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Radiochemical synthesis and biodistribution of a novel maxi-K potassium channel opener
Nuclear Medicine and Biology 29 (2002) 55–59
Radiochemical synthesis and biodistribution of a novel maxi-K potassium channel opener Dale O. Kiesewettera,*, Elaine M. Jagodaa, John E. Starrett Jr.b, Valentin K. Gribkoffb, Piyasena Hewawasamb, Nugahally Srinivasb, Daniel Salazarb, William C. Eckelmana a
Positron Emission Tomography Department, Clinical Center, National Institutes of Health, 10 Center Drive, Bethesda, MD 20892-1180, USA b Bristol-Myers Squibb Pharmaceutical Research Institute, Wallingford, CT and Lawrenceville, NJ, USA Received 30 April 2001; received in revised form 13 July 2001; accepted 10 August 2001
Abstract The racemate 1, ((⫹/-)-(5-Chloro-2-methoxyphenyl)-1,3-dihydro-3-fluoro-6-(trifluoromethyl)- 2H-indol-2-one), is a potent, specific and novel opener of cloned large-conductance, calcium-activated (maxi-K) potassium channels. One of its enantiomers, BMS-204352 (MaxiPostTM), is undergoing clinical evaluation for efficacy in patients with suspected acute stroke. In the current study, we have prepared [18F]-labeled 1 using a silver assisted nucleophilic substitution to examine its distribution and disposition in the rat, with particular emphasis on the brain. Biodistribution studies in rats confirm that brain uptake is rapid and occurs at high levels, and indicate that a major fraction of the compound in the brain does not accumulate by a specific, saturable mechanism. © 2002 Elsevier Science Inc. All rights reserved. Keywords: PET; Biodistribution; Brain; Maxi-K; MaxiPost; BMS-204352
1. Introduction More than 500,000 cases of stroke are reported in the U.S. each year, of which 70% of patients survive, and 55% of the survivors have some degree of permanent impairment. While many avenues have been explored for pharmacological intervention in acute stroke [3,5], thus far only a single form of therapy, thrombolysis, has been shown to be effective in improving outcome of acute stroke [4]. A novel approach for neuroprotective therapy in acute stroke is development of drugs that specifically open calcium-dependent neuronal potassium channels. The non-radiolabeled racemate 1 (Scheme 1, (⫹/-)-(5-Chloro-2-methoxyphenyl)-1,3dihydro-3-fluoro-6-(trifluoromethyl)-2H-indol-2-one) was discovered as an opener of large conductance calcium-activated potassium channels (maxi-K) with neuroprotective effects in animal models of stroke [7]. Subsequently, the single enantiomer BMS204352 ((3S)-(5-chloro-2-methoxyphenyl)-1,3-dihydro-3-fluoro6-trifluoromethyl-2H-indol-2-one) was isolated and found to open maxi-K potassium channels and demonstrated significant neuroprotective properties [7]. BMS-204352 (also * Corresponding author. Tel.: ⫹1-301-435-2229; fax: ⫹1-301-4023521. E-mail address: [email protected] (D.O. Kiesewetter).
known as MaxiPostTM) is currently undergoing worldwide efficacy trials in patients with suspected acute stroke. The present study was designed to determine if the in vivo biodistribution and pharmacokinetic properties of racemic 1 were consistent with its pharmacological actions. Positron emission tomography (PET), a three-dimensional imaging technique, can be applied to the study of pharmacokinetics in vivo. With the ultimate goal of having PET available for studies in higher primates and humans, we developed a radiosynthesis of 1 incorporating the positron emitting radionuclide F-18. The radiolabeled compound was evaluated in rats for a specific uptake mechanism into the brain.
2. Methods 2.1. (⫾)-3-chloro-3-(5-chloro-2-methoxyphenyl)-1,3dihydro-6-(trifluoromethyl)-2H-indol-2-one [3] (⫾)-3-(5-chloro-2-methoxyphenyl)-1,3-dihydro-3-hydroxy6-(trifluoromethyl)-2H-indol-2-one (2) was prepared as previously described [8]. Thionyl chloride (0.613 mL, 8.4 mmol, 6 equiv) was added to a -78 °C solution of (⫾)-3-(5-chloro-2methoxyphenyl)-1,3-dihydro-3-hydroxy- 6-(trifluoromethyl)2H- indol-2-one (2) (0.500 g, 1.4 mmol) and triethylamine
0969-8051/02/$ – see front matter © 2002 Elsevier Science Inc. All rights reserved. PII: S 0 9 6 9 - 8 0 5 1 ( 0 1 ) 0 0 2 8 1 - 5
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D.O. Kiesewetter et al / Nuclear Medicine and Biology 29 (2002) 55–59
Scheme 1. Preparation of [18F]- 1.
(0.726 mL, 8.4 mmol, 6 equiv) in dichloromethane (25 mL). The cold bath was removed and the reaction mixture was allowed to warm to room temperature. After 3 hr, the resulting mixture was cooled to 0 °C and carefully quenched by the addition of an aqueous solution of 0.1 N hydrochloric acid (10 mL). The contents were poured into water (100 mL) and extracted with ethyl acetate (3 x 50 mL). The combined organic layer was washed with brine (25 mL), dried (MgSO4), filtered and concentrated in vacuo. The crude residue was purified using silica gel column chromatography (4/1, hexanes/ ethyl acetate) to provided 325 mg (62% yield) of chloroindolone 3 as an off-white solid. The solid was re-crystallized from ethyl acetate/hexane. 1H NMR (400 MHz, DMSO-d6) 11.24 (1 H, s), 7.91 (1 H, d, J ⫽ 2.6), 7.52 (1 H, dd, J ⫽ 8.8, 2.6), 7.30 –7.24 (2 H, m), 7.17 (1 H, s), 7.07 (1 H, d, J ⫽ 8.8), 3.33 (3 H, s); MS-DCI m/z 376 (MH⫹); Anal. Calcd for C16H10Cl2F3NO2: C, 51.08; H, 2.67; N, 3.72. Found: C, 51.11; H, 2.53; N, 3.72. 2.2. Carrier-added radiochemical synthesis of [18F]-1 A 1 mL V-vial was charged with 20 L water containing 3 mol of Me4NF. To this was added 200 L of [18F]fluoride (33.6 mCi) in water. The water was evaporated under a stream of argon. Three portions of 200 L of CH3CN were added and each in turn evaporated in order to remove residual water as the azeotrope with CH3CN. The vial was then placed in a shallow beaker of tap water for about 1 min. To the dried [18F]fluoride was added 1) 100 L CH3CN, 2) a solution of chloroindolone 3 (1.9 mg in 200 L CH3CN), and 3) a solution of silver trifluoromethanesulfonate (3 mol in 30 L CH3CN). The solution was vortex-mixed thoroughly. The vial was capped and heated in a 110 °C block for 30 min. The vial was then cooled briefly in a shallow beaker of water. The contents of the vial were transferred onto a short (about 5 mm) column of silica gel in a Pasteur pipet. The vial was rinsed with 100 L CH3CN and the rinse added to the silica gel column. The solution was pushed through the column and an additional 300 L of CH3CN was added to elute the column. The eluate was diluted with 200 L of water and injected onto a Beckman ODS semipreparative HPLC column (9.4 x
250 mm) which was eluted with 60% CH3CN 40% water at 7 mL/min. The desired product eluted at about 9 –10 min. The collected fraction was reduced in volume by about one-half under an argon stream, loaded onto a BondElut C-18 (3 mL size), and washed with 2 mL water. The product was eluted from the BondElut C-18 with 1 mL ethanol. The ethanol was diluted with phosphate buffered saline for injection into rats. The product was obtained in 15% yield at end of synthesis (EOS). The procedure required 67 min. 2.3. No-carrier-added synthesis of [18F]-1 This synthesis required only minor changes to the above procedure. To a one mL V-vial containing 400 L of water containing [18F]fluoride was added 20 L water containing 3 mol of Me4NOH. The water was evaporated under a stream of argon. Three portions of 200 L of CH3CN were added and each in turn evaporated. The vial was then placed in a shallow beaker of tap water for about 1 min. To the dried [18F]fluoride was added, 1) 100 L CH3CN, 2) a solution of 3 (1.9 mg in 200 L CH3CN), and 3) a solution of silver trifluoromethanesulfonate (6 mol in 60 L CH3CN). The solution was vortex-mixed thoroughly. The reaction was continued as for the carrier-add reaction above. In two consecutive syntheses the yields were 1% EOS in 75 min and 0.7% EOS in 70 min. 2.4. Analysis of radiolabeled compounds Radiochemical purity was determined by TLC (Whatman silica gel LK6DF, elute with 1:1 ethyl acetate: hexane Rf ⫽ 0.4) and HPLC (Axxiom C-18 4.6 x 250 mm, eluting with 60% CH3CN, 40% water at 1.5 mL/min). Identity was confirmed on HPLC by re-injection and subsequently coinjection with authentic standard, 1, and observing co-elution. The authentic standard was prepared as previously described [8]. The product elutes at about 6.7– 6.8 min. The substrate (3), if present, would elute at about 8.0 min.
D.O. Kiesewetter et al / Nuclear Medicine and Biology 29 (2002) 55–59
Fig. 1. The time course of the biodistribution of
2.5. Biodistribution studies in rat All animal studies were performed under a protocol approved by the NIH Clinical Center Animal Care and Use Committee. Awake rats were separated into control and test groups of five or six individuals. One group was injected
Fig. 2. Biodistribution of
18
18
57
F in rats after injection of carrier-added [18F]-1.
intravenously (tail vein) with only [18F]-1 (50 Ci). The second group received the [18F]-1 (50 Ci) and co-injected unlabeled BMS-204352. Following the designated period of uptake, the rats were sacrificed. The brains were removed and immediately placed in 0.3 M sucrose on ice. The brains were dissected on ice; the blood and various tissues were
F in rats 30 min after injection of no-carrier-added [18F]-1 with and without 50 nmol of unlabeled BMS-204352.
58
D.O. Kiesewetter et al / Nuclear Medicine and Biology 29 (2002) 55–59
Fig. 3. Biodistribution of
18
F in rats at 30 min after injection of no-carrier-added [18F]-1 with and without 500 nmol of unlabeled BMS-204352.
excised from each animal and weighed. Gamma counting assessed the radioactivity concentration in each tissue, and the percent injected dose per gram tissue was calculated.
3. Results and discussion BMS-204352 has demonstrated the capacity for attenuating cerebral edema and motor impairment following experimental brain injury in rats. Doses of 0.03 mg (80 nmol)/kg reduced cerebral edema and doses of 0.1 mg (300 nmol)/kg improved neurologic motor function in these studies [2]. We wanted to determine if the observed properties were the results of the binding of the compound to a specific site in the brain. Both BMS-204352 and its racemate (1) show similar activity in assays of neuroprotection following brain injury [6]. The synthetic approach for preparation of the unlabeled authentic material was not amenable to the radiochemical synthesis, as it employed diethylaminosulfur trifluoride (DAST) as the flourinating reagent [8]. We wished to prepare the material with high specific activity in an attempt to determine if the uptake mechanism is a saturable, specific process. In order to obtain the [18F]-1 product with sufficiently high specific activity, we envisioned incorporation of the [18F] label by employing [18F]fluoride as the fluoride source. The radiosynthesis of [18F]-1 proceeded as shown in Scheme 1. We sought to employ a nucleophilic substitution on the rather inaccessible tertiary halogen. The chloro precursor 3 was prepared from alcohol 2 by treatment with
thionyl chloride. Substitution of the tertiary halogen with fluoride was quite difficult. Under the usual conditions, K222 and K2CO3, no fluoride incorporation was observed. Fluoride incorporation was achieved by increasing the Sn1 character of the reaction by the addition of silver triflate. Tetramethyl ammonium hydroxide was used as the base to minimize the precipitation of silver salts. Silver triflate was chosen because of the low nucleophilicity of the triflate anion. Without the addition of the silver salt no incorporation of radiofluorine was observed. Because of the need to develop Sn1 character, we decided not to attempt to use a single-enantiomer precursor, as racemization would occur. The product was purified by preparative HPLC and isolated from the eluate using a C-18 BondElut column. Even with the best conditions, the yields were approximately 1% at EOS (end of synthesis), based on radioactivity used at the beginning. With the addition of carrier fluoride, the yields could be increased to about 15%. The radiochemical purity was determined by HPLC and TLC. The specific activity was not measured. However, in the no-carried-added synthesis, we expect specific activities to be between 1 and 2 Ci/mol at EOS. In the carrier-added synthesis, the specific activity was 7 mCi/mol at EOS based on the ratio of radioactivity to added carrier fluoride. 3.1. Biodistribution studies Biodistribution studies were conducted in awake rats. We used the product from a carrier-added study to conduct a time course of brain uptake. Radioactivity uptake in the brain was high and peaked prior to 15 min (Fig. 1). At least
D.O. Kiesewetter et al / Nuclear Medicine and Biology 29 (2002) 55–59
half of the radioactivity washed out of all tissues examined by 60 min with the exception of the femur. Uptake increased in the femur with time indicating some defluorination of the compound by metabolic processes. To minimize any blood flow effects we selected 30 min for our coinjection studies. Using no-carrier added [18F]-1, we evaluated the effect of co-injection of 50 nmol per rat (⬃200 nmol/kg) and 500 nmol per rat (⬃2000 nmol/kg) of unlabeled BMS-204352 along with the radiolabeled product. These concentrations of compound are at or above the effective concentration in the neuroprotective studies. In the control group uptake was homogeneous in all brain regions examined. This homogeneity is at odds with published Bmax values in rat brain quantified by ligand binding to synaptic plasma membrane vesicles from various brain regions [9]. The potassium channel is highest in the frontal cortex, olfactory tubercle, and hippocampus; somewhat lower in the striatum and thalamus; and very low in the brain stem and spinal cord. The concentration ranged from 191 fmol/mg protein in frontal cortex to 18 fmol/mg protein in the brain stem. Studies of mRNA for channel proteins showed heterogeneity of expression throughout the brain [1], but the protein concentration and the mRNA concentration were not always correlated [9]. No difference in the uptake of any brain region was observed with co-injection of 50 nmol per rat compared to the control group (Fig. 2). At 500 nmol per rat, a small increase in uptake was observed (Fig. 3) which might be due to changes in blood flow caused by the large amount of drug administered. Since no reduction in regional brain uptake was observed in these co-injection experiments, brain uptake was unlikely to be predominately mediated by a saturable, specific mechanism.
4. Conclusion We have prepared [18F]-1 in carrier-added and no-carrier-added forms in high radiochemical purity using a silver ion assisted substitution of a tertiary chloride. The radiochemical yield is modest under carrier-added conditions and poor under no-carrier-added conditions. The compound is rapidly and widely distributed and uptake into the brain was rapid and occurred at high levels. The brain uptake was unlikely to be mediated by a saturable, specific uptake process because there was no demonstrable inhibition with
59
increasing mass dose. This study supports the concept of employing [18F]-1 in PET imaging in humans as a marker for the determination of blood brain barrier transport of BMS-204352, but not to follow a saturable binding process. Acknowledgments The authors acknowledge the contributions of the staff of the NIH cyclotron facility for radionuclide production. References [1] C.-P. Chang, S.I. Dworetzky, J. Wang, M.E. Goldstein, Differential expression of the a and b subunits of large-conductance calciumactivated potassium channel: implication for channel diversity. Mol. Brain Res. 45 (1997) 33– 40. [2] J.A. Cheney, J.D. Weisser, F.M. Bareyre, H.L. Laurer, K.E. Saatman, R. Raghupathi, V. Gribkoff, J.E. Starrett Jr., T.K. McIntosh, The maxi-K channel opener BMS-204352 attenuates regional cerebral edema and neurologic motor impairment after experimental brain injury. J. Cereb. Blood Flow Metab. 21 (2001) 396 – 403. [3] P.W. Duncan, H.S. Jorgensen, D.T. Wade, Outcome measures in acute stroke trials: a systematic review and some recommendations to improve practice, Stroke 31 (2000) 1429 –1438. [4] M. Fisher, Antithrombotic and thrombolytic therapy for ischemic stroke, J. Thromb. Thrombolysis 7 (1999) 165–169. [5] M. Fisher, S. Finklestein, Pharmacological approaches to stroke recovery, Cerebrovasc. Dis. 9 (Suppl. 5) (1999) 29 –32. [6] V.K. Gribkoff, J.E. Starrett Jr., S.I. Dworetzky, P. Hewawasam, C.G. Boissard, D.A. Cook, S.W. Frantz, K. Heman, J.R. Hibbard, K. Huston, G. Johnson, B.S. Krishnan, G.G. Kinney, L.A. Lombardo, N.A. Meanwell, P.B. Molinoff, R.A. Myers, S.L. Moon, A. Ortiz, L. Pajor, R.L. Pieschl, D.J. Post-Munson, L.J. Signor, N. Srinivas, M.T. Taber, G. Thalody, J.T. Trojnacki, H. Wiener, K. Yeleswaram, S.W. Yeola, Targeting acute ischemic stroke with a calcium-sensitive opener of maxi-K potassium channels, Nat. Med. 7 (2001) 471– 477. [7] P. Hewawasam, V.K. Gribkoff, S.I. Dworetzky, A.A. Ortiz, G.G. Kinney, C.G. Boissard, D.J. Post-Munson, J.T. Trojnacki, K. Huston, L.G. Signor, L.A. Lombardo, S.A. Reid, J.R. Hibbard, R.A. Myers, S.L. Moon, H.L. Wiener, G. Thalody, K. Yeleswaram, L.M. Pajor, J.O. Knipe, N.A. Meanwell, G. Johnson, P.B. Molinoff, J.E. Starrett, Q. Gao, Discovery of openers of large conductance, calcium activated potassium (maxi-K) channels: A new approach to stroke neuroprotection, Abstr. Pap. Am. Chem. S. 219: 320-MEDI, Part, 2 (2000). [8] P. Hewawasam, N.A. Meanwell, V.K. Gribkoff, (1996) 3-Substituted oxindole derivatives as potassium channel modulators U.S. Patent 5,565,483. [9] H.-G. Knaus, C. Schwarzer, R.O.A. Koch, A. Eberhart, G.J. Kaczorowski, H. Glossmann, F. Wunder, O. Pongs, M.L. Garcia, G. Sperk, Distribution of high-conductance Ca2⫹-activated K⫹ channels in rat brain: targeting to axons and nerve terminals, J. Neurosci. 16 (3) (1996) 955–963.
Radiochemical synthesis and biodistribution of a novel maxi-K potassium channel opener Dale O. Kiesewettera,*, Elaine M. Jagodaa, John E. Starrett Jr.b, Valentin K. Gribkoffb, Piyasena Hewawasamb, Nugahally Srinivasb, Daniel Salazarb, William C. Eckelmana a
Positron Emission Tomography Department, Clinical Center, National Institutes of Health, 10 Center Drive, Bethesda, MD 20892-1180, USA b Bristol-Myers Squibb Pharmaceutical Research Institute, Wallingford, CT and Lawrenceville, NJ, USA Received 30 April 2001; received in revised form 13 July 2001; accepted 10 August 2001
Abstract The racemate 1, ((⫹/-)-(5-Chloro-2-methoxyphenyl)-1,3-dihydro-3-fluoro-6-(trifluoromethyl)- 2H-indol-2-one), is a potent, specific and novel opener of cloned large-conductance, calcium-activated (maxi-K) potassium channels. One of its enantiomers, BMS-204352 (MaxiPostTM), is undergoing clinical evaluation for efficacy in patients with suspected acute stroke. In the current study, we have prepared [18F]-labeled 1 using a silver assisted nucleophilic substitution to examine its distribution and disposition in the rat, with particular emphasis on the brain. Biodistribution studies in rats confirm that brain uptake is rapid and occurs at high levels, and indicate that a major fraction of the compound in the brain does not accumulate by a specific, saturable mechanism. © 2002 Elsevier Science Inc. All rights reserved. Keywords: PET; Biodistribution; Brain; Maxi-K; MaxiPost; BMS-204352
1. Introduction More than 500,000 cases of stroke are reported in the U.S. each year, of which 70% of patients survive, and 55% of the survivors have some degree of permanent impairment. While many avenues have been explored for pharmacological intervention in acute stroke [3,5], thus far only a single form of therapy, thrombolysis, has been shown to be effective in improving outcome of acute stroke [4]. A novel approach for neuroprotective therapy in acute stroke is development of drugs that specifically open calcium-dependent neuronal potassium channels. The non-radiolabeled racemate 1 (Scheme 1, (⫹/-)-(5-Chloro-2-methoxyphenyl)-1,3dihydro-3-fluoro-6-(trifluoromethyl)-2H-indol-2-one) was discovered as an opener of large conductance calcium-activated potassium channels (maxi-K) with neuroprotective effects in animal models of stroke [7]. Subsequently, the single enantiomer BMS204352 ((3S)-(5-chloro-2-methoxyphenyl)-1,3-dihydro-3-fluoro6-trifluoromethyl-2H-indol-2-one) was isolated and found to open maxi-K potassium channels and demonstrated significant neuroprotective properties [7]. BMS-204352 (also * Corresponding author. Tel.: ⫹1-301-435-2229; fax: ⫹1-301-4023521. E-mail address: [email protected] (D.O. Kiesewetter).
known as MaxiPostTM) is currently undergoing worldwide efficacy trials in patients with suspected acute stroke. The present study was designed to determine if the in vivo biodistribution and pharmacokinetic properties of racemic 1 were consistent with its pharmacological actions. Positron emission tomography (PET), a three-dimensional imaging technique, can be applied to the study of pharmacokinetics in vivo. With the ultimate goal of having PET available for studies in higher primates and humans, we developed a radiosynthesis of 1 incorporating the positron emitting radionuclide F-18. The radiolabeled compound was evaluated in rats for a specific uptake mechanism into the brain.
2. Methods 2.1. (⫾)-3-chloro-3-(5-chloro-2-methoxyphenyl)-1,3dihydro-6-(trifluoromethyl)-2H-indol-2-one [3] (⫾)-3-(5-chloro-2-methoxyphenyl)-1,3-dihydro-3-hydroxy6-(trifluoromethyl)-2H-indol-2-one (2) was prepared as previously described [8]. Thionyl chloride (0.613 mL, 8.4 mmol, 6 equiv) was added to a -78 °C solution of (⫾)-3-(5-chloro-2methoxyphenyl)-1,3-dihydro-3-hydroxy- 6-(trifluoromethyl)2H- indol-2-one (2) (0.500 g, 1.4 mmol) and triethylamine
0969-8051/02/$ – see front matter © 2002 Elsevier Science Inc. All rights reserved. PII: S 0 9 6 9 - 8 0 5 1 ( 0 1 ) 0 0 2 8 1 - 5
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D.O. Kiesewetter et al / Nuclear Medicine and Biology 29 (2002) 55–59
Scheme 1. Preparation of [18F]- 1.
(0.726 mL, 8.4 mmol, 6 equiv) in dichloromethane (25 mL). The cold bath was removed and the reaction mixture was allowed to warm to room temperature. After 3 hr, the resulting mixture was cooled to 0 °C and carefully quenched by the addition of an aqueous solution of 0.1 N hydrochloric acid (10 mL). The contents were poured into water (100 mL) and extracted with ethyl acetate (3 x 50 mL). The combined organic layer was washed with brine (25 mL), dried (MgSO4), filtered and concentrated in vacuo. The crude residue was purified using silica gel column chromatography (4/1, hexanes/ ethyl acetate) to provided 325 mg (62% yield) of chloroindolone 3 as an off-white solid. The solid was re-crystallized from ethyl acetate/hexane. 1H NMR (400 MHz, DMSO-d6) 11.24 (1 H, s), 7.91 (1 H, d, J ⫽ 2.6), 7.52 (1 H, dd, J ⫽ 8.8, 2.6), 7.30 –7.24 (2 H, m), 7.17 (1 H, s), 7.07 (1 H, d, J ⫽ 8.8), 3.33 (3 H, s); MS-DCI m/z 376 (MH⫹); Anal. Calcd for C16H10Cl2F3NO2: C, 51.08; H, 2.67; N, 3.72. Found: C, 51.11; H, 2.53; N, 3.72. 2.2. Carrier-added radiochemical synthesis of [18F]-1 A 1 mL V-vial was charged with 20 L water containing 3 mol of Me4NF. To this was added 200 L of [18F]fluoride (33.6 mCi) in water. The water was evaporated under a stream of argon. Three portions of 200 L of CH3CN were added and each in turn evaporated in order to remove residual water as the azeotrope with CH3CN. The vial was then placed in a shallow beaker of tap water for about 1 min. To the dried [18F]fluoride was added 1) 100 L CH3CN, 2) a solution of chloroindolone 3 (1.9 mg in 200 L CH3CN), and 3) a solution of silver trifluoromethanesulfonate (3 mol in 30 L CH3CN). The solution was vortex-mixed thoroughly. The vial was capped and heated in a 110 °C block for 30 min. The vial was then cooled briefly in a shallow beaker of water. The contents of the vial were transferred onto a short (about 5 mm) column of silica gel in a Pasteur pipet. The vial was rinsed with 100 L CH3CN and the rinse added to the silica gel column. The solution was pushed through the column and an additional 300 L of CH3CN was added to elute the column. The eluate was diluted with 200 L of water and injected onto a Beckman ODS semipreparative HPLC column (9.4 x
250 mm) which was eluted with 60% CH3CN 40% water at 7 mL/min. The desired product eluted at about 9 –10 min. The collected fraction was reduced in volume by about one-half under an argon stream, loaded onto a BondElut C-18 (3 mL size), and washed with 2 mL water. The product was eluted from the BondElut C-18 with 1 mL ethanol. The ethanol was diluted with phosphate buffered saline for injection into rats. The product was obtained in 15% yield at end of synthesis (EOS). The procedure required 67 min. 2.3. No-carrier-added synthesis of [18F]-1 This synthesis required only minor changes to the above procedure. To a one mL V-vial containing 400 L of water containing [18F]fluoride was added 20 L water containing 3 mol of Me4NOH. The water was evaporated under a stream of argon. Three portions of 200 L of CH3CN were added and each in turn evaporated. The vial was then placed in a shallow beaker of tap water for about 1 min. To the dried [18F]fluoride was added, 1) 100 L CH3CN, 2) a solution of 3 (1.9 mg in 200 L CH3CN), and 3) a solution of silver trifluoromethanesulfonate (6 mol in 60 L CH3CN). The solution was vortex-mixed thoroughly. The reaction was continued as for the carrier-add reaction above. In two consecutive syntheses the yields were 1% EOS in 75 min and 0.7% EOS in 70 min. 2.4. Analysis of radiolabeled compounds Radiochemical purity was determined by TLC (Whatman silica gel LK6DF, elute with 1:1 ethyl acetate: hexane Rf ⫽ 0.4) and HPLC (Axxiom C-18 4.6 x 250 mm, eluting with 60% CH3CN, 40% water at 1.5 mL/min). Identity was confirmed on HPLC by re-injection and subsequently coinjection with authentic standard, 1, and observing co-elution. The authentic standard was prepared as previously described [8]. The product elutes at about 6.7– 6.8 min. The substrate (3), if present, would elute at about 8.0 min.
D.O. Kiesewetter et al / Nuclear Medicine and Biology 29 (2002) 55–59
Fig. 1. The time course of the biodistribution of
2.5. Biodistribution studies in rat All animal studies were performed under a protocol approved by the NIH Clinical Center Animal Care and Use Committee. Awake rats were separated into control and test groups of five or six individuals. One group was injected
Fig. 2. Biodistribution of
18
18
57
F in rats after injection of carrier-added [18F]-1.
intravenously (tail vein) with only [18F]-1 (50 Ci). The second group received the [18F]-1 (50 Ci) and co-injected unlabeled BMS-204352. Following the designated period of uptake, the rats were sacrificed. The brains were removed and immediately placed in 0.3 M sucrose on ice. The brains were dissected on ice; the blood and various tissues were
F in rats 30 min after injection of no-carrier-added [18F]-1 with and without 50 nmol of unlabeled BMS-204352.
58
D.O. Kiesewetter et al / Nuclear Medicine and Biology 29 (2002) 55–59
Fig. 3. Biodistribution of
18
F in rats at 30 min after injection of no-carrier-added [18F]-1 with and without 500 nmol of unlabeled BMS-204352.
excised from each animal and weighed. Gamma counting assessed the radioactivity concentration in each tissue, and the percent injected dose per gram tissue was calculated.
3. Results and discussion BMS-204352 has demonstrated the capacity for attenuating cerebral edema and motor impairment following experimental brain injury in rats. Doses of 0.03 mg (80 nmol)/kg reduced cerebral edema and doses of 0.1 mg (300 nmol)/kg improved neurologic motor function in these studies [2]. We wanted to determine if the observed properties were the results of the binding of the compound to a specific site in the brain. Both BMS-204352 and its racemate (1) show similar activity in assays of neuroprotection following brain injury [6]. The synthetic approach for preparation of the unlabeled authentic material was not amenable to the radiochemical synthesis, as it employed diethylaminosulfur trifluoride (DAST) as the flourinating reagent [8]. We wished to prepare the material with high specific activity in an attempt to determine if the uptake mechanism is a saturable, specific process. In order to obtain the [18F]-1 product with sufficiently high specific activity, we envisioned incorporation of the [18F] label by employing [18F]fluoride as the fluoride source. The radiosynthesis of [18F]-1 proceeded as shown in Scheme 1. We sought to employ a nucleophilic substitution on the rather inaccessible tertiary halogen. The chloro precursor 3 was prepared from alcohol 2 by treatment with
thionyl chloride. Substitution of the tertiary halogen with fluoride was quite difficult. Under the usual conditions, K222 and K2CO3, no fluoride incorporation was observed. Fluoride incorporation was achieved by increasing the Sn1 character of the reaction by the addition of silver triflate. Tetramethyl ammonium hydroxide was used as the base to minimize the precipitation of silver salts. Silver triflate was chosen because of the low nucleophilicity of the triflate anion. Without the addition of the silver salt no incorporation of radiofluorine was observed. Because of the need to develop Sn1 character, we decided not to attempt to use a single-enantiomer precursor, as racemization would occur. The product was purified by preparative HPLC and isolated from the eluate using a C-18 BondElut column. Even with the best conditions, the yields were approximately 1% at EOS (end of synthesis), based on radioactivity used at the beginning. With the addition of carrier fluoride, the yields could be increased to about 15%. The radiochemical purity was determined by HPLC and TLC. The specific activity was not measured. However, in the no-carried-added synthesis, we expect specific activities to be between 1 and 2 Ci/mol at EOS. In the carrier-added synthesis, the specific activity was 7 mCi/mol at EOS based on the ratio of radioactivity to added carrier fluoride. 3.1. Biodistribution studies Biodistribution studies were conducted in awake rats. We used the product from a carrier-added study to conduct a time course of brain uptake. Radioactivity uptake in the brain was high and peaked prior to 15 min (Fig. 1). At least
D.O. Kiesewetter et al / Nuclear Medicine and Biology 29 (2002) 55–59
half of the radioactivity washed out of all tissues examined by 60 min with the exception of the femur. Uptake increased in the femur with time indicating some defluorination of the compound by metabolic processes. To minimize any blood flow effects we selected 30 min for our coinjection studies. Using no-carrier added [18F]-1, we evaluated the effect of co-injection of 50 nmol per rat (⬃200 nmol/kg) and 500 nmol per rat (⬃2000 nmol/kg) of unlabeled BMS-204352 along with the radiolabeled product. These concentrations of compound are at or above the effective concentration in the neuroprotective studies. In the control group uptake was homogeneous in all brain regions examined. This homogeneity is at odds with published Bmax values in rat brain quantified by ligand binding to synaptic plasma membrane vesicles from various brain regions [9]. The potassium channel is highest in the frontal cortex, olfactory tubercle, and hippocampus; somewhat lower in the striatum and thalamus; and very low in the brain stem and spinal cord. The concentration ranged from 191 fmol/mg protein in frontal cortex to 18 fmol/mg protein in the brain stem. Studies of mRNA for channel proteins showed heterogeneity of expression throughout the brain [1], but the protein concentration and the mRNA concentration were not always correlated [9]. No difference in the uptake of any brain region was observed with co-injection of 50 nmol per rat compared to the control group (Fig. 2). At 500 nmol per rat, a small increase in uptake was observed (Fig. 3) which might be due to changes in blood flow caused by the large amount of drug administered. Since no reduction in regional brain uptake was observed in these co-injection experiments, brain uptake was unlikely to be predominately mediated by a saturable, specific mechanism.
4. Conclusion We have prepared [18F]-1 in carrier-added and no-carrier-added forms in high radiochemical purity using a silver ion assisted substitution of a tertiary chloride. The radiochemical yield is modest under carrier-added conditions and poor under no-carrier-added conditions. The compound is rapidly and widely distributed and uptake into the brain was rapid and occurred at high levels. The brain uptake was unlikely to be mediated by a saturable, specific uptake process because there was no demonstrable inhibition with
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increasing mass dose. This study supports the concept of employing [18F]-1 in PET imaging in humans as a marker for the determination of blood brain barrier transport of BMS-204352, but not to follow a saturable binding process. Acknowledgments The authors acknowledge the contributions of the staff of the NIH cyclotron facility for radionuclide production. References [1] C.-P. Chang, S.I. Dworetzky, J. Wang, M.E. Goldstein, Differential expression of the a and b subunits of large-conductance calciumactivated potassium channel: implication for channel diversity. Mol. Brain Res. 45 (1997) 33– 40. [2] J.A. Cheney, J.D. Weisser, F.M. Bareyre, H.L. Laurer, K.E. Saatman, R. Raghupathi, V. Gribkoff, J.E. Starrett Jr., T.K. McIntosh, The maxi-K channel opener BMS-204352 attenuates regional cerebral edema and neurologic motor impairment after experimental brain injury. J. Cereb. Blood Flow Metab. 21 (2001) 396 – 403. [3] P.W. Duncan, H.S. Jorgensen, D.T. Wade, Outcome measures in acute stroke trials: a systematic review and some recommendations to improve practice, Stroke 31 (2000) 1429 –1438. [4] M. Fisher, Antithrombotic and thrombolytic therapy for ischemic stroke, J. Thromb. Thrombolysis 7 (1999) 165–169. [5] M. Fisher, S. Finklestein, Pharmacological approaches to stroke recovery, Cerebrovasc. Dis. 9 (Suppl. 5) (1999) 29 –32. [6] V.K. Gribkoff, J.E. Starrett Jr., S.I. Dworetzky, P. Hewawasam, C.G. Boissard, D.A. Cook, S.W. Frantz, K. Heman, J.R. Hibbard, K. Huston, G. Johnson, B.S. Krishnan, G.G. Kinney, L.A. Lombardo, N.A. Meanwell, P.B. Molinoff, R.A. Myers, S.L. Moon, A. Ortiz, L. Pajor, R.L. Pieschl, D.J. Post-Munson, L.J. Signor, N. Srinivas, M.T. Taber, G. Thalody, J.T. Trojnacki, H. Wiener, K. Yeleswaram, S.W. Yeola, Targeting acute ischemic stroke with a calcium-sensitive opener of maxi-K potassium channels, Nat. Med. 7 (2001) 471– 477. [7] P. Hewawasam, V.K. Gribkoff, S.I. Dworetzky, A.A. Ortiz, G.G. Kinney, C.G. Boissard, D.J. Post-Munson, J.T. Trojnacki, K. Huston, L.G. Signor, L.A. Lombardo, S.A. Reid, J.R. Hibbard, R.A. Myers, S.L. Moon, H.L. Wiener, G. Thalody, K. Yeleswaram, L.M. Pajor, J.O. Knipe, N.A. Meanwell, G. Johnson, P.B. Molinoff, J.E. Starrett, Q. Gao, Discovery of openers of large conductance, calcium activated potassium (maxi-K) channels: A new approach to stroke neuroprotection, Abstr. Pap. Am. Chem. S. 219: 320-MEDI, Part, 2 (2000). [8] P. Hewawasam, N.A. Meanwell, V.K. Gribkoff, (1996) 3-Substituted oxindole derivatives as potassium channel modulators U.S. Patent 5,565,483. [9] H.-G. Knaus, C. Schwarzer, R.O.A. Koch, A. Eberhart, G.J. Kaczorowski, H. Glossmann, F. Wunder, O. Pongs, M.L. Garcia, G. Sperk, Distribution of high-conductance Ca2⫹-activated K⫹ channels in rat brain: targeting to axons and nerve terminals, J. Neurosci. 16 (3) (1996) 955–963.