Synthesis and In Vitro Biological Studies of Heterocyclic Benzimidazole Derivatives as Potential Therapeutics for Trichinellosis

Abstract

Here we presented the synthesis of two groups of heterocyclic benzimidazole derivatives—methanimines 4a–c and hydrazones 6a–c. In vitro biological activity screening of the compounds was performed on isolated encapsulated muscle larvae of Trichinella spiralis. All tested compounds showed higher efficacy than albendazole, with compound 4a demonstrating activity comparable to ivermectin. Structure–activity relationship (SAR) analysis revealed that methanimines 4a–c, containing a thiophene moiety, were more effective than their hydrazone counterparts, highlighting the beneficial synergy between benzimidazole and thiophene pharmacophores. However, replacing the -N=CH- linker in compound 4a with -NH-N=CH- (as in compound 6a) led to a 23% reduction in activity, suggesting that methaniamines possess superior larvicidal potency under equivalent structural conditions. The ability of the studied compounds to interfere with the tubulin polymerization was studied spectrophotometrically on purified porcine brain. Of note, the tested benzimidazoles 4a–b and 6a–b had no discernible effect on tubulin polymerization. An in silico study of the physicochemical and pharmacokinetic characteristics of the novel synthesized heterocyclic benzimidazoles showed that they were characterized by a significant degree of drug-likeness and optimal properties for antineurotrichinellosis agents.

1. Introduction

In recent years, our research interest has focused on synthesizing new benzimidazole derivatives as potential anthelmintic agents against Trichinella spiralis (T.spiralis), which causes trichinellosis [1,2,3]. This is supported by the fact that the current drugs used to treat this zoonotic disease—the benzimidazole anthelmintics albendazole and mebendazole—are highly effective in the initial phase of infection. They expel infective intestinal larvae and adult worms from the small intestinal epithelium of the host, inhibit females from producing newborn larvae, and kill larvae during the migration stage [4]. However, they are only moderately effective against encapsulated larvae in muscle tissue. These drugs also have poor bioavailability and numerous adverse effects, including headaches, nausea, vomiting, fever, and elevated liver enzymes. Patients with pre-existing liver damage or dysfunction are especially vulnerable to more severe side effects, including leukopenia, anemia, thrombocytopenia, and pancytopenia [5]. In addition, there is increasing evidence of emerging resistance to benzimidazole anthelmintics [6,7].

The most severe complications of Trichinella infection, however, are associated with involvement of the central nervous system (CNS) [8]. The presence of Trichinella larvae in the CNS causes damage to blood capillaries such as vasculitis and perivasculitis, which are responsible for diffuse or focal lesions, in addition to granulomatous and allergic inflammatory reactions [9]. In this context, neurotrichinellosis is among the most serious complications of severe trichinellosis, along with myocarditis and thromboembolic events, and is often associated with fatal outcomes. Treatment of neurotrichinellosis requires the use of antiparasitic drugs that can cross the blood–brain barrier. Unfortunately, albendazole and mebendazole do not meet this requirement. Therefore, there is a need for novel and safe anti-Trichinella drugs, especially with efficacy against parasites localized in the CNS.

Our previous studies have shown that simple structural modifications of the key benzimidazole scaffold, especially the addition of another heterocycle, such as 1,3,5-triazine [3], thiophene [3], 1,3-benzodioxole [1,3,10], indole [3], etc., is a successful design strategy for new hybrid molecules with synergistic activity against T. spiralis. Among the newly synthesized compounds with the highest larvicidal efficacy (58.41% at 50 μg/mL concentration after 24 h incubation), the fused triazinobenzimidazole with thiophen-2-yl moiety stands out [3].

On the other hand, the thiophene ring is a key structural motif in numerous antiparasitic compounds, demonstrating potent activity against parasites, including Leishmania [11,12,13], Plasmodium [14,15], and Trypanosoma spp. [16,17]. In addition, numerous literary sources confirm the importance of the thiophene scaffold as a potential agent for the central nervous system [18,19].

In light of recent findings, the synthesis of hybrid molecules combining both benzimidazole and thiophene pharmacophores is emerging as a promising strategy for finding new antiparasitic agents to treat neurotrichinellosis.

In the present study, we describe the synthesis of two groups of benzimidazole derivatives containing a thiophene or other heteroaromatic fragment: methanimines and hydrazones. We then discuss how small structural differences affect the activity of these compounds on the isolated muscle larvae of the parasite T. spiralis. In addition to testing the anthelmintic activity of the benzimidazoles, we also predicted their physicochemical properties and drug-likeness using an in silico approach to further explore the polypharmacology of these benzimidazole derivatives. The next phase of this study involved carrying out tubulin polymerization inhibition assays to clarify the possible antiparasitic action mechanism. Summing up, the results of this complex investigation pave the way for further extensive exploration of the compounds’ anthelmintic activity against T. spiralis and in-depth studies of other possible mechanisms of action.

2. Materials and Methods

2.1. General Procedures

All commercially available solvents and reagents—thiophene-2-carboxaldehyde, 98+%; pyridine-4-carboxaldehyde, 97%; and furan-2-carboxaldehyde, 99%—were purchased from Alfa-Aesar (Heysham, UK). Reference compounds albendazole, ≥98% and ivermectin were obtained from Sigma-Aldrich (Steinheim, Germany). The course of reactions and the purity of products were tracked by thin layer chromatography (TLC) on pre-coated silica gel plates ALUGRAM SIL G/UV254, 0.20 mm thick (Macherey-Nagel, Düren, Germany), in benzene/methanol (3:1, v/v) as eluent and visualized using UV light (CAMAG UV Lamp 4, Muttenz, Switzerland). The melting points (mp) were determined using an Electrothermal AZ 9000 3MK4 apparatus (Stone, Staffordshire, UK) and were uncorrected. Infrared (IR) spectra of the compounds in a solid state were obtained on a Bruker Tensor 27 FT spectrometer (Billerica, MA, USA) in ATR (attenuated total reflectance) mode with a diamond crystal accessory. The spectra were referenced to air as a background by accumulating 64 scans, at a resolution of 2 cm⁻¹. Air was used as the background reference, and spectra were collected by averaging 64 scans at a resolution of 2 cm⁻¹. The ¹H and ¹³C NMR spectra were acquired at room temperature (303 K) using a Bruker Avance 400 MHz spectrometer (Billerica, MA, USA) with DMSO-d₆ as the solvent. Chemical shifts (δ) are reported in parts per million (ppm). Coupling constants J are given in Hertz (Hz), and spin multiplicities are given as singlet (s), broad singlet (br s), doublet (d), doublet of doublets (dd), triplet (t), triplet of doublets (td) and multiplet (m). The mass spectra were recorded using a Thermo Scientific Q Exactive Plus Hybrid Quadrupole-Orbitrap Mass Spectrometer (Waltham, MA, USA) Thermo Scientific (HESI HRMS) in positive mode. The spectra were processed using the Thermo Scientific FreeStyle program version 1.8 SP1 (Thermo Fisher Scientific Inc., Waltham, MA, USA). The microanalyses for C, H, and N were performed on PerkineElmer elemental analyzer, and satisfactory results within ±0.4% calculated values were recorded for the novel compounds.

2.2. Chemistry

The starting 1-(un)substituted-benzimidazol-2-yl-sulfonic acids 1a–c, 1-(un)substituted-benzimidazol-2-yl-amines 2a–c, and 1H-benzimidazole-2-yl-hydrazine 3 were synthesized according to the reaction conditions described in our previous publications [1,20].

2.2.1. General Procedures for the Synthesis of Compounds 4a–c

General Procedure 1 [21]

A solution of 1-(un)substituted-benzimidazol-2-yl-amine (1 mmol) in anhydrous ethanol (5 mL) and benzene (1 mL) was prepared, to which the appropriate aldehyde (1.2 mmol) and a few drops of glacial acetic acid were added. The reaction mixture was refluxed for 8 h. Upon completion, the solvents were partially removed under reduced pressure, and the resulting residue was allowed to crystallize. The solid product was filtrated, dried, and recrystallized from suitable solvents.

General Procedure 2 [22]

To a solution of 1-(un)alkyl-benzimidazol-2-yl-amine (4 mmol) and the corresponding aldehyde (4 mmol) in 6 mL of ethanol, 6 mL of 5% aqueous NaOH was added dropwise with stirring. The reaction mixture was stirred at room temperature for 2 h. The resulting precipitate was collected by filtration and purified by recrystallization from appropriate solvents.

2.2.2. General Procedures for the Synthesis of Compounds 6a–c [1]

A mixture of 1H-benzimidazol-2-yl-hydrazine 5 (0.01 mol) and appropriate heteroaromatic aldehyde (0.01 mol) in ethanol (99%, 20 mL) was refluxed on a water bath for about 3 h and cooled. The crystalline solid which precipitated during the reaction, was filtered and recrystallized from ethanol.

2.2.3. Compound Data

• (E)-N-(1H-benzimidazol-2-yl)-1-(thiophen-2-yl)methanimine (4a):

mp 224–225 °C, Rf = 0.539, IR (KBr) υ/ cm⁻¹: 3410, 3100, 3232, 1582, 1045, 740. ¹H NMR (400 MHz, DMSO-d6): 12.67 (br s, 1H, NH), 9.58 (s, 1H, N=CH), 7.98 (m, 2H), 7.49 (d, 2H, J = 51.63 Hz,), 7.31 (t, 1H, J = 4.48 Hz), 7.17 (m, 2H). ¹³C NMR (150 MHz, DMSO-d6) δ (ppm): 158.6, 155.9, 141.8, 137.3, 133.9, 129.3, 128.7, 122.3, 118.8. HRMS (ESI) m/z: Calcd. for: C₁₂H₉N₃S: 227.0523; Found: 228.0585 [M + H]⁺. Anal. Calcd. for C₁₂H₉N₃S: C, 63.41; H, 3.99; N, 18.49; Found: C, 62.91; H, 4.39; N, 18.19.

• (E)-N-(1-methyl-1H-benzimidazol-2-yl)-1-(thiophen-2-yl)methanimine (4b):

mp 115–117 °C, Rf = 0.584; IR (KBr) υ/ cm⁻¹: 3096, 1616, 1045, 735,¹H NMR (400 MHz, DMSO-d6): 9.54 (s, 1H, N-CH), 7.20–7.70 (m, 4H), 6.90–6.83 (bd, 2H), 3.64 (s, 3H). HRMS (ESI) m/z: Calcd. for: C₁₃H₁₁N₃S: 241.0779; Found: 242.0742 [M + H]⁺. Anal. Calcd. for C₁₃H₁₁N₃S: C, 64.71; H, 4.59; N, 17.41; Found: C, 64.61; H, 4.83; N, 17.46.

• (E)-N-(1-ethyl-1H-benzimidazol-2-yl)-1-(thiophen-2-yl)methanimine (4c):

mp 104–105 °C, Rf = 0.563; IR (KBr) υ/ cm⁻¹: 3064, 1615, 1046, 734. ¹H NMR (400 MHz, DMSO-d6): 9.60 (d, 1H, J = 0.80 Hz), 7.99 (m, 2H), 7.57 (m, 2H), 7.32 (m, 1H), 7.22 (m, 2H), 4.37 (q, 2H, J = 7.11 Hz), 1.35 (t, 3H, J = 7.14 Hz). ¹³C NMR (150 MHz, DMSO-d6) δ (ppm): 158.6, 154.5, 142.0, 141.8, 137.5, 134.8, 134.3, 129.4, 122.6, 122.4, 119.17, 110.6, 37.6, 15.5. HRMS (ESI) m/z: Calcd. for C₁₄H₁₃N₃S: 255.0836; Found: 256.0896 [M + H]⁺. Anal. Calcd. for C₁₄H₁₃N₃S: C, 65.86; H, 5.13; N, 16.46; Found: C, 65.62; H, 5.35; N, 16.22.

• (E)-2-(2-(thiophen-2-ylmethylene)hydrazinyl)-1H-benzimidazole (6a):

mp 267–268 °C, Rf = 0.563; ¹H NMR (400 MHz, DMSO-d6): 11.32 (bs, 1H, NH), 8.25 (s, 1H), 7.57 (m, 2H), 7.57 (dt, 1H, J = 0.99 Hz, J = 4.99 Hz), 7.34 (dd, 1H, J = 0.43 Hz, J = 3.59 Hz), 7.22 (m, 2H), 7.10 (dd, 1H, J = 3.58 Hz, J = 4.94 Hz), 6.95 (m, 2H). ¹³C NMR (150 MHz, DMSO-d6) δ (ppm): 153.6, 140.3, 137.1, 128.7, 128.1, 127.5. HRMS (ESI) m/z: Calcd. for C₁₂H₁₀N₄S: 242.0632; Found: 243.0694 [M + H]⁺. Anal. Calcd. for C₁₂H₁₀N₄S: C, 59.48; H, 4.16; N, 23.12; Found: C, 59.63; H, 3.80; N, 23.26.

• (E)-2-(2-(furan-2-ylmethylene)hydrazinyl)-1H-benzimidazole (6b):

mp 230–231 °C, Rf = 0.620; IR (KBr) υ/ cm⁻¹:1653, 1239, 1010, 730. ¹H NMR (400 MHz, DMSO-d6): 11.40 (bs, 1H, NH), 7.96 (s, 1H), 7.78 (dd, 1H, J = 0.68 Hz, J = 1.91 Hz), 7.22 (t, 2H, J = 4.47 Hz), 6.95 (m, 2H) 6.80 (dd, 1H, J = 0.34 Hz, J = 1.75 Hz), 6.62 (dd, 1H, J = 1.89 Hz, J = 3.33 Hz). HRMS (ESI) m/z: Calcd. for C₁₂H₁₀N₄O: 226.0860; Found: 227.0922 [M + H]⁺. Anal. Calcd. for C₁₂H₁₀N₄O: C, 63.71; H, 4.46; N, 24.76, Found: C, 63.35; H, 4.21; N, 24.95.

• (E)-2-(2-(pyridin-4-ylmethylene)hydrazinyl)-1H-benzimidazole (6c):

mp 294–295 °C, Rf = 0.500; IR (KBr) υ/ cm⁻¹: 3056, 1637, 812, 721. ¹H NMR (400 MHz, DMSO-d6): 11.72 (bs, 1H, NH), 8.59 (dd, 2H, J = 1.63 Hz, J = 4.36 Hz),7.99 (s, 1H), 7.76 (dd, 2H, J = 1.54 Hz, J = 4.43 Hz), 7.26 (m, 2H),7.00 (m, 2H). HRMS (ESI) m/z: Calcd. for C₁₃H₁₁N₅: 237.1020; Found: 238.1083 [M + H]⁺. Anal. Calcd. for C₁₃H₁₁N₅: C, 65.81; H, 4.67; N, 29.52, Found: C, 65.45; H, 4.34; N, 29.72.

2.3. Photostability

The study on the photostability of the examined compounds was conducted in a solar simulator SUNTEST CPS equipment (Heraeus, Hanau, Germany), supplied with an arc air-cooled Xenon lamp (Hanau, 1.1 kW, 765 Wm²), at ambient temperature. The irradiation was performed in methanol, acetonitrile, and DMSO solutions at a concentration of 5 × 10⁻⁵ mol/L. The photodegradation was monitored spectrophotometrically. The UV–Vis spectra were recorded with a Hewlett-Packard 8452 A spectrophotometer (Agilent Technologies, Inc., Santa Clara, CA, USA) in 1.0 × 1.0 cm quartz cells. The concentration of the samples was 50 mM for all compounds. The measurements in different solvents were performed at 25.0 ± 0.1 °C. A blank sample with the corresponding solvent was used to calibrate the instrument.

2.4. Parasitological Study

The encapsulated T. spiralis muscle larvae were provided by the National Center for Infectious and Parasitic Diseases, Department of Parasitology and Tropical Medicine, Sofia, Bulgaria.

The in vitro parasitological assay was performed as previously described [1,3]. The activities of the tested derivatives 4a–c and 6a–c dissolved in DMSO were evaluated in vitro using 100 muscle larvae per 1 mL of physiological solution, at concentrations of 50 μg/mL, and 100 μg/mL. Albendazole and ivermectin were used as positive controls in this assay, and a negative control containing only the ML in DMSO was included in the assay and subjected to the same conditions as the experimental cultures. The samples were then incubated at 37 °C in an atmosphere containing 5% CO₂ for 24 and 48 h. The viability of the parasite larvae was determined by direct microscopy.

The efficacy percentage of the benzimidazoles studied on T. spiralis larval viability was calculated as the average of three independent experiments [1], calculated as follows:

Efficacy % = (the number of dead parasite larvae/total parasite larvae) × 100%

All data are expressed as the mean and standard deviation (mean ± SD). Statistically significant differences compared to albendazole, parasitic larvae only, and various compounds are reported [23].

2.5. In Vitro Tubulin Polymerization Assay

Tubulin polymerization inhibition was assayed with a commercial assay kit of purified porcine tubulin (cat. number BK006P, Cytoskeleton, Denver, CO, USA), according to a previously described protocol [1]. Fresh buffer solution (PB-GTP) containing polymerization buffer (100 µL volume of 3 mg·mL⁻¹ tubulin in 80 mM piperazine-N, N′-bis (2-ethanesulfonic acid) (PIPES) pH 6.9, 0.5 mM EGTA, 2 mM MgCl, 1 mM GTP, 10% glycerol) was prepared from the stock solutions provided in the kit. Then, the stock solution of pure bovine tubulin (240 µM) dissolved in the PB-GTP was diluted four-fold to obtain a 60 µM tubulin concentration before adding the tested compounds. Stock solutions of the test samples were prepared at a concentration of 700 µM in dimethyl sulfoxide (DMSO). Paclitaxel (700 µM), nocodazole (700 µM) and albendazole (700 µM) in DMSO were used as reference compounds. The test and control samples were adjusted to a final concentration of 70 µM by dissolution in PB-GTP. In a 96-well plate kept on ice 60 µL of 60 µM tubulin and 10 µL of 70 µM of the test samples or positive control were applied in each well. The spontaneous polymerization of tubulin was studied by the addition of 10 µL buffer to 60 µL of 60 µM tubulin. The reaction microplate was incubated at 37 °C in the thermostated spectrophotometer chamber. The turbidity of the mixtures was monitored at 340 nm every 66 s. for 120 min and compared to nocodazole and albendazole as negative controls and paclitaxel as a positive control. Turbidity was registered with an MRX A2000 microplate reader (KLab, Daejeon, Republic of Korea) equipped with software for kinetic measurements.

Turbidity (absorbance) readings were used to determine the extent of polymerization:

[% inhibition = (1 − A₃₄₀ sample/A₃₄₀ control) × 100]

2.6. In Silico Analysis

In silico analysis of the newly synthesized benzimidazole compounds, as well as the benzimidazole-based anthelmintic albendazole, were performed using the SwissADME web tool (accessed on 10 April 2025) [24], which allows the assessment of pharmacokinetics and drug similarity.

3. Results and Discussion

3.1. Synthesis

Schiff bases 4a–c under study were synthesized from the reactions of 1-(un)alkyl-benzimidazol-2-yl-amine with heteroaromatic aldehydes 3a–c using two different synthetic approaches, as shown in Figure 1.

The first synthetic approach follows the recommended methodology by Nawrocka, W. et al. [21] for the synthesis of azomethines, involving the reflux of the amine with the corresponding heterocyclic aldehyde in a 5:1 mixture of anhydrous ethanol and benzene, in the presence of a catalytic amount of glacial acetic acid, for 8 h.

In contrast, the second approach [22] was carried out at room temperature (~20 °C) in ethanol with 5% aqueous sodium hydroxide, with a reaction time of only 2 h. The target compounds 4a–c were obtained in comparable yields (69–76%), regardless of the difference in the reaction conditions used. Adding a 5% aqueous NaOH solution to the reaction mixture proved to be a more efficient approach, allowing the reaction to proceed smoothly at room temperature with a significantly shorter reaction time.

For the preparation of hydrazones 6a–c, 2-hydrazino-1H-benzimidazole 5 was reacted with the respective heterocyclic aldehydes 3a–c in ethanol under reflux [1].

The spectral characteristics and elemental analysis results detailed in the Experimental section are consistent with the proposed structures of the azomethines 4a–c and the hydrazones 6a–c. The IR spectra of compounds showed a strong absorption band at 1582–1616 cm⁻¹ characteristic of the group C=N. The presence of the imine proton was confirmed by the ¹H NMR spectra of all methanimines 4a–c, which display a one-proton singlet at δ 9.54–9.60 ppm, while in the ¹H NMR spectra of hydrazones 6a–c the imine proton resonates at δ 7.96–8.59 ppm. A one-proton singlet at 12.58 ppm was assigned to the imidazole group NH of 4a. The NH protons from the benzimidazole heterocycle appear in the ¹H NMR spectra of hydrazones 6a–c as a broadened singlet at 11.32–11.72 ppm. The product identification of hydrazone derivatives is described in more detail in a previous publication [1].

3.2. Photophysical Evaluation

The stability of the organic compounds is an important characteristic regarding their practical application. Under normal conditions, oxidation, particularly photooxidation, is the major factor contributing to molecular degradation. This is why the present work also involved studying the photostability of 4a–c and 6a–c. For this purpose, solutions of 4a–c and 6a–c in methanol, acetonitrile, and DMSO at concentration 5 × 10⁻⁵ mol/L were subjected to irradiation with UV light in a SUNTEST instrument (AMETEK, Linsengericht-Altenhasslau, Germany). The kinetics of photostability were monitored spectrophotometrically. All compounds under study showed negligible changes in their absorption spectra during irradiation in all three solvents. Figure 2 shows the absorption spectra of compound 6a in DMSO after irradiation, which are similar to those of the other compounds. The results clearly demonstrate the high stability of the studied benzimidazoles under the experimental conditions used in this work, which is consistent with previous reports for compounds of the same nature [25] and their suitability for future biological applications.

3.3. Anthelmintic Activity

In the present study, the effect of the synthesized benzimidazole derivatives on the viability of T. spiralis ML was evaluated in comparison to albendazole and ivermectin, following the standard procedure [1,3]. The results, presented in

Table 1. In vitro activity of tested compounds against Trichinella spiralis muscle larvae ^a.

Compound	Substituents		Concentration 50 (100) µg/mL in µM	Efficacy (%) After 24 h		Efficacy (%) After 48 h
	R	Het		50 µg/mL	100 µg/mL	50 µg/mL	100 µg/mL
4a	H	thiophen-2-yl	0.220 (0.440)	54.5 ± 0.52 b	68.9 ± 0.53	71.5 ± 3.21 c	80.5 ± 0.33 d
4b	CH3	thiophen-2-yl	0.207 (0.414)	50.9 ± 0.35	63.8 ± 1.82	68.6 ± 1.03	74.0 ± 0.48 d
4c	C2H5	thiophen-2-yl	0.196 (0.392)	48.4 ± 1.57	61.7 ± 0.51	58.9 ± 0.55	73.4 ± 0.63 c
6a	H	thiophen-2-yl	0.206 (0.413)	25.4 ± 0.58 c	28.9 ± 0.39	53.8 ± 0.34	57.5 ± 1.02
6b	H	furan-2-yl	0.221 (0.442)	20.4± 0.45	28.0 ± 0.67	22.8 ± 0.05	38.4 ± 0.67
6c	H	pyridin-4-yl	0.210 (0.421)	11.4 ± 2.48	13.5 ± 1.11	18.5 ± 0.57	25.5 ± 1.61 b
Albendazole			0.188 (0.377)	10.5 ± 0.62	10.7 ± 0.32	14.5 ± 0.26	15.7 ± 0.34
Ivermectin			0.057 (0.114)	45.5 ± 3.96	48.9 ± 0.01	62.1 ± 0.04	78.3 ± 0.86 c

^a Positive Control (untreated)–97.6 ± 1.14 viable parasitic larvae after 24 h, 95.8 ± 1.48 viable parasitic larvae after 48 h; ^b p < 0.05; ^c p < 0.001; ^d p < 0.0001.

, showed that the tested benzimidazoles reduced the viability of parasitic larvae in a time- and dose-dependent manner.

The benzimidazole-thiophene compounds exhibited high larvicidal activity, confirming our initial expectations. The methenamine analogue 4a resulted in a significant reduction (p ≤ 0.001) in the mean number of viable larvae count, with an efficacy of 54.5% at a concentration of 50 μg/mL after 24 h of incubation compared to the control group (untreated larvae), which had a mean viable larvae count of 97.6 ± 1.14. This efficacy is higher than that observed for ivermectin (45.5%) and albendazole (10.5%) under these experimental conditions (p ≤ 0.05 versus albendazole). The in vitro study also revealed a significant reduction (p ≤ 0.001 versus albendazole) in the average number of T. spiralis ML after 48 h of incubation with 100 μg/mL of compound 4a, with an efficacy of 80.5%.

The introduction of an alkyl substituent at the first position of the benzimidazole nucleus (4b and 4c) leads to a slight decrease in activity. For example, compound 4c (R = C₂H₅) showed lower activity against T. spiralis ML (73.4% at 100 µg/mL after 48h) compared to that of its methyl derivative 4b (R = CH₃) (74.0%). It is interesting to note that the addition of a nitrogen atom to the linker that connects the benzimidazole and thiophene parts of the molecule (compound 4a, -N=CH- vs. compound 6a, -NH-N=CH-) results in a 23% reduction in anthelmintic activity. Compound 4a was distinguished for 80.5% efficacy at a concentration of 100 µg/mL (p ≤ 0.0001 versus albendazole), while derivative 6a showed a 57.5% larvicidal effect (p ≤ 0.001 versus albendazole) after 48-h exposure at the same concentration (see Table 1). The benzimidazolyl-2-hydrazones of furan-2-carboxaldehyde and pyridine-4-carboxaldehyde (compounds 6b and 6c), on the other hand, showed less pronounced effects against the parasites than a thiophene derivative 6a, but were comparable to those of the clinically approved anthelmintic albendazole. These results are consistent with findings from our previous antiparasitic study on benzimidazole compounds bearing a heterocyclic moiety [3]. They pave the way for further in-depth studies regarding the anti-T. spiralis activity of benzimidazole-thiophene Schiff base compounds.

3.4. In Vitro Tubulin Inhibition Assay

Benzimidazole anthelmintics (such as albendazole and mebendazole) are known to inhibit tubulin polymerization in parasitic worms, causing their immobilization, cessation of reproduction, and death [7]. These agents bind to nematode tubulin with 250–400 times greater affinity than to mammalian tubulin, indicating selective toxicity to the parasites.

In view of establishing the potential mechanism of anti-trichinella action, the ability of the synthesized compounds 4a, 4b and 6a–c to interfere with tubulin polymerization was assayed in vitro. The effect of the compounds was tested on porcine tubulin at a 10 µM concentration and compared to paclitaxel, albendazole and nocodazole as reference drugs (Figure 3 and Supplementary Materials).

The measured kinetic curves were approximated to sigmoidal or bi-sigmuidal curves and fitted by Boltzmann equations (

Table 2. In vitro effect on tubulin polymerization of compounds 4a–b and 6a–c compared with reference drugs paclitaxel, nocodazole and albendazole.

Compound 1	Half-Time of Aggregation χ0, min	Slope Factor of the Boltzmann Fit dχ, min	Lag Time 2, min	Curve/Equation
tubulin (spontaneous polymerization)	17.6	11.6	No lag time	Sigmoidal/Boltzmann
albendazole	32.4	17.6	No lag time	Sigmoidal/Boltzmann
nocodazole	24.1	15.9	No lag time	Sigmoidal/Boltzmann
paclitaxel	7.4	3.9	No lag time	Sigmoidal/Boltzmann
4a	12.9	7.1	No lag time	Sigmoidal/Boltzmann
4b	15.9	9.96	No lag time	Sigmoidal/Boltzmann
6a	14.8	9.8	No lag time	Sigmoidal/Bolzman
6b	15.9	10.4	No lag time	Sigmoidal/Boltzmann
6c	20 (1 st) 65 (2 nd)		Between 0–10 min and 45–55 min	Bi-Sigmoidal/Boltzmann-dual curve

¹ final concentration of all compounds and standards is 10 µM; ² lag time = χ₀ − 2dχ.

). The spontaneous tubulin polymerization was characterized with a half-time of aggregation χ₀ of 17.6 min (Table 2), while in the presence of paclitaxel the half-time of aggregation was considerably shorter—of 7.4 min. In contrast, the presence of both nocodazole and albendazole, which are tubulin polymerization inhibitors, resulted in increased half-time of aggregation—32.4 and 24.1 min, respectively.

Four of the tested compounds—4a, 4b, 6a, and 6b—slightly promoted the tubulin aggregation, showing half-times of 12–16 min (Table 2). Hydrazone 6c showed a two-step polymerization curve (Figure 3f). In the tubulin assembly assay such a type of curve is not a typical pattern, and it is usually interpreted as a sign of more complex or multi-phase interaction with the tubulin or the microtubule polymerization machinery. Similar kinetic behavior was reported for paclitaxel at specific concentrations, epothilones and some synthetic tubulin polymerization stabilizers. The tubulin polymerization inhibition effect of compound 6c is exerted through two lag phases—between 0 and 10 min and between 45 and 55 min.

The slope factor of the Boltzmann fit (dχ) describes the rate of the aggregation growth, i.e., the smaller the dχ, the faster the aggregation growth. In the studied series of compounds, slope factors between 7 and 10.4 min were observed, which is only slightly faster than the spontaneous polymerization of tubulin.

The observed modulation effect of the studied compounds shows a different trend from those established for the larvicidal effect against T. Spiralis ML. Notably, compound 6c, showing the most pronounced tubulin polymerization inhibition, did not manifested marked anti-trichinella efficacy, suggesting that alternative mechanistic properties are likely to be involved in the antiparasitic effect. One possible route to be considered is modulation of mitochondrial function through ultrastructural alterations in mitochondria [26] or inhibition of certain mitochondria membrane enzymes [27].

3.5. In Silico Analysis of Physicochemical Properties, Pharmacokinetics, and Drug-likeness

To gain further insight into the potential of newly synthesized benzimidazole derivatives as candidates for the treatment of trichinosis infection, we analyzed these compounds using the web bioinformatics tool SwissADME [24]. The parameters describing the physicochemical, pharmacokinetic, and medicinal chemical properties of the compounds and of albendazole are presented in

Table 3. Calculated molecular properties of compounds 4a–c and 6a–c for assessment of the drug-likeness.

Compound	MW a	TPSA b	Natoms c	HBA d	HBD e	Nviol f	NRotB g	logP h
4a	227.28	69.28	16	2	1	0	2	3.01
4b	241.31	58.42	17	2	0	0	2	3.19
4c	255.34	58.42	18	2	0	0	3	3.43
6a	242.30	81.31	17	2	2	0	3	2.58
6b	226.23	66.21	17	3	2	0	3	1.90
6c	237.26	65.96	18	3	2	0	3	1.80
Albendazole	265.33	92.31	18	3	2	0	6	1.62

^a Molecular weight; ^b Topological polar surface area; ^c Number of nonhydrogen atoms; ^d Number of hydrogen-bond acceptors (O and N atoms); ^e Number of hydrogen-bond donors (OH and NH groups); ^f Number of ‘Rule of five’ violations; ^g Number of rotatable bonds; ^h Consensus Log Po/w, calculated as an average of the five available methods for logP prediction.

and Table 4.

The data from the evaluation of drug-likeness (Table 3) indicate that all synthesized compounds comply with Lipinski’s Rule [28]. Their physicochemical properties—in particular, fewer than five hydrogen bond donors, fewer than 10 acceptors, molecular weight below 500, and logP below 5—suggest a potential for high bioavailability, similar to those observed for albendazole. Based on average logP values below 4 (Table 3), calculated using five different predictive methods [29], all benzimidazoles demonstrate potential for good absorption and bioavailability. Methanimines 4a–c and hydrazone 6a, containing a thiophene moiety, showed higher logP values. The topological polar surface area (TPSA) is below 140 Å 2 for all benzimidzole derivatives, so they are expected to exhibit good intestinal absorption. To predict the oral bioavailability of thr compounds under study, we estimated their percentage absorption (%ABS), which depends on the TPSA that is used to calculate % ABS [25]. All compounds have %ABS > 60% (Table 4) indicating that they are expected to be orally bioavailable. Solubility data for the benzimidazoles tested (values from −3.08 to −3.94, Table 4) indicate that they are sufficiently soluble and can be used as oral drug candidates [30].

An interesting prediction suggested by the SwissADME tool is the ability of compounds (TPSA < 70 Ų, Table 2) except 6a (TPSA = 81.31 Ų) to penetrate the blood–brain barrier (BBB) (Table 4). This makes them suitable candidates for the treatment of severe trichinellosis infection, which can involve multiple organs, including the brain. Brain damage in trichinellosis can manifest as either diffuse cerebral damage or focal neurological deficits [31]. Penetration of the BBB is a key factor in central nervous system (CNS) drug delivery, as it blocks the entry of large molecules and over 98% of small drug candidates [32].

Another important prediction of the SwissADME tool concerns the lack of interaction of the compounds in this study with the permeability transition glycoprotein (P-gp) (Table 4). P-glycoprotein plays a crucial role in transporting various substrates across cellular membranes and is key to many physiological processes. In particular, their over-expression is a common response to chemotherapy in tumour cells and to treatment with macrocyclic lactones (including ivermectin) in nematodes. This overexpression can lead to treatment failure by reducing the concentration of therapeutic agents at the target site. It is also well-known that P-gp also contributes to resistance to some anthelmintics [33]. Nare et al. reported that some benzimidazoles interact specifically with P-gp and have been shown to be substrates for P-glycoprotein-mediated drug efflux [34]. However, albendazole and fenbendazole have been shown to have no effect on P-gp activity [35]. None of the compounds tested (4a–c and 6a–c) also interacted with P-gp (Table 4), suggesting a lower risk of efflux-related resistance mechanisms that often compromise drug efficacy.

Furthermore, the presence of substructures giving false positive biological output, i.e., showing a potent response in assays irrespective of the protein target, commonly referred to as PAINS (Pan Assay Interference Compounds), was also evaluated. This analysis showed that heterocyclic benzimidazoles 4a–c and 6a–c are devoid of this property (PAINS = 0, Table 4).

In addition, the properties of methanimines 4a–c and hydrazones 6a–c were evaluated using the Brain Or IntestinaL EstimateD (BOILED-Egg) method and the bioavailability radar of the SwissADME tool. The boiled-egg images (Figure 4) were obtained by considering the parameters WLOGP and TPSA reported in Table 3, which are a lipophilic index and a measure of apparent polarity, respectively. These graphs allow an easy understanding and visualization of the two parameters: the passive absorption at the GI tract (white area) and the ability to permeate the BBB (yellow area). As can be seen, all compounds except compound 6a and albendazole were estimated to be able to cross the BBB, as would be required to reach Trichinella larvae localized in the brain. The red colour of the dots indicates that all of the benzimidazoles tested are theoretically not substrates of P-gp, as previously estimated in Table 4.

Table 4. In silico estimated physicochemical parameters of the heterocyclic benzimidazoles 4a–c, 6a–c, and albendazole.

Compound	WLOGP a	% ABS b	XLOGP3 c	Log S d (ESOL)	Csp 3 e	GI abs f	P-gp sub g	BBB h	PAINS i
4a	3.13	85.1	3.13	−3.74	0.00	High	No	Yes	0
4b	3.39	88.9	3.09	−3.76	0.08	High	No	Yes	0
4c	3.87	88.9	3.39	−3.94	0.14	High	No	Yes	0
6a	2.88	81.0	3.45	−3.93	0.00	High	No	No	0
6b	2.41	86.2	2.84	−3.44	0.00	High	No	Yes	0
6c	2.21	86.3	2.14	−3.08	0.00	High	No	Yes	0
Albendazole	3.05	77.2	2.81	−3.23	0.33	High	No	No	0

^a Water Partition Coefficient; ^b % of Absorption = 109−0.345 × TPSA [25]; ^c Predicts the logP value of a query compound by using the known logP value of a reference compound as a starting point [36]; ^d Indicator of the aqueous solubility of a compound; ^e The Fraction of Carbon atoms in sp3 hybridization; ^f Gastrointestinal Absorption; ^g Transmembrane Glycoprotein; ^h Blood–brain Barrier Permeability; ⁱ Pan-Assay Interference Compounds.

Table 4. In silico estimated physicochemical parameters of the heterocyclic benzimidazoles 4a–c, 6a–c, and albendazole.

Compound	WLOGP a	% ABS b	XLOGP3 c	Log S d (ESOL)	Csp 3 e	GI abs f	P-gp sub g	BBB h	PAINS i
4a	3.13	85.1	3.13	−3.74	0.00	High	No	Yes	0
4b	3.39	88.9	3.09	−3.76	0.08	High	No	Yes	0
4c	3.87	88.9	3.39	−3.94	0.14	High	No	Yes	0
6a	2.88	81.0	3.45	−3.93	0.00	High	No	No	0
6b	2.41	86.2	2.84	−3.44	0.00	High	No	Yes	0
6c	2.21	86.3	2.14	−3.08	0.00	High	No	Yes	0
Albendazole	3.05	77.2	2.81	−3.23	0.33	High	No	No	0

^a Water Partition Coefficient; ^b % of Absorption = 109−0.345 × TPSA [25]; ^c Predicts the logP value of a query compound by using the known logP value of a reference compound as a starting point [36]; ^d Indicator of the aqueous solubility of a compound; ^e The Fraction of Carbon atoms in sp3 hybridization; ^f Gastrointestinal Absorption; ^g Transmembrane Glycoprotein; ^h Blood–brain Barrier Permeability; ⁱ Pan-Assay Interference Compounds.

The bioavailability radars presented in the Supplementary Materials offer the drug-likeness depiction of the selected compounds. In the graphs, the pink-coloured area includes the optimal range of each physical–chemical property (lipophilicity, size, polarity, solubility, saturation and flexibility). Based on these results, it can be concluded that the compounds from this study meet all the requirements for drug similarity, with the sole exception of the saturation values (carbon atoms in sp³ hybridization).

4. Conclusions

In conclusion, we have performed a synthesis of two groups of heterocyclic benzimidazole derivatives—methanimines 4a–c and hydrazones 6a–c—as potential therapeutics for trichinellosis. Among the various methods available for the synthesis of azomethines of 2-aminobenzimidazole, we applied two different approaches and compared them in terms of reaction time and product yield. The addition of a 5% aqueous NaOH solution to the reaction mixture proved to be the more efficient method, as the reaction proceeded smoothly at room temperature within 2 h. Notably, both methods afforded the final products 4a–c in comparable yields. Newly synthesized benzimidazoles, being heterocyclic moieties, were evaluated for their in vitro anthelmintic activity against isolated T. spiralis ML. Similar to our previous results the tested benzimidazoles exhibited higher activity than albendazole at studied concentrations. The compound 4a showed remarkable efficacy, comparable to that of the commercial anthelmintic drug ivermectin—a 54.5% larvicidal effect at a concentration of 50 μg/mL after 24 h and an 80.5% effectiveness at a concentration of 100 μg/mL after 48 h of incubation. In addition, an SAR of the derivatives in both chemical series was discussed. The tested methaniamines 4a–c, bearing thiophene moiety, exhibited greater larvicidal activity than the hydrazone analogs. This suggests that the synergistic integration of benzimidazole and thiophene pharmacophores within one structure enhances antiparasitic efficacy. However, there was a 23% decrease in anthelmintic activity when the -N=CH- linker connecting the benzimidazole to the thiophene core in compound 4a was replaced by -NH-N=CH- in hydrazone 6a. This result suggests that methanimines have enhanced T. Spiralis larvicidal activity compared to hydrazones when all other structural features are held constant. Studies on the ability of the investigated benzimidazole derivatives to modulate microtubule polymerization show that all of the tested compounds, except compound 6a, exhibit only a minor effect on tubulin polymerization. This result suggests that alternative mechanistic properties, such as modulation of mitochondrial function through ultrastructural changes in mitochondria, or inhibition of certain enzymes in the mitochondrial membrane, must be considered in further studies of the anti-trichinella effect. The preliminary in silico evaluation of the physicochemical, pharmacokinetic and medicinal chemistry parameters of the compounds suggested the overall druglikeness, bioavailability and ability to cross the BBB of five of the synthesized heterocyclic benzimidazoles, making them suitable for future applications as neutothrichinellosis agents.