Synthesis, in vitro and computational studies of protein tyrosine phosphatase 1B inhibition of a small library of 2-arylsulfonylaminobenzothiazoles with antihyperglycemic activity

Article abstract of DOI:10.1016/j.bmc.2009.03.042

The 2-arylsulfonylaminobenzothiazole derivatives 1-27 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 4 and 16 are rapid reversible (mixed-type) inhibitors of PTP-1B with IC₅₀ values in the low micromolar range. The most active compounds (4 and 16) were docked into the crystal structure of PTP-1B. Docking results indicate potential hydrogen bond interactions between the nitro group in both compounds and the catalytic amino acid residues Arg 221 and Ser 216. Both compounds were evaluated for their in vivo antihyperglycemic activity in a type 2 diabetes mellitus rat model, showing significant lowering of plasma glucose concentration, during the 7 h post-intragastric administration.

Full text of DOI:10.1016/j.bmc.2009.03.042

Bioorganic & Medicinal Chemistry 17 (2009) 3332–3341
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis, in vitro and computational studies of protein tyrosine phosphatase
1B inhibition of a small library of 2-arylsulfonylaminobenzothiazoles
with antihyperglycemic activity
b,
c
d
Gabriel Navarrete-Vazquez a, , Paolo Paoli , Ismael León-Rivera , Rafael Villalobos-Molina ,
Jose Luis Medina-Franco e, Rolffy Ortiz-Andrade a,f, Samuel Estrada-Soto a, Guido Camici b,
Daniel Diaz-Coutiño c, Itzell Gallardo-Ortiz d, Karina Martinez-Mayorga e, Hermenegilda Moreno-Díaz a
*
*
a Facultad de Farmacia, Universidad Autónoma del Estado de Morelos, Cuernavaca, Morelos 62209, 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 Unidad de Biomedicina, FES Iztacala, Universidad Nacional Autónoma de México, Tlalnepantla, México 54090, Mexico
e Torrey Pines Institute for Molecular Studies, Port St. Lucie, FL 34987, USA
f Facultad de Química, Universidad Autónoma de Yucatán, Mérida, Yucatán 97150, Mexico
a r t i c l e i n f o
a b s t r a c t
Article history:
The 2-arylsulfonylaminobenzothiazole derivatives 1–27 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 4 and 16 are rapid reversible (mixed-type) inhibitors of PTP-1B with IC50 val-
ues in the low micromolar range. The most active compounds (4 and 16) were docked into the crystal
structure of PTP-1B. Docking results indicate potential hydrogen bond interactions between the nitro
group in both compounds and the catalytic amino acid residues Arg 221 and Ser 216. Both com-
pounds were evaluated for their in vivo antihyperglycemic activity in a type 2 diabetes mellitus rat
model, showing significant lowering of plasma glucose concentration, during the 7 h post-intragastric
administration.
Received 10 February 2009
Revised 13 March 2009
Accepted 20 March 2009
Available online 26 March 2009
Keywords:
Benzothiazoles
Protein tyrosine phosphatase (PTP-1B)
Diabetes
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
On the other hand, 11b-HSD1 catalyzes the conversion of inac-
tive cortisone into the active hormone cortisol, which is a potent
Type 2 diabetes mellitus (T2DM) is a progressive disease of met-
abolic deregulation characterized by insulin resistance in peripheral
tissues (liver, muscle, and adipose), and impaired insulin secretion
by the pancreas.1 Obesity is one of the major risk factors for develop-
ing T2DM.2 Current therapies to combat both diseases are limited
and often ineffective, and there is an urgent need for improved drugs
that can help reverse disease progression.3
The enzymes protein tyrosine phosphatase PTP-1D and 11b-
hydroxysteroid dehydrogenase type 1 (11b-HSD1) are both novel
attractive therapeutic targets for the treatment of diabetes and
obesity.4 PTP-1D is an intracellular enzyme that has been in-
volved in down-regulation of receptor tyrosine kinase activity
following stimulation of the insulin or leptin receptors. Recently
reports have demonstrated that mice lacking PTP-1D have en-
hanced insulin sensitivity.5 Inhibition of PTP-1D would result in
maintenance of an activated insulin receptor and hence prolong
insulin signaling.
functional antagonist of insulin action, and promotes gluconeogen-
esis in the liver, potentially leading to raised blood glucose concen-
tration in diabetes.6
Inhibition of both enzymes has been considered as attractive
therapeutic targets for T2DM, obesity and metabolic syndrome.4
Recently, our group reported a series of seven benzothiazoles re-
lated to this work, which were weak inhibitors of 11b-HSD1.7
However, the compounds displayed appreciable in vivo activity
in T2DM murine model, comparable to glibenclamide. In order to
find an alternative mode of action of this class of compounds, we
report in this article the one-step synthesis of an extended library
of 2-arylsulfonylaminobenzothiazole derivatives, their in vitro
inhibitory activity on the PTP-1D, and the in vivo antihyperglyce-
mic effect observed with the most active compounds.
2. Results and discussion
2.1. Chemistry
* Corresponding authors. Tel./fax: +52 777 3297089 (G.N.-V.).
E-mail addresses: gabriel_navarrete@uaem.mx (G. Navarrete-Vazquez), paolo.
paoli@unifi.it (P. Paoli).
The small library of title compounds (1–27), was designed using
a matrix of four benzothiazole and six arylsulfonylchlorides. Com-
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.03.042

G. Navarrete-Vazquez et al. / Bioorg. Med. Chem. 17 (2009) 3332–3341
3333
pounds were synthesized starting from 2-aminobenzothiazole-6-
substituted (28–33), via a coupling reaction with arylsulfonyl
chlorides 34–40, in the presence of a catalytic amount of 4-dimeth-
ylaminopyridine and triethylamine.7 Title compounds were recov-
ered with 26–90% yields (Table 1). Compounds were purified by
recrystallization or by column chromatography. Compounds 4, 6,
11, 16, 18 and 26 afforded the best yields; all of them contain elec-
tron-withdrawing groups attached at position four respect to the
phenyl sulfonamide moiety. On the other hand, compounds 1,
13–15, 17, 19, 20, 22 and 25, which bear electron-donating groups
at that position, had the lowest yields.
logically active drugs included in the database of software named
Prediction of Activity Spectra for Substances (PASS).9–12 Results
presented in Table 1 describe two biological activities taken from
PASS software: antiobesity and antidiabetic effects. Pa values esti-
mated for antiobesity activity were ranging between 0.80 and
0.89. These results indicated that compounds exhibited chemical
structures similar to known antiobesity drugs, and are likely to
reveal this activity. Pa-estimated antidiabetic activity values were
determined ranged between 0.59 and 0.72, which also indicates
that chemical structures of compounds 1–27 exhibited levels of
similarity to those of known antidiabetic drugs (i.e., sulfonylu-
reas). The predictions do not include a specific target as PTP-1D
or 11 b-HSD1.
The chemical structures of the synthesized compounds were
confirmed on the basis of their spectral data (NMR and mass spec-
tra), and their purity ascertained by microanalysis. Physical con-
stants of the title compounds are shown in Table 1.
2.2. In vitro biological assay
In the nuclear magnetic resonance spectra (1H NMR; d ppm),
the signals of the respective protons of the compounds were veri-
fied on the basis of their chemical shifts, multiplicities, and cou-
pling constants. The aromatic region of the 1H NMR spectrum
contained an ABX pattern signals ranging from d 6.94–6.99 (dd,
Jm = 2.2–2.6; Jo = 8.8 Hz), 7.18–7.23 (d, Jo = 8.8 Hz), and 7.41–
7.60 (d, Jm = 2.2–2.6 Hz) attributable to H-5, H-4, and H-7, of the
benzothiazole 6-substituted structure, respectively. Compounds
6, 7, 10, 14, 21 and 23 have been previously reported.7 Compound
3 was purified by recrystallization. A sample crystal was obtained
and diffracted by X-ray analysis, confirming the proposed
structure.8
To test the ability of the compounds to inhibit PTP-1D, all com-
pounds were dissolved in DMSO to prepare initial solutions
(20 mM, final concentration), aliquots of each initial solution were
diluted to 40 lM with the assay buffer containing 2 mM p-nitro-
phenyl phosphate (pNPP); the mixture was incubated at 37 °C,
and the reaction was started by adding an appropriate enzyme ali-
quot.13 The results showed that two compounds (4 and 16)
strongly reduce the PTP-1D enzyme activity (Fig. 1). Other com-
pounds, such as 10, 18, 23, and 27, are weaker inhibitors. It is inter-
esting to note that all of the above compounds, with exception of
18, bear a nitro group in their structure.
Before the establishment of an in vitro assay, we obtained pre-
dictive values concerning biological activities by comparing the
chemical structures of the compounds designed (1–27), with
structures or substructures of more than 46,000 well-known bio-
Considering the results of the preliminary PTP-1D inhibition
analysis, we performed additional tests on the more potent com-
pounds 4 and 16. To verify if these compounds were reversible
inhibitors, appropriate aliquots of PTP-1D were incubated in the
Table 1
Synthesis, physicochemical data and predictive values of biological activities calculated with PASS for derivatives 1–27
O
S
O
O
S
O
N
S
N
S
CH2Cl2, DMAP (cat)
TEA, 40 ºC
NH2
N
H
Cl
R2
R2
+
R1
R1
28-33
34-40
1-27
Compound
R1
R2
MW
Mp (°C)
Unoptimized yield (%)
Antiobesity effect (Pa/Pi)
Antidiabetic effect (Pa/Pi)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
–CH3
–CH3
–CH3
–CH3
–CH3
–CH3
–OCH3
–OCH3
–OCH3
–OCH3
–OCH3
–OCH3
–OCH2CH3
–OCH2CH3
–OCH2CH3
–OCH2CH3
–OCH2CH3
–OCH2CH3
–NO2
–NO2
–NO2
–NO2
–NO2
–NO2
–NO2
–F
–Cl
–H
–CH3
–OCH3
–NO2
–NHCOCH3
–Cl
–H
–CH3
–OCH3
–NO2
–NHCOCH3
–Cl
–H
–CH3
–OCH3
–NO2
–NHCOCH3
–Cl
–H
–CH3
–OCH3
–NO2
–NHCOCH3
–Cl
304
318
334
349
361
338
320
334
350
365
377
354
334
348
365
379
391
368
335
349
365
380
392
369
353
353
369
254.7–257.6
235.9 (dec.)
260.2–262.3
226.3–227.5
276.3–278.6
265.6–266.9
252.9–253.2
215.2–216.9
230.7–232.9
236.9–238.2
245.3–249.9
162.1–163.8
270.0–271.0
228.4–230.3
220.3–222.2
247.9–248.9
262.3–263.6
151.2–153.8
241.5–248.9
178.1–179.6
187.1–189.3
203.1–205.4
230.2–233.1
218.9–2237
163.3–164.1
259.3–262.1
244.9–249.5
26.0
37.7
48.8
90.1
52.2
85.9
39.4
33.3
69.3
66.1
75.6
51.0
41.2
47.1
46.5
71.2
40.3
85.1
37.6
18.8
33.8
47.4
53.3
35.6
34.4
81.1
58.2
0.892–0.005
0.897–0.005
0.853–0.006
0.835–0.006
0.819–0.006
0.884–0.005
0.889–0.005
0.863–0.006
0.894–0.005
0.833–0.006
0.809–0.006
0.882–0.005
0.880–0.005
0.858–0.006
0.858–0.006
0.817–0.006
0.813–0.006
0.872–0.006
0.888–0.005
0.860–0.006
0.845–0.006
0.893–0.005
0.809–0.006
0.880–0.005
0.861–0.006
0.836–0.006
0.881–0.005
0.719–0.006
0.727–0.006
0.675–0.007
0.653–0.007
0.633–0.007
0.705–0.006
0.716–0.006
0.684–0.007
0.724–0.006
0.654–0.007
0.628–0.007
0.703–0.006
0.709–0.006
0.686–0.007
0.685–0.007
0.651–0.007
0.640–0.007
0.697–0.006
0.679–0.007
0.640–0.007
0.631–0.007
0.687–0.007
0.595–0.007
0.666–0.007
0.638–0.007
0.638–0.007
0.697–0.006
–F
–NO2
–NO2

3334
G. Navarrete-Vazquez et al. / Bioorg. Med. Chem. 17 (2009) 3332–3341
120
100
80
60
40
20
0
C
1
2
3
4
5
6
7
8
9
10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27
Compounds
Figure 1. Inhibition of PTP-1B enzyme activity. Inhibition tests were assayed using a fixed substrate concentration, 2 mM pNPP, corresponding to the Km value of the enzyme
measured at pH 7.0. For each inhibitor the final concentration was 40 M. Each test (final volume 1 ml) was started with 0.16 g of PTP-1B. Control experiment (C) was
l
l
carried out using DMSO. All tests were conducted in quadruplicate. The data represent the means S.E.M. Residual activity, expressed as%, is defined as the ratio between
enzyme activity measured in the presence of the inhibitor and that of the control.
presence of a 125-fold molar excess of inhibitor for 1 h at two dif-
fering temperatures, 4 °C and 37 °C. Control experiments were con-
ducted adding DMSO instead of inhibitor. After this interval time,
the enzyme solutions were diluted 400-fold and the residual en-
zyme activity was assayed. The results are shown in Figure 2. In
all cases the recovery of the activity was almost complete, suggest-
ing that 4 and 16 behave as reversible inhibitors.
To assess the ability of compounds 4 and 16 to behave as slow-
binding inhibitors, we studied the time-course of the PTP-1D-cata-
lyzed hydrolysis of p-nitrophenyl phosphate in the presence of dif-
ferent inhibitor concentrations, monitoring continuously the
release of p-nitrophenol at 400 nm. For both compounds we found
that initial rates decrease with increasing inhibitor concentrations,
and curves maintain linear monophasic behaviors as time pro-
gresses, indicating that the rapid equilibrium conditions occur in
the formation of enzyme-inhibitor complexes. Identical behavior
was found pre-incubating PTP-1D with inhibitors for 30 min before
the activity assay (data not shown). Taken together our results sug-
gest that both association and dissociation of inhibitors to the en-
zyme are rapid events, excluding the hypothesis that these
compounds are slow-binding inhibitors.
of each inhibitor examined since IC50 is greater than fivefold over
enzyme concentration (10 nM in all assays) and the approximation
that [inhibitor] free = [inhibitor] total is valid.
To verify if some compounds act as partial inhibitors, we mea-
sured therate of PTP-1D-catalyzed hydrolysis of p-nitrophenylphos-
phate at high substrate concentration (12.5-fold the Km of the
enzyme) in the presence of increasing concentrations of each com-
pound. The results obtained for compounds 4 and 16 are reported
in Figure 4. We observed that, by increasing the inhibitor concentra-
tion, the hydrolysis rate is reduced near to zero, suggesting that the
binding of inhibitor to enzyme produces an inactive EI complex.
To determine the kinetic mechanism of inhibition, we analyzed
experimental data by the double reciprocal plot method. For each
compound, we measured the initial hydrolysis rate using a range
of substrate final concentrations (0.5–40 mM), in the presence of
increasing concentrations of each compound (Figs. 5–8).
The reciprocal plots for 4 and 16 showed straight lines that
intersect each other in the left panel, suggesting that they are
mixed-type inhibitors. This is confirmed by the fact that increasing
inhibitor concentration is accompanied by the increase of Km val-
ues and the decrease of Vmax values (see Figs. 5–8). Wiesmann
et al. reported a study on the inhibition of PTP-1B with a similar
class of compounds. They also observed a similar inhibition type.14
For both mixed-type inhibitors we considered the model:
Figures 3A and B show the concentration–response plots by
compound 4 and compound 16, respectively. The calculated IC50
values reported in Table 2 are in the low lM range. Thus our assay
system is operated under non-tight binding conditions in the case
Ks
k2
+
+
P
E
+
I
S
ES
+
E
120
100
80
60
40
20
0
I
Ki
αKi
EI +
S
ESI
αKs
Thus we expected that the inhibitor binds to the free enzyme, E,
and to the enzyme-substrate complex, ES. In this model EI has low-
er affinity for S than E (a > 1), and ESI is non productive. We calcu-
lated the value of Ki (mixed-type) replotting the slope of the
straight lines of the double reciprocal plot versus [I]. The results
obtained in this experiment are reported in Table 3.
DMSO
4
16
2.3. Docking studies with PTP-1D
Figure 2. Inhibition reversibility assay. Aliquots of PTP-1B were incubated in the
presence of 100 M of each compound for 1 h at 4 °C (black bar) or 37 °C (red bar).
Then, the enzyme was diluted 400-fold with the assay solution to measure the
residual activity (37 °C, 5 mM pNPP). Control experiments were carried out adding
DMSO. All tests were performed in triplicate. The data represent the mean S.E.M.
l
In order to gain an insight into the binding mode of 4 and 16,
compounds were docked into the ligand binding pocket of human
PTP-1D (PDB entry 2F71).15 Docking was performed with the pro-

G. Navarrete-Vazquez et al. / Bioorg. Med. Chem. 17 (2009) 3332–3341
3335
1.2
1.2
1.0
0.8
0.6
0.4
0.2
0.0
A
B
1.0
0.8
0.6
0.4
0.2
0.0
0.01
0.1
1
10
100
1000
1E-3
0.01
0.1
1
10
100
1000
[Compound 4] μM
[Compound 16] μM
Figure 3. The IC50 values were determined by plotting the relative activity of PTP-1B versus inhibitor concentration (4 graph A; 16 graph B). For each inhibitor, 15–18
different inhibitor concentrations were used. All tests were conducted in quadruplicate. The data represent the means S.E.M.
gram AUTODOCK 3.016 using an automated flexible protocol as de-
Table 2
IC50 values for compounds 4, and 16
scribed in Section 4. Before docking 4 and 16, the docking protocol
was validated predicting the binding mode of the crystallographic
1,2,3,4-tetrahydroisoquinolinyl sulfamic acid.15 AUTODOCK success-
fully predicted the binding mode of crystallographic ligand with
RMS deviation of 1.09 Å.
Compound
IC50 (lM)
4
16
19.5 1.0
40.9 2.8
After docking 4 and 16, the binding pockets of the 2-arylsulfo-
nylamino benzothiazoles were fully optimized. Figures 9A and B
depict the top ranked binding modes after energy minimization.
Figures 10A and B show the corresponding interaction diagram
of the optimized models generated with the program Molecular
Operating Environment (MOE).17 According to the optimized com-
plexes, the residues Tyr46, Arg47, Val49, Asp181, Phe182, Cys215,
Ser216, Ala217, Arg221 and Gln262 are common to the binding
pocket of 4 and 16. Interestingly, Asp181, Cys215 and Arg221 are
catalytic residues.18 In both molecules, the nitro group at the aryl-
sulfonylamino moiety is directed to the catalytic residues and it is
immersed in a highly positive region.19
2.4. In vivo antihyperglycemic activity of 4 and 16
Compounds 4 and 16 were evaluated for in vivo antihyperglyce-
mic activity using a STZ-nicotinamide rat model of T2DM.21
Glibenclamide was taken as positive control.7 The antihyperglyce-
mic activity of both compounds was determined using a 100 mg/kg
single dose. Compound 4 demonstrated important antihyperglyce-
mic activity, by lowering glycemia ranging from 27% to 35%. The
effect was consistent during the 7 h of experiment (Fig. 11). Com-
pound 16 showed similar effects than glibenclamide, lowering the
glycemia until 60% in this model of T2DM. The most pronounced
effect of both compounds was observed during the first 5 h of
post-intragastric administration of tested compounds, maintaining
it until the 7th-h. These behavior could be related to pancreatic
effects.
Figure 4. 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 lM. The symbols are: 4, j; 16, s. All tests were performed in
quadruplicate. The data represent the mean S.E.M.
3. Conclusion
We have synthesized a small library of 2-arylsulfonylamino-
benzothiazoles and evaluated these compounds for their protein
tyrosine phosphatase-1D inhibitory activity. Several compounds of
this series have shown significant PTP-1D inhibitory activity, in par-
ticular those that bear nitro group at position 4 of the sulfonamide
moiety. Compounds 4 and 16 have exhibited most promising activ-
ity as mixed-type inhibitors of PTP-1D. The in vivo anti-hyperglyce-
mic activities of these compounds make them a suitable leads to
develop new chemical entities for potential use in the treatment of
T2DM.
Figure 5. Compound 4: double reciprocal plot (1/v vs 1/[S]). The concentrations of
inhibitor were: 0
lM, h; 8 lM, d; 12 lM, 4; 16 lM, .; 20 lM, s. All tests were
conducted in quadruplicate. The data represent the means S.E.M.

3336
G. Navarrete-Vazquez et al. / Bioorg. Med. Chem. 17 (2009) 3332–3341
Figure 6. Dependence of main kinetic parameters Km and Vmax from the concentration of compound 4. The concentrations of inhibitor used were: 0, 8, 12, 16 and 20
data represent the Km or Vmax values S.E.M.
lM. The
Table 3
The inhibition constants for compounds 4 and 16
Compound
Ki (l
M)
a
Inhibition type
4
16
7.1 1.0
20.4 3.7
4.8
2.5
Linear mixed-type
Linear mixed-type
The data represent Ki values S.E.M.
ments. Chemical shifts are given in ppm relative to tetramethylsil-
ane (Me4Si, d = 0) in DMSO-d6; J values are given in hertz. The
following abbreviations are used: s, singlet; d, doublet; q, quartet;
dd, doublet of doublet; t, triplet; m, multiplet; br s, broad signal.
MS were recorded on a JEOL JMS-700 spectrometer by Fast Atom
Bombarded [FAB (+)]. Predictive values of antidiabetic and antiobe-
sity activities were also investigated using the chemistry software
server PASS (http://195.178.207.233/PASS/).
Starting materials 6-substituted-1,3-benzothiazol-2-amines
(28–33), and 4-substituted-benzenesulfonyl chlorides (34–40),
were commercially available from Aldrich and used without
purification.
Figure 7. Compound 16: double reciprocal plot (1/v vs 1/[S]). The concentrations of
inhibitor used were: 0 M, d; 5 M, s; 10 M, j; 20 M, h; 30
were conducted in quadruplicate. The data represent the means S.E.M.
l
l
l
l
lM, N. All tests
4. Experimental
4.1. Chemistry
4.1.1. General method of synthesis of derivatives 1–27
To
a solution of 4-substituted benzenesulfonyl chloride
(0.006 mol, 1.2 equiv) were added triethylamine (1.1 equiv), and
a catalytic amount of 4-dimethylaminopyridine (DMAP) The reac-
tion mixture was stirred at room temperature for 15 min. After, a
solution of 2-amino-6-substituted benzothiazole (0.0030 mol)
was added droopingly. The reaction mixture was stirred at 40 °C
for 6–10 h. After complete conversion as indicated by TLC, the sol-
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 INOVA 4008 (200 MHz) and
13C NMR (50.28 MHz) instrument, and Varian Mercury 200 instru-
Figure 8. Dependence of main kinetic parameters Km and Vmax from the concentration of compound 16. The concentrations of inhibitor used were: 0, 5, 10, 20, and 30
The data represent the Km or Vmax values S.E.M.
lM.

G. Navarrete-Vazquez et al. / Bioorg. Med. Chem. 17 (2009) 3332–3341
3337
Figure 9. Predicted binding mode of 4 (A) and 16 (B), into the catalytic site of PTP-1D. Amino acid residues common to the binding pocket of both molecules (within 4.5 Å) are
labeled. Figure generated with the program VMD 1.8.6.20
Figure 10. Two-dimensional interaction map of 4 (A) and 16 (B), docked into the PTP-1D catalytic pocket. Amino acid residues at 4.5 Å of the ligand are shown.
70
70
60
50
40
30
20
60
50
40
30
20
ISS
ISS
16
Glibenclamide
4
Glibenclamide
10
10
0
0
-10
-20
-30
-40
-50
-60
-70
-80
-10
-20
-30
-40
-50
-60
-70
-80
*
*
*
*
*
*
*
*
-1
0
1
2
3
4
5
6
7
8
-1
0
1
2
3
4
5
6
7
8
Time after administration (h)
Time after administration (h)
*
Figure 11. Effect of a single dose of 4 and 16 (100 mg/kg; intragastric, n = 5) in streptozotocin-nicotinamide rat model of diabetes. ISS: Isotonic saline solution. p < 0.05
versus ISS group.

3338
G. Navarrete-Vazquez et al. / Bioorg. Med. Chem. 17 (2009) 3332–3341
vent was removed in vacuo, the residue was neutralized with sat-
urated NaHCO3 solution, and the aqueous layer was extracted with
ethyl acetate (3 ꢀ 15 mL), washed with water (3 ꢀ 20 mL), and
dried over anhydrous Na2SO4. The solvent was evaporated in vacuo
and the precipitated solids were recrystallized from an appropriate
solvent or purified by column chromatography.
266.9 °C. 1H NMR (200 MHz, DMSO-d6): d 7.2 (s, 1H, H-7), 7.6 (m,
4H, H-20, H-30, H-50, H-60), 7.9 (m, 2H, H-4, H-5) ppm. 13C NMR
(50.28 MHz, DMSO-d6): d 20.8 (CH3), 112.5 (C-7), 122.4 (C-4),
124.7 (C-7a), 127.7 (C-20, C-60), 128.1 (C-5), 129.1 (C-30, C-50),
133.3 (C-6), 133.9 (C-40), 137.0 (C-3a), 140.8 (C-10), 166.9 (C-2)
ppm. MS (FAB+): m/z 339 (M+H)+; Anal. Calcd for C14H11ClN2O2S2:
C, 49.63; H, 3.27; N, 8.27. Found: C, 49.02; H, 3.13; N, 8.26.
4.1.1.1. N-(6-Methyl-1,3-benzothiazol-2-yl) benzenesulfon-
amide (1). White solid, yield 0.48 g (26.0%). Mp 254.7–257.6 °C.
1H NMR (200 MHz, DMSO-d6): d 2.3 (s, 3H, CH3), 7.2 (s, 2H, H-30,
H-50), 7.5–7.6 (m, 3H, H7, H20,H60, J = 2.0, 5.8), 7.8–7.9 (br s, 2H,
H-4, H-50) ppm. 13C NMR (50.28 MHz, DMSO-d6):d 20.8 (CH3),
112.4 (C-7), 122.4 (C-4), 124.7 (C-7a), 125.7 (C-20, C60), 128.0 (C-
5), 129.0 (C-30, C-50), 132.3 (C-40), 133.1 (C-6), 133.9 (C-10), 141.9
(C-3a), 166.7 (C-2) ppm; MS (FAB+): m/z 305 (M+H)+; Anal. Calcd
for C14H12N2O2S2: C, 55.24; H, 3.97; N, 9.20. Found: C, 55.18; H,
4.08; N, 8.87.
4.1.1.7. N-(6-Methoxy-1,3-benzothiazol-2-yl) benzenesulfon-
amide (7). White solid, Yield 0.69 g (39.4%) of a white solid. Mp
252.9–253.2 °C 1H NMR (200 MHz, DMSO-d6): d 6.9 (dd, 1H, H-5,
J = 8.8, J = 2.2 Hz), 7.2 (d, 1H, H-4, J = 8.8 Hz), 7.4 (d, 1H, H-7,
J = 2.2 Hz), 7.4–7.6 (m, 3H, H-0, H-40, H-50), 7.8 (m, 2H, H-20, H-60)
ppm. 13C NMR (50.28 MHz, DMSO-d6): d 55.7 (CH3O), 106.9 (C-7),
113.4 (C-5), 114.6 (C-4), 125.6 (C-20, C-60), 125.9 (C-7a), 129.1 (C-
30, C-50), 129.7 (C-40), 132.2 (C-10), 142.0 (C-4a), 155.9 (C-6),
166.4 (C-2). MS (FAB+): m/z 321 (M+H)+; Anal. Calcd for
C14H12N2O3S2: C, 52.48; H, 3.78; N, 8.74. Found: C, 52.29; H,
3.65; N, 8.98.
4.1.1.2. 4-Methyl-N-(6-methyl-1,3-benzothiazol-2-yl)benzene-
sulfonamide (2). White solid, yield 0.73 g (37.7%) Mp 235.9 (dec)
°C. 1H NMR (200 MHz, DMSO-d6): d 2.3 (s, 3H, CH3), 2.3 (s, 3H, CH3),
7.1–7.2 (m, 2H, H-30, H-50, J = 1.2, 8.0), 7.1 (d, 2H, H20, H60, J = 7.6),
7.5 (d, 2H, H-4, H-5 J = 8.0) ppm. 13C NMR (50.28 MHz, DMSO-d6): d
20.4 (CH3), 20.6 (CH3),106.5 (C-7), 112.9 (C-4), 121.6 (C-7a), 125.4
(C-20, C-60), 125.5 (C-5), 128.9 (C-30, C-50), 131.9 (C-10), 136.12 (C-
6), 139.4 (C-40), 141.6 (C-3a), 166.1 (C-2) ppm; MS(FAB+): m/z
319 (M+H)+; Anal. Calcd for C15H14N2O2S2: C, 56.58; H, 4.43; N,
8.80. Found: C, 57.10; H, 4.69; N,9.86.
4.1.1.8. N-(6-Methoxy-1,3-benzothiazol-2-yl)-4-methylbenzene-
sulfonamide (8). Beige solid, yield 0.61 g (33.3%) Mp 215.2–
216.9 °C. 1H NMR (200 MHz, DMSO-d6): d 2.3 (s, 3H, CH3), 3.8 (s,
3H, OCH3), 6.9 (dd, 1H, H5, J = 2.6, 8.8 Hz), 7.2 (d, 1H, H4,
J = 8.8 Hz), 7.3 (d, 2H, H30, H50, J = 8.0 Hz), 7.4 (d, 1H, H7,
J = 2.6 Hz), 7.7 (d, 2H, H20, H60, J = 8.4 Hz) ppm. 13C NMR
(50.28 MHz, DMSO-d6): d 20.9 (CH3), 55.7 (OCH3), 106.9 (C-7),
113.4 (C-5), 114.6 (C-4), 125.7 (C-20, C-60), 125.9 (C-7a), 129.4 (C-
30, C-50), 129.8 (C-10), 139.2 (C-3a), 142.4 (C-40), 155.9 (C-6), 166.2
(C-2) ppm; MS(FAB+): m/z 335 (M+H)+; Anal. Calcd for
C15H14N2O3S2: C, 53.87; H, 4.22; N, 8.38. Found: C, 53.63; H, 4.11;
N, 8.00.
4.1.1.3. 4-Methoxy-N-(6-methyl-1,3-benzothiazol-2-yl)benzene-
sulfonamide (3). White solid, yield 1.08 g (48.8%) Mp 260.2–
262.3 °C. 1H NMR (200 MHz, DMSO-d6): d 2.3 (s, 3H, CH3), 3.8 (s,
3H, –OCH3), 7.1 (d, 2H, H-30, H-50, J = 8.0), 7.2 (m, 2H, H20, H60), 7.6
(br s, 1H, H-7), 7.8 (d, 2H, H4, H5, J = 8.8) ppm. 13C NMR
(50.28 MHz, DMSO-d6): d 20.4 (CH3), 55.2 (OCH3), 111.9 (C-7),
113.7 (C-30, C-50) 121.9 (C-4), 124.3 (C-7a), 127.5 (C-20, C-60), 127.6
(C-5), 132.6 (10), 133.4 (C-6), 133.6 (C-3a), 161.6 (C-40), 165.9 (C-2)
ppm; MS(FAB+): m/z 335 (M+H)+; Anal. Calcd for C15H14N2O3S2: C,
53.87; H, 4.24; N, 8.38. Found: C, 53.51; H, 4.35; N, 8.24.
4.1.1.9. 4-Methoxy-N-(6-methoxy-1,3-benzothiazol -2-yl)ben-
zenesulfonamide (9). Beige solid, yield 1.34 g (69.3%) Mp 230.7–
232.9 °C. 1H NMR (200 MHz, DMSO-d6): d 3.8 (s, 3H, OCH3), 3.8 (s,
3H, OCH3), 6.9 (dd, 1H, H5, J = 2.2, 8.8 Hz), 7.0–7.1 (m, 2H, H30, H50,
J = 1.8, 7.0 Hz), 7.2 (d, 1H, H4, J = 8.8 Hz), 7.4 (d, 1H, H7, J = 2.2 Hz),
7.7–7.8 (m, 2H, H20, H60, J = 2.2, 7.0 Hz) ppm. 13C NMR (50.28 MHz,
DMSO-d6): d 65.2 (OCH3), 65.3 (OCH3), 116.6 (C-7), 122.9 (C-5),
123.8 (C-30, C-50), 124.2 (C-4), 135.5 (C-7a), 137.4 (C-20, C-60),
139.4 (C-10), 143.5 (C-3a), 165.5 (C-6), 171.6 (C-40), 175.6 (C-2)
ppm; MS(FAB+): m/z 351 (M+H)+; Anal. Calcd for C15H14N2O4S2:
C, 51.14; H, 4.03; N, 7.99. Found: C, 50.99; H, 4.190; N, 7.91.
4.1.1.4. N-(6-Methyl-1,3-benzothiazol-2-yl)-4-nitro benzenesul-
fonamide (4). Beige solid, yield 1.92 g (90.1%) of beige solid. Mp
226.3–227.5 °C. 1H NMR (200 MHz, DMSO-d6): d 2.3 (s, 3H, CH3),
7.2 (s, 2H, H-4, H-5), 7.6 (s, 1H, H-7), 8.1 (d, 2H, H-20, H-60,
J = 8.8 Hz), 8.3 (d, 2H, H-30, H-50, J = 8.8 Hz) ppm. 13C NMR
(50.28 MHz, DMSO-d6): d 20.7 (CH3), 112.8 (C-7), 122.4 (C-4),
124.4 (C-30, C-50), 124.9 (C-7a), 127.2 (C-20, C-60), 128.2 (C-5),
133.4 (C-6), 134.1 (C-10), 147.3 (C-3a), 149.3 (C-40), 167.3 (C-2)
ppm; MS (FAB+): m/z 350 (M+H)+; Anal. Calcd for C14H11N3O4S2:
C, 48.13; H, 3.17; N, 12.03. Found: C, 47.38; H, 3.13; N, 12.16.
4.1.1.10. N-(6-Methoxy-1,3-benzothiazol-2-yl)-4-nitrobenzene-
sulfonamide (10). Yield 1.33 g (66.1%) of a yellow solid. Mp
236.9–238.2 °C. 1H NMR (200 MHz, DMSO-d6):
d 3.8 (s, 3H,
CH3O), 7.0 (dd, 1H, H-5 J = 8.8, J = 2.2 Hz), 7.2 (d, 1H, H-4,
J = 8.8 Hz), 7.5 (d, 1H, H-7, J = 2.2 Hz), 8.1 (d, 2H, H-20, H-60,
J = 8.4 Hz), 8.4 (d, 2H, H-30, H-50, J = 8.8 Hz) ppm; 13C NMR
(50.28 MHz, DMSO-d6) d 55.7 (CH3), 106.9 (C-7), 113.8 (C-5),
114.9 (C-4), 124.4 (C-30, C-50), 126.0 (C-7a), 127.2 (C-20, C-60),
129.7 (C-3a), 147.3 (C-10), 149.3 (C-40), 156.2 (C-6), 167.0 (C-2)
ppm; MS (FAB+): m/z 366 (M+H)+; Anal. Calcd for C14H11N3O5S2:
C, 46.02; H, 3.03; N, 11.50;. Found: C, 45.75; H, 2.93; N, 11.77.
4.1.1.5. N-(4-{[(6-Methyl-1,3-benzothiazol-2-yl) amino]sulfo-
nyl}phenyl)acetamide (5). White solid, yield 1.15 g (52.2%)
Mp 276.3–278.6 °C. 1H NMR (200 MHz, DMSO-d6): d 2.1 (s,
3H, NHCOCH3), 3.4 (s, 3H, CH3), 3.7 (s, 1H, NH), 7.3–7.4 (dd,
1H, H-5, J = 1.2, 8.8 Hz), 7.5–7.8 (m, 5H, H20, H-30, H-50, H-60,
H-7), 8.2 (d, 1H, H-4, J = 8.8) ppm. 13C NMR (50.28 MHz,
DMSO-d6): d 20.5 (NHCOCH3), 24.3 (CH3), 115.5 (C-7), 118.1
(C-4), 118.2 (C-30, C-50) 122.7 (C-7a), 127.1 (C-20, C-60), 129.9
(C-5), 135.4 (C-10), 143.4 (C-40), 145.4 (C-3a), 168.8 (C-2),
169.1 (NHCOCH3), ppm; MS (FAB+): m/z 362 (M+H)+; Anal.
Calcd for C16H15N3O3S2: C, 53.17; H, 4.18; N, 11.63. Found:
C, 51.13; H, 4.30; N, 11.00.
4.1.1.11. N-(4-{[(6-Methoxy-1,3-benzothiazol-2-yl)amino]sulfo-
nyl}phenyl)acetamide (11). Beige solid, yield 1.57 g (75.6%) Mp
245.3–249.9 °C. 1H NMR (200 MHz, DMSO-d6): d 2.1 (s, 3H, NHC-
OCH3), 3.8 (s, 3H, OCH3), 5.8 (s, 1H, NH), 7.0 (dd, 1H, H5, J = 2.6,
8.8 Hz), 7.2 (d, 1H, H4, J = 8.8 Hz), 7.4 (d, 1H, H7, J = 2.4 Hz), 7.5–
7.8 (m, 4H, H20, H30, H50, H60, J = 1.8, 2.6, 9.2 Hz), 10.3 (s, 1H, NHC-
OCH3) ppm. 13C NMR (50.28 MHz, DMSO-d6): d 24.1 (NHCOCH3),
55.7 (OCH3), 107.0 (C-7), 113.4 (C-5), 114.6 (C-4), 118.4 (C-30, C-
50), 126.0 (C-7a), 126.9 (C-20, C-60), 129.8 (C-10) 135.7 (C-40),
4.1.1.6. 4-Chloro-N-(6-methyl-1,3-benzothiazol-2-yl)benzene-
sulfonamide (6). Yield 5.33 g (85.9%) of a white solid. Mp 265.6–

G. Navarrete-Vazquez et al. / Bioorg. Med. Chem. 17 (2009) 3332–3341
3339
142.6 (C-3a), 155.9 (C-6), 166.1 (NHCOCH3), 168.8 (C-2) ppm;
MS(FAB+): m/z 378 (M+H)+; Anal. Calcd for C16H15N3O4S2: C,
50.91; H, 4.01; N, 11.13. Found: C, 49.42; H, 4.21; N, 10.19.
4.1.1.17. N-(4-{[(6-Ethoxy-1,3-benzothiazol-2-yl)amino]sulfo-
nyl}phenyl)acetamida (17). White solid, yield 0.88 g (40.3%) Mp
262.3–263.6 °C. 1H NMR (200 MHz, DMSO-d6):
1.3 (t, 3H,
d
OCH2CH3), 2.1 (t, 3H,NHCOCH3), 4.0 (q, 2H, OCH2CH3), 5.8 (s, 1H,
NH), 6.9 (dd, 1H, H-5, J = 2.4, 8.8 Hz), 7.2 (d, 1H, H-4, J = 8.8), 7.4
(d, 1H, H-7, J = 2.2 Hz), 7.6–7.8 (m, 4H, H-20, H-30, H-50, H-60,
J = 3.2, 8.8 Hz), 10.3 (s, 1H, NH) ppm. 13C NMR (50.28 MHz,
DMSO-d6): d 24.1 (OCH2CH3), 30.7 (NHCOCH3), 63.7 (OCH2CH3),
107.5 (C-7), 113.5 (C-5), 115.0 (C-4), 118.4 (C-30, C-50), 126.9 (C-
30, C-50), 130.0 (C-7a), 135.8 (C-10), 142.6 (C-3a),142.8 (C-40),
155.1 (C-6), 166.1 (C-2), 168.8 (NHCOCH3) ppm; MS(FAB+): m/z
392 (M+H)+; Anal. Calcd for C17H17N3O4S2: C, 52.16; H, 4.38; N,
10.73. Found: C, 51.29; H, 4.51; N, 10.22.
4.1.1.12. 4-Chloro-N-(6-methoxy-1,3-benzothiazol-2-yl)benzene-
sulfonamide (12). Beige solid, yield 0.99 g (51.0%) Mp 162.1–
163.8 °C. 1H NMR (200 MHz, DMSO-d6): d 3.8 (s, 3H, OCH3), 7.0
(dd, 1H, H-5, J = 2.6, 8.8 Hz), 2.3 (d, 1H, H-4, J = 8.8 Hz), 7.3 (d,
1H, H-7, J = 2.6 Hz), 7.6–7.7 (m, 2H, H-20, H-60), 7.8–7.9 (m, 2H,
H-30, H-50) ppm. 13C NMR (50.28 MHz, DMSO-d6): d 55.7 (OCH3),
106.9 (C-7), 113.7 (C-5), 114,7 (C-4), 127.6 (C-20, C-60), 129.1 (C-
30, C-50), 129.9 (C-7a), 137.0 (C-40), 140.9 (C-3a), 156.0 (C-6),
166.5 (C-2) ppm; MS(FAB+): m/z 355 (M+H)+; Anal. Calcd for
C14H11N2O3S2Cl: C,47.39; H, 3.12; N, 8.89. Found: C, 47.68; H,
3.31; N, 8.90.
4.1.1.18. 4-Chloro-N-(6-ethoxy-1,3-benzothiazol-2-yl)benzene-
sulfonamide (18). White solid, yield 1.75 g (85.01%) Mp 151.2–
153.8 °C. 1H NMR (200 MHz, DMSO-d6): d 3.0 (s, 3H, OCH2CH3),
4.0 (q, 2H, OCH2CH3), 7.0 (dd, 1H, H-5, J = 2.6, 7.8 Hz) 7.3 (d,
1H, H-4, J = 8.8 Hz), 7.4 (s, 1H, H-5), 7.6 (d, 2H, H-20, H-60,
J = 8.8 Hz), 7.8 (d, 2H, H-30, H-50, J = 7.8 Hz) ppm. 13C NMR
(50.28 MHz, DMSO-d6) d: 14.6 (OCH2CH3), 63.7 (OCH2CH3),
107.5 (C-7), 113.7 (C-5), 115.1 (C-4), 125.9 (C-7a), 127.6 (C-20,
C-60),129.1 (C-30y C-50), 136.9 (C-40), 140.9 (C-3a), 155.24 (C-6),
166.4 (C-2) ppm; MS(FAB+): m/z 369 (M+H)+; Anal. Calcd for
C15H13N2O3S2Cl: C, 48.84; H, 3.55; N, 7.59. Found: C, 48.32; H,
3.56; N, 7.45.
4.1.1.13. N-(6-Ethoxy-1,3-benzothiazol-2-yl)benzenesulfon-
amide (13). White solid, yield 0.39 g (41.2%) Mp 270.0–271.0 °C.
1H NMR (200 MHz, DMSO-d6): d 1.2 (s, 3H, CH3), 3.9 (q, 2H,
OCH2CH3), 5.7 (s, 1H, NH), 6.9 (dd, 1H, H-5, J = 2.6, 8.8 Hz), 7.1 (d,
1H, H-4, J = 8.8), 7.3 (d, 1H, H-7, J = 2.2 Hz), 7.4–7.5 (m, 3H, H-30,
H-40, H-50), 7.7–7.8 (m, 2H, H-20, H-60, J = 1.4, 8.0 Hz) ppm. 13C
NMR (50.28 MHz, DMSO-d6): d 14.6 (OCH2CH3), 63.7 (OCH2CH3),
107.6 (C-7), 113.5 (C-5), 115.1 (C-4), 125.7 (C-20, C-60), 125.9 (C-
7a), 129.0 (C-30, C-50), 129.7 (C-40), 132.2 (C-10), 142.0 (C-3a),
155.2 (C-6), 166.4 (C-2) ppm; MS(FAB+): m/z 335 (M+H)+; Anal.
Calcd for C15H14N2O3S2: C, 53.87; H, 4.22; N, 8.38. Found: C,
53.67; H, 4.15; N, 8.56.
4.1.1.19. benzenesulfonamide (19)(19). Yellow solid, yield
0.64 g (37.6%) Mp 241.5–248.9 °C. 1H NMR (200 MHz, DMSO-d6):
d 7.4 (d, 1H, H4, J = 9.2 Hz), 7.5–7.6 (m, 3H, H30, H40, H50), 7.8–7.9
(m, 2H, H20, H60), 8.2 (dd, 1H, H5, J = 2.6, 8.8 Hz), 8.8 (d, 1H, H7,
J = 1.8 Hz) ppm. 13C NMR (50.28 MHz, DMSO-d6): d 112.8 (C-7),
119.3 (C-5), 123.2 (C-4), 125.8 (C-20, C-60), 126.7 (C-7a), 129.2 (C-
30, C-50), 132.7 (C-40) 141.3 (C-10), 141.6 (C-3a), 142.9 (C-6), 168.3
(C-2) ppm; MS(FAB+): m/z 336 (M+H)+; Anal. Calcd for
C13H9N3O4S2: C, 46.56; H, 2.70; N, 12.53. Found: C, 46.26; H,
2.90; N, 11.89.
4.1.1.14. N-(6-Ethoxy-1,3-benzothiazol-2-yl)-4-methylbenzene-
sulfonamide (14). Yield 0.92 g (47.1%) of beige solid. Mp 228.4–
230.3 °C. 1H NMR (200 MHz, DMSO-d6): d 1.3 (t, 3H, CH3), 2.3 (s,
3H, CH3O), 4.0 (q, 2H, CH2O), 6.9 (dd, 1H, H-5, J = 8.8. J = 2.6 Hz),
7.2 (d, 1H, H-4, J = 8.8 Hz), 7.3 (d, 2H, H-30, H-50, J = 8.0 Hz), 7.4
(d, 1H, H-7, J = 2.6 Hz), 7.7 (d, 2H, H-30, H-70, J = 8.2 Hz) ppm; 13C
NMR (50.28 MHz, DMSO-d6) d 14.3 (CH3), 20.6 (CH3), 63.3 (CH2),
107.1 (C-7), 113.1 (C-4), 114.6 (C-7a), 125.3 (C-20, C-60), 125.5 (C-
5), 129.0 (C-30, C-50), 130.0 (C-6), 138.8 (C-10), 142.0 (C-40), 154.7
(C-3a), 165.8 (C-2) ppm. MS (FAB+): m/z 349 (M+H)+; Anal. Calcd.
for C16H16N2O3S2: C, 55.15; H, 4.63; N, 8.04. Found: C, 54.20; H,
4.40; N, 8.04.
4.1.1.20. 4-Methyl-N-(6-nitro-1,3-benzothiazol-2-yl)benzene-
sulfonamide (20). Yellow solid, yield 0.34 g (18.8%) Mp 178.1–
179.6 °C. 1H NMR (200 MHz, DMSO-d6): d 2.1 (s, 3H, CH3), 5.8 (s,
1H, N-H), 7.3 (d, 1H, H-4, J = 8.4 Hz), 7. 5 (dd, 1H, H-5, J = 2.2,
8.4 Hz), 7.9 (d, 1H, H-7, J = 1.8 Hz), 8.1 (d, 2H, H-30, H-50, J =
8.8 Hz), 8.4 (d, 2H, H20, H60, J = 8.8 Hz) ppm. 13C NMR
(50.28 MHz, DMSO-d6): d 20.7 (CH3), 114.4 (C-7), 122.4 (C-4),
124.5 (C-20, C-60), 126.8 (C-5), 127.3 (C-30, C-50), 127.4 (C-7a),
127.9 (C-10), 135.3 (C-6), 147.1 (C-40), 149.4 (C-3a), 167.8 (C-2)
ppm; MS(FAB+): m/z 350 (M+H)+; Anal. Calcd for C14H11N3O4S2:
C, 48.13; H, 3.17; N, 12.03. Found: C, 49.00; H, 3.17; N, 12.00
4.1.1.15. N-(6-Ethoxy-1,3-benzothiazol-2-yl)-4-methoxybenzene-
sulfonamide (15). Beige solid, yield 1.5 g (46.5%) Mp 220.3–
222.2 °C. 1H NMR (200 MHz, DMSO-d6): d 1.3 (t, 3H, OCH2CH3),
3.8 (s, 3H, OCH3), 4.0 (q, 2H, OCH2CH3), 6.9 (dd, 1H, H5, J = 2.2,
8.8 Hz), 7.1 (d, 2H, H30, H50, J = 8.8 Hz), 7.2 (d, 1H, H4, J = 8.8 Hz),
7.4 (d, 1H, H7, J = 2.4 Hz), 7.8 (d, 2H, H20, H60, J = 8.4 Hz) ppm. 13C
NMR (50.28 MHz, DMSO-d6): d 14.6 (OCH2CH3), 55.6 (OCH3), 63.7
(OCH2CH3), 107.5 (C-7), 113.4 (C-5), 114.1 (C-30, C-50), 115.0 (C-
4), 125.9 (C-7a), 127.8 (C-20, C-60), 133.9 (C-10), 155.1 (C-3a),
161.9 (C-6), 166.0 (C-40), 186.2 (C-2) ppm; MS(FAB+): m/z 366
(M+H)+; Anal. Calcd for C16H16N2O4S2: C, 52.73; H, 4.43; N, 7.69.
Found: C, 52.41; H, 4.56, N,7.50.
4.1.1.21. 4-Methoxy-N-(6-nitro-1,3-benzothiazol-2-yl)benzene
sulfonamide (21). Yield 0.63 g (33.8%) of yellow solid. Mp 187.1–
189.3 °C. 1H NMR (200 MHz, DMSO-d6): d 3.8 (s, 3H, CH3O), 7.1 (d,
2H, H-30, H-50, J = 8.0), 7.8 (d, 2H, H20, H60, J = 8.4), 8.2 (sa, 2H, H-4,
H-5), 8.8 (s, 1H, H-7) ppm. 13C NMR (50.28 MHz, DMSO-d6): d 55.7
(CH3), 114.3 (C-30, C-50), 119.1 (C-7), 123.1 (C-5), 126.3 (C-4), 126.9
(C-7a), 127.9 (C-20, C-60), 133.1 (C-10), 138.9 (C-6), 142.8 (C-3a),
162.3 (C-40), 167.8 (C-2) ppm; MS (FAB+): m/z 366 (M+H)+; Anal.
Calcd for C14H11N3O5S2: C, 46.02; H, 3.03; N, 11.50. Found: C,
46.49; H, 3.83; N, 10.94.
4.1.1.16.
N-(6-Ethoxy-1,3-benzothiazol-2-yl)-4-nitrobenzene
sulfonamide (16). Yield 1.51 g (71.2%) of a yellow solid. Mp
247.9–248.9 °C. 1H NMR (200 MHz, DMSO-d6): d 1.3 (t, 3H, CH3),
4.0 (q, 2H, CH2O), 7.0 (dd, 1H, H-5, J = 8.8, J = 2.6 Hz), 7.2 (d, 1H,
H-4, J = 8.8 Hz), 7.5 (d, 1H, H-7, J = 2.2 Hz), 8.0–8.1 (m, 2H, H-20,
H-60), 8.3- 8.4 (m, 2H, H-30, H-50) ppm; 13C NMR (50.28 MHz,
DMSO-d6) d 14.3 (CH3), 63.4 (CH2O), 107.2 (C-4), 113.5 (C-7),
124.1 (C-30, C-50), 126.9 (C-20, C-60), 129.3 (C-5), 147.0 (C-7a),
148.9 (C-6), 155.1 (C-10), 166.7 (C-40), 171.9 (C-3a), 172.8 (C-2)
ppm; MS (FAB+): m/z 380 (M+H)+; Anal. Calcd For C15H13N3O5S2:
C, 47.48; H, 3.45; N, 11.08. Found: C, 46.88; H, 3.33; N, 11.26.
4.1.1.22. 4-Nitro-N-(6-nitro-1,3-benzothiazol-2-yl)benzenesul-
fonamide (22). Yellow solid, yield 0.92 g (47.4%) Mp 203.1–
205.4 °C. 1H NMR (200 MHz, DMSO-d6):
d 7.5 (d, 1H, H-5,
J = 8.8 Hz), 8.1 (d, 2H, H-20, H-60, J = 8.4 Hz), 8.3 (d, 1H, H-4,
J = 8.8 Hz), 8.4 (d, 2H, H-30, H-50, J = 8.4 Hz), 8.9 (s, 1H, H-7) ppm.

3340
G. Navarrete-Vazquez et al. / Bioorg. Med. Chem. 17 (2009) 3332–3341
13C NMR (50.28 MHz, DMSO-d6): d 113.3 (C-7), 119.4 (C-4), 123.3
(C-5), 124.6 (C-30, C-50), 126.3 (C-7a), 127.4 (C-20, C-60), 141.8
(C-6), 143.2 (C-10), 146.7 (C-40), 149.6 (C-3a), 169.3 (C-2) ppm;
MS(FAB+): m/z 381 (M+H)+; Anal. Calcd for C13H8N4O6S2:
C, 41.05; H, 2.12; N, 14.73. Found: C, 40.83; H, 2.40; N, 13.90.
4.2. Biological assays
4.2.1. PTP-1B expression and purification
All experiments were conducted using human recombinant
PTP-1D. Briefly, the complete sequence of PTP-1D was cloned in
the pGEX-2T bacterial expression vector downstream the GST se-
quence. This vector was used to transform Escherichia coli TB1
strain cells. The recombinant fusion protein was purified from bac-
terial lysate using a single-step affinity chromatography. The solu-
tion containing purified fusion protein was treated with thrombin
for 3 h at 37 °C. Then the PTP-1D was purified from GST and throm-
bin by gel filtration on a Superdex G75 column. The purity of PTP-
1D preparation was assessed by SDS–polyacrylamide gel
electrophoresis.
4.1.1.23. N-(4-{[(6-Nitro-1,3-benzothiazol-2-yl)amino]sulfonyl}-
phenyl)acetamide (23). Yield 1.07 g (53.3%) of a yellow solid.
Mp 230.2–233.1 °C. 1H NMR (200 MHz, DMSO-d6): d 2.9 (s, 3H,
CH3CO), 7.8 (d, 2H, H-4, H-5, J = 8.8), 8.5 (s, 1H, NH), 8.7 (s,
4H, H-20, H-30, H-50, H-60), 9.1 (s, 1H, H-7) ppm. 13C NMR
(50.28 MHz, DMSO-d6): d 23.9 (CH3), 116.9 (C-7),116.9 (C-5),
117.7 (C-30,C-50), 118.0 (C-4), 121.9 (C-7a), 122.0 (C-20, C-60),
131.6 (C-10), 140.7 (C-40), 153.6 (C-6), 158.5 (C-3a), 163.3 (CO),
171.8 (C-2) ppm; MS(FAB+): m/z 393 (M+H)+; Anal. Calcd for
C15H12N4O5S2: C, 45.91; H, 3.08; N, 14.28. Found: C, 45.91; H,
3.08; N, 14.28.
4.2.2. Enzyme assays13
All assays were carried out at 37 °C. The substrate (p-nitrophe-
nylphosphate) was dissolved in 0.075 M of b,b-dimethylglutarate
buffer pH 7.0, containing 1 mM EDTA and 1 mM dithiothreitol.
The final volume was 1 ml. The reactions were initiated by adding
4.1.1.24. 4-Chloro-N-(6-nitro-1,3-benzothiazol-2-yl)benzene-
sulfonamide (24). Yellow solid, yield 0.67 g (35.6%). Mp 218.9–
1
2237 °C. H NMR (200 MHz, DMSO-d6): d 7.6–7.7 (M, 2H, H-30, H-
aliquots of the enzyme (0.16 lg for each test), and stopped at
50, J= 2.2, 8.8 Hz), 7.8–7.9 (m, 2H, H-20, H-60, J = 2.2, 8.8 Hz), 8.3
(dd, 2H, H-4, H-5, J = 2.2, 8.8 Hz), 8.9 (d, 1H, H-7, J = 2.6 Hz) ppm.
13C NMR (50.28 MHz, DMSO-d6): d 112.9 (C-7), 119.3 (C-5), 123.2
(C-4), 126.2 (C-7a), 127.8 (C-20, C-60), 129.3 (C-30, C-50), 137.5 (C-
40), 140.2 (C-10), 141.6 (C-6), 143.1 (C-3a), 168.6 (C-2) ppm;
MS(FAB+): m/z 370 (M+H)+; Anal. Calcd for C13H8N3O4S2Cl: C,
44.22; H, 2.18; N, 11.36. Found: C, 42.08; H, 2.30; N, 11.22.
appropriate times with 4 ml of 1 M KOH. The released p-nitrophe-
nol was determined by reading the absorbance at 400 nm
(e )
= 18,000 Mꢁ1 cmꢁ1). The main kinetic parameters (Km and Vmax
were determined by measuring the initial rates using eight differ-
ent substrate concentrations in the 0.5–40 mM range. Experimen-
tal data were analyzed using the Michaelis-Menten equation and a
nonlinear fitting program (FIGSYS).
To evaluate the inhibition power of compounds 4 and 16 we
calculated the IC50 value using a fixed substrate concentration cor-
responding to the Km of enzyme and varying inhibitor concentra-
tions. The IC50 value was calculated by fitting experimental data
with a non-nonlinear fitting program (FIGSYS, Biosoft, UK), using
the equation:
4.1.1.25. 4-Fluoro-N-(6-nitro-1,3-benzothiazol-2-yl)benzene-
sulfonamide (25). Yellow solid, yield 0.62 g (34.4%) Mp 163.3–
164.1 °C. 1H NMR (200 MHz, DMSO-d6):
d 7.3 (d, 1H, H-4,
J = 8.4 Hz), 7.5 (dd, 1H, H-5, J = 2.2, 8.4 Hz), 7.9 (d, 1H, H-7,
J = 1.8 Hz), 8.1 (d, 2H, H-30, H-50, J = 8.8 Hz), 8.4 (d, 2H, H-20, H-60,
J = 8.8 Hz) ppm. 13C NMR (50.28 MHz, DMSO-d6): d 116.1–116.5
(C-30, C-50, J = 22.7 Hz), 117.8 (C-7), 120.2 (C-5), 122.7 (C-4),
125.4 (C-7a), 128.7- 128.9 (C-20,C-60, J = 9.5 Hz), 135.3 (C-10),
137.7–143.0 (C-40, J = 262.5 Hz), 145.7 (C-6), 155.1 (C-3a), 174.5
(C-2) ppm; MS(FAB+): m/z 354 (M+H)+; Anal. Calcd for
C13H8N3O4S2F: C, 44.19; H, 2.28; N, 11.89. Found: C, 44.41; H,
2.19; N, 11.62.
Max ꢁ Min
Y ¼
slope þ Min
ꢀ
ꢁ
x
1 þ
IC50
where ‘y’ is vi/vo, that is, 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.
4.1.1.26. N-(6-Fluoro-1,3-benzothiazol-2-yl)-4-nitrobenzene-
sulfonamide (26). Yellow solid, 0.83 g (81.1%) Mp 259.3–
262.1 °C. 1H NMR (200 MHz, DMSO-d6): d 7.19–7.35 (m, 2H, H-
4, H-7), 7.78 (dd, 1H, H-50, J = 1.0, 8.0 Hz), 8.05–8.09 (m, 2H,
H-30, H-50, J = 1.2 y 8.8 Hz), 8.33–8.37 (m, 2H, H-20, H-60, J = 1.2,
8.8 ppm. 13C NMR (50.28 MHz, DMSO-d6): d 108.1 (C-7), 113.9
(C-5), 121.4 (C-30, C50), 123.4 (C-4), 126.1 (C-7a), 128.2 (C-20,
C-60), 144.6 (C-3a), 145.8 (C-10), 151.6 (C-40), 158.6 (C-6), 174.5
(C-2) ppm; MS(FAB+): m/z 354 (M+H)+; Anal. Calcd for
C13H8N3O4S2F: C, 44.19; H, 2.28; N, 11.89. Found: C, 43.77; H,
2.30; N,11.48.
4.2.3. Animals
Male Wistar rats weighing 200–250 g bodyweight 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. Ani-
mals 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 Experimenta-
tion and Care (SAGARPA, NOM-062-ZOO-1999, México), and ap-
proved by the Institutional Animal Care and Use Committee
based on US National Institute of Health publication (No. 85-23,
revised 1985).
4.1.1.27. N-(6-Chloro-1,3-benzothiazol-2-yl)-4-nitrobenzene-
sulfonamide (27). Yellow solid, yield 1.16 g (58.2%) Mp 244.9–
249.5 °C. 1H NMR (200 MHz, DMSO-d6):
d 7.30 (d, 1H, H4,
4.2.4. Induction of diabetes
J = 8.8 Hz), 7.44 (dd, 1H, H5, J = 1.8, 8.4 Hz), 7.99 (d, 1H,
J = 1.8 Hz), 8.07–8.12 (m, 2H, H20, H60, J = 1.8, 8.8 Hz), 8.35- 8.39
(m, 2H, H30, H50, J = 8.8 Hz) ppm. 13C NMR (50.28 MHz, DMSO-
d6): d 114.34 (C-7), 112.41 (C-4), 124.51 (C-30, C-50), 126.73 (C-5),
127.29 (C-20, C-60), 127.39 (C-7a), 127.86 (C-6), 135.23 (C-10),
147.03 (C-3a), 149.41 (C-40), 167.75 (C-2) ppm; MS(FAB+): m/z
370 (M+H)+; Anal. Calcd for C13H11N2O2S2Cl: C, 44.22; H, 2.18; N,
11.36. Found: C, 44.20; H, 2.18; N, 11.35.
Streptozotocin (STZ) was dissolved in citrate buffer (pH 4.5) and
nicotinamide was dissolved in normal physiological saline solu-
tion. T2DM was induced in overnight fasted rats by a single intra-
peritoneal injection of 65 mg/kg streptozotocin, 15 min after the
i.p. administration of 110 mg/kg of nicotinamide.7,21 Hyperglyce-
mia was confirmed by the elevated glucose concentration in plas-
ma, determined at 72 h by glucometer. The animals with blood

G. Navarrete-Vazquez et al. / Bioorg. Med. Chem. 17 (2009) 3332–3341
3341
glucose concentration higher 250 mg/dL, were used for the antidi-
abetic screening.
mode found by AUTODOCK in complex with the binding pocket of
PTP-1D was subject to full energy minimization using the MMFF94
force field implemented in MOE until the gradient 0.05 was
reached. During minimization, residue atoms within 8 Å from the
ligand were free to move (other atoms were fixed).
4.2.5. In vivo antidiabetic assay (T2DM model)7
The diabetic animals were divided into groups of five animals
each (n = 5). Rats of experimental groups were administered a sus-
pension of the compounds 1–7 (prepared in 1% Tween 80) orally
(100 mg/kg body weight). Control group animals were also fed
with 1% Tween 80. Glibenclamide (5 mg/kg) was used as hypogly-
cemic reference drug. Blood samples were collected from the cau-
dal 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 commercial glucometer
(Accutrend GCT, RocheÒ). The percentage variation of glycemia for
each group was calculated in relation to initial (0 h) level, accord-
ing to: %Variation of glycemia = [(Gx ꢁ G0)/G0] ꢀ 100, where G0
were initial glycemia values and Gx were the glycemia values at
+1, +3, +5 and +7 h, respectively.7 All values were expressed as
mean S.E.M. Statistical significance was estimated by analysis of
variance (ANOVA), p < 0.05 and p < 0.01 implies significance.
Acknowledgments
This work was supported in part by grants from CONACYT, pro-
ject 55591, given to G. Navarrete Vazquez. P. Paoli and G. Camici
are grateful to the Italian MIUR for financial support (FIRB 2003-
Project RBNE03FMCJ_007). H. Moreno-Diaz acknowledges the fel-
lowships awarded by CONACyT to carry out graduate studies. J.L.
Medina-Franco and K. Martínez-Mayorga are grateful to the State
of Florida. Authors are grateful to AUTODOCK and VMD developers for
providing the software.
References and notes
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4.2.6. Docking
Molecular Operating Environment (MOE)17 2007 was used for
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The crystal structure was obtained from the Protein Data Bank22
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