Introduction

Leishmaniasis is a neglected infectious disease caused by over 20 identified protozoans belonging to the genus Leishmania within the Trypanosomatidae family, affecting both humans and animals1,2,3 Leishmaniasis is transmitted by the bite of female phlebotomine sandflies carrying the protozoan. Once transmitted, the parasite enters the body through macrophages located in organs such as the spleen, liver, and bone marrow. Leishmania parasites exhibit dimorphism, with amastigotes inhabiting the mononuclear phagocytic system of mammalian hosts and promastigotes in the vector’s digestive organs1,4,5,6.

Various Leishmania spp. induce diverse clinical presentations of the disease, which are categorized into three primary types: cutaneous leishmaniasis (CL), mucocutaneous leishmaniasis (MCL), visceral leishmaniasis (VL, also referred to as kala-azar)3,7,8. CL, the most prevalent type in numerous affected regions, encompasses a range of diseases with diverse clinical presentations, spanning from minor cutaneous nodules to extensive mucosal tissue damage1,9.MCL is characterized by the invasion of the laryngeal or naso-oropharyngeal mucosa, leading to the formation of ulcers or lesions10,11. VL is the most severe form, characterized by parasites’ invasion of vital organs. This condition presents as a debilitating disease with symptoms including prolonged fever, splenomegaly, pancytopenia, and hypergammaglobulinemia. Patients usually experience a gradual onset of symptoms over several months, and if left untreated, the outcome can be fatal1,12.

Annually, between 700,000 and 1 million new leishmaniasis cases are reported globally13. Leishmaniasis is endemic in over 70 countries worldwide, with 90% of cases concentrated in Afghanistan, Algeria, Brazil, Pakistan, Peru, Saudi Arabia, and Syria. Visceral leishmaniasis is prevalent in 65 countries, with the majority (90%) of cases occurring in agricultural areas and among the impoverished populations of five countries: Bangladesh, Brazil, India, Sudan, and Nepal1,8,14,15,16,17. Human-induced ecological disruptions have contributed to the expansion of leishmaniasis beyond its natural ecotopes. This environmental instability directly impacts the level of human exposure to sandfly vectors, further exacerbating the spread of the disease18.

The traditional treatment approach for leishmaniasis necessitates the use of toxic and poorly tolerated drugs such as Sodium stibogluconate (Pentostam), amphotericin B, pentamidine, meglumine antimoniate (Glucantime), miltefosine, and paromomycin (Fig. 1). However, these medications come with several drawbacks, including adverse effects, high costs, limited efficacy, development of drug resistance, and the requirement for multiple painful injections1,2,19,20,21,22,23. Due to the absence of effective vaccines for preventing leishmaniasis and the limitations of current medications, there is a crucial need for further research to develop new therapeutic agents to enhance the treatment outcomes of individuals with the disease21.

Fig. 1
figure 1

Chemical structures of some current medication agents of leishmaniasis.

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Benzopyran, also referred to as chromene, is a heterocyclic framework characterized by the fusion of a phenyl ring with an oxine ring. This structural motif is a pivotal pharmacophore in various natural compounds, including alkaloids, anthocyanins, flavonoids, polyphenols, and tocopherols24,25. The high lipophilicity of chromene derivatives facilitates their penetration into the cell membrane, enhancing the druggability of synthesized molecules26. The synthesis of chromene derivatives has attracted considerable attention recently, given their remarkable biological and pharmacological properties. Extensive literature reviews indicate that many pharmacologically active compounds contain fused chromenes, exhibiting various biological activities such as anti-proliferative, anti-cancer, antiviral, and antimicrobial effects. Moreover, these compounds find potential applications in the treatment of neurodegenerative diseases such as Alzheimer’s disease and schizophrenia24,25,27,28,29,30,31,32,33.

In a study by Shafi et al.., compounds featuring the β-nitrostyrene scaffold demonstrated promising inhibitory effects against both promastigote and amastigote forms of Leishmania donovani at nanomolar concentrations34. The structural similarity between the 3-nitro-chromene scaffold and the β-nitrostyrene scaffold, along with the encouraging effects of β-nitrostyrene derivatives on leishmaniasis, prompted us to utilize this scaffold in designing potential anti-leishmanial compounds. Previously, these scaffold’s derivatives exhibited cytotoxic effects in MCF-7, T-47D, and MDA-MB-231 cell lines35. This article discusses the synthesis, in silico modeling, and in vitro studies of 15 derivatives of 3-nitro-chromene as potent compounds for treating leishmaniasis.

Methods & materials

Chemistry

The synthesis of β-nitrostyrenes 3a-o was initially conducted using the Henry reaction. This involved reacting various benzaldehydes 2a-o with nitromethane in the presence of methanol and sodium hydroxide. The required β-nitrostyrenes 3a-o were produced with high yields and minimal or no by-product. Compounds with the benzyloxy group are obtained from the benzaldehyde and benzyl chloride derivatives reaction in the presence of K₂CO₃, MeCN, KI, and DMF, as shown in Fig. 2-A. The β-nitrostyrenes 3a-o underwent a reaction with salicylaldehyde in the presence of a 1,4-diazabicyclo[2.2.2]octane (DABCO), resulting in the formation of the corresponding final compounds, 2-aryl-3-nitro-2 H-chromenes 4a-o (Fig. 2-B)36. As detailed in the experimental section, the final product’s structure was confirmed through melting point analysis, FT-IR spectroscopy, 1H NMR and 13C NMR. In this study, we utilized commercially sourced reagents and solvents from Merck Chemical, which were applied as received without any additional purification.

Fig. 2
figure 2

(A) Synthesis of R-benxyloxy benzaldehydes 2i-o from hydroxybenzaldehyde and benzyl chlorides 1i-o. (B) Synthesis of 2-aryl-3-nitro-2 H-chromenes 4i-o from β-nitrostyrenes 3a-o and benzaldehydes 2a-o.

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General procedure for the preparation of r-benzyloxy benzaldehydes 2i-o

One mmol of hydroxybenzaldehyde was mixed with 1.1 mmol of benzyl chloride derivatives (1i-o). Subsequently, 2 g of potassium carbonate, 10 mL of acetonitrile, a small amount of potassium iodide, and 5 mL of DMF were added. The mixture was then refluxed at a temperature of 100–110 °C, stirring for several hours. After ensuring the complete consumption of the benzyl chloride derivatives, the mixture was placed in an ice water bath on the stirrer. After a few minutes, ice water was gradually added to induce precipitation. If no precipitate formed, ethyl acetate and 10% sodium hydroxide were added to the mixture in a separatory funnel to achieve phase separation. The organic phase was then dried with sodium sulfate until it became clumpy and filtered, and the solvent was removed using a rotary evaporator at 90 °C. Recrystallization was subsequently performed. The final product’s structure was confirmed by melting point and TLC analysis.

General procedure for the preparation of β-nitrostyrenes 3a-o

Initially, 5 mmol of aldehyde (2a-o) and 15 to 20 mL of methanol were combined in a flask. Once the aldehyde was fully dissolved, nitromethane (in an amount of 1.5 to 2 times that of the aldehyde) was added to the aldehyde-methanol solution. The flask was then placed on a stirrer in an ice bath at 0 °C. After a short interval, 0.5 mL of a precooled sodium hydroxide solution (made by dissolving 2.1 g of sodium hydroxide in 5 mL of water) was added dropwise to the reaction mixture. Following the sodium hydroxide addition, the reaction temperature was maintained between 10 and 15 °C, and the mixture was stirred in the ice bath for 3 to 4 h. The reaction progress was monitored using thin-layer chromatography (TLC). After 3 to 4 h, 3 mL of ice water was added to the reaction mixture, which was then gradually added to a beaker containing an acid solution (prepared by mixing 1 mL of HCl with 1.5 mL of water). Once a precipitate formed, it was filtered, washed with a small amount of water, and dried. The final product’s structure was confirmed by melting point determination and TLC analysis.

General procedure for the preparation of 2-aryl-3-nitro-2 H-chromenes 4a-o

Initially, 1 mmol of β-nitrostyrene (3a-o) and 1.2 mmol of salicylaldehyde were combined in a flask. Then, 10 mL of acetonitrile and 2 g of DABCO were added. The mixture was stirred in a warm water bath at 40 °C. After several hours, ensuring the complete consumption of the nitrostyrene compounds, ethyl acetate, and 10% sodium hydroxide were added to the mixture in a separatory funnel to achieve phase separation. The aqueous phase was then treated with sodium sulfate and filtered. The solvent was removed using a rotary evaporator at 90 °C. During the slow addition of water, a precipitate formed. The final product’s structure was confirmed by melting point and spectroscopic methods (IR and NMR).

Initially, 1 mmol of β-nitrostyrene and 1.2 mmol of salicylaldehyde were combined in a flask. Then, 10 mL of acetonitrile and 2 g of DABCO were added. The mixture was stirred in a warm water bath at 40 °C. After several hours, ensuring the complete consumption of the β-nitrostyrene, ethyl acetate, and 10% sodium hydroxide were added to the mixture in a separatory funnel to achieve phase separation. The aqueous phase was then treated with sodium sulfate and filtered. The solvent was removed using a rotary evaporator at 90 °C. During the slow addition of water, a precipitate formed. The final product’s structure was confirmed by melting point and spectroscopic methods (FT-IR and 1H and 13C NMR).

Structural analysis

Melting points were determined using a Kofler hot-stage apparatus (Keison, Chelmsford, UK). 1H and 13C NMR spectra were recorded on Bruker 400 or 500 spectrometers, with chemical shifts (δ) reported in ppm relative to TMS as an internal standard. Coupling constants (J) are reported in Hz, and spin multiplicities are indicated as s (singlet), d (doublet), t (triplet), and m (multiplet). IR spectra were obtained using a Nicolet Magna FT-IR 550 spectrophotometer with potassium bromide disks. Mass spectra were recorded on an Agilent Technology (HP) mass spectrometer operating at an ionization potential of 70 eV.

Biology

Preparation of serial concentrations of test compounds

For each synthesized compound, serial solutions with 12.5, 25, 50, and 100 µg/ml concentrations were prepared for further evaluation of their anti-leishmanial effects.

Cell lines and cell culture

This study used promastigote cells of the standard strain L. tropica and human monocytes (THP-1) that differentiation to macrophages. The cell lines were purchased from the Iranian Biological Resource Center (IBRC, Tehran, Iran). The cells were cultured in RPMI 1640 medium (GibcoeBRL, UK) enriched with 10% fetal bovine serum (FBS) and 5% penicillin (both from GibcoeBRL, UK) at 24 °C for L. tropica and 37 °C for THP-1 cells, with 5% CO2 ​in an incubator.

MTT assay

The effects of various concentrations of the synthesized compounds on the metabolic activity of L. tropica promastigote and macrophages cells were evaluated using the MTT colorimetric assay. MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] is a yellow tetrazolium salt that undergoes reduction by the metabolic activities of living cells via mitochondrial dehydrogenase enzymes. This reduction process results in the formation of insoluble purple formazan crystals. Subsequently, these crystals are dissolved in a suitable solvent (such as DMSO) and quantified using spectrophotometric methods. The amount of formazan crystals formed can indicate the percentage of metabolic activity and cell viability37,38. In brief, The cells were harvested in a complete growth medium to achieve a total cell count of 5 × 105 cells/ml (L. tropica promastigotes); the cell suspension was seeded in 96-well plates, and the plates were incubated overnight in an incubator. 24 h after seeding, 100 µL of medium containing 10 µl of different concentrations of the test compounds was pipetted into each well in triplicate. Every plate included three control wells (cells without test compounds) and three blank wells (medium containing 1% DMSO) serving as negative controls for cell viability. After 72 h of treatment,10 µL of MTT solution was added to each well and incubated for 4 h. Subsequently, the culture medium was replaced with 100 µL of DMSO to dissolve the formazan crystals. Using a multi-well plate reader, the Absorbance was measured at 490 nm. The IC50 values, compared to the control, were calculated using nonlinear regression analysis and expressed as mean ± SD.

Amastigote inhibition assay

In order to evaluate the amastigote inhibition, two coverslips were placed in each of the 12 cm plates, and then 100 µl of human macrophage THP-1 cells suspension containing a count of 50,000 cells were added to each coverslip. Then, the plates were incubated in a 37 ◦C incubator with 5% CO2 for 4 h. After removing the supernatant on the coverslips, the counted promastigotes in the growth stationary phase were added to coverslips in plates two and onward using a Neubauer chamber, at a minimum of 5 times the number of macrophages, i.e., 250,000 parasites in 100 µl of culture medium per coverslip. Following a 24-hour incubation period, which allowed the metacyclic promastigotes to invade the macrophage cells, solutions of the test compounds were added to the coverslips from the third plate onward in triplicate. The first plate served as a negative control (medium containing macrophages without parasites and drugs), and the second plate served as a positive control (medium containing macrophages with parasites and standard drug Glucantime). Following the incubation for 72 h, the contents of the coverslips were emptied and dried thoroughly. Once dried, they were fixed with methanol and stained with Giemsa dye. Drug action was assessed by counting amastigotes (the average number of parasites per cell) in the cells by examining 100 cells. Every test was done thrice.

Calculation of the combination index (CI) and evaluation of the synergistic/antagonistic effects of the synthesized compounds

We additionally assessed the combination index (CI) utilizing the formula [CI= (D)/(Dx)1 + (D)/(Dx)2], wherein (Dx)1 and (Dx)2 denote the individual dosages of the drug and MA that elicit a 50% inhibition of growth, and (D) represents the concentration of the drug and MA amalgamation that achieves an equivalent effect. The proportion of 1:1 ratio between the compounds and the standard drug evaluated. The CI values of less than 1, equal to 1, and greater than 1 signify synergistic interactions, additive effects, and antagonistic relationships, respectively. Furthermore, to analyze the drug interactions, isobologram analysis was performed. The IC50 concentrations for the individual dosages of MA and drug were plotted on the x and y axes, correspondingly. The line denoting additivity was constructed by connecting these two coordinates. The theoretical IC50 was computed as [Theoretical IC50 (IC50 MA/2) + (IC50 drug/2)]. Ultimately, the presence of synergy, additivity, or antagonism is discerned based on the positioning of the combination IC50 relative to the line, specifically when it is situated below, on, or above the line, respectively39.

Selectivity index (SI)

The selectivity index (SI) is calculated from the comparison of the drug’s effect on the disease agent (amastigote) and the host cell (macrophage) (CC50/IC50 ≥ 1 = SI).

Statistical analysis

Data analysis was performed using SPSS version 20 software. Differences between control groups or among different concentrations of synthesized compounds were analyzed using analysis of variance (ANOVA) and t-test. IC50 and CC50 values were calculated using probit analysis in SPSS. Data were presented as mean ± standard deviation (SD). Statistical significance was defined at a level of p < 0.05.

Molecular docking

All computational analyses were performed on a 64-bit Windows system with an Intel Core i7-12700 H CPU operating at 2.30 GHz and a GeForce RTX 3050 Ti graphics processor. Ligands selected from in vitro evaluations were initially visualized and depicted as 2D structures using ChemDraw 19.1 software. These structures were then imported into the Molecular Operating Environment (MOE 2019) software for subsequent optimization40. The receptors were obtained in PDB format from the RCSB41 Protein Data Bank (https://www.rcsb.org) andsubsequently imported into the MOE software environment. From this database, 16 key proteins integral to the life cycle of Leishmania were meticulously selected (Table 1). The initial steps involved automatically preparing both the ligand and receptor using QuickPrep. Any minor inconsistencies were subsequently corrected with the “structure preparation” tool42. Finally, the “energy minimize” tool was applied, utilizing the Amber10: EHT forcefield, an R-field value of 1:80, and a cut-off range of9,10,11 to refine the 3D structures of the ligands and optimize the receptor concerning atomic energy levels. This standardized approach was uniformly applied across all molecular docking procedures involving different receptors43,44.

Table 1 List of selected receptors alongside their PDB ID, origin, function and resoulution.
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Throughout all procedures, PDB files were utilized, each including a default primary ligand that acted as a molecular docking control alongside meglumine antimoniate, and facilitated the identification of the receptor’s binding site. To optimize computational efficiency and reduce processing time, identical chains of each protein were excluded from the analysis whenever they did not contribute structural variations to the functional site. This approach allowed us to streamline the computational load without compromising the accuracy of the functional site assessment, ensuring that resources were allocated effectively toward relevant structural interactions. The binding site was determined using the “Site Finder” tool, with careful consideration given to alignment with prior research and the default ligand present in the receptor. The most prominent site was generally identified as the primary site and designated as “Dummies.” When multiple binding sites are detected, the compatibility with the primary ligand serves as the principal criterion for selecting the functional site. In cases where the receptor does not contain a primary ligand, the largest identified pocket is designated as the functional site. This method ensures a systematic and rational approach to site selection, prioritizing ligand compatibility or spatial capacity as appropriate. The molecular docking procedure was performed after the preparation of ligands and receptors and the identification of the primary binding site. In this process, “Receptor atoms” were selected within the receptor section, while “Dummies” were designated within the site section. The placement and refinement settings were configured as “Triangle Matcher” and “Induced Fit,” respectively. For each ligand, both scoring methods were specified as “London dG,” with a pose count of 120:1, indicating that only the top results were reported45.

Results & discussions

Chemistry

Following the culmination of the synthetic procedures, a total of 15 compounds were successfully synthesized and subsequently gathered. It is essential to note that the synthesis of the mentioned compounds has been optimized through the accumulation of extensive experiences and modifications in this field (Table 2).

Table 2 The synthesized compounds 4a-o.
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Biology

The results of the evaluation of the anti-leishmanial activity of synthesized compounds on L. tropica amastigotes are shown in Table 3. The standard drug, glucantime, has the highest anti-leishmanial activity (with IC50 = 119.3 ± 3.2) compared to the test compounds. Among the test compounds, compound 4 m has the highest activity (IC50 = 142.3 ± 10.7 µg/mL), while compound 4j has the lowest anti-leishmanial activity (IC50 = 181.3 ± 6.7 µg/mL).

Synthesized nitrochromene compounds, in combination with glucantime, significantly reduced the average number of amastigotes per macrophage compared to glucantime alone. In combination therapy, all test compounds demonstrated lower IC50 values compared to glucantime, indicating higher activity. The highest activities in the combination of glucantime and nitrochromene compounds were related to compounds 4d and 4n, with IC50 values of 73.4 ± 1.9 7 µg/mL and 74.3 ± 2.07 µg/mL, respectively. Additionally, the lowest activity was related to compound 4j with an IC50 value of 102.3 ± 2.47 µg/mL.

Glucantime has a CC50 value of 598.3 ± 13.77 µg/mL against treated macrophage cells. The compounds 4 m, 4o and 4 h has the highest antileishmanial activity in single mode with IC50 values of 142.3 ± 10.7 µg/mL, 144.3 ± 7.1 µg/mL and 147.3 ± 5.0 µg/mL respectively. In combination therapy 4d, 4n, and 4o has the highest antileishmanial activity with IC50 values of 73.4 ± 1.9 µg/mL, 74.3 ± 2.0 µg/mL and 79.4 ± 3.1 µg/mL respectively.

The lower toxicity related to 4 L as alone usage (CC50: 651.3 ± 14.8 µg/mL) and in combination therapy is related to 4 h with CC50 value of 573.2 ± 14.1 µg/mL.

The evaluation of combination therapy with glucantime and synthesized nitrochromene compounds showed that the CI index is less than 1, which is recognized as a synergistic effect. These results are consistent with previous studies, which showed that combination therapy with glucantime is more efficient than glucantime alone against L. tropica46.

This study determined the Selectivity Index (SI) for each test compound, both in single and combination therapy. All SI values were above 1. In fact, this state indicates that there is greater toxicity to the parasites when compared to the toxicity to the host cell. A higher SI index, closer to 10, indicates better and more specific drug performance. Among the synthesized nitrochromene compounds, compound 4 m exhibited the highest SI index in single therapy (4.37), while compound 4d demonstrated the highest SI index in combination therapy (7.92).

The SI as a marker of toxicity demonstrated that 4d, 4n and 4o are highly safe for mammalian macrophages as the central harboring cell of the Leishmania parasites. Similarly, Glucantime® as an approved drug has long been used against various disease conditions, including leishmaniasis, and proved relatively safe in vitro assays (SI = 5.01). The combined therapy overcome the limitations of the drugs available in the clinic, such as the occurrence of species or strains resistant to treatment, reduction of treatment toxicity to the patient, among others.

The studies conducted on the synthesized compounds revealed that their inhibitory activities on promastigotes were lower than those of meglumine antimonate. However, compounds 4k, 4 m, 4n, and 4o exhibited effects comparable to meglumine antimonate. Regarding the combined effect, most of the compounds demonstrated a synergistic effect superior to meglumine antimonate monotherapy, with compounds 4k, 4 L, 4 m, 4n, and 4o exhibiting the most pronounced synergistic effects (Table 4).

Table 3 Evaluating the IC50 values of synthesized nitrochromene compounds alone and in combination with MA against amastigote and promastigote forms of L. Tropica in comparison with MA (glucantime) as positive control drug and the CC50 values of the drugs on macrophage using the SI index.
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Table 4 Individual and combined effects of the synthesized compounds in combination with meglumine antimonate (MA) on promastigote forms of the parasite.
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Molecular docking

Subsequent to in vitro investigations, molecular docking experiments were carried out with the synthesized compounds and all 16 specified receptors of Leishmania. A comprehensive set of data points for each docking procedure was documented based on the evaluation of energy levels and the formation of chemical bonds. These scores are represented with indicator colors in Table 5. Although docking scores alone do not definitively confirm receptor inhibition or activation, these investigations provide valuable insights into the potential mechanisms by which these compounds may exert inhibitory effects. According to the data presented in Table 5, several notable high-probability outcomes are evident, particularly the scores associated with the 1XTP (a SAM-dependent methyltransferase) and 4GED (peroxidase-cytochrome C complex) receptors, which exhibit the highest degree of prominence. While benzyloxy derivatives may possess an advantage in binding affinity across various targets, the overall binding strength or compatibility within these active sites remains relatively moderate. A more detailed investigation into these targets could elucidate the potential synergistic effects of these compounds. Moreover, according to these results, ligands 4k, 4 L, 4 m, 4n, and 4o showed notable scores against 1XTP, 2WSA, 3MHU, 4G5D, 4GED (second site), 5WB5, and 6QD9 receptors.

S-adenosylmethionine (SAM) is a ubiquitous molecule in living organisms, serving a critical role in metabolism as a cofactor for various enzymes. Among these, methyltransferases (MTases) constitute a prominent group of SAM-dependent enzymes, mediating the transfer of methyl groups from SAM to carbon, oxygen, nitrogen, and sulfur atoms in small-molecule secondary metabolites and macromolecules, including proteins and nucleic acids. MTases have attracted significant interest in biomedical research due to their vital role in the epigenetic regulation of macromolecules and the biosynthesis of natural products with diverse pharmacological properties. However, another class of SAM-dependent enzymes, sharing core domains with MTases, is capable of catalyzing non-methylation reactions and possesses a wide range of functions47. In trypanosomatid parasites, the synthesis of endogenous sterols requires the transfer of a methyl group from SAM to the C24 position of sterol intermediates, leading to the production of ergostane-based sterols. This process is catalyzed by sterol methyltransferase (SMT), which facilitates the methylation of the C24 position on the sterol side chain, thereby distinguishing the biosynthesis of ergosterol from that of cholesterol. In Leishmania major promastigotes, SMT is localized within the endoplasmic reticulum and endocytic vesicles. Importantly, mammals do not possess orthologs of SMT, making this protein an attractive target for the development of anti-leishmanial drugs or vaccines48.

As part of the host’s protective mechanisms, reactive oxygen species, including hydrogen peroxide, are produced to combat infections. In response, Leishmania major produces a peroxidase enzyme known as L. major peroxidase (LmP), which aids in shielding the parasite from oxidative stress. LmP is a heme peroxidase that catalyzes the peroxidation of mitochondrial cytochrome C (LmCytc), using it as its substrate. Inhibition of this enzyme complex would render the parasite more vulnerable to the reactive oxygen species produced by the host, thereby compromising its ability to withstand oxidative stress49,50.

Molecular interactions between ligands and receptors with the best docking scores are illustrated in detail in Fig. 3. Within the main pocket 1XTP, a π-π stacking interaction is observed between tryptophan residues (Trp57) and the intermediate arene ring of the small molecule. The amino acids phenylalanine (Phe), tyrosine (Tyr), and especially tryptophan (Trp) are frequently involved in π interactions. Supramolecular π-stacking is an important phenomenon across various scientific disciplines; however, its strength is generally considered to be similar to that of Van der Waals forces and multipole–multipole interactions. For this particular target, the docking scores of the benzyloxy compounds (exclusively 4k and 4o) approach the score of the primary ligand, which has been previously confirmed to inhibit this protein effectively. This suggests that the benzyloxy compounds may possess inhibitory potential comparable to that of the primary ligand, reinforcing their suitability as candidates for further investigation. The closeness of their scores to the primary ligand supports the idea that these compounds might achieve similar binding efficiency or interaction profiles within the active site, indicating promising efficacy in inhibiting the target protein.

In the second binding site of 4GED, along with a π-H stacking interaction involving serine (SerB92), numerous donor and acceptor hydrogen bonds are present. These interactions contribute to a more favorable and stable fit of the ligand within the enzyme’s active site. The precise spatial positioning of the ligands within the confined space of the active site in both enzymes is remarkable and is clearly depicted in the 3D representation shown in Fig. 4. The increased spatial crowding introduced by the benzyloxy chain seems to improve its fitting within the receptor pockets, especially when compared to other synthesized compounds with smaller molecular sizes.

The variability in the statistical data makes it challenging to establish a precise correlation between the IC₅₀ values and the docking scores. As mentioned earlier, based on IC₅₀ values, all tested compounds demonstrated weaker performance than the control compound, MA, in monotherapy. However, in combined treatments, each compound showed a better effect than MA alone, suggesting a potential synergistic advantage in combination therapy. Among the tested compounds, 4 h, 4 m, 4n, and 4o exhibited the best IC₅₀ values, with all but 4 h achieving acceptable docking scores. This indicates that while compound 4 h performs well in vitro, further refinement may be needed to enhance its docking affinity, or it may act through alternative binding interactions not fully captured in the docking assessment.

Table 5 The docking scores related to each ligand-receptor complex (PL: primary ligand).
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Fig. 3
figure 3

Ligand interactions of compound 4k with 1XTP (left) and 4o with the second site of 4GED (right). Both are well placed in the pocket space and form donor and acceptor hydrogen bonds alongside strong H- π and π- π stackings.

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Fig. 4
figure 4

3D schematic of the placement of ligand 4k (green) in 1XTP (left) and 4o (yellow) in the second site of 4GED (right). This figure is illustrated utilizing Maestro 12.8 application of Schrödinger suite.

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Conclusion

In the context of this study, a thorough series of fifteen compounds was synthesized using the Henry reaction. Following in vitro assessments and computational analyses, substantial evidence indicates that these compounds may serve as active agents against both L. tropica amastigotes and promastigotes. In addition to their inhibitory properties, the compounds under investigation demonstrated a notably intriguing feature: they exhibited synergistic interactions with glucantime for inhibiting both amastigotes and promastigotes. Our findings indicate that combination therapies generally exhibit superior efficacy compared to monotherapy. While the synthesized nitrochromene derivatives exhibit promising biological activity against Leishmania, it is important to emphasize that their efficacy has not yet been definitively established. The current findings indicate potential, yet further research is required to fully elucidate the therapeutic capabilities of these compounds.

Moreover, the utilization of in silico studies revealed that these compounds, exclusively compounds bearing the nitrochromene pharmacophore in conjunction with the benzyloxy functional group, can block essential proteins of Leishmania such as 1XTP (a SAM-dependent methyltransferase) and 4GED (peroxidase-cytochrome C complex) receptors. This proposition arises from the expectation that these compounds could undergo more extensive investigation, which would elucidate their inhibitory properties as well as their potential for synergistic interactions. Additionally, given the versatility and adaptability of these compounds within the binding pockets of the examined receptors, particularly the 4GED receptor, it is possible to identify potential avenues for derivatization to enhance their binding characteristics. Furthermore, it is prudent to evaluate the effects of these compounds on various Leishmania species to determine their potential broad-spectrum efficacy in addressing this global health challenge.

Notes

Spectra (FT-IR, 1H NMR, 13C NMR) related to all compounds have been reviewed in detail in a separate supplementary file. In the following, characterization and analytical data of synthesized compounds will be introduced.

2-(2-bromophenyl)-3-nitro-2 H-chromene (4a)

Yield 77%; mp lit. 115.5-116.5 oC51; mp 118–120 oC; FT-IR (KBr) (ῡmax, cm− 1): 3073 (C-HAr), 2828 (C-H), 1645 (C = C), 1569 (C = CAr), 1510 & 1332 (NO2), 1220 (C-OAr), 1118(C-O), 512 (C-Br).

2-(3-chlorophenyl)-3-nitro-2 H-chromene (4b)

Yield 68%; mp lit. not reported; mp 90–92 oC; FT-IR (KBr) (ῡmax, cm− 1): 3062 (C-HAr), 2959 (C-H), 1650 (C = C), 1569 (C = CAr), 1521 & 1332 (NO2), 1222 (C-OAr), 1119(C-O), 804 (C-Cl).

2-(2-chlorophenyl)-3-nitro-2 H-chromene (4c)

Yield 71%; mp lit. 90–92 oC52; mp 89–91 oC; FT-IR (KBr) (ῡmax, cm− 1): 3074 (C-HAr), 2952 (C-H), 1603 (C = C), 1603 (C = CAr), 1512 & 1320 (NO2), 1222 (C-OAr), 1119 (C-O), 695 (C-Cl).

2-(4-methoxyphenyl)-3-nitro-2 H-chromene (4d)

Yield 68%; mp lit. 158–160 oC52; mp 153–155 oC; FT-IR (KBr) (ῡmax, cm− 1): 3070 (C-HAr), 2905 (C-H), 1649 (C = C), 1605 (C = CAr), 1512 & 1328 (NO2), 1222 (C-OAr), 1065 (CSP3-O), 749 (C-Cl).

2-(3-bromophenyl)-3-nitro-2 H-chromene (4e)

Yield 81%; mp lit. 114.5-115.551 oC; mp 113–115 oC; FT-IR (KBr) (ῡmax, cm− 1): 3082 (C-HAr), 2958 (C-H), 1650 (C = C), 1603 (C = CAr), 1518 & 1328 (NO2), 1222 (C-OAr), 1119 (C-O), 697 (C-Cl).

3-nitro-2-(p-tolyl)-2 H-chromene (4f)

Yield 71%; mp lit. 120–122 oC35; 125–127 oC; FT-IR (KBr) (ῡmax, cm− 1): 3071 (C-HAr), 2928 (C-H), 1649 (C = C), 1603 (C = CAr), 1510 & 1323 (NO2), 1226 (C-OAr), 1119 (C-O).

2-(4-chlorophenyl)-3-nitro-2 H-chromene (4 g)

Yield 68%; mp lit. 133–134 oC53; 133–137 oC; FT-IR (KBr) (ῡmax, cm− 1): 3080 (C-HAr), 2968 (C-H), 1639 (C = C), 1603 (C = CAr), 1487 & 1327 (NO2), 1223 (C-OAr), 1119 (C-O), 547 (C-Cl).

2-(4-fluorophenyl)-3-nitro-2 H-chromene (4 h)

Yield 51%; mp lit. 93–95 oC54; 92–93 oC; FT-IR (KBr) (ῡmax, cm− 1): 3067 (C-HAr), 2968 (C-H), 1650 (C = C), 1602 (C = CAr), 1504 & 1320 (NO2), 1231 (C-OAr), 1118 (C-O), 1067(C-F).

2-(2-((2-chlorobenzyl)oxy)phenyl)-3-nitro-2 H-chromene (4i)

Yield 83%; mp lit. not reported; mp 123–125 oC; FT-IR (KBr) (ῡmax, cm− 1): 3070 (C-HAr), 2935 (C-H), 1643 (C = C), 1604 (C = CAr), 1505 & 1321 (NO2), 1220 (CAr-O), 1030 (C-O), 750 (C-Cl); 1H NMR (DMSO, 300 MHz, d6) δ (ppm): 8.41 (s, 1 H, H4-Chromene), 7.67 (m, 1 H, CHAr), 7.55 (m, 2 H, CHAr), 7.45–7.30 (m, 4 H, CHAr), 7.21–7.12 (m, 2 H, CHAr), 7.00 (t, 1 H, J = 9HZ, CHAr), 6.90 (s, 1 H, H2-Chromene), 6.87 ( t, 1 H, J = 9HZ, CHAr), 6.69 (d, 1 H, J = 9HZ, CHAr), 5.34 (d, 1 H, J = 12HZ, CHSP3), 5.27 (d, 1 H, J = 12HZ, CHSP3). 13C NMR (DMSO, 75 MHz, d6) δ (ppm): 156.09, 153.32, 140.25, 134.77, 134.57, 133.05, 131.66, 131.55, 131.01, 130.34, 130.30, 129.93, 128.15, 127.88, 124.82, 122.78, 122.39, 118.51, 116.99, 113.67, 68.71, 67.70.

2-(4-((4-fluorobenzyl)oxy)phenyl)-3-nitro-2 H-chromene (4j)

Yield 72%; mp lit. not reported; mp 98–100 oC; FT-IR (KBr) (ῡmax, cm− 1): 3070 (C-HAr), 2929 (C-H), 1648 (C = C), 1604 (C = CAr), 1510 & 1326 (NO2), 1222 (CAr-O), 1067 (C-O), 1050 (C-F); 1H NMR (DMSO, 300 MHz, d6) δ (ppm): 8.41 (s, 1 H, H4-Chromene), 7.61 (d, 1 H, J = 6HZ, CHAr), 7.46 (m, 2 H, CHAr), 7.35 (t, 1 H, J = 9HZ, CHAr), 7.30 (d, 2 H, J = 9HZ, CHAr), 7.19 (t, 2 H, J = 9HZ, CHAr), 7.03 (t, 1 H, J = 6HZ, CHAr), 6.98 (d, 2 H, J = 9HZ, CHAr), 6.85 (d, 1 H, J = 6HZ, CHAr), 6.59 (s, 1 H, H2-Chromene), 5.03 (s, 2 H, CH2O). 13C NMR (DMSO, 75 MHz, d6) δ (ppm): 163.87, 160.64, 159.53, 153.21, 141.10, 134.84, 133.45, 133.41, 131.58, 130.48, 130.37, 129.42, 129.05, 122.90, 118.64, 117.23, 115.85, 115.57, 115.46, 73.51, 68.98.

2-(3-((4-methylbenzyl)oxy)phenyl)-3-nitro-2 H-chromene (4k)

Yield 77%; mp lit. not reported; mp 125–127 oC; FT-IR (KBr) (ῡmax, cm− 1): 3075 (C-HAr), 2921 (C-H), 1647 (C = C), 1606 (C = CAr), 1508 & 1327 (NO2), 1221 (C-OAr), 1072 (C-O); 1H NMR (DMSO, 300 MHz, d6) δ (ppm): 8.41 (s, 1 H, H4-Chromene) ,7.61(d, 1 H, J = 6HZ, CHAr), 7.38 (t, 1 H, J = 6HZ, CHAr), 7.28 (d, 2 H, J = 6HZ, CHAr), 7.24 (s, 1 H, CHAr), 7.17 (d, 2 H, J = 6HZ, CHAr),7.06 (t, 1 H, J = 6HZ, CHAr), 6.99–6.85 (m, 4 H, CHAr), 6.61 (s, 1 H, H2-Chromene), 4.99 (s, 2 H, CH2O), 2.30 (s, 3 H, CH3). 13C NMR (DMSO, 75 MHz, d6) δ (ppm): 159.05, 153.20, 142.76, 140.83, 138.57, 134.95, 134.12- 131.69, 130.65, 130.57, 129.45, 128.43, 123.04, 119.62, 118.53, 117.18, 115.87, 114.28, 73.64, 69.63, 21.24.

2-(4-((4-methylbenzyl)oxy)phenyl)-3-nitro-2 H-chromene (4 L)

Yield 74%; mp lit. not reported; mp 113–116 oC; FT-IR (KBr) (ῡmax, cm− 1): 3075 (C-HAr), 2924 (C-H), 1651 (C = C), 1604 (C = CAr), 1514 & 1322 (NO2), 1224 (C-OAr), 1064 (C-O); 1H NMR (DMSO, 300 MHz, d6) δ (ppm): 8.41 (s, 1 H, H4-Chromene), 7.61 (d, 1 H, J = 6HZ, CHAr), 7.36 (t, 1 H, J = 9HZ, CHAr), 7.30 (d, 4 H, J = 9HZ, CHAr), 7.17 (d, 2 H, J = 9HZ, CHAr), 7.05 (t, 1 H, J = 6HZ, CHAr), 6.95 (d, 2 H, J = 9HZ, CHAr), 6.84 (d, 1 H J = 6HZ, CHAr), 6.57 (s, 1 H, H2-Chromene), 5.00 (s, 2 H, CH2O), 2.28 (s, 3 H, CH3). 13C NMR (DMSO, 75 MHz, d6) δ (ppm): 159.63, 153.18, 141.11, 137.61, 134.88, 134.18, 131.60, 130.39, 129.24, 129.03, 128.29, 122.92, 118.64, 117.24, 115.48, 73.46, 69.57, 21.21.

2-(4-((4-chlorobenzyl)oxy)phenyl)-3-nitro-2 H-chromene (4 m)

Yield 91%; mp lit. not reported; mp 118–120 oC; FT-IR (KBr) (ῡmax, cm− 1): 3079 (C-HAr), 2925 (C-H), 1639 (C = C), 1603 (C = CAr), 1506 & 1326 (NO2), 1220 (C-OAr), 1049 (C-O), 763 (C-Cl); 1 H NMR (DMSO, 300 MHz, d6) δ (ppm): 8.41 (s, 1 H, H4-Chromene), 7.61 (dd, 1 H, J = 1HZ, 9HZ, CHAr), 7.43 (brs, 4 H, CHAr), 7.37 (dd, 1 H, J = 9HZ, 1HZ, CHAr), 7.31 (d, 2 H, J = 9HZ, CHAr), 7.04 (t, J = 9HZ, 1 H, CHAr), 6.96 (d, 2 H, J = 9HZ, CHAr), 6.85 (d, 1 H, J = 9HZ, CHAr), 6.58 (s, 1 H, H2-Chromene), 5.06 (s, 2 H, CH2O). 13C NMR (DMSO, 75 MHz, d6) δ (ppm): 159.45, 153.20, 141.12, 136.32, 134.89, 132.94, 131.62, 130.43, 130.00, 129.50, 129.09, 128.93, 122.94, 121.65, 117.26, 115.52, 73.47, 68.85.

2-(3-((4-chlorobenzyl)oxy)phenyl)-3-nitro-2 H-chromene (4n)

Yield 79%; mp lit. not reported; mp 115–117 oC; FT-IR (KBr) (ῡmax, cm− 1): 3075 (C-HAr), 2920 (C-H), 1647 (C = C), 1606 (C = CAr), 1509 & 1327 (NO2), 1220 (C-OAr), 1073 (C-O), 778 (C-Cl); 1H NMR (DMSO, 300 MHz, d6) δ (ppm): 8.41 (d, 1 H, J = 3HZ, H4-Chromene), 7.61 (d, 1 H, J = 9HZ, CHAr), 7.47–7.42 (m, 4 H, CHAr), 7.36 (d, 1 H, J = 9HZ, CHAr), 7.28 (m, 1 H, CHAr), 7.07 (d, 1 H, J = 6HZ, CHAr), 7.01 (d, 2 H, J = 6HZ, CHAr), 6.95 (d, 1 H, J = 6HZ, CHAr), 6.86(d, 1 H, J = 6HZ, CHAr), 6.60 (d, 1 H, J = 3HZ, CHAr). 13C NMR (DMSO, 75 MHz, d6) δ (ppm): 158.83- 153.17- 140.79- 138.61- 136.24- 134.94- 132.93- 131.69- 130.67- 130.63- 130.08- 128.91- 123.05- 119.83- 118.51- 117.16- 115.91- 114.24- 73.60- 68.85- 40.75- 40.48- 40.20- 39.92- 39.64- 39.36- 39.09.

3-nitro-2-(3-((4-nitrobenzyl)oxy)phenyl)-2 H-chromene (4o)

Yield 69%; mp lit. not reported; mp 147–149 oC; FT-IR (KBr) (ῡmax, cm− 1): 3072 (C-HAr), 2894 (C-H), 1651 (C = C), 1603; (C = CAr), 1513 & 1318 (NO2), 1180 (CAr-O), 1053 (C-O); 1H NMR (DMSO, 300 MHz, d6) δ (ppm): 8.41 (s, 1 H, H4-Chromene), 8.22 (d, 2 H, J = 9HZ, CHAr), 7.66 (d, 2 H, J = 9HZ, CHAr), 7.59 (d, J = 6HZ, 1 H, CHAr), 7.32 (t, 2 H, J = 6HZ, CHAr), 7.02 (brs, 4 H, CHAr), 6.84 (d, 1 H, J = 6HZ, CHAr), 6.61 (s, 1 H, H2-Chromene), 5.24 (s, 2 H, CH2O). 13C NMR (DMSO, 75 MHz, d6) δ (ppm): 158.61, 153.15,147.42, 145.18, 140.75, 137.71, 134.91, 131.69, 130.69, 128.73, 128.62, 124.57, 123.01, 120.11, 118.47, 117.13, 116.02, 114.22, 73.60, 68.53.