Document OExrbgExjnVVEz2N9gBo790Qw

J U * -A BEST COPY AVAII ABLE S y n t h e s i s ami Cli;i rae: L e r i / . . i t i un u f F C - 1 4 3 - 14 C ARJ3GOH56 December 28, 1979 Conducted at: During: Synthesis Conducted by: Specific Activity and Radiochemical 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 ^k^-r' Jn/& F. E. Behr, Ph.D. Date Research Specialist Commercial Chemicals Division D. Johiisin Date Senior ^ochemical Pharmacologist Riker Laboratories, Inc. i `M i o R. E. Ober, Ph.D. Tiate Manager, Drug Metabolism Riker Laboratories, Inc. 002870 Summary 14 The synthesis of a 20.0 g lot of FC-143" C is described (carbonyl carbon is labeled). The specific activity was determined to be 0.507 0.012 pCi/mg. Radiochemical purity determination showed the FC-l43_ ^ C to be at least 98% pure; the FC-l43_ ^ C was found suitable for metabolism studies. Introduct ion A series of experiments is being planned to investigate the metabolism of FC-143 and the possibility of affecting the rate of elimination of FC-143-^C from the body. To facilitate these experiments, carbon-14 labeled FC-1A3 was synthesized and characterized. The synthetic pathway illustrated in Figure 1 is described. A. Synthesis of FC-143~^C I. Fractional Distillation of 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 filtration to yield 434.0 gms for distillation. Three fractions were collected (Table 1). Fractions 1 and 2 (389.3 g) * were combined and will be used for the preparation of C^F^j-CC^NH^. II. Hydrolysis and Stabilization of Distilled Cell Drainings from R-3265-B 111. Into a 500 ml three-necked round-bottomed flask which had been equipped with a mechanical stirrer, Tru-Bore stirrer assembly, addition funnel, condenser, and thermometer was placed the combined cell drainings, Fractions 1 and 2, 389.3 g. A pre mixed solution of NaOH (68.Ag) and water (68.4 g) was added to the flask through a pressure-equalized addition funnel over 45 minutes at a rate sufficient to keep the temperature between 52-55C. The mixture was heated to reflux (85C) for 9 hours. After the 9 hour reaction time, the pH was found to be > 12. The mixture was cooled to 65C and a mixture of water (257*0 ml) and 96% H2S0j+ (94.3 ml) was added slowly to the flask contents. The final pH was < 1 (Congo red paper). The acidified mixture was heated at 80C for 30 minutes, cooled to 65C, and the bottom fluorochemical phase was separated. The yield was 356.7 g (91.6% mass yield). JU Fractional Distillation of CyFj^i^H The apparatus consisted of a 500 ml three-necked round-bottomed flask, a small packed heated distillation column, pot thermometer, take-off head, a collection assembly, -78C cold trap, magnetic stirrer assembly, and a mercury manometer. The base-stabilized radioactive fluorochemical (356.7 g ) , 96% sulfuric acid (12.0 ml), and Super-Cel (0.7 g) was added to the distillation flask. The 002871 BEST COPY AVAI1 ABLE water aspirator vacuum was adjusted to 200 mm and the low boiling materials were removed. As the head temperature dropped below J0C 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 CyFj^CO-H (See Table II). Fraction 6, M . O g, was refractionated to give 95.0% CyFirCOoH (see Table III), Fraction 3, (21.3 g, 97.5% CyF^io^H), and Fraction k , (13-^ 9, 96.08% C-7jfF1^5CCO0^2HL1)' were combine^for use in preparation of FC- 11*3- 11C. IV. Fractions 3 and k (30.0 g) were dissolved in FC-80 (90 ml). 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 solid at 65C for 1 hour. Analysis by gas chromatography showed an assay of 98.8% C^Fj .(^CH,. The yield was 31.1 g , The following L numbers were assigned: L-l585... FC- 11*3" ' , 100% solids, 20.0 g; L-l586, fractionated CyFj^t^H, NB 5231*3:8-30, Fraction h , O . k g. 11 B. Specific Activity Determination of FC-ll3~ C Three standard solutions of FC-ll*3" ^ C -- (L-l*585, see Synthesis Section) were prepared by weighing-- I, 10.56 mg; II, 25-28 mg; and III, 20.06 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 mi croppettors^- were used to aliquot six 10 pi and six 50 pi aliquots of each solution directly into scintillation counting vials. To three of the vials containing 10 pi aliquots and to three containing 50 pi aliquots 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 pCi/mg was calculated for each vial. The data are shown in Table k . The overall average specific activity based on the 36 replicates (12 from each weighing) was 0.507 0.012 pCi/mg. The mean pCi/mg found for each of the primary weighings was within 1.5% of the overall mean. The means between Aquasol and MTSS were within 0.12%, and the means between 10 pi and 50 pi aliquots were within 2.0%. Riker Isotope Number **59. Weighings were accomplished on a 5-place Mettler H64 electronic balance which had been recently calibrated by 3M Metrology. -- L/l Micropipettor, Lab Industries, Berkeley, CA. -- New England Nuclear, Boston, Mass. -- Modified TSS:25.2 g 1.01 g Dimethyl P0P0P and 3-8 liters toluene. G02872 |cr |o> 14 C. Radiochemical Purity Analysis of FC-l43~ 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 S6F 250 micron pre-scored Uni plates-- . Plates were routinely developed in 10" x 12" x 4" thin-layer tanks-- fitted with glass lids and lined with saturation pads-b-. 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 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 radioactivity peaks corresponding to separated components in the material applied to the plate. I1. Solution Used for Radiochemical Purity Analysis The solution used to streak the thin-layer plates was prepared by placing 30.39 mg of FC-143-^C (Riker Isotope Inventory Number 459) 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 in a narrow four cm long streak to each plate. III. Results and Discussion Since the chemical identity of the FC-143- is well established by synthesis and gas chromatography (see section A, this report), unlabeled FC-143 was not co-chromatographed with the FC-143- **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, the radiochemical purity of F C - ^ S - '^C is at least 98%. The FC-143- **C is suitable for metabolism studies. a_ Analtech, 75 Blue Hen Drive, Newark, Delaware. b Supelco Inc., Bellefonte, Pennsylvania. 002873 Acknowledgement The authors gratefully acknowledge the gas chromatographic analysis of the carbon-1^ labeled cell drainings, fractionated acid, and FC-lA3" ^ 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 C0C1. 002874 List of Tables and Figures Table 1: Table 2: Table 3: Data from One Plate Distillation of Cell Run R-3265-B. NB 509^2 p. 53 * Data from Fractional Distillation of C F CO H. NB 52343 p. 5,6 . 7 15 2 Data for Refractionation of C F CO H. NB 52343 p.8 . 7 15 2 14 Table 4: Specific Activity Determination of FC-143- C. NB 51807 p. 28. Table 5: Thin-Layer Chromatography Systems for FC-143-14C. NB 51807 p. 35- 14 Figure 1: Pathway for Synthesis of FC-143- C. NB 52343 p . k , 5, 6, 8, 9- Figure 2: Thin-Layer Radiochromatogram, Plate 1. NB 51807 p. 11, 39. Figure 3: Thin-Layer Radiochromatogram, Plate 2. NB 51807 p. 11, 39. Figure 4: Thin-Layer Radiochromatogram, Plate 3. NB 51807 p. 11, 39. Figure 5 : Thin-Layer Radiochromatogram, Plate 4. NB 51807 p. 15, b0. Figure 6: Thin-Layer Radiochromatogram, Plate 5. NB 51807 p. 15, b0. C-02875 Table 1 Data from One Plate Distillation of Cell Run, R-3265-B Fr bp C WT (g) a LB's " Assay GLC (Area Percent) C 7F16 C8CE " C F COF 7 15 1 82-89 2 90-92 3 92-110 Bottoms -- 215.6 173.7 12.2 22.4 5.51 -- -- 31.97 46.92 10.24 4.32 66.93 25.90 -- 30.37 50.87 Mostly High Boilers HB *s - 0 0 17.88 ic r L B 's :lowboilers -- unidentified but with retention times < C , F . . 7 16 CgCE: CQ cylic ether - various perfluorobutyltetrahydrofurans and isomers. 5 HB 1s -materials boiling higher than C^F^COF. C02S76 Table 2 * Data from Fractional Distillation of C^F^CC^H Fr bpC 1 <35 (200mm) 2 35-40 3 26-47 (10mm) 4 98-101 5 101-103 6 104-109 (7mm) WT(g) 102.3 103.6 2.4 2.1 1.2 44.0 Inerts 97.98 99.98 2.18 537 -- -- Assay C5FllC02li ------ -- 9.41 13.19 15.85 5.19 C 7F15C02H -- -- 87.9 79.8 78.8 90.3 GLC analysis of FR #6 showed about 3.9% high boiling materials . 002877 -9- Tabie 3 * Data for Refractionation of C^F CX^H 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 %) C7f 15CO: 89.03 97.26 97.54 96.08 Assay for input to refractionatiori was 94.35% C^F^^CO^H analyzed as F15C02CH3 ' 092878 BEST COPY AVAIl ABLE SpuciTic AcLiviLy Determination oi' 14 FC-143- C Solution I 10.56ma/10.0ml 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 Q.50650.0124 Solution II 25.28mg/10.0ml pCi/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 0.514510.0114 Solution III 2 0 ,06mg/1 0 .0ml pCi/mg 0.4966 0.4922 0.4965 0.4982 0.5007 0.5084 0.4948 0.5061 0.5041 0.4973 0.4999 0.5098 0.500410.0056 Comparison of Scintillation Solvents Aquasol 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 0.507410.0137 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 0.506810.0093 Comparison of lOpl and 50pl Aliquots 10pl pCi/mg 50pl pCi/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 0.502110.0109 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 0.512110.0101 Overall average = 0.50710.0115pCi/mg 002879 -II- Tnblo 5 Thin-Layer Chromatography Systems for FC-143-14C Plate No. 1 2 3 4 5 Solvent System^- b K^of 14 FC-143- C 100 chloroform 100 acetone 0.03 100 chloroform 100 methanol Q 2 acetic acid- 0.67 100 butanol 10 water 10 acetic acid-^ 0.60 130 chloroform 50 methanol c 5 ammonium hydroxide- 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. -- Rj is of major (>98%) peak on the thin-layer chromatography plate, c -- Acetic acid and ammonium hydroxide were concentrated. C028S0 Fij-ure 1 BEST COPY AYAH ABIE Pathway for Synthesis of FC-143-^C C-7JK15rCOCl ECF 11J-' Prepared by Pathfinder Laboratories C7F15COF One-Plate__ Distillation I + NaOH 1) Heat _ _ 2) "U2S04" II Fractional Distillation h i + nh3 -F-C-----8--0-------------- > C7F15COF R-3256-B c 7f 15c o f C 7F15C 0 2H II Refractionation C.F^O.H > 95% III Assay NH42C 7F15 FC-143-14C - *-Denotes position of Carbon-14 a 14 FC-143- C, Ammonium perfluorooctanoate 002831 ') > -13- 90 85 80 75 70 65 60 55 50 45 40 35 30 25 20 15 10 5 0, Distance from Origin (CM) C02SS2 Percent of Total CPM on Plate 70. 60. 50 40 30 20 10 0 - I'i- Figure 3 Thin-layer Radiochromatograro of FC-143-14C, Plate No. 2 BESTEMMI (CM) C02SS3 I Figure 4 Thin-layer Radiochromatogram of FC-143-1AC, Plate No. 3 BEST COPT ky Distance from Origin (CM) 002834 Percent of Total CPM on Plate 70. 60 50 . 40 3020 10 0J -16Figure 5 ui.gi m Thin-layer Radiochromatogram of FC-143-14C, Plate No. 4 SGF Uniplate: 150 chloroform 50 methanol 5 ammonium hydroxide Total CPM on Plate = 105,567 Distance from Origin (CM) 002885 Percent of Total CPM on Plate 60 _ 50 _ 40 30 20 _ 10 -17- Figure 6 Thin-layer Kadiochromatogram of FC-143- C, Plate No. 5 Distance from Origin (CM) 002886