Aryl aryl methyl thio arenes prevent multidrug-resistant malaria in mouse by promoting oxidative stress in parasites

Free Radical Biology and Medicine 53 (2012) 129–142

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Original Contribution

Aryl aryl methyl thio arenes prevent multidrug-resistant malaria in mouse by promoting oxidative stress in parasites Manish Goyal a, Priyanka Singh b, Athar Alam a, Sajal Kumar Das b, Mohd Shameel Iqbal a, Sumanta Dey a, Samik Bindu a, Chinmay Pal a, Sanjit Kumar Das b, Gautam Panda b,n, Uday Bandyopadhyay a,n a b

Division of Infectious Diseases and Immunology, CSIR–Indian Institute of Chemical Biology, 4, Raja S. C. Mullick Road, Jadavpur, Kolkata 700032, West Bengal, India Medicinal and Process Chemistry Division, CSIR–Central Drug Research Institute, Chattar Manzil Palace, M. G. Marg, Lucknow 226001, UP, India

a r t i c l e i n f o

abstract

Article history: Received 31 August 2011 Received in revised form 18 April 2012 Accepted 23 April 2012 Available online 3 May 2012

We have synthesized a new series of aryl aryl methyl thio arenes (AAMTAs) and evaluated antimalarial activity in vitro and in vivo against drug-resistant malaria. These compounds interact with free heme, inhibit hemozoin formation, and prevent Plasmodium falciparum growth in vitro in a concentrationdependent manner. These compounds concentration dependently promote oxidative stress in Plasmodium falciparum as evident from the generation of intraparasitic oxidants, protein carbonyls, and lipid peroxidation products. Furthermore, AAMTAs deplete intraparasite GSH levels, which is essential for antioxidant defense and survival during intraerythrocytic stages. These compounds displayed potent antimalarial activity not only in vitro but also in vivo against multidrug-resistant Plasmodium yoelii dose dependently in a mouse model. The mixtures of enantiomers of AAMTAs containing 3-pyridyl rings were found to be more efficient in providing antimalarial activity. Efforts have been made to synthesize achiral AAMTAs 17–23 and among them, compound 18 showed significant antimalarial activity in vivo & 2012 Elsevier Inc. All rights reserved.

Keywords: Oxidative stress Hydroxyl radical Malaria Aryl aryl methyl thio arenes (AAMTA) Antimalarial activity Drug resistance

Introduction Malaria is a major health problem in developing countries. According to the World Health Organization (WHO), it infects more than 300 million people per year and causes more than one million deaths annually, mostly among young children in the age group of less than 5 years [1]. Malaria is reemerging as the biggest infectious killer and is currently a first-priority tropical disease [2,3]. The management of malaria is becoming extremely difficult due to limitations associated with vaccine development [3,4], vector control, and increasing spread of resistance against established antimalarial drugs [5,6]. Therefore, it is necessary to search for new antimalarial drug, which will be effective against multidrug-resistant parasites in vivo. The intraerythrocytic stages of malaria parasites are mainly responsible for clinical manifestations associated with the malaria [7]. During intraerythrocytic stages, malaria parasites

Abbreviations: AAMTAs, aryl aryl methyl thio arenes; H2O2, hydrogen peroxide; OH, hydroxyl radical; PBN, phenyl-a-tert-butyl-nitrone; P. falciparum, Plasmodium falciparum; IC50, Inhibitory concentration required to inhibit 50% hemozoin formation; EC50, Inhibitory concentration required to inhibit 50% P. falciparum growth; KD, dissociation constant n Corresponding authors. Fax: þ 91 33 24735197. E-mail addresses: [email protected] (G. Panda), [email protected] (U. Bandyopadhyay). 

0891-5849/$ - see front matter & 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.freeradbiomed.2012.04.028

degrade vast amounts of hemoglobin inside food vacuoles and release globin along with large quantities of toxic pro-oxidantfree heme [8–10]. During the process of oxidation of oxyhemoglobin [oxyHb (Fe þ 2)] to methemoglobin [met-Hb (Fe þ 3)] electrons are liberated, resulting in the formation of superoxide anions (O2  ). O2  is either dismutated by P. falciparum cytosolic superoxide dismutase (PfSOD1) to yield hydrogen peroxide (H2O2) or can react with H2O2 to form hydroxyl radicals (OH). Furthermore, the interaction between heme and intracellular H2O2 releases iron (Fe þ 3), which can generate oxidants via the Fenton reaction [8,9]. Therefore, free heme (Fe þ 3) offers a major toxic insult to the parasite, which if allowed to accumulate may reach to 300–500 mM [2,11,12] and lead to the development of oxidative stress [13,14]. Free heme (Fe þ 3) can intercalate in the membrane causing changes in membrane permeability and lipid organization and induces lipid peroxidation of parasite membrane [15,16], which ultimately leads to parasite death. The malaria parasite is very much susceptible to oxidative stress during intraerythrocytic stages [8,9,17]. This free heme not only causes oxidative stress to parasites but the host cell is also equally susceptible, as observed from changes in RBC membrane fluidity, possibly due to changes of membrane lipid composition, lysing erythrocytes and inducing cell death [16,18–23]. It also inhibits key metabolic enzymes as well as redox-active molecules (glutathione) and perturbs cellular redox balance [24,25]. However, parasites have unique rescue mechanisms against free heme

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toxicity by converting it into less toxic hemozoin [8,9,26]. Therefore, molecules which interact with heme and interfere in its conversion into hemozoin are effective antimalarials [8,9,14,26]. Apart from hemozoin formation, to endure oxidative burden, malaria parasites also contain diverse antioxidants such as glutathione and thioredoxin-dependent proteins, superoxide dismutase, g-glutamyl-cysteine synthetase, glutathione reductase, glutathione-S-transferase, thioredoxin reductase (TrxR), thioredoxins thioredoxin-dependent peroxidases (TPx), and peroxiredoxin [8,17]. Apart from these antioxidant proteins, P. falciparum macrophage migration inhibitory factor (PfMIF) also exhibits thioredoxin (Trx)-like oxidoreductase activity and may also contribute to reduce the oxidative burden [27]. Numerous drugs exhibit potent antimalarial activity by targeting these crucial enzymes or preventing hemozoin formation and thereby promoting oxidative stress [9,28–30]. Trisubstituted methane (TRSM) has the capability to interact with heme and this class of molecules also inhibits hemozoin formation and offers antimalarial activity [28,30–32]. Here, we report the synthesis of a large number of TRSM derivatives, aryl aryl methyl thio arenes (AAMTAs) and evaluation of their antimalarial activities in vitro and in vivo. The data indicate that these molecules interact with free heme, inhibit hemozoin formation, promote oxidative stress in parasites, and prevent P. falciparum growth. These AAMTAs also show antimalarial activity in vivo against a multidrugresistant strain of parasite (MDR strain, P. yoelli) in a BALB/c mouse model.

Chemical synthesis Detailed methods for synthesis and characterization of all aryl aryl methyl thio arenes derivatives are provided separately (Fig. 1 and see the additional information in the supplementary data).

Parasite culture P. falciparum was cultured according to the method of Trager and Jensen [33,34]. In brief, parasite culture was maintained at a hematocrit level of 5% in complete RPMI 1640 medium supplemented with 25 mM Hepes, 50 mg ml  1 gentamycin, 370 mM hypoxanthine, and 0.5% (w/v) AlbuMaxII in tissue culture flasks with loose screw caps. The medium was regularly changed with fresh medium once a day. The growth of P. falciparum was regularly checked after Giemsa staining of thin blood smears. In vivo growth of P. yoelii was maintained in BALB/c mice. Male BALB/c mice (20–25 g) were inoculated intraperitoneally with P. yoelii (MDR strain) as described earlier [18–20]. Parasitemia was monitored by preparing thin smears of blood and subsequent Giemsa staining. Experiments on animals were conducted after obtaining permission from the animal ethics committee and in accordance with the institutional guidelines for the care and the use of laboratory animal.

Preparation of parasite lysate Materials and methods Materials Hemin, RPMI 1640, saponin, SDS, chloroquine, glutathione (GSH), thiobarbituric acid (TBA), trichloroacetic acid (TCA), a-phenyl-n-tert-butyl-nitrone (PBN), dichlorofluorescein diacetate (DCF-DA), fetal calf serum (FCS), manitol, dimethyl sulfoxide (DMSO), penicillin, streptomycin, actinomycin D, and 2,4-dinitrophenylhydrazine were purchased from Sigma (St. Louis, MO, USA). Albumax II was procured from Life Technologies (USA). Giemsa stain was purchased from Qualigens Fine Chemicals (India). [3H]Hypoxanthine was purchased from Amersham Biosciences (USA). Cell Proliferation Reagent WST-1 was procured from Roche Applied Science. All other chemicals were of analytical grade purity.

Parasites (P. falciparum and P. yoelii) were isolated as described [33,35]. In brief, erythrocytes with either  10% parasitemia (P. falciparum) or 50% parasitemia (P. yoelii, from infected mice blood) were centrifuged at 800 g for 5 min, washed twice, and resuspended in cold phosphate-buffered saline (PBS, 137 mM NaCl, 2.7 mM KCl, 5.3 mM Na2HPO4, and 1.8 mM KH2PO4). For erythrocytes lysis, an equal volume of 0.5% saponin in PBS (final concentration 0.25%) was added to the erythrocyte suspension and kept on ice for 15 min. It was further centrifuged at 1300g for 5 min to obtain parasite pellet. The pellet was subsequently washed with PBS thrice and either used immediately or kept at  80 1C. The isolated parasite was lysed in PBS by mild sonication (30 s pulse, bath-type sonicator) at 4 1C and the lysate was then stored at –20 1C for further use. Protein content of the parasite lysate was estimated by the method of Lowry et al. [36].

Fig. 1. Scheme of synthesis of aryl aryl methyl thio arenes (4–16) along with achiral AAMTAs (17–23). Reagents and conditions are as follows: (a) dry THF, rt, 1 h; (b) conc H2SO4, dry benzene, reflux, 0.5 h or anhydrous AlCl3, dry benzene, rt, 0.5 h.

M. Goyal et al. / Free Radical Biology and Medicine 53 (2012) 129–142

Heme interaction studies The interaction of the synthesized AAMTAs with heme was analyzed by differential optical spectroscopy as described earlier [28,29]. In brief, reaction assembled in a total volume of 1 ml containing heme (1 mM) in 100 mM sodium acetate buffer, pH 5.2, and differential optical spectra were recorded using a Perkin Elmer Lamda 15 UV/VIS spectrophotometer at 2571 1C with quartz cells of 1 cm light path. Binding of AAMTAs to native heme was monitored at different concentrations (1–20 mM) of AAMTAs. For difference spectra of heme –AAMTAs versus the heme, both the reference and the sample cuvettes were filled with 1 ml of heme solution (1 mM) to provide the baseline trace. It is followed by the addition of different concentrations of AAMTAs (1–20 mM) to the sample cuvette along with concomitant addition of the same volume of DMSO (used to dissolve the AAMTAs, 0.5– 10 ml) to the reference cuvette. The contents were mixed well before the spectrum was recorded. Soret spectrum without AAMTAs and after successive addition of AAMTAs was recorded immediately. The equilibrium dissociation constant (KD) for complex formation was calculated from the expression as described by Schejter et al. [37]. 1=DA ¼ ðK D =DAa Þ1=S þ1=DAa where KD is the dissociation constant of the heme–AAMTAs complex, S is the concentration of AAMTAs, DA is the observed absorption changes at a particular wavelength, and DAa is the absorption changes at saturating concentration of the ligand (AAMTAs). Assay of hemozoin (b-hematin) formation In vitro hemozoin (b-hematin) formation was assayed as described earlier [38–40]. In brief, the reaction was assembled in a final volume of 1 ml containing 100 mM sodium acetate buffer, pH 5.2, 100 mM hemin, parasite lysate from P. yoelli (20 mg) along with or without different concentrations of AAMTAs (1–100 mM). The reaction without AAMTAs was treated as control. The reaction was started by the addition of hemin and incubated for 12 h at 37 1C to allow the conversion of hemin in to hemozoin. The reaction was terminated by centrifugation at 15,000g for 10 min at room temperature. The reaction pellet was washed thrice with 100 mM Tris buffer, pH 7.8, containing 2.5% SDS and finally with 100 mM bicarbonate buffer, pH 9.2. The resultant insoluble pellet (hemozoin) was then solubilized in 50 ml of 2 N NaOH and diluted further to 1 ml with 2.5% SDS. The absorbance of the solution was recorded at 400 nm using a Perkin Elmer Lamda 15 UV/VIS spectrophotometer at 2571 1C with quartz cells of 1 cm light path. Extinction coefficient of 91 mM  1 cm  1 was used to quantitate the amount of free heme converted to hemozoin and expressed as nanomole of hemozoin formed per milligram of parasite lysate [38]. Measurement of intraparasitic oxidants Intraparasitic oxidant production was monitored by measuring the fluorescent dichlorofluorescein (DCF) oxidation product of nonfluorescent probe dichlorofluorescein diacetate and hydroxyl radical (OH) after AAMTA treatment at different concentrations [41–44]. To check the effect of AAMTAs on oxidant production, P. falciparum was cultured in the presence or absence of different concentrations (1–20 mM) of AAMTAs for a period of 48 h. The culture was then further incubated for a period of 30 min in complete RPMI medium containing 10 mM 20 ,70 -dichlorofluorescein diacetate. The culture was subsequently washed thrice with PBS and the parasite was isolated from control (without AAMTAs

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treatment) and treated groups as stated above. Isolated parasites were lysed by mild sonication (5-s pulse, bath-type sonicator) at 4 1C and DCF-reactive products were measured in control or AAMTA-treated parasites. Fluorescent intensities were recorded from the lysate in a Hitachi F-7000 fluorescence spectrofluorometer in a 5-mm path-length quartz cell in a total volume of 1 ml at wavelength 502 and 523 nm for excitation and emission, respectively. DCF-reactive products were measured as relative fluorescence and expressed as fluorescence intensity per milligram of parasite lysate. Hydroxyl radical (OH) generated as a consequence of oxidative stress in the P. falciparum after AAMTA treatment at different concentrations was also measured using dimethyl sulfoxide as  OH scavenger as described earlier [43,44]. In brief, P. falciparum culture (200 ml) (4% parasitemia) was grown in multiwell plates in the presence or absence of different concentrations of AAMTAs containing DMSO (the final concentration of DMSO was maintained at 1%) for 48 h. The parasite alone (without DMSO and AAMTAs) was used as control, whereas parasite with only DMSO (1%) was used as negative control. DMSO was added in each time along with the specific concentrations of AAMTAs when the medium was changed (once in 24 h). After 48 h, the culture was centrifuged at 800 g for 5 min, washed, and resuspended in cold PBS. The parasite was isolated as described above and the isolated parasite was lysed in deionized water and processed for the extraction of methanesulfinic acid formed by the reaction of  OH with DMSO. Methanesulfinic acid formed was allowed to react with Fast Blue BB salt and the intensity of the resulting yellow chromophore was measured at 425 nm using benzenesulfinic acid as standard to quantitate the amount of OH formed and expressed as nanomole of OH per milligram of parasite lysate. Measurement of reduced glutathione (GSH) GSH content of control and AAMTA-exposed parasite was measured by using a fluorometric method using the commercially available glutathione assay kit (BioVision, Mountain View, CA, USA). In order to measure intraparasitic reduced glutathione concentration, P. falciparum (4% parasitemia) was cultured in the presence or absence of different concentrations of AAMTAs. To perform the experiments, essentially the same protocol was followed as described in the kit with slight modifications. In brief, the assay kit provided probe o-phthalaldehyde (OPA) which reacts with GSH but not with GSSG and generating fluorescence to specifically quantify GSH. After 48 h of AAMTA treatment (1–20 mM), the culture was washed twice with PBS and subsequently parasite was isolated. Isolated parasite was sonicated in 200 ml of assay buffer using a sonicator (9 s on/10 s off cycle for a period of 60 s) and centrifuged at 10,000g for 20 min to obtain clear lysate and glutathione was stabilized with 6 N perchloric acid. After that 10 ml of OPA was mixed with supernatant and incubated at room temperature for 40 min according to the manufacturer’s instruction. Samples were then read on a fluorescence plate reader equipped with Ex/Em¼340/450 nm (Hitachi F7000, Tokyo, Japan). GSH was used as a standard to quantitate the reduced glutathione level and expressed as nanomole of GSH per milligram of parasite lysate. Assessment of lipid peroxidation and protein carbonyl formation in P. falciparum To analyze the oxidative stress induced by AAMTAs, we also checked the formation of lipid peroxidation and protein carbonyl formation in P. falciparum. Oxidative stress results in the formation of highly reactive and unstable lipid hydroperoxides. The unstable peroxides after decomposition produced malondialdehyde (MDA),

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48 h. The percentage growth inhibition was calculated by considering CPM of control sample (without any treatment) as 100% survival. CPM observed in blank sample was subtracted to eliminate the background signal. Dose-response curves were constructed to obtain the EC50 values. All experimental data were derived from at least 3 independent experiments.

which can be quantified colorimetrically following its controlled reaction with thiobarbituric acid. P. falciparum culture (4% parasitemia) was treated with or without different concentrations of AAMTAs (1–20 mM) for 48 h. After treatment, parasite was isolated to prepare parasite lysate and the lipid peroxidation product (as thiobarbituric acid-reactive substances, TBARS) from lysate was measured as described earlier [44,45]. In brief, parasite lysate was treated with 1 ml TCA (trichloroacetic acid)–TBA (thiobarbituric acid) mixture in 1 N HCl and incubated 15 min at 100 1C. After incubation, it was cooled and centrifuged (4000 rpm for 10 min). After the centifugation, supernatant was collected and absorbance was measured at 535 nm using a Shimadzu UV/VIS spectrophotometer. Tetraethoxypropane was used as a standard and formation of lipid peroxide in parasite membrane was expressed as nanomole formed per milligram of protein. Formation of protein carbonyl was measured as described previously [46]. In brief, P. falciparum culture (4% parasitemia) was treated with different concentrations of AAMTAs for 48 h and after that parasite lysate was prepared from isolated parasite. Parasite lysate was treated with 10% trichloroacetic acid for protein precipitation and allowed to react with 0.5 ml of 10 mM 2,4-dinitrophenylhydrazine for 1 h. After that, the protein was washed thrice with a mixture of ethanol:ethylacetate (1:1), dissolved in 0.6 ml of a solution containing 6 M guanidine-HCl in 20 mM potassium phosphate adjusted to pH 2.3 with trifluoroacetic acid. The solution was centrifuged, and the supernatant was used for the measurement of carbonyl content by following the absorbance at 362 nm.

The antimalarial efficacy of AAMTAs in vivo was evaluated in a rodent model as described earlier [28,29]. In brief, BALB/c mice were infected by MDR (chloroquine, mefloquine, and halofantrine) strain of P. yoelii (N-67) and subsequently treated intraperitoneally at three dose levels (5, 10, 25 mg/kg body weight) of AAMTAs. For each dose level, a group of six mice (25 75 g) was inoculated with 1  105 parasitized RBCs on Day 0 and AAMTAs were administered on Days 2, 3, 4, and 5 of postinfection. The treatment was continued at each dose level from Day 2 to Day 5. An aqueous suspension (emulsion) of drugs was prepared in ground-nut oil so as to obtain the required drug dose per animal in 0.2 ml volume. The efficacy of active AAMTAs was assessed by continuous monitoring of the effects of the drugs on percentage parasitemia and animal survival. Level of parasitemia in mice was monitored by Giemsa staining of thin blood smears every day. The mean value resulting for each group of mice was used to calculate the percentage of suppression in parasitemia with respect to the vehicle control group. a,b-arteether was used as a positive control at a dose level of 50 mg/kg/day body weight.

In vitro antimalarial activity

Toxicity assay

P. falciparum growth inhibition following AAMTA treatment was studied by using [3H]hypoxanthine uptake as described previously [47]. Parasites were cultured in vitro and synchronized by using 5% aqueous D-sorbitol to achieve ring stages as described earlier [48]. In brief, P. falciparum culture was centrifuged at 500g for 5 min to pellet the cells. The supernatant media were discarded and packed cells were suspended in 5 vol of 5% Dsorbitol and allowed to stand for 15–20 min at 37 1C. The cells were washed twice with complete RPMI medium (without albumax II) to remove sorbitol and cell debris. Pellet was suspended to complete RPMI 1640 medium into a final hematocrit of 2–3%. Synchronization of the culture was confirmed by microscopic examination of Giemsa-stained thin smear slides. To check the antimalarial activity of AAMTA compounds, the ring synchronized P. falciparum (parasitemia 0.5–1%) was cultured in multiwell (200 ml/well) plates in the presence or absence of different concentrations of AAMTAs (0.1–4 mM). Parasite culture without any treatment was used as control, whereas DMSO and chloroquine (5 nM–1 mM) were used as negative and positive controls, respectively. After 48 h, [3H]hypoxanthine (0.7mCi/well) was added in each well and further incubated for 48 h to monitor parasite viability by measuring incorporation of free [3H]hypoxanthine in parasite nucleic acids. After 48 h, culture was harvested and washed thrice in PBS. Packed RBCs were centrifuged and dissolved in 100 ml 3 N NaOH, kept at 37 1C for 6 h, and then added in (10 ml/vial) scintillation fluid (PPO, 4 g; POPOP, 200 mg; naphthalene, 60 g; ethylene glycol, 20 ml; methanol 100 ml in 1 liter of 1,4-dioxane). After 12 h of addition, [3H]hypoxanthine uptake was measured using a b-scintillation counter. In order to access the effect of hydroxyl radical scavengers on AAMTAinduced growth inhibition, P. falciparum growth was measured by following [3H]hypoxanthine uptake in the presence or absence of hydroxyl radical scavengers (PBN and mannitol). P. falciparum culture (4% parasitemia) was treated with AAMTAs (4.0 mM) alone or AAMTAs along with PBN (50 mM) or mannitol (10 mM) for

Drug-induced hemolysis is a relatively rare but serious toxicity. Therefore, the hemolytic activity of AAMTAs was evaluated to screen for toxic hemolytic effects at sufficiently high concentrations of AAMTAs, if any. To evaluate in vitro hemolysis, hemoglobin release following AAMTAs exposure was measured. Freshly isolated RBCs (human) were (1  105) incubated in 96-well plates either in the presence of DMSO (control), saponin (0.5%) or in the presence of different concentrations of AAMTAs (1–1000 mM) in complete RPMI (CRPMI) medium at 5% hematocrit level for 4 h at 37 1C. After incubation, cells were pelleted by centrifugation at 13,000 rpm for 15 min and supernatant was used for measuring the release of hemoglobin. Percentage hemoglobin release was measured by measuring OD at 540 nm. All hemolysis data points were presented as the percentage of the complete hemolysis. The hemoglobin released by saponin (0.5%) was considered as 100% hemolysis. In vitro cytotoxicity of AAMTAs was also evaluated using human leukemic (U937) and liver cancer (HepG2) cell lines. The cytotoxic effects of different concentrations of AAMTAs on U937 and HepG2 cell lines were evaluated as described earlier [49]. In brief, cells were obtained from culture medium by centrifugation at 500g for 5 min. The cell pellets were resuspended and loaded in a hemocytometer for cell count before plating of the cells. Cells were seeded in a 96-well flat-bottomed tissue culture microplate at a concentration of 1  105 cells per well and incubated for 24 h. After incubation, the medium was removed from all wells of the microplate and then fresh 200 ml MEM (minimal essential media) was added for each well along with different concentrations of AAMTAs (1 mM–1 mM) and incubated for 24 h at 37 1C in a humidified atmosphere containing 5% CO2. To analyze timedependent toxicity of AAMTAs, U937 and HepG2 cells were incubated in the presence and absence of AAMTAs (100 mM) for different time periods (6, 12, 18, and 24 h) as well. DMSO (negative control) and actinomycin D (25 mM) were used as positive control. Before addition to the culture medium, the AAMTAs were dissolved in DMSO and followed by serial dilution to achieve the desired

In vivo antimalarial activity

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concentration. The final concentration of DMSO used in the corresponding wells did not exceed 1% (v/v). Control cultures had the same concentration of solvent alone (1% DMSO) and used as a negative control. Cell viability was measured using WST-1 (watersoluble tetrazolium salts) following the manufacturer’s protocol. In brief, at the end of incubation, 10 ml of cell proliferation reagent WST-1 was added to each well and incubated for 3 h. After incubation, the plates were thoroughly shaken on an ELISA shaker for 30 s. The absorbance of each well was recorded at 450 nm using an ELISA reader (Beckman Coulter, CA, USA) with a reference serving as blank and subtracted to eliminate the background absorbance. The viability of control cells was considered as 100% and was measured from the absorbance value. The viability of treated cells was calculated similarly from the absorbance values and expressed as percentage of control viability. Dose–response curves were constructed to obtain the IC50 values. In order to test the toxic effects of AAMTAs when administered in vivo at therapeutic dosages, serological markers of liver, kidney, and muscle damage were assayed. For each dose level, a group of six BALB/c mice (2575 g) was administrated AAMTAs intraperitoneally for 4 days. The suspension (emulsion) of drugs was prepared in ground-nut oil so as to obtain the required drug dose per animal in 0.2 ml volume. Mice treated with ground-nut oil alone were used as control. The animals were acclimated to optimal conditions of temperature (2572 1C) and light/dark cycle (12 h each) before initiation of drug administration. All animals were sacrificed by cervical dislocation on Day 5 of study and liver was taken out after perfusion with normal saline and a part of it was kept at -80 1C until further analysis. Prior to sacrifice blood was taken out from cardiac puncture from each animal and left undisturbed for 30 min for serum separation. Serum was separated by centrifugation at 600g for 5 min and kept at  20 1C. For liver function assay, enzyme activities of alanine transaminase (ALT), aspartate transaminase (AST), and alkaline phosphatase (ALP) were measured. We also measured the total amount of bilirubin (TBIL) and conjugated or direct bilirubin (DBIL) to analyze liver damage. These assays served as parameters to evaluate the extent of hemolysis and liver damage. Creatinine in serum serves as a parameter for kidney injury. Therefore, we measured the level of creatinine in serum of control and drug-treated mice. All assays were performed by using kits purchased from Randox Laboratories Ltd. (Ardmore, Diamond Road, Crumlin, Co. Antrim, UK). The manufacturer’s instructions were strictly followed. To analyze the effect of drugs in muscle we measured the activity of creatine kinase (CK) in serum of control and drug-treated mice. Serum creatine kinase activity was analyzed using a creatine kinase assay kit (BioAssay Systems, Hayward, CA, USA), following the manufacturer’s protocol.

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anhydrous AlCl3 at room or reflux temperature furnished AAMTAs 4–23 (Fig. 1). Failure of purifying one of the enantiomers of 3b along with its cost for large-scale production prompted us to access achiral AAMTAs. Consequently, to have antimalarial activity of achiral AAMTAs, we specifically replaced pyridine rings of racemic AAMTAs by substituted aryls. Thus, syntheses of achiral AAMTAs containing substituted benzene rings 17–23 were undertaken. The compounds 17–23 were synthesized (Fig. 1) essentially following a similar reaction sequence as described (supplementary material). The yields of the synthesized compounds along with their calculated CSLogP values are given in (Table 1). AAMTAs interact with heme and inhibit hemozoin (b-hematin) formation The interaction of AAMTAs with heme was followed at pH 5.2, approximating the pH of the parasite food vacuole to mimic the cellular milieu inside the parasite. The binding of AAMTAs to heme was studied by optical differential spectroscopy and the binding affinity of these compounds was calculated (Fig. 2 and Table 2). The apparent KD values for the binding of AAMTAs to heme were calculated from the plot of 1/DA362 against 1/[AAMTAs] are shown in the insets (Fig. 2). KD values of heme interaction were compared and out of various AAMTAs, compounds 7, 9, 10, 12, 13, 14, and 16 showed better heme interaction at a KD value in the range of 4–8 mM (Table 2). Antimalarial drugs such as chloroquine and amodiaquine (type 1 blood schizontocides) act by forming complexes with heme (the hydroxo or aqua complex of ferriprotoporphyrin IX (Fe (III) PPIX). Such interaction with heme leads to the inhibition of hemozoin formation. Inhibition of hemozoin formation by antimalarial drugs is attributed mainly to the interaction with free heme [9,28,29]. Therefore, we further checked whether interaction of AAMTAs with heme could inhibit hemozoin formation and offer antimalarial activity. The data clearly indicated that AAMTAs inhibited hemozoin formation in a concentration-dependent manner (Fig. 3 and Table 2). Among AAMTAs, compounds 7, 9, 10, 12, 13, and 16 showed much higher activity (IC50 5–10 mM) in inhibiting hemozoin formation as observed from dose– response curves used to derive the IC50 values (Fig. 3). Comparative analyses of the effect of AAMTAs on hemeozoin inhibition with that of other well-established type 1 blood schizontocides drugs (chloroquine, mefloquine, and halofantrine) revealed that IC50 values for the inhibition of hemozoin formation in vitro were much lower compared to well established type 1 blood schizontocides drugs (Table 2).

Statistical analysis

AAMTAs promote oxidative stress in P. falciparum

Statistical analysis was performed by using the Student t test or analysis of variance. P value of o0.05 was considered as statistically significant for all the experiment.

Compounds, which interact with free heme and inhibit hemozoin formation, may allow the accumulation of pro-oxidant free heme to exhibit antimalarial activity [9,28,29]. Accumulation of free heme (Fe þ 3) produced vast amounts of cellular oxidant and developed oxidative stress. Therefore, selected AAMTAs (7, 9, 10, 12, 13, and 16), based on their superior heme interacting property and ability to inhibit hemozoin formation, were further tested in P. falciparum culture to evaluate oxidative stress. DCFH-DA can be used for measurement of redox-active iron signaling and cellular oxidants generation [41]. Therefore, we used DCFH-DA as redox indicator probe to measure the generation of oxidant through redox-active iron signaling. The data indicated that AAMTA treatment induced significant generation of intraparasitic oxidant level as measured by DCF-reactive product (Fig. 4A). Among AAMTAs tested, compounds 9, 10, 12, and 16 were shown to be highly potent (Fig. 4A). We have also check the formation of

Results Chemistry Synthesis of aryl aryl methyl thio arenes requires a series of carbinols on which addition of nucleophiles (aryl or heteroaryl thiols) may be performed. Toward this objective, carbinols 3a–m were synthesized by Grignard reaction of arylmagnesium bromide 1a–f with arylcarbaldehydes 2a–f. Treatment of carbinols 3a–m with different aryl or heteroaryl thiols in the presence of catalytic amounts of conc H2SO4 under reflux condition or with

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Table 1 Structure, yields, and CS log P values of synthesized aryl arylmethyl thio arenes (AAMTAs). Compound (AAMTAs)

CS log P Mean7 SEM

% Yield

4

3.897 0.8

78

5

4.017 0.8

68

6

3.457 1.0

63

7

3.417 1.0

61

8

5.207 1.2

64

9

5.077 1.1

64

5.17 1.3

64

10

Structure

Table 1 (continued ) Compound (AAMTAs)

Structure

CS log P Mean 7SEM

% Yield

18n

5.17 7 0.89

76

19n

4.31 7 1.15

78

20n

4.37 0.84

79

21n

4.93 7 1.1

71

22n

4.097 0.89

70

23n

4.64 7 1.07

68

The CS log P values of the AAMTAs were calculated by using the property prediction software CSPredict (ChemSilico LLC, Tewksbury, MA). n Achiral aryl aryl methyl thio arenes (AAMTAs 17–23 containing substituted benzene rings).

11

5.667 0.9

68

12

5.137 1.4

64

13

5.157 1.0

60

14

5.257 1.1

81

15

5.187 1.4

64

16

3.097 0.9

60

17n

4.37 0.83

70

hydroxyl radical (OH) inside parasites after treating the parasite with different concentrations of AAMTAs. AAMTAs also induced the generation of highly reactive OH in a concentration-dependent manner (Fig. 4B), when compared with the control (without DMSO and AAMTAs). DMSO (1%) alone did not have any significant effect on parasite growth as well as OH generation. GSH plays an essential role in antioxidant defense mechanisms by maintaining the redox state of protein thiol (–SH) groups, reducing the lipid peroxides, and allowing degradation of free heme [50]. Apart from this, GSH is also involved in termination of radical-based chain reactions where single electron is transferred from disulfide radical [51]. Generally, oxidative stress significantly reduced the cellular GSH level by oxidizing GSH to GSSG. Because AAMTAs promote oxidative stress, we check the level of GSH in P. falciparum after the exposure of selected AAMTAs at varying concentrations under culture conditions. The data showed that AAMTAs significantly decreased the GSH level in P. falciparum in a concentration-dependent fashion (Fig. 5) and out of these compounds, 7, 10, 12, and 16 were found to be effective. This decreased GSH level therefore may disturb cellular redox homeostasis and cause oxidative damage to critical biomolecules, such as lipids and proteins, leading to lipid peroxidation and protein carbonylation, respectively. Parasites when treated with varying concentrations of AAMTAs also induced lipid peroxidation [measured as thiobarbituric acid-reactive substances (TBARS)] (Fig. 6A) and protein oxidation (carbonyl formation) (Fig. 6B). Among selected AAMTAs, compounds 7, 10, 12, and 16 showed significant formation of lipid peroxide and protein carbonyl (Fig. 6A and B).

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Fig. 2. Interaction of AAMTAs with heme. Interaction of AAMTAs with heme was studied by using optical differential Soret spectroscopy at different concentrations of AAMTAs (1–20 mM) (A to H). Compound names are indicated in brackets. Inset shows the plot of 1/D360 nm vs 1/[AAMTAs] used to calculate KD values of AAMTA–heme interaction.

AAMTAs act as potent antimalarial The data so far presented indicate that AAMTAs interact with free heme, inhibit hemozoin formation, deplete cellular GSH level, and oxidatively damage biomolecules. In order to assess whether the oxidative stress induced by AAMTAs is responsible for the inhibition of parasite growth and development, the antimalarial

activity of synthesized AAMTAs was evaluated by following [3H]hypoxantine uptake. The synthesized AAMTAs showed significant antimalarial activity (Table 3) and among AAMTAs, compounds 7, 10, 12, and 16 were found to be very potent as observed from EC50 values (42.0 mM) derived from dose response curves (Fig. 7). Chloroquine was used as positive control, whereas DMSO (concentration used to dissolve AAMTAs) alone

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Table 2 Heme interaction and inhibition of hemozoin formation. Compound (AAMTAs)

Heme binding KD (mM)

Inhibition of hemozoin formation, IC50 (mM)

4 5 6 7 8 9 10 11 12 13 14 15 16 Chloroquinen Mefeloquinen Halofantrinen 4an 4bn 4en

12 71.9 14.4 7 1.4 12.3 7 1.2 6.25 7 0.8 8.25 7 0.9 7.67 7 1.5 4.76 7 0.6 15.5 7 2.1 4.26 7 0.4 7.22 7 0.3 5.11 7 0.5 8.33 7 1.1 4 657 0.8 ND ND ND 20 74 26 76 12 73

947 4.5 567 4.5 707 5.4 57 0.24 5.47 0.3 107 0.9 5.97 0.7 587 5.9 5.27 0.4 57 0.98 207 1.2 237 1.9 57 0.4 24.4 46.9 184.9 187 4 267 5 207 4

Differential optical Soret spectroscopy was performed in order to determine AAMTA–hemin interaction and KD values were calculated. In vitro hemozoin formation was determined in the presence and absence of AAMTAs and values were calculated from the dose response curve (Fig. 7). KD ¼dissociation constant for AAMTAs–heme interaction, IC50 ¼ inhibitory concentration required to inhibit 50% hemozoin formation; ND, not done. All experiments were performed in triplicate. Data are shown as mean 7 SEM (n ¼6). n

See Refs. [14,28].

Fig. 3. Inhibition of hemozoin formation by AAMTAs. AAMTAs inhibit hemozoin formation and favor the accumulation of free heme in parasite. Hemozoin formation was measured in the presence or absence of AAMTAs as described under Materials and methods. Each experiment was performed in triplicate. Data are shown as mean 7SEM. nPo 0.001 versus control (n¼ 6).

was used as negative control. EC50 values of chloroquine were calculated and checked with the reported values (Table 3) [52], while DMSO did not affect significantly parasite growth (data not shown). Structure–function analysis of synthesized AAMTAs reveals that compounds 7, 12, and 16 having 3-pyridyl rings showed more activity than compound 13 having a 2-pyridyl ring. Interestingly, introducing methoxy and thiomethoxy groups on benzene rings at meta and para positions showed better antimalarial activity as evident from compounds 7, 12, and 16 (para isomer) and compound 10 (meta isomer) in comparison with

compound 13 having these substituents at the ortho position. This is possibly due to better exposition of p-methoxy and p-thiomethoxy moieties of central pharmacophore skeleton. On the other hand, replacement of benzoxazole moiety in compound 7 with 2-pyridyl in compound 16 does not have much effect on antimalarial activity. In order to assess whether the oxidative stress induced by AAMTAs is actually responsible for the inhibition of parasite growth and development, we further checked the effect of different antioxidants or OH scavengers, such as mannitol and spin traps like phenyl-a-tert-butyl-nitrone (PBN) on AAMTAinduced death of P. falciparum. Results indicated that OH scavengers significantly protected P. falciparum from AAMTAinduced parasite growth inhibition (Fig. 8) and suggest that the development of oxidative stress may be the antimalarial mode of action of AAMTAs.

In vivo antimalarial activity of AAMTAs against multidrug-resistant (MDR) malaria parasite P. yoelii In vitro antimalarial activity of AAMTAs encouraged us to evaluate the effect of AAMTAs against multidrug-resistant rodent malaria parasite P. yoelii under in vivo condition. We selected the most active in vitro screened AAMTAs for in vivo evaluation of antimalarial activity. AAMTAs dose dependently showed antimalarial activity under in vivo conditions and compounds 7, 12, and 16 were found to be highly active (Table 4). At a dose of 25 mg/kg body weight, compounds 7, 12, and 16 not only suppressed the mean parasitemia by 81, 90, and 85%, respectively, but also showed the significant survival of the infected mice as evidenced from te number of surviving mice in treated groups (Table 4). However, compounds 10 and 13 were comparatively less effective when compared with the dose–response antimalarial efficacy (Table 4). Synthesized AAMTAs are racemic in nature and therefore are mixtures of enantiomers. It is difficult to understand which form of enantiomer (R or S) is biologically active. Moreover, the resolution into one of the pure enantiomers will not be very feasible for large-scale synthesis (Fig. 1). Hence, primary emphasis was given to the generation of achiral molecules. Out of seven achiral molecules (compounds 17–23), only compound 17 and 18 showed good antimalarial activity. Compound 18 was found to be better than 17, which suppressed the mean parasitemia by 82% (at a dose of 25 mg/kg body weight) and moderately cured the infected mice when compared with the other AAMTAS (Table 4). Analysis of in vivo data of achiral molecules 17–23 suggests that incorporation of methoxy and thiomethoxy substituent on benzene rings did not enhance significant antimalarial activity (Tables 1 and 4). The achiral 17 and 18 containing unsubstituted benzene rings exhibited significant antimalarial activity in vivo compared to their substituted benzene ones (methoxy and thiomethoxy at any position of benzene ring). Significant enhancement in antimalarial activity has been observed by change of thiol moiety from 2-pyridyl to benzoxazole, as compound 17 suppressed the (Day 6) mean parasitemia by 60%, and compound 18 by 82%, at dose levels of 25 mg/kg. However, if we compare the antimalarial activity of all racemic and achiral AAMTAs in vivo, compounds 7, 12, 16, and 18 showed very significant antimalarial activity. It is worth noting that these new series showed antimalarial activity in vivo in the MDR strain of P. yoelii, where chloroquine, mefloquine, and halofantrine were ineffective at the same dose (25 mg/kg) (Table 5). AAMTAs, at a dose of 25 mg/kg showed in vivo parasite suppression in the range of 60–90%, whereas a,b-arteether showed 100% parasite suppression (Table 5).

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Fig. 4. AAMTAs induce the generation of oxidants in P. falciparum. (A) AAMTAs induce the redox-active iron signaling and oxidant generation (A) and OH (B). The levels of DCF-reactive products and OH in P. falciparum were measured at different concentrations of AAMTAs as described under Materials and methods. Each experiment was performed in triplicate. Values are expressed as nmol of product formed per mg of parasite lysate. C represents control group. Data are shown as mean 7 SEM. *Po 0.001 versus control, #Po 0.002 versus control (n¼ 6).

Fig. 5. AAMTA treatment depletes GSH level in P. falciparum. Cellular GSH content in P. falciparum was measured after the treatment of different concentrations of AAMTAs as indicated. GSH content was measured as described under Materials and methods. Each experiment was performed in triplicate. C represents the control group. Values are expressed as nmol of product formed per mg of parasite lysate. Data are shown as mean 7 SEM. *Po 0.001 versus control, **P o 0.002 versus control, #P o 0.01 versus control (n ¼ 6).

Toxicity studies So far, the data clearly indicated that achiral 18 and a mixture of enantiomers 7, 12, and 16 showed very good antimalarial activity under in vivo conditions. However, synthesized AAMTAs may also cause toxicity or damage to host cells. To eliminate this possibility, we evaluated the toxicity of active achiral 18 and mixture of enantiomers 7, 12, and 16 under in vitro and in vivo using a variety of experiments. To analyze any toxic effects exerted by AAMTAs on noninfected red blood cells, hemolysis assay was performed (Fig. 9). The in vitro hemolysis indicates drug-induced hemoglobin release (as an indicator of red blood cell lysis) following drug exposure. The data documented that compounds 7, 12, 16, and 18 did not exert any toxic effect on red blood cells at  100-fold higher concentrations of the effective antimalarial concentration used. However, compound 10 showed comparatively higher toxicity (Fig. 9).

In order to evaluate the cytotoxicity of selected active AAMTAs, cytotoxicity assays were also performed using leukemic cell (U937) and liver cell lines (HepG2). Cells were incubated with 1 mM to 1.0 mM AAMTAs, and the cell viability was determined by following the percentage of viable cells at varying concentrations of AAMTAs (1, 10, 100, 500, and 1000 mM) at 24 h (Fig. 10A and B) of treatment analyzed by WST-1 assay. The active achiral 18 and a mixture of enantiomers 7, 12, and 16 did not show any significant cytotoxicity as observed from IC50 values derived from dose response curves (Fig. 10 and Table 6). Time-dependent effects of AAMTAs on U937 and HepG2 cells were analyzed following cell viability in the presence and absence of AAMTAs for different time periods (Fig. 10C and D). Actinomycin D (anticancer drug), used as positive control, caused a significant mortality with a 20% survival in 24 h at 25 mM concentration, while DMSO (1%) did not exert any significant toxicity (Fig. 10C and D). In order to analyze that the concentrations used in cells are comparable with the concentrations used with the parasites, we have considered a selectivity index (SI). The selectivity index showed a comparison of compound therapeutic effects (antimalarial effect, IC50) to the concentration that causes toxicity (cytotoxicity IC50). Selectivity index of compounds 7, 12, and 16 is in the range of 130–590, which is greater than 100, suggesting that these compounds are not cytotoxic to both U937 and HepG2 cell lines (Table 6). In vivo toxicity studies of AAMTAs at a therapeutic dosage were done to assess the effect of these molecules on normal biochemical parameters (serological markers) of liver, kidney, and muscle function in BALB/c mouse (Table 7). AAMTAs, when administrated in vivo (25 mg/kg body weight for 4 days), did not significantly affect clinically important enzyme levels such as ALT, AST, TBIL, DBIL, creatinine, and CK (Table 7). However, perhaps compounds 12, 16, and 18 might be superior compared with compound 7 with regard to serological markers, when compared with the control (Table 7).

Discussion In this study, we have synthesized a new series of TRSM derivatives, aryl aryl methyl thio arenes and presented evidence that these compounds interact with heme and inhibit hemozoin formation. These compounds exhibit antimalarial activity and promote oxidative stress in erythrocytic parasites. We have also presented evidence that selective in vitro active AAMTAs show

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Fig. 6. AAMTAs develop oxidative stress in P. falciparum. The generation of lipid peroxide (A) and protein carbonyl (B) at different concentrations of AAMTAs as described under Materials and methods. Each experiment was performed in triplicate. C represents the control group. Data are shown as mean 7SEM. *P o 0.001 versus control, # P o 0.002 versus control (n ¼6).

Table 3 In vitro antimalarial activity of synthesized aryl aryl methyl thio arenes (AAMTAs). Compound (AAMTAs)

Antimalarial activity (inhibition of hypoxanthine uptake), EC50 (mM)

4 5 6 7 8 9 10 11 12 13 14 15 16 Chloroquinen Mefeloquinen Halofantrinen 4an 4bn 4en

44 44 4.2 7 0.4 1.45 7 0.08 44 3.4 7 0.29 1 70.08 44 1.95 7 0.09 3 70.2 44 44 1.50 7 0.07 0.015 0.042 0.010 5.0 71 8.0 72 4.0 70.5

In vitro antimalarial activity was determined following [3H]hypoxanthine uptake using synchronized P. falciparum culture, in the presence and absence of AAMTAs (0.1–4.0 mM).The EC50 (effective concentration required to inhibit 50% P. falciparum growth) values were calculated from the dose response curve (Fig. 7). Parasite culture without any treatment was used as control, whereas DMSO and chloroquine were used as negative and positive controls respectively. All experiments were performed in triplicate. Data are shown as mean 7SEM (n¼ 6). n

See Refs. [14,28,52].

antimalarial activity in vivo against a multidrug-resistant strain of parasite (MDR strain, P. yoelli) without any significant toxicity at the effective antimalarial dose. Several conventional approaches have been suggested during the recent past to discover new therapies against malaria including optimization and developing analogs of existing drugs, testing of compounds from natural products, identifying resistancereversal agents, and exploiting combination of chemotherapeutic approaches [53]. These approaches were successful in producing clinically useful drugs; however, discovering new antimalarial drug against multidrug-resistant and rapidly mutating malarial parasites is warranted. Analysis of antimalarial drug-resistant Plasmodium through genetic, molecular, and pharmacological approaches has shown that different targets are resistant to mutation on their key enzymes or transporters [5]. These includes

Fig. 7. Effect of AAMTAs on P. falciparum growth. (A) AAMTAs inhibit parasite growth as measured by [3H]hypoxanthine uptake. [3H]Hypoxanthine uptake was measured in the presence or absence of AAMTAs as described under Materials and methods. Each experiment was performed in triplicate. Data are shown as mean 7SEM. *Po 0.001 versus control, #P o0.01 versus control (n ¼6).

chloroquine, atovaquone, antifolates (pyrimethamine and proguanil), sulfonamides, and sulfones [5]. Artemisinin and their derivatives, the safest treatment against multidrug-resistant P. falciparum malaria, are also not the exception of drug resistance today [54]. Thus, identification or design of new drugs against resistant malaria parasite is now mandatory. Hemozoin formation is a validated target with the highest significance because it seems to be a chemical/physical process and therefore this target is not easily susceptible toward resistance by mutation. Till now, the inhibition of hemozoin formation by novel heme-interacting molecule is considered to be an effective way to handle drug-resistant parasites. Keeping the above concept in mind, we synthesized a series of AAMTAs, a new class of TRSMs with significant antimalarial activity against drug-resistant malaria parasites (shows high level of virulence and produces 100% lethal infection) in vivo. Trisubstituted methanes (TRSMs) with or without sulfur spacers have been reported to exhibit various biological activities such as antibreast

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cancer [55], antitubercular [56–61], antiproliferative [62], and antiplasmodial [28,30–32]. These synthesized AAMTAs were screened initially based on their interaction with free heme. These compounds showed good heme interaction under conditions similar to parasitic food vacuoles. AAMTAs also inhibited hemozoin formation, which may allow the accumulation of free heme and initiated the iron-mediated redox signaling in parasite. Studies showed the role of redox-active iron in intracellular oxidation of DCFH to DCF. Therefore, we used dichlorofluorescein diacetate to measure redox-active iron signaling [41]. The data indicated that AAMTA treatments leads to induction of redox-

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active free iron signaling and oxidant generation as depicted from increased DCF-reactive products. The Fenton reaction is the basic underlying mechanism involved in the hydroxyl radical (OH) production through interaction of iron and H2O2 [63,64]. Therefore, we measured OH production in parasites after AAMTA treatment in culture and these compounds also produced OH. GSH depletion results in serious oxidative burden in parasites due to less efficient detoxification of free heme and inactivation of many enzymes as well as oxidation of protein and lipid after AAMTA treatment. Results suggested the significant reduction in glutathione level with the concomitant increment in protein Table 5 Comparative analysis of in vivo antimalarial activity of AAMTAs with known antimalarials against MDR Strain (P. yoelli).

Fig. 8. Effect of hydroxyl radical scavengers on AAMTA-induced inhibition of P. falciparum growth. P. falciparum growth was measured by following [3H]hypoxanthine uptake in the presence or absence of hydroxyl radical scavengers (PBN and mannitol) during AAMTAs treatment as described under Materials and methods. P. falciparum culture (4% parasitemia) was treated with AAMTAs (4.0 mM) alone or AAMTAs along with PBN (50 mM) or mannitol (10 mM) for 48 h. Each experiment was performed in triplicate. Data are shown as mean7SEM. **Po0.001 versus control, *Po0.01 versus treated (n¼ 6).

Compound

% suppressiona

Chloroquine Mefloquine Halofantrine a,b  Arteether 4an 4bn 4en 7 10 12 13 16 17 18

NE NE NE 100 307 5 307 4 657 6 817 9 607 5 907 8 607 5 857 8 607 5 827 4

NE, not effective when tested in vivo. a Percentage suppression (Day 6) was calculated at dose 25 mg/kg body weight 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 7SEM (n¼ 6). n As reported earlier, Ref. [28].

Table 4 In vivo antimalarial activity of the selective AAMTAs at different doses with time Compound

Dose (mg/kg)

Control

a/b arteether 7

10

12

13

16

17*

18*

50 5 10 25 5 10 25 5 10 25 5 10 25 5 10 25 5 10 25 5 10 25

% parasitemiaa Day 2

Day 4

Day 6

Day 8

37 0.23 37 0.3 37 0.27 37 0.26 27 0.19 37 0.32 3.57 0.4 2.97 0.3 2.67 0.24 2.97 0.31 2.57 0.23 2.97 0.27 2.97 0.32 2.57 0.21 37 0.33 2.57 0.2 2.77 0.2 3.27 0.27 37 0.3 37 0.21 3.57 0.27 2.97 0.3 2.87 0.24

8 7 0.9 2 7 0.11 6 7 0.5 5 7 0.4 5 7 0.6 7.9 7 0.8 5 7 0.52 4 7 0.23 4 7 0.34 3.5 7 0.23 3 7 0.2 9 7 0.76 7 7 0.6 6 7 0.56 5 7 0.35 4 7 0.26 4 .6 7 0.3 8 7 0.5 8 7 0.6 107 0.67 8 7 0.26 6 7 0.34 6 7 0.56

40 7 3.6 0 18 7 2.3 971 7 7 0.8 26 7 2.9 19.5 7 1.4 16 7 1.5 18.9 7 2.2 8.2 7 0.6 4.4 7 0.6 23 7 1.9 17 7 1.4 15.2 7 2.1 14.8 7 1.8 8 7 0.67 6 7 0.8 25 7 2.4 19 7 1.6 15.3 7 1.4 18.4 7 2 12 7 1.2 7 7 0.5

557 6.23 0 257 3.4 247 2.8 177 1.5 417 3.5 307 3 257 1.8 157 1.2 117 0.7 57 0.56 357 2.3 237 1.3 217 1.7 207 1.2 137 0.87 77 0.65 407 3.2 347 2.9 307 2.7 257 2.4 207 1.7 147 0.98

Mean % suppression of parasitemiaa

Survival rate

0 100 54 76.0 72 78.0 81 79.0 34 73.0 51 75.0 60 75.0 55 7 7.0 79 77.0 90 78.0 43 74.0 57 75.0 60 75.0 62 77.0 78 76.0 85 78.0 38 74.0 55 76.0 60 75.0 50 76.0 76 75.2 82 74.0

0/6 6/6 0/6 2/6 5/6 0/6 0/6 1/6 0/6 4/6 6/6 0/6 0/6 1/6 0/6 3/6 5/6 0/6 0/6 3/6 0/6 0/6 3/6

BALB/c mice were infected by MDR (chloroquine, mefloquine, and halofantrine) strain of P. yoelii and subsequently treated intraperitoneally with AAMTAs (5, 10, and 25 mg/kg body weight) to follow parasitemia with time. a Percentage suppression of parasitemia at Day 6 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 7 SEM (n¼6). Survival number was counted at Day 15 (in control group all mice were dead at Day 9). n Achiral aryl aryl methyl thio arenes.

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carbonyl and lipid peroxidation products. Furthermore, impaired parasitic growth as a consequence of AAMTA-mediated oxidative stress was regained by antioxidant treatment using PBN and mannitol. Apart from hemozoin formation inhibition, other ways of oxidative stress include inhibition of key redox regulatory proteins such as glutathione, thioredoxin and glutathione reductase, peroxidase, peroxiredoxin, and superoxide reductase [8]. However, we cannot exclude the possibility of the inhibition of these targets, which can modulate cellular redox state and therefore generation of oxidative stress after the treatment of AAMTAs. We also tested the effect of these potent antimalarial AAMTAs in vivo against the MDR strain of P. yoelli in a mouse model. In this Table 6 In vitro cytotoxicity of selected active AAMTAs against different cell lines.

Fig. 9. Hemolytic effect of AAMTAs. Hemolytic activity of AAMTAs was evaluated as described under Materials and methods. Freshly isolated human RBCs (1  105) were incubated in a 96-well plate either in the presence of DMSO (control) or saponin (0.5%) or different concentrations of AAMTAs as indicated. Hemoglobin release as a result of RBC damage was measured by following the absorbance at 540 nm. The hemoglobin released by saponin (0.5%) was considered as 100% hemolysis. Each experiment was performed in triplicate. Data are shown as mean 7 SEM. *Po 0.001 versus control, **P o 0.002 (n¼ 6).

AAMTAs

U 937 cell line IC50 (mM)a

U 937/NF-54 SIb

HepG2 cell line IC50 (mM)a

HepG2/NF-54 SIb

7 12 16 18

200 712 300 720 380 722 430 730

137.93 153.84 253.33 ND

342 7 23 450 7 32 890 7 35 950 7 32

235.86 230.76 593.33 ND

ND, not determined. a Each experiment is performed in triplicate and data are shown as mean 7 SEM (n¼ 6). b SI, selectivity index, calculated as cytotoxicity IC50 /antiplasmodial IC50.

Fig. 10. In vitro cytotoxicity of selected active AAMTAs against different cell lines. (A–D) The effect of AAMTAs on survival of U937 and HepG2 cells was evaluated by following WST-1(water-soluble tetrazolium salts) as described under Materials and methods. U937 cells (A) and HepG2 cells (B) were treated with varying concentrations of AAMTAs (1–1000 mM) for 24 h and dose response curves were plotted. In order to analyze time-dependent effects of AAMTAs, U937 cells (C) and HepG2 cells (D) were treated with AAMTAs (100 mM) for varying time points (6, 12, 18, and 24 h). Actinomycin D (25 mM) was used as positive control, whereas DMSO (1%) was used as negative control. The color absorbance of each well was recorded at 450 nm and was used to determine growth relative to control cells. Each experiment was performed in triplicate. Data are shown as mean 7 SEM. *P o 0.001 versus control. **P o0.002 versus control, #Po 0.05 versus control (n¼ 6).

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Table 7 Evaluation of in vivo toxicity of selective active AAMTAs by measuring the serological markers for liver, kidney, and muscles damage. Experimental groups

Control 7 12 16 18

Liver functiona

Kidney functiona

Muscle functiona

ALT (U/L)

AST (U/L)

ALP (U/L)

TBIL(mg/dl)

DBIL(mg/dl)

Cretinine (mg/dl)

CK(U/L)

18.067 1.2 18.87 2.5 18.96 7 1.37 19.75 7 1.4 16.78 7 2.5

43.1 7 3.5 50.79 7 2.65 49.96 7 4.8 40.67 7 4.98 48.56 7 3.65

102.93 79.54 117.01 79.42 110.32 78.47 98.74 78.36 112.01 710.45

1.14 7 0.18 1.62 7 0.34 1.16 7 0.39 1.32 7 0.33 1.18 7 0.31

0.23 7 0.014 0.41 7 0.024 0.21 7 0.09 0.29 7 0.024 0.22 7 0.18

0.52 7 0.045 0.72 7 0.039 0.49 7 0.037 0.63 7 0.038 0.62 7 0.045

10.5 70.86 9.7 71.1 8.5 70.95 9.7 71.2 9.96 70.9

Liver, kidney, and muscle function tests were performed in mice at Day 5 with or without administration of AAMTAs (4 days) at therapeutic dose 25 mg/kg body weight. The activity of the ALT, AST, CK, and ALP enzymes in serum is expressed in Units/liter (U/L), whereas that of TBIL, DBIL, and creatinine is expressed in mg/dl. ALT, alanine transaminase; AST, aspartate transaminase; ALP, alkaline phosphatise; TBIL, total amount of bilirubin; DBIL, conjugated or direct bilirubin; CK, creatine kinase. a

All experiments were performed in triplicate and data are shown as mean 7SEM (n¼ 6).

model artimisinin/a,b-arteether provides effective protection to the treated mice at a dose Z5 mg/kg. Therefore, we have tested AAMTAs at doses (5, 10, and 25 mg/kg body weight) comparable or higher than the effective dose of artimisinin/a,b-arteether for in vivo efficacy studies [65–68]. Compounds 7, 12, 16, and 18 (achiral) appeared to be potentially active during in vivo testing (suppressing mean parasitemia 81–90% at dose 25 mg/kg body weight). Specific modifications in the substitution pattern, like compounds having a 3-pyridyl ring, were more active than those with a 2-pyridyl ring. In addition, compounds having methoxy and thiomethoxy at para and meta positions of the benzene ring showed more activity than those having these substituents at ortho positions, presumably due to the better exposition of substituents at para positions. Toxicity studies of selected active AAMTAs suggest that these compounds are safe and efficacious for treating malaria in a mouse model. In conclusion, we have designed a novel class of heme-interacting compounds, which offers antimalarial activity in vivo against multidrug-resistant malaria. Thus, AAMTAs offer an opportunity and bear potential for animalarial therapy.

Acknowledgments The authors acknowledge Department of Science & Technology, New Delhi, for funding this project. Manish Goyal, Priyanka Singh, Sanjit and Sajal Kumar Das thank CSIR for providing fellowships. Priyanka Singh, Sanjit Kumar Das, and Sajal Kumar Das contributed equally to synthesize compounds described in this paper. We also thank Dr. S.K Puri (Division of Parasitology, CSIR-Central Drug Research Institute, Lucknow) for providing P. falciparum and P. yoelli culture. This has CSIR-CDRI communication No. 8245.

Appendix A. Supplementary information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.freeradbiomed. 2012.04.028.

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