Document Jvx9ywaeodpjE887ELB9Nj6ZZ

Synthesis and Characterization of FC-95- 14 c November 2, 1979 Conducted at: During: Synthesis Conducted by: Specific Activity and Radiochemical Purity Determination 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 Report by: DD . Johr)@ I M.S. Date @ eniioorrjbB0@i@kodhemical Pharmacologist Riker Laboratories, Inc. Specific Activity and Radiochemical Characterization Section Reviewed by: Senior Research Chemist Commercial Chemicals Division c@t"W I R. E. Ober, Ph.D. Manager, Drug Metabolism Riker Laboratories, Inc. /7L Date 2. Sunwary The synthesis of a 20.0 9 lot of FC-95-14C (carbon-14 label a to sulfur atom) is described. The specific activity is 0.459 0.008 UCi/mg. Thin-layer and column chromatography showed the pC-95-14C to be at least 99% radiochemically pure. The FC-95-14C was found suitable for metabolism studies. 3. Introduction A series of experiments is planned to investigate the metabolism of rc-95 (a sulfonic acid salt) and the possibility of FC-807 being biotransformed to FC-95 and/or the alcohol (FM-3422). To facilitate these experiments, carbon-14 labeled FC-95 was synthesized and characterized. The synthetic pathway illustrated in Figure 1 is described. 4. A. Synthesis of FC-95-14C I. Fractional Distillation of c7 F 15 CF 2 so 2 F (Step 1) Il. The crude cell drainings from cell run R-3256-B were filtered through glass wool to remove the cell tars. The fluorochemical product was washed twice with cold, dilute saturated KHCO and twice with water; the product was dried over silica gel. A yield of 293.7g of base- washed cell drainings was obtained from cell run R-3256-B. GLC analysis showed 71.1% Ca F 17 SO 2 F. One hundred sixty grams of the basewashed cell drainings was purified by fractional distillation (see Table 1) . Analysis for percent C F SO F was done by gas chroma- 872 tography (area percent). Fractions 11-1@ were combined to give 98.3 g of product which was found to contain 98.15% C 8F 17 so 2F. Preparation of FC-95- 14 C (KO S*CF c F ) 3 2 7 15 Water (31.1g) and potassium hydroxide pellets, (85% assay, 15.53g) were added to a 500 ml three-necked round-bottomed flask which had been equipped with a reflux condenser, thermometer, an air-driven mechanical stirrer, Tru-Bore@ stirrer assembly and a small, pressureequalized addition funnel. The flask contents were heated to 78-BOOC. Fractional C F *CF SO F (27.0g, 98.15% assay) was added dropwise 7 A5tio2 f2 through the ad i n unnel at a rate sufficient to maintain the temperature between 80-830C. Gummy, white solids were formed during the addition. Upon completion of the addition of the radioactive perfluorooctane-sulfonyl fluoride, the mixture was heated at 850C for 3 hours. At the end of the three hour reaction time, the gummy mass had broken into smaller solid pieces. Water (10.8 ml) was added. The pH of the solution was - 13.5. The upper aqueous phase was removed (aspirator) and the solids were washed successively with water (54.0 ml) at 45-500C. The solids were washed once more with water (17.8 ml), stirred at a high agitation rate, and the upper aqueous phase was discarded. Isopropanol (19.15g) and water (17.0g) were added to the solid product, and the mixture was heated at reflux temperature for one hour. All of the solids had dissolved after heating the mixture for one hour. The flask contents were poured into a glass evaporating dish. The flask was washed with isopropanol (24.7 ml)-water (17.0 ml). Concentration of the product was done on a steam bath. A final drying of the potassium perfluorooctane sulfonate was done at 600C for 3 hours in a vacuum desiccator. The dry powder was triturated once with Freon 113 to remove small traces of silicon oil impurity used as stirrer lubricant The dried product (27.8g) was assigned the following i4 L number: FC-95- C, 20.0g, L-4544. FC-95 normally is produced from one-plate distilled perfluorooctanesulfonyl fluoride (assay 66-67%). The high assay C F *CF SO F (98.15%) 7 15 2 2 will also be used to prepare FC-807-14C. 5. 14 B. Specific Activity Determination of FC-95- c Three standard solutions of FC-95-14C@@ (L-4544, see Synthesis Section) were prepared by weighingb- 1, 25.00 mg; 11, 26.84 mg; and, 111, 26.79 mg into three 25 ml class A volumetric flasks. Solution I was prepared with methanol and solutions II and III were prepared with a 1:1 mixture (volume:vol=e) of water and methanol. The volumes were adjusted to 25 ml with the appropriate solvent and mixed by inverting by hand. Calibrated micropipettorsc were used to aliquot six 10 Ul and six 50 ul aliquots of each solution directly into scintillation counting vials. To three of the vials containing 10 ul aliquots and to three containing 50 ul aliaunts from each primary solution, 1 ml of water and 15 ml of Aquasol@d were added. The remaining vials were prepared with 2.5 ml of methanol and 7.5 mi of MTSSI. 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 UCi/mg was calculated for each vial. The data are shown in Table 2. The mean UCi/mg found for each of the weighings was within 2% of each of the other means. The means between Aquasol(Dand MTSS were within 1.2% and the means between 10 ul and 50ul were within 1.3%. The overall average specific activity based on the 36 replicates (12 from each primary weighing) was 0.459 0.0080 UCi/mg. 14 C. Radiochemical Analysis of FC-95- c I. Thin-Layer Chromatography Systems and Carbon-14 Analysis The analysis of radiochemical purity was carried out with a variety of thin-layer chromatography systems using either SGF 250 micron pre-scored Uniplates=.or pre-adsorbent SGF 250 micron pre-scored Uniplates,.f-. Plates were routinely developed in 10"xl2"x4" thin-layer tanksz fitted with glass lids and lined with saturation pads.-g.Plates 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. a Riker Isotope Number 442. 11Weighings were accomplished on a 5-place Mettler H64 electronic balance which had been recently calibrated by 3m Metrology. c L/I Micropipettor, Lab Industries, Berkeley, California. New England Nuclear, Boston, Massachusetts. Modified TSS:25.2 g PPO, 1.Olg Dimethyl POPOP and 3.8 liters toluene. Analtech, 75 Blue Hen Drive, Newark, Delaware. Supelco Inc., Bellefonte, Pennsylvania. 6. The scraped silica gel segments were collected in scintillation vials containing 2.5 ml 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 segment x 100 sum of cpm on plate carbon-14 content in segment. The p e r c e n t 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. II. Solutions Used for Radiochemical Purity Analysis Solution 1: The FC-95- 14 C made from 25.00 mg methanol described in the section on Specific this reportwas designated as Solution 1. 14 of FC-95- C in 25.0 ml Activity Determination, Solution 2: 28.12 mg of FC-95- 14 C, L-4544, (Riker Isotope Inventory Number 442) was placed into a 10 ml volumetric flask and the volume adjusted to 10 ml with methanol. The solution was inverted several times to ensure mixing. 14 III. Column Chromatography of FC-95- c A silicic acid (Unisil2@)column was prepared by pouring a chloroform slurry of silicic acid into a 14mm I.D. glass col@@b to a height of 21 cm. A 1.0 ml aliquot of Solution 2 was applied to the top of the column. Three successive 200 ml fractions were collected after applying 200 ml of CHC1 3 , Fraction 1; 200 ml of chloroform-methanol 1: 1 (volume: volume), Fraction 2; and 200 ml of methanol, Fraction 3. Each of the 200 ml fractions was evaporated with a rotating evaporator, transferred to a 12 ml centrifuge tube, evaporated to near dryness with a stream of nitrogen, and reconstituted with 0.20 ml of chloroform-methanol 1:1 (volume:volume). Fifty microliters was applied to thin-layer plates and analyzed as described previously. IV. Results and Discussion Since the chemical identity of the FC-95- 14 C is well established by synthesis (see synthesis of FC-95-14C, this report), unlabeled FC-95 was not co-chromatographed with the labeled FC-95-14C. The results of the radiochemical purity analysis of FC-95-14C by thin-layer chromatography are shown in Table 3 and in Figures 2-5. These analyses did not discern any impurities in FC-95-14C. Thin-layer analysiz of three column fractions from a silicic acid column showed 99.1% of the radioactivity recovered in Fraction 2 (1:1 chloroform-methanol) (see Figure 6). Fraction 3 (methanol) contained 0.8 per cent of the Unisil, Activated Silicic Acid, 100-200 mesh. Clarkson Chemical Company, Inc., Williamsport, Pennsylvania. Column was fitted with a sintered-glass base and stopcock. 7. recovered radioactivity (see Figure 7). The impurity contained in Fraction 3 has a different R (0.40) from that of FC-95 (0.27) in the same solvent systems. The iffmpuritywas not identified. 14 Overall, the radiochemical purity of FC-95- C is at least 99%. The FC-95-14C is suitable for metabolism studies. Acknowledgement The authors gratefully acknowledge the gas chromatographic analysis of the carbon-14 labeled C F CE SO F fractionsdone by Mr. Todd 7.15 1. 2 . Mathisen of Co-m rcial Chemicals ivision and the assistance of John C. Hansen and Larry Headrick during the electrochemical fluorination of labeled C F CF so F. 7 is 2 2 8. List of Tables and Figures Table 1: Data from Fractional c 7 F 15 *CF 2 so F. 2 NB 50942 p. 34,35. Distillation of Base-washed Table 2: Table 3: Specific Activity Determination of FC-95-14 C. NB 51312 p. 33. 14 Thin-Layer Chromatography Systems for FC-95- C. NB 51807 p. 21. Figure 1. Pathway for Synthesis of FC-95_14C. NB 50942 p. 36. Figure 2: Thin-Layer Radiochromatogram NB 51807 p. 4. Plate 1. Figure 3: Thin-Layer Radiochromatogram NB 51807 p. 4. Plate 2. Figure 4: Thin-Layer Radiochromatogram NB 51807 p. 5. Plate 3. Figure 5: Thin-Layer Radiochromatogram NB 51807 p. 5. Plate 4. Figure 6: Thin-Layer Radiochromatogram Silicic Acid Column Eluent, (Chloroform-Methanol). NB 51807 p. 18. 14 of FC-95- c Fraction 2 Figure 7: Thin-Layer Radiochromatogram 14 of FC-95- c Silicic Acid Column Eluent, Fraction 3 (Methanol) NB 51807 p. 18. 9. Table 1 Data from Fractional Distillation of Base-Washed c F CF so F 7 is 2 2 Fraction bP 0C Yield (g) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Bottoms < 40 < so 80-83 83-92 92-94 94-97 97-104 104-108 108-118 118-123 126-128 128-130 131- 33 138- 40 > 140 ----- Blend FR 11-16 4.3 10.8 8.7 4.1 5.7 4.3 1.8 2.5 1.8 2.7 9.4 7.1 9.4 31.2 16.0 25.2 --- LB'S.@ 99.3 95.3 80.99 73.55 63.63 50.23 41.92 22.02 12.16 6.17 7.23 0.49 --- ----- CSF,B Area %2@ C6Fl3SO2F C7Fl5So2F C8F 17SO2 F c HBIS 2.30 8.82 12.46 15.39 18.54 21.18 18.90 15.03 11.12 5.44 1.93 ----------- 0.12 0.95 1.55 2.17 3.09 3.80 5.27 5.82 5.84 5.33 4.39 0.43 1.52 0.22 0.058 --- 2.20 7.86 13.36 18.94 27.52 32.42 53.51 66.66 76.78 87.53 95.66 96.64 97.92 99.55 93.22 5.13 98.15 ----- --------------0.46 0.49 0.09 --64.0 Area percent was determined by gas chromatographic analysis. Low boiling non-functional fluorocarbons below C8 F 18* High-boiling,unidentified fluorocarbons boiling above C8 F 17 so 2F. 10. Table 2 14 Specific Activity Determination of PC-95- C Solution I (25.00 mg/25.0 ml) pci/mg X + S.D. 0.4535 0.4558 0.4582 0.4658 0.4816 0.4736 0.4521 0.4637 0.4589 0.4637 0.4653 0.4685 0.4634 + 0.0085 Comparison of Primary Solutions Solution II (26.84 mg/25.0-mi) lici/mg 0.4610 0.4530 0.4535 0.4652 0.4607 0.4611 0.4565 0.4422 0.4486 0.4540 0.4576 0.4518 0.4554 + 0.0063 Solution III (26.79 mg/25.0 mi) ]'Ci/mg 0.4660 0.4647 0.4598 0.4532 0.4554 0.4644 0.4425 0.4576 0.4567 0.4625 0.4490 0.4513 0.4569 + 0.0071 Comparison of Scintillation Solvents MTSS lici/mg Aguasol pci/mg 0.4658 0.4816 0.4736 0.4637 0.4653 0.4685 0.4652 0.4607 0.4611 0.4540 0.4576 0.4518 0.4532 0.4554 0.4644 0.4625 0.4490 0.4513 + S.t). 0.4614 0.0084 0.4535 0.4558 0.4582 0.4521 0.4637 0.4589 0.4610 0.4530 0.4535 0.4565 0.4422 0.4486 0.4660 0.4647 0.4598 0.4425 0.4576 0.4567 0.4558 0.0067 Comparison of 10 pl and 50 ul Aliquots -10 ul pci/mg 50 pl lici/mg 0.4535 0.4558 0.4582 0.4658 0.4816 0.4736 0.4610 0.4530 0.4535 0.4652 0.4607 0.4611 0.4660 0.4647 0.4598 0.4532 0.4554 0.4644 0.4615 + 0.0076 0.4521 0.4637 0.4589 0.4637 0.4653 0.4653 0.4685 0.4565 0.4422 0.4486 0.4576 0.4518 0.4425 0.4576 0.4567 0.4625 0.4490 0.4513 0.4557 0.0075 Thin-Layer Table Chromatography 3 Systems 14 for FC-95- C Plate No. 1 2 3 4 5 Type of Plate Uniplate Uniplate Uniplate Uniplate Pre-adsorbent Uniplate Solvent Systeff6.-a- R f of F( 50 chloroform 50 acetone 100 chloroform 100 methanol 2 acetic acid c loo butanol 10 water 10 acetic acid c 150 chloroform 50 methanol 5 ammonium hydroxidec loo butanol 10 water 10 acetic acid C 0.17 0.70 0.67 0.30 0.67 Solvents were prepared volume:volume; a loo ml aliquot of solvent mixture was added to chromatography tank. b is of only radioactive peak on plate. -f C Acetic acid and ammonium hydroxide were concentrated. 12. Figure 1 Pathway for Synthesis of FC-95- 14 c c H CH SO F2@ 7 is 2 2 C 7F isCF so 2 2F Assay 71.1% ECF '!wC F CF so F HF 7 15 2 2 R-3256-B Fractional Distillaf-i-on-30c 7 F isCF 2 so 2 F Assay 98.15% I + 2KOH H 20 BOOC -110 KO SCF C F 3 2 7 15 FC-95-14Cb Denotes position of Carbon-14. 2@C H CH so 2F was prepared by Pathfinder Laboratories. 7 is 2 FC-95 _14C_,_ potassium perfluorooctanesulfonate. 60. 50. 40, 0 30. 0 E- 4-4 0 4-1 r_ 20. Q) u 10. 13. Figure 2 Thin-layer Radiochromatogram of FC-95-14C, Plate No. 1 SGF Uniplate: 100 chloroform 100 acetone Total CPM on Plate 39770 0 0 1 2 3 4 5 6 7 8 9 lo 11 12 13 14 1S Distance from Origin (CM) 14. Figure 3 Thin-layer Radiochromatogram of FC-95-14C, Plate No. 2 SGF Uniplate: 100 chloroform 100 methanol 2 acetic acid 60. Total CPNI on Plate 41058 so 40- 0 m: Cd 30. 41 0 E- 0 -W r_ 20. 0 u 10- 0 0 1 2 34 io 11 12 13 14 15 Distance from Origin (CM) 15. Figure 4 Thin-layer Radiochromatogram of FC-95-14C, Plate No. 3 SGF Uniplate: 100 butanol 10 water 10 acetic acid 60. Total CPM on Plate 38549 50- 40- 30. 20. 10. 0. 0 12 56 78 iO 11 i2 i3 i4 15 Distance from Origin (CM) 60. so. (D ce 40 0 30. ct -W 0 E. L4-4 0 20 0 u 10 16. Figure 5 Thin-layer Radiochromatogram of FC-95-14C,l Plate No. 4 SGF Uniplate: 1SO chloroform 50 methanol 5 ammonium hydroxide Total CPM on Plate 40842 0 0 1 '3 5 6 7 8 9 10 11 12 13 14 15 Distance from Origin (CM) 17. Figure 6 Thin-layer Radiochromatogram of FC-95- 14 C Silicic Acid Column Eluent, Fraction 2 (Chloroform-Methanol) Pre-adsorbent SGF Uniplate: 100 chloroform 35 methanol 5 ammonium hydroxide 60. Total CPM on Plate 429.9720 so. 40. 0 04 u r-4 30. ce +i 0 E- t4-4 0 4-1 r_ 20. CL) u 10. 0. 0 1 2 34 5 67 9 10 11 12 13 14 is Distance from Origin (CM) 18. Figure 7 14 Thin-layer Radiochromatogram of FC-95- C Silicic Acid Column Eluent, Fraction 3 (Methanol) Pre-adsorbent SGF Uniplate: 100 chloroform 60. 35 methanol 5 ammonium hydroxide Total CPM on Plate 3565 so. Cd 40 0 u V.-I 30. ct 41 0 E- 4-4 0 r_ a) 20. u 10. 0 T:!:% -10 f1 12 13 14 -15 Distance from Origin (CM)