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

Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Al-Neelain University, Khartoum, Sudan Department of Pharmaceutical Chemistry, College of Pharmacy, Jouf University, Sakaka, Saudi Arabia Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Omdurman Islamic University, Khartoum, Sudan Department of Pharmaceutics, College of Pharmacy, Jouf University, Sakaka, Saudi Arabia Department of Pharmaceutics, Faculty of Pharmacy, University of Khartoum, Khartoum, Sudan Institute of Endemic Diseases, University of Khartoum, Khartoum, Sudan Department of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Khartoum, Khartoum, Sudan


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].e 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].
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].
e 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].e 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].
ere 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.e treatment is mainly based on organic pentavalent antimonial compounds, pentamidine, amphotericin B, miltefosine, and few other drugs [14,15].e 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.e 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.e 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].eir biological activity includes antileishmanial, antimalarial, antibacterial, antifungal, anthelmintic, cardiotonic, anticonvulsant, antiinflammatory, analgesic, and antitumor activity [22][23][24][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][27][28][29][30][31][32][33][34][35].
e current work aims at synthesizing some quinoline derivatives and at investigating their antileishmanial activity.

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.e 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 UVlamp or iodine.
Infrared (IR) spectra were recorded as KBr disk using Shimadzu IR apparatus at the Research Center, College of Science, Khartoum University, Sudan.e data are given in υ ′ (cm −1 ).NMR spectra were determined in DMSO-d6 and recorded on NMR spectrophotometer (500 MHz) at Kyoto University, Japan.e 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.
e 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 %).

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.e solution was neutralized with 1 M hydrochloric acid.e precipitate was filtered, washed with water, dried, and recrystallized from ethanol (Scheme 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.

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.e medium was changed every 3 days until promastigotes reached their stationary phase.

In Vitro Evaluation of the Antileishmanial Activity.
Promastigote density was adjusted to 2 × 10 6 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.e plates were incubated at 25 °C for 24 hours.Parasites were counted by using a hemocytometer.e IC 50 for each compound was determined and manipulated statistically.e result is represented in Table 1.
Solvent molecules and cocrystallized ligands were removed from the proteins, and hydrogen atoms were added.
e 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].
e compounds set was docked on the previous targetbinding sites by using AutoDock Vina software in PyRx [43,44].e 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.e docking poses were ranked according to their docking scores as free energy of binding, and the results were exported for further analysis.e results are represented in Table 2.

Drug-Likeness Assessment.
e 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).

Biological Activity.
e parasite viability curve of compounds Q1, Q4, Q5, and Q12 is shown in Figure 1. e IC 50 of the synthesized quinolines and positive controls are given in Table 1.

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.

Discussion
VL is considered as one of the leading causes of morbidity and mortality in tropical countries.e 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][46][47][48][49][50][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][53][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-4carboxylic 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.e synthesized compounds were purified by recrystallization from ethanol.e purity of these compounds was ensured by TLC.
e molecular structures of the synthesized quinoline-4carboxylic acids were confirmed on the basis of their spectral data which were consistent with the reported data [54][55][56].
e infrared spectra of all synthesized compounds showed a broad band at 3419-3300 cm −1 characteristic 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 3021.80-3096.98cm −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 1 H 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.e 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 -CH 3 and -OCH 3 protons, respectively.A highly deshielded singlet at Journal of Chemistry 9.95, 10.02, and 9.78 for compounds Q7, Q8, and Q13, respectively, corresponding to phenolic OH was observed.
e 13 C 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].
e tested parasite was subjected to different concentration levels of the synthesized compounds to determine IC 50 , the compound concentration causing 50% inhibition of the parasitic growth.e 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 IC 50 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   5). is 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 (2phenylsubstituted quinoline) to be active is between 4.20 and 4.50.It can be proposed that the presence of an electronwithdrawing 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.
In general, for all synthesized compounds together, there is no constant pattern that correlates the activity with the calculated log P values.is 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.e synthetic quinolines were docked into 5 leishmanial targets that have been previously identified as potential drug targets.
e 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.us, 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.

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.

Table 3 :
49ug-likeness assessment by Molinspiration for compound Q1.IC 50 values 17.19 and 14.70 µg/mL, respectively.Interestingly, compound Q1 (IC 50 � 1.49 µg/mL) was found to be five times more potent than the standard drug sodium stibogluconate (IC 50 � 8.06 µg/mL) and ten times more potent than amphotericin B (IC 50 � 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 IC 50 is higher than other analogues with comparable lipophilicity (Q5 IC 50 � 103 µg/mL compared with Q4 clogP: 4.48 with IC 50 � 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 IC 50 of these analogues theoretically.However, the activity of Q6 (IC 50 � 84.49µg/ mL) is much lower than those of Q4 and Q12 (clogP � 4.48, IC 50 � 27.03 and clogP � 4.38, IC 50 � 17.19 µg/mL, respectively).It is worth noting that compound Q6 violates one of the Lipinski's rule parameters (Q6 clogP > with