Cyclooctene compositions and uses thereof

Abstract

The present disclosure provides a compound of formula (I) [Formula 1] TIFF2026500338000155.tif27165 or a pharmaceutically acceptable salt thereof, and uses thereof, for example, in assessing the biodistribution and / or concentration of a biomolecule in a subject.

Description

[Technical Field]

[0001] Related Applications This application claims the benefit under 35 U.S.C. § 119 of U.S. Provisional Application No. 63 / 433,107, filed December 16, 2022, U.S. Provisional Application No. 63 / 461,029, filed April 21, 2023, and U.S. Provisional Application No. 62 / 061,233, filed October 9, 2023, the entire contents of which are incorporated herein by reference.

[0002] The present disclosure generally relates to compositions and methods for assessing the distribution and / or concentration of a biomolecule, such as an antisense oligonucleotide, an antibody, or a gene therapy agent, in a subject. [Background technology]

[0003] Biomolecules such as antisense oligonucleotides (ASOs), antibodies, or gene therapy agents have proven highly effective in treating various diseases in subjects. To ensure their proper distribution and kinetics, it is necessary to develop imaging tools that are compatible with these biomolecules.

[0004] While directly radiolabeled biomolecules have proven effective, their application in human subjects has been limited by challenges associated with intrathecal administration of radiotracers and the constraints on longer imaging time points imposed by the half-life of radioisotopes. Pretargeted imaging ("PTI") using inverse electron demand Diels-Alder ("IEDDA") click chemistry has recently been applied to CNS compartments using unconventional therapeutic modalities. Cook, BE, demonstrated that click-responsive PET tracers can be developed to cross the blood-brain barrier and undergo in vivo click reactions within the brain and spinal cord 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 the need for further development of this tetrazine in nonhuman primates ("NHPs"), prompting the search for alternative strategies.

[0005] Therefore, there is a need to discover new imaging modalities that are more suitable for clinical use, ie, have low non-specific binding, fast clearance, etc. Summary of the Invention

[0006] This application relates to compounds, compositions, and methods for determining the distribution and / or concentration of a biomolecule (e.g., an ASO, an antibody, a gene therapy agent, or a nanoparticle) in a subject. The present disclosure provides compounds and compositions that are useful for assessing the distribution and dynamics of a biomolecule (e.g., an ASO, an antibody, a gene therapy agent, or a nanoparticle) in a subject. Also covered are methods for assessing the concentration of an antisense oligonucleotide in a desired region (e.g., the brain, the spinal cord, etc.) in a subject.

[0007] In one aspect, the present disclosure provides a compound of formula (I') or a pharmaceutically acceptable salt thereof: [ka] In the formula, R 1 , R2 , X 1 , Y 1 , W, and [ka] is as defined herein.

[0008] 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 instances, the compound of formula (I) is CNS permeable (i.e., it can pass through the blood-brain barrier). In certain instances, the compound of formula (I) is 18 F-labeled Compound 1. In some cases, the pharmaceutically acceptable carrier is phosphate buffered saline. In some cases, the pharmaceutically acceptable carrier is artificial cerebrospinal fluid (a-CSF). In some cases, the pharmaceutically acceptable carrier is sterile water for injection.

[0009] In another aspect, the present disclosure relates to a method for determining the distribution of a biomolecule in a subject, the method comprising administering the biomolecule to the subject, and subsequently administering to the subject a compound of the present disclosure, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of the present disclosure, wherein the biomolecule binds to the compound in vivo, and the method further comprises imaging the distribution of the biomolecule in the subject.

[0010] 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.

[0011] In other cases, the biomolecule of the present disclosure is an antibody (monovalent whole antibody, bispecific whole antibody), an antigen-binding fragment (Fab, Fab', F(ab)2, scFv, sc(Fv)2, diabody, nanobody), peptide, or nucleic acid (e.g., antisense oligonucleotide, siRNA, miRNA, shRNA, or aptamer).

[0012] 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. In practicing or testing the present invention, methods and materials similar or equivalent to those described herein can be used, among which 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 merely illustrative and are not limiting of the present invention.

[0013] Other features and advantages of the invention will be apparent from the following detailed description, and from the claims. [Brief explanation of the drawings]

[0014] [Figure 1] We demonstrate the second-generation PTI procedure of this disclosure, which utilizes in vivo inverse electron-demand Diels-Alder [4+2] (IEDDA) cycloaddition click ligation of radiolabeled trans-cyclooctene with a 1,2,4,5 tetrazine-conjugated biomolecule. [Figure 2] Dynamic PET scans in rats show rapid washout from the brain in animals administered a control ASO (approximately 0.18 SUV at 20 min), while ASO-methyltetrazine (compound 21) cohorts receiving three different doses (750 μg, 500 μg, and 250 μg) maintained activity in the brain (0.50–1.0 SUV at 20 min). [Figure 3A] PET / CT images show rats administered either Malat1 ASO (left) or compound 21 (right), followed 24 hours later by administration of 18F-labeled compound 1, and imaged 0-20 min. pI shows a click reaction with compound 21 compared to the control ASO. This suggests that quantitative imaging of compound 21 concentration is feasible. [Figure 3B]Graph showing ASO concentration in brain subregions relative to PET SUV normalized to blood activity. [Figure 3C] Graph showing ASO concentrations in the thalamus and hypothalamus relative to PET SUV normalized to blood activity. [Figure 3D] Graph showing ASO concentration in the cerebellum relative to PET SUV normalized to blood activity. [Figure 4] 1A-1C are PET / CT images showing specific uptake of tracer in the brain and spinal cord in rats treated with the second generation PTI procedure of the present disclosure. [Figure 5] PET / CT images showing the biodistribution of 18F-labeled Compound 1 after IV administration in rats 1 (A) and 2 (B). [Figure 6] Time activity curves and model fits of 18F-labeled Compound 1 in SUVs in rat brain from 0 to 60 minutes after tracer injection for rats 1 (A) and 2 (B). [Figure 7] PET / CT images showing the biodistribution of 18F-labeled Compound 1 after IV administration 24 hours after IT administration of ASO control in rats 8 (A), 9 (B), and 11 (C). [Figure 8] Time-radioactivity curves and model fits of Compound 1 in SUV in rat brain from 0 to 20 min after tracer injection 24 h after IT administration of ASO control in rats 8 (A), 9 (B), and 11 (C). [Figure 9] PET / CT images showing the biodistribution of 18F-labeled Compound 1 after IV administration 24 hours after IT administration of Compound 21 (750 μg) in rats 4 (A), 5 (B), 7 (C), 12 (D), and 19 (E). [Figure 10] Time activity curves and model fits of 18F-labeled compound 1 in SUVs in rat brain from 0 to 20 min after tracer injection 24 h after IT administration of compound 21 (750 μg) in rats 4 (A), 5 (B), 7 (C), 12 (D), and 19 (E). [Figure 11] PET / CT images showing the biodistribution of 18F-labeled Compound 1 after IV administration 24 hours after IT administration of Compound 21 (250 μg) in rats 14 (A), 16 (B), and 17 (C). [Figure 12] Time activity curves and model fits of 18F-labeled compound 1 in SUVs in rat brain from 0 to 20 min after tracer injection 24 h after IT administration of compound 21 (250 μg) in rats 14 (A), 16 (B), and 17 (C). [Figure 13] PET / CT images showing the biodistribution of 18F-labeled Compound 1 after IV administration 24 hours after IT administration of Compound 21 (500 μg) in rats 13 (A), 15 (B), 18 (C), and 20 (D). [Figure 14] Time activity curves and model fits of 18F-labeled compound 1 in SUVs in rat brain from 0 to 20 min after tracer injection 24 h after IT administration of compound 21 (500 μg) in rats 13 (A), 15 (B), 18 (C), and 20 (D). [Figure 15] Time activity curves and model fits of 18F-labeled Compound 1 in SUVs in rat tissues from 0 to 20 min after tracer injection 24 h after IT administration of ASO control in rats 8 (A), 9 (B), and 11 (C). [Figure 16] Time activity curves and model fits of 18F-labeled compound 1 in SUVs in rat tissues from 0 to 20 min after tracer injection 24 h after IT administration of compound 21 (750 μg) in rats 4 (A), 5 (B), 7 (C), 12 (D), and 19 (E). [Figure 17] Time activity curves and model fits of 18F-labeled compound 1 in SUVs in rat tissues from 0 to 20 min after tracer injection 24 h after IT administration of compound 21 (250 μg) in rats 14 (A), 16 (B), and 17 (C). [Figure 18]Time activity curves and model fits of 18F-labeled compound 1 in SUVs in rat tissues from 0 to 20 min after tracer injection 24 h after IT administration of compound 21 (500 μg) in rats 13 (A), 15 (B), 18 (C), and 20 (D). [Figure 19A] 1 is a reference PET image of 18F-labeled Compound 1 in cynomolgus monkeys. [Figure 19B] 1 is an exemplary baseline PET scan of 18F-labeled Compound 1 in cynomolgus monkeys over the periods 0-30 minutes and 0-120 minutes. [Figure 20] 1 is a graph showing the time-activity curve of Compound 1 in NHP brain. Baseline (blue, 18F-labeled Compound 1) and pre-treatment (red, pre-treatment with non-radiolabeled Compound 1) indicate dose overlap. [Figure 21] 1 shows the chemical structure of metastasis-associated lung adenocarcinoma transcript 1 ("MALAT1") with a C6 amine linker. [Figure 22] The chemical structures of three MALAT1-ASO-Tz analogs are shown. [Figure 23] FIG. 1 is a schematic diagram of the reaction solution, including volumes relative to the reaction plate and crush plate. [Figure 24] 1 is a graph showing the disappearance of a test compound over time in the presence of liver microsomes. [Figure 25] FIG. 1 is a schematic diagram of plasma and PBS buffer solution containing volumes for the RED plate and aliquoted for the crush plate. [Figure 26] 1 is a graph showing in vitro autoradiography of rats administered 500 μg of Compound 21 IT and naive rats. [Figure 27]Figure 1 shows the stability of compound 21. Longitudinal stability and clearance of compound 21 in whole rat brain after IT administration as measured by LC-MS. The graph shows total ASO concentrations (gray triangles), including the parent compound and any degradation products affected by the linker or tetrazine, compound 21 with unchanged linker and tetrazine mass (red squares), and compound 21-TCO-DBCO (blue circles), in which compound 21 was reactive to the TCO-DBCO ligand. [Figure 28] 1 shows the plasma parent fraction of 18F-labeled Compound 1 for each PET scan in NHPs and an exemplary radio-HPLC chromatogram 30 minutes after injection of NHP4. [Figure 29] From left to right, the graphs show an exemplary baseline scan with 18F-labeled Compound 1, an exemplary non-radioactive self-blocking scan with 1.0 mg / kg Compound 1, and pretargeting with 20 mg of Compound 21 administered IT, followed 24 hours later by tracer (NHP4 and NHP5). PET imaging is shown in NHPs. Images are summed from 60 to 120 minutes post-injection. SUV stills ranging from 0 to 2 are overlaid with a T1-weighted MRI of a standard template for cynomolgus monkeys at a transparency of 0.7. The SUV images are smoothed and masked with a 2 mm FWHM to show only values ​​within the brain mask template. [Figure 30] 1 shows pre-targeted PET imaging in cynomolgus monkeys without application of a brain mask. [Figure 31] Figure 1 shows the results of a metabolite identification study for compound 2. The table on the left shows the percentage of each isolated metabolite derived from hepatocytes of the different species tested. [Figure 32] Time-activity curves are shown for whole-brain PET regions of interest in NHPs. DETAILED DESCRIPTION OF THE INVENTION

[0015] The present disclosure relates, in part, to compounds and compositions useful for "pretargeting." "Pretargeting" separates the delivery of a radiolabeled compound / radioligand from the delivery of a modified biomolecule (e.g., an antibody, nanoparticle, gene therapy agent, ASO). These compounds and compositions can be used in vivo, for example, to assess the distribution and / or concentration of a biomolecule (e.g., an antibody, nanoparticle, gene therapy agent, ASO) in a subject. In some instances, the compounds and compositions are used to assess the distribution and / or concentration of a biomolecule (e.g., an antibody, nanoparticle, gene therapy agent, ASO) in the brain and / or spinal cord of a subject.

[0016] 1. Pretargeting Medical diagnosis and therapy commonly utilize imaging agents. Such agents can be useful, for example, to determine whether a therapeutic agent has reached its intended target, as well as to determine the location and / or concentration of a therapeutic or diagnostic agent. However, existing methods can be problematic. For example, the relatively slow pharmacokinetics of certain biomolecules used for imaging necessitates that the attached radiolabel have a half-life of several days. This is because, if the distribution of the biomolecule is to be assessed over a longer time period, sufficient radiation must remain for successful imaging. In some cases, this leads to high radioactivity concentrations and corresponding radiation doses in non-target organs. To circumvent these problems, an alternative approach called "pretargeting" has emerged, in which a radiolabeled compound or radioligand (e.g., radiolabeled cyclooctene) and a modified biomolecule (e.g., antibody, nanoparticle, gene therapy agent, ASO) are delivered to a subject separately.

[0017] Pretargeting methods generally involve the following steps: first, injection into a subject of a modified biomolecule (e.g., antibody, nanoparticle, gene therapy agent, ASO) that binds to or localizes to a target of interest but also has the ability to bind to a radioligand; second, the slow accumulation of the modified biomolecule (e.g., antibody, nanoparticle, gene therapy agent, ASO) at the site of the target and the accompanying clearance of the modified biomolecule (e.g., antibody, nanoparticle, gene therapy agent, ASO) or targeting agent from the blood; third, injection into the bloodstream of a radiolabeled compound or radioligand (e.g., a small molecule radioligand); and fourth, binding of the radiolabeled compound or radioligand to the biomolecule (e.g., antibody, nanoparticle, gene therapy agent, ASO), followed by rapid clearance of excess radioactivity. In some cases, an additional step is added prior to 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.

[0018] In one embodiment, the present disclosure provides compounds and compositions that can be used to penetrate the blood-brain barrier and thus have utility for pretargeting in the central nervous system.

[0019] An exemplary embodiment of applying pretargeting to a subject is shown in Figure 1B. As can be seen in this example, pretargeting separates the delivery of radioactivity from the delivery of biomolecules (e.g., antibodies, nanoparticles, gene therapy agents, ASOs). Biomolecules (e.g., antibodies, nanoparticles, gene therapy agents, ASOs) are modified with 1,2,4,5 tetrazine (Tz) and administered to a subject, while radioligands contain cyclooctene and are intravenously injected. Tz and cyclooctene undergo an inverse electron demand Diels-Alder (IEDDA) reaction in vivo, covalently binding the radioligand to the biomolecule (e.g., antibody, nanoparticle, gene therapy agent, ASO).

[0020] 2. Compound In a first embodiment, the present disclosure provides a compound of formula (I): [ka] or a pharmaceutically acceptable salt thereof, wherein: [ka] is a single bond indicating that the cyclooctene is trans or cis, W is a radiolabeled moiety containing a radioisotope; Y 1 is -CH2-, Ar, 4- to 10-membered heterocyclyl, @ -SO2-Ar- @@ ,or @ -C(=O)-Ar- @@ where Y 1 or Y 1 In the group represented by the formula (I), the Ar or 4-10 membered heterocyclyl may be one or more R Y and optionally substituted by @ teeth, [ka] and indicate the point where it connects to @@ indicates the point of connection with W, where Ar is a 6- to 10-membered aryl or a 5- to 10-membered heteroaryl; R Y is halogen, C 1-6 alkyl, or oxo (optionally); [ka] is the bond, -(CH2) n -,Het, & -C(=O)-Het- && , & -C 1-6 Alkylenyl-C(=O)-Het- && , & -C(=O)-C 1-6 Alkylenyl-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 a 4- to 10-membered carbocyclyl, and R L is hydrogen or C 1-6 alkyl, n is 1, 2, or 3; & is X 1 and indicate the point where it connects to && is Y 1 and indicate the point where it connects to X 1 is a bond, -O-, -CH2-, -N(R X )-, ^ -(CH2)2O- ^^ ,or ^ -(CH2)2N(R X )- ^^ where R X is hydrogen or C 1-6 is alkyl, ^ indicates the point of connection with cyclooctene, ^^ teeth, [ka] and indicate the point where it connects to R 1 is hydrogen or -OH, R 2 is hydrogen, or X 1 and R 2 together with the atoms to which they are attached form a moiety represented by formula A, [ka] wherein Z is a bond, —O—, or —NH—; [ka] indicates the point of connection with cyclooctene, [ka] teeth, [ka] and indicate the point where it connects to Here, the heterocyclyl contains 1 to 3 heteroatoms selected from oxygen, nitrogen, and sulfur, and the heteroaryl contains 1 to 4 heteroatoms selected from oxygen, nitrogen, and sulfur.

[0021] In a second embodiment, the present disclosure provides a compound according to the first embodiment or a pharmaceutically acceptable salt thereof, wherein the compound has the formula (IIA): [ka] The definitions of the variables are provided in the first embodiment.

[0022] In a third embodiment, the present disclosure provides a compound according to the first embodiment, or a pharmaceutically acceptable salt thereof, wherein the compound has the formula (IIB): [ka] The definitions of the variables are provided in the first embodiment.

[0023] In a fourth embodiment, the present disclosure provides a compound according to any one of the first to third embodiments, or a pharmaceutically acceptable salt thereof, wherein W is 18 F, 11 C moiety, chelation 68 Ga, one or more 18 C replaced by F 1-6 Alkyl or one or more 18 C replaced by F 1-6 and alkoxyl. The definitions of the remaining variables are as provided in any one of the first to third embodiments.

[0024] In a fifth embodiment, the present disclosure provides a compound according to any one of the first to fourth embodiments, or a pharmaceutically acceptable salt thereof, wherein W is 18 F, 1 piece 18 C replaced by F 1-4 Alkyl or one 18 C replaced by F 1-4 and alkoxyl. The definitions of the remaining variables are as provided in any one of the first to fourth embodiments.

[0025] In a sixth embodiment, the present disclosure provides a compound according to any one of the first to fifth embodiments, or a pharmaceutically acceptable salt thereof, wherein W is 18 F, -CH2 18 F, or -OCH2CH2 18 F. The definitions of the remaining variables are provided in any one of the first to fifth embodiments.

[0026] In a seventh embodiment, the present disclosure provides a compound according to any one of the first to sixth embodiments, or a pharmaceutically acceptable salt thereof, wherein: Y 1 is Ar, 6- to 9-membered heterocyclyl, @ -SO2-Ar- @@ ,or @ -C(=O)-Ar- @@ where Y 1 or Y 1In the group represented by the formula, the Ar or 6- to 9-membered heterocyclyl may be substituted with 1 to 3 R Y and optionally substituted by Ar is phenyl or 6-membered heteroaryl; R Y is halogen, C 1-4 alkyl, or oxo (optionally). The definitions of the remaining variables are provided in any one of the first to sixth embodiments.

[0027] In an eighth embodiment, the present disclosure provides a compound according to any one of the first to seventh embodiments, or a pharmaceutically acceptable salt thereof, wherein Y 1 teeth, [ka] The remaining variable definitions are provided in any one of the first to seventh embodiments.

[0028] In a ninth embodiment, the present disclosure provides a compound according to any one of the first to eighth embodiments, or a pharmaceutically acceptable salt thereof, wherein: [ka] is a bond, -(CH2) n -,Het, & -C(=O)-Het- && , & -C 1-4 Alkylenyl-C(=O)-Het- && , & -C(=O)-C 1-4 Alkylenyl-Het- && , -NH-Het-NH-, -NH-Cyc-NH-, -NH-, & -NR L -Het- && ,or & -Het-NR L - &&wherein Het is a 4- to 8-membered monocyclic heterocyclyl, a 6- to 9-membered spiroheterocyclyl, or a 6- to 8-membered bridged heterocyclyl; Cyc is a 4- to 8-membered cycloalkyl; and R L is hydrogen or C 1-4 alkyl, and n is 1 or 2. The definitions of the remaining variables are provided in any one of the first to eighth embodiments.

[0029] In a tenth embodiment, the present disclosure provides a compound according to any one of the first to ninth embodiments, or a pharmaceutically acceptable salt thereof, wherein: [ka] is a bond, -CH2-, Het, & -C(=O)-Het- && , & -C 1-2 Alkylenyl-C(=O)-Het- && , & -C(=O)-C 1-2 Alkylenyl-Het- && , -NH-Het-NH-, -NH-Cyc-NH-, -NH-, & -NR L -Het- && ,or & -Het-NR L - && wherein Het is a 4- to 6-membered monocyclic heterocyclyl, a 6- to 7-membered spiroheterocyclyl, or a 7- to 8-membered bridged heterocyclyl; Cyc is a 4- to 6-membered monocyclic cycloalkyl; and R L is hydrogen or C 1-2 alkyl. The definitions of the remaining variables are provided in any one of the first to ninth embodiments.

[0030] In an eleventh embodiment, the present disclosure provides a compound according to any one of the first to tenth embodiments, or a pharmaceutically acceptable salt thereof, wherein: [ka] teeth, [ka] The remaining variable definitions are provided in any one of the first to tenth embodiments.

[0031] In a twelfth embodiment, the present disclosure provides a compound according to any one of the first to eleventh embodiments, or a pharmaceutically acceptable salt thereof, wherein X 1 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 to eleventh embodiments.

[0032] In a thirteenth embodiment, the present disclosure provides a compound according to any one of the first to eleventh embodiments, or a pharmaceutically acceptable salt thereof, wherein X 1 -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 to eleventh embodiments.

[0033] In a fourteenth embodiment, the present disclosure provides a compound according to any one of the first to eleventh embodiments, or a pharmaceutically acceptable salt thereof, wherein X 1 and R 2 together with the atoms to which they are attached, have the formula A; [ka] forming a portion represented by wherein Z is -O- or -NH-. Definitions of the remaining variables are provided in any one of the first to eleventh embodiments.

[0034] In a fifteenth embodiment, the present disclosure provides a compound according to any one of the first, third, seventh to eleventh, and fourteenth embodiments, or a pharmaceutically acceptable salt thereof, wherein the compound has the formula (II): [ka] The remaining variable definitions are provided in any one of the first, third, seventh to eleventh, and fourteenth embodiments.

[0035] In a sixteenth embodiment, the present disclosure provides a compound according to the fifteenth embodiment, or a pharmaceutically acceptable salt thereof, wherein Y 1 teeth, [ka] The remaining variable definitions are provided in the fifteenth embodiment.

[0036] In a seventeenth embodiment, the present disclosure provides a compound according to the fifteenth or sixteenth embodiment, or a pharmaceutically acceptable salt thereof, wherein Y 1 teeth, [ka] The definitions of the remaining variables are provided in the fifteenth or sixteenth embodiments.

[0037] In an eighteenth embodiment, the present disclosure provides a compound according to any one of the fifteenth to seventeenth embodiments, or a pharmaceutically acceptable salt thereof, wherein: [ka] teeth, [ka] The remaining variable definitions are provided in the fifteenth through seventeenth embodiments.

[0038] In a nineteenth embodiment, the present disclosure provides a compound according to any one of the fifteenth to eighteenth embodiments, or a pharmaceutically acceptable salt thereof, wherein: [ka] teeth, [ka] or [ka] The definitions of the remaining variables are provided in the fifteenth through eighteenth embodiments.

[0039] In a twentieth embodiment, the present disclosure provides a compound according to any one of the fifteenth to nineteenth embodiments, or a pharmaceutically acceptable salt thereof, wherein the compound is [ka] The definitions of the remaining variables are provided in the fifteenth through nineteenth embodiments.

[0040] 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 has formula (III): [ka] wherein: W is 18 F or -CH2 18 F, Y 1 is a 6-membered heteroaryl; [ka] is a bond, Het, & -C(=O)-Het- &&, & -C 1-4 Alkylenyl-C(=O)-Het- && , & -C(=O)-C 1-4 Alkylenyl-Het- && wherein Het is a 4- to 6-membered heterocyclyl; X 1 is a bond, -O-, or -N(R X )-, wherein R X is hydrogen or C 1-4 and alkyl. Definitions of the remaining variables are provided in the twenty-first embodiment.

[0041] In a twenty-second embodiment, the present disclosure provides a compound according to the twenty-first embodiment, or a pharmaceutically acceptable salt thereof, wherein: Y 1 is pyridyl, [ka] teeth, join, [ka] is selected from the group consisting of X 1 is a bond, —O—, —NH—, or —N(CH3)—. Definitions for the remaining variables are provided in the twenty-first embodiment.

[0042] In a twenty-third embodiment, the present disclosure provides a compound according to the first or third embodiment, or a pharmaceutically acceptable salt thereof, wherein the compound has formula (IV): [ka] wherein: W is 18 F or -OCH2CH2 18 F, Y 1 is a 6- to 9-membered heterocyclyl, or @ -C(=O)-Ar- @@ where Y 1or Y 1 In the group represented by the formula, the Ar or 6- to 9-membered heterocyclyl may be substituted with 1 to 3 R Y and optionally substituted by Ar is phenyl or 6-membered heteroaryl; R Y is C 1-4 alkyl or oxo (optionally); [ka] is a bond, -CH2-, or & C(=O)-Het- && wherein Het is a 6-8 membered heterocyclyl; X 1 is -CH2-, ^ -(CH2)2O- ^^ , ^ -(CH2)2NH- ^^ ,or ^ -(CH2)2N(CH3)- ^^ The definitions of the remaining variables are provided in the first or third embodiments.

[0043] In a twenty-fourth embodiment, the present disclosure provides a compound according to the twenty-third embodiment, or a pharmaceutically acceptable salt thereof, wherein: Y 1 teeth, [ka] is selected from the group consisting of [ka] teeth, bond, -CH2-, [ka] and [ka] The remaining variable definitions are provided in the twenty-third embodiment.

[0044] 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 18 F, Y 1 teeth [ka] and [ka] is a bond, X 1 teeth, ^ -(CH2)2NH- ^^ ,or ^ -(CH2)2N(CH3)- ^^ The definitions of the remaining variables are provided in the 23rd or 24th embodiments.

[0045] In one embodiment, the present disclosure provides a compound selected from the compounds disclosed in the Examples and Table 1, a pharmaceutically acceptable salt or stereoisomer thereof. [Table 1-1] [Table 1-2] [Table 1-3]

[0046] Antisense oligonucleotide (ASO)-1,2,4,5-tetrazine fusion ("ASO-Tz") Provided herein are antisense oligonucleotides (ASOs) linked to 1,2,4,5-tetrazine ("ASO-Tz"). The 1,2,4,5-tetrazine can be directly linked to the ASO. In some cases, 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 linked to the ASO at the 5' end of the ASO, either directly or indirectly. The 1,2,4,5-tetrazine can also be linked to the ASO at the 3' end of the ASO, either directly or indirectly. In some cases, the ASO is linked to a linker (as defined herein) via a phosphorothioate bond.

[0047] In some embodiments, 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 stretches of nucleic acid that bind to ribonucleic acid (RNA), thereby altering or reducing expression of the target RNA. They can not only reduce protein expression by degradation of the target transcript, but also restore protein expression or modify proteins by interfering with pre-mRNA splicing. The present disclosure encompasses both types of ASOs. In certain instances, the ASO of the present disclosure is a "gapmer." Such ASOs act primarily by selectively cleaving mRNAs with complementary sites via an RNase H-dependent mechanism. They have a central region that supports RNase H activity, flanked by chemically modified termini that increase affinity and / or reduce susceptibility to nucleases. In some instances, the ASO of the present disclosure is a splice-switching oligonucleotide (SSO) (e.g., nusinersen). SSOs are generally fully modified to eliminate RNase H activity during splicing and allow interaction with nuclear pre-mRNA. They can be designed to bind to 5' or 3' splice junctions or to intra-exon splicing enhancer or silencer sites. By binding to such sites, they can modify splicing, for example, by promoting exon selective use, exon exclusion, or exon inclusion. [Table 2] "o" is a phosphodiester internucleoside linkage, and the absence of "o" indicates a phosphorothioate internucleoside linkage.

[0048] In some instances, the antisense oligonucleotide consists of 12 to 20 nucleosides (e.g., 12, 13, 14, 15, 16, 17, 18, 19, 20). In some cases, the antisense oligonucleotide is capable of crossing the blood-brain barrier. In some cases, the antisense oligonucleotide is useful for treating a neurodegenerative disorder. In some cases, the antisense oligonucleotide is useful for treating 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.

[0049] In one embodiment, the present disclosure provides ASO-Tz represented by Formula 1A, 1B, or 1C, a pharmaceutically acceptable salt, or stereoisomer thereof: [ka] wherein L is a bond or a linker, i.e., the Tz moiety is conjugated to the ASO directly or via a linker, such as one of the linkers described in this disclosure.

[0050] In one embodiment, the present disclosure provides ASO-Tz represented by formula 1D, 1E, or 1F, a pharmaceutically acceptable salt or stereoisomer thereof: [ka] wherein ASO is an antisense oligonucleotide connected via its 5'-terminal group (e.g., phosphate -OP(=O)(OH)-O-*, or phosphorothioate -OP(=S)(OH)-O-*, where -O-* indicates the point of attachment of the 5'-OH group of the oligonucleotide) to a C6 amine linker.

[0051] In one embodiment, the antisense oligonucleotide comprises 1 to 100 nucleotides. In one embodiment, the antisense oligonucleotide comprises 2 to 50 nucleotides. In one embodiment, the antisense oligonucleotide comprises 2 to 30 nucleotides. In one embodiment, the antisense oligonucleotide comprises 2 to 20 nucleotides. In one embodiment, the antisense oligonucleotide comprises 20 nucleotides.

[0052] In one embodiment, the present disclosure provides an ASO-Tz selected from the compounds disclosed in Table 2, a pharmaceutically acceptable salt or stereoisomer thereof. [Table 3]

[0053] 3.Definition The term "about" in the context of quantity, 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 1 week before and after the 4 month point. 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 temperatures means + / - 3°C.

[0054] The term "radioisotope," as used herein, refers to an isotope of an element that is known to undergo natural radioactive decay. Examples of radioisotopes include: 3 H, 14 C. 32 P, 35 S, 18 F, 36Examples of radioisotopes include Cl, and isotopes whose decay modes are identified in VS Shirley & CM 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 selected based on the medical use of the radiolabeled pharmaceutical agent. When the radioisotope is a metallic radioisotope, a chelating agent is typically used to bind the metallic radioisotope to the remainder of the molecule. When the radioisotope is a non-metallic radioisotope, the non-metallic radioisotope is typically bound to the remainder of the molecule directly or via a linker.

[0055] The term "halo" or "halogen," as used herein, refers to fluoride, chloride, bromide, or iodide.

[0056] The term “alkyl” used alone or as part of a larger moiety such as “alkoxy” or “haloalkyl” refers to a group of the formula —C n H (2n+1) "Ci_6 alkyl" refers to a straight-chain or branched-chain monovalent saturated aliphatic hydrocarbon radical. Unless otherwise specified, an alkyl group typically has 1 to 20, 1 to 10, or 1 to 6 carbon atoms. In some embodiments, an alkyl group has 1 to 6 atoms, i.e., a Ci_6 alkyl. As used herein, a "Ci_6 alkyl" group refers to a radical having 1 to 6 carbon atoms in a linear or branched arrangement. Examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, hexyl, and the like. In some embodiments, an alkyl group has 1 to 4 carbon atoms, i.e., a C 1-4 In some embodiments, the alkyl group has 1 to 3 carbon atoms, i.e., C 1-3 It is alkyl.

[0057] The terms "alkoxy" or "alkoxyl," as used herein, refer to an O-alkyl group, where alkyl is as defined above.

[0058] The term "haloalkyl" refers to an alkyl optionally substituted with one or more halogen atoms. In one embodiment, the alkyl can be substituted with 1 to 3 halogen atoms. Examples of haloalkyl include, but are not limited to, trifluoromethyl, trichloromethyl, pentafluoroethyl, and the like.

[0059] As used herein, the term "alkylene" refers to a group of the formula -C n H 2n - means a straight or branched chain divalent hydrocarbon radical of the formula: -. Non-limiting examples include ethylene and propylene.

[0060] The term "carbocyclyl" refers to any stable non-aromatic hydrocarbon ring containing 3 to 12 carbocyclyl members.

[0061] In one embodiment, a carbocyclyl is a 3-, 4-, 5-, 6-, 7-, or 8-membered monocyclic or bicyclic hydrocarbon ring or a 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 may 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, a carbocyclyl is intended to include bridged rings, fused rings, 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 can be fused together so 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.

[0062] The term "bridged carbocyclyl" refers to a 5- to 12-membered polycyclic carbocyclyl group in which any two rings in the group share two non-linking atoms, and the rings may have one or more double bonds but do not have a completely conjugated pi-electron system. Representative examples of bridged carbocyclyls include, but are not limited to, the following groups: [ka]

[0063] The term "cycloalkyl" refers to a monocyclic, bicyclic, tricyclic, or polycyclic saturated hydrocarbon group having 3 to 12 ring carbons. In one embodiment, a cycloalkyl can 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, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. A cycloalkyl can include multiple fused and / or bridged rings. Non-limiting examples of fused / bridged cycloalkyls 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, etc. Cycloalkyl also includes spirocyclic rings (e.g., spirocyclic bicyclos in which the two rings are connected through only 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.

[0064] The term "heterocyclyl" or "heterocyclic" refers to the radical of a 3- to 12-membered non-aromatic ring system having ring carbon atoms and 1 to 4 ring heteroatoms, where each heteroatom is independently selected from nitrogen, quaternary nitrogen, nitrogen oxide (e.g., NO), oxygen, and sulfur, including sulfoxide and sulfone (a "3- to 12-membered heterocyclyl"). In some embodiments, a heterocyclyl group is a 3- to 7-membered non-aromatic ring system having ring carbon atoms and 1 to 4 ring heteroatoms, where each heteroatom is independently selected from nitrogen, oxygen, and sulfur (a "3- to 7-membered heterocyclyl"). In heterocyclyl groups containing one or more nitrogen atoms, the point of attachment may be at a carbon atom or a nitrogen atom, where valence allows. Heterocyclyl groups can be either monocyclic ("monocyclic heterocyclyl") or polycyclic (e.g., bicyclic ("bicyclic heterocyclyl") or tricyclic ("tricyclic heterocyclyl"); polycyclic ring systems include fused, bridged, or spirocyclic 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 contain heteroatoms in one or more rings in the polycyclic ring system. Substituents can be present on one or more rings in the polycyclic ring system.

[0065] Spiroheterocyclyl refers to a 5-12 membered polycyclic heterocyclyl having rings connected through a common carbon atom (called a spiroatom), wherein the rings have one or more heteroatoms selected from the group consisting of nitrogen, quaternary nitrogen, nitrogen oxide (e.g., NO), oxygen, and sulfur, including sulfoxide and sulfone, with the remaining ring atoms being C, and one or more rings may contain one or more double bonds, and none of the rings has a fully conjugated pi-electron system. Representative examples of spiroheterocyclyl include the following groups: [ka] These include, but are not limited to:

[0066] Fused heterocyclyl refers to a 5-12 membered polycyclic heterocyclyl group in which each ring in the group shares an adjacent pair of carbon atoms with another ring in the group, one or more rings may contain one or more double bonds, but none of the rings has a completely conjugated pi-electron system, and the rings have one or more heteroatoms selected from the group consisting of nitrogen, quaternary nitrogen, nitrogen oxide (e.g., NO), oxygen, and sulfur, including sulfoxide and sulfone, with the remaining ring atoms being C. Representative examples of fused heterocyclyls include the following groups: [ka] These include, but are not limited to:

[0067] Bridged heterocyclyl refers to a 5-12 membered polycyclic heterocyclyl group in which any two rings in the group share two non-linking atoms, the rings may contain one or more double bonds but do not have a fully conjugated pi-electron system, and the rings have one or more heteroatoms selected from the group consisting of nitrogen, quaternary nitrogen, nitrogen oxide (e.g., NO), oxygen, and sulfur, including sulfoxide and sulfone, as ring atoms, with the remaining ring atoms being C. Representative examples of bridged heterocyclyls include the following groups: [ka] These include, but are not limited to:

[0068] In general, a carbocyclyl, cycloalkyl, or heterocyclyl can be unsubstituted or, valence permitting, substituted with one or more substituents, where 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, and optionally, alkyl substituents can be further substituted.

[0069] The term "aryl" refers to a 6- to 10-membered all-carbon monocyclic or polycyclic fused ring (a "fused" ring system means that each ring in the system shares an adjacent pair of carbon atoms with another ring in the group) group having a completely conjugated pi-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.

[0070] The term "heteroaryl," as used herein, refers to a monocyclic or polycyclic (e.g., bicyclic) aromatic hydrocarbon in which at least one of the ring carbon atoms is replaced with a heteroatom independently selected from oxygen, nitrogen, and sulfur. Preferably, heteroaryl refers to a C 1 -C 2 -C 3 -C 4 -C 5 -C 6 -C 7 -C 8 -C 9 -C 10 -C 11 -C 12 -C 13 -C 14 -C 15 -C 16 -C 17 -C 18 -C 19 -C 20 -C 21 -C 22 -C 23 -C 24 -C 25 -C 26 -C 27 -C 28 -C 29 -C 30 -C 31 -C 32 -C 33 -C 40 -C 41 -C 22 -C 34 -C 42 -C 25 -C 35 -C 43 -C 26 -C 36 -C 44 -C 27 -C 37 -C 45 -C 28 -C 29 -C 38 -C 45 -C 29 -C 39 -C 46 -C 29 -C 38 -C 47 -C 29 -C 39 -C 48 -C 49 -C 50 -C 51 -C 52 -C 53 -C 54 -C 55 -C 56 -C 57 -C 58 -C 59 -C 60 -C 61 -C 62 -C 63 -C 64 -C 65 -C 66 -C 67 -C 68 -C 70 -C 71 -C 72 -C 73 -C 74 -C 75 -C 76 -C 77 -C 78 -C 79 -C 80 5-10 Heteroaryl groups are based on aryl. Heteroaryl groups can be attached through a ring carbon atom or, where valence allows, through a ring nitrogen atom. In general, heteroaryls can be unsubstituted or substituted with one or more substituents, where valence allows. Exemplary substituents include, but are not limited to, halogen, OH, alkyl, alkoxyl, and amino (e.g., NH, NH alkyl, N(alkyl)), and optionally, alkyl can be further substituted.

[0071] Examples of 5- to 6-membered monocyclic 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., For example, 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, quinolinyl, benzotriazolyl, benzothiazolyl, benzoxazolyl, benzimidazolyl, isoquinolinyl, indolyl, isoindolyl, acridinyl, or benzisoxazolyl. A "substituted heteroaryl group" is substituted at any one or more substitutable ring atoms, which are ring carbon or ring nitrogen atoms to which hydrogen is bonded.

[0072] 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 indicates that any portion of the moiety known to those of skill in the art to be available for substitution can be substituted, including one or more substituents. When multiple substituents are present, each substituent can be independently selected. Means for such substitution are known in the art and / or taught by this disclosure. An optional substituent can be any substituent suitable for attachment to that moiety.

[0073] If suitable substituents are not specifically recited, exemplary substituents include C1-5 alkyl, C1-5 hydroxyalkyl, C1-5 haloalkyl, C1-5 alkoxy, C1-5 haloalkoxy, halogen, hydroxyl, cyano, amino, -CN, -NO2, -OR c1 , -NR a1 R b1 , -S(O) i R a1 , -NR a1 S(O) i R b1 , -S(O) i NR a1 R b1 , -C(=O)OR a1 , -OC(=O)OR a1 , -C(=S)OR a1 , -O(C=S)R a1 , -C(=O)NR a1 R b1 , -NR a1 C(=O)R b1 , -C(=S)NR a1 R b1 , -C(=O)R a1 , -C(=S)R a1 , N.R. a1 C(=S)R b1 , -O(C=O)NR a1 R b1 , -NR a1(C=S)OR b1 , -O(C=S)NR a1 R b1 , -NR a1 (C=O)NR a1 R b1 , -NR a1 (C=S)NR a1 R b1 , phenyl, or 5-6 membered heteroaryl. a1 and each R b1 is -H, and hydroxyl or independently selected from C alkyl optionally substituted with C alkoxy; c1 is -H, C1-5 haloalkyl, or C1-5 alkyl, where C1-5 alkyl is optionally substituted with hydroxyl or C1-C3 alkoxy.

[0074] The term "biomolecule," as used herein, refers to a molecule capable of binding to a biological target, such as an antigen. Biological molecules according to various embodiments of the present disclosure include, but are not limited to, antibodies, peptibodies, fusion proteins, ASOs, gene therapy agents, nanoparticles, muteins (i.e., mutant proteins), multispecific proteins, bispecific proteins, and biologically active fragments, analogs, derivatives, and variants, and biosimilars thereof.

[0075] [ka] The symbol as used herein refers to the point at which the moieties are attached.

[0076] The term "nucleoside," as used herein, refers to a molecule composed of a nucleobase and a ribose, deoxyribose, or a modified or locked ribose, such as LNA, or a modified or locked deoxyribose. The nucleobase is attached to a glycosidic carbon atom (1' position of the pentose), and internucleotide bonds (e.g., phosphate) are formed between the 3' oxygen or sulfur atom and, preferably, the 3' oxygen atom of the nucleoside, and between the 5' oxygen or sulfur atom and, preferably, the 5' oxygen atom of an adjacent nucleoside, while the internucleotide bonds do not belong to the nucleoside.

[0077] The term "nucleotide," as used herein, refers to a molecule composed of a nucleobase; a ribose, a deoxyribose, or a modified or locked ribose, such as LNA, or a modified or locked deoxyribose; and an internucleotide linkage, such as a phosphate. The nucleobase is attached to a glycosidic carbon atom (the 1' position of the pentose), and the internucleotide linkages are formed between the 3' oxygen or sulfur atom, preferably the 3' oxygen atom of the nucleotide, and between the 5' oxygen or sulfur atom, preferably the 5' oxygen atom of an adjacent nucleotide, while the internucleotide linkages are part of the nucleotide.

[0078] The term "DNA nucleotide" or "2-deoxyribonucleotide," as used herein, encompasses a DNA monomer comprising a 2-deoxyribose unit linked through its first carbon to a nitrogenous base selected from the group consisting of A, C, T, and G, and linked through its fifth carbon to a phosphate group or terminal group. As used herein, the term "DNA nucleotide" refers to a naturally occurring DNA nucleotide or a DNA nucleotide with a modified base, sugar, or phosphate-linked subunit.

[0079] The term "RNA nucleotide" or "ribonucleotide," as used herein, encompasses an RNA monomer comprising a ribose unit linked through its first carbon to a nitrogenous base selected from the group consisting of A, C, G, and U, and linked through its fifth carbon to a phosphate group or terminal group. As used herein, the term "RNA nucleotide" refers to naturally occurring RNA nucleotides or RNA nucleotides with modified base, sugar, or phosphate-linked subunits.

[0080] The term "nucleobase," abbreviated herein as "B," refers to the five standard nucleotide bases, adenine (A), thymine (T), guanine (G), cytosine (C), and uracil (U), as well as modifications or analogs thereof, or analogs capable of forming Watson-Crick base pairs with bases in a complementary strand. Modified nucleobases include 5-methylcytosine (C*), 5-hydroxymethylcytosine, N-methylcytosine (N*), 5-methylcytosine (N*), 5-hydroxymethylcytosine (N*), 5- ... 4-methylcytosine, xanthine, hypoxanthine, 7-deazaxanthine, 2-aminoadenine, 6-methyladenine, 6-methylguanine, 6-methyladenine, 6-methylguanine, 2-propyladenine, 2-propylguanine, 6-carboxyuracil, 5-halouracil, 5,6-dihydrouracil, 5-halocytosine, 5-propynyluracil, 5-propynylcytosine, 6-azauracil, 6- Azacytosine, 6-azathymine, 5-uracil (pseudouracil), 4-thiouracil, 8-fluoroadenine, 8-chloroadenine, 8-bromoadenine, 8-iodoadenine, 8-aminoadenine, 8-thioladenine, 8-thioalkyladenine, 8-hydroxyladenine, 8-fluoroguanine, 8-chloroguanine, 8-bromoguanine, 8-iodoguanine, 8-aminoguanine, 8-thio Other synthetic and natural nucleobases include 5-methylguanine, 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, with 5-methylcytosine and / or 2-aminoadenine substitutions being preferred since these modifications have been shown to increase the stability of nucleic acid duplexes. The preferred antisense oligonucleotide of the present disclosure can contain nucleobase analogs.The nucleobase of only one nucleotide unit of the antisense oligonucleotide can be replaced with a nucleobase analog, or two, three, four, five, or even all of the nucleobases in the antisense oligonucleotide can be replaced with a nucleobase analog.

[0081] When referring to a sequence of nucleotides or monomers, it is understood that the sequence refers to a sequence of bases, such as A, T, G, C, or U. The representation of an antisense oligonucleotide by the letter codes A, T, G, C, and U can be understood to mean that the antisense oligonucleotide can contain any of the nucleobases disclosed herein, any of the 3'-terminal groups disclosed herein, any of the 5'-terminal groups disclosed herein, and any of the internucleotide linkages (also referred to as internucleotide bridges) 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.

[0082] 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, allergic response, etc., and commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, S. M. Berge et al. describe pharmacologically acceptable salts in J. Pharm. Sci., 1977, 66, 1-19.

[0083] Pharmaceutically acceptable salts of the compounds of any one of the above formulas include acid addition and base salts.

[0084] Pharmaceutically acceptable salts of the compounds disclosed herein are included in the present teachings. Compounds having a basic group can form pharmaceutically acceptable salts with pharmaceutically acceptable acid(s). Suitable pharmaceutically acceptable acid addition salts of the compounds described herein include salts with inorganic acids (such as hydrochloric acid, hydrobromic acid, phosphoric acid, metaphosphoric acid, nitric acid, and sulfuric acid) and organic acids (such as acetic acid, benzenesulfonic acid, benzoic acid, ethanesulfonic acid, methanesulfonic acid, and succinic acid). Compounds of the present teachings that contain an acidic group, such as a carboxylic acid, can form pharmaceutically acceptable salts with pharmaceutically acceptable base(s). Suitable pharmaceutically acceptable base salts include ammonium salts, alkali metal salts (such as sodium salts and potassium salts), and alkaline earth metal salts (such as magnesium salts and calcium salts).

[0085] Pharmaceutically acceptable salts of compounds of any one of the above formulas can be prepared in three ways: (i) reacting a compound of any one of the above formulas with a desired acid or base; (ii) removing an acid- or base-labile protecting group from a suitable precursor of a compound of any one of the above formulae, or ring-opening a suitable cyclic precursor, such as a lactone or lactam, with a desired acid or base; or (iii) may be prepared by one or more of converting one salt of a compound of any one of the above formulas to another salt by reaction with an appropriate acid or base or by use of a suitable ion exchange column.

[0086] All three reactions are typically carried out in solution. The resulting salt precipitates and can be collected by filtration or recovered by evaporation of the solvent. The degree of ionization in the resulting salt can vary from completely ionized to nearly non-ionized.

[0087] Compounds of any one of the above formulas and their pharmaceutically acceptable salts may exist in unsolvated and solvated forms.

[0088] Stereoisomers and other variations Compounds of any one of the above formulas may exhibit one or more types of isomerism (e.g., optical isomers, geometric isomers, or tautomers). Such variations are intended to be implicit in any compound of any one of the above formulas defined by reference to its structural features and are therefore within the scope of this disclosure.

[0089] Compounds with one or more chiral centers can exist in various stereoisomeric forms, i.e., each chiral center can have an R or S configuration, or a mixture of both. Stereoisomers are compounds that differ only in 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 that have two or more chiral centers and are not identical or mirror images of each other.

[0090] If a compound is designated in its chemical name to represent a single enantiomer (e.g., when the configuration is indicated by "R" or "S" in the chemical name) or its structure (e.g., when the configuration is indicated by a "wedge" bond), unless otherwise indicated, 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 of the mixture of the named or designated enantiomers divided by the total weight of the mixture of both enantiomers.

[0091] Where the stereochemistry of a disclosed compound is named or depicted by a structure, and the named or depicted structure encompasses multiple stereoisomers (e.g., as in the case of a diastereomeric pair), it is understood to include one of the encompassed stereoisomers or any mixture of the encompassed stereoisomers. It is further understood that the stereoisomeric purity of the named or depicted stereoisomer is at least 60%, 70%, 80%, 90%, 99%, or 99.9% by weight. Stereoisomeric purity in this case is determined by dividing the total weight of the mixture of stereoisomers encompassed by the name or structure by the total weight of the mixture of all stereoisomers.

[0092] When two stereoisomers are designated by their chemical names or structures and the chemical names or structures are connected by "and," a mixture of the two stereoisomers is intended.

[0093] When two stereoisomers are depicted by their chemical names or structures and the chemical names or structures are connected by "or", either one or the other of the two stereoisomers is intended, but not both.

[0094] When disclosed compounds having chiral centers are shown with a structure that does not indicate the configuration at that chiral center, the structure is meant to encompass compounds in which the chiral center is in the S configuration, compounds in which the chiral center is in the R configuration, or compounds in which the chiral center is a mixture of R and S configurations. When disclosed compounds having chiral centers are shown with a chemical name that does not indicate the configuration at that chiral center with "S" or "R," the name is meant to encompass compounds in which the chiral center is in the S configuration, compounds in which the chiral center is in the R configuration, or compounds in which the chiral center is a mixture of R and S configurations.

[0095] A racemic mixture means 50% of one enantiomer and 50% of the corresponding enantiomer. When a compound with one chiral center is named or drawn by a structure without indicating the stereochemistry of the chiral center, the name or structure is understood to encompass both possible enantiomeric forms of the compound (e.g., both enantiomerically pure, enantiomerically enriched, or racemic). When a compound with two or more chiral centers is named or drawn by a structure without indicating the stereochemistry of the chiral centers, the name or structure is understood to encompass all possible diastereomeric forms of the compound (e.g., diastereomerically pure, diastereomerically enriched, and equimolar mixtures of one or more diastereomers (e.g., racemic mixtures)).

[0096] The term "geometric isomer" refers to isomers that differ in the orientation of substituent atoms with respect to a carbon-carbon double bond, a carbocyclic ring, or a bridged bicycle. Substituent atoms (other than hydrogen) on either side of a carbon-carbon double bond can be in either the E or Z configuration according to the Cahn-Ingold-Prelog precedence rules. In the "E" configuration, the highest priority substituents are on opposite sides of the carbon-carbon double bond. In the "Z" configuration, the highest priority substituents are on the same side of the carbon-carbon double bond.

[0097] Substituents around a carbon-carbon double bond may be referred to as "cis" or "trans," with "cis" representing substituents on the same side of the double bond and "trans" representing substituents on opposite sides of the double bond. For example, trans-cyclooctene is represented by the following structure: [ka] On the other hand, cis-cyclooctene has the following structure: [ka] It is expressed by:

[0098] The arrangement of substituents around a carbocyclic ring may also be referred to as "cis" or "trans." The term "cis" refers to substituents on the same side in relation to the plane of the ring, and the term "trans" refers to substituents on opposite sides in relation to the plane of the ring. Mixtures of compounds having substituents arranged on both the same and opposite sides in relation to the plane of the ring are termed "cis / trans."

[0099] Tautomeric isomerism ("tautomerism") can occur where structural isomers are interconvertible via a low energy barrier. This can take the form of proton tautomerism in compounds of any one of the above formulae containing an imino, keto, or oxime group, or so-called valence tautomerism in compounds containing an aromatic moiety. That is, a compound can exhibit more than one form of isomerism.

[0100] In certain cases, tautomeric forms of the disclosed compounds exist, for example, the tautomeric structures shown below: [ka]

[0101] When a geometric isomer is designated by name or structure, it is understood that the named or depicted isomer is present in greater amount than another isomer, and that the geometric isomer purity of the named or depicted geometric isomer is greater than 50% by weight, e.g., at least 60%, 70%, 80%, 90%, 99%, or 99.9% pure by weight. Geometric isomer purity is determined by dividing the weight of the named or depicted geometric isomer in a mixture by the total weight of all geometric isomers in the mixture.

[0102] Cis / trans isomers can be separated by conventional techniques well known to those skilled in the art, such as chromatography and fractional crystallization.

[0103] Conventional techniques for preparing / isolating individual enantiomers / diastereomers include chiral synthesis from suitable optically pure precursors or resolution of the racemate (or racemate of a salt or derivative) using, for example, chiral high-performance liquid chromatography (HPLC). Alternatively, the racemate (or racemic precursor) may be reacted with a suitable optically active compound, for example, an alcohol, or, if a compound of any one of the above formulas contains an acidic or basic moiety, with a base or acid such as 1-phenylethylamine or tartaric acid. The resulting diastereomeric mixture can be separated by chromatography and / or fractional crystallization, and one or both of the diastereoisomers can be converted to the corresponding pure enantiomer(s) by means well known to those skilled in the art. Chiral compounds of any one of the above formulas (and their chiral precursors) can be obtained in enantiomerically enriched form using chromatography, typically HPLC, on an asymmetric resin with a mobile phase consisting of 0-50% by volume, typically 2%-20% isopropanol, and a hydrocarbon, typically heptane or hexane, containing 0-5% by volume of an alkylamine, typically 0.1% diethylamine. Concentration of the eluate yields the enriched mixture. Chiral chromatography using subcritical and supercritical fluids can also 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 are available from Chiral Technologies, Inc., West Chester, Pa., USA, a subsidiary of Daicel® Chemical Industries, Ltd., Tokyo, Japan.

[0104] Although compounds of any one of the above formulas are depicted herein in a single tautomeric form, it is emphasized that all possible tautomeric forms are included within the scope of the present disclosure.

[0105] 4. Administration and Dosage Typically, the compounds of the present disclosure can be administered as the compound itself or, alternatively, as a pharmaceutically acceptable salt. For purposes of administration and dosage, the compound itself or a pharmaceutically acceptable salt thereof will simply be referred to as a compound of the present disclosure.

[0106] The compounds of the present disclosure are administered by any suitable route in the form of a pharmaceutical composition adapted to such a route, in a dose effective for the intended treatment. The compounds of the present disclosure can be administered orally, rectally, vaginally, or parenterally.

[0107] The compounds of the present disclosure can be administered orally, which may involve swallowing, so that the compound enters the gastrointestinal tract, or by buccal or sublingual administration by which the compound enters the blood stream directly from the mouth.

[0108] In another embodiment, the compounds of the present disclosure may also be administered directly into the bloodstream, muscle, or an internal organ. Suitable means for parenteral administration include intravenous, intraarterial, 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 administration regimen for the compounds of the present disclosure and / or compositions containing the compounds is based on various 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 administration regimen can vary widely. In one embodiment, the total daily dose of the compounds of the present disclosure is typically about 0.001 to about 100 mg / kg (i.e., mg of compound of the present disclosure per kg of body weight) for treatment of the indicated conditions discussed herein.

[0109] Suitable subjects according to the present disclosure include mammalian subjects, including non-human mammals, for example, primates, rodents (mouse, rat, hamster, rabbit, etc.). In one embodiment, a human is a suitable subject. Human subjects may be of either gender and at any stage of development.

[0110] 5. Pharmaceutical Compositions In another embodiment, the present disclosure includes pharmaceutical compositions. Such pharmaceutical compositions include the disclosed compounds, pharmaceutically acceptable salts, or stereoisomers thereof, together with a pharmaceutically acceptable carrier or excipient. Other pharmacologically active substances may also be present.

[0111] As used herein, "pharmaceutically acceptable carriers or excipients" includes any and all physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. Examples of pharmaceutically acceptable carriers include one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, and the like, and combinations thereof; isotonic agents, for example, sugars, sodium chloride, or polyalcohols such as mannitol or sorbitol, may be included 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.

[0112] The compositions of the present disclosure may be in a variety of forms, including, 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.

[0113] In another embodiment, the present disclosure comprises a parenteral dosage form.

[0114] "Parenteral administration" includes, for example, subcutaneous injection, intravenous injection, intraperitoneal injection, intramuscular injection, and infusion. Injectable preparations (i.e., sterile injectable aqueous or oily suspensions) can be formulated according to known techniques using suitable dispersing agents, wetting agents, and / or suspending agents.

[0115] Typical compositions are in the form of injectable or infusible solutions, such as compositions similar to those commonly used for passive immunization of humans with antibodies. One mode of administration is parenteral (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular). In another embodiment, the antibody is administered by intravenous infusion or injection. In yet another embodiment, the antibody is administered by intramuscular or subcutaneous injection.

[0116] Oral administration of solid dosage forms can be provided in separate 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, oral administration can be in powder or granular form.In another embodiment, oral dosage forms are sublingual, such as lozenges.In such solid dosage forms, the compound of any one of the above formulas is usually combined with one or more adjuvants.Such capsules or tablets can contain controlled-release formulations.In the case of capsules, tablets, and pills, dosage forms can also contain buffering agents or be prepared with enteric coatings.

[0117] In another embodiment, oral administration can be in liquid dosage form.Liquid dosage forms for oral administration include, for example, pharmaceutically acceptable emulsions, solutions, suspensions, syrups and elixirs, which contain inert diluents commonly used in the art (e.g., water).Such compositions can also contain auxiliary agents such as wetting agents, emulsifying agents, suspending agents, flavoring agents (e.g., sweeteners), and / or flavoring agents.

[0118] The present disclosure also provides pharmaceutical compositions comprising the radiolabeled cyclooctene compounds described herein. In certain instances, such pharmaceutical compositions comprise or consist of sterile saline and the radiolabeled cyclooctene compounds described herein. In some instances, such pharmaceutical compositions are sterile, buffered, isotonic solutions. In some instances, the pharmaceutical compositions are preservative-free.

[0119] The radiolabeled cyclooctene compounds described herein may be admixed with pharmaceutically acceptable active and / or inactive substances for the preparation of pharmaceutical compositions or formulations. The compositions and methods for formulating pharmaceutical compositions depend on several criteria, including, but not limited to, the route of administration, the extent of the disease, or the dose to be administered.

[0120] 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 instances, the radiolabeled cyclooctene compounds described herein are formulated in phosphate buffered saline (PBS). In other instances, the radiolabeled cyclooctene compounds described herein are formulated in artificial cerebrospinal fluid (a-CSF). In yet other instances, the radiolabeled cyclooctene compounds described herein are formulated in sterile water for injection.

[0121] For intranasal administration or inhalation administration, the compound of the present disclosure can be conveniently delivered in the form of solution or suspension from a pump spray container that is pushed or pumped by the patient, or in the form of aerosol spray from a pressurized container or nebulizer with the use of suitable propellant.The formulation suitable for intranasal administration is typically administered in the form of dry powder from a dry powder inhaler (either alone, as a mixture (for example, dry blend with lactose), or as mixed component particles (for example, mixed with phospholipids such as phosphatidylcholine)), or as aerosol spray from a pressurized container, pump, spray, atomizer (preferably, electrohydrodynamic atomizer to generate fine mist), or nebulizer with or without the use of suitable propellant such as 1,1,1,2-tetrafluoroethane or 1,1,1,2,3,3,3-heptafluoropropane.For intranasal administration, powder can contain bioadhesive agent, for example, chitosan or cyclodextrin.

[0122] In another embodiment, the present disclosure includes a rectal dosage form. Suitable rectal dosage forms may be, for example, in the form of a suppository. Cocoa butter is a traditional suppository base, although various alternatives may be used as appropriate.

[0123] Other carrier materials and dosage forms known in the pharmaceutical art may also be used. The pharmaceutical compositions of the present disclosure may be prepared by any of the well-known techniques of pharmacy, including effective formulation and administration procedures.

[0124] The above considerations regarding effective formulation and administration procedures are well known in the art and are described in standard textbooks. Drug formulations are described, for example, in Hoover, John E., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1975; Liberman et al., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, NY, 1980; and Kibbe et al., Eds., Handbook of Pharmaceutical Excipients ( 3rd Ed.), American Pharmaceutical Association, Washington, 1999.

[0125] 6. Methods for assessing biomolecule distribution The present disclosure features methods for assessing the distribution of a biomolecule (e.g., an antibody, nanoparticle, gene therapy agent, ASO) in a subject (e.g., a human). In some instances, the distribution of the biomolecule (e.g., an antibody, nanoparticle, gene therapy agent, ASO) is investigated in the brain and / or spinal cord of the subject. The method involves administering a modified biomolecule (e.g., an antibody, nanoparticle, gene therapy agent, ASO) to the subject. In some embodiments, the modified biomolecule (e.g., an antibody, nanoparticle, gene therapy agent, ASO) is a biomolecule (e.g., an antibody, nanoparticle, gene therapy agent, ASO) conjugated to methyltetrazine. In some embodiments, the modified biomolecule (e.g., an antibody, nanoparticle, gene therapy agent, ASO) is administered intravenously. In some embodiments, the modified biomolecule (e.g., an antibody, nanoparticle, gene therapy agent, ASO) is administered intrathecally, particularly in cases where the distribution of the biomolecule (e.g., an antibody, nanoparticle, gene therapy agent, ASO) is investigated in the brain and / or spinal cord. In some embodiments, the modified biomolecule (e.g., antibody, nanoparticle, gene therapy agent, ASO) is formulated in PBS or a-CSF. The method further involves administering to a subject a radiolabeled cyclooctene compound described herein. In some embodiments, the cyclooctene compound is radiolabeled with any radionuclide or radioisotope known in the art for diagnostic imaging (as described herein). In some embodiments, the cyclooctene compound is radiolabeled with a radionuclide that decays exclusively or almost exclusively by electron emission. In some embodiments, the cyclooctene compound is radiolabeled with a radionuclide having a short half-life (less than 12 hours) or a medium half-life (about 12-18 hours). In some embodiments, the cyclooctene compound is radiolabeled with fluorine-18, carbon-11, or gallium-68. In some embodiments, the cyclooctene compound is radiolabeled with fluorine-18. In some cases, the cyclooctene compound is a compound as described, for example, 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 bound 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 chelating agent 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.

[0126] The timing of administration of the radiolabeled cyclooctene compound depends on the half-life of the biomolecule (e.g., antibody, nanoparticle, gene therapy agent, ASO). In some embodiments, the radiolabeled cyclooctene compound is administered within 24 hours, up to 1 day (including), up to 2 days (including), up to 3 days (including), up to 4 days (including), up to 5 days (including), up to 6 days (including), up to 7 days (including), up to 8 days (including), up to 9 days (including), or up to 12 hours (including), or up to 14 days (including), or up to 16 days (including), or up to 18 ... The subject is administered up to 10 days (including the same day), up to 11 days (including the same day), up to 12 days (including the same day), up to 13 days (including the same day), up to 14 days (including the same day), up to 15 days (including the same day), up to 16 days (including the same day), up to 17 days (including the same day), up to 18 days (including the same day), up to 19 days (including the same day), or up to 20 days (including the same day). In certain instances, the radiolabeled cyclooctene compound is administered about 1 to about 2 days, about 1 to about 3 days, about 1 to about 4 days, about 1 to about 5 days, about 1 to about 6 days, about 1 to about 7 days, about 1 to about 10 days, about 1 to about 14 days, about 1 to about 20 days, about 1 to about 24 days, or about 1 to about 32 days after administration of the modified biomolecule (e.g., antibody, nanoparticle, gene therapy agent, ASO). In one instance, the radiolabeled cyclooctene compound is administered to the subject about 24 hours after administration of the modified biomolecule (e.g., antibody, nanoparticle, gene therapy agent, ASO). In some cases, 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 administration of the modified biomolecule (e.g., antibody, nanoparticle, gene therapy agent, ASO).

[0127] In some cases, an additional step is added prior to injection of the radioligand, specifically the administration of a clearance agent designed to accelerate the removal of residual biomolecules (i.e., any targeting agent not bound to the target) from the bloodstream.

[0128] In certain instances, the subject is imaged after administration of the radiolabeled cyclooctene compound. In certain instances, the subject is imaged after administration of the radiolabeled cyclooctene compound based on the PK of the radiolabeled cyclooctene compound. In some instances, the imaging is performed 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 instances, the imaging is performed about 30 minutes to about 1 hour, about 1 hour to about 1.5 hours, about 2 hours, about 3 hours, about 4 hours, or about 5 hours after injection of the radiotracer. In certain instances, imaging is performed by any suitable imaging modality 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 imaging techniques.

[0129] In certain instances, the subject includes any human or non-human mammal, hi certain non-limiting embodiments, the subject is a non-human primate, sheep, dog, cat, rabbit, horse, cow, or rodent.

[0130] In certain instances, the subject is a human subject. In certain instances, the human subject is a pediatric patient. In certain instances, the human subject is an infant. In certain instances, the human subject is an adult patient (i.e., 18 years of age or older). In some instances, the human subject has a CNS disorder. In certain instances, the CNS disorder is a synucleinopathy or tauopathy. In some instances, 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.

[0131] In some cases, the distribution of a biomolecule (e.g., antibody, nanoparticle, gene therapy agent, ASO) is assessed within the CNS (e.g., cortex, striatum, thalamus, substantia nigra, cerebellum) of a human subject.

[0132] 7. Methods for investigating biomolecule concentrations Also addressed are methods for measuring the concentration of a biomolecule (e.g., an antibody, nanoparticle, gene therapy agent, ASO) in a target region (e.g., the brain and / or spinal cord) of a subject (e.g., a human). The method involves administering a modified biomolecule (e.g., an antibody, nanoparticle, gene therapy agent, ASO). In some instances, the modified biomolecule (e.g., an antibody, nanoparticle, gene therapy agent, ASO) conjugated to methyltetrazine is administered intravenously. In certain instances, particularly when the distribution of the ASO is assessed within the brain and / or spinal cord, the methyltetrazine-conjugated ASO is administered intrathecally. In certain instances, the methyltetrazine-conjugated ASO 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-penetrating compound. In some instances, the cyclooctene compound is radiolabeled with a radionuclide that decays exclusively or almost exclusively via positron emission tomography (PET). In some cases, the cyclooctene compound is radiolabeled with a radionuclide having a short half-life (less than 12 hours) or a medium half-life (about 12-18 hours). In some cases, the cyclooctene compound is radiolabeled with fluorine-18, carbon-11, or gallium-68. In some cases, the cyclooctene compound does not contain a chelator. In some instances, the radiolabeled cyclooctene compound is administered intravenously. In some instances, the radiolabeled cyclooctene compound is formulated in PBS or a-CSF. In some cases, an additional step is added before injection of the radioligand, specifically, administration of an efflux 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 the tissue concentration of the biomolecule (e.g., antibody, nanoparticle, gene therapy agent, ASO) in the subject (e.g., the subject's brain and / or spinal cord).

[0133] The timing of administration of the radiolabeled cyclooctene compound depends on the respective half-life of the biomolecule (e.g., antibody, nanoparticle, gene therapy agent, ASO). In some instances, the radiolabeled cyclooctene compound is administered within 24 hours, up to 1 day (including the same day), up to 2 days (including the same day), up to 3 days (including the same day), up to 4 days (including the same day), up to 5 days (including the same day), up to 6 days (including the same day), up to 7 days (including the same day), up to 8 days (including the same day), up to 9 days (including the same day) after administration of the modified biomolecule (e.g., antibody, nanoparticle, gene therapy agent, ASO). The subject is administered the medicament of claim 1 by up to 10 days (including the same day), up to 11 days (including the same day), up to 12 days (including the same day), up to 13 days (including the same day), up to 14 days (including the same day), up to 15 days (including the same day), up to 16 days (including the same day), up to 17 days (including the same day), up to 18 days (including the same day), up to 19 days (including the same day), or up to 20 days (including the same day). In certain embodiments, the radiolabeled cyclooctene compound is administered about 1 to about 2 days, about 1 to about 3 days, about 1 to about 4 days, about 1 to about 5 days, about 1 to about 6 days, about 1 to about 7 days, about 1 to about 10 days, about 1 to about 14 days, about 1 to about 20 days, about 1 to about 24 days, or about 1 to about 32 days after administration of the modified biomolecule (e.g., antibody, nanoparticle, gene therapy agent, ASO). In one example, the radiolabeled cyclooctene compound is administered to the subject about 24 hours after administration of the ASO conjugated to trans-cyclooctene. In some cases, 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 administration of the modified biomolecule (e.g., antibody, nanoparticle, gene therapy agent, ASO).

[0134] In some instances, imaging is performed 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 instances, imaging is performed about 30 minutes to about 1 hour, about 1 hour to about 1.5 hours, about 2 hours, about 3 hours, about 4 hours, or about 5 hours after injection of the radiotracer. In certain instances, imaging is performed by any suitable 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 imaging technique.

[0135] From the imaging data, an uptake value of the radiolabeled cyclooctene compound for each region of interest can be calculated, which can then be applied to either an equation or an empirically compiled reference lookup table to obtain the corresponding concentration of the biomolecule (e.g., antibody, nanoparticle, gene therapy agent, ASO) in the tissue.

[0136] In some cases, the concentration of a biomolecule (e.g., antibody, nanoparticle, gene therapy agent, ASO) is assessed within the subject's CNS (e.g., cortex, striatum, thalamus, substantia nigra, cerebellum).

[0137] In certain instances, the subject is a human subject. In certain instances, the human subject is a pediatric patient. In certain instances, the human subject is an infant. In certain instances, the human subject is an adult patient (i.e., 18 years of age or older). In some instances, the human subject has a CNS disorder. In certain instances, the CNS disorder is a synucleinopathy or tauopathy. In some instances, 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.

[0138] 8. Kit The present disclosure further provides kits that can be used to practice the methods disclosed herein. For example, the kits can include at least one targeting probe (e.g., a modified biomolecule (e.g., an antibody, a nanoparticle, a gene therapy agent, an ASO)) and / or at least one labeled probe (e.g., a radiolabeled cyclooctene compound described herein). In certain embodiments, the kits can optionally include instructions for using the kit for molecular imaging. In certain cases, the kits can further include an administration device, such as a syringe and / or a catheter and / or an introducer sheath.

[0139] The present disclosure further provides a kit for preparing a targeting probe and / or a labeling probe. In certain cases, the kit of the present invention contains a targeting probe (in dry or liquid form) and / or a labeling probe (in dry or liquid form) for application to a biological material. When the probe is provided in dry form, the kit may contain a suitable buffer or solvent for preparing a solution or composition.

[0140] The following examples are provided to more fully illustrate the claimed invention and should not be construed as limiting the scope of the invention. To the extent that specific materials are mentioned, they are for illustrative purposes only and are not intended to limit the invention. Those skilled in the art may develop equivalent means or reactants without the exercise of inventive capacity and without departing from the scope of the invention. [Table 4]

[0141] Example 1A - 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 ( 18 Preparation of F-labeled compound 1) [ka]

[0142] 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 [ka]

[0143] 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) was added 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) (Fairhall, Jessica M., et al. Bioorganic & Medicinal Chemistry, incorporated herein by reference in its entirety). (Prepared according to the procedure described in 2021,46,116361) was added, and the mixture was stirred at rt for 1 h. Volatiles were removed under reduced pressure, and the mixture was subjected to silica gel flash chromatography (0→50% EtOAc in heptane). After pooling and concentration of 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 μmol, 42.76% yield) was obtained as a pale yellow oil. 1 H 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).

[0144] 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 [ka]

[0145] 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 μmol) in MeOH (3 mL), a solution of NaOH (52.58 mg, 1.31 mmol) in water (1 mL) was added, and the mixture was heated to 60° C. for 1 h. The mixture was cooled to rt, and the volatile organics were removed under reduced pressure. The mixture was diluted with water and extracted three times with DCM. The combined organics were passed through a plug of magnesium sulfate and concentrated to give a colorless film, which was used without further purification.

[0146] 1 H 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).

[0147] Step c: Preparation of 2,3,5,6-tetrafluorophenyl 6-(fluoro-18F)nicotinate [ka]

[0148] [ 18 fluoride, [F] 18 A 2.0 mL bolus of [O] water was delivered to the synthesis module and trapped with Sep-Pak QMA Carbonate Plus Light (46 mg, Waters). The active material was eluted from the cartridge into reaction vial-1 using tetrabutylammonium bicarbonate solution (TBAHCO3, 0.5 mL, 0.075 M; diluted with 0.5 mL of MeCN). The solution was azeotropically dried at 120 °C under N2 flow and vacuum by the successive addition of two 1 mL portions of acetonitrile. After cooling the reactor to 40 °C, FPyTFP precursor (10 mg) dissolved in 1 mL of t-butanol and anhydrous acetonitrile (8 / 2, v / v) was dried. 18 The labeling reaction was carried out 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 of water to rinse the reactor. The diluted solution was loaded onto an Oasis® MCX cartridge SPE cartridge (pre-conditioned with 10 mL of MeCN, followed by 10 mL of water). The SPE was washed with 10 mL of water and eluted into a second reaction vial using 1 mL of MeCN to obtain 2,3,5,6-tetrafluorophenyl 6-(fluoro- 18 F) The nicotinate was obtained without further purification.

[0149] 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 [ka]

[0150] 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 of MeCN) was then added to [ 18 [F]FPy was added to the TFP solution, and the coupling reaction was carried out at 40 °C for 10 min. The crude reaction mixture was diluted using 2 mL of water and injected into a semi-preparative HPLC for purification through an ACE C18 HPLC column (10 μm, 10 x 250 mm) eluted with HO (MeCN / water, 95 / 5 v / v) in a 5 mL HPLC loop (semi-preparative HPLC method: 60 / 40 v / v ratio, 5.5 mL / min, RT = 21 min). The fractions corresponding to the title compound were collected and diluted in water (30 mL) containing 450 mg of sodium ascorbate. The solution was loaded onto a Classic C18 SPE cartridge (Waters) and purified at 5 mL min. -1 The cartridge was washed with water (10 mL) containing 50 mg of sodium ascorbate. The title compound was eluted from 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. The tocopherol formed a suspension upon dilution with PBS. Therefore, separate dose aliquots were filtered before each injection using an Acrodisc® sterile filter. The chemical and radiochemical purity of the title compound was determined by HPLC (QC HPLC method: Eclipse XDB-C18 column (5 μm, 4.6 × 150 mm) eluting with 50 mM ammonium acetate / MeCN in a 65 / 35 v / v ratio at 1.5 mL / min). The percentage of title compound in solution was determined by reacting a 100 μL volume with an excess of test tetrazine (MeTz-BzI-NH2.HCl; 0.1–0.4 mg). The percentage of title compound in solution ranged from 0.4% to 14%.

[0151] 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) [ka]

[0152] To a vial containing 3,8-diazabicyclo[3.2.1]octan-8-yl-(6-fluoro-3-pyridyl)methanone (43.39 mg, 184.44 μmol) and TEA (155.53 mg, 1.54 mmol, 214.22 μL) in DCM (1 mL) was added 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 μmol), and the mixture was stirred at RT for 1 h. The mixture was concentrated under reduced pressure, and the crude residue was subjected to silica gel flash chromatography (0→80% EtOAc in heptane). After pooling and concentration of 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 μmol, 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, 3H) 1.79 - 1.95 (m, 4 H) 1.54 - 1.69 (m, 4 H) 1.46 (br s, 1 H).

[0153] 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) [ka]

[0154] 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 μmol), 3,8-diazabicyclo[3.2.1]octan-8-yl-(6-fluoro-3-pyridyl)methanone (38.31 mg, 110.00 μmol, TFA), and TEA (101.19 mg, 1.00 mmol, 139.38 μL) in DCM (1 mL) was stirred at rt for 1 h and then concentrated under reduced pressure. The crude mixture was taken up in DMSO, filtered, and passed through a PSR for purification. Purification was achieved by reverse-phase HPLC on a Waters Sunfire Prep C18 5µm OBD 19x100mm column using a 5% to 60% MeCN gradient in water (ammonium hydroxide modifier). 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 (31.5mg, 70.71µmol, 70.71% yield) as a clear film (a 10:1 mixture of syn and anti diastereomers). 1 H 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).

[0155] Example 3 - Preparation of 2-[(4Z)-cyclooctat-4-en-1-yl]oxy-6-(fluoromethyl)pyridine (Compound 3) [ka]

[0156] At 0 °C, KHMDS (1 M, 185.89 μL) was added dropwise to a vial containing (4Z)-cyclooct-4-en-1-ol (29.32 mg, 232.37 μmol) and 2-fluoro-6-(fluoromethyl)pyridine (20 mg, 154.91 μmol) in THF (1 mL). With each addition of KHMDS, the solution gradually darkened. After the addition was complete, TLC indicated that the reaction was complete. The mixture was passed through a silica plug, eluted with DCM, and the eluate was concentrated. The residue was subjected to flash chromatography (0 → 10% EtOAc in heptane), and after pooling and concentration of the appropriate fractions, 2-[(4Z)-cyclooct-4-en-1-yl]oxy-6-(fluoromethyl)pyridine (31.9 mg, 135.57 μmol, 87.52% yield) was obtained as a colorless oil. LCMS: m / z = 128 (M cyclooctene) + , cyclooctene retention time = 1.06 min. 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).

[0157] Example 4 - Preparation of N-[(4Z)-cyclooct-4-en-1-yl]-6-(fluoromethyl)pyridin-2-amine (Compound 4) [ka]

[0158] A vial containing (4Z)-cyclooct-4-en-1-amine (29.09 mg, 185.89 μmol), 2-fluoro-6-(fluoromethyl)pyridine (20 mg, 154.91 μmol), KCO (42.82 mg, 309.82 μmol), and NMP (1 mL) was heated to 150° C. for 18 h. After this time, the mixture was allowed to cool to rt and then diluted with water and EtOAc. The organic material was separated and washed three times with water, then with brine. The organic material was then dried over magnesium sulfate, filtered, concentrated, and subjected to silica flash chromatography (0→50% EtOAc in heptane). After pooling and concentration of 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) +

[0159] Example 5 - Preparation of 1-[(4Z)-cyclooct-4-en-1-yl]-4-[6-(fluoromethyl)-2-pyridyl]piperazine (Compound 5) [ka]

[0160] To a scintillation vial containing 1-[(4Z)-cyclooct-4-en-1-yl]piperazine (24.54 mg, 126.31 μmol), 2-bromo-6-(fluoromethyl)pyridine (20 mg, 105.26 μmol), sodium tert-butoxide (15.17 mg, 157.88 μmol), and dioxane (1 mL) was added RuPhos Pd G3 (4.40 mg, 5.26 μmol), and the mixture was heated to 80 °C for 4 h. The mixture was filtered through a Celite plug, and the Celite plug was rinsed with DCM. The eluate was concentrated, and the crude residue was subjected to silica flash chromatography (0 to 20%, 3:1 EtOAc:EtOH in heptane). After pooling and concentration of 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) + .

[0161] Example 6 - Preparation of rac-[(1S,4Z)-cyclooct-4-en-1-yl]4-(5-fluoro-2-pyridyl)piperazine-1-carboxylate (Compound 6) [ka]

[0162] A vial containing rac-[(1R,4Z)-cyclooct-4-en-1-yl](2,5-dioxopyrrolidin-1-yl)carbonate (50 mg, 187.07 μmol), 1-(5-fluoro-2-pyridyl)piperazine (40.68 mg, 224.49 μmol), DCM (1 mL), and triethylamine (37.86 mg, 374.14 μmol, 52.15 μL) was stirred at rt for 1 h. The mixture was concentrated and taken up in DMSO, filtered, and subjected to purification by reverse-phase HPLC (10 to 90% MeCN in water, 0.1% TFA). After pooling and lyophilization of the desired fractions, rac-[(1S,4Z)-cyclooct-4-en-1-yl]4-(5-fluoro-2-pyridyl)piperazine-1-carboxylate (47.9 mg, 107.30 μmol, 57.36% yield, trifluoroacetate) was obtained as a colorless oil. LCMS: m / z = 334 (m+H) + . 1 H 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). 19 F NMR (376 MHz, DMSO-d6) δ ppm -74.93 (s, 3 F), -143.01 (s, 1 F).

[0163] 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) [ka]

[0164] A vial containing 1-(5-fluoro-2-pyridyl)piperazine (79.73 mg, 440.00 μmol), 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 μL), and DCM (2 mL) was treated with T3P (763.63 mg, 1.20 mmol, 713.68 μL, 50% purity) and stirred for 48 h. The reaction was diluted with saturated aqueous sodium bicarbonate and extracted three times with DCM. The combined organics were dried over magnesium sulfate, filtered, and concentrated. The crude residue was subjected to flash chromatography (0 to 80% EtOAc in heptane) to afford, after pooling and concentration of the appropriate fractions, an impure mixture containing an unidentified by-product. This mixture was subjected to purification by preparative HPLC (10→90%, MeCN in water, 0.1% TFA modifier) ​​to give, 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) as a colorless oil. LCMS: m / z = 348 (M+H). + . 1 H 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, 3H), 1.49 - 1.60 (m, 1 H), 1.41 (dtt, J=14.1, 9.3, 4.7 Hz, 1 H).

[0165] Example 8 - Preparation of rac-2-[[(1S,4Z)-cyclooct-4-en-1-yl]-methylamino]-1-[4-(5-fluoro-2-pyridyl)piperazin-1-yl]ethanone (Compound 8) [ka]

[0166] To a vial containing 1-(5-fluoro-2-pyridyl)piperazine (105.12 mg, 580.11 μmol), rac-2-[[(1S,4Z)-cyclooct-4-en-1-yl]-methylamino]acetic acid (150 mg, 483.42 μmol, trifluoroacetic acid), DCM (3 mL), and TEA (146.75 mg, 1.45 mmol, 202.14 μL) was charged T3P (461.45 mg, 725.13 μmol, 431.26 μL, 50% purity), and the mixture was allowed to stir at rt for 48 h. The reaction was diluted with DCM and sodium carbonate and stirred vigorously. The phases were separated, and the aqueous phase was washed twice with DCM. The combined organics were dried over magnesium sulfate, filtered, and concentrated. After pooling and concentration of the appropriate fractions, rac-2-[[(1S,4Z)-cyclooct-4-en-1-yl]-methylamino]-1-[4-(5-fluoro-2-pyridyl)piperazin-1-yl]ethanone (105 mg, 291.29 μmol, 60.26% yield) was obtained as a light tan oil. LCMS: m / z = 361 (M+H) + . 1 H 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).

[0167] Example 9 - Preparation of rac-N-[(1S,4Z)-cyclooct-4-en-1-yl]-2-[4-(5-fluoro-2-pyridyl)piperazin-1-yl]-N-methylacetamide (Compound 9) [ka]

[0168] To a vial containing rac-(1S,4Z)-N-methylcyclooct-4-en-1-amine (12.80 mg, 91.96 μmol), 2-[4-(5-fluoro-2-pyridyl)piperazin-1-yl]acetic acid (20 mg, 83.60 μmol), TEA (42.30 mg, 417.98 μmol, 58.26 μL), and DMF (1 mL) was charged T3P (106.39 mg, 167.19 μmol, 99.43 μL, 50% purity), and the mixture was allowed to stir at rt overnight. The mixture was diluted with saturated aqueous sodium carbonate and extracted with EtOAc. The organic material was collected, dried over magnesium sulfate, filtered, and concentrated. The crude residue was subjected to silica flash chromatography (0→100% EtOAc in heptane) and the appropriate fractions were pooled and, after cooling, afforded rac-N-[(1S,4Z)-cyclooct-4-en-1-yl]-2-[4-(5-fluoro-2-pyridyl)piperazin-1-yl]-N-methylacetamide (11.2 mg, 31.07 μmol, 37.17% yield) as a white crystalline solid.

[0169] 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) + .

[0170] 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) [ka]

[0171] Step a: Preparation of (R,Z)-2-(1-hydroxycyclooct-4-en-1-yl)acetic acid [ka]

[0172] rac-Methyl (R,Z)-2-(1-hydroxycyclooct-4-en-1-yl)acetate (1.0 g, 5.04 mmol, 1.0 equiv) was dissolved in DCE (20 mL). MeSnOH (2.74 g, 15.13 mmol, 3.0 equiv) was added, and the mixture was heated at 80 °C for 5 h under N2. TLC (PE:EA = 1:1) analysis showed the reaction was complete. After completion of the reaction, the mixture was concentrated under reduced pressure, and the residue was taken up in EA (50 mL). The organic layer was washed with hydrochloric acid (5%) (30 mL × 3). Then, the organic layer was washed with brine (40 mL) and dried over Na2SO4. The solvent was removed under reduced pressure to give rac-(R,Z)-2-(1-hydroxycyclooct-4-en-1-yl)acetic acid (1.67 g, crude) as a pale yellow oil. 1 H NMR: (400 MHz, 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).

[0173] Step b: Preparation of rac-2,5-dioxopyrrolidin-1-yl(R,Z)-2-(1-hydroxycyclooct-4-en-1-yl)acetic acid [ka] To a solution of rac-(R,Z)-2-(1-hydroxycyclooct-4-en-1-yl)acetic acid (800.0 mg, 4.34 mmol, 1.0 equiv) in DCM (8 mL) was added 1-hydroxypyrrolidine-2,5-dione (749.63 mg, 6.51 mmol, 1.5 equiv) and DIC (822.00 mg, 6.51 mmol, 1.01 mL, 1.5 equiv). The mixture was stirred at 20 °C for 16 h. TLC (PE:EA = 1:1) showed complete consumption of the starting material. The mixture was concentrated and purified by Combi Flash (EA in PE, 0% → 27% to 30%) to give rac-2,5-dioxopyrrolidin-1-yl(R,Z)-2-(1-hydroxycyclooct-4-en-1-yl)acetic acid (638.4 mg, 47.04% yield) as a white solid. 1 H 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).

[0174] 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 [ka]

[0175] 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 equiv.) was added TEA (719.44 mg, 7.11 mmol, 990.96 μL, 10.0 equiv.) and rac-2,5-dioxopyrrolidin-1-yl(R,Z)-2-(1-hydroxycyclooct-4-en-1-yl)acetic acid (200.00 mg, 710.98 μmol, 1.0 equiv.). The mixture was stirred at 25 °C for 4 h. LCMS indicated that the desired product was found. The mixture was concentrated and purified by Combi Flash (EA in PE, 0% to 63%) to give the crude product, which was confirmed by LCMS. The crude product was concentrated, dissolved in MeOH, and purified by preparative HPLC (Column: Welch Xtimate C18 150x25mmx5µm; Conditions: water (NH4HCO3)-ACN; Start B 23, End B 53; Gradient time (min): 11; 100% B Retention time (min): 2; Flow rate (mL / min): 25; Injection 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 a white solid. LCMS: (M+H + : 402.1). HPLC: (Purity: 100.00%). 1 H 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).

[0176] 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) [ka]

[0177] Step a: Preparation of rac-methyl (R,Z)-2-(1-hydroxycyclooct-4-en-1-yl)acetate [ka]

[0178] To a solution of methyl acetate (1.79 g, 24.16 mmol, 1.5 equiv) in THF (5 mL) was added LiHMDS (1 M, 24.16 mL, 1.5 equiv) at −78° C. under N2. The mixture was stirred at −78° C. for 30 minutes. The mixture was added dropwise to 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 that the starting material was completely consumed. The reaction mixture was quenched by adding saturated NH4Cl (10 mL). After stirring at 20° C. for 10 minutes, the mixture was diluted with water (50 mL). The mixture was extracted with EtOAc (30 mL × 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 a 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).

[0179] Step b: Preparation of rac-(R,Z)-1-(2-hydroxyethyl)cyclooct-4-en-1-ol [ka]

[0180] To a solution of LiAlH (287.13 mg, 7.57 mmol, 1.5 equiv) in THF (3 mL) was added a solution of rac-methyl (R,Z)-2-(1-hydroxycyclooct-4-en-1-yl)acetate (1 g, 5.04 mmol, 1.0 equiv) in THF (10 mL) at 0 °C under N. The reaction mixture was stirred at 0 °C for 30 min. TLC (PE:EA = 4:1) showed that a new spot was observed. After cooling the reaction mixture to 0 °C, 0.3 mL of HO was added, followed by 0.9 mL of 15% aqueous NaOH solution and 0.3 mL of HO to quench the reaction mixture. After stirring at 20 °C for 30 min, 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 a yellow oil. 1 H 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).

[0181] Step c: Preparation of rac-(R,Z)-2-(1-hydroxycyclooct-4-en-1-yl)ethyl (4-nitrophenyl) carbonate [ka]

[0182] A solution of 4-nitrophenyl carbonochloridate (997.78 mg, 4.95 mmol, 1.2 equiv) in DCM (6 mL) was added dropwise via syringe to a mixture of rac-(R,Z)-1-(2-hydroxyethyl)cyclooct-4-en-1-ol (702.3 mg, 4.13 mmol, 1.0 equiv) and pyridine (815.75 mg, 10.31 mmol, 2.5 equiv) in DCM (12 mL). The mixture was stirred for 16 h at 20 °C. Several spots were observed on TLC (PE / EA = 2 / 1), followed by quenching with the addition of saturated NH4Cl. The resulting phases were separated, and the aqueous layer was extracted with DCM (30 mL × 3). The combined organic layers were dried over Na2SO4, filtered, and concentrated to a residue. The crude residue was purified by Combi Flash (EA in PE, 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 a yellow oil. LCMS: (2M+Na + : 693.2). 1 H 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).

[0183] 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 [ka]

[0184] 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 equiv) in DCM (5 mL) was added DIPEA (96.35 mg, 745.49 μmol, 129.85 μL, 2.5 equiv). The mixture was stirred at 20° C. for 10 min. To 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 equiv). The mixture was stirred at 20° C. for 16 h. LCMS indicated that the desired product was found. The mixture was concentrated and purified by Combi Flash (EA in PE, 0% to 60%) to give the crude product, which was confirmed by LCMS. The crude product was dissolved in MeOH and purified by preparative HPLC (Column: Welch Xtimate C18 150x25mmx5µm; Conditions: water (NH4HCO3)-ACN; Start B 28, End B 58; Gradient time (min): 11; 100% B Retention time (min): 2; Flow rate (mL / min): 25; Injection 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), 2.41-2.31 (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).

[0185] 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) [ka]

[0186] Step a: Preparation of tert-butyl 4-(4-(2-fluoroethoxy)benzoyl)piperazine-1-carboxylate [ka]

[0187] To a solution of 4-(2-fluoroethoxy)benzoic acid (320.0 mg, 1.74 mmol, 1.0 equiv) in DCM (4 mL) was added tert-butyl piperazine-1-carboxylate (388.35 mg, 2.09 mmol, 1.2 equiv), TEA (527.48 mg, 5.21 mmol, 726.55 μL, 3.0 equiv), and T3P (829.30 mg, 2.61 mmol, 668.79 μL, 1.5 equiv). The mixture was stirred at 15° C. for 16 h. LCMS indicated the desired product was obtained. The mixture was concentrated to give a residue, which was purified by Combi Flash (EA in PE, 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. 1 H 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).

[0188] Step b: Preparation of (4-(2-fluoroethoxy)phenyl)(piperazin-1-yl)methanone [ka]

[0189] A solution of tert-butyl 4-(4-(2-fluoroethoxy)benzoyl)piperazine-1-carboxylate (260 mg, 738.65 μmol, 1.0 equiv) in HCl / EA (4 M, 5 mL, 27.08 equiv) was added. The mixture was stirred at 20° C. for 1 h. LCMS showed that the desired product was obtained. The reaction mixture was concentrated to give (4-(2-fluoroethoxy)phenyl)(piperazin-1-yl)methanone (198.5 mg, crude, hydrochloride salt) as a white solid. LCMS: (M+H +: 253.2).

[0190] Step c: Preparation of rac-(R,Z)-2-(1-hydroxycyclooct-4-en-1-yl)ethyl 4-(4-(2-fluoroethoxy)benzoyl)piperazine-1-carboxylate [ka]

[0191] To a solution of (4-(2-fluoroethoxy)phenyl)(piperazin-1-yl)methanone (169.27 mg, 670.94 μmol, 1.5 equiv) in DCM (5 mL) was added DIPEA (144.52 mg, 1.12 mmol, 194.77 μL, 2.5 equiv). The mixture was stirred at 20° C. for 10 min. To the reaction mixture was added rac-(R,Z)-2-(1-hydroxycyclooct-4-en-1-yl)ethyl (4-nitrophenyl)carbonate (150.00 mg, 447.29 μmol, 1.0 equiv). The mixture was stirred at 20° C. for 16 h. LCMS showed that the desired product was found. The product was concentrated, dissolved in MeOH, and purified by preparative HPLC (Column: Welch Xtimate C18 150x25mmx5µm; Conditions: water (NH4HCO3)-ACN; Start B 32, End B 62; Gradient time (min): 11; 100% B Retention time (min): 2; Flow rate (mL / min): 25; Injection 8) 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 a white solid. 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), 3 1-3.44 (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).

[0192] 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) [ka]

[0193] Step a: Preparation of methyl 4-(2-fluoroethoxy)-3-methylbenzoate [ka]

[0194] To a solution of methyl 4-hydroxy-3-methylbenzoate (1 g, 6.02 mmol, 1.0 equiv.) in MeCN (10 mL) was added 1-fluoro-2-iodoethane (2.09 g, 12.04 mmol, 2.0 equiv.) and K2CO3 (2.50 g, 18.05 mmol, 3.0 equiv.). The mixture was stirred at 75 °C for 16 h. LCMS showed that the starting material was completely consumed and a new peak with the desired MS was detected. The reaction solution was concentrated to give a residue, which was purified by Combi Flash eluting with EtOAc in PE (0% to 30%) to give methyl 4-(2-fluoroethoxy)-3-methylbenzoate (1.2 g, 93.96% yield) as a white solid. 1 H 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).

[0195] Step b: Preparation of 4-(2-fluoroethoxy)-3-methylbenzoic acid [ka]

[0196] To a solution of methyl 4-(2-fluoroethoxy)-3-methylbenzoate (1 g, 4.71 mmol, 1.0 equiv) in MeOH (20 mL) and water (10 mL) was added NaOH (753.89 mg, 18.85 mmol, 4.0 equiv). The mixture was stirred at 20 °C for 16 h. LCMS showed complete consumption of the starting material and a new peak with the desired MS was detected. The reaction was acidified with HCl (4 M) to pH = 4 and diluted with HO. The resulting precipitate was filtered, washed with HO and hexane, and concentrated in vacuo to give 4-(2-fluoroethoxy)-3-methylbenzoic acid (930 mg, 99.58% yield) as a 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).

[0197] Step c: Preparation of tert-butyl 4-(4-(2-fluoroethoxy)-3-methylbenzoyl)piperazine-1-carboxylate [ka]

[0198] To a solution of 4-(2-fluoroethoxy)-3-methylbenzoic acid (930 mg, 4.69 mmol, 1.0 equiv) in DCM (15 mL) was added tert-butyl piperazine-1-carboxylate (1.75 g, 9.38 mmol, 2.0 equiv), TEA (949.66 mg, 9.38 mmol, 1.31 mL, 2.0 equiv), and T3P (2.24 g, 7.04 mmol, 1.5 equiv). The mixture was stirred at 25 °C for 16 h. LCMS showed that the desired product was obtained. The mixture was concentrated, and then water (20 mL) was added. The mixture was extracted with EtOAc (20 mL × 3). The combined organic layers were washed with brine (20 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure to give a residue that was purified by Combi Flash eluting with EtOAc in PE (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: (400 MHz, 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).

[0199] Step d: Preparation of (4-(2-fluoroethoxy)-3-methylphenyl)(piperazin-1-yl)methanone [ka]

[0200] A solution of tert-butyl 4-(4-(2-fluoroethoxy)-3-methylbenzoyl)piperazine-1-carboxylate (1.4 g, 3.82 mmol, 1.0 equiv) in DCM (15 mL) was added dropwise to a solution of HCl / EA (4 M, 20 mL) at 25 °C over 1 h. LCMS showed that the starting material was consumed and the desired product was detected. The reaction mixture was concentrated to give crude (4-(2-fluoroethoxy)-3-methylphenyl)(piperazin-1-yl)methanone (1 g, 98.28% yield) as a white solid, which was used directly in the next step. LCMS: (M+H+: 267.1).

[0201] Step e: Preparation of (R,Z)-2-(1-hydroxycyclooct-4-en-1-yl)ethyl 4-(4-(2-fluoroethoxy)-3-methylbenzoyl)piperazine-1-carboxylate [ka]

[0202] To a solution of (4-(2-fluoroethoxy)-3-methylphenyl)(piperazin-1-yl)methanone (150 mg, 563.25 μmol, 1.0 equiv) 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 equiv) and DIPEA (145.59 mg, 1.13 mmol, 196.22 μL, 2.0 equiv). The mixture was stirred at 20 °C for 16 h. LCMS indicated that the desired product was obtained. Water (10 mL) was added, and the mixture was extracted with DCM (10 mL × 3). The organic material was washed with brine (15 mL), dried over Na2SO4, filtered, and concentrated to give crude material, which was purified by preparative HPLC (Column: Boston Prime C18 150*30 mm*5 um, Conditions: Water (NH3H2O+NH4HCO3)-ACN Start B 41, End B 71 Gradient time (min) 10, 100% B Retention 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 a yellow oil. LCMS: (M+Na + : 485.3). HPLC: (Purity: 97.25%). 1 H NMR: (500MHz, DMSO-d6) δ = 7.27-7.20 (m, 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).

[0203] Example 14 - Preparation of rac-(R,Z)-4-(2-fluoroethoxy)-N-(2-(1-hydroxycyclooct-4-en-1-yl)ethyl)-3-methylbenzamide (Compound 14) [ka]

[0204] Step a: Preparation of methyl 4-(2-fluoroethoxy)-3-methylbenzoate [ka]

[0205] To a solution of methyl 4-hydroxy-3-methylbenzoate (1.00 g, 6.02 mmol, 1.0 equiv) in MeCN (20 mL) was added 1-fluoro-2-iodoethane (2.09 g, 12.04 mmol, 2.0 equiv). KCO (2.50 g, 18.05 mmol, 3.0 equiv) was then added to the mixture at 20 °C. The reaction was stirred at 75 °C for 16 hours. TLC showed the reaction was complete. The reaction solution was concentrated to give a residue, which was purified by Combi Flash eluting with EtOAc in PE (0% to 30%) to give 4-(2-fluoroethoxy)-3-methylbenzoate (1.2 g, 93.96% yield) as a white solid. 1 H 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).

[0206] Step b: Preparation of 4-(2-fluoroethoxy)-3-methylbenzoic acid [ka]

[0207] To a solution of methyl 4-(2-fluoroethoxy)-3-methylbenzoate (1.2 g, 5.65 mmol, 1.0 equiv) in HO (10 mL) and MeOH (20 mL) was added NaOH (904.67 mg, 22.62 mmol, 4.0 equiv) at 20 °C. The mixture was stirred at 20 °C for 16 h. LCMS indicated that the starting material had been consumed and the desired product had been detected. The reaction was acidified to pH 4 with concentrated hydrochloric acid and diluted with HO. The resulting precipitate was filtered, washed with HO and hexanes, and concentrated under reduced pressure to give 4-(2-fluoroethoxy)-3-methylbenzoic acid (946.3 mg, 84.44% yield) as a white solid. LCMS: (M+H + : 199.2).

[0208] Step c: Preparation of rac-(R,Z)-4-(2-fluoroethoxy)-N-(2-(1-hydroxycyclooct-4-en-1-yl)ethyl)-3-methylbenzamide [ka]

[0209] To a solution of rac-(R,Z)-1-(2-aminoethyl)cyclooct-4-en-1-ol (500.00 mg, 2.95 mmol, 1.0 equiv) in DCM (20 mL) was added 4-(2-fluoroethoxy)-3-methylbenzoic acid (585.45 g, 2.95 mmol, 1.0 equiv), DMAP (72.18 mg, 590.80 μmol, 0.2 equiv), and EDCI (849.42 mg, 4.33 mmol, 1.5 equiv) 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 h. LCMS showed that the desired product was obtained. Water (60 mL) was added to the reaction mixture, and it was extracted with DCM (20 mL × 3). The organic layer was washed with brine and dried over Na2SO4. The reaction solution was concentrated to give a residue, which was purified by Combi Flash eluting with EtOAc in PE (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 a yellow oil. A portion of the product (80.00 mg, 228.94 μmol, 1.0 equiv) was dissolved in MeOH (3 mL). The residue was purified by preparative HPLC (column: Boston Prime C18 150x30mmx5µm; conditions: water (NH3H2O ​​+ NH4HCO3)-ACN; start 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 a 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).

[0210] Example 15 - Preparation of rac-(R,Z)-4-(2-fluoroethoxy)-2-(2-(1-hydroxycyclooct-4-en-1-yl)ethyl)isoindolin-1-one (Compound 15) [ka]

[0211] To a solution of 4-(2-fluoroethoxy)isoindolin-1-one (78.60 mg, 402.68 μmol, 0.5 equiv) in DMF (2 mL) was added NaH (64.42 mg, 1.61 mmol, 60% purity, 2.0 equiv) at 25° C. under N for 30 min. Then, rac-(R,Z)-2-(1-hydroxycyclooct-4-en-1-yl)ethyl methanesulfonate (200.00 mg, 805.35 μmol, 1.0 equiv) in DMF (1 mL) was added at 25° C. for 5 h. LCMS showed that the desired product was found. The reaction mixture was quenched with HO (1 mL) and filtered to obtain a filtrate, which was purified by preparative HPLC (column: Welch Xtimate C18 150*25 mm*5 um, conditions: water (NH4HCO3)-ACN start B 33, end B 60 gradient time (min) 11, 100% B retention 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, yield 4.82%) 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).

[0212] Example 16 - Preparation of rac-(R,Z)-5-(2-fluoroethoxy)-2-(2-(1-hydroxycyclooct-4-en-1-yl)ethyl)isoindolin-1-one (Compound 16) [ka]

[0213] To a solution of 5-(2-fluoroethoxy)isoindolin-1-one (80.00 mg, 409.86 μmol, 1.0 equiv) 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 equiv) and CsCO (400.62 mg, 1.23 mmol, 3.0 equiv) at 20° C. The mixture was stirred at 90° C. under N for 3 h. The residue was purified by preparative HPLC (column: Welch Xtimate C18 150x25mmx5µm; conditions: water (NH4HCO3)-ACN; start 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).

[0214] Example 17 - Preparation of rac-(S,E)-6-fluoro-N-(2-(1-hydroxycyclooct-4-en-1-yl)ethyl)-4-methylnicotinamide (Compound 17) [ka]

[0215] Step a: rac-(S,E)-1-(2-aminoethyl)cyclooct-4-en-1-ol [ka]

[0216] The 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 herein by reference in its entirety.

[0217] Step b: Preparation of rac-(S,E)-6-fluoro-N-(2-(1-hydroxycyclooct-4-en-1-yl)ethyl)-4-methylnicotinamide [ka]

[0218] 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-methylpyridine-3-carboxylic acid (37 mg, 236 μmol) and T3P (1.68 M, 236 μmol, 141 μL). The mixture was stirred overnight at room temperature. The resulting material was injected directly onto silica gel and purified by flash chromatography using a 0 to 100% EtOAc-heptane gradient. After evaporation of organic material below room temperature to avoid decomposition, the title compound was obtained. Ionization corresponds to -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).

[0219] Example 18 - Preparation of rac-(S,E)-6-fluoro-N-(2-(1-hydroxycyclooct-4-en-1-yl)ethyl)-N,4-dimethylnicotinamide (Compound 18) [ka]

[0220] Step a: Preparation of rac-(S,E)-6-fluoro-N-(2-(1-hydroxycyclooct-4-en-1-yl)ethyl)-N,4-dimethylnicotinamide [ka]

[0221] To a solution of rac-6-fluoro-N-[2-[(1S,4Z)-1-hydroxycyclooct-4-en-1-yl]ethyl]-4-methylpyridine-3-carboxamide (12 mg, 39 μmol) in DMF (2 mL) was added NaH (5 mg, 118 μmol, 60% purity) at 0° C. The mixture was stirred at 0° C. for 15 minutes, and CHI (9 mg, 59 μmol, 4 μL) was added at 0° C. The mixture was stirred at 0° C. for 3 hours, then treated with a saturated solution of ammonium chloride while warming to room temperature and extracted with EtOAc. The organic material was concentrated below room temperature to avoid decomposition, then loaded onto silica and purified by flash chromatography using a 0 to 100% EtOAc-heptane gradient to give 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).

[0222] Example 19 - Preparation of rac-(S,Z)-6-fluoro-N-(2-(1-hydroxycyclooct-4-en-1-yl)ethyl)-N,4-dimethylnicotinamide (Compound 19) [ka]

[0223] 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 equiv) in DMF (2 mL) was added NaH (39.16 mg, 979.19 μmol, 60% purity, 3.00 equiv) at 0° C. The mixture was stirred at 0° C. for 15 min, and CHI (69.49 mg, 489.60 μmol, 1.50 equiv) was added at 0° C. The mixture was stirred at 0° C. for 2 h. LCMS showed that the starting material was consumed and the desired MS was detected. The reaction mixture was concentrated to give crude material, which was purified by preparative HPLC (Column: Welch Xtimate C18 150x25mmx5µm; Conditions: water (NH4HCO3)-ACN, Start B 25, End B 54, Gradient time (min) 11, 100% B Retention time (min) 2, Flow rate (mL / min) 25, Injection 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 a 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).

[0224] Example 20 - Preparation of rac-(S,Z)-6-fluoro-N-(2-(1-hydroxycyclooct-4-en-1-yl)ethyl)-4-methylnicotinamide (Compound 20) [ka]

[0225] To a mixture of 6-fluoro-4-methylnicotinic acid (100 mg, 644.64 μmol, 1.00 equiv) and DCM (3 mL), DIEA (249.94 mg, 1.93 mmol, 336.85 μL, 3.00 equiv) and HATU (294.13 mg, 773.56 μmol, 1.20 equiv) were added at 20° C. The mixture was stirred at 20° C. for 15 minutes, and rac-(S,Z)-1-(2-aminoethyl)cyclooct-4-en-1-ol (163.67 mg, 966.95 μmol, 1.50 equiv) was added at 20° C. The mixture was stirred at 20° C. for 1 hour. LCMS showed the reaction was complete. The reaction mixture was concentrated to give the crude material, which was purified by preparative HPLC (Column: Waters X bridge BEH C18 100x30 mmx10 μm; Conditions: water (NH4HCO3)-ACN, start B 26, end B 56, gradient time (min) 11, 100% B hold time (min) 3, flow rate (mL / min) 50, injection 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 a 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).

[0226] Example 21 General procedure for the synthesis of MALAT1-ASO-Tz [ka]

[0227] To a 1 mL vial containing MALAT1 C6 amine (4.2 mg, 0.572 μmol, chemical structure shown in FIG. 21) dispensed from a 44 mg / mL stock solution in water (95 μL dispensed) with 5 μL of NaHCO3 (1 M), tetrazine NHS ester (24 equiv.) was added in two portions as a solution in DMSO (15 mg / mL). The second aliquot was added approximately 5 minutes after the first, and the reaction was allowed to stir for 1 hour. After this period, the reaction was confirmed to be complete by HPLC.

[0228] The contents of the mixture were loaded into an Amicon centrifugal filter (3000 MW cutoff), and the mixture was diluted to a total volume of 5 mL with water and then sedimented at 4000 RPM for 30 minutes. After centrifugal filtration, the total volume of the mixture was approximately 100 μL. The mixture was once again diluted to 5 mL and subjected to a second precipitation. The resulting solution was modified and lyophilized to yield the desired ASO-Tz conjugate as a pale pink solid.

[0229] Compounds 21-23 were prepared using the general procedure described above. [Table 5]

[0230] Biological Example 1 a. Brain and plasma protein binding: Individual test articles were spiked into 1500 μL aliquots of various plasma samples at a final concentration of 1 μM. 300 μL of these solutions were added to the donor chamber of the RED device (RED tube). An identical 300 μL solution was spiked with warfarin (1 μM) as a positive control. 500 μL aliquots (100 mM potassium phosphate, 150 mM NaCl, pH 7.4 (phosphate buffer)) were added to the corresponding recipient side of the RED device insert. Test compounds were incubated with rat and human plasma, and rat brain homogenates were shaken (200 rpm) in an incubator at 37°C for 6 hours in the presence of 5% CO2. Individual 96-well 2.0 mL assay blocks from Corning (Corning, NY) containing the same mixtures of test articles in plasma or homogenates as described above were incubated at 37°C for 6 hours for stability experiments.

[0231] For the 1 μM test article and positive control incubations, 50 μL aliquots of plasma samples were removed from the RED donor compartment (for protein binding experiments) and the corresponding 2.0 mL assay block wells (for plasma stability experiments) within 10 minutes (0 h time point) and 6 hours after addition to provide controls for the plasma stability experiments.

[0232] Aliquots of 50 μL of matrix were removed from the RED donor compartment. These aliquots were transferred to separate 96-well plates containing 50 μL of phosphate buffer and 200 μL of CPDPX (25 ng / mL) (internal standard) in 1:1 methanol / acetonitrile. An additional 50 μL aliquot from each RED device receiver compartment was collected after 6 hours and transferred to separate 96-well plate wells containing 50 μL of the appropriate matrix mixture and 200 μL of the internal standard. All samples were vortex mixed for 30 seconds and centrifuged at 3900 rpm for 10 minutes. All matrix and buffer samples were matrix-matched.

[0233] Fifty microliters of sample supernatant was transferred to individual wells of a 96-well plate containing 200 μL of 0.1% formic acid in 90 / 10 water / acetonitrile and gently vortexed. Plasma protein binding, including stability assay samples, was analyzed in duplicate. Standard curves for compounds of the present disclosure and warfarin ranged from 5 nM to 1500 nM. Calibration curves were prepared in 1:1 plasma mixtures (rat, dog, monkey, and human). All standards were matrix-matched and underwent the same preparation procedure as the samples. For sample analysis, all samples were directly injected into the LC / MS / MS system.

[0234] b.MDR1-MDCK: The assay uses human MDR1-transfected MDCK cells (an NIH cell line licensed from AbsorptionSystems). Compounds were tested at a concentration of 1 μM prepared in transport buffer (Hank's balanced salt solution containing HEPES). MDR1-MDCK cells were cultured in 96-well Transwell insert plates (Corning, NY, USA) for 7 days. Prior to the assay, the insert plates were washed and TEER (transepithelial electrical resistance) was measured. These plates were loaded with 85 μL of test compound solution for AB transport and 260 μL for BA transport into each donor compartment. Receiver buffer (1 μM) in each receiver compartment was added. The volumes of the donor compartment (transport buffer supplemented with 10% BSA) and receiver compartment (transport buffer supplemented with 10% BSA) were 250 μL and 75 μL, respectively. A 10 μL sample was taken from the donor compartment (time point T = 0). The assay plate was incubated for 120 min. At 120 min (time point T = 120), samples were taken from each donor (10 μL) and receiver (50 μL) compartment. 40 μL of transport buffer containing BSA was added to the donor sample, followed by the addition of crash solution (acetonitrile containing internal standard, 110 μL) to all samples. After centrifugation, 50 μL of the supernatant was transferred to another plate and mixed with 50 μL of water. The samples were analyzed using an LC-MS / MS integrated with a high-throughput injection system.

[0235] The test substance / internal standard area ratio is used to estimate the apparent permeability (Papp) and efflux ratio based on the following equation:

[0236] Papp = (dCr / dt) x Vr / (A x CE) Mass balance = 100 x ((VrxCrfinal) + (VdxCdfinal)) / (VdxCE) During the ceremony, dCr / dt is uM s1 is the slope of the cumulative concentration in the receiver compartment versus time in units of V r is the volume of the receiving compartment in cm V d is the volume of the feed compartment in cm3, A is the area of ​​the insert (0.143 cm for 96-well inserts) 2 ) CE is the estimated experimental concentration of the dosing solution (time = 0), 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.

[0237] c. Metabolic stability to liver microsomes i. Method Assay Summary: [Table 6]

[0238] 1. Prepare a 100X stock solution of the test article in the solvent solution. The initial 10 mM test article is diluted to 100 μM by adding 2.5 μL to a total volume of 250 μL of solvent solution (3:1 acetonitrile / water). If the test article is suspected to be insoluble, 100% acetonitrile may be used, provided the final reaction concentration of any organic solvent is less than 1%. These solutions form the "compound" plates in Figure 23.

[0239] 2. Frozen microsomes (20 mg / mL, Sekisui / Xenotech, Kansus City, KS) are thawed in a warm water (37°C) bath and adjusted to a concentration of 0.5 mg / mL in phosphate / MgCl2 buffer. Prepare a working buffer solution by adding 330 μL of MgCl2 (1 M) to each 100 mL of phosphate buffer (100 mM potassium phosphate, 150 mM sodium chloride, pH 7.4). Warm the solution to 37°C. Adjust the volume based on the scale of the assay. Prepare microsome solution by adding 2.5 mL of microsomes (20 mg / mL) to 100 mL of warmed phosphate / MgCl2 buffer. Adjust volume based on assay scale. Use the following microsome preparations containing over 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 is designated as the "LM" plate in Figure 23. When using slot plates, multiple species of microsomes can be prepared together.

[0240] 3. Prepare a 10 mM NADPH solution (nicotinamide adenine dinucleotide phosphate, Sigma-Aldrich, N1630) in phosphate / MgCl2 buffer. Prepare phosphate / MgCl2 buffer as described in step 2. Prepare NADPH solution by adding 74.44 mg of NADPH to each 10 mL of warmed phosphate / MgCl2 buffer. Adjust weight and volume based on assay scale. Heat to 37°C. This solution is the "NADPH" plate in Figure 23.

[0241] 4. Prepare a chilled "crash" solution of the internal standard in the solvent solution. Prepare a 50 ng / mL solution of the internal standard, CPDPX (8-cyclopentyl-1,3-dipropylxanthine, Sigma-Aldrich, C101) in a 1:1 acetonitrile / methanol solvent solution. Store on ice or in the refrigerator at 4°C. The solution becomes the "crushed" plate shown in Figure 23.

[0242] 5. Transfer 445 μL of microsome solution (0.5 mg / mL) to the reaction plate followed by the addition of 5 μL of test article (100 μM) and mix thoroughly. Maintain the reaction plate at 37°C throughout the assay.

[0243] 6. Start the reaction by adding 50 μL of NADPH solution (10 mM) to the reaction plate and mix thoroughly.

[0244] 7. From the "crash" solvent solution of the internal standard (CPDPX), transfer 160 μL to the final crash plate. Transfer 40 μL from the reaction plate to the crash plate and mix thoroughly. The first 40 μL sample crashed from the reaction plate represents time zero. At 5, 10, 15, 25, and 40 minutes after time zero, five additional aliquots of 40 μL are removed and transferred to their own individual crash plates containing 160 μL of crash solution, including the internal standard, for a total of six final crash plates.

[0245] 8. After the assay is complete, centrifuge the final crush plate at approximately 4000 rpm (3200 x g) for 10 minutes in a large benchtop centrifuge and refrigerate (4°C).

[0246] 9. Transfer an aliquot of the supernatant from the centrifuged crash plate to the LC-MS / MS ready plate. 50 μL of supernatant from each well of the centrifuged crash plate is transferred to a clean analytical sample plate containing 0.1% formic acid (200 μL), mixed, and then directly injected into the LC / MS / MS system for sample analysis. The volume and diluent composition can be adjusted based on the sensitivity of the instrument (LC-MS / MS) and the sensitivity, solubility, and polarity of the test substance to ensure adequate signal and retention of the test substance within the linear limits of the instrument.

[0247] ii. Data Processing The ln peak area ratio (compound peak area / internal standard peak area) is plotted against time and the slope (slope) of the line is measured. Figure 24 is an exemplary graph.

[0248] The following parameters are measured: Elimination rate constant: k = (-slope) Half-life: t 1 / 2 (min)=0.693 / k Assay Protein Content: V (μL / mg) = incubation volume (μL) / protein in incubation (mg)

[0249] Using the above parameters, the inherent clearance (CL int ) is represented as follows: CL int (μL / min / mg protein) = V x 0.693 / t 1 / 2 Intrinsic clearance reflects the ability of microsomes to metabolize a drug. To reliably measure metabolic rates, the amount of compound consumed during the microsomal stability assay must exceed 10%. Metabolic rates below 10% are reported as approximate values. When extending in vitro metabolic stability data, the amount of protein per gram of liver is a key factor that directly reflects the calculation of intrinsic clearance values ​​for the whole organ.

[0250] iii. Data reporting and interpretation Classification bands are typically used to categorize compounds into low, intermediate, or high clearance. [Table 7]

[0251] iv. Validation Dataset The metabolic competition of the test system is verified by including positive control compounds with known stability under the assay conditions described above. The table below shows a list of control compounds for specific species of microsomes. [Table 8]

[0252] The table below shows the CLint.exp (μL / min / mg protein) for a validation set of clinical drugs in multiple animal species in microsomes. [Table 9]

[0253] d.LogD Assay Summary: Thermodynamic Definition: LogD was measured by Analiza using the partition coefficient in a buffer-octanol system at equilibrium using an automated and miniaturized version of the gold standard shake flask method. 17

[0254] Partitioning: Analiza's standard two-phase plate was used for octanol / buffer partitioning. Assay partition plates were prepared using octanol equilibrated with phosphate-buffered saline (PBS) (pH 7.4). This buffer spanned a wide pH range and provided a uniform ionic composition. DMSO stock solution was added to each partition plate to achieve a final concentration of 10% DMSO in singlet plates. Plates were sealed, vortexed in Analiza's custom-designed deep-well plate mixer, and centrifuged to aid phase sedimentation. Assays were performed on an ADW workstation using chemiluminescent nitrogen detection.

[0255] Calculation of Results: The detector's equimolar nitrogen response was calibrated using standards spanning the instrument's dynamic range, from 0.08 to 4500 μg / mL nitrogen. Both the top and bottom phases were quantified against this calibration curve, and the logarithm of the ratio of the concentration in the top phase to the concentration in the bottom phase was calculated as Log D. In addition to the Log D values ​​observed directly in the presence and absence of DMSO in the partition system, the observed Log D values ​​were adjusted for a corrected Log D* based on the Analyza's working corrected Log D.

[0256] The calculated Log D and Log D* values ​​are corrected for background nitrogen in DMSO and octanol-buffer two-phase systems.

[0257] e.Kpuu assay In vivo To assess the brain-to-plasma partition coefficient (Kp), animals were infused intravenously with the dosing solution at a constant flow rate for 4 to 24 hours. Blood samples were collected continuously during the infusion, and CSF and brain samples were collected at the end of the infusion.

[0258] To evaluate PK characteristics, animals were administered the dosing solution via oral gavage or parenteral routes. Blood samples were collected post-dose. Other biological samples, such as tissue, bile, urine, and feces, can be collected during or at the end of the study, as needed.

[0259] All animal experiments were performed in accordance with in-house approved animal protocols.

[0260] bioanalysis Tissue samples were typically homogenized in phosphate-buffered saline (PBS) using a bead raptor.

[0261] To prevent nonspecific binding, CSF samples were routinely diluted with 8% BSA in PBS. Artificial CSF (aCSF) was used as a surrogate matrix.

[0262] When necessary, dosing solutions were spiked into plasma for analysis.

[0263] Calibration curves were generated by spiking analytes into blank matrix, which was processed alongside plasma, tissue, homogenate, and / or CSF samples by protein precipitation in appropriate organic solvents (e.g., acetonitrile and methanol) containing common analog internal standards (e.g., verapamil, chrysin, glyburide). Matrix matching was used when analyzing multiple matrices in the same run. Samples above the upper limit of quantitation (ULOQ) had to be diluted to the calibration range using a pre- or post-extraction dilution approach.

[0264] Processed samples were analyzed by LC-MS / MS using an appropriate method that performed within acceptable sensitivity, selectivity, precision, and accuracy. For an analytical run to be approved, at least 75% of the calibration curves of the duplicate calibration curves had to be within 20% of the nominal concentration.

[0265] Where necessary, compound or study-specific bioanalytical methods that deviate from typical procedures may be used, and this will be documented in the study-specific protocol included with the data upload.

[0266] Penalty kick Plasma concentrations were analyzed by noncompartmental analysis (NCA) using "linear up-log-down" fitting to generate essential PK parameters, including but not limited to, volume of distribution (Vd), maximum concentration (Cmax), time to reach maximum concentration (Tmax), area under the curve (AUC), half-life (t1 / 2), clearance (CL), and bioavailability (F). When dosing solution analysis was performed, PK parameters were normalized to the adjusted dose.

[0267] Brain concentrations were compared with plasma concentrations at corresponding time points to calculate partition coefficients (Kp).

[0268] The unbound drug partition coefficient (Kpuu), defined as the fraction of unbound drug partition across the blood-brain barrier, was calculated using the following equation:

number

[0269] Determination of the unbound fraction (Fu): The unbound fraction of test compound was determined based on the protocol described below.

[0270] 1) Dilute the initial 10 mM test substance to 125 μM by adding 5 μL to a total volume of 395 μL of solvent solution (100% acetonitrile) in a 96-well plate (Waters 186002481 Milford, MA). Ensure the compound is in solution.

[0271] 2) Thaw frozen (rat, human, mouse, dog, and / or monkey) plasma (BIOIVT, Westbury, NY) in a warm (37 °C) water bath and warm the PBS buffer.

[0272] Dilute the 125 μM test substance solution in a 2 mL 96-well plate (Costar 3961) by adding 8 μL to a final volume of 992 μL plasma to give a final concentration of 1 μM. Mix thoroughly. This spiked plasma solution is shown in Figure 25.

[0273] 3) Prepare a chilled "crash" solution of the internal standard in the solvent solution. Pipette 200 µL of a 25 ng / mL solution of the internal standard, CPDPX (8-cyclopentyl-1,3-dipropylxanthine, Sigma-Aldrich, C101) in a 1:1 acetonitrile / methanol solvent solution into a 1 mL 96-well plate. Store on ice or in the refrigerator at 4°C. The solution becomes the "crushed" plate shown in Figure 25.

[0274] 4) From the remaining spiked plasma, remove 50 μL of each plasma sample (T=0 h) and place it in a crush plate containing 200 μL. To match the matrix, add 50 μL of blank buffer to the crushed sample (similar to the PPB samples). Keep the remaining spiked plasma at 37°C for 4 hours. 5) Transfer 500 μL of warmed PBS buffer to the white side of the RED device (Thermo Scientific, Rockford IL, baseplate cat# 89811, insert cat# 89810) and transfer 300 μL of spiked plasma to the corresponding red ring side of the RED device.

[0275] 6) Cover all RED device plates with lids and transfer to a 37°C incubator in a 5% CO2 environment, shaking at 200 rpm for 4 hours.

[0276] 7) Reaction completion after 4 hours: Add 50 μL of sample (plasma or buffer sample) and 50 μL of opposing blank matrix (blank buffer added to plasma samples, blank plasma added to buffer samples) to the crush plate (same as above) containing 200 μL. Mix the crush plate well. From the remaining spiked plasma, remove 50 μL of each plasma sample (T=4 h) and place it on the crush plate. To match the matrix, add 50 μL of blank buffer to the crushed samples (as with the protein-binding samples). Centrifuge the crushed plate at 3900 rpm for 10 minutes at 4°C (Eppendorf Centrifuge 5810R, Hamburg, Germany).

[0277] 8) LC / MS / MS sample preparation: Using Tecan's PPB96~384pretty method, inject 30 μL of supernatant from the crash plate into an LC / MS containing 120 μL of 0.1% formic acid in 90:10 water:acetonitrile.

[0278] The volume and diluent composition can be adjusted based on the sensitivity of the instrument (LC-MS / MS) and the sensitivity, solubility, and polarity of the test substance to ensure adequate signal and retention of the test substance within the linear limits of the instrument.

[0279] 9) Standard curve · Plasma and buffer are used to generate a standard curve of pooled test substances treated in the same manner as the reaction samples.

[0280] 10) Data processing and analysis Multiquant is selected as the application used to process the PPB data.

[0281] equation: Formula 1. Calculation of %Free(%PPBunb) %Free = (PAR in buffer side / PAR in plasma side) * 100 PAR-Peak Area Ratio (PAR) Fu=%Free / 100 Fu = uncombined fraction Equation 2. Final calculation using dilution factor (D) This dilution factor formula is used only when tissue or plasma is diluted.

[0282] f. Data Summary The biological data collected for the compounds of the present disclosure in the above assays are summarized in the table below. Compound A is a tool compound, a cis-cyclooctene analog of MICA-213 (see Ruivo, E., et al. ACS Omega 2020, 5, 9, 4449-4456). Compound A and MICA-21 show comparable results in physicochemical and pharmacokinetic properties, i.e., plasma protein binding, brain tissue binding, and in vitro microsomal clearance. [Table 10]

[0283] Biological Example 2 - In the Naive Rat Brain 18 Investigating the distribution and kinetics of F-labeled compound 1

[0284] Animal models: 1. Naive male Sprague Dawley rats (N=17, 307±41 g) were supplied by Charles River.

[0285] 2. All rats were treated at baseline (N=2) or 24 hours after intrathecal (IT) administration of Compound 21 or ASO control (N=15). 18 Receive an intravenous (IV) administration of F-labeled Compound 1.

[0286] 3. A summary of animal weights and detailed study observations can be found in the "Raw Study Data Section" below.

[0287] Imaging methods: Obtaining the Standard 1. Naive male Sprague Dawley rats (N = 2) were placed under peripheral anesthesia (isoflurane approximately 1.5–3%, 1 L oxygen min). 2. Cannulae were placed in the arteries and veins. 3. Body temperature was monitored and maintained throughout the experiment, and respiratory rate was monitored. 4. 18After IV injection of F-labeled Compound 1, dynamic PET scans were performed for 60 minutes in each rat, with the field of view focused on the brain and upper body. 5. Arterial blood samples were collected consecutively for the first 1–2 min, followed by individual samples at 5, 15, 30, 45, and 60 min, and metabolite analysis was performed on the extracted plasma to generate tracer input functions for kinetic analysis. 6. At the end of the scan, the rats were euthanized. 7. Regions of interest were defined and time activity curves (TACs) were generated in eight brain regions (frontal cortex, striatum, cortex, hypothalamus, thalamus, hippocampus, cerebellum, and whole brain). 8. Different compartmental models were explored and used to generate volume of distribution (VT) estimates. The most appropriate model, as defined by Akaike Information Criterion (AIC), goodness of fit, and robustness of outcome parameters, was selected (MA1 with t* set to 15 min).

[0288] Obtained after administration 1. Recovery Under anesthesia (isoflurane approximately 1.5–3%, 1 L oxygen min), naive male Sprague Dawley rats (N=15) were anesthetized and a catheter was inserted into the spinal canal for IT administration (30 μL dose, followed by a 40 μL saline flush) of ASO control or compound 21 as follows: 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 peripheral anesthesia (approximately 1.5–3% isoflurane in 1 L oxygen min −1 ) and a cannula was placed in the vein. 3. Body temperature was monitored and maintained throughout the experiment, and respiratory rate was monitored. 4. 18 Following IV injection of F-labeled Compound 1, dynamic PET scans were acquired for 20 minutes in each rat, with the field of view focused on the brain and upper body. 5. At the end of the scan (blood and plasma), exsanguinated arterial blood samples were collected for radioactivity determination. 6. Regions of interest were defined and time activity curves (TACs) were generated in eight brain regions (frontal cortex, striatum, cortex, hypothalamus, thalamus, hippocampus, cerebellum, and whole brain), as well as the heart, liver, muscle, neck, and thoracic spine.

[0289] Ex vivo tissue collection procedure 1. After the post-dose scan, the animals were euthanized by exsanguination followed by cervical dislocation. 2. For animals scanned after dosing, 10 tissue samples were collected: brain regions (frontal cortex, striatum, hippocampus, thalamus + hypothalamus, cerebellum), spinal cord (cervical, thoracic, lumbar), liver, and kidney.

[0290] Sample characteristics: 1. Tissue samples collected after the post-dose scan were placed into Lysing matrix D tubes, weighed, flash frozen in liquid nitrogen, and stored at -80°C for further analysis. [Table 11]

[0291] result (without IT administration, 18 Figure 5 shows summed images from 0 to 60 min of rats scanned at baseline (after IV injection of F-labeled Compound 1). Brain uptake was not evident in these images. Similarly, the time activity curves (TACs) in Figure 6 show peak concentrations (approximately 3 SUV) in the first 5 min after injection, followed by rapid clearance and low activity (0.10-0.16 SUV, Table A) after 10 min in both rats. [Table 12]

[0292] Regional brain volume of distribution (VT) was measured as the outcome measure using the model found to be the best fit (multilinear analysis MA1). VT values ​​were approximately 1 in all brain regions, confirming the tracer's ability to cross the blood-brain barrier and diffuse homogeneously into brain tissue (Table B). [Table 13]

[0293] The sum of images from 0 to 20 min (Figure 7) and quantification of tracer concentrations in rat brains (Figure 8) for subjects IT injected with ASO control showed similar results to those in rats scanned at baseline. 18 The biodistribution of F-labeled Compound 1 is shown.

[0294] 18 For rats that were IT injected with various doses of Compound 21 prior to F-labeled Compound 1 scanning, tracer concentrations in the brain were found to be greatest for the high- and medium-dose Compound 21-treated groups, up to 0.64 and 0.67 SUV, respectively (Figures 9-14 and Table C). [Table 14]

[0295] 18 A heterogeneous distribution of F-labeled Compound 1 was observed in the brain of animals treated with Compound 21, with greater uptake in the cerebellum and hippocampus. Similarly, peripheral tissue distribution was consistent across groups, showing similar uptake in liver, heart, and muscle, as well as increased uptake in both the thoracic and cervical spine relative to the post-Compound 21 group (Figures 15-18 and Table D). [Table 15]

[0296] conclusion The purpose of this study was to evaluate the biodistribution of 18F-labeled Compound 1 in the brain of naive rats under baseline conditions and after IT administration of either an ASO control or increasing doses of Compound 21. Baseline scans demonstrated that the tracer was brain-penetrant and exhibited rapid plasma and tissue kinetics conducive to robust quantification. Rats administered the ASO control exhibited similar tracer biodistribution to that in rats scanned at baseline. Groups injected with various doses of Compound 21 exhibited heterogeneous distribution within the brain. The highest brain concentrations were found in animals administered the highest and intermediate doses of Compound 21. It should be noted that within-group variability was observed, which may be due to Compound 21 distribution after IT administration. Finally, the liver was the peripheral tissue with the greatest tracer uptake in all animals.

[0297] Biological Example 3 For cynomolgus monkeys, 18 F-labeled Compound 1 was administered i.v. and baseline scans were acquired from PET at 0-120 min post-injection. 18 1 shows baseline PET imaging of F-labeled Compound 1, with rapid clearance and brain uptake (C max Approximately 6% ID).

[0298] To measure non-specific binding of the tracer in the brain, a second cohort was first pretreated with a 1 mg / kg dose of non-radiolabeled Compound 1, followed 5 minutes later by pretreatment with the PET tracer. 18 FIG. 20 shows that the baseline time activity curve ("TAC") of F-labeled Compound 1 closely overlaps with the pretreatment TAC, suggesting that there is no measurable nonspecific binding of the tracer in the brain.

[0299] Biological Example 4 - In vitro and ex vivo characterization of compound 21 I. Quantification of Compound 21 in Tissues Separate stock solutions of MALAT1 ASO and compound 21 were prepared at a concentration of 1 mg / mL. Standard and quality control (QC) working solutions were prepared by diluting the stock solutions with ASO diluent (25 mM HFIP, 15 mM DMCHA, 100 μM EDTA, and 0.05% rat plasma in water:ACN (90:10% (v / v))). The working solutions were spiked into a control matrix to obtain calibration standards in the range of 1 to 1000 ng / mL.

[0300] Rat brain samples were weighed and homogenized with 9x (v / w) cell lysis loading buffer using an MP Biomedicals FastPrep-24 homogenizer to obtain a 10x dilution of brain homogenate. 200 μL aliquots of calibration standards, QC samples, and brain samples were added to a 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 washed three times consecutively. Finally, the beads were transferred to a preheated elution plate (containing 200 μL of ASO diluent with 25 ng / mL internal standard) and vigorously agitated at 90°C for 15 minutes to generate hybridization extracts for LC-MS / MS analysis. To analyze the reactive compound 21, 500 μM TCO-PEG4-DBCO (10 μL) was added to each well to conjugate with compound 21. After incubation at room temperature for 120 min, the samples were analyzed by LC-MS / MS.

[0301] An ExionLC AD UHPLC system equipped with a 1.7 μm transparent Oligo-XT 100 Å 50 x 2.1 mm column was used for the chromatographic separation of MALAT1 ASO, compound 21, compound 21-TCO-PEG4-DBCO, and the internal standard. The initial conditions were 2% mobile phase B, 98% mobile phase A (0.45 mL / min), and 100% mobile phase C (0.05 mL / min). The initial concentration was maintained for 0.2 min, and the separation gradient was ramped to 20% B within 5.5 min, while mobile phase C was maintained at 100% at 0.05 mL / min. After the gradient, the mobile phase was set to the initial conditions and equilibrated for 1 min. The column temperature was set to 60 °C. The injection volume was 10 μL. MS / MS detection was performed using a Sciex QTRAP 6500+ mass spectrometer equipped with an electrospray ionization (ESI) source in negative ion mode using multiple reaction monitoring (MRM). Optimized ion source parameters included a curtain gas of 40, a high collision gas, an ion spray voltage of -3500 V, a temperature of 550 °C, ion source gas 1 at 60, and ion source gas 2 at 60. MRM transitions were 793.1-95 for Malat ASO, 754.7-95 for compound 21, 819.1-95 for compound 21-TCO-PEG4-DBCO, and 879.5-95 for the internal standard.

[0302] II. Cellular uptake of compound 21 and immunofluorescence staining The following protocol was adapted from Cook, et al. (2022) (4). Briefly, HeLa cells were cultured in Eagle's Minimum Essential Medium (EMEM) containing 10% fetal bovine serum and incubated at 37°C and 5% CO2.

[0303] Cells were cultured in 4-well cell culture slides (ThermoFisher) for 24 hours, and a PBS stock solution of compound 21 was added to the cell culture medium to a final concentration of 5 μM. The cells were again incubated for 24 hours. Cells were then fixed in 4% PFA in PBS for 15 minutes at room temperature (RT) and permeabilized using 0.1% Triton X-100 before staining. Primary antibodies were diluted in PBST (EEA1 at 1:100; LAMP1 at 1:200), each added to a single well (100 μL) and incubated at 37°C for 45 minutes. Secondary antibodies (goat anti-rabbit IgG-FITC) were diluted 1:60 in PBST-BSA. TCO-Cy5 (click chemistry tool #1089) was also added to this solution to a final concentration of 1 μM. 100 μL of this solution was added to each well and incubated at RT for 1 hour in the dark. 200 μL of a 1× stock solution of phalloidin-AF568 was added to each well and incubated for 20 minutes at room temperature. Wells were washed in PBS and then fixed with 30 μL of Prolong Gold + DAPI and allowed to harden overnight at room temperature in the dark.

[0304] Confocal imaging data were acquired using a fully motorized Zeiss Axio Observer Z1 (Carl Zeiss, Jena, Germany) inverted imaging system using a spinning disk confocal scanning unit CSU-W1 (Yokogawa) equipped with a 63x objective and two Hamamatsu ORCA-Flash 4.0 v2 sCMOS cameras for simultaneous dual-channel acquisition. Solid-state lasers (405, 488, 561, 647, and 725 nm) were coupled to the spinning head via optical fiber. 3D stacks were acquired using a Piezo PZ-2150 XYZ motorized stage. Images were acquired, reviewed, aligned, processed, and exported for presentation and image analysis using Slidebook (Intelligent Imaging Innovations, Dencer, USA).

[0305] III. Ex vivo autoradiography of compound 21 Compound 21 (500 μg in 30 μL of aCSF, 40 μL flush) was administered it to naive female Sprague-Dawley rats (n=4).Rats were allowed to recover for 24 hours, after which they were euthanized by CO2 inhalation, and their brains were removed and frozen on crushed dry ice.These were then sectioned into 10 micrometer slices and mounted on glass slides.

[0306] The sections were saved for autoradiography and were not fixed in PFA. 18 The cells were incubated with a solution of F-labeled Compound 1 (5 nM) for 15 min, followed by extensive washing with saline. The blocking solution contained 5 μM of non-radioactive Compound 1 in addition to the radioactive tracer.

[0307] After washing and development on a phosphor plate, the signal distribution showed high uptake in the brain periphery and meninges, with slow diffusion toward the midbrain (Figure 26). Furthermore, this binding signal was eliminated when incubating tissue with a solution containing the homologous block and when incubating the tracer with brain sections from naive rats. Overall, this indicates that tracer binding is highly specific for the presence of compound 21.

[0308] IV. Longitudinal distribution and stability of compound 21 in rat brain Compound 21 (500 μg in 30 μL of aCSF, 40 μL flush) was administered it to naive female Sprague-Dawley rats (n=10). The rats were allowed to recover for 24, 48, 96, 168, or 336 hours, after which they were euthanized by CO2 inhalation, and their brains were removed, bisected into left and right hemispheres, and frozen on crushed dry ice. The right hemisphere was then sectioned into 10-micrometer slices and fixed on glass slides, while the left hemisphere was homogenized and analyzed by LC-MS / MS. Brain sections were stained with TCO-Cy5 as described above.

[0309] The compound 21 signal was found to decrease from 7.07 μg / mL on day 1 to 0.87 μg / mL on day 7 and to 0.10 μg / mL on day 14 (FIG. 27). Notably, the concentration of compound 21 (including unreactive species) remained essentially identical to that of compound 21-TCO-PEG4-DBCO, indicating that there was no measurable degradation of MeTz. Any instability in the compound is likely due to some loss of MeTz or the linker as a whole, rather than oxidation or reduction of the tetrazine.

[0310] Biological Example 5 - Pre-targeted PET imaging in NHPs This study was carried out in accordance with the Karolinska Institutet's "Guidelines for planning, conducting and documenting experimental research."

[0311] For the baseline experiment, two female cynomolgus nonhuman primates (NHPs) were used (NHP1, 5.2 kg; NHP2, 6.0 kg). NHPs were housed at the Astrid Fagraeus laboratory (KM-F), Comparative Medicine Department, Karolinska Instituitet (Solna, Sweden). PET experiments were performed using a MultiScan LFER 150 PET / CT system (Mediso Ltd.). Anesthesia was initiated with intramuscular ketamine injection (10 mg / kg) and maintained after tracheal intubation with a mixture of sevoflurane, oxygen, and medical air. Oxygen saturation, heart and respiratory rates, and blood pressure were continuously monitored throughout the scan. Body temperature was maintained with a Bair Hugger-Model 505 (Arizant Healthcare Inc., MN) and monitored with an esophageal thermometer. The head was immobilized with a fixation device throughout the scan, and fluid balance was maintained by continuous infusion of Ringer Acetate. 18F-labeled Compound 1 was injected as an intravenous bolus (158 MBq in NHP1 and 152 MBq in NHP2). Brain radioactivity was measured continuously for 125 min according to a preprogrammed series of 35 frames. For both NHPs, arterial blood sampling using a chronic indwelling catheter placed in the femoral artery was performed at different time points for blood and plasma radioactivity measurements and for radiometabolite HPLC analysis.

[0312] Whole-brain time-activity curves showed rapid brain uptake, followed by washout during the scan, with approximately 90% clearance (<0.5 SUV) by 60 minutes pi. Arterial blood samples were collected at individual time points throughout the scan and analyzed by radio-HPLC for the percent parent fraction. The parent tracer was rapidly metabolized in vivo, with approximately 30% remaining by 15 minutes pi. Importantly, elution of two major radioactive metabolites was observed prior to the parent compound, indicating increased polarity and a reduced likelihood of brain entry and confusion with the PET signal.

[0313] Although very little signal was observed to be retained in the brain, self-blocking experiments in two additional NHPs confirmed the lack of specific binding in the absence of compound 21. 18 NHPs were administered 1 mg / kg of Compound 1 as a self-block before injection with F-labeled Compound 1. No changes in tracer kinetics were observed after self-blocking, indicating no detectable off-target binding in the brain (Figure 32).

[0314] For experiments involving IT administration, one female (NHP4, 4.2 kg) and one male (NHP5, 7.0 kg) NHP were used. Intrathecal administration of Compound 21 was performed. 18The study was performed 24 hours before administration of F-labeled Compound 1. After sedation with intramuscular injection of ketamine (10 mg / kg), animals were positioned in a ventral lateral position and a lumbar puncture was performed at 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 aCSF (Bio-Techne) and injected in a volume of 2.4 mL over 2 to 6 minutes. PET experiments were performed as described above. Simultaneously with the start of PET data collection, 18 F-labeled Compound 1 was infused as an intravenous bolus (145 MBq in NHP4 and 142 MBq in NHP5). Venous blood sampling in NHP4 and arterial blood sampling in NHP5 using a chronic indwelling catheter placed in the femoral artery were performed at different time points for measurement of blood and plasma radioactivity and for radiometabolite analysis.

[0315] The summed PET images from all PET acquisitions were manually co-registered to T1-weighted brain magnetic resonance (MR) images, and regions of interest (ROIs) were manually delineated for the whole brain, occipital cortex, caudate nucleus, putamen, ventral striatum, frontal cortex, white matter, thalamus, cerebellum, and hippocampus. Time-activity curves for brain regions were generated from the dynamic PET data after application of co-registration parameters to the ROIs. Regional uptake was calculated as the standardized uptake value (SUV), defined as uptake (Bq / mL) / injected radioactivity (Bq) × body weight (g).

[0316] Figure 29 underwent the following processing to best highlight tracer uptake and distribution within the brain. To create a static volume, dynamic frames were averaged between 60 and 120 minutes post-injection, and SUV was 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)). Furthermore, 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 were then linearly resampled from natural space to template space, and a Gaussian blur kernel of 2 mm full width at half maximum (FWHM) was applied. Finally, the SUV image was masked using a template brain mask to show only values ​​within the brain. Figure 30 shows brain PET processed as described above, but without the brain mask applied.

[0317] Greater uptake of the PET tracer in the brain was observed in both animals receiving compound 21 (Figure 28). Importantly, the distribution of the PET signal was primarily located in the cortical gray matter, which is expected given that the ASO was delivered intrathecally and is very similar to the distribution observed in rats.

[0318] Biological Example 6 - Metabolite Identification of 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) was incubated in DMEM containing 1 million cells / mL for 1 hour (human) or 0.5 hours (rat and monkey) at 37°C and 5% CO2. The reaction was stopped using a 1:1 addition of cold acetonitrile. After centrifugation, the supernatant was diluted 1:3 with water for analysis. LC-MS was performed using a Waters UPLC and Sciex 5600 Triple-TOF MS equipped 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 to 55% over 5 min at 0.45 mL / min, followed by a wash with 90% B. A HCD collision energy of 35 was used and data were processed using Sciex PeakView and MetabolitePilo software. Figure 31 summarizes the results of the metabolite identification study for compound 2. The table on the left shows the percentage of each isolated metabolite derived from hepatocytes of the different species tested.

[0319] Other embodiments While the present invention has been described in connection with the detailed description, the above description is intended to be illustrative and not limiting of the scope of the invention, which is defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the claims.

Claims

1. Formula (I): 【Chemistry 1】 or a pharmaceutically acceptable salt thereof, wherein 【Chemistry 2】 is a single bond indicating that the cyclooctene is trans or cis, W is a radiolabeled moiety containing a radioisotope; Y １ is -CH ２ -, Ar, 4- to 10-membered heterocyclyl, ＠ -SO ２ -Ar- ＠＠ ,or ＠ -C(=O)-Ar- ＠＠ wherein Y １ or Y １ In the group represented by the formula: Ｙ and optionally substituted by ＠ teeth, 【Transformation 3】 and indicate the point where it connects to ＠＠ indicates the point of connection with W, where Ar is a 6- to 10-membered aryl or a 5- to 10-membered heteroaryl; R Ｙ is halogen, C １－６ alkyl, or oxo (optionally); 【Chemistry 4】 is a bond, -(CH ２ ) ｎ -, Het, & -C(=O)-Het- && , & -C １－６ Alkylenyl-C(═O)-Het- && , & -C(=O)-C １－６ Alkylenyl-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 a 4- to 10-membered carbocyclyl, and R Ｌ is hydrogen or C １－６ alkyl, n is 1, 2, or 3; & is X １ and indicate the point where it connects to && Is Y １ and indicate the point where it connects to X １ is a bond, —O—, —CH ２ -, -N(R Ｘ ) -, ＾ - (CH ２ ) ２ O- ＾＾ ,or ＾ - (CH ２ ) ２ N (R Ｘ )- ＾＾ wherein R Ｘ is hydrogen or C １－６ is alkyl, ＾ indicates the point of connection with cyclooctene, ＾＾ teeth, 【Transformation 5】 and indicate the point where it connects to R １ is hydrogen or —OH, R ２ is hydrogen, or X １ and R ２ together with the atoms to which they are attached form a moiety represented by formula A, 【Transformation 6】 wherein Z is a bond, —O—, or —NH—; 【Transformation 7】 indicates the point of connection with cyclooctene, 【Transformation 8】 teeth, 【Chemistry 9】 and indicate the point where it connects to wherein the heterocyclyl contains 1 to 3 heteroatoms selected from oxygen, nitrogen, and sulfur, and the heteroaryl contains 1 to 4 heteroatoms selected from oxygen, nitrogen, and sulfur.

2. The compound has the formula (IIA): 【Chemistry 10】 2. The compound of claim 1, wherein:

3. The compound has formula (IIB): 【Chemistry 11】 2. The compound of claim 1, wherein:

4. W is １８ F. １１ C part, chelation ６８ Ga, one or more １８ C substituted by F １－６ alkyl, or one or more １８ C substituted by F １－６ The compound of any one of claims 1 to 3, or a pharmaceutically acceptable salt thereof, which is alkoxyl.

5. W is １８ F, one １８ C substituted by F １－４ Alkyl or one １８ C substituted by F １－４ The compound of any one of claims 1 to 4, or a pharmaceutically acceptable salt thereof, which is alkoxyl.

6. W is １８ F, -CH ２ １８ F, or -OCH ２ CH ２ １８ The compound according to any one of claims 1 to 5, wherein R is H, or a pharmaceutically acceptable salt thereof.

7. Y １ is Ar, 6- to 9-membered heterocyclyl, ＠ -SO ２ -Ar- ＠＠ ,or ＠ -C(=O)-Ar- ＠＠ wherein Y １ or Y １ In the group represented by the formula: Ｙ and optionally substituted by Ar is phenyl or 6-membered heteroaryl; R Ｙ is halogen, C １－４ 7. The compound of any one of claims 1 to 6, or a pharmaceutically acceptable salt thereof, which is alkyl, or oxo (optionally).

8. Y １ teeth, 【Chemistry 12】 8. The compound according to any one of claims 1 to 7, selected from the group consisting of: or a pharmaceutically acceptable salt thereof. 【Request Item 9】 【Chemistry 13】 is a bond, -(CH ２ ) ｎ -, Het, & -C(=O)-Het- && , & -C １－４ Alkylenyl-C(═O)-Het- && , & -C(=O)-C １－４ Alkylenyl-Het- && , -NH-Het-NH-, -NH-Cyc-NH-, -NH-, & -NR Ｌ -Het- && ,or & -Het-NR Ｌ - && wherein Het is a 4-8 membered monocyclic heterocyclyl, a 6-9 membered spiroheterocyclyl, or a 6-8 membered bridged heterocyclyl, Cyc is a 4-8 membered cycloalkyl, and R Ｌ is hydrogen or C １－４ The compound according to any one of claims 1 to 8, wherein n is alkyl and n is 1 or 2, or a pharmaceutically acceptable salt thereof. 【Request Item 10】 【Chemistry 14】 is a bond, -CH ２ -, Het, & -C(=O)-Het- && , & -C １－２ Alkylenyl-C(═O)-Het- && , & -C(=O)-C １－２ Alkylenyl-Het- && , -NH-Het-NH-, -NH-Cyc-NH-, -NH-, & -NR Ｌ -Het- && ,or & -Het-NR Ｌ - && wherein Het is a 4- to 6-membered monocyclic heterocyclyl, a 6- to 7-membered spiroheterocyclyl, or a 7- to 8-membered bridged heterocyclyl; Cyc is a 4- to 6-membered monocyclic cycloalkyl; and R Ｌ is hydrogen or C １－２ The compound of any one of claims 1 to 8, or a pharmaceutically acceptable salt thereof, wherein R is alkyl. 【Request Item 11】 【Chemistry 15】 teeth, 【Chemistry 16】 11. The compound according to any one of claims 1 to 10, or a pharmaceutically acceptable salt thereof, selected from the group consisting of:

12. X １ is a bond, —O—, —CH ２ -, -NH-, -N(CH ３ ) -, ＾ - (CH ２ ) ２ O- ＾＾ , ＾ - (CH ２ ) ２ NH- ＾＾ ,or ＾ - (CH ２ ) ２ N (CH ３ )- ＾＾ 12. The compound according to any one of claims 1 to 11, wherein:

13. X １ is -O-, -NH-, -N(CH ３ ) -, ＾ - (CH ２ ) ２ O- ＾＾ , ＾ - (CH ２ ) ２ NH- ＾＾ ,or ＾ - (CH ２ ) ２ N (CH ３ )- ＾＾ 12. The compound according to any one of claims 1 to 11, wherein:

14. X １ and R ２ together with the atoms to which they are attached, form the formula A: 【Chemistry 17】 forming a portion represented by 12. The compound according to any one of claims 1 to 11, wherein Z is -O- or -NH-, or a pharmaceutically acceptable salt thereof.

15. The compound has the formula (II): [Chemistry 18] The compound according to any one of claims 1, 3, 7 to 11, and 14, or a pharmaceutically acceptable salt thereof, wherein

16. Y １ teeth, 【Chemistry 19】 16. The compound of claim 15, selected from the group consisting of: or a pharmaceutically acceptable salt thereof.

17. Y １ teeth, 【Chemistry 20】 17. The compound of claim 15 or 16, wherein: 【Request Item 18】 【Chemistry 21】 teeth, 【Chemistry 22】 18. The compound according to any one of claims 15 to 17, or a pharmaceutically acceptable salt thereof, selected from the group consisting of: 【Request Item 19】 【Chemistry 23】 teeth, 【Chemistry 24】 or 【Chemistry 25】 19. The compound according to any one of claims 15 to 18, wherein:

20. The compound is 【Chemistry 26】 20. The compound according to any one of claims 15 to 19, wherein:

21. The compound has the formula (III): 【Chemistry 27】 wherein: W is １８ F or -CH ２ １８ F, Y １ is a 6-membered heteroaryl; 【Chemistry 28】 is a bond, Het, & -C(=O)-Het- && , & -C １－４ Alkylenyl-C(═O)-Het- && , & -C(=O)-C １－４ Alkylenyl-Het- && wherein Het is a 4-6 membered heterocyclyl; X １ is a bond, —O—, or —N(R Ｘ )—, wherein R Ｘ is hydrogen or C １－４ 10. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein R is alkyl.

22. Y １ is pyridyl, 【Chemistry 29】 teeth, join, 【Transformation 30】 is selected from the group consisting of X １ represents a bond, —O—, —NH—, or —N(CH ３ 22. The compound of claim 21,

23. The compound has formula (IV): 【Chemistry 31】 wherein: W is １８ F or -OCH ２ CH ２ １８ F, Y １ is a 6- to 9-membered heterocyclyl, or ＠ -C(=O)-Ar- ＠＠ wherein Y １ or Y １ In the group represented by the formula: Ｙ and optionally substituted by Ar is phenyl or 6-membered heteroaryl; R Ｙ is C １－４ alkyl or oxo (optionally); 【Chemistry 32】 is a bond, -CH ２ -,or & C(=O)-Het- && wherein Het is a 6-8 membered heterocyclyl; X １ is -CH ２ -, ＾ - (CH ２ ) ２ O- ＾＾ , ＾ - (CH ２ ) ２ NH- ＾＾ ,or ＾ - (CH ２ ) ２ N (CH ３ )- ＾＾ 4. The compound of claim 1 or 3, wherein:

24. Y １ teeth, 【Transformation 33】 is selected from the group consisting of 【Transformation 34】 teeth, Combination, -CH ２ -、 【Chemistry 35】 and 【Transformation 36】 24. The compound of claim 23, selected from the group consisting of: or a pharmaceutically acceptable salt thereof.

25. W is １８ F, Y １ teeth 【Chemistry 37】 and 【Transformation 38】 is a bond, X １ teeth, ＾ - (CH ２ ) ２ NH- ＾＾ ,or ＾ - (CH ２ ) ２ N (CH ３ )- ＾＾ 25. The compound of claim 23 or 24, wherein:

26. A compound set forth in Table 1, or a pharmaceutically acceptable salt thereof.

27. A pharmaceutical composition comprising a compound according to any one of claims 1 to 26, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or excipient.

28. 1. A method for measuring the distribution of a biomolecule in a subject, the method comprising: (i) administering the biomolecule to the subject; (ii) administering to the subject a compound according to any one of claims 1 to 26 or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to claim 27, wherein the biomolecule binds to the compound in vivo; and (iii) imaging the distribution of the biomolecule in the subject; and The method comprising:

29. 29. The method of claim 28, wherein the biomolecule is an antisense oligonucleotide, an antibody, a gene therapy agent, or a nanoparticle.

30. 30. The method of claim 29, wherein the biomolecule is an antisense oligonucleotide.

31. The method according to any one of claims 28 to 30, wherein the method measures the distribution of a biomolecule in the brain and / or spinal cord of a subject.

32. The method according to any one of claims 28 to 31, wherein the biomolecule and the compound according to any one of claims 1 to 26, or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition according to claim 27, are administered simultaneously to a subject.

33. The method according to any one of claims 28 to 31, wherein the biomolecule and compound according to any one of claims 1 to 26, or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition according to claim 27, is administered individually to a subject.

34. The method according to any one of claims 28 to 33, wherein the biomolecule is administered intrathecally to the subject, and the compound according to any one of claims 1 to 26, or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition according to claim 27, is administered intravenously to the subject.

35. The method according to any one of claims 28 to 34, wherein the imaging is performed by PET / CT.

36. The method according to any one of claims 28 to 34, wherein the imaging is performed by SPECT.

37. The method of any one of claims 28 to 36, wherein the subject is a human.

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