Synthesis , Antiplasmodial and Antitrypanosomal Evaluation of a Series of Novel 2-Oxoquinoline-based Thiosemicarbazone Derivatives

Herein a series of novel thiosemicarbazones (TSCs) derived from 2-oxoquinoline scaffold is reported, and the target compounds have been successfully synthesized and characterized using standard spectroscopic techniques. The in vitro biological activities of synthesized molecules were evaluated against Plasmodium falciparum malaria parasites (strain 3D7), Trypanosoma brucei brucei parasites (strain 427) and HeLa cells. All the compounds displayed modest or no activity at a concentration of 20 μM and percentage viability of >50 % was often observed. Except for compound 9o, none of the final compounds exhibited cytotoxic effects against HeLa cells at 20 μM.


Introduction
Malaria, an infectious parasitic disease, is a major health risk in many developing countries worldwide. 1Despite tremendous progress over the last two decades, in 2017 there were 216 million cases of malaria infection, with an estimated 445 000 deaths, 90 % of which occur in sub-Saharan Africa. 1,2Currently, it is estimated that almost 3.2 billion people globally are at risk of contracting the disease. 3This is further aggravated by the widespread drug and multidrug resistant Plasmodium falciparum parasite, the main cause of infection in humans, to almost all antimalarial drugs that are in clinical use. 4 In the absence of an effective malaria vaccine, the need to discover and develop new antimalarial drugs, with unique structural motifs and new mode of action, that are safe and effective against highly resistant parasites is imperative. 4,5n other hand, human African trypanosomiasis (HAT), commonly referred to as a sleeping sickness, is caused by protozoan parasites of the genus Trypanosoma, and the two species that are transmitted to humans by blood-feeding tsetse flies (Glossina spp.) are Trypanosoma brucei gambiense and Trypanosoma brucei rhodesiense. 6,7Up to 70 million people, in various parts of the 36 countries in Africa where the disease is endemic, are at risk of infection. 6While the cases of HAT in Africa have been reasonably low, in 2015 an estimated 3000 new infections of East and West African trypanosomiasis were reported to the World Health Organisation (WHO). 8Regrettably, in pregnant women or those of child-bearing age, the disease causes infertility and abortion, and it is invariably fatal if left untreated. 8Currently, only a handful of drugs are available for the treatment of HAT, and are utilized based on the causative trypanosome species and stage of the disease. 8For example, pentamidine and suramin are recommended for treatment of the acute initial stage of T. b. gambiense, while a combination of nifurtimox-eflornithine and melarsoprol are deployed for the secondary stage of the disease.For T. b. rhodesiense, suramin is a preferred drug for treatment of the initial stage of the disease, while melarsoprol is reserved for secondary stage chemotherapy. 9However; these drugs have shortcomings and some of them are associated with lifethreatening side effects that have prompted the scientific community to search for new compounds with desirable safety margins and drug-like properties to replace them.
Thiosemicarbazones are a class of compounds which have enjoyed significant attention due to their broad-spectrum of biological activities, including antibacterial, antiprotozoal, antifungal, antiviral and antitumour activity. 10Quinoline and related derivatives, on the other hand, are useful compounds with diverse pharmaceutical applications, and some have even reached markets for treatment of various ailments. 113][14][15] For example, oxoquinoline-derived thiosemicarbazones I and III (Fig. 1) were found to exhibit good antiproliferative activity against the HCT116 cell line. 16n our pursuit of developing biologically relevant molecules, that could address some of the problems associated with infections caused by protozoan parasites, we are interested in exploring a class of 2-oxoquinoline-derived thiosemicarbazone derivatives as antiplasmodial and antitrypanosomal agents.To the best of our knowledge, there has been no report on the antiplasmodial and antitrypanosomal properties of these compounds in literature.To this end, in this study we report on the synthesis of quinoline-derived thiosemicarbazones and their in vitro bioassay screening against P. falciparum 3D7 strain and T. b. brucei (strain 427) as well as cytotoxicity evaluation against HeLa cell lines.
Similarly, reacting the key intermediates 4a-c with substituted benzylbromides and 7-chloro-N-(2-chloropropyl)quinolin-4-amine yielded quinoline aldehydes 7a-f and 8b in moderate yields, respectively.Compound 8a could not be isolated in its pure form, and instead it was reacted with thiosemicarbazide (step viii) as crude product to form the desired quinolinederived thiosemicarbazone 9n.5][26] All the intermediates and target compounds were fully characterized by analytical and spectroscopic techniques.

In Vitro Biological Evaluation
The prepared target compounds were evaluated in vitro for antiplasmodial activity against the chloroquine sensitive (CQS) 3D7 P. falciparum, the trypanosomal subspecies responsible for nagana T. b. brucei, and for cytotoxicity evaluation using a human cervix adenocarcinoma (HeLa) cell line.Chloroquine (CQ) was included as a positive control for P. falciparum and pentamidine (PMD) was employed for T. b. brucei assays while emetine (EMT) was a positive control drug for HeLa cells.The screening assay was done using the malaria parasite lactate dehydrogenase Pf(pLDH), T. b. brucei and HeLa cell resazurin assays that were performed in duplicates using 20 mM final concentrations of each compound.The percentage cell viability results upon exposure of P. falciparum, T. b. brucei and HeLa cells to the compounds are displayed in Table 2.
The tested compounds (Table 2) exhibited no cytotoxic effects (percentage viability >64 %) as measured by the viability of HeLa cell lines at a concentration of 20 µM, the exception is compound 9o which reduced HeLa cell viability to 6.6 %.Excluding compounds 9m, 9n and 9o, which reduced the percentage parasite viability to below 20 %, none of the tested target compounds displayed desirable potency at the concentration of 20 µM.These compounds were evaluated further to determine the corresponding IC 50 values (Fig. 2) against the 3D7 strain of the parasite P. falciparum.And while 9m emerged as inactive, compounds 9n and 9o displayed notable activity with the corresponding IC 50 values of 2.09 and 1.79 µM, respectively.These data suggest that the observed antiplasmodial activity may be related to the presence of aminoquinoline moiety in each molecule, which is known to bind with haem, thus preventing the formation of haemozoin.However, the corresponding quinoline aldehyde intermediates 6c and 8b were ineffective (data not included) at the maximum tested concentration mM) suggesting that enhanced activity of 9n and 9o could be attributed to the contribution of the thiosemicarbazone and 4-aminoquinoline fragments. 27imilarly, in terms of antitrypanosomal activity, none of the tested compounds exhibited appreciable activity except compounds 9n and 9o, which reduced the viability of trypanosomes (T.b. brucei) at 20 µM to 12.7 % and 12.8 %, respectively.Since compound 9o showed a significant cytotoxic effect at 20 µM, only compound 9n was further screened to determine the corresponding IC 50 value (Fig. 3).Despite significant growth inhibition as measured by the viability of trypanosomes, compound 9n was found to be inactive with IC 50 value of 167.7 µM.

Conclusion
In summary, a series of thiosemicarbazone derivatives 9a-r based on the 2-oxoquinoline structural motif have been prepared in moderate to excellent yields and their structural integrity confirmed using various spectroscopic techniques.Despite the poor antiplasmodial and antitrypanosomal activity of the majority of the tested compounds, the promising potency of 9n and 9o provides an avenue for further in-depth investigation of these bi-quinoline thiosemicarbazone compounds as a new family of quinoline-based anti-infective agents.Apart from compounds 9m, 9n and 9o, which exhibited weak to good activity with IC 50 (T.b. brucei) = 167.7 µM and IC 50 s (Pf) = 2.09 and 1.79 µM values, the rest of compounds were inactive and parasite percentage viabilities of >50 % were often observed.As determined by the HeLa cell line, the majority of these compounds showed no significant toxicity.

General
All commercially available chemicals and reagents were purchased from Sigma-Aldrich (Pty) Ltd and Merck (Pty) Ltd, and were used without further purification unless stated otherwise.The progress of reactions were monitored by analytical thin layer chromatography (TLC) using Merck F 254 silica gel plates (supported on aluminium), which were visualized under ultraviolet (UV 254 and 366 nm) light or, where necessary, stained in iodine flask.The crude compounds were purified by flash column chromatography using Merck Kieselgel 60 Å: 70-230 silica gel mesh or by preparative thin-layer chromatography (PTLC) using Merck 60GF 254 silica gel coated on glass plates (2.0 × 200 × 200 mm).The 1 H and 13 CNMR spectra were recorded on either a Bruker Fourier 300 or a 400 MHz spectrometer.Spectra were recorded in deuterated solvents: CDCl  formed on Elementar Analysensysteme varioMICRO V1.6.2GmbH analysis system.

General Procedure for Synthesis of Thiosemicarbazone Compounds 9a-r
An appropriate starting 2-oxoquinoline-3-carbaldehyde (0.081 g, 0.5 ), and thiosemicarbazide (0.5 mmol) were mixed in methanol (25 mL) and heated to 80 °C for 10 h.After reaction completion, the resulting product was allowed to cool to ambient temperature and resulted in the formation of a precipitate, which was filtered, washed with ice-cold MeOH and dried to give the desired products.

In Vitro Antitrypanosomal and Cytotoxicity Assays
The HeLa cells (Cellonex) were cultured using method described by Oderinlo et al. and Adeyemi et al. 30,31 Trypanosoma brucei brucei 427 trypomastigotes were cultured in Iscove's Modified Dulbecco's Medium (IMDM; Lonza) supplemented with 10 % fetal calf serum, HMI-9 supplement, 32 hypoxanthine and penicillin/streptomycin at 37 °C in a 5 % CO 2 incubator.Serial dilutions of test compounds were incubated with the parasites in 96-well plates for 24 h and residual parasite viability in the wells determined by adding 20 µL of 0.54 mM resazurin in phosphate buffered saline (PBS) and incubating for an additional 24 h.Reduction of resazurin to resorufin by viable parasites was assessed by fluorescence readings (excitation 560 nm, emission 590 nm) in a Spectramax M3 plate reader.Fluorescence readings were converted to % parasite viability relative to the average readings obtained from untreated control wells.IC 50 values were determined by plotting % viability vs. log[compound] and performing non-linear regression using GraphPad Prism (v.5.02) software. 30,31

In Vitro Antiplasmodial Assay
Activity was determined against the 3D7 chloroquinesensitive strain of P. falciparum.Parasites were maintained in continuous culture using the method of Trager and Jensen 33 with modifications.Growth medium consisted of RPMI 1640 containing 25 mM HEPES, and further supplemented with 0.5 % (w/v) Albumax II, 22 mM glucose, 0.65 mM hypoxanthine, 0.05 mg mL -1 gentamicin and 2-4 % (v/v) human erythrocytes. Compounds were prepared as 20 mM stock solutions in dimethyl sulfoxide, sonicated for 10 minutes to enhance solubility and stored at -20 °C until use.To assess antimalarial activity, compounds were diluted to a final concentration of 20 µM in culture medium, added to parasite cultures (2 % parasitaemia, 1 % haematocrit) in 96 well plates and incubated for 48 h at 37 °C under an atmosphere of 5 % CO 2 , 5 % O 2 , 90 % N 2. Parasite viability was assessed using the parasite lactate dehydrogenase assay described by Makler et al. 34 Wells containing uninfected erythrocytes were used as negative controls (0 % parasite viability) and untreated parasite-infected wells as positive controls (100 % parasite viability).To determine IC 50 -values, parasite cultures were incubated with 3-fold serial dilutions of test compounds and non-linear regression analysis carried out on dose-response plots of % parasite viability vs. log[compound] using GraphPad Prism (v.5.02) software.

Figure 1
Figure 1 Chemical structures of biologically active quinoline-derived thiosemicarbazone derivatives (I and II) based on 2-oxo-quinoline structural motif.

Figure 3
Figure 3 Plot of percentage antitrypanosomal activity against log concentration for compound 9n and the standard drug, pentamidine.

Table 2 :
In vitro antiplasmodial and antitrypanosomal activity, and cytotoxicity evaluation of target compounds 9a-r.The IC 50 values (in µM) obtained with the standard drug compounds CQ, PMD and EMT are also shown.
Figure2Plot of percentage antiplasmodial activity against log concentration for compounds 9m, 9n, 9o and the standard drug, chloroquine.