The synthesis and structural characterization of the seventeen (17), thiazolidinones derivatives (TZDs) has been reported in detail elsewhere [13]. Briefly, the synthesis employed molecular hybridization between a 2-amino-5-nitrothiazole moiety and thiazolidin-4-one. This was followed by exploration of structure-activity relationships (SARs) by addition of a substituted benzylidene moiety on the thiazolidin-4-one, to obtain a library of seventeen (17) thiazolidinone derivatives using Knoevenagel condensation. The structures of target compounds were determined using Fourier-transform infrared spectroscopy (FTIR), ¹H and ¹³C-NMR, and high-resolution mass spectra. The structures of the compounds are shown in Figure 1.

Figure 1.Chemical structures of the thiazolodinone derivatives [13].

Stock solutions of compounds (30 mM) were prepared by dissolving in Dimethyl sulfoxide (DMSO), (Sigma Aldrich, Germany), vortexed (Labnet VX 100, USA) for homogeneity and kept at −20˚C for subsequent use. Complete culture medium (CCM) was prepared by supplementing RPMI-1640 with L-glutamine (BioConcept, Switzerland), 5% newborn calf serum (SIGMA, USA), 200 units/mL penicillin, 200 µg/mL streptomycin, and 0.25 µg/mL amphotericin B (SIGMA, USA), pH 7.4.

Adult worms were isolated as described [14][15]. Briefly, fresh pieces of umbilical cattle skin rich in palpable nodules were obtained from slaughterhouses in Douala, thoroughly washed, cleaned and processed. The dampness of the skin was reduced by dabbing with a dry towel and then spread on a sterile wooden board in a laminar flow hood and disinfected with 95% and 70% ethanol consecutively. Worm masses were dissected out of the nodules and each was immersed in 2 mL CCM per well in a 12-well plate (BioConcept, Switzerland; NUNC, USA). The plates were incubated overnight at 37˚C, 5% CO₂ (HERACELL-150i, USA), and worm viability and sterility of cultures were evaluated using an inverted microscope (Nikon Eclipse TS100, China). Before use in primary and secondary screens, 1 mL of CCM was added to each well, giving a volume of 3 mL.

O. ochengi microfilariae (MFs) were isolated from infected umbilical cattle skin with palpable nodules as described [14][15]. The skin was carefully cleaned, shaved, and sterilized as previously described. Skin slivers were cut out, placed in CCM for two hours at room temperature for MFs to emerge. The emerged highly motile MFs were concentrated by centrifugation (2000 rpm, 10 min), re-suspended in CCM and distributed into wells (about 15 MFs/100 μL of CCM/well) of the 96-well microtitre plate that contained a layer of monkey kidney epithelial (LLCMK2) cells. Their viability and sterility were assessed for 24 hours before compounds were added [14][15].

Mammalian (monkey kidney epithelial) cells (LLCMK2; ATCC, Virginia, USA), were cultured to confluence in CCM at 37˚C and 5% CO₂ as described [14][15]. The medium was decanted and cells dislodged using 0.5 mM EDTA and 0.125% trypsin in incomplete culture medium (ICM), re-suspended in 10 mL of CCM and centrifuged (560 g, 10 mins) to eliminate the trypsin. The cells were then transferred into 96-well plates (100 µL/well) and incubated as above. LLCMK2 cells served as a feeder layer for microfilariae cultures and were also used for cytotoxicity studies [14].

2.6.1. Primary Screen on Adult Worms

This was done as reported [14] to identify active compounds. The stock solution (30 mM) of each compound was diluted to four times the final concentration with CCM; then 1 mL was added per well into 12-well plates containing worm masses submerged in 3 mL making a final volume of 4 mL (DMSO concentration < 2%), a final concentration of 30 µM. Positive control wells contained auranofin at (30 µM) while negative controls contained the diluent (≤2% DMSO in CCM). All wells were set up in triplicate and the plates were incubated as above for five and seven days, for adult male (AM) and female (AF) worm assays, respectively, then worm viability was evaluated. Adult male worm viability was assessed based on motility using an inverted microscope on the following scale: 100% (complete inhibition of motility), 75% (only head or tail of worm shaking occasionally), 50% (whole worm motile, but sluggishly), 25% (only little change in motility), to 0% (no observable change in motility).

Thereafter, the worm masses were incubated in 500 μL of 0.5 mg/mL MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) for 30 minutes and female worm viability was evaluated by visual estimation of the percentage inhibition of formazan formation (blue coloration); from 100% parasite killing (no blue formazan coloration seen), 90%, 75%, 50%, 25%, to 0% (the entire worm appears blue as in the negative control). A compound was deemed active if there was ≥90% inhibition of male worm motility or of formazan formation; moderately active with 50% - 89% inhibition of male worm motility or of formazan formation; and inactive with < 50% inhibition of worm motility or formazan formation.

2.6.2. Secondary Screen on Adult Worms

The assay was repeated to confirm activity established in the primary screen, ascertain the compounds’ dose-dependent response and determine their 50% inhibitory concentrations (IC₅₀ value). Compounds with 90% - 100% inhibitory activities on both male and female adult worms at the primary screen were selected for secondary screens. The assay was set up at concentrations of 0.1 - 30 µM in triplicate and plates incubated as above. Activities of the compounds were determined using the same methods as above.

2.6.3. Primary Screen on Microfilariae

A primary screen was performed similarly as above to identify active compounds [8][14] with minor adjustments. The monkey kidney epithelial cells were cultured to confluence as mentioned above, the medium was decanted quickly and the adhered cells were used as feeder layer. Microfilariae (MFs) in 100 µL of fresh CCM were added to each well of the 96-well plate (Greiner Bio-One, Austria) and incubated overnight. Before compound addition, sterility of the culture was verified and 50 µL of fresh CCM was added to each well. Each compound was added (50 μL per test well) at a single final concentration of 100 µM. Wells were set up in duplicate. Amocarzine (30 µM) and ≤2% DMSO in CCM were included as positive and negative controls, respectively and incubated as above with daily evaluation of worm viability by microscopy. Culturing was terminated after 120 hours. The percentage motility inhibition was determined daily as 100% (all MF immotile), 75% (only head or tail of MF shaking, occasionally), 50% (whole body of MF motile but sluggishly or with difficulties), 25% (almost vigorous motility), 0% (vigorous motility). A compound was considered active on the MFs if there was 90% to 100% mean reduction in MFs motility; moderately active between 50% - 89% mean reduction in motility and inactive if less than 50% compared to untreated controls.

2.6.4. Secondary Screen on Microfilariae

This was carried out to confirm the activity in the primary screen and determine the IC₅₀ values. Compounds with ≥90% inhibitory activity in the primary screen were tested at 0.03 to 100 µM using a feeder layer as above. Each test solution (50 µL) was added into a 96-well plate that contained MFs in 150 µL of CCM and the plate was incubated. The MFs motility was scored daily, cultures were stopped 120 hours later, and compound activities were recorded as above.

Cytotoxicity of 12 compounds with ≥90% inhibition in the primary screen was evaluated using monkey kidney epithelial cells [8][14]. Briefly, the cells were cultured in CCM to confluence as described above, the medium was decanted and the cells were rinsed twice with ICM. About 3000 cells/100µL in CCM were seeded in duplicate in a 96-well flat-bottom microtitre plate (Greiner Bio-one, Austria) and incubated for three days until fully confluent. Cells were examined by microscopy for confluence, the medium was discarded by flipping the plate and drying on a tissue, and 150 µL of fresh medium (CCM) was added per well. Then 50 µL of test compound (8 final concentrations: 0.03 µM to 100 µM), and controls were added to corresponding wells in duplicate and incubated for 5 days. Thereafter, the medium was discarded, the cells were washed by shaking twice in ICM for five minutes at 600 rpm (IKA Labortechnik KS125 basic shaker), and 100 μL of MTT (1 mg/mL ICM) was added and then incubated for 2 hours as above. Post incubation, the MTT solution was discarded and 100 μL of DMSO was added to dissolve the formazan precipitate, then homogenized and optical densities were read at 630 nm using an ELISA plate reader (Sinothinker SK201, China). Percentage inhibition was calculated using the formula below:

The cytotoxic concentration (CC₅₀) was determined as described above, while selectivity index was determined using the formula below:

This was done for three compounds (5,7 and16), selected based on the lowest IC₅₀ values and highest SI values on adult worms; 7 was the most active (lowest IC₅₀) and most selective (highest SI) among the nine compounds active against all three worm stages in the primary screen. The test was conducted as previously described [16][17] and in accordance with guidelines of the Organization for Economic Cooperation and Development, version 423 [18] and ARRIVE, on animal research. Ethical clearance was obtained from the university’s Institutional Animal Care and Use Committee (No. UB-IACUC Nº 18/2025 issued on 20 June 2025). BALB/c mice (20) were handled and processed as described in previous works [16][19], then separated into four groups (3 test groups and a control group) with five mice per group. One mouse was fasted overnight (with water), weighed, administered 2000 mg/kg of compound, fasted for a further 2 hours and observed till 24 hours with food provided. Following the survival of the treated mouse, the others were treated the same and observed for 14 days (with food and water), then weighed. The mice were again fasted overnight, anesthetized with ketamine/xylazine (90/10 mg/kg); blood was collected by retro-orbital bleeding, allowed to coagulate then centrifuged (2000 rpm × 5 mins). The serum was used for biochemical analysis of two markers of hepatocellular damage, alanine aminotransferase (ALT) and aspartate aminotransferase (AST), and renal damage (urea and creatinine), using test kits (Chronolab, Spain) according to the manufacturer’s procedure.

The average percentage inhibition from secondary screens was used to determine the IC₅₀ values of active compounds by nonlinear regression in Graphpad Prism 5.0 software and Excel 2019. Dose-response curves were also plotted using Graphpad Prism. CC₅₀ values were determined from average percentage inhibition of compounds on cells using Graphpad Prism. Biochemical data of control and test mice were compared using an unpaired t-test. Statistical significance was set at P < 0.05.

Out of the 17 thiazolidinone derivatives (TZDs) tested in the primary screen, 12 and 9 compounds showed high anti-onchocerca activity (≥90% inhibition), against adult male (AM) and adult female (AF) worms respectively (Table 1). All compounds showed activity against at least one worm stage. Meanwhile, 1 and 5 compounds were highly active only against AM and MFs respectively. Two were active against both AM and MF only, and 9 were active against all three stages (Table 1). No compound was active on adults only.

In the secondary screen, the IC₅₀ values of compounds ranged from 0.098 to 8.5 µM against AM and 0.30 to 15.38 µM against AF. Among the 12 compounds tested against AM, compound3 was the most active with an IC₅₀ of 0.0988 µM. Of the 9 compounds tested against AF, compound5 was the most active with an IC₅₀of 0.3029 µM. The macrofilaricidal activity of each compound showed a dose-dependent response for adult worms (Figure 2). Seven compounds had lower IC₅₀s against AM compared to AF. Only 2 compounds had lower IC₅₀s against AF than AM (Table 2).

Table 1.Inhibition of Onchocerca ochengi motility by thiazolodinone derivatives at 100 µM.

A: all stages; AM: Adult male; AF: Adult female; MFs: Microfilariae. Positive controls Auranofin, Amorcazine.

Figure 2.Dose-response curves with IC₅₀s of the most active thiazolidinones: (a) compound 3 and (b) compound 5 on O. ochengi adult male and female worms, respectively.

Table 2.Cytotoxicity, filaricidal activity and selectivity index (SI) values of active thiazolodinone derivatives on O. ochengi worm stages.

Cutoff for cytotoxicity, CC₅₀: < 10 µM. NA: Not highly active, -: Not determined. Some key values are shown in bold.

Out of the 17 TZDs, 8 hits against AM and 4 hits against AF were found based on the following criteria: ≥90% inhibition, IC₅₀ < 10 µM, cytotoxicity (CC₅₀) > 10 µM and a selectivity index (SI) ≥ 10 (Table 1and Table 2) [20]-[22]. The classification of these compounds as hits is supported by data from a study of a large library of over 2000 drugs in clinical use where ≥50% inhibition at 10 µM was considered a hit [20]. The AM shares three of the four hits on AF (compounds1, 5and16). To the best of our knowledge, this is likely the first report on the activity of thiazolidinone derivatives against a causative agent of onchocerciasis, O. ochengi, albeit in cattle. Compound3, the most active (IC₅₀ = 0.098), was also highly selective (SI = 231) against AM; but was only moderately active (IC₅₀ = 7.17 µM), with low selectivity (SI = 3.18) against AF. Compound 3 also demonstrated 100% inhibition against microfilariae (MFs) at 100 µM. Compound5, the most active against AF (IC₅₀ = 0.302 µM) showed similar activity against AM. TZDs which are macrofilaricidal against both adult stages have great potential as killing the adult stages will consequently lead to the absence of MFs which contribute significantly to the devastating pathology of human onchocerciasis. When compared to data from a previous study, 12 compounds had either a lower or comparable IC₅₀ to Albendazole (15.7 µM), Levamisole (5.1 µM) and Ivermectin (1.3 µM) against AM O. ochengi, while 8 compounds had a similar trend against AF [23], As earlier mentioned, Ivermectin is only microfilaricidal alongside other limitations and acts mainly by inhibiting glutamate-gated chloride channels in invertebrates leading to paralysis [24]. The TZD hits which are highly active against adult stages are likely acting on different targets to that of ivermectin hence producing their effects by different mechanisms. These hits therefore have the potential to overcome the limitations of ivermectin.

Out of the 17 TZDs, 16 (except compound15), showed high microfilaricidal activity (≥90% inhibition at 100 µM) against MFs in the primary screen. The IC₅₀ was determined for only two compounds,13 and17, with the values 28.9992 µM and 5.6465 µM respectively. Though almost all the TZDs were highly active against MFs (Table 1), IC₅₀s were not determined for most of them due to scarcity of MFs following treatment of bovine onchocerciasis with ivermectin in the study area. Ivermectin is used in veterinary medicine to kill MFs of O. ochengi, the bovine parasite, in order to improve animal health and beef productivity [24]. This was a major setback to identifying broad-acting TZDs which will be pursued as a priority in future work.

Further studies of the 9 compounds with high activity against all three worm stages may afford broad-acting anti-onchocerca agents which could be used to interrupt transmission and hence eradication of onchocerciasis.

A previous study had reported macrofilaridical activity against AM and microfilaricidal activity for 7 thienylazoryl dyes [8]. Studies on the anthelmintic activity of synthetic thiazolidinones are uncommon. A study [25] reported high activity of some novel thiozolidine-2,4-dione derivatives against adult earth-worm Pheretima posthuma.

In terms of SARs, the unsubstituted benzylidene moiety (parent compound), (Figure 1), possesses high activity (compound1, R = H), against all three worm stages with very low IC₅₀s (0.32 to 1.17 µM) and high SI values (19.1 to 68.2). This activity is modulated following substitution on this moiety. Considering compounds active against AM, substitution with electron-withdrawing groups in the para-position of the benzylidene moiety [3 (R = 4-Cl) and16 (R = 3,4-Cl)], greatly enhances activity. Hence, compound3was the most active of all the TZDs. Compounds2 (R = 4-F) and 11 (R = 3-Br), which are also electron-withdrawing had relatively high activities while7 (R = 4-OH) and 10 (R = 3-Cl), had relatively lower activities. The substitutions in the ortho- and meta-positions in compounds5 (R = 2-Me), and 9 (R = 3-F), respectively, did not alter activity compared to the parent compound1. Substituents in the ortho-position 2 in 13 (R = 2-Cl) and electron-donating groups [compounds15 (R = 2,4,6-MeO) and17 (R = 4-Me)], significantly decreased activity.

Considering the SARs for AF, the contrary to AM was observed whereby electron-donating substituents in the para-position (compound7, R = 4-OH) and ortho-position-2 (5, R = 2-Me), greatly enhanced activity compared to compound1. The electron-withdrawing substituents [Compounds 2 (R = 4-F), 3 (R = 4-Cl), 11 (R = 3-Br), 13 (R = 2-Cl), 15 (R = 2,4,6-MeO), and 16 (R = 3,4-Cl)] decreased activity. Compound 19 (R = Me) did not enhance activity because it is weakly electron-donating. The plausible basis for the difference in the pattern of activity between AM and AF is likely that the adult stages have different target sites for the active compounds. Further evidence of acting at a target site is the sigmoid dose-dependent profiles in Figure 2. These targets need to be identified in future work.

Of the 12 active compounds tested, the CC₅₀ values of 9 compounds on monkey kidney epithelial cells were above the cut-off value for cytotoxicity of pure compounds (CC₅₀ < 10 µM; according to other works [21][22]. This indicates they were non-toxic against this cell line. Three compounds,11, 13 and17 had CC₅₀ values <10 µM indicating cytotoxicity (Table 2). In terms of selectivity, compound16 had the highest selectivity index (SI) of 607.5 while the most active compounds with the lowest IC₅₀s, against AM (3) and against AF, (5) had relatively high SI values of 231 and 76 respectively (Table 2). The high SI values indicate a very low risk of harmful effects if used in the treatment of human onchocerciasis.

There was an increase in body weight of animals but the difference between control and test mice was not significant (compounds5, p = 0.637;7, p = 0.604 and16, p = 0.487). No adverse effects and no mortality were observed. There was also no significant difference between control and test mice for all biochemical parameters (ALT, AST, urea and creatinine), as all p-values were > 0.05,i.e., compound5 (p = 0.594 - 0.867), 7 (p = 0.141 - 0.999), and16 (p = 0.053 - 0.564), Table 3.

Table 3.Effects of highly active thiazolodinone derivatives on liver and kidney function in acute toxicity test BALB/c mice.

Dose: 2000 mk/kg observed for 14 days. P values for mean ± SD ALT, AST, urea and creatinine: 5, P = 0.867, 0.594, 0.709, 0.778; 7, P = 0.387, 0.141, 0.999, 0.596 and 16, P = 0.053, 0.412, 0.307, 0.564, respectively.

The overall absence of harmful effects in the acute toxicity is consistent with the high selectivity of the tested compounds. Abnormally high levels of biochemical markers in serum are an indication of tissue damage in the corresponding organ. Toxicity studies of TZDs are few and mostly focused on anticancer screening. A study [26] reported moderate anticancer activity for some TZDs against three cancer cell lines, but they were not toxic to Vero cells. Another study [27] reported high inhibitory activity (IC₅₀ = 0.27310 - 0.533 µM) on aldose reductase for a series of 2,4-thiazolidinediones with CC₅₀ values < 10 µM on L929 fibroblasts; despite the CC₅₀s being low and below the cut-off for cytotoxicity (≤10 µM) [21][22], the compounds were considered safe because their SI values were relatively high.

As a main strength, this is the first study, to the best of our knowledge, to demonstrate anti-onchocerca activity for TZDs with hits found in this class of compounds. However, there are some limitations to the study. ThoughO. ochengi is genetically a close relative of the human parasite, O. volvulus, theO. ochengiin vitro model does not exactly replicate the activity of the TZDs on O. volvulus. The mechanisms of action of the potent compounds were not determined in this study and secondary screen was not done for most compounds active against microfilariae to permit a better appraisal of their activity on the three worm stages. Acute toxicity test needs to be done for the other highly active compounds in order to identify the safest ones, and extended to repeated-dosing (sub-acute) with histopathological analysis for a better assessment of the toxicity of the active compound on long-term use.