Antimalarial activity of small-molecule benzothiazole hydrazones

Article abstract of DOI:10.1128/AAC.01575-15

We synthesized a new series of conjugated hydrazones that were found to be active against malaria parasite in vitro, as well as in vivo in a murine model. These hydrazones concentration-dependently chelated free iron and offered antimalarial activity. Upo

Full text of DOI:10.1128/AAC.01575-15

AAC Accepted Manuscript Posted Online 2 May 2016
Antimicrob. Agents Chemother. doi:10.1128/AAC.01575-15
Copyright © 2016, American Society for Microbiology. All Rights Reserved.
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Antimalarial activity of small molecule benzothiazole hydrazones
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Souvik Sarkar ¶‡, Asim A. Siddiqui ¶‡, Shubhra J. Saha , Rudranil De , Somnath Mazumder ,
Chinmoy Banerjee ‡, Mohd. S. Iqbal ‡, Shiladitya Nag ‡, Susanta Adhikari §, and Uday
Bandyopadhyay ‡*
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‡Division of Infectious Diseases and Immunology, CSIR-Indian Institute of Chemical Biology, 4,
Raja S. C. Mullick Road, Jadavpur, Kolkata 700032, West Bengal, India.
§Department of Chemistry, University of Calcutta, 92, A. P. C. Road, Kolkata 700 009, West Bengal,
India.
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¶These authors contributed equally.
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*Address correspondence to this author at the Division of Infectious Diseases and Immunology,
CSIR-Indian Institute of Chemical Biology, 4, Raja S. C. Mullick Road, Jadavpur, Kolkata 700032,
West Bengal, India. Tel: 91-33-24995735, Fax: 91-33-4730284.
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E-mail: ubandyo_1964@yahoo.com, udayb@iicb.res.in
Running title: Antimalarial benzothiazole hydrazone
ABSTRACT
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We synthesized a new series of conjugated hydrazones that were found to be active against
malaria parasite in vitro as well as in vivo in murine model. These hydrazones concentration-
dependently chelated free iron and offered antimalarial activity. Upon screening of the synthesized
hydrazones, compound 5f was found to be the most active iron chelator as well as antiplasmodial.
Compound 5f also interacted with free heme (KD = 1.17 ± 0.8 µM), an iron containing tetrapyrrole
released after hemoglobin digestion by the parasite and inhibited heme polymerisation by parasite
lysate. Structure activity relationship studies indicated that nitrogen and sulphur substituted five-
membered aromatic ring present within the benzothiazole hydrazones might be responsible for their
antimalarial activity. The dose-dependent antimalarial and heme polymerization inhibitory activities
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of the lead compound 5f were further validated by following [3H]-hypoxanthine incorporation and
hemozoin formation in parasite respectively. It is worth mentioning that compound 5f exhibited
antiplasmodial activity in vitro against chloroquine/pyrimethamine resistant strain of P. falciparum
(K1). We also evaluated in vivo antimalarial activity of compound 5f in a murine model where a lethal
multiple drug resistant (MDR) strain Plasmodium yoelii was used to infect Swiss albino mice.
Compound 5f significantly suppressed the growth of parasite and the infected mice experienced
longer life span upon treatment with this compound. During in vitro and in vivo toxicity assays,
compound 5f showed minimal alteration in biochemical and hematological parameters compared to
control. In conclusion, we identified a new class of hydrazone with therapeutic potential against
malaria.
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INTRODUCTION
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Malaria is one of the foremost widespread lethal diseases in the world. Nearly 40% of the global
population is affected by malaria and it has become a major public health concern in the developing
countries. According to World Health Organisation, with 198 million reported cases and around
584000 deaths in 2013, most of which comprised of children below 5 years of age, Malaria poses a
serious threat to a large chunk of the human population. Malaria has evolved as a gigantic infectious
killer and its control has become a high precedence assignment globally (1-2). P. falciparum is the
deadliest among the Plasmodium species that causes malaria in human. With an alarming increase in
the emergence of drug resistant parasites especially the MDR strains, established antimalarials have
become more and more ineffective thereby instigating active exploration for developing newer
substitutes to confront the disease. In this regard new generation of in vivo active antimalarial is the
need of time (3-5). Recognition and design of new chemical scaffolds particularly influencing the
validated biological targets crucial for parasite survival and disease development might prove
instrumental in achieving the aforesaid goals (5). With advancement in research on the biology of
Plasmodium, many potential drug targets have been identified and worked upon; most of which
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belong to the asexual blood stages. In this context, iron chelation in parasites has proved promising;
however it is largely underutilized.
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To cause infection, nearly all protozoa, fungi, and bacteria obtain growth-essential iron from their
hosts. Iron is required for the development of the parasite since many enzymes of the plasmodial
metabolic pathways (like δ-aminolevulinate synthase, responsible for the de novo synthesis of heme,
or ribonucleotide reductase, involved in DNA synthesis) depend on the presence of this element (6).
Deprivation of utilizable iron by chelation is a proficient approach to arrest parasite growth and
associated infection (7). A strong iron chelator, deferoxamine (DFO), reportedly diminishes the
activity of Fe (III)-containing enzymes in parasitic protozoa (8-11). Therefore, iron chelators have
recently gained importance as potent anti-malarial compounds. It is proposed that iron chelator
antimalarials mainly arrest iron-dependent ribonucleotide reductase-mediated production of
nucleotides, although the precise mechanistic details of antiparasitic action are still unclear (12).
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The intra-erythrocytic stages during malaria are mainly responsible for the clinical symptoms
related to malaria. During that stage, malaria parasite digests enormous quantities of hemoglobin
inside the food vacuole and thereby releasing high quantities of toxic free heme (13-16). Free heme
can change membrane permeability by intercalating into the cell membrane and also may induce
oxidative stress (17-18) which may lead to parasite death. However, by converting heme into less
toxic hemozoin parasites cleverly evade the toxicity of free heme (13-14, 19). Oxidative stress-
mediated parasite death through the prevention of hemozoin formation is also an accepted therapeutic
strategy against malaria (20). Heme contains an iron atom at the center of its tetrapyrrole ring;
therefore, iron chelating molecule bears a possibility to interact with free heme through its iron.
Furthermore, it is worth mentioning that this sort of heme interacting molecule may inhibit hemozoin
formation to induce parasite death (20-21). Therefore, a small molecule, which can chelate free iron
as well as prevent hemozoin formation through free heme binding, would be a novel dual target
antimalarial agent. Here, we report the designing, synthesis and antimalarial activities of conjugated
hydrazones. These molecules chelated free iron, interacted with free heme, inhibited hemozoin
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formation and prevented P. falciparum growth in vitro. Compound 5f (the lead molecule) also showed
antimalarial activity in vivo against the MDR strain of P. yoelii in Swiss albino mice.
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MATERIALS AND METHODS
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Chemicals. RPMI1640, Hemin, saponin, dimethyl sulfoxide (DMSO), SDS, sodium bicarbonate,
tripyridyl triazine (TPTZ), Saponin, D-glucose and Giemsa stain were purchased from Sigma
(St.Louis, MO, USA). Albumax II, gentamycin, penicillin, streptomycin, fetal bovine serum (FBS)
were purchased from GIBCO (Life Technologies, USA). [3H]-hypoxanthine was purchased from
PerkinElmer (USA). All other chemicals were of analytical grade purity.
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Chemical synthesis. General procedure for the synthesis of hydrazone compounds (1-5 (a-f)). To
a solution of the corresponding hydrazine (30 mmol) in MeOH (50 ml), 1.1 equivalents of the
appropriate aldehyde dissolved in MeOH were added at room temperature. After proper mixing of the
solution, stirring was continued overnight. The solid products were isolated and purified by column
chromatography. The structures of hydrazones were confirmed by NMR and mass spectral analysis.
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Iron chelating activity in vitro. The iron chelating activity of the synthesized compound was
monitored by following the protocol as described earlier (22).The assay was carried out in optically
clear 1 ml quartz cuvette containing 20 mM phosphate buffer (pH 7.4) and Fe(II) (10 µM).
Compounds 5a-f were added with increasing concentrations (1–20 µM) with the instant addition of
tripyridyl triazine (TPTZ) (20 µM) in small volumes to the sample cuvette. The same volume of
DMSO was added to the reference cuvette concomitantly (Compounds were dissolved in DMSO).
Assay system without compound was considered as a control set. A well-known iron chelator
deferoxamine was used as a positive control. Iron chelating capability of compounds (5a-f) at various
concentrations was monitored immediately after each addition by following the drop of absorbance of
Fe(II)-TPTZ complex at 595 nm in a Shimadzu UV/VIS spectrophotometer and expressed as
concentration required to drop 50% of the original absorbance.
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Parasite culture in vitro and in vivo parasite maintenance. P. falciparum 3D7 strain
(chloroquine sensitive) and K1 strain (chloroquine and pyrimethamine resistant) were cultured as
mentioned earlier with some modifications (23-24). In brief, parasite culture was maintained in RPMI
1640 medium supplemented with 25 mM HEPES, 1.76 g per litre sodium bicarbonate, 2 g per litre D-
glucose, 50 µg per ml gentamycin, 370 µM hypoxanthine, and 0.5% (w/v) AlbuMax II at a hematocrit
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level of 5% in culture flasks inside of a sealed candle jars temperature maintained at 37 C. The
culture medium was exchanged with fresh medium at 24 hours interval. P. falciparum growth was
regularly monitored by Giemsa staining of thin blood smears. In vivo malaria was maintained by
intra-peritoneal inoculation of P. yoelii (N-67, MDR strain) in male Swiss albino mice (20–25g) as
described earlier (25). Giemsa staining of thin blood smears was used to quantify parasitemia. Animal
experiments were conducted in accordance with the institutional guidelines and permission from the
animal ethics committee registered with the CPCSEA, India (Permit Number: 147/1999/CPCSEA).
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In vitro antimalarial activity by SYBR green assay. SYBR green assay was performed in 96-
well plate as mentioned earlier (26). In brief, parasite culture with 4% hematocrit and 1% parasitemia
in synchronized ring stage was dispensed 96-well plate with increasing concentration of compounds
in three sets and kept in candle jar in a total volume of 100 µl per well. After 48 hours the cells were
lysed in 100 µl lysis buffer (20 mM Tris pH 7.5, 5 mM EDTA, 0.008% Saponin (wt/vol.), 0.08%
Triton-X 100 (vol/vol) and 2X SYBR Green nucleic I acid stain) in each well by pipetting and
incubated in dark at room temperature for 1 hour. Deferoxamine, a standard iron chelator, was used as
a positive control. Artesunate, a water-soluble semi-synthetic derivative of artemisinin and
chloroquine were used as positive controls to check the antimalarial activity. Fluorescence intensities
were determined upon excitation at 485 nm and following the emission at 530 nm using Hitachi F-
7000 fluorimeter.
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Fe(III)-compound 5f complex formation. Fe(III)-compound complex was prepared using the
method as mentioned earlier (27). Ligand (compound 5f, 2 mmol) was dissolved in 45 ml dry
methanol with subsequent addition of Et3N (2 mmol) to the solution. After complete dissolution of
ligand, a methanolic solution of FeCl3.6H2O (2.75 mmol) was added drop wise to the ligand solution
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and the resulting mixture was refluxed for 1 hour. Progression of the reaction was monitored by
checking through TLC. Upon completion of the reaction, the reaction mixture was allowed to come to
room temperature and precipitated product was filtered off and washed with methanol several times
followed by water and then acetone. The complex obtained was subjected to IR spectroscopy for
characterization.
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Heme interaction studies. The interaction of hydrazone derivatives with heme was evaluated by
differential optical spectroscopy as mentioned before (28). In brief, assay system was carried out with
heme (1 µM) in 100 mM sodium acetate buffer, pH 5.2 in a final volume of 1ml and differential
optical spectra were recorded using quartz cells of 1 cm light path in Shimadzu 15UV/VIS
spectrophotometer at 25 ± 1 0C. Native heme binding of hydrazones was documented with increasing
concentrations (1–20 µM) of hydrazones. Differential spectroscopy of heme versus heme–hydrazones
interaction was performed by the addition of heme solution (1 µM) in both the reference and the
sample cuvettes to obtain the baseline trace at the final volume of 1 ml. This step is followed by the
subsequent addition of increasing concentrations of hydrazones (1–20 µM) to the sample cuvette
along with the concomitant addition of the same volume of DMSO (used to dissolve the hydrazones,
0.5–10 µl) to the reference cuvette. Soret spectrum of thoroughly mixed contents was recorded
without hydrazones and after successive addition of hydrazones immediately. The equilibrium
dissociation constant (KD) for heme-hydrazone complex formation was evaluated by the equation
described by Schejter et al. (28).
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1/ΔA = (KD/ΔA∞) 1/S +1/ ΔA∞
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Where S is the concentration of hydrazones, ΔA is the absorption changes at a particular wavelength
and at saturating concentration of the ligand (hydrazones), absorption changes are expressed as ΔA∞.
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Preparation of parasite lysate. The parasite (P. yoelii) lysate was prepared as mentioned earlier
(29-30). In brief, erythrocytes (from P. yoelii infected mice blood) were collected and centrifuged at
800×g for 5 min. Pellet was washed twice and reconstituted in cold phosphate-buffered saline (PBS).
For the lysis of erythrocytes, PBS containing an equal volume of 0.5% saponin (final concentration
0.25%) was mixed well with erythrocyte suspension in PBS and allowed to stand for 15 min in ice.
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The mixture was then centrifuged at 1300×g for 5 min to get parasite pellet. The pellet was rinsed
thrice with cold PBS and kept at –80 0C for further use. Parasite pellet was then lysed in PBS by mild
sonication (30 s pulse in water bath-type sonicator) at the ice-cold condition and the protein content of
the lysate was quantified. The lysate obtained was stored at –20 0C for further use.
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Hemozoin (β-hematin) formation in vitro by parasite lysate and in P. falciparum culture.
Formation of hemozoin (β-hematin) in the presence of parasite lysate was evaluated as mentioned
earlier with some modification (31-33). In brief, the reaction was carried out in the presence of 100
mM hemin, parasite lysate (20 µg) and different concentrations of hydrazones (1–100 µM) in a final
volume of 1 ml of sodium acetate buffer (100 mM, pH 5.2). The reaction in the absence of hydrazones
was considered as control. The reaction was carried out at 37 0C for 12 hours to allow the conversion
of hemin into hemozoin. After 12 hours, the reaction mixture was centrifuged at 15,000×g for 15 min
at room temperature. Obtained pellet was rinsed three times with Tris buffer (100 mM, pH 7.8,
containing 2.5% SDS) and once in bicarbonate buffer (100 mM, pH 9.2). The resultant insoluble
pellet (hemozoin) was then dissolved using 100 µl of 2N NaOH and diluted with 0.9 ml of 2.5% SDS.
The absorbance of the solution was recorded at 400 nm using Shimadzu 15UV/VIS
spectrophotometer at 25±1 0C with quartz cells of 1cm light path.
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Efficacy of the lead compound was further tested for its activity against hemozoin formation in P.
falciparum in culture at various concentrations. Parasite in culture medium was incubated with the
compound for 48 hours, and then the cells were collected by centrifugation and treated with 0.01%
saponin in phosphate buffered saline (PBS) to release the parasite and lyse the uninfected RBCs.
Parasite pellet was washed with PBS thrice and lysed by sonication. Total protein content of all
samples was estimated. Free heme from parasite lysate was removed by washing with 2.5% SDS and
0.1 M sodium bicarbonate (pH 9.1) with subsequent discard of the supernatant after centrifugation.
The remaining pellet was re-suspended in 1 ml of 0.1 N NaOH and absorbance was measured at 400
nm.
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[3H]-hypoxanthine incorporation assay to follow antimalarial activity in vitro. The inhibition
of P. falciparum growth by the compound was confirmed by following [3H]-hypoxanthine uptake as
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described previously (20, 34). The synchronized ring stage of the parasite (parasitemia 1%) was
cultured in multi-well (200 µl/well) plates with different concentrations of the compound in triplicate.
Parasite with DMSO alone was used as vehicle controls. After 24 hours, [3H]-hypoxanthine (5
µCi/well) was added to each well and the plate was kept in an incubator for another 24 hours to allow
incorporation of free [3H]-hypoxanthine in parasite nucleic acids. After treatment parasite was
pelleted and washed thrice in PBS. Packed RBCs pellet was washed and dissolved in 100 µl 3N
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NaOH and kept at 37 C for 6 hours. The lysate content was then dissolved in 10 ml of scintillation
fluid (PPO, 4g; POPOP, 200mg; naphthalene, 60g; ethylene glycol, 20ml; methanol 100 ml in 1 liter
of 1,4-dioxane) and transferred to scintillation vials. The level of incorporated [3H]-hypoxanthine was
assessed using the Perkin Elmer (Tricarb 2810TR) liquid scintillation counter. Radioactivity of the
experimental samples was expressed as disintegrations/min (CPM), which was proportional to the
amount of [3H]-hypoxanthine incorporated into the DNA.
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Evaluation of antimalarial activity of compound 5f in Swiss albino mice. The in vivo
antimalarial efficacy of compound 5f was assessed in a murine malaria model as mentioned before
(20). In brief, MDR (chloroquine, mefloquine, and halofantrine resistant) strain of P. yoelii (N-67)
was used to infect Swiss albino mice. Compound 5f was dissolved in alkaline water and administered
intra-peritoneally at three different doses (10, 25, 50 mg.kg-1 body weight). For each set of treatment,
a group of six mice (each weighing 25 ± 5 g) was inoculated with approximate 1×106 parasitized
RBCs on Day 0 and compound 5f were administered from day 4 to day 7 post-infection. The
treatment was done at each of the three indicated doses from Day 4 to Day 7. The effectiveness of
compound 5f was evaluated by constant observation of parasitemia and animal survival every day.
Giemsa staining of thin blood smears was used to measure the level of parasitemia in mice. A drop of
blood was extracted from the tail vein of the mice and smeared on a glass slide to obtain a thin smear.
The slide was air-dried and fixed in absolute methanol for 20 sec. Giemsa stain working solution of
was prepared by diluting it in phosphate buffer pH 7.2 in the ratio 1:3. Fixed and air-dried blood
smear was stained for 30 min and washed under running tap water. Slides were viewed and pictured at
a resolution of 100X under oil immersion lens. The mean value of percentage parasitemia observed
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from an individual set of mice was used to analyze the percentage inhibition of parasitemia compared
to the vehicle control group. As a positive control α/β-arteether (an ethyl ether derivative of
artemisinin) was used at a dose of 50 mg.kg-1.day-1 body weight.
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The suppressive activity of compound 5f against P. yoelii was also evaluated in vivo by following
Peter’s 4-day test (35) in Swiss albino mice with slight modification. In brief, blood from an infected
donor mouse was diluted with isotonic saline to yield an inoculum containing 2 ×107 infected
erythrocytes per ml. On the first day (D0), thirty mice were inoculated intraperitoneally with 0.2 ml of
the inoculum before being randomly divided into five treatment groups of six mice each. After three
hours of inoculation, three experimental groups of mice were treated with compound 5f
intraperitoneally, at three different doses (10, 25, or 50 mg.kg-1.day-1) for four days (D0 to D3)
respectively. The fourth group was treated with α/β-arteether which was used as a positive control at a
dose of 50 mg.kg-1.day-1. The fifth group was considered as ‘control’. On the fifth day (D4), 24 h after
the last treatment, a thin smear was prepared from the tail blood of each mouse and stained with
Giemsa’s stain to determine the percent parasitemia (by determining the number of parasitized
erythrocytes per 300 erythrocytes in random fields). Parasitemia was determined by light microscopy
using a 100X objective lens. The suppression of average percentage parasitemia was calculated as
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A-B
% parasitemia suppression =
× 100
A
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Where, A is the average percentage parasitemia in the negative control group and B is the average
percentage parasitemia in the test group.
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In vitro hemolysis assay. The cytotoxic effect of compound 5f on RBCs was evaluated by
hemolysis assay as described earlier with some modifications (36). The freshly obtained RBCs were
washed with 3 volumes of PBS (pH 7.4) thrice. Hemolysis assay was performed at 2% hematocrit
with different concentrations of drug in a final volume of 0.3ml and incubated at 37 °C for 2 h and 12
h. Saponin (0.05% in PBS) was used as a positive control whereas 0.2% DMSO was used as vehicle
control. After incubation, tubes were centrifuged at 1000×g for 10 min and the absorbance of the
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supernatants were taken over 300-700 nm wavelength range. The absorbance of the supernatants was
also measured at 540 nm in ELISA plate reader (µQuant, BioTek) to measure the amount of
hemoglobin released upon RBC lysis. The experiment was performed in triplicate.
In vitro cytotoxic effect of compound 5f against mammalian cells. The cytotoxicity of
compound 5f was further assessed using human liver cell line (HepG2) and human embryonic kidney
293 (HEK293) cells, cultured in RPMI 1640 and DMEM respectively, supplemented with 10% heat-
inactivated fetal bovine serum and antibiotics, in a 5% CO2 atmosphere at 37 0C. When confluent, the
cells were washed with respective culture media, trypsinized and re-plated in the flat-bottom 24-well
plate (5 × 104 cells/well) for cell adherence as described earlier (37). The compound 5f was added to
each well at various concentrations (1-50 µM) and the plate was further incubated for 24 h. MTT [3-
(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide] (1 mg/mL in PBS; 500 µL/well) was
added and incubated for 3 h under the same culture conditions. The supernatant was carefully
removed and 500 µL of DMSO was added with mixing to dissolve the formazan crystals formed. The
optical density was determined at 570. Cell viability was expressed as the percentage relative to
control.
Evaluation of in vivo toxicity of compound 5f by following biochemical and hematological
parameters. To check any toxic effect of compound 5f on liver, serum levels of aspartate
aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), gamma-
glutamyltransferase (GGT) and bilirubin (total, conjugated and unconjugated) were measured using
kits from Span Diagnostics Limited (Surat, India) (38). Further to check any toxic effect of
compound 5f on kidney, serum urea and creatinine levels were analyzed using kits from Span
Diagnostics Limited (Surat, India) (38). To follow the effect of compound 5f on other blood
parameters, hemoglobin content was measured along with a total count of blood cells and differential
count of white blood cells (WBCs) from mice blood exposed to compound 5f (50 mg.kg-1.day-1) for 4
successive days. Six animals were taken in each set and all the animals from all the sets were
sacrificed on the 5th day. The packed cell volume was determined using a conventional method by
microcapillary centrifugation.
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Statistical analysis. For IC50 determination, each experiment was performed three times in
triplicate. The data were expressed as the mean of the three experiments and the corresponding SD.
Statistically significant differences between the groups were determined by the Student’s t-test, in
GraphPad Prism 4. Values of p < 0.005 were considered significant.
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269 RESULTS
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Chemistry. The hydrazones were synthesized via conjugation of differentially substituted
aldehydes and hydrazines (Fig. 1). Synthesized compounds were purified by column chromatography
using the ethyl acetate in hexane solvent gradient. All the mass, 1H and 13C NMR spectra of
benzothiazole hydrazones (BHs) were consistent with the anticipated product structure (Supplemental
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material for reviewers' information only). Other compounds were characterized by the mass, H and
13C NMR spectroscopies (data not shown). All the 30 compounds were synthesized by following a
similar reaction condition as described (Fig. 1). The percent yield of the synthesized compounds was
given in parentheses (Fig. 1). All these synthesized compounds were then screened to explore whether
they can chelate free iron and exhibit any antiplasmodial activity.
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Iron chelation and antimalarial activity of synthesized hydrazones. Synthesized hydrazones
were screened for Fe(II) chelating activity by measuring the decrease of Fe(II)-TPTZ colour in the
presence of increasing concentrations of compounds at 595 nm. Absorbance was decreased due to
Fe(II)-hydrazone interaction which minimizes the Fe(II)-TPTZ complexation, thus inhibiting colour
formation. All the synthesized compounds showed iron chelating activity (Table 1). Data revealed that
BHs exhibited better iron chelating activity compared to other synthesized series of compounds
(compound 1-4 (a-f)) (Table 1). Comparison of iron chelating activity in terms of drop of 50%
absorbance at 595 nm of synthesized compounds showed that methoxylated hydrazones had less
binding affinity compared to hydrazones containing free hydroxyl groups (Table 1). Deferoxamine
was used as a reference compound (Table 1).
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We were then interested in evaluating the effect of these compounds on growth of P. falciparum
by following SYBR green assay. This dye binds to DNA and gives fluorescence depending on the
number of parasites present in the culture. Data indicated that among the synthesized hydrazones, free
hydroxyl group containing hydrazones inhibited SYBR green fluorescence significantly indicating
inhibition of P. falciparum growth in culture whereas methoxylated hydrazones did not inhibit
parasite growth (Table 1). IC50 data indicated that benzothiazole hydrazones (BHs) (compound 5a-f)
were more efficient in inhibiting P. falciparum growth compared to other synthesized compounds
(compound 1-4 (a-f)). Furthermore, among the BHs compounds, 5f was found to be the most effective
antiplasmodial (Table 1). Deferoxamine, a standard iron chelator also exhibited antiplasmodial
activity in vitro (Table 1). Known antimalarials, chloroquine and artesunate, were used as positive
controls (Table 1).
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Structure activity relationship study. Based on the antimalarial activity of synthesized
hydrazones we attempted to search for the active functional group behind their activity. Previous
results showed that among the synthesized molecules BHs were most effective in chelating iron and
against P. falciparum growth compared to other hydrazone derivatives (compound 1-4 (a-f)). Various
hydrazones derived from aromatic hydrazines bearing differential substitution like chloro-fluoro (1a-
f), bromo (2a-f), cyano (3a-f) and hydroxyl (4a-f) could not enhance their antimalarial activity. Hence,
extra conjugation of electron withdrawing group (-CN) or electron donating group (-OH) along with
substitution of halogen groups on aromatic ring did not show any enhanced activity of hydrazones. In
contrast, benzothiazole hydrazine derived molecules showed higher antimalarial as well as iron
chelating activity (Table 1). Conjugation of simple aromatic system fused with nitrogen and sulphur
substituted five membered ring might be the probable factor behind the improved activity of BHs.
Among the BHs methoxy-substituted aldehydes were ineffective as evident from the data of iron
chelation, heme binding and in vitro antimalarial activities (Table 1 and 2). 3,5- and 3,4-hydroxy
substituted BHs were found to be the more effective whereas only 4-hydroxy substituted BH was
futile against P. falciparum (Table 1). When iron chelating and antiplasmodial activities were
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compared, compound 5f (3,4-hydroxy substituted benzothiazole) was found to be the most efficient
one.
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Compound 5f interacts with Fe(III). To further understand the interaction of hydrazone with
Fe(III), we did IR spectroscopy of the compound 5f alone upon forming its complex with Fe(III). The
IR spectrum of compound 5f exhibited prominent peaks corresponding to the two O-H groups (peak
corresponding to bonds marked with (d)) at 3466.41 cm-1 (stretch) and one hydrazone N-H (peak
corresponding to bonds marked with (b)) at 3245.81 cm-1 (stretch) (Fig. 2A). A higher frequency
stretching of aromatic 5-membered C=N (peak corresponding to bond marked with (a)) appeared at
1602.60 cm-1 (stretch) and hydrazone C=N (peak corresponding to bond marked with (c)) appeared at
1562.03 cm-1 (stretch) (Fig. 2A). In contrast, the complex as a black precipitate exhibited a broad peak
at 3418.17 cm-1 instead of two distinct peaks of O-H and N-H present in compound 5f. IR peak
corresponding to bond (b) (N-H stretching) at 3245.81 cm-1 was totally disappeared in the complex.
The position of peaks of compound 5f at 1602.60 cm-1, 1513.32 cm-1, 1458.23 cm-1 were shifted in
compound 5f-Fe(III) complex at1627.21 cm-1, 1532.94 cm-1, 1467.74 cm-1 respectively indicating the
involvement these specific bonds for iron complexation (Fig. 2B). Stretching frequency at 1562.03
cm-1 (peak corresponding to bond marked with (c)) in parent compound was disappeared in Fe(III)-
compound 5f complex indicating the alteration of hydrazone C=N (Fig. 2B).
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Benzothiazole hydrazones (BHs) interact with heme. Heme is an iron containing tetrapyrrole
and because hydrazones can interact with iron, we became interested to test heme interacting property
of BHs. The binding of BHs to heme was evaluated by differential optical spectroscopy and
subsequently, binding constants were determined (Fig. 3 and Table 2). The approximate KD values for
the binding of BHs to heme were analyzed from the plot of 1/Δλmax against 1/[BHs] (Fig. 3, insets).
Compound 5f exhibited the highest affinity to heme (KD = 1.17 ± 0.8 µM) as evident from the
comparative analysis of the dissociation constant (KD) of heme interaction of various BHs, (Table 2).
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BHs inhibit hemozoin (β-hematin) formation by parasite lysate in vitro. Heme-interacting
molecule may inhibit hemozoin formation (39-40). Thus, to check the effect of these molecules on
hemozoin formation, we measured hemozoin formation in vitro by parasite lysate in presence or
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absence of different BHs. Data revealed that BHs in a concentration-dependent manner inhibited
hemozoin formation (Fig. 4). Dose-response studies indicated that among BHs, compound 5f
followed by compound 5d and 5e showed potent activity against hemozoin formation (Fig. 4).
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Compound 5f inhibits hemozoin (β-hematin) formation and [3H]-hypoxanthine uptake in P.
falciparum. Compound 5f being the most active compound in the series of BHs, its efficacy in
inhibition of hemozoin formation was further evaluated in parasite in culture. P. falciparum was
treated with increasing concentrations of compound 5f and kept for 48 h. Data indicated that
inhibition of hemozoin formation in P. falciparum was significantly increased with the increasing
concentrations of compound 5f (Fig. 5A). Compound 5f, concentration-dependently, not only
inhibited hemozoin formation but also P. falciparum growth as evident from [3H]-hypoxanthine
incorporation into parasitic DNA (Fig. 5B). Treatment with vehicle (DMSO) did not show any effect
on parasite growth (data not shown).
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Iron supplementation inhibits the antiplasmodial activity of compound 5f in vitro in P.
falciparum culture. Next, we were interested to see the effect of iron supplementation on the
antiplasmodial activity of compound 5f in vitro in P. falciparum culture. We performed iron
supplementation assay with various concentration of FeCl3 (1-100 µM) against fixed concentration of
compound 5f (25 µM, in which around 75% inhibition of parasite growth was observed). Iron
supplementation offered although not complete but significant protection against the antiplasmodial of
compound 5f. The data indicated that the antiplasmodial activity of compound 5f was gradually
decreased in the presence of increasing concentration of FeCl3 (Fig. 6). No significant change in the
parasite growth was observed when iron is supplemented in the absence of compound 5f (data not
shown).
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In vitro antimalarial activity of compound 5f against chloroquine resistant P. falciparum K1
strain. Now we were interested to evaluate the antimalarial activity of compound 5f against a
chloroquine and pyrimethamine resistant strain of P. falciparum (K1 strain) in vitro. Interestingly,
compound 5f offered significant antiplasmodial activity in a concentration- dependent fashion (Fig.
7). The IC50 of compound 5f was found to be 14.17 ± 1.34 µM. Both artesunate and chloroquine were
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used as reference drugs to validate the resistant strain of P. falciparum. While artesunate (100 nM)
inhibited the growth of parasite but chloroquine (100 nM) which was effective against 3D7 strain,
failed to inhibit the growth of this parasite (Fig. 7).
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In vivo antimalarial activity of compound 5f against multidrug-resistant (MDR) malaria
parasite P. yoelii. Next we were interested to explore in vivo antimalarial activity of the lead
compound 5f against lethal MDR (P. yoelii) strain in murine model. Post-infection administration of
compound 5f (from day 4 to day 7) showed dose-dependent antimalarial activity in vivo (Table 3).
Compound 5f not only suppressed the mean parasitemia by 31, 57, and 74% at dosage of 10, 25 and
50 mg.kg-1 respectively but also significantly increased the survival of the infected mice as evident
from the number of living mice in treated groups compared to infected control (Table 3).
Administration of compound 5f did not show any mortality on non-infected mice at the highest
concentration (50 mg.kg-1) we used for antimalarial evaluation (data not shown). The suppression of
parasite growth by compound 5f in Swiss albino mice was evaluated by counting parasitemia on the
slides of thin blood smears under the light microscope during infection. Occurrence of lethal P. yoelii
parasite on day 7 was measured in infected control, α/β-arteether (50 mg.kg-1) treated group and the
experimental group treated with increasing concentrations of compound 5f (Table 3). Compound 5f
showed antimalarial activity against MDR strain of P. yoelii, where existing halofantrine, mefloquine,
and chloroquine were futile as described earlier (20). At a dose of 50 mg.kg-1 α/β-arteether showed
100% parasite clearance, whereas compound 5f showed parasite suppression in the range of 60–90%
(Table 3). Antimalarial activity of compound 5f against P. yoelii was also evaluated in vivo by
following parasite growth suppression after performing Peter’s 4-day test. Administration of
compound 5f (intraperitoneal) produced dose-dependent chemosuppression (Table 4). Significant
reduction of parasitemia was observed in treated mice compared to vehicle treated control. α/β-
arteether was used as a positive control (Table 4).
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Cytotoxicity analysis of compound 5f. After evaluating the antimalarial activity of compound
5f, we were keen to check the cytotoxic activity of this compound 5f in the host. Therefore, we
monitored hemolysis by measuring the release of hemoglobin from RBC in presence/absence of
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various concentrations of compound 5f. Data revealed that compound 5f did not produce any
significant hemolysis at concentrations (0-100 µM) (Fig. 8A). Saponin was used as a positive control.
We also checked the release of hemoglobin in presence or absence of compound 5f (100 µM) by
Soret spectroscopy (Fig 8A, inset). Spectral analysis also revealed that the compound did not cause
significant hemolysis (Fig 8A, inset) compared to saponin. We also assessed the cytotoxic effect of
compound 5f in mammalian HepG2 and HEK 293 cells by MTT reduction assay. Data indicated that
compound 5f did not produce any significant toxicity to HepG2 as well as HEK 293 cells at
concentrations even higher than the concentration required for exhibiting in vitro antimalarial activity
(Fig 8B).
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Biochemical and hematological evaluation. Having performed all the in vitro toxicity assays,
we next checked any probable in vivo toxicity of compound 5f by measuring biochemical as well as
hematological parameters in mice after compound 5f treatment for four successive days. Data
revealed that compound 5f did not alter the levels of ALT, AST, GGT, total bilirubin, conjugated
bilirubin and unconjugated bilirubin indicating no hepato-toxicity upon treatment with an effective
dose of compound 5f (Table 5). The serum level of both creatinine and urea in the presence of
compound 5f was found to be close to the range of normal control level (Table 5), which indicated
that this compound did not significantly affect renal functions at the effective dose. Hematological
parameters were also assessed for possible toxicity of compound 5f in vivo. Compound 5f did not
produce any significant change in hemogram of treated mice compared to control (Table 6).
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DISCUSSION
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In this study, we report the synthesis of a series of hydrazone derivatives, which exhibited iron
chelating and antiplasmodial activities in vitro. We also provide evidence that compound 5f, a lead
molecule, exhibited a high free heme binding property and significantly inhibited hemozoin formation
in P. falciparum. Moreover, compound 5f offered antimalarial activity in vivo against lethal MDR
strain of P. yoelii in mouse model.
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Iron is crucial for many biochemical reactions involved in the growth and multiplication of the
malaria parasite that can be repressed by iron chelators. A number of biochemical studies have
indicated that deprivation of iron would adversely affect the activity of ribonucleotide reductase,
cytochrome and other vital cellular functions, which in turn inhibit the growth of the malaria parasite
(16, 21). Parasite digests host hemoglobin and as a consequence free heme is released, which is very
toxic to the parasite (3, 14, 41). However, parasite converts toxic free heme into a less toxic hemozoin
for its own survival (42). Iron chelators may interact with free heme through the iron present at its
center. This kind of interaction may subsequently hinder hemozoin formation from free heme, which
in turn may lead to inhibition of parasite growth. Among the synthesized molecules, hydroxylated
hydrazones showed more free iron chelating activity compared to the methoxylated hydrazones.
Hydroxyl group contains labile hydrogen atom, which may be replaced or can take part in conjugation
during chelation with iron whereas in case of methoxy group, the C-O bond is too strong to be
replaced in the normal physiological condition. We found that introduction of polar or ionizable
groups within compounds rendered them more active for free iron chelation. To find the outcome of
iron chelation on parasite health, antimalarial activity of synthesized hydrazones was screened by
SYBR green assay. Highest inhibition was obtained for the hydroxylated compounds of heterocyclic
BHs. The data further suggested the importance of ionizable hydroxyl groups on iron chelation and its
consecutive effect on parasite growth. Among the synthesized hydrazones, compound 5f was found to
be the most active compound. Interaction of compound 5f with Fe(III) was further evaluated by
complex formation and subsequent identification by infrared spectroscopy. IR spectroscopy is a
spectroscopic tool which provides the valuable information on alteration in bond vibration when
compound bind to a metal compared to original compound. Omission and shifting of specific
stretching frequencies corresponding to particular bonds in complex provide indication towards their
interaction with metal. We evaluated the alterations in bond vibration to explore the involvement of
specific bonds of the lead molecule (compound 5f) when it forms a complex with Fe(III). Owing to
structural similarity of BHs they are expected to interact with Fe(III) in the similar way and reducing
the need for testing compound-Fe(III) complex formation with other BHs. Thus, spectral changes in
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Fe(III)-compound 5f complex occurred as a consequence of re-orientation of bonds in compound 5f in
the presence of Fe(III).
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Compound, which interacts with or chelates iron, may also interact with iron present in free heme
and inhibition of hemozoin formation by novel heme-interacting molecule is considered to be an
effective way to prevent the growth of malaria parasites (43-44). Hence, synthesized active BHs were
screened against heme binding activity by differential spectroscopy. Compound 5f showed the lowest
dissociation constant (KD) for heme interaction as well as maximum inhibitory power against
hemozoin formation. To see the impact of the inhibition of hemozoin formation in parasite growth, we
concurrently measured the content of hemozoin in parasite and growth of parasite at different
concentrations of compound 5f. Concentration-dependent inhibition of both hemozoin formation and
parasite growth was observed with compound 5f. At this point, we were interested to see the effect of
iron supplementation on antimalarial activity of compound 5f. Iron supplementation was supposed to
produce free iron-compound 5f complex decreasing the affinity of compound 5f towards free cellular
iron. Iron supplementation significantly reduced the antimalarial activity of compound 5f. However,
the inhibition of antimalarial activity upon iron supplementation was not absolute because compound
5f might have some other mechanisms that inhibit parasite growth. Inhibition of hemozoin formation
may be one of such ways as compound 5f exhibited a significant inhibition of hemozoin formation.
We observed that deferoxamine showed higher iron chelation activity but less antimalarial activity
than compound 5f. The reason behind this may be the inability of deferoxamine to inhibit hemozoin
formation (45). We further checked the antimalarial activity of compound 5f against resistant P.
falciparum K1 strain. Compound 5f significantly inhibited P. falciparum growth both in chloroquine
sensitive and chloroquine/pyrimethamine resistant strains. In vitro active antiplasmodial molecules
may be active or inactive in vivo because under in vivo conditions there are many other factors that
may play a significant part in deciding drug activity. The drug may encounter absorption problem or it
may undergo degradation by host cells. Moreover, a drug must be specific in targeting parasite
without harming host cells. The advantage of small molecules as the drug is their lesser probability of
degradation in physiological conditions. In vivo drug testing is important because it shows the effects
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of a new compound on a living subject. On the other hand, development of resistance in parasite
against the most effective drugs in use is a serious issue and demands immediate attention (46).
Therefore, after confirming activity of compound 5f in chloroquine sensitive and resistant P.
falciparum strains, we used a multiple drug resistant P. yoellii parasite for in vivo drug testing. We
performed animal drug testing under high parasitimic conditions to determine the drug efficacy when
the number of parasite is higher enough to cause physiological stress in the host (47). Moreover, in
such conditions, we were able to compare the lead molecule with the frontline anti-malarial α/β-
arteether that is known to be effective in a similar situation (47). Compound 5f significantly
suppressed the parasite growth. The activity of this molecule against drug resistant parasites pointed
out its efficacy in avoiding common mechanisms of drug resistance present in malaria parasite. After
the evaluation of antimalarial activity of compound 5f, we measured both in vitro and in vivo toxicity
of this compound before claiming its therapeutic potential as a drug. Hemolysis of host RBCs by any
compound may also lead to the death of malaria parasite. To check whether compound 5f has got any
hemolytic activity we performed hemolysis assay. The data indicated that compound 5f was non-
hemolytic; thus, the antimalarial activity is not due to RBC lysis. Furthermore, we evaluated the
cytotoxicity of the compound 5f in HepG2 as well as HEK293 cells where no significant change in
cell viability was observed indicating the non-toxic nature of compound 5f in vitro against
mammalian cells. Since xenobiotic metabolism is mostly attributed to liver and kidney therefore
evaluation of liver and kidney functions was extremely relevant to check any potential in vivo toxicity
of compound 5f. Therefore, mice were treated for 4 successive days with compound 5f whereupon
serum levels of AST, ALT, ALP, GGT, bilirubin, creatinine and urea along with hematological
parameters were stringently measured. All the biochemicals as well as hematological parameters were
found to be well comparable to the control (healthy) mice indicating negligible or non-toxic nature of
compound 5f. Thus, we identified a lead compound 5f, active in vivo against resistant and highly
lethal P. yoelii and have the potential to be developed as a novel antimalarial.
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The effectiveness of current antimalarial treatments is threatened by the naturally occurring evolution
of the parasites that allow selection of resistant strains. Existing antimalarial drugs mostly contain a
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few basic chemotypes against which malaria parasite has become resistant (48). As the threat of
antimalarial drug resistance grows, there is growing pressure that demands imperative discovery of
new antimalarial drugs that are able to overcome these issues. Here, we have identified benzothiazole
hydrazones as a new chemotype that can prevent the growth of malaria parasite. This chemotype may
be used as a scaffold to design next generation antimalarials. Proper investigation of its structure
activity relationship and its suitable optimization by side chain modifications may result in better
bioavailability and improved efficacy. Thus, compound 5f is a lead chemotype, which has shown
antimalarial activity both in vitro and in vivo against resistant parasite can be modified into effective
compound by fragment-based modification or combination therapy with artemisinin and other well-
known antimalarials.
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ACKNOWLEDGMENTS
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This work was supported by Council of Scientific and Industrial Research, New Delhi, through
grants (SPlenDID, BSC 0104) and offering fellowship to Souvik Sarkar and Asim Azhar Siddiqui to
carry out this work. The financial support from DST (J. C. Bose Fellowship) is also acknowledged.
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Sonnet P, Mullie C. 2011. In vitro antimalarial activity of ICL670: a further proof of the
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hemin. Exp Parasitol 128:26-31.
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Mita T, Tanabe K, Kita K. 2009. Spread and evolution of Plasmodium falciparum drug
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Dey S, Mazumder S, Siddiqui AA, Iqbal MS, Banerjee C, Sarkar S, De R, Goyal M,
Bindu S, Bandyopadhyay U. 2014. Association of heme oxygenase 1 with the restoration of
liver function after damage in murine malaria by Plasmodium yoelii. Infect Immun 82:3113-
3126.
48.
Calderon F, Wilson DM, Gamo FJ. 2013. Antimalarial drug discovery: recent progress and
future directions. Prog Med Chem 52:97-151.
651
652
653
654
655
656
657
658
659
660
661
662
25

663
LEGENDS
664
665
666
Fig.1. Synthesis of conjugated arylhydrazones. Hydrazones were synthesised using methanol at
room temperature. Different substitutions with percent yield of the respective compounds are
mentioned in parentheses.
667
668
669
670
Fig. 2. IR spectra of compound 5f and its iron complex. IR spectra of (A) compound 5f (B)
compound 5f + Fe(III). Structure of compound 5f with the bonds responsible for IR peaks is
assigned with alphabetical notation in the inset under (A). Arrows indicate the change of IR peaks
due to chelation.
671
672
673
674
675
Fig. 3. Interaction of benzothiazole hydrazones with heme. (A-F) Interaction of benzothiazole
hydrazones (Compound 5a-f) with heme was studied by using optical differential Soret spectroscopy
at different concentrations (1–20 µM) (i, 1 µM; f, 20 µM). Compound names are indicated in
brackets. The inset shows the plot of 1/λmax(nm) vs. 1/[compounds 5a-f] used to calculate KD values of
benzothiazole hydrazones-heme interaction.
676
677
678
679
680
681
Fig. 4. Inhibition of hemozoin formation by benzothiazole hydrazones. Compounds (5a-f) inhibit
hemozoin formation and favour the accumulation of free heme in the parasite. Hemozoin formation
was measured in the presence or absence of benzothiazole hydrazones as described under
“MATERIALS AND METHODS”. Values are normalized by use of the upper and the lower plateaus
of the best-fit curve as 100 and 0% inhibition of hemozoin formation, respectively. Each experiment
was performed in triplicate. Data are shown as means ± S.D.
682
683
684
685
686
687
688
Fig. 5. Dose-dependent inhibition of hemozoin formation and [3H]-hypoxanthine uptake by
compound 5f. (A) Hemozoin formation was measured from P. falciparum culture with increasing
dose of compound 5f. Data are presented as % inhibition of hemozoin formation compared to control.
Data are the mean ± S.D. of three independent experiments (**, p < 0.05 versus control). (B) P.
falciparum growth was monitored by following the incorporation of [3H]-hypoxanthine into parasite
DNA and expressed as average radioactive count per minute (CPM) in a liquid scintillation counter.
Data are the mean ± S.D. of three independent experiments (*, p < 0.5; **, p < 0.05 versus control).
26

689
690
691
692
693
694
695
Fig. 6. Effect of iron supplementation on the antimalarial activity of compound 5f in vitro. The
effect of compound 5f (25 µM) on P. falciparum growth was followed in presence of increasing
concentrations of FeCl3 (0-100 µM). After 48 hours of treatment, parasite was lysed in 100 µl lysis
buffer containing 2X SYBR Green nucleic I acid stain and incubated for 1 hour. Data were collected
upon excitation at 485 nm and following the emission at 530 nm in terms of fluorescence intensities.
The experiment was performed in triplicate. Data are shown as means ± S.D. (*, p < 0.5; **, p < 0.05
versus control).
696
697
698
699
700
701
Fig. 7. Antimalarial activity compound 5f in chloroquine and pyrimethamine resistant P.
falciparum (K1 strain). The growth of chloroquine and pyrimethamine resistant P. falciparum K1
strain was evaluated in the presence of different concentrations of compound 5f (1-50 µM).
Artesunate (100 nM) and chloroquine (100 nM) were used as a positive and negative control,
respectively. The experiment was performed in triplicate. Data are shown as means ± S.D. (**, p <
0.05; ***, p < 0.005 versus control).
702
703
704
705
706
707
708
Fig. 8. Compound 5f did not show cytotoxic effect in vitro. (A) RBC lysis was measured in
presence of compound 5f in various concentrations (0-100 µM) at 2 h and 12 h. Inset; Soret spectra of
supernatant of untreated RBCs and RBCs incubated with saponin/compound 5f (100 µM) at the time
points 2 h and 12 h has been provided in the range of 300-700 nm. (B) The viability of HepG2 and
HEK293 cell was assessed by MTT assay of triplicate cultures, expressed as percentage of untreated
control. Data represent mean ± SD (**, p < 0.05 versus 0 µM in HepG2; ##, p < 0.05 versus 0 µM in
HEK293).
27

Table 1.
Iron chelating and in vitro antimalarial activities of synthesized conjugated hydrazone derivatives.
Iron chelating activity
In vitro antimalarial
Compound
(50% drop in TPTZ-Fe(II) activity (SYBR green assay)
absorbance)
(µM)a
IC50
(µM)a
1a
1b
1c
1d
1e
1f
83.86 ± 5.30
75.24 ± 8.57
>100
>100
11.73 ± 2.73
9.73 ± 1.89
NA
NA
NA
59.48 ± 9.55
71.33 ± 6.93
42.96 ± 3.63
2a
2b
2c
2d
2e
2f
83.66 ± 9.33
>100
>100
>100
13.90 ± 2.82
15.12 ± 4.15
NA
NA
NA
62.16 ± 8.54
78.85 ± 7.22
65.56 ± 4.91
3a
3b
3c
3d
3e
3f
65.24 ± 10.95
53.58 ± 7.30
>100
>100
8.94 ± 3.14
12.26 ± 4.63
NA
NA
NA
>100
>100
89.44 ± 10.74
4a
4b
4c
4d
4e
4f
21.37 ± 4.15
12.48 ± 1.68
>100
>100
2.45 ± 0.99
2.51 ± 1.97
NA
NA
NA
92.98 ± 6.93
>100
76.55 ± 9.16
5a
5b
5c
5d
5e
5f
8.28 ± 3.22
6.10 ± 1.81
43.73 ± 5.89
78.11 ± 8.25
2.35 ± 0.35
1.87 ± 0.24
0.72 ± 0.19
ND
>100
NA
NA
27.11 ± 2.87
53.87 ± 4.32
16.95 ± 1.19
27.13 ± 2.03
0.020 ± 0.0012
0.015 ± 0.0009
Deferoxamine
Chloroquine
Artesunate
ND
a Values are means ± standard errors of the means of a single parallel determination run in triplicate.
ND: Not determined

Table 2.
Heme binding activity of benzothiazole hydrazones (BHs)
Compound
Heme binding
KD (µM)
5a
5b
5c
5d
5e
5f
9.69 ± 1.8
9.23 ± 2.3
11.19 ± 4.1
5.58 ± 1.2
3.03 ± 0.9
1.17 ± 0.8
Differential optical Soret spectroscopy was performed in order to determine BHs–heme interaction
and KD values were calculated from 1/λmax(nm) vs 1/[compounds 5a-f] (Fig. 3). KD = dissociation
constant for compound-heme interaction. All experiments were performed in triplicate. Data are
shown as mean ± S.D. (n=3).

Table 3.
Antimalarial activity of compound 5f against MDR strain of P. yoelii in vivo in mice model.
Compound
Dose
(mg/kg)
% Parasitemia
Day 6
% Suppression
of parasitemiaa
(Mean ± S.D.)
Survival rate
Day 2
2.5 ± 0.3
3.5 ± 1.1
2.8 ± 0.8
2.6 ± 1.3
3.2 ± 0.4
Day 4
Day 7
58.6 ± 9.5
40.3 ± 3.3
24.8 ± 2.6
14.8 ± 2.6
0
Control
5f
13.3 ± 2.2
15.8 ± 0.9
14.9 ± 1.9
14.1 ± 1.4
14.2 ± 1.4
42.5 ± 4.5
28.7 ± 2.9
18.6 ± 3.1
12.6 ± 3.1
6.5 ± 0.7
0
0/6
0/6
2/6
5/6
6/6
10
25
50
31.2 ± 3.6
57.7 ± 6.4
74.7 ± 8.3
100
α/β arteether
Swiss albino mice (six mice in each group) were infected by MDR (chloroquine, mefloquine, and
halofantrine) strain of P. yoelii and subsequently treated with compound 5f (10, 25 and 50 mg/kg
body weight) from Day 4 to Day 7 intra-peritoneally to follow parasitemia with time.
a
Percentage suppression of parasitemia at Day 7 was calculated as [(C–T)/C]×100, where C is the
parasitemia in the control group and T is the parasitemia in the treated group. Data represent the mean
± S.D. (n = 6). Survival number was counted at Day 10 (in control group all mice were dead at Day
7).