Evaluation of Anthelmintic and Anti-Inflammatory Activity of 1,2,4-Triazole Derivatives

Parasitic diseases, caused by intestinal helminths, remain a very serious problem in both human and veterinary medicine. While searching for new nematicides we examined a series of 1,2,4-triazole derivatives 9–22, obtained during reactions of N3-substituted amidrazones with itaconic anhydride. Two groups of compounds, 9–16 and 17–22, differed in the position of the double bond on the methacrylic acid moiety. The toxicity of derivatives 9–22 and the anti-inflammatory activity of 12 and 19–22 were studied on peripheral blood mononuclear cells (PBMC). Antiproliferative activity of compounds 12 and 19–22 was tested cytometrically in PBMC cultures stimulated by phytohemagglutinin. The influence of derivatives 12 and 19–22 on the TNF-α, IL-6, IL-10 and IFN-γ production was determined by ELISA in lipopolysaccharide-stimulated PBMC cultures. Anthelmintic activity of compounds 10–22 was studied in the Rhabditis sp. nematodes model. Most compounds (11–22) proved to be non-toxic to human PBMC. Derivatives 19–22 showed anti-inflammatory activity by inhibiting the proliferation of lymphocytes. Moreover, compounds 12 and 19–22 significantly reduced the production of TNF-α and derivatives 19–21 decreased the level of INF-γ. The strongest anti-inflammatory activity was observed for compound 21. Compounds 12 and 14 demonstrated anthelmintic activity higher than albendazole and may become promising candidates for anthelmintic drugs.


Introduction
Parasitic diseases, including intestinal parasitic infections, are caused by various species of parasites, and constitute a serious health and socioeconomic problem. Globally, it is estimated that infections caused by intestinal parasites affect 3.5 billion people, 450 million of whom are symptomatic and have health problems as a direct result of the disease. Nematodes are largely responsible for these diseases, including Ascaris lumbricoides, Trichiuris trichiuria, Ancylostoma duodenale and Strongyloides stercoralis [1][2][3][4][5][6]. Diseases caused by intestinal nematodes can cause, among others, abdominal pain, dehydration, weight loss, anemia, intestinal obstruction, disorders of mental and physical development, malabsorption, inflammation and in severe cases, if left untreated, these can even lead to death [6][7][8].
Currently, a narrow group of drugs is used in the treatment of parasitic diseases caused by helminths, mainly albendazole, mebendazole and semi-synthetic ivermectin. Anthelmintic agents are characterized by a variety of structures and mechanisms of action, which makes it difficult to develop a simple route in the search for new drugs [9][10][11][12][13][14][15][16].
Although there is an urgent need for new antiparasitic drugs, including anthelmintics, a new generation of drugs is still not available. This lack of investment in research is mainly caused by the fact that there is no interest from the pharmaceutical industry, as most parasitic diseases occur in poor countries and there is a high risk of no return on investment [17]. However, an increase in helminths' resistance to available drugs has been observed in recent years [18][19][20]. Therefore, it is important to conduct scientific research using both natural and synthetic chemical compounds to obtain new and effective anthelmintic drugs. Many plants have drawn attention as a potential source of anthelmintic substances [9,10,21,22]. The anthelmintic activity of synthetic chemical compounds has also been demonstrated, including that of thiosemicarbazide derivatives [23,24]. Our research is in line with the trend moving towards discovering new synthetic anthelmintic compounds. Currently, research concerning the bioactivity of triazole derivatives is very popular. Many different biological activities of 1,2,4-triazole derivatives have been described in the literature, e.g., antibacterial, antifungal, antiviral and antitumor [25][26][27][28][29]. However, there are very few reports on the anthelmintic activity of triazole derivatives and these are mainly studies conducted on earthworms, not nematodes specifically [30][31][32].
In our previous publication, we described some 1,2,4-triazole derivatives which possessed methacrylic acid moieties, promising anti-inflammatory activity and low toxicity towards human lymphocytes [41]. Continuing our study on amidrazone derivatives, we took up the idea that compounds possessing a 1,2,4-triazole ring similar to (but containing one extra nitrogen atom) the diazole moiety present in anthelmintic drugs like albendazole or mebendazole could also be active against nematodes. The aim of this study was to determine the anthelmintic and anti-inflammatory activities of newly synthesized and already known 1,2,4-triazole derivatives and the anti-inflammatory activity of the new compounds.

The Synthesis of 9-22
1,2,4-triazole derivatives 9-16 were obtained following previously established procedures via the reaction of amidrazones 1-8 [42] with itaconic anhydride [41]. Consequently, isomerization of compounds 9-14 was undertaken in alkalic solution, leading to derivatives 17-22 (Scheme 1). Considerably higher yields of isomerization were obtained for compounds 20 and 21, which may suggest that a 4-pyridine ring, found in the R 1 position, facilitates this reaction. In contrast, no isomerization products were obtained following the reactions of 1,2,4-triazole derivatives 15 and 16, which may suggest that the presence of a 4-nitrophenyl substituent hinders this reaction. Structures of new compounds were confirmed by 1 H NMR, 13

Biological Assays In Vitro
Human PBMC represented a tissue source in the biological part of the presented studies. Interestingly, PBMC are widely used as a physiological model for immunological research of parasitic infections, such as those caused by Toxoplasma gondii and others [43]. The main structural difference between derivatives 9-14 and 17-22 was the presence of a methylidene CH 2 = moiety attached to propanoic acid in compounds 9-14 and the presence of a methyl (CH 3 ) group attached to (E)-propenoic acid in compounds 17-22. Derivatives 17-22 were also more thermodynamically stable than compounds 9-14 which is beneficial for pharmacological use. The influence of this structural difference on anthelmintic and anti-inflammatory activity was the subject of further biological research.

Biological Assays In Vitro
Human PBMC represented a tissue source in the biological part of the presented studies. Interestingly, PBMC are widely used as a physiological model for immunological research of parasitic infections, such as those caused by Toxoplasma gondii and others [43]. PBMC are routinely isolated from blood samples (as we describe previously in [44]) and then used in several different tests to determine the toxicity, anti-proliferative activity and anti-inflammatory activity of studied compounds. We used the cytometric method to evaluate the proliferation response to phytohemagglutinin (PHA) and immunoezymatic tests to verify the levels of proinflammatory cytokines after lipopolysaccharide (LPS) stimulation. The cytokines from the early immune response that were tested include: TNF-α, IL-6 and IL-10. Additionally, we verified the level of cytokines from adaptive immunity-namely, IFN-γ. IFN-γ is a very important cytokine which takes part in immune responses against parasites [45].

Toxicity of Compounds 9-22
Most of the studied 1,2,4-triazole derivatives 11-22 were characterized by low toxicity towards PBMC at a concentration range up to 100 µg/mL (the range of toxicity at the highest compound concentration was 6.60-28.96% of apoptotic and necrotic cells, Figure S21, Supplementary Materials). Only derivatives 9 and 10 possessed a higher toxicity (only 52.7 and 43.40% of viable cells in 24 h PBMC culture, respectively). To compare the results, we also prepared the PBMC culture with a nonsteroidal anti-inflammatory drug-ibuprofen (the reference control); the toxicity level was 29.53% of apoptotic and necrotic cells [41]. It should be noted that all compounds that had a higher toxicity level than 30% were excluded from our research. In our previous study, PBMC cultures treated with compounds 9-11 and 13-18 showed 71.04-82.39% viable cells [41]. The percentage of viable cells for new compounds 12, 19-22 was in the range of 90.56-93.40%, which is higher than the control culture containing the same concentration of DMSO (87.27%). For comparison, five by six 1H-pyrrole-2,5-dione derivatives (obtained from the same amidrazones 1-8 as compounds 11-22) showed about 79-92% of viable cells [46]. In general, derivatives 17-22 were less toxic than 9-16 while compounds 10 and 18 possessing phenyl and 2-pyridine rings were the most toxic within those two groups.

Antiproliferative Activity of 12 and 19-22
Flow cytometry assay was used to find the cytotoxic potential of compounds upon proliferation of PHA-induced PBMC. Compounds 19, 21 and IBU (reference drug) were inhibitory at a concentration of 50 µg/mL. All compounds (12,(19)(20)(21)(22) as well as IBU inhibited the proliferation of PBMC at a concentration of 100 µg/mL (the strongest inhibitory effect was observed for IBU and compound 19 causing 77.0% and 62.4% of inhibition, respectively; Figure 1). Significant inhibition of PBMC proliferation was also observed for previously studied compounds 13 and 17-18 (nearly 100% at a dose of 100 µg/mL and 80% at 50 µg/mL) [41]. None of the compounds (12 and 19-22) inhibited PBMC proliferation at the lowest concentration of 10 µg/mL. The influence of newly synthetized compounds and IBU was studied on a PBMC model. PBMC were induced by LPS (an endotoxin from gram-negative bacteria used to elicit an inflammatory response in vitro). Stimulating PBMC with LPS produces a high level of pro-inflammatory cytokines such as TNF-α, IL-6, IFN-γ and anti-inflammatory cytokines IL-10 [47].

The Influence of Compounds 12 and 19-22 on Cytokine Production
The influence of newly synthetized compounds and IBU was studied on a PBMC model. PBMC were induced by LPS (an endotoxin from gram-negative bacteria used to elicit an inflammatory response in vitro). Stimulating PBMC with LPS produces a high level of pro-inflammatory cytokines such as TNF-α, IL-6, IFN-γ and anti-inflammatory cytokines IL-10 [47].

TNF-α
IBU significantly reduced the level of TNF-α in PBMC stimulated by LPS at all used doses (by about 99%). Compounds 12 and 19-22 significantly inhibited the production of TNF-α only at a concentration of 100 µg/mL ( Figure 2). The strongest activity was observed for 21 (about 84% of inhibition), while compounds 19, 20 and 22 caused about 71-74% of inhibition. Inhibitory effects were also observed for derivatives 20-21 at concentrations of 50 µg/mL. Compound 12 inhibited the production of TNF-α only at the highest dose (about 50% of inhibition). Derivatives 13 and 17-18 revealed stronger suppression of TNF-α production, especially in low doses (nearly 100% at 10 µg/mL, about 75% at 50 µg/mL and about 50% at 100 µg/mL) [41]. The influence of newly synthetized compounds and IBU was studied on a PBMC model. PBMC were induced by LPS (an endotoxin from gram-negative bacteria used to elicit an inflammatory response in vitro). Stimulating PBMC with LPS produces a high level of pro-inflammatory cytokines such as TNF-α, IL-6, IFN-γ and anti-inflammatory cytokines IL-10 [47].

IL-6
Compounds 12 and 19-22 had no significant effect on IL-6 production with the exception of compound 21, which elevated IL-6 production at a concentration of 100 µg/mL ( Figure 3). This result is in agreement with our previous studies-compounds 9-11 and 13-18 demonstrated no significant influence on IL-6 production [41]. Compounds 12 and 19-22 had no significant effect on IL-6 production with the exception of compound 21, which elevated IL-6 production at a concentration of 100 µg/mL ( Figure 3). This result is in agreement with our previous studies-compounds 9-11 and 13-18 demonstrated no significant influence on IL-6 production [41].

IL-10
In the case of LPS-induced IL-10 production, neither of the compounds significantly increased the release of this cytokine. However, an inhibition of IL-10 production was observed at a concentration of 100 µg/mL for 12, 19, 20, 22 (about 71-83% inhibition) and IBU (92.3% inhibition) as well as for 22 at a concentration of 50 µg/mL ( Figure 4). It is noteworthy that compound 21 inhibited the production of IL-10 the least.

INF-γ
Newly synthetized compound 19 (high dose), 20 (low dose) and 21 (middle and high dose) significantly reduced the production of IFN-γ ( Figure 5). Interestingly, using a similar model, researchers have shown that this factor is very important for the transmission

IL-10
In the case of LPS-induced IL-10 production, neither of the compounds significantly increased the release of this cytokine. However, an inhibition of IL-10 production was observed at a concentration of 100 µg/mL for 12, 19, 20, 22 (about 71-83% inhibition) and IBU (92.3% inhibition) as well as for 22 at a concentration of 50 µg/mL ( Figure 4). It is noteworthy that compound 21 inhibited the production of IL-10 the least. Compounds 12 and 19-22 had no significant effect on IL-6 production with the exception of compound 21, which elevated IL-6 production at a concentration of 100 µg/mL ( Figure 3). This result is in agreement with our previous studies-compounds 9-11 and 13-18 demonstrated no significant influence on IL-6 production [41].

IL-10
In the case of LPS-induced IL-10 production, neither of the compounds significantly increased the release of this cytokine. However, an inhibition of IL-10 production was observed at a concentration of 100 µg/mL for 12, 19, 20, 22 (about 71-83% inhibition) and IBU (92.3% inhibition) as well as for 22 at a concentration of 50 µg/mL (Figure 4). It is noteworthy that compound 21 inhibited the production of IL-10 the least.

INF-γ
Newly synthetized compound 19 (high dose), 20 (low dose) and 21 (middle and high dose) significantly reduced the production of IFN-γ ( Figure 5). Interestingly, using a similar model, researchers have shown that this factor is very important for the transmission of some parasites, because it increases the expression of an adhesion marker such as intercellular adhesion molecule-1 (ICAM-1) [48]. Thus, it can be concluded that the reduction of IFN-γ production is beneficial in counteracting parasitic infections. of some parasites, because it increases the expression of an adhesion marker such as intercellular adhesion molecule-1 (ICAM-1) [48]. Thus, it can be concluded that the reduction of IFN-γ production is beneficial in counteracting parasitic infections.

Discussion
Continuing our previous research on 1,2,4-triazole derivatives obtained during the reaction between N 3 -substituted amidrazones and itaconic anhydride [41], we examined two groups of compounds, 9-16 and 17-22, differing in the position of the double bond in the side chain of methylacrylic acid. Anthelmintic and anti-inflammatory activities of obtained compounds were studied to determine the influence of their structure on their biological properties.
Derivatives 11-22 were not toxic to human PBMC at a concentration of 100 µg/mL in a physiological model. In our previous study, derivatives 13 and 17-18 revealed the strongest anti-inflammatory activity among compounds 11 and 13-18 by inhibiting both TNF-α production and proliferation of PBMC [41]. As a continuation of this work, we extended the research on the anti-inflammatory activity of derivatives 12 and 19-22. Compounds 12 and 19-22 (at concentration of 100 µg/mL) and derivatives 20-21 (50 µg/mL) significantly inhibited production of TNF-α in LPS-stimulated PBMC (but were less effective than the IBU, Figure 2). Neither of the studied compounds lowered the level of IL-6 ( Figure 3). All compounds (12 and 19-22, except for 21) showed a similar inhibitory effect on the synthesis of anti-inflammatory IL-10 as IBU (Figure 4). Compounds 19-22 (at a concentration of 100 µg/mL) and derivatives 19 and 21 (50 µg/mL) significantly inhibited lymphocyte proliferation in a similar way to reference IBU (Figure 1). Comparing the results with earlier studies, we confirmed that derivatives 17-22 (which possess a methyl  [38,49] (p < 0.05 *, p < 0.01 **, p < 0.001 ***).

Discussion
Continuing our previous research on 1,2,4-triazole derivatives obtained during the reaction between N 3 -substituted amidrazones and itaconic anhydride [41], we examined two groups of compounds, 9-16 and 17-22, differing in the position of the double bond in the side chain of methylacrylic acid. Anthelmintic and anti-inflammatory activities of obtained compounds were studied to determine the influence of their structure on their biological properties.
Derivatives 11-22 were not toxic to human PBMC at a concentration of 100 µg/mL in a physiological model. In our previous study, derivatives 13 and 17-18 revealed the strongest anti-inflammatory activity among compounds 11 and 13-18 by inhibiting both TNF-α production and proliferation of PBMC [41]. As a continuation of this work, we extended the research on the anti-inflammatory activity of derivatives 12 and 19-22. Compounds 12 and 19-22 (at concentration of 100 µg/mL) and derivatives 20-21 (50 µg/mL) significantly inhibited production of TNF-α in LPS-stimulated PBMC (but were less effective than the IBU, Figure 2). Neither of the studied compounds lowered the level of IL-6 ( Figure 3). All compounds (12 and 19-22, except for 21) showed a similar inhibitory effect on the synthesis of anti-inflammatory IL-10 as IBU (Figure 4). Compounds 19-22 (at a concentration of 100 µg/mL) and derivatives 19 and 21 (50 µg/mL) significantly inhibited lymphocyte proliferation in a similar way to reference IBU (Figure 1). Comparing the results with earlier studies, we confirmed that derivatives 17-22 (which possess a methyl group in propenoic acid moiety) have stronger antiproliferative properties in PHA-induced PBMC cultures than compounds 11-16 (which contain a CH 2 = group in a propanoic acid moiety) [41].
We suspected that compound 21, which contains the same 4-pyridyl and 4-methyphenyl substituents as 13 (the strongest anti-inflammatory agent among 11 and 13-16), and the favorable position of the double bond will be the most active derivative in the current study. In fact, compound 21 was the only one among derivatives 19-22 which inhibited lymphocyte proliferation and the production of pro-inflammatory cytokines (TNF-α and IFN-γ) at two doses: 50 µg/mL and 100 µg/mL in mitogen-stimulated PBMC cultures. Moreover, derivative 21 was the only one among compounds 17-22 that did not significantly decrease the production of the anti-inflammatory cytokine IL-10 at all studied concentrations. Summing up, compound 21 has shown the most promising properties among derivatives 19-22 and it is worth future research as anti-inflammatory agent.
Afterwards, we studied the anthelmintic potential of compounds 11-22 on Rhabditis sp. nematodes. Our research model was similar to the Caenorhabditis elegans often used in research [50]. Rhabditis sp. nematodes were used, among others, to determine the anthelmintic activity of the new rhodanine derivatives of cinnamaldehyde [49], naphthalimide-conjugates of boron clusters [51]-and thiosemicarbazide derivatives [24].
According to our knowledge, there are only few reports in the literature on the anthelmintic activity of 1,2,4-triazole derivatives. In previous research on Indian worm Pheritima postuma activity of 1,2,4-triazole, the strongest anthelmintic activity showed derivatives possessing 2,4-dichlorophenyl [52], 4-chlorophenyl or 4-nitrophenyl moieties [53]. The diversity of substituents present in the most active compounds indicates various possibilities for the modification of potential nematicidal agents.
Among 1,2,4-triazole derivatives 11-22 we studied in the Rhabditis model, compound 12 showed about eight times greater nematocidal activity than albendazole and compound 14 was approximately three times more active than the reference drug. When analyzing the results of the anthelmintic activity, we have also been able to detect a relationship between the structure and antiparasitic potential of studied compounds. The derivatives which possess a methylidene group CH 2 =, like 12 and 14, showed stronger anthelmintic activity than compounds obtained by their isomerization (20 and 22). Among the second group of 1,2,4-triazole derivatives (17)(18)(19)(20)(21)(22), only one compound 18 at a single dose 1.1 µg/µL possessed significant anthelmintic activity (Figure 7c), which indicates a lower nematocidal potential of derivatives 17-22. We observed that compounds possessing a 4-nitrophenyl (15 and 16) or 2-pyridine moiety at the R 2 position (17) were devoid of anthelmintic activity. However, the phenyl substituent at the R 2 position could be connected with the anthelmintic activity of compounds 12 and 18. The influence of the substituent at the R 1 position on anthelmintic activity is not so clear, however, the presence of a 2-or 4-pyridyl substituent at the R 1 position in conjunction with the phenyl or 4-methylphenyl substituent at R 2 seems to be important.
It's worth noting that the 1,2,4-triazole ring present in studied compounds 11-22 may interact with the same molecular target as albendazole (colchicine binding side). Our reference anthelmintic drug has three nitrogen atoms in a similar configuration to the compounds we studied. The structural advantage of compound 12 above other compounds could be the 4-pyridyl substituent (R 1 ), which would detach with a similar purpose to the sulfur atom present in albendazole. The phenyl substituent in the R 2 position provides better lipophilicity than the compounds 9, 15-17 having an additional nitrogen atom or nitro group, none of which showed nematocidal activity. Another important element of the structure of derivative 12 seems to be the presence of the CH 2 = group in a methacrylic acid moiety also present in the second nematocidal compound 14.
A limited number of publications on the anthelmintic properties of triazoles indicate that further research is required. The next scientific step will be to conduct research in experimental in vivo models and to determine the molecular mechanism of action of 1,2,4-triazole derivatives 12 and 14.

Materials and Methods
All reagents and solvents were acquired from Sigma-Aldrich (Burlington, MA, USA) or Avantor Performance Materials Poland S.A (Gliwice, Poland). 1 H NMR and 13 C NMR spectra were recorded on Bruker Avance spectrophotometers (300, 400 or 700 MHz) in DMSO-d6 using TMS as an internal standard. Elemental analyses were performed on a Vario MACRO CHN analyzer (ELEMENTAR Analysensysteme GmbH, Langenselbold, Germany). Melting points were determined on a Mel TEMP 1002D apparatus and are given uncorrected. All reactions were controlled by TLC chromatography.

General Methods of Synthesis
Compounds 9-22 were synthesized according to established general procedures [41]: Amidrazones 1-8 were obtained from corresponding thioamides and 80% aqueous hydrazine hydrate solution [42]. 0.01 mol of amidrazones 1-8 and 0.01 mol (1.12 g) of itaconic anhydride were dissolved in 100 mL of anhydrous diethyl ether and left in room temperature for 7 days. Obtained crude solids 9-16 were filtered off and purified by crystallization from a methanol−water mixture (1:1) [41,54]. Compounds 17-22 were obtained by heating 0.25 g of derivatives 9-14 at boiling point with 20 mL of 2% NaOH aqueous solution for 2 h. After cooling down, the reaction mixture was filtered off. A 1% hydrochloric acid solution was added dropwise to the obtained filtrate for as long as precipitation was observed. Obtained crude solids of 17-22 were filtered off and purified by crystallization from water or from water with the addition of methanol [41,54]. The characteristics of the new compounds 12 and 19-22 are available in the Supplementary Materials ( 1 H NMR and 13 C NMR spectral data on Figures S1-S10, HRMS data on Figures S11-S15, HPLC data on Figures S16-S20). Compounds 9-11 and 13-18 were characterized in our previous publications [41,54,55].

Peripheral Blood Mononuclear Cells Preparation
Peripheral blood mononuclear cells (PBMC) were isolated immediately from fresh blood as previously described [43]. After isolation, PBMC were used to conduct experiments which assessed toxicity of compounds and their impact on proliferation and cytokine production.
The cell suspensions (1 × 10 6 cells/mL) in culture medium (5% fetal bovine serum-FBS-Euroclone SpA, Pero, Ml, Italy, in RPMI 1640-Biomed Lublin, Lublin, Poland) were added to 5 mL tubes (Falcon ® Round Bottom Polystyrene Tubes, Corning, NY, USA) in all experiments. Compounds 9-22 and ibuprofen (IBU) were initially dissolved in dimethyl sulfoxide (DMSO, Sigma-Aldrich) and afterwards, were added in appropriate amounts to the cell culture. Final concentrations of compounds and IBU in the cultures were 10, 50 and 100 µg/mL in all biological assays. The maximum concentration of DMSO in the individual assay was lower than 0.5%. These concentrations allowed us to avoid cell culture toxicity. The ibuprofen was selected as the reafference medicine generally used as nonsteroidal anti-inflammatory drugs (positive control). Additionally, control samples contained DMSO in the highest used dose (negative control) were prepared.

Toxicity of Compounds 9-22
Obtained PBMC were subjected to a 24 h culture (37 • C at 5% CO 2 atmosphere) with the studied compounds. After stimulation, the cells were washed once (centrifuged at 400× g at 4 • C for 5 min) with PBS (phosphate-buffered saline; Biomed Lublin, Lublin, Poland) and then used for staining with fluorescein isothiocyanate (FITC)-labeled Annexin V and propidium iodide (Annexin V Apoptosis Detection Kit I, Becton Dickinson, Franklin Lakes, NJ, USA). This procedure has been described previously [43].

Antiproliferative Activity of 12 and 19-22
Lymphocyte proliferation was evaluated by BD Horizon™ Violet Proliferation Dye 450 (VPD450, BD Pharmingen, San Diego, CA, USA). This dye is used for flow cytometric monitoring of cell division. VPD450-stained cells were cultured for 72 h with the mitogen, phytohemagglutinin (PHA, Sigma-Aldrich, 1 µg/mL, positive control), and/or increasing concentrations of compounds 12 and 19-22 in DMSO (10, 50 and 100 µg/mL). Control samples contained DMSO in the highest used dose (negative control). Thereafter, culture tubes were centrifuged at 400× g at room temperature for 5 min and washed once in PBS. The cell palette was suspended in PBS and analyzed by flow cytometry (FACSCanto II flow cytometer, Becton Dickinson, Franklin Lakes, NJ, USA). Twenty thousand events were collected and analyzed with FlowJo software (v 7.6.1, Tree Star, Ashland, OR, USA).

The Influence of 12 and 19-22 on Cytokine Production
The in vitro effect on cytokine production was measured by the enzyme-linked immunosorbent assay (ELISA). The assay was conducted as described earlier [41]. PBMC were cultured with lipopolysaccharide (LPS, from E. coli, O55:B5, Sigma-Aldrich, 1 µg/mL, positive control) and/or increasing concentrations of compounds. Cytokine levels (TNF-α, IL-6, IL-10 and IFN-γ) were measured by means of commercially available ELISA kits (Du-oSet, BD Bioscience, San Diego, CA, USA) according to the manufacturer's instructions. The samples were analyzed with iEMS Reader MF (Labsystems, Vanta, Finland). The contents of the analyzed cytokines were calculated by Ascent Software AscSW26 (London, UK).

Data Analysis
Data was analyzed in Statistica 13.3 software (StatSoft, Cracow, Poland) and graphed in Excel 2016. All p-values represent nonparametric Mann−Whitney U test.

Anthelmintic Activity of 10-22
Due to their very low solubility in water, test compounds 10-22 were suspended in 60% glycerol. To obtain a homogeneous emulsion, sonication was performed using an ultrasonic homogenizer (Hielscher Ultrasound Technology). The culturing of nematodes and the methodology of testing nematocidal properties was developed by the Department of Biology and Genetics of the Medical University of Lublin (patent No. PL232918, Bogucka-Kocka A., Kołodziej P., 2019). The culturing was carried out for 4-5 days at room temperature. All development stages of the nematodes were observed.
Owing to their high anthelmintic potential, which was proved to be higher than that of the recommended drug albendazole, we have selected compounds 12 and 14 as potential candidates for further research. Derivative 12 also showed anti-inflammatory activity (by inhibiting TNF-α production), which constitutes an additional advantage. Interestingly, the clinically used anthelmintic drug ivermectin also has an anti-inflammatory effect [56]. Thanks to their anthelmintic and anti-inflammatory properties, the selected compounds may, in the future, be used in the treatment of parasitic diseases caused by intestinal nematodes in humans.

Patents
Part of the results of the work were used to prepare the Polish patent application No. P.434865.