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Article

Biological Effects of Novel Synthetic Guanidine Derivatives Targeting Leishmania (Viannia) braziliensis

by
Geovane Dias-Lopes
1,2,†,
Luana Ribeiro Dos Anjos
3,†,
Sara Maria Xavier da Cruz
2,
Cauã Dias Abrão
3,
Maria Eduarda Pinto Gonçalves
2,
Franklin Souza-Silva
4,5,
Anna Fabisikova
6,
Eduardo Rene Perez González
3,* and
Carlos Roberto Alves
2,*
1
Departamento de Ciências Biomédicas e Saúde, Instituto de Biologia Roberto Alcântara Gomes, Universidade do Estado do Rio de Janeiro, Rua Arízio Gomes da Costa 186—Jardim Flamboyant, Cabo Frio 28905-320, RJ, Brazil
2
Laboratório de Biologia Molecular e Doenças Endêmicas, Instituto Oswaldo Cruz, Fundação Oswaldo Cruz, Avenida Brazil 4365—Manguinhos, Rio de Janeiro 21040-900, RJ, Brazil
3
Laboratório de Química Orgânica Fina, Departamento de Química e Bioquímica, Escola de Ciências e Tecnologia, Universidade Estadual Paulista, Rua Roberto Simonsen, 305—Centro Educacional, Presidente Prudente 19060-080, SP, Brazil
4
Centro de Desenvolvimento Tecnológico em Saúde, Fundação Oswaldo Cruz, Avenida Brazil, 4365—Manguinhos, Rio de Janeiro 21040-900, RJ, Brazil
5
Laboratório de Pesquisa Pré-Clínica, Universidade Iguaçu, Avenida Abílio Augusto Távora 2134, Dom Rodrigo, Nova Iguaçu 26260-045, RJ, Brazil
6
Mass Spectrometry Centre, Faculty of Chemistry, University of Vienna, Währinger Straße 38, A-1090 Vienna, Austria
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Molecules 2026, 31(4), 629; https://doi.org/10.3390/molecules31040629
Submission received: 12 January 2026 / Revised: 23 January 2026 / Accepted: 7 February 2026 / Published: 12 February 2026
(This article belongs to the Special Issue Chemical Compounds or Agents Against Parasites, Bacteria, and Neglected Tropical Diseases)
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Abstract

Leishmaniasis remains an important neglected tropical disease, and current treatments are limited by toxicity, resistance, and low bioavailability. In this study, novel guanidine derivatives were evaluated through an integrated approach, combining in silico physicochemical profiling with in vitro biological assays using Leishmania (Viannia) braziliensis, the etiological agent of American Tegumentary Leishmaniasis (ATL). Most compounds exhibited favorable drug-like properties, though variations in lipophilicity and solubility influenced biological performance. Among the tested molecules, FURL-G5 emerged as the most promising candidate, showing potent activity against promastigote forms and low cytotoxicity in murine macrophages, resulting in high selectivity indices (SI > 10), comparable to those of LQOF-G1, a compound with previously established leishmanicidal effects. These compounds were also tested on intracellular amastigotes, drastically reducing the infection rate of macrophages. The integration of an in silico approach and biological validation enabled rational compound prioritization and supports the early-stage development of these scaffolds. Overall, this study reinforces the potential of guanidine-based compounds as leads for innovative ATL drug discovery and demonstrates the value of multidisciplinary strategies for identifying selective and safe therapeutic candidates.

Graphical Abstract

1. Introduction

Leishmaniasis comprises a group of parasitic diseases caused by protozoa of the genus Leishmania, nearly twenty species of which are pathogenic to humans [1,2]. The infection presents heterogeneous clinical outcomes, including cutaneous (CL), mucocutaneous (MCL), and visceral (VL) leishmaniasis forms, whose expression depends on both the parasite species and the host’s immune profile [3]. In South America, Leishmania (Viannia) braziliensis stands out as the predominant etiological agent of American Tegumentary Leishmaniasis (ATL), producing a broad spectrum of clinical manifestations, including localized cutaneous, mucosal, and disseminated disease [2,4,5].
The current therapeutic landscape remains limited, as available drugs are few, toxic, and often associated with poor patient compliance [6]. Treatments approved for leishmaniasis mainly include pentavalent antimonials, amphotericin B, and miltefosine [2]. Despite their widespread use as first-line options, antimonial formulations are associated with serious adverse reactions and the growing emergence of resistant Leishmania strains, often resulting in therapeutic failure [7,8]. Resistance to other compounds has also been documented [9,10,11,12]. Consequently, the search for new chemical entities with improved efficacy and tolerability remains an urgent necessity. In this context, in vitro assays are crucial for compound screening and mechanistic evaluation, helping to identify promising therapeutic candidates [13,14].
Within the structural classes investigated for antileishmanial activity, guanidine-based compounds have attracted considerable attention. The guanidine functional group constitutes a pharmacophoric motif of high biological relevance, exemplified by drugs such as metformin (antidiabetic) and guanabenz (antihypertensive) [15,16]. Derivatives containing this scaffold have demonstrated activity against several protozoan pathogens, including Leishmania spp. and Trypanosoma spp. [17,18,19], as well as antibacterial and antifungal effects [20,21]. Remarkably, a group of ten N,N′,N″-trisubstituted guanidines exhibited pronounced in vitro activity against Leishmania (Leishmania) amazonensis [22]. Subsequent structural optimization efforts have produced derivatives with improved pharmacological properties and enhanced leishmanicidal potential [23,24,25,26].
Based upon previous evidence of the leishmanicidal potential, this study aimed to further explore guanidine’s activity against L. (V.) braziliensis. To this end, structural modifications were introduced, including hydrophobic groups in the guanidine scaffold, such as dichlorobenzylamine, furoyl and p-fluoroaniline (Figure 1). These groups were already reported to be active against Leishmania (L.) infantum [27]. The structural characterization, physicochemical properties, leishmanicidal effect and cytotoxicity were performed. The results reveal that specific substituent patterns markedly modulate antiparasitic activity, offering valuable insights into the structure-activity relationships (SAR) of this compound class and supporting their potential as therapeutic leads for ATL.

2. Results

2.1. Synthesis and Characterization

The synthesis of the new compounds was carried out following the methodology previously employed for benzoylguanidines (LQOF-series) using furoyl chloride (FURL- series) as starting material (Figure 2). Furoyl isothiocyanate was then reacted with p-bromoaniline or p-chloroaniline to synthesize the corresponding thioureas (FURL-TA, and FURL-TB). In the sequence, the synthesis of the guanidines (FURL-G1, -G2, -G3, -G4, and -G5) was made by the reaction of intermediate thioureas with 2,4-dichlorobenzylamine, 3,4-dichlorobenzylamine, or benzylamine. The thioureas and guanidine compounds were characterized by LC-UV/MS, ESI(+)-MS, ESI(+)-MS/MS, and NMR. Detailed characterization, the spectra and chromatograms are available in the Supplementary File: High-resolution ESI-Qq-TOF mass spectrum of compounds (Figures S1–S7); HPLC analysis (Figures S8–S14); and Nuclear Magnetic Resonance (Figures S15–S31).
The FURL-G1 and FURL-G2 compounds were planned to have the furoyl, p-fluoraniline and dichlorobenzylamine groups. The FURL-TA and FURL-G3 (with p-bromoaniline), and FURL-TB and FURL-G4 (with p-chloroanilin) were planned. Finally, FURL-G5, which contains the p-fluoroaniline group and benzylamine without chlorine substituents, was planned. Supplementary Table S1 presents all the planned structures, their molar masses, melting points and solubility, together with the previously published LQOF-G1 molecule, which was also evaluated for comparison. LQOF-G1 was used as a reference compound because, among the benzoylguanidine derivatives previously described in L. (L.) amazonensis [22] and L. (V.) braziliensis [25], it presented the highest selectivity index (SI) values in the latter.

2.2. In Silico Evaluation of Physicochemical Properties of Guanidine Derivatives

The SMILES files of the guanidine derivative molecules were used to evaluate the physicochemical properties (Table 1). These properties were crucial for assessing the potential of the guanidine derivatives as therapeutic agents.
The compounds exhibit molecular weights ranging from 280.73 g/mol (FURL-TB) to 467.15 g/mol (FURL-G3), with the compounds in the FURL-G series having the highest molecular mass. The lipophilicity (cLogP) of the compounds also varies considerably, ranging from 3.06 (FURL-TB) to 5.75 (FURL-G3), with the compounds in the FURL-G series exhibiting higher cLogP values, except the FURL-G5 compound. In contrast, the aqueous solubility (cLogS) follows an inverse trend, ranging from −3.93 (FURL-TB) to −6.55 (FURL-G3). Regarding hydrogen bond interactions, most compounds have 2 or 3 donors and 2 or 3 acceptors. The topological polar surface area (TPSA) ranged from 54.27 Å2 to 103.93 Å2, with the FURL-T compounds showing the lowest values. The drug-likeness index ranged from 0.30 (LQOF-G1) to 0.83 (FURL-TA and FURL-TB), with the FURL-T compounds being the closest to typical drug-like characteristics. Finally, the number of rotatable bonds (RB) ranged from 5 to 8, reflecting moderate molecular flexibility, which may be relevant for molecular interactions.
Furthermore, bioavailability data were evaluated using a radar chart (Figure 3). The physicochemical characteristics represented in the chart fall within the acceptable range for each axis, suggesting favorable properties for potential drug candidates. The only exception was the saturation parameter, which indicated a low sp3 hybridization fraction among the analyzed compounds.

2.3. Evaluation of Leishmanicidal Effect and Cytotoxicity of Guanidine Derivatives

First, eight guanidine compounds (50 µM to 0.195 µM) were assessed for their effectiveness against the L. (V.) braziliensis promastigotes and cytotoxicity on murine peritoneal macrophages. After obtaining the IC50 and CC50 values, it was possible to calculate the SI of the evaluated compounds (Table 2).
The IC50 values ranged from 3.47 ± 0.52 µM to 15.36 ± 0.56 µM, with the lowest values observed in the compounds LQOF-G1 (3.47 ± 0.52 µM), FURL-G1 (6.83 ± 0.32 µM) and FURL-G5 (7.17 ± 0.41 µM) (Table 2). It is important to note that the compounds FURL-TA, FURL-TB and FURL-G4 had no effect on promastigotes in tested concentrations.
Regarding the macrophage assays, the CC50 values ranged from 17.17 ± 3.69 µM to 164.8 ± 11.59 µM, with the highest values observed in the compounds LQOFG-1 (164.8 ± 11.59 µM) and FURL-G5 (124.4 ± 5.65 µM) (Table 2). The compounds FURL-TA and FURL-TB had no effect on peritoneal macrophages in tested concentrations. Amphotericin B was used as a positive control in assays, and despite being cytotoxic in peritoneal macrophages (72.61 ± 4.40 µM), the low IC50 (0.26 ± 0.01 µM) in promastigotes provides a high SI (279.26). As the compounds were prepared in 100% DMSO, control assays were performed using DMSO at the same final dilution corresponding to the highest concentration of the tested compounds (5% DMSO). Under these conditions, no significant differences were observed compared to the untreated control.
These assays aimed to identify the most active and selective compounds against the parasite. Compounds (LQOFG-1 and FURL-G5) that presented SI values greater than 10 were used in intracellular amastigote assays.

2.4. Assessment of LQOF-G1 and FURL-G5 Compounds in Intracellular Amastigotes

Compounds with SI values greater than 10 (LQOF-G1 and FURL-G5) were tested at three concentrations (10, 5 and 2.5 µM) in peritoneal macrophages infected with L. (V.) braziliensis. The endocytic index (EI) was used as a comparison parameter and showed that both compounds reduced parasite load (Figure 4). The greatest reductions were observed at 10 μM FURL-G5 (90.7%), followed by 10 μM LQOF-G1 (83.7%) (Figure 4). At the lowest concentration evaluated (2.5 µM), the compounds FURL-G5 (49.6%) and LQOF-G1 (47.1%) show a significant reduction (Figure 4). Representative images of the controls and infections treated with 10 μM LQOF-G1 and 10 μM FURL-G5 were obtained to highlight the impact of the compounds on intracellular amastigotes (Figure 5).

3. Discussion

Due to their diverse chemical and pharmacological properties, guanidine-containing compounds have been crucial for drug design and development. These molecules have shown great promise as anti-inflammatory agents, antithrombotics, antidiabetics, and chemotherapeutics [28,29]. Motivated by the promising antileishmanial activity previously reported for several N,N′,N″-trisubstituted guanidine compounds against species such as L. (L.) amazonensis, L. (L.) major, L. (L.) infantum, and L. (V.) braziliensis [22,23,24,25,26], this study reinforces the potential of guanidine derivatives as leishmanicidal agents and expands the portfolio of this class of compounds as therapeutic leads for the treatment of leishmaniasis.
Novel treatment options for leishmaniasis face persistent challenges, particularly related to the low solubility of many new compounds [30]. Establishing parameters such as dose, route, frequency, and duration of administration requires prior knowledge of solubility, pharmacokinetic, and pharmacodynamic profiles, information that is often limited or unavailable for experimental molecules. In such cases, computational analysis becomes a valuable tool to anticipate drug behavior and support early decision-making. Accordingly, based on the molecular structures of FURL-T series (FURL-TA and FURL-TB) and FURL-G series (FURL-G1, FURL-G2, FURL-G3, FURL-G4, and FURL-G5), their physicochemical and pharmacokinetic properties were assessed using in silico approaches to estimate their drug-likeness and potential as therapeutic candidates against leishmaniasis.
Therefore, the prediction of physicochemical properties is essential for new therapeutic candidates, as it provides vital early-stage insights for successful drug development [31,32]. The in silico data provided here on the physicochemical properties of guanidine derivatives show relevant insights into their drug-like behavior and potential as therapeutic candidates against L. (V.) braziliensis. Most compounds exhibited molecular weights and lipophilicity (cLogP) within the acceptable range for oral drugs, which is in line with Lipinski’s Rule of Five [33]. However, compounds in the FURL-G series, particularly FURL-G3, displayed higher molecular weights and cLogP values, suggesting an increase in hydrophobic character that may negatively influence aqueous solubility, as observed by their lower cLogS values [33].
Bioavailability radar plots confirmed that most compounds fall within the ideal physicochemical space for oral bioavailability. Nonetheless, a consistent deviation was observed in the saturation axis, indicating a low sp3 hybridization fraction. This reflects a high degree of molecular planarity and aromaticity, characteristics often associated with poor solubility and lower metabolic flexibility [33]. This could partially explain the limited activity or cytotoxicity of some of the tested compounds. Interestingly, the FURL-T series presented more favorable drug-likeness indices, with FURL-TA and FURL-TB scoring the highest (0.83), possibly due to their balanced TPSA, hydrogen bonding capacity, and moderate molecular weight. Despite this, these two compounds showed no leishmanicidal activity against promastigotes of L. (V.) braziliensis, suggesting that favorable physicochemical parameters alone are not sufficient to ensure bioactivity [34]. In fact, in another study, the other compound from the FURL-T series also showed no in vitro biological activity against three species of Leishmania [26]. Conversely, FURL-G5 and LQOF-G1 compounds, which presented slightly higher lipophilicity and lower solubility, performed well in both cytotoxicity and efficacy assays, suggesting that a moderate deviation from ideal parameters can still yield biologically relevant molecules, especially when supported by acceptable ADMET profiles [35].
In the synthesis of the new compounds, structural modifications including 2,4- or 3,4-dichlorobenzylamines and furoyl were performed with the aim of enhancing leishmanicidal activity. Dichlorobenzylamines present biological activities, including antimycobacterial, algicidal and antifungal [36,37,38]. In addition, compounds with 2,4-dichlorobenzylamine have also been highlighted with antitumor immune responses, controlling the growth of lung tumors [39]. However, among the evaluated compounds, those containing these groups (FURL-G2, -G3, and -G4), except for FURL-G1, did not exhibit the lowest IC50 values, indicating that the presence of these chemical moieties did not necessarily enhance the leishmanicidal potency of the molecules.
The furan derivatives, which are important compounds in pharmaceutical chemistry, display a wide range of biological activities, including antimicrobial, antifungal, anti-inflammatory, and anticancer properties [40]. Among several drugs containing a furane moiety in their structure, Nifurtimox is a nitrofuran used for the treatment of Chagas disease, caused by the protozoan Trypanosoma cruzi [41]. In the present study, the compounds FURL-G1 and FURL-G5, which also contain a furan ring, exhibited the lowest IC50 values, suggesting a possible contribution of this group to their leishmanicidal activity.
Furthermore, previous studies on N,N′,N″-trisubstituted benzoylguanidines have reported greater activity of compounds that have bromine or chlorine atoms rather than fluorine [22,25]. In contrast, in the present study, FURL-G5 exhibited higher SI. In contemporary drug development, the systematic screening of compounds with different substituents is a standard strategy, and the incorporation of fluorine atoms has become a routine step in optimization processes [42]. Fluorinated substituents bound to sp2-hybridized carbons can enhance the lipophilicity of drug molecules, thereby improving their interaction with biological membranes [43]. Moreover, fluorinated molecules have demonstrated notable microbicidal properties; for example, voriconazole, a fluorinated triazole antifungal, has been approved for the treatment of invasive aspergillosis and refractory infections with species of Pseudallescheria/Scedosporium and Fusarium [44].
Despite these favorable chemical features, not all guanidine-based derivatives exhibited acceptable toxicity profiles to proceed to the leishmanicidal activity assays, possibly because they interfered with vital metabolic pathways of the host cells. Leishmania spp. alternates between promastigote (the form found in the sandfly gut) and intracellular amastigote (the form present in the mammalian host) [2]. Despite not being the target of treatment, promastigotes are commonly utilized in high-throughput drug screening because they offer a simpler and faster in vitro system [45,46,47]. The biological assays revealed that LQOF-G1 and FURL-G5 were the most promising candidates, combining significant antipromastigote activity and low cytotoxicity in murine macrophages, with high selectivity indices (SI > 10). The current results for both the promastigote IC50 and SI of LQOF-G1 are consistent with those reported previously to L. (V.) braziliensis, confirming previous evaluations of this compound class [25].
Evaluating the drugs against intracellular amastigotes is essential to confirm their ability to eliminate parasites within host cells and to generate biologically relevant data [48,49,50]. Accordingly, the findings in promastigotes were further supported by their efficacy against intracellular amastigotes, with parasite load reductions exceeding 90% at 10 μM for both LQOF-G1 and FURL-G5, highlighting their potential for further in vivo validation. Overall, the results reinforce the value of guanidine derivatives as a chemical scaffold for antileishmanial drug discovery and highlight the effectiveness of combining computational and experimental approaches in early-stage drug development.

4. Materials and Methods

4.1. Guanidine-Derived Compounds Series

4.1.1. Synthesis of the Compounds

The synthesis of the compounds was performed by previously reported methods described below [22]. The compound series LQOF- was also reported [22], and the novel compounds are identified as FURL-T and FURL-G series. Commercial benzoyl chloride, furoyl chloride, ammonium isothiocyanate, 4-nitroaniline, 4-fluoraniline, 4-bromoaniline, 4-chloroaniline, benzylamine, 2,4-dichlorobenzylamine, 3,4-dichlorobenzylamine, triethylamine, pentahydrated bismuth nitrate and the solvents N,N-dimethylformamide, acetonitrile and dichloromethane were obtained from Sigma Aldrich and were used without previous purification.

4.1.2. General Procedure for the Synthesis of Thiourea and Guanidine Compounds

Thioureas were synthesized as intermediate compounds for the synthesis of guanidines. Initially, furoyl isothiocyanate was made through the reaction between furoyl chloride and ammonium isothiocyanate; the product was reacted with the selected anilines and thus thioureas were obtained (Figure 2). Finally, guanidines were synthesized through the reaction between thioureas and benzylamines (Figure 2).

4.1.3. HRESIMS and HPLC-UV

High-resolution electrospray ionization mass spectra (HRESIMS) in positive ion mode in the range of m/z 80–1600 were recorded on a timsTOF fleX mass spectrometer (Bruker Daltonics, Bremen, Germany) by direct infusion. The sum formulae of the detected ions were determined using Bruker Compass DataAnalysis 5.3 based on the mass accuracy (Δm/z ≤ 5 ppm) and isotopic pattern matching (SmartFormula algorithm). UV-chromatograms in the range of 190–400 nm have been obtained on a Vanquish Horizon UHPLC system (Thermo Fisher Scientific, Waltham, MA, USA) using an Acclaim 120 C18, 150 mm × 21 mm, 3 μm HPLC column (Thermo Fisher Scientific). Water and acetonitrile/water (9:1), both modified with 0.1% formic acid, were used as mobile phases A and B, respectively. The sample components were separated and eluted with a linear gradient from 5% to 100% B in 15 min followed by an isocratic column cleaning (5 min at 100% B) and a re-equilibration step (5 min at 5% B). The flow rate was 0.4 mL/min, and the column oven temperature was set to 25 °C except for BENG-7, which gave a better peak shape at 40 °C. The purity was determined from the UV chromatogram (254 nm) as the ratio of the peak area of the compound to the total peak area (i.e., the sum of the areas of all peaks that were not present in the solvent blank).
The compound FURL-G5 was analyzed using a Shimadzu HPLC-DAD instrument, employing a C-18 column and Methanol/H2O-85/15 as solvent; the samples were diluted in HPLC-grade methanol. The mass spectrum for this compound was acquired by direct introduction into a GCMS-QP2010 PLUS gas chromatography mass spectrometer (Shimadzu, Kyoto, Japan). The parameters used for these analyses were: interface temperature: 240 °C; ionization chamber temperature: 250 °C; solvent cutoff time: 0.25 min; initial time: 0.30 min; final time: 40.0 min; ionization chamber temperature program: initial temperature: 50 °C, with heating from 20 °C·min−1 to 350 °C and a holding time of 10 min.

4.1.4. NMR Spectroscopy

The 1H and 13C NMR spectra in solution were recorded using an AVANCE-III HD-500 MHz NMR spectrometer. The analyses were conducted with 5–15 mg of the sample either dissolved in CDCl3 with a purity of 99.8 atom % D, containing 0.03% (v/v) tetramethylsilane (TMS) or in DMSO-d6 with a purity of 99.9 atom % D. Chemical shifts (δ) were referenced to TMS or the solvent signals, respectively. The 1H NMR data are presented as follows: chemical shifts, multiplicity (s—singlet, d—doublet, dd—double doublet, t—triplet, dt—double triplet, tt—triple triplet, qua—quartet, qu—quintet, m—multiplet, br s—broad singlet), coupling constants (J) in Hertz (Hz), and peak integrals.

4.1.5. Physical Chemical Characterization Data of the Compounds

N-((4-bromophenyl)carbamoyl)furan-2-carboxamide (FURL-TA). White solid; yield 89.43%; mp: 139.8–140 °C. 1H NMR (500 MHz, CDCl3) δ: 12.21 (s, 1H), 9.20 (s, 1H), 7.64 (t, J = 7.5 Hz, 2H), 7.54 (d, J = 10 Hz, 2H), 7.40 (d, J = 2.85, 1H) 6.65 (m, 1H), 1.57 (s, 1H). 13C (125 MHz, CDCl3) δ: 177.98 (C), 156.76 (C), 146.60 (CH), 144.82 (C), 136.66 (C), 132.00 (2CH), 125.58 (2CH), 120.01 (C), 119.19 (CH), 113.51 (CH). HPLC (254 nm): Rt 12.53 min, purity 99.40%. HRESIMS m/z 324.9642 [M + H]+ (calcd for C12H10BrN2O2S+, m/z 324.9641, Δ = −0.2 ppm).
N-((4-chlorophenyl)carbamoyl)furan-2-carboxamide (FURL-TB). White solid; yield 74.12%; mp: 127.0–127.3 °C. 1H NMR (500 MHz, CDCl3) δ: 12.31 (s, 1H), 9.20 (s, 1H), 7.64 (t, J = 7.5, 2H), 7.39 (d, J = 5 Hz, 1H), 7.37 (d, J = 5 Hz, 2H), 6.63 (m, 1H), 1.66 (s, 1H). 13C (125 MHz, CDCl3) δ: 178.08 (C), 156.77 (C), 146.61 (CH), 144.82 (C), 136.16 (C), 132.15 (C), 129.02 (2CH), 125.33 (2CH), 119.17 (CH), 113.49 (CH). HPLC (254 nm): Rt 12.20 min, purity 99.40%. HRESIMS m/z 281.0147 [M + H]+ (calcd for C12H10ClN2O2S+, m/z 281.0146, Δ = −0.3 ppm).
(Z)-N-furoyl-N-(2,4-dichlorobenzyl)-N-(4-fluorophenyl)guanidine (FURL-G1). Yellow solid; yield 88.31%; mp: 91.1–93.2 °C. 1H NMR (500 MHz, CDCl3) δ: 11.63 (s, 1H), 7.55 (s, 1H), 7.48 (d, J = 10 Hz, 1H), 7.38 (s, 1H), 7.23 (d, J = 5 Hz, 3H), 7.13 (s, 3H), 6.48 (s, 1H), 5.25 (s, 1H), 4.68 (s, 2H). 13C (125 MHz, CDCl3) δ: 169.45 (C), 162.50 (C), 158.76 (C), 152.35 (C), 145.09 (2CH), 134.22 (CH), 131.56 (C), 129.36 (3CH), 128.02 (C), 127.39 (2C), 117.18 (CH), 117.00 (CH), 115.25 (CH), 111.58 (CH), 42.61 (CH2). 19F NMR (470 MHz, CDCl3) δ: −113.33 (F). HPLC (254 nm): Rt 12.35 min, purity 96.41%. HRESIMS m/z 406.0520 [M + H]+ (calcd for C19H15Cl2FN3O2+, m/z 406.0520, Δ = 0.0 ppm).
(Z)-N-furoyl-N-(3,4-dichlorobenzyl)-N-(4-fluorophenyl)guanidine (FURL-G2). Yellow liquid; yield 81.65%. 1H NMR (500 MHz, CDCl3) δ: 11.63 (s, 1H), 7.53 (s, 1H), 7.44 (s, 1H), 7.40 (d, J = 10 Hz, 1H), 7.19 (d, J = 5 Hz, 2H), 7.11 (s, 4H), 6.47 (s, 1H), 5.09 (s, 1H), 4.61 (s, 2H). 13C (125 MHz, CDCl3) δ: 169.53 (C), 158.84 (C), 152.27 (C), 145.25 (C), 138.72 (CH), 132.70 (C), 130.68 (2CH), 129.75 (2CH), 128.19 (C), 127.00 (CH), 117.04 (CH), 115.47 (CH), 111.63 (CH), 44.04 (CH2). 19F NMR (470 MHz, CDCl3) δ: −113,15 (F). HPLC (254 nm): Rt 12.03 min, purity 93.15%. HRESIMS m/z 406.0520 [M + H]+ (calcd for C19H15Cl2FN3O2+, m/z 406.0520, Δ = 0.0 ppm).
(Z)-N-furoyl-N-(2,4-dichlorobenzyl)-N-(4-bromophenyl)guanidine (FURL-G3). Beige solid; yield 78.86%; mp: 119.9–121.2 °C. 1H NMR (500 MHz, CDCl3) δ: 11.70 (s, 1H), 7.52 (s, 3H), 7.47 (d, J = 10 Hz, 1H), 7.38 (d, J = 2.1 Hz, 1H), 7.23 (dd, J = 2.05, 1H), 7.14 (s, 1H), 7.10 (s, 1H), 6.48 (m, 1H), 5.37 (s, 1H), 4.68 (s, 1H). 13C (125 MHz, CDCl3) δ: 169.39 (C), 158.25 (C), 152.27 (C), 145.29 (CH), 134.68 (C), 134.16 (2CH), 133.22 (CH), 131.53 (C), 129.39 (2CH), 127.36 (2CH), 115.51 (CH), 111.70 (CH), 42.62 (CH2). HPLC (254 nm) Rt 13.81 min, purity 98.6%. HRESIMS m/z 465.9721 [M + H]+ (calcd for C19H15BrCl2N3O2+, m/z 465.9719, Δ = −0.5 ppm).
(Z)-N-furoyl-N-(2,4-dichlorobenzyl)-N-(4-chlorophenyl)guanidine (FURL-G4). Beige solid; yield 69.82%; mp: 116.2–117.6 °C. 1H NMR (500 MHz, CDCl3) δ: 11.71 (s, 1H), 7.54 (s, 1H), 7.47 (d, J = 10 Hz, 1H), 7.38 (d, J = 2.05 Hz, 4H), 7.23 (dd, J = 4.3 Hz, 1H), 7.18 (s, 1H), 7.13 (s, 1H), 6.48 (s, 1H), 5.37 (s, 1H), 4.69 (s, 2H). 13C (125 MHz, CDCl3) δ: 169.43 (C), 158.33 (C), 152.29 (C), 145.15 (CH), 134.15 (2C), 131.57 (CH), 130.28 (2CH), 129.37 (2CH), 127.40 (CH), 126.98 (CH), 115.37 (CH), 111.61 (CH), 42.64 (CH2). HPLC (254 nm) Rt 13.54 min, purity 98.8%. HRESIMS m/z 422.0226 [M + H]+ (calcd for C19H15Cl3N3O2+, m/z 422.0224, Δ = −0.5 ppm).
(Z)-N-furoyl-N-benzyl-N-(4-fluorophenyl)guanidine (FURL-G5). White solid; yield 76.55%; mp: 105.8–106.6 °C. 1H NMR (500 MHz, CDCl3) δ: 11.69 (s, 1H), 7.53 (s, 1H), 7.32 (m, 4H), 7.22 (m, 2H), 7.13 (s, 2H), 7.08 (t, J = 7.5 Hz, 1H), 6.46 (s, 2H), 5.03 (s, 1H), 4.69 (s, 2H). 13C (125 MHz, CDCl3) δ: 169.54 (C), 158.93 (C), 152.45 (C), 145.04 (CH), 138.15 (C), 131.65 (C), 128.83 (2CH), 127.73 (CH), 127.61 (2CH), 117.03 (CH), 116.88 (CH), 115.25 (2CH), 111.51 (2CH), 45.27 (CH2). 19F NMR (470 MHz, CDCl3) δ: −113.59 (F). HPLC (1285 nm) R t 9.42 min, purity 99.67%. EI-MS (m/z 337).

4.2. Physicochemical Profiling of Guanidine Derivatives

The physicochemical parameters were calculated based on the Simplified Molecular Input Line Entry System (SMILES) representations of each molecule, generated using ACD/ChemSketch (version 2025). The physicochemical profiles of the guanidine derivatives were assessed as previously assessed [48], using the SwissADME (http://www.swissadme.ch/, accessed on 21 September 2025) and ADMET-AI (https://admet.ai.greenstonebio.com/, accessed on 21 September 2025) web servers.

4.3. Parasite Culture and Maintenance

Promastigotes of the L. (V.) braziliensis Thor strain (MCAN/BR/1998/R619) were cultured at 26 °C in Schneider’s insect medium (pH 7.2) (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 20% (v/v) fetal bovine serum (FBS) (Gibco, Thermo Fisher Scientific, Waltham, MA, USA), 100 IU/mL penicillin and 100 μg/mL streptomycin (Sigma-Aldrich Chemical Co., St. Louis, MO, USA).

4.4. Biological Effect on Promastigotes

To assess the in vitro biological effect of the synthesized guanidine derivatives on L. (V.) braziliensis promastigotes, eight compounds were dissolved previously in DMSO at a concentration of 16 mM. The resazurin-based viability assay was performed as previously described [49] to determine the half-maximal inhibitory concentration (IC50 value). Briefly, promastigotes in the logarithmic grown phase were seeded in 96-well plates at a density of 2 × 106 cells per well in 100 µL of Schneider’s medium supplemented with 20% of FBS and treated with test compounds at concentrations ranging from 50 µM to 0.195 µM. The plates were incubated for 24 h at 26 °C. Amphotericin B (Bristol-Myers Squibb, Lawrence, NJ, USA), tested at concentrations ranging from 40 µM to 0.0078 µM, and 5% DMSO (v/v) were used as positive and vehicle controls, respectively. Subsequently, 10 µL of resazurin solution at 0.015% (Sigma-Aldrich Chemical Co., St. Louis, MO, USA) was added to each well, followed by a 4 h incubation at 26 °C. Fluorescence was measured using a FlexStation® 3 (Molecular Devices Inc., San Jose, CA, USA) with excitation at 560 nm and emission at 590 nm. Data analysis was performed using GraphPad Prism version 8.0.1 (GraphPad Software, San Diego, CA, USA, www.graphpad.com).

4.5. Cytotoxicity of Compounds to BALB/c Mice Macrophages

To determine the cytotoxicity of the guanidine derivatives, murine peritoneal macrophages were obtained as previously described [50]. Female BALB/c mice (5–7 weeks old, weighing approximately 22 g) were supplied by the Instituto de Ciência e Tecnologia em Biomodelos da Fundação Oswaldo Cruz (ICTB, Fiocruz/RJ) for the experimental infections. To isolate macrophages, female BALB/c mice received an intraperitoneal injection of Brewer thioglycolate medium (5 mL). After 72 h, RPMI 1640 medium was injected into the peritoneal cavity, and cells were collected by aspiration. Samples were then centrifuged at 520× g for 5 min at 4 °C [50]. Then, the peritoneal macrophages were seeded at 5 × 104 per well in a 96-well cell culture plate containing RPMI 1640 medium supplemented with 10% FBS and allowed to adhere for 24 h at 37 °C in 5% CO2. Adherent macrophages were incubated in 100 µL of RPMI 1640 medium supplemented with 10% FBS with compounds in concentrations ranging from 800 µM to 12.5 µM for 24 h at 37 °C in 5% CO2. Amphotericin B (Bristol-Myers Squibb, Lawrence, NJ, USA) in concentrations ranging from 400 µM to 3.125 µM were used as controls. Additional control assays were included by treating the cell cultures with 5% DMSO (v/v). Macrophage viability was determined by the addition of 10 µL of resazurin to each well and incubated for 4 h at 37 °C in 5% CO2. Fluorescence signals were acquired with a FlexStation® 3 (Molecular Devices Inc.) using 560 nm excitation and 590 nm emission. Statistical analysis was carried out using GraphPad Prism software, version 8.0.1 (GraphPad Software, San Diego, CA, USA, www.graphpad.com).

4.6. Assessing Activity Against Intracellular Amastigotes

To determine the effect of compounds on intracellular amastigotes, murine peritoneal macrophages obtained as previously described [50] were seeded at 1 × 105 per well in LabTek wells containing RPMI 1640 medium supplemented with 10% FBS and allowed to adhere for 24 h at 37 °C in 5% CO2. Adherent macrophages were infected with stationary-phase promastigotes of L. (V.) braziliensis Thor strain at a ratio of 10:1 (parasite: macrophage) with 2 h interaction at 37 °C in 5% CO2. The non-internalized parasites were removed by washing, and infected cells were incubated in a fresh RPMI 1640 medium supplemented with 10% FBS. The total time of infection was 24 h. Then, infected peritoneal macrophages were treated with different concentrations of compounds (10 μM, 5 μM and 2.5 μM) or 5% DMSO (v/v) for 24 h. Finally, macrophages in the LabTek slides were stained using the Panoptic and observed using a Panthera L Optical Microscope (Motic, Xiamen, China). The assays were performed in triplicate. To determine the percentage of infected macrophages and the number of parasites per cell, a total of 100 macrophages was analyzed per slide chamber. These values were used to estimate the EI as follows: EI = (% of infected macrophages) × (mean number of parasites in each infected macrophage).

5. Conclusions

This study demonstrates the potential of guanidine-based compounds as antileishmanial agents through a comprehensive evaluation that integrates in silico physicochemical profiling, pharmacokinetic and toxicity prediction, and in vitro biological validation. Among the tested molecules, LQOF-G1 and FURL-G5 stood out due to their potent activity against both promastigote and intracellular amastigote forms of L. (V.) braziliensis, coupled with low cytotoxicity and favorable selectivity indices.
The physicochemical and bioavailability analyses confirmed that most derivatives fall within the acceptable drug-like space, although a low saturation index (fraction sp3) indicated potential areas for structural optimization. Importantly, the integration of computational tools with biological screening enabled an efficient and rational prioritization of candidates.
Considering the current scope of the study, the technological readiness level (TRL) can be estimated at TRL 3, corresponding to analytical and experimental proof of concept (https://www.excellenting.com/drug_discovery_trl/, accessed on 29 October 2025). While the findings provide promising leads, further investigations, including in vivo efficacy, pharmacokinetics, and toxicity studies, are needed to advance these compounds toward clinical development.
Collectively, these results support the hypothesis that certain guanidine derivatives exhibit promising antileishmanial activity with an acceptable safety profile. The combination of physicochemical and biological data reinforces the potential of LQOF-G1 and FURL-G5 as lead compounds for further optimization and development.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/molecules31040629/s1, Table S1: Structure and physical properties of new planned compounds and LQOF-G1; Figure S1: High-resolution ESI-Qq-TOF mass spectrum of FURL-TA (top) and the simulated isotopic pattern of its [M+H]+ ion (bottom); Figure S2: High-resolution ESI-Qq-TOF mass spectrum of FURL-TB (top) and the simulated isotopic pattern of its [M+H]+ ion (bottom); Figure S3: High-resolution ESI-Qq-TOF mass spectrum of FURL-G1 (top) and the simulated isotopic pattern of its [M+H]+ ion (bottom); Figure S4: High-resolution ESI-Qq-TOF mass spectrum of FURL-G2 (top) and the simulated isotopic pattern of its [M+H]+ ion (bottom); Figure S5: High-resolution ESI-Qq-TOF mass spectrum of FURL-G3 (top) and the simulated isotopic pattern of its [M+H]+ ion (bottom); Figure S6: High-resolution ESI-Qq-TOF mass spectrum of FURL-G4 (top) and the simulated isotopic pattern of its [M+H]+ ion (bottom); Figure S7: High-resolution ESI-TOF-MS mass spectrum of FURL-G5; HPLC-UV AND HPLC-DAD; Figure S8: HPLC-UV chromatogram (254 nm) of FURL-TA; Figure S9: HPLC-UV chromatogram (254 nm) of FURL-TB; Figure S10: HPLC-UV chromatogram (254 nm) of FURL-G1; Figure S11: HPLC-UV chromatogram (254 nm) of FURL-G2; Figure S12: HPLC-UV chromatogram (254 nm) of FURL-G3; Figure S13: HPLC-UV chromatogram (254 nm) of FURL-G4; Figure S14: HPLC-DAD chromatogram (285 nm) of FURL-G5; Figure S15: 1H NMR Spectrum of FURL-TA in CDCl3 at 25 °C; Figure S16:13C NMR Spectrum of FURL-TA in CDCl3 at 25 °C; Figure S17: 1H NMR Spectrum of FURL-TB in CDCl3 at 25 °C; Figure S18: 13C NMR Spectrum of FURL-TB in CDCl3 at 25 °C; Figure S19: 1H NMR Spectrum of FURL-G1 in CDCl3 at 25 °C; Figure S20:13C NMR Spectrum of FURL-G1 in CDCl3 at 25 °C; Figure S21: 19F NMR Spectrum of FURL-G1 in CDCl3 at 25 °C; Figure S22: 1H NMR Spectrum of FURL-G2 in CDCl3 at 25 °C; Figure S23:13C NMR Spectrum of FURL-G2 in CDCl3 at 25 °C; Figure S24:19F NMR Spectrum of FURL-G2 in CDCl3 at 25 °C; Figure S25:1H NMR Spectrum of FURL-G3 in CDCl3 at 25 °C; Figure S26: 13C NMR Spectrum of FURL-G3 in CDCl3 at 25 °C; Figure S27: 1H NMR Spectrum of FURL-G4 in CDCl3 at 25 °C; Figure S28: 13C NMR Spectrum of FURL-G4 in CDCl3 at 25 °C; Figure S29: 1H NMR Spectrum of FURL-G5 in CDCl3 at 25 °C; Figure S30: 13C NMR Spectrum of FURL-G5 in CDCl3 at 25 °C; Figure S31:19F NMR Spectrum of FURL-G5 in CDCl3 at 25 °C.

Author Contributions

G.D.-L.: Conceptualization, formal analysis, methodology, and original draft and writing—review & editing. L.R.D.A.: Methodology and writing—review & editing. S.M.X.d.C.: Methodology. C.D.A.: Methodology. M.E.P.G.: Methodology. F.S.-S.: Methodology and writing—review & editing. A.F.: Methodology. E.R.P.G.: Funding acquisition, investigation, project administration, resources, supervision, original draft and writing—review & editing. C.R.A.: Funding acquisition, investigation, project administration, resources, supervision, original draft and writing—review & editing. All authors have read and agreed to the published version of the manuscript.

Funding

This study was financed in part by the Conselho Nacional de Desenvolvimento Científico e Tecnológico—Brazil (CNPq:305287/2023-0; 441634/2024-9), Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro—Brazil (FAPERJ: E-26/010.000983/2019; E-26/203.931/2024; E-26/210.712/2025), Fundação de Amparo à Pesquisa do Estado de São Paulo—Brazil (FAPESP APR: 2021/02595-8 and PhD scholarship 2025/01056-7), and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior—Brazil (CAPES)—Finance Code 001. Carlos R. Alves is a research fellow at CNPq and CNE/FAPERJ.

Institutional Review Board Statement

The animal study protocol was approved by the Committee for the Ethical Use at Animals of Instituto Oswaldo Cruz (CEUA-IOC: L-014/2023), approved on 6 October 2023. Access to this parasite, the L. (V.) braziliensis Thor strain (MCAN/BR/1998/R619), was registered in the Brazilian System of Genetic Resource Management and Associated Traditional Knowledge (SisGen) under number A41DBDD (approved on 16 October 2018).

Data Availability Statement

The original contributions presented in this study are included in the article/Supplementary Materials. Further inquiries can be directed to the corresponding authors.

Acknowledgments

The authors acknowledge the support from the Culture Medium Platform at the Instituto Oswaldo Cruz—Fundação Oswaldo Cruz.

Conflicts of Interest

The authors declare they have no actual or potential competing financial interests.

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Molecules 31 00629 g001
Figure 1. Structural modifications in the guanidine scaffold. Initially designed guanidines and their groups with potential biological activity: p-fluoroaniline (green), dichlorobenzylamine (pink), and furan (yellow).
Figure 1. Structural modifications in the guanidine scaffold. Initially designed guanidines and their groups with potential biological activity: p-fluoroaniline (green), dichlorobenzylamine (pink), and furan (yellow).
Molecules 31 00629 g001
Molecules 31 00629 g002
Figure 2. Scheme of synthetic route for the FURL compounds TA, TB, and G1–G5. (a) ACN, 75 °C, 1 h; (b) ACN, 75 °C, 2 h; (c) DMF, Bi(NO3)3∙5H2O, Et3N, 120 °C, 24 h.
Figure 2. Scheme of synthetic route for the FURL compounds TA, TB, and G1–G5. (a) ACN, 75 °C, 1 h; (b) ACN, 75 °C, 2 h; (c) DMF, Bi(NO3)3∙5H2O, Et3N, 120 °C, 24 h.
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Molecules 31 00629 g003
Figure 3. Bioavailability radar plots of the guanidine derivatives. The pink area represents the optimal range for each property: lipophilicity (LIPO_XLOGP3 between −0.7 and +5.0), size (SIZE_MW between 150 and 500 g/mol), polarity (POLAR_TPSA between 20 and 130 Å2), solubility (INSOLU_log S not lower than −6), saturation (INSAT_sp3 hybridization fraction ≥ 0.25), and flexibility (FLEX_ ≤ 9 rotatable bonds). The guanidine derivatives (LQOF-G1, FURL-TA, FURL-TB, FURL-G1, FURL-G2, FURL-G3, FURL-G4 and FURL-G5) are shown as bidimensional representations.
Figure 3. Bioavailability radar plots of the guanidine derivatives. The pink area represents the optimal range for each property: lipophilicity (LIPO_XLOGP3 between −0.7 and +5.0), size (SIZE_MW between 150 and 500 g/mol), polarity (POLAR_TPSA between 20 and 130 Å2), solubility (INSOLU_log S not lower than −6), saturation (INSAT_sp3 hybridization fraction ≥ 0.25), and flexibility (FLEX_ ≤ 9 rotatable bonds). The guanidine derivatives (LQOF-G1, FURL-TA, FURL-TB, FURL-G1, FURL-G2, FURL-G3, FURL-G4 and FURL-G5) are shown as bidimensional representations.
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Figure 4. Effects of LQOFG-1 and FURL-G5 guanidine compounds on the endocytic index (EI) of the Leishmania (V.) braziliensis amastigotes in mice peritoneal macrophages for 24 h. Control cultures were treated with RPMI 1640 medium only or with 5% of DMSO (v:v), respectively. The results are expressed as the mean and standard deviation of three assays. All concentration points of both compounds showed statistical significance from controls: (****) p ≤ 0.0001.
Figure 4. Effects of LQOFG-1 and FURL-G5 guanidine compounds on the endocytic index (EI) of the Leishmania (V.) braziliensis amastigotes in mice peritoneal macrophages for 24 h. Control cultures were treated with RPMI 1640 medium only or with 5% of DMSO (v:v), respectively. The results are expressed as the mean and standard deviation of three assays. All concentration points of both compounds showed statistical significance from controls: (****) p ≤ 0.0001.
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Molecules 31 00629 g005
Figure 5. In vitro infection of peritoneal macrophages with Leishmania (V.) braziliensis Thor strain and treated with guanidine derivatives. Peritoneal macrophages were seeded and infected with metacyclic promastigotes at a ratio of 10:1 (parasite: macrophage) for 24 h. The figure shows images obtained from light microscopy of control (A) and treated with 10 μM LQOFG-1 (B) and 10 μM FURL-G5 (C) after 24 h, stained using the Panoptic. Scale bar: 50 μm.
Figure 5. In vitro infection of peritoneal macrophages with Leishmania (V.) braziliensis Thor strain and treated with guanidine derivatives. Peritoneal macrophages were seeded and infected with metacyclic promastigotes at a ratio of 10:1 (parasite: macrophage) for 24 h. The figure shows images obtained from light microscopy of control (A) and treated with 10 μM LQOFG-1 (B) and 10 μM FURL-G5 (C) after 24 h, stained using the Panoptic. Scale bar: 50 μm.
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Table 1. Key physicochemical features of guanidine derivatives.
Table 1. Key physicochemical features of guanidine derivatives.
CompoundsMW (g/mol)cLogPcLogSHBAHBDTPSA (Å2)Drug LikenessRB
LQOF-G1374.143.99−4.963296.630.308
FURL-TA325.183.17−4.253254.270.835
FURL-TB280.733.06−3.933254.270.835
FURL-G1406.245.12−5.802266.630.477
FURL-G2406.245.12−5.802266.630.477
FURL-G3467.155.75−6.552266.630.387
FURL-G4422.705.64−6.232266.630.427
FURL-G5337.353.82−4.612266.630.567
MW—molecular weight, cLogP—octanol–water partition coefficient (lipophilicity), cLogS—water solubility, HBA—number of hydrogen bond acceptors, HBD—number of hydrogen bond donors, TPSA—topological polar surface area, RB—rotatable bond.
Table 2. In vitro activity of guanidine derivatives in L. (V.) braziliensis promastigotes and murine peritoneal macrophages.
Table 2. In vitro activity of guanidine derivatives in L. (V.) braziliensis promastigotes and murine peritoneal macrophages.
CompoundPromastigote IC50
(µM)		Peritoneal Macrophage CC50
(µM)		SI
LQOF-G13.47 ± 0.52164.8 ± 11.5947.49
FURL-TA>50>800-
FURL-TB>50>800-
FURL-G16.83 ± 0.3264.86 ± 5.649.49
FURL-G215.36 ± 0.5623.30 ± 2.421.51
FURL-G310.49 ± 1.4919.06 ± 1.201.81
FURL-G4>5017.17 ± 3.69-
FURL-G57.17 ± 0.41124.4 ± 5.6517.35
Amphotericin B0.26 ± 0.0172.61 ± 4.40279.26
NA—no activity; SI—selectivity index.
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Dias-Lopes, G.; Dos Anjos, L.R.; Cruz, S.M.X.d.; Abrão, C.D.; Gonçalves, M.E.P.; Souza-Silva, F.; Fabisikova, A.; González, E.R.P.; Alves, C.R. Biological Effects of Novel Synthetic Guanidine Derivatives Targeting Leishmania (Viannia) braziliensis. Molecules 2026, 31, 629. https://doi.org/10.3390/molecules31040629

AMA Style

Dias-Lopes G, Dos Anjos LR, Cruz SMXd, Abrão CD, Gonçalves MEP, Souza-Silva F, Fabisikova A, González ERP, Alves CR. Biological Effects of Novel Synthetic Guanidine Derivatives Targeting Leishmania (Viannia) braziliensis. Molecules. 2026; 31(4):629. https://doi.org/10.3390/molecules31040629

Chicago/Turabian Style

Dias-Lopes, Geovane, Luana Ribeiro Dos Anjos, Sara Maria Xavier da Cruz, Cauã Dias Abrão, Maria Eduarda Pinto Gonçalves, Franklin Souza-Silva, Anna Fabisikova, Eduardo Rene Perez González, and Carlos Roberto Alves. 2026. "Biological Effects of Novel Synthetic Guanidine Derivatives Targeting Leishmania (Viannia) braziliensis" Molecules 31, no. 4: 629. https://doi.org/10.3390/molecules31040629

APA Style

Dias-Lopes, G., Dos Anjos, L. R., Cruz, S. M. X. d., Abrão, C. D., Gonçalves, M. E. P., Souza-Silva, F., Fabisikova, A., González, E. R. P., & Alves, C. R. (2026). Biological Effects of Novel Synthetic Guanidine Derivatives Targeting Leishmania (Viannia) braziliensis. Molecules, 31(4), 629. https://doi.org/10.3390/molecules31040629

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