Deuterium and fluorine-18 eabeeed geutamine composition

Abstract

The present disclosure provides a composition comprising a compound having the structure (A) wherein each of H1, H2, H3, H4, H5, H6, and H7 is independently hydrogen or a deuterium-enriched -H site; and wherein at least one of H1, H2, H3, H4, H5, H6, and H7 is a deuterium-enriched -H site, or a salt or ester thereof.

Description

Docket: 92669-A-PCT / GJG / YXDEUTERIUM AND FLUORINE-18 LABELED GLUTAMINE COMPOSITION, SYNTHETIC METHODS, AND METHODS OF USE AS IMAGING AGENT

[0001] Throughout this application, various publications are referenced, including referenced in parenthesis. 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 APPLICATIONS

[0002] This application claims the benefit of U. S. Provisional Application No. 63 / 756,889 filed February 11, 2025 and U. S. Provisional Application No. 63 / 742,945 filed January 8, 2025, the contents of which are hereby incorporated by reference.BACKGROUND OF THE INVENTION

[0003] Positron emission tomography (PET) is a clinical and research imaging technique for the non-invasive investigation of biochemical and molecular events in living organisms (Fowler 1997 and Miller 2008). The tremendous success of [18F]fluoro-2 -deoxy -D-glucose ([18F]FDG) (Fig. 1, A) based on PET imaging for various (oncological, neurological and cardiovascular) diseases diagnosis in the past decades has poised FDG-PET as one of the most powerful molecular imaging modalities (Weber 2000 and Hoh 2007). The mechanism of FDG-PET is based upon the upregulation of the PI3K / Akt / mTor pathway, which can boost aerobic glycolysis and support tumor proliferation (Warburg effect) (Vander 2009). Glutamine is the second most abundant nutrient in the human body and is found in blood as well as stored in the skeletal muscles in high concentration (0.5 - 1.0 mmol / L). It plays various critical functions such as a substrate for DNA and protein synthesis, a primary source of fuel for cells lining the inside of the small intestine and rapidly dividing immune cells, a regulator of acid-base balance in the kidney by producing ammonium (Cruzat 2018 and Newsholme 1985). In the brain, the glutamine-glutamate shunt is a critical pathway to control the inhibitory and excitatory' neuronal signals (Cooper 2016). The ample proofs have shown that glutaminolysis can be upregulated by overexpression of the oncogene c-Myc (Myc) and play a significant role in tumor growth and metabolism (Fig. 2 and 6) (Wise 2008, Wise 2010 and Yang 2017).

[0004] Based upon this mechanism, a series of radioactive and stable isotope labeled glutamines, L-5-[11C]glutamine ([11C]Gln, Fig. 1, B), L-[5-13C-4-D2]glutamine (Fig. 1, C) and four stereoisomers of 4-[18F]fluoroglutamine (representative isomer (2S,4R)-4-[18F]fluoroglutamine. 4-[18F]FGln, as shown in Fig.1, D) had been synthesized and evaluated for tumor imaging purpose (Qu 2012, and Qu 2011). Among them, fluorine-18 (18F, T1 / 2 = 110 min) labeled glutamine analog 4-[18F]FGln has been explored broadlythrough various clinical and pre-clinical research and demonstrated a great potential for imaging gliomas, neuroblastoma and brain metastasis, assessing ASCT2 expression in lung cancer, predicting response to BRAFV600E-targeted therapy in preclinical models of colon cancer, detecting glutamine pool size changes in triple-negative breast cancer (TNBC) and imaging 4-[18F]FGln accumulation in breast cancer, imaging glutamine metabolism in murine models of hepatocellular carcinoma (HCC) and nonalcoholic steatohepatitis progression, exploiting glutamine consumption in atherosclerotic lesions and addiction in multiple myeloma (MM) cells, indicating bone marrow metabolism dysfunctions, as well as following the changes in clear cell renal cell carcinoma (ccRCC) metabolism in vivo, finding metabolically-targeted osteosarcoma therapy imaging biomarkers, and detecting KRAS mutation for noninvasive PDAC diagnosis etc. (Reference Nos.15-39).

[0005] There is growing evidence to show that transport of 4-[18F]FGln across cell membranes mainly relies on alanine-serine-cysteine preferring transporter 2 (ASCT2, or SLC1A5), which is frequently found to be highly upregulated in many tumor cells. Once taken up by tumor cells, 4-[18F]FGln behaves similar to [18F]FDG and mainly stays unaltered, minimally metabolized to 4-[18F]fluoroglutamic acid (Fig. 2) (Lieberman 2011, Hassanein 2016, Zhou 2017, and Cooper 2012). This characteristic clearly adds more weight for developing 4-[18F]FGln as a cancer imaging agent. The other structural characteristic of this molecule, i.e. fluorine- 18 attached to the carbon-skeleton of molecule at C-4 which is a-position to a carbonyl group (C-5), makes18F-C(4) bond vulnerable as compared to a normal sp3C-F bond. Several reports have shown that 4-[18F]FGln has propensity toward defluorination in vivo and apparent bone uptake is observed both in animals and humans (Thompson 2015, Wu 2014 and Chen Lieberamn 2019). This limitation may dampen the interest in using this radiotracer for cancer imaging, especially for metastatic cancers which tumor has spread from original sites to lymph system and / or bone marrow. In order to slow down the in vivo defluorination process and lessen the interference from free [18F]fluoride ion, there is an unmet demand to develop novel18F-labeled glutamine analogs with better in vivo stability while maintaining comparable cancer cell uptake avidity.BRIEF SUMMARY OF THE INVENTION

[0006] The present discourse provides a composition comprising a compound having the structure:wherein each of H1, H2, H3, H4, H5, H6, and H7is independently hydrogen or a deuterium-enriched -H site; andwherein at least one of H1, H2, H3, H4, H5, H6, and H7is a deuterium-enriched -H site,or a salt or ester thereof.

[0007] The present discourse provides a method for positron emission tomography (PET) imaging of a subject, comprising:(a) administering to the subject a composition comprising a compound having the structure:wherein each of Hi, H2, H3, EE, H5, He, and H7is independently hydrogen or a deuterium - enriched -H site; andwherein at least one of H1, H2, H3, H4, H5, H6, and H7is a deuterium-enriched -H site,or a salt or ester thereof;(b) detecting positrons emitted from the compound or the composition; and(c) constructing a PET image of the spatial distribution of the compound or the composition.

[0008] The present discourse provides a process of preparing a compound of formula (VI):wherein D is a deuterium-enriched -H site:wherein the process comprises:(a) reacting a compound of formula (I) with an oxidizing agent in a baseD D Oll(X Jc' JLuD DNHBoc (I),to produce a compound of formula (II)'Y Or-BuD NHBoc (JI);(b) reacting the compound of formula (II) with isocyanide in an acid to produce an intermediate, then reacting the intermediate with thiourea in a base to produce a compound of formula (III)O D D Q(III);(c) reacting the compound of formula (III) with a protecting agent in a base to produce a compound of formula (IV)O D D OTmob^‘Or-rs< J u NHBoc (IV);(d) reacting the compound of formula (IV) with an18F labeled salt in a base to produce a compound of formula (V)'Ot(V); ande) reacting the compound of formula (V) with a de-protecting agent to thereby produce the compound of formula (VI).BRIEF DESCRIPTION OF THE DRAWINGS

[0009] FIG. 1. Structures of (A). [18F]FDG; (B). L-5-[11C]glutamine; (C). L-[5-13C-4-D2]glutamine; (D). (2S,4R)-4-[18F]fluoroglutamine (4-[18F]FGln).

[0010] FIG. 2. Representative FDA approved deuterated drugs and PET radiotracers. (A) First FDA approved deuterated drug Deutetrabenazine in 2017; (B) FDA approved deuterated drug Deucravacitinib in 2022; (C) [nC]L-deprenyl-d2; (D) (25’,47?)-[4-18F-3,3,4-D3]fluoroglutamine (4-[18F]FGln-ds).

[0011] FIG. 3. Cellular uptake of 4-[18F]FGln and 4-[18F]FGln-d3. (A) Time-dependent uptake of 4-[18F]FGln and 4-[18F]FGln-d3in 9L / lacZ cells (n=3). (B) Uptake of 4-[18F]FGln and 4-[18F]FGln-d3by 9L / lacZ cells in the presence of BCH (n = 3), MeAIB (n = 3), L-Ser (n = 3), and L-Gln (n = 3).

[0012] FIG. 4. PET imaging of 4-[18F]FGln and 4-[18F]FGln-d3in 9L / lacZ tumor-bearing rats. Representative dynamic PET images of 4-[18F]FGln (n=3) and 4-[18F]FGln-d3(n=3) in 9L / lacZ tumorbearing rats at 30, 60, and 120 min postinjection.

[0013] FIG. 5. ROI analysis of PET imaging for 4-[18F]FGln and 4-[18F]FGln-d3in 9L / lacZ-tumor-bearing rats. Time-activity curves of (A) tumor, (B) bone, (C) muscle, and (D) blood for 4-[18F]FGln (n=3) and 4-[18F]FGln-d3(n=3).

[0014] FIG. 6. Simplified mechanism of increased glutaminolysis in tumor cells and accumulation of 4-[18F]FGln in tumor cells. FDG / PET - a paradigmatic modality for cancer imaging; its mechanism - up-regulated oncogene PI3K / Akt in cancer cells - increased aerobic glycolysis (Warburg Effect). Up-regulation of oncogene c-Myc: increased glutaminolysis - Gln / PET new modality for cancer imaging.

[0015] FIG. 7. Potential labeling site for isotope labeled glutamine analogs, such as18F-Gln Targets: 4-[18F]FGlnsnC-Gln Target.

[0016] FIG.8. A. Chemical structures ofnC-Gln,18F-(2S,4R) &18F-(2S,4S)4FGln. B. In vitro uptake studies of18F-(2S,4R) &18F-(2S,4S)4FGln in SF-188bcl-xL cell line.3H-L-Gln and18F-FDG were used as references.

[0017] FIG. 9. a-position to carbonyl-5C, relatively labile in vivo defluorination tendency.

[0018] FIG. 10. A. Small animal imaging of18F-(2S,4R) 4FGln in F344 9L (glioma tumor) after intravenous injection. Data represent images from summed 2 h scan. Images are shown in transverse, coronal, and sagittal views. Arrows represent location of tumors. B. Small animal PET time-activity curve for18F-(2S,4R)-4FGln after intravenous injection into F344 rat bearing xenografted 9L tumor on left shoulder.

[0019] FIG. 11.18F-4-FGln shows uptake in human gliomas undergoing progression. Clear bone uptake in both animal models and humans (including axial uptake), with free fluorine- 18 detected in the blood of all patients, implying that in vivo defluorination occurs. (A-F) images from glioma patient. A. Tl-weighted MR image with contrast enhancement from a 42-y-old iDHlm oligodendroglioma patient showing tumor with minimal gadolinium enhancement (red arrows) along surgical cavity (indicated by white dotted line). B. Fusion18F-4-FGln PET / CT showing18F-4-FGln uptake in areas corresponding to tumor (red arrows). C.18F-4-FGln PET showing high uptake in tumor with minimal uptake in surrounding brain. D. CT scan used to generate PET / CT fusion image in B. E.18F-FDG PET image from same patient showing high background brain avidity and tumor uptake in posterior part of tumor (3 red arrows), but not in anterior portion (2 red arrows). F. Time activity curve of18F-4-FGln: indicating standard uptake values (SUV) corresponding to tumor (black squares) and blood (clear circles).

[0020] FIG. 12. [’8F]FGln has great potential to be used to monitor inflammation in addition to cancer.

[0021] FIG. 13. Synthesis of (2S,4R)-4-[18F]fluoroglutamine ([18F]FGln). The total production time ranged from 86 to 110 min. Starting with 9.1-15.2 GBq (245-410 mCi)18F activity, 166-411 mBq (4.5-11.1 mCi) [18F]FGln was produced at the end of synthesis (EOS) with the radiochemical purities of the final product range from 89% to 94%.

[0022] FIG. 14. The improved metabolic stability: deuterium kinetic isotope effect (DKIE). KIE= KH / KD. Potential energy of a C-H and C-D bond demonstrating: the zero-point energy of a deuterated isotopologue (ED°) is lower than the zero-point energy of a protonated molecule (EH°), due to the increased mass of the D atom. The transition state energy remains relatively the same due to the near identical electronic properties of both molecules. The result is that a greater difference in energy (ΔED) is required to dissociate the C-D bond, compared to the energy difference (ΔEH) required to dissociate a C-H bond, despite the transition state potential energy of both deuterated and non-deuterated molecules being the same. This phenomenon accounts for the improved metabolic stability of deuterated radiopharmaceuticals.

[0023] FIG. 15. (a) Severe erythema and swelling (arrow) were observed in the left hind paw with carrageenan-induced paw edema (CIPE) compared to the right hind paw. (b) Severe erythema and swelling (arrow) were observed in the left hind paw with collagen-induced arthritis (CIA) compared to the right hind paw. (c) [18F]FGln uptake in the injected hind paw (arrow) was higher compared to the non-injected hind paw by 83% in the CIPE model, (d) [18F]FGln uptake in the hind paw with severe inflammation (arrow) was higher compared to the averaged controls by 143% in the CIA model. In contrast, the contralateral hind paw without inflammation did not show increased uptake, (e) [18F]FGln time-activity curves from 0 to 90 min showed consistent findings with the averaged PET images in (c and d). (f) The average uptake from 0 to 90 min in the paws with severe inflammation (score 4) did not show significant difference between themodels (P = 0.831). Four non-injected right hind paws of the CIPE models are presented as controls, (g) The average uptake from 0 to 90 min and inflammation severity score in CIA rats showed significant positive correlation (r = 0.88, P = 0.009). (h) Hematoxylin & Eosin (H & E) staining of the hind paw of a normal rat. (i) H & E staining of the hind paw with severe inflammation in a rat with CIA. Profound synovial inflammation was observed in the joint. [18F]FGln PET images (c and d) are presented as maximum intensity projection images averaged from 10 to 40 min. The organs in red are urinary bladders filled with radioactive urine. CIA = Collagen-Induced Arthritis; CIPE = Carrageenan-Induced Paw Edema: B = bone; BM = bone marrow; C = cartilage; S = synovium; SI = synovial inflammation.

[0024] FIG. 16. [18F]FGln uptake was increased in both acute and chronic inflammatory conditions in the rat paw edema and arthritis models. Particularly, in rats with the CIA models. [18F]FGln uptake level showed significant positive correlation with the severity of induced inflammation. It was suggested the utility of [18F]FGln as a potential metabolic imaging marker for inflammatory conditions.

[0025] FIG. 17. Synthesis of tosylate precursor and19F-standard compounds.

[0026] FIG. 18. Radiolabeling method.

[0027] FIG. 19. Synthesis of deuterated homoserine intermediate 11.

[0028] FIG. 20. Synthesis of deuterated and fluorinated (2S,4R)-4-fluoroglutamines. 4-[18F]FGln-d3and 4-FGln-d3DETAILED DESCRIPTION OF THE INVENTION

[0029] The present discourse provides a composition comprising a compound having the structure:wherein each of Hi, H2, H3, H4, H5, He, and H7 is independently hydrogen or a deuterium - enriched -H site; andwherein at least one of Hi, H2, H3, H4, Hs, He, and H7is a deuterium-enriched -H site,or a salt or ester thereof.

[0030] In some embodiments, wherein in the compound, at least two of H1, H2, H3, H4, H5, H6, and H7are deuterium-enriched -H sites.

[0031] In some embodiments, wherein in the compound, at least three of H1, H2, H3, H4, H5, H6, and H7are deuterium-enriched -H sites.

[0032] In some embodiments, wherein in the compound, at least four of H1, H2, H3, H4, H5, H6, and H7are deuterium-enriched -H sites.

[0033] In some embodiments, wherein in the compound, at least three of H1, H2, H3, H4, H5, H6, and H7are deuterium-enriched -H sites.

[0034] In some embodiments, wherein in the compound, three of H1, H2, H3, H4, H5, H6, and H7are deuterium-enriched -H sites.

[0035] In some embodiments, wherein in the compound, one of H1, H2, and H3is a deuterium-enriched -H site.

[0036] In some embodiments, wherein in the compound, two of Hi, H2, and H3are deuterium-enriched -H sites.

[0037] In some embodiments, wherein in the compound, one of H4, H5, H6, and H7is a deuterium-enriched -H site.

[0038] In some embodiments, wherein in the compound, two of H4, H5, H6, and H7are deuterium-enriched -H sites.

[0039] In some embodiments, wherein in the compound, three of H4, H5, H6, and H7are deuterium-enriched -H sites.

[0040] In some embodiments, wherein in the compound, one of H1, H2, and H3is deuterium-enriched -H site; and two of H4, H5, H6, and H7are deuterium-enriched -H sites.

[0041] In some embodiments, wherein in the compound, one of H3, H4, and H5is deuterium-enriched -H sites.

[0042] In some embodiments, wherein in the compound, two of H3, H4, and H5are deuterium-enriched -H sites.

[0043] In some embodiments, wherein in the compound, H3, H4, and H5are deuterium-enriched -H sites.

[0044] In some embodiments, wherein in the compound, H3, H4, and H5are deuterium-enriched -H sites and the level of deuterium at the deuterium-enriched -H site is 0.02% to 100%.

[0045] In some embodiments, wherein in the compound, H3, H4, and H5are deuterium-enriched -H sites and the level of deuterium at the deuterium-enriched -H site is 20%-100%, 50%-100%, 70%-100%, 90%-100%, 97%-100%, or 99%-100%.

[0046] In some embodiments, wherein in the compound, H3, H4, and H5are deuterium-enriched -H sites and the level of deuterium at the deuterium-enriched -H site is no less than 50%.

[0047] In some embodiments, wherein in the compound, H3, H4, and H5are deuterium-enriched -H sites and the level of deuterium at the deuterium-enriched -H site is no less than 70%.

[0048] In some embodiments, wherein in the compound, H3, H4, and H5are deuterium-enriched -H sites and the level of deuterium at the deuterium-enriched -H site is no less than 90%.

[0049] In some embodiments, wherein in the compound, H3, H4, and H5are deuterium-enriched -H sites and the level of deuterium at the deuterium-enriched -H site is no less than 97%.

[0050] In some embodiments, wherein in the compound, H3, H4, and H5are deuterium-enriched -H sites and the level of deuterium at the deuterium-enriched -H site is no less than 99%.

[0051] In some embodiments, wherein in the compound, H3, H4, and H5are deuterium-enriched -H sites and the proportion of molecules having deuterium at the H3, H4, and H5 position is substantially greater than 0.0156% of molecules in the composition.

[0052] In some embodiments, the compound has the structure:HxN OH IHwherein H is hydrogen and D represents a deuterium -enriched -H site.

[0053] In some embodiments, the compound has the structure:OH

[0054] In some embodiments, the composition further comprises one or more compound having structure

[0055] In some embodiments, the composition further comprises F NH2

[0056] In some embodiments, the molar ratio betweenin the composition is from 99: 1 to 1:99.O D D O OHH18f D

[0057] In some embodiments, the molar ratio betweenH H and^2 in the composition is from 95:5 to 50:50.OH H18F D

[0058] In some embodiments, the molar ratio betweenH H andin the composition is from 95:5 to 60:40.O D D oH18F D

[0059] In some embodiments, the molar ratio betweenH H andH2N OHNH, in the composition is from 90:10 to 70:30.

[0060] In some embodiments, the molar ratio betweenand^2 in the composition is from 99: 10 to 80:20.

[0061] In some embodiments, the composition further comprises one or more pharmaceutically acceptable carriers.

[0062] The present disclosure provides a method of detecting the presence of inflammatory cells and / or tumor cells in a subject by administering the composition disclosed herein to the subject, wherein the method comprises determining if an amount of the compound or the composition is present in the subject after a period after administration.

[0063] Tire present disclosure provides a method of detecting the location of inflammatory cells and / or tumor cells in a subject by administering the composition disclosed herein to the subject, wherein the method comprises determining if an amount of the compound or the composition is present in the subject after a period after administration.

[0064] The present disclosure provides a method of imaging inflammatory cells and / or tumor cells in a subject by administering the composition disclosed herein to the subject, wherein the method comprisesdetermining if an amount of the compound or the composition is present in the subject after a period after administration.

[0065] In some embodiment, the period after administration is 1, 2, 3 or 4 hours.

[0066] In some embodiment, the period after administration is 1. 2, or 3 hours.

[0067] In some embodiment, the period after administration is 1, or 2 hours.

[0068] In some embodiments, wherein the inflammatory cells are caused by bacteria infection, viral infection, injuries, toxins, or autoimmune triggers.

[0069] In some embodiments, wherein the tumor cells are cancerous cells.

[0070] Tire present disclosure provides a method of imaging cells expressing glutamine in a subject, comprising administering the composition disclosed herein to the subject.

[0071] Tire present disclosure provides a method for positron emission tomography (PET) imaging of a subject, comprising:(a) administering to the subject the composition disclosed herein:(b) detecting positrons emitted from the compound or the composition; and(c) constructing a PET image of the spatial distribution of the compound or the composition.

[0072] The present disclosure provides a method of diagnosing a disease in a subject, wherein the method comprises performing the PET imaging disclosed herein to the subject.

[0073] In some embodiments, wherein the disease is gliomas, neuroblastoma or brain metastasis, lung cancer, colon cancer, triple-negative breast cancer (TNBC), breast cancer, hepatocellular carcinoma, nonalcoholic steatohepatitis (NASH), atherosclerotic lesions, multiple myeloma (MM), clear cell renal cell carcinoma (ccRCC), osteosarcoma, pancreatic ductal adenocarcinoma (PDAC), inflammatory arthritis, or myositis.

[0074] In some embodiments, wherein the subject is a mammal.

[0075] In some embodiments, the mammal is human.

[0076] The present disclosure provides a process of preparing a compound of formula (VI):O D D OH2N><X' y \)H18F D NH2(VI),wherein D is a deuterium-enriched -H site:wherein the process comprises:(a) reacting a compound of formula (I) with an oxidizing agent in a baseD D O Y O / -BuD D NHBoc (J).to produce a compound of formula (II)D D O\ 'CK AOr-BuD NHBoc (n);(b) reacting the compound of formula (II) with isocyanide in an acid to produce an intennediate, then reacting the intermediate with thiourea in a base to produce a compound of formula (III)HD' -D0HHO D NHBOC (III);(c) reacting the compound of formula (III) with a protecting agent in a base to produce a compound of formula (IV)o D D oTmob^N X Y Ot- H-isu u NHBOC (IV);(d) reacting the compound of formula (IV) with an18F labeled salt in a base to produce a compound of formula (V)Ot(V); ande) reacting the compound of formula (V) with a de-protecting agent to thereby produce the compound of formula (VI).

[0077] In some embodiments, wherein the compound of formula (I) has the structure:D D 0HCk 'c'O / -BuO NHBoc

[0078] In some embodiments, wherein the compound of formula (II) has the structure:D \ / D OO ("Ot-BuD NHBoc

[0079] In some embodiments, wherein the compound of formula (III) has the structure: o D p o Tmobx J-L Jx.N y O / -BuHHO D NHBoc

[0080] In some embodiments, wherein the compound of formula (IV) has the structure:O D D OTmob N jO / ’BuH TsO D NHBoc

[0081] In some embodiments, wherein the compound of formula (V) has tire structure:O D D OTmob^N'^S<CY ^-BuH 18F D NHBOC

[0082] In some embodiments, wherein the oxidizing agent in step (a) is Dess-Martin periodinane (DMP), pyridinium chlorochromate (PCC), swem oxidation reagents, or 2,2,6,6-Tetramethylpiperidine-l- oxyl (TEMP).

[0083] In some embodiments, the oxidizing agent in step (a) is DMP.

[0084] In some embodiments, the base in step (a) is NaHCCh. KHCOs, NH4HCO3, NazCCE, or CaCCE.

[0085] In some embodiments, the base in step (a) is NaHCCE.

[0086] In some embodiments, the isocyanide in step (b) is trimethoxybenzyl isocyanide(TmobNC), dimethoxybenzyl isocyanides, monomethoxybenzyl isocyanides, 4-methoxybenzyl isocyanide.

[0087] In some embodiments, the isocyanide in step (b) is TmobNC.

[0088] In some embodiments, the acid in step (b) is chloroacetic acid (CAA), bromoacetic acid, iodoacetic acid, fluoroacetic acid, dichloroacetic acid, or trichloroacetic acid.

[0089] In some embodiments, the acid in step (b) is CAA.

[0090] In some embodiments, the base in step (b) is NaHCCE. KHCO3, NH4HCO3, Na2CO3, or CaCOs.

[0091] In some embodiments, the base in step (b) is NaHCCE

[0092] In some embodiments, the protecting agent in step (c) is tosyl chloride (TsCl), mesyl chloride (MsCl), nosyl chloride (NsCl), besyl chloride, ortriflyl chloride (TfCl).

[0093] In some embodiments, the protecting agent in step (c) is TsCl.

[0094] In some embodiments, the base in step (c) is triethylamine (EtsN), tributylamine, tripropylamine, N, N-diisopropylamine, pyridine, or N-methylmorpholine.

[0095] In some embodiments, the base in step (c) is EtsN.

[0096] In some embodiments, step (c) is conducted in the presence of a catalyst, preferably, the catalyst is 4-dimethylaminopyridine (DMAP), 4-pyrrolidinopyridine (PPY), 4-dimethylaminopyridine N-oxide (D MAP-O), 4-(N, N-diethylamino)pyridine, N-methylimidazole (NMI), 1,2,4-triazole, or imidazole.

[0097] In some embodiments, the catalyst is DMAP.

[0098] In some embodiments, the18F labeled salt in step (d) is18F labeled potassium fluoride ([18F]KF),18F labeled sodium fluoride ([18F]NaF),18F labeled ammonium fluoride ([18F]NH4F),18F labeled lithium fluoride ([18F]LiF),18F labeled cesium fluoride ([18F]CsF), or18F labeled tetrabutylammonium fluoride ([18F]TBAF).

[0099] In some embodiments, the18F labeled salt in step (d) is [18F]KF.

[0100] In some embodiments, the base in step (d) is KHCOs or K2CO3.

[0101] In some embodiments, the base in step (d) is KHCO3.

[0102] In some embodiments, the de-protecting agent in step (e) is trifluoroacetic acid (TFA), anisole, dichloroacetic acid (DCA), trichloroacetic acid, or hexafluoroisopropanol (HFIP).

[0103] In some embodiments, the de-protecting agent in step (e) is TFA and anisole.IJD' -DIJT"’“b'N;S<CYAO,. B0Ho D NHBoc )=O

[0104] In some embodiments, the intermediate in step (b) is:Cl?D' -DuTn, Ob'N'kXCY^O / -B1,H0 ’ t> NHBoc >=O

[0105] In some embodiments, the intennediate in step (b) is Clo D D O Tmolx ^C^N O / -BuII O D NHBoc 7=0

[0106] In some embodiments, the intermediate in step (b) is Cl

[0107] In some embodiments, steps (a), (b), (c), and (d) are each conducted in the presence of one or more solvents.

[0108] In some embodiments, steps (a), (b), and (c) are conducted in one pot.

[0109] In some embodiments, steps (a), (b), (c), and (d) are conducted in one pot.

[0110] In some embodiments, steps (a), (b), (c), (d) and (e) are conducted in one pot.

[0111] In some embodiments, the solvent is DCM, EtOH, tetrahydrofuran (THF), acetonitrile (MeCN).

[0112] In some embodiments, the solvent in steps (a), (b) and (c) is DMP, and the solvent in step (d) is MeCN.

[0113] In some embodiments, step (d) further comprises adding a cyclic polyether, preferably, the cyclic polyether is IS-cro\\n-6. 15-crown-5, 12-crown-4, dicyclohexano-18-crown-6, benzo- 18-crown-6, or 21 -crown-7.

[0114] In some embodiments, the cyclic polyether is 18-crown-6.

[0115] In some embodiments, step (a) is conducted at room temperature.

[0116] In some embodiments, step (a) is conducted for a period from 1 to 5 hours; preferably, from 1 to 4 hours; more preferably, from 2 to 3 hours.

[0117] In some embodiments, in step (b), the compound of formula (II) is reacted with isocyanide at room temperature.

[0118] In some embodiments, in step (b), the compound of formula (II) is reacted with isocyanide for a period from 5 to 30 hours; preferably, from 10 to 20 hours; more preferably from 15 to 17 hours.

[0119] In some embodiments, in step (b), the intermediate is reacted with thiourea at a temperature from 30 to70 °C; preferably, from 40 to70 °C; more preferably, from 40 to 60 °C; more preferably, from 45 to 55 °C.

[0120] In some embodiments, in step (b), the intermediate is reacted with thiourea for a period from 2 to 20 hours; preferably, from 3 to 10 hours; more preferably from 4 to 6 hours.

[0121] In some embodiments, step (c) is conducted at a temperature from 0 to 30 °C; preferably, from 0 to 25 °C.

[0122] In some embodiments, step (d) is conducted at a temperature from 30 to 90 °C; preferably, from 40 to 80 °C; more preferably, from 50 to 80 °C; more preferably, from 60 to 80 °C.

[0123] In some embodiments, step (e) is conducted at a temperature from 30 to 90 °C; preferably, from 40 to 80 °C; more preferably, from 50 to 70 °C; more preferably, from 55to 65 °C.

[0124] The present disclosure provides a process for preparing a compound of formula I,D D O HO-. c'Y Or-BuD D NHBoc (I).wherein D is a deuterium -enriched -H site;wherein the process comprises:OBnN N(a) reacting OBn N OBnwith a deuterium enriched haloalcohol in an acid to produce a compound of formula (VII);D DD- / C-C \-DRj()15n(VII), wherein Ri is halogen;OPh> N OtBu(b) reacting tire compound of formula (VII) withPh in a base to produce an intemrediate; then reacting the intemrediate with an acid to produce a compound of formula (VIII)BnO(VIII);(c) reacting the compound of formula (VIII) with a protecting agent to produce a compound of formula (IX)BnO(IX); and(d) reacting the compound of formula (IX) with a de-protecting agent in an alcohol to produce the compound of formula (I).

[0125] In some embodiments, wherein after step (b) and before step (c), S enantiomer of formula (VIII) is isolated for further reaction; wherein the S enantiomer of formula (VIII) has the structure:D2 HBnCK X. XC y O / -BuD2NH2

[0126] In some embodiments, the S enantiomer of formula (VIII) is isolated by HPLC.BnO DD N OtBuPh

[0127] In some embodiments, wherein the intemiediate isPh

[0128] In some embodiments, the intennediateis

[0129] In some embodiments, Ri is F, Br, Cl. I, preferably. Ri is F.D D O HCk C'y Ji Ot-Bu

[0130] In some embodiments, the compound of formula (I) has the structure: D D NHBocBnO

[0131] In some embodiments, the compound of formula (IX) has tire structure:

[0132] In some embodiments, the acid in step (a) is trifluoromethanesulfonic acid (TfOH); nonafluorobutanesulfonic acid, perfluoroethane sulfonic acid, perfluoropropanesulfonic acid, fluorosulfuric acid, methanesulfonic acid, or p-toluenesulfonic acid.

[0133] In some embodiments, the acid in step (a) is TfOH.

[0134] In some embodiments, step (a) is conducted in the presence of a solvent, preferably, the solvent is 1,4-dioxane, tetrahydrofuran, 1,3 -dioxolane, dimethoxyethane, ethylene glycol dimethyl ether, diethylene glycol dimethyl ether, or diglyme.

[0135] In some embodiments, the solvent is 1,4-dioxane.

[0136] In some embodiments, the base in step (b) is THF, lithium diisopropylamide, sodium hydride, potassium tert-butoxide, sodium methoxide, or potassium hydride.

[0137] In some embodiments, the base in step (b) is THF.

[0138] In some embodiments, the acid is step (b) is citric acid, tartaric acid, malic acid, ascorbic acid, oxalic acid, or lactic acid.

[0139] In some embodiments, the acid is step (b) is citric acid.

[0140] In some embodiments, step (b) further comprises adding a base, preferably, the base is t-BuOK, sodium hydride, lithium diisopropylamide, potassium bis(trimethylsilyl)amide, sodium methoxide, or potassium tert-amylate.

[0141] In some embodiments, steps (a) and (b) are conducted in one pot.

[0142] In some embodiments, steps (c) and (d) are conducted in one pot.

[0143] In some embodiments, steps (a) and (b) are conducted in one pot and steps (c) and (d) are conducted in another pot.

[0144] In some embodiments, the base is t-BuOK.

[0145] In some embodiments, the protecting agent in step (c) is (BocfiO. di-tert-butyl dicarbonate, or tert-butyl chloroformate.

[0146] In some embodiments, the protecting agent in step (c) is (Boc O.

[0147] In some embodiments, step (c) is conducted in the presence of a solvent, preferably, the solvent is 1,4-dioxane, tetrahydrofuran. 1,3-dioxolane. dimethoxyethane, ethylene glycol dimethyl ether, di ethylene glycol dimethyl ether, or diglyme.

[0148] In some embodiments, the solvent is 1,4-dioxane.

[0149] In some embodiments, the de-protecting agent in step (d) is palladium on carbon (Pd / C), platinum on carbon, Raney nickel, palladium hydroxide on carbon, or rhodium on carbon.

[0150] In some embodiments, the de-protecting agent in step (d) is Pd / C.

[0151] In some embodiments, the alcohol in step (d) is EtOH, methanol, propanol, isopropanol, butanol, or tert-butanol.

[0152] In some embodiments, the alcohol in step (d)is EtOH.

[0153] In some embodiments, step (a) is conducted at room temperature.

[0154] In some embodiments, step (a) is conducted for a period from 10 to 30 hours; preferably, from 15 to 20 hours; more preferably from 15 to 18 hours.

[0155] In some embodiments, step (a) is conducted with molecular sieves.

[0156] In some embodiments, in step (b), the compound of formula (VII) is first reacted with OPh >=N OtBu Ph at a temperature from -70 °C to 0 °C, then reacted at room temperature; preferably, atroom temperature for a period from 10 to 30 hours; more preferably, from 15 to 20 hours; more preferably from 1 to 18 hours.

[0157] In some embodiments, the intermediate is reacted with the acid at room temperature.

[0158] In some embodiments, the intermediate is reacted with the acid for a period from 10 to 30 hours; preferably, from 15 to 20 hours; more preferably from 15 to 18 hours.

[0159] In some embodiments, step (c) is conducted at room temperature.

[0160] In some embodiments, step (c) is conducted for a period from 10 to 30 hours; preferably, from 15 to 20 hours; more preferably from 15 to 18 hour.

[0161] In some embodiments, step (d) is conducted in hydrogen.

[0162] In some embodiments, step (d) is conducted at room temperature.

[0163] In some embodiments, step (d) is conducted for a period from 10 to 30 hours; preferably, from 15 to 20 hours; more preferably from 15 to 18 hours.

[0164] Tire present invention provides a PET imaging system comprising a PET imaging device, and radiotracers comprising the composition disclosed herein.

[0165] The present invention provides a package for use in PET imaging which comprises the composition of any one of claims 1-21.

[0166] The present invention provides use of the composition disclosed herein in(a) detecting the presence of inflammatory cells and / or tumor cells in a subject;(b) detecting the location of inflammatory cells and / or tumor cells in a subject;(c) imaging inflammatory cells and / or tumor cells in a subject;(d) imaging cells expressing glutamine in a subject;(e) PET imaging of a subject; and / or(f) diagnosing a disease in a subject.

[0167] Tire present invention provides use of the composition disclosed herein in(a) detecting the presence of inflammatory cells and / or tumor cells in a subject;(b) detecting the location of inflammatory cells and / or tumor cells in a subject;(c) imaging inflammatory cells and / or tumor cells in a subject;(d) imaging cells expressing glutamine in a subject;(e) PET imaging of a subject; and / or(f) diagnosing a disease in a subject.

[0168] Except where otherwise specified, the structure of a compound of this invention includes an asymmetric 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.

[0169] Except where otherwise specified, the subject invention is intended to include all isotopes of atoms 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.

[0170] It will be noted that any notations of a carbon in structures throughout this application, when used 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.

[0171] It will also be noted that any notations of a hydrogen (H) in structures throughout this application, when used without further notation, are intended to represent all isotopes of hydrogen, such as ’H,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.

[0172] Isotopically-labeled compounds can generally be prepared by conventional techniques known to those skilled in the art using appropriate isotopically-labeled reagents in place of the non-labeled reagents employed.

[0173] Deuterium (2H or D) is a stable, non-radioactive isotope of hydrogen and has an atomic weight of 2.0144. Hydrogen atom in a compound naturally occurs as a mixture of the isotopes H (hydrogen or protium), D (2H or deuterium), and T (3H or tritium). Hie natural abundance of deuterium is 0.0156%. Thus, in a composition comprising molecules of a naturally occurring compound, the level of deuterium at a particular hydrogen atom site in that compound is expected to be 0.0156%. Thus, a composition comprisinga 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.

[0174] As used herein, a hydrogen at a specific site in a compound is '‘deuterium -enriched” if the amount 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 tire 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.

[0175] In the compounds used in the method of the present invention, the substituents may be substituted or unsubstituted, unless specifically defined otherwise.

[0176] In tire compounds used in tire method of the present invention, alkyl, heteroalkyl, monocycle, bicycle, aryl, hctcroaryl 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.

[0177] It is understood that substituents and substitution patterns on the compounds used in the method of 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.

[0178] In choosing the compounds used in the method of the present invention, one of ordinary skill in the art will recognize that the various substituents, i.e. Ri, R2, etc. are to be chosen in conformity with well-known principles of chemical structure connectivity.

[0179] Tire compounds used in the method of the present invention may be prepared by techniques well known in organic synthesis and familiar to a practitioner ordinarily skilled in the art. However, these may not be tire only means by which to synthesize or obtain the desired compounds.

[0180] The compounds used in the method of the present invention may be prepared by techniques described in Vogel's Textbook of Practical Organic Chemistry, A. I. Vogel, A. R. Tatchell, B. S. Fumis. 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.

[0181] The various R groups attached to the aromatic rings of the compounds disclosed herein may be added 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.

[0182] Another aspect of the invention comprises a compound used in the method of the present invention as a pharmaceutical composition.

[0183] As used herein, tire tenn “pharmacally active agent” means any substance or compound suitable for administration to a subject and furnishes biological activity or other direct effect in tire 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 arc hereby incorporated by reference. Pharmacally 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 docs not interfere with tire pharmaceutically active agent’s biological activity or effect.

[0184] Tire compounds used in the method of the present invention may be in a salt form. As used herein, a “salt” is a salt of tire 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, the 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. Tire 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 the sodium, potassium, or lithium salts, and the like. The term "pharmacally acceptable salt" in this respect, refers to the relatively non-toxic, inorganic and organic acid or base addition salts of compounds of the present invention. These salts can be prepared in situ during the final isolation and purification of the compoundsof the invention, or by separately reacting 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 the 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",. Pharm. Set. 66:1-19).

[0185] As used herein, "treating" means preventing, slowing, halting, or reversing the progression of a disease. Treating may also mean improving one or more symptoms of a disease.

[0186] The compounds used in the method of the present invention may be administered in various forms, 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 tire 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.

[0187] As used herein, a "pharmaceutically acceptable carrier" is a pharmacally acceptable solvent, suspending agent or vehicle, for delivering tire 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.

[0188] The dosage of the compounds administered in treatment will vary depending upon factors such as 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.

[0189] A dosage unit of the compounds used in the method of the present invention may comprise a single 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 fonns well known to those of ordinary skill in the pharmaceutical arts.

[0190] The compounds used in the method of the present invention can be administered in a mixture with 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 fonn 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. Capsule 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 fonns 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. |01911 Techniques and compositions for making dosage forms useful in the present invention are described in the following references: 7 Modem Pharmaceutics, Chapters 9 and 10 (Banker & Rhodes, Editors, 1979); Pharmaceutical Dosage Forms: Tablets (Lieberman et al. 1981); Ansel, Introduction to Pharmaceutical Dosage Fonns 2nd Edition (1976); Remington's Pharmacal 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 Florwood Books in the Biological Sciences. Scries in Pharmacal Technology; J. G. Hardy, S. S. Davis, Clive G. Wilson, Eds.); Modem Pharmacs Drugs and the Pharmaceutical Sciences. Vol 40 (Gilbert S. Banker, Christopher T. Rhodes, Eds.). All of the aforementioned publications are incorporated by reference herein.

[0192] 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, com 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.

[0193] The compounds used in the method of the present invention may also be administered in the form 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.

[0194] The compounds used in the method of the present invention may also be coupled to soluble polymers 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.

[0195] Gelatin capsules may contain active ingredient compounds and powdered carriers, such as lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, and tire 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.

[0196] For oral administration in liquid dosage form, the oral drug components are combined with any oral, non-toxic, pharmaceutically acceptable inert carriers 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.

[0197] Liquid dosage fonns for oral administration can contain coloring and flavoring to increase patient 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, I7th ed., 1989, a standard reference text in this field.

[0198] The compounds used in the method of the present invention may also be administered in intranasal 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 transdennal delivery system, the dosage administration will generally be continuous rather than intennittent throughout the dosage regimen.

[0199] Parenteral and intravenous fonns may also include minerals and other materials to make them compatible with the type of injection or delivery system chosen.

[0200] Each embodiment disclosed herein is contemplated as being applicable to each of the other disclosed 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.

[0201] 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.

[0202] Unless otherwise defined, all technical and / or scientific terms used herein have the same meaning 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 tire practiceor 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.

[0203] 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.

[0204] It should be understood that the terms “a” and “an” as used above and elsewhere herein refer to “one or more” of the enumerated components. It will be clear to one of ordinary skill in the art that the 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.

[0205] For purposes of better understanding the present teachings and in no way limiting the scope of the 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 arc approximations that may vary’ depending upon the desired properties sought to be obtained. At the very least, each numerical parameter should at least be constmed in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0206] 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

[0207] For the foregoing embodiments, each embodiment disclosed herein is contemplated as being applicable to each of the other disclosed embodiments.

[0208] As used herein, all headings are simply for organization and are not intended to limit the disclosure 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.

[0209] Additional objects, advantages, and novel features of the present invention will become apparent 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.

[0210] It is appreciated that certain features of the invention, which are. for clarity, described in the context of separate embodiments, may also be provided in combination in 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.

[0211] Examples are provided below to facilitate a more complete understanding of the invention. Hie following 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

[0212] Materials and Methods

[0213] Organic chemistry: All reagents used were commercial products and were used without further purification unless otherwise indicated. Reactions were carried out under an atmosphere of nitrogen. Analytical thin layer chromatography was carried out using aluminum-backed plates coated with Merck Kieselgel 60 GF254. Chromatography was performed using silica gel (70-230 mesh, Sigma-Aldrich). 'H NMR spectra were obtained either at 400, 500 or 700 MHz;13C NMR spectra were recorded either at 125 or 175 MHz;19F NMR spectra were recorded at 376 MHz; and2H NMR spectra were obtained at 61 MHz (Bruker Nanobay 400, Avance III 500 and Avance III 700 spectrometers). Chemical shifts are reported as 5 values (parts per million) relative to remaining protons in deuterated solvent. Coupling constants are reported in hertz. The multiplicity is defined by s (singlet), d (doublet), t (triplet), q (quartet), p (pentet), br (broad) or m (multiplet). HPLC analyses were performed on Jasco LC-4000 series. LRMS and HRMS wereobtained at Agilent 6110 Quadrapole LC / MS and Agilent LC-UV-TOF instruments, respectively. Optical rotation values were measured on Anton Paar MCP 100 polarimeter.

[0214] tert-Butyl (<9J-benzyl-3,3,4,4-tetradeutero)homoserinate (9a,b)D D OBn D4 — (-D Br > OH D D D D oD) (D +)=< TfOH, 1,4-dioxane Br OBn D OBn 4A MS, rt, 16 h 7 7’ TriBOT, 5 i. f-BuOK, THF, -70 to 0 °C then rt 16 h ii 10% CitricAcid, THF, rt, 16 h57% over two steps

[0215] Step i. To a solution of 2-bromoethanol-<746 (0.73 mL, 10 mmol), in 1,4-dioxane (50 mL) was added triazine 5 (1.6 g, 4 mmol) and 4 A MS (1.25 g) followed by TfOH (0.18 mL, 2 mmol) and stirred at room temperature for 1.5 h under N2atmosphere. At this point, triazine (1.4 g, 3.5 mmol) was added to the reaction mixture and stirred at room temperature for 16 h under N2atmosphere. The solvent was removed under reduced pressure and the residue was purified over silica gel column chromatography using 5% EtOAc in hexanes to afford a mixture of ( 1, 1,2, 2-<74-2-bromoethoxy )benzene (7) and (l,2,2-< A-vinyl)benzyl ether (7’) in the ratio 9.4: 1 (2.275 g) as a pale-yellow oil.

[0216] Step ii. In a flame-dried 250 mL round-bottomed flask a solution of / -butyl N-(diphenylmethylene) glycinate 8 (1.77 g, 6 mmol) in THF (30 mL) was prepared and cooled down to -70 °C under N2atmosphere. To this, a solution of KO / BLI (0.9 g, 6 mmol) in THF (6 mL) was added and stirred at 0 °C for 30 min. Then a solution of mixture from step 1 (1.137 g) in THF (8 mL) was added to the mixture and stirred at room temperature for 16 h. Tire solvent was removed under reduced pressure and to the residue was added saturated NaHCOs (100 mL) and then extracted with CH2C12(3 x 75 mL). All organic layers were combined, washed with brine (10 mL), dried with anhydrous Na2SC>4, filtered, and concentrated under reduced pressure to afford a red oil. Another reaction was also conducted on the same scale simultaneously.

[0217] Step Hi. This residue from the two batches of step ii was dissolved in THF (15 mL). To this solution, was added 10% Citric Acid (15 mL) dropwise and stirred overnight at room temperature. The mixture was acidified with 0.5 M HC1 (20 mL), and then washed with tert-butylmethyl ether (TBME, 50 mL). The organic phase was extracted with water (2 x 50 mL). The combined aqueous layers were washed with TBME (4x50 mL) to remove the cleaved benzophenone. The aqueous layer was then basified with solid Na2COs to adjust PH = 9 and extracted with CH2CI2 (3 x 50 mL). Hie combined organic extracts were dried with anhydrous Na2SO4 and concentrated in vacuo. The crude product was purified by silica gel column chromatography using 1-3% MeOH in CH2CI2 to give the title compounds 9a and 9b (1.39 g, 51%) as a colorless liquid. 'HNMR (400 MHz, CDCL) <57.37-7.31 (m, 4H), 7.31-7.27 (m, 1H), 4.51 (s, 2H), 3.49 (s, 3H). 1.44 (s, 9H). HRMS ealed for C15H20D4NO3 [M+H]+, 270.2002; found, 270.2001.

[0218] Chiral Separation of (2S)-tert-Butyl (<9J-benzyl-3,3,4,4-tetradeutero)homoserinate (9a) and (2R)- / c 7-Butyl (OJ-benzyl-3,3,4,4-tetradeutero)homoserinate (9b).

[0219] The chiral separation of enantiomers 9a and 9b was carried out on Chiralpak AY-H column (250x20 mm, 5 pm) with the mobile phase (EtOH: 0.1% DIPEA in hexanes = 4: 1), and monitored at 254 nm with flow rate 8.0 mL / min (Rt-9b: 10.82 min; Rt-9a: 15.39 min). A solution of racemic mixture 9a, b (16 mg) in mobile phase (0.8 mL) was used in each injection. A total of 1.765 g (6.552 mmol) of the racemic mixture 9a, b was separated in multiple injections under the aforementioned conditions and both enantiomers 9b and 9a were collected between 10.5-13 min and 14.7-19 min, respectively. All the fractions corresponding to the enantiomer were combined and concentrated under reduced pressure and then dried under high vacuum for several hours until DIPEA completely evaporated to give 9a (873.6 mg, 49%) as a colorless liquid and 9b (854.3 mg, 48%) as a colorless liquid.

[0220] Compound 9a: HPLC Purity: >99% (Rt = 9.692 min; column: Amylose-2 (250 x 4.6 mm), UV detector, 254 nm, 20 / 80 / 0.1 EtOH / Hexanes / DIPEA; flow rate: 1.0 mL / min). ’H NMR (700 MHz, CDCL) <57.36-7.31 (m, 4H), 7.31-7.27 (m, 1H), 4.51 (s, 2H), 3.50 (s, 3H), 1.44 (s, 9H).2HNMR(61 MHz, CDCL) 53.58 (s, 2D), 2.06 (s. ID), 1.75 (s, ID).13C NMR (175 MHz, CDCl3) δ175.2, 138.5, 128.6, 127.9, 127.8, 81.3, 73.2, 66.4 (m), 52.8. 33.8 (m), 28.2. HRMS ealed for C15H20D4NO3 [M+H]+. 270.2002; found,23270.2001. [α]D = 5.233° (c = 0.168, EtOH).

[0221] Compound 9b: HPLC Purity: >99% (Rt = 6.383 min; column: Amylose-2 (250 x 4.6 mm), UV detector, 254 nm, 20 / 80 / 0.1 EtOH / Hexanes / DIPEA; flow rate: 1.0 mL / min). ’H NMR (700 MHz, CDCL) 57.36-7.31 (m, 4H), 7.31-7.26 (m, 1H), 4.50 (s, 2H), 3.48 (s, 3H), 1.44 (s, 9H).2HNMR(61 MHz, CDCL) 53.57 (s, 2D). 2.04 (s. ID), 1.74 (s, ID).13C NMR (175 MHz, CDCl3) δ175.3, 138.4. 128.5, 127.8, 127.7,81.0, 73.1, 66.3 (dt, J = 43.0, 21.5 Hz), 52.6, 33.8 (dt, J = 39.1, 19.4 Hz), 28.1. HRMS calcd for C15H20D4NO3 [M+H]+, 270.2002; found, 270.2000. [α]D = -15.476° (c = 0.172, EtOH).

[0222] (2S)-tert-Butyl (O2-benzyl-N-(tert-butoxycarbonyl)-3,3,4,4-tetradeutero)homoserinate (10)D2?BnCL „C y Of-Bu°2NHBoc

[0223] (2S)-tert-Butyl (O2-benzyl-N-(tert-butoxycarbonyl)-3,3,4,4-tetradeutero)homoserinate (10)

[0224] To a solution of compound 9a (852 mg, 3.163 mmol) in 1,4-dioxane (32 mL), (Boc)2O (828.3 mg, 3.795 mmol) was added and stirred at room temperature for 16 h. Tire mixture was concentrated under reduced pressure and the crude product was purified by silica gel column chromatography using 10% EtOAc in hexanes to give compound 10 (1.168 g, quantitative), as a colorless viscous liquid.1H NMR (700 MHz, CDCh) <57.37-7.31 (m, 4H). 7.31-7.26 (m. 1H), 5.39 (d. 1H. J = 7.8 Hz), 4.48 (ABq, 5AB= 0.02, JAB = 11.9 HZ, 2H), 4.27 (d, 1H, J= 8.0 Hz), 1.44 (s, 9H), 1.43 (s, 9H).2HNMR (61 MHz, CDCh) <53.50 (s, 2D), 2.06 (s, ID), 1.91 (s, ID).13C NMR (175 MHz, CDCh) <5171.8, 155.7, 138.3, 128.6, 128.0, 127.9, 81.9, 79.6, 73.4. 66.2 (m), 52.4. 31.5 (m), 28.6. 28.2. HRMS calcd for C20H28D4NO5 [M+H]+, 370.2526;25found, 370.2523. [α]D = -26.556° (c = 0.9, EtOH).

[0225] (2S)-4-fert-Butyl-2-(tert-butoxycarbonylamino)-3,3,4,4-tetradeutero-4-hydroxybutanoate (11)9■C^D2NHBoc

[0226] In a flame-dried 500 mL two-neck round-bottomed flask a solution of L-homoserinate 10 (1.168 g, 3.161 mmol) in EtOH (234 mL) was prepared to which 10% Pd / C (234 mg) was added and then sealed. The flask was degassed by connecting to a vacuum line. After the solvent in the flask started bubbling, the vacuum was cut off and the flask was filled with H2 gas using a balloon filled with H2 gas. This process was repeated 3 times and then the reaction was stirred overnight under H2atmosphere. The mixture was filtered through a short plug of silica gel and washed with MeOH (100 mL). The filtrate was concentrated under reduced pressure and dried under high vacuum to give compound 11 (844.1 mg, 96%), as a light yellowish liquid. 'H NMR (700 MHz, CDCh) < 5.35 (d, 1H, J= 7.2 Hz), 4.32 (d. 1H. J= 7.2 Hz), 3.42 (br s, 1H), 1.45 (s, 9H), 1.43 (s, 9H).2HNMR (61 MHz, CDC13) <53.62 (s, 2D), 2.07 (s, ID), 1.49 (s, ID).13C NMR (175 MHz, CDCh) <5172.2, 156.8, 82.5, 80.5, 57.6 (dt, J= 43.3, 21.7 Hz), 50.9, 35.8 (dt, J= 38.6, 19.4 Hz), 28.4, 28.2. HRMS calcd for C13H22D4NO5 [M+H]+: 280.2057, found: 280.2058. [α]D =-27.453° (c = 1.06, EtOH).

[0227] (2S)-4-tert-Butyl-2-(tert-butoxycarbonylamino)-3,3,4-trideutero-4-oxobutanoate (12)D NHBoc

[0228] In a 100 mL round botomed flask, a solution of alcohol 11 (465 mg, 1.66 mmol) in CH2CI2 (8.3 mL) was prepared to which solid NaHCO3 (1.4 g, 16.6 mmol) followed by a solution of DMP ( 1.06 g, 2.497 mmol) in CH2CI2 (8.3 mL) were added. The reaction was stirred at room temperature for 1 h. To the reaction mixture, 1 M Na2S2O3 (14 mL) was added the resulting biphasic mixture was vigorously stirred for 5 min and then sat NaHCO3 solution (4.6 mL) was added. Tire mixture was diluted with deionized H2O (20 mL) and extracted with CH2CI2 (3 x 50 mL). Tire combined organic layer was dried over anhydrous Na2SC>4. filtered and concentrated under reduced pressure. Hie crude product was purified by silica gel column chromatography using 5% EtOAc in CH2Cl2 followed by 10% EtOAc in CH2Cl2 to give aldehyde 12 (351.9 mg, 77% yield). 'HNMR (400 MHz, CDC13) <55.35 (d, 1H, J= 6.8 Hz), 4.45 (s, 1H, J= 7.7 Hz), 1.42 (s, 9H), 1.41 (s, 9H).2HNMR(61 MHz, CDCI3) <59.78 (s, ID), 2.92 (s, ID), 2.01 (br s, ID).13CNMR (175 MHz, CD3CN) <5200.5, 171.4, 156.5. 82.6, 80.1, 52.2, 50.2. 28.5, 28.1. HRMS calcd for C13H21D3NO5 [M+H]+, 277.1837; found, 277.1834. [α]D = -31.5° (c = 1.5, EtOH).

[0229] (2S, 4S)- / c77-Butyl 2-(tert-butoxycarbonylamino)-3,3,4-trideutero-4-hydroxy-5-oxo-5-(2,4,6-tri methoxybenzylamino)pentanoate (13a) and (2S, 4R) -tert-butyl 2-(tert-butoxycarbonylamino)-3,3,4-trideutero-4-hydroxy-5-oxo-5-(2,4,6-trimethoxybenzylamino)pentanoate (13b)O n OH O NHBocCi' llo

[0230] Step i: (2S)-tert-Butyl-2-(tert-butoxycarbonylamino)-4-(2-chloroacetoxy)-3.3.4-trideutero-5-oxo-5-(2,4,6-trimethoxybenzylamino)pentanoate In a 50 mL round bottomed flask, a solution of aldehyde 12 (350 mg, 1.267 mmol) in CH2CI2 (9.9 mL) was prepared to which 2,4,6-trimethoxybenzyl isocyanide (288.7 mg, 1.393 mmol) followed by monochloroacetic acid (131.7 mg, 1.393 mmol) were added. The reaction was stirred at room temperature for 24 h. Hie mixture was concentrated under reduced pressure and the crude product was purified by silica gel column chromatography using 20% EtOAc in hexanes followed by 40% EtOAc in hexanes to give the title compound (550.9 mg. 75%) as a white solid. ’H NMR(700 MHz, CDCh) <56.84-6.75 (br s, 1H), 6.47 (t, 1H, J= 4.9 Hz), 6.11 (s, 4H), 5.23 (d, 1H, J= 7.6 Hz), 5.03 (d, 1H, J= 9.3 Hz), 4.59 (dd, 1H,= 13.9 Hz, J2= 5.8 Hz), 4.54-4.41 (m, 2H), 4.34 (dd, 2H, Jj = 13.7 Hz, J2= 4.5 Hz), 4.23 (d, 1H, J= 8.0 Hz), 4.18-4.13 (m, 2H), 4.11-4.06 (m, 2H), 3.81 (s, 18H), 1.45 (s, 18H), 1.42 (s, 9H). 1.39 (s. 9H).2HNMR (61 MHz, CDCh) <55.10 (br s, ID), 2.34 (br s, ID). 2.08 (br s, ID).13C NMR (175 MHz, CDCh) <5 171.2, 171.1, 168.0, 167.6, 166.6, 166.3, 161.23, 161.18, 159.4, 155.63, 155.61, 106.3, 106.1, 90.7, 82.8, 82.7, 80.3, 80.2, 72.1 (brm), 71.9 (brm), 56.0, 55.57, 55.55, 51.1, 50.8, 41.0, 40.8, 34.1 (br m), 32.6, 32.5, 28.5, 28.16, 28.15. HRMS calcd for C26H37D3ClN2O10[M+H]+, 578.2554; found, 578.2551.

[0231] Step ii: In a 20 mL round bottom flask, to a solution of above 2-chloroacetyl ester (550 mg, 0.951 mmol) in a solvent of EtOH / THF (1 / 1, v / v, 8.4 mL), thiourea (217.3 mg, 2.854 mmol), and NaHCO3(239.8 mg, 2.854 mmol) were added and the mixture was stirred at 50 °C for 1.5 h. The mixture was concentrated under reduced pressure and the crude product was purified by CombiFlash (24 g Silica, Redisep Column, flow rate 3 mL / min, 210 nm) using 30-40% EtOAc in Hexanes to give compound 13a (178 mg, 37%) as a white solid, a mixture of 13a and 13b (101.2 mg, 21%) and compound 13b (115 mg, 24%) as a colorless viscous liquid.0 if n O:< 2 9. / I. G X.N 'X Y vGFki TrfK> SK.,..Jsv+ h -X I Ot-feuH0 QH13a

[0232] Compound 13a:]H NMR (700 MHz, CDCh) <5 7.24 (br s, 1H), 6.11 (s, 2H), 5.44 (d, J= 7.6 Hz, 1H), 4.88 (s, 1H), 4.52 (dd, J= 13.7, 5.6 Hz, 1H), 4.42 (dd. J= 13.7 Hz, 5.3 Hz, 1H), 4.29 (d, J= 7.7 Hz, 1H), 3.81 (s, 6H), 3.80 (s, 3H), 1.44 (s, 9H), 1.41 (s, 9H).2H NMR (61 MHz, CDCh) <53.98 (s, ID), 1.94 (s, 2D).13C NMR (175 MHz, CDCh) <5 171.9, 171.4, 161.0, 159.5, 157.5, 106.7, 90.7, 82.9, 80.9, 68.4-67.6 (m), 56.0, 55.5, 50.8, 39.0-38.1 (m), 31.9, 28.4, 28.1. HRMS calcd for C24H35D3N2O9[M+H]+,2502.2838; found, 502.2833. [α]D = -11.0° (c = 0.1, CH2C12).

[0233] Compound 13b:!H NMR (700 MHz, CDCh) < 7.26 (br s. 1H), 6.10 (s, 2H), 5.45 (s, 1H), 4.56 (dd, 1H, Ji = 13.2 Hz, J2= 5.2 Hz), 4.37 (dd, 1H, Jj = 13.7 Hz, J2= 4.6 Hz), 4.18 (d, 1H, J= 6.8 Hz), 4.07 (br s, 1H), 3.793 (s, 6H), 3.790 (s, 3H), 1.44 (s, 9H), 1.41 (s, 9H).2H NMR (61 MHz, CDCh) <54.06 (s, ID), 2.33 (s, ID), 1.87 (s. ID).13C NMR (175 MHz, CDCh) <5 172.4, 171.5, 161.0, 159.5, 156.4, 106.7, 90.6, 82.7, 80.5. 69.3 (t. J= 20.8 Hz), 55.9, 55.5. 51.7. 38.6-37.6 (m), 32.3, 28.4, 28.1. HRMS calcd for 25C24H36D3N2O9[M+H]+, 502.2838; found, 502.2827. [α]D = 9.821° (c = 0.112, CH2C12).

[0234] (2S, 4R)-tert-butyl-2-(tert-butoxycarbonylamino)-3,3,4-trideutero-5-oxo-4-(tosyloxy)-5- (2,4,6-trimethoxybenzylamino)pentanoate (14)Tmotu 9 D2J?N"\^CY ^Ot-BuHTSO D NHBOC

[0235] In an 8 mL vial, to a solution of alcohol 13a (42.5 mg. 84.7 pmol) in CH2CI2 (0.78 mL) at 0 °C, Et3N (59 pL, 0.424 mmol), TsCl (32.3 mg, 0.170 mmol) and DMAP (1.0 mg, 8.5 pmol) were added and the mixture was stirred at 0 °C for 15 min. The mixture was brought to room temperature and stirred 3 h. It was diluted with CH2CI2 (30 mL) and washed with deionized water (3 x 20 mL), brine solution (20 mL), dried with anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The crude material was purified by silica gel column chromatography using 30% EtOAc in hexanes followed by 40% EtOAc in hexanes to give tosylate compound 14 (41.6 mg, 75%) as a white solid. HPLC purity: 95.0%, [Major peak: Rt = 14.6 min; minor peaks: 10.0 and 19.2 min] column: Cellulose-1 (250 x 4.6 mm), UV detector, 210 nm, 7% 2-propanol in hexanes; flow7rate: 1.0 mL / min], 'HNMR (400 MHz, CDCL) <57.74 (d, 2H, J = 7.8 Hz), 7.25 (d. 2H, J= 7.6 Hz), 6.87 (br s. 1H), 6.13 (s, 2H), 5.09 (d, 1H, J = 8.2 Hz). 4.40 (d, 2H, J= 5.0 Hz). 4.00 (d, 1H, J = 8.3 Hz), 3.83 (s, 3H). 3.82 (s, 6H). 2.41 (s, 3H). 1.43 (s. 9H), 1.42 (s. 9H).2H NMR (61 MHz, CDCL) <54.88 (s, ID), 2.19 (s, 2D).13C NMR (175 MHz, CDC13) <5170.7, 167.3, 161.2, 159.4, 155.5, 145.6, 132.8, 130.2, 128.1, 105.9, 90.7, 82.2, 79.9, 77.2, 56.0, 55.6, 51.3, 34.7-33.5 (m), 32.6, 28.5, 28.1, 21.9. HRMS calcd for C32H43D3N2O11S [M+H]+, 656.2927; found, 656.2925. [α]D = -27.961°(c = 1.03, MeOH).

[0236] (2S, 4R)-tert-butyl-2-(tert-butoxycarbonylamino)-3, 3, 4-trideutero-4-fluoro-5 -oxo-5 -(2,4,6-trimethoxy benzylamino )pentanoate (15)Tmotu j? j?N |' A<CY ^Ot-Bu_1 O •* > F N IHBoc

[0237] To a stirred solution of tris(dimethylamino)sulfonium difluorotrimethyl-silicate (TASL, 0.21 g, 0.7625 mmol) in 1:1 THF / CH. CI, (0.5 mL) was added Et3N*(HF)3(5 pL) dropwise. After that, tosylate 14 (0.100 g, 0.1525 mmol), THF (0.25 mL) and above “neutralized” TASF solution were added one by one to a 5 mL two-neck flask equipped with a reflux condenser. The mixture was heated to 50 °C by oil bath for 16 h. The reaction mixture was cooled to room temperature and then diluted with EtOAc. The resultant solution was washed with half saturated NaHCO3, water and brine subsequently. The EtOAc phase was collected, dried by Na2SO4. filtered, and concentrated under reduced pressure. The crude material was purified by silica gel (60-200 pm, 70-230 mesh) column chromatography using 30% EtOAc in Hexanesfollowed by 40% EtOAc in Hexanes to give the title compound 15 (56.3 mg). *H NMR (400 MHz, CDCL) 36.70 (br s, 1H), 6.13 (s, 2H), 4.57 (ddd, 1H, J! = 22.7 Hz, J2= 16.7 Hz, J3= 5.8 Hz), 4.47-4.34 (m, 2 H), 3.82 (s, 9 H), 1.45 (s, 9 H), 1.43 (s, 9H). HRMS calcd for C24H34D3FN2O8[M+H]+: 504.2795; found: 504.2794.

[0238] (2S, 4R)-2,5-Diamino-3,3,4-trideutero-4-fluoro-5-oxopentanoic acid (16)O D2OH2N'J\^'CV^OHD F NH2

[0239] To a mixture of 15 (56.3 mg, 0.1118 mmol) with dimethyl sulfide (30 pL) cooled with an ice bath (0 °C), trifluoroacetic acid (TFA, 1.5 mL) was added dropwise. After the addition, the reaction was brought to room temperature and stirred for 2.5 h. Tire solution was evaporated reduced pressure to remove most TFA. The residue was dissolved in H2O (5 mL) and washed with CH2CI2 (3 mL x 2). The aqueous part was cooled in an ice bath and neutralized to pH = 7 by slow addition of ice-cold 5% aqueous ammonia. The resultant liquid was lyophilized to give a white solid. The solid was washed several times by trituration with MeOH and 50%EtOH solution to give the final product 4-FGln-d316 (5 mg, 20% yield over two steps) as off-white solid. HPLC purity: 95.9%, [Major peak: R = 14.6 min; minor peaks: 11.8 and 18.1 min] column: Chirex 3126 (D)-pencillamine (250 x 4.6 mm), UV detector, 254 nm, 1.0 mM Cu(OAc)2 solution; flow rate: l. O mL / min, column temperature 10 °C], ’H NMR (400 MHz, D2O) 33.97 (s, 1H).2HNMR(61 MHz, CD3OD) <5 5.41-5.01 (m, 0.5D), 4.52-4.12 (m, 0.5D). 2.71-2.05 (m, 2D).13C NMR (175 MHz, D2O + trace CD3OD) 3 179.6, 174.8, 174.1, 89.9 (m), 88.9 (m), 52.8, 33.7 (m).19F NMR (376 MHz, D2O) 325-190.33. HRMS calcd for C5H7D3FN2O3[M+H]+: 168.0858; found: 168.0853. [α]D = -17.278° (c = 0.2, H2O).

[0240] Radiochemistry: All reagents were purchased from Sigma-Aldrich or Fisher Scientific unless otherw ise mentioned. All three cartridges used for radiofluorination chemistry were purchased from Waters Corporation (The Sep-Pak Light Accell™ Plus QMA Carbonate, Sep-Pak™ Light C 18 cartridges, Sep-Pak® Light Alumina N cartridges). Non-carrier-added [18F]fluoride was produced in-house by bombarding of H218O (98 atom%, NUCMEDCOR) w ith beam (50 pA) in a 5 - 25 min range of time using an 16.5 MeV cyclotron (GE, PETtrace 800). Both cartridges (Alumina-N Light and Sep-Pak C18 Light cartridges) w ere preconditioned with 5.0 mL ethanol and 10 mL water (HPLC grade) just before the use. Radioactivity measurements were performed using a Capintec CRC 55t or 55tR PET dose calibrator. Analytical radio-HPLC analysis was performed on a JASCO HPLC system using an Eckert & Ziegler Flow-Count radio-HPLC detecting system equipped with a diode detector.

[0241] The first step of radiosynthesis was operated in a GE TRACERLab FX2N synthesis module (Scheme 3) following below sequences:

[0242] (1). The [18F]fluoride in [18O]water (2.5 mb) was transferred from the cyclotron target to a GE synthesizer and the radioactivity was trapped on a QMA carbonate cartridge.

[0243] (2). The radioactivity trapped in the cartridge was eluted into the reactor with an 18-crown- 6 / KHCO3 (8.0 mg / 1.44 mg) aqueous MeOH solution (0.18mL / 1.0 mL).

[0244] (3). The reactor was heated to 70 °C under vacuum with a stream of helium for 7 min and drying was continued for a further 1 min at 100 °C.

[0245] (4) After cooling to 40 °C, a solution of tosylate precursor (5.0 mg) in MeCN (anhydrous, 0.60 mL) was added to the reactor, and then heated to 70 °C for 15 min.

[0246] (5). The reaction mixture was cooled below 40 °C and was diluted with HPLC solvent (2.0 mL) and water (0.6 mL). The diluted solution was passed through a light Al-N cartridge and was injected onto the semi-prep HPLC for purification (Agilent XDB-C18 column, 250 x 9.4 mm, 5 pM; mobile phase: 73.5% MeOH / 26.5% H2O (0.1% V / V HCO2H); 4 mL / min. UV detector set at 210 nm).

[0247] (6). The portion of product-containing radioactive peak (Rt~ 16.1 min) was collected and diluted with water (25 mL). This aqueous solution was next passed through a C 18 light cartridge and washed with w ater (10 mL). The radioactive product was eluted from the cartridge with EtOH (1.0 mL) into a V-shape microwave reaction tube (0.5 - 2.0 mL).

[0248] The second step acidic deprotection via manual operation:

[0249] (7). Hie radioactive intermediate collected in V-shape tube was transferred out from hot cell, the nitrogen stream was used to evaporate solvent from V-shape tube under a gentle heating condition (75 °C).

[0250] (8). Cool reaction tube to r.t., add TFA / anisole reagent (0.5 / 0.005 mL) to reaction tube, heat reaction mixture at 60 °C for 5 min; Remove reaction tube from heater, cool it, insert a vent needle, blow out most of the liquid in the reaction vial with gentle N2flow7(2 -3 psi).

[0251] (9), Agl 1-A8 ion retardation resin purification: cool reaction tube with ice water bath, add 1.0 mL of H2O into reaction tube, transfer this reaction mixture to the Agl 1-A8 column, drain liquid out and collect it as fraction 01; add 2 × 2 mL H2O for elution to get fraction 02; finally, add 2 mL H2O and collect as fraction 03.

[0252] (10). To fraction 02, add NaOAc reagent (4 mL, 2.36%) to form isocratic NaOAc (1.18%) solution, filter this solution through 0.22 gm filter and collect into a sterile vial (20 mL) to give the final product 4-[18F]FGln-d3 or 4-[18F]FGln.

[0253] (11). Final product radiochemical purity analysis: the final product sample (20 uL) was injected into analytical HPLC for radiochemical purity analysis (JASCO, column: Chirex 3126(D-Pencillamine), 250 mm x 4.6 mm; mobile phase: 1 mM Cu(OAc)2 solution; Flow rate: 1 mL / min; UV: 254 nm; column was cooled at 10 °C)

[0254] Culturing of 9L / lacZ Cells

[0255] Tire cell culturing method was followed from a previously published protocol.509L / 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 medium was renewed 2-3 times per week.

[0256] In Vitro Cellular Uptake of (2S,47?)-4-[18F] Fluoroglutamine and (2, S'.4 / ?)-4- [’8F]Fluoroglutamine-d3

[0257] Time-dependent uptake experiment

[0258] 9L / lacZ cells were detached from the T-75 flask with trypsin-EDTA solution, centrifuged, washed with DPBS (Ca2+, Mg2+added), and resuspended in DPBS (Ca2+, Mg2+added). One mL aliquots of 9L / lacZ cells (1 xlO6cells / mL) were seeded in a 1.5 mL conical tubes. Each three replicates of 9L / lacZ cells were incubated with (2S,4R)-4-[18F] fluoroglutamine (176 kBq) or (2. S'.4 / ?)-4-|18F]fluoroglutamine-d3 (240 kBq) together with selected inhibitors or substrates at 37 °C for 5 min, 30 min, 60 min and 120 min. The cells were then centrifuged at 3700 x g for 5 min, after which the supernatant was carefully removed, and the cells pellets were washed with 1 mL DPBS (No Ca2+and Mg2+added) and centrifuged again at 3700 x g for 5 min. The washing step was performed three times. Cell pellets were lysed with 60 pL of cell lysis buffer containing protease inhibitor and then vortexed for 30 s, and seeded on ice for 10 min. Hie process was repeated three times. The radioactivity of each sample was determined using a gamma-counter (WIZARD 2480) and stored at -80 °C. On the next day, the cell pellets were lysed with Cell Lytic M (catalog no. C2978 Sigma-Aldrich) supplemented with a protease inhibitor (catalog no. A32955 ThermoFisher Scientific). Lysates were centrifuged at 13,000 x g for 45 min at 4 °C, and the total proteincontent of the supernatant was quantified using a Pierce Bicinchoninic Acid (BCA) Protein Assay kit (cat. #23225 ThermoFisher Scientific). The results are expressed as %ID / mg protein.

[0259] Transporter characterization experiment

[0260] 9L / lacZ cells were detached from the T-75 flask with trypsin-EDTA solution, centrifuged, washed with DPBS (Ca2+, Mg2+added), and resuspended in DPBS (Ca2+and Mg2+added). One mL aliquots of 9L / lacZ cells (1 x 106cells / mL) were seeded in a 1.5 mL conical tubes. 9L / lacZ cells were incubated with (2S,4R)-4-[18F]FGln (176 kBq) or (2S,4R)-4-[18F]FGln-d3 (204 kBq)together with selected inhibitors or substrates at 37 °C for 1 hour. For inhibiting system L, 9L / lacZ cells were incubated with 2-amino-bicyclo[2.2.1]-heptane-2-carboxylic acid (BCH) at 0.5 mM, 1 mM and 5 mM. For inhibiting system A, 9L / lacZ cells were incubated with a-(Methylamino)isobutyric acid (MeAlB) at 0.5 mM. 1 mM and 5 mM. For inhibiting system ASC, 9L / lacZ cells were incubated with L-Ser at 0.5 mM, 1 mM and 5 mM. For inhibiting system ASC N, 9L / lacZ cells were incubated with L-Gln at 0.5 mM, 1 mM and 5 mM. The cells were then centrifuged at 3700 * g for 5 min, after which the supernatant was carefully removed, and the cells pellets were washed with 1 mL DPBS (No Ca2+and Mg2+added) and centrifuged again at 3700 x g for 5 min. The washing step was performed three times, after which the radioactivity of each sample w as determined using a gamma-counter (WIZARD 2480). The results are normalized to the control replicates analyzed by a students’ two-tailed t test.

[0261] 9L / lacZ Tumor-Bearing Rats

[0262] All animal work was approved by the Institutional Animal Use and Care Committee of Stony- Brook University (IACUC2022-00050). Fisher rats (SAS FISCH, RT11V, female) were ordered from Charles River and housed in a maximum isolation room for a w eek in the Division of Laboratory Animal Resources at Stony Brook University. Tire Fisher rats were then subcutaneously injected with 1 x 1069L / lacZ cells (0.1 mL of PBS, pH=7.4) in the right flank. Tire rats w ere monitored closely until a palpable tumor was present. The tumor volume was measured two or three times a week and quantified using the formula V = L x W2 / 2, where L is the largest dimension of the tumor, and W is the other dimension. After 8-9 weeks, the tumor volume reached around 500 mm3, and the rats were used for imaging experiments.

[0263] In vivo PET / CT Imaging

[0264] Nontumor-bearing Fisher rats were anesthetized by the inhalation of 2.5-3% isoflurane with oxygen as a carrier. PET and CT scans were obtained using a Siemens Inveon Multi-Modality preclinical microPET / CT / SPECT scanner. Catheters were placed in the tail veins of the rats after which small animals were positioned at the center of the scanner. Rats underwent a 8 min CT scan. Subsequently, 0.2-1 mL of formulated tracers (6.5-18.7 MBq of (2S,4R)-4-[18F]FGln) were carefully injected through the catheters intothe rats followed by 0.25 mL saline while simultaneously initiating the PET scan. Rats were closely monitored during the 120 min dynamic PET scan. Two days later, PET / CT imaging of (2S,4R)-4-[18F]FGln (13.3-14.8 MBq) was performed in the same group of rats by following the above protocol. 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 the PET reconstruction. PET volumes of interest (VOIs) and representative PET / CT images were generated by PMOD version 4.3 (PMOD Technologies Ltd), and the standardized uptake value (SUV) of tissue / organ was calculated. Errors in the averaged SUV values are reported as the standard deviation.

[0265] Table 1. In vivo PET imaging - comparison of 4-[18F]FGln and 4-[18F]FGln-d3 in 9L / lacZ tumor-bearing rats4-[18F]FGIn 4-[18F]FGln-d3Tumor uptake 0.61 ± 0.07 0.51 ± 0.14Tumor-to-muscle ratio 1.18 ± 0.24 0.92 ± 0.33Blood uptake @ 120 min 2.83 ± 0.38 2.14 ± 0.28postinjectionBlood elimination half-life 2.07 min 2.08 minTracer radiochemical purity 92.6% 87.0%Note: imaging experiments, 4-[18F]FGln at first day and 4-[18F]FGln-ds at the third day

[0266] Pharmacokinetics

[0267] Tire 120 min dynamic PET data were binned into designated time frames, followed by reconstruction using the OSEM 3D method. Attenuation correction was performed using tire reconstructed CT data during PET reconstruction. PET VOIs and representative PET / CT images were generated by PMOD version 4.3 (PMOD Technologies Ltd), and the SUV of tissue was calculated. Errors in the averaged SUV values were reported as the standard deviation. A nonlinear 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).

[0268] Statistical Analysis

[0269] Statistical analysis and nonlinear regression were performed using GraphPad Prism 9.1.0 (GraphPad Software). Quantitative data are expressed as the mean ± the standard deviation 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.

[0270] Chemistry: In the original synthesis of 4-[18F]FGln reported in 2011, a partially protected homoserine 1 was first synthesized and then converted to aldehyde 2 via a Dess-Martin selective oxidation. Next, the three-component Passerini reaction was employed to assemble a C-4 position being functionalized glutamine derivative and further transformation provided the stereospecific tosylate 3 as a fluorination precursor. Finally, the desired 4-[lsF]FGln [18F] 4 was synthesized via a mild fluorination and deprotection protocol (Scheme 1) (Qu 2011).18-Crown-6 NHBoc DMP TmobNC, CAA KHCOyCHKF 0 0 Oxidation DCM. r.t. MeCN, 70 °C H2N OH HO Passerini Rxn TFA / Anisole 60 °C NH24-['iF]FGIn, [1SF]4Scheme 1. Original reported method for synthesis of 4-[18F]FGln, [18F]4.

[0271] To approach desired deuterated 4-[18F]FGln counterpart 4-[18F]FGln-d3, the deuterated homoserine derivative 11 was assigned as the first synthetic target compound. After the initial attempts of synthesis this compound via the stereospecific or stereo-enriched manner, which only presented us with less satisfied results, a six-step method was designed and proceeded to provide us with this deuterated homoserine intermediate 11 (Scheme 2, Fig. 19). Firstly, an O-benzylating reagent, TriBOT 5, was synthesized with in a 40% yield following the report from Yamada et al,51and this reagent was next being used to provide benzoxy group attached tetra-deuterated alkylbromide intermediate 7. Next, it was reacted with tert-butyl N-(diphenylmethylene)-glycinate 8 using t-BuOK as base and the following deprotection using 10% citric acid gave a racemic mixture homoserine derivative 9a, b in 51% yield. Tire chiral preparative HPLC method using Chiral AY-H column successfully separated two isomers and, therefore free amine 9a in 48% yield was obtained. Following Boc N-protection and catalytic debenzylation two-step reactions provided us with a tetra-deuterated homoserine compound 11 in 96% yield.D D OBn D^DBr6OH D D D DD / (D +TfOH, 1,4-dioxane Br OBn D OBn 4A MS, rt, 16 h 7 7’ TriBOT, 5 i. f-BuOK, THF, -70 to 0 °C then rt 16 h ii. 10% Citric Acid, THF, rt, 16 h Of-Bu 51% over two steps 9a, bD-010% Pd / C, H2(1 atm) BnO^C EtOH, rt, 16 h, 96% C Of-Bu HPLC separation °2NH2Chiralpak AY-H9b+D29 (Boc)2O, 1,4-dioxane D29 9a, 48% BnO. / . CKAni P, rt, 16 h, qualitativeC Af Of-Bun 1 BnO'G'C-Y^Ot-B.2NHBoc NH 1029aScheme 2. Synthesis of deuterated homoserine intermediate 11.

[0272] With tetradeuterated homoserine 11 in hands, the synthetic protocol reported for 4-FGln synthesis was next employed to provide 4-FGln-d3 15 as the standard compound (Scheme 3, Fig.20): 1). controlled Dess-Martin oxidation to give 72% of aldehyde 12; 2). Passerini three-component reaction and dechloroacetylation to afford (2.8'.7, S')-alcohol 13a in 28% overall yield; 3). Tosylation to present 75% of (25',4> S)-tosylate 14; 4). Finally, TASF and EtsN*3HF combined nucleophilic fluorination and following global deprotection reactions using TFA / DMS provided desired 5 mg (20% overall yield) of 4-FGln-ds 16 as the standard compound.

[0273] Radiochemistry: In the past years, the synthesis and production of 4-[18F]-FGln have been well-explored and the fully automated production process had been reported by several research facilities to replace initially reported manual synthesis method with the use of different commercial synthesizers (Zhang 2016, Li 2016 and Zhang 2019). Based on existing instruments and specific conditions at the radiochemistry facilities, a semi-automated production protocol was chosen to synthesize both 4-[18F]-FGln-ds and 4-[18F]-FGln tracers. First of all, the intermediate [18F] 15 was synthesized following reported method with minimal modification using GE TRACERLab FXNPro as the synthesis module.52After the radiofluorination, semi-preparative HPLC purification and concentration using a C 18 light cartridge, the [18F]15 activity was eluted with ethanol (1 mL) to the V-shape Biotage microwave vial. Although next acidic deprotection reaction can also be processed in an automated manner, the volatile and high corrosive properties of trifluoroacetic acid potentially could contaminate the synthesis module, shorten the tubing’sand valves’ working lives and cause the tedious post-cleanup work of synthesizer. From this point of view, the second deprotection reaction was conducted following the original report with a manual operation manner (Qu 2011). Once all solvent was removed via an azeotropic evaporation, a series of operation, including acidic full deprotection, purification using a Ag 11 -A8 resin column, formulation with the addition of sodium acetate salt and the sterilization via filtration, provided the desired the isotonic and sterilized 4- [18F]FGln-ds ([18F]16) product which is ready for further biological evaluation purpose. The whole production process took around 90 - 115 min with an overall chemical yield of 3.6%-12.9% (non-decay corrected), and the radiochemical purity ranged from 81%-90%. The biological study used 4-[18F]FGln [18F]4 was also synthesized in the same process as above described, with the radiochemical purity ranged from 88.6% to 92.6%.D-01) TmobNC, CAA, DCM O o HO _ ^O C rt, 16 h, Yield: 75% Tmob Ot-Bu f-Bu -; - ► Of-Bu +Tmob'N^ Ot-Bu DMP, NaHCO:32) Thiourea, NaHCO NHBoc DCM, rt, 2 h D NHBoc3NHBocHD OH NHBoc EtOH / THF, 50 °C, 5 h 11 Yield: 72% 12 13a 13b 13a, Yield: 37%TsCI, DCM EtoN / DMAP. 0 °C to rt o D, TFA / Anisole Yield: 75%SO °C Tmob H2N i OH N Of-Bu MeCN, 18-Crown-S H KHCO3 / [1r’F]KF, 70 °C >0 4-^FJFGln-dj, [^FJIB [18F]15 D22%sO D O O o O Tosylate, 14 TFA / MezS, rt Tnob TASF / Et3N-3HF H2N OH Of-Bu DCM / THF, 40 °C NH2Two-steps: NHBoc 4-FGIn-dj, 16Yield: 20% 15 Scheme 3. Synthesis of deuterated and fluorinated (2S,- / 7?)-4-fluoroglutamines, 4-[18F]FGln-d3 and 4-FGln- d3.

[0274] In Vitro Cellular Uptake: Once obtained 4-[18F]FGln-ds, a rat gliosarcoma cell line, 9L / lacZ was used to compare the cellular uptake of the radiotracer with 4-[18F]FGln ([18F]4) (Fig. 3). 9L / lacZ cells were incubated with 4-[18F]FGln or 4-[18F]FGln-d3 for 5, 30, 60, 120 min to compare their specific uptake at each time point. In vitro results demonstrated that 4-[18F]FGln and 4-[18F]FGln-d3 were gradually taken up into 9L / lacZ cells over 120 min. The maximum uptake of 4-[18F]FGln and 4-[18F]FGln-d was 28.4 %ID / mg and 44.7 %ID / mg at 120 min, respectively.

[0275] Previous reports have confirmed that 4-[18F]FGln can be transported into 9L / lacZ cells via system L (Na+independent), ASC and ASC N (Na dependent) amino acid transporters. Since system A was found to be not responsible for the cellular uptake of 4-[18F]FGln, 4-[l8F]FGln-d3 was either not dose- dependently blocked with 0.5, 1, and 5 mM a-(methylamino)isobutyric acid (MeAIB, system A inhibitor).The uptake of 4-[18F]FGln-d3 was reduced from 16% (0.5 mM) to 38% (5 mM) with 2-aminobicyclo-(2,2, l)-heptane-2 -carboxylic acid (BCH, system L inhibitor), from 11% (0.5 mM) to 65% (5 mM) with L-Ser (system ASC substrate), and from 71% (0.5 mM) to 80% (5 mM) with L-Gln (system ASC N substrate), which was comparable to 4-[18F]FGln. As expected, the deuteration at C3 and C4 positions did not make impacts on the cellular uptake of 4-[18F]FGln-d3 as observed for L-[18F]FAla and L-[18F]FAla-d3.50Taken together, 4-[18F]FGln-d3 demonstrated a similar uptake manner to 4-[18F]FGln. 4-[18F]FGln-d3 was transported into 9L / lacZ cells partially via system L while predominantly via system ASC and ASC N.

[0276] [18F]FGln is a metabolic imaging marker for inflammation

[0277] Biological Models Selection

[0278] The carrageenan-induced paw edema (CIPE) model for local and acute inflammation: Carrageenan is a seaweed-derived sulfated polysaccharide that has been extensively used to induce inflammation in experimental animal models to study novel anti-inflammatory and analgesic drugs. In the CIPE model, carrageenan injected into the rodents' hind paws causes a classical innate immune response characterized by paw edema, neutrophil migration, and pain.

[0279] The collagen-induced arthritis (CIA) model for systemic and chronic inflammation: CIA is an experimental model of autoimmune arthritis and has been shown to exhibit similar histological, immunological, and clinical characteristics and genetic linkage to human rheumatoid arthritis (RA). This model has been widely used in studies on the pathogenesis of RA and the preclinical evaluation of novel therapeutics for RA.

[0280] Table 2 A qualitative scoring system used to assess the severity of paw inflammation Score Condition0 No evidence of erythema and swelling1 Erythema and mild sw elling confined to the tarsals or ankle joint2 Erythema and mild sw elling extending from the ankle to the tarsals3 Erythema and moderate swelling extending from the ankle to metatarsal joints4 Erythema and severe swelling encompass the ankle, foot, and digits, or ankylosis of the limb

[0281] Biological Models Generation

[0282] The CIPE model (n=4, Lewis female rats, Charles River) was generated by injecting 200 pL of 3% carrageenan solution into the left hind paw subplantarly under general anesthesia with isoflurane three hours before the PET scan.

[0283] The CIA model (n=4, Lewis female rats, Charles River) was generated by injecting collagen emulsion subcutaneously at the tail base 3-4 weeks before the PET scan. The injected animal was monitored daily for the development of arthritis.

[0284] Table 3. Summary of [18F]FGln micro-PET findings and the severity of inflammation. Model and # Foot (L / R) %Increase of AUC in Inflammation Severity time-activity curve ScoreCIPE #1 L (injected) 52 4R N / A 0CIPE #2 L (injected) 74 4R N / A 0CIPE #3 L (injected) 61 4R N / A 0CIPE #4 L (injected) 83 4R N / A 0CIA #1 L (injected) 173 4R -7 0CIA #2 L (injected) 69 4R 33 2CIA #3 L (injected) 54 4R 75 4CIA #4 L (injected) 69 4R 60 4

[0285] In vivo PET imaging - comparison of 4-[18F]FGln and 4-[18F]FGln-d3 in 9L / lacZ tumorbearing rats: A 120-min Dynamic PET imaging was performed in 9L / lacZ tumor-bearing rats to compare the biodistribution of 4-[18F]FGln-ds and 4-[18F]FGln (Fig.4). ROI analysis indicated that the tumor uptake of both radiotracers reached the maximum rapidly and decreased over the time (Fig. 5). 4-[18F]FGln-d3 demonstrated the comparable tumor uptake (0.51 ± 0.14) to 4-[18F]FGln (0.61 ± 0.07. P > 0.05). The similar tumor-to-muscle contrast was observed for 4-[18F]FGln-ds (0.92 ± 0.33) and 4-[18F]FGln (1.18 ± 0.24) as well (P > 0.05). The slightly lower tumor uptake and tumor-to-muscle contrast data of 4-[18F]FGln-d3 could be due to several varying factors from the experiments, such as the lower batch radiochemical purity of 4-[18F]FGln-d3 comparing with 4-[18F]FGln (87.0% vs 92.6%), and the change of tumor environment in the different days of imaging (4-[18F]FGln at first day and 4-[18F]FGln-d3 at the third day). Tire blood elimination half-lives of 4-[18F]FGln-d3 (2.08 min; 95% Cl, 1.52-2.97 min) and 4-[18F]FGln (2.07 min; 95% Cl. 1.56-2.84 min) were similar to each other as well. The bone uptake of both radiotracers increased gradually while the bone uptake of 4-[18F]FGln-d (2.14 ± 0.28) was significantly lower than 4-[18F]FGln (2.83 ± 0.38) at 120 min postinjection. As expected, the applied deuteration strategy successfully improved the resistance of 4-[18F]FGln-d3 to in vivo defluorination while not influencing its tumor uptake. Bone metastasis is one of tire common features observed in various cancers such as breast and prostate cancer. It was reported that the high nonspecific bone uptake of [18F]PSMA-1007 might lead to the false positive diagnosis of bone metastasis in prostate cancer (Ouvrard 2024 and Seifert 2023). Thus, the reduced nonspecific bone uptake of 4-[18F]FGln-ds could potentially offer high-quality PET images to doctors for a more accurate diagnosis, particularly bone metastasis (Klenner 2021).DISCUSSION

[0286] In the drug discovery and development, the replacement of hydrogen atom with deuterium has become a valuable approach for improving the phannacokinetic and / or toxicity properties, which is based on the mechanism of deuterium kinetic isotope effect (DKIE) (Fig. 14) (Klenner 2021, Di Martino 2023 and Timmins 2017). The recent U. S. FDA approved deuterated drugs such as Austedo and Deucravacitinib are such examples (Fig. 2, A & B). In the radiopharmaceutical research areas, the concept of DKIE was also employed even back to three decades ago while [nC]L-deprenyl-d2 was developed to replace its parent compound [nC]L-deprenyl for achieving better in vivo stability in human as MAO B imaging agent (Fig.3, C) (Fowler 1995. Fowler 1988 and Fowler 1987). Moreover, this strategy is still actively employed in the development of radiopharmaceuticals for PET imaging (Xiao 2021 and Li 2024). It was hypothesized that the substitution of hydrogen with deuterium in the molecule skeleton of 4-[18F]FGln might improve its in vivo metabolic stability, while maintain similar in vivo imaging properties. Urerefore, a deuterated 4-[18F]FGln counterpart molecule, (2X4A)-[4-18F-3,3,4 -d3] fluoroglutamine (4-[18F]FGln-d3, Fig. 2, D) wasdesigned with expectation as the next generation of PET tracer for imaging the glutamine metabolism. Herein, the first synthesis of 4-[18F]FGln-ds and its head-to-head biological comparison with 4-[18F]FGln was reported.

[0287] The present disclosure relates to the development of novel radiochemistry methodologies that enable rapid, highly efficient, and practically amenable carbon- 11 and fluorine- 18 labeling, as well as the synthesis of radiolabeled bioorganic molecules, including amino acids, oligopeptides, and lipids, for use as positron emission tomography (PET) imaging agents in the diagnosis and evaluation of chronic diseases. In particular, the disclosure describes the design, synthesis, and biological evaluation of radiolabeled bioactive organic molecules for brain and cancer imaging, with an emphasis on18F-labeled glutamine derivatives (Fig. 7). The development of18F-labeled glutamines is exemplified by (2S,4R)-4-[18F]fluoroglutamine ([18F]FGln), which has been investigated as a PET tracer for cancer imaging. In addition,lf:F-(2. S. - / / ?)4-fliiorogliita inc demonstrates an incorporation profile similar to the one observed for3H-1 -glutamine. As indicated by Lieberman, a significant percentage ofl 8F-(2. S, - / / ?)4-fluoroglutaminc was incorporated into protein after 2 h. At 30 and 120 min, the protein incorporation ofl8F-(2. S', - / / ?)4-fluoroglutamine in 9L cells was 29% and 72% and 12% and 62% in SF188 cells, respectively (Fig. 10 and 11). Recent studies further demonstrate the utility of [18F]FGln as a metabolic imaging marker for inflammation (Figs. 8, 9, 15, and 16). Synthesis of [18F]FGln is shown in Figs. 13, 17 and 18.

[0288] To address limitations in in vivo stability, the present disclosure also describes the synthesis and evaluation of18F- and2H-labeled glutamine analogs, including 4-[18F]FGln-d3, exhibiting improved metabolic stability. While18F-fluorodeoxyglucose (FDG) PET remains a paradigmatic modality for cancer imaging based on up-regulated oncogene PI3K / Akt signaling and enhanced aerobic glycolysis (the Warburg effect), oncogene c-Myc-driven increases in glutaminolysis provide a complementary metabolic pathway, thereby establishing glutamine-based PET imaging as a new and valuable modality for cancer diagnosis and characterization.

[0289] Amino acids (AAs) are essential building blocks for the synthesis of peptides, proteins, and other nitrogen-containing bioactive compounds such as nucleosides, creatine, and some human neurotransmitters. There is ample evidence of the heavy metabolic dependence of major inflammatory cells on glutamine (Fig. 12). Specifically, glutaminolysis, a conversion process from glutamine into glutamate, aspartate, and alanine, is an essential metabolic step to provide energy sources for the proliferation of inflammatory cells and their reactions against external or internal pathogen.

[0290] A new synthetic pathway was developed to prepare the deuterium and fluorine-18 dual isotope labeled glutamine, 4-[18F]FGln-ds, including a six-step preparation process to synthesize tetradeuterated homoserine intermediate, and thereafter next six-steps transformation to provide us with radiolabeling usedtosylate precursor and19F-standard. A semi -automated radiosynthesis protocol was established to synthesize 4-[18F]FGln-d3 in acceptable yield. In vitro cell uptake and in vivo murine animal imaging study results displayed that 4-[18F]FGln-d3 has similar cell uptake maimer, comparable tumor uptake and tumor-to-muscle ratio as the counterpart tracer 4-[18F]FGln, but clearly decreased radioactivity bone uptake at 120 min postinjection.

[0291] The newly discovered results warrant further investigation of 4-[l8F]FGln-d3 as the next generation of PET tracer for probing the in vivo glutamine metabolic process. The present disclosure developed a practical synthetic pathway. The present disclosure synthesized fluorine- 19 and deuterium labeled stereospecific 4-FGln-D3 compounds as “cold” standards and tosylate leaving group attached glutamine analogs as radiofluorination precursor(s). The most difficult issue is to synthesize radiolabeling precursor(s) in high stereochemical purity (single isomer amount > 98%). Such problem was solved by resorting to chiral prep-HPLC separation method that will provide us with desired stereo-isomer as precursors for radiofluorination. Hie present disclosure also developed a radiofluorination method. Various radiolabeling approaches were tested and enough radioactive 4-[18F]FGln-D3 product with desired quality (> 10% radiochemical yield; >90% radiochemical purity was obtained with good chemical and stereochemical purity). The biggest challenge =is to obtain radioactive product in good stereochemical purity (generally the desired stereospecific compound, such as (2S,4R) stereoisomer, should be more than 80%).

[0292] The deuterium and fluorine-18 dual-isotope labeled fluoroglutamine, (2S,4R)-[4-18F -3,3,4 -ds] fluoroglutamine (4-[18F]FGln-d3), were designed, synthesized, and biologically evaluated for attaining novel glutamine metabolic imaging agent with improved in vivo stability. The tetradeuterated homoserine 11 intermediate was first synthesized via a six-step synthetic pathway including a chiral HPLC separation process. Next, (2S,4R) tosylate precursor 14 was prepared following the reference reported method, and 4-[18F]FGln-d3 was successfully prepared using a semi-automated production process. After that, The head-to-head comparison of 4-[18F]FGln-d3 with its parent compound 4-[18F]FGln through in vitro cell uptake and in vivo murine animal imaging studies illustrated that the new tracer 4-[18F]FGln-d3 has similar cell uptake manner, comparable tumor uptake and tumor-to-muscle ratio as 4-[18F]FGln, but clearly decreased radioactivity bone uptake at 120 min postinjection. Overall, the data indicate the better in vivo stability of 4-[18F]FGln-d3 comparing with its counterpart 4-[18F]FGln, which warrants the further investigation of this new7radiotracer as the next generation of PET imaging agent for probing the in vivo glutamine metabolic process.

[0293] In summary, a new synthetic pathway was developed to prepare the deuterium and fluorine-18 dual isotopes labeled glutamine, 4-[18F]FGln-d3. After the successful synthesis of the key intermediate,tetradeuterated homoserine 11, via a six-step preparation process, the next six-step transformation following previous reported method for 4-FGln synthesis provided us with (2S,4S) form tosylate 14 as the labeling precursor and (2S.4R) form of 4-FGln-ds 16 as the standard compound. Next, a semi-automated radiosynthesis method was resorted to provided us with the desired new tracer 4-[18F]FGln-ds. Following in vitro cell uptake and in vivo murine animal imaging study results displayed that 4-[lsF]FGln-d3 has similar cell uptake manner, comparable tumor uptake and tumor-to-muscle ratio as the counterpart tracer 4-[18F]FGln, but clearly decreased radioactivity bone uptake at 120 min postinjection. All in all, the newly discovered results warrant the further investigation of 4-[18F]FGln-d3 as the next generation of PET tracer for probing the in vivo glutamine metabolic process.REFERENCES1) Fowler, J. S.; Wolf, A. P. Working against Time: Rapid Radiotracer Synthesis and Imaging the Human Brain. Acc. Chem. Res. 1997, 50 (4), 181-188. 10.1021 / AR960068C. DOI: 10.1021 / AR960068C.(2) Miller, P. W.; Long, N. J.; Vilar, R.; Gee, A. D. Synthesis of 11C, 18F, 150, and 13N radiolabels for positron emission tomography. Angewandte Chemie, International Edition 2008, 47 (47), 8998-9033, 10.1002 / anie.200800222. DOI: 10.1002 / ame.200800222.(3) Weber, W. A.; Schwaiger, M.; Avril, N. Quantitative assessment of tumor metabolism using FDG-PET imaging. Nuclear Medicine and Biology 2000, 27 (7), 683-687, 10.1016 / S0969-8051(00)00141-4. DOI: 10.1016 / S0969-8051 (00)00141 -4.(4) Hoh. C. K. Clinical use of FDG PET. 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Claims

CLAIMSWhat is claimed is:A composition comprising a compound having the structure:o H4H5ON X I0HHO18F H3N- Hr H7wherein each of Hi, H, H, Hi, H, H, and H7is independently hydrogen or a deuterium - enriched -H site; andwherein at least one of Hi, H2, H3, H, H, H, and H7is a deuterium-enriched -H site.or a salt or ester thereof.

2. The composition of claim 1, wherein in the compound,(a) at least two of Hi. H. H,. H4, H5, H„ and H? are deuterium -enriched -H sites;(b) at least three of Hi, H2, H, H4, H, H, and Hare deuterium -enriched -H sites; or(c) at least four of Hi, H2, H3, H4, H5, H, and H7are deuterium -enriched -H sites;preferably, at least three of Hi, H2, H3, H4, H5, Hs, and H7are deuterium-enriched -H sites;more preferably, three of Hi, H2, H3, Hi, H5, Hs, and H / are deuterium -enriched - H sites.

3. The composition of any one of claims 1-2, wherein in the compound,(a) one of Hi, H2, and H3is a deuterium-enriched -H site; or(b) two of Hi, H2, and Hare deuterium-enriched -H sites; and / or(c) one of H. H. H, and H7is a deuterium-enriched -H site;(d) two of H, H. H, and Hare deuterium-enriched -H sites; or(e) three of H, H, H, and Hare deuterium-enriched -H sites;preferably, one of Hi, H, and H is deuterium-enriched -H site; and two of H, H, H, and Hare deuterium-enriched -H sites.

4. The composition of any one of claims 1-3, wherein in the compound, one of H3, H4, and Hs is deuterium -enriched -H sites; or H,. H4. and H are deuterium-enriched -H sites.

5. Tire composition of any one of claims 1 -4, wherein in the compound, H3, H4, and H5 are deuterium- enriched -H sites and the level of deuterium at the deuterium-enriched -H site is 0.02% to 100%.

6. The composition of any one of claims 1-5, wherein in the compound, H3, H4, and Hs are deuterium- enriched -H sites and the level of deuterium at the deuterium-enriched -H site is 20%-100%, 50%- 100%, 70%-100%, 90%-100%, 97%-100%, or 99%-100%.

7. The composition of any one of claims 1-6, wherein in the compound, H3. H4, and H5are deuterium- enriched -H sites and the level of deuterium at the deuterium-enriched -H site is no less than 50%, no less than 70%, no less than 90%, no less than 97% or no less than 99%.

8. Tire composition of any one of claims 1-7, wherein in the compound, H3, H4, and H5are deuterium- enriched -H sites and the proportion of molecules having deuterium at the H3, H4, and H5 position is substantially greater than 0.0156% of molecules in the composition.

9. The composition of any one of claims 1-8, wherein the compound has the structure:wherein H is hydrogen and D represents a deuterium-enriched -H site.

10. The composition of any one of claims 1-9, wherein the compound has the structure:O D D OH, XN X OHH18F D, N.H H11. Tire composition of any one of claims 1-10 further comprising one or more compound having structurepreferably, further comprising12. Tire composition of claim 11, wherein the molar ratio betweenw- in the composition is from 99:1 to 1:99; preferably, from 95:5 to 50:50: more preferably from 95:5 to 60:40; more preferably from 90:10 to 70:30; more preferably from 99: 10 to 80:20.

13. The composition of any one of claims 1-12, further comprising one or more pharmaceutically acceptable carriers.

14. A method of detecting the presence of inflammatory cells and / or tumor cells in a subject by administering the composition of any one of claims 1-13 to the subject,wherein the method comprises determining if an amount of the compound or the composition is present in the subject after a period after administration.

15. A method of detecting the location of inflammatory cells and / or tumor cells in a subject by administering the composition of any one of claims 1-13 to the subject,wherein the method comprises determining if an amount of the compound or the composition is present in the subject after a period after administration.

16. A method of imaging inflammatory cells and / or tumor cells in a subject by administering the composition of any one of claims 1-13 to the subject,wherein the method comprises determining if an amount of the compound or the composition is present in the subject after a period after administration.

17. The method of any one of claims 14-16, wherein the inflammatory cells are caused by bacteria infection, viral infection, injuries, toxins, or autoimmune triggers.

18. Tire method of any one of claims 14-16, wherein the tumor cells are cancerous cells.

19. A method of imaging cells expressing glutamine in a subject, comprising administering the composition of any one of claims 1-13 to the subject.

20. A method for positron emission tomography (PET) imaging of a subject, comprising:(a) administering to the subject the composition of- any one of claims 1-13;(b) detecting positrons emitted from the compound or the composition; and(c) constructing a PET image of the spatial distribution of the compound or the composition.

21. A method of diagnosing a disease in a subject, wherein the method comprises performing the PET imaging of claim 20 to the subject.

22. Tire method of claim 21, wherein the disease is gliomas, neuroblastoma orbrain metastasis, lung cancer, colon cancer, triple-negative breast cancer (TNBC). breast cancer, hepatocellular carcinoma, nonalcoholic steatohepatitis (NASH), atherosclerotic lesions, multiple myeloma (MM), clear cell renal cell carcinoma (ccRCC), osteosarcoma, pancreatic ductal adenocarcinoma (PDAC), inflammatory arthritis, or myositis.

23. Tire method of any one of claims 14-22, wherein the subject is a mammal, preferably, the mammal is human.

24. A process of preparing a compound of formula (VI):o D D OH2N Y ]0HI 8F D NH2 (VI)wherein D is a deuterium -enriched -H site;wherein the process comprises:(a) reacting a compound of formula (I) with an oxidizing agent in a baseD D O HCk JXO / -BuD D NHBOC (I),to produce a compound of formula (II)D D OCk X'Y y Or-BuD NHBoc (II);(b) reacting the compound of formula (II) with isocyanide in an acid to produce an intermediate, then reacting the intermediate with thiourea in a base to produce a compound of formula (III)O D O OTmob~,N y Ot-BuHHO D NHBOC (m);(c) reacting the compound of formula (III) with a protecting agent in a base to produce a compound of formula (IV)o D D oTinob^N X T Ot-BuTsO D NHBOC (IV);(d) reacting the compound of formula (IV) with an18F labeled salt in a base to produce a compound of formula (V)O D D OTmob. -zCN Y Ot-BuFDNHBoc (V); and(e) reacting the compound of formula (V) with a de-protecting agent to thereby produce the compound of formula (VI).

25. The process of claim 24, wherein the(a) compound of formula (I) has the structure:D D OHCk AO / -BuD D NHBoc(b) compound of formula (II) has tire structure:D NHBoc(c) compound of formula (III) has the structure:O D D OTmob n 'c' n" N V^Ot-BuHHC) D NHBOC(d) compound of formula (IV) has tire structure:OTm°KNA^(■j O / -B11HTsO D NHBoc; and / or(e) compound of formula (V) has the structure:O D D OTmobxN X y O / -B11H 18F D NHBOC26. The process of any one of claims 24-25. wherein(a) the oxidizing agent in step (a) is Dess-Martin periodinane (DMP). pyridinium chlorochromate (PCC), swem oxidation reagents, or 2,2, 6, 6-Tetram ethylpiperidine- 1-oxyl (TEMP); preferably, the oxidizing agent in step (a) is DMP;(b) the base in step (a) is NaHCOs, KHCO3, NH4HCO3, ISfeCOs, or CaCOs; preferably, the base in step (a) is NaHCCE;(c) the isocyanide in step (b) is trimethoxybenzyl isocyanide(TmobNC), dimethoxybenzyl isocyanides, monomethoxybenzyl isocyanides. 4-methoxybenzyl isocyanide; preferably, the isocyanide in step (b) is TmobNC;(d) the acid in step (b) is chloroacetic acid (CAA), bromoacetic acid, iodoacetic acid, fluoroacetic acid, dichloroacetic acid, or trichloroacetic acid; preferably, the acid in step (b) is CAA; (e) the base in step (b) is NaHCO3. KHCO3, NH4HCO3, Na2CO3, or CaCO3, preferably, the base in step (b) is NaHCOs;(f) the protecting agent in step (c) is tosyl chloride (TsCl), mesyl chloride (MsCl), nosyl chloride (NsCl), besyl chloride, ortriflyl chloride (TfCl); preferably, the protecting agent in step (c) is TsCl;(g) the base in step (c) is triethylamine (EtsN), tributylamine, tripropylamine, N, N- diisopropylamine. pyridine, orN-methylmorpholine; preferably, the base in step (c) is Et3N; (h) step (c) is conducted in the presence of a catalyst, preferably, the catalyst is 4- dimethylaminopyridine (DMAP), 4-pyrrolidinopyridine (PPY), 4-dimethylaminopyridine N- oxide (DMAP-0), 4-(N, N-diethylamino)pyridine, N-methylimidazole (NMI), 1,2,4-triazole, or imidazole; more preferably, the catalyst is DMAP;(i) the18F labeled salt in step (d) is18F labeled potassium fluoride ([18F]KF),18F labeled sodium fluoride ([18F]NaF),18F labeled ammonium fluoride ([18F]NH4F).18F labeled lithium fluoride ([18F]LiF),18F labeled cesium fluoride ([18F]CsF). or18F labeled tetrabutylammonium fluoride ([18F]TBAF); preferably, the18F labeled salt in step (d) is [18F]KF;(j) the base in step (d) is KHCOs or K2CO3; preferably, the base in step (d) is KHCO3; and / or (k) the de-protecting agent in step (e) is trifluoroacetic acid (TFA), anisole, dichloroacetic acid (DCA), trichloroacetic acid, or hexafluoroisopropanol (HFIP); preferably, the de-protecting agent in step (e) is TFA and anisole.

27. The process of any one of claims 24-26, wherein the intermediate in step (b) is:; preferably, the intermediate in step (b) is?D'-DuHo, D NHBocACl more preferably, the intermediate in step (b) isO D D OTmobN X Y Ot-Buo D NHBOC>=OCl28. The process of any one of claims 24-27, wherein(a) steps (a), (b), (c), and (d) are each conducted in the presence of one or more solvents; preferably the solvent is DCM, EtOH, tetrahydrofuran (THF), acetonitrile (MeCN); more preferably, the solvent in steps (a), (b) and (c) is DMP, and the solvent in step (d) is MeCN; and / or(b) step (d) further comprises adding a cyclic polyether, preferably, the cyclic polyether is 18- crown-6, 15-crown-5, 12-crown-4, dicyclohexano-18-crown-6. benzo- 18-crown-6, or 21- crown-7, preferably, the cyclic polyether is 18-crown-6.

29. Tire process of any one of claims 24-28, wherein(a) step (a) is conducted at room temperature;(b) step (a) is conducted for a period from 1 to 5 hours; preferably, from 1 to 4 hours; more preferably, from 2 to 3 hours:(c) in step (b), the compound of formula (II) is reacted with isocyanide at room temperature; (d) in step (b), the compound of formula (II) is reacted with isocyanide for a period from 5 to 30 hours; preferably, from 10 to 20 hours; more preferably from 15 to 17 hours;(e) in step (b), the intermediate is reacted with thiourea at a temperature from 30 to70 °C;preferably, from 40 to70 " C; more preferably, from 40 to 60 °C; more preferably, from 45 to 55 °C:(f) in step (b), the intermediate is reacted with thiourea for a period from 2 to 20 hours; preferably.from 3 to 10 hours; more preferably from 4 to 6 hours;(g) step (c) is conducted at a temperature from 0 to 30 °C; preferably, from 0 to 25 °C;(h) step (d) is conducted at a temperature from 30 to 90 °C; preferably, from 40 to 80 °C; more preferably, from 50 to 80 °C; more preferably, from 60 to 80 °C; and / or(i) step (e) is conducted at a temperature from 30 to 90 °C; preferably, from 40 to 80 °C; more preferably, from 50 to 70 °C; more preferably, from 55to 65 °C.

30. A process for preparing a compound of formula I,D D O HCX Jc' JIor- BuNHBoc (I).wherein D is a deuterium -enriched -H site;wherein the process comprises:OBn(e) reacting OBn N OBnwlth a deuterium enriched haloalcohol in an acid to produce a compound of formula (VII);D,DD-C-C-DRi OBn (vii)wherein Ri is halogen;, OPh / — N OtBu(f) reacting the compound of formula (VII) with Ph in a base to produce an intermediate; then reacting the intermediate with an acid to produce a compound of formula (VIII)BnO(VIII);(g) reacting the compound of formula (VIII) with a protecting agent to produce a compound of formula (IX)D2HBnCk XX XC Y Or-BuD, I- NHBoc (IX); and(h) reacting the compound of formula (IX) with a de-protecting agent in an alcohol to produce the compound of formula (I).

31. The process of claim 30, wherein after step (b) and before step (c), S enantiomer of formula (VIII) is isolated for further reaction; wherein the S enantiomer of formula (VIII) has the structure:

32. The process of claim 31, wherein the S enantiomer of formula (VIII) is isolated by HPLC.

33. The process of any one of claims 30-32, wherein the intermediate isN OtBuPhPh, preferably, the intermediate is34. The process of any one of claims 30-33. wherein(a) Ri is F, Br, Cl, 1, preferably, Ri is F;(b) the compound of formula (I) has the structure:D D O HCk XX Ayy Or-BuD D NHBoc and / or(c) the compound of formula (IX) has the structure:D, OBnCC^C'Ot-Bu2NHBoc35. Tire process of any one of claims 30-34, wherein(a) the acid in step (a) is trifluoromethanesulfonic acid (TfOH); nonafluorobutane sulfonic acid, perfluoroethanesulfonic acid, perfluoropropane sulfonic acid, fluorosulfuric acid, methanesulfonic acid, or p-toluenesulfonic acid; preferably, the acid in step (a) is TfOH; (b) step (a) is conducted in the presence of a solvent, preferably, the solvent is 1,4-dioxane, tetrahydrofuran, 1,3-dioxolane, dimethoxyethane, ethylene glycol dimethyl ether, diethylene glycol dimethyl ether, or diglyme; more preferably, the solvent is 1,4-dioxane;(c) the base in step (b) is THF, lithium diisopropylamide, sodium hydride, potassium tert-butoxide, sodium methoxide, or potassium hydride; preferably, the base in step (b) is THF;(d) the acid is step (b) is citric acid, tartaric acid, malic acid, ascorbic acid, oxalic acid, or lactic acid: preferably, the acid is step (b) is citric acid;(e) step (b) further comprises adding a base, preferably, the base is t-BuOK, sodium hydride, lithium diisopropylamide, potassium bis(trimethylsilyl)amide, sodium methoxide, or potassium tert-amylate, more preferably, the base is t-BuOK;(f) the protecting agent in step (c) is (Boc)2O, di-tert-butyl dicarbonate, or tert-butyl chloroformate; preferably, tire protecting agent in step (c) is (Boc)2O;(g) step (c) is conducted in the presence of a solvent, preferably, the solvent is 1.4-dioxane, tetrahydrofuran, 1,3-dioxolane, dimethoxyethane, ethylene glycol dimethyl ether, diethylene glycol dimethyl ether, or diglyme; preferably, the solvent is 1,4-dioxane;(h) the de-protecting agent in step (d) is palladium on carbon (Pd / C), platinum on carbon, Raney nickel, palladium hydroxide on carbon, or rhodium on carbon; more preferably, the deprotecting agent in step (d) is Pd / C; and / or(i) the alcohol in step (d) is EtOH. methanol, propanol, isopropanol, butanol, or tert-butanol;preferably, the alcohol in step (d)is EtOH.

36. Tire process of any one of claims 30-35, wherein(a) step (a) is conducted at room temperature;(j) step (a) is conducted for a period from 10 to 30 hours; preferably, from 1 to 20 hours; more preferably from 15 to 18 hours;(b) step (a) is conducted with molecular sieves;, O p\ >=N OtBu (c) in step (b), the compound of formula (VII) is first reacted withPhat a temperature from -70 " C to 0 °C. then reacted at room temperature; preferably, at room temperature for a period from 10 to 30 hours; more preferably, from 15 to 20 hours; more preferably from 15 to 18 hours;(d) the intermediate is reacted with the acid at room temperature;(e) the intermediate is reacted with the acid for a period from 10 to 30 hours; preferably, from 15 to 20 hours; more preferably from 15 to 18 hours;(f) step (c) is conducted at room temperature;(g) step (c) is conducted for a period from 10 to 30 hours; preferably, from 15 to 20 hours; more preferably from 15 to 18 hours;(h) step (d) is conducted in hydrogen;(i) step (d) is conducted at room temperature; and / or(j) step (d) is conducted for a period from 10 to 30 hours; preferably, from 15 to 20 hours; more preferably from 15 to 18 hours.

37. A PET imaging system comprising a PET imaging device, and radiotracers comprising the composition of any one of claims 1-21.

38. A package for use in PET imaging which comprises the composition of any one of claims 1-21.

39. Use of the composition of any one of claims 1-21 in(a) detecting the presence of inflammatory cells and / or tumor cells in a subject;(b) detecting the location of inflammatory cells and / or tumor cells in a subject:(c) imaging inflammatory cells and / or tumor cells in a subject;(d) imaging cells expressing glutamine in a subject;(e) PET imaging of a subject; and / or(f) diagnosing a disease in a subject.

40. The composition of any one of claims 1-21 for use in(a) detecting the presence of inflammatory cells and / or tumor cells in a subject;(b) detecting the location of inflammatory cells and / or tumor cells in a subject;(c) imaging inflammatory cells and / or tumor cells in a subject;(d) imaging cells expressing glutamine in a subject;(c) PET imaging of a subject; and / or(f) diagnosing a disease in a subject.

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