Document ZnmNLkxJX28xx3xvDNV3exBa8

AR33&-0268 " -% li.rtR 7 iil 3M Synthesis and Characterization of N-Ethyl FOSE- 14C December 11, 1980 Conducted at: During: Commercial Chemicals Division and Riker Laboratories, Inc. 3M St. Paul, Minnesota 55144 April 1979 to July, 1979 Synthesis Conducted by: F.E. Behr Specific Activity and Radiochemical - J.D. Johnson and S.J. Gibson Purity Determination by: Report by: */(k! J.D. Johuhn Date Senior Biochemical Pharmacologist Riker Laboratories, Inc. F.E. Behr, Ph.D. Date Research Specialist Commercial Chemicals Division Specific Activity and Radiochemical Characterization Section Reviewed by: R.E. Ober, Ph.D. Date Manager, Drug Metabolism Riker Laboratories, Inc. 0*03457 -2Summary The synthesis of a lO.Q^g lot of 2-N-ethylperfluorooctanesulfamido ethanol (N-Ethyl FOSE- C), L-4545, is described (carbcn^tto sulfur atcm is labeled). The specific activity was determined to be 0.483 0.020^yCi/my. artiochemical purity determination showed the N-Ethyl FOSS- C to be at least 98% pure. The N-Ethyl FOSE was found suitable for metabolism studies. Introduction A series of experiments has been started to investigate the metabolism of N-Ethyl FOSE- C. To facilitate these experiments, carbon-14 labeled N-Ethyl FOSE was synthesized and characterized. The synthetic pathway illustrated in Figure I is described. 003458 -3- A . S y n L l i u s i s o f N - E t F U S E - 14C ( L --4 5 4 5 ) . I. Fractionation of C- ^^CF^SOJF (Step 1) . The fractional distillation of crude cell drainings from cell run r -3256-b has been previously described (1). GLC- analysis showed 98.15% C8F17S02F. II. Amidation of C7F1^*CF^S0^F (Step 2). Into a tared 250 ml three-necked round-bottomed flask was added C7F 15*CF2S0_F (69.0 g). The flask was fitted with an inlet and exitgas Bubbling assembly, a glass dip tube, condenser, thermo meter and a drying tube. Anhydrous ethylamine was introduced to the heated fluorochemical at 60C until excess CjHj-NH, was observed at the exit side of the reaction apparatus. A total of 46.3 g of C2H,jNH2 was lost from the cylinder mass. -The reaction mixture was stirred at 70C for 1.5 hrs. Dry nitrogen gas was used to de-gas the reaction mixture. The reaction mix ture was treated by the addition of sulfuric acid (10%, 75 ml). The mixture was heated at 50C for 0.25 hrs and the upper aqueous phase was discarded (pH 4-5). Additional sulfuric acid (10%, 75 ml) was added and the mixture was stirred at 70C for 0.25 hr. Upon cooling, the acidic aqueous phase (pH <1, c. ongo rod paper) was decanted fran the solid fluorochemical phase. The fluorochemical phase was washed with deicnized water until the pH of the wash solution was 4.5 to 5.0. Four water washes were needed. The fluorochemical was heated at 70 C for 0.25 hr at reduced pressure (vacuum pump) to remove residual water. A crude yield of 67.7 g (95.2%) was obtained. III. Reaction of N-Ethyl perfluorooctanesulfcnamide with Ethylene Chlorchydrin (Step 3). Into a pre-tared 200 ml three-necked round-bottaned flask, which had been equipped with a distillation head and condenser, thermo meter, an additional funnel and a magnetic stirring bar, was placed the crude radioactive sulfonamide (67.7 g) from Step 2; ethylene glycol (33.9 ml, RM- 3017) and 25% sodium methoxide in methanol (36.6 g) was added to the ethylene glycol-sulfonamide mixture. Methanol (10 ml) was used to wash the addition funnel. The mixture was heated to 55 C to effect the formation of sodium N-ethyl perfluorooctanesulfcnand.de and to start the distillation of methanol. The residual methanol was removed with water aspir ator vacuum and, finally, the mixture was heated at 70C and 3-4 mm vacuum. The mixture was cooled to 48C, the vacuum was broken and anhydrous powdered sodium carbonate (3.8 g), ethylene chlorohydrin (17.0 ml) and ethylene glycol (33.0 ml) were added rapidly. A vigorous reaction occurred as the mixture was heated to 60C. Quality Control Manual called for 96.0% min. RM * Raw Material. 003459 itr ipi -4- Aftcr the initial exothermic: reaction had subsided* the mixture was heated first to 90C for 1.0 hr and then the temperature of the mixture was raised to 115 C for 5 hrs. The mixture was cooled to 80C and the mixture was washed successively with water, brine (three times) and the residual water was stripped by vacuum at 75C. Crude alcohol (72.7 g) was vacuum distilled to yield two fractions: FR #1, 57.3 g, b.p. _ 5 142-144 and FR #2, 7.6 cr. b.r>. 3_ . n 146-148. GLC analysis - snowed FR #1 and #2 to be > 99.9% Cof ._S0_n ^ C2H5 O JL/ Z ^ ^ CH2CH20H (see reference 2). The non-distillable material (5.0 g) was discarded as radioactive waste material. FR #1 and #2 were combined and melted to give N-Et FOSE- .(Step 3) in 64.9 gm yield. A ten gram sample was taken from the total sample and was assigned the following des ignation: L-4545, Carbon-14 labeled N-Et FOSE. B. Specific Activity Determination of N-Ethyl FOSE-14C. Three standard solutions of N-Ethyl FOSE-14c-b (L-4545 see Synthesis Section) were prepared by weighing I 17.47 mg, II 37.55 mg, and III 25.08 mg into three 10 ml class A volumetric flasks-. The volumes were adjusted to 10 ml with methanol and the flasks were 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 hi aliquots and to three containing 50 yl aliquots from each primary solution, 1 ml of water and 15 ml of Aquasol<fc were added. The re- ^ maining vials were prepared with 2.5 ml of methanol and 7.5 ml of MTSS-. Analyses of three standard solutions allow conparison of three primary weighings. Analyses of 10 yl and 50 yl aliquots from each solution allow conparison of the precision of the micropipettors. The use of two different scintillation solvents allows comparison of solubility. (MTSS is toluene based and Aquasol is a water gel.) 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 1. The overall average specific activity based on the 36 replicates (12 from each weighing) was 0.4830.020 yCi/mg. The mean yCi/mg found for each of the primary weighings was within 1.2% of the overall mean. The mean yCi/mg between Aquasol and MTSS were within 4.1%, and the mean yCi/mg between 10 yl and 50 yl aliquots were within 0.7%. f'0u4S0 - 2-N-Ethylperfluorooctanesulfonamido ethanol, b - Riker Isotope Number 468. 0 - Weighings were accomplished on a 5-place Mettler H64 electronic balance which had recently been calibrated by 3M Metrology. d- L/I Micropippetor, Lab Industries, Berkeley, CA. e - New England Nuclear, Boston, Mass. - Modified TSS: 25.2 g PPO, 1.01 g Dimethyl POPOP and 3.8 1 toluene. c. K .id iot:ln'm L(M I. I `u r i Ly An.'ityr;i. o f N - IS t h y l f o s is - 1 4 c x . Thin-hayor chromatography Systems and Carbon-14 Analysis The analysis of radiochemical purity was carried out with a variety of thin-layer systems using SGP 250 micron pre-scored Uniplates-. Plates were routinely developed in 10" x 12" thin-laygr tanksfitted with glass lids and lined with saturation pads-. 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. The scraped silica gel segments were collected in scintillation vials containing 2.5 ml of methanol. To the methanol, 7.5 ml of NTSS was added. The samples were mixed by shaking and cooled in the dark. 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: sum of cpm on plate x 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 radioactivity peaks corresponding to separated components in the material applied to the plate. II. Column Chromatography System A 14 mm column was packed to 21 cm with a slurry of silicic acidin chloroform. A one ml aliquot of N-Ethyl POSE- 4C test solution (see next section C-III) was applied to the column. The column was eluted successively with 200 ml of chloroform, 200 ml of 1:1 chloroform-methanol, and 200 ml of methanol. The three fractions were evaporated to dryness, reconstituted in 200 yl of 1:1 chloro form-methanol and streaked on three separate 5 x 20 cm plates. The plates (No. 6, 7, and 8) were developed in the same solvent system as Plate No. 5. III. Solution Used for Radiochemical Purity Analysis The solution used to streak the thin-layer plates was prepared by placing 26.16 mg of N-Ethyl FOSE- c (Riker Isotope Inventory Number 468) into a 10.0 ml class A volumetric flask, adjusting the volume to 10.0 ml with methanol, and mixing by hand by inverting. Fifty yl of this solution was applied to each plate. One ml of this solution was applied to the silicic acid column. IV. Results and Discussion Since the chemical identity of the N-Ethyl FOSE-14C is well established by synthesis and gas chromatography (see Section A, this report), unlabeled N-Ethyl FOSE was not co-chromatographed with the e o a 461 - Analtech, 75 Blue Hen Drive, Newark, Delaware. u - Supelco Inc., Beliefonte, Pennsylvania. - Unisil, activated silicic acid 100/120 mesh, Clarkson Chemical Company, Inc., Williamsport, Pennsylvania. -6- 14 N-Ethyl FOSE- C. The results of the thin-layer radiochemical purity analysis are shown in Table 1 and Figures 1-5. Small amounts of impurity (<2%^4were present either at the origin or just before the N-Ethyl FOSE- c peak in Plates No. 1-5. The impurities were separated by the column; 99.2% of the radioactivity recovered was eluted with chloroform, 0.7% with 1:1 chloroform-methanol and 0.1% with methanol. Plate No. 6 is a ragiochromatogram of the chloroform fraction. The N-Ethyl FOSE- c peak contained 99.3% of the total radioactivity recovered from the plate. Thus, this analysis indicates a radiochemical purity of 98.5% (99.3% of the___ total in the fraction [99.2%] = 98.5%). The presence of small amounts of impurities is demonstrated for the other two column fractions by Plates No. 7 and 8. No attempt was made to identify these minor impurities. Overall, the radiochemical purity of N-Ethyl FOSE- is at least 98%. This is in agreement with the GLC analysis (see Section A, this report). The N-Ethyl FOSE- c is suitable for metabolism studies. Acknowledgement The authors gratefully acknowledge the gas chromatographic analysis of the carbon-14 labeled cell drainings and N-Ethyl FOSE- C 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._SCLF. o i/ Z C0u4b2 -7References 1. Johnson JD and Behr PE: Synthesis and Characterization of FC-95-14C (Report) November 2, 1979. 2. Analytical Report Number 14557. COci 4 S 3 -8- Table 1 Specific Activity Determination of N-Ethyl FOSE- 14C Solution I 17.47 mq/10.0 ml pCi/mg 0.4854 0.4832 0.4811 0.4822 0.4827 0.4790 0.4935 0.5008 0.5080 0.4297 0.4958 0.4100 0.4776+0.0287 Solution II 37.55 mq/10.0 ml pCi/mg 0.5048 0.4986 0.5000 0.4533 0.4749 0.4635 0.4864 0.4953 0.4900 0.4843 0.4920 0.4773 0.4850+0.0154 Solution III 25.08 mq/10.0 ml pCi/mg 0.4828 0.4848 0.4853 0.4749 0.4709 0.4853 0.5065 0.4955 0.4946 0.4833 0.4916 0.4939 0.48750.0096 Aquasol pCi/mg 0.4854 0.4832 0.4811 0.4935 0.5008 0.5080 0.4986 0.5048 0.5000 0.4864 0.4953 0.4900 0.4828 0.4848 0.4853 0.5065 0.4955 0.4946 0.49310.0087 MTSS pCi/mg 0.4822 0.4827 0.4790 0.4297 0.4958 0.4100 0.4533 0.4749 0.4635 0.4843 0.4920 0.4773 0.4749 0.4709 0.4853 0.4833 0.4916 0.4939 0.473510.0225 10 pi pCi/mg 0.4854 0.4832 0.4811 0.4822 0.4827 0.4790 0.4986 0.5048 0.50Q0 0.4533 0.4749 0.4635 0.4828 0.4848 0.4853 0.4749 0.4709 0.4853 0.481810.0123 50 yl PCi/mg 0.4935 0.5008 0.5080 0.4297 0.4958 0.4100 0.4864 0.4953 0.4900 0.4843 0.4920 0.4773 0.5065 0.4955 0.4946 0.4833 0.4916 0.4939 0.484910.0251 Overall average - 0.48340.0195 See text (Section B) for description of sample prparation. G03464 -9- List of Tables and Figures Table 1 : Table 2: Figure 1: Figure 2 : Figure 3: Figure 4 : Figure 5: Figure 6: Figure 7: Figure 8: Figure 9: Specific Activity Determination of N-Ethyl FOSE-14C. NB 51807 p. 41, 43. Thin-layer Chromatography Systems for N-Ethyl FOSE-14C. NB 51807 p. 14,15. Synthetic Pathway for Synthesis of N-Ethyl FOSE-14C. NB 50942 p. 38-42. Thin-layer Radiochromatogram of N-Ethyl FOSE-14C, Plate No. 1. NB 51807 p. 14. Thin-layer Radiochromatogram of N-Ethyl FOSE-14C, Plate No. 2 NB 51807 p. 14. Thin-layer Radiochromatogram of N-Ethyl FOSE-14C, Plate No. 3 NB 51807 p. 14. Thin-layer Radiochromatogram of N-Ethyl FOSE-14C, Plate No. 4 NB 51807 p. 15. Thin-layer Radiochromatogram of N-Ethyl FOSE-14C, Plate No. 5 NB 51807 p. 15. Thin-layer Radiochromatogram of Chloroform Column Fraction N-Ethyl FOSE- C, Plate No. 6 NB 51807 p. 16,17. Thin-layer Radiochromatogram of lil Chloroform-Methanol Fraction of N-Ethyl FOSE- C, Plate No. 7 NB 51807 p. 16,17. Thin-layer Radiocbromatogram of Methanol Column Fraction of N-Ethyl FOSE-- C , Plate No. 8 NB 51807 p. 16,17. 003465 -1U- Table 2 Thin-Layer Chromatography Systems for N-Ethyl FOSE-14C Plate No. 1 2 3 4 5 Sol.vent S,,yst^em-a --b _ R,fof N-Ethyl F0SE-14C 100 chloroform 100 acetone 100 chloroform 100 methanol 2 acetic acid- 100 butanol 10 water 10 acetic acid- 150 chloroform 50 methanol 5 ammonium hydroxide- 100 chloroform 35 methanol 5 ammonium hydroxide- 0.67 0.77 0.73 0.80 0.70 - Solvents were prepared volume: volume; a 100 ml aliquot of solvent mixture was added to the chromatography tank. jj - Rf is of major (>98%) peak on the thin-layer chromatography plate. Q - Acetic acid and ammonium hydroxide were concentrated. C034S6 -11- Figure 1 14 a Synthetic Pathway for Synthesis of N-Ethyl FOSE- C- Step 1 C2H15 CH2S02F ECF HF C F 5 CF SO F Fractional r Distillation C7F15 CF2SQ2F C7F15 CF2S02F 98.15% Step 2 * 60 * ^ C2H5 c7f15 CF2S02F + 2C2H5NH2 -- ----> C?F15 CF2S02-N ' ^H Step 3 C_H_ C7F15*CF2S02-NX 2 5 H 1) NaOCH. -------------- > C-F 2) C!CH2CH2OH C-2H5 CF SO,-N N CH2-CH2-OH N-Ethyl FOSE-14C *Denotes position of carbon-14 label. ^N-Ethyl FOSE is 2-N-ethylperfluorooctanesulfonamido ethanol. C-03 467 Percent of Total CPM on Plate 70 60 50* 40- -12Figuro 2 Thin-layer Radiochromatogram of N-Ethyl FOSE- C, Plate No. 1 . . 100 chloroform o w Uuj.pJ.uLo: 100 aceto n e Total CPM on Plate 88,572 30' 20. 10* 0 f c v - r- *" " o 12 3 4 5 6 Distance from Origin (CM) 03468 Percent of Total CPM on Plate 70 . 60 50 40 30 20 * 10 0J -U - Distance from Origin (CM) C034S9 Percent of Total CPM on Plate 70 * f>0 ' 50 -14Figure 4 Thin-layer Radiochromatogram of N-Ethyl FOSE- C, Plate No. 3 SGF Uniplate: 100 butanol 40 30 20" 10- 0 Distance from Origin (CM) coa 470 70 -15Figure 5 Percent of Total CPM on Plate Distance from Origin (CM) C03471 Percent of Total CPM on Plate 70 i 60 1 504 401 30 H 20 J 101 0-* (CM) 003472 Percent of Total CPM on Plate 70" 60* 50. 40 30 20 100J -17Piqre 7 Thin-layer Radiochromatogram of Chloroform column fraction N-Ethyl FOSE- 4C, Plate No. 6 (CM) 003473 Percent of Total CPM on Plate 70 60 5040302010" 0 -* -18- _ Figure 8 Thin-layer Radiochromatogram of 11 ChlorjJormMethanol Column Fraction of N-Ethyl FOSE- C, Plate No. 7 SGF Update: 100 chloroform 35 methanol 5 ammonium hydroxide Total CPM on Plate ** 2,594 Distance from Origin (CM) C-0 3 4 7 4 -lyFigure y Thin-layer Radiochromatogram of Methanol Column Fraction of N-Ethyl FOSE- C, Plate No. 8 SOF Unipiate: 100 chloroform 35 methanol 5 ammonium hydroxide Total CPM on Plate = 354 Distance from Origin (CM) C05475 Distribution List M.T. Case S.F. Chang G. J. Conard H-E. Freier L.I. Harrison J.D. LaZerte L.J. Magill R.A. Nelson R.E. Ober R.A. Prokop A.L. Rosenthal F.A. bel P. Venkateswarlu H. A. Vogel Drug Metabolism Central File Tech Communications 218-2 218-2 218-2 201-1S 218-2 236-1 223-6S 218-1 218-2 236-3B 230-3 220-2E 236-3A 236-3B 218-2 201-2CN C0J476