Granzyme B directional imaging and therapy

Abstract

Compounds of Formula (I) and Formula (II) that are capable of binding to Granzyme B. Also provided herein are pharmaceutical compositions comprising such compounds, e.g., for use in imaging Granzyme B and / or treating immune dysregulation.

Description

[Technical Field]

[0001] Related applications This application claims the benefit as of the filing date of U.S. Provisional Application No. 63 / 036,918, filed on 9 June 2020, which is incorporated herein by reference in its entirety.

[0002] This disclosure relates to compounds useful for imaging techniques, and more specifically, to compounds useful for imaging granzyme B using medical imaging techniques, including positron emission tomography. [Background technology]

[0003] Granzyme B is the most commonly found serine protease in the granules of natural killer cells and cytotoxic T cells. Granzyme B is released along with the pore-forming protein perforin at the immunological synapse formed between T cells and their targets. Subsequently, some of the released granzyme B enters cancer cells, primarily through perforin pores, where it activates multiple substrates that lead to the activation of the caspase cascade. As a downstream effector of tumor cytotoxic T cells, granzyme B is used as an early biomarker for tumors responding to immunotherapy.

[0004] There is a need to develop novel compounds that function as effective granzyme B imaging agents, as well as therapies to treat immunomodulatory disorders such as cancer. [Overview of the Initiative]

[0005] This application provides specific compounds capable of targeting granzyme B, and their use as imaging agents or therapeutic agents.

[0006] In one embodiment, the present disclosure features a compound of formula (I) and a pharmaceutically acceptable salt thereof. [ka]

[0007] In equation (I), A is the chelate site, X is -CH2C(NH)-, -CH2C(O)-, -CH2C(S)-, -NHC(NH)-, -NHC(O)-, -NHC(S)-, -OC(NH)-, -OC(O)-, and -OC(S)-, and optionally X is -CH2C(O)- or -NHC(S)-, L is a peptide linker that comprehensively has 1 to 6 amino acid residues. R 1 H or C 1-6 Alkyl (for example, methyl), R 2 C 1-6 Alkyl or C 3-6 It is a cycloalkyl group.

[0008] In some examples, R 1 It can be H. In other examples, R 1 It can be methyl.

[0009] In some embodiments, the compound, or a pharmaceutically acceptable salt thereof, is of formula (Ia). [ka]

[0010] In some embodiments, L comprehensively comprises 1 to 3 (1, 2, or 3) amino acid residues. In one example, L comprises 3 amino acid residues.

[0011] In some cases, the compound, or a pharmaceutically acceptable salt thereof, is of formula (Ia-A). [ka]

[0012] Alternatively, the compound, or a pharmaceutically acceptable salt thereof, is of formula (Ia-B). [ka]

[0013] In any of the compounds of formula (I) disclosed herein, the chelate moiety A can be 1,4,7-triazacyclononane-N,N’,N’’-triacetic acid (NOTA). Alternatively, the chelate moiety A can be 1,4,7-triazacyclononane-4,7-diyl diacetic acid (NODA).

[0014] In a specific example, the compound or its pharmaceutically acceptable salt is any of Compounds 1 to 22 listed in Table 1 below.

[0015] In another aspect, the present disclosure features a compound of formula (II), or a pharmaceutically acceptable salt thereof.

Chemical formula

[0016] In formula (II), M is a metal or a metal linked to a radioisotope, A is a chelating moiety that chelates the metal, X is selected from -CH2C(NH)-, -CH2C(O)-, -CH2C(S)-, -NHC(NH)-, -NHC(O)-, -NHC(S)-, -OC(NH)-, -OC(O)-, and -OC(S)-, and optionally, X is -CH2C(NH)- or -NHC(S)-, L is a peptide linker that inclusively has 1 to 6 amino acid residues, R 1 is H or C 1-6 alkyl (e.g., methyl), R 2 is C 1-6 alkyl or C 3-6 cycloalkyl.

[0017] In some examples, R 1 can be H. In other examples, R 1 can be methyl.

[0018] In some embodiments, the compound, or a pharmaceutically acceptable salt thereof, is of formula (IIa). [ka]

[0019] In some embodiments, L comprehensively comprises 1 to 3 amino acid residues (1, 2, or 3). In one example, L comprises 3 amino acid residues.

[0020] In some cases, the compound, or a pharmaceutically acceptable salt thereof, is of formula (IIa-A). [ka]

[0021] In other examples, the compound, or a pharmaceutically acceptable salt thereof, is of formula (IIa-B). [ka]

[0022] In any of the formula (II) compounds disclosed herein, the chelate moiety may be 1,4,7-triazacyclononane-N,N',N'' triacetic acid (NOTA). In other examples, the chelate moiety may be 1,4,7-triazacyclononane-4,7-diyldiacetic acid (NODA).

[0023] Alternatively or additionally, the metal M in any of the formula (II) compounds disclosed herein may be a radioactive isotope of Ga. For example, a radioactive isotope of Ga is: 68 It is Ga. In other embodiments, the metal M in the formula (II) compound disclosed herein may be Al linked to a radioactive isotope. In one example, the radioactive isotope is 18 It is F.

[0024] Specific examples of compounds of formula (II) provided herein include any of the compounds listed in Table 2 below.

[0025] In another embodiment, the Disclosure provides a composition comprising either a compound of formula (I) or formula (II) disclosed herein, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

[0026] Furthermore, this disclosure provides a kit comprising (i) a compound of formula (I) or formula (II) disclosed herein, or a pharmaceutically acceptable salt thereof, and (ii) one or more additional therapeutic agents. Also within the scope of this disclosure are combination therapies comprising a compound of formula (I) or formula (II) disclosed herein, or a pharmaceutically acceptable salt thereof, and one or more additional therapeutic agents.

[0027] In another embodiment, the present disclosure features a method for treating an immunomodulatory disorder, the method comprising administering to a subject in need of such treatment a compound of formula (II) disclosed herein and optionally one or more additional therapeutic agents.

[0028] Examples of immunomodulatory disorders include, but are not limited to, autoimmune disorders, inflammatory disorders, skin disorders, cancer, and cardiovascular disorders. One example of an immunomodulatory disorder is cancer.

[0029] Exemplary therapeutic agents disclosed herein include, but are not limited to, anti-inflammatory agents, steroids, immunotherapeutic agents, chemotherapeutic agents, and therapeutic antibodies.

[0030] In another embodiment, the Disclosure features a method for imaging granzyme B in cells or tissues, samples, or cell or tissue samples. This method may include contacting cells or tissues, samples, or cell or tissue samples with a compound of formula (II) disclosed herein, or a pharmaceutically acceptable salt thereof, and imaging the cells or tissues, samples, or cell or tissue samples using a suitable imaging technique to image granzyme B in the cells or tissues, samples, or cell or tissue samples. In some examples, the compound of formula (II) is a radioisotope. 18 F or 68 Contains Ga.

[0031] Furthermore, within the scope of this disclosure are (a) a pharmaceutical composition comprising a compound of formula (I) or formula (II) and one or more therapeutic agents optionally disclosed herein for use in imaging granzyme B or in the treatment of immunomodulatory disorders, or (b) a compound of formula (I) or formula (II), either alone or in combination with one or more therapeutic agents, for the manufacture of a drug for use in imaging granzyme B or in the treatment of a target disease disclosed herein.

[0032] Details of one or more embodiments of the present invention are described in the following description. Other features or advantages of the present invention will become apparent from the following drawings and detailed descriptions of some embodiments, as well as from the appended claims. [Brief explanation of the drawing]

[0033] The following drawings form part of this specification and are included to further illustrate certain aspects of the disclosure, and this disclosure can be better understood by referring to the drawings in conjunction with a detailed description of the specific embodiments presented herein.

[0034] [Figure 1] This shows the semi-preparative HPLC purification chromatogram of the compound 18F-7-Al peak 2 via radiosynthesis using an ORA Neptis radiosynthesis system. [Figure 2]The analytical HPLC chromatogram of peak 2 of compound 18F-7-Al is shown. [Figure 3] The semi-preparative HPLC chromatograms of peaks 1 and 2 of the compound 18F-7-Al, combined via radiosynthesis using an ORA Neptis radiosynthesis system, are shown. [Figure 4] The analytical HPLC chromatogram combining peaks 1 and 2 of compound 18F-7-Al is shown. [Figure 5A-5B] The semi-preparative HPLC purification chromatograms combining peaks 1 and 2 of compound 18F-20-Al are shown. Figure 5A: HPLC Rad. Figure 5B: HPLC UV. [Figure 6A-6B] The analytical HPLC chromatograms combining peaks 1 and 2 of compound 18F-20-Al are shown. Figure 6A: HPLC Rad. Figure 6B: HPLC UV. [Modes for carrying out the invention]

[0035] Cancer immunotherapy represents a significant advance in cancer therapy in recent years. Antibodies targeting immune checkpoints such as programmed cell death protein 1 (PD-1) and cytotoxic T lymphocyte-associated protein 4 (CTLA-4) have been approved for positive outcomes in some patients. Research in the field of immuno-oncology continues, and strategies involving CAR-T cells, vaccines, small molecules, and antibodies are being developed. Despite the promise of these therapies, they are not panaceas. These immunotherapies can be associated with significant adverse events, are expensive, and have response rates typically ranging from 20 to 50%, meaning that the majority of patients do not respond to therapy. Furthermore, response can be difficult to determine an individual patient's response to therapy using conventional methods, as it is often associated with immune cell infiltration, which can cause responsive tumors to grow on anatomical imaging (e.g., CT, MRI) and show increased avidity on FDG-PET imaging due to the influx of metabolically active immune cells. Given the limitations of current imaging techniques, clinical studies of cancer immunotherapy typically use overall survival, as opposed to progression-free survival, as the study endpoint.

[0036] Granzyme B, a downstream marker of cytotoxic T cell activity, can serve as a novel biomarker for evaluating the effectiveness of cancer immunotherapy. Granzyme B expression within tumors can be evaluated not only as a measure of the presence or absence of CTLs, but also as an effector protein released by active T cells, thus explaining the problem of T cell depletion that makes it difficult to achieve assessment of the presence of CTLs.

[0037] This application provides certain specific compounds, such as compounds of formula (I) and (II), that exhibit high binding affinity to granzyme B. Such compounds may function as granzyme B imaging agents and may be used to identify patients who respond to immunotherapy agents. Such compounds may also be used for therapeutic purposes, for example, in combination with one or more therapeutic agents, such as immunotherapies.

[0038] definition It should be understood that the terms used herein are for the purpose of describing specific embodiments and are not intended to limit them. Furthermore, any methods, devices, and materials similar to or equivalent to those described herein may be used in carrying out or testing the present invention, but preferred methods, devices, and materials are described herein. In addition to the foregoing, when used herein and in the appended claims, the following terms have the meanings given unless otherwise specified. "Amino" refers to the -NH2 radical. "Cyano" refers to the -CN radical. "Hydroxyl" refers to the -OH radical. "Imino" refers to the =NH substituent. "Nitro" refers to the -NO2 radical. "Oxo" refers to an =O substituent. "Thioxo" refers to the =S substituent. "Trifluoromethyl" refers to the -CF3 radical. "Alkyl" refers to a linear, saturated, acyclic, monovalent hydrocarbon radical having 1 to 6 carbon atoms, or a branched, saturated, acyclic, monovalent hydrocarbon radical, which is bonded to the rest of the molecule by single bonds, such as methyl, ethyl, n-propyl, 1-methylethyl (isopropyl), n-butyl, n-pentyl, 1,1-dimethylethyl (t-butyl), 3-methylpentyl, 2-methylpentyl, etc. The alkyl portion may be unsubstituted, or it may be optionally substituted. Optionally substituted alkyl radicals, depending on their valence, can be independently classified as halo, cyano, nitro, aryl, cycloalkyl, heterocyclyl, heteroaryl, oxo, trimethylsilanyl, -OR 3 -OC(O)-R 3 , -N(R 3 )2, -C(O)R 4 , -C(O)OR 3 ,-C(O)N(R 3 )2, -N(R 3 )C(O)OR 5 , -N(R 3 )C(O)R 5 , -N(R3 )S(O)tR 5 (wherein t is 1 or 2), -S(O) t Ure 5 (wherein t is 1 or 2), -S(O) p R 5 (wherein p is 0, 1, or 2) and -S(O)tN(R 3 An alkyl radical which is optionally substituted with 1, 2, 3, 4, or 5 substituents selected from the group consisting of )2 (wherein t is 1 or 2), and each R 3 R is independently hydrogen, alkyl, haloalkyl, cycloalkyl, cycloalkylalkyl, aryl, aralkyl, heterocyclyl, or heteroaryl, and each R 4 R is independently hydrogen, cycloalkyl, aryl, heterocyclyl, or heteroaryl, and each R 5 These are independently alkyl, haloalkyl, cycloalkyl, aryl, or heteroaryl. "Cycloalkyl" refers to a stable, non-aromatic monocyclic or polycyclic hydrocarbon radical having 3 to 15 carbon atoms, preferably 3 to 10 carbon atoms, and being saturated or unsaturated, with single bonds connecting it to the rest of the molecule. Polycyclic hydrocarbon radicals are bicyclic, tricyclic, or tetracyclic systems. Unsaturated cycloalkyls contain one, two, or three carbon-carbon double bonds and / or one carbon-carbon triple bond. Examples of monocyclic cycloalkyl radicals include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. Examples of polycyclic cycloalkyl radicals include adamantyl, norbornyl, and dekalinyl. The cycloalkyl moiety may be unsubstituted, or it may be optionally substituted. The optionally substituted cycloalkyls are independently alkyl, alkenyl, halo, haloalkyl, haloalkenyl, cyano, nitro, oxo, aryl, aralkyl, cycloalkyl, heterocyclyl, heteroaryl, and -R. 4 -OR 3 , -R 4 -OC(O)-R3 , -R 4 -N(R 3 )2, -R 4 -C(O)R 3 , R 4 -C(O)OR 3 , -R 4 -C(O)N(R 3 )2, -R 4 -N(R 3 )C(O)OR 5 , -R 4 -N(R 3 )C(O)R 5 , -R 4 -N(R 3 )S(O)tR 5 (In the formula, t is either 1 or 2), -R 4 -S(O)tOR 5 (In the formula, t is either 1 or 2), -R 4 -S(O)pR5 (where p is 0, 1, or 2), and -R 4 -S(O)tN(R 3 A cycloalkyl radical which is optionally substituted with 1, 2, 3, 4, or 5 substituents selected from the group consisting of )2 (wherein t is 1 or 2), and each R 3 R is independently hydrogen, alkyl, haloalkyl, cycloalkyl, aryl, heterocyclyl, or heteroaryl, and each R 4 These are independently, directly bonded, or linear or branched alkylene or alkenylene chains, and each R 5 These are independently alkyl, haloalkyl, cycloalkylalkyl, aryl, aralkyl, heterocyclyl, or heteroaryl.

[0039] In some embodiments, the preparation of the compound may involve the addition of an acid or base that affects, for example, the catalytic action of the desired reaction or the formation of a salt form such as an acid addition salt.

[0040] Exemplary acids may be inorganic or organic acids, and include, but are not limited to, strong and weak acids. Some exemplary acids include hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, p-toluenesulfonic acid, 4-nitrobenzoic acid, methanesulfonic acid, benzenesulfonic acid, trifluoroacetic acid, and nitric acid. Some weak acids include, but are not limited to, acetic acid, propionic acid, butanoic acid, benzoic acid, tartaric acid, pentanoic acid, hexanoic acid, heptanoic acid, octanoic acid, nonanoic acid, and decanoic acid.

[0041] Examples of bases include lithium hydroxide, sodium hydroxide, potassium hydroxide, lithium carbonate, sodium carbonate, potassium carbonate, and sodium bicarbonate. Some examples of strong bases include, but are not limited to, hydroxides, alkoxides, metal amides, metal hydrides, metal dialkylamides, and arylamines. Alkoxides include lithium, sodium, and potassium salts of methyl, ethyl, and t-butyl oxides; metal amides include sodium amides, potassium amides, and lithium amides; metal hydrides include sodium hydride, potassium hydride, and lithium hydride; and metal dialkylamides include lithium, sodium, and potassium salts of methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, trimethylsilyl, and cyclohexyl-substituted amides.

[0042] As used herein, the term “pharmaceutically acceptable salt” refers to a derivative of a disclosed compound in which the parent compound is modified by converting an existing acidic or base moiety to its salt form. Examples of pharmaceutically acceptable salts include, but are not limited to, mineral salts or organic acid salts of basic residues such as amines, and alkali salts or organic salts of acidic residues such as carboxylic acids. The pharmaceutically acceptable salts of this application include, for example, conventional non-toxic salts of parent compounds formed from non-toxic inorganic or organic acids. The pharmaceutically acceptable salts of this application can be synthesized from parent compounds containing basic or acidic moieties by conventional chemical methods. Generally, such salts can be prepared by reacting the free acidic or base form of these compounds with a stoichiometric amount of a suitable base or acid in water, an organic solvent, or a mixture of the two, and generally preferred are non-aqueous media such as ether, ethyl acetate, alcohol (e.g., methanol, ethanol, isopropanol, or butanol), or acetonitrile (MeCN). A list of suitable salts can be found in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa., 1985, p. 1418 and Journal of Pharmaceutical Science, 66, 2 (1977). Conventional methods for preparing salt forms are described, for example, in Handbook of Pharmaceutical Salts: Properties, Selection, and Use, Wiley-VCH, 2002.

[0043] In some embodiments, the compounds or salts thereof provided to the present invention are substantially isolated. "Substantially isolated" means that the compounds are at least partially or substantially separated from the environment in which they were formed or detected. Partial isolation may include, for example, a composition concentrated in the compounds provided herein. Substantial isolation may include a composition containing at least about 50% by weight, at least about 60% by weight, at least about 70% by weight, at least about 80% by weight, at least about 90% by weight, at least about 95% by weight, at least about 97% by weight, or at least about 99% by weight of the compounds or salts thereof provided herein. Methods for isolating compounds and salts thereof are commonplace in the art.

[0044] As used herein, the terms “ambient temperature” and “room temperature” or “rt” are understood in the art and generally refer to, for example, the reaction temperature, i.e., the room temperature in which the reaction is carried out, e.g., about 20°C to about 30°C.

[0045] I. Granzyme B-targeted compounds Granzyme B target compounds disclosed herein, for example, compounds of formula (I) or formula (II), are provided herein. The compounds disclosed herein include the compounds themselves, pharmaceutically acceptable salts thereof, and stereoisomers thereof.

[0046] The compounds described herein may contain one or more chiral centers and therefore may exist in various isomeric forms, such as enantiomers and / or diastereomers. For example, the compounds described herein may be in the form of individual enantiomers, diastereomers or geometric isomers, or in the form of a mixture of stereoisomers, including a racemic mixture and a mixture concentrated into one or more stereoisomers. The isomers may be isolated from the mixture by methods known to those skilled in the art, including chiral high-pressure liquid chromatography (HPLC) and the formation and crystallization of chiral salts, or preferred isomers may be prepared by asymmetric synthesis. See, for example, Jacques et al., Enantiomers, Racemates and Resolutions (Wiley Interscience, New York, 1981), Wilen et al., Tetrahedron 33:2725 (1977), Eliel, Stereochemistry of Carbon Compounds (McGraw-Hill, NY, 1962), and Wilen, Tables of Resolving Agents and Optical Resolutions p.268 (ELEliel, Ed., Univ. of Notre Dame Press, Notre Dame, IN 1972). This disclosure further encompasses the compounds described herein as individual isomers that are substantially free from other isomers, or as mixtures of various isomers.

[0047] A. Compounds of formula (I) In some embodiments, the present disclosure provides compounds of formula (I) shown below that can bind to granzyme B with high binding affinity. [ka]

[0048] In formula (I), A is the chelate moiety. The chelate moiety is a molecule or ion that can function as a polydentate ligand for a metal ion. For example, a molecule having multiple atoms with available lone pairs (including, but not limited to, nitrogen and oxygen) can function as a chelate moiety. The chelate moiety can be linear (e.g., EDTA), cyclic (macrocyclic molecules, e.g., DOTA, including porphyrins), and may include macrocyclics (macrocyas) that are generally known in the art. The chelate moiety may have 2, 3, 4, 5, or 6 functional groups (e.g., amines, amides, hydroxyls, carboxylic acids, etc.) that have lone pairs available for coordination with the metal. Examples of chelate moieties for use in the granzyme B target compounds disclosed herein include 1,4,7-triazaicyclonan triacetic acid (NOTA), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), 1,4,7-triazaicyclonan-1-glutaric acid-4,7-diacetic acid (NODAGA), ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), cyclohexyl-1,2-diaminetetraacetic acid (CDTA), and ethylene glycol-0,0'-bis(2-aminoethyl)-N,N,N',N'tetraacetic acid (E Examples include, but are not limited to, GTA, N,N-bis(hydroxybenzyl)-ethylenediamine-N,N'-diacetic acid (HBED), triethylenetetraminehexaacetic acid (TTHA), hydroxydiaminetriacetic acid (HEDTA), 1,4,8,11-tetraazacyclotetradecane-N,N',N'',N'''-tetraacetic acid (TETA), 1,4,7,10-tetraaza-1,4,7,10-tetra-(2-carbamoylmethyl)-cyclododecane (TCMC), 1,4,7-triazacyclononane-4,7-diyldiacetic acid (NODA), and desferrioxamine B (DFO).In some embodiments, the chelating agent is selected from the group consisting of 1,4,7-triazacyclononane triacetic acid (NOTA), 1,4,7,10-tetrazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), 1,4,7-triazacyclononane-4,7-diyldiacetic acid (NODA), and 1,4,7-triazacyclononane-1-glutaric acid-4,7-diacetic acid (NODAGA). In some embodiments, the chelating agent is 1,4,7-triazacyclononane triacetic acid (NOTA). In other embodiments, the chelating agent is 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA). In some embodiments, the chelating agent is 1,4,7-triazacyclononane-4,7-diyldiacetic acid (NODA).

[0049] X can be -CH2C(NH)-, -CH2C(O)-, -CH2C(S)-, -NHC(NH)-, -NHC(O)-, -NHC(S)-, -OC(NH)-, -OC(O)-, or -OC(S)-. In one example, X is -CH2C(O)-. In another example, X is -NHC(S)-.

[0050] L can be a peptide linker having a total of 1 to 6 amino acid residues. In some examples, L contains a total of 1 to 3 amino acid residues. In other examples, L contains a total of 4 to 6 amino acid residues. In one example, L contains 1 amino acid residue. In another example, L contains 2 amino acids. In yet another example, L contains 3 amino acid residues. Or, L contains 4 amino acid residues. In yet another example, L contains 5 amino acid residues. Or, L contains 6 amino acid residues.

[0051] Suitable amino acid residues within peptide linker L may include, but are not limited to, native and unnatural amino acid residues (including β-amino acid residues and D-amino acids). Amino acid residues can form chains via standard peptide bonds or by forming amide bonds with computable side chains (e.g., glutamic acid (e.g., D-Glu), aspartic acid). Exemplary peptide linkers include, but are not limited to, Glu-Gly-Gly, D-Glu-β-Ala-β-Ala, Gly-Gly, Gly, Glu-Gly, Glu, D-Glu, Arg-Gly, and Lys-Gly.

[0052] In some embodiments, R 1 In other embodiments, R 1 C 1-6 It is alkyl. For example, R 1 It can be methyl.

[0053] In some embodiments, R 2 C 1-6 It could be alkyl. Or, R 2 C 3-6 Cycloalkyl (e.g., branched or unbranched, substituted or unsubstituted) or C 3-6 It may be a cycloalkyl (e.g., cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl).

[0054] The hemiacetal unit in the compound of formula (I) may be in the open-chain aldehyde form. As a result, the compound of formula (I) may have the following structure. [ka]

[0055] In any of equations (I-1) to (I-6), A, X, L, R 1 , and R 2 Each of these is as described herein.

[0056] In some embodiments, R 1 H is R 2 is a C4 alkyl compound, such as the compound of formula (Ia). [ka]

[0057] In some specific examples, X is -CH2C(O)-, as in the compound of formula (Ia-A). [ka]

[0058] In other examples, X is -NHC(S)-, as in the compound of formula (Ia-B). [ka]

[0059] Examples of compounds that embody formula (I) are listed in Table 1. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4]

[0060] In some examples, the compounds of formula (I) are compounds 1, 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 17, or 18. In one specific example, the compound of formula (I) is compound 7.

[0061] B. Compounds of formula (II) In some embodiments, the present disclosure provides compounds of formula (II) shown below that can bind to granzyme B with high binding affinity and specificity. [ka]

[0062] In formula (II), M is a metal or a metal bound to a radioisotope. Suitable metals for use in this disclosure include those useful for imaging granzyme B, such as metals that are suitable radioimaging agents, and metals that can be bound to nonmetallic radioisotopes that are suitable radioimaging agents. Exemplary metallic radioisotopes are: 68 It is Ga. An example of a nonmetallic radioactive isotope is, 18 F is present, which can be conjugated with Al to fill the granzyme B-binding compounds disclosed herein.

[0063] A, X, L, R 1 , and R 2 Each of these is as defined herein. See, for example, the section referred to as the compound of formula (I) above.

[0064] Examples of compounds containing radioactive isotopes are listed in Table 2. [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4]

[0065] In some examples, the compound of formula (II) is compound 1-Al, 2-Al, 3-Al, 4-Al, 5-Al, 6-Al, 7-Al, 9-Al, 10-Al, 11-Al, 12-Al, 13-Al, 17-Al, or 18-Al. In one example, the compound of formula (II) is compound 7-Al, 18 It can be filled with F.

[0066] Furthermore, the scope of this disclosure includes variants of the compounds of formula (I) and formula (II) disclosed herein, in which the lactone ring in the compound of formula (I) or formula (II) may be substituted with other parts, such as an aryl ring or a heteroaryl ring. In certain embodiments, the variants may have the following structures. [ka]

[0067] The above mutant compounds are radioactive isotopes (for example, 18 The material can be filled with a metal that can conjugate with F). An example is shown below. [ka]

[0068] The above-mentioned compounds containing radioisotopes are useful as imaging agents in one or more methods provided herein. Furthermore, the radioisotope-containing compounds provided herein may also be useful in one or more therapeutic applications when administered to a subject in a therapeutically effective dose. For example, 18The compounds containing F may be useful as imaging agents (e.g., as non-toxic and / or non-therapeutic radioisotopes) when administered to a subject at low concentrations (e.g., 5 mCi). In some embodiments, this isotope may be toxic. As noted above, this application also includes pharmaceutically acceptable salts of the compounds described herein. The term “pharmaceutically acceptable” is used herein to mean these compounds, materials, compositions, and / or drug forms that are within the scope of sound medical judgment and are suitable for use in contact with human and animal tissues without excessive toxicity, irritation, allergic reactions, or other problems or complications, commensurate with a reasonable benefit / risk ratio.

[0069] This application also includes stereoisomers of compounds such as compound 7 and 7-Al described herein. See below. Stereoisomers arise from two chiral centers (ring-closed) or one chiral center (open chain) of the hemiacetal unit, as indicated by the dashed lines. [ka]

[0070] C. Granzyme B Target Compound Chemical Synthesis As can be understood, the compounds provided herein, including stereoisomers and their salts, may be prepared using known organic synthesis techniques and may be synthesized according to any of a number of possible synthetic routes.

[0071] The compounds disclosed herein, or pharmaceutically acceptable salts thereof, can be prepared by following the exemplary protocols described below. Suitable protecting groups for use in such synthesis are known in the art. See, for example, McOmie, Protective Groups in Organic Chemistry, (1973):98. Briefly, the synthesis of the granzyme B-targeting compounds disclosed herein comprises the following processes: A) attaching a desired tricyclic ring (e.g., in formula (I)) to a resin (e.g., by peptide coupling) and deprotecting the tricyclic ring; B) attaching one of the L-peptide linkers (which may be formed by repeatedly coupling amino acid residues to a desired length) to the resin-attached tricyclic ring to form a tricyclic peptide linker moiety; C) attaching the tricyclic peptide linker moiety to a chelation site to form the compound of formula (I), cleaving it to form a resin, and then optionally D) chelating a metal with the compound of formula (I) to form the compound of formula (II).

[0072] For example, in step A, the tricyclic rings in compounds of formulas (I) and (II) (e.g., Fmoc-Haic(2S,5S)-OH) can be bonded to a resin (e.g., H-Asp(OtBu)-H) and deprotected. Then, in step B, the free amine of the tricyclic ring can be bonded to an Fmoc-protected amino acid (e.g., isoleucine), and then Fmoc can be removed. The resulting free amine can be bonded to a desired number of amino acids using conventional peptide synthesis methods to reach the tricyclic peptide linker moiety. Fmoc protection and deprotection can be carried out in each round of amino acid residue addition. In step C, a chelate moiety having a terminal coupling group (e.g., a carboxylic acid, carbamothio-O-acid, isothiocyanate, or thiocyanate) can be bonded to the final free amine of the amino acid chain in the tricyclic peptide linker moiety to produce the compound of formula (I), which can be released from the resin and purified as needed. The compound of formula (I) thus prepared can then be chelated with a metal disclosed herein (e.g., Ga, Al) to produce the compound of formula (II). Exemplary examples for synthesizing exemplary compounds of formula (I) and formula (II) are provided in the following examples. In an alternative embodiment of step C, the compound may be cleaved from the resin before bonding to the chelate portion.

[0073] Many suitable imaging agents (e.g., ratio isotopes) are known in the art (see, for example, U.S. Patents 5,021,236, 4,938,948, and 4,472,509, each of which is incorporated herein by reference in whole). The radiolabeled compounds, or pharmaceutically acceptable salts thereof, provided herein can be prepared according to methods well known in the art. Synthetic methods for incorporating radioisotopes into organic compounds are well known in the art, and those skilled in the art will readily recognize other methods applicable to the compounds provided herein.

[0074] Those skilled in the art will understand that the processes described herein are not exclusive means for synthesizing the compounds provided herein, and that a broad repertoire of synthetic organic reactions is available for potentially use in the synthesis of the compounds provided herein. Those skilled in the art will know how to select and implement appropriate synthetic routes. Preferred synthetic methods for starting materials, intermediates, and products are found in Advances in Heterocyclic Chemistry, Vols. 1-107 (Elsevier, 1963-2012); Journal of Heterocyclic Chemistry Vols. 1-49 (Journal of Heterocyclic Chemistry, 1964-2012), Carreira, et al. (Ed.) Science of Synthesis, Vols. 1-48 (2001-2010) and Knowledge Updates KU2010 / 1-4; 2011 / 1-4; 2012 / 1-2 (Thieme, 2001-2012), Katritzky, et al. (Ed.) Comprehensive Organic Functional Group Transformations (Pergamon Press, 1996), and Katritzky et al. (Ed.) Comprehensive Organic Functional Group Transformations II (Elsevier, 2001-2012). nd Edition, 2004), Katritzky et al. (Ed.), Comprehensive Heterocyclic Chemistry (Pergamon Press, 1984), Katritzky et al., Comprehensive Heterocyclic Chemistry II, (Pergamon Press, 1996), Smith et al., March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, 6 thIt can be identified by referring to documents including the sources such as Ed. (Wiley, 2007), Trost et al. (Ed.), Comprehensive Organic Synthesis (Pergamon Press, 1991).

[0075] The reactions for preparing the compounds described herein can be carried out in a suitable solvent that can be readily selected by those skilled in the art of organic synthesis. A suitable solvent can be substantially non-reactive with the starting materials (reactants), intermediates, or products at the temperature at which the reaction is carried out (e.g., a temperature ranging from the freezing temperature to the boiling temperature of the solvent). A given reaction can be carried out in one solvent or a mixture of one or more solvents. Depending on a particular reaction step, those skilled in the art can select a solvent suitable for the particular reaction step.

[0076] The preparation of the compounds described herein can involve the protection and deprotection of various chemical groups. The need for protection and deprotection, as well as the selection of appropriate protecting groups, can be readily determined by those skilled in the art. The chemical nature of the protecting groups can be found, for example, in T.W. Greene and P.G.M. Wuts, Protective Groups in Organic Synthesis, 3 rd Ed., Wiley & Sons, Inc., New York (1999).

[0077] The reaction can be monitored according to any suitable method known in the art. For example, the formation of the product can be monitored by spectroscopic means such as nuclear magnetic resonance spectroscopy (e.g., 1 H or 13 C), infrared spectroscopy, spectrophotometry (e.g., UV-visible), mass spectrometry, or by chromatographic methods such as high performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LCMS), or thin layer chromatography (TLC). The compounds can be purified by those skilled in the art by various methods including high performance liquid chromatography (HPLC) and normal phase silica chromatography.

[0078] II. Pharmaceutical Compositions Any of the compounds of formula (I) and formula (II), or a pharmaceutically acceptable salt thereof, may be mixed with a pharmaceutically acceptable carrier to form a pharmaceutical composition for use in granzyme B imaging and / or for therapeutic purposes as disclosed herein. In some embodiments, pharmaceutical compositions are provided herein that, in combination with one or more pharmaceutically acceptable carriers (excipients), contain as an active ingredient a metal-containing compound (formula (II) compound) provided herein, or a pharmaceutically acceptable salt thereof. "Acceptable" means that the carrier must be compatible with the active ingredient of the composition (preferably able to stabilize the active ingredient) and not harmful to the subject being treated. Suitable carriers include microcrystalline cellulose, mannitol, glucose, skim milk powder, polyvinylpyrrolidone, and starch, or combinations thereof.

[0079] Some examples of suitable excipients include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, and methylcellulose. Further examples of pharmaceutical formulations may include, but are not limited to, lubricants, wetting agents, emulsifiers, and suspending agents such as talc, magnesium stearate, and mineral oil, preservatives such as methyl and propyl hydroxybenzoates, sweeteners, flavoring agents, or combinations thereof. For details on acceptable pharmaceutical compositions, see Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa., 1985, p. 1418.

[0080] Conventional methods known to those skilled in the medical field can be used to administer a pharmaceutical composition to a subject, depending on the type of disease being treated or the site of the disease. This composition may also be administered via other conventional routes, e.g., orally, parenterally, by inhalation spray, topically, rectally, nasally, orally, vaginally, or via an implanted reservoir. As used herein, the term “parenteral” includes subcutaneous, intradermal, intravenous, intramuscular, intra-articular, intra-arterial, intra-synovial, intrasternal, subarachnoid, intralesional, and intracranial injection or infusion techniques. Parenteral administration may be in the form of a single bolus dose or, for example, by a continuous perfusion pump. In addition, it may be administered to a subject via an injectable depot administration route, such as using depot-injectable or biodegradable materials and methods for 1, 3, or 6 months.

[0081] The injectable composition may contain various carriers such as vegetable oil, dimethylactamide, dimethylformamide, ethyl lactate, ethyl carbonate, isopropyl myristate, ethanol, and polyols (glycerol, propylene glycol, liquid polyethylene glycol, etc.). For intravenous injection, water-soluble antibodies may be administered by drip method, thereby injecting a pharmaceutical preparation containing the antibody and physiologically acceptable excipients. Examples of physiologically acceptable excipients include 5% dextrose, 0.9% saline, Ringer's solution, or other suitable excipients. Intramuscular preparations, for example, a sterile preparation of a suitable soluble salt form of the antibody, can be dissolved and administered with a pharmaceutical excipient such as water for injection, 0.9% saline, or 5% glucose solution.

[0082] For oral administration, the composition may take the form of tablets or capsules, prepared by conventional means using acceptable excipients such as binders (e.g., pre-gelatinized corn starch, polyvinylpyrrolidone, or hydroxypropyl methylcellulose), fillers (e.g., lactose, microcrystalline cellulose, or calcium hydrogen phosphate), lubricants (e.g., magnesium stearate, talc, or silica), disintegrants (e.g., potato starch or sodium glycolate), or wetting agents (e.g., sodium lauryl sulfate). The tablets may be coated by methods well known in the art.

[0083] In some embodiments, the compounds provided in the present invention, or pharmaceutically acceptable salts thereof, are suitable for parenteral administration. In some embodiments, the compounds, or pharmaceutically acceptable salts thereof, are suitable for intravenous administration.

[0084] Pharmaceutical compositions and formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids, and powders. Conventional pharmaceutical carriers, aqueous solutions, powders, or oily bases, and thickeners may be necessary or desirable.

[0085] In the preparation of the pharmaceutical compositions provided herein, the active ingredient is typically mixed with an excipient, diluted by the excipient, or encapsulated within such a carrier, for example, in the form of a capsule, pouch, paper, or other container. When the excipient functions as a diluent, it may be a solid, semi-solid, or liquid material that functions as a vehicle, carrier, or medium for the active ingredient.

[0086] Therefore, pharmaceutical compositions may be in the form of tablets, pills, powders, lozenges, pouches, cachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols (as solid or in a liquid medium), ointments, soft and hard gelatin capsules, suppositories, sterile injectable solutions, and sterile packaged powders.

[0087] III.How to use This application further provides a method for imaging granzyme B using one of the above-mentioned compounds or pharmaceutically acceptable salts thereof. In some embodiments, the method is an in vitro method. In some embodiments, the method is an in vivo method.

[0088] In some embodiments, the imaging method is performed on cells, tissues, cell samples, tissue samples, or subjects.

[0089] As used herein, the term “Subject” refers to any animal, including mammals and invertebrates. For example, mice, rats, other rodents, rabbits, dogs, cats, pigs, cattle, sheep, horses, primates, fish, and humans. In some embodiments, the subject is a human. In some embodiments, the subject is a mouse. In some embodiments, the subject is a fish (for example, a zebra).

[0090] This application further provides a method for imaging granzyme B in cells or tissues. i) Contacting cells or tissues with an effective amount of one of the above compounds or a pharmaceutically acceptable salt thereof, ii) imaging cells or tissues using a suitable imaging technique, thereby imaging granzyme B within the cells or tissues.

[0091] This application further provides a method for imaging granzyme B in a sample, cell sample, or tissue sample. i) Contacting a sample, cell sample, or tissue sample with an effective amount of one of the above compounds or a pharmaceutically acceptable salt thereof, ii) Including imaging a sample, cell sample, or tissue sample using a suitable imaging technique, thereby imaging granzyme B within the sample, cell sample, or tissue sample.

[0092] As used herein in the context of imaging, the term "sample" refers to a biological sample other than a cell or tissue sample obtained from a subject. Examples of samples include, but are not limited to, saliva, blood, and andrin.

[0093] This application further provides a method for imaging granzyme B in a subject, i) Administering an effective amount of one of the above compounds, or a pharmaceutically acceptable salt thereof, ii) Including imaging the object using a suitable imaging technique, thereby imaging granzyme B in the object.

[0094] This application further provides a method for imaging immune responses in cell or tissue samples. i) Contacting a cell or tissue sample with an effective amount of one of the above compounds or a pharmaceutically acceptable salt thereof, ii) Including imaging a cell or tissue sample using a suitable imaging technique, thereby imaging the immune response within the cell or tissue sample.

[0095] This application further provides a method for imaging the immune response in a subject. i) Administering an effective amount of one of the above compounds, or a pharmaceutically acceptable salt thereof, ii) Including imaging a subject using a suitable imaging technique, thereby imaging the immune response in the subject.

[0096] This application further provides a method for monitoring the treatment of a disease in the subject, i) Administering an effective amount of one of the above compounds, or a pharmaceutically acceptable salt thereof, ii) Including imaging the subject using a suitable imaging technique.

[0097] This application further provides a method for monitoring the immune response in the treatment of diseases in the subject, i) Administering an effective amount of one of the above compounds, or a pharmaceutically acceptable salt thereof, ii) Including imaging the subject using a suitable imaging technique.

[0098] In some embodiments, the methods provided herein further include waiting for a sufficient amount of time prior to imaging to allow the compound or a pharmaceutically acceptable salt thereof to accumulate in disease-related cellular or tissue sites (e.g., cellular or tissue sites in the subject).

[0099] In some embodiments, the methods provided herein further include waiting for a sufficient amount of time prior to imaging to allow the compound or a pharmaceutically acceptable salt thereof to bind to granzyme B at disease-related cellular or tissue sites (e.g., cellular or tissue sites in the subject).

[0100] In some embodiments, a sufficient time range is approximately 30 seconds to approximately 24 hours, for example, approximately 30 seconds to approximately 24 hours, approximately 30 seconds to approximately 12 hours, approximately 30 seconds to approximately 6 hours, approximately 30 seconds to approximately 2 hours, approximately 30 seconds to approximately 1 hour, approximately 30 seconds to approximately 30 minutes, approximately 30 seconds to approximately 10 minutes, approximately 10 minutes to approximately 24 hours, approximately 10 minutes to approximately 12 hours, approximately 10 minutes to approximately 6 hours, approximately 10 minutes to approximately 2 hours, approximately 10 minutes to approximately 1 hour, approximately 10 minutes to approximately 30 The timeframes are approximately minutes, 30 minutes to 24 hours, 30 minutes to 12 hours, 30 minutes to 6 hours, 30 minutes to 2 hours, 30 minutes to 1 hour, 1 hour to 24 hours, 1 hour to 12 hours, 1 hour to 6 hours, 1 hour to 2 hours, 2 hours to 24 hours, 2 hours to 12 hours, 2 hours to 6 hours, 6 hours to 24 hours, 6 hours to 12 hours, or 12 hours to 24 hours.

[0101] In some embodiments, the preferred imaging technique is a non-invasive imaging technique. In some embodiments, the preferred imaging technique is a minimally invasive imaging technique. As used herein, the term “minimally invasive imaging technique” includes imaging techniques using an internal probe or injection of one of the above compounds or a pharmaceutically acceptable salt thereof, or the use of a radiotracer via a syringe.

[0102] Exemplary imaging techniques include, but are not limited to, fluorescence fluoroscopy, X-ray imaging, magnetic resonance imaging (MRI), ultrasound imaging, photoacoustic imaging, thermography, tomography, echocardiography, positron emission tomography (PET), computed tomography (CT) imaging (PET-MRI), single-photon emission tomography (SPECT), and ultrasound imaging. In some embodiments, preferred imaging techniques are selected from the group consisting of PET imaging, PET-CT, PET-MRI, and SPECT.

[0103] In some embodiments, the preferred imaging technique is selected from the group consisting of PET imaging, computed tomography-based PET, and magnetic resonance imaging (MRI)-based PET. In some embodiments, the preferred imaging technique is the selected PET imaging.

[0104] In some embodiments, the diseases described herein are selected from the group consisting of autoimmune disorders, inflammatory disorders, skin disorders, cancers, and cardiovascular disorders. As used herein, the term “disease” is used interchangeably with the term “immunomodulation.”

[0105] In some embodiments, the disease is cancer. In some embodiments, cancer includes solid tumors. In some embodiments, cancer is a blood cancer (e.g., leukemia, lymphoma, etc.). In some embodiments, cancer is selected from the group consisting of brain cancer, breast cancer, cervical cancer, colorectal cancer, lung cancer, lymphoma, melanoma, bladder cancer, renal cell carcinoma, multiple myeloma, pancreatic cancer, and prostate cancer. In some embodiments, cancer is hairy cell leukemia, Kaposi's sarcoma, follicular lymphoma, chronic myeloid leukemia, cutaneous T-cell lymphoma, peripheral T-cell lymphoma, pre-T-cell lymphocytic leukemia, classical Hodgkin lymphoma, B-cell non-Hodgkin lymphoma, chronic lymphocytic leukemia, acute myeloid leukemia, myelodysplastic syndrome, primary myelofibrosis, post-essential thrombocythemia myelofibrosis, post-polycythemia myelofibrosis, melanoma, renal cell carcinoma, prostate cancer, non-small cell lung cancer, small cell lung cancer, glioblastoma, hepatocellular carcinoma, urinary epithelial carcinoma, esophageal cancer, gastroesophageal cancer, gastric cancer The cancer is selected from the group consisting of multiple myeloma, colon cancer, rectal cancer, head and neck squamous cell carcinoma, epithelial ovarian cancer (EOC), primary peritoneal cancer, fallopian tube cancer, HER2+ breast cancer, ER+ / PR+ / HER2- breast cancer, trinegative breast cancer, gastric cancer, pancreatic cancer, bladder cancer, Merkel cell carcinoma, nasopharyngeal cancer, adrenocortical carcinoma, meningioma, neuroblastoma, retinoblastoma, osteosarcoma, rhabdomyosarcoma, Ewing's sarcoma, liposarcoma, fibrosarcoma, leiomyosarcoma, peripheral neuroectodermal tumor, uterine squamous cell carcinoma, vaginal squamous cell carcinoma, and vulvar squamous cell carcinoma. In some embodiments, the cancer is colon cancer.

[0106] In some embodiments, the disease is graft-versus-host disease, rheumatoid arthritis, systemic lupus erythematosus, Hashimoto's thyroiditis, multiple sclerosis, myasthenia gravis, type 1 diabetes, uveitis, post-uveitis, allergic encephalomyelitis, glomerulonephritis, rheumatic fever, post-infectious glomerulonephritis, psoriasis, atopic dermatitis, contact dermatitis, eczematous dermatitis, seborrheic dermatitis, lichen planitis, pemphigus, bullous pemphigus, epidermolysis bullosa, urticaria, angioedema, vasculitis, erythema, cutaneous eosinophilia, lupus erythematosus, acne, alopecia, keratoconjunctivitis, vernal keratoconjunctivitis, uveitis associated with Behçet's disease, keratitis Herpetic keratitis, keratoconus, corneal epithelial atrophy, corneal exudate, pemphigus bulbosus, Mullen's ulcer, scleritis, Graves' ophthalmopathy, Vogt-Koyanagi-Harada syndrome, sarcoidosis, pollen allergy, reversible obstructive airway disease, bronchial asthma, allergic asthma, endogenous asthma, exogenous asthma, dust asthma, chronic or reflux asthma, late asthma and high-response airway asthma, bronchitis, gastric ulcer, vascular injury due to ischemic disease and thrombosis, ischemic bowel disease, inflammatory bowel disease, necrotizing enterocolitis, intestinal lesions associated with burns, colon disease, proctitis, eosinophilic gastroenteritis, masticatory cytosis, Crohn's disease, ulcerative colitis, hemiplegia Pain, rhinitis, interstitial retinitis, predisposition syndrome, hepatitis syndrome, secondary nephritis, polynephritis, myositis, Guillain-Barré syndrome, Meniere's disease, polyneuritis, mononeuritis, curative disease, hyperthyroidism, Bassodine disease, pure erythrocytosis, aplastic anemia, hypoplastic anemia, idiopathic thrombocytopenic purpura, autoimmune hemolytic anemia, agranulocytosis, pernicious anemia, megablastic anemia, annelosplasia, osteoporosis, sarcoidosis, fibropulmonary dysplasia, idiopathic interstitial pneumonia, dermatomyositis, albinism vulgaris, sclerosis vulgaris, photoallergic susceptibility, cutaneous T-cell lymphoma, arteriosclerosis Chemical diseases, atherosclerosis, aortitis syndrome, polyarteritis, nodular cardiomyopathy, sclerosis, Wegen's granuloma, Sjögren's syndrome, fatty anemia, anemic anemia, ovarian tumors, Arborone's disease, parenchymal anemia, nephritis, dermatitis, male pattern baldness, senile alopecia by preventing hair loss, senile alopecia by promoting hair germination and / or hair growth, muscular dystrophy, pyoderma, Sézary syndrome, Addison's disease, organ ischemia-reperfusion injury, transplant diseases, ischemic diseases, endotoxin shock, pseudomembranous colitis, drug or radiation-induced colitis, ischemic acute renal failure, chronic renal failure,Pulmonary oxygen or drug-induced toxemia, lung cancer, emphysema, cataracts, siderosis, retinitis pigmentosa, senile macular degeneration, vitreous scarring, alkaline corneal fever, erythema multiforme, linear IgA globular dermatitis and cementodermitis, gingivitis, periodontitis, sepsis, pancreatitis, aging, cancer development, carcinoma and low-baropathy metastasis, histamine or leukotriene-C4 release-related disorders, Behçet's disease, autoimmune hepatitis, primary biliary cirrhosis, sclerosing cholangitis, partial hepatectomy, acute hepatic necrosis, The group is selected from the following: necrosis due to toxins, viral hepatitis, shock, anoxia, B-virus hepatitis, non-A / non-B hepatitis, cirrhosis, alcoholic cirrhosis, liver failure, acute liver failure, delayed liver failure, acute on-cycle liver failure, cytomegalovirus infection, HCMV infection, AIDS, senile dementia, trauma, chronic bacterial infection, malignant tumors of the lymphatic system, acute lymphoblastic leukemia, chronic lymphoblastic leukemia, acute lymphoblastic lymphoma, and chronic lymphoblastic lymphoma.

[0107] In some embodiments, the disease is selected from the group consisting of systemic lupus erythematosus, chronic rheumatoid arthritis, type 1 diabetes, inflammatory bowel disease, biliary cirrhosis, uveitis, multiple sclerosis, Crohn's disease, ulcerative colitis, bullous pemphigus, sarcoidosis, psoriasis, autoimmune myositis, Wegener's granulomatosis, ichthyosis, Graves' ophthalmopathy, asthma, Schreroder's disease, and Sjögren's syndrome.

[0108] In some embodiments, the disease is selected from the group consisting of bone marrow rejection, organ transplant rejection, and graft-versus-host disease.

[0109] As used herein, the term “therapeutic dose” refers to the amount of the aforementioned active compound, or a pharmaceutically acceptable salt thereof, or pharmaceutical agent that elicits a desired biological or pharmaceutical response in a tissue, system, animal, individual, or human, as determined by a researcher, veterinarian, physician, or other clinician.

[0110] In some embodiments, one dose of the compound or a pharmaceutically acceptable salt thereof administered to a subject or individual is approximately 1 μg to approximately 2 g, for example, approximately 1 μg to approximately 2 g, approximately 1 μg to approximately 1000 mg, approximately 1 μg to approximately 500 mg, approximately 1 μg to approximately 100 mg, approximately 1 μg to approximately 50 mg, approximately 1 μg to approximately 1 mg, approximately 1 μg to approximately 500 μg, approximately 1 μg to approximately 100 μg, approximately 1 μg to approximately 10 μg, approximately 10 μg to approximately 2 g, for example, approximately 10 μg to approximately 2 g, approximately 10 μg to approximately 1000 mg, approximately 10 μg to approximately 500 mg, approximately 10 μg to approximately 100 mg, approximately 10 μg to approximately 50 mg, approximately 10 μg to approximately 1 mg, approximately 10 μg to approximately 500 μg, approximately 10 μg to approximately 100 μg, about 100μg to about 2g, for example, about 100μg to about 2g, about 100μg to about 1000mg, about 100μg to about 500mg, about 100μg to about 100 mg, about 100 μg to about 50 mg, about 100 μg to about 1 mg, about 100 μg to about 500 μg, about 500 μg to about 2 g, for example, about 500 μg to about 2 g, about 5 The dosages are approximately 00 μg to 1000 mg, approximately 500 μg to 500 mg, approximately 500 μg to 100 mg, approximately 500 μg to 50 mg, approximately 500 μg to 1 mg, approximately 1 mg to 2 g, approximately 1 mg to 1000 mg, approximately 1 mg to 500 mg, approximately 1 mg to 100 mg, approximately 1 mg to 50 mg, or approximately 50 mg to 500 mg.

[0111] As used herein, the terms “to treat” or “to cure” mean one or more of the following: (1) inhibiting a disease, for example, inhibiting a disease, condition or disorder in an individual experiencing or exhibiting the pathological or symptomatic aspects of a disease, condition or disorder (i.e., preventing further development of the pathology and / or symptoms); and (2) improving a disease, for example, improving a disease, condition or disorder in an individual experiencing or exhibiting the pathological or symptomatic aspects of a disease, condition or disorder (i.e., reversing the pathology and / or symptoms), such as reducing, decreasing or mitigating one or more of the symptoms of a disease.

[0112] Combination therapy When used in methods of treating diseases, the compounds of formula (II) provided herein, or pharmaceutically acceptable salts thereof, may be administered as therapeutic agents in combination with one or more additional therapeutic agents. Examples of additional therapeutic agents include, but are not limited to, anti-inflammatory agents, steroids, immunotherapeutic agents, chemotherapeutic agents, and therapeutic antibodies.

[0113] In some embodiments, additional therapeutic agents induce an immune response in a cell or tissue sample or subject.

[0114] This application further provides a method for treating the disease in the subject, a) This includes administering an effective amount of one of the above compounds or a pharmaceutically acceptable salt thereof to the subject.

[0115] In some embodiments, the subject is identified and / or diagnosed with a disease to be treated before step a). In some embodiments, the subject is identified and / or diagnosed with a disease to be treated after step a). For example, the disease to be treated is selected from the group consisting of autoimmune disorders, inflammatory disorders, skin disorders, cancers, and cardiovascular disorders as described herein.

[0116] In some embodiments, the subject has been treated with one or more immunotherapeutic agents prior to step a). In some embodiments, the disease has been determined to be resistant to one or more immunotherapeutic agents administered prior to step a).

[0117] In some embodiments, this method b) further comprising administering one or more additional therapeutic agents after administering an effective amount of one of the above compounds or a pharmaceutically acceptable salt thereof. In some embodiments, steps a) to b) are repeated multiple times.

[0118] In some embodiments, additional therapeutic agents are administered to the subject in a therapeutically effective dose.

[0119] In some embodiments, the therapeutic agent is an antibody. Exemplary antibodies for use in combination therapy include, but are not limited to, trastuzumab (e.g., anti-HER2), ranibizumab (e.g., anti-VEGF-A), bevacizumab (e.g., anti-VEGF), panitumumab (e.g., anti-EGFR), cetuximab (e.g., anti-EGFR), rituxan (anti-CD20), antibodies against c-MET, and antibody inhibitors of granzyme B (e.g., clone GB11, clone GrB-7, and NCL-L-gran-B), ipilimumab (anti-CTLA-4), nivolumab (anti-PD-1), pembrolizumab (anti-PD-1), atezolizumab (anti-PD-1), elotuzumab (anti-SLAM7), and daratumumab (anti-CD38).

[0120] In some embodiments, the additional therapeutic agent is a steroid. Exemplary steroids include corticosteroids, such as cortisone, dexamethasone, hydrocortisone, methylprednisolone, prednisolone, and prednisone.

[0121] In some embodiments, additional therapeutic agents are anti-inflammatory compounds. Exemplary anti-inflammatory compounds include aspirin, choline salicylate, celecoxib, diclofenac potassium, diclofenac sodium, diclofenac sodium with misoprostol, diflunisal, etodolac, fenoprofen, flurbiprofen, ibuprofen, ketoprofen, meclofenamet sodium, mefenamic acid, nabumetone, naproxen, naproxen sodium, oxaprozin, pyroxycan, rofecoxib, salsalate, sodium salicylate, sulindac, tolmetin sodium, and valdecoxib.

[0122] In some embodiments, additional therapeutic agents are chemotherapeutic agents. Exemplary chemotherapeutic agents include cell proliferation inhibitors, cisplatin, doxorubicin, taxol, etoposide, irinotecan, topotecan, paclitaxel, docetaxel, epotilon, tamoxifen, 5-fluorouracil, methotrexate, temozolomide, cyclophosphamide, SCH66336, R115777, L778,123, BMS214662, gefitinib, erlotinib hydrochloride, antibody against EGFR, imatinib mesylate, intron, ara-C, gemcitabine, uracil mustard, chlormethine, ifosfamide, melphalan, chlorambucil, pipobromane, triethylenemelamine, triethylenethiophosphoramine, busulfan, carmustine, lomustine, streptozocin, dacarbazine, floxuridine, cytarabine, 6-mercaptopurine, 6-thioguanine, fludarabine phosphate, oxaliplatin, folinic acid, pen Tostatin, vinblastine, vincristine, vindesine, bleomycin, dactinomycin, daunorubicin, doxorubicin, epirubicin, idarubicin, mitramycin, deoxycoformycin, mitomycin C, L-asparaginase, teniposide, 17α-ethinylestradiol, diethylstilbestrol, testosterone, prednisone, fluoxymesterone, dromostanolone propionic acid, testactone, megestrol acetate, methylprednisolone, methyltestosterone, prednisolone, Liamcinolone, Chlorotrianicene, Hydroxyprogesterone, Aminoglutethimide, Estramustine, Medroxyprogesterone Acetate, Leuprolide, Flutamide, Toremifene, Goserelin, Carboplatin, Hydroxyurea, Amsacrin, Procarbazin Lev, Mitotane, Mitoxantrone, Levamizole, Vinorelbine, Anastrozole, Letrozole, Capecitabine, Reloxafine, Hexamethylmelamine, Bevacizumab, Vexar, Velcade, Zevalin, Trisenox, Xeroda, Vinorelbine, Porfima Examples include, but are not limited to, elbicus, liposome, thiotepa, altretamine, melphalan, trastuzumab, fulvestrant, exemestane, ifosfamide, rituximab, C225, alemtuzumab, clopharabine, cladribine, aphidicolin, sunitinib, dasatinib, tezacitabine, Sml1, triapin, zidox, trimidox, amidocox, 3-AP, MDL-101, 731, bendamustine, ofatumumab, and GS-1101 (also known as CAL-101).

[0123] In some embodiments, the chemotherapeutic agent is an alkylating agent (e.g., busulfan, chlorambucil, cisplatin, cyclophosphamide (cytoxane), dacarbazine, ifosfamide, mechloretamine (mastozine), and melphalan), nitrosourea (e.g., carmustine, lomustine, semustine, and streptozocin), triazine (e.g., dacarbazine), antimetabolite (e.g., 5-fluorouracil (5-FU), cytarabine (Ara-C), fludarabine, gemcitabine, and methotrexate), purine analogs (e.g., 6-mercaptopurine, 6-thio Guanine and pentosteoxycocitin (2-deoxycocitin), cell division inhibitors (e.g., docetaxel, etoposide (VP16), tenoside, paclitoside, taxol, vinblastine, vincristine, and vinorelbine), antitumor antibiotics (e.g., bleomycin, dactinomycin, daunorubicin, doxorubicin, mitomycin, plicamycin, and idarubicin), platinum chemotherapeutic agents (e.g., cisplatin and carboplatin), anthracendions (e.g., mitoxantrone), toxins (e.g., lysine A chain (Burbage, Leukemia)). The following are selected from the group consisting of research, 21.7(1997):681-690, diphtheria toxin A (Massuda et al., Proceedings of the National Academy of Sciences, 94.26(1997):14701-14706; Lidor, American journal of obstetrics and gynecology, 177.3(1997):579-585), pertussis toxin A subunit, Escherichia coli enterotoxin A subunit, cholera toxin A subunit, and Pseudomonas toxin c-terminus), and gene therapy vectors (e.g., signaling proteins (e.g., Src, Abl, and Ras), Jun, Fos, and Myc).

[0124] In some embodiments, the additional therapeutic agent is an immunotherapy agent. Immunotherapy agents generally trigger immune effector cells and molecules that target and destroy cells (e.g., cancer cells). Immune effectors may be, for example, antibodies specific to markers on the surface of cells (e.g., tumor cells). Antibodies can function as therapeutic effectors on their own or they can mobilize other cells to kill cells. Various effector cells include, but are not limited to, cytotoxic T cells and NK cells.

[0125] Examples of immunotherapeutic agents include azathioprine, chlorambucil, cyclophosphamide, cyclosporine, daclizumab, infliximab, methotrexate, tacrolimus, immunostimulants (e.g., IL-2, IL-4, IL-12, GM-CSF, tumor necrosis factor; interferon alpha, beta, and gamma; F42K and other cytokine analogs; chemokines such as MIP-1, MIP-1β, MCP-1, RANTES, IL-8; or growth factors such as FLT3 ligand), antigenic peptides, polypeptides or proteins, or autologous or allogeneic tumor cell compositions (e.g., Ravindranath & Morton, International reviews of Examples of immunotherapies include, but are not limited to, those described in immunology, 7.4(1991):303-329, hormone therapy, corticosteroids, progestins (e.g., hydroxyprogesterone caproate, medroxyprogesterone acetate, and megestrol acetate), estrogens (e.g., diethylstilbestrol and ethinylestradiol), antiestrogens (e.g., testosterone propionate and fluoxymesterone), antiandrogens (e.g., flutamide), and gonadotropin-stimulating hormone analogs (e.g., leuprolide). Additional immunotherapies are known in the art and can be found, for example, in Rosenberg et al, New England Journal of Medicine, 319.25(1988):1676-1680, and Rosenberg et al, Annals of surgery, 210.4(1989):474.

[0126] The therapeutic agents provided herein may be effective over a wide dose range and are generally administered in effective doses. However, it will be understood that the actual amount of therapeutic agent administered is usually determined by a physician depending on relevant circumstances, including the condition being imaged, the route of administration selected, the compound actually administered, the age, weight, and response of the individual subject, and the severity of the subject's symptoms.

[0127] IV. Granzyme B Imaging and Therapy Kit This disclosure also includes kits (e.g., drug packs) for granzyme B imaging and therapy. The kits provided may include containers (e.g., vials, ampoules, bottles, syringes, and / or dispenser packages, or other suitable containers) in which the pharmaceutical compositions disclosed herein may be placed. In some embodiments, the kits provided may optionally further include a second container containing a pharmaceutical excipient for dilution or suspension of the pharmaceutical compositions. In some embodiments, the pharmaceutical compositions provided in the first and second containers are combined to form a single unit dosage form. In some embodiments, the kit may include an additional container containing one or more additional therapeutic agents disclosed herein, such as anti-inflammatory agents, steroids, immunotherapeutic agents, chemotherapeutic agents, and therapeutic antibodies, as described in the section on combination therapy above.

[0128] In certain embodiments, the kit described herein further includes instructions for using the compounds or compositions contained in the kit. The kit described herein may include information required by regulatory agencies such as the U.S. Food and Drug Administration (FDA). In certain embodiments, the information included in the kit is prescription information. In certain embodiments, the kit and instructions are for imaging granzyme B and / or treating and / or reducing the risk of related target damage (e.g., those disclosed herein) in subjects requiring it. The kit described herein may include one or more additional agents described herein as separate compositions.

[0129] Alternative Embodiments The following describes alternative embodiments of the present disclosure. The following embodiments are not limiting and are intended to illustrate exemplary embodiments for the purpose of illustrating the usefulness of the present disclosure.

[0130] Embodiment 1: A compound, its stereoisomer, or a pharmaceutically acceptable salt thereof, wherein the compound is as follows: [ka] A compound, its stereoisomer, or a pharmaceutically acceptable salt thereof, wherein fluorine (F) is optionally a radioactive isotope and the hemiacetal unit is optionally in an open-chain aldehyde form. Embodiment 2: Compound I is the compound described in Embodiment 1, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, wherein compound I has the following structure. [ka] Embodiment 3: Radioactive isotopes 18 A compound containing F has the following structure: [ka] The compound according to Embodiment 1, its stereoisomer, or a pharmaceutically acceptable salt thereof, wherein the hemiacetal unit is optionally in an open-chain aldehyde form. Embodiment 4: Radioactive isotopes 18 The compound containing F is the compound described in Embodiment 1, its stereoisomer, or a pharmaceutically acceptable salt thereof, having the following structure. [ka] [ka] Embodiment 5: A compound, its stereoisomer, or a pharmaceutically acceptable salt thereof, wherein the compound is as follows: [ka] A compound, its stereoisomer, or a pharmaceutically acceptable salt thereof, wherein the hemiacetal unit is optionally in the open-chain aldehyde form. Embodiment 6: Compound II is the compound described in Embodiment 5, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, wherein compound II has the following structure. [ka] Embodiment 7: A pharmaceutical composition comprising the compound described in Embodiment 5, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier. Embodiment 8: A combination therapy comprising, as a therapeutic agent, the compound described in Embodiment 5, its stereoisomer, or a pharmaceutically acceptable salt thereof, and one or more additional therapeutic agents. Embodiment 9: The combination therapy according to Embodiment 8, wherein one or more additional therapeutic agents are selected from the group consisting of anti-inflammatory agents, steroids, immunotherapy agents, chemotherapeutic agents, and therapeutic antibodies. Embodiment 10: A method for imaging granzyme B in cells or tissues, samples, or cell or tissue samples, the method comprising contacting cells or tissues, samples, or cell or tissue samples with a compound described in any one of Embodiments 1 to 6, its stereoisomer, or a pharmaceutically acceptable salt thereof, and imaging the cells or tissues, samples, or cell or tissue samples using a suitable imaging technique, thereby imaging granzyme B in the cells or tissues, samples, or cell or tissue samples, wherein the compound is a radioisotope 18 A method containing F. Embodiment 11: A method for treating an immunomodulatory disorder in a subject requiring treatment, the method comprising administering to the subject a compound described in Embodiment 4, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof in an amount effective for treating the immunomodulatory disorder. Embodiment 12: The method according to Embodiment 11, wherein the immunomodulatory disorder is selected from the group consisting of autoimmune disorders, inflammatory disorders, skin disorders, cancer, and cardiovascular disorders. Embodiment 13: The method according to Embodiment 12, wherein the immunomodulatory disorder is cancer. Embodiment 14: A method for treating an immunomodulatory disorder in a subject requiring treatment, the method comprising administering to the subject the combination therapy described in claim 7 in an amount effective for treating the immunomodulatory disorder. Embodiment 15: The method according to Embodiment 14, wherein the immunomodulatory disorder is selected from the group consisting of autoimmune disorders, inflammatory disorders, skin disorders, cancer, and cardiovascular disorders. Embodiment 16: The method according to Embodiment 15, wherein the immunomodulatory disorder is cancer. Embodiment 17: A method for monitoring the immune response in the treatment of a disease in a subject, the method comprising administering to the subject an effective amount of the compound of Embodiment 1, its stereoisomer, or a pharmaceutically acceptable salt thereof, and imaging the subject using a suitable imaging technique.

[0131] Without further detail, those skilled in the art will be able to make the most of the present invention based on the above description. Accordingly, the following specific embodiments should be construed as merely illustrative and not to limit the remainder of this disclosure in any way. All publications referenced herein are incorporated by reference with respect to the purposes or subjects referenced herein.

[0132] Example 1: Synthesis and Characterization of Compound 7-Al [ka] Step A: Synthesis of compound 7. [ka] Tricyclic amino acid coupling and Fmoc deprotection: A solution of Fmoc-(2S,5S)-5-amino-1,2,4,5,6,7-hexahydroazepino[3,2,1-Hi]indole-4-one-2-carboxylic acid (2.5 equivalents, 1.23 g, 2.63 mmol) in DMF (43 mL) was added to H-Asp(OtBu)-H NovaSyn TG resin (1.05 mmol, resin-bound, 5 g, 0.21 mmol / g) in a calcination reaction vessel. Next, N,N'-diisopropylcarbodiimide (2.5 equivalents, 2.62 mmol, 1.8 M) and ethylcyanoglyoxalate-2-oxime (2.5 equivalents, 2.63 mmol, 0.9 M) in DMF were added, and the mixture was left overnight in an orbital shaker at ambient temperature. After 24 hours, the mixture was removed from the shaker and the solvent was drained. The resin was washed with DMF (3 × 50 ml), followed by dichloromethane (3 × 50 ml). The ninhydrin test was negative. The resin was treated with 20% piperidine in DMF (30 mL), and the mixture was stirred on a shaker table for 30 minutes. The solvent was then drained, and the resin was washed with DMF (3 × 30 mL). The resin was treated again with 20% piperidine in DMF (30 mL), and the mixture was stirred on a shaker table for 30 minutes. The solvent was then drained, and the resin was washed with DMF (3 × 50 ml), followed by dichloromethane (3 × 50 ml).

[0133] L-isoleucine coupling and Fmoc deprotection: A solution of (((9H-fluoren-9-yl)methoxy)carbonyl)-L-isoleucine (2.5 equivalents, 0.93 g, 2.63 mmol) in DMF (43 mL) was added to the resin from the previous step in a frit-treated reaction vessel. Then, N,N'-diisopropylcarbodiimide (2.5 equivalents, 2.62 mmol, 1.8 M) and ethylcyanoglyoxalate-2-oxime (2.5 equivalents, 2.63 mmol, 0.9 M) in DMF were added, and the mixture was left overnight in an orbital shaker at ambient temperature. The mixture was removed from the shaker, and the solvent was drained. The resin was washed with DMF (3 × 50 mL), followed by washing with dichloromethane (3 × 50 mL). The ninhydrin test was negative. The resin was treated with 20% piperidine in DMF (30 mL), and the mixture was stirred on a shaker table for 30 minutes. Next, the solvent was drained and the resin was washed with DMF (3 × 30 mL). The resin was treated again with 20% piperidine in DMF (30 mL), and the mixture was stirred on a shaker table for 30 minutes. Then the solvent was drained and the resin was washed with DMF (3 × 50 mL), followed by dichloromethane (3 × 50 mL).

[0134] Gly-Gly coupling and Fmoc deprotection: A solution of (((9H-fluoren-9-yl)methoxy)carbonyl)glycylglycine (2.5 equivalents, 0.93 g, 2.63 mmol) in DMF (43 mL) was added to the resin from the previous step in a frit-treated reaction vessel. Then, N,N'-diisopropylcarbodiimide (2.5 equivalents, 2.62 mmol, 1.8 M) and ethylcyanoglyoxalate-2-oxime (2.5 equivalents, 2.63 mmol, 0.9 M) in DMF were added, and the mixture was left overnight in an orbital shaker at ambient temperature. The mixture was removed from the shaker, and the solvent was drained. The resin was washed with DMF (3 × 50 mL), followed by washing with dichloromethane (3 × 50 mL). The ninhydrin test was negative. The resin was treated with 20% piperidine in DMF (30 mL), and the mixture was stirred on a shaker table for 30 minutes. Next, the solvent was drained and the resin was washed with DMF (3 × 30 mL). The resin was treated again with 20% piperidine in DMF (30 mL), and the mixture was stirred on a shaker table for 30 minutes. Then the solvent was drained and the resin was washed with DMF (3 × 50 mL), followed by dichloromethane (3 × 50 mL).

[0135] L-Glu coupling and Fmoc deprotection: A solution of (2S)-5-tert-butoxy-2-(9H-fluoren-9-ylmethoxycarbonylamino)-5-oxopentanoic acid (2.5 equivalents, 2.63 mmol) dissolved in DMF (43 mL) was added to the resin from the previous step in a frit-treated reaction vessel. Next, N,N'-diisopropylcarbodiimide (2.5 equivalents, 2.62 mmol, 1.8 M) in DMF and ethylcyanoglyoxalate-2-oxime (2.5 equivalents, 2.63 mmol, 0.9 M) in DMF were added, and the mixture was left overnight in an orbital shaker at ambient temperature. The mixture was removed from the shaker, and the solvent was drained. The resin was washed with DMF (3 × 50 ml), followed by washing with dichloromethane (3 × 50 ml). The ninhydrin test was negative. The resin was treated with 20% piperidine in 30 mL of DMF, and the mixture was stirred on a shaker table for 30 minutes. The solvent was then drained, and the resin was washed with 3 × 30 mL of DMF. The resin was treated again with 20% piperidine in 30 mL of DMF, and the mixture was stirred on a shaker table for 30 minutes. The solvent was then drained, and the resin was washed with 3 × 50 ml of DMF, followed by 3 × 50 ml of dichloromethane.

[0136] NODA Chelate Coupling: A solution of 2-[4-[[4,7-bis(2-tert-butoxy-2-oxo-ethyl)-1,4,7-triazonan-1-yl]methyl]phenyl]acetic acid (2 equivalents, 1.06 g, 2.10 mmol) in DMF (30 mL) was added to the resin (approximately 1.05 mmol) from the previous step in a frit-treated reaction vessel. Next, N,N'-diisopropylcarbodiimide (2 equivalents, 2.10 mmol, 1.8 M) and ethylcyanoglyoxalate-2-oxime (2 equivalents, 2.10 mmol, 0.9 M) in DMF were added, and the mixture was left in an orbital shaker at ambient temperature for 46 hours. The mixture was removed from the shaker, and the solvent was drained. The resin was washed with DMF (3 × 20 ml), followed by washing with dichloromethane (3 × 20 ml). The ninhydrin test was negative. The ingredients were stored in a vacuum oven at ambient temperature over the weekend.

[0137] Final cutting from the resin: The resin from the previous step (approximately 1.05 mmol) was treated with a mixture of trifluoroacetic acid (10 mL) and dichloromethane (30 mL), and the suspension was stirred overnight on a shaker table. The suspension was removed from the shaker table and the solvent was drained. The resin was washed with dichloromethane (3 × 25 mL) and then blow-dried under nitrogen. The resin was then left overnight in a vacuum oven at ambient temperature. The resin was transferred to a round-bottom flask and treated with a mixture of trifluoroacetic acid (0.6 mL, 8 mmol), water (24 mL), and acetonitrile (36 mL). The suspension was gently stirred and heated at 60°C for 1 hour. The heated suspension was filtered by vacuum filtration, and the resin was washed once with a mixture of water (24 mL) and acetonitrile (36 mL). The filtrate was then frozen at -78°C and freeze-dried to obtain 651.8 mg of a yellow solid as crude peptide.

[0138] Purification of crude peptides: A solution of 50 mg of crude peptide in 0.1% formic acid in H2O (1.0 mL) and acetonitrile (0.4 mL) was filtered through a cotton plug and rinsed with 0.1% formic acid in H2O (0.2 mL). The solution was then subjected to HPLC purification (Phenomenex Gemni C18 RP-HPLC prepared column, mobile phase: 10-40% H2O / acetonitrile, 0.1% formic acid). The fraction was collected and lyophilized to obtain compound 7 (12 mg) as a white, fluffy solid. ES / MS m / z 1077.5 (M+H) +

[0139] Step B: Synthesis of compound 7-Al A solution of compound 7 (crude peptide, 50 mg, ~0.046 mmol) in 100 mM NaF in H2O (1.1 mL, 2.0 equivalents, 0.11 mmol) and 20 mM AlCl3 in 0.1 M NaOAc (5.5 mL, 0.11 mmol) was first sonicated and then the mixture was heated at 105 °C for 30 minutes. The solution was then filtered and subjected directly to HPLC purification (Phenomenex Gemni C18 RP-HPLC preparative column, 0.1% formic acid in H2O / acetonitrile as mobile phase). Fractions were collected and lyophilized to give compound 7-Al(1:1) (13 mg) as an off-white fluffy solid. ES / MS m / z 1121.5 (M+H) +

[0140] Compound in Neptis Perform 18 Radiosynthesis of F-7-Al

Chemical formula

[0141] Next, the collected fractions were diluted with 0.5% (w / v) sodium ascorbate in an aqueous solution (about 30 mL), passed through a Waters Sep-Pak C18 Plus light cartridge (130 mg of adsorbent per cartridge, 55 - 105 μm, part number: 023501, pretreated with 5.0 mL of ethanol followed by 5 mL of water), the product retained on the cartridge was washed with 0.5% (w / v) sodium ascorbate in an aqueous solution (about 15 mL), and then eluted with 1.5 mL of EtOH (USP) into a final product vial containing 0.9% sodium chloride injection (USP) and sodium ascorbate (USP, 0.5% w / v). Then, the C18 cartridge was rinsed with an additional 3.5 mL of 0.9% sodium chloride injection (USP) and sodium ascorbate (USP, 0.5% w / v), and a 15.0 mL formulated product was obtained as 90% v / v 0.9% saline containing 10% v / v EtOH and 0.5% w / v sodium ascorbate.

[0142] To prepare the sterile product, the obtained product (in 90% v / v 0.9% saline containing 10% v / v ethanol and 0.5% w / v sodium ascorbate 18 F-7-Al) was sterile filtered through a 0.22 μm filter (e.g., Millipore part number SLGV033RS Millex® GV sterile filter) into a bulk product vial. Samples were taken from the bulk product vial for HPLC analysis. (Figures 2 and 4)

[0143] Analytical HPLC conditions: (Figure 2) Analytical column: Waters Xbridge BEH C-18 3.5 μm, 4.6 x 100 mm, flow rate = 1.2 mL / min, UV at 254 nm. Retention time 14 - 16 minutes. Mobile phase composition: A: 20 mM ammonium acetate in water, B: acetonitrile (HPLC grade) Gradient:

Table 3

[0144] Analytical HPLC conditions: (Figure 4) Analytical column: Agilent ZORBAX Eclipse XDB-C18 4.6x150mm, part number 993967-902, flow rate = 1.5 mL / min, UV at 254 nm. Gradient method -- Mobile phase: A: 20 mM ammonium acetate in water, B: Acetonitrile (HPLC grade) [Table 4]

[0145] Example 2: Characterization of granzyme B binding activity The above compounds were tested using the human granzyme B biochemical assay provided below. [Table 5] [Table 6]

[0146] Compound preparation: 1. Add DMSO to each compound vial to prepare a 10 mM compound stock solution, and store it at -20°C. 2. Thaw the 10 mM compound stock solution and prepare a 1 mM DMSO stock solution by adding 45 μl of DMSO to 5 μl of 10 mM compound stock solution. 3. The mM DMSO stock solution was sequentially diluted (3.16-fold) by adding 10 μl of 1 mM DMSO stock to 21.6 μl of DMSO and thoroughly mixed. Then, 10 μl of the resulting solution was added to 21.6 μl of DMSO and thoroughly mixed. This process was continued to create 11 dilution points for the assay standard inhibitor and 8 dilution points for the test compound in a 384-well polypropylene plate. Distribute 4.2 μl of each dilution into the assay preparation plate. 5. Next, each well is diluted 25-fold by adding 48 μl of assay buffer to 2 μl of the compound in the assay preparation plate to prepare a working stock of the compound.

[0147] Enzyme preparation: 1. Reconstitute the supplied granzyme B (human lymphocyte) enzyme to 1 mg / ml (approximately 31.25 μM) and keep 1 μl aliquots at -80°C. Dilute a 2.1 μl aliquot to 625 nM by adding 49 μl of assay buffer containing 0.1% BSA and gently mixing. A 3.10 nM enzyme-activated stock is prepared by adding an assay buffer containing 0.1% BSA.

[0148] Substrate preparation: 1. Reconstitute the supplied substrate (Ac-IETD-AFC) by adding DMSO to prepare a 10 mM stock solution, aliquot it, and store it at -80°C. A 2.4 mM substrate midstock is prepared by adding DMSO. 3. Prepare the substrate-activated stock, i.e., 800 μM, by adding assay buffer.

[0149] Assay protocol: Add 1.10 μl of serially diluted compound activating stock (starting dose 40 μM) from the assay preparation plate to the assay plate according to the plate map. 2. Add a positive control (40 μM standard inhibitor) and a negative control (4% DMSO buffer) to each well. Add 3.20 μl of the enzyme-activated stock to the assay plate and mix gently. 4. Incubate the plate at 22°C for 30 minutes, then rotate it at 130g for 1 minute. 5. After incubation, add 10 μl of substrate activating stock to each well and mix (keep the assay plate in the dark after adding the substrate). 6. Incubate the plate at 22 °C for 60 minutes and rotate it at 130 g for 1 minute. 7. Measure the fluorescence reading (RFU) (excitation: 400 nm / emission: 505 nm) after 60 minutes with an En Vision Multimode plate reader.

Table 7

[0150] Data analysis: 1. Analyze the RFU readings to calculate the percent inhibition by normalizing the readings in Microsoft Excel with the positive control and negative control as 100% and 0% effects, respectively. 2. Input the analyzed data into GraphPad prism5.0 software and generate a graph by obtaining the IC 50 value of each compound.

[0151] The structures and activities of exemplary compounds are shown in Table 6 below. These compounds showed high efficacy in inhibiting granzyme B as indicated by their IC 50 values included in the following table.

Table 8

[0152] <a Example 3: Synthesis of Exemplary Granzyme B Target Compounds The above synthetic method was used in the synthesis of the following compounds with the following general peptide synthesis procedures, and the exact mass of each was recorded. General peptide synthesis procedure: Using H-Asp(OtBu)-H resin, peptides were synthesized according to a standard Fmoc solid-phase peptide synthesis procedure. The final peptide was deprotected and cleaved from the resin according to a two-step procedure: 1) treated with either trifluoroacetic acid (TFA) for 2 hours at room temperature or TFA / dichloromethane (DCM) overnight at room temperature, then concentrated, and 2) treated with 0.1% TFA in acetonitrile / water (60:40) at 60°C for 1 hour. The crude peptide was concentrated or lyophilized and then subjected to preparative HPLC purification (0.1% formic acid or 0.1% TFA in water / acetonitrile mobile phase). The product-containing fraction was collected and lyophilized to obtain the peptide as a white, fluffy solid. [ka] ES / MS m / z 1091.5 (M+H) + . [ka] ES / MS m / z 1105.5(M+H) + . [ka] ES / MS m / z 1234.7 (M+H) + . [ka] ES / MS m / z 948.4(M+H) + . [ka] ES / MS m / z 891.5 (M+H) + . [ka] ES / MS m / z 1019.5(M+H) + . [ka] ES / MS m / z 1020.5(M+H) + . [ka] ES / MS m / z 963.5 (M+H) + . [ka] ES / MS m / z 963.5 (M+H) + . [ka] ES / MS m / z 977.5 (M+H) + [ka] ES / MS m / z 963.5 (M+H) + [ka] ES / MS m / z 1019.6(M+H) + [ka] ES / MS m / z 1047.6 (M+H) +

[0153] The following compounds were formed using the following general procedure: To a reaction vial containing the peptide precursor and a stirring rod, equivalent amounts of 20 mM AlCl3 in 0.1 M NaOAc (pH approximately 4.5) and 100 mM NaF in H2O (1.5–3.0 equivalents relative to the peptide) were added. Acetonitrile (0–34% of the total reaction volume) was then added. The mixture was heated at 100°C for 15–30 minutes. Acetonitrile was removed under reduced pressure, and the aqueous solution was purified by either a C18 ISCO column or a C18 HPLC preparatory column (using water and 0.1% formic acid in acetonitrile as eluent). Appropriate fractions were collected and lyophilized to obtain the peptide AlF complex as a white, fluffy solid. [ka]

[0154] Following a standard procedure, 1 (60 mg) was converted to 1-Al(1:1)(22 mg) as a white, fluffy solid. ES / MS m / z 1135.4(M+H) + [ka]

[0155] Following a standard procedure, 2 (16 mg) was converted to 2-Al(1:1)(8.6 mg) as a white, fluffy solid. ES / MS m / z 1149.5(M+H) + [ka]

[0156] Following a standard procedure, 18 (15 mg) was converted to 18-Al(1:1) (7.2 mg) as a white, fluffy solid. ES / MS m / z 1278.5 (M+H) + [ka]

[0157] Following a standard procedure, 4 (30 mg) was converted to 4-Al(1:1)(22 mg) as a white, fluffy solid. ES / MS m / z 935.4(M+H) + [ka]

[0158] Following a standard procedure, 11 (30 mg) was converted to 11-Al(1:1) (15 mg) as a white, fluffy solid. ES / MS m / z 1007.4(M+H) + [ka]

[0159] Following a standard procedure, 9 (30 mg) was converted to 9-Al(1:1)(24 mg) as a white, fluffy solid. ES / MS m / z 1007.4(M+H) + [ka]

[0160] Following a standard procedure, 10 (30 mg) was converted to 10-Al(1:1)(24 mg) as a white, fluffy solid. ES / MS m / z 1007.5(M+H) + [ka]

[0161] Following a standard procedure, 17 (30 mg) was converted to 17-Al(1:1)(23 mg) as a white, fluffy solid. ES / MS m / z 1021.5(M+H) + [ka]

[0162] Following a standard procedure, 5 (34 mg) was converted to 5-Al(1:1)(25 mg) as an off-white, fluffy solid. ES / MS m / z 1063.5(M+H) + [ka]

[0163] Following a standard procedure, 6 (30 mg) was converted to 6-Al(1:1)(25 mg) as a white, fluffy solid. ES / MS m / z 1064.5(M+H) + [ka]

[0164] Following a standard procedure, 12 (30 mg) was converted to 12-Al(1:1)(16 mg) as an off-white, fluffy solid. ES / MS m / z 1063.6(M+H) + [ka]

[0165] Following a standard procedure, 13 (31 mg) was converted to 13-Al(1:1)(23 mg) as an off-white, fluffy solid. ES / MS m / z 1091.5(M+H) + [ka]

[0166] Following a standard procedure, 3 (19 mg) was converted to 13-Al(1:1)(9 mg) as an off-white, fluffy solid. ES / MS m / z 992.5(M+H) +

[0167] Example 4: Radiation Synthesis Procedure General radiosynthesis of target compounds Typical 18 The F-GZB compound RCY is synthesized using a starting activity of 0.5–2.0 Ci in a synthesis time of 75 ± 10 minutes, with a concentration ranging from 5.6–63%. The precursor (e.g., 0.2–0.6 mg) is added to the reaction vial in an aqueous acetic acid / sodium acetate buffer (e.g., 200–400 μL, 1 mol / L, pH 3.0–5.0), AlCl3·6H2O (e.g., 34–82 μg, 100–240 nmol), and acetonitrile (e.g., 25–50% of the total volume of the reaction mixture). 18The fluoride activity is retained in treated [5 mL of 0.9% physiological saline, followed by 5 mL of WFI (water for injection)] anion exchange resin (e.g., Sep-Pak Accell Plus QMA Carbonate Plus Light cartridge, 46 mg of adsorbent per cartridge, 40 μm particle size, Waters part number 186004540). The retained [ 18 Elute the [F] fluoride from the cartridge into the reaction vial with 0.9% physiological saline (e.g., 0.5-0.8 mL). Heat the resulting mixture for a set time (e.g., 15 minutes) (e.g., 105°C), then cool it (e.g., 60°C) before diluting with water (e.g., 1.0-5.0 mL, HPLC grade). Pack the resulting crude product into a semi-preparative reverse-phase HPLC column (e.g., Agilent ZORBAX Eclipse XDB-C18, 5 μm, 9.4 mm × 250 mm, part number 990967-202) for purification (e.g., mobile phase containing aqueous acetonitrile solution (8-20%), pH 1-8). 18 The HPLC fraction containing the F-GZB compound is diluted with a 0.5% w / v sodium ascorbate aqueous solution (e.g., 30-50 mL) and then passed through a treated [5 mL ethanol (USP grade), followed by 5 mL of water (HPLC grade)] reverse-phase cartridge (e.g., Sep-Pak® Lite C18 cartridge, 130 mg adsorbent per cartridge, 55-105 μm particle size, Waters part number WAT0523501). 18 F-GZB is washed with a 0.5% w / v sodium ascorbate aqueous solution (e.g., 5-15 mL), and eluted from the cartridge using ethanol (e.g., 1.0-1.5 mL) from a formulation vial containing 0.5% w / v sodium ascorbate in 0.9% physiological saline (e.g., 6.0-10.0 mL). The C18 cartridge is then rinsed with an additional 0.5% w / v sodium ascorbate in 0.9% physiological saline (e.g., 3.0-3.5 mL), and the rinse solution is collected in the formulation vial. The strength of the product can be adjusted by adding a certain amount of diluent (90% v / v 0.9% physiological saline containing 10% v / v ethanol and 0.5% w / v sodium ascorbate).

[0168] To prepare the sterile product, the obtained product (containing 10% v / v ethanol and 0.5% w / v sodium ascorbate in 90% v / v 0.9% physiological saline) 18 F-GZB) is filtered into a bulk product vial through a 0.22 μm filter (e.g., Millex® GV sterile filter, Millipore part number SLGV033RS, Millex GV 25 mm sterile filter, Millipore part number SLGVV255F, and Millex LG 25 mm sterile filter, Millipore part number SLLG025SS).

[0169] Example 5: Competitive Binding Assay the purpose:[ 18 A competitive binding assay with human granzyme B was performed using [F]-20-Al to determine the IC50 of potential granzyme B ligands. 50 Determine the value material: 11. Granzyme B (Human Lymphocyte) Enzyme: Enzo Lifesciences, Catalog Number ALX-200-602-C010 12. Millipore MultiScreen HTS FB plate Millipore, catalog number MSFBN6B 13. Millipore MultiScreen HTS Millipore vacuum manifold, catalog number MSVMHTS00 14. Millipore MultiScreen (registered trademark) punch tip, Millipore, catalog number MADP19650 15. MultiScreen® Multi-Punch System, Millipore, Catalog Number MAMP09608 16.96-well 0.5 mL assay plate, Thermo Fisher, catalog number 12-565-502 17. Reaction buffer (10 mM HEPES, 150 mM NaCl, 1 mM EDTA, 0.01% BSA, pH 7.4)

[0170] method: Initial plate preparation (including fully bound and non-specific bound): 1. As described above, add 78 μL of reaction buffer to each well in the 96-well plate. 2. For wells used to examine total binding (defined as the binding of the radioactive ligand in the absence of competition), add 2 μL of DMSO to obtain a final DMSO concentration of 1% for all wells. 3. For wells used to investigate nonspecific binding (defined as the binding of a radioactive ligand in the presence of 10 μM 20-Al), 2 μL of 1 mM 20-Al is added to obtain a final concentration of 10 μM.

[0171] Enzyme preparation: 1. Dilute Granzyme B (GZB, human lymphocytes) to 0.5 μg / mL using the reaction buffer as described above. 2,100 μL of diluted human GzB at a concentration of 0.5 μg / mL was added to each well, yielding 50 ng per well, with a final concentration of 0.25 μg / mL.

[0172] Preparation of compounds: 1. Add DMSO or water to each compound vial to prepare a 1 mM compound stock solution. A 2.1 mM compound stock solution is sequentially diluted 1 / 2 logarithmically (3.16 times) by adding 18.5 μL of compound stock to 40 μL of DMSO, mixing up and down with a pipette, and then transferring 18.5 μL of the mixture to 40 μL of DMSO. This process is repeated to create 10 dilution points of the test compound. 3. For wells used to investigate ligand binding competition, 2 μL of each diluent is distributed to a 96-well plate prepared as described above to obtain a further 1 / 100 diluent.

[0173] Preparation of radioactive ligands: 1.200nM [ 18Prepare [F]-20-Al (10x radioactive ligand stock) in reaction buffer, aiming for approximately 2 million CPM per 20 μL of solution (input). Distribute 2.20 μL of radioactive ligand stock into each well to obtain a final concentration of 20 nM per well.

[0174] Incubation conditions, post-incubation sample processing, and data analysis: 1. All 96 wells have a final volume of 200 μL, and the assay plate is incubated at 37°C for 90 minutes. 2. All samples from the assay plate were pre-soaked in PBS buffer (pH 7.4) and filtered using a vacuum manifold. HTS Transfer to an FB plate. Add an additional 150 μL of PBS buffer to each well of the assay plate and mix to ensure the transfer of the remaining sample. 3. MultiScreen HTS The FB plate was washed three times with 150 μL of PBS buffer. 4. All filters are separated and transferred to individual tubes using MultiScreen punch tips and Millipore multi-punch equipment. 5. Place the sample set, 18 The analysis is performed using a Wizard2480 automated gamma counter [Perkin Elmer] via a specific F-specific profile. The values ​​are reported as decay-corrected counts per minute (CPM). 6. The obtained CPM values ​​are normalized and converted to inhibition percentages using the following formula:

number

[0175] The obtained inhibition percentage was then analyzed using GraphPad Prism 8.4.3 software, performing a 1-site-Fit logIC test. 50 The IC of each ligand is plotted using the formula, and50 The value was determined. Y = A + (BA) / (1 + 10) (x-LogIC50) ) Y = Inhibition % X = Logarithmic concentration (M) of the cold-competitive ligand A = Minimum Y (0%) B=Max Y(100%) LogIC 50 = Logarithmic concentration of cold-competitive ligand (M) midway between minimum and maximum Y

[0176] result: Table 7 below shows the IC252 50 List the values. [Table 9]

[0177] Example 6: Synthesis of 20-Al The following synthesis procedure is used for the preparation of compound 20 and 20-Al.

[0178] tert-butyl 2-[4-(2-tert-butoxy-2-oxo-ethyl)-1,4,7-triazonan-1-yl]acetic acid (20-a): 1,4,7-Triazonanane (21.61 g, 167.3 mmol, 1.0 equivalent) was dissolved in chloroform (360 ml), and the solution was cooled to -10°C in an ethanol / ice bath. Then, tert-butyl 2-bromoacetic acid (54.33 ml, 368.0 mmol, 2.2 equivalents) dissolved in chloroform (360 mL) was added dropwise over 4 hours. The mixture was then slowly warmed to ambient temperature. After stirring at ambient temperature overnight, the mixture was poured onto filter paper, and the filtrate was reduced under vacuum to obtain brown oil. The oil was purified on silica gel using 3%-12% 7N ammonia in methanol / methylene chloride as the solvent for 30 minutes, and the product was obtained as brown oil (16.53 g, 46.24 mmol, 28%). HRMS:C 18 H 35 N3O4(M+H) + In this case, the calculated value is 357.2628, and the measured value is 357.2619.

[0179] Methyl 2-[4-[[4,7-bis(2-tert-butoxy-2-oxo-ethyl)-1,4,7-triazonan-1-yl]methyl]phenyl]acetate(20-b): 16.53 g, 46.24 mmol, 1.0 equivalent of tert-butyl 2-[4-(2-tert-butoxy-2-oxo-ethyl)-1,4,7-triazonan-1-yl]acetate and 12.36 g, 50.86 mmol, 1.1 equivalents of methyl 2-[4-(bromomethyl)phenyl]acetate were combined in 300 mL of acetonitrile, and 24.2 ml, 138.7 mmol, 3.0 equivalents of N,N-diisopropylethylamine were added. The mixture was heated overnight at 50°C. LC-MS indicated that the reaction was complete, and the mixture was cooled to ambient temperature and stirred overnight. The solvent was then removed under vacuum to obtain a brown oil. The oil was partitioned into 150 ml of dichloromethane and 75 ml of water. The aqueous solution was removed, the organic matter was washed with water (75 mL), washed with brine (75 mL), dried on sodium sulfate, filtered, and concentrated under vacuum to obtain yellowish-brown foam. The foam was purified by SFC chromatography (Method: Chiralcel OD-H-5X15 cm, 20% methanol (0.5% N,N-dimethylethylamine) / carbon dioxide, 5 ml / min, 225 nm), the fraction containing the purified product was pooled, and concentrated under vacuum to obtain amber-colored oil (15.04 g, 28.94 mmol, 63%). HRMS:C 28 H 45 N3O6(M+H) + In this case, the calculated value is 519.3308, and the measured value is 519.3324.

[0180] 2-[4-[[4,7-bis(2-tert-butoxy-2-oxo-ethyl)-1,4,7-triazonan-1-yl]methyl]phenyl]acetic acid (20-c): Methyl 2-[4-[[4,7-bis(2-tert-butoxy-2-oxo-ethyl)-1,4,7-triazonan-1-yl]methyl]phenyl]acetate (15.04 g, 28.94 mmol, 1.0 equivalent) was dissolved in tetrahydrofuran (120 ml), and an aqueous lithium hydroxide solution (28.94 ml, 57.88 mmol, 2.0 equivalent, 2 M) was added. The mixture was stirred at ambient temperature for 22.5 hours. The solvent was then removed under vacuum to obtain foam. The foam was dissolved in water (40 mL), and the pH was carefully adjusted to approximately 7 using 2 M HCl. The aqueous solution was extracted six times with dichloromethane (100 ml), and the organic compounds were combined and reduced to the residue. The residue was taken up in a 3:1 chloroform / isopropanol (350 mL), and the turbid solution was poured onto filter paper. The filtrate was reduced under vacuum and left overnight in a vacuum oven at ambient temperature to obtain a yellow solid (10.77 g, 21.30 mmol, 74%). HRMS:C 27 H 43 N3O6(M+H) + In this case, the calculated value is 505.3152, and the measured value is 505.3167.

[0181] (9H-Fluoren-9-yl)methyl((3S,6S)-6-((((1H-1,2,3-triazole-4-yl)methyl)carbamoyl)-4-oxo-1,2,3,4,6,7-hexahydroazepino[3,2,1-hi]indole-3-yl)carbamate(20-d): (3S,6S)-3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-oxo-1,2,3,4,6,7-hexahydroazepino[3,2,1-hi]indole-6-carboxylic acid (8.0 g, 17.08 mmol, 1.0 equivalent), 1H-triazole-4-ylmethaneamine:hydrochloride (2.53 g, 18.78 mmol, 1.1 equivalent), and HATU (7.79 g, 20.49 mmol, 1.2 equivalents) were combined in N,N-dimethylformamide (80 mL), and then N,N-diisopropylethylamine (10.4 ml, 59.77 mmol, 3.5 equivalents) was added. The mixture was stirred overnight at ambient temperature, and then partitioned into ethyl acetate (200 ml) and brine (150 ml). The aqueous solution was removed, the organic matter was washed twice with brine (150 mL), dried over sodium sulfate, filtered, and concentrated under vacuum to obtain a brown oil. The oil was purified on silica gel using a 3%–5% methanol / methylene chloride gradient for 5.5 minutes, followed by holding in 5% methanol / methylene chloride for 3 minutes to obtain the product as a yellowish-brown solid (7.61 g, 13.90 mmol, 81%). HRMS:C 31 H 28 N6O4(M+H) + In this case, the calculated value is 548.2172, and the measured value is 548.2157.

[0182] (3S,6S)-N-(((1H-1,2,3-triazol-4-yl)methyl)-3-amino-4-oxo-1,2,3,4,6,7-hexahydroazepino[3,2,1-hi]indole-6-carboxamide(20-e): (9H-fluoren-9-yl)methyl((3S,6S)-6-(((1H-1,2,3-triazole-4-yl)methyl)carbamoyl)-4-oxo-1,2,3,4,6,7-hexahydroazepino[3,2,1-hi]indole-3-yl)carbamate (7.61 g, 13.9 mmol, 1.0 equivalent) was dissolved in tetrahydrofuran (120 mL), and dimethylamine (2 mol / L) in tetrahydrofuran (35.0 mL, 70.0 mmol, 5.05 equivalents) was added. The mixture was stirred overnight at ambient temperature. The following morning, the solvent was removed under vacuum, and the residue was sonicated in acetonitrile. The resulting suspension was stirred at ambient temperature for 2 hours, and the solid was collected by vacuum filtration. The cake was washed with acetonitrile, dried for 2 hours, and then left in a vacuum oven at ambient temperature for 64 hours to obtain an off-white solid product (3.69 g, 11.3 mmol, 82%). HRMS:C 16 H 18 N6O2(M+H) + In this case, the calculated value is 326.1491, and the measured value is 326.1482.

[0183] tert-butyl((2S,3S)-1-(((3S,6S)-6-((((1H-1,2,3-triazole-4-yl)methyl)carbamoyl)-4-oxo-1,2,3,4,6,7-hexahydroazepino[3,2,1-hi]indole-3-yl)amino)-3-methyl-1-oxopentan-2-yl)carbamate(20-f): Next, (3S,6S)-N-((1H-1,2,3-triazole-4-yl)methyl)-3-amino-4-oxo-1,2,3,4,6,7-hexahydroazepino[3,2,1-hi]indole-6-carboxamide (3.69 g, 11.3 mmol, 1.0 equivalent), (2S,3S)-2-(tert-butoxycarbonylamino)-3-methylpentanoic acid (3.14 g, 13.6 mmol, 1.2 equivalents), and HATU (5.16 g, 13.6 mmol, 1.2 equivalents) were added to N,N-dimethylformamide (50 mL), and then N,N-diisopropylethylamine (5.92 ml, 33.9 mmol, 3.0 equivalents) was added. The mixture was stirred overnight at ambient temperature. Next, the mixture was partitioned into ethyl acetate (200 ml) and brine (100 ml) and the aqueous solution was removed. The organic compound was then washed twice with brine (100 mL) and reduced under vacuum to obtain a yellowish-brown gel. The gel was dissolved in chloroform and exfoliated on silica gel (40 g) for purification. Using 5% methanol / dichloromethane as the solvent, the material was purified on a 330 g silica gel column to obtain 6.0 g of yellow foam. NMR showed that the material still contained residual DMF. The material was dissolved in xylene (100 ml) and the solvent was removed under vacuum. The residue was azeotropically analyzed twice more in xylene (100 mL) to obtain an off-white solid (5.55 g, 10.3 mmol, 91%). HRMS:C 27 H 37 N7O5(M+H) + In this case, the calculated value is 539.2856, and the measured value is 539.2851.

[0184] (3S,6S)-N-(((1H-1,2,3-triazole-4-yl)methyl)-3-((2S,3S)-2-amino-3-methylpentanamide)-4-oxo-1,2,3,4,6,7-hexahydroazepino[3,2,1-hi]indole-6-carboxamide (20-g): tert-butyl((2S,3S)-1-(((3S,6S)-6-(((1H-1,2,3-triazole-4-yl)methyl)carbamoyl)-4-oxo-1,2,3,4,6,7-hexahydroazepino[3,2,1-hi]indole-3-yl)amino)-3-methyl-1-oxopentan-2-yl)carbamate (5.55 g, 10.3 mmol, 1.0 equivalent) was dissolved in methylene chloride (50 mL), and then trifluoroacetic acid (15.6 ml, 206 mmol, 20.1 equivalents) was added. LC-MS after 2.5 hours at ambient temperature indicated that the reaction was complete. The solvent was removed under vacuum. The residue was partitioned between 3:1 chloroform / isopropanol (180 ml) and saturated sodium bicarbonate (100 ml). Organic matter was removed, and the aqueous solution was extracted twice with 3:1 chloroform / isopropanol (100 ml). The organic compounds were then mixed, washed with brine, dried over sodium sulfate, filtered, and concentrated under vacuum to a yellowish-brown solid (3.06 g, 6.96 mmol, 68%). HRMS:C 22 H 29 N7O3(M+H) + In this case, the calculated value is 439.2332, and the measured value is 439.2328.

[0185] tert-butyl(2-((2-(((2S,3S)-1-(((3S,6S)-6-((((1H-1,2,3-triazole-4-yl)methyl)carbamoyl)-4-oxo-1,2,3,4,6,7-hexahydroazepino[3,2,1-hi]indole-3-yl)amino)-3-methyl-1-oxopentan-2-yl)amino)-2-oxoethyl)amino)-2-oxoethyl)carbamate(20-h): (3S,6S)-N-((1H-1,2,3-triazole-4-yl)methyl)-3-((2S,3S)-2-amino-3-methylpentanamide)-4-oxo-1,2,3,4,6,7-hexahydroazepino[3,2,1-hi]indole-6-carboxamide (3.06 g, 6.96 mmol, 1.0 equivalent), 2-[2-(tert-butoxycarbonylamino)acetyl]amino]acetic acid (1.78 g, 7.66 mmol, 1.1 equivalent), and HATU (2.91 g, 7.65 mmol, 1.1 equivalent) were combined in N,N-dimethylformamide (50 mL), and then N,N-diisopropylethylamine (3.64 ml, 20.9 mmol, 3.0 equivalent) was added. The mixture was stirred overnight at ambient temperature, then partitioned into ethyl acetate (200 ml) and brine (100 ml). The aqueous solution was removed, and the organic matter was washed twice with brine (100 ml). The combined aqueous washing solutions were back-extracted three times with 3:1 chloroform / isopropanol (100 mL). All organic layers were mixed and reduced under vacuum to obtain an off-white gel. The gel was taken up with xylene (100 mL), and the solvent was removed under vacuum. This process was repeated two more times to remove persistent DMF. The resulting residue was dissolved in chloroform and exfoliated on silica gel (40 g) for purification. The material was purified over 35 minutes on a 330 g silica gel column using 5%-10% methanol / dichloromethane as the solvent. The fraction containing the product was pooled, concentrated under vacuum, and then left overnight in a vacuum oven at ambient temperature to obtain an off-white solid (3.50 g, 5.35 mmol, 77%). HRMS:C 31 H 43 N9O7(M+H) + In this case, the calculated value is 653.3285, and the measured value is 653.3270.

[0186] (3S,6S)-N-((1H-1,2,3-triazole-4-yl)methyl)-3-((2S,3S)-2-(2-(2-aminoacetamide)acetamide)-3-methylpentanamide)-4-oxo-1,2,3,4,6,7-hexahydroazepino[3,2,1-hi]indole-6-carboxamide trifluoroacetate (20-i): tert-butyl(2-((2-(((2S,3S)-1-(((3S,6S)-6-(((1H-1,2,3-triazole-4-yl)methyl)carbamoyl)-4-oxo-1,2,3,4,6,7-hexahydroazepino[3,2,1-hi]indole-3-yl)amino)-3-methyl-1-oxopentan-2-yl)amino)-2-oxoethyl)-2-oxoethyl)carbamate (3.50 g, 5.35 mmol, 1.0 equivalent) was suspended in methylene chloride (75 mL), and then trifluoroacetic acid (8.10 ml) was added. 107 mmol (20.0 equivalents) was added. LC-MS after 2.5 hours indicated that the reaction was complete. The solvent was removed under vacuum, and the resulting oil was sonicated in toluene. Then, the toluene was removed under vacuum. Azeotrope with toluene was repeated two more times, and then the material was left in a vacuum oven at ambient temperature for 64 hours to obtain a quantitative yield of the product as an off-white solid (3.57 g, 5.35 mmol, 100%). HRMS:C 26 H 35 N9O5(M+H) + In this case, the calculated value is 553.2761, and the measured value is 553.2775.

[0187] Di-tert-butyl 2,2'-(7-(4-(2-((2-((2S,3S)-1-(((3S,6S)-6-(((1H-1,2,3-triazol-4-yl)methyl)carbamoyl)-4-oxo-1,2,3,4,6,7-hexahydroazepino[3,2,1-hi]indole-3-yl)amino)-3-methyl-1-oxopentan-2-yl)amino)-2-oxoethyl)amino)-2-oxoethyl)2-oxoethyl)benzyl)-1,4,7-triazonan-1,4-diyl)diacetate(20-j): (3S,6S)-N-((1H-1,2,3-triazole-4-yl)methyl)-3-((2S,3S)-2-(2-aminoacetamide)acetamide)-3-methylpentanamide)-4-oxo-1,2,3,4,6,7-hexahydroazepino[3,2,1-hi]indole-6-carboxamide trifluoroacetate (3.47g, 5.20 mmol, 1.0 equivalent), and N,N-diisopropyl Ethylamine (3.63 ml, 20.8 mmol, 4.0 equivalents) was added to N,N-dimethylformamide (50 mL), followed by 2-[4-[4,7-bis(2-tert-butoxy-2-oxo-ethyl)-1,4,7-triazonan-1-yl]methyl]phenyl]acetic acid (3.15 g, 6.23 mmol, 1.2 equivalents), and then HATU (2.37 g, 6.23 mmol, 1.2 equivalents). The mixture was stirred overnight at ambient temperature and then diluted with toluene (150 ml). The solvent was removed under vacuum. Dimethylformamide was removed by repeating azeotropic mixing with toluene two more times. The resulting oil was dissolved in dichloromethane (200 ml) and peeled off on silica gel. The material was purified on a 330 g silica gel column using 10% (held for 25 minutes) to 20% (over 5 minutes) of 7N ammonia in methanol / dichloromethane. Appropriate fractions were pooled, concentrated under vacuum, and left in a vacuum oven at ambient temperature for 64 hours to obtain the product as a yellow foam (4.27 g, 4.10 mmol, 79%). HRMS:C 53 H 76 N 12 O 10 (M+H) + In this case, the calculated value is 1040.5807, and the measured value is 1040.5825.

[0188] (2,2'-(7-(4-(2-((2-((2S,3S)-1-(((3S,6S)-6-(((1H-1,2,3-triazol-4-yl)methyl)carbamoyl)-4-oxo-1,2,3,4,6,7-hexahydroazepino[3,2,1-hi]indole-3-yl)amino)-3-methyl-1-oxopentan-2-yl)amino)-2-oxoethyl)amino)-2-oxoethyl)amino)-2-oxoethyl)benzyl)-1,4,7-triazonan-1,4-diyl)diacetic acid (20): Next, di-tert-butyl 2,2'-(7-(4-(2-((2-(((2S,3S)-1-((3S,6S)-6-(((1H-1,2,3-triazole-4-yl)methyl)carbamoyl)-4-oxo-1,2,3,4,6,7-hexahydroazepino[3,2,1-hi]indole-3-yl)amino)-3-methyl-1-oxopentan-2-yl) (Amino)-2-oxoethyl)-2-oxoethyl)amino)-2-oxoethyl)benzyl)-1,4,7-triazonane-1,4-diyl)diacetate (4.05 g, 3.89 mmol, 1.0 equivalent) was suspended in 1,4-dioxane (300 ml), and hydrochloric acid (4 mol / L) in 1,4-dioxane (24.3 ml, 97.2 mmol, 25.0 equivalents) was added. LC-MS after 3 hours at ambient temperature showed approximately 10% of the mono-tert-butyl product. The mixture was then heated to 50°C, and after 5 hours, LC-MS showed approximately 56% of the desired product, approximately 31% of the mono-tert-butyl product, and approximately 13% of the remaining starting material. The mixture was cooled to ambient temperature and stirred overnight to prevent the formation of by-products. The following morning, the mixture was heated again to 50°C and monitored regularly throughout the day. LC-MS after 8 hours showed approximately 90% of the desired product and approximately 10% of the mono-tert-butyl product. Heating at 50°C was continued overnight. The following morning, LC-MS showed that approximately 5.5% of the mono-tert-butyl product remained. More hydrochloric acid (4 mol / L) was added in 1,4-dioxane (9.72 ml, 38.9 mmol, 10.0 equivalents), and LC-MS after 3 hours showed that approximately 5.1% of the mono-tert-butyl product remained. The temperature was then increased to 60°C and heating was continued for 3 hours. The mixture was then removed from the oil bath and cooled in an ice bath. During cooling, diethyl ether (450 mL) was added, and the mixture was stirred for 20 minutes. The solid was then removed by vacuum filtration through a sintered glass funnel. The cake was washed five times with diethyl ether (150 mL) and then left in a vacuum oven at 50°C for 64 hours.The material was purified by reverse-phase flash chromatography (C18, 275g, RediSep Gold) over 8.6 minutes using a gradient of 10.1% acetonitrile / water (w / 0.1% formic acid) to 26.2% acetonitrile / water (w / 0.1% formic acid), followed by retention in 26.2% acetonitrile / water (w / 0.1% formic acid) for 4.8 minutes. Appropriate fractions were pooled, frozen at -78°C, and lyophilized to obtain the product as an off-white, fluffy solid (2.29g, 2.47 mmol, 63%). HRMS:C. 45 H 60 N 12 O 10 (M+H) + In this case, the calculated value is 928.4555, and the measured value is 928.4580. [ka] 20-Al was prepared as follows.

[0189] Preparation of reaction solution: The solutions were prepared in vials or bottles using the reagents listed below. 1.0M AcOH in H2O: 1.8g of AcOH (30 mmol) in 30mL of H2O Solution obtained: S-092-1 1.0 M NaOAc in H2O: 2.46 g of NaOAc in 30 mL of H2O Solution obtained: S-092-2 0.20M AlCl3 in H2O: 1.45 g of AlCl3-6H2O in 30 mL of H2O Solution obtained: S-092-5 0.1M NaOAc in H2O: 20 mL 1.0 M NaOAc solution S-092-2 180 mL H2O Solution obtained: S-092-6 0.1M AcOH in H2O: 20 mL 1.0 M AcOH solution S-092-1 180 mL H2O Solution obtained: S-092-7 20 mM AlCl3 in 0.1 M NaOAc buffer: 40 mL 0.1M AcOH (S-092-7) 160 mL 0.1M NaOAc (S-092-6) The resulting solution had a pH of 5.2. It was prepared by adding 0.1 M NaOAc buffer and 22 mL of 0.2 M AlCl3 (S-092-5). pH=4.47 Solution obtained: S-092-8 0.1M NaF in H2O: 150 mL x 0.1 M = 15 mmol FW 42g / mol 0.63 g of NaF in 150 mL of H2O Solution obtained: S-092-9

[0190] Aluminum fluoride complexation: 2,2'-(7-(4-(2-((2-(((2S,3S)-1-(((3S,6S)-6-(((1H-1,2,3-triazole-4-yl)methyl)carbamoyl)-4-oxo-1,2,3,4,6,7-hexahydroazepino[3,2,1-hi]indole-3-yl)amino)-3-methyl-1-oxopentan-2-yl)amino)-2-oxoethyl)amino)-2-oxoethyl)amino)-2-oxoethyl)benzyl)-1,4,7-triazonan-1,4-diyl)diacetate (compound 20) was pre-weighed into nine clean vials. The reaction was carried out on the scale shown in Table 8 below. [Table 10]

[0191] Typical procedure for aluminum fluoride complexing: To a 40 mL pre-wash vial filled with compound 20 (150 mg, 0.1615 mmol), 20 mM AlCl3 in 0.1 M NaOAc (11.50 mL, 0.23 mmol) and 100 mM NaF in H2O (1.5 equivalents, 0.24 mmol) were added. The suspension was then sonicated to obtain a solution. The mixture was heated at 105°C for 30 minutes. The sample was then directly purified by C-18 reverse-phase chromatography (C18, 150 g gold) using H2O and 0.1% HCOOH in CH3CN. All clean fractions were combined, frozen, and lyophilized in a clean bottle.

[0192] Combination and analysis of the final batch (compound 20-Al, batch S-099): All samples (S-096, S-097, and S-098) were combined to form a single final batch. After lyophilization, compound 20-Al (batch S-099) was obtained as 1.23 g of a white, fluffy solid. The overall yield was calculated to be 87.7%. 19 F NMR (376.45 MHz, DMSO-d6) δ ppm: 169.68. HRMS (m / z): Observed value (M+H) + :973.4287, experimental monoisotopic mass:972.4215, theoretical monoisotopic mass:972.4198(C 45 H 59 AlFN 12 O 10 (Calculated value in this case).

[0193] compound 18 Radiation synthesis of F-20-Al [ka] Precursor (compound 20 or its formate), aluminum chloride (AlCl3), acid [acetic acid (AcOH) or hydrochloric acid (HCl)], sodium acetate (NaOAc), ethanol (EtOH) and [ 18[F] fluoride activity was added to the reaction vial and stored at 105°C for 15 minutes. The crude reaction mixture was then diluted with water [water for injection (WFI) or water for ion chromatography (WIC)] and packed into a semi-preparative HPLC column for purification. The purified compound was then obtained. 18 The HPLC fraction containing F-20-Al was directly collected or reconstituted using a Sep-Pak® Lite C18 cartridge into a 10% (v / v) EtOH formulation in 0.9% physiological saline containing approximately 3 mM phosphate and / or 0.5% (w / v) sodium ascorbate (NaAsc). 18 The typical radiochemical yield (RCY) for synthesizing F-20-Al varied from 22–56% (using 0.3–2.5 Ci initiation activity) over a synthesis time of 55–80 minutes.

[0194] Compound synthesis using the ORA Neptis radiation synthesis apparatus 18 Radiation synthesis of F-20-Al Compound 20 (0.35 mg, 376 nmol) dissolved in 1.4 mL of 71.5% (v / v) EtOH in WFI solution, and an aqueous solution of AlCl3 [43 μg (178 nmol) of AlCl3·6H2O dissolved in 0.5 mL of 16 mM HCl solution] were added to the reaction vial. [The solution was then held in a Sep-Pak Accell Plus QMA Carbonate Plus Light cartridge.] 18 [Fluoride activity (46 mg, 40 μm, Waters part number 186004540, pre-treated with 5 mL of 0.32 M NaOAc solution followed by 5 mL of WFI)] was eluted into a reaction vial containing the precursor and AlCl3 using 0.5 mL of 0.32 M NaOAc solution. The resulting mixture was heated at 105°C for 15 minutes, then cooled to 60°C, diluted, and quenched with 4.8 mL of WFI. The resulting crude reaction mixture was packed into a semi-preparative HPLC column for purification (Figures 5A and 5B). Purified compound 18The HPLC fraction (20 mL) containing F-20-Al was collected in a vial containing 6 mL of phosphate-buffered saline and 117 mg of NaAsc, yielding a solution of 10% (v / v) EtOH in 0.9% saline containing approximately 3 mmol of phosphate and 0.6 mmol of NaAsc (total volume 26 mL). The sample was removed from the product vial for HPLC analysis (Figures 6A and 6B). Compound 18 Typical RCY for F-20-Al radiosynthesis varied from 40–56% (n=11, using 0.5–2.7 Ci initiation activity) with a synthesis time of 60±5 minutes. Semi-preparative HPLC conditions: (Figures 5A and 5B) [Table 11] Analytical HPLC conditions: (Figures 6A and 6B) [Table 12]

[0195] GE TRACERlab FX F-N Compounds 18 Radiation synthesis of F-20-Al [ 18 The fluoride activity of [F] was retained in Sep-Pak Accell Plus QMA Carbonate Plus Light cartridges [(46 mg, 40 μm, Waters part number 186004540)] that had been pre-treated with either 2 mL of 0.9% physiological saline followed by 5 mL of WIC, or 5 mL of 0.32 M NaOAc solution followed by 5 mL of WFI. 18[F] fluoride was eluted from the cartridge into the reaction vial using either 0.8 mL of 0.32 M NaOAc solution or 0.9% physiological saline (pre-treated with Kirex). Then, precursor solution A {0.25 mg (196 nmol) of compound 20-Al (TFA salt) in 100 μL of 1 M AcOH / NaOAc aqueous buffer (pH 4.1) and AlCl3 solution [28.9 μg (120 nmol) of AlCl3·6H2O in 60 μL of 0.1 M AcOH / NaOAc pH 4 buffer and 1 mL of EtOH]} or solution B {150 μL of WFI, 43.4 μg (180 nmol) of AlCl3 solution of AlCl3·6H2O in 90 μL of WFI, 80 μL of 1N HCl, 110 μL of 1M Either NaOAc or 0.38 mg (298 nmol) of compound 20-Al (TFA salt) in 1 mL of EtOH was added to the reaction vial, the resulting mixture was heated at 105°C for 15 minutes, then cooled to 60°C, and diluted with 3.5 mL of WFI or WIC. The resulting crude reaction mixture was packed into a semi-preparative HPLC column for purification. Purified compound 18 The HPLC fraction containing F-20-Al was collected in a vial containing 40 mL of 0.5% (w / v) NaAsc aqueous solution and packed into a Sep-Pak® C18 Plus Light cartridge (130 mg, 55-105 μm, Waters part number WAT023501, pre-treated with 5 mL of EtOH followed by 5 mL of WFI). The retained compounds were... 18 F-20-Al was washed with 5 mL of 0.5% (w / v) NaAsc aqueous solution and eluted into a vial containing 7 mL of 0.5% (w / v) NaAsc in 0.9% physiological saline using 1 mL of EtOH. The C18 cartridge was rinsed with an additional 2 mL of 0.5% (w / v) NaAsc in 0.9% physiological saline to obtain a final solution of 10 mL of 10% (v / v) EtOH and 0.45% (w / v) NaAsc in 0.9% physiological saline. Compound 18 Typical RCY for F-20-Al radiosynthesis varied from 22–44% (n=19, using 0.3–1.4Ci initiation activity) over a synthesis time of 60±5 minutes.

[0196] compound 18Manual radiation synthesis of F-20-Al To an 8 mL reaction vial, 125 μg (100 nmol) of compound 20 (TFA salt) in 50 μL of 1 M AcOH / NaOAc aqueous buffer (pH 4.1), 14.5 μg (60 nmol) of AlCl3·6H2O in 30 μL of WFI, and 580 μL of EtOH or acetonitrile were added. 18 The fluoride activity [F] was retained in a Sep-Pak Accell Plus QMA Carbonate Plus Light cartridge [(46 mg, 40 μm, Waters part number 186004540)] pre-treated with 2 mL of 0.9% physiological saline followed by 5 mL of WIC. 18 The [F] fluoride was rinsed with 4 mL of WIC, and then eluted from the cartridge into a reaction vial using 0.5 mL of 0.18% physiological saline (prepared by mixing 0.4 mL of WIC with 0.1 mL of 0.9% physiological saline). The resulting mixture was heated at 105°C for 15 minutes, and then partially cooled (approximately 2 minutes) before adding 4.8 mL of WIC. The resulting unpurified material was packed into a semi-preparative HPLC column on an ORA Neptis radiosynthesis system for purification. Purified compound 18 The HPLC fraction containing F-20-Al was diluted with approximately 30 mL of WFI and packed into a Sep-Pak® C18 Plus Light cartridge (130 mg, 55-105 μm, Waters part number WAT023501, pre-treated with 5 mL of EtOH followed by 5 mL of WFI). The retained compounds were... 18 F-20-Al was washed with 10 mL of 0.5% (w / v) NaAsc aqueous solution and eluted into a vial containing 10 mL of 0.5% (w / v) NaAsc in 0.9% physiological saline using 1.8 mL of EtOH. The C18 cartridge was rinsed with an additional 3.5 mL of 0.5% (w / v) NaAsc in 0.9% physiological saline to obtain a final solution of 15 mL of 10% (v / v) EtOH and 0.45% (w / v) NaAsc in 0.9% physiological saline. Compound 18 Typical RCY for F-20-Al radiosynthesis varied from 37–44% (n=3, using 0.3–0.6 Ci initiation activity) over a synthesis time of 75±5 minutes.

[0197] Other Embodiments All features disclosed herein can be combined in any combination. Each feature disclosed herein can be replaced by an alternative feature that serves the same, equivalent, or similar purpose. Thus, unless otherwise expressly stated, each disclosed feature is merely a general example of an equivalent or similar feature.

[0198] Furthermore, from the above description, those skilled in the art can easily identify the essential features of this disclosure and make various changes and modifications to it to suit various uses and conditions without departing from its spirit and scope. Accordingly, other embodiments are also within the scope of the claims.

Claims

1. A compound, or a pharmaceutically acceptable salt thereof, wherein the compound is of formula (I), 【Chemistry 1】 During the ceremony, A is a chelate site which is 1,4,7-triazacyclononane-N,N',N'' triacetic acid (NOTA) or 1,4,7-triazacyclononane-4,7-diyldiacetic acid (NODA). X is -CH ２ It is C(O)- or -NHC(S)-, L is a peptide linker having 1 to 6 amino acid residues in total. R1 is H or methyl, R ２ However, C １－６ Alkyl or C ３－６ A cycloalkyl compound, or a pharmaceutically acceptable salt thereof.

2. The compound according to claim 1, or a pharmaceutically acceptable salt thereof, wherein the compound is of formula (Ia). 【Chemistry 2】

3. The compound according to claim 1 or claim 2, wherein L comprehensively comprises 1 to 3 amino acid residues, or a pharmaceutically acceptable salt thereof.

4. A compound according to any one of claims 1 to 3, wherein L has three amino acid residues, or a pharmaceutically acceptable salt thereof.

5. The compound according to claim 1, or a pharmaceutically acceptable salt thereof, wherein the compound is of formula (Ia-A). 【Transformation 3】

6. The compound according to claim 1, or a pharmaceutically acceptable salt thereof, wherein the compound is of formula (Ia-B). 【Chemistry 4】

7. The compound according to claim 1, or a pharmaceutically acceptable salt thereof, wherein the compound is as follows: 【Chemistry 5-1】 【Chemistry 5-2】 【Chemistry 5-3】 【Chemistry 5-4】

8. The compound according to claim 7, or a pharmaceutically acceptable salt thereof, wherein the compound is as follows: 【Transformation 6】

9. A compound, or a pharmaceutically acceptable salt thereof, wherein the compound is of formula (II), 【Transformation 7】 During the ceremony, M is a metal, or a metal linked to a radioactive isotope. A is a chelate moiety that chelates the metal, and the chelate moiety is 1,4,7-triazacyclononane-N,N',N'' triacetic acid (NOTA) or 1,4,7-triazacyclononane-4,7-diyldiacetic acid (NODA), X is -CH ２ It is C(NH)- or -NHC(S)-, L is a peptide linker having 1 to 6 amino acid residues in total. R1 is H or methyl, R ２ However, C １－６ Alkyl or C ３－６ A cycloalkyl compound, or a pharmaceutically acceptable salt thereof.

10. The compound according to claim 9, or a pharmaceutically acceptable salt thereof, wherein the compound is of formula (IIa). 【Transformation 8】

11. The compound according to claim 9 or claim 10, wherein L comprehensively comprises 1 to 3 amino acid residues, or a pharmaceutically acceptable salt thereof.

12. A compound according to any one of claims 9 to 11, wherein L has three amino acid residues, or a pharmaceutically acceptable salt thereof.

13. The compound according to claim 9, or a pharmaceutically acceptable salt thereof, wherein the compound is of formula (IIa-A). 【Chemistry 9】

14. The compound according to claim 9, or a pharmaceutically acceptable salt thereof, wherein the compound is of formula (IIa-B). 【Chemistry 10】

15. The compound according to any one of claims 9 to 14, or a pharmaceutically acceptable salt thereof, wherein the metal is a radioactive isotope of gallium (Ga).

16. The radioactive isotope of Ga is ６８ Ga, the compound according to claim 15, or a pharmaceutically acceptable salt thereof.

17. The compound according to any one of claims 9 to 14, or a pharmaceutically acceptable salt thereof, wherein the metal is aluminum (Al) linked to the radioactive isotope.

18. The aforementioned radioactive isotope, １８ The compound according to claim 17, or a pharmaceutically acceptable salt thereof, which is F.

19. The compound according to claim 9, or a pharmaceutically acceptable salt thereof, wherein the compound is as follows: 【Chemistry 11-1】 【Chemistry 11-2】 【Chemistry 11-3】 【Chemistry 11-4】

20. The compound according to claim 19, or a pharmaceutically acceptable salt thereof, wherein the compound is as follows: 【Chemistry 12】

21. A pharmaceutical composition comprising a compound according to any one of claims 9 to 20, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

22. (i) A compound according to any one of claims 9 to 20, or a pharmaceutically acceptable salt thereof, (ii) A kit comprising one or more additional therapeutic agents.

23. The kit according to claim 22, wherein the one or more additional therapeutic agents are selected from the group consisting of anti-inflammatory agents, steroids, immunotherapy agents, chemotherapeutic agents, and therapeutic antibodies.

24. A pharmaceutical composition for use in combination therapy with one or more additional therapeutic agents, comprising a compound according to any one of claims 9 to 20, or a pharmaceutically acceptable salt thereof, as a therapeutic agent.

25. The pharmaceutical composition according to claim 24, wherein one or more additional therapeutic agents are selected from the group consisting of anti-inflammatory agents, steroids, immunotherapy agents, chemotherapeutic agents, and therapeutic antibodies.

26. The pharmaceutical composition according to claim 24 or claim 25, comprising administering the compound and one or more additional therapeutic agents in an amount effective for treating the immunomodulatory disorder.

27. The pharmaceutical composition according to claim 26, wherein the immunomodulatory disorder is selected from the group consisting of autoimmune disorders, inflammatory disorders, skin disorders, cancer, and cardiovascular disorders.

28. The pharmaceutical composition according to claim 27, wherein the immunomodulatory disorder is cancer.

29. A method for imaging cells or tissues, samples, or granzyme B in cell or tissue samples, Contacting the cells or tissue, the sample, or the cell or tissue sample with a compound according to any one of claims 9 to 20, or a pharmaceutically acceptable salt thereof, This includes imaging the cells or tissue, the sample, and the cell or tissue sample using a suitable imaging technique, thereby imaging granzyme B in the cells or tissue, the sample, and the cell or tissue sample. The compound is 18F or ６８ A method containing the radioactive isotope Ga.

30. Use of a compound according to any one of claims 9 to 20, or a pharmaceutically acceptable salt thereof, in the preparation of a pharmaceutical for the treatment of an immunomodulatory disorder, wherein the compound or pharmaceutically acceptable salt is present in an amount effective for the treatment of the immunomodulatory disorder.

31. The use according to claim 30, wherein the immunomodulatory disorder is selected from the group consisting of autoimmune disorders, inflammatory disorders, skin disorders, cancer, and cardiovascular disorders.

32. The use according to claim 31, wherein the immunomodulatory disorder is cancer.

33. The use according to any one of claims 30 to 32, further comprising one or more additional therapeutic agents to the subject.

34. The use according to claim 33, wherein the one or more additional therapeutic agents are selected from the group consisting of anti-inflammatory agents, steroids, immunotherapy agents, chemotherapeutic agents, and therapeutic antibodies.

35. A method for monitoring the immune response in the treatment of a disease in a subject, the method comprising administering to the subject an effective amount of a compound according to any one of claims 9 to 20, or a pharmaceutically acceptable salt thereof, and imaging the subject using a suitable imaging technique.

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