Easy one-pot synthesis of multifunctionalized indole–pyrrole hybrids as a new class of antileishmanial agents

Abstract

A chemoselective one-pot synthesis of pharmaceutically prospective indole–pyrrole hybrids by the formal [3 + 2] cycloaddition of 3-cyanoacetyl indoles (CAIs) with 1,2-diaza-1,3-dienes (DDs) has been developed. The new indole–pyrrole hybrids were phenotypically screened for efficacy against Leishmania infantum promastigotes. The most active compounds 3c, 3d, and 3j showed IC₅₀ < 20 μM and moderate cytotoxicity, lower than miltefosine. Compound 3d was the most active with IC₅₀ = 9.6 μM and a selectivity index of 5. Consequently, 3d could be a new lead compound for the generation of a new class of antileishmanial hybrids.

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Introduction

Leishmaniasis, one of the most dangerous and neglected tropical diseases, results in a significant burden in terms of morbidity and mortality every year worldwide. It is caused by the protozoan parasites (genus Leishmania), which are transmitted by the bite of phlebotomine sandflies.¹ The disease has three different forms: visceral, cutaneous (the most common) and mucocutaneous. The visceral form can be fatal if untreated.² Leishmaniasis threatens more than 350 million people in large parts of Africa, the Middle East, South America, and Asia. Today, due to global warming and migration of populations, the endemic regions of the disease have increased to non-tropical areas, such as Mediterranean Europe, where the most common forms (visceral and cutaneous) are caused by L. infantum.³

Currently, no efficacious vaccine against leishmaniasis is available⁴ and the commercial drugs have several limitations such as toxicity or strong side effects, induction of resistance, administration route (intravenous), and high costs.⁵ Thus, the discovery and development of new antileishmanial agents represents an important objective for the global community.^6–9

Indole and pyrrole are two of the most important heterocycles and are ubiquitous in natural products and several drugs. In particular, several indole-based compounds have been proven active against leishmaniasis. For example, Singh and collaborators reported a series of N-(1-methyl-1H-indol-3-yl)methyleneamines active against L. major, among them, compound 6 (Fig. 1) was the most effective of this class with IC₅₀ = 0.57 mg mL⁻¹ (2.3 μM).¹⁰ Compound 7, an indole-2-hydrazone, showed a better activity than compound 6 against L. major promastigotes (IC₅₀ = 1.9 μM).¹¹ 3,3′-Diindolylmethane (DIM, Fig. 1) was reported by Roy and colleagues as potent L. donovani DNA topoisomerase I poison (IC₅₀ = 1.2 μM).¹² Very recently, our group reported a small library of DIM derivatives, and compound 8 was the most potent against L. infantum promastigotes with IC₅₀ = 2.7 μM. Moreover, 8 demonstrated its efficacy in the in vitro infection model (intracellular amastigotes), showing IC₅₀ of 6.8 μM.¹³

Fig. 1 Examples of indole- and pyrrole-based compounds and indole–pyrrole hybrids with antileishmanial activity.

Leishmanicidal activity of pyrrole-containing compounds is also well documented in the literature. In Fig. 1, we report four examples of differently substituted pyrroles. In detail, Allocco and coworkers demonstrated the potent in vitro activity of trisubstituted pyrrole 9 against L. major promastigotes (IC₅₀ = 0.6 μM) by the inhibition of casein kinase 1 of the protozoa.¹⁴ The trisubstituted pyrrole 10, an inhibitor of L. infantum trypanothione reductase, was active against L. donovani amastigotes (IC₅₀ = 13.8 μM).¹⁵ N-Pyridinyl-2-acyl pyrrole 11, reported in an interesting study by Santiago et al., showed activity against L. amazonensis and L. donovani promastigotes with IC₅₀ of 16.9 μM and 7.8 μM, respectively.¹⁶ The last example of a pyrrole-containing antileishmanial agent is compound 12 that showed IC₅₀ = 0.33 mg mL⁻¹ (1.2 μM) against L. major promastigotes.¹⁷

Among several strategies for the discovery of new hit and/or lead compounds, molecular hybridization has proven to be a good option.^18–20 Considering the importance of pyrrole and indole scaffolds, we envisaged that hybrid entities obtained by joining these two heterocycles should have a novel greater pharmacological potency and/or reduced compound toxicity than the individual starting moieties.^21–23 Literature already provides evidence regarding the antileishmanial activity of indole–pyrrole hybrids, but to the best of our knowledge, no information was reported about their mechanism of action.

In detail, an example is URB1483 (Fig. 1), described in our previous work,²⁴ which was selected from a phenotypic screening of an azole–bisindole chemical library and showed IC₅₀ = 3.7 μM against L. infantum with no quantifiable cytotoxicity in mammalian cells. Moreover, URB1483 reduced the infection index of both human and canine macrophages with an effect comparable to the clinically used drug pentamidine. In addition, we also hypothesized an inhibition of the topoisomerase IB of the parasite for the lead URB1483 by molecular modelling studies, but the compound resulted inactive against the isolated enzyme.²⁴ Another interesting example of a hybrid compound is the natural alkaloid (+)-spiroindimicin A, elegantly synthetized by the group of Zhang. (+)-Spiroindimicin A is a cyclic molecule containing a direct bond between the indole and pyrrole scaffolds that showed a very good activity against L. amazonensis promastigotes (IC₅₀ = 1.3 μM); however, the authors did not report any information about its mechanism of action.²⁵

Although considerable efforts have been made to obtain indole–pyrrole conjugates,^26–30 only a few have been dedicated to exploring multifunctionalized C3–C2′ indole linked pyrrole hybrids.^31–33 In view of our continued interest in fabricating heterocycles as antileishmanial compounds,²⁴ a novel series of densely functionalized indole–pyrrole hybrids was designed (Fig. 1), prepared and assessed for their in vitro anti-parasitic activity. Our strategy involves the construction of the pyrrole ring of bi-heterocyclic hybrids (3–5) by a formal [3 + 2] cycloaddition reaction of 3-cyanoacetyl indoles (1) with 1,2-diaza-1,3-dienes (2).

Result and discussion

Based on our previous work,^34,35 we initiated the study selecting 3-cyanoacetyl indole (1a) and 1,2-diaza-1,3-diene (2a) as model substrates to check the reaction progress. We registered a rapid and efficient formation of 1,4-adduct intermediate (I) as the main component from a mixture of 1a (0.4 mmol) and 2a (0.4 mmol) in THF (6 mL) at 0 °C upon addition of 20 mol% of sodium methoxide (0.08 mmol) (10 min, TLC monitoring) (Scheme 1). Although the pyrrole ring formation was previously obtained by Cu-assisted azacyclization,³⁵ our orienting experiments revealed that none of the copper salts tested [CuCl₂·2H₂O, CuCl₂, CuBr₂, Cu(OAc)₂, and Cu(TfO)₂] led to the desired biheterocycle 3a. In order to establish suitable reaction conditions for the direct assembly of indole–pyrrole hybrids under green conditions, we turned our attention to Amberlyst 15(H) (A-15), a commercially available heterogeneous sulfonic acid catalyst.³⁶ A-15 stands out among commonly used Brønsted acids because it is inexpensive, non-toxic, easily handleable, recoverable and recyclable. Thus, after the initial base promoted (MeONa) formation of the adduct I, A-15 (0.6 mmol) was subsequently added to the reaction mixture. To our great satisfaction, the in situ transformation of the intermediate proceeded smoothly, affording the desired product 3a with excellent isolated yield (12 h, 89%).

Scheme 1 One-pot two-step synthesis of indole–pyrrole hybrid 3a.

After establishing the reaction conditions for the rapid assembly of 3a, the reaction with indole derivatives having diverse substituents was carried out (Scheme 2).

Scheme 2 Substrate scope of highly functionalized indole–pyrrole hybrids 3. Reagents and conditions: (i) 1 (0.4 mmol), 2 (0.4 mmol), MeONa (0.08 mmol), THF, 0 °C; (ii) A-15 (1.5 eq.), r.t.

For example, substrates with methyl and halogen groups (Cl, Br, and F) at the 4-,5-,6-, and 7-position of the indole unit were positioned to give the corresponding product. Furthermore, 3-cyanoacetyl-4-benzyloxy indole reacted with DD 2a to provide the indole–pyrrole hybrid 3d, albeit in modest yield. On the other hand, the use of methyl indole-4-carboxylate furnished the relative hybrid compound 3h in excellent yield. While the use of a DD partner with ethyl ester protection at the N-terminus (2b) provided the desired compound 3i in lower yield than those from 2a, the introduction of a methyl group at the N1- and C2-position of the indole ring was well tolerated (3j and 3k). Moderate result in terms of yield was finally obtained by introducing both benzyloxy and methyl groups at C4 and N1 of the indole moiety, respectively (3l).

According to the experimental results and previous studies, a plausible mechanism is proposed (Scheme 3). Initially, a base-catalyzed Michael addition between 1 and 2 gives the adduct intermediate I, which undergoes an intramolecular pyrroline formation to furnish 2,3-dihydro-1H-pyrrol-2-ol intermediate II. Finally, this latter undergoes aromatization to give compound 3. Although an intramolecular nucleophilic attack of hydrazonic nitrogen at the cyano group via a path B mechanism to produce product 3′ from I could also be possible, we did not observe this occurrence. The compound 3 was obtained with exclusive chemoselectivity, suggesting that the reaction proceeds via a carbonyl function attack through a path A mechanism, a result which is supported by the previous literature data.^31,32

Scheme 3 Plausible mechanism for the formation of 3.

In addition, the presence of an alkoxycarbonyl amino residue at the pyrrole N-atom makes these biheterocycles excellent precursors for further synthetic manipulations such as N-Boc deprotection and N–N bond cleavage (Scheme 4).

Scheme 4 Synthetic transformations of 3a. (i) TFA (5 eq.), CH₂Cl₂, r.t.; (ii) (1) BrCH₂CO₂Et (1.5 eq.), Cs₂CO₃ (2.5 eq.), CH₃CN, 50 °C; (2) Cs₂CO₃ (2.5 eq.), CH₃CN, 80 °C.

Accordingly, compound 3a was subjected to removal of Boc protecting group by treatment with TFA,³⁷ to give 3-(1H-pyrrol-1-amine-2-yl)-1H-indole derivative 4a. For the N–N bond cleavage, the conversion to 5a was successfully obtained employing the Magnus conditions.³⁸

The in vitro anti-parasitic activity of all fourteen new indole–pyrrole hybrids was first evaluated on L. infantum MHOM/TN/80/IPT1 promastigotes at a single dose of 20 μM for 72 h. Only compounds 3j, 3c and 3d showed an anti-leishmanial activity >40% at this concentration. In particular, the non-substituted indole 3a did not show any inhibition of L. infantum promastigotes. Methylation of the indole ring in position 1 (compound 3j) and 7 (compound 3c) gave active compounds with inhibition of 47.6% and 44.1%, respectively (entries 2 and 5, Table 1). On the other hand, 2- and 5-methyl indole derivatives 3k and 3b did not show any activity. Halogenated derivatives 3e–g also showed no inhibition of the promastigote's viability. Position 6 was also explored by electron withdrawing (–COOMe, compound 3h) or donating (–OBn, compound 3d) substituents. Although, 3h was not active, 3d resulted the most active compound of the series with an inhibition of 63% of the protozoa viability. N-Methylation of 3d gave a less active compound with an inhibition of 35.6% of the promastigotes (compound 3l, entry 11), contrary to what was shown by compound 3a. Switching from Boc carbamate 3a to ethyl carbamate 3i showed no improvement in terms of activity (entry 12). Cleavage of N-Boc protecting group (compound 4a) or N–N bond (compound 5a) on the pyrrole ring did not give any active compounds (entries 13–14).

Table 1 Summary of indole–pyrrole hybrid activity on L. infantum promastigotes and THP-1 cells and corresponding selectivity indexes. IC₅₀ and CC₅₀ values are reported as mean and 95% CI, from duplicate experiments. Miltefosine (MILT) was used a positive control

	Cmp	Inhibition of L. infantum^a at 20 μM (%)	L. infantum^a IC50 (μM) (95% CI)	THP-1 CC50 (μM) (95% CI)	SI^b
a L. infantum MHOM/TN/80/IPT1 promastigotes.b Selectivity index = CC50/IC50.
1	3a	−31.6
2	3j	47.6	16.7 (15.1–18.8)	61.2 (58.6–63.5)	3.7
3	3k	−48.9
4	3b	−4.1
5	3c	44.1	20.7 (18.9–23.9)	65.2 (53.0–77.4)	3.2
6	3g	−29.8
7	3e	−15.6
8	3f	−75.7
9	3h	−13.9
10	3d	63.0	9.6 (8.6–10.7)	43.1 (36.2–51.3)	4.5
11	3l	35.6
12	3i	−91.5
13	4a	−49.8
14	5a	−46.6
15	MILT	97.8	3.7 (3.3–4.1)	36.6 (32.8–40.6)	9.9

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The most promising compounds 3j, 3c, and 3d were tested on L. infantum MHOM/TN/80/IPT1 promastigotes for 72 h with scalar dilution 1 : 2 and 2 : 3 (from 20 to 0.31 μM) to determine IC₅₀. While 3j and 3c showed IC₅₀ of 16.7 μM and 20.7 μM, compound 3d displayed IC₅₀ < 10 μM (9.6 μM, entry 10). Later, the exact cytotoxicity (CC₅₀) of these three most active compounds was evaluated on THP-1 cells as described in the methods. All three compounds showed moderate cytotoxicity (43 μM < CC₅₀ < 65 μM). The most active compound 3d displayed a cytotoxicity comparable to miltefosine (entries 10 and 15, Table 1).

Conclusions

Over the last 150 years of research, about 91% of small-molecule drugs were discovered through phenotypic screening. The data shows the importance of this type of research which could possibly counteract the actual productivity crisis of target-based drug discovery.³⁹ Along this line, a novel series of densely functionalized indole–pyrrole bi-heterocycles has been prepared and phenotypically screened against L. infantum promastigotes. The most active compounds 3d, 3j, and 3c showed IC₅₀ of 9.6, 16.7 and 20.7 μM, respectively. All these three compounds displayed moderate toxicity on human macrophage-like THP-1 cells comparable or lower than the reference drug miltefosine. Although the most potent compound 3d showed a moderate selectivity index (SI ≈ 5), it may represent a lead compound of a new class of multifunctionalized indole–pyrrole hybrid active against leishmaniasis. Even if at the present we do not have information about the biological target(s) of this class of molecules, this work does represent a further successful example of molecular hybridization strategy and consequent phenotypic screening for the discovery of new antiparasitic agents.

Experimental

Chemistry

General remarks. All the commercially available reagents and solvents were purchased from Sigma-Aldrich (St. Louis, MO, USA), BLDpharm (Shanghai, China), Fluorochem (Hadfield, UK), or TCI (Tokyo, Japan) and they were used without further purification. In particular, indoles (Ia–k), cyanoacetic acid (A) and acetic anhydride (B) were commercial materials; 3-cyanoacetyl indoles (CAIs) 1a–k and 1,2-diaza-1,3-dienes (DDs) 2a,b were prepared according to literature procedures (see ESI†). Chromatographic purification of compounds was carried out on silica gel (60–200 μm). TLC analysis was performed on Merck silica gel plates (silica gel 60 F254); compounds were visualized by exposure to UV light and by dipping the plates in 1% Ce(SO₄)·4H₂O, 2.5% (NH₄)₆Mo₇O₂₄·4H₂O in 10% sulphuric acid followed by heating on a hot plate. All ¹H NMR and ¹³C NMR spectra were recorded at 400 and 101 MHz, respectively, using DMSO-d₆ or CDCl₃ as solvent on a Bruker Ultrashield 400 spectrometer (Bruker, Billerica, MA, USA) and analyzed using TopSpin 1.3 (2013) software package. Chemical shifts (δ scale) are reported in parts per million (ppm) relative to the central peak of the solvent and are sorted in descending order within each group. The following abbreviations are used to describe peak patterns where appropriate: s = singlet, d = doublet, dd = doublet of doublet, t = triplet, q = quartet, sept = septet, m = multiplet and br = broad signal. All coupling constants (J value) are given in Hertz [Hz]. High-resolution mass spectra were performed by slow direct infusion (5 μL min⁻¹) of ≈0.1 μg mL⁻¹ solution (acetonitrile/0.1% aqueous formic acid 1 : 1) of new compounds, using Orbitrap Exploris 240 mass spectrometer (Thermo Scientific, Waltham, MA, USA) equipped with an ESI source; only molecular ions [M + H]⁺ are given. Melting points were determined by Buchi (Gallen, Switzerland) B-540 in open capillary tubes and are uncorrected.

General procedure for the one-pot two-step synthesis of indole–pyrrole hybrids 3a–l. To a stirred solution of the 3-cyanoacetyl indole 1 (0.4 mmol) in THF (2 mL) in the presence of a catalytic amount of sodium methoxide (0.08 mmol) was added dropwise a solution of azoalkene 2 (0.4 mmol) in THF (2 mL) at 0 °C. The mixture was magnetically stirred at 0 °C until consumption of the starting material (TLC check). Once the Michael addition was completed (the formation of two isomers of adduct intermediate as major components was revealed by TLC), Amberlyst 15(H) (1.5 equiv.) was added, and the reaction was stirred for an additional 12 hours. After completion of the reaction (monitored by TLC), the resin was filtered off, washed with THF and the solvent was removed under reduced pressure. The crude residue was purified by silica gel column chromatography using cyclohexane and EtOAc as an eluent to obtain the corresponding product 3. As an example, we report below the characterization of compound 3a. The characterization of the compounds 3b–l is reported in the ESI.†

Ethyl 1-((tert-butoxycarbonyl)amino)-4-cyano-5-(1H-indol-3-yl)-2-methyl-1H-pyrrole-3-carboxylate (3a). Compound 3a was isolated by column chromatography (ethyl acetate/cyclohexane 30 : 70) in 89% yield (145.4 mg); white solid; mp: 188–190 °C; ¹H NMR (400 MHz, DMSO-d₆) δ 11.73 (s, 1H), 10.56 (s, 1H), 7.53 (d, J = 2.8 Hz, 1H), 7.50 (d, J = 8.0 Hz, 2H), 7.20 (t, J = 7.6 Hz, 1H), 7.10 (t, J = 7.6 Hz, 1H), 4.30 (q, J = 7.2 Hz, 2H), 2.41 (s, 3H), 1.36 (s, 9H), 1.33 (t, J = 7.2 Hz, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ 162.8, 154.7, 139.0, 138.4, 136.3, 127.2, 126.0, 122.4, 120.3, 119.9, 116.0, 112.5, 110.2, 101.8, 90.4, 81.9, 60.5, 28.2, 14.5, 10.9; HRMS (ESI-Orbitrap, m/z): calcd for C₂₂H₂₅N₄O₄ [M + H]⁺ 409.1870; found 409.1882.

Ethyl 1-amino-4-cyano-5-(1H-indol-3-yl)-2-methyl-1H-pyrrole-3-carboxylate (4a). Compound 4a was prepared according to the literature procedure.³⁷ To a solution of 3a (81.7 mg, 0.2 mmol) in dichloromethane (2 mL) was added TFA (96.0 mg, 1.0 mmol) dropwise at 0 °C. The mixture was stirred at r.t. until TLC showed complete consumption of starting material. Water was added and the mixture was extracted with AcOEt (2 × 10 mL). The combined organic layer was washed with brine, separated, dried over Na₂SO₄ and filtered. After the solvent was removed under reduced pressure, the residue was purified by silica gel column chromatography (ethyl acetate/cyclohexane 40 : 60) to afford 4a as white solid (37.6 mg, 61%). Mp: 209–211 °C; ¹H NMR (400 MHz, DMSO-d₆) δ 11.65 (s, 1H), 7.75 (d, J = 2.3 Hz, 1H), 7.57 (d, J = 8.0 Hz, 1H), 7.49 (d, J = 8.0 Hz, 2H), 7.19 (t, J = 7.5 Hz, 1H), 7.11 (d, J = 7.5 Hz, 2H), 4.26 (q, J = 7.2 Hz, 2H), 1.31 (t, J = 7.2 Hz, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ 163.2, 139.1, 138.0, 136.3, 127.7, 126.2, 122.1, 120.5, 120.0, 116.9, 112.3, 109.9, 102.9, 88.9, 60.1, 14.6, 11.6. HRMS (ESI-Orbitrap, m/z): calcd for C₁₇H₁₇N₄O₂ [M + H]⁺ 309.1346; found 309.1341.

Ethyl 4-cyano-5-(1H-indol-3-yl)-2-methyl-1H-pyrrole-3-carboxylate (5a). Compound 5a was prepared according to a modified version of the Magnus method.³⁸ To a solution of 3a (81.7 mg, 0.2 mmol) in acetonitrile (5 mL), ethyl bromoacetate (0.033 mL, 0.3 mmol) and Cs₂CO₃ (162.9 mg, 0.5 mmol) were added. The mixture was stirred at 50 °C (oil bath) until the disappearance of the starting material (0.5 h, TLC check). The solvent was removed under vacuum, water (5 mL) was added, and the mixture was extracted with ethyl acetate (3 × 10 mL). The combined organic layer was dried over Na₂SO₄ and filtered. After the solvent was removed under reduced pressure, the residue was dissolved in acetonitrile (5 mL) and Cs₂CO₃ (162.9 mg, 0.5 mmol) was added. The mixture was stirred at 80 °C until TLC showed complete consumption of intermediate. The solvent was removed under vacuum, water (5 mL) was added, and the mixture was extracted with ethyl acetate (3 × 10 mL). The collected organic phase was washed with brine, dried over Na₂SO₄ and filtered. After the solvent was removed under vacuum, the residue was purified by column chromatography (ethyl acetate/cyclohexane 30 : 70) to afford compound 5a as a white solid (34.1 mg, 58% yield). Mp: 231–233 °C; ¹H NMR (400 MHz, DMSO-d₆) δ 12.11 (s, 1H), 11.65 (s, 1H), 7.75–7.71 (m, 2H), 7.50 (d, J = 8.0 Hz, 1H), 7.21 (t, J = 8.0 Hz, 1H), 7.14 (t, J = 8.0 Hz, 1H), 4.25 (q, J = 7.2 Hz, 2H), 2.51 (s, 3H), 1.31 (t, J = 7.2 Hz, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ 163.3, 137.9, 136.5, 136.0, 125.9, 125.0, 122.5, 120.2, 120.1, 117.4, 112.5, 112.0, 105.0, 89.1, 60.0, 14.7, 13.1. HRMS (ESI-Orbitrap, m/z): calcd for C₁₇H₁₆N₃O₂ [M + H]⁺ 294.1237; found 294.1252.

In vitro studies

Parasite and cell cultures. The WHO international reference strain L. infantum MHOM/TN/80/IPT1 (ATCC® 50134™) was routinely cultured in Evans's Modified Tobie Medium (EMTM) at 26–28 °C. To test the indole–pyrrole hybrid compounds, the parasites were maintained in RPMI-PY medium as described previously.¹³ The human monocytic cell line THP-1 (ECACC 88081201) was cultured in RPMI-1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS), 2 mM L-glutamine, 100 μg mL⁻¹ streptomycin, 100 U L⁻¹ penicillin, and maintained in a humidified incubator at 37 °C and 5% CO₂. All cell culture reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA).

L. infantum viability assay. The activity of indole–pyrrole hybrid compounds was first evaluated on late log/stationary phase L. infantum promastigotes resuspended in complete RPMI-PY medium in 96-well plates with a density of 2.5 × 10⁶ parasites per mL (100 μL per well). The anti-parasitic activity was initially investigated at a single dose of 20 μM of each compound for 72 h at 26 °C. Those compounds showing anti-parasitic activity >40% were further tested with scalar dilutions 1 : 2 or 2 : 3 (from 20 to 0.31 μM) on promastigotes to determine IC₅₀. As positive control, the anti-leishmanial drug miltefosine (Sigma-Aldrich, Milan, Italy) was included. All conditions were carried out in duplicate. The promastigotes viability was established using the CellTiter 96H aqueous non-radioactive cell proliferation assay (Promega, Madison, Wisconsin, USA), as previously reported.²⁴ The IC₅₀ values were determined using the nonlinear regression curves in GraphPad Prism 8.0 (GraphPad Software, Inc., San Diego, CA, USA). The equation used for data fitting was Y = 100/(1 + 10((log IC₅₀ − X) × hillslope)) (hillslope not constrained), where X is equal to the log of concentration and Y is the normalized response.

Evaluation of cytotoxicity on THP-1 cells. The cytotoxicity of indole–pyrrole hybrid compounds was evaluated on THP-1 cells seeded at a density of 5 × 10⁶ cells per mL, 100 μL per well in a 96-well plate and treated for 48 h with 20 ng mL⁻¹ phorbol myristic acid (PMA) to induce differentiation into macrophages-like cells. After cell adhesion, selected compounds were tested to determine CC₅₀ with scalar dilutions 1 : 2 (from 100 to 3.12 μM). Not-treated cells (negative control) and the anti-leishmanial drug miltefosine were included in each experiment. Each condition was carried out in duplicate. The CellTiter 96H Aqueous Non-Radioactive Cell Proliferation Assay (Promega) was performed to evaluate the selected compounds cytotoxicity, as described above. The selectivity index, calculated as the ratio between cytotoxicity in THP-1 and activity against L. infantum promastigotes, was included for each compound.

Statistical analysis. The evaluation of IC₅₀ in promastigotes and CC₅₀ in mammalian cells following indole–pyrrole hybrid compounds treatment was performed by nonlinear regression analysis and expressed as means and 95% confidence interval. All statistical tests were performed using GraphPad Prism version 8 (GraphPad Software, Inc., La Jolla, CA, USA). 277 A p-value ≤ 0.05 was considered significant.

Author contributions

Conceptualization: SL and GF; data curation: AD, FM and GM; funding acquisition: FM, LG, SL and GF; investigation: VC, AD, MGB and SM; methodology: AD, FM and GM; project administration: FM, LG, SL and GF; supervision: LG, SL and GF; writing – original draft: SL and GF; writing – review & editing: VC, AD, MGB, FM, GM, SL and GF.

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

The Authors thank Michela Raduazzo for her precious collaboration, Prof. Michele Menotta for HRMS characterization of the new compounds, and Prof. Francesca Ida Carducci (University of Urbino Carlo Bo) for editing the English language and style of the manuscript. The work was supported by the University of Urbino grant “DISB_GALLUZZI_PROG_SIC_ALIMENTARE_2021”, and the European Union – NextGenerationEU under the Italian Ministry of University and Research (MUR) National Innovation Ecosystem grant ECS00000041 - VITALITY - CUP H33C22000430006.

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Footnotes

† Electronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d4ra02790f

‡ These authors contributed equally to this work.

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