Synthesis, anti-diabetic and renoprotective activity of some new benzazole, thiazolidin-4-one and azetidin-2-one derivatives

Article abstract of DOI:10.21577/0103-5053.20170050

A new series of benzazole, thiazolidinone and azetidin-2-one derivatives incorporating to pyrazole moiety were synthesized by condensation of 1,3-diphenyl-1H-pyrazol-4-carboxaldehyde with different nucleophiles. The structures of the newly synthesized compounds were confirmed by analytical and spectral methods. Some of these compounds were evaluated for their anti-hyperglycemic and renoprotective activities. Three compounds showed remarkable anti-diabetic potency, whereas other five compounds showed significant renoprotective activity.

Full text of DOI:10.21577/0103-5053.20170050

http://dx.doi.org/10.21577/0103-5053.20170050
J. Braz. Chem. Soc., Vol. 28, No. 11, 2054-2063, 2017.
Printed in Brazil - ©2017 Sociedade Brasileira de Química
0103 - 5053 $6.00+0.00
Article
Synthesis, Anti-Diabetic and Renoprotective Activity of Some New Benzazole,
Thiazolidin-4-one and Azetidin-2-one Derivatives
Ahmed Abdou O. Abeed,a Mohamed Salah K. Youssef*,a and Rehab Hegazyb
aDepartment of Chemistry, Faculty of Science, Assiut University, 71516 Assiut, Egypt
bDepartment of Pharmacology, Medical Division, National Research Centre, 17156 Giza, Egypt
A new series of benzazole, thiazolidinone and azetidin-2-one derivatives incorporating to
pyrazole moiety were synthesized by condensation of 1,3-diphenyl-1H-pyrazol-4-carboxaldehyde
with different nucleophiles. The structures of the newly synthesized compounds were confirmed
by analytical and spectral methods. Some of these compounds were evaluated for their anti-
hyperglycemic and renoprotective activities. Three compounds showed remarkable anti-diabetic
potency, whereas other five compounds showed significant renoprotective activity.
Keywords: anti-diabetic, renoprotective, pyrazole, thiazolidine-4-one, azetidine-2-one
Introduction
failure.17 This pathogenic perception necessitates new
therapeutic considerations and new therapeutic goals for the
treatment of diabetes and its complications.Azetidin-2-ones
(Figure 1B) have been reported as having a potential
anti-inflammatory effect, as well as analgesic and anti-
tuberculosis activities.18,19
Diabetes mellitus is increasing in prevalence worldwide,
and its complication has grown into a public health
problem. It is a condition characterized by both insulin
resistance (poor tissue insulin sensitivity) and impaired
insulin secretion from pancreatic β-cells.1 Thiazolidine-
4-one ring (Figure 1A) system has appeared as a
significant heterocyclic system due to its extensive range
of pharmacological activities, and synthetic applications
for anti-hyperglycemic agents.2-4 Thiazolidinediones5-9 or
glitazones, ligands of the peroxisome-proliferator-activated
receptor-γ (PPAR-γ), are kind of anti-hyperglycemic agents
that reduce insulin resistance and improve insulin action,
thereby keeping normoglycemia and potentially preserving
β-cell function.10 Recent data suggest that they might also
have therapeutic potential in the treatment of inflammatory
diseases and certain cancers.11 There is increasing
evidence that inflammation is closely involved in the
pathogenesis of diabetes and associated complications.12,13
Therefore, drugs with anti-inflammatory properties such
as thiazolidinediones can possibly decrease the risk of
developing diabetes and diabetes-induced problems.14-16
Moreover, inflammation and proinflammatory cytokines
have been found to play a critical role in the development
of microvascular diabetic complications, including
nephropathy, the main cause of diabetes-induced renal
On the other hand, pyrazole derivatives are an important
class of heterocyclic compounds. Much attention has been
paid to compounds containing pyrazole ring (Figure 1C) due
to their biological activities, including anti-inflammatory,
anti-depressant, anti-influenza and anti-cancer activities.20-24
A pharmacophore hybridization method for the synthesis
of novel bioactive molecules is an efficient strategy, and it is
being used in current medicinal chemistry (Figure 1).
Hybridization of two diverse bioactive compounds
with complementary pharmacophoric purposes or with
different mechanisms of action frequently gives synergistic
effects.25,26 In sight of the above mentioned facts and in
extension of our interest in the synthesis of heterocycles
bearing pyrazole and/or thiazolidine moieties,27-30 to
discover new candidates that may be of importance in
designing novel, active, selective and less toxic anti-
diabetic and renoprotective drugs, we present herein the
synthesis and pharmacological evaluation of some novel
structural hybrids containing both the pyrazole moiety
with thiazolidin-4-one and azetidin-2-one ring systems
during various linkages. This pattern was suggested in an
attempt to investigate the influence of such hybridization
and structural variation on the anti-hyperglycemic and
*e-mail: salah_kamel2000@yahoo.com

Vol. 28, No. 11, 2017
Abeed et al.
2055
Figure 1. The pharmacophore hybridization process. (A) Thiazolidine-4-one ring; (B) azetidin-2-one; (C) pyrazole ring.
renoprotective activities, hoping to add some synergistic
biological significance to the target molecules.
2-(2-((1,3-Diphenyl-1H-pyrazol-4-yl)methylene)hydrazinyl)-
1H-benzimidazole (3a)
Yield: 70% (ethanol, brown powder); mp 196-197 °C;
1
Experimental
IR (KBr) ν / cm-1 3414, 3118, 3017, 1637; H NMR
(90 MHz, DMSO-d6) d 6.40 (s, 1H, CH=N), 7.00-8.00
(m, 14H, Ar-H), 8.20 (s, 1H, NH, D2O exchanged), 8.35
(s, 1H, pyrazole-H), 9.50 (s, 1H, benzimidazole-NH,
D2O exchanged). Anal. calcd. for C23H18N6 (378.43):
C, 73.00; H, 4.79; N, 22.21. Found: C, 72.91; H, 4.70;
N, 22.13.
All chemicals and reagents used in current study were of
analytical grade. Melting points were determined usingAPP.
Digital ST 15 melting point apparatus and are uncorrected.
Fourier transfrom infrared (FTIR) spectra were recorded on
a Pye-Unicam SP3-100 spectrophotometer in KBr pellet. 1H
and 13C nuclear magnetic resonance (NMR) spectra were
recorded on AVANCE-III (400 MHz) High Performance
FT-NMR Spectrometer (Bruker, Switzerland), JEOL
2-(2-((1,3-Diphenyl-1H-pyrazol-4-yl)methylene)hydrazinyl)
benzoxazole (3b)
1
JNM-LA 400 and some H NMR spectra were recorded
Yield: 71% (ethanol, pale orange crystals); mp
189-190 °C; IR (KBr) ν / cm-1 3409, 3052, 1638; 1H NMR
(90 MHz, DMSO-d6) d 6.38 (s, 1H, CH=N), 7.20-8.12
(m, 14H, Ar-H), 8.23 (s, 1H, NH, D2O exchanged), 8.30
(s, 1H, pyrazole-H). Anal. calcd. for C23H17N5O (379.41):
C, 72.81; H, 4.52; N, 18.46. Found: C, 72.73; H, 4.44;
N, 18.38.
on Varian EM-360L NMR Spectrophotometer (90 MHz)
(USA) in CDCl3, DMSO-d6 as solvent. Chemical shifts
(d) are reported in ppm and the coupling constants (J) are
reported in hertz. C, H, N and S analyses were performed
with a Vario EL C, H, N, S Analyzer.
General procedure for the synthesis of hydrazide derivatives
3a,b, 7a,b, and 11a,b
2-((1,3-Diphenyl-1’H-pyrazol-4-yl)methylidene-hydrazino-
acetyl)mercaptobenzimidazole (7a)
A mixture of 1, 3-diphenyl-1H-pyrazol-
4-carboxaldehyde (1) (0.74 g, 3 mmol), the corresponding
hydrazine (2a,b, 6a,b and 10a,b) (3 mmol), and 2-3 drops
glacial acetic acid in ethanol (20 mL) was refluxed on a
water bath for about 5 h. The solvent was removed and the
residue was recrystallized from the suitable solvent.
Yield: 72% (toluene, orange crystals); mp 179-180 °C;
IR (KBr) ν / cm-1 3379, 3157, 3049, 1677, 1637; 1H NMR
(90 MHz, DMSO-d6) d 4.35 (s, 2H, CH2), 6.35 (s, 1H,
CH=N), 7.15-8.10 (m, 14H, Ar-H), 8.25 (s, 1H, NH,
D2O exchanged), 8.35 (s, 1H, pyrazole-H), 9.48 (s, 1H,
benzimidazole-NH, D2O exchanged). Anal. calcd. for

2056 Synthesis, Anti-Diabetic and Renoprotective Activity of Some New Benzazole, Thiazolidin-4-one and Azetidin-2-one
J. Braz. Chem. Soc.
C25H20N6OS (452.53): C, 66.35; H, 4.45; N, 18.57; S, 7.09.
Found: C, 66.28; H, 4.38; N, 18.50; S, 6.99.
Ar-H), 8.29 (s, 1H, pyrazole-H), 8.58 (s, 1H, NH, D2O
exchanged), 9.50(s, 1H, benzimidazole-NH, D2Oexchanged).
Anal. calcd. for C25H20N6OS (452.53): C, 66.35; H, 4.45; N,
18.57; S, 7.09. Found: C, 66.27; H, 4.37; N, 18.50; S, 7.00.
2-((1,3-Diphenyl-1’H-pyrazol-4-yl)methylidene-hydrazino-
acetyl) mercaptobenzoxazole (7b)
Yield: 69% (ethanol, pale brown crystals); mp
194-195 °C; IR (KBr) ν / cm-1 3397, 3051, 1671, 1636;
1H NMR (90 MHz, DMSO-d6) d 4.37 (s, 2H, CH2), 6.38
(s, 1H, CH=N), 7.10-7.90 (m, 14H,Ar-H), 8.20 (s, 1H, NH,
D2O exchanged), 8.35 (s, 1H, pyrazole-H).Anal. calcd. for
C25H19N5O2S (453.52): C, 66.21; H, 4.22; N, 15.44; S, 7.07.
Found: C, 66.13; H, 4.14; N, 15.38; S, 7.00.
2-[2’-(1,3-Diphenyl-1H-pyrazol-4-yl)thiazolidine-5’-one]
hydrazinobenzoxazole (4b)
Yield 66% (ethanol, brown powder); mp 241-242 °C;
1
IR (KBr) ν / cm-1 3415, 3025, 1680; H NMR (90 MHz,
DMSO-d6) d 3.65 (s, 2H, thiazolidine-CH2), 5.10 (s, 1H,
CH), 7.05-8.10 (m, 14H, Ar-H), 8.30 (s, 1H, pyrazole-H),
8.50 (s, 1H, NH, D2O exchanged). Anal. calcd. for
C25H19N5O2S (453.52): C, 66.21; H, 4.22; N, 15.44; S, 7.07.
Found: C, 66.14; H, 4.12; N, 15.36; S, 7.00.
(Benzimidazol-2-ylsulfanyl) acetic acid ((1,3-diphenyl-
1’H-pyrazol-4-yl) methylidene) hydrazide (11a)
Yield 78% (toluene, white powder); mp 202-203 °C;
IR (KBr) ν / cm-1 3396, 3157, 3096, 1687, 1639; 1H NMR
(90 MHz, DMSO-d6) d 4.55 (s, 2H, CH2), 6.31 (s, 1H,
CH=N), 7.10-7.99 (m, 14H, Ar-H), 8.15 (s, 1H, NH,
D2O exchanged), 8.33 (s, 1H, pyrazole-H), 9.50 (s, 1H,
benzimidazole-NH, D2O exchanged). Anal. calcd. for
C25H20N6OS (452.53): C, 66.35; H, 4.45; N, 18.57; S, 7.09.
Found: C, 66.26; H, 4.36; N, 18.50; S, 7.01.
2-[2’-(2-(1,3-Diphenyl-1H-pyrazol-4-yl)thiazolidine-5-one)
acetylaminomercapto] benzimidazole (8a)
Yield 70% (dioxane, brown powder); mp 235-236 °C;
1
IR (KBr) ν / cm-1 3414, 3181, 1678, 1648; H NMR
(400 MHz, DMSO-d6) d 3.77 (s, 2H, thiazolidine-CH2),
4.13 (s, 2H, COCH2), 5.01 (s, 1H, thiazolidine-CH),
7.15-7.99 (m, 14H,Ar-H), 8.30 (s, 1H, pyrazole-H), 8.45 (s,
1H, NH, D2O exchanged), 9.55 (s, 1H, benzimidazole-NH,
D2O exchanged). Anal. calcd. for C27H22N6O2S2 (526.63):
C, 61.58; H, 4.21; N, 15.96; S, 12.18. Found: C, 61.50; H,
4.12; N, 15.90; S, 12.10.
(Benzoxazol-2-ylsulfanyl) acetic acid ((1,3-diphenyl-
1’H-pyrazol-4-yl) methylidene) hydrazide (11b)
Yield 79% (toluene, white powder); mp 190-191 °C;
1
IR (KBr) ν / cm-1 3395, 3050, 1675, 1635; H NMR
2-[2’-(2-(1,3-Diphenyl-1H-pyrazol-4-yl) thiazolidine-5-one)
acetylaminomercapto] benzoxazole (8b)
(90 MHz, DMSO-d6) d 4.53 (s, 2H, CH2), 6.38 (s, 1H,
CH=N), 7.13-8.00 (m, 14H, Ar-H), 8.11 (s, 1H, NH, D2O
exchanged), 8.36 (s, 1H, pyrazole-H). Anal. calcd. for
C25H19N5O2S (453.52): C, 66.21; H, 4.22; N, 15.44; S, 7.07.
Found: C, 66.13; H, 4.13; N, 15.36; S, 6.96.
Yield: 67% (dioxane, brown powder); mp 205-206 °C;
1
IR (KBr) ν / cm-1 3410, 3020, 1670, 1643; H NMR
(400 MHz, CDCl3) d 3.71 (s, 2H, thiazolidine-CH2), 4.08 (s,
2H, COCH2), 5.00 (s, 1H, thiazolidine-CH), 7.25-8.12 (m,
14H, Ar-H), 8.33 (s, 1H, pyrazole-H), 8.43 (s, 1H, NH, D2O
exchanged);13C NMR (100 MHz, CDCl3) d 37.4, 45.3, 51.3,
119.0, 121.8, 122.1, 125.3, 128.6, 131.3, 132.4, 135.3, 136.7,
139.8, 140.1, 141.5, 151.6, 152.9, 163.6, 174.3.Anal. calcd.
for C27H21N5O3S2 (527.62): C, 61.46; H, 4.01; N, 13.27; S,
12.15. Found: C, 61.40; H, 3.93; N, 13.20; S, 12.09.
General procedure for the synthesis of thiazolidin-5-ones
4a,b, 8a,b, and 12a,b
To a solution of the hydrazide (3a,b, 7a,b, or 11a,b)
(3 mmol) in DMF (20 mL) were added thioglycolic acid
(0.27 mL, 3 mmol) and ZnCl2 (0.3 g) and the reaction
mixture was refluxed for 8 h, cooled and poured into
crushed ice, the solid precipitate was filtered and washed
with sodium bicarbonate (10%). The product was dried and
recrystallized from the proper solvent.
1-[(Benzimidazol-2’’-yl)thioacetamidyl]-2-(1’,3’-diphenyl-
1’H-pyrazol-4’-yl) thiazolidin-5-one (12a)
Yield: 68% (toluene, white crystals); mp 230-231 °C;
IR (KBr) ν / cm-1 3415, 3215, 3019, 1670, 1650; 1H NMR
(90 MHz, DMSO-d6) d 3.99 (s, 2H, thiazolidine-CH2),
4.30 (s, 2H, CH2), 5.12 (s, 1H, thiazolidine-CH), 7.05-7.99
(m, 15H, Ar-H and pyrazole-H), 8.92 (s, 1H, NH, D2O
exchanged), 9.50 (s, 1H, benzimidazole-NH, D2O
exchanged). Anal. calcd. for C27H22N6O2S2 (526.63): C,
61.58; H, 4.21; N, 15.96; S, 12.18. Found: C, 61.49; H,
4.14; N, 15.90; S, 12.10.
2-[2’-(1,3-Diphenyl-1H-pyrazol-4-yl)thiazolidine-5’-one]
hydrazinobenzimidazole (4a)
Yield: 68% (ethanol, reddish brown powder);
mp 230-231 °C; IR (KBr) ν / cm-1 3427, 3237, 3037,
1676; H NMR (400 MHz, DMSO-d6) d 3.74 (s, 2H,
thiazolidine-CH2), 5.14 (s, 1H, CH), 7.19-7.99 (m, 14H,
1

Vol. 28, No. 11, 2017
Abeed et al.
2057
1-[(Benzoxazol-2’’-yl)thioacetamidyl]-2-(1’,3’-diphenyl-
1’H-pyrazol-4’-yl) thiazolidin-5-one (12b)
(d, 1H, CH-Cl), 7.18-7.80 (m, 14H, Ar-H), 8.31 (s, 1H,
pyrazole-H), 8.51 (s, 1H, NH, D2O exchanged), 9.56 (s,
1H, benzimidazole-NH, D2O exchanged). Anal. calcd.
for C27H21N6O2SCl (529.01): C, 61.30; H, 4.00; N, 15.89;
S, 6.06; Cl, 6.70. Found: C, 61.19; H, 3.93; N, 15.79; S,
5.94; Cl, 6.62.
Yield: 68% (ethanol, white crystals); mp 211-212 °C;
1
IR (KBr) ν / cm-1 3410, 3022, 1672, 16435; H NMR
(90 MHz, DMSO-d6) d 3.95 (s, 2H, thiazolidine-CH2), 4.18
(s, 2H, CH2), 5.12 (s, 1H, thiazolidine-CH), 7.02-7.98 (m,
14H, Ar-H), 8.30 (s, 1H, pyrazole-H), 8.41 (s, 1H, NH,
D2O exchanged). Anal. calcd. for C27H21N5O3S2 (527.62):
C, 61.46; H, 4.01; N, 13.27; S, 12.15. Found: C, 61.38; H,
3.92; N, 13.17; S, 12.01.
2-[2’-(4-(1,3-Diphenyl-1H-pyrazol-4-yl)-3-chloro-
2-oxoazetidine)acetylaminomercapto] benzoxazole (9b)
Yield: 65% (pale yellow crystals); mp 250-251 °C;
1
IR (KBr) ν / cm-1 3410, 3028, 1674, 1640; H NMR
General procedure for the synthesis of azetidin-2-ones
(90 MHz, DMSO-d6) d 4.10 (s, 2H, COCH2), 5.28 (d, 1H,
CH-N), 5.49 (d, 1H, CH-Cl), 7.10-7.98 (m, 15H, Ar-H and
pyrazole-H), 8.52 (s, 1H, NH, D2O exchanged).Anal. calcd.
for C27H20N5O3SCl (530.00): C, 61.19; H, 3.80; N, 13.21;
S, 6.05, Cl, 6.69. Found: C, 61.12; H, 3.72; N, 13.10; S,
5.96, Cl, 6.61.
5a,b, 9a,b, and 13a,b
A solution of hydrazides (3a,b, 7a,b, or 11a,b)
(3 mmol) in dioxane (20 mL) was added to a well stirred
mixture of chloroacetylchloride (0.24 mL, 3 mmol) and
triethylamine (3 mmol) in dioxane (10 mL) at 0-5 °C. The
reaction mixture was stirred for 3 h, and then refluxed for
8 h. The reaction mixture was cooled, poured into ice-cold
and the solid thus obtained was filtered, washed with water
and recrystallized from dioxane.
1-[(Benzimidazol-2’’-yl)thioacetamidyl]-3-chloro-
4-(1’,3’-diphenyl-1’H-pyrazol-4’-yl)azetidin-2-one (13a)
Yield: 60% (white crystals); mp 258-259 °C; IR (KBr)
ν / cm-1 3424, 3228, 3007, 1677, 1633; 1H NMR (400 MHz,
CDCl3) d 4.18 (s, 2H, COCH2), 5.33 (d, 1H, CH-N), 5.53
(d, 1H, CH-Cl), 7.03-7.99 (m, 14H, Ar-H), 8.34 (s, 1H,
pyrazole-H), 8.97 (s, 1H, NH, D2O exchanged), 9.54 (s, 1H,
benzimidazole-NH, D2O exchanged); 13C NMR (100 MHz,
CDCl3) d 33.5, 49.1, 59.1, 118.6, 121.9, 126.4, 129.1, 132.5,
136.5, 139.1, 143.6, 145.6, 149.7, 152.6, 154.6, 169.0,
173.2. Anal. calcd. for C27H21N6O2SCl (529.01): C, 61.30;
H, 4.00; N, 15.89; S, 6.06, Cl, 6.70. Found: C, 61.22; H,
3.92; N, 15.80; S, 5.95, Cl, 6.60.
2-[4’-(1,3-Diphenyl-1H-pyrazol-4-yl)-3’-chloro-
2’-oxoazetidine]hydrazinobenzimidazole (5a)
Yield: 61% (pale yellow powder); mp 225-226 °C; IR
(KBr) ν / cm-1 3440, 3257, 3024, 1755; 1H NMR (400 MHz,
CDCl3) d 5.18 (d, 1H, CH-N), 5.73 (d, 1H, CH-Cl),
7.18-7.94 (m, 14H, Ar-H), 8.37 (s, 1H, pyrazole-H), 8.48
(s, 1H, NH, D2O exchanged), 9.53 (s, 1H, benzimidazole-
NH, D2O exchanged); 13C NMR (100 MHz, CDCl3) d 43.4,
58.9, 119.4, 122.3, 122.4, 126.3, 129.7, 133.3, 136.3, 136.7,
139.7, 141.0, 141.3, 151.5, 152.5, 172.2. Anal. calcd. for
C25H19N6OCl (454.91): C, 66.01; H, 4.21; N, 18.47; Cl,
7.79. Found: C, 65.90; H, 4.11; N, 18.40; Cl, 7.65.
1-[(Benzoxazol-2’’-yl)thioacetamidyl]-3-chloro-
4-(1’,3’-diphenyl-1’H-pyrazol-4’-yl)azetidin-2-one (13b)
Yield: 64% (pale brown powder); mp 250-251 °C; IR
(KBr) ν / cm-1 3422, 3020, 1675, 1640; 1H NMR (400 MHz,
DMSO-d6) d 4.22 (s, 2H, COCH2), 5.30 (d, 1H, CH-N),
5.46 (d, 1H, CH-Cl), 7.05-8.00 (m, 14H, Ar-H), 8.25 (s,
1H, pyrazole-H), 8.92 (s, 1H, NH, D2O exchanged). Anal.
calcd. for C27H20N5O3SCl (530.00): C, 61.19; H, 3.80; Cl,
6.69; N, 13.21; S, 6.05. Found: C, 61.09; H, 3.71; Cl, 6.57;
N, 13.15; S, 5.96.
2-[4’-(1,3-Diphenyl-1H-pyrazol-4-yl)-3’-chloro-
2’-oxoazetidine]hydrazinobenzoxazole (5b)
Yield: 60% (pale orange crystals); mp 241-242 °C;
1
IR (KBr) ν / cm-1 3440, 3010, 1758; H NMR (90 MHz,
DMSO-d6) d 5.28 (d, 1H, CH-N), 5.50 (d, 1H, CH-Cl),
7.05-8.01 (m, 15H,Ar-H and pyrazole-H), 8.52 (s, 1H, NH,
D2O exchanged). Anal. calcd. for C25H18N5O2Cl (455.90):
C, 65.86; H, 3.98; N, 15.36; Cl, 7.78. Found: C, 65.75; H,
3.87; N, 15.26; Cl, 7.64.
Biological evaluation
Animals
2-[2’-(4-(1,3-Diphenyl-1H-pyrazol-4-yl)-3-chloro-
2-oxoazetidine)acetylaminomercapto]benzimidazole (9a)
Yield: 70% (orange powder); mp 230-231 °C; IR (KBr)
ν / cm-1 3416, 3209, 3080, 1688, 1645; 1H NMR (400 MHz,
CDCl3) d 4.11 (s, 2H, COCH2), 5.30 (d, 1H, CH-N), 5.51
Adult male albino Wistar rats, weighing 200-250 g,
were obtained from the animal house colony of National
Research Centre (NRC). The animals were maintained at
a normal temperature of 24 1 °C with a 12-12 h light-
dark cycle (light cycle, 7:00 am-7:00 pm). They were

2058 Synthesis, Anti-Diabetic and Renoprotective Activity of Some New Benzazole, Thiazolidin-4-one and Azetidin-2-one
J. Braz. Chem. Soc.
allowed free access to water and standard show ad libitum.
The animals were treated according to the national
and international ethics guidelines stated by the ethics
committee of NRC and all procedures and experiments
were performed according to its approved protocol.
Egypt). Immediately after blood sampling, animals were
sacrificed by cervical dislocation under ether anesthesia.
The two kidneys from each rat were immediately dissected
out, and rinsed with PBS to remove excess blood. Weighed
parts from both kidneys were homogenized (MPW-120
homogenizer, MED instruments, Poland) in PBS to
obtain 20% homogenate that stored overnight at ≤ −20 °C.
After two freeze-thaw cycles performed to break the
cell membranes, the homogenates were centrifuged for
5 min at 5000 × g using a cooling centrifuges (Sigma and
Laborzentrifugen, 2K15, Germany). The supernatant was
removed immediately and assayed for reduced glutathione
(GSH), lipid peroxides, measured as malondialdehyde
(MDA), and NOx (nitrite and nitrate, stable metabolites
of nitric oxide) contents using specific commercial reagent
kits (Biodiagnostic, Egypt).
Evaluation of the anti-diabetic effect of the tested compounds
against streptozotocin-induced hyperglycemia in rats
Hyperglycemia was induced in rats using a single
i.p. (intraperitoneal) injection of streptozotocin (STZ,
50 mg kg-1) dissolved in a citrate buffer (0.1 M, pH 4.5).31
After 48 hours, blood samples were withdrawn from the
retro-orbital venous plexus under light ether anesthesia
and the serum was separated by centrifugation for the
determination of glucose level. Only rats with serum
glucose levels more than 250 mg dL-1 were selected and
considered as hyperglycemic animals to be subjected to
further experimentation. Hyperglycemic rats were randomly
divided into five groups (6 rats each). One group served as
a hyperglycemic normal, while the other four groups were
treated with gliclazide (Glic, 5 mg kg-1 day-1, p.o.), as a
reference drug, or one of the tested compounds (7b, 12b,
and 13b), (50 mg kg-1 day-1, p.o.) for 10 consecutive days,
respectively. Drug treatment was started 48 h after STZ
injection (time at which hyperglycemia was confirmed).
In addition, a universal normal group which received only
the citrate buffer (6 rats) was used. Twenty-four hours after
the last dose of either drug treatment, the serum levels of
glucose, triglyceride (TG), and cholesterol are determined
using specific diagnostic kits (Stanbio, USA).
Statistical analysis
All the values are presented as means standard error
of the means (SE). Comparisons between different groups
were carried out using one-way analysis of variance
(ANOVA) followed by Tukey HSD (honest significant
difference) test for multiple comparisons.34 Graphpad
Prism software, version 5 (Inc., San Diego, USA) was
used to carry out these statistical tests. The difference was
considered significant when p < 0.05.
Results and Discussion
Synthesis
Evaluation of the renoprotective effects of the tested
compounds against renal ischemia/reperfusion-induced
acute renal failure in rats
Our synthetic strategy for a novel series of benzazole,
thiazolidin-4-one and azetidin-2-one derivatives bearing
1,3-diphenyl-1H-pyrazole moiety is illustrated in
Schemes 1-3. The required hydrazides 3a,b, 7a,b,
and 11a,b were synthesized by condensation of the
key aldehyde intermediate35 1 with the corresponding
hydrazine 2a,b, 6a,b and 10a,b on refluxing with ethanol
containing a few drops of acetic acid. The structure of the
compounds 3a,b, 7a,b, and 11a,b was assigned by spectral
data and elemental analysis (see Experimental section).
The hydrazide derivatives 3a,b, 7a,b, and 11a,b,
when treated with thioglycolic acid in the presence of
anhydrous ZnCl2, gave thiazolidine-4-one analogues
4a,b, 8a,b and 12a,b, respectively (Schemes 1-3). The
structures of latter compounds was assigned by IR, NMR
spectra and elemental analyses which are reliable with
the proposed thiazolidine-4-one structures. For example,
Eight groups of animals were used in this experiment;
each group consisted of six rats. The first group was
subjected to a sham operation and served as a normal group.
In the other seven groups, ischemia/reperfusion acute renal
failure (I/R ARF) was induced in anesthetized (ketamine/
xylazine) rats by clamping both renal pedicles for 45 min.
Reflow was visually confirmed, and to reduce abdominal
air, 4 mL warm normal saline was given intraperitoneally
before abdominal closure.32 For ten days prior to I/R,
groups 1 and 2 were treated with daily oral dose of saline,
while the other six groups were treated with daily oral
dose of 50 mg kg-1 day-1 of the tested compounds (3a,
3b, 5a, 7a, 8a, and 8b). Twenty-four hours following I/R,
blood samples were withdrawn from rats of all groups via
retro-orbital vein under light ether anesthesia.33 Serum
was used for estimation of serum urea, creatinine and total
protein levels, using specific diagnostic kits (Biodiagnostic,
1
the H NMR of these compounds showed signals at
d 3.65-3.99 ppm and 5.00-5.14 ppm due to CH2 and CH

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Abeed et al.
2059
Scheme 1. Reagents and conditions: (i) ethanol, AcOH, 5 h; (ii) thioglycolic acid, DMF, ZnCl2, reflux 8 h; (iii) chloroacetyl chloride, dioxane, TEA,
stirring then reflux 8 h.
Scheme 2. Reagents and conditions: (i) ethanol, AcOH, 5 h; (ii) thioglycolic acid, DMF, ZnCl2, reflux 8 h; (iii) chloroacetyl chloride, dioxane, TEA,
stirring then reflux 8 h.

2060 Synthesis, Anti-Diabetic and Renoprotective Activity of Some New Benzazole, Thiazolidin-4-one and Azetidin-2-one
J. Braz. Chem. Soc.
Scheme 3. Reagents and conditions: (i) ethanol, AcOH, 5 h; (ii) thioglycolic acid, DMF, ZnCl2, reflux 8 h; (iii) chloroacetyl chloride, dioxane, TEA,
stirring then reflux 8 h.
1
of thiazolidine ring. In the H NMR spectra of starting
hydrazides 3a,b, 7a,b, and 11a,b, the proton of −CH=N
appears at about d 6.31-6.40 ppm. But, in the spectra of
thiazolidin-4-ones 4a,b, 8a,b and 12a,b, this proton signal
(CH thiazolidine) is shifted slightly downfield in contrast
with hydrazides. The reason was due to the deshielding
affected by heterocycles.
was demonstrated 48 h after injection and persisted until the
end of the experiment. Oral treatment of hyperglycemic rats
with each of the tested compounds (7b, 12b, and 13b) for
10 days restored the normal serum glucose levels, a similar
effect to that produced by the treatment with the reference
anti-diabetic drug gliclazide (Glic) (Figure 2).
On the other hand, the cycloaddition reaction of
hydrazides 3a,b, 7a,b, and 11a,b with chloroacetyl chloride
in dioxane containing triethyl amine as a reaction mediator
gave azetidin-2-ones 5a,b, 9a,b, and 13a,b, respectively.
The 1H NMR spectra of these compounds showed the
signals at d 5.46-5.73 ppm due to the CH−Cl in the β-lactam
ring. Whereas the 13C NMR spectra of compounds 5a and
13a showed signals at d 58.9 and 59.1 ppm, respectively,
due to −CH−Cl. Moreover, signals at d 172.2 and
173.2 ppm are due to C=O cyclic in the β-lactam moiety.
Biological evaluation
Streptozotocin-induced hyperglycemia experiment
Figure 2. Serum glucose levels of compounds 7b, 12b and 13b. Normal:
rats treated with the vehicle; STZ: rats treated with STZ; STZ-Glic: rats
treated with STZ and Glic; STZ-T(x), rats treated with STZ and the tested
agent of the corresponding number (x). a: Significantly different from
Normal group at p < 0.05; b: significantly different from STZ group at
p < 0.05.
Serum glucose level
A single injection of streptozotocin (STZ) (50 mg kg-1,
i.p.) resulted in an elevation of the serum glucose level that

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Abeed et al.
2061
Serum cholesterol and triglyceride levels
total protein levels was observed in the rats subjected to I/R
as compared with those of normal group.Administration of
3a, 3b, 5a, 7a, and 8a significantly reduced the I/R-induced
elevation of serum urea and creatinine levels.
On the other hand, compound 8b reduced these levels,
but more than the normal. The normal level of serum total
protein was maintained in the rats pretreated with each of
the tested agents (Figure 4).
STZ-induced hyperglycemia in the present study was
related with an increase in the serum cholesterol and
triglyceride (TG) levels. Gliclazide (Glic) administration
restored the normal cholesterol and TG levels. Also,
normal serum cholesterol and TG levels were observed in
groups treated with 12b and 13b. The anti-cholesterolemic
effect of 7b was significantly less than that observed by
12b and 13b; however, it restored the normal serum TG
level (Figure 3).
Renal tissue biochemical analysis
Renal GSH content was significantly reduced following
I/R in rats. Restoration of normal renal GSH content was
observed in the group treated with compound 8a, while 3a
failed to improve the I/R-induced reduction of renal GSH
content. Treatment of rats with the other tested compounds
Renal ischemia/reperfusion experiment
Renal function markers
A significant elevation in serum urea, creatinine and
Figure 3. Serum cholesterol and triglyceride (TG) levels of compounds 7b, 12b and 13b. Normal: rats treated with the vehicle; STZ: rats treated with
streptozotocin; STZ-Glic: rats treated with streptozotocin and Glic; STZ-T(x): rats treated with STZ and the tested agent of the corresponding number (x).
a: Significantly different from normal group at p < 0.05; b: significantly different from STZ group at p < 0.05; c: significantly different from STZ-Glic
group at p < 0.05; d: significantly different from the other STZ-T(x) groups at p < 0.05.
Figure 4. Serum levels of urea, creatinine and total protein of compounds 3a, 3b, 5a, 7a, 8a, and 8b. Normal: rats with sham operation; I/R ARF: rats with
ischemia/reperfusion-induced acute renal failure; I/R-T(x): rats with ischemia/reperfusion-induced acute renal failure treated with the tested compounds
of the corresponding number (x). a: Significantly different from normal group at p < 0.05; b: significantly different from I/R ARF group at p < 0.05.

2062 Synthesis, Anti-Diabetic and Renoprotective Activity of Some New Benzazole, Thiazolidin-4-one and Azetidin-2-one
J. Braz. Chem. Soc.
Figure 5. Renal contents of GSH, MDA, and NOx of compounds 3a, 3b, 5a, 7a, 8a, and 8b. Normal: rats with sham operation; I/RARF: rats with ischemia/
reperfusion-induced acute renal failure; I/R-T(x): rats with ischemia/reperfusion-induced acute renal failure treated with the tested agent of the corresponding
number (x). a: Significantly different from normal group at p < 0.05; b: significantly different from I/R ARF group at p < 0.05.
resulted in renal GSH contents that are non-significantly
different from normal and I/R groups.
Acknowledgments
On the other hand, a significant elevation of renal MDA
content was detected following I/R. Treatment of rats with
all tested compounds significantly retrieved the altered
level of MDA. Interestingly, the renal content of MDA
in rats treated with 7a was remarkably lower than those
observed in the normal I/R 8b and I/R 8a groups. Moreover,
a significant decrease in renal NOx content was observed
in rats with I/R ARF. Treatment of rats with 5a, 8b, and 3b
has been found to restore the normal renal level of NOx.
Compounds 3a and 8a significantly improved the I/R-
induced reduction of NOx level, while 7a failed (Figure 5).
The authors are thankful to Assiut University for
providing the support to carry out this research work.
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Submitted: October 31, 2016
Published online: March 14, 2017