Synthesis, molecular docking, and antimycotic evaluation of some 3-acyl imidazo[1,2-a]pyrimidines

Article abstract of DOI:10.3390/molecules23030599

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.

Full text of DOI:10.3390/molecules23030599

molecules
Article
Synthesis, Molecular Docking, and Antimycotic
Evaluation of Some 3-Acyl Imidazo[1,2-a]pyrimidines
1
,
2,
1
Omar Gómez-García *, Dulce Andrade-Pavón *, Elena Campos-Aldrete ,
3
4
2
Ricardo Ballinas-Indilí , Alfonso Méndez-Tenorio , Lourdes Villa-Tanaca
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; camesol22@gmail.com
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; lourdesvilla@hotmail.com
Instituto de Química-UNAM, Circuito Exterior, Ciudad Universitaria, Coyoacán,
C.P. 04510 Ciudad de México, Mexico; ricardo_snf.49.ers@hotmail.com
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; amendezt@ipn.mx
2
3
4
*
Correspondence: jogomezga@ipn.mx (O.G.-G.); andrade_eclud88@hotmail.com (D.A.-P.); cecilio@unam.mx
(C.A.-T.); Tel.: +52-555-729-6000 (O.G.-G.); +52-555-729-6000 (D.A.-P.); +52-555-622-4464 (C.A.-T.)
Received: 7 February 2018; Accepted: 5 March 2018; Published: 7 March 2018
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 14 -demethylase enzyme (CYP51), essential for fungal growth of Candida species.
α
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.
Keywords: 3-benzoyl imidazo[1,2-a]pyrimidines; molecular docking; antimycotic activity; Lanosterol
1
4α-demethylase; Candida spp.
1
. Introduction
Imidazo[1,2-a]pyrimidine is an important core with a wide range of biological activity,
including antitumor [ ], antihyperglycemic [ ], antitubercular [ ], antiviral [ ], anti-HIV [ ],
and antiatherosclerosis [ ]. There are reports on the application of imidazo[1,2-a]pyridine derivatives
1
–
9
3
4
5
6,
7
8
as antifungal agents [10–12].
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
Molecules 2018, 23, 0; doi:10.3390/molecules23030599
www.mdpi.com/journal/molecules

Molecules 2018, 23, 0
2 of 17
belonging to the genus Candida, such as C. albicans, C. glabrata, C. tropicalis, and C. parapsilosis [15
]. 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 14 -demethylase (CYP51) enzyme, product of the
CYP51/ERG11) gene. Each of the CYP51 proteins has heme as its prosthetic group in the active site.
,
1
6
α
(
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
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
–20], making it necessary to seek alternatives for treatment of fungal infections.
,
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
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
–26]. Despite the fact that imidazo[1,2-a]pyrimidines have been prepared with
,
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 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 with different phenacyl
1
(
2
bromides under inert atmosphere at room temperature for three hours gave the 3-benzoyl derivatives in
synthetically useful yields (62–98%).

Molecules 2018, 23, 0
3 of 17
Scheme 1. Synthesis of 3-benzoylimidazo[1,2-a]pyrimidines 4a–j without conventional heating.
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).
Table 1. Synthesized 3-benzoyl imidazo[1,2-a]pyrimidines 4a–j.
Entry
Compound
R1
R2
Yield (%)
1
2
3
4
5
6
7
8
9
4a
4b
4c
4d
4e
4f
4g
4h
4i
H
OMe
H
H
H
H
H
H
H
H
OMe
OMe
Me
Ph
CN
NO2
Br
77
81
67
90
70
84
74
98
72
62
Cl
F
1
0
4j
H
1
The H NMR spectroscopic data are summarized in Table 2. Some compounds were analyzed in
CDCl and others in deuterated trifluoroacetic acid.
3
1
Table 2. H NMR spectral data for synthesized 3-benzoylimidazo[1,2-a]pyrimidines.
Compound
a *
b *
c *
H-2
H-5
H-6
H-7
H-12
H-13
H-14
H-15
H-16
4
4
4
8.41
8.39
8.37
10.0
9.88
9.90
7.23
7.18
7.17
8.84
8.78
8.78
7.90
7.43
7.88
7.57
3.94
7.00
7.65
3.96
3.90
7.57
6.96
7.00
7.90
7.54
7.88

Molecules 2018, 23, 0
4 of 17
Table 2. Cont.
Compound
H-2
H-5
H-6
H-7
H-12
H-13
H-14
H-15
H-16
4
4
4
4
d *
e **
f **
g **
h **
8.37
8.53
8.51
8.52
8.43
8.42
8.39
9.94
10.08
10.10
10.08
10.00
10.00
9.96
7.19
7.74
7.77
7.76
7.67
8.79
9.08
9.13
9.10
9.04
9.03
8.84
7.77
7.87
7.62
7.96
7.67
7.68
7.32
7.74
7.62
8.29
7.40
7.41
2.48
7.29–7.52
7.32
7.74
7.62
8.29
7.40
7.41
7.77
7.87
7.62
7.96
7.67
7.68
-
-
-
-
-
4
4
4
i **
j **
7.68
7.92–7.96
7.92–7.96 7.22–7.27
7.22–7.27 7.92–7.96
* 1
H NMR determined in CDCl3; ** H NMR ascertained in CF3COOD.
1
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).
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.
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).

Molecules 2018, 23, 0
5 of 17
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.
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 (
3.16 to 5.68 kcal/mol) or ketoconazole (48.93 to
comparable to those found in other studies for fluconazole [32
−6.11 to
−
9.43 kcal/mol) in all CYP51 of Candida spp. than that found for fluconazole
6.16 kcal/mol) (Table 3). The current results are
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).
Table 3. Docking results of 3-benzoyl imidazo[1,2-a]pyrimidines in the active site of CYP51 of
Candida spp.
Binding Energy (kcal/mol) inCYP51 from Candida spp.
Compound
A
B
C
D
E
F
G
Fluconazole
Ketoconazole 32.55
−4.44
−
−
−
−
−
−
−
4.56
1.95
−
−
4.38
6.16 −5.5
−
4.07
−
3.16
48.93
6.85
6.34
6.11
6.21
−
5.68
1.44
8.41
9.09
9.43
9.26
−4.99
3.92
4
a
−7.65
−8.06
−8.43
−8.02
−7.8
7.53 −7.1
−
−
−
−
−
7.21
7.79
7.89
7.87
7.19
−
−
−
−
−
−
−
−
−
−7.86
−8.52
−8.39
−8.45
−8.02
4
4
d
f
7.87
7.91
7.74
7.59
−
−
−
−
7.64
7.61
7.73
7.13
4
4
i
j
7.17 −8.5
Fluconazole and ketoconazole (reference compounds).
A, CYP51Ca; B, CYP51Cd; C, CYP51Cg; D, CYP51Cgui;
E, CYP51Cke; F, CYP51Ck; and G, CYP51Ct.
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).

Molecules 2018, 23, 0
6 of 17
Figure 3. Cont.

Molecules 2018, 23, 0
7 of 17
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 (( ), fluconazole; ( ), ketoconazole; ( ), 4a
), 4d; ( ), 4f; ( ), 4i; and ( ), 4j) within the active site of the enzyme. The coupling of the compounds
a
b
c
;
(
d
e
f
g
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.
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].

Molecules 2018, 23, 0
8 of 17
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.
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.
Table 4. Antifungal activity of compounds 4a, 4d, 4f, 4i, and 4j against the Candida species.
MIC50 (µg/mL)
Compound
A
B
C
D
E
F
G
Fluconazole
Ketoconazole
0.5
8
0.5
2
0.0625
0.25
0.0625
0.5
4
1
0.25
0.0625
0.0625
0.0625
1
0.5
0.25
4
1
1
2
0.5
0.25
0.0625
0.0312
0.25
0.125
0.0625
0.0625
0.0312
0.0625
0.25
4
a
0.125
0.125
0.25
0.25
0.0625
0.125
0.5
0.125
0.125
0.125
0.5
0.5
0.25
0.5
0.5
4
4
d
f
4
4
i
j
0.5
0.0312
Fluconazole and ketoconazole (reference compounds).
A
, Candida albicans (ATCC 10231);
, Candida guilliermondii (ATCC 6260);
ATCC 6358); and G, C. tropicalis (MYA-3404).
B
, Candida dubliniensis
(
(
CD36);
C, Candida glabrata (CBS138);
D
E, Candida kefyr; F, Candida krusei
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,
1
13
aromatization took place with the loss of dimethylamine. IR and NMR ( H and C) spectroscopy
as well as mass spectrometry analysis confirmed the structures of the synthesized compounds.
1
With the H 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.

Molecules 2018, 23, 0
9 of 17
The identification of compound 4b was unequivocally established. Its molecular weight was
13
found to be m/z = 283 (as determined by HREIMS). In the C 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 CF COOD were downfield compared to those analyzed in CDCl ,
3
3
due to the greater polarity of CF COOD versus CDCl . The most significant changes (over 1 ppm)
3
3
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,
9
0% 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
-benzoyl imidazo[1,2-a]pyrimidine derivatives act on the same target as the azoles [40]. Consequently,
3
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

Molecules 2018, 23, 0
10 of 17
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
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
,46], in C. guilliermondii [45,46], three of the compounds exhibited similar
[
,
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
caused by Candida spp.
,41,47,48] in the treatment of infections
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
1
13
without correction. By using chloroform-d and CF COOD, the H and C NMR spectra were recorded
3
1
13
on a Varian NMR system (Palo Alto, CA, USA) at 500 MHz ( H NMR) and 125 MHz ( C NMR),
as well as on a Bruker Advance III (Bruker Biospin, Ettlingen, Germany) at 300 MHz ( H NMR) and
5 MHz ( C NMR). Chemical shifts are given in parts per million with reference to internal TMS
1
13
7
(
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)
0
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 N atmosphere, was added
2
the appropriate phenacyl bromide (3a
The reaction was stirred for three hours at room temperature. Then the mixture was added to a flask
containing H O (30 mL) and extracted with EtOAc (3 20 mL). The organic extracts were combined
and dried (anhydrous Na SO ), and the solvent was removed under reduced pressure to give the
–j, 1 mmol) in anhydrous N,N-dimethylformamide (7 mL).
×
2
2
4
title compounds 4a
–
d
and 4j, which were further purified by column chromatography on silica gel
, once the
with a mixture of hexane and EtOAc as eluent. In the case of the phenacyl bromides 3e
–
i
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.

Molecules 2018, 23, 0
11 of 17
Imidazo[1,2-a]pyrimidin-3-yl(phenyl)methanone (4a). Synthesis of 4a furnished a yellow solid (0.17 g,
◦ ◦
2
3
7
1
7.5% yield); m.p. 234–235.5 C (lit. m.p. 234 C) [25]; IR (KBr) υmax 3134 (Csp -H), 3053 (Csp -H),
−
611 (C=O), 1513 (C=C), 1475 (C=C) cm ; H NMR (CDCl , 300 MHz) δ 10.0 (1H, dd, J = 7 Hz,
3
1 1
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),
.23 (1H, dd, J = 7 Hz, J = 4.5 Hz, H-6); 1 C NMR (CDCl , 75 MHz)
3
δ
185.1, 153.7, 151.4, 146.3, 138.2,
7
1
3
+
36.6, 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 C H N O, 223.0746).
13
9
3
(
(
1
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 (CH Cl ) υmax 2959.9 (Csp -H), 1615.5 (C=O), 1578.4 (C=C),
2 2
3
−
510.3 (C=C), 1470.9 (C=C), 1280.9 (C–O), 1264.7 (C–O) cm ; H NMR (CDCl , 300 MHz) δ 9.88 (1H,
3
1 1
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 (CDCl , 75 MHz)
δ
183.7, 153.3,
3
1
53.0, 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 C H N O , 284.1035).
15
13
3
3
Imidazo[1,2-a]pyrimidin-3-yl(4-methoxyphenyl)methanone (4c). Synthesis of 4c gave a light yellow solid
◦
3
(
1
0.17 g, 67.1% yield); m.p. 224–227 C; IR (CH Cl ) υmax 2915.8 (Csp -H), 1608.3 (C=O), 1575.1 (C=C),
2
1
2
−
1
515.9 (C=C), 1478.6 (C=C), 1256.7 (C–O) cm ; H NMR (CDCl , 300 MHz)
δ
9.90 (1H, dd, J = 6.9 Hz,
3
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);
1
3
C NMR (CDCl , 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;
3
DART-MS m/z 254 [M + H] (100); HREIMS m/z 254.0921 (calcd. for C H N O , 254.0929).
14
11
3
2
Imidazo[1,2-a]pyrimidin-3-yl(p-tolyl)methanone (4d). Synthesis of 4d delivered a white solid (0.21 g,
◦
2
3
9
1
0.3% yield); m.p. 208–209 C; IR (KBr) υmax 3134.7 (Csp -H), 2923.4 (Csp -H), 1611.8 (C=O),
−
513.8 (C=C), 1507(C=C)„ 1477 (C=C) cm ; H NMR (CDCl , 300 MHz) δ 9.94 (1H, dd, J = 6.9 Hz,
3
1 1
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);
1
3
C NMR (CDCl , 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;
3
+
EI-MS m/z 237 [M] (100); HREIMS m/z 237.0900 (calcd. for C H FN O, 237.0902).
14
11
3
[
1,1
'-biphenyl]-4-yl(imidazo[1,2-a]pyrimidin-3-yl)methanone (4e). Synthesis of 4e provided a light yellow
◦
2
solid (0.21 g, 70.2% yield); m.p. 244.5–247 C; IR (KBr) υmax 3133.3 (Csp -H), 1607.4 (C=O), 1510.1 (C=C),
1
−
1 1
478.2 (C=C) cm ; H NMR (CF COOD, 300 MHz)
δ
10.08 (1H, d, J = 6.6 Hz, H-5), 9.08 (1H, d,
3
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,
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); C NMR (CF COOD,
3
7
5 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,
+
1
17.4; DART-MS m/z 300 [M + H] (100); HREIMS m/z 300.1130 (calcd. for C H N O, 300.1136).
19
13
3
4
8
-(imidazo[1,2-a]pyrimidine-3-carbonyl)benzonitrile (4f). Synthesis of 4f generated a white solid (0.21 g,
◦
2
4.6% yield); m.p. 277–281 C; IR (KBr) υmax 3097.9, 3074 (Csp -H), 2230.3 (C
≡
N), 1629.3 (C=O),
−
1 1
1522.3 (C=C), 1476.0 (C=C) cm ; H NMR (CF COOD, 300 MHz)
δ
10.10 (1H, d, J = 6.9 Hz, H-5),
3
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 (CF COOD, 75 MHz)
δ
185.9, 161.4, 145.0, 140.2, 134.3,
3
+
1
33.8, 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 C H N O, 249.0776).
14 8 4
Imidazo[1,2-a]pyrimidin-3-yl(4-nitrophenyl)methanone (4g). Synthesis of 4g produced a light yellow
◦
2
solid (0.20 g, 74.5% yield); m.p. 233.2–233.7 C; IR (KBr) υmax 3113.7 (Csp -H), 1630 (C=O), 1598.2
−
1 1
(
C=C), 1521 (C=C), 1480.7 (C=C), 1349.7 (NO ) cm ; H NMR (CF COOD, 300 MHz)
δ
10.08 (1H, d,
2
3
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),

Molecules 2018, 23, 0
12 of 17
7
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 (CF COOD,
3
5 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 C H N O , 269.0674).
13
8
4
3
(
(
1
4-bromophenyl)(imidazo[1,2-a]pyrimidin-3-yl)methanone (4h). Synthesis of 4h resulted in a yellow solid
◦
2
3
0.29 g, 98% yield); m.p. 248–249 C; IR (KBr) υmax 3113.6 (Csp -H), 3043.6 (Csp -H), 1630 (C=O),
−
. H NMR (CF COOD, 300 MHz) δ 10.00 (1H, d, J = 6.9 Hz, H-5),
3
1
1
520 (C=C), 1480.7 (C=C) cm
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 (CDCl , 75 MHz)
δ
180.8, 161.1, 144.7, 143, 140, 133.9, 131.9, 130.8,
3
+
+
130.4, 122.2, 117.0; EI-MS m/z 302 [M + 2] (97), 300 [M] (100); HREIMS m/z 300.9851 (calcd. for
C H BrN O, 300.9851).
13
8
3
(
4-chlorophenyl)(imidazo[1,2-a]pyrimidin-3-yl)methanone (4i). Synthesis of 4i furnished a light yellow
◦
2
solid (0.18 g, 72.1% yield); m.p. 240–241 C; IR (KBr) υmax 3135 (Csp -H), 1613 (C=O), 1513 (C=C), 1513,
1
8
−
476 (C=C) cm . H NMR (CF COOD, 300 MHz) δ 10.00 (1H, d, J = 6.6 Hz, H-5), 9.03 (1H, s, H-7),
3
1 1
13
.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). C NMR
(
2
CDCl , 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
3
+
+
59 [M + 2] (33), 257 [M] (100); HREIMS m/z 257.0348 (calcd. for C H ClN O, 257.0356).
13
8
3
(
6
(
4-fluorophenyl)(imidazo[1,2-a]pyrimidin-3-yl)methanone (4j). Synthesis of 4j gave a white solid (0.15 g,
◦
2
2.2% yield); m.p. 256.5–257.5 C; IR (KBr) υmax 3133 (Csp -H), 1614 (C=O), 1513 (C=C), 1507, 1477
−
1
1
C=C) cm
.
H NMR (CDCl , 500 MHz) δ 9.96 (1H, dd, J = 7 Hz, J = 2 Hz, H-5), 8.84 (1H, dd,
3
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 C H FN O, 241.0651).
13
8
3
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).
Figure 5. 3D representation of the compounds used for biological activity and molecular docking.

Molecules 2018, 23, 0
13 of 17
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
3
in AutoDock Tools (ADT) [53]. The grid dimensions were 48
×
42
×
40 Å and the points were separated
by 0.375 Å. The following grid centers were calculated for CYP51 from C. albicans (CYP51 ; X =
−
47.731,
43.598, Y = −13.588 and Z = 25.836), C. glabrata
21.415), C. kefyr (CYP51
X = −31.107, Y = 69.159 and
Ca
Y = −13.422 and Z = 22.982), C. dubliniensis (CYP51Cd; X =
−
(
CYP51 ; X = −31.107, Y = 68.515 and Z =
−
;
Cke
Cg
Z = −19.206), C. krusei (CYP51Ckru; X =
−43.311, Y =
−
9.941 and Z = 25.384), and C. tropicalis (CYP51 ;
Ct
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
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.
±
0.1,
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

Molecules 2018, 23, 0
14 of 17
3
3
RPMI medium (at concentrations of 1
×
10 –5
×
10 ). The antifungal assay was performed with
L of yeast suspension. RPMI was
the latter dilution. The 96-well plates were inoculated with 100
µ
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.
References
1
.
Aeluri, R.; Alla, M.; Polepalli, S.; Jain, N. Synthesis and antiproliferative activity of imidazo[1,2-a]Pyrimidine
Mannich Bases. Eur. J. Med. Chem. 2015, 100, 18–23. [CrossRef] [PubMed]
2
.
Li, C.; Ai, J.; Zhang, D.; Peng, X.; Chen, X.; Gao, Z.; Su, Y.; Zhu, W.; Ji, Y.; Chen, X.; et al. Design,
synthesis, and biological evaluation of novel imidazo[1,2-a]pyridine derivatives as potent c-Met Inhibitor.
ACS Med. Chem. Lett. 2015, 6, 507–512. [CrossRef] [PubMed]
3
.
.
Kamal, A.; Reddy, J.S.; Ramaiah, M.J.; Dastagiri, D.; Bharathi, E.V.; Sagar, V.P.; Pushpavalli, S.N.; Ray, P.;
Pal-Bhadra, M. Design, synthesis and biological evaluation of imidazopyridine/pyrimidine-chalcone
derivatives as potential anticancer agents. Med. Chem. Commun. 2010, 1, 355–360. [CrossRef]
Meng, W.; Brigance, R.P.; Chao, H.J.; Fura, A.; Harrity, T.; Marcinkeviciene, J.; O’Connor, S.P.; Tamura, J.K.;
Xie, D.; Zhang, Y.; et al. Discovery of 6-(Aminomethyl)-5-(2,4-dichlorophenyl)-7-methylimidazo[1,2-a]
pyrimidine-2-carboxamides as Potent, Selective Dipeptidyl Peptidase-4 (DPP4) Inhibitors. J. Med. Chem. 2010, 53,
4
5620–5628. [CrossRef] [PubMed]
5
.
Moraski, G.C.; Markley, L.D.; Chang, M.; Cho, S.; Franzblau, S.G.; Hwang, C.H.; Boshoff, H.; Miller, M.J.
Generation and exploration of new classes of antitubercular agents: The optimization of oxazolines, oxazoles,
thiazolines, thiazoles to imidazo[1,2-a]pyridines and isomeric 5,6-fused scaffolds. Bioorg. Med. Chem. 2012
,
2
0, 2214–2220. [CrossRef] [PubMed]

Molecules 2018, 23, 0
15 of 17
6
.
.
Gueiffier, A.; Lhassani, M.; Elhakmaoui, A.; Snoeck, R.; Andrei, G.; Chavignon, O.; Teulade, J.C.; Kerbal, A.;
Essassi, E.M.; Debouzy, J.C.; et al. Synthesis of acyclo-C-nucleosides in the imidazo[1,2-a]pyridine and
pyrimidine series as antiviral agents. J. Med. Chem. 1996, 39, 2856–2859. [CrossRef] [PubMed]
Gueiffier, A.; Blache, Y.; Chapat, J.P.; Elhakmaoui, A.; Essassi, E.M.; Andrei, G.; Snoeck, R.;
De Clercq, E.; Chavignon, O.; Teulade, J.C.; et al. Synthesis and antiviral activity of 2 and 3-substituted
imidazo[1,2-a]pyrimidine. Nucleosides Nucleotides Nucleic Acids 1995, 14, 551–554. [CrossRef]
7
8
.
.
Moog, C.; Wick, A.; Le Ber, P.; Kirn, A.; Aubertin, A.M. Bicyclic imidazo derivatives, a new class of highly
selective inhibitors for the human immunodeficiency virus type 1. Antivir. Res. 1994, 24, 275–288. [CrossRef]
Chen, X.; Xu, W.; Wang, K.; Mo, M.; Zhang, W.; Du, L.; Yuan, X.; Xu, Y.; Wang, Y.; Shen, J. Discovery of a Novel
Series of Imidazo[1,2-a]pyrimidine derivatives as potent and orally bioavailable lipoprotein-associated
phospholipase A2 inhibitors. J. Med. Chem. 2015, 58, 8529–8541. [CrossRef] [PubMed]
9
1
1
1
1
0. Kishbaugh, T.L. Pyridines and Imidazopyridines with Medicinal Significance. Curr. Top. Med. Chem. 2016
6, 3274–3302. [CrossRef] [PubMed]
1. Cesur, Z.; Cesur, N.; Birteksöz, S.; Otük, G. Synthesis and biological evaluation of some new
imidazo[1,2-a]pyridines. Acta Chim. Slov. 2010, 57, 355–362. [PubMed]
2. Kaplancikli, Z.A.; Turan-Zitouni, G.; Ozdemir, A.; Revial, G. Synthesis and anticandidal activity of some
imidazopyridine derivates. J. Enzyme Inhib. Med. Chem. 2008, 23, 866–870. [CrossRef] [PubMed]
3. Cuenca-Estrella, M.; Bernal-Martínez, L.; Buitrago, M.J.; Castelli, M.V.; Gomez-Lopez, A.; Zaragoza, O.;
Rodriguez-Tudela, J.L. Update on the epidemiology and diagnosis of invasive fungal infection.
Int. J. Antimicrob. Agents 2008, 32 (Suppl. 2), 143–147. [CrossRef]
,
1
1
4. Olaechea, P.M.; Palomar, M.; León-Gil, C.; Álvarez-Lerma, F.; Jordá, R.; Nolla-Salas, J.; León-Regidor, M.A.;
EPCAN Study Group. Economic impact of Candida colonization and Candida infection in the critically ill
patient. Eur. J. Clin. Microbiol. Infect. Dis. 2004, 23, 323–330. [CrossRef] [PubMed]
1
1
1
5. Desai, J.V.; van de Veerdonk, F.L.; Lionakis, M.S. Understanding the role of host immune responses in
invasive candidiasis. Intensive Care Med. 2017. [CrossRef] [PubMed]
6. Kullberg, B.J.; Arendrup, M.C. Invasive Candidiasis. N. Engl. J. Med. 2015, 373, 1445–1456. [CrossRef]
[PubMed]
7. Rabelo, V.W.; Santos, T.F.; Terra, L.; Santana, M.V.; Castro, H.C.; Rodrigues, C.R.; Abreu, P.A. Targeting CYP51
for drug design by the contributions of molecular modeling. Fundam. Clin. Pharmacol. 2017, 31, 37–53.
[CrossRef] [PubMed]
1
1
8. Rezaei, Z.; Khabnadideh, S.; Zomorodian, K.; Pakshir, K.; Kashi, G.; Sanagoei, N.; Gholami, S. Design,
synthesis and antifungal activity of some new imidazole and triazole derivatives. Arch. Pharm. (Weinh.)
2
011, 344, 658–665. [CrossRef] [PubMed]
9. Gonçalves, S.S.; Souza, A.C.R.; Chowdhary, A.; Meis, J.F.; Colombo, A.L. Epidemiology and molecular
mechanisms of antifungal resistance in Candida and Aspergillus. Mycoses 2016, 59, 198–219. [CrossRef]
[
PubMed]
0. Masiá-Canuto, M.; Gutiérrez-Rodero, F. Antifungal drug resistance to azoles and polyenes. Lancet Infect. Dis.
002, 2, 550–563. [CrossRef]
1. Rival, Y.; Grassy, G.; Taudou, A.; Ecalle, R. Antifungal activity in vitro of some imidazo[1,2-a]pyrimidine
derivatives. Eur. J. Med. Chem. 1991, 26, 13–18. [CrossRef]
2
2
2
2
2
2
2. Rival, Y.; Taudou, A.; Ecalle, R. Synthesis and antifungal activity evaluation of 3-hydroxyimidazo [1,2-a]
pyridine and 3-hydroxyimidazo [1,2-a] pyrimidine derivatives. Farmaco 1993, 48, 857–869.
3. El Kazzouli, S.; Berteina-Raboin, S.; Mouaddib, A.; Guillaumet, G. Solid-phase synthesis of
imidazo[1,2-a]pyridines and imidazo[1,2-a]pyrimidines. Tetrahedron Lett. 2003, 44, 6265–6267. [CrossRef]
4. Cosimelli, B.; Laneri, S.; Ostacolo, C.; Sacchi, A.; Severi, E.; Porcù, E.; Rampazzo, E.; Moro, E.; Basso, G.;
Viola, G. Synthesis and biological evaluation of imidazo[1,2-a]pyrimidines and imidazo[1,2-a]pyridines as
new inhibitors of the Wnt/β-catenin signaling. Eur. J. Med. Chem. 2014, 83, 45–56. [CrossRef] [PubMed]
5. Ermolat’ev, D.S.; Giménez, V.N.; Babaev, E.V.; Van der Eycken, E. Efficient Pd(0)-mediated
microwave-assisted arylation of 2-substituted imidazo[1,2-a]pyrimidines. J. Comb. Chem. 2006, 8, 659–663.
2
2
[CrossRef] [PubMed]
6. Velázquez-Olvera, S.; Salgado-Zamora, H.; Velázquez-Ponce, M.; Campos-Aldrete, E.; Reyes-Arellano, A.;
Pérez-González, C. Fluorescent property of 3-hydroxymethyl imidazo[1,2-a]pyridine and pyrimidine
derivatives. Chem. Cent. J. 2012, 6, 83. [CrossRef] [PubMed]

Molecules 2018, 23, 0
16 of 17
2
2
2
3
3
3
7. Podergajs, S.; Stanovnik, B.; Tisler, M. A New Approach for the Synthesis of Fused Imidazoles: The synthesis
of 3-acyl-substituted imidazo[1,2-x]azines. Synthesis 1984, 3, 263–265. [CrossRef]
8. Shaaban, M.R. Facile Access to Novel 3-Acylimidazo[1,2-a]pyrimidines under Microwave Irradiation.
Heterocycles 2013, 87, 1775–1783. [CrossRef]
9. Gómez, O.; Salgado-Zamora, H.; Reyes, A.; Campos, M.E. A revised approach to the synthesis of 3-acyl
imidazo[l,2-a]pyridines. Heterocycl. Commun. 2010, 16, 99–104. [CrossRef]
0. Contreras-Moreira, B.; Fitzjohn, P.W.; Bates, P.A. Comparative modelling: An essential methodology for
protein structure prediction in the post-genomic era. Appl. Bioinform. 2002, 1, 177–190.
1. Shen, M.Y.; Sali, A. Statistical potential for assessment and prediction of protein structures. Protein Sci. 2006
5, 2507–2524. [CrossRef] [PubMed]
,
1
2. Yates, C.M.; Garvey, E.P.; Shaver, S.R.; Schotzinger, R.J.; Hoekstra, W.J. Design and optimization of
highly-selective, broad spectrum fungal CYP51 inhibitors. Bioorg. Med. Chem. Lett. 2017, 27, 3243–3248.
[CrossRef] [PubMed]
3
3. Hoekstra, W.J.; Garvey, E.P.; Moore, W.R.; Rafferty, S.W.; Yates, C.M.; Schotzinger, R.J. Design and
optimization of highly-selective fungal CYP51 inhibitors. Bioorg. Med. Chem. Lett. 2014, 24, 3455–3458.
[CrossRef] [PubMed]
3
3
4. Gao, P.; Cui, Y.L.; Wu, R.L. Molecular dynamic modeling of CYP51B in complex with azole inhibitors.
J. Biomol. Struct. Dyn. 2017, 28, 1–9. [CrossRef] [PubMed]
5. Cuenca-Estrella, M.; Lee-Yang, W.; Ciblak, M.A.; Arthington-Skaggs, B.A.; Mellado, E.; Warnock, D.W.;
Rodríguez-Tudela, J.L. Comparative evaluation of NCCLS M27-A and EUCAST broth microdilution
procedures for antifungal susceptibility testing of Candida species. Antimicrob. Agents Chemother. 2002
6, 3644–4647. [CrossRef] [PubMed]
,
4
3
6. Aggarwal, R.; Singh, G.; Sanz, D.; Claramunt, R.M.; Torralba, M.C.; Torres, M.R. NBS mediated one-pot
regioselective synthesis of 2,3-disubstitutedimidazo[1,2-a]pyridines and their unambiguous characterization
through 2D NMR and X-ray crystallography. Tetrahedron 2016, 72, 3832–3838. [CrossRef]
3
7. Horowitz, S.; Trievel, R.C. Carbon-Oxygen Hydrogen Bonding in Biological Structure and Function. J. Biol. Chem.
2012, 287, 41576–41582. [CrossRef] [PubMed]
3
8. Chen, W.; Lee, M.K.; Jefcoate, C.; Kim, S.C.; Chen, F.; Yu, J.H. Fungal cytochrome p450 monooxygenases:
Their distribution, structure, functions, family expansion, and evolutionary origin. Genome Biol. Evol. 2014, 6,
1
620–1634. [CrossRef] [PubMed]
9. Hovmöller, S.; Zhou, T.; Ohlson, T. Conformations of amino acids in proteins. Acta Crystallogr. D Biol. Crystallogr.
002, 58, 768–776. [CrossRef] [PubMed]
0. Ji, H.; Zhang, W.; Zhou, Y.; Zhang, M.; Zhu, J.; Song, Y.; Lü, J. A three-dimensional model of lanosterol
4alpha-demethylase of Candida albicans and its interaction with azole antifungals. J. Med. Chem. 2000, 43,
493–2505. [CrossRef] [PubMed]
3
4
2
1
2
4
4
4
1. Yu, S.; Chai, X.; Hu, H.; Yan, Y.; Guan, Z.; Zou, Y.; Sun, Q.; Wu, Q. Synthesis and antifungal evaluation of
novel triazole derivatives as inhibitors of cytochrome P450 14alpha-demethylase. Eur. J. Med. Chem. 2010, 45,
4
435–4445. [CrossRef] [PubMed]
2. Wu, J.; Ni, T.; Chai, X.; Wang, T.; Wang, H.; Chen, J.; Jin, Y.; Zhang, D.; Yu, S.; Jiang, Y. Molecular docking,
design, synthesis and antifungal activity study of novel triazole derivatives. Eur. J. Med. Chem. 2018, 143,
1
840–1846. [CrossRef] [PubMed]
3. Salazar, S.B.; Wang, C.; Munsterkotter, M.; Okamoto, M.; Takahashi-Nakaguchi, A.; Chibana, H.; Lopes, M.M.;
Guldener, U.; Butler, G.; Mira, N.P. Comparative Genomic and Transcriptomic Analyses Unveil Novel Features of
Azole Resistance and Adaptation to the Human Host in Candida Glabrata. FEMS Yeast Res. 2018, 18. [CrossRef]
[PubMed]
4
4
4
4. Mazari, W.; Boucherit-Otmani, Z.; Boucherit, K. In Vitro Susceptibility of Amphotericin-B, Voriconazole
and Caspofungin Against Candida Guilliermondii Biofilms, Isolated from Dentals Units Water Pipes, under
Different Growth Phases. J. Mycol. Med. 2015, 25, 57–62. [CrossRef] [PubMed]
5. Pfaller, M.A.; Boyken, L.; Hollis, R.J.; Kroeger, J.; Messer, S.A.; Tendolkar, S.; Diekema, D.J. Wild-Type MIC
Distributions and Epidemiological Cutoff Values for Posaconazole and Voriconazole and Candida spp. as
Determined by 24-Hour CLSI Broth Microdilution. J. Clin. Microbiol. 2011, 49, 630–637. [CrossRef] [PubMed]
6. Chryssanthou, E.; Cuenca-Estrella, M. Comparison of the Antifungal Susceptibility Testing Subcommittee
of the European Committee on Antibiotic Susceptibility Testing Proposed Standard and the E-Test with

Molecules 2018, 23, 0
17 of 17
the NCCLS Broth Microdilution Method for Voriconazole and Caspofungin Susceptibility Testing of Yeast
Species. J. Clin. Microbiol. 2002, 40, 3841–3844. [PubMed]
4
7. Chai, X.; Zhang, J.; Cao, Y.; Zou, Y.; Wu, Q.; Zhang, D.; Jiang, Y.; Sun, Q. New azoles with antifungal activity:
Design, synthesis, and molecular docking. Bioorg. Med. Chem. Lett. 2011, 21, 686–689. [CrossRef] [PubMed]
8. Al-Wabli, R.I.; Al-Ghamdi, A.R.; Ghabbour, H.A.; Al-Agamy, M.H.; Monica, J.C.; Joe, I.H.;
Attia, M.I. Synthesis, X-ray single crystal structure, molecular docking and DFT computations on
N-[(1E)-1-(2H-1,3-benzodioxol-5-yl)-3-(1H-imidazol-1-yl)propylidene]-hydroxylamine: A new potential
antifungal agent precursor. Molecules 2017, 22, 373. [CrossRef] [PubMed]
4
4
5
5
5
5
9. Webb, B.; Sali, A. Comparative protein structure modeling using Modeller. Curr. Protoc. Bioinform. 2014, 47,
5
–6. [CrossRef]
0. Berman, H.M.; Westbrook, J.; Feng, Z.; Gilliland, G.; Bhat, T.N.; Weissig, H.; Shindyalov, I.N.; Bourne, P.E.
The Protein Data Bank. Nucleic Acids Res. 2000, 28, 235–242. [CrossRef] [PubMed]
1. Dassault Systèmes BIOVIA, Discovery Studio Modeling Environment; Release 2017; Dassault Systèmes:
San Diego, CA, USA, 2016.
2. Laskowski, R.A. PDBsum: Summaries and analyses of PDB structures. Nucleic Acids Res. 2001, 29, 221–222.
[CrossRef] [PubMed]
3. Morris, G.M.; Huey, R.; Lindstrom, W.; Sanner, M.F.; Belew, R.K.; Goodsell, D.S.; Olson, A.J. Autodock4 and
AutoDockTools4: Automated docking with selective receptor flexiblity. J. Comput. Chem. 2009, 30, 2785–2791.
[CrossRef] [PubMed]
5
5
4. O’Boyle, N.M.; Banck, M.; James, C.A.; Morley, C.; Vandermeersch, T.; Hutchison, G.R. Open Babel: An open
chemical toolbox. J. Cheminf. 2011, 3, 33. [CrossRef] [PubMed]
5. Williams, C.J.; Headd, J.J.; Moriarty, N.W.; Prisant, M.G.; Videau, L.L.; Deis, L.N.; Verma, V.; Keedy, D.A.;
Hintze, B.J.; Chen, V.B.; et al. MolProbity: More and better reference data for improved all-atom structure
validation. Protein Sci. 2018, 27, 293–315. [CrossRef] [PubMed]
5
5
6. Hsin, J.; Arkhipov, A.; Yin, Y.; Stone, J.E.; Schulten, K. Using VMD: An introductory tutorial.
Curr. Protoc. Bioinform. 2008. [CrossRef]
7. Phillips, J.C.; Braun, R.; Wang, W.; Gumbart, J.; Tajkhorshid, E.; Villa, E.; Chipot, C.; Skeel, R.D.; Kalé, L.;
Schulten, K. Scalable molecular dynamics with NAMD. J. Comput. Chem. 2005, 26, 1781–1802. [CrossRef]
[PubMed]
Sample Availability: Samples of the compounds are not available from the authors.
©
2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).