Molecular modeling and synthesis of ZINC02765569 derivatives as protein tyrosine phosphatase 1B inhibitors: Lead optimization study

Article abstract of DOI:10.1007/s00044-012-0165-0

This article describes design, synthesis, and molecular modeling studies of the ZINC02765569 derivatives as potent protein tyrosine phosphatase 1B (PTP1B) inhibitors, which was previously reported as a vHTS hit (ZINC02765569) by our laboratory. Ten compou

Full text of DOI:10.1007/s00044-012-0165-0

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2013) 22:1618–1623
DOI 10.1007/s00044-012-0165-0
ORIGINAL RESEARCH
Molecular modeling and synthesis of ZINC02765569 derivatives
as protein tyrosine phosphatase 1B inhibitors: lead optimization
study
•
Prashant Joshi Girdhar Singh Deora
•
•
Vandana Rathore Arun K. Rawat
•
•
A. K. Srivastava Deepti Jain
Received: 22 April 2012 / Accepted: 16 June 2012 / Published online: 30 June 2012
Ó Springer Science+Business Media, LLC 2012
Abstract This article describes design, synthesis, and
molecular modeling studies of the ZINC02765569 deriva-
tives as potent protein tyrosine phosphatase 1B (PTP1B)
inhibitors, which was previously reported as a vHTS hit
(ZINC02765569) by our laboratory. Ten compounds were
synthesized and characterized by IR, MASS, and NMR
followed by in vitro screening for PTP1B inhibition and
glucose uptake in skeletal muscle L6 myotubes. The most
potent compound 3j shows 66.4 % in vitro PTP1B inhi-
bition and 39.6 % increase in glucose uptake. Glide was
used to study the nature of interactions governing binding
of designed molecules with active site of the PTP1B
enzyme.
Keywords Diabetes Á PTP1B inhibitors Á
ZINC02765569 derivatives Á Molecular modeling Á
Lead optimization study
Introduction
Diabetes mellitus (DM) is complex progressive disease,
considered one of the leading non-communicable meta-
bolic disorders, estimated to affect 300 million people
worldwide and 33 million people in India by the year 2025.
Indians alone may have about 1/5 of the global diabetic
population by 2025. Higher blood Glucose levels for
longtime may cause; slow wound healing, blurring of
vision, renal collapse, increased cardiovascular risk, and
may lead to premature death.
Prashant Joshi, Girdhar Singh Deora contributed equally to this study.
Diabetes mellitus is characterized by insulin deficiency
and insulin resistance, or, both (Goodman and Gilman,
2001). The effect of insulin on the tyrosine domain of the
insulin receptor (IR) for glucose metabolism is the result of
counter-action of tyrosine kinases versus tyrosine phos-
phatases (Zhang, 2003). Imbalance between these the two
enzymes’ activity disrupts normal cell function, therefore
entangled in diabetes, obesity, and cancer disorders
(Zhang, 2001; Lund et al., 2004; Seely et al., 1996; Ahmad
et al., 1995). Tyrosine kinases phosphorylate alpha subunit
of the insulin receptor, while the phosphatase family work
against maintaining homeostatic balance. Protein tyrosine
phosphatase 1B negatively regulates insulin signaling in
vivo by dephosphorylating important tyrosine residue at a-
subunit of the IR, assuaging receptor tyrosine kinase
activity. (Wang et al., 2001; Bandyopadhyay et al., 1997;
Ramachandran et al., 1992; Ahmad et al., 1997a, b; Kenner
et al., 1996; Chen et al., 1999). Inhibition of PTP1B is,
therefore, expected to improve insulin resistance in DM
Electronic supplementary material The online version of this
article (doi:10.1007/s00044-012-0165-0) contains supplementary
material, which is available to authorized users.
P. Joshi (&) Á G. S. Deora (&) Á D. Jain
School of Pharmaceutical Sciences,
Rajiv Gandhi Proudyogiki Vishwavidyalaya,
Airport Bypass Road, Gandhi Nagar, Bhopal 460 023, India
e-mail: prashant_pharm@rediffmail.com;
prashantm.pharm@gmail.com
G. S. Deora
e-mail: gsdeora84@gmail.com
G. S. Deora
Institute of Life Sciences, University of Hyderabad Campus,
Gachibowli, Hyderabad 500 046, India
V. Rathore
B. N. Girls College of Pharmacy, Udaipur 313 002, India
A. K. Rawat Á A. K. Srivastava
Division of Biochemistry, Central Drug Research Institute,
Lucknow 226 001, India
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Med Chem Res (2013) 22:1618–1623
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(Zhang and Lee, 2003; Blume and Hunter, 2001; Rotella,
2004). Further, many biochemical and genetic studies
justify this rationale; for example Chromosomal region
20q13.1, where the PTP1B gene is situated, is found as a
risk factor for obesity and type 2 diabetes (Lembertas et al.,
1997). Besides these, two different PTP1B knockout mice
studies in 1999 and 2000 show augmented insulin sensi-
tivity and reduced body weight gain following a high-fat
diet (Elchebly et al., 1999; Klaman et al., 2000). From
another study, it can be stated that, over-expression of a
PTP1B slice variant lead to improved plasma insulin level
(Sell and Reese, 1999). In similar surveillance, the
administration of PTP1B antisense Oligonucleotides to
diabetic, obese mice lead to reduce plasma glucose and
fetch insulin level to normal (Zinker et al., 2002).
Table 1 Classification of PTP1B inhibitors
Classification of PTP1B inhibitors
1. Competitive/Active site inhibitors
Peptidyl and peptido-mimetics
Phosphonates and fluoro-phosphonates
Carboxylic and a-keto carboxylic acids
Non-carboxylic acid derivatives: Tetrazoles, Catecholes,
Malononitrile and TZD isostere
2. Non-competitive/Allosteric Site inhibitors
a -Bromo acetophenones and pyridazines
Table 2, which upon further tailoring provide an opportu-
nity to develop better preclinical candidates with improved
overall efficacy and potency.
The key challenging issues in designing new PTP1B
inhibitors includes: first, incorporation of the hydrolytically
stable phosphotyrosine (pTyr) or phosphate mimics, in
appropriate PTP1B substrate (such as phosphonates, sele-
nates, and carboxylate); second, appropriate non-tyrosine
binding lipophillic aromatic domain was incorporated
which bind to allosteric site (to achieve selectivity and
higher bioavailability).
Experimental
Molecular modeling
For better understanding regarding binding mode of
ZINC02765569 derivatives at the molecular level, we
carried out molecular docking simulation of synthesized
molecules at the PTP1B ligand-binding site. The docking
studies of synthesized molecules (3b–k) were performed
using the Schrodinger software suite (Maestro, version 9.2,
2011). The molecules were sketched in 3D format using
build panel and were prepared for docking using LigPrep
application. The protein for docking study was taken from
Protein data Bank (PDB ID: 1XBO), was prepared by
removing solvent, adding hydrogens, and minimal mini-
mization in the presence of bound ligand (IX1) using
protein preparation wizard. Grids for molecular docking
were generated with bound co-crystallized ligand. Mole-
cules (3b–k) were docked using Glide in extra-precision
mode, with up to three poses saved per molecule.
The hurdles associated in development of negatively
charged phosphonates are bio-availability and selectivity;
may be because, inorganic phosphonates do not obey the
Lipinski principles, and PTP1B shares common homology
to other phosphatase (Evans and Jallal, 1999; Lipinski
et al., 2001; Blaskovich and Kim, 2002; Boutselis et al.,
2007). Therefore, intense search for phosphate mimics is
continuously having low molecular weight, low charge,
and improved cellular permeability, so that drug candidates
will have ideal drug-like properties (Hooft van Huijsduij-
nen et al., 2004). Therefore, in early 2000, carboxylic acid-
based anions proposed as PTP1B inhibitors and many of
them are in clinical phases like trodusquemine by Genaera.
The comprehensive research efforts in this thrust led to the
discovery and development of several PTP1B inhibitors
including diaryloxamic acid isothiazolidinedione, benzo-
furan and benzothiophene biphenyl, trodusquemine, and
many more in last decade, concise in (Table 1) (Bleasdale
et al., 2001; Burke et al., 1996a, b; Joshi et al., 2012a, b;
Chen and Seto 2002; Maccari et al., 2007; Liljebris et al.,
2002a, b; Malamas et al., 2000; Xin et al., 2003; Wrobel
et al., 2000; Fu et al., 2002; Seiner et al., 2007; Hussain
et al., 2008; and Doman et al., 2002).
Chemistry
Melting points determined by the open capillary method
using the VEEGO, programmable digital melting point
Table 2 Rationale for selection of the hit ZINC02765569 molecule
as Lead
Why ZINC02765569 selected as Lead?
Our previous research efforts led to identification of a
novel lead candidate, ZINC02765569, as a moderate
PTP1B inhibitor with good cell permeability (as estab-
lished by in vitro and in vivo experimental bioassay) by
vHTS of ZINC-database using GLIDE docking algorithm
(Joshi et al., 2012b). The rationales for considering the hit
ZINC02765569 as ideal lead molecule are mentioned in
Promising biological effects in DM
Novelty of the scaffold for PTP1B
Ease of synthesis
Amenability to structural derivatization
Good Lipinski profile
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Med Chem Res (2013) 22:1618–1623
apparatus and were uncorrected. TLC were carried out on
Pre-coated silica gel plates (F254 Merck) using chloro-
form–methanol (9:1) as solvent. FT-IR spectra were
obtained from FT-IR 470 plus spectrophotometer, made by
JASCO using KBr as the internal standard. MASS spectra
were recorded on Applied biosystems 3200 Q-Trap LC–
MS/MS. The NMR spectra were recorded on Bruker
(400 MHz) 1HNMR spectrophotometer in DMSO-D6.
Unless stated otherwise, all materials were procured from
commercial suppliers and used without further purification.
Common procedure for synthesis: first step involved
synthesis of the precursor chloroacetamido derivatives, by
reaction of amino benzoic acids with chloroacetyl chloride
in DMF. Second step entailed coupling the methylenic
carbon from precursor to thiol group of 2-mercaptobenz-
imidazoles in the presence of potassium carbonate as a base
(Joshi et al., 2012b). Scheme 1. Finally, all compounds
were either recrystallized from ethyl acetate: Methanol
[9:1] or purified by normal silica gel column chromatog-
raphy using chloroform–methanol in appropriate ratio.
expressed as picomoles/min/well. Metformin (500 lM)
and rosiglitazone (100 lM) were employed as standard.
In vitro PTP1B enzyme inhibition
PTP1B enzyme inhibitory activity was determined by
using the colorimetric, non-radioactive assay (PTP1B assay
kit, BML-AK 822, Enzo life sciences, USA). The test
compounds on PTP1B enzyme activity were determined by
incubating test compounds (10 lM) and standard Suramin
(10 lM) with human recombinant PTP1B enzyme and
determining the PTP1B activity using phosphate detection
reagent, Biomol red. The reaction was carried out in 96
well, flat-bottomed microtiter plates at 10 lm concentra-
tion using DMSO as Control. The detection of free phos-
phate released is based on classic Malachite green assay
(Martin et al., 1985). The percentage inhibition by test
compounds on PTP1B enzyme was calculated based on the
activity in the control tube (without inhibitor) as 100 %
from three autonomous sets of experiments.
Biological evaluation
Results and discussion
In vitro glucose uptake in differentiated myotubes
From initial molecular modeling studies and understanding
of synthetic organic chemistry, it ascertained that tailoring
in ZINC02765569 is possible at both head and tail sites as
depicted in Fig. 1. The position of carboxylic acid in
ZINC02765569, head may be either varied (as in com-
pounds 3a–3d) or additional polar features in head may be
incorporated (as in compounds 3b and 3d), to facilitate
additional interaction to catalytic site.
The glucose uptake was estimated by the reported method
of Klip et al., (1992). Skeletal muscle L6 myoblasts were
differentiated into post-confluency for 4–6 days until the
cells aligned to form myotubes. The myotubes were then
treated with standard metformin, rosiglitazone, and test
compounds in low glucose Dulbecco’s modified eagles
medium (DMEM) with 10 % fetal bovine serum (FBS) for
18 h. The myotubes were washed with phosphate-buffered
saline (PBS) and incubated for 3 h in serum-free DMEM,
followed by the Krebs–ringer phosphate hepes buffer
(KRPH) containing 0.5 % Bovine serum albumin (BSA)
and 100 nM Insulin for 30 min. The myotubes were then,
briefly washed with KRPH containing 0.5 % BSA. The
2-deoxy-D-[U-14C] glucose (2-DOG) (310 mCi/mmol;
Amersham biosciences, The UK) uptake was carried out in
KRPH buffer having 0.5 lCi of 2-DOG for 10 min and the
myotubes were washed thrice with cold PBS. The myotu-
bes were then lysed in 0.5 ml of 0.1 N NaOH followed by
radioactivity measurement (Beckman coulter, USA). All
assays were performed in triplicates, and results were
In addition, upon binding analysis of enzyme-inhibitor
complex and the literature knowledge on PTP1B,
ZINC02765569 tail was modified by either substituting
phenyl ring of benzimidazole (as in compounds 3e–3g) or
replacing cyclic NH group of benzimidazole ring by S
atom (as in compounds 3h–3k), to improve overall mole-
cule cellular permeability and bioavailability (Spark et al.,
2007). PTP1B inhibition studies revealed that most prom-
ising PTP1B inhibitors are compound 3f (62 %); 3i
(53.9 %); 3j (66.4 %) and 3k (56.2 %) at 10 lM conc. In
addition, designed compounds were also evaluated for the
in vitro glucose uptake assay in liver myotubes. Detailed
results are mention in the (Table 3), where Compound 3b
Scheme 1 Reagents and conditions: (i) Chloroacetyl-chloride, DMF, RT, 24 h (ii) 2-mercaptobenzimidazole, K2CO3, acetone, reflux, 4–6 h
123

Med Chem Res (2013) 22:1618–1623
1621
Table 3 Structure and biological activity data of synthesized
compounds
Code
R1
R2
X
% % PTP1B
Glucose inhibition
uptake
(at
(at
10 lM)
50 lM)
3a (ZINC02765569) 3-COOH
H
H
NH 18.5
NH 58.6
24.2
23.2
3b
5-COOH,
2-OH
3c
3d
4-COOH
H
H
NH 22.5
NH 28.7
18.7
Fig. 1 Design of novel PTP1B inhibitors
5-COOH,
2-Cl
35.82
3e
3f
5-COOH, 4-NO2 NH 37.6
2-Cl
36.56
62.68
20.89
5-COOH, 4-
2-Cl OCH3
NH 26.6
3g
5-COOH, 4-CH3 NH 31.4
2-Cl
3h
3i
3-COOH
H
H
S
S
59.2
29.2
24.2
53.9
5-COOH,
2-OH
3j
4-COOH
H
H
S
S
39.6
28.7
66.4
56.2
3k
5-COOH,
2-Cl
Suramin
Metformina(100 lM) –
Rosiglitazoneb
–
–
–
–
–
–
–
–
20
–
30.2
25.2
–
–
(500 lM)
a
b
Metformin and Rosiglitazone were evaluated at 100 and 500 lM
concentrations, respectively, for glucose uptake assay
their possible binding features and derive appropriate
SAR. Upon reassessment, it was found that all compounds
bind to catalytic domain in similar fashion to the
ZINC02765569 by H-bonding via carboxylic acid of
designed inhibitors to the Arg221, Cys215, and Ser217
(Fig. 2). Polar substituent such as hydroxyl group, in
ZINC02765569 shows additional H-bonding and improved
PTP1B inhibition (3b in comparison to 3a and 3i in com-
parison to 3h). Unlike hydroxyl group, incorporation of
halogen (Cl atom) does not show additional H-bonding to
PTP1B enzyme, but improved PTP1B inhibition and glu-
cose uptake may be due to favorable position of carboxylic
acid and increased lipophilicity (35.82 % enzyme inhibi-
tion and 28.7 % increase in glucose uptake by compound
3d in comparison to 3a which shows 24.2 % enzyme
inhibition and 18.5 increase in Glucose uptake). Therefore,
halogen-incorporated head group was selected constant and
further modifications were carried out on tail side of
ZINC02765569 by substituting phenyl ring through
methyl, methoxy, and nitro group. It was found that sub-
stitution on benzimidazole ring is favorable for PTP1B
inhibition and leads to constant increase in potency.
Fig. 2 Binding pattern of compound 3f (a) and 3j (b) at the catalytic
site of PTP1B enzyme
(58.6 %); 3 h (59.2 %) and 3j (39.6 %) are found
promising.
The results from the PTP1B inhibition studies were used
to re-assess the docking performance to gain insight into
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Similarly, in ZINC02765569 incorporation of benz-
thiazole fragment instead of benzimidazole increases the
potency consistently, as in compound 3i (53.9 %); 3j
(66.4 %); and 3 k (56.2 %); however, some of the binding
interaction of ligand with Asp48 and Gln262 residue
completely disappeared. However, this does not seem to
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bicity and favorable van der Waal interactions.
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Compounds 3b (58.6 %), 3e (37.6 %), 3 h (59.2 %),
and 3j (39.6) are potent inducer of normal glucose uptake
process in muscle myotubes and re-establishes the disease
implications. However, other compounds also stimulate
this process but there is little loss of enzyme inhibition
activity simultaneously.
Doman TN, McGovern SL, Witherbee BJ, Kasten TP, Kurumbail R,
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Conclusion
In this part of lead optimization process, molecule 3j was
found as potent PTP1B inhibitor and potential anti-hyper-
glycemic agent in glucose uptake assay by lead optimiza-
tion at head and tail sites of ZINC02765569. SAR suggests
that incorporation of additional polar features such as
hydroxyl and chlorine at head, substitution at benzimid-
azole phenyl ring and replacement of NH by S atom in tail
improve PTP1B inhibition significantly. Further lead
optimization is under pipeline.
Goodman LS, Gilman AG (2001) The pharmacological basis of
therapeutics, 10th edn. McGraw Hill Publications, New York,
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