Synthesis and in vitro Leishmania promastigote growth inhibition efficacy of novel 4(3 H )-quinazolinone derivatives

Molecular hybridization is an increasingly important strategy in rational drug design and development. A series of novel quinazolinone-triazole hybrids have been synthesized and their antileishmanial activity investigated. Derivatives ( E )-3-(prop-2-yn-1-yl)-2-styrylquinazolin-4(3 H )-one, and ( E )-3-{[1-(4-bromobenzyl)-1 H -1,2,3-triazol-4-yl]methyl}-2-styrylquinazolin-4(3 H )-one were observed to moderately inhibit the growth of promastigotes. An overall lack of significant antileishmanial activity may be attributable to the poor aqueous solubility of the derivatives. Future research endeavors will focus on potential remediation by investigating the anchoring of hydrophilic moieties to the quinazolinone scaffold.


Introduction
Leishmaniasis is a parasitic disease characterized by infection with an obligate intracellular parasite of the genus Leishmania. 1 The disease is primarily transmitted by the bite of an infected female phlebotomine sandfly. 2 Leishmaniasis is one of several infectious diseases of the tropics and sub-tropics that constitute a steadily increasing public health burden.This is the result of a variety of factors, including, inter alia, the coinfection with human immunodeficiency virus (HIV) 3 , and the migration of immigrants and refugees from areas of known endemicity. 4 Leishmaniasis is classified as a neglected tropical disease (NTD) due its low visibility amongst the public of high-income countries, and a paucity of research investment on the part of the pharmaceutical sector and policy makers. 5eishmaniasis is endemic to ninety-eight countries and territories, where an estimated one billion people are at risk of contracting this disease. 6There are over twenty Leishmania species, 7 of which only three are predominantly responsible for the three clinical forms of the disease in humans. 8These include: (i) L. major, which causes the most common form of the disease, i.e., cutaneous leishmaniasis (CL), which is seen to cause the development of ulcers and skin lesions across exposed areas of the body; 6 (ii) L. donovani, responsible for visceral leishmaniasis (VL), the most lethal form of the disease with a 95% fatality rate if left untreated, and which is typified by symptoms such as fever, weight loss, fatigue, and an enlarged spleen and liver 6,8 ; and (iii) L. braziliensis, which causes mucocutaneous leishmaniasis (MCL), and is characterized by a partial or complete loss of the mucous membranes in the mouth, nose and throat. 6,8In 2019 alone, around 600,000 -1 million new cases of cutaneous leishmaniasis, and some 50,000 -90,000 new cases of the visceral form of the disease, were reported to have occurred worldwide. 6he treatment of human leishmaniasis is limited to the chemotherapeutic use of only a handful of drugs, which include the pentavalent (Sb V ) antimonials (sodium stibogluconate and meglumine antimoniate), amphotericin B, miltefosine, pentamidine, and paromomycin (Figure 1).The use of these drugs, however, is negatively associated with treatment failure, the emergence of drug-resistant pathogenic strains in both experimental and clinical settings, as well as the occurrence of toxicity and adverse events. 9Altogether, these shortcomings serve to stress the need for new and effective leishmanicidal agents.
The development of novel chemical entities with entirely new mechanism(s) of action is expensive, time consuming and fraught with risk. 10It is, therefore, desirable, instead, to make use of drug development strategies that exploit structures with known biological activities.One such a strategy is molecular hybridization, a strategy in rational drug design and development wherein two or more pharmacophoric subunits, derived from structures with known biological activity, are joined into one single structure. 11The resulting hybrid is expected to have greater potency, efficacy and affinity compared to the respective parent molecules.The strategy has been utilized with great success to generate promising chemotherapeutic agents to prevent or treat tropical parasitic diseases, such as malaria, 12 trypanosomiasis and leishmaniasis. 13uinazolinones are a class of heterocycles comprised of fused nitrogen-containing rings that occur naturally in the form of some 200 alkaloids in plants, 14 as well as metabolites derived from microorganisms, such as Bacillus cereus. 15Synthetic, as well as semi-synthetic, quinazolinones are also extant in the scientific literature. 16This class of compounds has attracted considerable attention in medicinal chemistry owing to the ease of its synthesis, and the fact that it elicits a range of biological activities, including, inter alia, antimalarial, 17 antimycobacterial 18 and antileishmanial 19 activity.
1,2,3-Triazoles hold a significant importance in drug discovery for their ability to evoke a myriad of biological activities, chief among these being their antiprotozoal activities against the pathogens of diseases such as malaria, 20 leishmaniasis 21 and trypanosomiasis. 22They are readily synthesized by Huisgen cycloaddition 23 or copper-catalyzed alkyne-azide cycloaddition (CuAAC) reactions 24 between terminal alkynes and azides.In this study, a series of novel quinazolinone-triazole hybrids were synthesized, using a variety of synthetic routes, and their cytotoxicity profiles, as well as their ability to inhibit the growth of Leishmania promastigotes, were assessed in vitro.We, herein, report the syntheses and biological properties of these hybrids.
Third, the quinazolinone-triazole hybrids (4a-j) were produced using methods outlined in the literature. 27enzyl azides (3a-e) were synthesized through nucleophilic substitution reactions (S N 2) between commercial benzyl bromides and sodium azide (NaN 3 ).The benzyl azides obtained were oil-like liquids that were deemed suitable for use in subsequent reactions without further purification.These organic azides were then subjected to click chemistry with propargyl quinazolinones (2a,b) in the presence of sodium L-ascorbate and βcyclodextrin (CD), acting as reducing agent and phase-transfer catalyst, respectively, for 48-72 h to yield 4a-j in low to excellent yields (30 -89%) after purification by trituration with both EtOH and distilled H 2 O.
Successful syntheses of all compounds were confirmed by routine molecular characterization techniques, including infrared (IR), nuclear magnetic resonance (NMR) and high-resolution mass spectroscopy (HRMS).The 1 H and 13 C NMR spectra were inspected in order to identify peaks that are characteristic of quinazolinone, styryl and triazole moieties.The IR spectra were similarly investigated for the presence of characteristic absorptions that would allow for the identification of functional groups and substituents.
The 1 H spectra of compounds 1a,b bore a characteristic singlet peak around δ 12.00 ppm, indicative of proton H-3 of the amide of quinazolinone ring.The disappearance of this peak in the spectra of compounds 2a,b was confirmative of successful propargylation, further corroborated by the appearance of peaks that were indicative of propargyl protons.These included the doublet (d) of methylene CH 2 , H-1'' (ca.5.20 ppm) and the triplet (t) of alkyne proton H-3'' (ca.δ 3.24 ppm), respectively.Long-range coupling, 4 J 2.3-2.4 Hz, typical between alkyne protons was seen to occur between H-3'' and H-1'' in the spectra of 2a,b. 28The formation of the propargyl moiety was also indicated by the appearance of three additional peaks in the 13 C spectra of 2a,b.These peaks were representative of the propargyl carbons C-1'' (ca.δ 31.7 ppm), C-2'' (δ 79.0 ppm) and C-3'' (ca.δ 75.0 ppm).
The literature reports 3 J H -H 12-18 Hz values for vinylic hydrogens in trans-configuration, while those of the cis-configuration have 3 J H -H in the 0-12 Hz range. 28The 3 J H-1'-H-2' values of the vinylic protons in the styryl moiety were found in the 15 -16 Hz range, which suggested that the styryl moiety had occurred as the transisomer in all the quinazolinones synthesized.The J values of the proton H-5 (in the vicinity of the carbonyl) indicated o-coupling with proton H-6 ( 3 J H-5-H-6 6.7-7.9Hz) and m-coupling with proton H-7 ( 4 J H-5-H-7 0.8-1.3Hz). 28he spectra of intermediates 3a-e commonly had the singlets assigned to the resonance of methylene (CH 2 ) protons in the δ 4.50-4.30ppm region, and associated carbon in δ 54.9-53.5 ppm of the benzyl moiety.Additionally, the peaks resulting from the resonance of the aromatic protons were present in the spectra as doublets (d) or triplet (t) with an o-coupling ( 3 J H-3-H-4 7.4-8.5Hz) among them.For 3c, however, due to the fluoro-coupling, the signals of the protons adjacent to the fluorine resulted in a doublet of doublet (dd) with J 8.5, 5.3 Hz (o-coupling 3 J H-H 8.5 and F-coupling 3 J H-F 5. 3 Hz).
Further confirmation of the structures of compounds 1b, 2b and 4f-j was indicated by the presence of the singlets representative of the methoxy (OMe) protons (ca.δ 3.80 ppm) and carbon in the δ 55.0-51.6 ppm in their 1 H and 13 C spectra.Additional evidence of the structure of hybrid 4j was provided by the presence of the singlets attributed to the methyl (Me) protons, and the associated carbon at δ 2.22 and 20.6 ppm, in their respective NMR spectra.These peaks were also seen in the spectra of 4e at δ 2.25 for the methyl protons and at δ 20.1 ppm for the carbon atom.
Characteristic absorptions for carbonyl functional groups (C=O) (1672-1654 cm -1 , C=O stretching) and N-H portion of the amide (3186-3182 cm -1 , N-H stretching) were seen in the IR spectra of 1a,b.Additionally, absorptions were observed for the propargyl (3235-3209 cm -1 , ≡C-H stretching and 2115-2115 cm -1 , C≡C stretching) functional group in the IR spectra of 2a,b.Other absorptions, specifically those pertaining to the C=N (1642-1631 cm -1 , C=N stretching) functionality, could be seen in the spectra of most quinazolinones.The absorption resulting from N=N=N stretching of the azido (N 3 ) functional group was present in the spectra of the intermediates 3a-e as a strong band in the 2100-2090 cm -1 region.The disappearance of this band from the spectra of hybrids 4a-j is another telltale sign of the successful click chemistry.
The C=N stretching absorptions of compounds 4a, 4h and 4i, however, were not visible due to the overlapping with those of their respective carbonyl (C=O) functional groups.The C-H bending absorptions of the methylene bridges in compounds 2a,b and 4a-j occurred between 1473-1469 cm -1 .Nitro (NO 2 ) and alkyl aryl ether functional groups were observed to absorb ca.1519 cm -1 (N-O stretching), and 1256-1245 cm -1 (C-O stretching), respectively.Absorptions specific to the 1,2,3-triazole moiety were also observed; these included alkene =C-H stretching in the region of 3140-3108 cm -1 and C-N stretching in the 1348-1331 cm -1 range.

Physicochemical properties
The drug-likeness of the compounds was determined using Lipinski's rule of five, an index that is often adopted to predict whether a compound may be orally active in humans. 29Due consideration was given to oral activity as miltefosine is currently the only antileishmanial drug that is administered orally, and its use is threatened by toxicity and pathogenic resistance.This makes research into potentially orally active agents an imperative necessity.Thus, physicochemical properties, such as lipophilicity and aqueous solubility of the quinazolinone-triazole hybrids and their intermediates, were elaborated on and discussed as impactors of Lipinski's index (Table 1).a Calculated logP (consensus logP).b Predicted aqueous solubility, where log S is the logarithm of the amount of compound (in moles) able to dissolve in a liter of water.c ESOL = estimated aqueous solubility, calculated using a topological method 30 with log S scale: insoluble < -10 < poorly < -6 < moderately < -4 < soluble < -2 very soluble < 0 or more -highly soluble.d Calculated using a topological method. 31e Number of hydrogen bond donors (NH and OH groups).f Number of hydrogen bond acceptors (nitrogen and oxygen atoms).g Determined with reference to Lipinski's rule of five (MW ≤ 500 g/mol; cLogP ≤ 5; HBD ≤ 5; HBA ≤ 10; no more than one violation allowed). 29All values in this table were calculated using the SwissADME web tool, http://www.swissadme.ch 32s can be seen in Table 1, the synthesized compounds had the physicochemical properties well within the target ranges as set by Lipinski et al. 29 With the exception of 4g, none of the compounds violate Lipinski's rules, predicting these quinazolinones to be drug-like in nature, suitable for oral administration, and well absorbed in the gastrointestinal tract through passive diffusion.

Biological activity
A single-point screening of all synthesized compounds, in 100 μM concentration experiments, was performed to assess the antileishmanial activity against three strains of Leishmania promastigotes, with amphotericin B (AMB) serving as the reference drug.These strains were L. donovani strains (1S and 9515) and L. major strain IR-173.The Leishmania spp.were selected in order to determine the specificity of the synthesized compounds against L. major parasites that cause CL, and L. donovani parasites that communicate the more serious and debilitating VL. 33 In this study, the quinazolinone derivatives were screened against the L. promastigotes in a two-stage process, involving the determination of the parasite growth inhibition percentage at the single (100 μM) concentration, followed by the activity assessment through minimum inhibitory concentration (IC 50 ) determination, for those compounds that fulfilled the growth inhibition criteria to be screened further for anti-promastigote activity.The cytotoxicity profiles of the quinazolinones were evaluated, using Vero cells with emetine (EM), known for its high toxicity, as a positive control.The biological results are presented in Table 2.Only three compounds, 1a, 2a and 4b, were observed to possess activity against the L. donovani 1S strain.Substitution of a propargyl moiety at N-3 position of 1a (27%) effectively doubled the antileishmanial activity against the 1S strain, as evidenced by the activity of 2a (58%).The antileishmanial activity of 4b (22%) against L. donovani 1S was structure specific, as no other hybrid had activity against this strain.None of the compounds with a methoxy (CH 3 O) substituent had activity against this strain.
The replacement of H (R 1 ) in 1a (2%) with the electron-donating CH 3 O group was seen to have the effect of improving its activity against the L. donovani 9515 strain, as demonstrated by the markedly increased parasite growth-inhibitory activity of compound 1b (21%).Conversely, unsubstituted counterparts, i.e., 2a, 4b, 4d and 4e, were active against L. donovani 9515 promastigotes.The activities of 4b, 4d and 4e appeared to be structure specific, as no observable structure-activity relationship could be discerned between the net electronic effect [electron-withdrawing (EW) or electron-donating, (ED)] of their R 2 substituents and the ability to inhibit the growth of L. donovani 9515 promastigotes.
Amongst compounds 4a-j, the greatest inhibition of L. donovani 9515 promastigote growth was seen to be elicited by 4b (34%) that had no R 1 substituent and possessed a bromine as R 2 substituent.Overall, compound 2a (43%) was observed to be the most active quinazolinone against L. donovani 9515.L. major promastigotes appeared to be more susceptible to the synthesized compounds than were the L. donovani parasites.Most of the compounds were observed to exhibit some degree of growth inhibitory activity against L. major IR-173 strain promastigotes, with the exception of 2b, 4d and 4h.Overall, the greatest inhibitor of L. major IR-173 promastigote growth was observed to be hybrid 4b (47%).
The literature suggested a 70% parasite growth inhibition threshold at 10 μM for antileishmanial IC 50 determinations. 33We were compelled to use 100 μM in order to identify qualifying compounds as 10 μM screening produced nearly no inhibition.As no synthesized compounds had reached the 70% inhibition threshold, we subsequently also lowered, and arbitrarily set, a new threshold at 25% to allow for more quinazolinones qualifying for IC 50 determinations.Thus, the intermediate compounds 1b and 2a, and the hybrids 4b and 4e, all with parasite growth inhibition higher than 25% regardless of the strain, were selected for IC 50 determinations.They were also non-cytotoxic to Vero cells (IC 50 > 100 μM). 34They were also found to be inactive against all three Leishmania promastigotes strains with IC 50 >100 μM.
All synthesized compounds were screened as suspensions in aqueous growth medium.The poor aqueous solubility may have, in part, contributed to the lack of antileishmanial activity and cytotoxicity.

Conclusions
A small library of quinazolinone derivatives were synthesized in low to good yields (30 -89%) using a variety of synthetic methods, including cyclization, condensation, nucleophilic (S N 2) substitution and copper-catalyzed alkyne-azide cycloaddition (CuAAC) reactions.The in vitro antileishmanial activity and cytotoxicity of the compounds were determined against three strains of Leishmania promastigotes and Vero cells, respectively.
The compounds were nontoxic to the mammalian cells, but also had only moderate-to-no antileishmanial activities despite meeting predicted drug-like criteria.The complete lack of cytotoxicity and the inability to adequately inhibit promastigote growth may be attributed to the poor aqueous solubility of the compounds.Two derivatives stood out, however, as they were observed to possess moderate antileishmanial activity.These were 2a featuring styryl and propargyl moieties with 43 -58% growth inhibition of L. donovani, and hybrid 4b containing 4-methoxystyryl and propargyl moieties with 48% growth inhibition of L. major promastigotes.No conclusive structure-activity relationship could be deduced from this study.Future studies may include the investigation of more water-soluble derivatives in order to address the antileishmanial inactivity issue encountered in the study.

Experimental Section
General.All reagents were purchased from Sigma-Aldrich (Johannesburg, South Africa).Ammonium acetate and N,N-dimethylformamide (DMF) were purchased from Merck (Johannesburg, South Africa).All other solvents, and magnesium sulphate, were purchased from Associated Chemical Enterprises Acetone (Johannesburg, South Africa).All chemicals and reagents were reagent/analytical grade and were used without further purification.The 1 H and 13 C NMR spectra of all 14 compounds were recorded on a Bruker Avance III 600 spectrometer at frequencies of 600 MHz ( 1 H NMR) and 150.913 ( 13 C NMR), respectively, in deuterated dimethyl sulfoxide (DMSO-d 6 ) at room temperature (~25 °C) or, alternatively, 80 °C, where indicated.Chemical shifts in spectra obtained were expressed and reported in parts per million δ (ppm), with the residual protons of the deuterated solvents serving as the reference.J (coupling constant) values were expressed in Hertz (Hz).Abbreviations used to indicate splitting patterns were as follows: singlet (s), doublet (d), doublet of doublets (dd), triplet (t), triplet of doublets (td), and multiplet (m).HRMS spectra were recorded on a micrOTOF-Q II mass spectrometer, using an APCI source type heated at 200 °C.The software used to record the mass spectra was the Bruker Compass DataAnalaysis 4.0 software package.Scans were performed from 50-1600 m/z at a capillary voltage of 4500 volts (V), an end-plate-offset voltage of -500 V, with the nebulizer set at 1.6 Bar, and a collision cell radio frequency (RF) voltage of 100 volts peak to peak (Vpp).High-performance liquid chromatography (HPLC) was carried out on all compounds to determine their purity.Full description of the process can be found in the Supplementary Material file.Infrared (IR) spectra were recorded on a Bruker Alpha-P FTIR instrument system.Thin-layer chromatography (TLC) was performed using aluminium-backed silica gel plates from Merck (Johannesburg, South Africa) with a mean pore size of 60 Å with added fluorescent indicator (F254).TLC plates were visualized at 254 nm.Melting points of all compounds were performed on a Büchi melting point B-545 apparatus and are uncorrected.

Synthesis of 2-(4-substituted styryl)-4(3H)-quinazolinones 1a,b
Compounds 1a,b were synthesized through a known method reported by Kumar et al. 25 and illustrated in Scheme 1 (i -ii).The method was carried out as follows: To a mixture of isatoic anhydride (8.16 g, 0.05 mol) and ammonium acetate (3.85 g, 0.05 mol), in a flask outfitted with a magnetic stirrer, was added an equimolar amount of triethyl orthoacetate (8.11 g, 0.05 mol).The mixture was refluxed for 6 -8 h.Afterwards, the appropriate para-substituted benzaldehyde (0.05 mol) was added and refluxing was continued for another 6 -8 h until TLC monitoring showed completion of the reaction.The content of the flask was then suspended in a suitable amount of EtOH (25 mL), boiled and filtered.The crude residue was further purified by precipitation.It was heated ca.150 °C in DMF (100 mL) with stirring, and solubilized until a clear solution was obtained.The solution was then slowly poured into H 2 O (100 mL) with stirring at room temperature.This resulted in the precipitation of compounds 1a,b which were filtered off in a sintered glass funnel, washed with H 2 O (3 x 150 mL) and dried in oven at 90 o C.

Synthesis of 3-propynyl-2-(4-substituted styryl)-4(3H)-quinazolinones 2a,b
Compounds 2a-b were synthesized using a literature method 26 as illustrated in Scheme 1 (iii).The synthesis is described as follows: Propargyl bromide (2.86 g, 0.024 mol) was added to a suspension of the appropriate intermediate 2-(4-substituted styryl)-4(3H)-quinazolinone, 1a,b (0.02 mol) and potassium carbonate (33.17 g, 0.24 mol) in DMF (50 mL).The reaction mixture was allowed to stir at room temperature until TLC monitoring indicated complete disappearance of the intermediate (15 min -48 h).The content of the flask was then poured into distilled water at room temperature and stirred until complete dissolution of the potassium carbonate.This results in the formation of precipitate that was filtered off, washed with distilled water (3 x 50 mL), thoroughly dried, and recrystallized from acetone or EtOH to afford the target compound.

Synthesis of para-substituted benzyl azides 3a-e
Intermediates 3a-e were synthesized by employing a literature method 27 as illustrated in Scheme 1 (iv).The appropriate para-substituted benzyl bromide (0.01 mol) was dissolved in DMSO (20 mL) in a flask outfitted with a magnetic stirrer.Sodium azide (0.71 g, 0.011 mol) was slowly added to the flask and the reaction mixture was stirred for 10 -12 h at room temperature.Afterwards, the reaction was quenched with H 2 O (50 mL).The aqueous mixture was extracted with EtOAc (3 x 50 mL).The organic phase was then washed with brine (3 x 50 mL), dried overnight with MgSO 4 , and filtered off.The filtrate was evaporated under vacuum evaporator to produce 3a-e as oils which were used in subsequent syntheses without further purification.

Synthesis of quinazolinone-triazole hybrids 4a-j
The compounds were synthesized according to a literature method, 27      100%.
Cytotoxicity assay.The cytotoxicities of the synthesized compounds were evaluated in Vero cells (Cellonex, South Africa), using the resazurin assay method in Janse van Rensburg et al. 37

Table 1 .
Physicochemical properties and Lipinski's Rule of Five results of all synthesized quinazolinone derivatives as predicted by the SwissADME web tool, http://www.swissadme.ch

Table 2 .
In vitro anti-promastigote growth inhibition efficacy and cytotoxicity of quinazolinone derivatives at 100 μM concentration b represented as the mean ± the standard deviation (SD) from the triplicate biological experiments.c African green monkey kidney epithelial cells.AMB: Amphotericin B deoxycholate; EM: Emetine dihydrochloride hydrate.