Bioorganic & Medicinal Chemistry 24 (2016) 4492–4498

Contents lists available at ScienceDirect

Bioorganic & Medicinal Chemistry

journal homepage: www.elsevier.com/locate/bmc

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

sulfonamide derivatives
Thais B. Silva a,b, Alice M. R. Bernardino b, Maria de Lourdes G. Ferreira a, Kamilla R. Rogerio a,c,d,
Leonardo J. M. Carvalho c, Nubia Boechat a, Luiz C. S. Pinheiro a,⇑
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
b
Programa 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

a r t i c l e i n f o a b s t r a c t

Article history: Ten 1-phenyl-1H-pyrazolo[3,4-b]pyridine derivatives connected by a linker group to benzenesulfon-

Received 24 June 2016 amide moieties with different substituents in the 4-position were synthesized and assayed against
Revised 20 July 2016 Plasmodium falciparum. These ten compounds exhibited activity in vitro against the chloroquine-resistant
Accepted 22 July 2016
clone W2 with IC50 values ranging from 3.46 to 9.30 lM. The most active derivatives with substituent
Available online 25 July 2016
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
Keywords:
than those with a CN substituent. The 1H-pyrazolo[3,4-b]pyridine system appears to be promising for
Malaria
Pyrazolopyridine
further studies as an antimalarial for overcoming the burden of resistance in P. falciparum.
Sulfonamide Ó 2016 Elsevier Ltd. All rights reserved.
Plasmodium falciparum

1. Introduction against malaria, including a fixed dose of artemisinin-combined

Over the past fifteen years (2000 to 2015), there has been a sig-
nificant 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 period.1 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 min.1
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 drugs.2
Due to the high parasitic resistance exhibited by Plasmodium
falciparum to most available drugs, monotherapy is no longer used
to treat malaria. The WHO recommends the use of first-line drugs
⇑ Corresponding author. Tel.: +55 21 3977 2456; fax: +55 21 2560 2518.
E-mail address: lpinheiro@far.fiocruz.br (L.C.S. Pinheiro).
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 ACTs.3 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 derivative.3
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 artemisinins.4,5 In the recent literature, a
number of new antimalarial compounds in different states of
preclinical and clinical development have been described.6–8
Quinoline derivatives are still the dominate class of antimalarial
compounds;9,10 however, due to resistance to this class of drugs,
searching for other analogous compounds is extremely important.
The pyrazolopyridine ring is an isostere of quinoline, and the
literature has demonstrated that this system possesses anti-
inflammatory,11 anti-cancer,12 antibacterial13,14 and antiviral15–18

http://dx.doi.org/10.1016/j.bmc.2016.07.049
0968-0896/Ó 2016 Elsevier Ltd. All rights reserved.
 T. B. Silva et al. / Bioorg. Med. Chem. 24 (2016) 4492–4498
activities. The literature suggests that pyrazolopyridine is a poten-
tial system with antiparasitic activity against Trypanosoma cruzi,19
as well as antileishmanial20 and antimalarial activities.21,22
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 lM.23
The derivative 2-(trifluoromethyl)[1,2,4]triazolo[1,5-a]pyrim-
idine II was active against P. falciparum W2 with an
IC50 = 0.023 lM and showed no toxicity. In this work, the impor-
tance of the CF3 group at position 2 of the nucleus triazolo[1,5-a]
pyrimidine for increased activity was demonstrated.24
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 lM.25
We have recently synthesized a new class of hybrids of atorvas-
tatin (AVA) and aminoquinolines (primaquine and chloroquine
derivatives). The quinolinic moiety was connected to the pentasub-
stituted 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 lM.26
New derivatives of quinoline-sulfadoxine hybrids were planned
by molecular hybridization between the quinoline ring and the
benzenesulfonamide moiety present in chloroquine and sulfadox-
ine, which are drugs used in the treatment of malaria. All 15 com-
pounds 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 lM. 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
Plasmodium berghei malaria, compound V was the most active
and inhibited parasitemia by 49% on day 5 after inoculation, con-
tributing to the discovery of new prototypes with antimalarial
activity (Fig. 1).27
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-yl)amino)butyl)ben-
zenesulfonamides (1–10) were designed (Fig. 2).
H H
N H
O
OH
O
O H
H H
O
O N
O N CF3
O-
CF3
O
(I)
IC50 = 0.001 µM
O Cl
HN
Cl
N H
N O O
(IV)
IC 50 0.4 µM
F
Figure 1. Structures of compounds I–V.
4493
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.
2. Results and discussion
2.1. Chemistry
The synthetic route for preparing the N-(4-((1-phenyl-1H-pyra-
zolo[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 phenylhy-
drazine (11) (0.01 mol) and ethyl 2-cyano-3-ethoxyacrylate (12)
(0.01 mol), in ethanol under reflux. The derivative 5-amino-1-phe-
nyl-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 phospho-
ric 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-car-
boxylate (18) was prepared from diethyl 2-(((1-phenyl-1H-pyra-
zol-5-yl)amino)methylene)malonate (16) through a reaction with
phosphorous oxychloride under reflux in 84% yield as described
in the literature.16,20 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 precip-
itating from hexane with a yield of 84%.28 The derivative (22) was
refluxed with phosphorous oxychloride to produce (19) in 90%
yield.
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)
O
H
N
N
N N
N
F3 C
N
N Cl N
(II) (III)
IC 50 0.023 µM IC 50 1.4 µM
H
N N
HN S
Cl N (V)
IC 50 = 0.09 µM
4494 T. B. Silva et al. / Bioorg. Med. Chem. 24 (2016) 4492–4498

NH2
O
N S
HN O
O NH

O N
Cl N N
Cl
chloroquine sulfadoxine

H
N
HN S
O
O
isosteric ring
replacement
Cl N remained the group
linker benzenesulfonamide
(V)

R2

H
N
HN S
O
R1 O
N
N N R1 = CO2 Et, CN
R2 = H, F, Cl, CH 3
(1-10)

Figure 2. Rational approach to the design of compounds 1–10.

NHNH 2
CO2Et
R1
N NH 2 N
N N EtO CO2 Et N N
(i) NH 2 (ii) (iii) H CO2 Et
N
11 + 17 R 1 = CN
R1
+ (vii)
O
EtO CO 2Et 13 14 15 R 1 = CO2 Et;
12 R 1 = CN CN
16 R 1 = CO2 Et
CN 12 N
(iv) N
R2 N
H

NH2 Cl
H HN 22
N R1 (viii)
HN S (vi) R1 (v)
R1 O O N
N
N N N
N N
N N
R 1 = CO2 Et R1 = CN
20 R 1 = CO2 Et; 18 R 1 = CO2 Et;
1 R 2 = H; 6 R2 = H; 21 R 1 = CN 19 R 1 = CN
2 R 2 = CH 3; 7 R2 = CH3 ;
3 R 2 = F; 8 R2 = F;
4 R 2 = Cl; 9 R2 = Cl;
5 R 2 = OCH 3 10 R2 = OCH3

Scheme 1. Synthetic route used to prepare compounds 1–10. 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%.

were synthesized in 79% and 66% yield, respectively, by the nucle-
ophilic substitution reaction between 4-chloro-1-phenyl-1H-pyra-
zolo[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 tar-
get compounds N-(4-((1-phenyl-1H-pyrazolo[3,4-b]pyridin-4-yl)
amino)butyl)benzenesulfonamides (1–10) in 70–59% yield.27
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).
The values are summarized in Table 1. All the compounds that
presented activity showed IC50 values ranging from 3.46 to
9.30 lM in the anti-HPR2 assay, lower than that of sulfadoxine
(IC50 = 15.0 lM); however, these compounds were not more active
than chloroquine (IC50 = 0.55 lM).
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 lM (2)
and 3.60 lM (4 and 7). Compound 4 also showed the highest SI
values: >277.77.
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
 T. B. Silva et al. / Bioorg. Med. Chem. 24 (2016) 4492–4498 4495

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

Compounds Anti-P. falciparum activity IC50* (lM) Cytotoxicity to BGM cells MLD50** (lM) SI[MLD50/IC50]
1 R1 = CO2Et/R2 = H 6.52 ± 0.8 822.00 126.07
2 R1 = CO2Et/R2 = CH3 3.46 ± 0.25 596.00 172.25
3 R1 = CO2Et/R2 = F 4.12 ± 0.48 993.00 241.01
4 R1 = CO2Et/R2 = Cl 3.60 ± 2.04 >1000.00 >277.77
5 R1 = CO2Et/R2 = OCH3 9.30 ± 1.25 995.00 106.98
6 R1 = CN/R2 = H 4.20 ± 1.07 248.00 59.04
7 R1 = CN/R2 = CH3 3.60 ± 0.09 682.00 189.44
8 R1 = CN/R2 = F 9.30 ± 2.95 508.00 54.62
9 R1 = CN/R2 = Cl 7.50 ± 0.09 418.00 55.73
10 R1 = CN/R2 = OCH3 5.00 ± 1.15 498.00 99.60
20 R1 = CO2Et 2.10 ± 0.08 930.00 442.85
21 R1 = CN 5.00 ± 1.90 503.00 100.60
Chloroquine 0.55 385.00 700.00
Sulfadoxine 15.00 310.00 20.70
*
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.

of the intermediates 20 and 21 with IC50values of 2.10 and 5.00, chromatography was performed using silica gel 60
respectively. In fact, the amine 20 exhibited greater activity than (0.040–0.063 mm). The remaining reagents and solvents that were
the sulfonamides (1–10) and the highest SI value (442.85). used were of analytical grade.

3. Conclusions 4.2. Synthesis

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 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 lM. However, the observed activity was lower than
that of chloroquine and the quinolone-sulfonamide hybrid V.
Derivatives 2, 4 and 7 exhibited the lowest activity against
P. falciparum, with IC50 values of 3.46 lM (2) and 3.60 lM (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 lM and the high-
est SI value (442.85).
The 1H-pyrazolo[3,4-b]pyridine system appears to be promis-
ing for further studies as an antimalarial with the objective of over-
coming the burden of resistance in P. falciparum.
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 (d) 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 (mp) were determined using a Büchi model
B-545 apparatus. TLC (thin layer chromatography) was performed
using a silica gel F-254 glass plate (20
20 cm). Column
4.2.1. Procedure for preparing ethyl 5-amino-1-phenyl-1H-pyra-
zole-4-carboxylate (13)
A mixture of phenylhydrazine (9 mmol) and 10 mL of ethanol
was stirred and allowed to reflux for 1 h. Then, ethyl
(ethoxymethylene)cyanoacetate (9 mmol) dissolved in 10 mL of
ethanol was slowly added. The reaction mixture was refluxed for
20 min. 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.
Yield: 70%. Mp: 98 °C. IR (KBr. cm1): 3400–3140; 2995; 2912;
1677; 1621; 1552; 1528; 1280; 781–697. 1H NMR (400 MHz,
CDCl3, TMS, d 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, H40 );
7.55–7.48 (m, 4H, H20 , H30 , H50 , H60 ); 7.78 (s, 1H, H3). 13C NMR
(100 MHz, CDCl3, TMS, d in ppm): 14.5; 59.6; 96.2; 123.8; 128.1;
129.7; 137.6; 140.6; 149.0; 164.6. EI [M1] 230.2.
4.2.2. Procedure for preparing 1-phenyl-1H-pyrazol-5-amine
(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 h 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.
Yield: 86%. IR (KBr. cm1): 3418–3182; 1618; 1597; 1550;
760–694. 1H NMR (400 MHz, CDCl3, TMS, d in ppm): 3.84 (s, 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, H20 , H30 , H40 , H50 , H60 ). 13C NMR (100 MHz,
CDCl3, TMS, d in ppm): 90.6; 123.9; 127.4; 129.4; 138.6; 140.3;
144.7. EI [M1] 157.8.
4.2.3. General procedure for preparing diethyl 2-(((1-phenyl-1H-
pyrazol-5-yl)amino)methylene)malonate (16), and (E)-ethyl 2-cy-
ano-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
4496 T. B. Silva et al. / Bioorg. Med. Chem. 24 (2016) 4492–4498
2-cyano-3-ethoxyacrylate was maintained under reflux and stir-
ring for 2 h. After completion of the reaction, the reaction mixture
was poured into 50 mL of ice-cold water. The precipitate was col-
lected by filtration and washed with water to afford 16 and 17 in
yields of 88% and 86%, respectively.
4.2.3.1. Diethyl 2-(((1-phenyl-1H-pyrazol-5-yl)amino)methylene)-
malonate (16). Yield: 88%. Mp: 86 °C. IR (KBr. cm1): 3135;
3105; 2976; 2904; 1683; 1638; 1594; 1556; 1513; 1272. 1H NMR
(400 MHz, CDCl3, TMS, d 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, H20 , H30 , H40 , H50 , H60 ); 7.61 (d, 1H, J = 1.9 Hz, H3);
7.64 (d, 1H, J = 12.7 Hz, Hb); 10.89 (d, J = 12.4 Hz, NH). 13C NMR
(100 MHz, CDCl3, TMS, d 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 [M1] 328.2.
4.2.3.2. Ethyl 2-cyano-3-((1-phenyl-1H-pyrazol-5-yl)amino)acry-
late (17). Yield: 86%. Mp: 169 °C. IR (KBr. cm1): 3175–3140;
3068; 2992; 2939; 2212; 1667; 1621; 1559; 1511; 1238. 1H NMR
(400 MHz, CDCl3, TMS, d 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, H20 , H30 , H40 , H50 , H60 ); 7.61 (d, 1H, J = 1.9 Hz, H3); 8.22
(d, 1H, J = 12.9 Hz, Hb); 11.03 (d, J = 12.8 Hz, NH). 13C NMR
(100 MHz, CDCl3, TMS, d 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 [M1] 280.8.
4.2.4. Procedure for preparing ethyl 4-chloro-1-phenyl-1H-pyra
zolo[3,4-b]pyridine-5-carboxylate (18)
To 1 g (3 mmol) of derivative 16 was added 10 mL of phospho-
rus 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.
Yield: 84%. Mp: 97 °C. IR (KBr. cm1): 3084; 2982; 2942; 1718;
1591; 1463; 1178. 1H NMR (400 MHz, DMSO-d6, TMS, d 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, H40 ); 7.63–7.58 (m, 2H, H30 , H50 ); 8.18–8.14
(m, 2H, H20 , H60 ); 8.67 (s, 1H, H3); 9.09 (s, 1H, H6). 13C NMR
(100 MHz, DMSO-d6, TMS, d 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
[M1] 302.1.
4.2.5. Procedure for preparing 4-oxo-1-phenyl-4,7-dihydro-1H-
pyrazolo[3,4-b]pyridine-5-carbonitrile (22)
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 comple-
tion 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.
Yield: 71%. Mp: 223 °C. IR (KBr. cm1): 3124; 2229; 1661; 1583;
1497. 1H NMR (400 MHz, CDCl3, TMS, d in ppm): 7.45–7.41 (m, 1H,
H40 ); 7.61–7.57 (m, 2H, H30 , H50 ); 8.06 (d, 1H, J = 7.9 Hz, H20 , H60 );
8.53 (s, 1H, H3); 8.64 (s, 1H, H6). 13C NMR (100 MHz, CDCl3, TMS, d
in ppm): 91.5; 108.4; 116.1; 121.9; 127.1; 129.2; 134.2; 138.0;
151.8; 164.4. EI [M1] 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 Section 4.2.1.
Yield: 90%. Mp: 146 °C. IR (KBr. cm1): 3110; 2229; 1593; 1463.
1
H NMR (400 MHz, DMSO-d6, TMS, d in ppm): 7.47–7.43 (m, 1H,
H40 ); 7.63–7.59 (m, 2H, H30 , H50 ); 8.14–8.11 (m, 2H, H20 , H60 );
8.81 (s, 1H, H3); 9.08 (s, 1H, H6). 13C NMR (100 MHz, DMSO-d6,
TMS, d 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.
4.2.7. General procedure for preparing 4-[(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%.
4.2.7.1. Ethyl 4-[(4-aminobutyl)amino]-1-phenyl-1H-pyrazolo
[3,4-b]pyridine-5-carboxylate (20). Yield: 79%. Mp: 149 °C.
IR (KBr. cm1): 3271–3239; 2979; 2867; 1667; 1578–1473;
1266. 1H NMR (400 MHz, DMSO-d6, TMS, d 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, H40 ); 7.58–7.53 (m, 2H, H30 –H50 ); 8.19–8.16
(m, 2H, H20 –H60 ); 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, d 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) calcd
for C19H23N5O2 354.1852, found [M+1]+ 354.1930.
4.2.7.2. 4-[(4-Aminobutyl)amino]-1-phenyl-1H-pyrazolo[3,4-b]
pyridine-5-carbonitrile (21). Yield: 66%. Mp: 102 °C. IR
(KBr. cm1): 3302; 2939; 2861; 2214; 1581–1460. 1H NMR
(400 MHz, DMSO-d6, TMS, d in 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, H40 ); 7.54 (t, 2H, J = 7.6 Hz, H30 –H50 );
8.14 (d, 2H, J = 7.6 Hz, H20 –H60 ); 8.36 (s, 1H, H6); 8.53 (s, 1H,
H3). 13C NMR (100 MHz, DMSO-d6, TMS, d 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) calcd for C17H18N6 306.1593, found [M
+1]+ 307.1668.
4.2.8. General procedure for preparing N-(4-((1-phenyl-1H-pyra-
zolo[3,4-b]pyridine-4-yl)amino)butyl)benzenesulfonamides (1–
10)
The amine 20 or 21 (1.4 mmol) was treated with the appropri-
ate sulfonyl chloride (2.8 mmol) in MeOH as the solvent and TEA
(1 eq.). The reaction mixture was maintained under stirring at
70 °C for 24 h and was poured into ice-cold water (50 mL). The pre-
cipitate 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.
4.2.8.1. Ethyl 1-phenyl-4-((4-(phenylsulfonamido)butyl)amino)-
1H-pyrazolo[3,4-b]pyridine-5-carboxylate (1). Yield: 60%.
Mp: 52–54 °C. IR (KBr. cm1): 3290–3247; 2982–2872; 1682;
1582; 1370; 1181; 1266; 1156. 1H NMR (400 MHz, DMSO-d6,
TMS, d 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, H40 ); 7.62–7.54 (m, 5H, H30 –H50 , H400 , H300 –H500 or
H200 –H600 ); 7.68 (t, 1H, J = 5.8 Hz, SO2NH); 7.80–7.78 (m, 2H,
H200 –H600 or H300 –H500 ); 8.19–8.17 (m, 2H, H20 –H60 ); 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, d 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;
 T. B. Silva et al. / Bioorg. Med. Chem. 24 (2016) 4492–4498
140.4; 151.3; 152.3; 152.5; 167.7. HRMS (ESI) calcd for C25H28N5O4S
493.1784, found [M+1]+ 494.1871. HPLC: 98%.
4.2.8.2. Ethyl 4-((4-(4-methylphenylsulfonamido)butyl)amino)-1-
phenyl-1H-pyrazolo[3,4-b]pyridine-5-carboxylate (2). Yield:
66%. Mp: 130–132 °C. IR (KBr. cm1): 3293–3068; 2963–2851;
1682; 1578; 1328; 1268; 1161. 1H NMR (400 MHz, DMSO-d6, TMS,
d 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, H40 , H200 –H600 or H300 –H500 ), 7.59–7.54 (m, 3H, SO2NH,
H30 –H50 ); 7.67 (d, 2H, J = 8.2 Hz, H200 –H600 or H300 –H500 ); 8.19–8.17
(m, 2H, H20 –H60 ); 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, d 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) calcd for C26H29N5O4S 507.1940, found [M+1]+
508.2024. HPLC: 97%.
4.2.8.3. Ethyl 4-((4-(4-fluorophenylsulfonamido)butyl)amino)-1-
phenyl-1H-pyrazolo[3,4-b]pyridine-5-carboxylate (3). Yield:
59%. Mp: 160–162 °C. IR (KBr. cm1): 2987–2864; 1667; 1588;
1368; 1261; 1153. 1H NMR (400 MHz, DMSO-d6, TMS, d 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, H40 , H200 –H600 ); 7.55
(t, 2H, J = 7.7 Hz, H30 –H50 ); 7.71 (t, 1H, J = 5.9, SO2NH); 7.87–7.83
(m, 2H, H300 –H500 ); 8.19–8.17 (m, 2H, H20 –H60 ); 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, d in ppm): 14.1; 25.8; 26.3; 42.1; 44.0; 60.2; 99.3;
104.0; 116.2 (J = 22.5; C300 –C500 ); 121.3; 126.2; 128.9; 129.3
(J = 9.4; C200 –C600 ); 136.1; 136.8; 138.7; 151.4; 152.4; 152.5; 164.0
(J = 248.8; C400 ); 167.8. 19F NMR (376 MHz, DMSO-d6, TMS, d in
ppm): 107.14. HRMS (ESI) calcd for C25H26FN5O4S 511.1690, found
[M+1]+ 512.1764. HPLC: 100%.
4.2.8.4. 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. cm1): 3268; 2944–2869; 1666;
1581; 1324; 1267; 1157. 1H NMR (400 MHz, DMSO-d6, TMS, d 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, H40 );
7.56 (t, 2H, J = 7.9 Hz, H30 –H50 ); 7.64 (t, 2H, J = 8.6 Hz, H200 –H600 or
H300 –H500 ); 7.81–7.77 (m, 3H, H200 –H600 or H300 –H500 , SO2NH); 8.18
(d, 2H, J = 7.7 Hz, H20 –H60 ); 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, d 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) calcd for C25H26ClN5O4S 528.1394, found
[M+1]+ 528.1488. HPLC: 99.8%.
4.2.8.5. Ethyl 4-((4-(4-methoxyphenylsulfonamido)butyl)
amino)-1-phenyl-1H-pyrazolo[3,4-b]pyridine-5-carboxylate (5).
Yield: 60%. Mp: 165–167 °C. IR (KBr. cm1): 2960–2872; 1667;
1579; 1341; 1179; 1263; 1149. 1H NMR (400 MHz. DMSO-d6.
TMS. d 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, H200 –H600 or H300 –H500 ); 7.36 (t,
1H, J = 7.3 Hz, H40 ); 7.57–7.49 (m, 3H, H30 –H50 , SO2NH); 7.72 (d,
2H, J = 8.7 Hz, H200 –H600 or H300 –H500 ); 8.18 (d, 2H, J = 8.0 Hz,
H20 –H60 ); 8.48 (s, 1H, H3); 8.76 (s, 1H, H6); 9.06 (s, 1H, NH). 13C
NMR (100 MHz, DMSO-d6, TMS, d 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.
4497
HRMS (ESI) calcd for C26H29N5O5S 523.1889, found [M+1]+
524.1972. HPLC: 100%.
4.2.8.6. 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. cm1): 3305; 2941–2876; 2213; 1592–1422; 1320;
1152. 1H NMR (400 MHz, DMSO-d6, TMS, d 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, H40 ); 7.64–7.54 (m, 6H,
H30 –H50 , H400 , H200 –H600 or H300 –H500 , SO2NH); 7.79–7.77 (t, 2H,
H200 –H600 or H300 –H500 ); 8.14–8.12 (m, 2H, H20 –H60 ); 8.39 (s, 1H,
H6); 8.49 (s, 1H, H3). 13C NMR (100 MHz, DMSO-d6, TMS, d 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) calcd for C23H22N6O2S 446.1525, found [M+1]+
447.1609. HPLC: 98.3%.
4.2.8.7. 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. cm1): 3295; 2943–2874; 2207; 1592–
1455; 1322; 1153. 1H NMR (400 MHz, DMSO-d6, TMS, d in 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,
H40 , H200 –H600 or H300 –H500 ); 7.58–7.52 (m, 3H, H30 –H50 , SO2NH);
7.66 (d, 2H, J = 8.2 Hz, H200 –H600 or H300 –H500 ); 8.13 (d, 2H,
J = 7.7 Hz, H20 –H60 ); 8.39 (s, 1H, H6); 8.49 (s, 1H, H3). 13C NMR
(100 MHz, DMSO-d6, TMS, d 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) calcd for C24H24N6O2S 460.1681, found [M+1]+
461.1763. HPLC: 95.3%.
4.2.8.8. 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. cm1): 3285; 3150; 2869; 2221;
1590–1427; 1327; 1152. 1H NMR (400 MHz, DMSO-d6, TMS, d 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, H40 ,
H200 –H600 or H300 –H500 ); 7.58–7.54 (m, 2H, H30 –H5); 7.67 (t; 1H,
J = 5.8 Hz, SO2NH); 7.86–7.82 (m, 2H, H200 –H600 or H300 –H500 );
8.14–8.12 (m, 2H, H20 –H60 ); 8.38 (s, 1H, H6); 8.49 (s, 1H, H3). 13C
NMR (100 MHz, DMSO-d6, TMS, d in ppm): 25.9; 26.1; 42.2; 43.4;
116.2 (J = 22.5 Hz, C300 –C500 ); 118.5; 121.4; 123.3; 126.4; 128.9;
129.3 (J = 9.4 Hz, C200 –C600 ); 129.9; 134.8; 136.8 (J = 3.0 Hz, C100 );
138.5; 150.8; 151.4; 153.8; 156.5; 163.9 (J = 249.1 Hz, C400 ). 19F
NMR (376 MHz, DMSO-d6, TMS, d in ppm): 107.14. HRMS (ESI)
calcd for C23H21FN6O2S 464.1431, found [M+1]+ 465.1516. HPLC:
97.3%.
4.2.8.9. 4-Chloro-N-(4-((5-cyano-1-phenyl-1H-pyrazolo[3,4-b]
pyridin-4-yl)amino)butyl)benzenesulfonamide (9). Yield:
68%. Mp: 99–100 °C. IR (KBr. cm1): 3319; 3111; 2947–2875;
2214; 1578–1461; 1329; 1151. 1H NMR (400 MHz, DMSO-d6,
TMS, d 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, H40 ); 7.56 (t, 2H, J = 7.7 Hz, H30 –H50 ); 7.64 (d, 2H,
J = 8.6 Hz, H200 –H600 or H300 –H500 ); 7.78 (d, 2H, J = 8.6 Hz, H200 –H600
or H300 –H500 ); 8.13 (d, 2H, J = 7.8 Hz, H20 –H60 ); 8.39 (s, 1H, H6);
8.49 (s, 1H, H3). 13C NMR (100 MHz, DMSO-d6, TMS, d 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) calcd
for C23H21ClN6O2S 480.1135, found [M+1]+ 481.1216. HPLC: 91%.
4.2.8.10. 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. cm1): 3340; 3277; 2973–2873;
2211; 1596–1413; 1318; 1147. 1H NMR (400 MHz, DMSO-d6, TMS,
4498 T. B. Silva et al. / Bioorg. Med. Chem. 24 (2016) 4492–4498
d 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, H200 –H600 or H300 –H500 ); 7.39–7.35 (m, 1H, H40 ); 7.45 (t, 1H,
J = 5.9 Hz, SO2NH); 7.58–7.54 (m, 2H, H30 –H50 ); 7.72–7.68 (m, 2H,
H200 –H600 or H300 –H500 ); 8.14–8.12 (m, 2H, H20 –H60 ); 8.39 (s, 1H,
H6); 8.49 (s, 1H, H3). 13C NMR (100 MHz, DMSO-d6, TMS, d 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)
calcd for C24H24N6O3S 476.1631, found [M+1]+ 477.1715. HPLC:
95.3%.
4.3. Biological evaluation
4.3.1. Continuous cultures of P. falciparum-infected
erythrocytes and in vitro assays
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 forms.29,30
Parasitemia was adjusted to 0.05% and hematocrit to 1.5%; subse-
quently, cultures were placed in microplates together with the
drugs to be tested in serial dilutions (50–0.78 lM), as well as pos-
itive (chloroquine and sulfadoxine) and negative (no drug) con-
trols, and incubated for 72 h. The microplates were then
subjected to three cycles of freezing and thawing, and parasite
growth was measured through quantification of histidine-rich pro-
tein II (HRPII) using specific monoclonal antibodies in sandwich
ELISA.31 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, OriginLab Corporation,
Northampton, MA, USA).
4.3.2. Cell cultures and cytotoxicity tests
BGM cells were cultured in RPMI 1640 medium supplemented
with 10% fetal bovine serum (FBS) at a concentration of 5
103
cells per milliliter. Cells were added together with the test com-
pounds in serial dilution. The microplates were incubated for
24 h at 37 °C. Subsequently, MTT salt at a concentration of 3 mg/
mL was added after 3 h, the supernatant was removed, and the
dye present on the bottom of the plate wells was dissolved in
DMSO in a volume of 100 lL/well. The microplates were then read
using a Spectramax 190 spectrophotometer with a 570 nm filter.32
The determination of MLD50 was performed using dose response
curves with a nonlinear regression function.32 (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.
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’).
References and notes
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http://www.who.int/malaria/publications/world_malaria_report_2014/en/
Accessed: April/2016.
2. Severini, C.; Menegon, M. J. Glob. Antimicrob. Resist. 2015, 3, 58.
3. 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. White, N. J. J. Clin. Invest. 2004, 113, 1084.
5. Dondorp, A. M.; Yeung, S.; White, L.; Nguon, C.; Day, N. P.; Socheat, D.; von
Seidlein, L. Nat. Rev. Microbiol. 2010, 8, 272.
6. Schrader, F. C.; Barho, M.; Steiner, I.; Ortmann, R.; Schlitzer, M. Int. J. Med.
Microbiol. 2012, 302, 165.
7. Barnett, D. S.; Guy, R. K. Chem. Rev. 2014, 114, 11221.
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