Leishmaniasis remains one of the most burdensome parasitic diseases worldwide. According to World Health Organization estimates cited in the study, roughly 700,000 to 1 million new cases occur each year, with clinical outcomes ranging from disfiguring skin lesions to fatal visceral disease affecting the spleen, liver, and bone marrow. The parasites responsible, including Leishmania donovani, L. infantum, L. amazonensis, and L. braziliensis, are transmitted by sand flies and exist in distinct life-cycle stages: a motile promastigote form in the insect vector and an intracellular amastigote form that survives inside mammalian macrophages. Existing drugs such as miltefosine, amphotericin B, and pentavalent antimonials fall short of ideal profiles, and the clinical pipeline, while recently invigorated by candidates like LXE408 and DNDI-6174 developed through the Drugs for Neglected Diseases initiative, still needs new chemotypes with broad-spectrum coverage.

The research builds on a rational biological premise. Trypanosomatid parasites, the family that includes Leishmania, Trypanosoma cruzi, and Trypanosoma brucei, depend heavily on polyamine metabolism. Polyamines such as spermidine stabilize DNA, support translation, and underpin stress adaptation and cell proliferation. Critically, spermidine is also required to build trypanothione, a unique dithiol that replaces glutathione as the parasites’ major intracellular redox buffer. This trypanothione-dependent network is absent in mammalian cells, creating a pathogen-selective biochemical vulnerability. The clinical success of eflornithine, an inhibitor of ornithine decarboxylase, has long validated polyamine metabolism as a druggable target, and prior diamine-based scaffolds, from aminooxy analogues to N-arylspermidines, have shown antileishmanial activity.

Guided by this framework, the team, led by Guillermo R. Labadie of the Instituto de Química Rosario in Argentina, had previously assembled a first-generation library of thirty N,N′-disubstituted aliphatic diamines. Those compounds, made by one-pot reductive amination of aliphatic diamine linkers with substituted benzaldehydes, showed promising activity against the extracellular stages of T. cruzi, T. brucei, and L. donovani, and some later proved active against the clinically relevant intracellular stage of T. cruzi. The new study extends that chemistry into a second generation of fifty-two analogues, systematically varying both the length of the diamine linker, from three to twelve carbon atoms, and the identity of the terminal aromatic groups.

The synthetic design was deliberate. A 4-isopropyl substituent was introduced to probe the effect of increased hydrophobicity relative to the oxygenated benzyl groups explored earlier. A 4-nitrobenzaldehyde derivative was included to test whether a strong electron-withdrawing group might mimic the behavior of nitro-containing antiparasitic drugs such as nifurtimox, benznidazole, and fexinidazole, whose bioactivation in trypanosomatids relies on type I nitroreductases absent from mammalian hosts. Bulky aromatic systems, including 2-naphthyl and biphenyl aldehydes, were incorporated to examine the influence of extended π-surfaces and steric demand, while heteroaromatic aldehydes based on furan and pyrrole were chosen to modulate polarity and electronic distribution. Finally, a focused series of twelve derivatives built on para-halogenated and para-methyl benzaldehydes with C8, C10, and C12 linkers was synthesized to refine the structure–activity relationship around the most promising long-chain analogues.

Biological screening revealed clear patterns. Forty-five of the fifty-two compounds showed pIC50 values above 5.0 against at least one parasite species, and several exceeded pIC50 6.0, corresponding to submicromolar potency. The parasites differed in susceptibility: T. brucei was the most sensitive, followed by T. cruzi, with L. donovani generally less responsive. Sixteen compounds outperformed the reference drug nifurtimox against T. brucei, and seventeen did so against T. cruzi. Against L. donovani, only compound 3i, a biphenyl-substituted diamine with a three-carbon linker, surpassed amphotericin B, though 57 percent of the library beat pentamidine. The most active compound overall against T. brucei was 3i, with a pIC50 of 7.96 and a selectivity index of 591 relative to Vero cells.

Structure–activity analysis highlighted the interplay between linker length and aromatic substitution. Bulkier aromatic groups, such as naphthyl and biphenyl, tended to favor activity in analogues with shorter linkers of six carbons or fewer, while longer C10 and C12 linkers generally improved activity in the unsubstituted benzyl, para-halogenated, nitro, and heteroaromatic series. Within the para-fluoro series, activity increased consistently with chain length, and compound 12m, bearing a twelve-carbon linker, emerged as the most potent, with pIC50 values of 6.36 against L. donovani, 6.80 against T. cruzi, and 7.17 against T. brucei. The authors note that solubility limitations affected several of the bulkier derivatives, particularly in the T. cruzi and T. brucei assays, and caution that apparent trends in those series should be interpreted carefully.

Based on potency, selectivity, and chemical diversity, nine representative hits were advanced for expanded evaluation against L. amazonensis, L. braziliensis, and L. infantum, covering both promastigote and intracellular amastigote stages in a macrophage infection model. The results were striking: nearly all compounds showed pIC50 values above 6.0 against the amastigote stage across the three species, with generally higher potency against this clinically relevant intracellular form than against promastigotes. Compound 8e, a C8 linker bearing 3-methoxy-4-benzyloxy substituents, achieved the highest activity against L. amazonensis amastigotes at pIC50 7.70 and L. braziliensis amastigotes at pIC50 7.46. Compound 12m was strongest against L. infantum amastigotes at pIC50 7.40. The nitro analogue 10g showed a striking species-dependent profile, with pIC50 values of 7.40 against L. amazonensis and 7.10 against L. infantum but markedly reduced activity below 5.40 against L. braziliensis, a difference the authors speculate may reflect variation in nitroreductase expression or other intrinsic determinants of nitro-compound susceptibility, though they emphasize this hypothesis requires dedicated mechanistic study.

Selectivity was assessed against both Vero cells and bone-marrow-derived macrophages, the same host cells used in the intracellular assays. All compounds showed selectivity indices above 10 in Vero cells except 10g and 4e against L. braziliensis. In the more physiologically relevant BMDM counterscreen, compounds 8c, 12a, and 12m maintained selectivity indices above 10 against amastigotes of all three Leishmania species. The authors note that higher cytotoxicity in primary macrophages compared with immortalized epithelial cells likely reflects intrinsic biological differences, including the high endocytic and phagocytic activity of macrophages that may increase intracellular compound exposure. Combining potency, selectivity, and predicted physicochemical properties calculated with the SwissADME platform, compound 12a, the unsubstituted benzyl analogue with a twelve-carbon linker, emerged as the most balanced lead, complying with Lipinski’s rule and showing moderate predicted aqueous solubility. Compound 12m, with its para-fluoro substitution that may confer improved metabolic stability, represents an attractive complementary lead.

The authors acknowledge that the intracellular infection model cannot fully distinguish direct antiparasitic killing from host cell-mediated effects, and that further mechanistic work will be needed. They also note that for cutaneous leishmaniasis, topical administration could offer an alternative to oral delivery, potentially mitigating concerns about the poor aqueous solubility predicted for several analogues. Future optimization will focus on reducing lipophilicity while preserving potency and selectivity, through the incorporation of additional polar functional groups or a reduction in the aromatic content of the scaffold. For now, the study delivers a validated, synthetically accessible chemotype with demonstrated broad-spectrum activity against multiple Leishmania species and a clear structural map to guide the next round of lead optimization.

Subject of Research: Discovery of second-generation N,N′-disubstituted aliphatic diamines as potent antileishmanial hit compounds

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

Article References: Second-generation N,N′-disubstituted aliphatic diamines: discovery of potent hits for multi-species leishmaniasis treatment. (n.d.). https://doi.org/10.3389/fchbi.2026.1888764

Image Credits: AI Generated

DOI: 10.3389/fchbi.2026.1888764

Keywords: leishmaniasis, N,N′-disubstituted diamines, antiparasitic drug discovery, trypanosomatids, polyamine metabolism, structure-activity relationship, Leishmania amazonensis, Leishmania braziliensis, Leishmania infantum, intracellular amastigotes, neglected tropical diseases, medicinal chemistry