Synthesis, Characterization, and Antileishmanial Activity of Certain Quinoline-4-carboxylic Acids

Abstract

Leishmaniasis is a fatal neglected parasitic disease caused by protozoa of the genus Leishmania and transmitted to humans by different species of phlebotomine sandflies. The disease incidence continues to increase due to lack of vaccines and prophylactic drugs. Drugs commonly used for the treatment are frequently toxic and highly expensive. The problem of these drugs is further complicated by the development of resistance. Thus, there is an urgent need to develop new antileishmanial drug candidates. The aim of this study was to synthesize certain quinoline-4-carboxylic acids, confirm their chemical structures, and evaluate their antileishmanial activity. Pfitzinger reaction was employed to synthesize fifteen quinoline-4-carboxylic acids (Q1-Q15) by reacting equimolar mixtures of isatin derivatives and appropriate α-methyl ketone. The products were purified, and their respective chemical structures were deduced using various spectral tools (IR, MS, ¹H NMR, and ¹³C NMR). Then, they were investigated against L. donovani promastigote (clinical isolate) in different concentration levels (200 μg/mL to 1.56 μg/mL) against sodium stibogluconate and amphotericin B as positive controls. The IC₅₀ for each compound was determined and manipulated statistically. Among these compounds, Q1 (2-methylquinoline-4-carboxylic acid) was found to be the most active in terms of IC₅₀.

This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

1. Introduction

Leishmaniasis is a fatal neglected parasitic disease caused by obligate intramacrophage protozoa of the genus Leishmania and transmitted to humans by different species of phlebotomine sandflies [1]. Leishmaniasis is found in at least 88 countries, but most cases are observed in underdeveloped or developing countries [2]. According to the World Health Organization (WHO) estimations, 90% of global visceral leishmaniasis (VL) cases occurred in 6 countries: Bangladesh, Brazil, Ethiopia, India, South Sudan, and Sudan [3]. Around two-thirds of the VL cases every year in East African countries are reported from Sudan with estimated annual incidence of 15,700 to 30,300 [3, 4].

The causative agents of leishmaniasis belong to the genus Leishmania. Two different morphological forms appear during the life cycle of these parasites. One is the flagellated and the other is mobile promastigote, which lives in the midgut of the sandfly. Once inside the mammalian bloodstream, the promastigote converts into amastigote form, living inside the vertebrate macrophage cells [5, 6].

Leishmaniasis has traditionally been classified in three different clinical types, visceral (known as Kala-Azar) (VL), cutaneous (CL), and mucocutaneous leishmaniasis (MCL), which have different immunopathologies and degrees of morbidity and mortality [5, 7].

VL affects vital functions and organs of the body and represents the most severe clinical type, showing the second highest mortality rate for a parasitic disease, after malaria [5]. The disease affects approximately 12 million people around the world, primarily in developing regions [8]. Furthermore, it is endemic in many tropical and subtropical regions of the world [9]. The risk of leishmaniasis is further stressed out by the sharp increase of Leishmania/HIV coinfection in many parts of the world including European countries such as Spain, Italy, France, and Portugal [10, 11].

There are no approved vaccines or prophylactic drugs against any form of leishmaniasis [12, 13]; thus, the control and management of the disease depend mainly on chemotherapy. The treatment is mainly based on organic pentavalent antimonial compounds, pentamidine, amphotericin B, miltefosine, and few other drugs [14, 15]. The pentavalent antimonials have been the first-line antileishmanial agents for over 60 years, but their efficacy is reducing due to the emergence of resistance in some regions of the world [16, 17]. Amphotericin B is effective against VL but it is nephrotoxic, and its use requires a hospitalization. The liposomal formulation of amphotericin B is highly effective against VL and is less toxic than the normal one, but it is much more expensive than the other current antileishmanial drugs [8, 18, 19]. Other drugs like miltefosine, paromomycin, pentamidine, and fluconazole have shown some usefulness and could be a potential supplement in the drugs regimen [20].

Unfortunately, all drugs used currently for treatment of leishmaniasis are frequently toxic and highly expensive. The problem of these drugs is further complicated by the development of drugs resistance in various endemic regions of the world [14]. In light of the above, the development of new efficient and safer antileishmanial agents is urgently needed.

A huge number of studies have been conducted to evaluate the antileishmanial activity of various plant extracts and synthetic molecules. Among the classes of molecules that exhibit promising antileishmanial activity are quinolines. Quinolines are a structurally diverse group of compounds, with basic quinoline nucleus, that have a broad range of biological activity and are present in different natural and synthetic products [21]. Their biological activity includes antileishmanial, antimalarial, antibacterial, antifungal, anthelmintic, cardiotonic, anticonvulsant, anti-inflammatory, analgesic, and antitumor activity [22–25]. Numerous quinolines have been found to have a varying degree of activity against leishmaniasis. A huge number of publications disclosing promising compounds have been published in the last few years [26–35].

The current work aims at synthesizing some quinoline derivatives and at investigating their antileishmanial activity.

2. Materials and Methods

2.1. Chemistry

All chemicals used were of commercially available reagent grade and were used without further purification. Isatins were purchased from Sigma-Aldrich. Melting points were determined on Stuart melting point apparatus (Stuart Scientific, England) and were uncorrected. The reaction’s progress was monitored by precoated silica gel plates of 0.25 mm thickness obtained from SD Fine-Chem limited, India. Spots were visualized by using either UV-lamp or iodine.

Infrared (IR) spectra were recorded as KBr disk using Shimadzu IR apparatus at the Research Center, College of Science, Khartoum University, Sudan. The data are given in ${\upsilon }^{\prime }$ (cm⁻¹). NMR spectra were determined in DMSO-d6 and recorded on NMR spectrophotometer (500 MHz) at Kyoto University, Japan. The chemical shifts are expressed as δ values (ppm) relative to the internal standard, tetramethylsilane (TMS). Signals are indicated by the following abbreviations: s = singlet, bs = broad singlet, d = doublet, dd = doublet of doublet, t = triplet, q = quartet, and m = multiplet. The J constants were given in (Hz). Mass spectra were taken on EI-MS spectrometer at Kyoto University, Japan. Mass spectral data were given as m/z (intensity %).

2.1.1. General Procedure for the Synthesis of Target Molecules

A mixture of 0.5 g of the appropriate isatin derivative, 30 mL of 33% w/v aqueous potassium hydroxide, and equimolar amount of ketone was heated under reflux for 15–24 hours (the reaction progress was monitored by TLC using ethyl acetate : hexane mixture (3 : 2) as a mobile phase). After that, the reaction mixture was cooled and diluted with water. The solution was neutralized with 1 M hydrochloric acid. The precipitate was filtered, washed with water, dried, and recrystallized from ethanol (Scheme 1).

[figure omitted; refer to PDF]

2.2. Biological Evaluation

2.2.1. Preparation of the Synthesized Compounds and Standard Drugs Solutions

Sodium stibogluconate and amphotericin B (AmBisome) were used as positive controls. Stock solutions of the synthesized quinolines (10 mg/mL) were prepared by dissolving 10 mg of the tested compounds in 1 mL of DMSO. Each stock solution was further diluted with Roswell Park Memorial Institute (RPMI) complete media to obtain serial dilutions ranging from 200 μg/mL to 1.56 μg/mL.

2.2.2. Parasite Isolation and Cultivation

A confirmed positive VL patient was subjected to lymph node aspiration for parasite isolation. Biopsies were aseptically inoculated into Novy-MacNeal-Nicolle (NNN) medium. Cultures were incubated at 25°C, and parasite growth was monitored daily. Promastigotes were transferred into tissue culture flasks containing RPMI media supplemented with 10% fetal calf serum (FCS) and antibiotics, streptomycin and benzylpenicillin. The medium was changed every 3 days until promastigotes reached their stationary phase.

2.2.3. In Vitro Evaluation of the Antileishmanial Activity

Promastigote density was adjusted to 2 × 10⁶ parasites/mL using RPMI complete media. A volume of 100 μL from parasite culture was transferred into a 96-well flat-bottom culture plate. Various concentrations of tested compounds solutions were added (100 μL) in triplicates.

A negative control (1% DMSO) and positive controls (sodium stibogluconate and amphotericin B) were treated similarly. The plates were incubated at 25°C for 24 hours. Parasites were counted by using a hemocytometer. The IC₅₀ for each compound was determined and manipulated statistically. The result is represented in Table 1.

Table 1

IC₅₀ of the synthesized quinolines and positive controls.

Compound	IC50 (µg/mL)
Q1	1.49
Q2	205
Q3	207
Q4	27.03
Q5	103
Q6	84.49
Q7	210
Q8	108
Q9	215
Q10	204
Q11	101
Q12	17.19
Q13	212
Q14	31.90
Q15	208
SSG ${}^{\ast }$	8.06
AMP ${}^{\ast }$	14.70

${}^{\ast }$ SSG = sodium stibogluconate; AMP = amphotericin B.

2.3. Computational Studies

2.3.1. Molecular Docking

Molecular docking was carried out based on the crystal structures of Leishmania donovani pteridine reductase, PTR1 (PDB: 2xox with a resolution of 2.5 Å [36]), L. donovani mitogen-activated protein kinase, MapK (PDB: 4qny with a resolution of 2.26 Å [37]), L. donovani dihydroorotate dehydrogenase, DHODH (PDB: 3c61 with a resolution of 1.80 Å [38]), L. donovani N-myristoyltransferase, NMT (PDB: 2wuu with a resolution of 1.42 Å [39]) and L. donovani O-acetyl serine sulfhydrylase, AS (PDB: 3spx with a resolution of 1.79 Å [40]).

Solvent molecules and cocrystallized ligands were removed from the proteins, and hydrogen atoms were added. The site at which the cocrystallized ligand was present was chosen as the binding site. However, in the case of unavailability of cocrystallized ligand, the active site was determined using SiteFinder program embedded in Molecular Operating Environment (MOE). Preparation and energy minimization of each protein were performed with MOE (MMFF94 force field, gradient 0.01 kcal/mol Å2). Each ligand structure was built by ChemSketch and optimized using the MMFF force field on the MOE software [41, 42].

The compounds set was docked on the previous target-binding sites by using AutoDock Vina software in PyRx [43, 44]. The docking experiment was carried out between the energy-minimized ligands and the active site through a grid cube at the geometrical center of the active site. The docking poses were ranked according to their docking scores as free energy of binding, and the results were exported for further analysis. The results are represented in Table 2.

Table 2

Docking energies (kcal/mol) of the synthesized compounds.

	PTR1	NMT	MapK	AS	DHODH
Q1	−6.3	−6.9	−6.6	−7.1	−6.9
Q2	−7.9	−8.5	−7.7	−8.3	−8.3
Q3	−8.0	−8.7	−8.0	−8.6	−9.0
Q4	−8.1	−8.0	−7.8	−7.7	−9.0
Q5	−7.6	−8.7	−8.0	−8.6	−8.6
Q6	−7.7	−7.9	−7.9	−7.8	−8.1
Q7	−8.0	−8.4	−8.0	−8.5	−8.9
Q8	−8.1	−8.3	−8.0	−8.0	−8.7
Q9	−6.8	−7.3	−7.3	−8.2	−8.1
Q10	−8.4	−9.3	−8.2	−7.9	−9.3
Q11	−8.2	−8.6	−8.2	−7.8	−9.6
Q12	−7.6	−8.5	−8.3	−7.8	−9.3
Q13	−8.3	−8.6	−8.3	−7.8	−9.6
Q14	−8.9	−8.9	−8.9	−9.6	−10.4
Q15	−9.0	−8.3	−9.3	−8.9	−8.3
FMN ${}^{\ast }$	—	—	—	—	−9.7

${}^{\ast }$ FMN = flavin mononucleotide.

2.3.2. Drug-Likeness Assessment

The number of rotatable bonds, topological polar surface area (TPSA) and the number of hydrogen bond donors (HBD) and acceptors (HBA) for the most promising candidate of this study and clogP for all synthesized compounds were estimated using Molinspiration server (available at: http://www.molinspiration.com).

All the computational studies were carried out on a windows x64 operating system with 2.5 GHz Intel Core i5 processor and 8 GB (RAM).

3. Results

3.1. Spectral Data

3.1.1. 2-Methylquinoline-4-carboxylic Acid (Q1)

White odourless powder; yield%: 91%; m.p. : >300°C; IR ${\upsilon }^{\prime }$ cm⁻¹ (KBr): 1668.31 (C=O stretch); 2923.88 (aliphatic C–H stretch); 3080.11 (aromatic C–H stretch); and 3500–3250 (OH stretch).

3.1.2. 2-Phenylquinoline-4-carboxylic Acid (Q2)

White odourless powder; yield%: 46%; m.p.: 218–220°C; IR ${\upsilon }^{\prime }$ cm⁻¹ (KBr): 1704.96 (C=O stretch); 3035.75 (aromatic C–H stretch); and 3500–3250 (OH stretch). ¹H NMR-DMSO-d₆: δ ppm 7.6–8.8 (m, 10H, Ar-H); 14 (bs, 1H, -COOH); ¹³C NMR-DMSO-d₆: δ ppm 119.18, 123.47, 125.42, 127.25, 127.84, 129.04, 129.81, 130.04, 130.29, 137.62, 137.89, 148.41, 155.83 (Ar-C), and 167.65 (Ar-COOH).

3.1.3. 2-(4-Methoxyphenyl)quinoline-4-carboxylic Acid (Q3)

White odourless powder; yield%: 46%; m.p. :>300°C; IR ${\upsilon }^{\prime }$ cm⁻¹ (KBr): 1708.10 (C=O stretch); 2933.53 (aliphatic C–H stretch); 3078.18 (aromatic C–H stretch); and 3500–3250 (OH stretch). ¹H NMR-DMSO-d₆: δ ppm 3.85 (s, 3H, -OCH₃) and 7–8.8 (m, 9H, Ar-H). ¹³C NMR-DMSO-d₆: δ ppm 55.37 (-OCH₃), 114.40, 118.73, 123.11, 125.39, 127.37, 128.77, 129.53, 130.20, 130.28, 137.53, 148.35, 155.47, 160.97 (Ar-C), and 167.73 (Ar-COOH).

3.1.4. 6-Bromo2-(4-methoxyphenyl)quinoline4-carboxylic Acid (Q4)

Yellow odourless powder; yield%: 87%; m.p.: 250–252°C; IR ${\upsilon }^{\prime }$ cm⁻¹ (KBr): 1701.10 (C=O stretch); 2935.46 (aliphatic C–H stretch); 3074.32 (aromatic C–H stretch) and 3080–2800 (OH stretch); ¹H NMR-DMSO-d₆: δ ppm 3.85 (s, 3H, -OCH₃); 7–8.8 (m, 8H, Ar-H).; ¹³C NMR-DMSO-d₆: δ ppm 55.38 (-OCH₃); 114.44, 120.10, 120.64, 124.43, 127.55, 128.83, 129.90, 131.71, 133.14, 135.86, 147.13, 156.07, 161.14 (Ar-C), and 167.16 (Ar-COOH); EI-MS, Rel. Int: 77 (18%); 89 (19%); 107 (20%); 136 (68%); 137 (52%); 138 (26%); 154 (100%); 155 (24%); 192 (55%); 280 (54%); 307 (20%); 345 (12%); [M+] 358 (21%); and 360 (21%).

3.1.5. 2-(4-Bromophenyl)quinoline-4-carboxylic Acid (Q5)

Pale yellow odourless powder; yield%: 66%; m.p. :>300°C; IR ${\upsilon }^{\prime }$ cm⁻¹ (KBr): 1712.67 (C=O stretch); 3082.04 (C=O stretch); and 3400–2500 (OH stretch); ¹H NMR-DMSO-d₆: δ ppm 7.5–8.7 (m, 9H, Ar-H); ¹³C NMR-DMSO-d₆: δ ppm 118.98, 123.58, 123.87, 125.47, 128.07, 129.30, 129.74, 130.45, 131.97, 136.97, 137.89, 148.27, 154.66 (Ar-C), and 167.55 (Ar-COOH).

3.1.6. 6-Bromo2-(4-bromophenyl)quinoline4-carboxylic Acid (Q6)

Yellow odourless powder; yield%: 73%; m.p.: 272–274°C; IR ${\upsilon }^{\prime }$ cm⁻¹ (KBr): 1724.24 (C=O stretch); 3074.32 (aromatic C–H stretch); and 3400–2500 (OH stretch); ¹H NMR-DMSO-d₆: δ ppm 7.5–8.7 (m, 8H, Ar-H); ¹³C NMR-DMSO-d₆: δ ppm 120.28, 121.45, 124.09, 124.80, 127.58, 129.27, 131.88, 131.98, 133.40, 136.20, 136.56, 146.99, 155.22 (Ar-C), and 166.93 (Ar-COOH); EI-MS, Rel. Int: 77 (19%); 89 (23%); 107 (26%); 136 (94%); 137 (79%); 138 (39%); 154 (100%); 155 (35%); 192 (65%); 289 (15%); 307 (32%); 345 (15%); [M+] 407 (5%) and 409 (7%).

3.1.7. 2-(4-Hydroxyphenyl)quinoline-4-carboxylic Acid (Q7)

Yellow odourless powder; yield%: 82%; m.p.: >300°C; IR ${\upsilon }^{\prime }$ cm⁻¹ (KBr): 1712.67 (C=O stretch); 3132.18 (aromatic C–H stretch); and 3400–2500 (OH stretch); ¹H NMR-DMSO-d₆: δ ppm 6.8–8.4 (m, 9H, Ar-H); 9.95 (s, 1H, Ar-OH); ¹³C NMR- DMSO-d₆: δ ppm 115.81, 118.63, 122.98, 125.37, 127.15, 128.69, 128.84, 129.42, 130.11, 137.35, 148.33, 155.74, 159.51 (Ar-C), and 167.74 (Ar-COOH).

3.1.8. 6-Bromo-2-(4-hydroxylphenyl)quinoline-4-carboxylic Acid (Q8)

Brown odourless powder; Yield%: 90%; m.p.: >300°C; IR ${\upsilon }^{\prime }$ cm⁻¹ (KBr): 1718.46 (C=O stretch); 3101.32 (aromatic C–H stretch); and 3400–2500 (OH stretch); ¹H NMR-DMSO-d₆: δ ppm 6.8–8.4 (m, 8H, Ar–H); 10.02 (s, 1H, Ar-OH); ¹³C NMR-DMSO-d₆: δ ppm 115.89, 120.00, 120.40, 124.31, 127.55, 128.34, 128.96, 131.63, 133.11, 135.77, 147.15, 156.39, 159.78 (Ar-C), and 167.21 (Ar-COOH); EI-MS, Rel. Int: 45 (17%); 77 (19%); 89 (29%); 107 (22%); 136 (69%); 137 (51%); 138 (27%); 154 (100%); 155 (23%); 289 (14%); 307 (24%); [M+] 344 (23%) and 346 (23%).

3.1.9. 2-Methyl-6-nitroquinoline-4-carboxylic Acid (Q9)

Black odourless powder; yield%: 33%; m.p.: >300°C; IR ${\upsilon }^{\prime }$ cm⁻¹ (KBr): 1718.46 (C=O stretch); 3051.81 (aromatic C–H stretch); and 3400–2250 (OH stretch); ¹H NMR-DMSO-d₆: δ ppm 2.93 (s, 3H, -CH₃) and 6.6–8.4 (m, 4H, Ar-H).

3.1.10. 6-Nitro-2-phenylquinoline-4-carboxylic Acid (Q10)

Brown odourless powder; yield%: 56%; m.p. :>300°C; IR ${\upsilon }^{\prime }$ cm⁻¹ (KBr): 1708.81 (C=O stretch); 3082.04 (aromatic C–H stretch); and 3500–2400 (OH stretch); ¹H NMR-DMSO-d₆: δ ppm 7–9.7 (m, 9H, Ar-H).

3.1.11. 6-Bromo-2-(4-hydroxylphenyl)quinoline-4-carboxylic Acid (Q11)

Brown odourless powder; yield%: 77%; m.p.: >300°C; IR ${\upsilon }^{\prime }$ cm⁻¹ (KBr): 1681.81 (C=O stretch); 3101.32 (aromatic C–H stretch); and 3500–2500 (OH stretch) and ¹H NMR-DMSO-d₆: δ ppm 3.82 (s, 3H, -OCH₃); 6.8–8.4 (m, 8H, Ar–H); EI-MS, Rel. Int: 29 (16%), 39 (55%), 41 (36%) 43 (39%), 55 (48%), 57 (39%), 69 (36%), 89 (31%), 107 (30%), 136 (82%), 137 (57%), 138 (31%), 154 (100%), 155 (25%), 192 (37%), 295 (22%), 307 (15%), and 321 (5%).

3.1.12. 2-(4-Bromophenyl)-6-nitroquinoline-4-carboxylic Acid (Q12)

Brown odourless powder; yield%: 90%; m.p.: >300°C; IR ${\upsilon }^{\prime }$ cm⁻¹ (KBr): 1703.03 (C=O stretch); 3082.04 (aromatic C–H stretch); and 3400–2500 (OH stretch); ¹H NMR-DMSO-d₆: δ ppm 7.6–8.8 (m, 8H, Ar-H); ¹³C NMR- DMSO-d₆: δ ppm 121.27, 122.62, 123.57, 124.97, 129.66, 131.57, 131.73, 132.11, 136.08, 138.63, 145.69, 150.26, 158.20 (Ar-C), and 166.60 (Ar-COOH); EI-MS, Rel. Int: 77 (19%); 89 (21%); 107 (23%); 136 (84%); 137 (71%); 138 (35%); 154 (100%); 155 (31%); 289 (14%); 307 (29%); [M+] 372 (3%) and 374 (3%).

3.1.13. 2-(4-Hydroxyphenyl)-6-nitroquinoline-4-carboxylic Acid (Q13)

Brown odourless powder; yield%: 93%; m.p.: >300°C; IR ${\upsilon }^{\prime }$ cm⁻¹ (KBr): 1675.00 (C=O stretch); 3095.54 (aromatic C–H stretch) and 3500–2500(OH stretch); ¹H NMR-DMSO-d₆: δ ppm 7–8.8 (m, 8H, Ar-H), 9.78 (s, 1H, Ar-OH). EI-MS, Rel. Int: 41 (17%); 43 (21%); 55 (25%); 57 (23%); 69 (19%); 77 (18%); 89 (20%); 107 (22%); 136 (71%); 137 (61%); 138 (32%); 154 (100%); 155 (27%); 289 (10%); 307 (19%); 308 (6%) and 309 (2%).

3.1.14. 2-(6-Methoxynaphthalen-2-yl)quinoline-4-carboxylic Acid (Q14)

Brown odourless powder; yield%: 94%; m.p.: 280°C; IR ${\upsilon }^{\prime }$ cm⁻¹ (KBr): 1720.39 (C=O stretch); 2966.31 (aliphatic C–H stretch); 3062.75 (aromatic C–H stretch); and 3400–2500 (OH stretch); ¹H NMR-DMSO-d₆: δ ppm 3.90 (s, 3H, -OCH₃); 7.6–8.8 (m, 11H, Ar-H); 10.02 (s, 1H, Ar-OH); EI-MS, Rel. Int: 39 (11%); 43 (17%); 77 (11%); 89 (11%); 107 (9%); 136 (29%); 137 (19%); 138 (11%); 154 (39%); 185 (15%); 200 (45%); 201 (64%); 202 (11%); 329 (23%); and [M+] 330 (100%).

3.1.15. 6-Bromo-2-(6-methoxynaphthalen-2-yl)quinoline-4-carboxylic Acid (Q15)

Brown odourless powder; yield%: 94%; m.p.: >300°C; IR ${\upsilon }^{\prime }$ cm⁻¹ (KBr): 1714.60 (C=O stretch); 2972.74 (aliphatic C–H stretch); 3103.25 (aromatic C–H stretch); and 3500–2500 (OH stretch); ¹H NMR-DMSO-d₆: δ ppm 3.90 (s, 3H, -OCH₃); 7–9 (m, 10H, Ar-H), and 14.02 (bs, 1H, Ar-OH); EI-MS, Rel. Int: 39 (11%); 43 (17%); 77 (11%); 89 (11%); 107 (9%); 136 (29%); 137 (19%); 138 (11%); 154 (39%); 185 (15%); 200 (45%); 201 (64%); 202 (11%); 329 (23%); and [M+] 330 (100%).

3.2. Biological Activity

The parasite viability curve of compounds Q1, Q4, Q5, and Q12 is shown in Figure 1. The IC₅₀ of the synthesized quinolines and positive controls are given in Table 1.

[figures omitted; refer to PDF]

3.3. Molecular Docking and Drug-Likeness Studies

Docking energies (kcal/mol) of the synthesized compounds and drug-likeness assessment by Molinspiration for compound Q1 are given in Tables 2 and 3, respectively.

Table 3

Drug-likeness assessment by Molinspiration for compound Q1.

Compound	miLogP	MW	TPSA	HBA	HBD	nrotb
Q1	2.19	187.20	50.19	3	1	1

4. Discussion

VL is considered as one of the leading causes of morbidity and mortality in tropical countries. The current therapeutic options for leishmaniasis are limited and are fast shrinking due to the emergence of widespread resistance and toxicity. One of the essential approaches to develop a new candidate as an antileishmanial agent is to synthesize molecules possessing a known scaffold with a reported antileishmanial activity. One of these scaffolds is quinoline ring which has been used extensively in the literature to produce new compounds with potential antiparasitic agents [45–51]. As a consequence, the current work aims at synthesizing some quinoline derivatives and at investigating their antileishmanial activity.

Quinoline-4-carboxylic acids are compounds of general synthetic interest due to their wide range of biological activities [52–54]. Among several ways to synthesize them, Pfitzinger reaction; a reaction that offers an easy and suitable synthetic way to quinoline-4-carboxylic acids in good yields from readily available materials, shown in Scheme 1, was employed to synthesize the present fifteen quinoline-4-carboxylic acids (Q1-Q15) by reacting equimolar mixture of isatin derivatives and appropriate α-methyl ketone in aqueous potassium hydroxide (33% w/v) under reflux for 15–24 hours. Treatment of these salts with 1 M HCl afforded the target compounds.

TLC was used to monitor the chemical reactions progress, in which ethyl acetate: n-hexane (3 : 2) mixture was used as a solvent system. The synthesized compounds were purified by recrystallization from ethanol. The purity of these compounds was ensured by TLC. The percentage yields ranged from 30% to 95%.

The molecular structures of the synthesized quinoline-4-carboxylic acids were confirmed on the basis of their spectral data which were consistent with the reported data [54–56]. The infrared spectra of all synthesized compounds showed a broad band at 3419–3300 cm⁻¹characteristic to OH stretching of carboxylic acid in addition to the presence of a strong sharp band at 1680–1710 cm⁻¹ attributed to carboxylic C=O stretching. Bands around 1508–1640 cm⁻¹ and 3021.80–3096.98 cm⁻¹ corresponding to C=C and C–H aromatic stretching, respectively were also observed. While the synthesized compounds with a monosubstituted benzene ring showed two bands around 690 and 750 cm⁻¹ attributed to the out-of-plane aromatic C–H bending vibrations, compounds with para-disubstituted benzene ring revealed one band around 800–850 cm⁻¹.

For ¹H NMR, spectra of the synthesized compounds were found to be in consistence with the suggested structures. Furthermore, the number of the integrated protons in the spectra matched the expected number of aromatic protons in each case. The spectra showed a broad singlet at δ 14 ppm integrated to one proton most likely assigning to the most deshielded acidic COOH proton. Compounds Q3, Q4, Q9, Q11, Q14, and Q15 spectra displayed a singlet at δ 2.9 ppm (for compound Q9) and at 3.8 ppm (for the remaining) integrated to three protons corresponding to the –CH₃ and –OCH₃ protons, respectively. A highly deshielded singlet at 9.95, 10.02, and 9.78 for compounds Q7, Q8, and Q13, respectively, corresponding to phenolic OH was observed.

The ¹³C NMR spectra of all compounds displayed a signal resonating around δ 167 ppm, which disappeared in DEPT-90 and 135 spectra, relating to the carboxylic acid carbonyl carbon. Moreover, two signals resonating around δ 155 and δ 148 ppm, for all compounds that disappeared in DEPT spectra are attributed to the aromatic carbon atoms directly attached to quinoline’s nitrogen atom. As expected, all DEPT-135 spectra did not show a negative peak indicating the absence of methylene carbons in the synthesized compounds. As a result, the DEPT‐90 and DEPT-135 spectra found to be identical for those compounds that have no methyl groups.

Although the molecular ion peak [M+] varied in intensity, it was evidenced for all analyzed synthesized compounds. A special peak at m/z = 154, corresponding to a common fragment for all synthesized compounds, represented the base peak with an exception of compound Q14 in which [M+] was the base peak. Interestingly, the synthesized molecules with a bromo substituent displayed a characteristic 1 : 1 ratio between [M+]:[M+2], which is in agreement with the bromine natural isotopic distribution (50 : 50).

As a preliminary investigation, the antileishmanial effect of the synthesized quinolines was assessed on L. donovani promastigotes. L. donovani is the causative agent of VL which is the most severe form of leishmaniasis, especially in Sudan and other developing countries [57].

The tested parasite was subjected to different concentration levels of the synthesized compounds to determine IC₅₀, the compound concentration causing 50% inhibition of the parasitic growth. The results of the antileishmanial activity of the tested compounds along with the standard drugs as positive controls are presented in Table 1 and Figure 1.

Compounds Q4, Q5, Q6, and Q14 exhibited moderate to weak antileishmanial activity against L. donovani promastigotes, with IC₅₀ values 27.03, 103, 84.49, and 31.9 µg/mL, respectively (Table 1). Compound Q12 revealed a comparable activity to that of the standard amphotericin B with IC₅₀ values 17.19 and 14.70 µg/mL, respectively. Interestingly, compound Q1 (IC₅₀ = 1.49 µg/mL) was found to be five times more potent than the standard drug sodium stibogluconate (IC₅₀ = 8.06 µg/mL) and ten times more potent than amphotericin B (IC₅₀ = 14.70 µg/mL). However, the introduction of a nitro group at C-6 of the quinoline ring of compound Q1 abolishes the activity (Q9).

Regarding 2-phenyl quinoline-4-carboxylic acid series, there is a good correlation between the lipophilicity of these molecules and the biological activity. Nonetheless, compound Q5 has a good lipophilicity (clogP = 4.45), and its IC₅₀ is higher than other analogues with comparable lipophilicity (Q5 IC₅₀ = 103 µg/mL compared with Q4 clogP: 4.48 with IC₅₀ = 27.03 µg/mL), confirming that the presence of electron-withdrawing group in C-6 quinoline ring is essential for the activity of these compounds.

Among these compounds, compound Q6 has the highest lipophilicity (clogP = 5.23) with a bromosubstituent at C-6 suggesting that it has the lowest IC₅₀ of these analogues theoretically. However, the activity of Q6 (IC₅₀ = 84.49 µg/mL) is much lower than those of Q4 and Q12 (clogP = 4.48, IC₅₀ = 27.03 and clogP = 4.38, IC₅₀ = 17.19 µg/mL, respectively). It is worth noting that compound Q6 violates one of the Lipinski's rule parameters (Q6 clogP > 5). This violation may contribute to the reduction of the penetrative ability of this compound to the cell membrane of the parasite and thus decreasing its activity. It can reasonably be concluded that the optimum clogP of these compounds (2-phenylsubstituted quinoline) to be active is between 4.20 and 4.50. It can be proposed that the presence of an electron-withdrawing group in C-6 of the quinoline ring increases the electron-deficiency of this moiety making it a good candidate for a charge transfer interaction with the target.

The Q15 violation of Lipinski's rule (clogP> 5.64) may contribute to its weaker activity (IC₅₀ = 208 µg/mL) when compared to the 2-naphthyl analogue compound Q14 (IC₅₀ = 31.9 µg/mL).

In general, for all synthesized compounds together, there is no constant pattern that correlates the activity with the calculated log P values. This indicates that other factors may influence the activity. A molecular docking analysis has been carried out to assess the molecular interaction patterns of the synthetic compounds in L. donovani targets and to determine the best-ranking poses of the compounds that exhibit the best affinity values. The synthetic quinolines were docked into 5 leishmanial targets that have been previously identified as potential drug targets. The docking energies (kcal/mol) of the synthesized compounds are summarized in Table 2.

Knowing that promastigote is not an adequate biological stage to identify promising antileishmanial compounds, in silico studies were conducted to evaluate the activity of these compounds on selected targets known to be expressed in amastigotes and druggable to quinolines. It was hoped that promising docking results might be a potential evidence for the activity of these compounds against amastigotes. All compounds were docked nicely on the active site for each target with satisfactory binding energies (−6.3 to −10.4 kcal/mol).

Further evaluation of the most promising compound of this study (compound Q1) in terms of prediction of its oral bioavailability based on Lipinski’s rules and Veber’s parameters was conducted computationally using Molinspiration server [58, 59]. From these studies, compound Q1 was found to be in complete agreement with Lipinski's rule of five and Veber’s parameters. Thus, it is suggested to have a good GIT absorption and a good oral bioavailability (Table 3). Furthermore, the values of Lipinski's rule parameters and Veber’s parameters of this compound are in the lowest ranges making this compound a good candidate for further modification without major violations of these rules. With all these findings, compound Q1 could be envisioned as a promising and potential lead compound for antileishmanial drug discovery projects.

5. Conclusion

Fifteen 2-substituted quinoline-4-carboxylic acid analogues (Q1-Q15) were synthesized, their chemical structures were spectrally confirmed, and their activities were evaluated against L. donovani promastigotes using two standard drugs. Among these compounds, Q1 (2-methylquinoline-4-carboxylic acid) was found to be the most active. Virtual screening revealed that Q1 and possibly some other tested compounds might have strong interactions with important binding sites of multiple druggable targets in amastigotes.

Conflicts of Interest

The authors declare that they have no conflicts of interest.

Acknowledgments

The authors wish to thank Prof. Ken-ichi Yamada (Graduate School of Pharmaceutical Sciences, University of Tokushima, Japan) for his help.

References

[1] D. O. Santos, C. E. R. Coutinho, M. F. Madeira, "Leishmaniasis treatment-a challenge that remains: a review," Parasitology Research, vol. 103 no. 1,DOI: 10.1007/s00436-008-0943-2, 2008.

[2] M. Vannier-Santos, A. Martiny, W. Souza, "Cell biology of Leishmania spp.: invading and evading," Current Pharmaceutical Design, vol. 8 no. 4, pp. 297-318, DOI: 10.2174/1381612023396230, 2002.

[3] J. Alvar, I. D. Vélez, C. Bern, "Leishmaniasis worldwide and global estimates of its incidence," PLoS One, vol. 7 no. 5,DOI: 10.1371/journal.pone.0035671, 2012.

[4] W. Al-Salem, J. R. Herricks, P. J. Hotez, "A review of visceral leishmaniasis during the conflict in South Sudan and the consequences for East African countries," Parasites and Vectors, vol. 9 no. 1,DOI: 10.1186/s13071-016-1743-7, 2016.

[5] E. A. Cardoso, A. Rodrigues, G. C. De Carvalho, "Leishmaniasis: history, evolution of treatment and the need for new drugs," Current Biotechnology, vol. 3 no. 4,DOI: 10.2174/2211550104666150303231736, 2014.

[6] P. B. de Carvalho, E. I. Ferreira, "Leishmaniasis phytotherapy. Nature's leadership against an ancient disease," Fitoterapia, vol. 72 no. 6, pp. 599-618, DOI: 10.1016/s0367-326x(01)00301-x, 2001.

[7] H. W. Murray, J. D. Berman, C. R. Davies, N. G. Saravia, "Advances in leishmaniasis," The Lancet, vol. 366 no. 9496, pp. 1561-1577, DOI: 10.1016/s0140-6736(05)67629-5, 2005.

[8] J. V. Richard, K. A. Werbovetz, "New antileishmanial candidates and lead compounds," Current Opinion in Chemical Biology, vol. 14 no. 4, pp. 447-455, DOI: 10.1016/j.cbpa.2010.03.023, 2010.

[9] D. Pathak, M. Yadav, N. Siddiqui, S. Kushawah, "Antileishmanial agents: an updated review," Der Pharma Chemica, vol. 3 no. 1, pp. 239-249, 2011.

[10] P. Desjeux, J. Alvar, "Leishmania /HIV co-infections: epidemiology in Europe," Annals of Tropical Medicine and Parasitology, vol. 97 no. 1, pp. S3-S15, DOI: 10.1179/000349803225002499, 2003.

[11] J. Alvar, P. Aparicio, A. Aseffa, "The relationship between leishmaniasis and AIDS: the second 10 years," Clinical Microbiology Reviews, vol. 21 no. 2, pp. 334-359, DOI: 10.1128/cmr.00061-07, 2008.

[12] F. Modabber, "Leishmaniasis vaccines: past, present and future," International Journal of Antimicrobial Agents, vol. 36 no. 1,DOI: 10.1016/j.ijantimicag.2010.06.024, 2010.

[13] S. S. Soumya Neelagiri, I. Sravan Kumar, "Tools for antileishmanial drug discovery and drug development," Current Research in Phamaceutical Science, vol. 11 no. 2, pp. 22-28, 2010.

[14] B. Nare, J. Luba, L. W. Hardy, S. Beverley, "New approaches to Leishmania chemotherapy: pteridine reductase 1 (PTR1) as a target and modulator of antifolate sensitivity," Parasitology, vol. 114, pp. S101-S110, 1997.

[15] M. Ouellette, J. Drummelsmith, B. Papadopoulou, "Leishmaniasis: drugs in the clinic, resistance and new developments," Drug Resistance Updates, vol. 7 no. 4–5, pp. 257-266, DOI: 10.1016/j.drup.2004.07.002, 2004.

[16] H. Wang, Z. Yan, J. Geng, S. Kunz, T. Seebeck, H. Ke, "Crystal structure of the Leishmania major phosphodiesterase LmjPDEB1 and insight into the design of the parasite-selective inhibitors," Molecular Microbiology, vol. 66 no. 4, pp. 1029-1038, DOI: 10.1111/j.1365-2958.2007.05976.x, 2007.

[17] S. L. Croft, K. Seifert, V. Yardley, "Current scenario of drug development for leishmaniasis," Indian Journal of Medical Research, vol. 123 no. 3, pp. 399-410, 2006.

[18] S. L. Croft, G. H. Coombs, "Leishmaniasis- current chemotherapy and recent advances in the search for novel drugs," Trends in Parasitology, vol. 19 no. 11, pp. 502-508, DOI: 10.1016/j.pt.2003.09.008, 2003.

[19] P. J. Guerin, P. Olliaro, S. Sundar, "Visceral leishmaniasis: current status of control, diagnosis, and treatment, and a proposed research and development agenda," Lancet Infectious Diseases, vol. 2 no. 8, pp. 494-501, DOI: 10.1016/s1473-3099(02)00347-x, 2002.

[20] N. Singh, M. Kumar, R. K. Singh, "Leishmaniasis: current status of available drugs and new potential drug targets," Asian Pacific Journal of Tropical Medicine, vol. 5 no. 6, pp. 485-497, DOI: 10.1016/s1995-7645(12)60084-4, 2012.

[21] A. M. L. Carmo, F. M. C. Silva, P. A. Machado, "Synthesis of 4-aminoquinoline analogues and their platinum(II) complexes as new antileishmanial and antitubercular agents," Biomedicine and Pharmacotherapy, vol. 65 no. 3, pp. 204-209, DOI: 10.1016/j.biopha.2011.01.003, 2011.

[22] A. Marella, O. P. Tanwar, R. Saha, "Quinoline: a versatile heterocyclic," Saudi Pharmaceutical Journal, vol. 21 no. 1,DOI: 10.1016/j.jsps.2012.03.002, 2013.

[23] A. Zarghi, R. Ghodsi, E. Azizi, B. Daraie, M. Hedayati, O. G. Dadrass, "Synthesis and biological evaluation of new 4-carboxyl quinoline derivatives as cyclooxygenase-2 inhibitors," Bioorganic and Medicinal Chemistry, vol. 17 no. 14, pp. 5312-5317, DOI: 10.1016/j.bmc.2009.05.084, 2009.

[24] M. A. Fakhfakh, A. Fournet, E. Prina, "Synthesis and biological evaluation of substituted quinolines: potential treatment of protozoal and retroviral co-infections," Bioorganic and Medicinal Chemistry, vol. 11 no. 23, pp. 5013-5023, DOI: 10.1016/j.bmc.2003.09.007, 2003.

[25] A. Fournet, M. E. Ferreira, A. Rojas De Arias, "In vivo efficacy of oral and intralesional administration of 2-substituted quinolines in experimental treatment of new world cutaneous leishmaniasis caused by Leishmania amazonensis," Antimicrobial Agents and Chemotherapy, vol. 40 no. 11, pp. 2447-2451, DOI: 10.1128/aac.40.11.2447, 1996.

[26] V. S. Gopinath, J. Pinjari, R. T. Dere, "Design, synthesis and biological evaluation of 2-substituted quinolines as potential antileishmanial agents," European Journal of Medicinal Chemistry, vol. 69 no. 2013, pp. 527-536, DOI: 10.1016/j.ejmech.2013.08.028, 2013.

[27] H. Nakayama, J. Desrivot, C. Bories, "In vitro and in vivo antileishmanial efficacy of a new nitrilquinoline against Leishmania donovani," Biomedicine and Pharmacotherapy, vol. 61 no. 2–3, pp. 186-188, DOI: 10.1016/j.biopha.2007.02.001, 2007.

[28] S. B. Cammerer, C. Jimenez, S. Jones, "Quinuclidine derivatives as potential antiparasitics," Antimicrobial Agents and Chemotherapy, vol. 51 no. 11, pp. 4049-4061, DOI: 10.1128/aac.00205-07, 2007.

[29] L. Everson, P. Teixeira, D. S. Jr, E. Nunes, R. Körting, "In vitro antiprotozoal evaluation of zinc and copper complexes based on sulfonamides containing 8-aminoquinoline ligands," Letters in Drug Design and Discovery, vol. 7 no. 9, pp. 679-685, DOI: 10.2174/157018010792929586, 2010.

[30] L. E. da Silva, A. C. Joussef, L. K. Pacheco, D. G. da Silva, M. Steindel, R. A. Rebelo, "Synthesis and in vitro evaluation of leishmanicidal and trypanocidal activities of N-quinolin-8-yl-arylsulfonamides," Bioorganic and Medicinal Chemistry, vol. 15 no. 24, pp. 7553-7560, DOI: 10.1016/j.bmc.2007.09.007, 2007.

[31] G. Chakrabarti, A. Basu, P. P. Manna, S. B. Mahato, N. B. Mandal, S. Bandyopadhyay, "Indolylquinoline derivatives are cytotoxic to Leishmania donovani promastigotes and amastigotes in vitro and are effective in treating murine visceral leishmaniasis," Journal of Antimicrobial Chemotherapy, vol. 43, pp. 359-366, DOI: 10.1093/jac/43.3.359, 1999.

[32] P. Palit, P. Paira, A. Hazra, "Phase transfer catalyzed synthesis of bis-quinolines: antileishmanial activity in experimental visceral leishmaniasis and in vitro antibacterial evaluation," European Journal of Medicinal Chemistry, vol. 44 no. 2, pp. 845-853, DOI: 10.1016/j.ejmech.2008.04.014, 2009.

[33] L. Paloque, C. Hemmert, A. Valentin, H. Gornitzka, "Synthesis, characterization, and antileishmanial activities of gold(I) complexes involving quinoline functionalized N-heterocyclic carbenes," European Journal of Medicinal Chemistry, vol. 94, pp. 22-29, DOI: 10.1016/j.ejmech.2015.02.046, 2015.

[34] M. O. Faruk Khan, M. S. Levi, B. L. Tekwani, N. H. Wilson, R. F. Borne, "Synthesis of isoquinuclidine analogs of chloroquine: antimalarial and antileishmanial activity," Bioorganic and Medicinal Chemistry, vol. 15 no. 11, pp. 3919-3925, DOI: 10.1016/j.bmc.2006.11.024, 2007.

[35] K. Lackovic, J. P. Parisot, N. Sleebs, "Inhibitors of Leishmania GDP-mannose pyrophosphorylase identified by high-throughput screening of small-molecule chemical library," Antimicrobial Agents and Chemotherapy, vol. 54 no. 5, pp. 1712-1719, DOI: 10.1128/aac.01634-09, 2010.

[36] K. L. Barrack, L. B. Tulloch, L.-A. Burke, P. K. Fyfe, W. N. Hunter, "Structure of recombinant Leishmania donovani pteridine reductase reveals a disordered active site," Acta Crystallographica Section F Structural Biology and Crystallization Communications, vol. 67 no. 1, pp. 33-37, DOI: 10.1107/s174430911004724x, 2011.

[37] A. M. Wernimont, P. Loppnau, J. R Walker, "Crystal structure of MapK from Leishmania donovani , LDBPK_331470," Structure Of Mapk From Leishmania Donovani, 2014. https://www.rcsb.org/structure/4QNY

[38] T. L. Arakaki, E. A. Merritt, B. Ullman, Yates, "Crystal structure of dihydroorotate dehydrogenase from Leishmania donovani," Structural Genomics of Pathogenic Protozoa Consortium, 2008. https://www.rcsb.org/structure/3c61

[39] J. A. Brannigan, B. A. Smith, Z. Yu, "N-myristoyltransferase from Leishmania donovani : structural and functional characterisation of a potential drug target for visceral leishmaniasis," Journal of Molecular Biology, vol. 396 no. 4, pp. 985-999, DOI: 10.1016/j.jmb.2009.12.032, 2010.

[40] S. K., S. G. Isha Raj, "The narrow active-site cleft of O-acetylserine sulfhydrylase from Leishmania donovani allows complex formation with serine acetyltransferases with a range of C-terminal sequences," Acta Crystallographica Section D Biological Crystallography, vol. D68, pp. 909-919, DOI: 10.1107/s0907444912016459, 2012.

[41] MOE, "Molecular operating environment," Scientific Computing and Instrumentation, vol. 2009 no. 1, 2009.

[42] ACD/Structure lab, Version 15.2.5, 2015. http://www.acdlabs.com

[43] O. Trott, A. J. Olson, "AutoDock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading," Journal of Computational Chemistry, vol. 31 no. 2, pp. 455-461, DOI: 10.1002/jcc.21334, 2010.

[44] M. Mühlberg, V. Böhrsch, C. P. R. Hackenberger, "Chemical biology. Learning through case studies. Edited by herbert waldmann and petra janning," Angewandte Chemie International Edition, vol. 48 no. 44,DOI: 10.1002/anie.200904323, 2009.

[45] E. S. Coimbra, L. M. R. Antinarelli, N. P. Silva, "Quinoline derivatives: synthesis, leishmanicidal activity and involvement of mitochondrial oxidative stress as mechanism of action," Chemico-Biological Interactions, vol. 260, pp. 50-57, DOI: 10.1016/j.cbi.2016.10.017, 2016.

[46] R. Sharma, A. K. Pandey, R. Shivahare, K. Srivastava, S. Gupta, P. M. S. Chauhan, "Triazino indole-quinoline hybrid: a novel approach to antileishmanial agents," Bioorganic and Medicinal Chemistry Letters, vol. 24 no. 1, pp. 298-301, DOI: 10.1016/j.bmcl.2013.11.018, 2014.

[47] L. Paloque, P. Verhaeghe, M. Casanova, "Discovery of a new antileishmanial hit in 8-nitroquinoline series," European Journal of Medicinal Chemistry, vol. 54, pp. 75-86, DOI: 10.1016/j.ejmech.2012.04.029, 2012.

[48] M. A. I. Essa Ajmi Alodeani, Mohammad Arshad, "Antileishmanial activity and computational studies of some hydrazone derivatives possessing quinoline nucleus," European Journal of Pharmaceutical and Medical Research, vol. 2 no. 7, pp. 324-328, 2015.

[49] K. Kaur, S. R. Patel, P. Patil, "Synthesis, antimalarial, antileishmanial, antimicrobial, cytotoxicity, and methemoglobin (MetHB) formation activities of new 8-quinolinamines," Bioorganic and Medicinal Chemistry, vol. 15 no. 2, pp. 915-930, DOI: 10.1016/j.bmc.2006.10.036, 2007.

[50] R. Dietze, L. C. Valli, M. Grogl, "Phase 2 trial of WR6026, an orally administered 8-aminoquinoline, in the treatment of visceral leishmaniasis caused by Leishmania chagasi," American Journal of Tropical Medicine and Hygiene, vol. 65 no. 6, pp. 685-689, DOI: 10.4269/ajtmh.2001.65.685, 2001.

[51] P. M. Loiseau, S. Cojean, J. Schrével, "Sitamaquine as a putative antileishmanial drug candidate: from the mechanism of action to the risk of drug resistance," Parasite, vol. 18 no. 2, pp. 115-119, DOI: 10.1051/parasite/2011182115, 2011.

[52] M. S. T. Makki, A. Bakhotmah, M. Abdel-Rahman, "Highly efficient synthesis of novel fluorine bearing quinoline-4-carboxylic acid and the related compounds as amylolytic agents," International Journal of Organic Chemistry, vol. 2 no. 1, pp. 49-55, DOI: 10.4236/ijoc.2012.21009, 2012.

[53] J. Lloyd, D. E. Ryono, J. E. Bird, "Quinoline-4-carboxylic acids as angiotensin ii receptor antagonists," Bioorganic and Medicinal Chemistry Letters, vol. 4, pp. 195-200, DOI: 10.1016/s0960-894x(01)81146-x, 1994.

[54] G. C. Muscia, J. P. Carnevale, M. Bollini, S. E. Asís, "Microwave-assisted döbner synthesis of 2-phenylquinoline-4-carboxylic acids and their antiparasitic activities," Journal of Heterocyclic Chemistry, vol. 45 no. 2, pp. 611-614, DOI: 10.1002/jhet.5570450251, 2008.

[55] D. W. Rangnekar, G. R. Shenoy, "Synthesis of 2-aryl-6-substituted quinolines and their use as fluorescent brightening agents," Dyes and Pigments, vol. 8 no. 4, pp. 281-290, DOI: 10.1016/0143-7208(87)85018-0, 1987.

[56] A. E. M. S., S. A. Elhadi, "Synthesis of some 2-aryl- and 2,3-diaryl- quinolin-4-carboxylic acid derivatives," Synthetic Communications, vol. 41 no. 10, pp. 1435-1443, DOI: 10.1080/00397911.2010.486508, 2011.

[57] F. Meheus, M. Boelaert, A. A. Abuzaid, "The economic burden of visceral leishmaniasis in Sudan: an assessment of provider and household costs," American Journal of Tropical Medicine and Hygiene, vol. 89 no. 89, pp. 1146-1153, DOI: 10.4269/ajtmh.12-0585, 2013.

[58] C. A. Lipinski, F. Lombardo, B. W. Dominy, P. J. Feeney, "Experimental and computational approaches to estimate solubility and permeability in drug discovery and development settings," Advanced Drug Delivery Reviews, vol. 64,DOI: 10.1016/j.addr.2012.09.019, 2012.

[59] D. F. Veber, H.-Y. Johnson, H. Cheng, B. R. Smith, K. W. Ward, K. D. Kopple, "Molecular properties that influence the oral bioavailability of drug candidates," Journal of Medicinal Chemistry, vol. 45, pp. 2615-2623, DOI: 10.1021/jm020017n, 2002.