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. 2023 Oct 20;8(43):40613–40621. doi: 10.1021/acsomega.3c05441

Imidazo[1,2-a]pyrimidine as a New Antileishmanial Pharmacophore against Leishmania amazonensis Promastigotes and Amastigotes

Ravinder Kumar †, Rahul Singh †, Ayla das Chagas Almeida ‡, Juliana da Trindade Granato ‡, Ari Sérgio de Oliveira Lemos ‡, Kushvinder Kumar †, Madhuri T Patil §, Adilson D da Silva ∥, Ambadas B Rode ⊥, Elaine S Coimbra ‡,*, Deepak B Salunke †,#,*
PMCID: PMC10621021  PMID: 37929127

Abstract

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Leishmania poses a substantial threat to the human population all over the globe because of its visceral and cutaneous spread engendered by all 20 species. Unfortunately, the available drugs against leishmania are already hobbled with toxicity, prolonged treatment, and increasing instances of acquirement of resistance. Under these grave circumstances, the development of new drugs has become imperative to keep these harmful microbes at bay. To this end, a Groebke–Blackburn–Bienaymé multicomponent reaction-based library of different imidazo-fused heterocycles has been synthesized and screened against Leishmania amazonensis promastigotes and amastigotes. Among the library compounds, the imidazo-pyrimidine 24 has been found to be the most effective (inhibitory concentration of 50% (IC50) < 10 μM), with selective antileishmanial activity on amastigote forms, a stage of the parasite related to human disease. The compound 24 has exhibited an IC50 value of 6.63 μM, being ∼two times more active than miltefosine, a reference drug. Furthermore, this compound is >10 times more destructive to the intracellular parasites than host cells. The observed in vitro antileishmanial activity along with suitable in silico physicochemical and absorption, distribution, metabolism, excretion, and toxicity (ADMET) properties of compound 24 reinforce the imidazo-pyrimidine scaffold as a new antileishmanial pharmacophore and encourage further murine experimental leishmaniasis studies.

1. Introduction

Leishmaniasis is an endemic disease in 98 countries worldwide, transmitted to humans and other mammals by the bite of female phlebotomine sandflies, affecting mainly the Americas, East and North Africa, and West and Southeast Asia.1 The protozoan of the genus Leishmania is the etiological agent of leishmaniasis, a complex of clinical manifestations ranging from nonfatal cutaneous ulcerations to the disfiguring mucocutaneous form and the potential fatal visceral leishmaniasis.2 The pathology of this disease depends on several factors, such as the Leishmania species and the host’s immunobiological response.3

The therapies available for the treatment of leishmaniasis are very limited. Pentavalent antimonials, amphotericin B, pentamidine, and paromomycin, are currently available drugs. However, these medications are quite toxic and require long-term parenteral administration. Additionally, low therapeutic effectiveness in some endemic areas has been reported due to the emergence of resistant strains.4,5 Miltefosine, originally developed for the treatment of metastatic cutaneous breast carcinoma, is the only drug approved for the oral treatment of leishmaniasis. However, this medication is teratogenic and shows variability in efficacy.4 Therefore, the search for new drugs that are less toxic to the patient and more effective against leishmaniasis is required.

Nitrogen heterocycles are the key molecules to be explored in the field of leishmaniasis. Major classes of these heterocyclic moieties involve the quinoline, triazole, pyrazole, imidazole, indole, pyrimidine, β-carboline, quinoxaline, quinazoline, and benzimidazole. Among the various fused imidazole-based scaffolds, imidazo-pyridine has shown promising results against typanosomatida species, including Leishmania.6−9 In particular, 6,8-dibromo-3-nitro-2-phenyl sulfonyl methyl imidazo[1,2-a]-pyridine (1, Figure 1) has shown better antileishmanial activity than the marketed drug miltefosine and pentamidine against Leishmania donovani promastigotes.6 Antileishmanial screening of 2,3-diarylimidazo[1,2-a]pyridine series of compounds has resulted in a 2-phenyl-3-(p-tolyl)imidazo[1,2-a]pyridine (2) as the most potent analogue against amastigote stages of Leishmania major with inhibitory concentration (IC50) = 4 μM.7 A C2-, C3-, and C7-trisubstituted imidazo-pyridine (3) has also demonstrated promising antileishmanial activity against L. major (IC50 = 0.2 μM),8 whereas another imidazo-pyridine-based compound (4) is found to be active against Trypanosoma cruzi amastigotes (IC50 = 56.2 μM).9

Figure 1.

Figure 1

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Imidazo-pyridine-based molecules having antileishmanial activity.

The literature survey reveals the promising utility of a fused imidazo-pyridine scaffold10 for the discovery of new antileishmanial agents, and the active analogues demonstrate a diverse substitution tolerability at C2 and C3 positions as well as on the pyridine ring for the improved activity. Groebke–Blackburn–Bienaymé (GBB) multicomponent reaction (MCR) provides an easy access to construct a diverse set of imidazo-fused heterocycles in a single step utilizing different amidines, isonitriles, and aldehydes.11−19 In order to identify new antileishmanial agents, a new GBB-MCR-based library has been evaluated against Leishmania amazonensis promastigotes and amastigotes, and physicochemical and absorption, distribution, metabolism, excretion, and toxicity (ADMET) properties have been examined with a view to predict important information about imidazo-pyridine analogues.

2. Results and Discussion

A small library of imidazo-pyridines (compounds 5–12, Figure 2) was synthesized via GBB-MCR (Scheme 1) using 2-aminopyridine, aldehydes, and 4-(2-isocyanoethyl)morpholine.20

Figure 2.

Figure 2

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Antileishmanial activity and cytotoxicity of imidazo-pyridine analogues (in μM). Pro and ama mean promastigotes and amastigotes, respectively, of L. amazonensis (L.A.). Cytotoxicity is the toxic effect on murine macrophages. These results correspond to the average of three experiments performed in duplicate.

Scheme 1. General Synthetic Protocol for the Formation of Imidazo-Fused Heterocycles.

Scheme 1

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2.1. Reagent and Conditions

HCl in dioxane (4M), CH3CN, and MW (110 °C), 20 min. Morpholine is an important privileged structure due to its wide range of medicinal and pharmacological participations. Antiparasitic21 and specifically antileishmanial activities22−25 of morpholine-bearing compounds has motivated us to use 4-(2-isocyanoethyl)morpholine as a tool to attach 4-(2-aminoethyl)morpholine residue at the C3 position of our designed scaffold. The synthesized compound library has been screened against L. amazonensis promastigotes and amastigotes, and the toxicity has also been evaluated on macrophages (Figure 2).

Although the synthesized compounds (5–12, Figure 2) have shown no toxic effect on macrophages (cytotoxic concentration capable of inhibiting 50% (CC50) > 150 μM), unfortunately, none of them have shown any antileishmanial activity at the maximum concentration tested (100 μM). Considering the importance of pyrimidine along with the application of benzodioxole in medicinal chemistry, two more analogues (13 and 14, Figure 3) have been synthesized using 1,1,3,3-tetramethylbutyl isocyanide, benzodioxole aldehyde, and 2-aminopyrimidine or 2-aminopyridine components.

Figure 3.

Figure 3

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Antileishmanial activity and cytotoxicity of compounds 13 and 14 (in μM). Pro and ama mean promastigotes and amastigotes, respectively, of L. amazonensis (L.A.). Cytotoxicity is the toxic effect on murine macrophages. These results correspond to the average of three experiments performed in duplicate.

Interestingly, compound 14 has shown a positive result against L. amazonensis promastigotes (IC50 = 46.10 μM) suggesting the importance of the 3-(1,1,3,3-tetramethylbutylamine) residue at the C3 position of the imidazo[1,2-a]pyrimidine scaffold. It is noteworthy to observe that none of these active compounds showed any cytotoxicity to the macrophages. In the Leishmania species, the variation in sensitivity between promastigote and amastigote forms has been previously reported not only for new drugs but also for well-known antileishmanial drugs, such as amphotericin B, paromomycin, pentavalent antimonials, and miltefosine.26 The different drug susceptibility profiles are unclear, and they may be associated with many factors: (i) unlike promastigotes, amastigotes are intracellular parasites and found inside the macrophage parasitophorous vacuole, whose pH is acidic, and this can interfere in uptake/inactivation of the compound by host cell, (ii) different metabolic pathways of biological stages, and (iii) proteomic differences involved in important processes, among others.25−28

Based on these results and in order to obtain new compounds with promising antileishmanial activity, a series of new derivatives were synthesized with focus on compound 14, an imidazo-pyrimidine analogue (Table 1). First, changes were made at the C3 position of imidazo-pyrimidine by retaining the 1,3-benzodioxole residue. The activity was lost in each case (15–17, Table 1). Then, by preserving the 3-(1,1,3,3-tetramethylbutylamine) substitution at the C3 position of imidazo-pyrimidine, changes were made at the C2 position. Phenyl substitution (18, Table 1) in place of furfuryl at position 2 retained the activity against L. amazonensis promastigotes while showed more potency against the intramacrophage biological stages. The 4-chloro phenyl (19, Table 1) and biphenyl (20, Table 1) substituents enhanced the activity against both forms of the parasite. The compound with the 3-methoxy phenyl group (21, Table 1) showed an increase in the activity against both parasite forms, while the 3-hydroxy and 4-methoxy phenyl group (22, Table 1) was not tolerated for the antileishmanial activity. The C3-nitro phenyl-substituted analogue (23, Table 1) did not show any biological activity, but the 3-cyano phenyl analogue (24, Table 1) was found to be the most potent antileishmanial molecule in the series, with IC50 values <10 μM. Besides the capacity to inhibit the growth of promastigotes (IC50 = 8.41 μM), the compound 24 also exhibited good activity against intracellular amastigotes (IC50 = 6.63 μM), being more effective than miltefosine (IC50 = 15.05 and 12.52 μM, respectively), used as the reference drug. In addition, the lead compound 24 was 10 times more toxic to the parasite than to the host cell (CC50= 82.02 μM and selectivity index (SI) = 12.37), with a profile similar to that of miltefosine (CC50= 151.81 μM and SI = 12.12). It was interesting to note that in compound 24 along with a few more analogues (18, 22, and 25) when assayed on MCF-7, MDA-MB-231, and HEP-G2 cell lines at 25 μM, no any significant cytotoxicity was observed (Supporting Information Figure S65). The MTT assay was performed as reported by us earlier.29 Although these cell lines were mostly used for anticancer assays, they were cell lines of human origin, and therefore, these data also reinforced the negligible toxicity by compound 24 to mammalian cells. Further, the replacement of the 3-cyanophenyl group by the 3-pyridinyl group (25, Table 1) evaded the activity, but the 2-thiophenyl group (26, Table 1) showed an effect on L. amazonensis amastigotes only. 2-Butyl- and 2-octyl-substituted products (27 and 28, Table 1) demonstrated moderate activity against L. amazonensis promastigotes and amastigotes.

Table 1. Activity of Imidazo-Pyrimidine Analogues in L. amazonensis Promastigote and Amastigote Forms and Cytotoxicity on Murine Peritoneal Macrophages.

2.1.

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a

Values of CC50.

b

Values of IC50.

c

SI: CC50 in peritoneal macrophages/IC50 in intracellular amastigotes. ND means not determined. Miltefosine was used as the reference drug. These results correspond to the average of three experiments performed in duplicate.

In vitro results have stimulated future studies for assessing the activity of compounds in the in vivo model. However, it is important to predict the druggability of the new compound using computational tools. On the grounds that compound 24 has shown higher in vitro antileishmanial activity and a selective action (SI > 10), its physicochemical and ADMET properties are evaluated. The results obtained through the PKCSM software point out compound 24 with excellent oral bioavailability (Table 2), since it does not violate any of the parameters of the Lipinski’s rule of five (logP ≤ 5; MW ≤ 500, hydrogen bond acceptor (HBA) ≤ 5, and hydrogen bond donor (HBD) ≤ 10).30 These parameters are associated with a good solubility and intestinal permeability that comprise the first step of drug absorption.31

Table 2. In Silico Physicochemical Properties of 24.

compound molecular weight logP hydrogen acceptors hydrogen donors violations
24 347.466 4,89468 5 1 0
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For successful medications, ADMET profiles are critical, and the in silico pharmacokinetic prediction is a good tool in the early development process of the drug discovery.32 The ADMET profile of compound 24 by AdmetSAR has predicted a good intestinal absorption (Table 3), corroborating with the physicochemical analysis. In addition, favorable results are obtained for the topical administration of compound 24 (Table 3). This route reduces systemic adverse effects and can also be utilized to obtain regulated or delayed drug delivery.33 Also, the in silico prediction has revealed that the lead compound 24 is a substrate/inhibitor of several CYP450 enzymes, which are key enzymes in the liver.34 The CYP450 superfamily is the most prevalent phase-I drug-metabolizing enzyme, responsible for converting drugs into water-soluble compounds, facilitating their excretion by the kidney and/or liver.34 Thus, these data suggest that compound 24 is likely to undergo hepatic metabolism to be excreted later. The kidney, in turn, is a very important organ participating in the process of eliminating substances with organic cation transporters (OCTs) and multidrug and toxin extrusion proteins (MATEs), which are considered as the major transporters that secrete cationic drugs into the urine.35 Therefore, our results suggest that after hepatic metabolism, compound 24 can be excreted by OCTs in the kidney. Toxicology parameter is the last aspect in the ADMET profile examined, and the analysis has suggested that compound 24 exhibits liver and mutagenic toxicities. Thus, we consider that, if the oral route is chosen for in vivo model, these data should be better investigated in the preclinical trial phases. On the other hand, since no skin toxicity has been observed, a possible topical application can be explored.

Table 3. ADMET Profile of 24.

absorption intestinal absorption yes
absorption skin permeability yes
distribution BBB permeability yes
distribution CNS permeability yes
metabolism CYP2D6 substrate no
metabolism CYP3A4 substrate yes
metabolism CYP1A2 inhibitor yes
metabolism CYP2C19 inhibitor yes
metabolism CYP2C9 inhibitor yes
metabolism CYP2D6 inhibitor yes
metabolism CYP3A4 inhibitor no
excretion renal OCT2 substrate yes
toxicity AMES toxicity yes
Toxicity Hepatotoxicity Yes
Toxicity Skin Sensitization No
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Conclusions

In summary, twenty four imidazo-fused derivatives were synthesized and evaluated against L. amazonensis species. Among the compounds tested, compound 24 exhibited expressive activity against promastigote and amastigote forms of L. amazonensisand did not show significant toxic effects on mammalian cells. In addition, physicochemical and ADMET studies indicated that compound 24 could be used through oral or topical administration. These data stimulated further studies of the efficacy of this compound in murine experimental leishmaniasis.

4. Experimental Section

4.1. Chemistry

4.1.1. General

Commercially available reagents were used without any further purification. 2-Aminopyridine, 2-aminopyrimidine, and 2-aminothiazole were purchased from Sigma, while 2-aminopyrazine was purchased from Avra Synthesis. Further, aldehydes—benzaldehyde, butanal, 3-cyanobenzaldehyde, 2-hydroxybenzaldehyde, 3-hydroxy-4-methoxybenzaldehyde, 3-methoxybenzaldehyde, and thiopene-2-carbaldehyde—were also purchased from Avra Synthesis. In addition, aldehydes such as pyridine-3-carbaldehyde, pyridine-4-carbaldehyde, piperonal, 4-phenylbenzaldehyde, 3-nitrobenzaldehyde, and octanal were procured from Sigma, while 4-chlorobenzaldehyde and 4-(trifluoromethyl)benzaldehyde were procured from CDH and TCI, respectively. Furthermore, isonitriles—tert-butyl isonitrile, 1,1,3,3-tetramethylbutyl isonitrile, 4-(2-isocyanoethyl)morpholine isonitrile, and 4-methoxyphenyl isonitrile—were procured from Sigma. The catalyst HCL in dioxane was obtained from Sigma. The bulk solvents such as hexanes, ethyl acetate, dichloromethane, and MeOH were distilled before use. TLC was performed on Merck silica gel F254 aluminum sheets and viewed under UV light at 254 or 360 nm. Compounds were purified by column chromatography using a 230–400 mesh silica gel, and a MeOH–dichloromethane system was used as the mobile phase. 1H and 13C NMR spectra were recorded on Bruker Avance-II 400 or 500 MHz NMR spectrometers in CDCl3. Chemical shifts were reported in parts per million (ppm) relative to tetramethylsilane (TMS) as the internal standard. The abbreviations used in reporting spectra were as follows: s (singlet), d (doublet), t (triplet), m (multiple), dt (doublet of triplet), td (triplet of doublet), ddd (doublet of doublet of doublet), and br (broad). HPLC (Agilent 1260 Infinity-II LC system, Serial number DEABG06552; Column: Agilent 5 HC-C18(2) 150 × 4.6 mm) was used to confirm the purity (>98%) of the synthesized compounds. MS was reported on an Agilent 1290 LC/MSD single quad system or on Waters Alliance 2795, Q-TOF Micromass spectrometer. The Synthos 3000 MW reactor by Anton Paar was used for the GBB-MCRs.

4.1.2. Synthesis and Characterization of Compounds

General Procedure for the GBB Reaction

Amidine (200 mg, 1 equiv) was dissolved in acetonitrile (3 mL) in a microwave vessel, and aldehyde (1.5 equiv) was added to the reaction mixture followed by isonitrile (1.1 equiv). Catalytic amounts of HCL in dioxane (4M) were added to the reaction mixture. The reaction was allowed to occur in a microwave at 110 °C for 20 min in power-controlled mode (900 W). The crude compounds were concentrated on a rotary evaporator and subjected to column chromatography purification. The compounds were eluted with MeOH/DCM (0–5%) using a 230–400 mesh silica gel.

N-(2-morpholinoethyl)-2-(pyridin-3-yl)imidazo[1,2-a]pyridin-3-amine (6)

1H NMR (500 MHz, CDCl3) δ 9.27 (dd, J = 2.2, 0.7 Hz, 1H), 8.54 (dd, J = 4.8, 1.6 Hz, 1H), 8.41–8.29 (m, 1H), 8.12 (dt, J = 6.9, 1.0 Hz, 1H), 7.55 (d, J = 9.1 Hz, 1H), 7.37 (ddd, J = 7.9, 4.8, 0.7 Hz, 1H), 7.16 (ddd, J = 9.0, 6.7, 1.2 Hz, 1H), 6.82 (td, J = 6.8, 1.0 Hz, 1H), 3.97 (s, 1H), 3.80–3.62 (m, 4H), 3.22–2.99 (m, 2H), 2.61–2.50 (m, 2H), 2.50–2.33 (m, 4H). 13C NMR (126 MHz, CDCl3) δ 148.1, 148.0, 141.9, 134.3, 132.2, 130.5, 127.0, 124.3, 123.6, 122.4, 117.6, 112.0, 66.9, 58.30, 53.7, 44.2. MS-ESI [(M+H)+]: m/z calculated for C18H22N5O+: 324.2; found: 324.2.

N-(2-morpholinoethyl)-2-(pyridin-4-yl)imidazo[1,2-a]pyridin-3-amine (7)

1H NMR (500 MHz, CDCl3) δ 8.63 (dd, J = 4.6, 1.6 Hz, 2H), 8.11 (dt, J = 6.9, 1.1 Hz, 1H), 7.98 (dd, J = 4.6, 1.6 Hz, 2H), 7.56 (dt, J = 9.0, 0.9 Hz, 1H), 7.17 (ddd, J = 9.1, 6.6, 1.3 Hz, 1H), 6.82 (td, J = 6.8, 1.0 Hz, 1H), 4.00 (s, 1H), 3.82–3.66 (m, 4H), 3.16–3.08 (m, 2H), 2.59–2.55 (m, 2H), 2.53–2.42 (m, 4H). 13C NMR (126 MHz, CDCl3) δ 150.0, 142.0, 141.8, 131.8, 128.4, 124.7, 122.4, 120.9, 117.9, 112.2, 67.0, 58.3, 53.7, 44.1. MS-ESI [(M+H)+]: m/z calculated for C18H22N5O+: 324.2; found: 324.2.

2-(3-((2-morpholinoethyl)amino)imidazo[1,2-a]pyridin-2-yl)phenol (8)

1H NMR (500 MHz, CDCl3) δ 13.01 (s, 1H), 8.10 (dt, J = 6.9, 1.1 Hz, 1H), 7.92 (dd, J = 7.8, 1.6 Hz, 1H), 7.47–7.38 (m, 1H), 7.17–7.04 (m, 2H), 6.96 (dd, J = 8.2, 1.2 Hz, 1H), 6.87–6.70 (m, 2H), 3.81 (s, 1H), 3.75–3.56 (m, 4H), 3.03 (s, 2H), 2.64–2.50 (m, 2H), 2.45 (s, 4H). 13C NMR (126 MHz, CDCl3) δ: 158.0, 139.2, 134.2, 129.0, 125.7, 125.1, 124.5, 122.1, 118.7, 117.7, 117.3, 116.7, 112.3, 67.0, 58.5, 53.8, 43.8. MS-ESI [(M+H)+]: m/z calculated for C19H23N4O2+: 339.1816; found: 339.1961.

2-(3-methoxyphenyl)-N-(2-morpholinoethyl)imidazo[1,2-a]pyridin-3-amine (9)

1H NMR (500 MHz, CDCl3) δ 8.01 (d, J = 6.8 Hz, 1H), 7.57–7.39 (m, 3H), 7.25 (t, J = 7.9 Hz, 1H), 7.03 (ddd, J = 9.0, 6.7, 1.2 Hz, 1H), 6.84–6.74 (m, 1H), 6.69 (td, J = 6.8, 0.9 Hz, 1H), 3.99 (s, 1H), 3.79 (s, 3H), 3.66–3.56 (m, 4H), 3.04–2.89 (m, 2H), 2.44 (dd, J = 6.2, 4.8 Hz, 2H), 2.34 (s, 4H). 13C NMR (126 MHz, CDCl3) δ: 159.9, 141.2, 135.6, 134.6, 129.5, 126.7, 123.9, 122.4, 119.3, 117.4, 113.0, 112.7, 111.7, 66.9, 58.3, 55.3, 53.7, 44.0. MS-ESI [(M+H)+]: m/z calculated for C20H25N4O2+: 353.1972; found: 353.2070.

2-(benzo[d][1,3]dioxol-5-yl)-N-(2-morpholinoethyl)imidazo[1,2-a]pyridin-3-amine (10)

1H NMR (500 MHz, CDCl3) δ 8.09 (d, J = 6.8 Hz, 1H), 7.73–7.37 (m, 3H), 7.13 (ddd, J = 9.0, 6.7, 1.2 Hz, 1H), 6.89 (d, J = 8.1 Hz, 1H), 6.79 (td, J = 6.8, 1.0 Hz, 1H), 5.99 (s, 2H), 3.94 (s, 1H), 3.83–3.48 (m, 4H), 3.20–2.92 (m, 2H), 2.56 (dd, J = 6.3, 4.8 Hz, 2H), 2.47 (s, 4H).13C NMR (126 MHz, CDCl3) δ 148.0, 147.0, 141.1, 134.7, 128.4, 125.7, 123.8, 122.3, 120.8, 117.3, 111.7, 108.5, 107.5, 101.1, 67.0, 58.3, 53.7, 43.9, 29.7. MS-ESI [(M+H)+]: m/z calculated for C20H23N4O3+: 367.1765; found: 367.1915.

N-(2-morpholinoethyl)-2-(4-(trifluoromethyl)phenyl)imidazo[1,2-a]pyridin-3-amine (11)

1H NMR (500 MHz, CDCl3) δ 8.17 (d, J = 8.1 Hz, 2H), 8.10 (d, J = 6.9 Hz, 1H), 7.67 (d, J = 8.2 Hz, 2H), 7.55 (d, J = 9.1 Hz, 1H), 7.15 (ddd, J = 9.0, 6.6, 1.2 Hz, 1H), 6.80 (td, J = 6.8, 1.0 Hz, 1H), 3.95 (s, 1H), 3.84–3.60 (m, 4H), 3.08 (s, 2H), 2.68–2.50 (m, 2H), 2.49–2.26 (m, 4H). 13C NMR (126 MHz, CDCl3) δ 141.6, 138.1, 133.4, 131.0, 129.3, 129.0, 128.8, 128.5, 127.5, 127.3, 126.9, 125.5, 125.4, 125.4, 125.4, 124.3, 123.2, 122.4, 117.8, 111.9, 67.0, 58.3, 53.7, 44.1. MS-ESI [(M+H)+]: m/z calculated for C20H22F3N4O+: 391.2; found: 391.2.

3-(3-((2-morpholinoethyl)amino)imidazo[1,2-a]pyridin-2-yl)benzonitrile (12)

1H NMR (500 MHz, CDCl3) δ 8.41 (t, J = 1.4 Hz, 1H), 8.33 (dt, J = 7.7, 1.5 Hz, 1H), 8.12 (dt, J = 6.9, 1.0 Hz, 1H), 7.63–7.45 (m, 3H), 7.17 (ddd, J = 9.0, 6.7, 1.2 Hz, 1H), 6.82 (td, J = 6.8, 1.0 Hz, 1H), 3.87 (s, 1H), 3.79–3.63 (m, 4H), 3.11 (s, 2H), 2.67–2.53 (m, 2H), 2.53–2.35 (m, 4H). 13C NMR (126 MHz, CDCl3) δ 141.7, 135.9, 132.8, 130.9, 130.4, 130.3, 129.3, 127.0, 124.5, 122.4, 119.0, 117.8, 112.7, 112.1, 67.0, 58.4, 53.7, 44.1. MS-ESI [(M+H)+]: m/z calculated for C20H22N5O+: 348.2; found: 348.2.

2-(benzo[d][1,3]dioxol-5-yl)-N-(2,4,4-trimethylpentan-2-yl)imidazo[1,2-a]pyrimidin-3-amine (14)

1H NMR (500 MHz, CDCl3) δ 8.50 (dd, J = 6.8, 2.0 Hz, 1H), 8.46 (dd, J = 4.1, 2.0 Hz, 1H), 7.47 (d, J = 1.6 Hz, 1H), 7.39 (dd, J = 8.0, 1.7 Hz, 1H), 6.88 (dd, J = 7.9, 3.4 Hz, 1H), 6.82 (dd, J = 6.8, 4.1 Hz, 1H), 6.00 (s, 2H), 3.21 (s, 1H), 1.58 (s, 2H), 1.04 (s, 9H), 0.98 (s, 6H). 13C NMR (126 MHz, CDCl3) δ: 149.1, 147.7, 147.3, 144.9, 141.3, 131.0, 128.8, 122.4, 121.2, 109.2, 108.2, 107.7, 101.1, 60.9, 57.0, 31.8, 31.7, 29.0. ESI [(M+H)+]: m/z calculated for C21H27N4O2+: 367.2129; found: 367.2319.

2-(benzo[d][1,3]dioxol-5-yl)-N-(4-methoxyphenyl)imidazo[1,2-a]pyrimidin-3-amine (15)

1H NMR (500 MHz, CDCl3) δ 8.50 (dd, J = 4.1, 2.0 Hz, 1H), 8.08 (dd, J = 6.7, 2.1 Hz, 1H), 7.62–7.58 (m, 2H), 6.81–6.75 (m, 4H), 6.53 (d, J = 9.0 Hz, 2H), 5.95 (s, 2H), 5.55 (s, 1H), 3.74 (s, 3H). 13C NMR (126 MHz, CDCl3) δ: 154.0, 150.1, 148.0, 147.9, 145.5, 140.7, 137.8, 130.4, 127.1, 121.7, 116.7, 115.6, 114.7, 108.7, 108.5, 107.9, 101.2, 55.8. MS-ESI [(M+H)+]: m/z calculated for C20H17N4O3+: 361.1295; found: 361.1496.

2-(benzo[d][1,3]dioxol-5-yl)-N-(tert-butyl)imidazo[1,2-a]pyrimidin-3-amine (16)

1H NMR (500 MHz, CDCl3) δ 8.49 (dd, J = 6.8, 2.1 Hz, 1H), 8.45 (dd, J = 4.0, 2.0 Hz, 1H), 7.54 (d, J = 1.6 Hz, 1H), 7.49 (dd, J = 8.1, 1.7 Hz, 1H), 6.86 (d, J = 8.1 Hz, 1H), 6.80 (dd, J = 6.8, 4.1 Hz, 1H), 6.00 (s, 2H), 3.14 (s, 1H), 1.06 (s, 9H). 13C NMR (126 MHz, CDCl3) δ: 149.2, 147.6, 147.3, 144.9, 140.9, 130.9, 128.6, 122.3, 121.3, 108.9, 108.2, 107.7, 101.1, 56.6, 30.4. MS-ESI [(M+H)+]: calculated for C17H19N4O2+: 311.1: found: 311.1.

2-(benzo[d][1,3]dioxol-5-yl)-N-(2-morpholinoethyl)imidazo[1,2-a]pyrimidin-3-amine (17)

1H NMR (500 MHz, CDCl3) δ 8.95 (d, J = 1.4 Hz, 1H), 7.99 (dd, J = 4.6, 1.5 Hz, 1H), 7.83 (d, J = 4.6 Hz, 1H), 7.61–7.41 (m, 2H), 6.91 (d, J = 8.5 Hz, 1H), 6.02 (s, 2H), 4.12 (s, 1H), 3.83–3.59 (m, 4H), 3.10 (s, 2H), 2.64–2.51 (m, 2H), 2.47 (s, 4H). 13C NMR (126 MHz, CDCl3) δ: 148.2, 147.6, 143.2, 137.2, 136.4, 128.9, 127.6, 127.3, 121.2, 115.1, 108.7, 107.6, 101.3, 66.9, 58.2, 53.7, 43.6, 29.7. MS-ESI [(M+H)+]: m/z calculated for C19H22N5O3+: 368.1717; found: 368.1483.

2-phenyl-N-(2,4,4-trimethylpentan-2-yl)imidazo[1,2-a]pyrimidin-3-amine (18)

1H NMR (500 MHz, CDCl3) δ 8.54 (dd, J = 6.8, 2.0 Hz, 1H), 8.48 (dd, J = 4.0, 2.0 Hz, 1H), 7.96–7.83 (m, 2H), 7.44 (t, J = 7.6 Hz, 2H), 7.34 (t, J = 7.4 Hz, 1H), 6.83 (dd, J = 6.8, 4.1 Hz, 1H), 3.36 (s, 1H), 1.56 (s, 2H), 1.02 (s, 9H), 0.95 (s, 6H). 13C NMR (126 MHz, CDCl3) δ: 149.3, 145.1, 141.5, 134.7, 131.2, 128.6, 128.3, 127.9, 121.8, 107.8, 60.9, 56.9, 31.8, 31.7, 29.0. MS-ESI [(M+H)+]: m/z calculated for C20H27N4+: 323.2230; found: 323.2.

2-(4-chlorophenyl)-N-(2,4,4-trimethylpentan-2-yl)imidazo[1,2-a]pyrimidin-3-amine (19)

1H NMR (400 MHz, CDCl3) δ 8.27 (dd, J = 6.6, 1.9 Hz, 1H), 8.23 (dd, J = 4.3, 1.9 Hz, 1H), 7.53–7.46 (m, 2H), 7.43–7.36 (m, 2H), 6.71 (dd, J = 6.6, 4.4 Hz, 1H), 4.23 (s, 1H), 1.89 (s, 2H), 1.56 (s, 6H), 0.98 (s, 9H). 13C NMR (126 MHz, CDCl3) δ: 149.6, 145.1, 140.3, 133.7, 133.2, 131.2, 129.8, 128.5, 121.8, 107.9, 61.0, 57.0, 31.8, 31.7, 29.1. MS-ESI [(M+H)+]: m/z calculated for C20H26ClN4+: 357.1; found: 357.1.

2-([1,1′-biphenyl]-4-yl)-N-(2,4,4-trimethylpentan-2-yl)imidazo[1,2-a]pyrimidin-3-amine (20)

1H NMR (500 MHz, CDCl3) δ 8.54 (dd, J = 6.8, 2.0 Hz, 1H), 8.49 (dd, J = 4.0, 2.0 Hz, 1H), 8.02 (d, J = 8.4 Hz, 2H), 7.81–7.57 (m, 4H), 7.46 (t, J = 7.7 Hz, 2H), 7.35 (t, J = 7.4 Hz, 1H), 6.83 (dd, J = 6.8, 4.1 Hz, 1H), 3.32 (s, 1H), 1.60 (s, 2H), 1.04 (s, 9H), 0.99 (s, 6H). 13C NMR (126 MHz, CDCl3) δ: 149.3, 145.2, 141.1, 140.7, 140.5, 133.7, 131.1, 129.0, 128.8, 127.4, 127.0, 126.9, 121.9, 107.8, 61.0, 57.0, 31.9, 31.7, 29.1. MS-ESI [(M+H)+]: m/z calculated for C26H31N4+: 399.2; found: 399.3.

2-(3-methoxyphenyl)-N-(2,4,4-trimethylpentan-2-yl)imidazo[1,2-a]pyrimidin-3-amine (21)

1H NMR (400 MHz, CDCl3) δ 8.53 (dd, J = 6.8, 2.1 Hz, 1H), 8.49 (dd, J = 4.1, 2.1 Hz, 1H), 7.53 (dd, J = 2.5, 1.5 Hz, 1H), 7.46–7.40 (m, 1H), 7.34 (t, J = 7.9 Hz, 1H), 6.90 (ddd, J = 8.2, 2.6, 1.0 Hz, 1H), 6.84 (dd, J = 6.8, 4.1 Hz, 1H), 3.88 (s, 3H), 3.29 (s, 1H), 1.58 (s, 2H), 1.03 (s, 9H), 0.96 (s, 6H). 13C NMR (126 MHz, CDCl3) δ: 159.7, 149.3, 145.0, 141.3, 136.1, 131.2, 129.1, 121.9, 121.0, 114.2, 113.7, 107.8, 60.9, 56.9, 55.4, 31.8, 31.7, 29.0. MS-ESI [(M+H)+]: m/z calculated for C21H29N4O+: 353.2; found: 353.2.

2-methoxy-5-(3-((2,4,4-trimethylpentan-2-yl)amino)imidazo[1,2-a]pyrimidin-2-yl)phenol (22)

1H NMR (500 MHz, CDCl3) δ 8.51 (dd, J = 6.8, 2.0 Hz, 1H), 8.47 (dd, J = 4.0, 2.0 Hz, 1H), 7.53 (d, J = 2.0 Hz, 1H), 7.42 (dd, J = 8.3, 2.0 Hz, 1H), 6.91 (d, J = 8.4 Hz, 1H), 6.83 (dd, J = 6.8, 4.1 Hz, 1H), 3.92 (s, 3H), 3.29 (s, 1H), 1.57 (s, 2H), 1.03 (s, 9H), 0.96 (s, 6H). 13C NMR (126 MHz, CDCl3) δ: 149.0, 146.7, 145.5, 144.9, 141.3, 131.1, 127.9, 121.4, 120.7, 115.1, 110.7, 107.7, 60.9, 56.9, 55.9, 31.8, 31.7, 29.0. MS-ESI [(M+H)+]: m/z calculated for C21H29N4O2+: 369.2; found: 369.2.

2-(3-nitrophenyl)-N-(2,4,4-trimethylpentan-2-yl)imidazo[1,2-a]pyrimidin-3-amine (23)

1H NMR (400 MHz, CDCl3) δ 9.02–8.93 (m, 1H), 8.59–8.50 (m, 2H), 8.50–8.42 (m, 1H), 8.18 (ddd, J = 8.2, 2.3, 1.0 Hz, 1H), 7.62 (t, J = 8.0 Hz, 1H), 6.90 (dd, J = 6.7, 4.1 Hz, 1H), 3.19 (s, 1H), 1.65 (s, 2H), 1.05 (s, 9H), 1.02 (s, 6H). MS-ESI [(M+H)+]: m/z calculated for C20H26N5O2+: 368.2; found: 368.2.

3-(3-((2,4,4-trimethylpentan-2-yl)amino)imidazo[1,2-a]pyrimidin-2-yl)benzonitrile (24)

1H NMR (500 MHz, CDCl3) δ 8.59–8.47 (m, 2H), 8.36 (s, 1H), 8.28 (d, J = 7.8 Hz, 1H), 7.61 (d, J = 7.7 Hz, 1H), 7.55 (t, J = 7.8 Hz, 1H), 6.89 (dd, J = 6.5, 4.3 Hz, 1H), 3.22 (s, 1H), 1.60 (s, 2H), 1.04 (s, 9H), 0.99 (s, 6H). 13C NMR (126 MHz, CDCl3) δ: 150.2, 145.3, 139.1, 136.1, 132.7, 132.0, 131.3, 131.2, 129.2, 122.2, 118.8, 112.3, 108.3, 61.0, 57.1, 31.8, 31.7, 29.2. MS-ESI [(M+H)+]: m/z calculated for C21H26N5+: 348.2; found: 348.2.

2-(pyridin-3-yl)-N-(2,4,4-trimethylpentan-2-yl)imidazo[1,2-a]pyrimidin-3-amine (25)

1H NMR (400 MHz, CDCl3) δ 9.19 (d, J = 1.3 Hz, 1H), 8.58 (dd, J = 4.7, 1.3 Hz, 1H), 8.56–8.47 (m, 2H), 8.35 (dt, J = 7.9, 1.7 Hz, 1H), 7.41 (dd, J = 7.8, 4.8 Hz, 1H), 6.88 (dd, J = 6.6, 4.2 Hz, 1H), 3.28 (s, 1H), 1.59 (s, 2H), 1.03 (s, 9H), 0.99 (s, 6H). 13C NMR (126 MHz, CDCl3) δ: 149.9, 149.2, 148.7, 145.5, 138.5, 135.9, 131.3, 130.9, 123.5, 122.3, 108.1, 60.9, 57.0, 31.8, 31.7, 29.1. MS-ESI [(M+H)+]: m/z calculated for C19H26N5+: 324.2; found: 324.2.

2-(thiophen-2-yl)-N-(2,4,4-trimethylpentan-2-yl)imidazo[1,2-a]pyrimidin-3-amine (26)

1H NMR (400 MHz, CDCl3) δ 8.54–8.33 (m, 2H), 7.64 (dd, J = 3.6, 1.1 Hz, 1H), 7.35 (dd, J = 5.1, 1.1 Hz, 1H), 7.11 (dd, J = 5.1, 3.6 Hz, 1H), 6.81 (dd, J = 6.8, 4.1 Hz, 1H), 3.25 (s, 1H), 1.69 (s, 2H), 1.12 (s, 6H), 1.08 (s, 9H). MS-ESI [(M+H)+]: m/z calculated for C18H25N4S+: 329.1: found: 329.1.

2-butyl-N-(2,4,4-trimethylpentan-2-yl)imidazo[1,2-a]pyrimidin-3-amine (27)

1H NMR (400 MHz, CDCl3) δ 8.54–8.28 (m, 2H), 6.77 (dd, J = 6.7, 4.1 Hz, 1H), 3.32 (s, 1H), 2.82–2.69 (m, 2H), 1.79 (tt, J = 7.8, 6.8 Hz, 2H), 1.65 (s, 2H), 1.47–1.31 (m, 2H), 1.15 (s, 6H), 1.08 (s, 9H), 0.93 (t, J = 7.4 Hz, 3H). ESI [(M+H)+]: m/z calculated for C18H31N4+: 303.2: found: 303.2.

2-octyl-N-(2,4,4-trimethylpentan-2-yl)imidazo[1,2-a]pyrimidin-3-amine (28)

1H NMR (400 MHz, CDCl3) δ 8.50–8.31 (m, 2H), 6.79 (dd, J = 6.5, 4.3 Hz, 1H), 3.95 (s, 1H), 2.81–2.70 (m, 2H), 1.85–1.75 (m, 2H), 1.66 (s, 2H), 1.35–1.23 (m, 10H), 1.15 (s, 6H), 1.09 (s, 9H), 0.86 (t, J = 6.9 Hz, 3H). 13C NMR (126 MHz, CDCl3) δ 148.4, 145.1, 143.8, 130.7, 121.5, 107.4, 59.8, 56.7, 31.9, 31.9, 31.8, 31.6, 31.4, 31.4, 29.9, 29.5, 29.3, 29.3, 29.2, 28.0, 22.7, 14.1. ESI [(M+H)+]: m/z calculated for C22H39N4+: 359.3; found: 359.3.

4.2. Biological Experiments

4.2.1. Parasites

L. amazonensis-wild type (IFLA/BR/67/PH8) promastigotes were cultured in Warren’s medium (brain–heart infusion plus hemin and folic acid), enriched with 10% fetal bovine serum (FBS, CultiLab). L. amazonensis(IFLA/BR/67/PH8) promastigotes transfected with the red fluorescent protein (RFP) were kept in 199 medium (Himedia AT014) supplemented with hemin, FBS, MEM vitamin solution (Thermo Fisher 11120052), and penicillin and streptomycin solution. The cells were maintained in BOD incubators at 25 °C.

4.2.2. Antileishmanial Activity against Promastigotes of L. amazonensis

The toxicity of imidazo-pyrimidine analogues in L. amazonensis promastigotes was assessed by the colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolic bromide (MTT) method (Sigma Chemical Co., St. Louis, MO, United States).36 Briefly, promastigotes in the logarithmic growth phase were adjusted to a concentration of 2 × 106 cells/mL in a culture medium. The parasites were exposed to different concentrations of the compounds (6.25–100 μM), for 72 h at 25 °C. Then, the MTT solution (5 mg/mL) was added, and after 4 h of incubation, the plates were read on a spectrophotometer at 570 nm. Untreated promastigotes were used as the negative control, and miltefosine was used as the reference drug. The percentage of viable parasites was determined by comparing treated cells with the untreated control. The IC50 was calculated using the GraFit5 software (Erithacus Software Ltd., Horley, United Kingdom), using values obtained from three independent experiments.

4.2.3. Antileishmanial Activity against Amastigotes of L. amazonensis

Antileishmanial activity of compounds was evaluated in intracellular amastigotes of L. amazonensis RFP, by the fluorimetry method. To achieve this, macrophages were obtained from the peritoneal cavity of BALB/C mice after stimulus with thioglycolate medium (3%). Then, adherent macrophages were infected with stationary phase L. amazonensisRFP promastigotes at a ratio of 10:1 (parasite per cell). After 4h, the wells were washed to eliminate nonphagocyted parasites and treated with different concentrations of the compounds (12.5–100 μM). After 72 h of incubation at 33 °C with 5% CO2, the cells were lysed with deionized water, and the contents of the wells were transferred to black 96-well transparent bottom plates. The plates were read on a fluorimeter at corresponding wavelengths of 540/600 nm of excitation and emission. Untreated macrophages were used as the negative control, and miltefosine was used as the reference drug. The values were expressed in an IC50 of cell growth calculated using the average of three independent experiments. This protocol was approved by the Ethics Committee for Animal Research (CEUA) of Federal University of Juiz de Fora (no. 008/2018-CEUA).

4.2.4. Cytotoxicity in Murine Peritoneal Macrophages

The cytotoxicity of imidazo-pyrimidine analogues was determined by the MTT colorimetric method. Peritoneal macrophages were obtained as describe above. Then, adherent cells (2 × 106 cells/mL) were treated with different concentrations (6.25–150 μM) of the compounds. After 72 h, the cells were incubated with MTT (5 mg/mL) for 2 h, and then, the plates were read on a spectrophotometer at 570 nm. Untreated macrophages were used as negative controls, and miltefosine was used as the reference drug. The results were expressed in a CC50 of cell viability. This protocol was approved by the CEUA of Federal University of Juiz de Fora (no. 007/2018-CEUA).

4.3. In Silico Physicochemical, Pharmacokinetic, and Target Studies

The SMILE notation of compound 24 was obtained using ChemSketch software (ACD/Laboratories 2020.1.2). The SMILE notation was inserted into the free online platform PKCSM available at the address http://biosig.unimelb.edu.au/pkcsm/ to obtain data on physicochemical properties such as logP, molecular mass, HBD, and HBA sites. The results obtained were evaluated according to the Lipinski’s rule of five (logP ≤ 5; MW ≤ 500, HBA ≤ 5, and HBD ≤ 10). The ADMET profile was evaluated using the AdmetSAR software.32

Acknowledgments

Deepak B. Salunke is thankful to DBT New Delhi for the award of the Ramalingaswami Fellowship (BT/RLF/Re-entry/16/2013) and Science and Engineering Research Board (SERB), New Delhi for the Core Research Grant (CRG/2021/005467), Department of Science & Technology & Renewable Energy, Chandigarh Administration, Paryavaran Bhawan, Chandigarh for Short Term Research Project, and Indian Council of Medical Research, New Delhi for the ad hoc research project. M.T.P. is thankful to DST for the award of Women Scientist Scheme-A [SR/WOS-A/CS-132/2016 (G)]. SAIF/CIL of Panjab University is gratefully acknowledged. We are grateful to the Brazilian funding agencies, Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, Brazil), Fundação de Amparo a Pesquisa de Minas Gerais (FAPEMIG, Brazil), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES, Brazil), and Universidade Federal de Juiz de Fora (UFJF, Brazil). E.S.C. and A.D.S. are research productivity fellows from CNPq. The authors are grateful to the Center for Reproduction Biology (CBR/UFJF) for the animals.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.3c05441.

  • Copies of 1H NMR, 13C NMR, MS analysis for the synthesized compounds, HPLC purity analysis of the selected lead compounds and percent cell viability of compounds 18, 22, 24, and 25 measured using an MTT assay (PDF)

The authors declare no competing financial interest.

Supplementary Material

ao3c05441_si_001.pdf (3.2MB, pdf)

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