Design, synthesis and in vivo evaluation of novel glycosylated sulfonylureas as antihyperglycemic agents

Article abstract of DOI:10.3390/molecules201119676

Sulphonylurea compounds have versatile activities such as antidiabetic, diuretic, herbicide, oncolytic, antimalarial, antifungal and anticancer. The present study describes the design, synthesis and in vivo testing of novel glycosylated aryl sulfonylurea compounds as antihyperglycaemic agents in streptozocine-induced diabetic mice. The rational for the introduction of the glucosamine moiety is to enhance selective drug uptake by pancreatic β-cells in order to decrease the cardiotoxic side effect commonly associated with sulfonylurea agents. 2-Deoxy-2-(4-chlorophenylsulfonylurea)-D-glucopyranose was found to be the most potent antihyperglycaemic agents among the synthesized compounds in diabetic mice. This investigation indicates the importance of this novel class as potential antihyperglycaemic agents.

Full text of DOI:10.3390/molecules201119676

Article
Design, Synthesis and in Vivo Evaluation
of Novel Glycosylated Sulfonylureas as
Antihyperglycemic Agents
Ghadeer A. R. Y. Suaifan 1,*, Mayadah B. Shehadeh 1, Rula M. Darwish 2, Hebah Al-Ijel 1
and Vincenzo Abbate 3
Received: 17 September 2015 ; Accepted: 21 October 2015 ; Published: 6 November 2015
Academic Editor: Maria Emília de Sousa
1
Department of Pharmaceutical Sciences, Faculty of Pharmacy, The University of Jordan, Amman 11942,
Jordan; m.shehadeh@ju.edu.jo (M.B.S.); ghadeer_petra@yahoo.com (H.A.-I.)
Department of Pharmaceutics and Pharmaceutical Technology, Faculty of Pharmacy,
2
The University of Jordan, Amman 11942, Jordan; rulamdarwish@yahoo.com
Institute of Pharmaceutical Science, King’s College London, Franklin-Wilkins Building,
3
150 Stamford Street, London SE1 9NH, UK; vincenzo.abbate@kcl.ac.uk
*
Correspondence: gh.suaifan@ju.edu.jo; Tel.: +962-6-5355-000 (ext. 23312); Fax: +962-6-5355-522
Abstract: Sulphonylurea compounds have versatile activities such as antidiabetic, diuretic,
herbicide, oncolytic, antimalarial, antifungal and anticancer. The present study describes
the design, synthesis and in vivo testing of novel glycosylated aryl sulfonylurea compounds
as antihyperglycaemic agents in streptozocine-induced diabetic mice. The rational for the
introduction of the glucosamine moiety is to enhance selective drug uptake by pancreatic
β-cells in order to decrease the cardiotoxic side effect commonly associated with sulfonylurea
agents. 2-Deoxy-2-(4-chlorophenylsulfonylurea)-D-glucopyranose was found to be the most potent
antihyperglycaemic agents among the synthesized compounds in diabetic mice. This investigation
indicates the importance of this novel class as potential antihyperglycaemic agents.
Keywords: sulphonylurea; antihyperglycemic agents; glucosamine; streptozocine
1. Introduction
Diabetes mellitus (DM) is a major degenerative disease with a serious cause of maladies in
the 21st century [1]. The burden of diabetes is increasing globally, particularly in developing
countries. In 2012, an estimated 1.5 million deaths were directly caused by diabetes [2] and in 2014,
347 million diabetic cases have been diagnosed worldwide. Moreover, Word Health Organization
(WHO) estimated diabetes to be the 7th leading cause of death in 2030 [3].
DM is divided into three main types: Type I, Type II and Gestational diabetes. Type II diabetes
mellitus (T2DM) accounts for more than 90% of all diabetic cases [4]. T2DM is a heterogeneous disease
associated with both genetic and environmental causative factors including multiple defects in insulin
secretion and action [5,6]. Insulin is a hormone that moves glucose inside the cells to produce energy.
Upon inadequate insulin secretion, glucose level in the blood increases (hyperglycemia). Extended
period of hyperglycemia causes irreversible damage to the eyes, kidneys, nerves and heart [7].
Hyperglycemia can be controlled by the administration of insulin which suppresses glucose
production and augments glucose utilization. However, being ineffective upon oral administration,
short shelf life, requirement of refrigeration, and in the event of over dose-fatal hypoglycemia limits
insulin administration [8]. Despite extensive research efforts, only two classes of oral hypoglycemic
agents (sulfonylureas and biguanides) are presently available as alternatives. Sulfonylurea agents
Molecules 2015, 20, 20063–20078; doi:10.3390/molecules201119676
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Molecules 2015, 20, 20063–20078
act by increasing insulin release from β islet cells while biguanides act by reducing the excessive
hepatic glucose production. Tolbutamide (1, Figure 1) was the first generation sulphonylurea
drug developed and was later supplanted by the second generation (gliclazide, glipizide and
glibenclamide (glyburide)) and the third-generation agent Glimepiride (2, Figure 1) [9]. However,
these agents are associated with severe and sometimes fatal hypoglycemia, gastric disturbances
like nausea, vomiting, heartburn, anorexia and increased appetite [10,11]. Nevertheless, these
hypoglycemic agents are actively pursued since it is very difficult to maintain normoglycemia by any
means in patients with DM. Therefore, the discovery of new hypoglycemic scaffolds with minimum
side effects is still a challenge to medicinal chemists [12–14].
O
O
O
S
O
O
N
H
O
N
S
H
N
O
H
O
N
N
H
1
2
OH
O
HO
HO
O
OH
HN
N
O
N
3
Figure 1. Chemical structures of Tolbutamide (1); Glimepiride (2) and Streptozotocin (3).
The clinical and medicinal importance of the arylsulfonyl urea moiety has been previously
approved being an active pharmacophore, exhibiting various pharmacological activities. Literature
review revealed sulfonylurea compounds with diverse biological activities such as antihyperglycaemia
(e.g., glibenclamide) [15–17], diuretic (e.g., torasemide) [18], herbicide (e.g., chlosulfuron) [19–22],
oncolytic (e.g., sulofenur) [23], antimalarial [24] antifungal [25] and anticancer [16,26,27]. Moreover,
extensive research support the utilization of streptozotocin (STZ) (3, Figure 1), an atural
chemotherapeutic agent isolated from Streptomyces achromogenes [28], as a selective toxin to pancreatic
β-cells. Hence, it is utilized to create animal models of diabetes [29], or to treat pancreatic
carcinoma [30]. The selective uptake and toxic effect of STZ against β-cells were previously
unclear. However, recent studies have demonstrated that STZ is selectively transported by a glucose
transporter (GLUT2) expressed in the pancreatic β-islet cells. This selective uptake is attributed to
the presence of the glucosamine moiety acting as an analogue to N-acetylglucosamine, one of the cell
wall petidoglycans [29,31,32].
In light of the above, we report the development of novel glycosylated sulfonylurea scaffolds
by integrating the aryl sulfonamide with a glucosamine moiety to promote its selective uptake by
pancreatic β-cells and to minimize adverse effects. The novel glycosylated sulfonylurea compounds
were synthesized and evaluated for their hypoglycemic effect on normal (Group A) and diabetic
(Group B) mice in comparison with the potent sulfonylurea antihyperglycaemic drug, Glimepiride (2).
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Molecules 2015, 20, 20063–20078
2. Results and Discussion
2.1. Chemistry
Arylsulfonylurea derivatives were previously synthesized fromhazardous, irritant and
moisture sensitive reagents such as arylsulfonyl isocyanates [33–36].
Later on, safer and
more environmentally favorable methodologies have been developed to circumvent the use
1
of these stoichiometric bases [37–40]. For example, tolbutamide (N-arylsulfonyl-N -alkylureas)
was synthesized by reacting benzenesulfonamides with N-alkylthiocarbonates obtained by
selenium (or DMSO) assisted carbonylation of amines with carbon monoxide and sulfur [41,42].
Other methods used N,N-Dimethylaminopyridine (DMAP)-catalyzed reaction of amines with
di-tert-butyldicarbonate[(Boc)2O] for the synthesis of isocyanates, which upon in situ trapping
by an additional equivalent of amine, will produce unsymmetrical urea derivatives [21,43–47].
1
Alternatively, N,N -unsymmetrical ureas were synthesized via the action of lithium methylpiperazine
with the N-Boc-protected primary amines [48]. Other useful methods applied for the synthesis of
ureas utilized cationic carbamoyl imidazolium salts which are derived from carbonyl diimidazole
(CDI) [46]. However, it appears that (Boc)2O and CDI utilized in the above procedures were
synthesized from phosgene. Accordingly, our targeted glycosylated sulfonylureas were synthesized
according to the versatile, non-hazardous and practical synthesis of 4-dimethylaminopyridinium
N-(arylsulfonyl) carbamoylide intermediates [38].
1,3,4,6-Tetra-O-acetyl-2-amino-2-deoxy-D-glucose hydrochloride (6, Scheme 1) was prepared
from the commercially available D-glucosamine hydrochloride. Initially, the NH2 was protected by
p-anisaldehyde (4) followed by acetylation (5) and removal of the p-methoxybenzylidene group with
HCl in warm acetone (Scheme 1) [49].
OAc
OH
OH
AcO
HO
HO
AcO
HO
HO
OAc
OH
OH
i
ii
N
N
NH3 Cl-
+
D-(+)-Glucosamine
hydrochloride
OCH3
OCH3
5
iii
4
OAc
AcO
AcO
OAc
NH3 Cl-
6
+
(i) aq. NaOH, CH3O–C6H4–CHO; (ii) Py, Ac2O; (iii) acetone, 5 M HCl.
Scheme 1. Chemical synthesis of compound 6.
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Molecules 2015, 20, 20063–20078
On the other hand, the arylsulfonylcarbamoylides compounds were prepared as shown in
Scheme 2. Sulfonamides (7a–c, 1 molar equiv.) reacted at room temperature with diphenyl
carbonate (DPC, 1.1 molar equiv.) in the presence of DMAP (2 molar equiv.) in acetonitrile,
to generate the title 4-dimethylaminopyridinium N-(arylsulfonyl)carbamoylides 8a–c in 60%–68%
yields. Neither prolonged reaction time nor elevated temperature changed the reaction course.
Moreover, purification of the precipitated product was achieved by simple filtration from acetonitrile
soluble by-products. Subsequent washing with diethylether removed excess DMAP. The above
procedure worked well with phenylsulfonamide and its para-substituted congeners, such as p-methyl
and p-chloro-phenylsulfonamide. The proposed mechanism of reaction for the preparation of
compounds 8a–c involves the initial replacement of DPC phenoxy group followed by formation of
the pyridinium salt 8. After this, a phenol molecule will be lost giving rise to the final product 8a–c.
Carbamoylide compounds were reported to be stable at room temperature for at least six months
and indefinitely when refrigerated [21,38,50]. The stability of these highly polarizable adducts is
mainly due to the delocalization of the positive charge on the pyridine ring and the negative charge
on the arylsulfonylcarbamoyl moiety [21]. The chemical structure of compounds 8a–c was confirmed
1
´1
by IR and H-NMR spectra. The IR spectra showed a vibration signal at 1694–1711 cm for C=O.
Moreover, the 1H-NMR spectra of compound 8a showed the N–CH3 protons to resonate as a singlet at
3.21 ppm and a pair of doublets for the pyridinium ring protons appeared at 7.6 (3,5-CH) and 8.7 ppm
(2,6-CH). Notably, these results were consistent with spectral data analysis of similar compounds [38].
O
O
O
O
O
N
OAc
O
S
S
AcO
AcO
NH2
N
i
OAc
N
+
NH3 Cl-
+
R
R
6
7a-c
a; R= H
b; R= Cl
c; R= Me
8a-c
a; R= H
b; R= Cl
ii
c; R= Me
OH
O
OAc
O
HO
HO
AcO
AcO
O
O
OH
OAc
O
S
NH
O
S
NH
iii
O
NH
O
NH
10a-c
9a-c
a; R= H
b; R= Cl
c; R= Me
a; R= H
b; R= Cl
c; R= Me
R
R
(i) DPC, DMAP, MeCN; (ii) MeCN and Et3N; (iii) NaOMe, MeOH and Dowex 50WX8.
Scheme 2. Chemical synthesis of compound 10a–c.
Direct coupling of D-glucosamine hydrochloride with carbamoylides 8a–c under different
reaction conditions turned out to be impossible. Thus, O-acetylated D-glucosamine hydrochloride
was reacted with carbamoylides 8a–c in acetonitrile at elevated temperature to afford the desired
arylsulfonylureas 9a–c. In a typical reaction, a slight excess of compound 6 (1.5 molar equiv.) was
added in one portion to a solution of 8a–c and Et3N (1.6 molar equiv.). The reaction mixture was
refluxed for 5–30 min and then cooled to room temperature to afford sulfonylureas 9a–c which were
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Molecules 2015, 20, 20063–20078
easily separated from the reaction mixtures following in situ acidification with 1% aqueous HCl. The
identity of the newly synthesized arylsulfonylureas 9a–c was proven by spectroscopic analysis. The
´1
infrared spectrum exhibited a characteristic absorption band at 1710 cm in close resemblance to
those for the carbamoylides 8a–c.
The final O-deacetylated compounds 10a–c were prepared according to the Zemplén
procedure [51]. Following deacetylation, a mixture of products were always obtained, irrespective
of whether the reaction was carried out by means of NaOMe in methanol, (CH3)2NH in methanol
or K2CO3 in methanol-water solution [52]. The deprotection process was monitored by TLC using
thymol/sulfuric acid as a detection reagent. Neutralization with Dowex 50WX8-200 ion-exchange
resin afforded a crude mixture. Purification attempts by recrystalization using MeOH and acetone,
afforded a crude gummy white solid. Thus compounds 10a–c were purified by semi preparative
HPLC. Notably, analysis of their 1H-NMR spectra drew our attention to the presence of α:β anomers.
The thermodynamically more stable α-stereoisomer of glucosamine was predominated. The H-1
signal of the α-anomer appears at higher δ value compared to that of the β-anomer, owing to their
different equatorial and axial orientations. This observation complies with the empirical rules of
carbohydrates NMR spectroscopy [53].
2.2. In Vivo Evaluation
Administration of streptozotocin was previously reported to rapidly destroy pancreatic β-cells
resulting in impairment of glucose-stimulated insulin release and induction of insulin resistance, both
of which are associated with type II diabetes [54]. The antihyperglycaemic effect of different doses
of compounds 10a–c in normal (Group A) and STZ-induced (Group B) diabetic mice were assessed
at different time intervals. The percentage change of glucose level from the initial fasting glycemia is
shown in Figures 2 and 3.
80
Negtive Control
Compound 10b 60mg/kg b.wt
60
Compound 10a 60mg/kg b.wt
Compound 10c 60mg/kg b.wt
40
Glimepride 1mg/kg b.wt
20
A
0
0
30
60
90
120 150 180 210 240 270 300
-20
-40
Time (min)
Negtive Control
80
60
40
20
0
Compound 10b 30mg/kg b.wt
Compound 10a 30mg/kg b.wt
Compound 10c 30mg/kg b.wt
Glimepride 1mg/kg b.wt
B
0
30
60
90
120 150 180 210 240 270 300
-20
-40
Time (min)
Figure 2. Change in blood sugar level in fasting normal mice. (A) 10a–c (60 mg/kg b.wt); (B) 10a and
10b (30 mg/kg b.wt). Each bar results are the mean ˘ SEM for n = 5–8 rats per treatment group.
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Molecules 2015, 20, 20063–20078
Figure 2A shows the change in blood glucose level in control and experimental normal mice
received 10a–c (60mg/kg body weight (b.wt.)) and Glimepiride. A raise up pattern to the highest
blood glucose level was observed at 60-min time point of the test for the experimental, negative
and positive control mice. The peak blood glucose level in the experimental and standard control
mice was found to be lower than the negative control. In a comparable way, compound 10b and the
positive standard drug retained the blood glucose level to the fasting glycemia after 120 min. On the
other hand, compounds 10a and 10c where less potent and took longer time. Tested compounds
showed significant antihyperglycaemic effect over different time intervals when compared to the
negative untreated control mice and standard Glimepiride as listed in Table 1.
Table 1. P-value for SPSS results in normal mice.
Time (min)
Compound (Dose)
0
30
60
90
120
180
240
270
300
10a (60 mg/kg b.wt)
10a (30 mg/kg b.wt)
10b (60 mg/kg b.wt)
10b (30 mg/kg b.wt)
10b (7.5 mg/kg b.wt)
10c (60 mg/kg b.wt)
10c (30 mg/kg b.wt)
Glimepiride 1 mg/kg b.wt
10a (60 mg/kg b.wt)
10a (30 mg/kg b.wt)
10b (60 mg/kg b.wt)
10b (30 mg/kg b.wt)
10b (7.5 mg/kg b.wt)
10c (60 mg/kg b.wt)
10c (30 mg/kg b.wt)
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
***
-
-
-
-
-
-
-
-
-
-
*
*
*
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
**
**
*
-
*
-
*
-
*
*
*
-
*
-
*
**
**
**
*
-
-
-
***
*
**
*
***
**
-
*
**
*
-
*
-
-
*
-
*
*
-
-
**
*
*
-
-
-
*
-
**
-
-
-
A
B
**
-
-
*
*
*
*
*
-
-
-
-
-
**
**
**
**
-
-
-
-
-
*
(A) * p < 0.05, ** p < 0.05 and *** p < 0.001 compared to control (untreated mice); (B): * p < 0.05, ** p < 0.05 and
*** p < 0.001 compared to Glimepiride (Standard drug).
Figure 2B shows the change in blood glucose level in control and experimental mice received
10a–c (30 mg/kg b.wt) and the Glimepiride. Decreasing the dose 10b delayed the time requested
to revert back to the fasting glycemia by an hour (i.e., at 180 min-point). Notably, compound
10a was able to maintain low blood glucose level. In general, compounds 10a–c steadily exerted
antihyperglycaemic effect at the tested doses when compared to the negative control.
Diabetic Mice (Group B)
Figure 3A shows the change in blood glucose level in control and experimental diabetic mice
received 10a–c (60 mg/kg b.wt.) and the standard drug. Glimepiride induced significant (p < 0.005)
blood glucose reduction after 90 min. Compounds 10a and 10b at 60 mg/kg dose maintained lower
blood glucose level compared to the untreated mice (negative control) at different test intervals. It was
observed that the peak glycemia in the negative control mice rose from initial level to the peak value
at 60–90 min time point and remained almost steady after 90 min. Whereas, Glimepiride produced
a slight but significant (p < 0.05) fall in blood glucose level as shown in Table 2. Maximal drop
down in glucose level, below the initial glycemia, was observed in mice treated with 10 at 60 mg/kg
and 30 mg/kg (Figure 3A,B). Remarkably, compound 10b exhibited a significant antihyperglycaemic
effect at 60 mg/kg, 30 mg/kg and 7.5 mg/kg over different time intervals when compared to the
negative untreated control mice and standard Glimepiride as shown in Figure 3 and Table 2.
Compound 10a, at 60 mg/kg, exhibited slower onset of hypoglycaemic action when compared
to 10b as the blood glucose level retrieved back to the fasting glycemia after 180 min. On the
other hand, its antihyperglycemic effect was comparable with Glimepiride at different time intervals.
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Molecules 2015, 20, 20063–20078
Decreasing the dose of 10a to half (30 mg/kg) lowered its antihyperglycemic effect as shown in
Figure 3B. Compound 10c illustrated a shorter duration of action with a noticeable diminish in its
antihyperglycemic effect at 120 min time point.
Figure 3. Change of blood sugar level in fasting STZ diabetic mice. (A) 10a–c (60 mg/kg b.wt); (B)
10a and 10b (30 mg/kg b.wt); (C) 10b (60 mg/kg, 30 mg/kg and 7.5 mg/kg b.wt). Each bar results
are the mean ˘ SEM for n = 5–8 rats per treatment group.2.2.1. Normal Mice (Group A).
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Molecules 2015, 20, 20063–20078
Table 2. P-value for SPSS results in diabetic mice.
Time (min)
Compound (Dose)
0
30
60
90
120
150
180
10a (60 mg/kg b.wt)
10a (30 mg/kg b.wt)
10b(60 mg/kg b.wt)
***
-
***
-
-
-
**
-
-
***
-
***
*
-
-
**
-
*
***
-
***
-
***
-
***
-
***
-
***
***
**
**
**
-
***
***
***
**
**
-
***
***
***
***
***
-
***
***
**
***
***
-
***
***
**
***
***
-
A
10b (30 mg/kg b.wt)
10b (7.5 mg/kg b.wt)
10c (60 mg/kg b.wt)
Glimepiride 1 mg/kg b.wt
10a (60 mg/kg b.wt)
10a (30 mg/kg b.wt)
*
*
*
*
-
10b (60 mg/kg b.wt)
10b (30 mg/kg b.wt)
-
-
-
-
-
-
-
-
-
-
-
-
-
-
B
10b (7.5 mg/kg b.wt)
10c (60 mg/kg b.wt)
-
-
-
-
-
-
-
-
-
-
-
-
-
-
(A): * p < 0.05, ** p < 0.05 and *** p < 0.001 compared to control (untreated mice); (B): * p< 0.05, ** p < 0.05 and
*** p < 0.001 compared to Glimepiride (standard drug).
The synthesized compounds 10a–c were designed on the basis of Topliss scheme for aromatic
substituent in quantitative structure activity relationship (QSAR) which considers the hydrophobicity
and the electronic effect of various substituents on activity. Thus, the first analogue synthesized
(compound 10b) was the 4-chloro derivative which is more hydrophobic (positive π) and
electron-withdrawing (positive σ) than hydrogen. Alternatively, the other target (compound 10c)
has a methyl substituent which is an example for a substituent with a positive π and negative σ
values. Based on the in vivo biological evaluation of compound 10b, it is possible to propose that
hydrophobic electron withdrawing substituent would be good for activity. Therefore, future research
should address optimum substituent.
The targeted compounds 10a–c were designed to be tolbutamide analogues by merging the
pharmacophoric features of tolbutamide with glucosamine moiety to provide higher potency and
selectivity. In this study, glimepiride, the third generation sulfonylurea drug was used as a
potent antihypergylcemic positive control [55]. Accordingly, the synthesized compounds were
postulated to act primarily as tolbutamide by occupying sulphonylurea receptors (SUR) subunits of
the ATP-sensitive potassium channel in pancreatic β-cells. Following occupation, potassium channels
close and calcium channels open to enhance insulin secretion from the pancreatic β-cells. This results
in exocytosis of insulin from storage granules [56]. Clearly, ATP-sensitive potassium channels are also
found in cardiac, skeletal and smooth muscles. However, in these tissues the channels are composed
of different SUR subunits that confer different drug sensitivities, even though, suphonylureas could
have unfavourable cardiac effect [6]. Nevertheless, the presence of glucosamine moiety would assess
selective uptake by β-cells and thus minimizes the adverse effect on cardiac potassium channels and
so reduce its cardiotoxic side effect [56].
Based on our proposal, the synthesized compounds were expected to exhibit higher
antihyperglycaemic effect in normal mice model having intact pancreatic β-cells upon comparison
with diabetic mice model, where pancreatic cells were partially destroyed by streptozocine. On
the contrary, in vivo results illustrated that the tested compounds 10a–c excreted pronounced
antihyperglycaemic effect in diabetic mice model upon comparison with normal mice model, in
particular compound 10b. At this stage, it is hard to hypothesize a proofed rational for the difference
in the antihyperglycaemic effect observed and to suggest the exact mechanism of action of the
synthesised compounds. However, we tried to postulate a plausible mechanism of action. It
might be possible that the glycosylated sulfonylurea compounds exerted their hypoglcemic effect
by stimulating the residual pancreatic β-cell or through an extra pancreatic mechanism, probably by
exerting insulin mimetic action [57]. Previous studies on the molecular mechanism of extra pancreatic
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Molecules 2015, 20, 20063–20078
activity of some sulfonylureas suggested that these drugs could induce glucosetransporter-4
translocation from internal stores to the plasma membrane and activate the key metabolic enzymes,
glycogen synthase and glycerol-3-phosphate acyltransferase [57,58]. Moreover, a study in rats had
showed that glimebride stimulates glycogenesis. Furthermore, some sulphonylurea drugs were
found to stimulate lipogenesis in 3T3 adipocytes [57]. Therefore, it is conceivable to propose that
the hypoglycaemic effect may be attributed to an extra pancreatic mechanism in diabetic mice. This
proposal could be supported by previous literature perusal, which showed that the hypoglycaemic
effect of some sulfonylureas as tolbutamide is very unlikely to be via the stimulation of insulin
secretion from the pancreas, but, it might be caused by an inhibition of the release of glucose from the
liver. Clearly, further work should be conducted to explain the possible mechanisms of action.
3. Experimental Section
3.1. General Information
Reagent grade chemicals and solvents were purchased from Sigma-Aldrich (St. Louis, MO, USA)
and used without purification. TLC was performed on silica gel F254 plates (Macherey-Nagel Inc.,
Bethlehem, PA, USA). Melting points were measured in open capillary tubes, using a Stuart melting
point apparatus and by Differential Scanning Calorimetry (DSC). IR spectra were recorded as KBr
discs on a Bruker Optik GmbH (Bruker, Ettlingen, Germany). Optical rotation was measured at
ambient temperature using an AA-10 polarimeter (Optical Activity Ltd., Cambridgeshire, UK) in a
1
cell volume of 1 mL and specific rotation are given in 10´1 deg¨mL¨g´1. H-NMR (300 and 500 MHz)
and 13C-NMR (75 and 125 MHz) spectral data were recorded on Advance spectrometers (Bruker,
Fallanden, Switzerland) with DMSO-d6 or D2O as a solvent and TMS as an internal standard. J values
are in Hz. Elemental analyses were recorded on EuroEA 3000 elemental analyser (Milano, Italy). High
resolution mass spectra (HRMS) were acquired (in positive or negative mode) using electrospray ion
trap (ESI) technique by collision-induced dissociation on a Bruker APEX-4 (7-Tesla) instrument.
3.2. Chemistry
2-Deoxy-2-[p-methoxybenzylidene(amino)]-D-glucopyranose (4). p-Anisaldehyde (3 mL, 23 mmol) was
added to a freshly prepared aqueous solution of D-glucosaminehydrochloride (5 g, 23 mmol)
dissolved in 1 M NaOH (24 mL). The mixture was stirred until crystallization began and then
refrigerated overnight. Filtration, washing with cold water (20 mL) followed by EtOH:Et2O (1:1,
˝
50 mL) and drying afforded compound 4 (5.26 g, 77%) as a white solid: m.p. 161–163 C (lit. [49] m.p.
˝
´1
1643 cm´1 (N=C), 3317 cm (O–H).
165–166 C), IR ν
max
1,3,4,6-Tetra-O-Acetyl-2-deoxy-2-[p-methoxybenzylidene(amino)]β-D-glucopyranose (5).
Compound 4
(4.0 g, 13 mmol) was added to a cooled mixture of pyridine (22 mL) and Ac2O (12 mL). The mixture
was stirred for 1 h and then left overnight at room temperature. The yellow solution was poured into
cooled water (80 mL). Filtration, washing with cold water (50 mL) and drying afforded compound 5
20
˝
˝
(5.5 g, 88%) as a white solid: m.p. 180–182 C (lit. [49] m.p. 180–182 C); rαsD +93.0 (c 1.0, MeOH),
lit. [49] rαs2D0 +95 (H2O): IR νmax 1751 (C=O) cm´1 1643 (C=N) cm´1; 1H-NMR (DMSO-d6. 300 MHz)
δH 1.79 (3H, s, CH3CO), 1.99 (6H, s, CH3CO), 2.48 (3H, s, CH3CO), 3.33 (1H, m, H-2), 3.77 (3H, s,
CH3O), 3.96 (1H, d, JH-5–H-6 = 11.5 Hz, H-5), 4.22 (2H, m, H-6α, H-6β), 4.91 (1H, t, JH-4–H-3 = 9.4 Hz,
H-4), 5.38 (1H, t, JH-3–H-4 = 9.4 Hz, H-3), 6.04 (1H, d, JH-1–H-2 = 7.6 Hz, H-1), 6.95 (2H, d, J = 8.2 Hz,
Ar-H2), 7.77 (2 H, d, J = 8.2 Hz, Ar-H2), 8.26 (1H, s, NCH).
1,3,4,6-Tetra-O-acetyl-β-D-glucosamine hydrochloride (6). Compound 5 (4.0 g, 8.6 mmol) was dissolved
in warm acetone (36 mL) to which HCl (5 M, 2 mL) was added with the formation of a precipitate.
The mixture was cooled and then Et2O (36 mL) was added and stirred for 2 h. Filtration, washing
˝
with Et2O and drying afforded compound 6 (2.9 g, 79%) as a white solid: m.p. 231–233
C
20
˝
˝
´1
2939 cm broad
(lit. [49] m.p. 235 C); rαsD +36.6 (c 1.0, MeOH), lit. [49] +32 (H O): IR ν
max
2
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Molecules 2015, 20, 20063–20078
´1
1
(NH3Cl), 1751 cm (C=O); H-NMR (DMSO-d6. 300 MHz) δH 1.95 (6H, s, CH3CO), 2.00 (3H, s,
CH3CO), 2.14 (3H, s, CH3CO), 3.52 (1H, t, JH-2–H-3 = 9.7 Hz, H-2), 3.98 (2H, m, H-6, H-5), 4.15
(1H, dd, JH-6β–H-6α = 12.3 Hz, JH-6–H-5 = 4.1 Hz, H-6), 4.87 (1H, t, JH-4–H-3 = 9.8 Hz, H-4), 5.28 (1H, t,
JH-3–H-4 = 9.8 Hz, H-3), 5.84 (1H, d, JH-1–H-2 = 8.8 Hz, H-1), 8.26 (3 H, s, NH3Cl).
3.2.1. General Procedure for the Synthesis of N-(Arylsulfonyl)carbamoylides 8a–c
Benzene sulfonamide compounds 7a–c (33 mmol) and 4-(N,N-dimethylamino)pyridine
(66 mmol) and diphenylcarbonate (37 mmol) in acetonitrile (40 mL) was stirred and then allowed
to stand at room temperature overnight. Filtration, washing with MeOH (2 ˆ 15 mL) and drying
afforded compounds 8a–c.
3.2.2. Yields, Melting Points, Analytical and Spectroscopic Data of 8a–c
4-Dimethylaminopyridininum N-(benzenesulfonyl) carbamoylide (8a). This compound was prepared from
˝
benzenesulfonamide 7a (3.0 g); yield 3.5 g (60%); white solid; m.p. 214–216 C (lit. [38] m.p.
˝
´1 ´1 ´1 ´1
214–217 C); IR ν
3081 cm (NH); 1706 cm (C=O), 1646 cm (amide I), 1573 cm (amide
max
´1
II), 1257 cm
J = 7.6 Hz, 3,5-py), 7.3 (3H, m, Ar-H3), 7.8 (2H, m, Ar-H2), 8.7 (2H, d, J = 7.6 Hz, 2,6-py).
(S=O); 1H-NMR (DMSO-d6. 300 MHz) δH 3.21 (6H, s, CH3NCH3), 6.93 (2H, d,
4-Dimethylaminopyridininum N-(4-chlorophenylsulfonyl) carbamoylide (8b). This compound was
prepared from 4-chlorobenzenesulfonamide 7b (3.0 g); yield 3.5 g (65%); white solid; m.p. 220–222 C
˝
˝
´1
(lit. [38] m.p. 221–223 C); IR νmax 3091 cm´1 (NH); 1711 cm´1 (C=O), 1646 cm´1 (amide I), 1569 cm
(amide II), 1253 cm (S=O); 1H-NMR (DMSO-d6. 300 MHz) δH 3.24 (6H, s, CH3NCH3), 6.97 (2H, d,
J = 7.9 Hz, 3,5-py), 7.54 (2H, d, J = 8.5 Hz, 2, 6 Ar-H2), 7.83 (2H, d, J = 8.5 Hz, 3, 5 Ar-H2), 8.8 (2H, d,
J = 7.9 Hz, 2,6-py).
´1
4-Dimethylaminopyridininum N-(4-methylphenylsulfonyl) carbamoylide (8c).
prepared from p-toluenesulfonamide 7c (3.0 g); yield 3.9 g (68%); white solid; m.p. 216–220 C (lit. [38]
This compound was
˝
˝
m.p. 220 C); IR νmax 3092 cm´1 (NH); 1694 cm´1 (C=O), 1645 cm´1 (amide I), 1575 cm´1 (amide II),
´1
1
1260 cm (S=O); H-NMR (DMSO-d6. 300 MHz) δH 2.5 (3H, s, CH3), 3.24 (6H, s, CH3NCH3), 6.97
(2H, d, J = 7.9 Hz, 3,5-py), 7.54 (2H, d, J = 8.5 Hz, 2, 6 Ar-H2), 7.83 (2H, d, J = 8.5 Hz, 3, 5 Ar-H2), 8.8
(2H, d, J = 7.9 Hz, 2,6-py).
3.2.3. General Procedure for the Preparation of Arylsulfonylureates 9a–c
N-(Arylsulfonyl)carbamoylide 8a–c (5 mmol) and triethylamine (10 mmol) were added to a
solution of compound 6 (6 mmol) dissolved in acetonitrile (15 mL).The mixture was refluxed for
5–30 min and then allowed to cool down. The solution was then acidified to form a precipitate.
Filtration, washing and drying afforded compounds 9a–c.
1,3,4,6-Tetra-O-acetyl-2-deoxy-2-(benzenesulfonylurea)-D-glucopyranose (9a).
This compound was
prepared from compound 6 (2.0 g) and 8a (3.7 g); yield 2.7 g (80%); white solid; Decomposed at
20
˝
´1
´1
´1
210.7–215.7 C; rαsD +10.4 (c 1.0, MeCN); IR ν
3309 cm (NH); 1759 cm (C=O), 1666 cm
max
´1
´1
1
(amide I), 1550 cm (amide II), 1165 cm (S=O); H-NMR (DMSO-d6. 500 MHz) δH 1.69 (3H, s,
CH3CO), 1.83 (3H, s, CH3CO), 1.92 (6H, s, CH3CO), 3.74 (1H, m, H-2), 3.90 (2H, m, H-5, H-6), 4.10
(1H, d, JH-6–H-5 = 7.6 Hz, H-6), 4.79 (1H, t, JH-4–H-3 = 9.7 Hz, H-4) 5.26 (1H, t, JH-3–H-4 = 9.7 Hz, H-3), 5.77
(1H, d, JH-1–H-2 = 7.9 Hz, H-1), 6.50 (1H, d, J = 8.5 Hz, NH), 7.58 (3H, m, Ar-H3), 7.83 (2H, d, J = 6.7 Hz,
Ar-H2), 10.94 (1H, s, NH); 13C-NMR (DMSO-d6. 125 MHz) 20.80 (COCH3), 20.85 (COCH3), 20.86
(COCH3), 20.91 (COCH3), 20.99 (COCH3), 52.15 (C2), 61.95 (C6), 68.96 (C4), 71.75 (C5), 72.20 (C3),
91.23 (C1), 126.01 (Ar), 129.42 (Ar), 132.32 (Ar), 144.25 (CO), 169.20, 169.69, 170.00, 170.56 (COCH3 ˆ 4)
;
HMS HRMS (ESI+) m/z 553.10987 [M + Na]+ (C21H26N2NaO12S requires 553.11004).
1,3,4,6-Tetra-O-acetyl-2-deoxy-2-(4-chlorophenylsulfonylurea)-D-glucopyranose (9b). This compound was
prepared from compound 6 (2.0 g) and 8b (3.3 g); yield 2.6 g (80%); white solid; Decomposed at
20072

Molecules 2015, 20, 20063–20078
20
˝
´1 ´1 ´1
3309 cm (NH); 1759 cm (C=O), 1666 cm (amide
185.3 C; rαsD +11.4 (c 1.0, MeCN); IR ν
max
´1
´1
I), 1550 cm (amide II), 1165 cm (S=O); 1H-NMR (DMSO-d6. 300 MHz) δH 1.69 (3H, s, CH3CO),
1.83 (3H, s, CH3CO), 1.92 (6H, s, CH3CO), 3.74 (1H, m, H-2), 3.90 (2H, m, H-5, H-6), 4.10 (1H, d,
JH-6–H-5 = 7.6 Hz, H-6), 4.79 (1H, t, JH-4–H-3 = 9.7 Hz, H-4) 5.26 (1H, t, JH-3–H-4 = 9.7 Hz, H-3), 5.77
(1H, d, JH-1–H-2 = 7.9 Hz, H-1), 6.50 (1H, d, J = 8.5 Hz, NH), 7.58 (3H, m, Ar-H3), 7.83 (2H, d,
J = 6.7 Hz, Ar-H2), 10.94 (1H, s, NH); 13C-NMR (DMSO-d6. 125 MHz) 20.74 (COCH3), 20.82 (COCH3),
20.88 (COCH3), 20.92 (COCH3), 20.95 (COCH3), 53.15 (C2), 61.93 (C6), 68.56 (C4), 71.78 (C5), 72.97
(C3), 92.23 (C1), 128.07 (Ar), 129.54 (Ar), 129.61 (Ar), 137.04 (Ar), 143.40 (C4-Ar), 141.68 (CO), 169.70,
169.75, 170.01, 170.51 (COCH3 ˆ 4); Elem. Anal. for C21H26N2O12S% Cal. C, 44.65; H, 4.46; N, 4.96,
Found C, 44.93; H, 4.14; N, 5.84; MS HRMS (ESI+) m/z 587.06180[M + Na]+ (C21H25ClN2NaO12S
requires 587.07144).
1,3,4,6-Tetra-O-acetyl-2-deoxy-2-(4-methylphenylsulfonylurea)-D-glucopyranose (9c). This compound was
prepared from compound 6 (2.0 g) and 8b (3.5 g); yield 2.7 g (79%); white solid; Decomposed at
20
˝
´1
´1
´1
217.1–221.3 C; rαsD +11.8 (c 1.0, MeCN); IR ν
3309 cm (NH); 1759 cm (C=O), 1666 cm
max
´1
´1
1
(amide I), 1550 cm (amide II), 1165 cm (S=O); H-NMR (DMSO-d6. 300 MHz): δH 1.73 (3H, s,
CH3CO), 1.85 (3H, s, CH3CO), 1.96 (3H, s, CH3CO), 1.99(3H, s, CH3CO), 2.51 (3H, s, CH3), 3.76 (1H,
m, H-2), 3.95 (2H, d, JH-5–H-6 = 10.8 Hz, H-5, H-6), 4.16 (1H, m, H-6), 4.88 (1H, t, JH-4–H-3 = 9.5 Hz, H-4),
5.30 (1H, t, J
= 9.99 Hz, H-3), 5.82 (1H, d, JH-1–H-2 = 8.6 Hz, H-1), 6.50 (1H, d, J = 11 Hz, NH),
H-3–H-4
7.41 (2H, d, J = 8.1 Hz, Ar-H2), 7.76 (2H, d, J = 8.1 Hz, Ar-H2), 10.8 (1H, s, NH); 13C-NMR (DMSO-d6.
125 MHz) 20.52 (COCH3), 20.72 (COCH3), 20.83 (COCH3), 20.93 (COCH3), 21.44 (COCH3), 53.15 (C2),
61.95 (C6), 68.56 (C4), 71.75 (C5), 72.47 (C3), 92.23 (C1), 127.66 (C2, C6-Ar), 129.91 (C3, C5-Ar), 137.67
(C1-Ar), 144.25 (C4-Ar), 151.68 (CO), 169.20, 169.69, 170.00, 170.46 (COCH3 ˆ 4); Elem. Anal. for
C22H28N2O12S% Cal. C, 48.53; H, 5.18; N, 5.14, Found C, 47.59; H, 4.82; N, 4.99;MS HRMS (ESI+) m/z
543.12902 [M ´ H+]´ (C22H28N2O12S requires 543.12847).
3.2.4. General Procedure for Deacetylationof Compounds 10a–c
Compounds 9a–c (0.4 mmol) in MeOH (20 mL) were added to a solution of 250 mmol NaOMe
in MeOH (20 mL). The mixture was stirred and monitored by TLC (CHCl3–MeOH 9:1). The TLC was
sprayed with a sugar detection visualizing reagent solution (thymol(0.5 g)in ethanol (95 mL) and 97%
sulfuric acid (5 mL)). The TLC plate was then heated until a pink spot appeared. Neutralization with
Dowex 50WX8-200 ion-exchange resin, filtration and evaporation afforded a viscous residue, which
was re-dissolved in water, dried with anhydrous Na2SO4 and filtered. Purification by HPLC afforded
compound 10a–c as a fluffy white solid.
2-Deoxy-2-(benzenesulfonylurea)-D-glucopyranose (10a). This compound was prepared from compound
9a (37%): Decomposed at 241.1 C (DSC); rαs2D0 +10.7 (c 1.0, H2O): IR νmax 3376 cm´1 (OH); 2823 cm
˝
´1
´1
´1
´1
´1
1
(NH); 1592 cm (C=O), 1383 cm (amide I), 1352 cm (amide II), 1133 cm (S=O) H-NMR
(DMSO-d6. 500 MHz) δH 3.13 (1H, m, H-2), 3.51–3.64 (5H, m, H-3, H-4, H-5, H-6, H-6), 4.20 (1H,
M, H-1β), 4.41 and 4.57 (1H, b, 1-OHα and β), 4.9–5.10 (2H, M, H-1α and OH), 5.48 (1H, b, OH), 5.87
(1H, b, OH), 6.31 (0.6H, b, NH), 7.37 (3H, m, Ar-H3), 7.51 (0.4H, b, NH), 7.73 (2H, m, Ar-H2), 8.5 (1H,
s, NHC=O); 13C-NMR (DMSO-d6. 125 MHz) 61.69 (C6), 70.91, 71.71, 72.39, 72.78, 77.08, 83.79 (C1β),
91.75 (C1α), 126.78 (Ar), 126.88 (Ar), 128.00 (Ar), 128.05 (Ar),129.69 (Ar), 166.54 (CO); MS HRMS
(ESI+) m/z 385.06761 [M + Na]+ (C13H18N2NaO8S requires 385.06815).
2-Deoxy-2-(4-chlorophenylsulfonylurea)-D-glucopyranose (10b). This compound was prepared from
20
˝
´1
´1
compound 9b (71%): Decomposed at 189.4 C (DSC); rαsD +10.95 (c 1.0, H O): IR ν
3421 cm
max
2
´1
´1
´1
´1
(OH); 2831 cm (NH); 1601 cm (C=O), 13361 cm (amide I), 1245cm (amide II), 1135 cm
1
(S=O); H-NMR (DMSO-d6. 500 MHz) δH 3.10 (1H, m, H-2), 3.40–3.70 (5H, m, H-3, H-4, H-5, H-6,
H-6), 4.20 (1H, M, H-1β), 4.42 and 4.45 (1H, b, 1-OHα and β), 4.9–5.10 (2H, M, H-1α and OH), 5.40
(1H, b, OH), 5.89 (1H, b, OH), 6.41 (1H, b, NH), 7.42 (2H, d, J = 8.4 Hz, Ar-H2), 7.8 (2H, d, J = 8.4 Hz,
Ar-H2), 10.8 (1H, s, NHC=O); 13C-NMR (DMSO-d6. 125 MHz) 61.69 (C6), 70.92 (C2), 71.66 (C5), 72.41
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Molecules 2015, 20, 20063–20078
(C4), 77.06 (C3), 91.74 (C1α), 95.74 (C1β), 128.02 (Ar), 128.96 (Ar), 134.31 (Ar), 166.83 (CO); MS HRMS
(ESI+): m/z 419.02864 [M + Na]+ (C13H17ClN2NaO8S requires 419.02918).
2-Deoxy-2-(4-methylphenylsulfonylurea)-D-glucopyranose (10c). This compound was prepared from
20
˝
´1
´1
compound 9c (81%): Decomposed at 230.9 C (DSC); rαsD +11.2 (c 1.0, H O): IR ν
3421 cm
max
2
´1
´1
´1
´1
(OH); 2831 cm (NH); 1601 cm (C=O), 13361 cm (amide I), 1245cm (amide II), 1135 cm
(S=O); 1H-NMR (DMSO-d6. 500 MHz) δH 2.51 (3H, s, CH3), 2.54(1H, m, H-2), 2.72–3.23 (5H, m, H-3,
H-4, H-5, H-6, H-6), 4.20 (1H, M, H-1β), 4.42 and 4.87 (1H, b, 1-OHα and β), 4.9–5.12 (2 H, M, H-1α
and OH), 5.40 (1H, b, OH), 5.9 (1H, b, OH), 6.41 (1H, b, NH), 7.15 (2H, m, Hz, Ar-H2), 7.59 (2H, d,
J = 7.8 Hz, Ar-H2), 8.49 (1H, s, NH), 13C-NMR (DMSO-d6. 125 MHz) 23.28 (COCH3), 60.61 (C6), 69.94
(C2), 70.05 (C5), 71.54(C4), 75.84(C3), 91.53 (C1β), 93.74 (C1α), 126.08 (Ar), 129.36 (Ar), 142.87 (Ar),
142.90 (Ar), 171.07 (CO); MS HRMS (ESI+): m/z 399.08326 [M + Na]+ (C14H20ClN2NaO8S requires
399.08380).
3.3. In Vivo Testing
3.3.1. Chemicals
Streptozotocin (STZ) was purchased from Sigma-Aldrich, Glimperide was kindly donated by
Dr Ismail M. Khalifeh from Dar Al Dawa Development & Investment Companyr (Amman, Jordan).
3.3.2. Animals
A total of 90 Balb/cmale mice, weight between 20–30 g were used in all experiments. The
animals were purchased from the Animal House in Applied Science University. The in vivo testing
was conducted in the animal house of the Faculty of Medicine at The University of Jordan. All
animals were acclimatized for a week before use and were maintained in hygienic conditions at
room temperature, fed with standard pellets and tap water in accordance with the in-house ethical
guidelines for animal protection. The study was conducted after obtaining Institutional animal
ethical committee’s clearance by the Scientific Research Committee at the Faculty of Pharmacy, The
University of Jordan. Animals were deprived from food and water for 18 h before in vivo initial
glycemia determination. Blood glucose level from cut tail tips was determined using an Accu-Chekr
Active Glucose meter.
3.3.3. Oral Glucose Tolerance Test
Experimental mice were divided into two groups. Group A contained normal mice and group
B contained STZ-induced diabetic mice. In each group, mice were randomly divided into five cages
I–V (n = 6 mice per subgroup). Experimental mice were fasted overnight (18 h) and then, initial
glycemia was determined (0 min). After which, glimperide (standard antihyperglycaemic drug,
1 mg/kg b.wt.) and compounds 10a–c at different doses 60, 30 and 7.5 mg/kg b.wt. were dissolved in
water for injection and directly administered intraperitoneally. Control untreated mice received the
vehicle (water). At 30 min time point of the test, glucose (2 g/kg b.wt.) was administered orally via
intra-gastric intubation to all test mice groups [54].
3.3.4. Induction of Diabetes in Mice
Diabetes in group B mice was induced by single intraperitoneal (IP) injection of freshly prepared
STZ (200 mg/kg b.wt) in 200 µL 0.1 M citrate buffer pH 4.5. Mice were supplied orally with glucose
solution (2 g/kg) for 48 h after STZ injection in order to prevent hypoglycemia. After 7 days, blood
glucose level from cut tail tips was measured followed by daily measurement until autopsy. Mice
with permanent fasting blood glucose level (FBGL) above 280 mg/dL were considered as diabetic
and included in this study. Negative control mice were treated with the vehicle only [55,59].
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Molecules 2015, 20, 20063–20078
3.3.5. Blood Collection and Determination of Blood Glucose
Blood glucose level from cut tail tips was monitored using an Accu-Chekr Active
glucometer [59]. The percentage (%) change of glucose level from the initial glycaemia was calculated
using the following formula: % glycaemia change = (Gx´ Go)/Go ˆ 100. Go is the initial glycaemia
value at zero time after overnight fasting; Gx is the glycaemia value at x minutes after vehicle or tested
compounds administration [1,60,61].
3.3.6. Statistical Analysis
Experimental results were expressed as mean ˘ SEM. The data were analyzed by ANOVA
(p < 0.05) and means separated by Dunnett multiple range tests (by SPSS version 16 software, SPSS
Inc., Chicago, IL, USA).
3.4. High Performance Liquid Chromatography (HPLC)
An integrated HPLC system (Thermo Fisher Scientific, Waltham, MA, USA) equipped with a
Surveyor LC pump, Surveyor auto sampler, UV 6000 LP and Surveyor UV-VIS photodiode array
detector was used with a Hypersilr reverse phase C18 column (250 mm ˆ 4.6 mm, 5 µm). Mobile
phase was prepared with buffer, ACN, THF (40:50:10). Buffer was prepared by dissolving 7.1 g of
K2HPO4 in 1 L of water and the pH was adjusted to 5.0 with H3PO4. The mobile phase was filtered
through a 0.45 µm filter (Supelco, Bellefonte, PA, USA).The flow rate was 1.0 mL/min. The injection
˝
volume was 20 µL. Absorbance was monitored at 254 nm at 25 C [62]. Semi preparative RP-HPLC
was conducted on a Waters SymmetryPrep™ C8, 7 µm, 19 ˆ 300 mm column using water/ACN
gradients at a flow rate of 7 mL/min.
4. Conclusions
In the present study, we have synthesized and evaluated novel class of glycosylated aryl
sulfonylurea antidiabetic agents. Compounds 10a–c exhibited antihyperglycemic activity in
streptozotocin-induced diabetic mice. The percentage blood glucose reduction induced by the tested
compounds in streptozotocin-induced diabetic mice is greater than that observed in normal treated
mice. Although, assay of the changes in blood glucose level was a regular method for detecting
the hypoglycaemic effect of the tested compounds, there is still a need for future work such as the
assay of blood insulin level which could provide us with a more reliable explanation for the observed
activity. The highest antihyperglycemic activity was achieved by compound 10b. This investigation
thus indicates the importance of these novel compounds as potential lead candidates.
Acknowledgments: Ghadeer A.R.Y Suaifan would like to acknowledge Robert C. Hider for his valuable support
and useful assistance. The authors would also like to acknowledge the Deanship of the Scientific Research,
The University of Jordan (Grant Number: 1532, 1541), Hamdi Mango Center for Scientific Research and Daniel
Turnberg Fellowship committee for financial support.
Author Contributions: Ghadeer A.R.Y. Suaifan designed, synthesized and analyzed the spectra of the target
compounds. Ghadeer A.R.Y. Suaifan wrote and revised the manuscript. Mayadah B. Shehadeh and Rula M.
Darwish performed the in vivo studies. Vincenzo Abbate purified the synthesized compounds. Hebah Al-Ijel
assisted in SPSS and excel data presentation.
Conflicts of Interest: The authors declare no conflict of interest.
References
1.
Yeap, S.K.; Liang, W.S.; Beh, B.K.; Ho, W.Y.; Yousr, A.N.; Alitheen, N.B. In vivo antidiabetic and acute toxicity
of spray-dried Vernonia amygdalina water extract. Int. Food Res. J. 2013, 20, 613–616.
World Health Organization. Global Health Estimates: Deaths by Cause, Age, Sex and Country, 2000–2012.
Geneva, WHO, 2014; Available online: http://www.who.int/mediacentre/factsheets/fs312/en/(accessed
on 27 October 2015).
2.
20075

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3.
Global Status Report on Non Communicable Diseases 2010; World Health Organization: Geneva, Switzerland,
2011; Available online: http://www.who.int/nmh/publications/ncd_report2010/en/(accessed on 23
October 2015).
4.
5.
6.
7.
8.
Zimmet, P.; Alberti, K.; Shaw, J. Global and societal implications of the diabetes epidemic. Nature 2001, 414,
782–787. [CrossRef] [PubMed]
Defronzo, R.A.; Bonadonna, R.C.; Ferrannini, E. Pathogenesis of NIDDM-a balanced overview.
Diabetes Care 1992, 15, 318–368. [CrossRef] [PubMed]
Rendell, M. The role of sulphonylureas in the management of type 2 diabetes mellitus. Drugs 2004, 64,
1339–1358. [CrossRef] [PubMed]
Kumar, M.; Verma, D. Antidiabetic andantihyperlipidemic effect of Morinda citrofolia and Coccinia indica in
alloxan induced diabetic rats. Pharmacologyonline 2011, 2, 307–311.
Kasiviswanath, R.; Ramesh, A.; Kumar, K.E. Hypoglycemic and antihyperglycemic effect of Gmelina asiatica
Linn. in normal and in alloxan induced diabetic rats. Biol. Pharm. Bull. 2005, 28, 729–732. [CrossRef]
[PubMed]
9.
Prendergast, B.D. Glyburide and glipizide, second-generation oral sulfonylurea hypoglycemic agents.
Clin. Pharm. 1984, 3, 473–485. [PubMed]
10. Del Prato, S.; Pulizzi, N. The place of sulfonylureas in the therapy for type 2 diabetes mellitus. Metabolism
2006, 55, S20–S27. [CrossRef] [PubMed]
11. Shammi, G.; Jitendra, K.R.; Narang, R.K.; Rajesh, K.S. Sulfonyl ureas for antidiabetic therapy, an overview
for glipizide. Int. J. Pharm. Pharm. Sci. 2010, 2, 1–6.
12. Hosseinzadeh, N.; Seraj, S.; Bakhshi-Dezffoli, M.E.; Hasani, M.; Khoshneviszadeh, M.; Fallah-Bonekohal, S.;
Abdollahi, M.; Foroumadi, A.; Shafiee, A. Synthesis and antidiabetic evaluation of benzenesulfonamide
derivatives. Iran. J. Pharm. Res. 2013, 12, 325–330. [PubMed]
13. Faidallah, H.M.; Khan, K.A. Synthesis and biological evaluation of new barbituric and thiobarbituric acid
fluoro analogs of benzenesulfonamides as antidiabetic and antibacterial agents. J. Fluor. Chem. 2012, 142,
96–104. [CrossRef]
14. Mariappan, G.; Saha, B.P.; Datta, S.; Kumar, D.; Haldar, P.K. Design, synthesis and antidiabetic evaluation
of oxazolone derivatives. J. Chem. Sci. 2011, 123, 335–341. [CrossRef]
15. Iqbal, Z.; Hameed, S.; Ali, S.; Tehseen, Y.; Shahid, M.; Iqbal, J. Synthesis, characterization, hypoglycemic and
aldose reductase inhibition activity of arylsulfonylspiro[fluorene-9,51-imidazolidine]-“21,41-diones. Eur. J.
Med. Chem. 2015, 98, 127–138. [CrossRef] [PubMed]
16. Kharbanda, C.; Alam, M.S.; Hamid, H.; Javed, K.; Shafi, S.; Ali, Y.; Alam, P.; Pasha, M.A.; Dhulap, A.;
Bano, S.; et al. Novel benzenesulfonylureas containing thiophenylpyrazoline moiety as potential
antidiabetic and anticancer agents. Bioorg. Med. Chem. Lett. 2014, 24, 5298–5303. [CrossRef] [PubMed]
17. Zhang, H.B.; Zhang, Y.A.; Wu, G.Z.; Zhou, J.P.; Huang, W.L.; Hu, X.W. Synthesis and biological
evaluation of sulfonylurea and thiourea derivatives substituted with benzenesulfonamide groups as
potential hypoglycemic agents. Bioorg. Med. Chem. Lett. 2009, 19, 1740–1744. [CrossRef] [PubMed]
18. Wargo, K.A.; Banta, W.M. A comprehensive review of the loop diuretics: Should furosemide be first line?
Ann. Pharmacother. 2009, 43, 1836–1847. [CrossRef] [PubMed]
19. Guo, W.C.; Liu, X.H.; Li, Y.H.; Wang, S.H.; Li, Z.M. Synthesis and herbicidal activity of novel sulfonylureas
containing thiadiazol moiety. Chem. Res. Chin. Univ. 2008, 24, 32–35. [CrossRef]
20. Sohn, H.; Lee, K.S.; Ko, Y.K.; Ryu, J.W.; Woo, J.C.; Koo, D.W.; Shin, S.J.; Ahn, S.J.; Shin, A.R.;
Song, C.H.; et al. In vitro and ex vivo activity of new derivatives of acetohydroxyacid synthase inhibitors
against Mycobacterium tuberculosis and non-tuberculous mycobacteria. Int. J. Antimicrob. Agents 2008, 31,
567–571. [CrossRef] [PubMed]
21. Saczewski, F.; Kuchnio, A.; Samsel, M.; Lobocka, M.; Kiedrowska, A.; Lisewska, K.; Saczewski, J.;
Gdaniec, M.; Bednarski, P.J. Synthesis of novel aryl(heteroaryl)sulfonyl ureas of possible biological interest.
Molecules 2010, 15, 1113–1126. [CrossRef] [PubMed]
22. Galeazzi, R.; Marucchini, C.; Orena, M.; Zadra, C. Molecular structure and stereoelectronic properties of
herbicide sulphonylureas. Bioorg. Med. Chem. 2002, 10, 1019–1024. [CrossRef]
23. Rostom, S.A. Synthesis and in vitro antitumor evaluation of some indeno[1,2-c]pyrazol(in)es substituted
with sulfonamide, sulfonylurea(-thiourea) pharmacophores, and some derived thiazole ring systems.
Bioorg. Med. Chem. 2006, 14, 6475–6485. [CrossRef] [PubMed]
20076

Molecules 2015, 20, 20063–20078
24. Leon, C.; Rodrigues, J.; Gamboa de Dominguez, N.; Charris, J.; Gut, J.; Rosenthal, P.J.; Dominguez, J.N.
Synthesis and evaluation of sulfonylurea derivatives as novel antimalarials. Eur. J. Med. Chem. 2007, 42,
735–742. [CrossRef] [PubMed]
25. Mastrolorenzo, A.; Scozzafava, A.; Supuran, C.T. Antifungal activity of silver and zinc complexes of sulfa
drug derivatives incorporating arylsulfonylureido moieties. Eur. J. Pharm. Sci. 2000, 11, 99–107. [CrossRef]
26. Szafran´ski, K.; Sławin´ski, J. Synthesis of novel 1-(4-substituted pyridine-3-sulfonyl)-3-phenylureas with
potential anticancer activity. Molecules 2015, 20, 12029–12044. [CrossRef] [PubMed]
27. Sławin´ski, J.; Szafran´ski, K.; Vullo, D.; Supuran, C.T. Carbonic Anhydrase Inhibitors. Synthesis
of Heterocyclic 4-Substituted Pyridine-3-Sulfonamide Derivatives and Their Inhibition of the Human
Cytosolic Isozymes I and II and Transmembrane Tumor-Associated Isozymes IX and XII. Eur. J. Med. Chem.
2013, 69, 701–710. [CrossRef] [PubMed]
28. Vavra, J.J.; Deboer, C.; Dietz, A.; Hanka, L.J.; Sokolski, W.T. Streptozotocin, a new antibacterial antibiotic.
Antibiot. Annu. 1958, 7, 230–235.
29. Pathak, S.; Dorfmueller, H.C.; Borodkin, V.S.; van Aalten, D.M.F. Chemical dissection of the link between
streptozotocin, O-Glcnac, and pancreatic cell death. Chem. Biol. 2008, 15, 799–807. [CrossRef] [PubMed]
30. Brentjens, R.; Saltz, L. Islet cell tumors of the pancreas—The medical oncologist’s perspective. Surg. Clin.
N. Am. 2001, 81, 527–542. [CrossRef]
31. Ran, C.; Pantazopoulos, P.; Medarova, Z.; Moore, A. Synthesis and testing of beta-cell-specific
streptozotocin-derived near-infrared imaging probes. Angew. Chem. Int. Ed. 2007, 46, 8998–9001. [CrossRef]
[PubMed]
32. Konopka, J.B. N-acetylglucosamine (GlcNAc) functions in cell signaling. Scientifica 2012, 2012, 1–15.
[CrossRef] [PubMed]
33. King, C. Some reactions of p-toluenesulfonyl isocyanate. J. Org. Chem. 1960, 25, 352–356. [CrossRef]
34. Irie, H.; Nishimura, M.; Yoshida, M.; Ibuka, T. Lewis acid catalysed preparation of some carbamates and
sulphonylureas. Application to the determination of enantiomeric purity of chiral alcohols. J. Chem. Soc.
Perkin Trans. 1989, 1, 1209–1210. [CrossRef]
35. Cervelló, J.; Sastre, T. An improved method for the synthesis of sulfonylureas. Synthesis 1990, 1990, 221–222.
[CrossRef]
36. Andreani, A.; Rambaldi, M.; Leoni, A.; Locatelli, A.; Andreani, F.; Gehret, J.C. Synthesis of
imidazo[2,1-b]thiazoles as herbicides. Pharm. Acta Helv. 1996, 71, 247–252. [CrossRef]
37. McKay, M.J.; Nguyen, H.M. Recent developments in glycosyl urea synthesis. Carbohydr. Res. 2014, 385,
18–44. [CrossRef] [PubMed]
38. Saczewski, F.; Kornicka, A.; Brzozowski, Z. 4-dimethylaminopyridinium carbamoylides as stable and
non-hazardous substitutes of arylsulfonyl and heteroaryl isocyanates. Green Chem. 2006, 8, 647–656.
[CrossRef]
39. Abbas, M.A.; Hameed, S.; Farman, M.; Kressler, J.; Mahmood, N. Conjugates of degraded and oxidized
hydroxyethyl starch and sulfonylureas: Synthesis, characterization, and in vivo antidiabetic activity.
Bioconjugate Chem. 2015, 26, 120–127. [CrossRef] [PubMed]
40. Tan, D.; Strukil, V.; Mottillo, C.; Friscic, T. Mechanosynthesis of pharmaceutically relevant
sulfonyl-(thio)ureas. Chem. Commun. 2014, 50, 5248–5250. [CrossRef] [PubMed]
41. Mizuno, T.; Iwai, T.; Ishino, Y. Solvent-assisted thiocarboxylation of amines and alcohols with carbon
monoxide and sulfur under mild conditions. Tetrahedron 2005, 61, 9157–9163. [CrossRef]
42. Sonoda, N.; Mizuno, T.; Murakami, S.; Kondo, K.; Ogawa, A.; Ryu, I.; Kambe, N. Selenium-catalyzed
synthesis of S-alkyl thiocarbamates from amines, carbon monoxide, sulfur, and alkyl halides. Chem. Int.
Ed. Engl. 1989, 28, 452–453. [CrossRef]
43. Knolker, H.J.; Braxmeier, T.; Schlechtingen, G. A novel method for the synthesis of isocyanates under mild
conditions. Angew. Chem. Int. Ed. Engl. 1995, 34, 2497–2500. [CrossRef]
44. Knolker, H.J.; Braxmeier, T.; Schlechtingen, G. Isocyanates, Part 2. Synthesis of symmetrical and
unsymmetrical ureas by DMAP-catalyzed reaction of alkyl- and arylamines with di-tert-butyldicarbonate.
Synlett 1996, 1996, 502–504. [CrossRef]
45. Knolker, H.J.; Braxmeier, T. Isocyanates, Part 3. Synthesis of carbamates by DMAP-catalyzed reaction of
amines with di-tert-butyldicarbonate and alcohols. Tetrahedron Lett. 1996, 37, 5861–5864. [CrossRef]
20077

Molecules 2015, 20, 20063–20078
46. Grzyb, J.A.; Batey, R.A. Carbamoylimidazolium salts as diversification reagents: An application to the
synthesis of tertiary amides from carboxylic acids. Tetrahedron Lett. 2003, 44, 7485–7488. [CrossRef]
47. Ishii, H.; Goyal, M.; Ueda, M.; Takeuchi, K.; Asai, M. Oxidative carbonylation of phenol to diphenyl
carbonate catalyzed by Pd dinuclear complex bridged with pyridylphosphine ligand. J. Mol. Catal. A Chem.
1999, 148, 289–293. [CrossRef]
48. Liberek, B.; Melcer, A.; Osuch, A.; Wakiec, R.; Milewski, S.; Wisniewski, A. N-alkyl derivatives of
2-amino-2-deoxy-D-glucose. Carbohydr. Res. 2005, 340, 1876–1884. [CrossRef] [PubMed]
49. Myszka, H.; Bednarczyk, D.; Najder, M.; Kaca, W. Synthesis and induction of apoptosis in B cell
chronic leukemia by diosgenyl 2-amino-2-deoxy-β-D-glucopyranoside hydrochloride and its derivatives.
Carbohydr. Res. 2003, 338, 133–141. [CrossRef]
50. Sa˛czewski, J.; Gdaniec, M. The structure and theoretical study of 4-dimethylaminopyridinium
n-(arylsulfonyl)carbamoylides. J. Mol. Struct. 2009, 921, 13–17. [CrossRef]
51. Fairweather, J.K.; Liu, L.; Karoli, T.; Ferro, V. Synthesis of disaccharides containing 6-deoxy-α-L-talose as
potential heparan sulfate mimetics. Molecules 2012, 17, 9790–9802. [CrossRef] [PubMed]
52. Lamothe, M.; Perez, M.; Colovray-Gotteland, V.; Halazy, S. A simple one-pot preparation of
N,N1-unsymmetrical ureas from N-Boc protected primary anilines and amines. Synlett 1996, 1996, 507–508.
[CrossRef]
53. Fowler, P.; Bernet, B.; Vasella, A. A 1H-NMR spectroscopic investigation of the conformation of the
acetamido group in some derivatives of N-acetyl-D-allosamine and -D-glucosamine. Helv. Chim. Acta
1996, 79, 269–287. [CrossRef]
54. Stella, J.; Krishnamoorthy, P.; Mohamed, A.J. Hypoglycemic effect of Vitex agnus castus in streptozotocin
induced. Asian J. Biochem. Pharm. Res. 2011, 1, 206–212.
55. Mohamed, M.S.; Ali, S.A.; Abdelaziz, D.H.; Fathallah, S.S. Synthesis and evaluation of novel pyrroles and
pyrrolopyrimidines as anti-hyperglycemic agents. Biomed. Res. Int. 2014, 2014. [CrossRef] [PubMed]
56. Bryan, J.; Munoz, A.; Zhang, X.; Duefer, M.; Drews, G.; Krippeit-Drews, P.; Aguilar-Bryan, L. ABCC8
and ABCC9: Abc transporters that regulate K(+) channels. Pflugers Arch. 2007, 453, 703–718. [CrossRef]
[PubMed]
57. Muller, G.; Wied, S. The sulfonylurea drug, glimepiride, stimulates glucose-transport, glucose-transporter
translocation, and dephosphorylation in insulin-resistant rat adipocytes in vitro. Diabetes 1993, 42,
1852–1867. [CrossRef] [PubMed]
58. Bahr, M.; Vonholtey, M.; Muller, G.; Eckel, J. Direct stimulation of myocardial glucose-transport and glucose
transporter-1 (GLUT1) and GLUT4 protein expression by the sulfonylurea glimepiride. Endocrinology 1995,
136, 2547–2553. [PubMed]
59. Priyadarshini, L.; Mazumder, P.B.; Choudhury, M.D. Acute toxicity and oral glucosetolerance test of ethanol
and methanol extracts of antihyperglycaemic plant Cassia alata Linn. IOSR J. Pharm. Biol. Sci. 2014, 9, 43–46.
60. Kasabri, V.; Afifi, F.U.; Hamdan, I. In vitro and in vivo acute antihyperglycemic effects of five selected
indigenous plants from Jordan used in traditional medicine. J. Ethnopharmacol. 2011, 133, 888–896.
[CrossRef] [PubMed]
61. Jimenez, J.; Risco, S.; Ruiz, T.; Zarzuelo, A. Hypoglycemic activity of Salvia lavandulifolia. Planta Med. 1986,
52, 260–262. [CrossRef]
62. Nirupa, G.; Tripathi, U.M. RP-HPLC analytical method development and validation for simultaneous
estimation of three drugs: Glimepiride, pioglitazone, and metformin and its pharmaceutical dosage forms.
J. Chem. 2013, 2013. [CrossRef]
Sample Availability: Samples of the compounds 9a–c and 10a–c are available from the authors.
© 2015 by the authors; licensee MDPI, Basel, Switzerland. This article is an open
access article distributed under the terms and conditions of the Creative Commons by
Attribution (CC-BY) license (http://creativecommons.org/licenses/by/4.0/).
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