IBN AL- HAITHAM J. FO R PURE & APPL. SC I VO L. 23 (3) 2010 Synthesis, Characterization and Effect of bis-1,3,4- Oxadiazole Containing Glycine Moiety on the Activity of Some Transferase Enzymes. I. H. R. Tomi , A. H. J. Al-Qaisi*, Z. H. J. Al-Qaisi Department of Chemistry, College of Science, Al-Mustansiriya University *Department of Chemistry, College of Science, Al-Nahrain University Abstract We described herein the synthesis of novel bis-1,3,4-oxadiazole containing glycine moiety . N-{5-[5-(4-methoxyphenyl)-1,3,4-oxadiazole-2-yl-sulfanyl]-1,3,4-oxadiazole-2-yl-methyl}-4- methoxybenzamide was fully characterized by elemental analysis, FT-IR and 1 H NM R spectroscopy. Also this study was designed to show the effects of bis-oxadiazole compound on the activities of some transferase enzymes such as: GOT, GPT and γ-GT in sera. This compound demonstrated activation on GOT and GPT activities, inhibitory effects on the γ-GT activity. These effects increased with the increasing of the concentration of the compound. The causes of the increases and decreases in the enzymes activities are discussed. Introduction Derivatives of 1,3,4-oxadiazole constitute an important family of heterocyclic compounds [1]. Substituted 1,3,4-oxadiazole are considerable pharmaceutical and material interest, which are documented by a steadily increasing number of publications and patents, reported among these activities were: nervous system depressing [2], muscle relaxants [3], analgesic[4], herbicidal [5], hypoglycemic [6], antifungl [7], anti-inflammatory [8] and antibacterial [9] activities. In addition to that, derivatives of 1,3,4-oxadiazole are used in agriculture [10], photosensitizer [11] and liquid crystals [12]. Also, the derivatives of heterocyclic compounds were studied to show their effects on activities of some transferase enzymes. Dere et al [13] studied the biochemical analysis of (PQ) (1,1-dimethyl-4,4 \ -bipiridillium) on some transferase enzymes and they found some changes (increases or decreases) in these enzymes activities. Glutamate oxaloacetate transaminase (GOT) Enzyme EC 2.6.1.1: it is called also aspartate amino transfer (AST), it is one of the most important of transferase enzymes, catalyzes the transfer of the amino group of aspartate to α-ketoglutrate. GOT is widely distributed in human tissues; heart, liver, skeletal muscle and kidney. The optimum conditions of maximum enzyme activity are pH= 7.4 and temp. = 37 ° C. The clinical usefulness of the enzyme is largely restricted to the diagnosis of heart and liver diseases. Large amount of GOT may be released in to the blood. Very high levels are observed in acute liver disease while lesser elevation is seen in chronic liver disease. Glutamate pyruvate transaminase (GPT) Enzyme EC 2.6.1.2: it is also called alanine amino transferase (ALT) which is prevalent in mammalian tissue catalyzes the transfer of the amino group of alanine to α-ketoglutrate. GPT is found in a highest concentration in livers in sp ite of its active occurrence in skeletal muscles, heart and kidneys. The GPT activity in tissues is generally less than GOT. GPT level found to increase in the following diseases; infection hepatitis, liver cirrhosis and biliary cirrhosis, obstructive jaundice and liver cancer. Gamma glutamate transferase (γ-GT) Enzyme EC 2.3.2.2: it is also called gamma glutamate transpeptides (γ-GT) or (GGT), it is found in kidneys and liver and catalyzes the transfer of gamma-glutamyl group from glutathione to an amino acid. GGT levels are increased in most forms of liver disease, especially cholestasis. GGT, a plasma membrane- IHJPAS IBN AL- HAITHAM J. FO R PURE & APPL. SC I VO L. 23 (3) 2010 bound enzyme, provides the only activity capable to effect the hydrolysis of extra cellular glutathione (GSH), thus favoring the cellular utilization of its constituent amino acids. The common procedures of cyclization of oxadiazole ring is promoted by heat and anhydrous reagents like: thionyl chloride[14], phosphorous oxychloride [15], phosphorous pentoxide [16], triphenylphosphine [17] and triflic anhydride [18]. Also, Feray et al [19] showed the synthesis of the oxadiazole derivatives using the synthetic procedure based on the ring closure reactions of appropriate acid hydrazides with carbon disulfide in alkali media [20]. In the literature, we did not found any studies of the effect of oxadiazole derivatives on the activity of transferase enzymes. So, we studied here the effective of bis-1,3,4-oxadiazole compound containing sulfur atom between the two oxadiazole rings derived from N-protected glaycine attached at C-5 of the oxadiazole ring, on the activities of some tranferase enzymes in sera. This product might be potential compound for biological activity tests on transferase enzymes. A literature search revealed that no such oxadiazoles derived from amino acids have yet been prepared except Sebastiao et al [21] who synthesized some 3-aryl-1,2,4-oxadiazoles carrying protected L-alanine side chain. Result and Discussion Synthesis The characterization data of all compounds 1-7 are given in the experimental section. All the newly synthesized compounds gave satisfactory analysis for the proposed structures, which were confirmed on the basis of their elemental analysis, FT-IR and 1 H NM R data. Our synthetic strategy is show in scheme 1. Scheme 1. Synthesis of compounds (1-7) The first step was began to convert 4-methoxybenzoic acid (Anisic acid) to 4- methoxymethyl benzoate 1 by the standard esterfication method [22]. 4-Methoxybenzoyl hydrazine 2 was synthesized in good yield from the reaction of ester 1 with excess of hydrazine hydrate in ethanol. Compound 3 was synthesized by the ring closure reaction of acid hydrazide 2 with carbon disulfide in ethanolic KOH. The tautomeric equilibrium in compound 3 (3a and 3b) was investigated both for isolated and for solvated species considering the solution medium. Tsoleridis [23] studied the same equilibrium and he found that the thiol form (3b) is more stable in the gas phase. However, CH3O COOH H CH3O COOCH3 NH2-NH2.H2O CH3OH CH3O CH3O NN O S NH O NH2 NH2-NH2.H2O CH3O Cl O H2N CH2 COOH CH3O HN CH2 O POCl3 CH3O NN O S N N O CH2 NH OCH3 O COOH CH3O NH O NN O S O Et O ClCOOEt NH2 CS2 KOH CH3O CH3O NN O S H NN O S H Ethanol 1 2 Ethanol 3b 3a4 Ethanol5 6 5 + 6 7 + + Tout. IHJPAS IBN AL- HAITHAM J. FO R PURE & APPL. SC I VO L. 23 (3) 2010 this is contrary to the known experimental finding. On the basis of FT-IR and 1H NMR data, it is know that 5-(4-methoxyphenyl)-1,3,4-oxadiazole-2-thione (3a) exist in solution in the thione rather than the thiol form [24]. Parallel to experimental data, the thione tautomer is more stable than thiol in the solution. The equilibrium is even more favored to words the thione because the thione is better solvated than thiol. In the 1 H NMR spectrum of compound 3 (Fig. 1.), the broad peak at δ 14.38-14.78 of the NH proton was recorded, although it was very weak. It has been reported that the crystal structure of compound 3 correspond to the thione form [25], but the reaction conditions for the synthesis of compound 4 prove that compound 3 can be in the thiol form too. Finally, the crystal structure of compound 3 corresponded to the thione form, but they showed thiol – thione tautomerism in solution. . The mechanism [26] of ring closure of hydrazide by carbon disulfide may be outlined as follows in scheme 2. Scheme 2. The mechanism steps of formation of compound (3) An attempt to prepare the thio ester of oxadiazole ring 4 by the treatment of compound 3 with an excess of ethy l chloroformate was successful and the product obtained was identified by elemental analysis, FT-IR and 1H NM R, Figure 2. shows the 1H NM R spectrum of compound 4. The hydrazide of oxadiazole ring 5 was prepared in agood yield by the reaction of compound 4 with an excess of 80% hydrazine hydrate in ethanol. The peaks at δ 5.75 and 13.75 in 1 H NM R spectrum (Fig. 3.) are the good evidence for the formation of oxadiazole , s hydrazide 5. The compound 6 was synthesized by the reaction of 4-methoxybenzoyl chloride with glycine according to Steiger ,s p rocedure [27], to afford the corresponding hippuric acid 6. The 1H NMR spectrum of compound 6 was shown in figure 4. The hydrazide of oxadiazole ring 5 and compound 6 could be smoothly cyclidehydrated by boilig in phosphorus oxychloride affording the bis oxadiazole 7 in good yield. The 1H NM R spectra of this compound was shown in Figure 5. The mechanism of dehydration in the presence of POCl3 is depicted in the following steps: (Scheme 3). CH3O NN O SH Thiol form (3b) CH3O NN O S H Thione form (3a) A r N H O N H 2 S= C = S Ar C -H 2 S N K O H O O H N H C S K H C l SH Ar N H O C H 3O -H N C S A r C S K N O H O H N H C SK S A r N H O N H C S Ar C SK -H O H N O H N C S K S + A r = - +- + - +- + - A r C N O N C S K - + A r C N O N C S H - H H + - + - + - + + IHJPAS IBN AL- HAITHAM J. FO R PURE & APPL. SC I VO L. 23 (3) 2010 Scheme 3. The mechanism steps of formation of compound (7) Biological activity of Transferase enzymes (GOT, GPT and GGT) This research addresses investigation of the effects of compound (7) of GOT, GPT and γ- GT enzymes. The biochemical tests revealed that this compound caused inhibitory effects on γ-GT enzyme activity and activatory effects on GOT and GPT enzymes activities. Table 1. shows the effect of different concentrations of compound (7) on the activity of GOT, GPT and γ-GT enzymes in human serum. The normal value of the GOT and GPT enzyme activities were (17 and 16 U/L) respectively. The relationship between compound (7) concentrations versus and the activity of enzymes were shown in figure 6. These results observed that any increase in compound concentrations caused an increase in the percentage of activation of enzymes. The greater activation of compound (7) was demonstrated at concentration 10 -2 M (364.705%) for GOT and 10 -2 M (300%) for GPT as shown in figure 7. The normal value of the γ-GT enzyme activity was (6.545 U/L). The relationship between compound (7) concentration versus and the activity of enzyme was shown in figure 8(a). These results observed that any increase in compound concentrations caused an increase in the percentage of inhibition of enzyme. The greater inhibition of compound (7) was demonstrated at concentration 10 -5 M (55%) as shown in figure 8(b). Competitive, noncompetitive and uncompetitive inhibition can be easily distinguished with the use of double reciprocal plot of the Lineweaver-Burk plot. Two sets of rate determination CH3O NN O S N N O CH2 NH OCH3 O Ar O H O Ar NH-NH2 O POCl3 Ar C l O Ar Cl O C H3O NN O S Ar N H-NH O Ar O Ar Ar C N N H C O ArOH H+ Ar ArAr C N N H C Ar OH2 P.T -H3O O Ar C N N C O Ar N H CH2 O CH3O O H C N N H C O ArOH C N N H C OH ArOH C N N H C Ar OH Ar N H-NH O H3PO 4 Ar O 3 3 3 Ar = Ar = + + + + + + IHJPAS IBN AL- HAITHAM J. FO R PURE & APPL. SC I VO L. 23 (3) 2010 in which enzyme concentration was held constant, were carried out. In the first experiment the velocity of uninhibited enzyme was established, in the second experimental constant amount of inhibitor is included in each enzyme assay. Varieties of substances have the ability to reduce or eliminate the catalytic activity of specific enzyme [28]. Table 2. and figure 9(a). showed that the type of enzyme activation using Lineweaver-Burk plot for compound (7) on serum GOT activity. The Vmax and Km values determined with 10-2 M of compound (7) and without it. Vmax and Km values without compound (7) were 67 U/L, 200 M respectively. A liquate 10-2 M of compound (7) was noncompetitive activation for enzyme activity. Noncompetitive activation changed the Vmax of the enzyme but not the Km. When concentration of compound was 10 -2 M, the Vmax was 50 M. By using Lineweaver- Burk equation, the Ki values of enzyme for compound which was studied in different concentrations. The Ki of compound (7) in 10-2 M was 0.0129 M . Table 2. and figure 9(b). showed that the type of enzyme activation using Lineweaver-Burk plot for compound (7) on serum GPT activity. The Vmax and Km values determined with 10 -2 M of compound (7) and without it. Vmax and Km values without compound (7) were 100 U/L, 400 M respectively. A liquate 10-2 M of compound (7) was noncompetitive activation for enzyme activity. Noncompetitive activation changed the Vmax of the enzyme but not the Km. When concentration of compound was 10-2 M, the Vmax was 50 M. By using Lineweaver- Burk equation, the Ki values of enzyme for compound which was studied in different concentrations. The Ki of compound (7) in 10-2 M was 0.02 M . The enzymes play important role in amino acid metabolism and in urea and tricarboxylic acid cycles. We suggested that compound (7) molecule has (N- and O=) groups by which, it activates the active sides of amino acids of GOT and GPT enzymes by increasing affinity of active sides of enzymes to react with the substrates. Table 2. and figure 9(c). showed that the type of enzyme inhibitor using Lineweaver-Burk plot for compound (7) on serum γ-GT activity. The Vmax and Km values determined with 10-2 M of compound (7) and without it. Vmax and Km values without compound (7) were 37.037 U/L, 0.4 M respectively. A liquate 10-2 M of compound (7) was noncompetitive inhibition for enzyme activity. Competitive inhibition changed the Km of the enzyme but not the Vmax. When concentration of compound was 10 -2 M, the Km was 1 M. By using Lineweaver-Burk equation, the Ki values of enzyme for compound which was studied in different concentrations. The Ki of compound (7) in 10-2 M was 0.000263 M. Molecule of compound (7)have an interaction between the groups (N- and O=) with active sides of amino acids of γ- GT enzyme. Conclusion Novel bis-1,3,4-oxadiazole compound containing glycine moiety was prepared and structurally characterized using spectroscopic techniques. The synthetic route started from esterfication of anisic acid followed by reaction the ester with hydrazine hydrate. The acid hydrazied was converted to thione-thiol oxadiazole tautomer (3) by ring closure mechanism. The thio ester of oxadiazole (4) was prepared by the reaction of tautomer (3) with ethy l chloroformate. The acid hydrazide of oxadiazole (5) was synthesized by the same conditions for prepared compound (2). Compound (7) have been synthesized by dehydration mechanism in presence of POCl3. The biochemical studies revealed that the bis-oxadiazole caused activatory effects on GOT and GPT enzymes activities, and inhibitory effects on γ-GT enzyme activity. IHJPAS IBN AL- HAITHAM J. FO R PURE & APPL. SC I VO L. 23 (3) 2010 Experimental Materials and physical measurements. All starting materials and solvents were purchased from Aldrich and Fluka and used without further purification. Melting points were determined on Electrothermal capillary apparatus and are uncorrected, Elemental analysis (C, H, N) were carried out using a Perkin-Elmer model 2400 instrument in university of Al-albait, Amman, Jordan, FT-IR measurements were recorded on Shimadzu model FTIR-8400S in university of Al-Mustanseriya , department of chemistry, college of science, Baghdad, Iraq. 1 H NM R spectra were obtained with Bruker spectrophotometer model ultra shield at 300 M Hz. in university of Al-albait, Amman, Jordan. The compounds dissolved in DMSO-d6 solution with the TMS as internal standard. Note: in some 1 H NM R spectra, the peaks at δ 2.5 and 3.35 are for the solvent (DM SO-d6) and dissolved water in (DMSO-d6) respectively. Materials and methods of biological activity section. Effect of compound (7) on GOT and GPT activities: Colorimetric determination of GOT or GPT activity according to the following reactions: GOT L – Aspartate + α-ketoglutrate Oxaloacetic + glutmate GPT Alanine + α-ketoglutrate Pyruvate + glutmate The pyruvate or oxaloacetate formed was measured in its derived from 2,4- dinitrophenylhydrazine, which was absorbed at wave length 505 nm [29]. Effect of compound (7) on γ-GT activity: Kinetic colorimetric method for the determination of γ-GT activity was assayed by Persijn and Van Der Slik [30]. The principle of the method was measurement of the 5-amino-2-nitro benzoate form from reaction, which was absorbed at wave length 405 nm. γ- GT L-γ-glutamyl-3-carboxy-4-nitroanilide + glycylglycine L-γ-glutamyl-glycylglycine + 5-amino-2-nitro-benzoate. A stock solution (0.01 M) of compound (7) was prepared by dissolving it in ethanol/DMSO (10/1), and the following concentrations (10 -2, 10-3, 10-4, 10-5 M) were prepared by diluting with absolute ethanol. The enzymes GOT, GPT and GGT activities were measured in human serum by using the same methods of these enzymes with replacing 100µl of buffer with 100µl of compound (7). The inhibition percentage was calculated by comparing the activity with and without the compound (7) and under the same conditions, according to the equation: % Inhibition = 100 – 100 × (The activity in the presence of inhibitor) / (The activity in the absence of inhibitor) The activation percentage was calculated by comparing the activity with and without the activator and under the same conditions, according to the equation: % Activation = 100 × (The activity in the presence of activator) / (The activity in the absence of activator) – 100 A constant concentration of compound (7) (10 -2 M) was used with different substrate concentrations of (40, 80, 120, 160, 200) mmol/L for GOT and GPT, and (0.4, 0.8, 1.2, 1.62) mmol/L for γ-GT, to study the type of inhibition or activation. Buffers were used to prepare different substrates concentrations of these enzymes, GOT, GPT (phosphate buffer pH= 7.40, 100 mmol/L) and γ-GT (TRIS buffer pH= 8.25, 100 mmol/L). The enzymes activities were determined with and without compound (7), by using the Lineweaver-Burk equation and IHJPAS IBN AL- HAITHAM J. FO R PURE & APPL. SC I VO L. 23 (3) 2010 plotting 1/v against 1/[s] were evaluated values [31] ; ki, Apparent vmax (vmapp), Apperent km (kmapp), Type of inhibition or activation. Preparation methods and physical data of synthesized compounds (1-7) 4-Methoxymethyl benzoate (1) This compound was prepared by following the procedure described by Vogel [22]. Yield (95%); mp: 49-51 °C. 4-Methoxybenzoyl hydrazine (2) This compound was prepared by following the procedure by described by Smith [32]. Yield (91%); mp: 135-137 ° C. 5-(4-Methoxyphenyl)-1,3,4-oxadiazole-2-thione (3) To a solution of hydrazide 2 (1.66 g, 0.01 mol) in ethanol (20 mL), potassium hydroxide (0.56 g, 0.01 mol) in water (5 mL) and carbon disulfide (2 mL, 0.03 mol) were added. The reaction mixture was heated under reflux till the evolution of hydrogen sulfide ceased (approximately 5 h.), therefore, it was cooled, diluted with cold water (30 mL) and acidified with 10% hydrochloric acid. The solid that separated was collected by filtration, washed with water and recrystallized from ethanol. Yield (84%); mp: 199-201 °C; FT-IR (KBr disk, cm-1) 3217 (N-H), 1616 (C=N), 1253, 1080 (C-O-C); 1H NM R (DM SO-d6, 300 MHz, δ) 14.38- 14.78 (s, br. 1H, NH), 7.79-7.85 (dd, 2H, Ar.H), 7.11-7.16 (dd, 2H, Ar.H), 3.86 (s, 3H, OCH3). Anal. Calcd. For C9H8N2O2S (208 g/mol): C, 51.92; H, 3.85; N, 13.46. Found: C, 51.88; H, 3.80; N, 13.48. 5-(4-Methoxyphenyl)-1,3,4-oxadiazole-2-ethoxycarbonylsulfanyl (4) Compound 3 (2.08 g, 0.01 mol) in an excess of ethy l cloroformate (3mL) was heated under reflux for 5 h. After cooling the residue of ethy l chloroformate was removed under reduced pressure and the solid residue obtained was recrystallized from ethanol. Yield (79%); mp: 124-125 ° C; FT-IR (KBr disk, cm -1 ) 2980, 2937, 2841 (C-H aliph.), 1773 (C=O), 1626 (C=N), 1259, 1078 (C-O-C); 1H NMR (DM SO-d6, 300 MHz, δ) 7.88-7.85 (dd, 2H, Ar.H), 7.17-7.14 (dd, 2H, Ar.H), 4.48-4.41 (q, 2H, OCH2-), 3.86 (s, 3H, OCH3), 1.38-1.33 (t, 3H, CH3). Anal. Calcd. For C12H12N2O4S (280 g/mol): C, 51.43; H, 4.29; N, 10.00. Found: C, 51.44; H, 4.32; N, 10.03. 5-(4-Methoxyphenyl)-1,3,4-oxadiazole-2-hydrazinecarbonylsulfanyl (5) This compound was prepared by the same method described for compound 2. Yield (87%); mp: 222-224°C; FT-IR (KBr disk, cm-1) 3316, 3146 (N-H), 2935, 2920, 2851 (C-H aliph.) 1640 (C=O), 1610 (C=N), 1255, 1074 (C-O-C); 1 H NM R (DM SO-d6, 300 MHz, δ) 13.85- 13.75 (s, br. 1H, NH), 7.95-7.92 (dd, 2H, Ar.H), 7.05-7.02 (dd, 2H, Ar.H), 5.72 (s, 2H, NH2), 3.78 (s, 3H, OCH3). Anal. Calcd. For C10H10N4O3S (266 g/mol): C, 45.11; H, 3.76; N, 21.05. Found: C, 45.14; H, 3.77; N, 21.08. 4-(4-Methoxybenzenesulfonyl)-hippuric acid (6) Glysine (1.5g, 0.02 mol) in 1N sodium hydroxide solution (20 mL) was cooled at 0-5 °C and the cold solution was added dropwise to a solution of 4-methoxybenzoyl chloride (3.41g, 0.02 mol) in chloroform (30mL). The reaction mixture was continued under stirring for an additional 1 h. The aqueous layer was separated and acidified with 2N hydrochloric acid. The product 6 was collected by filtration and recrystallized from ethanol as colorless needles. Yield (83%); mp: 178-180 °C; FT-IR (KBr disk, cm-1) 3366 (N-H), 3190-2534 (O-H, carboxylic), 2933, 2847 (C-H aliph.) 1743 (C=O, acid), 1625 (C=O, amide); 1 H NMR CH3O COOCH3 CH3O NH O NH2 CH3O NN O SHCH3O NN O S H Tout CH3O HN CH2 O COOH IHJPAS IBN AL- HAITHAM J. FO R PURE & APPL. SC I VO L. 23 (3) 2010 (DM SO-d6, 300 MHz, δ) 8.65-8.63 (t , 1H, NH), 7.86-7.79 (dd, 2H, Ar.H), 6.98-6.95 (dd, 2H, Ar.H), 3.86-3.84 (d, 2H, CH2), 3.77 (s, 3H, OCH3). Anal. Calcd. For C10H11N1O4 (209 g/mol): C, 57.41; H, 5.26; N, 6.69. Found: C, 57.44; H, 5.23; N, 6.72. N-{5-[5-(4-methoxyphenyl)-1,3,4-oxadiazole-2-yl-sulfanyl]-1,3,4-oxadiazole-2-yl-methyl}- 4-methoxybenzamide (7) Compound 5 (2.66 g, 0.01 mol) and Compound 6 (2.09 g, 0.01 mol) were refluxed with phosphorous oxychloride (5mL) for 24 h. and the reaction mixture was then treated with ice water carefully and made basic by adding concentrated sodium bicarbonate solution. The resulting solid was filtered, dried and recrystallized by ethanol-DMSO (10/1). Yield (69%); mp: 193-195 °C; FT-IR (KBr disk, cm-1) 3362 (N-H), 2920, 2839 (C-H aliph.), 1620 (C=O, amide), 1610 (C=N), 1255, 1026 (C-O-C); 1H NM R (DM SO-d6, 300 MHz, δ) 8.29-8.22 (t, 1H, NH), 8.17-8.11 (dd, 2H, Ar.H), 7.88-7.81 (dd, 2H, Ar.H), 7.23-7.18 (dd, 2H, Ar.H), 7.09- 7.02 (dd, 2H, Ar.H) 3.87 (s, 3H, OCH3 attached to phenyl-oxadiazole), 3.81 (s, 3H, OCH3 attached to benzamide), 3.79-3.82 (d, 2H, CH2). Anal. Calcd. For C20H17N5O5S (439 g/mol): C, 54.67; H, 3.87; N, 15.94. Found: C, 54.71; H, 3.89; N, 15.98. Acknowledgment We thank Mr. Mohanad H. M. Masad (Al al-Bayt university, Jordan) for being helpful about doing the 1 H NM R spectra and Mrs. Zainab K. Mohammed Jawad (Al-Mustansiriya university, Chem. Dept.) about doing the FT-IR spectra. Also we are very grateful to Dr. Selma A. Abbass (Al-Mustansiriya university, Chem. Dept.) for performing the biological experiments. References 1. Hill, J.(1984)"1,3,4-Oxadiazoles in Comprehensive Heterocyclic Chemistry" Katrizky , A. R., Ress, C. W., Pergamon press: Oxford, New York, Toronto, Sydney, Paris and Frankfurt, 6: 427. 2. Maillard, J.; Vincent, M.; Morin, R.and Benard, M. (1962) Hypnotic and Sedative Drug, 2-(o-Hydroxyphenyl)-1,3,4-Oxadizole French Patent M 379, Chem. Abstr.. 57:15251. 3. Vousooghi, A. N.; Tabatabai, S. A.; Eezadeh, A. K.and Shafiee, A. 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(2000) Synth. Commun. A Mild Method for The Preparation of 1,3,4-Oxadiazoles: Triflic Anhydride Promoted Cyclization of Diacylhydrazines. 30:437. 19. Feray, A.; Zuhal, T.; Nuket, O. and Safiya, S. (2002) Synthesis and Electronic Structure of New Aryl- and Alkyl- Substituted 1,3,4-Oxadiazole-2-thione Derivatives. J. M ed. Chem. 26:159. 20. Koparir, M. ;Cetin, A. and Cansiz, A. (2005) 5-Furane-2yl[1,3,4]Oxadiazole-2-thiol, 5-Furan-2yl-4H[1,2,4] Triazole-3-thiol and Their Thiol-Thione Tautomerism. J. Med. Chem. 10:475. 21. Sebastiao, J. M.; Antonio, D. S.; Heron, L. L.and Srivastava, R. M. (1998) Synthesis of Some 3-Aryl-1,2,4-Oxadiazoles Carrying a Protected L-Alanine Side Chain. J. Med. Chem. 19(5):465. 22. Brian, S. F. ; Antony, J. H. ;Peter,W. G. S. and Austin,R. T. (1989)Vogel,s: Practical Organic Chemistry, 5 th ed., Longman Scientific Technical, New York, , P 1077. 23. Tsoleridis, C. A. ;Charistos, D. A. ;Vagenas, G. V. (1997). UV and MO Study on The Deprotonation of Some 2-Aryl-Δ 2 -1,3,4-Oxadiazole-5-thiones. J. Heterocyclic Chem 34:1715. 24. Horning, Muchowski, (1972) Five-membered Heterocyclic Thiones. Part I. 1,3,4- Oxadiazole-2-thione. Can. J. Chem. 50: 3079. 25. Ozturk, S. ;Akkurt, M.; Cansiz, A. ;Cetin, A. ;Sekerci, M.and Heinemann, F. W. (2004) 5-(Furan-2-yl)-1,3,4-Oxadiazole-2(3H)-thione. Acta. Cryst. E. E60 O322. 26. L. S. Ibrahimi, (1989) M.Sc Thesis, University of Baghdad, College of education Ibn Al-Haitham, Baghdad, Iraq. 27. Steiger, R. E. (1944). Benzolation of Amino Acids. J. Org. Chem 9: 396. 28. Satyanarayna,U. (2003) Biochemistry 2 nd ed. Books and Allied (P) LTD, India, pp 91- 95. 29. Reitman,S. and Frankel, S. (1957) Path. A Colorimetric Method for the Determination of Serum Glutamic Oxalacetic and Glutamic Pyruvic Transaminases. Am. J. Clin. 28, 56. 30. Persijn, Van Der Silk (1976) J. Chem. Clin. Biochem. A New Method for The Determination of Gamma-glutamyltransferase. 4, 421. IHJPAS IBN AL- HAITHAM J. FO R PURE & APPL. SC I VO L. 23 (3) 2010 31. Linweaver, H. Burke, D. (1934) The Determination of Enzyme Dissociation Constants. J. Am. Chem. Soc. 56: 658. 32. Smith,P. A. (1946) Organic Reactions 3: 366. Table (1): The effect of different concentrations of comp. (7) on the activity of GOT, GPT and γ-GT enzymes in human serum. No. Conc. [M] GOT activity U/L Activation % GPT activity U/L Activation % γ-GT Activity U/L Inhibition % 1 0 17 0.000 16 0.000 6.545 0.000 2 10-2 79 364.705 64 300.000 1.303 0.000 3 10-3 30 76.470 20 25.000 2.618 20.000 4 10-4 29 70.580 18 12.500 5.236 60.000 5 10-5 24 41.176 17 6.250 6.545 80.000 Table (2): The kinetic properties of GOT, GPT and γ-GT with comp. (7) Enzymes Kmap (M) Vmap (U/L) Ki (M) Type of effect GOT 100 50 0.0129 Noncompetitive GPT 400 50 0.02 Noncompetitive γ- GT 1 0.256 0.000263 Noncompetitive IHJPAS IBN AL- HAITHAM J. FO R PURE & APPL. SC I VO L. 23 (3) 2010 Figure 1. 1H NMR spectrum of compound 3 Figure 2. 1H NMR spectrum of co mpound 4 Figure 3. 1H NMR spectrum of compound 5 Figure 4. 1H NMR spectrum of co mpound 6 Figure 5. 1H NMR spectrum of compound 7 Figure 6. (a) The relationship between conc. of compound (7) and GOT enzyme activity. (b) The relationship between conc. ocompound (7) and GPT enzyme activity. IHJPAS IBN AL- HAITHAM J. FO R PURE & APPL. SC I VO L. 23 (3) 2010 Figure 7. (a) The percentage of activation GOT enzyme Figure 8. (a) The relationship between conc. of and compound (7) conc. compound(7) and GGT enzyme activity. (b) The percentage of activation GPT enzyme (b) The percentage of activation GGT and compound (7) conc. enzyme and compound (7) conc. Figure 9. Lineweaver-Burk plots for comp. (7) effects on (a) GOT, (b) GPT and (c) GGT IHJPAS 2010) 3( 23 مجلة ابن الھیثم للعلوم الصرفة والتطبیقیة المجلد فعالیة بعض فيتحضیر تأثیر حلقتي االوكسادیازول الحاویة على الكالیسین وتشخیصھا ودراستھا االنزیمات الناقلة ،زیاد حسین جواد القیسي * ،عالء حسین جواد القیسيایفان حمید روئیل تومي الجامعة المستنصریةالكیمیاء ،كلیة العلوم ، قسم جامعة النھرین قسم الكیمیاء ،كلیة العلوم ،* الجامعة المستنصریة قسم الكیمیاء، كلیة العلوم ، الخالصة ادیازول ومركـب الكالیسـین -4,3,1في هذا البحث حضر مركب جدید یحتوي على حلقتـین مـن -N-{5-[5اوكسـ (4-methoxyphenyl)-1,3,4-oxadiazole-2-yl-sulfanyl]-1,3,4- oxadiazole-2-yl-methyl}-4- methoxybenzamide .وطیــف االشــعة تحـت الحمــراء وطیــف ،طة التحلیــل الــدقیق للعناصـراشـخص هــذا المركــب بوسـ ادیازول الملتصــ. الــرنین النــووي المغناطیســي اثیر حلقتــي االوكســ ة بعــض فــيقة كــذلك صــممت هــذه الدراســة لبیــان تــ فعالیــ ـام بتنشـیط فعالیـة االنزیمــات . فــي مصـل الـدم GOT ،GPT ،GGT :مثـل ،االنزیمـات الناقلـة ووجــد ان هـذا المركـب قـ GOT وGPT بینما كان تاثیره مثبطا لفعالیة االنزیمGGT . فعالیـة االنزیمـات تـزداد بزیـادة فيووجد ان هذه التاثیرات الزیادة والنقصان في الفعالیة الحیاتیة لهذه االنزیمات بوجود هذا المركـب نوقشـت بالتفصـیل ان . تركیز مركب االوكسادیازول . في هذا البحث IHJPAS