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 De partment of Che mistry, Colle ge of S cience, Al-Mustansiriya Uni versity *De partment of Che mistry, Colle ge of S cience, Al-Nahrain University Abstract We described herein the sy nthesis of novel bis-1,3,4-oxadiazole containing glycine moiety . N-{5-[5-(4-methoxy p henyl)-1,3,4-oxadiazole-2-yl-sulfany l]-1,3,4-oxadiazole-2-yl-methy l}-4- methoxy benzamide was fully characterized by elemental analysis, FT -IR and 1 H NM R sp ectroscop y . Also this st udy was designed to show the effects of bis-oxadiazole comp ound on the activities of some transferase enzy mes such as: GOT, GPT and γ-GT in sera. This comp ound demonst rated activation on GOT and GPT activities, inhibitory effects on the γ-GT activity . These effects increased with the increasing of the concentration of the comp ound. The causes of the increases and decreases in the enzy mes activities are discussed. Introduction Derivatives of 1,3,4-oxadiazole constitut e an imp ortant family of heterocyclic comp ounds [1]. Subst itut ed 1,3,4-oxadiazole are considerable p harmaceutical and material interest, which are documented by a st eadily increasing number of p ublications and p atents, reported among these activities were: nervous sy st em depressing [2], muscle relaxants [3], analgesic[4], herbicidal [5], hy p oglycemic [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], p hotosensitizer [11] and liquid cryst als [12]. Also, the derivatives of heterocyclic comp ounds were st udied to show their effects on activities of some transferase enzy mes. Dere et al [13] st udied the biochemical analysis of (PQ) (1,1-dimethy l-4,4 \ -bipiridillium) on some transferase enzy mes and they found some changes (increases or decreases) in these enzy mes activities. Glutamate oxaloacetate transaminase (GOT) Enzy me EC 2.6.1.1: it is called also asp artate amino transfer (AST), it is one of the most imp ortant of transferase enzy mes, cataly zes the transfer of the amino group of asp artate to α-ketoglutrate. GOT is widely distributed in human tissues; heart, liver, skeletal muscle and kidney . The op timum conditions of maximum enzy me activity are p H= 7.4 and temp . = 37 ° C. The clinical usefulness of the enzy me 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 p y ruvate transaminase (GPT) Enzy me EC 2.6.1.2: it is also called alanine amino transferase (ALT) which is p revalent in mammalian tissue cataly zes 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 kidney s. 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) Enzy me EC 2.3.2.2: it is also called gamma glutamate transp eptides (γ-GT) or (GGT), it is found in kidney s and liver and cataly zes the transfer of gamma-glutamyl group from glutathione to an amino acid. GGT levels are increased in most forms of liver disease, esp ecially cholestasis. GGT, a plasma membrane- IHJPAS IBN AL- HAITHAM J . FO R PURE & APPL. SC I VO L. 23 (3) 2010 bound enzy me, p rovides the only activity cap able to effect the hy drolysis of extra cellular glutathione (GSH), thus favoring the cellular utilization of its const ituent amino acids. The common p rocedures of cyclization of oxadiazole ring is p romoted by heat and anhydrous reagents like: thionyl chloride[14], p hosp horous oxy chloride [15], p hosp horous p entoxide [16], triphenylphosp hine [17] and triflic anhydride [18]. Also, Feray et al [19] showed the sy nthesis of the oxadiazole derivatives using the sy nthetic procedure based on the ring closure reactions of app rop riate acid hy drazides with carbon disulfide in alkali media [20]. In the literature, we did not found any st udies of the effect of oxadiazole derivatives on the activity of transferase enzy mes. So, we st udied here the effective of bis-1,3,4-oxadiazole comp ound containing sulfur atom between the two oxadiazole rings derived from N-p rotected glay cine att ached at C-5 of the oxadiazole ring, on the activities of some tranferase enzy mes in sera. This p roduct might be p otential comp ound for biological activity tests on transferase enzy mes. A literature search revealed that no such oxadiazoles derived from amino acids have y et been p repared excep t Sebast iao et al [21] who sy nthesized some 3-aryl-1,2,4-oxadiazoles carry ing p rotected L-alanine side chain. Re sult and Discussion S ynthesi s The characterization data of all compounds 1-7 are given in the exp erimental section. All the newly sy nthesized comp ounds gave satisfactory analysis for the p rop osed st ructures, which were confirmed on the basis of their elemental analysis, FT -IR and 1 H NM R data. Our sy nthetic strategy is show in scheme 1. S cheme 1. S ynthesis of compounds (1-7) The first st ep was began to convert 4-methoxy benzoic acid (Anisic acid) to 4- methoxy methy l benzoate 1 by the st andard esterfication method [22]. 4-M ethoxy benzoy l hy drazine 2 was sy nthesized in good y ield from the reaction of ester 1 with excess of hy drazine hy drate in ethanol. Compound 3 was sy nthesized by the ring closure reaction of acid hydrazide 2 with carbon disulfide in ethanolic KOH. The tautomeric equilibrium in comp ound 3 (3a and 3b) was invest igated both for isolated and for solvated sp ecies considering the solution medium. Tsoleridis [23] st udied the same equilibrium and he found that t he thiol form (3b) is more st able in the gas p hase. However, CH3O CO OH H CH3O COOCH3 NH2-NH2.H2O CH3OH CH3O CH3O NN O S NH O NH2 NH2-NH2.H2O CH3O Cl O H2N CH 2 CO OH CH3O HN CH2 O POCl3 CH3O NN O S N N O CH2 NH O CH3 O CO OH CH3O NH O NN O S O Et O ClC OOEt NH 2 CS2 KOH CH3O CH3O NN O S H NN O S H Ethanol 1 2 Ethanol 3b 3a4 E thanol5 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 exp erimental finding. On t he basis of FT -IR and 1 H NM R data, it is know that 5-(4-methoxy p henyl)-1,3,4-oxadiazole-2-thione (3a) exist in solution in the thione rather than the thiol form [24]. Parallel to exp erimental data, the thione tautomer is more st able than thiol in the solution. The equilibrium is even more favored to words the thione because the thione is bett er solvated than thiol. In the 1 H NM R sp ectrum of comp ound 3 (Fig. 1.), the broad p eak at δ 14.38-14.78 of the NH p roton was recorded, although it was very weak. It has been reported that the cryst al st ructure of comp ound 3 corresp ond to the thione form [25], but the reaction conditions for the sy nthesis of comp ound 4 p rove that comp ound 3 can be in the thiol form too. Finally, the cryst al st ructure of comp ound 3 corresp onded to t he thione form, but they showed thiol – thione tautomerism in solution. . The mechanism [26] of ring closure of hy drazide by carbon disulfide may be outlined as follows in scheme 2. S cheme 2. The mechanism steps of formation of compound (3) An attemp t to p repare the thio ester of oxadiazole ring 4 by the treatment of comp ound 3 with an excess of ethy l chloroformate was successful and the p roduct obt ained was identified by elemental analysis, FT -IR and 1 H NM R, Figure 2. shows the 1 H NM R sp ectrum of comp ound 4. The hydrazide of oxadiazole ring 5 was p repared in agood y ield by the reaction of comp ound 4 with an excess of 80% hy drazine hydrate in ethanol. The peaks at δ 5.75 and 13.75 in 1 H NM R sp ectrum (Fig. 3.) are the good evidence for the formation of oxadiazole , s hy drazide 5. The comp ound 6 was sy nthesized by the reaction of 4-methoxy benzoy l chloride with glycine according to Steiger , s p rocedure [27], to afford the corresp onding hipp uric acid 6. The 1 H NM R sp ectrum of comp ound 6 was shown in figure 4. The hydrazide of oxadiazole ring 5 and comp ound 6 could be smoothly cyclidehy drated by boilig in p hosp horus oxy chloride affording the bis oxadiazole 7 in good y ield. The 1 H NM R sp ectra of this comp ound was shown in Figure 5. The mechanism of dehydration in the p resence of POCl3 is depicted in the following st eps: (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 A r C -H 2 S N K O H O O H N H C S K H C l S H A r N H O C H 3O -H N C S A r C S K N O H O H N H C S K S A r N H O N H C S A r C S K -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 S cheme 3. The mechanism st eps of formation of comp ound (7) Biological activity of Transfe rase enz ymes (GOT, GPT and GGT) This research addresses invest igation of the effects of comp ound (7) of GOT, GPT and γ- GT enzy mes. The biochemical tests revealed that t his comp ound caused inhibitory effects on γ-GT enzy me activity and activatory effects on GOT and GPT enzy mes activities. T able 1. shows t he effect of different concentrations of compound (7) on the activity of GOT, GPT and γ-GT enzy mes in human serum. The normal value of the GOT and GPT enzy me activities were (17 and 16 U/L) resp ectively. The relationship between comp ound (7) concentrations versus and the activity of enzy mes were shown in figure 6. These results observed that any increase in comp ound concentrations caused an increase in the p ercentage of activation of enzy mes. The greater activation of comp ound (7) was demonst rated 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 enzy me activity was (6.545 U/L). The relationship between comp ound (7) concentration versus and the activity of enzy me was shown in figure 8(a). These results observed that any increase in comp ound concentrations caused an increase in the p ercentage of inhibition of enzy me. The greater inhibition of comp ound (7) was demonst rated at concentration 10 -5 M (55%) as shown in figure 8(b). Competitive, noncomp etitive and uncompetitive inhibition can be easily distinguished with the use of double recip rocal plot of t he Lineweaver-Burk p lot. T wo sets of rate determination CH 3O NN O S N N O CH2 NH OC H3 O Ar O H O Ar NH -N H2 O POC l3 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 ArO H H + Ar ArAr C N N H C Ar OH 2 P .T -H3O O Ar C N N C O Ar N H CH2 O CH3O O H C N N H C O ArO H C N N H C O H ArO H C N N H C Ar O H 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 enzy me concentration was held constant, were carried out. In the first exp eriment the velocity of uninhibited enzy me was established, in the second exp erimental constant amount of inhibitor is included in each enzy me assay . Varieties of substances have the ability to reduce or eliminate the catalytic activity of sp ecific enzy me [28]. Table 2. and figure 9(a). showed that the ty p e of enzy me activation using Lineweaver-Burk p lot for compound (7) on serum GOT activity . The Vm ax and Km values determined with 10 -2 M of comp ound (7) and without it. Vm ax and Km values without comp ound (7) were 67 U/L, 200 M resp ectively. A liquate 10 -2 M of comp ound (7) was noncomp etitive activation for enzy me activity . Noncompetitive activation changed the Vm ax of the enzy me but not the Km . When concentration of comp ound was 10 -2 M , the Vm ax was 50 M . By using Lineweaver- Burk equation, the Ki values of enzy me for comp ound which was st udied in different concentrations. The Ki of comp ound (7) in 10 -2 M was 0.0129 M . Table 2. and figure 9(b). showed that the ty p e of enzy me activation using Lineweaver-Burk p lot for compound (7) on serum GPT activity . The Vm ax and Km values determined with 10 -2 M of comp ound (7) and without it. Vm ax and Km values without comp ound (7) were 100 U/L, 400 M resp ectively. A liquate 10 -2 M of comp ound (7) was noncomp etitive activation for enzy me activity . Noncompetitive activation changed the Vm ax of the enzy me but not the Km . When concentration of comp ound was 10 -2 M , the Vm ax was 50 M . By using Lineweaver- Burk equation, the Ki values of enzy me for comp ound which was st udied in different concentrations. The Ki of comp ound (7) in 10 -2 M was 0.02 M . The enzy mes p lay imp ortant role in amino acid metabolism and in urea and tricarboxy lic acid cy cles. We suggested that comp ound (7) molecule has (N- and O=) group s by which, it activates the active sides of amino acids of GOT and GPT enzy mes by increasing affinity of active sides of enzy mes t o react with t he subst rates. Table 2. and figure 9(c). showed that the ty p e of enzy me inhibitor using Lineweaver-Burk p lot for compound (7) on serum γ-GT activity . The Vm ax and Km values determined with 10 -2 M of comp ound (7) and without it. Vm ax and Km values without comp ound (7) were 37.037 U/L, 0.4 M resp ectively. A liquate 10 -2 M of comp ound (7) was noncompetitive inhibition for enzy me activity . Competitive inhibition changed the Km of the enzy me but not the Vm ax. When concentration of comp ound was 10 -2 M , the Km was 1 M . By using Lineweaver-Burk equation, the Ki values of enzy me for comp ound which was st udied in different concentrations. The Ki of comp ound (7) in 10 -2 M was 0.000263 M . M olecule of comp ound (7)have an interaction between the group s (N- and O=) with active sides of amino acids of γ- GT enzy me. Conclusion Novel bis-1,3,4-oxadiazole comp ound containing glycine moiety was p repared and st ructurally characterized using sp ectroscop ic techniques. The sy nthetic route st arted from esterfication of anisic acid followed by reaction the ester with hy drazine hy drate. The acid hy drazied was converted to thione-thiol oxadiazole tautomer (3) by ring closure mechanism. The thio ester of oxadiazole (4) was p repared by the reaction of tautomer (3) with ethy l chloroformate. The acid hy drazide of oxadiazole (5) was sy nthesized by the same conditions for p repared comp ound (2). Compound (7) have been sy nthesized by dehydration mechanism in p resence of POCl3. The biochemical st udies revealed that the bis-oxadiazole caused activatory effects on GOT and GPT enzy mes activities, and inhibitory effects on γ-GT enzy me activity . IHJPAS IBN AL- HAITHAM J . FO R PURE & APPL. SC I VO L. 23 (3) 2010 Experimental Materials and physical me asureme nts. All starting materials and solvents were purchased from Aldrich and Fluka and used without further p urification. M elting p oints were determined on Electrothermal cap illary app aratus and are uncorrected, Elemental analysis (C, H, N) were carried out using a Perkin-Elmer model 2400 inst rument in university of Al-albait, Amman, Jordan, FT -IR measurements were recorded on Shimadzu model FT IR-8400S in university of Al-M ust anseriy a , department of chemist ry , college of science, Baghdad, Iraq. 1 H NM R sp ectra were obtained with Bruker sp ectrop hotometer model ultra shield at 300 M Hz . in university of Al-albait, Amman, Jordan. The compounds dissolved in DM SO-d6 solution with t he TM S as internal st andard. Not e: in some 1 H NM R sp ectra, the p eaks at δ 2.5 and 3.35 are for the solvent (DM SO-d6) and dissolved water in (DM SO-d6) resp ectively. Materials and methods of biological activity se ction. Effect of compound (7) on GOT and GPT activities: Colorimetric determination of GOT or GPT activity according to the following reactions: GOT L – Asp artate + α-ketoglutrate Oxaloacetic + glutmate GP T Alanine + α-ketoglutrate Py ruvate + glutmate The p y ruvate or oxaloacetate formed was measured in its derived from 2,4- dinitrop henylhydrazine, 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 assay ed 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 comp ound (7) was p repared by dissolving it in ethanol/DMSO (10/1), and the following concentrations (10 -2 , 10 -3 , 10 -4 , 10 -5 M ) were p repared by diluting with absolute ethanol. The enzy mes GOT , GPT and GGT activities were measured in human serum by using the same methods of these enzy mes with rep lacing 100µl of buffer with 100µl of comp ound (7). The inhibition p ercentage was calculated by comp aring the activity with and without the compound (7) and under the same conditions, according to the equation: % Inhibition = 100 – 100 × (The activity in the p resence of inhibitor) / (The activity in the absence of inh ibitor) The activation p ercentage was calculated by comp aring 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 comp ound (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 st udy the ty p e of inhibition or activation. Buffers were used to p repare different subst rates concentrations of t hese enzy mes, GOT , GPT (p hosp hate buffer pH= 7.40, 100 mmol/L) and γ-GT (TRIS buffer p H= 8.25, 100 mmol/L). The enzy mes activities were determined with and without comp ound (7), by using the Lineweaver-Burk equation and IHJPAS IBN AL- HAITHAM J . FO R PURE & APPL. SC I VO L. 23 (3) 2010 p lott ing 1/v against 1/[s] were evaluated values [31] ; ki, Ap p arent vm ax (vm app), Ap p erent km (km app), Ty p e of inhibition or activation. Preparation methods and physical data of synthesi zed compounds (1-7) 4-Methoxymethyl benz oate (1) This comp ound was p repared by following the p rocedure described by Vogel [22]. Yield (95%); mp : 49-51 ° C. 4-Methoxybenz oyl hydrazine (2) This comp ound was p repared by following the procedure by described by Smith [32]. Yield (91%); mp : 135-137 ° C. 5-(4-Methoxyphe nyl )-1,3,4-oxadiazole-2-thi one (3) To a solution of hy drazide 2 (1.66 g, 0.01 mol) in ethanol (20 mL), p otassium hy droxide (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 hy drogen sulfide ceased (app roximately 5 h.), therefore, it was cooled, diluted with cold water (30 mL) and acidified with 10% hy drochloric acid. The solid that sep arated was collected by filtration, washed with water and recry st allized 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); 1 H NM R (DM SO-d6, 300 M Hz , 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-Methoxyphe nyl )-1,3,4-oxadiazole-2-ethoxycarbonyl sulfanyl (4) Compound 3 (2.08 g, 0.01 mol) in an excess of ethy l cloroformate (3mL) was heated under reflux for 5 h. Aft er cooling the residue of ethy l chloroformate was removed under reduced p ressure and the solid residue obtained was recry st allized from ethanol. Yield (79%); mp: 124-125 ° C; FT-IR (KBr disk, cm -1 ) 2980, 2937, 2841 (C-H alip h.), 1773 (C=O), 1626 (C=N), 1259, 1078 (C-O-C); 1 H NM R (DM SO-d6, 300 M Hz , 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-Methoxyphe nyl )-1,3,4-oxadiazole-2-hydrazine carbonyl sulfanyl (5) This comp ound was p repared by the same method described for comp ound 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 M Hz , 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-Methoxybenz ene sulfonyl )-hi ppuric acid (6) Gly sine (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-methoxy benzoy l chloride (3.41g, 0.02 mol) in chloroform (30mL). The reaction mixture was continued under st irring for an additional 1 h. The aqueous layer was separated and acidified with 2N hy drochloric acid. The p roduct 6 was collected by filtration and recry st allized from ethanol as colorless needles. Yield (83%); mp: 178-180 ° C; FT -IR (KBr disk, cm -1 ) 3366 (N-H), 3190-2534 (O-H, carboxy lic), 2933, 2847 (C-H alip h.) 1743 (C=O, acid), 1625 (C=O, amide); 1 H NM R CH 3O COOCH3 CH3O NH O NH2 CH3O NN O S HCH3 O 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 M Hz , 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 p hosp horous oxy chloride (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 recry st allized by ethanol-DM SO (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); 1 H NM R (DM SO-d6, 300 M Hz , 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 att ached to p henyl-oxadiazole), 3.81 (s, 3H, OCH3 att ached 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. Acknowledgme nt We thank M r. M ohanad H. M . M asad (Al al-Bayt university , Jordan) for being help ful about doing the 1 H NM R sp ectra and M rs. Zainab K. M ohammed Jawad (Al-M ust ansiriy a university , Chem. Dep t.) about doing the FT -IR sp ectra. Also we are very grateful to Dr. Selma A. Abbass (Al-M ust ansiriy a university , Chem. Dep t.) for p erforming the biological exp eriments. Refe rences 1. Hill, J.(1984)"1,3,4-Oxadiazoles in Comp rehensive Heterocyclic Chemistry " Katrizky , A. R., Ress, C. W., Pergamon p ress: Oxford, New York, Toronto, Sy dney, Paris and Frankfurt, 6: 427. 2. M aillard, J.; Vincent, M .; M orin, R.and Benard, M . (1962) Hy p notic and Sedative Drug, 2-(o-Hy droxy p henyl)-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. (2007) Sy nthesis, Ant iconvulsant and M uscle Relaxant Activities of Subst itut ed 1,3,4-oxadiazole, 1,3,4- thiadiazole and 1,2,4-triazole Acta. Chim. Slov. 54: 317. 4. M ishra, L., Said, M . K. It okawa, H. Takey a, K. (1995) Bioorg. M ed. Chem, Antitumor and Ant imicrobial Activities of Fe(II)/Fe(III) Complexes Derived From Some Heterocy clic Compounds 3 (9) :1241. 5. Kennedy, D. and Summers, L. (1981) Reduction Pot ential and Herbicidal Activity of 4,4-(1,3,4-Thiadiazole-2,5-diyl)-and4,4-(1,3,4-Oxadiazole-2,5-diy l)Bis(1- M ethy lpy ridinium)Diiodides. J. Heterocyclic Chem. XIV 409. 6. Girges, M . M . Arz neim-Forsch, (1994) Sy nthesis and Pharmacological Evalution of Novel Series of Sulfonate-est er Containing 1,3,4-Oxadiazole Derivatives with Ant icipated Hy p oglycimic Activity . Drug Res. 44, 490. 7. Xia, J.; Lu, H.; Gui,Y. and Zu, X. (2002) Sy nthesis, Fungicidal Activity , and 3D- QSAR of Py ridazinone-Subst itut ed 1,3,4-Oxadiazole and 1,3,4-Thiadiazole. J. Agric. Food Chem, 50:3757. 8. Suman, P. and Bahel, S. C. (1979) 5-Subst itut ed-1,3,4-Oxadiazoles and Related Compounds as Possible Fungicides. J. Indian Chem. Soc, 56 :712. 9. Tashfeen, A. ;Shahid, H.; Najim, A. ;Roberta, L.and Paolo, L. C. (2005) In Vitro Ant itumor and Ant iviral Activities of New Benzot hiazole and 1,3,4-Oxadiazole-2- thione Derivatives. Acta. Pharm. 58:135. IHJPAS IBN AL- HAITHAM J . FO R PURE & APPL. SC I VO L. 23 (3) 2010 10. Hodogaya, Chemical Co., LTD., Jp n. Tokky o Koho, 8027.042, (1980) Chem. Abstr. 1980, 93: 232718g. 11. Jai, F. W.; Ghassan, E. J.; Eugene, A. M . ;Jeff, A. ;Yadong, Z.; Paul, A. L.; Neal, R. A. P. and Nasser, Bernard, K. (1999) Oxadiazole M etal Complex for Organic Light- Emitt ing Diodes. Adv. M ater .11:15, 1266. 12. Parra, M . ;Hidalgo, P.; Carrasco, E.; Barbera, J. and Silvino, L. (2006) New 1,2,4- and 1,3,4-Oxadiazole M aterials: Sy nthesis, M esomorphic and Luminescence Prop erties. Liquid Cryst al, . 33( 8): 875. 13. Dere, E. and Polat, F. (2001) T he Effect of Paraquat on the Activity of Some Enzy mes in Different T issues of M ice (M us M usculus-Swiss Albino). Turk J. Biol. 25:323. 14. Al-Talib, M . ,Tastoush, H. and Odeh, N. (1990) A Convenient Sy nthesis of Alky l and Ary l Subst itut ed Bis-1,3,4-Oxadiazoles. Sy nth. Commun. 20:1811. 15. Theocharis, A. B. and Alexandrou, N. E. (1990) Sy nthesis and Sp ectral Data of 4,5- Bis[5-aryl-1,3,4-oxadiazole-2-yl]-1-Benzy l-1,2,3-Triazole.J.Heterocy clic Chem. 27:1685. 16. Carlsen, P. H. J.and Jorgensen, K. B. (1994) J. Heterocy clic Chem. Sy nthesis of Unsy mmetrically Subst itut ed 4H-1,2,4-Triazoles. J. Heterocy clic Chem. 31: 805. 17. Brown, P. ;Best , D. J.; Broom, N. J. P.; Cassels, R.; O , hanlon, P. J.; M itchell, T. J. ;Osborne, N. F. and Wilson, J. M . (1997) The Chemistry of Pseudomonic Acid. 18. Heterocy clic Replacment of The α,ß-Unsaturated Ester: Sy nthesis, M olecular M odelling, and Antibacterial Activity . J. M ed. Chem. 40: 2563. 18. Liras, S. ;Allen, M . P. and Segestein, B. E. (2000) Sy nth. Commun. A M ild M ethod for The Prep aration of 1,3,4-Oxadiazoles: Triflic Anhy dride Promoted Cy clization of Diacy lhydrazines. 30:437. 19. Feray, A.; Zuhal, T.; Nuket, O. and Safiy a, S. (2002) Sy nthesis and Electronic Structure of New Ary l- and Alky l- Subst itut ed 1,3,4-Oxadiazole-2-thione Derivatives. J. M ed. Chem. 26:159. 20. Kop arir, M . ;Cetin, A. and Cansiz, A. (2005) 5-Furane-2yl[1,3,4]Oxadiazole-2-thiol, 5-Furan-2y l-4H[1,2,4] Triazole-3-thiol and Their Thiol-Thione Tautomerism. J. M ed. Chem. 10:475. 21. Sebast iao, J. M .; Ant onio, D. S.; Heron, L. L.and Srivast ava, R. M . (1998) Sy nthesis of Some 3-Ary l-1,2,4-Oxadiazoles Carrying a Prot ected L-Alanine Side Chain. J. M ed. 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 M O Study on The Dep rotonation of Some 2-Ary l-Δ 2 -1,3,4-Oxadiazole-5-thiones. J. Heterocy clic Chem 34:1715. 24. Horning, M uchowski, (1972) Five-membered Heterocy clic Thiones. Part I. 1,3,4- Oxadiazole-2-thione. Can. J. Chem. 50: 3079. 25. Oz turk, S. ;Akkurt, M .; Cansiz, A. ;Cetin, A. ;Sekerci, M .and Heinemann, F. W. (2004) 5-(Furan-2-y l)-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. Saty anarayna,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 M ethod for the Determination of Serum Glutamic Oxalacetic and Glutamic Py ruvic Transaminases. Am. J. Clin. 28, 56. 30. Persijn, Van Der Silk (1976) J. Chem. Clin. Biochem. A New M ethod 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 Enzy me Dissociation Const ants. 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 enz ymes 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 36 4.705 64 30 0.000 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 ki ne tic propertie s of GOT, GPT and γ-GT with comp. (7) En z ymes Kma p (M) Vma p (U/L) Ki (M) Type of effect GOT 10 0 50 0.0129 Noncompet it ive GP T 40 0 50 0.02 Noncompetit ive γ- GT 1 0.256 0.000263 Noncompetit ive IHJPAS IBN AL- HAITHAM J . FO R PURE & APPL. SC I VO L. 23 (3) 2010 Fig ure 1. 1H NMR sp ectrum of compou nd 3 Fig ure 2. 1H NMR spectrum of co mpo und 4 Fig ure 3. 1H NMR sp ectrum of compou nd 5 Fig ure 4. 1H NMR sp ectrum of co mpo und 6 Figure 5. 1H NMR sp ectrum of compo un d 7 Fig ure 6. (a) The relat ionship b etw een co nc. of co mpo und (7) and GOT enzyme activity . (b) The relat ionship b etw een con c. ocompo und (7) and GP T enzyme acti vity. IHJPAS IBN AL- HAITHAM J . FO R PURE & APPL. SC I VO L. 23 (3) 2010 Figure 7. (a) The percentag e of activ ation GOT enzyme Figure 8. (a) The relationship betw een co nc. of and co mpou nd (7) conc. compoun d(7) and GGT enzyme activity. (b) The percen tage of acti vation GP T enzyme (b) The percen tage of acti vation GGT and compo und (7) con c. en zyme and compo und (7) conc. Figure 9. Lineweaver-Burk plots for comp. (7) effects o n (a) GO T, (b) GP T and (c) GG T 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 -N-{5-[5اوكسـادیازول ومركـب الكالیسـین -4,3,1في هذا البحث حضر مركب جدید یحتوي على حلقتـین مـن (4-methoxy p henyl)-1,3,4-oxadiazole-2-yl-sulfany l]-1,3,4- oxadiazole-2-yl-methy l}-4- methoxy benzamide .وطیــف االشــعة تحـت الحمــراء وطیــف ،طة التحلیــل الــدقیق للعناصـراشـخص هــذا المركــب بوسـ فعالیــة بعــض فــيقة كــذلك صــممت هــذه الدراســة لبیــان تــاثیر حلقتــي االوكســادیازول الملتصــ. الــرنین النــووي المغناطیســي ووجــد ان هـذا المركـب قــام بتنشـیط فعالیـة االنزیمــات . فــي مصـل الـدم GOT ،GPT ،GGT :مثـل ،االنزیمـات الناقلـة 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