (m, 2H, H2’-H6’); 8.56 (s, 1H, H3); 8.77 (s,
1H, H6); 9.13 (t; 1H, J = 5.2 Hz, NH). 13C NMR (100 MHz,
DMSO-d6, TMS,  in ppm): 14.0; 25.2; 25.7; 38.5; 44.0; 60.2;
99.3; 104.0; 121.3; 126.2; 128.9; 136.1; 138.7; 151.4; 152.4;
152.5; 167.8. HRMS (ESI) calc. for C19H23N5O2 354.1852, found
[M+1]+ 354.1930.
4.2.4. Procedure for preparing ethyl 4-chloro-1-
phenyl-1H-pyrazolo[3,4-b]pyridine-5-carboxylate
(18)
To 1 g (3 mmol) of derivative 16 was added 10 mL of
phosphorus oxychloride. The mixture was stirred under reflux for
6 h. Excess solvent was removed under reduced pressure, and the
resulting material was carefully added to 50 mL of crushed ice.
The mixture was then basified to pH 7 with NaOH (6 M aq.) and
stirred for 40 min. The mixture was diluted with water (30 mL)
and extracted with chloroform (3 × 30 mL). The combined
organic solution was washed with water (3 × 50 mL), dried
(anhydrous sodium sulfate), filtered, and concentrated under
vacuum to afford 14 in 86% yield.
4-[(4-Aminobutyl)amino]-1-phenyl-1H-
pyrazolo[3,4-b]pyridine-5-carbonitrile (21).
Yield: 66%. MP: 102°C. IR (KBr. cm-1): 3302; 2939; 2861;
1
2214; 1581-1460. H NMR (400 MHz, DMSO-d6, TMS,  in
Yield: 84%. MP: 97°C. IR (KBr. cm-1): 3084; 2982; 2942;
1718; 1591; 1463; 1178. 1H NMR (400 MHz, DMSO-d6, TMS, 
in ppm): 1.38 (t, 3H, J = 7.1 Hz, CH2CH3); 4.40 (q, 2H, J = 7.1
Hz, CH2 CH3); 7.45-7.41 (m, 1H, H4’); 7.63-7.58 (m, 2H, H3’,
H5’); 8.18-8.14 (m, 2H, H2’, H6’); 8.67 (s, 1H, H3); 9.09 (s, 1H,
H6). 13C NMR (100 MHz, DMSO-d6, TMS,  in ppm): 13.9;
61.6; 116.9; 119.1; 121.1; 126.7; 129.2; 134.4; 138.1; 138.1;
150.2; 151.6; 163.2. EI [M-1]- 302.1.
ppm): 1.55-1.50 (m; 1H, H10); 1.77-1.72 (m, 1H, H9); 2.64-2.60
(m, 1H, H11); 3.70-3.67 (m, 1H, H8); 7.38-7.34 (m, 1H, H4’);
7.54 (t, 2H, J = 7.6 Hz, H3’-H5’); 8.14 (d, 2H, J = 7.6 Hz, H2’-
H6’); 8.36 (s, 1H, H6); 8.53 (s, 1H, H3). 13C NMR (100 MHz,
DMSO-d6, TMS,  in ppm): 26.2; 29.7; 40.9; 118.5; 121.4;
126.4; 126.6; 128.9; 129.9; 138.5; 156.54; 157.7; 160.9. HRMS
(ESI) calc. for C17H18N6 306.1593, found [M+1]+ 307.1668.
4.2.8. General procedure for preparing N-(4-((1-
phenyl-1H-pyrazolo[3,4-b]pyridine-4-
yl)amino)butyl)benzenesulfonamides (1-10)
The amine 20 or 21 (1.4 mmol) was treated with the
appropriate sulfonyl chloride (2.8 mmol) in MeOH as the solvent
and TEA (1 eq.). The reaction mixture was maintained under
4.2.5. Procedure for preparing 4-oxo-1-phenyl-4,7-
dihydro-1H-pyrazolo[3,4-b]pyridine-5-carbonitrile
(22)

stirring at 70°C for 24 h and was poured into ice-cold water (50
mL). The precipitate was filtered and dried. The residual crude
product was purified via silica gel column chromatography using
a gradient mixture of chloroform/methanol. Compounds 1-10
were obtained as white solids in 59-70% yield.
1H, H3); 8.77 (s, 1H, H6); 9.07 (t, 1H, J = 5.1 Hz, NH). 13C
NMR (100 MHz, DMSO-d6, TMS,  in ppm): 14.1; 25.8; 26.3;
42.1; 44.0; 60.2; 99.3; 104.0; 121.3; 126.2; 128.3; 128.9; 129.2;
136.1; 137.1; 138.7; 139.3; 151.3; 152.4; 152.5; 167.8. HRMS
(ESI) calc. for C25H26ClN5O4S 528.1394, found [M+1]+
528.1488. HPLC: 99.8%.
Ethyl
1-phenyl-4-((4-(phenylsulfonamido)butyl)amino)-1H-
pyrazolo[3,4-b]pyridine-5-carboxylate (1). Yield: 60%. MP: 52-
Ethyl
4-((4-(4-methoxyphenylsulfonamido)butyl)amino)-1-
54°C. IR (KBr. cm-1): 3290-3247; 2982-2872; 1682; 1582; 1370;
phenyl-1H-pyrazolo[3,4-b]pyridine-5-carboxylate (5). Yield:
60%. MP: 165-167°C. IR (KBr. cm-1): 2960-2872; 1667; 1579;
1341; 1179; 1263; 1149. 1H NMR (400 MHz. DMSO-d6. TMS. 
in ppm): 1.34 (t, 3H, J = 7.0 Hz, CH2CH3); 1.60-1.54 (m, 2H,
H10); 1.72-1.68 (m, 2H, H9); 2.82-2.77 (m, 2H, H11); 3.64-3.63
(m, 2H, H8); 3.80 (s, 3H, OCH3); 4.31 (q, 2H, J = 7.0 Hz,
CH2CH3); 7.07 (d, 1H, J = 8.7 Hz, H2’’-H6’’or H3’’-H5’’); 7.36
(t, 1H, J = 7.3 Hz, H4’); 7.57-7.49 (m, 3H, H3’-H5’, SO2NH);
7.72 (d, 2H, J = 8.7 Hz, H2’’-H6’’ or H3’’-H5’’); 8.18 (d, 2H, J
= 8.0 Hz, H2’-H6’); 8.48 (s, 1H, H3); 8.76 (s, 1H, H6); 9.06 (s,
1H, NH). 13C NMR (100 MHz, DMSO-d6, TMS,  in ppm): 14.0;
25.8; 26.2; 42.0; 44.0; 55.4; 60.2; 99.3; 104.0; 114.2; 121.3;
126.9; 126.2; 128.5; 128.9; 132.1; 136.1; 138.7; 151.3; 152.4;
152.5; 161.9; 167.7. HRMS (ESI) calc. for C26H29N5O5S
523.1889, found [M+1]+ 524.1972. HPLC: 100%.
1
1181; 1266; 1156. H NMR (400 MHz, DMSO-d6, TMS,  in
ppm): 1.34 (t, 3H, J = 7.1 Hz, CH2CH3); 1.60-1.53 (m, 2H, H10);
1.74-1.67 (m, 2H, H9); 2.86-2.81 (m, 2H, H11); 3.67-3.63 (m,
2H, H8); 4.31 (q, 2H, J = 7.1 Hz, CH2CH3); 7.36 (t, 1H, J = 7.4
Hz, H4’); 7.62-7.54 (m, 5H, H3’-H5’, H4’’, H3’’-H5’’ or H2’’-
H6’’); 7.68 (t, 1H, J = 5.8 Hz, SO2NH); 7.80-7.78 (m, 2H, H2’’-
H6’’ or H3’’-H5’’); 8.19-8.17 (m, 2H, H2’-H6’); 8.49 (s, 1H,
H3); 8.76 (s, 1H, H6); 9.07 (t, 1H, J = 5.2, NH). 13C NMR (100
MHz, DMSO-d6, TMS,  in ppm): 14.1; 25.8; 26.4; 42.1; 44.0;
60.2; 99.3; 104.0; 121.3; 126.2; 126.3; 129.1; 129.9; 132.2;
136.1; 138.7; 140.4; 151.3; 152.3; 152.5; 167.7. HRMS (ESI)
calc. for C25H28N5O4S 493.1784, found [M+1]+ 494.1871. HPLC:
98%.
Ethyl
4-((4-(4-methylphenylsulfonamido)butyl)amino)-1-
phenyl-1H-pyrazolo[3,4-b]pyridine-5-carboxylate (2). Yield:
N-(4-((5-cyano-1-phenyl-1H-pyrazolo[3,4-b]pyridin-4-
yl)amino)butyl)benzenesulfonamide (6). Yield: 67%. MP: 158-
159°C. IR (KBr. cm-1): 3305; 2941-2876; 2213; 1592-1422;
66%. MP: 130-132°C. IR (KBr. cm-1): 3293-3068; 2963-2851;
1
1682; 1578; 1328; 1268; 1161. H NMR (400 MHz, DMSO-d6,
1
TMS,  in ppm): 1.34 (t, 3H, J = 7.1 Hz, CH2CH3); 1.59-1.53 (m,
2H, H10); 1.73-1.66 (m, 2H, H9); 2.34 (s, 3H, CH3); 2.83-2.78
(m, 2H, H11); 3.66-3.61 (m, 2H, H8); 4.31 (q, 2H, J = 7.0 Hz,
CH2CH3); 7.38-7.35 (m, 3H, H4’, H2’’-H6’’or H3’’-H5’’), 7.59-
7.54 (m, 3H, SO2NH, H3’-H5’); 7.67 (d, 2H, J = 8.2 Hz, H2’’-
H6’’ or H3’’-H5’’); 8.19-8.17 (m, 2H, H2’-H6’); 8.48 (s, 1H,
H3); 8.77 (s, 1H, H6); 9.06 (t, 1H, J = 5.2, NH). 13C NMR (100
MHz, DMSO-d6, TMS,  in ppm): 14.1; 20.8; 25.8; 26.3; 42.0;
44.0; 60.2; 99.3; 104.0; 121.3; 126.2; 126.4; 128.9; 129.5; 136.1;
137.5; 138.7; 142.4; 151.3; 152.4; 152.5; 167.7. HRMS (ESI)
calc. for C26H29N5O4S 507.1940, found [M+1]+ 508.2024. HPLC:
97%.
1320; 1152. H NMR (400 MHz, DMSO-d6, TMS,  in ppm):
1.55-1.48 (m, 2H, H10); 1.71-1.63 (m, 2H, H9); 2.83-2.78 (m,
2H, H11); 3.65-3.60 (m, 2H, H8); 7.39-7.35 (m, 1H, H4’); 7.64-
7.54 (m, 6H, H3’-H5’, H4’’, H2’’-H6’’ or H3’’-H5’’, SO2NH);
7.79-7.77 (t, 2H, H2’’-H6’’ or H3’’-H5’’); 8.14-8.12 (m, 2H,
H2’-H6’); 8.39 (s, 1H, H6); 8.49 (s, 1H, H3). 13C NMR (100
MHz, DMSO-d6, TMS,  in ppm): 25.9; 26.1; 41.9; 42.2; 43.4;
118.5; 121.4; 123.3; 126.3; 128.9; 129.0; 129.9; 132.2; 134.9;
138.5; 140.4; 150.8; 151.4; 153.8. HRMS (ESI) calc. for
C23H22N6O2S 446.1525, found [M+1]+ 447.1609. HPLC: 98.3%.
N-(4-((5-cyano-1-phenyl-1H-pyrazolo[3,4-b]pyridin-4-
yl)amino)butyl)-4-methylbenzenesulfonamide (7). Yield: 65%.
MP: 160-161°C. IR (KBr. cm-1): 3295; 2943-2874; 2207; 1592-
Ethyl
4-((4-(4-fluorophenylsulfonamido)butyl)amino)-1-
1
phenyl-1H-pyrazolo[3,4-b]pyridine-5-carboxylate (3). Yield:
1455; 1322; 1153. H NMR (400 MHz, DMSO-d6, TMS,  in
59%. MP: 160-162°C. IR (KBr. cm-1): 2987-2864; 1667; 1588;
ppm): 1.55-1.48 (m, 2H, H10); 1.69-1.63 (m, 2H, H9); 2.35 (s,
3H, CH3); 2.80-2.76 (m, 2H, H11); 3.65-3.59 (m, 2H, H8); 7.39-
7.35 (m, 3H, H4’, H2’’-H6’’ or H3’’-H5’’); 7.58-7.52 (m, 3H,
H3’-H5’, SO2NH); 7.66 (d, 2H, J = 8.2 Hz, H2’’-H6’’ or H3’’-
H5’’); 8.13 (d, 2H, J = 7.7 Hz, H2’-H6’); 8.39 (s, 1H, H6); 8.49
(s, 1H, H3). 13C NMR (100 MHz, DMSO-d6, TMS,  in ppm):
20.8; 26.1; 42.2; 43.4; 121.4; 126.4; 128.9; 129.5; 134.9; 137.5;
138.5; 142.4; 150.8; 151.4; 153.8. HRMS (ESI) calc. for
C24H24N6O2S 460.1681, found [M+1]+ 461.1763. HPLC: 95.3%.
1
1368; 1261; 1153. H NMR (400 MHz, DMSO-d6, TMS,  in
ppm): 1.34 (t, 3H, J = 7.1 Hz, CH2CH3); 1.60-1.53 (m, 2H, H10);
1.74-1.67 (m; 2H, H9); 2.87-2.82 (m, 2H, H11); 3.68-3.64 (m,
2H, H8); 4.31 (q, 2H, J = 7.0 Hz, CH2CH3); 7.43-7.34 (m, 3H,
H4’, H2’’-H6’’); 7.55 (t, 2H, J = 7.7 Hz, H3’-H5’); 7.71 (t, 1H, J
= 5.9, SO2NH); 7.87-7.83 (m, 2H, H3’’-H5’’); 8.19-8.17 (m, 2H,
H2’-H6’); 8.50 (s, 1H, H3); 8.77 (s, 1H, H6); 9.07 (t, 1H, J = 5.1
Hz, NH). 13C NMR (100 MHz, DMSO-d6, TMS,  in ppm): 14.1;
25.8; 26.3; 42.1; 44.0; 60.2; 99.3; 104.0; 116.2 (J = 22.5; C3’’-
C5’’); 121.3; 126.2; 128.9; 129.3 (J = 9.4; C2’’-C6’’); 136.1;
136.8; 138.7; 151.4; 152.4; 152.5; 164.0 (J = 248.8; C4’’); 167.8.
19F NMR (376 MHz, DMSO-d6, TMS,  in ppm): -107.14.
HRMS (ESI) calc. for C25H26FN5O4S 511.1690, found [M+1]+
512.1764. HPLC: 100%.
N-(4-((5-cyano-1-phenyl-1H-pyrazolo[3,4-b]pyridin-4-
yl)amino)butyl)-4-fluorobenzenesulfonamide (8). Yield: 63%.
MP: 128-129°C. IR (KBr. cm-1): 3285; 3150; 2869; 2221; 1590-
1
1427; 1327; 1152. H NMR (400 MHz, DMSO-d6, TMS,  in
ppm): 1.55-1.48 (m, 2H, H10); 1.71-1.64 (m, 2H, H9); 2.83-2.78
(m, 2H, H11); 3.65-3.60 (m, 2H, H8); 7.42-7.36 (m, 3H, H4’,
H2’’-H6’’ or H3’’-H5’’); 7.58-7.54 (m, 2H, H3’-H5); 7.67 (t;
1H, J = 5.8 Hz, SO2NH); 7.86-7.82 (m, 2H, H2’’-H6’’ or H3’’-
H5’’); 8.14-8.12 (m, 2H, H2’-H6’); 8.38 (s, 1H, H6); 8.49 (s, 1H,
H3). 13C NMR (100 MHz, DMSO-d6, TMS,  in ppm): 25.9;
26.1; 42.2; 43.4; 116.2 (J = 22.5 Hz, C3’’-C5’’); 118.5; 121.4;
123.3; 126.4; 128.9; 129.3 (J = 9.4 Hz, C2’’-C6’’); 129.9; 134.8;
136.8 (J = 3.0 Hz, C1’’); 138.5; 150.8; 151.4; 153.8; 156.5; 163.9
(J = 249.1 Hz, C4’’). 19F NMR (376 MHz, DMSO-d6, TMS,  in
ppm): -107.14. HRMS (ESI) calc. for C23H21FN6O2S 464.1431,
found [M+1]+ 465.1516. HPLC: 97.3%.
Ethyl
4-((4-(4-chlorophenylsulfonamido)butyl)amino)-1-
phenyl-1H-pyrazolo[3,4-b]pyridine-5-carboxylate (4). Yield:
70%. MP: 143-145°C. IR (KBr. cm-1): 3268; 2944-2869; 1666;
1581; 1324; 1267; 1157. 1H NMR (400 MHz, DMSO-d6, TMS, 
in ppm): 1.34 (t, 3H, J = 7.1 Hz, CH2CH3); 1.61-1.54 (m, 2H,
H10); 1.72-1.67 (m, 2H, H9); 2.87-2.83 (m, 2H, H11); 3.67-3.63
(m, 2H, H8); 4.31 (q, 2H, J = 7.0 Hz, CH2CH3); 7.36 (t, 1H, J =
7.4 Hz, H4’); 7.56 (t, 2H, J = 7.9 Hz, H3’-H5’); 7.64 (t, 2H, J =
8.6 Hz, H2’’-H6’’or H3’’-H5’’); 7.81-7.77 (m, 3H, H2’’-H6’’ or
H3’’-H5’’, SO2NH); 8.18 (d, 2H, J = 7.7 Hz, H2’-H6’); 8.50 (s,

4-Chloro-N-(4-((5-cyano-1-phenyl-1H-pyrazolo[3,4-
nonlinear regression function32. (Origin software, OriginLab
Corporation, Northampton, MA, USA). Selectivity index (SI)
was defined as the ratio of the MLD50 to the IC50 values of the
active compounds.
b]pyridin-4-yl)amino)butyl)benzenesulfonamide (9). Yield: 68%.
MP: 99-100°C. IR (KBr. cm-1): 3319; 3111; 2947-2875; 2214;
1
1578-1461; 1329; 1151. H NMR (400 MHz, DMSO-d6, TMS, 
in ppm):1.56-1.51 (m, 2H, H10); 1.70-1.64 (m, 2H, H9); 2.84-
2.80 (m, 2H, H11); 3.65-3.60 (m, 2H, H8); 7.37 (t, 1H, J = 7.4
Hz, H4’); 7.56 (t, 2H, J = 7.7 Hz, H3’-H5’); 7.64 (d, 2H, J = 8.6
Hz, H2’’-H6’’ or H3’’-H5’’); 7.78 (d, 2H, J = 8.6 Hz, H2’’-H6’’
or H3’’-H5’’); 8.13 (d, 2H, J = 7.8 Hz, H2’-H6’); 8.39 (s, 1H,
H6); 8.49 (s, 1H, H3). 13C NMR (100 MHz, DMSO-d6, TMS, 
in ppm): 25.9; 26.1; 42.2; 43.4; 118.5; 121.4; 126.4; 128.3;
128.9; 129.2; 134.9; 137.1; 138.5; 139.3; 150.8; 151.4; 153.9.
HRMS (ESI) calc. for C23H21ClN6O2S 480.1135, found [M+1]+
481.1216. HPLC: 91%.
Acknowledgments
The authors thank the Coordination of Improvement of Higher
Education (CAPES) and the National Council of R&D of Brazil
(CNPq) for the fellowships granted to the authors. We also thank
the Foundation for Research of the State of Rio de Janeiro
(FAPERJ), Technological Development Program on Products for
Health (PDTIS), for financial support. LJMC and NB is recipient
of research productivity fellowships from the CNPq and from
FAPERJ (“Cientista do Nosso Estado”).
N-(4-((5-cyano-1-phenyl-1H-pyrazolo[3,4-b]pyridin-4-
yl)amino)butyl)-4-methoxybenzenesulfonamide (10). Yield: 63%.
MP: 156-157°C. IR (KBr. cm-1): 3340; 3277; 2973-2873; 2211;
References and notes
1
1596-1413; 1318; 1147. H NMR (400 MHz, DMSO-d6, TMS, 
1.
World Health Organization (WHO), World Malaria Report 2014.
Available at:
in ppm):1.55-1.48 (m, 2H, H10); 1.70-1.63 (m, 2H, H9); 2.79-
2.74 (m, 2H, H11); 3.65-3.60 (m, 2H, H8); 3.81 (s, 3H, OCH3);
7.09-7.05 (m, 2H, H2’’-H6’’ or H3’’-H5’’); 7.39-7.35 (m, 1H,
H4’); 7.45 (t, 1H, J = 5.9 Hz, SO2NH); 7.58-7.54 (m, 2H, H3’-
H5’); 7.72-7.68 (m, 2H, H2’’-H6’’ or H3’’-H5’’); 8.14-8.12 (m,
2H, H2’-H6’); 8.39 (s, 1H, H6); 8.49 (s, 1H, H3). 13C NMR (100
MHz, DMSO-d6, TMS,  in ppm): 26.0; 26.1; 42.2; 43.4; 55.5;
114.2; 118.5; 121.4; 126.4; 128.5; 128.9;132.0; 134.9; 138.5;
150.7; 151.4; 161.9. HRMS (ESI) calc. for C24H24N6O3S
476.1631, found [M+1]+ 477.1715. HPLC: 95.3%.
http://www.who.int/malaria/publications/world_malaria_report_2
014/en/ Accessed: April/2016.
2.
3.
Severini, C. Menegon, M. J. Glob. Antimicrob. Resist. 2015, 3,
58–63.
World Health Organization(WHO), Guidelines for the treatment
of malaria 3rd ed, 2015. Available at:
http://www.who.int/malaria/publications/atoz/9789241549127/en
/ Accessed: April/2016.
4.
5.
White, N. J. J. Clin. Invest. 2004, 113, 1084–1092.
Dondorp, A. M., Yeung, S., White, L., Nguon, C., Day, N. P.,
Socheat, D., von Seidlein, L. Nat. Rev. Microbiol.2010, 8, 272–
280.
4.3 Biological evaluation
6.
Schrader, F. C.; Barho, M.; Steiner, I.; Ortmann, R.; Schlitzer,M.
Int. J. Med. Microbiol. 2012, 302, 165–171.
Barnett, D. S.; Guy, R. K. Chem. Rev. 2014, 114, 11221−11241.
Anthony, M. P.; Burrows, J. N.; Duparc, S.; JMoehrle J.; Wells,
T. N. C. Malaria Journal 2012, 11, 316.
4.3.1 Continuous cultures of P. falciparum-infected erythrocytes
and in vitro assays.
7.
8.
The cultivation of the P. falciparum W2 strain (chloroquine
resistant) was performed according to the protocol of Trager &
Jensen28 using human erythrocytes (A+) in RPMI 1640 medium
supplemented with human serum. Parasites were synchronized
with sorbitol in order to obtain the young (ring) parasite
forms29,30. Parasitemia was adjusted to 0.05% and hematocrit to
1.5%; subsequently, cultures were placed in microplates together
with the drugs to be tested in serial dilutions (50 – 0.78µM), as
well as positive (chloroquine and sulfadoxine) and negative (no
drug) controls, and incubated for 72 hours. The microplates were
then subjected to three cycles of freezing and thawing, and
parasite growth was measured through quantification of
histidine-rich protein II (HRPII) using specific monoclonal
antibodies in sandwich ELISA31. Absorbances were read at 450
nm using a SpectraMax 190 spectrophotometer, and the
determination of IC50 and SD was performed using dose-response
curves with a non-linear regression function. (Origin software,
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a
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Graphical Abstract
Design, synthesis and anti-P. falciparum
activity of pyrazolopyridine-sulfonamide
derivatives
Leave this area blank for abstract info.
Thais B. Silva, Alice M. R. Bernardino, Maria de Lourdes G. Ferreira, Kamilla R. Rogerio, Leonardo J. M. Carvalho, Nubia Boechat and Luiz C. S.
Pinheiro