Synthesis, conformational analysis and molecular docking studies on three novel dihydropyrimidine derivatives

Article abstract of DOI:10.1016/j.molstruc.2019.04.100

The dihydropyrimidine (DHPM)derivatives I, II and III were synthesized through Biginelli reaction and characterized by X-ray diffraction and vibrational spectroscopic analyses (Raman and FTIR), whereas a conformational study was conducted. The results sho

Full text of DOI:10.1016/j.molstruc.2019.04.100

Accepted Manuscript
Synthesis, conformational analysis and molecular docking studies on three novel
dihydropyrimidine derivatives
Lóide.O. Sallum, Wesley.F. Vaz, Nádia.M. Borges, Carlos.E.M. de Campos,
Adailton.J. Bortoluzzi, Chris.H.J. Franco, Luciana.M. Ramos, Hamilton.B. Napolitano
PII:
S0022-2860(19)30509-5
DOI:
https://doi.org/10.1016/j.molstruc.2019.04.100
MOLSTR 26472
Reference:
To appear in: Journal of Molecular Structure
Received Date: 7 December 2018
Accepted Date: 24 April 2019
Please cite this article as: Ló.O. Sallum, W.F. Vaz, Ná.M. Borges, C.E.M. de Campos, A.J. Bortoluzzi,
C.H.J. Franco, L.M. Ramos, H.B. Napolitano, Synthesis, conformational analysis and molecular docking
studies on three novel dihydropyrimidine derivatives, Journal of Molecular Structure (2019), doi: https://
doi.org/10.1016/j.molstruc.2019.04.100.
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ACCEPTED MANUSCRIPT
Synthesis, conformational analysis and molecular docking studies on three novel
dihydropyrimidine derivatives
a
a
a
b
Lóide O. Sallum , Wesley F. Vaz , Nádia M. Borges , Carlos E. M. de Campos , Adailton J.
b
c
a
a
Bortoluzzi , Chris H. J. Franco , Luciana M. Ramos and Hamilton B. Napolitano *
a
Universidade Estadual de Goiás, Anápolis, GO, Brazil
b
Universidade Federal de Santa Catarina, Florianópolis, SC, Brazil
c
Universidade Federal de Juiz de Fora, MG, Brazil
Abstract
The dihydropyrimidine (DHPM) derivatives I, II and III were synthesized through Biginelli
reaction and characterized by X-ray diffraction and vibrational spectroscopic analyses (Raman and
FTIR), whereas a conformational study was conducted. The results show that the DHPM I, II and
III belong to the triclinic and monoclinic systems. In a general way, the crystal structure of these
…
…
…
…
…
compounds are stabilized with N–H O, O–H O and N–H S interactions. The C–H π and π π
contacts were evaluated using the Hirshfeld surfaces. To evaluate eletronic properties of the DHPM
set, theoretical calculations, using B3LYP/6-31G(d,p) method were undertaken. Also, the calculated
infrared spectra with some global reactivity descriptors, the molecular electrostatic potential maps
and frontier molecular orbital energies were obtained for these compounds. Finally, molecular
docking was used to investigate the applicability of DHPM set as a tumor suppressor (L3MBTL1).
Keywords: dihydropyrimidine; X-ray diffraction; molecular docking.

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Introduction
Heterocyclic chemistry is important in modern organic chemistry research [1]. The Biginelli
2] reaction represents a classical approach for the preparation of 3,4-dihidropyrimidin-2(1H)-ones
[
by the one-pot condensation of an aromatic aldehyde, β-dicarbonyl and urea/thiourea in the
presence of a mineral acid catalyst [3], basic catalyst [4], SiO -CuCl [5], p-toluenesulfonic acid [6],
2
2
Keggin and Dawson-type polyoxometalates [7], β-Cyclodextrin-propyl sulfonic acid [8] and others
9]. Numerous multifunctionalized dihydropyrimidine derivatives were obtained through
[
cyclocondensation process, which increased their production considerably by using different
substituents on all three building blocks. For this specific heterocyclic frame the acronym DHPM
was adopted from the literature [3]. Heterocyclic DHPM have great importance in human biological
system, once they are an essential part of natural products, various pharmacologically active
molecules and nucleic acids. DNA & RNA base pairs (guanine, thymine, cytosine and adenine) are
constituted by heterocyclic DHPM such as purine, pyrimidine and others. These heterocyclic
DHPM can also be found in numerous drug candidates [9]. Although discovering a DHPM
compound is an arduous and time-consuming process their applications to medicinal chemistry and
its related fields are broad [10]. Some of the therapeutic and pharmacological properties of these
DHPM are: antiviral, anti-inflammatory, anticarcinogenic, antibacterial, antihypertensive, calcium
channel modulators, additionally, DHPM has promising application in the early suppression of
breast cancer cells [11–15].
Thus, a comparative structural and supramolecular arrangement study is proposed involving
5-acetyl-3,4-dihydro-4-(4-hydroxy-3-methoxyphenyl)-6-methyl-2(1H)-pyrimidinone (DHPM I),
the Ethyl-4-(4-hydroxy-3-methoxyphenyl)-6-methyl-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-
carboxylate (DHPM II) and 1-[1,2,3,4-tetrahydro-4-(4-hydroxy-3-methoxyphenyl)-6-methyl-2-
thioxo-5-pyrimidinyl]-ethanone (DHPM III). The geometry optimization, global reactivity
descriptors, molecular electrostatically potential and vibrational wavenumbers assignments for
DHPM I, II and III were calculated at the B3LYP/6-31G (d,p) level of theory. In addition,

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molecular docking analysis were made for these three compounds because of the biological activity
of DHPM I against human Lethal (3) malignant brain tumor-like protein 1, L3MBTL1.
Experimental and computational procedures
Synthesis and crystallization
DHPM I was synthesized by the one-pot reaction of acetylacetone (0.300g, 3.00 mmol), 4-
hydroxy-3-methoxybenzaldehyde (1.37 g, 9.00 mmol) and urea (0.18g, 3.00 mmol), whereas, to
obtain DHPM II, ethyl acetoacetate (0.39g, 3.00 mmol), 4-hydroxy-3-methoxybenzaldehyde (1.37
g, 9.00 mmol) and thiourea (0.22g, 3.00 mmol) were used. Finally, acetylacetone (0.300g, 3.00
mmol), 4-hydroxy-3-methoxybenzaldehyde (1.37 g, 9.00 mmol) and thiourea (0.22g, 3.00 mmol),
in the presence of MAI.Fe Cl (5mol%) and 1 mL of BMI.BF at 80ºC for 2 h, were used as a
2
7
4
model reaction [17] to obtain DHPM III, as shown in scheme 1.
OH
O
OCH3
O
H
X
2 7
MAI.Fe Cl (5mol/%)
O
+
R
+
HO
4
BMI.BF , 80ºC, 2h
H N
2
NH2
OCH3
R
NH
X
O
R = Me, OEt
X = O, S
N
H
Scheme 1. Chemical scheme of the synthesized DHPM: [DHPM I] R = Me, X = O; [DHPM II] R =
OEt, X = S and [DHPM III] R = Me, X = S.
The pure DHPM were obtained by filtering, drying and recrystallizing the precipitate with
ethanol. Melting point measurements (Table S1) and infrared spectroscopic were used to analyze
the purity of some samples. Infrared spectra were recorded on a PerkinElmer Frontier in the range
-1
4
000-400 cm using the KBr pellet technique. DHPM I: White powder, m.p. 240 °C; FT-IR (KBr,
-1
cm ): 3343, 3298, 1691, 1638, 1527, 1442, 1377, 1331, 1240, 1037, 756. DHPM II: White powder,
-1
m.p. 220-222 °C; FT-IR (KBr, cm ): 3416, 3181, 2998, 1685, 1587, 1522, 1483, 1378, 1180, 1025,

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-1
7
51. DHPM III: Yellow powder, m.p. 238-239 °C; FT-IR (KBr, cm ): 3468, 3189, 2996, 1629,
1587, 1504, 1456, 1373, 1193, 1125, 752.
Crystals of DHPM I, II and III were grown from ethanolic solutions by slow evaporation
method. Prismatic colorless crystals were obtained in a period of 15 days at room temperature
25ºC) with a semi-open bottle. To conduct the X-ray data collection, an appropriate single crystal
(
was selected from homogeneous crystalline samples of each DHPM compound.
Crystallographic characterization
A single crystal for DHPM I and III were selected and data were collected on a Rigaku
SuperNova diffractometer with AtlasS2 detector, using radiation MoKα (λ=0.71073 Å) at 293K. A
single crystal for DHPM II was selected and a Bruker APEX 2 diffractometer was utilized to collect
data, using CuKα (λ=1.54178 Å) at 110K. Direct methods were applied to solve these structures and
least square methods to refine them using SHELX [18] in WingGX package [19]. The ellipsoids
diagrams were generated from ORTEP [19] (Figure 1) and the packing diagrams were generating
using Mercury [20]. Geometrical calculations were done using PLATON [21]. The non-hydrogen
atoms were refined anisotropically. The Hydrogen bonded to Carbon and Nitrogen were placed
geometrically and refined using a riding model with distance of N – H = 0.86ꢀÅ and aromatic C – H
=
0.93ꢀÅ with Uiso(H) = 1.2 Ueq and 1.5 Ueq, respectively. The Crystallographic Information Files
(CIF) of for DHPM I, DHPM II and DHPM III were deposited in the Cambridge Structural Data
Base under codes 1882886, 1882887 and 1883842, respectively. Copies of the data can be obtained
free of charge via www.ccdc.cam.ac.uk.
Hirshfeld surface
The Hirshfeld surface (HS) consists of a spatial map which makes use of color coding for
representing the proximity of close contacts present around a certain molecule within a given
network [22]. Two models of electron density are the means of obtaining the HS; such models

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combine the overlapping of isolated molecules. These being illustrated by isolated spherical atoms,
together with another one of interacting molecules, where ꢁ ꢃꢄꢅ is determined a weight function
ꢂ
for each of the atoms in a molecule, as shown in Equation 1,
ꢂꢇ
ꢂꢇ
(Eq. 1)
ꢁꢂꢃꢄꢅ =ꢀꢆ ꢃꢄꢅ/ ꢈ
ꢆ ꢃꢄꢅ
ꢉ
ꢂ
ꢉ∈ꢊꢋꢌꢍꢎꢏꢌꢍ
ꢂꢇ
where ꢆ ꢃꢄꢅ represents the electrical densities of the various spherically averaged atoms, whereas
ꢉ
Equation 2 defines the electron density of an atomic fragment,
ꢆ ꢃꢄꢅ =ꢀꢁ ꢃꢄꢅꢆꢊꢋꢌꢃꢄꢅ
(Eq. 2)
ꢂ
ꢂ
ꢆꢊꢋꢌꢃꢄꢅ designates the molecular electron density. The normalized contact distance ꢐꢑꢋꢄꢊ could be
defined as the sum of the symmetric distances to the nearest atoms outside, ꢃꢐ ꢅ, and inside, (ꢐ )
ꢍ
ꢉ
of the surface. See Equation 3:
ꢐꢑꢋꢄꢊ = ꢃꢐ −ꢀꢄ ꢅ/ꢄ ꢒꢉ ꢐꢁ +ꢀꢃꢐ − ꢄꢍ ꢅ/ꢄ ꢒꢍ ꢐꢁ
ꢒꢐꢁ
ꢒꢐꢁ
(Eq. 3)
ꢉ
ꢉ
ꢍ
ꢒꢐꢁ
ꢉ
and ꢄ ꢒꢍ ꢐꢁ are the atoms’ van der Waals radii. Crystal Explorer 3.1 [23] was used to generate
ꢄ
HS intermolecular interactions and the 2D fingerprint plots.
Powder Diffraction and Raman
X-ray powder diffraction for DPHM I and II were performed on an Advance DaVinci D8
diffractometer using CuKα (1.54056 Å) with Bragg-Brentano θ-θ geometry and LynxEye linear
detector. The experimental data were collected in the 2θ range from 5° to 60°, in steps of 0.01° and
accumulation time ranging from 0.5s to 1s by steps. X-ray powder diffraction for DPHM II was
collected on a Xpert MPD diffractometer using CuKα (1.5418 Å) with Bragg-Brentano θ-θ
geometry and Xcelerator linear detector. The experimental data were collected in the 2θ range from
5° to 55°, in steps of 0.0167° and 3 scans with accumulation time of 19.685 s by step. Fourier-

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transform Raman spectroscopy for DPHM I and II were carry out using a Bruker RFS 100
3
+
spectrometer with Nd /YAG laser operating at 1064 nm excitation line (near infrared region) and
-1
CCD detector with spectral resolution of 4 cm . A good signal was obtained with 1024 scans.
Raman spectra of different regions of a DHPM II sample surface were collected with 20x objective
lens at room temperature using a PeakSeeker 785 (RAM–PRO–785) Raman system with a diode
laser of 785 nm and 100 mW at the source. The backscattered Raman radiation was dispersed with a
grating and focused on a Peltier-cooled charge-coupled device CCD detector allowing us to
-1
obtaining a spectral resolution of 6 cm . All spectra were recorded at room temperature in the
-1
spectral window of (from 200 to 2000) cm with same acquisition time (10 s).
Theoretical calculation
The theoretical calculations were made using the geometric parametres derived from the CIF
files of each structure. These structures were optimized by means of Density functional theory
(
DFT) using the hybrid functional B3LYP and 6-31G(d,p) basis set with the Gaussian 09 package
24]. The absence of imaginary frequencies confirm that were obtained strucutures in a minima of
[
energy, and so, it were used in the vibrational wavenumber calculations. The absence of imaginary
frequencies confirm that geometries are in a minima of energy, and so, can be used in the
vibrational calculations. The wavenumbers calculated were properly scaled [25] by the scale factor:
0.9620 and to assign the vibrational modes, the potential energy distribution analysis (PED) using
VEDA4 program [26] was used in combination with the normal mode animations in GaussView.
The same level of theory was used to calculate some global reactivity descriptors and to generate
the frontier molecular orbitals (FMO) and molecular electrostatic potential map (MEP) .
Molecular docking
Molecular docking is the most widely used tool for exploring the interactions between an
inhibitor molecule and the target protein. In our studies, the AutoDock-Vina software [27] was used

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to carry this out. The crystallographic structure was downloaded from the Protein Data Bank, PDB
(www.rcsb.org) [28]. Before docking, preparation of the ligand and receptor in Dock Prep [29] tool
was necessary. Amid the protein preparation procedure, all missing hydrogen atoms were included,
all water molecules and ligand erased, and the energy minimized utilizing Minimize Structure of
Chimera. Protein and ligand were managed through the general AMBER force field 12SB [30]. The
DHPM I, II and III were flexibly docked into the active site of the enzyme centered at the
3
geometric point (16.67 Å x 40.11 Å x 32.92 Å) and grid size of 17 Å . Each and every other setting
were kept as default setting.
Results and discussion
Solid State characterization
The ORTEP diagrams of DHPM I, II and III with their asymmetric units can be seen in
Figure 1. These molecules exist in the screw boat conformation with C4 and N1 forming the
flagpole atoms as described by Nayak et al. [31]. The carbonyl group C5=O2 appears in an s-cis
conformation with respect to the C2=C3 double bond [32]. Arbitrarily S configuration was chosen
for supramolecular analysis. The orientation of the methoxy group in the meta position is different
in these forms, for DHPM I methoxy group bounded in ring inverted meta position (C10), although
DHPM II and III bonded in ring standard meta position (C6).

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Figure 1. ORTEP diagram for (a) DHPM I, (b) DHPM II and (c) DHPM III. The ellipsoids are
represented at 50% of probability, for (a) and (c), while (b) is shown at 80% of probability.
Table 1 shows the main crystallographic parameters. The principal torsion angles in order to
describe the conformation differences among I, II and III are listed in Table 2. Table 3 lists the
intermolecular interactions observed in all crystal structures. The main bond lengths and bond
angles are depicted in Table S2.
Table 1. Crystallographic data and structure refinement for DHPM I, II and III.
Crystal data
DHPM I
DHPM II
DHPM III
Chemical formula
Molecular weight
Space group
C H N O
C H N O S
C H N O S
1
4
16
2
4
15 18
2
4
14 16
2
3
276.29
322.37
292.35
P2 /c
Pī
P2 /c
1
1
7
.2339 (2), 14.9870 (4),
2.3278 (4)
8.4527 (2), 9.2867 (3),
11.0363 (3)
12.3080 (14), 7.1963 (8),
16.9759 (19)
a, b, c (Å)
1
9
6.949 (1), 108.145 (1),
α, β, γ (°)
90, 92.161 (3), 90
90, 109.185 (12), 90
1
09.392 (1)
751.81 (4)
2
3
V (Å )
1335.57 (7)
4
1420.1 (3)
4
Z
Radiation type
Mo Kα
0.10
0.049
0.125
1.04
Cu Kα
2.10
0.029
0.074
1.05
Mo Kα
0.24
0.057
0.160
0.96
-
1
µ (mm )
2
2
R [F > 2σ(F )]
2
wR (F )
S
No. of reflections
No. of parameters
3609
185
0.28, -0.26
2539
203
0.29, -0.34
4971
187
0.26, -0.30
-
3
∆〉max, ∆〉min (e Å )
Table 2. Relevant experimental torsion angles (°) for DHPM I, II and III.
Torsion
DHPM I
-77.84 (14)
-98.78 (15)
122.87 (13)
156.86 (11)
102.62 (14)
65.60 (16)
DHPM II
-80.55 (15)
-93.01 (15)
167.12 (12)
156.11 (11)
99.29 (15)
106.82 (15)
-70.11 (16)
-15.95 (19)
DHPM III
-78.90 (4)
-94.10 (4)
137.70 (3)
157.30 (3)
100.60 (4)
79.40 (4)
C5
C5
C6
–
–
–
C2
C2
C5
–
–
–
C3
N2
C2
C2
C2
C5
C2
C2
–C12
–
C1
–
C3
C12
C4
N2
–
–
–
C3
C3
C2
C5
C5
–
–
N2
–
–
–
–
C5
–
C10
C6
–
–
N2
-112.54 (14)
-59.00 (17)
-98.80 (4)
-44.10 (4)
C10
–
–
–
C3
The crystal packing of DHPM is formed by the polar sheets and the functional group (if it is
involved in intermolecular interactions) present on the phenyl moiety (defined as ‘‘hydrophobic

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spacer’’) [16]. The DHPM I crystallized in the monoclinic system with space group P2 /c and Z=4
1
(Z’=1), which represents 4 molecules in the unit cell. The crystal packing of DHPM I is formed via
strong hydrogen bonds and weak interactions, where there are three potential hydrogen bond
donors, namely H1N, H2N, H4O and three potential acceptors, namely the carbonyl oxygen atom
O1, the hydroxyl oxygen O2 and the methoxy oxygen O3, respectively. These crystal packing of
…
DHPM I results in the formation of linear chain motifs utilizing N –H N O hydrogen bonds,
1
1
2
ꢔ
which can be described ꢓ (6), involving the ketone group generating strong motif in polar sheet
ꢔ
…
along the a axis (Figure 2a). While chain motif utilizing N –H N O hydrogen bonds (Figure 2b)
2
2
3
are responsible by generating motif in hydrophobic space involving the methoxy group along the c
ꢔ
axis, which can be described as ꢓ (7). Further, it is possible to observe an intermolecular hydrogen
ꢔ
…
ꢔ
bond of O –H O O , the graph set is given as ꢓ (10), which results in the generation of a zig zag
4
4
1
ꢔ
motif perpendicular to the ab plane (Figure 2c). Figure 2d shows the packing with the crystal
structure, S and R configurations are shown in stick and wireframe style, respectively.
Table 3. Hydrogen-bond geometry (Å, °) for DHPM I, II and III.
…
…
…
…
DHPM
I
D–H A
D–H
0.86
0.86
0.82
0.86
0.86
0.82
0.82
0.86
0.86
H A
D A
D–H A
Symmetry code
-1+x,y,z
x,1/2-y,-1/2+z
1+x,1/2-y,1/2+z
-x,-y,-z
1-x,-y,-z
2-x,1-y,1-z
1-x,1-y,-z
…
N1–H1N O2
N2–H2N O3
O4–H4O O1
N1–H1N S1
N2–H2N S1
O4–H4O O1
O3–H3O O2
N2–H2N S1
2.07
2.26
1.94
2.56
2.48
2.02
2.33
2.48
2.07
2.903
3.110
2.759
3.389
3.312
2.749
3.023
3.328
2.917
164.00
168.00
175.00
162.00
163.00
149.00
142.00
168.00
168.00
…
…
…
II
…
…
…
III
…
-x,2-y,-z
x,1+y,z
…
N1–H1N O1A
The packing diagram, in Figure 2, shows the motifs in the crystalline lattice. Previous
studies have been performed on these packing motifs of these DHPM [33,34].

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…
…
Figure 2. N–H O (a and b) and O–H O (c) intermolecular interactions and molecular packing (d)
of DHPM I.
It was observed that the molecular conformational and packing features with theoretical
calculation, powder diffraction and Raman did not study yet. It crystallizes in the triclinic system
with space group P
ī
and Z = 2, which represents two molecules in the unit cell. The crystal packing
…
ꢖ
consists of N–H S hydrogen bonded dimers form a ꢕ (8) [35-36] ring motif generating ribbons
ꢖ
…
along the a axis (Figure 3a). The hydroxyl group participates in strong interaction O –H O O
4
4
1
intermolecular hydrogen bonding generating dimers along the c axis, which can be described as
ꢖ
ꢗ (8) (Figure 3b). Figure 3c shows the packing diagram with motifs in the crystal structure. DHPM
ꢖ
II was characterized by X-ray powder diffraction and the results, shown in Figure S4, indicate that
the entire sample crystallizes in a single phase.

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…
…
Figure 3. N–H O (a and b) and O–H O (c) intermolecular interactions and molecular packing (d)
of DHPM I.
Similar to the DHPM I, the DHPM III crystallizes in the monoclinic system with space
group P2 /c and Z=4. The oxygen atom O1 derived from methoxy group finds itself disordered in
1
two occupation sites in the proportions 70:30. The crystal packing of DHPM III results in the
…
ꢖ
formation of dimeric motifs utilizing O –H O O hydrogen bonds, which can be described ꢗ (10),
3
3
2
ꢖ
involving the hydroxyl group generating strong motif in hydrophobic space (Figure 4a), while
…
dimeric motif utilizing N –H N S hydrogen bonds (Figure 4b) are responsible by generating
2
2
1
ꢖ
…
strong motif in polar sheets, which can be described as ꢗ (8). The intermolecular N -H N O
ꢖ
1
1
1A
ꢔ
hydrogen bonds, the graph set is given as ꢓ (6), link the molecules into polar sheet chains running
ꢔ
along the b axis (Figure 4c). Figure 4d shows the packing with motifs in the crystal structure, S and
R configurations are shown in stick and wireframe style, respectively. DHPM III was characterized
by X-ray powder diffraction and the results, shown in Figure S5, indicate that the entire sample
crystallizes in a single phase.

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…
…
Figure 4. N–H O (a and b) and O–H O (c) intermolecular interactions and molecular packing (d)
of DHPM III.
The HS mapped over ꢐꢑꢋꢄꢊ (ranging from -0.511 to 1.470 Å) is shown in Figure 5. The red
…
…
spot represent the H O (15.7%) and O H (18.5%) interactions of the DHPM I (Figure 5a). The
…
…
N H/H N interactions result in the fingerprint plot (Figure 5b and 5c). This analysis is very
helpful in the identification of the most dominant interactions among neighboring stacks [33]. In
…
…
…
…
Figure 5d, the red spots represent the H O (7.6%), O H (9.0%), H S (5.7%), S H (9.3%)
…
…
…
interactions of the DHPM II, whereas for the DHMP III, H O (9.7%), O H (10.9%) H S (6.2%)
…
and S H (10.1%) interactions can be observed (Figure 5e). Such interactions are seen in the ꢐ and
ꢍ
ꢐꢉ surfaces of the molecule.

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Figure 5. Hirshfeld surfaces ploted for DHPM I (a), (b) and (c); DHPM II (d) and DHPM III (e).
Dotted lines were used to represent hydrogen bonds.
…
The shape index HS is an important tool to signal hydrophobic interactions (π π and C–
…
…
H π interactions). The C–H π interaction from DHPM I, formed by the center of aromatic ring
characterized by C5–C10 (Cg1), is shown in Figure 6a and 6d. This interaction can be defined by

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…
large red depressions above the aromatic ring. The π π stacking shown in Figure 6b and 6e is
displayed on shape index of DHPM II by red and blue triangles. Shape index refers to phenyl rings,
which are maintained by hydrophobic Cg1–Cg1 interactions (the distance from one centroid to
…
another is 3.494 Å) granting further stability for the structures. The effect of C–H π interaction
from DHPM III involving Cg1 are shown in Figure 6c and 6f.

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…
…
Figure 6. Representation of C–H π and π π interactions of DHPM I (a), II (b) and III (c). Shape
…
index surfaces of DHPM I (d) and DHPM III (f) showing C–H π interactions and DHPM II (e)
…
evidencing π π interactions.
Figure 7 displays the 2D fingerprint plots of DHPM I, II and III, where most of the contact
…
is due to H H interaction, which adds up to 43.0 %, 47.5% and 43.6% of the HS of DHPM I, II
…
…
and III respectively. The contributions of O H/H O interactions, characterized by the spikes in the
bottom of the fingerprint plot, represent 34.2%, 16,6% and 20.6% of the HS in the DHPM I (Figure
…
…
7
1
a), II and III respectively. As for C H/H C interactions of DHPM I, II and III, it is observed
…
…
8.0%, 11.1% and 14.4% of the HS respectively. An important contribution of S H/H S
interactions, characterized by wings observed at the left and right-side bottom for DHPM II and III
(Figure 7c), reaches values of 16.6% and 16.3% of the HS, respectively. Another feature of DHPM
…
II is the presence of C C interactions, which is responsible for 2.8% of HS (Figure 7b).

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Figure 7. Fingerprint plots for DHPM I (a); DHPM II (b) and DHPM III (c).
The root of the mean squared (RMS) values, predicted using Mercury was 0.0189 for
DHPM I and II, while for DHPM I and III was 0.0124. Further for DHPM II and III was 0.0211 as
shown in Figure 8. The overlay of the tetrahydropyridimine ring is different in both the forms. The
o
six-membered tetrahydropyrimidine ring exhibits a dihedral twist of 82.77 with the aryl ring in
DHPM I. For the DHPM II, the six-membered tetrahydropyrimidine ring exhibits a dihedral twist
o
of 73.01 with the aryl ring. The rings of tetrahydropyrimidine and aryl are approximately
o
orthogonal to each other, the dihedral twist are at 89.76 in DHPM III. DHPM I, II and III also
differ by dihedral angles N2–C2–C5–C10, present in the tetrahydropyrimidine ring, the values are
65.60º, 106.82º and 79.30º, respectively (Table 2).
Figure 8. Overlay diagram of the molecular conformation in (a) DHPM I (purple), in (b) DHPM II
(black) and (c) DHPM III (green) in respect to the aromatic ring.
A further comparison of DHPM I with DHPM III results in the formation of dimeric motif
…
…
utilizing N –H N S hydrogen bonds and dimeric motifs being bridged by O –H O O
2
2
2
1
3
3
intermolecular hydrogen bonds involving the hydroxyl group, that made an strong motif in
hydrophobic space of DHPM III. Thus the crystal packing is different of DHPM I because of the
participation of the hydroxyl group generating edge-to-edge motif in hydrophobic space. These
intermolecular hydrogen bonds are responsible for the pre-organization of the DHPMs and
contribute to the stability of the crystalline lattice. As a result of the delocalization of the lone pair
of electrons from the nitrogen atoms N1 or N2 over the carbonyl group, the nitrogen and the oxygen

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atoms assume considerable positive and negative charges, respectively [16]. The sulfur atom
containing analogs of these Biginelli DHPM present similar behavior. The crystal structures object
of this study can be divided into two different supramolecular arrangements. The first, formed by
strong hydrogen bonds between polar functional groups, and the second built up using aryl ring
moieties.
Molecular Modeling
The calculated geometries of DHPM I and II were compared to experimental model by
structural overlapping (Figure 9) and the root of the mean squared deviation (RMSD) shows a good
agreement in terms of interatomic distances and bond angles in DHPM I, II and III. The RMSD
values are 0.0097, 0.0058 and 0.0090 for DHPM I, II and III, respectively, then DHPM III appears
to have a more flexible set of intermolecular interactions than DHPM I and II since the DFT
calculations consider the molecule in an environment free of interactions (gas phase).
Figure 9. Overlapping of DFT (black) and X-ray (gray) structures from DHPM I (a), II (b) and III
(c).
The HOMO and LUMO energy, obtained using DFT methods, was used to obtain some
global reactivity descriptors (Table 4) to develop a better explaining of the stability and reactivity of
DHPM I, II and III. In Figure 10 shows a graphical representation of HOMO and LUMO orbitals,
in DHPM I, II and III the HOMO is located at aromatic ring while LUMO is spread at
dihydropyrimidine ring.

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Figure 10. Theoretical HOMO-LUMO plots, energies and band gaps of DHPM I (a), II (b) and III
(c).
The evaluation of the global indicators, inherent electronic property, of a set of DHPM I, II
and III allows understand its potential for a chemical alteration and give good indexes to the
explanation of the system [37]. The values presented in Table 4 were obtained using the same
formulas proposed by DHANDAPANI et al. [33].
Table 4. The global reactivity descriptors of DHPM I, II and III.
Molecular properties
Ionization potential (I)
Electron affinity (A)
Chemical potential (µ)
Global hardness (η)
Softness (s)
Energy gap (eV)
Electrophilicity index (ω)
Dipole moment (Debye)
I
II
III
5.37
1.37
-3.37
2.00
0.50
4.00
2.84
3.67
5.52
1.52
-3.52
2.00
0.50
4.00
3.10
6.53
5.53
1.79
-3.66
1.87
0.53
3.74
3.58
5.59
The transfer of electrons between two molecules is driven by differences in chemical
potential, the values obtained in this study show a decreasing order of reactivity as DHPM III

ACCEPTED MANUSCRIPT
(
more reactive), II and I respectively. On the other hand, hardness could be understood like the
resistance of a system to transfer its charge, DHPM I and II has the same value while DHPM III
presents to be the easiest system to suffer transfer charge. In terms of electrophilicity the calculated
values determine that DHPM III is the most disposed of accept electrons from the outside
environment, that is a good electrophile, while the lower value of DHPM I indicates that as a good
nucleophilic species.
To understand the potential electrophilic and nucleophilic sites in DHPM I, II and III were
used the 3D MEP representation (Figure 11). In general, it is seen that the negative potential regions
(red), susceptible to electrophilic attack, are mostly limited over the oxygen atoms, moreover, in
DHPM II and III that appears over sulfur atoms too. On the other hand, positive regions (blue),
susceptible to nucleophilic attack, are restricted around hydrogen atoms of aromatic rings and
nitrogen atoms. This pattern is like that expressed involving that class of DHPM [33,38,39].
Figure 11. Molecular electrostatic potential maps of DHPM I (a), II (b) and III (c). The energy
range for each surface is show above it.
The experimental and calculated FT-IR spectra are shown in Figure 12 and the assigned
vibrational modes of main groups of DHPM I, II and III are proposed in Table 5. In Figures S6, S7
and S8, it are possible seen the main assignments to Raman spectra for DHPM I, II and III. To
correct the systematic overestimation by DFT methods in vibrational frequencies these values were
scaled by 0.962.

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Table 5. Theoretical and experimental vibrational assignments for DHPM I, II and III.
DHPM I
DHPM II
DHPM III
Scaled Freq . Exp. Freq.
a,b
b
a,b
b
a,b
b
Vibrational mode
ν (C=C)Ar
ν C=C
ν C=O
ν N-H
Scaled Freq . Exp. Freq . Scaled Freq . Exp. Freq.
1596; 1591
1606
1757; 1650
3508-3492
1294
1613; 1527
1608
1691; 1638
3293
1604; 1593
1615
1682
3504-3492
1228
1589; 1519
1587
1603; 1592
1605
1504; 1456
1587
1629
3293
1270
3468
1685
3181
1235
3416
1648
3499
1266
3625
ν O-CH3
ν O-H
1331
3350
3678
3625
ν CH3
3005-2899
2971-2934
3010-2910
2842
3008-2912
2945-2841
-
1
ν= stretching; a) Scale factor 0.962; b) cm
-1
For experimental spectra for DHPM I, it is possible noted two bands, at 1691 and 1638 cm ,
which are related to νC=O in heterocyclic and from the group in alkyl fraction, respectively.
-1
-1
Theoretical vibrational modes occur at 1757 cm and at 1650 cm , in relation to experimental data.
-1
In experimental Raman scattering, this bands occur with moderate intensity at 1685ꢀcm and at
-
1
-
1
1
1
1
642ꢀcm . For infrared data of the DHPM II, this mode appears at 1685 cm experimentally and at
-1
682 cm theoretically, where the νC=O belongs to ester group, while in DHPM III, this value is at
-
1
-1
629 cm (experimental) and at 1648 cm (DFT). For the Raman data for DHPM II and DHPM
-1
-1
III, it presents values of 1673 cm and 1627 cm , respectively, referring to the C=O bond.
-1
The stretching vibration in the spectra of vinyl group occurs at 1660–1600 cm and, in case
of conjugation, just like in DHPM I, II and III, it could be moved to lower frequencies [40]. In
-1
-1
DHPM I the presence of an infrared absorption band at 1608 cm and a calculated at 1606 cm
indicate the νC=C mode, and it shows up in experimental Raman as a band of strong intensity at
-1
-1
-1
1
603 cm , while in DHPM II and III these values are 1615 cm and 1605 cm , for experimental
-1
infrared spectra. In experimental Raman spectra it shows at 1607 cm for DHPM II and at 1606
-1
cm for DHPM III. Theoretical infrared spectra for DHPM II and III display absorption bands at
-1
-1
1
587 cm . The stretch C=C of an aromatic ring often occur [40] in pairs at 1600 to 1475 cm .
-1
-1
Experimentally these absorptions appear at 1613, 1527 cm (for DHPM I), 1589, 1519 cm (for
-1
-1
DHPM II), 1504 and 1456 cm (for DHPM III) while, theoretically, these values are 1596 cm ,
-1
-1
-1
-1
-1
1
591 cm , 1604 cm , 1593 cm , 1603 cm and 1592 cm , respectively. Experimental data for

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-1
-1
-1
Raman scattering shows this bands at 1469 cm and 1522 cm for DHPM I and at 1569 cm and
-1
-1
1
478 cm for DHPM II, whereas for DHPM III, this scattering bands show at 1586 cm and 1463
-
1
cm .
The methoxy stretching vibration, when it is attached to an aromatic ring, appears at the
-1
region [40] of 1310-1210 cm , experimental infrared spectra shows absorption peaks at 1331, 1235
-1
and 1270 cm for DHPM I, DHPM II, DHPM III, respectively. On the other hand, theoretical
-1
peaks appear at 1294, 1228 and 1266 cm , respectively. For DHPM I, experimental Raman spectra
-1
shows peaks at 1327, 1290 and 1240 cm , which are attributed to methoxy stretching vibration. For
-1
DHPM II, it shows up at 1373, 1330 and 1250 cm and for DHPM III in at 1331, 1274 and 1249
-
1
cm .
-1
The position of νO–H band, for primary phenols, appears around at 3630 cm , but
intermolecular hydrogen bonding weakens it, so shifting the band to lower frequency [40]. DFT
-1
-1
calculations assign the νO–H band at 3678 cm for DHPM I and at 3625 cm for DHPM II e III
…
while experimental bands are shifted to lower frequencies, due O H interactions, at 3350, 3416 and
-1
3
468 cm , respectively.

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Figure 12. Experimental (black) and theoretical (red) overlapped FT-IR spectrum of DHPM I (a),
II (b) and III (c).
The DHPM I studied in this work showed inhibitory activity against L3MBTL1 (IC = 89.1
5
0
nM [41]. L3MBTL1 is a protein “readers” of the histone code and play an important role in the
epigenetic regulation of gene expression, depletion of L3MBTL1 may trigger defects in DNA

ACCEPTED MANUSCRIPT
replication [42]. It was also observed that the L3MBTL1 gene is situated in a region normally
erased in a few myeloid malignancies, including intense myeloid leukemia [43]. These facts suggest
L3MBTL1 as a supposed tumor suppressor protein. L3MBTL1, belongs to a group of factor
containing malignant brain tumor (MBT) repeats having a tandem repeat of three MBT domains
(Figure 14). It is noteworthy that only the second MBT domain has been shown to bind to
methylated histone peptides [44]. We chose MBT2 repeat for our molecular docking research aimed
at to propose new promising ligands of L3MBTL1. The docking protocol was tested by redocking
process to reproduce the binding mode of the co-crystal structure (PDB code: 3P8H). The docking
protocol we employed predicted a similar conformation with a RMSD value of 0.76 Å. In redocking
simulations, RMSD, less than 2 Å validates a docking protocol [45,46]. Figure 13 shows how the
ligand 3-bromo-5-[(4-pyrrolidin-1-ylpiperidin-1-yl)carbonyl]pyridine (P8H) bound to the
L3MBTL1 MBT2 repeat analogous to the co-crystallized ligand. This shows that the parameters
used are consistent for the study of DHPM I, II and III capable of interacting with the same region
of the protein.
Figure 13. Redocking of co-crystallized ligand P8H (0.76 Å) inside MBT2 repeat (PDB code:
3P8H). Superposition between co-crystallized in blue and the best docking pose predicted in green
with the protein surface. The two-dimensional representation is showed on the right-hand side.
Molecular graphic was performed with the UCSF Chimera package.
Human L3MBTL1 has a deep, narrow pocket in the MBT2 domain. P8H ligand binds to this
…
…
pocket via hydrogen bonding (1.72 Å) with Asp355 residue and Cation π and π π stacking

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interactions with Tyr386 and Trp382 residues. This ligand was recently discovered as an inhibitor
of L3MBTL1 (IC = 6µM) [47]. Using the same docking protocol our DHPM I, II and III were
5
0
submited to molecular docking analysis into the L3MBTL1 structure (PDB code: 3P8H). After
docking procedures the DHPM I, II and III were ranked and the best results were found out by the
AutoDock Vina score. The interactions between DHPM I, II and III and the MBT2 repeat, reveal
that some key amino acids residues, such as Asn358, Leu361, Cys363, Trp382, Tyr386 and Asp
355, make good contacts, see Figure 14. The residue Asn358 forms H-bonding with DHPM I, II
and III, beyong to residue Asp355 forming the same hydrogen bond with the DHPM II like with
the P8H ligand. Due the chemical similarity DHPM I, II and III interacts with Tyr386 and Trp382
…
residues by π π stacking interactions. The ethoxy group of DHPM II forms hydrophobic
interactions with Met357. Hydrophobic contacts are also observed between the DHPM I and
Phe379. The S and O atoms in X position showed no interactions change. The DHPM II exhibited
the best interactions in MBT2.
The binding mode predicted by docking simulations was extremely valuable for
investigating the interactions between the DHPM I, II and III portrayed in this article and the
human Lethal (3) malignant brain tumor-like protein 1, the outcomes recommends the DHPM II
and III as potential candidates to L3MBTL1 inhibitors. Their results provided significant
information for the design and synthesis of novel L3MBTL1 inhibitors. The improvement of potent
L3MBTL1 inhibitors would fill as probes to more prominent comprehension the funcional role of
this protein.

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Figure 14. Docking results for DHPM I, II and III inside the MBT2 of L3MBTL1 crystallography
structure (PDB code: 3P8H).
Conclusions
In this research we describe the synthesis and characterization of 5-acetyl-3,4-dihydro-4-(4-
hydroxy-3-methoxyphenyl)-6-methyl-2(1H)-pyrimidinone (DHPM I), Ethyl-4-(4-hydroxy-3-
methoxyphenyl)-6-methyl-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate (DHPM II) and 1-
[
(
1,2,3,4-tetrahydro-4-(4-hydroxy-3-methoxyphenyl)-6-methyl-2-thioxo-5-pyrimidinyl]-ethanone
DHPM III). X-ray diffraction data confirmed, in all DHPM, that the ester moiety appears in an s-
cis configuration. The hydrogen bonds interactions originate different structural arrangements,
which are the chains, dimers and ribbon formations along with the entire crystal packing. A broad
vibrational analysis of DHPM I, II and III were done by B3LYP functional associated to 6-
31G(d,p) basis set. The frontier molecular orbital analysis show that the three compounds has
practically the same reactivity, being DHPM III slightly reactive. In a general way, the vibrational
wavenumbers analysis show good correspondence with experimental data. Moreover, theoretical

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calculations DHPM III as good electrophile, while DHPM I is the best nucleophilic species. In
addition, MEP analysis reveals that the regions near to oxygen and sulfur atoms are the most
susceptible to electrophilic attack. Molecular docking was successful used to predict the three novel
pyrimidine derivatives binding modes. The results suggest the DHPM II and III as potential
candidates to L3MBTL1 inhibitors.
Acknowledgements
The authors are grateful to the Coordenação de Aperfeiçoamento de Pessoal de Nível
Superior (CAPES), Financiadora de Estudos e Projetos (FINEP), Conselho Nacional de
Desenvolvimento Científico e Tecnológico (CNPq) and the Fundação de Amparo à Pesquisa do
Estado de Goiás (FAPEG) for financial support. This research was developed with the support of
the High Performance Computation Nucleus of UEG, State University of Goiás (UEG). Single
crystal X-ray diffraction data were collected at Physics Institute of Federal University of Santa
Catarina (UFSC) and Chemistry Department at Federal University of Juiz de Fora (UFJF).

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