EP4633683A1

EP4633683A1 - Cyclooctene compositions and uses thereof

Info

Publication number: EP4633683A1
Application number: EP23848601.3A
Authority: EP
Inventors: Thomas Charles II PICKEL; Emily Anne PETERSON; Brendon Ellery COOK; Maciej Adam KALISZCZAK
Original assignee: Biogen MA Inc
Current assignee: Biogen MA Inc
Priority date: 2022-12-16
Filing date: 2023-12-15
Publication date: 2025-10-22
Also published as: WO2024130105A1; JP2026500338A

Abstract

The present disclosure provides a compound of Formula (I) or a pharmaceutically acceptable salt thereof and its use in, e.g., evaluating the biodistribution and/or concentration of a biomolecule in a subject.

Description

Attorney Docket No.: 131734-01120 CYCLOOCTENE COMPOSITIONS AND USES THEREOF RELATED APPLICATIONS This application claims the benefit of the filing date, under 35 U.S.C. §119(e), of U.S. Provisional Application No.63/433,107, filed on December 16, 2022, U.S. Provisional Application No.63/461,029, filed on April 21, 2023, and U.S. Provisional Application No. 63/543,160, filed on October 9, 2023, the entire contents of which are incorporated here by reference. TECHNICAL FIELD This disclosure relates generally to compositions and methods for assessing the distribution and/or concentrations of biomolecules, e.g., antisense oligonucleotides, antibodies, or gene therapy agents, in a subject. BACKGROUND Biomolecules such as antisense oligonucleotides (ASOs), antibodies, or gene therapy agents have proven extremely efficacious in treating various diseases in a subject. To ensure their proper distribution and kinetics, it is necessary to develop imaging modalities that are compatible with these biomolecules. While directly radiolabeled biomolecules have proven effective, their application in human subjects has been limited by challenges relating to intrathecal administration of radiotracers and constraints on longer imaging time-points imposed by radioisotope half-life. Pretargeted imaging ("PTI") using Inverse electron demand Diels–Alder ("IEDDA") click chemistry has recently been applied in the CNS compartment using non-conventional therapeutic modalities. Cook B.E. demonstrated that click-reactive PET tracers can be developed to cross the blood-brain barrier and undergo an in vivo click reaction within the brain and spine of living animals. See Mol. Imaging Biol.2022 Dec; 24(6):940-949. This first-generation brain penetrant tetrazine exhibited slow clearance from the CNS and need additional developments in applying it in nonhuman primate ("NHP") studies, prompting exploration of an alternative strategy. Accordingly, there is a need to find new imaging modalities that are more suitable for clinical applications, i.e., low non-specific binding, fast clearance, etc. Attorney Docket No.: 131734-01120 SUMMARY This application relates to compounds, compositions, and methods for determining the distribution and/or concentrations of a biomolecule (e.g., ASOs, antibodies, gene therapy agents or nanoparticles) in a subject. This disclosure provides compounds and compositions that are useful in evaluating the distribution and kinetics of biomolecules (e.g., ASOs, antibodies, gene therapy agents or nanoparticles) in a subject. Also featured are methods for assessing the concentration of an antisense oligonucleotide in a desired region (e.g., brain, spinal cord, etc.) in a subject. In one aspect, the disclosure provides a compound of Formula (I), or a pharmaceutically acceptable salt thereof: (I), wherein R¹, R², X¹, Y¹, W, and are as defined herein. Also provided are pharmaceutical compositions comprising a compound of Formula (I) or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier or excipient. In certain cases, the compound of Formula (I) is CNS-penetrant (i.e., it can cross the blood brain barrier). In certain instances, the compound of Formula (I) is ¹⁸F labeled Compound 1. In some instances, the pharmaceutically acceptable carrier is phosphate buffered saline. In some instances, the pharmaceutically acceptable carrier is artificial cerebrospinal fluid (a-CSF). In some instances, the pharmaceutically acceptable carrier is sterile water for injection. In another aspect, the disclosure relates to a method of determining the distribution of a biomolecule in a subject. The method involves administering the biomolecule to the subject, followed by administering to the subject the compound of the present disclosure or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition of the present disclosure; wherein the biomolecule binds the compound in vivo. The method further includes imaging the distribution of the biomolecule in the subject. In some instances, of the above methods, the compound of Formula (I) is radiolabeled with a radiolabel selected from the group consisting of fluorine-18, carbon-11, and gallium- 68. In one instance, the compound of Formula (I) is radiolabeled with fluorine-18. Attorney Docket No.: 131734-01120 In another aspect, the biomolecule of the present disclosure is an antibody (a monovalent whole antibody, a bispecific whole antibody), an antigen-binding fragment (Fab, Fab’, F(ab)2, scFv, sc(Fv)2, diabody, nanobody), a peptide, or a nucleic acid (e.g., an antisense oligonucleotide, siRNA, miRNA, shRNA, or aptamer). Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the exemplary methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present application, including definitions, will control. The materials, methods, and examples are illustrative only and not intended to be limiting. Other features and advantages of the invention will be apparent from the following detailed description and from the claims. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows the second generation PTI procedure of the present disclosure, which utilizes an inverse electron-demand Diels-Alder [4 + 2] (IEDDA) cycloaddition click ligation between a radiolabeled trans-cyclooctene and a biomolecule attached with 1,2,4,5 tetrazine in vivo. Figure 2 shows dynamic PET scans in rat which demonstrated rapid washout from the brain in animals dosed with control ASO (~0.18 SUB at 20 minutes), while the ASO- methyltetrazine (Compound 21) cohorts with three different doses (750 µg, 500 µg, 250 µg) retained activity in the brain (0.50 ~ 1.0 SUV at 20 minutes). Figure 3A is a PET/CT image showing rats dosed with either Malat1 ASO (left) or Compound 21 (right) followed by ¹⁸F labeled Compound 124 hour later and imaged from 0- 20 minutes p.i. shows click reaction with Compound 21 compared to control ASO suggesting quantitative imaging of Compound 21 concentration is feasible. Figure 3B is a graph showing concentration of ASO in brain subregions versus PET SUV normalized to blood activity. Figure 3C is a graph showing concentration of ASO in thalamus and hypothalamus versus PET SUV normalized to blood activity. Attorney Docket No.: 131734-01120 Figure 3D is a graph showing concentration of ASO in cerebellum versus PET SUV normalized to blood activity. Figure 4 is a PET/CT image showing specific uptake of tracer in the brain and spinal cord in rats treated with the second generation PTI procedure in the present disclosure. Figure 5 are PET/CT images showing the biodistribution of ¹⁸F labeled Compound 1 after IV administration in Rats 1 (A) and 2 (B). Figure 6 are Time Activity Curves and model fits of ¹⁸F labeled Compound 1 in SUV in rat brain between 0 and 60 min after tracer injection, for rats 1 (A) and 2 (B). Figure 7 are PET/CT images showing the biodistribution of ¹⁸F labeled Compound 1 after IV administration, 24 h following IT administration of ASO control in Rats 8 (A), 9 (B) and 11 (C). Figure 8 are Time Activity Curves and model fits of Compound 1 in SUV in rat brain between 0 and 20 min after tracer injection, 24 h following IT administration of ASO control in Rats 8 (A), 9 (B) and 11 (C). Figure 9 are PET/CT images showing the biodistribution of ¹⁸F labeled Compound 1 after IV administration, 24 h following IT administration of Compound 21 (750 μg) in Rats 4 (A), 5 (B), 7 (C), 12 (D) and 19 (E). Figure 10 are Time Activity Curves and model fits of ¹⁸F labeled Compound 1 in SUV in rat brain between 0 and 20 min after tracer injection, 24 h following IT administration of Compound 21 (750 μg) in Rats 4 (A), 5 (B), 7 (C), 12 (D) and 19 (E). Figure 11 are PET/CT images showing the biodistribution of ¹⁸F labeled Compound 1 after IV administration, 24 h following IT administration of Compound 21 (250 μg) in Rats 14 (A), 16 (B) and 17 (C). Figure 12 are Time Activity Curves and model fits of ¹⁸F labeled Compound 1 in SUV in rat brain between 0 and 20 min after tracer injection, 24 h following IT administration of Compound 21 (250 μg) in Rats 14 (A), 16 (B) and 17 (C). Figure 13 are PET/CT images showing the biodistribution of ¹⁸F labeled Compound 1 after IV administration, 24 h following IT administration of Compound 21 (500 μg) in Rats 13 (A), 15 (B), 18 (C) and 20 (D). Figure 14 are Time Activity Curves and model fits of ¹⁸F labeled Compound 1 in SUV in rat brain between 0 and 20 min after tracer injection, 24 h following IT administration of Compound 21 (500 μg) in Rats 13 (A), 15 (B), 18 (C) and 20 (D). Attorney Docket No.: 131734-01120 Figure 15 are Time Activity Curves and model fits of ¹⁸F labeled Compound 1 in SUV in rat tissues between 0 and 20 min after tracer injection, 24 h following IT administration of ASO control in Rats 8 (A), 9 (B) and 11 (C). Figure 16 are Time Activity Curves and model fits of ¹⁸F labeled Compound 1 in SUV in rat tissues between 0 and 20 min after tracer injection, 24 h following IT administration of Compound 21 (750 μg) in Rats 4 (A), 5 (B), 7 (C), 12 (D) and 19 (E). Figure 17 are Time Activity Curves and model fits of ¹⁸F labeled Compound 1 in SUV in rat tissues between 0 and 20 min after tracer injection, 24 h following IT administration of Compound 21 (250 μg) in Rats 14 (A), 16 (B) and 17 (C). Figure 18 are Time Activity Curves and model fits of ¹⁸F labeled Compound 1 in SUV in rat tissues between 0 and 20 min after tracer injection, 24 h following IT administration of Compound 21 (500 μg) in Rats 13 (A), 15 (B), 18 (C) and 20 (D). Figure 19A is baseline PET imaging of ¹⁸F labeled Compound 1 in cyno monkeys. Figure 19B are representative baseline PET scans of ¹⁸F labeled Compound 1 in cyno monkeys over 0-30 min and 0-120 min. Figure 20 is a graph showing the time-activity curves of Compound 1 in a NHP brain. baseline (blue, ¹⁸F labeled Compound 1) and pretreatment (red, pretreated with non- radiolabeled Compound 1) show close overlap. Figure 21 shows the chemical structure of metastasis-associated lung adenocarcinoma transcript 1 ("MALAT1") with a C6 amine linker. Figure 22 shows the chemical structures of three MALAT1-ASO-Tz analogues. Figure 23 is schematic of reaction solutions including volumes to reaction plate and crash plates. Figure 24 is a graph showing test compound disappearance with time in the presence of liver microsomes. Figure 25 is a schematic of plasma and PBS buffer solutions including volumes to RED plates and aliquoted to crash plates. Figure 26 is a graph showing in vitro autoradiography of rat dosed IT with 500 µg of compound 21 vs. naïve rat. Figure 27 is a graph showing stability of Compound 21. Longitudinal stability and clearance of Compound 21 in whole rat brain after IT dosing as measured by LC-MS. Graph shows total ASO concentration (grey triangle) which includes parent compound and any degradation products where linker or tetrazine was affected, Compound 21 (red square) Attorney Docket No.: 131734-01120 where linker and tetrazine mass was unchanged, and Compound 21-TCO-DBCO (blue circle) in which Compound 21 was reactive to a TCO-DBCO ligand. Figure 28 shows plasma parent fraction of ¹⁸F labeled Compound 1 for each PET scan in NHP and a representative radioHPLC chromatogram from NHP430-minutes post- injection. Figure 29 shows pretargeted PET imaging in NHP. Left to right, representative baseline scan with ¹⁸F labeled Compound 1, representative non-radioactive self-block with 1.0 mg/kg Compound 1, pretargeted with 20 mg Compound 21 dosed IT followed 24 hours later with tracer (NHP4 and NHP5). Images are summed from 60-120 minutes post-injection. The static SUV images, in a range of 0-2, are overlaid on a standard template T1-weighted MRI of the cynomolgus monkey, with a transparency of 0.7. The SUV images are smoothed at 2 mm FWHM and masked to show only the values within the brain mask template. Figure 30 shows pretargeted PET imaging in cynomolgus monkeys without application of the brain mask. Figure 31 shows results from metabolite identification study for Compound 2. Table at left shows percentage of each isolated metabolite from the different species of hepatocytes tested. Figure 32 shows time-activity curves for whole-brain PET regions of interests in NHP. DETAILED DESCRIPTION This disclosure relates in part to compounds and compositions that are useful for “pretargeting.” “Pretargeting” separates the delivery of a radiolabelled compound/radioligand from that of a modified biomolecule (e.g., antibodies, nanoparticles, gene therapy agents, ASO). These compounds and compositions can be used in vivo, e.g., to evaluate distribution and/or concentration of a biomolecule (e.g., antibodies, nanoparticles, gene therapy agents, ASO) in a subject. In some cases, the compounds and compositions are used to evaluate the distribution and/or concentration of a biomolecule (e.g., antibodies, nanoparticles, gene therapy agents, ASO) in the brain and/or spinal cord of a subject. 1. Pretargeting Medical diagnosis and therapy routinely makes use of imaging agents. Such agents can be useful, e.g., to determine if a therapeutic agent has reached its intended target and to Attorney Docket No.: 131734-01120 determine the location and/or concentration of a therapeutic or diagnostic agent. Existing methods can however be problematic. For example, the relatively slow pharmacokinetics of certain biomolecules used for imaging require the attached radioactive label to have multiday half-lives because if distribution of the biomolecules is to be assessed at longer time -points there must remain sufficient radiation to image successfully. In some instances, this leads to high activity concentrations in and radiation doses to non-target organs. To circumvent these problems an alternative approach has emerged, that is referred to as “pretargeting” whereby a radiolabeled compound or radioligand (e.g., radiolabeled cyclooctene) and a modified biomolecule (e.g. antibodies, nanoparticles, gene therapy agents, ASO) are delivered separately to a subject. Pretargeted methods generally involve the following steps: first, the injection into the subject of a modified biomolecule (e.g., antibodies, nanoparticles, gene therapy agents, ASO) that binds or localizes to the target of interest but also has the ability to bind to a radioligand; second, the slow accumulation of the modified biomolecule (e.g. antibodies, nanoparticles, gene therapy agents, ASO) at the site of the target and concomitant clearance of the modified biomolecule (e.g. antibodies, nanoparticles, gene therapy agents, ASO) from the blood; third, the injection into the bloodstream of the radiolabeled compound or radioligand (e.g., a compound of Formula (I) of the present disclosure); and fourth the binding of the radiolabeled compound or radioligand to the modified biomolecule (e.g., antibodies, nanoparticles, gene therapy agents, ASO), followed by the rapid clearance of excess radioactivity. In some instances, an additional step is added before the injection of the radiolabeled compound or radioligand, specifically, the administration of a clearing agent designed to accelerate the removal of residual targeting agent from the bloodstream. In another aspect, the pharmacokinetics of the radiolabeled compound or radioligand not only reduces background radiation dose to non-target organs but also facilitates the use of radioisotopes with short half-lives that would normally be incompatible with such imaging. In one embodiment, this disclosure provides compounds and compositions that can be used for penetrating the blood brain barrier and thus having utility in pretargeting in the central nervous system. An illustrative example of applying pretargeting in a subject is depicted in Figure 1B. As can be seen in this example, pretargeting separates the delivery of the radioactivity from the biomolecule (e.g., antibodies, nanoparticles, gene therapy agents, ASO). The biomolecule (e.g., antibodies, nanoparticles, gene therapy agents, ASO) is modified with a Attorney Docket No.: 131734-01120 1,2,4,5 tetrazine (Tz) and is administered to a subject while the radioligand contains a cyclooctene and is injected intravenously. The Tz and cyclooctene undergo an inverse electron demand Diels-Alder (IEDDA) reaction in vivo, covalently binding the radioligand to the biomolecule (e.g., antibodies, nanoparticles, gene therapy agents, ASO). 2. Compounds In a first embodiment, the present disclosure provides a compound of Formula (I): (I), or a pharmaceutically acceptable salt thereof, wherein: is a single bond indicating cyclooctene is trans or cis; W is a radiolabel moiety comprising a radioisotope; Y¹ is -CH₂-, Ar, 4 to 10 membered heterocyclyl, ^@-SO₂-Ar-^@@, or ^@-C(=O)-Ar-^@@; wherein said Ar or 4 to 10 membered heterocyclyl represented by Y¹ or in the group represented by Y¹ is optionally substituted by one or more R^Y; ^@- indicates the point that connects with ; and ^@@- indicates the point that connects with W; wherein Ar is 6 to 10 membered aryl or 5 to 10 member heteroaryl; R^Y is halogen, C1-6alkyl, or oxo (as appropriate); is a bond, -(CH2)n-, Het, ^&-C(=O)-Het-^&&, ^&-C1-6alkylenyl-C(=O)-Het-^&&, ^&- C(=O)-C_1-6alkylenyl-Het-^&&, -NH-Het-NH-, -NH-Cyc-NH-, ^&-NR^L-Het-^&&, or ^&-Het-NR^L- ^&&; wherein Het is a 4 to 10 membered heterocyclyl; Cyc is 4-10 membered carbocyclyl; R^L is hydrogen or C1-6alkyl; n is 1, 2, or 3; ^&- indicates the point that connects with X¹; and ^&&- indicates the point that connects with Y¹; X¹ is a bond, - ^{^^}; wherein R^X is hydrogen or C1-6alkyl; ^{^}- indicates the point that connects with cyclooctene; and ^{^^}- indicates the point that connects with ; R¹ is hydrogen or -OH; R² is hydrogen; or X¹ and R², together with atoms to which they are attached form a moiety represented by formula A: Attorney Docket No.: 131734-01120 wherein Z is a bond, -O-, or -NH-; indicates the point that connects with cyclooctene; indicates the point that connects with ; wherein said heterocyclyl comprises 1-3 heteroatoms selected from oxygen, nitrogen, and sulfur; and said heteroaryl comprises 1-4 heteroatoms selected from oxygen, nitrogen, and sulfur. In a second embodiment, the present disclosure provides a compound according to the first embodiment, or a pharmaceutically acceptable salt thereof, wherein the compound is represented by formula (IIA): (IIA). The definitions of the variables are provided in the first embodiment. In a third embodiment, the present disclosure provides a compound according to the first embodiment, or a pharmaceutically acceptable salt thereof, wherein the compound is represented by formula (IIB): The definitions of the variables are provided in the first embodiment. In a fourth embodiment, the present disclosure provides a compound according to any one of the first through third embodiments, or a pharmaceutically acceptable salt thereof, wherein W is a ¹⁸F, ¹¹C-moiety, chelated ⁶⁸Ga, C_1-6alkyl substituted by one or more ¹⁸F, or C_1- ₆alkoxyl substituted by one or more ¹⁸F. The definitions of the remaining variables are provided in any one of the first through third embodiments. In a fifth embodiment, the present disclosure provides a compound according to any one of the first through fourth embodiments, or a pharmaceutically acceptable salt thereof, wherein W is ¹⁸F, C1-4alkyl substituted by one ¹⁸F, or C1-4alkoxyl substituted by one ¹⁸F. The Attorney Docket No.: 131734-01120 definitions of the remaining variables are provided in any one of the first through fourth embodiments. In a sixth embodiment, the present disclosure provides a compound according to any one of the first through fifth embodiments, or a pharmaceutically acceptable salt thereof, wherein W is ¹⁸F, -CH2¹⁸F, or –OCH2CH2¹⁸F. The definitions of the remaining variables are provided in any one of the first through fifth embodiments. In a seventh embodiment, the present disclosure provides a compound according to any one of the first through sixth embodiments, or a pharmaceutically acceptable salt thereof, wherein Y¹ is Ar, 6 to 9 membered heterocyclyl, ^@-SO₂-Ar-^@@, or ^@-C(=O)-Ar-^@@; wherein said Ar or 6 to 9 membered heterocyclyl represented by Y¹ or in the group represented by Y¹ is optionally substituted by one to three R^Y; wherein Ar is phenyl or 6-membered heteroaryl; R^Y is halogen, C1-4alkyl, or oxo (as appropriate). The definitions of the remaining variables are provided in any one of the first through sixth embodiments. In an eighth embodiment, the present disclosure provides a compound according to any one of the first through seventh embodiments, or a pharmaceutically acceptable salt thereof, wherein Y¹ is selected from the group consisting of: . The definitions of the remaining variables are provided in any one of the first through seventh embodiments. Attorney Docket No.: 131734-01120 In a ninth embodiment, the present disclosure provides a compound according to any one of the first through the eighth embodiments, or a pharmaceutically acceptable salt thereof, wherein is a bond, -(CH2)n-, Het, ^&-C(=O)-Het-^&&, ^&-C1-4alkylenyl-C(=O)- Het-^&&, ^&-C(=O)-C_1-4alkylenyl-Het-^&&, -NH-Het-NH-, -NH-Cyc-NH-, -NH-, ^&-NR^L-Het-^&&, or ^&-Het-NR^L-^&&; wherein Het is 4 to 8 membered monocyclic heterocyclyl, 6-9 membered spiro heterocyclyl, or 6-8 membered bridged heterocyclyl; Cyc is 4-8 membered cycloalkyl; R^L is hydrogen or C_1-4alkyl; n is 1 or 2. The definitions of the remaining variables are provided in any one of the first through eighth embodiments. In a tenth embodiment, the present disclosure provides a compound according to any one of the first through the ninth embodiments, or a pharmaceutically acceptable salt thereof, wherein is a bond, -CH₂ -, Het, ^&-C(=O)-Het-^&&, ^&-C_1-2alkylenyl-C(=O)-Het-^&&, ^&- C(=O)-C_1-2alkylenyl-Het-^&&, -NH-Het-NH-, -NH-Cyc-NH-, -NH-, ^&-NR^L-Het-^&&, or ^&-Het- NR^L-^&&; wherein Het is 4 to 6 membered monocyclic heterocyclyl, 6-7 membered spiro heterocyclyl, or 7-8 membered bridged heterocyclyl; Cyc is 4-6 membered monocyclic cycloalkyl; R^L is hydrogen or C_1-2alkyl. The definitions of the remaining variables are provided in any one of the first through ninth embodiments. In an eleventh embodiment, the present disclosure provides a compound according to any one of the first through the tenth embodiments, or a pharmaceutically acceptable salt thereof, wherein is selected from the group consisting of The definitions of the remaining variables are provided in any one of the first through tenth embodiments. Attorney Docket No.: 131734-01120 In a twelfth embodiment, the present disclosure provides a compound according to any one of the first through the eleventh embodiments, or a pharmaceutically acceptable salt thereof, wherein X¹ is a bond, -O-, -CH₂-, -NH-, -N(CH₃)-, ^{^}-(CH₂)₂O-^{^^}, ^{^}-(CH₂)₂NH-^{^^}, or ^{^}- (CH2)2N(CH3)-^{^^}. The definitions of the remaining variables are provided in any one of the first through the eleventh embodiments. In a thirteenth embodiment, the present disclosure provides a compound according to any one of the first through the eleventh embodiments, or a pharmaceutically acceptable salt thereof, wherein X¹ is -O-, -NH-, -N(CH3)-, ^{^}-(CH2)2O-^{^^}, ^{^}-(CH2)2NH-^{^^}, or ^{^}-(CH2)2N(CH3)- ^{^^}. The definitions of the remaining variables are provided in any one of the first through the eleventh embodiments. In a fourteenth embodiment, the present disclosure provides a compound according to any one of the first through the eleventh embodiments, or a pharmaceutically acceptable salt thereof, wherein X¹ and R², together with atoms to which they are attached form a moiety represented by formula A: wherein Z is -O- or -NH-. The definitions of the remaining variables are provided in any one of the first through the eleventh embodiments. In a fifteenth embodiment, the present disclosure provides a compound according to any one of the first, the third, the seventh through the eleventh, and the fourteenth embodiments, or a pharmaceutically acceptable salt thereof, wherein the compound is represented by Formula (II): The definitions of the remaining variables are provided in any one of the first, the third, the seventh through the eleventh, and the fourteenth embodiments. In a sixteenth embodiment, the present disclosure provides a compound according to the fifteenth embodiment, or a pharmaceutically acceptable salt thereof, wherein Y¹ is selected from the group consisting of Attorney Docket No.: 131734-01120 . The definitions of the remaining variables are provided in the fifteenth embodiment. In a seventeenth embodiment, the present disclosure provides a compound according to the fifteenth embodiment or the sixteenth embodiment, or a pharmaceutically acceptable salt thereof, wherein Y¹ is . The definitions of the remaining variables are provided in the fifteenth or the sixteenth embodiment. In an eighteenth embodiment, the present disclosure provides a compound according to any one of the fifteenth through seventeenth embodiments, or a pharmaceutically acceptable salt thereof, wherein is selected from the group consisting of . The definitions of the remaining variables are provided in the fifteenth through the seventeenth embodiments. In a nineteenth embodiment, the present disclosure provides a compound according to any one of the fifteenth embodiment through the eighteenth embodiments, or a pharmaceutically acceptable salt thereof, wherein is or Attorney Docket No.: 131734-01120 . The definitions of the remaining variables are provided in the fifteenth through the eighteenth embodiments. In a twentieth embodiment, the present disclosure provides a compound according to any one of the fifteenth through the nineteenth embodiments, or a pharmaceutically acceptable salt thereof, wherein the compound is . The definitions of the remaining variables are provided in the fifteenth through the nineteenth embodiments. In a twenty-first embodiment, the present disclosure provides a compound according to the first embodiment, or a pharmaceutically acceptable salt thereof, wherein the compound is represented by Formula (III): wherein W is ¹⁸F or -CH2¹⁸F; Y¹ is 6-membered heteroaryl; is a bond, Het, ^&-C(=O)-Het-^&&, ^&-C1-4alkylenyl-C(=O)-Het-^&&, ^&-C(=O)- C_1-4alkylenyl-Het-^&&; wherein Het is a 4 to 6 membered heterocyclyl; and X¹ is a bond, -O-, or -N(R^X)-; wherein R^X is hydrogen or C1-4alkyl. The definitions of the remaining variables are provided in the first embodiment. In a twenty-second embodiment, the present disclosure provides a compound according to the twenty-first embodiment, or a pharmaceutically acceptable salt thereof, wherein: Y¹ is pyridyl; Attorney Docket No.: 131734-01120 is selected from the group consisting of X¹ is a bond, -O-, -NH-, or –N(CH₃)-. The definitions of the remaining variables are provided in the twenty-first embodiment. In a twenty-third embodiment, the present disclosure provides a compound according to the first or the third embodiment, or a pharmaceutically acceptable salt thereof, wherein the compound is represented by Formula (IV): wherein W is ¹⁸F or –OCH₂CH₂ ¹⁸F; Y¹ is 6 to 9-membered heterocyclyl or ^@-C(=O)-Ar-^@@; wherein said Ar or 6 to 9- membered heterocyclyl represented by Y¹ or in the group represented by Y¹ is optionally substituted by one to three R^Y; wherein Ar is phenyl or 6-membered heteroaryl; R^Y is C1-4alkyl or oxo (as appropriate); is a bond, -CH2-, or ^&-C(=O)-Het-^&&; wherein Het is a 6 to 8 membered heterocyclyl; and X¹ is -CH₂-, ^{^}-(CH₂)₂O-^{^^}, ^{^}-(CH₂)₂NH-^{^^}, or ^{^}-(CH₂)₂N(CH₃)-^{^^}. The definitions of the remaining variables are provided in the first or the third embodiment. In a twenty-fourth embodiment, the present disclosure provides a compound according to the twenty-third embodiment, or a pharmaceutically acceptable salt thereof, wherein is selected from the group consisting of Attorney Docket No.: 131734-01120 ; and is selected from the group consisting of a bond, The definitions of the remaining variables are provided in the twenty-third embodiment. In a twenty-fifth embodiment, the present disclosure provides a compound according to the twenty-third or twenty-fourth embodiment, or a pharmaceutically acceptable salt thereof, wherein W is ¹⁸F; is a bond; and X¹ is ^{^}-(CH2)2NH-^{^^}, or ^{^}-(CH2)2N(CH3)-^{^^}. The definitions of the remaining variables are provided in the twenty-third or twenty-fourth embodiment. In one embodiment, the present disclosure provides a compound selected from the compounds disclosed in examples and Table 1, a pharmaceutically acceptable salt or a stereoisomer thereof.

Attorney Docket No.: 131734-01120 Table 1 Attorney Docket No.: 131734-01120 Attorney Docket No.: 131734-01120 ^# The F atom in the compounds of Table 1 is either F or its radioisotope ¹⁸F. Attorney Docket No.: 131734-01120 Antisense Oligonucleotide (ASO)-1,2,4,5 Tetrazine Fusions (“ASO-Tz”) Provided herein is an antisense oligonucleotide (ASO) linked to a 1,2,4,5-tetrazine (“ASO-Tz”). The 1,2,4,5-tetrazine can be directly linked to the ASO. In some instances, the 1,2,4,5-tetrazine is linked to the ASO via a linker, such as an alkylene linker. The 1,2,4,5- tetrazine can be either directly or indirectly linked to the ASO at the 5’-end of the ASO. The 1,2,4,5-tetrazine can also be either directly or indirectly linked to the ASO at the 3’-end of the ASO. In some instances, the ASO is linked to the linker (as defined herein) via a phosphorothioate linkage. In some aspects, the biomolecule of the present disclosure is an antisense oligonucleotide (“ASO”). According to the disclosure herein, the ASO can be any ASO known in the art. ASOs are synthetic single stranded strings of nucleic acids that bind to ribonucleic acid (RNA) and thereby alter or reduce expression of the target RNA. They can not only reduce expression of proteins by breakdown of the targeted transcript, but also restore protein expression or modify proteins through interference with pre-mRNA splicing. This disclosure encompasses ASOs of both types. In certain instances, the ASO of this disclosure is a “gapmer.” Such ASOs primarily act by selectively cleaving mRNAs that have complementary sites through an RNase H-dependent mechanism. They have a central region that supports RNase H activity flanked by chemically modified ends that increase affinity and/or reduce susceptibility to nucleases. In some instances, the ASO of this disclosure is a splice switching oligonucleotide (SSO) (e.g., nusinersen). SSOs are generally fully modified so as to ablate RNase H activity and allow interaction with nuclear pre-mRNA during splicing. They can be designed to bind to the 5’ or 3’ splice junctions or to exonic splicing enhancer or silencer sites. By binding to such sites they can modify splicing by, e.g., promoting alternative use of exons, exon exclusion, or exon inclusion. A non-limiting example of an ASO that is encompassed by this disclosure is provided below. wherein: the nucleoside labeled with * has a 2’-O-(2-methoxyethyl) (MOE) modification; Attorney Docket No.: 131734-01120 the “o” is a phosphodiester internucleoside linkage and the absence of “o” indicates a phosphorothioate internucleoside linkage. In some cases, the antisense oligonucleotide consists of 12 to 20 nucleosides (e.g., 12, 13, 14, 15, 16, 17, 18, 19, 20). In some instances, the antisense oligonucleotide is one that can cross the blood brain barrier. In some instances, the antisense oligonucleotide is one that is useful for the treatment of a neurodegenerative disorder. In some instances, the antisense oligonucleotide is one that is useful for the treatment of any one of: spinal muscular atrophy; amyotrophic lateral sclerosis, Alzheimer’s disease, Parkinson’s disease (familial or sporadic), frontotemporal dementia, myotonic dystrophy type 1, Huntington’s disease, Angelman syndrome, Creutzfeldt-Jakob disease, Spinocerebellar Ataxia Type 3, and Menkes disease. In one embodiment, the present disclosure provides an ASO-Tz represented by Formula 1A, 1B, or 1C, a pharmaceutically acceptable salt or a stereoisomer thereof, wherein L is a bond or a linker, i.e., the Tz moiety is conjugated directly to the ASO or via a linker, e.g., one of the linkers described in the present disclosure. In one embodiment, the present disclosure provides an ASO-Tz represented by Formula 1D, 1E, or 1F, a pharmaceutically acceptable salt or a stereoisomer thereof, Attorney Docket No.: 131734-01120 wherein ASO is an antisense oligonucleotideconnected with the C6 amine linker through its 5’-end group (e.g., phosphate –O-P(=O)(OH)-O-* or phosphorothioate –O-P(=S)(OH)-O-*, wherein –O-* indicates the attachment point of the 5’-OH group of the oligonucleotide). In one embodiment, the antisense oligonucleotide comprises 1-100 nucleotides. In one embodiment, the antisense oligonucleotide comprises 2-50 nucleotides. In one embodiment, the antisense oligonucleotide comprises 2-30 nucleotides. In one embodiment, the antisense oligonucleotide comprises 2-20 nucleotides. In one embodiment, the antisense oligonucleotide comprises 20 nucleotides. In one embodiment, the present disclosure provides an ASO-Tz selected from the compounds disclosed in Table 2, a pharmaceutically acceptable salt or a stereoisomer thereof. Table 2 Attorney Docket No.: 131734-01120 * indicates MOE, o indicates PO. The complete structures of compounds 21-23 are depicted in Figure 22. 3. Definitions The term “about” in the context of an amount, e.g., about X mg means +/- 10%, so “about 50 mg” encompasses 45 mg to 55 mg. The term “about” in the context of X days means +/-3 days, so “about 10 days” encompasses 7 to 13 days. The term “about” in the context of X months means +/- 1 week, so “about 4 months” encompasses a week before and after the 4 month mark. The term “about” in the context of X hours means +/-3 hours, so “about 10 hours” encompasses 7 to 13 hours. The term “about” in the context of X minutes means +/- 10 minutes, so “about 100 minutes” encompasses 90 to 110 minutes. The term “about” in the context of X temperature means +/- 3 °C. The term "radioisotope," as used herein, refers to an isotope of an element that is known to undergo spontaneous radioactive decay. Examples of radioisotopes include ³H, ¹⁴C, 3²P, ³⁵S, ¹⁸F, ³⁶Cl, and the like, as well as the isotopes for which a decay mode is identified in V. S. Shirley & C. M. Lederer, Isotopes Project, Nuclear Science Division, Lawrence Berkeley Laboratory, Table of Nuclides (January 1980). In some embodiments, the term “radioisotope” includes metallic and non-metallic radioisotopes. The radioisotope is chosen based on the medical application of the radiolabeled pharmaceutical agents. When the radioisotope is a metallic radioisotope, a chelator is typically employed to bind the metallic radioisotope to the rest of the molecule. When the radioisotope is a non-metallic radioisotope, the non-metallic radioisotope is typically linked directly, or via a linker, to the rest of the molecule. The term "halo" or "halogen," as used herein, refers to fluoride, chloride, bromide, or iodide. The term "alkyl" used alone or as part of a larger moiety, such as “alkoxy” or “haloalkyl” and the like, means saturated aliphatic straight-chain or branched monovalent hydrocarbon radical of formula -C_nH_(2n+1). Unless otherwise specified, an alkyl group typically has 1-20, 1-10 or 1-6 carbon atoms. In some embodiments, an alkyl group has 1-6 Attorney Docket No.: 131734-01120 carbon atoms, i.e. C1-6alkyl. As used herein, a “C1-6alkyl” group means a radical having from 1 to 6 carbon atoms in a linear or branched arrangement. Examples include methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, tert- butyl, n-pentyl, isopentyl, hexyl, and the like. In some embodiments, an alkyl group has 1-4 carbon atoms, i.e., C1-4alkyl. In some embodiments, an alkyl group has 1-3 carbon atoms, i.e., C1-3alkyl. The term "alkoxy" or “alkoxyl,” as used herein, refers to O-alkyl groups wherein alkyl is as defined above. The term "haloalkyl" means alkyl, as the case may be, substituted with one or more halogen atoms. In one embodiment, the alkyl can be substituted by one to three halogens. Examples of haloalkyl, include, but are not limited to, trifluoromethyl, trichloromethyl, pentafluoroethyl and the like. The term “alkylene” as used herein, means a straight or branched chain divalent hydrocarbon group of formula -C_nH_2n-. Non-limiting examples include ethylene, and propylene. The term “carbocyclyl” refers to any stable non-aromatic hydrocarbon ring having 3-12 membered carbocyclyl. In one embodiment, carbocyclyl is 3-, 4-, 5-, 6-, 7-, or 8- membered monocyclic or bicyclic or 7-, 8-, 9-, 10-, 11-, or 12-membered bicyclic or tricyclic hydrocarbon ring, any of which may be saturated, partially unsaturated, or unsaturated. Any substitutable ring atom can be substituted (e.g., by one or more substituents). Examples of such carbocycles include, but are not limited to, cyclopropyl, cyclobutyl, cyclobutenyl, cyclopentyl, cyclopentenyl, cyclohexyl, cycloheptenyl, cycloheptyl, cycloheptenyl, adamantyl, cyclooctyl, cyclooctenyl, and cyclooctadienyl. In one embodiment, carbocyclyl is intended to include, bridged, fused, and spirocyclic rings. In a spirocyclic carbocyclyl, one atom is common to two different rings. An example of a spirocyclic carbocyclyl is spiro[3.3]heptanyl. In a bridged carbocyclyl, the rings share at least two common non- adjacent atoms. Examples of bridged carbocyclyls include bicyclo[2.2.1]heptanyl, bicyclo[2.2.1]hept-2-enyl, and adamantanyl. In a fused-ring carbocyclyl system, two or more rings may be fused together, such that two rings share one common bond. Examples of two- or three-fused ring carbocyclyls include naphthalenyl, tetrahydronaphthalenyl (tetralinyl), indenyl, indanyl (dihydroindenyl), anthracenyl, phenanthrenyl, and decalinyl. The term "bridged carbocyclyl" refers to a 5 to 12 membered polycyclic carbocyclyl group, wherein any two rings in the group share two disconnected atoms, the rings can have one or more double bonds but have no completely conjugated π-electron system. Attorney Docket No.: 131734-01120 Representative examples of bridged carbocyclyl include, but are not limited to the following groups: The term “cycloalkyl” refers to a monocyclic, bicyclic, tricyclic, or polycyclic saturated hydrocarbon groups having 3 to 12 ring carbons. In one embodiment, cycloalkyl may have 3 to 7 or 3 to 6 ring carbons. Any substitutable ring atom can be substituted (e.g., by one or more substituents). Examples of cycloalkyl groups include, without limitation, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. Cycloalkyl may include multiple fused and/or bridged rings. Non-limiting examples of fused/bridged cycloalkyl include: bicyclo[1.1.0]butane, bicyclo[2.1.0]pentane, bicyclo[1.1.0]pentane, bicyclo[3.1.0]hexane, bicyclo[2.1.1]hexane, bicyclo[3.2.0]heptane, bicyclo[4.1.0]heptane, bicyclo[2.2.1]heptane, bicyclo[3.1.1]heptane, bicyclo[4.2.0]octane, bicyclo[3.2.1]octane, bicyclo[2.2.2]octane, and the like. Cycloalkyl also includes spirocyclic rings (e.g., spirocyclic bicycle wherein two rings are connected through just one atom). Non-limiting examples of spirocyclic cycloalkyls include spiro[2.2]pentane, spiro[2.5]octane, spiro[3.5]nonane, spiro[3.5]nonane, spiro[3.5]nonane, spiro[4.4]nonane, spiro[2.6]nonane, spiro[4.5]decane, spiro[3.6]decane, spiro[5.5]undecane, and the like. The term “heterocyclyl” or “heterocyclic” refers to a radical of a 3- to 12-membered non-aromatic ring system having ring carbon atoms and 1 to 4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, quaternary nitrogen, oxidized nitrogen (e.g., NO), oxygen, and sulfur, including sulfoxide and sulfone (“3-12 membered heterocyclyl”). In some embodiments, a heterocyclyl group is a 3-7 membered non-aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“3-7 membered heterocyclyl”). In heterocyclyl groups that contain one or more nitrogen atoms, the point of attachment can be a carbon or nitrogen atom, as valency permits. A heterocyclyl group can either be monocyclic (“monocyclic heterocyclyl”) or polycyclic (e.g., a bicyclic system (“bicyclic heterocyclyl”) or tricyclic system (“tricyclic heterocyclyl”); polycyclic ring systems include fused, bridged, or spiro ring systems). Exemplary monocyclic heterocyclyl groups include azetidinyl, oxetanyl, thietanyl, tetrahydrofuranyl, pyrrolidinyl, piperidinyl, tetrahydropyranyl, piperazinyl, morpholinyl, azepanyl, oxepanyl, thiepanyl, tetrahydropyridinyl, and the like. Heterocyclyl polycyclic ring systems can include heteroatoms in one or more rings in the Attorney Docket No.: 131734-01120 polycyclic ring system. Substituents may be present on one or more rings in the polycyclic ring system. Spiro heterocyclyl refers to 5 to 12 membered polycyclic heterocyclyl with rings connected through one common carbon atom (called as spiro atom), wherein said rings have one or more heteroatoms selected from the group consisting of nitrogen, quaternary nitrogen, oxidized nitrogen (e.g., NO), oxygen, and sulfur, including sulfoxide and sulfone, the remaining ring atoms being C, wherein one or more rings may contain one or more double bonds, but none of the rings has a completely conjugated pi-electron system. Representative examples of spiro heterocyclyl include, but are not limited to the following groups: . Fused heterocyclyl refers to a 5 to 12 membered polycyclic heterocyclyl group, wherein each ring in the group shares an adjacent pair of carbon atoms with another ring in the group, wherein one or more rings can contain one or more double bonds, but none of the rings has a completely conjugated π-electron system, and wherein said rings have one or more heteroatoms selected from the group consisting of nitrogen, quaternary nitrogen, oxidized nitrogen (e.g., NO), oxygen, and sulfur, including sulfoxide and sulfone, the remaining ring atoms being C. Representative examples of fused heterocyclyl include, but are not limited to the following groups: . Bridged heterocyclyl refers to a 5 to 12 membered polycyclic heterocyclyl group, wherein any two rings in the group share two disconnected atoms, the rings can have one or more double bonds but have no completely conjugated π-electron system, and the rings have one or more heteroatoms selected from the group consisting of nitrogen, quaternary nitrogen, oxidized nitrogen (e.g., NO), oxygen, and sulfur, including sulfoxide and sulfone as ring atoms, the remaining ring atoms being C. Representative examples of bridged heterocyclyl include, but are not limited to the following groups: Attorney Docket No.: 131734-01120 . Generally, the carbocyclyl, the cycloalkyl, or the heterocyclyl may be unsubstituted, or be substituted with one or more substituents as valency allows, wherein the substituents can be independently selected from a number of groups. Exemplary substituents include but are not limited to, oxo, -CN, halogen, alkyl and alkoxyl, optionally, the alkyl substitution may be further substituted. The term “aryl” refers to a 6 to 10 membered all-carbon monocyclic ring or a polycyclic fused ring (a “fused” ring system means that each ring in the system shares an adjacent pair of carbon atoms with other ring in the system) group, and has a completely conjugated π-electron system. The term “aryl” may be used interchangeably with the terms “aryl ring” “carbocyclic aromatic ring”, “aryl group” and “carbocyclic aromatic group”. Representative examples of aryl are phenyl and naphthyl. The term “heteroaryl,” as used herein, refers to a monocyclic or multicyclic (e.g., bicyclic) aromatic hydrocarbon in which at least one of the ring carbon atoms has been replaced with a heteroatom independently selected from oxygen, nitrogen and sulfur. Preferably, the heteroaryl is based on a C5-10 aryl with one or more of its ring carbon atoms replaced by the heteroatom. A heteroaryl group may be attached through a ring carbon atom or, where valency permits, through a ring nitrogen atom. Generally, the heteroaryl may be unsubstituted, or be substituted with one or more substituents as valency allows. Exemplary substituents include, but are not limited to, halogen, OH, alkyl, alkoxyl, and amino (e.g., NH₂, NHalkyl, N(alkyl)₂), optionally, the alkyl may be further substituted. Examples of monocyclic 5-6 membered heteroaryl groups include furanyl (e.g., 2- furanyl, 3-furanyl), imidazolyl (e.g., N-imidazolyl, 2-imidazolyl, 4-imidazolyl, 5-imidazolyl), isoxazolyl ( e.g., 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl), oxadiazolyl (e.g., 2-oxadiazolyl, 5-oxadiazolyl), oxazolyl (e.g., 2-oxazolyl, 4-oxazolyl, 5-oxazolyl), pyrazolyl (e.g., 3- pyrazolyl, 4-pyrazolyl), pyrrolyl (e.g., 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl), pyridyl (e.g., 2- pyridyl, 3-pyridyl, 4-pyridyl), pyrimidinyl (e.g., 2-pyrimidinyl, 4-pyrimidinyl, 5- pyrimidinyl), pyridazinyl (e.g., 3-pyridazinyl), thiazolyl (e.g., 2-thiazolyl, 4-thiazolyl, 5- thiazolyl), triazolyl (e.g., 2-triazolyl, 5-triazolyl), tetrazolyl (e.g., tetrazolyl), thienyl (e.g., 2- thienyl, 3-thienyl), pyrimidinyl, pyridinyl and pyridazinyl. Examples of polycyclic aromatic heteroaryl groups include carbazolyl, benzimidazolyl, benzothienyl, benzofuranyl, indolyl, Attorney Docket No.: 131734-01120 quinolinyl, benzotriazolyl, benzothiazolyl, benzoxazolyl, benzimidazolyl, isoquinolinyl, indolyl, isoindolyl, acridinyl, or benzisoxazolyl. A “substituted heteroaryl group” is substituted at any one or more substitutable ring atom, which is a ring carbon or ring nitrogen atom bonded to a hydrogen. As used herein, many moieties (e.g., alkyl, alkylene, cycloalkyl, aryl, heteroaryl, or heterocyclyl ) are referred to as being either “substituted” or “optionally substituted”. When a moiety is modified by one of these terms, unless otherwise noted, it denotes that any portion of the moiety that is known to one skilled in the art as being available for substitution can be substituted, which includes one or more substituents. Where if more than one substituent is present, then each substituent may be independently selected. Such means for substitution are well-known in the art and/or taught by the instant disclosure. The optional substituents can be any substituents that are suitable to attach to the moiety. Where suitable substituents are not specifically enumerated, exemplary substituents include, but are not limited to: C1-5alkyl, C1-5hydroxyalkyl, C1-5haloalkyl, C1-5alkoxy, C1-5 haloalkoxy, halogen, hydroxyl, cyano, amino, -CN, -NO2, -OR^c1, -NR^a1R^b1, -S(O)iR^a1, -NR^a1S(O)_iR^b1, -S(O)_iNR^a1R^b1, -C(=O)OR^a1, -OC(=O)OR^a1, -C(=S)OR^a1, -O(C=S)R^a1, -C(=O)NR^a1R^b1, -NR^a1C(=O)R^b1, -C(=S)NR^a1R^b1, -C(=O)R^a1, -C(=S)R^a1, NR^a1C(=S)R^b1, -O(C=O)NR^a1R^b1, -NR^a1(C=S)OR^b1, -O(C=S)NR^a1R^b1, -NR^a1(C=O)NR^a1R^b1, -NR^a1(C=S)NR^a1R^b1, phenyl, or 5-6 membered heteroaryl. Each R^a1 and each R^b1 are independently selected from –H and C_1-5alkyl, optionally substituted with hydroxyl or C1-3alkoxy; R^c1 is –H, C1-5haloalkyl or C1-5alkyl, wherein the C1-5alkyl is optionally substituted with hydroxyl or C1-C3alkoxy. The term "biomolecule," as used herein, refers to a molecule that is capable of binding to a biological target such as an antigen. A biomolecule, according to various embodiments of the disclosure, includes but is not limited to, an antibody, a peptibody, a fusion protein, an ASO, a gene therapy agent, a nanoparticle, a mutein (i.e., mutant protein), a multispecific protein, a bispecific protein, as well as biologically active fragments, analogs, derivatives and variants, as well as biosimilars, thereof. The symbol ,” as used herein, refers to the point where the moiety attaches. The term “nucleoside,” as used herein, refers to a molecule composed of a nucleobase and a pentose sugar moiety like ribose, desoxyribose or a modified or locked ribose or a modified or locked desoxyribose like the LNAs. A nucleobase is linked to the glycosidic carbon atom (position 1’ of the pentose) and an internucleotide linkage (e.g. a phosphate) is Attorney Docket No.: 131734-01120 formed between the 3’ oxygen or sulfur atom and preferably the 3’ oxygen atom of a nucleoside and the 5’ oxygen or sulfur atom and preferably the 5’ oxygen atom of the adjacent nucleoside, while the internucleotide linkage does not belong to the nucleoside. The term “nucleotide,” as used herein, refers to a molecule composed of a nucleobase, a pentose sugar moiety like ribose, desoxyribose or a modified or locked ribose or a modified or locked desoxyribose like the LNAs, and an internucleotide linkage, such as a phosphate. A nucleobase is linked to the glycosidic carbon atom (position 1’ of the pentose) and an internucleotide linkage is formed between the 3’ oxygen or sulfur atom and preferably the 3’ oxygen atom of a nucleotide and the 5’ oxygen or sulfur atom and preferably the 5’ oxygen atom of the adjacent nucleotide, while the internucleotide linkage is a part of the nucleotide. The term “DNA nucleotide” or “2-deoxyribonucleotide,” as used herein, encompasses a DNA monomer comprising a 2-deoxyribose unit which is bonded through its number one carbon to a nitrogenous base selected from the group consisting of A, C, T and G, and which is bonded through its number five carbon atom to a phosphate group or to a terminal group. The term “DNA nucleotide” used herein refers to a DNA nucleotide that exists in nature or a DNA nucleotide with a modified base, sugar, or phosphate binding subunit. The term “RNA nucleotide” or “ribonucleotide,” as used herein, encompasses a RNA monomer comprising a ribose unit which is bonded through its number one carbon to a nitrogenous base selected from the group consisting of A, C, G and U, and which is bonded through its number five carbon atom to a phosphate group or to a terminal group. The term “RNA nucleotide” used herein refers to an RNA nucleotide that exists in nature or an RNA nucleotide with a modified base, sugar, or phosphate binding subunit. The term “nucleobase” is herein abbreviated with “B” and refers to the five standard nucleotide bases adenine (A), thymine (T), guanine (G), cytosine (C), and uracil (U) as well as to modifications or analogues thereof or analogues with ability to form Watson-Crick base pair with bases in the complimentary strand. Modified nucleobases include other synthetic and natural nucleobases such as 5-methylcytosine (C*), 5-hydroxymethyl cytosine, N⁴- methylcytosine, xanthine, hypoxanthine, 7-deazaxanthine, 2-aminoadenine, 6-methyladenine, 6-methylguanine, 6-ethyladenine, 6-ethylguanine, 2-propyladenine, 2-propylguanine, 6- carboxyuracil, 5-halouracil, 5,6-dihydrouracil, 5-halocytosine, 5-propynyl uracil, 5-propynyl cytosine, 6-aza uracil, 6-aza cytosine, 6-aza thymine, 5-uracil (pseudouracil), 4-thiouracil, 8- fluoroadenine, 8-chloroadenine, 8-bromoadenine, 8-iodoadenine, 8-aminoadenine, 8- Attorney Docket No.: 131734-01120 thioladenine, 8-thioalkyladenine, 8-hydroxyladenine, 8-fluoroguanine, 8-chloroguanine, 8- bromoguanine, 8-iodoguanine, 8-aminoguanine, 8-thiolguanine, 8-thioalkylguanine, 8- hydroxylguanine, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, 5- trifluoromethyluracil, 5-fluorocytosine, 5-bromocytosine, 5-chlorocytosine, 5-iodocytosine, 5-trifluoromethylcytosine, 7-methylguanine, 7-methyladenine, 8-azaguanine, 8-azaadenine, 7-deazaguanine, 7-deazaadenine, 7-deaza-8-azaadenine, 3-deazaguanine, 3-deazaadenine, 2- thiouracil, 2-thiothymine and 2-thiocytosine etc., with 5-methylcytosine and/or 2- aminoadenine substitutions being preferred since these modifications have been shown to increase nucleic acid duplex stability. Preferred antisense-oligonucleotides of the present disclosure can comprise analogues of nucleobases. The nucleobase of only one nucleotide unit of the antisense-oligonucleotide could be replaced by an analogue of a nucleobase or two, three, four, five or even all nucleobases in an antisense-oligonucleotide could be replaced by analogues of nucleobases. It will be recognized that when referring to a sequence of nucleotides or monomers, it means the sequence of bases, such as A, T, G, C or U. The representation of the antisense- oligonucleotides by the letter code A, T, G, C and U is to be understood that said antisense- oligonucleotide may contain any the nucleobases as disclosed herein, any of the 3’ end groups as disclosed herein, any of the 5’ end groups as disclosed herein, and any of the internucleotide linkages (also referred to as internucleotide bridges) as disclosed herein. The nucleotides A, T, G, C and U can also be understood to include LNA nucleotides or non- LNA nucleotides. In some embodiments, the nucleotides are DNA nucleotides. Pharmaceutically Acceptable Salts The term “pharmaceutically acceptable salt” refers to a pharmaceutical salt that is, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, and allergic response, and is commensurate with a reasonable benefit/risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, S. M. Berge et al. describes pharmacologically acceptable salts in J. Pharm. Sci., 1977, 66, 1–19. Pharmaceutically acceptable salts of the compounds of any one of the formulae described above include acid addition and base salts. Included in the present teachings are pharmaceutically acceptable salts of the compounds disclosed herein. Compounds having basic groups can form pharmaceutically acceptable salts with pharmaceutically acceptable acid(s). Suitable pharmaceutically acceptable acid addition salts of the compounds described herein include salts of inorganic Attorney Docket No.: 131734-01120 acids (such as hydrochloric, hydrobromic, phosphoric, metaphosphoric, nitric, and sulfuric acids) and of organic acids (such as acetic, benzenesulfonic, benzoic, ethanesulfonic, methanesulfonic, and succinic acids). Compounds of the present teachings with acidic groups such as carboxylic acids can form pharmaceutically acceptable salts with pharmaceutically acceptable base(s). Suitable pharmaceutically acceptable basic salts include ammonium salts, alkali metal salts (such as sodium and potassium salts) and alkaline earth metal salts (such as magnesium and calcium salts). Pharmaceutically acceptable salts of compounds of any one of the formulae described above may be prepared by one or more of three methods: (i) by reacting the compound of any one of the formulae described above with the desired acid or base; (ii) by removing an acid- or base-labile protecting group from a suitable precursor of the compound of any one of the formulae described above or by ring-opening a suitable cyclic precursor, for example, a lactone or lactam, using the desired acid or base; or (iii) by converting one salt of the compound of any one of the formulae described above to another by reaction with an appropriate acid or base or by means of a suitable ion exchange column. All three reactions are typically carried out in solution. The resulting salt may precipitate out and be collected by filtration or may be recovered by evaporation of the solvent. The degree of ionisation in the resulting salt may vary from completely ionised to almost non-ionised. The compounds of any one of the formulae described above, and pharmaceutically acceptable salts thereof, may exist in unsolvated and solvated forms. Stereoisomers and Other Variations The compounds of any one of the formulae described above may exhibit one or more kinds of isomerism (e.g. optical, geometric or tautomeric isomerism). Such variation is implicit to the compounds of any one of the formulae described above defined as they are by reference to their structural features and therefore within the scope of the present disclosure. Compounds having one or more chiral centers can exist in various stereoisomeric forms, i.e., each chiral center can have an R or S configuration, or can be a mixture of both. Stereoisomers are compounds that differ only in their spatial arrangement. Stereoisomers include all diastereomeric and enantiomeric forms of a compound. Enantiomers are stereoisomers that are mirror images of each other. Diastereomers are stereoisomers having Attorney Docket No.: 131734-01120 two or more chiral centers that are not identifcal and are not mirror images of each other. When a compound is designated by its chemical name (e.g., where the configuration is indicated in the chemical name by “R” or “S”) or its structure (e.g., the configuration is indicated by “wedge” bonds) that indicates a single enantiomer, unless indicated otherwise, the compound is at least 60%, 70%, 80%, 90%, 99% or 99.9% optically pure (also referred to as “enantiomerically pure”). Optical purity is the weight in the mixture of the named or depicted enantiomer divided by the total weight in the mixture of both enantiomers. When the stereochemistry of a disclosed compound is named or depicted by structure, and the named or depicted structure encompasses more than one stereoisomer (e.g., as in a diastereomeric pair), it is to be understood that one of the encompassed stereoisomers or any mixture of the encompassed stereoisomers is included. It is to be further understood that the stereoisomeric purity of the named or depicted stereoisomers at least 60%, 70%, 80%, 90%, 99% or 99.9% by weight. The stereoisomeric purity in this case is determined by dividing the total weight in the mixture of the stereoisomers encompassed by the name or structure by the total weight in the mixture of all of the stereoisomers. When two stereoisomers are depicted by their chemical names or structures, and the chemical names or structures are connected by an “and”, a mixture of the two stereoisomers is intended. When two stereoisomers are depicted by their chemical names or structures, and the names or structures are connected by an “or”, one or the other of the two stereoisomers is intended, but not both. When a disclosed compound having a chiral center is depicted by a structure without showing a configuration at that chiral center, the structure is meant to encompass the compound with the S configuration at that chiral center, the compound with the R configuration at that chiral center, or the compound with a mixture of the R and S configuration at that chiral center. When a disclosed compound having a chiral center is depicted by its chemical name without indicating a configuration at that chiral center with “S” or “R”, the name is meant to encompass the compound with the S configuration at that chiral center, the compound with the R configuration at that chiral center or the compound with a mixture of the R and S configuration at that chiral center. Racemic mixture means 50% of one enantiomer and 50% of the corresponding enantiomer. When a compound with one chiral center is named or depicted without indicating the stereochemistry of the chiral center, it is understood that the name or structure encompasses both possible enantiomeric forms (e.g., both enantiomerically-pure, Attorney Docket No.: 131734-01120 enantiomerically-enriched or racemic) of the compound. When a compound with two or more chiral centers is named or depicted without indicating the stereochemistry of the chiral centers, it is understood that the name or structure encompasses all possible diasteriomeric forms (e.g., diastereomerically pure, diastereomerically enriched and equimolar mixtures of one or more diastereomers (e.g., racemic mixtures) of the compound. The term “geometric isomer” means isomers that differ in the orientation of substituent atoms in relationship to a carbon-carbon double bond, to a carbocyclic ring, or to a bridged bicyclic system. Substituent atoms (other than hydrogen) on each side of a carbon- carbon double bond may be in an E or Z configuration according to the Cahn-Ingold-Prelog priority rules. In the “E” configuration, the substituents having the highest priorities are on opposite sides in relationship to the carbon-carbon double bond. In the “Z” configuration, the substituents having the highest priorities are oriented on the same side in relationship to the carbon-carbon double bond. Substituents around a carbon-carbon double bond can also be referred to as “cis” or “trans,” where “cis” represents substituents on the same side of the double bond and “trans” represents substituents on opposite sides of the double bond. For example, trans-cyclooctene is represented by the following structure: , while the cis-cyclooctene is represented by the following structure: . The arrangement of substituents around a carbocyclic ring can also be designated as “cis” or “trans.” The term “cis” represents substituents on the same side of the plane of the ring, and the term “trans” represents substituents on opposite sides of the plane of the ring. Mixtures of compounds wherein the substituents are disposed on both the same and opposite sides of plane of the ring are designated “cis/trans.” Where structural isomers are interconvertible via a low energy barrier, tautomeric isomerism (“tautomerism”) can occur. This can take the form of proton tautomerism in compounds of any one of the formulae described above containing, for example, an imino, keto, or oxime group, or so-called valence tautomerism in compounds which contain an aromatic moiety. It follows that a single compound may exhibit more than one type of isomerism. Attorney Docket No.: 131734-01120 In certain instances tautomeric forms of the disclosed compounds exist, such as the tautomeric structures shown below: When a geometric isomer is depicted by name or structure, it is to be understood that the named or depicted isomer exists to a greater degree than another isomer, that is that the geometric isomeric purity of the named or depicted geometric isomer is greater than 50%, such as at least 60%, 70%, 80%, 90%, 99%, or 99.9% pure by weight. Geometric isomeric purity is determined by dividing the weight of the named or depicted geometric isomer in the mixture by the total weight of all of the geomeric isomers in the mixture. Cis/trans isomers may be separated by conventional techniques well known to those skilled in the art, for example, chromatography and fractional crystallization. Conventional techniques for the preparation/isolation of individual enantiomers/ diastereomers include chiral synthesis from a suitable optically pure precursor or resolution of the racemate (or the racemate of a salt or derivative) using, for example, chiral high pressure liquid chromatography (HPLC). Alternatively, the racemate (or a racemic precursor) may be reacted with a suitable optically active compound, for example, an alcohol, or, in the case where the compound of any one of the formulae described above contains an acidic or basic moiety, a base or acid such as 1-phenylethylamine or tartaric acid. The resulting diastereomeric mixture may be separated by chromatography and/or fractional crystallization and one or both of the diastereoisomers converted to the corresponding pure enantiomer(s) by means well known to a skilled person. Chiral compounds of any one of the formulae described above (and chiral precursors thereof) may be obtained in enantiomerically-enriched form using chromatography, typically HPLC, on an asymmetric resin with a mobile phase consisting of a hydrocarbon, typically heptane or hexane, containing from 0 to 50% by volume of isopropanol, typically from 2% to 20%, and from 0 to 5% by volume of an alkylamine, typically 0.1% diethylamine. Concentration of the eluate affords the enriched mixture. Chiral chromatography using sub-and supercritical fluids may be employed. Methods for chiral chromatography useful in some embodiments of the present disclosure are known in the art (see, for example, Smith, Roger M., Loughborough University, Loughborough, UK; Chromatographic Science Series (1998), 75 (Supercritical Fluid Chromatography with Packed Columns), pp.223-249 and references cited therein). Columns Attorney Docket No.: 131734-01120 can be obtained from Chiral Technologies, Inc, West Chester, Pa., USA, a subsidiary of Daicel^® Chemical Industries, Ltd., Tokyo, Japan. It must be emphasized that the compounds of any one of the formulae described above have been drawn herein in a single tautomeric form, all possible tautomeric forms are included within the scope of the present disclosure. 4. Administration and Dosing Typically, a compound of the present disclosure can be administered as compound per se, or alternatively, as a pharmaceutically acceptable salt. For administration and dosing purposes, the compound per se or pharmaceutically acceptable salt thereof will simply be referred to as the compounds of the present disclosure. The compounds of the present disclosure are administered by any suitable route in the form of a pharmaceutical composition adapted to such a route, and in a dose effective for the treatment intended. The compounds of the present disclosure may be administered orally, rectally, vaginally, or parenterally. The compounds of the present disclosure may be administered orally. Oral administration may involve swallowing, so that the compound enters the gastrointestinal tract, or buccal or sublingual administration may be employed by which the compound enters the bloodstream directly from the mouth. In another embodiment, the compounds of the present disclosure may also be administered directly into the bloodstream, into muscle, or into an internal organ. Suitable means for parenteral administration include intravenous, intra-arterial, intraperitoneal, intrathecal, intraventricular, intraurethral, intrasternal, intracranial, intramuscular and subcutaneous. Suitable devices for parenteral administration include needle (including microneedle) injectors, needle-free injectors and infusion techniques.The dosage regimen for the compounds of the present disclosure and/or compositions containing said compounds is based on a variety of factors, including the type, age, weight, sex and medical condition of the patient; the severity of the condition; the route of administration; and the activity of the particular compound employed. Thus the dosage regimen may vary widely. In one embodiment, the total daily dose of a compound of the present disclosure is typically from about 0.001 to about 100 mg/kg (i.e., mg compound of the present disclosure per kg body weight) for the treatment of the indicated conditions discussed herein. Suitable subjects according to the present disclosure include mammalian subjects, including non-human mammal such as primates, rodents (mice, rats, hamsters, rabbits etc). Attorney Docket No.: 131734-01120 In one embodiment, humans are suitable subjects. Human subjects may be of either gender and at any stage of development. 5. Pharmaceutical Compositions In another embodiment, the present disclosure comprises pharmaceutical compositions. Such pharmaceutical compositions comprise a compound of the present disclosure presented, a pharmaceutically acceptable salt, or a stereoisomer thereof with a pharmaceutically acceptable carrier or excipient. Other pharmacologically active substances can also be present. As used herein, “pharmaceutically acceptable carrier or excipient” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Examples of pharmaceutically acceptable carriers include one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol and the like, as well as combinations thereof, and may include isotonic agents, for example, sugars, sodium chloride, or polyalcohols such as mannitol, or sorbitol in the composition. Pharmaceutically acceptable substances such as wetting agents or minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives or buffers, which enhance the shelf life or effectiveness of the antibody or antibody portion. The compositions of present disclosure may be in a variety of forms. These include, for example, liquid, semi-solid and solid dosage forms, such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, tablets, pills, powders, liposomes and suppositories. The form depends on the intended mode of administration and therapeutic application. In another embodiment, the present disclosure comprises a parenteral dose form. “Parenteral administration” includes, for example, subcutaneous injections, intravenous injections, intraperitoneally, intramuscular injections, intrasternal injections, and infusion. Injectable preparations (i.e., sterile injectable aqueous or oleaginous suspensions) may be formulated according to the known art using suitable dispersing, wetting agents, and/or suspending agents. Typical compositions are in the form of injectable or infusible solutions, such as compositions similar to those used for passive immunization of humans with antibodies in general. One mode of administration is parenteral (e.g. intravenous, subcutaneous, intraperitoneal, intramuscular). In another embodiment, the antibody is administered by Attorney Docket No.: 131734-01120 intravenous infusion or injection. In yet another embodiment, the antibody is administered by intramuscular or subcutaneous injection. Oral administration of a solid dose form may be, for example, presented in discrete units, such as hard or soft capsules, pills, cachets, lozenges, or tablets, each containing a predetermined amount of at least one compound of the present disclosure. In another embodiment, the oral administration may be in a powder or granule form. In another embodiment, the oral dose form is sub-lingual, such as, for example, a lozenge. In such solid dosage forms, the compounds of any one of the formulae described above are ordinarily combined with one or more adjuvants. Such capsules or tablets may contain a controlled release formulation. In the case of capsules, tablets, and pills, the dosage forms also may comprise buffering agents or may be prepared with enteric coatings. In another embodiment, oral administration may be in a liquid dose form. Liquid dosage forms for oral administration include, for example, pharmaceutically acceptable emulsions, solutions, suspensions, syrups, and elixirs containing inert diluents commonly used in the art (e.g., water). Such compositions also may comprise adjuvants, such as wetting, emulsifying, suspending, flavoring (e.g., sweetening), and/or perfuming agents. The present disclosure also provides pharmaceutical compositions comprising a radiolabeled cyclooctene compound described herein. In certain instances, such pharmaceutical compositions comprise or consist of a sterile saline solution and a radiolabeled cyclooctene compound described herein. In some cases, such pharmaceutical compositions are sterile, buffered, isotonic solutions. In some cases, the pharmaceutical compositions are preservative-free. The radiolabeled cyclooctene compounds described herein may be admixed with pharmaceutically acceptable active and/or inert substances for the preparation of pharmaceutical compositions or formulations. Compositions and methods for the formulation of pharmaceutical compositions depend on a number of criteria, including, but not limited to, route of administration, extent of disease, or dose to be administered. In some instances, the radiolabeled cyclooctene compounds described herein are formulated for intravenous administration. In some instances, the radiolabeled cyclooctene compounds described herein are formulated for intrathecal administration. In certain cases, the radiolabeled cyclooctene compounds described herein are formulated in phosphate buffered saline (PBS). In other cases, the radiolabeled cyclooctene compounds described herein are formulated in artificial cerebrospinal fluid (a-CSF). In yet other cases, the Attorney Docket No.: 131734-01120 radiolabeled cyclooctene compounds described herein are formulated in sterile water for injection. For intranasal administration or administration by inhalation, the compounds of the present disclosure are conveniently delivered in the form of a solution or suspension from a pump spray container that is squeezed or pumped by the patient or as an aerosol spray presentation from a pressurized container or a nebulizer, with the use of a suitable propellant. Formulations suitable for intranasal administration are typically administered in the form of a dry powder (either alone, as a mixture, for example, in a dry blend with lactose, or as a mixed component particle, for example, mixed with phospholipids, such as phosphatidylcholine) from a dry powder inhaler or as an aerosol spray from a pressurized container, pump, spray, atomizer (preferably an atomizer using electrohydrodynamics to produce a fine mist), or nebulizer, with or without the use of a suitable propellant, such as 1,1,1,2-tetrafluoroethane or 1,1,1,2,3,3,3-heptafluoropropane. For intranasal use, the powder may comprise a bioadhesive agent, for example, chitosan or cyclodextrin. In another embodiment, the present disclosure comprises a rectal dose form. Such rectal dose form may be in the form of, for example, a suppository. Cocoa butter is a traditional suppository base, but various alternatives may be used as appropriate. Other carrier materials and modes of administration known in the pharmaceutical art may also be used. Pharmaceutical compositions of the present disclosure may be prepared by any of the well-known techniques of pharmacy, such as effective formulation and administration procedures. The above considerations in regard to effective formulations and administration procedures are well known in the art and are described in standard textbooks. Formulation of drugs is discussed in, for example, Hoover, John E., Remington’s Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1975; Liberman et al., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, N.Y., 1980; and Kibbe et al., Eds., Handbook of Pharmaceutical Excipients (3^rd Ed.), American Pharmaceutical Association, Washington, 1999. 6. Methods of Evaluating Biomolecule Distribution This disclosure features a method of evaluating the distribution of a biomolecule (e.g., antibodies, nanoparticles, gene therapy agents, ASO) in a subject (e.g., a human). In some cases, the distribution of the biomolecule (e.g., antibodies, nanoparticles, gene therapy agents, ASO) is assessed in the brain and/or spinal cord of the subject. The method involves Attorney Docket No.: 131734-01120 administering to the subject a modified biomolecule (e.g., antibodies, nanoparticles, gene therapy agents, ASO). In some embodiments, a modified biomolecule (e.g., antibodies, nanoparticles, gene therapy agents, ASO) is the biomolecule (e.g., antibodies, nanoparticles, gene therapy agents, ASO) linked with a methyltetrazine. In some embodiments, the modified biomolecule (e.g., antibodies, nanoparticles, gene therapy agents, ASO) is administered intravenously. In some embodiments, particularly where the distribution of the biomolecule (e.g., antibodies, nanoparticles, gene therapy agents, ASO) is assessed in the brain and/or spinal cord, the modified biomolecule (e.g., antibodies, nanoparticles, gene therapy agents, ASO) is administered intrathecally. In some embodiments, the modified biomolecule (e.g., antibodies, nanoparticles, gene therapy agents, ASO) is formulated in PBS or a-CSF. The method further involves administering a radiolabeled cyclooctene compound described herein to the subject. In some embodiments, the cyclooctene compound is radiolabeled with any radionuclide or radioisotope for diagnostic imaging (as described herein) known in the art. In some embodiments, the cyclooctene compound is radiolabeled with a radionuclide that decays exclusively or almost exclusively through positron emission. In some embodiments, the cyclooctene compound is radiolabeled with a radionuclide that has a short half-life (less than 12 hours) or a moderate half-life (about 12 to 18 hours). In some embodiments, the cyclooctene compound is radiolabeled with a fluorine-18, carbon-11, or gallium-68. In some embodiments, the cyclooctene compound is radiolabeled with fluorine- 18. In some instances, the cyclooctene compound is a compound described, e.g., a compound of Formula (I), (IIA), (IIB), (II), (III), or (IV), or a compound selected from Compounds 1- 20. In some embodiments, the radionuclide or radioisotope is covalently bonded to the radiolabeled compound/radioligand. In some embodiments, the radionuclide or radioisotope is bound to the radiolabeled compound/radioligand via a chelating moiety. The chelating moiety may be any suitable chelator known in the art (e.g., NOTA). In some embodiments, the radiolabeled cyclooctene compound is administered intravenously. In some embodiments, the radiolabeled cyclooctene compound is formulated in PBS or a-CSF. The timing of when the radiolabeled cyclooctene compound is administered depends on the half-life of the biomolecule (e.g., antibodies, nanoparticles, gene therapy agents, ASO). In some embodiments, the radiolabeled cyclooctene compound is administered to the subject within 24 hours, up to and including one day, up to and including two days, up to and including three days, up to and including four days, up to and including five days, up to and including six days, up to and including seven days, up to and including eight days, up to and including nine days, up to and including ten days, up to and including eleven days, up to and Attorney Docket No.: 131734-01120 including twelve days, up to and including thirteen days, up to and including fourteen days, up to and including fifteen days, up to and including sixteen days, up to and including seventeen days, up to and including eighteen days, up to and including nineteen days, or up to and including twenty days after administration of the modified biomolecule (e.g., antibodies, nanoparticles, gene therapy agents, ASO). In certain instances, the radiolabeled cyclooctene compound is administered between about one and about two days, between about one and about three days, between about one and about four days, between about one and about five days, between about one and about six days, between about one and about seven days, between about one and about ten days, between about one and about fourteen days, between about one and about twenty days, between about one and about twenty four days, or between about one and about thirty two days after administration of the modified biomolecule (e.g., antibodies, nanoparticles, gene therapy agents, ASO). In one instance, the radiolabeled cyclooctene compound is administered to the subject about 24 hours after the administration of the modified biomolecule (e.g., antibodies, nanoparticles, gene therapy agents, ASO). In some instances, the radiolabeled cyclooctene compound is administered to the subject at or about 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 hours after the administration of the modified biomolecule (e.g., antibodies, nanoparticles, gene therapy agents, ASO). In some instances, an additional step is added before the injection of the radioligand, specifically, the administration of a clearing agent designed to accelerate the removal of residual the biomolecule (i.e., any targeting agent that is not bound to a target) from the bloodstream. In certain instances, after the administration of the radiolabeled cyclooctene compound, the subject is imaged. In certain instances, after the administration of the radiolabeled cyclooctene compound, the subject is imaged based on the PK of the radiolabeled cyclooctene compound. In some cases, the imaging is done about 15 minutes, about 30 minutes, about 45 minutes, about 60 minutes, about 75 minutes, about 90 minutes, about 105 minutes, about 120 minutes, about 135 minutes, or about 150 minutes after injection of the radiotracer. In some cases, the imaging is done about half-an-hour to about 1 hour, about 1 hour to about one and a half hours, about two hours, about three hours, about four hours, or about five hours after injection of the radiotracer. In certain cases, imaging is conducted by any suitable diagnostic imaging method known in the art including but not limited to Positron Emission Tomography (PET), Positron emission tomography–computed tomography (PET-CT), Single Photon Emission Computed Tomography (SPECT), Single- Attorney Docket No.: 131734-01120 photon emission computed tomography (SPECT-CT), Planar gamma camera, X-ray CT, planar X-ray, Magnetic Resonance Imaging (MRI), optical imager, or other diagnostic imaging technique. In certain instances, the subject includes any human or non-human mammal. In certain non-limiting embodiments, the subject is a non-human primate, sheep, a dog, a cat, a rabbit, a horse, a cow, or a rodent. In certain instances, the subject is a human subject. In certain cases, the human subject is a pediatric patient. In certain cases, the human subject is an infant. In certain instances, the human subject is an adult patient (i.e., 18 years or older). In some cases, the human subject has a CNS disorder. In certain cases, the CNS disorder is a synucleinopathy or a tauopathy. In some cases, the CNS disorder is spinal muscular atrophy (SMA), amyotrophic lateral sclerosis (ALS), Parkinson’s disease, Alzheimer’s disease, Huntington’s disease, Angelman syndrome, frontotemporal dementia (FTD), Creutzfeldt-Jakob disease, spinocerebellar ataxia type 3 (SCA3), or Menkes disease. In some instances, the distribution of the biomolecule (e.g., antibodies, nanoparticles, gene therapy agents, ASO) is evaluated in the CNS (e.g., cortex, striatum, thalamus, substantia nigra, cerebellum) of the human subject. 7. Methods of Assessing Biomolecule Concentration Also featured are methods for determining the concentration of a biomolecule (e.g., antibodies, nanoparticles, gene therapy agents, ASO) in a target region (e.g., the brain and/or spinal cord) of a subject (e.g., human). The method involves administering a modified biomolecule (e.g., antibodies, nanoparticles, gene therapy agents, ASO). In some cases, the modified biomolecule (e.g., antibodies, nanoparticles, gene therapy agents, ASO) linked to a methyltetrazine is administered intravenously. In certain cases, particularly where the distribution of the ASO is assessed in the brain and/or spinal cord, the ASO linked to a methyltetrazine is administered intrathecally. In certain cases, the ASO linked to a methyltetrazine is formulated in PBS or a-CSF. Following this administration, the subject is administered a radiolabeled cyclooctene compound described herein. In some instances, the radiolabeled cyclooctene compound is a central nervous system penetrant compound. In some instances, the cyclooctene compound is radiolabeled with a radionuclide that decays exclusively or almost exclusively through positron emission. In some instances, the cyclooctene compound is radiolabeled with a radionuclide that has a short half-life (less than Attorney Docket No.: 131734-01120 12 hours) or a moderate half-life (about 12 to 18 hours). In some instances, the cyclooctene compound is radiolabeled with a fluorine-18, carbon-11, or gallium-68. In some instances, the cyclooctene compound does not include a chelator. In some cases, the radiolabeled cyclooctene compound is administered intravenously. In certain cases, the radiolabeled cyclooctene compound is formulated in PBS or a-CSF. In some instances, an additional step is added before the injection of the radioligand, specifically, the administration of a clearing agent designed to accelerate the removal of residual targeting agent from the bloodstream. The method further involves imaging the distribution of the biomolecule in the subject and deriving a tissue concentration of the biomolecule (e.g., antibodies, nanoparticles, gene therapy agents, ASO) in the subject (e.g., brain and/or spinal cord of the subject). The timing of when the radiolabeled cyclooctene compound is administered depends on the half-life of each of the biomolecule (e.g., antibodies, nanoparticles, gene therapy agents, ASO). In some cases, the radiolabeled cyclooctene compound is administered to the subject within 24 hours, up to and including one day, up to and including two days, up to and including three days, up to and including four days, up to and including five days, up to and including six days, up to and including seven days, up to and including eight days, up to and including nine days, up to and including ten days, up to and including eleven days, up to and including twelve days, up to and including thirteen days, up to and including fourteen days, up to and including fifteen days, up to and including sixteen days, up to and including seventeen days, up to and including eighteen days, up to and including nineteen days, or up to and including twenty days after administration of the modified biomolecule (e.g., antibodies, nanoparticles, gene therapy agents, ASO). In certain embodiments, the radiolabeled cyclooctene compound is administered between about one and about two days, between about one and about three days, between about one and about four days, between about one and about five days, between about one and about six days, between about one and about seven days, between about one and about ten days, between about one and about fourteen days, between about one and about twenty days, between about one and about twenty four days, or between about one and about thirty two days after administration of the modified biomolecule (e.g., antibodies, nanoparticles, gene therapy agents, ASO). In one instance, the radiolabeled cyclooctene compound is administered to the subject about 24 hours after the administration of the ASO linked to a trans-cyclooctene. In some instances, the radiolabeled cyclooctene compound is administered to the subject at or about 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 hours after the Attorney Docket No.: 131734-01120 administration of the modified biomolecule (e.g., antibodies, nanoparticles, gene therapy agents, ASO). In some cases, the imaging is done about 15 minutes, about 30 minutes, about 45 minutes, about 60 minutes, about 75 minutes, about 90 minutes, about 105 minutes, about 120 minutes, about 135 minutes, or about 150 minutes after injection of the radiotracer. In some cases, the imaging is done about half-an-hour to about 1 hour, about 1 hour to about one and a half hours, about two hours, about three hours, about four hours, or about five hours after injection of the radiotracer. In certain cases, imaging is conducted by any suitable diagnostic imaging method known in the art including but not limited to Positron Emission Tomography (PET), Positron emission tomography–computed tomography (PET-CT), Single Photon Emission Computed Tomography (SPECT), Single-photon emission computed tomography (SPECT-CT), Planar gamma camera, X-ray CT, planar X-ray, Magnetic Resonance Imaging (MRI), optical imager, or other diagnostic imaging technique. From the imaging data, an uptake value of radiolabeled cyclooctene compound for each region-of-interest can be calculated. This value can then be applied to either an equation or a reference lookup table that has been assembled empirically to provide a corresponding concentration of the biomolecule (e.g., antibodies, nanoparticles, gene therapy agents, ASO) in the tissue. In some instances, the concentration of the biomolecule (e.g., antibodies, nanoparticles, gene therapy agents, ASO) is evaluated in the CNS (e.g., cortex, striatum, thalamus, substantia nigra, cerebellum) of the subject. In certain instances, the subject is a human subject. In certain cases, the human subject is a pediatric patient. In certain cases, the human subject is an infant. In certain cases, the human subject is an adult patient (i.e., 18 years or older). In some cases, the human subject has a CNS disorder. In certain cases, the CNS disorder is a synucleinopathy or a tauopathy. In some cases, the CNS disorder is spinal muscular atrophy (SMA), amyotrophic lateral sclerosis (ALS), Parkinson’s disease, Alzheimer’s disease, Huntington’s disease, Angelman syndrome, frontotemporal dementia (FTD), Creutzfeldt-Jakob disease, spinocerebellar ataxia type 3 (SCA3), or Menkes disease. 8. Kits The disclosure further provides kits that can be used to practice the methods disclosed herein. For example, a kit can comprise at least one targeting probe (e.g., a modified Attorney Docket No.: 131734-01120 biomolecule (e.g., antibodies, nanoparticles, gene therapy agents, ASO)) and/or at least one labeling probe (e.g., a radiolabeled cyclooctene compound described herein). In certain embodiments, a kit can optionally comprise instructions on how to use the kit for molecular imaging. In certain instances, a kit can further comprise an administration device such as a syringe and/or catheter and/or introducer sheath. The disclosure further provides kits for preparing the targeting probe and/or labeling probe. In certain instances, the kit of the present invention contains the targeting probe (in dry or liquid form) and/or the labeling probe (in dry or liquid form) for application on the biomaterial. When the probe is provided in dry form, the kit can contain the appropriate buffer or solvent to prepare a solution or composition. The following examples are provided to better illustrate the claimed invention and are not to be interpreted as limiting the scope of the invention. To the extent that specific materials are mentioned, it is merely for purposes of illustration and is not intended to limit the invention. One skilled in the art can develop equivalent means or reactants without the exercise of inventive capacity and without departing from the scope of the invention. EXAMPLES Attorney Docket No.: 131734-01120 Example 1A – Preparation of rac-((2s,3aR,9aS,E)-3a,4,5,8,9,9a- hexahydrocycloocta[d][1,3]dioxol-2-yl)methyl 8-(6-(fluoro-18F)nicotinoyl)-3,8- diazabicyclo[3.2.1]octane-3-carboxylate (¹⁸F labeled Compound 1) Step a: Preparation of rac-((2R,3aR,9aS,E)-3a,4,5,6,9,9a-hexahydrocycloocta[d][1,3]dioxol- 2-yl)methyl 8-(2,2,2-trifluoroacetyl)-3,8-diazabicyclo[3.2.1]octane-3-carboxylate To a vial containing 1-(3,8-diazabicyclo[3.2.1]octan-8-yl)-2,2,2-trifluoro-ethanone (118.48 mg, 368.88 umol, TFA) and NEt3 (311.05 mg, 3.07 mmol, 428.45 uL) in DCM (2 mL), rac- [(3aR,6E,9aS)-3a,4,5,8,9,9a-hexahydrocycloocta[d][1,3]dioxol-2-yl]methyl (2,5- dioxopyrrolidin-1-yl) carbonate (100 mg, 307.40 umol) (prepared according to the procedure described in Fairhall, Jessica M., et al. Bioorganic & Medicinal Chemistry 2021, 46, 116361, which is incorporated by reference herein in its entirety) was added and the mixture was Attorney Docket No.: 131734-01120 stirred at rt for 1 hour. The volatiles were removed under reduced pressure and the mixture was subjected to flash chromatography on silica gel (0-50% EtOAc in heptane). After pooling and concentrating the appropriate fractions, rac-[(3aR,6E,9aS)-3a,4,5,8,9,9a- hexahydrocycloocta[d][1,3]dioxol-2-yl]methyl 8-(2,2,2-trifluoroacetyl)-3,8- diazabicyclo[3.2.1]octane-3-carboxylate (55 mg, 131.45 umol, 42.76% yield) was obtained as a pale yellow oil.¹H NMR (500 MHz, METHANOL-d4) δ ppm 5.51 - 5.72 (m, 2 H) 4.88 - 4.94 (m, 1 H) 4.63 - 4.77 (m, 1 H) 4.40 - 4.57 (m, 1 H) 3.89 - 4.21 (m, 6 H) 3.05 - 3.23 (m, 2 H) 2.33 - 2.43 (m, 1 H) 2.21 - 2.30 (m, 1 H) 2.04 - 2.21 (m, 3 H) 1.46 - 2.00 (m, 7 H). Step b: Preparation of rac-((2R,3aR,9aS,E)-3a,4,5,6,9,9a-hexahydrocycloocta[d][1,3]dioxol- 2-yl)methyl 3,8-diazabicyclo[3.2.1]octane-3-carboxylate To a vial containing rac-[(3aR,6E,9aS)-3a,4,5,8,9,9a-hexahydrocycloocta[d][1,3]dioxol-2- yl]methyl 8-(2,2,2-trifluoroacetyl)-3,8-diazabicyclo[3.2.1]octane-3-carboxylate (55 mg, 131.45 umol) in MeOH (3 mL) was added NaOH (52.58 mg, 1.31 mmol) in water (1 mL), and the mixture was heated to 60 °C for 1 hour. The mixture was cooled to rt and the volatile organics were removed under reduced pressure. The mixture was diluted with water and extracted with three portions of DCM. The combined organics were passed over a plug of magnesium sulfate and concentrated to afford a colorless film that was used without further purification. ¹H NMR (500 MHz, ACETONITRILE-d3) δ ppm 5.50 - 5.70 (m, 2 H), 5.14 (t, J=4.0 Hz, 1 H), 4.84 (t, J=3.7 Hz, 1 H), 3.86 - 4.11 (m, 4 H), 3.55 - 3.70 (m, 2 H), 3.36 (br d, J=13.4 Hz, 2 H), 2.92 (br dd, J=42.7, 12.2 Hz, 2 H), 2.29 - 2.39 (m, 1 H), 2.00 - 2.26 (m, 4 H), 1.81 - 1.92 (m, 1 H), 1.45 - 1.74 (m, 7 H). Step c: Preparation of 2,3,5,6-tetrafluorophenyl 6-(fluoro-18F)nicotinate Attorney Docket No.: 131734-01120 [¹⁸F]fluoride was delivered to the synthesis module in a 2.0 mL bolus of [¹⁸O]water and trapped on a Sep-Pak QMA Carbonate Plus Light (46 mg, Waters). A tetrabutylammonium hydrogen carbonate solution (TBAHCO₃, 0.5 mL, 0.075 M; diluted with 0.5 mL MeCN) was used to elute the activity off the cartridge into reaction vial-1. The solution was azeotropically dried with two consecutive additions of 1 mL of acetonitrile, at 120 °C under N2 flow and vacuum. After cooling the reactor to 40 °C, the FPyTFP precursor (10 mg), dissolved in 1 mL of t-Butanol and anhydrous Acetonitrile (8/2, v/v), was added to the dried [¹⁸F]fluoride. The labelling reaction was performed at 70 °C for 10 minutes. The reactor was cooled to room temperature and the crude reaction mixture was transferred to a dilution bottle containing 25 mL of water, followed by 3 mL water used to rinse the reactor. The diluted solution was loaded onto an Oasis® MCX cartridge SPE cartridge (previously conditioned with 10 mL MeCN followed by 10 mL water). The SPE was washed with 10 mL water and eluted into a second reaction vial using 1 mL of MeCN to afford 2,3,5,6-tetrafluorophenyl 6-(fluoro- ¹⁸F)nicotinate without further purification. Step d: Preparation of rac-((2s,3aR,9aS,E)-3a,4,5,8,9,9a-hexahydrocycloocta[d][1,3]dioxol- 2-yl)methyl 8-(6-(fluoro-18F)nicotinoyl)-3,8-diazabicyclo[3.2.1]octane-3-carboxylate A solution of rac-((2R,3aR,9aS,E)-3a,4,5,6,9,9a-hexahydrocycloocta[d][1,3]dioxol-2- yl)methyl 3,8-diazabicyclo[3.2.1]octane-3-carboxylate (10 mg in 0.5 mL MeCN) was then added to the [¹⁸F]FPyTFP solution, and the coupling reaction was performed at 40 °C for 10 min. The crude reaction mixture was diluted using 2 mL of water and injected onto the semi- preparative HPLC for purification, via a 5 mL HPLC loop (semi-preparative HPLC method: ACE C18 HPLC column (10 μm, 10 x 250 mm) eluted with H2O/(MeCN/water, 95/5 v/v) at 60/40 v/v ratio, 5.5 mL/min, RT=21min). The fraction corresponding to the title compound was collected and diluted in 30 mL of water containing 450 mg sodium ascorbate. The solution was loaded onto a classic C18 SPE cartridge (Waters) and washed with 10 mL of Attorney Docket No.: 131734-01120 water containing 5 mL.min^-1 sodium ascorbate. The title compound was eluted off the cartridge using a solution of propylene glycol (PEG) and EtOH (3 mL, 7/3 v/v) containing 50 mg of tocopherol, followed by 7 mL of PBS. Tocopherol formed a suspension upon dilution with PBS, therefore aliquots of the isolated dose were filtered prior to each injection by using an Acrodisc® sterilizing filter. The chemical and radiochemical purity of the title compound are determined by HPLC (QC HPLC method: Eclipse XDB-C18 column (5 μm, 4.6 x 150 mm) eluted with ammonium acetate 50 mM/MeCN at 65/35 v/v ratio, 1.5 mL/min). The percentage of the title compound in the solution was determined by reacting with an excess of a tetrazine (mixing 100 μL of the isolated dose with an excess of a test tetrazine (MeTz-BzI- NH2.HCl; 0.1-0.4 mg)). The percentage of the title compound in the solution was ranged from 0.4% to 14%. Example 1B – Preparation of rac-((2s,3aR,9aS,E)-3a,4,5,8,9,9a- hexahydrocycloocta[d][1,3]dioxol-2-yl)methyl 8-(6-(fluoro)nicotinoyl)-3,8- diazabicyclo[3.2.1]octane-3-carboxylate (Compound 1) To a vial containing 3,8-diazabicyclo[3.2.1]octan-8-yl-(6-fluoro-3-pyridyl)methanone (43.39 mg, 184.44 umol) and TEA (155.53 mg, 1.54 mmol, 214.22 uL) in DCM (1 mL), rac- [(3aR,6E,9aS)-3a,4,5,8,9,9a-hexahydrocycloocta[d][1,3]dioxol-2-yl]methyl (2,5- dioxopyrrolidin-1-yl) carbonate (50.00 mg, 153.70 umol) was added and the mixture was stirred at rt for 1 hour. The mixture was concentrated under reduced pressure and the crude residue was subjected to flash chromatography on silica gel (0-80% EtOAc in heptane). After pooling and concentrating the appropriate fractions, [(3aR,6E,9aS)-3a,4,5,8,9,9a- hexahydrocycloocta[d][1,3]dioxol-2-yl]methyl 8-(6-fluoropyridine-3-carbonyl)-3,8- diazabicyclo[3.2.1]octane-3-carboxylate (36.5 mg, 81.93 umol, 53.31% yield) was obtained as a colorless oil. LCMS: m/z = 446 (M+H)⁺.¹H NMR (500 MHz, DMSO-d6) δ ppm 8.42 (d, J=2.4 Hz, 1 H) 8.14 (td, J=8.1, 2.1 Hz, 1 H) 7.29 (dd, J=8.5, 2.4 Hz, 1 H) 5.52 - 5.61 (m, 2 H) 4.84 (br s, 1 H) 4.68 (br s, 1 H) 3.60 - 4.16 (m, 8 H) 3.19 (br d, J=11.6 Hz, 1 H) 3.09 (br d, J=12.2 Hz, 1 H) 2.29 (dt, J=13.6, 6.9 Hz, 1 H) 2.03 - 2.21 (m, 3 H) 1.79 - 1.95 (m, 4 H) 1.54 - 1.69 (m, 4 H) 1.46 (br s, 1 H). Attorney Docket No.: 131734-01120 Example 2 - Preparation of (meso)-[(3aR,6Z,9aS)-3a,4,5,8,9,9a- hexahydrocycloocta[d][1,3]dioxol-2-yl]methyl 8-(6-fluoropyridine-3-carbonyl)-3,8- diazabicyclo[3.2.1]octane-3-carboxylate (Compound 2) A vial containing (meso)-[(3aR,6Z,9aS)-3a,4,5,8,9,9a-hexahydrocycloocta[d][1,3]dioxol-2- yl]methyl (2,5-dioxopyrrolidin-1-yl) carbonate (32.53 mg, 100 umol), 3,8- diazabicyclo[3.2.1]octan-8-yl-(6-fluoro-3-pyridyl)methanone (38.31 mg, 110.00 umol, TFA), and TEA (101.19 mg, 1.00 mmol, 139.38 uL) in DCM (1 mL) was stirred at rt for 1 hour, then concentrated under reduced pressure. The crude mixture was taken up in DMSO, filtered, and submitted to the PSR for purification. Purification was accomplished via reversed phase HPLC on a Waters Sunfire Prep C185 um OBD 19x100mm column with a 5- 60% MeCN in water gradient (ammonium hydroxide modifier). The appropriate fractions were collected and lyophilized to give (meso)-[(3aR,6Z,9aS)-3a,4,5,8,9,9a- hexahydrocycloocta[d][1,3]dioxol-2-yl]methyl 8-(6-fluoropyridine-3-carbonyl)-3,8- diazabicyclo[3.2.1]octane-3-carboxylate (31.5 mg, 70.71 umol, 70.71% yield) as a transparent film. (10:1 mixture of syn and anti diastereomers).¹H NMR (500 MHz, DMSO- d6) δ ppm 8.42 (d, J=2.4 Hz, 1 H) 8.14 (td, J=7.9, 2.4 Hz, 1 H) 7.29 (dd, J=8.5, 2.4 Hz, 1 H) 5.54 - 5.63 (m, 2 H) 4.92 (br s, 1 H) 4.69 (br s, 1 H) 3.92 - 4.22 (m, 5 H) 3.76 (br d, J=55.5 Hz, 2 H) 3.06 - 3.25 (m, 2 H) 2.53 - 2.65 (m, 1 H) 2.35 - 2.46 (m, 2 H) 1.95 - 2.05 (m, 4 H) 1.81 - 1.92 (m, 4 H) 1.62 (br s, 2 H).¹⁹F NMR (470 MHz, DMSO-d6) δ ppm -66.15 (br s, 1 F). Example 3 - Preparation of 2-[(4Z)-cyclooct-4-en-1-yl]oxy-6-(fluoromethyl)pyridine (Compound 3) Attorney Docket No.: 131734-01120 To a vial containing (4Z)-cyclooct-4-en-1-ol (29.32 mg, 232.37 umol) and 2-fluoro-6- (fluoromethyl)pyridine (20 mg, 154.91 umol) in THF (1 mL) at 0 °C, KHMDS (1 M, 185.89 uL) was added in a dropwise manner. Each drop of KHMDS caused the solution to progressively darken. After complete addition, TLC suggested the reaction was complete. The mixture was passed through a plug of silica, eluted with DCM, and the eluent was concentrated. The residue was subjected to flash chromatography (0 to 10% EtOAc in heptane), and after pooling and concentrating the appropriate fractions, 2-[(4Z)-cyclooct-4- en-1-yl]oxy-6-(fluoromethyl)pyridine (31.9 mg, 135.57 umol, 87.52% yield) was obtained as a colorless oil. LCMS: m/z = 128 (M-cyclooctene)⁺, retention time = 1.06 minutes.1H NMR (400 MHz, CHLOROFORM-d) δ ppm 7.59 (t, J=7.8 Hz, 1 H), 6.96 (d, J=7.3 Hz, 1 H), 6.61 (d, J=8.3 Hz, 1 H), 5.64 - 5.79 (m, 2 H), 5.35 (d, J=46.9 Hz, 2 H), 5.01 - 5.11 (m, 1 H), 2.36 - 2.47 (m, 1 H), 2.00 - 2.28 (m, 4 H), 1.59 - 1.97 (m, 5 H). Example 4 - Preparation of N-[(4Z)-cyclooct-4-en-1-yl]-6-(fluoromethyl)pyridin-2-amine (Compound 4) A vial containing (4Z)-cyclooct-4-en-1-amine (29.09 mg, 185.89 umol) , 2-fluoro-6- (fluoromethyl)pyridine (20 mg, 154.91 umol) , K₂CO₃ (42.82 mg, 309.82 umol) , and NMP (1 mL) was heated to 150 °C for 18 hours. After this time, the mixture was allowd to cool to Attorney Docket No.: 131734-01120 rt, then diluted with water and EtOAc. The organics were separated and washed with three portions of water, then washed with brine. The organics were then dried over magnesium sulfate, filtered, concentrated, and subjected to flash chromatography on silica (0-50% EtOAc in heptane). After pooling and concentrating the appropriate fractions, N-[(4Z)-cyclooct-4- en-1-yl]-6-(fluoromethyl)pyridin-2-amine (9.1 mg, 38.84 umol, 25.07% yield) was obtained as a pale yellow oil.1H NMR (400 MHz, CHLOROFORM-d) δ ppm 7.46 (t, J=7.9 Hz, 1 H) 6.67 (d, J=7.3 Hz, 1 H) 6.24 (d, J=8.3 Hz, 1 H) 5.66 - 5.80 (m, 2 H) 5.26 (d, J=47.2 Hz, 2 H) 4.63 (br d, J=6.5 Hz, 1 H) 3.64 (br s, 1 H) 2.08 - 2.58 (m, 5 H) 1.44 - 2.02 (m, 5 H). LCMS: m/z = 235 (M+H)⁺ Example 5 - Preparation of 1-[(4Z)-cyclooct-4-en-1-yl]-4-[6-(fluoromethyl)-2- pyridyl]piperazine (Compound 5) To a scintillation vial containing 1-[(4Z)-cyclooct-4-en-1-yl]piperazine (24.54 mg, 126.31 umol) , 2-bromo-6-(fluoromethyl)pyridine (20 mg, 105.26 umol) , Sodium tert-butoxide (15.17 mg, 157.88 umol) , and Dioxane (1 mL) , RuPhos Pd G3 (4.40 mg, 5.26 umol) was added and the mixture was heated to 80 °C for 4 hours. The mixture was filtered over a plug of celite, and the celite plug was rinsed with DCM. The eluent was concentrated, and the crude resdidue was subjected to flash chromatography on silica (0-20% 3:1 EtOAc:EtOH in heptane). After pooling and concentrating the appropriate fractions, 1-[(4Z)-cyclooct-4-en-1- yl]-4-[6-(fluoromethyl)-2-pyridyl]piperazine (23.9 mg, 78.77 umol, 74.84% yield) was obtained as an amber oil.1H NMR (400 MHz, CHLOROFORM-d) δ ppm 7.48 - 7.55 (m, 1 H), 6.75 (d, J=7.3 Hz, 1 H), 6.56 (d, J=8.5 Hz, 1 H), 5.61 - 5.75 (m, 2 H), 5.31 (d, J=47.2 Hz, 2 H), 3.53 (br t, J=5.0 Hz, 4 H), 2.54 - 2.79 (m, 6 H), 2.06 - 2.42 (m, 5 H), 1.64 - 1.95 (m, 2 H), 1.35 - 1.54 (m, 2 H). LCMS: m/z = 304 (M+H)⁺. Example 6 - Preparation of rac-[(1S,4Z)-cyclooct-4-en-1-yl] 4-(5-fluoro-2- pyridyl)piperazine-1-carboxylate (Compound 6) Attorney Docket No.: 131734-01120 A vial containing rac-[(1R,4Z)-cyclooct-4-en-1-yl] (2,5-dioxopyrrolidin-1-yl) carbonate (50 mg, 187.07 umol) , 1-(5-fluoro-2-pyridyl)piperazine (40.68 mg, 224.49 umol) , DCM (1 mL) , and triethylamine (37.86 mg, 374.14 umol, 52.15 uL) was stirred at rt for 1 hour. The mixture was concentrated and taken up in DMSO, then filtered and subjected to purification by reverse phase HPLC (10-90% MeCN in water, 0.1% TFA). After pooling and lyophilizing the desired fractions, rac-[(1S,4Z)-cyclooct-4-en-1-yl] 4-(5-fluoro-2-pyridyl)piperazine-1- carboxylate (47.9 mg, 107.30 umol, 57.36% yield, Trifluoroacetate) was obtained as a colorless oil. LCMS: m/z = 334 (m+H)⁺.¹H NMR (400 MHz, DMSO-d₆) δ ppm 8.10 (d, J=3.3 Hz, 1 H), 7.47 - 7.58 (m, 1 H), 6.89 (dd, J=9.3, 3.5 Hz, 1 H), 5.54 - 5.73 (m, 2 H), 4.66 (td, J=9.0, 4.4 Hz, 1 H), 3.43 (br s, 8 H), 2.22 - 2.40 (m, 1 H), 1.98 - 2.18 (m, 3 H), 1.75 - 1.92 (m, 2 H), 1.39 - 1.72 (m, 4 H).¹⁹F NMR (376 MHz, DMSO-d6) δ ppm -74.93 (s, 3 F), - 143.01 (s, 1 F). Example 7 - Preparation of rac-2-[(1S,4Z)-cyclooct-4-en-1-yl]oxy-1-[4-(5-fluoro-2- pyridyl)piperazin-1-yl]ethanone (Compound 7) A vial containing 1-(5-fluoro-2-pyridyl)piperazine (79.73 mg, 440.00 umol), rac-2-[(1S,4Z)- cyclooct-4-en-1-yl]oxyacetic acid (73.69 mg, 0.4 mmol), TEA (202.38 mg, 2.00 mmol, 278.76 uL) and DCM (2 mL) was treated with T3P (763.63 mg, 1.20 mmol, 713.68 uL, 50% purity) and stirred for 48 hours. The reaction was diluted with saturated aqueous sodium bicarbonate, and extracted with three portions of DCM. The combined organics were dried over magnesium sulfate, filtered, and concentrated. The crude residue was subjected to flash Attorney Docket No.: 131734-01120 chromatography (0-80% EtOAc in heptane), and after pooling and concentrating the appropriate fractions, the desired amide was obtained as an impure mixture with unidentified byproducts. This mixture was subjected to purification by preparative HPLC (10-90% MeCN in water, 0.1% TFA modifier), and after lyophilization of the appropriate fractions, rac-2- [(1S,4Z)-cyclooct-4-en-1-yl]oxy-1-[4-(5-fluoro-2-pyridyl)piperazin-1-yl]ethanone (17.8 mg, 38.66 umol, 9.66% yield, Trifluoroacetate) was obtained as a colorless oil. LCMS: m/z = 348 (M+H)⁺.¹H NMR (400 MHz, CHLOROFORM-d) δ ppm 9.14 (br s, 2 H), 8.14 (d, J=2.0 Hz, 1 H), 7.54 (ddd, J=9.7, 7.1, 3.0 Hz, 1 H), 6.83 (dd, J=9.5, 3.5 Hz, 1 H), 5.51 - 5.73 (m, 2 H), 4.10 - 4.25 (m, 2 H), 3.53 - 3.85 (m, 8 H), 3.41 - 3.50 (m, 1 H), 2.26 - 2.46 (m, 1 H), 1.92 - 2.22 (m, 4 H), 1.64 - 1.87 (m, 3 H), 1.49 - 1.60 (m, 1 H), 1.41 (dtt, J=14.1, 9.3, 4.7 Hz, 1 H). Example 8 - Preparation of rac-2-[[(1S,4Z)-cyclooct-4-en-1-yl]-methyl-amino]-1-[4-(5- fluoro-2-pyridyl)piperazin-1-yl]ethanone (Compound 8) A vial containing 1-(5-fluoro-2-pyridyl)piperazine (105.12 mg, 580.11 umol) , rac-2- [[(1S,4Z)-cyclooct-4-en-1-yl]-methyl-amino]acetic acid (150 mg, 483.42 umol, Trifluoroacetate) , DCM (3 mL) , and TEA (146.75 mg, 1.45 mmol, 202.14 uL) was charged with T3P (461.45 mg, 725.13 umol, 431.26 uL, 50% purity) , and the mixture was allowed to stir at rt over 48 hours. The reaction was diluted with DCM and sodium carbonate, and stirred vigorously. The phases were separated, and the aqueous phase was washed with two portions of DCM. The combined organics were dried over magnesium suflate, filtered, and concentrated. The crude residue was purified by flash chromatography on silica (100% EtOAc), and after pooling and concentrating the appropriate fractions rac-2-[[(1S,4Z)- cyclooct-4-en-1-yl]-methyl-amino]-1-[4-(5-fluoro-2-pyridyl)piperazin-1-yl]ethanone (105 mg, 291.29 umol, 60.26% yield) was obtained as a pale tan oil. LCMS: m/z = 361 (M+H)⁺. ¹H NMR (400 MHz, CHLOROFORM-d) δ ppm 8.07 (d, J=3.0 Hz, 1 H), 7.28 - 7.34 (m, 1 H), 6.64 (dd, J=9.3, 3.3 Hz, 1 H), 5.59 - 5.70 (m, 2 H), 3.63 - 3.89 (m, 4 H), 3.37 - 3.60 (m, 4 H), 3.25 (m, J=20.8 Hz, 2 H), 2.62 (br s, 1 H), 2.04 - 2.38 (m, 7 H), 1.61 - 1.87 (m, 3 H), 1.30 - 1.52 (m, 3 H). Attorney Docket No.: 131734-01120 Example 9 - Preparation of rac-N-[(1S,4Z)-cyclooct-4-en-1-yl]-2-[4-(5-fluoro-2- pyridyl)piperazin-1-yl]-N-methyl-acetamide (Compound 9) A vial containing rac-(1S,4Z)-N-methylcyclooct-4-en-1-amine (12.80 mg, 91.96 umol) , 2- [4-(5-fluoro-2-pyridyl)piperazin-1-yl]acetic acid (20 mg, 83.60 umol) , TEA (42.30 mg, 417.98 umol, 58.26 uL) , and DMF (1 mL) was charged with T3P (106.39 mg, 167.19 umol, 99.43 uL, 50% purity) and allowed to stir overnight at rt. The mixture was diluted with saturated aqueous sodium carbonate and extracted with EtOAc. The organics were collected, dried over magnesium sulfate, filtered, and concentrated. The crude residue was subjected to flash chromatography on silica (0-100% EtOAc in heptane), and after pooling and concentrating the appropriate fractions, rac-N-[(1S,4Z)-cyclooct-4-en-1-yl]-2-[4-(5-fluoro-2- pyridyl)piperazin-1-yl]-N-methyl-acetamide (11.2 mg, 31.07 umol, 37.17% yield) was obtained as a white, crystalline solid. ¹H NMR (400 MHz, CHLOROFORM-d) - 2:1 mixture of rotamers - δ ppm 7.96 - 8.01 (m, 1 H), 7.14 - 7.23 (m, 2 H), 6.51 - 6.59 (m, 1 H), 5.52 - 5.70 (m, 2 H), 4.44 - 4.56 (m, 1 H), 4.19 (dq, J=11.5, 4.0 Hz, 1 H), 3.36 - 3.48 (m, 4 H), 3.06 - 3.17 (m, 2 H), 2.82 (s, 1 H), 2.71 (s, 2 H), 2.58 - 2.66 (m, 1 H), 2.51 - 2.58 (m, 3 H), 1.93 - 2.35 (m, 5 H), 1.46 - 1.76 (m, 5 H) LCMS: m/z = 361 (M+H)⁺. Example 10 - Preparation of rac-1-(8-(6-fluoronicotinoyl)-3,8-diazabicyclo[3.2.1]octan-3- yl)-2-((R,Z)-1-hydroxycyclooct-4-en-1-yl)ethan-1-one (Compound 10) Step a: Preparation of (R,Z)-2-(1-hydroxycyclooct-4-en-1-yl)acetic acid Attorney Docket No.: 131734-01120 rac-Methyl (R,Z)-2-(1-hydroxycyclooct-4-en-1-yl)acetate (1.0 g, 5.04 mmol, 1.0 eq.) was dissolved in DCE (20 mL) and after addition of Me3SnOH (2.74 g, 15.13 mmol, 3.0 eq.), the mixture was heated at 80 °C for 5 hours under N₂. TLC (PE: EA = 1: 1) analysis indicated a complete reaction. After completion of the reaction, the mixture was concentrated in vacuo, and the residue was taken up in EA (50 mL). The organic layer was washed with aqueous HCl (5%) (30 mL x 3). The organic layer was then washed with brine (40 mL) and dried over Na₂SO₄. Removal of the solvent in vacuo afforded rac-(R,Z)-2-(1-hydroxycyclooct-4-en-1- yl)acetic acid (1.67 g, crude) as light yellow oil.¹H NMR: (400MHz, CHLOROFORM-d) δ = 5.87-5.77 (m, 1H), 5.70-5.60 (m, 1H), 2.57 (m, 2H), 2.32-2.21 (m, 4H), 1.93-1.84 (m, 3H), 1.80-1.73 (m, 1H), 1.71-1.59 (m, 2H). Step b: Preparation of rac-2,5-dioxopyrrolidin-1-yl (R,Z)-2-(1-hydroxycyclooct-4-en-1- yl)acetate To a solution of rac-(R,Z)-2-(1-hydroxycyclooct-4-en-1-yl)acetic acid (800.0 mg, 4.34 mmol, 1.0 eq.) in DCM (8 mL) was added 1-hydroxypyrrolidine-2,5-dione (749.63 mg, 6.51 mmol, 1.5 eq.) and DIC (822.00 mg, 6.51 mmol, 1.01 mL, 1.5 eq.). The mixture was stirred at 20 °C for 16 hours. TLC (PE: EA = 1: 1) showed the starting material was consumed completely. The mixture was concentrated and purified by Combi Flsah (EA in PE from 0 % to 27 % ~ 30 %) to give rac-2,5-dioxopyrrolidin-1-yl (R,Z)-2-(1-hydroxycyclooct-4-en-1-yl)acetate (638.4 mg, 47.04% yield) as a white solid.¹H NMR: (400MHz, METHANOL-d₄) δ = 5.70 (m, 1H), 5.55-5.45 (m, 1H), 2.83 (s, 4H), 2.80 (d, J=1.2 Hz, 2H), 2.47-2.37 (m, 1H), 2.34-2.22 (m, 2H), 2.18 - 2.07 (m, 2H), 1.94-1.81 (m, 3H), 1.75-1.58 (m, 2H). Step c: Preparation of rac-1-(8-(6-fluoronicotinoyl)-3,8-diazabicyclo[3.2.1]octan-3-yl)-2- ((R,Z)-1-hydroxycyclooct-4-en-1-yl)ethan-1-one Attorney Docket No.: 131734-01120 To a solution of (3,8-diazabicyclo[3.2.1]octan-8-yl)(6-fluoropyridin-3-yl)methanone (334.52 mg, 1.42 mmol, 2.0 eq.) in DCM (5 mL) was added TEA (719.44 mg, 7.11 mmol, 990.96 μL, 10.0 eq.) and rac-2,5-dioxopyrrolidin-1-yl (R,Z)-2-(1-hydroxycyclooct-4-en-1-yl)acetate (200.00 mg, 710.98 μmol, 1.0 eq.). The mixture was stirred at 25 °C for 4 hours. LCMS showed the desired MS was found. The mixture was concentrated and purified by Combi Flash (EA in PE from 0 % to 63 %) to give the crude product confirmed with LCMS. The crude product was concentrated and dissolved in MeOH then purified by Prep-HPLC (Column: Welch Xtimate C18150 x 25 mm x 5 μm; Condition: water( NH₄HCO₃)-ACN; Begin B 23, End B 53; Gradient Time(min) 11; 100%B Hold Time(min) 2; Flow Rate(mL/min) 25; Injections 1) to give rac-1-(8-(6-fluoronicotinoyl)-3,8- diazabicyclo[3.2.1]octan-3-yl)-2-((R,Z)-1-hydroxycyclooct-4-en-1-yl)ethan-1-one (15.5 mg, 5.43% yield) as white solid. LCMS: (M+H⁺: 402.1). HPLC: (Purity: 100.00 %).¹H NMR: (500MHz, METHANOL-d4) δ = 8.43 (d, J = 2.0 Hz, 1H), 8.14-8.11 (m, 1H), 7.20-7.17 (m, 1H), 5.70 (m, 1H), 5.55-5.45 (m, 1H), 4.51-4.28 (m, 1H), 4.23-3.89 (m, 2H), 3.45 (s, 1H), 2.96 (s, 1H), 2.71-2.48 (m, 2H), 2.40 (m, 1H), 2.26 (s, 2H), 2.15-2.07 (m, 1H), 2.05-1.65 (m, 10H), 1.55 (m, 1H). Example 11 - Preparation of rac-2-((R,Z)-1-hydroxycyclooct-4-en-1-yl)ethyl 8-(6- fluoronicotinoyl)-3,8-diazabicyclo[3.2.1]octane-3-carboxylate (Compound 11) Step a: Preparation of rac-methyl (R,Z)-2-(1-hydroxycyclooct-4-en-1-yl)acetate Attorney Docket No.: 131734-01120 To a solution of methyl acetate (1.79 g, 24.16 mmol, 1.5 eq.) in THF (5 mL) was added LiHMDS (1 M, 24.16 mL, 1.5 eq,) at -78 °C under N2. The mixture was stirred at -78 °C for 30 mins. The mixture was added dropwise into a solution of (Z)-cyclooct-4-en-1-one (2.0 g, 16.11 mmol) in THF (10 mL). The reaction mixture was stirred at -78 °C for 3 hours under N2. TLC (PE: EA = 4: 1) showed the starting material was consumed completely. The reaction mixture was quenched by addition of saturated NH₄Cl (10 mL). After being stirred at 20 °C for 10 mins, the mixture was diluted with water (50 mL). The mixture was extracted with EtOAc (30 mL x 3). The combined organic layers were washed with brine, dried over Na2SO4, filtered and concentrated to give rac-methyl (R,Z)-2-(1-hydroxycyclooct-4-en-1- yl)acetate (2.8 g, crude) as colorless oil.¹H NMR: (400MHz, CDCl₃) δ = 5.73 (m, 1H), 5.61- 5.45 (m, 1H), 3.80-3.66 (m, 3H), 2.56-2.47 (m, 2H), 2.44-2.34 (m, 1H), 2.28-2.17 (m, 2H), 2.15-2.05 (m, 1H), 1.89-1.79 (m, 3H), 1.73-1.64 (m, 2H), 1.56-1.45 (m, 1H). Step b: Preparation of rac-(R,Z)-1-(2-hydroxyethyl)cyclooct-4-en-1-ol A solution of LiAlH₄ (287.13 mg, 7.57 mmol, 1.5 eq.) in THF (3 mL) was added dropwise a solution of rac-methyl (R,Z)-2-(1-hydroxycyclooct-4-en-1-yl)acetate (1 g, 5.04 mmol, 1.0 eq.) in THF (10 mL) at 0 °C under N₂. The reaction mixture was stirred at 0 °C for 30 mins. TLC (PE: EA = 4: 1) showed new spot was observed. After the reaction mixture was cooled to 0 °C, the reaction mixture was quenched by addition of 0.3 mL of H2O, followed by 0.9 mL of 15% aqueous NaOH and 0.3 mL H2O. After being stirred at 20 °C for 30 mins, the solid was removed by filtration. The filtrate was concentrated to give rac-(R,Z)-1-(2- hydroxyethyl)cyclooct-4-en-1-ol (802.3 mg, crude) as yellow oil.¹H NMR: (400MHz, CDCl3) δ = 5.85-5.76 (m, 1H), 5.68-5.58 (m, 1H), 3.95-3.78 (m, 2H), 2.31-2.13 (m, 4H), 1.91-1.85 (m, 3H), 1.75 (m, 1H), 1.73-1.58 (m, 4H). Step c: Preparation of rac-(R,Z)-2-(1-hydroxycyclooct-4-en-1-yl)ethyl (4-nitrophenyl) carbonate Attorney Docket No.: 131734-01120 A solution of 4-nitrophenyl carbonochloridate (997.78 mg, 4.95 mmol, 1.2 eq.) in DCM (6 mL) was added dropwise by syringe to a mixture of rac-(R,Z)-1-(2-hydroxyethyl)cyclooct-4- en-1-ol (702.3 mg, 4.13 mmol, 1.0 eq.) and pyridine (815.75 mg, 10.31 mmol, 2.5 eq.) in DCM (12 mL). The mixture was stirred for 16 hours at 20 °C. Several spots were shown on TLC (PE/EA = 2/1) and then quenched by addition of saturated NH4Cl. The resultant layers were separated and the aqueous layer was extracted with DCM (30 mL x 3). The combined organic layers were dried over Na₂SO₄, filtered, and concentrated into a residue. The crude residue was purified by Combi Flash (EA in PE from 0 % to 15 %) to give rac-(R,Z)-2-(1- hydroxycyclooct-4-en-1-yl)ethyl (4-nitrophenyl) carbonate (908.5 mg, 56.43% yield) as yellow oil. LCMS: (2M+Na⁺: 693.2).¹H NMR: (400MHz, CDCl₃) δ = 8.20-8.17 (m, 2H), 6.93-6.91 (m, 2H), 5.88-5.73 (m, 1H), 5.71-5.53 (m, 1H), 4.58-4.24 (m, 2H), 2.38-2.23 (m, 3H), 2.17 (m, 1H), 2.00-1.78 (m, 6H), 1.72-1.67 (m, 2H). Step d: Preparation of rac-2-((R,Z)-1-hydroxycyclooct-4-en-1-yl)ethyl 8-(6- fluoronicotinoyl)-3,8-diazabicyclo[3.2.1]octane-3-carboxylate To a solution of (3,8-diazabicyclo[3.2.1]octan-8-yl)(6-fluoropyridin-3-yl)methanone (210.46 mg, 894.59 μmol, 3.0 eq.) in DCM (5 mL) was added DIPEA (96.35 mg, 745.49 μmol, 129.85 μL, 2.5 eq.). The mixture was stirred at 20 °C for 10 mins. The reaction mixture was added rac-(R,Z)-2-(1-hydroxycyclooct-4-en-1-yl)ethyl (4-nitrophenyl) carbonate (100.00 mg, 298.20 μmol, 1.0 eq.). The mixture was stirred at 20 °C for 16 hours. LCMS showed the desired MS was found. The mixture was concentrated and purified by Combi Flash (EA in PE from 0 % to 60 %) to give the crude product confirmed with LCMS. The crude product was dissolved in MeOH then purified by Prep-HPLC (Column: Welch Xtimate C18150 x 25 mm x 5 μm; Condition: water( NH₄HCO₃)-ACN; Begin B 28, End B 58; Gradient Time(min) 11; 100%B Hold Time(min) 2; Flow Rate(mL/min) 25; Injections 1) to give rac-2-((R,Z)-1- hydroxycyclooct-4-en-1-yl)ethyl 8-(6-fluoronicotinoyl)-3,8-diazabicyclo[3.2.1]octane-3- carboxylate (9.2 mg, 7.15% yield) as an off-white solid. LCMS: (M+H⁺: 432.2). HPLC: (Purity: 100.00 %).¹H NMR: (400MHz, METHANOL-d₄) δ = 8.42 (d, J = 2.4 Hz, 1H), 8.14- 8.09 (m, 1H), 7.19 (dd, J1 = 2.4 Hz, J2 = 8.8 Hz, 1H), 5.71-5.64 (m, 1H), 5.52-5.43 (m, 1H), 4.78 (s, 1H), 4.29 (s, 2H), 4.20-4.06 (m, 1H), 4.02-3.83 (m, 2H), 3.28-3.06 (m, 2H), 2.41- Attorney Docket No.: 131734-01120 2.31 (m, 2H), 2.19-2.10 (m, 2H), 2.05-1.95 (m, 3H), 1.86 (s, 2H), 1.82-1.74 (m, 3H), 1.62 (s, 3H), 1.29 (s, 1H). Example 12 - Preparation of rac-(R,Z)-2-(1-hydroxycyclooct-4-en-1-yl)ethyl 4-(4-(2- fluoroethoxy)benzoyl)piperazine-1-carboxylate (Compound 12) Step a: Preparation of tert-butyl 4-(4-(2-fluoroethoxy)benzoyl)piperazine-1-carboxylate To a solution of 4-(2-fluoroethoxy)benzoic acid (320.0 mg, 1.74 mmol, 1.0 eq.) in DCM (4 mL) was added tert-butyl piperazine-1-carboxylate (388.35 mg, 2.09 mmol, 1.2 eq.), TEA (527.48 mg, 5.21 mmol, 726.55 μL, 3.0 eq.) and T3P (829.30 mg, 2.61 mmol, 668.79 μL, 1.5 eq.). The mixture was stirred at 15 °C for 16 hours. LCMS showed the desired MS was found. The mixture was concentrated to give a residue, which was purified by Combi Flash (EA in PE from 0 % to 35 %) to give tert-butyl 4-(4-(2-fluoroethoxy)benzoyl)piperazine-1- carboxylate (260.3 mg, 42.23% yield) as a white solid.¹H NMR: (400MHz, CDCl3) δ ppm = 7.45-7.35 (m, 2H), 7.01-6.92 (m, 2H), 4.84 (dd, J₁ = 4.0 Hz, J₂ = 5.2 Hz, 1H), 4.72 (dd, J₁ = 4.0, J₂ = 5.6 Hz, 1H), 4.31-4.19 (m, 2H), 3.71-3.39 (m, 8H), 1.48 (s, 9H). Step b: Preparation of (4-(2-fluoroethoxy)phenyl)(piperazin-1-yl)methanone To a solution of tert-butyl 4-(4-(2-fluoroethoxy)benzoyl)piperazine-1-carboxylate (260.3 mg, 738.65 μmol, 1.0 eq.) in HCl/EA (4 M, 5 mL, 27.08 eq.). The mixture was stirred 20 °C for 1 hour. LCMS showed the desired MS was found. The reaction mixture was concentrated to give (4-(2-fluoroethoxy)phenyl)(piperazin-1-yl)methanone (198.5 mg, crude, hydrochloride) as a white solid. LCMS: (M+H⁺: 253.2). Step c: Preparation of rac-(R,Z)-2-(1-hydroxycyclooct-4-en-1-yl)ethyl 4-(4-(2- fluoroethoxy)benzoyl)piperazine-1-carboxylate Attorney Docket No.: 131734-01120 To a solution of (4-(2-fluoroethoxy)phenyl)(piperazin-1-yl)methanone (169.27 mg, 670.94 μmol, 1.5 eq.) in DCM (5 mL) was added DIPEA (144.52 mg, 1.12 mmol, 194.77 μL, 2.5 eq.). The mixture was stirred at 20 °C for 10 mins. The reaction mixture was added rac- (R,Z)-2-(1-hydroxycyclooct-4-en-1-yl)ethyl (4-nitrophenyl) carbonate (150.0 mg, 447.29 μmol, 1.0 eq.). The mixture was stirred at 20 °C for 16 hours. LCMS showed the desired MS was found. The mixture was concentrated and dissolved in MeOH then purified by Prep- HPLC (Column: Welch Xtimate C18150 x 25 mm x 5 μm; Condition: water (NH4HCO3)- ACN; Begin B 32, End B 62; Gradient Time (min) 11; 100%B Hold Time (min) 2; Flow Rate (mL/min) 25; Injections 8) to give rac-(R,Z)-2-(1-hydroxycyclooct-4-en-1-yl)ethyl 4-(4-(2- fluoroethoxy)benzoyl)piperazine-1-carboxylate (167.06 mg, 83.27% yield) as light yellow oil. LCMS: (M+Na⁺: 471.2). HPLC: (Purity: 100.00 %).¹H NMR: (400MHz, CDCl₃) δ ppm = 7.45-7.35 (m, 2H), 7.00-6.91 (m, 2H), 5.78 (m, 1H), 5.64-5.54 (m, 1H), 4.84 (dd, J₁ = 4.4 Hz, J2 =5.6 Hz, 1H), 4.72 (dd, J1 = 4.4, J2 = 5.6 Hz, 1H), 4.35-4.27 (m, 3H), 4.24-4.20 (m, 1H), 31-3.44 (m, 8H), 2.33-2.22 (m, 3H), 2.20-2.11 (m, 1H), 1.95-1.86 (m, 2H), 1.83-1.73 (m, 3H), 1.68 -1.59 (m, 3H). Example 13 - Preparation of rac-(R,Z)-2-(1-hydroxycyclooct-4-en-1-yl)ethyl 4-(4-(2- fluoroethoxy)-3-methylbenzoyl)piperazine-1-carboxylate (Compound 13) Step a: Preparation of methyl 4-(2-fluoroethoxy)-3-methylbenzoate To a solution of methyl 4-hydroxy-3-methylbenzoate (1 g, 6.02 mmol, 1.0 eq.) in MeCN (10 mL) was added 1-fluoro-2-iodoethane (2.09 g, 12.04 mmol, 2.0 eq.) and K₂CO₃ (2.50 g, Attorney Docket No.: 131734-01120 18.05 mmol, 3.0 eq.). The mixture was stirred at 75 °C for 16 hours. LCMS showed the starting material was consumed completely and new peak with desired MS was detected. The reaction solution was concentrated to get the residue, which was purified by Combi flash eluting with EtOAc in PE from 0% to 30% to give methyl 4-(2-fluoroethoxy)-3- methylbenzoate (1.2 g, 93.96% yield) as a white solid.¹H NMR: (400MHz, CDCl3) δ = 7.98- 7.75 (m, 2H), 6.82 (d, J = 8.4 Hz, 1H), 4.92-4.69 (m, 2H), 4.39-4.21 (m, 2H), 3.89 (s, 3H), 2.28 (s, 3H). Step b: Preparation of 4-(2-fluoroethoxy)-3-methylbenzoic acid To a solution of methyl 4-(2-fluoroethoxy)-3-methylbenzoate (1 g, 4.71 mmol, 1.0 eq.) in MeOH (20 mL) and water (10 mL) was added NaOH (753.89 mg, 18.85 mmol, 4.0 eq.) at 20 °C. The mixture was stirred at 20 °C for 16 hours. LCMS showed the starting material was consumed completely and new peak with desired MS was detected. The reaction was acidified with HCl (4M) to pH = 4 and diluted with H₂O. The resulting precipitate was filtered, washed with H2O and hexanes, and concentrated in vacuum to give 4-(2- fluoroethoxy)-3-methylbenzoic acid (930 mg, 99.58% yield) as colorless oil.¹H NMR: (400MHz, DMSO-d6) δ = 12.59 (s, 1H), 7.85-7.66 (m, 2H), 7.04 (d, J = 8.4 Hz, 1H), 4.85- 4.71 (m, 2H), 4.38-4.27 (m, 2H), 2.20 (s, 3H). Step c: Preparation of tert-butyl 4-(4-(2-fluoroethoxy)-3-methylbenzoyl)piperazine-1- carboxylate To a solution of 4-(2-fluoroethoxy)-3-methylbenzoic acid (930 mg, 4.69 mmol, 1.0 eq.) in DCM (15 mL), tert-butyl piperazine-1-carboxylate (1.75 g, 9.38 mmol, 2.0 eq.), TEA (949.66 mg, 9.38 mmol, 1.31 mL, 2.0 eq.) and T3P (2.24 g, 7.04 mmol, 1.5 eq.). The mixture was stirred at 25 °C for 16 hours. LCMS showed the desired MS was found. The mixture was concentrated and then water (20 mL) was added. The mixture was extracted with EtOAc (20 mL x 3). The combined organic layers were washed with brine (20 mL), dried over Na₂SO₄, filtered and concentrated in vacuo to give the residue, which was purified by Combi flash Attorney Docket No.: 131734-01120 eluting with EtOAc in PE from 0% to 50% to give tert-butyl 4-(4-(2-fluoroethoxy)-3- methylbenzoyl)piperazine-1-carboxylate (1.4 g, 81.42% yield) as a white solid.1H NMR: (400MHz, CDCl3) δ = 7.26-7.19 (m, 2H), 6.82 (d, J = 8.0 Hz, 1H), 4.87-4.71 (m, 2H), 4.31- 4.19 (m, 2H), 3.69-3.38 (m, 8H), 2.27 (s, 3H), 1.48 (s, 9H). Step d: Preparation of (4-(2-fluoroethoxy)-3-methylphenyl)(piperazin-1-yl)methanone A solution of tert-butyl 4-(4-(2-fluoroethoxy)-3-methylbenzoyl)piperazine-1-carboxylate (1.4 g, 3.82 mmol, 1.0 eq.) in DCM (15 mL) was added dropwise into a solution HCl/EA (4 M, 20 mL) at 25 ℃ for 1 h. LCMS showed that the starting material was consumed and the desired product was detected. The reaction mixture was concentrated to give the crude (4-(2- fluoroethoxy)-3-methylphenyl)(piperazin-1-yl)methanone (1 g, 98.28% yield) as a white solid, which was used to next step directly. LCMS: (M+H+: 267.1). Step e: Preparation of (R,Z)-2-(1-hydroxycyclooct-4-en-1-yl)ethyl 4-(4-(2-fluoroethoxy)-3- methylbenzoyl)piperazine-1-carboxylate To a solution of (4-(2-fluoroethoxy)-3-methylphenyl)(piperazin-1-yl)methanone (150 mg, 563.25 µmol, 1.0 eq.) in DCM (5 mL) was added rac-(R,Z)-2-(1-hydroxycyclooct-4-en-1- yl)ethyl (4-nitrophenyl) carbonate (94.44 mg, 281.63 µmol, 0.5 eq.) and DIPEA (145.59 mg, 1.13 mmol, 196.22 µL, 2.0 eq.). The mixture was stirred at 20 °C for 16 hours. LCMS showed the desired MS was found. Water (10 ml) was added and the mixture was extracted with DCM (10 ml x 3). The organics were washed with brine (15 ml), dried over Na2SO4, filtered and concentrated to get a crude, which was purified by prep-HPLC(Column: Boston Prime C18150*30mm*5um, Condition: water(NH3H2O+NH4HCO3)-ACN Begin B 41, End B 71 Gradient Time (min) 10, 100%B Hold Time (min) 2 Flow Rate (ml/min) 25) to give (R,Z)-2-(1-hydroxycyclooct-4-en-1-yl)ethyl 4-(4-(2-fluoroethoxy)-3- methylbenzoyl)piperazine-1-carboxylate (70.1 mg, 26.17% yield) as yellow oil. LCMS: (M+Na⁺: 485.3). HPLC: (Purity: 97.25%).¹H NMR: (500MHz, DMSO-d6) δ = 7.27-7.20 (m, Attorney Docket No.: 131734-01120 2H), 7.00 (d, J = 9.0 Hz, 1H), 5.70-5.58 (m, 1H), 5.48-5.36 (m, 1H), 4.83-4.70 (m, 2H), 4.33- 4.25 (m, 2H), 4.21-4.13 (m, 3H), 3.61-3.36 (m, 8H), 2.34-2.23 (m, 2H), 2.19 (s, 3H), 2.10- 2.02 (m, 2H), 1.84-1.82 (m, 1H), 1.76-1.68 (m, 2H), 1.65-1.57 (m, 2H), 1.54-1.43 (m, 3H). Example 14 - Preparation of rac-(R,Z)-4-(2-fluoroethoxy)-N-(2-(1-hydroxycyclooct-4-en-1- yl)ethyl)-3-methylbenzamide (Compound 14) Step a: Preparation of methyl 4-(2-fluoroethoxy)-3-methylbenzoate To a solution of methyl 4-hydroxy-3-methylbenzoate (1.00 g, 6.02 mmol, 1.0 eq.) in MeCN (20 mL) was added 1-fluoro-2-iodoethane (2.09 g, 12.04 mmol, 2.0 eq.) at 20 °C. Then K₂CO₃ (2.50 g, 18.05 mmol, 3.0 eq.) was added to the mixture at 20 °C. The reaction was stirred at 75 °C for 16 hours. TLC showed the reaction was completed. The reaction solution was concentrated to get the residue, which was purified by Combi flash eluting with EtOAc in PE from 0% to 30% to give 4-(2-fluoroethoxy)-3-methylbenzoate (1.2 g, 93.96% yield) as a white solid.¹H NMR: (400 MHz, CDCl₃) δ = 7.90-7.86 (m, 2H), 6.82 (d, J = 8.8 Hz, 1H), 4.87-4.73 (m, 2H), 4.33-4.24 (m, 2H), 3.81 (s, 3H), 2.28 (s, 3H). Step b: Preparation of 4-(2-fluoroethoxy)-3-methylbenzoic acid To a solution of methyl 4-(2-fluoroethoxy)-3-methylbenzoate (1.2 g, 5.65 mmol, 1.0 eq.) in H2O (10 mL) and MeOH (20 mL) was added NaOH (904.67 mg, 22.62 mmol, 4.0 eq.) at 20 °C. The mixture was stirred at 20 °C for 16 hours. LCMS showed that the starting material was consumed and the desired product was detected. The reaction was acidified with concentrated HCl (aq) to pH 4 and diluted with H2O. The resulting precipitate was filtered, Attorney Docket No.: 131734-01120 washed with H2O and hexanes, and concentrated in vacuo to give 4-(2-fluoroethoxy)-3- methylbenzoic acid (946.3 mg, 84.44% yield) as a white solid. LCMS: (M+H⁺: 199.2). Step c: Preparation of rac-(R,Z)-4-(2-fluoroethoxy)-N-(2-(1-hydroxycyclooct-4-en-1- yl)ethyl)-3-methylbenzamide To a solution of rac-(R,Z)-1-(2-aminoethyl)cyclooct-4-en-1-ol (500.00 mg, 2.95 mmol, 1.0 eq.) in DCM (20 mL) was added 4-(2-fluoroethoxy)-3-methylbenzoic acid (585.45 g, 2.95 mmol, 1.0 eq.), DMAP (72.18 mg, 590.80 μmol, 0.2 eq.) and EDCI (849.42 mg, 4.33 mmol, 1.5 eq.) at 20°C. Then TEA (5.91 mmol, 823.45 μL) was added to the mixture at 20 °C. The reaction was stirred at 20 °C for 4 hours. LCMS showed the desired MS was found. The reaction mixture was added water (60 mL), and extracted with DCM (20 mL x 3). The organic layer was washed with brine, and dried over Na₂SO₄.The reaction solution was concentrated to get the residue, which was purified by Combi flash eluting with EtOAc in PE from 0% to 60% to give rac-(R,Z)-4-(2-fluoroethoxy)-N-(2-(1-hydroxycyclooct-4-en-1- yl)ethyl)-3-methylbenzamide (550 mg, 53.28% yield) as yellow oil. A portion of product (80.00 mg, 228.94μmol, 1.0 eq.) was dissolved in MeOH (3 mL). The residue was purified by Prep-HPLC (Column: Boston Prime C18150 x 30 mm x 5 μm; Condition: water(NH₃H₂O+NH₄HCO₃)-ACN; Begin B 44, End B 74; Gradient Time(min) 10; 100% B Hold Time(min) 2; Flow Rate(mL/min) 25) to give (R,Z)-4-(2-fluoroethoxy)-N-(2-(1- hydroxycyclooct-4-en-1-yl)ethyl)-3-methylbenzamide (56.1 mg, 70.13% yield) as yellow oil. LCMS: (M+H⁺: 350.2). HPLC: (Purity: 100.00 %).¹H NMR: (400MHz, CDCl₃) δ = 7.62- 7.59 (m, 2H), 6.81 (d, J = 8.0 Hz, 1H), 5.88-5.82 (m, 1H), 5.73-5.66 (m, 1H), 4.86-4.84 (m, 1H), 4.74-4.72 (m, 1H), 4.30 -4.28 (m, 1H), 4.23-4.21 (m, 1H), 3.63-3.58 (m, 2H), 2.39-2.32 (m, 2H), 2.28 (s, 3H), 2.23 -2.20 (m, 1H), 2.20-2.12 (m, 1H), 1.92-1.86 (m, 3H), 1.80-1.76 (m, 2H), 1.74-1.63 (m, 3H). Example 15 - Preparation of rac-(R,Z)-4-(2-fluoroethoxy)-2-(2-(1-hydroxycyclooct-4-en-1- yl)ethyl)isoindolin-1-one (Compound 15) Attorney Docket No.: 131734-01120 To a solution of 4-(2-fluoroethoxy)isoindolin-1-one (78.60 mg, 402.68 µmol, 0.5 eq.) in DMF (2 mL) was added NaH (64.42 mg, 1.61 mmol, 60% purity, 2.0 eq.) at 25 °C under N2 for 30 min. Then rac-(R,Z)-2-(1-hydroxycyclooct-4-en-1-yl)ethyl methanesulfonate (200.00 mg, 805.35 µmol, 1.0 eq.) in DMF (1mL) was added at 25 °C for 5 hours. LCMS showed the desired MS was found. The reaction mixture was quenched with H₂O (1 mL) and filtered to get a filtrate, which was purified by prep-HPLC (Column: Welch Xtimate C18 150*25mm*5um, Condition: water (NH4HCO3)-ACN Begin B 33, End B 60 Gradient Time (min) 11, 100%B Hold Time (min) 2 Flow Rate (ml/min) 25) to give rac-(R,Z)-4-(2- fluoroethoxy)-2-(2-(1-hydroxycyclooct-4-en-1-yl)ethyl)isoindolin-1-one (13.5 mg, 4.82% yield) as a yellow solid. LCMS: (M +H⁺: 348.1). HPLC: (Purity: 100%).1H NMR: (400MHz, MeOD-d4) δ = 7.47 (t, J = 7.8 Hz, 1H), 7.37 (d, J = 7.2 Hz, 1H), 7.19 (d, J = 6.4 Hz, 1H), 5.76-5.62 (m, 1H), 5.56-5.41 (m, 1H), 4.86-4.80 (m, 1H), 4.74-4.69 (m, 1H), 4.50 (s, 2H), 4.44-4.38 (m, 1H), 4.36-4.31 (m, 1H), 3.81-3.74 (m, 2H), 2.43-2.34 (m, 2H), 2.24- 2.11 (m, 2H), 2.07-2.00 (m, 1H), 1.91-1.83 (m, 3H), 1.81-1.75 (m, 1H), 1.70-1.63 (m, 3H). Example 16 - Preparation of rac-(R,Z)-5-(2-fluoroethoxy)-2-(2-(1-hydroxycyclooct-4-en-1- yl)ethyl)isoindolin-1-one (Compound 16) To a solution of 5-(2-fluoroethoxy)isoindolin-1-one (80.00 mg, 409.86 μmol, 1.0 eq.) in DMF (3 mL) was added rac-(R,Z)-2-(1-hydroxycyclooct-4-en-1-yl)ethyl methanesulfonate (203.57 mg, 819.71 μmol, 2.0 eq.) and Cs2CO3 (400.62 mg, 1.23 mmol, 3.0 eq.) at 20 °C. The mixture was stirred at 90°C under N2 for 3 hours. The residue was purified by Prep-HPLC Attorney Docket No.: 131734-01120 (Column: Welch Xtimate C18150 x 25 mm x 5 μm; Condition: water(NH4HCO3)-ACN; Begin B 31, End B 60; Gradient Time(min) 11; 100% B Hold Time(min) 2; Flow Rate(mL/min) 25) to give rac-(R,Z)-5-(2-fluoroethoxy)-2-(2-(1-hydroxycyclooct-4-en-1- yl)ethyl)isoindolin-1-one (18.8 mg, 13.20% yield) as a yellow solid. LCMS: (M+H⁺: 348.2). HPLC: (Purity: 100.00 %).1H NMR: (400MHz, METHANOL-d4) δ = 7.67 (d, J = 8.4 Hz, 1H), 7.15 (s, 1H), 7.09-7.06 (m, 1H), 5.72-5.67 (m, 1H), 5.52-5.45 (m, 1H), 4.82-4.68 (m, 2H), 4.49 (s, 2H), 4.35-4.26 (m, 2H), 3.76-3.71 (m, 2H), 2.41-2.34 (m, 2H), 2.20-2.14 (m, 2H), 2.06-1.99 (m, 1H), 1.87-1.83 (m, 3H), 1.80-1.75 (m, 1H), 1.68-1.63 (m, 3H). Example 17 - Preparation of rac-(S,E)-6-fluoro-N-(2-(1-hydroxycyclooct-4-en-1-yl)ethyl)-4- methylnicotinamide (Compound Preparation of rac-(S,E)-1-(2-aminoethyl)cyclooct-4-en-1-ol was described in Fox, J. et al Angewandte Chemie, 2021, 60(27), 14975-14980, which is incorporated by reference herein in its entirety. Step b: Preparation of rac-(S,E)-6-fluoro-N-(2-(1-hydroxycyclooct-4-en-1-yl)ethyl)-4- methylnicotinamide 5-ax-Hydroxy-5-eq-(2-aminoethyl)-trans-cyclooctene (40 mg, 236 µmol) was dissolved in DMF (1 mL). DIPEA (91 mg, 709 µmol, 123 µL) was added followed by 6-fluoro-4-methyl- pyridine-3-carboxylic acid (37 mg, 236 µmol) and T3P (1.68 M, 236 µmol, 141 µL). The mixture was stirred at room temp overnight. The resulting was injected directly on silica to be purified by flash chromatography using a gradient of 0-100% EtOAc-heptane to afford the title compound after evaporating the organics at room temp or below to avoid degradation. Attorney Docket No.: 131734-01120 The ionization corresponds to the -OH fragmentation. MS: m/z 289.3 [M-OH]; RT: 0.68 min. 1H NMR (500 MHz, BENZENE-d6) δ ppm 1.19 - 1.39 (m, 4 H) 1.52 - 1.60 (m, 3 H) 1.62 (s, 1 H) 1.64 - 1.75 (m, 2 H) 1.91 - 1.97 (m, 1 H) 2.06 - 2.13 (m, 1 H) 2.17 - 2.29 (m, 3 H) 3.25 - 3.39 (m, 2 H) 5.29 - 5.46 (m, 2 H) 6.08 (br s, 1 H) 6.16 (s, 1 H) 8.14 (s, 1 H). Example 18 – Preparation of rac-(S,E)-6-fluoro-N-(2-(1-hydroxycyclooct-4-en-1-yl)ethyl)- N,4-dimethylnicotinamide (Compound 18) Step a: Preparation of rac-(S,E)-6-fluoro-N-(2-(1-hydroxycyclooct-4-en-1-yl)ethyl)-N,4- dimethylnicotinamide To a solution of rac-6-fluoro-N-[2-[(1S,4Z)-1-hydroxycyclooct-4-en-1-yl]ethyl]-4-methyl- pyridine-3-carboxamide (12 mg, 39 umol) in DMF (2 mL) was added NaH (5 mg, 118 umol, 60% purity) at 0°C. The mixture was stirred at 0°C for 15 mins and CH3I (9 mg, 59 umol, 4 uL) was added at 0°C. The mixture was stirred at 0°C for 3 hrs, then treated with a saturated solution of ammonium chloride while allowing to return to room temp and extracted with EtOAc. The organics were concentrated at room temp or below to avoid degradation and then injected on silica and purified by flash chromatography using a gradient of 0-100% EtOAc- Heptane to afford the title compound. MS: m/z 322.3 [M+H]; RT: 0.72 min.1H NMR (500 MHz, BENZENE-d6) δ ppm 1.39 - 1.51 (m, 3 H) 1.62 - 1.74 (m, 2 H) 1.76 - 1.91 (m, 5 H) 1.96 (s, 1 H) 2.00 - 2.12 (m, 3 H) 2.18 - 2.35 (m, 1 H) 2.50 (qd, J=11.95, 4.73 Hz, 1 H) 2.79 (s, 1 H) 2.86 (br t, J=7.48 Hz, 1 H) 3.19 - 3.30 (m, 1 H) 3.35 - 3.48 (m, 1 H) 5.17 - 5.34 (m, 1 H) 5.50 (ddd, J=15.56, 11.75, 3.20 Hz, 1 H) 5.70 (ddd, J=15.56, 11.14, 3.81 Hz, 1 H) 6.11 - 6.22 (m, 1 H) 7.82 - 8.04 (m, 1 H). Example 19 - Preparation of rac-(S,Z)-6-fluoro-N-(2-(1-hydroxycyclooct-4-en-1-yl)ethyl)- N,4-dimethylnicotinamide (Compound 19) Attorney Docket No.: 131734-01120 To a solution of rac-(S,Z)-6-fluoro-N-(2-(1-hydroxycyclooct-4-en-1-yl)ethyl)-4- methylnicotinamide (100 mg, 326.40 μmol, 1.00 eq.) in DMF (2 mL) was added NaH (39.16 mg, 979.19 μmol, 60% purity, 3.00 eq.) at 0 °C. The mixture was stirred at 0 °C for 15 mins and CH₃I (69.49 mg, 489.60 μmol, 1.50 eq.) was added at 0 °C. The mixture was stirred at 0 °C for 2 hrs. LCMS showed that the starting material was consumed and the desired MS was detected. The reaction mixture was concentrated to give a crude, which was purified by prep- HPLC (Column: Welch X timate C18150 x 25 mm x 5 μm; Condition: water (NH₄HCO₃)- ACN, Begin B 25, End B 54, Gradient Time (min) 11, 100%B Hold Time (min) 2, Flow Rate (mL/min) 25, Injections 1.) to give rac-(S,Z)-6-fluoro-N-(2-(1-hydroxycyclooct-4-en-1- yl)ethyl)-N,4-dimethylnicotinamide (22.6 mg, 21.61% yield) as yellow oil. LCMS: (M+H+: 321.1). HPLC: (Purity: 100%).1H NMR: (500 MHz, DMSO-d6) δ = 8.08 (d, J = 19.0 Hz, 1H), 7.16 (d, J = 7.0 Hz, 1H), 5.67-5.56 (m, 1H), 5.45-5.36 (m, 1H), 4.21-4.07 (m, 1H), 3.60- 3.57 (m, 1H), 3.53-3.51 (m, 1H), 3.00-2.76 (m, 3H), 2.28-2.27 (m, 3H), 2.14-2.11 (m, 3H), 1.89-1.84 (m, 1H), 1.72-1.65 (m, 2H), 1.57-1.54 (m, 2H), 1.43-1.08 (m, 4H). Example 20 - Preparation of rac-(S,Z)-6-fluoro-N-(2-(1-hydroxycyclooct-4-en-1-yl)ethyl)-4- methylnicotinamide (Compound 20) To a mixture of 6-fluoro-4-methylnicotinic acid (100 mg, 644.64 μmol, 1.00 eq.) in DCM (3 mL) was added DIEA (249.94 mg, 1.93 mmol, 336.85 μL, 3.00 eq.) and HATU (294.13 mg, 773.56 μmol, 1.20 eq.) at 20 °C. The mixture was stirred at 20 °C for 15 mins and rac-(S,Z)- 1-(2-aminoethyl)cyclooct-4-en-1-ol (163.67 mg, 966.95 μmol, 1.50 eq.) was added at 20 °C. The mixture was stirred at 20 °C for 1 h. LCMS showed the reaction was completed. The reaction mixture was concentrated to give a crude, which was purified by prep-HPLC Attorney Docket No.: 131734-01120 (Column: Waters X bridge BEH C18100 x 30 mm x 10 μm; Condition: water (NH4HCO3)- ACN, Begin B 26, End B 56, Gradient Time (min) 11, 100%B Hold Time (min) 3, Flow Rate (mL/min) 50, Injections 4.) to give rac-(S,Z)-6-fluoro-N-(2-(1-hydroxycyclooct-4-en-1- yl)ethyl)-4-methylnicotinamide (37.4 mg, 18.94% yield) as yellow oil. LCMS: (2M+H+: 613.3). HPLC: (Purity: 100%).1H NMR: (400 MHz, METHANOL-d4) δ = 8.20 (s, 1H), 7.02 (s, 1H), 5.74-5.68 (m, 1H), 5.54-5.47 (m, 1H), 3.55-3.51 (m, 2H), 2.50 (s, 3H), 2.41- 2.34 (m, 2H), 2.19-2.16 (m, 2H), 2.05-2.00 (m, 1H), 1.87-1.82 (m, 4H), 1.68-1.65 (m, 3H). Example 21 General Procedure for the Synthesis of MALAT1-ASO-Tz To a 1 mL vial containing MALAT1 C6 amine (4.2 mg, 0.572 umol, chemical structure depicted in Figure 21) dispensed from a 44 mg/mL stock solution in water (95 uL dispensed), along with 5 uL of 1M NaHCO₃, Tetrazine NHS ester (24 equivalents) was added as a solution in DMSO (15 mg/mL) in two portions. The second aliquot was added approximately 5 minutes after the first, and the reaction was allowed to stir for 1 hour. Following this period, HPLC confirmed that the reaction was complete. The contents of the mixture were loaded into an amicon centrifugal filter (3000 MW cutoff) and the mixture was diluted to 5 mL total volume with water, then spun down at 4000 RPM for 30 minutes. Following centrifugal spin filtering, the total volume of the mixture was approximately 100 uL. The mixture was diluted to 5 mL once more and spun down a second time. The resulting solution was collected and lyophilized to afford the desired ASO-Tz conjugate as a light pink solid. Attorney Docket No.: 131734-01120 Compounds 21-23 were prepared using the general procedure described above. Biologic Example 1 a. Brain and Plasma Protein Binding: Individual test articles were each spiked into separate 1500 uL aliquots of various plasma at final concentrations of 1 µM. Three hundred microliters of these solutions were added to donor chambers in the RED device (RED tubes). Identical 300 uL solutions were spiked with warfarin (1 uM) as the positive control. Five hundred microliter aliquots of 100 mM potassium phosphate 150 mM NaCl, pH 7.4 (phosphate buffer) was added to the corresponding receiving side of the RED device inserts. Test compounds were incubated in rat and human plasma, and rat brain homogenate at 37 ^C for 6 h in an incubator with shaking (200 rpm) in the presence of 5% CO2. A separate 96 well 2.0 mL assay block from Corning (Corning, NY) with identical mixtures of test article in plasma or homogenate to those described above was incubated at 37°C for 6 h for the stability experiment. For the 1 µM test article and positive control incubations, aliquots of 50 ^L of plasma samples were removed from the RED donor compartment (for the protein binding experiment) and from the corresponding 2.0 mL assay block well (for the plasma stability experiment) within 10 minutes of addition (0 h time point) and after 6 h to provide the control for the plasma stability experiment. Aliquots of 50 ^L of matrix were removed from the RED donor compartment. These aliquots were transferred to an individual 96 well plate containing 50 ^L of the phosphate buffer and 200 ^L of 25 ng/mL of CPDPX in 1:1 methanol/acetonitrile (internal standard). An additional 50 µL aliquot from each RED device receiver compartment was removed after 6 h and were transferred into individual 96 well plate wells containing 50 µL of appropriate matrix mixture and 200 ^L of internal standard. All samples were vortex mixed for 30 Attorney Docket No.: 131734-01120 seconds and centrifuged at 3900 rpm for 10 minutes. All matrix and buffer samples were matrix matched. Fifty microliters of sample supernatants were transferred into individual wells in a 96 well plate containing 200 µL of 0.1% formic acid in 90/10 water/acetonitrile and vortex mixed gently. The plasma protein binding including stability assay samples were analyzed in duplicate. The standard curve for compounds of this disclosure and warfarin, ranged from 5nM to 1500nM. The calibration curve was prepared in a 1:1 plasma mixture (rat, dog, monkey, and human All standards were matrix matched and went through the same preparation procedure as the samples. All samples were directly injected into the LC/MS/MS system for sample analysis. b. MDR1-MDCK: The assay makes use of human MDR1 transfected MDCK cells (NIH cell line in- licensed from Absorption Systems, PA, USA). The compounds were tested at 1 µM concentration prepared in transport buffer (Hank’s balanced salt solution with HEPES). MDR1-MDCK cell were cultured for 7 days in 96-well transwell insert plates (Corning, NY, USA). Insert plates were washed before the assay and TEER (Trans epithelial electric resistance) was measured. These plates were loaded with test compound solution 85 uL for A- B transport and 260 uL for B-A transport in the respective donor compartment. The volume of receiver buffer (Transport buffer supplemented with 1% BSA) in the respective receiver compartment was 250 and 75 µL respectively.10 µL samples were taken from donor compartment (T=0 timepoint). Assay plates were incubated for 120 minutes. At 120 minutes (T=120 timepoint) samples from respective donor (10 µL) and receiver (50 µL) compartments was taken. After addition of 40 µL transport buffer with BSA to donor samples, crash solution (acetonitrile with internal standard, 110 µL) was added to all samples. After centrifugation 50 µL supernatant was transferred to separate plate and mixed with 50 µL water. Samples were analyzed using LC-MS/MS coupled with high throughput injection system. Analyte/internal standard area ratios were used for apparent permeability (Papp), efflux ratio and mass recovery estimation based on equations below. Papp = (dCr /dt) x Vr / (A x CE) Mass balance = 100 x ((Vr x Crfinal) + (Vd x Cdfinal)) / (Vd x CE) Attorney Docket No.: 131734-01120 Where: dCr /dt is the slope of cumulative concentration in the receiver compartment versus time in uM ^s1 Vr is the volume of the receiver compartment in cm³ Vd is the volume of the donor compartment in cm³ A is the area of the insert (0.143 cm² for 96-well insert) CE is the estimated experimental concentration (Time = 0) of the dosing solution Crfinal is the concentration of the receiver at the end of the incubation period Cdfinal is the concentration of the donor at the end of the incubation period. c. Metabolic Stability over Liver Microsomes i. Method Assay summary: 1. Prepare 100X stocks of the test articles in a solvent solution. Attorney Docket No.: 131734-01120 ^ Dilute initial 10mM test article to 100µM by adding 2.5µL to a total volume of 250µL solvent solution (3:1 acentonitrile/water). ^ If the test article appears insoluble, 100% acetonitrile may be used as long as the final reaction concentration of any organic solvent is <1%. ^ These solutions become the ‘Cmpds’ plate in Figure 23. Thaw frozen microsomes (20mg/mL, Sekisui/Xenotech, Kansus City, KS) in a warm (37°C) water bath and adjust concentration to 0.5mg/mL in a phosphate/MgCl₂ buffer. ^ Prepare working buffer solution by addidng 330µL 1M MgCl2 to every 100mL of phosphate buffer (100mM potassium phosphate, 150mM sodium chloride, pH 7.4). Warm solution to 37°C. Adjust volumes based on magnitude of assay. ^ Prepare microsome solution by adding 2.5mL microsomes (20mg/mL) to every 100mL of warmed phosphate/MgCl₂ buffer. Adjust volumes based on magnitude of assay. ^ Use following microsome preparations containing >50 donor pools: mouse (pooled, male, CD-1), rat (pooled, male, Sprague-Dawley), dog (pooled, male, Beagle), monkey (pooled, male, cynomolgus) and human (pooled, mixed). ^ This solution becomes the ‘LMs’ plate in Figure 23. If using a slotted plate, multiple species of microsomes may be prepared together. Prepare a 10mM solution of NADPH (Nicotinamide adenine dinucleotide phosphate, Sigma-Aldrich, N1630) in phosphate/MgCl₂ buffer. ^ Prepare phosphate/MgCl2 buffer as described in step 2. ^ Prepare NADPH solution by the addition of 74.44mg NADPH to every 10mL of warmed phosphate/MgCl2 buffer. Adjust weights and volumes based on assay’s magnitude. ^ Warm to 37°C. ^ This solution becomes the ‘NADPH’ plate in Figure 23. Prepare a chilled ‘crash’ solution of internal standard in a solvent solution. Attorney Docket No.: 131734-01120 ^ Prepare a 50ng/mL solution of internal standard, CPDPX (8-Cyclopentyl-1,3- dipropylxanthine, Sigma-Aldrich, C101) in 1:1 acetotitrile/methanol solvent solution. ^ Chill on ice or refrigerate at 4°C. ^ This solution becomes the ‘Crash’ plate in Figure 23. Transfer 445µL of the microsome solution (0.5mg/mL) to the reaction plate followed by the addition of 5µL of the test articles (100µM), mix well. ^ Maintain reaction plate at 37°C throughout assay. Initiate reaction with the addition of 50µL of the NADPH solution (10mM) to the reaction plate, mix well. Transfer 160µL from the ‘Crash’ solvent solution with internal standard (CPDPX) to a final crash plate. Transfer 40µL from the reaction plate to the crash plate, mix well. ^ The first 40µL sample crashed from the reaction plate becomes time zero. Five additional aliquots of 40µL are removed after 5, 10, 15, 25 and 40 minutes from time zero into their own individual crash plates containing 160µL of the crash solution with internal standard for a total of 6 final crash plates. After assay completion, final crash plates are centrifuged refrigerated (4°C) for 10 minutes at around 4000rpm (3200 x g) on most benchtop centrifuges. Transfer aliquots of supernant from the centrifuged crash plates to LC-MS/MS ready plates. ^ Transfer 50µL of supernatant from each well of the centrifuged crash plates to a clean analytical sample plate containing 200µL of 0.1% formic acid, mix, then directly injected onto an LC/MS/MS system for sample analysis. ^ Volumes and diluent composition may be adjusted based on instrument (LC- MS/MS) sensitivity and test article sensitivity, solubility and polarity to ensure adequate signal and retention of test articles within the linear limitation of the instrument. Attorney Docket No.: 131734-01120 ii. Data Processing The ln peak area ratio (compound peak area/internal standard peak area) is plotted against time and the gradient (slope) of the line determined. Figure 24 is a representative graph. Following parameters are determined; Elimination rate constant: k = (-gradient) Half-life: t1/2 (min) = 0.693 / k Assay protein content: V (µL/mg) = volume of incubation (µL) / protein in the incubation (mg) Using above parameters, intrinsic clearance (CLint) is expressed as; CLint (µL/min/mg protein) = V x 0.693 / t_1/2 Intrinsic clearance reflects the capacity of microsomes to metabolize a drug. The amount of compound consumed during the microsome stability assay should be greater than 10% in order to measure rates of metabolism confidently. Metabolism rates ≤ 10% will be reported as an approximation. If scaling in vitro metabolic stability data, the amount of protein per gram of liver is a key factor that directly affects the calcualtion of intrinsic clearance values for the whole organ. iii. Data Reporting and Interpretation Classification bands typically used for categorizing compounds into low, medium or high clearance. iv. Validation Data Set The metabolic competency of the test system is validated by the inclusion of positive control compounds with a known stability under the above assay conditions. The table below show a list of control compounds for specific species of microsomes. Attorney Docket No.: 131734-01120 The table below shows CLint.exp (µL/min/mg protein) of a validation set of clinical drugs in multiple animal species of microsomes. d. Log D Assay Summary: Log D was measured by Analiza using its thermodynamic definition: the partition coefficient in buffer-octanol system in equilibrium using an automated and miniaturized version of the gold standard shake-flask method. ^ Sample Requirements: ^ Compounds must contain at least 1 nitrogen atom ^ 30 μL of 10mM DMSO stock ^ Octanol/Water 2 phase system: Octanol in equilibrium with Phosphate Buffered Saline (PBS) ^ pH: 7.4 Partitioning: For octanol/buffer partitioning, Analiza’s standard two phase system plates was used. Octanol in equilibrium with phosphate buffered saline (PBS), pH 7.4 was used to prepare partitioning plates for the assay. This buffer provided uniform ionic composition across a wide pH range. DMSO stock solutions was added to each partitioning plate to a final concentration of 10% DMSO in singleton. The plates were sealed, vortexed on Analiza’s specially designed deepwell plate mixer, and centrifuged to aid in phase settling. The assay was conducted on the ADW workstation using chemiluminescent nitrogen detection. Calculation of Results: The equimolar nitrogen response of the detector was calibrated using standards which span the dynamic range of the instrument from 0.08 to 4500 μg/ml nitrogen. Both the top and bottom phases were quantified with respect to this calibration curve and the logarithm of the ratio of the concentration in the top phase to the concentration in the bottom phase were calculated as Log D. In addition to the directly observed Log D value, the observed Log D value were adjusted to a corrected Log D* based upon Analiza’s work correlating Log D in the presence and absence of DMSO in the partitioning system. Attorney Docket No.: 131734-01120 The calculated Log D and Log D* values are corrected for background nitrogen in the DMSO and octanol-buffer 2-phase systems. e. Kpuu Assay In vivo For the brain-to-plasma partition coefficient (Kp) evaluation, a dosing solution was intravenously infused into animals at a constant flow rate for 4 to 24 h. Blood samples were serially collected during infusion, and CSF and brain samples were harvested at the end of infusion. For characterization of PK properties, a dosing solution was administered to animals via oral gavage or parenteral routes. Blood samples were collected after administration. Other biological samples, including tissue, bile, urine, and feces, can be collected during or at the end of the study if necessary. All the animal experiments were conducted in accordance with the internally approved animal protocols. Bioanalysis Tissue samples were typically homogenized in phosphate buffer saline (PBS) using a bead ruptor. CSF samples were typically diluted with 8% BSA in PBS to prevent from non- specific binding. Artificial CSF (aCSF) is used as the surrogate matrix. Dosing solutions were spiked into plasma for analysis when needed. Calibration curves were prepared by spiking the analyte(s) into blank matrices, which were processed together with plasma, tissue homogenate and/or CSF samples by protein precipitation using a proper organic solvent (e.g. acetonitrile and methanol) containing generic analogue internal standards (e.g. verapamil, chrysin and glyburide). Matrix matching was used when analyzing multiple matrices in the same run. Samples above the upper limit of quantitation (ULOQ) needed to be diluted into the calibration range using either a pre- extraction or post-extraction dilution approach. Processed samples were analyzed by LC-MS/MS using a proper method performing within the acceptable sensitivity, selectivity, precision and accuracy. For an analytical run to be accepted, over 75% of the calibration standards in the dual calibration curves needed to be within 20% of the nominal concentrations. Attorney Docket No.: 131734-01120 Compound- or study-specific bioanalytical methods that deviate from the typical procedure might be used when necessary, which will be documented in a study specific protocol included in the data upload. PK Plasma concentrations were analyzed by non-compartmental analysis (NCA) using a “Linear up log down” fitting to generate basic PK parameters that include but are not limited to volume of distribution (Vd), maximal concentration (Cmax), time to reach maximal concentration (Tmax), area under the curve (AUC), half-life (t1/2), clearance (CL) and bioavailability (F). The PK parameters were normalized to the adjusted dose when dosing solution analysis was conducted. Brain concentrations were compared against plasma concentrations at the corresponding timepoint for the calculation of partition coefficient (Kp). Unbound drug partition coefficient (Kpuu), defined as the ratio of unbound drug partition across the blood-brain barrier, was calculated using the equation below: C_b: measured total drug concentration in brain F_ub: unbound drug fraction in brain Cp: measured total drug concentration in plasma Fup: unbound drug fraction in plasma Compound- or study-specific PK analysis that deviates from the typical procedure might be used when necessary, which will be documented in a study specific protocol included in the data upload. Determination of Fraction Unbound (Fu): The unbound fraction of the test compound was determined based on the protocols described below. 1) Dilute initial 10mM test article to 125μM by adding 5μL to a total volume of 395μL solvent solution (100% acetonitrile) in a 1mL 96-well plate (Waters 186002481 Milford, MA). Ensure that the compounds are in solution. 2) Thaw frozen (rat, human, mouse, dog, and/or monkey) plasma (BIOIVT, Westbury, NY) in and warm the PBS buffer in a warm (37°C) water bath. Attorney Docket No.: 131734-01120 • Dilute the 125uM test article solutions by adding 8μL to a final volume of 992μL of plasma to make a final concentration of 1μM in a 2mL 96 well plate (Costar 3961). Mix thoroughly. • This spiked plasma solution is shown in Figure 25. 3) Prepare a chilled ‘crash’ solution of internal standard in a solvent solution. • Pipette 200μL of 25ng/mL solution of internal standard, CPDPX (8-Cyclopentyl- 1,3-dipropylxanthine, Sigma-Aldrich, C101) in 1:1 acetonitrile/methanol solvent solution into a 1mL 96-well plate. • Chill on ice or refrigerate at 4°C. • This solution becomes the ‘Crash’ plate in Figure 25. 4) From remaining spiked plasma, remove 50μL (T=0h) of each plasma sample and place into a crash plate which contains 200μL. To matrix match, add 50μL of blank buffer to the crashed sample (similar to PPB samples). Maintain remaining spiked plasma at 37˚C for 4h time point 5) Transfer 500μL of the warmed PBS buffer to the white side of the RED device (Thermo Scientific, Rockford IL, baseplate cat# 89811, insert cat# 89810) and 300 μL of the spiked plasma to the corresponding red ring side of the RED device. 6) Cover all the RED device plates with a lid and transfer them to a 37˚C incubator with 5% CO2 environment and shake at 200 rpm for 4 hours. 7) Reaction Termination after 4 hours: • Add 50μL of sample (plasma or buffer sample) and 50μL of the opposite blank matrix (add blank buffer to the plasma samples and blank plasma to the buffer samples) to crash plates (same as above) to the crash plate containing 200μL. Mix crash plate thoroughly. • From remaining spiked plasma, remove 50μL (T=4h) of each plasma sample and place into a crash plate. To matrix match, add 50μL of blank buffer to the crashed sample (similar to the protein binding samples). • Centrifuge crash plate at 3900rpm for 10 minutes at 4˚C (Eppendorf Centrifuge 5810R, Hamburg, Germany) 8) Sample preparation of LC/MS/MS: • Transfer 30μL of supernatant from the crash plates to 384-well plates containing 120μL of 0.1 formic acid in 90:10 water:acetonitrile using the PPB 96 to 384 pretty method on the Tecan. Inject into the LC/MS. Attorney Docket No.: 131734-01120 • Volumes and diluent composition may be adjusted based on instrument (LC- MS/MS) sensitivity and test article sensitivity, solubility, and polarity to ensure adequate signal and retention of test articles within the linear limitation of the instrument. 9) Standard Curve • Prepare standard curve of pooled test articles treated in a similar fashion as the reaction samples using plasma and buffer. 10) Data processing and analysis Multiquant will be chosen application used to process the data for PPB. Equations: Equation 1. Calculation of %Free (% PPBunb) % Free = (PAR of buffer side/PAR of plasma side)*100 PAR – Peak area ratio (PAR) Fu = % Free / 100 Fu = fraction unbound Equation 2. Final calculation using dilution factor (D) This dilution factor formula is used only if tissue or plasma is diluted. f. Data Summary The biological data collected in the above assays for the compounds of the disclosure are summarized in the table below. Compound A is a tool compound that is a cis-cyclooctene analogue of MICA-213 (See Ruivo, E., et al. ACS Omega 2020, 5, 9, 4449–4456). Compound A and MICA-21 demonstrate comparable results in their physicochemical or pharmacokinetic properties, i.e., plasma protein binding, brain tissue binding, and in vitro microsomal clearance. Attorney Docket No.: 131734-01120 Attorney Docket No.: 131734-01120 Biologic Example 2 - Assess Distribution and Kinetics of ¹⁸F labeled Compound 1 in Naïve Rat Brain Animal Model: 1. Naïve male Sprague Dawley rats (N = 17, 307 ± 41g) were supplied by Charles River. 2. All rats receive intravenous (IV) administration of ¹⁸F labeled Compound 1 at baseline (N = 2) or 24h following intrathecal (IT) administration of Compound 21 or ASO control (N = 15). 3. A summary of animal body weights and detailed study observations can be found in Raw Study Data Section below. Imaging Methods: Baseline acquisition 1. Naïve male Sprague Dawley rats (N = 2) were placed under terminal anesthesia (isoflurane ca.1.5 – 3%, 1 L oxygen∙min-1). 2. Cannulae were placed in an artery and a vein. 3. Body temperature was monitored and maintained, and the respiration rate was monitored throughout the experiment. 4. A dynamic PET scan of 60 minutes was carried out on each of the rats after IV injection of ¹⁸F labeled Compound 1, with a field of view focused on the brain and upper body. 5. Arterial blood samples were collected continuously for the first 1 – 2 minutes, followed by discrete samples at 5, 15, 30, 45 and 60 minutes and metabolite analysis was carried out on the extracted plasma to generate the tracer input function for kinetics analysis. 6. The rats were euthanized at the end of the scans. 7. Regions of interest were defined over 8 brain regions (frontal cortex, striatum, cortex, hypothalamus, thalamus, hippocampus, cerebellum and whole brain) to generate the time activity curves (TACs). 8. Different compartmental models were explored and used to generate the volume of distribution (VT) estimates. The most appropriate model was selected (MA1 with t* set to 15 min), as defined by the Akaike Information Criterion (AIC), goodness of fit and robustness of the outcome parameters. Attorney Docket No.: 131734-01120 Postdose acquisition 1. Naïve male Sprague Dawley rats (N = 15) were anesthetized under recovery anesthesia (isoflurane ca.1.5 – 3%, 1 L oxygen∙min-1) and a catheter inserted into the spinal canal for IT administration (30 μL dose followed by 40 μL saline flush) of ASO control or Compound 21 as follow: a. ASO control (N = 3), b. High dose (750 μg) of Compound 21 (N = 5), c. Low dose (250 μg) of Compound 21 (N = 3), d. Medium dose (500 μg) of Compound 21 (N = 4). 2. 24 h after IT administration, rats were placed under terminal anesthesia (isoflurane ca. 1.5-3%, 1 L oxygen.min-1) and a cannula was placed in a vein. 3. Body temperature was monitored and maintained, and the respiration rate was monitored throughout the experiment. 4. A dynamic PET scan of 20 minutes was acquired on each of the rats after IV injection of ¹⁸F labeled Compound 1, with a field of view focused on the brain and upper body. 5. Exsanguinated arterial blood sample was collected for radioactivity measurement at the end of the scan (in blood and plasma). 6. Regions of interest were defined over 8 brain regions (frontal cortex, striatum, cortex, hypothalamus, thalamus, hippocampus, cerebellum and whole brain) as well as heart, liver, muscle, cervical and thoracic spine, to generate time activity curves (TACs). Ex vivo tissue collection procedure: 1. Following the postdose scans, animals were euthanized by exsanguination followed by cervical dislocation. 2. For animals scanned at postdose, 10 tissue samples were collected: brain regions (frontal cortex, striatum, hippocampus, thalamus+ hypothalamus, cerebellum), spinal cord (cervical, thoracic, lumbar), liver and kidneys. Sample Disposition: 1. Tissue samples collected following postdose scans were placed into Lysing matrix D tubes, weighed, flash frozen in liquid nitrogen, stored at -80 ºC for further analysis. Attorney Docket No.: 131734-01120 Study Design Summary Table: Results Summed Images from 0 to 60 min of rats scanned at baseline (following IV injection of ¹⁸F labeled Compound 1 without IT dosing) are shown in Figure 5. Brain uptake could not be confirmed on these images. Similarly, the Time Activity Curves (TAC) in Figure 6 showed a peak of concentration (ca.3 SUV) in the 5 first minutes following the injection, followed by a rapid clearance, and low activity was observed in both rats after 10 min (between 0.10 and 0.16 SUV, Table A). Table A Concentration of ¹⁸F labeled Compound 1 in SUV in rat brain, as determined by PET imaging at baseline (average 10 – 60 min) The regional brain volumes of distribution (VT) were determined as outcome measure using the model found to be most suitable (multilinear analysis MA1). VT values were around 1 in all brain regions confirming the tracer ability to cross the blood-brain barrier and homogeneously diffuse to the brain tissues (Table B). Attorney Docket No.: 131734-01120 Table B Volume of distribution (VT) of ¹⁸F labeled Compound 1 in rat brain, estimated from MA1 modelling as determined by PET imaging at baseline (average 10 – 60 min) Summed Images from 0 to 20 min of subjects injected IT with the ASO control (Figure 7) and quantification of the tracer concentration in rat brain (Figure 8) showed a similar biodistribution of ¹⁸F labeled Compound 1 to that in the rats scanned at baseline. For rats injected IT with different doses of Compound 21 prior to ¹⁸F labeled Compound 1 scans, the tracer concentration in the brain was found to be the highest for the group treated with the high and medium doses of Compound 21 with up to 0.64 and 0.67 SUV respectively (Figure 9 to Figure 14 and Table C). Table C Average concentration (5 – 20 min) of ¹⁸F labeled Compound 1 in SUV in rat brain after IV injection, 24 h following IT administration of either ASO control, high (750 μg), low (250 μg) or medium (500 μg) dose of Compound 21. Heterologous distribution of ¹⁸F labeled Compound 1 was observed within the brain of animals dosed with Compound 21, with higher uptake in cerebellum and in hippocampus. Similarly, distribution in peripheral tissues showed similar uptake in liver, heart and muscle across groups, and an increase uptake in both thoracic and cervical spines for the post Compound 21 groups (Figure 15 to Figure 18 and Table D). Attorney Docket No.: 131734-01120 Table D Average concentration (5 – 20 min) of ¹⁸F labeled Compound 1 in SUV in rat tissues after IV injection, 24 h following IT administration of either ASO control, high (750 μg), low (250 μg) or medium (500 μg) dose of Compound 21 Conclusions This study aimed to evaluate the biodistribution of ¹⁸F labeled Compound 1 in naïve rat brain under baseline conditions, and following IT administration of either ASO control or escalating doses of Compound 21. Baseline scans showed that the tracer was brain penetrant and displayed fast plasma and tissue kinetics conducive to robust quantification. Rats administered with ASO control showed a similar biodistribution of tracer to that in rats scanned at baseline. Groups injected with different doses of Compound 21 showed a heterogenous distribution within the brain. Highest brain concentration was found to be for the animals administered with the highest and medium doses of Compound 21. It must be noted that some intragroup variability, which may be due to Compound 21 distribution following IT administration. Finally, liver was the peripheral tissue with the highest tracer uptake in all animals. Biologic Example 3 Cynomolgus monkeys were dosed i.v. with ¹⁸F labeled Compound 1 and baseline scans acquired by PET from 0-120 minutes post-injection. Figure 19A and B illustrate the baseline PET imaging of ¹⁸F labeled Compound 1 in the cynomolgus monkeys, which demonstrates a brain uptake (Cmax ~6 %ID) with rapid clearance. To determine non-specific binding of tracer in the brain, a second cohort was first pre- treated with a 1 mg/kg dose of non-radiolabeled Compound 1, followed 5 minutes later by PET tracer. Figure 20 shows that the baseline of time-activity curve ("TAC") of ¹⁸F labeled Attorney Docket No.: 131734-01120 Compound 1 closely overlaps with the pretreatment TAC, which suggests no measurable non-specific binding of tracer in the brain. Bioglogic Example 4 - In Vitro and Ex Vivo Characterization of Compound 21 I. Quantification of Compound 21 in Tissue Individual stock solution of MALAT1 ASO and Compound 21 were prepared at the concentration of 1 mg/mL. Standard and quality control (QC) working solutions were prepared by dilutions of stock solutions using ASO diluent (25 mM HFIP, 15 mM DMCHA, 100 µM EDTA, and 0.05 % rat plasma in water: ACN 90:10% v/v). Working solutions were spiked into blank matrix to yield calibration standards within the range of 1 – 1000 ng/mL. Rat brain samples were weighed and homogenized in 9-fold (v/w) of lysis-loading buffer using a MP Biomedicals FastPrep-24 homogenizer, yielding 10-fold diluted brain homogenates. An aliquot of 200 µL calibration standards, QC samples and brain samples were added to the KingFisher 96 deep-well plate.15 µL of probe-conjugated magnetic bead suspension was added to each sample to capture MALAT1 ASO and Compound 21. The beads were transferred and sequentially washed three times. Finally, the beads were transferred to a pre-heated elution plate containing 200 µL ASO diluent with 25 ng/mL internal standard and rigorously agitated for 15 min at 90 °C to generate the hybridization extract for LC-MS/MS analysis. For the analysis of reactive Compound 21, 10 µL of 500 µM TCO-PEG4-DBCO was added to each well to conjugate with Compound 21. After incubation at room temperature for 120 hours, the samples were analyzed by LC-MS/MS. An ExionLC AD UHPLC system with a Clarity 1.7 µm Oligo-XT 100 Å 50 × 2.1 mm column was used for the chromatographic separation of MALAT1 ASO, Compound 21, Compound 21-TCO-PEG4-DBCO and internal standard. The initial condition was 2% of mobile phase B, 98% of mobile phase A at 0.45 mL/min and 100% of mobile phase C at 0.05 mL/min. The initial condition was held for 0.2 min, the separation gradient ramped to 20% B within 5.5 min, while mobile phase C kept at 100% at 0.05 mL/min. After the gradient, the mobile phase was set to the initial condition and equilibrated for 1 min. Column temperature was set to 60 °C. The injection volume was 10 µL. MS/MS detection was conducted using a Sciex QTRAP 6500+ mass spectrometer equipped with an electrospray ionization (ESI) source at negative ion mode using multiple reaction monitoring (MRM). The optimized ion source parameters included curtain gas at 40, collision gas at high, ionspray voltage at -3500 V, temperature at 550 °C, ion source gas 1 at 60, ion source gas 2 at 60. MRM transitions Attorney Docket No.: 131734-01120 were 793.1 to 95 for Malat1 ASO, 754.7 to 95 for Compound 21, 819.1 to 95 for Compound 21-TCO-PEG4-DBCO, and 879.5 to 95 for the internal standard. II. Cell Uptake and Immunofluorescence Staining of Compound 21 The following protocol was adapted from Cook, et al.2022 (4). Summarized in brief, HeLa cells were cultured in Eagle's Minimum Essential Medium (EMEM) with 10% fetal bovine serum, and incubated at 37 °C, 5% CO₂. Cells were cultured for 24 hours in a 4-well cell culture slide (ThermoFisher) and stock solution of Compound 21 in PBS was added to a final concentration of 5 µM in the cell media. The cells were incubated again for 24 h. Cells were then fixed with 4% PFA solution in PBS for 15 min at room temperature (RT) and permeabilized using a 0.1% Triton X-100 solution prior to staining. Primary antibodies were diluted in PBST (EEA1 at 1:100; LAMP1 at 1:200) and each on added to a single well (100 µL) and incubated for 45 min at 37 °C. The secondary antibody (Goat anti-rabbit IgG-FITC) was diluted 1:60 in PBST-BSA. Also to this solution was added TCO-Cy5 (Click Chemistry Tools #1089) to a final concentration of 1 µM.100 µL of this solution was added to each well and incubated in the dark at RT for 1 h. 200 µL of 1x stock solution of Phalloidin-AF568 was added to each well and incubated for 20 min at RT. Wells were washed in PBS and then mounted using 30 µL of Prolong Gold + DAPI and allowed to cure overnight at RT in the dark. Confocal imaging data was acquired using a fully motorized Zeiss Axio Observer Z1 (Carl Zeiss, Jena, Germany) inverted imaging system using a spinning disk confocal scanner unit CSU-W1 (Yokogawa), equipped with a 63x objective lens and two Hamamatsu ORCA- Flash4.0 v2 sCMOS cameras for 2 channel simultaneous acquisition. Solid-state lasers (405, 488, 561, 647 and 725 nm) were coupled to the spinning head through a fiber optic. A piezo PZ-2150 XYZ motorized stage was used to acquire 3-D stacks. Slidebook (Intelligent Imaging Innovations, Dencer, USA) was used to acquire, view, scale, process, and export images for publication and for image analysis. III. Ex Vivo Autoradiography of Compound 21 Compound 21 (500 µg in 30 µL aCSF, 40 µL flush) was dosed i.t. in naïve female Sprague-Dawley rats (n=4). Rats were allowed to recover for 24 hours, after which they were euthanized by CO₂ inhalation and brains removed and frozen on crushed dry ice. They were then sectioned to 10 micron slices and mounted on glass slides. Sections were set aside for autoradiography and not fixed in PFA. These sections were then incubated with a solution of ¹⁸F labeled Compound 1 (5 nM) for 15 min before Attorney Docket No.: 131734-01120 being thoroughly washed with saline. Blocking solutions included 5 µM non-radioactive Compound 1 in addition to the radioactive tracer. After washing and developing on a phosphor plate, the distribution of signal shows high uptake on the periphery and meninges of the brain, with a slow diffusion towards the midbrain (Fig.26). Furthermore, this binding signal is eliminated when tissue is incubated with a solution containing a homologous block, as well as when tracer is incubated with brain sections from a naïve rat. Overall, this demonstrates that binding of the tracer is highly specific to the presence of Compound 21. IV. Longitudinal Distribution and Stability of Compound 21 in Rat Brain Compound 21 (500 µg in 30 µL aCSF, 40 µL flush) was dosed i.t. in naïve female Sprague-Dawley rats (n=10). Rats were allowed to recover for 24, 48, 96, 168, or 336 hours, after which they were euthanized by CO2 inhalation and brains removed, bisected into left and right hemispheres, and frozen on crushed dry ice. Right hemispheres were then sectioned to 10 micron slices and mounted on glass slides, while left hemispheres were homogenized and analyzed by LC-MS/MS. Brain sections were stained with TCO-Cy5 as described above. It was found that Compound 21 signal decreased from 7.07 µg/mL at day 1 to 0.87 µg/mL at day 7, and 0.10 µg/mL at day 14 (Fig.27). Notably, the concentration of Compound 21 (which includes non-reactive species) remained essentially identical to the concentration of Compound 21-TCO-PEG4-DBCO, indicating that there was no measurable decomposition of the MeTz. Any instability in the compound is likely due to some loss of the MeTz or linker altogether rather than oxidation or reduction of the tetrazine. Bioglogic Example 5 - Pretargeted PET Imaging in NHP The study was performed according to “Guidelines for planning, conducting and documenting experimental research” of Karolinska Institutet. For baseline experiments, two female cynomolgus non-human primates (NHPs) were used (NHP1, 5.2 kg; NHP2, 6.0 kg). The NHPs were housed in the Astrid Fagraeus laboratory (KM-F), Comparative Medicine Department at Karolinska Instituitet (Solna, Sweden). PET experiments were conducted using the MultiScan LFER 150 PET/CT system (Mediso Ltd.). Anesthesia was initiated by an intramuscular ketamine injection (10 mg/kg) and maintained by the administration of a mixture of sevoflurane, oxygen, and medical air after endotracheal intubation. Oxygen saturation, heart and respiratory rates, and blood pressure were continuously monitored all through the scan. Body temperature was maintained by a Bair Hugger-Model 505 (Arizant Healthcare Inc., MN) and monitored with an Attorney Docket No.: 131734-01120 esophageal thermometer. The head was immobilized throughout scanning with a fixation device and fluid balance was maintained by a continuous infusion of Ringer Acetate.¹⁸F labeled Compound 1 was injected as an intravenous bolus (158 MBq in NHP1, 152 MBq in NHP2) simultaneously with the start of PET data acquisition. Brain radioactivity was measured continuously for 125 minutes according to a preprogrammed series of 35 frames. For both NHPs, arterial blood sampling using a permanent catheter placed into the femoral artery, were performed at different time points for the measurement of blood and plasma radioactivity and radiometabolite HPLC analysis. Whole brain time-activity curves show rapid uptake in the brain followed by washout over the duration of the scan, with ~90% clearance by 60-minutes p.i. (<0.5 SUV). Arterial blood samples were taken at discrete timepoints throughout the duration of the scan and analyzed for percent parent fraction by radioHPLC (Fig.26). The parent tracer metabolizes quickly in vivo, with ~30% remaining at 15 minutes p.i. Importantly, the two major radiometabolites observed elute before the parent compounds, indicating increased polarity and a low likelihood of entering the brain to confound PET signal. While there was very little observed signal being retained in the brain, a self-blocking experiment was performed on an additional two NHPs to confirm the lack of specific binding in the absence of Compound 21. NHPs were dosed with 1 mg/kg Compound 1 as a self-block before injection of the tracer ¹⁸F labeled Compound 1. There was no observed change in tracer kinetics following self-block, indicating that there is no detectable off-target binding in the brain (Fig.32). For experiments including IT administration, one female (NHP4, 4.2 kg) and one male (NHP5, 7.0 kg) NHP were used. The intrathecal administration of the Compound 21 dosing was performed 24 h prior to ¹⁸F labeled Compound 1 administration. Following sedation with an intramuscular injection of ketamine (10 mg/kg), animals were positioned in abdominal recumbency, and a lumbar puncture was performed in the L4/L5 or L5/L6 intervertebral space using a 25G spinal needle with an introducer. Compound 21 (20 mg per animal) was dissolved in the aCSF (Bio-Techne) and injected over 2-6 minutes in volume of 2.4 mL. The PET experiment was carried out as described above.¹⁸F labeled Compound 1 was injected as an intravenous bolus (145 MBq in NHP4, 142 MBq in NHP5) simultaneously with the start of PET data acquisition. Venous blood sampling in NHP4 and arterial blood sampling in NHP5, using a permanent catheter placed into the femoral artery, were performed at different time points for the measurement of blood and plasma radioactivity and radiometabolite analysis. Attorney Docket No.: 131734-01120 Summed PET images of the whole PET acquisition were coregistered manually to the T1-weighted brain magnetic resonance (MR) image and regions of interest (ROI) were delineated manually for the whole brain, occipital cortex, caudate, putamen, ventral striatum, frontal cortex, white matter, thalamus, cerebellum, and hippocampus. The time-activity curves of brain regions were generated from dynamic PET data after the application of coregistration parameters to ROIs. Regional uptake was calculated as standardized uptake value (SUV), which is defined as uptake (Bq/mL)/injected radioactivity (Bq) × body weight (g). Fig.29 underwent the following processing to best highlight tracer uptake and distribution within the brain. Dynamic frames were averaged from 60-120 minutes post- injection to create static volumes, and SUVs were calculated as described above. For each NHP, registration was then performed between the static SUV volume and the T1-weighted MRI using Advanced Neuroimaging Tools (See Avants, B., et al., A reproducible evaluation of ANTs similarity metric performance in brain image registration. Neuroimage 54, 2033- 2044 (2011). In addition, each NHP MRI was registered to a template T1-weighted MRI generated from 18 cynomolgus monkeys (See Frey, S., et al., An MRI based average macaque monkey stereotaxic atlas and space (MNI monkey space). NeuroImage 55, 1435- 1442 (2011)). Each registration was visually inspected to confirm accurate alignment. The SUV data was then linearly resampled from native space to the template space and a Gaussian blurring kernel of 2 mm full width at half maximum (FWHM) was applied. Lastly, the template brain mask was used to mask the SUV images to show only the values within the brain. Fig.30 displays brain PET as processed above but without application of the brain mask. Higher uptake of PET tracer in the brain was observed in both animals receiving doses of Compound 21 (Fig.28). Critically, the distribution of PET signal is located primarily in cortical grey matter, which is expected given that the ASO was delivered intrathecally and is very similar to the observed distribution in rats. Biologic Example 6 - Metabolite Identification for Compound 2 In vitro metabolite profiling was performed in human, rat, and cynomolgus monkey hepatocytes (Lonza PN HUCS50P and RSCS01, Sekisui Xenotech PN PPCH2000). Compound 2 (10 µM) wasss incubated in DMEM containing 1 million cells/mL for 1 hour (human) or 0.5 hours (rat and monkey) at 37°C under 5% CO₂. The reaction was stopped using a 1:1 addition of ice-cold acetonitrile. After centrifugation, the supernatant was diluted Attorney Docket No.: 131734-01120 1:3 with water for analysis. LC-MS was performed using a Waters UPLC and a Sciex 5600 Triple-TOF MS, with a Waters Acquity HSS T3 column (50 mm x 2.1 µm, 1.8 µm particle size) at 50˚C with a 20 µL injection volume. A linear gradient of 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B) was used from 5 - 55% B over 5 min, at 0.45 mL/min, followed by a wash of 90% B. HCD collision energy of 35 was used, and data was processed using Sciex PeakView and MetabolitePilot software. Figure 31 summarizes results from metabolite identification study for Compound 2. Table at left shows percentage of each isolated metabolite from the different species of hepatocytes tested. Other Embodiments While aspects of the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

Attorney Docket No.: 131734-01120 CLAIMS What is claimed is: 1. A compound of Formula (I): (I), or a pharmaceutically acceptable salt thereof, wherein: is a single bond indicating cyclooctene is trans or cis; W is a radiolabel moiety comprising a radioisotope; Y¹ is -CH₂-, Ar, 4 to 10 membered heterocyclyl, ^@-SO₂-Ar-^@@, or ^@-C(=O)-Ar-^@@; wherein said Ar or 4 to 10 membered heterocyclyl represented by Y¹ or in the group represented by Y¹ is optionally substituted by one or more R^Y; ^@- indicates the point that connects with ; and ^@@- indicates the point that connects with W; wherein Ar is 6 to 10 membered aryl or 5 to 10 member heteroaryl; R^Y is halogen, C1-6alkyl, or oxo (as appropriate); is a bond, -(CH2)n-, Het, ^&-C(=O)-Het-^&&, ^&-C1-6alkylenyl-C(=O)-Het-^&&, ^&- C(=O)-C_1-6alkylenyl-Het-^&&, -NH-Het-NH-, -NH-Cyc-NH-, ^&-NR^L-Het-^&&, or ^&-Het-NR^L- ^&&; wherein Het is a 4 to 10 membered heterocyclyl; Cyc is 4-10 membered carbocyclyl; R^L is hydrogen or C1-6alkyl; n is 1, 2, or 3; ^&- indicates the point that connects with X¹; and ^&&- indicates the point that connects with Y¹; X¹ is a bond, -O-, -CH₂-, -N(R^X)-, ^{^}-(CH₂)₂O-^{^^}, or ^{^}-(CH₂)₂N(R^X) -^{^^}; wherein R^X is hydrogen or C1-6alkyl; ^{^}- indicates the point that connects with cyclooctene; and ^{^^}- indicates the point that connects with ; R¹ is hydrogen or -OH; R² is hydrogen; or X¹ and R², together with atoms to which they are attached form a moiety represented by formula A: wherein Z is a bond, -O-, or -NH-; indicates the point that connects with cyclooctene; Attorney Docket No.: 131734-01120 indicates the point that connects with ; wherein said heterocyclyl comprises 1-3 heteroatoms selected from oxygen, nitrogen, and sulfur; and said heteroaryl comprises 1-4 heteroatoms selected from oxygen, nitrogen, and sulfur. 2. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein the compound is represented by formula (IIA): (IIA). 3. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein the compound is represented by formula (IIB): 4. The compound of any one of claims 1 to 3, or a pharmaceutically acceptable salt thereof, wherein W is a ¹⁸F, ¹¹C-moiety, chelated ⁶⁸Ga, C1-6alkyl substituted by one or more ¹⁸F, or C_1-6alkoxyl substituted by one or more ¹⁸F. 5. The compound of any one of claims 1 to 4, or a pharmaceutically acceptable salt thereof, wherein W is ¹⁸F, C_1-4alkyl substituted by one ¹⁸F, or C_1-4alkoxyl substituted by one ¹⁸F. 6. The compound of any one of claims 1 to 5, or a pharmaceutically acceptable salt thereof, wherein W is ¹⁸F, -CH₂ ¹⁸F, or –OCH₂CH₂ ¹⁸F. 7. The compound of any one of claims 1 to 6, or a pharmaceutically acceptable salt thereof, wherein Y¹ is Ar, 6 to 9 membered heterocyclyl, ^@-SO2-Ar-^@@, or ^@-C(=O)-Ar-^@@; wherein said Ar or 6 to 9 membered heterocyclyl represented by Y¹ or in the group represented by Y¹ is optionally substituted by one to three R^Y; wherein Attorney Docket No.: 131734-01120 Ar is phenyl or 6-membered heteroaryl; R^Y is halogen, C1-4alkyl, or oxo (as appropriate). 8. The compound of any one of claims 1 to 7, or a pharmaceutically acceptable salt thereof, wherein Y¹ is selected from the group consisting of: . 9. The compound of any one of claims 1 to 8, or a pharmaceutically acceptable salt thereof, wherein is a bond, -(CH₂)_n-, Het, ^&-C(=O)-Het-^&&, ^&-C_1-4alkylenyl-C(=O)- Het-^&&, ^&-C(=O)-C1-4alkylenyl-Het-^&&, -NH-Het-NH-, -NH-Cyc-NH-, -NH-, ^&-NR^L-Het-^&&, or ^&-Het-NR^L-^&&; wherein Het is 4 to 8 membered monocyclic heterocyclyl, 6-9 membered spiro heterocyclyl, or 6-8 membered bridged heterocyclyl; Cyc is 4-8 membered cycloalkyl; R^L is hydrogen or C1-4alkyl; n is 1 or 2. 10. The compound of any one of claims 1 to 9, or a pharmaceutically acceptable salt thereof, wherein is a bond, -CH₂ -, Het, ^&-C(=O)-Het-^&&, ^&-C_1-2alkylenyl-C(=O)- Het-^&&, ^&-C(=O)-C1-2alkylenyl-Het-^&&, -NH-Het-NH-, -NH-Cyc-NH-, -NH-, ^&-NR^L-Het-^&&, or ^&-Het-NR^L-^&&; wherein Het is 4 to 6 membered monocyclic heterocyclyl, 6-7 membered spiro heterocyclyl, or 7-8 membered bridged heterocyclyl; Cyc is 4-6 membered monocyclic cycloalkyl; R^L is hydrogen or C1-2alkyl. Attorney Docket No.: 131734-01120 11. The compound of any one of claims 1 to 10, or a pharmaceutically acceptable salt thereof, wherein is selected from the group consisting of 12. The compound of any one of claims 1 to 11, or a pharmaceutically acceptable salt thereof, wherein X¹ is a bond, -O-, -CH₂-, -NH-, -N(CH₃)-, ^{^}-(CH₂)₂O-^{^^}, ^{^}-(CH₂)₂NH-^{^^}, or ^{^}- (CH₂)₂N(CH₃)-^{^^}. 13. The compound of any one of claims 1 to 11, or a pharmaceutically acceptable salt thereof, wherein X¹ is -O-, -NH-, -N(CH₃)-, ^{^}-(CH₂)₂O-^{^^}, ^{^}-(CH₂)₂NH-^{^^}, or ^{^}-(CH₂)₂N(CH₃)- ^{^^}. 14. The compound of any one of claims 1 to 11, or a pharmaceutically acceptable salt thereof, wherein X¹ and R², together with atoms to which they are attached form a moiety represented by formula A: wherein Z is -O- or -NH-. Attorney Docket No.: 131734-01120 15. The compound of any one of claims 1, 3, 7-11, and 14, or a pharmaceutically acceptable salt thereof, wherein the compound is represented by Formula (II): 16. The compound of claim 15, or a pharmaceutically acceptable salt thereof, wherein Y¹ is selected from the group consisting of . 17. The compound of claim 15 or 16, or a pharmaceutically acceptable salt thereof, wherein Y¹ is . 18. The compound of any one of claims 15 to 17, or a pharmaceutically acceptable salt thereof, wherein is selected from the group consisting of . 19. The compound of any one of claims 15 to 18, or a pharmaceutically acceptable salt thereof, wherein Attorney Docket No.: 131734-01120 20. The compound of any one of claims 15-19, or a pharmaceutically acceptable salt thereof, wherein the compound is . 21. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein the compound is represented by Formula (III): wherein W is ¹⁸F or -CH2¹⁸F; Y¹ is 6-membered heteroaryl; is a bond, Het, ^&-C(=O)-Het-^&&, ^&-C1-4alkylenyl-C(=O)-Het-^&&, ^&-C(=O)- C_1-4alkylenyl-Het-^&&; wherein Het is a 4 to 6 membered heterocyclyl; and X¹ is a bond, -O-, or -N(R^X)-; wherein R^X is hydrogen or C1-4alkyl. 22. The compound of claim 21, or a pharmaceutically acceptable salt thereof, wherein: Y¹ is pyridyl; is selected from the group consisting of X¹ is a bond, -O-, -NH-, or –N(CH₃)-. Attorney Docket No.: 131734-01120 23. The compound of claim 1 or 3, or a pharmaceutically acceptable salt thereof, wherein the compound is represented by Formula (IV): wherein W is ¹⁸F or –OCH2CH2¹⁸F; Y¹ is 6 to 9-membered heterocyclyl or ^@-C(=O)-Ar-^@@; wherein said Ar or 6 to 9- membered heterocyclyl represented by Y¹ or in the group represented by Y¹ is optionally substituted by one to three R^Y; wherein Ar is phenyl or 6-membered heteroaryl; R^Y is C_1-4alkyl or oxo (as appropriate); is a bond, -CH₂-, or ^&-C(=O)-Het-^&&; wherein Het is a 6 to 8 membered heterocyclyl; and X¹ is -CH2-, ^{^}-(CH2)2O-^{^^}, ^{^}-(CH2)2NH-^{^^}, or ^{^}-(CH2)2N(CH3)-^{^^}. 24. The compound of claim 23, or a pharmaceutically acceptable salt thereof, wherein Y¹ is selected from the group consisting of ; and is selected from the group consisting of 25. The compound of claim 23 or 24, or a pharmaceutically acceptable salt thereof, wherein W is ¹⁸F; Attorney Docket No.: 131734-01120 is a bond; and X¹ is ^{^}-(CH2)2NH-^{^^}, or ^{^}-(CH2)2N(CH3)-^{^^} 26. A compound of Table 1, or a pharmaceutically acceptable salt thereof. 27. A pharmaceutical composition comprising the compound of any one of claims 1-26, or a pharmaceutically acceptable salt, and a pharmaceutically acceptable carrier or excipient. 28. A method of determining the distribution of a biomolecule in a subject, the method comprising: (i) administering the biomolecule to the subject; (ii) administering to the subject the compound of any one of claims 1-26 or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition of claim 27; wherein the biomolecule binds the compound in vivo; and (iii) imaging the distribution of the biomolecule in the subject. 29. The method of claim 28, wherein the biomolecule is an antisense oligonucleotide, an antibody, a gene therapy agent, or a nanoparticle. 30. The method of claim 29, wherein the biomolecule is an antisense oligonucleotide. 31. The method of any one of claims 28-30, wherein the method determines the distribution of a biomolecule in the brain and/or spinal cord of a subject. 32. The method of any one of claims 28-31, wherein the biomolecule and the compound of any one of claims 1-26 or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition of claim 27, are administered to a subject simultaneously. Attorney Docket No.: 131734-01120 33. The method of any one of claims 28-31, wherein the biomolecule and the compound of any one of claims 1-26 or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition of claim 27, are administered to a subject separately. 34. The method of any one of claims 28-33, wherein the biomolecule is administered to the subject intrathecally and the compound of any one of claims 1-26 or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition of claim 27 is administered to the subject intravenously. 35. The method of any one of claims 28-34, wherein the imaging is performed by PET/CT. 36. The method of any one of claims 28-34, wherein the imaging is performed by SPECT. 37. The method of any one of claims 28-36, wherein the subject is a human.