18f labeled alanine compositions and methods of detecting chronic disease using same

Abstract

The present invention provides a compound having the structure: wherein when the compound is then at least one of D1, D2, and D3 is a deuterium -enriched -H site; or wherein when the compound is then at least two of D1, D2, and D3 are deuterium-enriched -H sites; or a pharmaceutically acceptable salt or ester thereof.

Description

Docket: 92522-A-PCT / GJG / YX18F LABELED ALANINE COMPOSITIONS AND METHODS OF DETECTING CHRONIC DISEASE USING SAME

[0001] Throughout this application, various publications are referenced, including referenced inparenthesis. The disclosures of all publications mentioned in this application in their entireties are hereby incorporated by reference into this application in order to provide additional description of the art to which this invention pertains and of the features in the art which can be employed with this invention. CROSS-REFERENCE TO RELATED APPLICATION

[0002] This application claims the benefit of U.S. Provisional Application No.63 / 562,527 filed March7, 2024, the contents of which is hereby incorporated by reference. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0003] This invention was made with government support under EB031785, EB024549, andEB029860 awarded by National Institutes of Health. The government has certain rights in the invention. BACKGROUND OF THE INVENTION

[0004] Bacterial infections continue to have a major impact on global health that is exacerbated by theemergence of drug-resistant strains of pathogenic bacteria (Antimicrobial Resistance 2022 and Thompson 2022). The appropriate and effective treatment of bacterial infection relies on rapid and accurate diagnostic methods which in many cases is challenging (Polvoy, 2020, Ordonez, 2019, Jain 2017 and Parker 2020). For example, a biopsy could be difficult for patients with deep-seated infection and inaccurate for patients with chronic infection due to low bacterial burden and sample heterogeneity (Jain 2017, Parker 2020, and Jakobsen, 2021). Structural imaging methods such as computed tomography (CT) and magnetic resonance imaging (MRI) have been applied to the clinical management of infected patients (Plvoy, 2020, Ordonez, 2019, and Jain 2017). However, these imaging techniques rely on the detection of abnormal fluids within tissues and organs, which is associated with increased vasodilation and vascular permeability and not specific to bacterial infection (Pober 2014).

[0005] Positron emission tomography (PET) is a sensitive non-invasive imaging modality that iswidely used for monitoring metabolic processes and diagnosing disease. This method is used to detect bacterial infection, however, the currently used radiotracer, [¹⁸F]fluorodeoxyglucose ([18F]FDG), is not specific for infection since it images the enhanced glucose metabolism in active inflammatory cells instead of bacteria (Vaidyanathan 2015 and Pijl 2021). Therefore, bacteria-specific radiotracers are needed that can accurately differentiate infection from inflammation.

[0006] In this regard, several 11C- and 18F-labeled PET imaging agents have been developed forbacterial infection that target different aspects of bacterial metabolism (Mota 2020 and Kleynhans 2020). This includes 2-Deoxy-2-[18F]fluoro-D-sorbitol ([18F]FDS) which is selectively for Gram-negative bacteria Enterobacteriaceae and can accurately distinguish bacterial infection from inflammation (Figure 1) (Weinstein 2014). In addition, [11C]Para-aminobenzoic acid ([11C]PABA), 2-[18F]fluor-para- aminobenzoic acid ([18F]FPABA) are PET imaging agents that target folate metabolism and are taken up by Gram-positive and Gram-negative bacteria (Figure 1) (Mutch 2018, Zhang 2018 and Li 2020).

[0007] Amino acids are essential building blocks for the synthesis of peptides, proteins, and othernitrogen-containing bioactive compounds such as nucleosides, creatine, and some human neurotransmitters (Ling 2023 and Rose 2019). Amino acids also play many different roles in cancer development where they can function as alternative fuels, biosynthetic materials, redox balance mediators, and epigenetic and posttranscriptional regulators (Vettore 2020). Therefore, targeting enhanced amino acid metabolism is an attractive strategy to develop novel antineoplastic drugs (Lieu 2020 and Wei 2020).

[0008] L-Alanine is the second most abundant amino acid in human plasma and is synthesized byalanine aminotransferases from pyruvate and glutamate in the glucose-alanine cycle (Choi 2019 and Reitzer 2004). Compared with other well-studied amino acids such as L-glutamine, L-alanine is relatively underexplored in cancer (Choi 2019). Recently, some evidence has shown that alanine metabolism might be essential for cancer development. Indeed, L-alanine has been found to serve as an energy source and play a role in the tricarboxylic acid cycle (TCA) cycle as well as glucose metabolism when glucose is limited, for example in non-small cell lung cancer (Hodakoski 2019). Moreover, it has been found that pancreatic ductal adenocarcinomas can utilize L-alanine secreted by pancreatic stellate cells as an alternate carbon source (Sousa 2016). Interestingly, a metabolite analysis has identified L-alanine as a potential metabolic biomarker for glioma detection with high predictive accuracy (Firdous 2021).BRIEF SUMMARY OF THE INVENTION

[0009] The present invention provides a compound having the structure:, wherein when the compound is, then at least one of D1, D2, and D3is a-H site; or wherein when the compound is , then at least two of D1, D2, and D3 are-H sites; or a pharmaceutically acceptable salt or ester thereof.

[0010] The present invention provides a process of producing a compound having the followingstructure: ,wherein at least one of D1, D2, and D3-H site; the process comprises: (a) conducting a radio-fluorination reaction to the compound of formula (I)(I) to obtain a compound of formula(b) adding an acid.

[0011] The present invention provides a compound of formula III having the structure:(III), wherein at least one of D1, D2, and D3 is a deuterium-enriched -H site; the process comprises: (a) reacting to a compound of formula (I) with a fluorine-containing acid or a salt thereof,(I); (b) adding an acid to produce a compound of formula (II):(c) adding an acid to produce a.

[0012] The present inventiontarget cells in a subject comprisingadministering an effective amount of the compound having the structure: , wherein when the compound is,then at least one of D1, D2, and D3 is a -H site; or wherein when the compound is, then at least two of D1, D2, and D3 are deut -H sites; to the subject and imaging the subject with a molecular imaging device to detect the compound or the composition in the subject.BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1. 18F-labeled amino acids. (A) Approved 18F-labeled amino acids for oncology. (B)18F-Labeled L-alanine derivatives. (C)18F-labeled amino acids synthesized and studied in the present invention.

[0014] Figure 2. Antibiotics and Radiotracers for Bacteria Infection. (A) Non-amino acid derivedradiotracers. (B) D-amino acid derived radiotracers. (C) D-Alanine derived antibiotics. (D) D-amino acid- based radiotracers studied in the present invention.

[0015] Figure 3. Conditions explored for radiofluorination. (A)THF. (B) 1,4-Dioxane. (C) MeCN.(D) DMSO. (E) tert-Butanol. (F) tert-Amyl alcohol.

[0016] Figure 4. Bacterial uptake of D-[18F]FAla and D-[18F]FAla-d3. (A)The uptake of D-[18F]FAla and D-[18F]FAla-d3 in live E. coli (n=3), heat-killed E. coli (n=3), and live E. coli in the presence of 5 mM D-Ala (n=3), 1 mM D-Ala (n=3), and 0.5 mM D-Ala (n=3). (B)The uptake of D-[18F]FAla and D-[18F]FAla-d3 in live S. aureus (n=3), heat-killed S. aureus (n=3), and live S.aureus in the presence of 5 mM D-Ala (n=3), 1 mM D-Ala (n=3), and 0.5 mM D-Ala (n=3). Data were analyzed using the Student’s two-tailed t test, where **** indicates a P value < 0.0001, *** indicates a P value < 0.001, ** indicates a P value < 0.01, * indicates a P value < 0.05.

[0017] Figure 5. In vivo bioluminescent imaging and PET imaging of D-[18F]FAla and D-[18F]FAla-d3 in a rat S. Aureus infection model. (A) Bioluminescent image of a rat S. Aureus infection model for D- [18F]FAla on the PET imaging day. (B) Dynamic PET images of D-[18F]FAla (n=4) in the rat from (A) at 15, 20, 30, and 60 min post-injection. (C) Bioluminescent image of a rat S. Aureus infection model for D- [18F]FAla-d3 on the PET imaging day. (D) Dynamic PET images of D-[18F]FAla-d3 (n=4) in the rat from (C) at 15, 20, 30, and 60 min post-injection.

[0018] Figure 6. Cellular uptake of L-[18F]FAla and L-[18F]FAla-d3. (A)Time-dependent uptake ofL-[18F]FAla-d3 in 9L / lacZ (n=3), Mia Paca-2 (n=3), and U87MG (n=3) cells . (B) Uptake of L-[18F]FAla by 9L / lacZ cells in the presence of BCH (n=3), MeAIB (n=3), L-Ala (n=3), L-Ser (n=3), and L-Cys (n=3). Data were analyzed using the Student’s two-tailed t test, where**** indicates a P value ˂ 0.0001.

[0019] Figure 7. PET imaging and ex vivo biodistribution of L-[18F]FAla and L-[18F]FAla-d3 innon-tumor-bearing athymic nude mice. (A) Time-activity curves of bone, kidney, liver, and muscle for L-[18F]FAla (n=4). (B) Time-activity curves of bone, kidney, liver, and muscle for L-[18F]FAla-d3(n=4). (C) Ex vivo biodistribution 110 min post-injection with L-[18F]FAla (n=5) or L-[18F]FAla-d3(n=5) in non- tumor-bearing athymic nude mice, where SI= small intestines and LI= large intestines (D) Representativedynamic PET / CT images of L-[18F]FAla (n=4) and L-[18F]FAla-d3(n=4) in non-tumor-bearing athymic nude mice.

[0020] Figure 8. PET imaging of L-[18F]FAla, L-[18F]FAla-d3, D-[18F]FAla-d3 in U87MG tumor-bearing mice. Representative dynamic PET / CT images of L-[18F]FAla (n=4), L-[18F]FAla-d3(n=4), and D-[18F]FAla-d3(n=4) in U87MG tumor-bearing athymic nude mice at 15, 20, 30 and 60 min post-injection.

[0021] Figure 9. ROI analysis of PET imaging and ex vivo biodistribution of L-[18F]FAla, L-[18F]FAla-d3, and D-[18F]FAla-d3in U87MG tumor-bearing mice. (A) Time-activity curves of tumor and muscle for L-[18F]FAla (n=4). (B) Time-activity curves of tumor and muscle for L-[18F]FAla-d3(n=4). (C) Time-activity curves of tumor and muscle for D-[18F]FAla-d3 (n=4). (D) Time-activity curves of bone, kidney, and liver for L-[18F]FAla (n=4). (E) Time-activity curves of bone, kidney, and liver for L-[18F]FAla- d3 (n=4). (F) Time-activity curves of bone, kidney, and liver for D-[18F]FAla-d3 (n=4). (G) Ex vivo biodistribution of L-[18F]FAla-d3 (n=4) and D-[18F]FAla-d3 (n=4) at 110 min post-injection in U87MG tumor-bearing mice, where SI= small intestines and LI= large intestines. Data was analyzed using a Student’s two-tailed t test, where **** indicates a P value ˂ 0.0001, *** indicates a P value ˂ 0.001, ** indicates a P value ˂ 0.01, and * indicates a P value ˂ 0.05.

[0022] Figure 10. ROI analysis of PET imaging and ex vivo biodistribution of D-[18F]FAla, D-[18F]FAla-d3 in a rat S. Aureus infection model. (A) Time-activity curves of infected muscle (IM), inflamed muscle (IFM), healthy muscle (HM), bone, and brain for D-[18F]FAla (n=4). (B) Time-activity curves of infected muscle (IM), inflamed muscle (IFM), healthy muscle (HM), bone, and brain for D-[18F]FAla-d3 (n=4). (C) Time-activity curves of kidney and liver for D-[18F]FAla (n=4). (D) Time-activity curves of kidney and liver for D-[18F]FAla-d3 (n=4). (E) Comparison of the uptake of D-[18F]FAla and D-[18F]FAla- d3 in infected muscle (IM), inflamed muscle (IFM), and healthy muscle (HM) at 10-15 min post injection. (F) Ex vivo biodistribution 110 min post-injection of D-[18F]FAla (n=4) or D-[18F]FAla-d3 (n=4) in infected muscle (IM), inflamed muscle (IFM), and healthy muscle (HM). Data were analyzed using a Student’s two- tailed t test, where **** indicates a P value ˂ 0.0001, *** indicates a P value ˂ 0.001, ** indicates a P value ˂ 0.01, * indicates a P value ˂ 0.05, and ns indicates a P value > 0.05.

[0023] Figure 11A. Analytical HPLC analysis of non-radioactive D-FAla and D-FAla-d3.(A)Analytical HPLC of D-FAla. (B) Analytical HPLC of D-FAla-d3. Figure 11B Analytical HPLCanalysis of D-[18F]FAla and D-[18F]FAla-d3. (A) Quality control of D-[18F]FAla (tR=18.3 min). (B) Spikeinjection of D-[18F]FAla (tR=18.3 min) and nonradioactive L-FAla (3.3 μg, tR=11.3 min) and D-FAla (0.8μg, tR=18.2 min). (C) Quality control of D-[18F]FAla-d3(tR=18.2 min). (D) Spike injection of D-[18F]FAla-d3(tR=18.2 min) and nonradioactive L-FAla-d3(2.6 μg, tR=11.3 min)and D-FAla-d3 (1.3 μg, tR=18.1 min).Figure 11C. Shelf stability analysis of D-[18F]FAla and D-[18F]FAla-d3 using analytical HPLC.(A)Quality control of D-[18F]FAla at 0 hour (tR=18.3 min) and 3 hour (tR=18.3 min). (B) Quality control of D-[18F]FAla-d3at 0 hour (tR=18.2 min) and 3 hour (tR=18.3 min).

[0024] Figure 12. Analytical HPLC analysis of L-[18F]FAla, L-[18F]FAla-d3, and D-[18F]FAla-d3.(A) Quality control of L-[18F]FAla (tR=11.1 min). (B) Spike injection of L-[18F]FAla (tR=11.5 min) and nonradioactive L-FAla (3.3 μg, tR=11.5 min) and D-FAla (0.8 μg, tR=18.2 min). (C) Quality control of L- [18F]FAla-d3(tR=11.2 min). (D) Spike injection of L-[18F]FAla-d3(tR=11.5 min) and nonradioactive L-FAla- d3(2.6 μg, tR=11.4 min) and D-FAla-d3(1.3 μg, tR=18.1 min). (E) Quality control of D-[18F]FAla-d3(tR=18.2 min). (F) Spike injection of D-[18F]FAla-d3(tR=18.2 min) and nonradioactive L-FAla-d3(2.6 μg, tR=11.3 min)and D-FAla-d3 (1.3 μg, tR=18.1 min).

[0025] Figure 13. Shelf stability analysis of D-[18F]FAla and D-[18F]FAla-d3 using analyticalHPLC. (A) Quality control of L-[18F]FAla at 0 hour (tR=11.1 min) and 3 hour (tR=11.1 min). (B) Quality control of L-[18F]FAla-d3 at 0 hour (tR=11.1 min) and 3 hour (tR=11.1 min). (C) Quality control of D- [18F]FAla-d3 at 0 hour (tR=18.2 min) and 3 hour (tR=18.3 min).DETAILED DESCRIPTION OF THE INVENTION

[0027] The present invention provides a compound having the following structure:, wherein when the compound is, then at least one of D1, D2, and D3 is a-H site; or wherein when the compound is , then at least two of D1, D2, and D3are-H sites; or a pharmaceutically acceptable salt or ester thereof.

[0028] The present invention provides a compound having the following structure:.

[0029] In some embodiments, at leastare deuterium-enriched -H sites.

[0030] In some embodiments, all of D1, D2, and D3 are deuterium-enriched -H sites.

[0031] In some embodiments, the level of deuterium at the deuterium-enriched -H site of thecompound is about 20-100%.

[0032] In some embodiments, the level of deuterium at the deuterium-enriched -H site of thecompound is about 70-100%.

[0033] In some embodiments, the level of deuterium at the deuterium-enriched -H site of thecompound is about 90-100%

[0034] In some embodiments, the level of deuterium at the deuterium-enriched -H site of thecompound is about 97-100%.

[0035] In some embodiments, the level of deuterium at the deuterium-enriched -H site of thecompound is about 99-100%.

[0036] In some embodiments, the level of deuterium at the deuterium-enriched -H site of thecompound is no less than 50%.

[0037] In some embodiments, the level of deuterium at the deuterium-enriched -H site of thecompound is no less than 70%.

[0038] In some embodiments, the level of deuterium at the deuterium-enriched -H site of thecompound is no less than 90%.

[0039] In some embodiments, the level of deuterium at the deuterium-enriched -H site of thecompound is no less than 99%.

[0040] In some embodiments, each of D1, D2, and D3 is deuterium.

[0041] The present invention provides a compound having the following structure:.

[0042] In some embodiments, all of D1,enriched -H sites.

[0043] In some embodiments, the level of deuterium at the deuterium-enriched -H site of thecompound is about 20-100%.

[0044] In some embodiments, the level of deuterium at the deuterium-enriched -H site of thecompound is about 70-100%.

[0045] In some embodiments, the level of deuterium at the deuterium-enriched -H site of thecompound is about 90-100%

[0046] In some embodiments, the level of deuterium at the deuterium-enriched -H site of thecompound is about 97-100%.

[0047] In some embodiments, the level of deuterium at the deuterium-enriched -H site of thecompound is about 99-100%.

[0048] In some embodiments, the level of deuterium at the deuterium-enriched -H site of thecompound is no less than 50%.

[0049] In some embodiments, the level of deuterium at the deuterium-enriched -H site of thecompound is no less than 70%.

[0050] In some embodiments, the level of deuterium at the deuterium-enriched -H site of thecompound is no less than 90%.

[0051] In some embodiments, the level of deuterium at the deuterium-enriched -H site of thecompound is no less than 99%.

[0052] In some embodiments, each of D1, D2, and D3 is deuterium.

[0053] The present invention provides a composition comprising a compound having the followingstructure: , wherein when the compound is, then at least one of D1, D2, and D3is a-H site; or wherein when the compound is ,then at least two of D1, D2, and D3 are -H sites, or a pharmaceutically acceptable salt or ester thereof, and a pharmaceutically acceptable carrier.

[0054] In some embodiments, the compound has the following structure:.

[0055] In some embodiments, at least two of D1, D2, and D3 are deuterium-enriched -H sites.

[0056] In some embodiments, all of D1, D2, and D3 are deuterium-enriched -H sites.

[0057] In some embodiments, the level of deuterium at the deuterium-enriched -H site of thecompound is about 20-100%.

[0058] In some embodiments, the level of deuterium at the deuterium-enriched -H site of thecompound is about 70-100%.

[0059] In some embodiments, the level of deuterium at the deuterium-enriched -H site of thecompound is about 90-100%

[0060] In some embodiments, the level of deuterium at the deuterium-enriched -H site of thecompound is about 97-100%.

[0061] In some embodiments, the level of deuterium at the deuterium-enriched -H site of thecompound is about 99-100%.

[0062] In some embodiments, the level of deuterium at the deuterium-enriched -H site of thecompound is no less than 50%.

[0063] In some embodiments, the level of deuterium at the deuterium-enriched -H site of thecompound is no less than 70%.

[0064] In some embodiments, the level of deuterium at the deuterium-enriched -H site of thecompound is no less than 90%.

[0065] In some embodiments, the level of deuterium at the deuterium-enriched -H site of thecompound is no less than 99%.

[0066] In some embodiments, each of D1, D2, and D3 is deuterium.

[0067] In some embodiments, the compound has the following structure:.

[0068] In some embodiments, all of D1, are enriched -H sites.

[0069] In some embodiments, the level of deuterium at the deuterium-enriched -H site of thecompound is about 20-100%.

[0070] In some embodiments, the level of deuterium at the deuterium-enriched -H site of thecompound is about 70-100%.

[0071] In some embodiments, the level of deuterium at the deuterium-enriched -H site of thecompound is about 90-100%

[0072] In some embodiments, the level of deuterium at the deuterium-enriched -H site of thecompound is about 97-100%.

[0073] In some embodiments, the level of deuterium at the deuterium-enriched -H site of thecompound is about 99-100%.

[0074] In some embodiments, the level of deuterium at the deuterium-enriched -H site of thecompound is no less than 50%.

[0075] In some embodiments, the level of deuterium at the deuterium-enriched -H site of thecompound is no less than 70%.

[0076] In some embodiments, the level of deuterium at the deuterium-enriched -H site of thecompound is no less than 90%.

[0077] In some embodiments, the level of deuterium at the deuterium-enriched -H site of thecompound is no less than 99%.

[0078] In some embodiments, each of D1, D2, and D3 is deuterium.

[0079] In some embodiments, the pharmaceutically acceptable carrier is ascorbic acid.

[0080] In some embodiments, the composition further comprises a pharmaceutically active agent.

[0081] In some embodiments, the composition further comprises the following compound:.

[0082] In some embodiments, the amount in the composition is about 1%-90%;preferably, about 1%-80%; more preferably,more preferably, about 1%-60%; more preferably, about 1%-50%; more preferably, about 5%-50%; more preferably, about 5%-40%; more preferably, about 5%-30%; more preferably, about 5%-20%; more preferably, about 5%-10%; more preferably about 5%.

[0083] In some embodiments, the amount of in the composition is less than 60%,preferably, less than 50%; more preferably, less than 40%; more preferably, less than 30%; more preferably, less than 20%; more preferably, less than 10%; more preferably, less than 5%; more preferably, less than1%.

[0084] In some embodiments, the composition further comprises the following compound:.

[0085] In some embodiments, thethe following compound:.

[0086] In some embodiments, theratio amongin the composition is 1:2:1.

[0087] amongin the composition is 1:1:2.

[0088] amongin the composition is 2:1:1.

[0089] amongin the composition is 1:2:2.

[0090] among

[0091] In some embodiments, the concentration by weight ratio amongin the composition is 2:2:1.

[0092] amongin the composition is 1:1:1.

[0093] amongin the composition is 1:1.3:1.2.

[0094] a compound having the structure:.

[0095] In some embodiments, the amount in the composition is about 1%-90%;preferably, about 1%-80%; more preferably, more preferably, about 1%-60%; more preferably, about 1%-50%; more preferably, about 5%-50%; more preferably, about 5%-40%; more preferably, about 5%-30%; more preferably, about 5%-20%; more preferably, about 5%-10%; more preferably about 5%.

[0096] In some embodiments, the amount in the composition is less than 60%,preferably, less than 50%; more preferably, lessless than 30%; more preferably, less than 20%; more preferably, less than 10%; more preferably, less than 5%; more preferably, less than1%.

[0097] In some embodiments, the molar ratio is about1:50 to 100:1; preferably, about 1:10 to 100:1; moreabout 1:4 to 99:1; more preferably, about 1:3 to 98:1 more preferably, about 1:2 to 97:1 more preferably, about1:1 to 96:1 more preferably, about 1:1 to 95:1 more preferably, about 2:1 to 95:2 more preferably, about 3:1 to 95:2; more preferably, about 4:1 to 95:3; more preferably, about 5:1 to 95:4; more preferably, about 6:1 to 95:5.

[0098] In some embodiments, the weight ratio isabout 1:50 to 100:1; preferably, about 1:10 to 100:1; moreabout 1:4 to 99:1; more preferably, about 1:3 to 98:1 more about 1:1 to 96:1 more preferably, about 1:1 to 95:1 more preferably, about 2:1 to 95:2 more preferably, about 3:1 to 95:2; more preferably, about 4:1 to 95:3; more preferably, about 5:1 to 95:4; more preferably, about 6:1 to 95:5.

[0099] In some embodiments, the molar ratio is about1:50 to 100:1; preferably, about 1:10 to 100:1; more about1:4 to 99:1; more preferably, about 1:3 to 98:1 more preferably, about 1:2 to 97:1 more preferably, about 1:1 to 96:1 more preferably, about 1:1 to 95:1 more preferably, about 2:1 to 95:2 more preferably, about 3:1 to 95:2; more preferably, about 4:1 to 95:3; more preferably, about 5:1 to 95:4; more preferably, about 6:1 to 95:5.

[0100] In some embodiments, the weight ratio isabout 1:50 to 100:1; preferably, about 1:10 to 100:1; moreabout 1:4 to 99:1; more preferably, about 1:3 to 98:1 more preferably, about 1:2 to 97:1 more preferably, about 1:1 to 96:1 more preferably, about 1:1 to 95:1 more preferably, about 2:1 to 95:2 more preferably, about 3:1 to 95:2; more preferably, about 4:1 to 95:3; more preferably, about 5:1 to 95:4; more preferably, about 6:1 to 95:5.

[0101] In some embodiments, the molar ratio is about1:100 to 100: 1; preferably, about 1:90 to 90: 1; morepreferably, about 1:70 to 70: 1; more preferably, about 1:60 to 60: 1 more preferably, about 1:50 to 50: 1 more preferably, about 1:40 to 40: 1 more preferably, about 1:30 to 30: 1 more preferably, about 1:20 to 20:1more preferably, about 1:10 to 10:1; more preferably, about 1:5 to 5:1; more preferably, about 1:2 to 2:1; more preferably, about 1:1.2 to 1.2:1.

[0102] In some embodiments, the weight ratio isabout 1:100 to 100: 1; preferably, about 1:90 to 90: 1; moreabout 1:70 to 70: 1; more preferably, about 1:60 to 60: 1 more preferably, about 1:40 to 40: 1 more preferably, about 1:30 to 30: 1 more preferably, about 1:20 to 20:1 more preferably, about 1:10 to 10:1; more preferably, about 1:5 to 5:1; more preferably, about 1:2 to 2:1; more preferably, about 1:1.2 to 1.2:1.

[0103] In some embodiments, the weight percentage ofin the composition is at least 70%.

[0104] in the composition is at least 80%.

[0105] in the composition is at least 90%.

[0106] in the composition is at least 95%.

[0107] in the composition is at least 97%.

[0108] in the composition is at least 98%.

[0109] in the composition is at least 99%.

[0110] in the composition is at least 99.5%.

[0111] in the composition is at least 99.9%.

[0112] and a pharmaceutically acceptable carrier.

[0113] In somepharmaceutically acceptable carrier is ascorbic acid.

[0114] In some embodiments, the composition further comprises the following compound:.

[0115] In some embodiments, the.

[0116] In some embodiments, thebetween

[0117] In some embodiments, the concentration ratio by weight between

[0118] ratio by weight between

[0119] ratio by weight between

[0120] ratio by weight between

[0121] ratio by weight between

[0122] ratio by weight between

[0123] ratio by weight between

[0124] ratio by weight between

[0125] ratio by weight between1.

[0126] on ratio by weight between

[0127] ratio by weight betweenand is 1:3.

[0128] ratio by weight between

[0129] ratio by weight between

[0130] ratio by weight between

[0131] ratio by weight between

[0132] ratio by weight between

[0133] ratio by weight between9.

[0134] on ratio by weight between

[0135] further comprises a compound having the structure:.

[0136] In some embodiments, the weight percentage ofthe composition is at least 70%.

[0137] ofthe composition is at least 80%.

[0138] ofthe composition is at least 90%.

[0139] ofthe composition is at least 95%.

[0140] ofthe composition is at least 97%.

[0141] ofin the composition is at least 98%.

[0142] ercentage ofthe composition is at least 99%.

[0143] ofthe composition is at least 99.5%.

[0144] ofthe composition is at least 99.9%.

[0145] of producing a compound of formula (III) having thefollowing structure: (III), wherein at least one of D1, D2, and D3is a deuterium-enriched -H site; the process comprises: (a) reacting a compound of formula (I) with a fluorine-containing acid or a salt thereof,(I); (b) adding an acid to produce a compound of formula (II):(II); (c) adding an acid to produce a compound of formula (III):.

[0146] In some embodiments, the compound of formula (I) has the following structure.

[0147] In some embodiments, the compound of formula (II) has the following structure:.

[0148] In some embodiments, thehas the following structure:.

[0149] In some embodiments, thehas the following structure:.

[0150] In some embodiments, thehas the following structure:.

[0151] In some embodiments, the compound of formula (III) has the following structure:.

[0152] In some embodiments, theis KF, CaF2, NaF, AlF3, MgF2, ZnF2, FeF2or FeF.

[0153] In some embodiments, the fluorine-containing salt is KF or NaF.

[0154] In some embodiments, the fluorine-containing salt is KF.

[0155] In some embodiments, step (a) further comprises adding 18-Crown-6, tert-butanol; andacetonitrile (MeCN).

[0156] In some embodiments, the molar ratio between tert-butanol and acetonitrile is 10:1.

[0157] In some embodiments, the molar ratio between tert-butanol and acetonitrile is 9:1.

[0158] In some embodiments, the molar ratio between tert-butanol and acetonitrile is 8:1.

[0159] In some embodiments, the molar ratio between tert-butanol and acetonitrile is 7:1.

[0160] In some embodiments, the molar ratio between tert-butanol and acetonitrile is 6:1.

[0161] In some embodiments, the molar ratio between tert-butanol and acetonitrile is 5:1.

[0162] In some embodiments, the molar ratio between tert-butanol and acetonitrile is 4:1.

[0163] In some embodiments, the reaction in step (a) is conducted at a temperature of about 50-110°C.

[0164] In some embodiments, the reaction in step (a) is conducted at a temperature of about 60-100°C.

[0165] In some embodiments, the reaction in step (a) is conducted at a temperature of about 70-90 °C.

[0166] In some embodiments, the reaction in step (a) is conducted at a temperature of about 80 °C.

[0167] In some embodiments, the reaction in step (a) is conducted for about 10-50 minutes.

[0168] In some embodiments, the reaction in step (a) is conducted for about 10-40 minutes.

[0169] In some embodiments, the reaction in step (a) is conducted for about 10-30 minutes.

[0170] In some embodiments, the reaction in step (a) is conducted for about 20 minutes.

[0171] In some embodiments, the acid in step (b) is an inorganic acid.

[0172] In some embodiments, the acid in step (b) is H2SO4.

[0173] In some embodiments, the acid in step (b) is about 10%-50% H2SO4 in Dichloromethane(DCM).

[0174] In some embodiments, the acid in step (b) is about 10%-40% H2SO4 in DCM.

[0175] In some embodiments, the acid in step (b) is about 10%-30% H2SO4 in DCM .

[0176] In some embodiments, the acid in step (b) is about 20% H2SO4 in DCM .

[0177] In some embodiments, the reaction in step (b) is conducted at a temperature of about 10-50 °C.

[0178] In some embodiments, the reaction in step (b) is conducted at a temperature of about 20-40 °C.

[0179] In some embodiments, the reaction in step (b) is conducted at a temperature of about 20-30 °C.

[0180] In some embodiments, the reaction in step (b) is conducted at a temperature of about 25 °C.

[0181] In some embodiments, the reaction in step (b) is conducted for about 1 hour to 5 hours.

[0182] In some embodiments, the reaction in step (b) is conducted for about 1 hour to 4 hours.

[0183] In some embodiments, the reaction in step (b) is conducted for about 1 hour to 3 hours.

[0184] In some embodiments, the reaction in step (b) is conducted for about 2 hours.

[0185] In some embodiments, the acid in step (c) is Trifluoroacetic acid (TFA).

[0186] In some embodiments, TFA is dissolved in DCM.

[0187] In some embodiments, the reaction in step (c) is conducted at a temperature of about -10-10°C.

[0188] In some embodiments, the reaction in step (c) is conducted at a temperature of about -5-5 °C.

[0189] In some embodiments, the reaction in step (c) is conducted at a temperature of about -3-3 °C.

[0190] In some embodiments, the reaction in step (c) is conducted at a temperature of about 0 °C.

[0191] In some embodiments, the reaction in step (c) is conducted for about 5 minutes to 60 minutes.

[0192] In some embodiments, the reaction in step (c) is conducted for about 5 minutes to 50 minutes.

[0193] In some embodiments, the reaction in step (c) is conducted for about 5 minutes to 40 minutes.

[0194] In some embodiments, the reaction in step (c) is conducted for about 5 minutes to 30 minutes.

[0195] In some embodiments, the reaction in step (c) is conducted for about 10 minutes to 20 minutes.

[0196] In some embodiments, the reaction in step (c) is conducted for about 15 minutes.

[0197] In some embodiments, the compound of formula (III) is stored at room temperature for a periodof 5-24 hours.

[0198] In some embodiments, the compound of formula (III) is stored at room temperature for a periodof 5-20 hours.

[0199] In some embodiments, the compound of formula (III) is stored at room temperature for a periodof 5-15 hours.

[0200] In some embodiments, the compound of formula (III) is stored at room temperature for a periodof 10-15 hours.

[0201] In some embodiments, the compound of formula (III) is stored at room temperature for a periodof 12 hours.’

[0202] The present invention provides a process of producing a compound having the followingstructure: , wherein at least one of D1, D2, and D3-H site; the process comprises: (a) conducting a radio-fluorination reaction to the compound of formula (I)(I) to obtain a compound of formula(b) adding an acid.

[0203] In some embodiments, the acid in step (b) removes the -tBu, -SO3H and Boc groups of formula(IV).

[0204] In some embodiments, the acid in step (b) remove -tBu, -SO3H and Boc groups of formula (IV)in a one batch manner.

[0205] In some embodiments, the reaction in step (b) is conducted in a single pot.

[0206] In some embodiments, the reaction in step (a) and the reaction step (b) are carried out indifferent pots.

[0207] In some embodiments, the radio-fluorination reaction is conducted [18F] fluoride ion ([18F]F-),4,7,13,16,21,24-Hexaoxa-1,10-diazabicyclo[8.8.8]hexacosane (Kryptofix 222), K2CO3, and tert-amyl alcohol.

[0208] In some embodiments, the radio-fluorination reaction comprising adding an acid, preferably,the acid is inorganic acid; more preferably, the acid is formic acid.

[0209] In some embodiments, the reaction in step (a) is conducted at a temperature of about 50-130°C.

[0210] In some embodiments, the reaction in step (a) is conducted at a temperature of about 60-120°C.

[0211] In some embodiments, the reaction in step (a) is conducted at a temperature of about 70-110°C.

[0212] In some embodiments, the reaction in step (a) is conducted at a temperature of about 80-100°C.

[0213] In some embodiments, the reaction in step (a) is conducted at a temperature of about 90 °C.

[0214] In some embodiments, the reaction in step (a) is conducted for about 1-20 minutes.

[0215] In some embodiments, the reaction in step (a) is conducted for about 1-15 minutes.

[0216] In some embodiments, the reaction in step (a) is conducted for about 5-15 minutes.

[0217] In some embodiments, the reaction in step (a) is conducted for about 10 minutes.

[0218] In some embodiments, the acid in step (b) is an inorganic acid.

[0219] In some embodiments, the acid in step (b) is HCl.

[0220] In some embodiments, the concentration of HCl is about 0.5-10M.

[0221] In some embodiments, the concentration of HCl is about 0.5-8.5M.

[0222] In some embodiments, the concentration of HCl is about 0.5-6.5M.

[0223] In some embodiments, the concentration of HCl is about 1-4.5M.

[0224] In some embodiments, the concentration of HCl is about 2.5M.

[0225] In some embodiments, the reaction in step (b) is conducted at a temperature of about 80-140°C.

[0226] In some embodiments, the reaction in step (b) is conducted at a temperature of about 90-130°C.

[0227] In some embodiments, the reaction in step (b) is conducted at a temperature of about 100-120°C.

[0228] In some embodiments, the reaction in step (b) is conducted at a temperature of about 105-115°C.

[0229] In some embodiments, the reaction in step (b) is conducted at a temperature of about 110 °C.

[0230] In some embodiments, the reaction in step (b) is conducted for about 1-20 minutes.

[0231] In some embodiments, the reaction in step (b) is conducted for about 1-15 minutes.

[0232] In some embodiments, the reaction in step (b) is conducted for about 1-10 minutes.

[0233] In some embodiments, the reaction in step (b) is conducted for about 5 minutes.

[0234] In some embodiments, the compound of formula (I) has the following structure.

[0235] In some embodiments, thehas the following structure.

[0236] In some embodiments, the compoundhas the following structure.

[0237] In some embodiments, thehas the following structure.

[0238] In some embodiments, thethe following steps to produce thecompound of formula (I): (a) reacting a compound having the structure with a deuterium labeling agent, to obtain a compound having the; (b) adding Boc2O to obtain a compound h e:; (c) adding N, N'-Diisopropylcarbodiimide (DIC) to obtain a compound having the structure:; (d) adding SOCl2 to obtain a compoundstructure:; (e) adding NaIO4 to obtain the

[0239] In some .

[0240] In some .

[0241] In some .

[0242] In some embodiments,.

[0243] In some embodiments, has the .

[0244] In some embodiment .

[0245] In some embodiments, has the structure of .

[0246] In some embodiments, has the structure of .

[0247] In some .

[0248] In some has the .\

[0249] In someagent is

[0250] In some embodiments, step (a) further comprises adding H2 and a catalyst of Rutheniumnanoparticles on a carbon surface (Ru / C).

[0251] In some embodiments, the reaction in step (a) is conducted at a temperature of about 50-110°C.

[0252] In some embodiments, the reaction in step (a) is conducted at a temperature of about 60-100°C.

[0253] In some embodiments, the reaction in step (a) is conducted at a temperature of about 70-90 °C.

[0254] In some embodiments, the reaction in step (a) is conducted at a temperature of about 80 °C.

[0255] In some embodiments, the reaction in step (a) is conducted for about 1-10 days.

[0256] In some embodiments, the reaction in step (a) is conducted for about 1-8 days.

[0257] In some embodiments, the reaction in step (a) is conducted for about 1-6 days.

[0258] In some embodiments, the reaction in step (a) is conducted for about 1-4 days.

[0259] In some embodiments, the reaction in step (a) is conducted for about 1-3 days.

[0260] In some embodiments, the reaction in step (a) is conducted for about 2 days.

[0261] In some embodiments, step (b) further comprises adding NaOH and 1,4-dioxane.

[0262] In some embodiments, the reaction in step (b) is conducted at a temperature of about 10-50 °C.

[0263] In some embodiments, the reaction in step (b) is conducted at a temperature of about 20-40 °C.

[0264] In some embodiments, the reaction in step (b) is conducted at a temperature of about 20-30 °C.

[0265] In some embodiments, the reaction in step (b) is conducted at a temperature of about 25 °C.

[0266] In some embodiments, the reaction in step (b) is stored at room temperature for a period of 5-24 hours.

[0267] In some embodiments, the reaction in step (b) is stored at room temperature for a period of 5-20 hours.

[0268] In some embodiments, the reaction in step (b) is stored at room temperature for a period 5-15hours.

[0269] In some embodiments, the reaction in step (b) is stored at room temperature for a period of 10-15 hours.

[0270] In some embodiments, the reaction in step (b) is stored at room temperature for a period of 12hours.

[0271] In some embodiments, step (c) further comprises adding DCM.

[0272] In some embodiments, the reaction in step (c) is conducted at a temperature of about -10-10°C; preferably, the reaction in step (c) is conducted at a temperature of about -5-5 °C; more preferably, the reaction in step (c) is conducted at a temperature of about -3-3 °C; more preferably, the reaction in step (c) is conducted at a temperature of about 0 °C.

[0273] In some embodiments, the reaction in step (c) is conducted for about 10-60 minutes; preferably,the reaction in step (c) is conducted for about 10-50 minutes; more preferably, the reaction in step (c) is conducted for about 20-40 minutes; more preferably, the reaction in step (c) is conducted for about 30 minutes.

[0274] In some embodiments, the solution obtained in step (c) is further stored at a temperature ofabout 10-50 °C; preferably, the solution obtained in step (c) is further stored at a temperature of about 20- 40 °C; more preferably, the solution obtained in step (c) is further stored at a temperature of about 20-30 °C; more preferably, the solution obtained in step (c) is further stored at a temperature of about 25 °C.

[0275] In some embodiments, the solution obtained in step (c) is further stored at room temperaturefor a period of 5-24 hours; preferably, the solution obtained in step (c) is further stored at room temperature for a period of 5-20 hours; more preferably, the solution obtained in step (c) is further stored at room temperature for a period 5-15 hours; more preferably, the solution obtained in step (c) is further stored at room temperature for a period of 10-15 hours; more preferably, the solution obtained in step (c) is further stored at room temperature for a period of 12 hours.

[0276] In some embodiments, step (d) further comprises adding MeCN at a temperature of about -80-0 °C; preferably, step (d) further comprises adding MeCN at a temperature of about -60 - -20 °C; more preferably, step (d) further comprises adding MeCN at a temperature of about -50 - -30 °C; more preferably, step (d) further comprises adding MeCN at a temperature of about -40 °C.

[0277] In some embodiments, step (d) further comprises adding MeCN with SOCl2 at a temperatureof about -80 to 0 °C; preferably, step (d) further comprises adding MeCN with SOCl2 at a temperature of about -60 to -20 °C; more preferably, step (d) further comprises adding MeCN with SOCl2 at a temperature of about -50 to -30 °C; more preferably, step (d) further comprises adding MeCN with SOCl2 at a temperature of about -40 °C.

[0278] In some embodiments, step (d) further comprises adding MeCN and pyridine at a temperatureof about -80 to 0 °C; preferably, step (d) further comprises adding MeCN and pyridine at a temperature of about -60 to -20 °C; more preferably, step (d) further comprises adding MeCN and pyridine at a temperature of about -50 to -30 °C; more preferably, step (d) further comprises adding MeCN and pyridine at a temperature of about -40°C.

[0279] In some embodiments, the compound obtained in step (d) is further stored at a temperature ofabout 10-50 °C; preferably, the solution obtained in step (d) is further stored at a temperature of about 20- 40 °C; more preferably, the solution obtained in step (d) is further stored at a temperature of about 20-30 °C; more preferably, the solution obtained in step (d) is further stored at a temperature of about 25 °C.

[0280] In some embodiments, the compound obtained in step (d) is further stored at room temperaturefor a period of 10-70 minutes; preferably, the solution obtained in step (d) is further stored at room temperature for a period of 20-60 minutes; more preferably, the solution obtained in step (d) is further stored at room temperature for a period 30-50 minutes; more preferably, the solution obtained in step (d) is further stored at room temperature for a period of 40 minutes.

[0281] In some embodiments, step (e) further comprises adding RuCl3·H2O, and MeCN.

[0282] In some embodiments, the reaction in step (e) is conducted at a temperature of about -10-10°C; preferably, the reaction in step (e) is conducted at a temperature of about -5-5 °C; more preferably, thereaction in step (e) is conducted at a temperature of about -3-3 °C; more preferably, the reaction in step (e) is conducted at a temperature of about 0 °C.

[0283] In some embodiments, the reaction in step (e) is conducted for about 1 hour to 5 hours;preferably, the reaction in step (e) is conducted for about 1 hour to 4 hours; more preferably, the reaction in step (e) is conducted for about 1 hour to 3 hours; more preferably, the reaction in step (e) is conducted for about 2 hours.

[0284] In some embodiments, the present invention provides a method of detecting target cells in asubject comprising administering an effective amount of the compound disclosed herein or the composition disclosed herein to the subject, and imaging the subject with a molecular imaging device to detect the compound or the composition in the subject.

[0285] In some embodiments, the present invention provides a method of imaging target cells in asubject comprising: (a) administering to the subject an effective amount of the compound disclosed herein or thecomposition disclosed herein, wherein the compound or the composition specifically accumulates at the target cells in the subject; (b) detecting in the subject the location of the compound or the composition; and(c) obtaining an image of the target cells in the subject based on the location of the compound orthe composition in the subject.

[0286] In some embodiments, the present invention provides a method of detecting the presence oftarget cells in a subject which comprises determining if an amount of the compound disclosed herein or the composition disclosed herein is present in the subject at a period of time after administration of the compound or the composition to the subject, thereby detecting the presence of the target cells based on the amount of the compound or the composition determined to be present in the subject.

[0287] In some embodiments, the period of time is about 5-150 minutes; preferably, the period of timeis about 10-120 minutes; more preferably, the period of time is about 10-90 minutes; more preferably, the period of time is about 20-60 minutes; more preferably, the period of time is about 20-50 minutes; more preferably, the period of time is about 30-40 minutes.

[0288] In some embodiments, the present invention provides a method of imaging tumors in a subjectby administering to the subject an effective amount of the compound disclosed herein or the composition disclosed herein.

[0289] In some embodiments, the present invention provides a method of detecting bacterial infectionin a subject by administering to the subject an effective amount of the compound disclosed herein or the composition disclosed herein.

[0290] In some embodiments, the bacterial infection is caused by gram-negative or gram-positivebacteria; preferably, the bacterial infection is caused by S. Aureus, Escherichia coli, Mannheimia haemotytica, Pasteurella multocida, Histophilus somni, Actinobacillus pleuropneumoniae, Salmonella enteritidis, Salmonella gallinahum, Lawsonia intracellularis, Brachyspira hyodysenteriae, Brachyspira pilosicoli, Acinetobacter baumannii, Acinetobacter spp., Citrobacter spp., Enterobacter aerogenes, Enterobacter cloacae, Klebsiella oxytoca, Klebsiella pneumoniae, Serratia marcescens, Stenotrophomonas maltophilia, and Pseudomonas aeruginosa.

[0291] In some embodiments, the bacterial infection is caused by gram-negative or gram-positivebacteria; preferably, the bacterial infection is caused by E. coli or S. Aureus.

[0292] In some embodiments, the present invention provides a use of the compound disclosed hereinor the composition disclosed herein in imaging tumors in a subject and / or detecting bacterial infection in a subject.

[0293] In some embodiments, the present invention provides a compound for use in imaging tumorsin a subject and / or detecting bacterial infection in a subject

[0294] In some embodiments, the present invention includes a pharmaceutically acceptable salt of anyof the above compounds of the present invention.

[0295] In some embodiments, a salt of the compound of the present invention is used in any of theabove methods, uses, packages or compositions.

[0296] In some embodiments, a pharmaceutically acceptable salt of the compound of the presentinvention is used in any of the above methods, uses, packages or compositions.

[0297] In some embodiments, an ester of the compound of the present invention is used in any of theabove methods, uses, packages or compositions.

[0298] Any of the above compounds may be used in any of the disclosed methods, uses, packages orpharmaceutical compositions.

[0299] Any of the compounds used in the disclosed methods, uses, packages or pharmaceuticalcompositions may be replaced with any other compound disclosed in the present invention.

[0300] Any of the above generic compounds may be used in any of the disclosed methods, uses,packages or compositions.

[0301] A person skilled in the art may use the techniques disclosed therein to prepare compoundswhich are not enriched in deuterium and thereafter use the techniques disclosed herein to prepare deuterium analogs thereof.

[0302] Except where otherwise specified, the structure of a compound of this invention includes anasymmetric carbon atom, it is understood that the compound occurs as a racemate, racemic mixture, scalemic mixtures and isolated single enantiomers. All such isomeric forms of these compounds are expressly included in this invention. Except where otherwise specified, each stereogenic carbon may be of the R or S configuration. It is to be understood accordingly that the isomers arising from such asymmetry (e.g., all enantiomers and diastereomers) are included within the scope of this invention, unless indicated otherwise. Such isomers can be obtained in substantially pure form by classical separation techniques and by stereochemically controlled synthesis, such as those described in "Enantiomers, Racemates and Resolutions" by J. Jacques, A. Collet and S. Wilen, Pub. John Wiley & Sons, NY, 1981. For example, the resolution may be carried out by preparative chromatography on a chiral column.

[0303] Except where otherwise specified, the subject invention is intended to include all isotopes ofatoms occurring on the compounds disclosed herein. Isotopes include those atoms having the same atomic number but different mass numbers. By way of general example and without limitation, isotopes of hydrogen include tritium and deuterium. Isotopes of carbon include C-13 and C-14.

[0304] It will be noted that any notations of a carbon in structures throughout this application, whenused without further notation, are intended to represent all isotopes of carbon, such as12C,13C, or14C. Furthermore, any compounds containing13C or14C may specifically have the structure of any of the compounds disclosed herein.

[0305] It will also be noted that any notations of hydrogen (H) in structures throughout this application,when used without further notation, are intended to represent all isotopes of hydrogen, such as1H,2H (D), or3H (T) except where otherwise specified. Furthermore, any compounds containing2H or3H may specifically have the structure of any of the compounds disclosed herein except where otherwise specified.

[0306] Isotopically-labeled compounds can generally be prepared by conventional techniques knownto those skilled in the art using appropriate isotopically-labeled reagents in place of the non-labeled reagents employed.

[0307] Deuterium (2H or D) is a stable, non-radioactive isotope of hydrogen and has an atomic weightof 2.0144. Hydrogen atom in a compound naturally occurs as a mixture of isotopes1H (hydrogen or protium), D (2H or deuterium), and T (3H or tritium). The natural abundance of deuterium is 0.0156%. Thus, in a composition comprising molecules of a naturally occurring compound, the level of deuterium at aparticular hydrogen atom site in that compound is expected to be 0.0156%. Thus, a composition comprising a compound with a level of deuterium at any site of hydrogen atom in the compound that has been enriched to be greater than its natural abundance of 0.0156% is novel over its naturally occurring counterpart.

[0308] As used herein, a hydrogen at a specific site in a compound is “deuterium-enriched” if theamount of deuterium at the specific site in the compound is more than the abundance of deuterium naturally occurring at that specific site in view of all of the molecules of the compound in a defined universe such as a composition or sample. Naturally occurring as used above refers to the abundance of deuterium which would be present at a relevant site in a compound if the compound was prepared without any affirmative step to enrich the abundance of deuterium. Thus, at a "deuterium-enriched” site in a compound, the abundance of deuterium at that site can range from more than 0.0156% to 100%. Examples of ways to obtain a deuterium-enriched site in a compound are exchanging hydrogen with deuterium or synthesizing the compound with deuterium-enriched starting materials.

[0309] In the compounds used in the method of the present invention, the substituents may besubstituted or unsubstituted, unless specifically defined otherwise.

[0310] In the compounds used in the method of the present invention, alkyl, heteroalkyl, monocycle,bicycle, aryl, heteroaryl and heterocycle groups can be further substituted by replacing one or more hydrogen atoms with alternative non-hydrogen groups. These include, but are not limited to, halo, hydroxy, mercapto, amino, carboxy, cyano and carbamoyl.

[0311] It is understood that substituents and substitution patterns on the compounds used in the methodof the present invention can be selected by one of ordinary skill in the art to provide compounds that are chemically stable and that can be readily synthesized by techniques known in the art from readily available starting materials. If a substituent is itself substituted with more than one group, it is understood that these multiple groups may be on the same carbon or on different carbons, so long as a stable structure results.

[0312] In choosing the compounds used in the method of the present invention, one of the ordinaryskill in the art will recognize that the various substituents, i.e. R1, R2, etc. are to be chosen in conformity with well-known principles of chemical structure connectivity.

[0313] The compounds used in the method of the present invention may be prepared by techniqueswell known in organic synthesis and familiar to a practitioner ordinarily skilled in the art. However, these may not be the only means by which to synthesize or obtain the desired compounds.

[0314] The compounds used in the method of the present invention may be prepared by techniquesdescribed in Vogel’s Textbook of Practical Organic Chemistry, A.I. Vogel, A.R. Tatchell, B.S. Furnis, A.J. Hannaford, P.W.G. Smith, (Prentice Hall) 5thEdition (1996), March's Advanced Organic Chemistry:Reactions, Mechanisms, and Structure, Michael B. Smith, Jerry March, (Wiley-Interscience) 5thEdition (2007), and references therein, which are incorporated by reference herein. However, these may not be the only means by which to synthesize or obtain the desired compounds.

[0315] The various R groups attached to the aromatic rings of the compounds disclosed herein may beadded to the rings by standard procedures, for example those set forth in Advanced Organic Chemistry: Part B: Reactions and Synthesis, Francis Carey and Richard Sundberg, (Springer) 5th ed. Edition. (2007), the content of which is hereby incorporated by reference.

[0316] Another aspect of the invention comprises a compound used in the method of the presentinvention as a pharmaceutical composition.

[0317] As used herein, the term “pharmaceutically active agent” means any substance or compoundsuitable for administration to a subject and furnishes biological activity or other direct effect in the treatment, cure, mitigation, diagnosis, or prevention of disease, or affects the structure or any function of the subject. Pharmaceutically active agents include, but are not limited to, substances and compounds described in the Physicians’ Desk Reference (PDR Network, LLC; 64th edition; November 15, 2009) and “Approved Drug Products with Therapeutic Equivalence Evaluations” (U.S. Department of Health and Human Services, 30thedition, 2010), which are hereby incorporated by reference. Pharmaceutically active agents which have pendant carboxylic acid groups may be modified in accordance with the present invention using standard esterification reactions and methods readily available and known to those having ordinary skill in the art of chemical synthesis. Where a pharmaceutically active agent does not possess a carboxylic acid group, the ordinarily skilled artisan will be able to design and incorporate a carboxylic acid group into the pharmaceutically active agent where esterification may subsequently be carried out so long as the modification does not interfere with the pharmaceutically active agent’s biological activity or effect.

[0318] The compounds used in the method of the present invention may be in salt form. As usedherein, a “salt” is a salt of the instant compounds which has been modified by making acid or base salts of the compounds. In the case of compounds used to treat a disease or medical disorder, salt is pharmaceutically acceptable. Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as phenols; alkali or organic salts of acidic residues such as carboxylic acids. The salts can be made using an organic or inorganic acid. Such acid salts are chlorides, bromides, sulfates, nitrates, phosphates, sulfonates, formates, tartrates, maleates, malates, citrates, benzoates, salicylates, ascorbates, and the like. Phenolate salts are the sodium, potassium, or lithium salts, and the like. Carboxylate salts are sodium, potassium, or lithium salts, and the like. The term "pharmaceutically acceptable salt" in this respect, refers to the relatively non-toxic, inorganic and organic acid or base addition salts of compounds of the presentinvention. These salts can be prepared in situ during the final isolation and purification of the compounds of the invention, or by separately reacting to a purified compound of the invention in its free base or free acid form with a suitable organic or inorganic acid or base, and isolating the salt thus formed. Representative salts include hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate, valerate, oleate, palmitate, stearate, laurate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, napthylate, mesylate, glucoheptonate, lactobionate, and laurylsulphonate salts and the like. (See, e.g., Berge et al. (1977) "Pharmaceutical Salts", J. Pharm. Sci.66:1-19).

[0319] As used herein, "treating" means preventing, slowing, halting, or reversing the progression ofa disease. Treating may also mean improving one or more symptoms of a disease.

[0320] The compounds used in the method of the present invention may be administered in variousforms, including those detailed herein. The treatment with the compound may be a component of a combination therapy or an adjunct therapy, i.e. the subject or patient in need of the drug is treated or given another drug for the disease in conjunction with one or more of the instant compounds. This combination therapy can be sequential therapy where the patient is treated first with one drug and then the other or the two drugs are given simultaneously. These can be administered independently by the same route or by two or more different routes of administration depending on the dosage forms employed.

[0321] As used herein, a "pharmaceutically acceptable carrier" is a pharmaceutically acceptablesolvent, suspending agent or vehicle, for delivering the instant compounds to the animal or human. The carrier may be liquid or solid and is selected with the planned manner of administration in mind. Liposomes are also a pharmaceutically acceptable carrier, as are capsules, coatings and various syringes.

[0322] The dosage of the compounds administered in treatment will vary depending upon factors suchas the pharmacodynamic characteristics of a specific chemotherapeutic agent and its mode and route of administration; the age, sex, metabolic rate, absorptive efficiency, health and weight of the recipient; the nature and extent of the symptoms; the kind of concurrent treatment being administered; the frequency of treatment with; and the desired therapeutic effect.

[0323] A dosage unit of the compounds used in the method of the present invention may comprise asingle compound or mixtures thereof with additional agents. The compounds can be administered in oral dosage forms as tablets, capsules, pills, powders, granules, elixirs, tinctures, suspensions, syrups, and emulsions. The compounds may also be administered in intravenous (bolus or infusion), intraperitoneal, subcutaneous, or intramuscular form, or introduced directly, e.g. by injection, topical application, or other methods, into or onto a site of disease, all using dosage forms well known to those of ordinary skill in the pharmaceutical arts.

[0324] The compounds used in the method of the present invention can be administered in admixturewith suitable pharmaceutical diluents, extenders, excipients, or carriers (collectively referred to herein as a pharmaceutically acceptable carrier) suitably selected with respect to the intended form of administration and as consistent with conventional pharmaceutical practices. The unit will be in a form suitable for oral, rectal, topical, intravenous or direct injection or parenteral administration. The compounds can be administered alone or mixed with a pharmaceutically acceptable carrier. This carrier can be a solid or liquid, and the type of carrier is generally chosen based on the type of administration being used. The active agent can be co-administered in the form of a tablet or capsule, liposome, as an agglomerated powder or in a liquid form. Examples of suitable solid carriers include lactose, sucrose, gelatin and agar. Capsules or tablets can be easily formulated and can be made easy to swallow or chew; other solid forms include granules, and bulk powders. Tablets may contain suitable binders, lubricants, diluents, disintegrating agents, coloring agents, flavoring agents, flow-inducing agents, and melting agents. Examples of suitable liquid dosage forms include solutions or suspensions in water, pharmaceutically acceptable fats and oils, alcohols or other organic solvents, including esters, emulsions, syrups or elixirs, suspensions, solutions and / or suspensions reconstituted from non-effervescent granules and effervescent preparations reconstituted from effervescent granules. Such liquid dosage forms may contain, for example, suitable solvents, preservatives, emulsifying agents, suspending agents, diluents, sweeteners, thickeners, and melting agents. Oral dosage forms optionally contain flavorants and coloring agents. Parenteral and intravenous forms may also include minerals and other materials to make them compatible with the type of injection or delivery system chosen.

[0325] Techniques and compositions for making dosage forms useful in the present invention aredescribed in the following references: 7 Modern Pharmaceutics, Chapters 9 and 10 (Banker & Rhodes, Editors, 1979); Pharmaceutical Dosage Forms: Tablets (Lieberman et al. 1981); Ansel, Introduction to Pharmaceutical Dosage Forms 2nd Edition (1976); Remington's Pharmaceutical Sciences, 17th ed. (Mack Publishing Company, Easton, Pa., 1985); Advances in Pharmaceutical Sciences (David Ganderton, Trevor Jones, Eds., 1992); Advances in Pharmaceutical Sciences Vol. 7. (David Ganderton, Trevor Jones, James McGinity, Eds., 1995); Aqueous Polymeric Coatings for Pharmaceutical Dosage Forms (Drugs and the Pharmaceutical Sciences, Series 36 (James McGinity, Ed., 1989); Pharmaceutical Particulate Carriers: Therapeutic Applications: Drugs and the Pharmaceutical Sciences, Vol 61 (Alain Rolland, Ed., 1993); Drug Delivery to the Gastrointestinal Tract (Ellis Horwood Books in the Biological Sciences. Series in Pharmaceutical Technology; J. G. Hardy, S. S. Davis, Clive G. Wilson, Eds.); Modem Pharmaceutics Drugs and the Pharmaceutical Sciences, Vol 40 (Gilbert S. Banker, Christopher T. Rhodes, Eds.). All of the aforementioned publications are incorporated by reference herein.

[0326] Tablets may contain suitable binders, lubricants, disintegrating agents, coloring agents,flavoring agents, flow-inducing agents, and melting agents. For instance, for oral administration in the dosage unit form of a tablet or capsule, the active drug component can be combined with an oral, non-toxic, pharmaceutically acceptable, inert carrier such as lactose, gelatin, agar, starch, sucrose, glucose, methyl cellulose, magnesium stearate, dicalcium phosphate, calcium sulfate, mannitol, sorbitol and the like. Suitable binders include starch, gelatin, natural sugars such as glucose or beta-lactose, corn sweeteners, natural and synthetic gums such as acacia, tragacanth, or sodium alginate, carboxymethylcellulose, polyethylene glycol, waxes, and the like. Lubricants used in these dosage forms include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, and the like. Disintegrators include, without limitation, starch, methyl cellulose, agar, bentonite, xanthan gum, and the like.

[0327] The compounds used in the method of the present invention may also be administered in theform of liposome delivery systems, such as small unilamellar vesicles, large unilamellar vesicles, and multilamellar vesicles. Liposomes can be formed from a variety of phospholipids, such as cholesterol, stearylamine, or phosphatidylcholines. The compounds may be administered as components of tissue- targeted emulsions.

[0328] The compounds used in the method of the present invention may also be coupled to solublepolymers as targetable drug carriers or as a prodrug. Such polymers include polyvinylpyrrolidone, pyran copolymer, polyhydroxylpropylmethacrylamide-phenol, polyhydroxyethylasparta-midephenol, or polyethyleneoxide-polylysine substituted with palmitoyl residues. Furthermore, the compounds may be coupled to a class of biodegradable polymers useful in achieving controlled release of a drug, for example, polylactic acid, polyglycolic acid, copolymers of polylactic and polyglycolic acid, polyepsilon caprolactone, polyhydroxy butyric acid, polyorthoesters, polyacetals, polydihydropyrans, polycyanoacylates, and crosslinked or amphipathic block copolymers of hydrogels.

[0329] Gelatin capsules may contain active ingredient compounds and powdered carriers, such aslactose, starch, cellulose derivatives, magnesium stearate, stearic acid, and the like. Similar diluents can be used to make compressed tablets. Both tablets and capsules can be manufactured as immediate release products or as sustained release products to provide for continuous release of medication over a period of hours. Compressed tablets can be sugar coated or film coated to mask any unpleasant taste and protect the tablet from the atmosphere, or enteric coated for selective disintegration in the gastrointestinal tract.

[0330] For oral administration in liquid dosage form, the oral drug components are combined with anyoral, non-toxic, pharmaceutically acceptable inert carrier such as ethanol, glycerol, water, and the like. Examples of suitable liquid dosage forms include solutions or suspensions in water, pharmaceuticallyacceptable fats and oils, alcohols or other organic solvents, including esters, emulsions, syrups or elixirs, suspensions, solutions and / or suspensions reconstituted from non-effervescent granules and effervescent preparations reconstituted from effervescent granules. Such liquid dosage forms may contain, for example, suitable solvents, preservatives, emulsifying agents, suspending agents, diluents, sweeteners, thickeners, and melting agents.

[0331] Liquid dosage forms for oral administration can contain coloring and flavoring to increasepatient acceptance. In general, water, a suitable oil, saline, aqueous dextrose (glucose), and related sugar solutions and glycols such as propylene glycol or polyethylene glycols are suitable carriers for parenteral solutions. Solutions for parenteral administration preferably contain a water-soluble salt of the active ingredient, suitable stabilizing agents, and if necessary, buffer substances. Antioxidizing agents such as sodium bisulfite, sodium sulfite, or ascorbic acid, either alone or combined, are suitable stabilizing agents. Also used are citric acid and its salts and sodium EDTA. In addition, parenteral solutions can contain preservatives, such as benzalkonium chloride, methyl- or propyl-paraben, and chlorobutanol. Suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences, 17th ed., 1989, a standard reference text in this field.

[0332] The compounds used in the method of the present invention may also be administered inintranasal form via use of suitable intranasal vehicles, or via transdermal routes, using those forms of transdermal skin patches well known to those of ordinary skill in that art. To be administered in the form of a transdermal delivery system, the dosage administration will generally be continuous rather than intermittent throughout the dosage regimen.

[0333] Parenteral and intravenous forms may also include minerals and other materials to make themcompatible with the type of injection or delivery system chosen.

[0334] Each embodiment disclosed herein is contemplated as being applicable to each of the otherdisclosed embodiments. Thus, all combinations of the various elements described herein are within the scope of the invention. Any of the disclosed generic or specific compounds may be applicable to any of the disclosed compositions, processes or methods.

[0335] This invention will be better understood by reference to the Experimental Details which follow,but those skilled in the art will readily appreciate that the specific experiments detailed are only illustrative of the invention as described more fully in the claims, which follow thereafter. Definitions

[0336] ABBREVIATIONS

[0337] PET, positron emission tomography

[0338] ASC, alanine-serine-cysteine

[0339] TCA, tricarboxylic acid

[0340] CT, computed tomography

[0341] MRI, magnetic resonance imaging

[0342] [18F]FDOPA, 3,4-dihydroxy-6-18F-fluoro-l-phenylalanine

[0343] [18F]fluciclovine, anti-1-amino-3-18F-fluorocyclobutane-1-carboxylic acid

[0344] L-[18F]FAla, L-[18F]fluoroalanine

[0345] L-[18F]FAla-d3, L-[18F]fluoroalanine-d3

[0346] D-[18F]FAla-d3, D-[18F]fluoroalanine-d3

[0347] [18F]FAMP, 2-amino-3-18F-2-methylpropanoic acid

[0348] [18F]N-MeFAMP, 3-18F-2-methyl-2-(methylamino)propanoic acid

[0349] L-[18F]FMA, 3-(1-[18F]fluoromethyl)-L-alanine

[0350] [18F]Ala-BF3, [18F]trifluorobborate-derived alanine

[0351] RCY, radiochemical yield

[0352] SOCl2, thionyl chloride

[0353] MeCN, acetonitrile

[0354] r.t., room temperature

[0355] RuCl₃·H₂O, ruthenium(III) chloride hydrate

[0356] NaIO4, sodium periodate

[0357] Ru / C, ruthenium on carbon

[0358] D2O, deuterium oxide

[0359] Boc2O, di-tert-butyl decarbonate

[0360] NaOH, sodium hydroxide

[0361] DIC, N,N'-Diisopropylcarbodiimide

[0362] DCM, dichloromethane

[0363] TFA, trifluoroacetic acid

[0364] KF, potassium fluoride

[0365] H2SO4, sulfuric acid

[0366] 18-crown-6, 1,4,7,10,13,16-hexaoxacyclooctadecane

[0367] K2CO3, potassium carbonate

[0368] THF, tetrahydrofuran

[0369] DMSO, dimethyl sulfoxide

[0370] HCl, hydrochloric acid

[0371] BCH, 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid

[0372] MeAIB, α-(methylamino)isobutyric acid

[0373] HPLC, high-performance liquid chromatography

[0374] ROI, regions of interest

[0375] NMR, nuclear magnetic resonance spectroscopy

[0376] HRMS, high-resolution mass spectra

[0377] QTOF MS, quadrupole time-of-flight mass spectrometer

[0378] UHPLC, ultra-high performance liquid chromatography

[0379] ES+, electrospray positive

[0380] TLC, thin-layer chromatography

[0381] EtOAc, ethyl acetate

[0382] MgSO4, magnesium sulfate

[0383] NaHCO3, sodium bicarbonate

[0384] PVDF, polyvinylidene difluoride

[0385] SiO2, silica

[0386] QMA, quaternary methyl ammonium

[0387] HLB, hydrophilic lipophilic balanced

[0388] Et2O, diethyl ether

[0389] EDTA, trypsin-ethylenediaminetetraacetic acid

[0390] DPBS, Dulbecco’s phosphate buffered saline

[0391] DMEM, Dulbecco’s modified Eagle’s medium

[0392] EMEM, Eagle’s Minimum Essential Medium

[0393] FBS, fetal bovine serum

[0394] BCA, bicinchoninic acid

[0395] PBS, phosphate buffered saline

[0396] SPECT, single-photon emission computed tomography

[0397] %ID / cc, percentage of injected dose per cubic centimeter

[0398] %ID / g, percentage of injected dose per gram

[0399] SD, standard deviation

[0400] OSEM, ordered subset expectation maximization

[0401] VOIs, volume of interests

[0402] Unless otherwise defined, all technical and / or scientific terms used herein have the samemeaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the invention, exemplary methods and / or materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting.

[0403] In the discussion unless otherwise stated, adjectives such as “substantially” and “about”modifying a condition or relationship characteristic of a feature or features of an embodiment of the invention, are understood to mean that the condition or characteristic is defined to within tolerances that are acceptable for operation of the embodiment for an application for which it is intended. In embodiments, about means within a standard deviation using measurements generally acceptable in the art. In embodiments, about means a range extending to + / - 10% of the specified value. In embodiments, about includes the specified value. Unless otherwise indicated, the word “or” in the specification and claims is considered to be the inclusive “or” rather than the exclusive or, and indicates at least one of and any combination of items it conjoins.

[0404] It should be understood that the terms “a” and “an” as used above and elsewhere herein referto “one or more” of the enumerated components. It will be clear to one of the ordinary skill in the art thatthe use of the singular includes the plural unless specifically stated otherwise. Therefore, the terms “a,” “an” and “at least one” are used interchangeably in this application.

[0405] For purposes of better understanding the present teachings and in no way limiting the scope ofthe teachings, unless otherwise indicated, all numbers expressing quantities, percentages or proportions, and other numerical values used in the specification and claims, are to be understood as being modified in all instances by the term “about.” Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained. At the very least, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0406] In the description and claims of the present application, each of the verbs, “comprise,”“include” and “have” and conjugates thereof, are used to indicate that the object or objects of the verb are not necessarily a complete listing of components, elements or parts of the subject or subjects of the verb. Other terms as used herein are meant to be defined by their well-known meanings in the art. General

[0407] For the foregoing embodiments, each embodiment disclosed herein is contemplated as beingapplicable to each of the other disclosed embodiments.

[0408] As used herein, all headings are simply for organization and are not intended to limit thedisclosure in any manner. The content of any individual section may be equally applicable to all sections. All combinations of the various elements disclosed herein are within the scope of the invention.

[0409] Additional objects, advantages, and novel features of the present invention will becomeapparent to one ordinarily skilled in the art upon examination of the following examples, which are not intended to be limiting. Additionally, each of the various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below finds experimental support in the following examples.

[0410] It is appreciated that certain features of the invention, which are, for clarity, described in thecontext of separate embodiments, may also be provided in combination with a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements.

[0411] Examples are provided below to facilitate a more complete understanding of the invention. Thefollowing examples illustrate the exemplary modes of making and practicing the invention. However, the scope of the invention is not limited to specific embodiments disclosed in these Examples, which are for purposes of illustration only. EXAMPLES

[0412] General Information

[0413] All chemicals were used directly as received from commercial suppliers (Fisher, VWR, Sigma-Aldrich, Ambeed, 1Clickchemistry, Cambridge Isotope Laboratories). Crude products were purified on a Teledyne ISCO CombiFlash NextGen 100 system.1H nuclear magnetic resonance spectroscopy (NMR) and13C NMR spectra were recorded on a Bruker Avance III Console 400 MHz, Bruker Avance III Console 500 MHz, or Bruker Ascend™ 700 MHz spectrometers.19F NMR spectra were recorded on Bruker Avance III Console 400 MHz. Chemical shifts are expressed in δ ppm referenced to the residual solvent peak (CDCl3, δ = 7.26 ppm; D2O, δ = 4.79 ppm). Abbreviations used in describing peak signals are as follows: br, broad signal; s, singlet; d, doublet; dd, doublet of doublets; t, triplet; q, quartet; m, multiplet. High- resolution mass spectra (HRMS) were recorded on a Bruker Impact II quadrupole time-of-flight mass spectrometer (QTOF MS) equipped with an Agilent 1290 Infinity II ultra-high performance liquid chromatography (UHPLC) in electrospray positive (ES+) modes. The [18F]fluoride was produced on a cyclotron by the18O(p,n)18F reaction at the Kavita and Lalit Bahl Molecular Imaging Laboratory, Stony Brook Medicine. Radio-HPLC experiments were performed on a Jasco HPLC system equipped with PU- 2089s plus pumps, a UV-2075 plus UV detector, and an Eckert & Ziegler flow-count model-106 radioactivity detector. Radiochemical yields were determined using a dose-calibrator (Biodex Atomlab 500 or CRC-55tPET). Radio-TLC experiments were performed on an Eckert & Ziegler AR2000. The gamma counter was PerkinElmer Wizard 2480 instrument. Example 1 D-FAla analogue

[0414] D-serine-2,3,3-d3 (2)

[0415] Synthesis of Precursors and Nonradioactive References

[0416] D-sulfamidate precursor 9 was readily prepared from D-serine 1 via a four-step step synthesisroute with an overall yield of 37%. (Scheme 1). Deuterated D-sulfamidate precursor 10 was obtained similarly with the addition of an initial deuteration step, and in an overall yield of 23%. After precursors 9 and 10 were prepared, the optically pure enantiomers of D-fluoroalanine 13 and D-fluoroalanine-d314 were synthesized through nucleophilic fluorination followed by acidic deprotection. The optical purities of bothfluorinated amino acids were >99% as determined by chiral high-performance liquid chromatography (HPLC) (Figure 11A).

[0417] Scheme 1. Synthesis of precursors and nonradioactive references a(b)DIC, DCM, 0 °C, 30 min and then r.t., overnight. (c) 1. SOCl2, MeCN, -40 °C, 1 h; 2. pyridine, MeCN, - 40 °C, 1 h and then r.t., 40 min. (d) RuCl3·H2O, NaIO4, MeCN / H2O, 0 °C, 2 h. (e) 1. KF, 18-Crown-6, t- butanol / MeCN (4:1), 80 °C, 20 min; 2. 20% H2SO4, DCM, r.t., 2h. (f) TFA, DCM, 0 °C, 15 min then r.t., overnight.

[0419] Radiochemical Synthesis of D-[18F]FAla and D-[18F]FAla-d3

[0420] D-[18F]FAla and D-[18F]FAla-d3 were prepared in 2 steps by following a previously reportedprotocol with a decay-corrected radiochemical yield (RCY) of 18.9 ± 3.5 (n = 3) and 21.1 ± 2.5% (n = 3), respectively (Scheme 2). Both tracers with radiochemical purities (>95%) demonstrated good bench stability (Figure 11B). After incubation in saline (pH=7-8) at room temperature up to 3-4h, the radiochemical purities were still >95% (Figure 11C).

[0421] Scheme 2. Radiosynthesis of D-[18F]FAla and D-[18F]FAla-d3a

[0422] aReaction materials and condition: (a) [18F]F-, Kryptofix 2.2.2., K2CO3, tert-amyl alcohol, 90°C, 10 min. (b) 2.5 M HCl, 110 °C, 5 min.

[0423] The method was adapted from a previously published protocol with a slight modification.355wt.% Ru / C (210 mg, 40% wt) was added to a solution of compound 9 (525 mg, 5 mmol, 1.0 equiv.) in D2O(10 mL) in a 25-mL flask which was evacuated and then filled with hydrogen from a balloon. The reaction mixture was stirred at 80 °C overnight and the flask was then refilled with hydrogen balloon and stirring continued for 24 h. The reaction mixture was then filtered through a 0.45 µm PVDF filter, concentrated, and lyophilized. This reaction and the subsequent workup procedures were performed 3 additional times to obtain 0.4 g of compound 10 as a light orange powder. Yield: 74%. Deuteration: 99% [ES-MS] (ESI+) calculated for C3H4D3NO3 [M + H]+109.0687; found 109.0687.1H NMR (400 MHz, Deuterium Oxide) δ 3.97 – 3.92 (m, 0.03H), 3.85 (s, 0.01H), 2.09 (acetic acid reference, s, 3H).13C NMR (176 MHz, Deuterium Oxide) δ 172.78, 60.26 – 59.21 (m), 56.43 – 55.27 (m).

[0424] Boc-D-serine-tBu (5)

[0425] Step 1: Di-tert-butyl dicarbonate (764 mg, 3.42 mmol, 1.2 equiv.) was added to a solution ofcommercially available D-serine 1 (300 mg, 2.85 mmol, 1.0 equiv.) in 1,4-dioxane (6 mL) and 1M aqueous NaOH (5.7 mL, 5.7 mmol, 2.0 equiv.), at 0 °C. The reaction mixture was stirred at 0 °C for 30 min and then stirred at r.t. overnight, after which it was diluted with ethyl acetate (EtOAc, 100 mL) and extracted with H2O (100 mL × 3). The aqueous phase was acidified to pH 1 with ice-cold 15% aqueous HCl and extracted with EtOAc (100 mL × 3). The combined organic portions were dried over MgSO4, filtered, and concentrated to obtain 0.58 g of crude compound 3 as a light-yellow oil which was directly used for the next step without further purification. The crude compound 3 was directly used for the next step without further purification. Yield: 98%. [ES-MS] (ESI+) calculated for C8H15NO5 [M + Na]+228.0842; found 228.0839.

[0426] Step 2: N,N'-Diisopropylcarbodiimide (DIC, 1.68 g, 8.4 mmol, 3 equiv.) was added dropwiseto a solution of compound 3 (580 mg, 2.8 mmol, 1.0 equiv.) in dichloromethane (DCM, 6 mL) at 0 °C. The reaction mixture was stirred at 0 °C for 30 min and then at r.t. overnight after which it was diluted with hexanes (8 mL) and filtered through celite. The crude product was concentrated and purified by column chromatography (SiO2; 30% Hexanes / EtOAc) to obtain 502 mg of compound 5 as a colorless oil. Yield: 68%. [ES-MS] (ESI+) calculated for C12H23NO5[M + Na]+284.1468; found 284.1464.1H NMR (700 MHz, Chloroform-d) δ 5.41 (br, 1H), 4.25 (s, 1H), 3.89 (d, J = 4.0 Hz, 2H), 1.48 (s, 9H), 1.45 (s, 9H).13C NMR (176 MHz, CDCl3) δ 169.72, 155.90, 82.69, 80.52, 64.19, 56.36, 28.31, 28.01.

[0427] Boc-D-serine-tBu-2,3,3-d3 (6)

[0428] Compound 6 was prepared with the same procedure as the preparation of compound 5 exceptthat compound 2 was used as the starting material.

[0429] Step 1: Yield: 80%. [ES-MS] (ESI+) calculated for C8H12D3NO5 [M + H]+ 209.1211; found209.1210.

[0430] Step 2: Yield: 58%. [ES-MS] (ESI+) calculated for C12H20D3NO5 [M + H]+ 265.1837; found265.1836.1H NMR (700 MHz, Chloroform-d) δ 5.42 (br, 1H), 4.24 (s, 0.02H), 3.86 (s, 0.03H), 1.48 (s, 9H), 1.44 (s, 9H).13C NMR (176 MHz, Chloroform-d) δ 169.78, 155.90, 82.64, 80.13, 63.43 (dd, J = 43.7, 21.6 Hz), 57.19 – 54.20 (m), 28.31, 28.01.

[0431] Di-tert-butyl (R)-1,2,3-oxathiazolidine-3,4-dicarboxylate-4-d 2,2-dioxide (9)

[0432] Step 1: Acetonitrile (MeCN, 8 mL) and thionyl chloride (0.35 g, 2.9 mmol, 212 µL, 1.5 equiv.)were added to a 50-mL reaction flask at -40 °C under nitrogen, followed by the addition of a solution of 5 (502 mg, 1.92 mmol, 1.0 equiv.) in MeCN (8 mL). The reaction mixture was stirred at -40 °C for 1 h followed by the addition of pyridine (0.77 g, 9.6 mmol, 0.77 mL, 5.0 equiv). After further stirring at -40 °C for 1 h then at r.t. for 40 min, the reaction mixture was poured onto ice-water (50 mL) and then extracted with EtOAc (100 mL × 3). The combined organic portions were subsequently washed with aqueous HCl (1 M, 50 mL× 2, ice-cold), and saturated NaHCO3 (50 mL, ice-cold), dried over anhydrous MgSO4, filtered, concentrated, and then purified by column chromatography (SiO2; 15% Hexanes / EtOAc) to obtain 450 mg of compound 7 as a colorless oil. Yield: 76%. [ES-MS] (ESI+) calculated for C12H21NO6S [M + H]+308.1162; found 308.1162.

[0433] Step 2: RuCl3•H2O (8.2 mg, 0.04 mmol, 0.03 equiv), NaIO4 (0.436 g, 2 mmol, 1.5 equiv) andwater (8 ml) were added to a solution of compound 7 (0.42 g, 1.37 mmol, 1.0 equiv) in MeCN (8 mL), After stirring at 0 °C for 2 h, the reaction mixture was extracted with diethyl ether (Et2O, 50 mL × 3). The combined organic portions were washed with saturated aqueous NaHCO3 (25 mL, ice-cold), dried over anhydrous MgSO4, filtered, and further purified by column chromatography (SiO2; 20% Hexanes / EtOAc) to obtain 0.324 g of compound 9 as white solid powder. Yield: 73%. [ES-MS] (ESI+) calculated for C12H21NO7S [M + NH4]+341.1377; found 341.1377.1H NMR (400 MHz, Chloroform-d) δ 4.76 – 4.70 (m, 1H), 4.67 – 4.60 (m, 2H), 1.56 (s, 9H), 1.51 (s, 9H).13C NMR (126 MHz, CDCl3) δ 166.00, 148.08, 85.88, 84.49, 67.77, 58.12, 27.90, 27.82.

[0434] Di-tert-butyl (R)-1,2,3-oxathiazolidine-3,4-dicarboxylate-4,5,5-d32,2-dioxide (10)

[0435] Compound 10 was prepared following the same procedures as that used for compound 9 exceptthat compound 6 was used.

[0436] Step 1: Compound 8 yield: 78%. [ES-MS] (ESI+) calculated for C12H18D3NO6S [M + H]+311.1351; found 311.1350.

[0437] Step 2: Compound 10 yield: 86%. Deuteration: 98%. [ES-MS] (ESI+) calculated forC12H18D3NO7S [M + NH4]+344.1565; found 344.1559.1H NMR (400 MHz, Chloroform-d) δ 4.68 (d, J = 31.5 Hz, 0.03H), 4.61 (s, 0.03H), 1.56 (s, 9H), 1.51 (s, 9H).13C NMR (126 MHz, Chloroform-d) δ 165.99, 148.08, 85.87, 84.48, 67.01 (d, J = 24.8 Hz), 59.45 – 54.85 (m), 27.90, 27.82.

[0438] Tert-butyl (S)-2-((tert-butoxycarbonyl)amino)-3-fluoropropanoate (11)

[0439] MeCN (2 mL) was added to a solution of KF (80 mg, 1.38 mmol, 2 equiv.) and 18-crown-6(183 mg, 0.69 mmol, 1 equiv.) in Milli-Q water (0.3 mL). The solvent was azeotropically evaporated under nitrogen at 80 °C, after which MeCN (2 mL × 2) was then added to the reaction flask, evaporated, and vacuumed for 15 min to afford a dry residue. Compound 9 (224 mg, 0.69 mmol, 1 equiv.) in t- butanol / MeCN (4:1, 12 mL) was added to the dry residue, and the reaction mixture was stirred at 80 °C for 20 min. The solvent was evaporated and the remaining solid was dissolved in DCM (7 mL) and 20% H2SO4 (7 mL) after which the resulting mixture was stirred vigorously at r.t. for 2 h. The aqueous sulfuric acid layer was obtained using a separating funnel, washed with DCM (20 mL × 2) and the combined organic layers were then washed with an ice-cold saturated NaHCO3 solution to adjust the pH to 7-8. The aqueous layer was again obtained using a separating funnel and then washed with DCM (20 mL × 2). After drying over anhydrous MgSO4, the crude mixture was filtered, concentrated, and purified by column chromatography (SiO2; 15% Hexanes / EtOAc) to obtain 128 mg of compound 11 as colorless crystals. Yield: 70%. [ES-MS] (ESI+) calculated for C12H22FNO4 [M + H]+264.1606; found 264.1608.1H NMR (700 MHz, Chloroform-d) δ 5.36 (d, J = 8.0 Hz, 1H), 4.69 (dddd, J = 113.6, 47.9, 9.3, 2.5 Hz, 2H), 4.43 –4.29 (m, 1H), 1.49 (s, 9H), 1.45 (s, 9H). 13C NMR (176 MHz, Chloroform-d) δ 168.11 (d, J = 4.6 Hz),155.26, 83.90 (d, J = 172.4 Hz), 83.01, 80.15, 54.90 (d, J = 19.8 Hz), 28.30, 27.96.

[0440] Tert-butyl (S)-2-((tert-butoxycarbonyl)amino)-3-fluoropropanoate-2,3,3-d3 (12)

[0441] Compound 12 was prepared using the same procedures as those used for the preparation ofcompound 11 except that compound 10 was used as the starting material.

[0442] Compound 12, yield: 81%. [ES-MS] (ESI+) calculated for C12H19D3FNO4 [M + H]+ 267.1794;found 267.1795.1H NMR (700 MHz, Chloroform-d) δ 5.35 (br, 1H), 4.67 (dd, J = 111.2, 47.0 Hz, 0.04H), 4.36 (dd, J = 34.4, 8.0 Hz, 0.01H), 1.49 (s, 8H), 1.45 (s, 9H).13C NMR (176 MHz, Chloroform-d) δ 168.12 (d, J = 4.6 Hz), 155.25, 83.00, 80.15, 54.48 (q, J = 20.5 Hz), 28.30, 27.96.

[0443] (S)-2-amino-3-fluoropropanoic acid (13)

[0444] TFA (2.5 mL, 32.67 mmol, 172 equiv.) was added dropwise to a solution of compound 11 (50mg, 0.19 mmol, 1 equiv.) in DCM (2.5 mL) at 0 °C. The reaction mixture was stirred at 0 °C for 15 min and then at r.t. overnight, after which it was evaporated using a flow of nitrogen. The residue was dissolved in Milli-Q water (3 mL) and washed with diethyl ether (5 mL x 3). The aqueous layer was frozen in liquid nitrogen and lyophilized to obtain 36 mg of white solid. Ethanol (0.5 mL) was added to the white solid and the mixture was centrifuged after which the ethanol was carefully removed. This procedure was repeated three times. The residue was dried under the vacuum to give 12 mg of compound 13. A portion of the compound 13 aqueous solutions was then subjected to analytical HPLC purity analysis. Chromatography was performed using a Phenomenex Chirex 3126 (D)-penicillamine column (5 μm, 250 mm × 4.6 mm), and isocratic elution for 25 min at 1 mL / min, with 1 mM Cu(OAc)2 in water as the mobile phase and detection at 254 nm. Yield: 59%. [ES-MS] (ESI+) calculated for C3H6FNO2 [M + H]+108.0455; found 108.0457.1H NMR (400 MHz, Deuterium Oxide) δ 5.01 – 4.81 (m, 2H), 4.09 (ddd, J = 29.5, 4.8, 2.9 Hz, 1H).13C NMR (126 MHz, Deuterium Oxide) δ 170.43 (d, J = 6.3 Hz), 81.97 (d, J = 169.4 Hz), 54.92 (d, J = 19.7 Hz).19F NMR (376 MHz, D2O) δ -229.17. tR= 16.8 min and purity: > 99% determined by Chiral HPLC.

[0445] (S)-2-amino-3-fluoropropanoic-2,3,3-d3 acid (14)

[0446] Compound 14 was prepared using the same procedures as those used for the preparation ofcompound 13 except that compound 12 was used as the starting material.

[0447] Compound 14, Yield:77%. Deuteration: 99% [ES-MS] (ESI+) calculated for C3H3D3FNO2 [M+ H]+111.0644; found 111.0646.1H NMR (400 MHz, Deuterium Oxide) δ 4.92 (d, J = 15.2 Hz, 2H), 4.09 (d, J = 29.5 Hz, 1H), 2.10 (s, acetic acid reference, 3H).13C NMR (126 MHz, Deuterium Oxide) δ 170.45 (d, J = 6.1 Hz), 82.80 – 80.03 (m), 56.15 – 53.11 (m).19F NMR (376 MHz, Deuterium Oxide) δ -229.89 – -230.11 (m), -230.31 (p, J = 7.1 Hz), -230.48 – -230.75 (m). tR= 16.7 min and purity: > 99% determined by Chiral HPLC.

[0448] Manual Radiosynthesis

[0449] Aqueous [18F]fluoride (1.85-3.70 GBq) in H2O was passed through a preconditioned WatersQMA cartridge. The trapped18F-radioactivity was then eluted with a solution containing 6 mg of Kryptofix(K2.2.2) and 1 mg of K2CO3 in MeCN (1 mL) into the first reaction vial. The solvent was azeotropicallyevaporated under a stream of nitrogen. MeCN (1 mL × 2) was then added, and the solvent again evaporated under a stream of nitrogen. The residue was finally vacuumed for 3 min to afford a wax-like mixture. Tert- Amyl alcohol (0.5 mL) containing 2 mg of compound 9 or 10 was added to the solid residue, and the reaction mixture was stirred and heated at 90 °C for 10 min, cooled in the air for 30 sec, and then dilutedwith 2% aqueous formic acid (5 mL). The resulting mixture was loaded onto a preconditioned Waters Alumina N light cartridge and the eluate was collected in a Falcon tube (50 mL). A second portion of the 2% aqueous formic acid mixture (5 mL) was loaded onto the same cartridge and the eluate collected into the same Falcon tube (50 mL). The combined eluate was loaded onto a preconditioned Waters C18 long cartridge and an additional portion of the 2% aqueous formic acid mixture (5 mL) was loaded onto the same cartridge. The radioactive product was then eluted from the cartridge into the second reaction vial by flowing diethyl ether (5 mL) in reverse. After most of the solvent had been removed by a steam of nitrogen, HCl (4 N, 0.65 mL) was added to the residue and the reaction mixture was stirred at 110 °C for 5 min and subsequently cooled in the air for 30 sec. The aqueous mixture was then loaded onto an 8 mm × 60 mm Ag11-A8 resin column (2.5 g resin). Three portions of H2O (1 mL × 3) were passed through the column and collected separately. The second and third portions of the eluate were combined, diluted with aqueous 5% NaCl (0.44 mL), and passed through a membrane filter (Millipore Millex GV, 0.22 μm) into a vented sterile vial. A portion of the product solution was then subjected to analytical HPLC for quality control analysis. Chromatography was performed using a Phenomenex Chirex 3126 (D)-penicillamine column (5 μm, 250 mm × 4.6 mm), and isocratic elution for 25 min at 1 mL / min, with 1 mM CuCl2 in water as the mobile phase and detection at 254 nm. A portion of the product solution was also spiked with a non- radioactive standard and analyzed by HPLC as described above for spike injection analysis.

[0450] Bacterial Uptake of D-[18F]FAla and D-[18F]FAla-d3:

[0451] The method was slightly adapted from a previously published protocol.15, 23 The E. coli (ATCC8739) and S. aureus (ATCC 35556) strains were purchased from ATCC. Using a sterile loop, 10 mL of Luria-Bertani (LB) broth containing 100 μg / mL kanamycin or 10 mL of tryptic soy broth (TSB) was inoculated with S. aureus or E.coli from a glycerol stock. Both bacteria were aerobically grown overnight at 37 °C and with shaking at 250 RPM. The following day, 150 μL of each overnight culture was used to inoculate 10 mL LB. Both bacteria were cultured at 37 °C and 250 RPM for 3-4 h to achieve exponential phase growth until the OD600 reached 1.2-1.4. The bacteria cells were then pelleted via centrifugation at 3900 rpm for 10 min and washed 3 times with Dulbecco’s phosphate buffered saline (DPBS, Ca2+, Mg2+ added) before being resuspended back to their original volume. One mL of E. coli (1.1 x 109CFU) or S. aureus (4.2 x 108CFU) was aliquoted in 1.5 mL Eppendorf tubes and heated at 90 °C for 30 min to prepareheat-killed bacteria. The E. coli and S. aureus samples were incubated with D-[18F]FAla (290 kBq) or D-[18F]FAla-d3(300 kBq) together with selected inhibitors or substrates at 37 °C for 1 h. Blocking studies were conducted in triplicate by incubating the E. coli or S. aureus bacteria with 0.5, 1 and 5 mM D-alanine. The bacteria were then centrifuged at 11,000 rpm for 5 min, after which the supernatant was carefully removed, and the bacterial pellets were washed with 1 mL DPBS (No Ca2+, Mg2+added) and centrifugedagain at 11,000 rpm for 5 min. The washing step was performed three times, after which the radioactivity of each sample was determined using a gamma-counter (WIZARD 2480).

[0452] In Vitro Stability

[0453] Shelf Stability of D-[18F]FAla or D-[18F]FAla-d3

[0454] D-[18F]FAla and D-[18F]FAla-d3 were formulated in saline (pH 7-7.5) and incubated at r.t. for3.5 h. Subsequently, 20 µL of each radiotracer in saline was analyzed by HPLC as described above for radiochemical purity analysis.

[0455] Soft-Tissue Infection and Inflammation Model

[0456] All animal work was approved by the Institutional Animal Use and Care Committee at StonyBrook University (589413). The method was adapted from a previously published protocol with a slight modification.15, 23S. aureus Xen29 derived from ATCC 12600 was purchased from PerkinElmer (119240). Using a sterile loop, 10 mL LB containing 100 μg / mL kanamycin was inoculated with S. aureus Xen29 from a glycerol stock. The culture was aerobically grown overnight at 37 °C with shaking at 250 RPM. The next day, 150 μL of the overnight culture was used to inoculate 10 mL of fresh LB containing 100 μg / mL of kanamycin. The bacteria were shaken at 37 °C and 250 RPM for 4 h until the OD600 was ~1.2. Then, the bacteria were pelleted via centrifugation at 3900 rpm for 10 min, washed 3 times with DPBS (Ca2+, Mg2+added), and resuspended in DPBS (Ca2+, Mg2+added). One mL of S. aureus Xen29 (7.2 x 108CFU / mL) was aliquoted into one 1.5 mL Eppendorf tube, centrifuged and resuspended in 250 µL of DPBS (Ca2+, Mg2+added). A second 2 mL aliquot of S. aureus Xen29 (7.2 x 108CFU / mL) was aliquoted in a separate 1.5 mL conical tube, centrifuged, resuspended in 50 µL of DPBS (Ca2+, Mg2+added), and then incubated in boiling water for 30 min to prepare the heat-killed sample. The right triceps was inoculated with 50 µL of live S. aureus Xen29 (~108CFU) to induce infection while the left triceps was inoculated with 50 µL of heat-killed S. aureus Xen29 (~109CFU) to induce inflammation. Rats were monitored by bioluminescence imaging starting on the second day post inoculation. High levels of bioluminescence were normally observed two to three days after inoculation at which time animals were selected for PET imaging.

[0457] Bioluminescence Imaging

[0458] Rats were anesthetized by inhalation with 2.5% isoflurane using oxygen as a carrier and imagedusing an IVIS Lumina II imaging system. The following parameters were used for all bioluminescence imaging scans: field of view, 12.5 cm; subject height 1.5 cm; exposure time, 5 min; binning number, 8; f1 / stop value, 1; emission filter, open; excitation filter, blocked. Image analysis was performed with Living Image software. The signal intensity of the region of interest (ROI) was determined in units of radiance (p / s / cm2 / sr), which is the number of photons per second per square centimeter of tissue per steradian.

[0459] In Vivo PET / CT Imaging

[0460] Rats were anesthetized by inhalation of 2% isoflurane with oxygen as a carrier. PET and CTscans were obtained using a Siemens Inveon Multi-Modality pre-clinical microPET / CT / SPECT scanner. Catheters were placed into the tail veins of rats, after which the animals were positioned at the center of the PET scanner. Subsequently, 0.4 mL-0.8 mL of formulated tracers (25.38-41.37 MBq of D-[18F]FAla and 17.84-28.68 MBq of D-[18F]FAla-d3) were carefully injected through the catheters into the mice followed by 0.2 mL saline while simultaneously initiating the PET scan. Rats were closely monitored during the 90 min dynamic PET scan and then were moved and positioned for an 8-min CT scan. The PET data were binned into designated time frames, followed by reconstruction using the OSEM 3D method. Attenuation correction was performed using the reconstructed CT data during PET reconstruction. PET volume of interests (VOIs) and representative PET / CT images were generated by Amide version 1.0.4. (Fedora Project), and the percentage of injected dose per cubic centimeter (%ID / cc) of tissue was calculated. Errors in the averaged %ID / cc values are reported as the standard deviation.

[0461] Ex Vivo Biodistribution

[0462] Rats were euthanized by cervical dislocation at 110 min post-injection after the 90-mindynamic PET imaging scan. Major organs and tissues including infected muscle, inflamed muscle, healthy muscle, lungs, heart, kidneys, liver, stomach, small intestine, large intestine, bone, brain, and blood were collected. The radioactivity of these organs and tissues was quantified with a gamma counter (WIZARD 2480). Counts per minute were converted into nCi using a Sodium-22 reference phantom. Values were decay-corrected to the time of injection, and the percentage of injected dose per gram (%ID / g) of each tissue was calculated. After gamma counting, infected muscles and inflamed muscles were homogenized in PBS at 4 °C, serially diluted, and plated on LB agar. After overnight incubation at 37 °C, CFUs were quantified by enumeration.

[0463] Pharmacokinetics

[0464] The 90-min dynamic PET data were binned into designated time frames, followed byreconstruction using the OSEM 3D method. Attenuation correction was performed using the reconstructed CT data during PET reconstruction. PET VOIs and representative PET / CT images were generated by Amide version 1.0.4 (Fedora Project), and %ID / cc of tissue was calculated. Errors in the averaged %ID / cc values are reported as the standard deviation. A non-linear regression analysis of the dynamic PET data from the heart was performed and fit a one-phase decay model to determine the blood half-life and other pharmacokinetic properties of the radiotracer using GraphPad Prism version 9 (GraphPad Software, Inc., San Diego, CA).

[0465] Statistical Analysis

[0466] Statistical analysis and nonlinear regression were performed using GraphPad Prism 9.1.0(GraphPad Software). Quantitative data are expressed as the mean ± SD unless otherwise stated. Mean values were analyzed using the Student’s two-tailed unpaired t test and P values of < 0.05 were considered statistically significant. Example 2 L-FAla analogue

[0467] Synthesis of Precursors and Nonradioactive Reference Compounds

[0468] By following the synthetic route for the precursors of L-[11C]Asn (Xu, 2018), cyclic L-sulfamidate 3 was readily prepared starting from commercially available Boc-L-Ser-OtBu 1 via a 2-step synthesis with an overall yield of 64% (Scheme 3).

[0469] Scheme 3. Synthesis of Di-tert-butyl (S)-1,2,3-oxathiazolidine-3,4-dicarboxylate 2,2-dioxide(3)ah; 2. pyridine, MeCN, -40°C, 1 h and then r.t., 40 min. (b) RuCl3·H2O, NaIO4, MeCN / H2O, 0 °C, 2 h.

[0471] L-Ser-d3 L-5 was acquired from a commercial source while D-Ser-d3 D-5 was obtained usinga previously reported hydrogen / deuterium exchange reaction with a Ru / C catalyst under a hydrogenatmosphere (Scheme 2).38 With L-Ser-d3 L-5 and D-Ser-d3 D-5 in hand, cyclic deuterated sulfamidateprecursors L-9 or D-9 were prepared with an overall yield of 40% yield and 31%, respectively, and 98% deuteration. (Scheme 2).

[0472] Scheme 4. Synthesis of Di-tert-butyl (R) or (S)-1,2,3-oxathiazolidine-3,4-dicarboxylate-4,5,5-d32,2-dioxide (9)a, , , 80 °C, 2 d. (b) Boc2O, 1 MNaOH, 1,4-dioxane / H2O, r.t., overnight (c) DIC, DCM, 0 °C, 30 min and then r.t., overnight. (d) 1. SOCl2, MeCN, -40 °C, 1 h; 2. pyridine, MeCN, -40 °C, 1 h and then r.t., 40 min. (e) RuCl3·H2O, NaIO4, MeCN / H2O, 0 °C, 2 h. (f) 1. KF, 18-Crown-6, tert-butanol / MeCN (4 : 1), 80 °C, 20 min; 2. 20% H2SO4, DCM, r.t., 2 h. (g) TFA, DCM, 0 °C, 15 min then r.t., overnight

[0474] After three sulfamidate precursors were obtained, the synthesis of the non-radioactive referencecompounds was performed through fluorination followed by deprotection (Scheme 4). Inspired by previously reported nucleophilic displacement reactions with sulfamidates (Zeng 2017 and Zeng 2020), fluorinated intermediate 10 was prepared in an 80% yield prepared by nucleophilic monofluoroination of L-sulfamidate 3 with KF and 18-Crown-6 in tert-butanol / MeCN followed by the removal of hydrosulfite in 20% H2SO4 / DCM. Once the key intermediate 10 was obtained, optically pure nonradioactive L-3- fluoroalanine 12 was acquired using a mild deprotection condition with 1:1 TFA / DCM at room temperature overnight. The L and D- enantiomers of 3-fluoroalanine-d3L-13 and D-13 were obtained the same way using the deuterated intermediate L-11 and D-11, respectively. The optical purity of all three fluorinated amino acids was >99% determined by chiral HPLC (Figure 1).

[0475] Scheme 4. Synthesis of (R)-2-amino-3-fluoropropanoic acid (12) or deuterated (R) or (S)-2-amino-3-fluoropropanoic acid (13)a, 20 min; 2. 20%H2SO4, DCM, r.t., 2h. (b) TFA, DCM, 0 °C, 15 min then r.t., overnight.

[0477] Radiochemistry

[0478] With precursors and nonradioactive deuterated and nondeuterated 3-fluoroalanine in hand, L-[18F]FAla was initially synthesized by radiofluorination and deprotection, inspired by the radiosynthesis of L-[11C]Asp (Xu 2018). The radiofluorination reaction conditions, including reaction temperature, solvent, and reaction times, were screened at a fixed amount of Kryptofix 2.2.2. and K2CO3(Figure 3). It is clear that the yield of radiofluorination quickly reached a plateau at all conditions that were investigated. For instance, after 15 min the fluorination yields were 18% at 75 °C in THF, 41% at 120 °C in 1,4-dioxane, 38% at 90 °C in MeCN, and only 12% at 90 °C or 105 °C in DMSO. Interestingly, radiofluorination was most successful with moderately polar solvents (1,4-dioxane and MeCN) compared to the less polar solvent (THF) or more polar solvent (DMSO). Additionally, radiofluorination yields dramatically increased in moderately polar, protic solvents such as tert-butanol and tert-amyl alcohol. This is consistent with previously reported observations, which stated that using tertiary alcohols as the reaction solvent could dramatically increase the nucleophilicity of fluorine-18 in radiotracer synthesis (Kim 2006). Based on these experiments, we decided to use tert-amyl alcohol as the reaction solvent and heat at 90 °C for 10 min as the optimum conditions for radiofluorination.

[0479] To purify the 18F-labeled intermediate, the radiofluorination reaction mixture was firstquenched with 2% aqueous formic acid and then passed through an alumina N cartridge to remove unreacted18F fluoride. We then attempted to concentrate the18F-labeled intermediate using either a C18 short plus cartridge or an HLB short plus cartridge, however only 28-30% of the activity was trapped using either approach. After reducing the amount of Kryptofix 2.2.2. (14.4 mg to 6.0 mg) and K2CO3 (3.0 mg to 1.0 mg) and switching to a C18 long cartridge, the radiofluorination yield was about 72 ± 14% (n = 4) and 59% of the radioactivity was trapped by the cartridge. Although MeCN (1 mL) was able to elute > 90% of the18F-labeled intermediates from the cartridge, these intermediates were not thermostable when azeotropically dried at 80 °C. Therefore, Et2O (5 mL) was used to elute the intermediates from the cartridge and the elute was dried at r.t. using a stream of nitrogen. After most solvent had been removed, HCl (2.5M, 0.65 mL) was added to the vial and the mixture was heated to 110 °C for 5 min. The reaction mixture was then cooled to r.t. and purified by passing through an Ag11A8 resin column to remove the acid. Fractions containing radioactivity were collected and formulated to give a 0.9% NaCl aqueous solution. The overall RCY (decay corrected) of L-[18F]FAla, L-[18F]FAla-d3, and D-[18F]FAla-d3prepared through this two-step method were 17.0 ± 2.6% (n = 3), 22.5 ± 2.2% (n = 3), and 21.1 ± 2.5% (n = 3), respectively (Scheme 5). Compared with previously reported <1% non-decay corrected RCY of 3-[18F]FAla, the corresponding non-decay corrected RCY of L-[18F]FAla using sulfamidate precursors dramatically increased to ~12%.28The radiochemical purity of all three tracers was >98%. Formulated L-[18F]FAla, L- [18F]FAla-d3, and D-[18F]FAla-d3 were incubated at r.t. and analyzed by analytical radio-HPLC to determine their shelf stability which showed that all three tracers were stable under ambient conditions: The radiochemical purity of L-[18F]FAla, L-[18F]FAla-d3, and D-[18F]FAla-d3 was still more than 98% for up to 3 h.

[0480] Scheme 5. Radiosynthesis of L-[18F]FAla, L-[18F]FAla-d3, and D-[18F]FAla-d3.a10 min.(b) 2.5 M HCl, 110 °C, 5 min.

[0482] Di-tert-butyl (S)-1,2,3-oxathiazolidine-3,4-dicarboxylate 2,2-dioxide (3)

[0483] Compound 3 was synthesized using a method adapted from a previously published protocol.

[0484] Step 1: Acetonitrile (MeCN, 14 mL) and thionyl chloride (0.63 g, 5.2 mmol, 385 µL, 1.5 equiv.)were placed in a reaction flask that was cooled to -40 °C under nitrogen, to which a solution of compound 1 (0.91 g, 3.5 mmol, 1.0 equiv.) in MeCN (14 mL) was added. The reaction mixture was stirred at -40 °C for 1 h, pyridine (1.4 mL, 17.4 mmol, 5.0 equiv) was then added and after stirring at -40 °C for a further 1 h and then at r.t. for 40 min, the reaction mixture was poured into ice-water (10 mL). The mixture was extracted with ethyl acetate (EtOAc, 100 mL × 3) and the combined organic portions were washed subsequently with aqueous HCl (1 M, 50 mL× 2, ice-cold), and saturated NaHCO3 (50 mL, ice-cold). After drying over anhydrous MgSO4, the crude mixture was filtered and concentrated to give compound 2 (815mg, 76% yield) as a colorless oil. This crude compound was used directly for the next step without furtherpurification. [ES-MS] (ESI+) calculated for C12H21NO6S [M + H]+308.1162; found 308.1163.

[0485] Step 2: To a solution of compound 2 (0.816 g, 2.65 mmol, 1.0 equiv) in MeCN (10 mL), wasadded RuCl3•H2O (16 mg, 0.08 mmol, 0.03 equiv), NaIO4(0.848 g, 4 mmol, 1.5 equiv) and water (10 ml). After stirring at 0 °C for 2 h, the reaction mixture was extracted with diethyl ether (Et2O, 50 mL × 3). The combined organic portions were washed with saturated aqueous NaHCO3(25 mL, ice-cold), dried over MgSO4, filtered, and further purified by column chromatography (SiO2; 15% hexanes / EtOAc) to obtain 720 mg of compound 3 as a white solid powder (yield: 84%). [ES-MS] (ESI+) calculated for C12H21NO7S [M + NH4]+341.1377; found 341.1370.1H NMR (400 MHz, Chloroform-d) δ 4.76 – 4.70 (m, 1H), 4.67 – 4.60 (m, 2H), 1.56 (s, 9H), 1.51 (s, 9H).13C NMR (101 MHz, CDCl3) δ 166.15, 148.23, 86.02, 84.64, 67.90, 58.27, 28.04, 27.97.

[0486] D-serine-2,3,3-d3 (D-5)

[0487] Compound D-5 was synthesized using a method adapted from a previously published protocol38 A solution of compound 4 (525 mg, 5 mmol, 1.0 equiv.) in D2O (10 mL) was placed in a 25-mL flask,and 5 wt.% Ru / C (210 mg, 40% wt) was added. The 25-mL flask was vacuumed and then filled with a hydrogen balloon. The reaction mixture was stirred at 80 °C overnight. The flask was refilled with a new hydrogen balloon on the second day and stirred at 80 °C for the third day. The reaction mixture was filtered through a 0.45 µm PVDF filter, concentrated, and lyophilized. This reaction and the following workup procedures were performed 3 additional times to obtain 0.4 g of compound D-5 as a light orange powder (yield: 74%). Deuteration:99% [ES-MS] (ESI+) calculated for C3H4D3NO3 [M + H]+109.0687; found 109.0687.1H NMR (400 MHz, Deuterium Oxide) δ 3.97 – 3.92 (m, 0.03H), 3.85 (s, 0.01H), 2.09 (acetic acid reference, s, 3H).13C NMR (176 MHz, Deuterium Oxide) δ 172.78, 60.26 – 59.21 (m), 56.43 – 55.27 (m).

[0488] Boc-L-serine-tBu-2,3,3-d3 (L-7)

[0489] Step 1: Di-tert-butyl dicarbonate (1.2 g, 5.52 mmol, 1.2 equiv.) was added to a solution ofcommercially available L-serine-2,3,3-d3 L-5 (500 mg, 4.62 mmol, 1.0 equiv.) in1,4-dioxane (9 mL) and aqueous NaOH (1.0 M, 9.2 mL, 2.0 equiv.) at 0 °C. The reaction mixture was stirred at 0 °C for 30 min, and then at r.t. overnight, after which EtOAc (100 mL) was added. After extracting with H2O (100 mL × 3) the aqueous phase was acidified to pH = 1 with aqueous HCl (ice-cold, 15%) and extracted with EtOAc (100 mL × 3). The combined organic portions were dried over MgSO4, filtered, and concentrated to obtain 0.94 g of crude compound L-6 as a light-yellow oil. The crude compound L-6 was used directly for the next step without further purification. Yield: 98%. [ES-MS] (ESI+) calculated for C8H12D3NO5[M + H]+209.1211; found 209.1208.

[0490] Step 2: N,N'-Diisopropylcarbodiimide (DIC, 1.7g, 13.5 mmol, 3.0 equiv.) was added dropwiseto a solution of compound L-6 (940 mg, 4.51 mmol, 1.0 equiv.) in dichloromethane (DCM, 10 mL) at 0 °C. The reaction mixture was stirred at 0 °C for 30 min and then at r.t. overnight after which hexanes (15 mL) were added and the mixture filtered through celite. The crude product was concentrated and purified by column chromatography (SiO2; 30% hexanes / EtOAc) to obtain 788 mg of compound L-7 as a colorless oil. Yield: 66%. [ES-MS] (ESI+) calculated for C12H20D3NO5[M + H]+265.1837; found 265.1830.1H NMR (400 MHz, Chloroform-d) δ 5.39 (br, 1H), 4.24 (s, 0.05H), 4.00 – 3.90 (m, 0.01H), 1.48 (s, 9H), 1.45 (s, 9H).13C NMR (176 MHz, Chloroform-d) δ 169.84, 155.89, 82.57, 80.09, 64.94 – 62.03 (m), 57.19 – 52.99 (m), 28.30, 28.00.

[0491] Boc-D-serine-tBu-2,3,3-d3 (D-7)

[0492] Compound D-7 was prepared using the same procedure as those used for the preparation ofcompound L-7 but using compound D-5 as the starting material.

[0493] Step 1: Yield: 80%. [ES-MS] (ESI+) calculated for C8H12D3NO5 [M + H]+ 209.1211; found209.1210.

[0494] Step 2: Yield: 58%. [ES-MS] (ESI+) calculated for C12H20D3NO5 [M + H]+ 265.1837; found265.1836.1H NMR (700 MHz, Chloroform-d) δ 5.42 (br, 1H), 4.24 (s, 0.02H), 3.86 (s, 0.03H), 1.48 (s, 9H), 1.44 (s, 9H).13C NMR (176 MHz, Chloroform-d) δ 169.78, 155.90, 82.64, 80.13, 63.43 (dd, J = 43.7, 21.6 Hz), 57.19 – 54.20 (m), 28.31, 28.01.

[0495] (R) or (S)-Di-tert-butyl-1,2,3-oxathiazolidine-3,4-dicarboxylate-4,5,5-d32,2-dioxide (L-9 andD-9)

[0496] Compounds L-9 and D-9 were prepared using the same procedure as those used for thepreparation of compound 3 except that compounds L-7 and D-9 were used as the starting materials.

[0497] Step 1: L-8 Yield: 77%. [ES-MS] (ESI+) calculated for C12H18D3NO6S [M + CH3OH + H]+343.1613; found 343.1613.

[0498] D-8 Yield: 78%. [ES-MS] (ESI+) calculated for C12H18D3NO6S [M + H]+ 311.1351; found311.1350.

[0499] Step 2: L-9 Yield: 76%. Deuteration: 98%. [ES-MS] (ESI+) calculated for C12H18D3NO7S [M+ NH4]+344.1565; found 344.1563.1H NMR (400 MHz, Chloroform-d) δ 4.64 (s, 0.05H), 1.56 (s, 9H), 1.51 (s, 9H).13C NMR (101 MHz, CDCl3) δ 166.13, 148.23, 86.01, 84.62, 28.04, 27.97, 1.17.

[0500] D-9 Yield: 86%. Deuteration: 98%. [ES-MS] (ESI+) calculated for C12H18D3NO7S [M + NH4]+344.1565; found 344.1559.1H NMR (400 MHz, Chloroform-d) δ 4.68 (d, J = 31.5 Hz, 0.03H), 4.61 (s,0.03H), 1.56 (s, 9H), 1.51 (s, 9H).13C NMR (126 MHz, Chloroform-d) δ 165.99, 148.08, 85.87, 84.48, 67.01 (d, J = 24.8 Hz), 59.45 – 54.85 (m), 27.90, 27.82.

[0501] Tert-butyl (R)-2-((tert-butoxycarbonyl)amino)-3-fluoropropanoate (10)

[0502] MeCN (3 mL) was added to a solution of KF (180 mg, 3.1 mmol, 2 equiv.) and 18-crown-6(410 mg, 1.55 mmol, 1 equiv.) in MilliQ water (0.5 mL) in a 50-mL flask. The solvent was azeotropically evaporated under a stream of nitrogen at 80 °C, after which MeCN (3 mL × 2) was then added to the reaction flask, evaporated, and vacuumed for 15 min to afford a dry residue. Precursor 3 (500 mg, 1.55 mmol, 1 equiv.) in tert-butanol / MeCN (4:1, 30 mL) was added to the dry residue, and the reaction mixture was stirred at 80 °C for 20 min. The solvent was then evaporated, and the remaining solid residue was dissolved in DCM (16 mL) and 20% H2SO4 (16 mL). The aqueous sulfuric acid layer was obtained using a separating funnel, washed with DCM (30 mL × 2) and the combined organic layers were then washed with ice-cold saturated NaHCO3 solutions to adjust pH to 7-8. The aqueous layer was again obtained using a separating funnel and then washed with DCM (30 mL × 2). After drying over anhydrous MgSO4, the crude mixture was filtered, concentrated, and purified by column chromatography (SiO2; 15% hexanes / EtOAc) to obtain 325 mg of compound 10 as a colorless crystalline solid. Yield: 80%. [ES-MS] (ESI+) calculated for C12H22FNO4 [M + H]+264.1606; found 264.1603.1H NMR (700 MHz, Chloroform-d) δ 5.36 (d, J = 8.1 Hz, 1H), 4.69 (dddd, J = 113.7, 47.9, 9.3, 2.5 Hz, 2H), 4.48 – 4.21 (m, 1H), 1.49 (s, 9H), 1.45 (s, 9H).13C NMR (176 MHz, Chloroform-d) δ 168.11 (d, J = 4.6 Hz), 155.25, 83.90 (d, J = 172.4 Hz), 83.00, 80.15, 54.90 (d, J = 19.6 Hz), 28.30, 27.96.

[0503] (R) or (S)-tert-butyl -2-((tert-butoxycarbonyl)amino)-3-fluoropropanoate-2,3,3-d3 (L-11 andD-11)

[0504] Compounds L-11 and D-11 were prepared using the same procedures as those used for thepreparation of compound 10 except that compounds L-9 and D-9 were used as the starting materials.

[0505] L-11 Yield: 88%. [ES-MS] (ESI+) calculated for C12H19D3FNO4 [M + H]+ 267.1794; found267.1795.1H NMR (700 MHz, Chloroform-d) δ 5.35 (br, 1H), 4.67 (dd, J = 111.3, 47.1 Hz, 0H), 4.36 (dd, J = 34.3, 8.0 Hz, 0.03H), 1.49 (s, 9H), 1.45 (s, 9H).13C NMR (176 MHz, Chloroform-d) δ 168.11 (d, J = 4.6 Hz), 155.25, 82.99, 80.1455.02 – 53.97 (m), 28.30, 27.96.

[0506] D-11 Yield: 81%. [ES-MS] (ESI+) calculated for C12H19D3FNO4 [M + H]+ 267.1794; found267.1795.1H NMR (700 MHz, Chloroform-d) δ 5.35 (br, 1H), 4.67 (dd, J = 111.2, 47.0 Hz, 0.04H), 4.36 (dd, J = 34.4, 8.0 Hz, 0.01H), 1.49 (s, 8H), 1.45 (s, 9H).13C NMR (176 MHz, Chloroform-d) δ 168.12 (d, J = 4.6 Hz), 155.25, 83.00, 80.15, 54.48 (q, J = 20.5 Hz), 28.30, 27.96.

[0507] (R)-2-amino-3-fluoropropanoic acid (12)

[0508] TFA (2.5 mL, 32.67 mmol, 172 equiv.) was added dropwise to a solution of compound 11 (50mg, 0.19 mmol, 1 equiv.) in DCM (2.5 mL) at 0 °C. The reaction mixture was stirred at 0 °C for 15 min and then at r.t. overnight, after which it was evaporated using a flow of nitrogen. The residue was dissolved in Milli-Q water (3 mL) and washed with diethyl ether (5 mL x 3). The aqueous layer was frozen in liquid nitrogen and lyophilized to obtain 32 mg of white solid. Ethanol (0.5 mL) was added to the white solids and the mixture was centrifuged after that. The ethanol was carefully removed. This procedure was repeated three times. The residue was dried on the vacuum to yield 13 mg of compound 12. A portion of the compound 12 aqueous solution was then subjected to analytical HPLC purity analysis. Chromatography was performed using a Phenomenex Chirex 3126 (D)-penicillamine column (5 μm, 250 mm × 4.6 mm), and isocratic elution for 25 min at 1 mL / min, with 1 mM Cu(OAc)2 in water as the mobile phase and detection at 254 nm. Yield: 64%. [ES-MS] (ESI+) calculated for C3H6FNO2 [M + H]+108.0455; found 108.0456.1H NMR (700 MHz, Deuterium Oxide) δ 4.94 – 4.81 (m, 2H), 4.08 (ddd, J = 29.5, 4.9, 2.7 Hz, 1H).19F NMR (376 MHz, D2O) δ -229.16. tR= 10.8 min and purity: >99% determined by chiral HPLC.

[0509] (R) or (S)-2-amino-3-fluoropropanoic-2,3,3-d3 acid (L-13 and D-13)

[0510] Compounds L-13 and D-13 were prepared using the same procedures as those used for thepreparation of compound 12 except that compounds L-11 and D-11 were used as the starting materials.

[0511] L-13 Yield: 72%. Deuteration: 99% [ES-MS] (ESI+) calculated for C3H3D3FNO2 [M + H]+111.0644; found 111.0645.1H NMR (400 MHz, Deuterium Oxide) δ 4.09 (d, J = 29.5 Hz, 0.03H), 2.10 (s, acetic acid reference, 3H).13C NMR (176 MHz, Deuterium Oxide) δ 170.44 (d, J = 6.1 Hz), 81.30 (dp, J = 167.7, 23.8 Hz), 55.19 – 53.66 (m).19F NMR (376 MHz, Deuterium Oxide) δ -230.32 (p, J = 7.1 Hz), - 230.32 – -230.98 (m). tR= 10.7 min and purity: > 99% determined by Chiral HPLC.

[0512] D-13 Yield:77% Deuteration: 99% [ES-MS] (ESI+) calculated for C3H3D3FNO2 [M + H]+111.0644; found 111.0646.1H NMR (400 MHz, Deuterium Oxide) δ 4.92 (d, J = 15.2 Hz, 2H), 4.09 (d, J = 29.5 Hz, 1H), 2.10 (s, acetic acid reference, 3H).13C NMR (126 MHz, Deuterium Oxide) δ 170.45 (d, J = 6.1 Hz), 82.80 – 80.03 (m), 56.15 – 53.11 (m).19F NMR (376 MHz, Deuterium Oxide) δ -229.89 – - 230.11 (m), -230.31 (p, J = 7.1 Hz), -230.48 – -230.75 (m). tR= 16.7 min and purity: > 99% determined by Chiral HPLC.

[0513] Radiosynthesis

[0514] Aqueous [18F]fluoride (1.85-3.70 GBq) was passed through a preconditioned Waters QMAcartridge to trap [18F]fluoride ions. The trapped [18F]fluoride ion radioactivity was eluted with water (0.4mL) dampened MeCN (0.96 mL) solution containing Kryptofix (K2.2.2, 6 mg) and K2CO3 (1 mg). Thesolvent was azeotropically evaporated under a stream of nitrogen. MeCN (1 mL × 2) was then added to thereaction vessel, evaporated, and vacuumed for 3 min to afford a dry residue. Tert-Amyl alcohol (0.5 mL) containing 2 mg of compound 3 or 8 was added to the solid residue, and the reaction mixture was stirred and heated at 90 °C for 10 min, cooled in the air for 30 sec, and diluted with 2% aqueous formic acid (5 mL). The resulting mixture was loaded onto a preconditioned Waters Alumina-N light cartridge and the eluate was collected in a Falcon tube (50 mL). Another portion of the 2% aqueous formic acid solutions (5 mL) was loaded onto the same cartridge and the eluate was collected into the same Falcon tube (50 mL). The combined eluates were loaded onto a preconditioned Waters C18 long cartridge and an additional portion of the 2% aqueous formic acid solutions (5 mL) was loaded onto the same cartridge. The intermediate was then eluted with Et2O (5 mL) reversely from the cartridge into a second reaction vial. After most of the solvents were evaporated under a stream of nitrogen, HCl (4 N, 0.65 mL) was added to the residue. The reaction mixture was stirred at 110 °C for 5 min and subsequently cooled in the air for 30 sec. The reaction mixture was then loaded onto an 8 mm x 60 mm Ag11-A8 resin column (2.5 g resin). Three portions of H2O (1 mL × 3) were passed through the resin column and collected separately. The 2ndand 3rdportions of the eluate were combined, diluted with aqueous 5% NaCl (0.44 mL), and passed through a membrane filter (Millipore Millex GV, 0.22 μm) into a vented sterile vial. A portion of the product solution was then subjected to analytical HPLC for quality control analysis. Chromatography was performed using a Phenomenex Chirex 3126 (D)-penicillamine column (5 μm, 250 mm × 4.6 mm), and isocratic elution for 25 min at 1 mL / min, with 1 mM CuCl2 in water as the mobile phase and detection at 254 nm. A portion of the product solution was also spiked with a non-radioactive standard and analyzed by HPLC as described above for spike injection analysis.

[0515] Radiofluorination Optimization of L-[18F]FAla

[0516] Aqueous [18F]fluoride (0.37-0.74 GBq) in H2O was trapped on a preconditioned WatersQMA cartridge and then eluted with MeCN (1 mL) containing 14.4 mg of Kryptofix (K2.2.2) and 3 mg ofK2CO3. The solvent was azeotropically evaporated under nitrogen. MeCN (1 mL × 2) was then added to the reaction vessel, evaporated, and vacuumed for 3 min to afford a dry residue. THF, 1,4-dioxane, MeCN, DMSO, tert-butanol, and tert-Amyl alcohol (0.5 mL) containing 5 mg of compound 3 was added to the solid residue. The reaction mixture was stirred and heated at 60-120 °C. The reaction mixture was cooled for 30 seconds in the air and spotted with a capillary tube (0.5 mm diameter) on silica gel plates at 3, 8, 15, 25, and 40 min. The silica gel plates were dried, developed with 100% methanol, and dried again for radio- TLC analysis.

[0517] Culturing of 9L / lacZ, Mia Paca-2 and U87MG Cells

[0518] 9L / lacZ cells (rat nitrosourea-induced gliosarcoma cell line) were purchased from ATCC (cat#CRL-2200). Cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) purchased from Gibco(cat# 10569-010) which includes 4.5 g / L D-glucose and 110 mg / L sodium pyruvate. Media was supplemented with 10% fetal bovine serum (FBS, cat. #MT35010CV, ThermoFisher Scientific) and 1% penicillin / streptomycin (10,000 U / mL) (cat. #15140122, ThermoFisher Scientific) at 37 °C and 5% CO2. Cell density was maintained between 105and 106cells / mL, and the media was renewed 2-3 times per week.

[0519] U87MG cells (human glioblastoma) were purchased from ATCC (cat. #HTB-14). Cells weregrown in Eagle’s Minimum Essential Medium (EMEM) purchased from Quality Biological (cat. #112-018- 101) which includes L-glutamine. Media was supplemented with 10% fetal bovine serum (FBS, cat. #MT35010CV, ThermoFisher Scientific) and 1% penicillin / streptomycin (10,000 U / mL) (cat. #15140122, ThermoFisher Scientific) at 37 °C and 5% CO2. Cell density was maintained between 105and 106cells / mL, and the media was renewed 2-3 times per week.

[0520] Mia PaCa-2 cells (human epithelial pancreatic carcinoma) were purchased from ATCC (cat#CRL-1420). Cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) purchased from Gibco (cat# 10569-010) which includes 4.5 g / L D-glucose and 110 mg / L sodium pyruvate. Media was supplemented with 10% fetal bovine serum (FBS, cat. #MT35010CV, ThermoFisher Scientific) and 1% penicillin / streptomycin (10,000 U / mL) (cat. #15140122, ThermoFisher Scientific) at 37 °C and 5% CO2. Cell density was maintained between 105and 106cells / mL, and the media was renewed 2-3 times per week.

[0521] In Vitro Cellular Uptake of L-[18F]FAla and L-[18F]FAla-d3

[0522] Time-dependent Uptake of L-[18F]FAla and L-[18F]FAla-d3 in 9L / lacZ, U87MG, and Mia Paca-2 Cells :

[0523] 9L / lacZ, U87MG, and Mia Paca-2 cells were detached from the T-75 flask with trypsin-ethylenediaminetetraacetic acid (EDTA) solution, centrifuged, washed with Dulbecco’s phosphate buffered saline (DPBS, Ca2+, Mg2+added), and resuspended in DPBS (Ca2+, Mg2+added). One mL aliquot of cells (1 ×106cells / mL) was seeded in a 1.5 mL conical tube. Three replicates of each cell line were incubated with L-[18F]FAla (248 kBq) or L-[18F]FAla-d3 (253 kBq) at 37 °C for 5 min, 30 min, 60 min and 120 min. The cells were then centrifuged at 3,700 × g for 5 min, after which the supernatant was carefully removed, and the cell pellets were washed with 1 mL DPBS (No Ca2+, Mg2+added) and centrifuged again at 3,700 × g for 5 min. The washing step was performed three times. Cell pellets were lysed with 60 μL of cell lysis buffer containing a protease inhibitor, vortexed for 30 s, and seeded on ice for 10 min. This process was repeated three times. The radioactivity of each sample was determined using a gamma counter (WIZARD 2480) and stored under −80 °C. The total protein concentration of each cell lysate was measured by BCA assay. The results are expressed as %ID / 100 µg protein.

[0524] Transporter Characterization of L-[18F]FAla and L-[18F]FAla-d3 in 9L / lacZ and U87MG Cells:

[0525] 9L / lacZ and U87MG cells were detached from the T-75 flasks with trypsin-EDTA solution,centrifuged, washed with DPBS (Ca2+, Mg2+added, cat. #14040 ThermoFisher Scientific), and resuspended in DPBS (Ca2+, Mg2+added). One mL aliquot of cells (1 ×106cells / mL) was seeded in a 1.5 mL conicaltube. Cells were incubated with L-[18F]FAla (131 kBq) or L-[18F]FAla-d3 (187 kBq) together with selectedinhibitors or substrates at 37 °C for 1 hour. For inhibiting system L, cells were incubated with a 5 mM solution of 2-amino-bicyclo[2.2.1]-heptane-2-carboxylic acid (BCH). For inhibiting system A, cells were incubated with a 5 mM solution of α-(methylamino)isobutyric acid (MeAIB). For inhibiting system ASC, cells were incubated with a 5 mM solution of L-Ala or L-Ser. The cells were then centrifuged at 3,700 × g for 5 min, after which the supernatant was carefully removed, and the cell pellets were washed with 1 mL DPBS (No Ca2+, Mg2+added, cat. #14190 ThermoFisher Scientific) and centrifuged again at 3,700 × g for 5 min. The washing step was performed three times, after which the radioactivity of each sample was measured using a gamma counter (WIZARD 2480). Cell pellets were lysed with 60 μL of cell lysis buffer containing a protease inhibitor, vortexed for 30 s, and seeded on ice for 10 min. The process was repeated three times. The radioactivity of each sample was determined using a gamma counter (WIZARD 2480) and stored under −80 °C. The total protein concentration of each cell lysate was measured by a BCA assay.

[0526] BCA Assay

[0527] The cell pellets were lysed with Cell Lytic M (cat. #C2978 Sigma-Aldrich) supplemented witha protease inhibitor (cat. #A32955 ThermoFisher Scientific). Lysates were centrifuged at 13,000 × g for 45 min at 4 °C, and the total protein content of the supernatant was quantified using a Pierce Bicinchoninic Acid (BCA) Protein Assay kit (cat. #23225 ThermoFisher Scientific).

[0528] In Vitro Stability

[0529] Shelf Stability of L-[18F]FAla, L-[18F]FAla-d3, and D-[18F]FAla-d3

[0530] L-[18F]FAla and L-[18F]FAla-d3 were formulated in saline (pH = 7-7.5) and incubated at r.t. for3 h. Subsequently, 20 µL of each radiotracer in saline was analyzed by HPLC as described above for radiochemical purity analysis.

[0531] U87MG Tumor-Bearing Mice

[0532] All animal work was approved by the Institutional Animal Use and Care Committee at StonyBrook University (IACUC2023-00007). Athymic nude mice (Crl: NU(NCr)-Foxn1nu, female, 22-28 days old) were ordered from Charles River and housed in a maximum isolation room for a week in the Division of Laboratory Animal Resources at Stony Brook University. The method was slightly adapted from a previously published protocol.49The athymic nude mice were then subcutaneously injected with 4×106U87MG cells (0.1 mL 1:1 Matrigel / PBS) in the left flank. The mice were monitored closely until a palpabletumor was present. The tumor volume was measured two or three times a week and quantified using the formula V = L × W2 / 2, where L is the largest dimension of the tumor and W is the other dimension. After 5-6 weeks, the tumor volume reached around 181-521 mm3, and the mice were used for imaging experiments.

[0533] In Vivo PET / CT Imaging

[0534] Non-tumor-bearing thymic nude mice or U87MG xenografts were anesthetized by inhalationof 2% isoflurane with oxygen as a carrier. PET and CT scans were obtained using a Siemens Inveon Multi- Modality pre-clinical microPET / CT / SPECT scanner. Catheters were placed into the tail veins of the mice, after which small animals were positioned at the center of the PET scanner. Subsequently, 0.2 mL-0.4 mL formulated tracers (5.67-6.86 MBq of L-[18F]FAla and 5.48-6.13 MBq of L-[18F]FAla-d3 for non-tumor- bearing thymic nude mice; 5.82-7.78 MBq of L-[18F]FAla, 5.69-6.89 MBq of L-[18F]FAla-d3, and 6.45- 8.84 MBq of D-[18F]FAla-d3 for U87MG tumor-bearing mice)were carefully injected through the catheters into the mice followed by 0.2 mL saline while simultaneously initiating the PET scan. Mice were closely monitored during the 90-minute dynamic PET scan and then were moved and positioned for an 8-minute CT scan. The PET data were binned into designated time frames, followed by reconstruction using the OSEM 3D method. Attenuation correction was performed using the reconstructed CT data during PET reconstruction. PET volume of interest (VOIs) and representative PET / CT images were generated by Amide version 1.0.4. (Fedora Project), and the percentage of injected dose per cubic centimeter (%ID / cc) of tissue was calculated. Errors in the averaged %ID / cc values are reported as the standard deviation.

[0535] Ex Vivo Biodistribution

[0536] Non-tumor-bearing athymic nude mice or U87MG tumor-bearing mice were euthanized bycervical dislocation at 110 min post-injection after the 90-min dynamic PET imaging scan. Major organs and tissues including lungs, heart, kidneys, livers, stomach, small intestines, large intestines, muscle, bone, brain, and blood were collected for non-tumor-bearing athymic nude mice, and tumors were collected as well for tumor-bearing mice. The radioactivity in these organs and tissues was quantified with a gamma counter (WIZARD 2480). Counts per minute were converted into nCi using a Sodium-22 reference phantom. Values were decay-corrected to the time of injection, and the percentage of injected dose per gram (%ID / g) of each tissue was calculated.

[0537] Pharmacokinetics

[0538] The 90-min dynamic PET data were binned into designated time frames, followed byreconstruction using the OSEM 3D method. Attenuation correction was performed using the reconstructed CT data during PET reconstruction. PET VOIs and representative PET / CT images were generated byAmide version 1.0.4 (Fedora Project), and %ID / cc of tissue was calculated. Errors in the averaged %ID / cc values were reported as the standard deviation. A non-linear regression analysis of the dynamic PET data from the heart was performed and fit a one-phase decay model to determine the blood half-life and other pharmacokinetic properties of the radiotracer using GraphPad Prism version 9 (GraphPad Software, Inc., San Diego, CA).

[0539] Statistical Analysis

[0540] Statistical analysis and nonlinear regression were performed using GraphPad Prism 9.1.0(GraphPad Software). Quantitative data are expressed as the mean ± SD unless otherwise stated. Mean values were analyzed using the Student’s two-tailed unpaired t test and P values of < 0.05 were considered statistically significant. Results and Discussion

[0541] D-FAla analogue

[0542] Uptake in E. coli and S. Aureus

[0543] To determine the specificity of D-[18F]FAla and D-[18F]FAla-d3 for bacteria, uptake studieswere performed in E. coli and S. aureus two strains of bacteria that are commonly found in human infections (Kaper 2004 and Tong 2015). In each case the uptake was compared between live and heat-killed bacteria. For D-[18F]FAla and D-[18F]FAla-d3, the uptake in heat-killed E. coli was reduced to 4% and 2% of the uptake in live E. coli, respectively (Figure 3). Similarly, the uptake of D-[18F]FAla and D-[18F]FAla-d3 in heat-killed S. aureus was decreased to 41% and 31% of that observed in live bacteria, respectively. In addition, uptake in live bacteria was blocked in a dose-dependent manner by D-alanine. For D-[18F]FAla and D-[18F]FAla-d3, uptake in E. coli was blocked from ~80 to 95% by 0.5-5 mM D-alanine. In addition, 0.5-5 mM D-alanine blocked the uptake of D-[18F]FAla in S. aureus from ~15 to 57% and the uptake of D- [18F]FAla-d3 from 42 to 68%.

[0544] The difference in uptake between live and heat-killed bacteria and the impact of D-alanine onuptake indicates that specific uptake mechanisms exist in both E. coli and S. aureus, and suggest that D- [18F]FAla and D-[18F]FAla-d3could function as imaging agents in both Gram-negative and Gram-positive bacteria as observed for D-[11C]Ala (Parker 2020). However, the differential uptake of the radiotracers in S. aureus, either between live and heat-killed cells or by the presence of D-alanine suggests that a non- specific uptake mechanism is present in S. aureus, in contrast to previous studies on D-[11C]Ala (Parker 2020). These results suggested that the specific uptake mechanism of18F-labeled alanine analogs might be different from D-[11C]Ala in S. aureus versus E. coli.

[0545] PET Imaging Studies and Ex Vivo Biodistribution in A Rat S. Aureus Infection Model

[0546] The ability of D-[18F]FAla and D-[18F]FAla-d3 to detect bacterial infection in vivo wasevaluated in a soft-tissue model of S. aureus Xen29 infection (Parker 2020). The right triceps was inoculated with live S. aureus Xen29 to induce infection while the left triceps was inoculated with heat-killed S. aureus Xen29 to induce sterile inflammation in order to determine whether these radiotracers candifferentiate infection from inflammation. Animals with high levels of bioluminescence (~1.2 x 106p / sec / cm2 / sr) in the right triceps were then subjected to a 90-min dynamic PET imaging study following injection of D-[18F]FAla or D-[18F]FAla-d3. (Figure 5).

[0547] Region of interest (ROI) analysis demonstrated that both D-[18F]FAla and D-[18F]FAla-d3 weretaken up by infected muscle within a few minutes after the injection of tracers, followed by a gradual decline over 90 min (Figure 10). The blood half-lives of D-[18F]FAla and D-[18F]FAla-d3 were 1.28 min (95% Cl, 0.74-2.50 min) and 2.23 min (95% Cl, 1.09-5.30 min), respectively. The uptake of D-[18F]FAla and D- [18F]FAla-d3 in inflamed muscle and healthy muscle reached a maximum at 15 min post injection and then plateaued up to 90 min. The uptake at 15 min post injection of D-[18F]FAla was 0.78 ± 0.07 %ID / cc in infected muscle, which was significantly greater than that observed in inflamed muscle (0.42 ± 0.10 %ID / cc) and healthy muscle (0.38 ± 0.05 %ID / cc). Similarly, the uptake at 15 min post injection of D- [18F]FAla-d3 was 0.64 ± 0.03 %ID / cc in in infected muscle, which again was significantly greater than that observed in inflamed muscle (0.38 ± 0.07 %ID / cc) and healthy muscle (0.32 ± 0.06 %ID / cc). The ratio of the uptake at 15 min post-injection for infected muscle compared to either inflamed muscle or healthy muscle was 1.92 ± 0.42, and 2.01 ± 0.32, respectively. As for D-[18F]FAla-d3, the ratio of infection-to- inflammation and infection-to-healthy muscle at 15 min post injection was 1.77 ± 0.32 and 2.08 ± 0.43,respectively. Compared with D-[18F]FAla and D-[18F]FAla-d3, D-[11C]Ala was reported to have a higherratio of infection-to-inflammation (~3.5) at 60 min post injection in a soft-tissue mouse model of S. aureus Xen29 infection(Parker 2020). These ratios can be compared with infection-to-inflammation uptake ratios of 2.6 for [11C]PABA in mice, and 5.52 for [18F]FPABA in rats, 60 min post injection (Mutch 2018 and Zhang 2018). A head-to-head comparative imaging study is required to determine whether the differences in observed uptake between the radiotracers are significant or not.

[0548] High levels of uptake were observed in the kidneys for both radiotracers a few minutes postinjection followed by a slow decrease in signal, consistent with renal clearance of D-[18F]FAla and D- [18F]FAla-d3, while bone uptake increased over time. Although deuteration was expected to reduce the metabolism of D-[18F]FAla-d3, particularly the defluorination rate, the bone uptake of D-[18F]FAla-d3(1.10 ± 0.17 %ID / cc) was similar to D-[18F]FAla (0.85 ± 0.09 %ID / cc). D-Fluoroalanine is known to be oxidized to fluoropyruvate by D-amino acid oxidase followed by reduction to fluorolactate, which results in the liberation of inorganic fluoride (Schnellmann 2014). It was previously reported that deuteration of D-fluoroalanine reduced the oxidation rate of fludalanine by 2-3 fold both in vitro and in vivo, while the defluorination rate was less than 50% that of D-fluoroalanine (Darland 1986 and Schnellmann 2014). Therefore, it was surprising to observe similar high levels of bone uptake for both D-[18F]FAla-d3and D- [18F]FAla, indicating that deuteration did not reduce the rate of in vivo defluorination. It was speculated that the variation in metabolism between D-fluoroalanine / fludalanine and the radiotracers might stem from differences in the route of administration and dose levels for the nonradioactive compounds (oral gavage, micromole amounts) compared to the radiotracers (intravenous injection, picomole to nanomole amounts). Alternatively, the differential rates of defluorination might not be observed until greater than 90 min post injection, which is longer than our PET imaging scan time. A future radiometabolite study will investigate these possibilities.

[0549] Following PET imaging, the rats were euthanized to harvest major tissues and organs for exvivo biodistribution analysis (Figure 10). The uptake of D-[18F]FAla and D-[18F]FAla-d3 in infected muscle (0.53 ± 0.07 %ID / g for D-[18F]FAla; 0.41 ± 0.05 %ID / g for D-[18F]FAla-d3) was significantly higher than that in inflamed muscle (0.41 ± 0.07 %ID / g for D-[18F]FAla; 0.31 ± 0.04 %ID / g for D-[18F]FAla-d3) or healthy muscle (0.34 ± 0.04 %ID / g for D-[18F]FAla; 0.28 ± 0.04 %ID / g for D-[18F]FAla-d3). There was not a notable difference in inflamed muscle, healthy muscle, and bone uptake between the two tracers. The infected muscle and inflamed muscle were also homogenized for the quantification of the bacterial burden. The bacterial burden of S. aureus Xen29 was determined to be ~107colony-forming units (CFU) / ml, which is lower than 108CFU / ml often observed in clinical samples of acute infection, which indicates that D- [18F]FAla and D-[18F]FAla-d3 have sufficient high sensitivity for use in humans (Konig 1998). Taken together, the studies suggest that D-[18F]FAla and D-[18F]FAla-d3 are comparable and are both effective PET imaging agents for detecting bacterial infection.

[0550] L-FAla analogue

[0551] Cellular uptake of L-[18F]FAla and L-[18F]FAla-d3

[0552] Three cell lines were used to evaluate the uptake of L-[18F]FAla and L-[18F]FAla-d3, including9L / lacZ, (rat gliosarcoma), U87MG (human glioma), and Mia Paca-2 (human pancreatic cancer). The cells were incubated with the radiotracers for 5, 30, 60, and 120 min to measure the cellular uptake at specific time points. The results demonstrated that the uptake of L-[18F]FAla and L-[18F]FAla-d3quickly reached a maximum and then gradually decreased with time in each cancer cell line (Figure 3). The maximum uptake of L-[18F]FAla reached 5.6%ID / 100 µg protein in 9L / lacZ cells at 30 min, 1.56%ID / 100 µg protein in Mia Paca-2 cells at 5 min, and 11.5 %ID / 100 µg protein in U87MG cells at 5 min, while the maximum uptake of L-[18F]FAla-d3was 8.6 %ID / 100 µg protein in 9L / lacZ cells at 5 min, 4.0 %ID / 100 µg protein in Mia Paca-2 cells at 30 min, and 14.4 %ID / 100 µg protein in U87MG cells at 5 min, respectively. Radiolabeledamino acids are taken up into tumor cells through various amino acid transporter systems including system L (leucine preferring, sodium-independent), system A (alanine preferring, sodium-dependent), and system ASC (alanine, serine, and cysteine preferring, sodium-dependent) (McConathy 2008). In order to determine the amino acid transporters used by L-[18F]FAla and L-[18F]FAla-d3, cellular blocking studies were performed in 9L / lacZ and U87MG cells with specific amino acid transporter inhibitors. The cellular uptake of L-[18F]FAla was blocked 88% with 5 mM L-Ala and 81% with 5 mM L-Ser in 9L / lacZ cells, 78% with 5 mM L-Ala and 73% with L-Ser in U87MG cells, while L-[18F]FAla-d3uptake was blocked 87% with 5 mM L-Ala and 77% with 5 mM L-Ser in 9L / lacZ cells, 88% with 5 mM L-Ala and 80% with L-Ser in U87MG cells, respectively. Moreover, the cellular uptake of L-[18F]FAla and L-[18F]FAla-d3 was not blocked by the system L inhibitor, 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid (BCH), or the system A inhibitor, α-(methylamino)isobutyric acid (MeAIB). These data suggest that system ASC amino acid transporters are responsible for the uptake of L-[18F]FAla and L-[18F]FAla-d3 into tumor cells, consistent with previous reports on the uptake of L-[14C]alanine in U87MG cells (Gauthier-Coles, G 2021). System L played a minor role in L-[18F]FMA but not in L-[18F]FAla and L-[18F]FAla-d3 uptake, which indicates that extension of the side chain on radiolabeled L-alanine might impact which transporter is utilized (Wang 2012). The maximum uptake of L-[18F]FAla and L-[18F]FAla-d3 in brain tumor cells (9L / lacZ and U87MG) was much higher than pancreatic cancer cells (Mia Paca-2), which might indicate that a larger number of system ASC amino acid transporters are present in brain tumor cells.

[0553] PET imaging and ex vivo biodistribution in non-tumor-bearing athymic nude mice

[0554] In vivo evaluation of L-[18F]FAla and L-[18F]FAla-d3 was first performed in immunodeficientnon-tumor-bearing athymic nude mice (Figure 10). A 90-min dynamic PET scan demonstrated that both L-[18F]FAla and L-[18F]FAla-d3 quickly reached a maximum uptake of 1.68 ± 0.27 %ID / cc and 1.59 ± 0.47 %ID / cc, respectively, in the muscle at 15 min post-injection which gradually dropped to 0.56 ± 0.36 %ID / cc and 0.65 ± 0.23 %ID / cc at 90 min post-injection, respectively. The blood half-lives of L-[18F]FAla (0.79 min; 95% Cl, 0.54-1.25 min) and L-[18F]FAla-d3 (0.73 min; 95% Cl, 0.51-1.09 min) were similar, and there was no significant difference in bone uptake between L-[18F]FAla and L-[18F]FAla-d3. The bone uptake gradually increased with time and eventually reached a maximum level at 90 min post-injection of ~26% for both L-[18F]FAla and L-[18F]FAla-d3. The bone uptake is similar to that reported by David et al. for L- [18F]FAla and is consistent with in vivo defluorination (Yang 11993). The uptake of L-[18F]FAla and L- [18F]FAla-d3in the kidney and liver quickly reached a maximum at the beginning of the PET scan and then gradually decreased. After 110 min post-injection, all mice were sacrificed, and tissues and organs were harvested for ex vivo biodistribution analysis. The uptake of L-[18F]FAla and L-[18F]FAla-d3in the bonewas 17.93 ± 4.41 %ID / g and 16.39 ± 4.30 %ID / g, respectively, while the uptake in other tissues and organs was approximately 1 %ID / g or less for each radiotracer, consistent with the PET imaging data.

[0555] PET Imaging Studies and Ex Vivo Biodistribution in U87MG Tumor-bearing Mice

[0556] Dynamic PET imaging was also used to assess the biodistribution of L-[18F]FAla and L-[18F]FAla-d3in U87MG tumor-bearing athymic nude mice (Figure 5). Analysis of the regions of interest (ROI) demonstrated that both L-[18F]FAla and L-[18F]FAla-d3reached a maximum tumor uptake of ~2-3 %ID / cc at 15 min post-injection and gradually decreased until 90 min post-injection (Figure 9). The tumor uptake of L-[18F]FAla-d3was 1.3-fold higher than L-[18F]FAla at 15 min post-injection (P ˂ 0.05) while the muscle uptake of L-[18F]FAla was similar to L-[18F]FAla-d3 at 15 min post-injection (Table 1, P > 0.05). Therefore, the tumor-to-muscle ratio of L-[18F]FAla-d3 was higher than of L-[18F]FAla at 15 min post-injection (P ˂ 0.05). Bone, kidney, and liver uptake in U87MG tumor-bearing mice was similar to non-tumor bearing mice.

[0557] Table 1. PET Imaging Uptake of Tracers of in Tissues of U87MG Tumor-Bearing MiceaTissue L-[18F]FAla L-[18F]FAla-d3 D-[18F]FAla-d3Muscle 1.23 ± 0.13 1.24 ± 0.09 1.45 ± 0.22 T / M 1.77 ± 0.44 2.30 ± 0.29 1.07 ± 0.18 a Data were reported as %ID / cc ± SD (n=4) at 15 min post-injection. Data was analyzed by two-tailed t test, where a P value ˂ 0.05 is considered statistically significant.

[0558] The enhanced tumor uptake and tumor-to-muscle ratio of L-[18F]FAla-d3 compared to L-[18F]FAla indicates that deuteration has improved the performance of the radiotracer. This could be due to a direct effect on in vivo defluorination, a common problem for [18F]fluoroalkyl-labeled radiotracers, but it is not consistent with the observed bone uptake difference between these two radiotracers (Klenner 2021). Therefore, it was hypothesized that it might be due to an effect on other metabolic processes specific for L- alanine by improving the metabolic resistance of L-[18F]FAla-d3 to certain enzymes, which was commonly observed in other deuterated radiotracers (Ding 1995 and Leyton 2009). For instance, it is well-known that L-alanine is converted into pyruvate by alanine aminotransferase for use in the TCA cycle in tumor cells (Sousa 2016). Thus, the increased tumor uptake of L-[18F]FAla-d3 might be due to the enhanced metabolic stability to alanine aminotransferase by deuteration.

[0559] Although D-amino acids were generally thought to have no relevance in cancer, there is nowincreasing evidence that they may play a role. For instance, some D-amino acids such as D-alanine and D-proline are present at significantly higher levels in the bodily fluids of patients with gastric cancer (Nagata 2007). More recently, the concentration of D-aspartic acid, D-serine, and D-alanine were also found to be significantly higher in breast cancer cells (MCF-7) than normal breast tissue cells (MCF-10A) (Du 2019 and Du 2020). The role of D-amino acids such as D-alanine in cancer requires further exploration, and therefore D-[18F]FAla-d3was also evaluated in U87MG tumor-bearing mice (Figure 5) (Murtas 2023). The blood half-life of L-[18F]FAla-d3and D-[18F]FAla-d3were 0.59 min (95% Cl, 0.41-0.90 min) and 0.74 min (95% Cl, 0.50-1.16 min), respectively, while the tumor uptake of L-[18F]FAla-d3was 1.9-fold greater thanD-[18F]FAla-d3 at 15 min post-injection (Table 1, P ˂ 0.0001). The uptake of D-[18F]FAla-d3 in tumors wassimilar to the uptake in muscle 15 min post-injection (P > 0.05), indicating that D-[18F]FAla-d3 was not tumor-specific. The bone uptake of D-[18F]FAla-d3 was ~ 2%ID / cc over the 90-min PET scan, while the kidney and liver uptake of D-[18F]FAla-d3 gradually decreased over 90 min. The results of the ex vivo biodistribution studies are consistent with PET imaging which showed that the bone uptake of L-[18F]FAla- d3 was 10.80 ± 2.85 %ID / g, which was significantly greater than the uptake of D-[18F]FAla-d3 (1.73 ± 0.26 %ID / g). The highest uptake of D-[18F]FAla-d3 (7.44 ± 3.16 %ID / g) among the other organs and tissues studied was in the kidneys, which was consistent with the PET imaging observations. CONCLUSION

[0560] Positron emission tomography (PET) is a non-invasive and highly sensitive molecular imagingtechnique in nuclear medicine, which enables the in vivo physiological processes to be visualized and quantified with radiotracers in real time (Ametamey 2008). Recently, radiolabeled amino acids have been proven to be a class of effective imaging agents for cancer detection by targeting overexpressed amino acid transporters in cancer cells (Jain 2023, McConathy 2008 and Jager 2001). In particular, amino acid PET has shown great value in the clinical management of brain tumors and also provides more information on tumor metabolism than anatomic imaging methods such as computed tomography (CT) and magnetic resonance imaging (MRI) (Galldiks 2023, Verger 2021, Soni 2023, Galldiks 2017 and Zhang 2020). Compared with other PET radionuclides, fluorine-18 (t1 / 2 = 110 min) is the most commonly used radioisotope for radiolabeling amino acids due to its favorable physical and chemical properties such as clean positron emission (97% β+), short positron range (<0.3 mm), and suitable mean energy (250 keV) during decay (Conti 2016).18F-labeled amino acids have been widely used in clinical oncology (wang 2022, Qi, 2017, and Sun 2017). In 2009, 3,4-dihydroxy-6-[18F]fluoro-l-phenylalanine ([18F]FDOPA) was approved in Europe for the evaluation of recurrent brain tumors, while in 2016, an L-leucine analog, anti- 1-amino-3-[18F]fluorocyclobutane-1-carboxylic acid ([18F]fluciclovine) was approved by the FDA in the U.S. for use in suspected recurrent prostate cancer patients (Figure 1) (Kratochwil 2014, Bencherer 2004, Bogsrud 2018 and Ley 1983).

[0561] Given the importance of L-alanine in various cancers and the desire to elucidate the role of L-alanine metabolism in tumorigenesis, many efforts have been made to synthesize and characterize18F- labeled L-alanine analogs (Figure 1). In 1983, Ley explored the synthesis of 3-[18F]fluoroalanine through two different routes but with no success (Ley 1983). A decade later, David et al. reported that 3- [18F]fluoroalanine could be synthesized starting from tosylated Boc-protected serine methyl ester via a 2- step radiosynthesis method (Yang 1993). Unfortunately, the extremely low radiochemical yield (RCY, non- decay corrected, <1%) and lack of characterization of the optical purity prevented further exploration of this radiotracer. Only 5 µCi of radiotracers were given to each tumor-bearing rat for time-dependent ex vivo biodistribution experiments and no PET imaging studies were reported. In addition, the high bone uptake (> 4%ID / g at 30 min post-injection) indicated that 3-[18F]fluoroalanine was susceptible to in vivo defluorination. Subsequently, the Goodman group reported the synthesis of fluorine-18 labeled α- methylated fluoroalanine analogs, 2-amino-3-[18F]fluoro-2-methylpropanoic acid ([18F]FAMP) and 3- [18F]fluoro-2-methyl-2-(methylamino)propanoic acid ([18F]N-MeFAMP), in 2002 and then the synthesis of the pure enantiomers in 2010 (McConathy 2002, and Yu 2010). In addition, in 2012 Limin et al. reported several18F-labeled L-alanine derivatives coupled to alkyl chains of various lengths as potential tumor imaging agents such as 3-(1-[18F]fluoromethyl)-L-alanine ([18F]FMA), and in 2018 Hui et al. replaced the carboxylate group of L-alanine with an isosteric trifluoroborate group to produce [18F]trifluorobborate- derived alanine ([18F]Ala-BF3) for gastric cancer imaging (Wang 2012 and Liu 2018). While each of these reports represents promising progress in the development of radiolabeled L-alanine analogs, labeling L- alanine directly with fluorine-18, such as in 3-[18F]fluoroalanine, will cause the least perturbation in structure and is likely to be the best approach to directly probe L-alanine metabolism. However, 3- [18F]fluoroalanine is susceptible to defluorination and consequently, we decided to deuterate the radiotracer based on the knowledge that deuteration is often used to modify the in vivo metabolism of drugs and radiopharmaceuticals, and that this might improve the metabolic stability of18F-labeled L-fluoroalanine (Figure 1) (Klenner 2021 and Di Martino 2023).

[0562] Cyclic sulfamidates have been used as electrophiles in a variety of nucleophilic reactions, andpreviously a five-membered cyclic sulfamidate based on L-serine was used as the precursor to synthesize L-4-[11C]asparagine (L-[11C]Asn) via a [11C]cyanide ring opening nucleophilic reaction (Baldwin 1990, Terry 1987 and Xu 2018). The present invention synthesized optically pure L-[18F]fluoroalanine (L- [18F]FAla) with an enhanced RCY starting from the same cyclic sulfamidate precursor. The deuterated analog L-[3-18F]fluoroalanine-d3(L-[18F]FAla-d3) and its D-enantiomer, D-[18F]Fluoroalanine-d3(D- [18F]FAla-d3) were also prepared via the same method with similar RCY and radiochemical purity. Subsequent evaluation of these tracers demonstrated that L-[18F]FAla-d3is a specific superior tumor imaging agent in comparison with L-[18F]FAla.

[0563] In addition to glucose and folate metabolism, D-amino acid metabolism also plays a pivotalrole in bacterial growth (Miyamoto 2021 and Cava 2011). In particular, D-amino acids are building blocks for the peptidoglycan muropeptides which are essential components of the bacterial cell wall (Dorr 2019 and Egan 2017). Therefore, the incorporation of radiolabeled D-amino acids into the bacterial cell wall could be an attractive strategy for developing bacteria-specific imaging agents (Figure 1). In 2017, Neumann et al. reported that D-methyl-11C-methionine (D-[11C]Met) was able to specifically detect bacteria by replacing the D-alanine at the terminus of peptidoglycan muropeptides (Neumann 2017). Subsequently, first-in-human PET imaging demonstrated that the safe use of D-[11C]Met could be safely administered to patients with suspected prosthetic join infections (Polvoy 2022). In addition to D-methionine, D-alanine is the most common D-amino acid for bacteria peptidoglycan (Miyamoto 2021 and Cava 2011). Moreover, the sequence of D-alanine-D-alanine is highly conserved at the terminal of muropeptides in most bacteria (Egan 2017). In 2020, Parker et al. characterized D-[3-11C]alanine (D-[11C]Ala) as a highly sensitive bacteria-specific imaging agent by incorporating it into the cell wall in a broad scope of Gram-negative and Gram-positive bacteria (Parker 2020). In 2021, Renick et al. demonstrated that the hydrolyzed D-5- [11C]glutamine (D-[11C]Gln) was incorporated into peptidoglycan muropeptides to selectively image bacteria (Renick 2021).

[0564] PET imaging with radiolabeled amino acids has primarily focused on 11C-labeled D-aminoacids. Carbon-11 has a relatively short half-life (20.4 min), and an on-site cyclotron is required for the preparation of carbon-11 radiopharmaceuticals (Ametamey 2008). In contrast,18F-labeled tracers can be prepared using18F-fluoride produced off-site. The present invention explored the use of18F-labeled D- amino acids for imaging bacteria, examining how the introduction of fluorine impacted uptake and imaging, and whether the extended imaging time-window based on the longer half-life of fluorine-18 (110 min) conveyed an advantage. Given the successful use of D-[11C]Ala in bacterial infection imaging,18F-labeled D-alanine was synthesized.

[0565] Merck had previously reported the synthesis of D-fluoroalanine and the deuterium-labeledanalog, fludalanine (MK641) which had reduced in vivo defluorination (Figure 1) (Kollonitsch 1973, Kollonitsch 1976, Wise 1984, Darland 1986, and Schnellmann 2014). Although a clinical trial to explore the antibiotic properties of MK641 in combination with pentizidone was halted due to safety issues, radiotracers contain only microdoses of compound (Herschman 2003). The present invention synthesized18F-labeled D-fluoroalanine (D-[18F]FAla) and D-fluoralanine-d3(D-[18F]FAla-d3) and investigated their potential as effective PET imaging agents for bacterial infection (Figure 1). D-[18F]FAla and D-[18F]FAla- d3showed specific uptake in both Gram-negative (Escherichia coli) and Gram-positive bacteria (Staphylococcus aureus). D-[18F]FAla and D-[18F]FAla-d3were able to detect infection with anapproximate 2-fold difference between infection and inflammation in a soft-tissue model of S. aureus infection. To our knowledge, this is the first application of using18F-labeled D-amino acids to image bacteria.

[0566] D-amino acids such as D-alanine are substrates for bacterial peptidoglycan biosynthesis andare selectively taken up by bacteria and not mammalian cells. Consequently, D-amino acid metabolism is an attractive target for antibiotic discovery and the development of bacteria-specific imaging agents. D- fluoroalanine and the deuterium-labeled analog fludalanine (MK641) were originally explored as antibiotics by Merck. The present invention evaluate the ability of a fluorine-18 labeled analog of D- fluoroalanine, D-[3-18F]fluoroalanine (D-[18F]FAla) and the deuterated analog, D-[3-18F]fluoroalanine-d3 (D-[18F]FAla-d3) to image Staphylococcus aureus infection. The radiotracers were synthesized using sulfamidate precursors in high radiochemical yield (>18%, decay corrected) and radiochemical purity (>95%), and both D-[18F]FAla and D-[18F]FAla-d3 showed specific uptake in S. aureus as well as Escherichia coli that was blocked dose-dependently by D-alanine. In addition, both D-[18F]FAla and D- [18F]FAla-d3 were able to accurately differentiate S. aureus infection from inflammation in a soft-tissue model.

[0567] To fully explore the potential of 18F-labeled L-fluoroalanine for imaging cancer and otherchronic diseases, a simple and mild radiosynthesis method has been established to produce optically pure L-[3-18F]fluoroalanine (L-[18F]FAla), using a serine-derivatized five-member ring sulfamidate as the radiofluorination precursor. A deuterated analog, L-[3-18F]fluoroalanine-d3 (L-[18F]FAla-d3), was also prepared which was expected to have increased metabolic stability. Both L-[18F]FAla and L-[18F]FAla-d3 were rapidly taken up by 9L / LacZ, Mia Paca-2, and U87MG cells and were shown to be substrates for the alanine-serine-cysteine (ASC) amino acid transporter. The ability of L-[18F]FAla, L-[18F]FAla-d3, and the D-enantiomer, D-[18F]FAla-d3, to image tumors was evaluated in U87MG tumor-bearing mice. L- [18F]FAla-d3 had enhanced tumor uptake (2.85 ± 0.39 %ID / cc) compared to L-[18F]FAla (2.17 ± 0.62 %ID / cc), while D-[18F]FAla-d3 was not specifically taken up by the tumors. Overall, the results warrant further development of L-[18F]FAla-d3 as a clinical cancer imaging agent.

[0568] A two-step protocol utilizing five-membered ring sulfamidates as radio-nucleophilicfluorination precursors, was used to prepare three18F-labeled alanine analogs, L-[18F]FAla, L-[18F]FAla-d3and D-[18F]FAla-d3, all in >10% non-decay corrected yield with high radiochemical purity (>95%). The in vitro cell uptake and in vivo imaging studies show that both L-[18F]FAla and L-[18F]FAla-d3are transported into tumor cells via the ASC amino acid transporter and are effective tumor imaging agents in U87MG tumor-bearing mice. In contrast, D-[18F]FAla-d3is not taken up by tumors. Collectively, the data show thatL-[18F]FAla-d3has enhanced tumor uptake compared to L-[18F]FAla, suggesting that L-[18F]FAla-d3will be a valuable clinical imaging tool to explore the role of alanine in cancer biology.

[0569] The present invention developed an efficient method to stereo-specifically synthesize D-[18F]FAla and D-[18F]FAla-d3using a D-sulfamidate precursor. D-[18F]FAla and D-[18F]FAla-d3are taken up specifically in vitro by both E. coli (Gram-negative) and S. aureus (Gram-positive). D-[18F]FAla and D- [18F]FAla-d3show similar ability to differentiate infection from inflammation with an approximate 2-fold difference. A fully automated synthesis method for D-[18F]FAla and D-[18F]FAla-d3will be developed for future preclinical studies and clinical translation. D-[18F]FAla and D-[18F]FAla-d3are anticipated to be powerful tools for localizing and monitoring bacterial infection to improve the clinical management of infected patients.REFERENCES 1. Antimicrobial Resistance, C., Global burden of bacterial antimicrobial resistance in 2019: asystematic analysis. Lancet 2022, 399 (10325), 629-655. DOI: 10.1016 / S0140-6736(21)02724-0.2. 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DOI: 10.1021 / acsptsci.2c00215.

Claims

CLAIMS What is claimed is:

1. A compound having the structure:, wherein when the compound is, then at least one of D1, D2, and D3is a-H site; or wherein when the compound is , then at least two of D1, D2, and D3 are-H sites; or a pharmaceutically acceptable salt or ester thereof.

2. The compound of claim 1 having the following structure:.

3. The compound of claim 2, wherein at least two of D1, D2, and D3 are deuterium-enriched -H sites;preferably, all of D1, D2, and D3are deuterium-enriched -H sites.

4. The compound of any one of claims 2-3, wherein the level of deuterium at the deuterium-enriched-H site of the compound is about 20-100%; preferably, about 50-100%, more preferably, about 70- 100%, more preferably about 90-100%, more preferably, about 97-100%, more preferably, about 99-100%.

5. The compound of any one of claims 2-4, wherein the level of deuterium at the deuterium-enriched-H site of the compound is no less than 50%, preferably, no less than 70%, more preferably, no less than 90%; more preferably, no less than 99%.

6. The compound of any one of claims 2-5, wherein each of D1, D2, and D3 is deuterium.

7. The compound of claim 1 having the structure:.

8. The compound of claim 7, wherein all of D1, D2, and D3 are deuterium-enriched -H sites.

9. The compound of any one of claims 7-8, wherein the level of deuterium at the deuterium-enriched-H site of the compound is about 20-100%; preferably, about 50-100%, more preferably, about 70- 100%, more preferably about 90-100%, more preferably, about 97-100%, more preferably, about 99-100%.

10. The compound of any one of claims 7-9, wherein the level of deuterium at the deuterium-enriched-H site of the compound is no less than 50%, preferably, no less than 70%, more preferably, no less than 90%; more preferably, no less than 99%.

11. The compound of any one of claims 7-10, wherein each of D1, D2, and D3 is deuterium.

12. A composition comprising the compound of any one of claims 1-11 and a pharmaceuticalacceptable carrier or a pharmaceutical active agent.

13. The composition of claim 12, further .

14. The composition of claim 13, wherein the amount of in the composition is about1%-90%; preferably, about 1%-80%; more preferably, about 1%-70%; more preferably, about 1%- 60%; more preferably, about 1%-50%; more preferably, about 5%-50%; more preferably, about 5%-40%; more preferably, about 5%-30%; more preferably, about 5%-20%; more preferably, about 5%-10%; more preferably about 5%.

15. The composition of any one of claims 13-14, wherein the amount is less than60%, preferably, less than 50%; more preferably, less than 40%;less than 30%; more preferably, less than 20%; more preferably, less than 10%; more preferably, less than 5%; more preferably, less than1%.

16. The composition of any one of claims 12-15, further .

17. The composition of claim 16, wherein the amount in the composition is about1%-90%; preferably, about 1%-80%; moremore preferably, about 1%- 60%; more preferably, about 1%-50%; more preferably, about 5%-50%; more preferably, about 5%-40%; more preferably, about 5%-30%; more preferably, about 5%-20%; more preferably, about 5%-10%; more preferably about 5%.

18. The composition of any one of claims 16-17, wherein the amount is less than60%, preferably, less than 50%; more preferably, less than 40%; more preferably, less than 30%; more preferably, less than 20%; more preferably, less than 10%; more preferably, less than 5%; more preferably, less than1%.

19. The composition of any one of claims 12-18, wherein the molar ratio betweenis about 1:50 to 100:1; preferably, about 1:10 to 100:1; more preferably, about 1:5 about 1:4 to 99:1; more preferably, about 1:3 to 98:1 more preferably,more preferably, about 1:1 to 96:1 more preferably, about 1:1 to 95:1 more preferably, about 2:1 to 95:2 more preferably, about 3:1 to 95:2; more preferably, about 4:1 to 95:3; more preferably, about 5:1 to 95:4; more preferably, about 6:1 to 95:5.

20. The composition of any one of claims 12-18, wherein the molar ratiois about 1:50 to 100:1; preferably, about 1:10 to 100:1; more preferably, about 1:5about 1:4 to 99:1; more preferably, about 1:3 to 98:1 more preferably, about 1:2 to 97:1 more preferably, about 1:1 to 96:1 more preferably, about 1:1 to 95:1 more preferably, about 2:1 to 95:2 more preferably, about 3:1 to 95:2; more preferably, about 4:1 to 95:3; more preferably, about 5:1 to 95:4; more preferably, about 6:1 to 95:5.

21. The composition of any one of claims 12-18, wherein the molar ratiois about 1:100 to 100:1; preferably, about 1:90 to 90: 1; more preferably,preferably, about 1:70 to 70: 1; more preferably, about 1:60 to 60:1 more preferably, about 1:50 to 50: 1 more preferably, about 1:40 to 40: 1 more preferably, about 1:30 to 30: 1 more preferably, about 1:20 to 20:1 more preferably, about 1:10 to 10:1; more preferably, about 1:5 to 5:1; more preferably, about 1:2 to 2:1; more preferably, about 1:1.2 to 1.2:1.

22. A process of producing a compound of formula III having the structure:(III),wherein at least one of D1, D2, and D3is a deuterium-enriched -H site; the process comprises: (a) reacting to a compound of formula (I) with a fluorine-containing acid or a salt thereof,(I); (b) adding an acid to produce a compound of formula (II):(II); and (c) adding an acid to produce a compound of formula (III):.

23. A process of producing a compound having the following structure:, wherein at least one of D1, D2, and D3 is a deuterium-enriched -H site; the process comprises: (a) conducting a radio-fluorination reaction to the compound of formula (I)(I) to obtain a compound of formula(b) adding an acid.

24. The process of claim 22, wherein(a) the compound of formula (I) has the structure; (b) the compound of formula (II) has(c) the compound of formula.

25. The process of claim 22, wherein(a) the compound of formula (I) has the structure:; (b) the compound of formula (II) has the structure:(c) the compound of formula.

26. The process of claim 22, wherein(a) the fluorine-containing salt is KF, CaF2, NaF, AlF3, MgF2, ZnF2, FeF2 or FeF3; preferably, thefluorine-containing salt is KF or NaF; more preferably, the fluorine-containing salt is KF; (b) step (a) further comprises adding 18-Crown-6, tert-butanol; and acetonitrile (MeCN);preferably, the molar ratio between tert-butanol and acetonitrile is from about 10:1 to 1:10; more preferably, the molar ratio between tert-butanol and acetonitrile is about 9:1 to 1:9; more preferably, the molar ratio between tert-butanol and acetonitrile is about 8:1 to 1:8; more preferably, the molar ratio between tert-butanol and acetonitrile is about 7:1 to about 1:7; morepreferably, the molar ratio between tert-butanol and acetonitrile is about 6:1 to 1:6; more preferably, the molar ratio between tert-butanol and acetonitrile is about 5:1 to 1:5; more preferably, the molar ratio between tert-butanol and acetonitrile is about 4:1;(c) the reaction in step (a) is conducted at a temperature of about 50-110 °C; preferably, thereaction in step (a) is conducted at a temperature of about 60-100 °C; more preferably, the reaction in step (a) is conducted at a temperature of about 70-90 °C; more preferably, the reaction in step (a) is conducted at a temperature of about 80 °C;(d) the reaction in step (a) is conducted for about 10-50 minutes; preferably, the reaction in step(a) is conducted for about 10-40 minutes; more preferably, the reaction in step (a) is conducted for about 10-30 minutes; more preferably, the reaction in step (a) is conducted for about 20 minutes;(e) the acid in step (b) is an inorganic acid, preferably a strong inorganic acid, more preferably, theacid is H2SO4; preferably, the acid in step (b) is about 10%-50% H2SO4 in Dichloromethane (DCM); more preferably, the acid in step (b) is about 10%-40% H2SO4 in DCM; more preferably, the acid in step (b) is about 10%-30% H2SO4 in DCM ; more preferably, the acid instep (b) is about 20% H2SO4 in DCM ;(f) the reaction in step (b) is conducted at a temperature of about 10-50 °C; preferably, the reactionin step (b) is conducted at a temperature of about 20-40 °C; more preferably, the reaction in step (b) is conducted at a temperature of about 20-30 °C; more preferably, the reaction in step (b) is conducted at a temperature of about 25 °C;(g) the reaction in step (b) is conducted for about 1 hour to 5 hours; preferably, the reaction in step(b) is conducted for about 1 hour to 4 hours; more preferably, the reaction in step (b) is conducted for about 1 hour to 3 hours; more preferably, the reaction in step (b) is conducted for about 2 hours;(h) the acid in step (c) is an organic acid, preferably, the organic acid is Trifluoroacetic acid (TFA);preferably, TFA is dissolved in DCM;(i) the reaction in step (c) is conducted at a temperature of about -10-10 °C; preferably, the reactionin step (c) is conducted at a temperature of about -5-5 °C; more preferably, the reaction in step (c) is conducted at a temperature of about -3-3 °C; more preferably, the reaction in step (c) is conducted at a temperature of about 0 °C;(j) the reaction in step (c) is conducted for about 5 minutes to 60 minutes; preferably, the reactionin step (c) is conducted for about 5 minutes to 50 minutes; more preferably, the reaction in step (c) is conducted for about 5 minutes to 40 minutes; more preferably, the reaction in step (c) is conducted for about 5 minutes to 30 minutes; more preferably, the reaction in step (c) isconducted for about 10 minutes to 20 minutes; more preferably, the reaction in step (c) is conducted for about 15 minutes; and / or (k) the process further comprises storing the compound of formula (III) at room temperature for aperiod of 5-24 hours; preferably, for a period of 5-20 hours; more preferably, for a period of 5- 15 hours; more preferably, for a period of 10-15 hours; more preferably, for a period of 12 hours.

27. The process of claim 23, wherein(a) the radio-fluorination reaction is conducted with [18F] fluoride ion ([18F]F-), preferably, theradio-fluorination reaction is also conducted with 4,7,13,16,21,24-Hexaoxa-1,10- diazabicyclo[8.8.8]hexacosane (Kryptofix 222), K2CO3, and tert-amyl alcohol; (b) the reaction in step (a) is conducted at a temperature of about 50-130 °C; preferably, thereaction in step (a) is conducted at a temperature of about 60-120 °C; more preferably, the reaction in step (a) is conducted at a temperature of about 70-110 °C; more preferably, the reaction in step (a) is conducted at a temperature of about 80- °C; more preferably, thereaction in step (a) is conducted at a temperature of about 90 °C; (c) the reaction in step (a) is conducted for about 1-20 minutes; preferably, the reaction in step (a)is conducted for about 1-15 minutes; more preferably, the reaction in step (a) is conducted for about 5-15 minutes; more preferably, the reaction in step (a) is conducted for about 10 minutes; (d) the acid in step (b) is an inorganic acid; preferably, the inorganic acid is HCl; more preferably,the concentration of HCl is about 0.5-10M; more preferably, the concentration of HCl is about 0.5-8.5M; more preferably, the concentration of HCl is about 0.5-6.5M; more preferably, the concentration of HCl is about 1-4.5M; more preferably, the concentration of HCl is about 2.5M; (e) the reaction in step (b) is conducted at a temperature of about 80-140 °C; preferably, thereaction in step (b) is conducted at a temperature of about 90-130 °C; more preferably, the reaction in step (b) is conducted at a temperature of about 100-120 °C; more preferably, the reaction in step (b) is conducted at a temperature of about 105-115 °C; more preferably, the reaction in step (b) is conducted at a temperature of about 110 °C; and / or (f) the reaction in step (b) is conducted for about 1-20 minutes; preferably, the reaction in step (b)is conducted for about 1-15 minutes; more preferably, the reaction in step (b) is conducted for about 1-10 minutes; more preferably, the reaction in step (b) is conducted for about 5 minutes.

28. The process of claim 23, wherein(a) the compound of formula (I) has the structure; (b) the compound of formula (IV) has the structure.

29. The process of claim 23, wherein(a) the compound of formula (I) has the structure; (b) the compound of formula (IV).

30. The process of any one of claims 22-29, further comprises the following steps to produce thecompound of formula (I):(a) reacting to a compound having the structure with a deuterium labeling agent, to obtain a compound having the;(b) adding Boc2O to obtain a compound having the structure:;(c) adding N, N'-Diisopropylcarbodiimide (DIC) to obtain a compound having the structure:;(d) adding SOCl2 to obtain a compound having the following structure:; and(e) adding NaIO4 to obtain the31. The process of claim 30, wherein(a) ;;;and(e).

32. The process of claim 30, wherein;(b) ;(c) ;(d) ; and(e) .

33. The process of any one of claims 30-32, wherein(a) the deuterium labeling agent is D2O;(b) step (a) further comprises adding H2 and a catalyst of Ruthenium nanoparticles on a carbonsurface (Ru / C); (c) the reaction in step (a) is conducted at a temperature of about 50-110 °C; preferably, thereaction in step (a) is conducted at a temperature of about 60-100 °C; more preferably, the reaction in step (a) is conducted at a temperature of about 70-90 °C; more preferably, the reaction in step (a) is conducted at a temperature of about 80 °C; (d) the reaction in step (a) is conducted for about 1-10 days; preferably, the reaction in step (a) isconducted for about 1-8 days; more preferably, the reaction in step (a) is conducted for about 1-6 days; more preferably, the reaction in step (a) is conducted for about 1-4 days; more preferably, the reaction in step (a) is conducted for about 1-3 days; more preferably, the reaction in step (a) is conducted for about 2 days; (e) step (b) further comprises adding NaOH and 1,4-dioxane;(f) the reaction in step (b) is conducted at a temperature of about 10-50 °C; preferably, the reactionin step (b) is conducted at a temperature of about 20-40 °C; more preferably, the reaction in step (b) is conducted at a temperature of about 20-30 °C; more preferably, the reaction in step (b) is conducted at a temperature of about 25 °C; (g) the reaction in step (b) is stored at room temperature for a period of 5-24 hours; preferably, thereaction in step (b) is stored at room temperature for a period of 5-20 hours; more preferably,the reaction in step (b) is stored at room temperature for a period 5-15 hours; more preferably, the reaction in step (b) is stored at room temperature for a period of 10-15 hours; more preferably, the reaction in step (b) is stored at room temperature for a period of 12 hours;(h) step (c) further comprises adding DCM;(i) the reaction in step (c) is conducted at a temperature of about -10-10 °C; preferably, the reactionin step (c) is conducted at a temperature of about -5-5 °C; more preferably, the reaction in step (c) is conducted at a temperature of about -3-3 °C; more preferably, the reaction in step (c) is conducted at a temperature of about 0 °C;(j) the reaction in step (c) is conducted for about 10-60 minutes; preferably, the reaction in step(c) is conducted for about 10-50 minutes; more preferably, the reaction in step (c) is conducted for about 20-40 minutes; more preferably, the reaction in step (c) is conducted for about 30 minutes;(k) the compound obtained in step (c) is further stored at a temperature of about 10-50 °C;preferably, the compound obtained in step (c) is further stored at a temperature of about 20-40 °C; more preferably, the compound obtained in step (c) is further stored at a temperature of about 20-30 °C; more preferably, the compound obtained in step (c) is further stored at a temperature of about 25 °C;(l) the compound obtained in step (c) is further stored at room temperature for a period of 5-24hours; preferably, the compound obtained in step (c) is further stored at room temperature for a period of 5-20 hours; more preferably, the compound obtained in step (c) is further stored at room temperature for a period 5-15 hours; more preferably, the compound obtained in step (c) is further stored at room temperature for a period of 10-15 hours; more preferably, the compound obtained in step (c) is further stored at room temperature for a period of 12 hours;(m) step (d) further comprises adding MeCN at a temperature of about -80-0 °C; preferably, step(d) further comprises adding MeCN at a temperature of about -60 - -20 °C; more preferably, step (d) further comprises adding MeCN at a temperature of about -50 - -30 °C; more preferably, step (d) further comprises adding MeCN at a temperature of about -40 °C;(n) step (d) further comprises adding MeCN with SOCl2 at a temperature of about -80 to 0 °C;preferably, step (d) further comprises adding MeCN with SOCl2at a temperature of about -60 to -20 °C; more preferably, step (d) further comprises adding MeCN with SOCl2at a temperature of about -50 to -30 °C; more preferably, step (d) further comprises adding MeCN with SOCl2at a temperature of about -40 °C;(o) step (d) further comprises adding MeCN and pyridine at a temperature of about -80 to 0 °C;preferably, step (d) further comprises adding MeCN and pyridine at a temperature of about -60to -20 °C; more preferably, step (d) further comprises adding MeCN and pyridine at a temperature of about -50 to -30 °C; more preferably, step (d) further comprises adding MeCN and pyridine at a temperature of about -40°C; (p) the compound obtained in step (d) is further stored at a temperature of about 10-50 °C;preferably, the compound obtained in step (d) is further stored at a temperature of about 20-40 °C; more preferably, the compound obtained in step (d) is further stored at a temperature of about 20-30 °C; more preferably, the compound obtained in step (d) is further stored at a temperature of about 25 °C; (q) the compound obtained in step (d) is further stored at room temperature for a period of 10-70minutes; preferably, the compound obtained in step (d) is further stored at room temperature for a period of 20-60 minutes; more preferably, the compound obtained in step (d) is further stored at room temperature for a period 30-50 minutes; more preferably, the compound obtained in step (d) is further stored at room temperature for a period of 40 minutes; (r) step (e) further comprises adding RuCl3·H2O, and MeCN;(s) the reaction in step (e) is conducted at a temperature of about -10-10 °C; preferably, the reactionin step (e) is conducted at a temperature of about -5-5 °C; more preferably, the reaction in step (e) is conducted at a temperature of about -3-3 °C; more preferably, the reaction in step (e) is conducted at a temperature of about 0 °C; and / or (t) the reaction in step (e) is conducted for about 1 hour to 5 hours; preferably, the reaction in step(e) is conducted for about 1 hour to 4 hours; more preferably, the reaction in step (e) is conducted for about 1 hour to 3 hours; more preferably, the reaction in step (e) is conducted for about 2 hours.

34. A method of detecting target cells in a subject comprising administering an effective amount of thecompound of any one of claims 1-11 or the composition of any one of claims 12-21 to the subject, and imaging the subject with a molecular imaging device to detect the compound or the composition in the subject.

35. A method of imaging target cells in a subject comprising:(a) administering to the subject an effective amount of the compound of any one of claims 1-11 orthe composition of any one of claims 12-21, wherein the compound or the composition specifically accumulates at the target cells in the subject; (b) detecting in the subject the location of the compound or the composition; and(c) obtaining an image of the target cells in the subject based on the location of the compound orthe composition in the subject.

36. A method of detecting the presence of target cells in a subject, the method comprises determiningif an amount of the compound of any one of claims 1-11 or the composition of any one of claims 12-21 is present in the subject for a period after administration of the compound or the composition to the subject, thereby detecting the presence of the target cells based on the amount of the compound or the composition determined to be present in the subject.

37. The method of claim 36, wherein the period is about 5-150 minutes; preferably, the period is about10-120 minutes; more preferably, the period is about 10-90 minutes; more preferably, the period is about 20-60 minutes; more preferably, the period is about 20-50 minutes; more preferably, the period is about 30-40 minutes.

38. A method of imaging tumors in a subject by administering to the subject an effective amount of thecompound of any one of claims 1-11 or the composition of any one of claims 12-21.

39. A method of detecting bacterial infection in a subject by administering to the subject an effectiveamount of the compound of any one of claims 1-11 or the composition of any one of claims 12-21.

40. The method of claim 39, wherein the bacterial infection is caused by gram-negative or gram-positive bacteria; preferably, the bacterial infection is caused by S. Aureus, Escherichia coli, Mannheimia haemotytica, Pasteurella multocida, Histophilus somni, Actinobacillus pleuropneumoniae, Salmonella enteritidis, Salmonella gallinahum, Lawsonia intracellularis, Brachyspira hyodysenteriae, Brachyspira pilosicoli, Acinetobacter baumannii, Acinetobacter spp., Citrobacter spp., Enterobacter aerogenes, Enterobacter cloacae, Klebsiella oxytoca, Klebsiella pneumoniae, Serratia marcescens, Stenotrophomonas maltophilia, and Pseudomonas aeruginosa; more preferably, caused the bacterial infection is caused by E. coli or S. Aureus.

41. The compound of any one of claims 1-11 or the composition of any one of claims 12-21 for use inimaging tumors in a subject and / or detecting bacterial infection in a subject.

42. Use of the compound of any one of claims 1-11 or the composition of any one of claims 12-21 inimaging tumors in a subject and / or detecting bacterial infection in a subject.

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