Design, synthesis and molecular modelling studies of novel 3-acetamido-4-methyl benzoic acid derivatives as inhibitors of protein tyrosine phosphatase 1B

Article abstract of DOI:10.1016/j.ejmech.2013.10.030

A novel series of 3-acetamido-4-methyl benzoic acid derivatives designed on the basis of vHTS hit ZINC02765569 were synthesized and screened for PTP1B inhibitory activity. The most potent compounds 3-(1-(5-methoxy-1H-benzo[d] imidazol-2-ylthio)acetamido)-4-methyl benzoic acid (10c, IC₅₀ 8.2 μM) and 3-(2-(benzo[d]thiazol-2-ylthio)acetamido)-4-methyl benzoic acid (10e, IC₅₀ 8.3 μM) showed maximum PTP1B inhibitory activity. Docking studies were also performed to understand the nature of interactions governing the binding mode of the designed molecules within the active site of the PTP1B enzyme.

Full text of DOI:10.1016/j.ejmech.2013.10.030

European Journal of Medicinal Chemistry 70 (2013) 469e476
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Short communication
Design, synthesis and molecular modelling studies of novel
3-acetamido-4-methyl benzoic acid derivatives as inhibitors of protein
tyrosine phosphatase 1B
,
,
,1
Monika Rakse a 2, Chandrabose Karthikeyan a 2, Girdhar Singh Deora a
,
N.S.H.N. Moorthy a b, Vandana Rathore c, Arun K. Rawat d, A.K. Srivastava d,
,
,
Piyush Trivedi a
*
a CADD Laboratory, School of Pharmaceutical Sciences, Rajiv Gandhi Proudyogiki Vishwavidyalaya, Airport Bypass Road, Gandhi Nagar, Bhopal, MP 462036,
India
b Department of Chemistry and Biochemistry, Faculty of Sciences, University of Porto, 687, Rua de Campo Alegre, Porto 4169-007, Portugal
c Dr. Reddy’s Institute of Life Sciences, University of Hyderabad Campus, Gachibowli, Hyderabad 500046, India
d Division of Biochemistry, Central Drug Research Institute, Lucknow, UP 226001, India
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel series of 3-acetamido-4-methyl benzoic acid derivatives designed on the basis of vHTS hit
ZINC02765569 were synthesized and screened for PTP1B inhibitory activity. The most potent compounds
Received 4 March 2013
Received in revised form
9 October 2013
Accepted 11 October 2013
Available online 23 October 2013
3-(1-(5-methoxy-1H-benzo[d]imidazol-2-ylthio)acetamido)-4-methyl benzoic acid (10c, IC50 8.2
mM)
and 3-(2-(benzo[d]thiazol-2-ylthio)acetamido)-4-methyl benzoic acid (10e, IC50 8.3 M) showed
m
maximum PTP1B inhibitory activity. Docking studies were also performed to understand the nature of
interactions governing the binding mode of the designed molecules within the active site of the PTP1B
enzyme.
Keywords:
Diabetes
Ó 2013 Elsevier Masson SAS. All rights reserved.
PTP1B
Acetamido benzoic acid derivatives
Molecular modelling
Lead optimization
1. Introduction
generated by two laboratories, with PTP1B knockout mice, have
demonstrated that the targeted disruption of the PTP1B gene in
Diabetes mellitus is a complex metabolic syndrome, has a major
human health concern over the world and is estimated to affect 300
million people by the year 2025. Indians alone may have about 1/5
of the global diabetic population by 2025 [1e4] due to hereditary,
ageing, poor diet, obesity and sedentary life style [5]. Severe dia-
betes may leads to poor wound healing, blurring vision, kidney
damage, cardiovascular diseases and premature death [6]. Protein
tyrosine phosphatase 1B (PTP1B) have emerged as a promising
therapeutic target of diabetes mellitus as it down regulates insulin
transduction mediated by receptor tyrosine kinases such as insulin
receptor and epidermal growth factor [7,8]. Studies independently
mice resulted in enhanced insulin sensitivity. Thus, PTP1B is
considered as an attractive therapeutic target for type-II diabetes
[9,10].
The crystal structure of PTP1B enzyme is well explored [11,12]
and the main structural feature of PTP1B enzyme is the catalytic
site which contains two aryl phosphate-binding sites: a high-
affinity catalytic site (containing the nucleophile cysteine residue,
Cys215) and a low-affinity non-catalytic site (demarcated by Arg24
and Arg254 residues). The inhibitor binding with high-affinity
catalytic site is deemed to be significant for its potency whereas
the low-affinity non-catalytic site has been frequently exploited by
medicinal chemist for enhancing the ligand binding affinity and
selectivity for inhibition of PTP1B over other PTPs.
* Corresponding author. Tel.: þ91 755 2678883; fax: þ91 755 2742001.
E-mail addresses: piyush.trivedi@rgtu.net, drtrivedislab@gmail.com (P. Trivedi).
Current address: The University of Queensland, School of Pharmacy, Brisbane,
The catalytic site of PTP1B enzyme accommodates pTyr, which
contains two negative charges at physiological pH [13]. Because of
the electrostatic properties of the enzyme active site, the most
commonly adopted strategy in the design of PTP1B inhibitors is to
1
Qld 4072, Australia.
2
These authors contributed equally to this work.
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.10.030

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M. Rakse et al. / European Journal of Medicinal Chemistry 70 (2013) 469e476
Fig. 1. Chemical Structures of some reported PTP1B inhibitors (1e7) and compound synthesized in the study (8).
insert non-hydrolysable pTyr-mimetic groups that are able to
replicate the interactions of pTyr residue with the active site of the
enzyme onto different optimal templates. Most of previously used
pTyr mimetic groups such as phosphonates [14], carboxylic acids
[15], sulphonamides [16], sulphanamic acids [17] have proven lack
of cell permeability and oral bioavailability because of the strong
negative charge carried by the pTyr mimetics as well as their high
molecular weight [18]. Hence, there is an urgent need to discover
new and orally bioavailable PTP1B inhibitors that can combat type-
II diabetes. Several efforts aimed at developing potent and
bioavailable PTP1B inhibitors integrating a carboxylic group as pTyr
mimetic have been recorded in literature. Some examples of car-
boxylic acids endowed with cellular activity and/or oral availability
are (2-hydroxyphenoxy) acetic acid derivatives (1) [19], isoxazole
carboxylic acid derivatives (2) [20], 2-aryl-3,3,3-trifluoro-2-
hydroxypropionic acids (3) [21], heterocyclic carboxylic acid de-
rivatives (4) [22], 4-[(5-arylidene-2-arylimino-4-oxo-3-thiazoli
dinyl)methyl]benzoic acids (5) [23] and Thiophene carboxylic
acid derivatives (6) [24] (Fig. 1).
PTP1B enzyme through a network of hydrogen bonds with the
residues Cys215, Ser216 and Arg221 of catalytic site and the
benzimidazole moiety (ring B) is accommodated inside the addi-
tional aryl phosphate binding site showing strong hydrogen
bonding interaction with the residues Asp48 and Gln262 (Fig. 2). It
has been previously described that inhibitor interaction with amino
acid residues specifically Asp48 confers selectivity for PTP1B over
More recently, our laboratory reported the discovery of 3-(2-
(1H-benzo[d]imidazol-2-ylthio)acetamido)benzoic acid (7) as in-
hibitor of PTP1B enzyme through a high throughput virtual
screening of Zinc database [25]. The compound (ZINC02765569)
presented modest PTP1B inhibition at 10
mM concentration and
good cellular permeability. Docking studies of the identified
molecule revealed that the molecule shows all the relevant in-
teractions essential for PTP1B inhibition. The acetamido benzoic
acid moiety (ring A) of the molecule binds at the catalytic site of
Fig. 2. Docked molecular model of ZINC02765569 in the active site of PTP1B enzyme.

M. Rakse et al. / European Journal of Medicinal Chemistry 70 (2013) 469e476
471
Table 2
other PTPs as this site and the channel communicating to the cat-
In vitro PTP1B enzyme inhibitory activity of the compounds (15aeg).
alytic appear to be conserved in PTP1B [26]. These promising re-
sults alongwith the feasibility for structural derivatization of
ZINC02765569 led us to expand our efforts in the synthesis of new
diversely substituted 3-(2-(1H-benzo[d]imidazol-2-ylthio)acet-
amido)benzoic acid derivatives as potential PTP1B inhibitors. More
particularly, we were interested in structural manipulations at ring
A and ring B in order to derive 3-(2-(1H-benzo[d]imidazol-2-
ylthio)acetamido)benzoic acid analogues with improved PTP1B
inhibitory potency.
COOH
O
R
S
O
N
H
N
CH3
N
Compound
R
% PTP1B inhibition
(at 10 M)
IC50 (mM)
m
Our immediate goal was to identify the sites for structural
manipulation through molecular docking studies. To this end, the
results derived from docking of the lead compound ZINC02765569
with the active site of PTP1B enzyme were utilized. From the pre-
liminary analysis of the inhibitor binding mode predicted by
docking studies (Fig. 2), it was ascertained that a methyl substitu-
tion at position 5 in the ring A would be beneficial to facilitate
additional interactions with the residue Tyr45 in the catalytic site. It
was also hypothesized from the orientation of the benzimidazole
ring in the non-catalytic site that substitution in the benzo ring of
benzimidazole and replacement of benzimidazole ring with phenyl
oxadiazole would be beneficial in terms of harnessing additional
polar and hydrophobic interactions with amino acid residues in
second aryl binding site.
15a
2,3-di-Cl
2-Cl
e
29.85
27.46
43.28
38.80
47.01
51.49
50.74
24.7
15b
e
15c
2,4-di-Cl
2-OH
e
15d
e
15e
4-OH
10.3
8.9
9.7
e
15f
4-CH3
15g
3,4,5-tri-OCH3
e
ZINC02765569
Prompted by these findings, two series of compounds (10aee,
15aeg) (Tables 1 and 2) were prepared and their inhibitory activity
against the PTP1B enzyme was assessed. The results from the
binding assays were then used to re-evaluate the docking results in
order to gain insight into their possible binding modes, allowing
the development of more active compounds in the future.
required
a longer synthetic sequence included esterification,
cyclization and elimination reactions, as shown in Scheme 2.
Initially, the substituted 5-phenyl-1,3,4-oxadiazole-2-thiol de-
rivatives were synthesized in three steps according to the reported
procedure [30,31] using appropriate benzoic acids. The synthesized
5-phenyl-1,3,4-oxadiazole-2-thiol derivatives were subsequently
linked to 3-(2-chloroacetamido)-4-methylbenzoic acid to give the
desired 3-(2-(5-phenyl-1,3,4-oxadiazol-2-ylthio)acetamido)-4-met
hylbenzoic acid derivatives (15aeg).
2. Result and discussion
2.1. Chemistry
Synthesis of the designed compounds (10aee, 15aeg) was
accomplished by using known synthetic approaches [27e31]. The
3-(2-(1H-benzo[d]imidazol-2-ylthio)acetamido)benzoic acid de-
rivatives (10aec) alongwith 3-(2-(benzo[d]oxazol-2-ylthio)acet-
amido)-4-methylbenzoic acid (10d) and 3-(2-(benzo[d]thiazol-2-
ylthio)acetamido)-4-methylbenzoic acid (10e) were prepared as
outlined in Scheme 1. The synthesis began with chloroacetylation
of 3-amino-4-methylbenzoic acid (7) in DMF under basic condition
to afford 3-(2-chloroacetamido)-4-methylbenzoic acid (7). The
obtained 3-(2-chloroacetamido)-4-methylbenzoic acid was then
reacted with 1H-benzo[d]imidazole-2-thiols, benzo[d]oxazole-2-
thiol and benzo[d]thiazole-2-thiol in acetone in the presence of
anhydrous potassium carbonate to give the requisite compounds
(10aee). The preparation of 3-(2-(5-phenyl-1,3,4-oxadiazol-2-
ylthio)acetamido)-4-methylbenzoic acid derivatives (15aeg)
2.2. In vitro PTP1B inhibition
The effect of synthesized compounds on protein tyrosine
phosphatase inhibition was studied using colorimetric, non-
radioactive PTP1B tyrosine phosphatase drug discovery kit-BML-
AK 822 from Enzo Life Sciences, USA. The assay was done accord-
ing to the Kit manufacturer’s protocol. The results of the enzymatic
assay are shown in Tables 1 and 2 In general, the compounds are
moderate to potent inhibitors of PTP1B with the exception of
compound 10b and 15b, which are significantly less active toward
PTP1B. The unsubstituted benzimidazole derivative showed almost
twice the potency exhibited by the lead compound ZINC02765569
which confirms the positive influence of the methyl substitution in
benzoic acid moiety to PTP1B inhibition. Incorporation of electron
withdrawing group in the fifth position of phenyl ring (10b)
resulted in decreased PTP1B inhibitory potency whereas an elec-
tron releasing methoxy substitution (10c) in the same position
improved PTP1B inhibitory activity. The best active compounds in
the series are 10c, and 10e which showed PTP1B inhibitory activity
greater than 50%. Interestingly, benzothiazole derivative (10e) has
found to be equipotent with the benzimidazole derivative with
methoxy substitution at fifth position of the phenyl ring (10c).
However, replacement of the benzimadazole ring (10a) with a
benzoxazole ring (10d) leads to drop in PTP1B inhibitory potency.
Further, the compounds showing around 50% inhibition of PTP1B
enzyme are further tested using same PTP1B drug discovery kit-
BML-AK 822 at six different concentrations for calculation of their
IC50 value. The derived data has been analysed on Microsoft excel
software and the IC50 value is calculated (Tables 1 and 2) from the
Table 1
In vitro PTP1B enzyme inhibitory activity of the compounds (10aee).
O
HOOC
X
N
R
NH
S
CH3
Compound
R
X
% PTP1B inhibition (at 10
m
M)
IC50 (mM)
10a
10b
10c
10d
10e
H
NH
NH
NH
O
47.76
26.86
57.46
32.83
57.46
15.2
e
5-NO2
5-OCH3
H
8.2
e
H
S
8.5

472
M. Rakse et al. / European Journal of Medicinal Chemistry 70 (2013) 469e476
Cl
H3C
H2N
H
N
HOOC
N
a
HS
R
+
X
COOH
O
CH3
(X=NH,S,O)
7
9a-e
8
b
H3C
N
X
S
HN
O
R
COOH
10a-e
Scheme 1. Reagent and conditions: (a) DMF, chloroacetyl chloride, RT, 24 h; (b) K2CO3, acetone, reflux, 6 h.
R
R
R
a
b
H2NHNOC
HOOC
C2H5OOC
13a-g
12a-g
11a-g
c
COOH
Cl
H
N
HS
O
HOOC
R
O
N
R
d
S
O
+
N
N
H
O
CH3
N
CH3
N
8
14a-g
15a-g
Scheme 2. Reagent and conditions: (a) ethanol, reflux, 8 h; (b) NH2NH2.H2O, reflux, 6 h; (c) CS2, ethanol, reflux, 5 h; (d) K2CO3, acetone, reflux, 6 h.
values of percent inhibition obtained at different concentrations of
the compounds.
Compound 15g with 3,4,5 trimethoxy substitution in the phenyl
ring also showed comparable potency to that of compound 15f.
Both the compounds manifest around 50% enzyme inhibition at
However, majority of the compounds in the phenyl oxadiazole
series showed good PTP1B inhibitory potency except for com-
pound 15b which suggest that phenyl oxadiazole moiety is well
tolerated at second aryl binding site of PTP1B enzyme. The most
potent compound of the series 15f bears a methyl group in the
para position of the phenyl ring attached to the oxadiazole moiety.
10
mM concentration. A chlorine at ortho position (15b) of the
phenyl ring resulted in drastic decrease in PTP1B inhibitory po-
tency. Multiple chlorine substitutions in the phenyl ring favours
PTP1B inhibitory activity if it is in ortho and para positions of the
phenyl ring (15c) whereas it is detrimental when it is present in
ortho and meta position of the phenyl ring (15a). Hydroxyl sub-
stituent at para position (15e) moderately enhanced PTP1B
inhibitory activity relative to hydroxyl group in ortho position
(15d).
Furthermore, the most active compound 10c was screened for
general toxicity studies using zebrafish embryos. The embryos were
grown in the presence of the compound for 24, 48 and 72 h. Control
embryos were incubated in 0.1% DMSO. Finally, embryos were
observed using visible light microscopy at regular intervals after
compound treatment to document general toxicity related effects
such as developmental delays, deformations, oedema and death.
The results indicate that tested compound 10c having no any sig-
nificant toxic effect on zebrafish embryos.
2.3. Molecular modelling
The docking studies reveal a common binding orientation of all
the synthesized compounds in the catalytic binding pocket of
PTP1B (Fig. 3). The benzoic acid moiety plays an important role in
the binding, as both the oxygen atoms of carboxylic group are
Fig. 3. Binding orientation of synthesized molecules at the catalytic site of PTP1B.

M. Rakse et al. / European Journal of Medicinal Chemistry 70 (2013) 469e476
473
Fig. 4. Binding mode and interaction of synthesized compounds 10c (a), 10e (b), 15f (c) and 15g (d) with catalytic site residues of PTP1B.
involved in hydrogen bonding interactions with amino acid resi-
dues Cys215, Ser216, Gly220 and Arg221 at the catalytic site as
4. Experimental section
shown in Fig. 4. A
pe
p
stacking interaction is also observed be-
4.1. Chemistry
tween phenyl ring of the benzoic acid moiety and the phenyl ring
of the amino acid residue Phe182 in all the compounds. In addi-
tion, the methyl group in the benzoic acid moiety shows hydro-
phobic interaction with phenyl ring of the Tyr45 residue and the
NH group of the acetamido benzoic acid moiety is involved in the
hydrogen bonding interaction with the carbonyl oxygen atom of
Gln262 amino acid. In addition to the above interactions, the ni-
trogen atom of benzimidazole/benzothiazole/benzoxazole rings of
10aee and oxadiazole ring of 15aeg formed hydrogen bonding
with Gln262 residue of the additional aryl phosphate binding site.
Most notably, the benzimidazole derivatives 10aec showed extra
hydrogen bonding interaction between the NH group of imidazole
and Asp48. The most potent compound of the series 10c shows an
additional hydrogen bonding interaction with the Arg254 residue
of the additional aryl phosphate binding site of PTP1B enzyme
(Fig. 4a).
Melting points were determined by Open Capillary Method
using VEEGO, Programmable Digital Melting Point Apparatus and
are uncorrected. Unless stated otherwise, all materials obtained
from commercial suppliers were used without further purification.
TLC controls were carried out on precoated silica gel plates (F254
Merck) using Chloroform-Methanol (9:1) as solvents. IR spectra
were recorded on FT-IR 470 plus spectrophotometer (JASCO) using
KBr as the internal standard (vmax in cmꢀ1). 1H and 13C NMR spectra
were recorded in DMSO on a Bruker 400 MHz spectrometer using
tetramethylsilane (TMS) as the internal reference (chemical shift
was measured in d ppm). Mass spectra (ESI-MS) were measured on
Applied Biosystems 3200 Q-TRAP LC-MS/MS. The progress of the
reaction and the purity of the synthesized compounds were veri-
fied on ascending thin layer chromatography (TLC) plates coated
with silica gel G (Merck). An iodine chamber and UV lamp were
used for the visualization of the TLC spots.
3. Conclusion
4.1.1. Procedure for the preparation of 3-(2-chloro acetamido) 4-
methyl benzoic acid (8)
In conclusion, we describe herein structure guided optimization
of
a
lead molecule (3-(2-(1H-benzo[d]imidazol-2-ylthio)acet-
3-Amino, 4-methyl benzoic acid (7) (0.151 g, 1 mmol) was dis-
solved in DMF (10 mL) and chloroacetyl chloride (0.225 g, 2 mmol)
was added drop wise to the reaction mixture for 20 min. The re-
action mixture was stirred for 24 h, at room temperature and on
completion of the reaction, the reaction mixture was poured in to
crushed ice and stirred. Precipitated product was filtered and
washed with water to remove the traces of acetic acid. 1H NMR
amido)benzoic acid) obtained through virtual high throughput
screening. Incorporation of a methyl group vicinal to amino group
on ring A and substitution on phenyl ring of benzimidazole ring
(ring B) in the lead molecule provided two fold improvement in the
PTP1B inhibitory potency of these compounds. Further, the results
of the study also established that phenyl oxadiazole moiety is well
tolerated in the second aryl phosphate binding site of PTP1B
enzyme. Molecular interaction pattern of studied compounds with
the receptor has successfully generated with the help of docking
simulations and the results indicate that the compounds show all
the relevant interactions requisite for PTP1B inhibition. Further
medicinal chemistry efforts are in progress to develop more
structurally diverse analogues from this compound to optimize the
PTP1B inhibitory potency and establish structure activity relation-
ship which will help in the development of the studied acetamido
benzoic acid derivatives into probable antidiabetic drug candidates.
(DMSO-d6)
d (ppm) 9.6193 (s, 1H, NHCO), 8.0857 (s, 1H, AreCH),
7.7321 (d, 1H, AreCH, J ¼ 7.88 Hz), 7.2883 (d, 1H, AreCH,
J ¼ 7.96 Hz), 4.2393 (s, 2H, CH2), 2.3158 (s, 3H, CH3).
4.1.2. General procedure for the preparation of 3-(2-(1H-benzo[d]
imidazol-2-ylthio)acetamido)-4-methylbenzoic acids (10a-e)
A mixture of 3-(2-chloroacetamido)-4-methyl benzoic acid (8)
(0.227 g, 1 mmol), substituted 2-mercapto benzimidazoles (9aec)
or benzo[d]oxazole-2-thiol (9d) or benzo[d]thiazole-2-thiol (9e)
(1 mmol) and K2CO3 (0.207 g, 1.5 mmol) was refluxed in acetone

474
M. Rakse et al. / European Journal of Medicinal Chemistry 70 (2013) 469e476
(20 mL) for 5e6 h. Excess of solvent was evaporated at room
temperature, concentrated the reaction mixture and diluted with
water (about 200 mL). The product precipitated out on acidification
with dilute hydrochloric acid was filtered, thoroughly washed with
cold water.
129.2, 134.5, 136.5, 137.2, 152.5, 166.1, 166.6, 168.1. MS (ESI) m/z:
356.6 (M ꢀ H).
4.1.3. General procedure for the preparation of substituted aromatic
ethyl esters (12aeg)
A mixture of substituted benzoic acids (11aeg) (0.001 M, 2.12 g)
and ethanol (20 mL) were heated under reflux until the benzoic
acid was dissolved in ethanol then few drops of concentrated H2SO4
was added to the mixture and reflux for 8 h. The resulting mixture
was cooled to room temperature and a saturated solution of sodium
bicarbonate was added to the mixture to neutralise the benzoic
acid. The precipitated product was filtered and washed with water
and dried. The dried product was recrystallized with ethanol.
4.1.2.1. 3-(2-(1H-benzo[d]imidazol-2-ylthio)acetamido)-4-
methylbenzoic acid, 10a. Yield 44.05%, Mp 245 ꢁC. IR (KBr, cmꢀ1
)
1698.24 (CO st. of COOH), 3250.25 (OH st. of COOH), 2869.20 (CH3
st), 1618.23 (amide CO st.), 758.34 (CeS st.), 3389.68 (NH st.),
3060.91 (Ar CeH st.), 1580.23 (Ar CeC st.). 1H NMR (400 HMz,
DMSO-d6)
d (ppm): 12.4158 (s broad peak, 1H, COOH), 10.1371 (s,
1H, NHCO), 7.6811 (m, 3H, AreCH), 7.4409 (m, 2H, AreCH), 7.1378
(m, 2H, AreCH), 7.0849 (m, 1H, AreCH), 4.5788 (s, 2H, CH2), 2.3156
(s, 3H, CH3). 13C NMR (100 MHz, DMSO-d6)
d
17.5, 36.5, 110.5, 115.8,
4.1.4. General procedure for the preparation of substituted
benzohydrazide (13aeg)
123.8, 125.6, 125.9, 129.4, 135.2, 136.8, 139.1, 147.8, 165.3, 168.7. MS
(ESI) m/z: 339.05 (M ꢀ H).
A mixture of substituted ethyl benzoate (12aeg) (0.1 M) and
hydrazine hydrate (0.3 M) was heated under reflux for 30 min.
Ethanol (20 mL) was added to the refluxing mixture as a solvent in
order to homogenize the solution, the resulting mixture was
further allowed for 6 h. Excess of ethanol was distilled out and the
content was allowed to cool. The crystals formed were filtered and
washed thoroughly with water and dried.
4.1.2.2. 4-Methyl-3-(2-(5-nitro-1H-Benzo[d]imidazol-2-ylthio)acꢀe1t-
amido)benzoic acid, 10b. Yield 85.23%, Mp 191 ꢁC. IR (KBr, cm
)
1695.31 (CO st. of COOH), 3197.76 (OH st. of COOH), 2889.17 (CH3
st.), 1620.09 (amide CO st.), 757.97 (CeS st.), 3444.63 (NH st.),
3105.18 (Ar CeH st.),1583.45 (Ar CeC st.),1340.43 (NO2 st.). 1H NMR
(400 HMz, DMSO-d6) d (ppm): 9.973 (s,1H, NHCO), 8.321 (s,1H, Are
CH), 8.119 (s, 1H, AreCH), 8.076 (d, 1H, AreCH, J ¼ 8.8 Hz), 7.633
4.1.5. General procedure for the preparation of substituted 5-
phenyl-1,3,4-oxadiazole-2-thiol (14aeg)
(d,2H, AreCH, J ¼ 12.8 Hz), 7.33 (d, 1H, AreCH, J ¼ 8.0 Hz), 2.296 (s,
3H, CH3), 4.364 (s, 2H, CH2). 13C NMR (100 MHz, DMSO-d6)
d
17.8,
To a solution of substituted benzohydrazide (13aeg) (0.01 M) in
ethanol (15 mL) at 0 ꢁC, carbon disulphide (2 mL) and potassium
hydroxide (0.6 g) were added, and the reaction mixture was
refluxed until the evolution of H2S gas ceased (around 12 h). Excess
solvents were evaporated under reduced pressure and the residue
was dissolved in water and then acidified with dilute hydrochloric
acid (10%) to pH 5. The precipitate was filtered off, dried and
crystallized from ethanol.
37.2, 109.5, 111.7, 115.9, 118.3, 125.1, 125.9, 129.4, 136.1, 136.8, 137.9,
139.8, 143.4, 147.8, 166.3, 169.7. MS (ESI) m/z: 384.6 (M ꢀ H).
4.1.2.3. 3-(2-(5-Methoxy-1H-benzo[d]imidazol-2-ylthio)acetamidꢀo1)-
4-methylbenzoic acid, 10c. Yield 67.54%, Mp 221.5 ꢁC. IR (KBr, cm
)
1695.31 (CO st. of COOH), 3255.62 (OH st. of COOH), 2837.09 (CH3
st.), 1649.02 (amide CO st.), 761.83 (CeS st.), 3452.34 (NH st.),
3020.32 (Ar CeH st.), 1581.52 (Ar CeC st.), 1157.21 (OCH3 st.). 1H
NMR (400 MHz, DMSO-d6)
d
(ppm): 12.354 (s,1H, COOH),10.255 (s,
4.1.6. General procedure for the preparation of substituted 4-
methyl-3-(2-(5-phenyl-1,3,4-oxadiazol-2-ylthio)acetamido)benzoic
acid (15aeg)
1H, NHCO), 8.372 (s, 1H, AreCH), 6.5e8.0 (m, 5H, AreCH), 4.020 (s,
2H, CH2), 3.755 (s, 3H, OCH3), 2.251 (s, 3H, CH3). 13C NMR (100 MHz,
DMSO-d6)
d
17.8, 37.5, 56.3, 100.9, 109.8, 111.2, 117.9, 125.6, 125.9,
A mixture of 3-(2-chloroacetamido)-4-methyl benzoic acid (8)
(0.227 g, 1 mmol), substituted 5-phenyl-1,3,4-oxadiazole-2-thiol
(14aeg) (1 mmol) and K2CO3 (0.207 g, 1.5 mmol) was refluxed in
acetone (20 mL) for 5e6 h. The excess of solvent was evaporated at
room temperature, concentrated the reaction mixture and diluted
with water (about 200 mL). The product precipitated out on acid-
ification with dilute hydrochloric acid was filtered and thoroughly
washed with cold water.
129.8, 133.0, 136.8,137.7, 139.9, 147.8, 153.2, 165.3, 168.7. MS (ESI) m/
z: 369.6 (M ꢀ H).
4.1.2.4. 3-(2-Benzo[d]oxazol-2-ylthio)acetamido)-4-methylbenzoic
acid, 10d. Yield 43.85%, Mp 235.5 ꢁC, IR (KBr, cmꢀ1). 1706.88 (CO st.
of COOH), 3286.48 (OH st. of COOH), 2854.45 (CH3 st.), 1650.95
(amide CO st.), 754.12 (CeS st.), 3359.77 (NH st.), 3055.03 (Ar CeH
st.), 1581.52 (Ar CeC st.). 1H NMR (400 MHz, DMSO-d6)
d
(ppm):12.6405 (s, 1H, COOH), 9.7768 (s, 1H, NHCO), 8.4080 (s,
4.1.6.1. 3-(2-(5-(2,3-Dichlorophenyl)-1,3,4-oxadiazol-2-ylthio)acet-
amido)-4-methylbenzoic acid, 15a. Yield 57.20%, Mp 236.5 ꢁC. IR
(KBr, cmꢀ1) 1697 (CO st. of COOH), 3201.61 (OH st. of COOH),
2931.60 (CH3 st.), 1650.95 (amide CO st.), 760.89 (CeS st.), 3463.92
(NH st.), 3074.32 (Ar CeH st.), 1596.95 (Ar CeC st.), 1049.20 (Cl st.).
1H, AreCH), 7.8420 (d, 1H, AreCH, J ¼ 8.72 Hz), 7.6330 (d, 2H, Are
CH, J ¼ 12.8 Hz), 7.472 (t, 1H, AreCH, J ¼ 7.6 Hz), 7.1070 (d, 1H, Are
CH, J ¼ 7.6 Hz), 7.270 (t, 1H, AreCH, J ¼ 7.6 Hz), 4.1108 (s, 2H, CH2),
2.251 (s, 3H, CH3). 13C NMR (100 MHz, DMSO-d6)
d 17.5, 37.8, 109.8,
110.8, 119.5, 123.9, 124.9, 125.3, 125.9, 129.4, 136.9, 137.8, 139.9,
1H NMR (400 MHz, DMSO-d6)
d (ppm): 10.8162 (s, 1H, NHCO),
141.8, 152.2, 166.3, 168.1. (ESI) m/z: 340.8 (M ꢀ H).
7.9803 (m, 1H, AreCH), 7.8874 (m, 1H, AreCH), 7.4776 (m, 1H, Are
CH), 7.2918 (m, 3H, AreCH), 7.5793 (d, 1H, AreCH, J ¼ 7.84 Hz),
4.1923 (s, 2H, CH2), 2.3411 (s, 3H, CH3). 13C NMR (100 MHz, DMSO-
4.1.2.5. 2-Benzo[d]thiazol-2-ylthio)acetamido)-4-methylbenzoic
acid, 10e. Yield 87.75%, Mp 194 ꢁC, IR (KBr, cmꢀ1), 1693.38 (CO st. of
COOH), 3272.98 (OH st. of COOH), 2852.52 (CH3 st.), 1656.74 (amide
CO st.), 767.97 (CeS st.), 3411.84 (NH st.), 3056.96 (Ar CeH st.),
d6)
d 17.2, 38.2, 109.6, 125.4, 125.8, 127.0, 127.8, 129.5, 130.3, 131.3,
134.1, 136.5, 137.2, 138.4, 166.1, 166.6, 167.3, 168.3. MS (ESI) m/z:
435.6 (M ꢀ H).
1579.59 (Ar CeC st.).1H NMR (400 MHz, DMSO-d6)
d (ppm): 9.508
(s,1H, NHCO), 8.194 (s, 1H, AreCH), 7.781 (d, 2H, AreCH,
J ¼ 14.4 Hz), 7.6435 (d, 1H, AreCH, J ¼ 7.6 Hz), 7.471 (t,1H, AreCH,
J ¼ 7.6 Hz), 7.279 (t, 1H, AreCH, J ¼ 7.6 Hz), 7.170 (d, 1H, AreCH,
J ¼ 7.6 Hz), 4.235 (s, 2H, CH2), 2.230 (s, 3H, CH3). 13C NMR (100 MHz,
4.1.6.2. 3-(2-(5-(2-Chlorophenyl)-1,3,4-oxadiazol-2-ylthio)acet-
amido)-4-methylbenzoic acid, 15b. Yield 52.10%, Mp 194 ꢁC. IR (KBr,
cm-1) 1718.45 (CO st. of COOH), 3207.40 (OH st. of COOH), 2979.82
(CH3 st.), 1654.81 (amide CO st.), 756.04 (CeS st.), 3388.70 (NH st.),
1596.95 (Ar CeC st.), 1037.63 (Cl st.). 1H NMR (400 MHz, DMSO-d6)
DMSO-d6)
d 17.8, 38.2, 109.6, 121.5, 121.8, 124.3, 125.5, 125.6, 125.8,

M. Rakse et al. / European Journal of Medicinal Chemistry 70 (2013) 469e476
475
d
(ppm): 12.447 (s,1H, COOH), 9.742 (s,1H, NHCO), 8.083 (s, 1H, Are
CH), 7.1e8.0 (m, 6H, AreCH), 4.302 (s, 2H, CH2), 2.272 (s, 3H, CH3).
13C NMR (100 MHz, DMSO-d6)
17.3, 38.2,109.6,125.5,125.9,127.2,
DMSO-d6) d 17.3, 38.4, 56.4, 61.0, 104.3, 109.6, 120.6, 124.9, 125.4,
129.3, 135.6, 137.8, 139.3, 152.8, 164.3, 166.3, 167.2, 168.2. MS (ESI)
m/z: 457.6 (M ꢀ H).
d
127.8, 129.6, 129.9, 131.5, 134.5, 136.5, 137.2, 138.4, 166.2, 166.5,
167.3, 168.3. MS (ESI) m/z: 401.6 (M ꢀ H).
4.2. In vitro PTP1B enzyme inhibition
4.1.6.3. 3-(2-(5-(2,4-Dichlorophenyl)-1,3,4-oxadiazol-2-ylthio)acet-
amido)-4-methylbenzoic acid, 15c. Yield 80.01%, Mp 257.5 ꢁC. IR
(KBr, cmꢀ1) 1691.46 (CO st. of COOH), 3226.69 (OH st. of COOH),
2995.25 (CH3 st.), 1643.46 (amide CO st.), 761.83 (CeS st.), 3461.99
(NH st.), 3082.04 (Ar CeH st.), 1596.95 (Ar CeC st.), 1033.17 (Cl st.).
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 studied by incubating these with human recombinant
PTP1B enzyme and determined the PTP1B activity using phosphate
detection reagent, Biomol red. The reaction was carried out in 96
1H NMR (400 MHz, DMSO-d6)
d
(ppm): 10.7706 (s, 1H, NHCO)),
7.9819 (d, 2H, AreCH, J ¼ 7.92 Hz), 7.4715 (m, 4H, AreCH), 4.1909 (s,
2H, CH2), 2.33374 (s, 3H, CH3). 13C NMR (100 MHz, DMSO-d6)
17.8,
well, flat-bottomed microtiter plates at 10 mM concentration using
d
DMSO as control. Further, those compounds show around 50% in-
hibitions were tested at six different concentrations for the calcu-
lation of IC50 values. The detection of free phosphate released is
based on classic Malachite green assay [32]. The percentage inhi-
bition by test compounds on PTP1B enzyme was calculated based
on the activity in the control tube (without inhibitor) as 100% from
three independent sets of experiments.
38.3, 109.4, 125.4, 125.9, 127.4, 127.8, 129.6, 130.4, 131.5, 134.6, 136.8,
137.4, 138.6, 166.3, 166.6, 167.3, 168.3. MS (ESI) m/z: 437.8 (M ꢀ H).
4.1.6.4. 3-(2-(5-(2-Hydroxyphenyl)-1,3,4-oxadiazol-2-ylthio)acet-
amido)-4-methylbenzoic acid, 15d. Yield 60.36%, Mp 257.5 ꢁC. IR
(KBr, cmꢀ1) 1689.53 (CO st. of COOH), 3228.62 (OH st. of COOH),
2923.88 (CH3 st.), 1645.17 (amide CO st.), 752.19 (CeS st.), 3382.91
(NH st.), 3072.39 (Ar CeH st.), 1552.59 (Ar CeC st.), 1356.51 (OH st.).
4.3. Evaluation of in vivo toxicity using zebrafish embryos
1H NMR (400 MHz, DMSO-d6)
d (ppm): 11.7969 (s broad peak, 1H,
OH), 10.9380 (s, 1H, NHCO), 7.9758 (d, 1H, AreCH, J ¼ 7.88 Hz),
7.9082 (d, 1H, AreCH, J ¼ 1.64 Hz), 7.8378 (d, 1H, AreCH,
J ¼ 4.58 Hz), 7.4771 (d, 1H, AreCH, J ¼ 8.04 Hz), 7.3481 (m, 1H, Are
CH), 6.9293 (d, 1H, AreCH, J ¼ 8.12 Hz), 6.8748 (t, 1H, AreCH,
J ¼ 7.72 Hz), 4.2406 (s, 2H, CH2), 2.3359 (s, 3H, CH3). 13C NMR
All procedures using zebrafish were in accordance with ethical
guidelines for animal use and followed those published by the NIH.
An indigenous wild type adult zebrafish strain from India was used
for this study (obtained from Vikrant Aquaculture, Mumbai, India).
They were maintained in a recirculation system using purified ELIX
System (Millipore, Billerica, US) grade water containing 200 mg/l
sea salt at 28 ꢁC under a 14:10 h light and dark cycle. Fish were fed
three times daily with a combination of freshly hatched live brine
shrimp and dry food. Males and females were kept in separate
tanks for four days before they were allowed to spawn. On day five,
300e400 embryos were obtained by natural mating of both adult
sexes (3:2 female and male ratios) in a breeding tank setup. Em-
bryos were collected and raised in 60 mm Petri dishes containing
E3 medium (5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2, and 0.33 mM
MgSO4) for 24 h. Compound stock was diluted in E3 medium to
(100 MHz, DMSO-d6)
d 17.3, 38.4, 109.6, 118.2, 121.8, 125.5, 125.3,
126.5,129.2,129.9,131.5,136.4,137.2,155.3,166.1,166.3,167.1,168.5.
MS (ESI) m/z: 383.8 (M ꢀ H).
4.1.6.5. 3-(2-(5-(4-Hydroxyphenyl)-1,3,4-oxadiazol-2-ylthio)acet-
amido)-4-methylbenzoic acid, 15e. Yield 59.05%, Mp 230.5 ꢁC. IR
(KBr, cmꢀ1) 1695.31 (CO st. of COOH), 3272.98 (OH st. of COOH),
2829.38 (CH3 st.), 1662.52 (amide CO st.), 763.76 (CeS st.), 3385.42
(NH st.), 3028.03 (Ar CeH st.), 1581.52 (Ar CeC st.), 1374.15 (OH st.).
1H NMR (400 MHz, DMSO-d6)
d (ppm): 8.4369 (d, 1H, AreCH,
J ¼ 1.52 Hz), 7.7174 (d, 1H, AreCH, J ¼ 7.84 Hz), 7.3674 (d, 2H, Are
CH, J ¼ 8.4 Hz), 7.3211 (d, 1H, AreCH, J ¼ 7.88 Hz), 7.1962 (m, 1H,
AreCH), 7.1050 (m, 1H, AreCH), 4.1379 (s, 1H, CH2), 2.4161 (s, 3H,
obtain final concentrations of 10
bryos at 24 h post fertilization were dechorionated with 500
Pronase K for 10 min. Six embryos per well were placed in 24-well
plates containing E3 medium and compounds added to a final
m
M in 0.1% DMSO. Zebrafish em-
mg/ml,
CH3). 13C NMR (100 MHz, DMSO-d6)
d
17.3, 38.4, 109.6, 116.6, 116.8,
118.8, 125.3, 125.9, 129.2, 136.7, 137.3, 158.3, 166.4, 166.6, 167.1,
concentration of 10 mM (in 0.1% DMSO). The embryos were grown
168.5. MS (ESI) m/z: 383.6 (M ꢀ H).
in the presence of the compound for 24 h, 48 h and 72 h. Control
embryos were incubated in 0.1% DMSO. Finally, embryos were
observed using visible light microscopy at regular intervals after
compound treatment to document general toxicity-related effects.
4.1.6.6. 4-Methyl-3-(2-(5-p-tolyl-1,3,4-oxadiazol-2-ylthio)acꢀe1t-
amido)benzoic acid, 15f. Yield 89.03%, Mp 246 ꢁC. IR (KBr, cm
)
1693.88 (CO st. of COOH), 3220.90 (OH st. of COOH), 2999.10 (CH3
st.), 1645.17 (amide CO st.), 754.12 (CeS st.), 3542.99 (NH st.),
1539.09 (Ar CeC st.), 1741 (C6H5CH3 st.).1H NMR (400 MHz, DMSO-
4.4. Molecular modelling
d6)
d
(ppm): 10.597 (s, 1H, NHCO), 7.0e8.0 (m, 7H, AreCH), 4.102 (s,
17.3,
For better understanding of binding mode of substituted 3-
acetamido-4-methyl benzoic acid derivatives at the molecular
level, we carried out molecular docking simulations of synthesized
molecules (10aee and 15aeg) at the PTP1B catalytic ligand binding
site. The docking simulations of synthesized compounds (10aee
and 15aeg) were performed using Maestro, version 9.2 imple-
mented from Schrodinger software suite [33]. The ligands 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) and prepared by
removing solvent, adding hydrogen and further minimization in
the presence of bound ligand (IX1) using protein preparation
wizard. Grids for molecular docking were generated with bound
co-crystallized ligand. For the validation of docking parameters the
co-crystal ligand (IX1) was re-docked at the catalytic site of protein
2H, CH2), 2.329 (s, 6H, CH3). 13C NMR (100 MHz, DMSO-d6)
d
21.7, 38.4, 109.6, 123.1, 125.3, 125.8, 126.7, 127.8, 129.2, 131.9, 136.7,
137.3, 164.4, 166.6, 167.1, 168.8. MS (ESI) m/z: 381.8 (M ꢀ H).
4.1.6.7. 4-Methyl-3-(2-(5-(3,4,5-trimethoxyphenyl)-1,3,4-oxadiazol-
2-ylthio)acetamido) benzoic acid, 15g. Yield 54.70%, Mp 163.5 ꢁC. IR
(KBr, cmꢀ1) 1718.46 (CO st. of COOH), 3311.55 (OH st. of COOH),
2945.10 (CH3 st.), 1654.81 (amide CO st.), 751.03 (CeS st.), 3496.70
(NH st.), 1589.23 (Ar CeC st.), 1128.28 (OCH3 st.). 1H NMR (400 MHz,
DMSO-d6)
d (ppm): 10.7274 (s broad peak, 1H, COOH), 9.7648 (s,
1H, NHCO), 8.0864 (s, 1H, AreCH), 7.8527 (s, 1H, AreCH), 7.6501 (d,
1H, AreCH, J ¼ 7.48 Hz), 7.4099 (d, 1H, AreCH, J ¼ 8.04 Hz), 7.2233
(d,1H, AreCH, J ¼ 7.92 Hz), 4.2744 (s, 2H, CH2), 3.8046 (s, 6H, OCH3),
3.7720 (s, 3H, OCH3), 2.2754 (s, 3H, CH3). 13C NMR (100 MHz,

476
M. Rakse et al. / European Journal of Medicinal Chemistry 70 (2013) 469e476
[17] K.K.D. Amarasinghe, A.G. Evidokimov, K. Xu, C.M. Clark, M.B. Maier,
A. Srivastava, A.-O. Colson, G.S. Gerwe, G.E. Stake, B.W. Howard, M.E. Pokross,
J.L. Gray, K.G. Peters, Design and synthesis of potent, non-peptidic inhibitors
of HPTPb, Bioorg. Med. Chem. Lett. 16 (2006) 4252e4256.
[18] S. Zhang, Z.Y. Zhang, PTP1B as a drug target: recent developments in PTP1B
inhibitor discovery, Drug Discov. Today 12 (2007) 373e381.
and the RMSD between co-crystal and re-docked pose was found to
be 0.255 A. All compounds (10aee and 14aeg) were docked using
Glide extra-precision (XP) mode, with up to three poses saved per
molecule.
ꢀ
[19] Z. Xin, G. Liu, C. Abad-Zapatero, Z. Pei, B.G. Szczepankiewicz, X. Li, T. Zhang,
C.W. Hutchins, P.J. Hajduk, S.J. Ballaron, M.A. Stashko, T.H. Lubben,
Acknowledgements
J.M. Trevillyan, M.R. Jirousek, Identification of
a monoacid-based, cell
permeable, selective inhibitor of protein tyrosine phosphatase 1B, Bioorg.
Med. Chem. Lett. 13 (2003) 3947e3950.
The authors gratefully acknowledge the Central Instrumenta-
tion Facility; Jamia Hamdard Deemed University, New Delhi, India
for the NMR spectral analysis of the compounds used in this study.
The authors AKR and C. Karthikeyan are recipients of a senior
research fellowship of CSIR, New Delhi. GSD wishes to thank AICTE,
New Delhi for a postgraduate fellowship.
[20] H. Zhao, G. Liu, Z. Xin, M.D. Serby, Z. Pei, B.G. Szczepankiewicz, P.J. Hajduk,
C. Abad-Zapatero, C.W. Hutchins, T.H. Lubben, S.J. Ballaron, D.L. Haasch,
W. Kaszubska, C.M. Rondinone, J.M. Trevillyan, M.R. Jirousek, Isoxazole car-
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