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

Authors: Assem Barakat; Hazem A. Ghabbour; Abdullah Mohammed Al-Majid; Qurat-ul-ain; Rehan Imad; Kulsoom Javaid; Nimra Naveed Shaikh; Sammer Yousuf; M. Iqbal Choudhary; Abdul Wadood
Journal: Journal of Chemistry
 (2016)

---

## Abstract

A series of barbiturates derivatives synthesized and screened for different set of bioassays are described. The molecular structures of compounds5a,5d, and5f were solved by single-crystal X-ray diffraction techniques. The results of bioassay show that compounds4a,4b,4c,4d,4e,4f, and4g are potent antioxidants in comparison to the tested standards, butylated hydroxytoluene (BHT), and N-acetylcysteine. Compounds4a–4e (I
C
50
=
101.8
±
0.8
–
124.4
±
4.4
μ
M) and4g (I
C
50
=
104.1
±
1.9
μ
M) were more potent antioxidants than the standard (BHT, I
C
50
=
128.8
±
2.1
μ
M). The enzyme inhibition potential of these compounds was also evaluated,in vitro, against thymidine phosphorylase, α-glucosidase, and β-glucuronidase enzymes. Compounds4c,4h, 4
o,4p,4q, 5f, and5m were found to be potent α-glucosidase inhibitors and showed more activity than the standard drug acarbose, whereas compounds4v,and5h were found to be potent thymidine phosphorylase inhibitors, more active than the standard drug, 7-deazaxanthine. All barbiturates derivatives (4a–4x,4z, and5a–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, except4y which was cytotoxic against all the cell lines.


---

## Body

## 1. Introduction

Derivatives of 1,3-dimethylbarbituric acid including pyrimidine-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, antispasmodic, anticancer, matrix metalloproteinase inhibitors, anticonvulsants, 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, and sodium pentothal (Figure 1,1–5) [10–13]. The interesting mechanism of action of barbital and phenobarbital against epilepsy made them attractive targets for medicinal chemists. They 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) which are peroxyls (ROO•), superoxides (O
2
•
-), hydroxyls (HO•), alkoxyls (RO•), and nitric oxides (NO•) [15], while hypochlorous acid (HOCl), hydrogen peroxide (H2O2), and singlet oxygen (O2) are classified as nonradical ROS. The ROS may react in various environments [15]. However overproduction of ROS species has been linked to cell membrane damage, protein and enzyme modifications, and DNA damage within the body [16]. These 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. Compound10 was found to possess several hundredfold more activity against nitric oxide radical (IC50 values of 69
±
1.66 μM) than the standard drug ascorbic acid (IC50 = 618
±
2.0 μM).Figure 1
Structure of biologically active barbituric acid derivatives.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 newN-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].The 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 ofα-glucosidase as potential antidiabetic agents.The 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. These compounds were evaluated for their antioxidant activity by using DDPH scavenging assay, as well as forin vitroenzyme inhibition activities against alpha-glucosidase, thymidine phosphorylase, and β-glucuronidase enzymes.


## 2. Materials and Methods

The 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.


### 2.1. General Procedure for Aldol Condensation Michael Addition for the Synthesis of4 and5 (GP1)

A mixture of aldehyde3 (1.5 mmol) and1 and2 (3 mmol) as well as Et2NH (1.5 mmol, 155 μL) in 3 mL of degassed H2O (bubbling nitrogen through the water) was stirred at room temperature for 1–5 h until TLC showed complete disappearance of the reactants. The precipitate was removed by filtration and washed with ether (3
×
20 mL). The solid product was dried to afford pure products4 and5.The full characterization of the synthesized compounds4 and5and the X-ray crystal data of5a,5d, and5f 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

The samples of the synthesized compound were screened against DPPH radical,β-glucuronidase inhibition assay, thymidine phosphorylase inhibition assay, and α-glucosidase inhibition assay and full methods descriptions are provided in Supplementary Materials.


## 2.1. General Procedure for Aldol Condensation Michael Addition for the Synthesis of4 and5 (GP1)

A mixture of aldehyde3 (1.5 mmol) and1 and2 (3 mmol) as well as Et2NH (1.5 mmol, 155 μL) in 3 mL of degassed H2O (bubbling nitrogen through the water) was stirred at room temperature for 1–5 h until TLC showed complete disappearance of the reactants. The precipitate was removed by filtration and washed with ether (3
×
20 mL). The solid product was dried to afford pure products4 and5.The full characterization of the synthesized compounds4 and5and the X-ray crystal data of5a,5d, and5f 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

The samples of the synthesized compound were screened against DPPH radical,β-glucuronidase inhibition assay, thymidine phosphorylase inhibition assay, and α-glucosidase inhibition assay and full methods descriptions are provided in Supplementary Materials.


## 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 importancevia one-pot multicomponent reactions (MCRs) and avoiding organic solvents in organic synthesis [17, 19, 20]. This 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 adducts4a–4yand5a–5m were obtained in quantitative yields by simple filtration [17–20]. Products4s,4t, and5g were identified as new products (Scheme 1).Scheme 1
Synthesis of the target compounds.


### 3.2. X-Ray Diffraction

The molecular structures of compounds5a,5d, and5f were unambiguously deduced by single-crystal X-ray diffraction technique (Figure 2). The crystals of the synthesized molecules5a, 5d, and5f were grown by slow diffusion of diethyl ether solution of pure samples5a,5d, and5f in DCM/EtOH at room temperature followed by allowing standing for 24 h. The structures were resolved by direct methods by using the SHELXL97 program [21, 22] in the SHELXTL-plus package and refined by a full-matrix least-squares procedure on F
2 using SHELXS97. Diffraction data were collected on a Bruker APEX-II CCD diffractometer (Bruker AXS GmbH). The crystal structure and refinement data of compounds5a, 5d, and5f are listed in Table 1. The final atomic coordinates for all atoms and complete listing of bond distance and angles are presented in supplementary information. ORTEP drawings of final X-ray model of compounds5a, 5d, and5f with the atomic numbering scheme are presented in Figure 2, while crystal packing presentation of compounds5a,5d, and5f is shown in Figure 3.Table 1
The crystal and experimental data of compounds5a, 5d, and 5f.
5a
5d
5f
Empirical formula
C23H26NO6·C4H12N·H2O
C24H29O5·C4H12N
C23H26NO6·C4H12N
Formula weight
504.61
471.62
486.59
Temperature
100 K
100 K
100 K
Wavelength
MoKα radiation, λ = 0.71073 Å
MoKα radiation, λ = 0.71073 Å
MoKα radiation, λ = 0.71073 Å
Crystal system
Orthorhombic
Monoclinic
Monoclinic
Space group
P212121
P21/n
P21/n
a
11.6155 (5) Å
10.2288 (5) Å
10.1137 (4) Å
b
12.0514 (5) Å
18.0048 (7) Å
18.1225 (7) Å
c
19.3665 (7) Å
15.0893 (7) Å
15.4853 (6) Å
β
90.00°
106.425 (2)°
106.730 (1)°
Volume
2710.98 (19) Å3
2665.6 (2) Å3
2718.09 (18) Å3
Z
4
4
4
Calculated density
1.236 Mg m−3
1.175 Mg m−3
1.189 Mg m−3
Absorption coefficient
0.09 mm−1
0.08 mm−1
0.08 mm−1
F (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
θ range
2.4–24.2°
2.2–29.7°
2.2–32.1°
Reflections collected
28531
67389
25488
Reflections unique
4501
6194
4829
Independent reflections
6224
11203
6233
Parameters
350
326
334
R
i
n
t
0.090
0.122
0.053
R [F
2 > 2σ(F
2)]
0.054
0.069
0.055
w
R (F
2)
0.109
0.156
0.132
Goodness of fit
1.05
1.01
1.09
Max/minρ e Å−3
0.42 and −0.27
0.43 and −0.30
0.29 and −0.26
CCDC number
1444058
1443783
1444050Figure 2
ORTEP diagram of5a,5d, and5f. Displacement ellipsoids are plotted at the 40% probability level for non-H atoms.Figure 3
Molecular packing of5a,5d, and5f viewed approximately three-dimensional network, along b axis and three-dimensional network, respectively. Intermolecular interaction is drawn as dashed lines.Compound5a crystallizes in orthorhombic crystal system in a space group ofP212121; on the other hand, compounds5d and5f crystalize in monoclinic crystal system in a space group ofP21/n. It was observed that the compound5a has hydrogen bonding between the C5-O2⋯H⋯O4-C13, while compounds5d and5f exist only in the enol form. There are two strong intramolecular hydrogen bonds on compound5a between O4—H1O4⋯O2 and N2—H2N2⋯O1, while compounds5d and5f have no intramolecular hydrogen bonding. The crystal structures of the tested compounds5a,5d, and5f were formed by different hydrogen bonds between the molecules and diethyl amine and water solvent molecules in case of5a to form network structures. Crystallographic data for the solved structures5a,5d, and5f were deposited to the Cambridge Crystallographic Data Center as supplementary 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. Biological Activity Evaluation

All synthesized diethyl ammonium salts of barbiturates havingbis(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-dimethyl-2,6-dioxo-1,2,3,6-tetrahydropyrimidin-4-olate (4m–4z) as well asbis(2-hydroxy-4,4-dimethyl-6-oxocyclohex-1-ene) (5a–5m) as basic nuclei were screened for their antioxidant potentialin 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. These derivatives4a–4z and5a–5m were also evaluated forin vitroenzyme inhibition activities against α-glucosidase, thymidine phosphorylase, and β
-glucuronidase enzymes. The results are summarized in Table 2.Table 2
Result ofin vitrobiological evaluation of the synthesized compounds.
Number
DPPH radical scavenging assayIC50  ± SEMa (µM)
Anticancer/cytotoxicity assayIC50  ±  SEM (µM)
α-Glucosidase inhibitionIC50  ± SEM (µM)
Thymidine phosphorylase inhibitionIC50  ± SEM (µM)
β-Glucuronidase inhibitionIC50  ± SEM (µM)
PC3b
Helac
MCF-7d
3T3
4a
118.8 ± 3.1
>30
>30
NA
>30
NA
NA
204.81 ± 6.0
4b
117 ± 2.4
>30
>30
NA
>30
NA
NA
NA
4c
111.9 ± 1.9
>30
>30
NA
>30
355 ± 10
NA
172.1 ± 2.1
4d
124.4 ± 4.4
>30
>30
NA
>30
NA
230.9 ± 0.8
NA
4e
101.1 ± 0.8
>30
>30
NA
>30
NA
NA
338.9 ± 6.4
4f
148.6 ± 2.6
>30
>30
NA
>30
NA
217.9 ± 2.0
NA
4g
104.1 ± 1.9
>30
>30
NA
>30
NA
NA
NA
4h
133.6 ± 5.7
>30
>30
NA
>30
230 ± 3.9
NA
NA
4i
384.9 ± 6.6
>30
>30
NA
>30
NA
NA
NA
4j
384.3 ± 8.8
>30
>30
NA
>30
NA
NA
NA
4k
NA
>30
>30
NA
>30
NA
182.6 ± 1.0
NA
4l
397.9 ± 0.1
>30
>30
NA
>30
NA
NA
NA
4m
NA
>30
>30
NA
>30
NA
160.2 ± 4.5
NA
4n
NA
>30
>30
NA
>30
NA
226.9 ± 1.5
NA
4o
NA
>30
>30
NA
>30
300.9 ± 6.5
NA
262.1 ± 4.3
4p
NA
>30
>30
NA
>30
186.8 ± 8.1
242.0 ± 1.5
NA
4q
NA
>30
>30
NA
>30
214.8 ± 7.2
141.3 ± 1.5
292.17 ± 1.0
4r
NA
>30
>30
NA
>30
201 ± 5.5
168 ± 1.2
262.1 ± 4.4
4s
439.6 ± 10.1
>30
>30
NA
>30
NA
NA
NA
4t
278.2 ± 10.0
>30
>30
NA
>30
NA
NA
NA
4v
133.2 ± 4.7
>30
>30
NA
>30
NA
57.6 ± 1.2
NA
4w
411.6 ± 0.6
>30
>30
NA
>30
NA
NA
143.5 ± 1.2
4x
388.8 ± 7.9
>30
>30
NA
>30
NA
239 ± 2.0
NA
4y
NA
10.71 ± 0.3
3.47 ± 0.25
4.9 ± 0.01
3.20 ± 0.2
NA
NA
NA
4z
223.0 ± 3.9
>30
>30
NA
>30
NA
55.8 ± 1.5
NA
5a
461.6 ± 8.8
>30
>30
NA
>30
NA
NA
NA
5b
NA
>30
>30
NA
>30
NA
216.9 ± 0.5
NA
5c
NA
>30
>30
NA
>30
NA
161.4 ± 1.5
NA
5d
NA
>30
>30
NA
>30
NA
60.5 ± 1.87
NA
5e
289 ± 0.9
>30
>30
NA
>30
NA
NA
NA
5f
NA
>30
>30
NA
>30
238 ± 9.3
NA
185.0 ± 2.5
5g
270.4 ±  2.3
>30
>30
NA
>30
NA
186.6 ± 1.2
NA
5h
NA
>30
>30
NA
>30
NA
56.9 ± 1.6
NA
5i
NA
>30
>30
NA
>30
NA
NA
323.6 ± 6.4
5j
NA
>30
>30
NA
>30
NA
318 ± 2.5
NA
5k
127.6 ± 1.6
>30
>30
NA
>30
NA
166.4 ± 1.2
NA
5l
169.7 ± 9.3
>30
>30
NA
>30
NA
178.1 ± 2.1
387.5 ± 7.0
5m
NA
>30
>30
NA
>30
453.4 ± 4.2
147.1 ± 1.2
NA
STD.
eBHT (28.8 ± 2.1)
Doxorubicin
Doxorubicin
Doxorubicin
Cycloheximide
Acarbose
7-Deazaxanthine
D-Saccharic acid 1-4 lactone
N-acetyl cysteine (107.6 ± 2.8)
0.912 ± 0.12
0.506 ± 0.15
1.56 ± 0.05
0.26 ± 0.1
840 ± 1.73
41 ± 1.64
45.75 ± 2.16
aSEM: standard error of mean, bPC3: human prostate cancer cell line, cHeLa: Henrietta Lacks cervical cancer cell line, dMCF-7: Michigan Cancer Foundation-7 breast cancer cell lines, and eBHT: butylated hydroxytoluene.


#### 3.3.1. Antioxidant Activity (DPPH Radical Scavenging Assay)

Among series of compounds (4a–4h) havingbis(6-hydroxy-1,3-dimethyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidin-5-yl) ring as basic nucleus, compounds4a (IC50 = 118.8
±
2.1 μM),4b (IC50 = 117
±
2.4 μM),4c (IC50 = 111.9
±
1.9 μM),4d (IC50 = 124.4
±
4.4 μM),4e (IC50 = 101.1
±
0.8 μM),4f(IC50 = 148.6
±
2.6 μM), and4g (IC50 = 104.1
±
1.9 μM) were found to be potent antioxidants in comparison to the standards BHT (IC50 = 128.8
±
2.1 μM) andN-acetylcysteine (IC50 = 107.6
±
2.8 μM). Compounds4a–4e (IC50 = 101.8
±
0.8
–
124.4
±
4.4 μM) were found to be more active compared to the standard BHT (IC50 = 128.8
±
2.1 μM). The meta-bromo-substituted phenyl ring containing compound4e(IC50 = 101.1
±
0.8 μM) was the most potent member of the series (4a–4e), followed by thepara-methyl-substituted phenyl ring containing compound4g (IC50 = 104.1
±
1.9 μM). A gradual decrease in activity was observed for compounds4c (IC50 = 119.9
±
1.9 μM) and4a(IC50 = 118.8
±
3.1 μM) havingpara-nitro- andpara-aldehyde-substituted phenyl ring attached to centralbis(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 compound4b (IC50 = 117
±
3.1 μM) havingmeta-substituted phenyl ring instead ofpara-methyl-substituted phenyl ring (4g, IC50 = 104.1
±
1.9 μM). The electron donatingpara-hydroxyl-substituted phenyl ring containing compound4f (IC50 = 148.6
±
2.6 μM) was found to be a last active member of the series. However, a considerable increase in activity was observed for compound4d (IC50 = 124.4
±
4.4 μM) having apara-methoxy group instead of hydroxyl functionality on phenyl ring, whereas naphthalene ring containing barbiturate4h (IC50 = 133.6
±
5.7
μM) was also found to be a weak antioxidant agent against DPPH radicals.Among second series of the tested compounds4i–4l havingbis(6-hydroxypyrimidine-2,4(1H,3H)-dione as backbone, compounds havingpara-methyl- (4i, IC50 = 384.9
±
6.6 μM),para-chloro- (4j, IC50 = 384.3
±
8.8 μM), and para-methoxy- (4l, IC50 = 397.9
±
0.9 μM) substituted phenyl ring attached to the centralbis(6-hydroxypyrimidine-2,4(1H,3H)-dione) showed better antioxidant activity as compared to BHT (IC50 = 128.8
±
2.1 μM) andN-acetylcysteine (IC50 = 107.6
±
2.8 μM), whereas naphthalene containing compound4l was found to be inactive.(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 compounds4m–4z were also evaluated for their antioxidant potentialin vitro in DPPH radical scavenging assay. These compounds were found to be significant to weak inhibitors of free radicals (IC50 = 133.2
±
4.7
–
439.6
±
10.1 μM) except compounds4m–4r and4y which were found to be inactive.Similarlybis(2-hydroxy-4,4-dimethyl-6-oxocyclohex-1-ene) (5a–5h) and 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) (5i–5k) rings containing compounds were also evaluated for their antioxidant potential by using DPPH radical scavenging assay. Para-bromo-substituted phenyl ring containing compound5k(IC50 = 127.6
±
1.6 μM) was found as a potent antioxidant agent against the tested standards BHT (IC50 = 128.8
±
2.1 μM). The compound showed similar antioxidant potential as BHT and showed a significant antioxidant agent againstN-acetyl cysteine (IC50 = 107.6
±
2.8 μM). The gradual and drastic decrease in activity was for compounds5l (IC50 = 169.7
±
5.3 μM),5g (IC50 = 270.4
±
2.3 μM),5e (IC50 = 289
±
0.9 μM), and5a (IC50 = 461.6
±
8.8 μM) having para-methyl-, para-aldehyde-, ortho-chloro-, and ortho-nitro-substituted phenyl rings attached to the central barbiturate moieties. Compounds5b–5d,5f, and5h–5j were found to be inactive.On the basis of the observed antioxidant abilities of the above five different series of pyridinium adducts, it was concluded thatbis(6-hydroxy-1,3-dimethyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidin-5-yl) ring containing compounds4a–4h are the most active one. The activity may be due to the methyl substituted nitrogen atoms of the tetrahydropyrimidine 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 the presence of electron donating (-OH (4f), -OCH3 (4d), and -CH3 (4b,4g)), electron withdrawing (-NO2 (4a) and -CHO (4c)), and halogen (-Br (4e)) substituents on the phenyl ring attached to thebis(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. Cytotoxic Activity

All synthesized diethyl ammonium salts of barbiturates (4a–4z and5a–5m) were evaluated for theirin 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-tetrahydropyrimidin-4-olate ring containing compounds4y, which was found to be significantly toxic against all the cell lines (PC3, IC50 = 10.71
±
0.26 μM, HeLa, IC50 = 3.47
±
0.3 μM, MCF-7, IC50 = 4.9
±
0.01 μM, and 3T3, IC50 = 3.20
±
0.2 μM). Doxorubicin was used as a standard drug to compare the activities against PC3 (IC50 = 1.70
±
0.29 μM), HeLa (IC50 = 0.51
±
0.15 μM), and MCF-3 (IC50 = 0.92
±
0.01 μM) cancer cell lines, while cycloheximide (IC50 = 0.26
±
0.12 μM) was used as a standard-in 3T3 cell line (Table 2).


#### 3.3.3.α-Glucosidase Inhibition Activity

Synthesized diethyl ammonium salts of barbiturates,4a–4z and5a–5m, were also evaluated for theirin vitro  α-glucosidase inhibition activity, in comparison to the standard drug, acarbose (IC50 = 841
±
1.73 μM). Among dimethyl ammonium salts (4a–4h), nitro-substituted phenyl ring containing4c (IC50 = 355
±
10 μM) and naphthalene ring containing4h (IC50 = 230
±
3.92 μM) were found to be potent inhibitors of α-glucosidase enzyme and showed more activity than acarbose (IC50 = 841
±
1.73 μM). 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),para-OCH3- (40, IC50 = 300.9
±
6.5 μM),para-chloro- (4p, IC50 = 186.8
±
0.81 μM),para-bromo- (4q, IC50 = 214.8
±
0.72 μM), andmeta-bromo- (4r, IC50 = 201
±
5.5 μM) substituted phenyl ring containing pyridinium adducts were found to be potent α-glucosidase inhibitors and showed more activity than standard acarbose (IC50 = 841
±
1.73 μM). Other members of the series (4m-4n and4s–4z) werefound to be inactive. The results showed the substitution effects of various functionalities of phenyl ring on the potential activity of tested series. Thepara-bromo-substituted phenyl ring containing compound4q (IC50 = 214.8
±
0.72 μM) showed 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 compounds4n,4t,4v, and4x, respectively. The presence of ortho-nitro-,ortho-choloro-, andpara-choloro-substituted phenyl ring also contributed towards inactivity, as observed for compounds4t,4w, and4y.Similarlybis(2-hydroxy-4,4-dimethyl-6-oxocyclohex-1-ene) ring containing diethyl ammonium pyridinium salts (5a–5h) were also evaluated for theirin vitro  α-glucosidase enzyme inhibition activity. The series nitro-substituted phenyl ring containing5fwas found to be the only potent α-glucosidase inhibitor. All other compounds (5a–5e and5g-5h) were found to be inactive.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-dioxo-1,2,3,4-tetrahydropyrimidin-5-yl)methyl) central moiety attached with substituted phenyl ring, the benzaldehyde containing5m (IC50 = 453.4 ± 4.2 μM) was found to be the only potent inhibitor compound in comparison to against the standard drug acarbose (IC50 = 841
±
1.73 μM). All other compounds were found to be inactive. The results are summarized in Table 2.


#### 3.3.4. Thymidine Phosphorylase Inhibition Activity

Five different series of diethyamonium salts of barbiturates,4a–4g,4i–4k,4m–4z,5a–5h, and5i–5m, were also evaluated for theirin vitrothymidine phosphorylase inhibition. 7-Deazaxanthine was used as a standard inhibitor (IC50 = 41
±
1.46 μM). Phenol and naphthalene rings 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 moiety containing compounds4v (IC50 = 57.6
±
1.20 μM) and4z(IC50 = 55.8
±
1.50 μM) were found to be the potent thymidine phosphorylase inhibitors. However, a drastic decrease in activity was observed whenpara-hydroxy group on phenyl ring was replaced with methyl (4n, IC50 = 226.9
±
1
.
50 μM), chloro (4n, IC50 = 242
±
1
.
5
μM), bromo (4q, IC50 = 141.3
±
1
.
5 μM), and aldehyde (4x, IC50 = 239
±
2.0 μM) groups, whereas the compounds4o,4s,4t,4w, and4y havepara-methoxy-,ortho-nitro-,ortho-N,N-dimethyl amine-, ortho-, para-dichloro-, andortho-ortho-dichloro-substituted phenyl ring, respectively.Among series of compounds (5a–5h) havingbis(2-hydroxy-4,4-dimethyl-6-oxocyclohex-1-ene) ring as central moiety, electron donating hydroxy- and methoxy-substituted phenyl ring containing compounds5h(IC50 = 56.9
±
1.6 μM) and5d (IC50 = 60.5
±
1.87 μM), respectively, were found as potent inhibitors of thymidine phosphorylase enzyme against the tested standard 7-deazaxanthine which was (IC50 = 41
±
1.46 μM) used as standard. Again a drastic decrease in activity was observed for compounds5b (IC50 = 216.9
±
0.5
μM),5c (IC50 = 161.4
±
1.5 μM), and5g (IC50 = 186.6
±
1.2 μM) havingmeta-methyl-,ortho-methyl-, andpara-aldehyde-substituted benzene ring respectively, instead ofpara-hydroxyl phenyl ring. The replacement ofortho-methyl with chloro-group makes the compound5e completely inactive. Similarly the replacement of electron donatingpara-hydroxy-group of phenyl ring with electron withdrawing nitro-functionality also contributed towards inactivity of compound, as observed for5f. Phenol substitutedbis(2-hydroxy-4,4-dimethyl-6-oxocyclohex-1-ene) ring containing5h (IC50 = 56.9 ± 1.6 μM) was found to be the potent thymidine phosphorylase inhibitors.All members of the 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-dioxo-1,2,3,4-tetrahydropyrimidin-5-yl)methyl) central moiety attached with substituted phenyl ring were found to be weak inhibitors of thymidine phosphorylase enzyme against the tested standard compound 7-deazaxanthine (IC50 = 41
±
1
.
46 μM) with IC50 values 318
±
2.5 μM, 166.4
±
1
.
2 μM, 178.1
±
2
.
1 μM, and 147.1
±
1
.
23 μM for5j,5k,5l, and5m, respectively, whereas para-chloro-substituted phenyl ring containing compound5i was found to be inactive. The results are summarized in Table 1.


#### 3.3.5.β-Glucuronidase Inhibition Activity

Compounds4a–4g,4i–4k,4m–4z,5a–5h, and5i–5m were also evaluated for theirin vitro β-glucuronidase inhibitory activity by using D-saccharic acid 1,4-lactone as standard inhibitor (IC50 = 45.75
±
2.16 μM). Compounds4a (IC50 = 204.8
±
6.01 μM),4c (IC50 = 172.1
±
2.1 μM),4e (IC50 = 338.9
±
6.42 μM),4o (IC50 = 262.1
±
4.35 μM),4q (IC50 = 292.2
±
1.03 μM),4r (IC50 = 262.1
±
4.4 μM),4w (IC50 = 143.5
±
1.24 μM),5f (IC50 = 185
±
2.5 μM),5i (IC50 = 223.6
±
6.4 μM), and5l (IC50 = 387.5
±
7.0 μM) showed very weak β-glucuronidase inhibition potential, whereas pyridinium adducts4a–4c,4e,4g–4j,4l,4o,4s-4t,4w,4y,5a,5e-5f, and5i were found to be inactive (Table 2).


### 3.4. Molecular Docking Studies

#### 3.4.1. Binding Interactions ofα-Glucosidase Enzyme

To define inhibitors interactions and their specificity, the docking study of the most active compound4p against α-glucosidase 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 α-glucosidase. The 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). The presence of polar and electron rich center in the form of pyrimidinedione and chlorobenzene groups of the compound 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 compound4p were also observed.Figure 4
Three-dimensional representation of compound4p with target macromolecule α-glucosidase.


#### 3.4.2. Binding Interactions of Thymidine Phosphorylase Enzyme

To explore the binding modes of these newly synthesized compounds in the active site of thymidine phosphorylase, the most active compound4v was docked against this enzyme. The docking results showed that compound4v well accommodated in the active site of thymidine phosphorylase (Figure 5). The active site residue Ala 206 established a hydrogen bond through its lone pair of 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.Figure 5
Three-dimensional representation of compound4v with target macromolecule thymidine phosphorylase.


#### 3.4.3. Binding Interaction ofβ-Glucuronidase Enzyme

The predicted binding mode of most active compound4w against β-glucuronidase showed that a hydrogen bond is formed between the lone pair of oxygen of cyclohexanedione component of the compound and imidazole hydrogen of the important 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 compound4w and the active site residues were also observed. The pyrimidinedione moiety in this case was observed to be passive regarding interactions.Figure 6
Three-dimensional representation of compound4w with target enzyme β-glucuronidase.


## 3.1. Chemistry

We have been focusing on the development of highly expedient methods for the synthesis of diverse heterocyclic compounds of biological importancevia one-pot multicomponent reactions (MCRs) and avoiding organic solvents in organic synthesis [17, 19, 20]. This 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 adducts4a–4yand5a–5m were obtained in quantitative yields by simple filtration [17–20]. Products4s,4t, and5g were identified as new products (Scheme 1).Scheme 1
Synthesis of the target compounds.


## 3.2. X-Ray Diffraction

The molecular structures of compounds5a,5d, and5f were unambiguously deduced by single-crystal X-ray diffraction technique (Figure 2). The crystals of the synthesized molecules5a, 5d, and5f were grown by slow diffusion of diethyl ether solution of pure samples5a,5d, and5f in DCM/EtOH at room temperature followed by allowing standing for 24 h. The structures were resolved by direct methods by using the SHELXL97 program [21, 22] in the SHELXTL-plus package and refined by a full-matrix least-squares procedure on F
2 using SHELXS97. Diffraction data were collected on a Bruker APEX-II CCD diffractometer (Bruker AXS GmbH). The crystal structure and refinement data of compounds5a, 5d, and5f are listed in Table 1. The final atomic coordinates for all atoms and complete listing of bond distance and angles are presented in supplementary information. ORTEP drawings of final X-ray model of compounds5a, 5d, and5f with the atomic numbering scheme are presented in Figure 2, while crystal packing presentation of compounds5a,5d, and5f is shown in Figure 3.Table 1
The crystal and experimental data of compounds5a, 5d, and 5f.
5a
5d
5f
Empirical formula
C23H26NO6·C4H12N·H2O
C24H29O5·C4H12N
C23H26NO6·C4H12N
Formula weight
504.61
471.62
486.59
Temperature
100 K
100 K
100 K
Wavelength
MoKα radiation, λ = 0.71073 Å
MoKα radiation, λ = 0.71073 Å
MoKα radiation, λ = 0.71073 Å
Crystal system
Orthorhombic
Monoclinic
Monoclinic
Space group
P212121
P21/n
P21/n
a
11.6155 (5) Å
10.2288 (5) Å
10.1137 (4) Å
b
12.0514 (5) Å
18.0048 (7) Å
18.1225 (7) Å
c
19.3665 (7) Å
15.0893 (7) Å
15.4853 (6) Å
β
90.00°
106.425 (2)°
106.730 (1)°
Volume
2710.98 (19) Å3
2665.6 (2) Å3
2718.09 (18) Å3
Z
4
4
4
Calculated density
1.236 Mg m−3
1.175 Mg m−3
1.189 Mg m−3
Absorption coefficient
0.09 mm−1
0.08 mm−1
0.08 mm−1
F (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
θ range
2.4–24.2°
2.2–29.7°
2.2–32.1°
Reflections collected
28531
67389
25488
Reflections unique
4501
6194
4829
Independent reflections
6224
11203
6233
Parameters
350
326
334
R
i
n
t
0.090
0.122
0.053
R [F
2 > 2σ(F
2)]
0.054
0.069
0.055
w
R (F
2)
0.109
0.156
0.132
Goodness of fit
1.05
1.01
1.09
Max/minρ e Å−3
0.42 and −0.27
0.43 and −0.30
0.29 and −0.26
CCDC number
1444058
1443783
1444050Figure 2
ORTEP diagram of5a,5d, and5f. Displacement ellipsoids are plotted at the 40% probability level for non-H atoms.Figure 3
Molecular packing of5a,5d, and5f viewed approximately three-dimensional network, along b axis and three-dimensional network, respectively. Intermolecular interaction is drawn as dashed lines.Compound5a crystallizes in orthorhombic crystal system in a space group ofP212121; on the other hand, compounds5d and5f crystalize in monoclinic crystal system in a space group ofP21/n. It was observed that the compound5a has hydrogen bonding between the C5-O2⋯H⋯O4-C13, while compounds5d and5f exist only in the enol form. There are two strong intramolecular hydrogen bonds on compound5a between O4—H1O4⋯O2 and N2—H2N2⋯O1, while compounds5d and5f have no intramolecular hydrogen bonding. The crystal structures of the tested compounds5a,5d, and5f were formed by different hydrogen bonds between the molecules and diethyl amine and water solvent molecules in case of5a to form network structures. Crystallographic data for the solved structures5a,5d, and5f were deposited to the Cambridge Crystallographic Data Center as supplementary 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. Biological Activity Evaluation

All synthesized diethyl ammonium salts of barbiturates havingbis(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-dimethyl-2,6-dioxo-1,2,3,6-tetrahydropyrimidin-4-olate (4m–4z) as well asbis(2-hydroxy-4,4-dimethyl-6-oxocyclohex-1-ene) (5a–5m) as basic nuclei were screened for their antioxidant potentialin 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. These derivatives4a–4z and5a–5m were also evaluated forin vitroenzyme inhibition activities against α-glucosidase, thymidine phosphorylase, and β
-glucuronidase enzymes. The results are summarized in Table 2.Table 2
Result ofin vitrobiological evaluation of the synthesized compounds.
Number
DPPH radical scavenging assayIC50  ± SEMa (µM)
Anticancer/cytotoxicity assayIC50  ±  SEM (µM)
α-Glucosidase inhibitionIC50  ± SEM (µM)
Thymidine phosphorylase inhibitionIC50  ± SEM (µM)
β-Glucuronidase inhibitionIC50  ± SEM (µM)
PC3b
Helac
MCF-7d
3T3
4a
118.8 ± 3.1
>30
>30
NA
>30
NA
NA
204.81 ± 6.0
4b
117 ± 2.4
>30
>30
NA
>30
NA
NA
NA
4c
111.9 ± 1.9
>30
>30
NA
>30
355 ± 10
NA
172.1 ± 2.1
4d
124.4 ± 4.4
>30
>30
NA
>30
NA
230.9 ± 0.8
NA
4e
101.1 ± 0.8
>30
>30
NA
>30
NA
NA
338.9 ± 6.4
4f
148.6 ± 2.6
>30
>30
NA
>30
NA
217.9 ± 2.0
NA
4g
104.1 ± 1.9
>30
>30
NA
>30
NA
NA
NA
4h
133.6 ± 5.7
>30
>30
NA
>30
230 ± 3.9
NA
NA
4i
384.9 ± 6.6
>30
>30
NA
>30
NA
NA
NA
4j
384.3 ± 8.8
>30
>30
NA
>30
NA
NA
NA
4k
NA
>30
>30
NA
>30
NA
182.6 ± 1.0
NA
4l
397.9 ± 0.1
>30
>30
NA
>30
NA
NA
NA
4m
NA
>30
>30
NA
>30
NA
160.2 ± 4.5
NA
4n
NA
>30
>30
NA
>30
NA
226.9 ± 1.5
NA
4o
NA
>30
>30
NA
>30
300.9 ± 6.5
NA
262.1 ± 4.3
4p
NA
>30
>30
NA
>30
186.8 ± 8.1
242.0 ± 1.5
NA
4q
NA
>30
>30
NA
>30
214.8 ± 7.2
141.3 ± 1.5
292.17 ± 1.0
4r
NA
>30
>30
NA
>30
201 ± 5.5
168 ± 1.2
262.1 ± 4.4
4s
439.6 ± 10.1
>30
>30
NA
>30
NA
NA
NA
4t
278.2 ± 10.0
>30
>30
NA
>30
NA
NA
NA
4v
133.2 ± 4.7
>30
>30
NA
>30
NA
57.6 ± 1.2
NA
4w
411.6 ± 0.6
>30
>30
NA
>30
NA
NA
143.5 ± 1.2
4x
388.8 ± 7.9
>30
>30
NA
>30
NA
239 ± 2.0
NA
4y
NA
10.71 ± 0.3
3.47 ± 0.25
4.9 ± 0.01
3.20 ± 0.2
NA
NA
NA
4z
223.0 ± 3.9
>30
>30
NA
>30
NA
55.8 ± 1.5
NA
5a
461.6 ± 8.8
>30
>30
NA
>30
NA
NA
NA
5b
NA
>30
>30
NA
>30
NA
216.9 ± 0.5
NA
5c
NA
>30
>30
NA
>30
NA
161.4 ± 1.5
NA
5d
NA
>30
>30
NA
>30
NA
60.5 ± 1.87
NA
5e
289 ± 0.9
>30
>30
NA
>30
NA
NA
NA
5f
NA
>30
>30
NA
>30
238 ± 9.3
NA
185.0 ± 2.5
5g
270.4 ±  2.3
>30
>30
NA
>30
NA
186.6 ± 1.2
NA
5h
NA
>30
>30
NA
>30
NA
56.9 ± 1.6
NA
5i
NA
>30
>30
NA
>30
NA
NA
323.6 ± 6.4
5j
NA
>30
>30
NA
>30
NA
318 ± 2.5
NA
5k
127.6 ± 1.6
>30
>30
NA
>30
NA
166.4 ± 1.2
NA
5l
169.7 ± 9.3
>30
>30
NA
>30
NA
178.1 ± 2.1
387.5 ± 7.0
5m
NA
>30
>30
NA
>30
453.4 ± 4.2
147.1 ± 1.2
NA
STD.
eBHT (28.8 ± 2.1)
Doxorubicin
Doxorubicin
Doxorubicin
Cycloheximide
Acarbose
7-Deazaxanthine
D-Saccharic acid 1-4 lactone
N-acetyl cysteine (107.6 ± 2.8)
0.912 ± 0.12
0.506 ± 0.15
1.56 ± 0.05
0.26 ± 0.1
840 ± 1.73
41 ± 1.64
45.75 ± 2.16
aSEM: standard error of mean, bPC3: human prostate cancer cell line, cHeLa: Henrietta Lacks cervical cancer cell line, dMCF-7: Michigan Cancer Foundation-7 breast cancer cell lines, and eBHT: butylated hydroxytoluene.


### 3.3.1. Antioxidant Activity (DPPH Radical Scavenging Assay)

Among series of compounds (4a–4h) havingbis(6-hydroxy-1,3-dimethyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidin-5-yl) ring as basic nucleus, compounds4a (IC50 = 118.8
±
2.1 μM),4b (IC50 = 117
±
2.4 μM),4c (IC50 = 111.9
±
1.9 μM),4d (IC50 = 124.4
±
4.4 μM),4e (IC50 = 101.1
±
0.8 μM),4f(IC50 = 148.6
±
2.6 μM), and4g (IC50 = 104.1
±
1.9 μM) were found to be potent antioxidants in comparison to the standards BHT (IC50 = 128.8
±
2.1 μM) andN-acetylcysteine (IC50 = 107.6
±
2.8 μM). Compounds4a–4e (IC50 = 101.8
±
0.8
–
124.4
±
4.4 μM) were found to be more active compared to the standard BHT (IC50 = 128.8
±
2.1 μM). The meta-bromo-substituted phenyl ring containing compound4e(IC50 = 101.1
±
0.8 μM) was the most potent member of the series (4a–4e), followed by thepara-methyl-substituted phenyl ring containing compound4g (IC50 = 104.1
±
1.9 μM). A gradual decrease in activity was observed for compounds4c (IC50 = 119.9
±
1.9 μM) and4a(IC50 = 118.8
±
3.1 μM) havingpara-nitro- andpara-aldehyde-substituted phenyl ring attached to centralbis(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 compound4b (IC50 = 117
±
3.1 μM) havingmeta-substituted phenyl ring instead ofpara-methyl-substituted phenyl ring (4g, IC50 = 104.1
±
1.9 μM). The electron donatingpara-hydroxyl-substituted phenyl ring containing compound4f (IC50 = 148.6
±
2.6 μM) was found to be a last active member of the series. However, a considerable increase in activity was observed for compound4d (IC50 = 124.4
±
4.4 μM) having apara-methoxy group instead of hydroxyl functionality on phenyl ring, whereas naphthalene ring containing barbiturate4h (IC50 = 133.6
±
5.7
μM) was also found to be a weak antioxidant agent against DPPH radicals.Among second series of the tested compounds4i–4l havingbis(6-hydroxypyrimidine-2,4(1H,3H)-dione as backbone, compounds havingpara-methyl- (4i, IC50 = 384.9
±
6.6 μM),para-chloro- (4j, IC50 = 384.3
±
8.8 μM), and para-methoxy- (4l, IC50 = 397.9
±
0.9 μM) substituted phenyl ring attached to the centralbis(6-hydroxypyrimidine-2,4(1H,3H)-dione) showed better antioxidant activity as compared to BHT (IC50 = 128.8
±
2.1 μM) andN-acetylcysteine (IC50 = 107.6
±
2.8 μM), whereas naphthalene containing compound4l was found to be inactive.(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 compounds4m–4z were also evaluated for their antioxidant potentialin vitro in DPPH radical scavenging assay. These compounds were found to be significant to weak inhibitors of free radicals (IC50 = 133.2
±
4.7
–
439.6
±
10.1 μM) except compounds4m–4r and4y which were found to be inactive.Similarlybis(2-hydroxy-4,4-dimethyl-6-oxocyclohex-1-ene) (5a–5h) and 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) (5i–5k) rings containing compounds were also evaluated for their antioxidant potential by using DPPH radical scavenging assay. Para-bromo-substituted phenyl ring containing compound5k(IC50 = 127.6
±
1.6 μM) was found as a potent antioxidant agent against the tested standards BHT (IC50 = 128.8
±
2.1 μM). The compound showed similar antioxidant potential as BHT and showed a significant antioxidant agent againstN-acetyl cysteine (IC50 = 107.6
±
2.8 μM). The gradual and drastic decrease in activity was for compounds5l (IC50 = 169.7
±
5.3 μM),5g (IC50 = 270.4
±
2.3 μM),5e (IC50 = 289
±
0.9 μM), and5a (IC50 = 461.6
±
8.8 μM) having para-methyl-, para-aldehyde-, ortho-chloro-, and ortho-nitro-substituted phenyl rings attached to the central barbiturate moieties. Compounds5b–5d,5f, and5h–5j were found to be inactive.On the basis of the observed antioxidant abilities of the above five different series of pyridinium adducts, it was concluded thatbis(6-hydroxy-1,3-dimethyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidin-5-yl) ring containing compounds4a–4h are the most active one. The activity may be due to the methyl substituted nitrogen atoms of the tetrahydropyrimidine 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 the presence of electron donating (-OH (4f), -OCH3 (4d), and -CH3 (4b,4g)), electron withdrawing (-NO2 (4a) and -CHO (4c)), and halogen (-Br (4e)) substituents on the phenyl ring attached to thebis(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. Cytotoxic Activity

All synthesized diethyl ammonium salts of barbiturates (4a–4z and5a–5m) were evaluated for theirin 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-tetrahydropyrimidin-4-olate ring containing compounds4y, which was found to be significantly toxic against all the cell lines (PC3, IC50 = 10.71
±
0.26 μM, HeLa, IC50 = 3.47
±
0.3 μM, MCF-7, IC50 = 4.9
±
0.01 μM, and 3T3, IC50 = 3.20
±
0.2 μM). Doxorubicin was used as a standard drug to compare the activities against PC3 (IC50 = 1.70
±
0.29 μM), HeLa (IC50 = 0.51
±
0.15 μM), and MCF-3 (IC50 = 0.92
±
0.01 μM) cancer cell lines, while cycloheximide (IC50 = 0.26
±
0.12 μM) was used as a standard-in 3T3 cell line (Table 2).


### 3.3.3.α-Glucosidase Inhibition Activity

Synthesized diethyl ammonium salts of barbiturates,4a–4z and5a–5m, were also evaluated for theirin vitro  α-glucosidase inhibition activity, in comparison to the standard drug, acarbose (IC50 = 841
±
1.73 μM). Among dimethyl ammonium salts (4a–4h), nitro-substituted phenyl ring containing4c (IC50 = 355
±
10 μM) and naphthalene ring containing4h (IC50 = 230
±
3.92 μM) were found to be potent inhibitors of α-glucosidase enzyme and showed more activity than acarbose (IC50 = 841
±
1.73 μM). 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),para-OCH3- (40, IC50 = 300.9
±
6.5 μM),para-chloro- (4p, IC50 = 186.8
±
0.81 μM),para-bromo- (4q, IC50 = 214.8
±
0.72 μM), andmeta-bromo- (4r, IC50 = 201
±
5.5 μM) substituted phenyl ring containing pyridinium adducts were found to be potent α-glucosidase inhibitors and showed more activity than standard acarbose (IC50 = 841
±
1.73 μM). Other members of the series (4m-4n and4s–4z) werefound to be inactive. The results showed the substitution effects of various functionalities of phenyl ring on the potential activity of tested series. Thepara-bromo-substituted phenyl ring containing compound4q (IC50 = 214.8
±
0.72 μM) showed 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 compounds4n,4t,4v, and4x, respectively. The presence of ortho-nitro-,ortho-choloro-, andpara-choloro-substituted phenyl ring also contributed towards inactivity, as observed for compounds4t,4w, and4y.Similarlybis(2-hydroxy-4,4-dimethyl-6-oxocyclohex-1-ene) ring containing diethyl ammonium pyridinium salts (5a–5h) were also evaluated for theirin vitro  α-glucosidase enzyme inhibition activity. The series nitro-substituted phenyl ring containing5fwas found to be the only potent α-glucosidase inhibitor. All other compounds (5a–5e and5g-5h) were found to be inactive.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-dioxo-1,2,3,4-tetrahydropyrimidin-5-yl)methyl) central moiety attached with substituted phenyl ring, the benzaldehyde containing5m (IC50 = 453.4 ± 4.2 μM) was found to be the only potent inhibitor compound in comparison to against the standard drug acarbose (IC50 = 841
±
1.73 μM). All other compounds were found to be inactive. The results are summarized in Table 2.


### 3.3.4. Thymidine Phosphorylase Inhibition Activity

Five different series of diethyamonium salts of barbiturates,4a–4g,4i–4k,4m–4z,5a–5h, and5i–5m, were also evaluated for theirin vitrothymidine phosphorylase inhibition. 7-Deazaxanthine was used as a standard inhibitor (IC50 = 41
±
1.46 μM). Phenol and naphthalene rings 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 moiety containing compounds4v (IC50 = 57.6
±
1.20 μM) and4z(IC50 = 55.8
±
1.50 μM) were found to be the potent thymidine phosphorylase inhibitors. However, a drastic decrease in activity was observed whenpara-hydroxy group on phenyl ring was replaced with methyl (4n, IC50 = 226.9
±
1
.
50 μM), chloro (4n, IC50 = 242
±
1
.
5
μM), bromo (4q, IC50 = 141.3
±
1
.
5 μM), and aldehyde (4x, IC50 = 239
±
2.0 μM) groups, whereas the compounds4o,4s,4t,4w, and4y havepara-methoxy-,ortho-nitro-,ortho-N,N-dimethyl amine-, ortho-, para-dichloro-, andortho-ortho-dichloro-substituted phenyl ring, respectively.Among series of compounds (5a–5h) havingbis(2-hydroxy-4,4-dimethyl-6-oxocyclohex-1-ene) ring as central moiety, electron donating hydroxy- and methoxy-substituted phenyl ring containing compounds5h(IC50 = 56.9
±
1.6 μM) and5d (IC50 = 60.5
±
1.87 μM), respectively, were found as potent inhibitors of thymidine phosphorylase enzyme against the tested standard 7-deazaxanthine which was (IC50 = 41
±
1.46 μM) used as standard. Again a drastic decrease in activity was observed for compounds5b (IC50 = 216.9
±
0.5
μM),5c (IC50 = 161.4
±
1.5 μM), and5g (IC50 = 186.6
±
1.2 μM) havingmeta-methyl-,ortho-methyl-, andpara-aldehyde-substituted benzene ring respectively, instead ofpara-hydroxyl phenyl ring. The replacement ofortho-methyl with chloro-group makes the compound5e completely inactive. Similarly the replacement of electron donatingpara-hydroxy-group of phenyl ring with electron withdrawing nitro-functionality also contributed towards inactivity of compound, as observed for5f. Phenol substitutedbis(2-hydroxy-4,4-dimethyl-6-oxocyclohex-1-ene) ring containing5h (IC50 = 56.9 ± 1.6 μM) was found to be the potent thymidine phosphorylase inhibitors.All members of the 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-dioxo-1,2,3,4-tetrahydropyrimidin-5-yl)methyl) central moiety attached with substituted phenyl ring were found to be weak inhibitors of thymidine phosphorylase enzyme against the tested standard compound 7-deazaxanthine (IC50 = 41
±
1
.
46 μM) with IC50 values 318
±
2.5 μM, 166.4
±
1
.
2 μM, 178.1
±
2
.
1 μM, and 147.1
±
1
.
23 μM for5j,5k,5l, and5m, respectively, whereas para-chloro-substituted phenyl ring containing compound5i was found to be inactive. The results are summarized in Table 1.


### 3.3.5.β-Glucuronidase Inhibition Activity

Compounds4a–4g,4i–4k,4m–4z,5a–5h, and5i–5m were also evaluated for theirin vitro β-glucuronidase inhibitory activity by using D-saccharic acid 1,4-lactone as standard inhibitor (IC50 = 45.75
±
2.16 μM). Compounds4a (IC50 = 204.8
±
6.01 μM),4c (IC50 = 172.1
±
2.1 μM),4e (IC50 = 338.9
±
6.42 μM),4o (IC50 = 262.1
±
4.35 μM),4q (IC50 = 292.2
±
1.03 μM),4r (IC50 = 262.1
±
4.4 μM),4w (IC50 = 143.5
±
1.24 μM),5f (IC50 = 185
±
2.5 μM),5i (IC50 = 223.6
±
6.4 μM), and5l (IC50 = 387.5
±
7.0 μM) showed very weak β-glucuronidase inhibition potential, whereas pyridinium adducts4a–4c,4e,4g–4j,4l,4o,4s-4t,4w,4y,5a,5e-5f, and5i were found to be inactive (Table 2).


## 3.3.1. Antioxidant Activity (DPPH Radical Scavenging Assay)

Among series of compounds (4a–4h) havingbis(6-hydroxy-1,3-dimethyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidin-5-yl) ring as basic nucleus, compounds4a (IC50 = 118.8
±
2.1 μM),4b (IC50 = 117
±
2.4 μM),4c (IC50 = 111.9
±
1.9 μM),4d (IC50 = 124.4
±
4.4 μM),4e (IC50 = 101.1
±
0.8 μM),4f(IC50 = 148.6
±
2.6 μM), and4g (IC50 = 104.1
±
1.9 μM) were found to be potent antioxidants in comparison to the standards BHT (IC50 = 128.8
±
2.1 μM) andN-acetylcysteine (IC50 = 107.6
±
2.8 μM). Compounds4a–4e (IC50 = 101.8
±
0.8
–
124.4
±
4.4 μM) were found to be more active compared to the standard BHT (IC50 = 128.8
±
2.1 μM). The meta-bromo-substituted phenyl ring containing compound4e(IC50 = 101.1
±
0.8 μM) was the most potent member of the series (4a–4e), followed by thepara-methyl-substituted phenyl ring containing compound4g (IC50 = 104.1
±
1.9 μM). A gradual decrease in activity was observed for compounds4c (IC50 = 119.9
±
1.9 μM) and4a(IC50 = 118.8
±
3.1 μM) havingpara-nitro- andpara-aldehyde-substituted phenyl ring attached to centralbis(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 compound4b (IC50 = 117
±
3.1 μM) havingmeta-substituted phenyl ring instead ofpara-methyl-substituted phenyl ring (4g, IC50 = 104.1
±
1.9 μM). The electron donatingpara-hydroxyl-substituted phenyl ring containing compound4f (IC50 = 148.6
±
2.6 μM) was found to be a last active member of the series. However, a considerable increase in activity was observed for compound4d (IC50 = 124.4
±
4.4 μM) having apara-methoxy group instead of hydroxyl functionality on phenyl ring, whereas naphthalene ring containing barbiturate4h (IC50 = 133.6
±
5.7
μM) was also found to be a weak antioxidant agent against DPPH radicals.Among second series of the tested compounds4i–4l havingbis(6-hydroxypyrimidine-2,4(1H,3H)-dione as backbone, compounds havingpara-methyl- (4i, IC50 = 384.9
±
6.6 μM),para-chloro- (4j, IC50 = 384.3
±
8.8 μM), and para-methoxy- (4l, IC50 = 397.9
±
0.9 μM) substituted phenyl ring attached to the centralbis(6-hydroxypyrimidine-2,4(1H,3H)-dione) showed better antioxidant activity as compared to BHT (IC50 = 128.8
±
2.1 μM) andN-acetylcysteine (IC50 = 107.6
±
2.8 μM), whereas naphthalene containing compound4l was found to be inactive.(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 compounds4m–4z were also evaluated for their antioxidant potentialin vitro in DPPH radical scavenging assay. These compounds were found to be significant to weak inhibitors of free radicals (IC50 = 133.2
±
4.7
–
439.6
±
10.1 μM) except compounds4m–4r and4y which were found to be inactive.Similarlybis(2-hydroxy-4,4-dimethyl-6-oxocyclohex-1-ene) (5a–5h) and 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) (5i–5k) rings containing compounds were also evaluated for their antioxidant potential by using DPPH radical scavenging assay. Para-bromo-substituted phenyl ring containing compound5k(IC50 = 127.6
±
1.6 μM) was found as a potent antioxidant agent against the tested standards BHT (IC50 = 128.8
±
2.1 μM). The compound showed similar antioxidant potential as BHT and showed a significant antioxidant agent againstN-acetyl cysteine (IC50 = 107.6
±
2.8 μM). The gradual and drastic decrease in activity was for compounds5l (IC50 = 169.7
±
5.3 μM),5g (IC50 = 270.4
±
2.3 μM),5e (IC50 = 289
±
0.9 μM), and5a (IC50 = 461.6
±
8.8 μM) having para-methyl-, para-aldehyde-, ortho-chloro-, and ortho-nitro-substituted phenyl rings attached to the central barbiturate moieties. Compounds5b–5d,5f, and5h–5j were found to be inactive.On the basis of the observed antioxidant abilities of the above five different series of pyridinium adducts, it was concluded thatbis(6-hydroxy-1,3-dimethyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidin-5-yl) ring containing compounds4a–4h are the most active one. The activity may be due to the methyl substituted nitrogen atoms of the tetrahydropyrimidine 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 the presence of electron donating (-OH (4f), -OCH3 (4d), and -CH3 (4b,4g)), electron withdrawing (-NO2 (4a) and -CHO (4c)), and halogen (-Br (4e)) substituents on the phenyl ring attached to thebis(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. Cytotoxic Activity

All synthesized diethyl ammonium salts of barbiturates (4a–4z and5a–5m) were evaluated for theirin 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-tetrahydropyrimidin-4-olate ring containing compounds4y, which was found to be significantly toxic against all the cell lines (PC3, IC50 = 10.71
±
0.26 μM, HeLa, IC50 = 3.47
±
0.3 μM, MCF-7, IC50 = 4.9
±
0.01 μM, and 3T3, IC50 = 3.20
±
0.2 μM). Doxorubicin was used as a standard drug to compare the activities against PC3 (IC50 = 1.70
±
0.29 μM), HeLa (IC50 = 0.51
±
0.15 μM), and MCF-3 (IC50 = 0.92
±
0.01 μM) cancer cell lines, while cycloheximide (IC50 = 0.26
±
0.12 μM) was used as a standard-in 3T3 cell line (Table 2).


## 3.3.3.α-Glucosidase Inhibition Activity

Synthesized diethyl ammonium salts of barbiturates,4a–4z and5a–5m, were also evaluated for theirin vitro  α-glucosidase inhibition activity, in comparison to the standard drug, acarbose (IC50 = 841
±
1.73 μM). Among dimethyl ammonium salts (4a–4h), nitro-substituted phenyl ring containing4c (IC50 = 355
±
10 μM) and naphthalene ring containing4h (IC50 = 230
±
3.92 μM) were found to be potent inhibitors of α-glucosidase enzyme and showed more activity than acarbose (IC50 = 841
±
1.73 μM). 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),para-OCH3- (40, IC50 = 300.9
±
6.5 μM),para-chloro- (4p, IC50 = 186.8
±
0.81 μM),para-bromo- (4q, IC50 = 214.8
±
0.72 μM), andmeta-bromo- (4r, IC50 = 201
±
5.5 μM) substituted phenyl ring containing pyridinium adducts were found to be potent α-glucosidase inhibitors and showed more activity than standard acarbose (IC50 = 841
±
1.73 μM). Other members of the series (4m-4n and4s–4z) werefound to be inactive. The results showed the substitution effects of various functionalities of phenyl ring on the potential activity of tested series. Thepara-bromo-substituted phenyl ring containing compound4q (IC50 = 214.8
±
0.72 μM) showed 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 compounds4n,4t,4v, and4x, respectively. The presence of ortho-nitro-,ortho-choloro-, andpara-choloro-substituted phenyl ring also contributed towards inactivity, as observed for compounds4t,4w, and4y.Similarlybis(2-hydroxy-4,4-dimethyl-6-oxocyclohex-1-ene) ring containing diethyl ammonium pyridinium salts (5a–5h) were also evaluated for theirin vitro  α-glucosidase enzyme inhibition activity. The series nitro-substituted phenyl ring containing5fwas found to be the only potent α-glucosidase inhibitor. All other compounds (5a–5e and5g-5h) were found to be inactive.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-dioxo-1,2,3,4-tetrahydropyrimidin-5-yl)methyl) central moiety attached with substituted phenyl ring, the benzaldehyde containing5m (IC50 = 453.4 ± 4.2 μM) was found to be the only potent inhibitor compound in comparison to against the standard drug acarbose (IC50 = 841
±
1.73 μM). All other compounds were found to be inactive. The results are summarized in Table 2.


## 3.3.4. Thymidine Phosphorylase Inhibition Activity

Five different series of diethyamonium salts of barbiturates,4a–4g,4i–4k,4m–4z,5a–5h, and5i–5m, were also evaluated for theirin vitrothymidine phosphorylase inhibition. 7-Deazaxanthine was used as a standard inhibitor (IC50 = 41
±
1.46 μM). Phenol and naphthalene rings 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 moiety containing compounds4v (IC50 = 57.6
±
1.20 μM) and4z(IC50 = 55.8
±
1.50 μM) were found to be the potent thymidine phosphorylase inhibitors. However, a drastic decrease in activity was observed whenpara-hydroxy group on phenyl ring was replaced with methyl (4n, IC50 = 226.9
±
1
.
50 μM), chloro (4n, IC50 = 242
±
1
.
5
μM), bromo (4q, IC50 = 141.3
±
1
.
5 μM), and aldehyde (4x, IC50 = 239
±
2.0 μM) groups, whereas the compounds4o,4s,4t,4w, and4y havepara-methoxy-,ortho-nitro-,ortho-N,N-dimethyl amine-, ortho-, para-dichloro-, andortho-ortho-dichloro-substituted phenyl ring, respectively.Among series of compounds (5a–5h) havingbis(2-hydroxy-4,4-dimethyl-6-oxocyclohex-1-ene) ring as central moiety, electron donating hydroxy- and methoxy-substituted phenyl ring containing compounds5h(IC50 = 56.9
±
1.6 μM) and5d (IC50 = 60.5
±
1.87 μM), respectively, were found as potent inhibitors of thymidine phosphorylase enzyme against the tested standard 7-deazaxanthine which was (IC50 = 41
±
1.46 μM) used as standard. Again a drastic decrease in activity was observed for compounds5b (IC50 = 216.9
±
0.5
μM),5c (IC50 = 161.4
±
1.5 μM), and5g (IC50 = 186.6
±
1.2 μM) havingmeta-methyl-,ortho-methyl-, andpara-aldehyde-substituted benzene ring respectively, instead ofpara-hydroxyl phenyl ring. The replacement ofortho-methyl with chloro-group makes the compound5e completely inactive. Similarly the replacement of electron donatingpara-hydroxy-group of phenyl ring with electron withdrawing nitro-functionality also contributed towards inactivity of compound, as observed for5f. Phenol substitutedbis(2-hydroxy-4,4-dimethyl-6-oxocyclohex-1-ene) ring containing5h (IC50 = 56.9 ± 1.6 μM) was found to be the potent thymidine phosphorylase inhibitors.All members of the 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-dioxo-1,2,3,4-tetrahydropyrimidin-5-yl)methyl) central moiety attached with substituted phenyl ring were found to be weak inhibitors of thymidine phosphorylase enzyme against the tested standard compound 7-deazaxanthine (IC50 = 41
±
1
.
46 μM) with IC50 values 318
±
2.5 μM, 166.4
±
1
.
2 μM, 178.1
±
2
.
1 μM, and 147.1
±
1
.
23 μM for5j,5k,5l, and5m, respectively, whereas para-chloro-substituted phenyl ring containing compound5i was found to be inactive. The results are summarized in Table 1.


## 3.3.5.β-Glucuronidase Inhibition Activity

Compounds4a–4g,4i–4k,4m–4z,5a–5h, and5i–5m were also evaluated for theirin vitro β-glucuronidase inhibitory activity by using D-saccharic acid 1,4-lactone as standard inhibitor (IC50 = 45.75
±
2.16 μM). Compounds4a (IC50 = 204.8
±
6.01 μM),4c (IC50 = 172.1
±
2.1 μM),4e (IC50 = 338.9
±
6.42 μM),4o (IC50 = 262.1
±
4.35 μM),4q (IC50 = 292.2
±
1.03 μM),4r (IC50 = 262.1
±
4.4 μM),4w (IC50 = 143.5
±
1.24 μM),5f (IC50 = 185
±
2.5 μM),5i (IC50 = 223.6
±
6.4 μM), and5l (IC50 = 387.5
±
7.0 μM) showed very weak β-glucuronidase inhibition potential, whereas pyridinium adducts4a–4c,4e,4g–4j,4l,4o,4s-4t,4w,4y,5a,5e-5f, and5i were found to be inactive (Table 2).


## 3.4. Molecular Docking Studies

### 3.4.1. Binding Interactions ofα-Glucosidase Enzyme

To define inhibitors interactions and their specificity, the docking study of the most active compound4p against α-glucosidase 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 α-glucosidase. The 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). The presence of polar and electron rich center in the form of pyrimidinedione and chlorobenzene groups of the compound 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 compound4p were also observed.Figure 4
Three-dimensional representation of compound4p with target macromolecule α-glucosidase.


### 3.4.2. Binding Interactions of Thymidine Phosphorylase Enzyme

To explore the binding modes of these newly synthesized compounds in the active site of thymidine phosphorylase, the most active compound4v was docked against this enzyme. The docking results showed that compound4v well accommodated in the active site of thymidine phosphorylase (Figure 5). The active site residue Ala 206 established a hydrogen bond through its lone pair of 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.Figure 5
Three-dimensional representation of compound4v with target macromolecule thymidine phosphorylase.


### 3.4.3. Binding Interaction ofβ-Glucuronidase Enzyme

The predicted binding mode of most active compound4w against β-glucuronidase showed that a hydrogen bond is formed between the lone pair of oxygen of cyclohexanedione component of the compound and imidazole hydrogen of the important 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 compound4w and the active site residues were also observed. The pyrimidinedione moiety in this case was observed to be passive regarding interactions.Figure 6
Three-dimensional representation of compound4w with target enzyme β-glucuronidase.


## 3.4.1. Binding Interactions ofα-Glucosidase Enzyme

To define inhibitors interactions and their specificity, the docking study of the most active compound4p against α-glucosidase 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 α-glucosidase. The 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). The presence of polar and electron rich center in the form of pyrimidinedione and chlorobenzene groups of the compound 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 compound4p were also observed.Figure 4
Three-dimensional representation of compound4p with target macromolecule α-glucosidase.


## 3.4.2. Binding Interactions of Thymidine Phosphorylase Enzyme

To explore the binding modes of these newly synthesized compounds in the active site of thymidine phosphorylase, the most active compound4v was docked against this enzyme. The docking results showed that compound4v well accommodated in the active site of thymidine phosphorylase (Figure 5). The active site residue Ala 206 established a hydrogen bond through its lone pair of 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.Figure 5
Three-dimensional representation of compound4v with target macromolecule thymidine phosphorylase.


## 3.4.3. Binding Interaction ofβ-Glucuronidase Enzyme

The predicted binding mode of most active compound4w against β-glucuronidase showed that a hydrogen bond is formed between the lone pair of oxygen of cyclohexanedione component of the compound and imidazole hydrogen of the important 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 compound4w and the active site residues were also observed. The pyrimidinedione moiety in this case was observed to be passive regarding interactions.Figure 6
Three-dimensional representation of compound4w with target enzyme β-glucuronidase.


## 4. Conclusions

In conclusion, barbiturate salts were synthesized by one-pot multicomponent reactionsvia tandem Aldol/Michael addition reactions between barbituric acid and dimedone with aldehydes, mediated by aqueous diethylamine. The molecular structures of5a,5d, and5f were deduced by single-crystal X-ray diffraction techniques. All of the synthesized compounds were evaluated for their antioxidant potentialin vitro by using DPPH radical scavenging assay as well as enzyme inhibition activities against α-glucosidase, thymidine phosphorylase, and β-glucuronidase enzymes. Compounds4a–4g (IC50 = 101.8
±
0.8
–
124.4
±
4.4 μM) were found to be the potent antioxidants as compared to the standards, BHT (IC50 = 128.8
±
2.1 μM), andN-acetylcysteine (IC50 = 107.6
±
2.8 μM). Compound4p (IC50 = 186.8
±
8.1 μM) was found to be a potent α-glucosidase inhibitor. Compound4v (IC50 = 8.7
±
1.2 μM) was found to be a potent thymidine 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.



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*Source: 8517243-2016-05-11.xml*