High activity pharmaceutical formulations of f-18 flornaptitril and processes of preparation thereof

Abstract

This invention provides a pharmaceutical formulation that has 0.5-10 Ci (Curies) total activity of 2-(1-{6-[(2-[F-18]fluoroethyl)(methyl)amino]-2-naphthyl }ethylidene)malononitrile (F-18 Flornaptitril) that is suitable for separation into individual doses for use in medical imaging. The invention also includes a process for preparing Curie-scale pharmaceutical formulations of F- 18 Flornaptitril. The process minimizes radiolytic decomposition during the fluorination reaction to facilitate synthesis of F-18 Flornaptitril pharmaceutical compositions with Curie-scale activity levels.

Description

[0001] HIGH ACTIVITY PHARMACEUTICAL FORMULATIONS OF F-18 FLORNAPTITRIL AND PROCESSES OF PREPARATION THEREOF

[0002] CROSS REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of provisional U.S. Application No. 63 / 659,232 filed June 12, 2024 and provisional U.S. Application No. 63 / 807,165 filed May 16, 2025. The entirety of these applications is hereby incorporated by reference for all purposes.

[0004] FIELD OF THE INVENTION

[0005] This invention provides a pharmaceutical formulation that has 0.5-10 Ci (Curie), for example 0.75 to 5 Ci, or 0.8 to 2 Ci total activity of 2-(l-{6-[(2-[F-18]fluoroethyl)(methyl)amino]- 2-naphthyl}ethylidene)malononitrile (18F-FDDNP, also known by its USAN-designated name F- 18 Flornaptitril) that is suitable for division into multiple unit-dose patient-specific amounts of radioactivity for use in medical imaging. The invention also includes a process for preparing multiCurie pharmaceutical formulations of F-18 Flornaptitril. The process minimizes radiolytic decomposition during the fluorination reaction to facilitate synthesis of F-18 Flornaptitril pharmaceutical compositions with multi-Curie activity levels.

[0006] BACKGROUND OF THE INVENTION

[0007] Chronic Traumatic Encephalopathy (CTE) is a devastating neurodegenerative disease which is known to result from traumatic brain injury (McKee, A. et al. “The Neuropathology of Chronic Traumatic Encephalopathy” 2005, Brain Pathology, 25, 350-364). Traumatic brain injury resulting in CTE was first described in a study of professional boxers in 1928 and has since been observed in members of the military and professional sports players (Martland, H. “Punch Drunk” 1928, JAMA, 91, 1103-1107). Athletes exposed to sub-concussive and concussive injury are at risk, as are military personnel exposed to even a single blast (Lucke-Wold, B. et al. “Linking Traumatic Brain Injury to Chronic Traumatic Encephalopathy: Identification of Potential Mechanisms Leading to Neurofibrillary Tangle Development” 2014 Journal of Neurotrauma 31, 1129-1138). CTE can lead to behavioral and mood changes, memory loss, cognitive impairment, and dementia. These symptoms can significantly impact quality of life.

[0008] The neuropathology of CTE is primarily the result of aggregation of the protein tau. Tau protein is normally associated with microtubules where it facilitates the outgrowth of neurites. The phosphorylation of tau is tightly controlled by kinases and phosphatases. Traumatic brain injury causes the tau protein to dissociate from microtubules, exposing additional phosphorylation sites and ultimately leading to hyperphosphorylation. Hyperphosphorylated tau protein becomes insoluble and aggregates. These aggregates are poorly dephosphorylated, resulting in increasing levels of aggregated tau protein as neurofibrillary tangles (NFTs). The NFTs can propagate to additional neurons. The propagation of NFTs leads to the progressive neurodegenerative nature of CTE. A staging system based on the degree and location of tau deposition has been proposed to define the severity of CTE (McKee et al. “The spectrum of disease in chronic traumatic encephalopathy” Brain 136, 43-64), however the McKee staging system is based on postmortem analysis of brain tissue. Despite the impacts during a patient’s life, the presence and severity of CTE can currently only be diagnosed postmortem.

[0009] NFTs are also associated with Alzheimer’s Disease, as is beta amyloid plaque. Both NFTs and beta amyloid have been observed in the brains of individuals with AD. Beta amyloid often appears early on in AD development with the presence of NFTs occurring later in parallel with decline in cognitive function both most often occurring in different regions of brain than occurs in CTE neuropathology. Positron emission tomography (PET), typically employed in conjunction with computed tomography (CT; PET-CT), is an imaging technique which can be used to determine both structural features and biological processes in a living patient. PET detects the gamma rays produced when a positron emitted by a radiotracer annihilates with an electron in the patient’s body. PET radiotracers incorporate a radioactive isotope (e g.nC,18F,68Ga, or64Cu) and are typically designed to specifically bind to a protein target.

[0010] High activity18F is typically generated in a cyclotron, through the proton bombardment of18O-enriched water. The cyclotron accelerates protons to sufficiently high energy levels (-11-18 MeV) which can then react with the nucleus of oxygen-18. During this reaction, the proton knocks out a neutron in the18O nucleus and replaces it, thereby turning the18O into18F.

[0011] 18O + p - ►18F + n

[0012] 18F is a radioactive isotope of fluorine that has a half-life of approximately 110 minutes. The short half-life means that once the18F has been formed, it is a challenge to rapidly synthesize, purify, formulate, deliver, and administer the radiopharmaceutical to patients before naturally occurring radioactive decay processes consume significant amounts of18F.18F decays primarily through positron emission, which is the source of positrons for the annihilation process that produces the necessary emissions for imaging in positron emission tomography.

[0013] 18F decays by emitting a positron that travels a very short distance before it reacts with an electron in the environment. The distance the positron travels is typically less than 1 millimeter. The positron immediately reacts with an electron in an annihilation event, turning mass into energy with the creation of two photons of electromagnetic energy that are each 180 degrees to the other, thus traveling away from the annihilation in precise opposite directions. These two photons are gamma rays and possess high energy (511 keV). If the annihilation event occurs within a patient who is placed in a PET scanner, these two high energy photons can be detected by the PET scanner. The PET scanner is a series of detectors configured circumferentially about the patient. Using a computer-assisted algorithm-based imaging process, and the fact that positrons travel only short distances before the annihilation events occur, the specific location of the annihilations (and correspondingly the pathophysiological location of the decaying PET radiopharmaceutical) within the patient can be calculated from the two opposing gamma photons and its precise location determined.

[0014] PET radioligands have been developed to image either beta amyloid or tau deposits associated with Alzheimer's Disease, yet none of these radioligands have been successfully used to image CTE-associated tau deposits (Varlow, C. et al. “Evaluation of Tau Radiotracers in Chronic Traumatic Encephalopathy” 2023, J. Nucl. Med. 64:460-465). Indeed, in 2020 the U.S. Food and Drug Administration approved flortaucipir F-l 8 (Tauvid™) for use in evaluating tau deposits in Alzheimer's Disease patients. However, the FDA specifically states that flortaucipir is not indicated for use in evaluation of patients with possible CTE. Preliminary clinical and non- clinical investigations of flortaucipir suggest differences in tau conformation and distribution that limit flortaucipir binding to CTE-associated tau protein (Eli Lilly Press Release “Lilly Receives U.S. FDA Approval of TAUVID™ (flortaucipir F 18 injection) for Use in Patients Being Evaluated for Alzheimer's Disease, May 28, 2020).

[0015] F-18 Flornaptitril is a valuable PET tracer that has been shown to image CTE-associated tau protein ante-mortem (Small, G. et al. “PET Scanning of Brain Tau in Retired National Football League Players: Preliminary Findings” 2013, Am. J. Ger. Psych., 21(2) 138-144; WO 2000 / 010614; and Barrio, J. et al. “In vivo characterization of chronic traumatic encephalopathy using [F-18]FDDNP PET brain imaging” 2015, PNAS, 112(16)).

[0016] F-18 Flornaptitril

[0017] F-18 Flornaptitril can detect the presence of tau protein aggregates and amyloid plaques (Small, G. et al. “PET of Brain Amyloid and Tau in Mild Cognitive Impairment, 2006, NEJM, 355:25, 2652-2663). Investigational PET scans using F-18 Flornaptitril have shown that F-18 Flornaptitril has the potential to differentiate between CTE and other neurodegenerative diseases such as Alzheimer’s disease. In early CTE, tau deposits are localized in the limbic system including subcortical structures, limbic medial temporal lobe structures, and frontal cortex. The regions of the brain where tau aggregation begins in CTE are consistent with studies showing that these regions bear the maximum forces in American football players receiving concussions. These regions are also where processing of emotions, mood and behavior occur, consistent with symptoms of the disease. In contrast, the tau deposits in Alzheimer’s disease start in the hippocampus and spread through the cortex. (Barrio, J. et al. “In vivo characterization of chronic traumatic encephalopathy using [F-18]FDDNP PET brain imaging” 2015, PNAS, 112(16)). Additionally, detailed confirmation of the specificity of F-18 Flornaptitril was obtained in a patient in very advanced stages of Alzheimer’s Disease who underwent PET scanning and died shortly thereafter. Autopsy data on the brain of that patient confirmed the presence of B-amyloid plaques and NFTs in the medial temporal and lateral temporal lobes, areas corresponding to F-18 Flornaptitril uptake on the PET scan, as consistent with Alzheimer’s disease (Omalu B et al. “Postmortem autopsy confirmation of ante-mortem [F-18]FDDNP-PET scans in a football player with chronic traumatic encephalopathy.” Neurosurgery. 2018 Feb 1;82(2):237-246. Doi : 10.1093 / neuros / nyx536.

[0018] Diagnostic ability to distinguish between these two neurodegenerative diseases, which can present similarly in early stages as a clinical presentation of mild cognitive impairment (MCI), would be advantageous for patients and healthcare professionals by enabling precision in predicting the specific type of neurodegeneration.

[0019] Liu et al. describe an improved synthesis of F-18 Flornaptitril (Liu et al. “High-Yield, Automated Radiosynthesis of [18F]FDDNP for Animal or Human Administration” 2007, Mol. Imaging Biol. 9, 6-16). Liu et al. found the purity of the starting material to be a critical parameter in achieving high yield radiofluorination. Additional reported syntheses of F-18 Flornaptitril include Vercouille, J. et al. “Automated production of [18F]FDDNP using a TRACERlab MXFDG” 2010, J. Label Compd. Radiopharm 53, 208-212; WO 2000 / 010614, assigned to the Regents of the University of California; and U.S. PatentNos. 10,377,701 and 10,626,083, assigned to the Regents of the University of California.

[0020] Regents of the University of California, in U.S. Patent No. 10,377,701, describe an automated synthesis of F-18 Flornaptitril which follows the steps shown in the flowchart below:

[0021] U.S. Patent No. 10,377,701 describes the production of high specific activity F-18 Flornaptitril (between 1-5 Ci / pmol specific activity, but not greater than 0.3 Ci total activity), for use on small scale laboratory animal studies and not for commercial scale production that is required to support human clinical and commercial use. Specific activity is the amount of radioactive isotope for a given sample, for instance the amount of radioactive isotope per gram or per mole of material. A highly efficient labeling reaction can produce material with high specific activity, even if it is performed on a small amount of material (and thus low total activity).

[0022] The summary of U.S. Patent No. 10,377,701 purports to describe the synthesis of up to 100 doses of F-18 Flornaptitril of 0.010 Ci each (or 1 Ci total). However, the examples assert a maximum decay-corrected amount of F-18 Flornaptitril produced of only 0.5291 Ci. Notwithstanding, the decay-corrected amount is not the amount of radiotracer that can be administered to the human patient. A decay-corrected amount is the purely hypothetical amount of material that would have been produced had the radioisotope not decayed at all. The specification of U.S. Patent No. 10,377,701 states that the claimed process takes 100 minutes to perform, which is nearly an entire half-life of18F (ti / 2 for18F is 110 minutes). Therefore, the maximum amount of F-18 Flornaptitril in a batch produced by the process that can actually be administered to a patient is only 0.281 Ci. While this amount may be useful on laboratory scale for animal model experiments, or a small number of clinical doses but only if used quickly after manufacture, it is an insufficient amount of radiotracer for commercial scale production that is required to support larger numbers of doses for human clinical and commercial use particularly when clinical sites exist at some distance, and hours, from the manufacturing and dispensing sites.

[0023] For these reasons, clinical and commercial use of PET radiotracers necessitates synthesis of high total activity concentrations (as opposed to specific activity) of solutions of radiotracer, which can be portioned into multiple doses for human infusion. Since the half-life of18F is short (110 minutes), it is important to synthesize a quantity of radioligand sufficient that it will still be sufficiently active to be tested for quality, formulated, transported to a clinic, administered to a patient, and the patient imaged before the radioactivity decays below a useful level.

[0024] CTE impacts a very large and varied population of people who experience concussive or percussive brain injuries. Patients should not have to wait until death for a diagnosis. This includes people involved in car or other kinds of accidents, those who serve or have served in the military, and athletes from youth to amateur to professional level. It is critical to provide a large-scale commercial process to prepare high total activity F-18 Flornaptitril to assess CTE in this large and varied population while they are living and can take either therapeutic or life-changing steps to accommodate the disease. The current lab scale procedures are insufficient to accomplish this.

[0025] Thus, it is an object of the invention to provide a pharmaceutical formulation with high Curie total activity of F-18 Flornaptitril that is suitable for separation into individual doses for use in human medical imaging.

[0026] It is another object of the invention to provide an efficient, large-scale synthesis of F-18 Flornaptitril to enable clinical and commercial use of this important diagnostic, which results in the preparation of a pharmaceutical formulation with high Curie total activity of F-18 Flornaptitril suitable for separation into individual doses for use in medical imaging. SUMMARY OF THE INVENTION

[0027] High activity pharmaceutical formulations of F-18 Flornaptitril and a method of manufacturing high activity pharmaceutical formulations of F-18 Flornaptitril are provided. The high activity pharmaceutical formulations described herein are stable in the desired form for use for example for up to ten hours after manufacture.

[0028] Commercial scale synthesis of18F radiotracers is extremely challenging since the half-life of18F is less than two hours. Once the18F has been formed (typically in a cyclotron), it immediately begins to decay. All of the steps (including synthesis, purification, formulation, quality control testing, delivery to the patient, administration to the patient, and imaging the patient) must be carried out before the18F decays to unusable levels. A flow chart highlighting the complexity of the radioligand production process is shown in FIG. 7. To facilitate commercial use of a radiotracer, it must be produced at high activity levels to allow for eventual decay. However, high activity levels can lead to higher levels of byproducts resulting from concentrated radioactivity. Synthesis of F-18 Flornaptitril has never been reported on a commercial scale.

[0029] In certain embodiments, the amount of radioactivity of the pharmaceutical formulation comprising F-18 Flornaptitril contains from about 0.5 Ci to about 10 Ci depending upon the time of manufacture and the time of desired clinical use. In certain embodiments, the pharmaceutical formulation comprising F-18 Flornaptitril contains from about 1 Ci to about 5 Ci, for example from about 0.5 Ci to about 1.5 Ci or from about 0.75 to about 2.0 Ci total activity of F-18 Flornaptitril. In certain embodiments, the pharmaceutical formulation comprising F-18 Flornaptitril contains from about 0.5 Ci to about 3.5 Ci total activity of F-18 Flornaptitril (or even about 2.0 to about 3.0, or about 2.0 to about 2.5 Ci).

[0030] In certain other embodiments, the pharmaceutical formulation comprises from about 0.5 to about 5 Ci total activity of F-18 Flornaptitril, for example from about 0.5 Ci to about 3.5 Ci of F- 18 Flornaptitril. In certain embodiments, the pharmaceutical formulation comprises from about 0.75 to about 3 Ci of F-18 Flornaptitril. In certain embodiments, the pharmaceutical formulation comprises from about 0.75 to about 1.5, from about 0.8 to about 1.2, or from about 0.8 to about 2.0 Ci of F-18 Flornaptitril. In certain embodiments, the pharmaceutical formulation comprises from about 0.75 to about 2.75, from about 0.8 to about 2.5, or from about 0.85 to about 1.5 Ci of F-18 Flornaptitril. The advantageous process of manufacture described herein facilitates production of large scale, multi-Curie quantities of F-18 Flornaptitril to accommodate anticipated volumes of clinical doses. Pharmaceutical compositions with multi-Curie activity can enable production of multiple individual human doses at a nearby clinical site or for clinical sites at some distance from the manufacturing and distribution facilities, as will be practically and essentially required for commercial use of the radioligand.

[0031] The process invention advantageously introduces a new step, which is the incorporation of a means to reduce radiolysis during the reactions necessary to manufacture of F-18 Flornaptitril, as further described below. The means to reduce radiolysis is used in the reaction itself to reduce degradation of the F-18 Flornaptitril as well as to inhibit actions by other components that adversely affect the total activity of the final product.

[0032] Gamma rays produced by18F decay have sufficient energy to ionize organic molecules and can form many complex reaction products through a process known as radiolytic decomposition. For example, the ionization of a simple molecule such as water is shown below. Many of the reaction products of radiolytic decomposition are themselves highly reactive and their secondary reactions further contribute to the decomposition process. This degradation process is exaggerated as the scale of the radiochemistry manufacturing reaction increases and creates an impediment to the production of high activity F-18 Flornaptitril product.

[0033] Scheme 1. Radiolysis of Water

[0034] Radical species contain an unpaired electron and can react rapidly with other molecules. For example, a hydroxyl radical (shown in the scheme below) can abstract a hydrogen atom from an organic compound (represented by R-H) to form water and an organic radical (R-) The resulting organic radical (R‘) can then react with another molecule, generating additional reaction byproducts. These byproducts can propagate the radical chain or be oxidized or reduced back to a species with paired electrons.

[0035] Scheme 2. Radical Propagation

[0036] R-H + *OH - - R* + HOH PET radioligands, in particular those with aromatic amines such as F-18 Flornaptitril, can be susceptible to radiolytic decomposition (radiolysis) (Scott, P. J. H. et al. “Studies into Radiolytic Decomposition of Fluorine- 18 Labeled Radiopharmaceuticals for Positron Emission Tomography” 2009 Appl. Radiat. Isot. 67(1), 88-94). Radiolysis can become more pronounced with increasing amounts of radioactivity or higher specific activities (MacGregor et al. “Fluorine- 18-N-Methylspiroperidol: Radiolytic Decomposition as a Consequence of High Specific Activity and High Dose Levels” 1987 J. Nucl. Med. 28, 60-67), which is why large scale F-18 Flornaptitril preparation for commercial or clinical trial is so challenging. Even on small scale, F-18 Flornaptitril is susceptible to radiolysis, and use of antioxidants in the purification and formulation buffers to stabilize it have been reported (Klok, R.P. et al. “Synthesis of 2-(l,l,-dicyanopropen-2- yl)-6-(2-[18F]-fluoroethyl)-methylamino-naphthalene ([18F]FDDNP”, 2008, Appl. Radiat. Isot. 66, 203-207).

[0037] Radiolysis can occur (i) during the radiochemistry manufacturing reaction and / or (ii) during the purification and formulation process. Certain progress has already been made on minimizing radiolysis of F-18 Flornaptitril during the purification and formulation process, by using antioxidants such as ascorbic acid and / or sodium ascorbate, ethanol and / or ammonium acetate. In contrast, radiolysis of F-18 Flornaptitril during the radiolabeling reaction itself remains a large hurdle to clinical and commercial use of this radiotracer.

[0038] The present invention provides that the use of certain radiostabilizers as described herein in the fluorination reaction can facilitate synthesis of high activity F-18 Flornaptitril by quenching radiolysis reactions. The present invention indicates that antioxidants used in the purification and formulation of F-18 Flornaptitril (e.g. ascorbic acid), have different requirements from radiostabilizers used in the reaction process. For example, addition of a formulation antioxidant such as ascorbic acid into the radiolabeling reaction can be detrimental to the radiolabeling yield. Ascorbic acid can result in protonation of the18F-fluoride, rendering it nonnucleophilic and hampering reactivity with the precursor molecule.

[0039] General Process of Manufacture of High Activity F-18 Flornaptitril

[0040] The general process for manufacturing high activity F-18 Flornaptitril can comprise, for example, the following steps: trapping18F fluoride ion on an anion-exchange solid phase; eluting the18F fluoride ion into a reaction vessel having a cryptand solution by passing a cation solution optionally followed by water through the anion-exchange solid phase, wherein an18F fluoride / cryptand complex is formed; drying the18F fluoride / cryptand complex in the reaction vessel; reacting the dried18F fluoride ion / cryptand complex with a compound of Formula I (see Scheme 3 below) in an anhydrous organic solvent with a radiostabilizer, for example selected from those described herein, to form a reaction product; passing the reaction product through a solid phase extraction cartridge and into an injection vessel; injecting the reaction product contained in the injection vessel to an HPLC column; collecting a fraction containing F-18 Flornaptitril from the HPLC column in a dilution vessel; diluting the collected F-18 Flornaptitril contained in the dilution vessel with water; passing the diluted F-18 Flornaptitril through a solid-phase extraction cartridge and eluting F-18 Flornaptitril with ethanol or other suitable organic solvent; and formulating the F-18 Flornaptitril optionally with at least one suitable antioxidant and / or optionally with at least one excipient to form the final product.

[0041] In a principal aspect of the invention, a method of manufacturing high activity F-18 Flornaptitril formulations comprises reacting a compound of Formula I with18F-fluoride (produced in a cyclotron) in the presence of a selected radiostabilizer, a carbonate base, and a cryptand ligand (Scheme 3). The selected reaction additive radiostabilizer described herein may for example belong to a class selected from a persistent radical, a nitrone, an aniline, a phenol or a diarylalkene, as detailed below.

[0042] Scheme 3. Synthesis of F-18 Flornaptitril , ,

[0043] Formula I wherein R1is selected from the group consisting of Ci ealkyl, Ci-ehaloalkyl, and aryl optionally substituted with one or two substituents selected from the group consisting of Ci-ealkyl, Ci-ehaloalkyl, halogen, and nitro. In certain embodiments, R1is selected from methyl (forming a mesylate), trifluoromethyl (forming a triflate), para-methylphenyl (forming a tosylate), and parabromophenyl (forming a brosylate). In certain embodiments, R1is p-methylphenyl. In certain embodiments, R1is p-bromophenyl. In certain embodiments, R1is methyl.

[0044] In certain embodiments, the radiostabilizer is selected from 2,2,6,6-tetramethylpiperidin- l-yl)oxyl (TEMPO), TEMPOL, DMPO, diphenylethylene, and 2,6-di-tert-butyl-a-(3,5-di-tert- butyl-4-oxo-2,5-cyclohexadien-l-ylidene)-p-tolyloxy, free radical (galvinoxyl radical). In certain embodiments, the radiostabilizer is TEMPO.

[0045] In certain embodiments, the radiostabilizer is an aniline selected from the group consisting

[0046] In certain embodiments, the radiostabilizer is a phenol selected from the group consisting

[0047] In certain embodiments, the radiostabilizer is selected from di phenyl ethylene (phenylstyrene), dinapthylethylene, or dipyridylethylene.

[0048] In certain embodiments, the carbonate base is potassium carbonate, cesium carbonate, or tetraalkylammonium carbonate (e.g. tetrabutylammonium carbonate). In certain embodiments, the cryptand ligand is 4,7,13,16,21,24-hexaoxa-l,10-diazabicyclo[8.8.8]hexacosane (Kryptofix® K222). A cryptand ligand tightly binds the fluoride counterion (i.e. potassium) which can substantially increase the reactivity of the fluoride ion.

[0049] In certain embodiments, the solvent is a polar organic solvent, for example an aprotic polar organic solvent. Polar aprotic solvents include but are not limited to acetonitrile (ACN), dimethyl sulfoxide (DMSO), dimethylacetamide (DMA), n-methyl pyrrolidinone (NMP), and propionitrile. In certain embodiments, the aprotic organic solvent is selected from ACN and DMSO. In certain embodiments, the polar organic solvent is tert-butanol. The reaction can be performed at any temperature where the18F-fluoride can react with the compound of Formula I. However, the reaction can for example be performed at elevated temperature to increase the rate of reaction. In certain embodiments, the reaction is performed at from about 70°C to about 120°C, from about 80°C to about 115°C, or from about 90°C to about 110°C. In certain embodiments, the reaction is performed at from about 90°C to about 95°C, from about 95°C to about 100°C, from about 100°C to about 105°C, or from about 105°C to about 110°C. In certain embodiments, the reaction is performed at about 80°C, 85°C, 90°C, 95°C, 100°C, 105°C, or 110°C.

[0050] In certain embodiments, the reagents of the reaction described herein are added to a reaction vial which is then placed in a heated bath, for example an oil bath. The reaction does not immediately reach the temperature of the oil bath once the vial is placed in the bath. Furthermore, the temperature of the bath may temporarily decrease when the reaction vial (for example a reaction vial at room temperature) is added. A larger volume of oil or heating element can be used to minimize the temperature fluctuation resulting from addition of the reaction vial. In certain embodiments, the heated bath is set to a temperature from about 70°C to about 120°C, from about 80°C to about 115°C, or from about 90°C to about 110°C. In certain embodiments, the heated bath is set to a temperature of about 80°C, 85°C, 90°C, 95°C, 100°C, 105°C, or 110°C. In other embodiments the reaction is heated with a hot plate, in a reactor, or with any other means for heating that achieves the desired results.

[0051] Rapid fluorination of the precursor molecule (a compound of Formula I) minimizes the amount of decay which occurs before the radioligand reaches the patient. In certain embodiments, the reaction may be performed, for example, during less than one half-life of18F or even less than three-quarters or half or one-quarter of a half-life of18F. In certain embodiments, the reaction is performed until the starting material is completely consumed. In certain embodiments, the reaction is performed for a specific amount of time. In certain embodiments, the reaction is performed for less than 35 minutes, less than 25 minutes, less than 15 minutes, less than 10 minutes, or less than 5 minutes. In certain embodiments, the reaction is performed for between 5 and 35 minutes, between 5 and 25 minutes, between 10 and 35 minutes, between 10 and 25 minutes, or between 10 and 20 minutes. In certain embodiments, the reaction is performed for about 15 minutes.

[0052] It is advantageous to rapidly synthesize the radioligand because following synthesis, the radioligand must be purified, formulated, tested for quality, transported to a clinic, administered to a patient, and the patient imaged before the radioactivity decays to below a useful level. For example, purification and formulation of F-18 Flornaptitril may require, for example, at least about half, three quarters, one, two or more half-lives (110 minutes) after the radiosynthesis reaction is finished. Delivery may require, for example, up to 2, 3, 4, or 5 additional half-lives (up to about 4, 5, 6, 7, 8, 9, or 10 hours). The amount of F-18 Flornaptitril for use in imaging after 5 half-lives is ~3% of the amount that was synthesized.

[0053] In certain embodiments, a method of manufacturing high activity F-18 Flornaptitril formulations comprises reacting a compound of Formula I with18F-fluoride (produced in a cyclotron) in the presence of 2,2,6,6-Tetramethylpiperidin-l-yl)oxyl (TEMPO), potassium carbonate, and Kryptofix® K222, shown in Scheme 4 below.

[0054] Scheme 4. Synthesis of F-18 Flornaptitril

[0055] Formula I

[0056] In certain embodiments, a method of manufacturing high activity F-18 Flornaptitril formulations comprises reacting a compound of Formula I with18F-fluoride (produced in a cyclotron) in the presence of diphenylethylene, potassium carbonate, and Kryptofix® K222, shown in Scheme 5 below.

[0057] Scheme 5. Synthesis of F-18 Flornaptitril

[0058] Formula I

[0059] In certain embodiments, from about 1 to about 10 equivalents of the radiostabilizer (relative to the compound of Formula I) is used in the reaction. In certain embodiments, about 5 equivalents of the radiostabilizer is used in the reaction. In certain embodiments, from about 1 to about 100, from about 1 to about 50, from about 1 to about 30, from about 1 to about 25, from about 1 to about 20, from about 1 to about 15, from about 1 to about 10, or from about 2.5 to about 7.5 equivalents of the radiostabilizer is used in the reaction.

[0060] In certain embodiments, the method of manufacturing high activity F-18 Flornaptitril pharmaceutical formulations further includes purification of the reaction mixture, for example to remove the cryptand ligand. In certain embodiments, the reaction mixture comprising high activity F-18 Flornaptitril is purified using HPLC. In certain embodiments, the purification process reduces the level of cryptand ligand to less than about 4 mg / mL, less than about 2.5 mg / mL, less than about 1 mg / mL, less than about 0.1 mg / mL, or less than 0.05 mg / mL. In certain embodiments, an antioxidant stabilizer is used during purification of the reaction mixture, for example ethanol or ascorbic acid or ascorbate salts.

[0061] In certain embodiments, the method of manufacturing a high activity F-18 Flornaptitril pharmaceutical formulation further includes diluting the purified solution of F- 18 Flornaptitril with a pharmaceutically acceptable carrier, for example saline. In certain embodiments, the pharmaceutical formulation includes optionally one or more additional pharmaceutically acceptable excipients, for example an antioxidant, surfactant, solubilizer, or plasma protein.

[0062] The present invention thus includes at least the following features:

[0063] 1) A pharmaceutical formulation comprising from about 0.5 to about 10 Ci total activity of F-18 Flornaptitril, for example from about 0.75 Ci to about 5 Ci of F-18 Flornaptitril.

[0064] 2) A pharmaceutical formulation comprising from about 0.75 to about 3.5 Ci of F-18 Flornaptitril.

[0065] 3) A pharmaceutical formulation comprising from about 0.75 to about 3, from about 0.8 to about

[0066] 2, or from about 0.8 to about 1.5 Ci of F-18 Flornaptitril.

[0067] 4) A pharmaceutical formulation comprising from about 1.5 Ci to about 3.5 Ci, from about 2.0 Ci to about 3.0 Ci, or from about 2.0 Ci to about 2.5 Ci total activity of F-18 Flornaptitril.

[0068] 5) A pharmaceutical formulation comprising from about 0.8 to about 1.1 Ci of F-18 Flornaptitril.

[0069] 6) The pharmaceutical formulation of (1-5), wherein the antioxidant formulation stabilizer is selected from sodium ascorbate, ascorbic acid, sodium citrate, citric acid, ethanol, and gentisic acid.

[0070] 7) The pharmaceutical formulation of (1-5), comprising human serum albumin. 8) A process of preparing a high activity pharmaceutical formulation of F-18 Flornaptitril comprising: a) reacting a compound of Formula I, or a salt thereof, with18F -fluoride in the presence of a radiostabilizer described herein, a carbonate base, and a cryptand ligand: Formula I wherein R1is selected from the group consisting of methyl, Ci-shaloalkyl, and aryl optionally substituted with one or two substituents selected from the group consisting of Ci-6alkyl, Ci-ehaloalkyl, halogen, and nitro; b) purification of the F-18 Flornaptitril from the reaction mixture; and c) preparing a pharmaceutical formulation with the purified F-18 Flornaptitril and an antioxidant stabilizer; wherein the pharmaceutical formulation comprises from about 0.5 to about 5 Ci, from about 0.75 to about 3 Ci, from about 0.8 to about 2 Ci or from about 0.8 to about 1.5 Ci of F-18 Flornaptitril.

[0071] 9) The process of (8), wherein from about 1 mg to about 8 mg, about 2 mg to about 6 mg, or about 3 mg to about 5 mg of carbonate base, for example potassium, cesuim, ammonium, or tetrabutylammonium carbonate is used.

[0072] 10) The process of (8), wherein from about 5 mg to about 30 mg, about 10 mg to about 30 mg, about 5 mg to about 25 mg, or about 10 mg to about 25 mg of the cryptand ligand, for example Kryptofix® K222 is used.

[0073] 11) The process of (8), wherein step (b) comprises HPLC purification.

[0074] 12) The process of (11), wherein the HPLC purification uses at least about 40% aqueous mobile phase.

[0075] 13) The process of any one of (8)-( 12), wherein the reaction is performed in the dark.

[0076] 14) The process of any one of (8)-(12), wherein the pharmaceutical formulation comprises less than about 10, 8, 6, 4, 2, or 1 mg / mL of cryptand ligand.

[0077] 15) The process of any one of (8)-(12), wherein the pharmaceutical formulation comprises less than about 0.25, 0.20, 0.15, 0.10, or 0.05 mg / mL of cryptand ligand. 16) The process of any one of (8)-(l 4), wherein the radiostabilizer is a persistent radical, a nitrone, an aniline, a phenol, or a diarylalkene for example as described herein.

[0078] 17) The process of (11), wherein the radiostabilizer is a persistent radical selected from TEMPO,

[0079] 5.5-dimethyl-l-pyrroline N-oxide (DMPO), and 2,6-di-tert-butyl-a-(3,5-di-tert-butyl-4-oxo-

[0080] 2.5-cyclohexadien-l-ylidene)-p-tolyloxy, free radical (galvinoxyl radical).

[0081] 18) The process of (16) wherein the radiostabilizer is TEMPO.

[0082] 19) The process of any one of (8)-(l 8), wherein from about 1 to about 100 equivalents, from about 1 to about 50 equivalents, from about 1 to about 25 equivalents, from about 1 to about 20 equivalents, from about 1 to about 15 equivalents, from about 1 to about 10 equivalents, from about 2.5 to about 7.5 equivalents, or from about 4 to about 6 equivalents of radiostabilizer are used.

[0083] 20) The process of any one of (8)-(l 9), wherein about 5 equivalents of radiostabilizer are used.

[0084] 21) The process of any one of (8)-(20), wherein one or more of steps (a)-(c) is performed using an automated synthesizer.

[0085] 22) A manufacture of high activity F-18 Flomaptitril comprising the following steps: trapping18F fluoride ion on an anion-exchange solid phase; eluting the18F fluoride ion into a reaction vessel having a cryptand solution by passing a cation solution optionally followed by water through the anion-exchange solid phase, wherein an18F fluoride / cryptand complex is formed; drying the18F fluoride / cryptand complex in the reaction vessel; reacting the dried18F fluoride ion / cryptand complex with a compound of Formula I (see Scheme 3 below) in an anhydrous organic solvent with a radiostabilizer selected from those described herein, to form a reaction product, wherein the radiostabilizer is selected from persistent radical, nitrone, aniline, phenol, and a diarylalkene; passing the reaction product through a solid phase extraction cartridge and into an injection vessel; injecting the reaction product contained in the injection vessel to an HPLC column; collecting a fraction containing F-18 Flomaptitril from the HPLC column in a dilution vessel; diluting the collected F-18 Flomaptitril contained in the dilution vessel with water; passing the diluted F-l 8 Flomaptitril through a solid-phase extraction cartridge and eluting F-18 Flomaptitril with ethanol or other suitable organic solvent; and formulating the F-18 Flomaptitril with suitable antioxidants and excipients to form a final product.

[0086] 23) A process of preparing a high activity pharmaceutical formulation of F-18 Flomaptitril comprising: a) reacting a compound of Formula I, or a salt thereof, with18F -fluoride in the presence of a carbonate base and a cryptand ligand: Formula I wherein R1is selected from the group consisting of methyl, Ci-shaloalkyl, and aryl optionally substituted with one or two substituents selected from the group consisting of Ci-galkyl, Ci-ehaloalkyl, halogen, and nitro; b) purification of the F-18 Flomaptitril from the reaction mixture; and c) preparing a pharmaceutical formulation with the purified F-18 Flomaptitril and an antioxidant stabilizer; wherein the pharmaceutical formulation comprises from about 0.5 to about 5 Ci, from about 0.75 to about 3 Ci, from about 0.8 to about 2 Ci or from about 0.8 to about 1.5 Ci of F-18 Flomaptitril.

[0087] BRIEF DESCRIPTION OF THE FIGURES

[0088] FIG. 1 depicts the synthesis of high activity F-18 Flomaptitril.

[0089] FIG. 2 is a schematic depiction of an automated module for performing Step (a) of the process of preparing a high activity pharmaceutical formulation of F-18 Flomaptitril.

[0090] FIG. 3 is a schematic depiction of an automated module for performing Step (b) of the process of preparing a high activity pharmaceutical formulation of F-18 Flomaptitril.

[0091] FIG. 4 is a schematic depiction of an automated module for performing Step (c) of the process of preparing a high activity pharmaceutical formulation of F-18 Flomaptitril.

[0092] FIG. 5 depicts a GE Healthcare FASTlab™ 2 automated synthesizer. FIG. 6 depicts F-18 Flornaptitril.

[0093] FIG. 7 depicts a flow chart of a non-limiting exemplary process for large-scale multi-Curie manufacturing of F-18 Flornaptitril. [18F]-Fluoride is received from a commercial supplier or synchrotron. The [18F]-fluoride is then introduced to the automated synthesizer where it is trapped on a solid phase cartridge, eluted with a solution of potassium carbonate / K222 and then azeotropically dried. A solution of Compound 1 and TEMPO is added to a solution of dried [18F]- fluoride in acetonitrile. The reaction is heated to effect radiolabeling, cooled, and diluted with acetonitrile. The reaction mixture is purified by HPLC, and the fraction containing the F-18 Flornaptitril is trapped on a cartridge, and the cartridge is rinsed with aqueous solvent. The F-18 Flornaptitril is then eluted with ethanol and the other excipients are added. The formulation is filtered through a sterilizing filter and sent to quality control. If the batch passes quality control tests, it is released for transport to clinical imaging sites. The multidose vial of F-18 Flornaptitril can be divided into approximately 10-20 unit dosage forms at the clinical site and administered to patients for PET-CT scans.

[0094] FIG. 8 depicts the atom numbering system for Compound 1 as described in Example 1.

[0095] FIG. 9 depicts the 'HNMR spectrum for Compound 1 as described in Example 1.

[0096] FIG. 10 depicts the COSY NMR spectrum for Compound 1 as described in Example 1. FIG. 11 depicts the NOSY NMR spectrum for Compound 1 as described in Example 1. FIG. 12 depicts the HMBC NMR spectrum for Compound 1 as described in Example 1.

[0097] DETAILED DESCRIPTION OF THE INVENTION

[0098] High activity pharmaceutical formulations of F-18 Flornaptitril and a method of manufacturing high activity pharmaceutical formulations of F-18 Flornaptitril are provided.

[0099] In certain embodiments, the activity of the pharmaceutical formulation comprising F-18 Flornaptitril contains from about 0.5 Ci to about 10 Ci, for example from about 0.8 Ci to about 2.0 Ci total activity of F-18 Flornaptitril.

[0100] In certain embodiments, the pharmaceutical formulation comprises from about 0.6 to about 2, from about 0.75 to about 1.8, from about 0.8 to about 1.5, or from about 0.85 to about 1.3 Ci of F-18 Flornaptitril. In certain embodiments, the pharmaceutical formulation comprises from about 1.3 to about 5, from about 1.4 to about 4.5, from about 1.5 to about 4.0, from about 1.6 to about 3.5, or from about 1.7 to about 3.0 Ci of F-18 Flornaptitril. In certain embodiments, the pharmaceutical formulation comprises from about 1 .5 to about 4, from about 1 .6 to about 3.5, from about 1.7 to about 3.25, from about 1.8 to about 3, from about 1.9 to about 2.75 or from about 2 to about 2.5 Ci of F-18 Flornaptitril.

[0101] In certain embodiments, the pharmaceutical formulation comprising F-18 Flornaptitril contains from about 0.75 Ci to about 1.5 Ci total activity of F-18 Flornaptitril (or even about 0.8 to about 1.2, or about 0.8 to about 1.1 Ci).

[0102] A process of manufacturing high activity pharmaceutical formulations of F-18 Flornaptitril is provided. The method comprises a reaction step that includes an adequate amount of a radiostabilizer as further described herein to effectively quench disadvantageous radiolysis during the reaction, a purification step, and a formulation step, as described further herein. High activity pharmaceutical formulations of F-18 Flornaptitril enable the centralized production of F-18 Flornaptitril doses for imaging neurodegenerative diseases.

[0103] To avoid substantial decay of the18F, it is advantageous for the radiolabeling reaction to occur rapidly. In certain embodiments, the radiolabeling reaction is performed at elevated temperature in minimal solvent volume to facilitate rapid production of F-18 Flornaptitril. However, these same conditions also facilitate rapid production of radiolysis products and other byproducts. The invention teaches that a radiostabilizer as described herein, including but not limited to a persistent radical, nitrone, aniline, or phenol, as further described herein, is added to the reaction to minimize radiolysis and improve yield.

[0104] In principal embodiments, the invention includes: d) Reacting a compound of Formula I, or a salt thereof: Formula I wherein R1is selected from the group consisting of methyl, Ci-ehaloalkyl, and aryl optionally substituted with one or two substituents selected from the group consisting of Ci-ealkyl, Ci-ehaloalkyl, halogen, and nitro; with18F -fluoride, a radiostabilizer, a carbonate base, and a cryptand ligand; e) Purification of the F-18 Flornaptitril from the reaction mixture; and f) Preparing a pharmaceutical formulation with the purified F-18 Flornaptitril and an antioxidant stabilizer; wherein the pharmaceutical formulation comprises from about 1.5 to about 3.5 Ci of F-18 Flornaptitril. In certain embodiments, one, two, or three of (a)-(c) are performed under conditions that exclude light. Conditions that exclude light include those wherein the reaction is exposed to significantly less than normal laboratory ambient lighting, up to and including complete or substantial exclusion of light. For example, conditions that exclude light include wrapping the reaction in foil, such as aluminum foil, and / or turning off any lights in the fume hood or hot cell in which the reaction is carried out. In certain embodiments, one, two, or three of (a)-(c) are carried out in amber glass. In certain embodiments, one, two, or three of (a)-(c) are carried out wherein the reaction vessel in wrapped in aluminum foil.

[0105] Step (a)

[0106] In certain embodiments, the compound of Formula I is 2-((6-( 1,1 -di cyanoprop- l-en-2- yl)naphthalen-2-yl)(methyl)amino)ethyl 4-methylbenzenesulfonate: or a salt thereof.

[0107] In certain embodiments, the compound of Formula I is selected from the group consisting , .

[0108] In certain embodiments R1is aryl optionally substituted with one or two substituents selected from the group consisting of Ci-ealkyl, Ci-ehaloalkyl, halogen, and nitro. In certain embodiments, R1is phenyl substituted with one Ci-6alkyl group. In certain embodiments, R1is phenyl substituted with one halogen. In certain embodiments R1is phenyl substituted with one nitro group. In certain embodiments, R1is Ci-ealkyl. In certain embodiments R1is ethyl.

[0109] Radiostabilizer

[0110] The radiostabilizer in the radiolabeling reaction is an important factor in the synthesis of high activity F-18 Flornaptitril. See, for example, WO 2005 / 061415, assigned to GE Healthcare Ltd., as well as U.S. Patent No. 9,567,372; 9,796,641; and 11,565,257. Flornaptitril is a reactive compound that includes a malononitrile (a doubly activated Michael acceptor). Additionally, the precursor compound of Formula I used to produce18F-Flornaptitril has a sulfonyl leaving group as well as the malononitrile.

[0111] Nitrone Radiostabilizers

[0112] In certain embodiments, the radiostabilizer is a nitrone, for example as described herein. Nitrones can react with and trap reactive radical species, schematically represented in the figure below. In certain embodiments, the radiostabilizer is a nitrone selected from N-tert-butyl-a- phenylnitrone (PBN), a-4-pyridyl-l-oxide-N-t-butylnitrone (POBN), 5,5-dimethylpyrroline-N- oxide (DMPO), 3, 4-dihydro-2-methyl- 1,1 -dimethylethyl ester-2H-pyrrole-2-carboxylic acid-1- oxide (BMPO), 3,4-dihydro-2-methyl-2H-pyrrole-2-carboxylic acid 1 -oxide, ethyl ester (EMPO). In certain embodiments, the radiostabilizer is a nitrone selected from

[0113] In certain embodiments, the radiostabilizer is a nitrone selected from salt thereof.

[0114] In certain embodiments, the radiostabilizer is a nitrone selected from

[0115] Aniline Radiostabilizers

[0116] In certain embodiments, the radiostabilizer is an aniline, for example as described herein. Anilines can be readily oxidized, thereby intercepting the reactive radical species. Aniline radical cation can then couple to form a neutral side product species (Tran, H. J. Mater. Chem. 2011, 21, 3534-3550). For example, aniline can form diphenylhydrazine as shown in the scheme below.

[0117] In certain embodiments, the radiostabilizer is an aniline compound of Formula II: salt thereof; wherein

[0118] R3a, R3b, and R3care independently selected from hydrogen, halogen, Ci-6alkyl, C(O)OH, C(O)OCi-6alkyl, and C(O)N(R4)2; and

[0119] R4is selected from hydrogen, Ci-ealkyl, aryl, -Ci-ealkyl-aryl, heterocycle, and heteroaryl.

[0120] In certain embodiments, R3ais hydrogen,

[0121] In certain embodiments, R3bis C(O)OH.

[0122] In certain embodiments, R3bis hydrogen,

[0123] In certain embodiments, R3cis hydrogen.

[0124] In certain embodiments, the aniline radiostabilizer is selected from the group consisting of salt thereof.

[0125] In certain embodiments, the aniline radiostabilizer is selected from the group

[0126] In certain embodiments, the aniline radiostabilizer is selected from the group consisting of In certain embodiments, the aniline radiostabilizer is selected from the group consisting of

[0127] In certain embodiments, the aniline radiostabilizer is selected from the group consisting of

[0128] Phenol Radioslabili. :ers

[0129] In certain embodiments, the radiostabilizer is a phenol, for example as described herein.

[0130] Phenol compounds can be oxidized by radical species to form quinones (Scheme below):

[0131] The oxidation of phenol to a quinone proceeds stepwise. The first one electron oxidation results in the phenol radical cation (middle). This species can further react with a hydroxyl radical to form a quinone (right). It can be advantageous to use a phenol radiostabilizer because it can be oxidized twice, therefore requiring a lower concentration in the reaction mixture. In certain embodiments, the radiostabilizer is a phenol compound of Formula III: R23is selected from hydrogen, OH, OR4, CH2OH, and CH2OR4;

[0132] R3a, R3b, R3Cand R3dare independently selected from hydrogen, halogen, Ci-ealkyl,

[0133] C(O)OH, C(O)OCi-6alkyl, and C(O)N(R4)2; and

[0134] R4is selected from hydrogen, Ci ealkyl, aryl, -Ci ealkyl-aryl, heterocycle, and heteroaryl.

[0135] In certain embodiments, the phenol radiostabilizer is selected from the group consisting of

[0136] In certain embodiments, the phenol radiostabilizer is selected from the group consisting of, thereof. In certain embodiments, the phenol radiostabilizer is selected from the group consisting of, salt thereof.

[0137] Diarylalkene Radiostabilizers In certain embodiments, the radiostabilizer is a diarylalkene, for example phenyl styrene.

[0138] Diarylalkenes react with highly reactive radical species according to the scheme below.

[0139] The resulting benzyl radical is stabilized by resonance delocalization, which decreases its reactivity and can minimize reaction byproduct formation. In certain embodiments, the radiostabilizer is a diarylalkene selected from diphenylethylene (phenyl styrene), dinapthylethylene, or dipyridylethylene.

[0140] In certain embodiments, the diarylalkene radiostabilizer is selected from

[0141]

[0142] Persistent Radical Radiostabilizers

[0143] Persistent radicals are a class of compounds with a stable radical, for example an aminoxyl radical. One example of a persistent radical radiostabilizer is 2,2,6,6-tetramethylpiperidin-l- yl)oxyl (TEMPO). The TEMPO radical is a sterically hindered aminoxyl radical with no alpha hydrogen atoms. As a result, TEMPO is a commercially available and shelf stable radical compound. Persistent radical radiostabilizers can couple with a reactive radical formed in the radiolabeling reaction through radical-radical coupling. TEMPO is exemplified in the scheme below. In certain embodiments, the radiostabilizer is selected from TEMPO, 5,5-dimethyl-l- pyrroline N-oxide (DMPO), and 2,6-di-tert-butyl-a-(3,5-di-tert-butyl-4-oxo-2,5-cyclohexadien-l- ylidene)-p-tolyloxy, free radical (galvinoxyl radical). In certain embodiments, the radiostabilizer is TEMPO.

[0144] In certain embodiments, the radiostabilizer is a persistent radical compound of Formula IV: salt thereof, wherein R?is selected from OR4, C(O)R4, N(R4)2, oxo, and CN.

[0145] In certain embodiments, the radiostabilizer compound of Formula IV is selected from

[0146] In certain embodiments, the radiostabilizer compound of Formula IV is selected from salt thereof. In certain embodiments, the radiostabilizer compound of Formula IV is selected from, salt thereof.

[0147] The radiostabilizer is used in the process of preparing high activity pharmaceutical formulations of F-18 Flomaptitril in an amount which minimizes the amount of radiolytic decomposition. In certain embodiments, from about 0.1 to about 5 equivalents of radiostabilizer are used. In certain embodiments, about 5 equivalents of radiostabilizer are used. In certain embodiments, from about 1 to about 100 equivalents of radiostabilizer are used. In certain embodiments, from about 1 to about 10 equivalents of radiostabilizer are used. In certain embodiments, from about 0.1 to about 1.5 equivalent of radiostabilizer is used. In certain embodiments, from about 0.25 to about 0.75 equivalents of radiostabilizer are used. In certain embodiments, about 0.25, 0.5, 0.75, or 1 equivalent of radiostabilizer are used. In certain embodiments, about 0.5 equivalent of radiostabilizer is used.

[0148] In certain embodiments, about 5 equivalents of TEMPO (about 3.3 mg) is used.

[0149] In certain embodiments, from about 0.1 mg to about 10 mg of TEMPO is used. In certain embodiments, from about 1 to about 10 mg of TEMPO is used. In certain embodiments, from about 1 to about 5 mg of TEMPO is used. In certain embodiments, about 1, 1.5, 2, 2.5, 3, 3.1, 3.1, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10 mg of TEMPO is used.

[0150] Definitions

[0151] Compounds are described using standard nomenclature. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which this invention belongs. The use of examples, or exemplary language (e g., “such as”), is intended merely to better illustrate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. The terms “a” and “an” do not denote a limitation of quantity, but rather denote the presence of at least one of the referenced item. The term “or” means “and / or”.

[0152] The term “about”, as used herein, refers to a range that includes up to 10% less than and up to 10% greater than the stated value. For example, “about 100 milligrams” includes all values from 90 milligrams to 110 milligrams.

[0153] The term “substituted”, as used herein, means that any one or more hydrogens on the designated atom or group is replaced with a moiety selected from the indicated group, provided that the designated atom’s normal valence is not exceeded and the resulting compound is stable. Combinations of substituents and / or variables are permissible only if such combinations result in stable compounds or useful synthetic intermediates.

[0154] A dash ("-") that is not between two letters or symbols is used to indicate a point of attachment for a substituent. For example, -Ci -ealkyl -aryl is attached through the alkyl group, not the aryl group (e.g. a benzyl group). “18F”, “F-18”, or“[18F]” indicate the radioactive isotope Fluorine-18. When “18F”, “F-18”, or “[ 18F]” is used as a prefix, it indicates that the fluorine atom in the compound is the isotopic form, e.g. “[18F]Fluoride”. “18F-” and “F-18 Fluoride”, and the like all describe the radioactive fluorine-18 as an anion.

[0155] “F-18 Flornaptitril” or “18F-FDDNP” is 2-(l-(6-((2-(fluoro- 18F)ethyl)(methyl)amino)naphthalen-2-yl)ethylidene)malononitrile:

[0156] F-18 Flornaptitril (18F-FDDNP)

[0157] “Curie” abbreviated as “Ci” is aunit of radioactivity. A Curie is defined as 3.7xlO10decays per second. The Curie is used as a unit of the amount of radioactivity because the probability of decay for each radionuclide is a fixed physical constant, thus a given amount of a radionuclide will have a corresponding amount of decay. Unless otherwise noted, the radioactivity amounts described herein are non decay-corrected amounts at the end of synthesis, i.e. the actual amount of radioligand available for imaging purposes.

[0158] An alternative unit for radioactive decay (and thus radioactivity) is the becquerel (Bq), which is defined as one decay per second. Since one decay per second is a small amount, a prefix such as giga- (109) or mega- (106) is commonly used with the unit becquerel (i.e. gigabecquerel (GBq) or megabecquerel (MBq)). A Curie can be too large a unit to describe a particular quantity of radionuclide, for example an individual dose of a radioligand, and therefore millicurie (mCi), or 0.001 Ci, can be used.

[0159] As a becquerel is one decay per second and a Curie is 3.7xlO10decays per second, the unit conversion is 3.7xlO10, for example 1 Ci is equal to 37 GBq. One GBq is equal to 0.027 Ci.

[0160] The term “cryptand” or “cryptand ligand”, as used herein, describes a multidentate ligand for a cation which is typically cyclic. In certain embodiments, the cryptand ligand is a cyclic polyether, for example 18-crown-6. In certain embodiments, the cryptand ligand is a cyclic polyether polyamine, for example l-aza-18-crown-6, l,4,10,13-Tetraoxa-7,16- di azacyclooctadecane, 4,7,13,16,21-Pentaoxa-l,10-diazabicyclo[8.8.5]tricosane (Kryptofix® 221), or l ,10-diaza-4,7,13,16,21,24-hexaoxabicyclo[8.8.8]hexacosane (Kryptofix® K222). In certain embodiments, the cryptand ligand is l,10-diaza-4,7, 13, 16,21,24- hexaoxabicyclo[8.8.8]hexacosane (Kryptofix® K222):

[0161] Fluoride ion can form strong ion pairing interactions with cations in solution, decreasing its nucleophilicity. It can be advantageous to isolate the18F-fluoride in solution to increase the rate of fluorination reaction, in particular when the cationic counterion is potassium.

[0162] In certain embodiments, the cryptand ligand is a crown ether is used to bind the cation and isolate the18F-fluoride ion. In certain embodiments, 18-crown-6 is used to bind a potassium cation.

[0163] In certain embodiments, 15-crown-5 is used to bind a sodium cation.

[0164] In certain embodiments, Kryptofix® K222 is used to bind a potassium cation.

[0165] 18F-

[0166] “Antioxidant stabilizer” is an antioxidant compound added to the pharmaceutical formulation to prevent oxidative degradation of F-18 Flornaptitril. Nonlimiting examples of antioxidant stabilizers include ascorbic acid, sodium ascorbate, citric acid, sodium citrate, ethanol, benzoic acid, gentisic acid, ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, calcium stearate, potassium metabisulfate, propyl gallate, sodium metabisulfate, sodium thiosulfate, vitamin E (tocopherol), cyanidin chloride, lutein, lycopene, malvidin chloride, sodium sulfite, malic acid, methionine, tartaric acid, capsorubin, fumaric acid, pelargonidin chloride, peonidin chloride, sodium hydroxymethanesulfinate, and thymol. “Radiostabilizer” is a compound that can react with radical species formed in the fluorination reaction, thereby decreasing the amount of radiolytic decomposition. In certain embodiments, the radiostabilizer increases the yield of the fluorination reaction. In certain embodiments, the radiostabilizer is a persistent radial, a nitrone, an aniline or a phenol, as described herein. In certain embodiments, an aniline radiostabilizer is an aniline compound of Formula II. In certain embodiments, a phenol radiostabilizer is a phenol compound of Formula III. In certain embodiments, a persistent radical radiostabilizer is a persistent radical compound of Formula IV.

[0167] “Solid phase extraction” refers to the process of isolating compounds based on their affinity for a solid medium, for example C18-functionalized silica. In certain embodiments of solid phase extraction, a mixture of compounds is applied to the top of a solid phase extraction cartridge of C18-functionalized silica (for example, a Sep-Pak® cartridge) in a minimal amount of solvent. The cartridge is then washed with an aqueous solvent. The hydrophobic organic compounds are retained by the cartridge, and the salts and polar organic compounds are eluted off the cartridge. The cartridge is then washed with an organic solvent (for example acetonitrile), which elutes the hydrophobic organic compounds.

[0168] “Automated synthesizers” for use in synthesis of radiotracers are commercially available from suppliers including GE Healthcare, Trasis, and Sofie Biosciences. Nonlimiting examples include the GE Healthcare TRACERlab series (e.g. TRACERlab FX2, TRACERlab FX2 N, TRACERlab, and FX N Pro) and GE Healthcare FASTlab series (e.g. FASTlab and FASTlab 2) of automated synthesizers. Use of automated synthesizers in the manufacture of PET radiotracers is described in Alexoff, D.L. “Chapter 8: Automation for the Synthesis and Application of PET Radiopharmaceuticals” 2002, Handbook of Radiopharmaceuticals: Radiochemistry and Applications .

[0169] Automated synthesizers are modular systems which allow configuration of various synthetic operations onto an automated platform. These systems can be used with a dedicated cassette comprising the reagents and tubing to synthesize a specific radioligand or can be set up manually, for example for syntheses which do not have a commercially available cassette. Automated radiosynthesizers for18F-radioligand production typically include a phase separation unit for isolation of18F from the18O target water, a series of valves which control the flow of reagents and products, a heating and cooling system for reaction temperature control, and a purification unit. In certain embodiments, the automated synthesizer is set up inside of a hot cell. Before radioactivity is present, a technician loads the starting material solutions and then can seal the hot cell.18F fluoride can then be transferred from the cyclotron into the hot cell and the reagent manipulations for the radiosynthesis are performed by the automated synthesizer according to the programmed sequence. The automated synthesizer can perform any reaction quenching and / or extraction steps followed by purification. In certain embodiments, purification is performed using an HPLC module. In certain embodiments, the automated synthesizer can then isolate the product from the HPLC eluent and perform the steps required for formulation.

[0170] In certain embodiments, an automated synthesizer can be set up according to FIG. 2-4 and description herein for the synthesis of F-18 Flornaptitril in an automated or semi-automated manner.

[0171] Even an automated synthesis may require input from a technician, for example preparing reagent solutions for the automated synthesizer to use. A semi -automated synthesis is a synthesis requiring substantial input from a technician, for example using an automated synthesizer for the radiolabeling reaction followed by manual solid phase extraction, HPLC purification, and / or formulation.

[0172] GE TRACERlab FX2 N

[0173] The TRACERlab FX2 N is a fully automated two-reactor system for easy and efficient production of18F tracers starting with [18F]Fluoride from a cyclotron via a nucleophilic reaction. TRACERlab FX2 N combines flexibility and productivity. The synthesis sequence can be user defined in order to produce the desired tracer. It includes an HPLC purification and formulation system.

[0174] The first step is to define the process and to set up an appropriate sequence. After preparing the starting material, the production runs automatically. It performs the synthesis, purification and the formulation of the final solution. Once this is done, the tracer is prepared without requiring operator interaction when radioactivity is present. The [18F]Fluoride can be transferred automatically from a cyclotron [18O]water target into the TRACERlab FX2 N. The final radiochemical batch is dispensed into a product container, which may be at a separate location (e.g. outside of a hot cell). In-process diagnostic capability allows for the measurement of important process parameters with a frequency up to one measurement per second. These recorded data can be displayed as a graph and can be used for diagnostics in a “fingerprint” manner. In addition the time for each phase in the process and the chromatogram of the purification process are recorded. After synthesis, an automatic cleaning program is used to prepare for the next production. During this procedure the reaction vials can be autoclaved.

[0175] GEFASTLab 2

[0176] FASTlab 2 is an automated radiosynthesizer with a unique integrated cassette-based design together with GMP quality components (e.g. vials and cartridges) which enable and ease the regulatory reliability and compliance in tracer production. FASTlab 2 can include an integrated HPLC module which allows for automated purification of intermediates and final products through two columns. The FASTlab 2 can also include a formulation system which enables higher dilution volume and faster mixing compared to other automated synthesizers using a traditional cassette.

[0177] “Specific activity” is the amount of radioactive isotope for a given sample, for instance the amount of radioactive isotope per gram or per mole of material. During synthesis of PET tracers, contamination by sources of naturally occurring non-radioactive [19F]fluoride leads to a final product comprising a mixture of18F- and19F-labeled molecules. Specific activity describes the ratio of radioactivity (i.e., quantity of18F-labeled molecules) to the total quantity of radioactive plus non-radioactive (i.e.,19F-labeled) molecules. A highly efficient labeling reaction can produce material with high specific activity, even if it is performed on a small amount of material (and thus low total activity). In certain embodiments the specific activity of the pharmaceutical formulations described herein is from about 0.5 to about 10 Ci / pmol. In certain embodiments, the specific activity of the pharmaceutical formulations described herein is from about 1 to about 5 Ci / pmol. For example, a formulation with a specific activity of 3 Ci / pmol will contain 3 Ci of activity for 293 pg of Flomaptitril (molecular weight of 293 g / mol).

[0178] “Total activity” is the total amount of radioactive material, regardless of the specific activity of the material. For example, a formulation can have high total activity and a low specific activity if a portion of the precursor reacts with19F to generate non-radioactive F-19 Flomaptitril. This may occur, for example, if the H218O used during bombardment contains trace19F. A formulation can have a low total activity and a high specific activity if the labeling reaction is highly efficient but performed on a small amount of precursor. A high total activity is advantageous for centralized manufacturing of a radiotracer and distribution to healthcare clinics for use in PET scanning.

[0179] “Alkyl” is a branched, straight chain, or cyclic saturated aliphatic hydrocarbon group. In certain embodiments, the alkyl contains from 1 to about 8 carbon atoms, more generally from 1 to about 6 carbon atoms, from 1 to about 4 carbon atoms, or from 1 to 3 carbon atoms. In certain embodiments, the alkyl contains from 1 to about 6 carbon atoms. The specified ranges as used herein indicate an alkyl group which is considered to explicitly disclose as individual species each member of the range described as a unique species. For example, the term Ci ealkyl as used herein indicates a straight or branched alkyl group having from 1, 2, 3, 4, 5, or 6 carbon atoms and also a carbocyclic alkyl group of 3, 4, 5, or 6 carbon atoms and is intended to mean that each of these is described as an independent species. For example, the term Ci-4alkyl as used herein indicates a straight or branched alkyl group having from 1, 2, 3, or 4 carbon atoms and is intended to mean that each of these is described as an independent species. Examples of alkyl include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, t-butyl, n-pentyl, isopentyl, tert-pentyl, neopentyl, n-hexyl, 2-methylpentane, 3 -methylpentane, 2,2-dimethylbutane, 2,3-dimethylbutane, and hexyl.

[0180] “Halogen” includes fluorine, chlorine, bromine and iodine. In certain embodiments, “halogen” includes fluorine, chlorine, and bromine.

[0181] “Haloalkyl” indicates both branched and straight-chain alkyl groups substituted with 1 or more halogen atoms, up to the maximum allowable number of halogen atoms. In certain embodiments, the haloalkyl is a branched or straight-chain alkyl group substituted with 1 or more fluorine groups. Examples of haloalkyl include, but are not limited to, trifluoromethyl, monofluorom ethyl, difluoromethyl, pentafluoroethyl, heptafluoropropyl, and nonafluorobutyl. In certain embodiments, haloalkyl is nonafluorobutyl.

[0182] “Aryl" indicates an aromatic group containing only carbon in the aromatic ring or rings. In certain embodiments, the aryl group contains 1 to 2 separate or fused rings and is 6 to 10 ring atoms, without heteroatoms as ring members. The term “aryl” includes groups where a saturated or partially unsaturated carbocycle group is fused with an aromatic ring. The term “aryl” also includes groups where a saturated or partially unsaturated heterocycle group is fused with an aromatic ring so long as the attachment point is the aromatic ring. Such compounds may include aryl rings fused to a 4 to 7 or a 5 to 7-membered saturated or partially unsaturated cyclic group that optionally contains 1, 2 or 3 heteroatoms independently selected from N, O, B, P, Si and S, to form, for example, a 3,4-methylenedioxyphenyl group. Aryl groups include, for example, phenyl and naphthyl, including 1 -naphthyl and 2-naphthyl. The term “carrier” applied to pharmaceutical compositions / combinations of the invention refers to a diluent, excipient, or vehicle with which an active compound is provided.

[0183] A “pharmaceutically acceptable excipient” means an excipient that is useful in preparing a pharmaceutical composition / combination that is generally safe, acceptable for human consumption, and neither biologically nor otherwise inappropriate for administration to a human.

[0184] Reaction Conditions

[0185] To avoid substantial decay of the18F, it is advantageous for the radiolabeling reaction to occur rapidly. In certain embodiments, the radiolabeling reaction is performed at elevated temperature in minimal solvent volume to facilitate rapid production of F-18 Flornaptitril.

[0186] In certain embodiments, the reaction is performed at a temperature that facilitates conversion of at least about 60%, 70%, 80%, 85%, 90%, 95% or even 99% of the Compound of Formula I within about 20 minutes. In certain embodiments, the reaction is performed at from about 70°C to about 120°C. In certain embodiments, the reaction is performed at from about 80°C to about 115°C degrees. In certain embodiments, the reaction is performed at from about 85°C to about 105°C degrees. In certain embodiments, the reaction is performed at about 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, or 120°C. In certain embodiments, the reaction is heated for less than about 45, 40, 35, 30, 25, 20, 18, 16, 14, 12, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 minute. In certain embodiments, the reaction is heated for less than about 20, 15, or 10 minutes. In certain embodiments the reaction is heated for about 15 minutes. In certain embodiments, the reaction is heated for from about 5 to about 35 minutes, from about 10 to about 25 minutes, or from about 15 to about 20 minutes.

[0187] Once the reaction is complete, it is advantageous to limit further reactivity and potential decomposition. In certain embodiments, the reaction is cooled by passing an inert gas through the reaction vessel and then quenched. In certain embodiments, the reaction is quenched with water. In certain embodiments, the reaction is quenched with an acidic solution, for example 0.1M HC1. Quenching the reaction with an acidic solution may prevent hydrolysis of the malononitrile group in F-18 Flornaptitril. In certain embodiments, the reaction is quenched with a pre-cooled solution, for example a solution that is from about 0°C to about 10°C. In certain embodiments, the precooled solution comprises ammonium acetate, sodium ascorbate, or both. In certain embodiments, the reaction is cooled to about 70, 60, 50, 40, 30, or 25°C, and passed through a column, including but not limited to neutral alumina. In certain embodiments, the neutral alumina column is washed with acetonitrile to elute the product.

[0188] Embodiments of Step (a)

[0189] Preparation ofI8F-fluoride

[0190] In certain embodiments18F-fluoride is produced through the18O(p,n)18F nuclear reaction. A target of18O-enriched water is bombarded with high energy protons from a cyclotron to generate18F. In certain embodiments, from about 0.5 Ci to about 20 Ci of18F-fluoride is produced. In certain embodiments, from about 1.5 Ci to about 20 Ci of18F-fluoride is produced. In certain embodiments, from about 3.0 to about 18 Ci, from about 5.0 to about 15 Ci, or from about 7.5 to about 12.5 Ci of18F-fluoride is produced. In certain embodiments, about 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 Ci of18F -fluoride is produced. The solution of18F-fluoride can then be sent to a “hot cell” to be used in a fluorination reaction. A hot cell is an enclosure for performing synthetic radiochemistry which has shielding to protect the technician. The shielding can be made of concrete, lead, leaded glass, steel, or depleted uranium.

[0191] Trap and Release of18F-fluoride

[0192] In certain embodiments, once transported to the hot cell, the18F-fluoride solution from the cyclotron is trapped on an anion exchange column. This isolates the18F from the18O water target and can allow for reuse of the isotopically enriched water. The18F-fluoride in solution of18O water is passed through a column comprising a resin with cationic groups. The18F-fluoride interacts with the cations in the resin and is bound, while the18O water can flow through. The18F-fluoride can then be eluted using a salt solution. In one non-limiting example, the18F-fluoride is eluted from the anion exchange resin with an aqueous solution of potassium carbonate. The carbonate ion can displace the18F-fluoride and a solution of18F-potassium fluoride is eluted from the anion exchange column.

[0193] In certain embodiments, the anion exchange column is a quaternary ammonium- functionalized resin, such as a quaternary methylammonium (QMA) cartridge. In certain embodiments, the anion exchange column is functionalized with pyridinium-, tetrabutylammonium-, piperazine-, or phosphazine-functionalized resin. Preconditioning the anion exchange resin can improve the recovery of18F-fluoride. In certain embodiments, the anion exchange resin is preconditioned with a carbonate solution.

[0194] In certain embodiments, the18F-fluoride is eluted with a salt solution comprising carbonate ions. In certain embodiments, the solution is aqueous. In certain embodiments, the carbonate solution has an organic cosolvent, for example acetonitrile or methanol. In certain embodiments, the carbonate solution compri ses up to about 1 %, 2%, 5%, 10%, 15%, or 20% acetonitrile in water. In certain embodiments, the carbonate solution comprises from about 20% to about 50% acetonitrile, about 50% to about 75% acetonitrile, or from about 70% to about 95% acetonitrile. In certain embodiments, the carbonate solution comprises up to about 1%, 2%, 5%, 10%, 15%, or 20% methanol in water. In certain embodiments, the carbonate solution comprises from about 20% to about 50% methanol, about 50% to about 75% methanol, or from about 70% to about 95% methanol. In certain embodiments, the carbonate solution is a solution of sodium, lithium, potassium, calcium, cesium, tetraalkylammonium or ammonium carbonate. In certain embodiments, the carbonate solution is a solution of potassium, cesium, or tetrabutylammonium carbonate. In certain embodiments, the carbonate solution is a solution of potassium carbonate. In certain embodiments, the carbonate solution further comprises the cryptand ligand.

[0195] Additives in the salt solution can improve the recovery of18F-fluoride. In certain embodiments, the salt solution comprises one or more additives selected from potassium oxalate, potassium triflate, potassium dihydrogen phosphate, potassium bicarbonate, tetrabutylammonium hydroxide, tetrabutyl ammonium bicarbonate, tetrabutyl ammonium methanesulfonate, tetrabutylammonium triflate, and tetraethylammonium bicarbonate.

[0196] Preparation of anhydrous18F-fluoride

[0197] Fluoride ion in aqueous solution readily forms a cage-like structure of hydrogen bonds, termed a solvation sphere. This solvation sphere substantially reduces the reactivity of18F. Therefore, the water in the salt solution used to elute the18F -fluoride in certain embodiments rigorously removed to encourage reactivity of fluoride with the precursor compound. In certain embodiments, the water is removed through azeotropic drying, for example with acetonitrile. Water and acetonitrile form an azeotrope that boils at 80°C, which facilitates drying of the18F- fluoride solution. In certain embodiments, the aqueous solution of18F-fluoride eluted from the anion exchange resin is azeotropically dried with acetonitrile. In certain embodiments, flow of an inert gas is passed through the headspace of the reaction vessel and the vessel is heated to at least 80°C. The reaction is heated until the solvent evaporates, cooled, and then more acetonitrile is added. This process can be repeated until the water is removed, for example after 1, 2, 3, 4, 5, 6, or more azeotropic drying cycles. Other solvents that form a lower-boiling azeotrope, for example benzene, can also be used to azeotropically remove water. In certain embodiments, the water is evaporated without addition of a cosolvent.

[0198] Cryptand ligand

[0199] Fluoride ion can also form strong ion pairs with cations in solution, decreasing the nucleophilicity. It can be advantageous to isolate the18F-fluoride in solution to increase the rate of fluorination reaction, in particular when the cation present is potassium. In certain embodiments, no cryptand ligand is used in the reaction.

[0200] In certain embodiments, the cryptand ligand is a crown ether used to bind the cation and isolate the18F-fluoride ion. In certain embodiments, 18-crown-6 is used to bind a potassium cation.

[0201] In certain embodiments, 15-crown-5 is used to bind a sodium cation.

[0202] In certain embodiments, the cryptand ligand is a cyclic polyether polyamine, for example 1 -aza- 18-crown-6, 1,4,10,13 -Tetraoxa-7, 16-diazacyclooctadecane, 4,7,13,16,21 -Pentaoxa- 1,10- diazabicyclo[8.8.5]tricosane (Kryptofix® 221), or l,10-diaza-4,7,13,16,21,24- hexaoxabicyclo[8.8.8]hexacosane (Kryptofix® K222). In certain embodiments, the cryptand ligand is l,10-diaza-4,7,13,16,21,24-hexaoxabicyclo[8.8.8]hexacosane (Kryptofix® K222):

[0203] In certain embodiments, Kryptofix® K222 is used to bind a potassium cation: 18F-

[0204] In certain embodiments, the cryptand ligand is added to the salt solution used to elute the18F-fluoride. In certain embodiments, the cryptand ligand is added to the solvent used for azeotropic drying of the18F-fluoride.

[0205] Embodiments of the process of Step (a)

[0206] Al . In certain embodiments, the process of Step (a) comprises: , ,

[0207] Formula I i). Trapping from about 5 to about 20 Ci of18F-fluoride on a solid phase; ii). Eluting the18F-fluoride from the solid phase with a carbonate base; iii). Collecting the eluent into a vial; iv). Azeotropically drying the resulting solution; v). Adding a compound of Formula I and a radiostabilizer; vi). Reacting the mixture; and vii). Cooling and quenching the reaction.

[0208] A2. The process of embodiment Al, wherein the radiostabilizer is selected from the group consisting of a persistent radical compound, a nitrone, an aniline of Formula II, a phenol of Formula III, and a diarylalkene.

[0209] A3. The process of embodiment Al or A2, wherein the radiostabilizer is a persistent radical compound.

[0210] A4. The process of any one of embodiments Al -A3, wherein the radiostabilizer is TEMPO.

[0211] A5. The process of any one of embodiments Al -A3, wherein the radiostabilizer is galvinoxyl radical. A6. The process of embodiment Al or A2, wherein the radiostabilizer is a nitrone.

[0212] A7. The process of embodiment Al or A6, wherein the radiostabilizer is selected from N-tert- butyl-a-phenylnitrone (PBN), a-4-pyridyl-l-oxide-N-t-butylnitrone (POBN), 5,5- dimethylpyrroline-N-oxide (DMPO), 3, 4-dihydro-2-methyl- 1,1 -dimethylethyl ester-2H- pyrrole-2-carboxylic acid- 1 -oxide (BMPO), 3,4-dihydro-2-methyl-2H-pyrrole-2- carboxylic acid 1 -oxide, ethyl ester (EMPO).

[0213] A8. The process of embodiment Al or A2, wherein the radiostabilizer is an aniline of Formula: salt thereof; wherein

[0214] R2is selected from NH2, NHR4, and N(R4)2;

[0215] R3a, R3b, and R3care independently selected from hydrogen, halogen, Ci-ealkyl, C(O)OH, C(O)OCi-6alkyl, and C(O)N(R4)2; and

[0216] R4is selected from hydrogen, Ci-ealkyl, aryl, -Ci-salkyl-aryl, heterocycle, and heteroaryl.

[0217] A9. The process of embodiment A8, wherein the radiostabilizer is selected from

[0218] A10. The process of embodiment Al or A2, wherein the radiostabilizer is a phenol of Formula salt thereof; wherein

[0219] R22is OH;

[0220] R23is selected from hydrogen, OH, OR4, CH2OH, and CH2OR4;

[0221] R3a, R3b, R3Cand R3dare independently selected from hydrogen, halogen, Ci-ealkyl, C(O)OH, C(O)OCi-6alkyl, and C(O)N(R4)2; and

[0222] R4is selected from hydrogen, Ci-ealkyl, aryl, -Ci-ealkyl-aryl, heterocycle, and heteroaryl. Al l. The process of embodiment Al 1, wherein the radiostabilizer is selected from

[0223] A12. The process of embodiment Al or A2, wherein the radiostabilizer is a diarylalkene.

[0224] A13. The process of embodiment A12, wherein the radiostabilizer is diphenylethylene.

[0225] A14. The process of any one of embodiments Al-1 A2, wherein R1is 4-CH3-phenyl.

[0226] A15. The process of any one of embodiments A1-A12, wherein R1is 4-Br-phenyl.

[0227] A16. The process of any one of embodiments A1-A12, wherein R1is 4-NO2-phenyl.

[0228] A17. The process of any one of embodiments A1-A12, wherein R1is 2-NO2-phenyl.

[0229] A18. The process of any one of embodiments A1-A12, wherein R1is CH3.

[0230] A19. The process of any one of embodiments A1-A12, wherein R1is (CF2)3-CF3.

[0231] A20. The process of any one of embodiments Al -Al 9, wherein the solid phase in (i) is an anion exchange resin.

[0232] A21. The process of any one of embodiments A1-A20, wherein the anion exchange resin is QMA resin.

[0233] A22. The process of any one of embodiments A1-A21, wherein the18F is eluted in (ii) with an aqueous acetonitrile solution comprising a carbonate base.

[0234] A23. The process of embodiment A22, wherein the aqueous acetonitrile solution is from about 20% to about 50% acetonitrile, about 50% to about 75% acetonitrile, or from about 70% to about 95% acetonitrile.

[0235] A24. The process of embodiment A22, wherein the aqueous acetonitrile solution is from about 70% to about 95% acetonitrile.

[0236] A25. The process of embodiment A22, wherein the aqueous acetonitrile solution is up to about 20%, up to about 10%, up to about 5%, up to about 2% or up to about 1% acetonitrile.

[0237] A26. The process of any one of embodiments A1-A25, wherein the18F is eluted in (ii) with a solution comprising potassium carbonate. A27. The process of any one of embodiments A1-A25, wherein the18F is eluted in (ii) with a solution comprising cesium carbonate.

[0238] A28. The process of any one of embodiments A1-A27, wherein the18F is eluted in (ii) with a solution comprising a cryptand ligand.

[0239] A29. The process of any one of embodiments A1-A27, wherein the eluent in (iii) is collected into a vial containing a cryptand ligand.

[0240] A30. The process of embodiment A28 or A29, wherein the cryptand ligand is Kryptofix® K222.

[0241] A31. The process of any one of embodiments A1-A30, wherein the eluent of (iii) is azeotropically dried in (iv) with acetonitrile.

[0242] A32. The process of embodiment A31, wherein the azeotropic drying step is repeated three times.

[0243] A33. The process of any one of embodiments A1-A32, wherein the compound of Formula I is added in (v) as a solution in acetonitrile.

[0244] A34. The process of any one of embodiments A1-A33, wherein the reaction in (vi) is heated to from about 80°C to about 120°C.

[0245] A35. The process of any one of embodiments A1-A33, wherein the reaction in (vi) is heated to from about 90°C to about 110°C.

[0246] A36. The process of any one of embodiments A1-A33, wherein the reaction in (vi) is heated to about 90°C.

[0247] A37. The process of any one of embodiments A1-A33, wherein the reaction in (vi) is heated to about 95°C.

[0248] A38. The process of any one of embodiments A1-A33, wherein the reaction in (vi) is heated to about 100°C.

[0249] A39. The process of any one of embodiments A1-A33, wherein the reaction in (vi) is heated to about 105°C.

[0250] A40. The process of any one of embodiments A1-A33, wherein the reaction in (vi) is heated to about 110°C.

[0251] A41. The process of any one of embodiments A1-A33, wherein the reaction in (vi) is heated to about 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, or 105°C.

[0252] A42. The process of any one of embodiments A1-A41, wherein the reaction in (vi) is reacted for up to 55 minutes. A43. The process of any one of embodiments A 1-A41, wherein the reaction in (vi) is reacted for up to 45 minutes.

[0253] A44. The process of any one of embodiments A1-A41, wherein the reaction in (vi) is reacted for up to 35 minutes.

[0254] A45. The process of any one of embodiments A1-A41, wherein the reaction in (vi) is reacted for up to 25 minutes.

[0255] A46. The process of any one of embodiments A1-A41, wherein the reaction in (vi) is reacted for from about 5 minutes to about 20 minutes.

[0256] A47. The process of any one of embodiments A1-A41, wherein the reaction in (vi) is reacted for about 5 minutes.

[0257] A48. The process of any one of embodiments A1-A41, wherein the reaction in (vi) is reacted for about 10 minutes.

[0258] A49. The process of any one of embodiments A1-A41, wherein the reaction in (vi) is reacted for about 15 minutes.

[0259] A50. The process of any one of embodiments A1-A41, wherein the reaction in (vi) is reacted for about 20 minutes.

[0260] A51. The process of any one of embodiments A1-A41, wherein the reaction in (vi) is reacted for about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 minutes.

[0261] A52. The process of any one of embodiments A1-A51, wherein the reaction is cooled in (vii) by passing a stream of inert gas through the reaction vial headspace.

[0262] A53. The process of embodiment A52, wherein the inert gas is selected from helium, nitrogen, and argon.

[0263] A54. The process of any one of embodiments A1-A53, wherein the reaction is quenched in (vii) with an aqueous solution.

[0264] A55. The process of any one of embodiments A1-A53, wherein the reaction is quenched in (vii) with an acidic aqueous solution.

[0265] A56. The process of any one of embodiments A1-A55, wherein the reaction is quenched in (vii) with aqueous hydrochloric acid.

[0266] A57. The process of embodiment A56, wherein the aqueous hydrochloric acid is from about 0.01 to about IM hydrochloric acid. A58. The process of any one of embodiments A1-A55, wherein the reaction is quenched in (vii) with ammonium acetate.

[0267] A59. The process of embodiment A58, wherein the ammonium acetate is from about 0.01M to about IM ammonium acetate.

[0268] A60. The process of any one of embodiments A1-A54, wherein the reaction is quenched in (vii) with water and acetonitrile.

[0269] A61. The process of any one of embodiments A54-A60, wherein the aqueous solution used to quench the reaction is pre-cooled to from about 0°C to about 10°C.

[0270] A62. The process of any one of embodiments A53-A61, wherein the reaction is passed through a cartridge containing neutral alumina before quenching.

[0271] A63. The process of any one of embodiments A1-A62, wherein from about 1 to about 25 mg of carbonate base is used.

[0272] A64. The process of any one of embodiments A1-A62, wherein from about 2 to about 8 mg or carbonate base is used.

[0273] A65. The process of any one of embodiments A1-A62, wherein about 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, or 6 mg of carbonate base is used.

[0274] A66. The process of any one of embodiments A1-A62, wherein from about 3 to about 5 mg of carbonate base is used.

[0275] A67. The process of any one of embodiments A1-A62, wherein about 4 mg of carbonate base is used.

[0276] A68. The process of any one of embodiments A28-A67, wherein from about 8 to about 30 mg of cryptand ligand is used.

[0277] A69. The process of any one of embodiments A28-A67, wherein from about 8 to about 20 mg of cryptand ligand is used.

[0278] A70. The process of any one of embodiments A28-A67, wherein from about 10 to about 20 mg of cryptand ligand is used.

[0279] A71. The process of any one of embodiments A28-A67, wherein from about 16 to about 20 mg of cryptand ligand is used.

[0280] A72. The process of any one of embodiments A28-A67, wherein about 16, 17, 18, 19, or 20 mg of cryptand ligand is used. A73. The process of any one of embodiments A28-A67, wherein about 18 mg of cryptand ligand is used.

[0281] Additional embodiments of the process of Step (a)

[0282] In certain embodiments, the process of Step (a) comprises:

[0283] Formula I

[0284] 1. Trapping18F-fluoride on an anion exchange resin;

[0285] 2. Eluting the18F-fluoride with an aqueous solution of potassium carbonate;

[0286] 3. Collecting the eluent into a vial containing an acetonitrile solution of Kryptofix® K222;

[0287] 4. Azeotropically drying the solution;

[0288] 5. Adding a compound of Formula I and TEMPO in an acetonitrile solution;

[0289] 6. Reacting the mixture at about 90-110°C for about 5-20 minutes; and

[0290] 7. Cooling and then quenching the reaction.

[0291] In certain embodiments, the process of Step (a) comprises:

[0292] Formula I

[0293] 1. Trapping18F-fluoride on an anion exchange resin;

[0294] 2. Eluting the18F-fluoride with a solution of potassium carbonate and Kryptofix® K222 in about 10% acetonitrile in water;

[0295] 3. Azeotropically drying the solution;

[0296] 4. Adding a compound of Formula I and TEMPO in an acetonitrile solution;

[0297] 5. Reacting the mixture at about 90-1 10°C for about 5-20 minutes; and

[0298] 6. Cooling and then quenching the reaction. In certain embodiments, the process of Step (a) comprises:

[0299] Formula I Trapping18F-fluoride on an anion exchange resin; Eluting the18F-fluoride with a solution of potassium carbonate and Kryptofix® K222 in about 95% acetonitrile in water; Azeotropically drying the solution; Adding a compound of Formula I and TEMPO in an acetonitrile solution; Reacting the mixture at about 90-110°C for about 5-20 minutes; and Cooling and then quenching the reaction.

[0300] In certain embodiments, the process of Step (a) comprises:

[0301] Formula I Trapping18F-fluoride on an anion exchange resin; Eluting the18F-fluoride with a solution of potassium carbonate and Kryptofix® K222 in about 10% acetonitrile in water; Azeotropically drying the solution three times; Adding a compound of Formula I and TEMPO in an acetonitrile solution; Reacting the mixture at about 95°C for about 5-20 minutes; and Cooling and then quenching the reaction. In certain embodiments, the process of Step (a) comprises:

[0302] Formula I

[0303] 1. Trapping18F-fluoride on an anion exchange resin;

[0304] 2. Eluting the18F-fluoride with a solution of potassium carbonate and Kryptofix® K222 in about 10% acetonitrile in water;

[0305] 3. Azeotropically drying the solution three times;

[0306] 4. Adding a compound of Formula I and TEMPO in an acetonitrile solution;

[0307] 5. Reacting the mixture at about 95°C for about 10-15 minutes; and

[0308] 6. Cooling and then quenching the reaction.

[0309] In certain embodiments, the process of Step (a) comprises:

[0310] Formula I

[0311] 1. Trapping18F-fluoride on an anion exchange resin;

[0312] 2. Eluting the18F-fluoride with a solution of potassium carbonate and Kryptofix® K222 in about 10% acetonitrile in water;

[0313] 3. Azeotropically drying the solution three times;

[0314] 4. Adding a compound of Formula I and TEMPO (about 2: 1 precursocradiostabilizer) in an acetonitrile solution;

[0315] 5. Reacting the mixture at about 95°C for about 10-15 minutes; and

[0316] 6. Cooling and then quenching the reaction. In certain embodiments, the process of Step (a) comprises:

[0317] Formula I

[0318] 1. Trapping18F-fluoride on an anion exchange resin;

[0319] 2. Eluting the18F-fluoride with a solution of potassium carbonate and Kryptofix® K222 in about 10% acetonitrile in water;

[0320] 3. Azeotropically drying the solution three times;

[0321] 4. Adding a compound of Formula I and from about 1 to 100 equivalents of TEMPO in an acetonitrile solution;

[0322] 5. Reacting the mixture at about 95°C for about 10-15 minutes; and

[0323] 6. Cooling and then quenching the reaction.

[0324] In certain embodiments, the process of Step (a) comprises:

[0325] Formula I

[0326] 1. Trapping18F-fluoride on an anion exchange resin;

[0327] 2. Eluting the18F-fluoride with a solution of potassium carbonate and Kryptofix® K222 in about 10% acetonitrile in water;

[0328] 3. Azeotropically drying the solution three times;

[0329] 4. Adding a compound of Formula I and from about 2 to about 20 equivalents of TEMPO in an acetonitrile solution;

[0330] 5. Reacting the mixture at about 95°C for about 10-15 minutes; and

[0331] 6. Cooling and then quenching the reaction. In certain embodiments, the process of Step (a) comprises:

[0332] Formula I

[0333] 1. Trapping from from about 2.5 to about 7.5 Ci of18F-fluoride on an anion exchange resin;

[0334] 2. Eluting the18F-fluoride with a solution of from about 1 to about 5 mg of potassium carbonate and from about 12 to about 20 mg of Kryptofix® K222 in about 10% acetonitrile in water;

[0335] 3. Azeotropically drying the solution three times;

[0336] 4. Adding a compound of Formula I and from about 2 to about 20 equivalents of TEMPO in an acetonitrile solution;

[0337] 5. Reacting the mixture at about 95°C for about 10-15 minutes; and

[0338] 6. Cooling and then quenching the reaction.

[0339] In certain embodiments, the process of Step (a) comprises:

[0340] Formula I

[0341] 1. Trapping from from about 2.5 to about 7.5 Ci of18F-fluoride on an anion exchange resin;

[0342] 2. Eluting the18F-fluoride with a solution of from about 1 to about 5 mg of potassium carbonate and from about 12 to about 20 mg of Kryptofix® K222 in about 10% acetonitrile in water;

[0343] 3. Azeotropically drying the solution three times;

[0344] 4. Adding a compound of Formula I and from about 1 mg to about 10 mg of TEMPO in an acetonitrile solution;

[0345] 5. Reacting the mixture at about 95°C for about 10-15 minutes; and

[0346] 6. Cooling and then quenching the reaction. In certain embodiments, the process of Step (a) comprises:

[0347] Formula I

[0348] 1. Trapping from about 3.5 Ci of18F-fluoride on an anion exchange resin;

[0349] 2. Eluting the18F-fluoride with a solution of about 4 mg of potassium carbonate and about 18 mg of Kryptofix® K222 in about 10% acetonitrile in water;

[0350] 3. Azeotropically drying the solution three times;

[0351] 4. Adding a compound of Formula I and from about 3 to about 4 mg of TEMPO in an acetonitrile solution;

[0352] 5. Reacting the mixture at about 95°C for about 10-15 minutes; and

[0353] 6. Cooling and then quenching the reaction.

[0354] In certain embodiments, the process of Step (a) comprises:

[0355] Formula I

[0356] 7. Trapping from about 3.5 Ci of18F-fluoride on an anion exchange resin;

[0357] 8. Eluting the18F-fluoride with a solution of about 4 mg of potassium carbonate and about 18 mg of Kryptofix® K222 in about 10% acetonitrile in water;

[0358] 9. Azeotropically drying the solution three times;

[0359] 10. Adding a compound of Formula I and about 3.3 mg of TEMPO in an acetonitrile solution;

[0360] 11. Reacting the mixture at about 95°C for about 10-15 minutes; and

[0361] 12. Cooling and then quenching the reaction.

[0362] Step (b)

[0363] In certain embodiments, purification of the reaction mixture from Step (a) is performed by solid phase extraction. Solid phase extraction facilitates separation of the water-soluble salts (e.g. potassium fluoride and potassium carbonate) from the organic compounds (e.g. F-18 Flomaptitril) in the reaction mixture. In certain embodiments, the solid phase extraction media is a C18 cartridge. In certain embodiments, the solid phase extraction media is an alumina cartridge.

[0364] In certain embodiments, the reaction mixture is concentrated by passing an inert gas (such as helium, nitrogen, or argon) over the reaction mixture before purification by solid phase extraction. The concentrated reaction mixture can be loaded onto solid phase extraction media, flushed with aqueous solvent, and then eluted with an organic solvent. Suitable solid phase extraction media useful in the process described herein includes but is not limited to Cl 8 Sep- Pak®. In certain embodiments, the solid phase extraction media is flushed with from about 1 mL of aqueous solvent to about 50 mL of aqueous solvent. In certain embodiments, the solid phase extraction media is flushed with from about 5 mL to about 40 mL, from about 10 mL to about 30 mL, or from about 15 to about 25 mL of aqueous solvent, or about 10, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22 24, 26, 28, or 30 mL of aqueous solvent. In certain embodiments, the solid phase extraction media is flushed with from about 1 mL to about 20 mL, from about 2 mL to about 10 mL, or from about 3 mL to about 5 mL of aqueous solvent. In certain embodiments, the F-l 8 Flomaptitril is eluted from the solid phase extraction media with from about 0. 1 mL to about 20 mL of organic solvent. In certain embodiments, the F-l 8 Flomaptitril is eluted from the solid phase extraction media with from about 0.5 to about 5 mL of organic solvent. In certain embodiments, the F-18 Flomaptitril is eluted from the solid phase extraction media with about 0.5, 0.75, 1, 1.1, 1.25, 1.5, 1.75, 2, 2.5, 3, 3.5, 4, 4.5 or 5 mL of organic solvent. In certain embodiments, the F-18 Flomaptitril is eluted from the solid phase extraction media with from about 0.25 mL to about 15 mL, from about 0.3 mL to about 10 mL, from about 0.4 to about 7.5 mL, from about 0.5 to about 5 mL, from about 0.75 to about 3 mL, or from about 1 to about 2 mL of organic solvent.

[0365] In certain embodiments, the aqueous solution for flushing the solid phase extraction media is water. In certain embodiments, the aqueous solution for flushing the solid phase extraction media comprises ascorbic acid, sodium ascorbate, ammonium acetate, acetic acid, sodium citrate, citric acid, sodium chloride, or potassium chloride. In certain embodiments, the aqueous solution for flushing the solid phase extraction media comprises ascorbic acid and sodium ascorbate.

[0366] Suitable organic solvents for elution of the product include methanol, ethanol, dichloromethane, tetrahydrofuran, and acetonitrile. In certain embodiments, the organic solvent used to elute the F-18 Flomaptitril from the solid phase extraction media is ethanol. In certain embodiments, purification of the reaction mixture from Step (a) is performed by HPLC. In certain embodiments, the mobile phase for HPLC purification of the reaction mixture from Step (a) comprises acetonitrile, ethanol, sodium ascorbate, ascorbic acid, sodium citrate, or citric acid.

[0367] In certain embodiments, solid phase extraction is used in combination with HPLC to purify F-18 Flornaptitril from the reaction mixture. For example, the reaction mixture can be purified by solid phase extraction to remove inorganic impurities, followed by purification by HPLC to separate the F-18 Flornaptitril from organic impurities.

[0368] In certain embodiments, the reaction mixture from Step (a) is purified by solid phase extraction followed by HPLC. The reaction mixture is loaded onto solid phase extraction media, washed with an aqueous solvent, the F-18 Flornaptitril is eluted with an organic solvent, and the eluate is optionally concentrated and then injected into a HPLC system. In certain embodiments, the F-18 Flornaptitril is eluted with ethanol. In certain embodiments, the ethanol eluate is concentrated before injection into a HPLC system.

[0369] In certain embodiments, the F-18 Flornaptitril is isolated after HPLC by solid phase extraction. For example, the reaction mixture or solid phase extraction eluate is purified by HPLC, then the fraction or fractions containing F-18 Flornaptitril are diluted with water and applied to a solid phase extraction cartridge. The F-18 Flornaptitril is then eluted from the solid phase extraction cartridge using an organic solvent such as ethanol. Isolation of the product from the HPLC fractions using solid phase extraction facilitates the rapid production of concentrated solutions of F-18 Flornaptitril for formulation.

[0370] Step (c)

[0371] A high activity pharmaceutical formulation of F-18 Flornaptitril can be prepared from the purified high activity F-18 Flornaptitril of Step (b). In certain embodiments, the pharmaceutical formulation comprises sodium ascorbate. In certain embodiments, the pharmaceutical formulation comprises human serum albumin. In certain embodiments, the pharmaceutical formulation comprises sodium ascorbate, ascorbic acid, sodium citrate, citric acid, acetate, ammonium acetate or acetic acid.

[0372] In certain embodiments, the pharmaceutical formulation comprises a solubilizing excipient, for example those described in Strickley, R. et al. “Solubilizing excipients in oral and injectable formulations” 2004, Pharm Res. 21(2), 201-230. Tn certain embodiments, the solubilizing excipient is selected from polysorbate (polysorbatum) 20, polysorbate 40, polysorbate 60, and polysorbate 80. In certain embodiments, the solubilizing excipient is selected from a- tocopherol Solutol HS 15, sorbitan monooleate, poloxamer 407, Labrafil M-1944CS, Labrafil M- 2125CS, Labrasol, Gellucire 44 / 14, Softigen 767, and mono- and di-fatty acid esters of PEG 300, 400, or 1750. In certain embodiments, the solubilizing excipient is selected from polyethylene glycol 300, polyethylene glycol 400, ethanol, propylene glycol, and glycerin.

[0373] Cryptand ligands are known to cause convulsions and apnea (Yu, S. “Review of18F-FDG synthesis and quality control” 2006, Biomed. Imaging In terv. 2(4):e57), thus for formulation into a dose intended for human use it is advantageous to remove the cryptand ligand. In certain embodiments, the pharmaceutical formulation of F-18 Flomaptitril contains less than about 4, 3, 2, 1, 0.5, 0.1, 0.05, or 0.01 mg / mL of cryptand ligand. In certain embodiments, the pharmaceutical formulation contains less than 2.2 mg / mL of cryptand ligand. In certain embodiments, the pharmaceutical formulation comprises less than 0.05 mg / mL of cryptand ligand. In certain embodiments, the pharmaceutical formulation comprises from about 0.005 mg / mL to about 4 mg / mL, from about 0.005 to about 1 mg / mL, from about 0.005 to about 0.5 mg / mL, from about 0.005 to about 0.1 mg / mL, or from about 0.005 to about 0.05 mg / mL of cryptand ligand.

[0374] In certain embodiments, the pharmaceutical formulation is filtered through a sterile filter.

[0375] In certain embodiments, the reaction is performed with a compound of Formula X in place of the compound of Formula I: R44is selected from alkyl and aryl;

[0376] R55is a radical selected from the group consisting of -NH2, -OH, -SH, -NH-alkyl, -NHR44, -NH-alkyenyl-R44, -O-alkyl, -O-alkylenyl-R44, -S-alkyl, and -S-alkylenyl-R44;

[0377] R66is a radical selected from the group consisting of -CN, -COOH, -C(O)O-alkyl, -C(O)O- alkylenyl-R44, -C(O)-alkyl, -C(O)-alkylenyl-R44, -C(O)-halogen, -C(O)NH-alkyl, -C(O)NH- alkylenyl-R44and -C(0)NH2;

[0378] R77is a radical selected from the group consisting of O, NH, and S; and

[0379] R8is N, O or S.

[0380] Additional Embodiments of the Process

[0381] Bl. A process of preparing a high activity pharmaceutical formulation of F-18 Flornaptitril comprising: a) Reacting a compound of Formula I, or a salt thereof, with18F-fluoride in the presence of a radiostabilizer, a carbonate base, and a cryptand ligand: Formula I wherein R1is selected from the group consisting of methyl, Ci-ehaloalkyl, and aryl optionally substituted with one or two substituents selected from the group consisting of Ci-ealkyl, Ci-ehaloalkyl, halogen, and nitro; b) Purification of the F-18 Flornaptitril from the reaction mixture; and c) Preparing a pharmaceutical formulation with the purified F-18 Flornaptitril and an antioxidant stabilizer; wherein the pharmaceutical formulation comprises from about 1.5 to about 5 Ci of F-18 Flornaptitril. B2. The process of embodiment Bl, wherein Step (a) comprises: , ,

[0382] Formula I viii). Trapping from about 5 to about 20 Ci of18F-fluoride on a solid phase; ix). Eluting the18F-fluoride from the solid phase with a carbonate base; x). Collecting the eluent into a vial; xi). Azeotropically drying the resulting solution; xii). Adding a compound of Formula I and a radiostabilizer; xiii). Reacting the mixture; and xiv). Cooling and quenching the reaction.

[0383] B3. The process of embodiment B 1 or B2, wherein the radiostabilizer is selected from the group consisting of a persistent radical compound, a nitrone, an aniline of Formula II, a phenol of Formula III, and a diarylalkene.

[0384] B4. The process of any one of embodiments B1-B3, wherein the radiostabilizer is a persistent radical compound.

[0385] B5. The process of any one of embodiments B1-B4, wherein the radiostabilizer is TEMPO.

[0386] B6. The process of any one of embodiments B1-B4, wherein the radiostabilizer is galvinoxyl radical.

[0387] B7. The process of any one of embodiments B1-B3, wherein the radiostabilizer is a nitrone.

[0388] B8. The process of embodiment B6, wherein the radiostabilizer is selected from N-tert-butyl- a-phenylnitrone (PBN), a-4-pyridyl-l-oxide-N-t-butylnitrone (POBN), 5,5- dimethylpyrroline-N-oxide (DMPO), 3, 4-dihydro-2-methyl- 1,1 -dimethylethyl ester-2H- pyrrole-2-carboxylic acid- 1 -oxide (BMPO), 3,4-dihydro-2-methyl-2H-pyrrole-2- carboxylic acid 1 -oxide, ethyl ester (EMPO). B9. The process of any one of embodiments B1-B3, wherein the radiostabilizer is an aniline of Formula: salt thereof; wherein

[0389] R2is selected from NH2, NHR4, and N(R4)2;

[0390] R3a, R3b, and R3eare independently selected from hydrogen, halogen, Ci-ealkyl, C(O)OH, C(O)OCi-6alkyl, and C(O)N(R4)2; and

[0391] R4is selected from hydrogen, Ci-ealkyl, aryl, -Ci ealkyl-aryl, heterocycle, and heteroaryl.

[0392] BIO. The process of embodiment B9, wherein the radiostabilizer is selected from

[0393] Bl 1. The process of any one of embodiments Bl - B3, wherein the radiostabilizer is a phenol of Formula: salt thereof; wherein

[0394] R22is OH;

[0395] R23is selected from hydrogen, OH, OR4, CH2OH, and CH2OR4;

[0396] R3a, R3b, R3Cand R3dare independently selected from hydrogen, halogen, Ci-6alkyl, C(O)OH, C(O)OCi-6alkyl, and C(O)N(R4)2; and

[0397] R4is selected from hydrogen, Ci-ealkyl, aryl, -Ci-ealkyl-aryl, heterocycle, and heteroaryl. B12. The process of embodiment Bl 1, wherein the radiostabilizer is selected from

[0398] B13. The process of any one of embodiments B1-B3, wherein the radiostabilizer is a diarlyalkene.

[0399] B14. The process of embodiment B13, wherein the radiostabilizer is diphenylethylene.

[0400] B15. The process of any one of embodiments B1-B14, wherein R1is 4-CH3-phenyl.

[0401] B16. The process of any one of embodiments B1-B14, wherein R1is 4-Br-phenyl.

[0402] B17. The process of any one of embodiments B1-B14, wherein R1is 4-NC>2-phenyl.

[0403] B18. The process of any one of embodiments B1-B14, wherein R1is 2-NO2-phenyl.

[0404] B19. The process of any one of embodiments B1-B14, wherein R1is CH3.

[0405] B20. The process of any one of embodiments B1-B14, wherein R1is (CF2)3-CF3.

[0406] B21. The process of any one of embodiments B2-B20, wherein the solid phase in (i) is an anion exchange resin.

[0407] B22. The process of embodiment B21, wherein the anion exchange resin is QMA resin.

[0408] B23. The process of any one of embodiments B2-B22, wherein the18F is eluted in (ii) with an aqueous acetonitrile solution comprising a carbonate base.

[0409] B24. The process of embodiment B23, wherein the aqueous acetonitrile solution is from about 20% to about 50% acetonitrile, about 50% to about 75% acetonitrile, or from about 70% to about 95% acetonitrile.

[0410] B25. The process of embodiment B23, wherein the aqueous acetonitrile solution is from about 70% to about 95% acetonitrile.

[0411] B26. The process of embodiment B23, wherein the aqueous acetonitrile solution is up to about 20%, up to about 10%, up to about 5%, up to about 2% or up to about 1% acetonitrile.

[0412] B27. The process of any one of embodiments B2-B26, wherein the18F is eluted in (ii) with a solution comprising potassium carbonate. B28. The process of any one of embodiments B2-B26, wherein the18F is eluted in (ii) with a solution comprising cesium carbonate.

[0413] B29. The process of any one of embodiments B2-B26, wherein the18F is eluted in (ii) with a solution comprising a cryptand ligand.

[0414] B30. The process of any one of embodiments B2-B26, wherein the eluent in (iii) is collected into a vial containing a cryptand ligand.

[0415] B31. The process of embodiment B29 or B30, wherein the cryptand ligand is Kryptofix® K222.

[0416] B32. The process of any one of embodiments B2-B31, wherein the eluent of (iii) is azeotropically dried in (iv) with acetonitrile.

[0417] B33. The process of embodiment B32, wherein the azeotropic drying step is repeated three times.

[0418] B34. The process of any one of embodiments B2-B33, wherein the compound of Formula I is added in (v) as a solution in acetonitrile.

[0419] B35. The process of any one of embodiments B2-B34, wherein the reaction in (vi) is heated to from about 80°C to about 120°C.

[0420] B36. The process of any one of embodiments B2-B34, wherein the reaction in (vi) is heated to from about 90°C to about 110°C.

[0421] B37. The process of any one of embodiments B2-B34, wherein the reaction in (vi) is heated to about 90°C.

[0422] B38. The process of any one of embodiments B2-B34, wherein the reaction in (vi) is heated to about 95°C.

[0423] B39. The process of any one of embodiments B2-B34, wherein the reaction in (vi) is heated to about 100°C.

[0424] B40. The process of any one of embodiments B2-B34, wherein the reaction in (vi) is heated to about 105°C.

[0425] B41. The process of any one of embodiments B2-B34, wherein the reaction in (vi) is heated to about 110°C.

[0426] B42. The process of any one of embodiments B2-B34, wherein the reaction in (vi) is heated to about 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, or 105°C.

[0427] B43. The process of any one of embodiments B2-B42, wherein the reaction in (vi) is reacted for up to 55 minutes. B44. The process of any one of embodiments B2-B42, wherein the reaction in (vi) is reacted for up to 45 minutes.

[0428] B45. The process of any one of embodiments B2-B42, wherein the reaction in (vi) is reacted for up to 35 minutes.

[0429] B46. The process of any one of embodiments B2-B42, wherein the reaction in (vi) is reacted for up to 25 minutes.

[0430] B47. The process of any one of embodiments B2-B42, wherein the reaction in (vi) is reacted for from about 5 minutes to about 20 minutes.

[0431] B48. The process of any one of embodiments B2-B42, wherein the reaction in (vi) is reacted for about 5 minutes.

[0432] B49. The process of any one of embodiments B2-B42, wherein the reaction in (vi) is reacted for about 10 minutes.

[0433] B50. The process of any one of embodiments B2-B42, wherein the reaction in (vi) is reacted for about 15 minutes.

[0434] B51. The process of any one of embodiments B2-B42, wherein the reaction in (vi) is reacted for about 20 minutes.

[0435] B52. The process of any one of embodiments B2-B42, wherein the reaction in (vi) is reacted for about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 minutes.

[0436] B53. The process of any one of embodiments B2-B52, wherein the reaction is cooled in (vii) by passing a stream of inert gas through the reaction vial headspace.

[0437] B54. The process of embodiment B53, wherein the inert gas is selected from helium, nitrogen, and argon.

[0438] B55. The process of any one of embodiments B2-B54, wherein the reaction is quenched in (vii) with an aqueous solution.

[0439] B56. The process of any one of embodiments B2-B54, wherein the reaction is quenched in (vii) with an acidic aqueous solution.

[0440] B57. The process of any one of embodiments B2-B56, wherein the reaction is quenched in (vii) with aqueous hydrochloric acid.

[0441] B58. The process of embodiment B57, wherein the aqueous hydrochloric acid is from about 0.01 to about IM hydrochloric acid. B59. The process of any one of embodiments B2-B56, wherein the reaction is quenched in (vii) with ammonium acetate.

[0442] B60. The process of embodiment B59, wherein the ammonium acetate is from about 0.0 IM to about IM ammonium acetate.

[0443] B61. The process of any one of embodiments B2-B55, wherein the reaction is quenched in (vii) with water and acetonitrile.

[0444] B62. The process of any one of embodiments B55-B61, wherein the aqueous solution used to quench the reaction is pre-cooled to from about 0°C to about 10°C.

[0445] B63. The process of any one of embodiments B55-B62, wherein the reaction is passed through a cartridge containing neutral alumina before quenching.

[0446] B64. The process of any one of embodiments Bl -B63, wherein the F-18 Flornaptitril is purified by solid phase extraction (SPE).

[0447] B65. The process of embodiment B64, wherein the solid phase extraction is performed with C 18 solid phase.

[0448] B66. The process of embodiments B64 or B65, wherein the F-18 Flornaptitril is eluted with ethanol or acetonitrile.

[0449] B67. The process of any one of embodiments Bl -B63, wherein the F-18 Flornaptitril is purified by high performance liquid chromatography (HPLC).

[0450] B68. The process of any one of embodiments Bl -B67, wherein the F-18 Flornaptitril is purified by SPE followed by HPLC.

[0451] B69. The process of any one of embodiments B1-B68, wherein the pharmaceutical formulation of purified F-18 Flornaptitril in Step (c) comprises from about 1.5 Ci to about 2.5 Ci of F-18 Flornaptitril.

[0452] B70. The process of any one of embodiments B1-B68, wherein the pharmaceutical formulation of purified F-18 Flornaptitril in Step (c) comprises from about 2 Ci to about 2.5 Ci of F-18 Flornaptitril.

[0453] B71. The process of any one of embodiments B1-B68, wherein the pharmaceutical formulation of purified F-18 Flornaptitril in Step (c) comprises about 1.5 Ci of F-18 Flornaptitril.

[0454] B72. The process of any one of embodiments B1-B68, wherein the pharmaceutical formulation of purified F-18 Flornaptitril in Step (c) comprises about 2 Ci of F-18 Flornaptitril. B73. The process of any one of embodiments Bl -B68, wherein the pharmaceutical formulation of purified F-18 Flornaptitril in Step (c) comprises about 2.5 Ci of F-18 Flornaptitril.

[0455] B74. The process of any one of embodiments B1-B68, wherein the pharmaceutical formulation of purified F-18 Flornaptitril in Step (c) comprises about 3 Ci of F-18 Flornaptitril.

[0456] B75. The process of any one of embodiments B1-B68, wherein the pharmaceutical formulation of purified F-18 Flornaptitril in Step (c) comprises about 3.5 Ci of F-18 Flornaptitril.

[0457] B76. The process of any one of embodiments B1-B75, wherein the pharmaceutical formulation of purified F-18 Flornaptitril in Step (c) comprises less than about 4 mg / mL of cryptand ligand.

[0458] B77. The process of any one of embodiments B1-B75, wherein the pharmaceutical formulation of purified F-18 Flornaptitril in Step (c) comprises less than about 2 mg / mL of cryptand ligand.

[0459] B78. The process of any one of embodiments B1-B75, wherein the pharmaceutical formulation of purified F-18 Flornaptitril in Step (c) comprises less than about 1 mg / mL of cryptand ligand.

[0460] B79. The process of any one of embodiments B1-B75, wherein the pharmaceutical formulation of purified F-18 Flornaptitril in Step (c) comprises less than about 0.5 mg / mL of cryptand ligand.

[0461] B80. The process of any one of embodiments B1-B75, wherein the pharmaceutical formulation of purified F-18 Flornaptitril in Step (c) comprises less than about 0.1 mg / mL of cryptand ligand.

[0462] B81. The process of any one of embodiments B1-B75, wherein the pharmaceutical formulation of purified F-18 Flornaptitril in Step (c) comprises less than about 0.05 mg / mL of cryptand ligand.

[0463] B82. The process of any one of embodiments B1-B75, wherein the pharmaceutical formulation of purified F-18 Flornaptitril in Step (c) comprises from about 0.005 mg / mL to about 4 mg / mL of cryptand ligand.

[0464] B83. The process of any one of embodiments B1-B75, wherein the pharmaceutical formulation of purified F-18 Flornaptitril in Step (c) comprises from about 0.005 mg / mL to about 2 mg / mL of cryptand ligand.

[0465] B84. The process of any one of embodiments B1-B75, wherein the pharmaceutical formulation of purified F-18 Flornaptitril in Step (c) comprises from about 0.005 mg / mL to about 1 mg / mL of cryptand ligand. B85. The process of any one of embodiments Bl -B75, wherein the pharmaceutical formulation of purified F-18 Flornaptitril in Step (c) comprises from about 0.005 mg / mL to about 0.5 mg / mL of cryptand ligand.

[0466] B86. The process of any one of embodiments B1-B75, wherein the pharmaceutical formulation of purified F-18 Flornaptitril in Step (c) comprises from about 0.005 mg / mL to about 0.1 mg / mL of cryptand ligand.

[0467] B87. The process of any one of embodiments B1-B75, wherein the pharmaceutical formulation of purified F-18 Flornaptitril in Step (c) comprises from about 0.005 mg / mL to about 0.05 mg / mL of cryptand ligand.

[0468] B88. The process of any one of embodiments B1-B87, wherein the antioxidant stabilizer is selected from ascorbic acid, sodium ascorbate, citric acid, sodium citrate, ethanol, benzoic acid, gentisic acid, ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, calcium stearate, potassium metabi sulfate, propyl gallate, sodium metabisulfate, sodium thiosulfate, and vitamin E (tocopherol).

[0469] B89. The process of any one of embodiments B1-B88, wherein the antioxidant stabilizer is selected from ascorbic acid, sodium ascorbate, citric acid, sodium citrate, ethanol, benzoic acid, and gentisic acid.

[0470] B90. The process of any one of embodiments B1-B89, wherein the antioxidant stabilizer is selected from ascorbic acid, sodium ascorbate, citric acid, sodium citrate, and ethanol.

[0471] B91. The process of any one of embodiments B1-B90, wherein the antioxidant stabilizer is selected from ascorbic acid, sodium ascorbate, citric acid, sodium citrate, and ethanol.

[0472] B92. The process of any one of embodiments B1-B91, wherein the pharmaceutical formulation of purified F-18 Flornaptitril in Step (c) comprises saline.

[0473] B93. The process of any one of embodiments B1-B92, wherein the pharmaceutical formulation of purified F-18 Flornaptitril in Step (c) comprises plasma protein.

[0474] B94. The process of any one of embodiments B1-B93, wherein the pharmaceutical formulation of purified F-18 Flornaptitril in Step (c) comprises human serum albumin.

[0475] B95. The process of any one of embodiments B1-B94, wherein the pharmaceutical formulation is filtered through a sterile filter.

[0476] B96. The process of any one of embodiments B1-B95, wherein Step (a) is performed on an automated synthesizer. B97. The process of any one of embodiments B1 -B96, wherein Step (b) is performed on an automated synthesizer.

[0477] B98. The process of any one of embodiments B1-B97, wherein Step (c) is performed on an automated synthesizer.

[0478] B99. The process of any one of embodiments B1-B98, wherein two of Steps (a)-(c) are performed on an automated synthesizer.

[0479] Bl 00. The process of any one of embodiments B1-B99, wherein Steps (a)-(c) are performed on an automated synthesizer.

[0480] Automated Radiosynthesis

[0481] In certain embodiments, one or more steps of the process of preparing high activity pharmaceutical formulations of F-18 Flomaptitril is performed using a semi-automated or automated process to carry out one of the synthetic reactions described herein. It can be advantageous to automate one or more steps of the process described herein to increase repeatability, decrease time of synthesis, or minimize the exposure of technicians to radiation. In certain embodiments, the automated synthesis is performed with minimal intervention of a technician. A person of skill in the art will appreciate that an automated synthesis does not imply that there is no intervention from a technician. For example, operating the controllers, measuring reagent quantities, or preparing reagent solutions can still be performed by a technician in an automated synthesis.

[0482] In certain embodiments, one or more steps of the synthesis are automated, and one or more steps of the synthesis are performed manually (i.e. a semi-automated synthesis). For example, a semi-automated synthesis includes a synthesis wherein Unit 1 is used for the automated radiofluorination step (Step (a)), but the subsequent purification and formulation steps are performed manually.

[0483] In certain embodiments, Step (a) of the process of preparation described herein is performed on an automated synthesizer. In certain embodiments, the automated synthesis of Step

[0484] (a) is performed on an automated synthesis module comprising Unit 1.

[0485] In certain embodiments, Step (b) of the process of preparation described herein is performed on an automated synthesizer. In certain embodiments, the automated synthesis of Step

[0486] (b) is performed on an automated synthesis module comprising Unit 2 and / or Unit 3. In certain embodiments, Step (c) of the process of preparation described herein is performed on an automated synthesizer. In certain embodiments, the automated synthesis of Step (c) is performed on an automated synthesis module comprising Unit 4.

[0487] In certain embodiments, Steps (a), (b), and (c) of the process of preparation described herein is performed on an automated synthesizer. In certain embodiments, the automated synthesis of Steps (a), (b), and (c) is performed on an automated synthesis module comprising Units 1-4.

[0488] FIGS. 2-4 illustrate schematic diagrams of the semi-automated module for use in the preparation of F-18 Flornaptitril. In certain embodiments the semi-automated module is set up in a “hot cell” found in a cyclotron or other radiochemistry laboratory. A hot cell is a dedicated enclosure that is used for the handling of radioactive materials. Hot cells are typically radiation shielded to prevent exposing the technician or operator to radiation from gamma ray emitters. Various types of concrete, lead, lead glass, steel, and depleted uranium can be used for shielding materials.

[0489] As explained herein, some components of the semi-automated module may be located outside the hot cell. These may include valve controllers (V1-V2) and oil bath controller of Unit 1, valve controllers (V3 and V4) of Unit 2, Unit 3, Unit 4, as well as input lines or tubing that are used to delivery various reagents and solvents to the module. The bolded black box surrounding Units 1 and 2 in FIG. 2 and FIG. 3 is a non-limiting embodiment of which operations are performed within the hot cell (inside the box) and which operations can be performed outside the hot cell. The controllers located outside the hot cell can be connected to the components within the hot cell through cables, shown as dashed lines in FIGS. 2-4.

[0490] As seen in FIGS. 2-4, the semi-automated module has four functional units (i.e., identified as Units 1-4), with each unit performing one of the steps of the radiosynthesis. Unit 1 performs step (a), Units 2 and 3 perform step (b), and Unit 4 performs step (c). It should be appreciated that Units 1-4 are not necessarily physically separate units; a single automated synthesis module is used for the synthesis. The Units 1-4 that are described herein are referred to as individual units that each performs different operations or processes in the synthesis and final formulation yet are part of a single overall module. The individual components of the units are connected to one another via tubing or lines, and the terminals of the tubing may be fitted with male or female Luer tips for connection with other components. The tubing and fitting components are typically made of an inert or substantially inert polymer (e.g., Polytetrafluoroethylene (PTFE) or polyether ether ketone (PEEK)). Multi-position valves V1-V6 can be actuated using solenoids to connect different input lines to the output line. Solenoid-actuated multi-position valves used in an automated synthesis module are typically constructed with inert or substantially inert polymers (e.g. PTFE and / or Viton®) for the wetted flow paths.

[0491] During the operation, reagents and solvents are added to the system remotely via tubing or lines by application of positive pressure from outside of the hot cell with an inert gas such as nitrogen, helium or argon gas. In alternative embodiments, reagents and solvents can be transferred into or between components of the automated module using a source of vacuum.

[0492] Described below are non-limiting embodiments of the individual units and their construction and function.

[0493] Unit 1: Automated Radi ofluorinati on Using a Radiostabilizer

[0494] This unit includes an automated pinch valve (FT) with a loop or flow path that is used for delivering [F-18]fluoride ion (18F’) to a resin cartridge 2 for subsequent elution. Valve VI is controlled using the VI valve controller (located outside the hot cell). In one embodiment, the resin cartridge 2 is an anion exchange resin cartridge that is prepared to isolate the18F‘ from proton irradiated [18O]water in order to conserve the [18O]water and reduce the amount of water that needs to be evaporated during the synthesis.

[0495] In one embodiment the resin cartridge 2 is made by loading 0.125 inch (inner diameter), 0.160 inch (outer diameter) PTFE tubing with BioRad® MP-1 resin (catalog #1411851 available from BioRad®, Hercules, Calif.). The BioRad® MP-1 resin is a macroporous strong anion exchange resin that uses a matrix of styrene divinylbenzene. The functional group is R — CH2N+(CH3)3. The BioRad® MP-1 resin is packed into the tubing using either gravity settling or pulling the resin in the tube with vacuum. The resin is retained in the tubing using a pair of frits (made from 70 pm pore polyethylene frit material) that are formed with an 0.125” inch hole punch. The frits secure the resin material inside the tube and permit the passage of fluid through the resin material. The ends of the tubing may be fitted with Luer fittings to finalize the formation of the resin cartridge 2.

[0496] When valve VI is off (normally open), the valve allows flow through the flow path indicated in solid flow arrows (FIG. 2) and pinches or closes the flow through the flow path indicated with empty flow arrows. The solid flow arrow path allows the solution of18F' in H218O to flow through resin cartridge 2 where the18F' is loaded onto the resin contained in the resin cartridge 2 while the H218O proceeds to the waste (Wl).

[0497] When valve VI is on, the valve pinches or closes the flow path indicated by solid flow arrows and opens the flow path indicated by empty flow arrows. The flow path indicated by empty arrows allows a solution of carbonate base to flow through the resin cartridge 2. The carbonate solution elutes the solution of18F- fluoride to the reaction vessel 3. In certain embodiments, the carbonate base is selected from potassium carbonate, cesium carbonate, or tetraalkylammonium carbonate (e.g. tetrabutylammonium carbonate. In certain embodiments, the carbonate base is potassium carbonate. The18F-potassium fluoride solution is collected in the fluorination reaction vessel 3 via a line made from PTFE.

[0498] In certain embodiments, the18F" that is trapped on the resin cartridge 2 is released by the addition of from about 0.1 to about 1 mL of carbonate base solution, for example about 0.2 mb, 0.3 mL, 0.4 mL, 0.5 mL or 0.6 mL of carbonate base solution in water (other potassium salts may also be used). In certain embodiments, the solution of carbonate base is a solution that is from about 0.1% to about 1% carbonate base, for example about 0.1%, 0.2%, 0.25%, 0.3%, 0.4%, or 0.5% carbonate base.

[0499] In certain embodiments, the18F' that is trapped on the resin cartridge 2 is released by the addition of from about 0.1 to about 1 mL of potassium carbonate solution, for example about 0.2 mL, 0.3 mL, 0.4 mL, 0.5 mL or 0.6 mL of potassium carbonate solution in water (other potassium salts may also be used). In certain embodiments, the solution of potassium carbonate is a solution that is from about 0.1% to about 1% potassium carbonate, for example about 0.1%, 0.2%, 0.25%, 0.3%, 0.4%, or 0.5% K2CC>3.

[0500] A heating oil bath 4 sits on a motorized jack platform 5 permits the bath to selectively contact reaction vessel 3. The motorized jack platform 5 comprises a heating element which heats the oil bath 4, and the motorized jack allows a technician or operator to raise the oil bath via the oil bath controller (located outside the hot cell), thereby immersing the reaction vessel 3 in the heated oil. The temperature of the oil bath is set to the desired reaction temperature, for example from about 90°C to about 110°C.

[0501] The glass fluorination reaction vessel 3 is positioned above the bath 4 with a silicone stopper carrying four PTFE lines. One line receives the18F-potassium fluoride solution from valve VI The second line connects the reaction vessel 3 with a multi-position valve F2, controlled by the V2 valve controller (located outside the hot cell). A third line connects the reaction vessel 3 with waste W2, through normally open valve 6, controlled by a valve controller (not shown). A fourth line connects the reaction vessel 3 with Unit 2. The fourth line includes a dip tube 7, which extends to the bottom of reaction vessel 3 and connects through a normally closed valve 8 to Unit

[0502] 2. When a positive pressure of nitrogen gas is applied to the reaction vessel 3 and valve 8 is opened, the contents of reaction vessel 3 can be pushed up through the dip tube 7 and into Unit 2.

[0503] Multi-position valve V2 is used to control the sequence of reagent addition to the reaction vessel 3. After the18F-potassium fluoride solution is collected in the reaction vessel 3, valve V2 is actuated to add acetonitrile to the reaction vessel 3. Next, valve V2 is actuated to flow nitrogen gas through the headspace of reaction vessel 3, through normally open valve 6, and out the line attached to waste W2. The pre-heated oil bath 4 is raised via the motorized jack platform 5 and the acetonitrile-water azeotrope is boiled until the volume has decreased by at least half. While maintaining contact with oil bath , valve V2 is actuated to add acetonitrile to reaction vessel 3 and then V2 is actuated to flow nitrogen gas through the headspace, carrying the solvent vapor into waste W2. The above sequence of acetonitrile addition followed by azeotropic removal of water is repeated at least two, three, four, or five times in total.

[0504] After azeotropic drying of the18F-potassium fluoride, valve V2 is actuated to add a solution of precursor, radiostabilizer, and cryptand ligand in anhydrous acetonitrile to the reaction vessel

[0505] 3. The pre-heated oil bath 4 is raised via the motorized jack platform 5 for the duration of the reaction, for example 5-15 minutes, and then lowered. The valve 8 is then opened via the valve controller (not shown) and then valve V2 actuated to nitrogen gas. A positive pressure of nitrogen gas in the headspace transfers the reaction mixture from the reaction vessel 3 through the dip tube 7 into Unit 2.

[0506] Unit 2: Solid phase extraction ofF-18 Flornaptitril

[0507] The reaction mixture is transferred from Unit I into quench vial 9, which is pre-loaded with an aqueous solution. The aqueous solution quenches the reaction and facilitates solid phase extraction of the F-18 Flornaptitril. In certain embodiments, the aqueous solution in quench vial 9 is a solution of dilute acid, for example HC1, ascorbic acid, or ammonium acetate. In certain embodiments, the aqueous solution in quench vial 9 is cooled, for example at from about 0°C to about 10°C. In certain embodiments, the solution of acid is from about 0.01M to about 0.25M, or about 0.1M.

[0508] Multi-position valve V3 is then actuated to open the center line (attached to quench vial 9 via dip tube 11), and then normally closed valve 10 is opened and positive pressure of nitrogen pushes the aqueous solution of F-18 Flornaptitril onto solid phase extraction cartridge 12. Nonlimiting examples of solid phase extraction cartridge media include alumina, C4-, C8-, Cl 8-, or phenyl-functionalized silica, SOLA resin, or urea-functionalized polystyrene. In certain embodiments, solid phase extraction cartridge 12 is a packed column of C18-functionalized silica, for example a Waters Sep-Pak® C18 cartridge.

[0509] Valve V3 is then actuated to the left position (solvent SI reservoir) and multi-position valve V4 is actuated to the waste position (collecting in W4). Solvent SI is then flowed through the solid phase extraction cartridge 12 to wash inorganic salts into the waste W4. In certain embodiments, solvent SI is an aqueous solvent, for example a dilute solution of sodium ascorbate or ascorbic acid. Next, valve V3 is actuated to the right position (solvent S2 reservoir) and valve V4 is actuated to direct the flow into Unit 3. Solvent S2 is flowed through the solid phase extraction cartridge 12 to elute the F-18 Flornaptitril off the extraction cartridge 12. In certain embodiments, solvent S2 is a water-miscible organic solvent such as methanol, ethanol, or acetonitrile. In certain embodiments, solvent S2 is ethanol.

[0510] Unit 3: Preparative HPLC

[0511] Unit 3 is a preparative HPLC system, for example an HPLC system comprising an HPLC Injection Module 13, an HPLC Pump 14, solvent reservoirs A and B, an HPLC column 15, a UV / Gamma Detector 16, a Fraction Collector 17, and a waste reservoir W5.

[0512] The HPLC Injection Module 13 comprises a sample loop and a multi-position valve. The F-18 Flornaptitril is eluted from solid phase extraction cartridge 12, through the multi-position valve and into the sample loop. The multiposition valve is then actuated using the HPLC controller to allow the HPLC Pump 14 o flow through the sample loop and onto the HPLC column 75. In certain non-limiting embodiments, the HPLC column 5 has a solid phase of C18-functionalized silica. The HPLC pump then pumps solvents S3 and / or S4 through the HPLC column 75.

[0513] In certain embodiments, solvent S3 is an aqueous solution such as water, optionally comprising a modifier. Non-limiting examples of modifiers include ammonium formate, sodium ascorbate, ascorbic acid, acetic acid, formic acid, and trifluoroacetic acid. Tn certain embodiments solvent S3 comprises about 0.01% to about 1% modifier, for example about 0.05%, 0.1%, 0.15%, 0.2% or 0.25% of a modifier. In certain embodiments, solvent S3 is water with sodium ascorbate modifier. In certain embodiments, solvent S3 is water with 0.1% sodium ascorbate modifier.

[0514] In certain embodiments, solvent S4 is a water-miscible organic solvent, optionally comprising a modifier. Water-miscible organic solvents include but are not limited to methanol, ethanol, tetrahydrofuran and acetonitrile. In certain embodiments, solvent S4 is acetonitrile. In certain embodiments, solvent S4 is a mixture of water-miscible organic solvents. Non-limiting examples of modifiers include ammonium formate, sodium ascorbate, ascorbic acid, acetic acid, formic acid, and trifluoroacetic acid. In certain embodiments solvent S3 comprises about 0.01% to about 1% modifier, for example about 0.05%, 0.1%, 0.15%, 0.2% or 0.25% of a modifier.

[0515] In certain embodiments, the HPLC pump 14 runs an isocratic (i.e. a static ratio of S3 and S4) method. In certain embodiments, the HPLC Pump 14 runs a gradient method (i.e. increasing amounts of solvent S4 over time). In certain embodiments, the HPLC Pump 14 runs a gradient of about 5% to about 95% S4, from about 20% to about 60% S4, or from about 25% to about 50% S4. In certain embodiments, the HPLC Pump 14 runs an isocratic method using from about 20 to about 70%, from about 40% to about 60% or from about 45% to 55% S4 (with the remaining percentage being S3). In certain embodiments, the HPLC Pump 14 runs an isocratic method using about 40%, 45%, 50%, 55%, or 65% S4.

[0516] The solvent S3 or S4 or mixture of S3 and S4 flows through the HPLC column 15, and the flow passes through the UV / Gamma Detector 16. After flowing through the UV / Gamma Detector 16, the flow passes into the Fraction Collector 17, where it is either collected in waste W5 or sent to Unit 4.

[0517] The UV / Gamma Detector 16 is comprised of a UV detector and a gamma detector. The UV detector measures the absorbance of ultraviolet light of solutes dissolved in the outflow of HPLC column 15. This allows a technician to determine (using the detector controller) when UV- absorbing compounds, for example F-18 Flornaptitril or a precursor compound have eluted from the column. The gamma detector measures emission of gamma radiation from solutes dissolved in the outflow of the HPLC column 15. The gamma detector allows a technician to determine when radioactive compounds, for example F-18 Flornaptitril have eluted from the column. When F-18 Flornaptitril elutes from HPLC column 15, a peak can be observed which is both UV absorbing and gamma emitting. The Fraction Collector can then be triggered using the fraction collector controller to divert the flow from the waste W5 and into Unit 4.

[0518] Unit 4: Isolation and Formulation

[0519] The HPLC fraction containing F-18 Flornaptitril is transferred from Unit 3 into vial 18, which is pre-loaded with an aqueous solution. The aqueous solution dilutes the fraction to facilitate solid phase extraction of the F-18 Flornaptitril. In certain embodiments, the aqueous solution in vial 18 is an aqueous solution of an antioxidant stabilizer selected from ascorbic acid, sodium ascorbate, citric acid, sodium citrate.

[0520] Multi-position valve V5 is then actuated to open the center line (attached to vial 18 via dip tube 19), and then normally closed valve 20 is opened and positive pressure of nitrogen pushes the aqueous solution of F-18 Flornaptitril onto solid phase extraction cartridge 21. In certain embodiments, solid phase extraction cartridge 21 is a packed column of C18-functionalized silica, for example a Waters Sep-Pak® Cl 8 cartridge.

[0521] Valve V5 is then actuated to the left position (solvent SI reservoir) and multi-position valve V6 is actuated to the waste position (collecting in W6). Solvent SI is then flowed through the solid phase extraction cartridge 21 to wash inorganic salts into the waste W6. In certain embodiments, solvent SI is an aqueous solvent, for example a dilute solution of sodium ascorbate or ascorbic acid. Next, valve V5 is actuated to the right position (solvent S2 reservoir) and valve V6 is actuated to direct the flow through normally open valve 22 into the formulation vial 23. Solvent ,S'2 is flowed through the solid phase extraction cartridge 21 to elute the F-18 Flornaptitril off the extraction cartridge 21. In certain embodiments, solvent S2 is ethanol.

[0522] Valve 22 is then closed and solvent S4 is then added to the formulation vial 23. In certain embodiments, solvent S4 is a pharmaceutically acceptable carrier. In certain embodiments, solvent S4 is saline, optionally with plasma proteins and antioxidant stabilizers. In certain embodiments, solvent S4 is saline with human serum albumin. In certain embodiments, solvent S4 is saline with sodium ascorbate, ascorbic acid, sodium citrate, and / or citric acid. In certain embodiments, solvent S4 is saline with human serum albumin, sodium ascorbate, and ascorbic acid.

[0523] Normally closed valve 24 is then opened and positive pressure of nitrogen passes the pharmaceutical formulation of F-18 Flornaptitril via dip tube 25 through filter 26 and into sterile vial 27. In certain embodiments, filter 26 is used for sterile filtration of the pharmaceutical formulation of F-18 Flornaptitril. Tn certain embodiments, filter 26 has a pore size from about 0.1 pM to about 0.50 pM, or less than about 0.45 pM, 0.25 pM, or 0.22 pM. In certain embodiments, sterile vial 27 contains from about 0.5 to about 3.5 Ci, or at least about 0.75, 0.8, 1.0, 1.2 or 1.5 Ci of F-18 Flornaptitril in a pharmaceutical formulation. In certain embodiments, sterile vial 27 contains about 1.0 Ci of F-18 Flornaptitril in a pharmaceutical formulation.

[0524] PHARMACEUTICAL FORMULATION

[0525] In certain aspects, the invention includes a pharmaceutical formulation comprising from about 0.5 to about 10 Ci total activity of F-18 Flornaptitril, for example from about 0.75 Ci to about 1.2 Ci of F-18 Flornaptitril. In certain embodiments, the high activity F-18 Flornaptitril pharmaceutical formulation is a multidose pharmaceutical formulation comprising at least about 10, 15, 20, 25, 30, 35, 4, 45, 50, 55, 60, 70, 80 and typically not more than 100 individual doses of F-18 Flornaptitril, wherein an individual dose has for example an activity of from about 0.008 to about 0.020 Ci. In certain embodiments, the high activity F-18 Flornaptitril pharmaceutical formulation is a multidose pharmaceutical formulation comprising between about 10 and about 250, between about 50 and about 200, between about 75 and about 175, or between about 75 and about 150 individual doses of F-18 Flornaptitril, wherein an individual dose has for example an activity of from about 0.008 to about 0.020 Ci.

[0526] In certain embodiments, the pharmaceutical formulation comprises purified of F-18 Flornaptitril with a pharmaceutically acceptable carrier, for example saline. In certain embodiments, the pharmaceutical formulation includes one or more additional pharmaceutically acceptable excipients, for example an eluent, diluent, solvent (solubilizer), buffer (including an antioxidant buffer), surfactant, or plasma protein (such as a plasma protein useful for stabilizing the F-18 Flornaptitril).

[0527] Representative optional carriers include phosphate buffered saline, water, solvent(s), a diluent, pH a modifying agent, a preservative, an antioxidant, a suspending agent, a wetting agent, a viscosity agent, a tonicity agent, a stabilizing agent, or combinations thereof. In some embodiments, the formulation is an aqueous formulation. Examples of aqueous formulations include, but are not limited to, an aqueous solution or suspension, such as saline, plasma, bone marrow aspirate, a buffer, such as Hank’s Buffered Salt Solution (HBSS), HEPES (4-(2- hy droxy ethyl)- 1 -piperazineethanesulfonic acid), Ringers buffer, ProVisc®, diluted ProVisc®, Pro Vise® diluted with PBS, Krebs buffer, Dulbecco’s PBS, normal PBS, sodium hyaluronate solution (HA, 5 mg / mL in PBS), citrate buffer, a simulated body fluid, plasma platelet concentrate and tissue culture medium or an aqueous solution or suspension comprising an organic solvent. Acceptable solutions include, for example, water, Ringer’s solution and isotonic sodium chloride solutions. The formulation may also be a sterile solution, suspension, or emulsion in a non-toxic diluent or solvent such as 1,3 -butanediol.

[0528] In certain embodiments, the pharmaceutical formulation comprises an antioxidant formulation stabilizer. In certain embodiments, the pharmaceutical formulation comprises an antioxidant formulation stabilizer selected from the group consisting of sodium ascorbate, ascorbic acid, sodium citrate, citric acid, ethanol, and gentisic acid.

[0529] In certain embodiments, the pharmaceutical formulation comprises a plasma protein. In certain embodiments, the pharmaceutical formulation comprises human serum albumin.

[0530] In certain embodiments, the pharmaceutical formulation comprises from about 0.5 to about 10 Ci total activity of F-18 Flornaptitril and one or more excipients selected from ethanol, sodium chloride, polysorbate-80, sodium ascorbate, and human serum albumin.

[0531] EXAMPLES

[0532] Table 1. Abbreviations

[0533]

[0534] Example 1. Synthesis of Precursor Compounds

[0535] Scheme 6. Synthesis of 2-(l-(6-((2-hydroxyethyl)(methyl)amino)naphthalen-2- yljethylidenejmalononilrile (Intermediate 1-E)

[0536] Step 1: 10.03g of 2-Acetyl-6-methoxynaphthalene (1-A) was slurried in 3.3L concentrated HC1. The mixture was heated to reflux for 2 hours. The solution was filtered while hot. The filtrate was cooled at 5 °C to precipitate the crude product. The solids were washed with cold water until the pH of the filtrate was neutral. The solids were dried under high vacuum at 30°C. A total of 6.6g of an off-white solid (l-(6-hydroxynaphthalen-2-yl)ethan-l-one; Intermediate 1-B) at 71% yield was obtained.

[0537] Step 2: 2.5g of Intermediate 1-B, 15.0g sodium metabisulfite, and 17.5mL methylaminoethanol were slurried in 115mL water. The mixture was sealed in a reactor and heated to 130°C for 3 days. The mixture eventually dissolved. As the solution cooled to room temperature a precipitate formed. Addition of 0.1% TFA in CHiCN dissolved the precipitate. Neat TFA was added to lower the pH = 2.8. The solution was purified by reverse-phase flash chromatography using 0.1% TFA in CHsCN / 0.1% TFA in H2O. The pH of the kept fractions was increased to 8.4 with 25% NaOH. The organic solvent was removed by rotary evaporation at 35 °C. Di chloromethane was used to extract the product from the remaining aqueous solution. The organic layers were combined, dried with Na2SO4, and filtered. The solvent was removed by rotary evaporation. The residue was dried under high vacuum to afford product (l-(6-((2-hydroxyethyl)(methyl)amino)naphthalen-2- yl)ethan-l-one; Intermediate 1-C). A total of 1.8g of a yellow solid in 55% yield was obtained. Step 3: 1 ,5g Intermediate 1-C was dissolved in 25mL dichloromethane and 12mL methanol. 2.0g malononitrile and 2.7g ammonium acetate were added. The mixture was heated to 40°C overnight. Dissolution occurred with heating. The solution was cooled to room temperature. The solvents were removed by rotary evaporation. The oily reddish-orange residue dissolved in 120mL 50 / 50 0.1% TFA in CH3CN / 0.1% TFA in H2O. Neat TFA was used to lower the pH to 2.8. The solution was purified by reverse-phase flash chromatography using 0.1% TFA in CH3CN / 0.1% TFA in H2O. The organic solvent was removed by rotary evaporation at 35 °C. Dichloromethane was used to extract the product from the remaining aqueous solution. The organic layers were combined, dried with Na2SO4 and filtered. The solvent was removed by rotary evaporation at 30°C. The residue was dried under high vacuum to afford product (2-(l-(6-((2- hydroxyethyl)(methyl)amino)naphthalen-2-yl)ethylidene)malononitrile; Intermediate 1-D) as a ruby red solid. A total of 1.5g in 80% yield was obtained.

[0538] Scheme 7. Synthesis of 2-((6-(l,l-dicyanoprop-l-en-2-yl)naphthalen-2-yl)(methyl)amino)ethyl 4- methylbenzenesulfonate (Compound 1)

[0539] 1.0g of DDNPTs-300 INT dissolved in 30mL pyridine and then cooled in an ice bath. 6.2g of toluenesulfonic anhydride was added. After 1 hour of stirring, 60mL methanol was added. The solution was allowed to stir 1 hour. The solvents, including the majority of the pyridine, were removed by rotary evaporation at 40-50°C. The residue was dissolved in 75mL 0.1% TFA in CH3CN. The solution was purified by reverse-phase flash chromatography using 0.1% TFA in CH3CN / 0.1% TFA in H2O. After each purification run, di chloromethane was added such that the total volume was tripled. The product was extracted into the lower organic layer. The organic layer was dried with Na2SC>4 and filtered. The solvent was removed by rotary evaporation at 30°C. A residue remained that was dried under high vacuum to afford the product as a reddish-orange solid. A total of 1.0g in 65% yield was obtained. NMR Spectroscopic Characterization of Precursor Compound 1

[0540] NMR spectra were acquired on a Bruker NEO 400 MHz (9.4 T) spectrometer using a 5 -mm PABBO probe operating at a Larmor frequency of 400.15 MHz and 100.63 MHz for 'H and13C, respectively. 33.8 mg of the sample was added in 0.75 ml of CDCh. The solvent was then transferred into a 5 mm NMR tube for subsequent analysis. The acquired spectra were processed using TopSpin GxP 4.1.4 and referenced to the chemical shift of the residual sol- vent peak (e.g., CDCh at 7.26 ppm and 77.06 ppm for 'll and13C). All ID spectra were obtained with the sample spinning at 20 Hz, while 2D NMR spectra were performed with spin- off. More detailed acquisition parameters were provided in Table 2. All NMR samples were prepared under ambient conditions. Atom numbering as described herein refers to the numbering system for Compound 1 depicted in Figure 8.

[0541] Table 2. NMR acquisition parameters.

[0542] DEPT (Distortionless Enhancement by Polarization Transfer), COSY (Correlation Spectroscopy). HSQC (Heteronnclear Single Quantum Coherence), HMBC (Heteronuclear Multiple Bond Correlation), NOESY (Nuclear Overhauser Effect Spectroscopy).

[0543] The ’H NMR spectrum of Compound 1 (Figure 9) was analyzed to initiate the chemical shift assignments. This analysis focused on chemical shifts, integration values, multiplicity, and ’H-'H coupling constants. Based on the spectral data, the compound's proton signals were categorized into three distinct spin systems: aromatic ring system (6.7 - 8.1 ppm), ethylene group (3.7 - 4.3 ppm), and methyl group (2.3 - 3.1 ppm). Methyl phenyl proton assignments

[0544] The methyl protons resonating at 2.3 ppm were assigned to position 32 on the DDNPTs-4 structure (FIG. 9). This assignment was corroborated by the observation of strong interactions between these methyl protons and the aromatic protons at positions 27, 28, 29, and 30, which appear at 7.17 and 7.66 ppm in the COSY spectrum (FIG. 10). Further refinement of the aromatic proton assignments was achieved by analyzing the relative strengths of the correlations between the methyl protons and the aromatic protons. The aromatic signals at 7.17 ppm exhibited a stronger correlation with the methyl protons (2.3 ppm) and were thus assigned to positions 29 and 30 (Figure 10). Consequently, the signals at 7.66 ppm were attributed to the protons at positions 27 and 28. The 2D NOESY NMR spectrum (FIG. 11) revealed a distinctive cross peak between the methyl protons (2.3 ppm) and the aromatic protons (7.17 ppm). This observation provided additional evidence for the spatial proximity of the CH3 group at position 32 to the aromatic protons at positions 29 and 30.

[0545] Ethyl amino proton assignments

[0546] Two distinct triplet signals were observed at 3.78 ppm and 4.25 ppm, characteristic of an ethylene moiety (FIG. 9). The signal at 4.25 ppm was assigned to the CH2 protons adjacent to the oxygen atom (position 17). This downfield shift is consistent with the deshielding effect exerted by the electronegative oxygen atom. Consequently, the signal at 3.78 ppm was attributed to the CH2 protons at position 16. The assignment was further corroborated by examining the relative strengths of correlations between the methyl protons at 3.05 ppm (position 15, see below for the assignment) and the two CH2 groups in the NOESY spectrum (FIG. 11). A stronger correlation observed between the CH3 protons and the CH2 protons resonating at 3.78 ppm, indicating the closer spatial proximity between the methyl group and the CH2 at position 16, compared to the CH2 at position 17.

[0547] The three prominent singlets below 3.1 ppm in the NMR spectrum (FIG. 9) correspond to methyl protons. The most deshielded methyl signal at 3.05 ppm is assigned to the N- methyl protons at position 15, due to the electron-withdrawing effect of the nitrogen atom. Consequently, the Ifl signal at 2.7 ppm arises from the methyl protons of the ethylidene group at position 12. Naphthyl proton assignments

[0548] Based on the integration, multiplicity, and coupling constants (refer to Table 3) from the IDrH NMR spectrum (FIG. 9), along with the connectivity information from the 2D COSY spectrum (FIG. 10) of DDNPTs-4, it was determined that the aromatic!H signals at 6.78, 7.05, and 7.72 ppm (spin system I) are coupled, as are the signals at 7.56, 7.64, and 8.0 ppm (spin system II). These spin systems correspond to protons at positions 3, 4, and 5 or at positions 7, 9, and 10. The strong cross-peaks (FIG. 11) between the methyl protons at 2.7 ppm (position 12) and the aromatic protons at 7.56 and 8.0 ppm indicate that the aromatic protons in spin system II are located at positions 7, 9, and 10, rather than positions 3, 4, and 5. Based on the coupling constants and multiplicity, the proton signals at 7.56, 7.64, and 8.0 ppm are assigned to positions 10, 9, and 7, respectively, while the signals at 6.78, 7.05, and 7.72 ppm are assigned to positions 4, 5, and 3, respectively.

[0549] CH, CH2, and CH- carbon assignments

[0550] According to the!H peak positions (Table 3), the corresponding CH, CH2, and CH3 carbons were unequivocally assigned using the 2D HSQC and DEPT 135 and 90 spectra.

[0551] Quaternary carbon assignments

[0552] Due to the absence of attached protons, quaternary carbons cannot be assigned using DEPT and HSQC data. However, HMBC spectra provide two- to four-bond ^H-^C connectivity, enabling the assignment of quaternary carbons. Compound 1 contains ten quaternary carbons located at positions 1, 2, 6, 8, 11, 13, 19, 21, 24, and 31. For instance, The methyl protons resonating at 2.3 ppm (position 32) exhibited strong cross-peaks with13C signals at 129.8 and 145.1 ppm in the HMBC spectrum (FIG. 12). The peak at 129.8 ppm was previously assigned to carbons at positions 29 and 30. Consequently, the only signal dis- playing a strong C-H correlation (two bonds) is the quaternary carbon at position 31 (145.1 ppm). Another example includes the CH3 protons at 3.05 ppm (position 15), which show strong cross-peaks with13C signals at 51.3 ppm and 148.2 ppm. Of the carbons, three bonds away from these methyl protons, the signal at 51.3 ppm is attributed to carbon at position 16 (confirmed by DEPT and HSQC). Consequently, the signal at 148.2 ppm corresponds to the quaternary carbon at position 6, which is also three bonds away from the methyl protons. Using this approach, all quaternary carbon assignments were successfully completed. In conclusion, the NMR analyses confirmed the chemical structure of Compound 1 . Table

[0553] 3 details 'H and13C chemical shift assignments, H multiplicity, and 'H-'H coupling constants.

[0554] Table 3. The!H and13C chemical shift assignments of Compound 1. aMultiplicity notation: s = singlet, d = doublet, dd = doublet of doublets, t = triplet.bJ = coupling constant between two spins. N / A and N / D represent ‘not available’ and ‘not determined’, respectively. Example 2. Synthesis of Precursor Compounds

[0555] Scheme 8. Synthesis of 2-((6-(l ,l-Dicyanoprop-l-en-2-yl)naphthalen-2-yl)(methyl)amino)ethyl 4- bromobenzene sulfonate (Compound 2)

[0556] A solution of 2-(l-(6-((2-hydroxyethyl)(methyl)amino)naphthalen-2-yl)ethylidene)malononitrile (Intermediate 1-D) and tri ethylamine (2.5 equivalents) is dissolved in di chloromethane and cooled to about 0°C. A solution of brosyl chloride (1.1 equivalent) in dichloromethane is added dropwise. The reaction is stirred for Ihr, then is warmed to room temperature. Water is added and the reaction mixture extracted with dichloromethane. The dichloromethane layer is separated, concentrated, and purified by silica chromatography to provide the title compound.

[0557] Scheme 9. Synthesis of 2-((6-(l,l-dicyanoprop-l-en-2-yl)naphthalen-2-yl)(methyl)amino)ethyl 4- nitrobenzenesidfonate (Compound 3)

[0558] A solution of 2-(l-(6-((2-hydroxyethyl)(methyl)amino)naphthalen-2-yl)ethylidene)malononitrile (Intermediate 1-D) and tri ethylamine (2.5 equivalents) is dissolved in dichloromethane and cooled to about 0°C. A solution of nosyl chloride (1.1 equivalent) in di chloromethane is added dropwise. The reaction is stirred for Ihr, then is warmed to room temperature. Water is added and the reaction mixture extracted with di chloromethane. The di chloromethane layer is separated, concentrated, and purified by silica chromatography to provide the title compound. Scheme 10. Synthesis of 2-((6-(l, l-dicyanoprop-l-en-2-yl)naphthalen-2-yl)(methyl)amino)ethyl methanesulfoncite (Compound 4) solution of 2-(l-(6-((2-hydroxyethyl)(methyl)amino)naphthalen-2-yl)ethylidene)malononitrile (Intermediate 1-D) and tri ethylamine (2.5 equivalents) is dissolved in di chloromethane and cooled to about 0°C. A solution of mesyl chloride (1.1 equivalent) in dichloromethane is added dropwise. The reaction is stirred for Ihr, then is warmed to room temperature. Water is added and the reaction mixture extracted with dichloromethane. The dichloromethane layer is separated, concentrated, and purified by silica chromatography to provide the title compound.

[0559] Scheme 11. Synthesis of 2-((6-(l,l-dicyanoprop-l-en-2-yl)naphthalen-2-yl)(methyl)amino)ethyl

[0560] 1,1,2,2,3,3,4,4,4-nonafhiorobutane-l-sulfonate (Compound 5)

[0561] A solution of 2-(l-(6-((2-hydroxyethyl)(methyl)amino)naphthalen-2-yl)ethylidene)malononitrile (Intermediate 1-D) and tri ethylamine (2.5 equivalents) is dissolved in di chloromethane and cooled to about 0°C. A solution of 1,1,2,2,3,3,4,4,4-nonafluorobutane-l-sulfonyl chloride (1.1 equivalent) in dichloromethane is added dropwise. The reaction is stirred for Ihr, then is warmed to room temperature. Water is added and the reaction mixture extracted with dichloromethane. The di chloromethane layer is separated, concentrated, and purified by silica chromatography to provide the title compound.

[0562] Purity of Precursor Compounds

[0563] The purity of the precursor compound (Compounds 1-5) is important for the process of preparation described herein. It is advantageous for the precursor compound to be free of impurities which may negatively affect the process. These impurities may arise from, for example, leftover starting materials, hydrolyzed sulfonyl chlorides, or reaction of the precursor with water.

[0564] HPLC Purification of Precursor Compounds

[0565] The precursor compound can be purified by normal phase HPLC chromatography. A solution of precursor (Compounds 1-5) is dissolved in minimal toluene and injected onto the column. The compound is eluted with 50%-90% ethyl acetate in hexanes. The fractions containing the product are concentrated under reduced pressure. The residue is redissolved in benzene and the mixture concentrated to azeotrope residual water. The residue is then sealed and stored at -80°C.

[0566] Recrystallization of Precursor Compounds

[0567] The precursor compound can alternatively be purified by recrystallization from a mixture of nonpolar solvents, for example benzene and hexanes. The precursor is dissolved in minimal benzene in a vial, and hexanes is slowly added. The solution is optionally cooled. The recrystallized product is then filtered and stored in a sealed vial at -80°C.

[0568] Example 3. Illustrative Timing of Production of F-18

[0569] Modern cyclotrons comprising newer targets can produce up to 20 Ci of F-18. The cyclotron ‘target’ is the metallic body that embodies the stable material that is ‘bombarded’ by protons produced by the cyclotron. In certain embodiments, the target is made from silver, titanium, tantalum, or niobium. The relevant reaction is proton bombardment of stable oxygen-18 to produce fluorine- 18 as the starting material which is then converted by radiochemical synthesis into F-18 Flornaptitril.

[0570] F-18 production, for example, may be started early in the morning to be used in the manufacture of F-18 Flornaptitril for imaging later that day. For example, the production of F-18 fluoride might start at midnight using approximately 2.0 hour runs on the cyclotron and produce up to 20 Ci of F-18. The 20 Ci of F-18 may be divided into multiple fractions. In certain embodiments, the starting activity used to prepare the high-activity pharmaceutical formulation of F-18 Flornaptitril is from about 5 Ci to about 20 Ci, from about 7 Ci to about 15 Ci, from about 7 Ci to about 10 Ci, or from about 5 Ci to about 10 Ci of F-18. Significant decay can occur from the early-hours of manufacturing until the dose(s) reach the clinical sites. Decay can also occur during the approximately one-hour manufacturing and quality control time points.

[0571] For example, a 20 Ci F-18 run cyclotron run starting at 2:00 AM, with approximately 70% radiochemical conversion at 3 :00 AM (yielding 14 Ci of ‘finished’ drug product) will only be slightly more than 3.5 Ci when converted to patient-specific, unit-doses at 8am and then thereafter less at times of dose calibration.

[0572] Table 4. A non-limiting example schedule of F-18 production Example 4. Radiosynthesis of F-18 Flornaptitril

[0573] General Preparation of18F-fluoride

[0574] 18F-Fluoride (8 Ci) is produced by the18O(p,n)18F nuclear reaction according to Example 3. An18O enriched water target is bombarded with protons. The18F is then trapped on an ion exchange column, for example a carbonate or trialkylamine cartridge.

[0575] General Radiosynthesis of F-18 Flornaptitril

[0576] The18F-fluoride is eluted from the cartridge by passing an aqueous solution of a potassium salt (e.g. K2CO3) through the cartridge. The eluent is collected in a vial pre-charged with cryptand ligand solution in acetonitrile. Positive pressure flow of inert gas (e.g. helium, nitrogen, or argon) is passed through the vial headspace and the vial is heated until the solvent evaporates. The residue is redissolved in acetonitrile and heated until the solvent evaporates. This process is repeated about three times to azeotropically remove water.

[0577] The dried residue is then dissolved in acetonitrile and a solution of precursor and radiostabilizer is added. The mixture is heated and stirred. The reaction is then cooled, diluted with aqueous sodium ascorbate and loaded onto an SPE cartridge. F-18 Flornaptitril is then eluted with minimal acetonitrile, and the solution is injected into HPLC. F-18 Flornaptitril is then eluted with acetonitrile:water (0.1% sodium ascorbate). The fractions containing the product are concentrated and then loaded onto an SPE cartridge. The product is then eluted with minimal ethanol. The ethanol eluent is then diluted with sodium ascorbate. The solution is then diluted with saline and human serum albumin is added.

[0578] Scheme 12, Radiosynthesis of F-18 Flornaptitril from 2-((6-(El-dicyanoprop-l-en-2- yl)naphthalen-2-yl)(methyl)amino)ethyl 4-methylbenzenesulfonate

[0579] No-carrier added18F-fluoride (8 Ci) is produced by the18O(p,n)18F nuclear reaction by proton bombardment of an18O-enriched water target using a cyclotron. The18F-fluoride is trapped on an anion exchange column to recover the18O water. The18F-fluoride is subsequently released by passing a 0.5% (weightvolume basis) aqueous solution (0.8 mb) of potassium carbonate into a glass reaction vessel pre-loaded with l,10-diaza-4,7, 13, 16,21,24- hexaoxabicyclo[8.8.8]hexacosane (Kryptofix® K222; 50 mg) dissolved in 2 mb of acetonitrile. The solution is evaporated at 115°C, and is then azeotroped three times with 1 mL of acetonitrile. A solution of 2-((6-( 1,1 -di cyanoprop- l-en-2-yl)naphthalen-2-yl)(methyl)amino)ethyl 4- methylbenzenesulfonate (20 mg) and TEMPO (7 mg, 0.5 equivalents per mmol tosylate precursor) in acetonitrile (2 mL) is then added. The reaction is heated to 95°C for 15 minutes and is then cooled with a nitrogen stream for 1 minute. The reaction is then passed through a neutral alumina cartridge (pre-rinsed with acetonitrile) and the cartridge rinsed with 1 mL additional acetonitrile. The acetonitrile eluent is then quenched with 1 mL of an ice-cold aqueous solution (0.1M ammonium acetate and 0.02M ascorbic acid in water). The quenched reaction mixture is then loaded onto a SPE catridge, eluted with 0.75 mL acetonitrile, the SPE catridge rinsed with 0.5 mL acetonitrile and the combined eluent and rinsate is injected into a preparative HPLC system (Cl 8). The F-18 Flomaptitril is then eluted with 50% ACbLFLO (mobile phase modified with 0.1M ammonium acetate and 0.02 M ascorbic acid). The outflow of the HPLC column is monitored by UV (254 nm) and radioactivity (flow through gamma detector). The HPLC fraction containing F- 18 Flomaptitril is diluted with water and loaded onto an SPE cartridge. The F-18 Flomaptitril is then eluted from the SPE cartridge with 3 mL of injection grade ethanol, the SPE cartridge rinsed with 1 mL injection grade ethanol, and the combined eluent and rinsate is combined in a glass vial. The ethanolic solution of F-18 Flomaptitril is diluted with saline containing sodium ascorbate (to a total volume of 25 mL) and 25% human serum albumin (20 mL). The pharmaceutical formulation comprising 2.5 Ci of F-18 Flomaptitril is then sterilized for injection by passing it through a 0.22 pm fdter into a multidose vial.

[0580] Scheme 13, Radiosynthesis of F-18 Flomaptitril from 2-((6-( 1,1 -di cyanoprop- l-en-2- yl)naphthalen-2-yl)(methyl)amino)ethyl 4-nitrobenzenesulfonate No-carrier added18F-fluoride (8 Ci) is produced by the18O(p,n)18F nuclear reaction by proton bombardment of an18O-enriched water target using a cyclotron. The18F-fluoride is trapped on an anion exchange column to recover the18O water. The18F-fluoride is subsequently released by passing a 0.5% (weight: volume basis) aqueous solution (0.8 mL) of potassium carbonate into a glass reaction vessel pre-loaded with l,10-diaza-4,7,13,16,21,24- hexaoxabicyclo[8.8.8]hexacosane (Kryptofix® K222; 50 mg) dissolved in 2 mL of acetonitrile. The solution is evaporated at 115°C, and is then azeotroped three times with 1 mL of acetonitrile. A solution of 2-((6-( 1,1 -di cyanoprop- l-en-2-yl)naphthalen-2-yl)(methyl)amino)ethyl 4- nitrobenzenesulfonate (43 mg) and TEMPO (7 mg, 0.5 equivalents per mmol nosyl precursor) in acetonitrile (2 mL) is then added. The reaction is heated to 90°C for 5 minutes and is then cooled with a nitrogen stream for 1 minute. The reaction is then passed through a neutral alumina cartridge (pre-rinsed with acetonitrile) and the cartridge rinsed with 1 mL additional acetonitrile. The acetonitrile eluent is then quenched with 1 mL of an ice-cold aqueous solution (0.1M ammonium acetate and 0.02M ascorbic acid in water). The quenched reaction mixture is then loaded onto a SPE catridge, eluted with 0.75 mL acetonitrile, the SPE catridge rinsed with 0.5 mL acetonitrile and the combined eluent and rinsate is injected into a preparative HPLC system (Cl 8). The F-18 Flomaptitril is then eluted with 50% ACNiELO (mobile phase modified with 0.1M ammonium acetate and 0.02 M ascorbic acid). The outflow of the HPLC column is monitored by UV (254 nm) and radioactivity (flow through gamma detector). The HPLC fraction containing F-18 Flomaptitril is diluted with water and loaded onto an SPE cartridge. The F-18 Flomaptitril is then eluted from the SPE cartridge with 3 mL of injection grade ethanol, the SPE cartridge rinsed with 1 mL injection grade ethanol, and the combined eluent and rinsate is combined in a glass vial. The ethanolic solution of F-18 Flomaptitril is diluted with saline containing sodium ascorbate (to a total volume of 25 mL) and 25% human semm albumin (20 mL). The pharmaceutical formulation comprising 2.5 Ci of F-18 Flomaptitril is then sterilized for injection by passing it through a 0.22 pm filter into a multidose vial. Example 5. Automated Radiosynthesis of F-18 Flornaptitril

[0581] No-carrier added18F-fluoride was produced by the18O(p,n)18F nuclear reaction by proton bombardment of an18O-enriched water target using a cyclotron. The [18F]fluoride was transferred onto and trapped on an ion exchange cartridge. It was then eluted with an aqueous acetonitrile solution of potassium carbonate (4.0 mg) andKryptofix 222 (18 mg) into the reaction vessel (RV1) of the TRACERlab® module. The solution was first evaporated by heating at 95°C for 3.6 min under vacuum and nitrogen flow. Acetonitrile (1 mL) was added to RV1 and the evaporation was continued under the same conditions for 2 min under vacuum and nitrogen flow. After a second addition of acetonitrile (1 mL), final evaporation was carried out at 95°C for 2 min under vacuum and nitrogen flow. The reactor was then cooled to 35°C.

[0582] A solution of the precursor (2.3 mg) and TEMPO (3.3 mg) in anhydrous acetonitrile was added to a reaction vial (RV1) and the reaction mixture was heated at 95°C for 15 min. The reactor was cooled to 35°C, diluted with anhydrous acetonitrile (1.4 mL) and the contents were passed through an alumina N solid-phase extraction (SPE) cartridge and transferred into the loop-loading vial (LLV) pre-loaded with 1.6 mL of aqueous mobile phase. The reactor was rinsed with anhydrous acetonitrile (2.0 mL) and the rinse was transferred into the LLV after passing through the alumina SPE cartridge. The contents of the LLV were transferred into the HPLC injector loop for purification.

[0583] Purification was performed by HPLC using a semi-preparative Waters SunFire Cl 8 OBD column (5 pm, 250 x 10 mm) and eluted with a mixture of acetonitrile / 10 mM ammonium acetate and 20 mM sodium ascorbate solution (60:40, v / v) at a flow rate of 5 mL / min. The product fraction was collected in Flask 1, containing 20 mL of sodium ascorbate (20 mg / mL) in water for injection (WFI). The diluted product mixture was passed through a tC18 SPE cartridge and the cartridge was rinsed with 6.0 mL of sodium ascorbate (20 mg / mL) in WFI. The radiolabeled product was eluted from the SPE cartridge with 1.5 mL of 200-proof USP grade ethanol into the formulation flask, pre-loaded with 3.0 mL of sodium ascorbate (117 mg / mL) and polysorbate 80 (PS80) (25 mg / mL) in saline plus 2.0 mL of saline. The cartridge was rinsed with 3.0 mL of sodium ascorbate (1 17 mg / mL) and PS80 (25 mg / mL) in saline and the rinse was mixed with the contents of the formulation flask. The resulting solution was transferred into a filter-vented amber intermediate vial, and 7.5 mL of 25% HSA was added. The contents of the intermediate vial were mixed then passed through a freshly conditioned sterilizing 0.22 pm membrane filter into a sterile, filter- vented amber vial (final product vial, FPV).

[0584] Example 6. Radiochemical Process

[0585] The following procedural runs were conducted using the automated radiosynthesis procedure of Example 5. Yields were calculated as non-decay corrected, based on the activity measured at the start of synthesis (SOS) — [18F]fluoride counted in the dose calibrator before transfer to the hot cell — and at the end of synthesis (EOS), when the final F-l 8 Flornaptitril product vial was measured. Decay corrected yields are provided in Table 8.

[0586] Table 5. Radiochemical Process Development

[0587] Table 6. Radiochemical Process Development Table 7. Radiochemical Process Development Table 8. Decay Corrected Yields of Radiosyntheses

[0588] While the conditions described in Barrio et al. (U.S. Patent No. 10,377,701) provided only an 11.3% decay corrected radiochemical yield (Run 1), the current HPLC purification and reaction condition improvements resulted in nearly a five-fold improvement in yield when starting with approximately 0.5 Ci of 18F-fluoride (Run 2 and 3).

[0589] The effect of a radiostabilizer (TEMPO) was tested starting with approximately 1.5 Ci of18F-fluoride (Runs 6-10). Prior experiments with multicurie quantities of18F-fluoride resulted in low and variable yields, however when the radiostabilizer was added, the reaction proceeded smoothly with yield of up to 48.4%. This advantageous effect was observed across a range of radiostabilizer concentrations (1-100 equivalents).

[0590] The reaction was then performed on approximately 3.5 Ci scale in triplicate (Runs 12-14), with an average decay corrected radiochemical yield of 43.2%. The advantageous process described herein allows for Curie scale production of F-18 Flornaptitril.

[0591] Example 7. Sterile Filtration

[0592] Previous handling of F-18 Flornaptitril revealed issues with sterile filter retention when passing the formulated product through a sterile filter. A total of six filters were evaluated by passing the formulated F-l 8 Flornaptitril solution through each filter, with radioactivity measured and calculations performed to assess filter retention. Significant product retention (>30%) was observed with four filters, while only two filters showed acceptable retention.

[0593] Table 9. F-18 Flornaptitril filter retention assessments

[0594] Example 8. Pharmaceutical Formulation

[0595] The excipients used in the formulation of F-18 Flornaptitril were sourced from commercial suppliers and accepted based on manufacturer’s certificate of analysis. All excipients were released according to compendial standards. Excipients are listed in Table 10.

[0596] F-18 Flornaptitril Injection was radiosynthesized from the drug substance precursor (Compound 1) by reaction with [18F]fluoride and formulated in physiological solution (ethanol < 10%, sodium ascorbate < 43 mg / mL, PS-80 < 9.2 mg / mL, albumin (human) < 118 mg / mL and saline 0.9%).

[0597] Ethanol was used to elute the labeled drug substance (F-18 Flornaptitril from a solid phase extraction cartridge during production. Sodium ascorbate is a buffer by virtue of its weak acidity and a stabilizer against radiolysis by virtue of its antioxidant properties. Isotonic 0.9% NaCl (normal saline) is a standard diluent. Polysorbate-80 acts as a surfactant, increasing solubility, and helps prevent adsorption. Human serum albumin is used as a stabilizer, offering protection against oxidative degradation and reducing the potential for degradant chemical reactions. Table 10. Nonlimiting Exemplary Composition of F-18 Flornaptitril Formulation

[0598] 1Based on a target fill volume of 17 mL per vial.

[0599] 2+ / - 10% (e.g., about)

[0600] 3Radioactivity at end of synthesis.

[0601] Example 9. Manufacturing Process for F-18 Flornaptritril

[0602] F-18 Flornaptitril Injection was manufactured according to the following narrative description using a commercial synthesizer, GE TRACERlab® FX-2N.

[0603] The radioactive drug product F-l 8 Flornaptitril Injection was manufactured in a continuous process from the tosylate drug substance precursor Compound 1 in acetonitrile (MeCN) in the presence of [18F]fluoride, potassium carbonate (K2CO3), and Kryptofix K222. The resulting radiolabeled product was purified by HPLC and formulated in a solution that is sterile, clear, colorless, and free of visible particulate matter.

[0604] [18F] -Fluoride was trapped on a QMA cartridge and then eluted with a Kryptofix® (K222) / potassium bicarbonate solution into the reaction vessel (RV1) of the TRACERlab™ FX2N module. After two rounds of azeotropic drying, the precursor solution (2.19 to 2.42 mg DDNPTs and approximately 3.3 mg TEMPO in MeCN) was added to the reaction vessel and reacted at 95°C for 15 mins. The reactor was cooled to 35°C and the solution was diluted with MeCN before transferring to a loop loading vial prefilled with aqueous mobile phase. The loop loading vial contents were loaded onto the HPLC loop and then injected for purification using a semipreparative Waters Sunfire C18 (5pm, 250 x 10 mm) and eluted with a mixture of acetonitrile (MeCN), ammonium acetate (NFEOAc), and sodium ascorbate solution (60% MeCN / 40% 10 mM NI EOAc & 20 mM sodium ascorbate) at a flow rate of 5 mL / min. The product fraction was collected in Flask 1, preloaded with 5 mg / mL sodium ascorbate in water for injection (WFI).

[0605] The diluted product mixture was then trapped on a tC18 solid-phase extraction cartridge, rinsed with diluted sodium ascorbate, and eluted with 1.5 mL of 200-proof USP grade ethanol into the formulation flask pre-loaded with 2.0 mL 0.9% saline and 3.0 mL of formulation base (sodium ascorbate and PS-80 in 0.9% saline). The solid phase extraction (SPE) cartridge was then rinsed with an additional 3.0 mL of formulation base. The resulting solution was transferred into an amber intermediate vial with 7.5 mL of 25% human serum albumin (HSA, 25%). The contents were finally passed through a sterilizing 0.20 pm membrane filter into the empty amber final product vial.

[0606] Quality control testing includes visual inspection of appearance, assessment of identity, chemical and radiochemical purity, product strength, sterile filter integrity, endotoxin content, sterility, residual solvents, ethanol content, and product pH. All tests, except inoculation of product in two media for sterility, were performed before the product is released. Inoculation was performed within 30 hours after end of synthesis.

[0607] All publications and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. Although the foregoing invention has been described in some detail by way of illustration and example for the purpose of clarity of understanding, it will be readily apparent to one of ordinary skill in the art in light of the teaching of this invention that certain changes or modifications may be made thereto without departing from the spirit or scope of the invention as defined in the appended claims. Additionally, those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments and methods described herein. Such equivalents are intended to be encompassed by the scope of the present application.

Claims

CLAIMSWe claim1. A pharmaceutical formulation comprising from about 0.5 to about 10 Ci of F-18 Flornaptitril in a pharmaceutically acceptable carrier.

2. The pharmaceutical formulation of claim 1, comprising from about 0.75 to about 3.5 Ci of F- 18 Flornaptitril in a pharmaceutically acceptable carrier.

3. The pharmaceutical formulation of claim 1, comprising from about 0.8 to about 2.0 Ci of F-18 Flornaptitril in a pharmaceutically acceptable carrier.

4. The pharmaceutical formulation of claim 1, comprising about 1.0 Ci of F-18 Flornaptitril in a pharmaceutically acceptable carrier.

5. The pharmaceutical formulation of any one of claims 1-4, wherein the formulation comprises an antioxidant stabilizer.

6. The pharmaceutical formulation of any one of claims 1 -5, wherein the is selected from ascorbic acid, sodium ascorbate, citric acid, sodium citrate, ethanol, benzoic acid, gentisic acid, ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, calcium stearate, potassium metabi sulfate, propyl gallate, sodium metabisulfate, sodium thiosulfate, and vitamin E (tocopherol).

7. The pharmaceutical formulation of any one of claims 1-5, wherein the antioxidant stabilizer is selected from ascorbic acid, sodium ascorbate, citric acid, sodium citrate, ethanol, benzoic acid, and gentisic acid.

8. The pharmaceutical formulation of any one of claims 1-5, wherein the antioxidant stabilizer is selected from ascorbic acid, sodium ascorbate, citric acid, sodium citrate, and ethanol.

9. The pharmaceutical formulation of any one of claims 1-5, wherein the antioxidant stabilizer is selected from ascorbic acid, sodium ascorbate, and ethanol.

10. The pharmaceutical formulation of any one of claims 1-5, wherein the antioxidant stabilizer is ascorbic acid, sodium ascorbate, and ethanol.

11. The pharmaceutical formulation of any one of claims 5-10, comprising from about 5% ethanol to about 20% ethanol.

12. The pharmaceutical formulation of any one of claims 5-10, comprising less than about 15% ethanol.

13. The pharmaceutical formulation of any one of claims 5-10, comprising about 10% ethanol.

14. The pharmaceutical formulation of any one of claims 5-13, comprising from about 20 to about 80 mg / mL sodium ascorbate.

15. The pharmaceutical formulation of any one of claims 5-13, comprising from about 30 to about 60 mg / mL sodium ascorbate.

16. The pharmaceutical formulation of any one of claims 5-13, comprising from about 40 to about 50 mg / mL of sodium ascorbate.

17. The pharmaceutical formulation of any one of claims 5-13, comprising about 43 mg / mL sodium ascorbate.

18. The pharmaceutical formulation of any one of claims 1-17, wherein the pharmaceutically acceptable carrier comprises saline.

19. The pharmaceutical formulation of any one of claims 1-18, wherein the pharmaceutically acceptable carrier comprises about 0.9% sodium chloride.

20. The pharmaceutical formulation of any one of claims 1-19, wherein the pharmaceutically acceptable carrier comprises human serum albumin.

21. The pharmaceutical formulation of any one of claims 1-19, wherein the pharmaceutically acceptable carrier comprises from about 10% to about 30% human serum albumin.

22. The pharmaceutical formulation of any one of claims 1-19, wherein the pharmaceutically acceptable carrier comprises about 10%, 15%, 20%, 25%, or 30% human serum albumin.

23. The pharmaceutical formulation of any one of claims 1-19, wherein the pharmaceutically acceptable carrier comprises about 25% human serum albumin.

24. The pharmaceutical formulation of any one of claims 1-19, wherein the pharmaceutically acceptable carrier comprises from about 110 to about 130 mg / mL of 20-30% human serum albumin.

25. The pharmaceutical formulation of any one of claims 1-19, wherein the pharmaceutically acceptable carrier comprises from about 110 to about 130 mg / mL of 25% human serum albumin.

26. The pharmaceutical formulation of any one of claims 1-19, wherein the pharmaceutically acceptable carrier comprises about 120 mg / mL of about 25% human serum albumin.

27. The pharmaceutical formulation of any one of claims 1-19, wherein the pharmaceutically acceptable carrier comprises about 118 mg / mL of about 25% human serum albumin.

28. The pharmaceutical formulation of any one of claims 1 -27, wherein the pharmaceutically acceptable carrier comprises a nonionic surfactant.

29. The pharmaceutical formulation of any one of claims 1-27, wherein the pharmaceutically acceptable carrier comprises a nonionic surfactant selected from polysorbate 20, polysorbate 40, polysorbate 60, and polysorbate 80.

30. The pharmaceutical formulation of any one of claims 1-27, wherein the pharmaceutically acceptable carrier comprises polysorbate 80.

31. The pharmaceutical formulation of any one of claims 1-30, wherein the pharmaceutically acceptable carrier comprises from about 1 to about 20 mg / mL of nonionic surfactant.

32. The pharmaceutical formulation of any one of claims 1-30, wherein the pharmaceutically acceptable carrier comprises from about 5 to about 15 mg / mL of nonionic surfactant.

33. The pharmaceutical formulation of any one of claims 1-30, wherein the pharmaceutically acceptable carrier comprises about 10 mg / mL of nonionic surfactant.

34. The pharmaceutical formulation of any one of claims 1-30, wherein the pharmaceutically acceptable carrier comprises about 10 mg / mL of polysorbate 80.

35. The pharmaceutical formulation of any one of claims 1-34, wherein the pharmaceutical formulation contains less than about 0.025 mg / mL of TEMPO.

36. The pharmaceutical formulation of any one of claims 1-34, wherein the pharmaceutical formulation contains less than about 0.05 mg / mL of cryptand ligand.

37. The pharmaceutical formulation of any one of claims 1-36, wherein the formulation is for intravenous administration.

38. A process of preparing a high activity pharmaceutical formulation of F-18 Flornaptitril comprising: a) Reacting a compound of Formula I, or a salt thereof, with18F-fluoride in the presence of a radiostabilizer, a carbonate base, and a cryptand ligand:wherein R1is selected from the group consisting of methyl, Ci-shaloalkyl, and aryl optionally substituted with one or two substituents selected from the group consisting of Ci-ealkyl, Ci-ehaloalkyl, halogen, and nitro; b) Purification of the F-18 Flornaptitril from the reaction mixture; and c) Preparing a pharmaceutical formulation with the purified F-18 Flornaptitril and an antioxidant stabilizer; wherein the pharmaceutical formulation comprises from about 0.5 to about 5 Ci ofF-18 Flornaptitril.

39. The process of claim 38, wherein Step (a) comprises:, ,Formula I i). Trapping from about 1 to about 5 Ci of18F-fluoride on a solid phase; ii). Eluting the18F-fluoride from the solid phase with a carbonate base; iii). Collecting the eluent into a vial; iv). Azeotropically drying the resulting solution; v). Adding a compound of Formula I and a radiostabilizer; vi). Reacting the mixture; and vii). Cooling and quenching the reaction.

40. The process of claim 38 or 39, wherein the radiostabilizer is selected from the group consisting of a persistent radical compound, a nitrone, an aniline of Formula II, a phenol of Formula III, and a diarylalkene.

41. The process of any one of claims 38-40, wherein the radiostabilizer is a persistent radical compound.

42. The process of any one of claims 38-41, wherein the radiostabilizer is TEMPO.

43. The process of any one of claims 38-41, wherein the radiostabilizer is galvinoxyl radical.

44. The process of any one of claims 38-40, wherein the radiostabilizer is a nitrone.

45. The process of claim 44, wherein the radiostabilizer is selected from N-tert-butyl-a- phenylnitrone (PBN), a-4-pyridyl-l-oxide-N-t-butylnitrone (POBN), 5,5- dimethylpyrroline-N-oxide (DMPO), 3, 4-dihydro-2-methyl- 1,1 -dimethylethyl ester-2H- pyrrole-2-carboxylic acid-l-oxide (BMPO), 3,4-dihydro-2-methyl-2H-pyrrole-2- carboxylic acid 1 -oxide, ethyl ester (EMPO).

46. The process of any one of claims 38-40, wherein the radiostabilizer is an aniline of Formula:salt thereof; whereinR2is selected from NH2, NHR4, and N(R4)2;R3a, R3b, and R3care independently selected from hydrogen, halogen, Ci-ealkyl, C(O)OH, C(O)OCi-6alkyl, and C(O)N(R4)2; andR4is selected from hydrogen, Ci-ealkyl, aryl, -Ci-salkyl-aryl, heterocycle, and heteroaryl.

47. The process of claim 46, wherein the radiostabilizer is selected fromsalt thereof.

48. The process of any one of claims 38-40, wherein the radiostabilizer is a phenol of Formula:salt thereof; whereinR22is OH;R23is selected from hydrogen, OH, OR4, CH2OH, and CH2OR4;R3a, R3b, R3Cand R3dare independently selected from hydrogen, halogen, Ci-ealkyl, C(O)OH, C(O)OCi-6alkyl, and C(O)N(R4)2; andR4is selected from hydrogen, Ci-ealkyl, aryl, -Ci-ealkyl-aryl, heterocycle, and heteroaryl.

49. The process of claim 48, wherein the radiostabilizer is selected from50. The process of any one of claims 38-40, wherein the radiostabilizer is a diarlyalkene.

51. The process of claim 50, wherein the radiostabilizer is diphenylethylene.

52. The process of any one of claims 38-50, wherein R1is 4-CH3-phenyl.

53. The process of any one of claims 38-50, wherein R1is 4-Br-phenyl.

54. The process of any one of claims 38-50, wherein R1is 4-NO2-phenyl.

55. The process of any one of claims 38-50, wherein R1is 2-NO2-phenyl.

56. The process of any one of claims 38-50, wherein R1is CH3.

57. The process of any one of claims 38-50, wherein R1is (CF2)3-CF3.

58. The process of any one of claims 39-57, wherein the solid phase in (i) is an anion exchange resin.

59. The process of claim 58, wherein the anion exchange resin is QMA resin.

60. The process of any one of claims 39-59, wherein the18F is eluted in (ii) with an acetonitrile solution comprising a carbonate base.

61. The process of any one of claims 39-60, wherein the18F is eluted in (ii) with a solution comprising potassium carbonate.

62. The process of any one of claims 39-60, wherein the18F is eluted in (ii) with a solution comprising cesium carbonate.

63. The process of any one of claims 39-60, wherein the18F is eluted in (ii) with a solution comprising a cryptand ligand.

64. The process of any one of claims 39-60, wherein the eluent in (iii) is collected into a vial containing a cryptand ligand.

65. The process of claim 63 or 64, wherein the cryptand ligand is Kryptofix® K222.

66. The process of any one of claims 39-65, wherein the eluent of (iii) is azeotropically dried in (iv) with acetonitrile.

67. The process of claim 66, wherein the azeotropic drying step is repeated three times.

68. The process of any one of claims 39-67, wherein the compound of Formula I is added in (v) as a solution in acetonitrile.

69. The process of any one of claims 39-67, wherein the compound of Formula I and the radiostabilizer are added in (v) as a solution in acetonitrile.

70. The process of any one of claims 39-69, wherein the reaction in (vi) is heated to from about 80°C to about 120°C.

71. The process of any one of claims 39-69, wherein the reaction in (vi) is heated to from about 90°C to about 110°C.

72. The process of any one of claims 39-69, wherein the reaction in (vi) is heated to about 90°C.

73. The process of any one of claims 39-69, wherein the reaction in (vi) is heated to about 95°C.

74. The process of any one of claims 39-69, wherein the reaction in (vi) is heated to about 100°C.

75. The process of any one of claims 39-69, wherein the reaction in (vi) is heated to about 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, or 105°C.

76. The process of any one of claims 39-75, wherein the reaction in (vi) is reacted for up to 35 minutes.

77. The process of any one of claims 39-75, wherein the reaction in (vi) is reacted for up to 25 minutes.

78. The process of any one of claims 39-75, wherein the reaction in (vi) is reacted for from about 5 minutes to about 20 minutes.

79. The process of any one of claims 39-75, wherein the reaction in (vi) is reacted for about 5 minutes.

80. The process of any one of claims 39-75, wherein the reaction in (vi) is reacted for about 10 minutes.

81. The process of any one of claims 39-75, wherein the reaction in (vi) is reacted for about 15 minutes.

82. The process of any one of claims 39-75, wherein the reaction in (vi) is reacted for about 20 minutes.

83. The process of any one of claims 39-82, wherein the reaction is cooled in (vii) by passing a stream of inert gas through the reaction vial headspace.

84. The process of claim 83, wherein the inert gas is selected from helium, nitrogen, and argon.

85. The process of any one of claims 39-84, wherein the reaction is quenched in (vii) with an aqueous solution.

86. The process of any one of claims 39-85, wherein the reaction is quenched in (vii) with an acidic aqueous solution.

87. The process of any one of claims 39-85, wherein the reaction is quenched in (vii) with aqueous hydrochloric acid.

88. The process of any one of claims 39-85, wherein the reaction is quenched in (vii) with ammonium acetate.

89. The process of claim 88, wherein the ammonium acetate is from about 0.0 IM to about IM ammonium acetate.

90. The process of any one of claims 39-89, wherein the reaction is quenched in (vii) with an acidic aqueous solution comprising sodium ascorbate.

91. The process of any one of claims 39-90, wherein the aqueous solution used to quench the reaction in (vii) is pre-cooled to from about 0°C to about 10°C.

92. The process of any one of claims 39-91, wherein the F-18 Flomaptitril is purified in (b) by high performance liquid chromatography (HPLC).

93. The process of claim 92, wherein the mobile phase comprises sodium ascorbate.

94. The process of claim 92, wherein the mobile phase comprises acetonitrile.

95. The process of claim 92, wherein the mobile phase comprises at least about 35% aqueous solvent.

96. The process of claim 92, wherein the mobile phase comprises about 40% aqueous solvent.

97. The process of claims 95 or 96, wherein the aqueous solvent comprises sodium ascorbate.

98. The process of any one of claims 39-97, wherein the F-18 Flomaptitril is purified in (b) by solid phase extraction.

99. The process of any one of claims 39-98, wherein the F-18 Flomaptitril is purified in (b) by solid phase extraction before HPLC.

100. The process of any one of claims 39-98, wherein the F-l 8 Flomaptitril is purified in (b) by solid phase extraction after HPLC.

101. The process of any one of claim 98-100, wherein the solid phase extraction is performed with C18 solid phase.

102. The process of any one of claim 98-101, wherein the F-18 Flomaptitril is eluted with ethanol or acetonitrile.

103. The process of any one of claims 39-102, wherein the pharmaceutical formulation of purified F-18 Flomaptitril in Step (c) comprises from about 0.5 Ci to about 2.5 Ci of F-18 Flomaptitril.

104. The process of any one of claims 39-102, wherein the pharmaceutical formulation of purified F-18 Flomaptitril in Step (c) comprises from about 0.8 Ci to about 2.0 Ci of F-18 Flomaptitril.

105. The process of any one of claims 39-102, wherein the pharmaceutical formulation of purified F-18 Flomaptitril in Step (c) comprises about 0.75 Ci of F-18 Flomaptitril.

106. The process of any one of claims 39-102, wherein the pharmaceutical formulation of purified F-18 Flomaptitril in Step (c) comprises about 0.8 Ci of F-18 Flomaptitril.

107. The process of any one of claims 39-102, wherein the pharmaceutical formulation of purified F-18 Flomaptitril in Step (c) comprises about 1.0 Ci of F-18 Flomaptitril.

108. The process of any one of claims 39-102, wherein the pharmaceutical formulation of purified F-18 Flomaptitril in Step (c) comprises about 1.2 Ci of F-18 Flomaptitril.

109. The process of any one of claims 39-102, wherein the pharmaceutical formulation of purified F-18 Flomaptitril in Step (c) comprises about 1.5 Ci of F-18 Flomaptitril.

110. The process of any one of claims 39-109, wherein the pharmaceutical formulation of purified F-18 Flomaptitril in Step (c) comprises less than about 0.05 mg / mL of cryptand ligand.

111. The process of any one of claims 39-109, wherein the pharmaceutical formulation of purified F-18 Flomaptitril in Step (c) comprises an antioxidant stabilizer is selected from ascorbic acid, sodium ascorbate, and ethanol.

112. The process of any one of claims 39-109, wherein the pharmaceutical formulation of purified F-18 Flomaptitril in Step (c) comprises from about 5% ethanol to about 20% ethanol.

113. The process of any one of claims 39-109, wherein the pharmaceutical formulation of purified F-18 Flomaptitril in Step (c) comprises less than about 15% ethanol.

114. The process of any one of claims 39-109, wherein the pharmaceutical formulation of purified F-18 Flomaptitril in Step (c) comprises about 10% ethanol.

115. The process of any one of claims 39-114, wherein the pharmaceutical formulation of purified F-18 Flomaptitril in Step (c) comprises from about 20 to about 80 mg / mL sodium ascorbate.

116. The process of any one of claims 39-114, wherein the pharmaceutical formulation of purified F-18 Flomaptitril in Step (c) comprises from about 30 to about 60 mg / mL sodium ascorbate.

117. The process of any one of claims 39-114, wherein the pharmaceutical formulation of purified F-18 Flomaptitril in Step (c) comprises from about 40 to about 50 mg / mL of sodium ascorbate.

118. The process of any one of claims 39-114, wherein the pharmaceutical formulation of purified F-18 Flomaptitril in Step (c) comprises about 43 mg / mL sodium ascorbate.

119. The process of any one of claims 39-118, wherein the pharmaceutical formulation of purified F-18 Flomaptitril in Step (c) comprises saline.

120. The process of any one of claims 39-118, wherein the pharmaceutical formulation of purified F-18 Flomaptitril in Step (c) comprises about 0.9% sodium chloride.

121. The process of any one of claims 39-120, wherein the pharmaceutical formulation of purified F-18 Flomaptitril in Step (c) comprises human serum albumin.

122. The process of any one of claims 39-120, wherein the pharmaceutical formulation of purified F-18 Flomaptitril in Step (c) comprises from about 10% to about 30% human serum albumin.

123. The process of any one of claims 39-120, wherein the pharmaceutical formulation of purified F-18 Flomaptitril in Step (c) comprises about 10%, 15%, 20%, 25%, or 30% human serum albumin.

124. The process of any one of claims 39-120, wherein the pharmaceutical formulation of purified F-18 Flomaptitril in Step (c) comprises about 25% human serum albumin.

125. The process of any one of claims 39-124, wherein the pharmaceutical formulation of purified F-18 Flomaptitril in Step (c) comprises from about 80 to about 150 mg / mL human serum albumin.

126. The process of any one of claims 39-124, wherein the pharmaceutical formulation of purified F-18 Flornaptitril in Step (c) comprises from about 90 to about 130 mg / mL human serum albumin.

127. The process of any one of claims 39-124, wherein the pharmaceutical formulation of purified F-18 Flornaptitril in Step (c) comprises from about 120 mg / mL human serum albumin.

128. The process of any one of claims 39-124, wherein the pharmaceutical formulation of purified F-18 Flornaptitril in Step (c) comprises from about 118 mg / mL human serum albumin.

129. The process of any one of claims 39-120, wherein the pharmaceutical formulation of purified F-18 Flornaptitril in Step (c) comprises from about 110 to about 130 mg / mL of 20- 30% human serum albumin.

130. The process of any one of claims 39-120, wherein the pharmaceutical formulation of purified F-18 Flornaptitril in Step (c) comprises from about 110 to about 130 mg / mL of 25% human serum albumin.

131. The process of any one of claims 39-120, wherein the pharmaceutical formulation of purified F-18 Flornaptitril in Step (c) comprises about 120 mg / mL of about 25% human serum albumin.

132. The process of any one of claims 39-131, wherein the pharmaceutical formulation of purified F-18 Flornaptitril in Step (c) comprises a nonionic surfactant.

133. The process of any one of claims 39-131, wherein the pharmaceutical formulation of purified F-18 Flornaptitril in Step (c) comprises a nonionic surfactant selected from polysorbate 20, polysorbate 40, polysorbate 60, and polysorbate 80.

134. The process of any one of claims 39-131, wherein the pharmaceutical formulation of purified F-18 Flornaptitril in Step (c) comprises polysorbate 80.

135. The process of any one of claims 39-131, wherein the pharmaceutical formulation of purified F-18 Flornaptitril in Step (c) comprises from about 1 to about 20 mg / mL of nonionic surfactant.

136. The process of any one of claims 39-131, wherein the pharmaceutical formulation of purified F-18 Flornaptitril in Step (c) comprises from about 5 to about 15 mg / mL of nonionic surfactant.

137. The process of any one of claims 39-131, wherein the pharmaceutical formulation of purified F-18 Flornaptitril in Step (c) comprises about 10 mg / mL of nonionic surfactant.

138. The process of any one of claims 39-131 , wherein the pharmaceutical formulation of purified F-18 Flomaptitril in Step (c) comprises about 10 mg / mL of polysorbate 80.

139. The process of any one of claims 39-138, wherein the pharmaceutical formulation of purified F-18 Flomaptitril in Step (c) contains less than about 0.025 mg / mL of TEMPO.

140. The process of any one of claims 39-138, wherein the pharmaceutical formulation of purified F-18 Flomaptitril in Step (c) contains less than about 0.05 mg / mL of cryptand ligand.

141. The process of any one of claims 39-140, wherein the pharmaceutical formulation of purified F-18 Flomaptitril in Step (c) is for intravenous administration.

142. The process of any one of claims 39-140, wherein the pharmaceutical formulation of purified F-18 Flomaptitril in Step (c) is filtered through a sterile filter.

143. The process of any one of claims 39-142, wherein Step (a) is performed on an automated synthesizer.

144. The process of any one of claims 39-142, wherein Step (b) is performed on an automated synthesizer.

145. The process of any one of claims 39-142, wherein Step (c) is performed on an automated synthesizer.

146. The process of any one of claims 39-142, wherein two of Steps (a)-(c) are performed on an automated synthesizer.

147. The process of any one of claims 39-142, wherein Steps (a)-(c) are performed on an automated synthesizer.

148. The process of any one of claims 39-142, wherein Steps (a)-(c) are performed on a GE Healthcare TRACERlab automated synthesizer.

149. The process of any one of claims 39-142, wherein Steps (a)-(c) are performed on a GE Healthcare FASTlab automated synthesizer.

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