Synthesis , Anti-Diabetic and Renoprotective Activity of Some New Benzazole , Thiazolidin-4-one and Azetidin-2one Derivatives

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 antihyperglycemic and renoprotective activities. Three compounds showed remarkable anti-diabetic potency, whereas other five compounds showed significant renoprotective activity.


Introduction
Diabetes mellitus is increasing in prevalence worldwide, and its complication has grown into a public health problem.3][4] Thiazolidinediones [5][6][7][8][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. 10Recent data suggest that they might also have therapeutic potential in the treatment of inflammatory diseases and certain cancers. 11There is increasing evidence that inflammation is closely involved in the pathogenesis of diabetes and associated complications. 12,13][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 failure. 17This 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 antituberculosis activities. 18,19n the other hand, pyrazole derivatives are an important class of heterocyclic compounds.][22][23][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. 25,26In sight of the above mentioned facts and in extension of our interest in the synthesis of heterocycles bearing pyrazole and/or thiazolidine moieties, [27][28][29][30] to discover new candidates that may be of importance in designing novel, active, selective and less toxic antidiabetic 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 Vol. 28, No. 11, 2017   renoprotective activities, hoping to add some synergistic biological significance to the target molecules.

Experimental
All chemicals and reagents used in current study were of analytical grade.Melting points were determined using APP.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. 1 H and 13 C nuclear magnetic resonance (NMR) spectra were recorded on AVANCE-III (400 MHz) High Performance FT-NMR Spectrometer (Bruker, Switzerland), JEOL JNM-LA 400 and some 1 H NMR spectra were recorded on Varian EM-360L NMR Spectrophotometer (90 MHz) (USA) in CDCl 3 , DMSO-d 6 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 A m i x t u r e o f 1 , 3 -d i p h e n y l -1 H -p y r a z o l -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.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 ZnCl 2 (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.

Animals
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 lightdark cycle (light cycle, 7:00 am-7:00 pm).They were 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.
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). 31fter 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).
Evaluation of the renoprotective effects of the tested compounds against renal ischemia/reperfusion-induced acute renal failure in rats 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. 32For 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. 33Serum was used for estimation of serum urea, creatinine and total protein levels, using specific diagnostic kits (Biodiagnostic, 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).

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. 34Graphpad 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.

Synthesis
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 intermediate 35  The hydrazide derivatives 3a,b, 7a,b, and 11a,b, when treated with thioglycolic acid in the presence of anhydrous ZnCl 2 , 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, the 1 H NMR of these compounds showed signals at d 3.65-3.99ppm and 5.00-5.14ppm due to CH 2 and CH A single injection of streptozotocin (STZ) (50 mg kg -1 , i.p.) resulted in an elevation of the serum glucose level that 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).Serum cholesterol and triglyceride levels 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 ischemia/reperfusion experiment Renal function markers
A significant elevation in serum urea, creatinine and 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).

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  resulted in renal GSH contents that are non-significantly different from normal and I/R groups.
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/Rinduced reduction of NOx level, while 7a failed (Figure 5).

Conclusions
In conclusion, the objective of the present study was to synthesize and evaluate the anti-diabetic and renoprotective activity of some new benzazole, thiazolidinone and azetidin-2-one derivatives incorporating 1,3-diphenylpyrazole moiety with the hope of discovering new structures leads serving as potent pharmacological agents.Our aim was verified by the synthesis of different groups of structure hybrids comprising basically the pyrazole moiety attached to benzazole, thiazolidinone, azetidin-2-one counter parts through various linkages of synthergistic purpose.

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).

Figure 2 .
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.

Figure 3 .
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 .
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.