Given the antileishmanial activity of natural 2-substituted quinoline alkaloids (Fournet et al., 1993; 1996), a library of 2-substituted quinolines was synthesized (Fakhfakh et al., 2003; Franck et al., 2004). Initial in vitro screening against L. amazonensis and L. infantum identified quinolines substituted in the 2-position by a three-carbon alkenyl side chain containing an aldehyde (1), hydroxy (2) or bromine substituent (3) as the most active compounds, with IC₅₀ values between 2 and 4 μM. By contrast, three carbon alkenyl side chains containing carboxylic acid, ester, amide, nitro or phenyl functionalities or larger alkenyl side chains displayed lower activity. To determine if this potent in vitro activity translated to a therapeutic effect, some derivatives were selected for in vivo studies. Compounds were administered by the oral route to a group of mice infected with L. amazonensis, L. infantum, and L. donovani. Interestingly, a disconnect was observed: compound 3, which was the most potent in vitro, showed no activity in vivo, whereas other derivatives with lower in vitro potency demonstrated a significant reduction (>80%) in parasite burden. Compounds 1 and 2 showed satisfactory activity in the visceral leishmaniasis models, with more than an 80% reduction of the parasite burden in the liver and close to 50% reduction in the spleen of mice infected with L. infantum and close to 50% reduction of the parasite burden in the liver of mice infected with L. donovani (Nakayama et al., 2005). Based on these results, the same group synthesized an α,β-unsaturated nitrile (4) with promising in vitro activity against L. donovani amastigotes and significant in vivo efficacy with a significant reduction in parasite burden in the liver (Nakayama et al., 2007).

The synthesis of a group of quinoline-2-carbohydrazides yielded compounds 5a and 5b as the most active against Leishmania (Viannia) panamensis (Coa et al., 2015). These compounds bear a 2-hydroxyphenyl moiety with a second hydroxy group at the 3- or 4- position of the aromatic ring, the presence of which is associated with the improvement of antileishmanial activity. Similar derivatives, with hydroxy groups replaced by methoxy functionalities, showed lower activity (Alodeani et al., 2015).

GDP-mannose pyrophosphorylase, which is involved in the biosynthetic pathway of glycoconjugates, has been recognized as an interesting target for the development of chemotherapeutic agents. Among several selected inhibitors, compound 6 emerged as the most promising antileishmanial agent, with an IC₅₀ of 1.06 μM on axenic amastigotes and 0.63 μM on the RAW264.7 model of L. donovani. However, its selectivity index (SI) of 2.4 was low, limiting its therapeutic potential (Mao et al., 2017).

The primary strength of C-2 substitution lies in the accessibility for introducing different groups, which allows for an easy structural optimization. Despite this, a disconnection between in vitro potency and in vivo efficacy is generally observed.

Based on previous studies describing the antileishmanial activity of 3-arylquinolines, this structure was selected as the lead to perform different substitution modifications at various positions (Katsuno et al., 2015; Ortiz et al., 2017). Some 3-substituted quinolines bearing alkenyl, alkynyl, and phenyl groups were synthesized and evaluated against L. amazonensis amastigotes. These analogues exerted low to negligible activity, suggesting this position is less favorable (Fakhfakh et al., 2003). Among a series of synthesized 3-arylamino quinolines, 3- and 4-fluorophenyl derivatives (7a-b) were effective against L. mexicana promastigotes (IC₅₀ = 41.9 μM) with low cytotoxicity (>100 μM) (Chanquia et al., 2019).

Synthesis of hybrid molecules constitutes an attractive strategy for obtaining pharmaceutical compounds, which involves the covalent combination of two biologically active pharmacophores (Viegas-Junior et al., 2007). Several quinoline-based hybrids have been shown to exhibit various activities, including antimalarial, antibacterial, antiviral, antitumoral, and anti-inflammatory (Ammar et al., 2021; Hu et al., 2017; Singh et al., 2022). From this perspective, a library of quinoline-thiazolidinone hybrids was synthesized to target methionine aminopeptidase, an important enzyme for the development of antiprotozoal agents. Compound 8 arose as a promising antileishmanial agent with a twenty-fold higher inhibitory activity against L. donovani aminopeptidase (IC₅₀ = 3.0 μM) compared to the human enzyme (IC₅₀ = 58.0 μM), a good drug-likeness profile, and low cytotoxicity (CC₅₀ > 150 μM) (Bhat et al., 2021).

Simple substitutions at C-3 have generally failed to produce potent compounds but the design of hybrid molecules with complex pharmacophores could yield more selective compounds. Additionally, a general lack of in vivo validation is observed.

Substitution on the benzene ring also presents alternatives for the development of new drugs. Based on the concept of molecular hybridization, two series of 6-substituted quinolines linked to either oxadiazole-thiosemicarbazides or 1,3,4-thiadiazoles were synthesized. The in vitro activity against L. major promastigotes was evaluated, showing excellent antileishmanial activity with an IC₅₀ in the submicromolar range. In the first series, fluorinated derivatives 9a and 9b were the most potent with IC₅₀ values of 0.10 μM and 0.15 μM, respectively (Taha et al., 2017). In the second series, dihydroxyphenyl derivatives (10a-d) were the most potent compounds, achieving IC₅₀ values as low as 0.04 µM (Almandil et al., 2019). Docking studies suggested a possible mechanism of action as these compounds are tightly fitted into the active site of pteridine reductase 1, a validated drug target in trypanosomatids. The presence and position of hydroxyl groups influenced antileishmanial activity, indicating that these groups afford polar interactions with residues from the active site of pteridine reductase 1. In the case of fluorinated derivatives, the one with the fluorine atom in the ortho position was the one that exhibited the most significant inhibition, demonstrating that the substitution in this position is essential for its activity.

The primary strength of the 6-substitution is the extremely high in vitro potency achieved through hybridization. In addition, the SAR is relatively clear, with electron-withdrawing and hydrogen-bond-donating groups significantly improving activity. The major limitation is the absence of amastigotes and cytotoxicity assays.

The 8-position of the quinoline ring is historically significant due to the antimalarial drug primaquine. A group of N-quinolin-8-yl-arylsulphonamides showed promising activity (Da Silva et al., 2007). Particularly, the dihalogenated compounds 11a-c were very effective against both promastigotes of L. amazonensis and L. chagasi, and significant selectivity indices (SI). More lipophilic derivatives without halogenated substituents have shown somewhat lower activity. Promising results were obtained from assays on the amastigote form of L. amazonensis: significant antileishmanial activity was observed with compounds 11a-c, with IC₅₀ < 1 µM. In contrast, substitution with a hydroxyl or phenyl group resulted in weaker antileishmanial activity against L. amazonensis or L. (V) panamensis amastigotes (Coa et al., 2020; Silva et al., 2018).

Chalcone, furochalcone, and chromone–quinoline hybrids via an alkyl linker have also been tested against L. (Viannia) panamensis (Coa et al., 2017; García et al., 2018). The antileishmanial activity was influenced by the length of the alkyl linker connecting the quinoline ring to its substituent. However, no clear correlation between antiprotozoal activity and alkyl chain length was established, as no consistent trend in activity relative to chain length was observed. Within the furanochalcone series, the derivative which features a three-carbon linker exhibited the highest activity against L. (V) panamensis amastigotes, but it showed high cytotoxicity. In the case of chromone-based series, compounds 12a and 12b, bearing two- and seven-carbon linkers respectively, showed the highest antileishmanial activity (IC₅₀ = 16.9 and 17.0 μM). However, these compounds also displayed considerable cytotoxicity, with SI values below 1. By contrast, compounds 12c and 12d were less active but exhibited low cytotoxicity, with SI values of 4.89 and 5.84, respectively. All chalcone-quinoline hybrids showed moderate activity and low SI.

The low cytotoxicity constitutes the main strength of the 8-substitution pattern. Additionally, some compounds showed very potent activity against the clinically relevant amastigote form.

Considering the groups and substitution positions that have led to compounds with higher activity, new quinolines have been developed, combining different substituents at various positions. For example, a series of 2-styrylquinolines with additional groups at the C-7 position was synthesized and evaluated in vitro against the amastigote form of L. donovani (Loiseau et al., 2011). Compound 13 emerged as the most promising candidate, with IC₅₀ = 1.2 µM and low toxicity (SI = 121.5). Additionally, it was observed that highly hydrophilic groups, such as COOH, dramatically decreased antileishmanial activity, whereas the presence of NO₂ groups improved the SI. Compound 2 was taken as the lead structure for the synthesis of a series of quinolines with a hydroxypropenyl group at the two-position. In vitro and in vivo activity against L. donovani was measured (Gopinath et al., 2013). It was observed that the presence of halogens improved metabolic stability compared to the reference compound, and with a morpholine group at position 4, compound 14 emerged as the best candidate with significantly enhanced antileishmanial activity (IC₅₀ = 0.22 μM, SI = 187). Furthermore, in vivo assays using a L. donovani/hamster model, its hydrochloride salt exhibited an 84% inhibition of parasite growth.

Considering the hybridization approach and the known antiparasitic activity of metronidazoles, a series of quinoline-metronidazole hybrids was synthesized and evaluated against L. donovani promastigotes and amastigotes (Upadhyay et al., 2019). Among these hybrids, compound 15 showed more potent activity against L. donovani both in vitro and in vivo and presented a high selectivity index and metabolic stability. Furthermore, the induction of apoptosis via mitochondrial membrane depolarization and an increase in the generation of reactive oxygen species was established as a possible dual mechanism of action. Compound 15 is a promising candidate both as a lead structure for the development of new antileishmanial agents and for clinical trials, taking into account its potency in vitro (IC₅₀ = 3.75 µM in amastigotes) and in vivo assays (>80% reduction of parasite burden in liver and spleen), low toxicity (SI = 57.9) and excellent pharmacokinetic properties.

Consequently, a series of 3-aryl and 3-heteroaryl-N ⁷,N ⁷-dimethylquinoline-2,7-diamine derivatives was synthesized. These compounds were evaluated against L. mexicana amastigotes, showing promising activity (IC₅₀ < 1 μM) (Hammill et al., 2021). According to SAR analysis, no significant variation in antileishmanial activity was observed upon substitution of the aromatic ring, and replacing the aryl group with six membered or bicyclic heterocycles also maintained the activity. However, substitution with five-membered heterocycles reduced activity, suggesting a minimum steric bulk requirement. Among the series, compounds 16 and 17 were the most active and were selected for in vivo experiments, but 16 exhibited toxicity, and 17 failed to suppress lesion progression in a murine model of cutaneous leishmaniasis.

Considering the antileishmanial activity of quinoline and 1,2,3-triazole scaffolds, a series of triazolyl 2-methyl-4-phenylquinoline-3-carboxylate derivatives was synthesized via click chemistry-based molecular hybridization approach and evaluated against L. donovani (Upadhyay et al., 2018). Despite good antileishmanial activity against promastigotes in most cases, only a few compounds showed significant inhibitory activity against intracellular amastigotes. Among them, compounds 18a-b and 19a-b were selected for their evaluation in a L. donovani/golden hamster model. Compounds 18a and 19a showed moderate activity, with 40% and 46% parasite inhibition, respectively, whereas 18b and 19b exhibited poor inhibitory activity.

Clioquinol (20), a dihalogenated 8-hydroxyquinoline, showed promising in vitro activity against L. infantum and L. amazonensis promastigotes and amastigotes, exhibiting low cytotoxicity against murine macrophages (CC₅₀ = 834.4 µM) and human erythrocytes (CC₅₀ = 1.5 × 10³ μM), with a higher selectivity index than amphotericin B. As no toxicity was observed when clioquinol was administered to BALB/c mice, it constitutes a promising candidate for subsequent in vivo studies (Tavares et al., 2018). Compound 21, a racemic 8-aminoquinoline derivative, has a strong effect on the mobility and morphology of L. mexicana promastigotes, exhibiting an IC₅₀ value of 1.03 µM, markedly lower compared to glucantime (Isaac-Márquez et al., 2010). Also, 21 exhibited negligible cytotoxicity toward HeLa cells over 120 h.

The obvious strength of polysubstitution is the possibility of designing more active and selective molecules, optimizing pharmacokinetic and pharmacodynamic properties, although at the cost of increased synthetic complexity.

The structures of the most representative compounds are shown in the Figure 1 at the end of the manuscript. The Table 1 summarizes the antileishmania activity data of the most promising quinolines, which exhibit low IC₅₀ values in vitro assays (<10 μM), or significant in vivo inhibition, or high selectivity indexes (SI > 10).

In summary, in the last decades, the development of new quinoline-derived compounds as potential chemotherapeutic agents for the treatment of leishmaniasis has increased. This remains a significant challenge due to multiple causes, such as the numerous species that cause the disease, diverse manifestations, resistance, and more. The quinoline framework interacts with different receptors, ion channels, and enzymes, making it a privileged scaffold in organic chemistry for the search for bioactive compounds; many natural and synthetic derivatives have been shown to exhibit a wide range of biological properties. In this mini-review, we presented an overview of the impact of substitution position on the antileishmanial properties of substituted quinolines.

Considering all examples mentioned, it can be observed that, among monosubstituted quinolines, 2-substitution results in compounds with greater in vitro (amastigotes) and in vivo activity; 3-substituted quinolines are the least active compounds; and 8-substitution led to compounds with good activity in vitro on amastigotes and high SI, but dependent on Leishmania species. In many cases, for the same type of derivatives, the inclusion of hydroxyl or halogen groups leads to improved antileishmanial activity. In the case of polysubstituted quinolines, substituents in position 2 and/or a hydroxyl group in position 8 seem to be key in the antileishmanial activity and excellent selectivity index values. Among the compounds described, 2, 4, 6, 11a-c, 13, 14, 15, and 20 are the most promising either due to their low IC₅₀ (<10 μM) values against amastigotes and/or high selectivity indexes (SI > 10) SI or good in vivo activity. However, a strict comparison is hindered due to several limitations, like: different Leishmania species with differences in drug sensitivity; in studies reporting very high potency on promastigotes, lack of data about assays against amastigotes, the clinically relevant form of the parasite; heterogeneity in experimental protocols; scarcity of information about molecular targets or action mechanisms. Future strategies should focus not only on a combination of substituents at diverse positions or synthesis of hydrid molecules, but also on systematic studies on structure-activity relationships and mechanisms of action.