Study of the Effect of Metal Complexes on Morphology and Viability of Embryonated Toxocara canis Eggs

Abstract

An organic salt and four metal complexes derived from azole were evaluated against embryonated Toxocara canis eggs (TCE). The new organic salt, (LH)⁺(FeCl₄)⁻, where L = 3,5-bis(3,5-dimethylpyrazole-1-ylmethyl)toluene (5), a potential environmental disinfectant, was isolated as an air-stable yellow solid and characterized by elemental analysis, electrical conductivity, mass spectrometry, and infrared and ultraviolet/visible spectroscopy. In addition, the structure of 5 was determined by single-crystal X-ray diffraction. Compound 2 showed high anti-TCE activity. Interestingly, these compounds showed little effect on hepatocytes, indicating that they are not cytotoxic. These results will assist in the design of anti-TCE compounds.

Introduction

Toxocariosis is an important zoonosis caused by larval forms of Toxocara (Pawlowski 2001, Divyamol et al. 2016). The nematodes responsible for this infection in humans are the parasites Toxocara cati and Toxocara canis; however, the increased frequency of human infections has been attributed to T. canis (Camilo et al. 2013). The infection arises when humans ingest the embryonated eggs of the parasite. T. canis eggs (TCE) are found throughout the environment and especially in soil due to their conformation and structural resistance to unfavorable environmental and chemical conditions. The most used substance and method for killing TCEs in the environment have been chlorine and ultraviolet radiation, respectively. However, they are not effective (Mun et al. 2009).

On the other hand, Magaña et al. (2016) reported that minerals such as Ag, Cu, Zn, Ti, and Fe showed activity against TCEs. Their results are attributed to structural injury caused by the mechanical stress from the interaction of the minerals with the surface of the TCEs. It has been shown that the combination of albendazole (ABZ) and Cu nanoparticles has potent activity against Setaria cervi, a filarial nematode (family Onchocercidae) that inhabits the peritoneum of bovine. In this research, it was concluded that a nanocomposite leads to greater DNA destruction and concomitant apoptosis than is caused by free ABZ (Zafar et al. 2016). Metal complex derivatives of azole have shown greater antimicrobial activities than free azoles, and the increase in the antimicrobial properties of these compounds is associated with their higher pharmacological lipophilicity (Gammal et al. 2015, Keshia et al. 2016).

The increase in the number of bioactive metal complexes bearing N-donor and other ligands is a positive and important advance in the search for antiparasitic and antimicrobial metal-containing drugs. Based on the great interest in the design of anti-TCE substances, we focused our attention on the use of metal complexes and compounds derived from azole. To the best of our knowledge, the use of complexes derived from azoles for altering the morphology and viability of TCEs has not yet been reported.

Materials and Methods

General procedures

The synthesis of the ligands and metal complexes was performed under an inert atmosphere (nitrogen) using standard Schlenk techniques, including the pretreatment of reagent-grade solvents, which were dried, distilled, and stored under the same inert atmosphere. These compounds were prepared according to previous reports: the ligand 3,5-bis(3,5-dimethylpyrazole-1-ylmethyl)toluene (L) (Hurtado et al. 2009), dichloro[(bdmpzm)] zinc (II) (1) (Cheng et al. 2006), dichloro[bdmpzm] copper (II) (2) (Potapov and Khlebnikov 2006), dichloro[bdmpzm] nickel (II) (3) (Reedijk and Jan Verbiest 1979, Castillo et al. 2016), and [(Co(bdmpzm)₂(NCS)₂] (4) (bdmpzm = bis(3,5-dimethyl-1-pyrazolyl)methane) (Machura et al. 2013, Andrea et al. 2017, Nagles et al. 2017).

To characterize the new organic salt, 1-(3-((3,5-dimethyl-1H-pyrazole-1-yl)methyl)-5-methylbenzyl)-3,5-dimethyl-1H-pyrazole-2-ium}FeCl₄ ⁻ (5), elemental analyses (C, H and N) were conducted with a Thermo Scientific™ FLASH 2000 CHNS/O Analyzer. Fourier transform infrared (FTIR) spectra were recorded on a Thermo Nicolet NEXUS FTIR spectrophotometer using KBr pellets. Mass spectrometry (HRMS) data were obtained on an Agilent Technologies Q-TOF 6520 spectrometer by electrospray ionization (ESI) in positive ion mode. Melting points were determined on a Mel-Temp^® 1101D apparatus in open capillary tubes, and they are presented uncorrected. Molar conductivity measurements were carried out in a CON 700OAKTON instrument using 1 mM solutions in DMSO

{1-(3-((3,5-Dimethyl-1H-pyrazole-1-yl)methyl)-5-methylbenzyl)-3,5-dimethyl-1H-pyrazole-2-ium}FeCl₄ ⁻ (5)

To Schlenk flask was added 3,5-bis(3,5-dimethylpyrazol-1-ylmethyl)toluene (L) (0.162 mmol, 50 mg), FeCl₃ (0.2 mmol, 32 mg), and acetonitrile (5 mL). The resulting mixture was stirred at room temperature for 1 h. The formed solid was isolated by filtration, washed with acetonitrile and diethyl ether, and dried under vacuum. Yield: 54.2 mg (66%). M.p.: 210–211°C. UV/vis λ_max nm (ɛ, L mol⁻¹ cm⁻¹): 284 (6600). FTIR (KBr, cm⁻¹): 3132, 2924, 2360, 1857, 1550, 1463, 1433, 1301, 794, 719, 682, 588, and 522. Analysis Calcd for the complex C₁₉H₂₅Cl₄FeN₄: C, 45.00; H, 4.97; and N, 11.05. Found: C, 45.00; H, 4.97; and N, 11.05. Λ_M = 837 μS.

X-ray structural determination of 5

Recrystallization of 5 by slow evaporation from a pentane/ethyl acetate (98:2) solution gave crystals of suitable size and quality for single-crystal X-ray diffraction analysis. All the non-hydrogen atoms were refined anisotropically. The hydrogen atoms were generated geometrically, placed in calculated positions (C-H = 0.93–0.97 Å), and included as riding contributions with isotropic displacement parameters set at 1.2–1.5 times the U_eq value of the parent atom. H atoms belonging to nitrogen atoms were located in difference density maps and were refined freely. The X-ray intensity data were measured at room temperature (298(2) K) in an Agilent SuperNova, Dual, Cu at zero, Atlas four-circle diffractometer equipped with a CCD plate detector. The crystal structure was refined using the SHELXL2014 program (Sheldrick 2015).

Anti-Toxocara canis eggs activity

Parasites and eggs

The nematodes were obtained from naturally infected pups at the canine control center in the city of Guadalupe, Nuevo Leon, México. To obtain the parasites, we administered a commercial dewormer (pyrantel pamoate, Pfizer) to the pups. Adult parasites were transported in a container with physiological saline solution at room temperature to the Laboratory of Parasitology of the Faculty of Veterinary Medicine and Animal Science of the Universidad Autonóma de Nuevo León. The female parasites were selected and the distal region of the uterus was dissected; this zone contains the largest number of fertile eggs that are needed for the experiment. The obtained eggs were placed in sterile containers that contained 5% sodium hypochlorite solution (Clorox^®) for 10 min and then were washed by centrifugation thrice with 5 mL of saline solution. We visualized the specimens with an inverted microscope (Zeiss Invertosko) every third day to determine the embryonic development and rule out possible bacterial or fungal contamination. The culture medium for egg maturation was 0.1 N H₂SO₄ (sulfuric acid) solution. When the number of eggs reached 1 × 10⁶, they were placed in 5 mL culture dishes and kept under constant hydration with the same H₂SO₄ solution until they reached maturity (45–60 days) (Tiersch et al. 2013).

In vitro activity

The in vitro assay was carried out in 96-well microplates (30 embryonated eggs/well) with the ligands (bdmpzm and L) and compounds 1–5. Piperazina, a standard anthelmintic, was used as a positive control, and DMSO was used as the solvent control. All tests were conducted in triplicate. The nematocidal activity was calculated in terms of the relative mobility (RM). Worm motility is a clear indication of its survivability, and this parameter is given by the motility index score (Zaridah et al. 2001). This method was originally developed by Kiuchi et al. (1987) and was modified by Reis et al. (2010) who added more scores, as shown in Table 1. The TCEs were exposed to different concentrations of ligands and compounds (5, 10, and 15 μM in DMSO). After 48 h, the larva mobility of the TCEs was examined under an inverted microscope; some specimens were randomly separated to identify the morphological changes that occurred in the internal and external structures of the organisms. A score corresponding to a specific type of movement was given to each larva. Larva mortality was defined as the number of larvae that received a score of zero (0) divided by the total number of larvae in each well (Table 2)

Table 1.

Criteria for Evaluating on the Effect of Ligands and Compounds on Toxocara canis Larvae

State of larvae (score)	(n)
Rapid movement with the whole body	5
Intermediate movement with the whole body	4
Slow movement with the whole body	3
Movement with only one part of the body	2
Stationary, but not dead	1
Dead	0

Mobility index (MI) = Σn Nn/ΣNn, where Nn: number of larvae with the score of n (1). Relative mobility (RM) = MI sample/MI control 100 (2).

Table 2.

Crystal Data and Experimental Details

Crystal data
Chemical formula	C₁₉H₂₅Cl₄FeN₄
M _r	507.08
Crystal system, space group	Monoclinic, P2₁/c
Temperature (K)	298
a, b, c (Å)	12.5434 (19), 12.9339 (19), 16.045 (2)
β (°)	104.757 (15)
V (Å³)	2517.1 (7)
Z	4
Radiation type	Mo Kα
μ (mm⁻¹)	1.03
Crystal size (mm)	0.31 × 0.13 × 0.12

Toxicity study of ligands and compounds

To establish a measure of cytotoxicity for the ligands and compounds, 96-well cell culture dishes were prepared with a murine cell line of hepatocyte BpRc1 (ATCC^® CRL-2217™). This cell line was cultured under optimum conditions for growth, that is, the cells were maintained at a temperature of 37°C, a relative humidity of ≈82%, and an atmosphere of 95% air and 5% CO₂ in an incubator (Thermo Scientific). The recommended culture media (GIBCO^®) supplemented with penicillin/streptomycin (GIBCO) and fetal bovine serum (FBS; GIBCO) were used. This cell line was maintained according to the information provided by the corresponding ATCC guide. The cell line was in contact with the compounds at the same concentrations used in the in vitro assay. Cells were seeded in 96-well tissue culture plates with 5 × 10⁴ cells/well, and 24 h after platting, they were supplemented in triplicate with each compound. All dilutions were prepared with fresh culture media, and plates were incubated for an additional 24 h. Cells in plates were washed with PBS pH = 7.4 to remove dead cells, and the surviving cells were then used to estimate the cytotoxic effect relative to a mock preparation. The neutral red method was used to determine the cell viability (Repetto et al. 2008).

Results and Discussion

Synthesis and characterization of {1-(3-((3,5-dimethyl-1H-pyrazole-1-yl)methyl)-5-methylbenzyl)-3,5-dimethyl-1H-pyrazole-2-ium}FeCl₄ ⁻ (5)

The organic salt was obtained in good yield after the reaction of L with anhydrous FeCl₃. It was found that the small amounts of HCl present in the iron chloride promote the formation of 5 with tetrachloroferrate as a counterion. The use of other solvents such as acetone and methanol did not impact the course of the reaction, but did have an important effect on the quantity of product obtained. It is presumed that more polar solvents favor the formation of 5, and for that reason, acetonitrile presented the best results. UV/vis spectroscopy, electrical conductivity, FT-IR spectrometry, and single-crystal X-ray diffraction analyses were used to characterize the isolated compound. Infrared spectrometry of the salt showed absorption bands that were not observed in the spectrum of the free ligand (3132, 1857 and 1662 cm⁻¹) (Supplementary Figs. S1–S3; Supplementary Data are available online at www.liebertpub.com/vbz), and these bands were attributed to the vibrations produced by the protonated pyrazole nitrogen in L. The mass spectrum (ESI-positive mode) showed two peaks: the first at a mass/charge ratio (m/z) of 309.2088, which coincides with the expected mass of the cation calculated for [C₁₉H₂₅N₄]⁺ (309.2074 m/z), and the second at 213.1383 (m/z), which corresponds to the mass of the cation [C₁₄H₁₇N₂]⁺ (213.1386 m/z) generated from the loss of the dimethyl pyrazolium ion from L (Supplementary Fig. S4).

Crystal structure of 5

The molecular structure is shown in Figure 1a. This organic–inorganic hybrid crystallizes in a monoclinic P2₁/c space group. The compound is characterized by the protonation of the N6 atom in one of the two pyrazole rings (N7−N6−C27−C26−C24) to form a NH⁺ group giving a residual positive charge. This phenomenon allows the interaction with a tetrahedral counterion FeCl₄ ⁻, forming an organic–inorganic ionic pair in the asymmetric unit with the Fe(III) surrounded by four Cl⁻. This sort of interactions has been already reported in similar compounds such as [(HL1)₂(ZnCl₄)].H₂O, [(HL1)₂(CuCl₄)], [(HL2)₂SnCl₆], and [(HL3)FeCl₄] (with L1 = 2-methylquinoline, L2 = 6-bromobenzo[d]thiazole-2-amine, and L3 = 5,7-dimethyl-1,8-naphthyridine-2-amine) (Jin and Wang 2012)

FIG. 1.

(a) The molecular structure of compound 5 showing anisotropic displacement ellipsoids drawn at the 20% probability level, and (b) the Hirshfeld surface mapped over d_norm . The contact points are labeled to indicate the atoms participating in the intermolecular interactions.

The molecular structure contains a central six-membered ring with trans five-membered ring substituents (with a dihedral angle of 49.7(3)° between them). The dihedral angles between each pyrazole ring and the central benzene ring are 84.7(2)° for the (N17−N18−C19−C21−C22) ring and 85.2(3)° for the (N7−N6−C27−C26−C24) ring. The FeCl₄ ⁻ counterion is in a tetrahedral arrangement and has an average volume of 5.394 Å³ and a mean tetrahedral quadratic elongation of λ = 1.000, which is characteristic of a nondistorted polyhedron (Robinson et al. 1971). The lack of distortion was confirmed by the observed Fe−Cl distances of Fe1−Cl2 2.2029(15), Fe1−Cl3 2.1777(15), Fe1−Cl4 2.1933(16), and Fe1−Cl5 2.190(2), which are almost equal.

The supramolecular assembly is mainly controlled by N6−H6···N18ⁱ (symmetry code: (i) x,1/2-y,1/2+z) hydrogen bonds with an H···N distance of 1.82(5)Å. However, the weaker interactions of C(16, 10)−H(16A, 10)···Cl(14, 13), with H···Cl distances of 2.83 Å and 2.85 Å, respectively, help to define the three-dimensional array. The N−H···N hydrogen bond is also observed in the Hirshfeld (HF) surface analysis performed using Crystal Explorer 3.1 software (Wolff et al. 2012) mapped over d_norm (analysis of the contact distances (d_i and d_e ) from the HF surface to the nearest atom inside and outside, respectively), where the bright red spots indicate the intermolecular interactions (Fig. 1b). A weak red spot was observed over the aromatic C−H···Cl interaction, indicating the weakness of the interaction.

In vitro anti-TCE activity

Metal azole complexes have shown antimicrobial and antiparasitic activities (Keshia et al. 2016, Hurtado et al. 2017). However, the efficacy of these compounds against TCEs has not been studied. In this work, we tested the anti-TCE activities of the ligands, metal complexes, and compounds derived from azole at different concentrations. The results showed that the free ligands do not have anti-TCE activity, probably because the azoles have higher antifungal activities since they inhibit some of the steps in the synthesis of ergosterol in fungal cell walls (Van den Bossche et al. 1983, Herbrecht 2014). While the RM of larval eggs was low after exposure to different concentrations of compounds 1–5 (Fig. 2), with the copper complex (2), lower mobilities were observed at 15 μM, which is a lower concentration than was required to achieve the same result with piperazine. In general, the anti-TCE effects were concentration dependent.

FIG. 2.

RMs of parasites within the egg exposed to compounds 1–5. RM, relative mobility.

This result is likely associated with the superior solubility, bioavailability, and contact with DNA through intermolecular interactions (An et al. 2008, Patel and Patel 2010). In addition, it is promising that increasing the lipophilicity of compounds lowers the permeability barrier of the TCEs and slows the normal cellular activity of the microorganisms, causing an increase in antiparasitic bioactivity.

The efficacy of the combination of copper oxide nanoparticles with ABZ has been shown; the enhanced synergistic cytotoxicity of the nanocomposites against nematodes resulted in a greater amount of DNA fragmentation than was caused by the free ABZ; and this fragmentation increases the generation of ROS and consequently apoptosis (Zafar et al. (2016). With 2, the cuticle of the larvae was degraded (Fig. 3).

FIG. 3.

Morphology of parasites within the egg (a) without exposure to the compounds and (b) after exposure to 2 (15 μM).

The morphology of the TCEs was another characteristic that was considered in this study. The control test showed a TCE with intact structures (Fig. 3a), and clear delineation, homogeneity, and differentiation were observed in each organism.

The TCEs exposed to compounds 1–5 were characterized by differences in their initial ovoid geometry, structural heterogeneity, and component variation of their external constituents; a representative example is shown in Figure 3b.

These observed structural changes may be due to the presence of the azole complexes since they inhibit some of the steps in the synthesis of tubulin in parasite cell walls. The exact mechanism by which the metal azole complexes generate this anti-TCE activity remains unclear.

Cytotoxicity

The cytotoxicities of these compounds toward hepatocytes cells were evaluated. In the cytotoxicity studies, the compounds did not show notable cytotoxicity in the first 24 h (Fig. 4). The viability of the hepatocytes was always higher than 60% for all compounds independent of the concentration.

FIG. 4.

Cytotoxicity of 1–5 toward hepatocytes. The figure shows the percentage of cell viability after exposure to different concentrations of the compounds.

This is the first time metal compounds derived from azole have been used against TCEs. Interestingly, the compounds did not show signs of cytotoxicity against the murine cell line; therefore, they could be potential anti-TCE substances.

Conclusion

In summary, we have synthesized and characterized a new organic salt derived from azole. The effects of this salt and four metal complexes derived from azole on the larval motility of Toxocara canis eggs (TCEs) were evaluated. The in vitro assays showed that the compounds in general have moderate anti-TCE activities. The low motility of the larvae and morphological damage indicated that compound 2 had more potent anti-TCE activity. In addition, the compounds showed little effect on hepatocytes, indicating that they are not cytotoxic. The metal complexes derived from azole are promising as lead compounds for the development of disinfectant solutions that can reduce environmental contamination by Toxocara canis eggs (TCE). Additional studies must be carried out to modify their structures and test them against the parasite in other growth stages to define the role of these compounds against various parasites.

Footnotes

Acknowledgments

J. Hurtado wishes to thank the Universidad de los Andes and the science faculty and Chemistry Department for providing funding. K. Vazquez wishes to thank the Universidad Autónoma de Nuevo Leon and Facultad de Medicina Veterinaria y Zootecnia for providing funding.

Ethical Approval

This article does not contain any studies with human participants performed by any of the authors.

Database Linking

CCDC 1565069 contains the supplementary crystallographic data for C₁₉H₂₅Cl₄FeN₄. These data can be obtained free of charge from the Cambridge Crystallographic Data Centre, Cambridge, UK.

Author Disclosure Statement

No conflicting financial interests exist.

References

Allen

, Wilson

, Drew

, Perfect

. Azole antifungals: 35 years of invasive fungal infection management. Expert Rev Anti Infect Ther, 2015; 13:787–798.

C-X

, Han

X-L

, Wang

P-B

, Zhang

Z-H

, et al. Synthesis, crystal structures, and biological activities of silver(I) and cobalt(II) complexes with an azole derivative ligand. Transition Met Chem, 2008; 33:835–842.

Andrea

, Sandoval-Rojas

, Laura Ibarra

, María Teresa Cortés

, et al. Synthesis and Characterization of a New Copper(II) polymer containing a thiocyanate bridge and its application in dopamine detection. Inorganica Chim Acta, 2017; 459:95–102.

Camilo

RÑ

, Selene

, Germán

, Lilia

, et al. Contamination and viability of eggs of Toxocara spp. in soil and feces collected from public parks, streets and dogs in Toluca. Rev Cient FCV-LUZ, 2013; 23:475–479.

Castillo

, Bello-Vieda

, Nuñez-Dallos

, Pastrana

, et al. Metal complex derivatives of Azole: A study on their synthesis, characterization, and antibacterial and antifungal activities. J Braz Chem Soc, 2016; 27:2334–2347.

Cheng

, Li

, Zang

, Lang

. Dichloro[(3,5-dimethyl-1H-pyrazol-1-yl)methane]zinc(II) and di-μ-chloro-bis{chloro[(3,5-dimethyl-1H-pyrazol-1-yl)methane]cadmium(II)}. Acta Cryst, 2006; C62:m74–m77.

Divyamol

, Jeyathilakan

, Abdul Basith

, Senthilkumar

. In vitro production of Toxocara canis excretory-secretory (TES) antigen. J Parasit Dis, 2016; 40:1038–1043.

Gammal

, Bekheit

, Tahoon

. Synthesis, Characterization and Biological Activity of 2-Acetylpyridine-α-Naphthoxyacetylhydrazone Its Metal Complexes. Spectrochim Acta A Mol Biomol Spectrosc, 2015; 135:597–607.

Herbrecht

. Voriconazole: Therapeutic review of a new azole antifungal. Expert Rev Anti Infect Ther, 2014; 4:485–497.

10.

Hurtado

, Ibarra

, Yepes

, García-Huertas

, et al. Synthesis, crystal structure, catalytic and anti-Trypanosoma cruzi activity of a new chromium(III) complex containing bis(3,5- dimethylpyrazol-1-yl)methane. J Mol Struct, 2017; 1146:365–372.

11.

Hurtado

, Mariana

Portaluppi

, Raúl

Quijada

, Rene

Rojas

, et al. Synthesis, characterization, and reactivity studies in ethylene polymerization of cyclometalated palladium(II) complexes containing terdentate ligands with N,C,N-donors. J Coord Chem, 2009; 62:2772–2781.

12.

Jin

, Wang

. Syntheses and molecular structures of four inorganic–organic hybrid compounds from N-containing aromatic bases and perchlorometallates of Zn, Cu, Sn, and Fe. J Coord Chem, 2012; 65:3188–3204.

13.

Keshia

, Nestor

, Nelson

, Homero

, et al. Metal complex derivatives of Azole: A study on their synthesis, characterization, and antibacterial and antifungal activities. J Braz Chem Soc, 2016; 27:1–14.

14.

Kiuchi

, Miyashita

, Tsuda

, Kondo

, et al. Studies on crude drugs effective on Visceral Larva Migrans. I. Identification of larvicidal principles in betel nuts. Chem Pharm Bull, 1987; 35:2880–2886.

15.

Machura

, Palion

, Penkala

, Gron

, et al. Thiocyanate manganese(II) and cobalt(II) complexes of bis(pyrazol-1-yl)methane and bis(3,5-dimethylpyrazol-1-yl)methane–Syntheses, spectroscopic characterization, X-ray structure and magnetic properties. Polyhedron, 2013; 56:189–199.

16.

Magaña

, Luna

, Barrera

, Orta de Velásquez

, et al. Effect of mineral aggregates on the morphology and viability of Toxocara canis eggs. Ecol Eng, 2016; 90:125–134.

17.

Mun

, Cho

, Kim

, Oh

, et al. Inactivation of Ascaris eggs in soil by microwave treatment compared to UV and ozone treatment. Chemosphere, 2009; 77:285–290.

18.

Nagles

, Ibarra

, Penagos

, Hurtado

, et al. Development of a novel electrochemical sensor based on cobalt(II) complex useful in the detection of dopamine in presence of ascorbic acid and uric acid. J Electroanal Chem, 2017; 788:38–43.

19.

Patel

, Patel

. Synthesis, Characterization, Metal Complexation Studies and Biological screening of some Newly Synthesized Metal Complexes of 1-(4-carboxy-3-hydroxy-N-isopropyl phenyl amino methyl) benzotriazole with Some Transition Metals. Int J Chem Tech Res, 2010; 2:1147–1152.

20.

Pawlowski

. Toxocariasis in humans: Clinical expression and treatment dilemma. J Helminthol, 2001; 75:299–305.

21.

Potapov

, Khlebnikov

. Synthesis of mixed-ligand copper(II) complexes containing bis(pyrazol-1-yl)methane ligands. Polyhedron, 2006; 25:2683–2690.

22.

Reedijk

, Jan Verbiest

. Coordination compounds derived from transition metal salts and bis(3,5-dimethylpyrazolyl)methane. Transition Met Chem, 1979; 4:239–243.

23.

Reis

, Trinca

, Ferreira

, Monsalve-Puello

, et al. Toxocara canis: Potential activity of natural products against second-stage larvae in vitro and in vivo. Exp Parasitol, 2010; 126:191–197.

24.

Repetto

, Del Peso

, Zurita

. Neutral red uptake assay for the estimation of cell viability/cytotoxicity. Nat Protoc, 2008; 3:1125–1131.

25.

Robinson

, Gibbs

, Ribbe

. Quadratic elongation: A quantitative measure of distortion in coordination polyhedra. Science, 1971; 172:567–570.

26.

Sheldrick

. Crystal structure refinement with SHELXL. Acta Cryst, 2015; C71:3–8.

27.

Tiersch

, Das

, Samson-Himmelstjerna

, Gauly

. The role of culture media on embrionation and subsequent infectivity of Capillaria obsignata eggs . Parasitol Res, 2013; 112:357–364.

28.

Van den Bossche

, Willemsens

, Cools

, Marichal

, et al. Hypothesis on the molecular basis of the antifungal activity of N-substituted imidazoles and triazoles. Biochem Soc Trans, 1983; 11:665–667.

29.

Wolff

, Grimwood

, McKinnon

, Turner

, et al. (2012) Crystal Explorer 3.0. University of Western Australia, Perth.

30.

Zafar

, Ahmad

. Copper(II) oxide nanoparticles augment antifilarial activity of Albendazole: In vitro synergistic apoptotic impact against filarial parasite Setaria cervi . Int J Pharm, 2016; 501:49–64.

31.

Zaridah

, Idid

, Omar

, Khozirah

. In vitro antifilarial effects of three plant species against adult worms of subperiodic Brugia malayi . J Ethnopharmacol, 2001; 78:79–84.

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