Synthesis, in vitro and in silico screening of ethyl 2-(6-substituted benzo[d]thiazol-2-ylamino)-2-oxoacetates as protein-tyrosine phosphatase 1B inhibitors

Article abstract of DOI:10.1016/j.ejmech.2012.04.025

The ethyl 2-(6-substituted benzo[d]thiazol-2-ylamino)-2-oxoacetate derivatives (OX 1-9) were prepared using a one-step reaction. The in vitro inhibitory activity of the compounds against protein tyrosine phosphatase 1B (PTP-1B) was evaluated. Compounds OX-(1, 6 and 7) were rapid reversible (mixed-type) inhibitors of PTP-1B with IC₅₀ values in the low micro-molar range. The most active compounds OX-(1, 6 and 7) were docked into the crystal structure of PTP-1B. Docking results indicate potential hydrogen bond interactions between the oxamate group in all compounds and the catalytic amino acid residues Arg221 and Ser216. The compounds were evaluated for their in vivo hypoglycemic activity, showing significant lowering of plasma glucose concentration in acute normoglycemic model and oral glucose tolerance test similarly at the effect exerted for hypoglycemic drug glibenclamide.

Full text of DOI:10.1016/j.ejmech.2012.04.025

European Journal of Medicinal Chemistry 53 (2012) 346e355
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis, in vitro and in silico screening of ethyl 2-(6-substituted benzo[d]
thiazol-2-ylamino)-2-oxoacetates as protein-tyrosine phosphatase 1B inhibitorsq
,
a
a
a
Gabriel Navarrete-Vazquez a , Marleth Ramírez-Martínez , Samuel Estrada-Soto , Carlos Nava-Zuazo ,
Paolo Paoli b, Guido Camici b, Jaime Escalante-García c, José L. Medina-Franco d, Fabian López-Vallejo d,
Rolffy Ortiz-Andrade e
*
a Facultad de Farmacia, Universidad Autónoma del Estado de Morelos, Av Universidad 1001, Chamilpa, Cuernavaca 62209, Morelos, Mexico
b Dipartimento di Scienze Biochimiche, Università degli Studi di Firenze, Firenze 50134, Italy
c Centro de Investigaciones Químicas, Universidad Autónoma del Estado de Morelos, Cuernavaca, Morelos 62209, Mexico
d Torrey Pines Institute for Molecular Studies, Port St. Lucie, FL 34987, USA
e Facultad de Química, Universidad Autónoma de Yucatán, Mérida, Yucatán 97000, Mexico
a r t i c l e i n f o
a b s t r a c t
Article history:
The ethyl 2-(6-substituted benzo[d]thiazol-2-ylamino)-2-oxoacetate derivatives (OX 1e9) were prepared
using a one-step reaction. The in vitro inhibitory activity of the compounds against protein tyrosine
phosphatase 1B (PTP-1B) was evaluated. Compounds OX-(1, 6 and 7) were rapid reversible (mixed-type)
inhibitors of PTP-1B with IC50 values in the low micro-molar range. The most active compounds OX-(1, 6
and 7) were docked into the crystal structure of PTP-1B. Docking results indicate potential hydrogen
bond interactions between the oxamate group in all compounds and the catalytic amino acid residues
Arg221 and Ser216. The compounds were evaluated for their in vivo hypoglycemic activity, showing
significant lowering of plasma glucose concentration in acute normoglycemic model and oral glucose
tolerance test similarly at the effect exerted for hypoglycemic drug glibenclamide.
Received 16 January 2012
Received in revised form
16 April 2012
Accepted 18 April 2012
Available online 26 April 2012
Keywords:
Diabetes
Protein tyrosine phosphatase (PTP-1B)
Benzothiazole
Ó 2012 Elsevier Masson SAS. All rights reserved.
1. Introduction
However, compounds displayed appreciable in vivo activity in
a
non-insulin-dependent diabetes mellitus murine model,
Diabetes mellitus is a chronic and progressive disease of meta-
bolic deregulation characterized by insulin resistance in peripheral
tissues (liver, muscle, and adipose), and impaired insulin secretion
by the pancreas [1].
comparable to glibenclamide [7]. In order to find new class of
compounds with hypoglycemic action, we report in this article the
one-step synthesis of ethyl 2-(6-substituted benzo[d]thiazol-2-
ylamino)-2-oxoacetate derivatives (OX-(1e9), Table 1), their
in vitro inhibitory activity on the PTP-1B, molecular docking of the
most active compounds as well as the in vivo hypoglycemic effect in
normoglycemic rats.
Type-2 diabetes and obesity are related with the deficiency in
insulin receptor signaling and is supposed to be recovered by the
inhibition of a certain protein-tyrosine phosphatases involved in
the down-regulation of insulin receptor, which causes prolonged
phosphorylation of the insulin receptor kinase [2e4]. The protein-
tyrosine phosphatase 1B (PTP-1B) plays a key role in cellular
signaling and in several human diseases, particularly diabetes and
obesity [5]. This enzyme is a novel attractive therapeutic target for
the treatment of diabetes [6]. Recently, our group reported a series
of 2-arylsulfonylaminobenzothiazoles, which were weak inhibitors
of PTP-1B (Fig. 1) [7].
2. Results and discussion
2.1. Chemistry
Compounds OX-1 to OX-8 were synthesized starting from 2-
amino-6-substituted benzo[d]thiazole (10e17), via
a coupling
reaction with ethyl chlorooxoacetate (18), in the presence of trie-
thylamine. Compound OX-9 was prepared by catalytic reduction of
compound OX-8. Title compounds were recovered with 44e98%
yields (Table 1). Compounds were purified by recrystalization or
by column chromatography. The chemical structures of the
synthesized compounds were confirmed on the basis of their
spectral data (NMR and mass spectra), and their purity ascertained
q
Taken in part from the M. Pharm. thesis of M. Ramírez-Martínez.
* Corresponding author. Tel./fax: þ52 777 329 7089.
E-mail address: gabriel_navarrete@uaem.mx (G. Navarrete-Vazquez).
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2012.04.025

G. Navarrete-Vazquez et al. / European Journal of Medicinal Chemistry 53 (2012) 346e355
347
Fig. 1. Structure of 2-arylsulfonylaminobenzothiazoles inhibitors of PTP-1B [7].
by microanalysis. Physical constants of the title compounds are
shown in Table 1.
In the nuclear magnetic resonance spectra (1H NMR;
d ppm), the
signals of the respective protons of the compounds were verified on
the basis of their chemical shifts, multiplicities, and coupling
constants. The aromatic region of the 1H NMR spectrum contained
an ABX pattern signals ranging from
d 7.06e8.24 ppm (dd,
Fig. 2. Inhibition of PTP-1B activity by OX-(1e9) compounds. Inhibition assays were
performed at 37 ꢀC and pH 7.0 at a fixed substrate final concentration (2.5 mM p-NPP),
which is a value close to that of Km for PTP-1B at pH 7.0. The final concentration of each
Jm ¼ 1.0e2.4; Jo ¼ 7.5e8.8 Hz), 7.61e7.89 (d, Jo ¼ 8.8 Hz), and
7.59e8.43 (d, Jm ¼ 1.1e2.6 Hz) attributable to H-5, H-4, and H-7, of
the benzothiazole-6-substituted structure, respectively. The signals
for ethyl oxamate were found ranging from
assignable to CH2, and 1.20e1.34 attributable to CH3.
compound in the reaction assay was 40
mM. Each assay (final volume 1 ml) was started
with 0.16 g of PTP-1B. Control experiment was carried out using DMSO. Each datum
m
d 3.80e4.32 ppm
represents the means ꢁ S.E.M. of four distinct assays. Residual activity, expressed as
percentage, is defined as the ratio between enzyme activity measured in the presence
of the inhibitor and that of the control.
2.2. In vitro PTP-1B inhibition assay
nitrophenyl phosphate in the presence of different inhibitor
concentrations, measuring the time-course of p-nitrophenol
release at 400 nm. In all cases we found that initial rates decrease
with increasing inhibitor concentrations, and curves maintain
linear monophasic behaviors as time progresses, indicating that the
rapid equilibrium conditions occur in the formation of
enzymeeinhibitor complexes. Identical behavior was found pre-
incubating PTP-1B with inhibitors for 30 min before the activity
assay. Taken together our results suggest that both association and
dissociation of inhibitors to the enzyme are fast events, excluding
the hypothesis that these compounds are slow-binding inhibitors.
To better evaluate the inhibitory power of these compounds, their
To test the ability of each compound to inhibit PTP-1B, aliquots
of stock solutions (dissolved in DMSO to prepare 20 mM) were
diluted with the assay buffer, containing 2.5 mM p-nitrophenyl
phosphate (p-NPP), to the final concentration of 40 mM. Samples
were incubated at 37 ꢀC, and the reactions were initiated by adding
an appropriate enzyme aliquot. Fig. 2 shows that compounds OX-1,
OX-6, and OX-7 strongly reduce the PTP-1B activity, whereas all
other compounds showed weaker activity.
Taking into account the results of the above preliminary anal-
yses, we performed additional assays on the most active
compounds OX-1, OX-6, and OX-7 to explore the type of inhibition.
Appropriate aliquots of PTP-1B were incubated in the presence of
a 125-fold molar excess of inhibitor for 1 h at two differing
temperature, 4 ꢀC and 37 ꢀC. Control experiments were performed
adding DMSO instead of the inhibitor solution. After this interval
time, the enzyme solutions were diluted 400-fold with the assay
solution, and the residual enzyme activity was measured. The
results, shown in the Fig. 3, demonstrate that the recovery of the
enzyme activity is almost complete in all cases, suggesting that all
OX-1, OX-6, and OX-7 act as reversible inhibitors.
IC50
values
were
determined.
Fig.
4
show
the
concentrationeresponse plots by the most active compounds OX-1,
OX-6, and OX-7. Table 2 summarizes the corresponding IC50 values,
which are in the low micro-molar range.
Thus, our assay system is operated under non-tight binding
conditions in the case of each inhibitor examined since IC50 is
greater than 5-fold over enzyme concentration (10 nM in all assays)
and the approximation that [inhibitor] free ¼ [inhibitor] total is
valid.
To assess the ability of OX-1, OX-6, and OX-7 to behave as slow-
binding inhibitors, we studied the PTP-1B catalyzed hydrolysis of p-
Table 1
Synthesis and physicochemical data for derivatives OX-(1e9).
Compd
R
MW
Mp (ꢀC)
Unoptimized yield (%)
OX-1
OX-2
OX-3
OX-4
OX-5
OX-6
OX-7
OX-8
OX-9
eCH3
eH
eOCH3
eOCH2CH3
eSO2CH3
eCl
eF
eNO2
eNH2
265
251
280
295
329
285
269
295
265
163e165
187e188
182e184
176e178
209e212
218e221
214e215
255(dec.)
282 (dec.)
52
49
45
44
98
47
63
80
92
Fig. 3. Inhibition reversibility assay. Aliquots of PTP-1B were incubated in the presence
of 100 m
M of each compound for 1 h at 4 ꢀC (black bar) or 37 ꢀC (white bar). Then the
enzyme was diluted 400-fold with the assay solution to measure the residual activity
(37 ꢀC, 5 mM pNPP final concentration). Control experiments were carried out adding
DMSO. All tests were performed in triplicate. The data represent the mean ꢁ S.E.M.

348
G. Navarrete-Vazquez et al. / European Journal of Medicinal Chemistry 53 (2012) 346e355
To verify if some compounds act as partial inhibitors, we
measured the rate of PTP-1B-catalyzed hydrolysis of p-nitrophenyl
phosphate at high substrate concentration (10-fold the Km value of
the enzyme) in the presence of increasing concentrations of each
compound. The results obtained for OX-1, OX-6, and OX-7 are
reported in Fig. 5. We can observe that, by increasing the inhibitor
concentration, the hydrolysis rate is reduced near to zero, sug-
gesting that the binding of inhibitor to enzyme produces an inac-
tive EI complex.
To determine the kinetic mechanism of inhibition, we studied
the dependence of enzyme main kinetic parameters (Km and Vmax
)
from the inhibitors concentration. For each compound, we
measured the initial hydrolysis rate using height substrate final
concentrations (0.5e40 mM range), in the presence of increasing
concentrations of each compound (Fig. 6). Experimental data was
analyzed by the double reciprocal plot method.
All reciprocal plots for OX-1, OX-6, and OX-7 show straight lines
that intersect each other in the left panel demonstrating that they
are mixed-type inhibitors. This is confirmed by the fact that
increasing inhibitor concentration is accompanied by the increase
of Km values and the decrease of Vmax (values Figs. 6e8).
To determine the Ki and the
a values for the mixed-type
inhibitors OX-1, OX-6, and OX-7 we considered the model repre-
sented in Scheme 1.
In this model, the inhibitor binds both the free enzyme, E, and
the enzymeesubstrate complex, ES. The EI has lower affinity for S
than E (a > 1), and ESI is non-productive. We calculated the value of
Ki (mixed-type) by replotting the slope of the straight lines of the
double reciprocal plot versus [I], using the following equation:
Km
Kt ꢂ Vmax
Km
slope ¼
ꢂ ½Iꢃ þ
Vmax
The results are reported in Table 3.
2.3. Molecular docking of compounds OX-(1, 6 and 7) with PTP-1B
In order to gain insight into the putative binding mode of the
most active compounds with PTP-1B, these compounds were
docked with a crystallographic structure of human PTP-1B using
AutoDock4 [11]. The crystallographic structure was obtained from
the Protein Data Bank (PDB), accession code 1C83 [8]. PTP-1B is co-
crystallized with 6-(oxalyl-amino)-1H-indole-5-carboxilic acid
which shows an extensive hydrogen bond network with residues in
the catalytic site. The catalytic residues, Arg221 and Asp181, play
a key role in the binding mode of the co-crystallized ligand (Fig. 9).
Fig. 4. The IC50 values were determined by plotting the relative activity of PTP-1B
versus inhibitor concentration. For each inhibitor, 15e20 different inhibitor concen-
trations were used. All tests were performed in quadruplicate. The data represent the
means ꢁ S.E.M.
Table 2
IC50 values for the most active compounds against
PTP-1B.
Compound
IC50 (mM)
OX-1
OX-6
OX-7
11.0 ꢁ 1.8
10.1 ꢁ 0.4
8.7 ꢁ 0.3
Fig. 5. The residual enzyme activity was measured at 37 ꢀC and pH 7.0 using 25 mM
pNPP as substrate. The inhibitors concentrations used were: 2.5, 5.0, 10, 25, 50, 100 and
200
mM. The symbols are: OX-1, C; OX-6, ,; OX-7, :. All tests were performed in
The data represent the IC50 value ꢁ S.E.M.
quadruplicate. The data represent the mean ꢁ S.E.M.

Fig. 6. [A] Double reciprocal plot of OX-1 (1/v versus 1/[S]). The concentrations of inhibitor used were: 0
mM, C; 5 mM, B; 10 mM, -; 15 mM, ,. All tests were performed in
quadruplicate. Dependence of main kinetic parameters Km [B] and Vmax [C] from the concentration of compound OX-1. The data represent the Km or Vmax values ꢁ S.E.M.
Fig. 7. [A] Compound OX-6: double reciprocal plot (1/v versus 1/[S]). The concentrations of inhibitor used were: 0 mM, C; 3 mM, B; 6 mM, :; 9 mM, ; 12 mM, -. All tests were
7
performed in quadruplicate. Dependence of main kinetic parameters Km [B] and Vmax [C] from the concentration of compound OX-6. The data represent the Km or Vmax
values ꢁ S.E.M.

350
G. Navarrete-Vazquez et al. / European Journal of Medicinal Chemistry 53 (2012) 346e355
Fig. 8. [A] Compound OX-7: double reciprocal plot (1/v versus 1/[S]). The concentrations of inhibitor used were: 0 mM, C; 3 mM, B 6 mM, :; 9 mM, ; 12 mM, -. All tests were
7
performed in quadruplicate. Dependence of main kinetic parameters Km [B] and Vmax [C] from the concentration of compound OX-7. The data represent the Km or Vmax
values ꢁ S.E.M.
Table 4 shows the predicted binding free energy of the co-
crystal ligand and benzothiazole derivatives along with their cor-
responding experimental Ki values. In general, the binding energies
are similar. This result is in line with the comparable experimental
activity of the three compounds. In addition, all compounds
showed better affinities than the crystal ligand.
Fig. 10 shows the binding mode of OX-1. It has a similar binding
as the co-crystal ligand and both form a similar hydrogen bond
network with the catalytic site.
Fig. 11 shows a comparison of the binding mode of the three
most active compounds with 6-(oxalyl-amino)-1H-indole-5-
carboxilic acid. The figures clearly show that the three benzothia-
zole derivatives have the same binding mode. In addition, all three
docked benzothiazole derivatives share the same binding pattern
and pharmacophoric interactions as the co-crystal ligand of PTP-1B
(1C83). It is notable the three-dimensional similarity of the binding
models of the benzothiazole derivatives and 6-(oxalyl-amino)-1H-
indole-5-carboxilic acid.
Fig. 12 depicts the 2D interactions maps of the 6-(oxalyl-amino)-
1H-indole-5-carboxilic acid and OX-1.
A three-dimensional
comparison of the binding conformation of the co-crystal ligand
and docked OX-1 is also shown. Hydrogen bond network is
conserved by OX-1 and analogs, with Gly220, Arg221, Asp181,
Ser216. As compared to the crystal ligand, OX-1 and analogs lack of
two important hydrogen bonds with Lys120 and Gln262. These
considerations could be taken into account for designing new
compounds. Interestingly, Asp181, Cys215 and Arg221 are catalytic
residues [9]. According to the models predicted by Autodock4, the
studied benzothiazole derivatives bind into the catalytic site of
PTP-1B.
2.4. In vivo hypoglycemic activity
Compounds OX-1 and OX-6 were evaluated for in vivo hypo-
glycemic activity using a normoglycemic rats. Glibenclamide was
taken as positive control [7]. The hypoglycemic activity of both
Ks
k2
+
+
P
E
+
I
S
ES
+
E
Table 3
I
The inhibition constants for compounds OX-1, OX-6, and OX-7.
Ki
αKi
Compound
Ki (
m
M)
a
Inhibition type
OX-1
OX-6
OX-7
6.2 ꢁ 0.7
7.9 ꢁ 0.6
7.9 ꢁ 0.6
4.5
3.8
1.8
Linear mixed-type
Linear mixed-type
Linear mixed-type
EI +
S
ESI
αKs
Scheme 1. Linear mixed-type inhibition.
Data represent Ki values ꢁ S.E.M.

G. Navarrete-Vazquez et al. / European Journal of Medicinal Chemistry 53 (2012) 346e355
351
Fig. 9. [A] Binding mode of the co-crystal ligand with PTP-1B showing an extensive hydrogen bonds network. Hydrogen bonds are depicted with yellow dashes. [B] Catalytic
binding site of PTB-1B. Surface in red shows the residues (Asp181, Ser216, Gly220 and Arg221) that participate in the hydrogen bond network. (For interpretation of the references
to color in this figure legend, the reader is referred to the web version of this article.)
Table 4
glibenclamide in both normoglycemic models, and indicate that
exerts a hypoglycemic effect of equal potency and efficacy that
repaglinide, confirming the hypoglycemic effects observed in the
acute normoglycemic model.
Docking results of the co-crystal ligand and benzothiazole derivatives into the
catalytic site of PTP-1B.
Compound
Binding energy
G (Kcal/mol)
Experimental inhibition
constant Ki (mM)
D
Co-crystal
ligand
OX-1
OX-6
OX-7
ꢄ6.01
14.0 [8]
3. Conclusion
ꢄ6.37
ꢄ6.35
ꢄ6.01
6.2
7.9
7.9
We report the synthesis of nine ethyl 2-(6-substituted benzo[d]
thiazol-2-ylamino)-2-oxoacetate derivatives which were obtained
with modest yields, and evaluated these compounds for their
protein tyrosine phosphatase 1B inhibitory activity. Several
compounds was determined using a 100 mg/kg single dose.
Compound OX-1 demonstrated significant hypoglycemic activity,
by lowering glycemia ranging from 26% to 37%. The effect was
consistent during the first 5 h of experiment (Fig. 13). Compound
OX-6 showed similar effects than glibenclamide during the first 3 h,
lowering the glycemia until 30%. The most pronounced effect of
both compounds was observed during the first 3e5 h of post-
intragastric administration.
2.5. Oral glucose tolerance test
Compounds OX-1 and OX-6 were tested in glucose-fed normal
rats, and they significantly prevent the rise of blood glucose (more
than 20%) when compared with control of glucose-fed normal rats,
repaglinide and glibenclamide (Fig. 14). Both compounds signifi-
cantly diminish hyperglycemic curve induced by overdose of
glucose (2 g/kg). This effect is similar to that exercised by
Fig. 11. [A] Docked benzothiazole derivatives and co-crystal ligand. They represent the
lowest energy conformation estimated by Autodock4. [B] Docked benzothiazole
derivatives into the catalytic binding site. Residues forming hydrogen bonds in red:
Asp181, Ser216, Gly220 and Arg221. Residues making van der Waals contacts in yellow:
Tyr46, Arg47, Val49 and Phe182. (For interpretation of the references to color in this
figure legend, the reader is referred to the web version of this article.)
Fig. 10. Binding model of OX-1 in the catalytic site of PTP-1B. Hydrogen bonds are
depicted with yellow dashes. (For interpretation of the references to color in this figure
legend, the reader is referred to the web version of this article.)

352
G. Navarrete-Vazquez et al. / European Journal of Medicinal Chemistry 53 (2012) 346e355
Fig. 12. 2D dimensional interaction maps of the crystal ligand and OX-1.
30
20
OX-1 (100 mg/Kg)
ISS +Tween 5%
Glibenclamide (10 mg/Kg)
10
*
0
-10
-20
-30
-40
-50
*
*
0
1
2
3
4
5
6
Time after administration (h)
7
30
20
OX-6 (100 mg/Kg)
ISS +Tween 5%
Glibenclamide (10 mg/Kg)
10
0
-10
-20
-30
-40
-50
*
*
0
1 2
Time after administration (h)
3
4
5
6
7
Fig. 14. Oral glucose tolerance test. Effect of a single dose of OX-1 and OX-6 (100 mg/
kg; intragastric route, n ¼ 5) in normoglycemic rats. ISS: Isotonic saline solution.
*p < 0.05 versus ISS group.
Fig. 13. Effect of a single dose of OX-1 and OX-6 (100 mg/kg; intragastric, n ¼ 5) in
normoglycemic rats. ISS: Isotonic saline solution. *p < 0.05 versus ISS group.

G. Navarrete-Vazquez et al. / European Journal of Medicinal Chemistry 53 (2012) 346e355
353
compounds of this series have shown significant PTP-1B inhibitory
activity in the low micro-molar range. Compounds OX-1, 6 and 7
exhibited the most promising activity as reversible and non-slow
binding mixed-type inhibitors of PTP-1B. Docking results indicate
potential hydrogen bond interactions between the oxamate group
in all compounds and the catalytic amino acid residues Arg221 and
Ser216. Compounds OX-1, 6 and 7 were two to four-times more
active than previously benzothiazole inhibitors synthesized by our
group [7].
MS/FABþ: m/z 251 (M
þ
Hþ). HRMS (FABþ): m/z 251.0483
[M þ H]þ (Calculated for C11H10N2O3SHþ 251.0490).
4.1.1.3. Ethyl 2-(6-methoxybenzo[d]thiazol-2-ylamino)-2-oxoacetate
(OX-3). Yield: 45%, mp: 182e184 ꢀC. 1H NMR (400 MHz, DMSO-
d6)
d
: 7.69 (1H, d, Jo ¼ 8.8 Hz, H-4), 7.61 (1H, d, Jm ¼ 2.4 Hz, H-7),
7.06 (1H, dd, Jm ¼ 2.4 Hz, Jm ¼ 2.4 Hz, Jo ¼ 8.8 Hz, H-5), 4.31 (2H,
q, CH2), 3.80 (3H, s, CH3O), 1.31 (3H, t, CH3) ppm; 13C NMR
(100 MHz, DMSO-d6)
d
: 159.0 (C-20), 156.8 (C-10), 156.4 (C-2),
The in vivo hypoglycemic activities of these compounds make
them a suitable leads to develop new chemical entities for potential
use in the treatment of diabetes.
155.4 (C-6), 141.8 (C-3a), 132.8 (C-7a) 121.2 (C-5), 115.3 (C-7),
104.7 (C-4), 62.6 (CH2), 55.6 (CH3O) 13.7 (CH3) ppm; MS/FABþ: m/
z
281 (M
þ
Hþ). HRMS (FABþ): m/z 281.0587 [M
þ
H]þ
(Calculated for C12H12N2O4SHþ 281.0596).
4. Experimental
4.1.1.4. Ethyl 2-(6-ethoxybenzo[d]thiazol-2-ylamino)-2-oxoacetate
(OX-4). Yield: 44%, mp: 176e178 ꢀC. 1H NMR (400 MHz, DMSO-
4.1. Chemistry
d6)
d
: 7.68 (1H, d, Jo ¼ 8.8 Hz, H-4), 7.59 (1H, d, Jm ¼ 2.2 Hz, H-7),
7.05 (1H, dd, Jm ¼ 2.2 Hz, Jm ¼ 2, Jo ¼ 8.8 Hz, H-5), 4.31 (2H, q,
Melting points were determined on an EZ-Melt MPA120 auto-
mated melting point apparatus from Stanford Research Systems
and are uncorrected. Reactions were monitored by TLC on 0.2 mm
precoated silica gel 60 F254 plates (E. Merck). 1H NMR spectra were
recorded on a Varian Oxford (400 MHz) and 13C NMR (100 MHz)
instrument. Chemical shifts are given in ppm relative to tetrame-
CH2), 4.06 (2H, q, CH2), 1.34 (3H, t, CH3), 1.31 (3H, t, CH3) ppm.;
13C NMR (100 MHz, DMSO-d6) : 168.4 (C-20), 159.0 (C-10), 156.8
d
(C-2), 155.6 (C-6), 141.7 (C-3a), 132.8 (C-7a) 121.2 (C-5), 115.6 (C-
7), 105.3 (C-4), 63.6 (CH2), 62.6 (CH2), 14.7 (CH3) 13.7 (CH3) ppm;
MS/FABþ: m/z 295 (M
þ
Hþ). HRMS (FABþ): m/z 295.0770
[M þ H]þ (Calculated for C13H14N2O4SHþ 295.0753).
thylsilane (Me4Si,
d
¼ 0) in DMSO-d6; J values are given in Hz. The
following abbreviations are used: s, singlet; d, doublet; q, quartet;
dd, doublet of doublet; t, triplet; m, multiplet; bs, broad signal. MS
were recorded on a JEOL JMS-700 spectrometer by Fast Atom
Bombarded [FAB (þ)]. Starting materials were commercially avail-
able from Aldrich and used without purification.
4.1.1.5. Ethyl
2-{6-(methylsulfonyl)benzo[d]thiazol-2-ylamino}-2-
oxoacetate (OX-5). Yield: 98%, mp: 209e212 ꢀC. 1H NMR
(400 MHz, DMSO-d6)
d: 8.14 (1H, s, H-7), 7.77 (1H, d, Jo ¼ 8 Hz,
H-4), 7.46 (1H, dd, Jo ¼ 8 Hz, Jm ¼ 1 Hz, H-5), 4.3 (2H, q, CH2),
2.49 (3H, s, CH3), 1.31 (3H, t, CH3) ppm; 13C NMR (100 MHz,
DMSO-d6) d
: 162.7 (C-20), 159.7 (C-10), 158.6 (C-2), 152.6 (C-3a),
4.1.1. General method of synthesis of ethyl 2-(6-substituted benzo
[d]thiazol-2-ylamino)-2-oxoacetates (1e8)
137.2 (C-6), 133.2 (C-7a), 126.2 (C-7) 123.5 (C-4), 122.2 (C-5), 64.1
(CH2), 45.1 (CH3), 15.0 (CH3) ppm; MS/FABþ: m/z 329 (M þ Hþ).
To
a
solution of 2-amino-6-substituted benzo[d]thiazole
HRMS (FABþ): m/z 329.0233 [M
þ
H]þ (Calculated for
(0.0015 mol) in dichloromethane was added triethylamine
(1.2 equiv.). The reaction mixture was stirred at 5 ꢀC for 15 min.
After that, a solution of ethyl chlorooxoacetate (0.0018 mol,
1.2 equiv.) was added droopingly. The reaction mixture was stirred
at room temperature for 9e32 h. After complete conversion as
indicated by TLC, the solvent was removed in vacuo, the residue was
neutralized with saturated NaHCO3 solution, and the aqueous layer
was extracted with ethyl acetate (3 ꢂ 15 mL), washed with water
(3 ꢂ 20 mL), and dried over anhydrous Na2SO4. The solvent was
evaporated in vacuo and the precipitated solids were recrystalized
C12H12N2O5S2Hþ 329.0266).
4.1.1.6. Ethyl
(OX-6). Yield: 47%, mp: 218e221 ꢀC. 1H NMR (400 MHz, DMSO-
d6)
: 8.13 (1H, s, H-7), 7.76 (1H, d, Jo ¼ 8.4 Hz, H-4), 7.48 (1H, dd,
Jm ¼ 1.2 Hz, Jo ¼ 8.4 Hz, H-5), 4.30 (2H, q, CH2), 1.30 (3H, t, CH3)
ppm; 13C NMR (100 MHz, DMSO-d6) : 159.9 (C-20), 159.5 (C-10),
2-(6-chlorobenzo[d]thiazol-2-ylamino)-2-oxoacetate
d
d
158.2 (C-2), 148.0 (C-3a), 134.3 (C-7a), 129.4 (C-5) 127.9 (C-6),
122.9 (C-4), 122.6 (C-7), 64.0 (CH2), 15.08 (CH3) ppm; MS/FABþ:
m/z 285 (M þ Hþ). HRMS (FABþ): m/z 285.0091 [M þ H]þ
(Calculated for C11H9ClN2O3SHþ 285.0101).
from
a mixture of ethanol:acetone or purified by column
chromatography.
4.1.1.7. Ethyl
(OX-7). Yield: 63%, mp: 214e215 ꢀC. 1H NMR (400 MHz, DMSO-
d6)
2-(6-fluorobenzo[d]thiazol-2-ylamino)-2-oxoacetate
4.1.1.1. Ethyl 2-(6-methylbenzo[d]thiazol-2-ylamino)-2-oxoacetate
(OX-1). Yield: 52%, mp: 163e165 ꢀC. 1H NMR (400 MHz, DMSO-
d
: 7.93 (1H, dd, Jm, HeH ¼ 2.6 Hz, Jo, HeF ¼ 8.72 Hz, H-7), 7.81
d6)
d
: 7.69 (1H, s, H-7), 7.65 (1H, d, Jo ¼ 8 Hz, H-4), 7.25 (1H, dd,
(1H, dd, Jo, HeH ¼ 8.92 Hz, Jm, HeF ¼ 4.8 Hz, H-4), 7.32 (1H, td, Jm,
Jo ¼ 7.5 Hz, Jm ¼ 1 Hz, H-5), 4.29 (2H, q, CH2), 2.38 (3H, s, CH3),
¼ 2.7 Hz, Jo, HeH ¼ 9.12 Hz, Jo, HeF ¼ 9.04 Hz, H-5), 4.32 (2H,
HeH
1.2 (3H, t, CH3) ppm; 13C NMR (100 MHz, DMSO-d6)
d: 160.2 (C-
q, CH2), 1.31 (3H, t, CH3) ppm; 13C NMR (100 MHz, DMSO-d6)
d:
20), 158.4 (C-10), 158.1 (C-2), 146.6 (C-3a), 134.9 (C-7a), 132.6 (C-6),
129.0 (C-5), 122.6 (C-7), 121.3 (C-4), 63.9 (CH2), 22.2 (CH3) 15.0
(CH3) ppm; MS/FABþ: m/z 265 (M þ Hþ). HRMS (FABþ): m/z
265.07062 [M þ H]þ (Calculated for C12H12N2O3SHþ 265.0647).
158.9 (d, J ¼ 239.24 Hz, C-6), 158.93 (C-20), 157.5 (C-10), 157.1 (d,
Jp ¼ 0.81 Hz, C-3a), 144.8 (C-2), 132.8 (d, Jm ¼ 11.39 Hz, C-7a),
122.0 (d, Jm ¼ 9.05 Hz, C-4), 114.6 (d, Jo ¼ 24.57 Hz, C-7), 108.3 (d,
Jo ¼ 26.69 Hz, C-5), 62.7 (CH2), 13.7 (CH3) ppm; MS/FABþ: m/z
269 (M þ Hþ). HRMS (FABþ): m/z 269.0392 [M þ H]þ (Calculated
for C11H9FN2O3SHþ 269.0396).
4.1.1.2. Ethyl
2-(benzo[d]thiazol-2-ylamino)-2-oxoacetate
(OX-
2). Yield: 49%, mp: 187e188 ꢀC. 1H NMR (400 MHz, DMSO-d6)
d:
8.03 (1H, d, Jo ¼ 7.88 Hz, H-7), 7.84 (1H, d, Jo ¼ 8.4 Hz, H-4), 7.47
(1H, m, Jo ¼ 7.6 Hz, Jo ¼ 7.6 Hz, Jm ¼ 1.1 Hz, H-6), 7.36 (1H, m,
Jo ¼ 7.3 Hz, Jo ¼ 7.3 Hz, Jm ¼ 1.1 Hz, H-5), 4.32 (2H, q, H-30), 1.32
4.1.1.8. Ethyl
(OX-8). Yield: 80%, mp: 255 ꢀC (dec). 1H NMR (400 MHz, DMSO-
d6)
: 9.03 (1H, s, H-7), 8.24 (1H, dd, Jo ¼ 8.8 Hz, Jm ¼ 1 Hz, H-5),
7.89 (1H, d, Jo ¼ 8.8 Hz, H-4), 4.30 (2H, t, CH2) 1.31 (3H, t, CH3)
ppm; 1C NMR (100 MHz, DMSO-d6) : 162.8 (C-20), 158.3 (C-10),
157.2 (C-2), 152.6 (C-3a), 143.2 (C-6), 132.1 (C-7a) 121.7 (C-4),
2-(6-nitrobenzo[d]thiazol-2-ylamino)-2-oxoacetate
d
(3H, t, H-40) ppm; 13C NMR (100 MHz, DMSO-d6) : 159.1 (C-20),
d
157.9 (C-10), 157.4 (C-2), 147.5 (C-3a), 131.4 (C-7a), 126.5 (C-6),
124.2 (C-5), 122.0 (C-7), 120.5 (C-4), 62.7 (CH2), 13.7 (CH3) ppm;
d

354
G. Navarrete-Vazquez et al. / European Journal of Medicinal Chemistry 53 (2012) 346e355
120.8 (C-7), 119.1 (C-5), 62.9 (CH2), 13.7 (CH3) ppm; MS/FABþ: m/z
296 (M þ Hþ). HRMS (FABþ): m/z 296.0393 [M þ H]þ (Calculated
for C11H9N3O5SHþ 296.0341).
Institutional Animal Care and Use Committee based on US National
Institute of Health publication (No. 85-23, revised 1985).
4.2.4. In vivo hypoglycemic assay
4.1.1.9. Ethyl
(OX-9). Yield: 90%, mp: 290 ꢀC (dec). 1H NMR (400 MHz, DMSO-
d6)
: 6.84 (1H, s, H-7), 6.60 (1H, dd, Jo ¼ 8.8 Hz, Jm ¼ 1 Hz, H-5),
7.59 (1H, d, Jo ¼ 8.8 Hz, H-4), 4.32 (2H, t, CH2) 1.34 (3H, t, CH3)
ppm; 13C NMR (100 MHz, DMSO-d6) : 158.2 (C-20), 156.5 (C-10),
150.4 (C-2), 148.4 (C-3a), 145.7 (C-6), 127.1 (C-7a) 122.1 (C-4),
113.8 (C-5), 103.8 (C-7), 63.2 (CH2), 14.7 (CH3) ppm; MS/FABþ: m/z
266 (M þ Hþ). HRMS (FABþ): m/z 266.0578 [M þ H]þ (Calculated
for C11H11N3O3SHþ 266.0593).
2-(6-aminobenzo[d]thiazol-2-ylamino)-2-oxoacetate
Animals were divided into groups of five animals each (n ¼ 5).
Experimental groups were orally treated with a single dose
(100 mg/kg body weight) of suspension of the compounds OX-1,
OX-6, and OX-7 (prepared in 5% Tween 80). Control group
animals were also fed with 5% Tween 80. Glibenclamide (10 mg/kg)
was used as hypoglycemic reference drug. Blood samples were
collected from the caudal vein at 0, 1, 3, 5, and 7 h after vehicle,
sample and drug administration. Blood glucose concentration was
estimated by enzymatic glucose oxidase method using a commer-
cial glucometer (Accutrend GCT, RocheÒ). The percentage variation
of glycemia for each group was calculated in relation to initial (0 h)
level, according to: %Variation of glycemia ¼ [(Gx ꢄ G0)/G0] ꢂ 100,
where G0 were initial glycemia values and Gx were the glycemia
values at þ1, þ3, þ5 and þ7 h, respectively. All values were
expressed as mean ꢁ S.E.M. Statistical significance was estimated
by analysis of variance (ANOVA), p < 0.05 implies significance.
d
d
4.2. Biological assays
4.2.1. PTP-1B expression and purification
All experiments were conducted using human recombinant
PTP-1B. Briefly, the complete sequence of PTP-1B was cloned in the
pGEX-2T bacterial expression vector downstream the GST
sequence. This vector was used to transform E. coli TB1 strain cells.
The recombinant fusion protein was purified from bacterial lysate
using a single-step affinity chromatography. The solution contain-
ing purified fusion protein was treated with thrombin for 3 h at
37 ꢀC. Then the PTP-1B was purified from GST and thrombin by gel
filtration on a Superdex G75 column. The purity of PTP-1B prepa-
ration was assessed by SDSepolyacrylamide gel electrophoresis.
4.2.5. Oral glucose tolerance test [11]
After a food overnight deprivation of 18 h, normoglycemic rats
(n ¼ 5) were subjected to an oral glucose tolerance test (OGTT) to
investigate the short-term hypoglycemic effect of the compounds.
Compounds OX-1 and OX-6 (100 mg/kg), vehicle, acarbose
(10 mg/kg), repaglinide (4 mg/kg) and glibenclamide (5 and
10 mg/kg) were administered to rats in the equal volume of
suspension, and 10 min after received a single dose of glucose (2 g/
kg) by de same route. Blood samples were collected from the tail tip
at 0 (before oral administration), 0.5, 1, 2, 3 and 4 h after vehicle,
positive controls and samples administration. Blood glucose
concentration was estimated as described.
4.2.2. Enzyme assays [10]
All assays were carried out at 37 ꢀC. The substrate (p-nitro-
phenylphosphate) was dissolved in 0.075 M of b,b-dimethylgluta-
rate buffer pH 7.0, containing 1 mM EDTA and 1 mM dithiothreitol.
The final volume was 1 mL. The reactions were initiated by adding
aliquots of the enzyme (0.16
appropriate times with 4 mL of 1 M KOH. The released p-nitro-
mg for each test), and stopped at
4.2.6. Docking
Molecular Operating Environment (MOE) 2009.10 was used for
ligand and protein preparation, and molecular structure viewing.
The crystal structure was obtained from the PDB with the accession
code 1C83 [8]. Docking calculations were conducted with Auto-
Dock, version 4.0 [12]. In short, AutoDock performs an automated
docking of the ligand with user-specified dihedral flexibility within
a protein rigid binding site. The program performs several runs in
each docking experiment. Each run provides one predicted binding
mode. All water molecules and 6-(oxalyl-amino)-1H-indole-5-
carboxilic acid (crystallographic ligand) were removed from the
crystallographic structure and all hydrogen atoms were added. For
all ligands and protein, Gasteiger charges were assigned and
nonpolar hydrogen atoms were merged. All torsions were allowed
to rotate during docking. The auxiliary program AutoGrid gener-
ated the grid maps. Each grid was centered at the crystallographic
coordinates of the crystallographic compound. The grid dimensions
phenol was determined by reading the absorbance at 400 nm
(
3
¼ 18,000 Mꢄ1 cmꢄ1). The main kinetic parameters (Km and Vmax
)
were determined by measuring the initial rates using eight
different substrate concentrations in the 0.5e40 mM range.
Experimental data were analyzed using the MichaeliseMenten
equation and a nonlinear fitting program (FigSys).
To evaluate the inhibition power of compounds OX-1, OX-6, and
OX-7 we calculated the IC50 value using a fixed substrate concen-
tration corresponding to the Km of enzyme and varying inhibitor
concentrations. The IC50 value was calculated by fitting experi-
mental data with a non-nonlinear fitting program (FigSys, Biosoft,
UK), using the equation:
Max ꢄ Min
y ¼
slope þ Min
ꢀ
ꢁ
x
1 þ
IC50
3
ꢀ
ꢀ
were 22.12 ꢂ 22.12 ꢂ 22.12 A with points separated by 0.375 A.
LennardeJones parameters 12e10 and 12e6, supplied with the
program, were used for modeling H-bonds and Van der Waals
interactions, respectively. The Lamarckian genetic algorithm was
applied for the search using default parameters. The number of
docking runs was 100. After docking, the 100 solutions were clus-
where “y” is vi/vo, i.e. the ratio between the activity measure in the
presence of the inhibitor (vi) and the activity of the control without
the inhibitor (vo). The parameter “x” is the inhibitor concentration.
4.2.3. Animals
ꢀ
Male Wistar rats weighing 200e250 g body weight were housed
at standard laboratory conditions and fed with a rodent pellet diet
and water ad libitum. They were maintained at room temperature
and at a photoperiod of 12 h day/night cycle. Animals described as
fasted were deprived of food for 18 h but had free access to water.
All animal procedures were conducted in accordance with our
Federal Regulations for Animal Experimentation and Care
(SAGARPA, NOM-062-ZOO-1999, México), and approved by the
tered into groups with RMS lower than 1.0 A. The clusters were
ranked by the lowest energy representative of each cluster. In order
to describe the ligand-binding pocket interactions, the top ranked
binding mode found by AutoDock in complex with the binding
pocket of PTP-1B was subject to full energy minimization using the
MMFF94 force field implemented in MOE until the gradient 0.05
was reached. PyMOL 1.0 [13] was used to generate the molecular
surface of docking models.

G. Navarrete-Vazquez et al. / European Journal of Medicinal Chemistry 53 (2012) 346e355
355
[4] A.P. Combs, Recent advances in the discovery of competitive protein tyrosine
phosphatase 1B inhibitors for the treatment of diabetes, obesity, and cancer,
J. Med. Chem. 53 (2010) 2333e2344.
[5] S. Zhang, Z.Y. Zhang, PTP1B as a drug target: recent developments in PTP1B
inhibitor discovery, Drug Discov. Today 12 (2007) 373e381.
[6] R. Sarabu, J. Tilley, Recent advances in therapeutic approaches to type 2 dia-
betes, Annu. Rep. Med. Chem. 40 (2005) 167e181.
[7] G. Navarrete-Vazquez, P. Paoli, I. León-Rivera, R. Villalobos-Molina,
J.L. Medina-Franco, R. Ortiz-Andrade, S. Estrada-Soto, G. Camici, D. Diaz-
Coutiño, I. Gallardo-Ortiz, K. Martinez-Mayorga, H. Moreno-Díaz, Synthesis,
in vitro and computational studies of protein tyrosine phosphatase
Acknowledgments
This work was supported in part by grants from CONACYT,
project 100608 (Ciencia basica-2008), and internal funds from
Facultad de Farmacia, UAEM, given to G. Navarrete Vazquez. P. Paoli
and G. Camici are grateful to the Ente Cassa di Risparmio di Firenze
for financial support. M. Ramirez-Martinez acknowledges the
fellowships awarded by CONACYT to carry out graduate studies, as
well as ECOES Santander Mexico and CUPIA, to carry out a research
stay at Universidad Autónoma de Yucatan. J.L. Medina-Franco and F.
Lopez-Vallejo are grateful to the State of Florida.
1B inhibition of
a small library of 2-arylsulfonylaminobenzothiazoles
with antihyperglycemic activity, Bioorg. Med. Chem. 17 (2009)
3332e3341.
[8] H.S. Andersen, L.F. Iversen, C.B. Jeppesen, S. Branner, K. Norris,
H.B. Rasmussen, K.B. Møller, N.P. Møller, 2-(Oxalylamino)-benzoic acid is
a general, competitive inhibitor of protein-tyrosine phosphatases, J. Biol.
Chem. 275 (2000) 7101e7108.
Appendix A. Supplementary data
[9] L. Bialy, H. Waldmann, Inhibitors of protein tyrosine phosphatases: next-
generation drugs? Angew. Chem. Int. Ed. 44 (2005) 3814e3839.
[10] R. Maccari, P. Paoli, R. Ottanà, M. Jacomelli, R. Ciurleo, G. Manao, T. Steindl,
T. Langer, M.G. Vigorita, G. Camici, 5-Arylidene-2, 4-thiazolidinediones as
inhibitors of protein tyrosine phosphatases, Bioorg. Med. Chem. 15 (2007)
5137e5149.
[11] R. Ortiz-Andrade, J.C. Sachez-Sagado, G. Navarrete-Vázquez, S.P. Webster,
M. Binnie, S. García-Jiménez, I. León-Rivera, P. Cigarroa-Vázquez, R. Villaobos-
Molina, S. Estrada-Soto, Antidiabetic and toxicologial evaluations of nar-
ingenin in normoglycemic and NIDMM rat models and its implications on
extra-pancreatic glucose regulation, Diabetes Obes. Metab. 10 (2008)
1097e1104.
[12] R. Huey, G.M. Morris, A.J. Olson, D.S. Goodsell, A semiempirical free energy
force field with charge-based desolvation, J. Comput. Chem. 28 (2007)
1145e1152.
[13] W.L. DeLano, The PyMOL Molecular Graphics System. DeLano Scientific LLC,
San Carlos, CA, USA. Available at: http://www.pymol.org.
Supplementary data related to this article can be found online at
doi:10.1016/j.ejmech.2012.04.025.
References
[1] L. Gang, Protein tyrosine phosphatase 1B inhibition: opportunities and chal-
lenges, Curr. Med. Chem. 10 (2003) 1407e1421.
[2] S. Gupta, G. Pandey, N. Rahuja, A.K. Srivastava, A.K. Saxena, Design, synthesis
and docking studies on phenoxy-3-piperazin-1-yl-propan-2-ol derivatives as
protein tyrosine phosphatase 1B inhibitors, Bioorg. Med. Chem. Lett. 20 (2010)
5732e5734.
[3] C. Ramachandran, B.P. Kennedy, Protein tyrosine phosphatase 1B: a novel
target for type
2 diabetes and obesity, Curr. Top. Med. Chem. 3 (2003)
749e757.