S. Chortani, M. Horchani, M. Znati et al.
Journal of Molecular Structure 1230 (2021) 129920
compound (in DMSO) was mixed with 0.5 mL of α-amylase solu-
tion (0.5 mg/mL) with 0.02 M sodium phosphate buffer (pH 6.9
with 0.006 M NaCl). The mixture was incubated at room temper-
ature for 10 min and 0.5 ml of starch solution (1%) in 0.02 M
sodium phosphate buffer (pH 6.9 with 0.006 M NaCl) was added.
The resulting mixture was incubated at room temperature for
10 min, and the reaction was terminated using 1 mL of dinitros-
alicylic acid color reagent. At this time, the test tubes were placed
in a water bath (100 °C and 5 min) and cooled until room tem-
perature was reached. The mixture was then diluted with 10 mL
of deionized water, and absorbance was determined at 540 nm.
The absorbances of blank (buffer and DMSO instead of the tested
compound and amylase solution) and control (buffer and DMSO
instead of the tested compound) samples were also determined.
Acarbose was used as a positive control. The inhibition of α-
amylase was calculated using the following equation:
Scheme 1. Synthetic pathway to benzopyrimidinone derivatives.
ANOVA. Means were separated at the 5% significance level by a
least significant difference test (Student’s test).
%
inhibition of α-Amylase
=
(Abscontrol
–
Abssample)
3. Results and discussion
/(Abscontrol) × 100
Where Abscontrol corresponds to the absorbance of the solu-
tion containing only α-amylase and the buffer instead of the com-
pound, and Abssample corresponds to the absorbance of the solution
in the presence of both tested compound and α-amylase. Com-
pound concentration providing 50% inhibition (IC50) was obtained
plotting the inhibition percentage against the concentrations of the
tested compound. The tests were carried out in triplicate.
3.1. Synthesis
The synthetic strategy adopted to obtain the target compounds
are depicted in Scheme 1.The synthesis of new benzopyrimidinone
derivatives was done by the chloroacylation of 2-aminobenzamide
1 using various acyl chloride on steam bath for one hour followed
by treatment with sodium carbonate solution [24]. The formed
compounds were characterized according to their spectral data.
In fact, the 1H NMR spectrum of compound 2b, as an exam-
ple, showsa doublet at δH 1.84 (J = 6.6 Hz) relative to the methyl
group and a quadruplet at δH 5.03 (J = 6.6 Hz) attributable to
the methane proton, both introduced by the 2-chloropropanoyl
chloride, and the presence a characteristic singlet at δH 12.54
assignable to the mobile proton –NH. The remarkable de-shielding
of the methine proton isexplained by the inductive attractor ef-
fect exerted by the neighboring chlorine atom and the two nitro-
gen atoms of the pyrimidinone ring (one hybridized sp2 attarctor,
and the other conjugated with the carbonyl function), thus leav-
ing a partial positive charge on the sp2 quaternary carbone directly
linked to the methine. Moreover, the exploration of the 13C NMR
spectrum allowed to notes the appearance of new signals at δC
21.7 and 55.4 due to carbons C9 and C10, respectively.
2.2.3. Molecular docking procedure
Molecular docking simulations were performed by the Auto
Dock 4.2 program package [15]. The optimization of all the geome-
tries of scaffolds was performed with ACD (3D viewer) software
structures of PDB (PDB: 4W93) was obtained from the RSCB pro-
tein data bank [16]. First, the water molecules were eliminated and
the missing hydrogens and Gasteiger charges were then added to
the system during the preparation of the receptor input file. In-
deed, Auto Dock Tools were used for the preparation of the cor-
responding ligand and protein files (PDBQT). Next, pre-calculation
of the grid maps was carry out using Auto Grid for saving a lot
of time during docking. Subsequently, the docking calculation was
˚
performed using a grid per map with 40 × 40 × 40 A points in ad-
Finally, the ES-HRMS of this compounds howed a pseudo-
molecular ion peak [M + H]+at m/z 209.0484 in agreement with
its molecular formula (C10H9ClN2O)+.
˚
dition to grid-point spacing of 0.375 A, which was centered on the
receptor in order to define the active site. After complex formation,
the visualization and analysis of interactions were performed us-
3.2. In vitro α-amylase inhibitory activity
The in vitro α-amylase inhibitory activity of compounds 2a-d
were assessed by measuring the inhibition percentage (PI%). As
shown in Table 1, the evaluated compounds exhibited significant
inhibitory potentials towards α-amylase with IC50 values rang-
ing from 60 to 31 μg/mL compared to acarbose used as a pos-
–
2.2.4. Computational details (DFT studies)
Density functional theory plays an important role in determin-
ing the molecular electronic structure by computer simulations.
The studied products were modelled with the Gauss View program
[17] and, later optimized with the Gaussian 09 program [18] by us-
ing the functional hybrid (Becke, three-parameter, Lee-Yang-Parr)
B3LYP with the 6–311++G(d, p) basis set [19, 20]. Thermodynamic
parameters and molecular electrostatic potentials were predicted
at the same level of theory. Additionally, reactivities and behav-
iors of the studied products were predicted by using calculations
of frontier orbitals and the chemical potential (μ) electronegativity
(χ), global softness (S), global hardness (η), global electrophilicity
index (ω) and nucleophilicity indexes (E) descriptors [21-23].
itive control (IC50=29.86
1.57 μg/mL). Compound 2a(-CH2 Cl),
was found to be the less active derivative with an IC50value of
60.07 1.89 μg/mL. On the other hand, it has been found that
the compound 2b (CH3 CH Cl) exhibited the highest α-amylase
inhibition activity with an IC50 value of 31.12 1.05 μg/mL, com-
–
–
parable to that of acarbose. The significant activity of compound
2b compared to that of its analogue 2a is certainly due to the
additional methyl group which, by its inductive donor effect and
its spatial arrangement in this position, could promote this ac-
–
tivity. The compound 2c (Cl-CH Cl) was found to be more ac-
2.2.5. Statistical analysis
tive than 2a. This finding clearly shows the contribution of the
two chlorine atoms to this activity. On the other hand, the com-
parison of the activity of derivative 2c with that of 2b shows
that the substitution of the methyl group in the latter by a sec-
ond chlorine atom (compound 2c) did not improve this activity
Data from the bioassay carried out were subjected to analysis
of variance (ANOVA) using SPSS 16.0 for Windows. The inhibition
data of α-amylase activity of compounds 2a-d have been trans-
formed using arcsin- square root (arcsin x) transformation before
√
3