F-18 labeled polyunsaturated fatty acids for imaging lipid metabolism dysregulation

Abstract

A radiofluorinated docosahexaenoic acid fatty acid and methods of making the same. Also disclosed are methods of using the radiofluorinated docosahexaenoic acid fatty acid for use in multimodal imaging, such as positron emission tomography (PET) scans.

Description

[0001] F-18 LABELED POLYUNSATURATED FATTY ACIDS FOR IMAGING LIPID METABOLISM DYSREGULATION

[0002] RELATED APPLICATIONS

[0003] This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Application No. 63 / 581,112, filed September 7, 2023, which is incorporated herein by reference.

[0004] GOVERNMENT SUPPORT

[0005] This invention was made with government support under grant no. R21AG056518, awarded by the (NIH / NIA) National Institute on Aging. The government has certain rights in the invention.

[0006] BACKGROUND OF THE INVENTION

[0007] Docosahexaenoic acid [22:6(n-3), DHA] is the most abundant co-3 polyunsaturated fatty acid in the brain where it regulates several important processes and serves as a precursor to bioactive mediators to resolve inflammation in neurons, microglia, and endothelial cells. However, the capacity of the brain to synthesize DHA locally is low, and the uptake of DHA from circulating lipid pools is essential to maintaining homeostatic levels. Brain DHA uptake is affected by various factors and is implicated in the progression of neurodegenerative disorders such as Alzheimer’s disease (AD).

[0008] The metabolism of brain lipids and the production of bioactive mediators are influenced by the apolipoprotein E4 (APOE4) allele, which is a major genetic risk factor for AD. APOE4 affects the catabolism, transport, and esterification of DHA in the brain, leading to changes in arachidonic acid (AA) and DHA signaling cascades. These changes are associated with increased brain inflammation and amyloid deposition and moreover, APOE4 modulates the associations between serum or plasma DHA levels and brain structure and function. Similarly, higher plasma DHA levels are associated with lower amyloid burden in cognitively normal individuals without APOE4, but not in those with APOE4. Therefore, APOE4 status is speculated to alter the brain delivery and effects of supplemental DHA. The optimal dose and duration of DHA supplementation for AD prevention are still unclear; some studies have suggested that larger doses (>2 g per day) of DHA may be needed for adequate brain bioavailability and that APOE4 carriers may have reduced response to supplementation compared to non-carriers. Further research is thus needed to elucidate the mechanisms and implications of APOE4-DHA interactions in AD pathogenesis and treatment. Radiolabeled DHA has not only been used to investigate factors that affect the uptake and metabolism of DHA through autoradiography of rodent brains but also has been employed in positron emission tomography (PET) imaging to assess DHA uptake in human brains. Using [rnC]-DHA PET, the regional and global incorporation rates (K*) of DHA into the human brain have been estimated and found that they were influenced by factors such as cerebral blood flow, unesterified plasma DHA concentration, and AP0E4 status. However, the use of [1-11C]- DHA as a PET tracer has some limitations, such as a short half-life (20 minutes) making clinical translation quite difficult.

[0009] Accordingly, there is a need for a new PET tracer with a longer in vivo half-life to provide clinicians with vital diagnostic information of patients. The present invention satisfies these needs.

[0010] SUMMARY OF THE INVENTION

[0011] In this disclosure,18F-labeled DHA was synthesized for in vivo brain DHA imaging. Previously, 20-[18F]fluoroarachidonic acid (20-[18F]FAA) was used to evaluate arachidonic acid (AA) uptake and kinetics in the brains of APOE4-targeted replacement mice through dynamic PET -MR imaging. Here, we report the synthesis of an to-fluorinated DHA PET probe, 22-[18F]fluorodocosahexaenoic acid (22-[18F]FDHA). We validated the observed uptake of the tracer into the brain by monitoring the changes in brain uptake kinetics through arecoline administration. The development of the 22-[18F]FDHA PET probe can address the hurdles related to the short-lived l-[nC]DHA PET probe. This development can provide more precise and readily applicable assessments of DHA incorporation into the brain.

[0012] Accordingly, the disclosure provides for a radiofluorinated docosahexaenoic acid fatty acid having structure wherein the radiofluorinated fatty acid is 22-[18F]FDHA. The disclosure also provides for compositions comprising the radiofluorinated fatty acid and at least one of a solvent and / or a pharmaceutically acceptable carrier, excipient, or diluent.

[0013] In another aspect, the disclosure provides for methods of synthesizing a radiofluorinated fatty acid comprising the steps of combining: (a) an omega-tosylated docosahexaenoic acid fatty acid methyl ester in a first nitrile solvent; and (b) a solution of tetra- w-butylammonium [18F]fluoride ([18F]TBAF) in a second nitrile solvent, at about 75 °C to about 95 °C for about 10 minutes to about 30 minutes, to form a [18F]fluorinated ester; and treating the [18F]fluorinated ester with a solvent comprising a strong base at about 75 °C to about 95 °C to hydrolyze the methyl ester into a free fatty acid; thereby providing the radiofluorinated fatty acid.

[0014] In another aspect, the disclosure provides for methods of positron emission tomography (PET) imaging comprising administering to a subject an effective amount of i) 22- [18F]fluorodocosahexaenoic acid ([18F]FDHA); ii) optionally, a muscarinic receptor agonist; iii) optionally, a pharmaceutically acceptable carrier, excipient, or diluent; and capturing one or more PET images of the subject.

[0015] In some aspects, the [18F]FDHA is administered as a composition comprising a pharmaceutically acceptable carrier, excipient, or diluent. In other aspect, the [18F]FDHA or a composition thereof is administered, either concurrently or sequentially with the muscarinic receptor agonist. In some aspects, the muscarinic receptor agonist is one or more of arecoline, bethanechol, cevimeline, pilocarpine, methacholine, xanomeline, acetylcholine, carbachol, muscarine, and oxotremorine.

[0016] These and other features and advantages of this invention will be more fully understood from the following detailed description of the invention taken together with the accompanying claims. It is noted that the scope of the claims is defined by the recitations therein and not by the specific discussion of features and advantages set forth in the present description.

[0017] BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The following drawings form part of the specification and are included to further demonstrate certain embodiments or various aspects of the invention. In some instances, embodiments of the invention can be best understood by referring to the accompanying drawings in combination with the detailed description presented herein. The description and accompanying drawings may highlight a certain specific example, or a certain aspect of the invention. However, one skilled in the art will understand that portions of the example or aspect may be used in combination with other examples or aspects of the invention.

[0019] Figure 1. Structures of polyunsaturated fatty acids AA and DHA with the corresponding co-fluorinated analogues.

[0020] Figure 2. Western blot quantitation of LPS-induced COX-2 induction in BV2 microglial cells, which attenuated by treating with DHA and 22-FDHA (*p < 0.05 and ***p < 0.001 by one-way ANOVA with Tukey’s post hoc test).

[0021] Figure 3. Ex vivo brain autoradiography of mice at 30 min after injection of (A) 22- [18F]FDHA (37 MBq) or (B) [18F]fluoride (41 MBq). Scale bar: 5 mm. Figure 4A-B. (A) Superimposed PET -MR images of a representative mouse brain at different time points. (B) Time-activity curve of a representative mouse after i.v. injection of 22-[18F]FDHA.

[0022] Figure 5A-C. (A) PET-MR images at the 9-10 min and 28-30 min frames for representative mice treated with saline or arecoline. (B) %ID / g over time for mice treated with saline or 15 mg / kg arecoline (n = 3 / group). The mean %ID / g values measured for mice treated with arecoline were not significantly higher than those of control mice. (C) Incorporation coefficients (K*) estimated by Irr2TCM using each mouse’s TAC as the output function. 22- [18F]FDHA T* estimates for mice treated with arecoline were significantly higher (**p < 0.01, Student’s / -test) than control mice.

[0023] Figure 6. PET images of a representative mouse after the injection of 22-[18F]FDHA at different time points. Normal nude mice (n = 3) were injected with a bolus of 22-[18F]FDHA (9-11 MBq) via the tail vein. PET images were acquired for 5 min at 15, 30, and 60 min p.i.

[0024] Figure 7A-B. 22-[18F]FDHA imaging of APOE4-TR mice, compared by diet. (A) Representative PET images of a transverse section of the brains of APOE4-TR fed either a DHA-deficient, high-fat diet or a DHA-rich diet. (B) Percent injected dose per gram (%ID / g) from data acquired 10-30 min p.i. of the tracer. Error bars are S.D.; * represents -value <0.05 (Student’s / -test).

[0025] DETAILED DESCRIPTION

[0026] Docosahexaenoic acid [22:6(n-3), DHA] is an co-3 polyunsaturated fatty acid that is important in regulating both the structure and function of neurons, microglia, and endothelial cells. Altered brain DHA uptake is implicated in the progression of neurodegenerative disorders such as Alzheimer’s disease (AD). Here, we report the synthesis of an coradiofluorinated analogue of DHA, 22-[18F]fluorodocosahexaenoic acid, [18F]FDHA as a PET probe for imaging the uptake of DHA into the brain. Using the nonradioactive analogue FDHA, we confirmed that fluorination of DHA at the co-position does not significantly alter the antiinflammatory effect of DHA in microglial cells. Through dynamic PET-MR studies in live mice, we then observed the accumulation of [18F]FDHA in the brain over time. We successfully estimated the incorporation coefficient (K*), a measure of uptake kinetics from time-activity data in the brain region using an image-derived input function. Finally, we also confirmed that intravenous administration of 15 mg / kg arecoline, a natural product known to elevate the DHA K* in rodents, results in significantly altered brain uptake kinetics using our imaging protocol. Our results demonstrate that [18F]FDHA is a promising PET tracer that can reveal the altered lipid metabolism in AD and other related disorders. This development would enable non- invasive and longitudinal studies of brain lipid dynamics and their role in neurodegeneration.

[0027] Definitions.

[0028] The following definitions are included to provide a clear and consistent understanding of the specification and claims. As used herein, the recited terms have the following meanings. All other terms and phrases used in this specification have their ordinary meanings as one of skill in the art would understand. Such ordinary meanings may be obtained by reference to technical dictionaries, such as Hawley ’s Condensed Chemical Dictionary 14thEdition, by R.J. Lewis, John Wiley & Sons, New York, N.Y., 2001.

[0029] References in the specification to "one embodiment", "an embodiment", etc., indicate that the embodiment described may include a particular aspect, feature, structure, moiety, or characteristic, but not every embodiment necessarily includes that aspect, feature, structure, moiety, or characteristic. Moreover, such phrases may, but do not necessarily, refer to the same embodiment referred to in other portions of the specification. Further, when a particular aspect, feature, structure, moiety, or characteristic is described in connection with an embodiment, it is within the knowledge of one skilled in the art to affect or connect such aspect, feature, structure, moiety, or characteristic with other embodiments, whether or not explicitly described.

[0030] The singular forms "a," "an," and "the" include plural reference unless the context clearly dictates otherwise. Thus, for example, a reference to "a compound" includes a plurality of such compounds, so that a compound X includes a plurality of compounds X. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for the use of exclusive terminology, such as "solely," "only," and the like, in connection with any element described herein, and / or the recitation of claim elements or use of "negative" limitations.

[0031] The term "and / or" means any one of the items, any combination of the items, or all of the items with which this term is associated. The phrases "one or more" and "at least one" are readily understood by one of skill in the art, particularly when read in context of its usage. For example, the phrase can mean one, two, three, four, five, six, ten, 100, or any upper limit approximately 10, 100, or 1000 times higher than a recited lower limit. For example, one or more substituents on a phenyl ring refers to one to five, or one to four, for example if the phenyl ring is disubstituted.

[0032] As will be understood by the skilled artisan, all numbers, including those expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth, are approximations and are understood as being optionally modified in all instances by the term "about." These values can vary depending upon the desired properties sought to be obtained by those skilled in the art utilizing the teachings of the descriptions herein. It is also understood that such values inherently contain variability necessarily resulting from the standard deviations found in their respective testing measurements. When values are expressed as approximations, by use of the antecedent "about," it will be understood that the particular value without the modifier "about" also forms a further aspect.

[0033] The term "about" can refer to a variation of ± 5%, ± 10%, ± 20%, or ± 25% of the value specified. For example, "about 50" percent can in some embodiments carry a variation from 45 to 55 percent, or as otherwise defined by a particular claim. For integer ranges, the term "about" can include one or two integers greater than and / or less than a recited integer at each end of the range. Unless indicated otherwise herein, the term "about" is intended to include values, e.g., weight percentages, proximate to the recited range that are equivalent in terms of the functionality of the individual ingredient, composition, or embodiment. The term about can also modify the end-points of a recited range as discussed above in this paragraph.

[0034] As will be understood by one skilled in the art, for any and all purposes, particularly in terms of providing a written description, all ranges recited herein also encompass any and all possible sub-ranges and combinations of sub-ranges thereof, as well as the individual values making up the range, particularly integer values. It is therefore understood that each unit between two particular units are also disclosed. For example, if 10 to 15 is disclosed, then 11, 12, 13, and 14 are also disclosed, individually, and as part of a range. A recited range (e.g., weight percentages or carbon groups) includes each specific value, integer, decimal, or identity within the range. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, or tenths. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art, all language such as "up to", "at least", "greater than", "less than", "more than", "or more", and the like, include the number recited and such terms refer to ranges that can be subsequently broken down into sub-ranges as discussed above. In the same manner, all ratios recited herein also include all sub-ratios falling within the broader ratio. Accordingly, specific values recited for radicals, substituents, and ranges, are for illustration only; they do not exclude other defined values or other values within defined ranges for radicals and substituents. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. One skilled in the art will also readily recognize that where members are grouped together in a common manner, such as in a Markush group, the invention encompasses not only the entire group listed as a whole, but each member of the group individually and all possible subgroups of the main group. Additionally, for all purposes, the invention encompasses not only the main group, but also the main group absent one or more of the group members. The invention therefore envisages the explicit exclusion of any one or more of members of a recited group. Accordingly, provisos may apply to any of the disclosed categories or embodiments whereby any one or more of the recited elements, species, or embodiments, may be excluded from such categories or embodiments, for example, for use in an explicit negative limitation.

[0035] The term "contacting" refers to the act of touching, making contact, or of bringing to immediate or close proximity, including at the cellular or molecular level, for example, to bring about a physiological reaction, a chemical reaction, or a physical change, e.g., in a solution, in a reaction mixture, in vitro, or in vivo.

[0036] An "effective amount" refers to an amount effective to treat a disease, disorder, and / or condition, or to bring about a recited effect. For example, an effective amount can be an amount effective to reduce the progression or severity of the condition or symptoms being treated. Determination of a therapeutically effective amount is well within the capacity of persons skilled in the art. The term "effective amount" is intended to include an amount of a compound described herein, or an amount of a combination of compounds described herein, e.g., that is effective to treat or prevent a disease or disorder, or to treat the symptoms of the disease or disorder, in a host. Thus, an "effective amount" generally means an amount that provides the desired effect. An appropriate "effective" amount in any individual case may be determined using techniques, such as a dose escalation study.

[0037] Alternatively, the terms "effective amount" or "therapeutically effective amount," as used herein, refer to a sufficient amount of an agent or a composition or combination of compositions being administered which will relieve to some extent one or more of the symptoms of the disease or condition being treated. The result can be reduction and / or alleviation of the signs, symptoms, or causes of a disease, or any other desired alteration of a biological system. For example, an "effective amount" for therapeutic uses is the amount of the composition comprising a compound as disclosed herein required to provide a clinically significant decrease in disease symptoms. An appropriate "effective" amount in any individual case may be determined using techniques, such as a dose escalation study. The dose could be administered in one or more administrations. However, the precise determination of what would be considered an effective dose may be based on factors individual to each patient, including, but not limited to, the patient's age, size, type or extent of disease, stage of the disease, route of administration of the compositions, the type or extent of supplemental therapy used, ongoing disease process and type of treatment desired (e.g., aggressive vs. conventional treatment).

[0038] As used herein, "subject" or “patient” means an individual having symptoms of, or at risk for, a disease or other malignancy. A patient may be human or non-human and may include, for example, animal strains or species used as “model systems” for research purposes, such a mouse model as described herein. Likewise, patient may include either adults or juveniles (e.g., children). Moreover, patient may mean any living organism, preferably a mammal (e.g., human or non-human) that may benefit from the administration of compositions contemplated herein. Examples of mammals include, but are not limited to, any member of the Mammalian class: humans, non-human primates such as chimpanzees, and other apes and monkey species; farm animals such as cattle, horses, sheep, goats, swine; domestic animals such as rabbits, dogs, and cats; laboratory animals including rodents, such as rats, mice and guinea pigs, and the like. Examples of non-mammals include, but are not limited to, birds, fish and the like. In one embodiment of the methods provided herein, the mammal is a human.

[0039] Wherever the term “comprising” is used herein, options are contemplated wherein the terms “consisting of’ or “consisting essentially of’ are used instead. As used herein, “comprising” is synonymous with "including," "containing," or "characterized by," and is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. As used herein, "consisting of' excludes any element, step, or ingredient not specified in the aspect element. As used herein, "consisting essentially of' does not exclude materials or steps that do not materially affect the basic and novel characteristics of the aspect. In each instance herein any of the terms "comprising", "consisting essentially of' and "consisting of' may be replaced with either of the other two terms. The disclosure illustratively described herein may be suitably practiced in the absence of any element or elements, limitation, or limitations not specifically disclosed herein.

[0040] Embodiments of the Invention.

[0041] Embodiments of the present disclosure provide for a radiofluorinated docosahexaenoic acid fatty acid having the formula (1): (see also Figure 1). The disclosure also provides for a composition comprising the radiofluorinated docosahexaenoic acid fatty acid of formula 1 in combination with a solvent or a pharmaceutically acceptable carrier.

[0042] The disclosure also provides for methods of synthesizing a radiofluorinated fatty acid comprising the steps of combining (a) an omega-tosylated docosahexaenoic acid fatty acid methyl ester in a first nitrile solvent and (b) a solution of tetra- / / -butylammonium [18F] fluoride ([18F]TBAF) in a second nitrile solvent to form a [18F] fluorinated ester; treating the [18F] fluorinated ester with a solvent comprising a strong base to hydrolyze the methyl ester into a free fatty acid; and optionally, isolating the free fatty acid, thereby providing the radiofluorinated fatty acid. Preferably, the radiofluorinated fatty acid is 22- [18F]fluorodocosahexaenoic acid ([18F]FDHA).

[0043] In other embodiments, methods of synthesizing a radiofluorinated fatty acid may comprise the steps of combining (a) an omega-tosylated docosahexaenoic acid fatty acid methyl ester in a first nitrile solvent and (b) a solution of tetra- / / -butylammonium [18F] fluoride ([18F]TBAF) in a second nitrile solvent, at about 70 °C to about 100 °C for about 5 minutes to about 40 minutes, to form a [18F] fluorinated ester; treating the [18F] fluorinated ester with a solvent comprising a strong base, at about 70 °C to about 100 °C, to hydrolyze the methyl ester into a free fatty acid; and optionally, isolating the free fatty acid, thereby providing the radiofluorinated fatty acid. Preferably, the radiofluorinated fatty acid is 22- [18F]fluorodocosahexaenoic acid ([18F]FDHA).

[0044] In still other embodiments, methods of synthesizing a radiofluorinated fatty acid may comprise the steps of combining (a) an omega-tosylated docosahexaenoic acid fatty acid methyl ester in a first nitrile solvent and (b) a solution of tetra- / / -butylammonium [18F] fluoride ([18F]TBAF) in a second nitrile solvent, at about 85 °C for about 20 minutes, to form a [18F] fluorinated ester; treating the [18F] fluorinated ester with 2M potassium hydroxide at about 85 °C to hydrolyze the methyl ester into a free fatty acid. The method can further include isolating the free fatty acid by reverse phase HPLC, thereby providing the radiofluorinated fatty acid. Preferably, the radiofluorinated fatty acid is 22-[18F]fluorodocosahexaenoic acid ([18F]FDHA).

[0045] In some embodiments, the combining steps of (a) and (b) are carried out at about 70 °C, about 72 °C, about 74 °C, about 76 °C, about 78 °C, about 80 °C, about 82 °C, about 84 °C, about 86 °C, about 88 °C, about 90 °C, about 92 °C, about 96 °C, about 96 °C, about 98 °C, or about 100 °C. In some embodiments, the combining steps of (a) and (b) are carried out at about 75 °C to about 95 °C for about 5 minutes to about 60 minutes, or about 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 35 minutes, about 40 minutes, about 45 minutes, about 50 minutes, about 55 minutes, or about 60 minutes.

[0046] In some embodiments, the step of treating the [18F] fluorinated ester with a solvent comprising a strong base is conducted at about 70 °C to about 100 °C, or about 70 °C, about 75 °C, about 80 °C, about 85 °C, about 90 °C, about 95 °C, or about 100 °C. In some embodiments, the treating step proceeds for about 5 minutes to about 60 minutes, or about 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 35 minutes, about 40 minutes, about 45 minutes, about 50 minutes, about 55 minutes, or about 60 minutes. In some embodiments, treating the [18F] fluorinated ester with a strong base occurs at about 85 °C for about 15 minutes.

[0047] In some embodiments, the first nitrile solvent and the second nitrile solvent comprise acetonitrile, benzonitrile, butyronitrile, 3 -methoxypropionitrile, propionitrile, 3,3’- oxydiprpionitrile, methoxyacetonitrile, pimelonitrile, phenylacetonitrile, ethylene glycol bis(propionitrile) ether, or glutaronitrile. In some embodiments, the first nitrile solvent and the second nitrile solvent comprise acetonitrile. In some embodiments, the first nitrile solvent and the second nitril solvent or the same or different.

[0048] In some embodiments, the solvent comprises a strong base comprises potassium hydroxide, lithium hydroxide, sodium hydroxide, rubidium hydroxide, cesium hydroxide, calcium hydroxide, strontium hydroxide, or barium hydroxide. In some embodiments, the strong base is potassium hydroxide. The strong base may be present in a concentration of about IM, 2M, 3M, 4M, or 5M.

[0049] In some embodiments, the omega-tosylated docosahexaenoic acid fatty acid methyl ester is:

[0050] In some embodiments, the tosylate is synthesized according to Scheme 1 and the radiofluorinated fatty acid is synthesized according to Scheme 2.

[0051] The disclosure also provides for a method of positron emission tomography (PET) imaging comprising administering to a subject an effective amount of: i) the 22-[18F]fluorodocosahexaenoic acid ([18F]FDHA)or a composition thereof to a subject; and ii) optionally, one or more muscarinic receptor agonist; wherein the muscarinic receptor agonist increases uptake of the radiofluorinated fatty acid compound; and optionally, capturing one or more PET images of the subject. In some embodiments, the muscarinic receptor agonist is one or more of arecoline, bethanechol, cevimeline, pilocarpine, methacholine, xanomeline, acetylcholine, carbachol, muscarine, and oxotremorine. Preferably, the muscarinic receptor agonist may be administered in an amount of about 0.1 mg / kg to about 50 mg / kg, or about 1 mg / kg to about 25 mg / kg, or about 5 mg / kg to about 20 mg / kg, or about 10 mg / kg to about 15 mg / kg.

[0052] As used herein, “PET” or “PET-scan” refers to positron emission tomography (PET) scanning using a molecular tracer. PET-scan is a nuclear medicine functional imaging technique that is widely used in the medical field to observe metabolic processes in the body as an aid to the diagnosis of disease.

[0053] The 22-[18F]FDHA disclosed herein can be used as a PET tracer for certain medical conditions, including, but not limited to, cancer, neurodegenerative diseases, autoimmunity inflammation, and bone marrow transplant.

[0054] The term “cancer” refers to a group of diseases characterized by abnormal and uncontrolled cell proliferation starting at one site (primary site) with the potential to invade and to spread to other sites (secondary sites, metastases) which differentiate cancer (malignant tumor) from benign tumor. Virtually all the organs can be affected, leading to more than 100 types of cancer that can affect humans. Cancers can result from many causes including genetic predisposition, viral infection, exposure to ionizing radiation, exposure to environmental pollutant, tobacco and or alcohol use, obesity, poor diet, lack of physical activity or any combination thereof. “Metastasis” refers to the biological process involved in the development of metastases. “Neoplasm” or “tumor” including grammatical variations thereof means new and abnormal growth of tissue, which may be benign or cancerous.

[0055] Exemplary cancers include breast cancer, non-small cell lung cancer, brain cancer, and osteosarcoma. Exemplary cancers also include, but are not limited to, Acute Lymphoblastic Leukemia, Adult; Acute Lymphoblastic Leukemia, Childhood; Acute Myeloid Leukemia, Adult; Adrenocortical Carcinoma; Adrenocortical Carcinoma, Childhood; AIDS-Related Lymphoma; AIDS-Related Malignancies; Anal Cancer; Astrocytoma, Childhood Cerebellar; Astrocytoma, Childhood Cerebral; Bile Duct Cancer, Extrahepatic; Bladder Cancer; Bladder Cancer, Childhood; Bone Cancer, Osteosarcoma / Malignant Fibrous Histiocytoma; Brain Stem Glioma, Childhood; Brain Tumor, Adult; Brain Tumor, Brain Stem Glioma, Childhood; Brain Tumor, Cerebellar Astrocytoma, Childhood; Brain Tumor, Cerebral Astrocytoma / Malignant Glioma, Childhood; Brain Tumor, Ependymoma, Childhood; Brain Tumor, Medulloblastoma, Childhood; Brain Tumor, Supratentorial Primitive Neuroectodermal Tumors, Childhood; Brain Tumor, Visual Pathway and Hypothalamic Glioma, Childhood; Brain Tumor, Childhood (Other); Breast Cancer; Breast Cancer and Pregnancy; Breast Cancer, Childhood; Breast Cancer, Male; Bronchial Adenomas / Carcinoids, Childhood: Carcinoid Tumor, Childhood; Carcinoid Tumor, Gastrointestinal; Carcinoma, Adrenocortical; Carcinoma, Islet Cell; Carcinoma of Unknown Primary; Central Nervous System Lymphoma, Primary; Cerebellar Astrocytoma, Childhood; Cerebral Astrocytoma / Malignant Glioma, Childhood; Cervical Cancer; Childhood Cancers; Chronic Lymphocytic Leukemia; Chronic Myelogenous Leukemia; Chronic Myeloproliferative Disorders; Clear Cell Sarcoma of Tendon Sheaths; Colon Cancer; Colorectal Cancer, Childhood; Cutaneous T-Cell Lymphoma; Endometrial Cancer; Ependymoma, Childhood; Epithelial Cancer, Ovarian; Esophageal Cancer; Esophageal Cancer, Childhood; Ewing's Family of Tumors; Extracranial Germ Cell Tumor, Childhood; Extragonadal Germ Cell Tumor; Extrahepatic Bile Duct Cancer; Eye Cancer, Intraocular Melanoma; Eye Cancer, Retinoblastoma; Gallbladder Cancer; Gastric (Stomach) Cancer; Gastric (Stomach) Cancer, Childhood; Gastrointestinal Carcinoid Tumor; Germ Cell Tumor, Extracranial, Childhood; Germ Cell Tumor, Extragonadal; Germ Cell Tumor, Ovarian; Gestational Trophoblastic Tumor; Glioma. Childhood Brain Stem; Glioma. Childhood Visual Pathway and Hypothalamic; Hairy Cell Leukemia; Head and Neck Cancer; Hepatocellular (Liver) Cancer, Adult (Primary); Hepatocellular (Liver) Cancer, Childhood (Primary); Hodgkin's Lymphoma, Adult; Hodgkin's Lymphoma, Childhood; Hodgkin's Lymphoma During Pregnancy; Hypopharyngeal Cancer; Hypothalamic and Visual Pathway Glioma, Childhood; Intraocular Melanoma; Islet Cell Carcinoma (Endocrine Pancreas); Kaposi's Sarcoma; Kidney Cancer; Laryngeal Cancer; Laryngeal Cancer, Childhood; Leukemia, Acute Lymphoblastic, Adult; Leukemia, Acute Lymphoblastic, Childhood; Leukemia, Acute Myeloid, Adult; Leukemia, Acute Myeloid, Childhood; Leukemia, Chronic Lymphocytic; Leukemia, Chronic Myelogenous; Leukemia, Hairy Cell; Lip and Oral Cavity Cancer; Liver Cancer, Adult (Primary); Liver Cancer, Childhood (Primary); Lung Cancer, Non-Small Cell; Lung Cancer, Small Cell; Lymphoblastic Leukemia, Adult Acute; Lymphoblastic Leukemia, Childhood Acute; Lymphocytic Leukemia, Chronic; Lymphoma, AIDS — Related; Lymphoma, Central Nervous System (Primary); Lymphoma, Cutaneous T-Cell; Lymphoma, Hodgkin's, Adult; Lymphoma, Hodgkin's; Childhood; Lymphoma, Hodgkin's During Pregnancy; Lymphoma, Non-Hodgkin's, Adult; Lymphoma, Non-Hodgkin's, Childhood; Lymphoma, Non-Hodgkin's During Pregnancy; Lymphoma, Primary Central Nervous System; Macroglobulinemia, Waldenstrom's; Male Breast Cancer; Malignant Mesothelioma, Adult; Malignant Mesothelioma, Childhood; Malignant Thymoma; Medulloblastoma, Childhood; Melanoma; Melanoma, Intraocular; Merkel Cell Carcinoma; Mesothelioma, Malignant; Metastatic Squamous Neck Cancer with Occult Primary; Multiple Endocrine Neoplasia Syndrome, Childhood; Multiple Myeloma / Plasma Cell Neoplasm; Mycosis Fungoides; Myelodysplasia Syndromes; Myelogenous Leukemia, Chronic; Myeloid Leukemia, Childhood Acute; Myeloma, Multiple; Myeloproliferative Disorders, Chronic; Nasal Cavity and Paranasal Sinus Cancer; Nasopharyngeal Cancer; Nasopharyngeal Cancer, Childhood; Neuroblastoma; Non-Hodgkin's Lymphoma, Adult; Non-Hodgkin's Lymphoma, Childhood; Non-Hodgkin's Lymphoma During Pregnancy; Non-Small Cell Lung Cancer; Oral Cancer, Childhood; Oral Cavity and Lip Cancer; Oropharyngeal Cancer; Osteosarcoma / Malignant Fibrous Histiocytoma of Bone; Ovarian Cancer, Childhood; Ovarian Epithelial Cancer; Ovarian Germ Cell Tumor; Ovarian Low Malignant Potential Tumor; Pancreatic Cancer; Pancreatic Cancer, Childhood', Pancreatic Cancer, Islet Cell; Paranasal Sinus and Nasal Cavity Cancer; Parathyroid Cancer; Penile Cancer; Pheochromocytoma; Pineal and Supratentorial Primitive Neuroectodermal Tumors, Childhood; Pituitary Tumor; Plasma Cell Neoplasm / Multiple Myeloma; Pleuropulmonary Blastoma; Pregnancy and Breast Cancer; Pregnancy and Hodgkin's Lymphoma; Pregnancy and Non-Hodgkin's Lymphoma; Primary Central Nervous System Lymphoma; Primary Liver Cancer, Adult; Primary Liver Cancer, Childhood; Prostate Cancer; Rectal Cancer; Renal Cell (Kidney) Cancer; Renal Cell Cancer, Childhood; Renal Pelvis and Ureter, Transitional Cell Cancer; Retinoblastoma; Rhabdomyosarcoma, Childhood; Salivary Gland Cancer; Salivary Gland'Cancer, Childhood; Sarcoma, Ewing's Family of Tumors; Sarcoma, Kaposi's; Sarcoma (Osteosarcoma) Malignant Fibrous Histiocytoma of Bone; Sarcoma, Rhabdomyosarcoma, Childhood; Sarcoma, Soft Tissue, Adult; Sarcoma, Soft Tissue, Childhood; Sezary Syndrome; Skin Cancer; Skin Cancer, Childhood; Skin Cancer (Melanoma); Skin Carcinoma, Merkel Cell; Small Cell Lung Cancer; Small Intestine Cancer; Soft Tissue Sarcoma, Adult; Soft Tissue Sarcoma, Childhood; Squamous Neck Cancer with Occult Primary, Metastatic; Stomach (Gastric) Cancer; Stomach (Gastric) Cancer, Childhood; Supratentorial Primitive Neuroectodermal Tumors, Childhood; T-Cell Lymphoma, Cutaneous; Testicular Cancer; Thymoma, Childhood; Thymoma, Malignant; Thyroid Cancer; Thyroid Cancer, Childhood; Transitional Cell Cancer of the Renal Pelvis and Ureter; Trophoblastic Tumor, Gestational; Unknown Primary Site, Cancer of, Childhood; Unusual Cancers of Childhood; Ureter and Renal Pelvis, Transitional Cell Cancer; Urethral Cancer; Uterine Sarcoma; Vaginal Cancer; Visual Pathway and Hypothalamic Glioma, Childhood; Vulvar Cancer; Waldenstrom's Macro globulinemia; and Wilms' Tumor.

[0056] “Cancer cell” or “tumor cell”, and grammatical equivalents refer to the total population of cells derived from a tumor or a pre-cancerous lesion, including both non tumorigenic cells, which comprise the bulk of the tumor population, and tumorigenic stem cells (cancer stem cells).

[0057] In some embodiments, the 22-[18F]FDHA may be used as a PET tracer for one or more neurodegenerative disorder. These may be one of acute disseminated encephalomyelitis, amyotrophic lateral sclerosis (ALS), retinitis pigmentosa, mild cognitive impairment, senile dementia, progressive supranuclear palsy, subcortical dementias, Wilson disease, multiple infarct disease, arteriosclerotic dementia, AIDS associated dementia, cerebellar degeneration, spinocerebellar degeneration syndromes, Friedreich's ataxia, ataxia telangiectasia, epilepsy- related brain damage, spinal cord injury, restless legs syndrome, striatonigral degeneration, cerebral vasculitis, mitochondrial encephalomyopathies, neuronal ceroid lipofuscinosis, spinal muscular atrophies, lysosomal storage disorders with central nervous system involvement, leukodystrophies, urea cycle defect disorders, hepatic encephalopathies, renal encephalopathies, metabolic encephalopathies, porphyria, bacterial meningitis, viral meningitis, meningoencephalitis, prion diseases, poisonings with neurotoxic compounds, Guillain Barre syndrome, chronic inflammatory neuropathies, polymyositis, dermatomyositis, or radiation-induced brain damage. Other neurodegenerative disorders include Alpha- mannosidosis, Cystinosis, Danon disease, Fabry disease, Farber disease, Fucosidosis, Galactosialidosis, Gaucher disease (Type I, Type II, Type III), GM1 gangliosidosis (infantile, juvenile and adult), I-Cell disease (Mucolipidosis II), Infantile Free Sialic Acid Storage Disease, Juvenile Hexosaminidase A Deficiency, Krabbe disease (Infantile and late onset), Lysosomal acid lipase deficiency (early and late), Metachromatic Leukodystrophy, PseudoHurler polydystrophy (Mucolipidosis IIIA), MPSI (Hurler Syndrome), MPS II (Hunter syndrome), Sanfilippo syndrome Type A (MPS III A), Sanfilippo syndrome Type B (MPS III B), Sanfilippo syndrome Type C (MPS III C), Sanfilippo syndrome Type D (MPS III D), Morquio Type A (MPS IVA), Morquio Type B (MPS IVB), MPS IX (Hyaluronidase Deficiency), MPS VI (Maroteaux-Lamy), MPS VII (Sly Syndrome), Mucolipidosis I (Sialidosis), Mucolipidosis IIIC, Mucolipidosis type IV, Multiple sulfatase deficiency, Niemann-Pick Disease, Type A, Niemann-Pick Disease, Type B, Niemann-Pick Disease, Type C, Neuronal Ceroid Lipofuscinoses, Pompe disease, Pycnodysostosis, Sandhoff disease (infantile, juvenile and adult), Schindler disease, Salla disease (Sialic Acid Storage Disease), Tay-Sachs disease, Wolman disease, chronic traumatic encephalopathy, Alzheimer's disease (AD), Parkinson disease (PD), Huntington disease (HD), Frontotemporal dementia (FTD-3 subtype), Charcot-Marie Tooth disease type 2B, Neuronal ceroid lipofuscinoses / Batten disease (NCL), Creutzfeldt- Jakob disease, Autosomal dominant Spastin hereditary spastic paraplegia (ADHSP), Chediak-Higashi syndrome (CHS), and Inclusion body myositis (IBM).

[0058] The compounds can be administered in various modes, e.g., orally, topically, or by injection. In some embodiments, the compounds (e.g., 22-[18F]FDHA) are administrated by injection or intravenously.

[0059] The terms “administration of’ and “administering a” compound should be understood to mean providing a compound of the disclosure or pharmaceutical composition to a subject. An exemplary administration route is intravenous administration. In general, administration routes include but are not limited to intracutaneous, subcutaneous, intravenous, intraperitoneal, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, transdermal, transtracheal, sub cuticular, intraarticulare, subcapsular, subarachnoid, intraspinal and intrasternal, oral, sublingual buccal, rectal, vaginal, nasal ocular administrations, as well infusion, inhalation, and nebulization. The phrases “parenteral administration” and “administered parenterally” as used herein means modes of administration other than enteral and topical administration. The compositions of the present invention may be processed in a number of ways depending on the anticipated application and appropriate delivery or administration of the pharmaceutical composition. For example, the compounds or compositions may be formulated for injection or intravenous administration.

[0060] In some embodiments, an effective amount of the 22-[18F]FDHA administered to a subject may be about 0.1 MBq, about 0.5 MBq, about 1 MBq, about 1.5 MBq, about 2 MBq, about 2.5 MBq, about 3 MBq, about 3.5 MBq, about 4 MBq, about 4.5 MBq, about 5 MBq, about 5.5 MBq, about 6 MBq, about 6.5 MBq, about 7 MBq, about 7.5 MBq, about 8 MBq, about 8.5 MBq, about 9 MBq, about 9.5 MBq, about 10 MBq, about 10.5 MBq, about 11 MBq, about 11.5 MBq, about 12 MBq, about 12.5 MBq, about 13 MBq, about 13.5 MBq, about 14 MBq, about 14.5 MBq, about 15 MBq, about 15.5 MBq, about 16 MBq, about 16.5 MBq, about 17 MBq, about 17.5 MBq, about 18 MBq, about 18.5 MBq, about 19 MBq, about 19.5 MBq, or about 20 MBq. In other embodiments, an effective amount of the 22-[18F]FDHA administered to a subject may be about 20 MBq to about 50 MBq.

[0061] In some embodiments, a composition for use as a PET tracer may comprise one or more buffers and / or one or more stability agents. For example, a buffer or one or more buffering agent may include phosphate buffered saline, saline, saline-sodium citrate, tris-acetate-EDTA, tris-borate-EDTA, tris-EDTA, 2-[4-(2-hydroxyethyl)piperazin-l-yl]ethane sulfonic acid (HEPES) phosphoric acid, citric acid, acetic acid, histidine, lactic acid, tromethamine, gluconic acid, aspartic acid, tartaric acid, succinic acid, malic acid, fumaric acid, sodium acetate, sodium phosphate, sodium citrate, potassium acetate, potassium phosphate, potassium citrate, and the like. The concentration of a buffer to be added to the composition can be between about 1 and 100 mM, such as between about 5 mM to about 50 mM. Suitable stabilizing agents include bovine serum albumin that may be present in a concentration of about 1 mg / ml, about 5 mg / ml, about 10 mg / ml, about 15 mg / ml, about 20 mg / ml, about 25 mg / ml, about 30 mg / ml, about 35 mg / ml, about 40 mg / ml, about 45 mg / ml, or about 50 mg / ml. In other embodiments, the stabilizing agent e.g., BSA) is present in an amount of between 1 mg / ml and about 150 mg / ml. In some embodiments, a composition may comprise or consist essentially of the compound 22- [18F]FDHA, about 5 mM to about 25 mM HEPES, and about 25 mg / ml to about 75mg / ml BSA.

[0062] Pharmaceutical Formulations.

[0063] The compounds described herein can be used to prepare therapeutic pharmaceutical compositions, for example, by combining the compounds with a pharmaceutically acceptable diluent, excipient, or carrier. The compounds may be added to a carrier in the form of a salt or solvate. For example, in cases where compounds are sufficiently basic or acidic to form stable nontoxic acid or base salts, administration of the compounds as salts may be appropriate. Examples of pharmaceutically acceptable salts are organic acid addition salts formed with acids that form a physiologically acceptable anion, for example, tosylate, methanesulfonate, acetate, citrate, malonate, tartrate, succinate, benzoate, ascorbate, a-ketoglutarate, and P- glycerophosphate. Suitable inorganic salts may also be formed, including hydrochloride, halide, sulfate, nitrate, bicarbonate, and carbonate salts.

[0064] Pharmaceutically acceptable salts may be obtained using standard procedures well known in the art, for example by reacting a sufficiently basic compound such as an amine with a suitable acid to provide a physiologically acceptable ionic compound. Alkali metal (for example, sodium, potassium or lithium) or alkaline earth metal (for example, calcium) salts of carboxylic acids can also be prepared by analogous methods.

[0065] The compounds of the formulas described herein can be formulated as pharmaceutical compositions and administered to a mammalian host, such as a human patient, in a variety of forms. The forms can be specifically adapted to a chosen route of administration, e.g., oral or parenteral administration, by intravenous, intramuscular, topical or subcutaneous routes.

[0066] The active compound may be administered intravenously or intraperitoneally by infusion or injection. Solutions of the active compound or its salts can be prepared in water, optionally mixed with a nontoxic surfactant. Dispersions can be prepared in glycerol, liquid polyethylene glycols, triacetin, or mixtures thereof, or in a pharmaceutically acceptable oil. Under ordinary conditions of storage and use, preparations may contain a preservative to prevent the growth of microorganisms.

[0067] Pharmaceutical dosage forms suitable for injection or infusion can include sterile aqueous solutions, dispersions, or sterile powders comprising the active ingredient adapted for the extemporaneous preparation of sterile injectable or infusible solutions or dispersions, optionally encapsulated in liposomes. The ultimate dosage form should be sterile, fluid and stable under the conditions of manufacture and storage. The liquid carrier or vehicle can be a solvent or liquid dispersion medium comprising, for example, water, ethanol, a polyol (for example, glycerol, propylene glycol, liquid polyethylene glycols, and the like), vegetable oils, nontoxic glyceryl esters, and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the formation of liposomes, by the maintenance of the required particle size in the case of dispersions, or by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and / or antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, buffers, or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by agents delaying absorption, for example, aluminum monostearate and / or gelatin.

[0068] Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in the appropriate solvent with various other ingredients enumerated above, as required, optionally followed by filter sterilization. In the case of sterile powders for the preparation of sterile injectable solutions, methods of preparation can include vacuum drying and freeze-drying techniques, which yield a powder of the active ingredient plus any additional desired ingredient present in the solution.

[0069] Useful dosages of the compounds described herein can be determined by comparing their in vitro activity, and in vivo activity in animal models. Methods for the extrapolation of effective dosages in mice, and other animals, to humans are known to the art; for example, see U.S. Patent No. 4,938,949 to Borch et al. The amount of a compound, or an active salt or derivative thereof, required for use in treatment will vary not only with the particular compound or salt selected but also with the route of administration, the nature of the condition being treated, and the age and condition of the patient, and will be ultimately at the discretion of an attendant physician or clinician.

[0070] In general, however, a suitable dose will be in the range of from about 0.5 to about 100 mg / kg, e.g., from about 10 to about 75 mg / kg of body weight per day, such as 3 to about 50 mg per kilogram body weight of the recipient per day, preferably in the range of 6 to 90 mg / kg / day, most preferably in the range of 15 to 60 mg / kg / day.

[0071] The compound is conveniently formulated in unit dosage form; for example, containing 5 to 1000 mg, conveniently 10 to 750 mg, most conveniently, 50 to 500 mg of active ingredient per unit dosage form. In one embodiment, the invention provides a composition comprising a compound of the invention formulated in such a unit dosage form.

[0072] The compound can be conveniently administered in a unit dosage form, for example, containing 5 to 1000 mg / m2, conveniently 10 to 750 mg / m2, most conveniently, 50 to 500 mg / m2of active ingredient per unit dosage form. The desired dose may conveniently be presented in a single dose or as divided doses administered at appropriate intervals, for example, as two, three, four or more sub-doses per day. The sub-dose itself may be further divided, e.g., into a number of discrete loosely spaced administrations.

[0073] The desired dose may conveniently be presented in a single dose or as divided doses administered at appropriate intervals, for example, as two, three, four or more sub-doses per day. The sub-dose itself may be further divided, e.g., into a number of discrete loosely spaced administrations.

[0074] Results and Discussion.

[0075] Synthesis of 22-Fluordocosahexaenoic Acid (FDHA).

[0076] For in vitro study of the DHA analogue, we first aimed to synthesize the nonradiolabeled fatty acid, 22-FDHA. We accomplished this starting from the methyl ester of a 22-hydroxylated DHA 10 (Scheme 1), which was prepared by closely following a previously reported method for the modular synthesis of co-hydroxylated PUFAs. Briefly, copper- catalyzed coupling of methyl 4-pentynoate and 4-chlorobutyn-l-ol to diyne 1, followed by bromination to 2, then another copper-catalyzed alkyne extension into 3, and a final bromination step afforded triyne 4. In parallel, the copper-catalyzed coupling of propargyl alcohol and 3 -bromo- 1 -(trimethyl silyl)- 1 -propyne to 5, followed by tosylation to 6, then another copper-catalyzed alkyne extension into 7, and a final desilylation step afforded triyne 8. After the copper-catalyzed coupling of 4 and 8, the hexayne 9 was obtained in modest yield.

[0077] Due to the instability of intermediate 9, a controlled partial hydrogenation was performed immediately using Lindlar’s catalyst in the presence of 2-methyl-2-butene under H2 gas at ambient pressure to obtain the all-cis derivative 10. The subsequent tosylation step using -toluenesulfonyl chloride under basic anhydrous conditions was performed using crude 10 containing undesired over-hydrogenated and incomplete partial-hydrogenated byproducts to obtain crude 11. However, we successfully isolated the desired tosylate precursor 11 using semipreparative reversed-phase HPLC.

[0078] Next, the purified tosylate 11 was subjected to nucleophilic fluorination (Scheme 2). The reaction was performed according to our previous method for the synthesis of fluorinated AA (20-FAA) using TBAF. While the desired fluorinated ester 13 was successfully obtained, only a modest yield (29%) was achieved due to the formation of the 1,2-elimination product 12 (Scheme 2) caused by the intrinsic basicity of TBAF. Finally, the desired compound, 22- FDHA, was obtained by hydrolyzing ester 13 using LiOH.

[0079] Scheme 1. Synthesis of fluorination precursor 11.

[0080] (a) Cui, Nal, CS2CO3, DMF, r.t., 83% yield; (b) CBr4, PPh3, DCM, 0 °C, 80% yield; (c) propargyl alcohol, Cui, Nal, CS2CO3, DMF, r.t., 65% yield; (d) CBr4, PPh3, DCM, 0 °C, 61% yield; (e) Cui, Nal, CS2CO3, DMF, r.t., 94% yield; (f) TS2O, pyridine, DCM, r.t., 60% yield; (g) 3-butyn-l-ol, Cui, Nal, CS2CO3, DMF, r.t., 48% yield; (h) TBAF, acetic acid, THF, 0 °C, 84% yield; (i) Cui, Nal, CS2CO3, DMF, r.t., 41% yield; (j) H2 (balloon), Lindlar’s catalyst, 4:2:2: 1 (v / v / v / v) 2-methyl-butene / EtOAc / methanol / pyridine; (k) TsCl, Et3N, DMAP, DCM, 0 °C to r.t., 13% yield over 2 steps. Scheme 2. Synthesis of the DHA analogues FDHA and [18F]FDHA.

[0081] (a) TBAF, THF, 0 °C to r.t., 29% yield; (b) LiOH, THF / water, r.t., 94% yield; (c) [18F]TBAF, MeCN, 85 °C, 20 min; (d) KOH, water / MeCN, 85 °C, 15 min.

[0082] Radiosynthesis of [18F]FDHA.

[0083] 22-[18F]FDHA was successfully prepared through radiofluorination (Scheme 2) with the tosylate 11 by following the procedure for the synthesis of the18F -fluorinated arachidonic acid, 20-[18F]FAA. Briefly, a solution of the tosylate 11 in anhydrous MeCN was treated with a solution of [18F]TBAF in MeCN at 85 °C for 20 min, and then the resulting mixture containing the fluorinated ester [18F] 13 was treated with 2 M KOH at 85 °C to hydrolyze the intermediate into the free fatty acid. The probe was isolated from the reaction mixture using a single pass semipreparative reverse-phase HPLC, affording 22-[18F]FDHA in high radiochemical purity (>99%), with 1.06-6.37% decay-corrected radiochemical yields. The total synthesis time was about 180 min, including the HPLC purification time. Specific activity was 18.0-117.4 GBq / pmol (0.486-3.172 Ci / pmol), which is similar to that previously reported for 20-[18F]FAA.

[0084] LogP and In Vitro Stability

[0085] The partition coefficient (logP) between 1-octanol and PBS of 22-[18F]FDHA was 0.76 ± 0.01. The stability in mouse serum at 37 °C at 1, 2, 4, 6, and 8 h and the percent purities were 98.92 ± 0.01, 98.86 ± 0.01, 98.77 ± 0.03, 98.61 ± 0.29, and 98.33 ± 0.45%, respectively. After analyzing the stability of the probe in the buffer formulation developed for use in animal imaging (5 mM HEPES in saline containing 50 mg / mL BSA), the percent purity of the probe after 5 h was 95.83 ± 0.23%. Bioequivalence Assay

[0086] We evaluated the ability of 22-FDHA to mimic DHA by probing whether fluorination at the co-position on DHA affects the biochemical role of the natural fatty acid. DHA is a precursor to various specialized pro-resolving mediators and is also anti-inflammatory; it is known to attenuate inflammatory responses of BV-2 microglial cells to LPS. Co-treatment of BV-2 cells with LPS and 6 pM DHA did not show significantly reduced COX-2 expression compared to the LPS-only controls (Figure 2). However, a significant reduction was observed for both DHA and 22-FDHA at 15 pM with no significant difference in COX-2 induction in LPS-treated microglia cotreated with either compound (p > 0.05). Therefore, it is likely that fluorination at the co-position on DHA can preserve the ability of DHA to be recognized, transported, and metabolized in the brain, making 22-[18F]FDHA a suitable probe for monitoring DHA uptake in vivo.

[0087] In Vivo Uptake and Stability.

[0088] To assess the nature of the radioactive species contributing to the activity in tissues, as a measure of the in vivo stability of 22-[18F]FDHA, Folch extraction was performed. In this assay, [18F]fhioride ions and low molecular-weight metabolites were extracted into the hydrophilic phase and 22-[18F]FDHA and lipid metabolites were extracted into the hydrophobic phase. Radioactivity partitioned into organic, aqueous, and interphase (pellet) fractions of blood and brain tissue obtained from mice injected with 22-[18F]FDHA was detected as shown in Table 1.

[0089] Table 1. Radioactivity in tissues and fractions obtained from Folch extraction in tissues of mice (n = 3) 30 min after i.v. injection with [18F]FDHA.

[0090] Blood was taken 30 min p.i. of tracer. Mean ± S.D., n = 3

[0091] A significant amount of radioactivity was detected in the hydrophilic phase obtained from both blood and brain samples. As observed on radio HPLC, two radioactive compounds were present in the urine collected 30 min p.i. The peak at 3.1 min, which constitutes approximately 60% of the radioactivity, corresponds to [18F]fhioride ions. The remaining 40% of the radioactivity was detected from the peak that appeared at 6.8 min, which likely corresponds to a metabolite that is significantly more hydrophilic than 22-[18F]FDHA. No peak corresponding to the parent probe 22-[18F]FDHA was present (data not shown) as we observed with 20-[18F]FAA, indicating that the probe had been cleared as hydrophilic metabolites.

[0092] Ex Vivo Analysis

[0093] We have confirmed through autoradiography (Figure 3) that activity can be detected in the brain tissue of a mouse 30 min p.i. with 22-[18F]FDHA. The uptake and distribution are distinct from those of the brain of another mouse injected with a similar amount of sodium [18F]fluoride in the same formulation. This suggests that the tracer is taken up and accreted into the brain within 30 min.

[0094] In addition, we conducted an in vivo uptake study by administering nonradioactive 22- FDHA via intravenous injection into C57BL / 6 mice. After tissue extraction from plasma, brain, and liver, we employed multiple reaction monitoring via LC-MS to successfully detect free 22- FDHA in these tissues. Following a single administration of 22-FDHA, the compound was detectable in both the plasma and brain after 30 min. Notably, a dose-dependent increase was observed in both compartments (Table 2), with plasma levels reaching 10 ng / mL and brain levels reaching 1 ng / g for the mouse receiving a 1 pg dose. Furthermore, the administration of a higher dosage of 22-FDHA resulted in a larger fraction being taken up by the brain, as evidenced by the elevated brain / plasma ratio (10.5% vs 5.6%) for the 1 pg dose compared to the 0.5 pg dose.

[0095] Table 2. 22-FDHA concentrations in tissues at 30 min after i.v. injection.

[0096] 22-FDHA Concentration 22-FDHA Tissue / Plasma Ratio

[0097] Plasma Brain Liver

[0098] , , , , , , , , Brain / Plasma Liver / Plasma

[0099] (ng / mL) (ng / g) (ng / g)

[0100] Mouse 1 ,410.16 1.07 0.28 10.5% 2.8%

[0101] (1 Pg)

[0102] Mouse 2 , , 3.60 0.20 0.16 5.6% 4.4%

[0103] (0.5 pg)

[0104] PET-MR Imaging of 22-[18F]FDHA Brain Uptake In Vivo

[0105] Like 20-[18F]FAA, ROI analysis of the dynamic PET images using 22-[18F]FDHA showed that the probe was mostly taken up in nonbrain regions such as the liver and kidneys, with only about 0.5% of the total probe [ca. 1.4% injected dose per gram (%ID / g), Figure 4A] entering the mouse brain after 30 min. In the brain ROI, we observed for 22-[18F]FDHA a pattern of a sharp increase followed by a slow, steady increase in radioactivity (Figure 4B). In our PET imaging experiments using normal nude mice, marginal increases in radioactivity were found in brain ROI at 60 min p.i. (ca. 1.6%ID / g, Figure 6).

[0106] Effect of Arecoline. Arecoline, a muscarinic acetylcholine receptor agonist, has been shown to increase d* for DHA (measured by autoradiography) in rodents through the activation of calcium-independent phospholipase A2 (iPLA?). DHA is preferentially cleaved from the 2-position of phospholipids by iPLA, and the activation of the enzyme is known to affect DHA cycling in the brain. Thus, we investigated the effect of arecoline administration on the K* estimate by 22-[18F]FDHA dynamic PET. Mice were first pretreated subcutaneously with methylatropine (4 mg / kg), to reduce systemic effects without affecting K*, and then intravenously injected with a bolus of 22-[18F]FDHA. During PET acquisition, mice were then intravenously injected with saline or arecoline in saline (15 mg / kg) at 10 min p.i. of the probe. The %ID / g measured in the brain ROI at the last time point (28-30 min) was not significantly different between control and arecoline-treated mice (Figure 5A, B). However, there was an apparent increase in the rate of uptake (which could be revealed by K* estimation) into the brains of mice after iv injection of arecoline.

[0107] Through kinetic modeling of the dynamic PET data, we found a significantly higher mean DHA T* estimate in mice injected with arecoline (Figure 5C and Table 3, 160% increase of mean T* value; p = 0.0016 by Student’s t test); increases ranging between 68 and 120% for rodents subcutaneously injected with arecoline were previously reported (Table 3). K* estimates for the control mice studied were much lower than those determined in unanesthetized mice by autoradiography using 1-[14C]DHA but comparable to values found for rats and to values estimated in humans by PET (Table 3). The lower values are attributed to the fact that we did not account for the metabolite fraction in the whole blood samples used to correct the IDIFs, and radioactivity in whole blood does not always equate radioactivity in plasma, which is a factor in the “scaling” of the T* estimates. In addition, the uptake of fatty acids was monitored in anesthetized mice.

[0108] Table 3. Estimates of the brain incorporation coefficient (K*).

[0109] K* (xio-3min'1)

[0110] Methods DHA Probes Models _A..

[0111] Control Arecoline

[0112] 4,5-[3H]DHA Rats 1.9 3.3

[0113] Autoradiography i-[i4C]DHA Mice 79.9 - 258.4 175.5 - 494.9

[0114] 1-[14C]DHA Rats 13.9 23.3 l-[nC]DHA Humans 4.0 rL I’lv"' 22-[18F]FDHA Mice 4.69 ± 1.09a12.20 ± 0.60aaMean ± S.D. (n =3) Accordingly, we have successfully developed, synthesized, and characterized a PET probe, 22-[18F]FDHA, designed for visualizing DHA uptake in the brains of mice. In our cellular studies using the non-radiolab eled 22-FDHA, we observed that fluorination did not impact the biological activity of DHA in vitro. Following a protocol that we have previously employed for PET-MR imaging with 20-[18F]FAA in mice, we were able to accurately estimate K* through dynamic PET-MR imaging with 22-[18F]FDHA. Moreover, we observed the anticipated increase in the K* value in mice treated with arecoline

[0115] Material and Methods.

[0116] All reagents and solvents were obtained from various commercial sources and used without further purification unless otherwise noted. All reactions were performed under nitrogen gas unless otherwise mentioned. Thin-layer chromatography (TLC) was performed on precoated silica gel 60 A plates with UV 254 indicator and visualized with either UV light or a KMnCh stain. Automated flash chromatography was performed using a Teledyne ISCO Combi / ’ / a.s / ? Rf 200 automatic chromatography system with RedhS'c prepacked gel cartridges. Nuclear magnetic resonance (NMR) spectra were recorded on a 400 MHz Oxford NMR AS400, a 400 MHz Varian Mercury, or a 500 MHz Varian VNMRS NMR spectrometer. Proton and13C NMR chemical shifts are reported in ppm (5) downfield from TMS using internal TMS or residual protonated solvent as reference (CHCh: 7.26 ppm for 'H and 77.16 ppm for13C). Fluorine- 19 NMR chemical shifts are reported in ppm downfield from CFCh using the deuterium lock channel as a reference. NMR data are reported as follows: chemical shift, multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, p = pentet, dt = triplet of doublets, dq = quartet of doublets, tt = triplet of triplets, m = multiplet), coupling constant (Jin Hz), and integration. Low-resolution mass spectra were obtained on an MSQ Plus single quadrupole mass spectrometer in positive mode with an electrospray ionization (ESI) ion source. High- resolution mass spectra were obtained on a Thermo Fisher Q-Exactive Orbitrap system in positive mode with an ESI ion source.

[0117] Synthetic Procedures

[0118] Synthesis of 22-FDHA. General synthetic methods for the synthesis of a common intermediate 11 (the tosylate precursor for fluorination, Scheme 1) and synthetic methods for 22-FDHA (Scheme 2) are fully described herein.

[0119] Radiosynthesis of 22-[I8F]FDHA. 22-[18F]FDHA was synthesized from the labeling precursor 11 (Scheme 1) and [18F]fluoride ion generated by the18O(p,n)18F nuclear reaction in a GE PETtrace 800 cyclotron, adapting a procedure on the synthesis of 20-[18F]FAA. [18F]fluoride ion in [18O]water was transferred through a preconditioned (10 mL ethanol followed by 10 mL water) anion exchange cartridge (QMA). The retained [18F]fluoride was eluted into a V-vial with 0.075 M aq. tetrabutylammonium bicarbonate solution (0.4 mL). Anhydrous MeCN (1 mL) was added to the V-vial and the resulting solution of [18F]TBAF was azeotropically dried at 100 °C with nitrogen flow by subsequent addition of 1 mL portions of anhydrous MeCN (3*).

[0120] A solution of 11 (ca. 1.5 mg) in anhydrous MeCN (0.5 mL) was added to the residue in the V-vial, and the reaction mixture was shaken and heated at 85 °C for 20 min. After cooling for 5 min, 2 M aq. KOH (0.5 mL) was added to the vial. The mixture was shaken and heated again at 85 °C for 15 min. After cooling, the mixture was then acidified with 1 M aq. oxalic acid (0.75 mL) and the crude reaction mixture was purified by semipreparative reversed-phase high-performance liquid chromatography (HPLC, Method C). After evaporating the fraction containing the product under reduced pressure, 22-[18F]FDHA was obtained. For use in the in vivo experiments, the 22-[18F]FDHA probe was formulated in 0.75 mL of freshly prepared 5 mM HEPES buffered saline containing 50 mg / mL fatty acid-free bovine serum albumin (BSA) by sonication.

[0121] Partition Coefficient. The 1-octanol-phosphate-buffered saline (PBS) partition coefficient was measured at room temperature (RT) and the value was designated as LogP. A solution of 22-[18F]FDHA (ca. 370 KBq) in 10 pL of PBS (pH = 7.4) was added to a microcentrifuge tube containing 500 pL of PBS (pH 7.4) and 500 pL of 1-octanol. The mixture was vortexed for 3 min and then centrifuged (12,000 x g) for 10 min. The PBS and 1-octanol layers (150 pL of each layer) were pipetted into separate gamma counter test tubes. The radioactivity was determined using a PerkinElmer 2480 WIZARD automatic gamma counter (PerkinElmer Inc., Waltham, MA). The partition coefficients of 1 -octanol -to-PBS were calculated as LogP = log ([organic-phase cpm] / [aqueous-phase cpm]). Measurements were carried out in quintuplicate.

[0122] Bioequivalence Assay

[0123] Cell Culture and Lysate Preparation. The immortalized microglial cell line BV-2 was grown and maintained in Dulbecco’s modified Eagle’s medium (DMEM, Corning, 10013CV) supplemented with 10% fetal bovine serum and 1% antibiotic-antimycotic. Seeded BV-2 cells (5 x 104) were stimulated with 200 ng lipopolysaccharide (LPS), then treated with DHA or 22- FDHA (6 or 15 pM) for 6 hr. Cells were then lysed in 60 pL of 1xRIPA buffer (Cell Signaling Technology, CST 9806) containing protease inhibitor cocktail (Sigma, P8340), phosphatase inhibitor cocktail (Sigma, P0044), and 4* Sample Buffer (Bio-Rad, 1610747). The cell lysates were then boiled for 5 min and collected for Western blot.

[0124] Cyclooxygenase-2 (COX-2) Western Blot. Proteins from the cell lysates were separated using 4-15% Criterion™ TGX™ Precast gels (Bio-Rad, 5671085), transferred onto a nitrocellulose membrane (Bio-Rad, 1704270) and blocked with 5% fat-free milk (Bio-Rad, 1706404) for 2 hr at RT. The membranes were then incubated with the primary antibody diluted in 5% BSA at 4 °C overnight. The membranes were then washed (3 x 5 min) with Tris-buffered saline with 0.1% Tween-20 (TBS-T) and incubated with horseradish peroxidase (HRP)- conjugated secondary antibody for 30 min at room temperature. After washing with TBS-T (3 x 5 min), the membranes were imaged using a Fujifilm LAS-4000 imager system, and protein was detected using chemiluminescent HRP substrate (Millipore, WBKLS0500). GelQuant.NET software was used for densitometric quantification. The following antibodies and dilution factors were used: COX-2 rabbit antibody (CST, 12282; 1 : 1000), actin rabbit antibody (CST, 4970; 1 : 1000), and HRP-linked anti-rabbit IgG (CST, 7074; 1 :2000).

[0125] 22-[18F]FDHA / n Vitro Stability

[0126] Formulation Stability. A solution of ca. 37 MBq of 22-[18F]FDHA in 0.25 mL of HEPES buffered saline (5 mM) containing fatty-acid-free BSA (50 mg / mL) was incubated at room temperature for 5 h. The sample was diluted with water and analyzed by HPLC (Method A) to determine the stability of the probe. Measurements were performed in duplicate.

[0127] Stability in Mouse Serum. Solutions of ca. 9.25-74 MBq of 22-[18F]FDHA in mouse serum (250 pL) were shaken at 37 °C for 1, 2, 4, 6, and 8 h, respectively. After adding trifluoroacetic acid (TFA, 10 pL), each mixture was vortexed for 3 min and centrifuged for 10 min (12,000 x g). The resulting supernatants were analyzed by HPLC (Method A). Measurements were performed in duplicate.

[0128] Animals. All animal studies were approved by the Institutional Animal Care and Use Committee at the University of Southern California. The female C57BL / 6J mice (6-month-old, weighing 21-30 g) used in the study were bred in the USC animal facility. Animals were housed with standardized 12 h light and dark cycles and had access to food and water ad libitum. Vivarium temperature was maintained between 22 °C and 24 °C and humidity was maintained between 50 and 60%.

[0129] 22-[18F]FDHA In Vivo Uptake and Stability

[0130] Probe Injection and Tissue Extraction. A modified procedure was performed on female C57BL / 6J mice (n = 3). Mice anesthetized with isoflurane (1.5-2%) were injected with the 22- [18F]FDHA (10.8 ± 1.4 MBq) in formulation through the tail vein. Blood samples were collected from the submandibular vein under anesthesia at 5 and 10 min post-injection (p.i.) and transferred to EDTA-coated microcentrifuge tubes. At 30 min p.i., the mouse was dissected under anesthesia, and urine was withdrawn from the bladder. Then, blood was obtained from the inferior vena cava and the mouse was euthanized by decapitation. The brain was extracted from the skull within 1 min and kept on ice until further processing. Urine was filtered through a 0.22 pm polyvinylidene difluoride filter and analyzed by HPLC (Method A).

[0131] Folch Extraction. A literature procedure according to DeGrado el al., J. NucL Med. 2010, 51 (8), 1310 was performed on plasma (obtained by centrifugation of blood sampled at 30 min p.i.) and brain tissue samples. A 2: 1 (v / v) mixture of chloroform / methanol (7 mL) was added at 0 °C to the tissue, which was then homogenized using a handheld homogenizer. The mixtures were sonicated for 20 s, and 40% aq. urea (1.75 mL) and 5% aq. H2SO4 (1.75 mL) were added. After additional sonication for 20 s, the mixtures were centrifugated (1,800 x g) for 10 min. Organic, aqueous, and interphase (pellet) fractions were isolated and radioactivity was determined by a gamma counter.

[0132] Ex Vivo Autoradiography. A female mouse was injected with 37 MBq of 22- [18F]FDHA in formulation via a tail vein under isoflurane anesthesia (1.5-2%). Another mouse was injected with 41 MBq [18F]fluoride in the same formulation as a control. Mice were kept under maintenance anesthesia for 30 min p.i. and were then euthanized. Blood (ca. 0.7 mL) was obtained from the heart and transferred to EDTA-coated microcentrifuge tubes. Each mouse was then perfused with cold PBS and the brain was extracted from the skull. One hemisphere was frozen in cryostat fluid and 20 pm sagittal sections were then obtained in a cryostat. Frozen brain sections were exposed to a phosphor storage screen (PerkinElmer Super Resolution phosphor screen) overnight. The screen was then read at 600 dpi (42 pm resolution) using a PerkinElmer Cyclone Plus Storage Phosphor Scanner with Opti Quant software.

[0133] 22-[18F]FDHA PET-MR Imaging and Effect of Arecoline

[0134] Tracer Injection, Arecoline Administration, and PET-MR Imaging. PET-MR image acquisition and reconstruction were performed on a 7T MRI scanner with an integrated PET scanner (MR solutions, Guildford, UK) as described previously except that the total scan time was reduced to 32 min (2 min PET baseline acquisition before tracer injection plus 30 min p.i.); list mode data were reconstructed and binned as follows: 4 frames of 30 s, 12 frames of 10 s, 6 frames of 30 s, 5 frames of 60 s, and 10 frames of 120 s.

[0135] Six female C57BL / 6J mice were divided into a control and a treatment group (n = 3 per group). Each mouse was first injected subcutaneously with saline (control group) or 4 mg / kg methylatropine in saline (treatment group), cannulated at the tail vein, and then placed in the scanner under isoflurane anesthesia (1.5-2%). After 5 min, PET acquisition was started and after another 2 min, each mouse was injected with 100 pL of formulated 22-[18F]FDHA (8.7 ± 1.4 MBq) through the installed cannula. After 10 min, mice were then injected with 100 pL of either saline (control group) or 15 mg / kg arecoline in saline (treatment group) through the same cannula, and image acquisition was continued for an additional 20 min. During PET acquisition, MR images (FSET2w, to define the brain and surrounding area, and respiratory -gated FLASH, to delineate the right ventricle of the heart) were obtained in tandem for defining regions of interest (ROIs). Images were processed using VivoQuant 4.0 (Invicro). PET and MR images were co-registered through the use of a PET / MR phantom. This phantom comprised a capillary tube filled with 10 pL of ~40 kBq of 22-[18F]FDHA in a solution, which was securely positioned on the mouse’s abdomen prior to imaging, serving as a reference point. Three- dimensional regions of interest (ROIs) were delineated through manual segmentation, with the MRI image serving as the anatomical reference. Subsequently, time-activity curves (TAC) were obtained for each mouse within the ROIs corresponding to the right ventricle and the brain.

[0136] Dynamic PET Kinetic Analysis. For K* estimation, an image-derived input function (IDIF), as a surrogate for the arterial input function, was determined for each mouse by correcting the TAC of the right ventricle ROI for myocardium spillover. Briefly, the shape of the TACs for each mouse was scaled to that of the venous input function (from measurements of whole blood radioactivity at 5, 10, and 30 min); this scaled IDIF was not corrected for radiotracer’s metabolites that do not cross the blood-brain barrier. Using the spillover-corrected IDIF and the brain TAC for each mouse, kinetic analysis was performed in MATLAB (version R2022b) using custom numerical fitting routines. First, the spillover-corrected IDIF was fitted into an 8-parameter model, which includes a linear interpolation of the data before the curve’s peak, and the sum of three decreasing exponentials after the peak. Then, kinetic microparameters (Ki, k , and U) for the irreversible two-tissue compartment model (Irr2TCM) as well as the blood volume fraction in the brain ROI (LB), were estimated from the brain TAC using the fitted IDIF. The value of the K* in the Irr2TCM, which indicates the net irreversible influx rate of the radiotracer into the brain tissue, was calculated for each set of mouse dynamic PET data from the fitted micro-parameters following Equation 1. 1 HPLC Methods

[0137] 2 Method A (Analytical HPLC)

[0138] Analytical reversed-phase high-performance liquid chromatography (HPLC) was carried out using a Thermo Scientific UltiMate 3000 HPLC system with a Phenom enex Luna C18(2) reversed-phase column (5 pm, 250 mm x 4.6 mm). The UV absorbance was recorded at 214 and 250 nm. Radioactivity was recorded using a Model 105 radio-detector (Carroll & Ramsey Associates, Berkeley, CA). The mobile phases were A, H2O with 0.1% TFA and B, MeCN with 0.1% TFA. The gradient was as follows: 0-1 min, 100% A; 1-11 min, 0-100% B; 11-16.5 min, 100% B; 16.5-17.5 min, 100-0% B; and 17.5-20 min, 100% A, at a flow rate of 1 mL / min.

[0139] 3 Method B (Semi-preparative HPLC)

[0140] Purification of the fluorination precursor 11 was conducted on a Thermo Scientific UltiMate 3000 HPLC system with a Phenomenex Luna Cl 8(2) reversed-phase column (5 pm, 250 mm x 10 mm). The UV absorbance was recorded at 214 and 254 nm. The mobile phases were A, H2O and B, MeCN. The gradient was as follows: 0-3 min, 100% A; 3-20 min, 0- 100% B; 20-27.5 min, 100% B; 27.5-30 min, 100-0% B; and 30-33 min, 100% A, at a flow rate of 3 mL / min.

[0141] 4 Method C (Semi-preparative HPLC)

[0142] Purification of 22-[18F]FDHA was performed on a Knauer HPLC system with an Azura P 6. IL HPG pump and BlueShadow 40D UV / VIS detector with a Phenomenex Luna C18(2) reversed-phase column (5 pm, 250 mm x 10 mm). The UV absorbance was recorded at 254 nm. Radioactivity was recorded using a Model 101 radio-detector (Carroll & Ramsey Associates, Berkeley, CA). The mobile phases were A, H2O and B, MeCN. The gradient was as follows: 0-3 min, 100% A; 3-20 min, 0-100% B; 20-27.5 min, 100% B; 27.5-30 min, 100- 0% B; and 30-33 min, 100% A, at a flow rate of 3 mL / min.

[0143] 5 Synthetic Procedures

[0144] 6 Methyl 9-Hydroxynona-4,7-diynoate (1).

[0145] A procedure from the Hwang et al., Bioorg. Med. Chem. Lett. 2017, 27 (3), 620-625 was modified as follows: to a solution of methyl 4-pentynoate (617.2 mg, 1.2 equiv) in anhyd. V,V-dimethylformamide (DMF, 10 mL) was added Nal (900.1 mg, 1.2 equiv), Cui (1.14 g, 1.2 equiv), and CS2CO3 (2.45 g, 1.5 equiv) with rapid stirring. To the mixture was then added a solution of 4-chlorobutyn-l-ol (523.0 mg,) in anhyd. DMF (2 mL). Stirring continued overnight, then the mixture was diluted with ethyl acetate (EtOAc, 40 mL). To this was then added a mixture of saturated aq. NH4Q (40 mL) and saturated aq. Na2S2Ch (10 mL). The biphasic mixture was separated, and the aqueous layer was then extracted with EtOAc (2 x 25 mL) and the combined organic layers were washed with water (20 mL), then saturated aq. NaCl (brine, 10 mL). The organic solution was then dried over anhyd. MgSO4 and evaporated under reduced pressure. The residue was loaded onto silica gel and purified by flash chromatography (12 g silica, 0-50% EtOAc in hexanes) to obtain diyne 1 as a yellow oil (746.6 mg, 83% yield).

[0146] TLC: Rt = 0.41 (1 : 1 EtOAc / hexanes). 'H NMR (500 MHz, CDCh): 5 4.24 (t, J = 2.2 Hz, 2H), 3.69 (s, 3H), 3.16 (p, J = 2.3 Hz, 2H), 2.57 - 2.42 (m, 4H).13C NMR (125 MHz, CDCh): 5 172.61, 80.48, 79.09, 78.71, 74.48, 51.94, 51.31, 33.44, 14.72, 9.95.

[0147] 7 Methyl 9-Bromonona-4,7-diynoate (2).

[0148] A procedure from Hwang et al., Bioorg. Med. Chem. Lett. 2017, 27 (3), 620-625 was modified as follows: to a solution of 1 (519.4 mg, 2.88 mmol) and CBn (1.44 mg, 1.5 equiv) in anhyd. dichloromethane (DCM, 7.5 mL), cooled in an ice-water bath, was added a solution of triphenylphosphine (PPI13, 1.14 mg, 1.5 equiv) in anhyd. DCM (2.5 mL) with stirring. After 1.5 h, the solution was evaporated under reduced pressure then the residue was loaded onto silica gel. After flash chromatography (12 g silica, 0-20% EtOAc in hexanes), bromide 2 was obtained as a yellow oil (559.4 mg, 80% yield).

[0149] TLC: Rt = 0.73 (1 : 1 EtOAc / hexanes). *H NMR (400 MHz, CDCh): 5 3.90 (t, J= 2.3 Hz, 2H), 3.70 (s, 3H), 3.20 (p, J = 2.4 Hz, 2H), 2.59 - 2.41 (m, 4H).13C NMR (100 MHz, CDCh): 5 172.56, 81.78, 79.36, 75.55, 73.90, 51.97, 33.39, 14.92, 14.73, 10.23.

[0150] 8 Methyl 12-Hydroxydodeca-4,7,10-triynoate (3).

[0151] Following a similar procedure for the synthesis of 1, the reaction of compound 2 (559.4 mg, 2.30 mmol), propargyl alcohol (195 mg, 2 equiv), Cui (658 mg, 1.2 equiv), Nal (520 mg, 1.2 equiv), and CS2CO3 (1.50 g, 1.5 equiv) in DMF (8 mL), followed by aqueous work-up and flash chromatography (12 g silica, 0-50% EtOAc in hexanes) afforded triyne 3 as a yellow oil (327.7 mg, 65% yield). TLC Rf = 0.42 (1 : 1 EtOAc / hexanes).1H NMR (500 MHz, CDCh): 54.25 (t, J= 2.1 Hz,

[0152] 2H), 3.69 (s, 3H), 3.19 (p, J= 2.3 Hz, 2H), 3.11 (p, J= 2.3 Hz, 2H), 2.55 - 2.44 (m, 4H).13C NMR (125 MHz, CDCh): 5 172.63, 80.09, 78.93, 78.89, 75.27, 74.69, 74.02, 51.94, 51.32, 33.48, 14.75, 9.99, 9.86. MS (ESI) m / z [M+H]+Calcd for C13H15O3 219.1; Found. 219.2.

[0153] 9 Methyl 12-Bromododeca-4,7,10-triynoate (4).

[0154] Following a similar procedure for the synthesis of 2, the reaction of compound 3 (327.7 mg, 1.5 mmol), CBn (745.1 mg, 1.5 equiv), and triphenylphosphine (707.3 mg, 1.5 equiv) in DCM (11 mL), followed by flash chromatography (4 g silica, 0-20% EtOAc in hexanes) afforded the bromide 4 as a yellow oil (259.1 mg, 61% yield).

[0155] TLC: Rt = 0.75 (1 : 1 hexane s / EtO Ac). 'H NMR (400 MHz, CDCh): 5 3.90 (t, J = 2.3 Hz, 2H), 3.70 (s, 3H), 3.22 (p, J= 2.3 Hz, 2H), 3.12 (p, J= 2.3 Hz, 2H), 2.57 - 2.42 (m, 4H).13C NMR (100 MHz, CDCh): 5 172.57, 81.37, 78.99, 75.75, 75.51, 74.59, 73.51, 51.95, 33.47, 14.83, 14.76, 10.27, 9.89

[0156] 10 6-(Trimethylsilyl)hexa-2,5-diyn-l-ol (5).

[0157] Following a similar procedure for the synthesis of 1, 3 -bromo- 1 -(trimethylsilyl)- 1- propyne (1.05 g, 3 mmol), propargyl alcohol (257.6 mg, 1.5 equiv), Cui (693.4 g, 1.2 equiv), Nal (546 mg, 1.2 equiv), and CS2CO3 (1.47 g, 1.5 equiv) in DMF (9 mL), followed by aqueous work-up and flash chromatography (12 g silica, 0-40% EtOAc in hexanes) afforded diyne 5 as a light-yellow oil (468.3 mg, 94% yield).

[0158] TLC: Rt = 0.63 (1 : 1 EtOAc / hexanes), Rf = 0.29 (1 :4 EtOAc / hexanes). 'H NMR (400 MHz, CDCh): 5 4.26 (t, J= 2.2 Hz, 2H), 3.25 (t, J= 2.2 Hz, 2H), 0.15 (s, 9H).13C NMR (100 MHz, CDCh): 5 99.47, 85.61, 79.81, 78.94, 51.36, 11.08, -0.01.

[0159] 11 6-(Trimethylsilyl)hexa-2,5-diyn-l-yl 4-methylbenzenesulfonate (6).

[0160] To a solution of 5 (410.0 mg, 2.47 mmol) in anhyd. DCM (8 mL), cooled in an icewater bath, was added pyridine (280 pL, 1.4 equiv) and / 2-toluenesulfonic anhydride (freshly recrystallized from hot EtOAc, 1.04 g, 1.3 equiv). The mixture was slowly wanned to room temperature. After 1 h, the mixture was partitioned between DCM (15 mL) and saturated aq. NH4Q (15 mL). The aqueous layer was extracted with DCM (2 x 10 mL) and the pooled organic solutions were washed with brine. The organic solution was dried over anhyd. Na2SO4, then evaporated under reduced pressure. The residue was loaded onto silica gel and after flash chromatography (12 g silica, 0-20% EtOAc in hexanes), tosylate 6 was obtained as a yellow oil (471.7 mg, 60% yield).

[0161] TLC: Rt = 0.43 (1 :4 EtOAc / hexanes), Rt = 0.74 (1 : 1 EtOAc / hexanes). 'H NMR (400 MHz, CDCh): 5 7.82 (d, J = 8.4 Hz, 2H), 7.35 (d, J= 7.9 Hz, 2H), 4.70 (t, J= 2.2 Hz, 2H), 3.11 (t, J= 2.2 Hz, 3H), 2.46 (s, 3H), 0.16 (s, 9H).13C NMR (100 MHz, CDCh): 5 145.15, 133.33, 129.94, 128.31, 98.27, 86.06, 83.60, 72.61, 58.32, 21.82, 11.08, -0.03.

[0162] 12 10-(Trimethylsilyl)deca-3,6,9-triyn-l-ol (7).

[0163] Following a similar procedure for the synthesis of 1, the reaction of compound 6 (1.25 g, 3.9 mmol), 3-butyn-l-ol (361.6 mg, 1.5 equiv), Cui (898 mg, 1.2 equiv), Nal (704 mg, 1.2 equiv), and CS2CO3 (1.91 g, 1.5 equiv) in DMF (12 mL), followed by aqueous work-up and flash chromatography (12 g silica, 0-30% EtOAc in hexanes) afforded triyne 7 as a yellow oil (409.9 mg, 48% yield).

[0164] TLC: Rt = 0.57 (1 : 1 EtOAc / hexanes), Rt = 0.19 (1 :4 EtOAc / hexanes). 'H NMR (400 MHz, CDCh): 5 3.70 (t, J = 6.2 Hz, 2H), 3.20 (t, J = 2.4 Hz, 2H), 3.16 (p, J = 2.4 Hz, 2H), 2.44 (tt, J= 6.2, 2.4 Hz, 2H), 1.77 (s, 1H), 0.16 (s, 9H).13C NMR (100 MHz, CDCh): 5 99.90, 85.35, 77.39, 76.24, 75.06, 74.19, 61.23, 23.24, 11.05, 10.00, 0.04.

[0165] 13 Deca-3,6,9-triyn-l-ol (8).

[0166] A procedure from Hwang et al., Bioorg. Med. Chem. Lett. 2017, 27 (3), 620-625 was modified as follows: compound 7 (409.9 mg, 1.88 mmol) was dissolved in anhyd. tetrahydrofuran (THF, 10 mL) and cooled in an ice-water bath. Acetic acid (65 pL, 0.6 equiv) was added to a 1 M solution of tetrabutylammonium fluoride (TBAF) in THF (2.25 mL, 1.2 equiv) and this mixture was then added dropwise with stirring over 5 min to the cooled THF solution of 7. The resulting brown solution was stirred for 1 h, and then partitioned between diethyl ether (40 mL) and cold water (40 mL). The aqueous layer was further extracted with diethyl ether (3 x 15 mL) and the pooled organic layers were washed with water (20 mL) and brine (20 mL). The organic solution was dried over anhyd. Na2SO4 and evaporated under reduced pressure. The residue was loaded onto silica gel and after flash chromatography (4 g silica, 0-40% EtOAc in hexanes), desilylated triyne 8 was obtained as a light-yellow oil (229.5 mg, 84% yield).

[0167] TLC: Rt = 0.51 (1 : 1 EtOAc / hexanes), Rt = 0.14 (1 :4 EtOAc / hexanes). 'H NMR (400 MHz, CDCh): 5 3.69 (t, J= 6.3 Hz, 2H), 3.18 - 3.11 (m, 4H), 2.43 (tt, J = 6.3, 2.3 Hz, 2H), 2.06 (t, J = 2.6 Hz, 1H), 1.99 (s, 1H).13C NMR (100 MHz, CDCh): 5 78.20, 77.48, 76.01, 75.33, 73.84, 68.97, 61.17, 23.17, 9.88, 9.69.

[0168] 14 Methyl (4Z,7Z, 10Z, 13Z, 16Z, 19Z)-22-(Tosyloxy)docosa-4,7, 10, 13, 16, 19-hexaenoate

[0169] (11).

[0170] Following a similar procedure for the synthesis of 1, the reaction of 4 (259.1 mg, 0.92 mmol), 8 (229.5 mg, 1.7 equiv), Cui (211.1 mg, 1.2 equiv), Nal (166.0 mg, 1.2 equiv), and CS2CO3 (452.0 mg, 1.5 equiv) in DMF (6 mL), followed by aqueous work-up and flash chromatography (12 g silica, 0-50% EtOAc in hexanes) afforded hexayne 9 as a yellow-orange solid (130.5 mg, 41% yield). Due to the instability of 9, it was used in the following step immediately without further characterization by NMR.

[0171] A procedure from Hwang et al., Bioorg. Med. Chem. Lett. 2017, 27 (3), 620-625 was modified as follows: to a flask charged with 9 (130.5 mg, 0.38 mmol) was added a mixture of methanol (4 mL), EtOAc (4 mL), and 2-methyl-2-butene (8 mL). The solution was immediately purged with N2 and to it was then added Lindlar’s catalyst (130.2 mg) and pyridine (2 mL). H2 gas was bubbled through the suspension for 1 min, which was then kept stirring under a constant positive-pressure atmosphere of H2 (balloon pressure, with a 26G needle through a rubber septum to vent) while monitoring the progress of the hydrogenation by TLC (1 : 1 EtOAc / hexanes). After 2 h, the mixture was filtered, and the filtrate was then partitioned between EtOAc (30 mL) and 1 M aq. HC1 (25 mL). The aqueous layer was further extracted with EtOAc (3 x i5mL), and the pooled organic solutions were washed with water (20 mL) and then brine (15 mL). The organic phase was then dried over anhyd. Na2SO4 and evaporated under reduced pressure yielding a brown oil. The residue was loaded onto silica gel and after flash chromatography (4 g silica, 0-25% EtOAc in hexanes), the hydrogenated product was obtained as a light-yellow oil (79.2 mg, 59% crude yield). Analytical HPLC of the isolated products showed that it was a mixture of the intended hexaene product 10 (ca. 70% by UV at 250 nm) with over-reduced and under-reduced derivatives; the mixture was used in the next step without further purification. In smaller-scale reactions, the crude product may be isolated from strongly colored impurities by taking the residue up in 1 :4 EtOAc / hexanes, passing the solution through a short silica plug, and eluting with 1 :4 EtOAc / hexanes.

[0172] A procedure was adapted from Van Valkenburgh el al., Prostaglandins Leukot. Essent. Fatty Acids 2022, 186, 102510 as follows: to a solution of the partially purified 10 (17.5 mg) in anhyd. DCM (3 mL), cooled in an ice-water bath, was added tri ethylamine (14 pL) and 4- (dimethylamino)pyridine (0.8 mg). Then, a solution of tosyl chloride (34.7 mg) in DCM (0.5 mL) was added to the mixture. After slowly warming to room temperature and then continuing stirring for 2 days, the solution was diluted with DCM (10 mL). The solution was then washed with cold water (10 mL), then with brine (5 mL). The organic solution was then dried over anhyd. Na2SO4 and evaporated under reduced pressure. The residue was loaded onto silica gel and partially purified by flash chromatography (4 g silica, 0-10% EtOAc in hexanes) to obtain the desired tosylated compound. After further purification of the isolated product by semipreparative HPLC (Method B), analytically pure hexaene tosylate 11 was obtained as a colorless oil (5.6 mg, 13% yield over 2 steps):

[0173] TLC: Rt = 0.37 (1 :4 EtOAc / hexanes), Rt = 0.14 (1 :9 EtOAc / hexanes), Rt = 0.03 (1 : 19 EtOAc / hexanes). Analytical HPLC (Method A): tT= 15.72 min. 'HNMR (400 MHz, CDCh): 5 7.79 (d, J= 8.2 Hz, 2H), 7.34 (d, J= 8.0 Hz, 2H), 5.53 - 5.21 (m, 12H), 4.02 (t, J= 6.9 Hz, 2H), 3.67 (s, 3H), 2.87 - 2.72 (m, 10H), 2.45 (s, 3H), 2.44 - 2.34 (m, 6H).13C NMR (100 MHz, CDCh): 5 173.69, 144.85, 133.33, 131.77, 129.96, 129.95, 129.45, 128.66, 128.44, 128.36, 128.28, 128.26, 128.14, 128.06, 128.05, 127.72, 123.50, 69.72, 51.72, 34.15, 27.26, 25.82, 25.78, 25.78, 25.76, 25.72, 22.94, 21.79. HRMS (ESI / QTOF) m / z: [M+H]+Calcd for C30H41O5S 513.2669; Found 513.2651.

[0174] 15 Methyl 22-Hydroxydocosa-4, 7,10, 13, 16, 19-hexaynoate (9).

[0175] TLC: Rf = 0.42 (1 : 1 EtOAc / hexanes). Analytical HPLC (Method A): tr= 12.31 min. MS (ESI) m / z: [M+H]+Calcd for C23H23O3 347.2; Found 347.2. 16 Methyl (4Z, 7Z, 10Z, 13Z,16Z, 19Z)-22-Hydroxydocosa-4, 7, 10, 13, 16, 19 -hexaenoate (10).

[0176] TLC: Rf = 0.69 (1 : 1 EtOAc / hexanes), Rf = 0.19 (1 :4 EtOAc / hexanes). Analytical HPLC (Method A): tT= 14.79 min. MS (ESI) m / z [M+H]+Calcd for C23H23O3 359.3; Found 359.3.

[0177] 17 Methyl (4Z,7Z,10Z,13Z,16Z,19Z)-22-Fluorodocosa-4,7,10,13,16,19-hexaenoate (13).

[0178] A procedure from Van Valkenburgh et al. , Prostaglandins Leukot. Essent. Fatty Acids 2022, 186, 102510 was modified as follows: to a solution of 11 (10.7 mg) in anhyd. THF (1.5 mL), cooled in an ice-water bath, was added 1 M TBAF in THF (87 pL, 4.2 equiv). The resulting orange solution was slowly warmed to room temperature with stirring overnight. The solution was evaporated under reduced pressure and the residue was taken up in DCM (5 mL). The solution was washed with water (1 mL) and then brine (1 mL). After drying the solution over anhyd. Na2SO4, the solvent was evaporated under reduced pressure and the residue was loaded onto silica gel. After flash chromatography (4 g silica, 0-5% EtOAc in hexanes), two products were isolated as colorless oils: an elimination product 12 (2.2 mg, 29% yield) and the intended substitution product 13 (2.1 mg, 29% yield):

[0179] TLC: Rf = 0.39 (1 :9 EtOAc / hexanes), Rf = 0.22 (1 : 19 EtOAc / hexanes). Analytical HPLC (Method A): tT= 15.49 min. 'H NMR (400 MHz, CDCI3): 5 5.58 - 5.29 (m, 12H), 4.43 (dt,2JFH = 47.1 Hz, HH = 6.5 Hz, 2H), 3.67 (s, 3H), 2.92 - 2.72 (m, 10H), 2.49 (dq, FH = 22.3 Hz,3. / HII = 6.8 Hz, 2H), 2.42 - 2.33 (m, 4H).19F NMR (376 MHz, CDCI3): 5 -216.81 (tt, HF = 47.0 Hz,3JHF = 23.4 Hz).

[0180] 18 Methyl (4Z, 7Z, 10Z, 13Z,16Z, 19Z)-Docosa-4, 7,10, 13,16, 19,21 -heptaenoate (12).

[0181] TLC: Rf = 0.46 (1 :9 EtOAc / hexanes), Rf = 0.28 (1 : 19 EtOAc / hexanes). Analytical HPLC (Method A): tT= 16.29 min. *HNMR (400 MHz, CDCI3): 5 6.66 (dt, J= 16.9, 10.6 Hz, 1H), 6.02 (t, J = 10.9 Hz, 1H), 5.48 - 5.32 (m, 11H), 5.21 (d, J = 16.8 Hz, 1H), 5.12 (d, J = 10.1 Hz, 1H), 3.67 (s, 3H), 2.97 (t, J= 6.8 Hz, 2H), 2.91 - 2.80 (m, 8H), 2.44 - 2.33 (m, 4H). 19 (4Z,7Z,10Z,13Z,16Z,19Z)-22-Fluorodocosa-4,7,10,13,16,19-hexaenoic acid (22-

[0182] FDHA).

[0183] A procedure from Van Valkenburgh et al. , Prostaglandins Leukot. Essent. Fatty Acids 2022, 186, 102510 was adapted as follows: to a solution of the methyl ester 13 (2.1 mg) in THF (0.85 mL) was added a solution of LiOH (1.1 mg, 8 equiv) in water (0.15 mL). After stirring overnight at room temperature, the mixture was diluted with water (1.0 mL), then the pH was adjusted to around 2 with 0.1 M aq. H2SO4. The aqueous mixture was then extracted with DCM (3 x 1mL) and the pooled organic solutions were washed with brine (1 mL), then dried over anhyd. MgSCh. The solution was evaporated under reduced pressure and the residue was loaded onto silica gel. After flash chromatography (4 g silica, 0-20 % EtOAc in hexanes), 22- FDHA in its free acid form was obtained as a colorless oil (1.9 mg, 94% yield):

[0184] TLC: Ri = 0.13 (1 :4 EtOAc / hexanes). Analytical HPLC (Method A): tr= 14.47 min. 'H NMR (500 MHz, CDCh): 5 5.63 - 5.25 (m, 12H), 4.43 (dt,2JFH =47.2 Hz,3. / HII = 6.5 Hz, 2H), 2.92 - 2.71 (m, 10H), 2.49 (dq,3. / i n = 23.0 Hz,3. / HII = 6.8 Hz, 2H), 2.42 - 2.37 (m, 4H).19F NMR (470 MHz, CDCh): 5 -216.75 (tt, HF = 47.0 Hz, HF = 23.4 Hz).13C NMR (100 MHz, CDCh): 5 177.70, 131.25, 129.66, 128.53, 128.41, 128.37, 128.32, 128.30, 128.23, 128.17, 127.99, 127.78, 124.08 (d,3. / i c = 7.3 Hz), 83.32 (d, FC = 167.9 Hz), 33.96, 28.81 (d, FC = 20.7 Hz), 25.86, 25.79, 25.78, 25.77, 25.73, 22.72. HRMS (ESEQTOF) m / z [M+H]+Calcd for C22H31FO2 347.2381; Found 347.2371.

[0185] 20 Ex Vivo LC-MS Analysis of 22-FDHA

[0186] Probe Injection and Tissue Extraction. A solution of 22-FDHA in acetonitrile was dried in a glass vial under a stream of filtered dry N2 gas, and then under vacuum. The residue was formulated in injection buffer to a concentration of 10 ng / mL. A female mouse was anesthetized with isoflurane, and then 1 pg of 22-FDHA was injected into the tail vein. The mouse was kept under maintenance anesthesia for 30 min after which it was dissected and euthanized by cardiac puncture. Approximately 500 pL of blood was extracted from the heart and transferred to an EDTA-coated microcentrifuge tube. After blood collection, the mouse was immediately perfused with cold PBS then the liver and brain were extracted. Tissues, including isolated plasma, brain, and liver, were stored at -80 °C until processing. Following the same procedure, another mouse was injected with 500 ng 22-[18F]FDHA, and the tissues were extracted at 30 min p.i. Plasma and Brain 22-FDHA Extraction. 22-FDHA was extracted from mouse plasma (100 pL) and brain tissue (-250 mg) and then quantified by liquid chromatography-tandem mass spectrometry (LC-MS / MS) using multiple reaction monitoring (MRM). Prior to extraction, mouse brains were cut in half through the midsagittal plane, and the left brain was used for analysis. Standard curve samples were prepared by spiking 3X stripped C57BL mouse plasma (BioIVT) with varied concentrations of synthetic 22-FDHA. To extract 22-FDHA and prevent lipid peroxidation, methanol (MeOH) containing 0.05% butylated hydroxytoluene and triphenylphosphine was added to plasma samples (200 pL) and brain tissues (500 pL). 50 pL rfs-DHA (50 ng / mL, Cayman) was added to each sample and used as the internal standard for quantification. To facilitate the extraction of 22-FDHA from plasma, samples were vortexed for 10 seconds and kept cool in ice for 10 min while brain tissues were homogenized. Brain samples were homogenized completely using a pre-cooled TissueLyser (Qiagen) at 30 Hz at 1.5-min intervals. Subsequently, plasma and brain samples were centrifuged at 10,000*g for 10 min at 4 °C. The supernatant was collected from each sample and diluted with water to obtain 10% MeOH solutions. Each sample was then subjected to solid phase extraction using Strata X 33pm Polymeric Reverse Phase cartridges (Phenomenex). Cartridges were preconditioned with MeOH (1 mL) followed by water (1 mL) before the diluted samples were eluted through and then washed with water (1 mL). 22-FDHA and the internal standard were eluted using 700 pL MeOH, which was then evaporated to dryness under a steady stream of filtered dry N2 gas. The dried residues were reconstituted with 50% aq. MeOH (50 pL) and centrifuged to remove any undissolved debris before transferring it to UV-resistant glass vials for LC-MS / MS analysis.

[0187] LC-MS / MS Analysis. 22-FDHA and t / s-DHA were separated and quantified in an Agilent 1290 UPLC system coupled to a Sciex QTRAP API6500+ system using ESI in negative ion mode. For adequate separation, a Poroshell 120 EC-Cis column (2.7 pm, 4.6 x 100 mm, Agilent) was used with the mobile phases: A, 0.01% formic acid in H2O; and B, 0.01% formic acid in MeOH. The gradient was as follows: 0-0.1 min, 20-50% B; 0.1-2 min, 50% B; 2-11 min, 50-80% B; 11-14.5 min, 80% B; 14.5-14.6 min, 80-98% B; 14.6-20 min, 98% B; 20-20.1 min, 98-20% B; 20.1-23 min, 20% B. An injection volume of 20 pL was used and the flow rate was held at 0.5 mL / min while the column temperature was set to 40 °C. Both 22-FDHA and t / s-DHA were detected using unique MRM signatures (Table 4) and data analysis and quantification were performed using Skyline 23.1. (Adams et al., J Proteome Res 2020, 19 (4), 1447-1458.) Table 4. LC-MS / MS Assay MRM Signatures

[0188] The following Examples are intended to illustrate the above invention and should not be construed as to narrow its scope. One skilled in the art will readily recognize that the Examples suggest many other ways in which the invention could be practiced. It should be understood that numerous variations and modifications may be made while remaining within the scope of the invention.

[0189] EXAMPLES

[0190] Example 1. F-18 labeled polyunsaturated fatty acids for imaging lipid metabolism dysregulation.

[0191] Methods.

[0192] Animals. All animal studies were approved by the Institutional Animal Care and Use Committee at the University of Southern California. The AP0E4 targeted replacement (TR) mice used in the study were bred in the USC animal facility. Animals were housed with standardized 12 h light and dark cycles and had access to food and water ad libitum. Vivarium temperature was maintained between 22 °C and 24 °C and humidity was maintained between 50 and 60%.

[0193] General Procedure for PET-MR Imaging. PET and MR images were acquired using a 7T MRI scanner with an integrated PET scanner (MR Solutions, Guildford, UK). Mice were anesthetized with isoflurane (1.5-2.5%) and respiration was monitored using a pneumatic pillow. Body temperature was maintained at 37 °C using a heated animal holder (Minerve, France). Two-dimensional Fast Spin Echo (FSE) transverse Tl-weighted MR images were acquired to define anatomy with the following parameters: TE / TR= 11 ms / 3556 ms, slice thickness = 1 mm, 1 average, echo train length = 4, field of view = 72 mm x 72 mm, matrix size = 512 x 256, flip angle = 90°. For PET, a list mode acquisition protocol was obtained from 2 min before to 30 min after bolus injection of 22-[18F]FDHA through a tail veil cannula (7.4 ± 1.9 MBq). Image data were corrected for intrascan radioactivity decay, detector nonuniformity, and random coincident noise. Three-dimensional ordered subsets expectationmaximization (3D-OSEM) reconstruction was performed, resulting in a 0.28 x 0.28 x 0.28 mm3voxel dimension, 34 mm field of view, 64 subsets, and 4 iterations. The acquisition protocol was as follows: 4 frames of 30 s, 12 frames of 10 s, 6 frames of 30 s, 5 frames of 60 s, and 10 frames of 120 s.

[0194] PET / MR Image Co-registration and Static PET Analysis. The last 10 frames (10-30 min) of the dynamic PET reconstruction were averaged. This PET image and the MR images were co-registered using a PET / MR phantom, which consisted of a capillary tube filled with 10 pL of ~40 kBq18F in solution, as a guide. Three-dimensional regions of interest (ROIs) were defined through manual segmentation using the MR image as the anatomical reference. For static analysis, the percent injected dose per gram (%ID / g) was calculated from the concentration in the brain ROI, calculated from the PET image data at 10-30 min, and the injected dose.

[0195] Results: Diet in APOE4-TR mice. To compare the effect of diet on a mouse model for Alzheimer’s disease, APOE4-TR mice were divided into two groups each fed a different diet for 12 months: one group was fed a high-fat, DHA-deficient diet (35-65 g at the time of the scan) and one group was fed a DHA-rich diet (27-46 g at the time of scan). Then, mice were imaged with the 22-[18F]FDHA tracer. Results for the static analysis from the dynamic PET data are shown in Figure 7A-B.

[0196] All publications, patents, and patent documents cited herein are incorporated by reference as though individually incorporated by reference, and in particular, Duro et al., ACS Chem. Neurosci. 2023, 14, 24, 4409-4418; U.S. Patent Publication Nos. 2021 / 0009624 to Chen et al., 2012 / 0053337 to Zibo et al., and International Patent Publication No. WO 2022 / 109244 to Chen et al. No limitations inconsistent with this disclosure are to be understood therefrom. The invention has been described with reference to various specific and preferred embodiments and techniques. However, many variations and modifications may be made while remaining within the spirit and scope of the invention.

[0197] While specific embodiments have been described above with reference to the disclosed embodiments and examples, such embodiments are only illustrative and do not limit the scope of the invention. Changes and modifications can be made in accordance with ordinary skill in the art without departing from the invention in its broader aspects as defined in the following claims.

Claims

CLAIMSWhat is claimed is:

1. A radiofluorinated docosahexaenoic acid fatty acid having structure (I):

2. A composition comprising the radiofluorinated docosahexaenoic acid fatty acid of claim 1; and at least one of a solvent or a pharmaceutically acceptable carrier.

3. The composition of claim 2, wherein the solvent or pharmaceutically acceptable carrier comprises bovine serum albumin (BSA) and 2-[4-(2-hydroxyethyl)piperazin-l- yl]ethane sulfonic acid (HEPES).

4. The composition of claim 3, wherein the BSA is present in the composition in an amount of about 25 mg / to about 75 mg / ml; the HEPES is present in the composition in an amount of about 1 mM to about 50 mM; and optionally, saline.

5. A method of synthesizing a radiofluorinated fatty acid comprising combining:(a) an omega-tosylated docosahexaenoic acid fatty acid methyl ester in a first nitrile solvent; and(b) a solution of tetra-w-butylammonium [18F]fluoride ([18F]TBAF) in a second nitrile solvent, at about 75 °C to about 95 °C for about 10 minutes to about 30 minutes, to form a [18F]fluorinated ester; and treating the [18F]fluorinated ester with a solvent comprising a strong base at about 75 °C to about 95 °C to hydrolyze the methyl ester into a free fatty acid; thereby providing the radiofluorinated fatty acid.

6. The method of claim 5, wherein the step of combining of (a) and (b) is carried out at about 85 °C for about 10 minutes to about 20 minutes.

7. The method of claim 5, wherein the treating step is carried out at about 85 °C for about 10 minutes to about 20 minutes.

8. The method of claim 5, wherein the first nitrile solvent and the second nitrile solvent comprise acetonitrile.

9. The method of claim 5, wherein the solvent comprising the strong base comprises potassium hydroxide in a concentration of about 1 M to about 5 M.

10. The method of claim 5, wherein the radiofluorinated fatty acid is 22- [18F]fluorodocosahexaenoic acid ([18F]FDHA).

11. The method of claim 5, wherein the omega-tosylated docosahexaenoic acid fatty acid methyl ester is:

12. The method of claim 5, further comprising purifying the free fatty acid by reverse phase high-performance liquid chromatography (HPLC) to provide the radiofluorinated fatty acid.

13. A method of positron emission tomography (PET) imaging comprising: administering to a subject an effective amount of: i) 22-[18F]fluorodocosahexaenoic acid ([18F]FDHA); ii) optionally, a muscarinic receptor agonist; iii) optionally, a pharmaceutically acceptable carrier, excipient, or diluent; and capturing one or more PET images of the subject.

14. The method of claim 13, wherein the muscarinic receptor agonist is administered to the subject concurrently or sequentially with the [18F]FDHA, wherein the muscarinic receptor agonist increases uptake of the [18F]FDHA.

15. The method of claim 13, wherein the muscarinic receptor agonist is arecoline.

16. The method of claim 13, wherein the pharmaceutically acceptable carrier, the excipient, or the diluent is administered with the [18F]FDHA) to the subject.

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