Novel potent and selective DPP-4 inhibitors: Design, synthesis and molecular docking study of dihydropyrimidine phthalimide hybrids

Article abstract of DOI:10.3390/ph14020144

Background: Dipeptidyl peptidase-4 (DPP-4) inhibitors have emerged as antihyperglycemic agents that improve glycemic control in type 2 diabetic patients, either as monotherapy or in combination with other antidiabetic drugs. Methods: A novel series of dihydropyrimidine phthalimide hybrids was synthesized and evaluated for their in vitro and in vivo DPP-4 inhibition activity and selectivity using alogliptin as reference. Oral glucose tolerance test was assessed in type 2 diabetic rats after chronic treatment with the synthesized hybrids ± metformin. Cytotoxicity and antioxidant assays were performed. Additionally, molecular docking study with DPP-4 and structure activity relationship of the novel hybrids were also studied. Results: Among the synthesized hybrids, 10g, 10i, 10e, 10d and 10b had stronger in vitro DPP-4 inhibitory activity than alogliptin. Moreover, an in vivo DPP-4 inhibition assay revealed that 10g and 10i have the strongest and the most extended blood DPP-4 inhibitory activity compared to alogliptin. In type 2 diabetic rats, hybrids 10g, 10i and 10e exhibited better glycemic control than alogliptin, an effect that further supported by metformin combination. Finally, 10j, 10e, 10h and 10d had the highest radical scavenging activity in DPPH assay. Conclusions: Hybrids 10g, 10i and 10e are potent DPP-4 inhibitors which may be beneficial for T2DM treatment.

Full text of DOI:10.3390/ph14020144

pharmaceuticals
Article
Novel Potent and Selective DPP-4 Inhibitors: Design, Synthesis
and Molecular Docking Study of Dihydropyrimidine
Phthalimide Hybrids
Ahmed A. E. Mourad 1 , Ahmed E. Khodir 2, Sameh Saber 3 and Mai A. E. Mourad 4,
*
1
Pharmacology and Toxicology Department, Faculty of Pharmacy, Port-Said University, Port-Said 42511, Egypt;
ahmed.mourad@yahoo.com
Pharmacology Department, Faculty of Pharmacy, Horus University, New Damietta 34518, Egypt;
akhodir@horus.edu.eg
Pharmacology Department, Faculty of Pharmacy, Delta University for Science and Technology, Gamasa City,
Mansoura, Dakahlia 11152, Egypt; sameh.mohamed@deltauniv.edu.eg
Medicinal Chemistry Department, Faculty of Pharmacy, Port-Said University, Port-Said 42511, Egypt
Correspondence: mai.mourad@yahoo.com
2
3
4
*
Abstract: Background: Dipeptidyl peptidase-4 (DPP-4) inhibitors have emerged as anti-hyperglycemic
agents that improve glycemic control in type 2 diabetic patients, either as monotherapy or in combi-
nation with other antidiabetic drugs. Methods: A novel series of dihydropyrimidine phthalimide
hybrids was synthesized and evaluated for their in vitro and in vivo DPP-4 inhibition activity and
selectivity using alogliptin as reference. Oral glucose tolerance test was assessed in type 2 diabetic
rats after chronic treatment with the synthesized hybrids
assays were performed. Additionally, molecular docking study with DPP-4 and structure activity
relationship of the novel hybrids were also studied. Results: Among the synthesized hybrids, 10g
10i 10e 10d and 10b had stronger in vitro DPP-4 inhibitory activity than alogliptin. Moreover, an
in vivo DPP-4 inhibition assay revealed that 10g and 10i have the strongest and the most extended
blood DPP-4 inhibitory activity compared to alogliptin. In type 2 diabetic rats, hybrids 10g, 10i and
10e exhibited better glycemic control than alogliptin, an effect that further supported by metformin
±
metformin. Cytotoxicity and antioxidant
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
,
Citation: Mourad, A.A.E.; Khodir,
A.E.; Saber, S.; Mourad, M.A.E. Novel
Potent and Selective DPP-4 Inhibitors:
Design, Synthesis and Molecular
Docking Study of Dihydropyrimidine
Phthalimide Hybrids. Pharmaceuticals
2021, 14, 144. https://doi.org/
10.3390/ph14020144
,
,
combination. Finally, 10j
assay. Conclusions: Hybrids 10g
,
10e
,
10h and 10d had the highest radical scavenging activity in DPPH
10i and 10e are potent DPP-4 inhibitors which may be beneficial
,
for T2DM treatment.
Academic Editor: Thierry Besson
Keywords: DPP-4 inhibitors; T2DM; OGTT; DPPH; dihydropyrimidine; phthalimide
Received: 6 January 2021
Accepted: 7 February 2021
Published: 11 February 2021
1. Introduction
Diabetes mellitus is a rapidly growing chronic disease that threatens human health
worldwide. T2DM is characterized by insulin resistance and impaired glucose homeostasis,
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations.
resulting in hyperglycemia [
ing sulfonylureas, meglitinides, thiazolidinediones, biguanides,
aimed at suppressing hepatic glucose output, stimulating insulin release, mitigating glu-
cose absorption, and increasing peripheral glucose utilization [ ]. Unfortunately, the
use of these drugs is associated with various side effects including weight gain and hy-
poglycaemia which deteriorate the optimum glycemic control over a longer time [ ].
1]. Currently, there are a number of antidiabetic drugs includ-
α
-glucosidase inhibitors
2,3
4,5
Copyright:
© 2021 by the authors.
Therefore, many newer approaches with novel mechanism of action have been emerged
for better T2DM management. Among these newer approaches, dipeptidyl peptidase-4
(DPP-4) inhibitors that gained extensive interest in T2DM treatment with proved long term
efficacy and better glycemic control. Additionally, DPP-4 inhibitors play an important
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
role in regeneration and differentiation of pancreatic
β-cells and also proved to be well
tolerated as well as decreasing the risk of hypoglycemia and cardiovascular side effects [
6
].
Pharmaceuticals 2021, 14, 144. https://doi.org/10.3390/ph14020144
https://www.mdpi.com/journal/pharmaceuticals

Pharmaceuticals 2021, 14, 144
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DPP-4 is widely expressed in most tissues and body fluids including the biliary tract,
kidney, gastrointestinal tract, uterus and liver [
signaling factor of incretin hormones (insulin-stimulating hormones, glucagon-like peptide
(GLP-1), and glucose-dependent insulinotropic polypeptide (GIP)) [ ]. These hormones
play a crucial role in promoting -cell proliferation, increasing insulin biosynthesis, re-
ducing -cell apoptosis [ 10]. As a consequence of rapid degradation by DPP-4, both
GLP-1 and GIP have very short half-life which is 1–2 minutes for the former and 7 minutes
for the latter [11 12]. Currently, there are several structurally diverse DPP-4 inhibitors
7]. It is the key regulatory enzyme and a
8
β
β
9,
,
some of them have been approved on the market such as sitagliptin, alogliptin, linagliptin,
gemigliptin, anagliptin and teneligliptin [13,14].
On the other hand, pyrimidines represented one of the most important versatile scaf-
folds in medicinal chemistry [15] that possess many activities including antidiabetic [16],
anti-inflammatory [17], anticancer [18], antibacterial [19], antihypertensive [20], antifun-
gal [21] and antitubercular [22]. Moreover, some of the synthesized N-methylated and
N-benzylated pyrimidinediones have shown in vitro DPP-4 inhibitory activity in sig-
nificant range [23]. In addition, excellent IC50 values were recorded for novel pyrim-
idinedione derivatives when evaluated for their DPP-4 inhibitory activity and in vivo
anti-hyperglycemic efficacy [24].
On the other hand, phthalimide-containing compounds are precious synthetic scaf-
folds that showed different biological activities such as: antihyperglycemic [25], anti-
inflammatory [26], antimicrobial [27], antihyperlipidimic [28], anticonvulsant [29], antivi-
ral [30] and anticancer effects [31]. Additionally, a novel series of potent DPP-4 inhibitors
bearing an N-phenylphthalimide skeleton has been synthesized and displayed higher
potency compared to a well-known DPP-4 specific inhibitor [32,33]. Recently, some non-
competitive DPP-4 inhibitors have been synthesized from a series of benzo[4,5]thieno[2,3-
d]pyrimidine phthalimide derivatives and have shown better activity compared to DPP-4
inhibitors synthesized from amine precursors [34].
Based on the aforementioned information, we aimed at combining pyrimidine and
phthalimide moieties into a single candidate template for the purpose of synergistic activity
(Figure 1). To better elucidate their DPP-4 inhibitory activity, our synthesized hybrids were
subjected to in vitro DPP inhibition assay utilizing human recombinant DPP-4, DPP-8 and
DPP-9; alogliptin used as a reference standard. Moreover, the effect of our compounds
on in vivo DPP-4 activity was assessed in SD rats, where DPP-4 activity was evaluated
in the blood over 2 days using alogliptin as a reference standard. Additionally, to gain
better understanding of the role of our compounds on T2DM, we studied the effect of
chronic treatment of the synthesized hybrids 10a–j in presence and absence of metformin
(MET) on HFD-induced T2DM in rats. Also, the target hybrids were tested for any possible
antioxidant activity using DPPH method. Finally, the effect of synthesized hybrids 10a–j
on cell viability was examined on normal hepatic LO2 cells using MTT assay, to check for
any cytotoxic effect. Furthermore, structure activity relationship and molecular docking
analysis of the titled hybrids were performed to get a distinct insight about the binding
mode and interactions of the compounds with DPP-4 active site.

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Figure 1. Diagram represents design of novel DPP-4 inhibitors.
2. Result and Discussion
2.1. Chemistry
The synthetic protocols applied to synthesize the target compounds 10a–j are illus-
trated in Scheme 1. Substituted acetophenone reacted with various aromatic aldehydes
in 10% sodium hydroxide solution following Claisen-Schmidt condensation conditions
to afford exclusively the E-chalcone isomers 3a–j in high yields [35]. These chalcones 3a–j
were further reacted with thiourea ( ) in the presence of sodium hydroxide in abs. ethanol
to give 4,6-diaryl-3,4-dihydropyrimidine-2(1H)-thiones 5a–j. The structures of compounds
5e and
were confirmed on the basis of their spectral data (FT-IR, 1H-NMR, 13C-NMR,
1
2
4
,
g,
i
j
MS) and elemental analyses (see Section 4).
Reduction of phthalimide gave phthalide (
6
) which on refluxing with potassium
). Reaction of with thionyl
) [36]. Treatment of 4,6-diaryl-
in dichloromethane, and in the presence
phthalimide in DMF afforded
chloride brought about -phthalimido-o-toluyl chloride (
3,4-dihydropyrimidine-2(1H)-thiones 5a–j with
α
-phthalimido-o-toluic acid (
8
8
α
9
9
of triethyl amine (TEA) as a base brought about the target compounds (4,6-diaryl-1,6-
dihydropyrimidin-2-yl)-2-((1,3-dioxoisoindolin-2-yl)methyl) benzothioates 10a–j. Notably,
the S-substituted product was isolated as nucleophilicity of sulphur is greater than nitrogen
and no N-substituted product was isolated. The structure of 10a–j hybrids were elucidated
on the bases of both spectral data (IR, 1H-NMR, 13C-NMR, MS) and elemental analyses.
In the IR spectra the following peaks were observed: 3476–3384 cm−1 (NH),
3071–2918 cm−1 (Ar-CH), 1774–1770 −c1m−1, 1725–1714 cm−1 (CO), 1677–1603 cm−1
(C=N), 1595–1502 cm−1, 1491–1467 cm (C=C). Beside the
NH2 side chains in the H-NMR spectra of the hybrids 10b
appear at 3.76, 2.34, 2.52, 6.19 and 6.17, respectively, the
δ
values of OCH3, CH3, Nme2,
10d 10e 10h and 10j which
δ values of the other functional
1
,
,
,
groups fall in the following ranges: NH:11.52–11.19 ppm; Ar-H: 8.51–6.75 ppm; HC=CH-Ar:
6.68–6.35 ppm; CH-Ar: 5.29–5.27 ppm; CH2: 5.08–5.01 ppm. Also, the 13C-NMR spectra of
some selected compounds were resolved in appropriate regions confirming the assigned
structures. Both mass spectra and elemental analyses of 10a–j hybrids are a further support
of their structures by giving correct molecular formula and molecular ion peaks.

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Scheme 1. Synthesis of the target compounds 10a–j. Reagents and conditions: (a) NaOH, dil. EtOH, rt; (b) NaOH, abs.
EtOH; (c) DMF, reflux; (d) SOCl2, reflux, dry benzene, distillation; (e) abs. dichloromethane, TEA, rt.
2.2. Biological Evaluation
2.2.1. In Vitro DPP Inhibition Assay
All the target hybrids listed in Table 1 were evaluated for the inhibition of human
recombinant DPP-4 and alogliptin was chosen as a reference compound. Additionally, all

Pharmaceuticals 2021, 14, 144
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the synthesized hybrids were tested for the inhibition of DPP-8 and DPP-9 which is closely
related to DPP-4 and whose inhibition is associated with great toxicity.
Table 1. DPP-4, DPP-8 and DPP-9 inhibitory activities of hybrids 10a–j and alogliptin.
Compound No.
DPP-4 IC50 (nM) *
DPP-8 IC50 (µM) *
DPP-9 IC50 (µM) *
10a
10b
10c
10d
10e
10f
10g
10h
10i
19.74 ± 1.27
3.78 ± 0.23
14.71 ± 0.82
2.19 ± 0.14
1.42 ± 0.11
31.52 ± 2.41
0.51 ± 0.03
10.37 ± 0.54
0.66 ± 0.04
7.35 ± 0.36
5.71 ± 0.21
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
10j
Alogliptin
* Data are means of four replicates with standard error (Mean ± SE).
Interstingly, hybrids 10g and 10i identified as the most potent DPP-4 inhibitors with
picomolar potency, exhibiting IC50 values of 0.51 and 0.66 nM, respectively; which repre-
sented 11.2, 8.6 fold superior activity than alogliptin. The potent DPP-4 inhibitory activity
of these compounds may be attributed to electronic behavior of the nitro group in ring B of
hybrid 10g and the small size of the furanyl group in ring B of hybrid 10i, which facilitated
the formation of hydrogen bond with DPP-4 active site.
Of considerable interest, the mesomeric effect raised from 4-Me2N group in ring B as
well as 4-bromo substitutions in ring A of hybrid 10e markedly boosted DPP-4 inhibition
resulted in low nanomolar potency activity (1.42 nM, 4 fold increased activity than that
of alogliptin). Moreover, introducing an electron donating groups (methyl group in ring
A and methoxy group in ring B) retained the potent DPP-4 inhibitory activity of hybrids
10b and 10d, whose activities were 1.5 fold and 2.6 fold the activity of alogliptin with IC50
values of 3.78, 2.19 nM, respectively. According to the basis of electron donating properties,
hybrid 10j expected to be the most potent DPP-4 inhibitor but on the contrary, the DPP-4
inhibitory potency is eroded (IC50 = 7.35 nM). The probable reason could be the worse fit of
the naphthyl group in ring B with more steric inhibition into the receptor pocket compared
to the phenyl ring in the other synthesized compounds. On the other hand, the enzyme
inhibition activity was dramatically decreased in the unsubstituted phenyl group and upon
introducing the electron withdrawing chlorine atom in 10h, 10c and hybrids 10f which
was confirmed by their high IC50 values compared to alogliptin. Out of these, it might be
concluded that the substituent electronic character and the steric volume of the phenyl ring
considerably influenced the DPP-4 inhibition activity.
On the other hand, all the synthesized hybrids targeted DPP-8 and DPP-9 at IC50
exceeding 100 µM which indicates that our compounds have superior selectivity for DPP-4
and neglected affinity toward DPP-8 and DPP-9.
2.2.2. Effects of Synthesized Hybrids (10a–j) on Cell Viability
The cytotoxic effect of the synthesized hybrids 10a–j was examined on normal hepatic
LO2 cells using MTT assay. The test was done using 3 different concentrations (10
30 M and 50 M) for each compound. As shown in (Figure 2a), hybrid 10c had the most
cytotoxic effect with 72.57% and 72.50% cell viability at 30 M and 50 M concentration,
respectively. However, hybrid 10i came in the second place with cell viability percent of
74.60 at 10 M concentration, followed by hybrid 10h with cell viability percent of 77.47 at
50 M concentration, as presented in (Figure 2b). While, the cell viability percent for other
µM,
µ
µ
µ
µ
µ
µ
synthesized hybrids ranged from 80.13 to 101.2. In conclusion, our compounds showed
weak cytotoxic effect against hepatic LO2 cells.

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(a)
(b)
Figure 2.
(
a
) Effect of hybrids 10a–e on viability of normal hepatic LO2 cells. (n = 5). (
b
) Effect of
hybrids 10f–j on viability of normal hepatic LO2 cells (n = 5). *** Significant from control group at
p < 0.001, ** Significant from control group at p < 0.01, * Significant from control group at p < 0.05.
2.2.3. Effect of Synthesized Hybrids (10a–j) on In Vivo DPP-4 Activity
The effect of the synthesized hybrids 10a–j on blood DPP-4 activity was investigated
in SD rats, as shown in (Figure 3a,b). The hybrids were administrated in a single oral dose
of 10 mg/kg and in vivo DPP-4 activity was evaluated over 2 days, using alogliptin as
reference compound. Notably, among all tested hybrids and aloglipin, hybrid 10g has
both the strongest and longest DPP-4 inhibitory action, with 18.45% and 47% DPP-4 blood
activity at 12 h and 24 h, respectively, followed by hybrid 10i with DPP-4 blood activity of
18.8% and 49.9% at 12 h and 24 h, respectively. While, alogliptin achieved DPP-4 blood
activity of 20.95% and 56.1% at 12 h and 24 h, respectively. Importantly, hybrids 10g and
10i also showed extended DPP-4 inhibitory activity at 48 h with blood DPP-4 activity of

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73.3% and 76%, respectively, while, alogliptin DPP-4 blood activity was 97.05%. Worthily,
hybrids 10g, 10i and 10e had the strongest in vitro and in vivo DPP-4 inhibiting activity.
(a)
(b)
Figure 3. (a). The in vivo DPP-4 activity of 10a–e hybrids and alogliptin within 48 h. (n = 3). (b) The
in vivo DPP-4 activity of 10f–j hybrids and alogliptin within 48 h. (n = 3).
2.2.4. Effect of Chronic Treatment of Compounds 10a–j with or without MET on
HFD-Induced Type 2 Diabetic rats
HFD significantly induced insulin resistance in SD rats as evident by extremely sig-
nificant increase in the AUC of OGTT in non-treated diabetic group compared to control
group, as shown in (Figure 4a,b). We studied the chronic effect of oral administration
of hybrids 10a–j at a dose of 10 mg/kg/day in absence and presence of MET, on insulin
resistance in type 2 diabetic rats. In absence of MET, hybrids 10g, 10i and 10e significantly
improved glucose tolerance above alogliptin, as evident by the reduction in the AUC
of OGTT when compared to non-treated diabetic group, (Figure 5a,b). Moreover, oral
administration of MET (150 mg/kg/day) together with hybrids 10a–j, further enhanced
insulin sensitivity with a profound reductions in AUC of OGTT almost in all treated groups.

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Interestingly, MET addition to 10g
,
10i and 10e-treated groups reduced AUC of OGTT by
22.93%, 21.7% and 22.82%, respectively. Accordingly, glucose tolerance in 10g/MET treated
group reached a normal level with AUC equals 13787 201 mg.min/dL compared to
14305 318 mg.min/dL for normal control group. Similarly, addition of MET to alogliptin
treated group reduced AUC of OGTT from 20835 146 mg.min/dL in alogliptin treated
±
±
±
group to 15451 ± 110 mg.min/dL in MET/alogliptin treated group.
(a)
(b)
Figure 4. (a) Chronic effect of hybrids 10a–j and alogliptin administration on blood glucose levels
during an OGTT in type 2 diabetic rats. Data are presented as mean SEM (n = 7). ( ) Chronic effect
±
b
of combined administration of 10a–j/MET, alogliptin/MET on blood glucose levels during an OGTT
in type 2 diabetic rats. Data are presented as mean ± SEM (n = 7).

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(a)
(b)
Figure 5. (a) Chronic effect of hybrids 10a–j and alogliptin administration on area under the curve
(AUC) of OGTT in type 2 diabetic rats. Data are presented as mean SEM (n = 7). *** Significant from
diabetic control group at p < 0.001, ** Significant from diabetic control group at p < 0.01. ( ) Chronic
effect of combined administration of 10a–j/MET, alogliptin/MET on area under the curve (AUC) of
OGTT in type 2 diabetic rats. Data are presented as mean SEM (n = 7). *** Significant from diabetic
±
b
±
control group at p < 0.001, * Significant from diabetic control group at p < 0.05.
2.2.5. Antioxidant Activity
The in vitro antioxidant activity of the synthesized hybrids 10a–j was tested depending
on DPPH method using ascorbic acid as standard. The results in Table 2 revealed that all the
synthesized compounds showed good antioxidant activity, wherein hybrids 10j
, 10e, 10h and
10d have the highest radical scavenging activity in DPPH assay at 100 µM concentration.

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Table 2. Antioxidant activity of hybrids 10a–j using the DPPH method.
Compound No. (100 µM)
% DPPH Scavenging *
10a
10b
10c
10d
10e
10f
10g
10h
10i
73.85 ± 2.46
74.28 ± 1.69
52.39 ± 0.93
80.16 ± 1.38
87.21 ± 2.58
57.63 ± 2.14
49.47 ± 1.08
85.74 ± 2.76
69.11 ± 1.44
93.52 ± 1.65
10j
* Data are means of four replicates with standard error (Mean ± SE).
2.2.6. Molecular Docking Study: Docking at the DPP-4 active site
To verify the protein-ligand binding properties and the key structural features required
for the inhibitory activity of the novel synthesized hybrids 10a–j with the protein DPP-4,
a molecular docking study was performed using the molecular docking program MOE.
The results of energy binding scores and binding interactions were depicted in Table 3 and
Figure 6. The 3D crystal structure of DPP-4 was obtained from the Protein Data Bank [PDB
code 3G0B, resolution 2.25 Å (www.rcsb.org, accessed on 10 February 2021)] and alogliptin
was used as a reference DPP-4 inhibitor. DPP-4 active site is formed of two main binding
pockets Figures S1 and S2 in addition to Figure S2 extensive subsite pocket. Figure S1
pocket is formed of highly hydrophobic residues Ser 630, Tyr 631, Tyr 547, Val 656, Trp 659,
Tyr 662, Tyr 666, Asn 710, Val 711 and His 740), Figure S2 pocket (formed by Glu 205 and
Glu 206 dyad and Arg 125) [37]. The Figure S2 pocket is surrounded by residues of Val
207, Ser 209, Arg 358, Phe 357, Lys 544, Trp 627, Trp 629 and Asp 708 which constitute the
Figure S2 extensive subsite [38].
Table 3. Types of binding interactions and energy scores (kcal/mol) for hybrids 10a–j and alogliptin at the DPP-4 active site.
Compound No.
10a
Types of Interactions
Energy Scores
−8.68
- One hydrogen bonding interaction with Arg 125 (3.14 Å).
- One π-cation interaction with Lys 554 (4.06 Å)
- One π-hydrogen bonding interaction with Trp 629 (4.23 Å).
10b
10c
−9.12
−7.74
- Two hydrogen bonding interaction with Arg 125 and Tyr 547 (3.18 Å, 2.77 Å).
- One π-hydrogen bonding interaction with His 126 (4.03 Å).
- Three hydrogen bonding interaction with Arg 125 (2.85 Å, 3.01 Å, 3.22 Å).
- One hydrogen bonding interaction with Tyr 457 (2.71 Å).
- One π-hydrogen bonding interaction with Tyr 547 (4.20 Å).
10d
−8.14
-Three hydrogen bonding interaction with Arg 125 (2.83 Å, 3.01 Å, 3.24 Å).
- One hydrogen bonding interaction with Tyr 547 (2.73 Å).
10e
10f
−8.15
−7.74
- One hydrogen bonding interaction with Asp 545 (3.14 Å).
- One π-cation interaction with Lys 554 (4.87 Å).
10g
10h
−9.45
−9.32
- Two hydrogen bonding interaction with Lys 554 and Glu 206 (2.97 Å and 3.09 Å).
- Two hydrogen bonding interaction with ASP 545 and VAL 546 (3.07 Å and 2.83 Å).
- Two hydrogen bonding interaction with Tyr 547 and Glu 206 (2.89 Å and 3.63 Å).
- Two hydrogen bonding interaction with Arg 125 (3.34 Å and 2.91 Å).
10i
−8.50

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Table 3. Cont.
Compound No.
10j
Types of Interactions
Energy Scores
- Three hydrogen bonding interaction with Glu 205, Glu 206 and Tyr 631 (3.16 Å,
3.12 Å and 3.53 Å).
- One H-π interaction with Trp 629 (4.33 Å).
- One π-H interaction with Tyr 456 (4.21 Å).
−7.72
−7.92
- Five hydrogen bonding interaction with Glu 205, Glu 206, Tyr 631, Arg 125 (2.78 Å
and 2.96 Å, 2.90 Å, 3.35 Å and 2.97 Å).
- Three ionic bond with Glu 206 and Glu 205 (2.96 Å, 2.90 Å and 3.95 Å).
Alogliptin
(a)
(b)
(c)
(d)
(e)
(f)
Figure 6. Cont.

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(g)
Figure 6. Docking and binding pattern of compounds 10g
(h)
(a,
b), 10i
(c,
d), 10e
(e,f
) and alogliptin (g,h) showing interactions
with different amino acid residues found in the active site of DPP-4 (PDB code: 3G0B).
The SiteMap analysis highlighted the importance of the Tyr 547, Lys 554, and Trp
629 residues as a top-ranked binding pocket of DPP-4 in the formation of the enzyme–
inhibitor complex [39]. Moreover, the hydroxyl group of Tyr 547 plays an oxyanion-
stabilizing role in the DPP-4 catalytic mechanism as well as Trp 629 residue forms catalytic
Figure S2 pocket in this protease [40]. The DPP-4 inhibitory activity is markedly increased
when the inhibitor can interact to Figures S1 and S2 pockets. Interestingly, studies suggested
that only few inhibitors can bind into Figure S2 extensive subsite and enhancing the
bioactivity of DPP-4 inhibitors through the interactions with Lys 554 so it is the target of
DPP-4 inhibitor selectivity [41].
It was obvious from the results depicted in Table 3 that nearly all of the novel hybrids
achieved energy binding scores higher than that of alogliptin. Additionally, hybrids 10b
,
10d 10e 10g 10i bind efficiently with the key amino acids located in the receptor pockets
,
,
,
which explained the potent DPP-4 inhibitory activity of these hybrids. Of considerable
interest, hybrid 10g recorded the highest energy binding score and the lowest in vitro DPP-4
IC50 inhibitory activity among the other synthesized hybrids via binding mainly to the
Figure S2 pocket and Figure S2 extensive subsite through formation of two strong hydrogen
bonds between both oxygen of the free nitro group and phthalimido carbonyl group with
Glu 206 and Lys 554 which have a vital role in the enhancement of DPP-4 affinity. However,
the binding mode showed that the phthalimido carbonyl oxygen and S atom of hybrid 10i
participated in a hydrogen-bonding network with Arg 125 and Glu 206 residues in Figure S2
pocket together with another strong hydrogen bond formed with Tyr 547 in Figure S1 pocket.
Similarly, compound 10d was buried in Figures S1 and S2 pockets via formation of four
hydrogen bonds as well as π-H bond with Arg 125 and Tyr 547.
Notably, one hydrogen bond is formed between one of the phthalimido carbonyl
oxygen of compound 10e and free OH group of Tyr 547 residue in Figure S1 pocket
together with another three hydrogen bonds between the another carbonyl oxygen and the
free NH2 of Arg 125 in Figure S2 pocket. However, hybrid 10b stacked with the side chain
of Lys 554 and Trp 629 in Figure S2 extensive subsite via weak π-cation and π-hydrogen
interactions which explained its higher IC50 value comparable to the previously mentioned
hybrids but its DPP-4 inhibitory activity still higher than that of alogliptin. On the other
hand, in Figure S2 pocket, the nitrogen of the free amino group of hybrid 10j is responsible
for binding with Glu 205 and Glu 206 residues through two hydrogen bonds together with
another hydrogen bond formed with Tyr 631 in Figure S1 pocket. Moreover, the phenyl ring
of that compound provided additional binding affinity with Figure S2 extensive subsite via
H-π interaction with Trp 629 residue while the naphthyl ring forms another π-H stacking
with Tyr 456 side chain.
However, the electron withdrawing effect of chlorine atom in hybrid 10c negatively
affects its binding with the receptor site and hence its DPP-4 inhibitory activity. In addition

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hybrid 10a binds weakly with the receptor site via only one hydrogen bonding with Arg 125.
Meanwhile, 10f and 10h exhibited weak DPP-4 inhibitory activity as they could not be able
to bind to the crucial amino acids responsible for the activity. Finally, it can be concluded
that most of the titled synthesized hybrids interact with DPP-4 protein in distinct manner
and thus are able to inhibit its activity efficiently.
3. Structure Activity Relationship
Based on the aforementioned results, it becomes clear that the electronic nature of the
substituents and the steric factor greatly affect the biological activity of the synthesized
hybrids, so the present SAR study focused on the effect of substituents on position 4 in the
aryl groups that located in positions 2 and 4 of dihydropyrimidine as in hybrids 10a–h, as
well as the furanyl and 1-naphthyl groups in hybrids 10i and 10j. It is worth noting that
positions 2 and 4 in dihydropyrimidine are equivalent due to the migration of hydrogens
and electron transition which is cleared in (Figure 7).
Ar
Ar
`H
H
N
O
N
O
N
Ar
S
N
H
Ar
S
N
N
O
O
O
O
Figure 7. Equivalence of positions 2 and 4 in dihydropyrimidine.
Moreover, the structures of most of the title compounds 10a–j contain a 4-chlorophenyl
group. Consequently, the compounds under investigation are related structurally to the
substituents attached to the other phenyl group. On the basis of the electron donating
effect of both the 1-naphthyl and 4-aminophenyl groups and the lipophilic character of the
latter, hybrid 10j should be the top ranked electron donor and consequently expected to be
the most potent DPP-4 inhibitor via formation of more strong H-bonds with the receptor
binding sites but on the contrary, the steric factor of the 1-naphthyl and 4-aminophenyl
groups hinder the efficient binding with the receptor binding site, indicating that the steric
factor is more effective, but it is still potent enough for inhibiting the enzyme activity as its
IC50 was slightly less than that of alogliptin. Interestingly, hybrid 10g achieved the most
potent DPP-4 inhibitory activity owing to the electron donating properties and the unique
electronic ionic behavior of the nitro group leading to higher ability to form strong H-bond
with Figure S2 extensive subsite as evidenced by its higher energy binding score.
Additionally, the DPP-4 inhibitory activity is markedly increased thanks to the lipophilic
character and the small size of the furanyl group in hybrid 10i which enhance its fit to the
receptor site, and accordingly its ability to form strong H- bond with the receptor site as
evidenced by its molecular docking results. On the other hand, the lipophilic electron
donating character of the methyl group, via hyperconjugative effect, in hybrid 10d resulted
in an increase of its ability to form more H-bonds which, in turn, reflected on the marked
difference in DPP-4 inhibitory IC50 values compared to that of the corresponding electron-
withdrawing chlorine-atom in hybrid 10c. Moreover, the mesomeric effect raised from
4-Me2N group as well as 4-bromo atom in 10e hybrid seemed to be the most effective factor

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than hyperconjugative effect of 4-methyl and mesomeric effect of 4-chloro in hybrid 10d
that led to stabilization of H-bonding and consequently better DPP-4 inhibiting activity.
Meanwhile, the lipophilic electron donating methoxy group of hybrid 10b experienced
much higher DPP-4 inhibiting activity than the hydrophilic electron-withdrawing chlorine-
atom hybrid 10f which exhibited much weaker binding interaction with the key amino acid
residue in the receptor site as evidenced by its high IC50 value. Finally, it is concluded from
the aforementioned SAR results that the electronic nature, the steric factor and lipophilicity
of the substituents greatly affected the biological activity of the system incorporated.
,
4. Materials and Methods
4.1. Animals
Male Sprague Dawley (SD) rats (6–7 weeks old and 100–120g body weight) were pur-
chased from Abu-rawash Animal House (Giza, Egypt). Rats were allowed to acclimatize
for one week with free access to food and water and kept under controlled laboratory
conditions of humidity and 12 h light-dark cycle. All experimental protocols were ap-
proved by the Research Ethics Committee of the Faculty of Pharmacy, Port-said University
(R 20.5.1527).
4.2. Experimental Design
After acclimatization period, rats were randomly divided into 25 groups, each of seven
rats. The first group was normal control group which maintained on normal rat chow for
the entire experiment period (16 weeks). The second group served as diabetic control group
in which T2DM was induced by HFD that consisted of casein (30%), raw beef fat (Suet,
40%), wheat flour (7%), glucose (10%), salt mixture (6%), vitamin mixture (3%), and bran
(4%), for 16 weeks. The remaining groups consumed HFD for 16 weeks, however, in the last
four weeks, HFD was administrated together with each compound from our synthesized
hybrids 10a–j at a dose of 10 mg/kg/day, orally, or with alogliptin (Takeda Pharmaceuticals,
Deerfield, IL, USA) at a dose of 20 mg/kg/day, orally, with and without MET (Alfa Aesar,
ThermoFisher Inc., Haverhill, MA, USA) at a dose of 150 mg/kg/day, orally.
4.3. In Vitro DPP-4, DPP-8 and DPP-9 Inhibition Assay
DPP-4 inhibition was assessed, as previously described [42,43], based on a continu-
ous fluorometric assay method in which the substrate Gly-Pro-AMC (Gly-Pro-7-amido-4-
methylcoumarin hydrobromide) was split by DPP-4 to release fluorescent aminomethyl
coumarin (AMC). The liberated AMC was continuously monitored using an excitation
wavelength of 360 nm and an emission wavelength of 460 nm using an EnVision mi-
croplate reader (PerkinElmer, Waltham, MA, USA). The reaction mixture consisted of
different concentrations of the synthesized hybrids 10a–j, 20
µU/µL of recombinant hu-
man DPP-4 (Sigma Aldrich, St. Louis, MO, USA), 10 M Gly-Pro-AMC (Sigma Aldrich,
µ
St. Louis, MO, USA) and assay buffer (150 mM NaCl, 25 mM 4-(2-hydroxyethyl)-1-
piperazineethanesulfonic acid (HEPES), 0.12 mg/ml bovine serum albumin (BSA)) and pH
of 7.5. The assay was carried out in quadruplicate. IC50 was calculated using GraphPad
Prism 8 software (San Diego, CA, USA). Similar to the DPP-4 assay, DPP-8 and DPP-9
inhibition assay was performed and the pH of the assay buffer was 8.
4.4. Cytotoxicity Test
We assessed any possible cytotoxic effect of our synthesized hybrids 10a–j on hepatic
LO2 cells using MTT assay. The cells, in DMEM (Gibco, Invitrogen), were seeded in 96 well
plates at density of 1
×
104/well. The synthesized compounds were added to the cells
and incubated for 48 h at 37 ◦C and 5% CO2. Then, MTT reagent was added and the cells
were re-incubated for further 4 h. After discarding the supernatants, the formed formazan
crystals were dissolved in DMSO. The intensity of color was assessed using microplate
reader (Bio-Rad Laboratories, Hercules, CA, USA) at 490 nm wavelength.

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4.5. In Vivo DPP-4 Inhibition Assay
The assay was performed using male SD rats (n = 3 per group), as previously de-
scribed [44]. The synthesized hybrids 10a–j were suspended in 0.5 carboxymethyl cellulose
(CMC) and administrated orally at a single dose of 10 mg/kg.
Blood samples were collected at time points 0, 6, 12, 24, 36 and 48 h. Plasma samples
were extracted and kept at
−
20 ◦C until analysis time. Similar to the in vitro DPP-4 assay
principle, plasma samples were mixed with equal volumes of Gly-Pro-AMC in assay buffer
(100 mM HEPES, 0.1 mg/ml BSA) and pH of 7.5. The liberated AMC was continuously
monitored using an excitation wavelength of 360 nm and an emission wavelength of
460 nm using EnVision microplate reader (PerkinElmer, Waltham, MA, USA). The data
were expressed as% blood DPP-4 activity.
4.6. In Vivo Oral Glucose Tolerance Test (OGTT) in SD Rats
At the end of treatment regimen that lasted for four weeks; using our synthesized
hybrids 10a–j with and without MET, rats were fasted overnight (12 h) with free access
to water. Oral glucose tolerance test (OGTT) was performed as previously described [45].
Briefly, blood samples were collected from retro-orbital sinuses veins using glass capil-
laries at 0 time, 30, 60, 90 and 120 min after oral glucose loading (2 g/kg) and glucose
concentration was determined with an automatic blood glucose meter (Accu-Chek; Roche
Diagnostics, Rotkreuz, Switzerland).
4.7. Antioxidant Activity (DPPH Method)
DPPH (1,1-diphenyl-2-picrylhydrazyl) free radical is stable at rt and has a purple
colour with a maximum absorption at 517 nm. This free radical is reduced by antioxidant
molecules into 1,1-diphenyl-2-picrylhydrazine with resultant discolouration of free radical;
this discolouration is stoichiometric with regard to the captured electrons. Accordingly, the
fluctuation in the absorbance is an indicator for the antioxidant activity. Our synthesized
hybrids 10a–j (100 µM) were mixed with DPPH (100 µM) and left for 20 min at rt, the
absorbance was measured at 517 nm.
4.8. Statistical Analysis
Results were expressed as means
±
standard error of the mean (SEM) and were ana-
lyzed for statistically significant differences using one-way analysis of variance (ANOVA).
P values less than 0.05, 0.01 and 0.001 were considered significant. GraphPad Prism® 8 was
used for statistical calculations (GraphPad Software, St. Louis, MO, USA).
4.9. Molecular Docking
Molecular docking studies of the synthesized hybrids 10a–j and alogliptin were
performed using MOE software. Ligands were built into the builder interface of the
MOE program and their energies were minimized until a root mean square deviation
(RMSD) gradient of 0.01 kcal/mol and root mean square (RMS) distance of 0.1 Å with
MMFF94
×
(Merck molecular force field 94 ) force-field and the partial charges were
×
automatically calculated. The X-ray crystallographic structure of DPP-4 (PDB code: 3G0B)
was downloaded from protein data bank (www.rcsb.org, accessed on 10 February 2021).
The enzymes were prepared, the hydrogens were added then the atoms connection and type
were checked with automatic correction. The obtained poses were studied and the poses
showed best ligand-enzyme interactions were selected and stored for energy calculations.
4.10. Chemistry
Melting points were determined by open tube capillary on a Mel-Temp hot stage
apparatus (Stuart, Staffordshire, UK) on the Celsius scale and are uncorrected. Reactions
were monitored by thin-layer chromatography on silica gel PF254 plates (Merck, Darmstadt,
Germany) using dichloromethane/ethyl acetate (9:1) as mobile phase, and spots were
detected with UV analyses lamp at 254 and 366 nm. IR spectra were recorded as KBr disks

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on a Fourier Transform (FT-IR) spectrophotometer (Bruker, Billerica, MA, USA). 1H-NMR
and 13C-NMR spectra were recordedout on a Bruker NMR spectrometer operating at
400 MHz and 100 MHz, respectively, using TMS as internal reference. Chemical shifts
(δ values) are given in parts per million (ppm) using CDCl3 (7.26) or DMSO-d6 (2.49) as
solvents and coupling constants (J) in Hz. Splitting patterns are designated as follows:
s, singlet; bs, broad singlet; d, doublet; t, triplet; m, multiplet. GCMS: Mass spectra was
recorded on a LCMS-QQQ instrument (Agilent, Santa Clara, CA, USA). Elemental analyses
were performed on a 2400 CHN elemental analyzer (Perkin–Elmer, Waltham, MA, USA)
and were within ± 0.4% of theoretical values unless otherwise specified.
4.11. Materials
Chemicals and solvents are of commercial grade, and purchased from Alfa Aesar
(Haverhill, MA, USA), Cambrian Chemicals (Ontario, CA), Sigma-Aldrich (St. Louis,
MO, USA), Acros Organics (Haverhill, MA, USA), Fluka (Gillingham, Dorset, UK), Merck
(Ontario, CA), and El-Nasr Pharmaceutical Chemical Company (Cairo, Egypt). Chemicals
were used without purification. Solvents were purified following the reported methods.
Substituted 1,3-diaryl-2-propene-1-ones
described elsewhere in the literature [46 51].
phthalimido-o-toluyl chloride ( ) were prepared according to the literature [36]. 4,6-Diaryl-
3,4-dihydropyrimidine-2(1H)-thiones: 5a 5b 5c 5f and 5h were synthesized following the
3
were prepared according to a procedure
–
α
-Phthalimido-o-toluic acid ( ) and α-
8
9
,
,
,
reported procedure in the literatures [52–55].
4.12. Synthesis
4.12.1. General Procedure for Synthesis of 4,6-diaryl-3,4-dihydropyrimidine-2(1H)-thiones
5d–g, i, j [53]
A mixture of 1,3-diaryl-2-propen-1-one derivatives
Claisen-Schmidt condensation between substituted acetophenones
and thiourea (2.404 mmol) and NaOH (2.404 mmol) in absolute ethanol (25 mL) was
3
(2.404 mmol), prepared by
1, benzaldehydes 2,
4
heated under reflux for 4–6 h. The reaction progress was monitored by TLC using
dichloromethane/ethyl acetate (9:1). The reaction mixture was cooled to rt and poured
onto crushed ice. The solid product was filtered off, washed with water and crystallized
from ethanol to yield the following pure titled compounds.
4-(4-Chlorophenyl)-6-(p-tolyl)-3,4-dihydropyrimidine-2(1H)-thione (5d)
Yield: 0.574 g, 76% (pale yellow solid); m.p. 138–9 ◦C; IR (KBr, cm−1
)
υ
: 3183, 2967
10.12 (s,
(Ar-CH), 1672 (C=N), 1551, 1462 (C=C), 1178 (C=S). 1H-NMR (CDCl3, 400 MHz):
δ
1H, 1NH), 9.54 (s, 1H, 1NH), 7.39 (d, 2H, J = 8.1 Hz, ArH), 7.31 (d, 4H, J = 8.1 Hz, ArH),
7.21(d, 2H, J = 8.1 Hz, ArH), 6.38 (d, 1H, J = 4.8 Hz, H5), 5.17 (d, 1H, J = 4.8 Hz, H4),
2.39 (s, 3H, OCH3). 13C-NMR (CDCl3, 100 MHz):
δ 175.5 (CS), 141.5, 140.0, 134.5, 133.7,
129,128.0, 127.7, 125.7, 124.9 (ArC), 99.3 (NH—C=C), 55.5 (CHAr), 21.2 (CH3). GCMS m/z
(rel. abundance): 314 (M.+, 100), 297 (13), 281 (34), 268 (26), 241 (54), 239 (43), 223 (63), 203
(30), 160 (19), 113 (19),100 (11). Anal. Calcd. for C17H15ClN2S (314.83); C, 64.85; H, 4.80; N,
8.90; S, 10.18. Found: C, 64.77; H, 5.01; N, 8.72; S, 10.33.
6-(4-Bromophenyl)-4-(4-(dimethylamino)phenyl)-3,4-dihydropyrimidine-2(1H)-thione (5e
)
Yield: 0.633 g, 68% (orange solid); m.p.112–3 ◦C; IR (KBr, cm−1
2925 (Ar-CH), 1679 (C=N), 1523, 1482 (C=C), 1174 (C=S). 1H-NMR (CDCl3, 400 MHz):
) υ: 3195 (NH), 3084,
δ
10.52 (s, 1H, 1NH), 9.76 (s, 1H, 1NH), 7.63 (d, 2H, J = 8.1 Hz, ArH), 7.57 (d, 2H, J = 8,1 Hz,
ArH), 7.32 (d, 2H, J = 8.1 Hz, ArH), 7.22 (d, 2H, J = 8.1 Hz, ArH), 6.34 (d, 1H, J = 4.8 Hz,
H5), 5.08 (d, 1H, J = 4.8 Hz, H4), 2.98 (s, 6H, NMe2). GCMS m/z (rel. abundance): 388 (M.+,
100), 372 (21), 355 (60), 332 (17), 301 (16), 268 (65), 232 (31), 186 (21), 157 (23), 120 (17). Anal.
Calcd. for C18H18BrN3S (388.32); C, 55.67; H, 4.67; N, 10.32; S, 8.26. Found: C, 55.93; H,
4.72; N, 10.11; S, 8.25.

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6-(4-Chlorophenyl)-4-phenyl-3,4-dihydropyrimidine-2(1H)-thione (5f)
Yield: 0.384 g, 66% (white solid); m.p. 162–3 ◦C; IR (KBr, cm−1
)
υ
: 3188 (NH), 3028,
2979 (Ar-CH), 1671 (C=N), 1569,1486 (C=C), 1190 (C=S). 1H-NMR (CDCl3, 400 MHz):
δ
10.00 (s, 1H, 1NH), 9.67 (s, 1H, 1NH), 7.70–7.27 (m, 17H, ArH), 6.72 (d, 1H, J = 4.8 Hz, H5),
5.24 (d, 1H, J = 4.8 Hz, H4). GCMS m/z (rel. abundance): 300 (M.+, 67), 285 (40), 254 (48),
225 (100), 219 (55), 189 (32), 161 (49), 137 (31), 103 (14). Anal. Calcd. for C16H13ClN2S
(300.81); C, 63.88; H, 4.36; N, 9.31; 10.66. Found: C, 64.06; H, 4.22; N, 10.41; S, 8.14.
6-(4-Chlorophenyl)-4-(4-nitrophenyl)-3,4-dihydropyrimidine-2(1H)-thione (5g)
Yield: 0.589 g, 71% (grey solid); m.p. 210–12 ◦C; IR (KBr, cm−1
)
υ
: 3155 (NH), 3070
10.28 (s,
(Ar-CH), 1678 (C=N), 1552,1493 (C=C), 1177 (C=S). 1H-NMR (CDCl3, 400 MHz):
δ
1H, 1NH), 9.72 (s, 1H, 1NH), 8.30 (d, 2H, J = 8.1 Hz, ArH), 8.10 (d, 2H, J = 8.1 Hz, ArH),
7.59 (d, 2H, J = 8.1 Hz, ArH), 7.42 (d, 2H, J = 8.1 Hz, ArH), 6.43 (d, 1H, J = 4.8 Hz, H5),
5.26 (d, 1H, J = 4.8 Hz, H4). 13C-NMR (CDCl3, 100 MHz):
δ 176.6 (CS), 144.5, 140.0, 135.0,
132.4, 1312.0, 130.0, 128.4, 126.2, 123.8 (ArC), 101.9 (NH-C=C), 52.0 (CHAr). GCMS m/z (rel.
abundance): 345 (M.+, 100), 312 (50), 310 (6), 270 (26), 255 (9), 223 (16), 190 (9), 176 (16), 134
(16), 122 (28). Anal. Calcd. for C16H12ClN3O2S (345.80); C, 55.57; H, 3.50; N, 12.15; S, 9.27.
Found: C, 55.33; H, 3.62; N, 12.23; S, 9.15.
6-(4-Chlorophenyl)-4-(furan-2-yl)-3,4-dihydropyrimidine-2(1H)-thione (5i)
Yield: 0.509 g, 73% (yellow solid); m.p. 165–7 ◦C; IR (KBr, cm−1
) υ: 3187 (NH), 3104,
2984 (Ar-CH), 1676 (C=N), 1571,1477 (C=C), 1186 (C=S). 1H-NMR (CDCl3, 400 MHz)
δ:
10.08 (s, 1H, 1NH), 9.86 (s, 1H, 1NH), 7.69–7.39 (m, 7H, 4 ArH & 3 furyl-H), 6.23 (d, 1H,
J = 4.8 Hz, H5), 5.35 (d, 1H, J = 4.8 Hz, H4). GCMS m/z (rel. abundance): 290 (M.+, 100),
257 (32), 235 (15), 215 (32), 190 (16), 179 (42), 159 (23), 131 (13), 115 (5). Anal. Calcd. for
C14H11ClN2OS (290.77); C, 57.83; H, 3.81; N, 9.63; S, 11.03. Found: C, 58.14; H, 3.69; N, 9.48;
S, 10.83.
6-(4-Aminophenyl)-4-(naphthalen-1-yl)-3,4-dihydropyrimidine-2(1H)-thione (5j)
Yield: 0.555 g, 73% (pale yellow solid); m.p. 155–6 ◦C; IR(KBr, cm−1
3051,2924 (Ar-CH), 1669 (C=N), 1559,1464 (C=C), 1177 (C=S). 1H-NMR (CDCl3, 400 MHz):
9.28 (s, 1H, 1NH), 9.23 (s, 1H, 1NH), 8.16 (d, 1H, J = 8.1 Hz, ArH), 7.88–7.53 (m, 6H, ArH),
) υ: 3217 (NH),
δ
7.43 (d, 2H, J = 8.1 Hz, ArH), 6.75 (d, 2H, J = 8.1 Hz, ArH), 6.68 (bs, 2H, NH2), 6.30 (d, 1H, J
= 4.8 Hz, H5), 5.30 (d, 1H, J = 4.8 Hz, H4). GCMS m/z (rel. abundance): 331 (M.+, 100), 298
(50), 274 (43), 256 (23), 239 (15), 204 (23), 160 (17), 136 (16), 119 (8), 112 (10). Anal. Calcd. for
C20H17N3S (331.43); C, 72.48; H, 5.17; N, 12.68; S, 9.67. Found: C, 72.22; H, 4.93; N, 12.53;
S, 9.9.82.
4.12.2. General Procedure for Synthesis of (4,6-diaryl-1,6-dihydropyrimidin-2-yl)-2-((1,3-
dioxoisoindolin-2-yl)methyl)benzothioates 10a–j
A solution of equimolar amounts of α-phthlimido-o-toluyl chloride (9, 0.668 mmol)
and an appropriate 4,6-diaryl-3,4-dihydropyrimidine-2(1H)-thione 5a–j (0.668 mmol) in
10 mL absolute dichloromethane was stirred at rt for 4–6 h. The progress of the reaction
was monitored periodically by TLC using dichloromethane/ethyl acetate (9:1) as mobile
phase. The reaction mixture was then treated with brine, extracted with ethyl acetate,
treated with dil. hydrochloric acid, sod. bicarbonate and finally washed with water. The
organic solvent was dried with anhydrous sodium sulfate and evaporated under vacuum.
The solid product was purified by crystallization from the appropriate solvent to give the
title hybrids 10a–j.

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S-(4,6-Diphenyl-1,6-dihydropyrimidin-2-yl)2-((1,3-dioxoisoindolin-2-
yl)methyl)benzothioate (10a)
◦
−1
) υ
Yield: 0.233 g, 66% (white solid, ethanol); m.p. 147–8 C; IR (KBr, cm
: 3399 (NH),
3071,3011 (Ar-CH), 1772,1716 (CO), 1603 (C=N), 1547,1491 (C=C). 1H-NMR (DMSO-d6,
400 MHz):
δ
11.46 (s, 1H, NH), 7.90–721 (m, 18 H, ArH), 6.42 (d, 1H, J = 4.8 Hz, CH=C-Ar),
189.7
5.12 (d, 1H, J = 4.8 Hz, CHAr), 5.06 (s, 2H, CH2). 13C-NMR (DMSO-d6, 100 MHz):
δ
(CO), 168.3 (2CO), 156.4, 144.0, 140.5, 136.0, 136.7, 135.5, 135.0, 132.0, 132.6, 131.4, 129.3,
128.4, 127.6, 127.0, 126.7, 126.3, 124.6, 124.0 (ArC), 107.8 (C=CH-Ar), 55.1 (CHAr). (GCMS
m/z (rel. abundance): 529 (M.+, 36), 452 (29), 392 (43), 338 (20), 280 (100), 265 (60), 236 (20),
223 (10), 173 (35), 146 (28). Anal. Calcd for C32H23N3O3S (529.61); C, 72.57; H, 4.38; N, 7.93;
S, 6.05. Found: C, 72.34; H, 4.30; N, 8.07; S, 6.23.
S-(4-(4-Chlorophenyl)-6-(4-methoxyphenyl)-1,6-dihydropyrimidin-2-yl)2-((1,3-
dioxoisoindolin-2-yl)methyl)benzothioate (10b)
◦
Yield: 0.278 g, 70% (pale yellow solid, ethanol); m.p. 142–3 C; IR (KBr, cm−1
3450 (NH), 3064,2918 (Ar-CH), 1772, 1725 (CO), 1676 (C=N), 1594,1489 (C=C). 1H-NMR
(DMSO-d6, 400 MHz): 11.49 (s, 1H, NH), 7.99–6.92 (m, 16 H, ArH), 6.28 (d, 1H, J= 4.8 Hz,
CH=C-Ar), 5.20 (d, 1H, J = 4.8 Hz, CHAr), 5.08 (s, 2H, CH2), 3.76 (s, 3H, OCH3). 13C-NMR
(DMSO-d6, 100 MHz): 188.5 (CO), 168.3 (2CO), 159.4, 145.0, 138.3, 137.8, 136.1, 135.6,
) υ:
δ
δ
135.9, 134.3, 133.7, 132.8, 132.2, 131.5, 129.3, 129.0, 128.5, 128.3, 127.3, 124.5, 120.6, 114.9
(ArC), 111.4 (C=CH-CHAr), 55.5 (CHAr). GCMS m/z (rel. abundance): 593 (M.+, 100), 562
(50), 462 (34), 382 (43), 355 (61), 329 (23), 264 (32), 186 (26), 160 (35), 132 (20). Anal. Calcd.
for C33H24ClN3O4S (594.08); C, 66.72; H, 4.07; N, 7.07; S, 5.40. Found: C, 66.39; H, 4.22; N,
8.11; S, 5.91.
S-(4,6-Bis(4-chlorophenyl)-1,6-dihydropyrimidin-2-yl)2-((1,3-dioxoisoindolin-2-
yl)methyl)benzothioate (10c)
Yield: 0.258 g, 65% (pale yellow solid, ethanol/chloroform); m.p. 118–19 ◦C; IR (KBr,
cm−1
)
υ
: 3432 (NH), 3064, 2964 (Ar-CH), 1772,1616 (CO), 1672 (C=N), 1592, 1489 (C=C).
1H NMR (DMSO-d6, 400 MHz):
δ
11.43 (s, 1H, NH), 7.92–7.19 (m, 16H, ArH), 6.42 (d, 1H,
J = 4.8 Hz, CH=C-Ar), 5.19 (d,1H, J = 4.8 Hz, CHAr), 5.06 (s, 2H, CH2). GCMS m/z (rel.
abundance): 598 (M.+, 100), 487 (86), 466 (20), 568 (37)521 (17), 398 (28), 358 (18), 302 (42),
266 (59), 275 (27), 190 (19), 160 (30), 147 (46), 132 (23). Anal. Calcd. for C32H21Cl2N3O3S
(598.50); C, 64.22; H, 3.54; N, 7.02; S, 5.36. Found: C, 64.00; H, 3.3.78; N, 7.21; S, 5.30.
S-(6-(4-Chlorophenyl)-4-(p-tolyl)-1,6-dihydropyrimidin-2-yl)2-((1,3-dioxoisoindolin-2-
yl)methyl)benzothioate (10d)
◦
Yield: 0.282 g, 73% (yellow solid, ethanol); m.p. 144–6 C; IR(KBr, cm−1
(NH), 3061,3041, 2922 (Ar-CH), 1771,1715 (CO), 1677 (C=N), 1545, 1490 (C=C). 1H-NMR
(DMSO-d6, 400 MHz): 11.45 (s, 1H, 1NH), 7.89–7.18 (m, 16H, ArH), 6.34 (d,1H, J = 4.8 Hz,
) υ: 3413
δ
CH=C-Ar), 5.20 (d,1H, J = 4.8 Hz, CH-Ar), 5.08 (s, 2H, CH2), 2.34 (s, 3H, CH3). GCMS m/z
(rel. abundance): 577.5 (M.+, 31), 487 (70), 466 (100), 457 (20), 385 (30), 375 (21), 314 (39), 297
(81), 264 (57), 217 (10), 160 (26), 146 (22), 132 (16). Anal. Calcd. for C33H24ClN3O3S (578.08);
C, 68.56; H, 4.18; N, 7.27; S, 5.55. Found: C, 68.84; H, 4.01; N, 7.37; S, 5.23.
S-(4-(4-Bromophenyl)-6-(4-(dimethylamino)phenyl)-1,6-dihydropyrimidin-2-yl)
2-((1,3-dioxoisoindolin-2-yl)methyl)benzothioate (10e)
◦
Yield: 0.304 g, 70% (brown solid, ethanol/chloroform); m.p. 135–6 C; IR (KBr,
−1
cm
)
υ
: 3476 (NH), 2920 (Ar-CH), 1770,1715 (CO), 1674 (C=N), 1578,1485(C=C). 1H-NMR
(DMSO-d6, 400 MHz):
δ
11.45 (s, 1H, NH), 8.08–6.75 (m, 16H, ArH), 6.34 (d, 1H, J = 4.8 Hz,
CH=C-Ar), 5.18 (d, 1H, J = 4.8 Hz, CHAr), 5.05 (s,2H, CH2), 2.52 (s, 6H, NMe2). GCMS m/z
(rel. abundance): 651 (M.+, 41), 569 (19), 546 (100), 531 (13), 519 (18), 415 (21), 362 (49), 302

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(36), 264 (30), 236 (18), 202 (12), 146 (16), 132 (24). Anal. Calcd. for C34H27BrN4O3S (651.57);
C, 62.67; H, 4.18; N, 8.60; S, 4.92. Found: C, 62.93; H, 4.28; N, 8.44; S, 4.88.
S-(4-(4-Chlorophenyl)-6-phenyl-1,6-dihydropyrimidin-2-yl)2-((1,3-dioxoisoindolin-2-
yl)methyl)benzothioate (10f)
◦
Yield: 0.282 g, 75% (pale yellow solid, ethanol); m.p. 143–4 C; IR (KBr, cm−1
3464 (NH), 3061, 2970 (Ar-CH), 1772,1716 (CO), 1677 (C=N), 1557,1490 (C=C). 1H-NMR
(DMSO-d6, 400 MHz): 11.42 {s, 1H, NH), 8.18–7.15 (m, 17H, ArH), 6.45 (d, 1H, J = 4.8 Hz,
) υ:
δ
CH=C-Ar), 5.16 (d, 1H, J = 4.8 Hz, CH-Ar), 5.01 (s,2H, CH2). GCMS m/z (rel. abundance);
563.5 (M.+, 100), 543 (48), 443 (43), 414 (38), 389 (18), 375 (36), 300 (28), 265 (31), 227 (24),
161 (36), 146 (27), 132 (17). Anal. Calcd. for C32H22ClN3O3S (564.05); C, 68.14; H, 3.93; N,
7.45; S, 5.68. Found: C, 67.83; H, 4.17; N, 7.27; S, 5.55.
S-(4-(4-Chlorophenyl)-6-(4-nitrophenyl)-1,6-dihydropyrimidin-2-yl)2-((1,3-
dioxoisoindolin-2-yl)methyl)benzothioate (10g)
◦
−1
) υ
Yield: 0.280 g, 69% (brown solid, ethanol/chloroform); m.p.161–3 C; IR (KBr, cm
3418 (NH), 3030,2926 (Ar-CH), 1774,1715 (CO), 1623 (C=N), 1521 (C=C). 1H-NMR (DMSO-
d6, 400 MHz): 11.52 (s, 1H, NH), 8.51–7.15 (m, 16H, ArH), 6.39 (d, 1H, J = 4.8 Hz, CH=C-
Ar), 5.21 (d, 1H, J = 4.8 Hz, CHAr), 5.02 (s, 2H, CH2). 13C-NMR (DMSO-d6, 100 MHz):
:
δ
δ
188.4 (CO), 168.3 (2CO), 157.5, 148.0, 146.1, 142.0, 137.9, 137.7, 135.3, 135.0, 133.7, 132.5,
131.8, 129.8, 129.0, 128.1, 127.4, 126.1, 125.1, 124.6, 124.5 (ArC), 112.0 (C=CH—CHAr), 45.2
(CHAr). GCMS m/z (rel. abundance): 608.5 (M.+, 50), 551 (17), 532 (25), 504 (55), 446 (31),
373 (100), 346 (22), 311 (25), 306 (9), 264 (39), 232 (21), 188 (31), 163 (16). Anal. Calcd. for
C32H21ClN4O5S (609.05); C, 63.11; H, 3.48; N, 9.20; S, 5.26. Found: C, 62.88; H, 3.59; N, 9.18;
S, 5.17.
S-(4-(4-Aminophenyl)-6-(4-chlorophenyl)-1,6-dihydropyrimidin-2-yl)2-((1,3-
dioxoisoindolin-2-yl)methyl)benzothioate (10h)
Yield: 0.285 g, 74% (orange solid, ethanol/chloroform); m.p. 185–7 ◦C; IR(KBr, cm−1
: 3430 (NH), 3060,2942 (Ar-CH), 1772,1716 (CO), 1677 (C=N), 1545,1490 (C=C). 1H-NMR
(DMSO-d6, 400 MHz): 11.48 (s, 1H, NH), 8.10–6.98 (m, 16H, ArH), 6.68 (d,1H, J = 4.8 Hz,
)
υ
δ
CH=C-Ar), 6.19 (bs, 2H, NH2), 5.23 (d, 1H, J = 4.8 Hz, CHAr), 5.03 (s, 2H, CH2). GCMS m/z
(rel. abundance): 578.5 (M.+, 47), 514 (9), 432 (22), 394 (11), 342 (100), 312 (49), 282 (46), 264
(34), 238 (27), 196 (17), 140 (14), 132 (11). Anal. Calcd. for C32H23ClN4O4S (579.07); C, 66.37;
H, 4.00; N, 9.68; S, 5.54. Found: C, 66.20; H, 3.83; N, 9.83; S, 5.50.
S-(4-(4-Chlorophenyl)-6-(furan-2-yl)-1,6-dihydropyrimidin-2-yl)2-((1,3-dioxoisoindolin-2-
yl)methyl)benzothioate (10i)
◦
Yield: 0.274 g, 74% (grey solid, ethanol); m.p. 146–8 C; IR (KBr, cm−1
(NH), 3064,2928 (Ar-CH), 1770,1716 (CO), 1666 (C=N), 1502 (C=C). 1H-NMR (DMSO-d6,
400 MHz): 11.19 (s,1H, NH), 7.92–7.09 (m, 12 ArH and 3 furanyl-H), 6.25 (d, 1H, J = 4.8 Hz,
) υ: 3464
δ
CH=C-Ar), 5.21 (d, 1H, J = 4.8 Hz, CHAr), 5.02 (s, 2H, CH2). GCMS m/z (rel. abundance):
553.5 (M.+, 100), 511 (11), 449 (41), 393 (20), 315 (44), 287 (18), 254 (23), 238 (26), 264 (20),
160 (21), 132 (16). Anal. Calcd. for C30H20ClN3O4S (554.02); C, 65.04; H, 3.64; N, 7.58; S,
5.79. Found: C, 65.33; H, 3.78; N, 7.42; S, 5.51.
S-(4-(4-Aminophenyl)-6-(naphthalen-1-yl)-1,6-dihydropyrimidin-2-yl)2-((1,3-
dioxoisoindolin-2-yl)methyl)benzothioate (10j)
◦
−1
) υ
Yield: 0.287 g, 74% (orange solid, ethanol); m.p. 176–8 C; IR (KBr, cm
3062,2975 (Ar-CH), 1771,1714 (CO), 1667 (C=N), 1598,1467 (C=C). 1H-NMR (DMSO-d6,
400 MHz): 11.52 (s, 1H, NH), 8.50–7.12 (m, 18H, ArH), 6.58 (d, 1H, J = 4.8 Hz, CH=C-Ar),
: 3384 (NH),
δ

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6.17 (bs, 2H, NH2), 5.23 (d, 1H, J = 4.8 Hz, CHAr), 5.05 (s, 2H, CH2). 13C-NMR (DMSO-d6,
100 MHz):
δ 188.1 (CO), 167.5 (2CO), 157.5, 145.3, 143.1, 137, 136.6, 135.8, 135.5, 135.3, 134.3,
134.0, 133.5, 132.5,132.3, 130.8, 129.7, 129.1, 128.9, 128.6, 127.8, 126.0, 125.5, 125.0, 124.0,
123.5 (ArC),113.3 (C=CH-Ar), 55.0 (CHAr). GCMS m/z (rel. abundance): 594 (M.+, 42), 475
(28), 434 (16), 358 (100), 330 (29), 317 (10), 264 (28), 232 (11), 127 (15), 133 (27), 104 (9). Anal.
Calcd. for C36H26N4O3S (594.68); C, 72.71; H, 4.41; N, 9.42; S, 5.39. Found: C, 72.63; H, 4.62;
N, 9.65; S, 5.23.
5. Conclusions
A novel series of dihydropyrimidine phthalimide hybrids was synthesized, charac-
terized and evaluated for their DPP-4 inhibitory activity, as well as, antioxidant activity.
Hybrids 10g, 10i, 10e, 10d and 10b showed superior DPP-4 inhibitory activity compared to
alogliptin, in the in vitro DPP-4 inhibition assay. Additionally, hybrids 10g and 10i have
the most potent and longest DPP-4 inhibitory activity among the synthesized hybrids
and alogliptin, in the in vivo study that carried on SD rats. Furthermore, investigation of
antihyperglycemic effect of our synthesized hybrids in HFD-induced type 2 diabetic rats,
revealed that hybrids 10g
, 10i and 10e exhibited better glycemic control than alogliptin;
this effect was further potentiated by addition of MET. Notably, glucose tolerance reached
a normal level in 10g/MET treated group. Finally, hybrids 10j, 10e, 10h and 10d showed
potent radical scavenging activity in DPPH assay.
Supplementary Materials: IR spectroscopy, 1H-NMR, 13C-NMR and mass spectroscopy of all the
synthesized compounds are available online at https://www.mdpi.com/1424-8247/14/2/144/s1,
Figure S1: IR spectrum of compound 5a, Figure S2: IR Spectroscopy of compound 5b, Figure S3:
IR Spectroscopy of compound 5c, Figure S4: IR Spectroscopy of compound 5d, Figure S5: IR Spec-
troscopy of compound 5e, Figure S6: IR Spectroscopy of compound 5f, Figure S7: IR Spectroscopy
of compound 5g, Figure S8: IR Spectroscopy of compound 5h, Figure S9: IR Spectroscopy of com-
pound 5i, Figure S10: IR Spectroscopy of compound 5j, Figure S11: IR Spectroscopy of compound
10a, Figure S12: IR Spectroscopy of compound 10b, Figure S13: IR Spectroscopy of compound
10c, Figure S14: IR Spectroscopy of compound 10d, Figure S15: IR Spectroscopy of compound
10e, Figure S16: IR Spectroscopy of compound 10f, Figure S17: IR Spectroscopy of compound 10g
,
,
,
Figure S18: IR Spectroscopy of compound 10h, Figure S19: IR Spectroscopy of compound 10i
1
Figure S20: IR Spectroscopy of compound 10j, Figure S21: H NMR Spectroscopy of Compound 5b
1
1
Figure S22: H-NMR spectrum of compound 5c, Figure S23: H NMR Spectroscopy of Compound
5d, Figure S24: 1H NMR Spectroscopy of Compound 5e, Figure S25: 1H NMR Spectroscopy of
Compound 5f, Figure S26: 1H NMR Spectroscopy of Compound 5g, Figure S27: 1H NMR Spec-
1
1
troscopy of Compound 5h, Figure S28: H NMR Spectroscopy of Compound 5i, Figure S29: H NMR
1
Spectroscopy of Compound 5j, Figure S30: H NMR Spectroscopy of Compound 10a, Figure S31:
1H NMR Spectroscopy of Compound 10b, Figure S32: 1H NMR Spectroscopy of Compound 10c
,
1
1
Figure S33: H NMR Spectroscopy of Compound 10d, Figure S34: H NMR Spectroscopy of Com-
1
1
pound 10e, Figure S35: H NMR Spectroscopy of Compound 10f, Figure S36: H NMR Spectroscopy
of Compound 10g, Figure S37: 1H NMR Spectroscopy of Compound 10h, Figure S38: 1H NMR
1
Spectroscopy of Compound 10i, Figure S39: H NMR Spectroscopy of Compound 10j, Figure S40:
13C NMR Spectroscopy of Compound 5a, Figure S41: 13C NMR Spectroscopy of Compound 5d
,
Figure S42: 13C NMR Spectroscopy of Compound 5g, Figure S43: 13C NMR Spectroscopy of Com-
pound 5h, Figure S44: 13C NMR Spectroscopy of Compound 10a, Figure S45: 13C NMR Spectroscopy
of Compound 10b, Figure S46: 13C NMR Spectroscopy of Compound 10d, Figure S47: 13C NMR
Spectroscopy of Compound 10g, Figure S48: 13C NMR Spectroscopy of Compound 10j, Figure S49:
Mass Spectroscopy of Compound 5b, Figure S50: Mass Spectroscopy of Compound 5c, Figure S51:
Mass Spectroscopy of Compound 5d, Figure S52: Mass Spectroscopy of Compound 5e, Figure S53:
Mass Spectroscopy of Compound 5f, Figure S54: Mass Spectroscopy of Compound 5g, Figure S55:
Mass Spectroscopy of Compound 5h, Figure S56: Mass Spectroscopy of Compound 5i, Figure S57:
Mass Spectroscopy of Compound 5j, Figure S58: Mass Spectroscopy of Compound 10a, Figure S59:
Mass Spectroscopy of Compound 10b, Figure S60: Mass Spectroscopy of Compound 10c, Figure S61:
Mass Spectroscopy of Compound 10d, Figure S62: Mass Spectroscopy of Compound 10e, Figure S63:
Mass Spectroscopy of Compound 10f, Figure S64: Mass Spectroscopy of Compound 10g, Figure S65:

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Mass Spectroscopy of Compound 10h, Figure S66: Mass Spectroscopy of Compound 10i, Figure S67:
Mass Spectroscopy of Compound 10j.
Author Contributions: Conceptualization, A.A.E.M. and M.A.E.M.; methodology, A.A.E.M.; soft-
ware, A.A.E.M. and S.S. validation, A.E.K., S.S. and A.A.E.M.; resources, A.A.E.M.; M.A.E.M.; S.S.
and A.E.K.; writing—review and editing, A.M., A.E.K. and M.M. All authors have read and agreed
to the published version of the manuscript.
Funding: This research received no external funding.
Institutional Review Board Statement: All the in vivo protocols were approved by animal research
ethics committee, faculty of pharmacy, Port-Said University, Egypt.
Informed Consent Statement: Not applicable.
Data Availability Statement: The data presented in this study are openly available at doi.
Conflicts of Interest: The authors declare no conflict of interest.
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