benzotriazole-5-carboxylic Acid (CBt-PMN) by Direct - ACS Publications

Article pubs.acs.org/jmc

Synthesis of 11C‑Labeled RXR Partial Agonist 1‑[(3,5,5,8,8Pentamethyl-5,6,7,8-tetrahydronaphthalen-2yl)amino]benzotriazole-5-carboxylic Acid (CBt-PMN) by Direct [11C]Carbon Dioxide Fixation via Organolithiation of Trialkyltin Precursor and PET Imaging Thereof Osamu Shibahara,† Masaki Watanabe,† Shoya Yamada,† Masaru Akehi,‡ Takanori Sasaki,‡ Akiya Akahoshi,‡ Takahisa Hanada,‡ Hiroyuki Hirano,§ Shunsuke Nakatani,† Hiromi Nishioka,† Yasuo Takeuchi,† and Hiroki Kakuta*,† †

Division of Pharmaceutical Sciences, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, 1-1-1, Tsushima-Naka, Kita-Ku, Okayama 700-8530, Japan ‡ Collaborative Research Center for OMIC, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, 2-5-1 Shikata-Cho, Kita-Ku, Okayama 700-8558, Japan § SHI Accelerator Service Ltd. 1-17-6 Osaki Shinagawa-Ku, Tokyo 141-0032, Japan S Supporting Information *

ABSTRACT: The retinoid X receptor (RXR) partial agonist 1-[(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydronaphthalen-2-yl)amino]benzotriazole-5-carboxylic acid (1; CBt-PMN, Emax = 75%, EC50 = 143 nM) is a candidate for treatment of central nervous system (CNS) diseases such as Alzheimer’s and Parkinson’s diseases based on reports that RXR-full agonist 4[1-(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydro-2-naphthyl)ethynyl]benzoic acid (bexarotene) shows therapeutic effects on these disease in rodent models. Here, we synthesized carbon-11-labeled ([11C]1) as a tracer for positron emission tomography (PET) and used it in a PET imaging study to examine the brain uptake and biodistribution of 1. We found that 11CO2 fixation after tin−lithium exchange at −20 °C afforded [11C]1. This methodology may also be useful for synthesizing 11CO2H-PET tracer derivatives of other compounds bearing π-rich heterocyclic rings. A PET/CT imaging study of [11C]1 in mice indicated 1 is distributed to the brain and is thus a candidate for treatment of CNS diseases.

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INTRODUCTION Retinoid X receptors (RXRs) are nuclear receptors that function as transcription factors with multiple roles, including regulation of development, cell differentiation, and metabolism. They act either as homodimers or as heterodimers with thyroid hormone receptor (TR), retinoic acid receptors (RARs), vitamin D receptor (VDR), peroxisome proliferator-activated receptors (PPARs), liver X receptors (LXRs), farnesoid X receptor (FXR), pregnane X receptor (PXR), or two members of the small nerve growth factor-induced clone B (NGFIB) subfamily, namely NG-FIB and NURR1.1 Among them, PPARs and LXRs function in the regulation of glucose/lipid metabolism,2,3 and activation of RXR/PPAR or RXR/LXR by RXR agonists is effective to treat type 2 diabetes4,5 or atherosclerosis in rodent models.6 On the other hand, the RXR agonist 4-[1-(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydro-2naphthyl)ethynyl]benzoic acid (bexarotene) (Emax = 100%, EC50 = 20 nM, Figure 1)7 is approved for the treatment of cutaneous T cell lymphoma (CTCL)8 in the US, EU, and © 2017 American Chemical Society

Figure 1. Chemical structures of RXR-full agonist bexarotene and RXR-partial agonist 1.

Japan. Interestingly, bexarotene has also been reported to show anti-Alzheimer’s disease9 or anti-Parkinson’s disease10 activity in rodent models. However, bexarotene is a RXR-full agonist, i.e., it activates RXR completely, and consequently it may cause adverse effects such as hypothyroidism, weight gain, triglyceride elevation, and hepatomegaly.11−15 Received: June 7, 2017 Published: July 28, 2017 7139

DOI: 10.1021/acs.jmedchem.7b00817 J. Med. Chem. 2017, 60, 7139−7145

Journal of Medicinal Chemistry

Article

Scheme 1a

Reagents and conditions: (a) DIEA, 1,4-dioxane, 200 °C, microwave, 6 h, 64%; (b) H2, Pd/C, EtOAc, rt, 6 h, 98%; (c) NaNO2, conc H2SO4, H2O, THF, rt, 1 h, 99%; (d) (1) n-BuLi/n-hexane, Et2O, −78 °C, (2) CO2; (e) (1) i-PrMgCl·LiCl, Mg, LiCl, THF, rt, 45 min, (2) CO2; (f) B2Pin2, PdCl2(dppf)·CH2Cl2, KOAc, 1,4-dioxane, 100 °C, 3 h, 97%; (g) TBAT, CuTC, TMEDA, NMP, CO2, 110 °C, 5 min; (h) (SnMe3)2, Pd(PPh3)4, toluene, 90 °C, 3 h, 70%; (i) (1) n-BuLi/n-hexane, Et2O, −20 °C, (2) CO2.

a

hindrance of the benzotriazole ring, we selected a [11C]carboxy group. This could be obtained using 11CO or 11CO2,19 but because the former method requires several extra steps of 11CO synthesis20 from 11CO2 and hydrolysis after ester synthesis,21,22 we decided to use 11CO2. Carboxylation using 11CO2 can be performed by using lithiated compounds or Grignard reagents derived from bromo or iodo precursors23 or by using boronate esters in the presence of CuTC as a catalyst, as reported for the synthesis of [11C]bexarotene.24 Thus, we first examined these reactions. Synthesis of precursors and the results of cold runs using dry ice as a 12CO2 gas source are shown in Scheme 1. Common intermediate 6 was synthesized by constructing a triazole ring from 5, which was obtained by hydrogenation of 4. Direct lithiation of bromo precursor 6 with n-BuLi failed because of the high reactivity of the π-rich triazole ring (Scheme 1d). Next, to produce Grignard reagent from 6, we attempted to use Mg with iodine and LiCl in THF but without success (Supporting Information, Table 1, entry 1). Thus, we examined i-PrMgCl· LiCl (so-called turbo-Grignard reagent), which has been used for the synthesis of polyfunctionalized organomagnesium reagents including triazo, aldehyde, or carboxyl ester groups (Scheme 1e).25,26 The results of investigation of various conditions such as reactants, temperature, and reaction time in cold runs using dry ice as a 12CO2 gas source are summarized in Supporting Information, Table 1. Single turbo-Grignard reagent and dry ice bubbling as a CO2 gas source failed to produce the target compound (Supporting Information, Table 1, entry 2). Next, we added the turbo-Grignard reagent at rt to a solution of 6 with Mg and LiCl in THF, followed by dry ice

We hypothesized that there is a threshold difference between the therapeutic and adverse effects of RXR activation, and therefore we were interested in RXR-partial agonists, which would activate RXR only moderately, or whose transcriptional efficacy would be limited because we considered that they might retain the desired activities but exhibit reduced side effects. We developed the RXR-partial agonist 1-[(3,5,5,8,8pentamethyl-5,6,7,8-tetrahydronaphthalen-2-yl)amino]benzotriazole-5-carboxylic acid (1; CBt-PMN, Figure 1)16 and found that it did indeed induce glucose tolerance and antiinsulin resistance without the severe side effects of bexarotene. Because we expected that 1 might have similar therapeutic effects to bexarotene in CNS diseases, we next wished to examine the brain uptake of 1. For this purpose, PET imaging is suitable because it can noninvasively detect the biodistribution of suitably labeled compounds with high spatiotemporal resolution.17,18 Therefore, we required a suitable tracer derivative of 1. For this purpose, we designed a 11C-labeled carboxy derivative [11C]1 (Figure 1). Taking the short lifetime of 11C (t1/2 = ca. 20 min) into account, we focused on direct carboxylation of a precursor bearing a π-rich triazole ring, using 11 CO2. In this article, we report the synthesis of [11C]1 via a trialkyltin precursor as well as application of the synthesized tracer to confirm brain distribution of [11C]1 in mice by PETCT imaging.

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RESULTS AND DISCUSSION A C atom could be inserted either at the carboxy group or at the methyl group on the benzene ring. Because of the steric 11

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Figure 2. Real-time changes in regions of interest (ROI) in PET images of brain (closed blue triangle), heart (closed pink circle), liver (closed red circle), kidney (closed green diamond), muscle (closed yellow square). Time course of [11C]1 administered iv was obtained from the mean pixel radioactivity in the ROI of the PET images acquired after administration. Data shown are the average (n = 3) ± SEM. (A) brain, (B) heart, (C) liver, (D) kidney, and (E) muscle.

Grignard intermediate produced by turbo-Grignard reaction appears to have low reactivity with CO2. So, we judged this approach was unsuitable for PET tracer synthesis. Therefore, we next considered a method using boronic acid ester precursor with CuTC as a catalyst, as reported for the synthesis of [11C]bexarotene.24 Boronic acid ester precursor 7 synthesized from 6 was mixed with TBAT, CuTC, and TMEDA in NMP at rt to afford a blue-colored solution. Into this solution was bubbled 12CO2 from dry ice, and the mixture was heated at 110 °C for 5 min to give 1. However, HPLC separation again revealed a byproduct with a retention time very close to that of 1 (Supporting Information, Figure 2a; retention times for 1 and the byproduct were 6.66 and 7.4 min, respectively). A hot run using 11CO2 instead of dry ice afforded [11C]1 in 99.6% radiochemical purity and 0.42% radiochemical yield. However, the isolated product was blue-colored due to contamination with copper ion and the operation was complicated, so we abandoned this approach too.

bubbling, and in this case, 1 was formed (Supporting Information, Table 1, entry 3). The turbo-Grignard reagent with either Mg or LiCl alone proved unsuccessful (Supporting Information, Table 1, entry 4, 5). Therefore, we decided to use THF, Mg, LiCl, and turbo-Grignard reagent as a solvent and reagents, but unfortunately, HPLC separation of the reaction mixture gave a byproduct with a retention time very close to that of 1 (Supporting Information, Figure 1a). Structure determination showed that the byproduct was the debrominated H derivative (Supporting Information, Figure 1b−e). To avoid production of the byproduct, we tried a lower temperature, but the reaction did not proceed below −15 °C even after 2 h (Supporting Information, Table 1, entries 8,9). When the above reaction conditions at rt were examined using 11 CO2, the desired [11C]1 was not detected at all (Supporting Information, Scheme, part a), possibly because the amount of 11 CO2 was quite small (88.42 GBq, ca. 258 pmol) compared to the case of using dry ice as a CO2 gas source. Because the yield of the byproduct was greater than that of 1 in the cold runs, the 7141



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Figure 3. Autoradiography of coronal sections of mouse brain after administration of [11C]1. Radiographic images were captured of the whole brain area, especially the internal capsule. The images go from the nose to the occipital region (top left to right, continued from bottom left to right). The presence of [11C]1 can be seen throughout the brain.

brain uptake of [11C]bexarotene in a baboon is high (the SUV ratio of brain (ca. 0.8) to heart (35) at approximately 90 s is about 0.02).24 In contrast, in the case of [11C]1, the %ID ratio of brain (ca. 0.6%) to heart (ca. 4.2%) was approximately 0.14, indicating that the brain distribution of [11C]1 is higher than that of [11C]bexarotene. To elucidate brain accumulation and the sites of accumulation of [11C]1, autoradiography was performed after PET imaging (Figure 3, Supporting Information, Figure 6). Although the mouse brain was too small to identify sites of accumulation region in detail, a broad distribution of [11C]1 was observed. Experiments with larger animals in the future may provide more detailed information. Real-time imaging, which is an advantage of PET imaging, enabled us to look at the in vivo kinetics of this compound. Figure 4 shows static capture images revealing remarkable differences during 0−1, 8−11, 17−20, 45−50 min after iv administration. From 0 to 1 min, immediately after intravenous administration, transition of the tracer from the heart to the liver was observed. After retention in the liver from 8 to 11 min, migration from liver to duodenum was observed, indicating biliary excretion (Supporting Information, Movies 1 and 2). At around 45−50 min post injection, the tracer had not moved to the lower digestive tract and the %ID of the heart was substantially constant, suggesting enterohepatic circulation of [11C]1. High uptake of [11C]1 through the blood−brain barrier was observed, presumably due to the lipophilicity of [11C]1.

On the basis of the idea of using a more lipophilic precursor than [11C]1 in order to make it easier to separate [11C]1 from byproducts, we focused on an alkyl-tin precursor, as reported for the lithiation of diazo compounds by Staubitz et al.27 Alkyltin precursor is lipophilic, and its polarity is quite different from that of [11C]1. Alkyl-tin precursor 8 was synthesized from 6 by coupling reaction with hexamethylditin in the presence of palladium catalyst (Scheme 1h). According to the report by Staubitz et al., we first used MeLi for a cold run. Although the target molecule 1 was obtained (Supporting Information, Table 2, entry 1), the hygroscopicity of MeLi made it unsuitable for microreaction to synthesize PET tracer. Thus, n-BuLi was selected instead of MeLi, and diethyl ether was used instead of THF to improve the solubility of 8 (Supporting Information, Table 2, entry 2). After lithiation of 8 with n-BuLi in diethyl ether at −20 °C, CO2 bubbling using dry ice afforded 1 without major byproduct formation (Supporting Information, Figure 4a). Because the byproduct produced by turbo-Grignard reaction was not formed, the lithio intermediate obtained by this method must be more reactive with CO2 than the Grignard intermediate. Reactions at lower temperature gave 1 in lower yield. MeTHF (used by Staubitz et al.) failed to undergo lithiation. Finally, a hot run using 11CO2 was performed with n-BuLi and diethyl ether as the solvent at −20 °C. After the lithiation of 8 and 11CO2 bubbling for 5 min, acidification with 2 mL of a solvent (MeOH/H2O = 90/10 containing 0.5% formic acid) followed by preparative HPLC (MeOH/H2O = 90/10 containing 0.1% formic acid) afforded [11C]1 in 99.6% radiochemical purity and 0.33% radiochemical yield in ca. 25 min after lithiation (including preparative HPLC and concentration) (Supporting Information, Scheme, part c, Figures 4b,c, and 5). Although the radiochemical yield is quite low, the operations are simple and should be readily adaptable to other π-rich hetero ring compounds, and we concluded that this method was suitable for synthesizing PET tracer. Isolated pure [11C]1 was collected in a 1.5 mL sample tube as an acetone solution and dried with a hair dryer. The residue was dissolved in a mixture of 7% sodium hydrogen carbonate solution (Meylon injection7%)/ethanol/saline = 3/1/6 at ca. 10 MBq/mouse. After administration via a tail vain under isoflurane anesthesia, PET scanning was performed for 50 min. The pharmacokinetics of the tracer was evaluated in terms of the percentage of injected dose (%ID) values, i.e., the radioactivity of each organ (Bq) per the radioactivity of the whole body (Bq). Comparing %ID of brain, heart, kidney, liver, and thigh muscle, brain uptake was the third largest, after liver and heart (Figure 2). Liang and Vasdev et al. concluded that

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CONCLUSION The RXR-partial agonist 1 is a candidate for treatment of CNS diseases such as dementia or Parkinson’s disease. To evaluate brain uptake of 1, we synthesized [11C]1 as a PET tracer by direct insertion of 11CO2 into a lithio precursor bearing a π-rich heterocyclic ring from alkyltin derivative 8. This method was easier and more convenient than other alternative approaches that we investigated. The major advantages of our new 11Clabeling method using a trialkyltin precursor are that it is easier to separate the desired molecule from others including the precursor and the byproducts, and the reaction can be performed at −20 °C. This is an important advance for PET tracer synthesis because we can use intermediates that would be unstable at the high temperatures required for the Cu-mediated method (ex. 110 °C). In this work, we focused on 11C-carboxyl labeling, but Strueben et al. have shown that the alkyl groups of trialkyltin precursor can be converted to other alkyl groups by using the appropriate alkyl halide,27 and this might also be the case for 11C-alkylation. A PET/CT imaging study of [11C]1 in 7142



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Determination of Production or Purity of [12C]1 by HPLC. The HPLC system used was a Shimadzu liquid chromatographic system (Kyoto, Japan) consisting of a LC-10AD pump, a SPD-10A VP UV− vis spectrophotometric detector, a CTO-10AS VP column oven, and a C-R5A Chromatopac. An Inertsil ODS-3 column (4.6 mm i.d. × 100 mm, 5 μm, GL Sciences, Tokyo, Japan) was used at a temperature of 40 °C; the mobile phase for [12C]1 was MeOH/H2O/HCO2H = 90/ 10/0.1% or MeOH/H2O/HCO2H = 85/15/0.1%. The flow rate was 0.7 mL/min. Identification of the products was confirmed by monitoring absorbance at 260 nm with a PDA detector. HPLC Conditions for Isolating PET Tracer. The HPLC system was a CFN-MPS-100 system equipped with an RI detector, consisting of a PU-2086 Plus pump and UV-2075 Plus UV−vis spectrophotometric detector (Sumitomo Heavy Industry, Osaka, Japan). Chromatographic isolation was carried out on a YMC Pack ODS-AM, (4.6 mm i.d. × 250 mm, 5 μm, YMC Co., Ltd., Kyoto, Japan) with a guard column of Inertsil ODS-3 (4.6 mm i.d. × 10 mm, 5 μm, GL Sciences), using methanol:H2O + 0.1% formic acid (90:10 v/v) as the mobile phase. The flow rate was 4.0 mL/min, and the absorbance at 260 nm was monitored. HPLC Conditions for Analyzing PET Tracer. The HPLC system was a Shimadzu liquid chromatographic system (Kyoto, Japan) consisting of a LC-10AD pump, a SPD-10AV UV−vis spectrophotometric detector, a CTO-10AS column oven, and a C-R5A Chromatopac. The chromatographic analyses were carried out on an Inertsil ODS-3 column (4.6 mm i.d. × 100 mm, 5 μm, GL Sciences, Tokyo, Japan) with a guard column of Inertsil ODS-3 (4.6 mm i.d. × 10 mm, 5 μm, GL Sciences) kept at 40 °C, using methanol:H2O + 0.1% formic acid (85:15 v/v) as the mobile phase. The flow rate was 0.7 mL/min, and the absorbance at 260 nm was monitored. N-(4-Bromo-2-nitrophenyl)-3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydronaphthalen-2-amine (4). Compound 216 (2.7 g, 12.4 mmol) and DIEA (2.39 mL, 13.7 mmol) were added to a solution of 3 (1.7 mL, 13.7 mmol, purchased from TCI) in 1,4-dioxane (12 mL). The reaction mixture was stirred at 200 °C under microwave irradiation for 6 h, then poured into H2O (80 mL) and extracted with EtOAc (3 × 80 mL). The organic layer was collected, washed with H2O (2 × 80 mL) and brine (80 mL), and dried over MgSO4. The solvent was evaporated under reduced pressure. The residue was purified by flash column chromatography (EtOAc/n-hexane = 1/20) to yield 4 (640 mg, 64%) as a red oil. 1H NMR (300 MHz, CDCl3) δ: 9.31 (s, 1H), 8.36 (d, J = 2.4 Hz, 1H), 7.38 (dd, J = 9.0, 2.4 Hz, 1H), 7.22 (s, 1H), 7.14 (s, 1H), 2.18 (s, 3H), 1.70 (s, 4H), 1.30 (s, 6H), 1.25 (s, 6H). FAB-MS m/z: 416, 418 [M]+. HRMS (FAB+) m/z: [M]+ calcd for C21H25 N2O279Br 416.1099, found 416.1058; calcd for C21H25N2O 81 Br 418.1079, found 418.1025. 4-Bromo-N1-(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydronaphthalen-2-yl) benzene-1,2-diamine (5). Tin chloride(II) (1.45 g, 7.67 mmol) and concentrated hydrochloric acid (300 μL) were added to a solution of 4 (640 mg, 1.53 mmol) in EtOH (15 mL). The reaction mixture was stirred at 80 °C for 4 h, then neutralized with 5 N NaOH and filtered through Celite. The Celite cake was washed with EtOAc. The combined filtrate and washing were evaporated under reduced pressure to afford a purple solid (670 mg, 99%), which showed a single spot (Rf = 0.4−0.5) on TLC (EtOAc/n-hexane = 1/10) and gave a yellow coloration with ninhydrin. This orange solid was directly used for the next reaction. FAB-MS m/z: 386, 388 [M]+. 5-Bromo-1-(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydronaphthalen-2-yl)-1H-benzo[d][1,2,3]triazole (6). Sodium nitrite (125 mg, 1.80 mmol) in H2O (2 mL, cooled on ice to under 5 °C) and 8% diluted sulfuric acid (6.5 mL, concentrated sulfuric acid, 500 μL, in H2O, 6 mL) were added to a solution of 5 (500 mg, 1.29 mmol) in THF (10 mL) with cooling on ice. The reaction mixture was stirred at rt for 1 h and then neutralized with 5 N NaOH (5 mL). The mixture was poured into H2O (80 mL) and extracted with EtOAc (3 × 80 mL). The organic layer was collected, washed with H2O (2 × 80 mL) and brine (80 mL), and dried over MgSO4. The solvent was evaporated under reduced pressure. The residue was purified by flash column chromatography (EtOAc/n-hexane = 1/40) to yield 6 (465 mg, 90%) as a beige gum. 1H NMR (300 MHz, CDCl3) δ: 8.27 (s, 1H), 7.54

Figure 4. Decay-corrected whole-body coronal PET/CT images of mice from static scans at 0−1, 8−11, 17−20, and 45−50 min after iv administration of [11C]1. To show time-dependent changes in target organs, the images go from dorsal to ventral side (top to bottom) at each time point. The tracer first migrates to the liver and then gradually transfers to the gallbladder and duodenum. H, G, L, and D indicate heart, gallbladder, liver, and duodenum, respectively.

mice indicated that 1 is distributed to the brain. Thus, 1 may be suitable for further development studies for treatment of CNS diseases.

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EXPERIMENTAL SECTION

Chemistry and Radiochemistry. General. 1H NMR (300 MHz) and 13C NMR (75 MHz) spectra were recorded on a Varian Mercury300 spectrometer. FAB-MS spectra (low and high resolution mass spectra) were measured on a JEOL JMS-700 mass spectrometer. 11CCarbon dioxide without added carrier was obtained from Cypris HM12S, Sumitomo Heavy Industries, Ltd. Reverse-phase extraction SepPak Accell Plus QMA cartridges (Waters) were obtained from Waters and were pretreated with ethanol and water prior to use. Syringe filters and Millex GS filter (pore size, 0.22 μm) were from Millipore Corporation. The purity of all tested compounds was >95%, as confirmed by HPLC. 7143



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(dd, J = 8.7, 1.8 Hz, 1H), 7.30 (s, 1H), 7.25 (d, J = 8.7 Hz, 1H), 7.22 (dd, J = 8.7, 1.8 Hz, 1H), 2.17 (s, 3H), 1.74 (s, 4H), 1.35 (s, 6H), 1.28 (s, 6H). 13C NMR (75 MHz, CDCl3) δ: 129.88, 129.32, 126.88, 115.54, 114.96, 114.01, 113.78, 112.35, 107.22, 105.16, 99.85, 94.26, 17.46, 17.34, 16.90, 16.82, 14.42, 14.38. FAB-MS m/z: 398, 400 [M + H]+. HRMS (FAB+) m/z: [M + H]+ calcd for C21H25N379Br 398.1232, found 398.1264; calcd for C21H25N381Br 400.1211, found 400.1207. 1-(3,5,5,8,8-Pentamethyl-5,6,7,8-tetrahydronaphthalen-2-yl)-5(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-benzo[d][1,2,3]triazole (7). [1,1′-Bis(diphenylphosphino)ferrocene]palladium(II) dichloride dichloromethane adduct (41 mg, 0.05 mmol), bis(pinacolato)diboron (381 mg, 1.5 mmol), and potassium acetate (147 mg, 1.5 mmol) were added to a solution of 6 (200 mg, 0.5 mmol) in 1,4-dioxane (8 mL). The reaction mixture was stirred at 100 °C under Ar for 3 h, then filtered through Celite, poured into H2O (50 mL), and extracted with EtOAc (3 × 50 mL). The organic layer was collected, washed with H2O (2 × 50 mL) and brine (50 mL), and dried over MgSO4. The solvent was evaporated under reduced pressure. The residue was purified by flash column chromatography (EtOAc/n-hexane = 1/20) to yield 7 (310 mg, 97%) as a white solid. 1 H NMR (300 MHz, CDCl3) δ: 8.64 (s, 1H), 7.90 (dd, J = 8.4, 1.8 Hz, 1H), 7.36 (d, J = 8.4, Hz, 1H), 7.33 (s, 1H), 7.28 (s, 1H), 2.05 (s, 3H), 1.74 (s, 4H), 1.39 (s, 6H), 1.35 (s, 6H), 1.27 (s, 12H). FAB-MS m/z: 446 [M + H]+. HRMS (FAB+) m/z: [M + H]+ calcd for C27H37N3O211B 446.2979, found 446.2930. 1-(3,5,5,8,8-Pentamethyl-5,6,7,8-tetrahydronaphthalen-2-yl)-5(trimethylstannyl)-1H-benzo[d][1,2,3]triazole (8). Hexamethylditin (7.5 mL, 0.036 mmol) and tetrakis(triphenylphosphine)palladium(0) (150 mg, 0.13 mmol) were added to a solution of 6 (300 mg, 0.63 mmol) in toluene (36 mL). The reaction mixture was stirred under reflux and held in an Ar atmosphere for 0.5 h, then filtered through Celite. The solvent was evaporated under reduced pressure, and the residue was purified by flash column chromatography (EtOAc/nhexane = 1/40) to yield 8 (210 mg, 71%) as a white solid. 1H NMR (300 MHz, CDCl3) δ: 8.28 (s, 1H), 7.58 (d, J = 8.0 Hz, 1H), 7.38 (dd, J = 8.0, 1.0 Hz, 1H), 7.34 (s, 1H), 7.27 (s, 1H), 2.09 (s, 3H), 1.74 (s, 4H), 1.36 (s, 6H), 1.27 (s, 6H), 0.38 (s, 9H). 13C NMR (75 MHz, CDCl3) δ: 146.78, 145.79, 144.04, 137.41, 134.18, 134.05, 132.85, 131.47, 129.61, 127.40, 124.67, 109.94, 34.936, 34.80, 34.26, 34.20, 31.82, 17.52, −9.300. FAB-MS m/z: 484 [M + H]+. HRMS (FAB+) m/z: [M + H]+ calcd for C24H34N3116Sn 480.1770, found 480.1808; calcd for C24H34N3118Sn 482.1769, found 482.1826; calcd for C24H34N3120Sn 484.1775, found 484.1775. 1-(3,5,5,8,8-Pentamethyl-5,6,7,8-tetrahydronaphthalen-2-yl)-1Hbenzo[d][1,2,3]triazole-5-carboxylic-11C acid ([11C]1, Supporting Information, Scheme, part b). Tetrabutylammonium triphenyldifluorosilane (4.7 mg, 8.8 μmol) and copper(I) 2-thiophenecarboxylate (1.7 mg, 8.8 μmol) were added to a solution of 7 (23.4 mg, 52.6 μmol) in N-methyl-2-pyrrolidone (400 μL). Then N,N,N′,N′-tetramethylethylenediamine (105 μL, 701 μmol) was added with shaking. The color changed to brilliant blue on contact with air. This solution was cooled to −20 °C and directly added to the reaction vessel. 11CO2 (approximately 23 GBq) was bubbled through the reaction mixture. After the reaction, a mixture of MeOH/H2O/formic acid (90/10/0.1% 1.5 mL) was added. The residue was purified by preparative HPLC (MeOH/H2O/formic acid = 90/10/0.1% 1.5 mL), and the solvent was evaporated under reduced pressure to yield [11C]1 (41.4 MBq). Radiochemical purity was 99.6%, and radiochemical yield was 0.42%. The product showed the same retention time as [12C]1 (MeOH/ H2O/formic acid = 85/15/0.1%). The solvent was removed from the collected fractions by rotary evaporation, and the radiolabeled product was reconstituted in EtOH (80 μL) and saline (100 μL) for in vivo experiments. 1-(3,5,5,8,8-Pentamethyl-5,6,7,8-tetrahydronaphthalen-2-yl)-1Hbenzo[d][1,2,3]triazole-5-carboxylic-11C acid ([11C]1, Supporting Information, Scheme, part c). A solution of 1.6 M n-BuLi in nhexane (16 μL, 25 μmol) was added directly to a reaction vessel, and the solvent was removed at −20 °C under a stream of nitrogen gas. A solution of 8 (12 mg, 25 μmol) in Et2O (500 μL) at rt was added to the reaction vessel, and 11CO2 (approximately 88.42 GBq) was

bubbled through the reaction mixture. After the reaction, MeOH/ H2O/formic acid (90/10/0.5% 1.5 mL) was added to reaction mixture. The residue was purified by preparative HPLC (MeOH/ H2O/formic acid = 90/10/0.1%), and the solvent was evaporated under reduced pressure to yield [11C]1 (123.1 MBq). Radiochemical purity was 99.6%, and radiochemical yield was 0.33%. The product [11C]1, which showed the same retention time as [12C]1, was isolated by analytical HPLC (MeOH/H2O/formic acid = 85/15/0.1%). The solvent was removed from the collected fractions by rotary evaporation, and the residue was collected in a 1.5 mL sample tube as an acetone solution and dried using a hair dryer. The radiolabeled product was reconstituted in a mixture of 7% sodium hydrogen carbonate solution (Meylon injection7%)/ethanol/saline (3/1/6) for in vivo experiments. In Vivo Studies. Small Animal PET and CT Imaging. Animal experiments were performed in accordance with institutional guidelines and a protocol approved by the Animal Research Committee of Okayama University. Four 6-week-old male ICR mice (25−30 g) were used. All mice were deprived of food for at least 2 h before PET scans but were allowed free access to water. For intravenous injection studies, a 29 gauge needle connected to a PE 10 catheter was inserted into a lateral tail vein for administration. All PET scans were performed using a ClairvivoPET (Shimadzu Co. Ltd., Kyoto, Japan) designed for laboratory animals. The animals were anesthetized by inhalation of 1.5%−2% isoflurane in room air, and their body temperature was maintained at 36 °C with a heating pad. Tracer (8− 12 MBq, 0.1−0.15 mL) was injected intravenously. A dynamic emission scan was acquired for 30 min in the list mode with an energy window of 400−650 keV, and 12 frames were collected in the following manner: 5 × 60 s, 5 × 180 s, and 1 × 300 s. Images were reconstructed using FORE-FBP. Regions of interest (ROIs) were drawn over the brain and heart in axial images on the basis of the corresponding CT image using PMOD 3.0 software (PMOD Technologies Inc.). Decay-corrected radioactivity was expressed as a percentage of the injected dose per milliliter of tissue (%ID/mL) or a percentage of the injected dose per tissue (%ID), and time-course data were plotted.

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.7b00817. NMR charts, HRMS data, 11CO2 hot run schemes, HPLC charts, LC-MS data, light or autoradiographic images of mouse brain coronal section, tables of optimization of reaction conditions (PDF) Molecular formula strings (CSV) PET movie 1 (AVI) PET movie 2 (AVI)

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AUTHOR INFORMATION

Corresponding Author

*Phone: +81-(0)86-251-7963. Fax: +81-(0)86-251-7926. Email: [email protected]. ORCID

Hiroki Kakuta: 0000-0002-3633-8121 Author Contributions

H.K. conceived and designed the project. O.S. synthesized compounds. O.S., M.W., S.Y., M.A., H.H., and H.K. synthesized PET tracer. O.S., H.N., and H.K. performed LRMS and HRMS. O.S., T.S., A.A., T.H., and T.H. performed PET scanning and ARG. O.S., M.W., and S.N. performed LC-MS. The manuscript was written by O.S., H.N., Y.T., and H.K. 7144



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Notes

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The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We are grateful to the Division of Instrumental Analysis, Okayama University, for the NMR and MS measurements. This work was partially supported by a subsidy to promote science and technology in prefectures where nuclear and other power plants are located (to H.K.) from the Ministry of Education, Culture, Sports, Science, and Technology (MEXT) of Japan, a Grant-in-Aid for the COE projects by MEXT, Japan, titled “Center of excellence for molecular and gene targeting therapies with microdose molecular imaging modalities” (to H.K.), and Takeda Science Foundation (to H.K.).

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ABBREVIATIONS USED CMC, carboxymethyl cellulose; CuTC, copper(I) 2-thiophenecarboxylate; EC50, half-maximal (50%) effective concentration; Emax, maximum efficacy; PET, positron emission tomography; ROI, region(s) of interest; RXR, retinoid X receptor; SEM, standard error of the mean; SUV, standard uptake values; TBAT, tetrabutylammonium triphenyldifluorosilane; TMEDA, N,N,N′,N′-tetramethylethylenediamine; NMP, N-methylpyrrolidone; %ID, the percentage of injected dose

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