Document 6wMg0wQ315Y2YLe01mr6KK15g

,A Synthesis and Characterization of N-Ethyl FOSE-14 c December 11, 1980 Conducted at: During: Commercial Chemicals Division and Riker Laboratories, Inc. 3H St. Paul, Minnesota 55144 April 1979 to July, 1979 Synthesis Conducted by: F.E. Behr Specific Activity and Radiochemical Purity Determination by: J.D. Johnson and S.i. Gibson Report by: Q. @ -3/(/,?/ J.DD.joJrohl n Date Se@Sjne-n.Lor ,@c crhemical Pharmacologist Riker LiVDoratories, Inc. F.E. Behr, Ph.D. Date Research Specialist Commercial Chemicals Division specific Activity and Radiochemical Characterization Section Reviewed by: .1 0A 3/17/-gt 1 R.E. Ober, Ph.D. Date Manager, Drug Metabolism Riker Laboratories, Inc. -2- The synthesis of a 10. @4g lot of 2-N-ethylperfluorooctanesulfamido ethanol (N-Ethyl POSE- C), L-4545, is described (carbcne4to sulfur atom is labeled). The specific activity was determined to be 0.483 0. 020,@Ci/nKj. liatliocliemicaplurity determination showed the N-Ethyl FOS@3- C to be at least 98% pure. The N-Ethyl FOSE was found suitable for metabolism studies. Introduction A series of e eriments has been started to investigate the metabolism of N-Ethyl POSE- fR C. To facilitate these experiments, carbon-14 labeled N-Etliyl FOSE was synthesized and characterized. The synthetic pathway illustrated in Figure I is described. -3- 1.1 A. -I;yziLli(jLoifL; N-Lt I-'USL- C (L-4545) 1. Fracticnation of C3Elr*CF 2SO 2F (Step 1). II. The fractional distillation of crude cell drainings from cell run R-3256-B has been previously described (1). GLC! analysis showed 98.15% C 8 F 17 so 2 F. Amidation of C@p *CF ij; ;ISO P (Step 2). Into a tared 250 ml three-necked round-bottomed flask was added c 7 F JS*CF2 SO F (69.0 g). The flask was fitted with an inlet and cxi gas bugbling assembly, a glass dip tube, condenser, thermometer and a drying tube. Anhydrous ethyla-ine was introduced to the heated fluorochemical at 600C until excess C2 H 5NH was observed at the exit side of the reaction apparatus. A iotal of 46.3 g of C2 H 5NH 2 was lost from the cylinder mass. The reaction mixture was stirred at 700C for 1.5 hrs. Dry nitrogen gas was used to de-gas the reaction mixture. The reaction mixture was treated by the addition of sulfuric acid (10%, 75 ml). The mixture was heated at 50*C 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 700C for 0.25 hr. Upon cooling, the acidic aqueous phase (pH <1, Congo red paper) was decanted from the solid fluorochemical phase. The fluorochemical phase was washed with deimized water until the pH of the wash solution was 4.5 to 5.0. Four water washes were needed. The fluorochemical was heated at 700C 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 22rfluorooctanesulfcnamide Chlorchydrin (Step 3). with Ethylene Into a pre-tared 200 ml three-necked round-bottcmed flask, which had been equipped with a distillation head and condenser, thermometer, an additional funnel and a magnetic stirring bar, was placed the crude radioactive b sulfcnamide (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-sulfmamide mixture. Methanol (10 ml) was used to wash the addition funnel. The rmixture was heated to 550C to effect the formation of sodium N-ethyl perfluorooctanesulfanamide and to start the distillation of methanol. The residual methanol was removed with water aspir- ator vacuum and, finally, the mixture was heated at 700C and 3-4 m vacuum. The mixture was cooled to 480C, 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 reacticn occurred as the mixture was heated to 600C. Quality Control Manual called for 96.0% min. b RM = Raw Material. -4- After tike irlitial exothermic reaction had subsided, the mixturc was heated first to 900C for 1.0 hr and then the tezqperat%=e of the mixture was raised to 115 OC for 5 hrs. The mixture was cooled to 80*C and the mixture was washed successively with water, brine (three times) and the residual water was stripped by vacuum at 75*C. Crude alcohol (72.7 g) was vacuum distilled to yield two fractions: FR #1, 57.3 g, b.p. 142-1440 and FR #2, 7.G g, b.1). .5 IL46-148*. GLC analysis sgowed FR #1 and #2 to 5.4. be > 99.9% C F SO N *-C 2 H 5 a 17 2 1% CH CH OH 22 (see reference 2). The non-distillable material (5.0 g) was discarded as radioactive waste material. FR #1 and #2 were ccmbined and melted to give N-Et FOSE2 (Step 3) in 64.9 gm yield. A ten gram sample was takr--nfrom tlte total sample and was assigned the following designation: L-4545, Carbon-14 labeled N-Et POSE, B. Specific Activity Determination of N-Ethyl FOSE- 14 C. Three standard solutions of N-Ethyl FOSE- 14 C b (L-4545 see Synthesis Section) were prepared by weighing 1 17.47 mg, 11 37.55 mg, and 111 25.08 mg into three 10 ml class A volumetric flaskse. The volumes were adjusted to 10 ml with methanol and the glasks were mixed by inverting by hand. Calibrated micropipettors- were used to aliquot six 10 pl and six 50 ol aliquots of each solution directly into scintillation counting vials. To three of the vials containing 10 ul aliquots and to three containing 50 lilaliquots from each primary solution, 1 ml of water and 15 ml of AquasoltA were added. The re- f maining vials were prepared with 2.5 ml of methanol and 7.5 ml of MTSS-. Analyses of three standard solutions allow comparison of three primary weighings. Analyses of 10 pl and 50 pl aliquots from each solution allow comparison of the precision of the micropipettors. The use of two different scintillation solvents allows comparison of solubility. (MTSS is toluene based and AquasollP 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 PCi/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 UCi/mg. The mean liCi/mg found for each of the primary weighings was within 1.2% of the overall mean. The mean UCi/mg between AquasolID and MTSS were within 4.1%, and the mean liCi/mg between 10 pl and 50 pl aliquots were within 0.7%. 2-N-Ethylperfluorooctanesulfonamido ethanol. Riker Isotope Number 468. Weighings were accomplished on a 5-place Mettler H64 electronic balance which had recently been calibrated by 3M Metrology. L/I Micropippetor, Lab Industries, Berkeley, CA. New England Nuclear, Boston, Mass. f Modified TSS: 25.2 g PPO, 1.01 g Dimethyl POPOP and 3.8 1 toluene. I'liriLy AiiiAty:-;L:c;ir N-I!,'Lliy.l1.,OS1L4-C I - 'I'Iiiii-iayuCrliromatography 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 Uniplat s a 9 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 wid(--sayments. 'Iltisecraping 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 MTSS was added. The samples were mixed by shaking and cooled in tl)c 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: Sem on segment sum of cpm on plate 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. Il. Column Chromatography System A 14 mm column was packed to 21 cm with a slurry ii silicic acide in chloroform. A one ml aliquot of N-Ethyl POSE- C test solution (see next section C-Ill) 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 drynessp reconstituted in 200 ul of 1:1 chloroform-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. S. III. Solution Used for Radiochemical Purity Analysis The solution used to streak the qin-layer plates was prepared by placing 26.16 mg of N-Ethyl FOSE- C (Riker Isotope inventory Number 468) into a 10.0 al class A volumetric flask, adjusting the volume to 10.0 ml with methanol, and mixing by hand by inverting. Fifty Pl of this solution was applied to each plate. one mi of this solution was applied to the silicic acid column. IV. Results and Discussion Since the chemical identity of the N-Fthyl POSE- 14 C is well established by synthesis and gas chromatography (see Section A, report), unlabeled N-Ethyl MSE was not co-chromatographed with this the Analtech, 75 Blue Hen Drive, Newark, Delaware. Supelco Inc., Bellefonte, Pennsylvania. Unisil, activated silicic acid 100/120 mesh, Clarkson Chemical Company, Inc., Williamsporti Pennsylvania. -6- N-Ethyl FOSE- 14 C. The results of the thin-layer radiochemical purity analysis are shown in Table 1 and Fig=es 1-5. Small amounts of impurity (<2%14 were present either at the origin or just before the N-Ethyl FOSE- C peak in Plates No. 1-5. The impurities were scl)arated 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 rT!i"-Iochromatogram 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 B. No attempt was made to identify these minor i urities. Overall, the radiochemical purity of N-Ethyl FOSE IN C is at least 98%. This is in agreement with tie GLC analysis (see Section A, this report). The N-Ethyl FOSE- C is suitable for metabolism studies. Acknowledgement The authors gratefully acknowledge the gas chromatographi analysis of the carbon-14 labeled cell drainings and N-Ethyl FOSE- T4 C done by Mr. Todd Hathisen of Comercial Chemicals Division. The authors gratefully acknowledge the assistance of John C. Hansen and Larry G. Headrick during the electrochemical fluorination of labeled C H SO F. 8 17 2 -7References 1. Johnson JD and Behr FE: Synthesis and Characterization (Report) November 2, 1979. 2. Analytical Report Number 14557. of FC-95-14 c I Table 14 Specific Activity Determination of N-Ethyl FOSE- c Solution I 17.47 mq/10.0 ml wci/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 0.47760.02ii7 Solution II 37.55 mg/10.0 ml uci/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 0.48500.0154 Solution III 25.08 mgllO.O ml lici/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 AguasolO" lici/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 iici/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 0.47350.0225 10 iil pci/mg 0.4854 0.4832 0.4811 0.4822 0.4827 0.4790 0.4986 0.5048 0.5000 0.45,33 0.4749 0.4635 0.4828 0.4848 0.4853 0.4749 0.4709 0.4853 0.48180.0123 50 pl lici/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 0.48490.0251 Overall average m 0.48340.0195 See text (Section B) for description of sample preparation. Table 1: Table 2: Figure 1: Figure 2: Figure 3: Figure 4: Figure 5: Figure 6: Figure 7: Figure 8: Figure 9: List of Tables and Figures Sl)ccific Activity Determination NB 51807 p. 41, 43. of N-Ethyl 14 FOSE- C. Thin-layer Chromatography NB 51807 p. 14,15. 14 Systems for N-Ethyl FOSE- C. Synthetic Pathway for Synthesis NB 50942 p. 38-42. of N-Ethyl 14 FOSE- C. Thin-layer Radiochromatogram of N-Ethyl Plate No. 1. NB 51807 p. 14. 14 FOSE- C, Thin-layer Radiochromatogram of N-Ethyl Plate No. 2 NB 51807 p. 14. 14 FOSE- C, Thin-layer Radiochromatogram of N-Ethyl Plate No. 3 NB 51807 p. 14. FOSE- 14 C, Thin-layer Radiochromatogram of N-Ethyl Plate No. 4 NB 51807 p. 15. 14 FOSE- C, Thin-layer Radiochromatogram of N-Ethyl Plate No. 5 NB 51807 p. 15. 14 FOSE- C, Thin-layer Raliochromatogram of Chloroform Column Fraction N-Ethyl FOSE- C, Plate No. 6 NB 51807 p. 16,17. Thin-layer Radiochromatogiam of lil Chloroform-Methanol Fraction of N-Ethyl FOSE- C, Plate No. 7 NB 51807 p. 16,17. Thin-layer Radioykromatogram of Methanol Column Fraction of N-Ethyl FOSE- C, Plate No. 8 NB 51807 p. 16,17. -lu- Thin-Layer Table 2 Chromatography Systems for N-Ethyl 14 FOSE- c Plate No. 1 2 3 4 5 Solvent System a b 14 R of N-Ethyl FOSE- C f 100 chloroform 100 acetone 100 chloroform 100 methanol c 2 acetic aci 100 butanol 1D water 10 acetic acid2 150 chloroform 50 methanol 5 ammonium hydroxidec 100 chloroform 35 methanol 5 ammonium hydroxidec 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. b R is of major (>98%) peak on the thin-layer chromatography plate. f 2 Acetic acid and am=nium hydroxide were concentrated. Figure 1 14c!@ !-.yrithvtiPcithway for Synthenis of N-Ethyl FOSE- Step 1 c 2 H 15 CH 2 so 2 F ECF > HF c 7 F 15 CF 2 so 2 F Fractional Distillation C F CF SO F 7 15 2 2 c 7F 15 CF 2 so 2 F 98.15% Step 2 600 c 71115 CF 2 SO 2 F + 2C 2H 5NH 2 C 7F 15 cH o 25 CF 2 so 2-N \ Step 3 c 7F 15 CF 2 so 2-N cH 25 H 1) NAOCH 3 2) CLCH CH OH 22 * . / c2H 5 c IF CF SO -N 7 15 2 2 \ CH 2-CH 2-OH 14 N-Ethyl FOSE- c *Denotes position of carbon-14 label. aN-Ethyl FOSE is 2-N-ethylperfluorooctanesulfonanLido ethanol. 70 60 - 4rJa , 50' r-14 r_ 0 a. u 4J 40- 0 p 44 0 4J 30' 20. -12- riqurc- 2 Thin-layer Raliochromatogram of N-Ethyl FOSE- C, Plate No. 1 100 chloroform 100 acetone Total CPM on Plate 86,572 10, 0 0 1 2 3 4 5 6 78 9 16 11 22 13 14 Distance from origin (CM) 70 60 50 4J 0 04 40 u 41 0 E.-4 k,14 0 4J 30 Figure 3 Thin-layer Raliochromatogram of N-Ethyl FOSE- C, Plate No. 2 SGF Uniplate: 100 chloroform 100 methanol 2 acetic acid Total CPM on Plate 82,187 20 10 0 0 12 3 4 5 67 Distance from Origin (CM) lb Zl i2 13 14 1.5 70. 4J 50 m 0 04 u 40' 4J 0 E- 44 0 4-1 30u $4 a) 04 20- 10. -14I-liclur4u Thiii-layer Raliochrnmatogram of N-Ethyl POSE- C, Plate No. 3 SGF Uniplate: 100 butanol 10 water 10 acetic acid Total CPM on Plate 89,696 0 01 2 3 - ----------------- 5 7 89 10 11 12 13 14 3:5 Distance from Origin (CM) 70' 60, 500 4J 0 E- 40- 44 0 4J 30' 20. -15Figure 5 Thin-layer Raliochromatogram of N-Ethyl ilose- C, Plate No. 4 SGF Uniplate: 150 chloroform 5U mltliariol 5 ammonium hydroxide Total CPM on Plate 91,164 10. oj 0 4 6 7 Distance from Origin (CM) 2 13 is 70 60" 50 0 u ,--1 ro 4 41 0 E- 44 0 4J r_ Q) u 30 @4 a) 20. 10, Figure 6 Thin-layer Raliochromatogram of N-Ethyl FOSE- C, Plate No. 5 SGF uniplate: 100 chloroform 35 methanol 5 ambonium hydroxide Total CPM on Plate 90,961 0 1 2 3 4 5 6 8 9 10 11 12 13 14' ;5 Distance from Origin (CM) 70 60 50 4J 0 2: 40 4.J 0 E- 44 0 30 -17Figure 7 Thin-layer Raliochromatogram of Chloroform column fraction N-Ethyl FOSE- C, Plate No. 6 SGF Uniplate: 100 chloroform 35 methanol 5 ammonium hydroxide Total CPM on Plate 372,209 20 10 0 ------ o 1 2 3 4 56 7 8 9 10 11 12 13- 14 Distance from Origin (CM) 70 60- so- a4 a 0 04 C) 1-4 fd 40. 4J 0 E-4 44 0 30, 20- 10, 0 Figure 8 Thin-layer Radiochromatogram of 1;1 Chlor T40rmMethanol 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 3 4 5 6 7 8 10 Distance from Origin (CM) 12 13 14 15 70 60 - 41 a4 50 r_ 0 m: a4 L) 0 40 E-4 44 0 30 Figure 9 Thin-layer Radiochromatogfim of Methanol Column Fraction of N-Ethyl FOSE- C, Plate No. 8 SUk' Uniplate: iOU chloroform 35 methanol 5 ammonium hydroxide Total CPM on Plate 354 20 10, 0 0 1 2 3 4 6 7 8 Distance from Origin (CM) 10 13. i2 @3 1'4 15 Distribution List M.T. Case S.F. Chang G.J. Conard H.E. Frei:er L.I. Harrison J.D. LaZerte L.J. Magill R.A. Nelson R.E. Ober R.A. Prokop A.L. Rosenthal F.A. Ubel P. Venkateswarlu H.A. Vogel Drug Metabolism Central File Tech Communications 218-2 218-2 218-2 201-lS 218-2 236-1 223-6S 218-1 218-2 236-3B 230-3 220-2E 236-3A 236-3B 218-2 201-2CN