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Frontiers in Chemical Biology ·

Volume 5 - 2026 | https://doi.org/10.3389/fchbi.2026.1888764

Second-generation N,N′-disubstituted aliphatic diamines: discovery of potent hits for multi-species leishmaniasis treatment

  • Alejandro I. Recio-Balsells1
  • Esteban A. Panozzo-Zenere1
  • Elizabeth M. Coser2
  • Bianca A. Ferreira2
  • Adriano Cappellazzo Coelho2
  • Shane R. Wilkinson3
  • Babu L. Tekwani4
  • Guillermo R. Labadie1,5*corresponding author
  1. 1Instituto de Química Rosario (IQUIR-CONICET-UNR), Universidad Nacional de Rosario, Rosario, Argentina
  2. 2Departamento de Biologia Animal, Instituto de Biologia, Universidade Estadual de Campinas (UNICAMP), Campinas, Brazil
  3. 3School of Biological and Chemical Sciences, Queen Mary University of London, London, United Kingdom
  4. 4National Center for Natural Products Research & Department of Biomolecular Sciences, School of Pharmacy, University of Mississippi, Oxford, MS, United States
  5. 5Departamento de Química Orgánica, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Rosario, Argentina
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Abstract

Kinetoplastid diseases remain a major global health challenge, highlighting the need for new antiparasitic chemotypes. N,N′ -disubstituted aliphatic diamines have emerged as a promising scaffold against trypanosomatid parasites. Building on our previous studies, we expanded the structure–activity relationship (SAR) of this chemotype through the synthesis of fifty-two analogues obtained by one-pot reductive amination of aliphatic diamine linkers with structurally diverse aromatic and heteroaromatic aldehydes. The compounds were first evaluated against the extracellular stages of Trypanosoma cruzi, Trypanosoma brucei, and Leishmania donovani. Several derivatives displayed pIC 50 values above 6.0 and Vero-cell selectivity index above 10. Based on these results, nine hits were further evaluated against the promastigote and amastigote stages of L. amazonensis, L. braziliensis, and L. infantum, responsible for cutaneous and visceral leishmaniasis. Among them, compounds 12a, 12m, and 8e showed potent activity against intracellular amastigotes with selectivity indices above 10, highlighting this scaffold as a promising starting point for antileishmanial drug discovery.

Keywords

1 Introduction

Diseases caused by protozoa of the family Trypanosomatidae remain a major global health challenge, particularly in tropical and subtropical regions where structural inequalities amplify their impact (World Health Organization WHO, 2021). Human African trypanosomiasis (HAT), Chagas disease, and leishmaniasis are caused by Trypanosoma brucei gambiense or T. b. rhodesiense, T. cruzi, and several Leishmania species, respectively. Although progress in surveillance and vector control has reduced the incidence of HAT, the burden of chronic Chagas disease and visceral leishmaniasis remains high. According to World Health Organization (WHO) estimates, approximately 700,000 to 1 million new cases of leishmaniasis occur annually (World Health Organization, 2026a), around 8 million people are infected with T. cruzi (World Health Organization, 2026b), and fewer than 1,000 new cases of HAT are reported each year (World Health Organization, 2026c).

Available treatments are limited by toxicity, prolonged administration, stage-specific efficacy, and emergence of drug resistance. In particular, therapeutic options for chronic Chagas disease and leishmaniasis remain suboptimal, highlighting the need for new molecular entities acting through validated and parasite-selective mechanisms (Pathak et al., 2023; Olías-Molero et al., 2021). The DNDi (2026), a non-profit research organization, has had a major impact on the development of therapies for neglected tropical diseases, including those caused by trypanosomatid parasites. This example highlights the importance of collaborative initiatives in advancing anti-trypanosomatid drug discovery, particularly for diseases that require substantial investment in late-stage clinical development (Ma et al., 2023). Regarding leishmaniasis, DNDi, in collaboration with other partners, has developed several promising small-molecule leads that have advanced to clinical stages, including LXE408 (Nagle et al., 2020), DNDI-6148 (Mowbray et al., 2021), DNDI-6899 (Thomas et al., 2019) and DNDI-6174 (Braillard et al., 2023). In contrast, the drug pipeline for Chagas disease has remained constrained. Nevertheless, the oxaborole scaffold is emerging as a promising source of clinical candidates, with the benzoxaborole DNDI-6148, initially developed for leishmaniasis, also demonstrating promising activity against T. cruzi (DNDi, 2026).

Leishmaniasis comprises a spectrum of diseases caused by several Leishmania species and manifests in three main clinical forms: cutaneous leishmaniasis, characterized by skin lesions; mucocutaneous leishmaniasis, affecting the mucosa of the nose, mouth, and throat; and visceral leishmaniasis, the most severe form, which affects the spleen, liver, and bone marrow and is fatal if left untreated. These manifestations are mainly associated with the infecting Leishmania species. For example, visceral leishmaniasis is primarily caused by L. donovani and L. infantum, whereas cutaneous and mucocutaneous forms are typically associated with species such as L. major, L. tropica, L. braziliensis, L. mexicana, and L. amazonensis (Selvapandiyan et al., 2025). These species also display distinct geographical distributions: L. major, L. tropica, and L. donovani are prevalent in Africa and Eurasia, while L. mexicana, L. amazonensis, and L. braziliensis are endemic in the Americas. In this context, target product profiles (TPPs) play a crucial role in guiding antiparasitic drug discovery efforts, emphasizing the importance of identifying lead molecules with activity against a broad spectrum of Leishmania species (DNDi, 2026; Rao et al., 2019).

Chemical biology approaches to drug discovery seek to exploit mechanistic vulnerabilities in pathogen metabolism through the rational design of small molecules capable of perturbing essential biological pathways. In trypanosomatids, polyamine homeostasis represents one such vulnerability, as polyamines play critical roles in DNA stabilization, translation, stress adaptation, and cell proliferation (Phillips, 2018). Importantly, spermidine is required for the biosynthesis of trypanothione, a unique dithiol that replaces glutathione as the major intracellular thiol in trypanosomatids (Fairlamb et al., 1985). This trypanothione-dependent redox network is absent in mammalian cells, creating a distinctive pathogen-selective biochemical node that can be selectively targeted. Moreover, the clinical success of eflornithine, an inhibitor of ornithine decarboxylase, validates polyamine metabolism as a tractable therapeutic target against trypanosomatids (Bacchi et al., 1980). Furthermore, several polyamine-based drug discovery programs have yielded compounds with antileishmanial activity including, 3-aminooxy-1-aminopropane (APA) (Singh et al., 2007), γ-guanidinooxypropylamine (GAPA) (Singh et al., 2008), spermine analogs (Mukhopadhyay and Madhubala, 1995), N-arylspermidines (Mollo et al., 2025) and pyridine heterocyclic diamines (Martín-Montes et al., 2021), among others (Figure 1).

Polyamine-based compounds active against *Leishmania spp*. Legend: *Ld: L. donovani, Lb: L. Braziliensis, Li: L. Infantum, (p):* promastigote, *(a)*: amastigote.
Figure 1. Polyamine-based compounds active against Leishmania spp. Legend: Ld: L. donovani, Lb: L. Braziliensis, Li: L. Infantum, (p): promastigote, (a): amastigote.

Collectively, these studies demonstrate that structurally diverse diamine-based scaffolds constitute a promising source of antitrypanosomatid agents and provide a rationale for the development of simplified synthetic analogues with improved drug-like properties. Inspired by this biological framework, we previously developed a library of N,N′-disubstituted aliphatic diamines designed as simplified and synthetically accessible polyamine-inspired scaffolds. Several compounds exhibited pIC50 values above 6.0 and SI values above 10 against the extracellular stages of T. cruzi, T. brucei, and L. donovani (Caminos et al., 2012). In subsequent studies, several diamines also exhibited potent activity against the clinically relevant intracellular amastigote stage of T. cruzi (Recio-Balsells et al., 2026). Furthermore, we reported the expanded synthesis of second-generation analogues and also characterized their activity against apicomplexan parasites (Recio-Balsells et al., 2025). These compounds exhibited submicromolar activity against resistant strains of Plasmodium falciparum. These findings established this scaffold as a valuable starting point for antiparasitic drug discovery.

Building on these findings, the aim of the present study was to define the antitrypanosomatid structure–activity relationships of this second-generation diamine library and to identify lead compounds for expanded evaluation against clinically relevant Leishmania species.

Accordingly, we evaluated the fifty-two second-generation N,N′-disubstituted diamines against the extracellular stages of T. cruzi, T. brucei, and L. donovani. Based on their antiparasitic activity, selectivity indices, and chemical diversity, nine analogues were advanced for further evaluation against the promastigote and amastigote stages of additional Leishmania species, including L. amazonensis, L. braziliensis, and L. infantum responsible for cutaneous and visceral leishmaniasis.

2 Results and discussion

2.1 Chemistry

We previously reported the synthesis of a library of thirty N,N′-disubstituted aliphatic diamines that demonstrated significant activity against trypanosomatid parasites (Supplementary Table S2, Supplementary Material) (Caminos et al., 2012). The synthetic strategy, based on a one-pot reductive amination between 1,ω-aliphatic diamines and substituted aromatic aldehydes, was described in detail in our earlier work and is therefore not reiterated here. Briefly, the parent collection incorporated aliphatic diamine chain lengths ranging from three to twelve carbon atoms (n = 3, 4, 6, 8, 10, 12) and benzaldehyde derivatives bearing oxygenated substituents such as methoxy and benzyloxy groups.

To further expand the chemical space within this scaffold, we introduced a new panel of aromatic and heteroaromatic aldehydes designed to systematically evaluate steric, electronic, and lipophilic effects (Scheme 1) (Recio-Balsells et al., 2025). A 4-isopropyl substituent was selected to evaluate the impact of increased hydrophobicity relative to the previously explored oxygenated derivatives. In contrast, 4-nitrobenzaldehyde was incorporated to assess the influence of a strong electron-withdrawing group and potential mechanistic parallels with nitro-containing antiparasitic agents such as nifurtimox, benznidazole, and fexinidazole, whose bioactivation in trypanosomatids involves type I nitroreductases absent in mammalian hosts (Wyllie et al., 2016).

*N,N*′-disubstituted aliphatic diamines studied in this work.
Figure 2. N,N′-disubstituted aliphatic diamines studied in this work.

Bulky aromatic systems, including 2-naphthyl and biphenyl aldehydes, were introduced to investigate the effect of extended π-surfaces and increased steric demand. Given the favorable activity observed for oxygenated derivatives in the initial series, 3,4-dibenzyloxybenzaldehyde was selected to evaluate the tolerance for more sterically demanding oxygen-containing substituents. Moreover, heteroaromatic aldehydes (furan-2-carboxaldehyde and pyrrole-2-carboxaldehyde) were incorporated to modulate polarity and electronic distribution while maintaining aromatic character. Finally, to further investigate the influence of aromatic substitution within the most promising long-chain analogues, a focused series of twelve derivatives was synthesized from para-halogenated (4-F, 4-Cl, and 4-Br) and para-methyl-substituted benzaldehydes using aliphatic 1,ω-diamines containing 8-, 10-, and 12-carbon linkers. This design was based on the favorable antitrypanosomatid activity previously observed for the structurally simple analogues 10a and 12a, which identified long-chain diamines as a promising platform for further structural diversification.

2.2 Biology

2.2.1 Activities against extracellular stages of T. cruzi, L. donovani and T. brucei

The collection of fifty-two N,N´-bis-benzyl aliphatic diamines (compounds 3f-3l, 4f-4l, 6f-6l, 8f-8p, 10f-10p and 12f-12p) was tested in vitro against trypanosomatids (T. brucei brucei bloodstream form [trypomastigotes], T. cruzi epimastigotes and L. donovani promastigotes). To assess mammalian cytotoxicity, Vero cell counterscreens were used as a first-pass assay for the complete library, whereas BMDM counterscreens were reserved for prioritized hit compounds. Vero cell data were previously reported, with only compounds 4f, 6f, 8f, 3i, 8n and 10n exhibiting cytotoxicity at concentrations below 4.76 μg mL−1, the highest concentration tested (Recio-Balsells et al., 2025). The biological evaluation revealed clear differences in potency among the different substitution patterns and linker lengths, enabling a detailed structure–activity relationship analysis.

Table 1; Figure 2 summarize the in vitro antiparasitic activity of the second-generation library against trypanosomatid species and cytotoxicity values, with several compounds exhibiting pIC50 values above 6.0 against one or more of the trypanosomatid species evaluated. The selectivity index (SI) was calculated as the ratio of the IC50 for mammalian cytotoxicity to the IC50 for antiparasitic activity.

TABLE 1. Antiparasitic activity of N,N′-disubstituted diamines.

IDnRLd (p) a pIC50Selectivity e Ld/CttTc (e) b pIC50Selectivity e Tc/CttTb c pIC50Selectivity e Tb/CttCtt d pIC50
3f34-Isopropyl6.25>25.16.28>26.66.72>74.2<4.85
4f44-Isopropyl6.1718.76.4334.37.544374.90
6f64-Isopropyl5.582.636.4115.47.542005.22
8f84-Isopropyl6.3113.56.4920.66.8547.15.18
10f104-Isopropyl5.69>5.286.19>16.86.70>54.5<4.96
12f124-Isopropyl5.24>1.76SP-SP-<4.99
3g34-Nitro4.70>0.695.09>1.68<4.50-<4.86
4g44-Nitro5.13>1.815.19>2.085.07>1.54<4.88
6g64-Nitro5.24>2.135.66>5.645.21>1.98<4.91
8g84-Nitro5.40>2.905.75>6.505.70>5.72<4.94
10g104-Nitro5.68>5.115.71>5.516.13>14.5<4.97
12g124-Nitro4.82>0.66SP-SP-<5.00
3h32-Naphtyl5.60>5.406.19>21.16.80>84.4<4.87
4h42-Naphtyl5.89>10.06.21>20.87.22>226<4.89
6h62-Naphtyl5.69>5.896.33>25.57.30>266<4.92
10h102-Naphtyl<4.50-SP-SP-<4.98
12h122-Naphtyl5.44>2.75SP-SP-<5.00
3i34-Biphenyl6.8243.86.4317.57.965915.19
4i44-Biphenyl6.23>19.0SP-SP-<4.95
6i64-Biphenyl5.99>10.4SP-SP-<4.97
8i84-Biphenyl5.42>2.61SP-SP-<5.00
10i104-Biphenyl5.33>2.00SP-SP-<5.03
12i124-Biphenyl4.71>0.45SP-SP-<5.05
3j33.4-diOBn5.68>3.326.12>9.396.62>28.8<5.15
4j43.4-diOBn5.56>2.51SP-SP-<5.16
6j63.4-diOBn5.67>3.094.73>0.356.24>11.4<5.18
8j83.4-diOBn5.44>1.736.15>9.046.13>8.66<5.19
10j103.4-diOBn5.08>0.73SP-SP-<5.21
12j123.4-diOBn4.81>0.38SP-SP-<5.23
3k32-Furanyl<4.50>0.18<4.50-<4.50-<4.69
4k42-Furanyl<4.50>0.174.80>1.205.16>2.75<4.72
6k62-Furanyl<4.50>0.375.22>2.845.16>2.47<4.76
8k82-Furanyl4.56>0.575.64>6.875.55>5.51<4.81
10k102-Furanyl4.79>0.895.80>8.945.74>7.81<4.84
12k122-Furanyl5.32>2.735.25>2.346.11>17.1<4.88
3L32-Pyrrolyl<4.50>0.12<4.50-<4.50-<4.69
4L42-Pyrrolyl<4.50>0.12<4.50-<4.50-<4.71
8L82-Pyrrolyl4.82>1.035.72>8.185.64>6.88<4.80
10L102-Pyrrolyl5.47>4.295.95>12.76.17>21.4<4.84
12L122-Pyrrolyl5.36>3.026.27>24.66.14>18.4<4.88
8m84-Fluoro5.33>2.806.17>19.75.94>11.6<4.88
10m104-Fluoro5.81>7.976.19>18.96.70>61.5<4.91
12m124-Fluoro6.36>26.56.80>71.37.17>170<4.94
8n84-Chloro5.755.006.2315.16.7044.55.05
10n104-Chloro5.876.696.1713.17.171335.05
12n124-Chloro5.72>5.94NT-NT-<4.95
8o84-Bromo5.36>2.276.24>17.36.64>42.9<5.01
10o104-Bromo5.43>2.516.00>9.337.11>120<5.03
12o124-Bromo5.95>7.825.82>5.787.10>111<5.05
8p84-Methyl5.62>5.606.32>28.16.62>56.3<4.87
10p104-Methyl5.88>9.546.21>20.27.06>142<4.90
12p124-Methyl5.71>5.945.99>11.47.08>140<4.94
​​Pentamidine5.21​​​​​​
​​Amphotericin B6.49​​​​​​
​​Nifurtimox​​5.68​5.77​​

Ld (p): L. donovani promastigotes pIC50 growth inhibition assay tested up to a maximum concentration of 100 μM.

Tc (e): T. cruzi epimastigotes pIC50 growth inhibition assay tested at maximum concentration of 30 μM.

Tb: T. brucei trypomastigotes pIC50 growth inhibition assay tested up to a maximum concentration of 30 μM.

Ctt: Cytotoxicity was performed against VERO cells up to a maximum concentration of 4.76 μg/mL.

Selectivity index (SI); SI = IC50 vero cells/IC50 parasites. NT: Not tested. All drug activity assays were performed in triplicate. SP: Solubility issues, some compounds presented solubility problems as soon as the solution in DMSO was in contact with the corresponding culture medium in activity assays against trypanosomatids (12f and 6-12i).

Range plot of the anti-trypanosomatid activity (pIC<sub>50</sub>) for all analogs on *T. cruzi* epimastigotes **(a)**, *T. brucei* trypomastigotes **(b)** *,* and *L. donovani* promastigotes **(c)**.
Figure 3. Range plot of the anti-trypanosomatid activity (pIC50) for all analogs on T. cruzi epimastigotes (a), T. brucei trypomastigotes (b) , and L. donovani promastigotes (c).

Within the new collection, forty-five compounds exhibited pIC50 values above 5.0 against at least one of the parasites evaluated (Figure 3). Of the 4-isopropyl series (f) three of the six analogues exhibited a potent activity (pIC50 values >6.0) against the three trypanosomatid species. Analysis of the 4-nitro series (g) revealed that only one (10g) displayed activity against all the trypanosomatids tested (pIC50 values > 5.5). Among the eighteen bulkier derivatives (series h, i, j), solubility issues were encountered in the T. cruzi and T. brucei assays. Nevertheless, 5 of these analogs displayed pIC50 values >5.5, with 3i being the most active compound of the library against T. brucei (pIC50 = 7.96). It should be noted that the interpretation of the SAR within these bulkier aromatic series is limited by the solubility issues observed for several analogues. Consequently, the apparent activity trends should be interpreted with caution, as differences in aqueous solubility may have contributed to the observed biological profiles. Regarding the heterocyclic derivatives (k, l), their activity was notably underwhelming. Within this category, only one furan (12k) and two pyrrole (10l, 12l) analogs displayed pIC50 values >6.0 for T. cruzi and/or T. brucei. The focused long-chain halogenated and methyl series (m, n, o and p), proved particularly successful being active against the three trypanosomatid species evaluated. Among these analogs, 12m displayed the highest activity against T. cruzi and L. donovani. Against T. brucei, compounds 12m and 10n were the most active members of the series.

Anti-trypanosomatid activity of the analogs with pIC<sub>50</sub> > 5.0.
Figure 4. Anti-trypanosomatid activity of the analogs with pIC50 > 5.0.

The influence of the diamine linker length on antitrypanosomatid activity appears to be a key determinant of biological activity. Among the evaluated series, the 4-isopropyl (f), naphthyl (h), and biphenyl (i) derivatives included some of the most potent compounds, which generally contained diamine linkers of six carbon atoms or fewer. However, interpretation of the SAR within the bulkier aromatic series should be made with caution because several analogues were affected by solubility limitations, particularly as the diamine linker length increased. In contrast, long-chain diamine analogues of the para-halogenated and para-methyl-substituted series (m, n, o, p) exhibited potent antitrypanosomatid activity without any apparent solubility issues. As for the heterocyclic series (k, l), derivatives containing diamine chains of 8 carbons or more displayed highest anti-trypanosomatid activities.

In a more detailed analysis, all isopropyl derivatives (except for 12f) yielded pIC50 values > 6.00 against T. brucei and T. cruzi (Figure 3). Particularly, 4f and 6f displayed significantly higher activity against T. brucei compared to the other analogs of the series with both compound´s exhibiting pIC50 values of 7.54. Regarding L. donovani, only 3 derivatives generated pIC50 values >6.00. Concerning cytotoxicity, all derivatives presented SI > 15 for T. brucei and T. cruzi, while for L. donovani, only 3f and 4f achieve this SI threshold.

The bulkier aromatic series also included several compounds with pIC50 values above 6.0, predominantly among analogues containing diamine linkers of six carbon atoms or fewer. Within the naphthyl series, compounds 4h and 6h displayed potent activity against T. brucei (pIC50 = 7.22 and 7.30, respectively) and also showed high activity against T. cruzi (pIC50 = 5.89 and 6.33).

For the biphenyl series, all compounds except 3i displayed solubility issues in the T. cruzi and T. brucei assays, limiting the interpretation of SAR within this series. Compound 3i exhibited the highest activity in the library against T. brucei (pIC50 = 7.96), T. cruzi (pIC50 = 6.43), and L. donovani (pIC50 = 6.82). However, this finding should not be overinterpreted as representative of the biphenyl series, since the remaining analogues were affected by solubility limitations that precluded a comprehensive SAR analysis. The 3,4-diOBn series (j) also displayed antitrypanosomatid activity, with the highest potency observed against T. cruzi and T. brucei. Within this series, compounds 3j, 4j, and 8j were the only analogues that did not present solubility issues in the T. cruzi and T. brucei assays. These compounds also exhibited pIC50 values above 6.0, except for 8j against T. cruzi.

As previously mentioned, the 4-nitro series (g) exhibited limited activity against trypanosomatids. Among these analogues, only compound 10g reached a pIC50 value above 6.00, specifically against T. brucei (pIC5 = 6.13). Nevertheless, the remaining analogues displayed moderate activity against T. cruzi, T. brucei, and L. donovani, with pIC50 values ranging from 4.70 to 5.75, except for compounds 3g and 12g. Within the nitro series, activity generally increased with linker length, reaching its maximum with the C10 analogue.

Most heterocyclic derivatives displayed relatively poor antitrypanosomatid activity; however, potency generally increased with increasing diamine linker length. Among the furan derivatives, 12k showed the highest activity against L. donovani and T. brucei with pIC50 values of 5.32 and 6.11, respectively. Against T. cruzi, 10k was the most active analogue (pIC50 = 5.80). Among the pyrrole derivatives, compounds 12l and 10l displayed the highest activity against T. brucei, with pIC50 values of 6.17 and 6.14, respectively. In addition, 12l also displayed potent activity against T. cruzi (pIC50 = 6.27).

The focused long-chain para-halogenated (m–o) and para-methyl-substituted (p) series displayed consistently high antitrypanosomatid activity, with pIC50 values ranging from 5.33 to 7.17. Among these analogs, ten exhibited pIC50 values above 6.0 against T. brucei, nine against T. cruzi, and one against L. donovani. Within the synthesized para-fluoro series (C8–C12), activity increased consistently with increasing diamine chain length, with compound 12m emerging as the most potent analogue, displaying pIC50 values of 6.36, 6.80, and 7.17 against L. donovani, T. cruzi, and T. brucei, respectively. In contrast, no consistent chain length-dependent trend was observed within the synthesized C8–C12 analogues of the para-chloro (n), para-bromo (o), or para-methyl (p) series.

Collectively, these results revealed distinct structure–activity relationship patterns across the different aromatic series, highlighting the influence of both aromatic substitution and diamine linker length on antitrypanosomatid potency. Overall, the diamine analogues displayed the highest activity against T. brucei, followed by T. cruzi and, to a lesser extent, L. donovani. Among the compounds tested, 16 exhibited superior potency against T. brucei relative to the reference drug nifurtimox (pIC50 = 5.77), while 17 outperformed nifurtimox against T. cruzi (pIC50 = 5.68). In the case of L. donovani, only compound 3i displayed greater activity than amphotericin B (pIC50 = 6.49), although 57% of the library exhibited higher activity than pentamidine (pIC50 = 5.21).

2.2.2 Potency of selected hits against promastigote and amastigote of Leishmania spp. and cytotoxicity assays in BMDMs

To further explore the antileishmanial potential of this scaffold, nine representative derivatives from the first- and second-generation N,N′-disubstituted aliphatic diamine libraries were advanced for expanded biological evaluation. The selected compounds captured the most promising activity profiles while preserving the structural diversity identified during the SAR analysis, and were chosen based on antiparasitic potency, selectivity indices, and chemical diversity. From the first-generation library, the a 4-benzyloxy derivative 4c, the most active compound against L. donovani promastigote, with a pIC50 of 7.51 and broad trypanosomatid activity. Compound 8c was additionally selected to evaluate the influence of a longer diamine linker while maintaining good antiparasitic activity. The 3-methoxy-4-benzyloxy series (e) also emerged as a promising chemotype, displaying pIC50 values above 6.0 against L. donovani promastigotes as well as the other trypanosomatids evaluated. Because biological activity was comparable among analogues bearing diamine linkers from C3 to C8, compounds 4e and 8e were selected to enable direct comparison with the corresponding 4-benzyloxy derivatives (4c and 8c). Finally, compounds 12a, 12m, and 10g were selected as representative long-chain analogues combining high antiparasitic potency, excellent selectivity indices, and structurally simple aromatic substitution patterns.

To determine the broader therapeutic potential of this scaffold, we expanded our evaluation to include both the promastigote and the clinically relevant intracellular amastigote stage across a panel of Leishmania spp., including L. amazonensis, L. braziliensis, and L. infantum. These experiments assessed intracellular activity in a macrophage infection model across multiple Leishmania species, providing an initial indication of the scaffold’s potential to target parasites associated with both cutaneous and visceral leishmaniasis. Biological evaluation of the selected hits is summarized in Table 2; Figure 4. Overall, the selected hits displayed higher activity against the amastigote stage compared to the promastigote stage. Notably, all compounds exhibited pIC50 values above 6.00 against the amastigote stage across the three Leishmania spp. tested, except for compounds 4e and 10g against L. braziliensis. Moreover, several compounds, 8e, 12m, 10g, and 6i, displayed pIC50 values above 7.0 against two species. Interestingly, the nitro-analog 10g exhibited pIC50 values of 7.40 and 7.10 against the amastigote stage of L. amazonensis and L. infantum, respectively, but showed a substantial decrease in activity (pIC50 < 5.40) against L. braziliensis. This species-dependent difference may reflect variations in nitroreductase expression or activity, or other intrinsic determinants of nitro-compound susceptibility. However, this hypothesis remains speculative and will require dedicated mechanistic studies to establish its biological basis. Distinct profiles in drug sensitivity among Leishmania species are a recognized phenomenon across multiple antileishmanial agents (Zauli-Nascimento et al., 2010; Coser et al., 2021). In contrast, against the promastigote stage, only four hits showed pIC50 values above 6.0 for L. amazonensis and L. infantum, and five for L. braziliensis. Overall, these results demonstrate that the selected diamine analogues retain high levels of activity against the clinically relevant intracellular amastigote stage across multiple Leishmania species, despite the generally lower potency observed against promastigotes.

TABLE 2. Biological evaluation of selected hits against a panel of Leishmania spp. Endemic in South America.

IdnRL. AmazonensisL. braziliensisL. infantum
(p)(a)(p)(a)(p)(a)
4c44-OBn6.746.776.286.666.446.59
8c84-OBn5.736.805.396.705.636.85
4e43-OMe,4-OBn6.476.856.215.846.226.89
8e83-OMe,4-OBn5.727.706.277.466.256.43
12a12H5.726.825.507.005.616.82
12m12F6.246.926.067.005.657.40
10g10NO25.507.405.39<5.405.517.10
6i6Biphenyl5.827.155.736.745.727.00
4h4Naphtyl6.127.526.026.895.586.77
Miltefosine​​4.775.714.465.554.755.89

Legend: (p), promastigotes; (a), amastigotes.

Selective hits activity against *Leishmania* spp. La(p): *L. amazonensis* promastigotes*,* La(a): *L. amazonensis* amastigotes*,* Lb(p): *L. braziliensis* promastigotes, Lb(a): *L. braziliensis amastigotes*, Li(p): *L. infantum* promastigotes*,* Li(a): *L. infantum* amastigotes.
Figure 5. Selective hits activity against Leishmania spp. La(p): L. amazonensis promastigotes, La(a): L. amazonensis amastigotes, Lb(p): L. braziliensis promastigotes, Lb(a): L. braziliensis amastigotes, Li(p): L. infantum promastigotes, Li(a): L. infantum amastigotes.

Among the selected 4-OBn derivatives, compound 4c exhibited comparable activity against the three Leishmania species at both parasite stages, with pIC50 values ranging from 6.20 to 6.77. In contrast, compound 8c displayed a clear preference for the amastigote stage, exhibiting slightly higher activity than 4c. Within the 3-OMe,4-OBn series, compound 8e displayed the highest activity among the selected hits against the amastigote stage of L. amazonensis and L. braziliensis, with pIC50 values of 7.70 and 7.46, respectively. Compared with 8c, this increase in activity suggests a favorable contribution of the 3-OMe substituent to antileishmanial activity. Nonetheless, 8e was less active against L. infantum amastigotes, although it still maintained a pIC50 value of 6.43. Compound 4e showed activity comparable to that of 4c, except for a pronounced decrease in activity against L. braziliensis amastigotes.

Among the long-chain analogues, compounds 12a and 12m displayed broadly comparable activity profiles, although the para-fluoro derivative 12m consistently exhibited higher potency. In particular, 12m displayed the highest activity against L. infantum amastigotes with a pIC50 of 7.40, as well as strong activity against L. braziliensis and L. amazonensis amastigotes, with pIC50 values of 7.00 and 6.92, respectively. The nitro derivative 10g exhibited outstanding activity against L. amazonensis and L. infantum amastigotes, but markedly lower activity against L. braziliensis.

The bulky aromatic derivatives 6i and 4h also displayed comparable activity profiles. Compound 4h was more active against L. amazonensis and L. braziliensis, whereas 6i exhibited higher potency against L. infantum.

Although these results demonstrate potent activity against intracellular amastigotes, it should be noted that, as with any intracellular infection model, these assays do not exclude a contribution of host cell-mediated parasite killing. Therefore, additional mechanistic studies will be required to distinguish direct antiparasitic activity from host-mediated effects.

Finally, to further assess compound selectivity under physiologically relevant conditions, the selected hit compounds were evaluated for cytotoxicity against BMDMs, the same host cells used in the intracellular amastigote assays. The selectivity index (SI) based on BMDMs was considered the primary selectivity metric for intracellular assays, whereas the SI determined in Vero cells was used as supportive information. Cytotoxicity data obtained in BMDMs, together with the previously determined values in Vero cells and the corresponding SI values, are summarized in Table 3. The SI values reported in Table 1 were derived from Vero cells using a maximum tested concentration of 4.76 μg mL−1 and should therefore be interpreted as lower bounds when the CC50 was not reached. All compounds displayed measurable cytotoxicity against BMDMs, with pEC50 values ranging from 5.52 to 6.25. Nevertheless, compounds 8c, 12a, and 12m exhibited SI values above 10 against the amastigote stage of the three species when compared with BMDMs. Regarding selectivity relative to Vero cells, all compounds showed SI values above 10, except for 10g and 4e against L. braziliensis, which is consistent with their lower antiparasitic activity against this species. The higher cytotoxicity observed in BMDMs compared with Vero cells may reflect intrinsic biological differences between primary macrophages and immortalized epithelial cells. Macrophages display high constitutive endocytic and phagocytic activity, which may increase intracellular exposure to the compounds, whereas Vero cells exhibit distinct physiological and uptake characteristics. Similar differences between primary cells and immortalized cell lines have been widely reported in antiparasitic drug discovery (Horvath et al., 2016). Accordingly, for intracellular infection assays, the selectivity indices calculated using BMDMs provide a more physiologically relevant assessment of compound selectivity than those obtained using Vero cells.

TABLE 3. Selectivity index of the selected hits against Leishmania spp.

CompoundBMDMs pEC50Vero cellspEC50SI La (a) BMDMSI Lb (a) BMDMSI Li (a) BMDMSI La (a) veroSI Lb (a) veroSI Li (a) vero
4c6.105.234.73.63.0734.326.522.4
8c5.52<5.0518.815.121.5>55.4>44.4>63.4
4e5.97<5.067.60.78.15>62.9>6.1>67.7
8e5.985.3452.530.02.83226.0129.112.2
12a5.67<4.9014.421.614.4>83.3>125.0>83.3
12m5.64<4.9418.922.756.8>95.0>114.0>285.0
10g5.75<4.9744.0-22.0>270.0->135.0
6i5.76<4.9724.69.617.6>151.4>58.9>106.0
4h6.25<4.8918.64.30.43>430.0>99.2>75.9
Miltefosine4.33-24.016.635.9---

La (a): L. amazonensis amastigotes. Lb (a): L. braziliensis amastigotes. Li (a): L. infantum amastigotes. SI: Selectivity index. EC50 (BMDMs, or Vero cells)/IC50 (amastigote). Red: SI < 1; Yellow 1≤IS < 10; Green ≥10.

The physicochemical properties of the second-generation library were previously estimated (Recio-Balsells et al., 2025). Table 4 summarizes the relevant physicochemical parameters of the selected hit compounds, as calculated using the SwissADME platform (Daina et al., 2017). Given the potent activity of nearly all selected compounds against the intracellular amastigote stage of the three Leishmania species, the selectivity indices together with the predicted physicochemical properties were used to prioritize the most promising hit compounds.

TABLE 4. Physicochemical parameters estimations of the selected hits.

CompoundLipinskiWater sol. (μM)#SI La (a) BMDMSI Lb (a) BMDMSI Li (a) BMDMSI ≥ 10
4c✓0.79 (PS)4.73.63.07✗
8c✗0.05 (PS)18.815.121.5✓
4e✓0.06 (PS)7.60.78.15✗
8e✗0.004 (PS)52.530.02.83✗
12a✓1.50 (MS)14.421.614.4✓
12m✓0.71 (PS)18.922.756.8✓
10g✓5.33 (MS)44.0-22.0✗
6i✓0.19 (PS)24.69.617.6✗
4h✓2.70 (MS)18.64.30.43✗

Water sol. (μM)#: ESOL water solubility estimation (Swiss ADME).

The LogS scale is defined as: insoluble (< −10), poorly soluble (−10 to −6), moderately soluble (−6 to −4), soluble (−4 to −2), very soluble (−2 to 0), and highly soluble (>0). MS: moderately soluble, PS: poorly soluble. SI: selective index.

La: L. amazonensis. Lb: L. braziliensis. Li: L. infantum.

Among the compounds exhibiting the highest selectivity índices (8c, 12a and 12m) the benzyl-unsubstituted analog 12a showed the most favorable profile, complying with the Lipinski rule and displaying an estimated moderate aqueous solubility. Compound 12m also complied with these criteria, although it was predicted to be poorly soluble. In contrast, 8c did not comply with Lipinski’s rule and was also predicted to have poor aqueous solubility. Importantly, it is important to note that for cutaneous leishmaniasis, topical administration could be considered as an alternative to oral delivery. Furthermore, the structure–activity relationship findings obtained in this study provide a valuable basis for a new round of scaffold optimization aimed at improving aqueous solubility while maintaining antileishmanial activity and selectivity.

Overall, compound 12a emerged as the most balanced hit, combining broad-spectrum antileishmanial activity, favorable selectivity indices, compliance with Lipinski’s rule, and the most favorable predicted physicochemical profile among the compounds evaluated. Nevertheless, compound 12m also represents an attractive lead, as it combined excellent antileishmanial potency and selectivity with a para-fluoro substitution that may confer improved metabolic stability, making it an appealing candidate for further biological evaluation. Accordingly, future optimization efforts will focus on reducing lipophilicity while preserving antileishmanial potency and selectivity through rational structural modifications, including the incorporation of additional polar functional groups and/or a reduction in the aromatic content of the scaffold.

3 Conclusions

In summary, the second-generation N,N′-disubstituted diamine library was evaluated against the extracellular stages of L. donovani, T. brucei, and T. cruzi. The screening identified several highly potent antitrypanosomatid analogues and established distinct structure–activity relationship (SAR) trends across the different aromatic series. Among the parasites evaluated, T. brucei was the most susceptible species, followed by T. cruzi, whereas L. donovani displayed comparatively lower sensitivity.

The newly synthesized compounds expanded the structure–activity relationship (SAR) landscape of this scaffold, revealing a clear interplay between diamine linker length and the structural characteristics of the terminal aromatic substituent. In general, several bulkier aromatic substituents favored activity in analogues bearing short diamine linkers, whereas longer linkers (particularly C10 and C12) were generally associated with improved activity in unsubstituted benzyl, para-halogenated, nitro, and heteroaromatic derivatives.

To further advance this scaffold within the antileishmanial drug discovery pipeline, nine representative compounds were selected for expanded evaluation against L. amazonensis, L. braziliensis, and L. infantum in both promastigote and intracellular amastigote stages. Several analogues displayed pIC50 values above 6.0 across all three species, with generally higher potency against the clinically relevant intracellular amastigote stage. Cytotoxicity assays performed in BMDMs, the corresponding host cells, identified compounds 8c, 12a, and 12m as the most selective members of the series, each exhibiting selectivity indices above 10.

Overall, these findings demonstrate the broad antitrypanosomatid, and particularly antileishmanial, potential of this diamine scaffold. Compound 12a emerged as the most balanced lead, combining broad-spectrum activity, favorable selectivity, and predicted drug-like properties, whereas compound 12m represents an attractive complementary lead owing to its excellent antileishmanial potency and the potential pharmacokinetic advantages associated with para-fluorine substitution. Together with compound 8c, these analogues provide a strong foundation for further lead optimization and preclinical evaluation within the Leishmania drug discovery pipeline.

4 Experimental section

4.1 Biological assays against parasites

4.1.1 In vitro activity against T. brucei brucei

T. brucei (MIT at 427 strain; clone 221a) bloodstream form trypomastigotes were seeded at 1 × 103 mL−1 in 200 μL modified Iscove´s medium containing different concentrations of the compounds. After incubation at 37 oC under a 5% CO2 atmosphere for 3 days, 20 μL Alamar blue (Biosource UK Ltd.) was added to each well and the plates were further incubated at 37 °C for 16 h. The fluorescence of each culture was determined using a Germini Fluorescent Plate reader (Molecular Devices) at an excitation wavelength of 530 nm, emission wavelength of 585 nm and a filter cut off at 550 nm, and the IC50 established. The color change resulting from the reduction of Alamar blue is proportional to the number of live cells, which was established following production of a standard curve.

4.1.2 In vitro activity against T. cruzi epimastigotes

T. cruzi (clone CL-Brener) epimastigotes were seeded at 5 × 105 mL−1 in 200 µL modified RPMI-1640 medium (without glutamate) (Martinez et al., 2023) containing different concentrations of diamine and 20 µL Alamar blue (Biosource UK Ltd.) was added to each well. After incubation at 27 °C for 14 days, the cell densities were determined by monitoring the fluorescence of each culture as described above and the IC50 value for each compound established.

4.1.3 In vitro activity against L. donovani promastigotes

Leishmania donovani promastigotes of the S1 Sudan strain (2 × 106 cell mL−1) were cultured at 26 °C in plastic flasks (25 cm2), containing RPMI-1640 medium (without sodium bicarbonate and sodium pyruvate) with 10% (v/v) fetal bovine serum. Subculture of L. donovani promastigotes twice a week, with highest concentration cells in the range of 20–25 × 106 promastigotes mL−1. Antileishmanial activity of the compounds was tested in vitro on a culture of L. donovani promastigotes. In a 96-well microplate the compounds with appropriate dilution were added to the promastigotes culture (2 × 106 cell mL-1) to get the final concentrations of 40, 8 and 1.6 μg mL−1. The plates were incubated at 26 °C for 72 h and growth was determined using Alamar blue assay (Mikus et al., 2000). Pentamidine and Amphotericin B were used as the standard antileishmanial and DMSO was used as negative control. IC50 values were obtained by non-linear regression of dose response logistic functions, using the Microsoft Excel-based plug-in XLfit. The assays were performed in triplicate.

4.1.4 In vitro activity against L. amazonensis, L. braziliensis and L. infantum promastigotes

Promastigotes of L. amazonensis (MHOM/BR/1973/M2269), L. braziliensis (MHOM/BR/94/H3227) and L. infantum (MHOM/BR/1972/LD) were grown at 25 °C in 199 medium (M199) (Sigma-Aldrich) supplemented with HEPES 40 mM [pH 7.4], adenine 0.1 mM, hemin 0.005%, 10% heat-inactivated fetal bovine serum and 100 μg mL−1 of penicillin/streptomycin (Kapler et al., 1990). For the cultivation of L. braziliensis and L. infantum, M199 was supplemented with 2% sterile human urine (Howard et al., 1991).

The susceptibility of Leishmania promastigotes to compounds was determined using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoline bromide) colorimetric assay, after incubation of 2 × 106 promastigotes per well in a 96-well plate in M199 (Sigma-Aldrich) for 24 h at 25 °C in the presence of increasing concentrations of the each compound (0, 0.07, 0.15, 0.31, 0.62, 1.25, 2.5 and 5 μM), as described previously (Caminos et al., 2012). The experiments were carried out in triplicate and at least three independent experiments were performed for each compound. The 50% effective concentration (IC50) was determined by sigmoidal regression curves obtained by the GraphPad Prism 7 software.

4.1.5 In vitro activity against L. amazonensis, L. braziliensis and L. infantum amastigotes

To determine the susceptibility of intracellular Leishmania amastigotes to each compound, bone-marrow derived macrophages (BMDMs) were differentiated as previously described (Zamboni and Rabinovitch, 2003). Macrophages were plated at a density of 3 × 105 macrophages per well in complete RPMI 1640 medium in 24-well plate. Late-stage promastigotes were used to infect macrophages at a macrophage/parasite ratio of 1:5 for L. amazonensis, 1:20 for and 1:30 for. Four hours after infection at 5% CO2 and temperature of 34 °C for L. amazonensis and L. braziliensis, while the temperature of 37 °C was used for L. infantum, non-internalized promastigotes were removed by washing with warmed PBS. Infected macrophages were maintained in RPMI 1640 medium at different concentrations of each compound (0.05, 0.1, 0.5, 1, 1.5, 2 and 4 μM) for 72 h, 5% CO2 and the corresponding temperature depending on the Leishmania species. Afterwards, macrophages were fixed with methanol and stained with Giemsa. The percentage of infection and number of amastigotes per infected macrophage were determined by counting at least 100 macrophages in three independent experiments that were used to determine IC50 values as described above.

4.2 Cytotoxicity evaluation against BMDMs

For cytotoxicity assays, BMDMs were obtained from female 4 to 5-weeks-old BALB/c mice. Protocol and procedures involving mice for isolation of BMDMs were approved by the Ethics Committee for Animal Experimentation of the Instituto de Biologia, Universidade Estadual de Campinas (UNICAMP) (protocol no. 5719-1/2021).

Macrophages in a 500 µL suspension (3 × 105 macrophages per well) were allowed to adhere to the bottom of 24-well plate for 24 h at 37 °C. After the initial incubation, the macrophages were incubated in complete RPMI 1640 medium with increasing concentrations of each compound used (0.625, 1.25, 2.5, 5, 10 and 20 μM) for 72 h. After this period, the RPMI medium containing each compound was removed and then 100 μL of trypsin was added and incubated for 15 min. Then, 400 μL of PBS was added and 10 μL of cell suspension was added to 10 μL of 0.4% Trypan Blue (Sigma Aldrich Corp., St. Louis, MO, USA). Ten microliters of the resuspension were plated in the Neubauer chamber, followed by counting the total number of viable and non-viable macrophages. Cells that acquired the blue dye were considered non-viable. Experiments were performed in duplicate and at least three independent experiments were performed for each compound. To be considered a low toxicity compound/drug, it must be, at least, 10 times more active against the parasite than for the host cell; in other words, the selectivity index (SI) (CC50 for mammalian cell line/EC50 of the parasite) must be ≥10 (Ioset et al., 2009).

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Supplementary material

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fchbi.2026.1888764/full#supplementary-material

Supplementary material Supplementary material at the source

Footnotes

Edited by: Elizabeth Igne Ferreira, University of São Paulo, Brazil

Reviewed by: Ramon Guerra De Oliveira, Brazilian Biosciences National Laboratory (LNBio), Brazil

Angel Lobo-Rojas, National Center for Research in Energy and Materials, Brazil

Statements

Conflict of interest

The author(s) declared that this work was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

The author GL declared that they were an editorial board member of Frontiers at the time of submission. This had no impact on the peer review process and the final decision.

Funding

The author(s) declared that financial support was received for this work and/or its publication. This work was supported in part by grants from the National Research Council of Argentina, CONICET (PIP 2021-2023 11220200101045CO to G.R.L.); Agencia Nacional de Promoción Científica y Tecnológica, ANPCyT-Argentina (ANPCyT PICT 2021-I-A-00512 awarded to G.R.L.); UNR (Universidad Nacional de Rosario 80020190300101UR); Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) (grant number 2016/21171-6) and the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) (grant number 405235/2021-6). The research leading to these results has, in part, received funding from the UK Research and Innovation via the Global Challenges Research Fund under grant agreement A Global Network for Neglected Tropical Diseases grant number MR/P027989/1. G.R.L. is a member of the scientific staff of CONICET.

Data availability

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

Ethics approval

Protocol and procedures involving mice for isolation of BMDMs were approved by the Ethics Committee for Animal Experimentation of the Instituto de Biologia, Universidade Estadual de Campinas (UNICAMP) (protocol no. 5719-1/2021).

Author contributions

AR-B: Data curation, Writing – original draft, Investigation, Formal Analysis, Writing – review and editing. EP-Z: Investigation, Writing – review and editing, Data curation, Formal Analysis. EC: Data curation, Methodology, Investigation, Writing – review and editing, Formal Analysis. BF: Writing – review and editing, Formal Analysis, Methodology, Investigation, Data curation. AC: Methodology, Writing – review and editing, Conceptualization, Data curation, Investigation, Formal Analysis, Funding acquisition, Resources, Project administration. SW: Resources, Funding acquisition, Formal Analysis, Writing – review and editing, Data curation, Conceptualization, Project administration, Investigation, Methodology. BT: Conceptualization, Investigation, Data curation, Writing – review and editing, Methodology, Resources, Funding acquisition, Formal Analysis, Project administration. GL: Funding acquisition, Resources, Formal Analysis, Project administration, Supervision, Data curation, Writing – review and editing, Methodology, Conceptualization, Writing – original draft.

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Citation

Recio-Balsells, A. I., et al. (2026). Second-generation N,N′-disubstituted aliphatic diamines: discovery of potent hits for multi-species leishmaniasis treatment. Frontiers in Chemical Biology, Volume 5. https://doi.org/
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5 - 2026

Copyright

Copyright © 2026 Recio-Balsells, Panozzo-Zenere, Coser, Ferreira, Coelho, Wilkinson, Tekwani and Labadie.

License: https://creativecommons.org/licenses/by/4.0/

*Correspondence: Guillermo R. Labadie

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