New hybrid trifluoromethylquinolines as antiplasmodium agents

Article abstract of DOI:10.1016/j.bmc.2019.01.044

Malaria remains a major public health problem worldwide, and it is responsible for high rates of morbidity and mortality. Resistance to current antimalarial drugs has been identified, and new drugs are urgently needed. In this study, we designed and synthesized seventeen novel quinolines based on the structures of mefloquine ((2,8-bis(trifluoromethyl)quinolin-4-yl)(piperidin-2-yl)methanol) and amodiaquine (4-((7-chloroquinolin-4-yl)amino)-2-((diethylamino)methyl)phenol) using ring bioisosteric replacement and molecular hybridization of the functional groups. The compounds were evaluated in vitro against Plasmodium falciparum and in vivo in mice infected with P. berghei. All derivatives presented anti-P. falciparum activity with IC₅₀ values ranging from 0.083 to 33.0 μM. The compound with the best anti-P. falciparum activity was N-(5-methyl-4H-1,2,4-triazol-3-yl)-2,8-bis(trifluoromethyl)quinolin-4-amine (12) which showed an IC₅₀ of 0.083 μM. The three most active compounds were selected for antimalarial activity tests against P. berghei-infected mice. Compound 12 was the most active on the 5th day after infection, reducing parasitemia by 66%, which is consistent with its in vitro activity. This is an important result as 12, a simpler molecule than mefloquine, does not contain the stereogenic center, and consequently, its synthesis in the laboratory is easier and less expensive. This system proved promising for the design of potential antimalarial compounds.

Full text of DOI:10.1016/j.bmc.2019.01.044

Accepted Manuscript
New hybrid trifluoromethylquinolines as antiplasmodium agents
Renata M.R.J. da Silva, Marilia O. Gandi, Jorge S. Mendonça, Alcione S.
Carvalho, Julia Penna Coutinho, Anna C.C. Aguiar, Antoniana U. Krettli, Nubia
Boechat
PII:
S0968-0896(18)31818-2
https://doi.org/10.1016/j.bmc.2019.01.044
BMC 14736
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
26 October 2018
21 January 2019
31 January 2019
Please cite this article as: da Silva, R.M.R., Gandi, M.O., Mendonça, J.S., Carvalho, A.S., Coutinho, J.P., Aguiar,
A.C.C., Krettli, A.U., Boechat, N., New hybrid trifluoromethylquinolines as antiplasmodium agents, Bioorganic &
Medicinal Chemistry (2019), doi: https://doi.org/10.1016/j.bmc.2019.01.044
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New hybrid trifluoromethylquinolines as antiplasmodium agents
Renata M. R. J. da Silvaa,b, Marilia O. Gandia,b,c, Jorge S. Mendonçab, Alcione S.
Carvalhob, Julia Penna Coutinhod, Anna C. C. Aguiard, Antoniana U. Krettlid and Nubia
Boechatb*
a Programa de Pós-Graduação Farmacologia e Química Medicinal, Instituto de Ciências Biomédicas, UFRJ, Av. Carlos Chagas,
373 - bl. K, 2º andar, sala 35 - Prédio do Centro de Ciências da Saúde, Cidade Universitária, Ilha do Fundão - Rio de Janeiro - RJ,
21941-902, Brazil
b Departamento de Síntese Orgânica, Instituto de Tecnologia em Fármacos -Farmanguinhos - Fiocruz, Rua Sizenando Nabuco, 100
Manguinhos, Rio de Janeiro - RJ, 21041-250, Brazil
c Faculdade de Farmácia Universidade Iguaçu-UNIG, Av. Abílio Augusto Távora, 2134. Nova Iguaçu – RJ, 26275-580, Brazil
dLaboratório de Malária, Instituto René Rachou, Fiocruz/MG, Avenida Augusto de Lima, 1715, Barro Preto, Belo Horizonte - MG,
30190-002, Brazil
keywords: malaria; P. falciparum; quinoline; hybrids; mefloquine; amodiaquine
Abstract
Malaria remains a major public health problem worldwide, and it is responsible for high rates of morbidity and mortality.
Resistance to current antimalarial drugs has been identified, and new drugs are urgently needed. In this study, we designed and
synthesized seventeen novel quinolines based on the structures of mefloquine ((2,8-bis(trifluoromethyl)quinolin-4-yl)(piperidin-2-
yl)methanol) and amodiaquine (4-((7-chloroquinolin-4-yl)amino)-2-((diethylamino)methyl)phenol) using ring bioisosteric
replacement and molecular hybridization of the functional groups. The compounds were evaluated in vitro against Plasmodium
falciparum and in vivo in mice infected with P. berghei. All derivatives presented anti-P. falciparum activity with IC50 values
ranging from 0.083 to 33.0 µM. The compound with the best anti-P. falciparum activity was N-(5-methyl-4H-1,2,4-triazol-3-yl)-2,8-
bis(trifluoromethyl)quinolin-4-amine (12) which showed an IC50 of 0.083 µM. The three most active compounds were selected for
antimalarial activity tests against P. berghei-infected mice. Compound 12 was the most active on the 5th day after infection,
reducing parasitemia by 66%, which is consistent with its in vitro activity. This is an important result as 12, a simpler molecule than
mefloquine, does not contain the stereogenic center, and consequently, its synthesis in the laboratory is easier and less expensive.
This system proved promising for the design of potential antimalarial compounds.
1. Introduction
Malaria is an important infectious disease globally, and approximately 3.2 billion people are at risk of
infection.1,2 In 2016, an estimated 216 million cases of malaria and 445 thousand deaths occurred worldwide.
However, US$ 2.7 billion have been invested in malaria control and elimination efforts globally by
governments of malaria endemic countries and international partners.1,2 For decades, the treatment of malaria
was based on chloroquine (CQ), which has rapid efficacy and low toxicity and is safe even for children and
pregnant women. A number of other quinolines, such as amodiaquine (AQ), primaquine (PQ) and mefloquine
(MQ), have been developed to treat chloroquine-resistant strains (CQR) of P. falciparum. However, the
parasite has developed resistance to most antimalarials around the world.3,4,5,6 Artemisinin and its derivatives,
such as artesunate (AS), have reached prominence in the antimalarial arsenal due to their efficacy with high
parasitic reduction rates.7 AS reduces parasitemia more quickly than any other therapeutic options, as it acts on
more phases of the parasite cycle, and it can be used against severe malaria in adults and children.8 AS has an
extremely short half-life, being almost completely eliminated in 24 h, and it is not used as a monotherapy since
at least seven days of treatment are needed to adequately eliminate all parasites from circulation.7,9 Artemisinin
derivatives are used in association with a quinoline compound, which has a high efficacy and a long half-life in
plasma. These combinations are known as artemisinin‐ based combination therapies (ACTs).9 The different
ACT combinations, such as artesunate-amodiaquine (ASAQ) and artesunate-mefloquine (ASMQ), share the
fact that they have high efficacy and excellent safety profiles, and they have been recommended by the WHO
(World Health Organization) as first-line treatments against P. falciparum.10 However, drug resistance has

significantly increased, and the emergence and spread of parasite strains resistant to ACTs highlight the need
for new drugs with different structural characteristics and modes of action.11,12,13,14,15,16
Hybridization is an important tool in medicinal chemistry and is based on combining two
pharmacophoric chemical entities into a single skeleton.17 Studies have been conducted using synthetic hybrid
compounds designed to have increased efficacy over quinoline derivatives such as AZT-chloroquinoline18 and
quinine-dihydroartemisinin.19 We have demonstrated the importance of some new compounds against P.
falciparum based on molecular hybrids, such as salts of mefloquine and artesunate,20 primaquine and
artesunate,21 2-(trifluoromethyl)[1,2,4]triazolo[1,5-a]pyrimidine,22 1H-1,2,3-triazole-quinoline,23 quinoline-
sulfonamide24 and aminoquinolines-atorvastatin.25
Thus, aminoquinolines are still attractive lead compounds in the search for new antimalarial drugs.18-29
The development of new bioactive substances by modifications of the chemical structure of a prototype using
molecular hybridization and bioisosterism as tools17,30 has become common, and this technique has been
successfully used in the search for compounds with higher activity and/or better pharmacological profiles.14-23
As an extension of our efforts to develop novel compounds to fight malaria,14-23 we present herein the synthesis
and biological evaluation of novel N-substituted-2,8-bis(trifluoromethyl)-quinolin-4-amines derivatives 1-14
and 4-substituted 2,8-bis(trifluoromethyl)quinoline derivatives 15-16.
2. Results and discussion
These novel quinolines were designed using molecular hybridization and ring bioisosterism as tools
for combining two pharmacophoric chemical entities into a single skeleton. The pharmacophoric subunit 2,8-
bis-(trifluoromethyl)quinoline, which is present in the structure of MQ (in blue), plus the aminoheteroaromatic
or aromatic function contained in AQ (in red) could generate potent antimalarial drugs (Fig. 1). An analysis of
the nature of the aliphatic, aromatic and heteroaromatic substituents in the AQ moiety (in red) will provide the
electronic and lipophilic properties to determine the distinct contributions of each fragment to the desired
activity profile of each member of this new class of compounds (Fig. 1A). On the other hand, we investigated
the influence of the absence of the NH group by preparing compounds 15 and 16, as shown in Fig. 1B.
Compound 17 was also prepared to compare the importance of 2,8-bis(trifluoromethyl)quinoline versus 7-
chloroquinoline in the antiplasmodial (Fig. 1C).

Figure 1. Rational approach to the design of N-substituted-2,8-bis(trifluoromethyl)-quinolin-4-amines
derivatives 1-14 in A, 4-substituted-2,8-bis(trifluoromethyl)quinoline 15-16 in B and 7-chloro-N-(pyridin-4-
yl)quinolin-4-amine (17) in C.
2.1 Chemistry
The synthetic route to prepare N-substituted-2,8-bis(trifluoromethyl)-quinolin-4-amines derivatives 1-
14 is shown in Scheme 1. The 2,8-bis(trifluoromethyl)quinoline core was prepared by cyclization of
commercially available 2-(trifluoromethyl) aniline (18) with ethyl 4,4,4-trifluoroacetoacetate in the presence of
polyphosphoric acid (PPA) to give 19 in 91% yield.31 Intermediate 19 was chlorinated with phosphorus
oxychloride at 80°C to obtain 4-chloro derivative 20 in 98% yield.32 The last step was an aromatic nucleophilic
substitution reaction between 4-chloro derivative 20 and the corresponding amine in NaH and DMSO to afford
desired products 1-16 in good yields.33,34
To obtain 7-chloro-N-(pyridin-4-yl)quinolin-4-amine (17), the nucleophilic substitution of 4-
aminopyridine with commercial 4,7-dichloroquinoline (21) was used to give 17 in 89% yield. The structures of
1
all the prepared compounds were confirmed by spectral analyses, namely, H, 19F and 13C NMR, FTIR and
mass spectrometry, and the obtained data were in full agreement with the proposed structures.

Scheme 1. Synthesis of N-substituted-2,8-bis(trifluoromethyl)-quinolin-4-amines derivatives 1-14, 4-
substituted-2,8-bis(trifluoromethyl)quinoline 15-16 and 7-chloro-N-(pyridin-4-yl)quinolin-4-amine (17).
2.2 Antiplasmodial assays
Studies have been conducted using synthetic compounds, which were designed to have the increased
efficacy of quinoline derivatives.35,36 A total of 16 quinoline derivatives containing a MQ pharmacophoric
entity in combination with an aromatic moiety from the lipophilic region of AQ were synthesized and
evaluated in vitro against the intraerythrocytic stages of CQ-resistant P. falciparum parasites (W2 clone). Their
efficacies were compared with those of MQ and AQ, and these results are shown in Table 1.

Table 1. In vitro activities (IC50 values) of synthesized compounds 1-17 and references antimalarials CQ, MQ
and AQ against a chloroquine-resistant Plasmodium falciparum clone (W2), cytotoxicity against the BGM cell
line (EC50), and selectivity index (SI), which is the ratio between the EC50 and IC50.
Compound
Code
IC50 ± SD [M]
R1
SI
EC50 BGM [M]
1
2
19.6±2.8
8.4±1.7
<44.8
>560
<2.29
>66.7
3
4
5
8.4±2
>559
>66.5
11.5
11.0±8.3
28.0±2.8
127±44
<22.5
<0.83
6
7
9.6±1.0
12.3
1.2
33.0±2.4
<18.9
<0.57
<2.67
8
9
16.0±0.0
24.1±9.4
<42.8
672
<27.88
27
10
11
12
13
14
4.8±1.0
129.8
<23.1
11±3.9
84.2±29
1.5
2.9±0.0
<8
0.083±0.006
11.5±1
132
7.3
0.8
1.8±1.0
15
16
31.5±2.9
>143
17.2±2.9
0.5
1680±131
<11.74
17
11.7±3
784.3
67
MQ
CQ
AQ
0.019±0.003
0.25±0.06
15±5.3
163
907±166
-
3628
-
0.006±0.002
The importance of the 2,8-bis(trifluoromethyl)quinoline moiety in MQ versus the 7-chloroquinoline
present in the CQ core was the first consideration investigated in this study. When the results of compounds 2
and 17 were compared, where the substitution pattern of the quinolinic ring was changed from 7-chlorine to
2,8-diCF3, respectively, 2 was equipotent than 17 demonstrating that substitution in the quinoline core was not
relevant for anti-plasmodium activity.
Another important factor was the presence of the quinoline-NH group in this class of molecules.
Compounds 15 (IC50 = 31.5 M) and 16 (IC50 >143 M), which are tertiary amines, were less active and
inactive, respectively. Comparing these biological results, we propose two hypotheses for the lack of anti-P.
falciparum activity of compounds that do not have an NH group between the trifluoromethylquinoline ring and
the hydrophobic region: (i) the cyclic tertiary amines of 15 and 16 lack conformational freedom because they

are more rigid than the other compounds, and this rigidity interferes with their ability to interact with the
biomacromolecule receptor; and (ii) the NH group could interact with the bioreceptor via hydrogen bonding.
The most active compound in the 2,8-bis-(trifluoromethyl)quinoline series was 12 (IC50 = 0.083 µM)
containing a 3-methyl-1,2,4-triazole substituent. Despite being less potent than MQ, the antiplasmodium
activity of 12 proved to be relevant as it is 3-fold more potent than CQ. Moreover, its chemical structure is
simpler than that of MQ as it does not contain a stereogenic center, and consequently, its synthesis in the
laboratory is easier and less expensive. If the 5-H or 5-CF3 in the triazole ring in derivatives 11 (IC50 = 2.9 µM)
and 13 (IC50 = 11.5 µM), respectively, were substituted, significantly lower activities were observed relative to
when 5-CH3 remained intact as it is in 12 (IC50 = 0.083 µM). The investigation of the isosteric replacement of
the 5-methyltriazole unit in 12 with a 5-methylthiadiazole unit in 14 (IC50 = 1.8 µM) proved to be deleterious to
the activity, showing the importance of the triazole nucleus in the antiplasmodial activity. This fact
corroborates previous literature results37,38,39 showing the importance of the triazole nucleus against the
Plasmodium parasite.
When comparing different substituents on the phenyl group, the following results were observed: 3,4-
di-OCH3 in 10 (IC50 = 4.8 µM); p-F in 8 (IC50 = 16.0 µM); p-OH in 9 (IC50 = 24.1); H in 5 (IC50 = 28.0 µM);
and o-CF3 in 7 (IC50 = 33.0 µM). The replacement of the phenyl group in 5 (IC50 = 28.0 µM) with a ß-naphthyl
group afforded 6 (IC50 = 9.6 µM), which was three times more active, showing the importance of lipophilicity
in this region to biological activity; however, it was not selective and showed high toxicity.
Concerning the location of the nitrogen atom on the pyridine, when the nitrogen was in the 4-position,
as in 2 (IC50 = 8.4 µM), the compound was 2.5 times more active than when the nitrogen was in the 2-position,
as in 1 (IC50 = 19.6 µM), and it was much more selective. Moreover, when we evaluated the effect of a
pyrimidine derivative, as in 3 (IC50 = 8.4 µM), with two adjacent nitrogen atoms, the compound showed
equipotent anti-P. falciparum activity and toxicity with 2. The LogP, LogD (pH 7.4 and 5.2) pKa were
calculated for all substances. However, it fails to correlate with the biological activity.
2.3 Antimalarial tests
Due to the high SI value of compounds 2, 3, and 12, they were selected for antimalarial activity tests
against P. berghei in experimentally infected mice. Compound 3 inhibited parasitemia by 61% on day 5 after
inoculation, whereas compound 2 was less active with a maximum of 25% parasitemia inhibition. Compound
12 was the most active on the 5th day after infection, reducing parasitemia by 66%, which is consistent with its
in vitro activity (Table 2).

Table 2. Antimalarial activity of the compounds that were most selective in vitro in mice infected with
Plasmodium berghei treated daily (doses of 50 mg/kg body weight) for three consecutive days. The
antimalarial mefloquine was used as a standard drug.
Survival
Parasitemia on daysa
Compound
(Mean ±
SD)
Dose
Mean ± SD (% Reduction)
(mg/kg)
5th
7th
9th
2
50
50
50
20
20
1.3 ± 0.9 (25)
0.9 ± 0.7 (61)
0.3 ± 0.2 (66)
12.3 ± 5.4 (42)
16.2 ± 11 (35)
15 ± 4.9 (30)
29.5 ± 19 (11)
22.9 ± 14 (31)
31 ± 13 (23)
6.3 ± 3.8 (84)
0.43 ±0.9 (99)
40.4 ± 12
15±2
13±4
19±4
24±3
>30
3
12
CQ
0.01± 0.02 (99)
0.04 ± 0.04 (98)
0.88 ± 0.3
0.14 ± 0.1 (99)
0.46 ± 0.6 (98)
21.5 ± 2.7
MQ
Non-treated
13±3
aReduction of parasitemia in relation to untreated controls; when < 30%, the compound was considered
inactive, 30-40% was partially active and > 40% was active. The data from compounds 2 and 3 are from one
experiment. The compound 12 was tested in two individual experiments.
3. Conclusions
We have successfully designed, synthesized and characterized a series of 17 novel quinolines 1-17
that are structural analogues of MQ and AQ. These compounds were obtained in only three steps with good
yields and are of great importance because they are simpler and less expensive to produce than MQ. Final costs
cannot be disregarded in the area of drug development, and this parameter depends on (i) the production
process of the specific drug, which is directly related to its structural complexity; (ii) less complex drugs
require fewer steps to produce and, consequently, reduce the consumption of reagents, solvents, energy, water,
and personnel hours and reduce safety concerns; and (iii) these new aminoquinolines were synthesized in only
three steps. Based on initial data reported in this paper, the selected potent analogues may represent new leads
for the development of synergistic drug partners in antimalarial combination therapies.
All compounds exhibited anti-P. falciparum activity in vitro against chloroquine-resistant parasites,
with IC50 values ranging from 0.083 to 33.0 µM. The compound in their series with the most potent anti-
plasmodium activity was N-(5-methyl-4H-1,2,4-triazole-(4-amino-(2,8-bis(trifluoromethyl)-quinoline) (12),
although less potent than MQ, it was 3-fold more potent than to CQ, and it is simpler and less expensive to
synthesize, as it lacks the stereogenic center present in MQ. Another relevant observation is that 2,8-diCF3
quinoline 2 was equipotent than 7-Cl quinoline 17 demonstrating that substitution in the quinoline core was not
relevant for anti-plasmodium activity. Furthermore, the absence of the quinoline-NH group, molecules of this
class are less active and more toxic (15 and 16). Comparing the lipophilic region of the phenyl substituent (5-
10) and different substituents on the phenyl ring, improvements in activity were observed when larger groups
were present in this region, as in 6 and 10; however, these compounds are toxic. In addition, the location of the
nitrogen atom in the pyridine core influences the activity; when the nitrogen is in position 4, the compound is
more active and selective. Finally, the 5-methyltriazole core provided superior antimalarial activity compared
with the other groups tested in this study, confirming that it is an important fragment for the development of

new antiparasitic drugs. The antimalarial activities of the compounds that were most selective in vitro (2, 3 and
12) in mice infected with Plasmodium berghei showed that 12 was the most active on the 5th day after
infection, and it reduced parasitemia by 66%, which was consistent with its in vitro activity. Ultimately, this
system proved promising for the design of potential antimalarial drugs.
4. Experimental
4.1 Chemistry
Reagents were purchased from Aldrich or Merck and were used without further purification. Thin-
layer chromatography separations were performed on silica gel plates (Merck, Kieselgel 60 F254), and the
spots were visualized using ultraviolet light (UV, 254 nm). Yields were determined after purification. Melting
points were obtained on a Buchi melting point apparatus model B-545 and are uncorrected. Infrared spectra
were obtained on a Shimadzu FTIR spectrophotometer model Prestige-21 through KBr reflectance. Gas
chromatography with mass spectrometry detection (GC-MS) was performed using an Agilent® Model 6890
Chromatograph with an Agilent® Model 5973 at 70 eV. The values of the fragmentation and molecular ions are
expressed as the mass/load (m/z) values. The column used was an Agilent® 122e5532 DB-5MS (5% diphenyl:
95% dimethylpolysiloxane), and analyses were performed using a temperature ramp of 50°C and 350°C. The
electron ionization mass spectrometry (ESI-MS, scan ES þ capillary (3.0 kV)/cone (30 V)/ extractor (1 V)/RF
lens (1.0 V)/source temperature (150°C)/desolvation temperature (300°C)) data were recorded using a
Micromass/Waters Spectrometer model: ZQ-4000. High-resolution mass spectrometry (HRMS) data were
1
obtained using an LC-MS Bruker Daltonics MicroTOF (time of flight analyzer). H, 13C and 19F nuclear
magnetic resonance (NMR) spectra were obtained at 400, 100 and 376 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. The coupling constants (J) are reported in hertz along with the apparent
peak multiplicities.
2,8-bis(trifluoromethyl)quinolin-4-ol (19)
To a solution of polyphosphoric acid (637.7 mmol) in 2-trifluoromethyl aniline (77.6 mmol) was
added ethyl 4,4,4-trifluoroacetoacetate (77.7 mmol). The reaction mixture was stirred for 3 hours at 150 °C.
Completion of the reaction was monitored by TLC. After, the reaction mixture was poured into iced distilled
water slowly with vigorous stirring to form yellow precipitate. The precipitate was vacuum filtered and washed
o
1
with cold distilled water to give yellow solid in 91% yield. m.p 128-130 C; H-NMR (400 MHZ; DMSO-d6,
ppm): δ 7.30 (s, 1H, H-3); 7.80 (t, 1H, H-6; J = 7.9 Hz); 8.28 (d, 1H, H-5; J = 7.2 Hz); 8.52 (d, 1H, H-7, J = 8.2
Hz); 13C NMR (100 MHz, DMSO-d6, ppm): δ 101.0 (C-3); 119.9 (C-4a); 121.9 (C-6); 122.6 (q, C-9, J = 273
Hz); 125.3 (q, C-10, J = 276 Hz); 127.3 (C-7); 130.0 (q, C-8, J = 58 Hz); 144.5 (C-5); 148.4 (q, C-2, J = 67
Hz); 163.3 (C-4); 19F-NMR (376 MHZ; DMSO-d6, ppm): δ -58.98 (s, 3F; F-10); -67.03 (s, 3F; F-9).
4-chloro-2,8-bis(trifluoromethyl)quinoline (20)
A mixture of 2,8-bis(trifluoromethyl)quinolin-4-ol (2g) and POCl3 (108.2 mmol) was heated at 80 °C
for 4 h. The reaction was monitored by TLC. After completion of the reaction was poured into iced distilled
water slowly with vigorous stirring to form dark brown precipitate. The precipitate was collected by filtration
to give yellow solid in 98% yield. m.p 45-46oC; GC-MS m/z (%): 299 (100); 280 (23); 230 (54); 210 (16);1H-

NMR (400 MHZ; DMSO-d6, ppm): δ 7.92 (s, 1H, H-3); 7.84 (t, 1H, H-6, J = 7.9 Hz); 8.28 (d, 1H, H-5, J = 7,2
Hz); 8.54 (d, 1H, H-7, J = 8.2 Hz); 19F-NMR (376 MHZ; MeOH, ppm): δ - 58.98 (s, 3F; F-10); -67.00 (s, 3F;
F-9).
General procedure for the synthesis of N-substituted-2,8-bis(trifluoromethyl)-quinolin-4-amines (1-16)
and 7-chloro-N-(pyridin-4-yl)quinolin-4-amine (17).
To a suspension of sodium hydride in mineral oil (80%, 3.33 mmol) in dry DMSO (2 ml) at 0 °C, the
appropriate amine (3.33 mmol) was slowly added. The mixture was stirred for 15 min at 0 C and then at room
temperature for 1 h. After this time, 4-chloro-2,8-bis(trifluoromethyl)quinoline or 4,7-dichloroquinoline (1
mmol) in dry DMSO (2 ml) was added to the reaction mixture, and it was stirred at room temperature for an
additional 24 h. The reaction was monitored by TLC. After it was complete, the reaction was poured into iced
distilled water to form a precipitate, which was isolated by filtration and dried. When no precipitation occurred,
the reaction mixture was extracted with ethyl acetate (3 x 20 ml), washed with water (3 x 15 ml), and dried
over anhydrous Na2SO4. The solvent was evaporated under vacuum and solid 1-17 precipitated in 56-87%
yields.
N-(pyridin-2-yl)-2,8-bis(trifluoromethyl)quinolin-4-amine (1)
Yellow solid; 77 % yield; m.p 170-172 °C; GC-MS m/z (%): 356 (100); 357 (80); 288 (87); 78 (16); 1H NMR
(400 MHz, DMSO-d6, ppm): δ 7.12-7.09 (m, H-5’, J = 5.2 Hz); 7.46 (d, 1H, H-6’); 7.86-7.82 (m, H-6; H-4’);
8.27 (d, 1H, H-5, J = 7.2 Hz); 8.43 (d, 1H, H-3’, J = 4.8 Hz); 8,95 (d, H-7, J = 8.6 Hz); 9.01 (s, H-3); 13C NMR
(100 MHz, DMSO-d6, ppm): δ 101.7 (C-3); 114.6 (C-5’); 118.1 (C-3’); 122.5 (q, C-9, J = 273 Hz); 122.6 (q, C-
10, J = 276 Hz); 123.4 (C-4a); 125.5 (C-6); 126.6 (q, C-8, J = 29 Hz); 127.4 (C-5); 129.2 (q, C-7, J = 5.1 Hz);
138.2 (C-4’); 144.3 (C-4); 147.2 (C-6’); 147.6 (q, C-2, J = 33.5 Hz); 148.0 (C-1’); 158.4 (C-8a); 19F-NMR (376
MHZ; DMSO-d6, ppm): - 58.79 (s, 3F; F-10); -66.94 (s, 3F; F-9); FTIR (KBr, cm−1): 3326 ( NH2); 2861 (
CH); 1618 (C=C);  1588( C=N); 1369 ( C-N); 1301 ( C-N); 1092, 1105 and 1119 ( CF); 724 ( CF3);
HRMS: calculated for C16H10F6N3: 358.0773, found: 358.0771.
N-(pyridin-4-yl)-2,8-bis(trifluoromethyl)quinolin-4-amine (2)
Yellow solid; 87 % yield; m.p 146-148 °C; GC-MS m/z (%): 357 (100); 358 (17); 288 (19); 78 (12);
1H NMR (400 MHz, DMSO-d6, ppm): δ 7.40 (d, 2H, H-1’; H-4’, J = 7.40 Hz); 7.65 (s, 1H, H-3); 7.88 (t, 1H,
H-6, J = 7.8 Hz); 8.32 (d, 1H, H-5, J = 7.2 Hz); 8.52 (d, 2H, H-2’, H-3’, J = 5.92 Hz); 8.74 (d, 1H, H-7, J = 8.5
Hz); 9.98 (1H, N-H). 13C NMR (100 MHz, DMSO-d6, ppm): δ 100.9 (C-3); 114.3 (C-2’, C-6’); 121.4 (q, C-9, J
= 273 Hz); 122.8 (q, C-10, J = 276 Hz); 125.2 (C-6); 126.4 (q, C-8, J = 29 Hz); 127.6 (C-5); 129.6 (q, C-7, J =
5.0 Hz); 144.3 (C-4); 147.4 (q, C-2, J = 33.5 Hz); 148.5 (C-1’); 150.8 (C-3’, C-5’). 19F-NMR (376 MHZ;
DMSO-d6, ppm): -58.84 (s, 3F, F-10); - 66.98 (s, 3F, F-9). HRMS: calculated for C16H10F6N3: 358.0773, found:
358.0771.
N-(pyrimidin-2-yl)-2,8-bis(trifluoromethyl)quinolin-4-amine (3)
Yellow solid; 87 % yield; m.p 118-120 °C; GC-MS m/z (%): 357 (100); 358 (76); 289 (59); 278 (5);
79 (6); 1H NMR (400 MHz, DMSO-d6, ppm): δ 7.20 (t, 1H, H-4’, J = 4.84 Hz); 7.83(t, 1H, H-6, J = 8 Hz); 7.30
(d, 1H, H-5, J = 7.2 Hz); 8.76 (d, 2H, H-3’, H-5’, J = 4.8 Hz); 8.90 (s, 1H, H-3); 9.00(d, 1H, H-7, J = 8,5 Hz);
9.98 (1H, N-H). 13C NMR (100 MHz, DMSO-d6, ppm): δ 104.6 (C-3); 115.6 (C-4’); 121.1 (C-4a); 121.4 (q, C-

9, J = 272 Hz); 122.4 (q, C-10, J = 274 Hz); 126.1 (C-6); 126.7 (q, C-8, J = 29 Hz); 127,9 (C-5); 129.5 (q, C-7,
J = 9.1 Hz); 143.9 (C-4); 146.4 (C-8a); 147.4 (q, C-2, J = 33.3 Hz); 158.4 (C-3’; C-5’); 159.3 (C-1’). 19F-NMR
(376 MHZ; DMSO-d6, ppm): -58.76 (s, 3F, F-10), - 66.83 (s, 3F, F-9); FTIR (KBr, cm−1): 3448 ( NH), 3236
( NH), 2923 ( CH), 1591 ( C=C), 1501-1533 ( NH), 1340-1252 ( C-N), 1189, 1113 e 994 ( CF), 756 (
CF3). HRMS: calculated for C15H9F6N4: 359,2412, found: 359.2410.
N-cyclohexyl-2,8-bis(trifluoromethyl)quinolin-4-amine (4)
Yellow solid; 79 % yield; m.p 84-85 °C; GC-MS m/z (%): 362 (68); 343 (15); 319 (100); 299 (18);
236 (10); 1H NMR (400 MHz, DMSO-d6, ppm): δ 1.41- 1.43 (ms, 10H, H-2’, H-3’, H-4’, H-5’, H-6’); 1.78 (bs,
1H, H-1’); 6.91 (s, 1H, H-3); 7.56 (d, 1H, NH, J = 7.8 Hz); 7.66 (t, 1H, H-6, J = 7.9 Hz); 8.14 (d, 1H, H-5, J =
7.2 Hz); 8.72 (d, 1H, H-7, J = 8.4 Hz); 13C NMR (100 MHz, DMSO-d6, ppm): δ 24.5 (C-4’); 25.2 (C-3’ C-5’);
31.5 (C-2’ C-6’); 51.8 (C-1’); 94.2 (C-3); 119.3 (C-4a); 120.9 (q, C-9, J = 263 Hz); 123.3 (q, C-10, J = 268
Hz); 124.1 (C-6); 126.3 (q, C-8, J= 28 Hz); 127.2 (C-5); 128.8 (q, C-7, J = 5 Hz); 144.0 (C-4); 147.8 (q, C-2, J
= 32.5 Hz); 151.2 (C-8a), 19F-NMR (376 MHZ; DMSO-d6, ppm): - 58.88 (s, 3F; F-10); -66.96 (s, 3F; F-9).
HRMS: calculated for C17H17F6N2: 363.3127, found: 363.3129.
N-phenyl-2,8-bis(trifluoromethyl)quinolin-4-amine (5)
Brown solid; 78 % yield; m.p 94-96 °C; GC-MS m/z (%): 356 (100); 335 (10); 288 (75); 217 (1); 1H
NMR (500 MHz, DMSO-d6, ppm): δ 7.07 (s, 1H, H-3), 7.32 (t, 1H, H-4’, J = 10 Hz), 7.44 (d, 2H, H-2’;H-6’, J
= 10 Hz), 7.52 (t, 2H, H-3’, H-5’, J = 10 Hz), 7.79 (t, 1H, H-6, J = 5.0 Hz), 8.25 (d, 1H, H-5, J = 5.0 Hz), 8.80
(d, 1H, H-7, J = 5.0 Hz) 9,79 (bs, 1H, NH); 13C NMR (100 MHz, DMSO-d6, ppm): δ 96.5 (C-3), 114.8 (C-4’),
120.0 (C-3’; C-5’), 121.4 (q, C-9, J = 273 Hz), 123.9 (q, C-10, J = 290 Hz), 125.3 (C-2’, C-6’), 125.8 (C-6),
126.5 (q, C-8, J = 28.7 Hz), 127.3 (C-5), 129.6 (q, C-7, J = 5.0 Hz), 130.1 (C-1’); 138.8 (C-4a), 144.3 (C-4),
147.6 (q, C-2, J = 33.7 Hz), 151.2 (C-8a). 19F-NMR (376 MHZ; DMSO-d6, ppm): -58.76 (s, 3F, F-10), - 66.83
(s, 3F, F-9); FTIR (KBr, cm−1): 3450 ( NH), 1580 ( C=C), 1502-1532 ( NH), 1363 ( C=N), 1315 ( C=N),
1106, 1132 e 1155 ( CF), 722 ( CF3); HRMS: calculated for C17H11F6N2: 357.0820, found: 357.0827.
N-(naphthalen-2-yl)-2,8-bis(trifluoromethyl)quinolin-4-amine (6)
1
White solid; 49 % yield; m.p 124-126 oC; GC-MS m/z (%): 406 (100), 336(36); H NMR (400 MHz,
DMSO-d6, ppm): δ 6.35 (s, 1H, H-3); 7.53 (t, 1H, H-3’, J = 7.1Hz); 7.59-7.70 (ms, 3H, H-6’, H-7’, H-2’); 7.84
(t, 1H, H-6, J = 7.9 Hz); 7.89 (d, 1H, H-4’, J = 8.3 Hz); 8.03 (d, 1H, H-5’, J = 7.6 Hz); 8.06 (d, 1H, H-8’, J =
8.1 Hz); 8.27 (d, 1H, H-5, J = 7.2 Hz); 9.01 (d, 1H, H-7, J = 8.4 Hz); 10.05 (bs, 1H, N-H); 13C NMR (100 MHz,
DMSO-d6, ppm): δ 96.4 (C-3); 119.6 (C-4a’); 122.6 (C-8a’); 122.7 (q, C-9, J = 263 Hz); 124.8 (q, C-10, J =
263 Hz); 125.3 (C-5’); 126.3 (C-3’); 126.40 (C-6); 126.7 (q, C-8, J = 28 Hz), 127.3-127.8 (C-6’, C-7’), 128.6
(C-2’), 129.4 (C-5), 129.5 (q, C-7, J = 5.1 Hz); 134.4-134.4 (C-4’, C-8’); 138.6 (C-4a); 144.1 (C-4); 147.9 (q,
C-2, J = 33.2 Hz); 152.9 (C-8a); 19F-NMR (376 MHZ; DMSO-d6, ppm): δ – 58.90 (s, 3F, F-10); - 67.38 (s, 3F,
F-9). HRMS: calculated for C21H13F6N2: 407.0977, found: 407.0961.
2,8-bis(trifluoromethyl)-N-(2-(trifluoromethyl)phenyl)quinolin-4-amine (7)
o
White solid; 76 % yield; m.p. 119-120 C; GC-MS m/z (%): 424 (100); 385 (31); 354 (59); 335
(25);315 (22); 1H NMR (400 MHz, DMSO-d6, ppm): δ 6.29 (s, 1H, H-3); 7.76 (ms, 2H, H-5’; H-6’, J = 7 Hz);
7.84 (t, 1H, H-4’, J = 8 Hz); 7.92 (t, 1-H, H-6, J = 7.5 Hz); 8.00 (d, 1H, H-3’, J = 8 Hz); 8.29 (d, 1H, H-5, J =

7.0 Hz); 8.86 (d, 1H, H-7, J = 8.5 Hz); 9.77 (bs, 1H, NH); 13C NMR (100 MHz, DMSO-d6, ppm): δ 96.7 (C-3);
122.3 (q, C-9, J = 273 Hz); 122.7 (q, C-7’ J = 236 Hz); 124.4 (q, C-10, J = 276 Hz), 125.6 (C-4’); 126.4 (C-6);
127.0 (q, C-8, J = 28.7 Hz); 128.2 (q, C-3’, J = 5.1 Hz); 128.8 (C-6’); 129.6 (q, C-7, J = 5.0 Hz); 131.4 (C-5);
134.7 (C-5’); 136.4 (q, C-2’, J = 31.2); 139.3 (C-4a); 143.9 (C-4); 147.7 (q, C-2, J = 33.1 Hz); 151.3 (C-1’);
152.9 (C-8a); 19F-NMR (376 MHZ; DMSO-d6, ppm): δ -58.96 (C-7’); -59.76 (C-10); -67.34 (C-9); FTIR (KBr,
cm−1): 3477 ( NH); 1584 ( C=C); 1506-1541 ( NH); 1310-1334 ( CF3); 1179; 1134 and 1118 ( CF); 758
( CF3). HRMS: calculated for C18H10F9N2: 425.0694, found: 425.0690.
N-(4-fluorophenyl)-2,8-bis(trifluoromethyl)quinolin-4-amine (8)
1
Brown solid; 87 % yield; m.p 121-122 °C; GC-MS m/z (%): 372 (100); 108 (62); H NMR (400
MHz, DMSO-d6, ppm): δ 6.97 (s, 1H, H-3); 7.34-7.40 (ms, H-2’; H-6’); 7.46-7.51 (ms, H-3’; H-5’), 7.80 (t, H-
6, J = 7,9 Hz), 8.25 (d, H-5, J = 7.2 Hz), 8.79 (d, H-7, J = 8.4 Hz); 9.75 (bs, 1H, NH); 13C NMR (100 MHz,
DMSO-d6, ppm): δ 96.2 (C-3); 116.7 (d, C-3’; C5’, J = 29 Hz); 119.8 (q, C-9, J = 273 Hz); 122.7 (q, C-10, J =
276 Hz); 125.2 (C-6); 126.5 (d, C-2’; C-6’ J = 8.0 Hz); 127.2 (q, C-8, J = 29 Hz); 129.4 (q, C-7, J = 5.4 Hz);
130.1 (C-5); 135.0 (C-4a); 144.2 (C-4); 147.4 (q, C-2, J = 32.9 Hz); 151.5 (C-8a ); 158.6 (C-1’); 161.03 (C-4’);
19F-NMR (376 MHZ; DMSO-d6, ppm): 58.94 (s, 3F, F-10); - 67.29 (s, 3F, F-9); -116.11 (s, 1F, F-4’); FTIR
(KBr, cm−1): 3299 ( NH); 1607 ( C=C);  1509-1547 ( NH); 1360 ( C-N); 1311 ( C-N); 1114, 1136 and
1136 ( CF); 729 ( CF3). HRMS: calculated for C17H10F7N2: 375.0726, found: 375.0717.
4-((2,8-bis(trifluoromethyl)quinolin-4-yl)amino)phenol (9)
Brown solid; 77 % yield; m.p 121-122 °C; GC-MS m/z (%):372 (100), 108 (62); 1H NMR (400 MHz,
DMSO-d6, ppm): δ 2.50 (bs, 1H, OH or NH); 5.30 (bs, 1H, NH or OH); 6.72 (d, 2H, H-2’;3’, J = 8,7 Hz); 6.86
(s, 1H, H-3); 7.05 (d, 2H, H-5’;6’, J = 8.7 Hz); 7.94 (t, 1H, H-6, J = 7.9 Hz); 8.40 (d, 1H, H-5, J = 7.2 Hz); 8.73
(d, 1H, H-7, J = 8.4 Hz); 13C NMR (100 MHz, DMSO-d6, ppm): δ 99.7 (C-3); 114.6 (C-3’); 119.5 (C-5’); 120.8
(q, C-9, J = 273 Hz); 121.5 (C-4’); 122.2 (q, C-10, J = 276 Hz); 126.2 (C-2’); 126.5 (C- 6’); 126.9 (C-6); 127.5
(q, C-8, J = 58 Hz); 127.7 (C-5); 130.5 (q, C-7, J = 5.0 Hz); 142.4 (C-4a); 144.0 (C-4); 147.4 (C-1’); 148.1 (q,
C-2, J = 33 Hz); 164.8 (C-8a); 19F-NMR (376 MHZ; DMSO-d6, ppm): - 58.91 (s, 3F, F-10); - 67.00 (s, 3F, F-
9); FTIR (KBr, cm−1): 3445 ( OH); 3369 ( NH); 1624 ( C=C); 1538-1580 ( NH); 1369 ( C-N); 1312 (
C-N); 1105; 1119 and 1142  CF); 722 ( CF3); HRMS: calculated for C17H11F6N2O: 373.0770, found:
373.0772.
N-(3,4-dimethoxyphenyl)-2,8-bis(trifluoromethyl)quinolin-4-amine (10)
Brown solid; 88 % yield; m.p 180-181 °C; GC-MS m/z (%):416 (100); 417 (20); 401 (80); 385 (16);
1H NMR (400 MHz, DMSO-d6, ppm): δ 3.76 (s, 3H, H4’); 3.81 (s, 3H, H3’); 6.94 (s, 1H, H-3), 6.96 (dd, 1H,
H6’, J = 2.3 Hz, J= 8.5 Hz); 7.02 (d, 1H, H2’, J = 2.3 Hz); 7,09 (d, 1H, H-5’, J = 8.5 Hz), 7.77 (t, 1H, H-6, J =
7.8 Hz), 8.24 (d, 1H, H-5, J = 7.1 Hz), 8.80 (d, 1H, H-7, J = 8.4 Hz); 9.65 (bs, 1H, NH); 13C NMR (100 MHz,
DMSO-d6, ppm): δ 55.6 (CH3); 96.1 (C-3); 122.8 (q, C-9, J = 263.6 Hz); 125.0 (q, C-10, J = 273.5 Hz); 125.5
(C-6); 126.6 (q, C-8, J = 58 Hz); 127.1 (C-5); 129.3 (q, C-7, J = 5.0 Hz); 131.4 (C-1’); 140.0 (C-4a); 143.4 (C-
5’); 144.2 (C-4); 146.0 (C-2’); 147.7 (q, C-2, J = 31 Hz); 149.5 (C-4’); 151.2 (C-8a); 151.9 (C-3’); 19F-NMR
(376 MHZ; DMSO-d6, ppm): -58.92 (s, 3F, F-10); - 67.29 (s, 3F, F-9). HRMS: calculated for C19H15F6N2O2:
417.1032, found: 417.1021.

N-(4H-1,2,4-triazol-3-yl)-2,8-bis(trifluoromethyl)quinolin-4-amine (11)
White solid; 67 % yield; m.p 150-152 °C; GC-MS m/z (%):347 (100), 305 (88), 278 (57), 251 (1); 1H
NMR (500 MHz, DMSO-d6, ppm): δ 6.14 (bs, 2H, NH); 7.97 (t, 1H, H-6, J = 7.9 Hz); 8,32 (s, 1H, H-3); 8.42
(d, 1H, H-5, J = 7.2 Hz); 9,01 (s, 1H, H-5’); 9.14 (d, 1H, H-7, J = 8.7 Hz); 13C NMR (125 MHz, DMSO-d6,
ppm): δ 110.3 (C-3); 119.7 (q, C-9, J = 273 Hz); 122.5 (q, C-10, J = 276 Hz); 126.5 (q, C-8, J = 30 Hz); 128.1
(C-6), 130.2 (C-5), 130.5 (q, C-7, J = 5.0 Hz), 143.2 (C-4a), 144.4 (C-4), 147.4 (q, C-2, J = 35 Hz), 151.5 (C-
5’), 156.5 (C-8a), 165.7 (C-3’). 19F-NMR (376 MHZ; DMSO-d6, ppm): -60.29 (s, 3F; F-10); -67.82 (s, 3F; F-
9); FTIR (KBr, cm−1): 3409 ( OH); 3334 ( NH); 3097 ( NH); 1628 ( C=C); 1521-1585 ( NH); 1376 ( C-
N); 1309 ( C-N); 1101; 1102; 1131  CF); 725 ( CF3); HRMS: calculated for C13H8F6N5: 348.2186, found:
348.2189.
N-(5-methyl-4H-1,2,4-triazol-3-yl)-2,8-bis(trifluoromethyl)quinolin-4-amine (12)
White solid; 67 % yield; m.p 172-173 °C; GC-MS m/z (%):361 (100), 342 (22), 278 (78), 251 (32);
1H NMR (500 MHz, MeOD, ppm): δ 2.34 (s, 3H, CH3); 3.31 (bs, 2H, NH); 7.92 (t, 1H, H-6, J = 7.5 Hz); 8.18
(s, 1H, H-3); 8.25 (d, 1H, H-5, J = 8.5 Hz); 8.36 (d, 1H, H-7, J = 7.5 Hz); 13C NMR (125 MHz, MeOD, ppm): δ
12.5 (CH3); 117.0 (C-3); 121.4 (q, C-9, J = 268 Hz); 123.4 (q, C-10, J = 273 Hz); 126.1 (C-6); 126.9 (q, C-8, J
= 30 Hz); 129.9 (C-5); 130.2 (q, C-7, J = 5 Hz); 145.2 (C-4); 146.3 (C-4a); 150.1 (q, C-2, J = 33.5 Hz); 151.2
(C-8a); 156.0 (C-3’); 165.4 (C-5’); 19F-NMR (376 MHZ; MeOD, ppm): -61.61(s, 3F; F-10), -69.40 (s, 3F; F-9).
HRMS: calculated for C14H10F6N5: 362.0834, found: 362.0851.
2,8-bis(trifluoromethyl)-N-(5-(trifluoromethyl)-4H-1,2,4-triazol-3-yl)quinolin-4-amine (13)
Yellow solid; 72 % yield; m.p 167-168 °C; GC-MS m/z (%): 415 (72), 345 (32), 278 (100), 209 (1);
1H NMR (500 MHz, MeOD, ppm): δ 3.3 (bs, 2H, N-H).7.95 (t, 1H, H-6, J = 7.9 Hz); 8.16 (d, 1H, H-5, J = 8.1
Hz); 8.22 (s, 1H, H-3); 8.38 (d, 1H, H-7, J = 8.2 Hz); 13C NMR (125 MHz, MeOD, ppm): δ 117.5 (C-3), 121.4
(q, C-9, J = 268 Hz), 123.4 (q, C-10, J = 273 Hz); 126.6 (C-6); 126.9 (q, C-8, J = 29 Hz); 129.3 (C-5); 130.1 (q,
C-6’, J = 276 Hz); 131.8 (q, C-7, J = 5 Hz); 144.1 (C-4); 146.5 (C-4a); 150.2 (q, C-2, J = 33.5 Hz), 154.3 (C-
3’), 159.5 (q, C-5’, J= 38.7 Hz); 19F-NMR (376 MHZ; MeOD, ppm): -61.59 (s, 3F; F-6’); -68.09 (s, 3F; F-10),
-69.41 (s, 3F, F-9). HRMS: calculated for C14H7F9N5: 416.2165, found: 416.2168.
N-(2,8-bis(trifluoromethyl)quinolin-4-yl)-5-methyl-1,3,4-thiadiazol-2-amine (14)
White solid; 42 % yield; m.p 124-126°C; GC-MS m/z (%): 378 (100); 359 (43); 337 (39); 310 (22);
1H NMR (400 MHz, DMSO-d6, ppm): δ 2.67 (s, 3H, CH3); 7.93 (t, 1H, H-6, J = 7,8 Hz); 8.34 (d, 1H, H-5, J =
7.20 Hz); 8.87 (d, 1H, H-7, J = 8.56 Hz); 9.07 (s, 1H, H-3), 11.21 (bs, 1H, N-H).13C NMR (100 MHz, DMSO-
d6, ppm): δ 15.1 (CH3); 102.7 (C-3); 121.3 (q, C-9, J = 273 Hz); 122.5 (q, C-10, J = 276 Hz); 126.6 (C-6);
126.9 (q, C-8, J = 31,4 Hz); 127.3 (C-5); 129.6 (q, C-7, J = 5.0 Hz); 143.7 (C-4a); 146.0 (C-4); 148.0 (q, C-2, J
= 33.5 Hz); 159.2 (C-2’); 163.2 (C-5’); 19F-NMR (376 MHZ; DMSO-d6, ppm): -58.75 (s, 3F; F-10); -66.98 (s,
3F; F-9); HRMS: calculated for C14H8F6N4NaS: 401.0266, found: 401.0274.
4-(piperazin-1-yl)-2,8-bis(trifluoromethyl)quinoline (15)
Yellow solid; 56% yield; m.p 133-134 °C; GC-MS m/z (%): 350 (100), 331 (30), 392 (100), 264 (28),
1
223 (100), 196 (33); H NMR (400 MHz, DMSO-d6, ppm): δ 2.99 (s, 4H, H-2’, H-6’); 3.27 (s, 4H, H-3’, H-
5’); 3.37 (bs, 1H, N-H); 7.36 (s, 1H, H-3); 7.80 (t, 1H, H-6, J = 7.8 Hz), 8.26 (d, 1H, H-5, J = 7.2 Hz), 8.37 (d,

1H, H-7, J = 8.4 Hz); 13C NMR (100 MHz, DMSO-d6, ppm): δ 45.3 (C-2’, C- 6’); 53.2 (C-3’C-5’); 104.6 (C-
3), 121.3 (q, C-10, J = 273 Hz); 122.7 (q, C-9, J = 276 Hz); 123.4 (C-4a); 125.8 (C-6); 126.9 (q, C-8, J = 28.5
Hz), 127.8 (C-5); 129.3 (q, C-7, J = 5.0 Hz); 139.2 (C-4a); 144.4 (C-4); 147.5 (q, C-2, J = 33.7 Hz); 159.4 (C-
8a); 19F-NMR (376 MHZ; DMSO-d6, ppm): δ 58.76 (s, 3F, F-10); - 66.65 (s, 3F, F-9); FTIR (KBr, cm−1):
3073 ( NH); 2984, 2954 ( C-H); 1591 ( C-N); 1299 ( CH2); 1033 ( C-N); 1097, 1112 and 1182( CF);
724 ( CF3; HRMS: calculated for C15H14F6N3: 350.1086, found: 350.1083.
4-(2,8-bis(trifluoromethyl)quinolin-4-yl)morpholine (16)
Yellow solid; 69% yield; m.p 198-200 °C; GC/MS m/z (%):349 (40); 330 (13); 307 (100); 238 (25);
1H NMR (400 MHz, DMSO-d6, ppm): δ 3.36 (t, 4H, C-3’, C-5’ J = 4.3 Hz); 3.91 (s, 4H, C-2’, C-6’ J = 4.5
Hz); 7.42 (s, 1H, H-3); 7.80 (t, 1H, H-6, J = 7.9 Hz); 8.27 (d, 1H, H-5, J = 7.2 Hz); 8.42 (d, 1H, H-7, J = 8.5
Hz); 13C NMR (100 MHz, DMSO-d6, ppm): δ 52.1 (C-3’, C-5’); 65.8 (C-2’, C-6’); 105.0 (C-3); 122.6 (q, C-
10, J = 276 Hz); 122.4 (q, C-9, J = 273 Hz); 123.4 (C-4a); 126.2 (C-6); 126,9 (q, C-8, J = 28.7 Hz); 129.3 (C-
5); 129.4 (q, C-7, J = 5.0 Hz); 144.3 (C-4); 147.6 (q, C-2, J = 33.0 Hz); 158.9 (C-8a); 19F-NMR (376 MHZ;
DMSO-d6, ppm): δ – 58.75 (s, 3F; F-10); -66.63 (s, 3F; F-9); FTIR (KBr, cm−1): 2952 ( NH); 2813 ( NH);
1590 ( NH); 1112 ( CH); 1097, 1112 and 1129 ( CF ); 724 ( CF3); HRMS: calculated for C15H12F6N2NaO:
373.0746, found: 373.0751.
7-chloro-N-(pyridin-4-yl)quinolin-4-amine (17)
Yellow solid; 89 % yield; m.p 157-158°C; 1H NMR (400 MHz, DMSO-d6, ppm): δ 7.26 (d, 2H, H-2’,
H-6’, J = 6.2 Hz); 7.38 (d, 1H, H-3, J = 5.2 Hz); 7.64 (d, 1H, H-6, J = 9.0 Hz); 7.98 (s, H-8); 8.34 (d, 1H, H-5,
3 J = 9.2 Hz); 8.40 (d, 2H, H-3’, H-5’, J = 6.2 Hz); 8.66 (d, 1H, H-2, 3 J = 5.2 Hz); 13C NMR (125 MHz,
MeOD, ppm): δ 106.6 (C-2’, C-6’); 112.9 (C-3); 119.7 (C-4a); 124.7 (C-6); 125.8 (C-5); 127.8 (C-8); 134.3 (C-
7); 145.0 (C-3’, C-5’); 148.3 (C-4); 149.6 (C-8a); 150.5 (C-2); 152.0 (C-1’); HRMS: calculated for
C14H11ClN3: 256.0636, found: 256.0624.
4.2 Biological evaluation
4.2.1 Continuous cultures and in vitro assays with P. falciparum-infected erythrocytes.
A CQ-resistant and MQ-sensitive P. falciparum clone W240 was cultivated as described41 in human
erythrocytes (A+) in complete medium (RPMI 1640 supplemented with 1% ALBUMAX II), and the tests
followed a protocol42 that was slightly modified as follows. Human erythrocytes (A+) were kindly donated by
the
Center
of
Hemotherapy
and
Hematology
of
Minas
Gerais
(HEMOMINAS-
http://www.hemominas.mg.gov.br) under the mutual cooperation term number 18/09.
The molecules were diluted in dimethyl sulfoxide (DMSO) (Sigma-Aldrich) to obtain a stock solution
of 10 mg/mL and further diluted to the specified concentrations with RPMI 1640 medium supplemented with
25 mM Hepes, 21 mM sodium bicarbonate, 11 mM glucose, 2% glutamine (Sigma-Aldrich), and 40 mg/L
gentamicin (Schering-Plough, Kenilworth, New Jersey, USA) to a final concentration of 0.02% DMSO for the
assays against P. falciparum.
The sorbitol-synchronized ring-stage parasites43 were adjusted for parasitemia and hematocrit,
according to the specifications of the test, and then distributed in 96-well microtiter plates (Corning, Santa
Clara, CA, USA). The plates had been prepared previously and contained dilutions of the test compounds (20
µL/ well). Triplicate tests were used for each dose. CQ, MQ and AQ, the standard antimalarials, were tested in

parallel each time. The drug activity was determined in comparison to untreated parasite cultures in complete
medium, as described previously.44 Parasite growth was measured through the anti-HRPII test45 in cultures
adjusted to 1.5% hematocrit and 0.05% parasitemia with monoclonal antibodies (MPFM-55A and MPFG-55P)
that were commercially acquired (ICLLABH, USA) and TMB chromogen (3,39,5,59-tetramethylbenzidine),
which was purchased from KPL (Gaithersburg, MD, USA). The reaction was stopped with the addition of 50
L/L of 1 M sulfuric acid, and the absorbance of each well was read at 450 nm using a spectrophotometer
(SpectraMax340PC384, Molecular Devices).
Sigmoid dose-response curves were generated with curve-fitting software (Microcal Origin Software
5.0, Inc.) and were used to determine the 50% inhibitory concentrations of parasite growth (IC50 values). The
activities were calculated by comparing the growth in the drug-exposed cultures and the drug-free control
cultures.
4.2.2 Cell Cultures and Cytotoxicity Tests
The monkey kidney cell line (BGM) was cultured in 75-cm2 sterile flasks with RPMI 1640
supplemented with 10% heat-inactivated fetal serum and 40 mg/L gentamicin in a 5% CO2 atmosphere at 37
°C. For the in vitro cytotoxicity experiments, the cell monolayer was trypsinized, washed with culture medium,
distributed in a flat-bottomed 96-well plate (5 × 103 cells/well), and finally incubated for 18 h at 37 °C to
achieve cell adherence.
For cytotoxicity testing, we used the (3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide
(MTT) assay described in the literature46. The BGM cells were incubated with 20 μL of the test compounds at
different concentrations (200–25 μg/mL) for 24 h under an atmosphere of 5% CO2 at 37°C. For the molecules
dilution a stock solution of 20 mg/mL in DMSO was made. Next, the compounds were solubilized in culture
medium RPMI 1640 supplemented with 10% heat-inactivated fetal serum.
For the MTT assay, which evaluates mitochondrial viability, 20 μL of MTT solution (5 mg/mL) was
added, and the plates were incubated for an additional 3 h. After incubation, the supernatant was carefully
removed from the wells, and 100 μL of DMSO was added and mixed thoroughly. Optical densities at 570 and
630 nm (background) were determined by an ELISA reader. Cell viability is expressed as the percentage of the
control absorbance obtained in untreated cells after subtracting the absorbance from the appropriate
background. Finally, half maximal effective concentration (EC50) was determined as previously described.47
The ratio between the EC50 and IC50 values was used to determine the selectivity index (SI).
4.2.3 P. berghei and antimalarial tests in mice
The suppressive test was performed, as described.48 The P. berghei NK65 strain used was maintained
through weekly blood passages, as described.49 For these experiments, groups of up to 30 mice were inoculated
i.p. with 1 × 105 infected erythrocytes, kept together for approximately 24 h, and then randomly distributed into
groups of five per cage. The mice were treated daily for three consecutive days with the compounds freshly
diluted in 3% DMSO (Sigma-Aldrich) in RPMI medium and administered orally at 50 mg/kg; the control
groups received either the drug vehicle or the antimalarials CQ and MQ at 20 mg/kg. On days 5, 7, and 9 after
the parasite inoculation, a blood sample was taken from the tail of each mouse and used to prepare thin smears
that were methanol-fixed, Giemsa-stained, and examined microscopically (1000X) to determine parasitemia.
The inhibition of parasite growth was determined relative to parasitemia in the untreated mice, which was

considered to be 100% parasite growth. Compounds reducing parasitemia by >40% were considered active,
between 30-40% were partially active, and by less than 30% were considered inactive.
Acknowledgments
This study was financed in part by the Coordenacao de Aperfeiçoamento de Pessoal de Nivel Superior - Brasil
(CAPES) - Finance Code 001. The authors thank the National Council for Scientific and Technological
Development (CNPq) for the fellowships granted to the authors and financial support of the projects. We are
also thankful to the Foundation for Research of the State of Rio de Janeiro (FAPERJ), Coordination of
Improvement of Higher-Level Personnel (CAPES), the Foundation for Research of the State of Minas Gerais
(FAPEMIG) and the Foundation for Scientific and Technological Development in Health (FIOTEC) for their
financial support.
References and notes
1. Malaria site: all about Malaria 2018 Available online: http://www.malariasite.com/ accessed in July 2018.
2. World Health Organization (WHO) 2018 Malaria http://www.who.int/en/news-room/fact-
sheets/depril/malaria accessed in July 2018.
3. Luthi, B.; Schlagenhauf, P., Travel Medicine and Infectious Disease, 2015, 13, 48.
4. Wu T.; Nagle A.S.; Chatterjee, A.K. Curr. Med. Chem. 2011, 18, 853.
5. Kaur, K; Jain, M.; Reddy, R.P.; Jain, R. Eur. J. Med. Chem. 2010 45, 3245.
6. Oduola, A.M.; Weatherly, N.F.; Bowdre, J.H.; Desjardins, R.E. Exp. Parasitol. 1988, 66, 86.
7.
http://www.who.int/malaria/areas/drug_resistance/en/ accessed in July 2018.
8. World Health Organization 2015. Third
World Health Organization (WHO) 2018 Malaria Drug resistance and response
edition.
Available:
http://apps.who.int/iris/bitstream/handle/10665/162441/9789241549127_eng.pdf
9. Nosten, F.; White, N.J. Am. J. Trop. Med. Hyg. 2007, 77, 181.
10. Sirima, S.B.; Ogutu, B.; Lusingu, J.P.A.; Mtoro, A.; Mrango, Z.; Ouedraogo, A.; Yaro, J. B.; Onyango, K.
O.; Gesase, S.; Mnkande, E.; Ngocho, J.S.; Ackermann, I.; Aubin, F.; Vanraes, J.; Strub, N.; Carn, G. The
Lancet, 2016, 16, 1123.
11. Rosenthal, P.J. Mol. Microbiol. 2013 89, 1025.
12. Ashley, E.A.; Dhorda M.; Fairhurst, R.M.; Amaratunga, C.; Lim, P.; Suon, S.; Sreng, S.; Anderson J.M.;
Mao, S.; Sam, B.; Sopha, C.; Chuor, C.M.; Nguon, C.; Sovannaroth, S.; Pukrittayakamee, S.; Jittamala, P.;
Chotivanich, K.; Chutasmit, K.; Suchatsoonthorn, C.; Runcharoen, R.; Hien, T.T.; Thuy-Nhien, N.T.; Thanh,
N.V.; Phu, N.H.; Htut, Y.; Han, K.T.; Aye, K.H.; Mokuolu, O.A.; Olaosebikan, R.R.; Folaranmi, O.O.;
Mayxay, M.; Khanthavong, M.; Hongvanthong, B.; Newton, P.N.; Onyamboko, M.A.; Fanello, C.I.; Tshefu,
A.K.; Mishra, N.; Valecha, N.; Phyo, A.P.; Nosten, F.; Yi, P.; Tripura, R.; Borrmann, S.; Bashraheil, M.;
Peshu, J.; Faiz, M.A.; Ghose, A.; Hossain, M.A.; Samad, R.; Rahman, M.R.; Hasan, M.M.; Islam, A.; Miotto,
O.; Amato, R.; MacInnis, B.; Stalker, J.; Kwiatkowski, D.P.; Bozdech, Z.; Jeeyapant, A.; Cheah, P.Y.;
Sakulthaew, T.; Chalk, J.; Intharabut, B.; Silamut, K.; Lee, S.J.; Vihokhern, B.; Kunasol, C.; Imwong, M.;
Tarning, J.; Taylor, W.J.; Yeung, S.; Woodrow, C.J. Flegg, J.A.; Das, D.; Smith, J.; Venkatesan, M.; Plowe,
C.V.; Stepniewska, K.; Guerin, P.J.; Dondorp, A.M.; Day, N.P.; White, N.J.; Tracking Resistance to
Artemisinin Collaboration (TRAC), N. Engl. J. Med. 2014. 371, 411.
13. Miotto, O.; Almagro-Garcia, J.; Manske, M.; Macinnis, B.; Campino, S.; Rockett, K.A.; Amaratunga, C.;
Lim, P.; Suon, S.; Sreng, S.; Anderson, J.M.; Duong, S.; Nguon, C.; Chuor, C.M.; Saunders, D.; Se, Y.; Lon,

C.; Fukuda, M.M.; Amenga-Etego, L.; Hodgson, A.V.; Asoala, V.; Imwong, M.; Takala-Harrison, S.; Nosten,
F.; Su, X.Z.; Ringwald, P.; Ariey, F.; Dolecek, C.; Hien, T.T.; Boni, M.F.; Thai, C.Q.; Amambua-Ngwa, A.;
Conway, D.J.; Djimdé, A.A.; Doumbo, O.K.; Zongo, I.; Ouedraogo, J.B.; Alcock, D.; Drury, E.; Auburn, S.;
Koch, O.; Sanders, M.; Hubbart, C.; Maslen, G.; Ruano-Rubio, V.; Jyothi, D.; Miles, A.; O'Brien, J.; Gamble,
C.; Oyola, S.O.; Rayner, J.C.; Newbold, C.I.; Berriman, M.; Spencer, C.C.; McVean, G.; Day, N.P.; White,
N.J.; Bethell, D.; Dondorp, A.M.; Plowe, C.V.; Fairhurst, R.M.; Kwiatkowski, D.P. Nat. Genet. 2013, 45, 648.
14. Takala-Harrison, S.; Jacob, C.G.; Arze, C.; Cummings, M.P.; Silva, J.C.; Dondorp, A. M.; Fukuda, M. M.;
Hien, T.T.; Mayxay, M.; Noedl, H.; Nosten, F.; Kyaw, M.P.; Nhien, N.T.; Imwong, M.; Bethell, D.; Se, Y.;
Lon, C.; Tyner, S.D.; Saunders, D.L.; Ariey, F.; Mercereau-Puijalon, O.; Menard, D.; Newton, P.N.;
Khanthavong, M.; Hongvanthong, B.; Starzengruber, P.; Fuehrer, H.P.; Swoboda, P.; Khan, W.A.; Phyo, A.P.;
Nyunt, M.M.; Nyunt, M.H.; Brown, T.S.; Adams, M.; Pepin, C.S.; Bailey, J.; Tan, J.C.; Ferdig, M.T.; Clark,
T.G.; Miotto, O.; MacInnis, B.; Kwiatkowski, D.P.; White, N.J.; Ringwald, P.; Plowe, C.V. J. Infect. Dis. 2015,
211, 670.
15. Ménard, D.; Khim, N.; Beghain, J.; Adegnika, A.A.; Shafiul-Alam, M.; Amodu, O.; Rahim-Awab, G.;
Barnadas, C.; Berry, A.; Boum, Y.; Bustos, M.D.; Cao, J.; Chen, J.H.; Collet, L.; Cui, L.; Thakur, G.D.; Dieye,
A.; Djallé, D.; Dorkenoo, M.A.; Eboumbou-Moukoko, C.E.; Espino, F.E.; Fandeur, T.; Ferreira-da-Cruz, M.F.;
Fola, A.A.; Fuehrer, H.P.; Hassan, A.M.; Herrera, S.; Hongvanthong, B.; Houzé, S.; Ibrahim, M.L.; Jahirul-
Karim, M.; Jiang, L.; Kano, S.; Ali-Khan, W.; Khanthavong, M.; Kremsner, P.G.; Lacerda, M.; Leang, R.;
Leelawong, M.; Li, M.; Lin, K.; Mazarati, J.B.; Ménard, S.; Morlais, I.; Muhindo-Mavoko, H.; Musset, L.; Na-
Bangchang, K.; Nambozi, M.; Niaré, K.; Noedl, H.; Ouédraogo, J.B.; Pillai, D.R.; Pradines, B.; Quang-Phuc,
B.; Ramharter, M.; Randrianarivelojosia, M.; Sattabongkot, J.; Sheikh-Omar, A.; Silué, K.D.; Sirima, S.B.;
Sutherland, C.; Syafruddin, D.; Tahar, R.; Tang, L.H.; Touré, O.A.; Tshibangu-wa-Tshibangu, P.; Vigan-
Womas, I.; Warsame, M.; Wini, L.; Zakeri, S.; Kim, S.; Eam, R.; Berne, L.; Khean, C.; Chy, S.; Ken, M.;
Loch, K.; Canier, L.; Duru, V.; Legrand, E.; Barale, J.C.; Stokes, B.; Straimer, J.; Witkowski. B.; Fidock, D.A.;
Rogier, C.; Ringwald, P.; Ariey, F.; Mercereau-Puijalon, O.; KARMA Consortium. N. Engl. J. Med. 2016, 374,
2453.
16. Cortopassi, W.A.; Franca, T.C.C.; Krettli, A.U. Expert. Opin. Drug. Discov. 2018, 13, 617.
17. Viegas-Junior, C.; Danuello, A.; Bolzani, V.S.; Barreiro, E.J.; Fraga, C.A.M. Curr. Med. Chem. 2007, 14,
1829.
18. Aminake, M.N.; Mahajan, A.; Kumar, V.; Hans, R.; Wiesner, L.; Taylor, D.; Kock, C.; Grobler, A.; Smith,
P.J.; Kirschner, M.; Rethwilm, A.; Pradel, G.; Chibale, K. Bioorg. Med. Chem., 2012, 20, 5277.
19. Walsh, J.J.; Coughlan, D.; Heneghan, N.; Gaynora, C.; Bell, A. Bioorg. Med. Chem. Lett., 2007, 17, 3599.
20. Varotti, F.P.; Botelho, A.C.C.; Andrade, A.A.; Paula, R.C.; Fagundes, E.M.S.; Valverde, A.; Mayer, L.M.
U.; Mendonca, J.S.; Souza, M.V.N.; Boechat, N.; Krettli, A.U. Antimicrob. Agents Chemother. 2008, 52, 3868.
21. Boechat, N.; Souza, M.V.N.; Valverde, A.l.; Krettli, A.U. US Patent 8802701B2, 2014.
22. Boechat, N.; Pinheiro, L.C.S.; Silva, T.S.; Aguiar, A.C.; Carvalho, A.S.; Bastos, M.M.; Costa, C.C.P.;
Pinheiro, S.; Pinto, A.C.; Mendonça, J.S.; Dutra, K.D.B.; Valverde, A.L.; Santos-Filho, O.A.; Krettli, A.U.
Molecules, 2012, 17, 8285.
23. Boechat, N.; Ferreira, M.L.G.; Pinheiro, L.C.S.; Jesus, A.M.L.; Leite, M.M.M.; Junior, C.C.S.; Aguiar,
A.C.C.; de Andrade, I.M.; Krettli, A.U. Chem. Biol. Drug Des. 2014, 84, 325
24. Pinheiro, L.C.S.; Boechat, N.; Ferreira, M.L.G.; Junior, C.C.S.; Jesus, A.M.L.; Leite, M.M.M.; Souza,
N.B.; Krettli, A.U. Bioorg. Med. Chem., 2015, 23, 5979.

25. Carvalho, R.C.C; Martins, W.A.; Silva, T.P.; Kaiser, C.R; Bastos, M.M.; Pinheiro, L.C.S.; Krettli, A.U.;
Boechat, N.A. Bioorg. Med. Chem. Lett. 2016, 26, 1881
26. Azeredo, L.F.S.P.; Coutinho, J.P.; Jabor, V.A.P.; Feliciano, P.R.; Nonato, M.C.; Kaiser, C.R.; Menezes,
C.M.S.; Hammes, A.S.O.; Caffarena, E.R.; Hoelz, L.V.B.; Souza, N.B.; Pereira, G.A.N.; Ceravolo, I.P.; Krettli,
A.U.; Boechat, N. Eur. J. Med. Chem. 2017, 123, 72.
27. Penna-Coutinho, J.; Almela, M.; Miguel-Blanco, C.; Herreros, E.; Sa, P.M.; Boechat, N.; Krettli, A.U.
Antimicrob. Agents Chemother. 2016, 60, 3145.
28. Silva, T.B.; Bernardino, A.M.R.; Ferreira, M.L.G.; Rogerio, K.R.; Carvalho, L.J.M.; Boechat, N.; Pinheiro,
L.C.S.; Bioorg. Med. Chem., 2016, 24, 4492.
29. Boechat, N.; Pinheiro, L.C.S.; Santos-Filho, O.A.; Silva, I.C. Molecules 2011, 16, 8083.
30. Meanwell, N. A. J. Med. Chem., 2011, 54, 2529.
31. Lutz, R.E.; Ohnmacht, C.J.; Patel, A.R.; J. Med. Chem., 1971, 14, 926.
32. Thomas, K.D.; Sumesh, E.; Pal, K.N.; Chowdhuty, I.H.; Adhikari, A.V.; Eur. J. Med. Chem., 2010, 45,
3374.
33. Rao, V.J.; Ramesh, P.; Kalyan, M.; Kavitha, P.; Kumar, D.A. Ind. J. Chem. 2012, 51B, 1411.
34. Wittlin, S.; Kuter, D.; Nsumiwa, S.; Egan, T. J.; Chibale, K. Bioorg. Med. Chem. 2013, 21, 3738.
35. Aguiar, A.C.C.; Panciera, M.; dos Santos, E.F.S.; Singh, M.K.; Garcia, M.L.; de Souza, G.E.; Nakabashi,
M.; Costa, J.L.; Garcia, C.R.S.; Oliva, G.; Correia, C.R.D.; Guido, R.V.C. Med. Chem., 2018, 61, 5547.
36. Mishra, M.; Mishra, V.K.; Kashaw, V.; Iyer, A.K.; Kashaw, S. K. Eur. J. Med. Chem. 2017, 125, 1300.
37. Kumar, S.; Saini, A.; Gut, J.; Rosenthal, P.J.; Raj, R.; Kumar, V. Eur. J. Med. Chem. 2017, 138, 993.
38. Asif, M. Mor. J. Chem. 2014, 2, 136.
39. Jarrahpour, A.; Shirvani, P.; Sinou, V.; Latour, C.; Brunel, J.M. Med. Chem. Res. 2016, 25, 149.
40. Oduola, A.M, Milhous W.K, Weatherly, N.F; Bowdre, J.H; Desjardins, R.E. Exp. Parasitol. 1988, 67, 354.
41. Trager, W, Jensen J.B. Science, 1976, 193, 673.
42. Andrade-Neto, V. F.; Goulart, M.O.F.; Filho, J.F.S.; Silva, M.J.; Pinto, M.C.F.R.; Pinto, A.V.; Zalis, M.G.;
Carvalho, L.H.; Krettli, A.U. Bioorg. Med. Chem. Lett., 2004, 14, 1145.
43. Lambros C, Vanderberg J. P. J. Parasitol. 1979, 65:418.
44. Desjardins, R.; Canfield, C.; Haynes, J.; Chulay, J. Antimicrob. Agents Chemother. 1979, 16, 710.
45. Noedl, H.; Wongsrichanalai, C.; Miller, R.; Myint, K.; Looareesuwan, S.; Sukthana, Y.; Wongchotigul, V.;
Kollaritsch, H.; Wiedermann, G.; Wernsdorfer, W. Exp. Parasitol. 2002, 102, 157.
46. Denizot, F.; Lang, R. J. Immunol. Methods. 1986, 89, 271.
47. Madureira, A.M.; Martins, A. P.; Gomes, M.; Paiva, J.; Cunha, A.P.; Rosário, V., J. Ethnopharmacol.
2002, 81, 23.
48. Peters, W.; Portus, J.H.; Robinson, B.L. Ann. Trop. Med. Parasitol. 1975, 69, 155.
49. Andrade-Neto, V.F.; Goulart, M.O.F.; Filho, J.F.S.; Silva, M.J.; Pinto, M.C.F.R.; Pinto, A.V.; Zalis, M.G.;
Carvalho, L. H.; Krettli, A. U. Bioorg. Med. Chem. Lett. 2004, 14, 1145.