Synthesis, SAR and docking studies of substituted aryl phenylthiazolyl phenylcarboxamide as potential protein tyrosine phosphatase 1B (PTP1B) inhibitors

Article abstract of DOI:10.1111/cbdd.13515

In our continued effort to discover novel PTP1B inhibitor with improved in vivo activity, we attempted to optimize our previously discovered lead compound by replacing the sulfonyl group with benzoyl group to yield compound II. Additional structural modif

Full text of DOI:10.1111/cbdd.13515

DR AMIT K GUPTA (Orcid ID : 0000-0002-2901-1075)
DR ANIL KUMAR SAXENA (Orcid ID : 0000-0002-0212-7246)
Article type : Research Article
Synthesis, SAR and Docking Studies of Substituted Aryl
phenylthiazolyl phenylcarboxamide as potential Protein
Tyrosine Phosphatase 1B (PTP1B) Inhibitors
Kanika Varshney[a],[e], Amit K. Gupta[b], Arun Rawat[c], Rohit Srivastava[c],
Akansha Mishra[c], Mridula Saxena[d], Arvind K. Srivastava[c], Sudha Jain*[e],
Anil K. Saxena*[a]
aMedicinal & Process Chemistry Division, Central Drug Research Institute, Lucknow,
226031, India.
bDepartment of Integrative Biology and Pharmacology, McGovern Medical School,
University of Texas Health Science Center at Houston, Houston, Texas 77030, USA.
cBiochemistry Division, Central Drug Research Institute, Lucknow, 226031, India.
dDepartment of Chemistry, Amity University, Lucknow, 226010, India.
eDepartment of Chemistry, Lucknow University, Lucknow, 226007, India.
*Corresponding authors: anilsak@gmail.com (AKS), jainsudha2@rediffmail.com (SJ)
Abstract
In our continued effort to discover novel PTP1B inhibitor with improved in vivo activity we
attempeted to optimize our previously discoved lead compound by replacing the sulfonyl
group with benzoyl group to yield compound II. Additional structural modifications were
performed on compound II to yield a series of 24 aryl phenylthiazolyl phenylcarboxamides
as potential PTP1B inhibitors. Of the 24 tested, 6 compounds showed good PTP1B inhibitory
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activity while compound 38 as the most promising one. The plausible PTP1B binding site
interaction of compound 38 showed favorable binding similar to known PTP1B binders and
suggest its selectivity towards PTP1B. Compound 38 also showed promising
antihyperglycemic, antidyslipidemic and insulin resistant reversal activities in vivo in STZ
model and db/db mice model. Altogether, the compound 38 present an excellent candidate for
future PTP1B targeted drug discovery.
Keywords: Type 2 diabetes • PTP1B • aryl thiazolyl phenyl benzamide • docking
1. Introduction
Protein-tyrosine phosphatase 1B (PTP1B) is a validated molecular target for the
development of novel insulin-sensitizer agents addressing both Type 2 diabetes and obesity.
PTP1B catalyses dephosphorylation of the activated insulin receptor (IR) as well as Janus
kinase 2 (JAK2) and thus negatively regulate insulin and leptin signalling pathways.[1-3] All
of PTPs share a high degree of structural similarity in the active site, pTyr (phosphotyrosine)-
binding pocket which poses a chanlenge to design PTP1B selective inhibitors.[4] In 2001,
Shen et al. successfully exploited the presence of a secondary PTP1B specific noncatalytic,
arylphosphate binding site located close to the active site to design selective bidentate PTP1B
inhibitors that can simultaneously occupy the active site and the nearby noncatalytic site.[5]
Since then number of PTP1B inhibitors targeting both of these binding sites are
Benzooxathiazole derivatives[6], Isothiazolidinone[7], Hydroxyphenylazole derivatives[8],
thiazolidine-2,4-dione derivative[9, 10], Thiazolyl derivatives[11] have been reported. These
inhibitors although show high PTP1B binding affinity but contain less cellular potency
because of poor membrane permeability which is associated with targeting active site by
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highly acidic phosphotyrosine mimetics.[1] In past we have synthesized novel substituted aryl
thiazolyl phenylsulphonamides as nonphosphorous small molecule inhibitors of PTP1B using
fragment-based approach, where compound I (73.6% PTP1B inhibition at 10µM) was found
to be the most active PTP1B inhibitor among the series (Figure 1).[12] In continuation of our
efforts to design more in vivo effective PTP1B inhibitors we herein report further lead
optimization of compound I using sub-structural approach to identify PTP1B inhibitors with
better in vitro and in vivo activities.
2. Method and Materials
2.1. Chemistry
Melting points were determined on an electrical heated m. p. apparatus /using silicon oil bath.
Reactions were monitored by thin layer chromatography on self-made plates of silica gel G
(Merck, India) or 0.25mm ready-made plates of silica gel 60F 254, (Merck, Darmstadt,
Germany). Column chromatography was performed on silica gel (Merck, 60 to 120mesh).
Infrared spectra (IR) were recorded on Perkin-FTIR model PC spectrophotometer with
frequency of absorptions reported in wave numbers. Mass were recorded on JEOL mass-
spectrometer with fragmentation pattern reported as values, 1H NMR was recorded on Bruker
spectrometer with a multinuclear inverse probe head with gradient at room temperature (298
K) using CDCl3 as solvent and tetramethylsilane (TMS) as internal standard. Chemical shifts
were described in parts per million (ppm) relative to TMS (0.00 ppm) using scale and
coupling constants were reported in hertz (Hz).
General method for synthesis of compound II
The key intermediate N-(2-(trifluoromethyl))-4, 5-dihydrothiazol-2-amine (3d) of compound
II was synthesized using 2-trifluoromethyl aniline according to the procedure mentioned in
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the earlier reported paper.[12] Further, the intermediate 3d on reaction with 4-
methoxybenzoylchloride gave the compound II.
General method for synthesis of compound 7-38
Substituted aniline (1a-c), on reaction with benzoyl isothiocyanate in dry benzene, resulted in
the formation of phenylcarbamothioyl benzamides, (2a-c) which on alkaline hydrolysis gave
the corresponding thioureas (3a-c). Condensation of the phenylthiourea with substituted α-
bromo-acetophenones (4-6) in THF at room temperature for half-an-hour resulted in the
formation of suspension, which was filtered and dried to afford substituted thiazol-2-amines
(7-14). These amines on reaction with substituted benzoyl chlorides (R4COCl) gave the
desired compounds 15-38 (Scheme 1).
2.2. In vitro PTP1B enzyme assay
All the synthesized compounds were evaluated for in vitro antihyperglycemic activity
against PTP1B using colorimetric, non-radioactive PTP1B tyrosine phosphatase drug
discovery kit BML-AK822 from Enzo Life Sciences, USA. We used human recombinant
PTP1B enzyme provided in the kit at five different concentration i.e. 0.3 µM, 1.0 µM, 3.0
µM, 5.0 µM and 10 µM and suramin as a control. IC50 value was calculated for the
compounds showing >50% inhibition at 10µM concentration. Other components of the kit
include substrate (IR5 insulin receptor residues), biomol red (phosphate determining reagent),
assay buffer, suramin (PTP1B inhibitor) and calibration standards. Assay was done according
to the Kit manufacturer’s protocol, in brief the reaction was carried out in 96 well flat-
bottomed microtiter plate by addition of assay buffer, solution of test compounds and diluted
PTP1B enzyme. Enzyme reaction was initiated by addition of 50µl of warmed 2x substrate
then incubated the plate at 300c for 30min. After incubation for 30 min. Reaction was
terminated by addition of 25 µl of biomol red reagent and mixed thoroughly by repeated
pipetting. Test compounds were dissolved in dimethyl sulfoxide (DMSO) and solution of
100µM concentration was prepared of which 10 µl solution was added in each reaction well
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to achieve final concentration of 10 µM in reaction mixture. Volumes and dilution of other
component were accordingly as instructed in the manual provided in the kit. PTP1B
phosphatase acting on the phosphopeptide substrate and release phosphate. The detection of
free phosphate released was based on classic Malachite green assay. After adding biomol red
to reaction wells after 30 minutes of incubation as described earlier the plate was incubated
for another 20 min to develop the colour. Absorbance was recorded at 620 nm on a
microplate reader. The percentage inhibition of PTP1b enzyme by test compounds was
calculated based on activity in the control tube (without inhibitor) taking as 100 % from three
independent set of experiments. The concentration of dimethyl sulfoxide (DMSO) in the test
well (1.0 %) had no demonstrable effect on PTP1b enzyme activity.
2.3. Molecular docking
To gain an insight about essential structural requirements for ligand interactions at the active
site of the PTP1B enzyme, the docking study was performed with the compound 38 at the
binding site of PTP1B crystal structure (PDB ID: 2ZMM)[13] using the standard precision
(SP) mode of Glide v6.7 of Schrödinger.[14] Ligands were prepared using LigPrep3.4 with
Epik3.2 at pH 7.0. The lowest energy conformation among the largest populated cluster
sharing common interactions was selected as the best binding pose for docked ligands.[15]
2.4. In vivo asaay
2.4.1. Anti-hyperglycemic activity
Streptozotocin induces anti-hyperglycemic activity screening was performed as reported in
ref. 16. Streptozotocin (STZ) is a broad spectrum antibiotic and selected to induce
experimental diabetes because of its greater selectivity of β-cells, lower mortality and
relatively longer half life (15 min) of STZ in the body. A solution of STZ (60 mg/kg) in 100
mM citrate buffer, pH 4.5 was prepared and calculated amount of the fresh solution dosed to
overnight fasted rats intraperitoneally. Two days later baseline blood glucose was drawn from
tail vein and glucose levels determined by glucostrips (Roche) to confirm the induction of
diabetes. Rats having hyperglycaemia of the range of 270 and 450 mg/dL were considered as
diabetic, selected and divided into groups of five animals each. One group used for normal
control receives only vehicle (gumacacia) and this group was considered as diabetic control.
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The blood glucose measured at this time was termed the baseline (0 min) blood glucose. Rats
of experimental groups were orally administered suspension of the desired test samples
(made in 1.0% gum acacia) at desired dose levels and the biguanide derivative Metformin
was used as standard antidiabetic drug and was always given at a dose of 100 mg/kg body
weight orally to the experimental group. After 30 min of drug treatment, blood glucose level
was again measured with glucometer. The blood glucose assessment were collected from tail
vein just prior administration of test sample i.e. 0, 30, 60, 90, 120, 180, 240, 300 and 1440
min post test sample administration. After 300 min the STZ treated animals were allowed to
feed over night to overcome drug induced hypoglycaemia. The animals were fed ad lib
during 5 to 24h of experiments. The average fall in AUC (area under curve) in experimental
group compared to control group was termed as % antihyperglycaemic activity. Statistical
analysis was done by Dunnett’s test.
2.4.2. db/db mice study
Male C57BL/Ks strain of mouse (db/db mouse) 10-12 weeks of age and around 40 3 g of
body weight was procured from the animal colony of the Institute. The animals were housed
four or five in a polypropylene cage in the animal house. The following norms were always
followed for animal room environment: temperature 23 2°C; humidity 50-60%; light 300
lux at floor level with regular 12h light cycle; noise level 50 decibel; ventilation 10-15 air
changes per hour. After randomization into groups, the mice were acclimatized for 2-3 days
in the new environment before initiation of experiment. Standard pellets were used as a basal
diet during the experimental period.
2.4.2.1. Antihyperglycaemic and Antidyslipidemic activity assessment in C57BL/Ks
strain of mouse (db/db mouse)
The animals were allocated into groups of 5 animals in each. Prior to start of the sample
feeding, a vehicle training period was followed from day -3 to day 0 during which all the
animals were given vehicle ( 1% gum acacia) at a dose volume of 10 mL/kg body weight. At
day 0 the animals having blood group level between 350 to 500 mg/dL were selected and
divided into three groups containing 5 animals in each. One group was considered as control
group while the other group was treatment group. The experimental group was given
suspensions of compound 154 and Pioglitazone at 30.0 and 10.0mg/kg body weight dose
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respectively. The control group was given an equal amount of vehicle. All the animals had
free access to fresh water and normal diet. Random blood glucose of each mouse was
checked daily at 10.00 pm. On day 10 and day 15 oral glucose tolerance (OGTT) test was
performed to study the effect of compound on glucose tolerance. Blood has been withdrawn
from the retroorbital plexus of mice eye for the estimation of lipid profile on DIALAB DTN-
410-K and insulin level by CALBIOTECH Insulin ELISA Kit. Body weight of each animal
was measured on alternate day for studying the effect of test sample on body weight. The
skeletal muscle from each mouse were quickly excised at the end of experiment under light
anesthesia and frozen at -80oC until further use.
2.4.2.2. Oral glucose and intraperitoneal insulin and pyruvate tolerance test
Oral glucose and pyruvate tolerance test were performed on 12h fasted mice while insulin
tolerance test was performed on 4h fasted mice. For OGTT mice were administered glucose
3.0 g/kg by gavages whereas for ITT and PTT, insulin (0.8U/kg) and pyruvate (2g/kg) was
injected intraperitoneally, respectively. Blood samples were obtained via tail nick at 0, 30, 60,
90 and 120 min during OGTT and PTT while at 0, 15, 30, 60, 90 and 120 time points in ITT.
Glucose was measured with the One Touch Ultra glucometer (Accu-Chek Sensor, Roche
Diagnostics).
2.4.3. Western blot analysis
Collected tissues and cells were homogenized into PBS containing 1% NP40, 5 mM
EDTA, phosphatase inhibitors and protease inhibitors cocktail (lysis buffer). Samples were
homogenized and incubated on ice for 15 min. Sample is then stored at – 80ºC and thawed at
37ºC in waterbath. Sample was then centrifuged at 16000 rpm at 4ºC. Then supernatant was
taken and quantified by Bradford assay. 40 μg protein (supernatant) of each incubation was
resolved on SDS-PAGE, transferred to nitrocellulose membranes and probed with p-Akt and
p-IRS1, Glut4 (Cell Signaling, MA, USA) and β-actin (Santa Cruz) was taken as the loading
control. Immunoreactive bands were visualized by Enhanced Chemiluminescence according
to manufacturer’s instructions (GE Healthcare, UK). Protein expression was evaluated by
densitometric analysis performed with Alpha DigiDoc 1201 software (Alpha Innotech
Corporation, CA, USA). The same size rectangle box was drawn surrounding each band and
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the intensity of each was analysed by the program after subtraction of the background
intensity.
2.4.4. Statistical analysis
The homeostatic model assessment (HOMA) was used to calculate relative insulin resistance
as follows: [Fasting blood glucose (mg/dL) × Fasting serum insulin (µLU/mL)]/405. All
statistical calculations were performed using Graph-Pad Prism version 3.02 for Windows
(GraphPad Software). Statistical analysis was carried out by Students t-test. Data was
expressed as mean +SE. The results are reported as mean values SEM.
3. Result and Discussion
3.1. Design and synthesis
PTP1B inhibitors containing thiazolyl and carboxyl group are known.[7, 9-11, 13] Therefore, we
replaced sulfonyl moiety in the compound I by benzoyl group (region B) to afford compound
II and to see its impact on the PTP1B inhibitory activity. (Figure 1)
Figure 1. Optimization of compound I.
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In the next step the dihydrothiazolyl moiety in compound II (Region A) was substituted by 5-
phenylthiazolyl group to investigate if the phenyl ring can occupy better space/ interactions at
the PTP1B enzyme active site than compound II. The systematic substitution by different
groups around region B and C was carried out for getting more insight for SAR. This led us
to synthesize a series of 24 substituted thiazolyl-N-phenyl-benzamide derivatives using
procedure described in scheme1.
These synthesized compounds were evaluated for PTP1B enzyme inhibitory activity.
Compounds more than 50% inhibition at 10 µM concentration were further evaluated at five
different concentrations to calculate their IC50 values ( Table1). Suramin (supplied in the
enzyme activity kit) was used as a reference standard. The antihyperglycemic activity
including PTP1B inhibitory activities of the thiazolyl-N-phenylcarboxamide derivatives are
also presented in Table 1.
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3.2. Structure activity relationship (SAR) of compounds tested
Initial SAR studies were focused on C5 position of thiazolyl ring (R3) to explore if binding
with the non-catalytic binding site of PTP1B could be enhanced. Therefore, as a starting point
-Me group was inserted at R3 position and was kept constant to see the effect of R, R1, R2 and
R4 substituent on the PTP1B inhibition.
Table 1: In vitro PTP1B enzyme inhibitory and in vivo antihyperglycemic activity in STZ model for
compound 15-38.
IC50
Blood glucose level b
Compd. No
R
R1
R2
R3
R4
n
% inhibition
at 10 µM
(µM)
0-5hr
0-24hr
II
CF3
H
H
F
H
H
-
4-OMePh
4-ClPh
-
48
15
Me
0
35.2 ( 3.4)
16
17
H
H
F
F
H
H
Me
Me
-CH2CH2Ph
-CH2Ph
0
0
33.0 ( 4.6)
60.3 ( 5.8)
8.0 ( 2.8) 14.1 ( 4.7)
23.2 ( 5.5)
18
19
H
H
F
F
H
H
OMe
OMe
4-ClPh
0
0
56.9 ( 3.8)
74.7 ( 6.7)
8.8 ( 2.2)
7.0 ( 2.4)
-CH2CH2Ph
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
H
F
H
OMe
H
-CH2Ph
0
0
0
0
0
0
0
0
0
0
0
0
0
2
2
2
2
2
9.8 ( 3.7)
H
F
H
4-ClPh
37.2 ( 9.7)
69.0 ( 5.8)
24.7 ( 4.7)
26.5 ( 4.2)
56.2 ( 6.2)
63.6 ( 6.8)
64.1 ( 7.7)
64.3 ( 7.9)
75.0 ( 8.8)
71.3 ( 9.2)
52.7 ( 6.6)
57.2 ( 8.6)
9.8 ( 3.8)
H
F
H
H
-CH2CH2Ph
-CH2Ph
6.9 ( 2.1) 2.44 ( 1.8)
11.7 ( 4.4)
H
F
H
H
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
H
H
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
H
Me
Me
Me
OMe
OMe
OMe
H
4-ClPh
H
-CH2CH2Ph
-CH2Ph
8.6 ( 2.1)
H
7.7 ( 1.5) 11.4 ( 3.6)
7.6 ( 1.7) 13.4 ( 3.8)
7.4 ( 2.8) 10.6 ( 2.5)
6.3 ( 2.1) 17.6 ( 1.9)
7.3 ( 3.5) 2.48 ( 1.8)
15.8 ( 4.2)
17.8 ( 3.9)
9.56 ( 2.8)
25.1* ( 2.8)
6.78 ( 2.7)
H
4-ClPh
H
-CH2CH2Ph
-CH2Ph
H
H
4-ClPh
H
H
-CH2CH2Ph
-CH2Ph
8.6 ( 4.5)
-
H
H
8.0 ( 3.8) 18.3 ( 4.1)
16.6 ( 4.8)
OMe
OMe
OMe
OMe
OMe
Me
Me
Me
OMe
OMe
4-ClPh
H
H
-CH2CH2Ph
-CH2Ph
24.6 ( 4.2)
21.6 ( 4.8)
14.6 ( 3.7)
69.9 ( 5.8)
H
H
H
H
4-ClPh
H
H
-CH2CH2Ph
6.9 ( 2.7) 6.10 ( 2.8)
13.2 ( 4.7)
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38
H
-
OMe
H
-
OMe
-CH2Ph
2
-
76.6 ( 7.6)
67.3 ( 6.8)
--
5.8 ( 1.8) 22.8 ( 3.7)*
24.3* ( 3.4)
23.7 ( 4.8) **
30.1 ( 4.8) **
Suramin
-
-
-
-
-
-
Sodium
-
-
orthovanadate
Values are mean SEM, n=4-6, p< 0.05 * p<0.01**, b % lowering at the dose of 100 mg/kg)
Compound 17 having R, R2 as H, R1 as fluoro, R3 as Me and R4 as CH2Ph showed 60.3%
PTP1B inhibition (IC50 = 8.0 µM). Substitution of R, R2 with OMe, R1 as H and keeping R4 as
CH2Ph leading to compound 26 resulted in a negligible increase in activity (63.6 % PTP1B
inhibition; IC50 = 7.7 µM). However, the substitution 2,5-(OMe)2Ph (R, R2=OMe, R1=H) in
compound 26 by 4-OMePhCH2CH2 (R, R2=H, R1=OMe, n=2) and keeping R4 as CH2Ph in
compound 35 resulted in a great decrease in the activity from 63% to 21.6% PTP1B
inhibition.
Further, the replacement of R4 group in compound 17 by 4-ClPh (compound 15) and by
CH2CH2Ph (compound 16) led to 35% and 33% reduction in PTP1B activity respectively as
compared to compound 17 (60.3% PTP1B inhibition). Similar trends were observed in
analogous compounds 24, 25 with 2,5-(OMe)2 (R, R2=OMe, R1=H) and compounds 33 and
34 with 4-Me (R, R2=H, R1=OMe, n=2) substitutions. To further explore the SAR, Me group
at R3 position in the above compounds was replaced by OMe group. In general, the
compounds with OMe at R3 position were more active in comparison to the compounds with
Me group except the compound 20 and 36 which showed less than 15% PTP1B inhibition.
The compound 38 (R, R2=H, R1, R3=OMe, n=2, R4=CH2Ph) was found to be the most active
compound of the series with 76.6% PTP1B inhibition (IC50 = 5.8 µM). The replacement of R,
R1 and R2 in compound 38 by R, R2 =OMe, R1=H in the compound 29 resulted into
negligible change in activity (75% PTP1B inhibition at 10 µM; IC50=6.3 µM) compared to
compound 38. However, insertion of the F group at R1 in place of H and R, R2=H in place of
OMe led to the substantial decrease in the activity and made compound 20 as least active
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compound of the series (9.8% PTP1B inhibition). Further, the replacement of CH2Ph group at
R4 position by CH2CH2Ph resulted in slight reduction in the activity as the compound 37 (R,
R2=H, R1=OMe, n=2, R3= OMe, R4=CH2CH2Ph, 69.9% PTP1B inhibition; IC50=6.9 µM); and
28 (R, R2=OMe, R1=H, R3=OMe, 64.3% PTP1B inhibition;IC50=7.34 µM);. In contrast to the
compounds 37 and 28, the compound 19 (R, R2=H, R1=F, R3=OMe and R4=CH2CH2Ph)
showed increased activity with 74.7% PTP1B inhibition (IC50=7.0 µM). Incorporation of 4-
ClPh group in the place of CH2CH2Ph at R4 position in compound 18 (R, R2=H, R1=F,
R3=OMe) showed decrease in activity with 56.9% PTP1B inhibition (IC50=8.6µM);. A similar
trend was observed in case of compound 27 (R, R2=OMe, R1=H, R3=OMe, 64.1% PTP1B
inhibition; IC50=7.55 µM); but in compound 36 (R, R2=H, R1=OMe, n=2, R3=OMe) which
showed highly reduced activity with 14.6% PTP1B inhibition in comparison to its analogous
compound. Removal of the 4-substituent at R3 position and keeping it as H (compound nos.
21, 31 and 32) resulted in slight decrease in the PTP1B inhibitory activity in comparison to
their corresponding 4-OMe and 4-Me analogues except compounds 22 and 30 which showed
slight increase in activity in comparison to corresponding analogues with 69% PTP1B
inhibition IC50=6.9 µM); and 71.3% PTP1B inhibition (IC50 = 7.33 µM) respectively.
3.3. Kinetics Measurements and Mechanism of Inhibition
Kinetic study was done to determine the type of inhibition of the compound 38. Enzyme
activity assay at different concentration of of compound 38 (0, 5, 10, and 20µM) with varying
concentration (40 to 140) µM of substrate IR5 (sequence from the insulin receptor ß subunit
domain provided in the kit) was performed and Lineweaver-Burk double reciprocal plot was
drawn. (Fig 2)
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Figure 2. Competitiive inhibitory profile of compound 38.
Figure 2 Plot shows intercept of aall lines obtained with 0µM, 5µM, 10µM and 20µM
compound concentration converging at Y-axis (1/Vmax ) where slope and X-axis intercept (-
1/Km ) vary with inhibitor concentraation. This suggests compound 38 follows a competitive
inhibition kinetics. The Km value for the substrate IR5 was calculated from this plot was
found to be 60.1µM. The value of Ki 8.59 µM was calculated from intercept on X-axis of this
replot which was determined by plottiing the slope values vs compound 38 concentratioon.
3.4. Molecular Modeling Studies
In past our lab has successfully exxploited structure-based computational approaches for
understanding the structure-activity relationships.[13, 17-20] To gain structural insight for further
optimization, we constructed the 3DD binding model of compound 38 to PTP1B using
molecular docking. The binding analyysis of the compound 38 revealed that 4-methoxyyphenyl
group linked with thiazolyl moeity waas buried deep inside of a hydrophobic cavity formed by
the residues Phe182, Ile219, and Alaa217 which was further stabilized by its π-π interaction

with Phe182, which in turn involved in the WPD loop closure. The thiazolyl group itself
made π-π interaction with Tyr46 (Figure 3) which is commonly observed in many known
PTP1B/inhibitor complexes.[14, 21-24] The amino acid residues Arg47 and Asp48 which are
close to the active site have been demonstrated important for selectivity of PTP1B because of
the point mutation at this residue present in other phosphatases such as LAR (Asn48) and
SHP-2 (Asn48).[25, 26] The phenyl acetyl group in the compound 38 showed potential to form
hydrogen bond interaction with Asp48 and was in close contact to Arg47 which suggest its
selectivity towards PTP1B. However, more experiments need to be done to confirm this
hypothesis. Water molecules also plays an important role in the stabilization of compound 38
through water mediated hydrogen bond interactions with amino acid residue Asp181 which is
consistent with co-crystallized ligand used in this study.[14] Because of these multiple
interactions, compound 38 positioned itself effectively at the PTP1B binding site to serve as
a potent inhibitor.
N262
F182
D48
I219
R47
V49
D181
Y46
A217
R221
Figure 3. Lowest energy docking pose of compound 38 (cyan) at the active site of PTP1B (PDB ID:
2ZMM).
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3.5. In vivo biological activity
3.5.1. Streptozotocin rat model study
Based on the high PTP1B inhibitory activity in vitro, a limited number of compounds with
more than 57% PTP1B inhibition were evaluated in vivo in Streptozotocin induced rat model
(STZ). The table 1 discribes the effect of the ten selected compounds 17, 22, 26-30, 32, 37,
38 along with the standard compound Sodium orthovanadate on decline in blood glucose
level of streptozotocin treated diabetic rats. Sodium orthovanadate was taken as positive
control. It is evident from the results that out of the ten compounds tested, only one
compound 38 showed a significant decline in blood glucose levels on streptozotocin induced
diabetic rats. This decline in blood glucose level was around 22.8 % (p<0.05) and 23.7 %
(p<0.01) during 0-5h and 0-24h, respectively (Figure 4A and B). The standard drug sodium
orthovanadate demonstrated maximum decline in blood glucose levels to the tune of 24.3 %
(p<0.05) during 0-5h and 30.1 % (p<0.01) during 0-24h, post treatment respectively on STZ-
induced diabetic rats at 100 mg/kg oral dose. The compounds 17, 22, 26-30, 32 and 37
though showed decline in blood glucose on STZ-induced diabetic rats at 100 mg/kg oral dose
level as shown in Table 1, however the effect was not found statistically significant in any of
the cases except the compound 38 where the decline in blood glucose was observed around
23.7 % during 0-24 h (Figure 4).
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Figure 4. A and B. Effect of compound 17, 22, 26-30, 32, 38 and Standard compound Sodium
orthovanadate on blood glucose levels of the streptozotocin treated diabetic rats at variouus time
intervals.
3.5.2. db/db mouse model study
3.5.2.1. Effect on hyperglycaemia
Compound 38 was further evaluated for antihyperglycaemic and antidyslipidemic activities in
C57BL/KsJ-db/db mice (Table 2). Antihyperglycaemic activity was carried out in vivo using
db/db mice by observing overall glucose lowering effect and also improvement on oral
glucose tolerance at 30mg/kg for the period of 15 days. Pioglitazone was taken as positive
control. It is evident from the Figure 5A that compound 38 exerted its effect on blood glucose
from day 5 while the significant effect was observed from day 7 and persisted till the end of
the experiment, whereas, Pioglitazone (at the dose of 10mg/kg) significantly declined the
random blood glucose from day 6 which persisted up to the end of the experiment as
compared to vehicle treated control group.

Table 2: Antihyperglycaemic and antidyslipidemic activity in db/db mice (% efficacy after
15 days)
Comp. Antihyperglycemic activity
no.
Antidyslipidemic activity
Insulin resistant reversal
OGTT
ITT
PTT
TG
CHOL
HDL
Fasting
blood
glucose
Serum
insulin
level
HOMA index
15 days
38
36.6 3.8
39.2 1.9
25.7 1.9
18.5 0.6
11.6 0.3
27.3 0.3
44.5 5.4
39.4 3.4
66.6 11.6
Pioglit 52.9 17.6
azone
62.4 7.3
35.8 2.1
12.6 0.6
9.2 0.8
11.1 0.8
64.7 6.6
46.1 1.8
70.3 10.6
3.5.2.2. Effect on oral glucose tolerance
To observe the effect of drug on glucose tolerance, an OGTT (oral glucose tolerance test)
conducted on day 14 during dosing in overnight fasted mice. After 14 days of consecutive
dosing an OGTT was carried out and the results showed that inhibition of PTP1B by the
compound 38 effectively resisted the rise in postprandial hyperglycaemia during 30 and 60
mins post glucose load and significantly enhanced the glucose clearance from blood at 60, 90
and 120 time points (Figure 5B) and an overall improvement of 36.6% (p<0.01) on glucose
disposal was calculated by AUC analysis (0-120min) with no observed hyperglycaemia,
whereas, reference drug Pioglitazone showed an improvement of 52.9% (p<0.01) on glucose
AUC as shown in Figure 5C. The fasting baseline blood glucose value at 0 time point was
also found lower in the compound 38 treated groups compared to the vehicle treated control
group at the corresponding time point because of the antihyperglycaemic effect. It is evident
from the graph that the compound 38 and Pioglitazone effectively declined the rise in
postprandial hyperglycaemia induced by glucose load of 3g/kg.
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Figure 5: Antihyperglycaemic profile and insulin resistance reversal profile of db/db mice; A.
Random blood glucose, B. Oral glucose tolerance, C. Glucose AUC (OGTT), D. ITT, E. AUC (ITT),
F. PTT, G. AUC (PTT), H. FBG, I. serum Insulin, J. HOMA-index.
To assess the whole-body insulin sensitivity, insulin tolerance test (ITT) was performed in
vehicle, compound 38 and pioglitazone treated mice. It was found that compound 38
significantly improved the insulin sensitivity at 15 and 30 min after a bolus of 0.75 units of
human insulin as shown in Figure 5D and area under the curve (AUC) analysis also showed a

39.2% improvement in the insulin tolerance (Figure 5E) as compared to vehicle treated
control group, whereas reference drug Pioglitazone improved the insulin sensitivity by 62.4%
(p<0.01). Further to assess the effect of the compound 38 on hepatic insulin sensitivity, the
pyruvate tolerance test was performed by intraperitoneally administrating pyruvate, a major
gluconeogenic substrate. The results of pyruvate tolerance test showed that the compound 38
significantly increased the insulin sensitivity and effectively resisted the rise in blood glucose
(Figure 5F) caused by a bolus of pyruvate injection as compared to vehicle treated control
group. The AUC analysis indicated an improvement of 25.7 and 35.8% (p<0.01) (Figure 5G)
on pyruvate tolerance by compound 38 and reference drug Pioglitazone, respectively as
compared to vehicle treated control group.
3.5.2.3. Effect on fasting blood glucose, serum insulin level, HOMA-index.
Insulin resistance is one of the characteristic features of db/db mice. Decreased insulin
sensitivity leads to hyperglycaemia, hyperinsulinemea. Treatment with the compound 38,
nearly normalizes the fasting blood glucose by 44.5% (p<0.01) and restores the altered
insulin level by 39.4% (p<0.01) in the treated diabetic mice as compared to vehicle treated
control group as shown in Figure 5H and 5I. Homeostatic model assessment (HOMA) is a
method used to quantify insulin resistance that is quantified on the basis of fasting blood
glucose and fasting serum insulin level. The treatment with the compound 38 significantly
improves the insulin resistance state by improving the HOMA-index by 66.6% (p<0.01)
(Figure 5J).
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3.5.2.4. Antidyslipidemic activity
Type 2 diabetes is known to be associated with several adverse cardiovascular risk factors,
including obesity, hypertension and serum lipid abnormalities, characterized mainly by
elevated serum total triglycerides and low levels of high-density lipoprotein (HDL)
cholesterol. Therefore, compound 38 was further tested for its effect on serum lipid profile.
The treatment of the compound 38 restored the altered serum lipid profile as evident from the
Figure6 (A, B and C). The compound 38 significantly lowerd the triglyceride and serum
cholesterol levels by 18.5% and 11.5 % (p<0.05) respectively and significantly increased
theserum HDL-cholesterol by 27.3.8% (p<0.05) in comparison to the standard drug
Pioglitazone which significantly lowered the serum triglyceride and cholesterol levels by
12.6% (p<0.05) and 9.7% respectively.
Excessive consumption of the food intake and body weight is responsible for the
development of obesity and it can be directly linked to type 2 diabetes. Further observation
on body weight during the antidiabetic activity in db/db mice study resulted in decrease of
body weight in mice during the treatment of compound 38 (Figure 6D).
Figure 6: Effect of compound 38 on serum lipid profile of db/db mice, A) Triglyceride; B)
Cholesterol; C) HDL-c; D) Body weight.

3.3.2.5. Western blot analysis of p-IRSI in skeletal muscle of db/db mice
Further, the inhibition of PTP1B by compound 38 was evident by western blot analysis as
shown in Figure7A. The compound 38 effectively inhibited the PTP1B which is evident with
increase of approximately 2.34 fold increase in the level of p-IRS-1 and 1.95 fold increase in
the level of p-Akt, a downstream receptor of insulin signaling, while the reference drug
rosiglitazone exhibited an increase of 3.21 and 2.52 folds in p-IRS1 and p-Akt respectively,
as compared to vehicle treated control group. (Figure7 B and C). The activation of IRS1 and
Akt by insulin resulted in the clearance of circulating blood glucose which is evident by the
significant increase in protein level of Glut4 in skeletal muscle. In the present study, it was
observed that the inhibition of PTP1B with compound 38 resulted in improved insulin
signaling and glucose homeostasis with an increase of approximately 2.1-fold in Glut4
(Figure 7D).
Figure 7: Inhibition of PTP1B by compound 38 improved insulin signaling in skeletal muscle of
db/db mice. A) Western blot analysis of p-IRSI, p-Akt and Glut4 in skeletal muscle of db/db mice, 40
µg of protein was resolved on SDS-PAGE; B) Evaluation for levels of p-IRS1; C) Evaluation for
levels of p-Akt; D) Evaluation for levels of Glut4. The experiments were repeated three times and
values are means SEM of three independent experiments. The blot shown were representatives of
the indicated groups and the densitometric analyses of the same are given below. *p< 0.05, **p <
0.01.

4. Conclusion
A series of aryl phenylthiazolyl phenylcarboxamide derivatives were synthesized and
evaluated against PTP1B enzyme. Among the twenty-five synthesized compounds, six
compounds showed good PTP1B inhibitory activity, namely 19 (IC50=7.0 μM), 22 (IC50=6.9
μM), 29 (IC50=6.3 μM), 30 (IC50=7.33 μM), 37 (IC50=6.9 μM) and 38 (IC50=5.8 μM).
Docking studies showed that the interacting residues with compound 38 at the active site are
consistent with known PTP1B inhibitors. Also interaction of compound 38 with Arg47 and
Asp48 may attribute to its selectivity over other homologous phosphatases. Compound 38
also showed promising antihyperglycaemic, antidyslipidemic and insulin resistant reversal
activities in vivo, in STZ model and db/db mice model. Thus, these studies may be helpful in
developing novel PTP1B inhibitors with improved pharmacological properties.
Acknowledgments
We thank Sophisticated Analytical Instruments Facility, CDRI for providing analytical data.
AKG is supported by a fellowship from the Gulf Coast Consortia, on the Computational
Cancer Biology Training Program (CPRIT Grant No. RP170593). The CDRI communication
number allotted to this manuscript is 9817.
The authors declare no conflict of interests.
Supporting Information
Method of preparation of intermediate compound 2a, 2b, 3a, 3b and 3c and Characterization
data for compound 7-38.
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Figure lengends
Figure 1. Optimization of compound I.
Figure 2. Competitive inhibitory profile of compound 38.
Figure 3. Lowest energy docking pose of compound 38 (cyan) at the active site of PTP1B
(PDB ID: 2ZMM).
Figure 4. A and B. Effect of compound 17, 22, 26-30, 32, 38 and Standard compound
Sodium orthovanadate on blood glucose levels of the streptozotocin treated diabetic rats at
various time intervals.
Figure 5: Antihyperglycaemic profile and insulin resistance reversal profile of db/db mice;
A. Random blood glucose, B. Oral glucose tolerance, C. Glucose AUC (OGTT), D. ITT, E.
AUC (ITT), F. PTT, G. AUC (PTT), H. FBG, I. serum Insulin, J. HOMA-index.
Figure 6: Effect of compound 38 on serum lipid profile of db/db mice, A) Triglyceride; B)
Cholesterol; C) HDL-c; D) Body weight.
Figure 7: Inhibition of PTP1B by compound 38 improved insulin signaling in skeletal
muscle of db/db mice. A) Western blot analysis of p-IRSI, p-Akt and Glut4 in skeletal muscle
of db/db mice, 40 µg of protein was resolved on SDS-PAGE; B) Evaluation for levels of p-
IRS1; C) Evaluation for levels of p-Akt; D) Evaluation for levels of Glut4. The experiments
were repeated three times and values are means SEM of three independent experiments.
The blot shown were representatives of the indicated groups and the densitometric analyses
of the same are given below. *p< 0.05, **p < 0.01.
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