Granzyme b-directed 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 the compounds, for example, for imaging and / or treating immunomodulatory abnormalities of granzyme B.

Description

[0001] Related Applications

[0002] This application claims the benefit of the filing date of U.S. Provisional Application No. 63 / 036,918 filed June 9, 2020, which is hereby incorporated by reference in its entirety. TECHNICAL FIELD

[0003] The present disclosure relates to compounds useful in imaging technology, and more specifically to compounds useful for imaging granzyme B using medical imaging, including positron emission tomography. BACKGROUND

[0004] Granzyme B is a serine protease most commonly found in granules of natural killer cells and cytotoxic T cells. Granzyme B is released at the immunological synapse formed between a T cell and its target, along with the pore-forming protein perforin. A portion of the released granzyme B then enters the cancer cell, primarily through the perforin pore, where it activates a variety of substrates, leading to caspase cascade activation. As a downstream effector of tumor cytotoxic T cells, granzyme B has been used as an early biomarker of tumor response to immunotherapy.

[0005] There is a need to develop new compounds that act as effective imaging agents for granzyme B, as well as therapies for treating immunomodulatory abnormalities such as cancer. SUMMARY

[0006] The present application provides specific compounds capable of targeting granzyme B, as well as their use as imaging agents or therapies.

[0007] In one aspect, the present disclosure features a compound of Formula (I):

[0008]

[0009] In Formula (I):

[0010] A is a chelating moiety;

[0011] X is -CH2C(NH)-, -CH2C(O)-, -CH2C(S)-, -NHC(NH)-, -NHC(O)-, -NHC(S)-, -OC(NH)-, -OC(O-), and -OC(S)-, optionally wherein X is -CH2C(O)- or -NHC(S)-;

[0012] L is a peptide linker having 1 to 6 amino acid residues, inclusive;

[0013] R 1 is H or C 1-6alkyl (e.g., methyl); and

[0014] R 2 is C 1-6 alkyl or C 3-6 cycloalkyl.

[0015] In some examples, R 1 may be H. In other examples, R 1 may be methyl.

[0016] In some embodiments, the compound or pharmaceutically acceptable salt thereof is of Formula (Ia):

[0017]

[0018] In some embodiments, L has 1-3 (1, 2, or 3) amino acid residues, inclusive. In one example, L comprises 3 amino acid residues.

[0019] In some examples, the compound or pharmaceutically acceptable salt thereof is of Formula (Ia-A):

[0020]

[0021] Alternatively, the compound or pharmaceutically acceptable salt thereof is of Formula (Ia-B):

[0022]

[0023] In any of the compounds of Formula (I) disclosed herein, the chelating moiety A can be 1,4,7-triazacyclononane-N,N',N"-triacetic acid (NOTA). Alternatively, the chelating moiety A can be 1,4,7-triazacyclononane-4,7-diyldiacetic acid (NODA).

[0024] In specific examples, the compound or pharmaceutically acceptable salt thereof is any one of Compounds 1-22 listed in Table 1 below.

[0025] In another aspect, the disclosure features a compound of Formula (II) and pharmaceutically acceptable salts thereof:

[0026]

[0027] In Formula (II):

[0028] M is a metal or a metal linked to a radioisotope;

[0029] A is a chelating moiety that chelates the metal;

[0030] X is selected from -CH2C(NH)-, -CH2C(O)-, -CH2C(S)-, -NHC(NH)-, -NHC(O)-, -NHC(S)-, -OC(NH)-, -OC(O-), and -OC(S)-, optionally wherein X is -CH2C(NH)- or -NHC(S)-;

[0031] L is a peptide linker having 1 to 6 amino acid residues, inclusive;

[0032] R 1 is H or C 1-6 alkyl (e.g., methyl); and

[0033] R 2 is C 1-6 alkyl or C 3-6 cycloalkyl.

[0034] In some examples, R 1 may be H. In other examples, R 1 may be methyl.

[0035] In some embodiments, the compound or pharmaceutically acceptable salt thereof has the formula (IIa):

[0036]

[0037] In some embodiments, L has 1 to 3 amino acid residues, inclusive (1, 2, or 3). In one example, L has 3 amino acid residues.

[0038] In some examples, the compound or pharmaceutically acceptable salt thereof has the formula (IIa-A):

[0039]

[0040] In other examples, the compound or pharmaceutically acceptable salt thereof has the formula (IIa-B):

[0041]

[0042] In any of the compounds of the formula (II) disclosed herein, the chelating moiety can be 1,4,7-triazacyclononane-N,N',N"-triacetic acid (NOTA). In other examples, the chelating moiety can be 1,4,7-triazacyclononane-4,7-diyldiacetic acid (NODA).

[0043] Alternatively or additionally, the metal M in any of the compounds of the formula (II) disclosed herein can be a radioisotope of Ga. In one example, the radioisotope of Ga is 68Ga. In other embodiments, the metal M in the compound of formula (II) disclosed herein may be Al, which is connected to a radioactive isotope. In one example, the radioactive isotope is... 18 F.

[0044] Specific examples of compounds of formula (II) provided herein include any of the compounds listed in Table 2 below.

[0045] In another aspect, this disclosure provides a composition comprising any compound of formula (I) or formula (II) disclosed herein, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

[0046] 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. Within the scope of this disclosure, there is also a combination therapy comprising a compound of formula (I) or formula (II) disclosed herein, or a pharmaceutically acceptable salt thereof, and one or more additional therapeutic agents.

[0047] In another aspect, this disclosure is characterized by a method for treating immune dysregulation, the method comprising administering to a subject in need a compound of formula (II) disclosed herein, and optionally one or more other therapeutic agents.

[0048] Exemplary immune dysregulation disorders include, but are not limited to, autoimmune diseases, inflammatory diseases, skin diseases, cancer, and cardiovascular diseases. In one instance, the immune dysregulation disorder is cancer.

[0049] The exemplary therapeutic agents disclosed herein include, but are not limited to, anti-inflammatory agents, steroids, immunotherapeutic agents, chemotherapy agents, and therapeutic antibodies.

[0050] In another aspect, this disclosure is characterized by a method for imaging granzyme B in cells or tissues, samples, or cell or tissue samples. The method may include contacting the cells or tissues, the sample, or the cell or tissue sample with a compound of formula (II) disclosed herein or a pharmaceutically acceptable salt thereof, and imaging the cells or tissues, the sample, or the cell or tissue sample using a suitable imaging technique, thereby imaging granzyme B in the cells or tissues, the sample, or the cell or tissue sample. In some instances, the compound of formula (II) contains a radioactive isotope. 18 F or 68 Ga.

[0051] Also within the scope of the present disclosure is: (a) a pharmaceutical composition comprising a compound of Formula (I) or Formula (II) disclosed herein and optionally one or more therapeutic agents, for use in imaging granzyme B or for use in treating immune dysregulation; or (b) any of the compounds of Formula (I) or Formula (II) alone or in combination with one or more therapeutic agents, for use in the manufacture of a medicament for imaging granzyme B or for use in treating a target disease disclosed herein.

[0052] The details of one or more embodiments of the application are set forth in the description below. Other features or advantages of the present application will become apparent from the following drawings and detailed description of several embodiments, and also from the appended claims. BRIEF DESCRIPTION OF DRAWINGS

[0053] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure. The application can be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0054] Figure 1 Depiction of compounds radiosynthesized by the ORA Neptis radiosynthesizer 18 Analytical HPLC chromatogram of F-7-A1 peak 2.

[0055] Figure 2 Depiction of compounds 18 Analytical HPLC chromatogram of F-7-A1 peak 2.

[0056] Figure 3 Depiction of compounds radiosynthesized by the ORA Neptis radiosynthesizer 18 Semi-preparative HPLC purification chromatogram of F-7-A1 peak 1 and peak 2.

[0057] Figure 4 Depiction of compounds 18 Analytical HPLC chromatogram of F-7-A1 peak 1 and peak 2.

[0058] Figure 5A and 5B Depiction of compounds 18 Semi-preparative HPLC purification chromatogram of F-20-A1 peak 1 and peak 2. Figure 5A : HPLC Rad. Figure 5B : HPLC UV.

[0059] Figure 6A and 6B Depiction of compounds 18Analytical HPLC chromatogram of F-20-Al peak 1 and peak 2. Figure 6A : HPLC Rad. Figure 6B : HPLC UV. DETAILED DESCRIPTION

[0060] In recent years, cancer immunotherapy has shown remarkable progress in cancer therapy. Antibodies against immune checkpoints such as programmed cell death protein 1 (PD-1) and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) have been approved and have produced positive results for some patients. Research in the field of immuno-oncology continues, with strategies including CAR-T cells, vaccines, small molecules, and antibodies being developed. Despite the promising outlook of these therapies, they are not a panacea. These immunotherapies can be associated with significant adverse events, are costly, and have a response rate of typically 20-50%, meaning that the majority of patients do not respond to the therapy. Furthermore, determining an individual patient’s response to therapy using conventional methods can be challenging, as the response is often associated with immune cell infiltration, which can cause the responding tumor to appear as growth on anatomical imaging such as CT, MRI, and to exhibit increased avidity for FDG-PET imaging due to influx of metabolically active immune cells. Given the constraints of current imaging techniques, clinical studies of cancer immunotherapy often use overall survival rather than progression-free survival as their study endpoint.

[0061] Granzyme B is a downstream marker of cytotoxic T cell activity that can serve as a novel biomarker for assessing the efficacy of cancer immunotherapy. Not only can expression of Granzyme B in a tumor be assessed to determine the presence or absence of CTLs, but Granzyme B can also serve as an effector protein that is released by active T cells, thus addressing the issue of T cell exhaustion that makes assessment of CTL presence difficult to accomplish.

[0062] The present application provides certain specific compounds, such as compounds of Formula (I) and compounds of Formula (II), that exhibit high binding affinity for Granzyme B. Such compounds can serve as Granzyme B imaging agents and can be used to identify patients that are responsive to immunotherapeutic agents. Such compounds can also be used for therapeutic purposes, e.g., in combination with one or more therapeutic agents such as immunotherapeutic agents.

[0063] DEFINITIONS

[0064] It should be understood that the terms used herein are for the purpose of describing particular embodiments and are not intended to be limiting. Further, although any methods, devices, and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, the preferred methods, devices, and materials are now described. Other than in the operating examples, or where otherwise indicated, all terms are to be understood as having their indicated meaning:

[0065] "Amino" refers to the -NH2group.

[0066] "Cyano" refers to the -CN group.

[0067] "Hydroxy" refers to the -OH group.

[0068] "Imino" refers to the =NH substituent.

[0069] "Nitro" refers to the -NO2group.

[0070] "Oxy" refers to the =O substituent.

[0071] "Sulfenyl" refers to the =S substituent.

[0072] "Trifluoromethyl" refers to the -CF3group.

[0073] "Alkyl" refers to a straight chain, saturated, acyclic, monovalent hydrocarbon group or a branched chain, saturated, acyclic, monovalent hydrocarbon group having one to six carbon atoms and attached to the rest of the molecule by a single bond, e.g., methyl, ethyl, n-propyl, 1-methylethyl (iso-propyl), n-butyl, n-pentyl, 1,1-dimethylethyl (tert-butyl), 3-methylpentyl, 2-methylpentyl, and the like. The alkyl moiety can be unsubstituted. Alternatively, the alkyl moiety can be optionally substituted. An optionally substituted alkyl is an alkyl that is optionally substituted with one, two, three, four, or five substituents independently selected from the group consisting of halo, cyano, nitro, aryl, cycloalkyl, heterocyclyl, heteroaryl, oxy, trimethylsilyl, -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(R 3 )S(O) t R 5(where t is 1 or 2), -S(O) t OR 5 (where t is 1 or 2), -S(O) p R 5 (where p is 0, 1, or 2) and -S(O) t N(R 3 )2(where t is 1 or 2), wherein each R 3 is independently hydrogen, alkyl, haloalkyl, cycloalkyl, cycloalkylalkyl, aryl, aralkyl, heterocyclyl, or heteroaryl; each R 4 is independently hydrogen, cycloalkyl, aryl, heterocyclyl, or heteroaryl; and each R 5 is independently alkyl, haloalkyl, cycloalkyl, aryl, heterocyclyl, or heteroaryl.

[0074] "Cycloalkyl" refers to a stable non-aromatic monocyclic or polycyclic hydrocarbon radical having three to fifteen carbon atoms, preferably having three to ten carbon atoms, and which is either saturated or unsaturated, and which is connected to the rest of the molecule by a single bond. Polycyclic hydrocarbon radicals are bicyclic, tricyclic, or tetracyclic systems. Unsaturated cycloalkyl groups contain one, two, or three carbon-carbon double bonds and / or one carbon-carbon triple bond. Monocyclic cycloalkyl groups include, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. Polycyclic cycloalkyl groups include, for example, adamantyl, norbornyl, decalinyl, and the like. The cycloalkyl moiety can be unsubstituted. Alternatively, the cycloalkyl moiety can be optionally substituted. An optionally substituted cycloalkyl is a cycloalkyl that is optionally substituted with one, two, three, four, or five substituents independently selected from the group consisting of alkyl, alkenyl, halo, haloalkyl, haloalkenyl, cyano, nitro, oxo, aryl, aralkyl, cycloalkyl, heterocyclyl, heteroaryl, -R 4 -OR 3 , -R 4 -OC(O)-R 3 , -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 (where t is 1 or 2), -R 4 -S(O) t OR 5 (where t is 1 or 2), -R 4 -S(O) p R 5 (where p is 0, 1, or 2) and -R 4 -S(O) t N(R 3 )2(where t is 1 or 2), wherein each R 3 is independently hydrogen, alkyl, haloalkyl, cycloalkyl, aryl, heterocyclyl, or heteroaryl; each R 4 is independently a direct bond or a straight- or branched-chain alkylene or alkenylene chain; and each R 5 is independently alkyl, haloalkyl, cycloalkyl, cycloalkylalkyl, aryl, aralkyl, heterocyclyl, or heteroaryl.

[0075] In some embodiments, the preparation of a compound can involve the addition of an acid or base to affect, for example, catalysis of a desired reaction or formation of a salt form such as an acid addition salt.

[0076] Exemplary acids can be inorganic or organic acids, and can include, but are not limited to, strong acids and weak acids. Some example 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, butyric acid, benzoic acid, tartaric acid, valeric acid, hexanoic acid, heptanoic acid, octanoic acid, nonanoic acid, and decanoic acid.

[0077] Exemplary bases include lithium hydroxide, sodium hydroxide, potassium hydroxide, lithium carbonate, sodium carbonate, potassium carbonate, and sodium bicarbonate. Some example strong bases include, but are not limited to, hydroxides, alkoxides, metal amides, metal hydrides, metal dialkylamides, and metal arylamines, wherein the alkoxides include lithium, sodium, and potassium salts of methyl, ethyl, and tert-butyloxide; the metal amides include sodium amide, potassium amide, and lithium amide; the metal hydrides include sodium hydride, potassium hydride, and lithium hydride; and the metal dialkylamides include lithium, sodium, and potassium salts of amides substituted with methyl, ethyl, n-propyl, i-propyl, n-butyl, t-butyl, trimethylsilyl, and cyclohexyl.

[0078] As used herein, the phrase "pharmaceutically acceptable salt" refers to a derivative of a disclosed compound in which the parent compound is modified by converting an existing acid or base moiety to its salt form. Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as carboxylic acids; and the like. The pharmaceutically acceptable salts of the present application include the conventional non-toxic salts of the parent compound formed, for example, from non-toxic inorganic or organic acids. As used herein, the pharmaceutically acceptable salts of the present application can be synthesized from the parent compound that contains a basic or acidic moiety by conventional chemical methods. Generally, such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent or in a mixture of both; usually, nonaqueous media like ether, ethyl acetate, alcohols (e.g., methanol, ethanol, isopropanol or butanol) or acetonitrile (MeCN) are preferred. Lists of suitable salts are found in, e.g., Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa., 1985, p. 1418 and Journal of Pharmaceutical Science, 66, 2 (1977). Conventional procedures for preparing salt forms in general are described in, e.g., Handbook of Pharmaceutical Salts: Properties, Selection, and Use, Wiley-VCH, 2002.

[0079] In some embodiments, the compounds provided herein, or salts thereof, are substantially isolated. By "substantially isolated" is meant that the compound is partially or substantially separated from the environment in which it was formed or detected. Partial separation can include, for example, a composition enriched in the compound provided herein. Substantial separation can include compositions 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 compound provided herein, or salts thereof. Methods for isolating compounds and salts thereof are routine in the art.

[0080] As used herein, the expressions "ambient temperature" and "room temperature" or "rt" are as understood in the art and generally refer to the temperature with respect to the room in which the reaction is carried out, e.g., a temperature of about 20 °C to about 30 °C, e.g., the temperature of the reaction.

[0081] I. Compounds targeting granzyme B

[0082] Provided herein are compounds targeting granzyme B disclosed herein, such as a compound of Formula (I) or a compound of Formula (II). The compounds disclosed herein encompass the compounds per se, their pharmaceutically acceptable salts, and their stereoisomers.

[0083] The compounds described herein can include one or more asymmetric centers and can thus occur as various isomers, such as enantiomers and / or diastereomers. For example, the compounds described herein can occur as individual enantiomers, diastereomers, or geometric isomers, or as mixtures of stereoisomers, including racemic mixtures and mixtures enriched in one or more stereoisomers. Isomers can be separated by methods known to those skilled in the art, including chiral high pressure liquid chromatography (HPLC) and formation and crystallization of chiral salts; or preferred isomers can be prepared by asymmetric synthesis. See, e.g., 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, pp. 268-273 (E. L. Eliel, Ed., Univ. of Notre Dame Press, Notre Dame, IN 1972). The present disclosure additionally encompasses the compounds described herein as individual isomers substantially free of other isomers and, alternatively, as mixtures of various isomers.

[0084] A. Compounds of Formula (I)

[0085] In some embodiments, the present disclosure provides compounds of Formula (I) as shown below, which are capable of binding to granzyme B with high binding affinity.

[0086]

[0087] In Formula (I), A is a chelating moiety. Chelating moieties are those molecules or ions that are capable of acting as polydentate ligands for metal ions. For example, molecules with multiple atoms with available lone pairs, including but not limited to nitrogen and oxygen, can act as chelating moieties. Chelating moieties can be linear (e.g., EDTA) or cyclic (including macrocycles, e.g., DOTA, porphyrins), and can involve macrocycles well known in the art. Chelating moieties can have 2, 3, 4, 5, or 6 functional groups with lone pairs that can coordinate to metals (e.g., amines, amides, hydroxyls, carboxylic acids, etc.). Exemplary chelating moieties for use with the cathepsin B targeting compounds disclosed herein include, but are not limited to, 1,4,7-triazacyclononane triacetic acid (NOTA), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), 1,4,7-triazacyclononane-1- glutaric acid-4,7-diacetic acid (NODAGA), ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), cyclohexyl-1,2-diaminetetraacetic acid (CDTA), ethyleneglycol-0,0'-bis(2-aminoethyl)-N,N,N',N'-tetraacetic acid (EGTA), N,N-bis(hydroxybenzyl)-ethylenediamine-N,N'-diacetic acid (HBED), triethylenetetraaminehexaacetic acid (TTHA), hydroxyethyldiaminetriacetic 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-diyl diacetic acid (NODA), and desferrioxamine B (DFO). In some embodiments, the chelator is selected from the group consisting of 1,4,7-triazacyclononane triacetic acid (NOTA), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), 1,4,7-triazacyclononane-4,7-diyl diacetic acid (NODA), and 1,4,7-triazacyclononane-1-glutaric acid-4,7-diacetic acid (NODAGA). In some embodiments, the chelator is 1,4,7-triazacyclononane triacetic acid (NOTA). In other embodiments, the chelator is 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA). In some embodiments, the chelator is 1,4,7-triazacyclononane-4,7-diyl diacetic acid (NODA).

[0088] 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)-.

[0089] L can be a peptide linker having 1 to 6 amino acid residues, inclusive. In some examples, L comprises 1 to 3 amino acid residues, inclusive. In other examples, L comprises 4 to 6 amino acid residues, inclusive. In one example, L comprises 1 amino acid residue. In another example, L comprises 2 amino acids. In yet another example, L comprises 3 amino acid residues. Alternatively, L comprises 4 amino acid residues. In still another example, L comprises 5 amino acid residues. Alternatively, L comprises 6 amino acid residues.

[0090] Compatible amino acid residues in the peptide linker L can include natural and unnatural amino acid residues, including beta-amino acid residues and D-amino acids, and are not limited to protienogenic amino acid residues. The amino acid residues can form a chain through standard peptide bonds or can form a chain through amide bonds with compatible 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-beta-Ala-beta-Ala, Gly-Gly, Gly, Glu-Gly, Glu, D-Glu, Arg-Gly, Lys-Gly.

[0091] In some embodiments, R 1 is H. In other embodiments, R 1 is C 1-6 alkyl. For example, R 1 may be methyl.

[0092] In some embodiments, R 2 may be C 1-6 alkyl. Alternatively, R 2 may be C 3-6 cycloalkyl (e.g., substituted or unsubstituted, branched or unbranched) or C 3-6 cycloalkyl (e.g., cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl).

[0093] The hemiacetal unit in the compound of Formula (I) can be in an open-chain aldehyde form. Thus, the compound of Formula (I) can have the following structure:

[0094]

[0095] In any of the items of formula (I-1) to (I-6), each of A, X, L, R 1 and R 2 is as described herein.

[0096] In some embodiments, R 1 is H and R 2 is C4alkyl, as in the compound of formula (la):

[0097]

[0098] In some specific examples, X is -CH2C(O)-, as in the compound of formula (la-A):

[0099]

[0100] In other specific examples, X is -NHC(S)-, as in the compound of formula (la-B):

[0101]

[0102] Exemplary compounds of formula (I) include those listed in Table 1.

[0103] Table 1: Exemplary compounds of formula (I)

[0104]

[0105]

[0106]

[0107]

[0108] In some examples, the compound of formula (I) is compound 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.

[0109] B. Compounds of formula (II)

[0110] In some embodiments, the present disclosure provides compounds of formula (II) as shown below, which are capable of binding to granzyme B with high binding affinity and specificity.

[0111]

[0112] In Formula (II), M is a metal or a metal linked to a radioisotope. Metals suitable for use in the present disclosure include those metals useful for imaging of cathepsin B, such as metals that are suitable radioimaging agents, as well as metals that can be conjugated to non-metallic radioisotopes that are suitable radioimaging agents. Exemplary metallic radioisotopes are 68 Ga. Exemplary non-metallic radioisotopes are 18 F, which can be conjugated to Al for loading into the cathepsin B binding compounds disclosed herein.

[0113] A, X, L, R 1 and R 2 each as defined herein. See, e.g., the section above entitled “Formula (I) Compounds.”

[0114] Exemplary compounds having a radioisotope include those in Table 2.

[0115] Table 2: Exemplary Formula (II) Compounds

[0116]

[0117]

[0118]

[0119]

[0120] In some examples, the Formula (II) compound 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 Formula (II) compound is Compound 7-Al, which can be loaded with 18 F.

[0121] Also within the scope of the present disclosure are variants of the Formula (I) compounds and Formula (II) compounds disclosed herein, wherein the lactone ring in the Formula (I) compound or Formula (II) compound can be substituted with other moieties, such as aryl rings, heteroaryl rings, and the like. In a particular embodiment, the variant can have the following structure:

[0122]

[0123] The above variant compounds can be loaded with a metal, which can be conjugated to a radioisotope (e.g., 18 F). One example is provided below:

[0124]

[0125] The above compounds containing a radioisotope are useful as imaging agents in one or more of the methods provided herein. Additionally, the radioisotope-containing compounds provided herein are also useful for one or more therapeutic applications when administered to a subject in a therapeutically effective amount. For example, the above compounds containing a radioisotope can be used as imaging agents (e.g., as non-toxic and / or non-therapeutic radioisotopes) when administered to a subject in low concentrations (e.g., 5 mCi). In some embodiments, the isotope can be toxic. As noted above, the present application also includes pharmaceutically acceptable salts of the compounds described herein. The phrase "pharmaceutically acceptable" is employed herein to refer to those compounds, materials, compositions, and / or dosage forms that are, within the scope of sound medical judgment, appropriate for use with human and animal tissues without undue toxicity, irritation, allergic response, or other problem or complication commensurate with a reasonable benefit / risk ratio. 18 The compounds of F can be used as imaging agents (e.g., as non-toxic and / or non-therapeutic radioisotopes). In some embodiments, the isotope can be toxic. As noted above, the present application also includes pharmaceutically acceptable salts of the compounds described herein. The phrase "pharmaceutically acceptable" is employed herein to refer to those compounds, materials, compositions, and / or dosage forms that are, within the scope of sound medical judgment, appropriate for use with human and animal tissues without undue toxicity, irritation, allergic response, or other problem or complication commensurate with a reasonable benefit / risk ratio.

[0126] The present application also includes stereoisomers of the compounds, such as stereoisomers of compounds 7 and 7-A1 as described herein. See below. The stereoisomers are produced by two chiral centers (closed ring) or one chiral center (open chain) of the hemiacetal unit, as indicated by the curved lines.

[0127]

[0128] C. Chemical synthesis of compounds targeting granzyme B

[0129] As will be appreciated, the compounds provided herein, including stereoisomers, and salts thereof, can be prepared using known organic synthesis techniques and can be synthesized according to any of a variety of possible synthetic routes.

[0130] The compounds disclosed herein, or pharmaceutically acceptable salts thereof, can be prepared by the exemplary schemes described below. Suitable protecting groups for use in such syntheses are known in the art. See, e.g., McOmie, Protective Groups in Organic Chemistry, (1973): 98. Briefly, the synthesis of the compounds targeting granzyme B disclosed herein can involve the following processes: A) coupling (e.g., peptide coupling) of a desired tricycle (e.g., in Formula (I)) to a resin, and deprotection of the tricycle; B) attachment of any peptide linker in the L peptide linker (which can be formed by repeated coupling of amino acid residues to a desired length) to the resin-bound tricycle to form a tricycle-peptide linker moiety; and C) coupling of the tricycle-peptide linker moiety to a chelating moiety to form a compound of Formula (I), which can be cleaved from the resin; and thereafter, optionally D) metalation with a compound of Formula (I) to form a compound of Formula (II).

[0131] For example, in Step A, the tricycle (e.g., Fmoc-Haic(2S,5S)-OH) present in the compound of formula (I) and (II) can be coupled to a resin (e.g., H-Asp(OtBu)-H) and deprotected. In Step B, the free amine of the tricycle can then be coupled to a Fmoc-protected amino acid (e.g., isoleucine), and the Fmoc can then be removed. The resulting free amine can be coupled to the desired number of amino acids using conventional peptide synthesis methods to give the tricycle peptide linker moiety. Fmoc protection and deprotection can be carried out in each amino acid residue addition round. In Step C, a chelating moiety having a terminal coupling group (e.g., carboxylic acid, thioamide O-acid, isothiocyanate, or thiocyanate) can be coupled to the final free amine of the amino acid chain in the tricycle-peptide linker moiety to give the compound of formula (I), which can be removed from the resin and purified, if desired. The compound of formula (I) thus prepared can then be chelated with a metal (e.g., Ga, Al) disclosed herein to give the compound of formula (II). Illustrative examples for the synthesis of exemplary compounds of formula (I) and (II) are provided in the following examples. In alternative embodiments of Step C, the compound can be cleaved from the resin prior to coupling to the chelating moiety.

[0132] Many suitable imaging agents (e.g., radioisotopes) are known in the art (see, e.g., U.S. Patents 5,021,236, 4,938,948, and 4,472,509, the disclosure of each of which is incorporated herein by reference in its entirety). The radiolabeled compounds provided herein, or pharmaceutically acceptable salts thereof, 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 one of ordinary skill in the art would readily recognize additional methods suitable for use with the compounds provided herein.

[0133] Those skilled in the art will appreciate that the methods described herein are not the only means by which the compounds provided herein can be synthesized, and that a wide range of synthetic organic reactions can potentially be used to synthesize the compounds provided herein. Those skilled in the art know how to choose and implement appropriate synthetic routes. Suitable synthetic methods for starting materials, intermediates, and products can be identified by reference to the literature, which includes the following reference sources: 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. (Eds.) Science of Synthesis, Vols. 1-48 (2001-2010) and Knowledge Updates KU 2010 / 1-4; 2011 / 1-4; 2012 / 1-2 (Thieme, 2001-2012); Katritzky, et al. (Eds.) Comprehensive Organic Functional Group Transformations, (Pergamon Press, 1996); Katritzky et al. (Eds.) Comprehensive Organic Functional Group Transformations II (Elsevier, 2nded., 2004); Katritzky et al. (Eds.), 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, 6thEd. (Wiley, 2007); Trost et al. (Eds.), Comprehensive Organic Synthesis (Pergamon Press, 1991).

[0134] The reactions for preparing the compounds described herein can be carried out in suitable solvents which can be readily selected by one of ordinary skill in the art of organic synthesis. Suitable solvents can be substantially non-reactive with the starting materials (reactants), the intermediates (if any), or the products under the conditions of the reaction. Reactions can be carried out in one solvent or a mixture of more than one solvent. The choice of a suitable solvent will depend, in part, on the nature of the particular reaction step.

[0135] The preparation of the compounds described herein can involve the protection and deprotection of various chemical groups. The need for protection and deprotection, and the selection of appropriate protecting groups can be readily determined by one skilled in the art. The chemistry of protecting groups can be found, for example, in T. W. Greene and P. G. M. Wuts, Protective Groups in Organic Synthesis, 3rd Ed., John Wiley & Sons, Inc., New York (1999).

[0136] The reactions can be monitored by any suitable means, in accordance with known practices in the art. For example, product formation can be monitored by spectroscopic means, such as nuclear magnetic resonance spectroscopy (e.g.,1H or13C), infrared spectroscopy, spectrophotometry (e.g., UV-vis), mass spectrometry, etc., or by chromatographic means, such as high-performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LCMS), or thin layer chromatography (TLC). 1 H or 13 C), infrared spectroscopy, spectrophotometry (e.g., UV-vis), mass spectrometry, etc., or by chromatographic means, such as high-performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LCMS), or thin layer chromatography (TLC). Compounds can be purified by various methods including high-performance liquid chromatography (HPLC) and normal phase silica gel chromatography by one skilled in the art.

[0137] II. Pharmaceutical Compositions

[0138] Any of the compounds of Formula (I) and Formula (II) or a pharmaceutically acceptable salt thereof can be mixed with a pharmaceutically acceptable carrier to form a pharmaceutical composition for the purposes of granzyme B imaging and / or treatment disclosed herein. In some embodiments, provided herein are pharmaceutical compositions comprising as an active ingredient a compound provided herein having a metal (Formula (II) compound) or a pharmaceutically acceptable salt thereof in combination with one or more pharmaceutically acceptable carriers (excipients). By "acceptable" is meant that the carrier must be compatible with the active ingredient of the composition (and preferably, capable of stabilizing the active ingredient) and not deleterious to the subject to be treated. Suitable carriers include microcrystalline cellulose, mannitol, dextrose, dried skim milk, polyvinylpyrrolidone, and starch or combinations thereof.

[0139] Some examples of suitable excipients include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starches, acacia, calcium phosphate, alginates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, and methyl cellulose. The therapeutic formulations can additionally include, but are not limited to, lubricating agents such as talc, magnesium stearate, and mineral oil; wetting agents; emulsifying and suspending agents; preservatives such as methyl and propylhydroxybenzoates; sweetening agents; flavoring agents; or combinations thereof. See Remington's Pharmaceutical Sciences, 17th Ed., Mack Publishing Company, Easton, Pa., 1985, p. 1418, for further information on acceptable pharmaceutical excipients.

[0140] The pharmaceutical composition can be administered to a subject using a conventional method known to those of ordinary skill in the medical art, depending on the type of disease to be treated or the site of the disease. This composition can also be administered by other conventional routes, for example, orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir. The term "parenterally" as used herein includes subcutaneous, intracutaneous, intravenous, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal, intralesional, and intracranial injection or infusion techniques. Parenteral administration can be in the form of a single bolus dose or can be, for example, by continuous perfusion pump. Additionally, the pharmaceutical composition can be administered to a subject by injectable depot routes, such as using 1-month, 3-month or 6-month depot injectable or biodegradable materials and methods.

[0141] Injectable compositions can contain various carriers, such as vegetable oils, dimethylacetamide, dimethylformamide, ethyl lactate, ethyl carbonate, isopropyl myristate, ethyl alcohol, and polyhydric alcohols (glycerol, propylene glycol, liquid polyethylene glycol, and the like). For intravenous injection, water soluble antibodies can be administered by the drip method, whereby a pharmaceutical formulation containing the antibody and a physiologically acceptable excipient is infused. The physiologically acceptable excipient can include, for example, 5% dextrose, 0.9% saline, Ringer's solution, or other suitable excipient. Intramuscular formulations, for example, sterile formulations of suitable soluble salt forms of the antibody, can be dissolved and administered in pharmaceutical excipients such as water for injection, 0.9% saline, or 5% glucose solution.

[0142] For buccal administration, the compositions can take the form of tablets or lozenges formulated in conventional manner; for example, with the active ingredient mixed with a non- irritating, flavour-improving binder such as sucrose, acacia, corn starch or gelatine, lubricated with magnesium stearate, and if desired, with the addition of suitable flavouring agents such as peppermint oil, vanillin or orange flavouring.

[0143] In some embodiments, the above compounds provided herein, or pharmaceutically acceptable salts thereof, are suitable for parenteral administration. In some embodiments, the above compounds, or pharmaceutically acceptable salts thereof, are suitable for intravenous administration.

[0144] Pharmaceutical compositions and formulations for topical administration can include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids, and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like can be necessary or desirable.

[0145] In making the pharmaceutical compositions provided herein, the active ingredient is typically mixed with an excipient, diluted by an excipient or enclosed within such a carrier in the form of, for example, a capsule, sachet, paper, or other container. When the excipient serves as a diluent, it can be a solid, semi-solid, or liquid material, which acts as a vehicle, a carrier or a medium for the active ingredient.

[0146] Thus, the pharmaceutical compositions can be in the form of tablets, pills, powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols (as solid or in liquid medium), ointments, soft and hard gelatin capsules, suppositories, sterile injectable solutions, and sterile packaged powders.

[0147] III. Methods of Use

[0148] The present application further provides a method of imaging granzyme B using one of the above compounds, or a pharmaceutically acceptable salt thereof. In some embodiments, the method is an in vitro method. In some embodiments, the method is an in vivo method.

[0149] In some embodiments, the method of imaging is performed in a cell, a tissue, a cell sample, a tissue sample, or a subject.

[0150] As used herein, the term“subject” refers to any animal, including mammals and invertebrates. For example, mice, rats, other rodents, rabbits, dogs, cats, pigs, cows, 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 (e.g., a zebrafish).

[0151] The application further provides a method of imaging granzyme B in a cell or tissue, the method comprising:

[0152] i) contacting the cell or tissue with an effective amount of one of the above compounds, or a pharmaceutically acceptable salt thereof; and

[0153] ii) imaging the cell or tissue with a suitable imaging technique, thereby imaging granzyme B in the cell or tissue.

[0154] The application further provides a method of imaging granzyme B in a sample, a cell sample, or a tissue sample, the method comprising:

[0155] i) contacting the sample, the cell sample, or tissue sample with an effective amount of one of the above compounds, or a pharmaceutically acceptable salt thereof; and

[0156] ii) imaging the sample, the cell sample, or tissue sample with a suitable imaging technique, thereby imaging granzyme B in the sample, the cell sample, or tissue sample.

[0157] 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. For example, samples include, but are not limited to, saliva, blood, and urine.

[0158] The application further provides a method of imaging granzyme B in a subject, the method comprising:

[0159] i) administering to the subject an effective amount of one of the above compounds, or a pharmaceutically acceptable salt thereof; and

[0160] ii) imaging the subject with a suitable imaging technique, thereby imaging granzyme B in the subject.

[0161] The application further provides a method of imaging an immune response in a cell or tissue sample, the method comprising:

[0162] i) contacting the cell or tissue sample with an effective amount of one of the above compounds, or a pharmaceutically acceptable salt thereof; and

[0163] ii) imaging the cell or tissue sample with a suitable imaging technique, thereby imaging the immune response in the cell or tissue sample.

[0164] The application further provides a method of imaging an immune response in a subject, the method comprising:

[0165] i) administering to the subject an effective amount of one of the above compounds, or a pharmaceutically acceptable salt thereof; and

[0166] ii) imaging the subject with a suitable imaging technique, thereby imaging the immune response in the subject.

[0167] The application further provides a method of monitoring treatment of a disease in a subject, the method comprising:

[0168] i) administering to the subject an effective amount of one of the above compounds, or a pharmaceutically acceptable salt thereof; and

[0169] ii) imaging the subject with a suitable imaging technique.

[0170] The application further provides a method of monitoring an immune response in treatment of a disease in a subject, the method comprising:

[0171] i) administering to the subject an effective amount of one of the above compounds, or a pharmaceutically acceptable salt thereof; and

[0172] ii) imaging the subject with a suitable imaging technique.

[0173] In some embodiments, the methods provided herein further comprise waiting for a time sufficient to allow the compound, or a pharmaceutically acceptable salt thereof, to accumulate at a cell site or tissue site associated with a disease (e.g., a cell site or tissue site of a subject) before imaging.

[0174] In some embodiments, the methods provided herein further comprise waiting for a time sufficient to allow the compound, or a pharmaceutically acceptable salt thereof, to bind to granzyme B at a cell site or tissue site associated with a disease (e.g., a cell site or tissue site of a subject) before imaging.

[0175] In some embodiments, the sufficient time is from about 30 seconds to about 24 hours, e.g., from about 30 seconds to about 24 hours, from about 30 seconds to about 12 hours, from about 30 seconds to about 6 hours, from about 30 seconds to about 2 hours, from about 30 seconds to about 1 hour, from about 30 seconds to about 30 minutes, from about 30 seconds to about 10 minutes, from about 10 minutes to about 24 hours, from about 10 minutes to about 12 hours, from about 10 minutes to about 6 hours, from about 10 minutes to about 2 hours, from about 10 minutes to about 1 hour, from about 10 minutes to about 30 minutes, from about 30 minutes to about 24 hours, from about 30 minutes to about 12 hours, from about 30 minutes to about 6 hours, from about 30 minutes to about 2 hours, from about 30 minutes to about 1 hour, from about 1 hour to about 24 hours, from about 1 hour to about 12 hours, from about 1 hour to about 6 hours, from about 1 hour to about 2 hours, from about 2 hours to about 24 hours, from about 2 hours to about 12 hours, from about 2 hours to about 6 hours, from about 6 hours to about 24 hours, from about 6 hours to about 12 hours, or from about 12 hours to about 24 hours.

[0176] In some embodiments, the suitable imaging technique is a non-invasive imaging technique. In some embodiments, the suitable imaging technique is a minimally invasive imaging technique. As used herein, the term “minimally invasive imaging technique” includes imaging techniques that employ use of an internal probe or injection of one of the above compounds or a pharmaceutically acceptable salt thereof or a radiotracer through a syringe.

[0177] Exemplary imaging techniques include, but are not limited to, fluoroscopic imaging, x-ray imaging, magnetic resonance imaging (MRI), ultrasound imaging, photoacoustic imaging, thermographic imaging, tomographic imaging, echocardiographic imaging, positron emission tomography (PET) imaging, PET-computed tomography (CT) imaging, PET-MRI, single photon emission computed tomography (SPECT), and ultrasound imaging. In some embodiments, the suitable imaging technique is selected from the group consisting of PET imaging, PET-CT, PET-MRI, and SPECT.

[0178] In some embodiments, the suitable imaging technique is selected from the group consisting of PET imaging, PET-computed tomography imaging, and PET-magnetic resonance imaging (MRI). In some embodiments, the suitable imaging technique is selected PET imaging.

[0179] In some embodiments, the disease described herein is selected from the group consisting of an autoimmune disorder, an inflammatory disorder, a skin disorder, a cancer, and a cardiovascular disorder. As used herein, the term “disease” is used interchangeably with the term “immunoregulatory abnormality.”

[0180] In some embodiments, the disease is cancer. In some embodiments, the cancer comprises a solid tumor. In some embodiments, the cancer is a hematological cancer (e.g., leukemia, lymphoma, etc.). In some embodiments, the 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, the cancer is selected from the group consisting of hairy cell leukemia, Kaposi's sarcoma, follicular lymphoma, chronic myelogenous leukemia, cutaneous T-cell lymphoma, peripheral T-cell lymphoma, T-cell prolymphocytic leukemia, Classical Hodgkin's lymphoma, B-cell non-Hodgkin's lymphoma, chronic lymphocytic leukemia, acute myeloid leukemia, myelodysplastic syndrome, primary myelofibrosis, post- polycythemia vera myelofibrosis, post-true red blood cell hyperplasia myelofibrosis, melanoma, renal cell carcinoma, prostate cancer, non-small cell lung cancer, small cell lung cancer, glioblastoma, hepatocellular carcinoma, urothelial carcinoma, esophageal carcinoma, gastroesophageal carcinoma, gastric carcinoma, 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, triple-negative breast cancer, gastric cancer, pancreatic cancer, bladder cancer, Merkel cell cancer, nasopharyngeal carcinoma, adrenocortical carcinoma, meningioma, neuroblastoma, retinoblastoma, osteosarcoma, rhabdomyosarcoma, Ewing's sarcoma, liposarcoma, fibrosarcoma, leiomyosarcoma, peripheral primitive neuroectodermal tumor, cervical squamous cell carcinoma, vaginal squamous cell carcinoma, and vulvar squamous cell carcinoma. In some embodiments, the cancer is colon cancer.

[0181] In some embodiments, the disease is selected from the group consisting of graft versus host disease, rheumatoid arthritis, systemic lupus erythematosus, Hashimoto's thyroiditis, multiple sclerosis, myasthenia gravis, type I diabetes mellitus, uveitis, posterior uveitis, allergic encephalomyelitis, glomerulonephritis, rheumatic fever, post-infective glomerulonephritis, psoriasis, atopic dermatitis, contact dermatitis, eczematous dermatitis, seborrhoeic dermatitis, lichen planus, pemphigus, bullous pemphigoid, epidermolysis bullosa, urticaria, angioedema, vasculitis, erythema, cutaneous eosinophilia, lupus erythematosus, acne, alopecia areata, catarrhal conjunctivitis, vernal conjunctivitis, uveitis associated with Behcet's disease, keratitis, herpetic keratitis, keratocous, corneal epithelial dystrophy, keratoleukoma, ocular pemphigus, Mooren's ulcer, scleritis, Grave's opthalmopathy, Vogt-Koyanagi-Harada syndrome, sarcoidosis, pollen allergy, reversible obstructive airway disease, bronchial asthma, allergic asthma, intrinsic asthma, extrinsic asthma, dust asthma, chronic or inveterate asthma, late asthma and airway hyperresponsiveness, bronchitis, gastric ulcer, vascular damage due to ischemic disease and thrombosis, ischemic bowel disease, inflammatory bowel disease, necrotizing enterocolitis, intestinal damage associated with thermal burns, coeliac disease, proctitis, eosinophilic gastroenteritis, mastocytosis, Crohn's disease, ulcerative colitis, migraine, rhinitis, eczema, interstitial nephritis, Goodpasture's syndrome, hemolytic-uremic syndrome, diabetic nephropathy, polymyositis, Guillain-Barre syndrome, Meniere's disease, polyneuritis, polyneuritis, mononeuritis, radiculopathy, hyperthyroidism, Basedow's disease, pure red cell anemia, aplastic anemia, hypoplastic anemia, idiopathic thrombocytopenic purpura, autoimmune hemolytic anemia, agranulocytosis, pernicious anemia, erythroblastopenia, osteoporosis, sarcoidosis, fibroid lung, idiopathic interstitial pneumonia, dermatomyositis, vitiligo vulgaris, ichthyosis vulgaris, sensitivity to light, cutaneous T-cell lymphoma, arteriosclerosis, atherosclerosis, Takayasu's arteritis syndrome, polyarteritis nodosa, cardiomyopathy, scleroderma, Wegener's granulomatosis, Sjogren's syndrome, Behcet's disease, primary biliary cirrhosis, chronic active hepatitis, autoimmune hemolytic anemia, autoimmune thrombocytopenia, myasthenia gravis, Graves' disease, Hashimoto's thyroiditis, type I diabetes mellitus, rheumatic heart disease, glomerulonephritis, autoimmune hepatitis, autoimmune uveitis, autoimmune neuritis, autoimmune cardiomyopathy, autoimmune myocarditis, autoimmune myocarditis, autoimmune myocarditis, autoimmune myocarditis, autoimmune myocarditis, autoimmune myocarditis, autoimmune myocarditis, autoimmune myocarditis, autoimmune myocarditis, autoimmune myocarditis, autoimmune myocarditis, autoimmune myocarditis, autoimmune myocarditis, autoimmune myocarditis, autoimmune myocarditis, autoimmune myocarditis, autoimmune myocarditis, autoimmune myocarditis, autoimmune myocarditis, autoimmune myocarditis, autoimmune myocarditis, autoimmune myocarditis, autoimmune myocarditis, autoimmune myocarditis, autoimmune myocarditis, autoimmune myocarditis, autoimmune myocarditis, autoimmune myocarditis, autoimmune myocarditis, autoimmune myocarditis, autoimmune myocarditis, autoimmune myocarditis, autoimmune myocarditis, autoimmune myocarditis, autoimmune myocarditis, autoimmune myocarditis, autoimmune myocarditis, autoimmune myocarditis, autoimmune myocarditis, autoimmune myocarditis, autoimmune myocarditis, autoimmune myocarditis, autoimmune myocarditis, autoimmune myocarditis, autoimmune myocarditis, autoimmunesyndrome), obesity, eosinophilic fasciitis, gum lesions, periodontal membrane, alveolar bone, odontoclastic lesions, glomerulonephritis, male pattern baldness, senile alopecia resulting from prevention of hair loss, senile alopecia resulting from providing hair germination and / or promoting hair production and hair growth, muscular dystrophy, pyoderma, Sezary's syndrome, Addison's disease, organ ischemia-reperfusion injury, graft disease, ischemic disease, endotoxic shock, pseudomembranous colitis, drug- or radiation-induced colitis, ischemic acute renal insufficiency, chronic renal insufficiency, pulmonary oxygen- or drug-induced toxicity, lung cancer, emphysema, cataracts, siderosis, retinitis pigmentosa, age-related macular degeneration, vitreous scarring, corneal alkali burn, erythema multiforme dermatitis, linear IgA bullous dermatosis and cement dermatitis, gingivitis, periodontitis, sepsis, pancreatitis, aging, carcinogenesis, cancer metastasis and hypobaropathy, diseases associated with histamine or leukotriene-C4 release, Behcet's disease, autoimmune hepatitis, primary biliary cirrhosis, sclerosing cholangitis, partial hepatectomy, acute liver necrosis, necrosis induced by toxins, viral hepatitis, shock, anoxia, hepatitis B, non-A / non-B hepatitis, cirrhosis, alcoholic cirrhosis, liver failure, fulminant liver failure, late-onset liver failure, acute and chronic liver failure, cytomegalovirus infection, HCMV infection, AIDS, senile dementia, trauma, chronic bacterial infection, lymphoid malignancy, acute lymphocytic leukemia, chronic lymphocytic leukemia, acute lymphocytic lymphoma, and chronic lymphocytic lymphoma.

[0182] In some embodiments, the disease is selected from the group consisting of systemic lupus erythematosus, chronic rheumatoid arthritis, type I diabetes mellitus, inflammatory bowel disease, biliary cirrhosis, uveititis, multiple sclerosis, Crohn's disease, ulcerative colitis, bullous pemphigoid, sarcoidosis, psoriasis, autoimmune myositis, Wegener's granulomatosis, ichthyosis, Grave's ophthalmopathy, asthma, scleroderma, and Sjogren's syndrome.

[0183] In some embodiments, the disease is selected from the group consisting of bone marrow rejection, organ transplant rejection, and graft versus host disease.

[0184] As used herein, the phrase "therapeutically effective amount" means the amount of an active compound or pharmaceutically acceptable salt or agent thereof that elicits the biological or medicinal response that is being sought in a tissue, system, animal, individual or human by a researcher, veterinarian, medical doctor or other clinician.

[0185] In some embodiments, the dose of one of the above compounds, or a pharmaceutically acceptable salt thereof, administered to a subject or individual is about 1 pg to about 2 g, for example, about 1 pg to about 2 g, about 1 pg to about 1000 mg, about 1 pg to about 500 mg, about 1 pg to about 100 mg, about 1 pg to about 50 mg, about 1 pg to about 1 mg, about 1 pg to about 500 pg, about 1 pg to about 100 pg, about 1 pg to about 10 pg, about 10 pg to about 2 g, for example, about 10 pg to about 2 g, about 10 pg to about 1000 mg, about 10 pg to about 500 mg, about 10 pg to about 100 mg, about 10 pg to about 50 mg, about 10 pg to about 1 mg, about 10 pg to about 500 pg, about 10 pg to about 100 pg, about 100 pg to about 2 g, for example, about 100 pg to about 2 g, about 100 pg to about 1000 mg, about 100 pg to about 500 mg, about 100 pg to about 100 mg, about 100 pg to about 50 mg, about 100 pg to about 1 mg, about 100 pg to about 500 pg, about 500 pg to about 2 g, for example, about 500 pg to about 2 g, about 500 pg to about 1000 mg, about 500 pg to about 500 mg, about 500 pg to about 100 mg, about 500 pg to about 50 mg, about 500 pg to about 1 mg, about 1 mg to about 2 g, about 1 mg to about 1000 mg, about 1 mg to about 500 mg, about 1 mg to about 100 mg, about 1 mg to 50 mg, or about 50 mg to about 500 mg.

[0186] As used herein, the term“treating” or“treatment” refers to one or more of: (1) inhibiting the disease; e.g., arresting the development of a disease, condition, or disorder in an individual who is experiencing or displaying the pathology or symptomatology of the disease, condition, or disorder (i.e., preventing further development of the pathology and / or symptomatology); and (2) relieving the disease; e.g., causing the regression of the pathology or symptomatology of a disease, condition, or disorder in an individual who is experiencing or displaying the pathology or symptomatology of the disease, condition, or disorder (i.e., reversing the pathology and / or symptomatology), such as reducing the severity of the disease or alleviating or relieving one or more symptoms of the disease.

[0187] Combination therapy

[0188] When used in a method of treating a disease, the above compounds of Formula (II) provided herein, or a pharmaceutically acceptable salt thereof, can be administered as a therapeutic agent 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.

[0189] In some embodiments, the additional therapeutic agent comprises inducing an immune response in a cell or tissue sample or in a subject.

[0190] The application further provides a method of treating a disease in a subject, the method comprising:

[0191] a) administering to the subject an effective amount of one of the above compounds, or a pharmaceutically acceptable salt thereof.

[0192] In some embodiments, the subject has been identified and / or diagnosed with the disease to be treated prior to step a). In some embodiments, the subject is identified and / or diagnosed with the disease to be treated after step a). For example, the disease to be treated is selected from the group consisting of the autoimmune disorders, inflammatory disorders, skin disorders, cancers, and cardiovascular disorders described herein.

[0193] 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).

[0194] In some embodiments, the method further comprises:

[0195] b) administering one or more additional therapeutic agents after administering the effective amount of one of the above compounds, or a pharmaceutically acceptable salt thereof. In some embodiments, steps a) - b) are repeated multiple times.

[0196] In some embodiments, the additional therapeutic agent is administered to the subject in a therapeutically effective amount.

[0197] 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 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).

[0198] In some embodiments, the additional therapeutic agent is a steroid. Exemplary steroids include corticosteroids such as cortisone, dexamethasone, hydrocortisone, methylprednisolone, prednisolone, and prednisone. In some embodiments, the additional therapeutic agent is a corticosteroid.

[0199] In some embodiments, the additional therapeutic agent is an anti-inflammatory compound. Exemplary anti-inflammatory compounds include aspirin, choline salicylate, celecoxib, diclofenac potassium, diclofenac sodium, diclofenac sodium with misoprostol, diflunisa, etodolac, fenoprofen, flurbiprofen, ibuprofen, ketoprofen, meclofenamate sodium, mefenamic acid, nabumetone, naproxen, naproxen sodium, oxaprozin, piroxican, rofecoxib, salicylic acid, sodium salicylate, sulindac, tolmetin sodium, and valdecoxib.

[0200] In some embodiments, the additional therapeutic agent is a chemotherapeutic agent. Exemplary chemotherapeutic agents include, but are not limited to, a cytostatic agent, cisplatin, doxorubicin, taxol, etoposide, irinotecan, topotecan, paclitaxel, docetaxel, epothilones, tamoxifen, 5-fluorouracil, methotrexate, temozolomide, cyclophosphamide, SCH 66336, R115777, L778,123, BMS 214662, gefitinib, erlotinib hydrochloride, an antibody to EGFR, imatinib mesylate, an intron, ara-C, gemcitabine, uracil mustard, chlormethine, ifosfamide, melphalan, chlorambucil, pipobroman, triethylenemelamine, triethylenethiophosphoramine, busulfan, carmustine, lomustine, streptozocin, dacarbazine, floxuridine, cytarabine, 6-mercaptopurine, 6-thioguanine, fludarabine phosphate, pentostatine, vinblastine, vincristine, etoposide, teniposide, daunorubicin, doxorubicin, epirubicin, idarubicin, deoxycoformycin, mitomycin C, dactinomycin, mitomycin, cisplatin, cyclosporin, cisplatin, ifosfamide, tamoxifen, clodronate, strontium 89, leuprolide, goserelin, megestrol, calusterone, dromostanolone propionate, testolactone, diethylstilbestrol, tamoxifen, toremifene, fulvestraol, exemestane, letrozole, aromatase inhibitors, medroxyprogesterone acetate, fluoxymesterone, 5-alpha reductase inhibitors, finasteride, dutasteride, bicalutamide, nilutamide, flutamide, megestrol acetate, leuprolide acetate, goserelin, triptorelin, gonadotropin-releasing hormone analogs, medroxyprogesterone acetate, megestrol acetate, leuprolide acetate, goserelin, triptorelin, gonadotropin-releasing hormone analogs, medroxyprogesterone acetate, megestrol acetate, leuprolide acetate, goserelin, triptorelin, gonadotropin-releasing hormone analogs, medroxyprogesterone acetate, megestrol acetate, leuprolide acetate, goserelin, triptorelin, gonadotropin-releasing hormone analogs, medroxyprogesterone acetate, megestrol acetate, leuprolide acetate, goserelin, triptorelin, gonadotropin-releasing hormone analogs, medroxyprogesterone acetate, megestrol acetate, leuprolide acetate, goserelin, triptorelin, gonadotropin-releasing hormone analogs, medroxyprogesterone acetate, megestrol acetate, leuprolide acetate, goserelin, triptorelin, gonadotropin-releasing hormone analogs, medroxyprogesterone acetate, megestrol acetate, leuprolide acetate, goserelin, triptorelin, gonadotropin-releasing hormone analogs, medroxyprogesterone acetate, megestrol acetate, leuprolide acetate, goserelin, triptorelin, gonadotropin-releasing hormone analogs, medroxyprogesterone acetate, megestrol acetate, leuprolide acetate, goserelin, triptorelin, gonadotropin-releasing hormone analogs, medroxyprogesterone acetate, megestrol acetate, leuprolide acetate, goserelin, triptorelin, gonadotropin-releasing hormone analogs, medroxyprogesterone acetate, megestrol acetphosphate), oxaliplatin, folinic acid, pentostatin, vinblastine, vincristine, vindesine, bleomycin, dactinomycin, daunorubicin, doxorubicin, epirubicin, idarubicin, mithramycin, deoxycoformycin, mitomycin-C, L-asparaginase, teniposide, 17α-ethinylestradiol, diethylstilbestrol, testosterone, prednisone, fluoxymesterone, dromostanolone propionate, testolactone, megestrol acetate, calusterone, dromostanolone propionate, testolactone, megestrol acetate, calusterone, dromostanolone propionate, testolactone, megestrol acetate, danazol, tamoxifen, toremifene, fulvestra, anastrozole, leuprolide, goserelin, megestrol acetate, medroxyprogesterone acetate, diethylstilbestrol, leuprolide acetate, flutamide, bicalutamide, nilutamide, estramustine, medroxyprogesterone acetate, megestrol acetate, estramustine, estradiol, estrone, estriol, estradiol cypionate, estradiol valerate, estradiol enanthate, estradiol benzoate, estrone sulfate, estriol glucuronide, estradiol dipropionate, estradiol cypionate, estradiol acetate, estradiol pivalate, estradiol hemisuccinate, estradiol hemiglucuronide, estradiol hemibutyrate, estradiol hemipivalate, estradiol hemisuccinate, estradiol hemiglucuronide, estradiol hemibutyrate, estradiol hemipivalate, estradiol hemisuccinate, estradiol hemiglucuronide, estradiol hemibutyrate, estradiol hemipivalate, estradiol hemisuccinate, estradiol hemiglucuronide, estradiol hemibutyrate, estradiol hemipivalate, estradiol hemisuccinate, estradiol hemiglucuronide, estradiol hemibutyrate, estradiol hemipivalate, estradiol hemisuccinate, estradiol hemiglucuronide, estradiol hemibutyrate, estradiol hemipivalate, estradiol hemisuccinateacetate), methylprednisolone, methyltestosterone, prednisolone, triamcinolone, chlorotrianisene, hydroxyprogesterone, aminoglutethimide, estramustine, medroxyprogesterone acetate, leuprolide, flutamide, toremifene, goserelin, carboplatin, hydroxyurea, amsacrine, procarbazine, mitotane, mitoxantrone, levamisole, vinorelbine, anastrazole, letrozole, capecitabine, reloxafen, hexamethylmelamine, bevacizumab, bexxar, velcade, zevalin, trisenox, xeloda, vinorelbine, porfimer, erbitux, liposomal, thiotepa, altretamine, melphalan, trastuzumab, fulvestrant, exemestane, ifosfamide, rituximab, C225, alemtuzumab, clofarabine, cladribine, aphidicolin, sunitinib, dasatinib, tazcitabine, Sml 1, triapine, didox, trimidox, amidox, 3-AP, MDL-101,731, bendamustine, ofatumumab, and GS-1101 (also known as CAL-101).

[0201] In some embodiments, the chemotherapeutic agent is selected from the group consisting of alkylating agents (e.g., busulfan, chlorambucil, cisplatin, cyclophosphamide (cytoxan), dacarbazine, ifosfamide, mustargen, and melphalan), nitrosoureas (e.g., carmustine, lomustine, semustine, and streptozocin), triazines (e.g., dacarbazine), antimetabolites (e.g., 5-fluorouracil (5-FU), cytarabine (Ara-C), fludarabine, gemcitabine, and methotrexate), purine analogs (e.g., 6-mercaptopurine, 6-thioguanine, and pentostatin (2-deoxycoformycin)), mitotic inhibitors (e.g., docetaxel, etoposide (VP 16), teniposide, taxol, taxotere, vinblastine, vincristine, and vinorelbine), antitumor antibiotics (e.g., bleomycin, dactinomycin, daunorubicin, doxorubicin, mitomycin, plicamycin, and idarubicin), platinum chemotherapeutics (e.g., cisplatin and carboplatin), anthracenediones (e.g., mitoxantrone), toxins (e.g., ricin A chain (Burbage, Leukemia 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, E. coli enterotoxin toxin A subunit, cholera toxin A subunit, and Pseudomonas toxin c-terminal), and gene therapy vectors (e.g., signal transduction proteins (e.g., Src, AbI, and Ras), Jun, Fos, and Myc).

[0202] In some embodiments, the additional therapeutic agent is an immunotherapeutic agent. Immunotherapeutic agents generally trigger immune effector cells and molecules to target and destroy cells (e.g., cancer cells). The immune effector can be, for example, an antibody specific for a marker on the surface of a cell (e.g., a tumor cell). The antibody alone can act as an effector of therapy, or it can recruit other cells to effect cell killing. Various effector cells include, but are not limited to, cytotoxic T cells and NK cells.

[0203] Exemplary immunotherapeutic agents include, but are not limited to, azathioprine, chlorambucil, cyclophosphamide, cyclosporine, daclizumab, infliximab, methotrexate, tacrolimus, immunostimulatory factors (e.g., IL-2, IL-4, IL-12, GM-CSF, tumor necrosis factor; interferons alpha, beta, and gamma; F42K and other cytokine analogs; chemokines such as MIP-1, MIP-1 beta, MCP-1, RANTES, IL-8; or growth factors such as FLT3 ligand), antigenic peptides, polypeptides, or proteins, or autologous or allogeneic tumor cell compositions (see, e.g., Ravindranath and Morton, International reviews of immunology, 7.4 (1991): 303-329), hormonal therapies, adrenocortical steroids, progestins (e.g., hydroxyprogesterone caproate, medroxyprogesterone acetate, and megestrol acetate), estrogens (e.g., diethylstilbestrol and ethinyl estradiol), antiestrogens (e.g., testosterone propionate and fluoxymesterone), antiandrogens (e.g., flutamide), and gonadotropin-releasing hormone analogs (e.g., leuprolide). Other immunotherapeutic agents are known in the art and can be found in, e.g., Rosenberg et al., New England Journal of Medicine, 319.25 (1988): 1676-1680; and Rosenberg et al., Annals of surgery, 210.4 (1989): 474.

[0204] The therapeutic agents provided herein can act over a wide dosage range and will generally be administered in an effective amount. It will be understood, however, that the amount of the therapeutic agent actually administered will usually be determined by a physician, according to the relevant circumstances, including the condition to be imaged, the chosen route of administration, the actual compound administered, the age, weight, and response of the individual subject, the severity of the subject's symptoms, and the like.

[0205] IV. Kits for granzyme B imaging and therapy

[0206] This disclosure also covers kits (e.g., drug kits) for granzyme B imaging and therapy. The provided kits may include containers (e.g., vials, ampoules, bottles, syringes, and / or dispenser kits or other suitable containers) in which the pharmaceutical compositions disclosed herein may be placed. In some embodiments, the provided kits may optionally further include a second container comprising a pharmaceutical excipient for diluting or suspending the pharmaceutical composition. In some embodiments, the pharmaceutical compositions in the first and second containers are combined to form a unit dosage form. In some embodiments, the kit may include additional containers comprising one or more other therapeutic agents disclosed herein, such as anti-inflammatory agents, steroids, immunotherapeutic agents, chemotherapy agents, and therapeutic antibodies as described in the Combination Therapies section above.

[0207] In some embodiments, the kits described herein further include instructions for use of the compounds or compositions contained in the kit. The kits described herein may also include information required by regulatory agencies, such as the U.S. Food and Drug Administration (FDA). In some embodiments, the information included in the kit is prescribing information. In some embodiments, the kit and instructions provide for imaging and / or treating granzyme B in subjects with a granzyme-related target condition (e.g., the target condition disclosed herein) and / or reducing the risk of said condition. The kits described herein may contain one or more additional pharmaceutical agents described herein as separate compositions.

[0208] Alternative embodiments

[0209] The following is a description of alternative embodiments of this disclosure. The following examples are non-limiting and are intended to describe exemplary embodiments to illustrate the utility of this disclosure.

[0210] Example 1: A compound, its stereoisomer, or a pharmaceutically acceptable salt thereof, wherein the compound is...

[0211]

[0212] The fluorine (F) is optionally a radioactive isotope and the hemiacetal unit is optionally in the form of an open-chain aldehyde.

[0213] Example 2: The compound according to Example 1, its stereoisomer or a pharmaceutically acceptable salt thereof, wherein compound I has the following structure:

[0214]

[0215]

[0216] Embodiment 3: The compound according to Embodiment 1, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, wherein the compound contains a radioisotope of F. 18 The compound of F has the following structure:

[0217] wherein the hemiacetal unit is optionally in open chain aldehyde form.

[0218] Embodiment 4: The compound according to Embodiment 1, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, wherein the compound contains a radioisotope of F. 18 The compound of F has the following structure:

[0219]

[0220]

[0221] Embodiment 5: A compound, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, wherein the compound is

[0222]

[0223] wherein the hemiacetal unit is optionally in open chain aldehyde form.

[0224] Embodiment 6: The compound according to Embodiment 5, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, wherein Compound II has the following structure:

[0225]

[0226]

[0227] Embodiment 7: A pharmaceutical composition comprising a compound according to Embodiment 5, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

[0228] Embodiment 8: A combination therapy comprising a compound according to Embodiment 5, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, and one or more additional therapeutic agents as therapeutic agents.

[0229] Embodiment 9: The combination therapy according to Embodiment 8, wherein the one or more additional therapeutic agents are selected from the group consisting of an anti-inflammatory agent, a steroid, an immunotherapeutic agent, a chemotherapeutic agent, and a therapeutic antibody.

[0230] Example 10: A method for imaging granzyme B in cells or tissues, samples, or cell or tissue samples, the method comprising contacting the cells or tissues, the sample, or the cell or tissue sample with a compound according to any one of Examples 1 to 6, its stereoisomers, or a pharmaceutically acceptable salt thereof, and imaging the cells or tissues, the sample, or the cell or tissue sample using a suitable imaging technique, thereby imaging granzyme B in the cells or tissues, the sample, or the cell or tissue sample, wherein the compound contains a radioactive isotope. 18 F.

[0231] Example 11: A method for treating an immune dysregulation in a subject in need, the method comprising administering to the subject, in an amount effective for treating the immune dysregulation, the compound according to Example 4, its stereoisomer, or a pharmaceutically acceptable salt thereof.

[0232] Example 12: The method according to Example 11, wherein the immune dysregulation is selected from the group consisting of: autoimmune diseases, inflammatory diseases, skin diseases, cancer and cardiovascular diseases.

[0233] Example 13: The method according to Example 12, wherein the immune regulation abnormality is cancer.

[0234] Example 14: A method for treating an immune dysregulation in a subject in need, the method comprising administering the combination therapy of claim 7 to the subject in an amount effective in treating the immune dysregulation.

[0235] Example 15: The method according to Example 14, wherein the immune dysregulation is selected from the group consisting of: autoimmune diseases, inflammatory diseases, skin diseases, cancer and cardiovascular diseases.

[0236] Example 16: The method according to Example 15, wherein the immune regulation abnormality is cancer.

[0237] Example 17: A method for monitoring the immune response during the treatment of a subject's disease, the method comprising administering to the subject an effective amount of the compound according to Example 1, its stereoisomer or a pharmaceutically acceptable salt thereof, and imaging the subject using a suitable imaging technique.

[0238] Without further elaboration, it is believed that those skilled in the art can utilize the present invention to the fullest extent based on the above description. Therefore, the following specific embodiments are to be interpreted as illustrative only and not as limiting the remainder of this disclosure in any way. All publications cited herein are incorporated by reference for the purposes or subject matter of this citation.

[0239] Example 1: Synthesis and characterization of compound 7-A1

[0240]

[0241] Step A: Synthesis of compound 7.

[0242]

[0243] Tricyclic amino acid coupling and Fmoc deprotection:

[0244] A solution of Fmoc-(2S,5S)-5-amino-l,2,4,5,6,7-hexahydroazepino[3,2,1- hi]indol-4-one-2-carboxylic acid (2.5 eq, 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 sintered reaction vessel. N,N'-diisopropylcarbodiimide (2.5 eq, 2.62 mmol, 1.8 M) in DMF and ethyl cyanoacetaldehyde-2-oxime (2.5 eq, 2.63 mmol, 0.9 M) in DMF were then added and the mixture was placed on an orbital shaker overnight at ambient temperature. After 24 hours, the mixture was removed from the shaker and the solvent drained. The resin was washed with DMF (3 x 50 ml) followed by dichloromethane (3 x 50 ml). The ninhydrin test was negative. The resin was treated with 20% piperidine in DMF (30 ml) and the mixture stirred on the shaker table for 30 minutes. The solvent was then drained and the resin washed with DMF (3 x 30 ml). The resin was again treated with 20% piperidine in DMF (30 ml) and the mixture stirred on the shaker table for 30 minutes. The solvent was then drained and the resin washed with DMF (3 x 50 ml) followed by dichloromethane (3 x 50 ml).

[0245] L-isoleucine coupling and Fmoc deprotection:

[0246] A solution of ((9H-fluoren-9-yl)methoxy)carbonyl)-L-isoleucine (2.5 eq, 0.93 g, 2.63 mmol) in DMF (43 mL) was added to the resin from the previous step in a sintered reaction vessel. N,N'-diisopropylcarbodiimide (2.5 eq, 2.62 mmol, 1.8 M) in DMF and ethyl cyanoacetoate-2-oxime (2.5 eq, 2.63 mmol, 0.9 M) in DMF were then added and the mixture was placed on an orbital shaker overnight at ambient temperature. The mixture was removed from the shaker and the solvent drained. The resin was washed with DMF (3 x 50 ml) before washing with dichloromethane (3 x 50 ml). The ninhydrin test was negative. The resin was treated with 20% piperidine in DMF (30 ml) and the mixture stirred on the shaker table for 30 minutes. The solvent was then drained and the resin washed with DMF (3 x 30 ml). The resin was again treated with 20% piperidine in DMF (30 ml) and the mixture stirred on the shaker table for 30 minutes. The solvent was then drained and the resin washed with DMF (3 x 50 ml) followed by dichloromethane (3 x 50 ml).

[0247] Gly-Gly coupling and Fmoc deprotection:

[0248] A solution of ((9H-fluoren-9-yl)methoxy)carbonyl)-L-isoleucine (2.5 eq, 0.93 g, 2.63 mmol) in DMF (43 mL) was added to the resin from the previous step in a sintered reaction vessel. N,N'-diisopropylcarbodiimide (2.5 eq, 2.62 mmol, 1.8 M) in DMF and ethyl cyanoacetoate-2-oxime (2.5 eq, 2.63 mmol, 0.9 M) in DMF were then added and the mixture was placed on an orbital shaker overnight at ambient temperature. The mixture was removed from the shaker and the solvent drained. The resin was washed with DMF (3 x 50 ml) before washing with dichloromethane (3 x 50 ml). The ninhydrin test was negative. The resin was treated with 20% piperidine in DMF (30 ml) and the mixture stirred on the shaker table for 30 minutes. The solvent was then drained and the resin washed with DMF (3 x 30 ml). The resin was again treated with 20% piperidine in DMF (30 ml) and the mixture stirred on the shaker table for 30 minutes. The solvent was then drained and the resin washed with DMF (3 x 50 ml) followed by dichloromethane (3 x 50 ml).

[0249] L-Glu coupling and Fmoc deprotection:

[0250] A solution of (2S)-5-tert-butoxy-2-(9H-fluoren-9-ylmethoxycarbonylamino)-5- oxo-pentanoic acid (2.5 eq, 2.63 mmol) dissolved in DMF (43 mL) was added to the resin from the previous step in a sintered reaction vessel. N,N'-diisopropylcarbodiimide (2.5 eq, 2.62 mmol, 1.8 M) in DMF and ethyl cyanoacetaldehyde-2-oxime (2.5 eq, 2.63 mmol, 0.9 M) in DMF were then added and the mixture was placed on an orbital shaker overnight at ambient temperature. The mixture was removed from the shaker and the solvent drained. The resin was washed with DMF (3 x 50 ml) before washing with dichloromethane (3 x 50 ml). The ninhydrin test was negative. The resin was treated with 20% piperidine in DMF (30 ml) and the mixture stirred on the shaker table for 30 minutes. The solvent was then drained and the resin washed with DMF (3 x 30 ml). The resin was again treated with 20% piperidine in DMF (30 ml) and the mixture stirred on the shaker table for 30 minutes. The solvent was then drained and the resin washed with DMF (3 x 50 ml) followed by dichloromethane (3 x 50 ml).

[0251] NODA chelation coupling:

[0252] 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 eq, 1.06 g, 2.10 mmol) in DMF (30 mL) was added to the resin from the previous step (about 1.05 mmol) in a sintered reaction vessel. N,N'-diisopropylcarbodiimide (2 eq, 2.10 mmol, 1.8 M) in DMF and ethyl cyanoacetaldehyde-2-oxime (2 eq, 2.10 mmol, 0.9 M) in DMF were then added and the mixture was placed on an orbital shaker for 46 hours at ambient temperature. The mixture was removed from the shaker and the solvent drained. The resin was washed with DMF (3 x 20 ml) before washing with dichloromethane (3 x 20 ml). The ninhydrin test was negative. The material was stored in a vacuum oven at ambient temperature over the weekend.

[0253] Final cleavage from the resin:

[0254] The resin from the previous step (about 1.05 mmol) was treated with a mixture of trifluoroacetic acid (10 mL) and dichloromethane (30 mL) and the suspension was stirred on a shaker table overnight. The suspension was removed from the shaker table and the solvent drained. The resin was washed with dichloromethane (3 x 25 ml) and then blown dry with nitrogen. The resin was then placed in a vacuum oven at ambient temperature overnight. 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 stirred gently and heated at 60 °C for 1 hour. The hot suspension was filtered by vacuum filtration and the resin was washed with a mixture of water (24 mL) and acetonitrile (36 mL) IX. The filtrate was then frozen at -78 °C and placed on a lyophilizer to yield 651.8 mg of a yellow solid as a crude peptide.

[0255] Purification of crude peptide:

[0256] A solution of 50 mg of crude peptide in H2O with 0.1% formic acid (1.0 mL) and acetonitrile (0.4 mL) was filtered through a cotton plug, rinsing with H2O with 0.1% formic acid (0.2 mL). The solution was then purified by HPLC (Phenomenex Gemni C18 RP-HPLC prep column, 10% to 40% H2O with 0.1% formic acid / acetonitrile as the mobile phase). Fractions were collected and lyophilized to give compound 7 as a white fluffy solid (12 mg). ES / MS m / z 1077.5 (M+H).

[0257] Step B: Synthesis of compound 7-Al

[0258] A solution of compound 7 (crude peptide, 50 mg, about 0.046 mmol) in 0.1 M NaOAc with 20 mM AlCl3(5.5 mL, 0.11 mmol) and H2O with 100 mM NaF (1.1 mL, 2.0 equiv, 0.11 mmol) was first sonicated and then the mixture was heated to 105 °C for 30 minutes. The solution was then filtered and directly purified by HPLC (Phenomenex Gemni C18 RP-HPLC prep column, H2O with 0.1% formic acid / acetonitrile as the mobile phase). Fractions were collected and lyophilized to give compound 7-Al (1 : 1) as a grayish white fluffy solid (13 mg). ES / MS m / z 1121.5 (M+H).

[0259] Compounds 18 Radiosynthesis of F-7-Al on Neptis Perform

[0260]

[0261] Typical yields of 18-22% (for pk2 only) and about 59% (for combined pk1 and pk2) can be synthesized on Neptis perform in 65 ± 5 minutes 18 F-7-Al. The product was >95% radiochemically pure and specific activity ranged from 497-2196 GBq / umol (12-53 mCi / ug). The reaction vessel was preloaded with precursor 7 [0.2 mL, 2 mg / mL in acetic acid-sodium acetate buffer (1.0 mol / L, pH = 3.5)], aluminum chloride solution (133 pL, 2.4 mM in water for injection), and acetonitrile (677 pL). The reaction was initiated by the addition of fluoride [0.5-2.0 Ci] and the reaction mixture was heated to 105 °C for 15 minutes. The reaction mixture was cooled to 60 °C and 4.6 mL of water (HPLC grade) was added to the reaction vessel. The diluted reaction mixture was then loaded onto a semi-preparative HPLC column (Agilent XDB-C18, 9.4 x 250 mm, Part Number 990967-202) for purification. Purification conditions are listed below: 18 F] fluoride (0.5-2.0 Ci) was retained on a Waters Sep-Pak Accell Plus QMA Carbonate Plus Light Cartridge (46 mg of adsorbent per cartridge, 40 pm particle size, Waters Part Number 186004540, pre-conditioned with 5 mL of saline (USP) followed by 5 mL of water for injection) and then eluted into the reaction vessel with saline (0.8 mL, USP). The resulting mixture was held at 105 °C for 15 minutes and then cooled to 60 °C. 4.6 mL of water (HPLC grade) was added to the reaction vessel and the diluted reaction mixture was then loaded onto a semi-preparative HPLC column (Agilent XDB-C18, 9.4 x 250 mm, Part Number 990967-202) for purification. Purification conditions are listed below:

[0262] 1) For preparation of peak 2 only:

[0263] Column - Agilent ZORBAX Eclipse XDB-C18 (Agilent ZORBAX Eclipse XDB-C18, Part Number 990967-202), 5 pm, 9.4 mm x 250 mm

[0264] Isocratic method - Purification mobile phase: 11% acetonitrile, 89% ammonium acetate / water solution (20 mM), flow rate = 4 mL / min, UV at 254 nm. Typical peak collection time ranged from about 28-30 minutes. Figure 1 ).

[0265] 2) For preparation of combined peak 1 and peak 2:

[0266] Column - Agilent ZORBAX Eclipse XDB-C18 (Part Number 990967-202), 5 μm, 9.4 mm x 250 mm

[0267] Gradient Method - Purification mobile phase: 8% acetonitrile in 20 mM ammonium acetate / water to 20% acetonitrile in 20 mM ammonium acetate / water over 24 minutes, flow rate = 4 mL / min, UV at 254 nm. Typical peak collection time range was about 20-22 minutes. Figure 3

[0268] The collected fractions were then diluted with 0.5% (w / v) sodium ascorbate in water (about 30 mL) and passed through a Waters Sep-Pak C18 Plus Light cartridge (130 mg of adsorbent per cartridge, 55-105 um, Part Number: 023501, pre-conditioned with 5.0 mL ethanol followed by 5 mL water), then the product retained on the cartridge was washed with 0.5% (w / v) sodium ascorbate in water (about 15 mL) and thereafter eluted with 1.5 mL EtOH (USP) into a final product vial containing 10.0 mL of Sodium Chloride Injection, USP 0.9% and sodium ascorbate (USP, 0.5 w / v%). The C18 cartridge was then rinsed with an additional 3.5 mL of Sodium Chloride Injection, USP 0.9% and sodium ascorbate (USP, 0.5 w / v%) to yield 15.0 mL of the formulated product, 10 v / v% EtOH and 90 v / v% 0.9% saline containing 0.5% w / v sodium ascorbate.

[0269] To prepare the sterile product, the product produced (in 10 v / v% ethanol and 90 v / v% 0.9% saline containing 0.5% w / v sodium ascorbate 18 F-7-Al) was sterile filtered into bulk product vials through a 0.22 μm filter (e.g. GV Sterile Filter, Millipore Part Number SLGV033RS). Samples were taken from the bulk product vials for HPLC analysis. Figure 2 and 4 )

[0270] Analytical HPLC Conditions: Figure 2

[0271] ​​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 min.

[0272] Mobile phase composition: A: water with 20 mM ammonium acetate, B: acetonitrile (HPLC grade)

[0273] Gradient:

[0274]

[0275] Analytical HPLC conditions: (Analytical column: Agilent ZORBAX Eclipse XDB-C18 4.6 x 150 mm, Part No. 993967-902; flow rate = 1.5 mL / min, UV at 254 nm) Figure 4 )

[0276] Analytical column: Agilent ZORBAX Eclipse XDB-C18 4.6 x 150 mm, Part No. 993967-902; flow rate = 1.5 mL / min, UV at 254 nm

[0277] Gradient method - mobile phase: A: water with 20 mM ammonium acetate, B: acetonitrile (HPLC grade)

[0278]

[0279] Example 2: Characterization of Granzyme B binding activity

[0280] The above compounds were tested using the human Granzyme B biochemical assay provided below.

[0281] Table 3: Assay parameters

[0282]

[0283] Materials :

[0284] 1. Granzyme B (human lymphocyte) enzyme: Enzo Lifesciences, Cat. No.

[0285] Cat. No. ALX-200-602-C010

[0286] 2. Substrate (Ac-IETD-AFC): Enzo Lifesciences, Cat. No.

[0287] ALX-260-110-M010

[0288] 3. Standard inhibitor (Compound 20 - Isomer 3): Synthesized in-house in TCG

[0289] 4. Dimethyl sulfoxide (DMSO): Sigma-Aldrich

[0290] Catalog number 41639

[0291] 5. HEPES: Gibco, catalog number 15630-080

[0292] 6. Calcium chloride: Sigma-Aldrich, catalog number C-5080

[0293] 7. Distilled water: Gibco, catalog number 15230-162

[0294] 8. Bovine serum albumin (BSA): Sigma-Aldrich, catalog number A3059

[0295] 9. Black transparent bottom PDL plate (384 wells): Greiner bio-one

[0296] Catalog number 781946

[0297] 10. Polypropylene sheet (384 holes): Corning Incorporated, catalog number 3657

[0298] Table 4: Composition of the determination buffer solution

[0299]

[0300]

[0301] Compound preparation:

[0302] 1. Add DMSO to the corresponding compound vial to prepare a 10 mM compound stock solution, which is stored at -20°C.

[0303] 2. Thaw the 10 mM compound stock solution and prepare a 1 mM DMSO compound stock solution by adding 45 μl DMSO to 5 μl of the 10 mM compound stock solution.

[0304] 3. Serially dilute the 1 mM DMSO stock solution (3.16-fold) by adding 10 μl of 1 mM DMSO stock solution to 21.6 μl of DMSO and mixing thoroughly. Then add 10 μl of the resulting solution to 21.6 μl of DMSO and mix thoroughly. Continue this process in 384-well polypropylene plates to produce 11 dilution points for determining the standard inhibitor and 8 dilution points for the test compound.

[0305] 4. Dispense 2 μl of each diluent into the assay-ready plate.

[0306] 5. Then, in the assay-ready plate, dilute each well 25-fold by adding 48 μl of assay buffer to 2 μl of the compound to prepare a working stock solution of the compound.

[0307] Enzyme preparation:

[0308] 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.

[0309] 2. Dilute 1 μl of aliquot sample to 625 nM by adding 49 μl of assay buffer containing 0.1% BSA and gently mixing.

[0310] 3. Prepare a 10 nM enzyme working stock solution by adding assay buffer containing 0.1% BSA.

[0311] Substrate preparation :

[0312] 1. The supplied substrate (Ac-IETD-AFC) was reconstructed by adding DMSO to prepare a 10 mM stock solution, which was aliquoted and stored at -80°C.

[0313] 2. Prepare a 4 mM substrate intermediate stock solution by adding DMSO.

[0314] 3. Prepare a substrate working stock solution of 800 μM by adding assay buffer.

[0315] Assay protocol :

[0316] 1. According to the plate plot, add 10 μl of serially diluted compound working stock solution (starting dose 40 μM) from the assay-ready plate to the assay plate.

[0317] 2. Add the positive control (40 μM standard inhibitor) and the negative control (4% DMSO buffer) to the corresponding wells.

[0318] 3. Add 20 μl of enzyme working stock solution to the assay plate and mix gently.

[0319] 4. Incubate the plate at 22°C for 30 minutes, and rotate it at 130g for 1 minute.

[0320] 5. After incubation, add 10 μl of substrate working stock solution to the corresponding well and mix (keep the assay plate in the dark after adding the substrate).

[0321] 6. Incubate the plate at 22°C for 60 minutes, and rotate it at 130g for 1 minute.

[0322] 7. After 60 minutes, extract fluorescence readings (RFU) in an Emerson multi-mode reader (Ex: 400nm / Em: 505nm).

[0323] Table 5: Final Measured Concentration

[0324] Reagents Final concentration Volume added Compound / standard / control 3.16-fold serial dilutions starting from 10 μΜ 10 μl Enzyme 5 nM 20 μl Substrate 200 μM 10 μl

[0325] Data analysis :

[0326] 1. Analyze RFU readings to calculate the percentage of inhibition by normalizing them in Microsoft Excel with positive and negative controls, which are considered as 100% and 0% effects, respectively.

[0327] 2. Graphs were generated by inputting the analyzed data into GraphPad Prism 5.0 software to obtain the IC50 value for each compound. 50 value.

[0328] Table 6 below shows the structures and activities of exemplary compounds. These compounds exhibit high potency in inhibiting granzyme B, as shown in the IC50 values ​​included in the table below. 50 The value indicated.

[0329] Table 6: IC50 of exemplary compounds 50 value

[0330]

[0331] Example 3: Synthesis of an exemplary compound targeting granzyme B

[0332] The above synthetic method, together with the following general peptide synthesis procedure, was used to synthesize the following compounds, and the exact mass of each compound was recorded:

[0333] General peptide synthesis procedure: Peptides are synthesized using H-Asp(OtBu)-H resin according to the standard Fmoc solid-phase peptide synthesis procedure. The final peptide is deprotected and cleaved from the resin in a two-step procedure: 1) Treatment with trifluoroacetic acid (TFA) at room temperature for 2 hours, or with TFA / dichloromethane (DCM) overnight at room temperature, followed by concentration; 2) Treatment with acetonitrile / water (60:40) containing 0.1% TFA at 60°C for 1 hour. The crude peptide is concentrated or lyophilized, and then purified by preparative HPLC (using a mobile phase of water / acetonitrile containing 0.1% formic acid or 0.1% TFA). The fraction containing the product is collected and lyophilized to obtain a white, fluffy solid peptide.

[0334]

[0335] ES / MS m / z 1091.5(M+H) + .

[0336]

[0337] ES / MS m / z 1105.5(M+H) + .

[0338]

[0339] ES / MS m / z 1234.7(M+H) + .

[0340]

[0341] ES / MS m / z 948.4(M+H) + .

[0342]

[0343] ES / MS m / z 891.5(M+H) + .

[0344]

[0345] ES / MS m / z 1019.5(M+H) + .

[0346]

[0347] ES / MS m / z 1020.5(M+H) + .

[0348]

[0349] ES / MS m / z 963.5(M+H) + .

[0350]

[0351] ES / MS m / z 963.5(M+H) + .

[0352]

[0353] ES / MS m / z 977.5(M+H) +

[0354]

[0355] ES / MS m / z 963.5(M+H) +

[0356]

[0357] ES / MS m / z 1019.6(M+H) +

[0358]

[0359] ES / MS m / z 1047.6(M+H) +

[0360] To form the following compound, use the following general procedure: Add equimolar amounts (1.5–3.0 equivalents relative to the peptide) of 0.1 M NaOAc (pH approximately 4.5) containing 20 mM AlCl3 and H2O containing 100 mM NaF to a reaction vial containing the peptide precursor and a stir bar. Then add acetonitrile (0–34% of the total reaction volume). Heat the mixture to 100 °C for 15–30 minutes. Remove the acetonitrile under reduced pressure and purify the aqueous solution by passing it through a C18 ISCO column or a C18 preparative HPLC column (using an aqueous solution containing 0.1% formic acid and acetonitrile as eluents). Collect the appropriate fractions and lyophilize to obtain the peptide AlF complex as a white, fluffy solid.

[0361]

[0362] Following standard procedure, 1 (60 mg) was converted into 1-Al(1:1) (22 mg), a white, fluffy solid. ES / MS m / z 1135.4 (M+H) +

[0363]

[0364] Following standard procedure, 2 (16 mg) was converted to 2-Al(1:1) (8.6 mg), a white, fluffy solid. ES / MS m / z 1149.5 (M+H) +

[0365]

[0366] Following standard procedure, 18 (15 mg) was converted to 18-Al (1:1) (7.2 mg), a white, fluffy solid. ES / MSm / z 1278.5 (M+H) +

[0367]

[0368] Following standard procedure, 4 (30 mg) was converted into 22 mg of 4-Al (1:1), a white, fluffy solid. ES / MS m / z 935.4 (M+H) +

[0369]

[0370] Following standard procedure, 11 (30 mg) was converted into 11-Al(1:1) (15 mg), a white, fluffy solid. ES / MSm / z 1007.4 (M+H) +

[0371]

[0372] Following standard procedure, 9 (30 mg) was converted into 9-Al (1:1) (24 mg), a white, fluffy solid. ES / MS m / z 1007.4 (M+H) +

[0373]

[0374] Following standard procedure, 10 (30 mg) was converted into 10-Al (1:1) (24 mg), a white, fluffy solid. ES / MSm / z 1007.5 (M+H) +

[0375]

[0376] Following standard procedure, 17 (30 mg) was converted into 17-Al (1:1) (23 mg), a white, fluffy solid. ES / MSm / z 1021.5 (M+H) +

[0377]

[0378] Following standard procedure, 5 (34 mg) was converted into 25 mg of 5-Al (1:1), a grayish-white, fluffy solid. ES / MSm / z 1063.5 (M+H) +

[0379]

[0380] Following standard procedure, 6 (30 mg) was converted to 25 mg of 6-Al (1:1), a white, fluffy solid. ES / MS m / z 1064.5 (M+H) +

[0381]

[0382] Following standard procedure, 12 (30 mg) was converted into 12-Al (1:1) (16 mg), a grayish-white, fluffy solid. ES / MSm / z 1063.6 (M+H) +

[0383]

[0384] Following standard procedure, 13 (31 mg) was converted into 13-Al (1:1) (23 mg), a grayish-white, fluffy solid. ES / MSm / z 1091.5 (M+H) +

[0385]

[0386] Following standard procedure, 3 (19 mg) was converted into 13-Al (1:1) (9 mg), a grayish-white, fluffy solid. ES / MSm / z 992.5 (M+H) +

[0387] Example 4: Radiosynthesis Program

[0388] General radiosynthesis of compounds of interest

[0389] Using 0.5–2.0 Ci as the initial activity, the typical synthesis time is 75 ± 10 minutes. 18 The RCY concentration of F-GZB compound ranges from 5.6% to 63%. An aqueous buffer solution of acetate / sodium acetate (e.g., 200-400 μL, 1 mol / L, pH 3.0-5.0) containing the precursor (e.g., 0.2-0.6 mg), AlCl3·6H2O (e.g., 34-82 μg, 100-240 nmol), and acetonitrile (e.g., 25-50% of the total reaction mixture volume) is added to the reaction vial. 18 F] Fluoride activity is retained on a conditioned anion exchange resin (e.g., Sep-Pak Accell Plus QMA Carbonate Plus lightweight filter cartridge, 46 mg adsorbent per cartridge, 40 μm particle size, Waters Part No. 186004540) [with 5 mL of 0.9% saline followed by 5 mL of WFI (water for injection)]. The retained [F] fluoride activity is further retained by passing 0.9% saline (e.g., 0.5–0.8 mL). 18F] Fluoride is eluted from the filter cartridge into a reaction vial. The resulting mixture is heated (e.g., 105°C) for a period of time (e.g., 15 minutes) and then cooled (e.g., 60°C) and diluted with water (e.g., 1.0–5.0 mL, HPLC grade). The resulting crude product is loaded onto a semi-preparative reversed-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., a mobile phase comprising an aqueous acetonitrile solution (8–20%), pH 1–8). The purified product is then purified by adding 0.5 w / v% sodium ascorbate aqueous solution (e.g., 30–50 mL). 18 The HPLC fraction of the F-GZB compound was diluted and then passed through a conditioned reverse-phase filter cartridge (e.g., with 5 mL of ethanol (USP grade) followed by 5 mL of water (HPLC grade)). Lightweight C18 filter cartridge, 130 mg adsorbent per cartridge, 55-105 μm particle size (Waters part number WAT0523501). Wash with 0.5 w / v % sodium ascorbate aqueous solution (e.g., 5-15 mL). 18 F-GZB was elute from the filter cartridge using ethanol (e.g., 1.0-1.5 mL) into a reconstitution vial containing 0.9% saline solution with 0.5% w / v sodium ascorbate (e.g., 6.0-10.0 mL). The C18 filter cartridge was then rinsed with another 0.9% saline solution containing 0.5% w / v sodium ascorbate (e.g., 3.0-3.5 mL), and the rinsing solution was collected in a reconstitution vial. The product strength could be adjusted by adding a certain amount of diluent (10 v / v ethanol and 90 v / v 0.9% saline solution containing 0.5 v / v sodium ascorbate).

[0390] To prepare sterile products, they are passed through a 0.22 μm filter (e.g., The Millex GV sterile filter, Millex part number SLGV033RS; the Millex GV 25mm sterile filter, Millex part number SLGVV255F; and the Millex LG 25mm sterile filter, Millex part number SLLG025SS) will produce the product (in 10 v / v % ethanol and 90 v / v 0.9% saline containing 0.5% w / v sodium ascorbate). 18 F-GZB) is aseptically filtered into small vials of bulk products.

[0391] Example 5: Competitive Binding Determination

[0392] Purpose: To use [ 18 F]-20-Al was used to perform a competitive binding assay with human granzyme B to determine the IC50 of potential granzyme B ligands.50 value

[0393] Material:

[0394] 11. Granulase B (human lymphocyte) enzyme: Enzo Life Sciences, catalog number ALX-200-602-C010

[0395] 12. Millipore MultiScreen HTS FB board (Millipore MultiScreen) HTS Millipore (FB plate), catalog number MSFBN6B

[0396] 13. Millipore MultiScreen HTS Vacuum manifold (Millipore MultiScreen) HTS Vacuummanifold, Millifold Corporation, Catalog No. MSVMHTS00

[0397] 14.Millipore Millipore tip punch tip) Millipore, catalog number MADP19650

[0398] 15. Millipore Multi-Drill System, Catalog No. MAMP09608

[0399] 16.96-well 0.5mL assay plate, Thermo Fisher Scientific, catalog number 12-565-502

[0400] 17. Reaction buffer (10mM HEPES, 150mM NaCl, 1mM EDTA, 0.01% BSA, pH 7.4)

[0401] method:

[0402] Initial plate preparation (including total and non-specific binding):

[0403] 1. Add 78 μL of reaction buffer as described above to each well in a 96-well plate.

[0404] 2. For wells used to check total binding (defined as binding of radioligands in the absence of competitors), add 2 μL of DMSO to produce a final DMSO concentration of 1% for all wells.

[0405] 3. For wells used to check for nonspecific binding (defined as binding of radioligands in the presence of 10 μM 20-Al), add 2 μL of 1 mM 20-Al to produce a final concentration of 10 μM.

[0406] Enzyme preparation:

[0407] 1. Dilute granzyme B (GZB, human lymphocytes) to 0.5 μg / mL using the reaction buffer described above.

[0408] 2. Add 100 μL of 0.5 μg / mL diluted human GzB to each well, making each well 50 ng and the final concentration 0.25 μg / mL.

[0409] Compound preparation:

[0410] 1. Add DMSO or water to the appropriate compound vial to prepare a 1 mM compound stock solution.

[0411] 2. The 1 mM compound stock solution was serially diluted (3.16-fold) by 1 / 2 log by adding 18.5 μL of the compound stock solution to 40 μL of DMSO via up-and-down pipetting, and then transferring the 18.5 μL mixture to 40 μL of DMSO. This process was repeated for the test compound to produce 10 dilution points.

[0412] 3. For wells used to detect ligand binding competition, 2 μL of each diluent prepared as described above is dispensed into 96-well plates to produce an additional 1 / 100 of the diluent.

[0413] Preparation of radioactive ligands:

[0414] 1. Prepare 200 nM [ ] in reaction buffer. 18 F]-20-Al (10x radioligand stock solution), with a target of approximately 2 million CPM per 20 μL solution (input).

[0415] 2. Dispense 20 μL of radioligand stock solution into each well to produce a final concentration of 20 nM per well.

[0416] Incubation conditions, post-incubation sample processing, and data analysis:

[0417] 1. The final volume of all 96 wells should be 200 μL, and the test plate should be incubated at 37°C for 90 minutes.

[0418] 2. Transfer all samples from the measurement plate to MultiScreen. HTSOn the FB plate, the plate had been pre-soaked in PBS buffer at pH 7.4 and filtered using a vacuum manifold. An additional 150 μL of PBS buffer was added to each well of the assay plate and combined to ensure transfer of any remaining sample.

[0419] 3. Wash MultiScreen with 150 μL PBS buffer. HTS FB board 3x.

[0420] 4. Use the Multiscreen punch tip and Millipore punch device to separate all filters and transfer them into individual tubes.

[0421] 5. The sample set was processed using a Wizard 2480 automatic gamma counter [PerkinElmer] via […]. 18 F] Specific spectra are analyzed. Values ​​are reported in the form of decay-corrected counts per minute (CPM).

[0422] 6. Normalize the resulting CPM values ​​and convert them to inhibition percentage using the following formula:

[0423]

[0424] *Under these assay conditions, the total binding fraction is typically less than 10% of the added radioactive ligands.

[0425] Using GraphPad Prism 8.4.3 software, a unit-point logIC fit was performed. 50 The formula plots the resulting inhibition values ​​(%) to determine the IC50 for each ligand. 50 value.

[0426] Y = A + (BA) / (1 + 10) (x-LogIC50) )

[0427] Y = Suppression %

[0428] X = Logarithm of the concentration of cold competitive ligands (M)

[0429] A = minimum Y (0%)

[0430] B = Maximum Y (100%)

[0431] LogIC 50 = Logarithm of the concentration of the cold competitive ligand at halfway between the minimum and maximum Y (M)

[0432] result:

[0433] Table 7 below lists the IC values ​​measured in competitive binding assays using 20-Al. 50 value.

[0434] Table 7: In GZB IC 50 IC50 of the competitor ligands tested in the assay 50 value.

[0435] Competitor ligand identification IC 50 Values (nM) 1-Al 6.2 2-Al 12 3-Al 2.1 4-Al 7.4 5-Al 1.5 6-Al 1.1 7-Al * ]] 4.5±0.82 9-Al 5.8 10-Al 1.2 11-Al 5.7 12-Al 2.9 13-Al 3.0 17-Al 4.5 18-Al 4.2

[0436] *For the control ligand 7-Al, the reported IC50 value is... 50 The values ​​are taken as the average of four studies (error reports are standard errors).

[0437] Example 6: Synthesis of 20-Al

[0438] Compounds 20 and 20-Al were prepared using the following synthetic steps. 。

[0439] 2-[4-(2-tert-butoxy-2-oxo-ethyl)-1,4,7-triazacyclononane-1-yl]tert-butyl acetate (20-a):

[0440] 1,4,7-Triazacyclononane (21.61 g, 167.3 mmol, 1.0 equivalent) was dissolved in chloroform (360 ml), and the solution was chilled to -10 °C in an ethanol / ice bath. 2-Tere-butyl bromoacetate (54.33 ml, 368.0 mmol, 2.2 equivalent) was dissolved in chloroform (360 ml) and added dropwise over 4 hours. The mixture was then slowly warmed to ambient temperature. After stirring overnight at ambient temperature, the mixture was poured off through filter paper, and the filtrate was concentrated under vacuum to produce a brown oil. The oil was purified on silica gel using methanol / methylene chloride containing 3% to 12% 7N ammonia as a solvent over 30 minutes to produce a product (16.53 g, 46.24 mmol, 28%) as a brown oil. HRMS: C 18 H 35 N3O4(M+H) + Calculated value: 357.2628, experimental value: 357.2619.

[0441] 2-[4-[[4,7-bis(2-tert-butoxy-2-oxo-ethyl)-1,4,7-triazacyclononane-1-yl]methyl]phenyl]methyl acetate (20-b):

[0442] 2-[4-(2-tert-butoxy-2-oxo-ethyl)-1,4,7-triazacyclononane-1-yl]tert-butyl acetate (16.53 g, 46.24 mmol, 1.0 equivalent) and 2-[4-(bromomethyl)phenyl]methyl acetate (12.36 g, 50.86 mmol, 1.1 equivalent) were combined in acetonitrile (300 mL), and N,N-diisopropylethylamine (24.2 mL, 138.7 mmol, 3.0 equivalent) was added. The mixture was heated overnight at 50 °C. LC-MS indicated the reaction was complete, and the mixture was cooled to ambient temperature and stirred overnight. The solvent was then removed under vacuum to produce a brown oil. The oil was partitioned between dichloromethane (150 mL) and water (75 mL). Water was removed, and the organic matter was washed with water (75 ml), followed by washing with brine (75 ml), dried over sodium sulfate, filtered, and concentrated under vacuum to produce a brown foam. The foam was purified by SFC chromatography (method: Chiralcel OD-H-5X 15 cm, 20% methanol (0.5% N,N-dimethylethylamine) / carbon dioxide, 5 mL / min, 225 nm). Fractions containing the product were combined and concentrated under vacuum to produce an amber oil (15.04 g, 28.94 mmol, 63%). HRMS: C 28 H 45 N3O6(M+H) + Calculated value: 519.3308, experimental value: 519.3324.

[0443] 2-[4-[[4,7-bis(2-tert-butoxy-2-oxo-ethyl)-1,4,7-triazacyclononane-1-yl]methyl]phenyl]acetic acid (20-c):

[0444] Methyl 2-[4-[[4,7-bis(2-tert-butoxy-2-oxo-ethyl)-1,4,7-triazacyclononane-1-yl]methyl]phenyl]acetate (15.04 g, 28.94 mmol, 1.0 equivalent) was dissolved in tetrahydrofuran (120 ml), and an aqueous solution of lithium hydroxide (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 produce foam. The foam was dissolved in water (40 ml), and the pH was carefully adjusted to approximately 7 using 2 M HCl. The water was extracted six times with dichloromethane (100 ml), and the organic matter was combined and concentrated to the residue. The residue was placed in 3:1 chloroform / isopropanol (350 ml), and the turbid solution was poured off through filter paper. The filtrate was concentrated under vacuum and placed in a vacuum oven at ambient temperature overnight to produce a yellow solid (10.77 g, 21.30 mmol, 74%). HRMS: C27 H 43 N3O6(M+H) + Calculated value: 505.3152, experimental value: 505.3167.

[0445] ((3S,6S)-6-(((1H-1,2,3-triazol-4-yl)methyl)carbamoyl)-4-oxo-1,2,3,4,6,7-hexahydroazapyro[3,2,1-hi]indol-3-yl)carbamate (9H-fluorene-9-yl)methyl ester (20-d):

[0446] (3S,6S)-3-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)-4-oxo-1,2,3,4,6,7-hexahydroazapyro[3,2,1-hi]indole-6-carboxylic acid (8.0 g, 17.08 mmol, 1.0 equivalent), 1H-triazol-4-ylmethylamine:hydrochloride (2.53 g, 18.78 mmol, 1.1 equivalent), and HATU (7.79 g, 20.49 mmol, 1.2 equivalent) were combined in N,N-dimethylformamide (80 mL), followed by the addition of N,N-diisopropylethylamine (10.4 mL, 59.77 mmol, 3.5 equivalent). The mixture was stirred overnight at ambient temperature and then partitioned between ethyl acetate (200 mL) and brine (150 mL). Water was removed, and the organic matter was washed twice with brine (150 ml), dried over sodium sulfate, filtered, and concentrated under vacuum to produce a brown oil. The oil was purified on silica gel using a gradient of 3% to 5% methanol / methylene chloride over 5.5 minutes, followed by holding at 5% methanol / methylene chloride for 3 minutes to produce a product as a brown solid (7.61 g, 13.90 mmol, 81%). HRMS: C 31 H 28 N6O4(M+H) + Calculated value: 548.2172, experimental value: 548.2157.

[0447] (3S,6S)-N-((1H-1,2,3-triazol-4-yl)methyl)-3-amino-4-oxo-1,2,3,4,6,7-hexahydroazapyro[3,2,1-hi]indole-6-carboxamide (20-e):

[0448] ((3S,6S)-6-(((1H-1,2,3-triazol-4-yl)methyl)carbamoyl)-4-oxo-1,2,3,4,6,7-hexahydroazapyro[3,2,1-hi]indol-3-yl)carbamate (9H-fluorene-9-yl)methyl ester (7.61 g, 13.9 mmol, 1.0 equivalent) was dissolved in tetrahydrofuran (120 mL), and tetrahydrofuran containing dimethylamine (2 mol / L) (35.0 mL, 70.0 mmol, 5.05 equivalent) was added. The mixture was stirred overnight at ambient temperature. The next 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 solids were then collected by vacuum filtration. The filter cake was washed with acetonitrile, pulled-dryed for 2 hours, and then placed in a vacuum oven at ambient temperature for 64 hours to produce a product as a grayish-white solid (3.69 g, 11.3 mmol, 82%). HRMS: C 16 H 18 N6O2(M+H) + Calculated value: 326.1491, experimental value: 326.1482.

[0449] ((2S,3S)-1-(((3S,6S)-6-(((1H-1,2,3-triazol-4-yl)methyl)carbamoyl)-4-oxo-1,2,3,4,6,7-hexahydroazapyro[3,2,1-hi]indol-3-yl)amino)-3-methyl-1-oxopentane-2-yl)tert-butyl carbamate (20-f):

[0450] (3S,6S)-N-((1H-1,2,3-triazol-4-yl)methyl)-3-amino-4-oxo-1,2,3,4,6,7-hexahydroazapyro[3,2,1-hi]indole-6-carboxamide (3.69 g, 11.3 mmol, 1.0 equivalent), (2S,3S)-2-(tert-butoxycarbonylamino)-3-methyl-valeric acid (3.14 g, 13.6 mmol, 1.2 equivalent), and HATU (5.16 g, 13.6 mmol, 1.2 equivalent) were combined in N,N-dimethylformamide (50 mL), followed by the addition of N,N-diisopropylethylamine (5.92 mL, 33.9 mmol, 3.0 equivalent). The mixture was stirred overnight at ambient temperature. The mixture was then fractionated between ethyl acetate (200 mL) and brine (100 mL), and the water was removed. The organic matter was then washed twice with brine (100 ml) and concentrated under vacuum to produce a brown gel. The gel was dissolved in chloroform and peeled off onto silica gel (40 g) for purification. The substance was purified on a 330 g silica gel column using 5% methanol / dichloromethane as a solvent to produce 6.0 g of yellow foam. NMR indicated that the substance still contained residual DMF. The substance was dissolved in xylene (100 ml) and the solvent was removed under vacuum. The residue was azeotropically treated more than twice in xylene (100 ml) to produce a grayish-white solid (5.55 g, 10.3 mmol, 91%). HRMS: C 27 H 37 N7O5(M+H) + Calculated value: 539.2856, experimental value: 539.2851.

[0451] (3S,6S)-N-((1H-1,2,3-triazol-4-yl)methyl)-3-((2S,3S)-2-amino-3-methylpentamido)-4-oxo-1,2,3,4,6,7-hexahydroazapyro[3,2,1-hi]indole-6-carboxamide (20-g):

[0452] ((2S,3S)-1-(((3S,6S)-6-(((1H-1,2,3-triazol-4-yl)methyl)carbamoyl)-4-oxo-1,2,3,4,6,7-hexahydroazapyro[3,2,1-hi]indol-3-yl)amino)-3-methyl-1-oxopentane-2-yl)carbamate tert-butyl (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 equivalent) was added. LCMS indicated the completion of the reaction after 2.5 hours at ambient temperature. 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 water was extracted twice with chloroform / isopropanol in a 3:1 ratio (100 ml). The organic matter was then combined, washed with brine, dried over sodium sulfate, filtered, and concentrated under vacuum to a brown solid (3.06 g, 6.96 mmol, 68%). HRMS: C 22 H 29 N7O3(M+H) + Calculated value: 439.2332, experimental value: 439.2328.

[0453] (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-hexahydroazapyro[3,2,1-hi]indol-3-yl)amino)-3-methyl-1-oxopentane-2-yl)amino)-2-oxoethyl)amino)-2-oxoethyl)tert-butyl carbamate (20-h):

[0454] (3S,6S)-N-((1H-1,2,3-triazol-4-yl)methyl)-3-((2S,3S)-2-amino-3-methylpentamido)-4-oxo-1,2,3,4,6,7-hexahydroazapyro[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), followed by the addition of N,N-diisopropylethylamine (3.64 mL, 20.9 mmol, 3.0 equivalent). The mixture was stirred overnight at ambient temperature and then partitioned between ethyl acetate (200 ml) and brine (100 ml). Water was removed, and the organics were washed twice with brine (100 ml). The aqueous washes were combined and back-extracted three times with 3:1 chloroform / isopropanol (100 ml). All organic layers were combined and concentrated under vacuum to produce a grayish-white gel. The gel was placed in xylene (100 ml) and the solvent was removed under vacuum. This process was repeated more than twice to remove persistent DMF. The resulting residue was dissolved in chloroform and stripped onto silica gel (40 g) for purification. The substance was purified over 35 minutes on a 330 g silica gel column using 5% to 10% methanol / dichloromethane as a solvent. Fractions containing the product were combined, concentrated under vacuum, and then placed in a vacuum oven at ambient temperature overnight to produce a grayish-white solid (3.50 g, 5.35 mmol, 77%). HRMS: C 31 H 43 N9O7(M+H) + Calculated value: 653.3285, experimental value: 653.3270.

[0455] (3S,6S)-N-((1H-1,2,3-triazol-4-yl)methyl)-3-((2S,3S)-2-(2-(2-aminoacetamido)acetamido)-3-methylpentamido)-4-oxo-1,2,3,4,6,7-hexahydroazapyro[3,2,1-hi]indole-6-methylcarboxamide trifluoroacetate (20-i):

[0456] (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-hexahydroazapheno[3,2,1-hi]indol-3-yl)amino)-3-methyl-1-oxopentane-2-yl)amino)-2-oxoethyl)amino)-2-oxoethyl)carbamate tert-butyl (3.50 g, 5.35 mmol, 1.0 equivalent) was suspended in methylene chloride (75 mL), and then trifluoroacetic acid (8.10 mL, 107 mmol, 20.0 equivalent) was added. LCMS indicated the reaction was complete at 2.5 h. The solvent was removed under vacuum, and the resulting oily substance was sonicated in toluene. The toluene was then removed under vacuum. The reaction was repeated twice or more with toluene, and then the substance was placed in a vacuum oven at ambient temperature for 64 hours to produce a quantitative yield of a grayish-white solid (3.57 g, 5.35 mmol, 100%). HRMS: C 26 H 35 N9O5(M+H) + Calculated value: 553.2761, experimental value: 553.2775.

[0457] 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-hexahydroazapyro[3,2,1-hi]indol-3-yl)amino)-3-methyl-1-oxopentane-2-yl)amino)-2-oxoethyl)amino)-2-oxoethyl)amino)-2-oxoethyl)benzyl)-1,4,7-triazacyclononane-1,4-diyl)ditert-butyl diacetate (20-j):

[0458] (3S,6S)-N-((1H-1,2,3-triazol-4-yl)methyl)-3-((2S,3S)-2-(2-(2-aminoacetamyl)acetamyl)-3-methylpentamido)-4-oxo-1,2,3,4,6,7-hexahydroazapyro[3,2,1-hi]indole-6-carboxamide trifluoroacetate (3.47 g, 5.20 mmol, 1.0 equivalent) and N,N-diisopropylethylamine ( 3.63 ml (20.8 mmol, 4.0 equivalents) was combined in N,N-dimethylformamide (50 mL), followed by the addition of 2-[4-[[4,7-bis(2-tert-butoxy-2-oxo-ethyl)-1,4,7-triazacyclononane-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. Azeotropic treatment with toluene was repeated more than twice to remove the dimethylformamide. The resulting oily substance was dissolved in dichloromethane (200 ml) and stripped onto silica gel. The substance was purified on a 330 g silica gel column using 10% (hold for 25 min) to 20% (over 5 min) methanol / dichloromethane containing 7N ammonia. Appropriate fractions were combined, concentrated under vacuum, and placed in a vacuum oven at ambient temperature for 64 hours to produce a yellow foamy product (4.27 g, 4.10 mmol, 79%). HRMS: C 53 H 76 N 12 O 10 (M+H) + Calculated value: 1040.5807, experimental value: 1040.5825.

[0459] (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-hexahydroazapyro[3,2,1-hi]indol-3-yl)amino)-3-methyl-1-oxopentane-2-yl)amino)-2-oxoethyl)amino)-2-oxoethyl)amino)-2-oxoethyl)benzyl)-1,4,7-triazacyclononane-1,4-diyl)diacetic acid(20):

[0460] 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-hexahydroazapyro[3,2,1-hi]indol-3-yl)amino)-3-methyl-1-oxopentane-2-yl)amino)-2-oxoethyl Di-tert-butyl diacetate (4.05 g, 3.89 mmol, 1.0 equivalent) was suspended in 1,4-dioxane (300 ml), and then 1,4-dioxane (24.3 ml, 97.2 mmol, 25.0 equivalent) containing hydrochloric acid (4 mol / L) was added. LCMS indicated approximately 10% mono-tert-butyl product after 3 hours at ambient temperature. The mixture was then heated to 50 °C, and LCMS indicated approximately 56% of the desired product, approximately 31% mono-tert-butyl product, and approximately 13% of the starting material remaining after 5 hours. The mixture was cooled to ambient temperature and stirred overnight to prevent the formation of any byproducts. The next morning, the mixture was reheated to 50 °C and monitored regularly throughout the day. LCMS indicated approximately 90% of the desired product and approximately 10% mono-tert-butyl product after 8 hours. Heating was continued overnight at 50°C. The next morning, LCMS indicated approximately 5.5% mono-tert-butyl product remaining. Further addition of 1,4-dioxane (9.72 ml, 38.9 mmol, 10.0 equivalent) containing hydrochloric acid (4 mol / L) was made, and LCMS indicated approximately 5.1% mono-tert-butyl product remaining after 3 hours. The temperature was then increased to 60°C and continued for 3 hours. The mixture was then removed from the oil bath and cooled in an ice bath. After cooling, diethyl ether (450 ml) was added and the mixture was stirred for 20 minutes. The solids were then removed by vacuum filtration through a sintered glass funnel. The filter cake was washed five times with diethyl ether (150 ml) and then placed in a vacuum oven at 50°C for 64 hours. The substance was purified by reversed-phase rapid chromatography (C18, 275 g, RediSep Gold) using a gradient of 10.1% acetonitrile / water (w / 0.1% formic acid) to 26.2% acetonitrile / water (w / 0.1% formic acid) over 8.6 min, followed by holding at 26.2% acetonitrile / water (w / 0.1% formic acid) for 4.8 min. Appropriate fractions were combined, frozen at -78 °C, and lyophilized to yield a product (2.29 g, 2.47 mmol, 63%) as a loose, off-white solid. HRMS: C 45 H 60 N 12 O 10 (M+H) +Calculated value: 928.4555, experimental value: 928.4580.

[0461]

[0462] The preparation of 20-Al is as follows.

[0463] Preparation of reaction solution:

[0464] Prepare solutions in vials or bottles using the reagents indicated below.

[0465] H2O containing 1.0M AcOH:

[0466] 30 mL of H2O containing 1.8 g AcOH (30 mmol)

[0467] The resulting solution: S-092-1

[0468] H2O containing 1.0M NaOAc:

[0469] 30mL H2O containing 2.46g NaOAc

[0470] The resulting solution: S-092-2

[0471] H2O containing 0.20M AlCl3:

[0472] 30 mL of H2O containing 1.45 g AlCl3-6H2O

[0473] The resulting solution: S-092-5

[0474] H₂O containing 0.1M NaOAc:

[0475] 20 mL 1.0 M NaOAc solution S-092-2

[0476] 180mL H2O

[0477] The resulting solution: S-092-6

[0478] H2O containing 0.1M AcOH:

[0479] 20 mL 1.0 M AcOH solution S-092-1

[0480] 180mL H2O

[0481] The resulting solution: S-092-7

[0482] 0.1M NaOAc buffer containing 20mM AlCl3:

[0483] 40mL 0.1M AcOH (S-092-7)

[0484] 160mL 0.1M NaOAc (S-092-6)

[0485] The resulting solution was: pH 5.2, 0.1M NaOAc buffer + 22mL 0.2M AlCl3 (S-092-5).

[0486] pH = 4.47

[0487] The resulting solution: S-092-8

[0488] H2O containing 0.1M NaF:

[0489] 150mL x 0.1M = 15mmol

[0490] FW 42g / mol

[0491] 150 mL of H₂O containing 0.63 g NaF:

[0492] The resulting solution: S-092-9

[0493] Aluminum fluoride complex:

[0494] 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-hexahydroazapyro[3,2,1-hi]indol-3-yl)amino)-3-methyl-1-oxopentane-2-yl)amino)-2-oxoethyl)amino)-2-oxoethyl)amino)-2-oxoethyl)benzyl)-1,4,7-triazacyclononane-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:

[0495] Table 8: Preparation of reaction solution

[0496]

[0497] Representative procedure for aluminum fluoride complexation:

[0498] To a 40 mL pre-washed vial containing compound 20 (150 mg, 0.1615 mmol), 0.1 M NaOAc (11.50 mL, 0.23 mmol) containing 20 mM AlCl3 and H2O (1.5 equivalent, 0.24 mmol) containing 100 mM NaF were added. The suspension was then sonicated to obtain a solution. The mixture was heated to 105 °C for 30 minutes. The sample was then purified directly by C-18 reversed-phase chromatography (C18, 150 g Gold) using H2O and CH3CN containing 0.1% HCOOH. All clean fractions were combined, frozen, and lyophilized in clean vials.

[0499] Final batch combination and analysis (compound 20-Al; batch S-099):

[0500] All samples (S-096, S-097, and S-098) were combined to form a single final batch. After lyophilization, 1.23 g of compound 20-Al (batch S-099) as a white, fluffy solid was obtained. The overall yield was calculated to be 87.7%. 19 F NMR (376.45MHz, DMSO-d6) δppm: 169.68. HRMS (m / z): Observed values ​​(M+H) + : 973.4287; Experimental monoisotope mass: 972.4215, Theoretical monoisotope mass: 972.4198 (C 45 H 59 AlFN 12 O 10 (Calculated value).

[0501] Radiosynthetic compounds 18 F-20-Al

[0502]

[0503] The precursor (compound 20 or its formate), aluminum chloride (AlCl3), acid [acetic acid (AcOH) or hydrochloric acid (HCl)], sodium acetate (NaOAc), ethanol (EtOH) and [ 18 The fluoride activity was added to the reaction vial and maintained 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 loaded onto a semi-preparative HPLC column for purification. The purified compound was then... 18 The HPLC fraction of F-20-Al was collected directly or used... The lightweight C18 filter cartridge is reconstituted as a formulation containing approximately 3 mM phosphate and / or 0.5% (w / v) sodium ascorbate (NaAsc) in 10% (w / v) EtOH in 0.9% saline. Synthetic compounds18 Typical radiochemical yields (RCY) of F-20-Al vary from 22% to 56% (using 0.3% to 2.5% starting activity) over a synthesis time of 55–80 minutes.

[0504] Radiosynthesis of compounds with ORA Neptis radiosynthesizer 18 F-20-Al

[0505] Compound 20 (0.35 mg, 376 nmol) dissolved in 1.4 mL of 71.5% (v / v) EtOH WFI solution and AlCl3 aqueous solution [43 μg (178 nmol) AlCl3·6H2O dissolved in 0.5 mL of 16 mM HCl solution] were added to the reaction vial. The [retained on Sep-Pak Accell Plus QMA Carbonate Plus Light Filter Cartridge [(46 mg, 40 μm, Waters Part No. 186004540, pre-conditioned with 5 mL of 0.32 M NaOAc solution followed by 5 mL of WFI)] were then added. 18 [F] Fluoride activity was eluted using 0.5 mL of 0.32 M NaOAc solution into a reaction vial containing the precursor and AlCl3. The resulting mixture was heated at 105 °C for 15 min, then cooled to 60 °C, diluted with 4.8 mL of WFI, and quenched. The resulting crude reaction mixture was loaded onto a semi-preparative HPLC column for purification. Figure 5A and 5B ). Containing purified compounds 18 The HPLC fraction (20 mL) of F-20-Al was collected in a vial containing 6 mL of phosphate-buffered saline and 117 mg of NaAsc, resulting in a solution (total volume 26 mL) of 10% (v / v) EtOH in 0.9% saline containing approximately 3 mmol of phosphate and 0.6 mmol of NaAsc. The sample was removed from the product vial for HPLC analysis. Figure 6A and 6B Compounds 18 Typical RCY for radiosynthesis of F-20-Al varies within 40-56% (n=11, using 0.5-2.7Ci initial activity) over a synthesis time of 60±5 minutes.

[0506] Semi-preparative HPLC conditions: ( Figure 5A and 5B )

[0507]

[0508] Analytical HPLC conditions: ( Figure 6A and 6B )

[0509]

[0510] With GE TRACERlab FX F-N Radiosynthetic compounds 18 F-20-Al

[0511] [ 18 F] Fluoride activity is retained on Sep-Pak Accell Plus QMA Carbonate Plus Lightweight Filter Cartridge [(46 mg, 40 μm, Waters Part No. 186004540), pre-conditioned with 2 mL 0.9% saline followed by 5 mL WIC or 5 mL 0.32 M NaOAc solution followed by 5 mL WFI]. The retained [F] fluoride activity is further enhanced by 0.8 mL 0.32 M NaOAc solution or 0.9% saline (pretreated with Chelex). 18 The fluoride was eluted from the filter cartridge into the reaction flask. Then, precursor solution A {100 μL of 1M AcOH / NaOAc aqueous buffer (pH 4.1) containing 0.25 mg (196 nmol) of compound 20-Al (TFA salt) and AlCl3 solution [60 μL of 0.1M AcOH / NaOAc pH 4 buffer containing 28.9 μg (120 nmol) AlCl3·6H2O and 1 mL of EtOH]} or solution B {150 μL of WFI containing 0.38 mg (298 nmol) of compound 20-Al (TFA salt), AlCl3·6H2O in 90 μL of WFI, 80 μL of 1N HCl, 110 μL of 1M NaOAc and 43.4 μg (180 nmol) of AlCl3 solution in 1 mL of EtOH} was added to the reaction vial. The resulting mixture was heated at 105 °C for 15 minutes and then cooled to 60 °C. Then, 3.5 mL of the solution was used as the final product. Dilute with WFI or WIC. Load the resulting crude reaction mixture onto a semi-preparative HPLC column for purification. The purified compound is then... 18 The HPLC fraction of F-20-Al was collected in a vial containing 40 mL of 0.5% (w / v) NaAsc aqueous solution and loaded into... C18Plus lightweight filter cartridge (130mg, 55-105μm, Waters part number WAT023501; pre-conditioned with 5mL EtOH followed by 5mL WFI). Retained compounds were washed with 5mL of 0.5% (w / v) NaAsc aqueous solution. 18F-20-Al was eluted with 1 mL of EtOH into a vial containing 7 mL of 0.9% saline solution with 0.5% (w / v) NaAsc. The C18 filter cartridge was then rinsed with another 2 mL of 0.9% saline solution containing 0.5% (w / v) NaAsc, resulting in a final solution of 10 mL of 10% (w / v) EtOH and 0.45% (w / v) NaAsc in 0.9% saline solution. 18 Typical RCY for F-20-Al radiosynthesis varies within 22-44% (n=19, using 0.3-1.4Ci initial activity) over a synthesis time of 60±5 minutes.

[0512] Artificially radiolabelled compounds 18 F-20-Al

[0513] Add 125 μg (100 nmol) of 1 M AcOH / NaOAc aqueous buffer (pH 4.1) containing compound 20 (TFA salt), 30 μL of WFI containing 14.5 μg (60 nmol) of AlCl3·6H2O, and 580 μL of EtOH or acetonitrile to an 8 mL reaction vial. 18 F] Fluoride activity was retained on the Sep-Pak Accell Plus QMA Carbonate Plus lightweight filter cartridge [(46 mg, 40 μm, Waters Part No. 186004540), pre-conditioned with 2 mL of 0.9% saline followed by 5 mL of WIC]. The retained [F] fluoride activity was retained after rinsing with 4 mL of WIC. 18 F] fluoride was then eluted from the filter cartridge into a reaction vial using 0.5 mL of 0.18% brine (prepared by mixing 0.4 mL of WIC with 0.1 mL of 0.9% brine). The resulting mixture was heated at 105 °C for 15 min and then partially cooled (approximately 2 min) before adding 4.8 mL of WIC. The resulting crude product was loaded onto a semi-preparative HPLC column on an ORA Neptis radiosynthesizer for purification. The purified compound was then... 18 The HPLC fraction of F-20-Al was diluted with approximately 30 mL of WFI and loaded into... C18Plus lightweight filter cartridge (130 mg, 55-105 μm, Waters part number WAT023501; pre-conditioned with 5 mL EtOH followed by 5 mL WFI). Retained compounds were washed with 10 mL of 0.5% (w / v) NaAsc aqueous solution. 18F-20-Al was eluted with 1.8 mL of EtOH into a vial containing 10 mL of 0.9% saline solution with 0.5% (w / v) NaAsc. The C18 filter cartridge was then rinsed with another 3.5 mL of 0.9% saline solution with 0.5% (w / v) NaAsc, resulting in a final solution of 15 mL of 10% (w / v) EtOH, 0.45% (w / v) NaAsc in 0.9% saline solution. 18 Typical RCY for F-20-Al radiosynthesis varies within 37-44% (n=3, using 0.3-0.6Ci initial activity) over a synthesis time of 75±5 minutes.

[0514] Other embodiments

[0515] All features disclosed in this specification can be combined in any combination. Each feature disclosed in this specification can be replaced by an alternative feature that serves the same, equivalent, or similar purpose. Therefore, unless otherwise expressly stated, each disclosed feature is merely an example of a series of equivalent or similar features.

[0516] Furthermore, from the above description, those skilled in the art can readily identify the basic features of this disclosure, and various changes and modifications can be made to adapt it to various uses and conditions without departing from its spirit and scope. Therefore, other embodiments are also within the scope of the claims.

Claims

1. A compound, or a pharmaceutically acceptable salt thereof, wherein the compound has Formula (la): wherein A is a chelating moiety that is 1,4,7-triazacyclononane-N,N',N"-triacetic acid or 1,4,7- triazacyclononane-4,7-diyl diacetic acid; X is -CH2C(O)- or -NHC(S)-; L is a peptide linker having from 1 to 6 amino acid residues, inclusive.

2. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein L has from 1 to 3 amino acid residues, inclusive.

3. The compound of claim 1 or claim 2, or a pharmaceutically acceptable salt thereof, wherein L has 3 amino acid residues.

4. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein the compound has Formula (la-A):

5. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein the compound has Formula (la-B):

6. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein the compound is:

7. The compound of claim 6, or a pharmaceutically acceptable salt thereof, wherein the compound is:

8. A compound, or a pharmaceutically acceptable salt thereof, wherein the compound has Formula (IIa): wherein M is a metal or a metal linked to a radioisotope; A is a chelating moiety that chelates the metal that is 1,4,7-triazacyclononane-N,N',N"-triacetic acid or 1,4,7-triazacyclononane-4,7-diyl diacetic acid; X is -CH2C(NH)- or -NHC(S)-; L is a peptide linker having from 1 to 6 amino acid residues, inclusive.

9. The compound of claim 8, or a pharmaceutically acceptable salt thereof, wherein L has from 1 to 3 amino acid residues, inclusive.

10. The compound of claim 8, or a pharmaceutically acceptable salt thereof, wherein L has 3 amino acid residues.

11. The compound of claim 8, or a pharmaceutically acceptable salt thereof, wherein the compound has Formula (IIa-A):

12. The compound of claim 8, or a pharmaceutically acceptable salt thereof, wherein the compound has Formula (IIa-B):

13. The compound of any one of claims 8 to 12, or a pharmaceutically acceptable salt thereof, wherein the metal is a radioisotope of gallium (Ga).

14. The compound of claim 13, or a pharmaceutically acceptable salt thereof, wherein the radioisotope of Ga is 68 Ga.

15. The compound of any one of claims 8 to 12, or a pharmaceutically acceptable salt thereof, wherein the metal is aluminum, which is linked to the radioisotope.

16. The compound of claim 15, or a pharmaceutically acceptable salt thereof, wherein the radioisotope is 18 F.

17. The compound of claim 8, or a pharmaceutically acceptable salt thereof, wherein the compound is:

18. The compound of claim 17, or a pharmaceutically acceptable salt thereof, wherein the compound is:

19. A pharmaceutical composition comprising a compound of any one of claims 8 to 18, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

20. A kit comprising: (i) a compound according to any one of claims 8 to 18, or a pharmaceutically acceptable salt thereof; and (ii) one or more additional therapeutic agents.

21. The kit of claim 20, wherein the one or more additional therapeutic agents are selected from the group consisting of anti-inflammatory agents, immunotherapeutic agents, and chemotherapeutic agents.

22. The kit of claim 20, wherein the one or more additional therapeutic agents are selected from the group consisting of steroids and therapeutic antibodies.

23. A product comprising a compound according to any one of claims 8 to 18, or a pharmaceutically acceptable salt thereof, as a therapeutic agent and one or more additional therapeutic agents.

24. The product of claim 23, wherein the one or more additional therapeutic agents are selected from the group consisting of anti-inflammatory agents, immunotherapeutic agents, and chemotherapeutic agents.

25. The product of claim 23, wherein the one or more additional therapeutic agents are selected from the group consisting of steroids and therapeutic antibodies.

26. Use of a compound of any one of claims 8 to 18, or a pharmaceutically acceptable salt thereof, in the manufacture of a reagent for imaging granzyme B in a cell or tissue, a sample, a cell or tissue sample, the imaging comprising: contacting the cell or tissue, the sample, the cell or tissue sample with the compound, or a pharmaceutically acceptable salt thereof, and imaging the cell or tissue, the sample, the cell or tissue sample with a suitable imaging technique, thereby imaging granzyme B in the cell or tissue, the sample, the cell or tissue sample; wherein the compound contains a radioisotope.

27. The use of claim 26, wherein the radioisotope is 18 F or 68 Ga.

28. Use of a compound of any one of claims 8 to 18, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for use in a method of treating an immune dysregulation in a subject in need thereof, the method comprising administering to the subject the compound, or a pharmaceutically acceptable salt thereof, in an amount effective to treat the immune dysregulation, wherein the immune dysregulation is a skin disorder and a cardiovascular disorder.

29. Use of a compound of any one of claims 8 to 18, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for use in a method of treating an immune dysregulation in a subject in need thereof, the method comprising administering to the subject the compound, or a pharmaceutically acceptable salt thereof, in an amount effective to treat the immune dysregulation, wherein the immune dysregulation is an autoimmune disorder selected from the group consisting of:

30. Use of a compound of any one of claims 8 to 18, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for use in a method of treating an immune dysregulation in a subject in need thereof, the method comprising administering to the subject the compound, or a pharmaceutically acceptable salt thereof, in an amount effective to treat the immune dysregulation, wherein the immune dysregulation is cancer.

31. Use of a compound of any one of claims 8-18, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for use in a method of treating an immune dysregulation in a subject in need thereof, the method comprising administering to the subject the compound, or a pharmaceutically acceptable salt thereof, in an amount effective to treat the immune dysregulation, wherein the immune dysregulation is an inflammatory condition.

32. The use of any one of claims 28-31, wherein the method further comprises administering to the subject one or more additional therapeutic agents.

33. The use of claim 32, wherein the one or more additional therapeutic agents are selected from the group consisting of: anti-inflammatory agents, immunotherapeutic agents, and chemotherapeutic agents.

34. The use of claim 32, wherein the one or more additional therapeutic agents are selected from the group consisting of: steroids and therapeutic antibodies.

35. Use of a compound of any one of claims 8-18, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for use in a method of monitoring an immune response in the treatment of a disease in a subject, the method comprising administering to the subject an effective amount of the compound, or a pharmaceutically acceptable salt thereof, and imaging the subject with a suitable imaging technique.

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