Synthesis, in-vitro α-glucosidase inhibition, antioxidant, in-vivo antidiabetic and molecular docking studies of pyrrolidine-2,5-dione and thiazolidine-2,4-dione derivatives

Article abstract of DOI:10.1016/j.bioorg.2019.103128

α-Glucosidase is considered as a therapeutic target for the treatment of type 2 diabetes mellitus (DM2). In current study, we synthesized pyrrolidine-2,5-dione (succinimide) and thiazolidine-2,4-dione derivatives and evaluated for their ability to inhibit

Full text of DOI:10.1016/j.bioorg.2019.103128

Accepted Manuscript
Synthesis, in-vitro α-glucosidase inhibition, antioxidant, in-vivo antidiabetic
and molecular docking studies of pyrrolidine-2,5-dione and thiazolidine-2,4-
dione derivatives
Fida Hussain, Zeeshan Khan, Muhammad Saeed Jan, Sajjad Ahmad, Ashfaq
Ahmad, Umer Rashid, Farhat Ullah, Muhammad Ayaz, Abdul Sadiq
PII:
S0045-2068(19)30760-6
https://doi.org/10.1016/j.bioorg.2019.103128
103128
DOI:
Article Number:
Reference:
YBIOO 103128
Bioorganic Chemistry
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10 May 2019
4 July 2019
17 July 2019
Please cite this article as: F. Hussain, Z. Khan, M. Saeed Jan, S. Ahmad, A. Ahmad, U. Rashid, F. Ullah, M. Ayaz,
A. Sadiq, Synthesis, in-vitro α-glucosidase inhibition, antioxidant, in-vivo antidiabetic and molecular docking
studies of pyrrolidine-2,5-dione and thiazolidine-2,4-dione derivatives, Bioorganic Chemistry (2019), doi: https://
doi.org/10.1016/j.bioorg.2019.103128
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Synthesis, in-vitro α-glucosidase inhibition, antioxidant, in-vivo
antidiabetic and molecular docking studies of pyrrolidine-2,5-dione
and thiazolidine-2,4-dione derivatives
Fida Hussain1, Zeeshan Khan2, Muhammad Saeed Jan1, Sajjad Ahmad1, Ashfaq
Ahmad1, Umer Rashid*2, Farhat Ullah1, Muhammad Ayaz1, Abdul Sadiq**1
1Department of Pharmacy, Faculty of Biological Sciences, University of Malakand,
Chakdara, 18000 Dir (L), KP, Pakistan.
2Department of Chemistry, COMSATS University Islamabad, Abbottabad Campus,
22060 Abbottabad, Pakistan.
Corresponding author (*): Email:, umerrashid@cuiatd.edu.pk (Umer Rashid)
Contact: Corresponding author (**): Email:, sadiquom@yahoo.com (Abdul Sadiq)
Contact: +92 (0)301-2297 102
1

Abstract
α-Glucosidase is considered as a therapeutic target for the treatment of type 2 diabetes mellitus
(DM2). In current study, we synthesized pyrrolidine-2,5-dione (succinimide) and thiazolidine-2,4-
dione derivatives and evaluated for their ability to inhibit α-Glucosidase. Pyrrolidine-2,5-dione
derivatives (11a-o) showed moderate to poor α-glucosidase inhibition. Compound 11o with the
IC50 value of 28.3 ±0.28 µM emerged as a good inhibitor of α-glucosidase. Thiazolidine-2,4-dione
and dihydropyrimidine (TZD-DHPM) hybrids (22a-c) showed excellent inhibitory activities. The
most active compound 22a displayed IC50 value of 0.98 ±0.008 µM. Other two compounds of this
series also showed activity in low micromolar range. The in-vivo antidiabetic study of three
compounds 11n, 11o and 22a were also determined using alloxan induced diabetes mice model.
Compounds 11o and 22a showed significant hypoglycemic effect compared to the reference drug.
In-vivo acute toxicity study showed the safety of these selected compounds. In-silico docking
studies were carried out to rationalize the in-vitro results. The binding modes and bioassay results
of TZD-DHPM hybrids showed that interactions with important residues appeared significant for
high potency.
Keywords: Pyrrolidine-2,5-dione; succinimide; cyanoacetate; thiazolidine-2,4-dione; α-
Glucosidase
2

Introduction
Diabetes is a growing chronic metabolic disorder. It is characterized by hyperglycemia which can
be detected by elevated blood glucose level. It is associated with long term dysfunction or failure
of various body organs, like kidneys, eyes and heart. Generally, there are two types of diabetes;
type-1 is due to deficiency of insulin production, while type-2 is due to insufficiency of insulin
action or resistance [1-4]. By 2030, the frequency of diabetes is projected to be double with a 69%
increase in developing countries and 20% increase between the grownups in developed countries.
According to the reports, globally 300 million people will be affected with diabetes by 2025 and
90% of the affected people possess type 2 diabetes [5]. Currently, commonly used marketed oral
hypoglycemic agents for type 2 are biguanides, sulfonylureas, thiazolidinediones, meglitinides and
α-glucosidase inhibitors [6-7]. However, all these classes of the drugs are associated with some
serious side or adverse effects such as increase food intake, cardiovascular mortality,
gastrointestinal discomfort and weight gain etc. Traditional plants or their bioactive compounds
are also currently used to treat hyperglycemia through various mechanism of actions [8]. These
herbal remedies are often considered free from side effects.
Among other therapeutic targets, inhibition of the activities of the key hydrolyzing enzymes linked
to type 2 diabetes is considered as a useful therapeutic approach [9-10]. The function of α-
glucosidase, an intestinal cell membrane enzyme, is to hydrolyze polysaccharides. Its competitive
inhibition facilitates the control of the blood sugar level. Hence, α-glucosidase inhibitors (synthetic
or natural) are new class of the drugs that can reduce the type 2 diabetes. Up till now, three α-
glucosidase inhibitors acarbose, miglitol and voglibose are in clinical use for the management of
diabetes are obtained from natural sources [11-13]. Traditional antidiabetic therapies from
medicinal plants can effectively control hyperglycemia. Majority of compounds obtained via
3

natural sources contain flavonoid, phenylpropanoid and terpene ring structures. However, recently
published data revealed that α,β-unsaturated lactone ring with a hydroxy substituent is also
considered important for α-glucosidase inhibition. Recently, a study carried out by Quan et al.
discovered that Momilactones A and B are good inhibitors of α-glucosidase and α-amylase and
can be further explored for antidiabetic therapy [14-15].
In diabetic condition, the electron transport chain and NADPH oxidase are activated producing the
reactive oxygen species (ROS) in the body [13]. In hyperglycemia, the ROS reduce insulin level
which control the gene transcription and ultimately cause apoptosis [14]. Overall, it is obvious that
ROS are produced under hyperglycemic condition which further implicate various disorders [15-
16]. The human immune system is constantly fighting to bring the free radicals to the controlled
level. However, the immune system fails to bring it to the normal level especially under the
condition of excessive production. Therefore, the use of antioxidant in combination with
antidiabetic drug will greatly help the immune system to combat the hyperglycemic condition.
Nitrile (cyano) containing compounds have reported to show a substantial role in therapeutic
drugs. The introduction of nitrile substituent is considered as important strategy to improve the
pharmacokinetic properties, polar and π-π interactions and metabolic stability [20]. Cyanoacetate
derivatives of succinimide (pyrrolidine-2,5-dione, 1) are important building-blocks for many
nitrogen-containing derivatives [21-22]. For asymmetric addition of cyanoacetates, efficient
catalysts are required. Thiazolidinedione (TZD, 2) is a known class of drugs for the management
of a number of diseases including diabetes [23--27]. Antidiabetic drugs rosiglitazone (3) and
pioglitazone (4) are the examples of thiazolidinedione containing drugs [28]. Structures of some
potent TZD core containing α-glucose containing inhibitors (5-6) are shown in Figure 1 [25].
4

Herein, we describe the synthesis of two closely related pyrrolidine-2,5-dione and thiazolidine-
2,4-dione derivatives. These derivatives were then evaluated for in-vitro α-glucosidase inhibition,
in-vivo cytotoxicity and blood glucose lowering and antioxidant potential.
O
S
N
S
O
O
NH
N
N
H
NH
O
O
O
O
1
2
3
O
S
O
O
N
N
NH
S
N
O
O
R
O
S
N
N
O
COOEt
N
O
O
N
4
5
6
H
Figure 1: Structures of pyrrolidine-2,5-dione (1), thiazolidinedione (2), rosiglitazone (3),
pioglitazone (4) and thiazolidinedione containing α-glucosidase inhibitors (5-6)
2. Results and discussion
2.1. Design strategy
Our design strategy involves the syntheses of cyanoacetate derivatives of pyrrolidine-2,5-dione
and
thiazolidine-2,4-dione-dihydropyrimidine (TDZ-DHPM) hybrids.
For the better
understanding of structure activity relationship (SAR), substitution at three different positions (R,
R1 and R2) of pyrrolidine-2,5-dione core was planned (7, Figure 2). For thiazolidine-2,4-dione
derivatives, we selected dihydropyrimidine (DHPM) scaffold (8, Figure 2).
5

R
O
Linker
SAR points
O
N
O
N R
CH3
O
S
N
O
H3C
R1
O
O
O
N
R2
H
O
O
DHPM
TZD
N
8
7
Figure 2: (A) Design strategy for the SAR studies of current research, (B) Generic structure of
thiazolidinedione class of anti-diabetic drugs.
2.2. Chemistry
We planned to use maleimide in this study as an acceptor [29]. Interestingly, both Michael and
cyclo-addition reactions are possible in the reaction of cyanoacetates and maleimides [30]. Both
reactions are straightforward methods to synthesize chiral substituted pyrrolidine-2,5-dione [31-
32]. Different cyanoacetate derivatives (11a-o, Scheme 1) of succinimides were prepared by
adding cyanoacetates to the different N-substituted maleimides in the occurrence of appropriate
organocatalyst, creatinine in combination with potassium hydroxide.
O
O
O
R1
N R
O
Organcatalyst
DCM (1M), rt
R2
N
R2
+
O
N R
O
O
R1
O
N
10
9
11a-o
Scheme 1: Synthesis of cyanoacetate derivatives of succinimides (11a-o)
6

Thiazolidine-2,4-dione derivatives 22a-c were obtained through a multistep protocol as shown in
Schemes 2 and 3. Biginelli products 17a-c were obtained by the reaction of 4-substituted
aldehydes (12-14), 1,3-dimethylurea (15) and ethyl acetoacetate (16) in dimethylformamide
(DMF) in the presence of trimethylsilyl chloride (TMSCl). These compounds were further reacted
with bromine in chloroform to yield 6-bromo-dihydropyrimidine-2-ones (18a-c) (Schemes 2).
Aldehydes 20a-c were synthesized by the reaction of 6-bromoderivatives (18a-c) and 4-
hydroxybenzaldehyde (19) in acetonitrile under basic conditions. These aldehydes undergo
Knoevenagel-type condensation with thiazolidine-2,4-dione (21) in absolute ethanol using
piperidine as base / condensing agent (Schemes 3). The resulting thiazolidine-2,4-dione
derivatives (22a-c) were obtained in fair yields.
R
R
O
O
O
O
TMSCl (4 equiv.)
DMF, rt, 45-47h
CH3
O
H3C
CH3
+
O
N
+
N
N
O
H
H
O
H
N
CH3
12-14
15
16
17a-c
R
R
O
O
Br2 / CHCl3
CH3
N
CH3
O
O
O
N
0oC; 10-12 hr
N
O
N
Br CH3
CH3
17a-c
18a-c
Scheme 2: Synthesis of dihydropyrimidines (17a-c) and their 6-bromo derivatives 18a-c.
7

R
N
R
O
O
O
CH3
O
K2CO3, KI
O
N
HO
CHO
OHC
+
N
CH3
O
N
Acetonitrile, Reflux
H3C
O
19
Br CH3
20a-c
18a-c
R
R
O
O
O
O
S
Piperidine
Abs. EtOH
O
+
N
H
OHC
N
O
CH3
N
O
CH3
S
O
N
N
H3C
H3C
O
O
O
O
N
H
20a-c
21
22a-c
Scheme 3: Synthesis of thizolidine2,4-dione and dihydropyrimidine hybrids (22a-c).
2.3. Inhibition of yeast α-Glucosidase
All the synthesized compounds were tested for their inhibitory potential against yeast α-
glucosidase. Acarbose served as a control drug in this experiment. The IC50 values of compounds
are tabulated in Table 1 and 2. Pyrrolidine-2,5-dione derivatives showed moderate activities.
Apparently, phenyl substituted (R1) and ethoxy (R2) containing compounds showed better
inhibition. Structure activity relationship (SAR) studies revealed that N-phenyl substituted
compounds (11e, 11j and 11o) showed good inhibition. Compound 11o with the IC50 of 28.3 ±0.28
µM emerged as a promising inhibitor of α-glucosidase. Cyclohexyl containing compounds also
showed good inhibition.
The activity results of derivatives of another five membered heterocyclic ring, thiazolidine-2,4-
dione are tabulated in Table 2. The three synthesized hybrids of dihydropyrimidine and
thiazolidine-2,4-dione (22a-c) have shown excellent inhibition of α-glucosidase. Unsubstituted
8

phenyl ring containing DHPM ring exhibited submicromolar activity (IC50 = 0.98 ±0.008 µM).
Other two compounds also showed activity in low micromolar range (Table 2).
Table 1: α-Glucosidase inhibitory potential of the synthesized compounds (11a-o).
O
O
N R
R2
O
R1
O
N
(11a-o)
Compound
R
R1
H
H
H
H
H
H
H
H
H
R2
IC50 (µM) + SEM
213.4 + 2.15
164.2 + 2.39
154.5 + 1.44
77.2 + 1.58
43.5 + 0.89
149.6 + 2.32
148.4 + 2.67
133.8 + 1.99
58.4 + 1.05
51.3 + 1.45
57.3 + 1.08
39.7 + 0.87
33.4 + 0.91
31.9 + 0.19
11a
11b
H
CH3
CH3
CH3
CH3
CH3
11c
11d
11e
11f
CH2CH3
Cyclohexyl
C6H5
CH3
CH2CH3
H
11g
11h
11i
CH2CH3
CH2CH3
CH2CH3
CH2CH3
CH2CH3
CH2CH3
CH2CH3
CH2CH3
CH2CH3
CH3
CH2CH3
Cyclohexyl
C6H5
11j
H
11k
11l
C6H5
H
C6H5
C6H5
C6H5
C6H5
CH3
11m
11n
11o
CH2CH3
Cyclohexyl
C6H5
28.3 + 0.28
10.6 + 0.10
Acarbose (standard drug)
9

Table 2: α-Glucosidase inhibitory potential of the synthesized compounds (22a-c)
R
O
O
N
CH3
O
S
N
H3C
O
O
O
N
H
(22a-c)
IC50 (µM) + SEM
Compound
R
H
0.98 + 0.008
1.09 + 0.05
6.10 + 0.10
10.6 + 0.10
22a
22b
22c
OCH3
Cl
Acarbose (standard drug)
2.4. Antioxidant study of pyrrolidine-2,5-dione derivatives (11a-o)
Apart from α-glucosidase inhibitory potential, all the synthesized compounds were tested for their
antioxidant potential by using 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2, 2'-Azino-Bis-3-
Ethylbenzothiazoline-6-Sulfonic Acid (ABTS) methods. From the pyrrolidine-2,5-dione
derivatives (11a-o) series, only 11c exhibited moderate activity in DPPH method. While, from
TZD-DHPM hybrids (22a-c) series, only chloro-substituted 22c showed moderate activity by
DPPH method. The results of antioxidant assays are shown in Table 3.
10

Table 3: Antioxidant assays of the synthesized pyrrolidine-2,5-dione derivatives (11a-o) and
TZD-DHPM hybrids (22a-c).
DPPH
IC50 (µg/mL)
ABTS
IC50 (µg/mL)
DPPH
IC50
(µg/mL)
ABTS
IC50 (µg/mL)
Compound
11a
Compound
11i
147.81
114.22
92.50
65.69
209.07
272.81
405.62
240.16
229.32
110.43
149.24
221.61
365.73
213.36
296.08
11b
11j
11c
11k
258.62
11d
11l
256.04
535.65
351.23
209.07
71.15
201.30
330.32
337.19
269.77
264.24
230.49
11e
11f
11g
11h
11m
11n
11o
746.48
176.59
272.20
>100
>100
-
-
89.59
0.035
-
22a
22b
22c
12.50
Standard
Ascorbic acid
In-vivo antidiabetic studies
Based on the in-vitro antidiabetic results, we selected compounds 11n, 11o and 22a for the in-vivo
antidiabetic assay. The selected compounds were initially screened for acute toxicity test. The
compounds were administered orally in a dose range of 250-2000 mg/kg body weight. After 3
days, no unusual clinical sign was observed.
The results of in-vivo hypoglycemic study are presented in Table 4. All the three selected
compounds (11n, 11o and 22a) revealed practical values in in-vivo results in comparison to the
hypoglycemic control drug glibenclamide. We tested the compounds in five different doses, i.e.
11

500, 250, 125, 62.5 and 31.25 µg/kg body weight. Among the three compounds, 11o and 22a were
successful in decreasing the blood glucose level better than reference drug glibenclamide.
Table 4: Result of in-vivo antidiabetic results of three most potent compounds.
Treatment
Conc/route
Glucose Level at varying time mmol/L
0 h
2 h
4 h
6 h
8 h
24 h
Group I
Oral/IP
5.23
5.26
5.27
5.30
5.31.
5.33
Normal saline
Group II
Tween 80
Group III
GB*
Oral/IP
Oral/IP
23.1
23.4
23.6
20.3
23.9
17.1
24.1
14.2
24.2
10.6
24.3
8.7**
1 (500 µg) Oral/IP
2(250 µg)
22.1
23.5
22.8
24.0
23.4
20.3
22.0
21.8
23.8
23.1
18.6
21.1
21.1
23.1
22.9
16.7
19.9
20.7
22.8
22.6
13.3
18.8
19.8
22.2
22.1
11.5
10.8
18.4
20.1
21.8
Group IV
Compound 11n
3(125 µg)
4(62.5 µg)
5(31.25 µg)
Group V
1 (500 µg) Oral/IP
2(250 µg)
21.9
20.5
19.4
21.5
23.6
16.6
17.4
18.5
20.7
22.8
13.7
13.7
16.3
20.1
21.7
11.7
11.4
15.9
19.3
21.1
10.2
10.2
15.2
18.5
20.7
7.2
8.1
Compound 11o
3(125 µg)
10.1
11.2
17.2
4(62.5 µg)
5(31.25 µg)
Group VI
1 (500 µg) Oral/IP
2(250 µg)
25.7
25.1
18.9
20.8
21.5
22.8
23.7
17.5
19.7
20.3
17.5
20.6
15.2
17.8
17.3
14.3
18.7
12.8
15.3
16.4
11.1
13.2
11.2
12.3
13.9
7.6
10.3
9.2
8.1
9.3
Compound 22a
3(125 µg)
4(62.5 µg)
5(31.25 µg)
*Control drug Glibenclamide 500 µg/ml. ** Bold values shown are for reference drug and compounds with better results than GB
2.5. Docking studies
We performed docking simulations on our previously reported homology modelled α-glucosidase
[33]. Molecular operating environment (MOE 2016.0802) software package was used for
simulations. The active site of the model comprised of catalytic triad residues: Asp214, Glu276
and Asp349 (Red spheres in Figure 1a). The other important amino acid residues in the active site
12

are shown in yellow spheres (Figure 1). First, we docked the standard acarbose into the binding
site of the homology modelled α-glucosidase. The lowest-energy binding-pose of the standard α-
glucosidase inhibitor acarbose is shown in Figure 1b.
Figure 2: (a) Homology modelled α-glucosidase shown in ribbon rendering. Catalytically
important residues (Asp214, Glu276 and Asp349) are shown as red spheres. While, the other active
site residues are represented as yellow spheres. (b) Three-dimensional (3-D) docking pose of
acarbose into the active site of homology modeled α-glucosidase.
The synthesized compounds were docked into the binding site of the homology modelled α-
glucosidase. All the synthesized pyrrolidine-2,5-dione derivatives showed binding affinities in the
range of -4 kcal/mol to -6 kcal/mol. The binding orientations of the active compound 11o of this
series is shown in Figure 3a. The 3-D interaction plot in Figure 3b shows that Phe157 forms π-π
stacking interactions with the phenyl ring. While, Arg312 forms two hydrogen bond interactions
with carbonyl oxygen and -NH of the pyrrolidine-2,5-dione ring (Figure 3b).
13

Figure 3: a) Three-dimensional (3-D) docking pose of compound 11o into the active site of
homology modeled α-glucosidase. Catalytic triad residues Asp214, Glu276 and Asp349 are shown
in red spheres; (b Close-up depiction of the lowest-energy three-dimensional (3-D) docking
interaction plot of 11o.
The binding modes and bioassay results of DHPM-TZD hybrids (22a-c) show that interactions
with important residues such as Phe157, Asp214, Glu276 and Arg312 appeared significant for
high potency. These larger structures also fitted well in the broad cleft to enhance the binding
affinities. The binding affinities of these three compounds are greater than -9 kcal/mol. The ribbon
diagram and three-dimensional interaction diagram of compounds 22a and 22b is shown in Figure
4a-d. The compounds exhibited not only hydrogen bond and π-π stacking interactions, but also π-
alkyl type of interactions also strengthen the ligand enzyme complex.
14

Figure 4: (a and c) Three-dimensional (3-D) docking pose of compound 15 into the active site of
homology modeled α-glucosidase. Catalytic triad residues Asp214, Glu276 and Asp349 are shown
in red spheres; (b and d) Close-up depiction of the lowest-energy three-dimensional (3-D) docking
pose of 15.
3. Conclusions
The identification of some new pyrrolidine-2,5-dione and thiazolidine-2,4-dione derivatives is
reported herein. Some compounds of pyrrolidine-2,5-dione series showed moderate inhibition.
Apparently, compounds with N-phenyl or C-phenyl substitution (R and R1 respectively) and
ethoxy (R2) showed better inhibition. While, thiazolidine-2,4-dione and dihydropyrimidine (TZD-
DHPM) hybrids (22a-c) showed excellent inhibitory activities. Moreover, we also subjected three
of the most potent compounds (11n, 11o and 22a) to the in-vivo studies. Our selected compounds
15

showed significant hypoglycemic effects better than the standard drug glibenclamide. In the in-
vivo acute toxicity study, no clinical unusual signs were observed. The in-vitro results were
rationalized by using our previously reported homology modelled α-Glucosidase. The binding
modes and bioassay results of active compounds showed that interactions with important residues
such as Phe157, Asp214, Glu276 and Arg312 appeared significant for high potency. The larger
structures also fitted well in the broad cleft to enhance the binding affinities. In future work, we
planned to use medicinal chemistry approaches to design pyrrolidine-2,5-dione conjugates at R2
ester with bioactive scaffolds. The chemical derivatization of thiazolidine-2,4-dione is also
planned.
4. Material and methods
4.1. General
All the reagents and solvents were purchased from standard commercial vendors and were used
1
without any further purification. H and 13C-NMR spectra were recorded in deuterated solvents on
JEOL ECX 400 NMR/100 MHz (for series 1) and Bruker spectrometer at 300 and 75 MHz
respectively (for series 2) using tetramethyl silane (TMS) as internal reference. Chemical shifts
are given in δ scale (ppm). The progress of all the reactions was monitored by TLC on 2.0 x 5.0
cm aluminum sheets pre-coated with silica gel 60F254 with a layer thickness of 0.25 mm (Merck).
LC-MS spectra were obtained using Agilent technologies 1200 series high performance liquid
chromatography comprising of G1315 DAD (diode array detector) and ion trap LCMS G2445D
SL. Final products were analyzed for their purity on Schimadzu system using C18 reversed phase
column and isocratic solvent system of water/methanol/TFA (10:90:1) at room temperature.
16

Biologically screened compounds are > 95 % pure as determined by HPLC. Elemental analyses
were conducted using LECO-932 CHNS Analyzer (LECO Corporation, USA).
4.2. Synthetic procedure for the synthesis of cyanoacetate derivatives of succinimides
To a stirred solution of cyano-acetates (2 mmol) in chloroform, different N-substituted maleimides
(1 mmol), creatinine and 20 mol% KOH were added at room temperature. After the completion of
the reaction (TLC), the reaction was quenched by adding sufficient amount of water (15 ml). The
chloroform portion was separated by using a separating funnel. The separation of the organic layer
was repeated three times (each 15 ml). After separation, the organic layer was dried by low vacuum
using rotary evaporator apparatus. The reaction mixture was then adsorbed at silica gel for loading
into the column for purification [34]. In column chromatography n-hexane and ethyl acetate were
used as solvent. The yield of the final product was calculated from the obtained pure product.
4.2.1. Methyl 2-cyano-2-(2,5-dioxopyrrolidin-3-yl)acetate (11a)
The reaction was completed in 19 hours and the color of the product was white with 92% isolated
1
yield. Rf = 0.43 (DCM/MeOH 5:1). H-NMR (400MHz, CDCl3): δ 8.72 (brs, 1H, NH), 4.66-4.41
(m, 2H, Hα-β), 3.91 (s, 3H, -OMe), 2.77 (dd, 1H, J = 5.93, 16.90 Hz, one proton of CH2 of
pyrrolidinedione ring), 2.52 (dd, 1H, J = 9.15, 16.90 Hz, one proton of CH2 of pyrrolidinedione
ring); 13C-NMR (100MHz, CDCl3): 175.21, 173.69, 164.74, 115.90, 60.38, 51.95, 47.85, 31.25;
HPLC purity = 98.5 %, TR = 6.2 min. Analysis calculated for C8H8N2O4: C, 48.98; H, 4.11; N,
14.28. Found: C, 48.92; H, 4.13; N, 14.30. LC-MS found (m/z) = 197.2 [M+H+].
4.2.2. Methyl 2-cyano-2-(1-methyl-2,5-dioxopyrrolidin-3-yl)acetate (11b)
The reaction was completed 24 hours and the color of the product was white with 92% isolated
1
yield. Rf = 0.48 (DCM/MeOH 5:1). H-NMR (400MHz, CDCl3): δ 4.63-4.37 (m, 2H, Hα-β), 3.97
17

(s, 3H, -OMe), 3.04 (s, 3H, -NCH3), 2.80 (dd,1H, J = 6.90, 16.50 Hz, one proton of CH2 of
pyrrolidinedione ring), 2.44 (dd, 1H, J = 5.50, 16.50 Hz, one proton of CH2 of pyrrolidinedione
ring); 13C-NMR (100MHz, CDCl3): 175.18, 173.11, 163.41, 113.77, 61.21, 51.80, 48.28, 30.74,
25.65; HPLC purity = 97.7 %, TR = 5.5 min. Analysis calculated for C9H10N2O4: C, 51.43; H,
4.80; N, 13.33. Found: C, 51.37; H, 4.81; N, 13.35; O, 30.46; LC-MS (m/z) = 211.2 [M+H+].
4.2.3. Methyl 2-cyano-2-(1-ethyl-2,5-dioxopyrrolidin-3-yl)acetate (11c)
The reaction was completed 21 hours and the color of the product was yellowish with 92% isolated
1
yield. Rf = 0.42 (DCM/MeOH 5:1). H-NMR (400MHz, CDCl3): δ 4.62-4.38 (m, 2H, Hα-β), 3.97
(s, 3H, -OMe), 3.04-3.02 (m, 1H, one proton of NCH2-), 2.87-2.84 (m,1H, one proton of NCH2-),
2.75 (dd, 1H, J = 5.40, 17.20 Hz, one proton of CH2 of pyrrolidinedione ring), 2.44 (dd, 1H, J =
8.30, 17.20 Hz, one proton of CH2 of pyrrolidinedione ring), 1.45(t,3H, J = 4.90 Hz, -NCH2CH3);
13C-NMR (100MHz, Chloroform-D): 174.91, 173.44, 164.07, 114.30, 62.48, 54.03, 47.05, 31.61,
25.73, 16.28; HPLC purity = 98.9 %, TR = 8.3 min. Analysis calculated for C10H12N2O4: C, 53.57;
H, 5.39; N, 12.49. Found: C, 53.61; H, 5.37; N, 12.46. LC-MS (m/z) = 225.3 [M+H+].
4.2.4. Methyl 2-cyano-2-(1-cyclohexyl-2,5-dioxopyrrolidin-3-yl)acetate (11d)
The reaction was completed 32 h and the color of the product was Yellowish with 93% isolated
1
yield. Rf = 0.51 (DCM/MeOH 5:1). H-NMR (400MHz, CDCl3): δ 4.57-4.34 (m,2H, Hα-β), 3.95
(s, 3H, -OMe), 3.08-2.94 (m,1H, NCH), 2.70 (dd,1H, J = 5.70, 16.80 Hz, one proton of CH2 of
pyrrolidinedione ring), 2.50 (dd, 1H, J = 9.10, 16.80 Hz, one proton of CH2 of pyrrolidinedione
ring), 1.98-1.84 (m, 2H, CH2 of cyclohexyl ring), 1.79-1.74 (m, 2H, CH2 of cyclohexyl ring), 1.63-
1.34 (m, 6H, - CH2CH2CH2- of cyclohexyl ring); 13C-NMR (100MHz, CDCl3): 175.48, 173.10,
164.27, 114.21, 62.93, 52.91, 48.12, 30.95, 27.92, 23.80, 23.61, 14.21, 7.73, 7.11; HPLC purity =
18

97.7 %, TR = 10.6 min. Analysis calculated for C14H18N2O4: C, 60.42; H, 6.52; N, 10.07. Found:
C, 60.31; H, 6.54; N, 10.09; LC-MS found for C14H18N2O4 (m/z) = 279.2 [M+H+].
4.2.5. Methyl 2-cyano-2-(2,5-dioxo-1-phenylpyrrolidin-3-yl)acetate (11e)
The reaction was completed 17 hours and the color of the product was white with 92% isolated
1
yield. Rf = 0.41 (DCM/MeOH 5:1). H-NMR (400MHz, CDCl3): δ 7.38-7.32 (m, 2H, ArH), 7.33-
7.24(m, 3H, ArH), 4.57.4.39 (m, 2H, Hα-β), 3.97 (s, 3H, -OMe), 2.78 (dd, 1H, J = 4.70, 17.10 Hz,
one proton of CH2 of pyrrolidinedione ring), 2.50 (dd, 1H, J = 9.10, 17.10 Hz, one proton of CH2
of pyrrolidinedione ring); 13C-NMR (100MHz, CDCl3): 174.83, 172.35, 164.63, 129.62, 129.56,
128.34, 127.84, 64.32, 52.73, 46.81, 31.24; HPLC purity = 95.1 %, TR = 11.8.2 min. Analysis
calculated for C14H12N2O4: C, 61.76; H, 4.44; N, 10.29; Found: C, 61.70; H, 4.46; N, 10.31. LC-
MS found for C14H12N2O4 (m/z) = 273.1 [M+H+].
4.2.6. Ethyl 2-cyano-2-(2,5-dioxopyrrolidin-3-yl)acetate (11f)
The reaction was completed 25 hours and the color of the product was white with 92% isolated
1
yield. Rf = 0.42 (DCM/MeOH 5:1). H-NMR (400MHz, CDCl3): δ 8.77 (brs, 1H, -NH), 4.63-4.33
(m, 4H, 2Hα-β, 2x OCH2), 2.74(dd,1H, J = 5.90, 16.77 Hz, one proton of CH2 of pyrrolidinedione
ring), 2.42 (dd, 1H, J = 4.40, 16.80 Hz, one proton of CH2 of pyrrolidinedione ring), 1.14(t,3H, J
= 3.7 Hz, CH2CH3); 13C-NMR (100MHz, Chloroform-D): 174.88, 172.95, 164.52, 115.90, 60.38,
51.95, 47.85, 31.25; LC-MS found for C9H10N2O4 (m/z) = 211.2 [M+H+]. HPLC purity = 96.2 %,
TR = 5.1 min. Analysis calculated for C9H10N2O4: C, 51.43; H, 4.80; N, 13.33. Found: C, 51.45;
H, 4.82; N, 13.31.
4.2.7. Ethyl 2-cyano-2-(1-methyl-2,5-dioxopyrrolidin-3-yl)acetate (11g)
19

The reaction was completed 29 hours and the color of the product was yellowish with 92% isolated
1
yield. Rf = 0.46 (DCM/MeOH 5:1). H-NMR (400MHz, CDCl3): δ 4.68-4.31 (m, 4H, 2Hα-β, 2x
OCH2), 3.06 (s, 3H, -NCH3), 2.72(dd,1H, J = 5.56, 18.20 Hz, one proton of CH2 of
pyrrolidinedione ring), 2.54(dd,1H, J = 5.30, 18.20 Hz, one proton of CH2 of pyrrolidinedione
ring), 1.11(t,3H, J = 5.60 Hz, -CH2CH3); 13C-NMR (100MHz, Chloroform-D): 175.10, 173.54,
165.95, 114.31, 62.05, 56.23, 50.62, 46.96, 30.95; Analysis calculated for C10H12N2O4: C, 53.57;
H, 5.39; N, 12.49. Found: C, 53.51; H, 5.41; N, 12.51.
4.2.8. Ethyl 2-cyano-2-(1-ethyl-2,5-dioxopyrrolidin-3-yl)acetate (11h)
The reaction was completed 13 hours and the color of the product was white with 94% isolated
1
yield. Rf = 0.53 (DCM/MeOH 5:1). H-NMR (400MHz, CDCl3): δ 4.70-4.35 (m, 4H, 2Hα-β, 2x
OCH2), 3.04-3.02 (m, 1H, NCH2), 2.98-2.92 (m, 1H, NCH2), 2.75(dd, 1H, J = 8.30, 17.20 Hz, one
proton of CH2 of pyrrolidinedione ring), 2.44 (dd,1H, J = 5.40, 17.20 Hz, one proton of CH2 of
pyrrolidinedione ring), 1.45(t,3H, J = 3.60 Hz, NCH2CH3), 1.23(t,3H, J = 3.90 Hz, OCH2CH3);
13C-NMR (100MHz, Chloroform-D): 174.24, 172.21, 165.32, 113.28, 62.64, 57.95, 51.54, 47.32,
26.65, 17.41, 14.52; LC-MS found for C11H14N2O4 (m/z) = 239.1 [M+H+]. Analysis calculated
for C11H14N2O4: C, 55.46; H, 5.92; N, 11.76. Found: C, 55.39; H, 5.94; N, 11.78.
4.2.9. Ethyl 2-cyano-2-(1-cyclohexyl-2,5-dioxopyrrolidin-3-yl)acetate (11i)
1
Rf = 0.49 (n-hexane/ethyl acetate, 4:1). H-NMR (400MHz, Chloroform-D): δ 4.45-4.10 (m, 4H,
(4H, 2Hα-β, 2x OCH2), 3.05-2.98(m, 1H NCH-), 2.74(dd,1H, J = 5.70, 16.80 Hz, one proton of
CH2 of pyrrolidinedione ring), 2.52 (dd, 1H, J = 9.10, 16.80 Hz, one proton of CH2 of
pyrrolidinedione ring), 2.04-1.97 (m, 1H, from CH2 of cyclohexyl ring), 1.96-1.79 (m, 2H, CH2 of
cyclohexyl ring), 1.76-1.40 (m, 7H, CH2 of cyclohexyl ring), 1.38-0.94 (m, 3H, - CH2CH2CH2- of
20

cyclohexyl ring); 13C-NMR (100MHz, CDCl3): 174.17, 172.43, 165.08, 113.63, 60.35, 51.26,
46.36, 31.94, 30.58, 29.69, 29.60, 29.52, 29.36, 14.13, 9.22, 8.54; HPLC purity = 97.3 %, TR =
14.1 min. Analysis calculated for C15H20N2O4: C, 61.63; H, 6.90; N, 9.58. Found: C, 61.70; H,
6.88; N, 9.56. LC-MS found for C15H20N2O4 (m/z) = 293.2 [M+H+].
4.2.10. Ethyl 2-cyano-2-(2,5-dioxo-1-phenylpyrrolidin-3-yl)acetate (11j)
1
Rf = 0.55 (n-hexane/ethylacetate, 4:1). H-NMR (400MHz, CDCl3): δ 7.38-7.31 (m,2H, ArH),
7.27-7.21 (m, 3H, ArH), 4.57-4.32 (m, 2H, 2Hα-β), 4.16 (q, 2H, J = 6.9 Hz, -COCH2), 2.79 (dd,
1H, J = 4.70, 17.10 Hz, one proton of CH2 of pyrrolidinedione ring), 2.50 (dd, 1H, J = 9.10, 17.10
Hz, one proton of CH2 of pyrrolidinedione ring); 1.08 (t,3H, J = 6.7 Hz, -CH2CH3). 13C-NMR
(100MHz, CDCl3): 177.3, 175.4, 165.2, 130.2(2), 125.6, 123.4 (2), 114.7, 62.3, 45.0, 34.6, 31.8,
30.5, 28.1, 15.6, 14.2; LC-MS (m/z) = 286.1 [M+H+]. Analysis calculated for C15H14N2O4: C,
62.93; H, 4.93; N, 9.79. Found: C, 62.88; H, 4.94; N, 9.81.
4.2.11. Ethyl 2-cyano-2-(2,5-dioxopyrrolidin-3-yl)-2-phenylacetate (11k)
1
Rf = 0.47 (n-hexane/ethylacetate, 4:1). H-NMR (400MHz, CDCl3): δ 8.79 (brs, 1H, NH), 7.60-
7.57 (m, 2H, ArH), 7.46-7.41 (m, 3H, ArH), 4.39-4.20 (m, 3H, one proton of CH of
pyrrolidinedione ring and two OCH2CH3), 2.67 (dd, 1H, J = 5.93, 16.40 Hz, one proton of CH2 of
pyrrolidinedione ring), 2.43 (dd, 1H, J = 4.30, 16.30 Hz, one proton of CH2 of pyrrolidinedione
ring), 1.27 (t, 3H, J = 6.90 Hz, OCH2CH3). 13C-NMR (100MHz, CDCl3): 174.72, 173.55, 165.12,
131.05, 129.72, 129.62, 125.89, 115.62, 63.85, 54.56, 47.76, 32.56, 13.52; HPLC purity = 96.8 %,
TR = 13.6 min. Analysis calculated for C15H14N2O4: C, 62.93; H, 4.93; N, 9.79. Found: C, 63.00;
H, 4.91; N, 9.77.
21

4.2.12. Ethyl 2-cyano-2-(1-methyl-2,5-dioxopyrrolidin-3-yl)-2-phenylacetate (11l)
1
Rf = 0.49 (n-hexane/ethylacetate, 4:1). H-NMR (400MHz, CDCl3): δ 7.65-7.60 (m, 2H, ArH),
7.51-7.44 (m, 3H, ArH), 4.40-4.22 (m, 3H, one proton of CH of pyrrolidinedione ring and two
OCH2CH3), 3.04 (s, 3H, NCH3), 2.70 (dd, 1H, J = 8.70, 17.10 Hz, one proton of CH2 of
pyrrolidinedione ring), 2.43 (dd, 1H, J = 4.30, 17.10 Hz, one proton of CH2 of pyrrolidinedione
ring), 1.30(t,3H, J = 7.60 Hz, OEt). 13C-NMR (100MHz, CDCl3): 174.72, 173.55, 165.12, 131.05,
129.72, 129.62, 125.89, 115.62, 63.85, 56.42, 50.12, 47.76, 32.56, 13.52; Analysis calculated for
C16H16N2O4: C, 63.99; H, 5.37; N, 9.33; O, 21.31; Found: C, 63.94; H, 5.36; N, 9.36; O, 21.34.
LCMS (m/z) = 301.2 [M+H+].
4.2.13. Ethyl 2-cyano-2-(1-ethyl-2,5-dioxopyrrolidin-3-yl)-2-phenylacetate (11m)
1
Rf = 0.57 (n-hexane/ethylacetate, 4:1). H-NMR (400MHz, CDCl3): δ 7.60-7.54 (m, 2H, ArH),
7.53-7.43 (m, 3H, ArH), 4.42-4.19 (m, 3H, one proton of CH of pyrrolidinedione ring and two
OCH2CH3), 3.15(q,2H J = 6.8 Hz), 2.66 (dd, 1H, J = 7.6, 15.90 Hz, one proton of CH2 of
pyrrolidinedione ring), 2.38 (dd, 1H, J = 4.70, 15.90 Hz, one proton of CH2 of pyrrolidinedione
ring), 1.32 (t, 3H, J = 6.80 Hz, NCH3), 1.20 (t, 3H, J = 7.30 Hz, OCH2CH3); 13C-NMR (100 MHz,
Chloroform-D): 174.72, 173.55, 165.12, 131.05, 129.72, 129.62, 125.89, 115.62, 63.85, 56.42,
50.12, 47.76, 32.56, 13.52; 13C NMR (100 MHz, CDCl3): 174.32 172.98 165.44 131.65
129.65129.10 126.87 114.98 63.55 56.32 47.65 32.65 14.02 13.57; LC-MS found for
C17H18N2O4 (m/z) = 315.1 [M+H+]. Analysis calculated for C17H18N2O4: C, 64.96; H, 5.77; N,
8.91; O, 20.36; Found: C, 65.01; H, 5.79; N, 8.88; O, 20.32.
22

4.2.14. Ethyl 2-cyano-2-(1-cyclohexyl-2,5-dioxopyrrolidin-3-yl)-2-phenylacetate (11n)
1
Rf = 0.55 (n-hexane/ethylacetate, 6:1). H-NMR (400MHz, CDCl3): δ 7.59-7.56 (m, 2H, ArH),
7.51-7.40 (m, 3H, ArH), 4.42-4.19 (m, 3H, one proton of CH of pyrrolidinedione ring and two
OCH2CH3), 3.12 (m, 1H, NCH), 2.72(dd, J = 8.10, 16.20 Hz, one proton of CH2 of
pyrrolidinedione ring, 1H), 2.48(dd, J = 5.60, 16.20 Hz, one proton of CH2 of pyrrolidinedione
ring, 1H), 2.08-2.05 (m, 2H), 1.99-1.92 (m, 3H, cyclohex ring), 1.79-1.35(m, 5H), 1.21-0.94 (m,
3H, OCH2CH3); 13C-NMR (100MHz, CDCl3): 174.55, 172.97, 164.87, 131.64, 129.98, 129.66,
127.02, 114.94, 62.56, 52.23, 46.66, 30.88, 22.54, 20.55, 16.55, 12.88, 9.88; Analysis calculated
for C21H24N2O4: C, 68.46; H, 6.57; N, 7.60; O, 17.37; Found: C, 68.53; H, 6.55; N, 7.58; O, 17.34.
4.2.15. Ethyl 2-cyano-2-(2,5-dioxo-1-phenylpyrrolidin-3-yl)-2-phenylacetate (11o)
Rf = 0.59 (n-hexane/ethylacetate, 6:1). 1H-NMR (400Mz, CDCl3): δ 7.70-7.61 (m, 2H, ArH), 7.47-
7.34 (m, 6H, ArH), 7.35-7.30 (m, 2H, ArH), 4.46-4.22 (m, 3H, one proton of CH of
pyrrolidinedione ring and two OCH2CH3), 4.82 (dd, 1H, J = 6.0 Hz, 16.40 Hz, one proton of CH2
of pyrrolidinedione ring), 2.52(dd,1H, J = 8.0 Hz, 16.40 Hz, one proton of CH2 of pyrrolidinedione
ring), 1.31(t,3H, J = 6.9 Hz, OCH2CH3); 13C-NMR (100MHz, CDCl3): 174.63, 173.59, 165.41,
129.17, 129.04, 128.90, 128.70, 128.68, 128.65, 128.00, 127.91, 115.07, 64.58, 54.99, 46.63,
31.12, 13.49; Analysis calculated for C21H18N2O4: C, 69.60; H, 5.01; N, 7.73. Found: C, 69.66; H,
5.00; N, 7.71. LC-MS found for C21H18N2O4 (m/z) = 363.1 [M+H+].
23

4.3. Generalmethod for the synthesis of N-substituted dihydropyrimidine-2-
ones (17a-c)
To a solution of 4-substituted aldehydes (20 mmol), ethyl acetoacetate (20 mmol) and 1,3-
dimethyl urea (22 mmol) in DMF, trimethylsilyl chloride was added drop-wise. The solution was
heated for 24 h at 70-80 oC. At complete TLC conversion, the solution was transferred into ice
cold water and stir for further 20-25 min. The resulting precipitates were washed with EtOH.
Finally, the crude product was recrystallized from ethyl acetate-n-hexane.
4.3.1. Ethyl 1,3,6-trimethyl-2-oxo-4-phenyl-1,2,3,4-tetrahydropyrimidine-5-carboxylate
(17a)
1
Yield = 79 %, H-NMR (300MHz, DMSO-d6): δ 7.10 (t,1H, ArH), 6.68-6.61 (m,4H, ArH), 5.04
(s, 1H, CH), 4.00(q, J = 7.2 Hz, -COCH2, 2H), 3.23 (s, 3H, Me), 3.05 (s, 3H, Me), 2.24 (s, 3H,
Me), 1.12 (t, J = 7.2 Hz, -CH2CH3, 3H).
4.3.2. Ethyl 4-(4-methoxyphenyl)-1,3,6-trimethyl-2-oxo-1,2,3,4-tetrahydropyrimidine-5-
carboxylate (17b)
1
Yield = 77.0 %, H-NMR (300MHz, DMSO-d6): δ 6.98 (d, J = 7.8 Hz, ArH, 2H), 6.79 (d, J = 7.8
Hz, ArH, 2H), 5.03(s, CH, 1H), 4.08 (q, J = 6.9 Hz, -COCH2, 2H), 3.84 (s, 3H, OMe), 3.23 (s, 3H,
Me), 3.04 (s, 3H, CH3), 2.25 (s, 3H, Me), 1.13(t, 3H, J = 7.1 Hz, -CH2CH3).
4.3.3. Ethyl 4-(4-chlorophenyl)-1,3,6-trimethyl-2-oxo-1,2,3,4-tetrahydropyrimidine-5-
carboxylate (17c)
Yield = 83.0 %, 1H-NMR (300MHz, DMSO-d6): δ 7.24 (d, J = 8.1 Hz, ArH, 2H), 7.01(d, J = 8.1
Hz, ArH, 2H), 5.02 (s, 1H, CH), 4.06 (q, 2H, J = 5.6Hz, -COCH2), 3.24 (s, 3H, Me), 3.06 (s, 3H,
Me), 2.26 (s, 3H, Me), 1.11 (t, J = 6.9 Hz, -CH2CH3, 3H).
24

4.4. Generalmethod for the synthesis 6-bromo-dihydropyrimidine-2-one
derivatives (18a-c)
For the synthesis of target derivatives (18a-c), we first synthesized 6-bromoderivatives according
to our previously reported method [35].
To a suspension of N-substituted DHPMs (17a-c) in chloroform, bromine was added dropwise
and then stirred with TLC monitoring time to time. When the reaction completed, the solvent
was evaporated and was used without purification.
4.5. Generalmethod for the synthesis dihydropyrimidine-2-one derivatives
(20a-c)
A solution of 4-hydroxybenzaldehyde (10 mmol), 6-bromo DHPMs (10 mmol), K2CO3 and few
crystals of KI were refluxed in acetonitrile for 4-6 h. The reaction completed and then the solvent
was evaporated. The product was suspended in water and extracted with ethyl acetate (2 x 20 mL).
Finally, the product was recyrstlized from DMF/EtOH.
4.5.1. Ethyl 6-((4-formylphenoxy)methyl)-1,3-dimethyl-2-oxo-4-phenyl-1,2,3,4-
tetrahydropyrimidine-5-carboxylate (20a)
1
Yield = 71.3 %, H-NMR (300MHz, DMSO-d6): δ 9.86(s, 1H, -CHO), 7.90 (d, J = 8.1 Hz, ArH,
2H), 7.13 (d, J = 8.4 Hz, ArH, 2H), 7.01-6.87 (m, 5H, Ar-H), 5.12 (s, 1H, CH), 4.90 (s, 2H, OCH2),
3.98 (q, J = 6.9 Hz, -COCH2, 2H), 3.23 (s, 3H, Me), 3.04 (s, 3H, Me), 1.10 (t, J = 7.2 Hz, -CH2CH3,
3H).
25

4.5.2. Ethyl 6-((4-formylphenoxy)methyl)-4-(4-methoxyphenyl)-1,3-dimethyl-2-oxo-1,2,3,4-
tetrahydropyrimidine-5-carboxylate (20b)
1
Yield = 68.7 %, H-NMR (300MHz, DMSO-d6): δ 9.88 (s, 1H, -CHO), 7.91(d, J = 6.7 Hz, ArH,
2H), 7.15(d, J = 6.5 Hz, ArH, 2H), 6.96 (d, J = 7.8 Hz, ArH, 2H), 6.80(d, J = 5.9 Hz, ArH, 2H),
5.13 (s, 1H, CH), 4.87 (s, 2H, OCH2), 4.06 (q, J = 7.0 Hz, -COCH2, 2H), 3.82 (s,3H, OCH3), 3.23
(s, 3H, Me), 3.04 (s, 3H, CH3), 1.13 (t, 3H, J = 6.9 Hz, -CH2CH3).
4.5.3. Ethyl 4-(4-chlorophenyl)-6-((4-formylphenoxy)methyl)-1,3-dimethyl-2-oxo-1,2,3,4-
tetrahydropyrimidine-5-carboxylate (20c)
1
Yield = 74.1 %, H-NMR (300MHz, DMSO-d6): δ 9.79 (s,1H, -CHO), 7.91 (d, J = 7.6 Hz, ArH,
2H), 7.25 (d, J = 7.6 Hz, ArH, 2H), 7.14 (d, J = 6.9 Hz, ArH, 2H), 7.02 (d, J = 7.0 Hz, ArH, 2H),
5.14 (s, 1H, CH), 4.89 (s, 2H, OCH2), 3.98 (q, J = 6.9 Hz, -COCH2, 2H), 3.20 (s, 3H, Me), 3.00
(s, 3H, Me), 1.11(t, J = 6.9 Hz, -CH2CH3, 3H).
4.6. Generalmethod for the synthesis thiazolidine-2,4-dione derivatives (22a-
c)
To a hot solution of DHPM aldehyde (5 mmol) in 20 mL of absolute ethanol, thiazolidine-2,4-
dione and few drops of piperidine were added. The mixture was refluxed for 24 h. The resulting
precipitates formed were recrystallized from DMF/EtOH.
4.6.1. Ethyl 6-((4-((2,4-dioxothiazolidin-5-ylidene)methyl)phenoxy)methyl)-1,3-dimethyl-2-
oxo-4-phenyl-1,2,3,4-tetrahydropyrimidine-5-carboxylate (22a)
1
Yield = 59.9 %, H-NMR (300MHz, DMSO-d6): δ 12.57 (s, 1H, CONHCO), 7.72 (s, 1H, CH),
7.18 (d, 2H, J = 8.4 Hz, ArH), 7.10-6.95 (m, 5H, ArH), 6.72 (d, 2H, J = 8.4 Hz, ArH), 5.12 (s, 1H,
CH), 4.85 (dd, 1H, J = 9.9 Hz, 6 Hz, OCH2), 4.02 (q, J = 7.0 Hz, -COCH2, 2H), 3.21 (s, 3H, Me),
26

3.03 (s, 3H, Me), 1.10 (t, J = 6.9 Hz, -CH2CH3, 3H). 13C-NMR (75 MHz, DMSO-d6): δ 170.4,
169.3, 168.1, 161.0, 151.3, 145.1, 144.8, 139.1, 129.1 (2), 128.6 (2), 128.2 (2), 127.8, 127.1, 116.7,
115.3 (2), 100.5, 73.1, 68.9, 59.6, 35.4, 34.3, 14.6. HPLC purity = 98.4 %, TR = 17.0 min. LC-MS
found for C26H25N3O6S (m/z) = 508.2 [M+H+]. Analysis calculated for C26H25N3O6S: C, 61.53;
H, 4.96; N, 8.28; S, 6.32; Found: C, 61.45; H, 4.94; N, 8.31; S, 6.35.
4.6.2. Ethyl 6-((4-((2,4-dioxothiazolidin-5-ylidene)methyl)phenoxy)methyl)-4-(4-methoxy-
phenyl)-1,3-dimethyl-2-oxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate (22b)
Yield =63.2%, 1H-NMR (300MHz, DMSO-d6): δ 12.55 (s, 1H, CONHCO), 7.73 (s, 1H, CH), 7.16
(d, J = 7.8 Hz, ArH, 2H), 6.98 (d, J = 7.5 Hz, ArH, 2H), 6.85 (d, J = 7.8 Hz, ArH, 2H), 6.73 (d, J
= 7.7 Hz, ArH, 2H), 5.11 (s, 1H, CH), 4.93 (d, 1H, J = 9.9 Hz, OCH2), 4.78 (d, 1H, J = 9.9 Hz,
OCH2), 4.01 (q, J = 7.0 Hz, -COCH2, 2H), 3.84 (s, 3H, OMe), 3.24 (s, 3H, Me), 3.02(s, 3H, Me),
1.12(t, J = 7.3 Hz, -CH2CH3, 3H). 13C-NMR (75MHz, DMSO-d6): δ 170.3, 169.3, 168.0, 161.3,
158.9, 151.5, 145.2, 144.7, 132.2, 130.1 (2), 128.3 (2), 127.8 (2), 116.8, 115.3 (2), 114.5 (2),100.4,
72.9, 69.0, 59.7, 56.2, 35.4, 34.2, 14.7. HPLC purity = 99.4 %, TR = 16.5 min. LC-MS found for
C27H27N3O7S (m/z) = 538.1 [M+H+]. Analysis calculated for C27H27N3O7S: C, 60.32; H, 5.06; N,
7.82; S, 5.96; Found: C, 60.41; H, 5.04; N, 7.79; S, 5.99.
4.6.3.
Ethyl
4-(4-chlorophenyl)-6-((4-((2,4-dioxothiazolidin-5-ylidene)methyl)phenoxy)
methyl)-1,3-dimethyl-2-oxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate (22c)
Yield = 67.4%, 1H-NMR (300MHz, DMSO-d6): δ 12.51 (s, 1H, CONHCO), 7.75 (s, 1H, CH), 7.31
(d, J = 7.5 Hz, ArH, 2H), 7.16(d, J = 609 Hz, ArH, 2H), 7.01(d, J = 6.9 Hz, ArH, 2H), 6.77(d, J =
7.1 Hz, ArH, 2H), 5.15 (s, 1H, CH), 4.92 (d, 1H, J = 9.3 Hz, OCH2), 4.79 (d, 1H, J = 9.3 Hz,
OCH2), 3.97 (q, J = 6.9 Hz, -COCH2, 2H), 3.20 (s, 3H, Me), 3.01 (s, 3H, CH3), 1.12 (t, 3H, J = 6.9
Hz, -CH2CH3). 13C-NMR (75 MHz, DMSO-d6): δ 170.2, 169.1, 168.2, 161.2, 151.4, 145.2, 144.6,
27

137.5, 134.1, 129.6 (2), 129.0 (2), 127.5, 127.0 (2), 116.6, 115.0 (2), 100.3, 73.0, 68.8, 60.0, 35.5,
34.3, 14.5. HPLC purity = 98.3%, TR = 19.6 min. LC-MS found for C26H24ClN3O6S (m/z) = 542.1
[M+H+]. Analysis calculated for C26H24ClN3O6S: C, 57.62; H, 4.46; N, 7.75; S, 5.92; Found: C,
57.70; H, 4.44; N, 7.74; S, 5.95.
4.7. In vitro α-Glucosidase inhibition study of synthesized compounds
The antidiabetic role of synthesized compounds was established by using α glucosidase inhibition
assay. Briefly the reaction mixture was comprised of 200 µL α-glucosidase solution, 1200 µL
phosphate buffer and 100 µL of various concentrations ranging from 62.5 to 1000 µg/ml of
synthesized compounds. Allowed to stand for 20 min at 37 centigrade. Added 800 µL of sodium
carbonate solution to the reaction mixture after cooling. Checked absorption of the reaction
mixture at 405 nm using UV visible spectrophotometer. Glucopyranoside was used as standard in
the experiment. The given formula was used to find out the percent inhibition of the αglucosidase
enzyme:
% Inhibition = Abs of Control – Abs of sample x 100
Abs of control
4.7. Antioxidant assays of the compounds
4.7.1. DPPH assay
The DPPH (1,1-diphenyl-2-picrylhydrazyl) assay was used to determine the antioxidant assay.
Serial dilutions of the compounds were prepared, i.e. 1000, 500, 250, 125, 62.5, 31.25, 15.60 and
7.80 µg/ml. The compounds' solution (0.1 ml) was added to the DPPH solution. After half an hour,
the absorbance was determined with double beam spectrophotometer at 517 nm. Ascorbic acid
28

was used as a positive control as per the protocols [36]. The percent DPPH activity was calculated
by the given formula;
Control absorbance − Sample absorbance
Percent DPPH activity =
× 100
Control absorbance
4.7.2. ABTS assay
The antioxidant activity was also determined with ABTS free radicals as per the proceure [37]. In
this assay, 7 mM of ABTS solution was prepared and was mixed with already prepared 2.45 mM
K2S2O4 solution. The mixture was stored for around 16 h in dark. The absorbance was measured
with double beam spectrophotometer at 734 nm. Ascorbic acid served as positive control in ABTS
assay also. The percent ABTS activity was calculated by the given formula;
Control absorbance − Sample absorbance
Percent ABTS activity =
× 100
Control absorbance
Estimation of IC50 values:
Concentrations of the synthesized compounds at which 50% of inhibition is observed (IC50), were
calculated among the inhibition percentages against the tested concentrations using the MS-Excel
program.
Statistical analysis:
All the assays were performed in triplicate and values were expressed as means+ standard error
means (SEM). Statistical analysis was performed by two-way analysis of difference (ANOVA),
followed by Bonferroni tests. The difference was measured to be statistically significant when the
p value ˂ 0.05.
29