The Synthesis Of Tritium Labeled Cyadox And Its Disposition In Rats And Chicken

Quinoxaline-N, N-dioxides were widely used as food additives in food animals, possessing broad antimicrobial spectrum and growth-promoting activity. Carbadox and olaquindox are the representatives of the compounds. However, carbadox and olaquindox have been recently forbidden or limited to use because of their side effects. It is important to develop lower toxic replacer of this type. Cyadox was a new product of quinoxaline-N, N-dioxides. The current researches on toxicology and clinical pharmacology of cyadox showed that cyadox has lower toxicity compared with its analogues, carbadox and olaquindox. Furthermore, Cyadox combined the advantages of quick absorption, rapid elimination, short-term residue in animal tissues and no accumulation toxicity. Radioactive isotope labeling method is important tool of determination of the total residues and metabolism studies and radioactive isotope is labeled at portion of most toxicological concern for the parent drug and its potential metabolites in animal tissues and excreta after metabolism. The objective of the present study was to synthesize a high radioactive purity and high specific activated, tritium labeled CYX and to characterize the tissue distribution and total residue depletion of CYX in rats and chicken, to examine the mass balance, study the route of elimination, to identify its metabolites in plasma, tissues and excreta.1. Preparation of tritium labeled CyadoxThe tritium labeling procedure applied in synthesis of 6-[3H]-CYX was started from Bromination, and followed by another 4 step reaction that Bromine-Tritium exchange reaction, Oxidation of 4-[3H]-ONA, Beirut reaction, Hydrazoning reaction. In this study, before starting of "hot test", we were carried out a "cold test" that the tritium was replaced by hydrogen to ensure all the reaction step was achieve a high yield in regular scale reaction. Then, modified regular scale reaction to microscale reaction and optimized it to achieve a high yield. Finally, based on optimized condition of microscale reaction, carried out the "hot test" and obtained tritium labeled CYX, and conducted a quality control analysis and stability study.Investigation of synthetic process of tritium labeled Cyadox:Objective of this section is to develop a synthetic process of tritium labeled Cyadox. The synthetic pathway of 6-[3H]-CYX was started from bromination of the ONA, and prepared 4-Br-ONA in yield of 76%. Second step was 10% Pd/C catalyzed Bromine-hydrogen exchange reaction that tritium was replaced by hydrogen to conduct a "cold test". Compound 4-[H]-ONA was isolated in 11% yield. Synthesis of [H]-BFO was used fresh sodium hypochlorite solution to stirred with 4-[H]-ONA, and obtained [H]-BFO yield of 95%. Synthesis of the 6-[H]-FQDDA and 6-[H]-CYX was straightforward and proceeded with yield of 57.68% and 90.23%, respectively. Cyanoacethlhydrazine is obtained by cyanoacetoacetate and hydrazine hydrate in anhydrous ethanol, yield of 92.2%.Microscale synthesis modification:Objective of this section is synthesize a high specific activated, radiolabeled CYX and for the purpose of this, synthesis of 4-[H]-ONA and all of the following three steps, were modified and improved to a microscale-synthesis level and each step of the following synthesis procedure was carried out at relatively high yield. For the synthesis of 4-[H]-ONA, reactant 4-Br-ONA was reduced to 20mg, and separation and purification was accomplished by preparative RP HPLC, obtained yield of 41.8%. In the following three steps, reactant 4-[H]-ONA, [H]-BFO and 6-[H]-FQDDA was reduced to 15mg,20mg and 10mg, yield of the reaction was 78%,42.3% and 92.5%, respectively.Preparation of the tritium labeled Cyadox:Objective of this section is based on the modified and optimized microscale reaction conditions and operation methods, synthesize a high specific activated 6-[3H]-CYX. Preparation of the 4-[3H]-ONA was accomplished by using tritium manifold system and Chemical purity of the product was≥99.5% on HPLC. Specific activity of obtained 4-[3H]-ONA was determined to be 6.18Ci/mmol. Without dilution, specific activity of the product was readily achieved 15Ci/mmol. Chemical purity of the 6-[3H]-CYX was≥99.0% on HPLC, which was>99% radiochemically pure, specific activity was determined to be 39.94mCi/mmol. Without dilution of the 4-[3H]-ONA, specific activity of the 6-[3H]-CYX was readily achieved 10Ci/mmol. Quality standards of the 6-[3H]-CYX was achieved requirement of ADME studies.2. Disposition of tritium labeled Cyadox in ratsThe objective of this section was to investigate the distribution, metabolism and excretion of CYX in rats. For excretion studies, a group of 6 rats (3 male and 3 female) were administered a single oral dose of 10mg/kg bw 6-[3H]-CYX. Urine and feces were collected every 24h from intact animals after the dose for 7d and plasma, tissues, carcass and cage rinse were collected after slaughter. Urine, feces, plasma, tissues, carcass and cage rinse were combusted and determined by LSC. On the other hand, extraction of urine, feces, plasma and tissues were determined by HPLC-LSC and LC-MS/MS. For distribution studies,20 rats were divided into five groups. Each groups consisted of 4 rats (2 male and 2 female), administered 6-[3H]-CYX (specific activity of 9044dpm/μg), orally, by gavage at 10mg/kg bw for 7 consecutive days, respectively. One group of two male and two female rats were randomly selected to be slaughtered at each time point (6h, 1d,3d,7d,14d) after withdrawal period. Plasma and tissues were combusted and determined by LSC. On the other hand, extraction of plasma and tissues were determined by HPLC-LSC and LC-MS/MS. Result showed that essentially the entire administered dose was recovered within 24h (91.22%), total mean recovery of the radioactivity was 96.19±1.27% from rats and feces were the major route for elimination that recovered 60.25±1.78% while recovered 34.75±1.04% from the urine after a single oral administration of 6-[3H]-CYX. Excretion of radioactivity was rapid and nearly complete in within 168h after dosing. Result of distribution studies showed that tissue distribution of 6-[3H]-CYX radioactivity was widespread and half-life time of the total radioactivity in liver was relatively longer than all of the other tissues, and liver can be determined as target tissue in rats. At 14d post dose, no drug related radioactivity was detected in all of the 17 tissues which detected by LSC. A total of 13 metabolites were identified by HPLC-LSC and ionspray LC-MS/MS in rats and the structures of eight new derivatives were proposed based on their accurate molecular masses. On the basis of the structures of the metabolites, four primary metabolic pathways of CYX were identified:reduction of the N�'O bond, cleavage of the side chain, hydrogenation on the C=N bond of side chain and conjugation reaction of Cy6. A total of 2,7,5,5 metabolites were found in plasma, liver, kidney and muscle, respectively. Unchanged CYX were no detectable in all of the tissues at all time points indicate that the CYX were has a relatively rapid turnover rate in the rats. The investigation of CYX disposition in rats can be expected to play a key role in food safety evaluations and will provide a basis for further studies.3. Dispositiion of tritium labeled Cyadox in chickenEarly research reported that CYX had a growth promotion effect in poultry. It was suggested that CYX was possible to use safely and effectively in poultry. As a new drug for food producing animals, food safety is a key parameter to evaluate the application foreground. The objective of this section was to investigate the distribution, metabolism and excretion of CYX in chicken. For excretion studies, a group of 6 chicken (3 male and 3 female) were administered a single oral dose of 6-[3H]-CYX, feces were collected every 24h from intact animals after the dose for 4d. Plasma, tissues, carcass and cage rinse were collected after slaughter. Samples were combusted and determined by LSC. On the other hand, extraction of feces, plasma and tissues were determined by HPLC-LSC. For total depletion studies,20 chicken were divided into five groups. Each groups consisted of 4 chicken (2 male and 2 female), administered 6-[3H]-CYX, orally, by gavage at 10mg/kg bw for 7 consecutive days, respectively. One group of two male and two female rats were randomly selected to be slaughtered at each time point (6h, Id,3d,7d,14d) after withdrawal period. Plasma and tissues were combusted and determined by LSC. On the other hand, extraction of plasma, liver, bile, muscle, kidney and fat were determined by HPLC-LSC. Result showed that excretion of radioactivity was rapid and nearly complete in within 96h after dosing and essentially the entire administered dose was recovered within 24h (90.70%). Total mean recovery of the radioactivity was 96.02±1.71% from rats and radioactivity of excreted from feces were 95.94±1.47% while recovered 0.078± 0.053% from the carcass after a single oral administration of 6-[3H]-CYX. Result showed that tissue distribution of 6-[jH]-CYX radioactivity was widespread and rapid elimination in chicken. Except for blood, heart, liver, lung and kidney, radioactivity of other tissues were below the limit of determination. At 14d post dose, no drug related radioactivity was detected in all of the tissues which detected by LSC. After 6h time point, concentration of total radioactivity residues was lower than safe concentration in chicken. Half-life time of the total radioactivity in liver was relatively longer than all of the other tissues, and liver can be determined as target tissue in chicken. HPLC-LSC detection indicated that except for great amount of CYX, also found Cy6、BDCYX and an unknown metabolite M. Cyl, Cy6 and few unknown radioactive compounds were detected in bile, liver, plasma, muscle, kidney and fat. The structures of CYX, Cyl and Cy6 were confirmed unambiguously by comparison of their chromatographic with those of the synthetic or purchased standards and others were will require further study to determine their structures.In conclusion,6-[3H]-CYX as prepared at high specific activity by catalytic bromine-tritium exchange of brominated O-nitroaniline and following three steps were accomplished by microscale-synthesis in high yield. Based on preparation of 6-[3H]-CYX, present study was to characterize the tissue distribution and total residue depletion of CYX in rats and chicken, to examine the mass balance, study the route of elimination, to identify its metabolites in plasma, tissues and excreta. In addition, tritium labeled O-nitroaniline that synthesized by this study can be used as starting material of tritium labeled quinoxaline-N,N-dioxides and quinoxaline derivatives.Stability of labeled O-nitroaniline and Cyadox were also investigated.4-[2H]-ONA was a byproduct of this study, and was can be also used as starting material of deuterium labeled quinoxaline-N,N-dioxides and quinoxaline derivatives. They were used as isotopic internal standard in LC-MS/MS quantitative methods. Result of 6-[3H]-CYX in rats and chicken study will provide comprehensive data to clarify the absorption, distribution, metabolism and excretion of CYX, and will contribute valuable information to the following mechanism study of CYX, the development of analytical method of residues. The proposed metabolic pathway of CYX in rats and chicken will provide a basis for further studies of the in vivo quantification and determination of major metabolites of this drug, to determine marker residue, and will improve the toxicological evaluation of CYX in target animals. Information generated from this study was also used to support the nonclinical safety evaluation of CYX.