Synthesis of protein tyrosine phosphatase 1B inhibitors: Model validation and docking studies

Article abstract of DOI:10.1016/j.bmcl.2009.02.058

The designed and synthesized 2-(4-methoxyphenyl) ethyl] acetamide derivatives (3a, 3b and 3c) were evaluated for their PTP1B inhibitory activity where they showed IC₅₀ values 69 μM, 87 μM and 71 μM, respectively. These results correlated well w

Full text of DOI:10.1016/j.bmcl.2009.02.058

Bioorganic & Medicinal Chemistry Letters 19 (2009) 2320–2323
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis of protein tyrosine phosphatase 1B inhibitors: Model validation
and docking studies
*
Anil K. Saxena , Gyanendra Pandey, Swati Gupta, Amar Bahadur Singh, Arvind K. Srivastava
Central Drug Research Institute, Medicinal and Process Chemistry, Chattar Manzil Palace, Mahatma Gandhi Marg, Lucknow, Uttar Pradesh 226 001, India
a r t i c l e i n f o
a b s t r a c t
Article history:
The designed and synthesized 2-(4-methoxyphenyl) ethyl] acetamide derivatives (3a, 3b and 3c) were
Received 6 October 2008
Revised 20 January 2009
Accepted 16 February 2009
Available online 21 February 2009
evaluated for their PTP1B inhibitory activity where they showed IC50 values 69 lM, 87 lM and 71 lM,
respectively. These results correlated well with the docking studies and in vivo screening of the com-
pounds for their antidiabetic activity in SLM and STZ models.
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Protein tyrosine phosphatases 1B
Docking
Sulfamic acid
Protein tyrosine phosphatase 1B (PTP-1B) has emerged as a new
drug target for the treatment of diabetes and obesity. Protein tyro-
sine phosphatases (PTPs) constitute a growing family (>90 mem-
bers) of receptor-like and cytoplasmic signal transducing
enzymes.1–4 These enzymes are classified on the basis of their cel-
lular localization and are characterized by their common 230 to
280 amino acid catalytic domain termed as PTP signature motif.5,6
Several PTPs viz PTP-1B, leukocyte antigen related tyrosine phos-
phatase (LAR), SH2 domain containing phosphotyrosine phospha-
Several three dimensional quantitative structure-activity relation-
ship (3D-QSAR) studies on the PTP-1B have been reported.17–21
As a part of our ongoing programme for targeting PTP-1B in
search of potential antidiabetic compounds, we developed 3D-
QSAR model on the peptidomimetic competitive inhibitors of
PTP-1B.21 These studies not only provided insights into the steric,
electrostatic, hydrophobic, and hydrogen bonding properties and
other structural features influencing the PTP-1B inhibitory activity
but also helped to gain an insight about the effect of different con-
former based alignments on the quality (in terms of leave one out
cross validation, q2; cross validation, r2, root mean square error,
RMSE) and predictiveness (in terms of r2pred) of the model. In order
to assess or validate the quality of the derived 3D-QSAR mode-
tase, PTPa and PTPe are capable of dephosphorylating the
tyrosine residues7,8 of the insulin receptor, and thereby attenuate
the tyrosine kinase activity. The phosphorylation (by protein tyro-
sine kinases) and dephosphorylation (by PTPs) of tyrosine residues
in proteins is recognized as an important cellular regulatory mech-
anism and many cellular functions could be artificially manipu-
lated exogenously by controlling the activities of these kinases
and phosphatases.9
l
21and to gain new lead molecules, the synthesis and in vitro
PTP-1B inhibitory activity of some N-[2-(4-Methoxy-
phenyl)ethyl]-acetamide derivatives which were predicted to
M along with their docking studies
show ꢀ75% inhibition at 100
l
The disruption of the above process is at the root of a variety of
disease states including cancer, inflammation and diabetes.10–14
Studies independently generated by two laboratories, with PTP-
1B knockout mice, have demonstrated that the targeted disruption
of the PTP-1B gene in mice resulted in enhanced insulin sensitivity
and decreased susceptibility to diet-induced obesity.15,16 Thus,
PTP-1B is considered as an attractive therapeutic target for type-
II diabetes as well as for obesity. It may thus provide a new thera-
peutic option to patients with at-risk obesity or type-II diabetes.
and in vivo antidiabetic activity are reported in this Letter.
The general route for the synthesis of N-[2-(4-methoxy-phenyl)
ethyl] acetamide derivatives is outlined in Scheme 1. Appropriately
H
NH2
N
R
a
O
MeO
3a, R=1-Methyl napthyl
MeO
(3a-3c)
3b, R=1-Methyl-2-nitro-phenyl
3c, R=1-Methyl phenoxy
* Corresponding author. Tel.: +91 522 2612411 18; fax: +91 522 2623405.
E-mail address: anilsak@gmail.com (A.K. Saxena).
Scheme 1. Reagent and conditions: (a) RCO2H, DIC, dry DCM, rt.
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.02.058

A. K. Saxena et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2320–2323
2321
substituted carboxylic acids were condensed with the 4-methoxy-
phenethylamine in the presence of diisopropyl carboximide (DIC)
in dry dichloromethane (DCM) to yield the required substituted
acetamide.27,28
crystal structure of PTP-1B with co-crystal of sulfamic acids deriv-
ative from Protein Data Bank (PDB) ID 2F70,25 with resolution
2.12 Å was downloaded for the present docking study because of
the similarity in the structure of 2F70 with amides 3a, 3b and 3c.
The docked poses were scored using GOLD score. Initially the pro-
tein was considered without ligand and water molecule for the
purpose of docking studies. The Protein-ligand complex of PTP-
1B (PDB-2F70) was minimised up to a gradient of 0.01 kcal/(mol)
and hydrogen were added using the CHARMm force field, available
in the software discovery studio 2.0.26 The minimised structure
was used for further docking analysis. On finishing point of docking
process the resulting conformation poses of N-[2-(4-methoxy-
phenyl)ethyl]acetamide derivatives in the binding sites of PTP-1B
were considered. Detailed binding pattern of inhibitor 3a exhibit-
ing the lowest IC50 value is shown in Figure 1. It is interesting to
note that the compound 3a takes the same pose as the sulfamic
acid (reference ligand) and also goes into the same active site of
PTP-1B. The oxygen of 4-methoxyphenyl group of 3a shows hydro-
gen bonding interaction with the –NH2 of Arg221 similar to the
hydrogen bonding interaction of carbonyl of reference ligand with
Arg221. The –NH group of compound 3a also shows hydrogen
bond interaction with water molecule similar to –NH in the sulfa-
mic acid. However unlike the sulfamic acid, it does not show close
interactions with the active site amino acids Ser216, Ala217,
Gly220 and Ile219 and with secondary binding site residues of
PTP-1B (Arg24, Arg254, Gln262). It has gold score value of 50.66.
The docking studies of the compound 3b which was lesser ac-
tive than 3a and 3c, show that 3b does not bind so well with cat-
alytic binding site and shows only one hydrogen bonding
interactions with Ile219. It has lower GOLD score value of 45.19
(Fig. 2), however it fits the secondary binding site of PTP-1B very
well. The –NH of the compound 3b shows hydrogen bonding inter-
action with the carbonyl group of Gln262 in the same manner as
shown by the –NH in the reference ligand. The nitro group of the
compound 3b shows interaction with Arg24 by hydrogen bonding.
Moreover in these interactions the phenyl group of compound 3b
also showed close interaction with Ile219 which is the key residue
of catalytic site.
As vanadate is a non-selective inhibitor of PTPs, and studies
have shown that treatment with vanadate can normalize blood
glucose level in diabetics,22 the PTP-1B inhibitory activity of N-
[2-(4-methoxyphenyl)ethyl]-acetamide derivatives was measured
at graded doses taking peroxovanadate as a control and the results
are summarized in Table 1. The effect of the test compounds on
protein tyrosine phosphatase was studied by pre-incubating PTP-
1B at the appropriate doses of the test chemicals in the reaction
system for 10 min and the residual protein tyrosine phosphatase
activity was determined according to the method of Goldstein
et al.23 The activity of PTPase was evaluated using p-nitrophenyl-
phosphate (PNPP) as the substrate.29
H
N
HO3SHN
NHSO3H
O
2F70
In order to get novel NCEs and to validate our earlier derived
best CoMFA model,21 three N-[2-(4-methoxy-phenyl)ethyl]ace
amide derivatives (3a–c), with considerably high predicted activity
1.33, 0.950 and 0.81 in term of legit transformation of percentage
inhibition were designed. These molecules were synthesized and
evaluated for their in vitro PTP-1B inhibitory as well as in vivo
ant diabetic activities where these compounds 3a, 3b and 3c
showed high PTP-1B inhibitory activity 76.9% (IC50 = 69
l
M),
62.5% (IC50 = 87
1).
lM) and 68.2% (IC50 = 71 lM) respectively (Table
Since the predicted activity for these compounds cannot be
compared in absolute terms with the observed activity due to
the difference in the screening model, hence the relative order of
the activity was compared. The observed in vitro PTP-1B inhibitory
activities of these three new compounds were nearly comparable
to their predicted activities by the 3D-QSAR model. The three com-
pounds (3a, 3b, and 3c) were predicted as 3a > 3b > 3c whereas ob-
served in vitro PTP-1B inhibitory activities of these compounds
were in the order 3a > 3c > 3b.
It is evident that the compound 3a, which was predicted in-sil-
ico to be the most active, has been observed to be the most active
(IC50 = 69 lM). However, the compound 3b, predicted to be the
next to the most active, was found to be the least active of the
three compounds. Nevertheless, all the three compounds 3a, 3b,
and 3c showed higher activity than the standard drug (Peroxo-
On the other hand the compound 3c with moderate activity
shows no interactions in the catalytic and non catalytic sites but
interacts only with one water molecule with the gold score value
of 49.85 (Fig. not shown). These studies suggested the activity or-
der of these compounds as 3a > 3c > 3b similar to the results
in vitro studies. Hence these three compounds were further evalu-
ated for their antidiabetic activities in the in vivo rodent model viz.
sucrose loaded rat model (SLM) and in streptozotocin-induced dia-
betic rat (STZ) model essentially according to the recently reported
procedure from our laboratory.30 The compounds 3a, 3b and 3c
showed 25.1%, 19.8% and 24.6% sugar lowering effect in the SLM
model (Fig. 3) and 21.4%, 17.5% and 20.6% sugar lowering effect
in the streptozotocin-induced diabetic rats (Fig. 4), respectively
at a dose of 100 mg/kg against the standard drug metformin
(30.2% in SLM and 27.6% in STZ model) (Table 1). It is interesting
vanadate, IC50 = 94.6 lM) in our test model.
In order to gain more insight into the binding mode of these
compounds with the enzyme of known X-ray structure, the dock-
ing studies were carried out using Genetic Optimisation for Ligand
Docking (GOLD) version 2.2 on windows based PC.24 The reported
Table 1
In vitro PTP-1B enzyme inhibitory and in vivo antihyperglycemic activity in SLM and STZ model for compound 3a–c
Compounds
% Inhibition at 100
Observed
l
M
IC50 (
l
M)
Anti- hyperglycemic activity (%) at 100
STZ
lM
Predicteda
SLM
3a
3b
3c
76.9
62.5
68.2
56
95.5
90.2
85.0
—
69 ( 0.20)
87( 0.49
74 ( 0.30)
94.6( 0.40)
—
25.1
19.8
24.6
—
24.1
17.5
20.6
—
Peroxovanadate
Metformin
—
—
27.2
23.9
a
Predicted from the 3D QSAR model.

2322
A. K. Saxena et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2320–2323
Comp.3a
Comp.3c
Control
Comp.3b
Metformin
600
500
400
300
200
100
0
24h
0
30 60 90 120 150 180 210 240 270 300
Time (min)
Figure 1. Pose attained by the 3a (green) and the control ligand (red) in the active
site of the PTP1B (PDB ID 2F70).
Figure 4. Blood glucose levels in STZ-induced diabetic rats before, and up to 24 h
after administration of vehicle, compound 3a, 3b, 3c and metformin.
to note that the in vivo activity of all the three compounds in both
the models was also in the same order 3a > 3c > 3b as found
in vitro and in docking studies.
It is pertinent to note that the three compounds 3a, 3b and 3c
which were predicted to show >75% PTP-1B inhibition at 100 lM
dose by our 3D QSAR model did show high activity but the order
of activity predicted to be 3a > 3b > 3c differed and was found to
be 3a > 3c > 3b .It may be due to the consideration of single point
observation data that too at high fixed molar concentration
(100 lM) because the observed order of activity corresponded well
with the IC50 value data determined for these compounds as well
as with the docking results in terms of gold score values and
in vivo antidiabetic activity data in both SLM and STZ models. It
may be inferred from the docking score results 3a > 3c > 3b that
the hydrogen bonding interactions at the active site residues
Arg221 (3a) has more influence on binding than the interaction
with Ile219 (3b).The observed activity of 3c is higher than 3b
and lesser than 3a suggest that the hydrogen bonding interaction
with water molecule provide better binding affinity than the
hydrogen bonding interaction with secondary site residue Arg24.
Figure 2. Interaction of the 3b (green) with the amino acids in the active site of
PTP-1B (PDB ID 2F70).
Acknowledgements
Authors are thankful to Messers Z. Ali and A.S. Kushwaha for the
technical assistance; SAIF, Lucknow, for providing spectroscopic
data and Network project on Diabetes mellitus new drug discovery
R&D for the financial supports. One of the authors SG is thankful to
ICMR, New Delhi for the award of senior research fellowship.
Comp.3a
Comp.3c
Control
Comp.3b
metformin
150
125
100
75
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N, 4.38. Found: C, 79.01; H, 7.00; N, 4.40. 1H NMR (CDCl3, 200 MHz): d 2.46–
2.53 (t, J = 6.6 Hz, 2H), 3.29–3.39 (m, 2H), 3.73 (s, 3H), 3.99 (s, 2H), 5.70 (br s,
1H), 6.54–6.64 (m, 3H), 7.25–7.56 (m, 5H), 7.79–7.92 (m, 3H); IR (KBr): 777,
1247, 1605, 3296; FAB-MS: m/z (M+1)+ 320; Compounds 3b and 3c were
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synthesized
similarly.N-[2-(4-Methoxyphenyl)ethyl]-2-(2-nitrophenyl)-
acetamide (3b): Yield: 0.700 g (50%); mp: 104 °C. Anal. Calcd for C17H18N2O4:
C, 64.96; H, 5.73; N, 8.91. Found: C, 64.98; H, 5.76; N, 8.93. 1H NMR (CDCl3,
200 MHz): d 2.72-2.76 (t, J = 6.80 Hz, 2H), 3.46–3.49 (m, 2H), 3.78 (s, 5H), 6.77–
6.81 (d, J = 8.6 Hz, 2H), 7.01–7.06 (d, J = 8.6 Hz, 2H), 7.44–7.48 (d, J = 7.2 Hz 2H),
7.50 (m, 1H), 8.01 (m, 1H); IR (KBr): 713, 1243, 1609, 3298; FAB-MS: m/z
(M+1)+ 315.N-[2-(4-Methoxyphenyl) ethyl]-2-phenoxy-acetamide (3c): Yield:
61%; mp: 112 °C. Anal. Calcd for C17H19NO3: C, 71.57; H, 6.66; N, 4.91.
Found: C, 71.59; H, 6.69; N, 4.93. 1H NMR (CDCl3, 200 MHz): d 2.01 (br s 1H),
2.77-2.80 (m, 2H), 3.54-3.57 (m, 2H), 3.78 (s, 3H), 4.46 (s, 2H), 6.79–6.87 (m,
4H), 7.02–7.08 (m, 3H), 7.26–7.31 (m, 2H); IR (KBr): 753, 1242, 1655, 3345;
FAB-MS: m/z (M+1)+286.
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apparatus. Infrared spectra (IR) were recorded on Perkin-FTIR model PC
spectrophotometer with frequency of absorptions reported in wave numbers.
MS were recorded on JEOL spectrometer. The NMR spectra were recorded on
Bruker DPX 200 MHz spectrometer.
28. General procedure: The general route for the synthesis of N-[2-(4-
methoxyphenyl)ethyl]-2-naphthalen-1-yl-acetamide (3a): The 4-methoxy
phenyl amine (0.468 ml, 0.0032 mol) in dry DCM was added to the stirred
solution of naphthalen-1-yl-acetic acid (0.600 g, 0.0032 mol) in dry
dichloromethane (DCM) and diisopropyl carboximide (DIC) at 0 °C. The
29. Assay mixture containing 10 mM PNPP in 50 mM HEPES buffer (pH 7.0), with
1 mM EDTA and DTT was made up to 1 ml. The reaction was stopped by the
addition of 500 ll of 0.1 N NaOH and absorbance was determined at 410 nm. A
molar extinction coefficient of 1.78 Â 104 MÀ1 cmÀ1 was used to calculate the
concentration of p-nitrophenolate ions produced in the reaction mixture. The
IC50 values were calculated from the% inhibition of the PTPase at five different
doses (10, 25, 50, 75 and 100 lM).
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