Monoalkylated barbiturate derivatives: X-ray crystal structure, theoretical studies, and biological activities

Article abstract of DOI:10.1016/j.molstruc.2017.04.017

Barbiturate derivatives are privileged structures with a broad range of pharmaceutical applications. We prepared a series of 5-monoalkylated barbiturate derivatives (3a–l) and evaluated, in?vitro, their antioxidant (DPPH assay), and α-glucosidase inhibito

Full text of DOI:10.1016/j.molstruc.2017.04.017

Accepted Manuscript
Monoalkylated barbiturate derivatives: X-ray crystal structure, theoretical studies, and
biological activities
Assem Barakat, Abdullah Mohammed Al-Majid, Saied M. Soliman, Mohammad
Shahidul Islam, Hussain Mansur Ghawas, Sammer Yousuf, M. Iqbal Choudhary,
Abdul Wadood
PII:
S0022-2860(17)30448-9
10.1016/j.molstruc.2017.04.017
MOLSTR 23639
DOI:
Reference:
To appear in: Journal of Molecular Structure
Received Date: 14 March 2017
Revised Date: 4 April 2017
Accepted Date: 6 April 2017
Please cite this article as: A. Barakat, A.M. Al-Majid, S.M. Soliman, M.S. Islam, H.M. Ghawas, S.
Yousuf, M.I. Choudhary, A. Wadood, Monoalkylated barbiturate derivatives: X-ray crystal structure,
theoretical studies, and biological activities, Journal of Molecular Structure (2017), doi: 10.1016/
j.molstruc.2017.04.017.
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Monoalkylated barbiturate derivatives: X-Ray crystal
structure, theoretical studies, and biological activities
Assem Barakat1,3,*, Abdullah Mohammed Al-Majid1, Saied M. Soliman2,3,
Mohammad Shahidul Islam1, Hussain Mansur Ghawas1, Sammer Yousuf3, M. Iqbal
Choudhary1,4, and Abdul Wadood5
1 Department of Chemistry, College of Science, King Saud University, P. O. Box 2455,
Riyadh 11451, Saudi Arabia.
2
Department of Chemistry, College of Science & Arts, King Abdulaziz University, P.O.
Box 344 Rabigh 21911, Saudi Arabia.
3
Department of Chemistry, Faculty of Science, Alexandria University, P.O. Box 426,
Ibrahimia, Alexandria 21321, Egypt.
4
H.E.J. Research Institute of Chemistry, International Center for Chemical Sciences,
University of Karachi, Karachi 75270, Pakistan.
5Department of Biochemistry, Abdul Wali Khan University, Mardan-23200, Pakistan.
(A.W)
* Correspondence: ambarakat@ksu.edu.sa; Tel.: +966-114-675-901; Fax:
+966-114-675-992.
Abstract: Barbiturate derivatives are privileged structures with a broad range of
pharmaceutical applications. We prepared a series of 5-monoalkylated barbiturate
derivatives (3a–l) and evaluated, in vitro, their antioxidant (DPPH assay), and
α-glucosidase inhibitory activities. Compounds 3a–l were synthesized via Michael
addition. The structure of compound 3k was determined using X-ray single-crystal
diffraction, and geometric parameters were calculated using density functional theory at
the B3LYP/6-311G(d,p) level of theory. Further, the structural analysis of 3k were also
investigated. Biological studies revealed that compounds 3b (IC50 = 133.1 ± 3.2 µM), 3d
(IC50 = 305 ± 7.7 µM), and 3e (IC50 = 184 ± 2.3 µM) have potent α-glucosidase enzyme
inhibitors and showed greater activity than the standard drug acarbose (IC50 = 841 ± 1.73
µM). Compounds 3a–3i were found to show weak antioxidant activity against

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1,1-diphenyl-2-picryl-hydrazyl (DPPH) radicals (IC50 = 91 ± 0.75 to 122 ± 1.0 µM) when
tested against a standard antioxidant, gallic acid (IC50 = 23 ± 0.43 µM).
Keywords: Barbituric acid; DFT; antioxidant (DPPH); α-Glucosidase Inhibitors.
1. Introduction
The 5-alkylbarbiturate structural motif has been found in several synthetic and natural
products with pharmaceutical activity such as antitumor, immunomodulating [1], sedative
[2], and anticonvulsant properties [3]. Additionally, several monoalkyl barbiturates have
been used in supramolecular chemistry [4], nonlinear optical studies [5], and manufacture of
dyes [6]. For example, 5-alkylbarbiturates inhibit matrix metalloproteinase (MMP)
completely [7], while the compound PNU-286607(-)-1 is an antibacterial agent [8] and
prevents mutant SOD1-dependent protein aggregation [9]. Thus, taking into consideration
the potential pharmaceutical activity of the 5-alkylbarbiturate structural system, the
construction of complex molecular frameworks incorporating this privileged structure is an
interesting goal. Toward this, different approaches have recently been disclosed, enabling the
construction of 5-monoalkylbarbiturates [10–17]. For example; 5-monoalkylated barbituric
acid derivative were identified as α-glucosidase inhibitors [18-21].
As part of our continuing research program, we report the synthesis of a series of
5-monoalkylbarbiturate derivatives and evaluate these in vitro with a DPPH radical
scavenging assay and for theirα-glucosidase inhibition. Quantum chemical calculations were
performed to analyze the structure and electronic and spectroscopic aspects of the studied
compounds in the framework of density functional theory (DFT) at the B3LYP/6-311G(d,p)
level of theory.
2. Results and discussion
2.1. Synthesis
The synthesis of the 5-monoalkylbarbiturate derivatives is depicted in Scheme 1.
Equimolar N,N-dimethyl barbituric acid (1) and chalcone derivatives (2a–l) in
dichloromethane (DCM) in the presence of diethylamine (NHEt2) base gave the desired
5-monoalkylbarbiturate derivatives (3a–l) in good yields. The yields of the
5-monoalkylbarbiturate derivatives were consistent with reported data [17]. Furthermore, the
molecular structure of 3k was confirmed by single crystal X-ray diffraction.

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Scheme 1. Preparation of 5-mono alkyl barbiturate derivatives 3a–l
O
R2
O
R1
O
NHEt2/DCM
30min/RT
O
N
N
R1
R2
+
O
83-95%
N
O
O
N
O
1
2a-l
3a-l
O
O
O
O
O
N
N
N
N
N
N
N
N
O
O
O
O
O
O
O
O
O2N
O2N
O
O
O
Br
3b, 86%
3c, 83%
3a, 95%
3d, 88%
O
O
O
N
O
O
N
N
N
N
N
N
O
N
O
O
O
O
O
O
S
O
F
O
F
O
F3C
O
3e, 85%
3f, 93%
3g, 87%
3h, 93%
CF3
CF3
O
O
O
O
N
O
N
N
O
N
N
N
N
O
N
O
O
O
O
O
Cl
O
Br
O
Cl
O
3i, 93%
O
3j, 89%
F
F
3l, 92%
3k, 95%
F
2.2. X-ray crystal structure of compound 3k
The solid-state structure of compound 3k was determined by single-crystal X-ray
diffraction. The crystal and structure refinement data, as well as selected geometric
parameters for compound 3k, are given in Tables 1 and 2. The asymmetric unit comprises
two independent molecules of 3k. The pyrimidine (N1/N3/C2/C4-C6), fluorophenyl
(C12-C17), and phenyl (C18-C23) rings are planar with a maximum root mean squared
deviation (RMSD) from the mean plane of 0.145(3) Å for C5 (Fig. 1). The two phenyl rings
(C12-C17 and C18-C23) were found to be twisted by 12.8(2)° and 37.7(2)°, respectively,
with respect to the plane of the pyrimidine ring. In the crystal, molecules are linked by
C7–H7B O6,
C7–H7C O4,
C8–H8B F1,
C13–H13A O4,
C37–H37A F2,

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C39–H39A O8, and C40–H40A O7 intermolecular interactions, resulting in a
three-dimensional network structure (Fig. 2 and Table 3) [22–24].
Fig. 1 ORTEP diagram of the compound 3k. Displacement ellipsoids are plotted at the 30%
probability level.
Fig. 2 Crystal packing showing intermolecular C---H….O hydrogen bonds as dashed lines.
Table 1.Crystal data and structure refinement for compound 3k
Crystal data
3K
Empirical Formula
Formula Weight
C42H38F2N4 O8
764.76

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Temperature (K)
Wavelength (Å)
Crystal system
Space group
293(2)
0.71073
Triclinic
P1
Unit cell dimensions
a = 7.9163(9) Å, α = 283.111(2)̊
b = 8.9021(10)Å, β = 287.831(2)
̊
c =24.079(3)Å, γ = 72.8270 (10)
̊
Volume (Å3)
1676.0(3)
2, 1.515
0.113
Z, catculated density (Mg.m-3)
Absorption coefficient (mm-1)
F(000)
800
Crystal size (mm)
0.59 x 0.52 x 0.22
Theta range for data collection 0.85º to 25.50 º
(o)
Limiting indices
-9≤h≤9, -10≤k≤10, -29≤l≤29
Reflections collected /unique
Completeness to theta
Refinement method
18870 / 6247 [R(int) = 0.0609]
25.50 (100 %)
Full-matrix least-squares on F2
6247 / 0 / 506
Data/ restraints/ parameters
Goodness of- fit on F2
Final R indices [I>2σ(I)]
R indices (all data)
0.981
R1 = 0.0625, wR2 = 0.1540
R1 = 0.1463, wR2 = 0.2077
Largest diff. peak and hole (e 0.239 and -0.249
A-3)
CCDC reference
1024287
Table 2 Selected geometric parameters (Å, °) of 3k
O1—C4 1.177(4) O3—C6
1.169(4)

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O2—C2
1.163(4)
1.174(4)
1.175(4)
1.317(5)
1.327(5)
1.408(4)
1.337(5)
1.317(4)
1.423(4)
1.329(5)
124.1(3)
117.9(4)
119.2(3)
123.8(3)
117.4(3)
118.5(4)
121.0(4)
118.0(4)
121.9(4)
120.2(4)
O4—C11
1.181(4)
1.154(4)
1.172(4)
1.313(4)
1.329(5)
1.318(5)
1.414(5)
1.328(4)
1.311(4)
1.418(4)
118.0(4)
123.2(4)
117.5(4)
118.8(3)
123.9(3)
117.5(3)
121.0(4)
120.2(4)
117.9(3)
119.1(4)
O5 —C24
O6 —C26
O7— C28
O8 —C34
F1 —C15
F2— C38
N1 —C6
N1 —C2
N1 —C7
N3 —C4
N3 —C2
N3 —C8
N25— C24
N25 —C26
N25— C30
N27 —C28
N27— C26
N27 —C31
C6 —N1— C2
C2 —N1— C7
C4— N3— C8
C24— N25 —C26
C26 —N25 —C30
C28— N27— C31
O2— C2— N1
N1 —C2 —N3
O1— C4 —C5
O4 —C11— C12
C6 —N1— C7
C4— N3 —C2
C2 —N3— C8
C24 —N25— C30
C28— N27 —C26
C26— N27— C31
O2— C2— N3
O1 —C4— N3
N3— C4— C5
O4 —C11— C10
Table 3.Hydrogen-bond geometry (Å, °) of 3k

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D—H···A
D—H
0.96
0.96
0.96
0.93
0.93
0.93
0.93
H···A
2.31
2.57
2.39
2.25
2.39
2.52
2.29
D···A
D—H···A
143
C7—H7B···O6i
C7—H7C···O4 ii
C8—H8B···F1 iii
C13—H13A···O4iv
C37—H37A···F2v
C39—H39A···O8i
C40—H40A···O7i
3.136(6)
3.360(5)
3.219(5)
3.096(5)
3.196(5)
3.406(5)
3.022(5)
140
145
152
144
159
136
Symmetry code: (i) -1+x,y,z, (ii) x,-1+y,z (iii) -x,2-y,-z, (iv) -x,3-y,-z, (v) -x,-1-y,1-z,
2.3. Computational details
Geometry optimization of 3k followed by frequency analysis was performed at the
B3LYP/6-311G(d,p) level of theory using Gaussian 03, revision C.01 [25]. The X-ray
structure coordinated taken from the crystallographic information file is used as starting input
for our calculations. All vibrational modes were positive, and no imaginary frequency modes
were detected. Time-dependent (TD)-DFT and gauge-including atomic orbital (GIAO) NMR
chemical shifts calculations at the optimized geometry were performed to predict the
1
electronic and NMR spectra of the studied compound. The 13C and H chemical shifts (δ)
were calculated using the GIAO isotropic magnetic shielding (IMS) values based on the
equation: CSx = IMSTMS - IMSx.
All calculations were made without symmetry restrictions. The optimized structure was
drawn with the aid of Chemcraft [26]. The frontier molecular orbital (FMO) pictures as well
as the molecular electrostatic potential (MEP) map were drawn using Gaussview 4.1 [27].
GaussSum 2.2 [28] was used to obtain the molecular orbital contributions of the studied
electronic transitions. The natural charges and the intramolecular charge transfer interaction
energies were deduced from natural bond orbital (NBO) analysis using NBO 3.1 [29]. The

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global chemical reactivity descriptors such as electronegativity (χ), chemical potential (ꢀ),
chemical hardness (η), global softness (S) and global electrophilicity index (ω) were
calculated using Eqs. (1)–(5) [30-34].
,
(1)
(2)
(3)
(4)
(5)
,
,
,
,
whereI and A are the ionization potential and electron affinity, respectively, which are
defined as the negative values of the EHOMO and ELUMO, respectively.
2.4. Structure
The minimum energy structure obtained from the geometry optimization of the X-ray
structure of 3k at the B3LYP/6-311G(d,p) level of theory is shown in Fig. 3. The molecular
point group is C1. The calculated and experimental bond distances and bond angles are
presented in Figs. S1–S2 and Table S1 (Supplementary Data). In this table, the statistical
parameters such as RMSD and correlation coefficients indicate a good agreement between
the calculated and experimental geometrical parameters. The maximum bond distance and
bond angle deviations from the experimental values are 0.087 Å and 3.4° for C30-C32 and
C12-C10-C21, respectively. The deviations of the calculated geometric parameters are
mainly due to phase differences between calculation and experiment. That is, the calculations
were performed on a single molecule in the gas phase, whereas, in a single crystal (as used for
the X-ray experiments) molecular packing effects are present. In the crystal, there are many
intermolecular interactions that affect the geometric parameters, resulting in deviations from
the gas-phase structure. Despite these differences, the geometric parameters correlate well
with the experimental data, and the bond distances offer a better agreement with the
experimental data than the bond angle values.

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Fig. 3The calculated molecular structure of 3k using B3LYP/6-311G(d,p) method.
2.5. Natural atomic charges
Most of the electronic properties of a molecule, such as the dipole moment, molecular
polarizability, electronic structure, and molecular reactivity, are related to the charge
distribution at the different atomic sites. The charges on the atoms of the compound were
calculated using the NBO method at the B3LYP/6-311G(d,p) level of theory. The results
are presented graphically in Fig. 4. Almost all the C-atoms of the phenyl rings have
negative natural charges. However, C32 has high positive charge, which is attributed to its
attachment to a highly electronegative F-atom. For the pyrimidinetrione ring, all C-atoms
(except C10) are electropositive because they are attached to either O and N-atoms or both.
The most electropositive C-atom is C8 (0.8500), and this is because C8 is bonded to three
highly electronegative atoms. Moreover, all H-atoms have positive charge densities, where
the most electropositive H-site is H11 (0.2915). The high positive charge density at H11
and the high negative charge density at C10 (-0.3900) are strong indications on the acidic
character of the C10-H11 bond.

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1
0.5
0
-0.5
-1
Fig. 4The natural charge distribution at the different atomic sites of the studied molecule.
2.6. Molecular electrostatic potential
An MEP map was calculated for the B3LYP/6-311G(d,p) optimized geometry (Fig.
S3; Supplementary Data). This three-dimensional map is colored according to the
electrostatic potential: deep red for electron rich regions and deep blue for electron poor
regions. The reddest regions are those most reactive to attack by an electrophile, while the
bluest regions are electrophilic sites. MEP maps can be used to determine how molecules
interact with one another [35, 36]. The MEP map shows that negative charge is
concentrated at the top of the carbonyl O-atoms as well as the F-atom. The positive regions
spread over the surface of the hydrogen atoms. It is clear also from this map that the phenyl
ring C-skeleton (yellow) is more electron rich than the pyrimidine trione core (blue). Hence,
the former favors electrophile attack, while the latter favors nucleophilic attack [37].
2.7. HOMO–LUMO analysis
The highest occupied molecular orbital (HOMO) and lowest occupied molecular
orbital (LUMO) are the FMOs. The HOMO energy is indicative of the ability of a molecule
to donate electrons. On the other hand, the LUMO energy gives an indication of the ability
of a molecule to accept electrons. Moreover, the HOMO-LUMO energy difference is
defined as the transition energy. This energy gap yields information on the ease of
electronic transitions. A large energy gap (∆E) indicates high stability with respect to
electron transfer. In addition, these FMOs are related to the electric and optical properties,

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as well as the chemical reactivity [38].For 3k, EHOMO = -7.0271 eV, ELUMO = -1.8806 eV,
and ∆E = 5.1465 eV.
The FMOs are shown in Fig. S4 (Supplementary data). Both HOMO and LUMO are
mainly distributed over the π-system of the molecule. The HOMO level is mainly localized
over the phenyl ring A and C26=O4 π-systems, while the LUMO is localized over ring B.
From this point of view, ring A acts as the electron rich fragment and acts as an electron
donor while ring B, which carries the very electronegative F-atom, is an electron acceptor.
Hence, the electronic transition could be described as a π-π* transition from ring A to B.
The energy of this electronic transition (5.1465 eV) is relatively high, indicating that the
studied compound is highly stable.
2.8. Chemical properties
Based on the HOMO and LUMO energies, the chemical reactivity descriptors such as
chemical potential (ꢀ), electronegativity (χ), chemical hardness (η), global softness (S), and
global electrophilicity index (ω) were calculated, and the results are presented in Table 4.
The chemical potential can be used to describe many electrical and nuclear properties [39],
and it reflects a molecule’s propensity for spontaneous chemical decomposition. Hence, if
the chemical potential is negative, a compound is stable and does not decompose
spontaneously [40]. The chemical hardness (ꢀ) indicates the ability of a molecular system
to resist polarization. High values of ꢀ indicate a less polarized system and, hence, greater
stability against electronic deformation under small perturbations during a chemical
reaction, and softness is the reverse of hardness. The electrophilicity index (ω) indicates the
ability of a molecule to gain electrons from the environment, that is, its ability to act as an
electrophile [41]. The high electrophilicity index indicates the high electrophilic character
of the studied compound, which is probably due to the presence of a large number of highly
electronegative atoms.
Table 4 The EHOMO, ELUMO and the derived chemical reactivity descriptors of 3k calculated
by B3LYP/6-31G(d,P) method.
Parameter
EHOMO
Energy (eV)
-7.0271

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ELUMO
-1.8806
- 4.4539
4.4539
2.5733
0.1943
chemical potential (ꢀ)
electronegativity (χ)
chemical hardness (η)
global softness (S)
global electrophilicity index (ω)
3.8544
2.9. Electronic spectra of the studied compound
The electronic spectrum of 3k was calculated using TD-DFT, and the results of the forty
singlet-singlet electronic transitions are given in Table S2 (Supplementary Data). Theoretical
and experimental ultraviolet-visible (UV-Vis) spectra are shown in Fig. S5 (Supplementary
Data). Experimentally, the spectrum of 3k in acetonitrile contains three electronic transition
bands at 203, 237, and 303 nm, as well as a shoulder at 265 nm. Theoretically, the two intense
electronic transitions were predicted at 203.9 nm (f = 0.1185) and 244.0 nm (f = 0.3377),
which agree with the experimental data. The most intense and longest wavelength band was
assigned to the H-2→L (80%) transition, while the shorter wavelength band arises from the
H-5→L (22%), H-2→L+2 (56%) excitations. The molecular orbitals involved in these
electronic transitions are shown in Fig. 5. Both electronic transitions are π→π* excitations.

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Fig. 5The molecular orbitals involved in the most important electronic transition bands
calculated using TD-DFT method at B3LYP/6-311G(d,p).
2.10. Natural bond orbital (NBO) analyses
The NBO analyses presented in this section show the most accurate Lewis structural
description of 3k. For each A-B bond, the electron occupancy (ED), the percentage electron
density at each atom (ED%), and the polarization coefficients of each atom forming the bond,
as well as the composition of the atomic orbitals in the bond hybrids, are presented in Table
S3 (Supplementary Data). For all C-H bonds, all H-atoms have 0% p-character. In contrast,
BD(2)C=C and BD(2)C=O π-NBOs have almost 100% p-character. Similarly, LP(1)N,
LP(2)F, LP(3)F, and LP(2)O NBOs have 100% p-character.
For 3k, the BD(1)C10-C21 with 1.9391 electrons has a 52.17% electron density at C10
(27.18% s) and 47.83% electron density at C21 (24.40% s). The high s-character at C10
indicates the high acidity of the C10-H11 bond. Moreover, the BD(1)C21-C23 NBO with
1.9710 electrons has 50.12% ED at C21 (27.01% s) and 49.88% ED at C23(29.50% s). The
BD(1)C21-C23 has almost two C-atoms of equal electron density and hence almost equal
polarization coefficients (0.7080 and 0.7063), while C10 of the BD(1)C10-C21 NBO has a
higher electron density than that of C21. As a result, C10 (0.7223) has a higher polarization
coefficient than that of C21 (0.6916). In addition, all C-O, C-N, and C-F bonds have higher

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polarization coefficients at the O, N, and F-atoms, respectively, compared to carbon. The
polarization coefficients at the O, N, and F atoms are 0.8091–0.8139, 0.7924–0.8025, and
0.8540, respectively. Fluorine has the highest electronegativity.
The stabilization energy (E(2)), which results from the delocalization of electrons among
bonds, is an important quantitative measure of the stability of a molecular system. Using
second-order perturbation theory and NBO analysis, the stabilization energies (E(2)) due to
the most important intramolecular charge transfer (ICT) interactions from the donor NBO to
the acceptor were determined, and these are given in Table 5. The stabilization energies (E(2))
arising from the π→π* ICT interactions range from 16.36 to 25.21 kcal/mol. Of these π→π*
ICT interactions, the strongest interaction is π(C28-C30)→ π*(C32-C33) in ring B, which
has an F-substituent. The presence of such a strongly electronegative atom at C32 increases
the ICT to the sπ*(C32-C33) antibonding NBO to a greater degree than the other ICTs
Moreover, the π(C27-C35)→π*(O4-C26) ICT has an E(2) value of 18.91 kcal/mol, indicating
strong electron delocalization from the π(C27-C35) NBO of ring A, which acts as a donor to
the π*-antibonding NBO of the carbonyl group (O4=C26).
The ICTs from the filled lone pair NBO to the adjacent σ*- or π*-NBOs have maximum
stabilization energies of 26.66 and 54.23 kcal/mol, respectively. The n → π * ICT
interactions stabilize the system more than the n→σ* ICT interactions, and the n→σ
*(C-N) ICT interactions are stronger than those of the n→σ*(C-C) ICTs.
Table 5. The most important intramolecular charge transfer interactions and their
stabilization energies deduced from the second order perturbation theory.
Donor NBO
Acceptor NBO
E(2)
(kcal/mol) NBO
LP(2)
O1
LP(2)
O1
Donor
Acceptor NBO
BD*(1)N7-C9
E(2)
(kcal/mol)
BD(2)C27-C35 BD*(2)O4-C26 18.91
BD(2)C27-C35 BD*(2)C28-C30 21.69
BD(2)C27-C35 BD*(2)C32-C33 18.42
26.66
BD*(1)C9-C10 19.27
LP(2)
O2
BD*(1)N6-C8
25.86

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LP(2)
BD(2)C28-C30 BD*(2)C32-C33 25.21
O2
BD*(1)N7-C8
25.49
BD*(1)N6-C12 25.85
BD*(1)C10-C12 18.10
BD*(1)C23-C26 19.36
LP(2)
O3
BD(2)C28-C30 BD*(2)C27-C35 17.48
BD(2)C32-C33 BD*(2)C27-C35 22.78
LP(2)
O3
LP(2)
O4
BD(2)C32-C33 BD*(2)C28-C30 16.36
BD(2)C37-C38 BD*(2)C40-C42 20.67
BD(2)C37-C38 BD*(2)C44-C46 20.23
BD(2)C40-C42 BD*(2)C37-C38 20.54
BD(2)C40-C42 BD*(2)C44-C46 21.04
BD(2)C44-C46 BD*(2)C37-C38 20.84
BD*(2)C40-
LP(2)O4 BD*(1)C26-C27 18.88
LP(3)F5 BD*(2)C32-C33 19.75
LP(1)N6 BD*(2)O2-C8
52.59
LP(1)N6 BD*(2)O3-C12 51.96
LP(1)N7 BD*(2)O1-C9
LP(1)N7 BD*(2)O2-C8
51.26
54.23
BD(2)C44-C46 C42
20.00
2.11. NMR spectral analysis
Isotropic chemical shifts are used for the identification of organic compounds. GIAO
1
calculations were used for the accurate prediction of the H and 13C isotropic chemical
shifts, and these are presented in Table 6. The chemical shifts of the aromatic carbons
overlap between 100 and 200 ppm [42]. Atom C32 has a higher chemical shift (calc. 176.01
pm and exp. 167.75 ppm) than the other aromatic carbons (calc. 119.80–149.12 ppm and
exp. 130.65–150.87 ppm). The high chemical shift of C32 is due to the deshielding effect of
the electronegative F-atom. The oxygen and nitrogen atoms are more electronegative than
the carbon atoms, so the carbonyl C-atoms were detected at higher chemical shifts
(150.87–196.03 ppm), which is consistent with the calculated values (calc. 156.59–202.30
ppm).
The 1H-chemical shifts of the aromatic ring usually appear in the region of 7 to 8 ppm.
In the present study, the aromatic protons were detected at 7.12–8.02 ppm, which is in good

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agreement with B3LYP theoretical values (7.15–8.62 ppm). The aliphatic protons have
lower chemical shifts than the aromatic ones. The experimentally measured aliphatic
1H-signals are in the range 3.02–4.36 ppm (calc. 2.29–4.83) ppm. Correlation graphs
between the experimental and theoretical NMR chemical shifts are shown in Figs. S6 and
S7 (Supplementary Data). The correlation equations shown below have high R2 values
(0.932–0.962, indicating a good agreement between the theoretical and experimental data.
δcalc = 0.996 δexp - 2.653
R² = 0.962
13C-NMR
(6)
δcalc = 0.899 δexp + 0.387 R² = 0.932
1H-NMR
(7)
13
Table 6 The calculated and experimental chemical shifts (1H and C) for 3k using GIAO
method at 6-311G(d,p) method.
Atom
δcalc
156.59
172.97
54.39
δexp
Atom
δcalc
4.70
4.28
2.41
2.65
4.27
2.59
2.29
4.38
4.83
2.89
8.62
7.25
7.15
8.08
7.39
7.38
7.39
7.44
δexp
3.96
3.09
3.09
3.09
3.02
3.02
3.02
4.36
4.03
3.48
8.02
7.24
7.24
8.02
7.12
7.12
7.24
7.12
C
8
9
150.87
197.11
52.86
H
11
14
15
16
18
19
20
22
24
25
29
31
34
36
39
41
43
45
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
10
12
13
17
21
23
26
27
28
30
32
33
35
37
38
40
174.60
28.73
167.75
28.07
29.22
27.95
50.79
40.18
44.58
44.33
202.30
138.59
137.58
120.95
176.01
119.80
136.28
149.12
136.46
134.20
196.03
137.97
115.83
130.74
167.75
130.65
115.61
150.87
128.68
128.35

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C
C
C
42
44
46
133.36
134.07
132.34
127.24
130.65
128.68
H
47
7.43
7.12
2.12. Infrared vibrational spectra
The studied molecule contains 47 atoms with a total of 135 vibrational modes. A
complete assignment of these modes is presented based on the total energy distribution
(TED) obtained from Veda 4 [43]. The results and their comparison with the experimental
values are listed in Table S4 (Supplementary Data). The theoretical and experimental IR
spectra of the studied molecule are shown in Figs. S8 and S9 (Supplementary data). The
calculated vibrational frequencies were scaled by 0.9670 to correct the calculated harmonic
frequencies [44]. Generally, the scaled infrared vibrational frequencies agreed well with the
experimental data. The most important modes are now presented based on the TED analysis.
2.12.1. C–H vibrations
The studied molecule has both aromatic and aliphatic C-H bonds. The aromatic C-H
stretching modes have higher wavenumbers than the aliphatic C–H stretching vibrations. In
the present case, the four C-H stretching modes of the phenyl ring B were predicted to have
the highest wavenumbers (3102–3087 cm-1), while, in the experimental spectrum, these
stretching modes were observed in the range of 3093–3072 cm-1. In contrast, the C-H
stretching modes of ring A were predicted by the TED analysis to occur at lower vibrational
wavenumbers than those for ring B. The aromatic C-H modes of ring A were calculated at
3086 and 3057–3079 cm-1 (exp. 3061 cm-1 and 2991–2946 cm-1). The presence of the
F-substituent increased the vibrational frequencies of the aromatic C-H modes.
The asymmetric C-H stretching modes of the N-CH3 groups were calculated at 3080 and
3029–2998 cm-1, while, in the experimental spectrum, these stretching modes were observed
at 3027, 2931, and 2920 cm-1. The high-frequency C-H mode of the N-CH3 group appeared
overlapped with the aromatic C-H modes. In contrast, the symmetric C-H modes of the
N-CH3 groups were predicted at a lower frequency of 2966 cm-1 (exp. 2892 cm-1) [45]. The
asymmetric and symmetric C-H stretching vibrations of the methylene group were predicted

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at 2998 and 2933 cm-1, respectively. The ν(C10-H11) and ν(C21-H22) stretches were calculated at
2947 (exp. 2844 cm-1) and 2983 cm-1, respectively. The acidic C-H bond of the ring carbons
appeared at very low wavenumbers in the IR spectra of the studied compounds. We found
C-H stretching modes are pure and have a high TED%.
The umbrella vibrational modes are considered the most important bending vibrations of
methyl groups. These modes usually detected in the range 1375 ± 10 cm-1. The studied
compound has two N-CH3 groups with umbrella vibrations appeared at a higher frequency
than expected, 1424 cm−1 (calc. 1423–1398 cm−1). Moreover, the asymmetric C-H bending
of the N-CH3 group was calculated at 1465–1449 cm−1 (exp. 1440 cm−1). The asymmetric
bending vibrations of the N-CH3 groups have higher wavenumbers than the symmetric
modes. Other bending vibrations were found mixed with the C-C, C-N, and other bending
vibrations (Table S4; Supplementary Data). The aromatic C-H in-plane bending modes were
found in this overlapping region, while the out of plane C-H modes of the aromatic structure
were predicted at lower frequencies of 976–696 cm-1 (exp. 969–705 cm-1)
2.12.2. Carbonyl vibrations
The carbonyl stretching vibration is considered one of the most intense bands in the IR
spectra. The studied compound has four carbonyl groups, so four C=O stretching modes were
predicted. The stretching vibrational wavenumbers of these carbonyl groups were calculated
at 1743, 1692, 1685, and 1676 cm-1. Experimentally, three (νC=O) bands were detected at
1730, 1685, and 1674 cm-1. The highest wavenumber band was predicted to have low
vibrational intensity both experimentally and theoretically. This mode was predicted using
the TED to arise from the symmetric C=O stretches of the three carbonyl groups. The
remaining νC=O modes were strong and assigned to the asymmetric stretching vibrations.
2.12.3. C-C, C-N, and C-F vibrations
The aromatic stretching C-C modes (νC-C) usually appears around 1600–1585 cm-1 and
1500–1400 cm-1. The TED analyses of 3k predicted the νC-C modes at 1589, 1572, 1570, and
1487 cm-1. The breathing modes of rings A and B were calculated at 984 and 808 cm-1,
respectively, while the out of plane ring vibrations for rings A and B were calculated at 404
and 410 cm-1, respectively. The C-N stretching modes of the pyrimidinetrione ring were

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calculated using TED analysis at 1354, 1255, and 1229 cm-1. The higher wavenumbers of
these modes indicated the C-N bonds have some double bond characters due to the electron
delocalization with the adjacent carbonyl groups. The aromatic νC-F stretching vibrations
usually appeared in the region 1400–100 cm-1. Experimentally this band was observed at
1216 cm-1, while it was calculated at 1220 cm-1. The CCF bending modes were calculated at
625, 425, and 387 cm-1 (exp. 619 and 439 cm-1).
2.13. Biological activity
The synthesized monoalkylated barbiturate derivatives 3a–l were screened in vitro for
their antioxidant activity (DPPH radical scavenging ability) and α-glucosidase enzyme
inhibition activity. The results of the bioassay studies are summarized in Table 7.
Table 7: Biological activity evaluation of compounds 3a-l
α-Glucosidase
DPPH Radical
Inhibition
#
Scavenging Assay
IC50±SEM[µM]
Assay
IC50±SEM[µM]
NA
314.127±2.4
91.223±0.75
107.241±0.83
93.149±0.63
112.084±0.55
92.245±0.79
92.094±1.3
3a
3b
3c
3d
3e
3f
133.1±3.2
NA
305±7.7
184±2.3
NA
NA
3g
3h
3i
124.109±0.49
118.634±1.02
122.662±1.05
116.440±0.28
114.6±0.9
NA
NA
NA
3j
NA
3k
3l
NA

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ST
D.
Gallic acid
Acarboses
840±1.73
23.436±0.43
2.13.1. Antioxidant activity (DPPH radical scavenging assay)
The synthesized monoalkylated barbiturate derivatives 3a–l were evaluated for their
antioxidant activity in vitro using the DPPH radical scavenging assay. These compounds
were found to be weak scavengers of free DPPH radicals (IC50 = 91 ± 0.75 – 122 ± 1µM)
compared to the standard, gallic acid (IC50 = 23 ± 0.43 µM). The 3-oxo-1,3-diphenylpropyl
substituted pyrimidine adduct 3a was found to be the least active member of the series (IC50 =
314 ± 2 µM). However a gradual increase in activity was observed for compounds 3h (IC50 =
124 ± 0.49 µM), 3j (IC50 = 122 ± 1µM), 3i (IC50 = 118 ± 1µM), 3k (IC50 = 116 ± 0.28µM), 3l
(IC50 = 114 ± 0.9 µM), 3e (IC50 = 112 ± 0.55 µM), 3c (IC50 = 107 ± 0.83 µM), 3b (IC50 =
91 ± 0.75 µM), 3d (IC50 = 93 ± 0.63 µM), 3f (IC50 = 92 ± 0.79 µM), and 3g (IC50 = 92 ±
1 µM), which contain fluoro, bromo, and chloro substituents on the phenyl rings of central
3-oxo-1,3-diphenylpropylpyrimidine backbone.
2.13.2. α-Glucosidase inhibition activity
The monoalkylated barbiturate derivatives 3a–l were also evaluated for their in vitro
α-glucosidase inhibiting activities compared to the standard drug acarbose (IC50
=
840 ± 1.73 µM). Compound 3a, which contains a diphenyl substituted 3-oxo-propyl chain,
was inactive against the enzyme. In contrast, high activities were observed for compounds
3b, 3d, and 3e, which contain para-nitrophenyl (3b, IC50 = 133.1 ± 3.2 µM),
meta-bromophenyl (3d, IC50 = 305 ± 7.7 µM), and thiophene rings (3e, IC50 = 184 ± 2.3 µM)
attached to the central 3-oxo-1,3-dipropyl substituted pyrimidine skeleton. These compounds
exhibited more activity than the tested standard acarbose (IC50 = 841 ± 1.73 µM). All other
compounds were found to be inactive. The complete absence of activity may be due to the
positional difference of substituents between different compounds, such as 3c, which has an
ortho-nitrophenyl ring instead of a para-nitrobenzene ring (3b). Interestingly all barbiturates
with fluoro or chloro-substituted phenyl rings (3f–3l) were inactive in this assay.

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2.14. Molecular docking
To predict the interaction of these newly synthesized compounds in the α-glucosidase
binding pocket, we docked the most potent compound (3b) with this enzyme [18, 46]. From
the docking simulation, we found that compound 3b fitted well into the α-glucosidase
binding cavity, forming interactions with the residues His 279 and Asn 241. His 279 forms an
arene-cation interaction with the phenyl moiety of the compound, whereas Asn 241 forms a
hydrogen bond with the carbonyl oxygen of pyrimidine moiety of the compound.
Additionally, several hydrophobic interactions between the compound and active site
residues (e.g., Phe 157, Ala 278, and Phe 311) were observed (Fig. 6).
In case of the second most active compound (compound 3e) in the series the docking
results showed that a hydrogen bond was established between the carbonyl oxygen atom of
the compound and nitrogen atom of the active site residue Asp 349 whereas the phenyl ring
of the compound formed an arene-cation interaction with active site residue Arg 312. A
number of hydrophobic interactions were also observed between the compound 3e and
hydrophobic residues present in the active site (e.g., Phe157 and Phe Phe311) (Fig. 7).
Fig. 6. Docking pose of the compound 3b in the active site of α-glucosidase enzyme

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Fig. 7. Docking pose of the compound 3e in the active site of α-glucosidase enzyme.
The docking result of the least active compound (compound 3d) of the series showed
only one interaction with the active site residues. This compound showed a hydrogen bond
with the active site residue Asn 347 but no anre-arene or arene-cation interaction were
observed (Fig. 8). This might be one of the reasons for this compound to show less activity as
compare to compound 3b and 3e.
In case of inactive compounds (compound 3h) no significant interaction with the active
site residues of the enzyme was observed (Fig. 9).
Fig. 8. Docking pose of the compound 3d in the active site of α-glucosidase enzyme.

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Fig. 9. Docking pose of the compound 3h in the active site of α-glucosidase enzyme.
3. Materials and Methods
3.1. General remarks
“All the chemicals were purchased from Aldrich, Sigma-Aldrich, and Fluka and were
used without further purification, unless otherwise stated. All melting points were measured
on a Gallenkamp melting point apparatus in open glass capillaries and are uncorrected. IR
spectra were measured on KBr pellets on a Nicolet 6700 FT-IR spectrophotometer. The
1
NMR spectra were recorded on a Varian Mercury Jeol-400 NMR spectrometer. H-NMR
(400 MHz) and 13C-NMR (100 MHz) were run in either deuterated dimethyl sulfoxide
(DMSO-d6) or deuterated chloroform (CDCl3). Chemical shifts (δ) are in ppm, and
J-coupling constants are given in Hz. Mass spectra were recorded on a Jeol JMS-600 H.
Elemental analysis was carried out on Elmer 2400 Elemental Analyzer in CHN mode. For
X-ray diffraction analysis, data were collected on a Bruker APEX-II D8 Venture
diffractometer with an area detector. The electronic spectrum of 3k was measured using a
Perkin Elmer, Lambda 35, UV/Vis spectrophotometer”.
3.2. General Michael addition reaction for the synthesis of pyrimidine derivatives 3a–l
(GP1).
The compounds were prepared using our previously reported method [17].
3.3. X- ray crystallography

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Crystals of compound 3k were obtained by the slow diffusion of a diethyl ether solution
of pure compound 3k in dichloromethane at room temperature for 24 h. Data were collected
on a Bruker APEX-II D8 Venture diffractometer equipped with a graphite monochromator
and Cu Kα radiation at 293(2)K. Cell refinement and data reduction were carried out using
Bruker SAINT. SHELXS-97 [22, 23] was used to solve the structure. The final refinement
was carried out by full matrix least squares refinement. Non-hydrogen atoms were refined
anisotropically. All the hydrogen atoms were placed in calculated positions.
3.4. Bioassays
3.4.1. DDPH radical scavenging assay:
A DPPH solution (95 µL, 300 µM) in ethanol was mixed with test solution (5 µl,
500 µM). The reaction was allowed to progress for 30 min at 37 °C, and the absorbance at
517 nm was monitored using a multiplate reader (SpectraMax340). Upon reduction, the color
of the solution faded from violet to pale yellow. Percent radical scavenging activity (% RSA)
was determined by comparison with a DMSO-containing control. The IC50 values of
compounds were calculated using the EZ-Fit enzyme kinetics program (Perrella Scientific
Inc., Amherst, MA, USA). N-Acetylcysteine, ascorbic acid, and BHA were used as reference
compounds [47].
3.4.2. In vitro α-glucosidase inhibition assay.
First, 135 µL of a 50 mM phosphate saline buffer pH (6.8) was dispensed in a 96-well
plate. Then, 20 µL of the test sample in 70% DMSO was dispensed into the wells.
Subsequently, 20 µL of the enzyme was added to the wells, and the plate was incubated for
15 min. After incubation, the plate was pre-read using the spectrophotometer. After the
pre-read, 25 µL of the substrate (ÞNPG) was added, and readings were taken again at 400 nm
for 30 minutes. Finally, a normal read was taken, and the percent inhibition was calculated
[48,49].
3.5. Molecular docking
Docking of the 5-monoalkylbarbiturate derivatives against α-glucosidase was carried out
using the Molecular Operating Environment (MOE 2010.11). The same protocol was used
for the protein, ligand preparation, and molecular docking, as described in our previous
papers [18–46].

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4. Conclusions
In summary, we have synthesized a series of 5-monoalkylbarbiturate derivatives via
Michael addition reaction mediated by NHEt2 in CH2Cl2. Using DFT calculations at the
B3LYP/6-311G(d,p) level of theory, we have optimized the X-ray structure. The final
optimized structure is in good agreement with the experimental structure. The structural
deviations between the models can be attributed to the differences between calculation and
experimental measurements, i.e., gas phase and solid state. The IR spectrum of the studied
compound was calculated and assigned based on the total energy distribution. The calculated
UV-Vis spectrum of 3k was compared with the experimental spectrum. The most intense and
longest wavelength band was assigned to the H-2→L (80%) transition. The molecular orbital
contribution showed that the most intense bands belong to π→π* transitions. A molecular
electrostatic potential map and global reactivity parameters were used to describe the
chemical reactivity of the studied molecule. The desired compounds were investigated in
vitro for antioxidant activity (DPPH), and α-glucosidase inhibition with promising results.
Supplementary Materials: General procedure of the Michael addition reaction for the
synthesis of pyrimidine derivatives 3a–l (GP1). Fig. S1 Comparison between the calculated
and experimental bond distances of 3k. Fig. S2 Comparison between the calculated and
experimental bond angles of 3k. Fig. S3 MEP map of 3k. Fig. S4 The HOMO and LUMO
energy levels of 3k calculated at the B3LYP/6-311G(d,p) level of theory. Fig. S5
Experimental (upper) and theoretical (lower) electronic spectra of 3k calculated using the
1
TD-DFT method. Fig. S6 Correlation graph between the experimental and calculated H
chemical shifts of 3k. Fig. S7 Correlation graph between the experimental and calculated 13C
chemical shifts of 3k. Fig. S8 The calculated scaled [45] IR vibrational spectra of 3k at the
B3LYP/6-311G(d,p) level of theory. Fig. S9 The FTIR spectra of 3k.
Acknowledgments: The authors would like to extend their sincere appreciation to the
Deanship of Scientific Research at King Saud University for providing funding to this
Research group NO (RGP -257).
Conflicts of Interest: “The authors declare no conflict of interest."

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