New Biguanides as Anti-Diabetic Agents (2017) - PDF

Arch. Pharm. Chem. Life Sci. 2017, 350, e1700183 ARCH PHARM Archiv der Pharmazie Full Paper New Biguanides as Anti-Diabetic Agents, Part II: Synthesis and Anti-Diabetic Properties Evaluation of...

Arch. Pharm. Chem. Life Sci. 2017, 350, e1700183 ARCH PHARM 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 Wahid M. Basyouni1, Samir Y. Abbas 1 , Mohamed F. El Shehry2, Khairy A.M. El-Bayouki1, Hanan F. Aly3, 3 3 Azza Arafa , and Mahmoud S. Soliman 1 National Research Centre, Department of Organometallic and Organometalloid Chemistry, Cairo, Egypt 2 National Research Centre, Department of Pesticide Chemistry, Cairo, Egypt 3 National Research Centre, Department of Therapeutic Chemistry, Cairo, Egypt 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 Diabetic disorder is one of the most serious public health such as aryl, benzo[1,3-d]dioxolyl, carboxamide, hydrazine, and problems overall the world [1, 2]. Biguanide derivatives were hydrazide moieties into the target compounds. Preliminary known for more than 100 years, confirmed to be potent broadly structure activity relationships revealed that incorporation of the applications in medicine [3, 4]. 1,1-Dimethylbiguanide (metfor- hydrazide moiety enhanced the anti-diabetic activity of the min) and phenylethylbiguanide (phenformin) have the ability to biguanide system. So, in continuation of our ongoing study in this reduce the blood glucose level through lowering of production field, the present study aimed to synthesize some new biguanide capacity of glucose which is produced by the liver. Metformin is a derivatives by incorporating the hydrazide moiety. 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 Results and discussion the insulin receptors in muscle tissue more sensitive [5, 6]. In our 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: [email protected] Additional correspondence: Prof. Wahid M. Basyouni, Fax: (þ202) 33370931 E-mail: [email protected] 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. ARCH PHARM Archiv der Pharmazie the desired hydrazide derivatives were synthesized as shown the enhancement of gluconeogensis and the utilized glucose in Scheme 1, where benzoic acid derivatives were subjected to level blood glucose is reduced. The present results (Table 1) esterification by heating with ethanol containing catalytic clearly indicated that diabetic rats exhibited significant amount of sulfuric acid. Heating of the latter esters with increase in blood glucose level with percentage increase hydrazine hydrate in ethanol under reflux afforded the reached to 293.96% as compared to normal control rats. required hydrazide derivatives. Upon heating the obtained A series of disubstituted biguanide hydrochloride salts were hydrazide derivatives with dicyandiamide (cyanoguanidine) synthesized which contain R1 and R2 at N-(1); in case of in ethanolic hydrochloric acid, the desired biguanide deriv- compounds 1–3, R1 is amide side chain ending with substituted atives 1–5 were afforded which separated from the reaction phenyl moiety, where the substituted phenyl moieties are 2- mixture in crystallized form with good yield and high purity bromophenyl (1), 3-chlorophenyl (2), 4-chlorophenyl (3), and (Scheme 1). Structure of the products 1–5 was elucidated by R2 ¼ hydrogen. In case of compound 6, R1 is (2-(4-chloro-2- careful studying of their spectral data. Thus, IR spectrum of methylphenoxy)acetyl)amino and R2 ¼ hydrogen. The standard the biguanide hydrochloride 1 (as representative example) drug (metformin), R1 ¼ R2 ¼ CH3. Hyperglycemic rats were showed multiple peaks at n: 3413, 3306, 3133 cm1 presum- treated orally with compounds 1–3 and 6 (2 mmol/kg/day) for ably owing to NH2 and NH groups. Two strong stretching evaluating their anti-diabetic effect in comparison to metfor- peaks at: 1704 and 1638 cm1 were observed for C –– O and C –– N min hydrochloride (2 mmol/kg/day) for 4 weeks. Treatment of functional groups, respectively. 1H NMR spectrum of 1 diabetic rats with different synthetic compounds showed exhibited signals in the range of 6.80–8.00 owing to nine marked amelioration in blood glucose level with different protons corresponding to four aromatic protons, amino percentage of improvements. Regarding the effect of group, and three NH protons. Amide NH proton was shifted each substituent on N-(1): The standard drug (metformin), to down field tend to give rise to a singlet at: d ¼ 10.48. R1 ¼ R2 ¼ CH3 showed 263.41% improvement. Introduction of 13 C NMR spectrum of the biguanide 1 has characteristic signal amide side chain ending with 2-bromophenyl at R1 and left it R2 at: 167.0 ppm corresponding to C –– O, beside the other without substitution as in case of compound 1 resulted in characteristic signals of aryl and C –– NH carbons. 221.86% improvement. Changing the substituent at phenyl Other type of hydrazide was synthesized. Ethyl 2-(4-chloro-2- moiety from 2-Br to 3-Cl as in compound 2 decreased the methylphenoxy)acetate was synthesized via esterification of 2- percentage of improvement to 189.61%. Changing the (4-chloro-2-methylphenoxy)acetic acid with ethanol in acidic substituent at phenyl moiety from 3-Cl to 4-Cl as in compound medium. Hydrazide derivative (2-(4-chloro-2-methylphenoxy)- 3 increased the percentage of improvement to 212.65%. acetohydrazide) was prepared by treating the latter ester with Introduction of (2-(4-chloro-2-methylphenoxy)acetyl)amino at hydrazine hydrate in ethanol under reflux (Scheme 1). Upon R1 and left it R2 without substitution resulted in the highest heating the obtained hydrazide derivative with dicyandiamide activity among all the compounds investigated in this study; in ethanolic hydrochloric acid, the substituted biguanide 6 was compound 6 showed the 236.31% of improvement. afforded with good yield. HPLC analysis: The retention times of compounds 1–6 were Liver function enzyme activities found to be around 2.408, 15.791, 18.000, 2.230, 14.998, and Diabetes mellitus is a major disease associated with disturban- 27.804 min, respectively. ces of the carbohydrate, fat and protein metabolism, affecting nearly 10% of the population. Aminotransferases (ALT and AST) Biological activity evaluation and ALP levels were significantly increased in STZ-treated Streptozocin-induced hyperglycemia animals, the increase in aminotransferases levels may be due to Streptozotocin induction causes pancreatic b-cells damage. In the cellular damage in the liver caused by STZ induction. addition, breakdown of glycogen from liver is responsible for Increased levels of serum ALP in pathological conditions 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 ARCH 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. R1 R2 Glucose (mg/dL) % Change % Improvement ve control 110.70  6.21 – – Diabetic rats 435.60  23.11 þ293.96 – 1 H 190.00  12.26 þ71.63 221.86 2 H 225.70  14.02 þ103.88 189.61 3 H 200.20  15.31 þ80.84 212.65 6 H 174.00  10.94 þ57.13 236.31 Metformin CH3 CH3 144.00  8.98 þ30.08 263.41 involving the liver and kidney was observed. Oxidative stress percentage of improvement 37.66, 37.64, 18.28 for AST, ALT, and alterations in glucose metabolism are important risk factors ALP, respectively. Changing the substituent at phenyl moiety for diabetes and its related complications. Advanced glycation from 3-Cl to 4-Cl as in 3 the percentage of improvement 41.84, end products (AGEs) and their carbonyl derivatives contribute 30.89, 17.16 for AST, ALT, ALP, respectively. Introduction of (2-(4- to the pathogenesis of diabetes by their interaction with chloro-2-methylphenoxy)acetyl)amino at R1 and left it R2 specific cell membrane receptors triggering to induce the without substitution resulted in the highest activities among expression of pro-inflammatory mediators and elicit oxidative all the compounds investigated in this study; compound 6 stress, which exacerbate diabetic complications. A significant showed the % improvement 64.85, 44.38, 38.53 for AST, ALT, elevation in liver function markers associated with insignificant ALP, respectively. change in total protein content as compared to the normal control group was illustrated. The high serum levels of these Lipid profiles studies enzymes after STZ treatment are associated with inflammation The levels of TG, TC, and TL in diabetic rats were increased in a and/or injury to liver cells, a condition known as hepatocellular significant way. Insulin activates lipoprotein lipase which liver injury and apoptosis. Hyperglycemia resulted in hepatol- hydrolyzes triglycerides. Insulin deficiency results in the ysis was reflected by increased blood serum aminotransferases failure of activate lipase enzyme, consequently causing as one of the consequences of diabetic complication. With hypertriglyceridemia. Hyperglycemic associated with signifi- respect to liver functional enzyme activities significant increase cant increase in the blood levels of triglycerides (TG), total in AST, ALT, and ALP enzyme activities in STZ-induced diabetic cholesterol (TC), and total lipid (TL) with percentages increase rats with percentages increase reached to 67.36, 50.00, and 88.26, 196.17, and 55.90%, respectively were recorded. As 40.92%, respectively, as compared to normal control group. shown in Table 2, treatment of diabetic rats with the As shown in Table 2, treatments of diabetic rats with the synthesized compounds showed marked amelioration in synthesized compounds 1–3 and 6 lead to improvements in TG, TC, and TL levels. The standard drug (metformin), enzyme activities. Regarding the effect of each substituent on N- R1 ¼ R2 ¼ CH3 showed 50.41% improvement for TL. Com- (1): The standard drug (metformin), R1 ¼ R2 ¼ CH3 showed 67.78, pound 6 with 2-(4-chloro-2-methylphenoxy)acetyl)amino 54.49, 35.62% improvement for AST, ALT, ALP, respectively. moiety at R1 resulted in the highest activity among all the Compound 1 with amide side chain ending with 2-bromophenyl compounds investigated in this study for TL. Compound 6 at R1 resulted in the percentage of improvement of 62.76, 47.75, exhibited activities greater than to the references drugs 39.43% for AST, ALT, ALP, respectively. Changing the substituent (56.33%). The other compounds displayed weak activities for at phenyl moiety from 2-Br to 3-Cl as in 2 decreased the TG and TC. 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. ARCH 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. mg/ mg/ mg/ no. R1 R2 U/L %b) U/L % U/L % dL % dL % dL % Normal 2.39 – 1.78 – 88.88 – 89.12 – 26.9 – 449.34 – Diabetic 4.00 – 2.67 – 125.3 – 167.78 – 79.67 – 700.54 – ratsa) 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 H 3.10 37.66 2.00 37.64 109.3 18.28 120.23 53.35 45.0 128.8 554.80 32.43 3 H 3.00 41.84 2.12 30.89 110.0 17.16 124.23 48.86 48.00 117.7 587.23 52.21 6 H 2.45 64.85 1.88 44.38 91.00 38.53 123.80 49.35 50.50 108.4 447.43 56.33 Metformin CH3 CH3 2.38 67.78 1.70 54.49 93.59 35.62 100.01 76.04 43.73 133.6 474.04 50.41 a) % Change to control of diabetic rats: AST (67.36), ALT (50.00), ALP (40.92), TG (88.26), TC (196.17), TL (55.90). b) % Improvement. Antioxidants studies improvement 51.89, 33.01, 33.89, 105.19, and 99.43 for Diabetic rats clearly demonstrated significant reduction in glutahione, superoxide dismutase, glutathione reductase, lipid glutathione-S-transferase (GST) in diabetic rats (45.56%), peroxide, and nitric oxide, respectively. The other compounds significant increase in superoxide dismutase (SOD) in diabetic displayed weak activities. rats with percentage increase 34.93% was observed (Table 3). Moreover, significant increase in lipid peroxide (MDA) for Adhesion molecules, inflammatory markers, and growth diabetic rats as compared to normal control with percentage factor evaluation increase reached to 118.37% was observed. In addition, nitric Adhesion molecules, inflammatory markers and growth oxide (NO) level showed significant increase in diabetic rats as factor were performed using a sandwich enzyme immuno- compared to normal control rats with percentage increase assay. As shown in Table 4, significant increase was noticed 102.54%. Regarding the effect of each substituent on N-(1): The in adhesion molecules ICAM and VCAM in diabetic rats. standard drug (metformin), R1 ¼ R2 ¼ CH3 showed percentage While, significant decrease in adhesion molecules in treated of improvement 56.96, 34.96, 33.23, 103.99, 3.21 for glutahione rats with the different chemical compounds, with the superoxide dismutase, glutathione reductase, lipid peroxide, greatest improvement was recorded for compound 6. nitric oxide, respectively. Introduction of 2-bromophenylamine Inflammatory marker, TNF-a, demonstrated significant or 2-(4-chloro-2-methylphenoxy)acetyl)amino moiety at R1 in increase in diabetic rats, while significant reduction in IL- case of compounds 1 and 6 resulted in the highest activity 10 was detected in diabetic rats as compared to normal among all the compounds investigated in this study. Com- control rats. Treatment of diabetic rats led to amelioration pounds 1 and 6 exhibited activities greater than to the reference in TNF-a and IL-10 levels with the most pronounced effect drug for most of tests. The other compounds displayed weak with compound 6. In addition, VEGF showed significant activities. Compound 1 exhibited percentage of improvement increase in diabetic rats as compared to normal control, 59.49, 32.19, 24.37, 107.85, 98.29 for glutahione, superoxide while treatment with the different chemical compounds dismutase, glutathione reductase, lipid peroxide, and nitric exhibited marked improvement with the greatest amelio- oxide, respectively. Compound 6 exhibited percentage of ration with compound 6. 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 ARCH 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- Superoxide Glutathione Lipid S-transferase dismutase reductase peroxide Nitric oxide (GST) (SOD) (GR) (MDA) (NO) Compd. mmol/ mmol/ mmol/ no. R1 R2 g %b) U/g % U/L % g % L % Normal 0.79 – 2920.7 – 121.87 – 865.8 – 19.3 – Diabetic 0.22 – 3941.2 – 70.70 – 1890 – 39.0 – ratsa) 1 H 0.69 59.49 3000.9 32.19 100.4 24.37 956.8 107.85 20.1 98.29 2 H 0.43 26.58 3481.0 15.96 93.12 18.39 1209 78.74 29.2 51.24 3 H 0.47 31.64 3200.2 25.37 98.60 22.89 1230 76.24 26.9 63.16 6 H 0.63 51.89 2976.9 33.01 112.0 33.89 979.9 105.19 19.9 99.43 Metformin CH3 CH3 0.67 56.96 2920.1 34.96 111.2 33.23 990.4 103.9 21.1 93.21 a) % Change to control of diabetic rats: GSH (45.56), SOD (34.93), MDA (118.37), and NO (102.54). b) % Improvement. Histopathological examination of the liver renal tubules and peritubular inflammatory cells infiltration. Microscopically, liver of diabetic rat showed cytoplasmic However, kidneys of diabetic rat treated with metformin vacuolization of hepatocytes, focal hepatic necrosis associ- showed slight congestion of glomerular tufts. Meanwhile, ated with inflammatory cells infiltration and hyperplasia of kidneys of diabetic rats treated with compounds 1, 2, and 3 epithelial lining of the bile duct. However, liver of diabetic revealed no histopathological changes. However, kidneys of rat treated with standard (metformin) revealed no changes diabetic rats treated with compound 6 revealed vacuolation except congestion of central vein). Moreover, liver of of epithelial lining of the renal tubules and endothelial diabetic rats treated with compounds 1, 2, 3, and 6 revealed lining of the glomerular tufts and slight congestion of more or less similar changes. Most examined sections glomerular tuft. Kidney of rat from control group showed revealed no changes except hydropic degeneration of the normal histological structure of renal parenchyma. hepatocytes. Liver of untreated rat control showed the Kidney of diabetic rat showed vacuolation of epithelial normal histological structure of hepatic lobule. Liver of lining of the renal tubules and endothelial lining glomerular diabetic rat showed cytoplasmic vacuolization of hepato- tufts. Kidney of diabetic rat showed eosinophilic protein cast cytes and focal hepatic necrosis associated with inflamma- in the lumen of renal tubules. Kidney of diabetic rat showed tory cells infiltration. Liver of diabetic rat showed peritubular inflammatory cells infiltration. Kidney of dia- hyperplasia of epithelial lining of the bile duct. Liver of betic rat treated with standard (metformin) showed slight diabetic rat treated with standard (metformin) showing congestion of glomerular tufts. Kidney of diabetic rat congestion of central vein. Liver of diabetic rat treated with treated with standard (metformin) showed slight congestion compounds 1–3 and 6 showed hydropic degeneration of of glomerular tufts. Kidney of diabetic rat treated with hepatocytes. compounds 1, 2, and 3 showed no histopathological changes. Kidney of diabetic rat treated with compound 6 showed Histopathological examination of the kidneys vacuolation of epithelial lining of the renal tubules and Examined kidney of diabetic rat showed vacuolation of endothelial lining of the glomerular tuft. Kidney of diabetic epithelial lining of the renal tubules and endothelial lining of rat treated with compound 6 showed slight congestion of the glomerular tufts, eosinophilic protein cast in the lumen of glomerular tuft. 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. ARCH PHARM Archiv der Pharmazie Table 4. Effect of chemical compounds adhesion molecules, inflammatory markers, and growth factor in STZ-induced diabetic rats. ICAM VCAM IL-10 VEGF TNF-a Groups R1 R2 (mg/mL) (mg/mL) (pg/mL) (pg/mL) (pg/mL) Control 3.5  0.2 8.2  0.3 55.9  3.1 150.2  9.5 90.2  5.9 Diabetic 28.5  1.2 32.5  2.1 33.1  1.2 262.5  15.5 251.9  10.2 rats 1 H 11.1  1.1 16.1  1.2 45.1  2.9 177.2  13.2 160.5  9.1 2 H 23.1  2.1 21.1  1.9 43.2  3.1 200.00  5.3 190.2  15.1 3 H 20.0  1.2 21.9  1.5 41.9  2.8 190.90  11.2 200.2  11.3 6 H 8.9  0.3 15.2  1.2 48.3  3.9 170.1  5.9 110.3  5.5 Metformin CH3 CH3 12.1  0.9 15.1  0.2 49.1  3.1 171.3  5.2 140.2  8.3 Histopathological examination of the pancreas Conclusion Pancreas of diabetic rat showed vacuolation of cells of islets of Langerhans, interacinar inflammatory cells infiltration, Biguanides are an intrinsically interesting class of compounds with and hyperplasia and thickening in the wall of pancreatic many known or potential applications. The synthesized duct. However, pancreas of diabetic rat treated with 1-substituted-amidebiguanide derivatives 1 and 6 showed standard (metformin) showed no changes except slight significant decrease in the elevated blood glucose level. Also, vacuolation of cells of islets of Langerhans. Pancreas of the antioxidant improvement of most of the synthesized products diabetic rat treated with compound 1 showed hyperplasia revealed percentages higher than that reported by metformin and hypertrophy of islets of Langerhans. No histopatho- hydrochloride were observed. A significant decrease in adhesion logical changes were noticed in pancreas of diabetic rats molecules in treated rats with the different chemical compounds, treated with compounds 1 or 3. However, pancreas of with the greatest improvement was recorded for compound 6. diabetic rat treated with compound 6 showed hyperplasia Histopathological study of the diabetic rats treated with 1- and hypertrophy of islets of Langerhans. Pancreas of rat substituted-amidebiguanide derivatives showed apparently from control showed the normal histological structure of healthy hepatic cords and blood sinusoids. Kidneys of the diabetic pancreatic parenchyma. Pancreas of diabetic rat showed rats treated with 1-substituted-amidebiguanide derivatives vacuolation of cells of islets of Langerhans. Pancreas of showed apparently healthy renal tubules and renal glomeruli diabetic rat showed interacinar inflammatory cells for their kidneys tissues. Also, pancreas of diabetic rats treated infiltration. with 1-substituted-amidebiguanide derivatives showing appar- Pancreas of diabetic rat showed hyperplasia and thickening in ently healthy pancreatic lobules with normal pancreatic acini and the wall of pancreatic duct. Pancreas of diabetic rat treated with well preserved b-cells. standard (metformin) showed slight vacuolation of cells of islets of Langerhans. Pancreas of diabetic rat treated with compound 1 showing no histopathological changes. Pancreas of diabetic rat Experimental treated with compound 2 showed hyperplasia and hypertrophy of islets of Langerhans. Pancreas of diabetic rat treated with Chemistry compound 3 showed no histopathological changes. Pancreas of General diabetic rat treated with compound 6 showed hyperplasia and All melting points are uncorrected and measured using hypertrophy of islets of Langerhans (Table 5). 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 ARCH PHARM Archiv der Pharmazie Table 5. Lesion score of diabetic and treated diabetic rats with tested compounds. Histopathological lesions Liver Kidneys Pancreas Compd. no. R1 R2 CVH FNH HD HBD CCV VIL HIL HPD ICI VELRT VCGT PICI Diabetes þþ þþ þþ þþþ þþ þþþ  þþþ þþ þþþ þþþ þþ 1 H   þ          2 H   þ          3 H   þ          6 H þ  þ   þ    þ þ  Metformin CH3 CH3     þ þ     þ  HCl 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 by filtration and washed with methanol to yield an initial crop Laboratory, National Research Centre, Cairo, Egypt. Infrared of biguanide hydrochloride. spectra (KBr-disc) were recorded using a Jasco FT/IR-300E spectrometer. 1H NMR and 13C NMR spectra were measured in 2-(2-Bromobenzoyl)-N- DMSO-d6 using Varian Mercury 500 MHz and Varian Gemini carbamimidoylhydrazinecarboximidamide hydrochloride 200 MHz with chemical shifts using TMS as standard solvent. (1) Mass spectra were recorded on a GC/MS Finnigan SSQ 7000 Yield 73%; mp 222–224°C; IR: n/cm1: 3413, 3306, 3133 (NH), spectrometer. Reactions were monitored by TLC on 0.25 mm 1704 (C –– O), 1638 (C –– N); 1H NMR: d/ppm: 6.80–8.00 (m, 9H, Merck Silica gel sheets (60 GF 354) (4  2 cm) and the spots 4Ar-H, NH2, 3NH), 9.44 (br, 1H, NH, D2O-exchangeable), 10.48 were detected with UV light. (br, 1H, CONH, D2O-exchangeable); 13C NMR: 120.1, 128.0 The InChI codes of the investigated compounds together (2C), 130.5, 132.2, 133.5, 160.1 (2CNH), 167.0 (C –– O); Anal. with some biological activity data are provided as Supporting calcd. for C9H12BrClN6O (335.59): C, 32.21; H, 3.60; N, 25.04; Information. Found: C, 32.54; H, 3.42; N, 24.87%. General procedure for the synthesis of the hydrochloride N-Carbamimidoyl-2-(3-chlorobenzoyl)- salt of 1-substituted-biguanide derivatives 1–6 hydrazinecarboximidamide hydrochloride (2) In a round-bottomed flask equipped with a magnetic stirrer, Yield 85%; mp 174–176°C; IR: n/cm1: 3382, 3342, 3184 (NH), arylhydrazide (0.1 mol) was added to aq. HCl (0.1 mol) in 50 mL 1687 (C –– O), 1646 (C –– N); 1H NMR: d/ppm: 6.80–8.00 (m, 9H, ethanol and the mixture was stirred at room temperature 4Ar-H, NH2, 3NH), 9.33 (br, 1H, NH, D2O-exchangeable), 10.77 until it became homogeneous. Dicyandiamide (0.1 mol) was (br, 1H, CONH, D2O-exchangeable); 13C NMR: 127.2, 128.3, then added, and the mixture was heated at reflux with 130.9 (2C), 132.0, 133.7, 160.3 (2C –– NH), 169.3 (C –– O); Anal. constant stirring for 12 h. The mixture was then cooled to calcd. for C9H12Cl2N6O (291.14): C, 37.13; H, 4.15; N, 28.87; room temperature, and the resulting crystals were separated 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. ARCH PHARM Archiv der Pharmazie N-Carbamimidoyl-2-(4-chlorobenzoyl)- hydrazinecarboximidamide hydrochloride (3) Biological activity evaluation Yield 86%; mp 189–191°C; IR: n/cm1: 3424, 3351, 3177 (NH), Anti-diabetic properties 1680 (C –– O), 1647 (C –– N); 1H NMR: d/ppm: 6.80–8.00 (m, 9H, Seventy male rats with an average weight of 220–250 g were 4Ar-H, NH2, 3NH); 9.35 (br, 1H, NH, D2O exchangeable), 10.77 obtained from animal house lab., National Research Centre, (br, 1H, CONH); 13C NMR: 128.9 (2CH), 130.4 (2CH), 131.4 (C), Dokki, Giza were used in this study. Animals were housed 137.5 (CCl), 160.8 (2C –– NH), 166.1 (C –– O); Anal. calcd. for under normal laboratory condition for 1 week before C9H12Cl2N6O (291.14): C, 37.13; H, 4.15; N, 28.87; Found: C, initiation of the biological experiments (adaptation period), 37.51; H, 4.03; N, 28.58%. 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 N-Carbamimidoyl-2-(4-hydroxybenzoyl)- water were supplied ad libitum and with free access to water. hydrazinecarboximidamide hydrochloride (4) The animals were divided randomly into seven main groups of Yield 84%; mp 225–227°C; IR: n/cm 1: 3381, 3218 (OH, NH), ten rats each to study the effect of the synthesized products as 1687 (C –– O), 1649 (C –– N); 1H NMR: d/ppm: 6.83 (d, 2H, well as metformin on the blood glucose level, liver function J ¼ 8.60 Hz, Ar-H), 7.06 (br, 2H, 2NH, D2 O-exchangeable), enzyme activities and lipid profile levels in STZ-induced 7.64 (br, 3H, NH 2 & NH), 7.78 (d, 2H, J ¼ 8.60 Hz, Ar-H), 9.30, diabetic rats as follows: 10.29, 10.49 (3br, 3H, 2NH & OH, D2 O-exchangeable); Group 1: Normal control rats. 13 C NMR: 115.5 (2C), 122.7, 130.1 (2C), 160.7 (2C –– NH), Group 2: Diabetes was induced by STZ. Each rat was injected 161.7, 167.1 (C –– O); Anal. calcd. for C 9H 13ClN 6O2 (272.69): intraperitoneally with a single dose of STZ (45 mg/kg body C, 39.64; H, 4.81; N, 30.82; Found: C, 39.32; H, 4.68; N, weight) dissolved in 0.01 M citrate buffer immediately before 30.54%. use. After injection, animals had free access for food and water and were given 5% glucose solution to drink overnight N-Carbamimidoyl-2-(4-methylbenzoyl)- to encounter hypoglycaemic shock. Animals were checked hydrazinecarboximidamide hydrochloride (5) daily for the presence of glycosuria. Animals were considered Yield 86%; IR: n/cm1: 3387, 3182 (NH), 1685 (C –– O), 1643 to be diabetic if glycosuria was present for 3 consecutive days. (C –– N); 1H NMR: d/ppm: 2.31 (s, 3H, CH3), 6.83–7.78 (m, 9H, 4Ar- After 3 days of STZ injection, fasting blood samples H & 3NH & NH2), 9.30, 10.31 (2br, 2H, 2NH); 13C NMR: 21.2, were obtained and fasting blood sugar was determined 115.4 (2C), 122.1, 130.8 (2C), 160.2 (2C –– NH), 161.0, 167.4 (C –– (>300 mg/dL). Hyperglycemic rats were used for the experi- O); Anal. calcd. for C10H15ClN6O (270.72): C, 44.37; H, 5.58; N, ment and classified as follows: 31.04; Found: C, 44.74; H, 5.49; N, 30.87%. Groups 3–6: Diabetic rats orally administered 2 mmol/kg body weight synthetic organic compounds for 30 days, N-Carbamimidoyl-2-(2-(4-chloro-2-methylphenoxy)- respectively. acetyl)hydrazine carboximidamide hydrochloride (6) Group 7: Diabetic rats orally administered anti-diabetic Yield 86%; mp 223–225°C; IR: n/cm1: 3300, 3167 (NH), 1670 metaformin reference drug 2 mmol/kg body weight daily for (C –– O), 1640 (C –– N); 1H NMR: d/ppm: 2.13 (s, 3H, CH3), 4.61 30 days. (s, 2H, OCH2), 6.60–7.40 (m, 9H, 3Ar-H, NH2, 4NH), 10.10 (br, 1H, CONH); 13C NMR: 16.42, 66.99, 113.78, 125.09, 126.82, Sample preparation 129.31, 130.54, 155.36, 167.12; Anal. calcd. for C11H16Cl2N6O2 After 30 days of treatments, rats were fasted overnight (335.19): 39.42; H, 4.81; N, 25.07; Found: C, 41.08; H, 5.04; N, (12–14 h), anesthetized by diethyl ether and blood 25.23%. collected by puncture of the sublingual vein in clean and dry test tube, left 10 min to clot and centrifuged at HPLC analysis 3000 rpm for serum separation. The separated serum was Samples were dissolved in 1 mL of HPLC grade MeOH/ used for biochemical analysis of blood glucose level, liver H2O (50:50) and centrifuged for 10 min at 4000 rpm before function enzyme activities, alanine and aspartate amino- HPLC analysis. Samples were analyzed on a Shimadzu LC-8A transferases (AST, ALT) and alkaline phosphatase (ALP), liquid chromatography system (Shimadzu) with LC solution lipid profile including, triglycerides (TG), total cholesterol software, SPD-M20A photodiode array detector (Shimadzu). (TC), and total lipid (TL). Liver in each exponential group A RESTEK (5 mm) C18 column was used (4.6 mm i.d.  was weighed and homogenized in 5–10 volumes of 150 mm). Elution was carried out with a gradient solvent appropriate medium using electrical homogenizer, cen- system at a flow rate of 1 mL/min. The mobile phase consisted trifuged at 3000 rpm for 15 min, the supernatants (10%) of MeOH (B) and 1% formic acid in water (A). The LC time were collected and placed in Eppendorff tubes, then program was as follows: 5–5% B (2 min), 5–20% B (5 min), stored at 80°C and used for determination of oxidative 20–30% B (3 min), 30–50% B (5 min), 50–98% B (8 min), stress markers (NO and MDA) as well as non-enzymatic 98–98% B (5 min), and 98–5% B (5 min), 5–5% B (2 min). The antioxidant (GSH), glutathione reductase (GR), and super- sample was injected into a volume of 40 mL and the eluate oxide dismutase (SOD). After blood collection, rats of each was monitored at 210 nm. group were sacrificed, the liver, kidney, and pancreas were 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 ARCH PHARM Archiv der Pharmazie removed immediately (a part was fixed in 10% formalin for shades of pink and red and the nuclei gave blue color. The histopathological examination). The non-hemolyzed, su- slides were examined and photographed under a light pernatant sera were quickly removed and kept at 80°C till microscope at a magnification power of 400. used for biochemical investigations of vascular markers Detailed histology data/photographs are provided in the including adhesion molecules, intercellular adhesion Supporting Information. molecule (ICAM-1) and vascular adhesion molecule (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. Liver function enzyme activities, J. S. Skyler, J. Med. Chem. 2004, 47, 4113–4117. alanine and aspartate amino tranferases (AST and ALT) as well P. Hogan, T. Dall, N. Plamen, Diabetes Care 2003, 26, as alkaline phosphatase (ALP) were determined in mice serum 917–932. according to the Reitman and Frankel and Belfield and A. R. Katritzky, S. R. Tala, A. Singh, ARKIVOC 2010, viii, Goldberg methods. Serum total lipids concentration was 76–96. determined according to the method of Zollner et al. , P. Ray, Chem. Rev. 1961, 61, 313–359. serum triglyceride (TG) concentration was determined A. Maric, Diabetol. Croat. 2010, 39, 95–104. according to the method of Fassati and Prencipe and W. E. Hume, T. Shingaki, T. Takashima, Y. Hashizume, serum total cholesterol concentration was estimated accord- T. Okauchi, Y. Katayama, E. Hayashinaka, Y. Wada, ing to the method of Allain et al.. Liver nitrite (NO) level H. Kusuhara, Y. Sugiyama, Y. Watanabe, Bioorg. Med. was estimated. GSH level was assayed in liver homoge- Chem. 2013, 21, 7584–7590. nate according to Beutler et al.’s method Liver MDA level S. Y. Abbas, W. M. Basyouni, K. A. M. El-Bayouk, was estimated according to the method of Satoh. GR was W. M. Tohamy, H. F. Aly, A. Arafa, M. S. Soliman, Drug determined according to the method of Goldberg and Res. 2017, 67 (10), 557–563. Spooner and SOD according to the Nishikimi et al.. S. Y. Abbas, W. M. Basyouni, K. A. M. El-Bayouki, Estimation of serum adhesionmolecules, ICAM-1 and VCAM-1, R. F. Abdel-Rahman, Drug Res. 2016, 66 (7), 377–383. VEGF, TNF-a, and IL-10 were estimated in sera by ELISA; a P. Trinder, J. Clin. Pathol. 1969, 22, 246–251. sandwich enzyme immunoassay. S. Reitman, S. Frankel, Am. J. Clin. Pathol. 1957, 28, 56–63. Calculations A. Belfield, D. Goldberg, Enzyme 1971, 12, 561–566. N. Zollner, K. Kirsch, Z. Ges. Exp. Med. 1962, 135, Mean of treated  Mean of control Percent of Change ¼  100 545–561. Mean of control P. Fossati, L. Prencipe, Clin. Chem. 1982, 28, 2077–2080. C. C. Allain, L. S. Poon, C. S. Chan, W. S. Richmond, Mean of disease  Mean of treated P. C. Fu, Clin. Chem. 1974, 20, 470–475. Percent of Improvement ¼  100 Mean of control H. Moshage, B. Kok, J. R. Huizenga, P. L. Jansen, Clin. Chem. 1995, 41, 892–896. E. Beutler, O. Duron, B. M. Kelly, J. Lab. Clin. Med. 1963, Histopathological analysis 61, 882–888. Liver, kidney, and pancreas slices were fixed instantaneously K. Satoh, Clin. Chim. Acta 1978, 90, 37–43. in buffer neutral formalin (10%) for 24 h for fixation then D. M. Goldberg, R. J. Spooner, Methods of Enzymatic processed in automatic processors, embedded in paraffin wax Analysis (Ed.: H. V. Bergmeyen), Verlag Chemie, Deer- (melting point 55–60°C), and paraffin blocks were obtained. field Beach, F1 1983, pp. 258–265. Sections of 6 mm thicknesses were prepared and stained with M. Nishikimi, N. A. Rao, K. Yagi, Biochem. Biophys. Res. haematoxylin and eosin (H&E) stain. The cytoplasm stained Commun. 1972, 46, 849–854. ß 2017 Deutsche Pharmazeutische Gesellschaft www.archpharm.com (9 of 9) e1700183