Introduction

Trypanosomatidae form a family of unicellular eukaryotic parasites from the order Kinetoplastida, phylum Euglenozoa, supergroup Excavates. Many human pathogens possess a number of species of kinds Leishmania and Trypanosoma. The life cycles of these organisms turn out to be very complex, constantly moving between insects and mammalian hosts (Guedes da Silva et al. 2017; Lepesheva et al. 2015).

Leishmaniasis is a common disease in many continents, each year 20,000 to 30,000 deaths and almost 1 million of new cases are reported, arriving to report oneself around 12 million infected people (Lepesheva et al. 2015). It is caused by unicellular eukaryotic organisms from genus Leishmania (Trypanosomatidae family). Analogous to other protozoan parasites, Leishmania has a complex life cycle, transmitted via the bite of female phlebotomine sandflies. The parasite exist in two forms namely promastigote in sandfly and amastigote in human macrophage (Bates 2007; Rodríguez et al. 2006). It is exhibited in three clinical form: visceral leishmaniasis (Leishmania donovani, Leishmania infantum/Leishmania chagasi), also known as kala-azar; cutaneous leishmaniasis caused by Leishmania major, L. donovani, Leishmania tropica, and Leishmania aethiopica; and muco-cutaneous leishmaniasis caused by Leishmania braziliensis.

American trypanosomiasis, more known as Chagas disease, is usually transmitted by the bites of triatomine insects, also known as “kissing bugs.” These insects or their feces carry the flagellate parasite Trypanosoma cruzi (T. cruzi) and can also contaminate foods such as fruits juices, resulting in foodborne. It is an endemic disease in Central and South America, a few cases occur occasionally in the southern USA. This disease has become a growing problem in non-endemic areas, where infections may not be recognized by many people, and where the few effective drugs may not be generally available (Lepesheva et al. 2015).

Human African trypanosomiasis, known commonly as sleeping sickness, is caused by infection with protozoans of the genus Trypanosoma. It is transmitted by their only vector tsetse fly (Glossina genus) bites, which have acquired their infection from human or other animals that contain in her organism other human parasites (WHO 2017). This disease has two clinical manifestations, Trypanosoma brucei gambiense (T.b. gambiense) and Trypanosoma brucei rhodesiense (T.b. rhodesiense). The T.b. gambiense causes a more chronic infection that is responsible for over 90% of cases, whereas T.b. rhodesiense causes an acute infection that is responsible for a smaller proportion of the overall human African trypanosomiasis disease burden. The disease develops rapidly and invades the central nervous system (WHO 2017).

The patient may exhibit some of the following symptoms: fevers, skin eruptions, irregular febrile episodes, headaches, malaise, exhaustion, anorexia, extreme thirst, muscle and joint pains, anemia, rash, coma, and ultimately death. Sleeping sickness is endemic in 36 Sub-Saharan African countries, with an 85% case reduction since year 2000 and less than 3000 cases in year 2015, a record low with around > 60 million people are at risk (WHO 2017).

Sterol 14α-demethylase (CYP51, EC.1.14.13.70) has been found as a vital target for the development of anti-fungal, anti-leishmania, anti-trypanosoma drugs and also for the design of cholesterol-lowering drugs. Crystal structures of protozoan sterol 14α-demethylases provide an opportunity for structure-directed development of such inhibitors. The enzyme is responsible for catalyzing the elimination of the alpha-methyl group of the sterol, which leads to the formation of cholesterol in vertebrates, the well-known ergosterol in fungi, and several ergosterol alkylated derivatives (phytosterol) for plants and protozoa (Buckner and Urbina 2012). The production of ergosterol in fungus and protozoa, unlike mammals, is lethal when it accumulates, affecting cytokinesis, stopping cell growth to cause death of the cell membrane (Roberts et al. 2003). Extensive work is performed already considering CYP51 as target. Silva et al. (Guedes da Silva et al. 2017) have analyzed the effectiveness of two classes of drugs using CYP51: VNI and VFV. Schel et al. (2016) have demonstrated the anti-fungal activity of VT-1161 and VT-1129, among many other application reports with the use of CYP51 target (Singh et al. 2015; Anusha et al. 2015).

Here, we report the synthesis, in vitro antiprotozoal activity, and cytotoxicity of 14 arylsubstituted imidazoles, as well as its molecular docking onto sterol 14α-demethylase (CYP51). We have chosen the CYP51 target because all analyzed compounds possess the principal pharmacophore features of CYP51 inhibitors reported by Vita et al., where they state that an active compound must contain a heterocycle with a nitrogen able to interact with the Fe atom present in the Heme group and having two hydrophobic groups near to it (De Vita et al. 2016).

Materials and methods

General procedure for the synthesis of substituted imidazoles

The arylsubstituted imidazoles were obtained by reaction of aromatic aldehydes, primary amines, benzils, and ammonium acetate (Fig. 1) catalyzed by boric acid (Shelke et al. 2009). All reagents used had commercial grade. Melting points were determined in open capillaries using a Digital Melting Point apparatus WRS-2A. 1H-NMR and 13C-NMR spectra were recorded with a Bruker DRX-600 spectrometer obtained in DMSO-d6 and MeOD solutions. The FT-IR spectra were recorded on a Shimadzu-FTIR Infrared spectrometer in KBr (νmax in cm−1). The reaction occurred at the maximum energy area in the reaction vessel, where was slightly lower than the level of the water and the temperature was controlled at 60 °C.

Fig. 1
figure 1

Synthesis of arylsubstituted imidazoles

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Reaction conditions are as follows: aldehyde 1 (1 mmol), amine 2 (1 mmol), ammonium acetate 3 (1.5 mmol), benzil 4 (1 mmol), H3BO3 (5 mol%), and propylene glycol (15 mL) heating under reflux for 6 h.

The products (5a-n) in Table 1 were confirmed by the analysis of spectra 1H-NMR, 13C-NMR, FT-IR, mass spectra, and the melting points obtained for each compounds.

Table 1 Chemical structure of arylsubstituted imidazole
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Antiparasitic activity

All compounds were in vitro tested against T. cruzi Tulahuen CL2, β-galactosidase strain (nifurtimox-sensitive), L. infantum (MHOM/MABE/67 amastigotes), and T. brucei (Squib-427 strain, suramin-sensitive) and T.b. rhodesiense (strain STIB-900).

Compound stock solutions were prepared in DMSO at 20 mM. The compounds were serially pre-diluted (2-fold or 4-fold) in DMSO followed by a further (intermediate) dilution in demineralized water to assure a final in-test DMSO concentration of < 1%. Test plates are identical for all screens and produced as a single batch. The results were expressed as % reduction in parasite burdens compared to control wells and an IC50 (50% inhibitory concentration) was calculated. The in vitro screening was carried out according to the previously described procedure (Cos et al. 2006).

Trypanosoma cruzi, Tulahuen CL2, β-galactosidase strain (nifurtimox-sensitive) was used. The strain was maintained on MRC-5SV2 (human lung fibroblast) cells in MEM medium, supplemented with 200 mM L-glutamine, 16.5 mM NaHCO3, and 5% inactivated fetal calf serum. All cultures and assays were conducted at 37 °C under an atmosphere of 5% CO2. Assays were performed in sterile 96-well microtiter plates, each well containing 10 μl of the watery compound dilutions together with 190 μl of MRC-5 cell/parasite inoculum (2.104 cells/ml + 2.105 parasites/ml). Parasite growth was compared to untreated-infected controls (100% growth) and non-infected controls (0% growth) after 7 days incubation. Parasite burdens were assessed after adding the substrate CPRG (chlorophenol red β-D-galactopyranoside): 50 μl/well of a stock solution containing 15.2 mg CPRG + 250 μl Nonidet in 100 ml PBS. The change in color was measured at 540 nm after 4 h incubation at 37 °C. Benznidazole was included as reference drug. The test compound is classified as inactive when the IC50 is higher than 30 μM. When IC50 lies between 30 and 5 μM, the compound is regarded as being moderate active. When the IC50 is lower than 5 μM, the compound is classified as highly active on the condition that it also demonstrates selective action (absence of cytotoxicity).

Trypanosoma brucei brucei Squib 427 strain (suramin-sensitive) and T.b. rhodesiense (strain STIB-900) were used for screening. The strains are maintained in Hirumi (HMI-9) medium, supplemented with 10% inactivated fetal calf serum. All cultures and assays were conducted at 37 °C under an atmosphere of 5% CO2. Assays were performed in sterile 96-well microtiter plates, each well containing 10 μl of the compound dilutions together with 190 μl of the parasite suspension (1.5 × 104 parasites/well—T.b. brucei, and 4 × 103 parasites/well—T.b. rhodesiense). Parasite growth was compared to untreated-infected (100% parasite growth) and uninfected controls (0% growth). After 3 days incubation, parasite growth was assessed fluorimetrically after addition of 50 ml resazurin per well. After 6 h (T.b. rhodesiense) or 24 h (T.b. brucei) at 37 °C, fluorescence was measured (λex 550 nm, λem 590 nm). Suramin (T.b. brucei and T.b. rhodesiense) was included as the reference drugs. The compound is classified as inactive when the IC50 is higher than 5 μM. When IC50 lies between 5 and 1 μM, the compound is regarded as being moderate active. When the IC50 is lower than 1 μM, the compound is classified as highly active on the condition that it also demonstrates selective action (absence of cytotoxicity).

Leishmania infantum MHOM/MA(BE)/67 was maintained in the golden hamster (Mesocricetus auratus) and spleen amastigotes were collected for infection. Primary peritoneal mouse macrophages (PMM) were used as host cells and were collected 2 days after peritoneal stimulation with a 2% potato starch suspension. Assays were performed in 96-well microtiter plates, each well containing 10 μl of the compound dilutions together with 190 μl of macrophage-parasite inoculum (3.105 cells + 3.106 parasites/well // RPMI-1640 + 5% FCSi). After 5 days incubation, total amastigote burdens were microscopically assessed after Giemsa staining. Miltefosine was included as reference drug. A test compound is classified as inactive when the IC50 is higher than 30 μM. When IC50 lies between 30 and 10 μM, the compound is regarded as moderately active. If the IC50 is lower than 10 μM, the compound is classified as highly active on the condition that it also demonstrates selective action (absence of cytotoxicity against primary peritoneal macrophages).

In vitro cytotoxicity evaluation on human fibroblasts (MRC-5 cell line)

MRC-5SV2 cells were cultured in MEM + Earl’s salts-medium, supplemented with L-glutamine, NaHCO3, and 5% inactivated fetal calf serum. All cultures and assays were conducted at 37 °C under an atmosphere of 5% CO2. Assays were performed in 96-well microtiter plates, each well containing 10 μl of the watery compound dilutions together with 190 μl of MRC-5SV2 inoculum (1.5 × 105 cells/ml). Cell growth was compared to untreated-control wells (100% cell growth) and medium-control wells (0% cell growth). After 3 days incubation, cell viability was assessed fluorimetrically after addition of resazurin (λex 550 nm, λem 590 nm). Tamoxifen was used as positive control. The compound is classified non-toxic when the IC50 is higher than 30 μM. Between 30 and 10 μM, the compound is regarded as moderately toxic. When the IC50 is lower than 10 μM, the compound is classified as highly toxic.

Molecular docking

Ligands and proteins preparation

Molecular modeling was performed using the high-performance computing capabilities of the Cluster of Chemistry Department of University of Oriente running the Linux operating system Debian 8.0 distribution. The pdb file was prepared using the software UCSF Chimera molecular graphic system, version 1.10.227 (Pettersen et al. 2004). Cytochrome P450 14α-sterol demethylase, CYP51, of the species T. cruzi, T. brucei, and L. infantum was selected: CYP51 of T. cruzi in complex with an inhibitor VNF ((4-(4-chlorophenyl)-N-[2-(1H-imidazol-1-yl)-1-phenylethyl]benzamide)—PDB ID: 3KSW (CYP51—T. cruzi) (Lepesheva et al. 2010) with a resolution of 3.05 Å, CYP51 of T. brucei bound to (S)-N-(3-(1H-indol-3-yl)-1-oxo-1-(pyridin-4-ylamino)propan-2-yl)-3,3′-difluoro-(1,1′-biphenyl)-4-carboxamide—PDB ID: 4BJK (CYP51—T. brucei) (Choi et al. 2013) with a resolution of 2.67 Å and CYP51 of L. infantum in complex with fluconazole—PDB ID: 3L4D (CYP51—L. infantum) (Hargrove et al. 2011) with resolution of 2.75 Å. The structures 3L4D and 4BJK contained identical domains; we selected chain A for the docking experiments. Structure 3KSW contained only one chain. Finally, the resulting prepared 3D structure of the proteins was saved as PDB file, using AutodockTools 1.5.6 (Sanner 1999).

Kollman united atom charges, solvation parameters, and polar hydrogens were added to the receptor. Since the ligands are not peptides, Gasteiger charges were assigned and non-polar hydrogens were merged. ChemBioDraw (Evans 2014) was used to draw and design the structures of arylsubstituted imidazoles (Fig. 1) taking into account reference Planche et al. (2009). ChemBio3D Ultra 12.0 (Evans 2014) was used to optimize the geometry, running a MMFF94 energy minimization of the 3D structures. The Gasteiger charge calculation method (Gasteiger and Marsili 1980) was used and partial charges were added to the ligand atoms prior to docking, via AutodockTools.

Identification of binding site residues

The binding site residues for CYP51—T. cruzi, CYP51—T. brucei, and CYP51—L. infantum were identified from the analysis of 3KSW, 4BJK, and 3L4D available in the RCSB Protein Data Bank. CYP51—L. infantum in the active site involves the amino acids Leu355, Met357, Leu358, Met459, Val212, Phe104, Met105, Tyr115, Ala114, Phe109, Gly282, Met283, Phe289, Leu129, Ala290, and Val460 (Hargrove et al. 2011), CYP51—T. cruzi involves the amino acids Tyr103, Met106, Phe110, Tyr116, Leu127, Ala297, Phe290, Ala291, Thr295, Leu356, Leu357, Met358, Met360, Val461, Met460, and Ala287 (Lepesheva et al. 2010), and CYP51—T. brucei involves the amino acids Tyr116, Tyr103, Met106, Phe105, Phe290, Ala287, and Phe110 (Choi et al. 2013) according to the references.

Grid box preparation and docking

Docking experiment were performed between arylsubstituted imidazoles and CYP51—T. cruzi and CYP51—L. infantum proteins. AutoDock requires pre-calculated grid maps, one for each atom type, present in the ligand being docked, as it stores the potential energy arising from the interaction with the macromolecule. This grid must surround the region of interest (active site) in the macromolecule.

Grid box parameters (Table 2) were set by using AutoDockTools (ADT), a free graphic user interface of MGL software packages (version 1.5.6rc3) (Sanner 1999).

Table 2 Grid box parameters selected for the target enzymes
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The molecular docking program AutoDock version 4.2 (Sanner 1999) was employed to perform the docking experiment. The Lamarckian Genetic Algorithm was used during the process to explore the best conformational space for the ligand with a population size of 50 individuals. The maximum number of generations and evaluations were set at 27,000 and 2,500,000, respectively. Other parameters were set as default. After complete execution of Autodock, 50 conformations of the ligands in complex with the receptor were obtained, which were ranked based on binding energy and inhibition constant (Ki).

To check the docking procedure and given the similar structure (imidazole and triazole rings) between the compounds here reported, we choose known CYP51 inhibitors, VNF ((R)-N-(2-(1H-imidazol-1-yl)-1-phenylethyl)-4′-chloro-[1,1′-biphenyl]-4-carboxamide) to CYP51—T. cruzi (Friggeri et al. 2014), ketoconazole (KET) for CYP51—T. brucei (Hargrove et al. 2012), and VFV ((R)-N-(1-(3,4′-difluorobiphenyl-4-yl)-2-(1H-imidazol-1-yl)ethyl)-4-(5-phenyl-1,3,4-oxadiazol-yl)benzamide) for CYP51—L. infantum (Lepesheva et al. 2015), which were rebuilt and redocked (Fig. 2).

Fig. 2
figure 2

Structure of control compounds VNF (a), KET (b), and VFV (c)

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Results and discussion

Synthesis and characterization of arylsubstituted imidazoles

Since arylsubstituted imidazoles have become increasingly useful and important in the pharmaceutical field, the development of clean, high-yielding, and environmentally friendly synthetic approaches is still desirable and much in demand. The synthesized imidazole derivatives were characterized after recrystallization from an appropriate solvent by recording their 1H-NMR, 13C-NMR, FT-IR, and MS spectra. The results of the synthesis are summarized in Table 3.

Table 3 Physical data and yields of arylsubstituted imidazoles
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According to the results shown in Table 3, all synthesized compounds have a similar melting point and good yield compared with the reference.

Spectral data results

(5a) 4-(1-benzyl-4,5-diphenyl-1H-imidazol-2-yl)-N,N-dimethylaniline. MW: 429.57 mp: 151–153 °C (Reported: 150–152 °C (Kantevari et al. 2006)). 1H-NMR (600 MHz, DMSO-d6): δ ppm 8.10–7.18 (m, 19H, Ar-H), 5,12 (s, 2H, CH2), 3.34 (s, 6H, CH3 × 2). 13C-NMR (150 MHz, DMSO-d6): δ ppm 155.2, 150.42, 141.3, 137.78, 131.51, 129.38, 128.94, 128.75, 128.60, 128.49, 128.35, 127.48, 126.38, 125.61, 118.42, 111.99(Carom), 47.68 (CH2), 41.02 (CH3x2). Mass spectrum (ES+). 430.23 [M + H]+, 387.18, 340.18, 297.13.

(5b) 4-(1-benzyl-4,5-diphenyl-1H-imidazol-2-yl) phenol. MW: 402.49 mp: 136–139 °C (Reported: 135–138 (Khalafi-Nezhad et al. 2016)). 1H-NMR (600 MHz, DMSO-d6): δ ppm 9.76 (s, 1H, OH), 6.74–7.46 (m, 19H), 5.11 (s, 2H, CH2), 13C-NMR (150 MHz, DMSO-d6): δ ppm 158.04, 153.84, 141.40, 137.57, 136.52, 134.75, 130.88, 130.07, 129.63, 128.80, 128.55, 128.10, 127.18, 126.10, 125.70, 121.57, 115.46 (Carom), 47.63(CH2). Mass spectrum (ES+). 403.18 [M + H]+, 313.13, 297.14.

(5c) 1-benzyl-2,4,5-triphenyl-1H-imidazole. MW: 386.50 mp: 158–160 °C (Reported: 155–158 (Khalafi-Nezhad et al. 2016)). 1H-NMR (600 MHz, DMSO-d6): δ ppm 6.75–7.69 (m, 20H, Ar-H), 5,13 (s, 2H, CH2). 13C-NMR (150 MHz, DMSO-d6) δ ppm 153.04, 141.33, 137.20, 137.36, 133,58, 131.12, 130.43, 129.7, 129.10, 127.49, 126.94, 126.18, 125.67, 125.26 (Carom), 47.73 (CH2). Mass spectrum (ES+). 387.18 [M + H]+ 297.13.

(5d) 1-benzyl-2-(4-methoxyphenyl)-4,5-diphenyl-1H-imidazole. MW: 416.52 mp: 165–168 °C (Reported: 167–169 (Khalafi-Nezhad et al. 2016)). 1H-NMR (600 MHz, DMSO-d6): δH ppm 6,75–8.01 (m, 17H, Ar-H), 5.65 (s, 2H, CH2), 3.81 (s, 3H, OCH3). 13C-NMR (150 MHz, DMSO-d6): 159.66, 153.70, 147.20, 141.30, 138.82, 137.49, 134.30, 130.70, 129.80, 129.60, 128.59, 127.66, 125.63, 123.20, 114.15(Carom), 55.26(CH3), 47.66(CH2). Mass spectrum (ES+). 417.19 [M + H]+ 327.14, 221.14.

(5e) 2-(3-nitrophenyl)-4,5-diphenyl-1-(m-tolyl)-1H-imidazole. MW: 431.50 mp: 165–166 °C (Reported: none reported found). 1H-NMR (600 MHz, DMSO-d6): δH ppm 7.10–8.80 (m, 18H, Ar-H), 2,35 (s, 3H, CH3). 13C-NMR (150 MHz, DMSO-d6): 149.80, 148.73, 146.04, 145.00, 143.97, 139.15, 138.12, 136.04, 134.86, 132.72, 131.00, 130.08, 129.99, 129.57, 127.83, 126.07, 123.16, 122.19, 118.82, 115.25(Carom), 21.4 (CH3). IR (KBr): υmax3058(C=Carom), 169(C=N), 1575(N-O), 1460 y 1502(C=Carom), 1335 (C-NO2). δmax 853(C-NAr-NO2), 710 y 810(C-Hm-sust) cm−1. Mass spectrum (ES+). 432.17 [M + H]+ 418.19, 342.12, 297.13, 221.13, 179.08.

(5f) 2-(4-nitrophenyl)-4,5-diphenyl-1-(o-tolyl)-1H-imidazole. MW: 431.50 mp: 235–236 °C (Reported: none reported found). 1H-NMR (600 MHz, DMSO-d6): δH ppm 7.12–8.69 (m, 18H, Ar-H), 2.33 (s, 3H, CH3). 13C-NMR (150 MHz, DMSO-d6): 149.63, 148.89, 144.32, 141.74, 137,36, 135.60, 132.30, 131.72, 130.36, 129.65, 129.53, 127.68, 126.93, 126.65, 124.10, 122.36, 117.76(Carom), 17.57(CH3). IR (KBr): υmax3080(C=Carom), 1685(C=N), 1580(N-O), 1500(C=Carom), 1345(C-NO2). δmax 860(C-NAr-NO2), 740(C-Ho-sust) cm−1. Mass spectrum (ES+). 432.17 [M + H]+ 387.18, 342.12, 297.14.

(5g) 2-(4-nitrophenyl)-4,5-diphenyl-1-(m-tolyl)-1H-imidazole. MW: 431.50 mp: 221–223 °C (Reported: none reported found). 1H-NMR (600 MHz, MeOH-d4): δH ppm 7.33–8.31 (m, 12H, Ar-H), 2.21 (s, 3H, CH3). 13C-NMR (150 MHz, DMSO-d6): 149.63, 148.89, 144.32, 141.74, 137,36, 135.60, 132.30, 131.72, 130.36, 129.65, 129.53, 127.68, 126.93, 126.65, 124.10, 122.36, 117.76(Carom), 17.57(CH3). IR (KBr): υmax 3058 (C=Carom), 1650 (C=N), 1599 (C=Carom), 1335 (C-NO2), 853 (C-NAr-NO2), 767 (C-Hm-sust) cm−1. Mass spectrum (ES+). 432.17 [M + H]+ 342.12, 179.09, 149.02.

(5h) 2-(4-nitrophenyl)-4,5-diphenyl-1-(p-tolyl)-1H-imidazole. mp: 219–222 °C (Reported: 219–220 °C (Kantevari et al. 2006)). MW: 431.50. 1H-NMR (600 MHz, MeOH-d4): δH ppm 7.32–8.21 (m, 18H, Ar-H), 2.14 (s, 3H, CH3). 13C-NMR (150 MHz, MeOH-d4): δH ppm, 148.81145.56, 139.16, 137.06, 133.29, 130.39, 129.72, 129.61, 129.52, 129.43, 128.92, 128.89, 127.28, 126.82, 125.21, 124.35, (Carom). 22.15(CH3). IR (KBr): υmax 3050 (C=Carom), 1615 (C=N), 1500 (C=Carom), 1338 (C-NO2), δmax 860 (C-NAr-NO2), 840 (C-Hp-sust) cm−1. Mass spectrum (ES+). 432.17 [M + H]+ 342.12, 301.14, 179.09, 149.02, 137.08.

(5i) 4-(2-(4-(dimethylamino)phenyl)-4,5-diphenyl-1H-imidazol-1-yl)benzenesulfonic acid. MW: 495.60 mp: 109–112 °C (Reported: none reported found). 1H-NMR (600 MHz, DMSO-d6): δH ppm 9.66 (s, 1H, OH), 6.47–7.97(m, 18H, Ar-H), 2.99 (s, 6H, CH3 × 2). 13C-NMR (150 MHz, DMSO-d6): 151.35, 145.87, 143.10, 139.60, 136.40, 130.69, 130.09, 128.72, 128.53, 128.31, 127.40, 126.88, 126.3, 126.21, 113.93, 112.49 (Carom), 40.01 (CH3x2) ppm. Mass spectrum (ES−). 495.14 [M-H]−, 338.17, 214.01, 172, 155.

(5j) 4-(4,5-diphenyl-1H-imidazol-2-yl)-N,N-dimethylaniline. MW: 339.44 mp: 255–257 °C (Reported: 256–259 °C (Eidi et al. 2016)). 1H-NMR (600 MHz, DMSO-d6): δH ppm 12.36 (s, 1H, NH), 6.80–7.97 (m, 14H, Ar-H), 2.99 (s, 6H, CH3x2). 13C-NMR (150 MHz, DMSO-d6): δH ppm 154.21, 150.36, 146.80, 128.44, 127.84, 126.56, 118.60, 112.03, 111.11(Carom), 43.45 (CH3x2). Mass spectrum (ES+). 340.18 [M + H]+, 297.14.

(5k) 4-(1-(furan-2-ylmethyl)-4,5-diphenyl-1H-imidazol-2-yl)-2-methoxyphenol. MW: 422.48 mp: 244–245 °C (Reported: none reported found). 1H-NMR (600 MHz, DMSO-d6): δH ppm 9.28 (s, 1H, OH), 6.85–7.72 (m, 16H, Ar-H, Furanyl), 4.46 (s, 2H, CH2), 3.83 (s, 3H, CH3). 13C-NMR (150 MHz, DMSO-d6): δH ppm 148.72, 147.77, 147.22, 146.08, 142.06, 138.62, 128.50, 128.74, 127.82, 127.52, 127.15, 121.77, 118.52, 115.70, 112.08, 110.92, 105.42, 109.46(Carom), 55.79 (CH3), 40.01 (CH2). Mass spectrum (ES+). 423.17 [M + H]+, 343.14, 297.14.

(5l) 4-(4,5-di(furan-2-yl)-1H-imidazol-2-yl)-3-methoxyphenol. MW: 276.30 mp: 189–190 °C (Reported: none reported found). 1H-NMR (600 MHz, DMSO-d6): δH ppm 3.87 (s, 3H, CH3), 9.33 (s, 1H, OH), 6.57–7.81 (m, 9H, Ar-H, Furanyl), 12.62 (s, 1H, NH). 13C-NMR (150 MHz, DMSO-d6): δH ppm 144.71, 142.46, 141.80, 129.33, 128.97, 121.29, 120.91, 118.91, 115.67, 112.81, 111.45, 108.18, 106.79(Carom), 55.84(CH3). Mass spectrum (ES+). 323.10 [M + H]+, 293.09, 201.08.

(5 m) 4,5-di(furan-2-yl)-2-phenyl-1H-imidazole. MW: 276.30 mp: 196–198 (Reported: 197–198 °C (Kang et al. 2011)). 1H-NMR (600 MHz, DMSO-d6): δH ppm 6.58–8.10 (m, 11H, Ar-H, Furanyl), 12.87 (s, 1H, NH). 13C-NMR (150 MHz, DMSO-d6): δH ppm 149.34, 146.32, 144.48, 142.69, 141.94, 129.89, 128.78, 119.42, 111.96, 106.98(Carom). Mass spectrum (ES+). 277.09 [M + H]+, 201.07.

(5n) 4-(4,5-di(furan-2-yl)-1-(furan-2-ylmethyl)-1H-imidazol-2-yl)phenol. MW: 392.46 mp: 155–158 (Reported: none reported found). 1H-NMR (600 MHz, DMSO-d6): δH ppm 9.79 (s, 1H, Ar-OH), 3.42 (s, 2H, CH2), 6.87–7.92 (m, 13H, Ar-H, Furanyl). 13C-NMR (150 MHz, DMSO-d6): δH ppm 158.50, 157.91, 153.67, 151.30, 146.19, 142.9, 142.1, 130.75, 128.48, 127.80, 126.98, 121.73, 115.96, 107.70(Carom), 37.80 (CH2). Mass spectrum (ES+). 373.12 [M + H]+ 293.09, 277.10.

Antiprotozoal and cytotoxic effects

The antiparasitic activities and the cytotoxicity of the test and reference compounds are listed in Table 4. All compounds were tested in vitro against the tripomastigotes stage of T.b. brucei and T.b. rhodesiense and amastigote stage of T. cruzi and L. infantum. The cytotoxicity of all arylsubstituted imidazoles against MRC-5 cell lines (human lung fibroblasts) was evaluated and the selectivity indices (SI) (SI = CC50 MRC-5 cells/IC50 protozoa) were calculated (Table 5) (Ehata et al. 2012).

Table 4 Antitrypanosomal, antileishmanial (IC50, μM ± SD), and cytotoxic (MRC-5) (CC50, μM ± SD) activities of arylsubstituted imidazoles. Biological experiments were performed in duplicate
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Table 5 Selectivity index (SI) of the arylsubstituted imidazole from T. cruzi, L. infantum, T.b. brucei, and T.b. rhodesiense
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The imidazoles 5a, 5c, 5e, 5f, 5g, 5i, 5j, 5k, and 5n exhibited IC50 values lower than 5 μM against T. cruzi. The ligands 5l, 5m, and 5h had a moderate activity with IC50 values of 18.19, 16.99, and 17.66 μM, respectively. Only the ligands 5d and 5b, both with similar structure (Table 1) substituted in position two of the arylsubstituted imidazole by a hydroxyl (OH) and a methoxy (OCH3), showed a low activity. The most active compounds 5a, 5f, 5g, and 5j showed lower IC50 values than the reference compound benznidazole (Table 4). However, only compound 5c with an IC50 of 4.07 μM presented a good selectivity towards T. cruzi with a SI of 9.49 (Table 5).

On the other hand, against T.b. brucei and T.b. rhodesiense, the active compounds were 5a, 5c, 5e, 5f, 5g, 5i, and 5j. The most active compound against T.b. brucei was 5e (IC50 = 2.11 μM), while for T.b. rhodesiense the most active compound was 5a with an IC50 = 0.86 μM. Both compounds were less active than the reference compounds suramin for T.b. brucei (0.03 μM) and T.b. rhodesiense (0.04 μM). For T.b. rhodesiense, the ligands with a good activity were 5h, 5k, 5l, and 5m, with IC50 values between 11.99 and 15.53 μM. Ligands 5k and 5l have a similar structure in position two of the imidazoles with a phenyl group joined to a hydroxyl (4-OH) substituent and a methoxyl (3-CH3O) group.

None of studied arylsubstituted imidazoles showed a higher activity than the reference drug suramin towards T.b. rhodesiense, demonstrating the lower sensitivity for this class of compounds (De Vita et al. 2016). Only the ligand 5a (IC50 = 0.86 μM) possessed an IC50 value comparable with the control drug suramin.

Compounds 5a, 5c, 5e, 5f, 5g, 5i, and 5j presented similar antileishmanial activity compared with the control drug miltefosine, but they showed a pronounced cytotoxicity on MRC-5 cells (Table 4), indicating the lack of selectivity towards L. infantum (Table 5).

Compound 5c had the highest selectivity to the Trypanosoma family with a SI of 9.49, 11.5, and 10.2 for T. cruzi, T.b. brucei, and T.b. rhodesiense, respectively (Table 5).

Molecular docking

The procedure adopted for the molecular docking was used for all designed arylsubstituted imidazoles (Table 1) with each target selected. The docked poses for each of the compounds have been evaluated and the pose with the lowest binding free energy and inhibition constant was thereby chosen (Table 6).

Table 6 Binding energy and inhibition constant of the arylsubstituted imidazoles and control CYP51 inhibitors VNF, KET, and VFV
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The lowest binding free energy (i.e., best docking score) and inhibition constant indicates the highest ligand/protein affinity. The docking studies were done in comparison with control drugs (i.e., known inhibitors of the target enzymes). The control drugs were redocked with each protein target chosen, VNF for CYP51—T. cruzi, VFV for CYP51—L. infantum, and KET for CYP51—T. brucei.

As shown in Table 6, the binding energies (ΔG) and inhibition constant (Ki) of the control drug VNF in the docking with CYP51—T. cruzi were found to be − 9.65 kcal/mol and 85.03 nM, respectively. However, the value for almost designed imidazoles was found to be higher than the VNF control, with ΔG value between − 9.25 and − 14.54 kcal/mol and low values of Ki. The ligand 5i shows the better values (ΔG = − 14.54 kcal/mol and Ki = 0.02 nM) and a similar conformation compared with the VNF control (Fig. 3a), other ligands showed better values and only the ligands 5l and 5m showed not accepted values of binding energies and inhibition constant compared with the control.

Fig. 3
figure 3

Superimposition of the best poses of the ligand + control drugs for CYP51—T. cruzi in a 5i (tan) + VNF (sky blue) and b 5f (tan) + KET (sky blue) for CYP51—T. brucei included in the binding pocket

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In the case of CYP51—T. brucei, the binding energy (ΔG) of the control drug ketoconazole was − 11.37 kcal/mol (Ki = 4.60 nM), exceeded only by the ligands 5e, 5f, 5g, and 5h with a binding energy of − 14.47, − 14.75, − 14.01, and − 12.56 kcal/mol, respectively, and Ki values very low compared with the KET control. The best ligand 5f showed similar conformation respect to KET control (Fig. 3b) and the ligands 5e, 5g, and 5h presented similar values (ΔG and Ki) to ligand 5f, due to the same interaction with the Heme group through the oxygen presented in the NO2 group (Fig. 4).

Fig. 4
figure 4

Similar interaction between ligands 5e, 5f, 5g, and 5h with the enzyme CYP51—T. brucei

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For enzyme CYP51—L. infantum with the control drug VFV, the binding energy was found to be − 10.29 kcal/mol with a Ki = 28.60 nM. The values of VFV are only exceeded by ligands 5a, 5b, 5c, 5d, 5e, 5k were found to have − 11.13, − 10.37, − 10.74, − 10.95, − 12.88, and − 10.61, respectively, for the binding energy and Ki values very low respect to the VFV control (Table 6). The best ligand was 5e and we can observe in Fig. 5 the superimposition between VFV and 5e, where both ligands interacted directly with the Heme group.

Fig. 5
figure 5

Superimposition of the best pose of the 5e (tan) + VFV (sky blue) included in the binding pocket of CYP51—L. infantum

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It should be noted that ligand 5e appeared to bind in the three receptor enzymes (CYP51—T. cruzi, CYP51—L. infantum, CYP51—T. brucei) and in all cases this ligand had lower ΔG values when compared to the control drugs. Some similar behavior occurs with the ligands 5a and 5k binding to CYP51—L. infantum and CYP51—T. brucei and 5f, 5g, 5h, and 5i binding with CYP51—T. cruzi and CYP51—T. brucei. All of them show better binding values than the control drugs. Our molecular docking studies revealed that all compounds (5a–n) in one way or another interacted with the 14 α-demethylase enzyme for each species (CYP51—T. cruzi, CYP51—L. infantum, CYP51—T. brucei) by hydrophobic interactions or azole-Heme coordination and π-π and π-cation interactions.

This study with CYP51—T. cruzi demonstrated that in some ligands (5a, 5b, 5c, 5d, 5j, 5k, 5m, and 5n) there are hydrophobic interactions with the Heme group (Fig. 6).

Fig. 6
figure 6

Docked structures of compound 5d in CYP51—T. cruzi; a two-dimensional representations of interaction by the Ligplot software (Wallace et al. 1996); b three-dimensional representation of the interaction the ligand (tan) and Heme group (cyan)

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We see also hydrogen bond interactions with amino acid residue Arg361 (5b, 5d, and 5n). All ligands showed hydrophobic interaction with Ala291, Ala287, Phe290, Phe110, Met106, Tyr116, Tyr103, Pro99, His420, Leu356, and Thr295. They shared some residues Ala291, Ala287, Phe290, Phe110, Met106, Tyr116, Tyr103, Leu356, and Thr295 involved in the active site according to Lepesheva et al. (2010). The ligands 5e, 5f, 5g, and 5h showed an electronic interaction between the NO2 group (electron pair of oxygen O33) and the Fe2+ of Hem488 group (Fig. 7); however for ligand 5i and 5l, the interaction with the Hem488 is with SO3H (electron pair of O20) for 5i and OCH3 (electron pair of O25) for 5l (Fig. 8).

Fig. 7
figure 7

Docked structures of compound 5g in the CYP51—T. cruzi; a 2D interaction by the Ligplot software; b 3D representation of the interaction the ligand (tan) and the heme group (cyan)

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Fig. 8
figure 8

Docked structures of compounds 5i (a) and 5l (b) in the CYP51—T. cruzi pocket represented using the Ligplot software

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The ligands 5e, 5f, 5g, and 5h also showed the interaction with the heme group. We could observe the hydrophobic interaction with Ala291, Ala287, Phe290, Phe110, Met106, Tyr116, Tyr103, Pro99, His420, Leu356, and Thr295 similar as seen with the ligands 5a, 5b, 5c, 5d, 5j, 5k, 5m, and 5n where they share the same residues involved in the active site.

In the case of CYP51—L. infantum, only the ligands 5e and 5h showed interaction between one of the NO2 group’s oxygens (O32) with the heme group (Fig. 9a) and in the ligand 5i complex the interaction was with the SO3H group’s oxygen atoms (Fig. 9b).

Fig. 9
figure 9

Docked structures of compounds 5e (a) and 5i (b) in the CYP51—L. infantum pocket represented using the Ligplot software

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On the other hand, the ligands 5a, 5b, 5c, 5d, 5j, 5k, 5l, 5m, and 5n were found to have hydrophobic interactions with Met359, Val212, Pro209, Me459, Val356, Met357, Val460, Thr294, Ala290, Leu355, Phe109, Met105, Phe289, Tyr115, Tyr102, Phe104, and Hem481 (Fig. 10). These ligands share some residues Leu355, Met357, Val212, Phe104, Met105, Tyr115, Phe109, and Ala290 involved in the active site according to Hargrove et al. (2011).

Fig. 10
figure 10

Docked structures of compound 5b in the CYP51—L. infantum; a 2D interaction by Ligplot software, b 3D representation of the interaction the ligand (tan) and Heme group (cyan)

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Compounds 5a, 5b, 5c, 5d, 5i, 5j, 5k, 5l, and 5m were found to establish hydrophobic interactions with CYP51—T. brucei (Fig. 11) that involved the residues Val461, Ala291, Phe290, Thr295, His294, Phe105, Tyr103, Met106, Tyr116, Leu127, Ala115, Ala287, Phe110, Hem1450, and His294. All those compounds (5a, 5b, 5c, 5d, 5i, 5j, 5k, 5l, and 5m) share some residues: Tyr116, Tyr103, Met106, Phe105, Phe290, Ala287, and Phe110 involved in the active site (Choi et al. 2013).

Fig. 11
figure 11

Docked structures of compound 5a in the CYP51—T. brucei; a 2D interaction by Ligplot software, b 3D representation of the interaction the ligand (tan) and Heme group (cyan)

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On the other hand, the ligands 5e, 5f, 5g, 5h, and 5n were observed to interact with the Heme group through the NO2 function for 5e, 5f, 5g, 5h and with OH for 5n (Fig. 12).

Fig. 12
figure 12

Docked structures of compounds 5f (a) and 5n (b) in the CYP51—T. brucei pocket represented using the Ligplot software

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These results reveal that the position of the substituent in the phenyl joined to the imidazole ring in the four positions affected strongly the orientation and the subsequently interaction of the imidazoles with the Heme iron atom in the binding site of the enzyme.

Conclusions

In conclusion, the present investigation has described the chemical synthesis and antiprotozoal, cytotoxic activities of several arylsubstituted imidazoles as well as their molecular docking in the active site of CYP51 of those protozoa. The reported results showed that all samples possessed interesting in vitro antiprotozoal activity at different extents. The ligand 5c was found to be non-toxic according to the study conducted with the MRC-5 cell line and the best selectivity index against T. cruzi, T.b. brucei, and T.b. rhodesiense. The main interactions, studied by molecular docking, are hydrophobic between the substituents in positions 1, 4, and 5 of the imidazolic rings and residues in the active site. Electrostatic interactions were identified between oxygen fundamentally present in NO2, SO3H, and OH groups and the Fe2+ ion of the Heme group. The reported results can partly justify and support the possible use of some of these imidazoles for the treatment of parasitic diseases such as sleeping sickness, leishmaniasis, and in some extent American trypanosomiasis named Chagas disease, with no apparent toxic effects in patients.