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. 2026 Oct 1;11(40):61199–61207. doi: 10.1021/acsomega.6c05944

4‑O‑Allyl Derivative of Natural Licarin A Exhibits Selective In Vitro Activity against Toxoplasma gondii Tachyzoites

Gabriel Candido Moura †, Stephanie Ortega Alves †, Ingrid de Oliveira Dias †, João Paulo dos Santos Fernandes ‡, Daniela da Costa Tristão §, João Henrique Ghilardi Lago §,*, Juliana Quero Reimão †,*
PMCID: PMC13670340  PMID: 42889921

Abstract

Toxoplasma gondii is an obligate intracellular protozoan responsible for toxoplasmosis, a widespread parasitic disease that poses a serious health threat, especially to immunocompromised patients and pregnant women. Current therapeutic options remain unsatisfactory because they are associated with adverse effects and do not eliminate latent infections, emphasizing the need for new antitoxoplasmic agents. In the present study, the natural neolignan (−)-licarin A (1), isolated from the Brazilian species Nectandra oppositifolia, together with three semisynthetic derivatives, 4-O-methyl (1a), 4-O-acetyl (1b), and 4-O-allyl (1c), were investigated for their in vitro activity against T. gondii tachyzoites. Antiparasitic activity (EC50) was determined using a β-galactosidase-based assay, while cytotoxicity (CC50) toward Human Foreskin Fibroblasts (HFF) was assessed by the resazurin assay. Among the tested compounds, derivative 1c exhibited the highest potency (EC50 = 3.49 μM) and lowest cytotoxicity (CC50 = 120.10 μM), resulting in the highest selectivity index (SI = 34.41). In silico ADMET analysis predicted favorable gastrointestinal absorption and blood–brain barrier permeability for all licarin derivatives. Mechanistic assays demonstrated that licarin A derivatives 1a–1c did not compromise parasite plasma membrane integrity but significantly reduced mitochondrial membrane potential, supporting an intracellular mode of action. Further functional analyses revealed that compound 1c inhibited intracellular parasite proliferation in a concentration-dependent manner and maintained significant antiparasitic activity after drug removal only at the highest concentration tested, without affecting parasite invasion or intracellular replication following pretreatment of extracellular tachyzoites. Overall, these findings identify the 4-O-allyl derivative of licarin A as the most selective compound of this series and provide additional evidence that semisynthetic modification of the licarin scaffold represents a promising strategy for discovering new therapeutic candidates against T. gondii.

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Introduction

Toxoplasma gondii is an obligate intracellular protozoan parasite with worldwide distribution and is the etiological agent of toxoplasmosis, one of the most prevalent parasitic infections in humans, affecting approximately one-third of the global population. In healthy individuals, infection generally remains asymptomatic or manifests as a mild, self-limiting illness. However, severe clinical outcomes may occur in immunocompromised patients, particularly following reactivation of latent tissue cysts, which can result in toxoplasmic encephalitis and other life-threatening complications. In addition, congenital toxoplasmosis remains a significant clinical challenge, as transplacental transmission can result in miscarriage, severe fetal abnormalities, and long-term neurological or ocular sequelae.

Despite its high prevalence and clinical relevance, the therapeutic options for toxoplasmosis remain limited and have changed little over the past decades. The standard therapeutic regimen combines pyrimethamine with sulfadiazine to inhibit folate biosynthesis in the parasite. However, this regimen is associated with important drawbacks, including significant toxicity, such as bone marrow suppression and hypersensitivity reactions, as well as poor tolerability in prolonged treatments. Importantly, these drugs are ineffective against the latent bradyzoite stage, precluding complete parasite clearance and allowing for disease reactivation. In this scenario, the identification of new compounds that are safer, more effective, and capable of targeting multiple parasite stages remains an urgent unmet need.

Natural products continue to represent an invaluable source of structurally diverse molecules for drug discovery, including the identification of new antiparasitic agents. Several clinically important drugs, such as quinine and artemisinin, originated from plant-derived compounds, highlighting the potential of natural scaffolds as sources of therapeutic agents. In this context, the exploration of plant secondary metabolites represents a rational strategy to identify molecules capable of interfering with essential and often parasite-specific metabolic pathways. Among these natural compounds, lignans and neolignans have attracted increasing attention due to their broad spectrum of biological activities, including antimicrobial and antiparasitic effects. − Within this class, the dihydrobenzofuran neolignan (−)-licarin A (1) has demonstrated promising activity against a variety of parasites, including Leishmania spp., Trypanosoma cruzi, and Schistosoma mansoni. ,, Semisynthetic derivatives of licarin A have also shown improved antiparasitic activity and selectivity against protozoan parasites compared with the parent compound, highlighting the potential of structural modifications to optimize the biological properties of this scaffold. In particular, the 4-O-allyl derivative exhibited enhanced activity against T. cruzi, whereas other derivatives have demonstrated potent activity against Leishmania infantum by inducing alterations in calcium homeostasis and mitochondrial bioenergetics. ,

More recently, a structurally related neolignan, licarin B, was reported to inhibit the proliferation of T. gondii tachyzoites by inducing mitochondrial dysfunction, autophagosome formation, and nuclear disorganization. , Although these findings support the potential of licarin-based compounds as anti-T. gondii agents, the antiparasitic activity of semisynthetic derivatives of licarin A against this parasite has not yet been systematically investigated. Therefore, it remains unclear whether chemical modifications of the licarin A scaffold can improve its antitoxoplasmic activity and selectivity while preserving its favorable pharmacological properties.

Considering the urgent need for novel anti-T. gondii therapies and the promising bioactivity of neolignan derivatives, the present study aimed to evaluate the in vitro antiparasitic activity and cytotoxicity of the natural dihydrobenzofuran neolignan (−)-licarin A (1), isolated from Nectandra oppositifolia, together with three semisynthetic analogues, namely 4-O-methyl (1a), 4-O-acetyl (1b), and 4-O-allyl (1c), as shown in Figure . Following the initial screening, the derivative exhibiting the highest selectivity was further investigated through functional assays and mitochondrial membrane potential analysis to provide additional insights into its biological effects on T. gondii. By comparing the antiparasitic activity of these structurally related molecules, this study contributes to the identification of promising lead compounds for the development of new therapeutic strategies against toxoplasmosis.

1.

1

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Structures of (−)-licarin A (1) and derivatives 1a–1c.

Results

The predicted pharmacokinetic and toxicity profiles of (−)-licarin A (1) and derivatives 1a–1c are summarized in Table . All compounds are expected to display high gastrointestinal absorption together with the capacity to permeate the blood–brain barrier, suggesting favorable oral bioavailability and potential distribution to the central nervous system. Regarding toxicity, all licarin derivatives were predicted to present low mutagenic potential and no irritant effects. However, high probabilities of tumorigenic and reproductive effects were predicted for all derivatives. Compared with the reference drug pyrimethamine, the licarin derivatives displayed a similar GI absorption and BBB permeability profile, while exhibiting lower predicted mutagenic and reproductive toxicity.

1. Predicted Pharmacokinetic and Toxicity Properties of (−)-Licarin A (1), Derivatives 1a–1c, and Pyrimethamine .

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a

Green indicates favorable predicted properties, corresponding to high gastrointestinal (GI) absorption, blood–brain barrier (BBB) permeability, and the absence of predicted mutagenic, tumorigenic, irritant, or reproductive toxicity. Red indicates unfavorable predicted properties, corresponding to low GI absorption, lack of BBB permeability, or the presence of predicted toxicity risks.

The antiparasitic activity of (−)-licarin A (1) and derivatives 1a–1c was determined using a β-galactosidase reporter assay. Cytotoxicity toward human foreskin fibroblasts (HFF) host cells was assessed by the resazurin viability assay, as summarized in Table .

2. Half-Maximal Effective Concentration (EC50) against T. gondii Intracellular Tachyzoites and Half-Maximal Cytotoxic Concentration (CC50) of (−)-Licarin A (1) and Its Derivatives 1a–1c and the Reference Drug Pyrimethamine.

compound EC50 (μM) ± SD against T. gondii tachyzoites CC50 (μM) ± SD in human foreskin fibroblasts selectivity index (CC50/EC50)
1 6.97 ± 2.36 41.96 ± 14.28 6.02
1a 5.46 ± 1.74 55.19 ± 12.46 10.11
1b 7.29 ± 1.88 52.30 ± 10.31 7.17
1c 3.49 ± 0.94 120.10 ± 24.23 34.41
Pyrimethamine 0.37 ± 0.03 >50 >135
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All tested compounds exhibited antiparasitic activity, with EC50 values ranging from 3.49 to 7.29 μM. Although compound 1c displayed the lowest EC50 value (3.49 ± 0.94 μM), the remaining derivatives also showed comparable micromolar activity, with EC50 values varying between 5.46 and 7.29 μM. Among the derivatives, the 4-O-allyl derivative (1c) was the most potent, followed by the 4-O-methyl derivative (1a), whereas the 4-O-acetyl derivative (1b) exhibited activity comparable to that of the naturally occurring compound 1.

Cytotoxicity analysis revealed similar toxicity profiles for 1 and derivatives 1a and 1b, with CC50 values of 41.96 ± 14.28, 55.19 ± 12.46, and 52.30 ± 10.31 μM, respectively. In contrast, 1c exhibited a higher CC50 value (120.10 ± 24.23 μM), suggesting that the presence of the allyl group not only enhances antiparasitic activity but also reduces cytotoxicity. Consequently, the superior selectivity of compound 1c resulted primarily from its lower cytotoxicity combined with the lowest EC50 among the tested derivatives, yielding the highest selectivity index (SI = 34.41). Nevertheless, the reference drug pyrimethamine showed superior potency and selectivity, with an EC50 value of 0.37 ± 0.03 μM and a SI > 135.

To investigate whether compounds 1a–1c affect parasite plasma membrane integrity, propidium iodide uptake assays were performed. As shown in Figure A, treatment with digitonin (positive control) resulted in a time-dependent increase in fluorescence intensity, consistent with progressive membrane permeabilization. In contrast, tachyzoites treated with (−)-licarin A and derivatives 1a–1c displayed stable fluorescence levels over time, comparable to the DMSO-treated negative control, indicating preservation of membrane integrity. Quantitative analysis at 30 min (Figure B) confirmed these observations, revealing a significant increase in fluorescence exclusively in the positive-control group (p < 0.01), whereas none of the licarin-treated groups differed significantly from the negative control.

2.

2

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Effect of (−)-licarin A (1) and derivatives 1a–1c on T. gondii plasma membrane integrity. (A) Time-course analysis of propidium iodide uptake in tachyzoites treated with licarin A (1) and derivatives 1a–1c or digitonin at concentrations equivalent to their respective EC50 values. Fluorescence intensity was recorded every minute. (B) Quantification of fluorescence intensity after 30 min of treatment. Digitonin and DMSO were used as positive and negative controls for membrane permeabilization, respectively. Statistical significance was calculated relative to the DMSO-treated negative control. ** indicates p < 0.01.

Considering that plasma membrane integrity was preserved after compound treatment, mitochondrial membrane potential was evaluated to investigate whether licarin derivatives affect parasite mitochondrial function. As shown in Figure , treatment with the positive control antimycin A caused a marked reduction in MitoTracker Red fluorescence compared with the DMSO control (p < 0.001), confirming mitochondrial depolarization. Likewise, treatment with 1 and derivatives 1b and 1c at their respective EC50 values significantly decreased fluorescence intensity relative to the negative control (p < 0.05), indicating disruption of mitochondrial membrane potential. In contrast, no significant alteration was observed for the 1a derivative. These results demonstrate that selected licarin derivatives affect mitochondrial function without causing detectable plasma membrane damage.

3.

3

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Effect of (−)-licarin A (1) and derivatives 1a–1c on T. gondii mitochondrial membrane potential. Bar graph showing the fluorescence intensity of tachyzoites labeled with MitoTracker Red FM (100 nM) after treatment with 1 and derivatives 1a–1c at their respective EC50 values, or antimycin A (10 μM). DMSO (0.01%) was used as the negative control. Statistical significance was determined relative to the DMSO-treated control (**p < 0.05; ***p < 0.001).

Given its superior selectivity, compound 1c was further evaluated in functional assays assessing parasite proliferation, reversibility of drug effect, and host cell invasion. As shown in Figure A, compound 1c significantly inhibited tachyzoite proliferation at concentrations of 30, 15, and 7.5 μM compared with the DMSO-treated control (p < 0.001). No differences were observed at concentrations of 3.75 μM or lower, despite the expected biological variability among independent experiments.

4.

4

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Functional characterization of the antiparasitic effects of the 4-O-allyl derivative (1c) against T. gondii. (A) Concentration-dependent inhibition of intracellular parasite proliferation. (B) Evaluation of parasite recovery following 24 h exposure to the compound and subsequent incubation for 48 h in compound-free medium. (C) Host-cell invasion assay performed using extracellular tachyzoites pre-exposed to 1c for 1 h before infection. (D) Intracellular replication of parasites pretreated prior to host-cell infection. Statistical comparisons were performed using the DMSO-treated group as reference (***p < 0.001).

In the reversibility assay (Figure B), only the highest concentration tested (30 μM) resulted in a significant reduction in parasite recovery following drug removal (p < 0.001), indicating that exposure to high concentrations of compound 1c produced sustained antiparasitic effects after drug withdrawal. Lower concentrations did not differ significantly from the DMSO-treated control.

Pretreatment of extracellular tachyzoites with compound 1c did not significantly influence their ability to invade host cells (Figure C). Likewise, intracellular replication assessed 72 h after infection (Figure D) remained unaffected, indicating that transient exposure of extracellular parasites to the compound is insufficient to impair subsequent invasion or intracellular multiplication.

Discussion

Neolignans comprise a structurally diverse group of plant-derived metabolites that have attracted considerable interest because of their broad spectrum of biological activities, including activity against both helminth and protozoan parasites. − In this context, (−)-licarin A (1) and several derivatives have been previously reported as promising scaffolds against parasites such as S. mansoni, , T. cruzi, ,, Leishmania spp., , and the structurally related neolignan licarin B has demonstrated activity against T. gondii, supporting the exploration of this chemical class as a valuable source of lead compounds for antiparasitic drug discovery. The present study expands the findings by providing the first systematic evaluation of semisynthetic licarin A derivatives against T. gondii, demonstrating that all tested compounds display antiparasitic activity, while the 4-O-allyl derivative (1c) exhibits the most favorable balance between efficacy and host cell safety.

The observed EC50 values in the low micromolar range are consistent with those reported for licarin A derivatives 1a–1c against other protozoan parasites. For instance, these derivatives exhibited EC50 values between 9 and 13 μM against intracellular amastigotes of L. infantum, whereas the 4-O-allyl derivative previously showed improved activity against bloodstream forms of T. cruzi. Although compound 1c exhibited the lowest EC50 in the present study, the differences in antiparasitic potency among the evaluated derivatives were relatively modest. In contrast, the substantially lower cytotoxicity of compound 1c resulted in a markedly higher selectivity index, which ultimately supported its selection for subsequent mechanistic investigations. This finding reinforces previous structure–activity relationship studies indicating that semisynthetic modifications of the licarin scaffold can improve its biological profile without substantially altering its antiparasitic potency.

The in silico ADMET analysis further supports the potential of this chemical class for antiparasitic drug discovery. All licarin derivatives were predicted to possess high gastrointestinal absorption together with the ability to penetrate the blood–brain barrier, two desirable properties for compounds intended to treat cerebral toxoplasmosis. Moreover, the predicted absence of mutagenicity and irritant effects represents a favorable safety feature, although the predicted tumorigenic and reproductive risks highlight the need for comprehensive toxicological evaluation in future preclinical studies. These computational predictions should be interpreted cautiously, as they require experimental validation.

In addition to potency, selectivity toward the parasite over host cells is a critical parameter for drug development. 4-O-allyl licarin A (1c) exhibited a markedly higher selectivity index compared to the parent compound (−)-licarin A (1), 4-O-methyl (1a) and 4-O-acetyl (1b) derivatives. Although the reference drug pyrimethamine remains significantly more potent and selective, the selectivity index obtained for compound 1c is within a range considered promising for hit compounds in early stage drug discovery.

Mechanistically, the absence of plasma membrane permeabilization in tachyzoites treated with licarin derivatives indicates that their antiparasitic effect is not mediated by direct membrane disruption. This finding is consistent with previous reports in Leishmania, in which structurally related licarin derivatives also failed to induce plasma membrane damage. To further investigate their intracellular effects, mitochondrial membrane potential was evaluated in the present study. (−)-Licarin A (1), compound 1b, and particularly compound 1c significantly reduced MitoTracker fluorescence, indicating disruption of mitochondrial membrane potential. Together with the preserved plasma membrane integrity, these findings suggest that mitochondrial dysfunction represents an early cellular event associated with the antiparasitic activity of these compounds.

Our results are also consistent with previous mechanistic studies in other protozoan parasites. In T. cruzi, licarin-derived compounds induced mitochondrial depolarization, oxidative stress, and alterations in ATP homeostasis, whereas in L. infantum they promoted mitochondrial dysfunction associated with calcium imbalance and bioenergetic impairment. Likewise, licarin B was previously shown to induce mitochondrial damage, autophagosome formation, and nuclear disorganization in T. gondii. Although the present study did not investigate ROS production, ATP depletion, or ultrastructural alterations, the observed mitochondrial depolarization strengthens the evidence that mitochondria constitute an important intracellular target of licarin-based compounds across different protozoan parasites.

The lack of effect on host cell invasion observed in the present study further indicates that compound 1c does not impair early host–parasite interactions. Likewise, pretreatment of extracellular parasites did not affect their subsequent intracellular proliferation, suggesting that brief exposure to the compound before infection is insufficient to induce irreversible damage. In contrast, continuous treatment of infected cells significantly reduced parasite proliferation, indicating that compound 1c exerts its antiparasitic activity predominantly after parasite internalization. Furthermore, the reversibility assay demonstrated that only the highest concentration tested maintained significant inhibition following compound removal, indicating that the sustained antiparasitic effect depends on drug concentration and exposure time rather than representing a fully irreversible mechanism of action.

Taken together, these findings indicate that semisynthetic modification of the licarin scaffold preserves antiparasitic activity while improving selectivity and provides additional evidence supporting mitochondrial dysfunction as a relevant cellular target. Nevertheless, complementary studies evaluating reactive oxygen species production, ATP levels, ultrastructural changes, and molecular targets will be required to obtain a comprehensive understanding of the mechanism of action of these compounds.

Conclusions

Collectively, the present results demonstrate that natural (−)-licarin A (1) and its semisynthetic derivatives 1a–1c exhibit consistent in vitro activity against T. gondii tachyzoites. Although all compounds displayed similar micromolar antiparasitic activity, the 4-O-allyl derivative (1c) combined the lowest cytotoxicity with the highest selectivity index, making it the most promising candidate for further investigation.

Mechanistic assays showed that the antiparasitic activity was not associated with plasma membrane disruption but was accompanied by mitochondrial membrane depolarization, supporting mitochondrial dysfunction as a potential mechanism involved in parasite killing. In addition, the predicted pharmacokinetic profile suggests favorable gastrointestinal absorption and blood–brain barrier permeability, characteristics that may be advantageous for future development of antitoxoplasmosis agents, although these predictions require experimental confirmation.

Future studies should focus on elucidating the precise molecular targets in T. gondii, validating the mitochondrial effects through complementary biochemical approaches, and evaluating pharmacokinetic properties, safety, and in vivo efficacy to advance these compounds toward preclinical development.

Methods

General Experimental Procedures

Column chromatographic separations were carried out on silica gel 60, and reaction progress was monitored by thin-layer chromatography (TLC) on silica gel plates containing a fluorescent indicator, with spots visualized under UV light at 254 nm. Stock solutions (10 mM) were prepared in dimethyl sulfoxide (DMSO) and diluted in culture medium to the desired concentrations immediately prior to use. All additional reagents were purchased from Sigma-Aldrich unless otherwise stated. NMR spectra were recorded on a Bruker Avance 300 spectrometer operating at 300 and 75 MHz for the 1H and 13C nuclei, respectively, using CDCl3 as the solvent and TMS as the internal standard. Chemical shifts (δ) are reported in ppm, and coupling constants (J) in Hz. HRESIMS data were acquired using a Bruker Daltonics MicroTOF QII mass spectrometer. Optical rotations were measured with a JASCO P-2000 polarimeter equipped with a sodium filter (λ = 589 nm).

Plant Material

Fresh leaves of N. oppositifolia were collected in June 2023 in the municipality of Artur Nogueira, São Paulo State, Brazil, from the same specimen previously investigated. The voucher specimen was compared with the reference material deposited at the Herbarium of the Institute of Biosciences, University of São Paulo, São Paulo, Brazil, under accession number SPF225339.

Isolation of (−)-Licarin A from N. oppositifolia

After drying and pulverization, the leaves of N. oppositifolia (600 g) were exhaustively extracted with hexanes at room temperature. The combined extracts were concentrated under reduced pressure to yield 65 g of crude hexane extract. The extract was subjected to silica gel column chromatography, using hexanes with increasing proportions of EtOAc as the mobile phase, affording six fractions (I–VI). Fraction IV (6.2 g) was further purified by silica gel column chromatography, eluted with hexanes/EtOAc (7:3), to afford 2.7 g of (−)-licarin A.

(−)-Licarin A (1)

[α]D24 −63 (c 0.11, MeOH). 1H NMR (CDCl3), δ/ppm: 6.97 (d, J = 1.4 Hz, H-2), 6.92 (dd, J = 7.8 and 1.4 Hz, H-6), 6.91 (d, J = 7.8 Hz, H-5), 6.79 (br s, H-2′), 6.77 (br s, H-6′), 6.36 (dd, J = 15.7 and 1.6 Hz, H-7′), 6.11 (dq, J = 15.7 and 6.6 Hz, H-8′), 5.63 (s, OH), 5.11 (d, J = 9.4 Hz, H-7), 3.90 (s, OCH3), 3.89 (s, OCH3), 3.45 (dq, J = 9.4 and 6.8 Hz, H-8), 1.87 (dd, J = 6.6 and 1.6 Hz, H-9′), 1.38 (d, J = 6.8 Hz, H-9). 13C NMR (CDCl3), δ/ppm: 146.7 (C-3), 146.6 (C-4′), 145.8 (C-4), 144.2 (C-5′), 132.2 (C-1′), 132.1 (C-1), 130.9 (C-7′), 123.5 (C-8′), 120.0 (C-6), 114.1 (C-5), 113.3 (C-2′), 113.2 (C-3′), 109.3 (C-6′), 108.9 (C-2), 93.8 (C-7), 56.1 (OCH3), 56.0 (OCH3), 45.6 (C-8), 18.4 (C-9′), 17.6 (C-9). HRESIMS m/z 325.8384 [M – H]− (calcd for C20H21O4 325.1439).

Procedure for the Preparation of Compound 1a

Compound 1 (250 mg) was treated with K2CO3 (2 equiv) and CH3I (2 equiv) in acetone (10 mL) under reflux for 7 h. The solvent was then removed under reduced pressure, and the residue was extracted with CH2Cl2 and washed with H2O (2 × 25 mL). The organic phase was dried over anhydrous Na2SO4 and concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography using n-hexane/EtOAc (9:1) as the eluent to afford 1a as a white amorphous solid in 98% yield.

4-O-Methyl licarin A (1a)

1H NMR (CDCl3) δ/ppm: 6.98 (d, J = 1.7 Hz, H-2), 6.88 (dd, J = 7.8 and 1.7 Hz, H-6), 6.95 (d, J = 7.8 Hz, H-5), 6.86 (d, J = 1.8 Hz, H-6′), 6.80 (d, J = 1.8 Hz, H-2′), 6.36 (dd, J = 15.7 and 1.3 Hz, H-7′), 6.10 (dq, J = 15.7 and 6.5 Hz, H-8′), 5.11 (d, J = 9.4 Hz, H-7), 3.91 (s, OCH3), 3.90 (s, OCH3), 3.88 (s, OCH3), 3.45 (dd, J = 9.4 and 6.8 Hz, H-8), 1.86 (dd, J = 6.8 and 1.3 Hz, H-9′), 1.38 (d, J = 6.8 Hz, H-9). 13C NMR (CDCl3) δ/ppm: 149.1 (C-4), 149.0 (C-5′), 146.6 (C-6′), 144.2 (C-3), 133.3 (C-4′), 132.6 (C-6), 132.2 (C-6′), 130.9 (C-7′), 123.5 (C-8′), 119.2 (C-1), 113.3 (C-2), 110.8 (C-3′), 109.5 (C-1′), 108.9 (C-5), 93.6 (C-7), 55.9 (OCH3), 55.8 (OCH3), 55.9 (OCH3), 45.5 (C-8), 18.4 (C-9′), 17.6 (C-9). HRESIMS m/z 341.1772 [M + H]+ (calcd. for C21H25O4 341.1753).

Procedure for the Preparation of Compound 1b

Compound 1 (350 mg) was treated with Et3N (1 equiv) and acetyl chloride (2 equiv) in CH2Cl2 (5 mL) and stirred at 0 °C for 4 h. The reaction mixture was then treated with 20 mL of 10% aqueous NaHCO3, and the organic phase was separated and washed with H2O (2 × 25 mL). The organic layer was dried over anhydrous Na2SO4 and concentrated under reduced pressure. The residue was purified by silica gel column chromatography using n-hexane/EtOAc (9:1) as the eluent to afford 1b as a white amorphous solid in 27% yield.

4-O-Acetyl Licarin A (1b)

1H NMR (CDCl3) δ/ppm: 7.00 (br s, H-6′), 6.98 (br s, H-2′), 6.88 (br s, H-2), 6.80 (br s, H-6), 6.78 (br s, H-5), 6.36 (dd, J = 15.7 and 1.5 Hz, H-7′), 6.10 (dq, J = 15.7 and 6.6 Hz, H-8′), 5.11 (d, J = 9.2 Hz, H-7), 3.46 (dd, J = 9.2 and 6.8 Hz, H-8), 1.87 (dd, J = 6.6 and 1.5 Hz, H-9′), 1.41 (d, J = 6.8 Hz, H-9), 3.90 (s, OCH3), 3.88 (s, OCH3), 2.31 (s, CH3). 13C NMR (CDCl3) δ/ppm: 169.0 (C = O), 151.2 (C-5′), 146.5 (C-4), 144.1 (C-6′), 139.6 (C-3), 139.3 (C-4′), 133.0 (C-6), 132.4 (C-6′), 130.9 (C-7′), 123.6 (C-8′), 122.7 (C-1), 118.7 (C-2), 113.4 (C-3′), 110.3 (C-1′), 109.3 (C-5), 93.1 (C-7), 55.9 (OCH3), 45.8 (C-8), 17.9 (C-9), 20.7 (CH3), 18.4 (C-9′). HRESIMS m/z 369.1608 [M + H]+ (calcd. for C22H25O5 369.1702).

Procedure for the Preparation of Compound 1c

Compound 1 (600 mg) was treated with K2CO3 (2 equiv) and allyl bromide (2 equiv) in acetone (15 mL) under reflux for 19 h. The solvent was then removed under reduced pressure, and the residue was extracted with CH2Cl2 and washed with H2O (2 × 25 mL). The organic phase was dried over anhydrous Na2SO4 and concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography using n-hexane/EtOAc (9:1) as the eluent to afford 1c as a white amorphous solid in 73% yield.

4-O-Allyl Licarin A (1c)

1H NMR (CDCl3) δ/ppm: 6.99 (d, J = 8.2 Hz, H-5), 6.93 (d, J = 1.9 Hz, H-2), 6.92 (dd, J = 8.2 and 1.9 Hz, H-6), 6.84 (d, J = 1.9 Hz, H-2′), 6.78 (d, J = 1.9 Hz, H-6′), 6.36 (dd, J = 15.7 and 1.5 Hz, H-7′), 6.09 (qdd, J = 10.6, 7.2, and 4.8 Hz, H-β), 6.04 – 6.16 (m, H-8′), 5.39 (dd, J = 17.3 and 1.5 Hz; H-γ), 5.11 (d, J = 9.4 Hz, H-7), 4.61 (dt, J = 5.4 and 1.4 Hz, H-α), 3.86 (s, OCH3), 3.89 (s, OCH3), 3.46 (dq, J = 9.4 and 6.8 Hz; H-8), 1.86 (dd, J = 6.6 and 1.5 Hz, H-9′), 1.38 (d, J = 6.8 Hz, H-9). 13C NMR (CDCl3) δ/ppm: 149.6 (C-5′), 148.1 (C-4′), 146.6 (C-6′), 144.1 (C-3), 133.3 (C-4), 133.2 (C-2), 133.0 (C-6), 132.3 (C-2′), 130.9 (C-7′), 123.5 (C-8′), 119.1 (C-1), 118.0 (C-γ), 113.3 (C-β), 113.1 (C-3′), 109.9 (C-1′), 109.2 (C-5), 93.6 (C-7), 69.9 (C-α), 55.9 (OCH3), 45.6 (C-8), 18.4 (C-9′), 17.7 (C-9). HRESIMS m/z 367.1920 [M + H]+ (calcd. for C23H26O4 367.1909).

Parasite and Host Cell Maintenance

The RH-2F1 strain of T. gondii, which constitutively expresses β-galactosidase and was originally generated by Seeber and Boothroyd, was propagated by continuous passage in human foreskin fibroblast (HFF) monolayers. HFF cells (ATCC SCRC-1041) were maintained in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 2 mM l-glutamine, and 40 μg/mL gentamicin (D10 medium). Cell cultures were incubated at 37 °C in a humidified incubator with 5% CO2, following previously established procedures. For parasite infection experiments, confluent HFF monolayers were switched to DMEM containing 2% FBS (D2 medium).

Determination of Half-Maximal Effective Concentration (EC50)

Antiparasitic activity was quantified by monitoring β-galactosidase activity as an indirect measure of intracellular parasite growth, according to a previously reported protocol. Immediately after inoculation, infected HFF monolayers were exposed to compound 1 or derivatives 1a–1c at concentrations ranging from 30 to 0.47 μM. Cultures were maintained for 72 h without removing extracellular parasites. At the end of the incubation period, cells were lysed for 15 min at room temperature with 100 μL of lysis buffer (100 mM HEPES, pH 7.5, 1 mM MgSO4, 0.1% Triton X-100, and 5 mM DTT). Enzymatic activity was then determined by adding 160 μL of assay buffer (100 mM sodium phosphate, pH 7.3, 102 mM β-mercaptoethanol, and 9 mM MgCl2) together with 40 μL of chlorophenol red-β-d-galactopyranoside (CPRG, 6.25 mM). Following a 30 min incubation at 37 °C, absorbance was recorded at 570 nm using a Varioskan LUX microplate reader (Thermo Scientific). Untreated infected cultures, vehicle-treated cultures (1% DMSO), pyrimethamine-treated cultures, and blank wells served as viability, vehicle, reference drug, and background controls, respectively. All assays were performed in duplicate in three independent experiments. Concentration–response curves were fitted by nonlinear regression using a four-parameter logistic model, and EC50 values were calculated with SkanIt software (Thermo Fisher Scientific).

Determination of Half-Maximal Cytotoxic Concentration (CC50)

Cytotoxicity was assessed in uninfected HFF monolayers using the resazurin reduction assay as previously described. After complete monolayer formation, cells were incubated with compounds at the same concentration range employed in the antiparasitic assay (30–0.47 μM) for 72 h. Resazurin was subsequently added to a final concentration of 100 μM, and the plates were incubated for an additional 4 h at 37 °C. Fluorescence was measured at excitation and emission wavelengths of 565 and 590 nm, respectively. Untreated cells, vehicle-treated cells (1% DMSO), pyrimethamine-treated cells, and blank wells were included as controls. Experiments were carried out in duplicate and independently repeated three times. CC50 values were obtained by fitting the data to a four-parameter nonlinear regression model using SkanIt software.

Plasma Membrane Integrity Assay

The integrity of the parasite plasma membrane following treatment with 1 and derivatives 1a–1c was examined using propidium iodide uptake, according to a previously reported method. Freshly released tachyzoites (3 × 106 parasites) were suspended in PBS and incubated for 30 min at 37 °C under 5% CO2 in the presence of each compound at its corresponding EC50 concentration. Digitonin (3 μM) was included as the positive control for membrane permeabilization, whereas parasites exposed to 0.01% DMSO served as the negative control. Fluorescence was monitored with a Varioskan LUX microplate reader (Thermo Fisher Scientific) using excitation and emission wavelengths of 504 and 524 nm, respectively.

Mitochondrial Membrane Potential Assay

Changes in mitochondrial membrane potential were investigated using the fluorescent probe MitoTracker Red FM. Freshly egressed tachyzoites (2 × 106 parasites per well) were incubated with compounds 1 and 1a–1c at concentrations equivalent to their respective EC50 values for 30 min at 37 °C in a humidified atmosphere containing 5% CO2. Parasites treated with 0.01% DMSO or antimycin A (10 μM) were used as negative and positive controls, respectively. After treatment, parasites were washed with PBS and stained with 100 nM MitoTracker Red FM for an additional 30 min under the same incubation conditions. Fluorescence intensity was then determined using a Varioskan LUX multimode microplate reader (Thermo Fisher Scientific) with excitation and emission wavelengths set at 603 and 651 nm. Each experiment included eight technical replicates and was independently repeated three times.

In Vitro Assays with 4-O-Allyl Licarin A (1c)

Parasite Proliferation Assay

HFF cells (1 × 104 cells/well) were seeded in D10 medium and allowed to adhere overnight. Monolayers were subsequently infected with T. gondii tachyzoites at an MOI of 1:1 and maintained for 3 h to permit parasite invasion. After removal of extracellular parasites by PBS washing, infected cells were exposed to compound 1c (30–0.47 μM) for 72 h. Parasite growth was quantified by measuring β-galactosidase activity using the procedure described above.

Reversibility Assay

To determine whether the inhibitory activity of compound 1c persisted after compound withdrawal, infected HFF monolayers were exposed to the compound for 24 h. The treatment medium was then replaced with fresh compound-free medium, and cultures were maintained for an additional 48 h. Parasite viability at the end of the experiment was quantified by the β-galactosidase assay.

Invasion and Intracellular Proliferation Assays Using Pretreated Tachyzoites

Extracellular tachyzoites were incubated with compound 1c (30–0.47 μM) for 1 h at 37 °C, washed thoroughly with PBS after centrifugation (1900g, 10 min), and immediately used to infect HFF monolayers at an MOI of 1:1. After 3 h, nonadherent parasites were removed by washing. Parasite invasion was quantified immediately after this step by β-galactosidase activity. To evaluate intracellular replication, parallel cultures were maintained for an additional 72 h before parasite growth was quantified using the same enzymatic assay.

In Silico ADMET Prediction

The pharmacokinetic and toxicity properties of 1 and derivatives 1a–1c were predicted using freely available computational platforms. Gastrointestinal absorption (GI), blood–brain barrier permeability (BBB), lipophilicity, and drug-likeness descriptors were predicted using SwissADME (http://www.swissadme.ch). Toxicological end points, including mutagenicity, tumorigenicity, irritant effects, and reproductive toxicity, were estimated using the OSIRIS Property Explorer. These analyses were used to compare the predicted pharmacokinetic behavior and safety profiles of the evaluated compounds.

Statistical Analysis

Experimental data are presented as mean ± SD obtained from at least three independent experiments. Statistical comparisons among groups were carried out by one-way ANOVA followed by Tukey’s multiple-comparison test, adopting p < 0.05 as the threshold for statistical significance. Dose–response curves and EC50/CC50 values were generated by fitting a four-parameter logistic nonlinear regression model using SkanIt software (Thermo Fisher Scientific).

Supplementary Material

ao6c05944_si_001.pdf (813.2KB, pdf)

Acknowledgments

The authors used ChatGPT (OpenAI) to improve the English language and readability of the manuscript. All scientific content, interpretation of the results, and final editorial decisions were performed by the authors, who carefully reviewed and approved the final version of the manuscript and assume full responsibility for its contents.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.6c05944.

  • HPLC analysis, HRESIMS and NMR spectra for compounds 1, 1a–1c; HPLC analysis of compound 1 (Figure S1); HRESIMS spectrum (negative mode) of compound 1 (Figure S2); 13C NMR spectrum of compound 1 (δ/ppm, 75 MHz, CDCl3) (Figure S3); 1H NMR spectrum of compound 1 (δ/ppm, 300 MHz, CDCl3) (Figure S4); HPLC analysis of compound 1a (Figure S5); HRESIMS spectrum (positive mode) of compound 1a (Figure S6); 13C NMR spectrum of compound 1a (δ/ppm, 75 MHz, CDCl3) (Figure S7); 1H NMR spectrum of compound 1a (δ/ppm, 300 MHz, CDCl3) (Figure S8); HPLC analysis of compound 1b (Figure S9); HRESIMS spectrum (positive mode) of compound 1b (Figure S10); 13C NMR spectrum of compound 1b (δ/ppm, 75 MHz, CDCl3) (Figure S11); 1H NMR spectrum of compound 1b (δ/ppm, 300 MHz, CDCl3) (Figure S12); HPLC analysis of compound 1c (Figure S13); HRESIMS spectrum (positive mode) of compound 1c (Figure S14); 13C NMR spectrum of compound 1c (δ/ppm, 75 MHz, CDCl3) (Figure S15); 1H NMR spectrum of compound 1c (δ/ppm, 300 MHz, CDCl3) (Figure S16) (PDF)

G.C.M., S.O.A., I.O.D., and D.C.T. performed the experimental investigation, analyzed the data, and prepared the original draft. J.P.S.F., J.H.G.L., and J.Q.R. conceived the study, supervised the project, curated the data, acquired funding, and critically revised the manuscript. All authors critically reviewed the final version of the manuscript and approved it for submission.

Financial support for this study was provided by the São Paulo State Research Support Foundation (FAPESP, grant 2018/18954–4, 2020/03399–5, 2025/18855-0, and 2025/26427–8) and the Brazilian National Research Council (CNPq, grants 307299/2026–0, 302348/2025-5, and 303820/2023–3). G.C.M. and I.O.D. received a fellowship from FAPESP (2023/18327–8 and 2024/22547–6), and S.O.A. was supported by a CAPES fellowship (88887.162302/2025–00). The Article Processing Charge for the publication of this research was funded by the Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior (CAPES), Brazil (ROR identifier: 00x0ma614).

The authors declare no competing financial interest.

References

  1. Smith N. C., Goulart C., Hayward J. A., Kupz A., Miller C. M., van Dooren G. G.. Control of Human Toxoplasmosis. Int. J. Parasitol. 2021;51(2):95–121. doi: 10.1016/j.ijpara.2020.11.001. [DOI] [PubMed] [Google Scholar]
  2. Dian S., Ganiem A. R., Ekawardhani S.. Cerebral Toxoplasmosis in HIV-Infected Patients: A Review. Pathog. Global Health. 2023;117(1):14–23. doi: 10.1080/20477724.2022.2083977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Dubey J. P., Murata F. H. A., Cerqueira-Cézar C. K., Kwok O. C. H., Villena I.. Congenital Toxoplasmosis in Humans: An Update of Worldwide Rate of Congenital Infections. Parasitology. 2021;148(12):1406–1416. doi: 10.1017/S0031182021001013. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Alday P. H., Doggett J. S.. Drugs in Development for Toxoplasmosis: Advances, Challenges, and Current Status. Drug Des. Dev. Ther. 2017;11:273–293. doi: 10.2147/DDDT.S60973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Dunay I. R., Gajurel K., Dhakal R., Liesenfeld O., Montoya J. G.. Treatment of Toxoplasmosis: Historical Perspective, Animal Models, and Current Clinical Practice. Clin. Microbiol. Rev. 2018;31(4):10.1128/cmr.00057–17. doi: 10.1128/cmr.00057-17. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Hajj R. E., Tawk L., Itani S., Hamie M., Ezzeddine J., El Sabban M., El Hajj H.. Toxoplasmosis: Current and Emerging Parasite Druggable Targets. Microorganisms. 2021;9(12):2531. doi: 10.3390/microorganisms9122531. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Rangel G. W., Llinás M.. Re-Envisioning Anti-Apicomplexan Parasite Drug Discovery Approaches. Front. Cell. Infect. Microbiol. 2021;11:691121. doi: 10.3389/fcimb.2021.691121. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Mao E. Y., Page S. W., Sleebs B. E., Gancheva M. R., Wilson D. W.. A Review of Natural Products as a Source of Next-Generation Drugs against Apicomplexan Parasites. npj Antimicrob. Resist. 2025;3(1):51. doi: 10.1038/s44259-025-00119-x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Jang W. Y., Kim M.-Y., Cho J. Y.. Antioxidant, Anti-Inflammatory, Anti-Menopausal, and Anti-Cancer Effects of Lignans and Their Metabolites. Int. J. Mol. Sci. 2022;23(24):15482. doi: 10.3390/ijms232415482. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Godínez-Chaparro B., Pérez-Gutiérrez S., Pérez-Ramos J., Heyerdahl-Viau I., Hernández-Vázquez L.. Synthesis and Biological Activities of Dehydrodiisoeugenol: A Review. Pharmaceuticals. 2022;15(11):1351. doi: 10.3390/ph15111351. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. de Castro Levatti E. V., Costa-Silva T. A., Morais T. R., Fernandes J. P. S., Lago J. H. G., Tempone A. G.. Lethal Action of Licarin A Derivatives in Leishmania (L.) Infantum: Imbalance of Calcium and Bioenergetic Metabolism. Biochimie. 2023;208:141–150. doi: 10.1016/j.biochi.2022.12.018. [DOI] [PubMed] [Google Scholar]
  12. Morais T. R., Conserva G. A. A., Varela M. T., Costa-Silva T. A., Thevenard F., Ponci V., Fortuna A., Falcão A. C., Tempone A. G., Fernandes J. P. S., Lago J. H. G.. Improving the Drug-Likeness of Inspiring Natural Products - Evaluation of the Antiparasitic Activity against Trypanosoma cruzi through Semi-Synthetic and Simplified Analogues of Licarin A. Sci. Rep. 2020;10(1):5467. doi: 10.1038/s41598-020-62352-w. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Gomez-Cansino R., Guzmán-Gutiérrez S. L., Campos-Lara M. G., Espitia-Pinzón C. I., Reyes-Chilpa R.. Natural Compounds from Mexican Medicinal Plants as Potential Drug Leads for Anti-Tuberculosis Drugs. An. Acad. Bras. Ciênc. 2017;89:31–43. doi: 10.1590/0001-3765201720160298. [DOI] [PubMed] [Google Scholar]
  14. Meleti V. R., Esperandim V. R., Flauzino L. G. B., Prizantelli A. H., de Lima Paula L. A., Magalhães L. G., Cunha W. R., da Silva Laurentiz R., da Rocha Pissurno A. P., Nanayakkara N. P. D., Pereira A. C., Bastos J. K., Parreira R. L. T., Orenha R. P., e Silva M. L. A.. (±)-Licarin A and Its Semi-Synthetic Derivatives: In Vitro and in Silico Evaluation of Trypanocidal and Schistosomicidal Activities. Acta Trop. 2020;202:105248. doi: 10.1016/j.actatropica.2019.105248. [DOI] [PubMed] [Google Scholar]
  15. Wijesekera K., Taki A. C., Byrne J. J., White J. M., Carroll A. R., Gasser R. B., Davis R. A.. Anthelmintic Activity of Selected Neolignans and Semisynthetic Derivatives from Styrax suberifolius . Tetrahedron. 2025;169:134366. doi: 10.1016/j.tet.2024.134366. [DOI] [Google Scholar]
  16. Brito J. R., Wilairatana P., Roquini D. B., Parra B. C., Gonçalves M. M., Souza D. C. S., Ferreira E. A., Salvadori M. C., Teixeira F. S., Lago J. H. G., de Moraes J.. Neolignans Isolated from Saururus cernuus L. (Saururaceae) Exhibit Efficacy against Schistosoma mansoni . Sci. Rep. 2022;12(1):19320. doi: 10.1038/s41598-022-23110-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Pospíšil J., Konrádová D., Strnad M.. Antileishmanial Activity of Lignans, Neolignans, and Other Plant Phenols. Prog. Chem. Org. Nat. Prod. 2021;115:115–176. doi: 10.1007/978-3-030-64853-4_3. [DOI] [PubMed] [Google Scholar]
  18. Cabral M. M. O., Barbosa-Filho J. M., Maia G. L. A., Chaves M. C. O., Braga M. V., De Souza W., Soares R. O. A.. Neolignans from Plants in Northeastern Brazil (Lauraceae) with Activity against Trypanosoma cruzi . Exp. Parasitol. 2010;124(3):319–324. doi: 10.1016/j.exppara.2009.11.007. [DOI] [PubMed] [Google Scholar]
  19. Latif A., Du Y., Dalal S. R., Fernández-Murga M. L., Merino E. F., Cassera M. B., Goetz M., Kingston D. G. I.. Bioactive Neolignans and Other Compounds from Magnolia grandiflora L.: Isolation and Antiplasmodial Activity. Chem. Biodiversity. 2017;14(9):e1700209. doi: 10.1002/cbdv.201700209. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Mengarda A. C., Silva M. P., Cirino M. E., Morais T. R., Conserva G. A. A., Lago J. H. G., de Moraes J.. Licarin A, a Neolignan Isolated from Nectandra oppositifolia Nees & Mart. (Lauraceae), Exhibited Moderate Preclinical Efficacy against Schistosoma mansoni Infection. Phytother. Res. 2021;35(9):5154–5162. doi: 10.1002/ptr.7184. [DOI] [PubMed] [Google Scholar]
  21. Pereira A. C., Magalhães L. G., Gonçalves U. O., Luz P. P., Moraes A. C. G., Rodrigues V., da Matta Guedes P. M., da Silva Filho A. A., Cunha W. R., Bastos J. K., Nanayakkara N. P. D., e Silva M. L. A.. Schistosomicidal and Trypanocidal Structure–Activity Relationships for (±)-Licarin A and Its (−)- and (+)-Enantiomers. Phytochemistry. 2011;72(11):1424–1430. doi: 10.1016/j.phytochem.2011.04.007. [DOI] [PubMed] [Google Scholar]
  22. Néris P. L., Caldas J. P. A., Rodrigues Y. K. S., Amorim F. M., Leite J. A., Rodrigues-Mascarenhas S., Barbosa-Filho J. M., Rodrigues L. C., Oliveira M. R.. Neolignan Licarin A Presents Effect against Leishmania (Leishmania) major Associated with Immunomodulation in Vitro . Exp. Parasitol. 2013;135(2):307–313. doi: 10.1016/j.exppara.2013.07.007. [DOI] [PubMed] [Google Scholar]
  23. Zhang J., Si H., Lv K., Qiu Y., Sun J., Bai Y., Li B., Zhou X., Zhang J.. Licarin-B Exhibits Activity Against the Toxoplasma gondii RH Strain by Damaging Mitochondria and Activating Autophagy. Front. Cell Dev. Biol. 2021;9:684393. doi: 10.3389/fcell.2021.684393. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Seeber F., Boothroyd J. C.. Escherichia coli beta-galactosidase as an in vitro and in vivo reporter enzyme and stable transfection marker in the intracellular protozoan parasite Toxoplasma gondii . Gene. 1996;169:39–45. doi: 10.1016/0378-1119(95)00786-5. [DOI] [PubMed] [Google Scholar]
  25. Wang, Q. ; Sibley, L. D. . Assays for Monitoring Toxoplasma gondii Infectivity in the Laboratory Mouse. In Methods Mol. Biol.; Springer, 2020; Vol. 2071, pp 99–116. [DOI] [PubMed] [Google Scholar]
  26. McFadden D. C., Seeber F., Boothroyd J. C.. Use of Toxoplasma gondii Expressing Beta-Galactosidase for Colorimetric Assessment of Drug Activity in Vitro. Antimicrob. Agents Chemother. 1997;41(9):1849–1853. doi: 10.1128/AAC.41.9.1849. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Moura G. C., Reimão J. Q.. Phenotypic Screening of the MMV Global Health Priority Box Identifies Selective Compounds with Anti-Toxoplasma gondii Activity. ACS Omega. 2025;10(28):31147–31152. doi: 10.1021/acsomega.5c05130. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. da Silva Bellini Ramos A. B., Torres T., dos Reis L. F. C., Lambert G. C., Colombo F. A., Marques M. J., Reimão J. Q.. Assessment of Nebivolol Efficacy in Experimental Models of Toxoplasmosis: Insights into Parasite Burden Reduction and Neuronal Protection. Parasitol. Res. 2024;123(8):303. doi: 10.1007/s00436-024-08318-7. [DOI] [PubMed] [Google Scholar]
  29. de Oliveira R. M., dos Santos N. C. L., Almeida M. P. O., Lima J. P. D. Jr, Souza G. D., Barbosa M. C., Faria G. V., Paschoalino M., Alves R. N., Gomes A. O., Reimão J. Q., Barbosa B. F., Teixeira S. C., Ferro E. A. V.. Novel Drug Candidates Targeting Toxoplasma gondii in Maternal–Fetal Interface Models. Front. Pharmacol. 2025;16:1673462. doi: 10.3389/fphar.2025.1673462. [DOI] [PMC free article] [PubMed] [Google Scholar]

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Supplementary Materials

ao6c05944_si_001.pdf (813.2KB, pdf)

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