S_NAr Radiofluorination with In Situ-Generated [¹⁸F]Tetramethylammonium Fluoride

Abstract

This report describes a method for the nucleophilic radiofluorination of (hetero)aryl chlorides, (hetero)aryl triflates, and nitroarenes using a combination of [¹⁸F]KF•K_2.2.2 and Me₄N HCO₃ for the in situ formation of a strongly nucleophilic fluorinating reagent (proposed to be [¹⁸F]Me₄NF). This method is applied to 24 substrates bearing diverse functional groups, and it generates [¹⁸F](hetero)aryl fluoride products in good to excellent radiochemical yields in the presence of ambient air/moisture. The reaction is applied to the preparation of ¹⁸F-labeled HQ-415 for potential (pre)clinical use.

Graphical Abstract

Introduction

(Hetero)aryl fluorides are common structural motifs in pharmaceutical scaffolds.^1,2 These fluorine-containing biologically active molecules offer opportunities for the simultaneous development of imaging agents for positron emission tomography (PET) by preparation of ¹⁸F isotopologs. To capitalize on this, there is an urgent need for late-stage synthetic methods for C(sp²)–¹⁸F bond formation.^1,3 In general, such methods remain challenging to develop due to the short half-life of ¹⁸F (~110 minutes), the low concentrations of ¹⁸F present during radiolabeling (typically nanomole scale), and the high sensitivity of many [¹⁸F]fluoride sources to water.⁴

One of the most common approaches for the formation of C(sp²)–¹⁸F bonds is nucleophilic aromatic (S_NAr) radiofluorination, which involves the reaction of electron-deficient (hetero)aryl halide or nitroarene precursors with [¹⁸F]fluoride. These precursors are attractive starting materials that are widely used in PET radiochemistry due to their stability, ready availability, and ease of separation from ¹⁸F-labeled products by HPLC.^4,5 Anhydrous alkali metal fluoride salts (e.g. [¹⁸F]KF or [¹⁸F]CsF) are common fluoride sources for S_NAr radiofluorination. However, these salts are hydroscopic and become much less nucleophilic upon hydration.^2b Moreover, the modest solubility of the salts as well as their tight ion pairing in organic solvents can result in low nucleophilicity and slow reaction rates. Consequently, these reactions can require elevated temperatures (often ≥150 °C), inclusion of additives like Kryptofix^® 2.2.2 (K_2.2.2), and relatively long reaction times to afford high radiochemical yields (RCYs).^4c,d Such requirements often limit functional group compatibility, lead to the formation of side products, and result in undesirably long times of synthesis during ¹⁸F labeling.

An attractive approach to mitigate these issues is utilization of tetraalkylammonium [¹⁸F]fluoride derivatives ([¹⁸F]R₄NF) for S_NAr fluorination. The corresponding ¹⁹F reagents exhibit dramatically enhanced solubility and nucleophilicity in S_NAr reactions relative to their alkali metal congeners (Scheme 1A).^5-10 Quaternary alkylammonium [¹⁸F]fluoride salts (most commonly [¹⁸F]Bu₄NF and [¹⁸F]Et₄NF) have found some application in fluorine-18 radiochemistry, including both S_NAr radiofluorination (Scheme 1B)¹¹ and in other approaches to late-stage fluorination.¹² For instance, in recent examples, Brichard and Aigbirhio^13a and Packard^13b independently reported the radiofluorination of nitro precursors with [¹⁸F]Et₄NF, while the Scott group employed [¹⁸F]Et₄NF to prepare [¹⁸F]FL2-b from a 2-chloropyridine precursor.¹⁴ While some of the newer approaches to late-stage radiofluorination have eliminated the need for azeotropic drying of quaternary alkylammonium [¹⁸F]fluorides,^12g for S_NAr [¹⁸F]R₄NF salts are most commonly prepared via solid-phase extraction methods that use aqueous solutions of quaternary alkylammonium salts to elute [¹⁸F]fluoride from a quaternary methylammonium cartridge. As such, these sequences require azeotropic drying of the eluted [¹⁸F]R₄NF prior to radiofluorination. However, the [¹⁸F]R₄NF salts tend to be very hygroscopic, which impedes efficient drying. In addition, salts containing R groups with β-hydrogen atoms can undergo competing decomposition via Hoffmann elimination both during drying and during S_NAr reactions at elevated temperatures. This problem is well-documented in ¹⁹F fluorination reactions.^8,9,15 While its contribution to radiofluorination is less definitively established (and in certain instances [¹⁸F]Bu₄NF has outperformed [¹⁸F]CsF and [¹⁸F]KF•K_2.2.2^11h), the loss of volatile ¹⁸F-species can be difficult to detect and quantify.^12l

Scheme 1.

S_NAr fluorination and radiofluorination with tetraalkylammonium fluoride salts

This elimination problem can be alleviated by moving to tetramethylammonium fluoride (Me₄NF), which lacks β-hydrogen atoms. Literature reports have shown that Me₄NF is thermally stable and can be dried (albeit over days at elevated temperatures)^6,16 to yield an anhydrous fluoride source that is highly nucleophilic in S_NAr reactions.¹⁶ Despite these advantages, S_NAr radiofluorination reactions with [¹⁸F]Me₄NF remain rare,^17,18 likely due to challenges with removal of [¹⁸O]H₂O (a process that is known to be slow and poorly reproducible in the ¹⁹F analogue^16,19). In this report, we describe the development of a convenient and reproducible in situ preparation of [¹⁸F]Me₄NF from [¹⁸F]KF and tetramethylammonium salts that circumvents the requirement for rigorous drying of [¹⁸F]Me₄NF (Scheme 1C). We employ this reagent for the S_NAr radiofluorination of a variety of electron deficient (hetero)aryl precursors, including HQ-415, a potential radioligand for metal-protein aggregates implicated in neurodegeneration. We show that the in situ procedure offers key advantages, including relatively low reaction temperatures (≤110 °C), short synthesis times, and compatibility with ambient moisture.

Results and Discussion

Our initial studies explored the development of a simple and reliable method for the preparation of [¹⁸F]Me₄NF. We first evaluated solid phase extraction approaches involving the direct elution of [¹⁸F]Me₄NF from a quaternary methylammonium cartridge using Me₄NHCO₃.¹³ However, there are key problems with such procedures. Elution with aqueous Me₄NHCO₃ using standard approaches^13,20 results in hydrated [¹⁸F]Me₄NF which, given the literature precedent of drying nonradioactive Me₄NF for hours to days,^4a is not compatible with azeotropic drying of short-lived ¹⁸F (8-12 min for [¹⁸F]KF²⁰). In an attempt to shorten the azeotropic drying time, we examined elution with non-aqueous solutions of tetramethylammonium salts. However, this resulted in poor recovery of fluoride [just 21–80% from [¹⁸F]fluoride in ¹⁸O target water, decay-corrected (see Supplementary Information, Table S2)].

These initial results suggested a need for an alternative synthetic approach to [¹⁸F]Me₄NF that circumvents the requirement for elution and azeotropic drying of this salt. Thus, we next pursued the in situ formation of [¹⁸F]Me₄NF from the combination of [¹⁸F]KF and a tetramethylammonium (Me₄N⁺) salt (Scheme 1c). This approach offers the advantages that the elution and azeotropic drying of [¹⁸F]KF is already highly optimized,²¹ allowing short (8-12 min) drying times and high (>95%) recovery of ¹⁸F from [¹⁸F]fluoride in ¹⁸O target water.

2-Chloroquinoline was employed as a model substrate for S_NAr radiofluorination with in situ generated [¹⁸F]Me₄NF. A [¹⁸F]KF•K_2.2.2 stock solution in DMF was prepared via azeotropic drying under our standard mild conditions (see Experimental Section). Briefly, [¹⁸F]fluoride was eluted into the reaction vessel using aqueous KOTf (5 mg in 0.5 mL water, 0.05 M) and K_2.2.2 in acetonitrile (15 mg, 1 mL, 0.04 M). [¹⁸F]KF•K_2.2.2 was then azeotropically dried by heating the reaction vessel at 100 °C while drawing vacuum for 6 min. The reaction vessel was subsequently subjected to an Ar stream and simultaneous vacuum draw for an additional 6 min to produce anhydrous [¹⁸F]KF•K_2.2.2. Notably, the use of KOTf for elution (as opposed to eluting with stronger bases like K₂CO₃) allows us to precisely control the amount of base present within a reaction mixture, and such customization has proven useful for other methodology development in our labs.^12d An aliquot of this stock solution [92.5-129.5 MBq (2500–3500 μCi) in 100 μL] was then added to a solution containing 2-chloroquinoline (50 μmol) and a tetramethylammonium salt (Me₄NX, 100 μmol) in a 4 mL vial. Reactions were heated at 100 °C for 30 min and then analyzed by radio-TLC. To benchmark reactivity in this system, we first carried out control reactions in the absence of Me₄NX additives (Table 1, entries 1–6). With just [¹⁸F]KF•K_2.2.2 as the [¹⁸F]fluoride source, no radiofluorinated product was detected under these conditions (Table 1, entry 1). Low radiochemical yields of 2-[¹⁸F]fluoroquinoline were obtained upon the addition of KHCO₃ or K₂CO₃ (<10% RCY, in each case; entries 2 and 3) or extra K_2.2.2 (8% RCY; entry 6).²³ In contrast, the addition of Me₄NHCO₃ resulted in 91% radiochemical yield of 2-[¹⁸F]fluoroquinoline (entry 8), indicating the formation of a dramatically more reactive [¹⁸F]fluoride source, which is proposed to be [¹⁸F]Me₄NF.

Table 1.

In situ formation of [¹⁸F]Me₄NF for the radiofluorination of 2-chloroquinoline^a

Entry	Me4NX	Additive	RCY (%)f
1	none	none	0
2	none	KHCO3	6
3	none	K2CO3	<1
4	none	NaHCO3	<1
5	none	KOTf	<1
6	none	K2.2.2	8
722	Me4NBF4	none	<1
8	Me4NOAc	none	17
9b	Me4NHCO3e	none	91
10b	Et4NHCO3	none	85
11c	Me4NHCO3, H2O	none	70
12d	Me4NHCO3	none	96

^aAzeotropically dried [¹⁸F]K¹⁸F with K_2.2.2 in DMF prepared in an GE TRACERlab FX_FN. 100 μL of the [¹⁸F]K¹⁸F/K_2.2.2 was used for each manual reaction (total reaction volume = 100 μL).

^bMe₄NX was added to the reaction vial on the bench top.

^c2 equiv of H₂O (1.8 μL, 100 μmol) added compared to approx. 0.00001 equiv of fluoride (92.5 MBq (2.5 mCi) assuming molar activity of 185 GBq/μmol (5 mCi/nmol), 0.5 nmol)

^d110 °C

^eUp to 6 equiv of Me₄NHCO₃ could be added without significant effect on RCY (see Table S4 in Supporting Information)

^fRadiochemical yield (RCY) was determined by radio-TLC, and the identity of the product was confirmed by radio-HPLC.

We next investigated a series of different Me₄NX salts, with X = Cl, Br, I, BF₄, OAc, and HCO₃). Among these, only Me₄NOAc (entry 8) and Me₄NHCO₃ (entry 9) afforded >10% radiochemical yield of 2-[¹⁸F]fluoroquinoline (Tables 1 and S5). Me₄NBF₄ gave poor (<1%) yield with this substrate (entry 7); however, for some other substrates it proved an effective tetramethylammonium source (see Supporting Information).²⁴ The radiofluorination of 2-chloroquinoline with [¹⁸F]KF•K_2.2.2/Me₄NHCO₃ proceeded in high radiochemical yield at 100 °C. An analogous effect was observed by adding Et₄NHCO₃ (presumably to form [¹⁸F]Et₄NF in situ), although in slightly diminished RCY (entry 10).The reactions proceed in the presence of ambient air/moisture. Indeed, the radiochemical yield remained >70% even upon the addition of 2 equiv of H₂O to the mixture (entry 11), indicating only modest sensitivity to moisture. Further optimization of the reaction temperature, time, concentration, and solvent (see Supporting Information) afforded optimal conditions as 50 μmol of substrate, 100 μmol of Me₄NHCO₃, 100 μL of DMF, 110 °C, resulting in 96% radiochemical yield of 2-[¹⁸F]fluoroquinoline (entry 12).

The scope of this transformation was next evaluated with a series of (hetero)aryl precursors containing standard chloride, nitro, or triflate leaving groups.²⁵ In all cases, the results with [¹⁸F]KF•K_2.2.2 (control, conditions A) were compared to those with [¹⁸F]KF•K_2.2.2 and Me₄NHCO₃ (conditions B) under otherwise identical conditions (at 100-100 °C).^23,24 As summarized in Figure 1-i, for 1¹⁸F–15¹⁸F the control reaction with [¹⁸F]KF•K_2.2.2 afforded low yield at 100-110 °C (ranging from 0–27% yield, Figure 1-i). Conversely, moderate to excellent RCY (up to 97%) was obtained upon addition of Me₄NX, even with the precursors that afforded low (or no) RCY with [¹⁸F]KF•K_2.2.2 (Figure 1-i). In other cases (16¹⁸F–24¹⁸F) the addition of Me₄NHCO₃ led to comparable yields as the control reactions with [¹⁸F]KF•K_2.2.2 (Figure 1-ii).

Figure 1. Substrate scope^{27, a,b}.

^aRCY was determined by radio-TLC and identity of final product was confirmed by HPLC. N.a. = not available. Reaction temperature was optimized to 110 °C for 6¹⁸F, 12¹⁸F, 13¹⁸F, and 21¹⁸F. All other reactions were conducted at 100 °C; ^b For data using Me₄NBF₄ as an additive, see Supporting Information.

This study also revealed that for pyridines bearing both nitro and chloro groups, ¹⁸F-fluorination at the 2-position is favored over the 3- and 5-positions, regardless of leaving group. For example, 2-chloropyridines were converted to 2-[¹⁸F]fluoropyridines in the presence of 3-nitro or 5-nitro substituents (9¹⁸F, and 23¹⁸F, respectively), while a 2-nitro group was substituted in the presence of a 3-chloro (4¹⁸F). In these cases, there was no evidence of ¹⁸F-labeled side products from competing substitution reactions. Conversely, in the case of pyridines containing both chloro and fluoro substituents, isotopic exchange was the dominant reaction pathway. Thus, 2-chloro-3-fluoropyridine and 2-chloro-6-fluoropyridine underwent isotopic exchange (6-91% RCY), while minimal quantities (≤9%) of the ¹⁸F-difluoropyridines were observed (Supporting Information, Figure S2).

Substituted pyridine, pyrazine, quinoline, and isoquinoline precursors all underwent efficient radiofluorination using this method. Less activated substrates, such as 2-chlorobenzonitrile and 4-chlorobenzophenone afforded modest yields of the radiofluorinated products 10¹⁸F and 12¹⁸F (16 and 9%, respectively). However, moving from the chloro- to the nitro- precursor led to a dramatic increase in the yield of 12¹⁸F (to 77%). It is also noteworthy that 2-chlorobenzonitrile was significantly more reactive than 4-chlorobenzonitrile, which afforded <5% yield of the radiofluorinated product 11¹⁸F under conditions A or B. A similar trend was observed for chloropyridine derivatives bearing electron withdrawing groups either ortho- or para- to the chlorine (for instance compare 8¹⁸F to 24¹⁸F and 16¹⁸F to 18¹⁸F). These results are consistent with the previously documented ortho effect in nucleophilic aromatic substitution.²⁶ A variety of less electrophilic precursors, including 2-chloro-4-aminopyridine, 3-chloroquinoline, and 3-chlorobenzonitrile, were also examined in this transformation. As expected based on the S_NAr literature, these all showed low reactivity, affording <1% yield of the targeted radiofluorinated products (Supplementary Information, Figure S3).

This method was next applied to the labeling of the 2-chloroquinoline moiety in Pre-HQ415 to form [¹⁸F]HQ415, a molecule of interest for PET imaging of the toxic TDP43 protein deposits implicated in amyotrophic lateral sclerosis (ALS).^28,29 As shown in Scheme 2, a deprotection of the Boc groups with 2 M HCl was incorporated into the manual one-pot synthesis. Using the combination of [¹⁸F]KF•K_2.2.2 and Me₄NHCO₃, the radiofluorination proceeded in 82 ± 2% (n = 3) radiochemical yield, and [¹⁸F]HQ415 was obtained in 56 ± 3% (n = 3) yield from the two step sequence. In contrast, the radiofluorination proceeded in just 10% RCY with [¹⁸F]KF•K_2.2.2 alone, again highlighting the advantages of in situ-generated [¹⁸F]Me₄NF for enhancing reactivity and yields in S_NAr fluorination reactions conducted at lower temperatures.

Scheme 2.

Synthesis of [¹⁸F]HQ415

Finally, the S_NAr radiofluorination of 2-chloroquinoline was translated to an automated method using 63 GBq (1.7 Ci) of [¹⁸F]KF•K_2.2.2 (Scheme 3). The reaction was upscaled with respect to the amount of the precursor, amount of Me₄NHCO₃, and reaction volume to simplify automation, as smaller volumes are not compatible with our automated synthesis modules). Conditions for automated runs were as follows: 100 μmol precursor, 500 μmol Me₄NHCO₃, and 0.5 mL reaction volume. The fully automated S_NAr radiofluorination with [¹⁸F]KF•K_2.2.2 and Me₄NHCO₃ afforded 2-[¹⁸F]fluoroquinoline (1¹⁸F) in 18 ± 4% radiochemical yield, with molar activity of 155.5 ± 66.4 GBq/μmol (4.2 ± 1.8 Ci/μmol) and radiochemical purity of greater than 99% (n = 4). Automated RCYs of late-stage fluorination reactions are frequently lower than those for the corresponding manual reactions,³⁰ and the present work is no exception. The reasons for this are currently unknown, but efforts are underway in our laboratories to investigate the problem via a systems approach using machine learning (ML).³¹ The data from this study will be incorporated into ML models and findings reported in due course. Lastly, one of the automated production batches was reformulated into 10% ethanol in saline using a C18 cartridge. We analyzed the formulated product for residual tetramethylammonium salts using recently developed TLC methods.³² This analysis shows levels below the accepted regulatory limits (not more than 2.6 mg/10 mL patient dose), promising results for translation of this methodology to the production of PET radiotracers for clinical use.

Scheme 3.

Automation of S_NAr radiofluorination with in situ Me₄N¹⁸F

Conclusions

In conclusion, this report describes a method for the S_NAr radiofluorination of (hetero)aryl chlorides and triflates as well as nitroarenes using a combination of [¹⁸F]KF•K_2.2.2 and Me₄NHCO₃ for the in situ formation of a strongly nucleophilic fluorination reagent (presumed to be [¹⁸F]Me₄NF). This method circumvents the need to independently synthesize and dry [¹⁸F]Me₄NF, steps that can be inefficient and low yielding. Instead, this approach enables rapid nucleophilic aromatic radiofluorination using a well-optimized [¹⁸F]fluoride source at moderate temperature (100-110 °C) in the presence of ambient air/moisture, offering a valuable approach for labeling temperature sensitive molecules. This method is operationally simple and proceeds reliably without rigorous exclusion of water to provide radiolabeled products in high RCYs. Furthermore, it is readily translated to automation. The method is effective for the late-stage nucleophilic radiofluorination of biologically active molecules, as highlighted by the synthesis of [¹⁸F]HQ415, a molecule of interest for PET imaging of the TDP43 deposits implicated in ALS. Overall, this work makes [¹⁸F]Me₄NF readily accessible to the PET community, enabling exploitation of its unique reactivity profile for the development of new radiochemistry methodology and the application to the synthesis of new PET radiotracers.

Experimental Section

Chemistry

Materials and Methods

All commercial products were used as received and reagents were stored under ambient conditions unless otherwise stated. Quinoline, pyridine, pyrazine, benzophenone, and benzonitrile precursors for the substrate scope as well as their fluorinated reference standards were purchased from commercial sources (Synthonix, Frontier Scientific, Oakwood Products, Acros Organics, Synthonix, Chem Impex, TCI America, Matrix Scientific, Alfa Aesar, Ark Pharm, or Sigma Aldrich) unless otherwise stated. Isopropyl 5-chloro-6-phenylpicolinate were prepared using previously described methods.¹⁰ Isopropyl 5-fluoro-6-phenylpicolinate and 4-fluoro-7-(trifluoromethyl)quinoline was prepared using previously described methods.¹⁶ 3-Ethoxy-4-methoxybenzaldehyde was purchased from Sigma Aldrich. The manipulation of solid reagents was conducted on the benchtop unless otherwise stated. Reactions were conducted under an ambient atmosphere unless otherwise stated. Reaction vessels were sealed with either a septum (flask) or a Teflon-lined cap (4 mL or 20 mL vial). Reactions conducted at elevated temperatures were heated on a hot plate using an aluminum heating block. The temperatures was regulated using an external thermocouple. TLC analyses were performed using TLC Silica gel 60 F₂₅₄ MS-grade plates. R_F values are reported based on standard silica gel plates with fluorescent indicator.

NMR spectra were obtained on a Varian vnmrs700 (699.76 MHz for ¹H; 175.95 MHz for ¹³C), a Varian vnmr500 (500.09 MHz for ¹H; 470.56 MHz for ¹⁹F; 125.75 MHz for ¹³C), or a Varian MR400 (400.53 MHz for ¹H; 376.87 MHz for ¹⁹F) spectrometer. All ¹³C NMR data presented are proton-decoupled ¹³C NMR spectra, unless noted otherwise. ¹H and ¹³C NMR chemical shifts (δ) are reported in parts per million (ppm) relative to TMS with the residual solvent peak used as an internal reference. ¹H and ¹⁹F NMR multiplicities are reported as follows: singlet (s), doublet (d), triplet (t), quartet (q), and multiplet (m). Melting point (mp) data were collected on an OptiMelt Automated Melting Point System and are uncorrected. High performance liquid chromatography (HPLC) was performed using a Shimadzu LC-2010A HT system equipped with a Bioscan B-FC-1000 radiation detector. High resolution mass spectroscopy (HRMS) was performed on an Agilent Technologies 6520 Accurate Mass Q-TOF LC/MS using electrospray ionization (ESI+).

Preparation and characterization of aryl chloride and aryl triflate precursors

Quinolin-2-yl trifluoromethanesulfonate.

In a modification of a published method,³³ triflic anhydride (3.10 g, 11 mmol, 1.1 equiv) was added dropwise to the corresponding phenol (1.45 g, 10 mmol, 1 equiv) in pyridine (10 mL) at 0 °C. The resulting solution was stirred for 10 min at 0 °C. The reaction was then allowed warm to ambient temperature and was stirred for an additional 12 h. The solution was diluted with diethyl ether (25 mL) and extracted with a 1 M solution of CuSO₄ (3 x 25 mL). The organic layer was washed with water (1 x 25 mL), dried over MgSO₄, and concentrated in vacuo. The aryl triflate was then purified via column chromatography on silica gel (95 : 5 hexanes/ethyl acetate). The product was isolated as a pale yellow oil (2.69 g, 97%).

R_f: 0.50 (9 : 1 hexanes/ethyl acetate).

¹H NMR (400 MHz, CDCl₃): δ 8.34 (d, J = 8.7 Hz, 1H), 8.05 (d, J = 8.5 Hz, 1H), 7.90 (dd, J = 8.2, 1.4 Hz, 1H), 7.80 (ddd, J = 8.5, 6.9, 1.4 Hz, 1H), 7.64 (ddd, J = 8.2, 7.0, 1.2 Hz, 1H), 7.24 (d, J = 8.8 Hz, 1H).

¹³C{¹H} NMR (176 MHz, CDCl₃): δ 153.8, 146.0, 142.0, 131.3, 129.0, 127.9, 127.8, 127.7, 118.8 (q, J = 320.5 Hz), 113.3.

¹⁹F NMR (377 MHz, CDCl₃): δ –73.00.

HRMS: (ESI) m/z [M+H]⁺ calcd. for C₁₀H₇F₃NO₃S⁺ 278.0093; found 278.0085.

8-(Benzyloxy)-2-chloroquinoline.

2-Chloro-4-hydroxyquinoline (121.2 mg, 0.67 mmol, 1.0 equiv), potassium carbonate (185.1 mg, 1.34 mmol, 2.0 equiv), potassium iodide (11.1 mg, 0.067 mmol, 0.1 equiv), and benzyl bromide (96 μL, 0.80 mmol, 1.2 equiv) were combined in acetonitrile (3 mL). The reaction mixture was heated at reflux for 24 h. After cooling to room temperature, solids were removed by filtration, and the filtrate was concentrated under reduced pressure. Purification via column chromatography on silica (85 : 15 hexanes/ethyl acetate) afforded the product as a white solid (142.4 mg, 79% yield).

R_f: 0.54 (8 : 2 hexanes/ethyl acetate).

m.p. 90.2-91.1 °C.

¹H NMR (700 MHz, CDCl₃): δ 8.06 (dd, J = 8.6, 1.4 Hz, 1H), 7.51 (s, 1H), 7.50 (s, 1H), 7.41 (dd, J = 8.6, 1.4 Hz, 1H), 7.39-7.34 (multiple peaks, 4H), 7.30 (m, 1H), 7.06 (dt, J = 6.6, 1.9 Hz, 1H), 5.45 (s, 2H).

¹³C{¹H} NMR (176 MHz, CDCl₃): δ 153.7, 150.0, 140.1, 138.9, 137.0, 128.7, 128.3, 127.9, 127.2, 127.0, 123.1, 119.7, 111.8, 71.0.

HRMS: (ESI) m/z [M+H]⁺ calcd. for C₁₆H₁₃ClNO⁺ 270.0680; found 270.0680.

2-Chloro-7-((3-ethoxy-4-methoxyphenyl)((4-methylpyridin-2-yl)amino)methyl)quinolin-8-ol (HQ415).

3-Ethoxy-4-methoxy-benzaldehyde (0.620 g, 3.45 mmol, 1 equiv) and 2-amino-4-methyl pyridine (0.373 g, 3.45 mmol, 1 equiv) was dissolved in EtOH (25 mL) at room temperature. 2-Chloro-5-hydroxyquinoline (0.622 g, 3.45 mmol, 1 equiv) was added. The reaction was stirred at room temperature for 14 d. The solvent was removed under vacuum. The crude product was diluted with DCM (30 mL). The organic solution was extracted with water (30 mL), the organic layer was collected, and the aqueous fraction was washed with dichloromethane (3 x 30 mL). The combined organic extracts were washed with brine (30 mL), dried over anhydrous magnesium sulfate, and concentrated under vacuum. The product was purified by column chromatography on silica gel (60 : 40 hexanes/ethyl acetate) afforded the product as a white solid (713 mg, 46%).

R_f: 0.29 (4 : 6 hexanes/ethyl acetate).

m.p. 77.6-79.6 °C.

¹H NMR (400 MHz, CDCl₃): δ 8.03 (d, J = 8.6 Hz, 1H), 7.96 (d, J = 5.2 Hz, 1H), 7.55 (d, J = 8.5 Hz, 1H), 7.35 (d, J = 8.5 Hz, 1H), 7.28 (d, J = 8.6 Hz, 1H), 6.99 (d, J = 2.1 Hz, 1H), 6.92 (dd, J = 8.4, 2.1 Hz, 1H), 6.79 (d, J = 8.3 Hz, 1H), 6.43 (d, J = 5.2 Hz, 1H), 6.33 (d, J = 6.4 Hz, 1H), 6.21 (s, 1H), 5.42 (d, J = 6.4 Hz, 1H), 4.02 (qd, J = 7.0, 2.3 Hz, 2H), 3.82 (s, 3H), 2.15 (s, 3H), 2.04 (s, 1H), 1.39 (t, J = 7.0 Hz, 3H).

¹³C{¹H} NMR (126 MHz, CDCl₃): δ 158.2, 149.4, 148.9, 148.6, 148.5, 148.4, 147.8, 139.0, 138.2, 134.4, 126.5, 126.3 122.8, 119.1, 118.1, 115.3, 112.1, 111.4, 107.5, 64.4, 56.0, 54.7, 21.4, 14.8.

HRMS: (ESI) m/z [M+H]⁺ calcd. for C₂₅H₂₅ClN₃O₃⁺ 450.1579; found 450.1576.

Tert-butyl ((8-((tert-butoxycarbonyl)oxy)-2-chloroquinolin-7-yl)(3-ethoxy-4-methoxyphenyl)methyl)(4-methylpyridin-2-yl)carbamate (pre-HQ415).

2-Chloro-7-((3-ethoxy-4-methoxyphenyl)((4-methylpyridin-2-yl)amino)methyl)quinolin-8-ol (31.4 mg, 0.0697 mmol, 1.0 equiv), Boc₂O (56.0 μL, 0.244 mmol, 3.5 equiv), and 4-dimethylaminopyridine (17.0 mg, 0.139 mmol, 2.0 equiv) were dissolved in tetrahydrofuran (0.5 mL). The reaction was stirred for 24 h at room temperature and then diluted with dichloromethane (10 mL). The organic solution was extracted with brine (10 mL), the organic layer were collected, and the aqueous fraction was washed with dichloromethane (3 x 10 mL). The combined organic extracts were washed again with brine (10 mL), dried over anhydrous magnesium sulfate, and concentrated under vacuum. Purification by column chromatography on silica gel (3 : 2 hexanes/ethyl acetate) afforded the product as a white solid (30.9 mg, 68% yield).

R_f: 0.89 (2 : 3 hexanes/ethyl acetate).

m.p. 80.3-82.4 °C.

¹H NMR (400 MHz, CDCl₃): δ 8.18 (d, J = 5.1 Hz, 1H), 8.02 (d, J = 8.6 Hz, 1H), 7.88 (d, J = 8.7 Hz, 1H), 7.55 (d, J = 8.7 Hz, 1H), 7.34 (d, J = 8.6 Hz, 1H), 7.12 (s, 1H), 6.99 (s, 1H), 6.95 (d, J = 2.1 Hz, 1H), 6.87 (dd, J = 8.4, 2.1 Hz, 1H), 6.77 (d, J = 4.6 Hz, 1H), 6.72 (d, J = 8.3 Hz, 1H), 3.96 (p, J = 6.9 Hz, 2H), 3.81 (s, 3H), 2.17 (s, 3H), 1.54 (s, 1H), 1.45 (s, 9H), 1.37 (t, J = 6.9 Hz, 3H), 1.31 (s, 9H).

¹³C{¹H} NMR (176 MHz, CDCl₃): δ 154.6, 154.0, 151.0, 150.9, 148.2, 147.7, 147.5, 144.4, 140.6, 138.5, 134.9, 131.6, 129.2, 126.9, 124.4, 123.7, 122.9, 122.4, 121.3, 113.7, 110.7, 83.4, 81.4, 64.2, 60.4, 56.0, 28.3, 27.6, 20.9, 14.8 (d, J = 2.3 Hz).

HRMS: (ESI) m/z [M+H]⁺ calcd. for C₃₅H₄₁ClN₃O₇⁺ 650.2628; found 650.2619.

Preparation and characterization of fluorinated standards

8-(Benzyloxy)-2-fluoroquinoline.

4-(Benzyloxy)-2-chloroquinoline (135.6 mg, 0.50 mmol, 1.0 equiv) and Me₄NF•t-AmylOH (1:0.95) (132.6 mg, 0.75 mmol, 1.5 equiv) were dissolved in anhydrous DMSO (2.5 mL). The reaction was stirred for 24 h at 80 °C. After cooling to room temperature, the mixture was diluted with dichloromethane (10 mL), and the organic solution was extracted with brine (10 mL). The organic layer was collected, and the aqueous fraction was washed with dichloromethane (3 x 10 mL). The combined organic extracts were then combined, washed with brine (10 mL), dried over anhydrous magnesium sulfate, and concentrated under reduced pressure. Purification by column chromatography on silica gel (4 : 1 hexanes/ethyl acetate) afforded the product as a light yellow solid (85.0 mg, 67% yield).

R_f: 0.71 (7 : 3 hexanes/ethyl acetate).

m.p. 68.6-69.7 °C.

¹H NMR (400 MHz, CDCl₃): δ 8.22 (t, J = 8.4 Hz, 1H), 7.53–7.49 (multiple peaks, 2H), 7.44–7.33 (multiple peaks, 4H), 7.30 (t, J = 7.3 Hz, 1H), 7.10 (td, J = 8.4, 2.2 Hz, 2H), 5.42 (s, 2H).

¹³C{¹H} NMR (126 MHz, CDCl₃): δ 160.7 (dd, J = 241.9, 3.7 Hz), 153.6 (d, J = 3.2 Hz), 142.1 (dd, J = 9.7, 3.6 Hz), 137.7 (dd, J = 15.3, 3.7 Hz), 136.9 (d, J = 3.9 Hz), 128.7 (d, J = 3.8 Hz), 128.2, 127.9 (d, J = 3.7 Hz), 127.1 (d, J = 3.8 Hz), 126.2 (d, J = 4.1 Hz), 119.7 (d, J = 3.7 Hz), 111.7 (d, J = 3.7 Hz), 110.6 (dd, J = 42.5, 3.7 Hz), 70.9 (d, J = 3.7 Hz).

¹⁹F NMR (377 MHz, CDCl₃): δ –61.07 (dd, J = 8.1, 3.1 Hz).

HRMS: (ESI) m/z [M+H]⁺ calcd. for C₁₆H₁₃FNO⁺ 254.0976; found 254.0982.

2-Fluoroquinolin-8-ol.

4-(benzyloxy)-2-fluoroquinoline (93.2mg, 0.367mmol, 1.0 equiv) was added to a 20mL vial and dissolved in DCM (1.5mL). A 1M solution of BBr₃ in DCM (0.147mL, 1.47mmol, 4.0 equiv) was added to the reaction dropwise. The reaction was capped and stirred for 24 hours at room temperature. After 24 hours, the reaction was diluted with DCM (10 mL) and brine (10 mL). The organic layer was collected and the aqueous fraction was washed 3 times with DCM (10 mL). The combined organic phases were then washed with brine (10 mL), dried over anhydrous magnesium sulfate, and concentrated under reduced pressure using a rotary evaporator. Purification by column chromatography (85 : 15 hexanes/ethyl acetate) afforded the product as a white solid (49.7 mg, 83% yield).

R_f: 0.64 (8 : 2 hexanes/ethyl acetate).

m.p. 54.6-55.4 °C.

¹H NMR (700 MHz, CDCl₃): δ 8.26 (dd, J = 8.8, 7.7 Hz, 1H), 7.45 (t, J = 7.9 Hz, 1H), 7.40 – 7.34 (multiple peaks, 2H), 7.23 (dd, J = 7.6, 1.3 Hz, 1H), 7.10 (dd, J = 8.8, 2.3 Hz, 1H).

¹³C{¹H} NMR (176 MHz, CDCl₃): δ 160.5 (d, J = 246.0 Hz), 151.3 (d, J = 2.1 Hz), 142.5 (d, J = 9.6 Hz), 135.3 (d, J = 13.3 Hz), 127.2 (d, J = 2.5 Hz), 127.1 (d, J = 2.0 Hz), 118.1, 112.1, 110.6 (d, J = 40.9 Hz).

¹⁹F NMR (377 MHz, CDCl₃): δ –64.33 (d, J = 7.5 Hz).

HRMS: (ESI) m/z [M+H]⁺ calcd. for C₉H₇FNO⁺ 164.0507; found 164.0506.

Tert-butyl ((8-((tert-butoxycarbonyl)oxy)-2-fluoroquinolin-7-yl)(3-ethoxy-4-methoxyphenyl)methyl)(4-methylpyridin-2-yl)carbamate (F-inter-HQ415).

7-((3-Ethoxy-4-methoxyphenyl)((4-methylpyridin-2-yl)amino)methyl)-2-fluoroquinolin-8-ol (23.6 mg, 0.054 mmol, 1.0 equiv), Boc₂O (124 μL, 0.54 mmol, 10.0 equiv), and 4-dimethylaminopyridine (26.4 mg, 0.216 mmol, 4.0 equiv) were dissolved in tetrahydrofuran (0.5 mL). The reaction was stirred for 48 h at room temperature and then diluted with DCM (10 mL). The organic solution was extracted with water (10 mL), the organic layer was collected, and the aqueous fraction was washed with dichloromethane (3 x 10 mL). The combined organic extracts were washed with brine (10 mL), dried over anhydrous magnesium sulfate, and concentrated under vacuum. Purification by column chromatography on silica gel (7 : 3 hexanes/ethyl acetate) afforded the product as a white solid (11.4 mg, 33% yield).

R_f: 0.62 (6 : 4 hexanes/ethyl acetate).

m.p. 67.6-68.9 °C.

¹H NMR (400 MHz, CDCl₃): δ 8.22–8.13 (multiple peaks, 2H), 7.86 (d, J = 8.6 Hz, 1H), 7.58 (d, J = 8.7 Hz, 1H), 7.10 (s, 1H), 7.04 (dd, J = 8.9, 2.6 Hz, 1H), 6.98 (s, 1H), 6.96 (d, J = 1.9 Hz, 1H), 6.88 (d, J = 9.2 Hz, 1H), 6.77 (d, J = 5.1 Hz, 1H), 6.72 (d, J = 8.3 Hz, 1H), 3.96 (p, J = 6.9 Hz, 2H), 3.81 (s, 3H), 2.16 (s, 3H), 1.57 (s, 1H), 1.43 (s, 9H), 1.37 (t, J = 6.9 Hz, 3H), 1.30 (s, 9H).

¹³C{¹H} NMR (176 MHz, CDCl₃): δ 161.2 (d, J = 243.5 Hz), 154.5, 154.0, 150.9, 148.2 (d, J = 6.0 Hz), 147.6, 147.4, 144.2, 141.7 (d, J = 9.7 Hz), 135.2, 131.6, 128.3, 126.99 (d, J = 1.4 Hz, 124.4, 123.7, 122.4, 121.4, 113.8, 110.8, 110.7, 110.5, 83.4, 81.3, 64.2, 60.4, 56.0, 28.4, 28.3, 27.6, 20.9, 14.8.

¹⁹F NMR (377 MHz, CDCl₃): δ –60.57 (d, J = 7.7 Hz).

HRMS: (ESI) m/z [M+H]⁺ calcd. for C₃₅H₄₁FN₃O₇⁺ 634.2923; found 643.2912.

7-((3-Ethoxy-4-methoxyphenyl)((4-methylpyridin-2-yl)amino)methyl)-2-fluoroquinolin-8-ol (F-HQ415).

3-Ethoxy-4-methoxybenzaldehyde (119.3 mg, 0.662 mmol, 1.0 equiv), 2-amino-4-methylpyridine (71.6 mg, 0.662 mmol, 1.0 equiv), and 2-fluoroquinolin-8-ol (108.0 mg, 0.662 mmol, 1.0 equiv) were dissolved in ethanol (2.6 mL). The reaction was stirred for 380 d at room temperature and then diluted with DCM (10 mL). The organic solution was extracted with brine (10 mL), the organic layer was collected, and the aqueous fraction was washed with dichloromethane (3 x 10 mL). The combined organic extracts were washed again with brine (10 mL), dried over anhydrous magnesium sulfate, and concentrated under vacuum. Purification by column chromatography on silica gel (3 : 2 hexanes/ethyl acetate) afforded the product as a white solid (44.4 mg, 15% yield).

R_f: 0.29 (2 : 3 hexanes/ethyl acetate).

m.p. 66.7-68.5 °C.

¹H NMR (400 MHz, CDCl₃): δ 8.19 (t, J = 8.3 Hz, 1H), 7.92 (d, J = 5.4 Hz, 1H), 7.53 (d, J = 8.5 Hz, 1H), 7.33 (d, J = 8.5 Hz, 1H), 7.06 (dd, J = 8.7, 2.2 Hz, 1H), 7.01 (d, J = 2.1 Hz, 1H), 6.94 (dd, J = 8.3, 2.1 Hz, 1H), 6.80 (d, J = 8.3 Hz, 1H), 6.43 (d, J = 5.4 Hz, 1H), 6.34 (d, J = 6.3 Hz, 1H), 6.25 (s, 1H), 5.91 (s, 1H), 4.03 (qd, J = 6.9, 2.2 Hz, 2H), 3.83 (s, 3H), 2.16 (s, 3H), 1.40 (t, J = 6.9 Hz, 3H).

¹³C{¹H} NMR (126 MHz, CDCl₃): δ 160.7 (d, J = 245.6 Hz), 158.2, 149.0, 148.6, 148.5, 148.4, 147.6, 142.2 (d, J = 9.5 Hz), 135.7 (d, J = 13.4 Hz), 134.4, 126.9, 126.3, 126.2 (d, J = 2.2 Hz), 119.0 118.2, 115.3, 112.1, 111.4, 110.3 (d, J = 41.1 Hz), 107.6, 64.4, 56.0, 54.6, 21.4, 14.8.

¹⁹F NMR (377 MHz, CDCl₃): δ –63.86 (dd, J = 7.5, 2.5 Hz).

HRMS: (ESI) m/z [M+H]⁺ calcd. for C₂₅H₂₅FN₃O₃⁺ 434.1874; found 434.1879.

Radiochemistry

Materials and methods.

HPLC grade acetonitrile, potassium trifluoromethanesulfonate, and anhydrous dimethylformamide were purchased from Fisher Scientific. Sodium bicarbonate, kryptofix^® 2.2.2 (K_2.2.2), and anhydrous acetonitrile were purchased from Sigma-Aldrich. Tetramethylammonium bicarbonate (Me₄NHCO₃) was purchased from Enamine. Tetramethylammonium tetrafluoroborate (Me₄NBF₄) was purchased from TCI America. Sterile product vials (10 mL) were purchased from Hollister-Stier. QMA-light and C18 Lite Sep-Paks were purchased from Waters Corporation. QMA-light Sep-Paks were flushed with isopropanol (10 mL), 0.5 M aqueous sodium bicarbonate (10 mL), and MilliQ water (10 mL) prior to use for the generation of K¹⁸F and in situ Me₄N¹⁸F. C18 Sep-Paks were flushed with ethanol (10 mL) and sterile water for injection (10 mL) prior to use. High performance liquid chromatography (HPLC) was performed using a Shimadzu LC-2010A HT system equipped with a Bioscan B-FC-1000 radiation detector. Radio-TLC analyses were performed using a Bioscan AR 2000 Radio-TLC scanner with EMD Millipore TLC silica gel 60 plates (3.0 cm wide x 6.5 cm long). Residual TMA levels were determined using TLC spot tests as previously described.³² Chromatography was performed using the following conditions:

Chromatography Conditions

HPLC Conditions A.

Solvent conditions: Gradient

0 – 4 min	5 % acetonitrile in water, 0.1 % (v/v) trifluoroacetic acid
4 – 6 min	25 % acetonitrile in water, 0.1 % (v/v) trifluoroacetic acid
6 – 19 min	95 % acetonitrile in water, 0.1 % (v/v) trifluoroacetic acid
19 – 25 min	5 % acetonitrile in water, 0.1 % (v/v) trifluoroacetic acid

Flow rate: 2 mL/min, running time: 25 min

Column: Phenomenex® Kinetex PFP column 250 × 4.6 mm, 5 μm

HPLC Conditions B.

Solvent: Isocratic 60 % acetonitrile in water, 0.1 % (v/v) trifluoroacetic acid

Flow rate: 2 mL/min, running time: 20 min

Column: Phenomenex® Luna PFP(2) column 250 × 4.6 mm, 5 μm

HPLC Conditions C.

Solvent: Isocratic 50 % acetonitrile in water, 0.1 % (v/v) trifluoroacetic acid

Flow rate: 5 mL/min, running time: 30 min

Column: Phenomenex® Luna PFP(2) column 250 × 10.0 mm, 5 μm

Radio-TLC method

Mobile phase: hexane/ethyl acetate (1/1)

Scanning time: 1 min

Scanning range: 60 mm – 125 mm

Spotting position of radioactive product: 65 mm

Generation of [¹⁸F]Fluoride

Generation of [¹⁸F]KF.

The [¹⁸F]fluoride was delivered to the automated synthesis module (TRACERLab FX_FN, GE) in a 1.5 mL bolus of ¹⁸O target water and was trapped on a preconditioned QMA-light Sep-Pak to remove ¹⁸O target water and other aqueous impurities. [¹⁸F]Fluoride was eluted into the reaction vessel using aqueous potassium trifluoromethanesulfonate (5 mg in 0.5 mL water, 0.05 M) with potassium carbonate (0.5 mg) and K_2.2.2 in acetonitrile (15 mg, 1 mL, 0.04 M). Azeotropic drying/evaporation was achieved by heating the reaction vessel at 100 °C while drawing vacuum for 6 min. The reaction vessel was then subjected to an argon stream and simultaneous vacuum draw for an additional 6 min to produce anhydrous [¹⁸F]KF•K_2.2.2. The reaction vessel was cooled to room temperature under an argon stream, and anhydrous DMF (6 mL) was added. The mixture was heated to 50 °C with stirring for 5 min to suspend the [¹⁸F]KF•K_2.2.2. The resulting solution was cooled to room temperature and was transferred to a sterile vial.

Generation of in situ [¹⁸F]Me₄NF.

[¹⁸F]KF•K_2.2.2 was prepared by the same protocol described above. A aliquot of [¹⁸F]KF•K_2.2.2 in DMF was added to a vial containing a tetramethylammonium salt (e.g. Me₄NHCO₃ or Me₄NBF₄, 100-300 μmol) and the substrate (20-50 μmol) in DMF.

Radiosynthesis of ¹⁸F-labeled molecules

Manual synthesis general procedure.

A 4 mL vial was charged with chloro-, nitro-, or triflate substrate (50 μmol) and tetramethylammonium salt (Me₄NHCO₃ or Me₄NBF₄) (100-300 μmol) on the bench top. An aliquot of [¹⁸F]KF•K_2.2.2 in DMF (100 μL, 92.5 – 129.5 MBq (2500–3500 μCi) of radioactivity) was used for each manual reaction. Total reaction volume was 100μL. The reaction vial was sealed with a Teflon-lined cap and heated in an aluminum block at 100-110 °C for 30 min. After 30 min, the reaction was cooled to room temperature, and the radiochemical yield (RCY, %) was determined by radio-TLC analysis using the following procedure. The crude reaction mixture was spotted onto a standard silica-coated glass TLC plate (EMD Millipore Corporation, TLC Silica-gel 60 F₂₅₄) and developed with hexanes:ethyl acetate (1:1) in a glass TLC chamber. The RCY was determined by dividing the integrated area of radiation under the fluorinated product spot by the total integrated area of radiation on the TLC plate. In reactions where radio-HPLC traces show multiple peaks, the RCY (determined by radio-TLC) was corrected by dividing the integrated area of radiation under the desired ¹⁸F-labeled product peak by the total integrated area of radiation on the analytical radio-HPLC (HPLC conditions A). To prepare samples for HPLC analysis, 80 μL of the reaction mixture was spiked with 20 μL of 2 mg/mL fluorinated standard solution in DMF. Eluent systems and columns used for HPLC analysis are described below.

$$\text{RCY}(\%)=\frac{\text{Integration of the radioproduct peak}}{\text{Sum of integration of all radiopeaks}}$$

Automated synthesis of 2-[¹⁸F]fluoroquinoline.

All loading operations were conducted under ambient atmosphere. Argon gas and vacuum were used for automated sample transfers. [¹⁸F]Fluoride was produced via the ¹⁸O(p, n)¹⁸F nuclear reaction using a General Electric (GE) PETTrace cyclotron (55 μA beam for 3 min generated ca. 63 GBq (1.7 Ci) of [¹⁸F]fluoride). [¹⁸F]KF•K_2.2.2 was prepared in a GE TRACERLab FX_FN automated synthesis module as described above. Dried [¹⁸F]KF•K_2.2.2 was prepared in the reactor pre-charged with tetramethylammonium bicarbonate (67.5 mg, 500 μmol, 5 equiv). A solution containing 2-chloroquinoline (16.4 mg, 100 μmol, 1 equiv) in 0.5 mL of anhydrous DMF was added to the reactor by applying Ar gas through the valve containing the anhydrous DMF. The open valves leading out of the reactor were closed, and the reaction mixture was stirred at 110 °C for 30 min. The mixture was cooled to 30 °C with compressed air cooling, and the resulting mixture was diluted with 1 mL of DI water and then with 3 mL of semi-preparative HPLC buffer (50% acetonitrile in water, 0.1 % (v/v) trifluoroacetic acid). The resulting solution was then loaded onto the HPLC injection loop and injected onto the semi-prep HPLC for purification by HPLC Conditions C described in Section 4.2.5. The peak for the ¹⁸F-labeled organic product was collected for 1 min (t_R = 8.4 min, collected volume: 5 mL) in a 10 mL sterile product vial. The dose vial was transferred out of the synthesis module. Final radiochemical yield and product identity were then determined using a Capintec dose calibrator and analytical HPLC (see Supplementary Information, Table S1).

Reformulation of 2-[¹⁸F]fluoroquinoline.

2-[¹⁸F]fluoroquinoline in HPLC mobile phase (5 mL) was diluted with water (40 mL) and passed through a C18 Lite Sep-Pak. The cartridge was washed with water (10 mL) and 2-[¹⁸F]fluoroquinoline was eluted into a sterile dose vial with ethanol (1 mL). Sterile saline (9 mL) was added to provide product formulated in 10 mL.

Molar activity of 2-[¹⁸F]fluoroquinoline.

A 10 μL aliquot was analyzed by HPLC, using HPLC Conditions B, and the area of the UV peak (280 nm) corresponding to the 2-fluoroquinoline standard (t_R = 8.9 min) was determined. The molar concentration (μmol/μL) of 2-fluoroquinoline in the sample was then determined by linear regression analysis against a standard curve generated from injection of identical volumes of solutions of known concentration of 2-fluoroquinoline. The concentration of activity was determined by dividing the total activity (4.588 – 7.474 GBq 12.4 – 20.2 ×10⁻² Ci) by the volume of the solution (5 mL). The end of synthesis (EOS) molar activity (Ci/μmol) is given by dividing the concentration of activity for 2-[¹⁸F]fluoroquinoline (GBq/μL or Ci/μL) by the molar concentration of the product (μmol/μL). EOS molar activity was found to be 155.5 ± 66.4 GBq/μmol (4.2 ± 1.8 Ci/μmol) for the high activity runs (n = 4, Table S1).

Synthesis of [¹⁸F]Inter-HQ415 followed by deprotection to provide [¹⁸F]HQ415.

A 4 mL vial was charged with pre-HQ415 (20 μmol) and tetramethylammonium bicarbonate (100 μmol) on the bench top. An aliquot of [¹⁸F]KF•K_2.2.2 in DMF (100 μL, 92.5 – 129.5 MBq (2500–3500 μCi) of radioactivity) was used for each manual reaction. Total reaction volume was 100μL. The reaction vial was sealed and heated in an aluminum block at 110 °C for 30 min. After 30 min, the reaction was cooled to rt, and 10 μL of the crude mixture was used for analysis [radiochemical yield (RCY, %) determined by radio-TLC analysis and analytical HPLC (HPLC Conditions A)]. Deprotection of the Boc groups was achieved by the addition of 2M aqueous HCl (200 μL) to the crude reaction mixture. The reaction was heated at 120 °C for 15 min to yield [¹⁸F]HQ415.