Synthesis, X-Ray Crystal Structures, Biological Evaluation, and Molecular Docking Studies of a Series of Barbiturate Derivatives

Article abstract of DOI:10.1155/2016/8517243

A series of barbiturates derivatives synthesized and screened for different set of bioassays are described. The molecular structures of compounds 5a, 5d, and 5f were solved by single-crystal X-ray diffraction techniques. The results of bioassay show that

Full text of DOI:10.1155/2016/8517243

Hindawi Publishing Corporation
Journal of Chemistry
Volume 2016, Article ID 8517243, 11 pages
http://dx.doi.org/10.1155/2016/8517243
Research Article
Synthesis, X-Ray Crystal Structures, Biological Evaluation, and
Molecular Docking Studies of a Series of Barbiturate Derivatives
Assem Barakat,1,2 Hazem A. Ghabbour,3,4 Abdullah Mohammed Al-Majid,1
Qurat-ul-ain,5 Rehan Imad,5 Kulsoom Javaid,5 Nimra Naveed Shaikh,5 Sammer Yousuf,5
M. Iqbal Choudhary,1,5 and Abdul Wadood6
1 Department of Chemistry, College of Science, King Saud University, P.O. Box 2455, Riyadh 11451, Saudi Arabia
2Department of Chemistry, Faculty of Science, Alexandria University, P.O. Box 426, Ibrahimia, Alexandria 21321, Egypt
3Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, P.O. Box 2457, Riyadh 11451, Saudi Arabia
4Department of Medicinal Chemistry, Faculty of Pharmacy, Mansoura University, Mansoura 35516, Egypt
5H.E.J. Research Institute of Chemistry, International Center for Chemical and Biological Sciences, University of Karachi,
Karachi 75270, Pakistan
6Department of Biochemistry, Abdul Wali Khan University, Mardan 23200, Pakistan
Correspondence should be addressed to Assem Barakat; ambarakat@ksu.edu.sa
Received 4 February 2016; Revised 6 April 2016; Accepted 10 April 2016
Academic Editor: Gustavo Portalone
Copyright © 2016 Assem Barakat et al. is is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
A series of barbiturates derivatives synthesized and screened for different set of bioassays are described. e molecular structures of
compounds 5a, 5d, and 5f were solved by single-crystal X-ray diffraction techniques. e results of bioassay show that compounds
4a, 4b, 4c, 4d, 4e, 4f, and 4g are potent antioxidants in comparison to the tested standards, butylated hydroxytoluene (BHT), and ꢀ-
acetylcysteine. Compounds 4a–4e (IC = 101.8 0.8–124.4 4.4 ꢁM) and 4g (IC = 104.1 1.9 ꢁM) were more potent antioxidants
50
50
than the standard (BHT, IC = 128.8 2.1 ꢁM). e enzyme inhibition potential of these compounds was also evaluated, in vitro,
50
against thymidine phosphorylase, ꢂ-glucosidase, and ꢃ-glucuronidase enzymes. Compounds 4c, 4h, 4o, 4p, 4q, 5f, and 5m were
found to be potent ꢂ-glucosidase inhibitors and showed more activity than the standard drug acarbose, whereas compounds 4v,
and 5h were found to be potent thymidine phosphorylase inhibitors, more active than the standard drug, 7-deazaxanthine. All
barbiturates derivatives (4a–4x, 4z, and 5a–5m) were found to be noncytotoxic against human prostate (PC-3), Henrietta Lacks
cervical (HeLa) and Michigan Cancer Foundation-7 breast (MCF-7) cancer cell lines, and 3T3 normal fibroblast cell line, except 4y
which was cytotoxic against all the cell lines.
1. Introduction
and sodium pentothal (Figure 1, 1–5) [10–13]. e interesting
mechanism of action of barbital and phenobarbital against
epilepsy made them attractive targets for medicinal chemists.
ey have also been investigated as anticancer and anti-
AIDS agents [14]. Reactive oxygen species (ROS) are oxygen-
centered free radicals such as reactive oxygen species (ROS)
Derivatives of 1,3-dimethylbarbituric acid including pyrim-
idine-2,4,6-trione (PYT) heterocycles play an important role
in the pharmaceutical chemistry [1, 2]. Barbituric acid and its
derivatives are well known as hypotensive, antibacterial, and
antisclerotic, hypnotic, sedative, anticonvulsant, antispas-
modic, anticancer, matrix metalloproteinase inhibitors, anti-
convulsants, and anti-inflammatory and anxiolytic agents, as
well as being used in local anesthetic drugs [2–9]. Barbituric
acid derivatives are also known for their use as clinical
drugs, such as Seconal, Veronal, phenobarbital, bucolome,
which are peroxyls (ROO∙), superoxides (O ∙−), hydroxyls
2
(HO∙), alkoxyls (RO∙), and nitric oxides (NO∙) [15], while
hypochlorous acid (HOCl), hydrogen peroxide (H O ), and
2
2
singlet oxygen (O ) are classified as nonradical ROS. e ROS
2
may react in various environments [15]. However overpro-
duction of ROS species has been linked to cell membrane

2
Journal of Chemistry
⊖
S
⊕
O
N
O
O
Na
HN
HN
NH
HN
N
HN
NH
O
OH
O
O
O
O
O
O
1 (Bucolome)
2 (Sodium pentothal) 3 (Seconal)
4 (Phenobarbital)
⊕
O
NH2Et2
H
⊖
O
HO
O
O
N
O
O
O
N
O
N
O
HN
NH
H
N
N
N
O
O
O
O
5 (Veronal)
6 (Hsp90 inhibitor)
NO2
O
O
Potent nitric oxide scavenger
HN
NH
10: IC50 = 69 1.66ꢀM
HN
O
NH
Std ascorbic acid IC50 = 618 2.0 ꢀM
O
O
O
O
N
O
Cl
NH
NH
N
O
O
N
NH
N
H
O
O
O
NO2
7 (Ro 28-2653, MMP inhibitor) 8 (EM20-25, BCL-2 inhibitor)
9 (TACE inhibitor)
Figure 1: Structure of biologically active barbituric acid derivatives.
damage, protein and enzyme modifications, and DNA dam-
age within the body [16]. ese types of changes accelerate
aging and play a causative role in a variety of degenerative
diseases, such as cancers, atherosclerosis, brain disfunction,
inflammation, shock, and heart diseases. Recently Barakat
et al. [17] have synthesized some novel salts of Michael adduct
derived from PYT and evaluated them for NO (nitric oxide)
scavenging activity. Compound 10 was found to possess
several hundredfold more activity against nitric oxide radical
scavenging assay, as well as for in vitro enzyme inhibition
activities against alpha-glucosidase, thymidine phosphory-
lase, and ꢃ-glucuronidase enzymes.
2. Materials and Methods
e chemical reagents used for the preparation of the target
compounds were commercially available and used without
further purification. 1H-NMR and 13C-NMR were measured
using Bruker Avance AV-600 NMR spectrometer, MS were
carried out with a Jeol, JMS-600 H instrument, and single-
crystal X-ray diffraction was collected using a Bruker SMART
APEX II CCD diffractometer.
(IC values of 69 1.66 ꢁM) than the standard drug ascorbic
50
acid (IC = 618 2.0 ꢁM).
50
Because of the important biological activities, PYT is
widely used as a synthon in the design of antitumor agents.
It has a high efficacy to form hydrogen bonding with drug
targets (6–9, Figure 1). Singh et al. have reported the activity
of a series of new N-benzyl indole-pyrimidine-2,4,6-trione
hybrids against a panel of 60 human tumor cell lines. Several
of these analogs were also found to be inhibitors of DNA
repair and replication stress response polymerases [18].
e discovery and development of effective antioxidants
capable of supplementing body’s antioxidant system is an
important area of health care research. Similarly research
efforts are focused on the identification of effective and safe
inhibitors of ꢂ-glucosidase as potential antidiabetic agents.
e focus in recent years is on the development of
resource-efficient, environmentally benign, and sustainable
chemistry practices. In continuation of our work in this
field, we describe here the synthesis of a series of previously
reported [17, 19, 20] and new PYT adducts. ese compounds
were evaluated for their antioxidant activity by using DDPH
2.1. General Procedure for Aldol Condensation Michael Addi-
tion for the Synthesis of 4 and 5 (GP1). A mixture of aldehyde
3 (1.5 mmol) and1 and 2 (3 mmol) as well as Et NH (1.5 mmol,
2
155 ꢁL) in 3 mL of degassed H O (bubbling nitrogen through
2
the water) was stirred at room temperature for 1–5 h until
TLC showed complete disappearance of the reactants. e
precipitate was removed by filtration and washed with ether
(3 × 20 mL). e solid product was dried to afford pure
products 4 and 5.
e full characterization of the synthesized compounds 4
and 5 and the X-ray crystal data of 5a, 5d, and 5f can be seen
in the Supplementary Materials (see supplementary materials
available online at http://dx.doi.org/10.1155/2016/8517243).
2.2. Biological Activity. e samples of the synthesized com-
pound were screened against DPPH radical, ꢃ-glucuronidase

Journal of Chemistry
3
04
C26
N2
C12
C13
C23
C27
C25
C24
C11
C4
C21
02
C5
C2
C5
01
C3
C4
C10
C9
C22
C1
C6
C1
C8
C20
C21
C20
01W
C7
01
C23
C3
C2
C6
C25
N1
03
03
C11
C10
C14
C9
C15
06
N1
C8
C26
C16
C22
C7
C19
C18
05
C12 C13
02
04
C15
C16
C14
C27
C17
05
C24
C28
C19
C18
C17
5a
5d
C4
02
C5
C21
C3
C22
03
C20
C9
C10
C11
C2
C6
C1
C7
C23
C8
C14
C13
C15
C16
C12
01
C26
C27
04
N2
C19
C18
C17
C25
C24
N1
05
06
5f
Figure 2: ORTEP diagram of 5a, 5d, and 5f. Displacement ellipsoids are plotted at the 40% probability level for non-H atoms.
inhibition assay, thymidine phosphorylase inhibition assay,
and ꢂ-glucosidase inhibition assay and full methods descrip-
tions are provided in Supplementary Materials.
Products 4s, 4t, and 5g were identified as new products
(Scheme 1).
3.2. X-Ray Diffraction. e molecular structures of com-
pounds 5a, 5d, and 5f were unambiguously deduced by
single-crystal X-ray diffraction technique (Figure 2). e
crystals of the synthesized molecules 5a, 5d, and 5f were
grown by slow diffusion of diethyl ether solution of pure
samples 5a, 5d, and 5f in DCM/EtOH at room temperature
followed by allowing standing for 24 h. e structures were
resolved by direct methods by using the SHELXL97 program
[21, 22] in the SHELXTL-plus package and refined by a
3. Results and Discussion
3.1. Chemistry. We have been focusing on the development
of highly expedient methods for the synthesis of diverse
heterocyclic compounds of biological importance via one-
pot multicomponent reactions (MCRs) and avoiding organic
solvents in organic synthesis [17, 19, 20]. is had led to
the development of several efficient, environmentally benign,
clean, and economical process technology (Green Chemistry
Concepts). In current reaction strategy, equimolar amounts
of barbituric acid (1) and dimedone (2) with aldehyde
(3) in the presence of aqueous diethylamine medium at
RT yielded salts of PYT adducts 4a–4y and 5a–5m were
obtained in quantitative yields by simple filtration [17–20].
2
full-matrix least-squares procedure on ꢄ using SHELXS97.
Diffraction data were collected on a Bruker APEX-II CCD
diffractometer (Bruker AXS GmbH). e crystal structure
and refinement data of compounds 5a, 5d, and 5f are listed
in Table 1. e final atomic coordinates for all atoms and
complete listing of bond distance and angles are presented

4
Journal of Chemistry
NH2Et2
⊕HO
⊖
O
N
O
N
O
O
R2
O
R1
R1
O
N
N
O
O
O
1a, b
2
H2O/NHEt2
RT
⊕
O
NH2Et2
HO
R1
N
R1
N
N
NH2Et2
⊕HO
R1
O
R1
⊖
O
⊖
O
N
N
O
O
O
2
2
O
O
O
N
1a, b
2
R1
R1
O
O
O
N
O
H2O/NHEt2
H2O/NHEt2
RT
R2
RT
3
R2
R2
⊕
NH2Et2
⊕HO
NH2Et2
HO
CH3
N
NH2Et2
⊕HO
⊖
O
CH3
N
N
O
H3C
H3C
O
N
⊖
O
H
N
H
N
⊖
O
O
O
O
O
N
NH
HN
CH3
H3C
O
O
R4
R1
R2
O
O
O
R1
R
R3
R
4m R1=R2=R3=R4=H
4a R=CHO, R1=H
4b R=H, R1=CH3
4c R=NO2, R1=H
4d R=OCH3, R1=H
4e R=H, R1=Br
4i R=CH3
4j R=Cl
4n R1=R2=R4=H, R3=CH3
4o R1=R2=R4=H, R3=OCH3
4p R1=R2=R4=H, R3=Cl
4q R1=R2=R4=H, R3=Br
4r R1=R3=R4=H, R2=Br
4s R1=NO2, R2=R3=R4=H
4t R1=R2=R4=H, R3=NMe2
4v R1=R2=R4=H, R3=OH
4w R1=R3=Cl, R2=R4=H
4x R1=R2=R4=H, R3=CHO
4y R1=R4=Cl, R2=R3=H
4k R=OCH3
NH2Et2
⊕
⊖
O
H3C
H3C
4f R=OH, R1=H
4g R=CH3, R1=H
O
N
HO
N
NH2Et
2 H
O
O
⊖
O
O
NH
N
O
⊕HO
H
N
HN
O
O
4z
NH2Et2
CH3
N
NH2Et2
CH3
N
N
O
O
⊕ HO
⊕
⊖
H
⊖
O
O
NH
O
N
4l
HO
N
O
O
N
CH3
N
H3C
O
O
O
CHO
5l
4h
NH2Et2
⊕ HO
CH3
CH3
⊖
O
NH2Et2
HO
H3C
H3C
⊕
H
N
⊖
O
H3C
H3C
O
NH
O
O
R1
R4
O
O
R2
R3
R
5a R1=NO2, R2=R3=R4=H
5b R1=R3=R4=H, R2=CH3
5c R1=R3=R4=CH3, R2=H
5d R1=R2=R4=H, R3=OCH3
5e R1=R4=Cl, R2=R3=H
5f R1=R2=R4=H, R3=NO2
5g R1=R2=R4=H, R3=CHO
5h R1=R2=R4=H, R3=OH
5i R=Cl
5j R=H
5k R=Br
5l R=CH3
5m R=CHO
Scheme 1: Synthesis of the target compounds.

Journal of Chemistry
5
Table 1: e crystal and experimental data of compounds 5a, 5d, and 5f.
5a
5d
5f
Empirical formula
Formula weight
Temperature
Wavelength
Crystal system
Space group
ꢆ
C H NO ⋅C H N⋅H O
C H O ⋅C H N
C H NO ⋅C H N
23 26
6
4
12
2
24 29
5
4
12
23 26
6
4
12
504.61
100 K
471.62
100 K
486.59
100 K
˚
˚
˚
Mo Kꢂ radiation, ꢅ = 0.71073 A
Mo Kꢂ radiation, ꢅ = 0.71073 A
Mo Kꢂ radiation, ꢅ = 0.71073 A
Orthorhombic
Monoclinic
Monoclinic
P2 2 2
11.6155 (5) A
12.0514 (5) A
P2 /n
10.2288 (5) A
18.0048 (7) A
P2 /n
10.1137 (4) A
18.1225 (7) A
1
1
1
1
1
˚
˚
˚
˚
˚
˚
˚
˚
˚
ꢇ
ꢈ
19.3665 (7) A
15.0893 (7) A
15.4853 (6) A
ꢃ
90.00∘
106.425 (2)∘
106.730 (1)∘
3
3
3
˚
˚
˚
Volume
2710.98 (19) A
2665.6 (2) A
2718.09 (18) A
ꢉ
4
4
4
Calculated density
Absorption coefficient
1.236 Mg m−3
1.175 Mg m−3
1.189 Mg m−3
0.09 mm−1
0.08 mm−1
0.08 mm−1
ꢄ (000)
1088
1024
1048
Crystal size
0.57 × 0.41 × 0.07 mm
0.66 × 0.33 × 0.25 mm
0.33 × 0.29 × 0.22 mm
ꢊ range
2.4–24.2∘
28531
4501
2.2–29.7∘
67389
6194
2.2–32.1∘
25488
4829
Reflections collected
Reflections unique
Independent reflections
Parameters
6224
350
11203
326
6233
334
ꢋ
0.090
0.054
0.109
0.122
0.069
0.156
0.053
0.055
0.132
int
R [F2 > 2ꢌ(F2)]
ꢍR (F2)
Goodness of fit
1.05
1.01
1.09
−3
˚
Max/min ꢎ e A
0.42 and −0.27
0.43 and −0.30
0.29 and −0.26
CCDC number
1444058
1443783
1444050
in supplementary information. ORTEP drawings of final X-
ray model of compounds 5a, 5d, and 5f with the atomic
numbering scheme are presented in Figure 2, while crystal
packing presentation of compounds 5a, 5d, and 5f is shown
in Figure 3.
3.3. Biological Activity Evaluation. All synthesized diethyl
ammonium salts of barbiturates having bis(6-hydroxy-1,3-
dimethyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidin-5-yl) (4a–
4h),bis(6-hydroxypyrimidine-2,4(1H,3H)-dione (4i–4l), and
(2-hydroxy-4,4-dimethyl-6-oxocyclohex-1-en-1-yl)-1,3-dim-
ethyl-2,6-dioxo-1,2,3,6-tetrahydropyrimidin-4-olate (4m–
4z) as well as bis(2-hydroxy-4,4-dimethyl-6-oxocyclohex-
1-ene) (5a–5m) as basic nuclei were screened for their
antioxidant potential in vitro by using DPPH radical
scavenging assay and cytotoxicity against PC-3, HeLa, and
MCF-7 cancer cell lines and 3T3 normal fibroblast cell lines.
ese derivatives 4a–4z and 5a–5m were also evaluated for
in vitro enzyme inhibition activities against ꢂ-glucosidase,
thymidine phosphorylase, and ꢃ-glucuronidase enzymes.
e results are summarized in Table 2.
Compound 5a crystallizes in orthorhombic crystal sys-
tem in a space group of P2 2 2 ; on the other hand, com-
1
1
1
pounds 5d and 5f crystalize in monoclinic crystal system in
a space group of P2 /n. It was observed that the compound
1
5a has hydrogen bonding between the C -O ⋅ ⋅ ⋅ H⋅ ⋅ ⋅ O -C ,
5
2
4
13
while compounds 5d and 5f exist only in the enol form. ere
are two strong intramolecular hydrogen bonds on compound
5a between O —H O ⋅ ⋅ ⋅ O and N —H N ⋅ ⋅ ⋅ O , while
4
1
4
2
2
2
2
1
compounds 5d and 5f have no intramolecular hydrogen
bonding. e crystal structures of the tested compounds 5a,
5d, and 5f were formed by different hydrogen bonds between
the molecules and diethyl amine and water solvent molecules
in case of 5a to form network structures. Crystallographic
data for the solved structures 5a, 5d, and 5f were deposited to
the Cambridge Crystallographic Data Center as supplemen-
tary publication numbers CCDC-1444058, CCDC-1443783,
and CCDC-1444050, respectively, which can be obtained free
of charge from the Cambridge Crystallographic Data Centre
via http://www.ccdc.cam.ac.uk/data request/cif.
3.3.1. Antioxidant Activity (DPPH Radical Scavenging Assay).
Among series of compounds (4a–4h) having bis(6-hydroxy-
1, 3-dimethyl-2, 4-dioxo-1, 2, 3, 4-tetrahydropyrimidin-5-yl)
ring as basic nucleus, compounds 4a (IC = 118.8 2.1 ꢁM),
50
4b (IC = 117 2.4 ꢁM), 4c (IC = 111.9 1.9 ꢁM), 4d
50
50
(IC = 124.4 4.4 ꢁM), 4e (IC = 101.1 0.8 ꢁM), 4f
50
50
(IC = 148.6 2.6 ꢁM), and 4g (IC = 104.1 1.9 ꢁM)
50
50
were found to be potent antioxidants in comparison to the

6
Journal of Chemistry
5d
5a
5f
Figure 3: Molecular packing of 5a, 5d, and 5f viewed approximately three-dimensional network, along ꢇ axis and three-dimensional network,
respectively. Intermolecular interaction is drawn as dashed lines.
standards BHT (IC = 128.8 2.1 ꢁM) and N-acetylcysteine
observed for compound 4d (IC = 124.4 4.4 ꢁM) having
50
50
(IC = 107.6
2.8 ꢁM). Compounds 4a–4e (IC
=
a para-methoxy group instead of hydroxyl functionality
50
50
101.8 0.8–124.4 4.4 ꢁM) were found to be more active
on phenyl ring, whereas naphthalene ring containing
compared to the standard BHT (IC = 128.8 2.1 ꢁM). e
barbiturate 4h (IC = 133.6 5.7 ꢁM) was also found to be
50
50
meta-bromo-substituted phenyl ring containing compound
a weak antioxidant agent against DPPH radicals.
4e (IC = 101.1 0.8 ꢁM) was the most potent member of
Among second series of the tested compounds 4i–4l
having bis(6-hydroxypyrimidine-2,4(1H,3H)-dione as back-
50
the series (4a–4e), followed by the para-methyl-substituted
phenyl ring containing compound 4g (IC = 104.1 1.9 ꢁM).
bone, compounds having para-methyl- (4i, IC = 384.9
50
50
A gradual decrease in activity was observed for compounds
6.6 ꢁM), para-chloro- (4j, IC = 384.3 8.8 ꢁM), and para-
50
4c (IC = 119.9 1.9 ꢁM) and 4a (IC = 118.8 3.1 ꢁM)
methoxy- (4l, IC = 397.9 0.9 ꢁM) substituted phenyl ring
50
50
50
having para-nitro- and para-aldehyde-substituted phenyl
ring attached to central bis(6-hydroxy-1,3-dimethyl-2,4-
dioxo-1,2,3,4-tetrahydropyrimidin-5-yl) ring. A decrease in
DPPH radical scavenging activity was due to positional effect
of methyl substituent on phenyl ring which was observed for
attached to the central bis(6-hydroxypyrimidine-2,4(1H,3H)-
dione) showed better antioxidant activity as compared to
BHT (IC = 128.8 2.1 ꢁM) and N-acetylcysteine (IC
=
50
50
107.6 2.8 ꢁM), whereas naphthalene containing compound
4l was found to be inactive.
compound 4b (IC = 117 3.1 ꢁM) having meta-substituted
(2-Hydroxy-4,4-dimethyl-6-oxocyclohex-1-en-1-yl)-1,3-
dimethyl-2,6-dioxo-1,2,3,6-tetrahydropyrimidin-4-olate ring
containing compounds 4m–4z were also evaluated for their
antioxidant potential in vitro in DPPH radical scavenging
assay. ese compounds were found to be significant to weak
50
phenyl ring instead of para-methyl-substituted phenyl ring
(4g, IC = 104.1 1.9 ꢁM). e electron donating para-
50
hydroxyl-substituted phenyl ring containing compound 4f
(IC = 148.6 2.6 ꢁM) was found to be a last active member
50
of the series. However, a considerable increase in activity was
inhibitors of free radicals (IC = 133.2 4.7–439.6 10.1 ꢁM)
50

Journal of Chemistry
7

8
Journal of Chemistry
except compounds 4m–4r and 4y which were found to be
inactive.
1.73 ꢁM). Among dimethyl ammonium salts (4a–4h), nitro-
substituted phenyl ring containing 4c (IC = 355 10 ꢁM)
50
Similarly bis(2-hydroxy-4,4-dimethyl-6-oxocyclohex-1-
and naphthalene ring containing 4h (IC = 230 3.92 ꢁM)
50
ene) (5a–5h) and 4-((6-hydroxy-1,3-dimethyl-2,4-dioxo-
were found to be potent inhibitors of ꢂ-glucosidase enzyme
1,2,3,4-tetrahydropyrimidin-5-yl)
(6-hydroxy-2,4-dioxo-
and showed more activity than acarbose (IC = 841
50
1,2,3,4-tetrahydropyrimidin-5-yl)methyl) (5i–5k) rings con-
taining compounds were also evaluated for their antioxidant
potential by using DPPH radical scavenging assay. Para-
bromo-substituted phenyl ring containing compound
1.73 ꢁM). All other compounds were found to be inactive.
Among diethyl ammonium salts having (2-hydroxy-
4,4-dimethyl-6-oxocyclohex-1-en-1-yl)-1,3-dimethyl-2,6-
dioxo-1,2,3,6-tetrahydropyrimidin-4-olate ring (4m–4z),
5k (IC = 127.6 1.6 ꢁM) was found as a potent anti-
para-OCH - (40, IC = 300.9
6.5 ꢁM), para-chloro-
50
3
50
oxidant agent against the tested standards BHT (IC
=
(4p, IC = 186.8
0.81 ꢁM), para-bromo- (4q, IC
=
50
50
50
128.8 2.1 ꢁM). e compound showed similar antioxidant
214.8 0.72 ꢁM), and meta-bromo- (4r, IC = 201 5.5 ꢁM)
50
potential as BHT and showed a significant antioxidant agent
substituted phenyl ring containing pyridinium adducts were
against N-acetyl cysteine (IC = 107.6
2.8 ꢁM). e
found to be potent ꢂ-glucosidase inhibitors and showed
50
gradual and drastic decrease in activity was for compounds
more activity than standard acarbose (IC = 841 1.73 ꢁM).
50
5l (IC = 169.7 5.3 ꢁM), 5g (IC = 270.4 2.3 ꢁM),
Other members of the series (4m-4n and 4s–4z) were found
to be inactive. e results showed the substitution effects of
various functionalities of phenyl ring on the potential activity
of tested series. e para-bromo-substituted phenyl ring
50
50
5e (IC = 289 0.9 ꢁM), and 5a (IC = 461.6 8.8 ꢁM)
50
50
having para-methyl-, para-aldehyde-, ortho-chloro-, and
ortho-nitro-substituted phenyl rings attached to the central
barbiturate moieties. Compounds 5b–5d, 5f, and 5h–5j were
found to be inactive.
containing compound 4q (IC = 214.8 0.72 ꢁM) showed
50
more activity than tested standard. However, the replacement
of bromo group with that of methyl, N,N-dimethyl, hydroxyl,
and aldehyde functionalities resulted in complete loss of
activity as observed in compounds 4n, 4t, 4v, and 4x,
respectively. e presence of ortho-nitro-, ortho-choloro-,
and para-choloro-substituted phenyl ring also contributed
towards inactivity, as observed for compounds 4t, 4w, and
4y.
On the basis of the observed antioxidant abilities of
the above five different series of pyridinium adducts, it
was concluded that bis(6-hydroxy-1,3-dimethyl-2,4-dioxo-
1,2,3,4-tetrahydropyrimidin-5-yl) ring containing com-
pounds 4a–4h are the most active one. e activity may be
due to the methyl substituted nitrogen atoms of the tetrahy-
dropyrimidine rings that facilitate the electronic conjugation
within the ring. However, the variation of antioxidant
properties within the most important series is may be due to
Similarly bis(2-hydroxy-4,4-dimethyl-6-oxocyclohex-1-
ene) ring containing diethyl ammonium pyridinium salts
(5a–5h) were also evaluated for their in vitro ꢂ-glucosidase
enzyme inhibition activity. e series nitro-substituted
phenyl ring containing 5f was found to be the only potent
ꢂ-glucosidase inhibitor. All other compounds (5a–5e and
5g-5h) were found to be inactive.
the presence of electron donating (-OH (4f), -OCH (4d),
and -CH (4b, 4g)), electron withdrawing (-NO (4a) and
-CHO (4c)), and halogen (-Br (4e)) substituents on the
phenyl ring attached to the bis(6-hydroxy-1,3-dimethyl-2,4-
dioxo-1,2,3,4-tetrahydropyrimidin-5-yl) moiety. However,
detailed studies are required to study the mechanism of
action of these compounds.
3
3
2
Among series of diethylaminium salts of pyridinium
adducts (5i–5m) having 4-((6-hydroxy-1,3-dimethyl-2,4-
dioxo-1,2,3,4-tetrahydropyrimidin-5-yl) (6-hydroxy-2,4-di-
oxo-1,2,3,4-tetrahydropyrimidin-5-yl)methyl) central moiety
attached with substituted phenyl ring, the benzaldehyde
3.3.2. Cytotoxic Activity. All synthesized diethyl ammonium
salts of barbiturates (4a–4z and 5a–5m) were evaluated for
their in vitro cytotoxicity against PC-3, HeLa, MCF-7 cancer,
and 3T3 normal cell lines. All compounds were found to be
noncytotoxic against PC-3, HeLa, MCF-7 cancer, and 3T3 cell
lines except dichloro-substituted (2-hydroxy-4,4-dimethyl-
6-oxocyclohex-1-en-1-yl)-1,3-dimethyl-2,6-dioxo-1,2,3,6-tet-
rahydropyrimidin-4-olate ring containing compounds 4y,
which was found to be significantly toxic against all the cell
lines (PC3, IC = 10.71 0.26 ꢁM, HeLa, IC = 3.47 0.3 ꢁM,
containing 5m (IC = 453.4 4.2 ꢁM) was found to be the
50
only potent inhibitor compound in comparison to against the
standard drug acarbose (IC = 841 1.73 ꢁM). All other com-
50
pounds were found to be inactive. e results are summarized
in Table 2.
3.3.4. ymidine Phosphorylase Inhibition Activity. Five dif-
ferent series of diethyamonium salts of barbiturates, 4a–
4g, 4i–4k, 4m–4z, 5a–5h, and 5i–5m, were also evalu-
ated for their in vitro thymidine phosphorylase inhibition.
50
50
MCF-7, IC = 4.9 0.01 ꢁM, and 3T3, IC = 3.20 0.2 ꢁM).
50
50
Doxorubicin was used as a standard drug to compare the
activities against PC3 (IC = 1.70 0.29 ꢁM), HeLa (IC
=
7-Deazaxanthine was used as a standard inhibitor (IC
50
50
50
0.51 0.15 ꢁM), and MCF-3 (IC = 0.92 0.01 ꢁM) cancer
= 41
1.46 ꢁM). Phenol and naphthalene rings sub-
50
cell lines, while cycloheximide (IC = 0.26 0.12 ꢁM) was
stituted (2-hydroxy-4,4-dimethyl-6-oxocyclohex-1-en-1-yl)-
1,3-dimethyl-2,6-dioxo-1,2,3,6-tetrahydropyrimidin-4-olate
50
used as a standard-in 3T3 cell line (Table 2).
moiety containing compounds 4v (IC = 57.6 1.20 ꢁM)
50
3.3.3. ꢂ-Glucosidase Inhibition Activity. Synthesized diethyl
and 4z (IC = 55.8 1.50 ꢁM) were found to be the potent
50
ammonium salts of barbiturates, 4a–4z and 5a–5m, were also
thymidine phosphorylase inhibitors. However, a drastic
evaluated for their in vitro ꢂ-glucosidase inhibition activity,
decrease in activity was observed when para-hydroxy
in comparison to the standard drug, acarbose (IC = 841
group on phenyl ring was replaced with methyl (4n, IC
50
50

Journal of Chemistry
9
= 226.9
1.50 ꢁM), chloro (4n, IC = 242
1.5 ꢁM),
50
bromo (4q, IC = 141.3
1.5 ꢁM), and aldehyde (4x,
50
IC = 239
2.0 ꢁM) groups, whereas the compounds
50
4o, 4s, 4t, 4w, and 4y have para-methoxy-, ortho-nitro-,
ortho-N,N-dimethyl amine-, ortho-, para-dichloro-, and
ortho-ortho-dichloro-substituted phenyl ring, respectively.
Among series of compounds (5a–5h) having bis(2-
hydroxy-4,4-dimethyl-6-oxocyclohex-1-ene) ring as central
moiety, electron donating hydroxy- and methoxy-substituted
phenyl ring containing compounds 5h (IC = 56.9 1.6 ꢁM)
50
and 5d (IC = 60.5 1.87 ꢁM), respectively, were found
50
as potent inhibitors of thymidine phosphorylase enzyme
against the tested standard 7-deazaxanthine which was
Figure 4: ree-dimensional representation of compound 4p with
target macromolecule ꢂ-glucosidase.
(IC = 41 1.46 ꢁM) used as standard. Again a drastic
50
decrease in activity was observed for compounds 5b (IC
50
= 216.9
0.5 ꢁM), 5c (IC = 161.4
1.5 ꢁM), and 5g
50
(IC = 186.6 1.2 ꢁM) having meta-methyl-, ortho-methyl-,
50
and para-aldehyde-substituted benzene ring respectively,
instead of para-hydroxyl phenyl ring. e replacement of
ortho-methyl with chloro-group makes the compound 5e
completely inactive. Similarly the replacement of electron
donating para-hydroxy-group of phenyl ring with electron
withdrawing nitro-functionality also contributed towards
inactivity of compound, as observed for 5f. Phenol substi-
tuted bis(2-hydroxy-4,4-dimethyl-6-oxocyclohex-1-ene) ring
containing 5h (IC = 56.9 1.6 ꢁM) was found to be the
50
potent thymidine phosphorylase inhibitors.
All members of the series of diethylaminium salts of pyri-
dinium adducts (5i–5m) having 4-((6-hydroxy-1,3-dimethyl-
2,4-dioxo-1,2,3,4-tetrahydropyrimidin-5-yl) (6-hydroxy-2,4-
dioxo-1,2,3,4-tetrahydropyrimidin-5-yl)methyl) central moi-
ety attached with substituted phenyl ring were found to be
weak inhibitors of thymidine phosphorylase enzyme against
Figure 5: ree-dimensional representation of compound 4v with
target macromolecule thymidine phosphorylase.
was carried out [23–25]. Ten different conformations of
protein-ligand interactions were generated and the top
ranked pose was selected to explore the binding interactions.
In our docking study, we have observed two interactions with
the active site residues of ꢂ-glucosidase. e polar imidazole
ring of His 239 showed hydrogen bond through its H atom
with the lone pair of oxygen of the pyrimidinedione moiety of
the compound, whereas Arg 312 formed hydrogen bond with
the OH group of the same moiety of compound (Figure 4).
e presence of polar and electron rich center in the form
of pyrimidinedione and chlorobenzene groups of the com-
pound was observed here as key factors for the interaction
between compound and protein. A number of hydrogen bond
donor and acceptor pairs with their suitable orientations
were observed in the interaction pose. Furthermore, several
hydrophobic interactions between the nonpolar active site
residues and the compound 4p were also observed.
the tested standard compound 7-deazaxanthine (IC = 41
50
1.46 ꢁM) with IC values 318 2.5 ꢁM, 166.4 1.2 ꢁM,
50
178.1 2.1 ꢁM, and 147.1 1.23 ꢁM for 5j, 5k, 5l, and 5m,
respectively, whereas para-chloro-substituted phenyl ring
containing compound 5i was found to be inactive. e results
are summarized in Table 1.
3.3.5. ꢃ-Glucuronidase Inhibition Activity. Compounds 4a–
4g, 4i–4k, 4m–4z, 5a–5h, and 5i–5m were also evaluated
for their in vitro ꢃ-glucuronidase inhibitory activity by using
D-saccharic acid 1,4-lactone as standard inhibitor (IC
=
50
45.75 2.16 ꢁM). Compounds 4a (IC = 204.8 6.01 ꢁM),
50
4c (IC = 172.1 2.1 ꢁM), 4e (IC = 338.9 6.42 ꢁM), 4o
50
50
(IC = 262.1 4.35 ꢁM), 4q (IC = 292.2 1.03 ꢁM), 4r
50
50
(IC = 262.1 4.4 ꢁM), 4w (IC = 143.5 1.24 ꢁM), 5f (IC
50
50
50
= 185 2.5 ꢁM), 5i (IC = 223.6 6.4 ꢁM), and 5l (IC
=
50
50
387.5 7.0 ꢁM) showed very weak ꢃ-glucuronidase inhibition
potential, whereas pyridinium adducts 4a–4c, 4e, 4g–4j, 4l,
4o, 4s-4t, 4w, 4y, 5a, 5e-5f, and 5i were found to be inactive
(Table 2).
3.4.2. Binding Interactions of ymidine Phosphorylase
Enzyme. To explore the binding modes of these newly syn-
thesized compounds in the active site of thymidine phos-
phorylase, the most active compound 4v was docked against
this enzyme. e docking results showed that compound
4v well accommodated in the active site of thymidine
phosphorylase (Figure 5). e active site residue Ala 206
established a hydrogen bond through its lone pair of
3.4. Molecular Docking Studies
3.4.1. Binding Interactions of ꢂ-Glucosidase Enzyme. To de-
fine inhibitors interactions and their specificity, the docking
study of the most active compound 4p against ꢂ-glucosidase

10
Journal of Chemistry
found to be a potent ꢂ-glucosidase inhibitor. Compound 4v
(IC = 8.7 1.2 ꢁM) was found to be a potent thymidine
50
phosphorylase inhibitor. Promising results indicate that these
compounds need to be investigated as antioxidant agents
to treat various associated oxidative disorders. Similarly
their enzyme inhibitory potential can be reported on drug
discovery program.
Competing Interests
e authors declare that there are no competing interests
regarding the publication of this paper.
Authors’ Contributions
Assem Barakat, Hazem A. Ghabbour, Abdullah Mohammed
Al-Majid, Qurat-ul-ain, Rehan Imad, Kulsoom Javaid, Nimra
Naveed Shaikh, Sammer Yousuf, M. Iqbal Choudhary, and
Abdul Wadood contributed equally to this work.
Figure 6: ree-dimensional representation of compound 4w with
target enzyme ꢃ-glucuronidase.
oxygen with the active hydrogen of hydroxyl moiety of
the pyrimidinedione group of the compound. However,
the substituted phenolic group and the other electron rich
centers of the pyrimidinedione showed poor behavior
regarding interaction which may depend on the nature and
orientation of the surrounding active site residues. Moreover,
several weak hydrophobic interactions between the active
site residues and the compound were also observed.
Acknowledgments
e authors would like to extend their sincere appreciation to
the Deanship of Scientific Research at King Saud University
for funding this Research Group no. (RGP-257-1435-1436).
References
[1] K. Undheim, T. Bennecke, A. R. Katritzky, C. W. Rees, and
E. F. V. Scriven, Comprehensive Heterocyclic Chemistry, vol. 6,
supplement 93, Elsevier Pergamon, Oxford, UK, 1996.
3.4.3. Binding Interaction of ꢃ-Glucuronidase Enzyme. e
predicted binding mode of most active compound 4w against
ꢃ-glucuronidase showed that a hydrogen bond is formed
between the lone pair of oxygen of cyclohexanedione compo-
nent of the compound and imidazole hydrogen of the impor-
tant active site residue, His 509. An arene-arene and arene-
cation interaction was also observed between the pi electronic
cloud of dichlorobenzene ring of compound and imidazole
ring of His 509 (Figure 6). Besides hydrogen bonding and
arene-arene interactions, several hydrophobic interactions
between compound 4w and the active site residues were
also observed. e pyrimidinedione moiety in this case was
observed to be passive regarding interactions.
[2] B. Taylor, Modern Medical Chemistry, Prentice Hall, New York,
NY, USA, 1994.
[3] R. Y. Levina and F. K. Velichko, “Advances in the chemistry of
barbituric acids,” Russian Chemical Reviews, vol. 29, no. 8, pp.
437–459, 1960.
[4] P. Sing and K. Paul, “A simple synthesis of 5-spirobarbituric
acids and transformations of spirocyclopropane barbiturates to
5-substitutated barbiturates,” Indian Journal of Chemistry B, vol.
45, pp. 247–251, 2006.
[5] X. Chen, K. Tanaka, and F. Yoneda, “Simple new method for the
synthesis of 5-deaza-10-oxaflavin, a potential organic oxidant,”
Chemical and Pharmaceutical Bulletin, vol. 38, no. 2, pp. 307–
311, 1990.
4. Conclusions
[6] L. R. Morgan, B. S. Jursic, C. L. Hooper, D. M. Neumann, K.
angaraj, and B. LeBlanc, “Anticancer activity for 4, 4ꢀ-di-
hydroxybenzophenone-2, 4-dinitrophenylhydrazone (A-007)
analogues and their abilities to interact with lymphoendothelial
cell surface markers,” Bioorganic & Medicinal Chemistry Letters,
vol. 12, no. 23, pp. 3407–3411, 2002.
[7] G. Orzalesi, P. Gratteri, and S. Selleri, “Synthesis of new 1,3-
dicyclohexyl barbituric acid derivatives with anti-inflammatory
potential activity,”European Journal of Medicinal Chemistry, vol.
25, no. 2, pp. 197–201, 1990.
In conclusion, barbiturate salts were synthesized by one-pot
multicomponent reactions via tandem Aldol/Michael addi-
tion reactions between barbituric acid and dimedone with
aldehydes, mediated by aqueous diethylamine. e molec-
ular structures of 5a, 5d, and 5f were deduced by single-
crystal X-ray diffraction techniques. All of the synthesized
compounds were evaluated for their antioxidant potential
in vitro by using DPPH radical scavenging assay as well as
enzyme inhibition activities against ꢂ-glucosidase, thymidine
phosphorylase, and ꢃ-glucuronidase enzymes. Compounds
[8] D. T. Puerta and S. M. Cohen, “A bioinorganic perspectives
on matrix metalloproteinase inhibition,” Current Topics in
Medicinal Chemistry, vol. 4, no. 15, pp. 1551–1573, 2004.
4a–4g (IC = 101.8 0.8–124.4 4.4 ꢁM) were found to
50
be the potent antioxidants as compared to the standards,
BHT (IC = 128.8 2.1 ꢁM), and N-acetylcysteine (IC
=
[9] M. E. Wolff, Burger’s Medicinal Chemistry and Drug Discovery,
50
50
107.6 2.8 ꢁM). Compound 4p (IC = 186.8 8.1 ꢁM) was
John Wiley & Sons, New York, NY, USA, 1997.
50

Journal of Chemistry
11
[10] E. Fischer and J. von Mering, “Ueber eine neue klasse von
schlafmitteln,” in Untersuchungen aus Verschiedenen Gebieten,
M. Bergmann, Ed., pp. 671–679, Springer, Berlin, Germany,
1924.
[11] W. J. Doran, “Barbituric acid hypnotics,” Medicinal Chemistry,
vol. 4, pp. 164–167, 1959.
[12] B. Bobranski, “Progress in chemistry of barbituric acid,” Wiad-
´
omosci Chemiczne, vol. 31, pp. 231–278, 1977.
[13] S. Senda, H. Izumi, and H. Fujimura, “Uracil derivaives and
related compounds. 6. Derivatives of 5-alkyle-2,4,6-trioxoper-
hydropyrimidine as antiphlogistics,” Arzneimittel Forschung,
vol. 17, no. 12, p. 151, 1967.
[14] N. Moussier, L. Bruche´, F. Viani, and M. Zanda, “Fluorinated
barbituric acid derivatives: synthesis and bio-activity,” Current
Organic Chemistry, vol. 7, no. 11, pp. 1071–1080, 2003.
[15] P.-G. Pietta, “Flavonoids as antioxidants,” Journal of Natural
Products, vol. 63, no. 7, pp. 1035–1042, 2000.
[16] N. Ramarathnam, T. Osawa, H. Ochi, and S. Kawakishi, “e
contribution of plant food antioxidants to human health,”
Trends in Food Science & Technology, vol. 6, no. 3, pp. 75–82,
1995.
[17] A. Barakat, A. M. Al-Majid, H. J. Al-Najjar et al., “Zwitterionic
pyrimidinium adducts as antioxidants with therapeutic poten-
tial as nitric oxide scavenger,” European Journal of Medicinal
Chemistry, vol. 84, pp. 146–154, 2014.
[18] P. Singh, M. Kaur, and P. Verma, “Design, synthesis and
anticancer activities of hybrids of indole and barbituric acids-
Identification of highly promising leads,” Bioorganic and Medic-
inal Chemistry Letters, vol. 19, no. 11, pp. 3054–3058, 2009.
[19] A. M. Al-Majid, A. Barakat, H. J. Al-Najjar, Y. N. Mabkhot, H.
A. Ghabbour, and H.-K. Fun, “Tandem aldol-michael reactions
in aqueous diethylamine medium: a greener and efficient
approach to bis-pyrimidine derivatives,” International Journal
of Molecular Sciences, vol. 14, no. 12, pp. 23762–23773, 2013.
[20] A. Barakat, A. M. Al-Majid, G. Lotfy et al., “Synthesis and dy-
namics studies of barbituric acid derivatives as urease inhibi-
tors,” Chemistry Central Journal, vol. 9, no. 1, article 63, 2015.
[21] G. M. Sheldrick, “A short history of SHELX,” Acta Crystallo-
graphica A, vol. 64, pp. 112–122, 2008.
[22] A. L. Spek, “Structure validation in chemical crystallography,”
Acta Crystallographica Section D: Biological Crystallography, vol.
65, no. 2, pp. 148–155, 2009.
[23] F. Rahim, K. Ullah, H. Ullah et al., “Triazinoindole analogs as
potent inhibitors of ꢂ-glucosidase: synthesis, biological evalua-
tion and molecular docking studies,” Bioorganic Chemistry, vol.
58, pp. 81–87, 2015.
[24] F. Rahim, F. Malik, H. Ullah et al., “Isatin based Schiff bases
as inhibitors of ꢂ-glucosidase: synthesis, characterization, in
vitro evaluation and molecular docking studies,” Bioorganic
Chemistry, vol. 60, article 1803, pp. 42–48, 2015.
[25] A. Barakat, A. M. Al-Majid, M. S. Islam et al., “Molecular struc-
ture investigation and biological evaluation of Michael adducts
derived from dimedone,” Research on Chemical Intermediates,
vol. 42, pp. 4041–4053, 2016.

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