Synthesis and evaluation of 1,ω-bis(1,2,3,5-thiatriazol-5-yl)alkanes as in vitro and in vivo α-amylase and lipase inhibitors

Article abstract of DOI:10.1002/ardp.201200312

Thionyl chloride reacts with 1,ω-bis-(1-tosylamidrazone)alkanes 1 to give a series of 1,ω-bis-(4-alkyl-2-tosyl-1,2,3,5-thiatriazol-5-yl)alkanes 2. All the newly synthesized compounds were characterized by IR, ¹H NMR, ¹³C NMR, elemental analysis, and ESI-MS spectral data. All the new compounds were screened for their inhibitory effect on key enzymes related to diabetes and obesity, such as α-amylase and lipase. In vitro and in vivo studies revealed that these thiatriazole derivatives exert an inhibitory action against these key enzymes. Moreover the administration of these compounds to surviving diabetic rats induced a significant decrease in plasma glucose level. Additively 2d significantly protected the liver-kidney functions and modulated lipid metabolism, which were evidenced by the decrease in aspartate transaminase (AST), alanine transaminase (ALT), and gamma-glutamyl transpeptidase (GGT) activities and creatinine, urea albumin, LDL-cholesterol and triglycerides levels as well as an increase in the HDL-cholesterol level in surviving diabetic rats. Overall, the findings of the current study indicate that 2d exhibits attractive properties and can, therefore, be considered for future application in the development of anti-diabetic and hypolipidemic drugs. A newly synthesized series of 1,ω-bis-(4-alkyl-2-tosyl-1,2,3,5- thiatriazol-5-yl)alkanes 2 were screened for their inhibitory effect on key enzymes related to diabetes and obesity, such as α-amylase and lipase. The administration of these compounds to surviving diabetic rats induced a significant decrease in plasma glucose levels. 2d significantly protected the liver-kidney functions and modulated lipid metabolism. Overall, compound 2d exhibited attractive properties, which make it interesting for future application in the development of anti-diabetic and hypolipidemic drugs. Copyright

Full text of DOI:10.1002/ardp.201200312

Arch. Pharm. Chem. Life Sci. 2013, 346, 321–329
321
Full Paper
Synthesis and Evaluation of 1,v-Bis(1,2,3,5-thiatriazol-
5-yl)alkanes as In Vitro and In Vivo a-Amylase and
Lipase Inhibitors
Salwa Hamzaoui1, Khaled Hamden2, Adel Ben Salem1, Maxime Mourer3,4
Jean-Bernard Regnouf-De-Vains3,4, and Mohamed Kossentini1
,
1
´
´ ´
`
Laboratoire de Chimie Appliquee: Heterocycles, Corps Gras et Polymeres Faculte des Sciences de Sfax,
´
´
Universite de Sfax, Sfax, Tunisia
2 Laboratory of Animal Ecophysiology, Faculty of Sciences of Sfax, University of Sfax, Sfax, Tunisia
3
´
Universite de Lorraine, SRSMC, UMR 7565; Groupe d’Etude des Vecteurs Supramoleculaire du
Medicament, Faculte de Pharmacie, Nancy, France
´
´
´
4
´
CNRS, SRSMC, UMR 7565, Groupe d’Etude des Vecteurs Supramoleculaire du Medicament,
´
´
Faculte de Pharmacie, Nancy, France
Thionyl chloride reacts with 1,v-bis-(1-tosylamidrazone)alkanes 1 to give a series of 1,v-bis-(4-alkyl-2-
tosyl-1,2,3,5-thiatriazol-5-yl)alkanes 2. All the newly synthesized compounds were characterized
by IR, 1H NMR, 13C NMR, elemental analysis, and ESI–MS spectral data. All the new compounds
were screened for their inhibitory effect on key enzymes related to diabetes and obesity, such as
a-amylase and lipase. In vitro and in vivo studies revealed that these thiatriazole derivatives exert
an inhibitory action against these key enzymes. Moreover the administration of these compounds
to surviving diabetic rats induced a significant decrease in plasma glucose level. Additively 2d
significantly protected the liver–kidney functions and modulated lipid metabolism, which were
evidenced by the decrease in aspartate transaminase (AST), alanine transaminase (ALT), and
gamma-glutamyl transpeptidase (GGT) activities and creatinine, urea albumin, LDL-cholesterol and
triglycerides levels as well as an increase in the HDL-cholesterol level in surviving diabetic rats.
Overall, the findings of the current study indicate that 2d exhibits attractive properties and can,
therefore, be considered for future application in the development of anti-diabetic and hypolipidemic
drugs.
Keywords: a-Amylase / 1,v-Bis-(thiatriazol-5-yl)alkanes / Diabetes / Lipase / Obesity
Received: August 8, 2012; Revised: December 24, 2012; Accepted: January 18, 2013
DOI 10.1002/ardp.201200312
Introduction
global mortality [1]. It is generally recognized that patients
with diabetes are at risk for numerous severe complications,
including diabetic hyperlipidemia, liver–kidney compli-
cations, and hypertension [2–4]. One of the therapeutic
approaches for decreasing hyperglycemia and obesity is
to retard absorption of glucose by the inhibition of
carbohydrate-hydrolyzing enzymes such as a-amylase and
a-glucosidase, in the digestive organs [5, 6]. Screening of
a-glycosidase inhibitors is becoming increasingly attractive
because a-glycosidases are responsible for the processing and
hydrolysis of complex carbohydrates into absorbable simple
sugar in the small intestine [2–5]. Inhibitors of these enzymes
are important molecular tools for glycobiology and can be
used to modulate cellular functions.
Diabetes mellitus is a major and growing public health
problem throughout the world, with an estimated worldwide
prevalence in 2008 of more than of 347 million people. This
disease is a heterogeneous disorder with varying incidence
and it is reported to constitute the 16th leading cause of
Correspondence: Professor Mohamed Kossentini, Laboratoire de
´
Chimie Appliquee, Heterocycles, Corps Gras et Polymeres, Faculte des
Sciences de Sfax, Universite de Sfax, 3018 Sfax, Tunisia.
E-mail: med_kossentini@yahoo.fr
Fax: 21674490320
´ ´
`
´
´
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322
S. Hamzaoui et al.
Arch. Pharm. Chem. Life Sci. 2013, 346, 321–329
On the other hand, five-membered heterocyclic com-
pounds, natural as well as synthetic, are important for their
biological activities. Compounds with thiatriazole rings are
of interest due to their broad spectrum of biological activi-
ties, such as antihypertensive [7], antibacterial [8], antifungal
[9], antitubercular [10], fungicidal [11], and anticancer activi-
ties [12]. Further, it has been reported that many biologically
active natural and synthetic products have interesting mol-
ecular symmetry [13]. Recently, some bis-triazole derivatives
endowed with antibacterial activities have been reported
from our laboratory [14]. Prompted by these observations
and in continuation of our search toward the synthesis of
new heterocyclic compounds with anticipated biological
activities, we synthesized a new series of compounds con-
taining two thiatriazoles rings and investigated the thera-
peutic action of these compounds on the diabetic status as
well as the activities of a-amylase and lipase with respect to
lipid profile and liver–kidney toxicity in alloxan induced
diabetic rats.
Experimental section. The formation of the cyclised products
2a–g was confirmed by IR, 1H NMR, 13C NMR, elemental
analysis, and ESI–MS. Assignment of selected characteristic
IR bands provides significant indications for the formation of
bis-thiatriazoles 2a–g. The absence of the band in the range of
3100–3250 cmꢀ1 confirmed the disappearance of the amino
groups, while the appearance of an intense band at around
1155 cmꢀ1 revealed the n (S O) stretch of thiatriazole rings;
–
–
the absorption band in the range of 1590–1610 cmꢀ1 was
1
–
attributed to the vibration of the –C N group. H NMR spec-
–
tra of compounds 2a–g performed in CDCl3 showed the
absence of the characteristics signals of NH groups at 6.5
and 8.7 ppm observed in the parent bis-amidrazones 1.
1
1
Interestingly, as shown by H, H COSY experiment (Fig. 1),
the N-CH2 resonance signals appeared as an ABm system
located between 3.30 and 3.80 ppm, indicating the non-
equivalence of these two protons, with respect to their
hindered environment. 13C NMR spectra showed resonance
signals in the range of 147–150 ppm corresponding to the
amidino carbons. Elemental analyses were consistent with
the proposed formula.
Results and discussion
In summary, all the synthesized compounds exhibited
satisfactory spectral data consistent with their molecular
structures.
Chemistry
Bis-amidrazones 1 were prepared according to the procedure
described in the literature [14]. These compounds have four
nucleophilic centers at positions 1,4 and 10,40 that are very
reactive face to compounds containing an electrophilic
center (e.g., thionyl chloride). In fact, the addition, in cold
conditions of two equiv. of thionyl chloride in dichloro-
methane with one equiv. of 1, in the presence of a slight
excess of anhydrous pyridine afforded, with a good yield, a
cyclized product identified as 1,v-bis-(4-alkyl-2-tosyl-1,2,3,5-
thiatriazol-5-yl)alkanes 2 (Scheme 1).
Biological activity
a-Amylase and lipase inhibition assay in vitro
Table 1 lists data for an initial series of key enzymes related
to diabetes and obesity as a-amylase and lipase inhibitors
having substitution on the thiatriazole ring. While all of
these compounds have IC50 values as a-amylase and lipase
inhibitors, relatively little variation in potency is observed.
It is noteworthy that compounds 2b, 2d, and 2g, the three
examples displaying a CH2CH3 substitution on the ring
increase the inhibitory action of these drugs against the
The characterisation data of 1,v-bis-(4-alkyl-1-oxo-2-tosyl-
1,2,3,5-thiatriazol-5-yl)alkanes 2a–g is presented in the
Ts
Ts
O
S
O
S
Ts
Ts
NH
4 N
HN
N
N
Cl
Cl
N
N
(CH2)n
S
N
C
+
2
N
R
N
N
(CH2)n
O
C
NH
HN
R
R
R
1
2
n
Compound
1a, 2a
R
Compound
1e, 2e
R
n
6
6
8
8
CH3
CH3
CH3
10
12
12
1b, 2b
C2H5
CH3
1f, 2f
1c, 2c
1g, 2g
C2H5
1d, 2d
C2H5
Scheme 1. Synthetic pathway of 1,v-bis-thiatriazoles derivatives 2a–h from 1,v-bis-amidrazones 1a–h.
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Arch. Pharm. Chem. Life Sci. 2013, 346, 321–329
Effect of Bisthiatriazoles on a-Amylase and Lipase
323
Figure 1. Partial 1H, 1H COSY spectrum of 2c
(CDCl3, 400 MHz, rt).
two enzymes. The largest inhibition of the activity of
a-amylase and lipase was observed in 2d, agreed by the
smaller IC50 in vitro and explaining the use of this compound
for the in vivo study.
glucose, TG and LDL-C and increase in LDH-C concentration
in the plasma of surviving diabetic rats (Fig. 2).
Liver functions of control and treated diabetic rats
Table 2 shows that the administration of 2d to surviving
diabetic rats seems to have reverted back this increase and
ameliorated all indices related to liver dysfunction induced
by diabetes. Further histological analyses confirmed the
positive effect of 2d. As compared to normal rats (Fig. 3A)
and to diabetic rats characterized by apparition of fatty cysts
(Fig. 3B), the histopathological study of 2d-treated diabetic
rats livers showed a neat decrease of the latter (Fig. 3D).
However, the administration of 2d to surviving diabetic rats
protects liver tissues.
a-Amylase and lipase activities in small intestine of control
and treated diabetic rats
The results revealed that diabetes induced a considerable
increase in the a-amylase and lipase activities in both the
small intestine and the plasma, which leads to a significant
increase of plasma glucose, TC and LDL-C and decrease in
HDL-C of diabetic rats. On the other hand, in 2d treated
diabetic rats, the activities of those enzymes underwent
considerable improvements, which leads to decrease of the
Table 1. In vitro a-amylase and lipase inhibition assay of thiatriazoles.
Compound
R
n
IC50 in mg/mL (mM)
against a-amylase
Compound
IC50 in mg/mL (mM)
against lipase
Acarbose (645.6)
2a (655.82)
2b (656.86)
2c (656.86)
2d (684.91)
2e (702.92)
2f (712.97)
2g (759.03)
14.88 (23.05)
94.32 (143.8)
84 (127.8)
82.96 (126.3)
65.16 (95.1)
94.22 (134.0)
91.33 (128.1)
69.25 (91.2)
Orlistat (495.73)
35.21 (71.0)
115.8 (176.5)
108.8 (165.6)
112.2 (170.8)
74.6 (108.9)
98.1 (139.5)
121.5 (170.4)
135.1 (178.0)
CH3
C2H5
CH3
C2H5
CH3
CH3
6
6
8
8
10
12
12
C2H5
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Arch. Pharm. Chem. Life Sci. 2013, 346, 321–329
20
18
16
14
12
10
8
*
3500
3000
2500
2000
1500
1000
500
*
*#@
*#@
#
#
6
4
2
0
0
Con
Diab
Diab +Acar Diab +2d
Con
Diab Diab +Acar Diab +2d
Groups
Groups
4
3,5
3
7
6
5
4
3
2
1
0
*
*
*#@
2,5
2
*#
*#
*#
1,5
1
0,5
0
Con
Diab
Diab +Acar Diab +2d
Con
Diab
Diab +Acar Diab +2d
Groups
Groups
25
*
20
15
10
5
*#@
*#
0
Con
Diab
Diab +Acar
2d
Groups
Figure 2. a-Amylase and lipase activities and plasma blood glucose level of control and experimental groups of rats. Values are statistically
presented as follows: ꢁp < 0.05 significant differences compared to controls. #p < 0.05 significant differences compared to diabetic rats
@p < 0.05 significant differences to diabetic rats treated with acarbose (Cont: control; Diab: diabetic; Diab þ Acar: diabetic rats treated with
acarbose; Diab þ 2d: diabetic rats treated with 2d).
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Arch. Pharm. Chem. Life Sci. 2013, 346, 321–329
Effect of Bisthiatriazoles on a-Amylase and Lipase
325
Table 2. Lipid profile, liver-kidney toxicity indices of control and experimental groups of rats.
Groups
Cont
Diab
DiabþAcar
Diabþ 2d
Serum (mmol/L)
T-Ch
TG
LDL-C
HDL-C
1.83 ꢂ 0.1
0.95 ꢂ 0.15
0.79 ꢂ 0.11
0.85 ꢂ 0.09
2.64 ꢂ 0.2ꢁ
1.77 ꢂ 0.14ꢁ
1.63 ꢂ 0.17ꢁ
0.65 ꢂ 0.05ꢁ
1.97 ꢂ 0.1ꢁ#
1.31 ꢂ 0.08ꢁ#
0.95 ꢂ 0.04ꢁ#
0.75 ꢂ 0.07#
2.18 ꢂ 0.13ꢁ#@
2.15 ꢂ 0.11ꢁ#@
0.93 ꢂ 0.02#
1.21 ꢂ 0.06ꢁ#@
Liver function
AST
ALT
T-Bili
D-Bili
GGT
82 ꢂ 6.59
52.5 ꢂ 9.25
1.52 ꢂ 0.13
0.18 ꢂ 0.01
3.25 ꢂ 0.5
185 ꢂ 11ꢁ
133 ꢂ 8.18ꢁ
2.58 ꢂ 0.18ꢁ
0.76 ꢂ 0.05ꢁ
5.5 ꢂ 0.57ꢁ
88 ꢂ 9#
69 ꢂ 69ꢁ#@
119 ꢂ 14ꢁ#@
1.78 ꢂ 0.12ꢁ#@
0.51 ꢂ 0.04ꢁ#@
4.12 ꢂ 0.61ꢁ#
58.3 ꢂ 4.5#
1.63 ꢂ 0.11#ꢁ#
0.31 ꢂ 0.08
4 ꢂ 0.81ꢁ#
Kidney function
Creatinine (mmol/L)
Urea (mmol/L)
40.8 ꢂ 1.52
6.64 ꢂ 0.35
1.41 ꢂ 5.08
49.26 ꢂ 0.25ꢁ
12.05 ꢂ 1.52ꢁ
148.79 ꢂ 13.81ꢁ
43.56 ꢂ 1.26ꢁ#
7.96 ꢂ 0.2ꢁ#
39.5 ꢂ 8.64#ꢁ#
8.71 ꢂ 0.54
Uric acid (mmol/L)
55.88 ꢂ 2.04ꢁ#
59.6 ꢂ 6.69ꢁ#
Values are statistically presented as follows: ꢁp < 0.05 significant differences compared to controls. #p < 0.05 significant differences
compared to diabetic rats. @p < 0.05 significant differences to diabetic rats treated with acarbose (Cont: control; Diab: diabetic;
Diab þ Acar: diabetic rats treated with acarbose; Diab þ 2d: diabetic rats treated with 2d).
Figure 3. Histopathological studies of liver in
the control and experimental groups of rats.
Section of the liver from a control rat showing
normal architecture (A); liver of diabetic rat
showing fatty cysts apparition in liver tissues
(B). Liver of diabetic rat treated with acarbose
(C) or 2d (D): protective effect is observed in
evidence by the absence of fatty cysts.
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Kidney functions of control and treated diabetic rats
vascular diseases, and liver–kidney dysfunction. For these
reasons, further studies are currently under progress in
our laboratories to more explore this anti-diabetic agent
and to make its application suitable for pharmaceutical
and medicinal application for the development of anti-
diabetic and hypocholesterolemic drugs.
Table 2 evidenced that the administration of 2d to surviving
diabetic rats seems to have reverted back this increase of
plasma creatinine, urea, and uric acid as compared to
untreated diabetic rats. The positive effect of 2d was confirmed
by histological analyses. In fact, in kidneys of diabetic rats
showing glomerular hypertrophy as compared to control rat
(Fig. 4A), administration of acarbose (Fig. 4C) and 2d (Fig. 4D) to
surviving diabetic rats showed a potential protective action,
evidenced by the reversion of glomerular size to normal.
Experimental
Chemistry
Melting points were determined with an Electrothermal 9100
apparatus and are uncorrected. IR spectra were recorded in KBr
pellets (n in cmꢀ1), on a Perkin–Elmer 100 infrared spectropho-
Conclusion
tometer whose precision is of 2 cmꢀ1 covering 400–4000 cmꢀ1
.
In summary, we prepared and evaluated a series of novel
1,v-bis-(4-alkyl-2-tosyl-1,2,3,5-thiatriazol-5-yl)alkanes for their
inhibitory effect of key enzymes related to diabetes and
obesity. Most of the compounds showed significant inhibi-
tory action against a-amylase and lipase, two enzymes impli-
cated in the development of diabetes and obesity. In
particular, the compound 2d showed promising inhibitor
activity against the two enzymes. It can allow to explore
new pathways for the treatment and prevention of diabetes
and related diseases, and can be considered as a potential
therapeutic agent for the treatment of diabetes, cardio-
The NMR spectra were recorded in CDCl3 on a Bruker Avance
spectrometer (300 MHz for 1H, 75 MHz for 13C). 1H and
13C chemical shifts are given on the d scale (ppm) and are
referenced to internal TMS. The multiplicities of the signals
are indicated by the following abbreviations: s: singlet, d:
doublet, t: triplet, q: quadruplet, m: multiplet and coupling
constants are expressed in Hertz (Hz). Mass spectra were acquired
on a Bruker micrOTOF-Q apparatus using electrospray ion trap
(ESI) technique by collision-induced dissociation (in positive or
negative mode). Elemental analyses were performed on a Thermo
Finnigan EA 1112 apparatus, both at the Service Commun
´
d’Analyses, Universite de Lorraine, Nancy.
Figure 4. (A) Normal rat kidney. (B) Diabetic rat
kidney: tubular epithelial damage messangial
and capillary proliferation. Kidney of diabetic rat
treated with acarbose (C) or 2d (D).
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Arch. Pharm. Chem. Life Sci. 2013, 346, 321–329
Effect of Bisthiatriazoles on a-Amylase and Lipase
327
The reactions and the purity of substances were monitored by
thin layer chromatography (TLC) (elution systems chloroform/
ethanol, 9:1) using aluminum sheets with silica gel 60 F254
Merck. All reactions, unless otherwise stated, were carried out
under nitrogen atmosphere in dry solvents under anhydrous
conditions. Bis-amidrazones 1 were synthesized according to
the literature procedures [14]. All other reagents were purchased
and used without purification.
1,8-Bis-(4-ethyl-1-oxo-2-tosyl-1,2,3,5-thiatriazol-5-yl)-
octane (2d)
1
–
–
–
Yield: 63%. Mp: 144–1468C. IR: 1151 (S O), 1598 (C N). H NMR
–
(CDCl3): 1.24 (m, 14 H, CH2CH3 and CH2), 1.71 (m, 4 H, CH2), 2.38
(s, 10 H, CH2CH3 and CH3 Ts), 3.49–3.65 (ABm, 4 H, CH2–N), 7.31
(d, 4 H, J ¼ 7.8 Hz, ArH Ts), 7.89 (d, 4 H, J ¼ 7.8 Hz, ArH Ts).
13C NMR (CDCl3): 10.5 (CH2CH3), 19.6 (CH2CH3), 21.8 (CH3 Ts),
26.5, 28.8, 30.2 (CH2), 42.8 (NCH2), 128.6, 129.5, 134.6, 145.3
(C Ar), 153.3 (C(4)). Anal. calcd. for C28H40N6O6S4 (684.91):
C 49.10; H 5.89; N 12.27; found: C 48.95; H 5.95; N 12.02. ESI–
MS (pos. mode): 685.19 [MþHþ]þ, 707.18 [MþNaþ]þ, 723.15
[MþKþ]þ.
Synthesis of bis-(4-alkyl-1-oxo-2-tosyl-1,2,4,3-thiatriazol-
5-yl)alkanes 2a–g
To a solution of 1.0 ꢃ 10ꢀ3 mole of bis-amidrazone 1 and
4.0 ꢃ 10ꢀ3 mole of pyridine with 20 mL of anhydrous dichloro-
methane (CH2Cl2), stirred magnetically in ice-cold water (H2O),
was added dropwise a solution of 2.0 ꢃ 10ꢀ3 mole of thionyl
chloride (SOCl2) with 10 mL of anhydrous dichloromethane. The
mixture was stirred during 48 h (TLC monitoring, SiO2, chloro-
form (CHCl3)/ethanol (C2H5OH) 9:1), then was washed three times
with 20 mL of distillated water. The dichloromethane layer was
recovered, dried and the solvent was evaporated to give the
resulting compound 2 precipitated soon by the addition of
diethyl ether. After filtration, the resulting compounds were
re-crystallized from methanol (CH3OH).
1,10-Bis-(4-methyl-1-oxo-2-tosyl-1,2,3,5-thiatriazol-5-yl)-
decan (2e)
1
–
–
–
Yield: 66%. Mp: 124–1268C. IR: 1596 (C N), 1152 (S O). H NMR
–
(CDCl3): 1.21 (m, 12 H, CH2), 1.63 (m, 4 H, CH2), 2.05 (s, 6 H, CH3),
2.34 (s, 6 H, CH3 Ts), 3.43–3.59 (ABm, 4 H, CH2–N), 7.23 (d, 4 H,
J ¼ 8.1 Hz, ArH Ts), 7.81 (d, 4 H, J ¼ 8.1 Hz, ArH Ts); 13C NMR
(CDCl3): 11.4 (CH3), 21.3 (CH3 Ts), 26.0, 28.4, 28.7, 39.8 (CH2), 42.7
(NCH2), 128.1, 129.1, 134.1, 144.8 (C Ar), 148.7 (C(4)). Anal. calcd.
for C28H40N6O6S4 ꢄ H2O (702.92): C 47.84; H 6.02; N 11.95; found:
C 47.94; H 5.86; N 11.82. ESI–MS (pos. mode): 685.19 [MþHþ]þ,
707.17 [MþNaþ]þ, 723.15 [MþKþ]þ.
1,6-Bis-(4-methyl-1-oxo-2-tosyl-1,2,3,5-thiatriazol-5-yl)-
hexane (2a)
1,12-Bis-(4-methyl-1-oxo-2-tosyl-1,2,3,5-thiatriazol-5-yl)-
dodecane (2f)
1
–
–
–
Yield: 55%. Mp: 174–1768C. IR: 1156 (S O), 1696 (C N); H NMR
–
(CDCl3): d 1.35 (m, 4 H, CH2), 1.71 (m, 4 H, CH2), 2.11 (s, 6 H, 2 CH3),
2.40 (s, 6 H, CH3 Ts), 3.49–3.65 (ABm, 4 H, CH2-N), 7.29 (d, 4 H,
J ¼ 8.4 Hz, ArH Ts), 7.87 (d, 4 H, J ¼ 8.4 Hz, ArH Ts). 13C NMR
(CDCl3): 11.2 (CH3), 21.8 (CH3 Ts), 26.2, 30.2 (CH2), 43.2 (NCH2),
128.6, 129.7, 134.6, 145.5 (C Ar), 149.2 (C(4)). Anal. calcd.
for C24H32N6O6S4 ꢄ H2O (655.82): C 43.95; H 5.22; N 12.81; found:
C 44.13; H 5.16; N 12.79. ESI–MS (pos. mode): 629.13 [MþHþ]þ,
651.11 [MþNaþ]þ, 667.08 [MþKþ]þ.
1
–
–
–
Yield: 70%. Mp: 140–1428C. IR: 1598 (C N), 1154 (S O), H NMR
–
(CDCl3): 1.23 (m, 16 H, CH2), 1.65 (m, 4 H, CH2), 2.12 (s, 6 H, CH3),
2.35 (s, 6 H, CH3 Ts), 3.45–3.62 (ABm, 4 H, CH2–N), 7.21 (d, 4 H,
J ¼ 7.8 Hz, ArH Ts), 7.88 (d, 4 H, J ¼ 7.8 Hz, ArH Ts). 13C NMR
(CDCl3): 11.9 (CH3), 21.7 (CH3 Ts), 26.6, 29.0, 29.3, 29.7, 30.3 (CH2),
43.3 (NCH2), 128.6, 129.6, 134.8, 145.2 (C Ar), 149.1 (C(4)).
Anal. calcd. for C30H44N6O6S4 (712.97):
N 11.79; found: C 50.96; H 6.28; N 11.85. ESI–MS (pos. mode):
C 50.54; H 6.22;
713.23 [MþHþ]þ, 735.21 [MþNaþ]þ, 751.18 [MþKþ]þ.
1,6-Bis-(4-ethyl-1-oxo-2-tosyl-1,2,3,5-thiatriazol-5-yl)-
hexane (2b)
1,12-Bis(4-ethyl-1-oxo-2-tosyl-1,2,3,5-thiatriazol-5-yl)-
dodecane (2g)
Yield: 60%. Mp: 160–1628C. IR: 1147 (S O),1595 (C N); 1H NMR
–
–
–
–
(CDCl3): 1.20 (t, 3 H, J ¼ 6.8 Hz, CH3), 1.35 (m, 4 H, CH2), 1.71
(m, 4 H, CH2), 2.38 (m, 10 H, CH3 Ts and CH2CH3), 3.50–3.61
(ABm, 4 H, CH2–N), 7.30 (d, 4 H, J ¼ 7.8 Hz, ArH Ts), 7.87 (d, 4 H,
J ¼ 7.8 Hz, ArH Ts). 13C NMR (CDCl3): 10.5 (CH2CH3), 19.6
(CH2CH3), 21.8 (CH3 Ts), 26.2, 30.2 (CH2), 42.7 (NCH2), 128.6,
129.6, 134.7, 145.3 (C Ar), 153.2 (C(4)). Anal. calcd.
for C26H36N6O6S4 (656.86): C 47.54; H 5.52; N 12.79; found: C
47.74; H 5.55; N 12.96. ESI–MS (pos. mode): 657.16 [MþHþ]þ,
679.15 [MþNaþ]þ, 695.12 [MþKþ]þ.
1
–
–
–
Yield: 67%. Mp: 133–1358C. IR: 1598 (C N), 1153 (S O). H NMR
–
(CDCl3): 1.23 (m, 16 H, CH2CH3 and CH2), 1.71 (m, 4 H, CH2),
2.41 (m, 10 H, CH2CH3 and CH3 (Ts)), 3.47–3.66 (ABm, 4 H, CH2–N),
7.29 (d, 4 H, J ¼ 8.1Hz, ArH Ts), 7.91 (d, 4 H, J ¼ 8.1 Hz, ArH Ts).
13C NMR (CDCl3): 10.5 (CH3), 19.6 (CH2CH3), 21.8 (CH3 Ts),
26.6, 29.0, 29.3, 29.7, 30.3 (CH2), 42.8 (NCH2), 128.6,
129.6, 134.8, 145.23 (C Ar), 153.3 (C(4)). Anal. calcd. for
C32H48N6O6S4 ꢄ H2O (759.03): C 50.63; H 6.63; N 11.07; found:
C 50.63; H 6.66; N 11.27. ESI–MS (pos. mode): 741.26 [MþHþ]þ,
763.24 [MþNaþ]þ, 779.21 [MþKþ]þ.
1,8-Bis-(4-methyl-1-oxo-2-tosyl-1,2,3,5-thiatriazol-5-yl)-
octane (2c)
1
Biology
Materials
–
–
–
Yield: 67%. Mp: 136–1388C. IR: 1170 (S O), 1595 (C N). H NMR
–
(CDCl3): 1.32 (m, 4 H, CH2), 1.58 (m, 4 H, CH2), 1.72 (m, 4 H, CH2),
2.13 (s, 6 H, CH3), 2.42 (s, 6 H, CH3 Ts), 3.48–3.69 (ABm,
4 H, CH2–N), 7.31 (d, 4 H, J ¼ 8.0 Hz, ArH Ts), 7.91 (d, 4 H,
J ¼ 8.0 Hz, ArH Ts). 13C NMR (CDCl3): 11.9 (CH3), 21.7 (CH3 Ts),
26.4, 28.8, 30.3 (CH2), 43.2 (NCH2), 128.6, 129.6, 134.7, 145.3 (C Ar),
149.0 (C(4)). Anal. calcd. for C26H36N6O6S4 (656.86): C 47.54; H
5.52; N 12.79; found: C 47.78; H 5.58; N 12.82. ESI–MS (pos. mode):
657.16 [MþHþ]þ, 679.15 [MþNaþ]þ, 695.12 [MþKþ]þ.
Porcine lipase and alpha-amylase, 4-methylumbelliferyl oleate
(4-MU oleate) and starch were obtained from Sigma (St. Louis,
MO, USA). Alloxan, was purchased from Sigma–Aldrich (St. Louis,
MO, USA), the GOD, HDL, TC, TG, aspartate transaminase (AST),
alanine transaminase (ALT), gamma-glutamyl transpeptidase
(GGT), T-bili and D-bili were from Biomaghreb analyticals
(Tunis, Tunisia). Orlistat and acarbose were purchased from
ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

328
S. Hamzaoui et al.
Arch. Pharm. Chem. Life Sci. 2013, 346, 321–329
Central Pharmacy (Sfax, Tunisia). All other chemicals used were
of analytical grade.
emulsified in sodium thioglycolate, lipase (0.4 unit) and the
sample was adjusted to pH 8.8, incubated at 258C. The inhibitory
activity of the sample was calculated from the titrant volume.
This was done in triplicate for each experiment.
Animals and treatments
The assays of the present study were conducted on adult male
Wistar rats, weighing 212 ꢂ 19 g, which were obtained from the
local Central Pharmacy, Tunisia. All rats were kept in an environ-
mentally controlled breeding room (temperature: 20 ꢂ 28C,
humidity: 60 ꢂ 5%, 12-h dark/light cycle) where they had stand-
ard diets and free access to tap water. The experimental protocols
were conducted in accordance with the guide for the care and
use of laboratory animals issued by the University of Sfax,
Tunisia, and approved by the Committee of Animal Ethics.
Diabetes was induced in rats by a single intraperitoneal injec-
tion of freshly prepared alloxan solution in normal saline at a
dose of 150 mg/kg body weight [6]. After 2 weeks, the rats whose
blood glucose was higher than 2 g/L were chosen for the exper-
iment. On the day the experiments started, and before treat-
ment, the rats were divided into four groups of eight animals,
each as follows:
AbsorbanceControl ꢀ Absorbancetest
PI ¼
ꢃ 100
Absorbancecontrol
Biochemical analysis
The mucosal small intestine of each rat was excised and the
lumen was flushed out several times with 0.9% NaCl. The
mucosal washing and the scraped mucosa were pooled, hom-
ogenized, and centrifuged (5000 ꢃ g, 15 min). The supernatant
was frozen and stored at ꢀ808C for further use in subsequent
enzymatic assays. The activity of a-amylase was obtained by
measuring the amount of glucose released from starch substrate
[15]. Lipase activity was assayed, with olive oil as substrate, using
the method described by Tietz and Fiereck [16]. For histochemical
procedures, tissue specimens of the liver and kidney were
obtained and fixed with 10% buffered formalin, and sub-
sequently embedded in paraffin. After that, the paraffin-
embedded samples were cut in sections (thickness, 5 mm) and
then stained with hematoxylin–eosin. The samples were then
examined using an Olympus CX41 light microscope.
Group 1: diabetic control rats at day 30 (Diab30)
Group 2: diabetic rats treated with acarbose by gastric gavage
route food (10 mg/kg of body weight/day during 30 days) and
termed [Diab–Acar].
Group 3: diabetic rats treated with 2d by gastric gavage route
food (65 mg/kg of body weight/day during 30 days) and termed
[Diab–2d].
Group 4: normal rats were used as controls, were scarified
at day 30, considered as referent non-diabetic rats at day 30
(Con30).
Statistical analysis
Data are presented as means ꢂ SD. Determinations were per-
formed from eight animals per group and differences were
examined by a one-way analysis of variance (ANOVA) followed
by the Fisher test (Stat View). The significance was accepted at
p < 0.05.
One month later, the rats were weighed and sacrificed by
decapitation, and their trunk blood collected. The serum was
prepared by centrifugation (1500 ꢃ g, 15 min, 48C).
We gratefully acknowledge the Ministry of Higher Education, Scientific
Research and Technology in Tunisia for financial support.
a-Amylase assay in vitro
The authors have declared no conflict of interest.
The in vitro a-amylase inhibition activity of all drugs was deter-
mined based on the spectrophotometric assay using acarbose as
the reference compound. The drugs were dissolved in DMSO
to give concentrations of 25, 50, and 100 mg/mL. The enzyme
a-amylase solution was prepared by mixing 3.246 mg of a-amylase
in 100 mL of 40 mM phosphate buffer, pH 6.9. Positive control,
acarbose was obtained by dissolving 50 mg in 50 mL of phosphate
buffer and diluted to get a concentration of 2.5 mg/mL. The essays
were conducted by mixing 80 mL of plant extract, 20 mL of
a-amylase solution and 1 mL of 2-chloro-4-nitrophenol-a-D-
maltotrioside (CNPG3) [16]. The mixture was incubated at 378C
for 5 min. The absorbance was measured at 405 nm. Similarly, a
control reaction was carried out without the drugs/acarbose.
Percentage inhibition (PI) was calculated by the expression:
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