Misato Takashima-Hirano a, Tadayuki Takashima a, Yumiko Katayama a, Yasuhiro Wada a,
Yuichi Sugiyama b, Yasuyoshi Watanabe a, Hisashi Doi a, Masaaki Suzuki a,⇑
a RIKEN Center for Molecular Imaging Science (CMIS), Kobe, Japan
b Department of Molecular Pharmacokinetics, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Japan
a b s t r a c t
Synthesis of [11C]celecoxib, a selective COX-2 inhibitor, and [11C]SC-62807, a major metabolite of celecoxib, were achieved and the potential of these PET probes for assessing the function of drug
transporter in biliary excretion was evaluated. The synthesis of [11C]celecoxib was achieved in one-pot by reacting [11C]methyl iodide with an excess of the corresponding pinacol borate precursor using Pd2(dba)3, P(o-tolyl)3, and K2CO3 (1:4:9) in DMF. The radiochemical yield of [11C]celecoxib was 63 ± 23% (decay-corrected, based on [11C]CH3I) (n = 7) with a specific radioactivity of 83 ± 23 GBq/lmol (n = 7). The average time of synthesis from end of bombardment including formulation was 30 min with >99% radiochemical purity. [11C]SC-62807 was synthesized from [11C]celecoxib by further rapid oxidation in the presence of excess KMnO4 with microwave irradiation. The radiochemical yield of [11C]SC-62807 was 55 ± 9% (n = 3) (decay-corrected, based on [11C]celecoxib) with a specific radioactivity of 39 ± 4 GBq/ lmol (n = 3). The average time of synthesis from [11C]celecoxib including formulation was 20 min and the radiochemical purity was >99%. PET studies in rats and the metabolite analyzes of [11C]celecoxib and [11C]SC-62807 showed largely different excretion processes, and consequently, [11C]SC-62807 was rapidly excreted via hepatobiliary excretion without further metabolism. [11C]SC-62807 was shown to have a high potential as a PET probe for evaluating drug transporter function in biliary excretion.
1. Introduction
It is now accepted that drug transporters play important roles in tissue distribution and excretion of drugs and their metabolites. Clinical studies have shown that variation in drug transporter activity caused by genetic polymorphisms or drug–drug interactions can affect the variability in therapeutic efficacy and the incidence of adverse effects.1,2 The information on the functional characteristics of drug transporters in vivo allows improvements in drug delivery or drug design by targeting specific transporter proteins.3 Accordingly, the methods that allow quantitative estimation of the tissue concentration of the drugs in vivo have been required for the investigation of such variations in the tissue distribution or disposition processes of drugs. Positron emission tomography (PET) is a powerful and widely accepted noninvasive method for molecular imaging in living systems.4–6 The high sensitivity and exceptional spatial-temporal resolution of PET make it particularly useful for in vivo estimation of the function of drug transporters in various tissues.7 Celecoxib (4-[5-(4-methylphenyl)-3-(trifluoromethyl)-1H-pyrazole- 1-yl]benzenesulfonamide) is a selective cyclooxygenase (COX)-2 inhibitor that has analgesic and anti-inflammatory effects in patients with rheumatoid arthritis, but has no effect on COX-1 activity at therapeutic plasma concentrations.8 Inhumans, celecoxib is extensively metabolized in the liver via sequential two-step oxidative pathways, initially to a hydroxymethyl metabolite (SC-60613), and upon subsequent further oxidationto a carboxylic acid metabolite (SC-62807) (Fig. 1).9,10 Themajority of celecoxib is excreted into the bile as SC-62807. In thiscontext, Wu et al. reported that SC-62807 is a substrate of drugtransporters, such as Organic Anion Transporting Polypeptide1B1 (OATP1B1) and Breast Cancer Resistance Protein (BCRP),which presumably mediate its hepatobiliary transport.11 Therefore,celecoxib or SC-62807 radiolabeled with a short-livedpositron-emitting radionuclide could be a potential PET probefor evaluating the function of these drug transporters in hepatobiliaryexcretion.Celecoxib and SC-62807 have two readily accessible possiblepositions for 11C and 18F radiolabeling, and Prabhakaran et al.reported the synthesis of both [11C]celecoxib and [18F]celecoxib.12,13 However, chemical and metabolic instabilities derivedfrom [18F]defluorination on the benzylic position of [18F]celecoxibresulted in undesirable bone imaging due to [18F]fluorine
generation. Therefore, 11C-labeling of celecoxib would be preferableto 18F-labeling.The palladium(0)-mediated rapid C–[11C]methylation reactiondeveloped by our group and based on the use of [11C]CH3I and tributylstannyl substrate is used in the synthesis of [11C]celecoxib(Scheme 1).14 Protection of the sulfonamide group is necessary topromote such a reaction. We recently reported a new type of palladium(0)-mediated rapid C–[11C]methylation that uses an organoboroncompound.15 This reaction proceeds in the presence ofPd2(dba)3, P(o-tolyl)3, and K2CO3 in DMF for 5 min under mild temperatureandwithout the use of a microwave.16,17 In general, organoboroncompounds exhibit higher reactivity in the coupling reactionthan organostannyl compounds,18,19 and are also less toxic. Accordingly,the synthesis of [11C]celecoxib reported draws extensivelyfrom our brief report15 and advances made since its publication.SC-62807 has a benzoic acid structure that can be labeled with11C. Efficient 11C labeling of a benzoic acid structure has been reportedthat involves: (1) [11C]CO insertion to the correspondingaryl halide,20 (2) reaction of the Grignard reagent with[11C]CO2,21 and (3) coupling of the [11C]cyanide ion and thecorresponding aryl halide, and hydrolysis.22 Here, we exploredthe novel possibility of 11C labeling using a combination of rapidC–[11C]methylation and rapid oxidation, starting with a commonorganoboron precursor to carry out the sequential transformationof [11C]celecoxib to [11C]SC-62807.This report describes the highlyefficient synthesis of [11C]celecoxiband as an expanding of rapid C–[11C]methylation, the synthesisof [11C]SC-62807 by applying it to rapid C–[11C]carboxylation.In addition, we describe evaluation of the in vivo behavior of each11C-labeled compound in rats using PET to determine whetherthese radiotracers enable the visualization of hepatobiliary excretionfor the quantitativeassessment of drug transporter function.2. Results and discussion2.1. Synthesis of [11C]celecoxib and [11C]SC-62807 PET probesThe synthesis of the pinacol borate precursor, (4-[5-[4-(4,4,5,5-tetramethyl-[1.3.2]dioxaborolan-2-yl)phenyl]-3-trifluoromethyl-1H-pyrazol-1-yl]-1-phenylsulfonamide, (2) was achieved asdescribed in Scheme 1. 4-[5-(4-Bromophenyl)-3-(trifluoromethy)-1H-pyrazol-1-yl]-1-phenylsulfonamide (5) was prepared from1-(4-bromophenyl)-4,4,4-trifluorobutane-1,3-dione and hydrazine(4), which was generated from aniline (3) in one-pot based onpreviously described method with minor modifications.12,23 Compound5 was converted into 2 using bis(pinacolato)diboron andPd catalyst in DMSO.24Synthesis of [11C]celecoxib was previously reported by Prabhakaranet al.,12 using an organotin compound with a sulfonamidegroup protected by a dimethoxytrityl (DMT) group as a precursorfor radiolabeling. The authors succeeded in synthesizing [11C]celecoxibby the rapid methylation reaction using a DMT-protectedstannyl precursor and [11C]CH3I in the presence of Pd2(dba)3 andP(o-tolyl)3 in DMF at 135 _C for 5 min followed by deprotection ofthe DMT group in the presence of trifluoroacetic acid (TFA) at60 _C for 5 min. Prabhakaran et al. also reported the reaction betweenthe organostannyl-precursor without the protecting groupand CH3I in the presence of Pd2(dba)3, P(o-tolyl)3, CuCl, and K2CO3in DMF, but the reaction yielded starting material together withundesired destannylated product.25 We concluded that in the synthesisof a short-lived PET probe, a direct one-step reaction withoutdeprotection is necessary to reduce the total synthesis time. In acontext related approach, we employed the rapid C–[11C]methylationeaction recently developed by our group that uses an organoboronprecursor. Thus, [11C]celecoxib was synthesized by treatingpinacol borate precursor (2) with [11C]CH3I in DMF inthe presenceof Pd2(dba)3/P(o-tolyl)3/K2CO3 (1:4:9) at 65 _C for 4 min (Scheme2). This reaction produced a 98% analytical yield by HPLC(Fig. 2A). The total synthesis time was 30 min from the end of bombardment(EOB). The average decay-corrected radiochemical yield(DCY) based on [11C]CH3I was 63 ± 23% (n = 7), and the specificradioactivity at the end of synthesis was 84 ± 23 GBq/lmol (n = 7)with >98% radiochemical purity (Fig. 2B). The chemical identity of[11C]celecoxib was confirmed by co-injection with the referencestandard celecoxib in analytical HPLC and it showed the sameretention time peaks on the UV and the radioactive chromatograms.Thus, we efficiently synthesized [11C]celecoxib from the correspondingpinacol borate precursor without the protection of as ulfonamide group.
Scheme 2 also illustrates the synthesis of 11C-labeled SC-62807 via [11C]celecoxib based on sequential rapid C–[11C]methylationand oxidation. [11C]celecoxib was dissolved in 0.2 M NaOH andtransferred to a reaction vessel containing KMnO4 (the amount of[11C]celecoxib transferred by this method was _20–60% of the synthesizedradioactivity). The reaction was carried out by setting thetime to 5 min in order to optimize the heating conditions (Table 1).Consequently, [11C]celecoxib was converted to [11C]SC-62807 in32 ± 18% (n = 5) DCY26 based on [11C]celecoxib (HPLC analyticalyield27: 55 ± 30% (n = 5)) by conventional heating at 120 _C. Theprocess had poor reproducibility, presumably due to the presenceof different concentrations of [11C]celecoxib in each reaction.Therefore, we used microwave irradiation as an alternative heatingmethod based on prior experiments in which microwave irradiationtended to enhance reactivity at low concentrations. Asexpected, microwave irradiation was extremely effective inincreasing both the yield and the reproducibility of the reaction.Finally, [11C]SC-62807 was efficiently synthesized by rapidoxidation of [11C]celecoxib at 140 _C for 5 min under microwaveirradiation with 55 ± 9% (n = 3) DCY based on [11C]celecoxib (HPLCanalytical yield: 87 ± 5% (n = 3)) (Fig. 3A). The total synthesis timewas 20 min from the [11C]celecoxib transfer, with a specificradioactivity at the end of synthesis of 39 ± 4 GBq/lmol (n = 3)>98% radiochemical purity (Fig. 3B). The chemical identity of[11C]SC-62807 was confirmed by co-injection with the reference
standard SC-62807 in analytical HPLC and it showed the sameretention time peaks on the UV and the radioactive chromatograms.Thus, we successfully synthesized [11C]celecoxib and[11C]SC-62807 by rapid C–[11C]methylation of the correspondingpinacol borate precursor without the protection of a sulfonamidegroup, and sequential combination rapid oxidation of the[11C]methyl group, respectively. The total time required for thissequential synthesis was 50 min from EOB.2.2. Construction of an automated two-step radiolabelingsystem for the synthesis of [11C]-SC-62807Figure 4 shows the radiolabeling system we developed to carryout two-step synthesis of [11C]SC-62807 using microwave irradiation.The system consists of a reactor for 11CH3I production usingan established method,28 two reactors for cooling and heating,one reactor for rapid oxidation under microwave irradiation (Initi-
ator™, Biotage), multiple reservoirs for the addition of reagents,column switching (six columns available), and four fraction collectors,two of which play the role of evaporators attached to theformulation system. Any combination of reactors and fractioncollectors can be chosen by changing the connections. Thisremote-controlled synthesis apparatus was utilized for the preparationof [11C]SC-62807 using the rapid sequential C–[11C]methylation–oxidation reaction. The automated multistep labelingsystem could be utilized for the synthesis of a wide range of11C-incorporated PET probes.2.3. PET study and radiometabolite analyzes of [11C]celecoxibTime course maximum intensity projection PET images of theabdominal region of a rat following administration of [11C]celecoxibare shown in Figure 5A. Radioactivity initially localized inthe liver, and subsequently part of the radioactivity moved to theintestine, although the amount of radioactivity in the entireabdominal region was relatively high. Radioactivity in the bloodgradually decreased, however, the radioactivity was still higher level(more than 1% of dose/ml blood) even at the end of the scan(Fig. 5B). Figure 5C shows representative radiochromatograms ofblood, liver, and bile extracts prepared after administration of[11C]celecoxib to rats. At 40 min after administration, [11C]celecoxibwas detected predominantly in the blood and the liver, anda very small amount of radiometabolites, [11C]SC-60613 (hydroxymethylform of celecoxib) and [11C]SC-62807 (carboxylic acidform), were detected in the blood and the liver, respectively. Incontrast, the [11C]SC-62807 radiometabolite was the major componentfound in the bile after administration of [11C]celecoxib. Thesepharmacokinetic profiles of [11C]celecoxib will make the analysisof biliary excretion difficult for the following reasons: (1) sincePET cannot discriminate between [11C]celecoxib and its radiometabolite,[11C]celecoxib PET images of hepatobiliary excretion willbe the result of two different pharmacokinetic functions, metabolismand biliary excretion of [11C]celecoxib, and (2) the very high concentration of [11C]celecoxib in the blood may affect the tissue concentration determined by PET image analysis. Therefore,[11C]celecoxib should not be used as a PET probe for the evaluatingdrug transporter function in biliary excretion.2.4. PET study and radiometabolite analyzes of [11C]SC-62807Figure 6A shows time course maximum intensity projection PETimages of radioactivity in the rat abdominal region followingadministration of [11C]SC-62807. Radioactivity localized primarilyin the liver and kidneys within 2 min after [11C]SC-62807 administration.By 60 min, radioactivity was localized in the intestine (derivedfrom bile excreted into the intestine) and the urinary bladder.Time–activity curves for blood and tissues in the abdominal regionof rats are shown in Figure 6B. The radioactivity in blood rapidlydecreased. A maximum of 35 ± 6% and 14 ± 1% of the dose wasdistributed in the liver and kidney, respectively, by 2 min postadministration,at which point the amount of radioactivity beganto decline rapidly. The radioactivity in the intestine (derived fromradioactivity in bile excreted into the intestine) and the urinarybladder (derived from radioactivity excreted into the urine) increaseduntil 60 min, reaching 58 ± 6% and 22 ± 3% of the dose,respectively. Metabolites of [11C]SC-62807 were not detected inthe blood, liver, bile, or urine within 40 min after administrationof [11C]SC-62807 (Fig. 6C). These results show that the radioactivityof [11C]SC-62807 is rapidly excreted via hepatobiliary and renalexcretion without further metabolism.There are some key transporters in hepatobiliary transporterincluding MRP2 (multidrug resistance-associated protein 2), BCRP,and OATPs. Some probes such as 99mTc-mebrofenin, N-[11C]acetylleukotrieneE4, and (15R)-16-m-tolyl-17,18,19,20-tetranorisocarbacyclin(15R-[11C]TIC-Me) for molecular imaging technology havebeen reported for evaluating the function of MRP2 or OATPs inhepatobiliary excretion.29–31 Compared to these probes, the studyusing [11C]SC-62807 may have the potentials for evaluating theother transporter BCRP at least in hepatobiliary transport and renalexcretion. In addition, [11C]SC-62807 is superior to the otherprobes in the use of diagnosis because of the rapid excretion withoutfurther metabolism, which may enable the functional analysismore simple. Further investigations using [11C]SC-62807 are ongoingto evaluate the function of these drug transporters usingknockout animal models or specific drug transporter inhibitors.
3. Conclusion
A novel procedure for the synthesis of a [11C]benzoic acid structurevia sequential rapid C–[11C]methylation and oxidation reactionswas established. The first step involves rapid and highlyefficient 11C-labeling of celecoxib from a pinacol boron precursor.The subsequent rapid oxidation under microwave irradiationproduces [11C]SC-62807. Microwave irradiation significantlyenhances both radiochemical yield and reproducibility. The protocolfor this two-step rapid radiosynthesis can be executed in a fully remote-controlled manner by using the radiolabeling system wedeveloped.PETimage analysis in parallel with radiometabolite analyzes ofrats indicated that [11C]celecoxib is not suitable for evaluating biliaryexcretion because it shows high blood concentrationand itsbiliary excretion includes the mixture of two different pharmacokineticfunctions, metabolism and biliary excretion of [11C]celecoxib.On the other hand, [11C]SC-62807 enables thevisualization ofhepatobiliary excretion as well as renal excretion without furthermetabolism, and therefore is a potentially useful PET probe forthe quantitative determination of the drug transporter. Furtherevaluation of [11C]SC-62807 in terms of its utility in functional analyzesof drug transporters in hepatobiliary and renal excretion is inprogress.
4. Materials and methods
4.1. Chemistry
All chemicals and solvents were purchased from Sigma–AldrichJapan (Tokyo, Japan), Wako Pure Chemical Industries (Osaka,Japan), Tokyo Kasei Kogyo (Tokyo, Japan), Nacalai Tesque (Kyoto,
Japan), and ABX (Radeberg, Germany), and were used withoutfurther purification. Celecoxib, SC-60613, and SC-62807 as coldstandards were purchased from Tronto Research Chemicals (NorthYork, Canada). Flash chromatography was performed on TeledyneIsco CombiFlash Companion (Lincoln, USA). Nuclear magnetic resonance(NMR) spectra were recorded on JEOL JNM-ECX400P spectrometer(Tokyo, Japan) at ambient temperature. The chemicalshifts are expressed in parts per million (ppm) downfield from tetramethylsilaneor in ppm relative to CHCl3 (d 7.26 in 1H NMR and77.0 in 13C NMR). Signal patterns are indicated as follows: s,singlet; d, doublet; t, tripled; q, quartet; m, multiplet; br, broad signal.Coupling constants (J values) are given in hertz (Hz). Massspectra (MS) was measured on a ThermoFinnigan LCQ ion trapmass spectrometer (San Jose, CA, USA) equipped with a turbo ESIion source. Carbon-11 was produced by an 14N(p,a)11C nuclearreaction using a CYPRIS HM-12S Cyclotron (Sumitomo HeavyIndustry, Tokyo, Japan). An automated radiolabeling system wasused for heating the reaction mixture, dilution, HPLC injection,fraction collection,evaporation, and sterile filtration. Purificationwith semipreparative HPLC was performed on a GL Science system(Tokyo, Japan). Microwave irradiation was carried out in a BiotageInitiator™(Tokyo, Japan) using a sealed vessel. Radioactivity wasquantified with an ATOMLAB™ 300 dose calibrator (Aloka, Tokyo,Japan). Analytical HPLC was performed on a Shimadzu system(Kyoto, Japan) equipped with pumps and a UV detector, and effluentradioactivity was measured with an RLC700 radio analyzer(Aloka). The columns used for analytical and semipreparative HPLCwere COSMOSIL C18 MS-II and AR-II (Nacalai Tesque), respectively.
4.1.1. 4-[5-(4-bromophenyl)-3-(trifluoromethy)-1H-pyrazol-1-yl]1-phenylsulfonamide (5)A solution of sodium nitrate (1.38 g, 20 mmol) was added into amixture of sulfanilamide (6.8 g, 20 mmol) in concd HCl (10 mL)and water (2.5 mL) over 15 min at _5 _C. The mixture was rapidlyadded to a cooled (_20 _C) solution of tin(II) chloride dyhydrate(10 g, 44 mmol) in conc. HCl (15 mL). The resulting mixture wasstirred for 1 h at room temperature. A solution of 1-(4-bromophenyl)-4,4,4-trifluorobutane-1,3-dione (4.19 g, 14.2 mmol) in ethanol(20 mL) was added. The mixture was stirred under reflux for 16 h.It was evaporated under the reduced pressure and the residue wasextracted with ethyl acetate (30 mL, three times). Organic layerwas washed with brine (30 mL), dried over sodium sulfate, filtered,and evaporated. The crude product was purified by flash chromatographyeluting with hexane/ethyl acetate = 2:1 to give the titlecompound (4.5 g, 71%) as a white solid. 1H NMR spectrum wasidentified with the data of Ref. 12.
4.1.2. 4-[5-[4-(4,4,5,5-Tetramethyl-[1.3.2]dioxaborolan-2-yl)-phenyl]-3-trifluoromethyl-1H-pyrazol-1-yl]-1-phenylsulfonamide(2)A mixture of 5 (2.23 g, 5.0 mmol), bis(pinacolato)diboron(1.40 g, 5.5mmol), [1,10-bis(diphenylphosphino)ferrocene]dichloropalladium(122 mg, 0.15 mmol), and potassium acetate (1.47 g,15 mmol) in anhydrous DMSO (20 mL) was stirred at 80 _C for3 h. It was partitioned quenched with water (50 mL) and then extractedwith diethylehter (50 mL, two times). Organic layer wasdried over sodium sulfate, filtered, and evaporated. The crudeproduct was purified by flash chromatography eluting with hexane/ethyl acetate = 4:1 to give the title compound (1.5 g, 40%) asa white solid. 1H NMR (400 MHz, CDCl3) d: 7.90 (d, J = 8.8 Hz,2H), 7.79 (d, J = 8.0 Hz, 2H), 7.46 (d, J = 8.8 Hz, 2H), 7.22(d,J = 8.0 Hz, 2H), 6.80 (s, 1H), 5.04 (br s, 2H), 1.35 (s, 12H). 13CNMR (100 MHz, CDCl3) d: 145.0, 144.4, 144.0, 142.4, 141.4, 135.3,131.0, 128.1, 127.6, 125.5, 121.0 (q, J = 268.7 Hz), 106.8, 84.2,24.6. MS (ESI): m/z 494.35 [M+H]+, 492.40 [M_H]_.4.1.3. Synthesis of [11C]celecoxib[11C]CO2 was converted to [11C]CH3I by treatment with lithiumaluminum hydride followed by hyrdoiodic acid using an automatedsynthesis system.28 [11C]CH3I was trapped in a solution ofpinacol borate precursor 2 (3.8 mg, 7.7 lmol), Pd2(dba)3 (2.9 mg,3.2 lmol), P(o-tolyl)3 (3.8 mg, 12.7 lmol), and K2CO3 (4.0 mg,28.9 lmol) in DMF (400 lL) at 30 _C. Next, the mixture was heatedto 65 _C for 4 min and then diluted with CH3CN (700 lL) and water(300 lL). After filtration through a 0.2 lm PVDF filter (Millipore),the resulting mixture was injected onto a preparative HPLC column(AR-II C18, 20 mm i.d. _ 250 mm, 5 lm (COSMOSIL, Nacalai Tesque)using a mobile phase of CH3CN/water = 70:30 at a flow rateof 10 mL/min with UV detection at 254 nm. The [11C]celecoxibretention time was 9.6 min. The desired fraction was collected ina flask and evaporated to dryness.For the subsequent oxidation reaction, [11C]celecoxib was dissolvedin 0.2 M NaOH, while for the isolation and radiochemicalformulation of [11C]celecoxib for use in the PET study, it was reconstitutedwith a mixture of polysorbate 80, propylene glycol, andsaline (0.1:1:10, v/v/v, 4.0 mL). The total synthesis time from EOBuntil formulation was 30 min. The decay-corrected radiochemicalyield was 63 ± 23% (n = 7) based on [11C]CH3I with a specific radioactivityof 83 ± 23 GBq/lmol (n = 7). The chemical identity of[11C]celecoxib was confirmed by co-injection with referencestandard celecoxib in analytical HPLC (column: AR-II C18,4.6 mm i.d. _ 100 mm, 5 lm (COSMOSIL, Nacalai Tesque); mobilephase: CH3CN/water (pH 7.4) = 65:35; flow rate: 1 mL/min; UVdetection: 254 nm; retention time: 3.8 min) and it showed thesame retention time peaks on the UV and the radioactive chromatograms.Both the chemical purity analyzed at 254 nm andhe radiochemical purity were always greater than 98%.
4.1.4. Synthesis of [11C]SC-62807
[11C]celecoxib in 0.2 M NaOH was mixed with potassium permanganate(20 mg). The resulting mixture was heated under microwaveirradiation to 140 _C for 5 min. The reaction was quenchedwith 30% sodium hydrogen sulfite aqueous solution (400 lL) andthen the mixture was acidified with 2 MHCl (200 lL). After dilutionwith CH3OH (600 lL), the mixture was injected onto a preparativeHPLC column (AR-II (COSMOSIL), C18, 10 mm i.d. _ 250 mm, 5 lmwith a mobile phase consisting of CH3CN and 0.2% HCOOH (60:40)and a flow rate of 6 mL/min with UV detection at 254 nm. The[11C]SC-62807 retention time was 4.4 min. The desired fractionwas collected in a flask and the organic solvent was removed underreduced pressure.For the radiochemical formulation of [11C]SC-62807 for use inPETanalyzes, it was reconstituted with a mixture of polysorbate80, propylene glycol, and saline (0.1:1:10, v/v/v, 2.0 mL). The totalsynthesis time until formulation from [11C]celecoxib was 20 min,thus, the total synthesis time from EOB was about 50 min. The decay-corrected radiochemical yield was 55 ± 9% (n = 3) with specificradioactivity of 39 ± 4 GBq/lmol (n = 3). The chemical identity of
[11C]SC-62807 was confirmed by co-injection with the referencestandard celecoxib in analytical HPLC (column: AR-II, 4.6 mm i.d. _100 mm, 5 lm; mobile phase: CH3CN and 0.2% HCOOH = 50:50;flow rate: 1 mL/min; UV detection: 254 nm) and it showed the sameretention time peaks on the UV and the radioactive chromatograms.The [11C]SC-62807 retention time was 4.2 min. Both the chemicalpurity analyzed at 254 nmand the radiochemical purity were greaterthan 99%.
4.2. Experimental animals
Male Sprague–Dawley (SD) rats weighing 210–290 g(7–8 weeks old, n = 2 or 3) were purchased from Japan SLC Inc.(Shizuoka, Japan). The animals were kept in a temperature- andlight-controlled environment and had ad libitum access to standardfood and tap water. All experimental protocols were approvedby the Ethics Committee on Animal Care and Use of the Center forMolecular Imaging Science in RIKEN, and were performed in accordancewith the Principles of Laboratory Animal Care (NIH publicationNo. 85-23, revised 1985).
4.3. PET studies
Rats were anesthetized with a mixture of 1.5% isoflurane and nitrousoxide/oxygen (7:3) and then placed on the PET scanner gantry(MicroPET Focus 220, Siemens Co., Ltd, Knoxville, TN, USA). ThePET scanner has a spatial resolution of 1.4 mm FWHM at the centerof the field of view, which is 220 mm in diameter with an axialextent 78 mm in length. After intravenous bolus injection of[11C]celecoxib (approximately 26–33 MBq per animal) or [11C]SC-62807 (approximately 11–20 MBq per animal) via a venouscatheter inserted into the tail vein, a 60-min emission scan wasperformed. The chemical amounts of [11C]celecoxib and [11C]SC-62807 contained in the bolus injections were calculated to be0.29–1.3 nmol/body (0.11–0.51 lg/body) and 0.20–1.4 nmol/body(0.10–0.72 lg/body), respectively. Arterial blood was sampled viathe cannulated femoral artery at the following time points: 10,20, 30, 40, and 50 s, and 1, 2, 5, 10, 20, 40, and 60 min after administrationof the radiotracers. Blood radioactivity was measuredusing a 1470 WIZARD_ Automatic Gamma Counter (PerkinElmer,Waltham, MA, USA). Emission data were acquired in list mode,and the data were reconstructed with standard 2D filtered backprojection (Ramp filter, cutoff frequency of 0.5 cycles per pixel).Region of interests (ROIs) were placed on liver, intestine, kidney,or urinary bladder using image processing software (Pmodver.3.0, PMOD Technologies Ltd, Zurich, Switzerland). Regional uptakeof radioactivity in the tissue and blood radioactivity were decay-corrected to the injection time and expressed as % dose/tissueor % dose/ml blood, normalized for injected radioactivity.
4.4. Radiometabolite analyzes
Rats were anesthetized with 1.5% isoflurane before administrationof the radiotracers. After intravenous injection of [11C]celecoxib(approximately 26–48 MBq per animal) or [11C]SC-62807(approximately 10–32 MBq per animal), blood, urine, and liversamples were taken at 10, 20, and 40 min post-administration.The liver was removed quickly and then homogenized. To samplebile, the bile duct was cannulated before administration of theradiotracer, and bile was collected over the periods 0–10, 10–20,and 20–40 min post-administration. A two-fold volume of CH3CNwas added to an aliquot of each sample and the resulting mixturewas centrifuged at 12,000 rpm for 2 min at 4 _C. The supernatantwas diluted with HPLC mobile phase and analyzed for intact radiotracersand metabolites using a Shimadzu HPLC system coupled toa NaI(Tl) positron detector UG-SCA30 (Universal Giken, Kanagawa,Japan). Chromatographic separation was carried out using a4.6 mm i.d. _ 50 mm Waters Atlantis T3 column (Waters, Milford,MA). The flow was 2.0 mL/min at an initial condition of 80% solventA (5% CH3CN in 10 mM CH3COONH4) and 20% solvent B (90%CH3CN in 10 mMCH3COONH4). Analytes were eluted using the followinggradient conditions: 0–0.5 min: 20% solvent B in solvent A;0.5–2.5 min: 20–100% solvent B in solvent A; 2.5–4 min: 100% solventB. Following analyte elution, the column was returned to 20%solvent B in solvent A over 2 min. The elution was monitored by UVabsorbance at 254 nm and coupled with NaI positron detection.The amount of radioactivity associated with each intact radiotracerand its metabolite was calculated as a percentage of the totalamount radioactivity.AcknowledgmentsThis work was supported in part by a consignment expense forthe Molecular Imaging Program on ‘‘Research Base for ExploringNew Drugs’’ from the Ministry of Education, Culture, Sports, Scienceand Technology (MEXT) of Japan. We thank Mr. MasahiroKurahashi (Sumitomo Heavy Industry Accelerator Service Ltd) foroperating the cyclotron, Takeshi Ito and Tomohide Handa (DainihonSeiki Co., Ltd) for design and production of the radiolabelingsystem, and Satoru Tanabe and Koji Yokota (Biotage, Japan) forincorporating the microwave apparatus into our system.References and notes
1. Shitara, Y.; Sato, H.; Sugiyama, Y. Annu. Rev. Pharmacol. Toxicol. 2005, 45, 689.
2. Maeda, K.; Sugiyama, Y. Drug Metab. Pharmacokinet. 2008, 23, 223.
3. Willmann, J. K.; van Bruggen, N.; Dinkelborg, L. M.; Gambhir, S. S. Nat. Rev. Drug.
Disc. 2008, 7, 591.
4. Ametamey, S. M.; Honer, M.; Schubiger, P. A. Chem. Rev. 2008, 108, 1501.
5. Fowler, J. S.; Wolf, A. P. Acc. Chem. Res. 1997, 30, 181.
6. Miller, P. W.; Long, N. J.; Vilar, R.; Gee, A. D. Angew. Chem., Int. Ed. 2008, 47,
8998.
7. Riemann, B.; Sch鋐ers, K. P.; Schober, O.; Sch鋐ers, M. Q. J. Nucl. Med. Mol.
Imaging 2008, 52, 215.
8. Penning, T. D.; Talley, J. J.; Bertenshaw, S. R.; Carter, J. S.; Collins, P. W.; Docter,
S.; Graneto, M. J.; Lee, L. F.; Malecha, J. W.; Miyashiro, J. M.; Rogers, R. S.; Rogier,
D. J.; Yu, S. S.; Anderson, G. D.; Burton, E. G.; Cogburn, J. N.; Gregory, S. A.;
Koboldt, C. M.; Perkins, W. E.; Seibert, K.; Veenhuizen, A. W.; Zhang, Y. Y.;
Isakson, P. C. J. Med. Chem. 1997, 40, 1347.
9. Paulson, S. K.; Zhang, J. Y.; Breau, A. P.; Hri bar, J. D.; Liu, N. W. K.; Jessen, S. M.;
Lawal, Y. M.; Cogburn, J. N.; Gresk, C. J.; Markos, C. S.; Maziasa, T. J.;
Schoenhard, G. L.; Burton, E. G. Drug. Metab. Dispos. 2000, 28, 514.
10. Paulson, S. K.; Zhang, J. Y.; Jessen, S. M.; Lawal, Y.; Liu, N. W. K.; Dudkowski, C.
M.; Wang, Y.-F.; Chang, M.; Yang, D.; Findlay, J. W. A.; Berge, M. A.; Markos, C.
S.; Breau, A. P.; Hribar, J. D.; Yuan, J. Xenobitica 2000, 30, 731.
11. Wu, C.; Kusuhara, H.; Sugiyama, Y.; Takashima, T.; Takashima-Hirano, M.; Doi,
H.; Suzuki, M.; Watanabe, Y. Jpn. Phamacol. Ther. 2009, 37, S37.
12. Prabhakaran, J.; Majo, V. J.; Simpson, N. R.; Van Heertum, R. K.; Mann, J. J.;
Kumar, J. S. D. J. Labelled Compd. Radiopharm. 2005, 48, 887.
13. Prabhakaran, J.; Underwood, M. D.; Parsey, R. V.; Arango, V.; Majo, V. J.;
Simpson, N. R.; Heertum, R. V.; Mann, J. J.; Kumar, J. S. D. Bioorg. Med. Chem.
2007, 15, 1802.
14. Suzuki, M.; Doi, H.; Bj鰎kman, M.; Andersson, Y.; L錸gstr鰉, B.; Watanabe, Y.;
Noyori, R. Chem. Eur. J. 1997, 3, 2039.
15. Doi, H.; Ban, I.; Nonomiya, A.; Sumi, K.; Kuang, C.; Hosoya, T.; Tsukada, H.;
Suzuki, M. Chem. Eur. J. 2009, 15, 4165.
16. Hostetler, E. D.; Terr, G. E.; Burns, H. D. J. Labelled Compd. Radiopharm. 2005, 48,
629.
17. Hamill, T. J.; Krause, S.; Ryan, C.; Bonnefous, C.; Govek, S.; Seiders, T. J.; Cosford,
N. D. P.; Roppe, J.; Kamenecka, T.; Patel, S.; Gibson, R. E.; Sanabria, S.; Riffel, K.;
Eng, W.; King, C.; Yang, Z.; Green, M. D.; O’Malley, S. S.; Hargreaves, R.; Burns,
H. D. Synapse 2005, 56, 205.
18. Aldous, D. J.; Bower, S.; Moorcroft, N.; Todd, M. Synlett 2001, 150.
19. Morris, G. A.; Nguyen, S. T. Tetrahedron Lett. 2001, 42, 2093.
20. L錸gstr鰉, B.; Itsenko, O.; Rahman, O. J. Labelled Comp. Radiopharm. 2007, 50,
794.
21. L錸gstr鰉, B.; Antoni, G.; Gullberg, P.; Halldin, C.; Malmborg, P.; N錱ren, K.;
Rimland, A.; Sv鋜d, H. J. Nucl. Med. 1987, 28, 1037.
22. Ponchant, M.; Hinnen, F.; Demphel, S.; Crouzel, C. Appl. Radiat. Isot. 1997, 48,
755.
23. Ando, K.; Kato, T.; Kawai, A.; Nonomura, T. WO9964415, 1999.
24. Ishiyama, T.; Murata, M.; Miyaura, N. J. Org. Chem. 1995, 60, 7508.
25. Further details concerning with the yield, purity, etc. have not been reported in
Ref. 6.
26. Decay-corrected yield (DCY) in the oxidation reaction was calculated based on
the radioactivity of [11C]celecoxib. In oxidation reaction, a part of
[11C]celecoxib which was isolated in the rapid C–[11C]methylation reaction
was used, therefore, DCY based on [11C]CH3I in oxidation reaction was not able
to calculate.
27. HPLC analytical yield indicates the reaction efficiency. The reaction efficient is
calculated by the peak area ratio of the desired [11C]methylated product
obtained by a radio-HPLC analysis of the reaction mixture. See Ref. 32.
28. L錸gstr鰉, B.; Antoni, G.; Gullberg, P.; Halldin, C.; Malmborg, P.; Nagren, K.;
Rimland, A.; Svard, H. J. Nucl. Med. 1987, 28, 1037.
29. Ghibellini, G.; Leslie, E. M.; Pollack, G. M.; Brouwer, L. R. Pharm. Res. 2008, 25,
1851.
30. Guhlmann, A.; Krauss, K.; Oberdorfer, F.; Siegel, T.; Scheuber, P. H.; Muller, J.;
Csuk-Glanzer, B.; Ziegler, S.; Ostertag, H.; Keppler, D. Hepatology 1995, 21,
1568.
31. Takashima, T.; Nagata, H.; Nakae, T.; Cui, Y.; Wada, Y.; Kitamura, S.; Doi, H.;
Suzuki, M.; Maeda, K.; Kusuhara, H.; Sugiyama, Y.; Watanabe, Y. J. Pharmacol.
Exp. Ther. 2010, 37, 314.
32. Suzuki, M.; Doi, H. J. Synth. Org. Chem. Jpn. 2010, 68, 1195.
