Document JJ9Vky2EjrRvky57wMygmdm8a

AR326-00059 Synthesis and Characterization of FC-95- 14, November 2, 1979 Conducted at: Commercial Chemicals Division and Riker Laboratories, Inc. 3M St. Paul, Minnesota 55101 During: April 1979 to July, 1979 Synthesis Conducted by: Specific Activity and Radiochemical Purity Determination by: F. E. Behr J. D. Johnson and S. J. Gibson Report by: Specific Activity and Radio chemical Characterization Section Reviewed by: Commercial Chemicals Division Manager, Drug Metabolism Riker Laboratories, Inc. 000019 2. Summary The synthesis of a 20.0 g lot of FC-95-14c (carbon-14 label a to sulfur atom) is described. The specific activity is 0.459 0.008 yCi/mg. Thin-layer and column chromatography showed the FC-95-^C to be at least 99% radiochemically pure. The FC-95-14c was found suitable for metabolism studies. 000020 Introduction 3. A series of experiments is planned to investigate the metabolism of FC-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. 000021 4. A. Synthesis of FC-95-14C * I. Fractional Distillation of C_/Fxb CFZSOZF (Step 1) 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 basewashed cell drainings was obtained from cell run R-3256-B. GLC analysis showed 71.1% CgF^SO^F. 0ne hundred sixty grams of the basewashed cell drainings was purified by fractional distillation (see Table 1). Analysis for p e r c e n t C F SO F was ^one hy gas chroma tography (area percent). Fractions 11-15 were combined to give 98.3 g of product which was found to contain 98.15% C F SO F. 8 XV 2 II. Preparation of FC-95-14C ( K O ^ C F ^ F ^ ) Water (31.lg) 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 78-80C. Fractional C F *CF SO^F (27.Og, 98.15% assay) was added dropwise through the addition funnel at a rate sufficient to maintain the tempera ture between 80-83C. Gummy, white solids were formed during the addi tion. Upon completion of the addition of the radioactive perfluorooctane-sulfonyl fluoride, the mixture was heated at 85C 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 45-50OC. 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.Og) 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 60C 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 L number: FC-95- C, 20.Og, 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%) will also be used to prepare FC-807-^C. 000022 5. 14 B. S p e c i f i c A c t i v i t y D e t e r m i n a t i o n of F C - 9 5 - C 14 a Three standard solutions of FC-95- C-- (L-4544, see Synthesis Section) were prepared by weighing*! I, 25.00 mg; II, 26.84 mg; and, III, 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:volume) of water and methanol. The volumes were adjusted to 25 ml with the appropriate solvent and mixed by inverting by hand. Calibrated micropipettors. were used to aliquot six 10 yl and six 50 yl aliquots of each solution directly into scintillation counting vials. To three of the vials containing 10 yl aliquots and to three containing 50 yl aliauots from each primary solution, 1 ml of water and 15 ml of Aquasol^. were added. The remaining vials were prepared with 2.5 ml of methanol and 7.5 ml 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 yCi/mg was calculated for each vial. The data are shown in Table 2. The mean yCi/mg found for each of the weighings was within 2% of each of the other means. The means between Aquasoland MTSS were within 1.2% and the means between 10 yl and 50yl were within 1.3%. The overall average specific activity based on the 36 replicates (12 from each primary weighing) was 0.459 0.0080 yCi/mg. C . Radiochemical Analysis of FC-95- 14C 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 Uniplatesi. or pre-adsorbent SGF 250 micron pre-scored Uniplatesf.. Plates were routinely developed in 10"xl2"x4" thin-layer tanks fitted with glass lids and lined with saturation padsj=. 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. Riker Isotope Number 442. 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, California. New England Nuclear, Boston, Massachusetts. Modified TSS:25.2 g PPO, l.Olg Dimethyl POPOP and 3.8 liters toluene. Analtech, 75 Blue Hen Drive, Newark, Delaware. Supelco Inc., Beliefonte, Pennsylvania. 000023 |iq |Hi |<i> |0j |o | t r | o 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 sum of cpm on plate 100 = % carbon-14 content in segment. The percent carbon-14 content of each segment was plotted versus segment number. This provided a thin-layer radiochromatogram showing the radio activity peaks corresponding to separated components in the material applied to the plate. II. Solutions Used for Radiochemical Purity Analysis Solution 1: The FC-95- 14C made from 25.00 mg of FC-95-14C in 25.0 ml methanol described in the section on Specific Activity Determination, this report, was designated as Solution 1. Solution 2: 28.12 mg of FC-95- 14C, 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 (Unisil-- ) column was prepared by pouring a chloroform slurry of silicic acid into a 14mm I.D. glass column^, 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 CHCl^, 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 14 Since the chemical identity of the FC-95- C is well established by synthesis (see synthesis of FC-OS-^C, this report) , unlabeled FC-95 was not co-chromatographed with the labeled FC-95-^C. The results of the radiochemical purity analysis of FC-95--*-4C by thin-layer chromatography are shown in Table 3 and in Figures 2-5. These analyses did not discern any impurities in FC-95-^4C. Thin-layer analysis 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. 000024 7. recovered radioactivity (see Figure 7). The impurity contained in Fraction 3 has a different (0.40) from that of FC-95 (0.27) in the same solvent systems. The impurity was not identified. Overall, the radiochemical purity of FC-95-14C 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 El S02F fractionsdone by Mr. Todd Mathisen of Commercial Chemicals "Division and the assistance of John C. Hansen and Larry Headrick during the electrochemical fluorination of labeled C^F^^CF2S02F. 000025 List of Tables and Figures Table 1: Data from Fractional Distillation of Base-washed C F *CF SO F. yr15 2 2 NB 50942 p. 34,35. 14 Table 2: Specific Activity Determination of FC-95- C. NB 51312 p. 33. 14 Table 3: Thin-Layer Chromatography Systems for FC-95- C. NB 51807 p. 21. Figure 1. Pathway for Synthesis of FC-95-^C. NB 50942 p. 36. Figure 2: Thin-Layer Radiochromatogram Plate 1. NB 51807 p. 4. Figure 3: Thin-Layer Radiochromatogram Plate 2. NB 51807 p. 4. Figure 4: Thin-Layer Radiochromatogram Plate 3. NB 51807 p. 5. Figure 5: Thin-Layer Radiochromatogram Plate 4. NB 51807 p. 5. Figure 6: Thin-Layer Radiochromatogram of FC-95-^C Silicic Acid Column Eluent, Fraction 2 (Chloroform-Methanol). NB 51807 p. 18. Figure 7: 14 Thin-Layer Radiochromatogram of FC-95- C Silicic Acid Column Eluent, Fraction 3 (Methanol) NB 51807 p. 18. 8 000026 Table 1 * Data from Fractional Distillation of Base-Washed C F , CF SO F ___________________________ _____________ 7 15 2 2 9 Fraction O bP C Yield (g) LB'S- C8F18 Area % L c 6f 13s 02f c 7f 15s 02f C8F17S02F HB'S -- 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Bottoms < 40 < 80 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 -- -- 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 "---- Blend FR 11-16 99.3 95.3 80.99 73.55 63.63 50.23 41.92 22.02 12.16 6.17 7.23 0.49 -- -- -- -- -- 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 a -- Area percent was determined by gas chromatographic analysis. -- Low boiling non-functional fluorocarbons below CQF 8 18 c -- High-boiling,unidentified fluorocarbons boiling above C F SO F. 8 17 2 000027 Table 2 14 Specific Activity Determination of FC-95- C 10. Comparison of Primary Solutions Solution I >.00 mg/25.0 ml) yCi/mg 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 Solution II (26.84 mg/25.0 ml) yCi/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 ml) yCi/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 UCi/mg Aquasol yci/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.D. 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 yl and 50 yl Aliquots 10 yl yci/mg 50 yl yCi/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 000028 Table 3 14 Thin-Layer Chromatography Systems for FC-95- C Plate No. 1 2 3 4 5 Type of Plate Uniplate Uniplate Uniplate Uniplate Pre-adsorbent Uniplate Solvent System^- b R^-- of 50 chloroform 50 acetone 0.17 100 chloroform 100 methanol 2 acetic acid-- 0.70 100 butanol 10 water 10 acetic acid -- 0.67 150 chloroform 50 methanol 0.30 5 ammonium hydroxide 100 butanol 10 water 10 acetic acid -- 0.67 g -- Solvents were prepared volume :volume; a 100 ml aliquot of solvent mixture was added to chromatography tank. -- R^ is of only radioactive peak on plate. -- Acetic acid and ammonium hydroxide were concentrated. 000029 Figure 1 Pathway for Synthesis of FC-95-F4C C7H 1 5 2 S 0 2F- C 7F 15CF2S 0 2F Assay 71.1% ECF * HF ^ C 7F 15CF2S0 2F R-3256-B Fractional ,,,, * ^ ppi-- ----------- C-F.. cCF 9SO~F Distillation 7 15 2 2 Assay 98.15% I I + 2K0H H-0 * ^ K 0 3SCF2c 7F i5 FC~95-14C-- o\ 12 * Denotes position of Carbon-14. a - * C 7H15CH 2S02F was prepared by Pathfinder Laboratories. V> 1 A -- FC-95- potassium perfluorooctanesulfonate. 000030 Figure 2 Thin-layer Radiochromatogram of F C - 9 5 - 1 4 C, Plate No. 1 13. SGF Uniplate: 100 chloroform 100 acetone 60Total CPM on Plate = 39770 50. Percent of Total CPM on Plate 1---- 1---- 1 13 14 15 000031 Percent of Total CPM on Plate Figure 3 Thin-layer Radiochromatogram of F C - 9 5 - 14 C, P l at e No. 2 SGF Uniplate: 100 chloroform 100 methanol 2 acetic acid 60. 50- 40 30. .20 10- 0 (CM) 14. 000032 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 15. Percent of Total CPM on Plate Distance from Origin (CM) T---- 1 ..'I 13 14 15 000033 Percent of Total CPM on Plate 60 . 50 40 30 . 20 . 10 . 0 16. Figure 5 Thin-layer Radiochromatogram of FC-95-14C, Plate No. 4 SGF Uniplate: 150 chloroform 50 methanol 5 ammonium hydroxide Total CPM on Plate = 40842 (CM) 000034 Percent o Total CPM on Plate Figure 6 .. Thin-layer Radiochromatogram of FC-95- 4C Silicic Acid Column Eluent, Fraction 2 (Chloroform-Methanol) 17. Pre-adsorbent SGF Uniplate: 100 chloroform 35 methanol 5 ammonium hydroxide 60. Total CPM on Plate = 429,720 5 0. 40. 30. .20 10- 04 (CM) 00003S Percent of Total CPM on Plate Figure 7 -..Thin-layer Radiochromatogram of FC-95- 4C Silicic Acid Column Eluent, Fraction 3 (Methanol) 18 . Pre-adsorbent SGF Uniplate: 100 chloroform 60. 35 methanol 5 ammonium hydroxide Total CPM on Plate = 3565 50 40 * 30. 20. 10. 0 (CM) 000036