Structural insights of three 2,4-disubstituted dihydropyrimidine-5-carbonitriles as potential dihydrofolate reductase inhibitors

Article abstract of DOI:10.3390/molecules26113286

In this report, we describe the structural characterization of three 2,4-disubstituted-dihydropyrimidine-5-carbonitrile derivatives, namely 2-{[(4-nitrophenyl)methyl]sulfanyl}-6-oxo-4-propyl-1,6-dihydropyrimidine-5-carbonitrile 1, 4-(2-methylpropyl)-2-{[(4-nitrophenyl)methyl]sulfanyl}-6-oxo-1,6-dihydropyrimidine-5-carbonitrile 2, and 2-[(2-ethoxyethyl)sulfanyl]-6-oxo-4-phenyl-1,6-dihydropyrimidine-5-carbonitrile monohydrate 3. An X-ray diffraction analysis revealed that these compounds were crystallized in the centrosymmetric space groups and adopt an L-shaped conformation. One of the compounds (3) crystallized with a water molecule. A cyclic motif (R₂² (8)) mediated by N–H···O hydrogen bond was formed in compounds 1 and 2, whereas the corresponding motif was not favorable, due to the water molecule, in compound 3. The crystal packing of these compounds was analyzed based on energy frameworks performed at the B3LYP/6-31G(d,p) level of theory. Various inter-contacts were characterized using the Hirshfeld surface and its associated 2D-fingerprint plots. Furthermore, a molecular docking simulation was carried out to assess the inhibitory potential of the title compounds against the human dihydrofolate reductase (DHFR) enzyme.

Full text of DOI:10.3390/molecules26113286

molecules
Article
Structural Insights of Three 2,4-Disubstituted
Dihydropyrimidine-5-carbonitriles as Potential Dihydrofolate
Reductase Inhibitors
Lamya H. Al-Wahaibi 1, Althaf Shaik 2 , Mohammed A. Elmorsy 3 , Mohammed S. M. Abdelbaky 4,
Santiago Garcia-Granda 4 , Subbiah Thamotharan 5, Vijay Thiruvenkatam 6 and Ali A. El-Emam 7,
*
1
Department of Chemistry, College of Sciences, Princess Nourah bint Abdulrahman University,
Riyadh 11671, Saudi Arabia; lhalwahaibi@pnu.edu.sa
Discipline of Chemistry, Indian Institute of Technology, Gandhinagar 382355, India; althaf.shaik@iitgn.ac.in
Department of Pharmaceutical Organic Chemistry, Faculty of Pharmacy, Mansoura University,
Mansoura 35516, Egypt; mwahhab95@gmail.com
2
3
4
5
6
Department of Physical and Analytical Chemistry, Faculty of Chemistry, Oviedo University-CINN,
33006 Oviedo, Spain; saidmohammed.uo@uniovi.es (M.S.M.A.); sgg@uniovi.es (S.G.-G.)
Biomolecular Crystallography Laboratory, Department of Bioinformatics, School of Chemical and
Biotechnology, SASTRA Deemed University, Thanjavur 613401, India; thamu@scbt.sastra.edu
Discipline of Biological Engineering, Indian Institute of Technology Gandhinagar, Gujarat 382355, India;
vijay@iitgn.ac.in
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
7
Department of Medicinal Chemistry, Faculty of Pharmacy, Mansoura University, Mansoura 35516, Egypt
Correspondence: elemam@mans.edu.eg; Tel.: +20-50-2258087
ꢀꢁꢂꢃꢄꢅꢆ
*
Citation: Al-Wahaibi, L.H.; Shaik, A.;
Elmorsy, M.A.; Abdelbaky, M.S.M.;
Garcia-Granda, S.; Thamotharan, S.;
Thiruvenkatam, V.; El-Emam, A.A.
Structural Insights of Three
Abstract: In this report, we describe the structural characterization of three 2,4-disubstituted-dihydropyrimidine-
5-carbonitrile derivatives, namely 2-{[(4-nitrophenyl)methyl]sulfanyl}-6-oxo-4-propyl-1,6-dihydropyrimidine-5-
carbonitrile
, and 2-[(2-ethoxyethyl)sulfanyl]-6-oxo-4-phenyl-1,6-dihydropyrimidine-5-carbonitrile monohydrate
diffraction analysis revealed that these compounds were crystallized in the centrosymmetric space
1, 4-(2-methylpropyl)-2-{[(4-nitrophenyl)methyl]sulfanyl}-6-oxo-1,6-dihydropyrimidine-5-carbonitrile
2
3. An X-ray
2,4-Disubstituted
Dihydropyrimidine-5-carbonitriles as
Potential Dihydrofolate Reductase
Inhibitors. Molecules 2021, 26, 3286.
https://doi.org/10.3390/
groups and adopt an L-shaped conformation. One of the compounds (3) crystallized with a water
molecule. A cyclic motif (R22(8)) mediated by N–H···O hydrogen bond was formed in compounds
1
and 2, whereas the corresponding motif was not favorable, due to the water molecule, in compound 3.
The crystal packing of these compounds was analyzed based on energy frameworks performed at
the B3LYP/6-31G(d,p) level of theory. Various inter-contacts were characterized using the Hirshfeld
surface and its associated 2D-fingerprint plots. Furthermore, a molecular docking simulation was
carried out to assess the inhibitory potential of the title compounds against the human dihydrofolate
reductase (DHFR) enzyme.
molecules26113286
Academic Editors: Rui Fausto,
Sylvia Turrell and Gulce Ogruc Ildiz
Received: 6 May 2021
Accepted: 26 May 2021
Published: 29 May 2021
Keywords: pyrimidine-5-carbonitriles; dihydrofolate reductase; crystal structure; DFT; Hirshfeld
surface analysis
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations.
1. Introduction
Pyrimidine moiety was early discovered as an important scaffold in several chemother-
apeutic agents [1]. The chemotherapeutic efficacy of pyrimidine-based drugs is attributed
to their inhibitory effect on the biosynthesis of vital enzymes responsible for nucleic
acids, such as thymidylate synthetase (TSase), thymidine phosphorylase (TPase), dihy-
drofolate reductase (DHFR), and reverse transcriptase (RTase). Several pyrimidine-based
drugs are currently marketed as antineoplastic agents for the treatment of different hu-
Copyright:
© 2021 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 (https://
creativecommons.org/licenses/by/
4.0/).
man cancers [2–6]. Potent antiviral activities against human immunodeficiency viruses
(HIV) [ 12], herpes simplex virus (HSV) [13], hepatitis B virus (HBV) [14], and SARS-CoV
7
–
virus [15] have been reported for numerous substituted pyrimidine derivatives. In addition,
pyrimidine-based dihydrofolate reductase (DHFR) inhibitors are currently used as clini-
cally useful chemotherapeutic agents [16
,17]. Trimethoprim, the prototype antibacterial
Molecules 2021, 26, 3286. https://doi.org/10.3390/molecules26113286
https://www.mdpi.com/journal/molecules

Molecules 2021, 26, 3286
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DHFR inhibitor was discovered to be a potent drug, mainly in the treatment of urinary
tract infections [18]. The next generation of DHFR inhibitors was further developed as
potent antibacterial drugs for the treatment of resistant respiratory tract infections [19
Pyrimidine-based DHFR inhibitors are also employed as efficient antiprotozoal agents
for the treatment of malaria [22 23], leishmaniasis [24], and trypanosomiasis [25]. In ad-
–21].
,
dition, several pyrimidine-5-carbonitrile derivatives were reported to display marked
antimicrobial activities [26–32].
In the present investigation, we report an in-depth experimental and theoretical study
of the structures of three 2,4-disubstituted dihydropyrimidine-5-carbonitrile derivatives in
a trial to explore the mechanism of their antimicrobial activity.
2. Results and Discussion
2.1. Chemical Synthesis
The dihydropyrimidine-5-carbonitriles
of the corresponding aldehydes with thiourea
the presence of anhydrous potassium to yield the intermediate 6-substituted-2-thiouracil-5-
carbonitriles 31], 32], and 33]. Compounds and were reacted with 4-nitrobenzyl
bromide and compound with 1-bromo-2-ethoxyethane in the presence of anhydrous
potassium carbonate to yield the target compounds 1, 2, and 3 (Scheme 1).
1,
2
, and
3
were synthesized via condensation
A
B
and ethyl cyanoacetate
C
, in ethanol, in
D
[
E
[
F
[
D
E
F
O
N
HN
O
Br
N
O2N
HN
S
N
O
K2CO3
DMF, r.t.
1
O2N
S
N
H
N
N
D
E
HN
Br
O
O
N
N
O
O2N
S
N
NH2
NH2
HN
1. K2CO3 / EtOH
2. H+
C
K2CO3
DMF, r.t.
2
O2N
S
S
N
H
R CHO
A
B
O
N
O
HN
O
Br
HN
O
S
N
K2CO3
DMF, r.t.
S
N
H
3
F
Scheme 1. Synthesis of compounds 1–3.
2.2. Crystal Structures
Single crystal X-ray diffraction was used to determine the crystal structures of com-
pounds . A summary of the crystallographic data and structure refinement parameters
is listed in Table 1. The Oak Ridge Thermal Ellipsoid Plot (ORTEP) representation at 50%
probability corresponding to the asymmetric units of , and is depicted in Figure 1.
1–3
1
,
2
3
The bond lengths and bond angles of the three structures were comparable to the related
reported structures [34–37].

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Figure 1. Thermal ellipsoid representation of compounds (
level with atom numbering scheme is shown, and (
a
)
1
, (
b
)
2
, and (
c
)
3
at 50% probability
d
) structural superimposition of compounds 1
(grey), 2 (green), and 3 (orange). The water oxygen in compound 3 is not shown for clarity.
Table 1. Crystal data and structure refinement parameters of compounds 1–3.
Compound 1
Compound 2
Compound 3
Empirical formula
C15H14N4O3S
330.36
C16H16N4O3S
344.39
293 (2)
Monoclinic
C2/c
18.3676 (7)
5.7996 (2)
31.8592 (12)
90
95.343 (4)
90
3379.0 (2)
8
1.354
C15H17N3O3S
319.38
Formula weight
Temperature (K)
Crystal system
Space group
a/Å
Monoclinic
P21/n
12.5792 (7)
9.5493 (5)
13.3158 (8)
90
106.432 (7)
90
1534.20 (16)
4
Triclinic
P-1
7.1456 (4)
10.8642 (6)
11.2566 (7)
104.672 (5)
108.159 (5)
95.421 (5)
788.84 (8)
2
b/Å
c/Å
◦
α/
β/
γ/
◦
◦
Volume/Å3
Z
Calculated density (g/cm3)
1.430
2.068
688
1.345
1.968
336
−1
1.899
1440
Absorption coefficient (mm
F(000)
)
Crystal size (mm3)
Radiation
0.16 × 0.12 × 0.08
0.16 × 0.05 × 0.05
Cu Kα (λ = 1.54184)
5.6 to 151.4
−22 ≤ h ≤ 22,
−7 ≤ k ≤ 6,
−39 ≤ l ≤ 40
16836
0.26 × 0.14 × 0.10
2Θ range for data collection
8.5 to 151.4
−15 ≤ h ≤ 15,
−11 ≤ k ≤ 11,
−16 ≤ l ≤ 16
15422
8.6 to 151.1
−8 ≤ h ≤ 8,
−13 ≤ k ≤ 13,
−13 ≤ l ≤ 11
12241
Index ranges
Reflections collected

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Table 1. Cont.
Compound 1
Compound 2
Compound 3
3163
3418
3220
[Rint = 0.0971,
Rsigma = 0.0654]
3163/0/213
1.030
[Rint = 0.1098,
Rsigma = 0.0941]
3418/2/211
0.998
[Rint = 0.0442,
Rsigma = 0.0377]
3220/2/212
1.035
Independent reflections
Data/restraints/parameter
Goodness-of-fit on F2
R1 = 0.0569,
wR2 = 0.1209
R1 = 0.1087,
wR2 = 0.1479
0.20/−0.25
2063317
R1 = 0.0766,
wR2 = 0.1777
R1 = 0.1565,
wR2 = 0.2358
0.52/−0.36
2063318
R1 = 0.0432,
wR2 = 0.1040
R1 = 0.0689,
wR2 = 0.1210
0.16/−0.18
2063320
Final R indices [I>2 σ(I)]
Final R indices (all data)
−3
Largest diff. peak and hole (e.Å
CCDC number
)
Full crystallographic data for compounds 1 (CCDC 2063317), 2 (CCDC 2063318) and
3 (CCDC 2063320) can be obtained free of charge from The Cambridge Crystallo-graphic
Data Centre at: www.ccdc.cam.ac.uk (Supplementary Materials).
Compound
1
crystallized in the monoclinic crystal system with the P21/n space group.
. The asymmetric unit of compound
Figure 1a depicts the ORTEP diagram of compound
1
1
contains one molecule in an L-shape molecular conformation, where the angle between the
4-nitrobenzyl moiety and 1,6-dihydropyrimidine-5-carbonitrile (C9-S1-C1) was found to be
◦
◦
102.37 . The dihedral angle formed between the nitrobenzyl and pyrimidine ring is 87.90 .
To understand the crystal packing, we created an energy framework that combines
pairwise intermolecular interaction energies with a graphical depiction of their magni-
tude [38]. The energy frameworks of
as shown in Figure 2a. As shown from this figure, the molecules of
columnar fashion. The molecular packing was mainly stabilized by a pair of N1–H···O1
NHO = 174◦) hydrogen bonds which form be-
1
were projected onto the crystallographic ac plane,
1
are packed in a
(H···O = 1.795 Å,
N
···O = 2.800 (2) Å, ∠
tween the NH and carbonyl groups of two centrosymmetrically-related pyrimidine rings
2
(Figure 2b). These hydrogen bonds led to a cyclic inversion dimer with a R2 (8) graph-set
motif. This hydrogen bond (represented as large cylindrical tubes) links the molecules in
the layer with molecules in an adjacent layer. The total intermolecular interaction energy
for this hydrogen bonded dimer is
−
78.2 kJ mol−1. In addition to this, a chalcogen bond
of the type C1–S1···S1 is present, with the distance and angle 3.363 (2) Å and 166.2 (1)◦,
respectively (Figure 2b). The role of the chalcogen bond in this structure is similar to that
of a N–H···O hydrogen bond. One of the oxygens (O2 atom) of the nitro group makes a
short N-O···π (centroid of the pyrimidine ring) interaction with the distance of 3.033 (2)
Å (Figure 2b). This interaction also bridges the adjacent ladders. The ladder-pattern is
formed by weak van der Waals-type interactions.
Compound
2 crystallized in the monoclinic crystal system with the space group C2/c.
The asymmetric unit contains one molecule, as shown in Figure 1b. The 4-nitrobenzyl
moiety and 1,6-dihydropyrimidine-5-carbonitrile are positioned in an L-shaped structure
similarly to compound 1. Molecules 2 are arranged as layers in the solid state, and these
layers run parallel to the crystallographic a axis. Furthermore, the adjacent layers are
interlinked by a short and directional C–H···O interaction (involving H15 from nitrobenzyl
and O3 of the nitro group), with H···O = 2.35 Å and
∠
CHO = 161◦ forming a double
layer. Moreover, the adjacent layers are also connected by a C– S···S chalcogen bond
(S1···S1 = 3.444 (2) Å and ∠C1-S1···S1 = 162 (1)◦).

Molecules 2021, 26, 3286
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Figure 2. (a) Packing diagram of compound 1 viewed along the crystallographic b-axis with the incorporation of energy
framework (electrostatic energy: red; dispersion energy: green;−a1nd net interaction energy: blue with the cylindrical size
of 80; interaction energy for molecular pairs less than 15 kJ mol has been omitted for clarity) and (b) molecular dimers
formed in the crystal structure of 1.
The adjacent double layers are further interconnected via cyclic N–H···O synthon
2
(involving NH and C=O of pyrimidine ring and R2 (8) graph-set motif) as observed in
1
. The N–H·◦··O hydrogen bonding geometry (H···O = 1.79 Å, N···O = 2.776 (1) Å and
∠
NHO = 163 ) is very similar to the structure of 1. We also noted that the above mentioned
C–H···O and N–H···O bonded motifs are connected alternately, leading to the formation of
a chain (Figure 3a).
Though the molecular arrangement of
crystal structure of , these two structures show a different 3D-topology of the energy
frameworks for electrostatic and net interaction energies (Figure 3b). However, the disper-
sion energy framework shows a similarity between compounds and . In the electrostatic
2 in solid-state is somewhat different from the
1
1
2
energy framework, the large vertical cylindrical tubes correspond to cyclic N–H···O hydro-
gen bonds, and small horizontal tubes bridge the large cylindrical tubes. These horizontal
tubes represent the intermolecular C–H···N interaction in which the nitrile N atom acts
as an acceptor (C13–H13···N3; H13···N3 = 2.60 Å and
∠
CHN = 122◦). Furthermore, the
adjacent ladder-like topology is interconnected by small vertical cylindrical tubes that
represent an intermolecular short and directional C15–H15···O3 interaction. The chalcogen
bond observed in this structure interlinks the large vertical cylindrical tubes diagonally
in each ladder-like topology and is driven by the dispersion origin. The chalcogen bond
and intermolecular C–H···N interaction combined to generate a supramolecular sheet, as
shown in Figure 4.

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Figure 3.
compound
(
a
2
) Supramolecular chain built by intermolecular N/C–H···O hydrogen bonds, and (
b) packing diagram of
viewed along the crystallographic b-axis with the incorporation of the energy framework (electrostatic energy:
red; dispersion energy: green and net interaction energy: blue with the cylindrical size of 80; interaction energy for molecular
−1
pairs less than 10 kJ mol has been omitted for clarity).
Figure 4. Supramolecular sheet built by intermolecular C–S···S chalcogen bond and C–H···N interaction in compound 2.
Compound
the space group P-1 (Figure 1c). The asymmetric unit contains one molecule of
water molecule. Unlike compounds and , compound has a 2-ethoxyethyl group instead
of a 4-nitrobenzyl moiety connected to the dihydropyrimidine ring via a thioether bridge.
However, compound
maintains an L-shape with a bond angle of 101.56◦ (C8-S1-C12)
3
crystallized as a monohydrated form in the triclininc crystal system with
3
and one
1
2
3
3
between the 2-ethoxyethylthio group and the 1,6-dihydropyrimidine-5-carbonitrile. Fur-
thermore, the 4-phenyl group is in the same plane of the 1,6-dihydropyrimidine ring, with
a torsional angle of 178.18◦ (C6-C1-C7-C10). In the asymmetric unit of
3, the pyrimidine
ring NH is involved in an intermolecular N–H···O (N1 . . . O3 = 2.710 Å) hydrogen bond
with the water oxygen atom. Due to the presence of the crystallization water molecules in
the crystal of 3, a cyclic N–H···O bonded synthon, as observed in 1 and 2, disappears.
The basic packing motif of
3 is the molecular stacking, which forms as a columnar
fashion along the ac plane, and the water molecules sandwiched between adjacent columns.
The molecular dimers formed in this structure are illustrated in Figure 5. The inversion-
related molecules (1
to-centroid distance between the phenyl and pyrimidine rings is 3.588 (2) Å (Figure 5a).
Different inversion-related molecules (2 x, 1 y, 1 z) of also produce a molecular
−
x, 1
−
y, 1
−
z) of 3 generate molecular stacking, and the centroid-
−
−
−
3

Molecules 2021, 26, 3286
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stacking, and the centroid-to-centroid separation of phenyl and pyrimidine rings is 3.638 (2)
Å. This molecular stacking is further stabilized by the intermolecular C–H···N interaction,
in which nitrile N atom acts as an acceptor (Figure 5b).
The water molecule is involved in three intermolecular interactions, of which two are
O–H···O type hydrogen bonds and the remaining one is the N–H···O type hydrogen bond
(H1···O3 = 1.72 Å, N1···O3 = 2.709 (2) Å and
∠
NHO = 167◦; symmetry operation:
−
x + 1,
−
y + 1,
Å and
O3···O2 = 2.832 (2) Å and
oxygens of carbonyl and ether moieties are involved as acceptors and water acts as a
−z + 2). In the O–H···O hydrogen bonds (H1w···O1 = 1.83 Å, O3···O1 = 2.766 (2)
◦
∠
OHO = 157 ; symmetry operation:
−
x + 1,
−
y + 1,
−
z + 2 and H2w···O2 = 1.86 Å,
x + 1,
y + 2, −z + 2)
∠
OHO = 169◦; symmetry operation:
−
−
donor. As shown in Figure 5c, the neighboring molecules of
3 are interlinked by two water
molecules, forming alternate R4 (12) and R4 (18) rings.
4
4
Figure 5. Different dimeric motifs formed by (a) π-stacking interaction, (b) π-stacking and C–H···N
interactions, and (
) N–H···O and O–H···O hydrogen bonds in the crystal structure of compound
c
3.
The energy framework of the crystal structure
3 viewed down the b axis is depicted in
Figure 6. As can be seen from this figure, the crystal packing of compound is predomi-
3
nantly dispersive in nature. The zigzag chains with small cylindrical tubes (electrostatic
energy component) run parallel to the crystallographic a axis. The adjacent zigzag chains
are further interconnected by small vertical tubes. These vertical tubes correspond to
intermolecular N–H···O and O–H···O hydrogen bonds. The horizontal tubes represent
the
π
-stacking interaction and C–H···N interaction. Overall, the energy framework of the
crystal packing analysis suggests that compounds
1 and 2 display a similar 3D topology of
the energy framework, whereas compound shows a different framework compared to
3
the other two compounds. These features suggest that compound
mechanical properties compared to the other two compounds.
3 may possess different

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Figure 6. Packing diagram of compound
3
viewed along the crystallographic b-axis with the in-
corporation of the energy framework (electrostatic energy: red; dispersion energy: green; and net
interaction energy: blue with the cylindrical size of 80; interaction energy for molecular pairs less
−1
than 10 kJ mol has been omitted for clarity).
2.3. Hirshfeld Surface and 2D Fingerprint Plot Analysis
The Hirshfeld surfaces (HS) of the three dihydropyrimidine derivatives are repre-
sented in Figure 7. In compound
the HS due to the cyclic N-H···O hydrogen bonds, while a C-S···S type chalcogen bond
shows a relatively less intense red spot. In compound , in addition to the above two
interactions in
, there is a C–H···O interaction in which the nitro oxygen is involved as
an acceptor, showing red spots on the HS. Due to the presence of the crystallization water
molecule in the crystal of
1, the most prominent features are the deep red spots on
2
1
3
, a cyclic N-H···O synthon has disappeared. However, the water
molecule makes three hydrogen bonds, including two Ow–H···O hydrogen bonds with
the carbonyl oxygen of pyrimidine and ester oxygen atoms, and the N–H···Ow hydrogen
bond with the amine group of the pyrimidine ring. These three intermolecular interactions
are shown as intense red areas on the HS.
The shape index of compounds
red triangles on the HS, indicating the absence of π···π interactions in these two compounds.
On the other hand, compound , having a phenyl ring at position 4, showed complementary
blue and red triangles (at the phenyl ring and dihydropyrimidine rings) on the shape index
1 and 2 showed no sign of complementary blue and
3
mapped over the Hirshfeld surface. Furthermore, the curvedness of compound
3 showed
a flat region in the same area, confirming the presence of a weak π···π stacking.

Molecules 2021, 26, 3286
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Figure 7. Hirshfeld surface of compounds 1 (a), 2 (b), and 3 (c) mapped with dnorm, and (d) shape index, and curvedness.
The main intermolecular interactions influencing the molecular packing of com-
pounds 1–3 were studied using the 2D fingerprint plots. The 2D fingerprint plots of
the compounds are shown in Figure 8.
Figure 8. The 2D fingerprint plots for different inter-contacts obtained from structures 1, 2, and 3.
It is noticeable that the O···H, N···H, C···H, and S···H contacts play a significant role in
crystal packing [39
–
42]. The O···H/H···O contacts are represented by a pair of sharp spikes
in all three compounds, and these contacts are symmetrical in 1–2 with a di + de distance of
~1.8 Å. In contrast, the corresponding contacts are not symmetrical in the 2D-FP due to the
presence of a water molecule in compound
3
. The distribution of H···H contacts markedly
varied between compounds 1–2 and . In the latter compound, a single spike with the
3
shortest contact is located at 2.0 Å. We also noted that there is a considerable difference
in the contact distribution pattern of C···H/H···C interactions in these compounds. The
shortest contact of these interactions is located at ~2.8, 2.7, and 3.0 Å in compounds 1, 2,

Molecules 2021, 26, 3286
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and
3, respectively, suggesting its strength. The strength of this interaction is nearly equal
in 1 and 2, whereas it is weak in nature in 3.
Similarly, the distribution pattern of N···H/H···N interactions also looks different in
these compounds, with the closest contact separation of ~2.7, 2.6, and 2.8 Å in compounds
1
,
2
, and
total HS area) of the chalcogen contact (S···S) is nearly the same in all three compounds.
However, there is a remarkable variation in the closest contact distance. The shortest S···
contact is at 3.4 Å in and , and the corresponding contact is located beyond 3.4 Å in
3, respectively. It is important to point out that the contribution (0.7–0.9% to the
S
1
2
3,
which indicates that the chalcogen bond plays a significant role in the stabilization of the
crystal structures of 1 and 2, rather than the structure of 3.
The relative contribution of various intermolecular interactions in the title dihydropy-
rimidine derivatives was also obtained from 2D fingerprint plots. In compound
important contribution towards crystal packing is from the O···H (25.4%), C···H (19.1%),
···H (13.5%), and S···H (6.6%) contacts. The O···H/H···O showed a dominant role in
the overall crystal packing of compound (Figure 9). Compound also showed similar
contributions for some of the inter-contacts as compound . The replacement of propyl
1, the most
N
1
2
1
group with an isobutyl group at position 4 of the dihydropyrimidine ring led to increasing
the H···H (25.8%) and O···H (27.9%) and decreasing the H···C (16.7%) contacts compared
to compound 1. Additionally, the nitro group in compound 2 also contributed towards
the increase in O···H/H···O contacts in the whole system (Figure 7). Compound 3 has a
maximum contribution from H···H (44.5%), followed by N···H (13.8%), O···H (13.4%), and
S
···H contacts (5.6%). On the other hand, the substitution of the phenyl ring at position
4 of the dihydropyrimidine ring led to a significant increase in the C···C (9.7%) contacts,
confirming the π . . . π interaction in this structure (Figure 9).
Figure 9. Relative contribution of various intermolecular interactions in the crystal packing of compounds 1, 2, and 3.
2.4. Molecular Docking Studies as Human Dihydrofolate Reductase (hDHFR) Inhibitors
Molecular docking studies were performed to predict the molecular interaction be-
tween the receptor and compounds
1,
2
, and
3, and possible binding poses and binding
energies. The crystal structures of compounds
1
,
2
, and prepared from LigPrep [43
3
]
were subjected to rigid docking against hDHFR using Glide docking, followed by an
induced fit docking protocol. Amino acid residues Ile-7, Leu-22, Phe-31, Phe-34, Arg-70,
and Val-115 are essential for the activity of hDHFR and DHFR inhibition studies [44]. Our
docking results (Table 2) identified compound
2 as a top binding ligand with a docking
score of 8.58 kcal/mol (Figure 10). The nitro group formed a π . . . cation interaction
−
with key residue Phe34, present in the alpha helix. Importantly, the nitrile group and
1,6-dihydropyrimidne enhance the binding energy with several water-mediated hydrogen
bonding interactions with Asn64 and Ser59. Compound
8.34 kcal/mol, it formed key interactions with essential residues Phe34, Val115, and
Phe34 in the active site. The 1,6-dihydropyrimidine ring formed a hydrogen bonding
1 showed a docking score of
−

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interaction with Val115 and π . . . π interaction with Phe34 residues. Unlike compound
2,
the nitro group of compound
1
formed a π . . . cation interaction with the Phe31 residue.
Additionally, the nitro and nitrile groups enhanced the binding affinity with hDHFR via
several water-mediated interactions with Val8, Gln35, and Asp64. Compound showed
a docking score of 7.34 kcal/mol, with hydrogen bonding interactions with Arg70 and
3
−
Gln35 residues in the active site. The oxygen of the carbonyl group formed a hydrogen bond
with residue Gln35 and the nitrile group formed an interaction with Arg70. Furthermore,
compound 3 and hDHFR complex were stabilized by water-mediated hydrogen-bonding
interactions with Asn64 (Figure 11).
Table 2. The docking scores of compounds 1, 2, and 3 against hDHFR after IFD.
No. of
Interactions
No. of H-Bonding
Residues
Compound
1
Docking Score
Glide Energy
Interacting Residues
Phe34 (pi.cation) Interaction
with Gln35, Asn64, and
Ser59 via water
H-bond: Val115, Phe34 (pi
. . . pi), Phe31 (pi . . . cation)
Interaction with Gln35,
Asn64, and Val8 via water
H-bond: Arg 70, and Gln35;
water-mediated interactions
with Asn4
−8.53 kcal/mol
−8.34 kcal/mol
−7.34 kcal/mol
−93.57
1
1
2
0
1
2
2
3
−84.82
−75.03
Figure 10. Binding pose of compound 2 (blue) in the active site of hDHFR, with a docking score
of 8.58 kcal/mol (hDHFR represented as an illustration in a green color; interacting residues and
ligands are represented in a tube model).
−

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Figure 11. Binding poses of (a) compounds 1 (orange), (b) 2 (blue), and (c) 3 (magenta) in the active site of hDHFR (active
site residues and water molecules are represented as tubes and ligands in a ball and stick model).
3. Materials and Methods
3.1. Synthesis and Crystallization
Compounds 1, 2, and 3 were prepared following the reaction sequences in Scheme 1. The
pure single crystals were obtained by slow evaporation of EtOH/CHCl3 (1:2, v/v) solution at
room temperature to yield the compounds as colorless transparent prism crystals.
2-{[(4-Nitrophenyl)methyl]sulfanyl}-6-oxo-4-propyl-1,6-dihydropyrimidine-5-carbonitrile
1
[
31]: Yield: 92%; M.p. 210–212 ◦C (EtOH); Mol. Formula (Mol. wt.): C15H14N4O3S (330.36).
4-(2-Methylpropyl)-2-{[(4-nitrophenyl)methyl]sulfanyl}-6-oxo-1,6-dihydropyrimidine-
5-carbonitrile 2 [
32]: Yield: 90%; M.p. 217–219 ◦C (EtOH/H2O); Mol. Formula (Mol. wt.):
C16H16N4O3S (344.39).
2-[(2-Ethoxyethyl)sulfanyl]-6-ox◦o-4-phenyl-1,6-dihydropyrimidine-5-carbonitrile monohy-
drate 3 [30]: Yield: 42%; M.p. 161–163 C (EtOH); Mol. Formula (Mol. wt.): C15H15N3O2S (301.36).
3.2. Single Crystal X-ray Diffraction Determination
Single crystals of compounds were used to measure the X-ray diffraction at room
temperature (293 K) on a Xcalibur, Ruby, Gemini diffractometer (Agilent Technologies,
Inc., Santa Clara, CA, USA) using a single wavelength X-ray source (Cu K radiation:
1–3
α
λ = 1.54184 Å). Pre-experiment, data collection, data reduction, and analytical absorption
correction were performed with the program suite CrysAlisPro (Rigaku Oxford Diffrac-
tion) [45]. The structures were solved with the Olex2 program [46], and the refinement
was performed with the SHELXL 2018/3 program [47]. In all three structures, the position
of the NH atoms was located from a difference Fourier map, and in compound
3, the
positions of water H atoms were also located from a difference map. In compound , EADP
2
constraints were applied to atoms C7, C8, C14, and C15 and DFIX restraints were applied
to C7–C8/C7–C9 bonds with 2.0 Å. Furthermore, SIMU restraints were applied to make

Molecules 2021, 26, 3286
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the atomic displacement parameter (ADP) values for these atoms more reasonable. In
compound 3, the O–H distances were restrainted to 0.90 (2) Å during the final refinement.
In all three structures, H atoms bound to C atoms were placed in geometrically idealized
positions (C–H = 0.93–0.98 Å) and were constrained to ride on their parent atoms with
Uiso(H) = 1.2Ueq(C). The positions of methyl H atoms were placed in calculated positions
(C–H = 0.96 Å), but were allowed to rotate about the C–C bonds and constrained to ride on
their parent atoms with Uiso(H) = 1.5Ueq(C).
3.3. Computational Details
3.3.1. Hirshfeld Surface Analysis
Hirshfeld surface and 2D fingerprint plots were generated using CrystalExplorer3.1 [42].
The Crystallographic Information Files (cif files) of compounds 1, 2, and 3 were used as
input files to visualize HS and 2D fingerprint plots. The normalized contact distance
(dnorm), mapped throughout the surface, is defined as:
di − rivdW
de − revdW
+
dnorm
=
revdW
rivdW
where de and di represent the distances from a point on the surface to the nearest nucleus
outside and inside the surface, respectively, and rvdW corresponds to the van der Waals
(vdW) radii of the atoms involved. The red-white-blue colors on the dnorm surface indicate
the interatomic distances are shorter than vdW (red), equal to vdW (white), and longer than
vdW (blue) [39]. Shape index and curvedness were generated to study π···π interaction in
the current derivatives. Complimentary blue and red triangle on the shape index and green
flat areas on the curvedness are distinctive features of π···π stacking [40]. Furthermore,
2D-fingerprint plots were used to compare the relative contribution of various non-covalent
contacts present in the molecular packing of the dihydropyrimidine derivatives [40
,41].
Furthermore, the energy frameworks for compounds were generated with a B3LYP/6-
1–3
31G(d,p) level of approximation using the CrystalExplorer-17.5 program [48].
3.3.2. Molecular Docking Studies
Protein Preparation and Grid Generation
The 3D structure coordinates of human dihydrofolate reductase (hDHFR) were re-
tried from RCSB (PDB ID: 1drf) [49], and refined using a protein preparation module in
Schrödinger suite [50]. In protein preparation, hydrogen atoms were added, water beyond
5 Å was removed, added missing side chain using prime, assigned pH 7.0
±
1, followed by
structure optimization and minimization steps. After the protein preparation, the resulting
structure was subjected to a grid generation protocol. Receptor grid was generated on the
folic acid binding site using Schrodinger suite to dock small molecules of interest.
Ligand Preparation
Structures of compounds 1–3, along with cocrystalized ligand (folic acid), were im-
ported into the Maestro suite for docking calculation. These ligands were subjected to
ligand preparation using LigPrep module [51]. Possible ring conformations, ionization
states, and tautomers were generated using the Maestro suite.
4. Conclusions
The crystal structures of three 2,4-disubstituted-dihydropyrimidine-5-carbonitrile
derivatives were determined at the room temperature. The structural analysis revealed
that compounds
1
and
2
are primarily stabilized by a N–H···O hydrogen bond and C–S···
S
N
chalcogen bond. In compound
2
, a short intermolecular C–H···O interaction and C–H···
interaction provided additional stabilization. The N–H···O hydrogen bond forms an R22(8)
cyclic synthon in both compounds
1 and 2. Due to the presence of a water molecule
in compound 3, the cyclic synthon disappears, and the water molecule participates in

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N–H···O and O–H···O hydrogen bonds. In addition, C–H···O and C–H···N interactions
are additionally stabilized in compound 3. The Hirshfeld analysis revealed variations in
different inter-contacts due to the presence of substituents in these compounds. The shape
index plot confirmed the -stacking interaction in compound . The energy framework
π
3
analysis showed that these compounds adopt distinct 3D-energy topologies. However, the
dispersion energy framework showed a similar feature in compounds 1 and 2. Molecular
docking analysis indicated that these three compounds showed an inhibitory potential
against the human DHFR enzyme.
Supplementary Materials: The Checkcif report of compounds 1–3 are available online.
Author Contributions: Conceptualization, A.A.E.-E.; methodology, L.H.A.-W., A.S., M.S.M.A. and
M.A.E.; software, V.T., S.T. and M.S.M.A.; validation, L.H.A.-W., M.A.E. and S.T.; formal analysis,
V.T., S.T. and M.S.M.A.; investigation, S.G.-G., V.T., A.S. and M.A.E.; data curation, S.G.-G. and
S.T.; writing—original draft preparation, A.A.E.-E.; writing—review and editing, A.A.E.-E., V.T. and
S.T.; supervision, L.H.A.-W. and A.A.E.-E.; project administration, A.A.E.-E.; funding acquisition,
L.H.A.-W. All authors have read and agreed to the published version of the manuscript.
Funding: This research was funded by the Deanship of Scientific Research at Princess Nourah bint
Abdulrahman University through the Fast-track Research Funding Program.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Acknowledgments: The DST-FIST for Schrodinger facility offered by the Indian institute of Technol-
ogy Gandhinagar, India, is greatly appreciated.
Conflicts of Interest: The authors declare no conflict of interest.
Sample Availability: Samples of compounds 1, 2 and 3 are available from the corresponding author.
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