M.G. Gündüz, C. Dengiz, E. Koçak Aslan et al.
Journal of Molecular Structure 1247 (2022) 131316
ture was poured into ice-water. The precipitated solid was filtered,
washed with water and used for the next step without any further
purification.
In silico prediction of physicochemical properties
Physicochemical descriptors and drug-likeness properties of the
General procedure for the synthesis of DHP-azole hybrids
The target compounds were achieved according to an un-
symmetrical Hantzsch reaction. Equimolar amounts of 1,3-
cyclohexanedione, ethyl acetoacetate and appropriate 4-azolyl ben-
zaldehyde were refluxed for 8h in absolute ethanol in the presence
of excess ammonium acetate. The precipitated solid, upon cooling,
was filtered and washed with ethanol.
Antimicrobial activity determination
Minimum inhibitory concentration (MIC) values of DHP-azole
hybrids were determined according to the standard broth micro-
dilution assays, following the Standards of the European Commit-
tee on Antimicrobial Susceptibility Testing [29] for Candida spp.
(Candida albicans ATCC 10231, Candida parapsilosis ATCC 22019,
Candida krusei ATCC 6258, Candida glabrata ATCC 2001), and ac-
cording to the standard broth micro-dilution assays, recommended
by the National Committee for Clinical Laboratory Standards (M07-
A8) [30] for bacteria (Methicillin-resistant Staphylococcus aureus
(MRSA) ATCC 43300, Staphylococcus aureus ATCC 25923, Escherichia
coli NCTC 9001). The tested compounds were dissolved in DMSO
at a concentration of 50 mg/mL, and the highest used concentra-
tion was 500 μg/mL. For the MIC assessment, the inoculums were
1 × 105 colony forming units (CFU/mL), for Candida species, and
5 × 105 CFU/mL for bacteria. The MIC value was recorded as the
lowest concentration that inhibited the growth after 24 h at 37°C,
by inoculation of 5 μL from each well (after 24 h incubation at
37°C) on Luria Agar (LA) plates for bacteria and Sabouraud agar
(SAB) plates for Candida spp. followed with further incubation at
37°C for 24 h.
Ethyl 4-(4-(1H-pyrazol-1-yl)phenyl)-2-methyl-5-oxo-1,4,5,6,7,8-
hexahydroquinoline-3-carboxylate (DHP-P): Pale yellow solid,
yield: 68%. m.p. 199°C. IR (ν, cm−1): 3288 (N-H), 1684 (C=O, es-
ter), 1644 (C=O, ketone), 1605 (C=N). 1H-NMR (δ, DMSO-d6): 1.14
(t, J = 7.1 Hz, 3H), 1.73-1.79 (m, 2H), 1.89-1.93 (m, 2H), 2.16-2.23
(m, 2H), 2.31 (s, 3H), 3.99 (q, J = 7.1 Hz, 2H), 4.93 (s, 1H), 6.50 (dd,
J = 2.4, 1.8 Hz, 1H), 7.24 (d, J = 8.6 Hz, 2H), 7.63 (d, J = 8.6 Hz, 2H),
7.69 (d, J = 1.4 Hz, 1H), 8.35-8.37 (m, 1H), 9.18 (s, 1H). 13C-NMR (δ,
DMSO-d6): 14.6, 18.7, 21.2, 26.6, 35.7, 37.1, 59.5, 103.7, 107.9, 111.4,
118.7, 128.0, 128.8, 138.1, 141.0, 145.6, 146.3, 151.9, 167.2, 195.1.
Anal. Calcd. for C22H23N3O3; C, 70.01; H, 6.14; N, 11.13. Found: C,
69.78; H, 6.29; N, 10.98.
Ethyl
4-(4-(1H-imidazol-1-yl)phenyl)-2-methyl-5-oxo-
1,4,5,6,7,8-hexahydroquinoline-3-carboxylate (DHP-I): Pale yellow
solid, yield: 28%. m.p. 264°C. IR (ν, cm−1): 3197 (N-H), 1694 (C=O,
ester), 1648 (C=O, ketone), 1621 (C=N). 1H-NMR (δ, DMSO-d6):
1.15 (t, J = 7.1 Hz, 3H), 1.73-1.78 (m, 2H), 1.89-1.94 (m, 2H), 2.17-
2.24 (m, 2H), 2.31 (s, 3H), 4.00 (q, J = 7.1 Hz, 2H), 4.94 (s, 1H),
7.05-7.09 (m, 1H), 7.26 (d, J = 8.6 Hz, 2H), 7.44 (d, J = 8.6 Hz, 2H),
7.63 (t, J = 1.3 Hz, 1H), 8.11-8.13 (m, 1H), 9.20 (s, 1H). 13C-NMR (δ,
DMSO-d6): 14.6, 18.7, 21.2, 26.5, 35.8, 37.1, 59.6, 103.3, 111.3, 118.6,
120.7, 129.1, 130.0, 135.2, 135.9, 145.7, 147.0, 152.0, 167.2, 195.1.
Anal. Calcd. for C22H23N3O3; C, 70.01; H, 6.14; N, 11.13. Found: C,
69.86; H, 6.25; N, 11.13.
Molecular docking
Three dimensional crystal structure of CYP51 from Candida albi-
cans was downloaded from RCSB Protein Data Bank (www.rcsb.org)
under the PDB code 5V5Z [31]. Chemical structures of the synthe-
sized compounds were drawn using ChemDraw and their energies
were minimized with MM2 force field. Molecular modeling stud-
ies were performed using AutoDock Software (v1.5.6, The Scripps
Research Institute, San Diego, CA) [32]. For validation of docking
parameters, the co-crystallized ligand of 5V5Z was redocked into
Ethyl
4-(4-(1H-1,2,4-triazol-1-yl)phenyl)-2-methyl-5-oxo-
1,4,5,6,7,8-hexahydroquinoline-3-carboxylate
(DHP-T): White
solid, yield: 32%. m.p. 255°C. IR (ν, cm−1): 3274 (N-H), 1698(C=O,
ester), 1650 (C=O, ketone), 1622 (C=N). 1H-NMR (δ, DMSO-d6):
1.14 (t, J = 7.1 Hz, 3H), 1.70-1.81 (m, 2H), 1.89-1.94 (m, 2H),
2.14-2.25 (m, 2H), 2.31 (s, 3H), 3.99 (q, J = 7.1 Hz, 2H), 4.95 (s,
1H), 7.31(d, J = 8.5 Hz, 2H), 7.65 (d, J = 8.5 Hz, 2H), 8.19 (s, 1H),
9.15 (s, 1H), 9.20 (s, 1H). 13C-NMR (δ, DMSO-d6): 14.6, 18.7, 21.2,
26.5, 36.0, 37.3, 59.6, 103.5, 111.2, 119.8, 129.1, 135.1, 142.6, 145.8,
148.0, 152.0, 152.6, 167.2, 195.1. Anal. Calcd. for C21H22N4O3; C,
66.65; H, 5.86; N, 14.81. Found: C, 66.34; H, 5.69; N, 14.86.
˚
the active site of the enzyme (0.96 A root-mean-square deviation).
The obtained binding modes were visualized using Maestro [33].
Results and discussion
Chemistry
In this study, azole carrying benzaldehydes were obtained
through the nucleophilic aromatic substitution reaction of 4-
fluorobenzaldehyde with various azole rings (pyrazole, imidazole
or 1,2,4-triazole). The reactions were performed initially applying
ultrasonic irradiation followed by heating in oil bath in DMSO in
the presence of potassium carbonate as the base (Scheme 1).
In these reactions, we benefited from the advantages of ultra-
sonic irradiation such as shortening the reaction times and avoid-
ing using excess amount of amine, extreme conditions or transi-
tion metal catalyst reported in the literature for the nucleophilic
aromatic substitutions of haloarenes with amines [34–36].
Theoretical studies
The molecular structures of DHP-P, DHP-I, and DHP-T were
optimized at the B3LYP/6-31++G(d,p) [24] level of theory by us-
ing the software package Gaussian 09. Solvation in EtOH was
applied using the conductor-like polarizable continuum model
(CPCM) [25]. All structures are confirmed ground-state minima
according to the analysis of their analytical frequencies com-
puted at the same level, which show no imaginary frequencies.
On these minima, the vertical transition energies were calcu-
lated by time-dependent density functional theory (TD-DFT) at the
CAM-B3LYP/6-31++G(d,p). The isosurfaces of the frontier orbitals
(shown at 0.02 a.u.) were computed at the B3LYP/6-31++G(d,p)
level of theory, again with the CPCM solvation model in EtOH. NLO
properties of DHP-P, DHP-I, and DHP-T were predicted by DFT cal-
culations using basis set CAM-B3LYP/6-31++G(d,p) level of theory
Subsequently, the target compounds, DHP-azole hybrids, were
synthesized by the reaction of dicarbonyl compounds (1,3-
cyclohexanedione and ethyl acetoacetate), appropriate 4-azolyl
benzaldehyde and ammonium acetate according to a modified
Hantzsch reaction as shown in Scheme 2. Although Schade et al.
reported the synthesis of pyrazole-carrying DHP as TGFβ inhibitor
before [37], unlike in the mentioned paper, we employed in-house
synthesized 4-(1H-pyrazol-1-yl)benzaldehyde for the achievement
of the target scaffold instead of using a commercial aldehyde.
4