Synthesis of novel amodiaquine analogs and evaluation of their in vitro and in vivo antimalarial activities

Azar Tahghighi; Arezoo Rafie Parhizgar; Safoura Karimi; Mahboubeh Irani

doi:10.4103/0972-9062.289395

RESEARCH ARTICLE

Year : 2019 | Volume : 56 | Issue : 3 | Page : 221-230

Synthesis of novel amodiaquine analogs and evaluation of their in vitro and in vivo antimalarial activities

Azar Tahghighi¹, Arezoo Rafie Parhizgar², Safoura Karimi², Mahboubeh Irani³
¹ Malaria and Vector Research Group, Biotechnology Research Center; Medicinal Chemistry Laboratory, Department of Clinical Research, Pasteur Institute of Iran, Tehran, Iran
² Malaria and Vector Research Group, Biotechnology Research Center, Pasteur Institute of Iran; Department of Medicinal Chemistry, Pharmaceutical Sciences Branch, Islamic Azad University, Tehran, Iran
³ Traditional Medicine and Materia Medica Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran

Date of Submission	04-Mar-2018
Date of Acceptance	14-Dec-2018
Date of Web Publication	09-Jul-2020

Correspondence Address:
Dr Azar Tahghighi
Medicinal Chemistry Laboratory, Department of Clinical Research, Pasteur Institute of Iran, Tehran
Iran

Source of Support: None, Conflict of Interest: None

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DOI: 10.4103/0972-9062.289395

Abstract

Background & objectives: Due to the rapid increase of drug resistance in Plasmodium parasites, there is a pressing need of developing new antiplasmodial drugs. In this study, new amodiaquine (AQ) analogs were synthesized, followed by an evaluation of their antiplasmodial activity.
Methods: A new series of quinoline derivatives containing N-alkyl (piperazin-1-yl)methyl benzamidine moiety was synthesized by reacting 4-[(4-(7-chloroquinolin-4-yl)piperazin-1-yl)methyl]benzonitrile with appropriate primary amines. The synthesized compounds were investigated for inhibitory activity by inhibition test of heme detoxification (ITHD). Their antiplasmodial activity was then evaluated using the classical 4-day suppressive test (Peter’s test) against Plasmodium berghei-infected mice (ANKA strain).
Results: The results showed that the percentage of heme detoxification inhibition in the active compounds was 90%. The most promising analogs, N-butyl-4-[(4-(7-chloroquinolin-4-yl)piperazin-1-yl)methyl]benzamidine (compound 1e), and 4-[(4-(7-chloroquinolin-4-yl)piperazin-1-yl)methyl)]-N-(4-methylpentan-2-yl)benzamidine (compound 1f) displayed 97.65 and 99.18% suppressions at the doses of 75 and 50 mg/kg/day, respectively. Further, the mean survival time of the mice treated with these compounds was higher than that of the negative control group.
Interpretation & conclusion: The newly synthesized amodiaquine analogs presented sufficient antiplasmodial activity with excellent suppressions and high in vitro heme detoxification inhibition. Higher mean survival time of the mice treated with synthetic compounds further confirmed the in vivo antimalarial activity of these new AQ analogs. Therefore, these compounds have the potential to replace common drugs from 4-aminoquinoline class. However, further investigations such as pharmacokinetic evaluations, cytotoxicity, toxicity, and formulation seem to be necessary.

Keywords: Amodiaquine; antimalarial activity; heme detoxification; malaria; Peter’s test; Plasmodium berghei; quinoline

How to cite this article:
Tahghighi A, Parhizgar AR, Karimi S, Irani M. Synthesis of novel amodiaquine analogs and evaluation of their in vitro and in vivo antimalarial activities. J Vector Borne Dis 2019;56:221-30

How to cite this URL:
Tahghighi A, Parhizgar AR, Karimi S, Irani M. Synthesis of novel amodiaquine analogs and evaluation of their in vitro and in vivo antimalarial activities. J Vector Borne Dis [serial online] 2019 [cited 2023 Mar 30];56:221-30. Available from: http://www.jvbd.org//text.asp?2019/56/3/221/289395

Introduction

With approximately 200 million of new yearly cases, 500,000 annual deaths, and despite many efforts to control, eliminate, and eradicate malaria, it yet remains one of the most serious diseases in the world^[1]. Malaria is caused by five different species of Plasmodium: Plasmodium falciparum, P. vivax, P. ovale, P. malariae, and P. knowlesi, which are transmitted by mosquitoes of the genus Anopheles. Plasmodium vivax is more widespread, but P. falciparum is the most lethal form of malaria, which has shown spread resistance to available antimalarial drugs^[2]. Moreover, the coinfection of malaria with other serious infections such as HIV results in severe health outcomes^[3]. Due to the lack of late-stage clinical trials, and pilot implementation studies for a vaccine (RTS,S/AS01), drug resistance, and insufficient control of mosquito vectors, malaria is considered as a big global challenge in the field of health^[1].

Drug therapy is one of the main methods of malaria control because there are various drugs that affect different stages (sexual and asexual) of the parasite’s life cycle. For several decades, chloroquine (CQ) was the most effective, safe, and widely available antimalarial drug for the prophylaxis and treatment of malaria, especially P. falciparum infection in most endemic countries^[4],[5]. It inhibits heme polymerization in the parasite’s food vacuole (FV), leading to parasite death by toxic-free heme accumulation^[6]. However, due to a mutation in the P. falciparum CQ resistance transporter (pfcrt) gene caused by efflux of the drug from the parasite FV, drug concentration and its effectiveness has been decreased^[7]. Though, it has been shown in Malawi that after eight years of CQ discontinuation^[8], sensitivity to the CQ recovered with restriction of mutation such as the elimination of pfcrt T76 mutation^[9]. Chloroquine is a well-known 4-aminoquinoline class of drug containing a quinoline ring, a diethylamino head, and a pentamidine chain as a linker. In this drug, flat het- ero-aromatic ring for connection to heme, and the diethylamino group for accumulation in FV are necessary factors for antimalarial activity.

Other drugs have also been designed and synthesized based on the 4-aminoquinoline pharmacophore of CQ. For example, amodiaquine (AQ) with an aromatic linker (p-hydroxy aniline) is more potent than CQ and effective against most CQ-resistant strains [Figure 1]. However, its agranulocytosis and hepatotoxicity have restricted its clinical use^[10] . A bisquinoline analog of CQ, piperaquine (PQ) [Figure 1], was made in the 1960s and widely used for the treatment of CQ-resistant P. falciparum strains in China and Indochina for about 20 yr^[10]. In the late 1980s PQ monotherapy was reduced due to reports of drug resistance^[10]. Therefore, its combination therapy with dihydroartemisinin (dihydroartemisinin-piperaquine) was recommended for the appropriate cure rate and delay in the development of resistance^[11].

Figure 1: Structures of chloroquine and its analogs (AQ, PQ, TQ, ISQ, GSK369796), pentamidine, furamidine, pafuramidine and designed compounds (1a–1f).

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Tebuquine [TQ] is an analog of AQ with p-chloro phenyl moiety at the 5D- position of the hydroxyaniline linker of AQ having higher activity compared with CQ and AQ^[10] [Figure 1]. In this regard, it is notable that TQ becomes toxic similar to AQ after its long-term administration. Isoquine [ISQ] and GSK369796, other isomers of AQ have been obtained with displacement of the 3'-aminomethyl and the 4'-hydroxyl groups of the AQ^[10] [Figure 1]. These analogs have less toxicity than AQ but are not acceptable due to the lack of an adequate dose compared to CQ.

A resistant strain of malaria to the strongest antimalarial drug, artemisinin, has been proposed in Cambodia^[12]. This strain, which is now called super-malaria, was subsequently detected from Thailand, Laos, and Vietnam. The researchers are concerned about the rapid spread of this strain to several regions of Asia and eventually to Africa. In this situation, drug discovery plays a vital role in the development of new preventive and therapeutic agents. In fact, global malaria action plan (GMAP) has an outlook for a malaria-free world^[13] by eliminating malaria in at least 35 countries and preventing a resurgence of malaria in all the countries that are malaria-free^[14] by 2030.

Nowadays, due to the importance of the 4-amino quinoline class of drugs, researchers have focused on the design of new quinoline-based antimalarial compounds with preservation of quinoline ring and replacement of pentamidine chain^[10]. In fact, the chemical modification of current drugs structure is one of the most important strategies to improve their activity. The best example is the discovery of CQ from quinine in order to reduce its toxicity. Hence, due to rapid spread of resistance to available antimalarial drugs and urgent need of designing and developing a new, more affordable and accessible antimalarial agent, in this study new AQ analogs were synthesized for the first time with N-alkyl-[(piperazin-1-yl) methyl] benzamidine moiety containing amine head, preserving the 4-amino quinoline fragment similar to the 4-aminoquinoline family of antimalarial drugs.

The final compounds (1a–1f) were synthesized from 4-[(4-(7-chloroquinolin-4-yl)piperazin-1-yl)methyl] benzonitrile (compound 4) as the main intermediate by facile routes [Scheme 1]. The synthesized compounds were investigated for inhibitory activity by inhibition test of heme detoxification (ITHD). The antiplasmodial activity of the compounds was assessed by Peter’s test in mice inoculated with P. berghei (ANKA strain). Also, the mean survival time of treated mice with the synthesized compounds was evaluated and compared to the negative control group.

Material & Methods

Chemical reagents and materials were purchased from Sigma-Aldrich Chemei GmbH (Switzerland) and solvents were purchased from Sumchun Chemical Co. Ltd. (Seoul, South Korea). The main intermediate 7-chloro-4- (piperazin-1-yl) quinolone (compound 3) was prepared according to the methods described in the literature with some modifications^[15]. Uncorrected melting points were determined on a Kofler hot stage microscope apparatus. The IR spectra were obtained on a Shimadzu (Japan) 470 spec- trophotometer (potassium bromide dicks).^[1]H-NMR and ¹³C-NMR spectra were recorded on a Varian (USA) Unity 500 spectrometer, and chemical shifts δ were reported in parts per million (ppm) relative to tetramethylsilane, as an internal standard. The mass spectra were run on a Finigan TSQ-70 spectrometer (Finigan, USA) at 70 eV. Elemental analyses were carried out on a CHN rapid elemental analyzer (GmbH VarioEL, Germany) for C, H, and N, and the results were recorded (within 0.4% of the theoretical values). Merck silica gel 60 F254 plates were used for analytical thin layer chromatography. The log P-value of the compounds was calculated using ACD/Chem Sketch freeware version. The experimental female BALB/c mice (6-8 weeks) were purchased from the Pasteur Institute of Iran, Tehran.

Synthesis of 7-chloro-4-(piperazin-1-yl) quinoline (compound 3)

The compounds 4,7-dichloroquinoline (compound 2) (5 g, 25.24 mmol) and piperazine (7 g, 81.26 mmol) in 25 ml of 2-propanole were refluxed for 74 h. The resulting product was purified using silica gel column chromatography eluted with CH₂Cl₂/petroleum ether.

Synthesis of 4-[(4-(7-chloroquinolin-4-yl)piperazin-1-yl methyl]benzonitrile (compound 4)

A mixture 7-chloro-4-(piperazin-1-yl) quinoline (compound 3) (3 g, 12.11 mmol) and 4-(chloromethyl) benzonitrile (2.75 g, 18.16 mmol) in 15 ml of methanol was refluxed for 96 h. The resulting product was purified using silica gel column chromatography eluted with EtO-AC/petroleum ether.

General procedure for the synthesis of the final compounds 1a–1f

A mixture of 4-[(4-(7-chloroquinolin-4-yl)piperazin- 1-yl)methyl]benzonitrile (compound 4) (1mmol), appropriate primary amine (1.5 mmol), and CuCl (1.3 mmol) in 10 ml methanol was refluxed for 24–48 h. The mixture of reaction was quenched with NaOH 30%, extracted with CH₂Cl₂ and concentrated under reduced pressure to obtain a dark green solid. The resulting produces were purified using silica gel column chromatography eluted with CH₂Cl₂/petroleum ether.

Inhibition test of heme detoxification (ITHD)

To study the inhibition of heme detoxification of samples, the ITHD method optimized by Mosaddegh et al^[16] was used. Hemin chloride was dissolved in DMSO. The solution was diluted freshly to 60 μ ml^[1] with 1 M acetate buffer (pH 4.8). Tween-20 was diluted to 0.012 gL^-1 with distilled water. Diluted hemin, tween-20, and the sample dissolved in DMSO were distributed in each well of a 96-well plate with a ratio of 9 : 9 : 2, respectively, in triplicate. The final tested concentration of samples in each well was 200 μg ml^[1]. The solvent (DMSO) was added to the negative control wells. The plate was incubated for 24 h at 60 ° C to allow for completing the reaction. Finally, it was read with a micro-ELISA reader at 405 nm. The results were calculated and expressed as a percentage of heme detoxification inhibition.

Acute toxicity of the compounds in mice

The synthetic compounds 1a–1f with concentrations of 10 to 200 mg/kg/day were administered intraperitoneally (ip) to three female BALB/c mice for five days. The signs of toxicity and mortality in each group were monitored daily.

Evaluation of antiplasmodial activity (Peter’s test)

The experimental female BALB/c mice were kept under standard conditions for 10 days to adopt the laboratory animal housing facilities. The antiplasmodial (schizontocidal) activity of synthetic compounds (1c–1f) was evaluated using the 4-day suppressive test against P. berghei infection in mice^[17],[18]. The stock of CQ-sensitive P. ber- ghei (ANKA) parasite (500 μl containing 25% P. berghei) was defrosted and injected into two female BALB/c mice. Next, five animals were selected and infected with P. berghei through passaging. A total of 75 mice were weighed (18–22 g) and randomized into seven groups. Each animal was inoculated ip with 1.5 × 10⁷-infected erythrocytes of P. berghei in PBS (200 μl) on the first day (D0) of the experiment. The compounds were solubilized in 20% DMSO and prediluted in PBS to make appropriate concentrations. The first treatment was accomplished 3 h after the mice were infected (D0) and treated daily for four consecutive days. Groups 1 and 2 were treated with compounds 1c and 1d (100, 75, and 50 mg/kg/day) by ip injection for four days, whereas groups 3 and 4 were treated with compounds 1e (75, 50, and 25 mg/kg/day) and 1f (50, 25, and 12.5 mg/ kg/day) [Table 1]. Mice groups 5 and 6 received PBS and 20% DMSO as negative controls while mice group 7 was treated by CQ (25 mg/kg/day) as a positive control for four days [Table 1]. On Day 4, tail blood smear was taken, stained with 10% Giemsa stain in phosphate buffer (pH 7.2) for 20 min, and then visualized under a microscope at 100 × magnifications to determine the parasitaemia level. The parasitized red blood cells (pRBCs) on at least 3000 RBCs were counted to calculate the percentage of parasitaemia (% Parasitaemia = Number of infected RBC/Total number of RBC × 100). The percentage of parasitaemia suppression for each group was evaluated by comparing the percentage of parasitaemia in negative controls with that in the treated group (Suppression % = Parasitaemia in negative control–Parasitaemia in treated group/Parasitaemia in negative control x 100). During the treatment, all the mice were weighed on Days 0 and 4. Furthermore, the mortality of mice was monitored daily during the experiment up to 40 days’ post-infection, and the mean survival rate of each group was calculated. Also, the dissection of the internal organs (spleen, liver, and kidney) was done on the seventh day of treatment for toxicity evaluation.

Table 1: The in vitro and in vivo activities of the synthetic compounds 1a–1f

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Statistical analysis

Control and test data in the present study were analyzed using SPSS (ver. 16.0). One-way ANOVA was used to test the statistical differences for three doses within a group, followed by LSD test for multiple comparisons. The p-value ≤0.05 was considered statistically significant.

Ethical statement

All the applicable and acceptable guidelines for the care and use of animals were followed in the study. The Research Committee and Institutional Ethics Committee of the Pasteur Institute of Iran, Tehran approved this project (No. 740).

Results

The in vitro and in vivo activities of the synthetic compounds 1a–1f are listed in [Table 1]. The synthetic compounds obtained with varying yields are indicated in [Table 2]. Antiplasmodial activity and toxicity assay of synthetic compounds 1c–1fare shown in [Figure 2]. The biotransformation of AQ and analogs, TQ, ISQ and GSK369796 to their toxic metabolites is shown in [Figure 3]. The structure-activity relationship (SAR) of AQ and new synthetic compounds are shown in [Figure 4]. The proposed mechanism of heme detoxification is presented in [Figure 5]. The spectral data of the synthesized compounds are listed in [Table 3].

Figure 2: Antiplasmodial activity and toxicity assay of synthetic compounds 1c–1f: (a) Suppressive activity of the synthetic compounds in Plasmodium berghei infected mice on Days 5 and 10, (b) Body weight on Days 1 and 5; and (c) hepatomegaly and splenomegaly on Day 7.

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Figure 3: Biotransformation of AQ, TQ, ISQ and GSK369796 to their toxic metabolites.

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Figure 4: The structure-activity relationship (SAR) of AQ and new synthetic compounds.

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Table 2: Melting point, yield and formula of the synthetic derivatives (4) and (1a–f)

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Table 3: The spectroscopic data of compounds 4 and 1a–1f

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Figure 5: The proposed mechanism of transferring chloroquine analogs to food vacuole of the parasite and detoxification.

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4-[(4-(7-chloroquinolin-4-yl)piperazin-1-yl)methyl] benzonitrile (compound 4)

The compound 4 as an intermediate compound was successfully synthesized with a yield of 60% and melting point (MP) >300 °C. It was obtained as a white solid.

N-methyl-4-[(4-(7-chloroquinolin-4-yl)piperazin-1-yl) methyl]benzamidine (compound 1a)

Compound 1a as a final product was successfully synthesized with a yield of 16% and MP >300 °C. It was obtained as a cream solid.

N-ethyl-4[(4-(7-chloroquinolin-4-yl)piperazin-1-yl) methyl]benzamidine (compound 1b)

Compound 1b as a final product was successfully synthesized with a yield of 15% and MP >300 °C. It was obtained as a dark cream solid.

N-propyl-4-[(4-(7-chloroquinolin-4-yl)piperazin-1-yl) methyl]benzamidine (compound 1c)

Compound 1c as a final product was successfully synthesized with a yield of 16% and MP >300 °C. It was obtained as a light brown solid.

N-isopropyl-4-[(4-(7-chloroquinolin-4-yl)piperazin- 1-yl)methyl]benzamidine (compound 1d)

Compound 1d as a final product was successfully synthesized with a yield of 18% and MP >300 °C. It was obtained as a light brown solid.

N-butyl-4-[(4-(7-chloroquinolin-4-yl)piperazin-1-yl) methyl]benzamidine (compound 1e)

Compound 1e as a final product was successfully synthesized with a yield of 19% and MP >300 °C. It was obtained as a cream solid.

4-[(4-(7-chloroquinolin-4-yl)piperazin-1-yl)methyl]- N-(4-methylpentan-2-yl)benzamidine (compound 1f)

Compound 1f as a final product was successfully synthesized with a yield of 21% and MP >300 °C. It was obtained as a dark cream solid.

Inhibition test of heme detoxification

The selected samples were investigated for their inhibitory potential by ITHD. If the percentage of heme detoxification inhibition was >90%, the assay was considered positive, whereas a value <90% indicated a negative result.

Acute toxicity of the compounds in mice

Based on the study, the optimum dose for compounds 1a–1b was 200 mg/kg/day and for 1c–1d it was 100 mg/ kg/day; while for compounds 1e and 1f dosages were75 and 50 mg/kg/day, respectively.

Evaluation of antiplasmodial activity (Peter’s test)

Pre-test for schizontocidal effect in early infection of compounds 1a–1f was done in highest dose and compounds 1a–1b did not had any antiparasitic activity in high dose of 200 mg/kg. Therefore, compounds 1c–1f was selected for Peter’s test. Compound 1c with propyl substituent showed a moderate suppression of parasitaemia (60.95%) in highest dose [Table 1]; [Figure 2]a; while, weak antiplasmodial activity was observed for compound 1d with isopropyl moiety (29.67% suppression of parasi- taemia) in dose of 100 mg/kg. But, compounds 1e and 1f with N-butyl and N-(4-methylpentan-2-yl) substitutions lead to 97.65 and 99.18% growth inhibition in doses of 75 and 50 mg/kg, respectively [Table 1], [Figure 2]a. During the treatment, all the mice were weighed on Days 0 and 4 and showed weight reduction [Figure 2]b. For the evaluation of toxicity, one of the mice in each group was randomly selected and dissected seven days after the treatment. The internal organs (spleen, liver, and kidney) were dissected, where an enlargement of the liver was noticed in the treated groups with compounds 1e–1f in compared with the control groups [Figure 2]c. The kidneys of the treated groups did not show any change and their spleen display a mild enlargement especially in high dose [Figure 2]c.

Statistical analysis

The results of statistical analysis between the groups demonstrated that there was a significant difference between all the groups except compound 1c in the dose (75 mg/kg/day), which did not show a significant difference in the dose 50 mg/kg/day (p >0.05). Also, the compound 1e did not show any difference between the doses of 75 and 50 mg/kg/day (p >0.05). The compound 1e also indicated that the difference between the treated groups and the control groups was statistically significant (p <0.05) except the compound 1d in the doses 75 and 50 mg/kg/ day (p >0.05).

Discussion

The number of natural and synthetic structures are effective in the treatment and prevention of malaria, such as quinoline-based antimalarial drugs, CQ, and AQ. In these drugs, the flat and heteroaromatic ring of quinoline forms a complex with heme which leads to heme detoxification and finally the death of the malaria parasite in the erythrocytic stage^[6]. Heme also affects cellular metabolism by inhibiting enzymes, peroxidizing membranes, and producing oxidative free radicals^[19].

Now, CQ as an efficacious, safe, and the affordable antimalarial drug is not considered effective in many malaria-endemic countries due to a mutation in the pfcrt gene^[7]. The AQ is a CQ analog containing a phenyl linker, which increases its activity against CQ resistant parasites. However, AQ metabolizes in the liver to amodiaquine quinone-imine (AQQI) in the presence of cyto-chrome–P450 as a toxic metabolite^[10] [Figure 3]. The high dose and long-term use of AQ lead to the depletion of glutathione levels and, eventually, liver toxicity. Also, AQQI forms an adduct with key human enzymes that are fatal for liver cells^[10] [Figure 3]. Many attempts have been made to synthesize various AQ analogs like ISQ, and TQ with a different linker to diminish its toxicity against various strains of P. falciparum and P. berghei^[20],[21]. Nevertheless, their development has been discontinued due to low drug safety compared with CQ. Due to AQ analogs problems, the need of a new, effective, safe, and the affordable alternative drug was felt.

In the present study, the new analogs of AQ were synthesized with the side chain of benzamidine, which is important moiety in antileishmanial drug, pentamidine^[22] [Figure 1]. Also, furamidine and its prodrug, pafuramidine with benzamidine moiety have shown strong activity against some diseases such as leishmaniasis, trypanosomiasis, and malaria, which are under phase–III clinical trials^[23] [Figure 1].

In the synthetic compounds 1a–1f with N-alkyl [(piperazin-1 -yl)methyl)]benzamidine substitution in 4-position of quinoline ring, the amine group in the head of the molecule and 7-chloro-4-amino quinoline were preserved like AQ [Figure 3]. The presence of amine group in antimalarial drugs is very important for accumulation in FV. Also, the presence of quinoline ring is a necessary factor for connection to heme. Phenyl linker of AQ was changed with a 4-benzyl piperazine moiety in new compounds. Then, inhibition of heme detoxification of samples was evaluated with the ITHD method and the synthetic compounds 1c–1f showed the best result^[16]. However, the compounds with a small alkyl group in their side chain (1a–1b) did not show any inhibitory activity which can be correlated to low lipophilicity and little accumulation in the parasite’s FV. The mechanistic studies have shown that CQ and its analogs interfere in the mechanism of heme polymerization with malaria parasites^{[24],[25],[26]}. Indeed, in the parasite’s FV, the dimers of haematin molecules form β-haematin crystals by the linking of central iron of one heme unit with the acid side chain of another heme [Figure 5]. Meanwhile, haemozoin is made of β-haematin units by hydrogen bonding. This pigment is safe for the parasite, and the process is essential to the survival of the malaria parasite^[25].The antiplasmodial drugs (quinoline analogs) inhibit heme polymerization, which results in the accumulation of free toxic heme in FV and parasite’s death. Therefore, the inhibition of haemozoin formation is an excellent drug target for the development of antimalarial drugs^[26]. It is assumed that the present synthetic compounds are also accumulated in FV and entrapped in its acidic form (protonated form). Therefore, these compounds can inhibit the formation of haemozoin [Figure 5].

The synthetic N-alkyl-4-[(4-(7-chloroquinolin-4-yl) piperazin-1-yl)methyl]benzamidine derivatives were evaluated using the classical 4-day suppressive test and satisfactory results were obtained in inhibiting the parasitemia of P. berghei infection in BALB/c mice. [Table 1] illustrates the mean percentage of parasitemia and the percentage of suppression for each group. Compounds 1e and 1f with N-butyl-benzamidine and N-(4-methylpen- tan-2-yl) substitutions lead to 97.65 and 99.18% growth inhibitions in doses of 75 and 50 mg/kg, respectively [Figure 2]a. It is noteworthy that the active compounds showed high in vivo suppression of parasitaemia at the low dosage ≤75 mg/kg/day by ip route against inoculated mouse with 1.5×10⁷ P. berghei-infected erythrocytes. The better activity of compounds 1e and 1f may be due to more lipophilic- ity and high accumulation in the parasite’s FV [Table 1]. On the other hand, amidine groups had a significant effect on inhibition of parasite growth, which may be related to the basic effect of the amidine group and the appropriate lipophilic substitutions on it. Indeed, compounds 1e and 1f with log P = 4.08 and 4.78, respectively, showed a high suppressive effect in their high dose [Table 1], [Figure 2]a; however, compound 1d (log P = 3.37) with isopropyl sub-stituent showed a low antiplasmodial activity with 29.67% growth inhibition in dose of 100 mg/kg/day. Therefore, it can be concluded that lipophilicity plays an important role in the biological activity of synthetic compounds.

All mice in the study showed weight reduction, which can be related to the lack of a 100% reduction of parasitaemia after treatment with synthetic compounds [Figure 2]b. The mean survival rate of each group was calculated and compounds 1e–1f showed the best mean survival rate in highest dose [Table 1]. Thus, the result of this study indicates that the synthetic AQ analogs may exhibit similar or better activity compared with AQ. In fact, the compounds didn’t have a hydroxyphenyl linker in AQ which causes toxicity and can selectively be used to treat drug-resistant malaria.

Conclusion

In this study, novel amodiaquine analogs with anti-plasmodial activity were synthesized by replacement of AQ linker with N-alkyl[(piperazin-1-yl)methyl)]benza- midine moiety. The most promising analogs 1e and 1f displayed excellent suppressions and high in vitro heme detoxification inhibition. Also, the mean survival time of the mice treated with synthetic compounds was higher than that of negative control groups, confirming in vivo antimalarial activity of these new AQ analogs. Therefore, we can conclude that these compounds have suitable potential to replace common drugs from 4-aminoquinoline class. However, further investigations such as pharmacokinetic evaluations, cytotoxicity, toxicity, and formulation seem to be necessary.

Conflicts of interest

The authors declare no conflict of interest

Acknowledgements

The authors would like to thank the Pasteur Institute of Iran (IPI), Tehran for funding this research (No. 740). The authors are grateful to Prof. S Zakeri [Malaria and Vector Research Group (MVRG), IPI] and Prof. HR Basseri (Department of Medical Entomology & Vector Control, School of Public Health, Tehran University of Medical Sciences, Tehran) for providing P. berghei (ANKA strain). Most parts of the project have been performed in MVRG, IPI, Tehran, Iran.

References

1.	World Malaria Report 2016. Geneva: World Health Organization 2016. Available from: http://apps.who.int/iris/bitstre am/10665/200018/1/9789241565158_eng.pdf?ua=1 (Accessed on December 15, 2016).
2.	Winzeler EA. Malaria research in the post-genomic era. Nature 2008; 455(7214): 751-6.
3.	Abu-Raddad LJ, Patnaik P, Kublin JG. Dual infection with HIV and malaria fuels the spread of both diseases in sub-Saharan Africa. Science 2006; 314(5805): 1603-6.
4.	Schlitzer M. Malaria chemotherapeutics—Part I: History of antimalarial drug development, currently used therapeutics, and drugs in clinical development. Chem Med Chem 2007; 2(7): 944-86.
5.	Mishra A, Batchu H, Srivastava K, Singh P, Shukla PK, Batra S. Synthesis and evaluation of new diaryl ether and quinoline hybrids as potential antiplasmodial and antimicrobial agents. Bioorg Med Chem Lett 2014; 24(7): 1719-23.
6.	Kumar S, Guha M, Choubey V, Maity P, Bandyopadhyay U. Antimalarial drugs inhibiting hemozoin β-hematin) formation: A mechanistic update. Life Sci 2007; 80(9): 813-28.
7.	Musonda CC, Whitlock GA, Witty MJ, Brun R, Kaiser M. Chloroquine-astemizole hybrids with potent in vitro and in vivo antiplasmodial activity. Bioorg Med Chem Lett 2009; 19(2): 481-4.
8.	Djimdé A, Doumbo OK, Cortese JF, Kayentao K, Doumbo S, Diourté Y, et al. A molecular marker for chloroquine-resistant falciparum malaria. N Engl J Med 2001; 344(4): 257-63.
9.	Laufer MK, Thesing PC, Eddington ND, Masonga R, Dzinjala-mala FK, Takala SL, et al. Return of chloroquine antimalarial efficacy in Malawi. N Engl J Med 2006; 355(19): 1959-66.
10.	Parhizgar AR, Tahghighi A. Introducing new antimalarial analogues of chloroquine and amodiaquine: A narrative review. Iran J Med Sci 2017; 42(2): 115-28.
11.	Saunders DL. Dihydroartemisinin-piperaquine failure in Cambodia. N Engl J Med 2014; 371: 484-5.
12.	Imwong M, Hien TT, Thuy-Nhien NT, Dondorp AM, White NJ. Spread of a single multidrug resistant malaria parasite lineage (PfPailin) to Vietnam. Lancet Infect Dis 2017; 17(10): 1022-3.
13.	The global malaria action plan. Geneva: The Roll Back Malaria partnership (WHO) 2008.
14.	Action RBM. Investment to defeat Malaria 2016-2030 (AIM)— for a malaria-free world. Geneva: World Health Organization 2015; pp. 274.
15.	Inam A, Siddiqui SM, Macedo TS, Moreira DRM, Leite ACL, Soares MBP, et al. Design, synthesis and biological evaluation of 3-[4-(7-chloroquinolin-4-yl)-piperazin-1-yl]-propionic acid hydrazones as antiprotozoal agents. Eur J Med Chem 2014; 75: 67-76.
16.	Mosaddegh M, Irani M, Esmaeili S. Inhibition test of heme detoxification (ITHD) as an approach for detecting antimalarial agents in medicinal plants. Res J Pharmacogn 2018; 5(1): 5-11.
17.	Teimouri A, Haghi AM, Nateghpour M, Farivar L, Hanifian H, Mavi SA, et al. Antimalarial efficacy of low molecular weight chitosan against Plasmodium berghei infection in mice. J Vector Borne Dis 2016; 53(4): 312-6.
18.	Tahghighi A, Karimi S, Parhizgar AR, Zakeri S. Synthesis and antiplasmodial activity of novel phenanthroline derivatives: An in vivo study. Iran J Basic Med Sci 2018; 21(2): 202-11.
19.	Rathore D, Jani D, Nagarkatti R, Kumar S. Heme detoxification and antimalarial drugs–Known mechanisms and future prospects. Drug Discov Today Ther Strateg 2006; 3(2): 153-8.
20.	Miroshnikova OV, Hudson TH, Gerena L, Kyle DE, Lin AJ. Synthesis and antimalarial activity of new isotebuquine analogues. J Med Chem 2007; 50(4): 889-96.
21.	O’Neill PM, Park BK, Shone AE, Maggs JL, Roberts P, Stocks PA, et al. Candidate selection and preclinical evaluation of N- tert-butyl isoquine (GSK369796), an affordable and effective 4-aminoquinoline antimalarial for the 21st century. J Med Chem 2009; 52(5): 1408-15.
22.	Sands M, Kron MA, Brown RB. Pentamidine: A review. Rev Infect Dis 1985; 7(5): 625-44.
23.	Soeiro M, Werbovetz K, Boykin D, Wilson W, Wang M, Hemphill A. Novel amidines and analogues as promising agents against intracellular parasites: A systematic review. Parasitology 2013; 140(8): 929-51.
24.	Sullivan DJ, Gluzman IY, Russell DG, Goldberg DE. On the molecular mechanism of chloroquine’s antimalarial action. Proc Natl Acad Sci 1996; 93(21): 11865-70.
25.	Coronado LM, Nadovich CT, Spadafora C. Malarial hemozoin: From target to tool. BBA-Gen Subjects 2014; 1840(6): 2032-41.
26.	Weissbuch I, Leiserowitz L. Interplay between malaria, crystalline hemozoin formation, and antimalarial drug action and design. Chem Rev 2008; 108(11): 4899-914.