Original article

Novel sulfonyl(thio)urea derivatives act efciently both as insulin

secretagogues and as insulinomimetic compounds
Alessandra Mascarello
a
, Marisa J

adna Silva Frederico

b
, Alisson Jhonathan Gomes Castro
b
,
Camila P. Mendes
b
, M

arcio Ferreira Dutra

c
, Viviane Mara Woehl
d
,
Rosendo Augusto Yunes
a
, F

atima Regina Mena Barreto Silva

b, *
, Ricardo Jos

e Nunes
a, **
a
Universidade Federal de Santa Catarina, Departamento de Qumica, Centro de Ci^encias Fsicas e Matem aticas, Campus Trindade, 88040-900 Florian opolis,
SC, Brazil
b
Universidade Federal de Santa Catarina, Departamento de Bioqumica, Centro de Ci^encias Biol ogicas, Campus Trindade, Cx. Postal 5069, 88040-970
Florian opolis, SC, Brazil
c
Universidade Federal de Santa Catarina, Departamento de Biologia Celular, Embriologia e Genetica, Centro de Ci^encias Biol ogicas, Campus Trindade,
88040-970 Florian opolis, SC, Brazil
d
Universidade Federal de Santa Catarina, Departamento de Ci^encias Morfol ogicas, Centro de Ci^encias Biol ogicas, Campus Trindade, Cx. Postal 5069, 88040-
970 Florian opolis, SC, Brazil
a r t i c l e i n f o
Article history:
Received 6 February 2014
Received in revised form
2 September 2014
Accepted 3 September 2014
Available online 6 September 2014
Keywords:
Sulfonylthioureas
Diabetes
Glibenclamide
Insulinomimetic
a b s t r a c t
Glibenclamide is widely used in the management of non-insulin dependent diabetes mellitus, but
numerous risks limit its use in therapy. In the search for novel structures that are safer and more efcient
than glibenclamide, we obtained new chemical analogs based on bioisosterism, through the treatment of
benzenesulfonamide derivatives with isothiocyanates and isocyanates, affording (thio)ureas with good
yield. We also veried the hypoglycemic activity, through an in vivo approach. Most of these synthesized
compounds improved glucose tolerance, and the mechanism of action of the best compound (7) suggests
that its effect is mediated by insulin secretion, while its hypoglycemic action is triggered by glucose
uptake involving GLUT4 expression and translocation through PI-3K and PKA activity and active de novo
protein synthesis in skeletal muscle. Taking all these factors together, sulfonylthiourea 7 acts as an insulin
secretagogue and insulinomimetic agent on glucose homeostasis, and does not exhibit toxicity in acute
treatment.
2014 Elsevier Masson SAS. All rights reserved.
1. Introduction
According to the last major study published by the World Health
Organization, one in ten adults suffers fromdiabetes worldwide [1].
Glibenclamide has been widely used in the management of non-
insulin dependent diabetes mellitus. Despite the extensive accep-
tance of this drug and several other sulfonylureas, numerous risks
limit its use in therapy. Glibenclamide is associated with hypogly-
cemia and weight gain, hyperinsulinemia, deterioration of the
pancreas, and increased risk of cardiovascular mortality (in long-
term treatments). Furthermore, diabetic patients have a higher
risk for developing long-term complications as neoplasias, neuro
and nephropathies [2].
Some studies were carried out on the structural modication of
the core structure of glibenclamide (see the structures in Scheme
1). Russ et al. [3] proposed modications on the A-ring and elimi-
nation of the B-ring from glibenclamide, and the results showed
that this new compound exhibits a selectivity prole that is quite
different from that of glibenclamide, with greater loss of afnity
toward SUR1 and a slight preference for SUR2A [3]. In another
work, Yuriev et al. [4] demonstrated, using theoretical predictions
from CoMFA (Comparative Molecular Field Analysis), the impor-
tance of certain structural elements for blocking the potassium
channels (K
ATP
), and proposed new glibenclamide analogs [4].
Schneider et al. [5] proposed the inclusion of a glucose molecule
as the substituent on the A-ring to improve its solubility, but the
glibenclamide-glycosylated analog did not increase afnity to SUR1
* Corresponding author. Departamento de Bioqumica, Centro de Ci^ encias Bio-
l ogicas, UFSC, Campus Universitario, Bairro Trindade, Cx Postal 5069, CEP: 88040-
970 Florian opolis, SC, Brazil.
** Corresponding author. Departamento de Qumica, Centro de Ci^ encias Fsicas e
Matem aticas, UFSC, Campus Universit ario, Bairro Trindade, Cx Postal 476, CEP:
88040-970 Florian opolis, SC, Brazil.
E-mail addresses: mena.barreto@ufsc.br, mena@mbox1.ufsc.br (F.R. Mena
Barreto Silva), nunes@qmc.ufsc.br (R.J. Nunes).
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
j ournal homepage: ht t p: / / www. el sevi er. com/ l ocat e/ ej mech
http://dx.doi.org/10.1016/j.ejmech.2014.09.007
0223-5234/ 2014 Elsevier Masson SAS. All rights reserved.
European Journal of Medicinal Chemistry 86 (2014) 491e501
(K
D
2.08 nM) compared with the non-glycosylated analog (K
D
0.38 nM) [5]. On the other hand, the insertion of a pregnenolone on
the A-ring from glibenclamide, proposed by Figueroa-Valverde
et al. [6], promoted greater hypoglycemic activity than glibencla-
mide [6].
Calderone et al. [7] changed the B-ring from glibenclamide with
nitrooxymethylbenzoate derivatives as substituents (which is a
hydroxylated active metabolite of glibenclamide), and the results
showed that its hypoglycemic activity is increased with additional
NO-donor effect, conferring properties that are useful for treating
diabetes-related cardiovascular disorders [7]. Zhang et al. [8] pro-
posed analogs based on the structure of two sulfonamides with
antiplatelet and nephroprotector action, forming hybrids with the
original structure of glibenclamide, and generating promising hy-
poglycemic compounds, mainly of sulfonylurea shown in Scheme 1
[8].
In recent years, our group has been involved in searching for
therapeutically useful molecules for diabetes [9e12]. In our latest
work in particular (Scheme 1), we proposed the strategy of syn-
thesis of the glibenclamide-fragment using the benzenesulfona-
mides that indicated the best substituent for increasing
hypoglycemic activity and anti-diabetic action. From these, it was
identied excellent (p-CH
3
, p-OCH
3
and 2,4,6-triisopropyl), good
(8-quinoline, 10-camphoryl and thiophenyl) and poor (H and p-F)
benzenesulfonamides as oral hypoglycemic agents. In addition, the
most active compound (p-CH
3
-substituted) also showed a potent
insulinogenic index and increased soleus muscle glycogen content
without exhibit tissue toxicity [12].
To investigate the new features on the structure of glibencla-
mide, our current strategy for obtaining chemical analogs is based
on bioisosterism, proposing the inclusion of thiourea instead of
original urea (classical), replacement of the carbonyl group with a
sulfonyl group (non-classical) and changing the substituents on the
A-ring and B-ring, to check the hypoglycemic activity. Additionally,
the antihyperglycemic and/or hypoglycemic effect of these new
sulfonylurea and thiourea derivatives were studied in vivo. The
effect of the best compound on glucose uptake was also investi-
gated in skeletal muscle, in order to highlight its mechanism of
action on glucose uptake.
2. Chemistry
Sulfonyl(thi)oureas 1e10 (Scheme 2) were prepared from the
reaction between the substituted benzenesulfonamide previously
synthesized [12] and isocyanate or isothiocyanate with hydrous
potassium carbonate in acetone, to afford sulfonyl(thio)ureas, with
yields ranging from 41 to 79%. All the compounds are novel
structures and were characterized by
1
H NMR,
13
C NMR, IR and
elemental analysis. Detailed spectral characterization of all the
compounds are presented in the experimental section.
3. Results and discussion
3.1. Effect of sulfonyl(thio)urea derivatives on serum glucose levels
As it can be seen in Table 1, oral administration of compound 1
(no substituents on the A-ring) showed an acute and lasting
decrease in serum glucose levels about 17%, 20% and 13% at 15, 30
and 60 min, respectively. Compound 2 (p-CH
3
-substituted) also
showed a slight reduction in glucose (approximately 14% and 17% at
Scheme 1. Structural modication on core of glibenclamide.
A. Mascarello et al. / European Journal of Medicinal Chemistry 86 (2014) 491e501 492
15 and 30 min, respectively), with a similar effect of sulfonylth-
iourea 4 (2,4,6-triisopropyl-substituted), but not as signicant as
compound 1. For the compound 8 (p-F-substituted on A-ring), it
showed no activity during this study.
Based on the excellent result obtained in our previous work
with the most active sulfonamide (p-OCH
3
-substituted on A-ring)
[12], we synthesized and evaluated the sulfonyl(thio)urea de-
rivatives that have the same substitution pattern on the A-ring (3, 9
and 10), but these did not show the excellent results obtained with
the original one [12]. The sulfonylthioureas 3 and 9 (phenyl and p-
Cl-phenyl as B-ring) showed good activity, lowering blood glucose
by approximately 20% (15 min), point that the potency is not
improved by the addition of a chloride atom on B-ring but that the
substitution on A-ring is crucial for the biological activity [3,6]. The
only sulfonylurea synthesized in this work, 10 (1-naphthyl as B-
ring), was inactive. These results are different from those showed
by Zhang et al. [8], who demonstrated that the most active com-
pounds are sulfonylurea instead, sulfonylthiourea. Regarding sul-
fonylthioureas with A-ring different of phenyl or phenyl
substituted, it can be seen on Table 1 that the acute effect of com-
pound 5 (8-quinolinyl as A-ring) shows a prole of glucose toler-
ance curve similar to that of glibenclamide, with serum glucose
lowering around 23% at 60 min. Treatment with sulfonylthiourea 6
(camphoryl as A-ring), signicantly reduced the levels of serum
glucose at between 15 and 180 min. Compound 7 with ring
substituted by a thiophenyl notably reduced the glycemia about
25% during the period studied, and also exhibited potency greater
than 78% when compared to the glibenclamide positive control
group, at 30 min. These results corroborate with our previous work
[12]. In those studies, sulfonamides substituted on A-ring by 8-
quinolinyl, thiophenyl and camphoryl groups exhibited good
activity but it was signicantly improved when the same sub-
stituents were included into sulfonylthioureas instead. So, the
compounds that improved the glucose tolerance were assayed for
insulin secretion, LDH activity, muscle and liver glycogen content.
For that, it was selected the compounds 1, 3, 5, 7 and 9 for these
measurements after in vivo treatments.
3.2. Effect of sulfonylthiourea derivatives on insulin secretion
Insulinotropic sulfonylureas are currently major drugs used
extensively in the treatment of patients with type 2 diabetes [13]
despite the well-known side-effects of this class of drugs [14].
Glibenclamide stimulates insulin secretion from pancreatic b-cells
[15]. As expected, glibenclamide signicantly increased insulin
secretion around 134%, 354% and 416% at 15, 30 and 60 min
respectively. According to Bessesen [16], the glucose or insulin area
under the curve after glucose stimulation is widely used to assess
the insulinogenic index (II) or insulin sensitivity. Glibenclamide
showed an insulinogenic index that was 3-fold higher than hy-
perglycemic control group. Among these compounds studied, sul-
fonylthiourea 5 increased insulin secretion around 180% higher
than the hyperglycemic control at 30 min. The II calculated for this
compound was about 50% (1.3) compared with the potent II for
glibenclamide. Notably, sulfonylthiourea 7 signicantly increased
the secretion of insulin around 105%, 158% and 247% throughout the
period evaluated, and presented an II around 1.6-fold higher than
the hyperglycemic control (Table 2). This result, associated with the
results of glycemia showed inTable 1, indicate that sulfonylthiourea
7 presents the best antihyperglycemic activity among the sulfo-
nylthioureas selected for this study.
Scheme 2. Synthesis of novel sulfonyl(thio)urea derivatives. (a) RSO
2
Cl, dry pyridine; (b) K
2
CO
3
, isocyanate or isothiocyanate, dry acetone.
Table 1
Effect of sulfonylthiourea derivatives (10 mg/kg) on serumglucose levels in fasted Wistar rats. Each value represents the mean S.E.M. of six determinations.
a
p 0.05,
b
p 0.01,
c
p
0.001 compared to hyperglycemic control group (4 g/kg).
Group Serum glucose levels (mg/dL) time (min)
0 15 30 60 180
Hyperglycemic 102.29 6.34 177.46 5.95 190.15 1.77 182.55 6.95 141.15 4.47
Sulfonylthiourea
1 102.13 1.40 146.02 3.27
c
151.62 5.90
c
158.27 4.94
c
134.44 3.82
2 111.06 6.88 151.25 3.63
c
157.14 2.48
c
173.51 5.28 152.60 0.50
3 109.55 2.38 150.98 7.05
c
144.75 5.80
c
154.79 4.04 124.70 2.51
4 110.42 2.34 149.51 4.77
c
168.72 7.49
b
152.76 1.56 142.08 4.63
5 114.24 1.71 147.97 3.11
c
160.22 4.81
c
140.08 4.63
c
139.63 2.92
6 112.25 2.79 151.58 9.07
c
160.61 3.12
c
149.03 3.50
a
149.35 0.10
7 105.45 6.06 150.23 1.61
c
141.60 5.10
c
140.96 3.45
a
139.26 5.98
8 116.53 6.28 148.80 10.57
b
165.70 6.61
a
165.30 2.70 146.40 5.66
9 103.70 2.30 141.45 6.48
c
158.69 2.76
c
151.74 3.75
a
139.54 0.43
10 114.70 3.03 155.68 5.98
b
156.40 4.58
c
157.90 1.24 143.60 6.42
Glibenclamide 112.06 5.61 140.49 11.13 135.24 5.67 128.34 5.44 124.28 3.87
A. Mascarello et al. / European Journal of Medicinal Chemistry 86 (2014) 491e501 493
3.3. Effect of sulfonylthiourea derivatives on glycogen content
Although the effects of glibenclamide on the liver are rather
controversial, the specic site of action at plasma membrane on
hepatocytes was reported [17]. Additional studies have suggested a
role of glibenclamide on glycogen deposition [18] and pronounced
gluconeogenesis inhibition [19,20]. It has also been reported that
glibenclamide increases glucose uptake on skeletal muscle [21].
However, the effect of this drug on glycogen deposition in skeletal
muscle deserves additional studies. Based on these reports, we also
analyzed the glycogen content on liver and muscle after in vivo
acute treatment with sulfonylthiourea derivatives. Table 3 shows
that neither glibenclamide nor compounds 1, 5 and 7 were able to
accumulate glycogen content in the liver, when compared with the
hyperglycemic group. According to these results in in vitro experi-
ments, Lopez-Alarcon et al. [19] found that glibenclamide caused a
transient and dose-dependent activation of glycogen phosphory-
lase in hepatocytes. On the other hand, compounds 3 and 9
signicantly increased the glycogen content in the liver to 714% and
475%, respectively.
The muscle glycogen content was remarkably increased around
200% with glibenclamide; compound 7 about 240%; and compound
9 about 330%, compared with the hyperglycemic group. However,
the compounds 1, 3 and 5 did not change the muscle glycogen
content. The increase in muscle glycogen content caused by com-
pound 7 and glibenclamide may be a result of increased insulin
secretion (Table 2). These results are in agreement with those
described for glibenclamide [15]. It may also be an insulinomimetic
effect of these compounds directly on skeletal muscle, since that
the compound 9 did not increase insulin secretion (Table 2).
3.4. Effect of sulfonylthiourea derivatives on LDH activity
Compound 7 improved the oral glucose tolerance curve,
exhibited a glibenclamide-like effect on insulin secretion during
the period studied, and signicantly accumulated glycogen content
in an insulin responsive tissue, skeletal muscle. As the question
arose about insulin secretagogues in b-cell toxicity is frequent, the
compounds 1, 3, 5, 7 and 9 were analyzed by measuring the in vitro
LDH activity. After 3 h of treatment by oral gavage, neither sulfo-
nylthiourea derivatives nor glibenclamide were able to increase
LDH release compared with euglycemic or hyperglycemic group
(Table 4). The lowcytotoxicity for a series of similar derivatives was
also demonstrated for in vitro antitumor activity in 60 human tu-
mor cell lines [22]. It was detected that the concentration (IC
50
) of
the compounds able to inhibit the growth tumor cells was around
10 fold higher compared with the dose used by us for in vivo and
in vitro treatments. Based on the results obtained so far, sulfo-
nylthiourea 7 showed outstanding results in the parameters
studied, therefore it was chosen to assess the ability to stimulate
glucose uptake in soleus muscle.
3.5. Effect of sulfonylthiourea 7 on glucose uptake in rat soleus
muscle
Glucose transport is a fundamental process for the cell survival.
Table 5 shows the in vitro effect of different doses (1, 10 and 100 mM)
sulfonylthiourea 7 on glucose uptake in rat soleus muscle. As it can
be observed, compound 7 stimulated
14
C-DG uptake to a remark-
able extent (around 100%) for each dose assayed, when compared
with the control group. Therefore, the intermediate dose (10 mM)
was chosen for further studies.
To investigate the mechanism by which compound 7 increases
glucose uptake in soleus muscle, the glucose uptake was measured
with/without wortmannin (100 nM), a specic inhibitor for PI-3K
[23]. The stimulatory effect of 7 on glucose uptake was abolished
when muscle was pre-treated with the inhibitor (Fig. 1). The PI-3K/
AKT pathway plays a crucial role in insulin stimulation on GLUT4
translocation [24]. In skeletal muscle, the stimulation of glucose
uptake is mostly attributed to increase the translocation and
redistribution of GLUT4 to the plasma membrane [25]. This result
Table 2
Effect of sulfonylthiourea derivatives (10 mg/kg) on seruminsulin levels in fasted Wistar rats. Each value represents the mean S.E.M. of four determinations.
a
p 0.05,
b
p 0.01,
c
p
0.001 compared to hyperglycemic control group (4 g/kg).
Group Serum insulin levels (ng/mL) time (min)
0 15 30 60 II (ng/mg)
d
Hyperglycemic 0.09 0.001 2.84 0.002 0.75 0.007 0.59 0.004 0.66
1 e 0.39 0.041 0.35 0.11 0.78 0.048 0.31
3 e 2.75 0.03 0.70 0.006 0.53 0.02 0.77
5 e 2.26 0.001 1.36 0.002
c
0.60 0.009 0.84
7 e 3.01 0.001
a
1.19 0.125
c
1.46 0.006
c
1.06
9 e 2.69 0.11 0.71 0.006 0.52 0.004 0.72
Glibenclamide e 3.83 0.430
a
2.66 0.001
b
2.46 0.070
c
1.98
d
Insulinogenic index.
Table 3
Effect of sulfonylthiourea derivatives (10 mg/kg) on glycogen content in fasted rats.
Each value represents the mean S.E.M. of four determinations.
a
p 0.05,
c
p 0.001,
compared to hyperglycemic control group (4 g/kg).
Group Muscle (mg/g of tissue) Liver (mg/g of tissue)
Hyperglycemic 8.60 1.00 14.60 1.27
1 6.93 0.99 10.66 0.31
3 7.24 2.71 104.25 3.65
c
5 9.93 1.30 11.33 0.58
7 20.69 2.18
c
7.68 1.29
9 28.51 6.7
a
69.35 7.3
c
Glibenclamide 17.46 1.80
c
8.71 1.49
Table 4
Effect of sulfonylthiourea derivatives (10 mg/kg) on extracel-
lular LDH activity in serum. Each value represents
mean S.E.M. of four determinations.
Group LDH (I.U/L)
Euglycemic 118.20 9.32
Hyperglycemic 75.63 7.08
Glibenclamide 41.08 8.16
1 68.09 1.07
3 108.01 3.98
5 71.30 5.96
7 72.19 5.43
9 73.92 4.27
I.U/L: International Unit/L.
A. Mascarello et al. / European Journal of Medicinal Chemistry 86 (2014) 491e501 494
indicates that PI-3K plays a role in the stimulatory effect of com-
pound 7 on glucose uptake.
Several reports have demonstrated an insulin-independent ef-
fect on glucose uptake mediated by the adrenoceptors, suggesting
that it may represent an alternative glucose uptake even in the
absence of insulin [26,27]. We therefore analyzed the inuence of
protein kinase A (PKA) on the stimulatory effect of sulfonylthiourea
7 on glucose uptake. When 10 mM H89, a selective inhibitor for PKA
[28], was added to the incubation medium, the stimulatory effect of
7 was completely inhibited (Fig. 2A). In rat skeletal muscle cell line
L6, the glucose uptake was increased by ^ a
2
-adrenoceptores acti-
vation [26]. ^ a
2
-Adrenoceptores are coupled to Gs and adenylate
cyclase and utilize cAMP and PKA to produce the metabolic effects
of catecholamines. In another study, the involvement of PI-3K in ^ a
2
-
adrenoceptor-stimulated glucose uptake was suggested through
the partial inhibition by wortmannin [26]. However, in the present
study, wortmannin completely abolished the glucose uptake
stimulated by sulfonylthiourea 7. The adrenaline-stimulated
translocation of the insulin-sensitive GLUT4 has been shown in
skeletal muscle [27]. Moreover, PI-3K has been demonstrated to be
involved in glucose uptake mediated by a
1
-adrenoceptorin L6 cells
[29] and also in white adipocytes in vitro [30]. Imamur et al. [28]
demonstrated that gliclazide, a second generation sulfonylurea,
stimulated 2-deoxy-[
3
H]-D-glucose (2DG) uptake, 24 h after treat-
ment, in a dose-dependent manner in cultured rat L6 myoblasts.
The same authors showed that gliclazide increased GLUT1 protein
synthesis and mRNA expression. Following that, 2DG uptake and
GLUT1 protein synthesis induced by gliclazide were completely
blocked by H89 and rp-cAMP (inhibitors for PKA), while gliclazide
increased the intracellular cAMP levels from 3 to 24 h after
treatment [28]. Taking these results together, it seems that PKA is
deeply involved in intracellular translocation, which may result in
an increase in glucose uptake. Also, it reinforces an insulinomimetic
effect of compound 7 since in its in vitro environment there is no
insulin secretion.
The involvement of protein kinase C (PKC) on glucose uptake
activation is well recognized in studies using pharmacological
agents [31e33]. Phorbol 12-myristate 13-acetate (PMA), an acti-
vator of conventional and novel PKC (c/nPKC), is not involved in
insulin-independent glucose uptake on skeletal muscle [33]. Fig. 2B
shows that there is no per se effect of 100 nM PMA [33] on glucose
uptake in skeletal muscle. However, it was able to partially
diminish the glucose uptake stimulated by compound 7. These re-
sults also demonstrate that for sulfonylthiourea 7 there is no
involvement of c/nPKC in the mechanism of action of glucose up-
take on skeletal muscle. Furthermore, it is interesting to note that
the participation of this isoform of PKC usually mediates glucose
uptake in adipocytes [31,32].
To examine whether the biological response for compound 7 is
caused by de novo protein synthesis-dependent process, 350 mM
cycloheximide [34] was used. Fig. 3 shows a marked reduction
(around 70%) on the stimulatory effect of compound 7 on glucose
uptake in the presence of cycloheximide, without altering the basal
uptake, and characterizing the role of this compound on protein
expression. To conrm these results, protein synthesis assays was
carried out in soleus muscles. l-[Ue
14
C]Leucine incorporation into
proteins was signicantly increased by 7, suggesting that this
compound is able to alter nuclear activity (Fig. 4A).
As shown in Fig. 4B 20 mM sulfonylthiourea 7 signicantly
increased the total immunocontent of GLUT4 on soleus muscle
compared with the control group, after in vitro incubation (60 min).
In the presence of cycloheximide, the stimulatory effect of sulfo-
nylthiourea 7 on the total immunocontent of GLUT4 was abolished.
In addition, the localization of GLUT4 with/without 7 after in vivo
and in vitro treatment was investigated, as shown in Fig. 5 in panels
A, B, C, D and E. The effect of compound 7 on immunolocalization of
GLUT4 after in vitro (panel C) and also after in vivo treatment (panel
D) was evident, as observed at plasma membrane (see arrows) in
soleus muscle. The presence of GLUT4 in the cell was markedly
increased by sulfonylthiourea 7 after both approaches analyzed
(panels C and D), compared with the respective control groups
(panels A and B). This qualitative analysis is according with the
effect of 7 on the total GLUT4 immunocontent in soleus muscle,
reinforcing the results shown in Fig. 4A, B. In line with these results,
it is known that the PI-3K/AKT pathway also promotes protein
synthesis and regulation of gene expression [24]. These results
indicate that glucose uptake increased by 7 require active de novo
protein synthesis, an event that may be, at least in part, dependent
on PI-3K.
4. Conclusion
We have investigated the new features on structure of the gli-
benclamide by obtaining novel chemical analogs based on bio-
isosterism, and determined the hypoglycemic activity through an
in vivo approach. Six compounds improved the glucose tolerance
curve (1, 3, 5, 6, 7 and 9) and the mechanism of action of the best
compound (7) was investigated. The antihyperglycemic effect of
this compound is mediated by insulin secretion and its hypogly-
cemic action is triggered by glucose uptake involving GLUT4
expression and translocation through PI-3K and PKA activity and de
novo protein synthesis in skeletal muscle. In addition, its compound
did not exhibit toxicity in its acute treatment. Taking all these re-
sults together, sulfonylthiourea 7 acts as an insulin secretagogue
and an insulinomimetic agent on glucose homeostasis.
Table 5
Effect of sulfonylthiourea 7 at different doses on
14
C-glucose uptake in skeletal
muscle. Each value represents mean S.E.M. of four determinations.
c
p 0.001,
compared to control group.
Group Glucose uptake
(nmol of glucose/mg protein)
Control 0.16 0.03
Sulfonylthiourea 7 (1 mM) 0.35 0.03
c
Sulfonylthiourea 7 (10 mM) 0.30 0.001
c
Sulfonylthiourea 7 (100 mM) 0.32 0.009
c
Fig. 1. Involvement of PI-3K on the stimulatory action of sulfonylthiourea 7 on
14
C-
glucose uptake in rat soleus muscle. 100 nM wortmannin was present in the pre-
incubation and incubation periods. Preincubation time 30 min; incubation
time 60 min. Values are expressed as mean S.E.M.; n 4 of 3 independent ex-
periments.
c
p 0.001 compared to control group;
#c
p compared to the sulfonylthiourea 7
group.
A. Mascarello et al. / European Journal of Medicinal Chemistry 86 (2014) 491e501 495
5. Experimental protocols
5.1. Materials
Glibenclamide, wortmannin, H89, phorbol 12-myristate 13-
acetate (PMA), cycloheximide and oyster glycogen type II were
purchased fromSigma Chemical Company (St. Louis, MO, USA). The
iodine reagents (CaCl
2
I
2
KI) were purchased from VETEC, Rio
de Janeiro, Brazil. The commercial kit used to determine the gly-
cemia and LDH was from Analisa (Belo Horizonte, MG, Brazil). The
enzyme-linked immunosorbent assay (ELISA) for quantitative
determination of rat insulin (catalog no. EZRMI-13K) was pur-
chased from Millipore (St Charles, MO, USA).[Ue
14
C]-2-deoxy-D-
glucose ([
14
C]-DG), specic activity 10.6 GBq/mmol, l-[Ue
14
C]
leucine and biodegradable scintillation liquid were obtained from
Fig. 2. Involvement of PKA and PKC on the stimulatory action of sulfonylthiourea 7 on
14
C-glucose uptake in rat soleus muscle. (A), 10 mM H89 (B), 100 nM PMA were present
in the preincubation and incubation periods. Preincubation time 30 min; incubation
time 60 min. Values are expressed as mean S.E.M.; n 4 of 3 independent ex-
periments.
b
p 0.01,
c
p 0.001, compared to the control group;
#a
p 0.05,
#c
p 0.001,
compared to the sulfonylthiourea 7 group.
Fig. 3. Involvement of proteinsynthesis onthe stimulatoryactionof sulfonylthiourea7on
14
C-glucose uptake in rat soleus muscle. 350 mM cycloheximide was present in pre-
incubation and incubation periods. Preincubation time 30 min; incubation
time60min. Values areexpressedas meanS.E.M.; n4of 3independent experiments.
c
p 0.001 compared to the control group;
#a
p compared to the sulfonylthiourea7 group.
Fig. 4. Effect of sulfonylthiourea 7 on in vitro incorporation of
14
C-leucine into proteins
and in total GLUT4 immunocontent of soleus muscle. (A), 10 mM sulfonylthiourea 7 was
present during the incubation period. Preincubation time 30 min; incubation
time 60 min. Values are expressed as mean S.E.M.; n 4 of 3 independent ex-
periments.
b
p 0.01 compared to control group. (B), 350 mM cycloheximide was present
during preincubation (30 min) and incubation (60 min) medium, in the presence or
absence of 10 mM sulfonylthiourea 7. Values are expressed as mean S.E.M.; n 4 of 3
independent experiments.
b
p 0.01 compared to control group and # compared to
sulfonylthiourea 7 group.
A. Mascarello et al. / European Journal of Medicinal Chemistry 86 (2014) 491e501 496
Perkin Elmer Life and Analytical Sciences, (Boston, MA, USA). Salts
and solvents were purchased from Merck

AG (Darmstadt.
Germany).
For the in vivo treatments, sulfonylthiourea derivatives were
dissolved in a solution of 0.3% Tween 80. In all experiments, the
vehicle (solution of 0.3% Tween 80) was not able to modify the
results under any experimental condition, basal and treated groups
(data not shown). For the in vitro experiments, sulfonylthiourea 7
was dissolved in dimethyl sulfoxide and added to both the control
and test media at a nal concentration not exceeding 0.1% (v/v).
5.2. Sulfonyl(thio)urea derivative synthesis
5.2.1. General procedure for preparation of sulfonyl(thio)ureas
The sulfonyl(thio)ureas were prepared using a methodology
adapted by Yuriev et al. [4] and Zhang et al. [8], from the reaction
between the substituted benzenesulfonamide previously synthe-
sized [12] and a hydrous potassium carbonate in acetone, under
reux for 2 h. The resulting mixture was reuxed for 16 h in acetone
with isocyanate or isothiocyanate. Ice was added to the reaction
and the mixture was acidied with hydrochloric acid solution,
yielding viscous slurry. The solids were obtained by adding diethyl
ether to the slurry. Pure compounds were recrystallized from
ethanol when necessary. The purity of the synthesized compounds
was analyzed by thin-layer chromatography (TLC) using Merck
silica pre-coated aluminum plates of 200 mm thickness, with sol-
vent systems of different polarities. The sulfonylthioureas are sol-
uble in dimethylsulfoxide and acetone. The solvents and reagents
used were purchased from Merck, SigmaeAldrich, Fluka and Vetec.
All sulfonylthioureas are novel compounds.
5.2.2. Physicochemical data on synthesized compounds
The structures were conrmed through melting points (m.p.),
1
H and
13
C Nuclear Magnetic Resonance (NMR) Spectroscopy,
Infrared Spectroscopy (IR) and Elementary Analyses (CHNS).
Melting points were determined with a Microqumica MGAPF-301
apparatus and are uncorrected. NMR (
1
H and
13
C) spectra were
recorded with a Varian Oxford AS-400 (400 MHz) instrument;
DMSO-d
6
was used as a solvent and tetramethylsilane as an internal
standard. IR spectra were recorded with an AbbBomen FTLA 2000
spectrometer on KBr disks. Elementary analyses were obtained
with a CHNS EA 1110. Percentages of C and H were in agreement
with the product formula (within 0.4% of theoretical values to C).
Fig. 5. (A), In vitro effect of 10 mM sulfonylthiourea 7 on total GLUT4 immunocontent in soleus muscle. 350 mM cycloheximide was present in the preincubation and incubation
periods. Preincubation time 30 min; incubation time 60 min. (B), In vitro (10 mM) and in vivo (10 mg/kg) effect of sulfonylthiourea 7 on GLUT4 localization (arrows) by
uorescence in soleus muscle. For in vitro analyses (panels A and C), preincubation time 30 min; incubation time 60 min in the presence of sulfonylthiourea 7. For in vivo
analyses (panels B and D), after oral gavage of sulfonylthiourea 7. Panel E, negative control slide shows immunonegative reaction in soleus muscle. Values are expressed as
mean S.E.M.; n 4 of 3 independent experiments.
A. Mascarello et al. / European Journal of Medicinal Chemistry 86 (2014) 491e501 497
1 e N-(Phenylcarbamothioyl)-4-(2-(phenylsulfonamido)
ethyl)benzenesulfonamide. Beige solid, m.p. 220e223

C;
1
HNMR
(DMSO-d) d 2.67 (NHeCH
2
eCH
2
, t); 2.94 (NHeCH
2
eCH
2
, q); 6.83 (t,
1H, NH); 7.15e7.11 (m, 4H, AreH); 7.77e7.55 (m, 10H, AreH); 8.94
(NH-thiourea).
13
C NMR (DMSO-d) d 35.75 (NHeCH
2
eCH
2
); 44.46
(NHeCH
2
eCH
2
); 120.66 (C2, C6); 121.76 (C4); 127.14 (C2
0
, C6
0
);
128.11 (C2
00
, C6
00
); 128.27 (C3, C5); 128.58 (C3
00
, C5
00
); 129.92 (C3
0
,
C5
0
); 133.06 (C4
00
); 141.01 (C1, C4
0
), 142.03 (C1
0
); 144.03 (C1
00
);
182.80 (C]S). IR (KBr) (n
max
/cm
1
) 3307 (NH); 1327 SO
2
as
; 1138
SO
2
s
; 1091 (C]S). Anal. Calcd for C
21
H
21
N
3
O
4
S
3
: C 53.03; H 4.45;
N 8.84; S 20.23. Exp.: C 52.89; H 4.21; N 8.36; S 19.85. Yield: 52%.
2 e 4-Methyl-N-(4-(N-(phenylcarbamothioyl)sulfamoyl)phe-
nethyl)-benzenesulfonamide. White solid, m.p. 225e227

C;
1
H
NMR (DMSO-d) d 2.37 (s, 3H, CH
3
); 2.68 (NHeCH
2
eCH
2
, t); 2.95
(NHeCH
2
eCH
2
, q); 6.84 (t, 1H, NH); 7.12e7.15 (m, 4H, AreH);
7.37e7.39 (m, 2H, AreH); 7.69e7.62 (m, 7H, AreH); 8.92 (NH-
thiourea).
13
C NMR (DMSO-d) d 21.40 (CH
3
); 35.45 (NHeCH
2
eCH
2
);
44.22(NHeCH
2
eCH
2
); 120.40 (C6); 121.52 (C2); 126.98 (C2
0
, C6
0
);
127.83 (C2
00
, C6
00
); 128.02 (C3
0
, C5
0
); 128.35 (C3, C5); 130.10 (C3
00
,
C5
00
, C4); 137.80 (C1
00
); 140.78 (C4
00
); 141.78 (C4
0
); 143.07 (C1);
143.81 (C1
0
); 182.59 (C]S). IR (KBr) (n
max
/cm
1
) 3308 (NH); 1313
SO
2
as
; 1138 SO
2
s
; 1091 (C]S). Anal. Calcd for C
22
H
23
N
3
O
4
S
3
: C
53.97; H4.73; N8.58; S 19.65. Exp.: C 54.01; H4.77; N8.25; S 19.09.
Yield: 59%.
3 e 4-Methoxy-N-(4-(N-(phenylcarbamothioyl)sulfamoyl)
phenethyl)-benzenesulfonamide. Light beige solid, m.p.
146e147

C;
1
H NMR (DMSO-d) d 2.67 (NHeCH
2
eCH
2
, t); 2.90
(NHeCH
2
eCH
2
, q); 3.80 (s, 3H, OCH
3
); 6.83 (t, 1H, H4); 7.08 (d,
J 8.0 Hz, 2H, H3
00
, H5
00
); 7.13 (m, 4H, H2
0
, H6
0
, H3, H5); 7.57 (t, 1H,
NH); 7.63 (d, J 8.0 Hz, 2H, H2
00
, H6
00
); 7.67 (m, 4H, H2, H6, H3
0
, H5
0
),
8.93 (NH-thiourea).
13
C NMR (DMSO-d) d 35.42 (NHeCH
2
eCH
2
);
44.22 (NHeCH
2
eCH
2
); 56.04 (OCH
3
); 114.78 (C3
00
, C5
00
); 120.55
(C6); 123.69 (C1
00
); 126.09 (C4); 127.85 (C2
0
, C6
0
); 128.05 (C2
00
, C6
00
);
128.37 (C3, C5); 129.12 (C3
0
, C5
0
, C1); 129.24 (C2); 129.65 (C4
0
);
132.30 (C1
0
); 162.51 (C4
00
); 184.05 (C]S). IR (KBr) (n
max
/cm
1
) 3283
(NH); 1314 SO
2
as
; 1154 SO
2
s
; 1088 (C]S). Anal. Calcd for
C
22
H
23
N
3
O
5
S
3
: C 52.56, H 4.58; N 8.31; S 19.02. Exp.: C 52.16; H
4.29; N 8.02; S 19.44. Yield: 71%.
4 e 2,4,6-Triisopropyl-N-(4-(N-(phenylcarbamothioyl)sulfa-
moyl)phenethyl)-benzenesulfonamide. White solid, m.p.
155e157

C;
1
H NMR (DMSO-d) d 1.13 (s, 3H, CH
3
); 1.14 (s, 6H, CH
3
);
1.16 (s, 3H, CH
3
); 1.17 (s, 3H, CH
3
); 1.19 (s, 3H, CH
3
); 2.81 (t, 2H,
NHeCH
2
eCH
2
); 2.90e2.85 (m, 1H, CH); 3.07 (NHeCH
2
eCH
2
, q);
4.06e4.12 (m, 2H, CH); 7.10e7.81 (m, 12H, AreH, NH); 10.08 (NH-
thiourea).
13
C NMR (DMSO-d) d 23.89 (2C, CH
3
); 25.16 (4C, CH
3
);
29.22 (2C, CH); 33.75 (1C, CH); 35.48 (NHeCH
2
eCH
2
); 43.18
(NHeCH
2
eCH
2
); 123.60 (C5
00
); 123.99 (C3
00
); 126.10 (C2
0
, C6
0
);
128.19 (C5); 128.87 (C3); 128.93 (C6); 129.27 (C2); 129.50 (C3
0
, C5
0
);
129.67 (C4); 133.60 (C1
00
); 137.91 (C1); 142.58 (C4
0
); 143.37 (C1
0
);
150.06 (C2
00
, C6
00
); 152.45 (C4
00
); 181.46 (C]S). IR (KBr) (n
max
/cm
1
)
3254 (NH); 1390 SO
2
as
; 1140 SO
2
s
; 1085 (C]S). Anal. Calcd for
C
30
H
39
N
3
O
4
S
3
: C 59.87; H 6.53; N 6.98; S 15.98. Exp.: C 60.04; H
6.74; N 7.01; S 15.50. Yield: 53%.
5 e N-(4-(N-(Phenylcarbamothioyl)sulfamoyl)phenethyl)
quinoline-8-sulfonamide. Beige solid, m.p. 200e202

C;
1
H NMR
(DMSO-d) d 2.66 (NHeCH
2
eCH
2
, t); 3.04 (NHeCH
2
eCH
2
, q); 6.85 (t,
1H, H4); 6.98 (d, J 8.0 Hz, 2H, H3, H5); 7.15e8.98 (m, 10H, AreH);
7.15 (m, 1H, NH); 7.57 (d, J 8.0 Hz, 2H, H2, H6); 8.89 (NH-thio-
urea).
13
C NMR (DMSO-d) d 35.25 (NHeCH
2
eCH
2
); 44.69
(NHeCH
2
eCH
2
); 120.59e151.96 (21C, AreC); 182.66 (C]S). IR
(KBr) (n
max
/cm
1
) 3289 (NH); 1387 SO
2
as
; 1129 SO
2
s
; 1082 (C]
S). Anal. Calcd for C24H22N4O4S3: C 54.73; H4.21; N10.64; S 18.27.
Exp.: C 54.44; H 4.50; N 10.22; S 18.72. Yield 41%.
6 e 4-(2-((7,7-Dimethyl-2-oxobicyclo[2.2.1]heptan-1-yl)
methylsulfonamido)ethyl)-N-(phenylcarbamothioyl)
benzenesulfonamide. Light yellowsolid, m.p. 146e147

C;
1
HNMR
(DMSO-d) d 0.77 (s, 3H, CH
3
); 0.99 (s, 3H, CH
3
); 1.37 (m, 1H, H
f
); 1.50
(m, 1H, H
g
); 1.87 (m, H
i
); 1.92 (m, H
h
); 2.02 (t, H
e
); 2.30 (m, H
c
, H
d
);
2.85 (t, 2H, NHeCH
2
eCH
2
); 2.88 (d, 1H, H
b
); 3.25 (NHeCH
2
eCH
2
,
q); 3.29 (d, 1H, H
a
); 6.88e7.87 (m, 10H, AreH, NH); 10.11 (NH-
thiourea).
13
C NMR (DMSO-d) d 19.74 (2C, CH
3
); 24.83 (C3
00
); 26.70
(C2
00
); 35.90 (NHeCH
2
eCH
2
); 42.39 (C4
00
, C5
00
); 44.13
(NHeCH
2
eCH
2
); 48.07 (C7
00
); 58.21 (C1
00
, eCH
2
SO
2
); 117.24 (C2);
123.69 (C6); 126.10 (C2
0
, C6
0
); 128.19 (C4); 128.92 (C3); 129.33 (C5);
129.58 (C1); 129.74 (C3
0
, C5
0
); 142.58 (C4
0
); 143.61 (C1
0
); 181.56
(C]S); 215.16 (C]O). IR (KBr) (n
max
/cm
1
) 3294 (NH); 1381
SO
2
as
; 1134 SO
2
s
; 1087 (C]S). Anal. Calcd for C
25
H
31
N
3
O
5
S
3
: C
54.62; H 5.68; N 7.64; S 17.50. Exp.: C 55.00; H 5.88; N 7.67; S 17.69.
Yield 68%.
7 e N-(4-(N-(Phenylcarbamothioyl)sulfamoyl)phenethyl)
thiophene-2-sulfonamide. Beige solid, m.p. 220e222

C;
1
H NMR
(DMSO-d) d 2.72 (NHeCH
2
eCH
2
, t); 3.05 (NHeCH
2
eCH
2
, q); 6.84 (t,
1H, H4); 7.13e7.18 (m, 5H, H3, H5, H2
0
, H6
0
, NH); 7.56 (dd, 1H, H5
00
);
7.65 (d, J 8.0 Hz, 2H, H2, H6); 7.68 (d, J 8.0 Hz, 2H, H3
0
, H5
0
); 7.89
(dd, 1H, H3
00
); 8.94 (NH-thiourea).
13
C NMR (DMSO-d) d 35.30
(NHeCH
2
eCH
2
); 44.44 (NHeCH
2
eCH
2
); 120.50 (C3
00
); 121.68 (C1
00
);
127.80 (C2, C6); 128.11 (C2
0
, C6
0
); 128.20 (C5
00
); 128.38 (C3
0
, C5
0
, C4);
132.03 (C3); 132.93 (C5); 140.83 (C4
00
); 141.59 (C4
0
); 141.76 (C1);
143.82 (C1
0
); 180.14 (C]S). IR (KBr) (n
max
/cm
1
) 3264 (NH); 1318
SO
2
as
; 1155 SO
2
s
; 1084 (C]S). Anal. Calcd for C
19
H
19
N
3
O
4
S
4
: C
47.38; H3.98; N 8.72; S 26.63. Exp.: C 47.66; H 3.62; N8.66; S 26.25.
Yield 72%.
8 e 4-Fluoro-N-(4-(N-(phenylcarbamothioyl)sulfamoyl)phe-
nethyl)-benzenesulfonamide. Light brown solid, m.p. 120e122

C;
1
H NMR (DMSO-d)d 2.79 (NHeCH
2
eCH
2
, t); 3.02 (NHeCH
2
eCH
2
,
q); 7.17 (t, 1H, H4); 7.31e7.85 (m, 14H, AreH, NH); 10.19 (NH-thio-
urea).
13
C NMR (DMSO-d) d 35.28 (NHeCH
2
eCH
2
); 43.97
(NHeCH
2
eCH
2
); 116.69 (C5
00
); 116.91 (C3
00
); 117.24 (C4); 123.69
(C6); 124.05 (C2); 126.08 (C2
0
, C6
0
); 128.16 (C2
00
); 128.90 (C6
00
);
129.31 (C5); 129.57 (C3); 129.6 (C3
0
, C5
0
); 129.87 (C4
0
); 129.97 (C1);
142.58 (C1
00
); 143.32 (C1
0
); 163.27 (C4
00
); 181.02 (C]S). IR (KBr)
(n
max
/cm
1
) 3288 (NH); 1331 SO
2
as
; 1152 SO
2
s
; 1087 (C]S).
Anal. Calcd for C
21
H
20
FN
3
O
4
S
3
: C 51.10; H4.08; N8.51; S 19.49. Exp.:
C 51.10; H 4.20; N 8.21; S 19.22. Yield 43%.
9 e N-(4-Chlorophenylcarbamothioyl)-4-(2-(4-
methoxyphenylsulfonamido)ethyl)-benzenesulfonamide. Light
yellow solid, m.p. 152e153

C;
1
H NMR (DMSO-d)d 2.78
(NHeCH
2
eCH
2
, t); 2.97 (NHeCH
2
eCH
2
, q); 3.83 (s, 3H, OCH
3
); 7.09
(d, J 8.0 Hz, 2H, H2, H6); 7.41e7.30 (m, 4H, H3
00
, H5
00
, H3, H5); 7.48
(d, J 12.0 Hz, 2H, H2
0
, H6
0
); 7.59 (m, 1H, NH); 7.70 (d, J 8.0 Hz,
2H, H2
00
, H6
00
); 7.84 (d, J 8.0 Hz, 2H, H3
0
, H5
0
); 10.18 (NH-thiourea).
13
C NMR (DMSO-d) d 35.26 (NHeCH
2
eCH
2
); 44.01 (NHeCH
2
eCH
2
);
56.04 (OCH
3
); 114.77 (C3
00
, C5
00
); 117.00 (C6
00
); 125.65 (C2
00
); 126.09
(C2
0
, C6
0
); 128.18 (C3); 128.83 (C5
0
); 129.11 (C3
0
, C5
0
, C1); 129.65
(C4); 129.65 (C2, C6); 132.27 (C1
00
); 142.55 (C4
0
); 143.46 (C1
0
);
162.54 (C4
00
); 180.25 (C]S). IR (KBr) (n
max
/cm
1
) 3272 (NH); 1329
SO
2
as
; 1149 SO
2
s
; 1085 (C]S). Anal. Calcd for C
22
H
22
ClN
3
O
5
S
3
: C
48.93; H 4.11; N 7.78; S 17.81. Exp: C 48.87; H 4.15; N 7.70; S 17.44.
Yield 79%.
10 e 4-Methoxy-N-(4-(N-(naphthalen-2-ylcarbamoyl)sulfa-
moyl)phenethyl)-benzenesulfonamide. White solid, m.p.
161e162

C;
1
H NMR (DMSO-d)d 2.75 (NHeCH
2
eCH
2
, t); 2.95
(NHeCH
2
eCH
2
, q); 3.83 (s, 3H, OCH
3
); 7.10 (d, J 8.0 Hz, 2H, H3
00
,
H5
00
); 7.19 (t, 1H, H6); 7.40e7.30 (m, 4H, H5, H10, H2
0
, H6
0
);
7.74e7.50 (m, 6H, H4, H7, H9, H3
0
, H5
0
, NH); 7.89 (d, J 8.0 Hz, 2H,
H2
00
, H6
00
); 8.06 (d, 8.0 Hz, 1H, H2); 8.82 (NH-urea).
13
C NMR
(DMSO-d) d 35.26 (NHeCH
2
eCH
2
); 44.02 (NHeCH
2
eCH
2
); 56.05
(OCH
3
); 107.85 (C2); 114.77 (C3
00
, C5
00
); 115.81 (C4); 122.74e129.65
(12C, AreC); 132.27 (C1
00
); 134.60 (C1); 142.56 (C4
0
); 143.47 (C1
0
);
145.09 (C]O); 162.54 (C4
00
). IR (KBr) (n
max
/cm
1
) 3305 (NH); 1316
A. Mascarello et al. / European Journal of Medicinal Chemistry 86 (2014) 491e501 498
SO
2
as
; 1155 SO
2
s
; 1670 (C]O). Anal. Calcd for C
26
H
25
N
3
O
6
S
2
: C
57.87; H 4.67; N 7.79; S 11.88. Exp: C 58.04; H 4.57; N 7.77; S 10.98.
Yield 77%.
5.3. Experimental animals
Male albino Wistar rats (180e210 g; 49 day-old) were bred in an
animal facility and housed in an air-conditioned room (approxi-
mately 21 2

C) with controlled lighting (lights on from 06:00 to
18:00 h). The rats were fed on pelleted food (Nuvital, Nuvilab CR1,
Curitiba, PR, Brazil), and tap water was available ad libitum. The
treated rats were deprived of food for at least 16 h but allowed free
access to water. All the animals were monitored and maintained in
accordance with the ethical recommendations of the local Ethical
Committee for Animal Use (Protocol CEUA-UFSC PP00414).
5.4. Studies of sulfonyl(thio)urea derivatives on the oral glucose
tolerance curve
Hyperglycemic rats were divided into different groups of four
rats (see Tables and Figures), controls and treated. Control groups
were randomly divided, as follows: vehicle (solution of 0.3% Tween
80); hyperglycemic control group rats received glucose (4 g/kg;
8.9 M), p.o. gavage. Treated rats that received glibenclamide (1, 10
and 100 mg/kg) and sulfonylthioureas derivatives (10 mg/kg), p.o.
gavage. Blood samples from the tail vein were collected and
centrifuged, and the serum glucose levels were measured just
before (zero), and at 15, 30, 60 and 180 min after treatment. After
centrifugation, serum samples also were used to determine insulin
levels or lactate dehydrogenase activity. Glycemia was determined
by the glucose oxidase method [35].
5.5. Insulin serum measurements
The insulin was measured by the enzyme-linked immunosor-
bent assay (ELISA), according to the manufacturer's instructions.
The range of values detected by this assay was: 0.63 ng/mL to
4.3 ng/mL. The intra- and inter-assay coefcients of variation (CV)
for insulin were 3.22 and 6.95 respectively, with a sensitivity of
0.2 ng/mL. All insulin levels were estimated by means of colori-
metric measurements at 450 nm with an ELISA plate reader
(Organon Teknika, Roseland, NJ, USA) by interpolation from a
standard curve. Samples were analyzed in duplicate and the results
were expressed as ng of insulin serum mL
1
[12]. The incremental
areas under the response curves (AUCs) were calculated. The
insulinogenic index (II) was calculated as the ratio between the
AUC
insulin
and AUC
glucose
(from 0 to 60 min) [12].
5.6. Skeletal muscle and liver glycogen measurements
Soleus muscle and liver were harvested from untreated hyper-
glycemic rats and treated with glibenclamide and sulfonylthioureas
derivatives (10 mg/kg), and were used for the assay of glycogen
content immediately after 3 h of treatment. Glycogen was isolated
from these tissues as described by Krisman [36], with minor
modications. The tissues were weighed, homogenized in 33% KOH
and boiled at 100

C for 20 min, with occasional stirring. After
cooling, 96% ethanol was added to the samples and heated to
boiling point, then cooled in an ice bath to aid the precipitation of
glycogen. The homogenates were centrifuged at 1300 g for
15 min, the supernatant was discarded and the pellets were
neutralized with saturated NH
4
Cl before being heated to 100

C for
5 min, washed and solubilized in water. Glycogen content was
determined by treatment with iodine reagent and the absorbance
was measured at 460 nm. The results are expressed as mg of
glycogen/g of tissue.
5.7. Lactate dehydrogenase assay (LDH assay)
The toxicity was assessed by measuring the release of the
cytosolic enzyme lactate dehydrogenase to the serumafter 180 min
of sulfonylthiourea derivatives (10 mg/kg) treatment. The LDH ac-
tivity was measured by the spectrophotometric method, the esti-
mated activity estimation was calculated by measuring the
oxidation of NADH, and the results were expressed as international
units per liter (I.U/L) [37].
5.8. On
14
C-glucose uptake in rat soleus muscle
For the [Ue
14
C]-2-deoxy-D-glucose (
14
C-DG) uptake experi-
ments, the soleus muscles of fasted rats were used. Slices of soleus
muscle were distributed (left and right alternately) between the
basal and treated groups. The muscles were dissected, pre-
incubated and incubated at 37

C in Krebs Ringer-bicarbonate (KRb)

buffer with a composition of 122 mM NaCl, 3 mM KCl, 1.2 mM
MgSO
4
, 1.3 mM CaCl
2
, 0.4 mM KH
2
PO
4
, 25 mM NaHCO
3
, 3 mM
glucose and bubbled with O
2
/CO
2
(95%:5%, v/v) until pH 7.4. First,
sulfonylthiourea 7 (1 mM, 10 mM, 100 mM) was added to the incu-
bation medium with
14
C-DG (0.1 mCi/mL) for 60 min, in order to
study the doseeresponse curve. Next, different drugs, such as
100 nM wortmannin, 10 mM N-[2-(p-bromocinnamylamino)ethyl]-
5-isoquinolinesulfonamide (H89), 100 nM phorbol 12-myristate
13-acetate (PMA) and 350 mM cycloheximide, were added to the
preincubation (30 min) and incubation (60 min) medium in the
presence or absence of sulfonylthiourea 7 10 mM with
14
C-DG
(0.1 mCi/mL), which was added to each sample during the incuba-
tion period only. After incubation, the muscle samples were ho-
mogenized in 0.5 N NaOH, and boiled at 100

C for 10 min, with
occasional stirring. Aliquots of 25 mL were taken from each sample
for radioactivity measurements in scintillation liquid Optiphase
Hisafe III (WallacOy, Turku, Finland) on an LKB rack beta liquid
scintillation spectrometer (model LS 6500; Multi-Purpose Scintil-
lation Counter-Beckman Coulter, Boston, USA) and 5 mL aliquots
were used for protein quantication by the Lowry method [38]. The
results were expressed as nmol glucose/mg of protein [34], with
modications.
5.9. Protein synthesis assays
For the protein synthesis, l-[Ue
14
C]-leucine incorporation into
proteins in soleus muscles from fasted rats was assayed. The soleus
muscles were removed and preincubated for 30 min in KRb buffer.
The buffer was bubbled with O
2
/CO
2
(95:5; v/v) up to pH 7.4. Sul-
fonylthiourea 7 (10 mM) was added to the incubation (60 min)
medium in KRb buffer, in the presence of l-[Ue
14
C]leucine (0.1 mCi/
mL). Incubation was stopped when cold trichloroacetic acid (TCA)
was added to a nal concentration of 7% and the samples were
processed as described by Menegaz et al. [39]. The total protein was
measured according to the method of Lowry et al. [38]. The results
of protein synthesis were expressed as cpm/mg protein.
5.10. Polyacrylamide gel electrophoresis and immunoblotting
analysis
For in vitro treatment, rat soleus muscles were preincubated
(30 min) with/without 350 mM cycloheximide and incubation
(60 min) medium, in the presence or absence of 10 mM sulfo-
nylthiourea 7. After incubation, these samples were homogenized
in a lysis solution containing 2 mM EDTA, 50 mM TriseHCl, pH
A. Mascarello et al. / European Journal of Medicinal Chemistry 86 (2014) 491e501 499
6.8, 4% (w/v) and the total protein was determined [40]. For the
electrophoresis analysis (whole homogenate), samples were dis-
solved in 25% (v/v) of a solution containing 40% glycerol, 5%
mercaptoethanol and 50 mM TriseHCl, at pH 6.8, and boiled for
3 min. Equal protein concentrations were loaded onto 12% poly-
acrylamide gels, analyzed by SDS-PAGE according to the discon-
tinuous system of Laemmli [41], and transferred to nitrocellulose
membranes for 1 h at 15 V in transfer buffer (48 mM Trizma,
39 mM glycine, 20% methanol and 0.25% SDS). The nitrocellulose
membranes were incubated for 2 h in blocking solution (TBS;
0.5 M NaCl, 20 mM Trizma, plus 5% defatted dried milk) and then
incubated overnight at 4

C with anti-GLUT-4 (1:500). Mem-
branes were incubated for 2 h with goat anti-mouse IgG (1:1000)
and immunoreactive bands were visualized using the Immobi-
lon Western chemiluminescence HRP substrate kit [38]. Auto-
radiograms were quantied by determining the optical densities
with OptiQuant version 02.00 software (Packard Instrument
Company).
5.10.1. Immunouorescence analysis
Soleus muscles from in vivo treatment with 10 mg/kg sulfo-
nylthiourea7 (harvested after 180 min of oral treatment) and from
in vitro treatment with/without 10 mM sulfonylthiourea 7 (60 min
of incubation) were xed in 4% p-formaldehyde, dehydrated and
embedded in parafn according to Dos Santos et al. [42]. Briey,
representative blocks of parafn-embedded tissues were cut in
5 mm, deparafnized, rehydrated and placed in 10 mM citrate
buffer for 8 min at 37

C for antigen retrieval. Sections were
submitted to a block solution of 3% BSA and 1% Igepal in PBS for
1 h at 37

C and incubated overnight at 7

C with mouse anti-
GLUT4 (1:500) in the same solution. After washing, sections
were incubated for 1 h at 37

C with anti-mouse conjugated to Cy3
(1:500) in the same solution and then counterstained with DAPI.
The sections were mounted in FluorSave. Qualitative analyses
were carried out of the distribution of GLUT4 in the tissue adapted
from Barros et al. [43].
5.11. Data and statistical analysis
The data were expressed as mean S.E.M. One-way analysis of
variance (ANOVA) followed by the Bonferroni post-test or unpaired
Student's t-test to determine the signicance of the differences
between groups. Differences were considered to be signicant at p
0.05.
Conict of interest
The authors declare no competing nancial interest.
Acknowledgments
This study was supported by grants and fellowships from the
Conselho Nacional de Desenvolvimento Cientco e Tecnol ogico-
Brasil (CNPq) (Grant 472071/2013-0), Coordena~ ao de Pessoal de
Nvel Superior (CAPES-PPG-Bioqumica) (Grant 554/07) and
Funda~ ao de Amparo a Pesquisa do Estado de Santa Catarina
(FAPESC) (Grant 745/000). We thank LAMEB I and II-CCB/UFSC for
the histology and uorescence microscopy facilities, and biologist
Chirle Ferreira and Gilberto Domingos Marloch for the technical
assistance.
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