Primaquine-thiazolidinones block malaria transmission and development of the liver exoerythrocytic forms

Article abstract of DOI:10.1186/s12936-017-1755-6

Background: Primaquine is an anti-malarial used to prevent Plasmodium vivax relapses and malaria transmission. However, PQ metabolites cause haemolysis in patients deficient in the enzyme glucose-6-phosphate dehydrogenase (G6PD). Fifteen PQ-thiazolidinone

Full text of DOI:10.1186/s12936-017-1755-6

et al. Malar J (2017) 16:110
DOI 10.1186/s12936-017-1755-6
Aguiar
Malaria Journal
Open Access
RESEARCH
Primaquine-thiazolidinones block
malaria transmission and development of the
liver exoerythrocytic forms
Anna Caroline C. Aguiar1,2,3, Flávio Jr. B. Figueiredo1, Patrícia D. Neuenfeldt4, Tony H. Katsuragawa5,
Bruna B. Drawanz4, Wilson Cunico4, Photini Sinnis3, Fidel Zavala3 and Antoniana U. Krettli1,2*
Abstract
Background: Primaquine is an anti-malarial used to prevent Plasmodium vivax relapses and malaria transmission.
However, PQ metabolites cause haemolysis in patients deficient in the enzyme glucose-6-phosphate dehydrogenase
(G6PD). Fifteen PQ-thiazolidinone derivatives, synthesized through one-post reactions from primaquine, areneal-
dehydes and mercaptoacetic acid, were evaluated in parallel in several biological assays, including ability to block
malaria transmission to mosquitoes.
Results: All primaquine derivatives (PQ-TZs) exhibited lower cell toxicity than primaquine; none caused haemolysis
to normal or G6PD-deficient human erythrocytes in vitro. Sera from mice pretreated with the test compounds thus
assumed to have drug metabolites, caused no in vitro haemolysis of human erythrocytes, whereas sera from mice
pretreated with primaquine did cause haemolysis. The ability of the PQ-TZs to block malaria transmission was evalu-
ated based on the oocyst production and percentage of mosquitoes infected after a blood meal in drug pre-treated
animals with experimental malaria caused by either Plasmodium gallinaceum or Plasmodium berghei; four and five
PQ-TZs significantly inhibited sporogony in avian and in rodent malaria, respectively. Selected PQ-TZs were tested for
their inhibitory activity on P. berghei liver stage development, in mice and in vitro, one compound (4m) caused a 3-day
delay in the malaria pre-patent period.
Conclusions: The compound 4m was the most promising, blocking malaria transmissions and reducing the number
of exoerythrocytic forms of P. berghei (EEFs) in hepatoma cells in vitro and in mice in vivo. The same compound also
caused a 3-day delay in the malaria pre-patent period.
Keywords: Malaria, Thiazolidinones, Sporogony, Plasmodium berghei, Plasmodium gallinaceum, Blocking malaria
transmission, Exoerythrocytic stages, sporozoites
against gametocytes; thus, it is used to block malaria
transmission in areas approaching malaria elimina-
tion and/or facing artemisinin resistance where it is
expected that transmission-blocking strategies will
help to slow down the spread of resistant parasites [4].
A single 0.75 mg/kg dose of PQ is effective as a game-
tocytocidal agent [1]. However, PQ has restrictions
because it is poorly tolerated and its metabolites cause
haemolysis in patients with glucose-6-phosphate dehy-
drogenase (G6PD) deficiency [5]. PQ is currently the
only agent approved for elimination of hypnozoites,
Background
Plasmodium vivax is the most widespread parasite
in Asia and Americas, with three billion people esti-
mated to be at risk of vivax malaria [1–3]. To improve
the malaria control programmes and decrease malaria
mortality worldwide, the World Health Organization
recommends the use of primaquine (PQ), which is
used to prevent P. vivax relapses [1]. PQ is also active
*Correspondence: akrettli@cpqrr.fiocruz.br
1 Centro de Pesquisas René Rachou-Fiocruz, Av. Augusto de Lima 1715,
Belo Horizonte, MG 30190-002, Brazil
Full list of author information is available at the end of the article
© The Author(s) 2017. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License
(http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium,
provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license,
and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/
publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

et al. Malar J (2017) 16:110
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Page 2 of 11
the dormant liver stages of P. vivax that cause the late
relapses [1].
Methods
Chemical synthesis
e discovery that PQ-imidazolidin-4-one derivatives
(Fig. 1a) exhibit transmission-blocking anti-malarial
activity [6] has led to the exploration of the aliphatic ter-
minal amino group of PQ as a precursor to the synthe-
sis of thiazolidinones (TZ) (Fig. 1b). e thiazolidinone
heterocyclic class has wide applications in the medicinal
chemistry field [7] with some thiazolidinone and thiazi-
nanone-chloroquine analogs displaying higher activity
against Plasmodium falciparum than chloroquine (CQ)
[8].
e target compounds (Fig. 2) were prepared as previ-
ously described [9]. Briefly, the 2-aryl-3-[4-(6-methox-
yquinolin-8-amino) pentyl]thiazolidin-4-one derivatives
(PQ-TZs) 4b-p were synthesized by multicomponent
one-pot reactions from PQ diphosphate 1, N,N-diiso-
propylethylamine (DIPEA), arenealdehydes 2b-p and
excess of the mercaptoacetic acid 3 in refluxing tolu-
ene for 2–4 h. Primaquine (PQ), used as a control in
all experiments, was primaquine bisphosphate (>98%)
from Sigma). Doses were calculated using the molecular
weight of the PQ-salt 455.34 g/mol.
Aiming to find candidates less toxic than PQ, a total of
15 PQ thiazolidinone derivatives (PQ-TZs) were synthe-
sized through modification of the amino-terminal group
[9] and evaluated for: (i) in vitro haemolytic activity
against G6PD normal or deficient erythrocytes; (ii) tox-
icity in vitro to hepatoma and kidney cells; (iii) blocking
effect on the malaria sporogonic cycle in mosquitoes and;
(iv) ability to interfere with the development of Plasmo-
dium berghei liver stages in vivo and in vitro. In all assays,
PQ was used as the control drug.
Human and animal ethical approvals
Human erythrocytes, normal or G6PD-deficien were col-
lected from blood donors under the auspices of the Ethics
Committee of “Centro de Pesquisa em Medicina Tropical
de Rondônia–CEPEM” (CAAE-007 59912.0.0000.0011;
October 9, 2012) as described in a previous publication
[10]. e written consent was obtained for each new
sample used in the present research.
Fig. 1 General structures of a PQ-imidazolidinones and b PQ-thiazolidinones (PQ-TZs)
Fig. 2 Synthetic route for PQ-TZs 4b–4p. Reaction conditions were as described [9]

et al. Malar J (2017) 16:110
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e animal studies were approved by the Ethics Com- between the MDL50 of PQ-TZ and PQ was calculated
mittee at the Instituto Osvaldo Cruz (FIOCRUZ, CEUA (PQ-TZ/PQ) for each drug.
LW-23/13), and, by the Johns Hopkins University Insti-
tutional Animal Care Committee (protocol number In vivo cytotoxicity assay
MO13H123). Mice and chickens were anaesthesized Selected compounds (4b, 4c, 4g, 4h, 4m, 4o, 4p) and
with acepromazine 2.5 mg/kg then sacrificed by cervi- PQ were administered to normal mice orally at a single
cal dislocation. e parasitaemia was monitored in each dose. Different doses were evaluated (100, 50 and 25 mg/
animal starting 2 days after parasite inoculation, to deter- kg). Animal survival was observed during the follow-
mine the malaria pre-patent period (in mice subjected ing 30 days as described [13]. When the mice developed
to sporozoite infections). e physical conditions of the malaria, they were euthanized soon after the first symp-
animals were evaluated daily. Mice were sacrificed 3 days toms appeared, as described above. e compounds were
after the beginning of the experiment for liver stage eval- diluted in RPMI with 3% of DMSO; the control group
uation; in other experiments, they were anaesthesized was treated with the same vehicle.
with acepromazine 2.5 mg/kg then sacrificed soon after
the malaria symptoms appeared, such as anaemic blood, Plasmodium berghei and Plasmodium gallinaceum
weight loss, ruffled fur, abnormal postural responses, sporogonic assays
reduced reflexes and reduced grip strength, coma, and Mice were inoculated intraperitoneally (i.p.) with 106 P.
convulsions. Infected chicken with an early infection berghei (ANKA strain) infected red blood cells. Parasitae-
were sacrificed immediately after they were used to mia was monitored using Giemsa-stained blood smear
blood-feed the mosquitoes twice in the same experiment, and examined for the presence of gametocytes. When
thus before the malaria symptoms began.
present, a blood sample was examined to determine the
presence of exflagellating gametocytes. Anopheles ste-
phensi mosquitoes were blood-feed from mice whose
In vitro cytotoxicity assay
e cytotoxicity of PQ and PQ-TZ compounds was blood showed more than two exflagellating centres per
assessed against cultures of: (i) a human hepatoma cell field. e same infected mice were used twice, at time
line (HepG2), received from the New University of Lis- 0 h, before drug administration (control), and 2 h after
bon; (ii) a mouse hepatocyte cell line (HEPA 1-6), pur- receiving the drug by gavage. e drug was adminis-
chased from ATCC; and (iii) a kidney cell line from tered right after the first feeding. e control and experi-
Buffalo Green Monkey Kidney (BGM) kindly provided mental mosquito groups were dissected 12 days after
by the Federal University of Minas Gerais. Cells were cul- the blood meal in mice. Midguts were removed, stained
tured in 75-cm2 sterile flasks in RPMI 1640 supplemented with 0.5% mercurochrome, and oocysts were counted by
with 10% heat-inactivated fetal calf serum and 40 mg/L light microscopy. Inhibition of sporogony was calculated
gentamicin, under 5% CO2 at 37 °C. e confluent cell based on the oocyst numbers in the control mosquitoes
monolayer was trypsinized, distributed in a flat-bot- (0 h) considered as 100% infection, and in the test mos-
tomed 96-well plate (5 × 103 cells/well), then incubated quitoes blood-fed 2 h after drug treatment.
for 18 h at 37 °C for cell adherence, as previously
Chickens (1 week old) were inoculated with P. gal-
described [11]. e compounds were diluted in RPMI linaceum-infected erythrocytes. When parasitaemia
at a final concentration of 0.2% DMSO, and 20 µL were was increasing but still below 10% they were fed-upon
added at various concentrations up to 1000 µg/ML. e by Aedes fluviatilis mosquitoes, which were dissected
same diluent solution (RPMI + 1% DMSO) was used as a at days 6–7 after the infectious blood meal. e control
control in the non-treated cells. Cells were incubated for mosquito group was exposed to the infected chickens
24 h under 5% CO2 at 37 °C, then incubated with MTT before drug treatment (time 0 h); the test animals were
[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium fed by mosquitoes twice, at time 0 h (control) and 4–6 h
bromide] solution (5 mg/mL; 20 µL/well) for 3 h, in the after drug treatment, as described previously [14, 15].
same culture conditions. e supernatant was removed,
In all cases, PQ-TZ derivatives were tested at
100 µL of DMSO was added to solubilize the formazan 50–100 mg/kg while PQ, which is more toxic, was tested
crystals formed, and the optical density was determined at 15 mg/kg (non-toxic). PQ was diluted in water and the
at 570 and 630 nm (background) (SpectraMax340PC384
,
PQ-TZs were diluted in RPMI with 3% of DMSO, the
Molecular Devices). Cell viability was expressed as the control group was tread with the same vehicle.
percentage of the control absorbance in the untreated
e criteria used to evaluate the drug ability to block
cells after subtracting the appropriate background. e malaria sporogony was based on examination of 20 mos-
minimum lethal dose for 50% of the cells (MLD50) was quitoes in each group. e percentage of infected mos-
determined as previously described [12]. A toxicity ratio quitoes (prevalence) and the mean number of oocysts per

et al. Malar J (2017) 16:110
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Page 4 of 11
group (intensity) were calculated. A compound was con- sequence [17]. Parasite growth inhibition was calculated
sidered partially active when the number of oocysts and by dividing the 18S rRNA copy number of the experi-
mosquitoes infected was reduced by at least 50%, in com- mental group by that of the untreated control group.
parison to the non-treated control, and as active when
100% reduction occurred.
Prepatent period of malaria and survival assays
Mice were treated with 4M PQ-TZ at a dose of 50 mg/
In vitro liver stage development of Plasmodium berghei
kg by oral route on days −3, −2, −1 and 0, where day
Plasmodium berghei (ANKA) sporozoites freshly isolated 0 corresponds to the day of sporozoite inoculation. Ten
from salivary glands of infected An. stephensi mosquitoes thousand salivary gland sporozoites were inoculated by
were counted in a haemocytometer and diluted to the intravenous route and the parasitaemia and survival were
appropriated concentration. Ten thousand sporozoites followed for 30 days. A total of 5 mice were used per
in 0.4 mL of complete Dulbecco modified Eagle medium group. As described above, the mice were anaesthesized
(DMEM) supplemented with 10% foetal bovine serum with acepromazine 2.5 mg/kg, then sacrificed soon after
(FBS) and 40 mg/L of penicillin/streptomycin, were the malaria symptoms appeared.
added to each well of a 6-well culture plate containing
semi-confluent Hepa 1-6 cells seeded 1 day earlier. Drugs In vitro haemolysis assay
at 10, 1 and 0.1 µM, freshly diluted in 0.05% DMSO Experiments were performed with both human and
were added to the plates 3 h after sporozoite infection mouse erythrocytes. Human erythrocytes (2% haemato-
of cell cultures. e vehicle was used as a control (0.05% crit) from normal or G6PD deficient donors were incu-
DMSO). e cultures were allowed to grow at 37 °C in bated with test and control compounds, diluted in a 0.2%
5% CO2 for 48 h. e culture medium was changed every (v/v) DMSO solution to 15–1000 µg/mL, at 37 °C for
12 h, and fresh compounds were added at the same con- 2 and 24 h in a shaking water bath. To look at whether
centration, to maintain inhibitor pressure throughout the metabolites of the PQ-TZs had haemolytic properties,
growth period. After 48 h, the cells were fixed with 4% blood samples were taken from the mice, by retro-orbital
paraformaldehyde, permeabilized with cold methanol blood collection, 15 min to 2 h after the oral treatment
overnight, blocked with 1% bovine serum albumin (BSA) of the animals with PQ (15 mg/kg), with the PQ-TZ
in PBS for 2 h, then incubated for 1 h with monoclonal (100 mg/kg); blood from not treated animals were used
antibody 3D11 [16], directed against the circumsporo- as a control. Twenty microliter of serum were incu-
zoite protein of P. berghei. e plates were washed three bated with human erythrocytes, during 2 h at 37 °C. e
times with PBS, 0.05% Tween 20 (PBST), incubated with mixtures were centrifuged at 1000g for 10 min, and the
secondary antibody coupled to Alexa 594 (Life Technolo- absorbance of the supernatants was measured at 540 nm
gies) for 1 h, washed three times with PBST, and mounted in an ELISA plate reader (SpectraMax340PC384, Molec-
using prolong reagent containing DAPI (Life Technolo- ular Devices). e haemolytic rate was calculated using
gies). Images of liver-stage parasites were captured using 0.05% saponin, as positive control for haemolysis of
a fluorescence microscope (Nikon 90i). e Pro-Plus pro- human erythrocytes, considered as 100% [18].
gram (Media Cybernetics) was used to measure the size
of the liver-stage parasites [16].
e diagnosis of G6PD deficiency was confirmed using
a technique based on the principle that haemoglobin oxi-
dized to methaemoglobin by the action of sodium nitrite
is converted by the enzyme in the presence of methylene
In vivo parasite liver load assay in rodent malaria
Mice were drug-treated by oral route, on days −3, 2, 1 blue [19]. e final brown colour demonstrates a sample
and 0, where day 0 corresponds to the day of sporozoite positive for G6PD deficiency, while a bright red colour is
infection by intravenous route. e parasite liver load indicative of a normal sample. In the present study, the
was evaluated 42 h after sporozoite inoculation, when the RBC samples used were of four patients with G6PD defi-
mice were euthanized, their livers were harvested, and ciency first diagnosed by one of the authors (THK), as
total RNA extracted using Trizol reagent (ermo Fisher described before [10].
Scientific, MA, USA). Reverse transcription with 4 μg
of RNA was performed to obtain cDNA. In a real-time Results
PCR mix of 50 μL, a cDNA equivalent of 0.5 μg RNA Primaquine derivatives are not as toxic as primaquine
was used. e real-time PCR mix also contained primers To evaluate the toxicity of PQ derivatives on nucleated
specific for the P. berghei 18S rRNA and SYBR green dye. cells in vitro, HepG2 cells, a human hepatoma cell line,
Real-time PCR was performed in a Bio-Rad I cycler, and and a monkey kidney cell line (BGM), were incubated
the copy numbers were calculated using a known amount with the compounds for 24 h. Cell viability was measured
of a standard plasmid having the amplification target by MTT, a colorimetric assay for assessing cell metabolic

et al. Malar J (2017) 16:110
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Table 1 Cytotoxicity of 15 primaquine derivatives (PQ-TZ
4b to 4p) to human hepatoma cells (HepG2) and monkey
kidney cells (BGM), as compared to primaquine
PQ-TZ tested were less toxic than PQ; the compounds 4d,
4j, 4k, 4l, 4m, 4o and 4p were 20 times less toxic than PQ
in HepG2 and over 30× less toxic to BGM cells (Table 1).
e toxicity of the PQ derivatives was also evaluated on
human erythrocytes, aiming to see if these compounds
have different levels of toxicity to normal erythrocytes
compared to G6PD deficient erythrocytes. Different
concentrations (15–1000 µg/mL) of PQ-TZs (4b, 4c, 4d,
4e, 4f, 4g, 4h, 4i, 4j, 4k, 4l, 4m, 4n, 4o and 4p) were incu-
bated with human erythrocytes, and the rate of haemoly-
sis evaluated. ese compounds were not haemolytic to
normal erythrocytes or to G6PD deficient erythrocytes,
while PQ was haemolytic, even at the lowest dose tested
(Table 2). In addition, sera from drug-treated mice were
tested for haemolysis, to determine if these PQ-TZs pro-
duce toxic metabolites as PQ does. Sera were collected
between 15 min up to 2 h after treatment of mice with
PQ (15 mg/kg) or with PQ-TZ (100 mg/kg), and incu-
bated with the normal or G6PD deficient erythrocytes for
2 h (Table 2).
Compound
R
Cytotoxicity (MDL50
in μM)
Toxicity ratios
(MDL50
)
PQ-TZ/PQ
HepG2
BGM
HepG2 BGM
4b
2-F
NT
NT
–
–
4c
3-F
1302 1 08 550 43
4.4
23.3
9.4
2.7
17
2.7
4d
4-F
≥6831
≥6831
≥2193
252 24
NT
33.8
10.8
1.2
4e
2-Cl
≥2193
4f
3-Cl
641 31
3984 116
4g
4-Cl
–
4h
2-NO2
3-NO2
4-NO2
1463 1 05 1312 145 6.3
6.5
4i
536 17
523 210
≥6651
≥6833
≥6651
≥6644
≥2242
≥6726
≥6651
202 35
2.3
2.6
4j
≥6651
28.5
29.3
28.5
28.5
9.6
32.9
33.8
32.9
32.9
11.0
33.3
32.9
–
4k
2-OCH3 ≥6833
3-OCH3 ≥6651
4-OCH3 ≥6644
3-CN
4-CN
4-CH3
4l
4m
4n
≥2242
≥6726
≥6651
All mice (total of three mice per group) survived until
the end of the experiment. After the blood collection,
they were anaesthesized with acepromazine 2.5 mg/kg
then sacrificed by cervical dislocation. Only sera from
PQ-treated mice caused haemolysis: about 48% haemol-
ysis was observed when sera were collected 30 min fol-
lowing treatment. Sera collected 120 min after treatment
with PQ had a lower haemolysis rate, likely from the
lower concentration of circulating toxic metabolites.
e in vivo toxicity of PQ and PQ-TZs compounds
was tested in mice receiving the drugs administered by
gavage, 5 mice per group. PQ in a single dose of 100 or
50 mg/kg was fatal on the second day for all mice. e
25 mg/kg dose was not toxic and all mice survived for up
to 30 days. Compounds 4c, 4g, 4m and 4o administered
as a single dose of 1000 mg/kg caused no death within
the 30 days of observation. e compounds 4b, 4m and
4p were given in a single dose of 500 mg/kg, and all
mice survived for up to 30 days, the last day of observa-
tion. e animals were anaesthetized with acepromazine
2.5 mg/kg and sacrificed by cervical dislocation on day
30th (end of the experiment).
4o
28.8
28.5
–
4p
Primaquine
233
5
Drug concentration that killed 50% of cells using the MTT assay as a readout
NT not tested
Table 2 Hemolysis (%) of normal and G6PD deficient
erythrocytes after 2 h incubation with sera from mice
dosed with primaquine (15 mg/kg) or PQTZ (100 mg/kg)
Erythrocyte
samples
Hemolysis (%) by time between mouse dosage
and serum collection
15 min 30 min
45 min
60 min
120 min
Primaquine
Normal
0
0
0
0
22 12** 32 10** 30 16** 10 11**
48 2** 54 12** 43 22** 36 13**
G6PD defi-
cient
PQTZs (4b, 4c, 4d, 4e, 4f, 4g, 4h, 4i, 4j, 4k, 4l, 4m, 4n, 4o and 4p)
Normal
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
G6PD defi-
cient
Not treated
Normal
Primaquine derivatives block Plasmodium transmission
to mosquitoes
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
G6PD defi-
cient
Primaquine PQ-TZ derivatives were tested for their abil-
ity to inhibit oocyst formation in both rodent and bird
malaria models. An. stephensi mosquitoes were allowed to
feed on P. berghei-infected mice immediately before drug
treatment of mice (control groups) or 2 h after treatment
(test group). Aedes mosquitoes were fed on P. gallinaceum
infected chickens (control groups) immediately before
or after drug-treatment (test groups). Mosquito midguts
Mean and standard deviation of three different animals
** Statistical differences as compared to not treated controls are indicated by an
asterisk (p < 0.05, by Mann–Whitney test)
activity and the drug concentration that killed 50% of the
cells (MDL50) was calculated. e MDL50 value of the PQ
derivatives was compared with PQ, showing that all 14

et al. Malar J (2017) 16:110
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Page 6 of 11
were evaluated for two parameters: (a) the presence of
oocysts and (b) the number of oocysts per mosquito, as
compared to the control mosquito groups fed on the same
animal immediately before drug treatment (considered as
100%). us, the effect of PQ and the test compounds on
both prevalence and intensity of infection could be quan-
tified and compared. e infected mice or chickens were
fed-upon by Anopheles or Aedes mosquitoes, respectively,
before drug treatment (time = 0 h) or after drug treat-
ment (2 h in mice or 6 h in chickens). All animals (mice or
chickens) after the mosquitoes fed on them were anaes-
thesized with acepromazine 2.5 mg/kg then sacrificed by
cervical dislocation. e mosquito midgut infection was
assessed 12 days later for P. berghei or 6–7 days later for
P. gallinaceum. By comparing oocyst numbers in mosqui-
toes blood-fed on the same animal before and after drug
administration, each animal served as its own control. In
the P. berghei model, among the nine PQ-TZs tested, five
(4b, 4c, 4g, 4m and 4o) completely inhibited oocyst forma-
tion. Four of these five active compounds were also active
in the P. gallinaceum model (4c, 4g, 4m and 4o). In con-
trast to its strong activity in the rodent model, compound
4b was not active in P. gallinaceum (Table 3). Compound
4m, the only one tested in lower doses (50 and 25 mg/kg)
significantly inhibited the sporogonic cycle (p < 0.001) of
P. galllinaceum (Fig. 3).
Fig. 3 Plasmodium gallinaceum oocyst numbers in midguts of Aedes
fluviatilis. Mosquitoes fed on the same infected chicken at time 0 h
(before treatment) or 6 h after treatment with compound 4m, which
was administered orally. For each indicated time point, 20 mosqui-
toes were dissected. All doses significantly reduced oocyst numbers
(p < 0.001), calculated by Mann–Whitney test
Inhibition of the exoerythrocytic stage development
in vitro and in vivo
Anti-malarial drugs are designed to target the sympto-
matic blood stages, usually not exhibiting activity against
exoerythrocytic parasites. PQ does eliminate the liver
stage parasites [1] and is the only licensed drug that elim-
inates hypnozoites, the dormant liver stages of P. vivax.
Selected PQ-TZs were tested for their ability to inhibit
the P. berghei early exoerythrocytic form (EEF) devel-
opment in vivo, the only EEF available for tests at our
laboratories.
Table 3 Oocyst number of P. gallinaceum in Aedes
and P. berghei in Anopheles. Mosquitoes were allowed
to blood feed on the infected vertebrate hosts before and
after treatment with primaquine derivatives (4b to 4p) or
primaquine (15 mg/kg)
Five mice were used per group. ey were treated by
oral route for three consecutive days (50 mg/kg per day)
with PQ-TZ 4f, 4h, 4i, 4j, 4n and 4m, then they were
inoculated by intravenous route with freshly isolated
sporozoites and 42 h later their parasite liver load (EEF)
was evaluated. e animals were anaesthetized with
acepromazine 2.5 mg/kg then sacrificed by cervical dis-
location, their livers were harvested and the parasites
measured by real time PCR. As shown in Fig. 4, com-
pound 4m reduced the liver parasite burden by 95% in
comparison with untreated controls. Nonetheless, PQ
was more active, inhibiting liver parasite burden by 75, 93
and 99%, at lower doses (3.2, 7.5 and 15 mg/kg respec-
tively). e other compounds were inactive (Additional
file 1).
Compound tested
(50 mg/kg)
% Aedes infected (%
reduction)
% Anopheles infected
(% reduction)
4b
80 (11%)
10 (89%)
80 (6%)
89 (0%)
95 (5%)
45 (50%)
90 (0%)
70 (13%)
75 (0%)
95 (5%)
84 (16%)
45 (55%)
95 (0%)
15 (85%)
75 (6%)
0 (100%)
0 (100%)
0 (100%)
86 (4%)
NT
4c
4d
4e
4f
NT
4g
0 (100%)
86 (0%)
NT
4h
4i
4j
NT
4k
NT
4l
NT
To determine whether the decrease in liver EEF seen
in PQ-TZ 4m treated mice was due to fewer parasites
becoming mature exoerythrocytic stages or to all para-
sites being unable to complete liver stage development,
mice were inoculated with P. berghei sporozoites and
treated with PQ or PQ-TZ 4m. All mice were followed
for development of the blood stage infection. Blood
4m
0 (100%)
71 (30%)
0 (100%)
86 (0%)
0 (100%)
4n
4o
4p
Primaquine
The % reduction of sporogony was calculated in relation to the control
mosquitoes blood fed on the same animal host immediately before treatment

et al. Malar J (2017) 16:110
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Page 7 of 11
smears prepared on day 3 post sporozoite infection, until was euthanized on day 8 due to appearance of malaria
day 15, had their parasitaemia determined in Giemsa- symptoms. Mice were anaesthetized with acepromazine
stained thin smears. Complete protection is defined 2.5 mg/kg and then sacrificed by cervical dislocation. e
as the absence of parasites in blood smears for 2 weeks other mice survived until the end of the experiment and
post-challenge with sporozoites. e onset of blood stage did not present malaria symptoms.
parasitaemia in mice treated with PQ-TZ 4m was delayed
We then used hepatocyte cultures to determine
by 3 days, compared to untreated controls. is result whether PQ-TZ 4m gave rise to fewer or smaller EEFs
indicates that PQ-TZ 4m reduced but did not completely in vitro. Hepatoma cultures infected with 10,000 P.
inhibit the development of infectious liver stage para- berghei sporozoites (ANKA strain) were incubated with
sites and that some hepatic EEF were still able to initi- different concentrations of 4m for 2 days, at which point
ate a blood stage infection. Nonetheless, this 3-day delay cultures were fixed and EEFs stained, counted and meas-
suggests that the sporozoite load was reduced by at least ured. e compound PQ-TZ 4m at 1 µM significantly
100-fold. Consequently, PQ-TZ 4m treated mice also inhibited both the number of P. berghei EEFs and their
showed increased survival compared to controls (Fig. 5a, size, in agreement with the in vivo results (Fig. 6).
b). All five animals treated with PQ survived until the end
of the experiment, when they were anesthetized with ace- Discussion
promazine 2.5 mg/kg and then sacrificed by cervical dis- PQ is used to prevent P. vivax relapses and to block
location. Six mice were euthanized before the end of the malaria transmission. However, the toxicity of the PQ
experiment soon after malaria symptoms appeared: four metabolites, especially the haemolytic activity to G6PD-
non-treated control mice were euthanized on day 7 and 1 deficient erythrocytes [20], limits its use in humans. Fur-
on day 9 post-infection, and one treated with PQ-TZ 4m thermore, primaquine causes gastrointestinal disorders
Fig. 4 Plasmodium berghei liver parasite burden 42 h after intravenous inoculation with sporozoites. Parasite numbers at 42 h post inoculation were
measured by quantitative PCR in mice treated with PQ (a) or PQ-TZ 4m (b). *p < 0.05 by Mann–Whitney test
Fig. 5 Effect of PQ-TZ on parasite burden and survival of infected mice. Mice (5 per group) were treated with PQ-TZ 4m (50 mg/kg) or primaquine
(15 mg/kg), or not treated prior to inoculation of 10,000 P. berghei sporozoites. Mice parasitaemia was followed by blood smears (a) as well as
survival (b) for 15 days (a)

et al. Malar J (2017) 16:110
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Page 8 of 11
such as nausea, dizziness, and vomiting in both normal receive a single dose of PQ (0.75 mg/Kg) orally to pre-
and G6PD deficient patients. Hypersensitivity reac- vent malaria transmission to mosquitoes [4, 25]. e
tions can also occur but are rare [20]. Tafenoquine is a primary motivation for the present work was to study
candidate to replace PQ that has been evaluated in clini- the toxicity and anti-parasite activity of PQ-TZ deriva-
cal trials. Tafenoquine was active in a single dose but it tives using rodent and avian malaria species. Pharma-
was also haemolytic for erythrocytes in G6PD-deficient cokinetic studies in humans showed that carboxy-PQ
patients [21, 22].
is the main PQ metabolite, a compound less active than
e role of G6PD in red cells is to provide reductive PQ [26]. e modification on the terminal amino group
potential in the form of NADPH [23]. In G6PD-deficient of PQ improved the PQ-TZs bioavailability by blocking
red cells, the NADPH supply is adequate in the steady this metabolism. Pro-drugs of PQ [27, 28], double-drugs
state but falls short upon exposure to PQ. It is possible [29, 30] and bis-PQ [31] have been studied to prevent this
that the PQ metabolite causes an oxidative challenge metabolism, as well as to reduce its toxicity in patients
that the G6PD normal red cells would tolerate, but not who are G6PD deficient. Some PQ analogues were also
the G6PD-deficient red cells. However, the stepwise synthesized as PQ-trioxaquines [30] and the chemical and
sequence of the events from oxidative attack to haemoly- enzymatic stable PQ-imidazolidinones [32, 33] (Fig. 1b), a
sis has not been fully elucidated [24].
novel type of 8-aminoquinoline anti-malarial agents.
Diverse biological activities have been described for
Aiming to decrease malaria transmission, patients with
malaria, regardless of the parasite species diagnosed, heterocyclic thiazolidinones, including antimicrobial
Fig. 6 Effect of PQ-TZ 4m on infected hepatocytes in vitro. Number (a) and size (b) of the exoerythrocytic forms of P. berghei (EEF) in cultures of
mouse hepatocytes (cell line Hepa1-6), in the presence of compound PQ-TZ 4m at 0.1, 1 and 10 µM concentrations. Representative EEFs in control
(c) or 10 µM PQ-TZ 4m-treated Hepa1-6 cells (d). The same image is shown for each in phase, stained with DAPI, and, stained with mAb 3D11
against the developing EEF. **p < 0.05 by Mann–Whitney test. Need size bars

et al. Malar J (2017) 16:110
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Page 9 of 11
[34, 35], anti-fungal [36], anti-inflammatory [37], anti- to the chicken and the mosquitoes blood meal of 4–6 h
tumour [38], anti-HIV [39], anti-Alzheimer’s disease compared to 2 h in the mice model. New studies have
as muscarinic receptor 1 agonist [40], and anti-malarial shown that Plasmodium yoelii nigeriensis is a viable
[41]. As for PQ-imidazolidinone derivatives, PQ-TZs are model system to study malaria transmission by the New
unlikely to act as pro-drugs because the thiazolidin-4-one World vectors Anopheles aquasali and Anopheles albi-
ring is stable at low pH. A simple test consisting of addi- manus [47]. However, the differences between mam-
tion of concentrated chloridric acid (1 mL) to PQ-TZs mal and avian models need to be better understood,
(50 mg) at 40 °C for 24 and 48 h showed the stability of especially because of the limitation of a good Anopheles
PQ-TZs. Moreover, by blocking the amino free group, vector for infect mice in Brazil, therefore limiting the
PQ-TZs compounds may prevent the formation of cyclic mosquito work to the P. gallinaceum model in Brazil.
pyrrolidine [42]. Indeed, as opposed to PQ, several PQ-
One PQ-TZ derivative (4m) inhibiting sporogony, also
TZs were tested in vitro on G6PD deficient and normal inhibited development of P. berghei liver stages in vitro
human erythrocytes and caused no haemolysis (Table 2).
(1 µM dose), and in vivo; a result which makes the chemi-
Several reports have described the protection of the cal group valuable. e ability of 4m to destroy the hyp-
quinoline ring at the 5 position, to avoid the formation of nozoites of P. vivax, as does PQ, has yet to be determined.
the toxic 5-hydroxy-PQ metabolite [27, 43–45]. Although
the structure of PQ-TZs does not directly influence the Conclusions
5-position of the quinoline ring, 5-hydroxy-PQ-TZ might e main findings described here include: (i) all the
be formed. However, others report that the modifica- 15 PQ-TZs tested were not toxic to mammalian cells
tion of primary amino group decreases toxicity through in vitro, unlike PQ, nor to mice treated with high doses
unknown mechanisms [45]. Indeed, most of PQ-TZs of the new drugs; (ii) the PQ-TZs caused no direct hae-
tested were significantly less toxic than PQ against human molysis to human RBCs deficient or not in the enzyme
hepatoma and monkey kidney cells in vitro, as well as to G6PD; (iii) most PQ-TZs displayed a lower toxicity than
normal and G6PD deficient human erythrocytes.
PQ to mammalian cell lines - human hepatoma cells and
ere is no significant influence of the nature of the aryl kidney cells from green monkey; (iv) compounds 4b, 4c,
substituent on the ability of PQ-TZs to inhibit Plasmodium 4g, 4m and 4o completely blocked the malaria sporogonic
sporogony in the mosquito host. However, the 4-position cycle of P. berghei in An. stephensi mosquitoes compared
seems to be important for drug activity. Accordingly, this to the control mosquitoes blood-fed in the same animal
position was substituted in three of the four most active immediately prior to drug treatment; (v) four PQ-TZs
compounds (4g, 4m and 4o) (Table 3), with compound 4g (4c, 4g, 4m and 4o) administered to chickens experi-
resulting more toxic than 4m and 4o. e compound 4m, mentally infected with P. gallinaceum also inhibited the
which contains a methoxy group, inhibited Plasmodium sporogonic cycle by 50–90% in Aedes mosquitoes blood-
sporogony at a lower dose of 25 mg/kg; the other com- fed after drug treatment; (vi) compound 4m reduced P.
pounds were not tested at lower doses.
gallinaceum sporogony at lower doses of 50 and 25 mg/
In spite of the irregular production of oocysts observed kg; (vi) compound 4m reduced the number of EEFs in
in all malaria models [46], there were statistically sig- hepatoma cells and in mice; (vii) compound 4m also
nificant differences among the groups of mosquitoes caused a 3-day delay in the malaria pre-patent period.
blood-fed in animals pre-treated with the compounds Whether these active PQ-TZs will become promising
considered active (4c, 4g, 4m and 4o). Although none of new drugs to help malaria control is an open question
the compounds tested completely inhibited oocyst for- that requires further pharmacological studies and tests
mation in P. gallinaceum, 4b, 4c, 4g, 4m and 4o inhibited against the hypnozoite forms aiming to cure the P. vivax
oocyst formation of P. berghei in Anopheles mosquitoes, late relapses.
as did PQ tested in parallel in both models. One possibil-
Additional file
ity to explain these results is a different PQ-TZs metabo-
lism in mammals versus avian hosts, making the mouse
Additional file 1: Table S1. Inhibition of Plasmodium gallinaceum
sporogony by PQ-TZ derivatives in Aedes mosquitoes blood fed 6 h after
oral treatment (6 h) with one compound dose in relation to control mos-
quitoes fed in the same chicken before treatment (0 h). Table S2. Inhibi-
tion of Plasmodium berghei sporogony by PQ-TZ derivatives in Anopheles
mosquitoes blood fed 2 h after oral treatment (2 h) with one dose of each
compound in relation to mosquitoes blood fed in the mouse before drug
treatment (0 h). Figure S1. Plasmodium berghei liver parasite burden 42 h
after intravenous inoculation with sporozoites, measured by quantitative
PCR in mice treated with PQ-TZ.
model more efficient at detecting active compounds.
Nevertheless, in both models PQ caused 100% inhibition
of sporogony.
All active compounds in the mice model were less
inhibitory against the bird malaria parasites, or they
caused a lower inhibition of the oocyst prevalence. ese
overall results could also be explained by the longer
period of time elapsed between drug administration

et al. Malar J (2017) 16:110
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Page 10 of 11
Abbreviations
7. Tripathi AC, Gupta SJ, Fatima GN, Sonar PK, Verma A, Saraf SK. 4-Thiazolidi-
nones: the advances continue. Eur J Med Chem. 2014;72:52–77.
8. Solomon VR, Haq W, Srivastava K, Puri SK, Katti SB. Synthesis and antima-
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BGM: kidney cell line from Buffalo Green Monkey Kidney; EEFs: exoerythrocytic
forms; G6PD: glucose-6-phosphate dehydrogenase; HEPA 1-6: mouse hepato-
cyte cell line; HepG2: human hepatoma cell line; MDL50: drug concentration
that killed 50% of the cells; PQ: primaquine; PQ-TZs: primaquine derivatives; Tz:
thiazolidinones.
Authors’ contributions
ACCA, FJBF, PS, FZ and AUK designed, analysed and interpreted the biological
experiments. BBD, PDN and WC synthesized the compounds studied. TK pro-
vided red blood cells from G6PD deficient donors. ACCA, WC, PS, FZ and AUK
wrote the manuscript. All authors read and approved the final manuscript.
10. Katsuragawa TH, Gil LHS, Stábile RG, Pires MG, Bonini-Domingos CR. Aval-
iação da incidência da deficiência de Glicose-6-Fosfato Desidrogenase
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Antimalarial activity of medicinal plants used in traditional medicine in
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Author details
1 Centro de Pesquisas René Rachou-Fiocruz, Av. Augusto de Lima 1715, Belo
Horizonte, MG 30190-002, Brazil. 2 Faculdade de Medicina, Universidade Fed-
eral de Minas Gerais, Av. Alfredo Balena, 190, Belo Horizonte, MG 30130-100,
Brazil. 3 Department of Molecular Microbiology and Immunology, Johns
Hopkins Bloomberg School of Public Health, 615 N Wolfe St., Baltimore, MD
21205, USA. 4 Laboratório de Química Aplicada à Bioativos, Centro de Ciências
Químicas, Farmacêuticas e de Alimentos, UFPel, Campus Universitário s/
no, Pelotas, RS 98001-970, Brazil. 5 Laboratório de Epidemiologia, Fundação
Osvaldo Cruz-Fiocruz Rondônia, Bairro Lagoa, Porto Velho, RO, Brazil.
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zoite invasion, migration, and development by microscopy and flow
cytometry. Methods Mol Biol. 2013;923:385–400.
Acknowledgements
We thank Dr. Luciano Moreira (FIOCRUZ-Minas) for the insectary facilities, and
to the Bloomberg Family Foundation for their support of the Insectary facilities
and Parasitology Core Facilities at the Johns Hopkins Malaria Research Institute
(JHMRI).
17. Bruña-Romero O, Hafalla JC, González-Aseguinolaza G, Sano G, Tsuji M,
Zavala F. Detection of malaria liver-stages in mice infected through the
bite of a single Anopheles mosquito using a highly sensitive real-time
PCR. Int J Parasitol. 2001;31:1499–502.
Competing interests
The authors declare that they have no competing interests.
Availability of data and materials
All data generated or analysed during this study are included in this published
article [and its supplementary information files].
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Funding
This work had financial support from the Conselho Nacional de Pesquisas e
Desenvolvimento (CNPq) Edital MCT/CNPq 09/2009 PRONEX, Rede de Malária
(305314/2009-2), MCT/CNPq/CT/MS/SCTIE/DECIT 034/2008 (575746/2008-4),
and Fundação de Amparo a Pesquisa de Minas Gerais-FAPEMIG (Universal
CBB-APQ-01692-11; and PRONEX Rede Malária). CNPq also provided fellow-
ships to the authors (AUK, ACCA, FJBF, WC, and BBD); The Universidade Federal
de Pelotas and CAPES provided fellowships to PDN. The work was also sup-
ported by NIH grant R01AI056840 (PS).
Received: 12 October 2016 Accepted: 26 February 2017
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