Methods and Compounds Using In-Loop Fluorination

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application for Patent, Serial No. 62/840,392 filed April 30, 2019, which is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The invention relates to compounds and methods of radiolabeling chemical compounds using a simple, efficient“in-loop” fluorination procedure.

Throughout this application, various publications are referenced within parentheses. Disclosures of these publications in their entireties are hereby incorporated by reference into this application to more fully describe the state of the art to which this invention pertains. Full bibliographic citations for these references may be found at the end of this application, preceding the claims.

BACKGROUND OF THE INVENTION

Fluorinated materials, that is, organic compounds or polymers containing a fluorine atom ( ¹⁸ F or ¹⁹ F) or a fluorinated moiety, possess unique biological, chemical and physical properties. Fluorinated materials find use in a wide variety of industries, such as electronics (for liquid crystal displays, photovoltaic cells), industrial and consumer coatings, aerospace, agriculture and agrochemicals, refrigerants, high energy density materials, explosives, and fuel sciences. In the life sciences, such as pharmaceuticals and medical imaging, polyfluorinated molecules are used in ¹⁹ F magnetic resonance imaging (MRI) and ¹⁸ F-labeled molecules are essential radiotracers for positron emission tomography (PET), a diagnostic tool for cancers and other diseases, and molecular imaging in drug development. The success of molecular imaging with positron-emission tomography (PET) depends on the

18 availability of selective molecular probes labeled with positron-emitters, such as fluorine- 18 ( F, tm = 109.7 min) or carbon-11 ( ⁿ C, tm = 20.3 min). ^{[1 3]} The captive solvent (“in-loop”) methodologies have become widely adopted for routine ⁿ C-radiotracer syntheses because of the simplicity, high radiochemical yields (RCY), speed, versatility and ease of automation. ^{[4 8]} For example, U.S. Patent No. 6,749, 830 B2, to Wilson et al. described the use of in-loop methodology to ^u C-methylate target substrates. This in-loop methodology for ^u C-labeled compounds involved trapping a radiolabeling reagent, such as [ ^u C]-iodomethane, directly in a standard HPLC loop coated with a precursor solution, allowing these reagents to react, directly injecting the reaction mixture onto a HPLC purification column and collecting the ⁿ C-radiolabeled product.

While this process has advantages for ^U C radiopharmaceutical production, however, isotopic labeling using the most common PET radionuclide, ¹⁸ F has not yet been achieved with this in-loop technology. Current methods of synthesizing ¹⁸ F-labeled radiotracers typically require an“in vial” process: introducing an ¹⁸ F-labeling agent into a vial containing solvent, precursor and optionally a catalyst, heating the components to allow them to react, quenching the reaction, then transfer of the materials to a solid phase extract, ion cartridge and/or HPLC for purification. This vial method has multiple steps, is time consuming, which reduces the overall RCY (radiochemical yield).

With the increasing demand for new ¹⁸ F-labeled PET radiotracers and their application in drug development involving multi-center clinical trials ^{[2 3]} , there is a pressing need for new and practical methods for the introduction of ¹⁸ F-labels into bioactive molecules and for more cost efficient and robust manufacturing processes for ¹⁸ F-labeled compounds and radiopharmaceuticals.

SUMMARY OF THE INVENTION

A new method to introduce an ¹⁸ F-label onto compounds of interest is presented here. Our invention solves the problems of the current in vial process, and results in streamlined manufacture of ¹⁸ F-labeled compounds and radiopharmaceuticals, which have use as radiopharmaceuticals for PET imaging. The method also has applications in ¹⁹ F-fluorination reactions.

A first aspect of the invention provides a method for ¹⁸ F-radiolabeling chemical compounds comprising the steps of reacting an [ ¹⁸ F] gas with a radiolabeling agent having a leaving group susceptible for nucleophilic substitution, to form a [ ¹⁸ F] radiolabeling, adding the [ ¹⁸ F] radiolabeling reagent to a precursor compound in an injection loop of a high performance liquid chromatograph (HPLC) without additional solid supports, to provide a reaction mixture comprising an ¹⁸ F radiolabeled compound, injecting the reaction mixture into the HPLC column and isolating the radiolabeled compound.

Another aspect of the invention provides a method for preparing ¹⁸ F -radiolabeled compounds wherein the [ ¹⁸ F] gas is [ ¹⁸ F]triflyl fluoride, the radiolabeling agent 1,4-dinitrobenzene to prepare the radiolabeling reagent by [ ¹⁸ F]l-fluoro-4-nitrobenzene.

Another aspect of the invention provides for methods of preparing [ ¹⁸ F] radiolabeling precursor chemical compounds by reacting [ ¹⁸ F]triflyl fluoride with 1,4-dinitrobenzene to form [ ¹⁸ F]l-fluoro-4-nitrobenzene radiolabeling reagent in an injection loop of a high performance liquid chromatograph (HPLC) without additional solid supports, adding a precursor compound to provide a reaction mixture comprising an ¹⁸ F radiolabeled compound; injecting the reaction mixture into the HPLC column; and isolating the radiolabeled compound.

Another aspect of the invention provides that precursor compounds are pharmaceuticals, drug candidates, and biomolecules. A further aspect provides that the precursor compounds are flortaucipir and FEPPA.

Another aspect of the invention provides for [ ¹⁸ F] radiolabeled compounds, formed by the process comprising reacting an [ ¹⁸ F] gas with a radiolabeling agent having a leaving group susceptible for nucleophilic substitution to form a [ ¹⁸ F] radiolabeling reagent, adding the [ ¹⁸ F] radiolabeling reagent to a precursor compound in an injection loop of a high performance liquid chromatograph (HPLC) without additional solid supports, to provide a reaction mixture comprising an ¹⁸ F radiolabeled compound; injecting the reaction mixture into the HPLC column; and isolating the radiolabeled compound.

Another aspect of the invention provides for the use the compounds produced by the methods of the invention as radiopharmaceuticals.

Another aspect of the invention provides that ¹⁸ F gas is selected from the group consisting of [ ¹⁸ F]triflyl fluoride, [ ¹⁸ F]F2, [ ¹⁸ F]acetyl hypofluorite (AcOHF), [ ¹⁸ F]fluorohaloalkanes, [ ¹⁸ F]ethene sulfonyl fluoride, [ ¹⁸ F]fluoroform, and ¹⁸ F[HF]

Another aspect of the invention provides the radiolabeling agent is selected from the group consisting of halogens, nitro, iodine(III)-based groups, tosyloxy-, trimethylammonium FPEB, SDM- 8, fallypride, FLB457, FLT, FDOPA,and 1,4-dinitrobenzene.

Another aspect of the invention provides that the [ ¹⁸ F] gas is [ ¹⁸ F]triflyl fluoride, the radiolabeling agent is 1,4-dinitrobenzene, the precursor compound is selected from the group consisting of flortaucipir and FEPPA.

Another aspect of the invention provides for formulating the ¹⁸ F radiolabeled compounds produced by the invention for use as PET imaging agents and further obtaining a PET image of an organ or tissue of interest using the ¹⁸ F radiolabeled compounds of the invention.

Another aspect of the invention provides a method for adding a ¹⁹ F fluorine label to a compound by reacting an [ ¹⁹ F] gas with a labeling agent to form a [ ¹⁹ F] labeling reagent, adding the [ ¹⁹ F] labeling reagent to a precursor compound in an injection loop of a high-performance liquid chromatograph (HPLC) without additional solid supports, to provide a reaction mixture comprising an ¹⁹ F labeled compound; injecting the reaction mixture into the HPLC column; and isolating the ¹⁹ F labeled compound.

Specific preferred embodiments of the invention will become evident from the following more detailed description of certain preferred embodiments and the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 is a schematic drawing of an“in-loop” ¹⁸ F-labeling system. All terms not defined in this specification are given their usual and customary definitions as used by one of ordinary skill in the field.

DETAILED DESCRIPTION OF THE INVENTION

We have developed a new method of“in-loop” Fluorine labeling of compounds. Our method is generally applicable to ¹⁹ F labeling of materials as further discussed herein, it is preferably useful in ¹⁸ F-labeling radiopharmaceutical chemistry, and more preferably for ¹⁸ F-labeling of materials for PET imaging.

While the process for ^U C- radiolabeling is well known, until recently no ¹⁸ F-fluoride sources existed that were suitable for use in the“in-loop” process.

We discovered a method for performing ¹⁸ F -radiolabel reactions in the loop of an HPLC system. Our method uses a volatile, condensable ¹⁸ F Fluorine gas, such as [ ¹⁸ F]triflyl fluoride, reacted with a compound such as 1,4-dinitrobenzene to form a radiolabeling compound, [ ¹⁸ F] l-fluoro-4- nitrobenzene, which is radiolabeling compound is further used for the production of ¹⁸ F -radiotracer compounds from precursor compounds, all in the loop of the HPLC system.

Our process allows for a reduction in the amount of precursor needed, which not only conserves valuable materials but also can facilitate chromatographic purification. Our process eliminates the time consuming, multistep process used to prepare these ¹⁸ F-labeled materials in vials and results in compatible RCY. Our invention provides a simple, efficient, and a reliable production of radiofluorinated compounds and radiopharmaceuticals.

Using this method, we introduced ¹⁸ F-labels onto precursors and two well-known radiopharmaceuticals, namely, [ ¹⁸ F]T807 (a.k.a. [ ¹⁸ F]AV-1451; flortaucipir) for imaging of tau protein, and [ ¹⁸ F]FEPPA ((7V-acetyl-/V-(2-[ ¹⁸ F]fluoroethoxyben-zyl)-2-phenoxy-5-pyridinamine)) for imaging the translocator protein 18 KDa. As shown in the examples, we synthesized and formulated the radiotracers [ ¹⁸ F]FEPPA and [ ¹⁸ F]T807 using an automated radiosynthesis module with non decay corrected radiochemical yields of 27% and 29% (relative [ ¹⁸ F]F ), respectively. In these cases, our“in-loop” radiofluorination methodology enabled us to obtain equal or superior yields compared with conventional reactions in a vial. The radiochemical purities were >99% and the molar activities were >350 GBq/pmol at the end-of-synthesis for both radiotracers.

Selection of Precursor Compounds. Generally, precursor compounds are compounds of interest for ¹⁸ F-labeling, such as pharmaceuticals, drug candidates, biomolecules that target biomarkers (such as amino acids, peptides, proteins), and the like. The compounds can be aromatic and aliphatic precursor compounds. Our method is particularly useful for precursor compounds that are fluorinated and purified by HPLC, such as the FDA-approved radiopharmacueticals, flutemetamol ¹⁸ F injection, (Vizamyl) and florbetaben ¹⁸ F injection (Neuaceq).

Selection of ¹⁸ F source. Volatile, condensable gaseous sources of ¹⁸ F are preferred. While the reaction of [ ¹⁸ F]triflyl fluoride (see Table 1, [ ¹⁸ F]2), ^[9]) is used in the examples, other volatile condensed gases containing ¹⁸ F (“radiofluorinated”) may be used in practicing this invention. Non limiting radiofluorinated gas examples include [ ¹⁸ F]F2, [ ¹⁸ F]acetyl hypofluorite (AcOHF), [ ¹⁸ F]fluorohaloalkanes,[ ¹⁸ F]ethene sulfonyl fluoride, [ ¹⁸ F]fluoroform, [ ¹⁸ F]HF, fluorinated gases and ¹⁸ F[HF]

Selection of Compounds for Radiolabeling Agents. Reaction of the ¹⁸ F source with suitable compounds to form the ¹⁸ F radiolabeling agent have a leaving group susceptible for nucleophilic substitution such as halogens, nitro, iodine(III)-based groups, tosyloxy-, trimethylammonium, etc. Examples of compounds suitable for reaction to form radiolabeling reactions include, FPEB, SDM- 8, fallypride, FLB457, FLT, FDOPA, etc. 1,4-dinitrobenzene is the preferred compound.

Selection of Base. Suitable bases for the ¹⁸ F-labeling for nucleophilic fluorinations are generally should be soluble in dry aprotic solvents such as DMF, DMSO CFpCN and DMA. Ionic liquids may also be used. The reactions can be conducted at room temperature or at elevated temperatures. In our examples, our method produced ¹⁸ F-labeled product in near quantitative yield with tetraethylammonium bicarbonate (TEAB) as the base (Table 1, entries 1-2). Similar RCY was obtained with Kryptofix 222 / potassium bicarbonate (KHCO3 / K222) as the base (Table 1, entry 3). Because KHCO3 / K222 is sparingly soluble in DMSO, the solution required mixing using a vortex over- night for complete dissolution. The organic base, 2-tert-butylimino-2-diethylamino-l,3- dimethylperhydro-l,3,2-diazaphosphorine (BEMP), also promotes the [ ¹⁸ F]3 formation (Table 1, entry 4), but a higher temperature was needed to reach completion (Table 1, entry 5).

Selection of Solvent. Preferred solvents for use as reaction media are anhydrous aprotic solvents with high viscosity and boiling points for two reasons: (i) solvents with high viscosity are commonly preferred for captive solvent methods, and; (ii), the preferred solvent must prevent issues related to pressure increases during loop heating. Non-limiting examples include DMSO, DMF, and others as discussed below, shown in the tables, and known to those skilled in the art.

“In Loop” System. A standard HPLC injection system 1, as shown in Fig. 1 can be used in practicing the methods of the invention and producing the radiotracer compounds. With reference to Fig. 1, the“in-loop” ¹⁸ F-fluorination method and system (1) consists of: (i) delivery of compound 10 ([ ¹⁸ F]2) under helium 11 and the initial trapping of gaseous Compound [ ¹⁸ F]2 10 via base-mediated conversion to dry [ ¹⁸ F]F , followed by, (ii)“in-loop” radiofluorination to form ¹⁸ F-labeled products via HPLC system 30. Importantly, these steps are performed directly in a stainless steel HPLC loop 20. For easy adoption of this method for routine radiopharmaceutical production, we integrated the “in-loop” method to a commercial radiosynthesis module to carry out automated HPLC purification and formulation of the labeled compounds. Standard HPLC injection valves 30, columns, and loops 20 may be used with the invention. Solid supports are not necessary in this method.

Additional loops and/or vials can be added to the system for multi-step reactions, subsequent reactions, and purification steps.

Using the system 1 shown in Fig. 1, Compound [ ¹⁸ F]2 (at 10 in Fig. 1) was prepared and delivered in a stream of helium gas (5 mL/min) 11 to a reaction vessel 12 as previously described. ^[9] Compound [ ¹⁸ F]2 is continuously distilled out from the reaction vessel into a second vial containing dry aprotic solvent 13, in which dry [ ¹⁸ F]fluoride is released in the presence of base, where it is then transferred by the HPLC system 30 (in a general representation, with solvent from HPLC pump shown at 50) to a pre-coated HPLC loop 20 (2,4-dinitrobenzene (6 pmol), base (2 - 6 pmol) dissolved in 200 pL anhydrous aprotic solvent) instead of being transferred to a vial. After heating (100 °C) the HPLC loop 20 with a heating source 40 for 10 minutes the crude reaction mixture was transferred using the HPLC 30 (5 mL/min for 2 min) to 42 an empty receiving vial or additional columns or analysis. Unreacted material proceeds through 60 for waste or other processing. The RCY shown in Table 1 is determined by radio-HPLC analysis of the crude reaction mixture.

Table 1.“In-loop” synthesis of [ ¹⁸ F] l-fluoro-4-nitrobenzene ([ ¹⁸ F]3) via [ ¹⁸ F]2

"in-ioop"

RCY of

Entry

Base r ¹⁸ Fl2 trapped (%) ^c Solvent I ¹⁸ F13 (%) ^d

1 TEAB >99 DMSO 96±l ^e

2 TEAB >99 DMF 94

3 KHCCE / K222 >99 DMSO 95

4 BEMP >99 DMSO 70

5 ^b BEMP >99 DMSO 93

aReaction conditions: 1,4-dinitrobenzene (6 pmol). Base (TEAB (2 pmol), KHCO3 / K222 (2 pmol), BEMP (6 pmol)), solvent (200 pL), and 100 °C for 10 min. ^b 130 °C was used instead of 100 °C. Percentage of [ ¹⁸ F]F ^_ left in-loop after entrapment. ^d Non-isolated radiochemical yield (RCY), based on radio-HPLC analysis of the crude product. ^e (n = 2); all other entries unless noted represent single experiments.

The reaction of /VpV-bi s(trifluorom ethyl sulfonyl)aniline (1) with aqueous [ ¹⁸ F]fluoride ([ ^{I X} F]F-) yielded [ ¹⁸ F]2 in high radiochemical yields (RCY, >95%). [ ¹⁸ F]2 is continuously distilled out from the reaction vessel into a second vial containing dry aprotic solvent in which dry [ ¹⁸ F]fluoride is released in the presence of base. This method was used to effectively label a range of aromatic as well as aliphatic compounds in moderate to high RCYs.

This“in-loop” [ ¹⁸ F]fluorination methodology was then applied for the syntheses of [ ¹⁸ F]T807 and [ ¹⁸ F]FEPPA (Table 2). The synthesis of [ ¹⁸ F]T807 have previously been reported by several research groups in RCYs ranging from 14 - 32%. ^[11 - ^15] [ ¹⁸ F]T807 is commonly produced via our two- step one-pot procedure ^[12] , which entails firstly the nucleophilic ¹⁸ F-fluorination of the nitro- or tetramethylammonium based precursor compound, followed by thermal boc-deprotection to form the desired product. Some laboratories employ an acidic promoted deprotection however, this is not required for the reaction and is not easily implemented on a standard HPLC loop, and therefore thermal deprotection was selected as the preferred route. We made one minor alteration to the standard conditions (Table 1, entry 1), by employing a higher temperature (130°C instead of 100°C) for the“in loop” reaction to promote thermal deprotection.

In these reactions, [ ¹⁸ F]T807 was obtained in a non-isolated RCY of 42% (Table 2, entry 1). Next, the concentration of base was evaluated (Table 2, entries 1 - 4). In general, larger amounts of base improved the RCY. The highest RCY was obtained at a base concentration of 10 mg/mL (52%, Table 2, entry 2). We and others have previously reported the“in-vial” radiosynthesis of the

[ ¹⁸ F]FEPPA, which generally proceeds in good radiochemical yield (RCY = 30-40 % ). ^[10 _’ ^16] However, using standard conditions for the“in-loop” ¹⁸ F-fluorination failed to generate any product (Table 2, entry 5). On the other hand, with KHCO ₃ /K ₂₂₂ as base and C¾CN as the solvent, a non isolated RCY of 45% was achieved (Table 2, entry 6). In a last attempt to further increase the yield, BEMP was used as the fluorination base, however, a decreased RCY was obtained (Table 2, entry 7).

Table 2.“In-loop” formation

_ [ ¹⁸ F1T807 _

[ ¹⁸ F]2 trapped RCY of

Entry Base (nmol) Solvent Temperature (°C) (%) ^b | ^l8 F|3 (%) ^c

1 TEAB (2) DMSO 130 >99 42

2 TEAB (10) DMSO 130 >99 52

3 TEAB (4) DMSO 130 >99 45

4 TEAB (6) DMSO 130 >99 50 5 TE B (2) >99 <1

6 KHCO3/K222 (2) CH3CN 80 >99 45

7 _ BEMP (6) _ CH3CN _ 80 _ >99 _ 12

aReaction conditions: precursor (FEPPA precursor (1.8 pmol), T807 precursor (1.25 pmol)), base (2 -10 pmol), solvent (200 pL), and 80 - 130 °C for 10 min. ^b Percentage [ ¹⁸ F]F left in-loop after entrapment. ^c Isolated radiochemical yield (RCY), based on radio-HPLC analysis of the crude product.

To further exemplify the utility this newly developed methodology, [ ¹⁸ F]T807 and [ ¹⁸ F]FEPPA, were isolated using semi-preparative HPLC followed by solid-phase extraction (SPE) to generate the final formulated product. To achieve this, after completed reaction, the crude reaction mixture was injected directly onto the HPLC column for purification. [ ¹⁸ F]T807 and [ ¹⁸ F]FEPPA were obtained in isolated and non-decay corrected RCYs (relative [ ¹⁸ F]F ) of 27% and 29%, respectively. The radiochemical purity (RCP) was >99% and the molar activity (A _m ) >350 GBq/pmol at the end-of-synthesis.

Once collected and purified, the ¹⁸ F -radiolabeled compounds are suitable for use as PET imaging agents in animal or human subjects using the appropriate PET imaging protocols as known by those who practice in the art. The ¹⁸ F -radiolabeled compounds can be formulated for administration by any method known to those in the art for PET imaging. For example, the product can be formulated using 10 mL of saline (0.9% sodium chloride) and finally passed through a sterile filter (Millex-GS, 0.22 pim) in preparation for IV injection prior to imaging the subject.

1 ⁹ F -Fluorine Labeling Chemistry

Our method also is applicable to ¹⁹ F -Fluorine chemistry. The in-loop reactions are performed in a fluoroplastic HPLC loop such as Teflon or FEP, or a stainless-steel loop or alternative-metal loop. For non-metal loops, the loop is passivated with F2 prior to use. In these reactions any fluorinated gas can be used in the reaction, including non-radiofluorinated versions of the gases listed above, and anhydrous HF, F2 (dilute to 100% concentration), acetyl hypofluorite (AcOF), halofluorocarbons, etc. are used.

As described in the example, reactions can be carried out in solution on a thin film of substrate, or in an acid or base medium, and using the loop for pharmaceutical or industrial chemistry applications.

EXAMPLES

The invention is further illustrated by the following examples, so that a person of ordinary skill in the art may obtain better understanding of the invention. These examples are only provided as exemplary to illustrate the invention and should not be interpreted to limit the invention.

Unless otherwise stated, all reagents were obtained from commercially available sources and used without further purification.

Example 1.

No-carrier-added [ ¹⁸ F]F production was performed using a MCI 7 cyclotron (Scanditronix). The ^ls O(p,n) ¹⁸ F reaction was employed in a liquid target containing 10% [ ¹⁸ 0]H ₂ 0 by bombardment with a 30 mA proton beam (16.4 MeV) for 30 min. The RCP and A _m of the isolated products was determined by reverse phase HPLC. Identification of all radioactive products was confirmed by co elution with the corresponding non-radioactive compound. The HPLC analysis was performed using a high-pressure isocratic pump (Shimadzu LC-20AT) and a variable wavelength UV-detector (l = 254 nm, Shimadzu SPD-20A) with a radioactivity detector (Frisk-tech, Bicron) connected in series. The system was controlled by PowerChrom chromatography software. A modified Tracerlab FX2 _C module (GE) equipped with a Cl 8 semi-preparative HPLC column as well as 5-mL HPLC loop was utilized for automated purification and“in-loop” reaction platform.

Example 2. “In-loop” fluorination of [ ¹⁸ F] l-fluoro-4-nitrobenzene ([ ¹⁸ F]3). Prior to the start-of-synthesis, the precursor compound, 2,4-dinitrobenzene (6 mihoΐ), cryptand and/or base (TEAB (2 pmol), K222 / KHCO3 (2 pmol), or BEMP (6 pmol)) dissolved in 200 pL solvent (DMF or DMSO) was loaded to the HPLC loop (5-mL). Cyclotron produced [ ¹⁸ F]F was loaded and concentrated a ion-exchange column (Chromafix) from one side and the accumulated [ ¹⁸ F]F was further eluted from the other side with 0.5 mL of aqueous 0.1 M K2SO4 solution into a 5-mL vial pre charged with 4-6 mg of N,N-bis(trifluoromethylsulfonyl)aniline (1, Table 1) in 1 mL of DMF. The resulting mixture was heated (50°C for 5 min, block heater, IKA) and the formed [ ¹⁸ F]triflyl fluoride ([ ¹⁸ F]2) was continuously blown out with helium (5 pL/min) from the vial, first through a drying column (P2O5) and finally into the pre-coated HPLC loop. [ ¹⁸ F]2 was trapped by base-mediated conversion to [ ¹⁸ F]F inside the HPLC loop. Unreacted [ ¹⁸ F]2 was subsequently trapped quantitatively by an ascarite trap (hydroxide-coated silica). The loop was then heated to the desired temperature for 10 min using a pre-heated oil bath. The crude reaction mixture was cooled to room temperature and transferred to an empty receiving vial using the HPLC pump (5 mL/min for 2 min, CH3CN-NH4CO2H (0.1 M) (30/70, v/v)). The non-isolated RCY was measured using radio-HPLC.

Example 3. Synthesis of [ ¹⁸ F]FEPPA.

Prior to the start-of-synthesis, FEPPA precursor (1.8 pmol), KHCO3 (2 pmol), K222 (2 pmol) dissolved in 200 pL CH3CN was loaded to the HPLC loop (5-mL) prior to the start-of-synthesis. The formed [ ¹⁸ F]2 was produced transferred into the pre-coated HPLC loop. The resulting mixture was heated to 80°C for 10 min. Following this reaction, the crude mixture was cooled to room temperature and injected onto an HPLC column for purification and subsequent formulation. [ ¹⁸ F]FEPPA was purified using a reverse-phase HPLC on a Luna C-18 column (250 mm x 7.8 mm, 10 pm, Phenomenex), and CH ₃ 0H-formic acid (0.5% in H2O) (50/50, v/v) was used as the eluting solvent at a flow rate of 7 mL/min. The eluent was monitored by a UV absorbance detector (l = 254 nm) in series with a GM tube radioactivity detector. The product was eluted at 20-22 min. The fraction of the desired compound was collected into a vial pre-filled with 25 mL sterile water and 2 mL of NaHCC solution (8.4% in H2O) directly from the HPLC column. The SPE was pushed by helium pressure (1 Bar) through a SPE cartridge (SepPak tC18, Waters). The SPE was first washed with 10 mL of sterile water and eluted using 1 mL of ethanol. The product was finally formulated using 10 mL of saline (0.9% sodium chloride). The RCP and A _m were determined by reverse phase HPLC (4.6 x 250 mm, 5 pm, Phenomenex) and was eluted with acetonitrile-MLCCkH (0.1 M) (50/50, v/v) at a flow rate of 3 mL/min (retention time = 4 - 5 min).

Example 4. Synthesis of [ ¹⁸ F]T807.

Prior to the start-of-synthesis, T807 precursor (1.25 pmol), TEAB (10 pmol) dissolved in 200 pL DMSO was loaded to the HPLC loop (5-mL) prior to the start-of-synthesis. The formed [ ¹⁸ F]2 produced was transferred into the pre-coated HPLC loop. The resulting mixture was heated to 130°C for 10 min. After this reaction, the crude mixture was cooled to room temperature and injected onto an HPLC column for purification and subsequent formulation. [ ¹⁸ F]T807 was purified using reverse- phase HPLC on a Luna C-18 column (100 mm x 21.2 mm, 5 pm, Phenomenex), and CH3OH- NH4CO2H (0.1 M) (50/50, v/v) was used as the eluting solvent at a flow rate of 9 mL/min. The eluent was monitored by a UV absorbance detector (l = 254 nm) in series with a GM tube radioactivity detector. The product was eluted at 31-33 min. The fraction of the desired compound was collected into a vial pre-filled with 25 mL sterile water and 2 mL of NaHCCb solution (8.4% in H2O) directly from the HPLC column. The SPE was pushed by helium pressure (1 Bar) through a SPE cartridge (SepPak tC18, Waters). The SPE was first washed with 10 mL of sterile water and eluted using 1 mL of ethanol. The product was finally formulated using 10 mL of saline (0.9% sodium chloride). The RCP and A _m were determined by reverse phase HPLC (4.6 x 250 mm, 5 pm, Phenomenex) and was eluted with CH ₃ CN-NH ₄ CO ₂ H (0.1 M) (30/70, v/v) at a flow rate of 3 mL/min (retention time = 4 - 5 min).

Example 5. ¹⁹ F Fluorination of L-Tyrosine using In-Loop Process.

Diluted F2 (0.5- 10% in Ne) to elemental 100% F2 (15-20 mihoΐ) is passed over a film of 100pL solution containing approximately 50 mihoΐ of an amino acid, such as _L -tyrosine to prepare fluoro- tyrosine, in an acidic solvent, at 65 °C for the reactions involving anhydrous HF or other solvent. The outlet gas from the reaction vessel is passed through a 0.1 M solution of NaOH.

Acetyl hypofluorite (as a fluorinating reagent) is produced by passing dilute F2 through a 2 in. x 1/4 in. o.d. x 1/8 in. i.d. FEP column packed with KOAc-2HOAc. The solvents, acid and/or basic media are removed or reduced prior to fluorination by pumping through an FEP U-tube cooled to 196°C or by flow of a dry inert gas such as N2.

Following the reaction, the in-loop method is employed to directly purify the crude reaction mixture in a loop (typical HPLC loop; 5 mL stainless steel; or fluoroplastic loop) or alternative chromatographic method.

Alternatively, following the reaction, the residue is dissolved in 5 ml of 0.1% HO Ac and evaporated and the process may be repeated. The final residue is dissolved in 2 ml of 0.1% HO Ac and filtered through a 0.45 mih filter prior to HPLC purification.

All references, patents, and patent publications described herein are incorporated by reference in total.

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