Authors: Yanbo Yu (aDepartment of Radiology, Washington University School of Medicine, St Louis, MO 63110, USA), Sandip B. Jadhav (aDepartment of Radiology, Washington University School of Medicine, St Louis, MO 63110, USA), Zhimin Xing (aDepartment of Radiology, Washington University School of Medicine, St Louis, MO 63110, USA), Hao Jiang (aDepartment of Radiology, Washington University School of Medicine, St Louis, MO 63110, USA), Lin Qiu (aDepartment of Radiology, Washington University School of Medicine, St Louis, MO 63110, USA), Tianyu Huang (aDepartment of Radiology, Washington University School of Medicine, St Louis, MO 63110, USA), Joel S. Perlmutter (aDepartment of Radiology, Washington University School of Medicine, St Louis, MO 63110, USA; bDepartment of Neurology and Neuroscience, Washington University School of Medicine, St Louis, MO 63110, USA), Zibo Li (cDepartment of Radiology, University of North Carolina at Chapel Hill, ChapelHill, NC 27599, USA), Zhude Tu (aDepartment of Radiology, Washington University School of Medicine, St Louis, MO 63110, USA)
Categories: Article, TRPC5, Fluorine -18, Radiolabeling, PET tracer
Source: Journal of fluorine chemistry
Authors: Yanbo Yu, Sandip B. Jadhav, Zhimin Xing, Hao Jiang, Lin Qiu, Tianyu Huang, Joel S. Perlmutter, Zibo Li, Zhude Tu
TRPC5 is a member of the mammalian transient receptor potential (TRP) channel superfamily and it has been implicated in various physiological and pathological mechanisms of neurological and psychiatric diseases. Fluorine-18 is one of the most widely used radionuclides for PET imaging due to its favorable chemical characteristics and nuclear-physical properties. Herein, we describe two complementary radiosynthetic approaches and preliminary in vivo evaluation for [^18^F]TZ78141 as a novel and promising fluorine-18 labeled radiotracer for imaging TRPC5. The latter strategy employed a ruthenium-mediated radiofluorination method, facilitating the rapid synthesis of the desired radiotracer with substantial advantages in simplicity and efficiency.
Transient Receptor Potential Canonical 5 (TRPC5) belongs to the TRPC subfamily that include seven members named TRPC1 through TRPC7. It is an important ion channel that influences calcium signaling, neuronal function, sensory perception, cardiovascular regulation, and disease pathophysiology [1,2]. TRPC5 initially was identified in the central nervous system (CNS) and has been implicated in a broad spectrum of physiological and pathological mechanisms underlying neurological and psychiatric diseases [3]. For instance, recent clinical studies have shown that the loss of TRPC5 can lead to various behavioral and neurological issues, including social and generalized anxiety, autism-related traits, and postpartum depression, emphasizing its critical role in maintaining normal brain function and emotional regulation [4]. Pharmaceutical companies have transferred several small molecules targeting TRPC5 into clinical trials, none of these drugs is commercially available yet [5]. Positron emission tomography (PET) is a noninvasive imaging technique that with suitable radiotracers can visualize and quantify various biological processes in vivo, including changes in protein expression [6]. Therefore, a radiotracer, binding specifically to the TRPC5, may permit direct quantification of TRPC5 in the living brain and open new options for diagnosis and monitoring therapeutic effectiveness targeting TRPC5 protein.
In our pioneer efforts, we have reported structurally distinct PET tracers designed for TRPC5 imaging labeled with carbon-11, named [^11^C]HC608 (IC50 = 6.2 nM) and [^11^C]HC070 (IC50 = 9.3 nM), respectively (Fig. 1) [7,8]. Compared to the half-life of carbon-11 (20 min), the longer half-life of fluorine-18 (110 min) allows for extended distribution times and enables the transport of radiotracers to PET centers within a 3- to 4 h driving distance from cyclotron facilities, thereby increasing cost effectiveness and the implementation of more extended imaging sessions. Besides, ^18^F is superior to most PET radionuclides due to its low maximum positron energy of 0.634 MeV, which minimizes positron tissue penetration and improves image resolution [9,10]. The xanthine-based analogue, TZ78141, was reported by Hydra Bio-sciences/Boehringer Ingelheim in 2020. In vitro calcium flux assay and whole-cell manual patched clamp assay showed this compound is a potent inhibitor of human TRPC5 with an IC50 value of 0.477 nM [11]. Moreover, from a chemical structure perspective, the inclusion of a fluorine atom in the aromatic ring enables efficient radiolabeling of the molecule with fluorine-18. We herein report our efforts on implementing two different F-18 radiosynthetic approaches to the TRPC5 radiotracer [^18^F]TZ78141 and its preliminary evaluation in nonhuman primate (Fig. 2).
Given that TZ78141 contains a fluorobenzyl moiety, we initially planned to adopt a risk-averse and indirect strategy for fluorine-18 labeling, employing [^18^F]fluorobenzyl halide as a labeling synthon to alkylate the amine substrate. Simultaneously, we explored the deoxyfluorination reaction starting from the phenol precursor.
The synthesis route of reference compound TZ78141, along with the amine precursor 11 and phenol precursor 12, is presented in Scheme 1. Briefly, 7-benzylxanthine 3 was obtained in two steps from nucleoside guanosine 1 according to the general method with 7-benzylguanosine 2 as an intermediate. Selective isopropylation of the more acidic NH group (N3H) in compound 3 to prepare 4 was achieved using sodium hydride for deprotonation, though some disubstituted byproducts were observed. The tetrahydropyranyl (THP) ether could be introduced to the xanthine ring to furnish 5 in 82% yield. Protecting group switch from benzyl to (trimethylsilyl)ethoxymethyl (SEM) for synthesis of 7 was realized before debenzylation over palladium metal catalysts with molecular hydrogen and subsequently treated with 2-(trimethylsilyl) ethoxymethyl chloride (SEM-Cl). The cross coupling of 7 with 3-bromo-6-cyclobutoxy-2-methylpyridine (8) under Pd/Cu catalysis gave the desired biaryl product 9 in 52% yield, which was subjected to consecutive deprotection of SEM and THP to provide free amine 11. Finally, both the reference compound TZ78141 and its phenol precursor 12 were afforded in good yields by alkylation of 11 in the presence of K2CO3.
Considering that [^18^F]TZ78141 bearing a fluorobenzyl moiety, we first describe a strategy toward the ^18^F-radiolabeling of this complex molecule employing [^18^F]BnBr as a key building block to facilitate the final alkylation process of substrate 11 (Fig. 3). The initial step for this reaction sequence was the preparation of 4-[^18^F]fluorobenzaldehyde (14) by nucleophilic aromatic substitution reaction. According to previous reports, the radiosynthesis of 4-[^18^F]fluorobenzaldehyde can be achieved from either ammonium or nitro precursors, yielding similar radiochemical labeling outcomes [12]. However, the shorter reaction time of the ammonium salt 13 (3 min) compared to the corresponding nitro compound (15 min) offers a substantial advantage to achieve high radioactivity and good yield at the end of the multiple step synthesis. More importantly, unlike the nitro precursor, ammonium salt 13 offers the additional advantage of being water-soluble. This solubility simplifies the purification of the ^18^F-fluorinated alcohol 15 during solid phase extraction (SPE) trapping, enabling easy removal of excess starting material. Therefore, the radiofluorination of trimethylammonium salt 13 was conducted in dimethyl sulfoxide with [^18^F]fluoride in the presence of Kryptofix 222 and potassium carbonate at 140 °C. The labeling efficiency was checked using radio-TLC and radio-HPLC, resulting in a radiochemical yield of approximately 90%. The radioactive [^18^F]fluorobenzaldehyde 14 obtained was near quantitatively reduced to the corresponding alcohol [^18^F]15 by the addition of an aqueous solution of NaBH4. The crude ^18^F-fluorinated alcohol solution was diluted in 1% Na-ascorbate solution (w/v, 50 mL) and adsorbed onto an Oasis PRiME HLB cartridge (335 mg Sorbent, Part No. 186,008,887, Waters). Notably, the ascorbate addition acts here as an antioxidant or free radical scavenger and it is crucial to prevent radiolysis for large-scale production [13].
Halogenation of benzylic alcohols, particularly bromination, can be effectively achieved through multiple methods for radiosynthesis applications. For example, phosphorus tribromide [14] and triphenylphosphine dibromide [15] were reported as good reagents for the radiosynthesis of alkyl halides from alcohols. However, the halogenation reagents may decompose with moisture thus solvent change is required to maintain the strict dry medium which is crucial for successful reaction implementation. Hydrobromic acid either in an organic medium (i.e., HBr in ether) [16] or in gaseous form [17] was also utilized previously to facilitate radiohalogenation conversion despite this corrosive gas (HBr) is arduous to handle and store. Alternatively, the reported on-column reaction with HBr solution appears attractive because it eliminates the need for solvent replacement during the process from solid-phase extraction (SPE) to bromination. Indeed, after a concentrated aqueous solution of hydrobromic acid (48%) was passed through the solid support, both the alcohol and HBr are trapped on the cartridge. Replacing the PRiME HLB cartridge with SPE Sep-Pak cartridges (C18, tC18) may cause substantial radioactivity loss after hydrobromic acid elution. Notably, heating the wetted support for 10 min at 70 °C yielded [^18^F]FBnBr ([^18^F]16) in a much higher radio conversion rate (70%) compared with the room temperature reaction (<10%). Before subsequent elution, the residual HBr on the Oasis Sep-Pak was eluted by 10 mL of deionized water. The [^18^F]fluorobenzyl bromide intermediate (16) trapped on the column could be eluted using pentane and transferred into a vial containing the precursor 11, K2CO3~ and DMF by passing through two stacked sodium sulfate Sep-Pak cartridges. The mixture solution was evaporated at RT to remove pentane before heating at 100 °C for 15 min. During this step, sufficient dryness is essential to prevent the potential hydrolysis of [^18^F]fluorobenzyl bromide to [^18^F] fluorobenzyl alcohol, which will suppress the subsequent alkylation reaction. Finally, the solution was cooled, quenched, and injected into the HPLC system to purify and formulate the radioactive product in 10% ethanol in saline. Within this approach, [^18^F]TZ78141 was achieved in approximate 200 min with a radiochemical yield of 5.7 ± 1.4% (decay corrected to the end of bombardment based on starting H[^18^F]F) and molar activity 159 ± 11 GBq/μmole (decay corrected to end of synthesis, n =3). The radiochemical purity was >99% and the chemical purity was >90%.
Although the above described indirect method to implement the F-18 radiosynthesis is reliable, yet its complexity makes production challenging and increases the likelihood of radiosynthesis failure. To overcome this shortcoming, developing radiosynthetic schemes that introduce the radionuclide in a late phase of the synthetic pathway (late-stage ^18^F-labeling) reduces the radiosynthesis time and avoids multiple purification steps. In 2017, Ritter and co-workers reported the first ^18^F-deoxyfluorination of phenols via an in situ prepared η6-coordinated ruthenium-phenol complex [18]. This highly effective approach was utilized for F-18 radiochemistry to make a diverse range of non-activated functionalized [^18^F]fluoro(hetero)arenes. This reaction offers several advantages over conventional nucleophilic ^18^F-fluorination strategies, including superior chemoselectivity, tolerance to moisture and air, and compatibility with a wide range of functional groups. As a result, this method may enable the synthesis of radioligands that were previously inaccessible using traditional techniques. More importantly, this methodology has been successfully applied to the cGMP compliant production of [^18^F]EKZ-001 and its clinical validation to quantify HDAC6 expression in the human brain [19,20]. Inspired by this, we decided to explore the strategy for the synthesis of [^18^F]TZ78141 starting with the corresponding phenol precursor.
Preparation of the required labeling precursor 12 for this protocol is facile, which could be achieved through benzylation reaction of amine precursor 11 and 4-(bromomethyl)phenol in a single step (Scheme 1). Fortunately, the heterocyclic systems and unprotected aliphatic alcohol that substrate 12 possessed were compatible with this ^18^F-fluorination process (Fig. 4). Azeotropic drying of [^18^F]fluoride and addition of potassium carbonate was found beneficial to improve the RCY compared with the direct elution of [^18^F]fluoride without drying during ^18^F-radiolabeling (15.8 vs. 10%) [21]. One key optimized synthesis feature is the decreased amount of required precursor from 8.7 μmol (described by Ritter et al. [18]) to 4.8 μmol, which produced much cleaner reaction profile of the radiolabeling reaction mixtures. The distinct polarity difference between the precursor and the aryl fluoride product, combined with the lower precursor loading, facilitates straightforward and reliable purification of the desired product (Fig. 5). In particular, the optimized reaction conditions (30 min heating at 160 °C) are harsher than those developed by Ritter et al [18]. Nevertheless, the high radiochemical yield of these conditions present meaningful advantages. Finally, [^18^F]TZ78141 was produced with 15.8 ± 2.7% radiochemical yield (decay corrected to the end of bombardment based on starting H[^18^F]F) and 140 ± 8 GBq/μmol molar activity within 120 min (decay-corrected to end of synthesis, n = 3). For all batches, radiochemical purity was over 99% and the chemical purity was over 90%.
Within the radiosynthesis success of [^18^F]TZ78141, PET brain imaging study of [^18^F]TZ78141 was conducted in male cynomolgus macaques to determine if [^18^F]TZ78141 can cross the blood brain barrier and have sufficient brain uptake in nonhuman primate. As shown in Fig.6, [^18^F]TZ78141 rapidly enters into the brain with the standardized uptake value (SUV) peaking at approximately 2.7 between 3- and 5 min post-injection. Rapid brain washout indicates reversible pharmacokinetics in the macaque brain.
Meanwhile, [^18^F]TZ78141 demonstrated the highest brain uptake, surpassing both [^11^C]HC070, that has a peak standardized uptake value of approximately 2.2 [8], and [^11^C]HC608, with a value around 0.9 [7]. [^18^F]TZ78141 brain uptake remains higher than the carbon-11 radiotracers from 5 to 120 min post-injection. The enhanced and sustained brain uptake profile suggests that [^18^F]TZ78141 has potential to be a more suitable radiotracer for mapping the TRPC5 expression in the brain.
These above results will facilitate future studies in evaluating [^18^F]TZ78141 as a promising radiotracer for quantitatively assessing TRPC5 expression in vivo. Further in vitro and in vivo biological characterizations of [^18^F]TZ78141 are currently underway in our project, and additional progress will be reported when it is ready.
The radiosynthesis of the first fluorine-18 labeled radiotracer [^18^F]TZ78141 that targets TRPC5 was completed using two distinct methods. The first, an indirect approach, involved [^18^F]FBnBr as the labeling synthon, where fluorine-18 was introduced via a prosthetic group and then conjugated to the amine precursor. The second, a direct late-stage method, employed an innovative ruthenium-mediated radiofluorination chemistry. This appealing approach converted the easily accessible phenol precursor into an η6 π-coordinated ruthenium–phenol complex in situ, facilitating the direct incorporation of fluorine-18. The latter strategy provides significant advantages in both simplicity and efficiency, allowing for the rapid preparation of the desired radiotracer with high radiochemical yield, as well as excellent chemical and radiochemical purities and molar activity. PET studies in the non-human primate brain revealed that [^18^F]TZ78141 exhibited the highest brain uptake (SUV) among the three radiotracers and had favorable brain washout kinetics.
All reagents and solvents used in the syntheses were of ACS or HPLC grade and were obtained from Sigma-Aldrich or other commercial suppliers without further purification. Thin-layer chromatography (TLC) was performed using 0.25 mm silica gel 60 F254 plates (EMD Chemicals Inc., Billerica, MA), with visualization achieved under UV light (254 nm) or by staining with potassium permanganate. Flash column chromatography was conducted using 230–400 mesh silica gel (SiliCycle Inc., Quebec, Canada) as the stationary phase.
Nuclear magnetic resonance (NMR) spectra were recorded on a Varian 400 MHz spectrometer, with CDCl3 as the solvent and tetramethylsilane (TMS) as the internal standard. Chemical shifts (δ) are reported in parts per million (ppm), and coupling constants (J) are expressed in hertz (Hz). The following abbreviations were used to describe NMR s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), and br. (broad). High-resolution mass spectra (HRMS, m/z) were obtained using a Bruker MaXis 4G Q-TOF mass spectrometer equipped with an electrospray ionization (ESI) source.
For radiochemistry, no-carrier-added aqueous [^18^F]fluoride was produced via the ^18^O(p,n)^18^F reaction by proton irradiation of 95% enriched ^18^O water using an RDS111 cyclotron (Siemens/CTI Molecular Imaging, Knoxville, TN). The radiochemical yield (RCY) refers to the percentage of the radioactive fraction that has been incorporated in comparison to the starting amount of activity are estimated from an aliquot of the reaction mixtures by radio-HPLC and radio-TLC. RCY may be non-decay corrected or decay corrected. For method A and method B, the radiochemical purities for final radioactive product were both greater than 95%, as confirmed by HPLC analysis using a UV detector and a gamma detector.
Molar activity (Am) was determined by the ratio between the amount of radioactivity and the sum of the quantity of the isotopically stable compound after a calibration curve acquired with an authentic standard.
Note: radioactivity chromatographs are offset by 0.2–0.3 min on account of the delay introduced by the spatial separation between the diode array detector and the radioactivity detector.
All animal experiments were conducted in accordance with protocols approved by Washington University’s Institutional Animal Care and Use Committee (IACUC) and were compliant with the US National Research Council’s Guide for the Care and Use of Laboratory Animals.
Compound 3 was prepared based on a reported procedure with modification [22]. To a mixture of guanosine (2.83 g, 10 mmol, 1.0 equiv) in DMSO (50 mL) was added benzyl bromide (2.05 g, 12 mmol, 1.2 equiv). The solution was stirred at 50 °C for 4 h before cooled to room temperature, then hydrochloric acid (10 mL, 2 mol/ mL) was added and the mixture was stirred at 70 °C for 2 h. The reaction was cooled to room temperature. The obtained precipitate was filtered, washed with water and ethanol, and dried under vacuum to give 7-benzylguanine (2) as a grey solid. 2 was redissolved in hot acetic acid (60 mL) and water (7 mL). After cooling to 50 °C, a solution of sodium nitrite (2.5 g, 36 mmol, 3.6 equiv) in water (4 mL) was added slowly with vigorous stirring. The solution was allowed to stir at 50 °C for 1 h and at room temperature for another 2 h. The solid was collected by filtration, washed with water, and dried in vacuo and crystallized from methanol to yield 1.45 g (60% yield for 2 steps) of pure 7-benzylxanthine (3) as a white solid, which was identical with previously reported literature. M. P.: 297–298 °C. ^1^H NMR (400 MHz, DMSO-d6) δ 11.62 (s, 1H), 10.91 (s, 1H), 8.14 (s, 1H), 7.42–7.17 (m, 5H), 5.41 (s, 2H). ^13^C NMR (100 Hz, DMSO-d6) δ 155.6, 151.3, 149.6, 142.7, 137.2, 128.7, 128.0, 127.6, 106.1, 48.9. HRMS (m/z): [M+H]^+^ calcd for C12H11N4O2 243.0882, found 243.0876.
To a stirred solution of 3 (1.2 g, 5 mmol, 1.0 equiv) in 10 mL DMF was added sodium hydride (60% dispersion in mineral oil, 600 mg, 15 mmol, 3.0 equiv), and the reaction mixture was stirred for 30 min at 0 °C. Then 2-iodopropane (4.25 g, 25 mmol, 5 equiv) was added. The reaction mixture was then allowed to stir for 4 h at 50 °C before cooling to room temperature and quenched by saturated aqueous NH4Cl solution. The aqueous and organic layers were separated, the aqueous layer was extracted by ethyl acetate. The combined organic layers were washed with brine and dried over Na2SO4 before concentrated, and purified by chromatography to afford the pure product as white solid (497 mg, ethyl acetate=2:1 to 1, 35% yield). M.P.: 205–206 °C. ^1^H NMR (400 MHz, CDCl3) δ 8.32 (s, 1H), 7.54 (s, 1H), 7.37–7.33 (m, 5H), 5.46 (s, 2H), 5.12 (sep, J = 6.9 Hz, 1H), 1.57 (d, J = 6.9 Hz, 6H). ^13^C NMR (100 Hz, CDCl3) δ 154.7, 150.4, 150.3, 140.6, 135.0, 129.1, 128.7, 128.3, 107.8, 50.3, 47.8, 19.7. HRMS (m/z): [M+H]^+^ calcd for C15H17N4O2 285.1352, found 285.1345.
A round-bottom flask was charged with 4 (426 mg, 1.5 mmol, 1.0 equiv), 2-(3-bromopropoxy)tetrahydro-2H-pyran (400 mg, 1.8 mmol, 1.2 equiv). The compounds were dissolved in DMF (10 mL) before adding K2CO3 (311 mg, 2.25 mmol, 1.5 equiv). Then the solution was heated at 60 °C for 4 h at which point the TLC analysis indicated complete consumption of the starting material. The solution was cooled and quenched by saturated aqueous NaCl solution. The aqueous layer was extracted with ethyl acetate and the combined organic layers were washed with brine, dried over Na2SO4, filtered, and concentrated in vacuo. The crude material was purified by silica gel column chromatography (eluent: hexanes/EtOAc = 2/1) to afford desired 5 as a colorless oil (524 mg, 82% yield). ^1^H NMR (400 MHz, CDCl3) δ 7.49 (s, 1H), 7.37–7.33 (m, 5H), 5.46–5.44 (m, 2H), 5.18–5.11 (m, 1H), 4.55–4.52 (m, 1H), 4.15–4.04 (m, 2H), 3.83–3.77 (m, 2H), 3.45–3.41 (m, 2H), 1.97–1.86 (m, 2H), 1.77–1.72 (m, 1H), 1.54–1.43 (m, 10H), 1.24–1.17 (m, 1H). ^13^C NMR (100 MHz, CDCl3) δ 155.2, 150.6, 148.3, 140.1, 135.4, 129.0, 128.6, 128.2, 107.5, 98.5, 65.4, 61.9, 50.1, 48.2, 39.1, 30.6, 28.2, 25.5, 19.7, 19.3. HRMS (m/z): [M+Na]^+^ calcd for C23H30N4O4Na 449.2165, found 449.2156.
To a 25 mL bottom flask, 5 (511 mg, 1.2 mmol) was dissolved in 10 mL EtOH before 10% Pd/C (0.1 g) were added. Then the reaction mixture was hydrogenated under an atmosphere of H2 and stirred at RT until complete (monitored by TLC, typically 3 h - 4h). Upon completion, the mixture was filtered through celite and concentrated in vacuo before purified by chromatography to afford the pure product as white solid (hexane: ethyl acetate=1:3 to 5, 274 mg, 68% yield). M.P.: 110–111 °C. ^1^H NMR (400 MHz, CDCl3) δ 13.00 (s, 1H), 7.80 (s, 1H), 5.27–5.13 (m, 1H), 4.58 (t, J = 3.4 Hz, 1H), 4.26–4.14 (m, 2H), 3.88–3.81 (m, 2H), 3.54–3.45 (m, 2H), 2.04–1.97 (m, 2H), 1.79–1.45 (m, 12H). ^13^C NMR (100 MHz, CDCl3) δ 156.4, 150.6, 148.6, 140.0, 129.2, 128.3, 107.7, 98.8, 65.6, 62.3, 48.9, 39.7, 30.8, 28.4, 25.6, 19.9. HRMS (m/z): [M+Na]^+^ calcd for C16H24N4O4Na 359.1695, found 359.1689.
To a solution of 6 (235 mg, 0.7 mmol, 1.0 equiv) in DMF (10 mL) was added K2CO3 (193 mg, 1.4 mmol, 2.0 equiv) and SEMCl (175 mg, 1.05 mol, 1.5 equiv). The reaction mixture was then stirred for 12 h at 50 °C. The resulting mixture was then poured into 10 mL of brine. The aqueous and organic layers were diluted by ethyl acetate and separated, the aqueous layer was extracted with ethyl acetate and the combined organic layers were washed with LiCl solution and brine. The organic layer was dried over Na2SO4 and concentrated under reduced pressure. The product was purified with chromatography on silica gel (eluent: hexanes/EtOAc = 1/1) to afford 7 as a colorless oil (277 mg, 85% yield). ^1^H NMR (400 MHz, CDCl3) δ 7.68 (s, 1H), 5.65 (s, 2H), 5.15 (sep, J = 6.8 Hz, 1H), 4.56–4.55 (m, 1H), 4.09–4.07 (m, 2H), 3.82–3.75 (m, 2H), 3.63 (t, J = 8.1 Hz, 2H), 3.46–3.41 (m, 2H), 1.96–1.89 (m, 2H), 1.79–1.69 (m, 1H), 1.66–1.60 (m, 1H), 1.54 (d, J = 6.8 Hz, 6H), 1.47–1.43 (m, 4H), 0.91 (t, J = 8.0 Hz, 2H), −0.06 (s, 9H). ^13^C NMR (100 MHz, CDCl3) δ 155.0, 150.6, 148.2, 140.8, 107.3, 98.4, 74.9, 67.1, 65.3, 61.9, 48.3, 39.1, 30.5, 28.1, 25.4, 19.7, 19.3, 17.7, −1.5. HRMS (m/z): [M+Na]^+^ calcd for C22H38N4O5SiNa 489.2509, found 489.2500.
Following a reported procedure [23]. A 25 mL round bottomed flask was charged with 3-bromo-6-fluoro-2-methylpyridine (567 mg, 3 mmol, 1.0 equiv) and Cs2CO3 (1.96 g, 6 mmol, 2 equiv). The mixture was dissolved 10 mL DMF before and cyclobutanol (259 mg, 3.6 mol, 1.2 equiv) added. The mixture was then heated at 90 °C for 4 h before cooled to RT and quenched by brine (10 mL). The quenched mixture was then diluted and extracted by ethyl acetate. The combined organics were then washed with LiCl solution and brine. The organic layer was then dried with Na2SO4, filtered, and concentrated in vacuo to give the crude, which was then purified via flash column chromatography (elute: hexane) to provide 8 as a colorless oil (643 mg, 89% yield). ^1^H NMR (400 MHz, CDCl3) δ 7.59 (d, J = 8.6 Hz, 1H), 6.39 (d, J = 8.6 Hz, 1H), 5.11–5.04 (m, 1H), 2.51 (s, 3H), 2.47–2.39 (m, 2H), 2.14–2.09 (m, 2H), 1.82 (q, J = 10.1 Hz, 1H), 1.65 (dq, J = 10.3, 8.2 Hz, 1H). ^13^C NMR (100 MHz, CDCl3) δ 161.3, 154.6, 142.0, 111.7, 109.1, 70.1, 30.6, 24.7, 13.5. HRMS (m/z): [M+H]^+^ calcd for C10H13BrNO 242.0181, found 242.0175.
To a 20 mL pressure vessel equipped with a stir bar was added 7 (298 mg, 0.64 mmol, 1.0 equiv), 8 (308 mg, 1.28 mmol, 2.0 equiv), Pd(OAc)2 (29 mg, 0.128 mmol, 20 mol%), PCy3 (72 mg, 0.256 mmol, 40 mol%), CuI (365 mg, 1.92 mmol, 3.0 equiv), K2CO3 (177 mg, 1.28 mmol, 2.0 equiv) before being capped with a septum. After evacuated and back-filled with Ar three times, the tube was charged with anhydrous DMF (4 mL) and THF (2 mL). The reaction was stirred at 160 °C under a sealed Ar atmosphere for 8 h before cooled to RT and filtered through Celite and concentrated. The crude product was diluted by ethyl acetate, washed with LiCl solution and brine, and dried over anhydrous Na2SO4. The concentrated residue was then subjected to column chromatography (Hexane: ethyl acetate=1:1, SiO2) to obtain 9 as a colorless oil (209 mg, 52% yield). ^1^H NMR (400 MHz, CDCl3) δ 7.74 (d, J = 8.5 Hz, 1H), 6.60 (d, J = 8.5 Hz, 1H), 5.49 (s, 2H), 5.25–5.16 (m, 2H), 4.61 (t, J = 3.2 Hz, 1H), 4.21–4.08 (m, 2H), 3.84 (dt, J = 9.7, 6.9 Hz, 2H), 3.66 (d, J = 8.4 Hz, 2H), 3.52–3.47 (m, 2H), 2.51–2.43 (m, 6H), 2.20–2.10 (m, 3H), 2.03–1.95 (m, 2H), 1.88–1.63 (m, 6H), 1.59 (d, J = 6.9 Hz, 6H), 0.88 (d, J = 8.3 Hz, 2H), −0.04 (s, 9H). ^13^C NMR (100 MHz, CDCl3) δ 173.8, 163.3, 157.5, 155.0, 151.4, 150.7, 147.6, 140.6, 115.9, 107.8, 107.2, 98.5, 73.2, 70.3, 66.8, 65.5, 62.0, 48.3, 39.2, 30.7, 30.6, 28.2, 25.5, 23.3, 19.8, 19.3, 18.0, 13.5, −1.44. HRMS (m/z): [M+H]^+^ calcd for C32H50N5O6Si 628.3530, found 628.3525.
To a stirred solution of 9 (188 mg, 0.3 mmol, 1.0 equiv) in THF (10 mL) was added TBAF (3 mL, 1M in THF). The reaction mixture was stirred at 80 °C for 10 h. After cooling to RT, the mixture was diluted with EtOAc, washed with H2O, and concentrated in vacuo. The crude material was redissolved in methanol and added 4-toluenesulfonic acid monohydrate (PTSA, 171 mg, 0.9 mmol, 3.0 equiv). The mixture was stirred at RT overnight before concentrated, and purified by chromatography (hexane: ethyl acetate=3:2 to 1) to afford the pure product as yellow solid (99 mg, 80% yield for 2 steps). M.P.: 231–232 °C. ^1^H NMR (400 MHz, CDCl3) δ 12.72 (s, 1H), 8.04 (d, J = 8.1 Hz, 1H), 6.62 (d, J = 8.2 Hz, 1H), 5.28–5.20 (m, 2H), 4.16 (s, 2H), 3.51 (s, 3H), 2.77 (s, 3H), 2.49–2.48 (m, 2H), 2.18–2.14 (m, 2H), 1.87–1.62 (m, 8H), 1.23 (s, 1H). ^13^C NMR (100 MHz, CDCl3) δ 163.0, 156.9, 155.8, 151.3, 151.0, 149.0, 139.6, 116.9, 108.1, 107.5, 70.4, 58.6, 49.0, 38.3, 30.9, 30.7, 24.4, 19.8, 13.5. HRMS (m/z): [M+H]^+^ calcd for C21H28N5O4 414.2141, found 414.2133.
To a stirred solution of 11 (21 mg, 0.05 mmol, 1.0 equiv) in dry DMF (10 mL) was added K2CO3 (14 mg, 0.1 mmol, 2.0 equiv) and 1-(bromomethyl)-4-fluorobenzene (12 mg, 0.06 mmol, 1.2 equiv). The reaction mixture was then allowed stirred for 4 h at 50 °C. Upon completion, the reaction solution was quenched by water and extracted by ethyl acetate. The organic phase was washed with LiCl solution and brine, dried over anhydrous Na2SO4 and concentrated in vacuo. the desired product was purified by chromatography on silica gel eluting with ethyl acetate = 1 to afford TZ78141 as a white solid (21 mg, 80% yield). M.P.: 74–75 °C. ^1^H NMR (400 MHz, CDCl3) δ 7.36 (d, J = 8.4 Hz, 1H), 6.95–6.87 (m, 4H), 6.58 (d, J = 8.4 Hz, 1H), 5.34 (s, 2H), 5.23–5.15 (m, 2H), 4.19 (t, J = 5.9 Hz, 2H), 3.54 (t, J = 5.3 Hz, 2H), 2.50–2.43 (m, 2H), 2.16–2.12 (m, 4H), 2.02 (s, 1H), 1.91–1.83 (m, 3H), 1.74–1.64 (m, 1H), 1.56 (d, J = 6.9 Hz, 6H), 1.23 (t, J = 7.1 Hz, 1H). ^19^F NMR (376 MHz, CDCl3) δ −113.31 (s). ^13^C NMR (100 MHz, CDCl3) δ 163.5, 162.4 (d, J = 247.6 Hz), 157.1, 155.8, 151.0, 150.8, 148.1, 140.1, 131.8 (d, J = 3.3 Hz), 129.5 (d, J = 8.2 Hz), 116.2, 115.7 (d, J = 21.7 Hz), 107.6, 107.4, 70.4, 58.6, 48.7, 48.1, 37.7, 30.9, 30.6, 22.7, 19.7, 13.5. HRMS (m/z): [M+H]^+^ calcd for C28H33FN5O4 522.2517, found 522.2506.
The procedure to prepare 12 was similar with TZ78141 while utilizing 11 (42 mg, 0.1 mmol, 1.0 equiv) 4-(bromomethyl)phenol as the benzylation reagents. The crude material was purified by silica gel column chromatography (hexanes:EtOAc 1) to afford compound 12 (39 mg, 75%) as a white solid. M.P.: 360–362 °C (decomposed). ^1^H NMR (400 MHz, CDCl3) δ 7.41 (d, J = 8.4 Hz, 1H), 7.01 (s, 1H), 6.82 (d, J = 8.5 Hz, 2H), 6.68 (d, J = 8.5 Hz, 2H), 6.60 (d, J = 8.4 Hz, 1H), 5.35–5.26 (m, 2H), 5.26–5.13 (m, 2H), 4.28–4.11 (m, 2H), 3.94 (s, 1H), 3.63–3.56 (m, 2H), 2.52–2.45 (m, 2H), 2.22–2.12 (m, 5H), 1.94–1.83 (m, 4H), 1.58 (d, J = 6.9 Hz, 6H). ^13^C NMR (100 MHz, CDCl3) δ 163.6, 157.4, 156.4, 156.1, 151.1, 151.1, 148.4, 140.5, 129.3, 127.7, 116.5, 115.7, 107.6, 107.6, 70.6, 70.6, 58.8, 49.0, 48.6, 37.9, 30.8, 22.9, 19.8, 13.7. HRMS (m/z): [M+H]^+^ calcd for C28H34N5O5 520.2560, found 522.2554.
[^18^F]fluoride produced by cyclotron was first trapped on an a preconditioned Sep-Pak light QMA cartridge (Part No. WAT023525, Waters) and eluted by K2CO3 solution (0.02 M). Then 1.5 mL of CH3CN solution of Kryptofix 222 (6.5 mg) was added to the glass V-vial containing the [^18^F]fluoride solution (~200 mCi, ~7.4 GBq). The solution was dried by three cycles of azeotropic evaporation with additional MeCN (2 × 1.0 mL) under a gentle stream of nitrogen gas at 100 °C. 4-N, N,N-Trimethylammonium benzaldehyde triflate (10 mg, 0.032 mmol) in DMSO (400 μL) was added to the dried residue, and the mixture was heated at 150 °C for 3 min and cooled to RT. NaBH4 (10 mg in 500 μL EtOH) were added to the reaction mixture. After reacted at RT for 5 min, the reaction medium was then diluted with 1% Na-ascorbate solution (2 mL). RCY was checked by radio-TLC and radio-HPLC and about 90% radiochemical yield could be afforded.
The crude labeled [^18^F]fluorobenzaldehyde was diluted in 1% Na-ascorbate solution (50 mL) and adsorbed onto an Oasis PRiME HLB cartridge (335 mg Sorbent, Part No. 186,008,887, Waters, preconditioned by rinsing with 10 mL H2O). The 4-[^18^F]-fluorobenzyl alcohol formed was not isolated but directly converted into the corresponding bromide derivative by eluting the Sep-Pak by 48% aqueous HBr (3 mL) within 2 min. The HLB Sep-Pak was removed and the cap was closed before heating in Armor^™^ Aluminum Bath Beads (70 °C) for 10 min to ensure complete on-column bromination. After cooling, the remaining HBr on the Sep-Pak was eluted by 10 mL of deionized water. The bromide compound [^18^F]benzyl bromide (FBnBr) intermediate was eluted with CH3CN (4.0 mL), which was diluted by 50 mL of Milli-Q water and passed through a Plus Short C-18 Sep-Pak cartridge (Part No. WAT020515, Waters). After using 20 mL of Milli-Q water to rinse the C-18 cartridge, the radioactive product (about 2.0 GBq) was eluted by pentane (3 mL) and separated to remove water phase and dried by Plus Long Na2SO4 Sep-Pak (x2). Finally, the pentane phase containing pure [^18^F]FBnBr was transferred into a second vial containing xanthine precursor 11 (1.5 mg), K2CO3 (3 mg) and DMF (0.3 mL), which was evaporated at RT to remove pentane before heating at 100 °C for 15 min. Then the mixture was cooled and quenched by 2.0 mL HPLC mobile phase (CH3CN/H2O, 70/30, v/v). After that, the diluted reaction mixture was loaded onto a reverse phase semi-preparative C-18 column (Phenomenex^®^ Luna^®^ C18(2), 10 mm ID × 250, 5 μm) with 70% MeCN in 0.1 M ammonium formate (pH 4.5) as a mobile phase, a flow rate of 4.0 mL/min and UV wavelength of 254 nm. The desired radioactive fraction (from 30 to 32 min) was collected into a vial that contained 50 mL of Milli-Q water, which was passed through a Plus Short C-18 Sep-Pak cartridge (Part No. WAT020515, Waters, preconditioned by rinsing with 5 mL EtOH followed by 10 mL H2O). After using 20 mL of Milli-Q water to rinse the C-18 cartridge, the radioactive product was eluted into a dose vial using 1.0 mL absolute ethanol and then 9.0 mL saline (v/v) to formulate dose solution for further study.
A 20 μL aliquot of the dose sample was analyzed by HPLC for verification against the cold standard compound TZ78141, assessing its chemical and radiochemical purity, as well as molar activity. The HPLC system featured a reverse-phase Agilent Zorbax SB-C18 column (4.6 mm × 250 mm) with UV detection at 254 nm. The mobile phase was CH3CN/0.1 M HCOONH₄ (98/2, v/v, pH 4.5) at a flow rate of 1.0 mL/min. Under these conditions, the retention time (tR) was 4.5 min, confirming the radiolabeled product as [^18^F]TZ78141.
A bolus of aqueous [^18^F]fluoride (200 mCi, CN solution of Kryptofix 222 (6–7 mg) was added to the conical glass vial. The solution was dried by three cycles of azeotropic evaporation with additional MeCN (2 × 1.0 mL) under a gentle stream of nitrogen gas at 100 °C.7.4 GBq) was transferred into a glass V-vial containing aqueous potassium carbonate solution (40 μL, 45 mg/mL). Then 1.5 mL of CH3
Phenol precursor 12 (4.8 μmol, 2.5 mg) and ruthenium complex (8 mg, 26 μmol) were dissolved in ethanol (50 μL) in a V glass vial and heated at 85 °C for 30 min. The vial was removed from the heating bath and allowed to stand for 3 min at 23 °C. To the vial, imidazolium chloride (N,N’-bis(2,6-diisopropylphenyl)-2-chloroimidazolium chloride, 14 mg, 26 μmol), 175 μL of MeCN and 175 μL of DMSO were added. The resulting solution was drawn into a 1.0 mL polypropylene syringe and transferred into the reaction vial containing the [^18^F]KF. The reaction vial, which contained 400 μL of the reaction mixture was sealed with a teflon-lined cap and was heated at 160 °C for 30 min. After cooling down, the reaction mixture was analyzed by radio-TLC and radio-HPLC before quenched by 2 mL of mobile phase and injected into the semi-preparative HPLC system for further purification.
The PET study was performed on a male macaque (9–10 kg) using a microPET Focus-220 scanner (Siemens Microsystems, Knoxville, TN) across multiple sessions. Anesthesia was induced with ketamine, secretions reduced by glycopyrrolate and maintained by inhalation 1–2% isoflurane in a mixture of 40% N2O and 60% O2. A 2 h dynamic scan (3 × 1 min, 4 × 2 min, 3 × 3 min, and 20 × 5 min frames) was performed after a bolus injection of the radiotracer (0.3–0.4 GBq) via a venous catheter. For quantitative analysis, PET images were co-registered with MRI scans. The three-dimensional region of interest (whole brain) was delineated on the MRI and then mapped onto the reconstructed PET images to extract time-activity curves. The activity measurements were standardized to body weight and the injected radioactivity dose to calculate the standardized uptake value (SUV).