Synthesis and Biological Evaluation of Imidazopyridine-Isatin Hybrids as Inhibitors of Leishmania major Growth

Introduction

Over 12 million individuals worldwide suffer from leishmaniasis, a neglected tropical disease that causes severe morbidity and over 50,000 deaths yearly. New anti-leishmanial medications are desperately needed, as evidenced by the paucity of research and control efforts despite the disease’s worldwide effect.^1,2 The internal parasite Leishmania, which is spread by phlebotomine sandflies, is the cause of the illness. After infecting macrophages, the parasite develops into amastigotes, which proliferate and avoid immune clearance, causing tissue damage. Depending on the species and host immunological response, the primary clinical manifestations include visceral leishmaniasis (VL), mucocutaneous (MCL), diffuse cutaneous (DCL), and cutaneous (CL).^3,4

Dihydrofolate reductase (DHFR) and thymidylate synthase (TS) are essential enzymes in the folate metabolic pathway, which the parasite depends on for growth. Drug resistance can result from pteridine reductase 1 (PTR1) compensating for DHFR inhibition, while DHFR facilitates DNA synthesis by converting dihydrofolate (DHF) to tetrahydrofolate (THF).^5,6 Pentavalent antimonials, amphotericin B, miltefosine, pentamidine, and paromomycin are examples of current anti-leishmanial medications that have drawbacks such as toxicity, decreased efficacy, resistance, and high cost.^7–9 These difficulties highlight the need for novel oral drugs that are more economical, efficient, and safe.

There have been reports of pyridine derivatives having anti-leishmanial properties. As an illustration, triazolopyridyl ketones (compounds I–II, Figure 1) shown strong micromolar action against various Leishmania species promastigotes.^10,11 The sulfone metabolite of fexinidazole (III, Figure 1) is one example of an imidazole derivative that has shown effectiveness against intracellular amastigotes.¹² Numerous pharmacologically active substances, such as antifungal, anticancer, and antileishmanial drugs (IV–VII, Figure 1), contain fused imidazo[1,2-a]pyridine scaffolds with low cytotoxicity and action against both promastigote and amastigote stages.^13–15

Figure 1 Selected heterocyclic derivatives as anti-leishmanial agents: pyridine (I–II), imidazole (III), and fused imidazo[1,2-a]pyridine (IV–VII) highlighted in yellow, and isatin-based derivatives (VIII–XI) with or without DHFR inhibitory activity highlighted in blue.

Significant therapeutic promise, including anti-leishmanial properties, has also been demonstrated by heterocyclic compounds generated from isatin (indoline-dione). Targeting DHFR and exhibiting low micromolar potency against promastigotes and intracellular amastigotes, a few derivatives (VIII–XI, Figure 1) demonstrated improved action and selectivity.^16–31

Building on these results, we postulated that by integrating the essential pharmacophoric characteristics of both scaffolds, covalently joining imidazo[1,2-a]pyridine and isatin via a hydrazide linker might produce hybrids with increased activity and selectivity. This led to the design and synthesis of a novel set of molecular hybrids (5a-h, 8a-d, 11a-d, Figure 2). In order to maximize interactions with the DHFR-TS and PTR1 targets, structural changes were made at the C2-position of imidazopyridine as well as at the C5/C7 and N1 of the isatin nucleus. This design approach is summarized in Figure 2.

Figure 2 Structural planning of the newly synthesized compounds.

Materials and Methods

Chemistry

An Electrothermal IA 9000 device that was not calibrated was used to detect melting points. All synthesized compounds’ high-resolution mass spectrometry (HR-TOF-ESI-MS) data were obtained using a JEOL JMS-700 device (Tokyo, Japan). At Tokushima Bunri University’s Faculty of Pharmaceutical Science in Japan, Bruker 400 MHz spectrometers were used to record the ¹H and ¹³C NMR spectra. Coupling constants (J) are expressed in Hz, and chemical shifts (δ) in ppm. Using an iodine–potassium spray and a chloroform/methanol (9.5:0.5, v/v) mobile phase, the reaction progress was tracked by TLC on silica gel 60 F₂₅₄ aluminum sheets (Merck). The preparation of compounds 2,³² 3,³² 7a-d,³³ and 10a-d³⁴ was done in accordance with previously published protocols. Supplementary Information contains the characterization information for the recently synthesized compounds.

General Procedure for the Synthesis of Target Compounds (5a-h, 8a-d, and 11a-d)

A solution of hydrazide 3 (48 mg, 0.25 mmol) in 10 mL of absolute ethanol with five drops of glacial acetic acid was made in a 25 mL round-bottom flask. Alkylated isatins 7a-d or 10a-d (0.25 mmol) and other isatins 4a-h were added to this solution. TLC was used to track the reaction mixture’s progress during its 10-hour reflux. Filtration was used to gather the precipitated solid, which was then dried and purified by recrystallization from an ethanol/DMF combination.

(Z)-2-Methyl-N’-(2-oxoindolin-3-ylidene)imidazo[1,2-a]pyridine-3-carbohydrazide (5a)

Yield: 90% (Yellowish powder); Melting point: > 300°C. ¹H NMR δ (ppm): 13.40 (s, 1H, NH of hydrazide), 11.29 (s, 1H, NH of isatin), 9.39 (dt, J = 7.0, 1.2 Hz, 1H, Aromatic-H), 7.69 (dt, J = 8.9, 1.2 Hz, 1H, Aromatic-H), 7.59 (dt, J = 7.4, 1.0 Hz, 1H, Aromatic-H), 7.57–7.52 (m, 1H, Aromatic-H), 7.37 (td, J = 7.7, 1.3 Hz, 1H, Aromatic-H), 7.19 (td, J = 6.9, 1.3 Hz, 1H, Aromatic-H), 7.10 (td, J = 7.6, 1.0 Hz, 1H, Aromatic-H), 6.96 (dt, J = 7.8, 0.9 Hz, 1H, Aromatic-H), 2.78 (s, 3H, CH₃). High-Resolution MS (ESI) m/z: [M+H]⁺ Theoretical 320.11420 and Observed 320.11401. Microanalysis (%) Theoretical (Observed) for C₁₇H₁₃N₅O₂: C, 63.94 (64.17); H, 4.10 (4.11); N, 21.93 (22.00)%.

(Z)-N’-(5-fluoro-2-oxoindolin-3-ylidene)-2-methylimidazo[1,2-a]pyridine-3-carbohydrazide (5b)

Yield: 76% (Yellowish powder); Melting point: > 300°C. ¹H NMR δ (ppm): 13.44 (s, 1H, NH of hydrazide), 11.32 (s, 1H, NH of isatin), 9.40 (d, J = 7.0 Hz, 1H, Aromatic-H), 7.72 (s, 1H, Aromatic-H), 7.59 (s, 1H, Aromatic-H), 7.42 (dd, J = 8.0, 2.7 Hz, 1H, Aromatic-H), 7.24 (ddd, J = 11.6, 6.5, 2.4 Hz, 2H, Aromatic-H), 6.98 (dd, J = 8.6, 4.2 Hz, 1H, Aromatic-H), 2.80 (s, 3H, CH₃). High-Resolution MS (ESI) m/z: [M+H]⁺ Theoretical 338.10478 and Observed 338.10468. Microanalysis (%) Theoretical (Observed) for C₁₇H₁₂FN₅O₂: C, 60.53 (60.29); H, 3.59 (3.57); N, 20.76 (20.81)%.

(Z)-N’-(7-Fluoro-2-oxoindolin-3-ylidene)-2-methylimidazo[1,2-a]pyridine-3-carbohydrazide (5c)

Yield: 70% (Yellowish powder); Melting point: > 300°C. ¹H NMR δ (ppm): 13.41 (s, 1H, NH of hydrazide), 11.84 (s, 1H, NH of isatin), 9.41 (dt, J = 7.0, 1.2 Hz, 1H, Aromatic-H), 7.73 (dt, J = 9.0, 1.2 Hz, 1H, Aromatic-H), 7.60–7.56 (m, 1H, Aromatic-H), 7.47 (dd, J = 7.5, 0.9 Hz, 1H, Aromatic-H), 7.33 (ddd, J = 10.5, 8.4, 1.0 Hz, 1H, Aromatic-H), 7.24–7.20 (m, 1H, Aromatic-H), 7.16–7.13 (m, 1H, Aromatic-H), 2.81 (s, 3H, CH₃). High-Resolution MS (ESI) m/z: [M+H]⁺ Theoretical 338.10478 and Observed 338.10458. Microanalysis (%) Theoretical (Observed) for C₁₇H₁₂FN₅O₂: C, 60.53 (60.77); H, 3.59 (3.58); N, 20.76 (20.70)%.

(Z)-N’-(5-Chloro-2-oxoindolin-3-ylidene)-2-methylimidazo[1,2-a]pyridine-3-carbohydrazide (5d)

Yield: 82% (Yellowish powder); Melting point: > 300°C. ¹H NMR δ (ppm): 13.39 (s, 1H, NH of hydrazide), 11.41 (s, 1H, NH of isatin), 9.42 (d, J = 6.9 Hz, 1H, Aromatic-H), 7.73 (d, J = 8.9 Hz, 1H, Aromatic-H), 7.61–7.55 (m, 2H, Aromatic-H), 7.44 (dd, J = 8.3, 2.2 Hz, 1H, Aromatic-H), 7.22 (t, J = 6.8 Hz, 1H, Aromatic-H), 7.00 (d, J = 8.3 Hz, 1H, Aromatic-H), 2.81 (s, 3H, CH₃). High-Resolution MS (ESI) m/z: [M+H]⁺/[M+H+2]⁺ Theoretical 354.07523/356.07228 and Observed 354.07513/356.07205. Microanalysis (%) Theoretical (Observed) for C₁₇H₁₂ClN₅O₂: C, 57.72 (57.95); H, 3.42 (3.40); N, 19.80 (19.77)%.

(Z)-N’-(5-Bromo-2-oxoindolin-3-ylidene)-2-methylimidazo[1,2-a]pyridine-3-carbohydrazide (5e)

Yield: 82% (Yellowish powder); Melting point: > 300°C. ¹H NMR δ (ppm): 13.43 (s, 1H, NH of hydrazide), 11.41 (s, 1H, NH of isatin), 9.41 (s, 1H, Aromatic-H), 7.57 (s, 2H, Aromatic-H), 7.21 (s, 2H, Aromatic-H), 6.85 (s, 2H, Aromatic-H), 2.80 (s, 3H, CH₃). High-Resolution MS (ESI) m/z: [M+H]⁺/[M+H+2]⁺ Theoretical 398.02471/400.02267 and Observed 398.02460/400.02240. Microanalysis (%) Theoretical (Observed) for C₁₇H₁₂BrN₅O₂: C, 51.27 (51.04); H, 3.04 (3.01); N, 17.59 (17.64)%.

(Z)-2-Methyl-N’-(5-methyl-2-oxoindolin-3-ylidene)imidazo[1,2-a]pyridine-3-carbohydrazide (5f)

Yield: 76% (Yellowish powder); Melting point: > 300°C. ¹H NMR δ (ppm): 13.43 (s, 1H, NH of hydrazide), 11.33 (s, 1H, NH of isatin), 9.41 (dt, J = 7.0, 1.2 Hz, 1H, Aromatic-H), 7.97 (s, 1H, Aromatic-H), 7.73 (dt, J = 2.5, 1.2 Hz, 1H, Aromatic-H), 7.60–7.57 (m, 1H, Aromatic-H), 7.44 (d, J = 1.7 Hz, 1H, Aromatic-H), 7.24 (dt, J = 4.1, 2.1 Hz, 1H, Aromatic-H), 6.87 (d, J = 7.9 Hz, 1H, Aromatic-H), 2.80 (s, 3H, CH₃), 2.31 (s, 3H, CH₃). High-Resolution MS (ESI) m/z: [M+H]⁺ Theoretical 334.12985 and Observed 334.12955. Microanalysis (%) Theoretical (Observed) for C₁₈H₁₅N₅O₂: C, 64.86 (65.07); H, 4.54 (4.55); N, 21.01 (20.98)%.

(Z)-2-Methyl-N’-(2-oxo-5-(trifluoromethoxy)indolin-3-ylidene)imidazo[1,2-a]pyridine-3-carbohydrazide (5g)

Yield: 69% (Yellowish powder); Melting point: > 300°C. ¹H NMR δ (ppm): 13.36 (s, 1H, NH of hydrazide), 11.46 (s, 1H, NH of isatin), 9.40 (d, J = 6.9 Hz, 1H, Aromatic-H), 7.70 (d, J = 8.9 Hz, 1H, Aromatic-H), 7.59–7.48 (m, 2H, Aromatic-H), 7.39 (d, J = 8.5 Hz, 1H, Aromatic-H), 7.21 (t, J = 6.8 Hz, 1H, Aromatic-H), 7.05 (d, J = 8.6 Hz, 1H, Aromatic-H), 2.80 (s, 3H, CH₃). High-Resolution MS (ESI) m/z: [M+H]⁺ Theoretical 404.09650 and Observed 404.09628. Microanalysis (%) Theoretical (Observed) for C₁₈H₁₂F₃N₅O₃: C, 53.60 (53.38); H, 3.00 (3.00); N, 17.36 (17.33)%.

(Z)-2-Methyl-N’-(5-nitro-2-oxoindolin-3-ylidene)imidazo[1,2-a]pyridine-3-carbohydrazide (5h)

Yield: 75% (Yellowish powder); Melting point: > 300°C. ¹H NMR δ (ppm): 13.24 (s, 1H, NH of hydrazide), 11.52 (s, 1H, NH of isatin), 9.15 (s, 1H, Aromatic-H), 9.04 (d, J = 2.2 Hz, 1H, Aromatic-H), 8.32 (dd, J = 8.7, 2.2 Hz, 1H, Aromatic-H), 7.72 (d, J = 8.9 Hz, 1H, Aromatic-H), 7.59–7.54 (m, 1H, Aromatic-H), 7.18 (t, J = 7.1 Hz, 1H, Aromatic-H), 7.11 (d, J = 8.6 Hz, 1H, Aromatic-H), 2.74 (s, 3H, CH₃). High-Resolution MS (ESI) m/z: [M+H]⁺ Theoretical 365.09928 and Observed 365.09909. Microanalysis (%) Theoretical (Observed) for C₁₇H₁₂N₆O₄: C, 56.05 (55.84); H, 3.32 (3.34); N, 17.57 (17.60)%.

(Z)-2-Methyl-N’-(1-methyl-2-oxoindolin-3-ylidene)imidazo[1,2-a]pyridine-3-carbohydrazide (8a)

Yield: 79% (Yellowish powder); Melting point: > 300°C. ¹H NMR δ (ppm): 13.30 (s, 1H, NH of hydrazide), 9.38 (d, J = 6.8 Hz, 1H, Aromatic-H), 7.67 (d, J = 8.6 Hz, 1H, Aromatic-H), 7.60 (d, J = 7.4 Hz, 1H, Aromatic-H), 7.53 (t, J = 7.9 Hz, 1H, Aromatic-H), 7.43 (t, J = 7.8 Hz, 1H, Aromatic-H), 7.16 (dt, J = 15.7, 7.3 Hz, 3H, Aromatic-H), 3.23 (s, 3H, CH₃), 2.80 (s, 3H, CH₃). High-Resolution MS (ESI) m/z: [M+H]⁺ Theoretical 334.12985 and Observed 334.12964. Microanalysis (%) Theoretical (Observed) for C₁₈H₁₅N₅O₂: C, 64.86 (65.11); H, 4.54 (4.56); N, 21.01 (21.07)%.

(Z)-N’-(1-ethyl-2-oxoindolin-3-ylidene)-2-methylimidazo[1,2-a]pyridine-3-carbohydrazide (8b)

Yield: 86% (Yellowish powder); Melting point: > 300°C. ¹H NMR δ (ppm): 13.30 (s, 1H, NH of hydrazide), 9.40 (dt, J = 7.0, 1.2 Hz, 1H, Aromatic-H), 7.68 (dt, J = 8.9, 1.2 Hz, 1H, Aromatic-H), 7.61 (dd, J = 7.5, 1.2 Hz, 1H, Aromatic-H), 7.54 (ddd, J = 8.9, 6.8, 1.3 Hz, 1H, Aromatic-H), 7.44 (td, J = 7.8, 1.3 Hz, 1H, Aromatic-H), 7.22–7.13 (m, 3H, Aromatic-H), 3.81 (q, J = 7.2 Hz, 2H, CH₂), 2.80 (s, 3H, CH₃), 1.22 (t, J = 7.2 Hz, 3H, CH₃). ¹³C NMR δ 160.80, 157.77, 148.55, 146.84, 142.82, 136.43, 131.87, 129.00, 128.37, 123.56, 121.01, 119.95, 116.92, 114.77, 114.26, 110.45, 40.67, 34.54, 17.29, 13.08. High-Resolution MS (ESI) m/z: [M+H]⁺ Theoretical 348.14550 and Observed 348.14526. Microanalysis (%) Theoretical (Observed) for C₁₉H₁₇N₅O₂: C, 65.69 (65.90); H, 4.93 (4.95); N, 20.16 (20.09)%.

(Z)-N’-(5-Chloro-1-methyl-2-oxoindolin-3-ylidene)-2-methylimidazo[1,2-a]pyridine-3-carbohydrazide (8c)

Yield: 69% (Yellowish powder); Melting point: > 300°C. High-Resolution MS (ESI) m/z: [M+H]⁺ Theoretical 368.09088 and Observed 368.09079. Microanalysis (%) Theoretical (Observed) for C₁₈H₁₄ClN₅O₂: C, 58.78 (60.01); H, 3.84 (3.85); N, 19.04 (18.99)%.

(Z)-N’-(5-Bromo-1-Methyl-2-Oxoindolin-3-Ylidene)-2-Methylimidazo[1,2-A]pyridine-3-Carbohydrazide (8d)

Yield: 68% (Yellowish powder); Melting point: > 300°C. ¹H NMR δ (ppm): 13.32 (s, 1H, NH of hydrazide), 9.43 (d, J = 6.8 Hz, 1H, Aromatic-H), 7.68 (d, J = 7.9 Hz, 1H, Aromatic-H), 7.60 (d, J = 7.9 Hz, 1H, Aromatic-H), 7.48 (d, J = 9.4 Hz, 1H, Aromatic-H), 7.20 (dt, J = 23.2, 10.8 Hz, 3H, Aromatic-H), 3.26 (s, 3H, CH₃), 2.83 (s, 3H, CH₃). High-Resolution MS (ESI) m/z: [M+H]⁺/[M+H+2]⁺ Theoretical 412.04036/414.03832 and Observed 412.04022/414.03806. Microanalysis (%) Theoretical (Observed) for C₁₈H₁₄BrN₅O₂: C, 52.44 (52.15); H, 3.42 (3.40); N, 16.99 (17.02)%.

(Z)-N’-(1-Benzyl-2-oxoindolin-3-ylidene)-2-methylimidazo[1,2-a]pyridine-3-carbohydrazide (11a)

Yield: 79% (Yellowish powder); Melting point: > 300°C. ¹H NMR δ (ppm): 13.31 (s, 1H, NH of hydrazide), 9.42 (dt, J = 7.0, 1.2 Hz, 1H, Aromatic-H), 7.70 (dt, J = 8.9, 1.2 Hz, 1H, Aromatic-H), 7.64 (dd, J = 7.6, 1.2 Hz, 1H, Aromatic-H), 7.55 (ddd, J = 8.9, 6.9, 1.3 Hz, 1H, Aromatic-H), 7.41–7.33 (m, 5H, Aromatic-H), 7.31–7.27 (m, 1H, Aromatic-H), 7.20 (td, J = 6.9, 1.3 Hz, 1H, Aromatic-H), 7.15 (td, J = 7.6, 0.9 Hz, 1H, Aromatic-H), 7.05 (d, J = 7.9 Hz, 1H, Aromatic-H), 5.03 (s, 2H, CH₂), 2.84 (s, 3H, CH₃). High-Resolution MS (ESI) m/z: [M+H]⁺ Theoretical 410.16115 and Observed 410.16092. Microanalysis (%) Theoretical (Observed) for C₂₄H₁₉N₅O₂: C, 70.40 (70.17); H, 4.68 (4.69); N, 17.10 (17.06)%.

(Z)-2-Methyl-N’-(2-oxo-1-phenethylindolin-3-ylidene)imidazo[1,2-a]pyridine-3-carbohydrazide (11b)

Yield: 65% (Yellowish powder); Melting point: > 300°C. High-Resolution MS (ESI) m/z: [M+H]⁺ Theoretical 424.17680 and Observed 424.17673. Microanalysis (%) Theoretical (Observed) for C₂₅H₂₁N₅O₂: C, 70.91 (71.12); H, 5.00 (4.98); N, 16.54 (16.59)%.

(Z)-N’-(1-Benzyl-5-chloro-2-oxoindolin-3-ylidene)-2-methylimidazo[1,2-a]pyridine-3-carbohydrazide (11c)

Yield: 80% (Yellowish powder); Melting point: > 300°C. ¹H NMR δ (ppm): 13.29 (s, 1H, NH of hydrazide), 9.44 (dt, J = 7.0, 1.2 Hz, 1H, Aromatic-H), 7.73 (dt, J = 8.9, 1.2 Hz, 1H, Aromatic-H), 7.64 (d, J = 2.2 Hz, 1H, Aromatic-H), 7.58 (ddd, J = 8.7, 6.8, 1.3 Hz, 1H, Aromatic-H), 7.45 (dd, J = 8.4, 2.2 Hz, 1H, Aromatic-H), 7.39–7.30 (m, 5H, Aromatic-H), 7.24–7.21 (m, 1H, Aromatic-H), 7.08 (d, J = 8.4 Hz, 1H, Aromatic-H), 5.05 (s, 2H, CH₂), 2.85 (s, 3H, CH₃). High-Resolution MS (ESI) m/z: [M+H]⁺ Theoretical 444.12218 and Observed 444.12195. Microanalysis (%) Theoretical (Observed) for C₂₄H₁₈ClN₅O₂: C, 64.94 (65.19); H, 4.09 (4.10); N, 15.78 (15.83)%.

(Z)-N’-(1-Benzyl-5-bromo-2-oxoindolin-3-ylidene)-2-methylimidazo[1,2-a]pyridine-3-carbohydrazide (11d)

Yield: 74% (Yellowish powder); Melting point: > 300°C. ¹H NMR δ (ppm): 13.24 (s, 1H, NH of hydrazide), 9.42 (d, J = 7.0 Hz, 1H, Aromatic-H), 7.69 (d, J = 6.0 Hz, 2H, Aromatic-H), 7.56 (t, J = 8.7 Hz, 2H, Aromatic-H), 7.39–7.27 (m, 5H, Aromatic-H), 7.20 (t, J = 7.2 Hz, 1H, Aromatic-H), 7.00 (d, J = 8.5 Hz, 1H, Aromatic-H), 5.03 (s, 2H, CH₂), 2.83 (s, 3H, CH₃). High-Resolution MS (ESI) m/z: [M+H]⁺/[M+H+2]⁺ Theoretical 488.07166/490.06962 and Observed 488.07184/490.06976. Microanalysis (%) Theoretical (Observed) for C₂₄H₁₈BrN₅O₂: C, 59.03 (58.88); H, 3.72 (3.70); N, 14.34 (14.38)%.

Biological Evaluation

In vitro Anti-Leishmanial Activity

Both promastigote and amastigote forms of L. major strain were used for the in vitro evaluation. All experiments were conducted as reported earlier.^35–39 Axenic amastigotes were produced applying the method described by Teixeira et al⁴⁰ Briefly, L. major parasites were cultured in tissue flasks containing RPMI- 1640 (Gibco, Invitrogen, Co., UK) and supplemented with 10% heat inactivated fetal calf serum (HIFGs), 100 IU penicillin (Sigma), 100 mg/mL streptomycin (Sigma) and 1% L-glutamine (Sigma). Different test compounds were dissolved in DMSO to a final concentration of 1 mg/mL. Fresh complete media was used to prepare threefold serial dilutions of the standard solutions, the test compounds and the reference drug miltefosine in appropriate concentrations. 100 mL culture media of 3×10⁶ promastigotes of L. major were seeded into each well of a 96 well flat bottom plate. Various concentrations of the newly synthesized compounds (10, 3.33, 1.11, 0.37, 0.12 and 0.04 mg/mL) were added to the promastigote culture media. Wells containing only promastigotes were regarded as positive controls, while negative control wells contained 1% DMSO and the culture media alone. After 24 hours, 10 mL of alamar blue was added to each well, followed by measurement of fluorescence intensity (at wavelengths of 540 and 630 nm) using ELISA plate reader after 48 hours.

For evaluation of anti-amastigote activity, the test compounds were serially diluted in a 96-well microtitre plate to a final test concentration of 0.04–10 mg/mL in 50 mL culture medium. This was followed by addition of 50 mL suspensions containing 2×103 cells/mL amastigotes to each well. The plates were then incubated at 31^◦C in a humidified environment under 5% CO₂ 72 hours. Following an incubation period of 68 hours, 10 mL of fluorochrome resazurin solution was added to each well. Absorbance of the resulting mixture was measured, after a total incubation time of 72 hours, using 37 Victor 3 Multilabel Counter, at excitation and emission wavelengths of 530 nm and 590 nm respectively. Finally, the IC₅₀ value for each compound was determined from sigmoidal dose-response curves using the software Graphpad prism 3.0 (GraphPad Software, San Diego, CA, USA); results were expressed as mean ± SD of triplicate experiments with each test concentration measured in duplicates.

Reversal of the Anti-Leishmanial Activities

All operations were carried out in accordance with published protocols, and an in vitro promastigote growth assay was carried out using previously known methodology.²⁷

In vitro Cytotoxicity Testing

The most active compounds were evaluated at concentrations ranging from 0 to 100 μM in 96-well plates, each containing 1×10⁵ cells per well. Plates were incubated for 72 h at 37 °C under 95% humidity and 5% CO₂. All experiments were conducted according to previously published methodologies.⁴¹ African green monkey Kidney (Vero cells) was obtained from Holding Company for Biological Products & Vaccines (VACSERA, Dokki, Giza, Egypt).

Physicochemical Factors and Pharmacokinetic Properties in silico Forecasts

Virtual platforms were used to predict the pharmacokinetic profiles, drug-likeness, and chemical and physicochemical properties. For a thorough evaluation of drug-likeness, ADMET-related characteristics, and possible toxicity hazards, ADMETlab 3.0, an online program, was utilized.^42,43

Molecular Docking

The molecular docking investigations were conducted using AutoDock Vina and MGLTools, and the results were visualized using Discovery Studio. AutoDock Vina predicts how tiny molecules will attach to their targets using the AMBER force field, a united-atom scoring function, and a global optimization method based on a gradient-based local search genetic algorithm.^44–47 The Protein Data Bank provided the X-ray crystal structures of Leishmania major PTR1 (PDB ID: 6RXC) and DHFR-TS (PDB ID: 1J3K), both of which were co-crystallized with inhibitors. MGLTools was used to prepare ligands and proteins and convert them to PDBQT format. The coordinates of the co-crystallized ligands were used to determine the active sites of both DHFR-TS and L. major PTR1, with a grid box size of 22 × 22×22 Å along the x, y, and z axes. RMSD values of 0.75 Å for DHFR-TS and 0.69 Å for L. major PTR1 were obtained by redocking the reference compounds into their corresponding binding sites in order to validate the docking methodology. Docking scores and binding interactions derived from 2D interaction diagrams were used to assess the final results.

Results and Discussion

Chemistry

The synthetic strategies for obtaining the sixteen final compounds (5a-h, 8a-d, and 11a-d) are outlined in Schemes 1 and 2. In Scheme 1, pyridin-2-amine 1 was heated under reflux with ethyl 2-chloro-3-oxobutanoate in abs. EtOH to furnish ethyl 2-methylimidazo[1,2-a]pyridine-3-carboxylate 2 (Figure 3), which is subjected to hydrazinolysis to produce the key intermediate 2-methylimidazo[1,2-a]pyridine-3-carbohydrazide 3.^32,48–51 The terminal amino of the key intermediate 3 was subsequently condensed with various ketone moieties of isatin analogues 4a-h through refluxing in glacial acetic acid to yield the desired compounds 5a-h.

Scheme 1 Synthesis of compounds 5a-h. Reagents and conditions: (i) Ethyl 2-chloroacetoacetate, abs. EtOH, reflux, 6 h; (ii) Hydrazine hydrate, abs. EtOH, reflux, 6 h; (iii) abs. EtOH, drops of gl. AcOH as a catalyst, reflux, 10 h.

Scheme 2 Synthesis of compounds 8a-d and 11a-d. Reagents and conditions: (i) Anh. K2CO3, dry DMF, reflux, 6 h; (ii) EtOH, gl. AcOH, reflux, 10 h.

Figure 3 Plausible cyclization mechanism and formation of intermediate 2.

In Scheme 2, selected isatin derivatives 4a,d,e was dissolved in dry DMF in presence of anhydrous potassium carbonate as a base^16,52–55 and subjected to alkylation with methyl bromide 6a, ethyl bromide 6b, phenylmethyl bromide 9a and 2-phenylethyl bromide 9b to get N-alkyl isatin derivatives 7a-d and 10a-d, which condensed with the previously prepared key intermediate 3 in glacial AcOH affording the target compounds 8a-d and 11a-d, respectively.

The structures of all synthesized compounds were unequivocally confirmed through comprehensive spectral data, such as NMR (Supplementary Figures S1–S15) and HRMS (Figures S16–S31), and microanalysis, which aligned perfectly with the proposed structural frameworks.

By using ¹H NMR analysis, the structural integrity of the target compounds 5a-h, 8a-d, and 11a-d was clarified. The spectra showed that the two NH₂ protons of the hydrazide 3 completely vanished at 4.56 ppm,⁵⁶ and that the newly created aromatic systems were responsible for an increase in aromatic proton signals. A notable downfield shift of one NH proton in intermediate 3, from 9.18 ppm⁵⁶ to 13.24–13.44 ppm, significantly supported the successful synthesis of the Schiff base moiety. The suggested structures were supported by complementary ¹³C NMR spectroscopy data, which showed a noticeable upfield shift of the ketonic carbonyl carbon signal of isatin derivatives, shifting from about 180 ppm to ~140 ppm across all produced compounds.

Theoretically, the produced compounds might form both single and mixed (E and Z) stereoisomers. However, a single stereoisomer (either E or Z) was detected in the target derivatives’ ¹H NMR spectra. The discovery of a single distinctive NH signal of the hydrazide moiety, which appeared around 13.24–13.44 ppm, served as proof of this. According to earlier reports, the creation of an intramolecular hydrogen bond with the carbonyl group of the indolinone ring is responsible for the significant downfield shift of this NH proton, resulting in a pseudo–six-membered ring structure.^57,58 The Z-configuration, which is thought to be the thermodynamically preferred form under the given reaction circumstances, is strongly supported by this spectrum discovery. Factors like reaction temperature, starting material concentration, and the product’s intrinsic stability can all be used to explain why this isomer forms preferentially.⁵⁹

The target compounds, including both chlorinated and brominated derivatives, showed molecular ion peaks and their distinctive isotopic patterns in high-resolution mass spectrometric analysis. Additionally, the estimated values for the suggested chemical formulae and the microanalysis results showed excellent agreement, with differences limited to ±0.4%.

Biological Evaluations

In vitro Anti-Leishmanial Activities

Using miltefosine as the reference medication, the anti-leishmanial potential of sixteen newly synthesized imidazopyridine–isatin hybrids (5a-h, 8a-d, and 11a-d) against both promastigote and amastigote forms of Leishmania major was assessed. Table 1 summarizes the IC₅₀ values, shown in Figures 4 and 5, which are the micromolar concentrations required to prevent 50% of parasite growth. The IC₅₀ dose–response curve for the representative compound 5b is presented in Supplementary Figure S32. In contrast to miltefosine (IC₅₀ = 7.83 μM), all examined drugs (IC₅₀ = 0.84–4.84 μM) showed remarkable effectiveness against the promastigote stage of L. major. Also, all synthesized compounds (IC₅₀ = 1.28 to 7.18 μM) showed a higher anti-leishmanial activity than the reference (IC₅₀ = 8.07 μM) except for 8d (IC₅₀ = 8.18 μM) against amastigotes. Specifically, three compounds (5b, 5c, and 5h) showed IC₅₀ values of 1.28 μM or less, indicating that they were 6 to 9 times more effective against promastigotes than miltefosine. They also showed notable anti-amastigote activity, approximately 4 to 6 times that of miltefosine. Also, promastigote forms were more susceptible to most of the newly synthesized compounds compared to the amastigote forms of the L. major parasite. While both L. major forms showed similar susceptibility to 5a, 11c, and 11d (IC₅₀ = 2.33 to 3.84 μM).

Table 1 Comparative in vitro Anti-Leishmanial Activity (IC50, μM) of Miltefosine and the Synthesized Compounds Against Leishmania major Promastigote Forms

Figure 4 Effect of alkylated and non-halogenated isatins on both forms of L. major. The green arrow indicates increased activity, and the red arrow indicates decreased activity.

Figure 5 Effect of halogenated isatins on both forms of L. major. The green arrow indicates increased activity, and the red arrow indicates decreased activity.

Based on alterations at either C5 or C7 of the isatin ring, the SAR of series 5a-h was assessed. As a result of fluorine’s electronegativity and lipophilicity, the C5-fluoro derivative 5b demonstrated the strongest anti-leishmanial activity against promastigotes (IC₅₀ = 0.84 ± 0.06 μM) and amastigotes (IC₅₀ = 1.28 ± 0.18 μM), followed by the C7-fluoro 5c. On the other hand, activity was reduced by bigger halogens like chlorine (5d) or bromine (5e) at C5, with 5e being the least active in this series. These results show that the anti-leishmanial potency is strongly influenced by the size and electronegativity of the halogen at C5.

Activity was affected by substitutions with strongly electronegative groups at C5. In comparison to the un-substituted 5a, the OCF₃ derivative 5g exhibited lower activity against promastigotes (IC₅₀ = 4.62 ± 0.32 μM) and amastigotes (IC₅₀ = 4.89 ± 0.26 μM), whereas the NO₂ derivative 5h showed higher activity against promastigotes (IC₅₀ = 1.28 ± 0.12 μM) but lower activity against amastigotes (IC₅₀ = 2.82 ± 0.12 μM). These effects most likely result from the substituents’ steric and electronic impacts on target interactions.

Introducing an electron-donating methyl group at C5 (5f) resulted in the lowest activity in the series (IC₅₀ = 4.81 μM for promastigotes; 5.82 μM for amastigotes). Overall, the activity of series 5a-h depended on the electronic nature and position of the substituent on the isatin ring. Electron-withdrawing and small electronegative groups, such as F (5b) and NO₂ (5h), generally enhanced activity, whereas larger or electron-donating groups (Cl, Br, OCF₃, CH₃) decreased it. Activity at C5 was superior to C7, as demonstrated by 5b (5-F) versus 5c (7-F).

N-Alkylation and C5 substitution of the isatin ring affected activity in the second series (8a-d). The most active N-methyl unsubstituted derivative was 8a (IC₅₀ = 2.47 ± 0.24 μM for promastigotes; 4.02 ± 0.28 μM for amastigotes), while activity was generally reduced by increasing the N-alkyl size to ethyl (8b) or adding electronegative substituents at C5 (8c: Cl; 8d: Br). Interestingly, 8d exhibited the least amount of activity against both parasite types in this series. These findings suggest that anti-leishmanial efficacy is negatively impacted by bulky N-alkyl groups or by combining N-alkylation with C5-electronegative substitutions.

The third series (11a-d) showed moderate to good activity when lipophilic aralkyl groups were added at the isatin ring’s N1. In this series, the N-benzyl derivative with a C5-chlorine substitution (11c) exhibited the maximum potency (IC₅₀ = 2.97 ± 0.22 μM), whereas the unsubstituted 11a and the C5-bromo derivative 11d were less active. These results imply that anti-leishmanial action is increased when N-aralkylation is combined with a moderately electronegative C5 substituent.

However, the same compound was the least active of the third series (IC₅₀ = 3.84 ± 0.22 μM) against promastigote forms, assuming that the presence of the large-sized electronegative group with lipophilic aralkyl side chain decreased the availability and penetration against this form.

The inverse was noticed for the N-phenethyl derivative 11b (IC₅₀ = 6.22 ± 0.34 μM) that showed a visible decrease in activity compared to the N-benzyl derivative 11a (IC₅₀ = 4.82 ± 0.32 μM) against amastigote forms. In particular, the activity of this series seemed to be dependent on both the size of the halogen and its electronegativity at C5 of the isatin moiety and on the size of the aralkyl group on N₁. Hence, the order of the decrease in the activity for amastigote forms was: Cl with N-benzyl > Br with N-benzyl > H with N-benzyl > H with N-phenethyl, and the order of the decrease in the activity for the promastigote forms was: Cl with N-benzyl > H with N-phenethyl > H with N-benzyl > Br with N-benzyl.

Reversal of Anti-Leishmanial Activity of the Most Potent Compound 5b by Folic Acid and Folinic Acid

Using the methodology described by Mendoza-Martínez et al,⁶⁰ the anti-folate mechanism of the most active molecule, 5b, was examined. The test compound’s impact on L. major promastigotes’ survival was assessed in this assay both with and without folic acid/folinic acid at doses higher than their IC₅₀ values. Trimethoprim decreased parasite survival to 72% as a positive control; supplementing with folic acid improved this to over 99%. Without folic acid, parasite survival for compound 5b dropped to 22%. This technique depends on folic acid’s capacity to compete for DHFR and PTR1 active sites and its function in DNA synthesis without the need for previous activation. The anti-leishmanial effect of 5b was reversed by the addition of folinic acid, resulting in survival rates ranging from 82% to 87% (Table 2). According to these findings, compound 5b’s anti-leishmanial action is likely due to an anti-folate mechanism that involves dual inhibition of DHFR and PTR1.

Table 2 In vitro Evaluation of Folate Pathway Inhibition Expressed as Percentage Survival*

In vitro Cytotoxic Activity

Potential anti-leishmanial drugs must have strong anti-amastigote action with little cytotoxic effects on host cells to be deemed successful.⁶¹ To evaluate its safety profile, the most active molecule, 5b, was put through a cytotoxicity test against African green monkey kidney (VERO) cells, as previously reported.⁶² In summary, the 50% cytotoxic concentration (CC₅₀), which is the quantity needed to kill 50% of fibroblast cells, was found after VERO cells were exposed to different doses of the substance for 72 hours (Table 3). According to Table 3, the selectivity index (SI) was determined by dividing the compound’s IC₅₀ value against the amastigote form (μM) by its CC₅₀ value (μM). One important metric for determining a proposed drug’s therapeutic window is the SI. High selectivity and safety for mammalian cells were indicated by the results, which showed that compound 5b’s CC₅₀ against the parasite was significantly higher than its IC₅₀. Interestingly, 5b’s SI value was almost 35 times higher than miltefosine’s.

Table 3 Compound 5b Exhibits Cytotoxic Activity (CC50) Against Normal VERO Cells and Computed Selectivity Indices (SI) Against Promastigote Forms of Leishmania major

Computational Studies

Predicted ADMET Studies

Good biochemical activity and advantageous ADMET characteristics (which include absorption, distribution, metabolism, excretion, and toxicity) are essential for a potential therapeutic candidate. Many online resources and computational techniques have been created recently to forecast the ADMET profiles of possible anti-cancer medication candidates.^63–65 Notably, researchers have embraced ADMETlab 3.0, a completely rebuilt and currently commercially available web service, for the in silico assessment of drug-like characteristics.^66–68 Using ADMETlab 3.0, the ADMET properties of compound 5b and miltefosine were predicted for the current investigation. Miltefosine met all of the ADMETlab 3.0 requirements, with the exception of the number of rotatable bonds (nRot), as shown in Figure 6. Compound 5b, in contrast, satisfied every assessed criterion, indicating a better ADMET profile and emphasizing its room for improvement.

Figure 6 A) Pharmacokinetic mapping of compound 5b and miltefosine obtained from ADMETlab 3.0. Orangish lines indicate the compound parameters, while bluish regions define the upper limit, and greenish regions define the lower limit.

Molecular Docking

Leishmanial DHFR-TS

To indicate the mechanism of the anti-leishmanial activity of the most active compounds via the anti-folate pathway, our consideration was mainly focused on the protein structure of dihydrofolate reductase-thymidylate synthase (DHFR-TS). The most active compound 5b showed an excellent and superior docking score (−16.5 Kcal/Mol) compared to the reference inhibitor (−13.4 Kcal/Mol). This anticipates favorable binding towards DHFR-TS and hence confirms the anti-folate mechanism validated by the in vitro experiment (Section: Reversal of the anti-leishmanial activity via folic and folinic acid). Perceiving the intermolecular interactions, compound 5b showed a strong binding affinity compared to the reference, consistent with the obtained in vitro results, where the docking pose of 5b showed several hydrogen bonds with Asn108, Ser111, and Leu164. Moreover, the methyl imidazo[1,2-a]pyridine ring was oriented into the hydrophobic pocket, forming several hydrophobic interactions with Met55, Phe58, Ile112, Phe116, and Leu164. In addition, the 5-fluoro-isatin moieties interacted with residues Leu40, Val45, and Leu46 through hydrophobic interactions Figure 7.

Figure 7 The 2D interaction between 5b and DHFR-TS PDB ID: (1j3k).

Leishmanial PTR1

Additionally, we conducted a docking experiment of the most active molecule 5b against the Leishmania major PTR1 (Lm-PTR1) enzyme in comparison to the reference Lm-PTR1 to elucidate the potential molecular mechanism underlying the anti-malarial activity. The docking study result of 5b was well matched with the in vitro enzymatic inhibition assay result. The tested compound 5b was able to properly dock into the enzyme binding site, achieving a docking score of −13.9 Kcal/Mol, similar to the reference inhibitor, having a docking score of −14.1 Kcal/Mol. Clearly, achieving a comparable binding mode for the reference inhibitor and establishing comparable key interactions with the amino acid residues inside the binding sites determined the enzymatic activity of the tested compound 5b. For the imidazo[1,2-a]pyridine moiety, the core ring interacted with Ser111 and NADP301 through hydrophobic interactions. In addition, the 2-CH₃ group formed a hydrophobic interaction with the Met179 amino acid residue. For the carbohydrazine linker, the C=O and the imino N=C groups contributed to the hydrogen bonding interactions with Ser111, Tyr194, Lys198, and NADP301. Moreover, the 5-fluoro-isatin moiety was involved in several hydrophobic interactions with Phe113 and Asp181 residues (Figure 8).

Figure 8 The 2D interaction between 5b and Leishmania major PTR1 (Lm-PTR1) ID: (6rxc).

Conclusions

In this work, a novel series of imidazo[1,2-a]pyridine–isatin hybrids (5a-h, 8a-d, and 11a-d) was designed and synthesized using a molecular hybridization technique as possible anti-leishmanial agents. Benchmarked against the reference medication miltefosine, the developed framework incorporates an imidazo[1,2-a]pyridine core that is connected to C5/C7- and/or N1-substituted isatin derivatives at the C3 position via a hydrazide spacer. All compounds showed greater activity against Leishmania major promastigotes (IC₅₀ = 0.84–4.84 μM vs. 7.83 μM) than miltefosine, according to in vitro evaluation. Most compounds were also better against amastigotes (IC₅₀ = 1.28–7.18 μM vs. 8.07 μM), with the exception of 8d (IC₅₀ = 8.18 μM). Using trimethoprim as a positive control, the most potent molecule, 5b, showed sub-micromolar potency and an anti-folate mechanism of action; in the presence of folic or folinic acid, its anti-leishmanial activity was eliminated. Compound 5b demonstrated exceptional safety with a high selectivity index (SI = 438.03) against normal VERO cells. The parasite selectivity of 5b was highlighted by molecular docking studies that showed substantial binding to the active sites of T. cruzi DHFR-TS and L. major pteridine reductase (PTR1), with no interaction in human DHFR. Additionally, positive physicochemical and pharmacokinetic characteristics were revealed by in silico research. This study is limited by the absence of in vivo assessment of the antileishmanial activity. However, this gap outlines a clear direction for future work, in which a thorough evaluation of efficacy, pharmacokinetics, and potential acute and chronic toxicities is needed to fully validate the therapeutic potential of the compounds. Overall, the obtained results position compound 5b as a strong antileishmanial candidate with promising biocompatibility and selectivity, warranting further investigation given the current scarcity and toxicity of available leishmaniasis therapies.