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Article

Synthesis, Molecular Docking, and Antimycotic Evaluation of Some 3-Acyl Imidazo[1,2-a]pyrimidines

by
Omar Gómez-García
1,*,
Dulce Andrade-Pavón
2,*,
Elena Campos-Aldrete
1,
Ricardo Ballinas-Indilí
3,
Alfonso Méndez-Tenorio
4,
Lourdes Villa-Tanaca
2 and
Cecilio Álvarez-Toledano
3,*
1
Departamento de Química Orgánica-Laboratorio de Síntesis de Fármacos Heterocíclicos, Escuela Nacional de Ciencias Biológicas-IPN, Prolongación de Carpio y Plan de Ayala S/N, Colonia Santo Tomás, C.P. 11340 Ciudad de México, Mexico
2
Departamento de Microbiología-Laboratorio de Biología Molecular de Bacterias y Levaduras, Escuela Nacional de Ciencias Biológicas-IPN, Prolongación de Carpio y Plan de Ayala S/N, Colonia Santo Tomás, C.P. 11340 Ciudad de México, Mexico
3
Instituto de Química-UNAM, Circuito Exterior, Ciudad Universitaria, Coyoacán, C.P. 04510 Ciudad de México, Mexico
4
Departamento de Bioquímica-Laboratorio de Biotecnología y Bioinformática Genómica, Escuela Nacional de Ciencias Biológicas-IPN, Prolongación de Carpio y Plan de Ayala S/N, Colonia Santo Tomás, C.P. 11340 Ciudad de México, Mexico
*
Authors to whom correspondence should be addressed.
Molecules 2018, 23(3), 599; https://doi.org/10.3390/molecules23030599
Submission received: 7 February 2018 / Revised: 2 March 2018 / Accepted: 5 March 2018 / Published: 7 March 2018
(This article belongs to the Section Medicinal Chemistry)
Browse Figures
Versions Notes

Abstract

:
A series of 3-benzoyl imidazo[1,2-a]pyrimidines, obtained from N-heteroarylformamidines in good yields, was tested in silico and in vitro for binding and inhibition of seven Candida species (Candida albicans (ATCC 10231), Candida dubliniensis (CD36), Candida glabrata (CBS138), Candida guilliermondii (ATCC 6260), Candida kefyr, Candida krusei (ATCC 6358) and Candida tropicalis (MYA-3404)). To predict binding mode and energy, each compound was docked in the active site of the lanosterol 14α-demethylase enzyme (CYP51), essential for fungal growth of Candida species. Antimycotic activity was evaluated as the 50% minimum inhibitory concentration (MIC50) for the test compounds and two reference drugs, ketoconazole and fluconazole. All test compounds had a better binding energy (range: −6.11 to −9.43 kcal/mol) than that found for the reference drugs (range: 48.93 to −6.16 kcal/mol). In general, the test compounds showed greater inhibitory activity of yeast growth than the reference drugs. Compounds 4j and 4f were the most active, indicating an important role in biological activity for the benzene ring with electron-withdrawing substituents. These compounds show the best MIC50 against C. guilliermondii and C. glabrata, respectively. The current findings suggest that the 3-benzoyl imidazo[1,2-a]pyrimidine derivatives, herein synthesized by an accessible methodology, are potential antifungal drugs.

Graphical Abstract

1. Introduction

Imidazo[1,2-a]pyrimidine is an important core with a wide range of biological activity, including antitumor [1,2,3], antihyperglycemic [4], antitubercular [5], antiviral [6,7], anti-HIV [8], and antiatherosclerosis [9]. There are reports on the application of imidazo[1,2-a]pyridine derivatives as antifungal agents [10,11,12].
Fungal infections have become a growing problem for patient health, implying great economic losses in hospitals [13,14]. Among the main fungal infections is invasive candidiasis, caused by species belonging to the genus Candida, such as C. albicans, C. glabrata, C. tropicalis, and C. parapsilosis [15,16]. A common treatment for these infections is the application of a broad-spectrum antifungal agent. Prominent among the drugs administered for this purpose are different azoles (triazoles and imidazoles), which act by inhibiting the lanosterol 14α-demethylase (CYP51) enzyme, product of the (CYP51/ERG11) gene. Each of the CYP51 proteins has heme as its prosthetic group in the active site. This group is dependent on cytochrome P450, a key enzyme in the synthesis of sterols. The latter compounds are an important component of the fungal membrane [17].
Previous studies have demonstrated the efficacy of azoles and their derivatives on various strains and clinical isolates of Candida ssp. However, a large number of strains have shown resistance to azoles [18,19,20], making it necessary to seek alternatives for treatment of fungal infections. Although there are reports on the application of imidazo[1,2-a]pyrimidine derivatives as antifungal agents [21,22], they still do not display the pharmacological profile sought. Hence, it is necessary to design and synthesize new compounds with a better pharmacological profile.
The most widely used method of synthesis for the imidazo[1,2-a]pyrimidine nucleus was developed by Tschichibabin, based on the reaction between 2-aminopyrimidine and an alpha haloketone [23,24,25,26]. Despite the fact that imidazo[1,2-a]pyrimidines have been prepared with diverse variations in methodology, synthesis of 3-benzoyl-substituted imidazo[1,2-a]pyrimidines without a substituent in position 2 are scarce [27,28]. The known methods for the latter compounds employ conventional heating and microwave irradiation, such as the synthesis of 3-benzoyl imidazo[1,2-a]pyridine analogs previously described by our group [29]. However, no reports exist, to our knowledge, of the synthesis of 3-benzoyl-substituted imidazo[1,2-a]pyrimidines without using conventional heating or microwave irradiation.
The modern design and synthesis of new drugs has become more efficient by analyzing potential drugs with computational tools in order to explore the functional groups that can improve biological activity. Nevertheless, we were unable to find any reports on molecular docking studies carried out to establish the binding mode of 3-benzoyl imidazo[1,2-a]pyrimidine derivatives to the active site of the CYP51 enzyme. Such information would be advantageous for the evaluation of these compounds as antifungal agents against Candida species.
The aim of the present study was to synthesize a series of 3-benzoyl imidazo[1,2-a]pyrimidine derivatives without conventional heating or microwave irradiation, and analyze these compounds by molecular docking studies to determine their binding mode and energy in relation to the active site of the CYP51 enzyme. In addition, the antifungal activity of the test compounds was ascertained in relation to seven different Candida species, and these data were compared to the inhibitory activity of two reference drugs (fluconazole and ketoconazole).

2. Results

2.1. Synthesis

Imidazo[1,2-a]pyrimidines were presently synthesized from N,N-dimethyl-N-pyrimidilformamidine, which is obtained in quantitative yield by condensation of 2-amino pyrimidine 1 and an excess of N,N-dimethylformamide dimethyl acetal under reflux conditions. The synthetic route herein employed (Scheme 1) is a variation on our previous work [29]. The treatment of amidine 2 with different phenacyl bromides under inert atmosphere at room temperature for three hours gave the 3-benzoyl derivatives in synthetically useful yields (62–98%).
Thus, imidazo[1,2-a]pyrimidine derivatives with a benzoyl group having electron-donating groups (4b–e) or electron-withdrawing groups (4f–j) were prepared and fully characterized (Table 1).
The 1H NMR spectroscopic data are summarized in Table 2. Some compounds were analyzed in CDCl3 and others in deuterated trifluoroacetic acid.

2.2. Modeling CYP51 from Candida spp.

The 3D structures of the different CYP51 of Candida spp. (CYP51Ca) were initially generated with the homology modeling technique [30], widely used for the generation of comparative models. Fifteen models were presently generated by each query sequence. The DOPE method was utilized to evaluate the quality of the models obtained [31], selecting the best model based on the lowest score. The structures generated show overlapping with the template, indicating a high percentage of identity (>60%) with the latter (Figure 1). Ramachandran plots were constructed for the CYP51 sequences from Candida spp., revealing that 91.3% of the residues were located in favorable regions for the CYP51 sequence of C. dubliniensis (CYP51Cd), 91.5% for C. guilliermondii (CYP51Cgui), 92.4% for C. kefyr (CYP51Cke), 89.4% for C. krusei (CYP51Ck), and 91.4% for C. tropicalis (CYP51Ct) (Supplementary Materials Figure S57).

2.3. Molecular Docking of 3-Benzoyl Imidazo[1,2-a]pyrimidines in the Active Site of CYP51 of Candida spp.

A docking study was carried out between the 3-benzoyl imidazo[1,2-a]pyrimidine derivatives and the CYP51 from Candida spp. to explore the binding mode of these compounds and compare them to the two reference drugs, fluconazole and ketoconazole. It was observed that the synthesized and reference compounds bind in a similar manner inside the active site of CYP51 from Candida spp. A representative sample portrays the binding mode of the test compounds to CYP51Ca (Figure 2).
Once the binding mode of imidazo[1,2-a]pyrimidine 3-benzoyl derivatives to the CYP51 of Candida spp. had been established, the affinity of each of these compounds to the distinct CYP51 models was analyzed. For this purpose, 100 different conformations were obtained for each compound, selecting the one with the lowest binding energy. Each of the 3-benzoyl imidazo[1,2-a]pyrimidine derivatives proved to have a better docking energy (−6.11 to −9.43 kcal/mol) in all CYP51 of Candida spp. than that found for fluconazole (−3.16 to −5.68 kcal/mol) or ketoconazole (48.93 to −6.16 kcal/mol) (Table 3). The current results are comparable to those found in other studies for fluconazole [32,33]. Of the five imidazo[1,2-a]pyrimidine derivatives with the best binding energies in CYP51Ck, 4f had the lowest value (−9.43 kcal/mol).
The interactions of ligands 4a, 4d, 4f, 4i and 4j with each of the CYP51 from Candida spp. (Figures S61–S103 of Supplementary Materials) were analyzed to determine the residues involved. Within the active site of each of the CYP51 enzymes, functional groups or rings in imidazo[1,2-a]pyrimidine derivatives and reference compounds (fluconazole and ketoconazole) exhibited binding with similar polar and non-polar amino acid residues, such as Tyr108, Gly283, Leu352, Thr287, Thr98, Met493, Tyr94 and Phe102.
Once the interactions between the compounds and each of the CYP51 of the Candida species had been examined, it was found that the CYP51 of Candida krusei displayed the best binding energies. Hence, CYP51Kru was utilized as the receptor model to illustrate the interactions of the five docked compounds (Figure 3a–g).
An extension the 2D model is provided to depict the interactions between the imidazo[1,2-a]pyrimidine derivative 4f and CYP51Ck (Figure 4). Interestingly, polar and non-polar amino acid residues are shared between the reference compounds and imidazo[1,2-a]pyrimidine derivatives. The most frequently shared residues were non-polar, including Tyr94, Leu97, Thr98, Phe102, Tyr108, Phe199, Gly273, Val274, Gly277, Gly278, and Leu344. This suggests an important role of the amino acid residues of a hydrophobic character in the binding mechanism of the compounds to CYP51Ck [34].

2.4. Susceptibility of Candida spp. to 3-Benzoylimidazo[1,2-a]pyrimidines

We evaluated the susceptibility of the Candida strains to the imidazo[1,2-a]pyrimidine derivatives by the microdilution method [35]. The MIC50 was calculated for each of the compounds according to materials and methods (Table 4), finding that the five test compounds have a value much lower than that determined for either fluconazole or ketoconazole. The imidazo[1,2-a]pyrimidine derivative 4f showed the lowest MIC50 values for C. dubliniensis, C. glabrata and C. krusei, while 4j had the lowest values for C. albicans and C. guilliermondii. For C. kefyr, only compound 4d presented a MIC50 slightly higher than the remaining 3-benzoyl imidazo[1,2-a]pyrimidine derivatives.

3. Discussion

The mechanistic pathway for the synthesis of imidazo[1,2-a]pyrimidines is herein reported. It involves the formation of the pyridinium salt followed by the intramolecular cyclization of the carbanion (adjacent to the quaternary nitrogen) on the amidine carbon via a 5-exo-trig process. Finally, aromatization took place with the loss of dimethylamine. IR and NMR (1H and 13C) spectroscopy as well as mass spectrometry analysis confirmed the structures of the synthesized compounds. With the 1H NMR spectrum of compounds 4a–j, an abnormal chemical shift to downfield of proton 5 (~9.88–10.10 ppm) was found. This effect, also observed in 3-acyl imidazo[1,2-a]pyridines [36], is due to the interaction of C-H····O with the oxygen of the carbonyl group.
The identification of compound 4b was unequivocally established. Its molecular weight was found to be m/z = 283 (as determined by HREIMS). In the 13C NMR spectrum, the conjugated carbonyl group is evidenced by the signal at 183.7 ppm. The HMBC experiment showed two correlations between C-8a and H-5, as well as another interaction of C-8a with H-2. Moreover, an important correlation existed between C-3 and H-2, an interaction that confirms the presence of a quaternary carbon. On the other hand, the carbonyl group exhibited two correlations, with H-12 and H-16. The proton resonances of 4e–i were shifted due to the solvent used for the dilution (Table 2). In all cases, the compounds examined in CF3COOD were downfield compared to those analyzed in CDCl3, due to the greater polarity of CF3COOD versus CDCl3. The most significant changes (over 1 ppm) were detected for the equivalent protons (H-13 and H-15) in 4c with respect to 4g, where displacement strongly depended both on the solvent [37] and the electron-withdrawing and electron-donor nature of the nitro and methoxy substituents.
To evaluate whether the compounds synthesized herein are linked to the CYP51 enzyme of yeasts, models of CYP51 of Candida spp., were generated. The CYP51 of various species of the Candida genus proved to have a high structural similarity, which indicates that this structure is conserved among species [38], and emphasizes the importance of CYP51 as a therapeutic target of new drugs with antifungal activity [34].
Ramachandran diagrams were constructed to allow for visualization of the energetically favorable regions that are established by the dihedral angles of a protein [39]. For each CYP51 analyzed, 90% of the residues fall within the favorable regions. Hence, the models obtained are of good quality and comparable to those employed elsewhere [40]. The docking studies revealed that the 3-benzoyl imidazo[1,2-a]pyrimidine derivatives act on the same target as the azoles [40]. Consequently, these derivatives likely exhibit a broad spectrum of antifungal action on various species of the Candida genus, similar to that found previously for other azoles [32].
According to the docking results (Table 3), 4f and 4d have the best binding energies in relation to CYP51Ca and CYP51Cd, 4i with respect to CYP51Cg (and to a lesser extent in CYP51Cgui and CYP51Ck), as well as 4a and 4j in relation to CYP51Cke. Thus, 3-benzoyl imidazo[1,2-a]pyrimidines bound with distinct affinity in each of the CYP51 presently analyzed. Moreover, the docking studies on the different CYP51 enzymes demonstrated that similar amino acid residues interact with groups in the structures of the five 3-benzoyl imidazo[1,2-a]pyrimidine derivatives and the reference compounds. These findings indicate that the test compounds likely have a mechanism similar to that reported for azoles [33,41]. Additionally, hydrophobic, hydrophilic, and electrostatic interactions were evidenced between different substituents in the test ligands, such as methyl and halogen (Cl or F). Therefore, these connections are favored in the para position of the benzene ring.
According to the results, the best binding energies with CYP51Ck were shown by the five imidazo[1,2-a] pyrimidine derivatives selected in this study. Compound 4f displayed the greatest affinity for the active site of the enzyme, which is due to the C–H ·····O interactions of the imidazo[1,2-a]pyrimidine ring with the polar side chain of Tyr108 and Gly273 C–H. The current affinity data imply that imidazo[1,2-a]pyrimidine derivatives inhibit CYP51Ck more effectively than other CYP51. We propose that the cyano substituent in the structure of 4f may play an important role in the binding mode of this compound to the CYP51Ck.
During the docking study, the heme group of the protein exhibited hydrophobic interactions with the heterocyclic rings (imidazole and pyrimidine) of the 3-benzoyl imidazo[1,2-a]pyrimidine compounds, as well as with the triazole and imidazole rings of fluconazole and ketoconazole, respectively. For imidazo[1,2-a]pyrimidine derivatives 4a and 4i, hydrogen bonds were detected between amino acid Tyr108 of CYP51Ck and H-5 of the pyrimidine ring. Moreover, halogen bonds (Cl) were observed between compound 4i and the Tyr94, Leu97, Phe199, and Phe102 residues. Also found was another hydrogen bond between the oxygen of the carbonyl group in compound 4j and the amino acid residue Tyr281. Hydrophobic interactions could be appreciated between Gly277 and the heterocyclic moiety in compound 4a and 4i, and between this residue and the benzene ring of compound 4d. For fluconazole, hydrogen bonds were identified between Gly277 and the triazole ring, and hydrophobic interactions between Tyr94 and the other triazole moiety. For ketoconazole, hydrogen bonds were formed with the side chain of Tyr108, and type π-alkyl hydrophobic interactions occurred between Leu344 and the benzene ring, as well as between Leu344 and the Cl substituent in the benzene ring. Another important interaction was between the chlorine in the benzene ring and Ser346 [17,34,42].
Regarding the susceptibility of the Candida species to inhibition, the test compounds had lower MIC50 values than fluconazole and ketoconazole. However, if these results are compared with those reported in the literature for another class of azoles, such as voriconazole, we observe that the MIC50 for this antifungal in C. krusei is 0.25 [35], similar to that found in compound 4f, for C. glabrata and C. albicans MIC50 of 0.125 [43] and 1 [44] were found, respectively, observing that in C. glabrata, four compounds showed an MIC50 similar or better than voriconazole, while in C. albicans, the five derivatives showed better results. Regarding C. kefyr, four of the compounds showed MIC50 equivalent to that reported in voriconazole (0.125) [45,46], in C. guilliermondii [45,46], three of the compounds exhibited similar MIC50 (0.0625), while derivative 4j obtained a better value than the reference compound, and finally, for C. tropicalis [45,46], compound 4i showed a similar value (0.0312). The comparison of the MIC50 of the imidazo[1,2-a]pyrimidine derivatives indicate that different classes of azoles exhibit different inhibitory effects on the seven species of Candida, although it is observed that some are better in some species than in others, and it is necessary to search for new drugs or to improve those already proposed, that can be used in combinatorial therapy with already known compounds with the aim of improving the MIC50 reported. Given the above, these observations suggest that 3-benzoyl imidazo[1,2-a]pyrimidines may offer an alternative to azoles [33,41,47,48] in the treatment of infections caused by Candida spp.

4. Materials and Methods

4.1. Chemicals and Instruments

All glassware was thoroughly oven-dried. Chemicals and solvents were purchased from commercial suppliers. Melting points were determined on a Melt Temp II apparatus and are reported without correction. By using chloroform-d and CF3COOD, the 1H and 13C NMR spectra were recorded on a Varian NMR system (Palo Alto, CA, USA) at 500 MHz (1H NMR) and 125 MHz (13C NMR), as well as on a Bruker Advance III (Bruker Biospin, Ettlingen, Germany) at 300 MHz (1H NMR) and 75 MHz (13C NMR). Chemical shifts are given in parts per million with reference to internal TMS (Sigma-Aldrich, San Luis, MO, USA). EI-MS spectra were recorded on a JEOL JMS-AX505 (Akishima, Tokyo, Japan) and a JEOL GCmate spectrometer (Akishima). IR spectra were obtained with a Perkin Elmer FT-IR SPECTRUM 2000 spectrophotometer (Waltham, MA, USA).

4.2. Synthesis of Imidazo[1,2-a]pyrimidin-3-yl(phenyl)methanones (3a–j)

To a solution of N,N-dimethyl-N′-(pyrimidin-2-il)formamidine (2, 1 mmol) in anhydrous N,N-dimethylformamide (7 mL), at room temperature and under N2 atmosphere, was added the appropriate phenacyl bromide (3a–j, 1 mmol) in anhydrous N,N-dimethylformamide (7 mL). The reaction was stirred for three hours at room temperature. Then the mixture was added to a flask containing H2O (30 mL) and extracted with EtOAc (3 × 20 mL). The organic extracts were combined and dried (anhydrous Na2SO4), and the solvent was removed under reduced pressure to give the title compounds 4a–d and 4j, which were further purified by column chromatography on silica gel with a mixture of hexane and EtOAc as eluent. In the case of the phenacyl bromides 3e–i, once the reaction was completed the mixture was added to a flask containing ice (30 g), then the product was precipitated, filtered under vacuum, and recrystallized from ethanol–water to deliver the title compounds 4e–i.
Imidazo[1,2-a]pyrimidin-3-yl(phenyl)methanone (4a). Synthesis of 4a furnished a yellow solid (0.17 g, 77.5% yield); m.p. 234–235.5 °C (lit. m.p. 234 °C) [25]; IR (KBr) υmax 3134 (Csp2-H), 3053 (Csp3-H), 1611 (C=O), 1513 (C=C), 1475 (C=C) cm−1; 1H NMR (CDCl3, 300 MHz) δ 10.0 (1H, dd, J = 7 Hz, J = 2.0 Hz, H-5), 8.84 (1H, d, J = 4.5 Hz, J = 2.0 Hz, H-7), 8.41 (1H, s, H-2), 7.9 (2H, dd, J = 7.5 Hz, J = 1.5 Hz, H-12, H-16), 7.65 (1H, tt, J = 7.5 Hz, J = 1.5 Hz, H-14), 7.57 (2H, t, J = 7.5 Hz, H-13, H-15), 7.23 (1H, dd, J = 7 Hz, J = 4.5 Hz, H-6); 13C NMR (CDCl3, 75 MHz) δ 185.1, 153.7, 151.4, 146.3, 138.2, 136.6, 132.6, 128.8, 128.7, 121.7, 111.3; DART-MS m/z 224 [M + H]+ (100); HREIMS m/z 223.0746 (calcd. for C13H9N3O, 223.0746).
(3,4-dimethoxyphenyl)(imidazo[1,2-a]pyrimidin-3-yl)methanone (4b). Synthesis of 4b afforded a white solid (0.23 g, 81.1% yield); m.p. 251.5–252.5 °C; IR (CH2Cl2) υmax 2959.9 (Csp3-H), 1615.5 (C=O), 1578.4 (C=C), 1510.3 (C=C), 1470.9 (C=C), 1280.9 (C–O), 1264.7 (C–O) cm−1; 1H NMR (CDCl3, 300 MHz) δ 9.88 (1H, dd, J = 6.9 Hz, J = 2.1 Hz, H-5), 8.78 (1H, dd, J = 4.2 Hz, J = 2.1 Hz, H-7), 8.39 (1H, s, H-2), 7.54 (2H, dd, J = 8.4 Hz, J = 2.1 Hz, H-16), 7.43 (1H, d, J = 1.8 Hz, H-12), 7.18 (1H, dd, J = 6.9 Hz, J = 4.2 Hz, H-6), 6.96 (1H, d, J = 8.4 Hz, H-15), 3.96 (3H, s, OMe), 3.94 (3H, s, OMe); 13C NMR (CDCl3, 75 MHz) δ 183.7, 153.3, 153.0, 151.2, 149.2, 145.4, 136.5, 130.8, 123.4, 121.7, 111.1, 111.0, 110.3, 56.1, 55.9; DART-MS m/z 284 [M + H]+ (100); HREIMS m/z 284.1031 (calcd. for C15H13N3O3, 284.1035).
Imidazo[1,2-a]pyrimidin-3-yl(4-methoxyphenyl)methanone (4c). Synthesis of 4c gave a light yellow solid (0.17 g, 67.1% yield); m.p. 224–227 °C; IR (CH2Cl2) υmax 2915.8 (Csp3-H), 1608.3 (C=O), 1575.1 (C=C), 1515.9 (C=C), 1478.6 (C=C), 1256.7 (C–O) cm−1; 1H NMR (CDCl3, 300 MHz) δ 9.90 (1H, dd, J = 6.9 Hz, J = 1.8 Hz, H-5), 8.78 (1H, dd, J = 4.2 Hz, J = 1.8 Hz, H-7), 8.37 (1H, s, H-2), 7.88 (2H, d, J = 8.7 Hz, H-12, H-16), 7.17 (1H, dd, J = 6.9 Hz, J = 4.2 Hz, H-6), 7.00 (2H, d, J = 8.7 Hz, H-13, H-15), 3.9 (3H, s, OMe); 13C NMR (CDCl3, 75 MHz) δ 183.8, 163.3, 153.4, 151.1, 145.3, 136.6, 131.1, 130.7. 121.7, 114.0, 111.0, 55.5; DART-MS m/z 254 [M + H]+ (100); HREIMS m/z 254.0921 (calcd. for C14H11N3O2, 254.0929).
Imidazo[1,2-a]pyrimidin-3-yl(p-tolyl)methanone (4d). Synthesis of 4d delivered a white solid (0.21 g, 90.3% yield); m.p. 208–209 °C; IR (KBr) υmax 3134.7 (Csp2-H), 2923.4 (Csp3-H), 1611.8 (C=O), 1513.8 (C=C), 1507(C=C),, 1477 (C=C) cm−1; 1H NMR (CDCl3, 300 MHz) δ 9.94 (1H, dd, J = 6.9 Hz, J = 1.8 Hz, H-5), 8.79 (1H, dd, J = 4.2 Hz, J = 1.8 Hz, H-7), 8.37 (1H, s, H-2), 7.77 (2H, d, J = 7.8 Hz, H-12, H-16), 7.32 (2H, d, J = 7.8 Hz, H-13, H-15), 7.19 (1H, d, J = 6.9 Hz, J = 4.2 Hz, H-6), 2.48 (3H, s, Me); 13C NMR (CDCl3, 75 MHz) δ 184.8, 153.4, 151.3, 145.9, 143.3, 136.6, 135.5, 129.4, 128.9, 121.7, 111.1, 21.6; EI-MS m/z 237 [M]+ (100); HREIMS m/z 237.0900 (calcd. for C14H11FN3O, 237.0902).
[1,1'-biphenyl]-4-yl(imidazo[1,2-a]pyrimidin-3-yl)methanone (4e). Synthesis of 4e provided a light yellow solid (0.21 g, 70.2% yield); m.p. 244.5–247 °C; IR (KBr) υmax 3133.3 (Csp2-H), 1607.4 (C=O), 1510.1 (C=C), 1478.2 (C=C) cm−1; 1H NMR (CF3COOD, 300 MHz) δ 10.08 (1H, d, J = 6.6 Hz, H-5), 9.08 (1H, d, J = 2.7 Hz, H-7), 8.53 (1H, s, H-2), 7.87 (2H, d, J = 7.8 Hz, H-12, H-16), 7.74 (3H, d, J = 7.8 Hz, H-6, H-13, H-15), 7.52 (2H, d, J = 7.8 Hz, H-18, H-22), 7.29-7.37 (3H, m, H-19, H-20, H-21); 13C NMR (CF3COOD, 75 MHz) δ 183.9, 161.6, 151.5, 144.9, 141.1, 140.0, 132.5, 130.7, 130.4, 129.6, 128.7, 127.7, 125.1, 122.1, 117.4; DART-MS m/z 300 [M + H]+ (100); HREIMS m/z 300.1130 (calcd. for C19H13N3O, 300.1136).
4-(imidazo[1,2-a]pyrimidine-3-carbonyl)benzonitrile (4f). Synthesis of 4f generated a white solid (0.21 g, 84.6% yield); m.p. 277–281 °C; IR (KBr) υmax 3097.9, 3074 (Csp2-H), 2230.3 (C≡N), 1629.3 (C=O), 1522.3 (C=C), 1476.0 (C=C) cm−1; 1H NMR (CF3COOD, 300 MHz) δ 10.10 (1H, d, J = 6.9 Hz, H-5), 9.13 (1H, d, J = 4.5 Hz, H-7), 8.51 (s, 1H, H-2), 7.77 (1H, dd, J = 6.9 Hz, J = 4.5 Hz, H-6), 7.62 (4H, d, J = 7.1 Hz, H-12, H-13, H-15, H-16); 13C NMR (CF3COOD, 75 MHz) δ 185.9, 161.4, 145.0, 140.2, 134.3, 133.8, 133.7, 132.3, 132.1, 130.9, 122.4, 117.2; DART-MS m/z 249 [M + H]+ (100); HREIMS m/z 249.0775 (calcd. for C14H8N4O, 249.0776).
Imidazo[1,2-a]pyrimidin-3-yl(4-nitrophenyl)methanone (4g). Synthesis of 4g produced a light yellow solid (0.20 g, 74.5% yield); m.p. 233.2–233.7 °C; IR (KBr) υmax 3113.7 (Csp2-H), 1630 (C=O), 1598.2 (C=C), 1521 (C=C), 1480.7 (C=C), 1349.7 (NO2) cm−1; 1H NMR (CF3COOD, 300 MHz) δ 10.08 (1H, d, J = 6.9 Hz, H-5), 9.10 (1H, d, J = 4.5 Hz, H-7), 8.52 (1H, s, H-2), 8.29 (2H, d, J = 8.4 Hz, H-13, H-15), 7.96 (2H, d, J = 8.4 Hz, H-12, H-16), 7.76 (1H, dd, J = 6.9 Hz, J = 4.5 Hz, H-6); 13C NMR (CF3COOD, 75 MHz) δ 183.9, 161.5, 151.4, 144.9, 141.0, 140.0, 132.5, 130.6, 125.1, 121.9, 117.3; DART-MS m/z 269 [M + H]+ (100); HREIMS m/z 269.0669 (calcd. for C13H8N4O3, 269.0674).
(4-bromophenyl)(imidazo[1,2-a]pyrimidin-3-yl)methanone (4h). Synthesis of 4h resulted in a yellow solid (0.29 g, 98% yield); m.p. 248–249 °C; IR (KBr) υmax 3113.6 (Csp2-H), 3043.6 (Csp3-H), 1630 (C=O), 1520 (C=C), 1480.7 (C=C) cm−1. 1H NMR (CF3COOD, 300 MHz) δ 10.00 (1H, d, J = 6.9 Hz, H-5), 9.04 (1H, d, J = 3.6 Hz, H-7), 8.43 (1H, s, H-2), 7.67 (3H, d, J = 7.5 Hz, H-6, H-12, H-16), 7.4 (2H, d, J = 7.5 Hz, H-13, H-15); 13C NMR (CDCl3, 75 MHz) δ 180.8, 161.1, 144.7, 143, 140, 133.9, 131.9, 130.8, 130.4, 122.2, 117.0; EI-MS m/z 302 [M + 2]+ (97), 300 [M]+ (100); HREIMS m/z 300.9851 (calcd. for C13H8BrN3O, 300.9851).
(4-chlorophenyl)(imidazo[1,2-a]pyrimidin-3-yl)methanone (4i). Synthesis of 4i furnished a light yellow solid (0.18 g, 72.1% yield); m.p. 240–241 °C; IR (KBr) υmax 3135 (Csp2-H), 1613 (C=O), 1513 (C=C), 1513, 1476 (C=C) cm−1. 1H NMR (CF3COOD, 300 MHz) δ 10.00 (1H, d, J = 6.6 Hz, H-5), 9.03 (1H, s, H-7), 8.42 (1H, s, H-2), 7.68 (3H, d, J = 7.8 Hz, H-6, H-12, H-16), 7.41 (2H, d, J = 7.8 Hz, H-13, H-15). 13C NMR (CDCl3, 75 MHz) δ 185.3, 161.2, 144.7, 143.4, 139.9, 133.7, 132.0, 130.8, 130.5, 122.3, 117.1; EI-MS m/z 259 [M + 2]+ (33), 257 [M]+ (100); HREIMS m/z 257.0348 (calcd. for C13H8ClN3O, 257.0356).
(4-fluorophenyl)(imidazo[1,2-a]pyrimidin-3-yl)methanone (4j). Synthesis of 4j gave a white solid (0.15 g, 62.2% yield); m.p. 256.5–257.5 °C; IR (KBr) υmax 3133 (Csp2-H), 1614 (C=O), 1513 (C=C), 1507, 1477 (C=C) cm−1. 1H NMR (CDCl3, 500 MHz) δ 9.96 (1H, dd, J = 7 Hz, J = 2 Hz, H-5), 8.84 (1H, dd, J = 4.5 Hz, J = 2.0 Hz, H-7), 8.39 (1H, s, H-2), 7.92–7.96 (2H, m, H-12, H-16), 7.22–7.27 (3H, m, H-6. H-13, H-15); EI-MS m/z 241 [M]+ (100); HREIMS m/z 241.0647 (calcd. for C13H8FN3O, 241.0651).

4.3. Microorganisms and Compounds Tested

The fungal strains included presently were C. albicans (ATCC 10231), C. dubliniensis (CD36), C. glabrata (CBS138), C. guilliermondii (ATCC 6260, also called Meyerozyma guilliermondi), C. krusei (ATCC 6358), C. tropicalis (MYA-3404), and C. kefyr ENCB-BMBL (also called Kluyveromyces marxianus), kindly provided by Lourdes Villa Tanaca of IPN-ENCB. The yeast strains were previously incubated in YPD medium (1% yeast extract, 2% casein peptone, and 2% dextrose) to verify their purity. The five 3-benzoyl imidazo[1,2-a]pyrimidines tested herein were selected based on their expected good inhibitory effect. Fluconazole (triazole) and ketoconazole (imidazole) were the reference compounds (Figure 5).

4.4. Homology Modeling

The amino acid sequences of lanosterol 14α-demethylase (CYP51) proteins of the Candida spp. were downloaded from the NCBI database: (C. dubliniensis XP_002420370.1), (C. guilliermondii XP_001484034.1), (C. kefyr AHL25033.1), (C. krusei XP_020542549.1), and (C. tropicalis XP_002550985.1). The 3D models were generated with the Modeller 9.10 program [49] (http://www.salilab.org/modeller/), using as a template the crystallized structures of CYP51 of C. glabrata (PDB: 5JLC) and C. albicans (PDB: 5V5Z), respectively, deposited in the protein data bank (PDB) [50] (http://www.rcsb.org./pdb). The structures were selected based on the highest percentage of identity with the downloaded sequences. An overlapping of the structures was constructed and displayed with Discovery 4.0 Client [51].
The quality of the models was evaluated with the DOPE (discrete optimized protein energy) method [31]. The model having the lowest DOPE score was selected for ligand–protein interaction studies. In addition, Ramachandran plots [39] were calculated by using the PDBsum database [52] for validation of the 3D structure.

4.5. Molecular Docking Studies

A ligand–protein interaction study was previously validated with molecular docking software Autodock version 4.0 (The Scripps Research Institute, La Jolla, CA, USA) [53]. The 2D structure of each ligand was sketched in editor chemical MedChem Designer 3.0 (http://www.simulations-plus.com/software/medchem-designer) and converted to 3D, mol2 format in the Open Babel GUI program [54]. Hydrogens were added to the models generated with the MolProbity program [55] and prepared with Visual Molecular Dynamics (VMD 1.9.1) [56]. All ions were added by utilizing the optimization Nanoscale Molecular Dynamics (NAMD) software program (Illinois University, Urbana and Champaign, IL. USA) [57]. The resulting structures were used for docking.
The selected test compounds and the reference compounds (fluconazole and ketoconazole) were docked in the active site of CYP51. For the preparation of docking, the following parameters were estimated in AutoDock Tools (ADT) [53]. The grid dimensions were 48 × 42 × 40 Å3 and the points were separated by 0.375 Å. The following grid centers were calculated for CYP51 from C. albicans (CYP51Ca; X = −47.731, Y = −13.422 and Z = 22.982), C. dubliniensis (CYP51Cd; X = −43.598, Y = −13.588 and Z = 25.836), C. glabrata (CYP51Cg; X = −31.107, Y = 68.515 and Z = −21.415), C. kefyr (CYP51Cke; X = −31.107, Y = 69.159 and Z = −19.206), C. krusei (CYP51Ckru; X = −43.311, Y = −9.941 and Z = 25.384), and C. tropicalis (CYP51Ct; X = −45.446, Y = −10.26 and Z = 23.625).
Random starting positions, orientations and torsion angles were established for all ligands. Default values of translation, quaternation, and torsion steps were employed for the simulation. The hybrid Lamarckian Genetic Algorithm (set with default parameters) was applied for minimization. The number of docking runs was 100. The docked model with the lowest binding energy was considered for all further simulations. Docking results were analyzed in AutoDockTools and edited in Discovery 4.0 Client [51].

4.6. Antifungal Activity Tests

The minimum inhibitory concentration (MIC50) was determined according to CLSI guidelines in the document M27-A3 for yeasts [35]. The preparation of the dilutions of the reference compounds and five 3-benzoyl imidazo[1,2-a]pyrimidines selected was carried out by the method of serial double additive dilutions. For the water-soluble compound (fluconazole), the concentrations tested were 64–0.125 μg/mL, using RPMI 1640 as diluent with glutamine and without sodium bicarbonate, buffered with morpholino propane sulfonic acid (MOPS) at 0.164 M, adjusted to pH 7 ± 0.1, and with 0.2% glucose. For the water insoluble antifungals (ketoconazole and the test compounds), the concentrations ranged from 16 to 0.0312 μg/mL, using DMSO as diluent.
For the preparation of the inoculum of Candida spp., the optical density was adjusted in a spectrophotometer (530 nm) to 0.5 McFarland. Subsequently, a 1:1000 dilution was made with RPMI medium (at concentrations of 1 × 103–5 × 103). The antifungal assay was performed with the latter dilution. The 96-well plates were inoculated with 100 µL of yeast suspension. RPMI was utilized as the sterility control and DMSO without antifungal as the growth control. The plates were incubated for 24 h at 37 °C. The optical density was determined in a Multiskan™ GO microplate spectrophotometer by agitation of the plates to obtain a homogeneous suspension, followed by a spectrophotometric reading at 530 nm. The MIC50 is the antifungal concentration whose optical density equals 50% of the growth in the control well. The value reported herein represents the average of three different experiments.

5. Conclusions

A series of 3-benzoyl imidazo[1,2-a]pyrimidines was synthesized and tested in silico and in vitro. By docking the test compounds in the active site of fungal CYP51, the binding mode and binding energy could be predicted in each case. The MIC50 was determined for each compound as well as for two reference compounds (fluconazole and ketoconazole), in order to evaluate the respective capacity for growth inhibition of distinct Candida species. The docking results show that for each species of Candida spp., the binding mode of each test compound shares at least three amino acid residues with the reference drugs. We analyzed and described the interactions of the electron-donor and electron-withdrawing substituents in the aromatic ring with key amino acid side chains in the active site of CYP450. Although the antifungal activity of imidazo[1,2-a]pyrimidines has been studied, the present findings should certainly be instrumental in the design and development of new antifungal drugs derived from 3-benzoyl imidazo[1,2-a]pyrimidines. Further research is needed for this purpose, and to test new derivatives in combination with conventional drugs in animal models, and possibly later, in clinical trials as promising candidates for antifungal therapy.

Supplementary Materials

The supplementary materials are available online. Figures S1–S103.

Acknowledgments

O.G.-G.would like to acknowledge CONACYT 252020, PAPIIT IN202917, SIP-IPN (Grants 20162041 and 20172002). We thank Bruce Allan Larsen for proofreading the manuscript and Fortunata Santoyo, Luis Velasco, Javier Pérez and Carmen García for recording IR and mass spectroscopy.

Author Contributions

O.G.-G. and D.A.-P. conceived and designed the experiments, O.G.-G, D.A.-P. and R.B.-I performed the experiments, E.C.-A., A.M.-T., C.A.-T. and L.V.-T. supervised the experiments. All authors discussed the results and commented on the manuscript.

Conflicts of Interest

The authors declare no conflict of interest.

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Sample Availability: Samples of the compounds are not available from the authors.
Molecules 23 00599 sch001 550
Scheme 1. Synthesis of 3-benzoylimidazo[1,2-a]pyrimidines 4a–j without conventional heating.
Scheme 1. Synthesis of 3-benzoylimidazo[1,2-a]pyrimidines 4a–j without conventional heating.
Molecules 23 00599 sch001
Molecules 23 00599 g001 550
Figure 1. Flat ribbon representation of the CYP51 of Candida spp. (a) Overlapping of CYP51Cd (purple), CYP51Cgui (yellow), CYP51Ck (orange), and CYP51Ct (blue) with CYP51Ca (PDB: 5V5Z) (green); (b) Overlapping of CYP51Cke (brown) with CYP51Cg (PDB: 5JLC) (cyan blue). The heme as the prosthetic group is shown in red (stick representation). For each of the models, moreover, the percentage of identity with their respective template is listed.
Figure 1. Flat ribbon representation of the CYP51 of Candida spp. (a) Overlapping of CYP51Cd (purple), CYP51Cgui (yellow), CYP51Ck (orange), and CYP51Ct (blue) with CYP51Ca (PDB: 5V5Z) (green); (b) Overlapping of CYP51Cke (brown) with CYP51Cg (PDB: 5JLC) (cyan blue). The heme as the prosthetic group is shown in red (stick representation). For each of the models, moreover, the percentage of identity with their respective template is listed.
Molecules 23 00599 g001
Molecules 23 00599 g002 550
Figure 2. Binding mode on CYP51Ca of the 3-benzoyl imidazo[1,2-a]pyrimidines and reference compounds. The five selected compounds and the reference compounds were overlaid on this receptor. CYP51Ca is shown as a flat ribbon, while the compounds are illustrated with a stick model. Accordingly, the heme group is depicted in red, fluconazole in cyan blue, the ketoconazole in orange, 4a in dark blue, 4d in pink, 4f in yellow, 4i in green, and 4j in purple.
Figure 2. Binding mode on CYP51Ca of the 3-benzoyl imidazo[1,2-a]pyrimidines and reference compounds. The five selected compounds and the reference compounds were overlaid on this receptor. CYP51Ca is shown as a flat ribbon, while the compounds are illustrated with a stick model. Accordingly, the heme group is depicted in red, fluconazole in cyan blue, the ketoconazole in orange, 4a in dark blue, 4d in pink, 4f in yellow, 4i in green, and 4j in purple.
Molecules 23 00599 g002
Molecules 23 00599 g003a 550Molecules 23 00599 g003b 550
Figure 3. Schematic representation of the interactions of the five 3-benzoyl imidazo[1,2-a]pyrimidines and the two reference compounds with CYP51Ck. The flat ribbon denotes CYP51Ck, and the stick model portrays the heme group in red and the compounds in gray ((a), fluconazole; (b), ketoconazole; (c), 4a; (d), 4d; (e), 4f; (f), 4i; and (g), 4j) within the active site of the enzyme. The coupling of the compounds with amino acid residues from the active site of CYP51Ck are illustrated in a 3D model. The 2D model shows hydrophilic interactions (green dotted lines), hydrophobic interactions (gray dotted lines) and electrostatic interactions (without lines). Polar amino acids (cyan blue) and non-polar ones (green) are displayed in the circle.
Figure 3. Schematic representation of the interactions of the five 3-benzoyl imidazo[1,2-a]pyrimidines and the two reference compounds with CYP51Ck. The flat ribbon denotes CYP51Ck, and the stick model portrays the heme group in red and the compounds in gray ((a), fluconazole; (b), ketoconazole; (c), 4a; (d), 4d; (e), 4f; (f), 4i; and (g), 4j) within the active site of the enzyme. The coupling of the compounds with amino acid residues from the active site of CYP51Ck are illustrated in a 3D model. The 2D model shows hydrophilic interactions (green dotted lines), hydrophobic interactions (gray dotted lines) and electrostatic interactions (without lines). Polar amino acids (cyan blue) and non-polar ones (green) are displayed in the circle.
Molecules 23 00599 g003aMolecules 23 00599 g003b
Molecules 23 00599 g004 550
Figure 4. Schematic representation of the interactions between compound 4f and CYP51Ck. The dashed lines indicate the amino acids in the five 3-benzoyl imidazo[1,2-a]pyrimidines and the two reference compounds.
Figure 4. Schematic representation of the interactions between compound 4f and CYP51Ck. The dashed lines indicate the amino acids in the five 3-benzoyl imidazo[1,2-a]pyrimidines and the two reference compounds.
Molecules 23 00599 g004
Molecules 23 00599 g005 550
Figure 5. 3D representation of the compounds used for biological activity and molecular docking.
Figure 5. 3D representation of the compounds used for biological activity and molecular docking.
Molecules 23 00599 g005
Table
Table 1. Synthesized 3-benzoyl imidazo[1,2-a]pyrimidines 4a–j.
Table 1. Synthesized 3-benzoyl imidazo[1,2-a]pyrimidines 4a–j.
Molecules 23 00599 i001
EntryCompoundR1R2Yield (%)
14aHH77
24bOMeOMe81
34cHOMe67
44dHMe90
54eHPh70
64fHCN84
74gHNO274
84hHBr98
94iHCl72
104jHF62
Table
Table 2. 1H NMR spectral data for synthesized 3-benzoylimidazo[1,2-a]pyrimidines.
Table 2. 1H NMR spectral data for synthesized 3-benzoylimidazo[1,2-a]pyrimidines.
Molecules 23 00599 i002
CompoundH-2H-5H-6H-7H-12H-13H-14H-15H-16
4a *8.4110.07.238.847.907.577.657.577.90
4b *8.399.887.188.787.433.943.966.967.54
4c *8.379.907.178.787.887.003.907.007.88
4d *8.379.947.198.797.777.322.487.327.77
4e **8.5310.087.749.087.877.747.29–7.527.747.87
4f **8.5110.107.779.137.627.62-7.627.62
4g **8.5210.087.769.107.968.29-8.297.96
4h **8.4310.007.679.047.677.40-7.407.67
4i **8.4210.007.689.037.687.41-7.417.68
4j **8.399.967.92–7.968.847.92–7.967.22–7.27-7.22–7.277.92–7.96
* 1H NMR determined in CDCl3; ** 1H NMR ascertained in CF3COOD.
Table
Table 3. Docking results of 3-benzoyl imidazo[1,2-a]pyrimidines in the active site of CYP51 of Candida spp.
Table 3. Docking results of 3-benzoyl imidazo[1,2-a]pyrimidines in the active site of CYP51 of Candida spp.
CompoundBinding Energy (kcal/mol) inCYP51 from Candida spp.
ABCDEFG
Fluconazole−4.44−4.56−4.38−4.07−3.16−5.68−4.99
Ketoconazole32.55−1.95−6.16−5.548.931.443.92
4a−7.65−7.53−7.1−7.21−6.85−8.41−7.86
4d−8.06−7.87−7.64−7.79−6.34−9.09−8.52
4f−8.43−7.91−7.61−7.89−6.11−9.43−8.39
4i−8.02−7.74−7.73−7.87−6.21−9.26−8.45
4j−7.8−7.59−7.13−7.19−7.17−8.5−8.02
Fluconazole and ketoconazole (reference compounds). A, CYP51Ca; B, CYP51Cd; C, CYP51Cg; D, CYP51Cgui; E, CYP51Cke; F, CYP51Ck; and G, CYP51Ct.
Table
Table 4. Antifungal activity of compounds 4a, 4d, 4f, 4i, and 4j against the Candida species.
Table 4. Antifungal activity of compounds 4a, 4d, 4f, 4i, and 4j against the Candida species.
CompoundMIC50 (µg/mL)
ABCDEFG
Fluconazole0.50.5410.541
Ketoconazole820.1250.250.2512
4a0.1250.06250.06250.06250.1250.50.5
4d0.1250.250.06250.06250.50.50.25
4f0.250.06250.03120.06250.1250.250.0625
4i0.250.50.062510.1250.50.0312
4j0.06250.50.250.03120.1250.50.25
Fluconazole and ketoconazole (reference compounds). A, Candida albicans (ATCC 10231); B, Candida dubliniensis (CD36); C, Candida glabrata (CBS138); D, Candida guilliermondii (ATCC 6260); E, Candida kefyr; F, Candida krusei (ATCC 6358); and G, C. tropicalis (MYA-3404).

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Gómez-García, O.; Andrade-Pavón, D.; Campos-Aldrete, E.; Ballinas-Indilí, R.; Méndez-Tenorio, A.; Villa-Tanaca, L.; Álvarez-Toledano, C. Synthesis, Molecular Docking, and Antimycotic Evaluation of Some 3-Acyl Imidazo[1,2-a]pyrimidines. Molecules 2018, 23, 599. https://doi.org/10.3390/molecules23030599

AMA Style

Gómez-García O, Andrade-Pavón D, Campos-Aldrete E, Ballinas-Indilí R, Méndez-Tenorio A, Villa-Tanaca L, Álvarez-Toledano C. Synthesis, Molecular Docking, and Antimycotic Evaluation of Some 3-Acyl Imidazo[1,2-a]pyrimidines. Molecules. 2018; 23(3):599. https://doi.org/10.3390/molecules23030599

Chicago/Turabian Style

Gómez-García, Omar, Dulce Andrade-Pavón, Elena Campos-Aldrete, Ricardo Ballinas-Indilí, Alfonso Méndez-Tenorio, Lourdes Villa-Tanaca, and Cecilio Álvarez-Toledano. 2018. "Synthesis, Molecular Docking, and Antimycotic Evaluation of Some 3-Acyl Imidazo[1,2-a]pyrimidines" Molecules 23, no. 3: 599. https://doi.org/10.3390/molecules23030599

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