Antiparasitic Activity of Fluorophenyl-Substituted Pyrimido[1,2-a]benzimidazoles

A series of fourteen pyrimido[1,2-a]benzimidazole compounds was prepared by straightforward heterocyclic chemistry and oxidation methods. The new pyrimidobenzimidazole derivative 2a with a 3-fluorophenyl substituent was identified as a new antiparasitic compound showing excellent activities against Leishmania major parasites. 2a was highly active against L. major promastigotes and amastigotes with EC50 values in the nanomolar concentration range. Compound 3b was less active than 2a against L. major, but more active against Toxoplasma gondii with considerable selectivity. Hence, two promising and selective antiparasitic drug candidates 2a and 3b for the treatment of two parasitic diseases were identified, which can be prepared by green chemistry methods using simple one-pot reactions and oxidation procedures, respectively.


Introduction
Heterocyclic organic compounds play a crucial role for the design of new drugs for the treatment of various human diseases. N-Heterocyclic drugs such as Vinca alkaloids, camptothecins and nucleotides (e.g., 5-fluorouracil) are currently applied for the treatment of cancer diseases [1]. Azacycles also perform well against numerous life-threatening infectious diseases. Ribavirin, zidovudine, remdesivir, and paxlovid are prominent examples of antiviral drugs [2]. In terms of protozoal parasite infections, quinoline alkaloids such as quinine, chloroquine, and mefloquine are applied for the treatment of malaria, while the nitroimidazole metronidazole is clinically used on patients suffering from amoebiasis or giardiasis [3,4]. Benznidazole (a drug against Chagas disease) and fexinidazol (for the treatment of sleeping sickness) are further antiparasitic imidazoles [5]. The related benzimidazole scaffold acts as an isoster of natural purine bases and is a crucial component of anthelmintic (albendazole, mebendazole) and antiviral drugs (maribavir) representing a prolific field of medicinal chemistry research [6]. Pyrimidobenzimidazoles are a class of N-heterocycles with fused benzimidazole and pyrimidine moieties. Members of this class exhibited antibacterial, antimalarial, anticancer, and antioxidant activities, and the synthesis of pyrimido[1,2-a]benzimidazole derivatives via atom economic multicomponent reactions appears to be especially promising for the design of new drug candidates against various human diseases [7].
Leishmaniasis is as a neglected tropical disease (NTD), and cutaneous leishmaniasis (CL) caused by various Leishmania species (e.g., L. major, L. tropica, L. mexicana, L. amazonensis, etc.) is the dominant leishmaniasis form with up to 1 million, mostly young, cases every year [8,9]. CL leads to painful and stigmatizing skin lesions, and most patients suffering from CL receive pentavalent antimonials, miltefosine, amphotericin, or pentamidine [9][10][11]. While some of these drugs are toxic (e.g., the antimonials), the formation of drug resistance poses another increasing problem, which requires the development of new antileishmanial agents. Toxoplasmosis is another neglected parasitic disease caused by apicomplexan Toxoplasma gondii parasites, which leads to severe infections among immune-compromised people and also demands the development of new drugs [12].
The pyrimidobenzimidazoles 1a-d were reported to possess moderate anticancer activities ( Figure 1) [13]. In the present report, we disclose a series of pyrimidobenzimidazoles and their promising activities against L. major and T. gondii parasites.

Chemistry
The known compounds 2c, 2d, and 3d were prepared following literature procedures and analyzed (see supplementary materials Figures S1-S33). The obtained analytical data were in line with published data of these compounds [13,14]. Synthetic procedures and analytical data of the new derivatives are given below. All starting compounds were purchased from the usual retailers and used without further purification. Column chromatography: silica gel 60 (230-400 mesh). Melting points (uncorrected), Electrothermal 9100; NMR spectra, Bruker Avance 300/500 spectrometer; chemical shifts are given in parts per million (δ) downfield from tetramethylsilane as internal standard; Mass spectra, Thermo Finnigan MAT 8500 (EI), UPLC/Orbitrap (ESI-HRMS).

Chemistry
The known compounds 2c, 2d, and 3d were prepared following literature procedures and analyzed (see Supplementary Materials Figures S1-S33). The obtained analytical data were in line with published data of these compounds [13,14]. Synthetic procedures and analytical data of the new derivatives are given below. All starting compounds were purchased from the usual retailers and used without further purification. Column chromatography: silica gel 60 (230-400 mesh). Melting points (uncorrected), Electrothermal 9100; NMR spectra, Bruker Avance 300/500 spectrometer; chemical shifts are given in parts per million (δ) downfield from tetramethylsilane as internal standard; Mass spectra, Thermo Finnigan MAT 8500 (EI), UPLC/Orbitrap (ESI-HRMS).

Leishmania Major Cell Isolation, Culture Conditions, and Assays
Promastigotes of L. major were isolated from a Saudi patient (February 2016) and maintained, at 26 • C, in Schneider's Drosophila medium (Invitrogen, Carlsbad, CA, USA) containing 10% heat inactivated fetal bovine serum (FBS, Invitrogen, USA) and antibiotics in a tissue culture flask with weekly transfers. Promastigotes were cryopreserved in liquid nitrogen at concentrations of 3 × 10 6 parasite/mL. Virulent L. major parasites were maintained by passing in female BALB/c mice by injecting hind footpads with 1 × 10 6 stationary-phase promastigotes. L. major amastigotes were isolated from the mice after 8 weeks. Isolated amastigotes were converted to promastigotes by cultivation, at 26 • C, in Schneider's medium supplemented with antibiotics and 10% FBS. Amastigotederived promastigotes, which had undergone less than five in vitro passages, were used for infection. BALB/c mice (male and female individuals) were obtained from Pharmaceutical College, King Saud University, Kingdom of Saudi Arabia, and maintained in specific pathogen-free facilities. The 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.
L. major promastigotes from logarithmic-phase were cultured in phenol red-free RPMI 1640 medium (Invitrogen, USA) with 10% FBS and suspended on 96-wells plates to yield were analyzed by using an ELISA reader at 570 nm. EC 50 values were calculated from three independent experiments [15].
For evaluation of the activity against amastigotes in macrophages, peritoneal macrophages were collected from female BALB/c mice (6-8 weeks of age) by aspiration. A total of 5 × 10 4 cells/well were placed into 96-well plates containing phenol red-free RPMI 1640 medium with 10% FBS and were incubated to promote cell adhesion, at 37 • C, for 4 h in 5% CO 2 atmosphere. Thereafter, the medium was discarded, and the cells were washed with phosphate-buffered saline (PBS). L. major promastigotes solution (200 µL at a ratio of 10 promastigotes:1 macrophage in RPMI 1640 medium with 10% FBS) was added to each well, and the plates were incubated for 24 h, at 37 • C, in humidified 5% CO 2 atmosphere to enable macrophage infection and amastigote differentiation. The infected cells were washed three times with PBS to remove the free promastigotes and overlaid with fresh phenol red-free RPMI 1640 medium containing test compounds (50, 25, 12.5, 6.25, 3.13, 1.65, and 0.75 µg mL −1 ), whereupon the cells were incubated, at 37 • C, for 72 h in humidified 5% CO 2 atmosphere. Cultures solely containing DMSO (1%) were used as negative controls, while wells containing cultures with decreasing concentrations of amphotericin B (reference compound, 50, 25, 12.5, 6.25, 3.13, 1.65, and 0.75 µg mL −1 ) were used as positive control. The percentage of infected macrophages was evaluated microscopically after the removal of the medium, washing, fixation, and Giemsa staining of the cells. Calculated EC 50 values were obtained from three independent experiments [15].

Toxoplasma Gondii Cell Line, Culture Conditions, and Assay
Serial passages of cells of the Vero cell line (ATCC ® CCL81™, Manassas, VA, USA) were applied for the cultivation of T. gondii tachyzoites of the RH strain (a gift from Dr. Saeed El-Ashram, State Key Laboratory for Agrobiotechnology, China Agricultural University, Beijing, China). Vero cells were cultured by using complete RPMI 1640 medium with heat-inactivated 10% FBS in a humidified 5% CO 2 atmosphere, at 37 • C. The 96-well plates (5 × 10 3 cells/well in 200 µL RPMI 1640 medium) were used for the cultivation of the Vero cells and then incubated, at 37 • C, and 5% CO 2 for one day, followed by removal of medium and washing the cells with PBS. Then, RPMI 1640 medium containing 2% FBS and T. gondii tachyzoites (RH strain) was given to the cells at a ratio of 5 (parasite):1 (Vero cells). After incubation, at 37 • C, and 5% CO 2 for 5 h, cells were washed with PBS and then treated as described below.
After incubation, at 37 • C, and 5% CO 2 for 72 h, the cells were washed with PBS, fixed in 10% formalin and stained with 1% toluidine blue. Examination of the cells and determination of the infection index (number of cells infected from 200 cells tested) of T. gondii were carried out with an inverted photomicroscope. The following equation was used for the calculation of the inhibition in %: where "I Control" refers to the infection index of untreated cells and "I Experimental" refers to the infection index of cells treated with test compounds.
Then, effects of test compounds on parasite growth were expressed as EC 50 (effective concentration at 50%) values. EC 50 values were obtained from three independent experiments [15,16].

In Vitro Cytotoxicity Assay
MTT assays were carried out for a cytotoxicity evaluation of the test compounds. Briefly, Vero cells were cultured in 96-well plates (5 × 10 3 cells/well/200 µL) for 24 h in RPMI 1640 medium with 10% FBS and 5% CO 2 , at 37 • C. Cells were washed with PBS, followed by treatment with test compounds for 72 h at varying concentrations (50, 25, 12.5, 6.25, 3.13, 1.65, and 0.75 µg mL −1 ) in 10% FBS medium. Cells treated solely with medium in 2% FBS were used as a negative control. The supernatant was discarded, 50 µL RPMI 1640 medium containing 14 µL MTT (5 mg mL −1 ) was added, and the cells were incubated for 4 h. The supernatant was removed again, and 150 µL DMSO was added in order to dissolve the formed formazan. A FLUOstar OPTIMA spectrophotometer was applied for colorimetric analysis (λ = 540 nm). The cytotoxicity was expressed by IC 50 values (concentration which caused a 50% reduction in viable cells). IC 50 values were calculated from three independent experiments [15].

Results
The 4-amino-3-cyano-2-aryl-1,2-dihydropyrimido[1,2-a]benzimidazoles 2a-f were obtained from the three-component reaction of 2-aminobenzimidazole, malononitrile, and the corresponding aryl aldehyde under "green" conditions in hot H 2 O (Scheme 1). Compounds 2c and 2d were published before and were added to this compound series for comparison purposes in the following antiparasitic tests [13,14]. The fluorophenyl-substituted derivatives 2a-d were used as starting materials for the synthesis of compounds 3a-d and 4a-d. Oxidation of 2a-d with p-chloranil generated the pyrimido[1,2-a]benzimidazoles 3a-d. Compounds 3a-c are new, while 3d was published before [13]. The reaction of 2a-d with 2,5-dimethoxytetrahydrofuran led to the new pyrroles 4a-d, which were prepared in order to evaluate the role of the 4-amino group of compounds 2a-d. Compounds 2a-f, 3a-d, and 4a-d were initially tested for their activity against T. gondii tachyzoites (Table 1). Compound 3b (IC50 = 2.8 μM) showed the highest activity against T. gondii, closely followed by 2a (IC50 = 4.59 μM). 3b was distinctly less toxic to the non-malignant Vero cells than 2a, and exhibited a selectivity index (SI) of 8.12. In contrast, 2a showed an activity against Vero cells similar to the activity against the T. gondii cells indicating no selectivity of this compound for T. gondii. The approved antiparasitic drug atovaquone (ATO) showed higher activity against T. gondii than 3b, but ATO was also more toxic to Vero cells than 3b. Compounds 2a-f, 3a-d, and 4a-d were initially tested for their activity against T. gondii tachyzoites (Table 1). Compound 3b (IC 50 = 2.8 µM) showed the highest activity against T. gondii, closely followed by 2a (IC 50 = 4.59 µM). 3b was distinctly less toxic to the nonmalignant Vero cells than 2a, and exhibited a selectivity index (SI) of 8.12. In contrast, 2a showed an activity against Vero cells similar to the activity against the T. gondii cells indicating no selectivity of this compound for T. gondii. The approved antiparasitic drug atovaquone (ATO) showed higher activity against T. gondii than 3b, but ATO was also more toxic to Vero cells than 3b. The activity of compounds 2a-f, 3a-d, and 4a-d against L. major promastigotes and amastigotes was also determined ( Table 2). Again, compound 2a performed well and showed excellent activities against L. major promastigotes and amastigotes (EC 50 = 0.2-0.4 µM), which were higher than the activities of the approved antileishmanial drug amphotericin B (AmB). A considerable selectivity for L. major was observed for compound 2a when compared with its activity against Vero cells (SI = 11.1 for promastigotes and 21.6 for amastigotes). Among the other compounds, only 2d, 3b, and 4c showed moderate activities against the L. major promastigotes (IC 50 = 13.7-15.2 µM).
In addition to Vero cells, the effects of selected test compounds on macrophages were investigated in order to obtain more information about the selectivity of the test compounds for the L. major amastigotes, which are growing within macrophages ( Table 2). The most active antileishmanial compound 2a was five times less active against macrophages (IC 50 = 0.98 µM) than against L. major amastigotes (EC 50 = 0.20 µM), indicating a moderate selectivity of this compound for amastigotes. Compounds 2b-d and 3a-d exhibited IC 50 values of 20-24 µM, while compounds 4a-d were distinctly less toxic, with IC 50 values of 40-47 µM. The fluorine-free compounds 2e and 2f were least toxic to macrophages, with IC 50 values of 64 µM and 86 µM, respectively. However, these compounds showed only low activity against L. major amastigotes, too.

Discussion
The test compounds used in this study were obtained in low-to-moderate yields. However, the synthesis of the 1,2-dihydropyrimido[1,2-a]benzimidazoles 2a-f was accomplished by using one-pot "green" conditions. "Green chemistry" is of growing importance as a sustainable way to prepare drug candidates for the treatment of cancer and infectious diseases [13,17,18]. Compounds 2a-f and 4a-d were obtained and tested as racemic mixtures, and an increase in activity might be achieved by separation of the enantiomers of the active derivatives followed by testing against T. gondii and L. major parasites. In addition, chiral synthetic procedures by using chiral catalysts or additives might be applied to obtain enantiopure test compounds for biological testing.
In terms of antiparasitic activities, the new 4-amino-3-cyano-2-(3,5-difluorophenyl)pyrimido[1,2-a]benzimidazole 3b exhibited a high activity against and distinct selectivity for T. gondii cells, while its activity against L. major parasites, Vero cells, and macrophages was considerably lower. The reasons for this selectivity for T. gondii remain to be elucidated and are likely based on differences in the biology of T. gondii cells and L. major/Vero/macrophage cells. Based on its high selectivity and reduced toxicity when compared with atovaquone, 3b might be considered for advanced biological testing using T. gondii parasite models in vitro and in vivo.
In contrast, very promising results were obtained from the evaluation of the new 4-amino-3-cyano-2-(3-fluorophenyl)-1,2-dihydropyrimido[1,2-a]benzimidazole 2a in L. major parasites. Both excellent activities against and considerable selectivities for L. major promastigotes and amastigotes were discovered. It is remarkable that 2a was more active than amphotericin B, and it showed slightly higher activity against L. major amastigotes than against promastigotes of this parasite. The high activity of 2a against L. major amastigotes is of special interest since many clinically approved antileishmanial drugs also perform well against intracellular/intra-macrophage amastigotes [19,20]. Assays with amastigotes cover host cell mechanisms and, thus, provide more information about the properties of test compounds within human cells and about the potential of an active test compound to become an antileishmanial drug in the future [21,22]. Thus, 2a has the potential to proceed to advanced stages of antileishmanial testing including in vivo experiments with suitable CL animal models. More detailed research about the mechanisms of action is warranted in order to identify the reason for the promising antileishmanial effects of 2a. The combination of compound 2a with approved antileishmanial drugs would also be useful in order to optimize the efficacy, and to reduce the minimal effective dosage and possible side-effects in future animal studies.
Structure-activity relationships were difficult to identify because only 2a and 3b showed reasonable antiparasitic activities. Slight modifications of 2a (oxidation to pyrimido[1,2-a]benzimidazole 3a, and modification of the 4-amino group to pyrrole 4a) quickly led to distinctly reduced activities. Similarly, the reduced 1,2-dihydropyrimido[1,2a]benzimidazole 2b and pyrrole 4b exhibited lower activities than their close analog 3b. However, the fluoro-substitution pattern is critical too, and 3-fluorophenyl and 3,5-difluorophenyl compounds seem to be preferred in terms of activity. The underlying parasite-specific reasons for these differences in activity remain to be elucidated. However, 3,5-difluorophenyl-substituted pyrans were found to be especially active against cancer cells based on ROS formation and interference with microtubule dynamics [23]. In contrast, the 3-bromo-4,5-dimethoxyphenyl motif of compound 2d exerted no antiparasitic activity, despite the well-documented apoptosis-inducing antitumor activities of this structural motif [24].

Conclusions
Two promising drug candidates against L. major and T. gondii were identified in this study. In particular, the high antileishmanial activity of 2a warrants further investigation in terms of possible mechanisms of action and its activity against other Leishmania species as well as Kinetoplastea parasites such as Trypanosoma.