Discovery of novel 1,3,5-triazine-thiazolidine-2,4-diones as dipeptidyl peptidase-4 inhibitors with antibacterial activity targeting the S1 pocket for the treatment of type 2 diabetes

Article abstract of DOI:10.1039/c4ra16903d

A novel series of 1,3,5-triazine-thiazolidine-2,4-diones was synthesized and characterized by a number of analytical and spectroscopic techniques. The molecules were screened for the in vitro inhibition of dipeptidyl peptidase-4 and compound 7a showed the most prominent inhibition with IC₅₀ = 6.25 μM. The other compounds showed considerable inhibition (IC₅₀ = 12.11-49.21 μM). Docking studies indicated that the lipophilic thiazolidine-2,4-dione fragment of ligand 7a was oriented towards the tight lipophilic cavity of the S1 pocket of the active site formed by residues such as Tyr631, Val656, Trp659, Tyr662, Tyr666 and Val711 via the formation of H-bonds with Tyr547. One of the amines present on the wings of the triazine formed a hydrogen bond with Glu205, a vital residue for the N-terminal recognition site with an efficient CDOCKER interaction energy. In a bacterial inhibition study, the entire set of compounds showed excellent activity and, in some instances, were found to be equipotent to the cefixime used as a standard.

Full text of DOI:10.1039/c4ra16903d

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Page 1 of 23
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DOI: 10.1039/C4RA16903D
Discovery of novel 1,3,5-triazine-thiazolidine-2,4-diones as dipeptidyl peptidase-4
(DPP - 4) inhibitor targeting S1 pocket for the treatment of type 2 diabetes along with
antibacterial activity
Jitendra Kumar Shrivastava,1 Pragya Dubey,1 Saumya Singh,1 Hans Raj Bhat,1 Mukesh Kumar Kumawat,2
Udaya Pratap Singh1*
1 Drug Design & Discovery Laboratory, Department of Pharmaceutical Sciences, Sam Higginbottom
Institute of Agriculture, Technology & Sciences, Allahabad, India, 211007
2 Anand College of Pharmacy, Agra, India, 282007
Graphical Abstract

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DOI: 10.1039/C4RA16903D
Discovery of novel 1,3,5-triazine-thiazolidine-2,4-diones as dipeptidyl peptidase-
4 (DPP - 4) inhibitor targeting S1 pocket for the treatment of type 2 diabetes
along with antibacterial activity
Jitendra Kumar Shrivastava,1 Pragya Dubey,1 Saumya Singh,1 Hans Raj Bhat,1 Mukesh Kumar
Kumawat,2 Udaya Pratap Singh1*
1 Drug Design & Discovery Laboratory, Department of Pharmaceutical Sciences, Sam
Higginbottom Institute of Agriculture, Technology & Sciences, Allahabad, India, 211007
2 Anand College of Pharmacy, Agra, India, 282007
Corresponding author
Udaya Pratap Singh
Drug Design & Discovery Laboratory, Department of Pharmaceutical Sciences, Sam Higginbottom Institute
of Agriculture, Technology & Sciences, Allahabad, India
E mail: udaysingh98@gmail.com; udaya.singh@shiats.edu.in

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Abstract
A novel series of 1,3,5ꢀtriazineꢀthiazolidineꢀ2,4ꢀdiones have been synthesized and characterized
with the aid of numerous analytical and spectroscopic techniques. These molecules were screened
for in vitro inhibition of dipeptidyl peptidaseꢀ4, where compound 7a showed most prominent
inhibition with IC50 = 6.25 µM. The rest of the compound showed considerable inhibition (IC50
=
12.11 – 49.21 µM). Docking studies indicate that, lipophillic thiazolidineꢀ2,4ꢀdione fragment of the
ligand 7a found oriented towards the tight liphophillic cavity of S1 pocket of the active site formed
by residues like, Tyr631, Val656, Trp659, Tyr662, Tyr666 and Val711 via formation of Hꢀbond
with Tyr547. Moreover, one of the amine present on the wings of triazine forms a hydrogen bond
with Glu205, a vital residue for the Nꢀterminal recognition site with efficient CDOCKER
interaction energy. In bacterial inhibition study, the entire set of compounds showed excellent
activity and even in some cases found equipotent to Cefixime as standard.

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Introduction
Diabetes is frequently concerned to by clinicians as diabetes mellitus, which further
classified into type 1 (previously known as insulinꢀdependent) and type 2 (formerly called nonꢀ
insulinꢀdependent). It depicts a group of metabolic diseases in which either the pancreas does not
make adequate insulin or when the body cannot effectively use the insulin it produces, or both.1 The
burden of disease is increasing globally, especially in developing nations which contribute more
than 80% of diabetes deaths.2 By an estimate, in 2012, 1.5 million deaths were induced by diabetes
and till 2030, it will be the 7th leading cause of morbidity and death rate across the world.3
In type 2 diabetic patients, besides classical insulinꢀbased treatment, inhibition of the serine
protease dipeptidyl peptidase 4 (DPPꢀ4) has proven to be an effective treatment for improving
glycemic control.4 DPPꢀ4 is also known as CD26 (cluster of differentiation 26 or Tꢀcell activation
antigen CD26) or is adenosine deaminase complexing protein 2 which selectively cleaves first two
amino acids of glucoseꢀdependent insulinotropic polypeptide (GIP) and glucagonꢀlike peptide
(GLPꢀ1) thereby makes it inactive. Accordingly, inhibition of DPPꢀ4 by a chemotherapeutic agent
may lead to increase the levels of circulating endogenous GLPꢀ1 by prolonging its halfꢀlife and
consequently enhances the beneficial effects of GLPꢀ1 in glucose dependent insulin secretion and βꢀ
cell restoration. The DPPꢀ4 inhibitor results in higher circulating concentrations of endogenous
GLPꢀ1 and subsequent decrease in plasma glucose, by enhancing βꢀcell glucoseꢀstimulated insulin
release and promoting insulin gene expression and its biosynthesis, fig 1.5
<Figure 1>
Imeglimin
((6R)ꢀ(+)ꢀ4ꢀdimethylaminoꢀ2ꢀiminoꢀ6methylꢀ1,2,5,6ꢀtetrahydroꢀ1,3,5ꢀtriazine
hydrochloride) is the first in a new tetrahydrotriazineꢀcontaining class of oral antidiabetic agents,
the glimins. It is currently in phase 2b clinical development.6 In preclinical studies, imeglimin has
been shown to reduce excessive hepatic glucose production, increase glucose uptake in skeletal
muscle, and improve insulin secretion in response to glucose via acting on the liver, muscle and the
pancreas, three key organs involved in the pathophysiology of type 2 diabetes. Imeglimin offers a
unique mechanism of action that targets the mitochondria and is compatible with drugs that counter
insulin resistance or enhance insulin secretion and βꢀcell protection.7
Thiazolidinꢀ2,4ꢀdiones (TZDs), a class of oral insulin sensitizing agents that improves
insulin resistance, are agonist of proxisome proliferator activated receptorꢀ γ (PPAR ꢀ γ). These
molecules amplify PPAR ꢀ γ expression in the adipose tissue thus increasing adipocytes and
subcutaneous adipose tissue mass. Increased PPAR ꢀ γ expression in the adipose tissue results in
increased fatty acid uptake and storage by increasing the transcription of fatty acid transport
proteinꢀ1 and acylꢀcoenzyme A synthetase. Decreased circulating free fatty acid levels protect β

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cells, the liver, and the skeletal muscle from their toxic effects, thus improving insulin sensitivity.8
Of TZDs, ciglitazone, pioglitazone (PGZ), rosiglitazone (RGZ), troglitazone (TGZ) (approved in
1997) and englitazone have been clinically examined.9
According to American Diabetes Association combinations of oral agents than monotherapy
have been used to attack the pathophysiology of diabetes at multiple points in cases where insulin
secretion is still moderate.10 Combination therapy includes treatment with two or more agents with
different, complementary mechanisms of action, for example, the combination of a
thiazolidinedione and a biguanide improves insulin sensitivity and lowers blood glucose through
complementary pathways, and therefore produces an additive effect. Nevertheless, sometimes, this
strategy fails because of incomplete and altered pharmacokinetics of the combined drugs which
affects their bioavailibity.11 Molecular hybridization is an innovative approach gaining attention
from medicinal chemists owing to resemblance with combination therapies where two diverse
pharmacophoric groups were joined. These drugs can indeed be more powerful than either of their
precursors because of dual drug targeting at more than one site and privileged activity when
compared to the individual agent.12
Considering the clinical implication of DPPꢀ4 inhibitors, advantages of molecular
hybridisation and antidiabetic potential of 1,3,5ꢀtriazine and thiazolidineꢀ2,4ꢀdiones, we report
design, synthesis and biological evaluation of novel hybrid 1,3,5ꢀtriazineꢀthiazolidineꢀ2,4ꢀdiones as
DPPꢀ4 inhibitor along with antibacterial activity.
Results and Discussion
Chemistry
In our approach, the synthesis of novel hybrid conjugates was accomplished in multiꢀstep
reaction as outlined in scheme 1. The synthesis of thiazolidinꢀ2,4ꢀdione (3) was achieved by
reacting chloro acetic acid (1) and thiourea (2) in the presence of water via formation of the
thiouronium salt, which later cyclises in the presence of an acid to afford the corresponding product
3 in good yield. Reaction of 2,4,6ꢀtri chloro 1,3,5ꢀtriazine (4) with distinguished amines 5 (aꢀk) in
the presence of activating base yielded mono chloro diꢀsubstituted 1,3,5ꢀtriazine 6 (aꢀk) upon
stirring for desired time, step 2. The clubbing of previously obtained product from two different
steps viz., 3 and 6 (aꢀk) leads to the generation of novel target hybrid conjugates 7 (aꢀk) via
nucleophilic reaction in the presence of K2CO3 as a base.
<SCHEME 1>
Inhibitory activity against DPP-4 in vitro
The synthesized 1,3,5ꢀtriazineꢀthiazolidineꢀ2,4ꢀdiones derivatives 7 (a–k) were evaluated in
vitro for DPPꢀ4 enzyme inhibitory activity, and results obtained are reported as micromolar
inhibitory concentration, IC50 (µM) along with standard (P 32 / 98) in table 1. In a comparison test,

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compound 7a containing hydrazine as substituent was identified as very potent analogue of the
series (IC50 = 6.25 µM). Whereas, the twofold decline in activity was reported on the introduction
of bulky aromatic substitution (IC50 = 12.11 µM), compound 7b. A marginal progress in activity
was indicated by compound 7c having oꢀNH2 as substituent on the phenyl. While on the
introduction of NO2 (compound 7d) in place of NH2 render compound less potent, and much more
decline in activity was reported on insertion of Cl (compound 7g). The optimization strategy has
been then focused to study the optimal placement of these functional groups. Compound 7e and 7f
having mꢀ and pꢀNO2 has been led to further decline in activity. Moreover, the same pattern of
activity was not being observed and showed comparative weak inhibition in the case of compounds
7h and 7i containing mꢀ and pꢀCl. A significant decline in activity was reported by compounds 7j
and 7k having pꢀF and a pꢀBr substituent, IC50 = 38.37 µM and 49.21 µM, respectively.
Structuralꢀactivity relationship study suggests that, the presence of substituent has a
substantial influence on the inhibitory activity. It has been clearly revealed that, the presence of
bulky substituent endangers the activity and further decrease was observed on the introduction of
the substituent. The pattern of the SAR has been clearly depicted in fig 2.
<Figure 2>
Molecular Docking Studies
Dipeptidyl peptidaseꢀ4 enzyme consists of two domains, a eightꢀstranded βꢀpropeller
domain at the Nꢀterminus and an α/βꢀhydrolase domain at the Cꢀterminus. The Xꢀray crystal
study of various inhibitors in complex with DPPꢀ4 revealed that the binding site of DPPꢀ4
mainly comprises three parts: a S1 pocket, the Nꢀterminal recognition region, and a S2
pocket.13 The S1 hydrophobic pocket comprising residues Tyr631, Val656, Trp659, Tyr662,
Tyr666 and Val711 adjacent to catalytic triad of active site. The active site is situated in the
hydrolase domain and comprised of catalytic triad residues Ser630, Asp708 and His740. The
Nꢀterminal recognition region is formed by Glu205, Glu206, and Tyr662, while the
hydrophobic S2 pocket, which is larger compared with S1, is surrounded by residues
Phe357, Arg358, Ser209, and Tyr666.
With inꢀvitro results inꢀhand, it is worthwhile to perform the molecular docking
study of most active compound 7a with DPPꢀ4 inhibitor to explicate key molecular
interactions (2FJP.pdb). Thereupon, as depicted in fig. 3, it has been found that one of the
amine present on the wings of triazine forms a hydrogen bond with Glu205, a vital residue
for the Nꢀterminal recognition site. In the new orientation, amine present on the other wing
protrudes towards the Ser630 and Tyr631 of S1 pocket by creating a hydrogen bond with
neighboring Tyr547. The lipophillic thiazolidineꢀ2,4ꢀdione fragment of the ligand found

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oriented towards the S1 pocket of the active site formed by residues like, Tyr631, Val656, Trp659,
Tyr662, Tyr666 and Val711. Additionally, the creation of one piꢀcation interaction was also
reported between 1,3,5ꢀtriazine and Arg105 of S1 pouch. Scoring parameters indicate that
compound 7a bind more firmly to the S1 pocket of the DPPꢀ4 by the virtue of efficient CDOCKER
interaction energy and CDOCKER energy. As a consequence of this tight binding, the residence
time of the 7a increases in DPPꢀ4 which may transform into higher inhibitory activity inꢀvitro. The
engulfment of compound 7a in the S1 pocket of DPPꢀ4 was found in accordance with the earlier
studies which states that, small fragments target tight liphophillic cavity of the S1 pocket.14
The docking of least active compound 7k in the DPPꢀ4 binding site revealed the reason for
its nonꢀactivity. It was found that, thiazolidineꢀ2,4ꢀdione fragment of ligand 7k positioned towards
the S2 pocket of binding site by making Hꢀbonds with Arg358 and Arg669 owing to its extensive
hydrophobic character, fig 4. While, it also create Hꢀbond with Glu205 of Nꢀterminal recognition
site with different CDOCKER energies.
The docking study suggested that optimum balance between hydrophobicꢀhydrophillic
character favors to increase the bioactivity (7a), while extensive hydrophibic character as seen in
compound 7k lead to loss of activity. It was also recommended that, for better activity, the size of
molecule should be kept small enough to fit into S1 pocket rather that big which detoriates the bioꢀ
activity.
<FIGURE 3>
<FIGURE 4>
Antibacterial activity
The immune dysfunction in diabetes patients due to hyperglycemic environment leads to
more frequent and/or serious infectious diseases, which potentially increases their morbimortality.15
However the efficacy of the drugs used to treat these infections, so called antibiotics has been
seriously compromised by the resistance and necessitates the discovery of newer agents.16 In
continuation of our research endeavours towards the discovery of novel antibacterial agents from
1,3,5ꢀtriazine derivatives,17 these constitutive derivatives were tested against panel of diverse
microorganisms.
The newly prepared compounds were screened for determination of their minimal inhibitory
concentration (MIC) against selected Gramꢀpositive organisms viz. Bacillus subtilis (NCIMꢀ2063),
Bacillus cereus (NCIMꢀ2156), Staphylococcus aureus (NCIMꢀ2079) and Gramꢀnegative organism
viz. Escherichia coli (NCIMꢀ2065), Proteus vulgaris (NCIMꢀ2027) and Pseudomonas aeruginosa
(NCIMꢀ2036), broth microdilution (in tubes) method of the Clinical and Laboratory Standards

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Institute (CLSI) with minor modifications using Cefixime as standard and results are
mentioned in table 3.
In a comparison test, the entire set of hybrid derivative showed moderate to excellent
activities against tested Gramꢀpositive and Gramꢀnegative microorganisms in comparison to
a standard. Compound 7a showed moderate to no activity against tested organisms. The
compound 7b showed equipotent activity to standard against E. coli and found moderately
active against the rest of the strains. The compound 7c showed improved activity in
comparison to standard against E.coli, B. Subtilis and S. aureus with moderate activity
against rest of the strains. The presence of NO2 (7d) renders compound more active against
all the strains except P. aeruginosa and P. vulgaris where it displayed moderate activity in
comparison to the previous analogue. The isomeric replacements of NO2, as seen in the case
of compound 7e and 7f does not display any significant change on the activity profile except
in the case of B. Subtilis (for 7f), where it showed almost no activity. In the case of P.
vulgaris, compound 7gꢀi showed considerable activity, while mild to no activity was
reported by compound 7j and 7k. Moreover, these compounds, i.e. 7 (gꢀj) showed moderate
activities against E. coli except compound 7k. In the case of P. aeruginosa, mꢀchloro
substituted analogue (7h) showed prominent inhibition than their isomeric counterparts,
compounds 7g and 7i. The compound 7j and 7k showed mild to no activity against P.
aeruginosa, respectively. It is noteworthy to mention that, against entire tested Gram
positive microorganism, halogen substituted compounds showed mild to moderate activity,
7 (gꢀk).
Experimental
Melting points of the synthesized compounds was determined in an open capillary tube
Hicon Melting point apparatus and are uncorrected. Thin layer chromatography (TLC) was
performed on silica gelꢀG coated plates to detect the completion of the reaction. The diverse mobile
phase was selected in different proportion according to the assumed polarity of the products. The
spots was visualized by exposure to the Iodine vapor. InfraꢀRed (IR) spectra were recorded in KBr
1
on Biored FTs spectrophotometer and the reported wave numbers are given in cmꢀ1. H NMR
spectra were recorded in DMSO on Bruker Model D9RXꢀ300MHz spectrometer. Chemical shifts
were reported as δ (ppm) relative to TMS as internal standard. Mass spectra were obtained on
Waters QꢀTOF MICROMA SS (LCꢀMS) .
General Procedure
Step 1
The synthesis of thiazolidinone (3) was performed according to a published procedure.18

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Step 2
The synthesis of diꢀsubstituted 1,3,5ꢀtriazines 6 (aꢀk) were performed in accordance with the earlier
reported procedure. 17a
Step 3
6ꢀChloroꢀN2,N4ꢀbis(substituted phenyl)ꢀ1,3,5ꢀtriazineꢀ2,4ꢀdiamine 6 (aꢀk) (0.1 mol) was added into
50 mL of dioxane at temperature 40–45 °C. A solution of thiazolidineꢀ2,4ꢀdione (3) (0.1 mol) in 35
mL dioxane was added slowly to the above solution and stirred for 2 h followed by dropꢀwise
addition of K2CO3 (0.1 mol), refluxed the reaction mixture at 120ꢀ135 °C for 3ꢀ6 h. The completion
of the reaction was monitored by TLC using benzene and ethyl acetate (9:1) as mobile phase. The
reaction mixture was washed thoroughly with water, filtered and further purified by column
chromatography to afford pure products 7 (aꢀk).
3ꢀ(4,6ꢀdihydrazinylꢀ1,3,5ꢀtriazinꢀ2ꢀyl)thiazolidineꢀ2,5ꢀdione, 7a
Dark brown powder; M.P.: >300 °C; FTꢀIR (νmax; cmꢀ1, in KBr): 3259.03 (ꢀNHꢀ Stretching),
1613.67 (ꢀC=O Stretching), 1599.12ꢀ1445.77(ꢀNH2 bending), 1385.41ꢀ1222.59(CꢀN Stretching),
687.11 (CꢀS Stretching); 1H NMR (300MHz, CDCl3ꢀd6, TMS) δ ppm: 2.52ꢀ2.54 (d, 4H, 2 x –NH2 ꢀ
hydrazine), 3.32ꢀ3.34 (d, 4H, 2 x –NHꢀ ꢀhydrazine), 3.59 (s, 2H, ꢀCH2ꢀ thiazolidine); 13C NMR
(400MHz, CDCl3) δ ppm: 163.84 (C=O, thiazolidine), 138.27 (C, triazine), 128.24 (C, triazine),
123.22 (C=O, thiazolidine), 78.34 (ꢀCH2ꢀ, thiazolidine); Mass Spectra (TOF MS ES+, m/z):
Calculated 256.05; Observed 257.20 (M+1).
3ꢀ(4,6ꢀbis(phenylamino)ꢀ1,3,5ꢀtriazinꢀ2ꢀyl)thiazolidineꢀ2,5ꢀdione, 7b
White powder; M.P.: >300 °C; FTꢀIR (νmax; cmꢀ1, in KBr): 3366.40 (ꢀNHꢀ Stretching), 1605.32 (ꢀ
C=O Stretching), 1573.76ꢀ1426.76 (ꢀNH2 bending), 1338.47ꢀ1238.92 (CꢀN Stretching), 667.75 (Cꢀ
S Stretching); 1H NMR (300MHz, CDCl3ꢀd6, TMS) δ ppm: 3.58 (s, 2H, 2 x ꢀNHꢀAr), 3.88 (s, 2H, ꢀ
CH2ꢀ thiazolidine), 6.59ꢀ6.61 (d, 4H, J=6Hz, 2 x ArꢀH), 6.96ꢀ7.02 (m, 2H, 2 x ArꢀH), 7.49ꢀ7.58 (m,
4H, 2 x ArꢀH). 13C NMR (400MHz, CDCl3) δ ppm: 163.99 (C=O, thiazolidine), 147.91 (C,
triazine), 140.81 (C, triazine), 135.37 (C=O, thiazolidine), 129.48 (2 x ꢀNHꢀC, Ar), 126.17(2 x C,
Ar), 119.80 (4 x C, Ar), 116.47 (4 x C, Ar), 109.67 (2 x C, Ar), 66.33 (ꢀCH2ꢀ, thiazolidine); Mass
Spectra (TOF MS, m/z): Calculated 378.09; Observed 139.1 (100%), 85.0 (35.86%), 379.3
(20.03%, M+1), 93.1 (17.69%).
3ꢀ(4,6ꢀbis(2ꢀaminophenylamino)ꢀ1,3,5ꢀtriazinꢀ2ꢀyl)thiazolidineꢀ2,5ꢀdione, 7c
Dark brown powder; M.P.: > 300 °C; FTꢀIR (νmax; cmꢀ1, in KBr): 3230.15 (ꢀNHꢀ Stretching),
1611.78 (ꢀC=O Stretching), 1566.21ꢀ1434.62 (ꢀNH2 bending), 1348.65ꢀ1249.96 (CꢀN Stretching),

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1
684.20 (CꢀS Stretching); H NMR (300MHz, CDCl3ꢀd6, TMS) δ ppm: 3.36 (s, 4H, 4 x –NH2), 3.56
(s, 2H, ꢀCH2ꢀ thiazolidine), 6.79ꢀ6.81 (m, 4H, 2 x ArꢀH), 7.01ꢀ7.07 (m, 4H, 2 x ArꢀH); 13C NMR
(400MHz, CDCl3) δ ppm: 163.65 (C=O, thiazolidine), 162.76 (C, triazine), 133.42 (2xC, triazine),
126.67 (C=O, thiazolidine), 126.03 (2 x ꢀNHꢀC, Ar), 124.43 (2 x NH2ꢀC, Ar),117.29 (4 x C, Ar),
112.15 (4 x C, Ar), 78.50 (2 x C, Ar), 65.38 (ꢀCH2ꢀ, thiazolidine); Mass Spectra (TOF MS, m/z):
Calculated 408.11; Observed 133.1 (100%), 189.2 (69.81%), 409.20 (09.17%, M+1), 134.1
(09.08%).
3ꢀ(4,6ꢀbis(2ꢀnitrophenylamino)ꢀ1,3,5ꢀtriazinꢀ2ꢀyl)thiazolidineꢀ2,5ꢀdione, 7d
Yellow powder; M.P.: 121ꢀ123 °C; FTꢀIR (νmax; cmꢀ1, in KBr): 3271.48 (ꢀNHꢀ Stretching), 1616.55
(ꢀC=O Stretching), 1566.12ꢀ1422.45(ꢀNH2 bending), 1398.80(N=O Stretching, ArꢀNO2), 1386.64ꢀ
1
1115.86(CꢀN Stretching), 819.61(CꢀN Stretching, ArꢀNO2), 617.06 (CꢀS Stretching); H NMR
(300MHz, CDCl3ꢀd6, TMS) δ ppm: 3.58 (s, 2H, 2 x ꢀNHꢀAr), 3.80 (s, 2H, ꢀCH2ꢀ thiazolidine), 7.36ꢀ
7.38 (d, 2H, J=6Hz, 2 x ArꢀH), 7.40ꢀ7.43 (t, 2H, J=9 Hz, 2 x ArꢀH), 7.47ꢀ7.49 (t, 2H, J=6Hz, 2 x
ArꢀH), 7.59ꢀ7.61 (d, 2H, J=6 Hz, 2 x ArꢀH); 13C NMR (400MHz, CDCl3) δ ppm: 163.75 (C=O,
thiazolidine), 162.16 (C, triazine), 131.00 (C, triazine), 122.67 (C=O, thiazolidine), 120.03 (2 x
NO2ꢀC, Ar), 113.15(2 x ꢀNHꢀC, Ar), 109.24(2 x C, Ar), 107.29 (2 x C, Ar), 84.15 (2 x C, Ar),
78.50 (2 x C, Ar), 66.33 (ꢀCH2ꢀ, thiazolidine); Mass Spectra (TOF MS, m/z): Calculated 468.06;
Observed 454.1 (100%), 469.10 (66.89%, M+1), 510.1 (64.90%), 113.0 (24.88%).
3ꢀ(4,6ꢀbis(3ꢀnitrophenylamino)ꢀ1,3,5ꢀtriazinꢀ2ꢀyl)thiazolidineꢀ2,5ꢀdione, 7e
Yellow powder; M.P.: > 300 °C; FTꢀIR (νmax; cmꢀ1, in KBr): 3198.08 (ꢀNHꢀ Stretching), 1646.10 (ꢀ
C=O Stretching), 1558.62ꢀ1456.79(ꢀNH2 bending), 1329.40(N=O Stretching, ArꢀNO2), 1287.17ꢀ
1
1195.20(CꢀN Stretching), 782.96 (CꢀN Stretching, ArꢀNO2), 615.40 (CꢀS Stretching); H NMR
(300MHz, CDCl3ꢀd6, TMS) δ ppm: 3.34 (s, 2H, 2 x ꢀNHꢀAr), 3.36 (s, 2H, ꢀCH2ꢀ thiazolidine), 6.82ꢀ
6.83 (d, 2H, J=3Hz, 2 x ArꢀH), 7.23ꢀ7.25 (t, 2H, J=6 Hz, 2 x ArꢀH), 7.39 (s, 2H, 2 x ArꢀH), 8.25ꢀ
8.27 (d, 2H, J=6 Hz, 2 x ArꢀH); 13C NMR (400MHz, CDCl3) δ ppm: 142.43 (C=O, thiazolidine),
134.35 (C, triazine), 131.52 (C, triazine), 123.25 (C=O, thiazolidine), 119.00 (2 x NO2ꢀC, Ar),
112.25 (2 x ꢀNHꢀC, Ar), 110.14 (2 x C, Ar), 108.25 (2 x C, Ar), 82.12 (2 x C, Ar), 78.40 (2 x C,
Ar), 42.36 (ꢀCH2ꢀ, thiazolidine); Mass Spectra (TOF MS, m/z): Calculated 468.06; Observed 175.0
(100%), 469.10 (40.85%, M+1), 259.0 (15.55%), 113.0 (14.55%).
3ꢀ(4,6ꢀbis(4ꢀnitrophenylamino)ꢀ1,3,5ꢀtriazinꢀ2ꢀyl)thiazolidineꢀ2,5ꢀdione, 7f
Yellow powder; M.P.: 221ꢀ223 °C; FTꢀIR (νmax; cmꢀ1, in KBr): 3341.72 (ꢀNHꢀ Stretching), 1622.48
(ꢀC=O Stretching), 1592.65ꢀ1426.44(ꢀNH2 bending), 1343.29(N=O Stretching, ArꢀNO2), 1281.37ꢀ
1
1008.75(CꢀN Stretching), 870.58 (CꢀN Stretching, ArꢀNO2), 729.41 (CꢀS Stretching); H NMR

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(300MHz, CDCl3ꢀd6, TMS) δ ppm: 3.33 (s, 2H, 2 x ꢀNHꢀAr), 3.36 (s, 2H, ꢀCH2ꢀ thiazolidine), 6.57ꢀ
6.61 (t, 4H, J= 12 Hz, 2 x ArꢀH), 6.99ꢀ7.02(d, 4H, J=12 Hz, 2 x ArꢀH); 13C NMR (400MHz, CDCl3)
δ ppm: 146.13 (C=O, thiazolidine), 135.35 (C, triazine), 130.32 (C, triazine), 125.24 (C=O,
thiazolidine), 119.04 (2 x ꢀNHꢀC, Ar), 115.23 (2 x NO2ꢀC, Ar), 79.12 (2 x C, Ar), 78.46 (2 x C, Ar),
40.16 (ꢀCH2ꢀ, thiazolidine); Mass Spectra (TOF MS, m/z): Calculated 468.06; Observed 121.1
(100%), 469.10 (91.11%, M+1), 85.0 (45.54%), 91.1 (29.93%).
3ꢀ(4,6ꢀbis(2ꢀchlorophenylamino)ꢀ1,3,5ꢀtriazinꢀ2ꢀyl)thiazolidineꢀ2,5ꢀdione, 7g
White Powder; M.P.: 286ꢀ288 °C; FTꢀIR (νmax; cmꢀ1, in KBr): 328.34 (ꢀNHꢀ Stretching), 1622.12 (ꢀ
C=O Stretching), 1586.58ꢀ1416.81(ꢀNH2 bending), 1298.13ꢀ1180.52(CꢀN Stretching), 1007.74(Cꢀ
Cl Stretching), 685.97 (CꢀS Stretching); 1H NMR (300MHz, CDCl3ꢀd6, TMS) δ ppm: 3.39 (s, 2H, 2
x ꢀNHꢀAr), 3.90(s, 2H, ꢀCH2ꢀ thiazolidine), 6.58ꢀ6.62 (m, 2H, 2 x ArꢀH), 6.99ꢀ7.02 (d, 2H, J=12
Hz, 2 x ArꢀH), 7.33ꢀ7.41 (m, 2H, 2 x ArꢀH), 7.91ꢀ7.95 (m, 2H, 2 x ArꢀH); 13C NMR (400MHz,
CDCl3) δ ppm: 164.95 (C=O, thiazolidine), 163.73 (C, triazine), 155.68 (C, triazine), 146.13 (C=O,
thiazolidine), 135.65 (2 x ꢀNHꢀC, Ar), 130.20 (2 x C, Ar), 126.35 (2 x ClꢀC, Ar), 125.32 (2 x C,
Ar), 124.67 (2 x C, Ar),119.33 (2 x C, Ar), 112.32 (ꢀCH2ꢀ, thiazolidine); Mass Spectra (TOF MS,
m/z): Calculated 446.01; Observed 386.2 (100%), 368.2 (90.99%), 447.20 (32.45%, M+1), 258.2
(25.36%).
3ꢀ(4,6ꢀbis(3ꢀchlorophenylamino)ꢀ1,3,5ꢀtriazinꢀ2ꢀyl)thiazolidineꢀ2,5ꢀdione, 7h
White crystal; M.P.: 245ꢀ247 °C; FTꢀIR (νmax; cmꢀ1, in KBr): 3374.84 (ꢀNHꢀ Stretching), 1613.45 (ꢀ
C=O Stretching), 1569.83ꢀ1486.44 (ꢀNH2 bending), 1423.52ꢀ1178.53 (CꢀN Stretching), 1090.62 (Cꢀ
Cl Stretching), 795.46 (CꢀS Stretching); 1H NMR (300MHz, CDCl3ꢀd6, TMS) δ ppm: 3.88 (s, 2H, ꢀ
CH2ꢀ thiazolidine), 3.77(s, 2H, 2 x ꢀNHꢀAr), 7.19 (s, 2H, 2 x ArꢀH), 7.21ꢀ7.22(d, 2H, J=3 Hz, 2 x
ArꢀH), 7.24ꢀ7.25(d, 2H, J=3 Hz, 2 x ArꢀH), 7.27ꢀ7.29(d, 2H, J=6 Hz, 2 x ArꢀH); 13C NMR
(400MHz, CDCl3) δ ppm: 163.73 (C=O, thiazolidine), 162.16 (C, triazine), 128.07 (C, triazine),
127.90 (C=O, thiazolidine), 122.16 (2 x ꢀNHꢀC, Ar), 78.70 (2 x C, Ar), 78.57 (2 x C, Ar), 78.37(2 x
C, Ar),78.04(2 x C, Ar), 40.21ꢀ39.16 (2 x ClꢀC, Ar), 38.85 (ꢀCH2ꢀ, thiazolidine); Mass Spectra
(TOF MS, m/z): Calculated 446.01; Observed 347.2 (100%), 349.2(63.92%), 447.10 (40.67%,
M+1), 368.1(37.74%).
3ꢀ(4,6ꢀbis(4ꢀchlorophenylamino)ꢀ1,3,5ꢀtriazinꢀ2ꢀyl)thiazolidineꢀ2,5ꢀdione, 7i
Light pink powder; M.P.: 281ꢀ283 °C; FTꢀIR (νmax; cmꢀ1, in KBr): 3399.59 (ꢀNHꢀ Stretching),
1620.96(ꢀC=O Stretching), 1580.88ꢀ1495.08(ꢀNH2 bending), 1431.00ꢀ1155.52(CꢀN Stretching),
1
986.80(CꢀCl Stretching), 792.14 (CꢀS Stretching); H NMR (300MHz, CDCl3ꢀd6, TMS) δ ppm:

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3.83 (s, 2H, 2 x ꢀNHꢀAr), 3.77 (s, 2H, ꢀCH2ꢀ thiazolidine), 6.99 (m, 4H, 2 x ArꢀH), 7.60ꢀ7.75(d, 4H,
J=4.5 Hz, 2 x ArꢀH). 13C NMR (400MHz, CDCl3) δ ppm: 163.83 (C=O, thiazolidine), 162.23 (C,
triazine), 135.58 (C, triazine), 134.54(C=O, thiazolidine), 122.81ꢀ121.98 (2 x ꢀNHꢀC, Ar), 115.23ꢀ
114.63 (4 x C, Ar), 79.16ꢀ78.50 (2 x ClꢀC, Ar), 40.12ꢀ38.87 (4 x C, Ar), 35.95 (ꢀCH2ꢀ, thiazolidine);
Mass Spectra (TOF MS, m/z): Calculated 446.01; Observed 316.2 (100%), 390.2 (51.65%), 315.2
(40.57%), 447.10 (30.09%, M+1), 372.2 (26.03%).
3ꢀ(4,6ꢀbis(4ꢀfluorophenylamino)ꢀ1,3,5ꢀtriazinꢀ2ꢀyl)thiazolidineꢀ2,5ꢀdione, 7j
White powder; M.P.: 123ꢀ125 °C; FTꢀIR (νmax; cmꢀ1, in KBr): 3297.56 (ꢀNHꢀ Stretching), 1619.16ꢀ
1600.60 (ꢀC=O Stretching), 1568.73ꢀ1480.86 (ꢀNH2 bending), 1426.75ꢀ1169.27 (CꢀN Stretching),
1
988.78 (CꢀF Stretching), 714.56 (CꢀS Stretching); H NMR (300MHz, CDCl3ꢀd6, TMS) δ ppm:
3.35 (s, 2H, 2 x ꢀNHꢀAr), 3.58 (s, 2H, ꢀCH2ꢀ thiazolidine), 7.08ꢀ7.11(d, 4H, J=9 Hz, 2 x ArꢀH),
7.31ꢀ7.35(d, 4H, J=12 Hz, 2 x ArꢀH); 13C NMR (400MHz, CDCl3) δ ppm: 163.84 (C=O,
thiazolidine), 139.69 (C, triazine), 133.15(C=O, thiazolidine), 130.01 (C, triazine), 123.07 (2 x FꢀC,
Ar), 120.29ꢀ119.06 (2 x ꢀNHꢀC, Ar), 79.16ꢀ78.50 (4 x C, Ar), 63.33 (4 x C, Ar), 40.15(ꢀCH2ꢀ,
thiazolidine); Mass Spectra (TOF MS, m/z): Calculated 414.07; Observed 85.0 (100%), 366.1
(88.11%), 415.1 (74.86%, M+1), 475.5 (54.79%).
3ꢀ(4,6ꢀbis(4ꢀbromophenylamino)ꢀ1,3,5ꢀtriazinꢀ2ꢀyl)thiazolidineꢀ2,5ꢀdione, 7k
Offꢀwhite crystals; M.P.: 167ꢀ169 °C; FTꢀIR (νmax; cmꢀ1, in KBr): 3388.40 (ꢀNHꢀ Stretching),
1586.84 (ꢀC=O Stretching), 1556.09ꢀ1434.25 (ꢀNH2 bending), 1372.76ꢀ1129.67 (CꢀN Stretching),
1
794.78 (CꢀBr Stretching), 729.98 (CꢀS Stretching); ); H NMR (300MHz, CDCl3ꢀd6, TMS) δ ppm:
3.45 (s, 2H, 2 x ꢀNHꢀAr), 3.68 (s, 2H, ꢀCH2ꢀ thiazolidine), 7.42ꢀ7.44(d, 4H, J=6 Hz, 2 x ArꢀH),
7.73ꢀ7.75(d, 4H, J=6 Hz, 2 x ArꢀH); 13C NMR (400MHz, CDCl3) δ ppm: 181.18 (C=O,
thiazolidine), 170.31(C, triazine), 162.96(C=O, thiazolidine), 135.17(C, triazine), 129.13ꢀ129.00(2
x ꢀNHꢀC, Ar), 127.39ꢀ126.73(4 x C, Ar), 79.21ꢀ78.55 (4 x C, Ar), 40.13ꢀ38.88 (2 x BrꢀC, Ar),
36.90(ꢀCH2ꢀ, thiazolidine); Mass Spectra (TOF MS, m/z): Calculated 535.91; Observed 347.2
(100%), 348.2 (74.59%), 537.10 (70.91%, M+1), 349.2 (70.83%)
Biological Activity
In vitro DPP-4 Inhibitory activity
The compounds synthesized were evaluated for in vitro DPPꢀ4 enzyme inhibition. The enzyme
assay was performed using DPPꢀ4 drug discovery kit (BMLꢀAK 499; Enzo Life Sciences, Plymouth
Meeting, PA, USA). The activity of test compounds was assayed using human recombinant DPPꢀ4
enzyme, chromogenic substrate (HꢀGlyꢀProꢀAMC, Km 114 µM), DPPꢀ4 inhibitor (P32/98), assay

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buffer and calibration standard as provided in the kit. The assay was performed using 96ꢀwell flatꢀ
bottomed microtitre plate followed by addition of assay buffer, DPPꢀ4 enzyme and chromogenic
substrate (HGlyꢀProꢀpNA). The assay principle and procedure was followed as per manufacturer’s
guidelines. Solutions of the test compounds were made in dimethyl sulphoxide (DMSO) at different
concentrations of 25, 50, 100, 200 µg/mL, and 20 µL were added to each well after further
dilutions. The plate was incubated at 37 °C for 10 min to allow enzymeꢀinhibitor reaction and read
continuously at Aꢀ405 nm using BioꢀRad Elisa Plate Reader. IC50 values were determined using
nonꢀlinear regression analysis.
Antibacterial Activity
All synthesized compounds were screened for their minimum inhibitory concentration (MIC,
ꢁg/mL) against selected Gramꢀpositive organisms viz. Bacillus subtilis (NCIMꢀ2063), Bacillus
cereus (NCIMꢀ2156), Staphylococcus aureus (NCIMꢀ2079) and Gramꢀnegative organism viz.
Escherichia coli (NCIMꢀ2065), Proteus vulgaris (NCIMꢀ2027) and Pseudomonas aeruginosa
(NCIMꢀ2036), by the broth dilution method as recommended by the National Committee for
Clinical Laboratory Standards with minor modifications.19 Cefixime was used as a standard
antibacterial agent. Solutions of the test compounds and reference drug were prepared in dimethyl
sulfoxide (DMSO) at concentrations of 125, 62.5, 31.25, 15.62, 7.81, 3.91, 1.95, 0.97 ꢁg/mL. Ten
tubes were made in parallel with the second set being used as MIC reference controls (16–24 h
visual). After sample preparation, the controls were placed in a 37 °C incubator and read for
macroscopic growth (clear or turbid) the next day. Into each tube, 0.8 mL of nutrient broth was
pipette (tubes 2–7), tube 1 (negative control) received 1.0 mL of nutrient broth and tube 10 (positive
control) received 0.9 mL of nutrient. Tube 1, the negative control, did not hold bacteria or
antibiotic. The positive control, tube 10, received 0.9 mL of nutrient broth since it contained
bacteria but not an antibiotic. The test compound was dissolved in DMSO (125 ꢁg/mL), 0.1 mL of
increasing concentration of the prepared test compounds which are serially diluted from tube 2 to
tube 9 (tube 2–9 containing 125, 62.5, 31.25, 15.62, 7.81, 3.91, 1.95, 0.97 ꢁg/mL, respectively).
After this process, each tube was inoculated with 0.1 mL of the bacterial suspension whose
concentration corresponded to 0.5 McFarland scale (9 × 108 cells/mL) and each bacterium was
incubated at 37 °C for 24 h at 150 rpm. The final volume in each tube was 1.0 mL. The incubation
chamber was kept humid. At the end of the incubation period, MIC values were recorded as the
lowest concentration of the substance that gave no visible turbidity, i.e. no growth of inoculated
bacteria.
Docking Study

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The computational studies were carried out with the Xꢀray crystal structure of DPPꢀ4 (PDB Id:
2FJP, Chain A) in complex with (2S,3S)ꢀ3ꢀAminoꢀ4ꢀ(3,3ꢀdifluoropyrrolidinꢀ1ꢀyl)ꢀN,Nꢀdimethylꢀ4ꢀ
oxoꢀ2ꢀ(4ꢀ[1,2,4]triazolo[1,5ꢀa]ꢀpyridinꢀ6ꢀylphenyl)butanamide: a selective alphaꢀamino amide
dipeptidyl peptidase IV inhibitor for the treatment of type 2 diabetes. Docking of the ligand 7a was
carried out in the binding pocket of DPPꢀ4 with CDOCKER protocol of DS 2.5 following the
protocol given elsewhere.20 CDOCKER is a gridꢀbased MDꢀsimulated, annealingꢀbased algorithm
that uses CHARMm. The receptor (protein) was held rigid, while the ligands were flexible during
the refinement.21,22 The basic strategy involves the generation of several initial ligand orientations in
the allosteric site of the target protein followed by MDꢀbased simulated annealing, and final
refinement by minimization
Acknowlwedgements
Authors are thankful to SHIATS for providing necessary infrastructural facilities. The support of
SAIF, Punjab University, India is highly appreciated for providing the spectral data of the
compounds synthesized herein.

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References
1. American Diabetes Association.. Report of the Expert Committee on the diagnosis and
classification of diabetes mellitus. Diabetes Care 1997, 20, 1183ꢀ1201.
2. Institute of Medicine (US) Committee on Preventing the Global Epidemic of Cardiovascular
Disease: Meeting the Challenges in Developing Countries; Fuster V, Kelly BB, editors.
Promoting Cardiovascular Health in the Developing World: A Critical Challenge to Achieve
Global Health. Washington (DC): National Academies Press (US); 2010. 2, Epidemiology of
Cardiovascular Disease. Available from: http://www.ncbi.nlm.nih.gov/books/NBK45688/.
3. J. E. Shaw, R. A. Sicree, P. Z. Zimmet, Diabetes Res. Clin. Pract., 2010, 87, 4ꢀ14.
4. C. H. S. McIntosh, Front Biosci. 2008, 13, 1753ꢀ1773.
5. (a) J. J. Holst, C. F. Deacon, Diabetes 1998, 47, 1663ꢀ1670 (b) M. Zander, S. Madsbad, J. L.
Madsen, Lancet 2002, 359, 824ꢀ830 (c) H.J. Mest, R. Mentlein, Diabetologia, 2005, 48,
616–620.
6. US/EU EudraCT number 2012ꢀ004045ꢀ33
7. P. Fouqueray, X. Leverve, E. Fontaine, M. Baquié, C. Wollheim, H. Lebovitz, S. Bozec, J.
Diabetes. Metab. 2011, 2, 126.
8. VS Jain, DK Vora, CS Ramaa, Bioorg Med Chem. 2013, 21, 1599ꢀ620.
9. B.B. Lohray, V.B. Lohray, Drug Discovery and Development In Chorghade, M. S., Ed.;
WileyꢀInterscience, 2007; pp 91–120. Vol. 2
10. American Diabetes Association. Standards of medical care in diabetes—2014. Diabetes
Care 2014, 37, S14–S80
11. G.M. Shenfield, Drugs 1982, 23, 462ꢀ80.
12. B. Meunier, Acc. Chem. Res. 2008, 41, 69–77
13. D. Kim, L. Wang, M. Beconi, G. J. Eiermann, M. H. Fisher, H. He, G. J. Hickey, J. E.
Kowalchick, B. Leiting, K. Lyons, F. Marsilio, M. E. McCann, R. A. Patel, A. Petrov, G.
Scapin, S. B. Patel, R. S. Roy, J. K. Wu, M. J. Wyvratt, B. B. Zhang, L. Zhu, N. A.
Thornberry, A. E. Weber, J. Med. Chem. 2005, 48, 141ꢀ151.
14. C. Rummey, S. Nordhoff, M. Thiemann, G. Metz, Bioorg Med Chem Lett., 2006, 16, 1405ꢀ
1409.
15. J. Casqueiro, C. Alves, Indian J Endocr Metab, 2012, 16, 27ꢀ36
16. J. Davies, D. Davies, Microbiol Mol Biol Rev 2010, 74, 417ꢀ433.
17. a) B. Singh, H.R. Bhat, M.K. Kumawat, U.P. Singh, Bioorg. Med. Chem. Lett., 2014, 24,
3321ꢀ3325. b) H.R. Bhat, U.P. Singh, P. Gahtori, S.K. Ghosh, K. Gogoi, A. Prakash, R.K.

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Singh, New J. Chem., 2013, 37, 2654ꢀ2662. c) S. Kumar, H.R. Bhat, M.K. Kumawat, U.P.
Singh, New J. Chem., 2013, 37, 581ꢀ584. d) H.R. Bhat, U.P. Singh, P. Gahtori, S.K. Ghosh,
K. Gogoi, A. Prakash, R.K. Singh, RSC Adv.., 2013, 3, 2942ꢀ2952. e) U.P. Singh, M. Pathak,
V. Dubey, H.R. Bhat, R.K. Singh, Chem. Biol. Drug Des., 2012, 80, 572ꢀ583. f) V. Dubey,
M. Pathak, H.R. Bhat, U.P. Singh, Chem. Biol. Drug Des., 2012, 80, 598ꢀ604. g) P. Gahtori,
S.K. Ghosh, P. Pratap, A. Prakash, K. Gogoi, H.R. Bhat, U.P. Singh, Exp. Parasitol., 2012,
130, 292ꢀ299. g) U.P. Singh, H.R. Bhat, P. Gahtori, J. Mycol. Méd., 2012, 22, 134ꢀ144.
18. K.R. Alagawadi,
S.G. Alegaon, Arabian. J. Chem., 2010, in press,
doi:10.1016/j.arabjc.2010.07.012.
19. National Committee for Clinical Laboratory Standards,Standard Methods for Dilution
Antimicrobial Susceptibility Test for Bacteria Which Grow Aerobically, Villanova, NCCLS,
1982, p. 242.
20. G. Kumari, Nutan, M. Modi, S.K. Gupta, R.K. Singh, Eur. J. Med. Chem., 2011, 46, 1181ꢀ
1188.
21. F.A. Momany, R. Rone, Comput. Chem., 1992, 13, 888–900
22. G. Wu, D.H. Robertson, C.L. Brooks, M. Vieth, J. Comput. Chem., 2003, 24, 1549–1562

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Figures and schemes legends
Figure 1: Classical role of Dipeptidyl peptidase-4 (DPP - 4) and implication of its inhibition by DPP-4
inhibitors.
Scheme 1: Synthesis of novel 1,3,5-triazine-thiazolidine-2,4-diones: Reagents and condition: a)
Conc. H2SO4, water, reflux; b) NaOH, stirring, 40-45 °C; c) K2CO3, reflux, 120-135 °C.
Figure 2: Structure-activity relationships of target analogues as DPP-4 Inhibitors.
Figure 3: The docked complex of compound 7a in the active of in the binding pocket of dipeptidyl peptidase
(DPP) 4.
Figure 4: The docked complex of compound 7k in the active of in the binding pocket of dipeptidyl peptidase
(DPP) 4.

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TABLES
Table 1: DPPꢀ4 inhibitory effect of novel 1,3,5ꢀtriazineꢀthiazolidineꢀ2,4ꢀdiones derivatives
Compound
IC50 (in µM)a
7a
7b
7c
7d
7e
7f
7g
7h
6.35
12.11
10.76
14.64
15.38
17.85
27.32
23.25
29.53
38.37
49.21
2.5
7i
7j
7k
P 32 /98 (Standard)
aIC50 represents inhibitory concentration determined by nonꢀlinear regression analysis using GRAPHPAD PRISM
software. Values are expressed as mean of three independent experiments
.
Table 2: Docking interaction and scoring of 7a (most active) and 7k (least active).
CDOCKER INTERACTION
ENERGY
CDOCKER
ENERGY
Hꢀbonding
residues
Piꢀcation
interaction
Compound
7a
31.90
44.31
Glu205, Tyr547
Arg125
Arg125
Arg358, Arg669,
Glu205
7k
44.27
43.51

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Table 3 Antibacterial activity of compound 7(aꢀk).
Minimum Inhibitory Concentration (MIC, in µg/mL)
P. vulgaris E.coli P. aeruginosa B. subtilis S. aureus B. cerus
Compound
7a
7b
7c
7d
7e
7f
7g
7h
7i
31.25
15.62
125
32.25
15.62
62.5
7.81
3.91
15.62
125
62.5
15.62
125
15.62
15.62
62.5
62.5
62.5
62.5
125
62.5
15.62
3.91
15.62
3.91
15.62
125
15.62
31.25
62.5
125
31.25
15.62
7.81
3.91
125
7.81
62.5
31.25
31.25
62.5
62.5
62.5
3.91
62.5
62.5
125
15.62
3.91
7.81
31.25
62.5
125
7.81
31.25
15.62
125
7.81
31.25
31.25
31.25
7.81
7j
7k
7.81
31.25
31.25
62.5
7.81
31.25
15.62
31.25
3.91
Cefixime
(Standard)
31.25

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FIGURES
Figure 1: Classical role of Dipeptidyl peptidase-4 (DPP - 4) and implication of its inhibition by DPP-4 inhibitors.

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Scheme 1: Synthesis of novel 1,3,5-triazine-thiazolidine-2,4-diones: Reagents and condition: a) Conc.
H2SO4, water, reflux; b) NaOH, stirring, 40-45 °C; c) K2CO3, reflux, 120-135 °C.

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Figure 2: Structure-activity relationships of target analogues as DPP-4 Inhibitors.

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Figure 3: The docked complex of compound 7a in the active of in the binding pocket of dipeptidyl peptidase (DPP)
4.
Figure 4: The docked complex of compound 7k in the active of in the binding pocket of dipeptidyl peptidase (DPP)
4.