Introduction

Parasitic worm infections, or helminthiasis, represent a substantial but often overlooked global health burden. Affecting over 2 billion individuals, primarily in tropical and subtropical regions, these infections disproportionately impact impoverished and marginalized populations with limited access to clean water and sanitation1. Helminthiasis contributes significantly to morbidity and mortality, and many emerging infectious diseases of parasitic origin are zoonotic, further complicating control efforts. Recognizing the urgent need for intervention, the World Health Organization (WHO) has included helminthic diseases in its 2021 roadmap, aiming to eliminate neglected diseases by 20302. Despite ongoing efforts, the arsenal of effective anthelmintic agents remains limited3.

Schistosomiasis, caused by the intravascular trematode of the genus Schistosoma, stands out as one of the most significant parasitic diseases globally, particularly in terms of public health and economic impact. Affecting millions in Africa, the Middle East, South America, and the Caribbean, S. mansoni infection leads to chronic morbidity due to the formation of granulomas and fibrosis from trapped eggs in human tissues4. While praziquantel is the only drug currently used in mass treatment programs5, its limitations, such as reduced efficacy against juvenile parasites and the emergence of drug resistance6, have highlighted the need for alternative therapies7.

Similarly, Angiostrongylus cantonensis, the rat lungworm, presents a growing zoonotic threat8, causing eosinophilic meningitis and other neurological complications in humans9. This nematode, prevalent in mollusk hosts, has spread across various continents, including Africa, Southeast Asia, and the Americas, with cases often linked to travel and misdiagnosed due to the mild symptoms10. Despite its public health importance, there is no effective anthelmintic treatment for A. cantonensis infections, underscoring the need for new therapeutic options11,12.

Transition metal complexes, especially those involving bioactive ligands, have emerged as a promising class of compounds in the search for novel antiparasitic drugs13. These complexes offer impressive chemical diversity and versatility, which depend on the metal of choice, its oxidation state, the number and type of coordinating ligands, and specific magnetic and/or optical properties14. This versatility allows for the rational design of compounds tailored to interact with specific biological targets, addressing the limitations of traditional anthelmintics.

Phenanthrenes, secondary metabolites produced by plants, serve as the chemical backbone for several biologically active compounds15,16. One such example is 1,10-phenanthroline-5,6-dione (phendione), a phenanthrene-based compound, whose metal complexes have shown significant potential due to their unique coordination chemistry and interactions with biological macromolecules17,18. Phendione and its metal complexes have been investigated for their antimicrobial and anticancer properties19,20, establishing them as promising candidates for antiparasitic research21,22. However, their potential against helminths remains largely unexplored.

Given the limited efficacy of current anthelmintic treatments and the pressing need for novel agents, this study investigates the potential antiparasitic properties of phendione and its copper, [Cu(phendione)3](ClO4)2·8H2O – Cu-phendione, and silver, [Ag(phendione)2](ClO4) – Ag-phendione, complexes. We evaluate their in vitro effects on S. mansoni and A. cantonensis and further explore their activity using Caenorhabditis elegans, a model organism for drug discovery. To assess their potential as therapeutic agents, we also performed cytotoxicity assays using Vero cells to determine their selectivity and safety profiles.

Results

The anthelmintic activities of phendione and its metal complexes (Cu-phendione and Ag-phendione) (Fig. 1) were evaluated alongside standard control drugs: praziquantel for S. mansoni, albendazole for A. cantonensis, doxorubicin for cytotoxicity in Vero cells, and ivermectin for C. elegans. Additionally, the simple metal salts AgNO₃ and CuSO₄·5 H₂O were included for comparison (Tables 1and Fig. 2).

Fig. 1
figure 1

Chemical structures of 1,10-phenanthroline-5,6-dione (phendione) and its metal complexes: Cu(phendione)₃(ClO₄)₂·8 H₂O (Cu-phendione) and [Ag(phendione)2](ClO4) (Ag-phendione).

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Table 1 Anthelmintic activity and toxicity of phendione and its metal-based complexes compared to standard drugs.
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Fig. 2
figure 2

Viability of adult Schistosoma mansoni worms after incubation with different concentrations of phendione and its metal complexes (Cu-phendione and Ag-phendione). Control groups included parasites incubated in drug-free medium, praziquantel (PZQ), metal salts (AgNO₃ and CuSO₄·5 H₂O), and 1% DMSO. Schistosomes were obtained from animals by perfusion 49 days post-infection. Each concentration was tested in triplicate, and worm viability was monitored for up to 72 h. Data represent the mean ± S.D. from at least three independent experiments performed in triplicate.

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Antiparasitic activity against Schistosoma mansoni

Cu-phendione exhibited the highest potency, with an EC50 of 2.3 µM against S. mansoni, outperforming both phendione (EC50 = 18.8 µM) and Ag-phendione (EC50 = 6.5 µM). While praziquantel remained the most effective control drug (EC50 = 1.2 µM), the potency of Cu-phendione was comparable. In contrast, the simple metal salts AgNO₃ and CuSO₄·5 H₂O showed no antischistosomal activity (Table 1). The negative control group (1% DMSO) displayed no significant alterations, with worms remaining motile throughout the entire incubation period (Fig. 2).

Antiparasitic activity against Angiostrongylus cantonensis L1 larvae

Cu-phendione exhibited strong activity against A. cantonensis (EC50 = 6.4 µM), outperforming phendione (EC50 = 25.6 µM) and demonstrating similar efficacy to albendazole (EC50 = 10.7 µM). Ag-phendione also showed good efficacy (EC50 = 12.7 µM) but was less active than Cu-phendione (Table 1). In contrast, the simple metal salts AgNO₃ and CuSO₄·5 H₂O showed no activity against A. cantonensis. Phendione treatment significantly reduced larval motility compared to the control group, while DMSO-treated larvae maintained normal activity throughout the assay (Fig. 3).

Fig. 3
figure 3

Viability of Angiostrongylus cantonensis L1 larvae during incubation with different concentrations of phendione and its metal complexes (Cu-phendione and Ag-phendione). Control groups included larvae incubated in drug-free medium, albendazole (ALB), metal salts (AgNO₃ and CuSO₄·5 H₂O), and 1% DMSO. Each concentration was tested in triplicate, and larval viability was monitored for up to 24 h. Data represent the mean ± S.D. from at least three independent experiments performed in triplicate.

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Toxicity and selectivity index

None of the tested compounds exhibited cytotoxicity in Vero cells at concentrations up to 200 µM (Fig. 4). In comparison, doxorubicin had a CC50 value of 9.6 µM, indicating that phendione and its metal complexes have significantly lower toxicity. The selectivity index (SI) values for Cu-phendione were particularly noteworthy, with SI > 86.9 for S. mansoni and SI > 31.2 for A. cantonensis, indicating high therapeutic potential. Ag-phendione also showed favorable SI values, with SI > 307 for S. mansoni and SI > 15.5 for A. cantonensis (Table 1).

Fig. 4
figure 4

Toxicity of phendione and its metal complexes (Cu-phendione and Ag-phendione) on Vero cells and Caenorhabditis elegans. Doxorubicin and ivermectin were used as reference drugs, while 1% DMSO-treated cells or C. elegans served as controls. Each concentration was tested in triplicate, with viability monitored over 72 h. Data represent the mean ± S.D. from at least three independent experiments performed in triplicate.

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The compounds were further evaluated for toxicity in C. elegans (Fig. 4). Cu-phendione exhibited an LD50 of 280 µM, significantly lower than ivermectin (LD50 = 4.1 µM), indicating lower toxicity. Similarly, Ag-phendione showed an LD50 of 167 µM, while phendione demonstrated the least toxicity (LD50 = 719 µM) (Table 1). These results suggest selective toxicity of the metal complexes toward parasitic helminths while minimizing the risk to non-target organisms.

Discussion

This study highlights the potent antiparasitic activity of phendione and its metal complexes, particularly Cu-phendione and Ag-phendione, against S. mansoni and A. cantonensis. These results emphasize the therapeutic potential of metallodrugs in addressing parasitic infections, especially considering the limitations of existing anthelmintic treatments. Importantly, the tested compounds exhibited selective toxicity, showing minimal impact on non-parasitic organisms like C. elegans and low cytotoxicity in Vero cells.

Among the tested compounds, Cu-phendione demonstrated the highest potency, with EC50 values comparable to those of praziquantel for S. mansoni and albendazole for A. cantonensis. Its enhanced efficacy compared to Ag-phendione aligns with previous studies indicating that copper complexes exhibit superior antiparasitic activity, such as against Trichomonas vaginalis21 and Leishmania braziliensis23. Notably, the inactivity of simple metal salts (AgNO₃ and CuSO₄·5 H₂O) reinforces the importance of metal-ligand coordination in optimizing biological activity.

The enhanced performance of Cu-phendione may be attributed to its involvement in parasite metabolism and oxidative stress, as previously reported in studies with S. mansoni24. Recent research has also identified peptidases as potential targets of Cu-phendione in Leishmania23 and T. vaginalis25, while metabolic disruption and altered membrane potential have been observed in Leishmania species treated with Cu-phendione22. These findings suggest that the coordination between the metal center and phendione enhances interactions with key biological targets, increasing the parasites’ susceptibility to treatment. Additionally, oxidative stress response signaling pathways may contribute to nematode death26,27. Further investigations are needed to elucidate the precise mechanisms underlying these interactions and optimize the design of future metallodrugs.

The schistosomicidal activity observed in this study surpasses many plant-derived products reported in the literature. For example, EC50 values reported for S. mansoni adult worms include 12.5 µM for verrucosin28, 26.1 µM for diterpene ent-kaur-16-en-19-oic acid29, 30 µM for neolignan licarin A30, 31.9 µM for dehydrodieugenol B31, 42.16 µM for carvacryl acetate32, 50 µM for cnicin33, licochalcone A34, 2-oxopopulifolic acid methyl ester, and 2-oxopopulifolic acid35, 56.8 µM for monoterpene carvacrol36, and 81.8 µM for flavonoid kaempferol37. Compared to the aforementioned compounds, which are obtained directly from plants, phendiones are obtained by synthesis and are easily obtained in large quantities. The superior performance of phendione-based complexes in this study underscores their potential as more effective alternatives to many of these plant-derived compounds. Additionally, unlike the aforementioned plant-derived compounds, which require extraction and purification processes, phendiones are synthetically produced and can be easily obtained in large quantities, making them more scalable and cost-effective for therapeutic applications.

In addition to their potent antiparasitic activity, the metal complexes exhibited high selectivity. The EC50 values were more than 40 times higher in C. elegans than in parasitic worms, demonstrating selective targeting. Furthermore, the compounds showed low cytotoxicity in Vero cells, with selectivity indices exceeding 86.9 for S. mansoni and 31.2 for A. cantonensis, reinforcing their favorable safety profiles. These findings align with previous studies reporting minimal toxicity of phendione derivatives in various cell lines21,38 and low toxicity in Galleria mellonella larvae38,39, as well as in mammalian models, including mice38 and hamsters40.

Despite the absence of toxicity observed in in vitro and in vivo models with phendione and its metal complexes, it is important to acknowledge that metal-based drugs also present certain limitations that must be addressed to realize their full therapeutic potential. These include concerns about potential toxicity, as metals can accumulate in tissues and cause adverse effects41. Furthermore, the stability of metal-based complexes under physiological conditions and their interactions with off-target biomolecules may compromise their efficacy and safety41,42. Future research should prioritize addressing these limitations through careful optimization of metal-ligand combinations, the development of targeted delivery systems, and comprehensive safety evaluations to ensure their viability as alternative therapeutics for parasitic diseases.

Finally, future research should focus on in vivo studies to validate the efficacy, pharmacokinetics, and safety of these compounds. Investigating potential synergistic effects with established drugs, such as praziquantel or albendazole, could further enhance therapeutic outcomes. Additionally, understanding the mechanisms of action in greater detail may provide insights for developing new metallodrugs with improved selectivity and efficacy. Such advancements are particularly critical in addressing the growing challenge of drug resistance in parasitic infections. Metal-based drugs, with their unique properties and diverse mechanisms, offer a promising alternative to overcome resistance issues, as they can target different biological pathways compared to traditional therapies. This versatility underscores their potential as a valuable addition to the arsenal against drug-resistant parasitic diseases.

Conclusions

This study demonstrates the potent antiparasitic activity of phendione and its metal complexes, particularly Cu-phendione, against two major helminth species. The selective toxicity observed, combined with low cytotoxicity in mammalian cells, underscores the therapeutic promise of these compounds. These findings provide a strong foundation for further in vivo studies and preclinical development, offering new opportunities to expand the limited arsenal of anthelmintic agents. Moreover, exploring synergistic combinations with existing drugs could enhance efficacy and mitigate drug resistance, contributing to more effective treatment strategies for parasitic infections.

Methods

Compounds

1,10-Phenanthroline-5,6-dione (phendione) and its metal complexes, [Ag(phendione)₂]ClO₄ (Ag-phendione) and Cu(phendione)₃(ClO₄)₂·8 H₂O (Cu-phendione) (Fig. 1), were synthesized following the protocols previously described43. Details of the synthesis and characterization of the compounds are provided in the Supplementary Information. These compounds were dissolved in dimethyl sulfoxide (DMSO) and stored at 4 °C until use. Praziquantel, albendazole, and ivermectin were generously provided by Ecovet Indústria Veterinária Ltda (São Paulo, Brazil), while doxorubicin was purchased from Sigma-Aldrich (St. Louis, MO, USA).

Maintenance of parasitic worms

The life cycle of Schistosoma mansoni (Belo Horizonte strain) was maintained at the Research Center on Neglected Diseases, Guarulhos University, using Biomphalaria glabrata snails as the intermediate host and Swiss mice (Mus musculus) as the definitive host. Similarly, the Angiostrongylus cantonensis (NPDN-AC strain) life cycle was maintained by alternating between B. glabrata snails and Wistar rats (Rattus norvegicus). All animals were housed under controlled conditions (22 °C, ~ 50% humidity) with ad libitum access to food and water.

In vitro assay with adult S. mansoni

Adult S. mansoni worms were obtained by hepatic portal vein perfusion from Swiss mice, 49 days post-infection, following established protocols44,45. The worms were washed in RPMI 1640 medium with antibiotics and transferred to 24-well plates, with one pair of adult worms per well. Each well contained 1 mL of RPMI 1640 medium enriched with 10% fetal bovine serum, penicillin (100 U/mL), and streptomycin (100 µg/mL).

Test compounds, praziquantel, and metal salts (AgNO₃ and CuSO₄·5 H₂O) were added at concentrations starting from 50 µM. DMSO (1%) served as the negative control. Plates were incubated at 37 °C with 5% CO₂ for up to 72 h, and worm viability was assessed at 0, 24, 48, and 72 h using an inverted microscope. Parasite death was determined by the absence of movement for at least one minute upon gentle mechanical stimulation46.

In vitro assay with A. cantonensis L1 larvae

First-stage larvae (L1) of A. cantonensis were isolated from the feces of Wistar rats, using the Rugai technique, and washed in RPMI 1640 medium with antibiotics. Approximately 100 larvae per well were transferred to 96-well plates12. The compounds, praziquantel, and metal salts were tested at concentrations starting from 50 µM, with incubation at 21 °C. Larval viability was assessed at 0 and 24 h under an inverted microscope. Movement was categorized as immobile, intermittent, slow, or highly active. Efficacy was defined as at least 60% of larvae becoming immobile within 24 h11,12.

Lethal toxicity assay in C. elegans

The Bristol N2 strain of Caenorhabditis elegans was cultured on nematode growth medium (NGM) plates seeded with Escherichia coli OP5047. L4-stage larvae were synchronized and transferred to 96-well plates containing M9 medium, with 60 larvae per well48. The compounds and ivermectin were tested at concentrations starting from 1,000 µM, with 1% DMSO as the negative control. Viability was assessed after 24 h of incubation at 21 °C by observing movement30. Lethal toxicity was defined as 60% or more of the larvae exhibiting complete immobility49.

Cytotoxicity assay in vero cells

Vero cells (ATCC CCL-81) were maintained in DMEM medium supplemented with 10% fetal bovine serum, penicillin (100 U/mL), and streptomycin (100 µg/mL) at 37 °C with 5% CO₂. The cells were seeded into 96-well plates at 2 × 10³ cells per well and incubated with the test compounds or doxorubicin at concentrations starting from 200 µM, with 1% DMSO as the negative control. Notably, the 200 µM concentration represents the maximal solubility of the compounds under the assay conditions and adheres to standard practices for selectivity index calculations50. Additionally, 1% DMSO was confirmed to be non-toxic to the cells. Cytotoxicity was assessed using the MTT assay51,52. After 72 h, MTT solution was added, followed by 3 h of incubation. Absorbance was measured at 595 nm using an Epoch Microplate Spectrophotometer (BioTek Instruments, Winooski, VT, USA). Assays were performed in triplicate and repeated three times. Selectivity index (SI) values were calculated as the ratio of the 50% cytotoxic concentration (CC50) in Vero cells to the 50% effective concentration (EC50) in the parasitic helminths53.

Data analysis

Statistical analyses were conducted using GraphPad Prism 8.0. EC50, CC50, and LD50 values were determined through sigmoidal dose-response curves48,54. Differences between groups were analyzed using one-way ANOVA followed by Tukey’s post-hoc test, with statistical significance set at P < 0.05.