Document Ev1q4En0zN5jL0xpEJx71jRG4

14 Of FC-14:3- C December 28, 1979 Conducted at: During: Synthesis Conducted by: Specific Activity and lzadiochemical Purity Determination by: Report by: Specific Activity and Radiochemical Characterization Section Reviewed by: Commercial Chemicals Division and Riker Laboratories., Inc. 3M St. Paul, Minnesota 55101 April 1979 to July, 1979 F. E. Behr J. D. Johnson and S. J. Gibson F. E. Behr, Ph.D. Research Specialist Commercial Chemicals Date Division D Jo@"on Date Senio*r q':@6chemical Pharmacologist Riker Laboratories, Inc. (F, R. E. Ober, Ph.D. Manager, Drug Metabolism Riker Laboratories, Inc. 'Date Summary 14 The synthesis of a 20.0 9 lot of FC-143- C is described (carbony) carbon is labeled). The specific activity was determined to be 0.507t 0.012 uCi/mg. Radiochemical purity determination showed the FC-143-14Cto be at least 98% pure; the FC-143-14C was found suitablefor metabolismstudies. Introduction A series of experiments is being planned to investigate the metabolism of FC-143 and the possibilityof affecting the rate of elimination of FC-143 4C from the body. To facilitate these experiments, carbon-14 labeled FC-143 was synthesized and characterized. The synthetic pathway illustratedin Figure I is described. A. Synthesis of FC-143- 14c 1. Fractional Distillationof Cell Run, R-3265-B Analysis by gas chromatography showed the cell drainings contained about 12.1% high boiling materials. The crude cell drainings were dried over anhydrous KF to remove HF. The solids were removed by filtrationto yield 434.0 gms for distillation. Three fractionswere collected (Table 1). Fractions I and 2 (389.3 g) * were combined and will be used for the preparationof C7 F 15C 02 NH 4* 11. Hydrolysis and Stabilizationof DistilledCell Drainings from R-3265-B Into a 500 mi three-necked round-bottomed flask which had been equipped with a mechanical stirrer, Tru-Bores stirrer assembly, addition funnel, condenser, and thermometer was placed the combined cell drainings, Fractions I and 2, 389.3 9. A premixed solution of NAOH (68.49) and water (68.4 9) was added to the flask through a pressure-equalizedaddition funnel over 45 minutes at a rate sufficient to keep the temperature between 52-55'C. The mixture was heated to reflux (85*C) for 9 hours. After the 9 hour reaction time, the pH was found to be > 12. The mixture was cooled to 65*C and a mixture of water (257.0 mi) and 96% H2SO4 (94.3 mi) was added slowly to the flask contents. The final pH was < I (Congo red paper). The acidified mixture was heated at 80*C for 30 minutes, cooled to 65*C, and the bottom fluorochemicalphase was separated. The yield was 356.7 g (91.6% mass yield). Ill. FractionaDlistillatiofn CF 7 15co 2H The apparatus consisted of a 500 mi three-necked round-bottomed flask, a small packed heated distillation column, pot thermometer, take-off head, a collection assembly, -78*C cold trap, magnetic stirrer assembly, and a mercury manometer. The base-stabilized radioactive fluorochemical (356.7 9), 96% sulfuric acid (12.0 ml), and Super-Cel (0.7 g) was added to the distillation flask. The water aspirator vacuum was adjusted to 200 mm and the low boiling iiiaterialswere reiiioved. As the head temperature dropped below 40*C at 200 mm vacuum, the distillation was continued with lower vacuum (10-20 mm). Fractions were collected, weighed, and analyzed by gas chromatography. The analysis was done by area percent and the assay reported was for percent C7F 15 co 2 H (See Table 11). Fraction 6, 44.0 g, was refractionated to give 95.0% C7 F 18 CO H (see Table 111). Fraction 3, (24.3 9, 97.5% C7 F@5 2HJ, and Fraction 4, (13.4_ 14C. 96.oB% c 7 F 15Co2H) were combine for use in preparation of FC 9143- IV. Fractions 3 and 4 (30.0 9) were dissolved in FC-80 (go mi). Anhydrous NH3 was added until the pH was greater than 8. The pH was rechecked after 2 hours. The slurry was filtered, and the product was air dried in the hood. The final product was prepared by pulverizing the air dried solid and drying the so] id at 65*C for I'hour. Analysis.@y gas chromatography !ihowed an assay of 98.8% C7 F 15 co 2 CH 3' The yield was 31.1 YA The following L numbers were assigned: L-4585..,F.C-143- C, 100% solids, 20.0 9; L-4586, fractionated C7 F 15 co 2H, NB 52343:8-30, Fraction 4, 0.4 g. 14 B. Specific Activity Determination of FC-143- c Three standard solutions f FC-143- 14 C2-(L-4585, see Synthesis Section) were prepared by weighing@9- 1, 10-56 mg; 11, 25.28 mg; and 111, 20.06 mg into three 10 mi class A volumetric flasks. The volumes were adjusted to 10 mi with methanol and the flasks were mixed by inverting by hand Calibrated micropipettorsr- were used to aliquot six 10 pi and six 50 t* il aliquots of each solution directly into scintillation counting vials. To three of the /ials containing 10 pi aliquots and to three containing 50 pi aliquo s from each primary solution, I ml of water and 15 ml of Aquaso]OD@were added. The remaining vials were prepared with 2.5 mi of methanol and 7.5 mi of MTSS!-. The samples were cooled to refrigerator temperature and allowed to equilibrate in the dark before they were counted two times at 5 minutes each with a Packard Model 3385 Liquid Scintillation Spectrometer. The counting efficiency was determined for each sample by the internal standard method. The averaged data were reduced to dpm and the pCi/mg was calculated for each via]. The data are shown in Table 4. The overall average specific activity based on the 36 replicates (12 from each weighing) was 0.507 0.012 liCi/mg. The mean pCi/mg found for each of the primary weighings was within 1.5% of the overall mean. The means between Aquaso]O and MTSS were within 0.12%, and the means between 10 pi and 50 PI aliquots were within 2.0%. 2- Riker Isotope Number 459. b Weighings were accomplished on a 5-place Mettler H64 electronic balance which had been recently calibrated by 3M Metrology. L/I Micropipettor, Lab Industries, Berkeley, CA. d New England Nuclear, Boston, Mass. Modified TSS:25.2 g 1.01 g Dimethyl POPOP and 3.8 liters toluene. 14 C. Radiochemical Purity Analysis of FC-143- c 1. Thin-Layer Chromatography Systems and Carbon-14 Analysis The analysis of radiochemical purity was carried out with a variety of thin-layer systems using SGF 250 micron pre-scored Uniplates2.. Plates were routinely developed in 10" x 12" x 4" thin-layer tanks-11-fittewdith glass lids and lined with saturation padsbPlates were allowed to develop 15 cm and were scraped laterally in 0.5 cm wide segments. The scraping was accomplished with a custom-made template and sharpened stainless steel spatula ground to exactly 0.5 cm width. The scraped silica gei segments were collected in scintillation vials containing 2.5 mi of methanol. To the methanol , 7.5 ml of MTSS was added. The samples were counted and the counts per minute (cpm) were corrected for background using a suitable blank (usually two 0.5 cm segments scraped from below the origin on the plate being assayed). The cpm were not corrected for efficiency. The carbon-14 content of each segment was expressed as percent of total carbon-14 on the plate: cpm on se2ment x 100 = % carbon-14 content in segment. sum of cpm on plate The percent carbon-14 content of each segment was plotted versus segment number. This provided a thin-layer radiochromatogram showing the radioactivity peaks corresponding to separated components in the material applied to the plate. li. Solution Used for Radiochemical Purity Analysis The solution used to streak 4he thin-layer plates was prepared by placing 30.39 mg of FC-143-1 C (Riker Isotope Inventory Number 459) into a 10.0 mi class A volumetric flask, adjusting the volume to 10.0 mi with methanol, and mixing by hand by inverting. Fifty vl of this solution was applied in a narrow four cm long streak to each plate. Ill. Results and Discussion Since the chemical identity of the FC-143-14C is well established by synthesis and gas chromatography (see section A, this repo@t), unlabeled FC-143 was not co-chromatographed with the FC-143- I c. The results of the radiochemical purity analysis are shown in Table 5 and in Figures 2-6. A small amount of impurity (< 2%) was apparent on Plate Number 2 (Figure 3). The other four solvent systems did not resolve this impurity. No attempt was made to identify the impurity found on Plate Number 2. Overall, on the basis of gas chromatography (section A, this report and thin-layer chromatography th radiochemical purity of FC-143- 14 C is at least 98%. The FC-143-1zC is !suitable for metabolism studies. Analtech, 75 Blue Hen Drive, Newark, Delaware. Supelco Inc., Bellefonte, Pennsylvania. Acknowledgement The authors gratefully acknowledge the gas chromatographic analysis of the carbon-14 labeled cell drainings, fractionated acid, and FC-143-14C done by Mr. Todd Mathisen of Commercial Chemicals Division. The authors gratefully acknowledge the assistance of John C. Hansen and Larry G. Headrick during the electrochemical fluorination of labeled C H coci. 7 15 List of Tables and Figures Table 1: Data from One Plate Distillation of Cell Run R-3265-B. NB 50942p. 53 Table 2: Data from Fractional Distillation of C F co H. NB 52343 P. 5,6 7 is 2 Table 3: Data for Refractionation of c F co H. 7 15 2 NB 52343 p.8 . Table 4: Specific Activity Determination of FC-143- 14 C. NB 51807 p. 28. Table 5: Thin-Layer Chromatography NB 51807 p. 35. 14 Systems for FC-143- C. 14 Figure 1: Pathway for Synthesis of FC-143- C. NB 52343 p.4, 5, 6, 8, 9. Figure 2: Thin-Layer Radiochromatogram, Plate 1. NB 51807 p. 14, 39. Figure 3: Thin-Layer Radiochromatogram, Plate 2. NB 51807 p. 14, 39. Figure 4: Thin-Layer Radiochromatogram, Plate 3. NB 51807 p. 14, 39. Figure 5: Thin-Layer Radiochromatogram, Plate 4. NB 51807 p. 15, 40. Figure 6: Thin-Layer Radiochromatogram, Plate 5. NB 51807 p. 15, 40. Table 1 Data from One Plate Distillation of Cell Run, R-3265-B Fr bp 0 c WT (g) LB's@2 Assay GLC (Area Percent) cF c CE 7 16 8 c 7 F isCOF 1 2 3 Bottoms 82-89 90-92 92-110 -- 215.6 173.7 12.2 22.4 5.51 -- 31.97 46.92 10.24 4.32 -- 66.93 30.37 25.90 50.87 Mostly high Boilers HB's 0 0 17.88 LB's :lowboilers -- unidentified but with retention times < C7F 16' b C CE: 8 c c@llicether - various perfluorobutyltetrahydrofurarisand isomers. 8 HB's:-,naterialbsoiling higher than C7F 15 COF. Table 2 Data from Fractional Distillation of C7F co H 15 2 Fr bp*C WT(g) 1 <35 (200mm) 102.3 2 35-40 103.6 3 26-47 (10mm) 2.4 4 98-101 2.1 5 101-103 1.2 6 104-109 (7mm) 44.0 Inerts 97.98 99.98 2.18 5.97 ----- Assay C5FllCU2il C6Fl3Co2H ----- 9.41 13.19 15.85 5.19 C 7F15CO 2R ----- 87.9 79.8 78.8 90.3 GLC analysis of FR #6 showed about 3.9% high boiling materials -9Table 3 Data for Refractionation of C7 F 15 co 2H Fr bp (mm) Yield (g) 1 67-71 (20) 1.5 2 71-73 (19) 3.7 3 72-74 (8) 24.3 4 62 (4) 13.4 42-9 Assay (Area %) C7Fl5Co2CH3 89.03 97.26 97.54 96.08 Assay for input to refractionation@'was 94.35% C7 F15 co2 H analyzed as C7F 15 co 2CH 3 S u L:if i c Ac L i v i Ly I)c Lu riiilia L iull U f I-C- 14 3- 14 c Solution I 10.56mg/lO.Oml pci/mg 0.4887 0.4898 0.5020 0.5061 0.4917 0.5038 0.5124 0.5122 0.5172 0.5070 0.5303 0.5163 0.50650.0124 Solution Il 25.28mg/lO.Oml lici/mg 0.4999 0.5292 0.5265 0.5067 0.5006 0.5006 0.5330 0.5142 0.5180 0.5193 0.5160 0.5101 0.51450.0114 Solution III 20.06m&/10.Onil PC:!/Mg 0.4966 0.4922 0.4965 0.4982 0.5007 0.5084 0.4948 0.5U61 0.5041 0.4973 0.4999 0.5098 0.500410.0056 Comparison of Scintillation Solvents Aguasole pci/mg -MTSS pci/mg 0.4887 0.4898 0.5020 0.5124 0.5122 0.5172 0.4999 0.5292 0.5265 0.5330 0.5142 0.5180 0.4966 0.4922 0.4965 0.4948 0.5061 0.5041 0.50740.0137 0.5061 0.4917 0.5038 0.5070 0.5303 0.5163 0.5067 0.5006 0.5006 0.5193 0.5160 0.5101 0.4982 0.5007 0.5084 0.4973 0.4999 0.5098 0.50680.0093 Overall average 0.50710.0115pCi/mg Comparison of 10pl and 50pl Aliquots 10)11 pci/mg 50ijl lici/mg 0.4887 0.4898 0.5020 0.5061 0.4917 0.5038 0.4999 0.5292 0.5265 0.5067 0.5006 0.5006 0.4966 0.4922 0.4965 0.4982 0.5007 0.5084 0.5021-+0.0109 0.5124 0.5122 0.5172 0.5070 0.5303 0.5163 0.5330 0.5142 0.5180 0.5193 0.5160 0.5101 0.4948 0.5061 0.5041 0.4973 0.4999 0.5098 0.51210.0101 14 Thin-Layer ClirottiatographySystems for FC-143- c Plate No. 1 2 3 4 5 SOIVL-nt a Sys Leur b 14 R fOL k-U-143- C 100 cliloroforni lUO acetone 0.03 100 chloroform 100 methanol c 2 acetic aci - 0.67 100 butanol 10 walur c 10 acetic acid-r- 0.60 150 chloroform 50 methanol c 5 ammonium hydroxi 0.27 100 chloroform 35 methanol c 5 ammonium hydroxide- 0.30 Solvents were prepared volume:volume; a 100 ml aliquot of solvent mixture was added to the chromatography tank. R f is of major (>98%) peak on the thin-layer chromatography plate. Acetic acid and ammonium hydroxide were concentrated. l,'igure1 14 Pathway for Syntliesis of FC-143- c c 7 11 15 cocl ECF 5T ------------ llrei)aredby Pathfinder Laboratories C F COF 7 15 Unu-I'laLL------------> Distillation c 7F 15 COF R-3256-B C F COF 7 15 I + NAOH 11 III + NH3 1) Heat > 2) 112-So4 Fractional Distillation- FC-80 > c7 F 15 co 2 H ii Refractionation > NH co c F 4 2 7 15 FC-143-14C' C F CO ii 7 15 2 > 95% 111 Assay *-Denotes position of Carbon-14 a FC-143- 14 C, Ammonium perfluorooctanoate 90 2 85 Tiiin-layer Radjochromatogram of FC-143-14C, Plate No. 1 80. 75 SGF Utiiplate: IOU (.-.Iilt)rol-oriti 100 acetone 70 Total CPM on Plate 99,892 65 60. 55 500 45 0 E 40 14-1 0 4-1 a 35 w u 30 25 20 1-5 10. 5 0 01 2 34 5 67 8 9 10 11 12 13 14 15 Distance from Origin (CK) Thin-layer Radiochromatogram of FC-143-14C, Plate No. 2 70. SGF Uniplate: 100 chloroform 60. 100 methanol 2 ;icetic acid Total CPM on Platc 103,222 50- cc CL4 40, r_ 0 30' 0 r_ @-4 20 10 0 0 1 2 3 4 5 6 78 9 10 11 12 13 14 15 Distance from Origin (CM) l-'igurL@4 Thin-layer lkadjochromatogram of FC-143-14C, Plate No. 3 70. SGF Uniplate: 100 I)uLailul 10 water 60. 10 acetic acid Total CPM on Plate 104, 442 50- 40, 30 E0. 20 a) 10, 0 01 2 34 56 7 8 9 10 11 12 13 14 15 Distance from Origin (CM) -16- I-igure 5 'riiin-layerRadiochromatogram of I-'C-143-14C,Plate No. 4 70. 60. SGF Uniplate: 150 chloroform 50 methanol 5 aijimoniuiiliyclruxidl! Total CPM on Plate 105,567 50 40. 30. 0 t2 20 10. 0 01 2 5 6 7 Distance from Origin (CM) iO i.1 12 13 i4 l@ Go_ 50 - 40 0 u EO 30 %W 0 u @4 a) a, 20 10 -17- Figure 6 'I'llirl-iayu.r_i!@diocliromatogroafm FC-143 C, Plate No. 5 SGF Uniplate: 100 Chloroform 35 muttiariol 5 ammonium hydroxid Total CPM on Plate 107,126 0 0 1 2 3 4 56 7 8 9 10 11 12 13 14 is Distance from Origin (CM)