Preliminary evaluation of thiazolidinone- and pyrazoline-related heterocyclic derivatives as potential antimalarial agents

© 2020 A. Kryshchyshyn et al.; Published by the Institute of Molecular Biology and Genetics, NAS of Ukraine on behalf of Biopolymers and Cell. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited


Introduction
The thiazolidinone based molecules had been widely studied and described as a fruitful source of novel drug-like molecules with a variety of pharmacological profiles [1][2][3][4]. Recently, the thiazolidinone/thiazole derivatives became interesting in the field of anti-parasitic agents search [5][6][7][8][9]. We had designed and synthesized a class of rhodanine derivatives -5-enamine-2-thioxo-4-thiazolidinone-3-carboxylic acids that showed [the] significant trypanocidal activity towards Trypanosoma brucei gambiense along with a good cytotoxicity profile against the myoblast derived cell line (L-6). The selectivity indices for these Bioorganic Chemistry ISSN 1993-6842 (on- compounds were within 158-1396.2 (calculated as the ratio of CC 50 to IC 50 ) designating this class of rhodanine-3-carboxylic acids as perspective in the search for antitrypanosomal agents [10]. For a number of related 5-benzylidenerhodanine-3-acetic acids the inhibitory activity against Trypanosoma brucei dolicholphosphate mannose synthase and glycosylphosphatidylinositol anchor was studied, these compounds also showed [the] in vitro trypanocidal activity against bloodstream forms [7]. There was also identified a row of hit-compounds among thiazolidinone/thiazoleimidazothiadiazole/phenyl-indole hybrids inhibiting growth of Trypanosoma brucei brucei and Trypanosoma gambiense at submicromolar concentrations. [11]. Encouraged by a significant trypanocidal activity of different thiazolidinone-and thiazole-based compounds we decided to study if these classes of small "drug-like molecules" possess the antimalarial activity.
Malaria is a parasitic infection of the genus Plasmodium, two of its species -Plasmodium falciparum and Plasmodium vivax account for more than 95 % of clinical cases and deaths. Although, in recent years, there has been a reduction in the numbers of deaths from malaria due to the efficiency of Artemisinin combination therapies (ACTs), the latter meet new challenges because of the emerging drug resistance [12]. Traditional directions in search for new antimalarial agents usually cover the study of various artemisinin analogs as well as different aminoquinoline derivatives [13,14]. Despite the fact, that Artemisinin combination therapies (ACTs) play a pivotal role in malaria control programmes as they remain the cornerstone of case management, it is im-portant to develop novel classes of active agents against artemisinin resistant strains of Plasmodium ssp. as well as targeting the multiple stages of the parasite life cycle.
Among various classes of organic compounds being investigated as potential agents to treat malaria, the study of thiazole based molecules indicated this heterocycle as a pharmacophore with antimalarial properties [15]. For example, a row of aminomethylthiazole pyrazole carboxamides showed good in vitro activity against P. falciparum and was orally effective in a P. berghei mouse model [16]. 2-(2-Hydrazinyl)thiazole derivatives with 2-pyridyl moiety inhibited [the] growth of blood stage P. falciparum (NF54) in submicromolar concentrations in vitro [17].
High-throughput screening of the AstraZeneca compound library against the asexual blood stage of Plasmodium falciparum led to identification of the active amino imidazole scaffold. Optimization of the latter yielded an orally bioavailable lead -2-aminoazabenzimidazole derivative with [the] nanomolar inhibitory activity against P. falciparum and efficiency in the humanized Pf/SCID model of malaria [18] (Fig. 1). The imidazolopiperazine derivatives, representing the next-generation antimalarial therapy with the clinical candidate KAF156, belong to the examples of the active antimalaria compounds bearing [6+5]-scaffolds. The latter is effective against Plasmodium falciparum drug-sensitive and drug-resistant strains in nanomolar concentrations targeting multiple life stages of the parasite like liver, ABS and gametocyte [19]. A series of tripeptides with different heterocycles in the side chains was tested for the falcipain-2 inhibitory activity as well as against Plasmodium falciparum (3D7 culture). Interestingly, the most active compounds in both assays and the less toxic contain indole fragment and different five-membered nitrogen containing heterocyclic moieties (pyrolidine and imidazole) [20].
One of the approaches to search for new antimalarials is the developing of agents with the modes of action distinct from the existing drugs. Thus, a spiroindolone derivative KAE609 (Cipargamin) is characterized by the fastest clearance rates in patients of any anti malarial yet (Fig. 1). Cipargamin targets the P-type Na + ATPase PfATP4, affecting Na + homeostasis in the parasite and as a result blocking [the] ABS development and transmission to mosquitoes [21]. Similar effects on intraerythrocytic Plasmodium falciparum caused a pyrazoleamide compound PA21A092 that could prevent parasite mating and therefore transmission by mosquitoes [22]. Another example of [6+5]-heterocyclic fragment implementation is an inhibitor of the mitochondrion-located DHODH (dihydroorotate dehydrogenase) DSM265 that showed the activity against both hepatic and ABS (intra-erythrocytic asexual blood stages) schizonts and was efficient even in single-dose regimens in human trials [23].
The development of novel small non-toxic molecules able to kill Leishamania and Plasmodium ssp. at different stages as well as to overcome multidrug resistance in treatment of the leishmaniasis and malaria is of great importance and remains a topical issue in parasitic diseases control. The attempts to develop the agents with dual inhibitory acti vity against both mentioned parasites had been also made. As follows, thiazolidinone and thiazole cores were utilized to obtain a series of molecules bearing thiazole/thiazolidinone cycle and pyrazole core within the hybrid pharmacophore approach. Although, pyrazolylthiosemicarbazones showed good antimalarial activity, their cyclization to thiazole and thiazolidinone increased and the hit-compounds had significant suppressive effect (> 90 %) against Plasmodium berghei in in vivo assays and even showed a better activity than chloroquine phosphate against chloroquine resistant (RKL9) strain of P. falciparum [24].

Chemistry
All chemicals were of the analytical grade and commercially available. All reagents and solvents were used without further purification and drying. Compounds 1,2 [11], 3 [25] and 4 [26] were synthesized as described previously. NMR spectra were determined with Varian Mercury 400 (400 MHz) spectrometer, in DMSO-d 6  General method for synthesis of 5- The mixture of 3-(4-hydroxyphenyl)thiosemicarbazide (0.01 mol), chloroacetic acid (0.01 mol), sodium acetate (0.02 mol) and appropriate oxocompound (0.03 mol) in the mixture of 5 mL of DMF and 10 mL of acetic acid was refluxed for 2 h. After cooling, the product of the reaction was filtered off and recrystallized from the mixture of DMF-acetic acid or DMF-ethanol.

Pharmacology
Antimalarial activity assay. P. falciparum strain FcB1/colombia was maintained continuously in culture on human erythrocytes as described by Trager and Jensen [27].

[The]
In vitro antiplasmodial activity was determined using a modification of the semi-automated microdilution technique [28]. Chloroquine diphosphate was used as a reference drug. Stock solutions of chloroquine diphosphate and test compounds were prepared in sterile, distilled water and DMSO, respectively. Drug solutions were serially diluted with the culture medium and added to asynchronous parasite cultures (1 % parasitemia and 1 % final hematocrite) in 96-well plates for 24 h, at 37 o C, prior to the addition of 0.5 ACi of [3H]hypoxanthine (1 to 5 Ci/mmol; Amersham, Les Ulis, France) per well, for 24 h. The growth inhibition for each drug concentration was determined by comparison of the radioactivity incorporated into the treated culture with that in the control culture (without drug) maintained on the same plate. The concentration causing 50 % inhibition (IC 50 ) was obtained from the drug concentration-response curve and the results were expressed as the mean of the standard deviations determined from se veral independent experiments. The DMSO concentration never exceeded 0.1 % and did not inhibit the parasite growth.
In general, all tested compounds possessed moderate and good antiplasmodial properties, although, it is rather complicated to outline any structure-activity peculiarities as the hit- . This pyrazoline containing molecule was also highly effective in the anticancer activity assay; at the micromolar concentrations it inhibited the growth of majority of the tested cancer cell lines and moreover showed certain selectivity towards Leukemia panel [25]. Such a promiscuous behaviour makes it a promising object within the concepts of polypharmacological approach and multitarget drugs design [29][30][31][32][33]. The studied 5-(Z)-arylidene-2-arylidenehydrazono-3-(4-hydroxyphenyl)-4-thiazolidinones 5-7 were characterized by the comparable IC 50 values proving [a] positive impact of combination of thiazolidinone core and hydrazine moiety on the antiparasitic activity [6]. On the other hand, such an impact strongly depends on other fragments in the molecule, e.g. the compond 1 bearing also a cinnamoyl fragment did not show high antimalarial activity. Interestingly, the compounds 1 and 2 earlier tested against Trypanosoma brucei brucei [11] showed analogous results regarding the antiplasmodial activity levels: i) compound 1 inhibited the growth of Trypanosoma brucei brucei by more than 90 % (by 83 % for Plasmodium falciparum) and was not active at 1 µg/mL; ii) the IC 50 calculated for compound 2 was 10.63 µM (comparing to 5.31 µM calculated in antimalarial assay). 3,7-Dithia-5,14-diazapentacyc lo[9.5.1.0 2,10 .0 4,8 .0 12,16 ]heptadecene 9 was cho-sen for the screening as an example of fused thiopyranothiazole scaffold that retains pharmacological profile of its precursors -5-ene-4-thiazolidinones, but at the same time does not keep the undesirable Michael acceptor properties [29]. Indeed, compound 9 showed a high rate of parasites growth inhibition at the concentration of 10µg/mL and the IC 50 value comparable with that for other derivatives.

Conclusions.
A versatile row of thiazolidinone and pyrazoline derivatives was studied against Plasmodium falciparum in the in vitro assay. The calculated IC 50 values were within 1.81-13.29 µM indicating the suitability of the described class of small molecules for the purposes of medicinal chemistry. For some of the investigated compounds a significant trypanocidal activity against Trypanosoma brucei brucei and Trypanosoma gambiense had been earlier established that can warrant the in-depth study of the above-mentioned molecules as promising antiparasitic agents.