N-alkylated thiazolidine-2,4-dione analogs as PTP1B inhibitors: synthesis, biological activity, and docking studies

Article abstract of DOI:10.1007/s00044-017-1823-z

Protein tyrosine phosphatase 1B enzyme has been found to be a negative regulator of insulin and leptin signaling pathway. It has gained considerable attention to medicinal chemists as a new therapeutic target for intervention in the treatment of type2 diabetes. A series of N-substituted-5-(thiophen-2-ylmethylene)thiazolidine-2,4-dione derivatives were synthesized and screened in vitro for protein tyrosine phosphatase 1B inhibitory activity and in vivo for anti-hyperglycaemic activity. The introduction of alkyl/halo alkyl moiety onto the amidic nitrogen of thiazolidine-2,4-dione ring was intended to enhance the inhibitor-enzyme affinity and hence, good protein tyrosine phosphatase 1B inhibition. The nature of interactions which governs the binding mode of ligands inside the active site of protein tyrosine phosphatase 1B was further studied by molecular docking simulation. Compound 7 was found to be a potent protein tyrosine phosphatase 1B inhibitor with IC₅₀ 9.96 μM. The synthesized compounds have also shown significant lowering of blood glucose level.

Full text of DOI:10.1007/s00044-017-1823-z

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-017-1823-z
ORIGINAL RESEARCH
N-alkylated thiazolidine-2,4-dione analogs as PTP1B inhibitors:
synthesis, biological activity, and docking studies
1 ●
1 ●
1
Manoj K. Mahapatra Rajnish Kumar Manoj Kumar
Received: 7 March 2016 / Accepted: 28 February 2017
©
Springer Science+Business Media New York 2017
Abstract Protein tyrosine phosphatase 1B enzyme has
been found to be a negative regulator of insulin and leptin
signaling pathway. It has gained considerable attention to
medicinal chemists as a new therapeutic target for inter-
vention in the treatment of type2 diabetes. A series of N-
substituted-5-(thiophen-2-ylmethylene)thiazolidine-2,4-
dione derivatives were synthesized and screened in vitro for
protein tyrosine phosphatase 1B inhibitory activity and
in vivo for anti-hyperglycaemic activity. The introduction
of alkyl/halo alkyl moiety onto the amidic nitrogen of
thiazolidine-2,4-dione ring was intended to enhance the
inhibitor-enzyme affinity and hence, good protein tyrosine
phosphatase 1B inhibition. The nature of interactions which
governs the binding mode of ligands inside the active site of
protein tyrosine phosphatase 1B was further studied by
molecular docking simulation. Compound 7 was found to
be a potent protein tyrosine phosphatase 1B inhibitor with
IC50 9.96 µM. The synthesized compounds have also shown
significant lowering of blood glucose level.
Introduction
Diabetes Atlas 2015, a recent study by International Dia-
betes Federation (IDF), has estimated the global occurrence
of diabetes to 415 million and another 318 million with
impaired glucose tolerance (IDF 2015). It was predicted to
become double globally from 171 million in 2000 to 366
million in 2030 with a maximum increase in India
(Kaveeshwar and Cornwall 2014; Wild et al. 2004).
Protein tyrosine phosphatase 1B (PTP1B) is one of the
prototypical members of protein tyrosine phosphatase (PTP)
super family and is expressed ubiquitously. It was the first
PTP enzyme to be purified from human placental tissue to
homogeneity (Tonks et al. 1988). PTP1B consists of 435
amino-acid residues and the active domains contained cat-
alytic loop (Cys215-Arg221), WPD loop (Trp179–Ser187)
and secondary aryl phosphate binding site (Arg24, Lys43-
Tyr46, Arg254, Met258, Gly259, Gln262, and Gln266)
(Johnson et al. 2002). Due to its pivotal role as a negative
regulator of the insulin and leptin signaling pathway, it has
been emerged as a legitimate therapeutic target for the
treatment of type2 diabetes and obesity, collectively known
as diabesity (Walchi et al. 2000; Byon et al. 1998; Colagiuri
●
●
●
Keywords PTP1B Diabetes N-alkylated TZD Docking
2010). Independent research from different laboratories has
shown that augmented insulin sensitivity, increased glucose
tolerance, and resistance to diet-induced obesity were
observed in PTP1B knockout mice (Elchebly et al. 1999;
Klaman et al. 2000).
Ertiprotafib and ISIS-113715 were withdrawn from
phase-II clinical trial studies due to undesirable side effects
Electronic supplementary material The online version of this article
doi:10.1007/s00044-017-1823-z) contains supplementary material,
which is available to authorized users.
(
(
Wang et al. 2014; Liu 2004). Despite the best efforts of
*
Manoj Kumar
manoj_uips@pu.ac.in
medicinal chemists, the endeavor for developing therapeutic
PTP1B inhibitors was mostly unsuccessful and a highly
conservative and cationic nature of PTP1B catalytic site was
attributed for this (Tang et al. 2013). Many small molecule
1
University Institute of Pharmaceutical Sciences, Panjab
University, Chandigarh 160014, India

Med Chem Res
inhibitors which bind to the active site with high affinity
were hydrophilic and thus shown inadequate in vivo activity
due to low cellular permeability (Wang et al. 2014). This
necessitated the imminent development of potent and
selective PTP1B inhibitors.
Various substituted thiazolidine-2,4-dione (TZD) deri-
vatives have been reported with significant PTP1B inhibi-
tory activity (Maccari et al. 2007; Ottana et al. 2009;
Bhattarai et al. 2009). Their study exhibited that insertion of
Material and methods
Chemistry
The melting points were recorded on Veego-540 melting
1
13
point apparatus and were uncorrected. H-NMR and C-
NMR was recorded on a Bruker AC-400F, 400 MHz
spectrometer for solutions in deuteriochloroform (CDCl3),
deuterated dimethylsulfoxide (DMSO-d6) and were repor-
ted in parts per million (ppm), downfield from tetra-
methylsilane as internal standard. Splitting patterns were
designated as- singlet (s); doublet (d); triplet (t); multiplet
(m). Chemical shift values were presented in parts per
million (ppm). Mass spectroscopy was conducted using
electrospray ionization mode in Micromass Q-TOF micro
(Waters) system. All reactions were monitored by TLC
(silica gel coated aluminium sheets). Synthesis of N-sub-
stituted 5-(thiophen-2-ylmethylene)thiazolidine-2,4-dione
derivatives has been outlined in Scheme 1.
5
-arylidene group improved receptor-ligand binding by
hydrophobic interaction with the residues of secondary non-
catalytic binding site. The side effects associated with TZD
derivatives were attributed to the acidic nature of amidic
proton of TZD nucleus, which resembles carboxylate anion
of the natural fatty acid ligands, enabling its binding to
peroxisome proliferator-activator receptor. Hence N-sub-
stituted TZD derivatives were expected to have lesser side
effects as compared to the currently used glitazones (Mac-
cari et al. 2007; Bhat et al. 2004). Moreover, various PTP1B
inhibitors
bearing
thiophene
scaffold
with
good potency have been reported (Wrobel et al. 1999;
Andersen et al. 2002; Moretto et al. 2006; Ye et al. 2010).
Therefore, it was envisaged to design ligands with alkyl
and haloalkyl substitution on amidic nitrogen of TZD
nucleus. The phenyl ring of 5-arylidene nucleus was
replaced by thiophene moiety. Most of the active-site-
directed PTP1B inhibitors reported till date have possessed
high charge density to serve as competitive inhibitors. Such
molecules were generally not druggable in nature and had
cell membrane permeability issues (Zhang and Zhang
2,4-Thiazolidinedione (1)
A mixture of chloroacetic acid (56.7 g, 0.6 mol) and
thiourea (45.6 g, 0.6 mol) was dissolved in water (125.0 ml),
stirred for 15 min. Conc. hydrochloric acid (60.0 ml) was
added dropwise to this solution. The reaction mixture was
refluxed for 12 h and then cooled in an ice bath. The product
separated as white needles was filtered, washed repeatedly
with water to remove traces of hydrochloric acid, dried and
recrystallized from methanol to yield 2,4-thiazolidinedione
(Sachan et al. 2007).
2
007). Herein, a series of small molecule inhibitors were
synthesized containing both thiophene and TZD nucleus
coupled by a vinylic linkage (Fig. 1). The amidic nitrogen
of TZD was substituted by alkyl/haloalkyl groups to
increase the lipophilicity and overcome the membrane
permeability problems. In the present investigation
synthesis, in vitro PTP1B inhibition and in vivo anti-
hyperglycaemic activity of N-substituted 5-(thiophen-2-
ylmethylene)thiazolidine-2,4-dione derivatives have been
reported.
White solid (Yield: 62.58 g, 88.2%; m. p. 123–125 °C)
R : 0.49 (chloroform:methanol:: 10:1); IR (KBr): 3132,
f
−
1 1
1739, 1662, and 615 cm ; H NMR (DMSO-d6, ppm): δ
1
3
11.98 (s, 1H, NH), δ 4.09 ppm (s, 2H, CH2); C NMR
(DMSO-d6, ppm): δ 173.54 (CO), δ 172.79 (CO), δ 35.65
(Aromatic C).
(Z)-5-(thiophen-2-ylmethylene)thiazolidine-2,4-dione (2)
A mixture of 2,4-thiazolidinedione (2.92 g, 25 mmol) and
anhydrous sodium acetate (2.04 g, 25 mmol) in glacial
acetic acid was refluxed at 110–120 °C for 20 min. 2-
Thiophenecarboxaldehyde (2.05 g, 25 mmol) was added
into the mixture and refluxed for 36 h by using Dean-Stark
apparatus to remove water from the reaction. The solvent
was evaporated and the residue was added into crushed ice
with stirring. The separated product was filtered, washed
with water and dried. It was crystallized from acetone
to yield (Z)-5-(thiophen-2-ylmethylene) thiazolidine-2,4-
dione.
Fig. 1 Design of final compounds

Med Chem Res
Scheme 1 Synthesis of N-
substituted 5-(thiophen-2-
ylmethylene)thiazolidine-2,4-
diones
Dark brown solid (Yield: 2.20 g, 42.79%; m.p. 238–240 °C)
131.85, 128.59, 126.22, and 119.59 (Aromatic C), δ 43.68
+
(
CH ), δ 21.17 (CH ), δ 11.18 (CH ); Mass (ES ): 253.4
2 2 3
+
+
R : 0.59 (hexane:ethylacetate:: 3:1); IR (KBr): 3129, 1735,
(M ), 254.4 (M + 1) .
f
−1 1
1
679, and 632 cm ; H NMR (DMSO-d6): δ 12.55 (s, 1H,
NH), δ 8.04 (s, 1H, =CH), δ 7.97 (d, 1H, J = 5.00 Hz,
Ar–H), δ 7.64 (d, 1H, J = 3.52 Hz, Ar–H), δ 7.27 (t, 1H, J
(Z)-3-(2-bromoethyl)-5-(thiophen-2-ylmethylene)
thiazolidine-2,4-dione (4)
1
3
=
4.36 Hz, Ar–H); C NMR (DMSO-d6): δ 167.06 (CO,
TZD), δ 166.96 (CO, TZD), δ 137.22 (=CH), δ 134.35,
32.89, 128.76, 125.14, and 120.91 (Aromatic C).
1
Light brown solid (yield: 59.41%; m.p. 150–152 °C); R :
f
0
1
.50 (hexane:ethylacetate = 4:1); IR (KBr): 3021, 2953,
−
1 1
739, 1675, 644, and 581 cm ; H NMR (CDCl ): δ 8.09
3
General procedure for synthesis of final compounds
3–12)
(
1
s, 1H, =CH), δ 7.68 (d, 1H, J = 5.00 Hz, Ar–H), δ 7.42 (d,
H, J = 3.60 Hz, Ar–H), δ 7.20 (t, 1H, J = 4.32 Hz, Ar–H),
δ 4.15 (t, 2H, J = 6.76 Hz, CH ), δ 3.59 (t, 2H, J = 6.80 Hz,
(
2
A mixture of (Z)-5-(thiophen-2-ylmethylene) thiazolidine-
,4-dione(1.47 g, 7 mmol) and anhydrous potassium car-
1
3
CH2); C NMR (CDCl ): δ 167.11 (CO, TZD), δ 165.67
3
2
(CO, TZD), δ 137.53 (=CH), δ 133.72, 132.33, 128.72,
bonate (1.93 g, 14 mmol) in acetone was refluxed for 45
min. Suitable alkyl halide (10.5 mmol) was added to the
reaction mixture and refluxed for 24 h. The mixture was
concentrated under reduced pressure and added into crushed
ice from which the product precipitated out. The separated
product was filtered, washed with water and dried. It was
crystallized from acetone to yield the final compounds.
1
27.12, and 118.74 (Aromatic C), δ 42.77 (CH ), δ 26.91
2
+
+
+
(CH ); mass (ES ): 318.3 (M ), 320.3 (M + 2) .
2
(
Z)-3-heptyl-5-(thiophen-2-ylmethylene)thiazolidine-2,4-
dione (5)
Light yellow solid (yield: 60.73%; m.p. 72–74 °C); R : 0.60
f
(
Z)-3-propyl-5-(thiophen-2-ylmethylene)thiazolidine-2,4-
(hexane:ethylacetate = 6:1); IR (KBr): 2923, 2856, 1732,
−
1
1
dione (3)
1668, and 609 cm ; H NMR (CDCl ): δ 8.04 (s, 1H,
3
=
CH), δ 7.64 (d, 1H, J = 5.04 Hz, Ar–H), δ 7.38 (d, 1H, J
Brown solid (yield: 61.01%; m.p. 90–92 °C); R : 0.56
= 3.76 Hz, Ar–H), δ 7.18 (t, 1H, J = 4.40 Hz, Ar–H), δ 3.72
(t, 2H, J = 7.48 Hz, CH ), δ 1.66 (m, 2H, CH ), δ 1.30 (m,
8H, CH ), δ 0.87 (m, 3H, CH ); C NMR (CDCl ): δ
f
(
hexane:ethylacetate = 5:1); IR (KBr): 2945, 2877, 1729,
2
2
668, and 605 cm− ; H NMR (CDCl ): δ 8.05 (s, 1H,
1
1
13
2 3 3
1
3
=
=
CH), δ 7.65 (d, 1H, J = 5.04 Hz, Ar–H), δ 7.39 (d, 1H, J
3.64 Hz, Ar–H), δ 7.18 (t, 1H, J = 4.36 Hz, Ar–H), δ 3.71
167.34 (CO, TZD), δ 166.17 (CO, TZD), δ 137.77 (=CH),
δ 133.24, 131.84, 128.58, 126.17, and 119.62 (Aromatic C),
δ 42.22 (CH ), δ 31.67 (CH ), δ 28.82 (CH ), δ 27.80
(
t, 2H, J = 4.28 Hz, CH ), δ 1.70 (m, 2H, CH ), δ 0.94 (t,
2 2
1
2
2
2
3
3
H, J = 7.44 Hz, CH3); C NMR (CDCl ): δ 167.41 (CO,
(CH ), δ 26.67 (CH ), δ 22.57 (CH ), δ 14.08 (CH ); mass
2 2 2 3
3
+
+
TZD), δ 166.22 (CO, TZD), δ 137.77 (=CH), δ 133.25,
(ES ): 310.4 (M + 1) .

Med Chem Res
(
Z)-3-(3-chloropropyl)-5-(thiophen-2-ylmethylene)
137.77 (=CH), δ 133.24, 131.84, 128.58, 126.17, and
119.61 (Aromatic C), δ 41.96 (CH ), δ 29.83 (CH ), δ 19.97
thiazolidine-2,4-dione (6)
2
+
2
+
(
CH ), δ 13.63 (CH ); mass (ES ): 268.4 (M + 1) .
2 3
Brown solid (yield: 63.29%; m.p. 135–137 °C); R : 0.54
f
(
1
hexane:ethylacetate = 4:1); IR (KBr): 3019, 2951, 1736,
667, 715, and 549 cm− ; H NMR (CDCl ): δ 8.07 (s, 1H,
1
1
(Z)-3-(3-bromopropyl)-5-(thiophen-2-ylmethylene)
3
=
=
CH), δ 7.67 (d, 1H, J = 5.04 Hz, Ar–H), δ 7.41 (d, 1H, J
3.68 Hz, Ar–H), δ 7.19 (t, 1H, J = 4.44 Hz, Ar–H), δ 3.91
thiazolidine-2,4-dione (10)
(
t, 2H, J = 6.96 Hz, CH ), δ 3.57 (t, 2H, J = 6.48 Hz, CH ),
Brown solid (yield: 63.41%; m.p. 130–132 °C); R : 0.50
2
2
f
1
3
δ 2.16 (m, 2H, CH2); C NMR (CDCl ): δ 167.36 (CO,
TZD), δ 166.05 (CO, TZD), δ 137.61 (=CH), δ 133.54,
(hexane:ethylacetate = 5:1); IR (KBr): 3018, 2948, 1737,
3
−
1 1
1669, 648, and 551 cm ; H NMR (CDCl ): δ 8.07 (s, 1H,
3
1
32.13, 128.67, 126.73, and 119.14 (Aromatic C), δ 41.82
=CH), δ 7.67 (d, 1H, J = 4.36 Hz, Ar–H), δ 7.41 (d, 1H,
J = 3.72 Hz, Ar–H), δ 7.19 (t, 1H, J = 4.44 Hz, Ar–H), δ
+
(
CH ), δ 39.77 (CH ), δ 30.66 (CH ); mass (ES ): 288.4
2 2 2
+
+
(
M ), 290.4 (M + 2) .
3.90 (t, 2H, J = 6.92 Hz, CH ), δ 3.41 (t, 2H, J = 6.68 Hz,
2
1
3
CH ), δ 2.25 (m, 2H, CH ); C NMR (CDCl ): δ 167.36
2
2
3
(
Z)-3-methyl-5-(thiophen-2-ylmethylene)thiazolidine-2,4-
(CO, TZD), δ 166.04 (CO, TZD), δ 137.60 (=CH), δ
dione (7)
133.57, 132.16, 128.68, 126.76, and 119.11 (Aromatic C), δ
+
4
0.79 (CH ), δ 30.82 (CH ), δ 29.48 (CH ); mass (ES ):
2
+
2
+
2
Light brown solid (yield: 74.19%; m.p. 173–175 °C); Rf:
332.2 (M ), 334.2 (M + 2) .
0
1
1
1
.52 (hexane:ethylacetate = 4:1); IR (KBr): 3015, 2943,
1 1
724, 1670, and 581 cm− ; H NMR (CDCl ): δ 8.07 (s,
3
H, =CH), δ 7.66 (d, 1H, J = 5.08 Hz, Ar–H), δ 7.40 (d,
H, J = 3.92 Hz, Ar–H), δ 7.19 (t, 1H, J = 4.44 Hz, Ar–H),
(Z)-3-pentyl-5-(thiophen-2-ylmethylene)thiazolidine-2,4-
dione (11)
1
3
δ 3.24 (s, 3H, CH3); C NMR (CDCl ): δ 167.46 (CO,
3
TZD), δ 166.26 (CO, TZD), δ 137.63 (=CH), δ 133.43,
Light brown solid (yield: 71.42%; m.p. 89–91 °C); R : 0.61
f
1
32.00, 128.63, 126.43, and 119.41 (Aromatic C), δ 28.07
(hexane:ethylacetate = 5:1); IR (KBr): 2928, 2864, 1734,
+
+
+
−1
1
(
CH ); mass (ES ): 226.4 (M ), 227.4 (M + 1) .
1667, and 607 cm ; H NMR (CDCl ): δ 7.97 (s, 1H,
3
3
=
CH), δ 7.56 (d, 1H, J = 5.04 Hz, Ar–H), δ 7.31 (d, 1H, J
(
Z)-3-ethyl-5-(thiophen-2-ylmethylene)thiazolidine-2,4-
= 2.82 Hz, Ar–H), δ 7.10 (t, 1H, J = 4.40 Hz, Ar–H), δ 3.65
(t, 2H, J = 7.52 Hz, CH ), δ 1.58 (m, 2H, CH ), 1.24 (m,
dione (8)
2
2
1
3
4
H, CH ), δ 0.82 (t, 3H, J = 6.84 Hz, CH ); C NMR
2
3
Brown solid (yield: 85.98%; m.p. 100–102 °C); R : 0.58
(CDCl ): δ 167.36 (CO, TZD), δ 166.18 (CO, TZD), δ
f
3
(
1
=
=
(
hexane:ethylacetate = 5:1); IR (KBr): 2946, 2879, 1731,
137.77 (=CH), δ 133.25, 131.84, 128.59, 126.19, and
119.62 (Aromatic C), δ 42.19 (CH ), δ 28.81 (CH ), δ 27.49
667, and 579 cm− ; H NMR (CDCl ): δ 8.06 (s, 1H,
1
1
3
2
2
+
CH), δ 7.65 (d, 1H, J = 4.24 Hz, Ar–H), δ 7.39 (d, 1H, J
3.64 Hz, Ar–H), δ 7.18 (t, 1H, J = 4.44 Hz, Ar–H), δ 3.81
(CH ), δ 22.25 (CH ), δ 13.93 (CH ); mass (ES ): 282.3
2 2 3
+
(M + 1) .
1
3
m, 2H, CH ), δ 1.27 (t, 3H, J = 7.20 Hz, CH ); C NMR
2
3
(
CDCl ): δ 167.25 (CO, TZD), δ 166.01 (CO, TZD), δ
3
1
1
37.74 (=CH), δ 133.30, 131.89, 128.61, 126.25, and
19.64 (Aromatic C), δ 37.22 (CH ), δ 13.17 (CH ); mass
(Z)-3-(5-bromopentyl)-5-(thiophen-2-ylmethylene)
thiazolidine-2,4-dione (12)
2
3
+
+
(
ES ): 240.4 (M + 1) .
Brown solid (yield: 62.35%; m.p. 75–77 °C); R : 0.53
f
(
Z)-3-butyl-5-(thiophen-2-ylmethylene)thiazolidine-2,4-
(hexane:ethylacetate = 5:1); IR (KBr): 2934, 2859, 1734,
−
1 1
dione (9)
1674, 645, and 548 cm ; H NMR (CDCl ): δ 8.05 (s, 1H,
3
=
CH), δ 7.65 (t, 1H, J = 5.00 Hz, Ar–H), δ 7.38 (m, 1H,
Brown solid (yield: 70.83%; m.p. 117–119 °C); R : 0.60
Ar–H), δ 7.18 (m, 1H, Ar–H), δ 3.74 (m, 2H, CH2),
f
(
1
hexane:ethylacetate = 5:1); IR (KBr): 2955, 2867, 1732,
δ 3.41 (m, 2H, CH ), δ 1.89 (m, 2H, CH ), δ 1.70 (m, 2H,
CH ), δ 1.49 (m, 2H, CH ); C NMR (CDCl ): δ 167.35
2 2 3
2
2
667, and 604 cm− ; H NMR (CDCl ): δ 7.96 (s, 1H,
1
1
13
3
=
=
CH), δ 7.56 (d, 1H, J = 5.12 Hz, Ar–H), δ 7.30 (d, 1H, J
3.72 Hz, Ar–H), δ 7.09 (t, 1H, J = 4.36 Hz, Ar–H), δ 3.66
(CO, TZD), δ 166.11 (CO, TZD), δ 137.72 (=CH), δ
133.32, 131.92, 128.61, 126.35, and 119.48 (Aromatic C),
δ 41.71 (CH ), δ 33.29 (CH ), δ 31.99 (CH ), δ 26.99
(
t, 2H, J = 7.36 Hz, CH ), δ 1.56 (m, 2H, CH ), δ 1.27 (m,
2 2
2
2
2
1
3
+
+
2
H, CH ), δ 0.86 (t, 3H, J = 7.32 Hz, CH ); C NMR
(CH ), δ 25.24 (CH ); mass (ES ): 360.2 (M ), 362.2
2 2
2
3
+
(
CDCl ): δ 167.34 (CO, TZD), δ 166.17 (CO, TZD), δ
(M + 2) .
3

Med Chem Res
In vitro PTP1B inhibition
days at a fixed time. The blood glucose level (mg/dl) was
monitored at 2, 4, 6, and 24 h during the treatment and
finally on 7th day. The percentage reduction in blood glu-
cose level was calculated with respect to the control group.
Statistical significance of differences between different
groups was determined by one-way analysis of variance
followed by Dunnett’s test (Li et al. 2012; Lakshminarayana
et al. 2010).
PTP1B tyrosine phosphatase drug discovery kit (BML-AK
8
22, Enzo life sciences, USA) was utilized for in vitro
testing of synthesized compounds against PTP1B. It
employed a colorimetric, non-radioactive method to mea-
sure the free phosphate released. The detection of free
phosphate released was measured by the principle of classic
Malachite green assay (Martin et al. 1985). Ten millimole
stock solution (in assay buffer containing 1% DMSO) of the
test compounds were prepared. Taking suitable aliquots
from this stock solution, five different concentrations were
made by dilutions. The test compounds along with suramin
Docking studies
The crystal structure of protein tyrosine phosphatise 1B
(2NT7) was downloaded from protein data bank. Using
protein preparation wizard, bond orders were assigned,
hydrogens were added, bonds to metals were deleted, the
formal charges on the metal and the neighboring atoms
were adjusted and finally waters that were more than 5 Å
distance were deleted. The protein structure was refined
with the help of restrained minimization. The receptor grid
was set up and generated from “receptor grid generation”
panel. The co-crystallized ligand was excluded for defining
the receptor structure. The grids were defined by keeping
the co-crystallized ligand as center and using the default
box size. Extra precision docking mode of Glide 5.8
(Schrodinger LLC, New York) was utilized for molecular
docking of low energy conformers into the active site grid.
The ligands were flexible but receptor was kept rigid except
during the docking. The final evaluation was done with
glide score (docking score) and single best pose was gen-
erated for every ligand (Friesner et al. 2004; Halgren et al.
2004; Friesner et al. 2006).
(
standard inhibitor) were incubated with human recombi-
nant PTP1B enzyme and then free phosphate was measured
using biomol red as phosphate detection reagent. A 96-well
microtiter plate was used for carrying out the assay using
1
9
0 mM DMSO as control. The reaction was carried out in
6-well, flat-bottomed microtiter plates at 10 mM con-
centration using DMSO as control. A phosphate standard
curve was prepared by taking six different concentrations of
standard inorganic phosphate. The optical density (OD620)
was measured by a microplate reader and the readings were
converted to nmol of phosphate with the help of previously
generated phosphate standard curve. The activity was cal-
culated as a % of control. IC50 values were determined as
concentration value for 50% activity by regression analysis
of the linear portion of the inhibition curves obtained from
three independent sets of experiments.
In vivo anti-hyperglycaemic activity
Albino mice of laca strain were used in the in vivo
experimental study conducted as per CPCSEA (Committee
for Prevention, Control and Supervision of Experimental
Animals) guidelines and duly approved by the Institutional
Animal Ethics Committee (PU/IAEC/S/14/91) at the central
animal house facilities of Panjab University, Chandigarh,
India. All the experimental animals were housed under
standard conditions with free access to food and water. A
single i.p. injection of alloxan in normal saline (185 mg/kg
body weight) was given for inducing diabetes. The mice
were given 5% glucose solution for 12 h to reduce hypo-
glycaemic shock. The mice were then starved for overnight
prior to treatment, but had access to drinking water.
Induction of diabetes was confirmed by measuring blood
collected from the tail vein of the mice using eBsensor
blood glucose meter (Visgeneer Inc. Taiwan). A homo-
genized suspension of the test compounds and pioglitazone
ADME studies
Qikprop 3.5 (Schrodinger LLC, New York) module was
utilized for prediction of physico-chemical descriptors and
pharmacokinetically important properties (Qikprop 2012).
Results and discussion
The designed compounds were synthesized by using
known synthetic methodologies as outlined in scheme 1.
TZD (1) was synthesized by condensing chloroacetic acid
and thiourea in presence of conc. hydrochloric acid.
Knoevenagel condensation of TZD and thiophene-2-
carboxaldehyde was carried out to afford (Z)-5-(thiophen-
2-ylmethylene)thiazolidine-2,4-dione (2). Compound 2 was
subjected to N-alkylation under basic conditions to afford
the final compounds (3–12). The synthesized compounds
(
standard drug) were prepared in 0.5% CMC and permis-
1
13
sible amount of Tween 80. The test compounds and stan-
dard drug were administered with the help of oral gavage to
the respective group of animals once daily for a period of 7
were characterized by IR, H-NMR, C-NMR and mass
spectroscopy. The exocyclic double bond was found to
be in Z-configuration in all the synthesized compounds

Med Chem Res
1
(
Luo et al. 2010). The H-NMR peak of vinylic proton
drug as well as synthesized compounds. The test com-
pounds were administered orally to the experimental ani-
mals for 7 days. The % reduction of blood sugar level for all
test compound treated groups showed similar profile
(Fig. 2), but was different from that of pioglitazone. The
elevated blood sugar level gradually decreased from 2 up to
6 h and then again increased when measured at 24 h. It was
observed that the peak effect of pioglitazone was at 4 h,
whereas the synthesized compounds showed most pro-
nounced effect at 6 h. It was evident from the in vivo results
that synthesized compounds have shown the hypoglycaemic
activity for a longer duration of time as compared to pio-
glitazone. After 7 days of treatment, the % reduction in
blood sugar level ranged from 15.43 to 32.50 as compared
to 31.19 of pioglitazone. Compound 5 and 10 were
observed to decrease the blood sugar level by 30.03 and
32.50%, respectively and were comparable to the in vivo
activity of standard drug pioglitazone. At 6 h the decrease in
blood sugar level was significantly greater in case of com-
pound 5, 7, and 10 than pioglitazone. A probability (P)
value of less than 0.05 indicated a statistically significant
difference between the treatment groups. However, it was
difficult to draw a brief SAR for PTP1B inhibition from the
in vivo results.
appeared within 7.90–8.09 ppm which confirmed the for-
mation of Z isomers.
Tyrosine phosphatase drug discovery kit (PTP1B) (Enzo
life sciences, USA) was used for in vitro screening of the
synthesized compounds against PTP1B taking suramin as
standard. The results of the PTP1B inhibition data were
summarized in Table 1. The synthesized compounds were
found to be moderate to weak inhibitors except compound 7
(
IC50 = 9.96 µM), which had activity comparable to that of
suramin (IC50 = 9.76 µM).
The IC50 values of synthesized compounds were ranged
from 9.96 to 73.30 µM. It was evident that compounds with
smaller substituent like methyl appeared to be more active
than molecules with relatively bulky substituents. Com-
pound 7 was twice potent than compound 8 and around
seven times more potent than compound 5 and 12. On
increasing the alkyl chain length, the activity was observed
to be decreased. Substitution of long chain alkyl groups like
heptane, bromopentane and pentane resulted in weak
PTP1B inhibitors. Molecular docking study was further
used for a better understanding of SAR of the synthesized
compounds based on their molecular interactions with the
binding residues of PTP1B.
In vivo anti-hyperglycaemic activity was carried out for
all the synthesized compounds by alloxan induced diabetic
mice model taking pioglitazone as positive control. A dose
of 30 mg/kg body weight was chosen for both the standard
All the ligands were docked into the binding pocket
of human PTP1B (PDB entry 2NT7) to gain insight into
the binding mode of the ligands. A common binding
orientation of all the ligands was observed inside the PTP1B
active site. The interactions and top ranked binding pose
of the active compound was depicted in Fig. 3 and Fig. 4,
respectively.
Table 1 Structure and PTP1B inhibitory activities of synthesized
In compound 7, the C-2 position and C-4 position car-
bonyl oxygen formed a H-bond with Arg221 residue of the
catalytic active site and Lys120 of the R loop respectively.
Lys 120 is one of the residues which forms the catalytic
cleft of the active site and also helps in tight binding of
ligand/substrate to the enzyme. Phosphotyrosine recogni-
tion loop (residues Lys43-Tyr46), an essential structural
component of the catalytic site, determines the depth of the
active site cleft and interacts with the aromatic ring of the
compounds
Compound
R
IC50 (µM)
3
4
5
6
7
8
9
CH
3
–CH
–CH
–(CH –CH
Cl–CH –CH
2
–CH
2
–
29.10
25.39
73.30
32.36
9.96
Br–CH
CH
2
2
–
20
10
control
pioglitazone
3
)
2 5
2
–
3
0
4
2
2
–CH
2
–
2
hr
4 hr
6 hr
24 hr
7rth day
5
CH
CH
CH
3
3
3
–
-10
-20
-30
6
–CH
2
–
20.48
43.63
31.89
64.37
69.87
9.76
7
8
–(CH
2
)
2
–CH
2
–
–
9
1
1
1
0
1
2
Br–(CH
2
)
2
–CH
–CH
–CH
2
–
10
-
-
40
50
1
1
CH –(CH
3
2
)
3
2
12
Br–(CH
)
2 4
2
–
Fig. 2 Graphical representation of in vivo anti-hyperglycaemic
activity data of synthesized compounds
Suramin
–

Med Chem Res
Conclusion
A series of N-substituted 5-(thiophen-2-ylmethylene)thia-
zolidine-2,4-dione derivatives with different alkyl and
haloalkyl groups substituted on the amidic nitrogen of TZD
nucleus were synthesized. The compounds were tested for
their in vitro PTP1B inhibitory activity and in vivo anti-
hyperglycaemic activity. (Z)-3-Methyl-5-(thiophen-2-ylme-
thylene)thiazolidine-2,4-dione (7) had emerged as a potent
PTP1B inhibitor having comparable activity as that of
suramin. The activity of compound 7 was attributed to the
smaller alkyl substituent (–CH ) and its low lipophilicity.
3
The in vitro data reflected the same observation as reported
by many researchers that no substitution or substitution of
negative ionizable groups like benzyl carboxyl favoured the
activity. The catalytic active site of PTP1B was hydrophilic
and positively charged, so it accommodates negatively
ionizable groups very well. Introduction of alkyl and
haloalkyl groups on amidic nitrogen had not favoured
PTP1B inhibitory activity except for the smallest methyl
substituent. This was also evident from docking simulation
where the amidic nitrogen of TZD ring was away from the
catalytic residues (His214-Arg221) due to lipophilic alkyl
chain. These preliminary results would be helpful in
designing and optimizing PTP1B inhibitors containing TZD
scaffold.
Fig. 3 2D representation of docking interactions of compound 7 with
binding site residues of PTP1B
Acknowledgments The authors gratefully acknowledge UGC
(University Grant Commission), New Delhi, India for providing UGC-
RFSMS fellowship to Manoj Kumar Mahapatra. The authors thank
Mr. Avtar Singh (SAIF, Panjab University, Chandigarh, India) for
doing the NMR study/characterization.
Compliance with ethical standards
Conflict of interest The authors declare that they have no competing
interests.
Fig. 4 3D binding pose of compound 7 inside the PTP1B binding site
References
substrate with its tyrosine residue. Pi-pi stacking was
observed between the thiophene ring and phenyl ring of
Tyr46 residue of the substrate recognition loop. Due to
N-substitution the amidic nitrogen of TZD ring was away
from the catalytic residues face (His214-Arg221). The
N-substitution did not allow formation of deprotonated
amidic nitrogen which could be easily accommodated
inside the positively charged catalytic active site.
A number of physico-chemically significant and phar-
maceutically relevant descriptors (QPlogPo/w, QPlogS,
QPPCaco, QPlogBB, % Human oral absorption etc.) were
predicted (Supplementary data). The pharmacokinetic
parameters were found to be within the acceptable range
defined for human use, exhibiting their druggable nature.
Andersen HS, Olsen OH, Iversen LF, Sørensen ALP, Mortensen SB,
Christensen MS, Branner S, Hansen TK, Lau JF, Jeppesen L,
Moran EJ, Su J, Bakir F, Judge L, Shahbaz M, Collins T, Vo T,
Newman MJ, Ripka WC, Møller NPH (2002) Discovery and
SAR of a novel selective and orally bioavailable nonpeptide
classical competitive inhibitor class of protein-tyrosine phospha-
tase 1B. J Med Chem 45:4443–4459
Bhat BA, Ponnala S, Sahu DP, Tiwari P, Tripathi BK, Srivastava AK
(2004) Synthesis and antihyperglycemic activity profiles of novel
thiazolidinedione derivatives. Bioorg Med Chem 12:5857–5864
Bhattarai BR, Kafle B, Hwang JS, Khadka D, Lee SM, Kang JS, Ham
SW, Han IO, Park H, Cho H (2009) Thiazolidinedione deriva-
tives as PTP1B inhibitors with antihyperglycemic and antiobesity
effects. Bioorg Med Chem Lett 19:6161–6165
Byon JCH, Kusari AB, Kusari J (1998) Protein-tyrosine phosphatase-
1
B acts as a negative regulator of insulin signal transduction. Mol
Cell Biochem 182:101–108

Med Chem Res
Colagiuri S (2010) Diabesity: therapeutic options. Diabetes Obes
Metab 12:463–473
Elchebly M, Payette P, Michaliszyn E, Cromlish W, Collins S, Loy
AL, Normandin D, Cheng A, Himms-Hagen J, Chan CC,
Ramachandran C, Gresser MJ, Tremblay ML, Kennedy BP
Maccari R, Paoli P, Ottana R, Jacomelli M, Ciurleo R, Manao G,
Steindl T, Langer T, Vigorita MG, Camici G (2007) 5-Arylidene-
2,4-thiazolidinediones as inhibitors of protein tyrosine phospha-
tases. Bioorg Med Chem 15:5137–5149
Martin B, Pallen CJ, Wang JH, Graves DJ (1985) Use of fluorinated
tyrosine phosphates to probe the substrate specificity of the low
molecular weight phosphatase activity of calcineurin. J Biol
Chem 260:14932–14937
(
1999) Increased insulin sensitivity and obesity resistance in mice
lacking the protein tyrosine phosphatase-1B gene. Science
83:1544–1548
2
Friesner RA, Banks JL, Murphy RB, Halgren TA, Klicic JJ, Mainz
DT, Repasky MP, Knoll EH, Shaw DE, Shelley M, Perry JK,
Francis P, Shenkin PS (2004) Glide: a new approach for rapid,
accurate docking and scoring. 1. Method and assessment of
docking accuracy. J Med Chem 47:1739–1749
Moretto AF, Kirincich SJ, Xu WX, Smith MJ, Wan ZK, Wilson DP,
Follows BC, Binnun E, Joseph-McCarthy D, Foreman K, Erbe
DV, Zhang YL, Tam SK, Tama SY, Lee J (2006) Bicyclic and
tricyclic thiophenes as protein tyrosine phosphatase 1B inhibitors.
Bioorg Med Chem 14:2162–2177
Friesner RA, Murphy RB, Repasky MP, Frye LL, Greenwood JR,
Halgren TA, Sanschagrin PC, Mainz DT (2006) Extra precision
glide: docking and scoring incorporating a model of hydrophobic
Ottana R, Maccari R, Ciurleo R, Paoli P, Jacomelli M, Manao G,
Camici G, Laggner C, Langer T (2009) 5-Arylidene-2-phenyli-
mino-4-thiazolidinones as PTP1B and LMW-PTP inhibitors.
Bioorg Med Chem 17:1928–1937
Schrödinger LLC (2012) QikProp, version 3.5. New York, NY
Sachan N, Kadam SS, Kulkarni VM (2007) Synthesis and anti-
hyperglycemic activity and QSAR of 5-benzylidine-2,4-thiazoli-
dinediones. Ind J Heterocycl Chem 17:57–62
Tang YB, Lu D, Chen Z, Hu C, Yang Y, Tian JY, Ye F, Wu L, Zhang
ZY, Xiao Z (2013) Design, synthesis and insulin-sensitising
effects of novel PTP1B inhibitors. Bioorg Med Chem Lett
23:2313–2318
enclosure for protein–ligand complexes.
9:6177–6196
J
Med Chem
4
Halgren TA, Murphy RB, Friesner RA, Beard HS, Frye LL, Pollard
WT, Banks JL (2004) Glide: a new approach for rapid, accurate
docking and scoring. 2. Enrichment factors in database screening.
J Med Chem 47:1750–1759
International Diabetes Federation (IDF) (2015) IDF Diabetes Atlas,
7
th edn. International Diabetes Federation, Brussels, Belgium,
http://www.diabetesatlas.org
Johnson TO, Ermolieff J, Jirousek MR (2002) Protein tyrosine
phosphatase 1B inhibitors for diabetes. Nat Rev Drug Discov
Tonks NK, Diltz CD, Fischer EH (1988) Purification of the major
protein-tyrosine-phosphatases of human placenta. J Biol Chem
263:6722–6730
1
:696–709
Kaveeshwar SA, Cornwall J (2014) The current state of diabetes
mellitus in India. Australas Med J 7:45–48
Walchi S, Curchod ML, Gobert RP, Arkinstall S, Huijsduijnen RH
(2000) Identification of tyrosine phosphatases that depho-
sphorylate the insulin receptor. A brute force approach based on
“substrate-trapping” mutants. J Biol Chem 275:9792–9796
Wang WL, Huang C, Gao LX, Tang CL, Wang JQ, Wu MC, Sheng L,
Chen HJ, Nan FJ, Li JY, Li J, Feng B (2014) Synthesis and
biological evaluation of novel bis-aromatic amides as novel
PTP1B inhibitors. Bioorg Med Chem Lett 24:1889–1894
Wild S, Roglic G, Green A, Sicree R, King H (2004) Global pre-
valence of diabetes: estimates for the year 2000 and projections
for 2030. Diabetes Care 27:1047–1053
Klaman LD, Boss O, Peroni OD, Kim JK, Martino JL, Zabolotny JM,
Moghal N, Lubkin M, Kim YB, Sharpe AH, Stricker-Krongrad
A, Shulman GI, Neel BG, Kahn BB (2000) Increased energy
expenditure, decreased adiposity, and tissue-specific insulin sen-
sitivity in protein-tyrosine phosphatase 1B-deficient mice. Mol
Cell Biol 20:5479–5489
Lakshminarayana N, Prasad YR, Gharat L, Thomas A, Narayanan S,
Raghurama A, Srinivasan CV, Gopalan B (2010) Synthesis and
evaluation of some novel dibenzo [b,d] furan carboxylic acids as
potential anti-diabetic agents. Eur J Med Chem 45:3709–3718
Li F, Wu X, Zou Y, Zhao T, Zhang M, Feng W, Yang L (2012)
Comparing anti-hyperglycemic activity and acute oral toxicity of
three different trivalent chromium complexes in mice. Food
Chem Toxicol 50:1623–1631
Wrobel JM, Sredy J, Moxham C, Dietrich A, Li Z, Sawicki DR,
Seestaller L, Wu L, Katz A, Sullivan D, Tio C, Zhang ZY (1999)
PTP1B inhibition and antihyperglycemic activity in the ob/ob
mouse model of novel 11-arylbenzo[b]naphtho[2,3-d]furans
and 11-arylbenzo[b]naphtho[2,3-d]thiophenes.
J Med Chem
Liu G (2004) Technology evaluation: ISIS-113715, Isis. Curr Opin
Mol Ther 6:331–336
Luo Y, Ma L, Zheng H, Chen L, Li R, He C, Yang S, Ye X, Chen Z,
Li Z, Gao Y, Han J, He G, Yang L, Wei Y (2010) Discovery of
42:3199–3202
Ye D, Zhang Y, Wang F, Zheng M, Zhang X, Luo X, Shen X, Jiang H,
Liu H (2010) Novel thiophene derivatives as PTP1B inhibitors
with selectivity and cellular activity. Bioorg Med Chem
18:1773–1782
(
Z)-5-(4-Methoxybenzylidene)thiazolidine-2,4-dione, a readily
available and orally active glitazone for the treatment of con-
canavalin a-induced acute liver injury of BALB/c mice. J Med
Chem 53:273–281
Zhang S, Zhang ZY (2007) PTP1B as a drug target: recent develop-
ments in PTP1B inhibitor discovery. Drug Discov Today
12:373–381