Antileishmanial and Antitoxoplasmal Activities of 1,4-Dihydropyridines

Abstract

We have synthesized 24 1,4-dihydropyridine compounds (1,4-DHPs) with different substituents at the aromatic ring by microwave-assisted one-pot Hantzsch multicomponent reaction and evaluated their in vitro activities against Toxoplasma gondii and Leishmania major. We have found that compound 9 ((±)-ethyl 2,7,7-trimethyl-4-(2-nitrophenyl)-5-oxo-1,4,5,6,7,8-hexahydroquinoline-3-carboxylate) is active against L. major amastigotes (IC₅₀ = 10.6 μM), but it is poorly selective for L. major over Vero cells (SI = 1.13) and macrophages (SI = 0.42). Among the evaluated 1,4-DHPs, compound 4 ((±)-ethyl 4-(4-(benzyloxy)­phenyl)-2,7,7-trimethyl-5-oxo-1,4,5,6,7,8-hexahydroquinoline-3-carboxylate) is the most active against T. gondii, providing the lowest IC₅₀ (3.1 μM) and the highest selectivity for this parasite (SI = 5.57) over Vero cells. Docking studies revealed that compound 4 has a high affinity for the T. gondii target (PDB ID: 4JBV). Furthermore, ADME-T predictions indicated that compound 4 meets the drug-likeness criteria without violating any Lipinski, Veger, or Egan’s rules.

1. Introduction

Leishmaniasis, a neglected tropical disease (NTD) that results in about 30,000 deaths annually, , is caused by protozoa of the genus Leishmania and is clinically subdivided into cutaneous leishmaniasis (CL), mucocutaneous leishmaniasis (MCL), and visceral leishmaniasis (VL). Leishmania major is one of the species that trigger CL, which affects up to 1 million individuals every year and can result in severe and disfiguring skin lesions, thereby posing the risk of stigmatization, especially among young patients. − Current treatment options for CL patients include antimonials, miltefosine, amphotericin B, and pentamidine. However, these drugs are toxic, not to mention that drug-resistant parasite forms have emerged, which calls for new potent antileishmanial drugs.

Toxoplasmosis, a zoonotic infectious disease caused by the apicomplexan protozoan Toxoplasma gondii, infects about one-third of the world’s human population. Immunocompromised individuals are particularly vulnerable to serious complications following infection by T. gondii. Currently, the available antiparasitic drugs for toxoplasmosis include sulfonamides (e.g., sulfadiazine), DHFR inhibitors (pyrimethamine, trimethoprim), and the naphthoquinone atovaquone. However, adverse effects such as nephrotoxicity, alongside the emergence of drug-resistant clinical isolates, highlight the urgent need for novel and effective therapeutic agents for the treatment and management of toxoplasmosis.

1,4-DHPs are known for their ability to block calcium channels. Nevertheless, over the past decade, several studies on their antiparasitic activity have been published. For example, the antileishmanial activity of 1,4-DHPs against Leishmania amazonensis, the main Leishmania species responsible for CL in the Americas, has been extensively reported. , Some studies on 1,4-DHPs have also demonstrated the antiparasitic effects against T. gondii.

As part of our interest in exploring the antiparasitic potential of natural and synthetic products, , and based on previous reports on the antiparasitic activities of 1,4-dihydropyridines (1,4-DHPs), ,, we have assessed the antiparasitic activities of 24 synthetic 1,4-DHPs against L. major and T. gondii.

2. Materials and Methods

2.1. Synthesis of Compounds 1–24

The 1,4-DHPs were synthesized according to the multicomponent one-pot methodology described in the literature. , In the general procedure, 2.0 mmol of dimedone (Aldrich), 2.0 mmol of ethyl acetoacetate (Aldrich), and 0.06 g (5.0 mol %) of ytterbium triflate (Aldrich), as reaction catalyst, were diluted in ethanol (5.0 mL). Subsequently, 2.0 mmol of benzaldehyde (Aldrich) and 2.0 mmol of ammonium acetate (Scientific Exodus) were added. All the reagents were added at room temperature. The reaction mixture was taken to the microwave reactor CEM FocusedMicrowave Synthesis System, model Discover (CEM Corp, Matthews, NC) set in the Power Time, where it was maintained for 30 min at a fixed power of 100 W. Compounds 1–24 were identified based on their NMR (¹H, ¹³C, and DEPT 135) and mass spectra (see Supporting Information). NMR experiments were performed on a Bruker Avance DRX400 spectrometer (Karlsruhe, Germany, 400.13 MHz for ¹H and 100.61 MHz for ¹³C). A direct 5 mm probe head (BBO) was used for ¹³C­{¹H} NMR experiments and an inverse 5 mm probe head (BBI) was used for other experiments. All compounds were dissolved in CDCl₃ using tetramethylsilane (TMS) as an internal reference to achieve concentrations in the range of 10–15 mg mL^–1. All the experiments were performed at 300 K.

Mass spectra were recorded on a triple quadrupole MS equipment (QqQ) Xevo TQS (Waters, Milford, MA, USA) equipped with Z-spray operating in the positive ion mode and Acquiti-H class UPLC system. The sample was dissolved in methanol/water (9:1, v/v) at a concentration of 0.5 mg mL^–1 and infused directly into the ESI source by using a Harvard Apparatus system (model 1746, Houston, MA, USA) at a flow rate of 5 μL min^–1. The capillary voltage was 3.20 kV, and the gas flow was 700 L/h (0.15 V). The desolvation temperature was set at 250 °C.

Compounds 1–16 were isolated as mixtures of enantiomers. All the tested compounds displayed purity higher than 95%, as confirmed by HPLC analyses. To this end, compounds 1–24 were dissolved in an acetonitrile/water 4:1 (v/v) solution to achieve a concentration of 0.5 mg mL^–1. These compounds were analyzed on a Shimadzu LC 6AD chromatograph fitted with a UV–vis SPD-20A operating at 270 nm and a DGU-20A5 degasser. A Phenomenex Luna C₁₈ 100 Å (5 μm × 250 mm × 4.6 mm) column was used. The injection volume was 500 μL. A mixture of acetonitrile/water 4:1 (v/v) was used as the mobile phase at a flow rate of 1 mL min^–1. The total time of each analysis was 1 h. All the HPLC chromatograms are given in the Supporting Information.

2.2. Anti-Toxoplasma Activity

Tachyzoites of the T. gondii RH strain were cultivated in Vero cells according to a published method. Complete RPMI 1640 medium (Invitrogen, USA) with 10% fetal bovine serum (FBS, Invitrogen, USA) was used to culture Vero cells in 96-well plates (5 × 10³ cells/well in 200 μL of medium), which were incubated at 37 °C and under 5% CO₂ for 24 h. This was followed by washing with phosphate-buffered saline (PBS) to remove the medium. Then, RPMI 1640 medium with 2% FBS containing T. gondii tachyzoites (RH strain) at a ratio of 5 (parasite): 1 (Vero cells) was added. After incubation at 37 °C and under 5% CO₂ for 5 h, the cells were washed with PBS and overlaid with medium containing one of the tested compounds or atovaquone (ATO) at 50, 25, 12.5, 6.25, 3.13, 1.65, 0.75, or 0.37 μg mL^–1.

After incubation at 37 °C and under 5% CO₂ for 72 h, the cells were stained with 1% toluidine before being examined under an inverted photomicroscope (MCD-400, Leica, Japan) to determine the T. gondii infection index (number of infected cells from 200 tested cells). The experiment was performed in triplicate.

2.3. Leishmania major Promastigotes and Amastigotes

L. major promastigotes were isolated and maintained according to a described method. BALB/c mice were injected at the hind footpads with 1 × 10⁶ L. major metacyclic promastigotes. L. major amastigotes were collected from the infected mice. After 8 weeks had elapsed since inoculation, Schneider’s medium (Invitrogen, USA) containing antibiotics and 10% FBS was used to transform isolated amastigotes into promastigotes by incubation at 26 °C. Amastigote-derived promastigotes with less than five in vitro passages were only used for infection.

To assess the activity of the compounds against L. major promastigotes, 10⁶ promastigotes/mL were cultured in 96-well plates in Schneider’s medium with 10% FBS. Then, the compounds or amphotericin B (AmB) were added to obtain the final concentrations (50, 25, 12.5, 6.25, 3.13, 1.65, 0.75, or 0.37 μg mL^–1). Negative control wells contained cultures with DMSO (1%) only. Plates were incubated at 26 °C for 72 h to evaluate the antiproliferative effect. The colorimetric MTT method was used to count viable promastigotes under a microplate absorbance spectrophotometer (xMark, Bio-Rad, USA) at 570 nm. The experiment was performed in triplicate.

Female BALB/c mouse peritoneal macrophages (6–8 weeks old) were used to culture L. major amastigotes to assess the activity of the compounds against amastigotes. Briefly, 1 mL of 3% Brewer’s thioglycollate medium/mouse was injected into the peritoneal cavity. After 4 days, the abdominal skin was removed to expose the peritoneal wall. Then, 3 mL of RPMI 1640 medium was injected before cells were collected by aspiration, whereupon 96-well plates containing RPMI 1640 medium with 10% FBS were used to culture 5 × 10⁴ cells/well, which were incubated at 37 °C under 5% CO₂ for 4 h. After washing with PBS, RPMI 1640 medium containing 5 × 10⁵ promastigotes were added to each well. Next, the cells were incubated at 37 °C under humidified 5% CO₂ for 24 h to enhance infection and differentiation of amastigotes. Washing with PBS several times removed free promastigotes. Fresh complete RPMI 1640 medium containing one of the tested compounds or AmB at the desired final concentration (50, 25, 12.5, 6.25, 3.13, 1.65, 0.75, or 0.37 μg mL^–1) was added and incubated at 37 °C under a humidified 5% CO₂ atmosphere for 72 h. DMSO (1%), only in complete RPMI media, was used as the negative control. The percentage of infected macrophages was assessed microscopically after the medium was removed, followed by washing with PBS, fixation with methanol, and staining with Giemsa. The reading was performed in triplicate. Handling of the laboratory animals followed the instructions and rules of the Committee of Research Ethics, Deanship of Scientific Research, Qassim University, permission number 20–03–20.

2.4. Cytotoxicity Assay

The MTT colorimetric assay was conducted to assess the cytotoxicity of the compounds according to a method described previously. Briefly, Vero cells or macrophages were cultured in 96-well plates (5 × 10³ cells/well/200 μL) in RPMI 1640 medium with 10% FBS at 37 °C and under 5% CO₂ for 24 h. Thereafter, PBS was used to wash the cells. Then, the compounds in RPMI complete medium with 10% FBS (at varying concentrations of 50, 25, 12.5, 6.25, 3.13, 1.65, 0.75, or 0.37 μg mL^–1) were added to 96-well plates and incubated for 72 h. Cells treated with a medium containing only DMSO (1%) were used as the negative control. After washing, MTT (1 mg mL^–1 in RPMI 1640 medium) was added to each well. The cells were incubated for 4 h, and the supernatant was removed. Next, 150 μL of DMSO was added, and a microplate absorbance spectrophotometer was applied for colorimetric analysis (λ = 540 nm). Cytotoxic effects were expressed by IC₅₀ values (concentration that caused a 50% reduction of viable cells). The experiment was performed in triplicate.

2.5. Molecular Docking

2.5.1. Ligands and Target Preparation

The geometry of compound 4 was optimized by using the Density Functional Theory (DFT) method. − The effect of the solvent (water) was considered by using CPCM. The exchange Becke three-parameter Lee–Yang–Parr correlation (B3LYP) level of theory in conjunction with dispersion correction D3, already implemented in Gaussian 16 and the base 6–311G (d, p), were employed.

2.5.2. Target Preparations

The crystal structure of calcium-dependent protein kinase-1 (PDB ID: 4JBV, resolution 1.95 Å) was obtained from the Protein Data Bank (PDB) (https://www.rcsb.org/). This T. gondii target (TgCDPK1) was selected based on the recent literature. ,

2.5.3. Molecular Docking Protocol and Validation

Docking calculations were performed on the selected proteins by using the “MOE Docking Option” implemented in the MOE software (Molecular Operating Environment (MOE) 2014.09). Therefore, the software was used to calculate the s-score energy and to predict interactions between proteins and compounds by keeping the macromolecule rigid and the compound flexible. The crystal structures of the studied proteins were simplified by removing water molecules, ions, cofactors, and native ligands from the PDB structures. The molecular docking simulation protocol used in this study has been detailed. , The molecular docking method is validated by redocking the crystallized ligands with their targets and calculating the RMSD (the RMSD of the resulting complex lies between 1 and 2 Å), which represents the accuracy and satisfaction of the docking method. The 2D Interaction diagrams between compound 4 and T. gondii target (4JBV). Furthermore, ADME-T predictions indicated that this compound meets the drug-likeness criteria without violating any Lipinski, V) were visualized by using the BIOVIA DS visualization package (Dassault Systèmes BIOVIA, Discovery Studio modeling environment, 2020).

2.5.4. Drug-Likeness Prediction and ADME-T

The number of hydrogen bond acceptors (nHA), the number of hydrogen bond donors (nHD), TPSA, nROT, MW, and LogP were obtained from the SwissADME server (http://www.swissadme.ch/ accessed on 15 April 2024). Besides that, Absorption, Distribution, Metabolism, Excretion, and Toxicity (ADME-T) were predicted by using the pkCSM server (http://biosig.unimelb.edu.au/pkcsm/prediction accessed on 15 April 2024).

3. Results

3.1. Synthesis of Compounds 1–24

We synthesized compounds 1–24 by a microwave-assisted one-pot Hantzsch multicomponent reaction between an aromatic aldehyde, ethyl acetoacetate, and a β-dicarbonyl compound (i.e., dimedone for the asymmetric compounds 1–16, and ethyl acetoacetate excess for the symmetric compounds 17–24) as previously reported (Scheme ). We employed ytterbium triflate (Yb­(OTf)₃) as a catalyst and ammonium acetate as a nitrogen donor. We used ethanol as the solvent, and the reaction time was 30 min. We isolated the product from the reaction mixture by vacuum filtration and purified it by recrystallization, to achieve yields of 15–40%.

1. Synthesis of Compounds 1–24 Through a Microwave-Assisted One-Pot Hantzsch Multicomponent Reaction.

3.2. Antileishmanial Activity of Compounds 1–24 against L. major

Table depicts the antileishmanial activity of compounds 1–24 against L. major promastigotes and amastigotes.

1. Effective Concentrations (EC₅₀, in μM) of Compounds 1–24 against Leishmania major Promastigotes (prom) and Amastigotes (ama) and Toxoplasma gondii; IC₅₀ (in μM) of Compounds 1–24 against Macrophages (Macro) and Vero (African Green Monkey Kidney Epithelial) Cells .

1,4-DHP	EC 50			IC 50			SI
	L. major		T. gondii	Vero	macro	L. major			Vero/T. gondii
	prom	ama				Vero/prom	Vero/prom	macro/ama
1	>61.3	>61.3	>61.3	>61.3	>61.3	n.d	n.d	n.d	n.d
2	>58.8	>58.8	>58.8	52.9 ± 9.8	47.8 ± 7.6	<0.90	<0.90	<0.81	<0.89
3	>56.3	>56.3	>56.3	49.8 ± 8.9	38.7 ± 6.8	<0.88	<0.88	<0.69	<1.13
4	>46.7	23.3 ± 3.4	3.1 ± 0.4	17.5 ± 3.0	25.1 ± 3.7	<0.37	<0.75	1.08	5.57
5	>54.4	>54.4	29.5 ± 4.1	49.4 ± 8.8	49.9 ± 7.4	<0.91	<0.91	0.92	1.67
6	>49.7	>49.7	23.4 ± 3.5	17.4 ± 2.9	23.4 ± 3.5	<0.35	<0.35	0.47	0.74
7	>58.2	>58.2	>58.2	>58.2	>58.2	n.d	n.d	n.d	n.d
8	>55.6	>55.6	>55.6	>55.6	>55.6	n.d	n.d	n.d	n.d
9	>58.2	10.6 ± 2.1	12.0 ± 1.9	4.5 ± 0.7	12.0 ± 1.9	<0.08	<0.42	1.13	0.37
10	>54.1	26.3 ± 3.7	16.4 ± 2.5	10.7 ± 2.1	24.5 ± 3.6	<0.20	<0.41	0.93	0.65
11	>54.0	>54.0	26.5 ± 3.6	45.1 ± 7.9	48.0 ± 8.1	<0.84	<0.89	0.89	1.70
12	47.5 ± 6.2	>60.6	30.3 ± 5.2	14.9 ± 2.6	42.5 ± 7.2	0.31	<0.25	0.70	0.49
13	>48.4	>48.4	>48.4	>48.4	>48.4	n.d	n.d	n.d	n.d
14	18.9 ± 2.9	21.0 ± 3.9	>48.4	22.8 ± 3.3	16.8 ± 3.1	0.89	1.09	0.8	<0.47
15	40.0 ± 6.4	>51.3	>51.3	>51.3	>51.3	1.28	n.d	n.d	n.d
16	>49.3	31.8 ± 5.4	8.1 ± 1.3	12.6 ± 2.1	29.9 ± 5.1	0.61	<0.40	0.94	1.55
17	>63.1	>63.1	49.8 ± 6.5	60.1 ± 10.6	>63.1	n.d	<0.95	n.d	1.21
18	>57.9	>57.9	>57.9	45.4 ± 7.8	>57.9	n.d	<0.78	n.d	<0.78
19	>47.8	>47.8	>47.8	33.5 ± 6.2	43.4 ± 7.5	0.70	<0.70	0.91	<0.70
20	>50.9	>50.9	>50.9	35.0 ± 5.9	44.8 ± 8.2	0.69	<0.69	0.88	<1.49
21	>59.9	>59.9	>59.9	>59.9	>59.9	n.d	n.d	n.d	n.d
22	46.5 ± 7.7	>55.6	17.4 ± 2.7	29.9 ± 4.4	46.5 ± 7.4	1.0	<0.54	0.84	1.72
23	>55.4	>55.4	29.8 ± 4.8	22.1 ± 3.2	40.5 ± 6.8	0.73	<0.40	0.73	0.74
24	42.7 ± 8.0	>49.6	>49.6	14.8 ± 2.6	34.1 ± 5.5	0.35	<0.30	0.69	<0.29
AmB	0.83 ± 0.2	0.47 ± 0.1		10.3 ± 1.2	8.1 ± 1.1	12.4	21.9	17.2
ATO			0.07 ± 0.01	9.5 ± 1.5					135

^aAmphotericin B (AmB) and atovaquone (ATO) were applied as positive controls. n.d.: not determined. Values are the means of at least three independent experiments ± SD and were obtained from concentration–response curves by calculating the percentage of treated cells compared to untreated controls after 72 h. ^bThe selectivity index (SI) (IC₅₀/EC₅₀) was calculated from the corresponding IC₅₀ against Vero cells and EC₅₀ against L. major promastigotes and T. gondii, or IC₅₀ against macrophages. Linear regression equation was used for the IC₅₀ and EC₅₀ calculations using Microsoft Excel program version 16.

Only five compounds (12, 14, 15, 22, and 24) were active (IC₅₀ < 50 μM) against L. major promastigotes. We obtained the lowest half-effective concentration (EC₅₀) for compound 14 (EC₅₀ = 18.9 μM). Similarly, only compounds 9 (EC₅₀ = 10.6 μM), 4 (EC₅₀ = 23.3 μM), 10 (EC₅₀ = 26.3 μM), 14 (EC₅₀ = 21.0 μM), and 16 (EC₅₀ = 31.8 μM) had EC₅₀ lower than 50 μM against L. major amastigotes. However, except for compound 4, the IC₅₀ of all the compounds against Vero cells resembled their EC₅₀ against L. major promastigotes and amastigotes, which resulted in a low selectivity index (i.e., the ratio between EC₅₀ and IC_50, which measures the window between cytotoxicity and biological activity) (SI < 1). Besides, compounds 4, 6, 9, 10, and 14 were the most toxic to macrophages, having the lowest IC₅₀ (25.1, 23.4, 12.0, 24.5, and 16.8 μM, respectively). Nevertheless, compound 9 was the most selective for L. major amastigotes (which grow within macrophages) over macrophages (SI = 1.13), but this selectivity was low as compared to the selectivity of amphotericin B (AmB).

3.3. Anti-T. gondii Activity of Compounds 1–24

Table lists the results of the anti-T. gondii activity of compounds 1–24. Only 11 compounds had EC₅₀ lower than 50 μM: 6 (23.4 μM), 9 (12.0 μM), 4 (3.1 μM), 5 (29.5 μM), 11 (26.5 μM), 10 (16.4 μM), 12 (30.3 μM), 16 (8.1 μM), 17 (49.8 μM), 23 (29.8 μM), and 22 (17.4 μM). On the other hand, Vero cells were slightly more sensitive to compounds 6, 9, 2, 3, 10, 14, 12, 20, 19, and 23 than T. gondii (Table ) as evidenced by their IC₅₀. Compound 4 gave the highest SI (SI = 5.57). Meanwhile, most compounds were not selective for T. gondii over Vero cells. Atovaquone (ATO), the positive control, had EC₅₀ of 0.07 μM and SI of 135.

3.4. Computational Approach

3.4.1. Ligands and Target Preparations

Figure illustrates the optimized geometry of compound 4.

1.

Optimized equilibrium structure of compound 4.

3.4.2. Score Energy and Best Poses Analysis

Table regroups the docking simulation results obtained for compound 4 with the pocket of the T. gondii protein. The energy score for the complex of compound 4 with the T. gondii target (PDB ID: 4JBV) was −7.174 kcal/mol (Table ).

2. Docking Results for the Binding of Compound 4 and Co-crystallized Ligand (ATO) to the Toxoplasma gondii (PDB ID: 4JBV) Target .

compound	S-Score (kcal/mol)	RMSD (Å)	bonds between atoms of compounds and active site residues
			atom of compound	involved receptor atoms	involved receptor	category	type
4	–7.174		H	O	LEU57	H-bond	conventional H-bond
				SD	MET112	other	Pi-sulfur
					LEU57	hydrophobic	Pi-alkyl
					VA65	hydrophobic	Pi-alkyl
		2.193			ALA78	hydrophobic	Pi-alkyl
					LEU181	hydrophobic	Pi-alkyl
					ILE194	hydrophobic	Pi-alkyl
					LYS80	hydrophobic	Pi-alkyl
					ILE194	hydrophobic	Pi-alkyl
cocrystallized ligand (ATO)	–10.891		O	HAD1	GLU129	H-bond	conventional H-bond
			NBF	OE2	GLU135	electrostatic	attractive charge
				SD	MET112	other	Pi-sulfur
				HG13	VA65	hydrophobic	Pi-sigma
			CAB		LEU114	hydrophobic	alkyl
			CAB		LEU126	hydrophobic	alkyl
			CAB		LEU198	hydrophobic	alkyl
			CAA		LEU103	hydrophobic	alkyl
			CAA		LEU198	hydrophobic	alkyl
		0.775			ALA78	hydrophobic	Pi-alkyl
					LEU181	hydrophobic	Pi-alkyl
					ILE194	hydrophobic	Pi-alkyl
					LEU157	hydrophobic	Pi-alkyl
					ALA78	hydrophobic	Pi-alkyl
					LEU181	hydrophobic	Pi-alkyl
					VA65	hydrophobic	Pi-alkyl
					LYS80	hydrophobic	Pi-alkyl
					MET112	hydrophobic	Pi-alkyl

^aATO: atovaquone.

Compound 4 established a strong conventional hydrogen bond with LEU57 residues (2.09 Å) and a Pi–Sulfur interaction with MET112 residues. Moreover, compound 4 established seven hydrophobic interactions (Pi-Alkyl) with LEU57, VA65, ALA78, LEU181, ILE194, and LYS80 residues (Table and Figure ).

2.

3D and 2D diagrams of interactions of the compound 4 (a) and its native ligand (b) with the active site residues of Toxoplasma gondii (PDB ID: 4JBV) target.

3.4.3. Physicochemical Parameters and ADME-T Prediction

We evaluated the drug-likeness of compound 4 by using ADME-T predictions and calculating physicochemical properties. Table summarizes the results.

3. ADME-T and Drug-likeness Properties of Compound 4 .

entry	TPSA (Å2)	n-ROTB	MW	MLog P	WLog P	n–ON acceptors	n–OHNH donors	Lipinski’s violations	Veber's violations	Egan's violations
	<140	<11	<500	≤5		<10	<5	≤1	≤1	≤1
4	64.63	7	445.55	3.36	4.90	4	1	accepted	accepted	accepted

	absorption		distribution		metabolism			excretion		toxicity
ADME-T	Caco2 (10–6 cm/s)	HIA %	CNS (Log PS)	BBB (Log BB)	CYP1A2 inhibitor	CYP2C19 inhibitor	CYP2D6 substrate	renal OCT2 substrate	total clearance (mL/min/kg)	AMES toxicity	hepatotoxicity
4	1.04	96.17	–1.77	0.002	YES	YES	NO	NO	0.484	NO	YES

^aTPSA: topological polar surface area, n-ROTB: number of rotatable bonds, MW: molecular weight, MLogP: logarithm of partition coefficient of compound between n-octanol and water, n-ON acceptors: number of hydrogen bond acceptors, n–OHNH donors: number of hydrogen bonds donors. Caco-2: colon adenocarcinoma, HIA: human intestinal absorption, CNS: central nervous system permeability, BBB: blood–brain barrier permeability. Renal OCT2 substrate: organic cation transporter 2.

Compound 4 had TPSA lower than 140 Å. Additionally, compound 6 had fewer than 5 n–OHNH donors and more than 10 n-ON acceptors. On the other hand, the molecular weight of this compound was lower than 500 g/mol, and its MLogP and WLogP were lower than 5. Besides, nROTB was less than 11.

Data presented in Table indicates that (1) Compound 4 and ATO had Caco-2 greater than −5.15 cm/s; (2) Compound 6 had HIA (96.17%) greater than 30%; (3) The logPS of compound 4 lay in the −3 < logPS < −2 range; (4) the logBB of compound 4 and ATO was 0.119 and 0.002, respectively (Table ); (5) Compound 4 inhibits CYP1A2 and CYP2C19 but not CYP2D6; (6) Neither compound 4 nor ATO are likely OCT2 substrates. Moreover, compound 4 has average excretion clearance (<5 mL/min/kg). (7) Compound 4 does not exhibit AMES toxicity, but it can be hepatotoxic.

4. Discussion

4.1. Synthesis of Compounds 1–24

We synthesized compounds 1– 24 by a “green” microwave-assisted one-pot multicomponent methodology. “Green chemistry” is on the rise as an environmentally friendly and sustainable way to prepare drug candidates to treat cancer and infectious diseases. The methodology used herein is attractive from the synthetic point of view: (1) compounds 1– 24 are obtained in only one synthetic step (i.e., a one-pot Hantzsch multicomponent reaction); (2) the methodology employs nontoxic solvents, (3) the reaction times are short (about 30 min), (4) the products are solid and easy to isolate (i.e., dismissing the need for time-demanding chromatographic steps), and yields range from 15% to 40% due to formation of byproducts (e.g., pyridines, 1,2-dihydropyridines, and acridine-1,8-diones, which were detected in the ethanol-soluble phase of recrystallization; data not shown).

4.2. Antileishmanial Activity of Compounds 1–24 against L. major

The antileishmanial activity of 1,4-DHPs against L. amazonensis has been extensively exploited. , While L. amazonensis is the main Leishmania species responsible for CL in the New World (i.e., South America), L. major is known to cause CL in the Old World (i.e., Central Asia, the Middle East, and Africa). Based on the different susceptibility of L. amazonensis to primary S-nitrosothiols and azithromycin, miltefosine, edelfosine, and amphotericin B compared to other Leishmania species, we investigated the in vitro antileishmanial activity of compounds 1–24 against L. major promastigotes and amastigotes.

In general, compounds with IC₅₀ lower than 10 μM, between 10 and 50 μM, between 50 and 100 μM, and higher than 100 μM are considered very active, active, moderately active, and inactive, respectively. Based on these criteria, we found that compounds 12, 14, 15, 22, and 24 are active against extracellular L. major promastigotes. Oliveira and co-workers reported that compounds 12, 14, 22, and 24 are inactive against L. amazonensis promastigotes. Conversely, among the most active compounds against L. amazonensis promastigotes (compounds 13, IC₅₀ = 24.62 μM) and amastigotes (compounds 7 and 9, IC₅₀ = 12.53 and 13.67 μM, respectively), only compound 9 (IC₅₀ = 13.67 μM) is active against L. major amastigotes at concentrations lower than 50 μM. Escobar and co-workers described different sensitivities of Leishmania promastigotes and amastigotes to miltefosine, edelfosine, and amphotericin B, which are among the drugs that are currently used to treat leishmaniasis. , Intrinsic variations in the sensitivity of these two Leishmania species to drugs have important implications for clinical treatments. On the other hand, among the compounds tested here, we found that only compounds 4, 9, 10, 14, and 16 are active against intracellular L. major amastigotes. Interestingly, we verified that none of the symmetric compounds (compounds 17– 24) are active against L. major amastigotes. When analyzed together, these data suggest that the presence of fluorine (in compound 9), a nitro (in compounds 10 and 22), or an aromatic ether (in compounds 4, 12, 14, 15, and 24) is essential for the antileishmanial activity against L. major promastigotes and amastigotes. Indeed, fluorophenyl has been identified as a key feature in the structure of other compounds that display potent antileishmanial activity against L. major, ,, such as 4-amino-2-(3-fluorophenyl)-1,2-dihydropyrimido­[1,2-a]­benzimidazole-3-carbonitrile. Similarly, nitroaromatic compounds 10 and 22 have also been reported to be effective against L. major. The antileishmanial activity of these compounds may be related to their ability to act as redox-active agents and to increase ROS (reactive oxygen species) generation in Leishmania parasites, to dissipate the mitochondrial potential. In the case of the methoxylated compounds 14 and 24, Genestra and co-workers demonstrated the role played by methoxy groups in the antileishmanial activity. However, only the presence of the methoxy group does not ensure antileishmanial activity, as evidenced by the inactivity of compounds 3, 11, 18, and 23. Indeed, combined with other structural features (e.g., the symmetry of the 1,4-DHP structure core), the number and location of these substituents at the aromatic ring may affect the antileishmanial activity significantly. Mohammadi-Ghalehbin reported a similar finding for n-aryl enamino amide derivatives. Compounds bearing benzyloxy and methylenodioxy substituents in their structures other than compounds 4 and 12 have been described as potent antileishmanial agents. ,

The selectivity index (SI) is an essential parameter when developing new chemotherapeutic agents: SI allows the toxicity of the tested compounds to normal cells to be evaluated and compared to the target cells and helps to predict their therapeutic potential. In the case of the antileishmanial activity, the IC₅₀ of compounds 1–24 against Vero cells is generally lower than the IC₅₀ of these compounds against L. major promastigotes and amastigotes and macrophages. This gives SI lower than 1, which shows poor selectivity for L. major promastigotes and amastigotes over Vero cells. Therefore, despite the promising activity of compounds 14 and 9 against L. major promastigotes and amastigotes, further docking studies were not carried out because of their poor selectivity over Vero cells.

4.3. Antitoxoplasmal Activity of Compounds 1–24 against T. gondii

Most of the 1,4-DHPs that proved active against T. gondii are asymmetric (i.e., derived from dimedone), also known as “polyhydroquinolines”. On the other hand, some symmetric 1,4-DHPs (commonly known as “Hantzsch esters”) were also active (IC₅₀ < 50 μM), such as compounds 17, 22, and 23. Therefore, the activity of 1,4-DHPs cannot be understood in terms of the type of 1,4-DHP only. On the other hand, the presence of particular substituents in specific positions of the aromatic ring was found to be important for the antitoxoplasmal activity of 1,4-DHPs, such as the groups 3-NO₂ (for compounds 10 and 22) and 3-OMe/4-OH (for compounds 11 and 23). Although these groups have also been found in the structure of other compounds active against T. gondii, their role in the antitoxoplasmal activity was not investigated more deeply in this study.

4.4. Computational Approach

Molecular docking is a computational strategy that predicts the precise alignment of a ligand molecule with a receptor protein, resulting in a stable complex formation. This alignment is essential for determining the binding affinity and strength of the ligand–protein interaction, accomplished through a scoring function. The interaction between a drug and its receptor lowers the overall free energy of the system, offering critical insights into the molecule’s affinity and activity, which are vital for advancing drug design and discovery. In this study, we have investigated the complex formed after docking of compound 4 with the T. gondii target (4JBV). Furthermore, ADME-T predictions indicated that this compound meets the drug-likeness criteria without violating any Lipinski, V). This target belongs to a family of Calcium-Dependent Protein Kinases (CDPKs) that regulate the complex life cycle of T. gondii, and it has been widely used in research for new therapeutics against T. gondii. The compound 4: 4JBV). Furthermore, ADME-T predictions indicated that this compound meets the drug-likeness criteria without violating any Lipinski, V complex had an energy score of −7.174 kcal/mol, which was slightly closer to the energy score of the native ligand (ATO) (energy score = −10.891 kcal/mol). A Pi-sulfur interaction with MET112 residues interaction and seven hydrophobic interactions Pi-Alkyl with LEU57, VA65, ALA78, LEU181, ILE194, and LYS80 residues was observed (Table and Figure ). Previous studies indicated that MET112, VA65, ILE194, and LYS80 residues are the key to the inhibition of T. gondii target (4JBV). Furthermore, ADME-T predictions indicated that this compound meets the drug-likeness criteria without violating any Lipinski, V). , Likewise, molecular docking results showed that the binding types of compound 4 with the T. gondii target (PDB ID: 4JBV) were similar to the native ligand (ATO), which is justified by the presence of several common residues.

The drug-like behavior of compound 4 was also estimated based on how physicochemical properties fit Lipinski’s “rule-of-five” (Table ). According to this rule, an orally active drug-like compound should not have more than one violation of the following criteria: hydrogen bond donors not greater than 5, hydrogen bond acceptors not greater than 10, molecular weight not greater than 500 Da, and an octanol–water partition coefficient (log P) not greater than 5. , As shown in Table , compound 4 complies with the drug-likeness criteria without violating Lipinski, Veber, or Egan’s rules. ,

The ADME-T (absorption, distribution, metabolism, excretion, and toxicity) properties of compound 4 were also predicted. Based on the data presented in Table , it is shown that (1) compound 4 has good human intestinal permeability (Caco-2 greater than −5.15 cm/s); (3) compound 4 can be easily absorbed by the gastrointestinal system after it is orally administered, as it has HIA (96.17%) greater than 30%; (4) compound 4 can penetrate the CNS (−3 < logPS < −2); (5) Compound 4 (logBB = 0.119) and ATO (logBB = 0.002) are poorly distributed in the brain (logBB < −10); (6) Compound 4 inhibits CYP1A2 and CYP2C19 but not CYP2D6; (7) Neither compound 4 nor ATO are likely OCT2 substrates (8) Compound 4 has average excretion clearance (<5 mL/min/kg). Based on the data presented in Table , it is also shown that compound 4 does not exhibit AMES toxicity, but it can be hepatotoxic.

5. Conclusions

We have found that seven 1,4-dihydropyridines display good antiparasitic activity against L. major promastigotes and amastigotes, albeit without selectivity for these parasites over Vero and macrophage cells. Compound 4 is active against T. gondii and about five times more selective for the parasite cells than for Vero cells. Docking studies revealed that compound 4 has a strong affinity for the T. gondii target (PDB ID: 4JBV). Furthermore, ADME-T predictions indicated that this compound meets the drug-likeness criteria without violating any Lipinski, Veber, or Egan rules.

Our results reinforce the antiparasitic potential of 1,4-DHPs. Structural modification-based strategies should be adopted in future works to increase the selectivity for T. gondii and L. major.

Glossary

Abbreviations

1

4-DHP 1,4-dihydropyridine

ADME-T

absorption, distribution, metabolism, excretion, and toxicity

AmB

amphotericin B

ATO

atovaquone

BALB

Bagg Albino Laboratory Bred

CL

cutaneous leishmaniasis

CPCM

conductor-like polarizable continuum model

DEPT

distortionless enhancement by polarization transfer

DFT

density functional theory

DHFR

dihydrofolate reduyctase

DMSO

dimethyl sulfoxide

EC₅₀

concentration causing 50% of the maximum effect

FBS

fetal bovine serum

IC₅₀

half inhibitory minimal concentration

LogP

logarithm of octanol/water partition coefficient

MCL

mucocutaneous leishmaniasis

MOE

molecular operating environment

MTT

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

MW

molecular weight

nHA

number of hydrogen bond acceptors

nHD

number of hydrogen bond donors

NMR

nuclear magnetic resonance

nROT

number of rotable bonds

NTD

neglected tropical diseases

PBS

phosphate-buffered saline

PDB

Protein Data Bank

RH

strain: right heart strain

RMSD

root mean square deviation

RPMI

Roswell Park Memorial Institute

SI

selectivity index

TPSA

topological polar surface area

VL

visceral leishmaniasis.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.5c04551.

• The following files are available free of charge. The NMR and MS spectra of compounds 1–24 (PDF)

T.A.S.O. and Y.R.R. synthesized and identified the compounds, W.S.K., T.A.K., and I.S.A.N. carried out the biological assays; I.D., S.R., and N.A. ran the docking studies; A.E.M.C., I.S.A.N., and R.B.S. supervised the synthesis, the biological tests, and the docking studies, respectively. The manuscript was written through the contributions of all authors. All authors have approved the final version of the manuscript.

This research was funded by the National Council for Scientific and Technological Development (CNPq), grant numbers (310648/2022-0 and 301417/2019-9). The Article Processing Charge for the publication of this research was funded by the Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior (CAPES), Brazil (ROR identifier: 00x0ma614).

The authors declare no competing financial interest.