Synthesis and characterisation of thiobarbituric acid enamine derivatives, and evaluation of their α-glucosidase inhibitory and anti-glycation activity

Article abstract of DOI:10.1080/14756366.2020.1737045

A new series of thiobarbituric (thiopyrimidine trione) enamine derivatives and its analogues barbituric acid derivatives was synthesised, characterised, and screen for in vitro evaluation of α-glucosidase enzyme inhibition and anti-glycation activity. This series of compounds were found to inhibit α-glucosidase activity in a reversible mixed-type manner with IC₅₀ between 264.07 ± 1.87 and 448.63 ± 2.46 μM. Molecular docking studies indicated that compounds of 3g, 3i, 3j, and 5 are located close to the active site of α-glucosidase, which may cover the active pocket, thereby inhibiting the binding of the substrate to the enzyme. Thiopyrimidine trione derivatives also inhibited the generation of advanced glycation end-products (AGEs), which cause long-term complications in diabetes. While, compounds 3a–k, 5, and 6 showed significant to moderate anti-glycation activity (IC₅₀ = 31.5 ± 0.81 to 554.76 ± 9.1 μM).

Full text of DOI:10.1080/14756366.2020.1737045

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: https://www.tandfonline.com/loi/ienz20
Synthesis and characterisation of thiobarbituric
acid enamine derivatives, and evaluation of their
α-glucosidase inhibitory and anti-glycation activity
M. Ali, Assem Barakat, Ayman El-Faham, Hessa H. Al-Rasheed, Kholoud
Dahlous, Abdullah Mohammed Al-Majid, Anamika Sharma, Sammer Yousuf,
Mehar Sanam, Zaheer Ul-Haq, M. Iqbal Choudhary, Beatriz G. de la Torre &
Fernando Albericio
To cite this article: M. Ali, Assem Barakat, Ayman El-Faham, Hessa H. Al-Rasheed, Kholoud
Dahlous, Abdullah Mohammed Al-Majid, Anamika Sharma, Sammer Yousuf, Mehar Sanam, Zaheer
Ul-Haq, M. Iqbal Choudhary, Beatriz G. de la Torre & Fernando Albericio (2020) Synthesis and
characterisation of thiobarbituric acid enamine derivatives, and evaluation of their α-glucosidase
inhibitory and anti-glycation activity, Journal of Enzyme Inhibition and Medicinal Chemistry, 35:1,
692-701, DOI: 10.1080/14756366.2020.1737045
To link to this article: https://doi.org/10.1080/14756366.2020.1737045
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JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
https://doi.org/10.1080/14756366.2020.1737045
RESEARCH PAPER
Synthesis and characterisation of thiobarbituric acid enamine derivatives, and
evaluation of their a-glucosidase inhibitory and anti-glycation activity
M. Alia, Assem Barakata,b, Ayman El-Fahama,b, Hessa H. Al-Rasheeda, Kholoud Dahlousa,
Abdullah Mohammed Al-Majida, Anamika Sharmac,d, Sammer Yousufe, Mehar Sanamf,
Zaheer Ul-Haqf, M. Iqbal Choudharye,f, Beatriz G. de la Torred and Fernando Albericioa,c,g
b
aDepartment of Chemistry, College of Science, King Saud University, Riyadh, Saudi Arabia; Department of Chemistry, Faculty of Science,
c
Alexandria University, Ibrahimia, Egypt; Peptide Science Laboratory, School of Chemistry and Physics, University of KwaZulu-Natal, Durban,
d
South Africa; KwaZulu-Natal Research Innovation and Sequencing Platform (KRISP), School of Laboratory Medicine and Medical Sciences,
e
College of Health Sciences, University of KwaZulu-Natal, Durban, South Africa; H.E.J. Research Institute of Chemistry, International Center for
f
Chemical and Biological Sciences, University of Karachi, Karachi, Pakistan; Dr. Panjwani Center for Molecular medicine and Drug Research,
g
International Center for Chemical and Biological Sciences, University of Karachi, Karachi, Pakistan; CIBER-BBN, Networking Centre on
Bioengineering, Biomaterials and Nanomedicine, and Department of Organic Chemistry, University of Barcelona, Barcelona, Spain
ABSTRACT
ARTICLE HISTORY
Received 26 December 2019
Revised 24 February 2020
Accepted 25 February 2020
A new series of thiobarbituric (thiopyrimidine trione) enamine derivatives and its analogues barbituric acid
derivatives was synthesised, characterised, and screen for in vitro evaluation of a-glucosidase enzyme
inhibition and anti-glycation activity. This series of compounds were found to inhibit a-glucosidase activity
in a reversible mixed-type manner with IC50 between 264.07 1.87 and 448.63 2.46 mM. Molecular dock-
ing studies indicated that compounds of 3g, 3i, 3j, and 5 are located close to the active site of a-glucosi-
dase, which may cover the active pocket, thereby inhibiting the binding of the substrate to the enzyme.
Thiopyrimidine trione derivatives also inhibited the generation of advanced glycation end-products (AGEs),
which cause long-term complications in diabetes. While, compounds 3a–k, 5, and 6 showed significant to
moderate anti-glycation activity (IC50 ¼ 31.5 0.81 to 554.76 9.1 mM).
KEYWORDS
Thiopyrimidine trione;
a-glucosidase inhibitor;
antiglycation; molecu-
lar docking
1. Introduction
and liver damage9. Therefore, an increasing interest in exploring
new drug candidates for glycosidase inhibition is needed10–12
.
Diabetes mellitus (DM) is a disease which caused by a breakdown
of carbohydrate metabolism, which plays a significant role in the
development of long-term diabetic complications. According to
the International Diabetes Federation, 693 million people will suf-
fer from this condition by 20451,2. DM can be categorised into
three types: Type I (T1DM); Type II (T2DM); and gestational (GDM).
About 80 ꢀ 90% of all DM patients are Type II (T2DM). Drug treat-
ments of T2DM aim to decrease hepatic glucose production,
enhance insulin action, and boost insulin secretion from b-pancre-
atic cells, or block a-glycosidase enzyme (carbohydrate digestive
enzymes)3–6. Therapeutic in individuals with this disease may lead
to various complications, including kidney disease, disorders of
the nervous system, leg amputation, heart disease and severe ret-
inopathy up to blindness7.
Carbohydrate digestive enzymes are found in the brush border
of the intestine. They catalyse the breaking down long-chain poly-
saccharides into absorbable monosaccharide units. Of these
enzymes, a-glucosidases, which play a key role in the digestion and
absorption of complex carbohydrates, and has emerged as target
to maintain postprandial blood glucose control. a-Glucosidase
inhibitors currently used to treat T2DM include acarbose (Precose),
voglibose, and miglitol8. However, these drugs are associated with
Barbituric acid (BA) derivatives have been reported to have
potential anti-hypertensive13, anti-cancer14, anti-convulsant15, anti-
inflammatory16, anti-psychotic17, and antitumor properties18–21
.
Recently, these derivatives have also been reported as anti-diabetic
agents22. On the other hand, thiobarbituric acid (TBA) analogues
has been described to exert anti-inflammatory16,23, immunotropic24,
anticonvulsant25, and anti-hypnotic25,26, anti-neoplastic27, and anti-
tumor activities28. De Belin et al.29 reported a number of TBA deriv-
atives as inhibitors of hypoxia-inducible factor 1 (HIF-1). Recently,
Barakat et al.30 described the synthesis of a new series of diethy-
lammonium salts of aryl substituted TBA derivatives as a-glycosi-
dase inhibitors. Therefore, given the relevance of TBA derivatives in
medicinal chemistry, the design of new molecules containing the
thiobarbituric moiety is an inspiring goal.
In continuation of our studies on the synthesis of biologically
active compounds22,30,31, herein, we synthesised 1,3-diethylthio-
barbiturate enamine derivatives and evaluated their in vitro a-glu-
cosidase inhibitory and anti-glycation activities. In addition,
molecular docking studies were performed to study the interac-
tions of the compounds with the catalytic site of the enzyme
using acarbose and evaluated their a-glucosidase inhibition cap-
several side effects, such as flatulence, stomach-ache, diarrhoea, acity and the anti-glycation properties.
CONTACT Assem Barakat
ambarakat@ksu.edu.sa; Ayman El-Faham
aelfaham@ksu.edu.sa
Department of Chemistry, College of Science, King Saud
University, Riyadh, Saudi Arabia
Supplemental data for this article can be accessed here.
ß 2020 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.

2
M. ALI ET AL.
Scheme 1. Synthetic route for the synthesis of 3a–k, 4a–d, 5, and 6.
to acarbose (IC50
¼
875.75 2.08 mM) and rutin (IC50
¼
2. Results and discussion
54.59 2.20 mM), as standard tested compounds (Table 1).
2.1. Synthesis of the target compounds
Enamine derivatives 2a32 and 2b33 were prepared by reacting the
The results summarised in Table 1 indicated that all the N,N0-
dimethylbarbituric-based enamine acid derivatives 4a–d were com-
commercially available compounds, 1,3-diethylthiobarbituric acid pletely inactive, while compounds 3a–k, 5, and 6 showed moderate
1a or 1,3-dimethylbarbituric acid 1b with DMF in the presence of to significant activity against protein glycation (IC50 ¼ 554.76 9.1 to
acetic anhydride as solvent for 2 h at 90 ꢁC to afford 2a and 2b,
31.5 0.81 mM). The dimeric moiety of TBA 6 via diaminobenezene
respectively as
a yellow crystalline solid in good yields.
linkage (IC50 ¼ 31.5 0.81 mM, Table 1) was the most protein glyca-
tion inhibitor in this series of compounds, and showed more activity
than the standard rutin (IC50 ¼ 54.59 2.20 mM). While, the dimeric
analogues of BA 5 (IC50 ¼ 554.76 9.1 mM) was the least active.
On the other hand, substituted phenyl with an electron-with-
drawing group such as a chlorine atom (a weak deactivating
group) at the ortho position, showed a better anti-glycation activ-
ity than the same atom at the para position. Therefore, the
change in the position had a remarkable effect on the anti-glyca-
tion activity34 (3 h vs 3f, Table 1). Halogen with a higher atomic
weight and weaker electron-withdrawing effect, such as iodine at
the ortho position, decreased the activity as compared to chlorine
at the same position (3i vs 3 h). This observation could be attrib-
uted to the negative inductive effect35,36. In contrast, a strong
electron-withdrawing group, such as sulphonic acid at ortho pos-
ition, decreased the activity compared to chlorine and iodine in
Compounds 2a32 and 2b33 were reacted with different amines in
ethanol at RT to afford the target products 3a–k and 4a–d,
respectively (Scheme 1) in excellent yields and purities, as
observed from their spectral data. The reaction of 2a (2 equiv.) or
its analogues 2b (2 equiv.) with the commercially available material
4-(aminomethyl)aniline (1 equiv.) under the same conditions
described above gave the dimeric products 6 and 5, respectively
as shown in Scheme 1. The structures of the products
obtained were deduced by 1H- and 13C-NMR spectra
(Supplementary material).
2.2. Biological activity
All the synthesised derivatives of TBA (3a–k), BA (4a–d), and the
dimeric analogues 5 and 6 were evaluated for their capacity to
inhibit a-glucosidase and protein glycation in vitro in comparison the ortho position (3j vs 3 h). Electron-donating group such as

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
3
Table 1. Result of in vitro a-glucosidase enzyme inhibitor and anti-glycation activities.
Anti-glycation assay
a-Glucosidase
Compound
Structure
IC50 SEM (mM)
88.57 0.37b
IC50 SEM (mM)
3a
NA
3b
3c
80.36 0.74b
NA
NA
82.22 4.36b
3d
3e
130.53 3.15b
77.28 0.72b
NA
NA
3f
81.74 1.39b
82.36 5.09b
NA
3g
397.45 0.98a
3h
3i
70.92 1.84b
75.13 0.65b
NA
264.07 1.87a
3j
101.92 1.7b
433.33 2.34a
3k
ND
ND
4a
4b
NA
NA
NA
NA
4c
NA
NA
(continued)

4
M. ALI ET AL.
Table 1. Continued.
Anti-glycation assay
a-Glucosidase
Compound
Structure
IC50 SEM (mM)
IC50 SEM (mM)
4d
NA
NA
5
554.76 9.1b
448.63 2.46a
6
31.5 0.81a
NA
Rutin
Acarbose
54.59 2.20
–
–
875.75 2.08
aSignificant activity.
bModerate activity.
ND: not determined; NA: not active.
methyl (a weak donating group) at the para position yielded were carried out to explore the binding modes of TBA derivatives
slightly better and a moderate activity as compared to the chlor- with a notorious a-glucosidase, such as that of Baker’s yeast
ine at the same position (3e vs 3f). On the other hand, replacing
the 4-methylphenyl 3e by 2-pyridylmethylene 3b or 3-methylpyr-
idyl 3c decreased the anti-glycation activity, and showed a com-
parable activity to compounds 3g and 3f as shown in Table 1.
Compound with pyrimidine benzenesulfonamide 3d moiety
decreased the activity, which is consistent with the result obtained
for 3j with a strong withdrawing group. While, compounds with
2-morpholinoethyl 3a and cyclohexyl 3g moieties showed moder-
ate activity against protein glycation.
The results summarised in Table 1 indicated, once again, that
none of the BA enamine derivatives showed any activity, while 3g,
3i, 3j, and 5 exerted a significant activity against a-glucosidase (IC50
¼ 264.07 1.87 to 448.63 2.46mM). Of the series of compounds,
thiopyrimidine trione derivative with higher atomic weight halogen,
such as iodine at the ortho position, was the most active, exhibiting
3.3-fold higher activity than the standard acarbose. Compounds
with a cyclohexyl ring 3g, sulphonic acid 3j, and the dimeric ana-
logue of BA 5 showed twice the activity of the standard drug. The
rest of the compounds did not show any activity.
(Saccharomyces cerevisiae). We used our previously built homology
model of a-glucosidase from the template (PDB ID: 3A4A)30.
Initially, the 3 D structures of all the ligands were built, proto-
nated, and minimised by means of the MMFF94x force field37, and
using the molecular operating environment (MOE)38 2018.04. All
recently synthesised TBA derivatives and a reference inhibitor
(acarbose) were docked into the active site of the receptor using
the default parameters in MOE. Each complex was visually ana-
lysed for ligand–protein interactions, and their images were pre-
pared using UCSF chimaera software39.
The top ranked conformer of TBA derivatives and standard
(acarbose) were selected based on docking score. The docking
score of the ligands 3g, 3i, 3j, and 5 and acarbose were ꢀ3.081,
ꢀ4.909, ꢀ5.19, ꢀ5.642, and ꢀ4.382, respectively. The docking
study revealed that the acarbose, and all the ligands accommo-
dated into the binding pocket of the C-terminal domain of a-glu-
cosidase. The clustering of standard and synthetic compounds at
the allosteric site of the C-terminal domain is shown in Figure 2.
Acarbose occupied a large cavity in the binding sites of a-glu-
cosidase due to its larger size, as compared to the synthetic com-
pounds. The oxygen functionality of acarbose formed two
hydrogen bonds with the active site residues, Arg212 and Arg439.
Ring structures were involved in the p–p interactions with Phe177,
His239, and Pro309. Moreover, residues Glu276, Glu304, and
Asp349 interacted hydrophobically with the ligand (Figure 3).
The carbonyl oxygen of the thiobarbituric ring of 3g, 3i, and
3j showed hydrogen bond interactions with crucial residue
Finally, the most two active compounds from the series are
shown in Figure 1. In conclusion, this work has demonstrated that
the core of TBA-based enamine derivatives is a privileged struc-
ture for anti-glycation and a-glucosidase inhibition and thus
deserves further investigation.
2.3. Molecular docking studies
Molecular docking provides significant insight into ligand-protein Arg212. Another hydrogen bond was observed between the nitro-
binding modes and mechanisms. Here, molecular docking studies gen atoms of 3g with Thr215. These compounds were further

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
5
Figure 1. Lead compounds 3i and 6 with promising activities.
Figure 2. Binding mode of thiobarbituric acid derivatives into the a-glucosidase binding cavity. For clarity, acarbose is shown in cyan. Compounds 3g, 3i, and 3j are
indicated in pink, and 5 in green. The part of the enzyme in the background is shown as surface model.
Figure 3. Interactions of acarbose with crucial residues of a-glucosidase.
stabilised through p–p interactions with the crucial active site resi- Tyr71. In the case of compound 3i, Phe157 was involved in form-
dues Tyr71, Phe157, and Phe177. Additionally, p–p interactions ing halogen–p interactions. Moreover, hydrophobic interactions
were observed with the thiol ring of 3j through Phe177 and with the active site residues Phe157, Thr215, Leu218, and Arg349

6
M. ALI ET AL.
Figure 4. The predicted binding interactions of compounds 3g, 3i, 3j, and 5 in the active site.
stabilised these compounds. In the case of 5, the 2,4,6-trione ring- position of substituent on phenyl ring (enamine moiety) has great
impact on the biological activity. In this regard, the moderate elec-
tron-withdrawing group, such as a chlorine atom at the ortho pos-
ition 3h showed greater activity compared to the same atom at the
para position 3f. On the other hand, the presence of iodine at the
ortho position decreased activity compared to chlorine in the same
position (3i vs 3h). The strong electron-withdrawing group, such as
sulphonic acid showed decrease in activity compared to weak elec-
tron-donating group like methyl (3e).
The dimeric thiobarbituric derivative 6 showed better anti-gly-
cation activity compared with the standard rutin, while thiobarbi-
turic ortho-iodo-enamine derivative 3i showed a positive effect as
a-glucosidase inhibitor compared to the standard acarbose.
Molecular docking studies indicated that compounds of 3g, 3i,
3j, and 5 are located close to the active site of a-glucosidase,
which may cover the active pocket, thereby inhibiting the binding
of the substrate to the enzyme.
bearing oxygen atom formed hydrogen bonds with His111 and
Arg212. Meanwhile, the amine functionality of the ligand also
formed a hydrogen bond with residue Arg312. The benzene ring
was involved in p–p interactions with Phe157 and Phe300. The
hydrophobic interaction with crucial residue Arg349 also contrib-
uted to the binding of 5 with a-glucosidase. The interaction dia-
grams of all the ligands are shown in Figure 4. The docking
results of 5 were in good agreement with experimental results,
thereby indicating that it could be a good candidate as a-glucosi-
dase inhibitor.
3. Conclusions
Several derivatives of barbitutic and thiobarbituric enamine deriva-
tives were synthesised, characterised, and screened for in vitro evalu-
ation of a-glucosidase enzyme inhibition and anti-glycation activity.
The results reveal that the four monomeric compounds 4a–d derived
from N,N0-dimethylbarbituric enamine derivatives showed no anti-gly-
cation activity, while compounds derived from N,N0-diethylthiobarbi-
turic enamine derivatives 3a–k exhibited moderate activity against
protein glycation with IC50 in the range 70–550 mM. The most potent
anti-glycation activity was showed by the dimeric product from N,N0-
diethylthiobarbituric enamine 6 with an IC50 of 31.5 mM, while the
dimeric analogue of N,N0-dimethylbarbituric enamine 5 showed less
activity with an IC50 of 554.8 mM. The reported series of compounds
were found to inhibit a-glucosidase activity in a reversible mixed-
This work has confirmed that the core of (thio)barbituric-based
enamine derivatives are a privileged structure, because in addition
of the previous described biological activity, they have shown
activity for anti-glycation and a-glucosidase inhibition.
4. Experimental
4.1. General methods
All melting points were determined using Mel-Temp apparatus
and are uncorrected. Thin layer chromatography (TLC) was per-
type manner with IC50 between 264 and 448 mM. The type and formed on silica gel (Kiesel gel G, Merck) and spots were detected

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
7
under UV light at 254 nm. FTIR Spectra were recorded in a KBr 4.2.4. 4-(((1,3-Diethyl-4,6-dioxo-2-thioxotetrahydropyrimidin-5(2H)-
matrix on a Bruker Tensor 37 FTIR spectrophotometer. 1H-NMR
ylidene)methyl)amino)-N-(pyrimidin-2-yl)benzenesulfonamide (3d)
spectra were recorded with a JEOL 400 MHz, 13C-NMR were
recorded using the JEOL spectrophotometers, and the chemical
shifts (d) are given in ppm.
Compound 3d was synthesised from 2a and sulphadiazine follow-
ing the general procedure, affording the product as a yellow pow-
der in 85% yield; mp 204 ꢁC; IR (KBr, cmꢀ1): 3421, 3116, 2958,
2860, 1618, 1591, 1508, 1440; 1H-NMR (DMSO-d6, d, ppm): 12.20
(d, 1H, J ¼ 14.0 Hz, NH), 8.72 (d, 1H, J ¼ 14.0 Hz, NH), 8.52 (d, 1H,
J ¼ 4.4 Hz, CH¼), 8.47 (d, 1H, J ¼ 8.8 Hz, Ar-H), 8.0 (d, 1H, J ¼ 8.8 Hz,
Ar-H), 7.79 (d, 2H, J ¼ 8.8 Hz, Ar-H), 7.10 (m, 1H, Ar-H), 6.57 (d, 2H,
J ¼ 8.8 Hz, Ar-H), 4.42 (m, 4H, 2CH2), 1.21 (m, 6H, 2CH3); 13C-NMR
(DMSO-d6, d, ppm): 178.9, 162.2, 160.5, 158.8, 157.8, 157.3, 154.3,
153.6, 142.4, 130.4, 129.8, 125.4,119.8, 116.1, 112.7, 95.7, 42.9, 42.4,
12.8, 12.7; LC/MS (ESI): 461.53 [M þ 1]þ; Anal. Calcd for
C19H20N6O4S2: C, 49.55; H, 4.38; N, 18.25; Found: C, 49.66; H, 4.50;
N, 18.41.
4.2. General procedure for the synthesis of 3a–k, 4a–d, 5, and 6
A solution of 2a32 or 2b33 (1 equiv.) was mixed with different
amines (1 equiv.) in MeOH (10 ml) and stirred at room tempera-
ture for 10–120 min (TLC 20% EtOAc/n-hexane). The solvent was
evaporated slowly, providing the corresponding solid products in
excellent yields and purities.
4.2.1. 1,3-Diethyl-5-(((2-morpholinoethyl)amino)methylene)-2-thio-
xodihydropyrimidine-4,6(1H,5H)-dione (3a)
4.2.5. 1,3-Diethyl-2-thioxo-5-((p-tolylamino)methylene)dihydropyri-
midine-4,6(1H,5H)-dione (3e)
Compound 3a was synthesised from 2a and 4–(2-aminoethyl)mor-
pholine following the general procedure, affording the product as
a yellow powder in 81% yield; mp 135 ꢁC; IR (KBr, cmꢀ1): 3420,
Compound 3e was synthesised from 2a and 4-methylanline uracil
following the general procedure, affording the product as a yellow
powder in 89% yield; mp 139 ꢁC; IR (KBr, cmꢀ1): 3448, 3215, 3169,
1
2999, 2960, 2908, 2870, 1624, 1591, 1456; H-NMR (CDCl3, d, ppm):
1
2953, 2866, 1595, 1570, 1554, 1476, 1435, 1440; H-NMR (CDCl3, d,
10.60 (brs, 1H, NH), 8.23 (d, 1H, J ¼ 14.8 Hz, CH¼), 4.50 (m, 4H,
2CH2), 3.72 (q, 2H, CH2), 3.54 (m, 4H, 2CH2), 2.60 (m, 2H, CH2), 2.50
(m, 4H, 2CH2), 1.25 (m, 6H, 2CH3); 13C-NMR (CDCl3 d, ppm): 179.1,
163.0, 161.2, 160.6, 93.0, 66.9, 57.6, 53.6, 47.1, 43.0, 42.3, 12.5, 12.4;
LC/MS (ESI): 341.44 [M þ 1]þ; Anal. Calcd for C15H24N4O3S: C, 52.92;
H, 7.11; N, 16.46; Found: C, 53.01; H, 7.25; N, 16.59.
ppm): 12.32 (d, 1H, J ¼ 13.8 Hz, NH), 8.70 (d, 1H, J ¼ 14.0 Hz, CH¼),
7.27 (d, 2H, J ¼ 8.0 Hz, Ar-H), 7.22 (dd, 2H, J ¼ 8.0 Hz, Ar-H), 4.60(m,
4H, 2CH2), 2.36 (s, 3H, CH3), 1.30 (m, 6H, 2CH3); 13C-NMR (CDCl3, d,
ppm): 178.8, 163.2, 160.9, 152.9, 137.3, 135.5, 130.9, 118.2, 94.6,
43.2, 42.5, 12.4, 12.2; LC/MS (ESI): 317.41 [M þ 1]þ; Anal. Calcd for
C16H19N3O2S: C, 60.55; H, 6.03; N, 13.24; Found: C, 60.32; H, 6.00;
N, 13.43.
4.2.2. 1,3-Diethyl-5-(((pyridin-2-ylmethyl)amino)methylene)-2-thio-
xodihydropyrimidine-4,6(1H,5H)-dione (3b)
4.2.6. 5-(((4-Chlorophenyl)amino)methylene)-1,3-diethyl-2-thioxodi-
hydropyrimidine-4,6(1H,5H)-dione (3f)
Compound 3b was synthesised from 2a and 2-picolylamine fol-
lowing the general procedure, affording the product as a pink
powder in 83% yield; mp 154 ꢁC; IR (KBr, cmꢀ1): 3215, 3045, 2958,
2908, 2866, 1614, 1598, 1544,1463; 1H-NMR (CDCl3 d, ppm): 11.01
(brs, 1H, NH), 8.63 (d, 1H, J ¼ 5.2 Hz, Ar-H) , 8.39 (d, 1H, J ¼ 14.0 Hz,
CH¼), 7.72 (t, 1H, J ¼ 8.8 Hz, Ar-H), 7.27 (d, 1H, J ¼ 8.8 Hz, Ar-H),
7.23 (m, 1H, Ar-H), 4.76 (d, 2H, J ¼ 3.6 Hz, CH2), 4.56 (m, 4H, 2CH2),
1.28–124 (m, 6H, J ¼ 16.4 Hz, 2CH3); 13C-NMR (CDCl3 d, ppm):
179.1, 163.0, 161.2, 160.7, 154.1, 150.3, 137.3, 123.5, 121.7, 93.5,
55.1, 43.0, 42.3, 12.5, 12.4; LC/MS (ESI): 319.40 [M þ 1]þ; Anal.
Calcd for C15H18N4O2S: C, 56.59; H, 5.70; N, 17.60; Found: C, 56.72;
H, 5.81; N, 17.78.
Compound 3f was synthesised from 2a and 4-chloroanline follow-
ing the general procedure, affording the product as a yellow pow-
der in 78% yield; mp 215 ꢁC; IR (KBr, cmꢀ1): 3302, 2958, 2908,
1
2866, 1614, 1587, 1545, 1504, 1438, 1409; H-NMR (CDCl3, d, ppm):
12.32 (d, 1H, J ¼ 14.0 Hz, NH), 8.65 (d, 1H, J ¼ 14.0 Hz, CH¼), 7.38
(d, 2H, J ¼ 8.0 Hz, Ar-H), 7.25 (d, 2H, J ¼ 8.8 Hz, Ar-H), 4.55 (m, 4H,
2CH2), 1.30 (t, 6H, J ¼ 7.9 Hz, 2CH3); 13C-NMR (CDCl3, d, ppm):
178.9, 163.2, 160.8, 152.8, 136.6, 132.6, 130.4, 119.4, 95.3, 43.2,
42.5, 12.5, 12.3; LC/MS (ESI): 338.82 [M þ 1]þ; Anal. Calcd for
C15H16ClN3O2S: C, 53.33; H, 4.77; N, 12.44; Found: C, 53.54; H, 4.80;
N, 12.63.
4.2.3. 1,3-Diethyl-5-(((4-methylpyridin-2-yl)amino)methylene)-2-thi- 4.2.7. 5-((Cyclohexylamino)methylene)-1,3-diethyl-2-thioxodihydro-
oxodihydro pyrimidine-4,6(1H,5H)-dione (3c) pyrimidine-4,6(1H,5H)-dione (3g)
Compound 3c was synthesised from 2a and 2-amino-4-picoline Compound 3g was synthesised from 2a and cyclohexylamine fol-
following the general procedure, affording the product as a light lowing the general procedure, affording the product as a white
yellow powder in 87% yield; mp 175 ꢁC; IR (KBr, cmꢀ1) 3215, 3157, powder in 84% yield; mp 105 ꢁC; IR (KBr, cmꢀ1): 3302, 2958, 2908,
1
3045, 2958, 2908, 2866, 1614, 1598, 1544, 1463; H-NMR (CDCl3 d, 2866, 1614, 1587, 1545, 1504, 1438, 1409; 1H-NMR (DMSO-d6, d,
ppm):12.25 (d, 1H, J ¼ 13.2 Hz, NH), 9.40 (d, 1H, J ¼ 13.2 Hz, CH¼), ppm): 10.61 (brs, 1H, NH), 8.25 (d, 1H, J ¼ 15.6 Hz, CH¼), 4.52 (m,
8.27 (d, 1H, J ¼ 5.2 Hz, Ar-H), 6.98 (d, 1H, J ¼ 5.2 Hz, Ar-H), 6.86(s, 4H, 2CH2), 3.40 (m, 1H, CH), 1.98 (m, 2H, CH2), 1.83 (m, 2H, CH2),
1H, Ar-H), 4.55 (m, 4H, 2CH2), 2.38 (s, 3H, CH3), 1.29 (m, 6H, 2CH3); 1.78 (m, 2H, CH2), 1.47 (m, 2H, 2CH2), 1.28 (m, 2H, 2CH3); 13C-NMR
13C-NMR (CDCl3 d, ppm):179.1, 163.3, 160.7, 152.5, 150.7, 149.6, (DMSO-d6, d, ppm): 179.0, 163.2, 161.3, 158.4, 92.6, 59.3, 42.9, 42.3,
149.0, 122.9, 113.6, 95.8, 43.2, 42.5, 21.2, 12.5, 12.4; LC/MS (ESI): 33.5, 24.9, 24.3, 12.5, 12.4; LC/MS (ESI): 310.43 [M þ 1]þ; Anal.
319.40 [M þ 1]þ; Anal. Calcd for C15H18N4O2S: C, 56.59; H, 5.70; N, Calcd for C15H23N3O2S: C, 58.23; H, 7.49; N, 13.58; Found: C, 58.39;
17.60; Found: C, 56.81; H, 5.78; N, 17.79.
H, 7.53; N, 13.78.

8
M. ALI ET AL.
4.2.8. 5-(((2-Chlorophenyl)amino)methylene)-1,3-diethyl-2-thioxodi- 4.2.12. 5-(((4-Chlorophenyl)amino)methylene)-1,3-dimethylpyrimi-
dine-2,4,6(1H,3H,5H)-trione (4a)
hydropyrimidine-4,6(1H,5H)-dione (3 h)
Compound 4a was synthesised from 2b and 4-chloroanline follow-
ing the general procedure, affording the product as a white pow-
der in 87% yield; mp 197 ꢁC; IR (KBr, cmꢀ1): 3302, 2958, 2908,
2866, 1614, 1587, 1545, 1504, 1438, 1409;1H-NMR (CDCl3, d,
ppm):12.00 (d, 1H, J ¼ 13.6 Hz, NH), 8.60 (d, 1H, J ¼ 14.0 Hz, CH¼),
7.36 (d, 2H, J ¼ 8.8 Hz, Ar-H), 7.16 (d, 2H, J ¼ 8.8 Hz, Ar-H), 3.32 (s,
6H, 2CH3); 13C-NMR (CDCl3, d, ppm): 165.1, 162.6, 151.8, 136.8,
132.1, 130.3,119.2, 93.4, 28.1, 27.4; LC/MS (ESI): 294.71 [M þ 1]þ;
Anal. for C13H12ClN3O3; Calcd: C, 53.16; H, 4.12; N, 14.31; Found: C,
53.15; H, 4.12; N, 14.33.
Compound 3h was synthesised from 2a and 2-chloroanline follow-
ing the general procedure, affording the product as a yellow pow-
der in 89% yield; mp 160 ꢁC; IR (KBr, cmꢀ1): 3302, 2958, 2908,
1
2866, 1614, 1587, 1545, 1504, 1438, 1409; H-NMR (CDCl3, d, ppm):
12.62 (d, 1H, J ¼ 13.2 Hz, NH), 8.73 (d, 1H, J ¼ 14.0 Hz, CH¼), 7.49
(m, 2H, Ar-H), 7.39 (t, 1H, J ¼ 7.9 Hz, Ar-H), 7.21 (t, 1H, J ¼ 8.0 Hz,
Ar-H), 4.56 (m, 4H, 2CH2), 1.30(m, 6H, 2CH3); 13C-NMR (CDCl3, d,
ppm): 178.9, 163.0, 160.9, 152.1, 135.3, 130.7, 128.5, 127.3, 125.0,
116.9, 96.1, 43.2, 42.6, 12.5, 12.4; LC/MS (ESI): 338.82 [M þ 1]þ;
Anal. Calcd for C15H16ClN3O2S: C, 53.33; H, 4.77; N, 12.44; Found:
C, 53.53; H, 4.92; N, 12.60.
4.2.13. 5-(((2-Chlorophenyl)amino)methylene)-1,3-dimethylpyrimi-
dine-2,4,6(1H,3H,5H)-trione (4b)
Compound 4b was synthesised from 2b and 2-chloroanline fol-
lowing the general procedure, affording the product as a white
powder in 87% yield; mp 202 ꢁC; IR (KBr, cmꢀ1): 3637, 3423, 3197,
4.2.9. 1,3-Diethyl-5-(((2-iodophenyl)amino)methylene)-2-thioxodi-
hydropyrimidine-4,6(1H,5H)-dione (3i)
Compound 3i was synthesised from 2a and 2-iodoanline following
the general procedure, affording the product as a yellow powder
in 83% yield; mp 175 ꢁC; IR (KBr, cmꢀ1): 3302, 2958, 2908, 2866,
1
2960, 2935, 1583, 1570, 1510, 1462; H-NMR (CDCl3, d, ppm): 12.44
(d, 1H, J ¼ 12.8 Hz, NH), 8.73 (d, 1H, J ¼ 13.2 Hz, CH¼), 7.48 (m, 2H,
Ar-H), 7.36 (t, 1H, J ¼ 7.2 Hz, Ar-H), 7.18 (m, 1H, Ar-H), 3.38 (s, 3H,
CH3), 3.36 (s, 3H, CH3); 13C-NMR (CDCl3, d, ppm): 164.9, 162.7,
151.9, 151.1, 135.4, 130.6, 128.5, 126.9, 124.7, 116.6, 94.4, 28.2,
27.5; LC/MS (ESI): 294.71 [M þ 1]þ; Anal. for C13H12ClN3O3; Calcd:
C, 53.16; H, 4.12; N, 14.31; Found: C, 53.17; H, 4.11; N, 14.29.
1
1614, 1587, 1545, 1504, 1438, 1409; H-NMR (CDCl3, d, ppm): 12.42
(d, 1H, J ¼ 13.2 Hz, NH), 8.67 (d, 1H, J ¼ 14.0 Hz, CH¼), 791 (d, 1H,
J ¼ 7.2 Hz, Ar-H), 7.45 (t, 1H, J ¼ 7.9 Hz, Ar-H), 7.36 (d, 1H,
J ¼ 8.0 Hz, Ar-H), 6.98 (t, 1H, J ¼ 7.2 Hz, Ar-H), 4.53 (m, 4H, 2CH2),
1.30 (m, 6H, 2CH3); 13C-NMR (CDCl3, d, ppm): 179.0, 162.8, 160.9,
153.0, 139.9, 130.0, 128.2,125.1, 117.7, 95.9, 89.9, 43.2, 42.5, 12.6,
12.4; LC/MS (ESI): 430.28 [M þ 1]þ; Anal. Calcd for C15H16IN3O2S: C,
41.97; H, 3.76; N, 9.79; Found: C, 41.88; H, 3.81; N, 10.01.
4.2.14.
5-(((2-Iodophenyl)amino)methylene)-1,3-dimethylpyrimi-
dine-2,4,6(1H,3H,5H)-trione (4c)
Compound 4c was synthesised from 2b and 2-iodoanline follow-
ing the general procedure, affording the product as a white pow-
der in 89% yield; mp 285 ꢁC; IR (KBr, cmꢀ1): 3086, 3053, 2953,
4.2.10. 2-(((1,3-Diethyl-4,6-dioxo-2-thioxotetrahydropyrimidin-5(2H)
-ylidene)methyl) amino)benzenesulfonic acid (3j)
1
2885,1598, 1560, 1516, 1463, 1371; H-NMR (CDCl3, d, ppm): 12.77
(d, 1H, J ¼ 14.8 Hz, NH), 8.55 (d, 1H, J ¼ 14.0 Hz, CH¼), 7.77 (d, 1H,
J ¼ 7.2 Hz, Ar-H), 7.57 (d, 1H, J ¼ 8.0 Hz, Ar-H), 7.46 (t, 1H,
J ¼ 7.6 Hz, Ar-H), 7.26 (t, 1H, J ¼ 7.2 Hz, Ar-H), 3.20 (s, 6H, 2CH3);
13C-NMR (CDCl3, d, ppm):163.3, 162.9, 152.2, 151.9, 138.3, 136.4,
131.3, 128.1, 125.9, 118.5, 93.9, 28.2, 27.6; LC/MS (ESI): 386.16
[M þ 1]þ; Anal. for C13H12IN3O3; Calcd: C, 40.54; H, 3.14; N, 10.91;
Found: C, 40.55; H, 3.15; N, 10.90.
Compound 3j was synthesised from 2a and 2-aminobenzenesul-
fonic acid following the general procedure, affording the product
as a yellow powder in 80% yield; mp 243 ꢁC; IR (KBr, cmꢀ1): 3302,
2958, 2908, 2866, 1614, 1587, 1545, 1504, 1438, 1409; 1H-NMR
(DMSO-d6, d, ppm): 13.01 (d, 1H, J ¼ 14.8 Hz, NH), 8.62 (d, 1H,
J ¼ 14.4 Hz, CH¼), 7.78 (d, 1H, J ¼ 7.9 Hz, Ar-H), 7.64 (d, 1H,
J ¼ 8.0 Hz, Ar-H), 7.49 (t, 1H, J ¼ 8.6 Hz, Ar-H), 7.30 (t, 1H, J ¼ 8.4 Hz,
Ar-H), 4.43 (m, 4H, 2CH2), 1.20 (m, 6H, 2CH3); 13C-NMR (DMSO-d6,
d, ppm): 178.9, 161.1, 160.9, 153.9, 138.6, 135.9, 130.7, 128.1,
124.6, 118.3, 95.3, 42.9, 42.2, 12.8. LC/MS (ESI): 384.44 [M þ 1]þ;
Anal. Calcd for C15H17N3O5S2: C, 46.99; H, 4.47; N, 10.96; Found: C,
47.09; H, 4.53; N, 11.13.
4.2.15. 1,3-Dimethyl-5-((pyridin-2-ylamino)methylene)pyrimidine-
2,4,6(1H,3H,5H)-trione (4d)
Compound 4d was synthesised from 2b and 2-aminopyridine fol-
lowing the general procedure, affording the product as a white
powder in 85% yield; mp 287–290 ꢁC; IR (KBr, cmꢀ1): 3420, 2999,
1
2960, 2908, 2870, 1624, 1591, 1456; H-NMR (CDCl3, d, ppm): 12.11
(brs, 1H, NH), 9.41 (d, 1H, J ¼ 13.2 Hz, CH¼), 8.43 (d, 1H, J ¼ 4.4 Hz,
Ar-H), 7.75 (dd, 1H, J ¼ 8.0, 2.4 Hz, Ar-H), 7.16 (t, 1H, J ¼ 7.6, Hz, Ar-
H), 6.99 (d, 2H, J ¼ 8.0 Hz, Ar-H), 3.36 (s, 6H, 2CH3); 13C-NMR
(CDCl3, d, ppm): 165.3, 162.6, 152.0, 151.3, 149.7, 149.3, 138.9,
121.4, 112.7, 94.3, 28.2, 27.5; LC/MS (ESI): 261.25 [M þ 1]þ; Anal. for
C12H12N4O3; Calcd: C, 55.38; H, 4.65; N, 21.53; Found: C, 55.38; H,
4.64; N, 21.51.
4.2.11. 1,3-diethyl-5-((pyridin-2-ylamino)methylene)-2-thioxodihy-
dropyrimidine-4,6(1H,5H)-dione (3k)
Compound 3k was synthesised from 2a and pyridin-2-amine fol-
lowing the general procedure, affording the product as a yellow
powder in 83% yield; mp 243 ꢁC; IR (KBr, cmꢀ1): 3302, 2958, 2908,
2866, 1614, 1587, 1545, 1504, 1438, 1409; 1H-NMR (DMSO-d6, d,
ppm): 13.03 (d, 1H, J ¼ 14.8 Hz, NH), 8.62 (d, 1H, J ¼ 14.4 Hz, CH¼),
7.76 (d, 1H, J ¼ 7.6 Hz, Ar-H), 7.63 (d, 1H, J ¼ 8.8 Hz, Ar-H), 7.49 (t,
1H, J ¼ 9.6 Hz, Ar-H), 7.28 (t, 1H, J ¼ 8.4 Hz, Ar-H), 4.46 (m, 4H,
4.2.16. 5-(((4-(((1,3-Dimethyl-2,4,6-trioxotetrahydropyrimidin-5(2H)-
2CH2), 1.22 (m, 6H, 2CH3); 13C-NMR (DMSO-d6, d, ppm): 178.4, ylidene)methyl) amino)benzyl)amino)methylene)-1,3-dimethylpyri-
160.5, 159.2, 138.1, 135.6, 130.7, 118.3, 94.6, 42.4, 41.7, 12.2. LC/MS
(ESI): 305.35 [M þ 1]þ; Anal. Calcd for C14H16N4O2S: C, 55.25; H,
5.30; N, 18.41; Found: C, 55.38; H, 5.41; N, 18.59.
midine-2,4,6(1H,3H,5H)-trione (5)
Compound 5 was synthesised from 2b (2 equiv.) and 4-aminoben-
zylamine (1 equiv.) following the general procedure, affording the

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
9
product as a white powder in 90% yield; mp 195 ꢁC; IR (KBr, 4.5. Calculation of inhibitory activity
cmꢀ1): 3302, 2958, 2908, 2866, 1614, 1587, 1545, 1504, 1438,
1409; 1H-NMR (DMSO-d6, d, ppm): 10.48 (brs, 2H, NH), 8.25(d, 2H,
J ¼ 14.0 Hz, 2CH¼), 7.01 (d, 2H, J ¼ 8.0 Hz, Ar-H), 6.55 (d, 2H,
J ¼ 9.0 Hz, Ar-H), 4.50 (d, J ¼ 6.4 Hz, 2H, CH2), 3.12 (s, 12H, 4CH3);
13C-NMR (DMSO-d6, d, ppm): 168.1, 165.5, 160.6, 151.9, 134.4,
118.7, 145.2, 95.3, 28.1, 27.4; LC/MS (ESI): 455.44 [M þ 1]þ; Anal.
Calcd for C21H22N6O6: C, 55.50; H, 4.88; N, 18.49; Found: C, 55.65;
H, 4.93; N, 18.70.
The percentage inhibition of advanced glycation end (AGEs) prod-
ucts formation by the test sample versus control was calculated
using the following formula:
The % inhibition of AGE formation
ꢂꢀ
ꢁꢃ
fluorescence of the test group
¼ 1ꢀ
ꢂ 100%
fluorescence of the control group
Acknowledgements
4.2.17. 5-(((4-(((1,3-Diethyl-4,6-dioxo-2-thioxotetrahydropyrimidin- The authors would like to extend their sincere appreciation to the
Deanship of Scientific Research at King Saud University for provid-
ing funding to this research group (RGP-234, Saudi Arabia).
5(2H)-ylidene)methyl)amino)benzyl)amino)methylene)-1,3-diethyl-
2-thioxodihydropyrimidine-4,6(1H,5H)-dione (6)
Compound 6 was synthesised from 2a (2 equiv.) and 4-aminoben-
zylamine (1 equiv.) following the general procedure, affording the
product as a yellow powder in 86% yield; mp 247 ꢁC; IR (KBr,
cmꢀ1): 3302, 2958, 2908, 2866, 1614, 1587, 1545, 1504, 1438,
Disclosure statement
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to
influence the work reported in this paper.
1
1409; H-NMR (CDCl3, d, ppm): 12.34 (d, 1H, J ¼ 13.6 Hz, NH), 10.77
(m, 1H, NH), 8.68 (d, 1H, J ¼ 14.0 Hz, CH¼), 8.29 (d, 1H, J ¼ 13.6 Hz,
CH¼), 7.39 (m, 4H, Ar-H), 4.65 (d, 2H, J ¼ 6.4 Hz, CH2), 4.46 (m, 8H,
4CH2), 1.35 (m, 12H, 4CH3); 13C-NMR (CDCl3, d, ppm): 179.0, 178.8,
163.3, 163.2, 161.0, 160.7, 160.4, 152.7, 138.4, 133.6, 129.7, 129.6,
118.9, 118.8, 95.4, 93.6, 53.6, 43.2, 43.0, 42.5, 42.4, 12.5, 12.4, 12.3,
12.2; LC/MS (ESI): 543.67 [M þ 1]þ; Anal. Calcd for C25H30N6O4S2: C,
55.33; H, 5.57; N, 15.49; Found: C, 55.54; H, 5.69; N, 15.66.
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