New Biguanides as Anti-Diabetic Agents, Part II: Synthesis and Anti-Diabetic Properties Evaluation of 1-Arylamidebiguanide Derivatives as Agents of Insulin Resistant Type II Diabetes

Article abstract of DOI:10.1002/ardp.201700183

New 1-arylamidebiguanide hydrochloride salts were synthesized via reaction of hydrazide derivatives with dicyandiamide in acidic medium. The structure of the obtained derivatives was characterized by spectroscopic and elemental analysis tools. The anti-diabetic properties of the synthesized compounds were determined. Oral treatment of hyperglycemic rats with the synthesized biguanide derivatives showed a significant decrease of the elevated glucose in comparison with the anti-diabetic standard drug, metformin. The effects of the synthesized biguanide derivatives on the diabetic properties regarding liver function enzyme activities (AST, ALT, and ALP), lipid profiles (TC, TG, and TL), lipid peroxide, and nitrous oxide as well as histopathological characteristics were investigated and discussed.

Full text of DOI:10.1002/ardp.201700183

ARC HH PHARM
Arch. Pharm. Chem. Life Sci. 2017, 350, e1700183
Archiv der Pharmazie
Full Paper
New Biguanides as Anti-Diabetic Agents, Part II: Synthesis and
Anti-Diabetic Properties Evaluation of 1-Arylamidebiguanide
Derivatives as Agents of Insulin Resistant Type II Diabetes
1
1
2
1
3
Wahid M. Basyouni , Samir Y. Abbas
, Mohamed F. El Shehry , Khairy A.M. El-Bayouki , Hanan F. Aly ,
3
3
Azza Arafa , and Mahmoud S. Soliman
1
National Research Centre, Department of Organometallic and Organometalloid Chemistry, Cairo, Egypt
National Research Centre, Department of Pesticide Chemistry, Cairo, Egypt
National Research Centre, Department of Therapeutic Chemistry, Cairo, Egypt
2
3
New 1-arylamidebiguanide hydrochloride salts were synthesized via reaction of hydrazide derivatives
with dicyandiamide in acidic medium. The structure of the obtained derivatives was characterized by
spectroscopic and elemental analysis tools. The anti-diabetic properties of the synthesized compounds
were determined. Oral treatment of hyperglycemic rats with the synthesized biguanide derivatives
showed a significant decrease of the elevated glucose in comparison with the anti-diabetic standard
drug, metformin. The effects of the synthesized biguanide derivatives on the diabetic properties
regarding liver function enzyme activities (AST, ALT, and ALP), lipid profiles (TC, TG, and TL), lipid
peroxide, and nitrous oxide as well as histopathological characteristics were investigated and discussed.
Keywords: Anti-diabetic drug / Biguanides / Diabetes mellitus / Metformin
Received: June 7, 2017; Revised: August 25, 2017; Accepted: September 11, 2017
DOI 10.1002/ardp.201700183
Additional supporting information may be found in the online version of this article at the publisher’s web-site.
:
Introduction
previous work in this area [7, 8], we synthesized series of 1-
substituted-biguanide derivatives containing various moieties
such as aryl, benzo[1,3-d]dioxolyl, carboxamide, hydrazine, and
hydrazide moieties into the target compounds. Preliminary
structure activity relationships revealed that incorporation of the
hydrazide moiety enhanced the anti-diabetic activity of the
biguanidesystem. So,incontinuationofourongoingstudyinthis
field, the present study aimed to synthesize some new biguanide
derivatives by incorporating the hydrazide moiety.
Diabetic disorder is one of the most serious public health
problems overall the world [1, 2]. Biguanide derivatives were
known for more than 100 years, confirmed to be potent broadly
applications in medicine [3, 4]. 1,1-Dimethylbiguanide (metfor-
min) and phenylethylbiguanide (phenformin) have the ability to
reduce the blood glucose level through lowering of production
capacity of glucose which is produced by the liver. Metformin is a
first-line treatment for humans with type II diabetes and
currently used as the potent anti-diabetic agent. Metformin
decreases the absorbed sugar quantity from the body and makes
the insulin receptors in muscle tissue more sensitive [5, 6]. In our
Results and discussion
Chemistry
The required hydrazide derivative precursors, which are not
commercially available as far as we aware were prepared. So,
Correspondence: Prof. Samir Y. Abbas, Organometallic and
Organometalloid Chemistry Department, National Research
Centre, Cairo 12622, Egypt.
E-mail: samiryoussef98@yahoo.com
Fax: (þ202) 33370931
Additional correspondence: Prof. Wahid M. Basyouni,
E-mail: w_basyouny2002@yahoo.com
ß 2017 Deutsche Pharmazeutische Gesellschaft
www.archpharm.com
(1 of 9) e1700183

Arch. Pharm. Chem. Life Sci. 2017, 350, e1700183
W. M. Basyouni et al.
ARC HH PHARM
Archiv der Pharmazie
the desired hydrazide derivatives were synthesized as shown
in Scheme 1, where benzoic acid derivatives were subjected to
esterification by heating with ethanol containing catalytic
amount of sulfuric acid. Heating of the latter esters with
hydrazine hydrate in ethanol under reflux afforded the
required hydrazide derivatives. Upon heating the obtained
hydrazide derivatives with dicyandiamide (cyanoguanidine)
in ethanolic hydrochloric acid, the desired biguanide deriv-
atives 1–5 were afforded which separated from the reaction
mixture in crystallized form with good yield and high purity
the enhancement of gluconeogensis and the utilized glucose
level blood glucose is reduced. The present results (Table 1)
clearly indicated that diabetic rats exhibited significant
increase in blood glucose level with percentage increase
reached to 293.96% as compared to normal control rats.
A series of disubstituted biguanide hydrochloride salts were
1 2
synthesized which contain R and R at N-(1); in case of
compounds 1–3, R is amide side chain ending with substituted
1
phenyl moiety, where the substituted phenyl moieties are 2-
bromophenyl (1), 3-chlorophenyl (2), 4-chlorophenyl (3), and
(
Scheme 1). Structure of the products 1–5 was elucidated by
R
2
¼ hydrogen. In case of compound 6, R
methylphenoxy)acetyl)aminoandR
¼ hydrogen.The standard
drug (metformin), R ¼ CH . Hyperglycemic rats were
¼ R
1
is (2-(4-chloro-2-
careful studying of their spectral data. Thus, IR spectrum of
the biguanide hydrochloride 1 (as representative example)
showed multiple peaks at n: 3413, 3306, 3133 cm presum-
ably owing to NH and NH groups. Two strong stretching
peaks at: 1704 and 1638 cm were observed for C–O and C–N
2
1
2
3
ꢀ
1
treated orally with compounds 1–3 and 6 (2 mmol/kg/day) for
evaluating their anti-diabetic effect in comparison to metfor-
min hydrochloride (2 mmol/kg/day) for 4 weeks. Treatment of
diabetic rats with different synthetic compounds showed
marked amelioration in blood glucose level with different
percentage of improvements. Regarding the effect of
each substituent on N-(1): The standard drug (metformin),
2
ꢀ
1
1
functional groups, respectively. H NMR spectrum of 1
exhibited signals in the range of 6.80–8.00 owing to nine
protons corresponding to four aromatic protons, amino
group, and three NH protons. Amide NH proton was shifted
to down field tend to give rise to a singlet at: d ¼ 10.48.
R
1
¼ R
2
¼ CH
3
showed 263.41% improvement. Introduction of
andleft it R
1
3
C NMR spectrum of the biguanide 1 has characteristic signal
amide side chain ending with 2-bromophenyl at R
1
2
at: 167.0 ppm corresponding to C–O, beside the other
characteristic signals of aryl and C–NH carbons.
Other type of hydrazide was synthesized. Ethyl 2-(4-chloro-2-
methylphenoxy)acetate was synthesized via esterification of 2-
without substitution as in case of compound 1 resulted in
221.86% improvement. Changing the substituent at phenyl
moiety from 2-Br to 3-Cl as in compound 2 decreased the
percentage of improvement to 189.61%. Changing the
substituent at phenyl moiety from 3-Cl to 4-Cl as in compound
3 increased the percentage of improvement to 212.65%.
Introduction of (2-(4-chloro-2-methylphenoxy)acetyl)amino at
R and left it R without substitution resulted in the highest
1 2
activity among all the compounds investigated in this study;
compound 6 showed the 236.31% of improvement.
(
4-chloro-2-methylphenoxy)acetic acid with ethanol in acidic
medium. Hydrazide derivative (2-(4-chloro-2-methylphenoxy)-
acetohydrazide) was prepared by treating the latter ester with
hydrazine hydrate in ethanol under reflux (Scheme 1). Upon
heating the obtained hydrazide derivative with dicyandiamide
in ethanolic hydrochloric acid, the substituted biguanide 6 was
afforded with good yield.
HPLC analysis: The retention times of compounds 1–6 were
found to be around 2.408, 15.791, 18.000, 2.230, 14.998, and
Liver function enzyme activities
Diabetes mellitus is a major disease associated with disturban-
ces of the carbohydrate, fat and protein metabolism, affecting
nearly10%ofthepopulation.Aminotransferases(ALTandAST)
and ALP levels were significantly increased in STZ-treated
animals, the increase in aminotransferases levels may be due to
the cellular damage in the liver caused by STZ induction.
Increased levels of serum ALP in pathological conditions
2
7.804 min, respectively.
Biological activity evaluation
Streptozocin-induced hyperglycemia
Streptozotocin induction causes pancreatic b-cells damage. In
addition, breakdown of glycogen from liver is responsible for
Scheme 1. Syntheses of hydrazide derivatives and the biguanide derivatives.
ß 2017 Deutsche Pharmazeutische Gesellschaft
www.archpharm.com
(2 of 9) e1700183

Arch. Pharm. Chem. Life Sci. 2017, 350, e1700183
New Biguanides as Anti-Diabetic Agents
ARC HH PHARM
Archiv der Pharmazie
Table 1. Effects of the compounds 1–3 and 6 as well as metformin standard drug on blood glucose level in STZ-induced
diabetic rats.
Compd. no.
ve control
Diabetic rats
1
R
1
R
2
Glucose (mg/dL)
% Change
% Improvement
ꢀ
110.70 ꢁ 6.21
435.60 ꢁ 23.11
190.00 ꢁ 12.26
–
–
–
þ293.96
þ71.63
H
221.86
2
3
H
H
225.70 ꢁ 14.02
200.20 ꢁ 15.31
þ103.88
þ80.84
189.61
212.65
6
H
174.00 ꢁ 10.94
144.00 ꢁ 8.98
þ57.13
þ30.08
236.31
263.41
Metformin
CH
3
CH
3
involving the liver and kidney was observed. Oxidative stress
andalterationsinglucosemetabolismareimportantriskfactors
for diabetes and its related complications. Advanced glycation
end products (AGEs) and their carbonyl derivatives contribute
to the pathogenesis of diabetes by their interaction with
specific cell membrane receptors triggering to induce the
expression of pro-inflammatory mediators and elicit oxidative
stress, which exacerbate diabetic complications. A significant
elevation in liver function markers associated with insignificant
change in total protein content as compared to the normal
control group was illustrated. The high serum levels of these
enzymes after STZ treatment are associated with inflammation
and/or injury to liver cells, a condition known as hepatocellular
liver injury and apoptosis. Hyperglycemia resulted in hepatol-
ysis was reflected by increased blood serum aminotransferases
as one of the consequences of diabetic complication. With
respect to liver functional enzyme activities significant increase
in AST, ALT, and ALP enzyme activities in STZ-induced diabetic
rats with percentages increase reached to 67.36, 50.00, and
percentage of improvement 37.66, 37.64, 18.28 for AST, ALT,
ALP, respectively. Changing the substituent at phenyl moiety
from 3-Cl to 4-Cl as in 3 the percentage of improvement 41.84,
30.89, 17.16 for AST, ALT, ALP, respectively. Introduction of (2-(4-
1 2
chloro-2-methylphenoxy)acetyl)amino at R and left it R
without substitution resulted in the highest activities among
all the compounds investigated in this study; compound 6
showed the % improvement 64.85, 44.38, 38.53 for AST, ALT,
ALP, respectively.
Lipid profiles studies
The levels of TG, TC, and TL in diabetic rats were increased in a
significant way. Insulin activates lipoprotein lipase which
hydrolyzes triglycerides. Insulin deficiency results in the
failure of activate lipase enzyme, consequently causing
hypertriglyceridemia. Hyperglycemic associated with signifi-
cant increase in the blood levels of triglycerides (TG), total
cholesterol (TC), and total lipid (TL) with percentages increase
88.26, 196.17, and 55.90%, respectively were recorded. As
shown in Table 2, treatment of diabetic rats with the
synthesized compounds showed marked amelioration in
TG, TC, and TL levels. The standard drug (metformin),
4
0.92%, respectively, as compared to normal control group.
As shown in Table 2, treatments of diabetic rats with the
synthesized compounds 1–3 and 6 lead to improvements in
enzyme activities. Regarding the effect of each substituent on N-
R
1
¼ R
pound
moiety at R
2
¼ CH
with 2-(4-chloro-2-methylphenoxy)acetyl)amino
resulted in the highest activity among all the
3
showed 50.41% improvement for TL. Com-
(
5
1): The standard drug (metformin), R
4.49, 35.62% improvement for AST, ALT, ALP, respectively.
Compound 1 with amide side chain ending with 2-bromophenyl
at R resulted in the percentage of improvement of 62.76, 47.75,
9.43%forAST,ALT,ALP,respectively.Changingthesubstituent
1
¼ R
2
¼ CH
3
showed 67.78,
6
1
compounds investigated in this study for TL. Compound 6
exhibited activities greater than to the references drugs
(56.33%). The other compounds displayed weak activities for
TG and TC.
1
3
at phenyl moiety from 2-Br to 3-Cl as in 2 decreased the
ß 2017 Deutsche Pharmazeutische Gesellschaft
www.archpharm.com
(3 of 9) e1700183

Arch. Pharm. Chem. Life Sci. 2017, 350, e1700183
W. M. Basyouni et al.
ARC HH PHARM
Archiv der Pharmazie
Table 2. Effects of compounds 1–3 and 6 as well as metformin drug on serum AST, ALT, ALP, TG, TC, and TL in STZ-
induced diabetic rats.
Liver function profiles
Lipids profiles
AST
ALT
ALP
TG
TC
TL
Compd.
no.
mg/
dL
mg/
dL
mg/
dL
b)
R
1
R
2
U/L
%
U/L
%
U/L
%
%
%
%
Normal
2.39
4.00
–
–
1.78
2.67
–
–
88.88
125.3
–
–
89.12
167.78
–
–
26.9
79.67
–
–
449.34
700.54
–
–
Diabetic
a)
rats
1
H
2.50
62.76
1.83
47.75
90.20 39.43
130.45 41.88
50.69
107.7
490.32 46.78
2
3
H
H
3.10
3.00
37.66
41.84
2.00
2.12
37.64
30.89
109.3
110.0
18.28
17.16
120.23 53.35
124.23 48.86
45.0
128.8
117.7
554.80 32.43
587.23 52.21
48.00
6
H
2.45
2.38
64.85
67.78
1.88
1.70
44.38
54.49
91.00 38.53
93.59 35.62
123.80 49.35
100.01 76.04
50.50
43.73
108.4
133.6
447.43 56.33
474.04 50.41
Metformin
CH
3
CH
3
a)
b)
%
%
Change to control of diabetic rats: AST (67.36), ALT (50.00), ALP (40.92), TG (88.26), TC (196.17), TL (55.90).
Improvement.
Antioxidants studies
improvement 51.89, 33.01, 33.89, 105.19, and 99.43 for
glutahione, superoxide dismutase, glutathione reductase, lipid
peroxide, and nitric oxide, respectively. The other compounds
displayed weak activities.
Diabetic rats clearly demonstrated significant reduction in
glutathione-S-transferase (GST) in diabetic rats (45.56%),
significant increase in superoxide dismutase (SOD) in diabetic
rats with percentage increase 34.93% was observed (Table 3).
Moreover, significant increase in lipid peroxide (MDA) for
diabetic rats as compared to normal control with percentage
increase reached to 118.37% was observed. In addition, nitric
oxide (NO) level showed significant increase in diabetic rats as
compared to normal control rats with percentage increase
Adhesion molecules, inflammatory markers, and growth
factor evaluation
Adhesion molecules, inflammatory markers and growth
factor were performed using a sandwich enzyme immuno-
assay. As shown in Table 4, significant increase was noticed
in adhesion molecules ICAM and VCAM in diabetic rats.
While, significant decrease in adhesion molecules in treated
rats with the different chemical compounds, with the
greatest improvement was recorded for compound 6.
Inflammatory marker, TNF-a, demonstrated significant
increase in diabetic rats, while significant reduction in IL-
10 was detected in diabetic rats as compared to normal
control rats. Treatment of diabetic rats led to amelioration
in TNF-a and IL-10 levels with the most pronounced effect
with compound 6. In addition, VEGF showed significant
increase in diabetic rats as compared to normal control,
while treatment with the different chemical compounds
exhibited marked improvement with the greatest amelio-
ration with compound 6.
1
02.54%. RegardingtheeffectofeachsubstituentonN-(1):The
standard drug (metformin), R showed percentage
1
¼ R
2
¼ CH
3
of improvement56.96, 34.96, 33.23, 103.99, 3.21forglutahione
superoxide dismutase, glutathione reductase, lipid peroxide,
nitric oxide, respectively. Introduction of 2-bromophenylamine
1
or 2-(4-chloro-2-methylphenoxy)acetyl)amino moiety at R in
case of compounds 1 and 6 resulted in the highest activity
among all the compounds investigated in this study. Com-
pounds1and6exhibitedactivitiesgreaterthantothereference
drug for most of tests. The other compounds displayed weak
activities. Compound 1 exhibited percentage of improvement
5
9.49, 32.19, 24.37, 107.85, 98.29 for glutahione, superoxide
dismutase, glutathione reductase, lipid peroxide, and nitric
oxide, respectively. Compound 6 exhibited percentage of
ß 2017 Deutsche Pharmazeutische Gesellschaft
www.archpharm.com
(4 of 9) e1700183

Arch. Pharm. Chem. Life Sci. 2017, 350, e1700183
New Biguanides as Anti-Diabetic Agents
ARC HH PHARM
Archiv der Pharmazie
Table 3. Comparative effects of compounds 1–3 and 6 as well as metformin drug on enzymatic and non-enzymatic
antioxidant as well as oxidative stress markers, lipid peroxide, and nitric oxide.
Glutathione-
S-transferase
Superoxide
dismutase
(SOD)
Glutathione
reductase
(GR)
Lipid
peroxide
(MDA)
Nitric oxide
(NO)
(
GST)
Compd.
no.
mmol/
g
mmol/
g
mmol/
L
b)
R
1
R
2
%
U/g
%
U/L
%
%
%
Normal
0.79
0.22
–
–
2920.7
3941.2
–
–
121.87
70.70
–
–
865.8
1890
–
–
19.3
39.0
–
–
Diabetic
a)
rats
1
H
0.69
59.49
3000.9
32.19
100.4
24.37
956.8
107.85
20.1
98.29
2
3
H
H
0.43
0.47
26.58
31.64
3481.0
3200.2
15.96
25.37
93.12
98.60
18.39
22.89
1209
1230
78.74
76.24
29.2
26.9
51.24
63.16
6
H
0.63
0.67
51.89
56.96
2976.9
2920.1
33.01
34.96
112.0
111.2
33.89
33.23
979.9
990.4
105.19
103.9
19.9
21.1
99.43
93.21
Metformin
CH
3
CH
3
a)
b)
%
%
Change to control of diabetic rats: GSH (45.56), SOD (34.93), MDA (118.37), and NO (102.54).
Improvement.
Histopathological examination of the liver
renal tubules and peritubular inflammatory cells infiltration.
However, kidneys of diabetic rat treated with metformin
showed slight congestion of glomerular tufts. Meanwhile,
kidneys of diabetic rats treated with compounds 1, 2, and 3
revealed no histopathological changes. However, kidneys of
diabetic rats treated with compound 6 revealed vacuolation
of epithelial lining of the renal tubules and endothelial
lining of the glomerular tufts and slight congestion of
glomerular tuft. Kidney of rat from control group showed
the normal histological structure of renal parenchyma.
Kidney of diabetic rat showed vacuolation of epithelial
lining of the renal tubules and endothelial lining glomerular
tufts. Kidney of diabetic rat showed eosinophilic protein cast
in the lumen of renal tubules. Kidney of diabetic rat showed
peritubular inflammatory cells infiltration. Kidney of dia-
betic rat treated with standard (metformin) showed slight
congestion of glomerular tufts. Kidney of diabetic rat
treated with standard (metformin) showed slight congestion
of glomerular tufts. Kidney of diabetic rat treated with
compounds 1, 2, and 3 showed no histopathological changes.
Kidney of diabetic rat treated with compound 6 showed
vacuolation of epithelial lining of the renal tubules and
endothelial lining of the glomerular tuft. Kidney of diabetic
rat treated with compound 6 showed slight congestion of
glomerular tuft.
Microscopically, liver of diabetic rat showed cytoplasmic
vacuolization of hepatocytes, focal hepatic necrosis associ-
ated with inflammatory cells infiltration and hyperplasia of
epithelial lining of the bile duct. However, liver of diabetic
rat treated with standard (metformin) revealed no changes
except congestion of central vein). Moreover, liver of
diabetic rats treated with compounds 1, 2, 3, and 6 revealed
more or less similar changes. Most examined sections
revealed no changes except hydropic degeneration of
hepatocytes. Liver of untreated rat control showed the
normal histological structure of hepatic lobule. Liver of
diabetic rat showed cytoplasmic vacuolization of hepato-
cytes and focal hepatic necrosis associated with inflamma-
tory cells infiltration. Liver of diabetic rat showed
hyperplasia of epithelial lining of the bile duct. Liver of
diabetic rat treated with standard (metformin) showing
congestion of central vein. Liver of diabetic rat treated with
compounds 1–3 and 6 showed hydropic degeneration of
hepatocytes.
Histopathological examination of the kidneys
Examined kidney of diabetic rat showed vacuolation of
epithelial lining of the renal tubules and endothelial lining of
the glomerular tufts, eosinophilic protein cast in the lumen of
ß 2017 Deutsche Pharmazeutische Gesellschaft
www.archpharm.com
(5 of 9) e1700183

Arch. Pharm. Chem. Life Sci. 2017, 350, e1700183
W. M. Basyouni et al.
ARC HH PHARM
Archiv der Pharmazie
Table 4. Effect of chemical compounds adhesion molecules, inflammatory markers, and growth factor in STZ-induced
diabetic rats.
ICAM
(mg/mL)
VCAM
(mg/mL)
IL-10
(pg/mL)
VEGF
(pg/mL)
TNF-a
(pg/mL)
Groups
R
1
R
2
Control
Diabetic
rats
3.5 ꢁ 0.2
8.2 ꢁ 0.3
55.9 ꢁ 3.1
33.1 ꢁ 1.2
150.2 ꢁ 9.5
90.2 ꢁ 5.9
28.5 ꢁ 1.2
32.5 ꢁ 2.1
262.5 ꢁ 15.5 251.9 ꢁ 10.2
177.2 ꢁ 13.2 160.5 ꢁ 9.1
1
H
11.1 ꢁ 1.1
16.1 ꢁ 1.2
45.1 ꢁ 2.9
2
3
H
H
23.1 ꢁ 2.1
20.0 ꢁ 1.2
21.1 ꢁ 1.9
21.9 ꢁ 1.5
43.2 ꢁ 3.1
41.9 ꢁ 2.8
200.00 ꢁ 5.3 190.2 ꢁ 15.1
190.90 ꢁ 11.2 200.2 ꢁ 11.3
6
H
8.9 ꢁ 0.3
15.2 ꢁ 1.2
15.1 ꢁ 0.2
48.3 ꢁ 3.9
49.1 ꢁ 3.1
170.1 ꢁ 5.9 110.3 ꢁ 5.5
171.3 ꢁ 5.2 140.2 ꢁ 8.3
Metformin
CH
3
CH
3
12.1 ꢁ 0.9
Histopathological examination of the pancreas
Conclusion
Pancreas of diabetic rat showed vacuolation of cells of islets
of Langerhans, interacinar inflammatory cells infiltration,
and hyperplasia and thickening in the wall of pancreatic
duct. However, pancreas of diabetic rat treated with
standard (metformin) showed no changes except slight
vacuolation of cells of islets of Langerhans. Pancreas of
diabetic rat treated with compound 1 showed hyperplasia
and hypertrophy of islets of Langerhans. No histopatho-
logical changes were noticed in pancreas of diabetic rats
treated with compounds 1 or 3. However, pancreas of
diabetic rat treated with compound 6 showed hyperplasia
and hypertrophy of islets of Langerhans. Pancreas of rat
from control showed the normal histological structure of
pancreatic parenchyma. Pancreas of diabetic rat showed
vacuolation of cells of islets of Langerhans. Pancreas of
diabetic rat showed interacinar inflammatory cells
infiltration.
Pancreas of diabetic rat showed hyperplasia and thickening in
the wall of pancreatic duct. Pancreas of diabetic rat treated with
standard (metformin) showed slight vacuolation of cells of islets
ofLangerhans.Pancreasofdiabeticrattreatedwithcompound1
showing no histopathological changes. Pancreas of diabetic rat
treated with compound 2 showed hyperplasia and hypertrophy
of islets of Langerhans. Pancreas of diabetic rat treated with
compound 3 showed no histopathological changes. Pancreas of
diabetic rat treated with compound 6 showed hyperplasia and
hypertrophy of islets of Langerhans (Table 5).
Biguanidesareanintrinsicallyinterestingclassofcompoundswith
many known or potential applications. The synthesized
1-substituted-amidebiguanide derivatives 1 and 6 showed
significant decrease in the elevated blood glucose level. Also,
theantioxidantimprovementofmostofthesynthesizedproducts
revealed percentages higher than that reported by metformin
hydrochloride were observed. A significant decrease in adhesion
molecules in treated rats with the different chemical compounds,
with the greatest improvement was recorded for compound 6.
Histopathological study of the diabetic rats treated with 1-
substituted-amidebiguanide derivatives showed apparently
healthy hepatic cords and blood sinusoids. Kidneys of the diabetic
rats treated with 1-substituted-amidebiguanide derivatives
showed apparently healthy renal tubules and renal glomeruli
for their kidneys tissues. Also, pancreas of diabetic rats treated
with 1-substituted-amidebiguanide derivatives showing appar-
ently healthy pancreatic lobules with normal pancreatic acini and
well preserved b-cells.
Experimental
Chemistry
General
All melting points are uncorrected and measured using
Electro-thermal IA 9100 apparatus, (Shimadzu, Tokyo, Japan).
ß 2017 Deutsche Pharmazeutische Gesellschaft
www.archpharm.com
(6 of 9) e1700183

Arch. Pharm. Chem. Life Sci. 2017, 350, e1700183
New Biguanides as Anti-Diabetic Agents
ARC HH PHARM
Archiv der Pharmazie
Table 5. Lesion score of diabetic and treated diabetic rats with tested compounds.
Histopathological lesions
Liver
Kidneys
Pancreas
Compd. no.
R
1
R
2
CVH FNH HD
HBD CCV VIL
HIL
HPD ICI
VELRT VCGT PICI
Diabetes
1
þþ
ꢀ
þþ
ꢀ
þþ
þ
þþþ þþ
þþþ
ꢀ
ꢀ
þþþ þþ
þþþ
ꢀ
þþþ
ꢀ
þþ
ꢀ
H
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
2
3
6
H
H
ꢀ
ꢀ
ꢀ
ꢀ
þ
þ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
H
þ
ꢀ
ꢀ
ꢀ
þ
ꢀ
ꢀ
ꢀ
ꢀ
þ
þ
þ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
þ
ꢀ
þ
þ
ꢀ
ꢀ
Metformin
HCl
CH
3
CH
3
CVH, cytoplasmic vacuolization of hepatocytes; FNH, focal necrosis of hepatocytes; HD, hydropic degeneration; HBD, hyperplasia
of bile duct; CCV, congestion of central veins; VIL, vacuolation of islets of Langerhans; HIL, hyperplasia of islets of Langerhans;
HPD, hyperplasia of pancreatic duct; ICI, inflammatory cells infiltration; VELRT, vacuolation of epithelial lining renal tubules;
VCGT, vacuolation and congestion of glomerular tufts; PICI, peritubular inflammatory cells infiltration.
Microanalyses were carried out by the Microanalytical
Laboratory, National Research Centre, Cairo, Egypt. Infrared
spectra (KBr-disc) were recorded using a Jasco FT/IR-300E
by filtration and washed with methanol to yield an initial crop
of biguanide hydrochloride.
1
13
spectrometer. H NMR and C NMR spectra were measured in
DMSO-d using Varian Mercury 500 MHz and Varian Gemini
00 MHz with chemical shifts using TMS as standard solvent.
2-(2-Bromobenzoyl)-N-
carbamimidoylhydrazinecarboximidamide hydrochloride
6
2
(1)
ꢀ
1
Mass spectra were recorded on a GC/MS Finnigan SSQ 7000
spectrometer. Reactions were monitored by TLC on 0.25 mm
Merck Silica gel sheets (60 GF 354) (4 ꢂ 2 cm) and the spots
were detected with UV light.
The InChI codes of the investigated compounds together
with some biological activity data are provided as Supporting
Information.
Yield 73%; mp 222–224°C; IR: n/cm : 3413, 3306, 3133 (NH),
1704 (C–O), 1638 (C–N); H NMR: d/ppm: 6.80–8.00 (m, 9H,
1
4Ar-H, NH
(br, 1H, CONH, D
(2C), 130.5, 132.2, 133.5, 160.1 (2CNH), 167.0 (C–O); Anal.
calcd. for C 12BrClN O (335.59): C, 32.21; H, 3.60; N, 25.04;
2 2
, 3NH), 9.44 (br, 1H, NH, D O-exchangeable), 10.48
1
3
2
O-exchangeable); C NMR: 120.1, 128.0
9
H
6
Found: C, 32.54; H, 3.42; N, 24.87%.
General procedure for the synthesis of the hydrochloride
salt of 1-substituted-biguanide derivatives 1–6
N-Carbamimidoyl-2-(3-chlorobenzoyl)-
hydrazinecarboximidamide hydrochloride (2)
ꢀ
1
In a round-bottomed flask equipped with a magnetic stirrer,
arylhydrazide (0.1 mol) was added to aq. HCl (0.1 mol) in 50 mL
ethanol and the mixture was stirred at room temperature
until it became homogeneous. Dicyandiamide (0.1 mol) was
then added, and the mixture was heated at reflux with
constant stirring for 12 h. The mixture was then cooled to
room temperature, and the resulting crystals were separated
Yield 85%; mp 174–176°C; IR: n/cm : 3382, 3342, 3184 (NH),
1
1687 (C–O), 1646 (C–N); H NMR: d/ppm: 6.80–8.00 (m, 9H,
4Ar-H, NH
(br, 1H, CONH, D
130.9 (2C), 132.0, 133.7, 160.3 (2C–NH), 169.3 (C–O); Anal.
calcd. for C O (291.14): C, 37.13; H, 4.15; N, 28.87;
2 2
, 3NH), 9.33 (br, 1H, NH, D O-exchangeable), 10.77
1
3
2
O-exchangeable); C NMR: 127.2, 128.3,
9 2 6
H12Cl N
Found: C, 36.94; H, 4.21; N, 28.67%.
ß 2017 Deutsche Pharmazeutische Gesellschaft
www.archpharm.com
(7 of 9) e1700183

Arch. Pharm. Chem. Life Sci. 2017, 350, e1700183
W. M. Basyouni et al.
ARC HH PHARM
Archiv der Pharmazie
N-Carbamimidoyl-2-(4-chlorobenzoyl)-
hydrazinecarboximidamide hydrochloride (3)
Biological activity evaluation
ꢀ
1
Yield 86%; mp 189–191°C; IR: n/cm : 3424, 3351, 3177 (NH),
Anti-diabetic properties
1
1
4
(
680 (C–O), 1647 (C–N); H NMR: d/ppm: 6.80–8.00 (m, 9H,
Seventy male rats with an average weight of 220–250 g were
obtained from animal house lab., National Research Centre,
Dokki, Giza were used in this study. Animals were housed
under normal laboratory condition for 1 week before
initiation of the biological experiments (adaptation period),
housed in a well-ventilated box (22 ꢁ 20°C) on a 12 h light and
dark cycle. Animals were fed with natural basal diet. Diets and
water were supplied ad libitum and with free access to water.
The animals were divided randomly into seven main groups of
ten rats each to study the effect of the synthesized products as
well as metformin on the blood glucose level, liver function
enzyme activities and lipid profile levels in STZ-induced
diabetic rats as follows:
Ar-H, NH , 3NH); 9.35 (br, 1H, NH, D O exchangeable), 10.77
2
2
13
br, 1H, CONH); C NMR: 128.9 (2CH), 130.4 (2CH), 131.4 (C),
37.5 (CCl), 160.8 (2C–NH), 166.1 (C–O); Anal. calcd. for
O (291.14): C, 37.13; H, 4.15; N, 28.87; Found: C,
7.51; H, 4.03; N, 28.58%.
1
9 2 6
C H12Cl N
3
N-Carbamimidoyl-2-(4-hydroxybenzoyl)-
hydrazinecarboximidamide hydrochloride (4)
ꢀ
1
Yield 84%; mp 225–227°C; IR: n/cm : 3381, 3218 (OH, NH),
1
1
687 (C –– O), 1649 (C –– N); H NMR: d/ppm: 6.83 (d, 2H,
O-exchangeable),
& NH), 7.78 (d, 2H, J ¼ 8.60 Hz, Ar-H), 9.30,
0.29, 10.49 (3br, 3H, 2NH & OH, D O-exchangeable);
C NMR: 115.5 (2C), 122.7, 130.1 (2C), 160.7 (2C –– NH),
61.7, 167.1 (C –– O); Anal. calcd. for C
13ClN (272.69):
J ¼ 8.60 Hz, Ar-H), 7.06 (br, 2H, 2NH, D
2
7
1
.64 (br, 3H, NH
2
2
Group 1: Normal control rats.
1
3
Group 2: Diabetes was induced by STZ. Each rat was injected
intraperitoneally with a single dose of STZ (45 mg/kg body
weight) dissolved in 0.01 M citrate buffer immediately before
use. After injection, animals had free access for food and
water and were given 5% glucose solution to drink overnight
to encounter hypoglycaemic shock. Animals were checked
daily for the presence of glycosuria. Animals were considered
to be diabetic if glycosuria was present for 3 consecutive days.
1
9
H
6 2
O
C, 39.64; H, 4.81; N, 30.82; Found: C, 39.32; H, 4.68; N,
0.54%.
3
N-Carbamimidoyl-2-(4-methylbenzoyl)-
hydrazinecarboximidamide hydrochloride (5)
ꢀ1
Yield 86%; IR: n/cm : 3387, 3182 (NH), 1685 (C–O), 1643
1
(
C–N); H NMR: d/ppm: 2.31 (s, 3H, CH
3
), 6.83–7.78 (m, 9H, 4Ar-
After 3 days of STZ injection, fasting blood samples
1
3
H & 3NH & NH
15.4 (2C), 122.1, 130.8 (2C), 160.2 (2C–NH), 161.0, 167.4 (C–
O); Anal. calcd. for C10 15ClN O (270.72): C, 44.37; H, 5.58; N,
1.04; Found: C, 44.74; H, 5.49; N, 30.87%.
2
), 9.30, 10.31 (2br, 2H, 2NH); C NMR: 21.2,
were obtained and fasting blood sugar was determined
(>300 mg/dL). Hyperglycemic rats were used for the experi-
ment and classified as follows:
Groups 3–6: Diabetic rats orally administered 2 mmol/kg
body weight synthetic organic compounds for 30 days,
respectively.
1
H
6
3
N-Carbamimidoyl-2-(2-(4-chloro-2-methylphenoxy)-
acetyl)hydrazine carboximidamide hydrochloride (6)
Group 7: Diabetic rats orally administered anti-diabetic
metaformin reference drug 2 mmol/kg body weight daily for
30 days.
ꢀ1
Yield 86%; mp 223–225°C; IR: n/cm : 3300, 3167 (NH), 1670
C–O), 1640 (C–N); 1H NMR: d/ppm: 2.13 (s, 3H, CH
(
(
3
), 4.61
, 4NH), 10.10 (br,
H, CONH); C NMR: 16.42, 66.99, 113.78, 125.09, 126.82,
29.31, 130.54, 155.36, 167.12; Anal. calcd. for C11
2 2
s, 2H, OCH ), 6.60–7.40 (m, 9H, 3Ar-H, NH
13
1
1
Sample preparation
H16Cl
2
N
6
O
2
After 30 days of treatments, rats were fasted overnight
(12–14 h), anesthetized by diethyl ether and blood
collected by puncture of the sublingual vein in clean
and dry test tube, left 10 min to clot and centrifuged at
3000 rpm for serum separation. The separated serum was
used for biochemical analysis of blood glucose level, liver
function enzyme activities, alanine and aspartate amino-
transferases (AST, ALT) and alkaline phosphatase (ALP),
lipid profile including, triglycerides (TG), total cholesterol
(TC), and total lipid (TL). Liver in each exponential group
was weighed and homogenized in 5–10 volumes of
appropriate medium using electrical homogenizer, cen-
trifuged at 3000 rpm for 15 min, the supernatants (10%)
were collected and placed in Eppendorff tubes, then
stored at ꢀ80°C and used for determination of oxidative
stress markers (NO and MDA) as well as non-enzymatic
antioxidant (GSH), glutathione reductase (GR), and super-
oxide dismutase (SOD). After blood collection, rats of each
group were sacrificed, the liver, kidney, and pancreas were
(
2
335.19): 39.42; H, 4.81; N, 25.07; Found: C, 41.08; H, 5.04; N,
5.23%.
HPLC analysis
Samples were dissolved in 1 mL of HPLC grade MeOH/
H O (50:50) and centrifuged for 10 min at 4000 rpm before
2
HPLC analysis. Samples were analyzed on a Shimadzu LC-8A
liquid chromatography system (Shimadzu) with LC solution
software, SPD-M20A photodiode array detector (Shimadzu).
A RESTEK (5 mm) C18 column was used (4.6 mm i.d. ꢂ
1
50 mm). Elution was carried out with a gradient solvent
system at a flow rate of 1 mL/min. The mobile phase consisted
of MeOH (B) and 1% formic acid in water (A). The LC time
program was as follows: 5–5% B (2 min), 5–20% B (5 min),
2
9
0–30% B (3 min), 30–50% B (5 min), 50–98% B (8 min),
8–98% B (5 min), and 98–5% B (5 min), 5–5% B (2 min). The
sample was injected into a volume of 40 mL and the eluate
was monitored at 210 nm.
ß 2017 Deutsche Pharmazeutische Gesellschaft
www.archpharm.com
(8 of 9) e1700183

Arch. Pharm. Chem. Life Sci. 2017, 350, e1700183
New Biguanides as Anti-Diabetic Agents
ARC HH PHARM
Archiv der Pharmazie
removed immediately (a part was fixed in 10% formalin for
histopathological examination). The non-hemolyzed, su-
pernatant sera were quickly removed and kept at ꢀ80°C till
used for biochemical investigations of vascular markers
including adhesion molecules, intercellular adhesion
molecule (ICAM-1) and vascular adhesion molecule
shades of pink and red and the nuclei gave blue color. The
slides were examined and photographed under a light
microscope at a magnification power of ꢂ400.
Detailed histology data/photographs are provided in the
Supporting Information.
(
VCAM-1), vascular endothelial growth factor (VEGF),
The authors have declared no conflict of interest.
inflammatory markers (TNF-a and IL-10).
Methods
References
Blood glucose level was measured in blood serum according
to the method of Trinder [9]. Liver function enzyme activities,
alanine and aspartate amino tranferases (AST and ALT) as well
as alkaline phosphatase (ALP) were determined in mice serum
according to the Reitman and Frankel [10] and Belfield and
Goldberg [11] methods. Serum total lipids concentration was
determined according to the method of Zollner et al. [12],
serum triglyceride (TG) concentration was determined
according to the method of Fassati and Prencipe [13] and
serum total cholesterol concentration was estimated accord-
ing to the method of Allain et al. [14]. Liver nitrite (NO) level
was estimated [15]. GSH level was assayed in liver homoge-
nate according to Beutler et al.’s method [16] Liver MDA level
was estimated according to the method of Satoh [17]. GR was
determined according to the method of Goldberg and
Spooner [18] and SOD according to the Nishikimi et al. [19].
Estimation of serum adhesionmolecules, ICAM-1 and VCAM-1,
VEGF, TNF-a, and IL-10 were estimated in sera by ELISA; a
sandwich enzyme immunoassay.
[1] J. S. Skyler, J. Med. Chem. 2004, 47, 4113–4117.
[2] P. Hogan, T. Dall, N. Plamen, Diabetes Care 2003, 26,
917–932.
[3] A. R. Katritzky, S. R. Tala, A. Singh, ARKIVOC 2010, viii,
76–96.
[4] P. Ray, Chem. Rev. 1961, 61, 313–359.
[5] A. Mari ꢀc , Diabetol. Croat. 2010, 39, 95–104.
[6] W. E. Hume, T. Shingaki, T. Takashima, Y. Hashizume,
T. Okauchi, Y. Katayama, E. Hayashinaka, Y. Wada,
H. Kusuhara, Y. Sugiyama, Y. Watanabe, Bioorg. Med.
Chem. 2013, 21, 7584–7590.
[7] S. Y. Abbas, W. M. Basyouni, K. A. M. El-Bayouk,
W. M. Tohamy, H. F. Aly, A. Arafa, M. S. Soliman, Drug
Res. 2017, 67 (10), 557–563.
[8] S. Y. Abbas, W. M. Basyouni, K. A. M. El-Bayouki,
R. F. Abdel-Rahman, Drug Res. 2016, 66 (7), 377–383.
[9] P. Trinder, J. Clin. Pathol. 1969, 22, 246–251.
[10] S. Reitman, S. Frankel, Am. J. Clin. Pathol. 1957, 28,
5
6–63.
[
[
11] A. Belfield, D. Goldberg, Enzyme 1971, 12, 561–566.
12] N. Zollner, K. Kirsch, Z. Ges. Exp. Med. 1962, 135,
545–561.
13] P. Fossati, L. Prencipe, Clin. Chem. 1982, 28, 2077–2080.
14] C. C. Allain, L. S. Poon, C. S. Chan, W. S. Richmond,
P. C. Fu, Clin. Chem. 1974, 20, 470–475.
Calculations
Mean of treated ꢀ Mean of control
Percent of Change ¼
ꢂ 100
Mean of control
[
[
Mean of disease ꢀ Mean of treated
Percent of Improvement ¼
ꢂ 100
Mean of control
[15] H. Moshage, B. Kok, J. R. Huizenga, P. L. Jansen, Clin.
Chem. 1995, 41, 892–896.
[
16] E. Beutler, O. Duron, B. M. Kelly, J. Lab. Clin. Med. 1963,
61, 882–888.
Histopathological analysis
Liver, kidney, and pancreas slices were fixed instantaneously
in buffer neutral formalin (10%) for 24 h for fixation then
processed in automatic processors, embedded in paraffin wax
[17] K. Satoh, Clin. Chim. Acta 1978, 90, 37–43.
[18] D. M. Goldberg, R. J. Spooner, Methods of Enzymatic
Analysis (Ed.: H. V. Bergmeyen), Verlag Chemie, Deer-
field Beach, F1 1983, pp. 258–265.
(
melting point 55–60°C), and paraffin blocks were obtained.
Sections of 6 mm thicknesses were prepared and stained with
haematoxylin and eosin (H&E) stain. The cytoplasm stained
[19] M. Nishikimi, N. A. Rao, K. Yagi, Biochem. Biophys. Res.
Commun. 1972, 46, 849–854.
ß 2017 Deutsche Pharmazeutische Gesellschaft
www.archpharm.com
(9 of 9) e1700183