Synthesis of pyrimidine-2,4,6-trione derivatives: Anti-oxidant, anti-cancer, α-glucosidase, β-glucuronidase inhibition and their molecular docking studies

Article abstract of DOI:10.1016/j.bioorg.2016.07.009

This paper describes a facile protocol, efficient, and environmentally benign for the synthesis a series of barbiturate acid substituted at C5 position 3a–o. The desired compounds subjected in vitro for different set of bioassays including against anti-oxidant (DPPH and super oxide scavenger assays), anti-cancer, α-glucosidase and β-glucuronidase inhibitions. Compound 3m (IC₅₀?=?22.9?±?0.5?μM) found to be potent α-glucosidase enzyme inhibitors and showed more activity than standard acarbose (IC₅₀?=?841?±?1.73?μM). Compound 3f (IC₅₀?=?86.9?±?4.33?μM) found to be moderate β-Glucuronidase enzyme inhibitors and showed activity comparatively less than the standard D-saccharic acid 1,4-lactone (IC₅₀?=?45.75?±?2.16?μM). Furthermore, in sillico investigation was carried out to investigate bonding mode of barbiturate acid derivatives.

Full text of DOI:10.1016/j.bioorg.2016.07.009

Accepted Manuscript
Synthesis of pyrimidine-2,4,6-trione derivatives: anti-oxidant, Anti-cancer, α-
glucosidase, ß-glucuronidase inhibition and their molecular docking studies
Assem Barakat, Mohammad Shahidul Islam, Abdullah Mohammed Al-Majid,
Hazem A. Ghabbour, Sammer Yousuf, Mahwish Ashraf, Nimra Naveed Shaikh,
M. Iqbal Choudhary, Ruqaiya Khalil, Zaheer Ul-Haq
PII:
S0045-2068(16)30132-8
DOI:
http://dx.doi.org/10.1016/j.bioorg.2016.07.009
YBIOO 1929
Reference:
Bioorganic Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
13 February 2016
5 July 2016
17 July 2016
Please cite this article as: A. Barakat, M.S. Islam, A.M. Al-Majid, H.A. Ghabbour, S. Yousuf, M. Ashraf, N.N.
Shaikh, M. Iqbal Choudhary, R. Khalil, Z. Ul-Haq, Synthesis of pyrimidine-2,4,6-trione derivatives: anti-oxidant,
Anti-cancer, α-glucosidase, ß-glucuronidase inhibition and their molecular docking studies, Bioorganic
Chemistry (2016), doi: http://dx.doi.org/10.1016/j.bioorg.2016.07.009
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Synthesis of pyrimidine-2,4,6-trione derivatives: anti-
oxidant, Anti-cancer, α-glucosidase, ß-glucuronidase
inhibition and their molecular docking studies
Assem Barakata,b,*, Mohammad Shahidul Islama, Abdullah Mohammed Al-Majida,
Hazem A. Ghabbourc, Sammer Yousuf,d Mahwish Ashraf,d Nimra Naveed Shaikh,d M.
Iqbal Choudharya,d, Ruqaiya Khalile, Zaheer Ul-Haqe
a
b
c
d
e
Department of Chemistry, College of Science, King Saud University, P. O. Box 2455,
Riyadh-11451, Saudi Arabia.
Department of Chemistry, Faculty of Science, Alexandria University, P. O. Box 426,
Ibrahimia, 21321 Alexandria, Egypt.
Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, P.
O. Box 2457, Riyadh 11451, Saudi Arabia.
H.E.J. Research Institute of Chemistry, International Center for Chemical Sciences,
University of Karachi, Karachi 75270, Pakistan.
Dr. Panjwani Center for Molecular Medicine and Drug Research, International Center for
Chemical and Biological Sciences, University of Karachi, Karachi 75270, Pakistan.
* Author to whom correspondence should be addressed; E-Mail: ambarakat@ksu.edu.sa.
Tel.: +966-11467-5884 (A.B.); Fax: +966-11467-5992 (A.B.).
Abstract
This paper describes a facile protocol, efficient, and environmentally benign for the synthesis
a series of barbiturate acid substituted at C5 position 3a-o.The desired compounds subjected

in vitro for different set of bioassays including against anti-oxidant (DPPH and super oxide
scavenger assays), anti-cancer, α-glucosidase and ß-glucuronidase inhibitions. Compound 3m
(IC50 = 22.9 0.5µM) found to be potent α-glucosidase enzyme inhibitors and showed more
activity than standard acarbose (IC50 = 841 1.73 µM). Compound 3f (IC50 = 86.9 4.33µM)
found to be moderate ß-Glucuronidase enzyme inhibitors and showed activity comparatively
less than the standard D-saccharic acid 1,4-lactone (IC50 = 45.75 2.16µM). Furthermore, in
sillico investigation was carried out to investigate bonding mode of barbiturate acid
derivatives.
Keywords: pyrimidine-2,4,6-trione; anti-oxidant; anti-cancer; α-Glucosidase inhibitor; ß-
Glucuronidase inhibitor.
1. Introduction
Type 2 diabetes is a serious chronic health condition with increasing prevalence worldwide.
If untreated, this metabolic condition can lead to a suite of devastating conditions including
blindness, kidney failure, and nerve damage [1]. Furthermore diabetes is associated with an
increased risk of developing cardiovascular diseases [1]. The current scientific evidence
indicates that there may be a multi-faceted link between oxidative stress, inflammation, and
hyperglycemia associated with type 2 diabetes, and obesity [2,3].Considering the chronic
nature of this disease and the limitations of current therapies such as high secondary failure
rates and adverse side effects, there remains a consensus for the need to develop and explore
new therapeutic agents that have the potential to combat this epidemic. Because intestinal α-
glucosidase plays a key role in the digestion and absorption of complex carbohydrates, the
inhibition of this enzyme is a metabolic target for managing diabetes by improving post-
prandial blood glucose control. Commercially available oral α-glucosidase inhibitors, such as
acarbose (Precose®) have been widely marketed and prescribed to treat type 2 diabetes.
These expensive medications, however, are often associated with a variety of undesirable

symptoms as a result of undigested starch into the colon. For instance, bloating, abdominal
discomfort, diarrhea, and flatulence were seen in nearly 20% of patients treated with acarbose
[4]. Consequently, a variety of anti-diabetes agents have been developed and prescribed in an
effort to delay intestinal carbohydrate absorption such as miglitol (Glycet®), voglibose, and
1-Deoxynojirimycin [5,6]. Recently, Barakat et al., have been synthesized and evaluated a
series of barbituric acid derivatives and has been compound 5 most active compound against
NO radical scavenger compared to the standard drug acrabose (Fig. 1) [7], compound 6 and 7
were also discovered as α-glucosidase inhibitor [8,9]. Hence, great interest is currently being
devoted to alternative α-glucosidase inhibitors derived from natural sources and foods that
may be effective, safe, tolerable, and less expensive.
Particularly intriguing is the substituted barbituric acid scaffold at the C5-position has been
found as a skeleton in a potentially bioactive agents. Several examples of the
monoalkylbarbiturate exhibit significant implementation the pharmaceutical field including
immunomodulating, antitumor [10], sedative [11], and anticonvulsant potentials [12]. PNU-
286607(-)-1 for example, was described as antibacterial agent [13], the 5-alkylbarbiturates
were discovered matrix metalloproteinase (MMP) inhibitor [14], and other also have shown
mutant SOD1-dependent protein aggregation [15].On the other hand, it has been shown
different applications in the non linear optical study [16], supramolecular chemistry [17], as
well as dyes manufacturing [18]. Thus, taking in mind the pharmacological function of the 5-
barbituric acid moiety, the construction of molecular analogous containing this synthon is a
very challenging goal. In these regards, a several protocols have been reported the synthesis
of 5-monoalkylbarbiturates [19-25].
In continuation of our studies [26-30], Research was conducted to investigate the effect of
barbituric acid compounds derived on α-glucosidase inhibitory activity and also, anti-oxidant

(DPPH and super oxide scavenger assays), anti-cancer and ß-glucuronidase inhibitions were
investigated.
2. Results and discussion
2.1.Chemistry
The synthetic route to the barbituric acid derivatives substituted at C5-position is depicted in
Scheme 1. Addition of barbituric acid analogous 1 into nitro styrene, 2a-k in water mediated
by NHEt2 as a base gave the target 5-monoalkylbarbiturates 3a-o in good to excellent yield
[30]. The Michael addition reaction of barbiturate anion which act as a Michael donor into
the nitro styrene which is a Michael acceptor furnished a Michael adduct with 2 stereo
generic centers. The 5-monoalkylbarbiturate derivatives were agreed with reported data.
Compounds 3j, 3m-o were identified as a new entities. Furthermore, the molecular structure
of 3j, 3n and 3o were confirmed by X-ray single crystal techniques.
Scheme 1: Synthesis of mono alkyl barbiturate derivatives 3a-o

O
O
O
O
HN
NH
H C
3
CH
O
H C
3
CH
O
3
3
H C
3
CH
O
N
H
N
H
N
H
N
H
3
N
H
N
H
O
O
H
H
O
O
O
Cl
NO
2
Fe
NO
NO
2
2
Cl
Cl
H CO
3
NO
2
Cl
3a
3b
3c
3d
, 90% yield
, 89% yield
, 91% yield
, 92% yield
O
O
O
O
H C
3
CH
HN
NH
3
HN
NH
HN
NH
N
H
N
H
O
O
H
O
O
H
O
H
O
O
O
H
H
H
NO
2
NO
NO
2
2
NO
H C
3
2
Cl
Cl
O N
2
Br
3e
3f
3g
, 92% yield
3h
, 94% yield
, 85% yield
O
, 87% yield
S
O
O
Et
Et
O
N
H
N
H C
3
CH
O
HN
NH
H C
3
CH
O
3
3
N
H
N
N
H
N
H
O
O
O
H
O
H
H
O
H
NO
2
NO
2
F C
3
NO
2
H CO
3
NO
2
Cl
H C
3
3i
3j
3k,
3l
, 96% yield
, 88% yield
, 92% yield
91% yield
O
S
O
H C
3
CH
O
Et
Et
N
H C
3
CH
O
3
3
N
H
N
H
N
H
N
H
N
H
O
O
O
H
O
NO
NO
NO
2
2
2
Cl
Cl
F
Br
3o
3m
3n
, 93% yield
, 84% yield
, 91% yield
2.2.Biological Activity
The promising biological activities of alkyl substituted pyridinium dione adducts prompted us
to investigate newly synthesized compounds having nitroethyl dialkyl pyridinium dione (3a-
3c, 3g, 3j-3o) and nitroethyl pyridinium dione (3e, 3f, 3h-3i) central moieties with
substituted phenyl (3a-3c and 3e-3o) and ferrocene (3d) rings attached to nirtoethyl chain for
their in vitro anti-oxidant (DPPH and superoxide anion radical scavenging) activity. These

pyridinium adducts were also evaluated for their α-glucusidase and β-glucuronidase
inhibition potential. Cytotoxicity against HeLa (cervical cancer), and 3T3 (mouse fibroblast)
cell lines, was also evaluated and promising results were obtained.
2.2.1. Antioxidant activity (DPPH and super oxide anion radical scavenging
activity)
Synthesized pyridinium adduct (3a-3o) were evaluated for their in vitro antioxidant activity
by using DPPH and superoxide anion radical scavenging, assays. Compounds 3a (IC50
=
112.7 7.7 μM) and 3g (IC50 = 116.9 1.95 μM) showed potent DPPH radicals scavenging
ability against standard inhibitor N-acetyl cystiene (IC50 = 105.9 1.2 μM). The activity may
be due to the presence of di chloro (3a) and a bromo (3g) substituents on phenyl ring attached
to central nitroethyl dialkyl pyridinium adduct. Both compounds found to be weak anti-
oxidant agenst against the second tested standard BHA (IC50 = 44.6 0.67 μM). The
pyridinium adducts 3b (IC50 = 342.8 2.02 μM), 3h (IC50 = 229.9 6.2 μM), 3i (IC50 = 345.6
5.6 μM), 3j (IC50 = 136.5 5.12 μM), 3m (IC50 = 137.5 5.6 μM), 3n (IC50 = 176.6 1.01 μM)
and 3o (IC50 = 155.7 1.2 μM), were found to be significant to weak antioxidant agents
against the tested standards N-acetyl cystiene (IC50 = 105.9 1.2 μM) and BHA (IC50 = 44.6
0.67 μM). Whereas compounds 3c-3f found to be inactive in DPPH radial scavenging assay.
All pyridinium adducts (3a-3o) were found to be in active in superoxide anion radicals
scavenging assay except compounds 3g (IC50 = 438.6 3.8 μM), 3j (IC50 = 112.6 0.94 μM),
3m (IC50 = 109.3 1.01 μM) and 3o (IC50 = 143.9 2.8 μM) which showed weak antioxidant
activity against the tested drug quercetin (IC50 = 94.1 1.1 μM). The results are summarized in
Table-1.

2.2.2. α-Glucosidase inhibition assay
Substituted phenyl derivatives of nitroethyl pyridinium dione (3a-3o) were evaluated for their
in vitro α-glucosidase inhibition activity in comparison to α-glucosidase inhibitor drug
acarbose (standard) and interesting results were obtained. Acarbose, used as a standard in this
study, is not particularly suited for the α-glucosidase enzyme. It is a potent inhibitor of many
other enzymes, such as α-amylase, which cleave internally in long chains of α-linked glucose
units. Whereas, the Baker's yeast α-glucosidase studied here cleaves terminally and has a
preference for shorter substrates, such as maltose, and molecules of similar size. Thus a
smaller inhibitor such as 1-deoxynojirimycin (DNT) or miglitol, would give a better
representation of the current state of available inhibitors for this class of enzyme. However it
is expensive to acquire sufficient DNJ to test, we have therefore dicided to use acarbose in
this study.
Compounds 3a (IC50 = 57.6 0.5 μM), 3b (IC50 = 211.5 2.3 μM), 3c (IC50 = 135.2 1.15 μM),
3d (IC50 = 66.3 0.5 μM), 3e (IC50 = 80.8 2.48 μM), 3h (IC50 = 415.4 4.3 μM), 3j (IC50 =
46.3 0.4 μM), 3k (IC50 = 205.5 5.53 μM), 3m (IC50 = 22.9 0.5 μM), and 3o (IC50
=
172.2 0.86 μM), were identified as potent α-glucosidase inhibitors, that showed more activity
than the standard drug acarbose (IC50 = 840 1.73 μM).
All tested compounds can be categorized in three series, having phenyl substituted nitro ethyl
chain containing pyrimidine dione (3d-3f, and 3h-3i), N,N-dimethyl pyrimidine dione (3a-3c,
3g, 3k-3l, and 3n-3o), and N,N-diethyl pyrimidine dione ring (3j and 3m).
Ortho, para di-chloro substituted phenyl ring containing compounds 3a (IC50 = 57.6 0.5
μM), 3e (IC50 = 80.8 2.48 μM) and 3m (IC50 = 22.9 0.5 μM) found to be the most active
member of the aforementioned three series. The decrease in activity due to positional effect

was observed for compound 3c (IC50 = 135.2 1.15 μM) having o,o dichloro substituted
phenyl ring attached to the nitro ethyl chain of N,N-dimethyl pyrimidine dione ring instead of
o,p-dichloro substituted phenyl ring (3a, IC50 = 57.6 0.5 μM). A further decrease in activity
observed for monochloro substituted phenyl ring containing 3k (IC50 = 205.5 5.53 μM) also
supported the initial inference that position and number of chlorine atom on phenyl ring is
directly influence the activity of N,N-dimethyl pyrimidine dione derivatives of tested series.
The positional effect was also observed in bromo substituted phenyl ring containing
compounds 3k and 3o of the same series. The meta bromo phenyl ring containing compound
3o (IC50 = 172.2 0.86 μM) found to be a potent inhibitor whereas 3k fund to be inactive.
Interesting results were obtained for compounds 3b and 3i, the meta methoxy phenyl ring
attached to nitroethyl chain of N,N-dimethyl pyrimidine dion and pyrimidine dione moieties,
respectively. N,N-dimethyl pyrimidine dion containing 3b (IC50 = 211.5 2.3 μM) found to
be a potent inhibitor whereas compound 3i found to be inactive. Similarly compound 3h (IC50
= 415.4 4.3 μM) having toluene substituted nitroethyl pyrimidine dione moiety found to be a
potent inhibitor of α-glucosidase enzyme whereas toluene substituted nitroethyl N,N-dimethyl
pyrimidine dione moiety containing 3l found to be inactive. Results are summarized in Table-
1.
Kinetics studies
Kinetic studies of substituted pyridinium dione inhibitors 3a, 3c, 3d, 3e, 3j, 3k, 3m, and 3o
was carried out. Type of inhibition was identified by using the Line-weaver Burk plots,
respectively secondary replot of Lineweaver-Burk plot and the Dixon plot.
Compounds 3a, 3c, 3d, 3j, 3k, and 3m with Ki values of 45, 62, 55.6, 43, 156, and 19 µM
respectively showed a non-competitive type of inhibition with no change in km, while the
Vmax of the enzyme decreased. Compound 3e with Ki of 14.8 µM showed a competitive type

of inhibition with no change in Vmax and an increase in km. Compound 3o, Ki = 126 µM,
showed un-competitive type of inhibition with decrease in km and Vmax. The standard
inhibitor acarbose showed a competitive inhibition of α-glucosidase enzyme with Ki value of
736 µM as shown in Table 1a.
2.2.3. -Glucuronidase inhibition assay
Series of substituted pyridinium dione compounds 3a-3o, was also evaluated for in vitro β-
glucuronidase inhibitory activity by using D-saccharic acid 1,4-lactone as a standard inhibitor
(IC50 = 45.75 2.16 μM). Compounds 3c (IC50 = 97.89 6.92 μM), 3d (IC50 = 291.9 4.04
μM), 3f (IC50 = 86.9 4.33 μM), 3i (IC50 = 188.2 1.87 μM), 3k (IC50 = 201.9 7.21 μM), and
3m (IC50 = 258.4 9.14 μM), were found to be weak inhibitors, whereas compounds 3a, 3b,
3e, 3g, 3h, 3j, 3l, 3m, 3n and 3o found to be in active. Results are summarized in Table-1.
Kinetics studies
Substituted pyridinium dione compounds 3c, 3d, 3f, 3i, 3k, and 3m inhibited the β-
glucuronidase enzyme in a concentration dependent manner. To identify the mechanism of
inhibition kinetics studies were performed. The type of inhibition were conducted by Line-
weaver Burk plots, secondary replot of Lineweaver-Burk plot, and the Dixon plot.
Compounds 3c and 3m with Ki values 150.3 and 232.18 µM, respectively showed a
competitive inhibition with the change in km, while the Vmax of β-glucuronidase enzyme
remains the same. While compounds 3d and 3f with Ki values 238.7 and 104 µM,
respectively showed a non-competitive inhibition with no change in km values. Compounds
3i and 3k with Ki values 149.68 and 282.89 µM respectively showed a uncompetitive
inhibition in which both km and Vmax of the enzyme decreasesd. The refernce compound D-
saccharic acid 1,4-lactone is a competitive inhibitor of β-glucuronidase with Ki value of 21.5
µM shown in Table 1b.

2.2.4. Cytotoxic activity
Phenyl substituted nitroethyl chain containing pyridinium adducts 3a-3o were also evaluated
for their in vitro cytotoxicity against HeLa (cervical cancer), and 3T3 normal cell lines. All
compounds found to be non-cytotoxic against cervical cancer HeLa cells except compound
3d (IC50 = 21.8 0.97 μM) and 3l (IC50 = 25.9 0.5 μM), which showed weak cytotoxicity
against the tested standard drug Doxorubicine (IC50 = 0.51 0.15 μM). The cytotoxicity may
be due to the presence of ferrocene (3d) and toulene (3l) moieties attached to the nitro ethyl
chain of pyridinium dione adduct.
The cytotoxic effect of compounds (3a-3o) against 3T3 cell line concluded with result that
compounds 3d (IC50 = 15.7 0.06 μM), 3g (IC50 = 26.3 0.5 μM), 3h (IC50 = 23.8 1.1 μM),
3i (IC50 = 25.4 4.4 μM), 3k (IC50 = 25.4 1.6 μM), 3l (IC50 = 12.8 1.7 μM), 3n (IC50 = 16.1
2.4 μM), and 3o (IC50 = 23.9 1.7 μM), showed significant to weak cytotoxcity against the
tested standard cycloheximide (IC50 = 0.26 0.12 μM). Whereas compounds 3a-3c, 3e-3f, 3j
and 3m found to be noncytotoxic in nature against 3T3 cell line (Table-1).
2.3.Computational studies
Molecular recognition is one of the basic features of virtually all the biological phenomena.
In case of a ligand-protein it can be defined as the ability of a ligand and a protein to form a
non-covalent complex. The understanding of this recognition event is a prerequisite for the
rational design of new potential drug molecules.
Molecular docking is one of the most frequently used methods in modern drug design, since,
it can predict, with a substantial degree of accuracy, the conformation of small-molecule
ligands within the appropriate target binding site [31]. In the present study we have taken
advantage of in sillico tools to investigate the molecular basis of inhibition of α-glucosidase
and β-glucoronidase exhibited by the newly synthesized barbituric acid derivatives.
We initiated our studies with the docking simulation of α-glucosidase. Although, α-
glucosidase of baker’s yeast (Saccharomyces cerevisiae) is the most extensively used enzyme

for biological assays, the structural information is still not available [32]. Therefore, docking
simulation has been carried out on structure obtained from homology modelling. For this purpose
SWISS-MODEL [33] was implemented providing yeast isomaltase (PDB 3AJ7) as template (72%
identity and 85 % similarity). Evaluation of Phi/Psi angles and rotamer energy pronounces the
accuracy of our proposed model (Fig.S21-21). Alignment of the two protein presented a root mean
square deviation of 1.3 Ǻ amongst each other.
In order to validate our docking protocol, we challenged the ability of the software to reproduce the
binding pose of ligand identified using crystallography. For this exercise, alpha glucosidase from
sugar beet was selected as previously mentioned by Taha et al. [34]. As previously suggested by
Nakrmura and co-workers, we have also used a truncated model for acarbose [35]. The
RMSD value of the heavy atoms of ligand between the native and simulated structure was
1.33 Ǻ for acarbose (fig 2). The observed error of deviation values underpin the ability of our
protocol to reproduce the experimental binding poses accurately.
The kinetic studies highlighted subtle differences in the inhibitory modes of barbituric acid
derivatives. As mentioned earlier, 3e displayed competitive inhibition, 3m, 3j, 3a, 3d, 3c and
3k presented non-competitive type of inhibition. Whereas, 3o was found to be un-competitive
inhibitor of α-glucosidase. The binding mode of barbituric derivatives were investigated to
have a deeper insight into their mechanism of inhibition. The pocket side identifier module in
MOE software suite was implemented to rationalize various druggable sites in the protein.
Apart from other allosteric sites, active site of the enzyme was identified as a part of a fairly
larger cavity (Fig. S23). We hypothesize the presence of this large cavity as a prime factor
governing different types of inhibition displayed by our ligands. The simulated binding mode
of compounds identifies a number of polar and apolar contacts between 3e and the protein
(Fig. 3). The relative potency of the compound can be attributed partially to the hydrogen
bond between SER144 and ligand. While, the π-cation interaction and π- arene stacking
mediated by HIS 239 and PHE157 respectively might have provided anchorage into the
cavity. The contact is further stabilized by special halogen bond was also found between Cl
and LYS155.
In order to identify the possible allosteric site occupied by the non-competitive inhibitors of
the series, blind docking was carried out. It is noteworthy that with the difference in docking
score compounds bind to more than one allosteric site. However, only the top ranked pose of
each compound having least energy was analyzed (table. S10). The predicted binding site of
3m is close to the catalytic triad, while the other non-competitive inhibitors bind to the

allosteric site distant from the active site. Both the ortho and para chloro substitutions on
benzene were found to be involved in halogen bond with the surrounding residues. The nitro
moiety also displayed hydrogen bond with the ARG312, the later also supported the ligand
establishing apolar contact. The di-chloro -benzene can be seen sandwiched between HIS157
and ARG312 (Fig. 4).
As depicted in fig. 5, 3k forms a halogen bond besides three hydrogen bonds within the
binding cavity. Both rings confers stability to the ligands establishing hydrophobic contacts
with TRP14 and ALA289. The nitro moiety held the ligand in pocket by special bidentate
interaction with TYR292 and GLU393.
In contrast to 3k, other non-competitive inhibitors (3j, 3a and 3c) interact with LYS 15 and
LYS12 forming hydrogen bonds. While 3a and 3c mediate interaction with one of the LYS,
3j displayed hydrogen bond with both LYS12 and LYS15, the distance being 2.9 and 3.2 Ǻ
respectively.
In order to enhance our understanding of binding mode of the series, we carried out short
explicit molecular dynamics simulation of 3m and 3e complexes. Production run revealed
overall stability of the ligand-protein complex. It was observed that the interactions displayed
by 3e remained fairly constant. However, nitro group twisting resulted in the development of
additional hydrophobic and polar contacts.
Figure 6 presents the top ranked binding mode of compound 3f and 3c targeting the active
site of human β-glucoronidase. In case of most active compound 3f, strong hydrogen bonding
network with the residues of active site has been observed. The hydrogen bond between
GLU540; the principle catalytic residue, rationalize the inhibitory potential of the ligand.
Similar to α-glucosidase, the nitro group of ligand display bidentate interaction with TYR
504 and LYS606. A similar bidentate interaction is observed between ASP 207 and ligand.
Moreover, the nitro-phenol ring forms hydrophobic interaction with benzene ring of TYR504
and TYR508. The simulated binding mode of compound 3c depicts the formation of
hydrogen bond between ARG 600 and ligand. The di-chlorine benzene ring of ligand display
hydrophobic contacts with ASP207.
3. Conclusion

We have synthesized a series of 5-monoalkylbarbiturate derivatives via Michael addition
reaction mediated by NHEt2 in water. Simple, efficient and concise protocol offered a
straightforward generation of the designed compounds. The synthesized compounds screened
for different sets of assay and provide promising results against α-glucosidase inhibitor. In
sillico analysis suggest that the formation of several polar and aploar contacts between the
barbituric acid derivatives and human α-glucosidase which in part define the potency of these
compounds. Docking simulation followed by molecular dynamics studies confirmed the
formation of stable hydrogen and halogen bonding between 3m and α-glucosidase. While, the
potency of compound 3f against β-glucoronidase is attributed to hydrogen bonding between
GLU540 along with other hotspot residues.
Acknowledgements
The authors would like to extend their sincere appreciation to the Deanship of Scientific
Research at king Saud University for its funding this Research group NO (RGP -257-1435-
1436).
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Fig.1. Anticancer agents, anti-oxidant bearing pyrimidine-2,4,6-trione moiety and α-
Glucosidase inhibiting drugs available in the market.

Fig. 2 Superimposed images of the native and simulated acarbose. The native structure from
PDB 3AJ4 is presented in tan whereas blue colour shows simulated pose.
Fig. 3 The predicted binding pose of compound 3e and the residues of modelled protein. The
dicloro ring of the ligand can be seen sandwiched in between HIS 329 and PHE157 making
apolar contacts stabilizing the ligand in pocket.

Fig. 4 Compound 3m present a number of polar and apolar contacts with the neighbouring
residue. The flipping of di-cloro benzene allows the formation of special halogen bonds.

Fig. 5 The inhibitory modes of compounds 3a (A), 3k(B), 3c(C) and 3j (D).
Fig. 6 The predicted binding modes of compound 3c and 3f in active site of β-glucoronidase
(PDB :1BHG)

Table 1: Biological activity evaluation of compounds 3a-o
IC50 SEM[µM]
S.
No
Compound
Antioxidant activities
Anticancer/Cytotoxicity Assay
α-Glucosidase
Inhibition
ß-Glucuronidase
Inhibition
DDPH
Super
Oxide
Hela
3T3
1
2
3a
3b
112.7 7.7
342.8 2.02
NA
NA
>30
>30
>30
>30
57.6 0.5
211.5 2.3
NA
NA
3
4
5
6
7
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m
3n
3o
NA
NA
NA
NA
NA
NA
NA
NA
438.6 3.8
NA
NA
112.6 0.94
NA
NA
109.3 1.01
NA
>30
21.819 0.965
>30
>30
15.689 0.066
>30
135.2 1.15
66.3 0.5
80.8 2.48
NA
NA
415.4 4.3
NA
46.3 0.4
205.5 5.53
NA
22.9 0.5
NA
172.2 0.86
Acarbose
840 1.73
97.89 6.92
291.9 4.04
NA
86.9 4.33
NA
NA
188.16 1.87
NA
201.95 7.21
NA
258.4 9.14
NA
>30
NA
NA
NA
NA
NA
25.4 0.5
NA
NA
>30
116.98 1.95
229.9 6.2
345.6 5.6
136.5 5.12
225.6 3.2
179.7 2.42
137.7 5.6
176.6 1.01
155.73 1.17
BHA
26.3 0.5
23.8 1.1
25.4 4.4
NA
25.4 1.6
12.8 1.7
NA
8
9
10
11
12
13
14
15
16.1 2.4
23.9 1.7
Cycloheximide
0.26 0.1
443.9 2.8
Quercetin
94.1 1.1
NA
Doxorubicin
0.506 0..15
NA
STD.
D-Saccharic acid
1-4 lactone
45.75 2.16
(44.6 0.67)
N-acetyl
cystiene
(105.9 1.2)
Table 1a: The Inhibition and Kinetic Parameter of α-Glucosidase Inhibitors
Compound
Km (mM)
0.90
Ki (µM)
45
Type of Inhibition
3a
3c
3d
3e
3j
3k
3m
Non-competitive
Non-competitive
Non-competitive
Competitive
Non-competitive
Non-competitive
Non-competitive
0.99
0.92
0.90
0.99
0.92
0.91
62
55.6
14.8
43
156
19
3o
0.97
0.9
126
736
Un-competitive
Competitive
Standard (Acarbose)

Table 1b: The Inhibition and Kinetic Parameter of β-Glucuronidase Inhibitors
Compound
Km
(mM)
0.27
0.27
0.27
0.27
0.27
0.27
0.22
Ki (µM) Type Of Inhibition
3c
3d
3f
3i
3k
150.3
238.7
104
149.68
282.89
232.18
21.5
Competitive
Noncompetitive
Noncompetitive
Uncompetitive
Uncompetitive
Competitive
3m
Standard : D-
Saccharic acid 1,
4-Lactone
Competitive
Table 2: Summary of important interactions displayed by variable groups of ligand with
residues of C terminal human α-glucosidase (MGAM).
Ligand
name
3m
Residue Name
Distance
Donor angle
Ligand atom
Type of
interaction
Hydrogen
Hydrogen
Pi-stacking
Halogen
Hydrogen
Hydrogen
Hydrogen
Hydrogen
Hydrophobic
Hydrophobic
Hydrophobic
Hydrophobic
Hydrogen
Hydrogen
Hydrogen
Hydrogen
Hydrophobic
Hydrophobic
hydrogen
TRP1388
THR1619
PHE1592
ARG1615
TRP1369
SER1425
ARG1510
THR1586
TYR1251
PHE1559
TYR1251
TRP1355
TRP1369
SER1425
ARG1510
THR1586
TRP1355
PHE1560
TRP1369
THR1586
3.09
3.07
3.70
3.26
2.68
131.10
131.5
O2
O2
165.6
129.6
171.2
Cl
3o
S3j
S3a
O2
O2
O2
O2
3.01
2.27,2.86
2.63
160.8,138.5
122.1
3.40
3.40
3.60
3.36
2.87
2.9
120.6
168.7
139,162
3.13,2.47
O2
O2
2.75
3.5
3.53
3.09
120.3
123.7
117.0
O2
O2
2.77
hydrogen

Graphical abstract
2
2
2
50
50
50
50

Highlights
• A series of pyrimidine-2,4,6-trione scaffold were synthesized.
• X-Ray crystal structure of 3j, 3n and 3o were reported.
• Compound 3m (IC50 = 22.9 0.5µM) was identified as α-glucusidase inhibitor.
• Compound 3f (IC50 = 86.9 4.33µM) was identified as β-glucuronidase inhibitor.
• Molecular docking was carried out to investigate bonding mode of barbiturate acid.