Compounds specific to Granzyme B and their use

Abstract

Compounds having the ability to bind to granzyme B and containing a radioactive moiety, such as the compound of formula (I), and pharmaceutical compositions containing the same. The use of the compound and pharmaceutical compositions in the treatment and / or imaging of cancer is also provided herein.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims the benefits of U.S. Provisional Application No. 63 / 506,718, filed on 7 June 2023, which is incorporated herein by reference in its entirety.

[0002] Technical field This disclosure relates to radioactive compounds useful as therapeutic agents, and more particularly to radioactive compounds useful for identifying granzyme B and eliminating cancer cells containing it. [Background technology]

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

[0004] There is a need to develop novel compounds that act as effective granzyme B imaging agents and therapies for treating immunomodulatory disorders such as cancer. [Means for solving the problem]

[0005] This application provides a radioactive compound capable of targeting granzyme B, and its use as a therapeutic agent for treating granzyme B-related diseases such as cancer.

[0006] In some embodiments, the Specified herein provides compounds having the structure of formula (I), or stereoisomers, tautomers, or salts thereof. [ka]

[0007] In formula (I), M represents the radioactive portion; A is the chelating portion that chelates the radioactive portion of M; X is selected from the group consisting of -CH2C(NH)-, -CH2C(O)-, -CH2C(S)-, -NHC(NH)-, -NHC(O)-, -NHC(S)-, -OC(NH)-, -OC(O)-, and -OC(S)-, and arbitrarily, X is either -CH2C(O)- or -NHC(S)-; Y is either CH or N; Z is -CH2-, -CH2C(NH)-, -CH2C(O)-, -CH2C(S)-, -NHC(NH)-, -NHC(O)-, -NHC(S)-, -OC(NH)-, -OC(O)-, or -OC(S)-; optionally, Z is -CH2- or -CH2C(O)-; L is a peptide linker having 1 to 6 amino acid residues (including both ends); R 1 H or C 1~6 It is alkyl, and optionally R 1 is H or methyl; and R 2 C 1~6 Alkyl or C 3~6 It is a cycloalkyl group.

[0008] In some embodiments, the compound is represented by formula (Ia): [ka] That is the case.

[0009] Any of the compounds of formula (I), for example, any of the compounds of formula (Ia), X can be -CH2C(O)-.

[0010] In some cases, the compound is of formula (Ib):

Chemical formula

[0011] Any of the compounds of formula (I) provided herein, for example, any of the compounds of formula (Ia) or formula (Ib), L can be a peptide having 1 to 5 amino acid residues (including 1, 2, 3, 4, or 5). In some cases, L can have one or more non-natural amino acid residues. Exemplary peptides of L are provided below: Glu-Gly-Gly, Glu-βAla-βAla, γGlu, D γGlu, γGlu-βAla, D Glu-βAla-βAla, D Glu-AEA, D Glu-AEEA-AEEA, D Glu- D Glu-AEA, D Glu- D Glu-βAla-βAla, βAla- D Glu-βAla, diacid-βAla-βAla, N-acid-βAla-βAla, or βAla-N-acid-βAla.

[0012] Any of the compounds of formula (I) provided herein, for example, either of formula (Ia) or formula (Ib), the chelating moiety A can be 1,4,7-triazacyclononane-N,N’,N’’-triacetic acid (NOTA), 1,4,7-triazacyclononane-4,7-diyl diacetic acid (NODA), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid (DO3A), a constrained chelator (RESCA), or MACROPA.

[0013] In any of the compounds of formula (I) provided herein, for example, in either the compounds of formula (Ia) or formula (Ib), the radioactive moiety of M may be a therapeutic radioisotope. Examples, but are not limited to, 67 Cu, 90 Y, 177 Lu, 225 Ac, 47 Sc, 131 I, 153 Sm, 161 Tb, 211 At, 212 Pb, 212 Bi, 223 Ra, or 227Th Examples include: 90 It is Y.

[0014] In some embodiments, the chelate portion is NOTA or DOTA in the compound provided herein, and the therapeutic radioisotope is 90 Y, 177 Lu, or 225 It is Ac. In other examples, the chelate portion is NODA, and the therapeutic radioisotope is 47 Sc or 67 It is Cu.

[0015] In some examples, the compounds disclosed herein have the following structure: [ka] (In the formula, M is 177 Lu, 90 Y, 225 Ac, or 213 (Bi) It has.

[0016] In other examples, the compounds disclosed herein have the following structure: [ka] (In the formula, M is 177 Lu, 90 Y,225 Ac, or 213 (Bi) It has.

[0017] Pharmaceutical compositions containing one or more compounds of the aforementioned formula (I) are also provided and fall within the scope of this disclosure.

[0018] In other embodiments, the Disclosure features a method for treating cancer in a subject, the method comprising administering to a subject in need of such treatment a pharmaceutical composition comprising an effective amount of any of the compounds of formula (I) disclosed herein, or similarly disclosed herein.

[0019] In some cases, subjects may be administered an immunotherapy agent before receiving the compound of formula (I). Examples of immunotherapy agents include, but are not limited to, immune checkpoint inhibitors (e.g., PD1 inhibitors such as anti-PD1 antibodies or anti-PD-L1 antibodies) or genetically engineered T cells expressing chimeric antigen receptors (CARs).

[0020] Alternatively, or in addition, an imaging agent may be administered to the subject for imaging of granzyme B.

[0021] In some examples, any of the methods disclosed herein may further include treating the subject with one or more additional therapeutic agents, such as one or more anti-inflammatory agents, steroids, immunotherapeutic agents, and / or chemotherapeutic agents.

[0022] The use of any of the compounds of formula (I) disclosed herein (e.g., compounds of formula (Ia) and formula (Ib)) for use in cancer therapy, as well as the use of any of the compounds of formula (I) for the manufacture of pharmaceuticals for cancer treatment, is also included within the scope of this disclosure.

[0023] Various aspects and embodiments are described in more detail below. Such aspects and embodiments can take many different forms, and the examples disclosed herein should not be construed as limiting; rather, these embodiments are provided to ensure that the disclosure is thorough and complete and that its scope is fully conveyed to those skilled in the art. [Modes for carrying out the invention]

[0024] Cancer immunotherapy has shown remarkable progress in cancer treatment in recent years. Antibodies directed at immune checkpoints such as programmed cell death protein 1 (PD-1) and cytotoxic T lymphocyte-associated protein 4 (CTLA-4) have been approved with positive results in some patients. Research in the field of immuno-oncology continues, and strategies include CAR-T cells, vaccines, small molecules, and antibodies under development. Despite the promise of these therapies, they are not panaceas. These immunotherapies can be associated with significant adverse events, and response rates are typically 20–50%. In other words, the majority of patients do not respond to therapy. Furthermore, determining an individual patient's response to therapy can be challenging using conventional methods, as responses are often associated with immune cell infiltration, which may appear to cause tumor growth on anatomical imaging (e.g., CT, MRI) or demonstrate increased binding affinity on FDG-PET imaging due to the influx of metabolically active immune cells. Given the limitations of current imaging technologies, clinical trials of cancer immunotherapy typically utilize overall survival as the trial endpoint, as opposed to progression-free survival.

[0025] Theranostics refer to a combination of diagnostic imaging and therapy for diagnosing and treating a target disease. Generally, the agents used in theranostic therapy include diagnostic imaging compounds and radiotherapeutic compounds. In some cases, the diagnostic imaging compound and the therapeutic compound may be the same molecule except for different radiolabels, one nuclide for imaging purposes and one nuclide for treatment purposes. In some cases, the diagnostic imaging compound and the therapeutic compound may have the same pharmacophore or different elements bound to the molecule, each with different radioisotopes, for imaging and treatment purposes.

[0026] Granzyme B, a downstream marker of cytotoxic T cell activity, may function as a novel biomarker for evaluating the efficacy of cancer immunotherapy. Granzyme B expression in tumors can be evaluated not only as a measure of the presence or absence of CTLs, but also as an effector protein released by active T cells, thus compensating for the challenge of T cell exhaustion that makes it difficult to achieve evaluation of the presence of CTLs. This disclosure provides certain specific compounds capable of binding to granzyme B (GZB), such as compounds of formula (I), such as compounds of formula (Ia) and formula (Ib), which exhibit high binding affinity to granzyme B. Such compounds may have a radioactive moiety, such as a radioisotope for therapeutic use. Such compounds have exhibited excellent features, including a good metabolic profile with little or no uptake into the gastrointestinal tract and complete renal clearance without the formation of metabolites. See International Patent Application PCT / US2022 / 081098. The relevant disclosures are incorporated by reference to the subject matter and purposes referred to herein.

[0027] Therefore, this specification provides granzyme B-targeted theranostic therapy for treating cancer in subjects requiring treatment. The theranostic therapy comprises a granzyme B-targeted therapeutic compound containing a therapeutic radioisotope disclosed herein, and optionally a granzyme B-targeted imaging compound. In some cases, the granzyme B-targeted therapeutic compound and the granzyme B-targeted imaging compound may be the same molecule loaded with different types of radioactive moieties, e.g., therapeutic radioisotope versus imaging radioisotope. In some embodiments, the subject of treatment with granzyme B-targeted theranostic therapy may have previously received immunotherapy, or is currently receiving immunotherapy. In some embodiments, the theranostic therapy disclosed herein may be administered to the subject concurrently with immunotherapy.

[0028] definition It should be understood that the technical terms used herein are for the purpose of describing specific embodiments and are not intended to limit them. Any methods, devices, and materials similar or equivalent to those described herein may be used in carrying out or testing the present invention, but preferred methods, devices, and materials are described herein. In addition to the foregoing, where used herein and in the appended claims, the following terms have the meanings indicated below unless otherwise specified:

[0029] "Amino" refers to the -NH2 group.

[0030] "Cyano" refers to the -CN group.

[0031] "Hydroxyl" refers to the -OH group.

[0032] "Imino" refers to the =NH substituent.

[0033] "Nitro" refers to the -NO2 group.

[0034] "Oxo" refers to an =O substituent.

[0035] "Thioxo" refers to the =S substituent.

[0036] "Trifluoromethyl" refers to the -CF3 group.

[0037] "Alkyl" refers to a linear, saturated, acyclic, monovalent hydrocarbon group or a branched, saturated, acyclic, monovalent hydrocarbon group having 1 to 6 carbon atoms and bonded to the remainder of the molecule by a single bond, such as methyl, ethyl, n-propyl, 1-methylethyl (iso-propyl), n-butyl, n-pentyl, 1,1-dimethylethyl (t-butyl), 3-methylpentyl-l,2-methylpentyl, etc. The alkyl portion does not have to be substituted. Alternatively, the alkyl portion may be optionally substituted. Optionally substituted alkyl groups include halo, cyano, nitro, aryl, cycloalkyl, heterocyclyl, heteroaryl, oxo, trimethylsilanyl, -OR 3 ,-OC(O)-R 3 , -N(R 3 )2, -C(O)R 4 , -C(O)OR 3 ,-C(O)N(R 3 )2, -N(R 3 )C(O)OR 5 , -N(R 3 )C(O)R 5 , -N(R 3 )S(O) t R 5 (wherein t is 1 or 2), -S(O) t Ure 5 (wherein t is 1 or 2), -S(O) p R 5 (wherein p is 0, 1, or 2) and -S(O) t N(R 3 )2 (wherein t is 1 or 2) (wherein each R 3 R is independently hydrogen, alkyl, haloalkyl, cycloalkyl, cycloalkylalkyl, aryl, aralkyl, heterocyclyl, or heteroaryl; each R 4R is independently hydrogen, cycloalkyl, aryl, heterocyclyl, or heteroaryl; each R 5 A alkyl group is substituted with 1, 2, 3, 4, or 5 substituents independently selected from the group consisting of alkyl, haloalkyl, cycloalkyl, aryl, heterocyclyl, or heteroaryl, as long as the valency is acceptable.

[0038] "Cycloalkyl" refers to a stable, non-aromatic monocyclic or polycyclic hydrocarbon group having 3 to 15 carbon atoms, preferably 3 to 10 carbon atoms, being saturated or unsaturated, and bonded to the remainder of the molecule by a single bond. Polycyclic hydrocarbon groups are bicyclic, tricyclic, or tetracyclic systems. Unsaturated cycloalkyls contain one, two, or three carbon-carbon double bonds and / or one carbon-carbon triple bond. Examples of monocyclic cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. Examples of polycyclic cycloalkyl groups include adamantyl, norbornyl, and dekalinyl. The cycloalkyl moiety may not be substituted. Alternatively, the cycloalkyl moiety may be optionally substituted. Optionally substituted cycloalkyls include alkyl, alkenyl, halo, haloalkyl, haloalkenyl, cyano, nitro, oxo, aryl, aralkyl, cycloalkyl, heterocyclyl, heteroaryl, and -R. 4 -OR 3 ,-R 4 -OC(O)-R 3 ,-R 4 -N(R 3 )2, -R 5 -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 4 ,-R 4 -N(R 3 )C(O)R 5 ,-R 4-N(R 3 )S(O) t R 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)(where each R 3 is, independently, hydrogen, alkyl, haloalkyl, cycloalkyl, aryl, heterocyclyl, or heteroaryl; each R 4 is, independently, a direct bond or a linear or branched alkylene chain or alkenylene chain; each R 5 is, independently, alkyl, haloalkyl, cycloalkyl, cycloalkylalkyl, aryl, aralkyl, heterocyclyl, or heteroaryl) and is a cycloalkyl group optionally substituted by 1, 2, 3, 4, or 5 substituents independently selected from the group consisting of).

[0039] The "chelating moiety" is a molecule or ion that can act as a polydentate ligand towards metal ions. For example, a molecule having a plurality of atoms with available lone pairs (including, but not limited to, nitrogen and oxygen) can act as a chelating moiety. The chelating moiety can be linear (e.g., EDTA) or cyclic (macrocyclic, including, for example, DOTA, porphyrin) and can include macrocycles generally known in the art. The chelating moiety may have 2, 3, 4, 5, or 6 functional groups (e.g., amines, amides, hydroxyls, carboxylic acids, etc.) with available lone pairs for coordinating to the metal.

[0040] In some embodiments, the preparation of the compounds involves the addition of an acid or a base and can affect, for example, the catalysis of the desired reaction or the formation of salt forms such as acid addition salts.

[0041] Exemplary acids may be inorganic or organic acids, and may include strong and weak acids, but are not limited to these. Some examples of 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 acetic acid, propionic acid, butanoic acid, benzoic acid, tartaric acid, pentanoic acid, hexanoic acid, heptanoic acid, octanoic acid, nonanoic acid, and decanoic acid.

[0042] Examples of bases include lithium hydroxide, sodium hydroxide, potassium hydroxide, lithium carbonate, sodium carbonate, potassium carbonate, and sodium bicarbonate. Some examples of strong bases, but not limited to, include hydroxides, alkoxides, metal amides, metal hydrides, metal dialkylamides, and arylamines, where alkoxides include lithium, sodium, and potassium salts of methyl, ethyl, and t-butyl oxides; metal amides include sodium amide, potassium amide, and lithium amide; metal hydrides include sodium hydride, potassium hydride, and lithium hydride; and metal dialkylamides include lithium, sodium, and potassium salts of methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, trimethylsilyl, and cyclohexyl-substituted amides.

[0043] As used herein, the term “pharmaceutically acceptable salt” refers to a derivative of a compound of the present disclosure obtained by modifying the parent compound by converting an existing acidic or base moiety to its salt form. Examples of pharmaceutically acceptable salts include, but are not limited to, inorganic or organic acid salts of basic residues such as amines; and alkali or organic salts of acidic residues such as carboxylic acids. Examples of pharmaceutically acceptable salts of the present application include conventional non-toxic salts of parent compounds formed from non-toxic inorganic or organic acids. Pharmaceutically acceptable salts of the present application can be synthesized from parent compounds containing a basic or acidic moiety by conventional chemical methods. Generally, such salts can be prepared by reacting the free acidic or free base form of these compounds with a stoichiometric amount of a suitable base or acid in water, an organic solvent, or a mixture thereof, generally preferred in aqueous media such as ether, ethyl acetate, alcohol (e.g., methanol, ethanol, iso-propanol, or butanol), or acetonitrile (MeCN). A list of preferred salts can be found in Remington's Pharmaceutical Sciences, 17 th This can be found in ed., Mack Publishing Company, Easton, Pa., 1985, p. 1418 and Journal of Pharmaceutical Science, 66, 2 (1977). Conventional methods for preparing salt forms are described, for example, in Handbook of Pharmaceutical Salts: Properties, Selection, and Use, Wiley-VCH, 2002.

[0044] In some embodiments, the compounds or salts thereof provided herein are substantially isolated. "Substantially isolated" means that the compound is at least partially or substantially separated from the environment in which it was formed or detected. Partial isolation may include, for example, a composition in which the compounds provided herein are concentrated. Substantial isolation may include a composition containing at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 97%, or at least about 99% of the compounds or salts thereof provided herein. Methods for isolating compounds and salts thereof are routinely performed in the art.

[0045] As used herein, the terms “ambient temperature” and “room temperature” or “rt” are understood in the art and generally refer to temperature, such as reaction temperature, i.e., the room temperature in which the reaction takes place, for example, about 20°C to about 30°C.

[0046] I. Compounds for targeting Granzyme B In some embodiments, compounds for targeting granzyme B, such as the compound of formula (I), are provided herein. The compounds disclosed herein include the compounds themselves, pharmaceutically acceptable salts thereof, and stereoisomers thereof.

[0047] The compounds described herein may contain one or more chiral centers and thus may exist in various isomeric forms, such as enantiomers and / or diastereomers. For example, the compounds described herein may be in the form of individual enantiomers, diastereomers or geometric isomers, or in the form of a mixture of stereoisomers, including racemic mixtures and mixtures rich in one or more stereoisomers. The isomers may be isolated from the mixture by methods known to those skilled in the art, including chiral high-performance liquid chromatography (HPLC) and the formation and crystallization of chiral salts; or preferred isomers may be prepared by asymmetric synthesis. See, for example, Jacques et al., Enantiomers, Racemates and Resolutions (Wiley Interscience, New York, 1981); Wilen et al., Tetrahedron 33:2725 (1977); Eliel, Stereochemistry of Carbon Compounds (McGraw-Hill, NY, 1962); and Wilen, Tables of Resolving Agents and Optical Resolutions p. 268 (ELEliel, Ed., Univ. of Notre Dame Press, Notre Dame, IN 1972). This disclosure further encompasses the compounds described herein as individual isomers substantially free from other isomers, and as mixtures of various isomers.

[0048] A. Compounds of formula (I) The GZB targeting compounds provided herein are of formula (I): [ka] Or it may have the structure of its stereoisomer, tautomer, or salt. In formula (I), M is the radioactive moiety; A is the chelating moiety that chelates the radioactive moiety; X is -CH2C(NH)-, -CH2C(O)-, -CH2C(S)-, -NHC(NH)-, -NHC(O)-, -NHC(S)-, -OC(NH)-, -OC(O)-, or -OC(S)-; Y is CH or N; Z is -CH2-, -CH2C(NH)-, -CH2C(O)-, -CH2C(S)-, -NHC(NH)-, -NHC(O)-, -NHC(S)-, -OC(NH)-, -OC(O)-, and -OC(S)-; L is a peptide linker having 1 to 6 amino acid residues (including both ends); R 1 H or C 1~6 Alkyl; R 2 C 1~6 Alkyl or C 3~6 It is a cycloalkyl group.

[0049] In some embodiments, R 1 In other embodiments, R 1 C 1~6 It is alkyl. In other embodiments, R 1 is a C1 alkyl group. In other embodiments, R 1 is a C2 alkyl group. In other embodiments, R 1 is a C3 alkyl group. In other embodiments, R 1 is a C4 alkyl group. In other embodiments, R 1 is a C5 alkyl group. In other embodiments, R 1 is a C6 alkyl group. In some specific embodiments, R 1 It is methyl (-CH3).

[0050] In some embodiments, R 2 C 1~6 It is alkyl. In some specific embodiments, R 2 C 3~6 It is alkyl. For example, R 2 C 3~6 Alkyl can be branched or unbranched, substituted or unsubstituted C 3~6It is alkyl. In some embodiments, R 2 is a C3 alkyl group. In some embodiments, R 2 is a C4 alkyl group. In some embodiments, R 2 is a C5 alkyl group. In some embodiments, R 2 is a C6 alkyl group. In some embodiments, R 2 It is sec-butyl (-CH(CH3)CH2CH3).

[0051] In some embodiments, R 2 C 3~6 It is cycloalkyl. In some embodiments, R 2 C 3~6 Cycloalkyls include cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl.

[0052] In one embodiment, the compound is of formula (Ia): [ka] It has the structure of [the object].

[0053] In another embodiment, the compound is of formula (Ib): [ka] It has the structure of [the object].

[0054] In some cases, X is -CH2C(NH)-. In some cases, X is -CH2C(O)-. In some cases, X is -CH2C(S)-. In some cases, X is -NHC(NH)-. In some cases, X is -NHC(O)-. In some cases, X is -NHC(S)-. In some cases, X is -OC(NH)-. In some cases, X is -OC(O)-. In some cases, X is -OC(S)-. In a specific example, X is -CH2C(O)-.

[0055] In some cases, Z is -CH2-. In some cases, Z is -CH2C(NH)-. In some cases, Z is -CH2C(O)-. In some cases, Z is -CH2C(S)-. In some cases, Z is -NHC(NH)-. In some cases, Z is -NHC(O)-. In some cases, Z is -NHC(S)-. In some cases, Z is -OC(NH)-. In some cases, Z is -OC(O)-. In some cases, Z is -OC(S)-.

[0056] In any of the compounds of formula (I), for example, any of the compounds of formula (Ia) and formula (9Ib), L is a peptide linker having 1 to 6 amino acid residues (including both ends). In some embodiments, L has 1 to 5 amino acid residues (including both ends). In some embodiments, L has 2 to 4 amino acid residues (including both ends). In some examples, L has 1 amino acid residue. In some examples, L has 2 amino acid residues. In some examples, L has 3 amino acid residues. In some examples, L has 4 amino acid residues. In some examples, L has 5 amino acid residues. In some examples, L has 6 amino acid residues.

[0057] In some embodiments, any amino acid residue in the L-peptide may be a standard proteinogenic amino acid (i.e., one of the 20 naturally occurring amino acid residues found in natural proteins), or a non-natural amino acid which may be a derivative of a natural protein or an isomer of a natural amino acid residue. As used herein, proteinogenic amino acid residues refer to the 20 naturally occurring amino acid residues which are building blocks for protein synthesis. The amino acid residues may form a chain via standard peptide bonds or by forming amide bonds with compatible side chains (e.g., glutamic acid (e.g., D-Glu), aspartic acid). Exemplary structures of non-natural amino acid residues that may be included in the L-linker are provided in Table 1 below.

[0058] [Table 1]

[0059] Exemplary amino acid sequences of L peptides include Glu-Gly-Gly, Glu-βAla-βAla, γGlu, D γGlu, γGlu-βAla, D Glu-βAla-βAla, D Glu-AEA, D Glu-AEEA-AEEA, D Glu- D Glu-AEA, D Glu- D Glu-βAla-βAla, βAla- D Examples include Glu-βAla, diacid-βAla-βAla, N-acid-βAla-βAla, and βAla-N-acid-βAla. In some embodiments, L has a Glu-Gly-Gly sequence. In some embodiments, L has a Glu-βAla-βAla sequence. In some embodiments, L has a γGlu sequence. In some embodiments, L is D It has a γGlu sequence. In some embodiments, L has a γGlu-βAla sequence. In some embodiments, L is D It has a Glu-βAla-βAla sequence. In some embodiments, L is D It has a Glu-AEA sequence. In some embodiments, L is D It has a Glu-AEEA-AEEA sequence. In some embodiments, L is D Glu- D It has a Glu-AEA sequence. In some embodiments, L is D Glu- D It has a Glu-βAla-βAla sequence. In some embodiments, L is βAla- DIt has a Glu-βAla sequence. In some embodiments, L has a diacid-βAla-βAla sequence. In some embodiments, L has an N-acid-βAla-βAla sequence. In some embodiments, L has a βAla-N-acid-βAla sequence. See Table 2 for the structures of these exemplary L linkers.

[0060] [Table 2]

[0061] [Table 3]

[0062] [Table 4]

[0063] In some embodiments, Y is CH forming a piperidine ring. In other embodiments, Y is N forming a piperazine ring.

[0064] In some specific examples, Z is -CH2-. In other specific examples, Z is -CH2C(O)-.

[0065] In some embodiments, the compounds of formula (I) provided herein may have one of the following structures of formula (Ib-A) or (Ib-B) (where variables A, L, and M are as defined herein): [ka]

[0066] In formula (I), A is the chelating moiety. The chelating moiety is a molecule or ion that can act as a polydentate ligand for a metal ion. For example, a molecule having multiple atoms with available lone pairs of electrons (including, but not limited to, nitrogen and oxygen) can act as a chelating moiety. The chelating moiety can be linear (e.g., EDTA) or cyclic (macroring, including DOTA and porphyrins), and may include macrorings commonly known in the art. The chelating moiety may have 2, 3, 4, 5, or 6 functional groups (e.g., amines, amides, hydroxyls, carboxylic acids, etc.) with lone pairs of electrons available to coordinate to the metal. Examples of chelating moieties for use in the granzyme B targeting compounds disclosed herein include, but are not limited to, 1,4,7-triazacyclononane triacetic acid (NOTA), 2-S-(4-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid (p-SCN-Bm-NOTA), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), 1,4,7-triazacyclononane-l-glutaric acid-4,7-diacetic acid (NODAGA), ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), and cyclohexyl-l,2-diaminetetraacetic acid (CDTA). Examples include ethylene glycol-0,0'-bis(2-aminoethyl)-N,N,N',N'-tetraacetic acid (EGTA), N,N-bis(hydroxybenzyl)-ethylenediamine-N,N'-diacetic acid (HBED), triethylenetetramine hexaacetic acid (TTHA), hydroxyethyldiamine triacetic acid (HEDTA), 1,4,8,11-tetraazacyclotetradecane-N,N',N”,N”'-tetraacetic acid (TETA), 1,4,7,10-tetraaza-1,4,7,10-tetra-(2-carbamoylmethyl)-cyclododecane (TCMC), 1,4,7-triazacyclononane-4,7-diyldiacetic acid (NODA), and desferrioxamine B (DFO).In some embodiments, the chelating agent is selected from the group consisting of 1,4,7-triazacyclononane triacetic acid (NOTA), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), 1,4,7-triazacyclononane-4,7-diyldiacetic acid (NODA), 1,4,7-triazacyclononane-1-glutaric acid-4,7-diacetic acid (NODAGA), a restraining complexing agent (RESCA), and MACROPA. In some embodiments, the chelating agent is 1,4,7-triazacyclononane triacetic acid (NOTA). In other embodiments, the chelating agent is 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA).

[0067] In one embodiment, the chelating portion A is 1,4,7-triazacyclononane-N,N',N''-triacetic acid (NOTA) or 1,4,7-triazacyclononane-4,7-diyldiacetic acid (NODA). In some embodiments, the chelating portion A is 1,4,7-triazacyclononane-N,N',N''-triacetic acid (NOTA). In some embodiments, 1,4,7-triazacyclononane-N,N',N''-triacetic acid (NOTA) of A has the following structure: [ka]

[0068] In some embodiments, the chelate portion A is 1,4,7-triazacyclononane-4,7-diyldiacetic acid (NODA). In some embodiments, 1,4,7-triazacyclononane-4,7-diyldiacetic acid (NODA) of A has the following structure: [ka]

[0069] In some embodiments, the chelating portion A is 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) having the following structure: [ka] In some embodiments, the chelating portion A is 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid (DO3A) having the following structure: [ka]

[0070] The selection of a suitable chelating agent to pair with a therapeutic radioisotope may follow conventional methods (see, for example, Sgouros et al., Nature Reviews, 19:589-608, 2020 and Poty et al., J. Nuclear Medicine, 59 (6): 878-884; 2018 (the relevant disclosures of each thereof are incorporated by reference for the subject matter and purposes referred herein)) or the guidance provided herein. In some cases, the compound of formula (I) may have pairs of chelating agents and therapeutic radioisotopes listed in Table 3 below. Such chelating agent-therapeutic radioisotope pairs may result in high loading efficiency of the therapeutic radioisotope.

[0071] [Table 5]

[0072] In some embodiments, the radioactive moiety of M in any of the compounds of formula (I) disclosed herein (e.g., compounds of formula (Ia) or compounds of formula (Ib), such as formula (Ib-A) or formula (Ib-B)) can be a therapeutic radioisotope. In some embodiments, the therapeutic radioisotope of M is 67 It is Cu. In some embodiments, the therapeutic radioactive isotope of M is 90 Y is the therapeutic radioactive isotope of M. 177 It is Lu. In some embodiments, the therapeutic radioactive isotope of M is 225It is Ac. In some embodiments, the therapeutic radioactive isotope of M is 47 It is Sc. In some embodiments, the therapeutic radioactive isotope of M is 131 I is the therapeutic radioactive isotope of M. 153 It is Sm. In some embodiments, the therapeutic radioactive isotope of M is 161 It is Tb. In some embodiments, the therapeutic radioactive isotope of M is 211 At. In some embodiments, the therapeutic radioactive isotope of M is 212 It is Pb. In some embodiments, the therapeutic radioactive isotope of M is 212 It is Bi. In some embodiments, the therapeutic radioactive isotope of M is 223 It is Ra. In some embodiments, the therapeutic radioactive isotope of M is 227 Th is the chelate portion in some specific embodiments, where the therapeutic radioisotope is NOTA or DOTA. 90 Y, 177 Lu, or 225 It is Ac. In some other embodiments, the chelater portion is NODA, and the therapeutic radioisotope is 47 Sc or 67 It is Cu.

[0073] Exemplary compounds of formula (I) are provided in Table 4 below.

[0074] [Table 6]

[0075] [Table 7]

[0076] This application also includes stereoisomers of compounds such as the stereoisomers of compounds 1-M and 4-M described herein and disclosed in Table 5 below. Stereoisomers arise from two chiral centers (ring-closed) or one chiral center (open chain) of the hemiacetal unit, as indicated by the wavy lines: [ka]

[0077] [Table 8]

[0078] [Table 9]

[0079] [Table 10]

[0080] [Table 11]

[0081] Exemplary compounds of formula Ib-B are disclosed in Table 6.

[0082] [Table 12]

[0083] [Table 13]

[0084] [Table 14]

[0085] [Table 15]

[0086] As shown in the following examples, the GZB-binding compounds disclosed herein, comprising either a piperidine ring or a piperazine ring linking the peptide linker and the chelating moiety, as well as a specific peptide linker structure, have demonstrated better in vivo binding to GZB and clearance profiles compared to other GZB-binding compounds. See International Application PCT / US2022 / 081125 and International Publication 2021 / 252644, the relevant disclosures of which are incorporated by reference to the subject matter and purposes referred herein.

[0087] Exemplary improved characteristics include improved pharmacokinetics (e.g., renal clearance), pharmacodynamics, and efficacy. In particular, improved pharmacokinetics can be seen in the absence of uptake into the gastrointestinal tract, the absence of radioactive metabolites in the urine, and / or superior renal clearance. See International Application PCT / US2022 / 081125.

[0088] The compounds described above, when containing radioisotopes such as therapeutic radioisotopes, are useful as theranostic agents in one or more of the methods provided herein for treating GZB-related diseases such as cancer. As noted above, this application also includes pharmaceutically acceptable salts of the compounds described herein. The term “pharmaceutically acceptable” is used herein to mean a compound, material, composition, and / or dosage form that is suitable for use in contact with human and animal tissues within the bounds of sound medical judgment, in proportion to a reasonable benefit-risk ratio, without excessive toxicity, irritation, allergic reactions, or other problems or complications.

[0089] Chemical synthesis of B. granzyme B targeting compounds As you may see, the compounds provided herein, including their stereoisomers and salts, can be prepared using known organic synthesis techniques and can also be synthesized according to any of a number of possible synthetic routes.

[0090] Compounds disclosed herein, or pharmaceutically acceptable salts thereof, can be prepared by following the exemplary protocols described below. Suitable protecting groups for use in such synthesis are known in the art. See, for example, McOmie, Protective Groups in Organic Chemistry, (1973):98.

[0091] General synthetic procedures and examples for the preparation of the peptide linker L, the Fmoc-Haic(2S,5S)-OH tricyclic moiety, and appropriate metal complexation with the chelate moiety can be found in International Application PCT / US2021 / 036661, filed on 9 June 2021, the relevant disclosures thereof are incorporated by reference to the subject matter and purposes referred herein.

[0092] Many suitable radioactive moieties (e.g., radioisotopes, e.g., therapeutic radioisotopes, e.g., those disclosed herein) are known in the art (see, for example, U.S. Patents 5,021,236; 4,938,948; and 4,472,509, each of which disclosures are incorporated herein by reference in whole). The radioconjugate compounds or pharmaceutically acceptable salts thereof provided herein can be prepared according to methods well known in the art. Synthetic methods for incorporating radioisotopes into organic compounds are well known in the art, and other methods applicable to the compounds provided herein would be readily apparent to those skilled in the art.

[0093] Those skilled in the art will recognize that the processes described herein are not exclusive means of synthesizing the compounds provided herein, and that a broad repertoire of synthetic organic reactions is available for potentially synthesizing the compounds provided herein. How to select and implement an appropriate synthetic route is well known to those skilled in the art. Preferred synthesis methods for starting materials, intermediates, and products can be identified by reference to the following sources, for example: Advances in Heterocyclic Chemistry, Vols. 1-107 (Elsevier, 1963-2012); Journal of Heterocyclic Chemistry Vols. 1-49 (Journal of Heterocyclic Chemistry, 1964-2012); Carreira, et al. (Ed.) Science of Synthesis, Vols. 1-48 (2001-2010) and Knowledge Updates KU2010 / 1-4; 2011 / 1-4; 2012 / 1-2 (Thieme, 2001-2012); Katritzky, et al. (Ed.) Comprehensive Organic Functional Group Transformations, (Pergamon Press, 1996); Katritzky et al. (Ed.); Comprehensive Organic Functional Group Transformations II (Elsevier, 2 nd Edition, 2004);Katritzky et al.(Ed.), Comprehensive Heterocyclic Chemistry (Pergamon Press, 1984);Katritzky et al., Comprehensive Heterocyclic Chemistry II, (Pergamon Press, 1996);Smith et al., March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, 6 thEd. (Wiley, 2007); Trost et al. (Ed.), Comprehensive Organic Synthesis (Pergamon Press, 1991).

[0094] The reactions for preparing the compounds described herein can be carried out in suitable solvents readily selectable by those skilled in the art of organic synthesis. Suitable solvents can be substantially inactive with respect to the starting materials (reactants), intermediates, or products at the reaction temperature (for example, a temperature that can range from the freezing temperature to the boiling temperature of the solvent). A given reaction can be carried out in one solvent or a mixture of two or more solvents. Depending on the specific reaction step, a solvent suitable for that particular step can be selected by those skilled in the art.

[0095] The preparation of the compounds described herein may 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 those skilled in the art. The chemistry of protecting groups can be found, for example, in TW Greene and PGM Wuts, Protective Groups in Organic Synthesis, 3. rd This can be found in Ed., Wiley & Sons, Inc., New York (1999).

[0096] The reaction can be monitored according to any preferred method known in the art. For example, product formation can be monitored by nuclear magnetic resonance spectroscopy (e.g., 1 H or 13 C) The compounds can be monitored by spectroscopic means such as infrared spectroscopy and spectrophotometric methods (e.g., UV-Vis), by mass spectrometry, or by chromatographic methods such as high-performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LCMS), and thin-layer chromatography (TLC). The compounds can be purified by those skilled in the art by various methods, including high-performance liquid chromatography (HPLC) and normal-phase silica chromatography.

[0097] II. Pharmaceutical Compositions Any compound of formula (I), such as the compound of formula (Ia) or the compounds of formula (Ib), such as formula (Ib-A) or formula (Ib-B), or any pharmaceutically acceptable salt thereof, may be mixed with a pharmaceutically acceptable carrier to form a pharmaceutical composition, for example, for therapeutic purposes as disclosed herein. In some embodiments, pharmaceutical compositions comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof as an active ingredient in combination with one or more pharmaceutically acceptable carriers (excipients) are provided herein. "Acceptable" means that the carrier is compatible with the active ingredient of the composition (preferably has the ability to stabilize the active ingredient) and is not harmful to the subject being treated. Suitable carriers include microcrystalline cellulose, mannitol, glucose, skim milk powder, polyvinylpyrrolidone, and starch, or combinations thereof.

[0098] Some examples of suitable excipients include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, and methylcellulose. Pharmaceutical formulations may additionally contain, but are not limited to, lubricants such as talc, magnesium stearate, and mineral oil; humectants; emulsifiers and suspending agents; preservatives such as methyl and propyl hydroxybenzoates; sweeteners; flavoring agents; or combinations thereof. For further information regarding acceptable pharmaceutical compositions, see Remington's Pharmaceutical Sciences, 17 th See ed., Mack Publishing Company, Easton, Pa., 1985, p. 1418.

[0099] The pharmaceutical composition may be administered to a subject using conventional methods known to those skilled in the art of pharmaceuticals, depending on the type of disease being treated or the site of the disease. The composition may also be administered via other conventional routes, such as orally, parenterally, inhaled spray, topically, rectally, nasally, buccally, vaginally, or via an implanted reservoir. The term "parenterally," as used herein, includes subcutaneous, intradermal, intravenous, intramuscular, intra-articular, intra-arterial, intra-synovial, intrasternal, intrathecal, intralesional, and intracranial injection or infusion techniques. Parenteral administration may be in the form of a single bolus dose or, for example, by a continuous perfusion pump. In addition, it may be administered to a subject via an injectable depot administration route, such as by using depot-injectable or biodegradable materials and methods for 1 month, 3 months, or 6 months.

[0100] The injectable composition may contain various carriers, such as vegetable oil, dimethylacetamide, dimethylformamide, ethyl lactate, ethyl carbonate, isopropyl myristate, ethanol, and polyols (glycerol, propylene glycol, liquid polyethylene glycol, etc.). For intravenous injection, water-soluble antibodies can be administered by drip method, thereby injecting a pharmaceutical preparation containing the antibody and physiologically acceptable excipients. Examples of physiologically acceptable excipients include 5% dextrose, 0.9% saline, Ringer's solution, or other suitable excipients. Intramuscular preparations, such as sterile preparations in a suitable soluble salt form of the antibody, can be administered dissolved in pharmaceutical excipients such as water for injection, 0.9% saline, or 5% glucose solution.

[0101] For oral administration, the composition may take the form of tablets or capsules prepared by conventional means using acceptable excipients such as binders (e.g., pre-gelatinized corn starch, polyvinylpyrrolidone, or hydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystalline cellulose, or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc, or silica); disintegrants (e.g., potato starch or sodium starch glycolate); and wetting agents (e.g., sodium lauryl sulfate). The tablets may be coated by methods well known in the art.

[0102] In some embodiments, the compounds or pharmaceutically acceptable salts thereof provided herein are suitable for parenteral administration. In some embodiments, the compounds or pharmaceutically acceptable salts thereof are suitable for intravenous administration.

[0103] Pharmaceutical compositions and formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids, and powders. Conventional pharmaceutical carriers, aqueous, powdered, or oily bases, and thickeners may be necessary or desirable.

[0104] When preparing the pharmaceutical compositions provided herein, the active ingredient is typically mixed with an excipient, diluted by an excipient, or encapsulated in such a carrier, for example, in the form of a capsule, sachet, paper, or other container. If the excipient acts as a diluent, it may be a solid, semi-solid, or liquid material acting as a vehicle, carrier, or medium for the active ingredient.

[0105] Therefore, pharmaceutical compositions can take the form of tablets, pills, powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols (as solids or in liquid media), ointments, soft and hard gelatin capsules, suppositories, sterile injection solutions, and sterile packaged powders.

[0106] III.How to use Any of the granzyme B-targeted therapeutic compounds of formula (I) disclosed herein can be used in the treatment of granzyme B-related diseases, for example, in cancer therapy.

[0107] To carry out the methods disclosed herein, an effective amount of a pharmaceutical composition containing a compound of formula (I) disclosed herein or a pharmaceutically acceptable salt thereof may be administered to a subject requiring treatment via a suitable route. In some embodiments, such methods may further include administering an effective amount of a granzyme B targeting agent, for example, one of the compounds of formula (I) disclosed herein or a pharmaceutically acceptable salt thereof, to the subject, which may be carried out prior to the administration of the therapeutic agent. In some embodiments, the subject may or may have previously received immunotherapy, for example, one disclosed herein.

[0108] As used herein, the term “Subject” refers to any mammal, such as a mouse, rat, other rodent, rabbit, dog, cat, pig, cattle, sheep, horse, non-human primate, or human. In some embodiments, the subject is human.

[0109] In some embodiments, the subjects are human patients with cancer. In some cases, the cancer is a solid tumor. Examples, but not limited to, include brain cancer, breast cancer (e.g., HER2+, ER+ / PR+ / PR+, HER2-, or triple-negative breast cancer), cervical cancer (e.g., squamous cell carcinoma of the cervix), colorectal cancer, lung cancer (e.g., non-small cell lung cancer or small cell lung cancer), melanoma, bladder cancer, renal cell carcinoma, multiple myeloma, pancreatic cancer, prostate cancer, glioblastoma, hepatocellular carcinoma, and urothelial carcinoma. These include esophageal carcinoma, gastroesophageal carcinoma, gastric cancer, head and neck squamous cell carcinoma, epithelial ovarian carcinoma (EOC), primary peritoneal cancer, Fallopian duct carcinoma, Merkel cell carcinoma, nasopharyngeal carcinoma, adrenocortical carcinoma, meningioma, neuroblastoma, retinoblastoma, osteosarcoma, rhabdomyosarcoma, Ewing's sarcoma, liposarcoma, fibrosarcoma, leiomyosarcoma, peripheral primitive neuroectodermal tumors, vaginal squamous cell carcinoma, and vulvar squamous cell carcinoma. In some cases, the cancer is colon cancer.

[0110] In other embodiments, the cancer is a blood cancer (e.g., leukemia, lymphoma, etc.). Examples include, but are not limited to, pilocytic cell leukemia, Kaposi's sarcoma, follicular lymphoma, chronic myeloid leukemia, cutaneous T-cell lymphoma, peripheral T-cell lymphoma, pre-T-cell lymphocyte leukemia, classical Hodgkin lymphoma, B-cell non-Hodgkin lymphoma, chronic lymphocytic leukemia, acute myeloid leukemia, myelodysplastic syndrome, primary myelofibrosis, post-essential thrombocythemia myelofibrosis, or post-hypercytic myelofibrosis.

[0111] As used herein, “effective dose” refers to the amount of each activator required to impart a therapeutic effect to a subject, either alone or in combination with one or more other activators. Determining whether the amount of antibody has achieved a therapeutic effect will be obvious to those skilled in the art. The effective dose varies, as recognized by those skilled in the art, depending on the detailed pathological condition under treatment, the severity of the condition, parameters including the individual patient’s age, health status, build, sex, and weight, the duration of treatment, the nature of any combination therapy, the specific route of administration, and similar factors within the scope of the knowledge and expertise of healthcare professionals. These factors are well known to those skilled in the art and can be addressed without going beyond routine experimentation. Generally, it is preferable to use the maximum dose of the individual components or their combinations, i.e., the highest safe dose determined by sound medical judgment.

[0112] In some cases, the maximum tolerated dose of a checkpoint inhibitor can be used in the manner disclosed herein. Alternatively, or in addition, any of the therapeutic granzyme B-targeting molecules of formula (I) disclosed herein can be used in the manner thereof.

[0113] Empirical considerations, such as half-life, will generally contribute to the determination of dosage. For example, antibodies compatible with the human immune system, such as humanized or fully human antibodies, may be used to prolong the half-life of the antibody and to prevent it from being attacked by the host immune system. The frequency of administration may be determined and adjusted over the duration of therapy, but is generally not essential, based on the treatment, and / or suppression, and / or recovery, and / or delay of the target disease / disorder. Alternatively, a sustained-release formulation of the antibody may be appropriate. Various formulations and devices for achieving sustained release are known in the art.

[0114] In some embodiments, the dose of one of the above compounds or a pharmaceutically acceptable salt thereof administered to a subject or individual is approximately 1 μg to approximately 2 g, for example, approximately 1 μg to approximately 2 g, approximately 1 μg to approximately 1000 mg, approximately 1 μg to approximately 500 mg, approximately 1 μg to approximately 100 mg, approximately 1 μg to approximately 50 mg, approximately 1 μg to approximately 1 mg, approximately 1 μg to approximately 500 μg, approximately 1 μg to approximately 100 μg, approximately 1 μg to approximately 10 μg, approximately 10 μg to approximately 2 g, for example, approximately 10 μg to approximately 2 g, approximately 10 μg to approximately 1000 mg, approximately 10 μg to approximately 500 mg, approximately 10 μg to approximately 100 mg, approximately 10 μg to approximately 50 mg, approximately 10 μg to approximately 1 mg, approximately 10 μg to approximately 500 μg, approximately 10 μg to approximately 100 μg, about 100μg to about 2g, for example, about 100μg to about 2g, about 100μg to about 1000mg, about 100μg to about 500mg, about 100μg to about 100 mg, about 100 μg to about 50 mg, about 100 μg to about 1 mg, about 100 μg to about 500 μg, about 500 μg to about 2 g, for example, about 500 μg to about 2 g, about 5 The dosages are approximately 00 μg to 1000 mg, approximately 500 μg to 500 mg, approximately 500 μg to 100 mg, approximately 500 μg to 50 mg, approximately 500 μg to 1 mg, approximately 1 mg to 2 g, approximately 1 mg to 1000 mg, approximately 1 mg to 500 mg, approximately 1 mg to 100 mg, approximately 1 mg to 50 mg, or approximately 50 mg to 500 mg.

[0115] As used herein, the terms “to treat” or “treatment” mean (1) inhibiting cancer; for example, inhibiting cancer in an individual experiencing or exhibiting cancer pathology or overall symptoms (i.e., preventing further development of pathology and / or overall symptoms); and (2) restoring cancer; for example, restoring cancer in an individual experiencing or exhibiting cancer pathology or symptoms (i.e., reversing pathology and / or overall symptoms), for example, reducing the severity of cancer or reducing or alleviating one or more symptoms of cancer.

[0116] In some embodiments, subjects can be identified and / or diagnosed as having cancer prior to administration of the therapeutic compound of formula (I). In some cases, subjects having the target cancer can be identified by routine medical examinations, e.g., laboratory tests, organ function tests, CT scans, or ultrasound. In some embodiments, subjects treated by the methods described herein may be human cancer patients who have undergone or are undergoing anti-cancer therapies, e.g., chemotherapy, radiotherapy, immunotherapy, or surgery.

[0117] Human patients subjected to any of the cancer therapies disclosed herein (e.g., theranostic therapies disclosed herein) may have a prior history of immunotherapy or may be subjected to immunotherapy in parallel. Immunotherapy agents generally trigger immune effector cells and molecules to target and destroy cells (e.g., cancer cells). Immune effectors may be, for example, antibodies specific to markers on the surface of cells (e.g., tumor cells). Antibodies may act as effectors of therapy on their own or recruit other cells to cause cell death. Various effector cells include, but are not limited to, cytotoxic T cells and NK cells.

[0118] Examples of immunotherapeutic agents include, but are not limited to, azathioprine, chlorambucil, cyclophosphamide, cyclosporine, daclizumab, infliximab, methotrexate, tacrolimus, immunostimulants (e.g., IL-2, IL-4, IL-12, GM-CSF, tumor necrosis factor; interferon alpha, beta, and gamma; F42K and other cytokine analogs; chemokines, e.g., MIP-1, MIP-1β, MCP-1, RANTES, IL-8; or growth factors, e.g., FLT3 ligand), antigenic peptides, polypeptides or proteins, or autologous or allogeneic tumor cell compositions (e.g., Ravindranath & Morton, International reviews of immunology, 7.4 (1991): See pages 303-329), hormone therapy, adrenocorticosteroids, progestins (e.g., hydroxyprogesterone caproate, medroxyprogesterone acetate, and megestrol acetate), estrogens (e.g., diethylstilbestrol and ethinylestradiol), antiestrogens (e.g., testosterone propionate and fluoxymesterone), antiandrogens (e.g., flutamide), and gonadotropin-releasing hormone analogs (e.g., leuprolide). Further immunotherapies are known in the art and can be found, for example, in Rosenberg et al, New England Journal of Medicine, 319.25 (1988): 1676-1680; and Rosenberg et al, Annals of surgery, 210.4 (1989): 474.

[0119] In some embodiments, the immunotherapy agent is an immune checkpoint inhibitor, such as a PD1 inhibitor (e.g., anti-PD-1 antibodies such as nivolumab, pembrolizumab, or cemiprimab; or PD-L1 antibodies such as atezolizumab, avelumab, or durvalumab), a CTLA-4 inhibitor (e.g., anti-CTLA-4 antibodies such as ipilimumab), or a LAG-3 inhibitor (e.g., anti-LAG-3 antibodies such as relatrimab). In other embodiments, the immunotherapy agent may be CAR-T cells, such as axicapbutagen siloleucel or brexcapbutagen autoleucel.

[0120] The therapeutic agents provided herein may be effective over a wide range of doses and are generally administered at an effective dose. However, it should be understood that the actual amount of therapeutic agent administered is usually determined by a physician in accordance with relevant circumstances, including the condition being imaged, the route of administration selected, the actual compound being administered, the age, weight, and response of the individual subject, and the severity of the subject's symptoms.

[0121] In some embodiments, any of the granzyme B-targeted theranostic compounds can be used in combination with anticancer therapies, such as those disclosed herein, for example, immunotherapy, chemotherapy, etc.

[0122] Alternatively, or in addition, the granzyme B-targeted theranostic compounds disclosed herein may be used in combination with granzyme B imaging agents. Any imaging compound known in the art that targets granzyme B may be used in the theranostic therapies disclosed herein.

[0123] Any granzyme B imaging agent known in the art can be used in the manner disclosed herein. Examples include those disclosed in U.S. Patent No. 11,559,590, International Publication No. 2021 / 252664, International Application No. PCT / US2022 / 081098, and International Application No. PCT / US2022 / 081125 (each of which relevant disclosures are incorporated by reference to the subject matter and purposes referred herein).

[0124] In some embodiments, the granzyme B imaging compound is represented by the following formula (II): [ka] The compound may be a stereoisomer, tautomer, or salt thereof. In formula (II), M is an imaging agent; A is a chelating moiety that chelates the imaging agent; B is an aryl, heteroaryl, cycloalkyl, or heterocyclyl (for example, B is a 6-membered ring); X is -CH2C(NH)-, -CH2C(O)-, -CH2C(S)-, -NHC(NH)-, -NHC(O)-, NHC(S)-, -OC(NH)-, -OC( O)-, or -OC(S)- (for example, -CH2C(O)- or -NHC(S)-; Z is -CH2-, -CH2C(NH)-, -CH2C(O)-, -CH2C(S)-, -NHC(NH)-, -NHC(O)-, -NHC(S)-, -OC(NH)-, -OC(O)-, or -OC(S)-; L is a peptide linker having 1 to 6 amino acid residues (including both ends); R 1 H or C 1~6 It is alkyl, and optionally R 1 is H or methyl; R 2 C 1~6 Alkyl or C 3~6 It is cycloalkyl; and R 3 C 1~6 It is alkyl.

[0125] In some examples, the granzyme B imaging compound is represented by the following formula (II-a): [ka] (In the formula, M, A, X, L, R 1 , R 2 (This is as defined above.) It may have a structure.

[0126] In some cases, the granzyme B imaging compound is represented by the following formula (II-B): [ka] (In the formula, M, Z, A, X, L, R 1 and R 2 (As defined above, Y is either CH or N) It may have a structure.

[0127] In any of the imaging agents disclosed herein, M may be a metal or a metal bound to a radioactive isotope suitable for imaging. Suitable metals for use in this disclosure include metals useful for imaging granzyme B, such as metals that are suitable radioactive imaging agents, and metals that can be bound to nonmetallic radioactive isotopes that are suitable radioactive imaging agents. Exemplary metallic radioactive isotopes are: 68 It is Ga. An example of a nonmetallic radioactive isotope is, 18 F is present, and it can be conjugated with Al for loading into the granzyme B binding compounds disclosed herein.

[0128] IV. Kits for Granzyme B-Targeted Theranostic Therapy Kits for granzyme B-targeted cancer therapy (e.g., pharmaceutical packs) are also included in this disclosure. The kits provided are disclosed in containers (e.g., vials, ampoules, bottles, syringes, and / or dispenser packages, or other suitable containers). In some embodiments, the kits provided may optionally further include a second container containing pharmaceutical excipients for dilution or suspension of the pharmaceutical composition. In some embodiments, the pharmaceutical compositions provided in the first and second containers are combined to form a single unit dosage form. In some embodiments, the kit may include an additional container containing one or more additional therapeutic agents disclosed herein, e.g., immunotherapeutic agents. In some embodiments, the kit may include a diagnostic imaging compound or a pharmaceutical composition thereof, and a therapeutic compound or a pharmaceutical composition thereof.

[0129] In certain embodiments, the kit described herein further includes instructions for using the compounds, drugs, and compositions contained in the kit. The kit described herein may also include information required by regulatory agencies such as the U.S. Food and Drug Administration (FDA). In certain embodiments, the information included in the kit is prescription information. In certain embodiments, the kit and instructions provide imaging and / or treatment of cancer and / or reduction of cancer risk of granzyme B in the target population. The kit described herein may include one or more additional pharmaceutical agents described herein as separate compositions.

[0130] Based on the above description, it is likely that those skilled in the art can make the most of the present invention without further detail. Therefore, the specific embodiments described below should be construed as merely illustrative and not limit the remainder of this disclosure in any way. All publications referenced herein are incorporated by reference for the purposes or subject matter referred to herein. [Examples]

[0131] Example 1: Synthesis of an exemplary compound of formula (I') This example provides an exemplary synthetic method for producing a precursor compound of formula (I') that lacks the radioactive moiety compared to the corresponding compound of formula (I).

[0132] Preparation of coupling partners for the chelation portion [ka] 2-(4-((4,7-bis(2-(tert-butoxy)-2-oxoethyl)-1,4,7-triazonan-1-yl)methyl)piperidine-1-yl)acetic acid [ka] Step 1: To a solution of piperidine-4-ylmethanol (90.0 g, 781 mmol) in MeCN (540 mL), benzyl 2-bromoacetate (179 g, 781 mmol, 123 mL) and K2CO3 (162 g, 1.17 mol) were added. The mixture was stirred at 20°C for 12 hours. TLC (petroleum ether:ethyl acetate = 0:1, R f A reading of 0.20 indicated that the starting material had been completely consumed. The mixture was filtered, and the filtrate was concentrated under vacuum. The residue was purified by column chromatography (SiO2, petroleum ether:ethyl acetate = 50:1 to 0:1). 2-(4-(hydroxymethyl)piperidine-1-yl)benzyl acetate (92.0 g, 349 mmol) was obtained as a yellow oil. 1 H NMR:(400MHz,CDCl3)δ7.35-7.37(m,5H),5.16(s,2H),3.49(d,J=6.4Hz,2H),3.26(s,2H),2.95(d,J=11 .2Hz,2H),2.16-2.22(m,2H),1.72(d,J=13.2Hz,2H),1.59(s,1H),1.49-1.55(m,1H),1.30-1.40(m,2H).

[0133] [ka] Step 2: To a solution of oxalyl chloride (88.7 g, 699 mmol, 61.2 mL) in DCM (460 mL), DMSO (68.2 g, 873 mmol, 68.2 mL) was added dropwise at -65°C, and the mixture was stirred at -65°C for 30 minutes. A solution of benzyl 2-(4-(hydroxymethyl)piperidine-1-yl)acetate (92.0 g, 349 mmol) in DCM (92.0 mL) was added dropwise at below -60°C, followed by the addition of TEA (177 g, 1.75 mol, 243 mL). The resulting mixture was stirred for a further 30 minutes at -65°C. TLC (dichloromethane:methanol = 10:1, R f A reading of 0.33 indicated that the starting material was completely consumed. The reaction mixture was quenched at 0°C by adding NaHCO3 (500 mL) and subsequently extracted once with DCM (100 mL). The combined organic layers were washed once with brine (100 mL), dried over anhydrous Na2SO4, filtered, and concentrated under vacuum. Benzyl 2-(4-formylpiperidine-1-yl)acetate (135 g, crude) was obtained as a yellow oil. 1 H NMR:400MHz, CDCl3δ9.65(d,J=1.2Hz,1H),7.33-7.38(m,5H),5.17(s,2H),3.29(s,2H),2.8 6-2.91(m,2H),2.34-2.40(m,2H),2.19-2.28(m,1H),1.90-1.95(m,2H),1.70-1.80(m,2H).

[0134] [ka] Step 3: At 0°C, 2,2'-(1,4,7-triazonan-1,4-diyl)di-tert-butyl acetate (32.0 g, 89.5 mmol) and AcOH (4.30 g, 71.6 mmol, 4.10 mL) were added to a mixture of 2-(4-formylpiperidine-1-yl)benzyl acetate (28.1 g, 107 mmol) in DCE (192 mL). The mixture was stirred at 0°C for 1 hour. Then, NaBH(OAc)3 (28.5 g, 134 mmol, 1.50 eq) was added little by little. The mixture was stirred at 20°C for 1 hour. The reaction mixture was quenched by adding NaHCO3 (200 mL) and then extracted five times with DCM (50.0 mL). The combined organic layers were washed once with brine (50.0 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain the residue. Crude products from two other reactions, starting with 30.0 g of 2,2'-(1,4,7-triazonan-1,4-diyl)diacetate di-tert-butyl and 32.0 g of 2,2'-(1,4,7-triazonan-1,4-diyl)diacetate di-tert-butyl, were combined for further purification. The combined crude products were triturated with SiO2 (120 mL) for 12 hours to obtain 51.0 g of 2,2'-(7-((1-(2-(benzyloxy)-2-oxoethyl)piperidine-4-yl)methyl)-1,4,7-triazonan-1,4-diyl)diacetate di-tert-butyl as a white solid. ES / MS m / z603.5(M+H) + .

[0135] [ka] Step 4: To a solution of 2,2'-(7-((1-(2-(benzyloxy)-2-oxoethyl)piperidine-4-yl)methyl)-1,4,7-triazonan-1,4-diyl)diacetate di-tert-butyl (51.0 g, 84.6 mmol) in EtOH (306 mL), Pd / C (5.10 g, 10% purity) was added under an N2 atmosphere. The suspension was degassed and purged three times with H2. The mixture was stirred under H2 (50 Psi) at 50°C for 4 hours. The mixture was filtered through Celite, and the filtrate was concentrated under vacuum. The crude product was triturated with MTBE (150 mL) for 4 hours. 2-(4-((4,7-bis(2-(tert-butoxy)-2-oxoethyl)-1,4,7-triazonan-1-yl)methyl)piperidine-1-yl)acetic acid (30.0 g, 56.9 mmol) was obtained as an off-white foam. ES / MS m / z513.4(M+H) + .

[0136] General peptide synthesis procedure: Peptides were synthesized using H-Asp(OtBu)-H resin following a standard Fmoc solid-phase peptide synthesis procedure. The final peptides were deprotected and cleaved from the resin following a two-step procedure: 1) Treatment with trifluoroacetic acid (TFA) for 2 hours at room temperature or TFA / dichloromethane (DCM) overnight at room temperature, followed by concentration; 2) 60 o Treatment with 0.1% TFA in acetonitrile / water (60:40) for 1 hour at 1°C ((https: / / www.emdmillipore.com / US / en / product / H-AspOtBu-H-NovaSyn-TG-resin,MDA_CHEM-856072#documentation)). The crude peptide was either concentrated or lyophilized, and then subjected to preparative HPLC purification (0.1% formic acid or 0.1% TFA in water / acetonitrile mobile phase). The product-containing fraction was collected and lyophilized to obtain the peptide as a white, fluffy solid.

[0137] [ka] compound 1 Following a general peptide synthesis procedure, 2,2'-(7-((1-((4R,15S,16S)-4-(2-carboxyethyl)-15-(((3S,6S)-6-((2-hydroxy-5-oxotetrahydrofuran-3-yl)carbamoyl)-4-oxo-1,2,3,4,6,7-hexahydroazepino[3,2,1-hi]indole-3-yl)carbamoyl)-16-methyl-2,5,9,13-tetraoxo-3,6,10,14-tetraazaoctadecyl)piperidine-4-yl)methyl)-1,4,7-triazonan-1,4-diyl)diacetic acid (36 mg) was prepared. ES / MS m / z1112.6(M+H) + .

[0138] [ka] compound 2 Following a general peptide synthesis procedure, 2,2'-(7-((1-((4S,13S,14S)-4-(2-carboxyethyl)-13-(((3S,6S)-6-((2-hydroxy-5-oxotetrahydrofuran-3-yl)carbamoyl)-4-oxo-1,2,3,4,6,7-hexahydroazepino[3,2,1-hi]indole-3-yl)carbamoyl)-14-methyl-2,5,8,11-tetraoxo-3,6,9,12-tetraazahexadecyl)piperidine-4-yl)methyl)-1,4,7-triazonan-1,4-diyl)diacetic acid (100 mg) was prepared. ES / MS m / z1084.5(M+H) + .

[0139] [ka] compound 3 According to the general peptide synthesis procedure, 2,2'-(7-((1-((15S,16S)-7-(carboxymethyl)-15-(((3S,6S)-6-((2-hydroxy-5-oxotetrahydrofuran-3-yl)carbamoyl)-4-oxo-1,2,3,4,6,7-hexahydroazepino[3,2,1-hi]indol-3-yl)carbamoyl)-16-methyl-2,6,9,13-tetraoxo-3,7,10,14-tetraazaoctadecyl)piperidin-4-yl)methyl)-1,4,7-triazonane-1,4-diyl)diacetic acid (49 mg) was prepared. ES / MS m / z1098.4(M+H) + .

[0140] [Chemical formula] Compound 4 According to the general peptide synthesis procedure, 2,2'-(7-((1-((4R,7R,16S,17S)-4,7-bis(2-carboxyethyl)-16-(((3S,6S)-6-((2-hydroxy-5-oxotetrahydrofuran-3-yl)carbamoyl)-4-oxo-1,2,3,4,6,7-hexahydroazepino[3,2,1-hi]indol-3-yl)carbamoyl)-17-methyl-2,5,8,14-tetraoxo-12-oxo-3,6,9,15-tetraazanonadecyl)piperidin-4-yl)methyl)-1,4,7-triazonane-1,4-diyl)diacetic acid (80 mg) was obtained. ES / MS m / z1200.6(M+H) + .

[0141] [Chemical formula] Compound 5 According to the general peptide synthesis procedure, 2,2'-(7-((1-((15S,16S)-3-(carboxymethyl)-15-(((3S,6S)-6-((2-hydroxy-5-oxotetrahydrofuran-3-yl)carbamoyl)-4-oxo-1,2,3,4,6,7-hexahydroazepino[3,2,1-hi]indol-3-yl)carbamoyl)-16-methyl-2,5,9,13-tetraoxo-3,6,10,14-tetraazaoctadecyl)piperidin-4-yl)methyl)-1,4,7-triazonane-1,4-diyl)diacetic acid (28 mg) was obtained. ES / MS m / z1098.6(M+H) + .

[0142]

Chem.

[0143]

Chem.

[0144] [ka] compound 8 Following a general peptide synthesis procedure, 2,2'-(7-(2-(4-((4S,13S,14S)-4-(2-carboxyethyl)-13-(((3S,6S)-6-((2-hydroxy-5-oxotetrahydrofuran-3-yl)carbamoyl)-4-oxo-1,2,3,4,6,7-hexahydroazepino[3,2,1-hi]indole-3-yl)carbamoyl)-14-methyl-2,5,8,11-tetraoxo-3,6,9,12-tetraazahexadecyl)piperazine-1-yl)-2-oxoethyl)-1,4,7-triazonan-1,4-diyl)diacetic acid (100 mg) was obtained. ES / MS m / z1113.5(M+H) + .

[0145] [ka] compound 9 Following a general peptide synthesis procedure, 2,2'-(7-(2-(4-((4R,13S,14S)-4-(2-carboxyethyl)-13-(((3S,6S)-6-((2-hydroxy-5-oxotetrahydrofuran-3-yl)carbamoyl)-4-oxo-1,2,3,4,6,7-hexahydroazepino[3,2,1-hi]indole-3-yl)carbamoyl)-14-methyl-2,5,11-trioxo-9-oxa-3,6,12-triazahexadecyl)piperazine-1-yl)-2-oxoethyl)-1,4,7-triazonan-1,4-diyl)diacetic acid (78 mg) was obtained. ES / MS m / z1100.5(M+H) + .

[0146] [ka] compound 10 Following a general peptide synthesis procedure, 2,2'-(7-(2-(4-((4R,7R,16S,17S)-4,7-bis(2-carboxyethyl)-16-(((3S,6S)-6-((2-hydroxy-5-oxotetrahydrofuran-3-yl)carbamoyl)-4-oxo-1,2,3,4,6,7-hexahydroazepino[3,2,1-hi]indole-3-yl)carbamoyl)-17-methyl-2,5,8,14-tetraoxo-12-oxa-3,6,9,15-tetraazanonadecyl)piperazine-1-yl)-2-oxoethyl)-1,4,7-triazonan-1,4-diyl)diacetic acid (22 mg) was obtained. ES / MS m / z1229.7(M+H) + .

[0147] [ka] compound 11 According to the general peptide synthesis procedure, 2,2'-(7-(2-(4-((4R,25S,26S)-4-(2-carboxyethyl)-25-(((3S,6S)-6-((2-hydroxy-5-oxotetrahydrofuran-3-yl)carbamoyl)-4-oxo-1,2,3,4,6,7-hexahydroazepino[3,2,1-hi]indol-3-yl)carbamoyl)-26-methyl-2,5,14,23-tetraoxo-9,12,18,21-tetraoxa-3,6,15,24-tetraazaoctacosyl)piperazin-1-yl)-2-oxoethyl)-1,4,7-triazonane-1,4-diyl)diacetic acid (46 mg) was obtained. ES / MS m / z 1289.6 (M+H) + .

[0148]

Chemical Structure

[0149]

Chemical Structure

[0150] [ka] compound 14 Following a general peptide synthesis procedure, 2,2'-(7-(2-(4-(2-(((1R)-1-carboxy-4-(((2S,3S)-1-(((3S,6S)-6-((2-hydroxy-5-oxotetrahydrofuran-3-yl)carbamoyl)-4-oxo-1,2,3,4,6,7-hexahydroazepino[3,2,1-hi]indole-3-yl)amino)-3-methyl-1-oxopentan-2-yl)amino)-4-oxobutyl)amino)-2-oxoethyl)piperazin-1-yl)-2-oxoethyl)-1,4,7-triazonan-1,4-diyl)diacetic acid (32 mg) was obtained. ES / MS m / z999.5(M+H) + .

[0151] [ka] compound 15 Following a general peptide synthesis procedure, 2-((2S)-2-(2-(4-(2-(4,7-bis(carboxymethyl)-1,4,7-triazonan-1-yl)acetyl)piperazine-1-yl)acetamide)-3-((3-((3-(((2S,3S)-1-(((3S,6S)-6-((2-hydroxy-5-oxotetrahydrofuran-3-yl)carbamoyl)-4-oxo-1,2,3,4,6,7-hexahydroazepino[3,2,1-hi]indole-3-yl)amino)-3-methyl-1-oxopentan-2-yl)amino)-3-oxopropyl)amino)-3-oxopropyl)amino)-3-oxopropyl)malonic acid (20 mg) was obtained. ES / MS m / z1185.6(M+H) + .

[0152] [ka] compound 16 Following a general peptide synthesis procedure, 2,2'-(7-(2-(4-((4R,7R,18S,19S)-4,7-bis(2-carboxyethyl)-18-(((3S,6S)-6-((2-hydroxy-5-oxotetrahydrofuran-3-yl)carbamoyl)-4-oxo-1,2,3,4,6,7-hexahydroazepino[3,2,1-hi]indole-3-yl)carbamoyl)-19-methyl-2,5,8,12,16-pentaoxo-3,6,9,13,17-pentazahenicosyl)piperazine-1-yl)-2-oxoethyl)-1,4,7-triazonan-1,4-diyl)diacetic acid (30 mg) was obtained. ES / MS m / z1270.6(M+H) + .

[0153] [ka] compound 17 Following a general peptide synthesis procedure, 2,2'-(7-(2-(4-(2-(((1S)-1-carboxy-4-(((2S,3S)-1-(((3S,6S)-6-((2-hydroxy-5-oxotetrahydrofuran-3-yl)carbamoyl)-4-oxo-1,2,3,4,6,7-hexahydroazepino[3,2,1-hi]indole-3-yl)amino)-3-methyl-1-oxopentan-2-yl)amino)-4-oxobutyl)amino)-2-oxoethyl)piperazin-1-yl)-2-oxoethyl)-1,4,7-triazonan-1,4-diyl)diacetic acid (30 mg) was obtained. ES / MS m / z999.5(M+H) + .

[0154] [ka] compound 18 Following a general peptide synthesis procedure, 2,2',2''-(10-(2-((1-((4R,15S,16S)-4-(2-carboxyethyl)-15-(((3S,6S)-6-((2-hydroxy-5-oxotetrahydrofuran-3-yl)carbamoyl)-4-oxo-1,2,3,4,6,7-hexahydroazepino[3,2,1-hi]indole-3-yl)carbamoyl)-16-methyl-2,5,9,13-tetraoxo-3,6,10,14-tetraazaoctadecyl)piperidine-4-yl)methoxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (30 mg) was obtained. ES / MS m / z1270.6(M+H) + .

[0155] Example 2: Synthesis of an exemplary compound of formula (I) General procedure for therapeutic isotope complexing A reaction vial containing a peptide precursor and a stirring bar is filled with a suitable amount of therapeutic radioisotope (e.g., 1.5 to 3.0 equivalents relative to the peptide) in the solvent. 90 Add Y) and stir. This will result in therapeutic isotope complexing.

[0156] [ka] Compound 1-M Compound 1-M is synthesized according to the general procedure for therapeutic isotope complexing.

[0157] [ka] Compound 2-M Compound 2-M is synthesized according to the general procedure for therapeutic isotope complexing.

[0158] [ka] Compound 3-M Compound 3-M is synthesized according to the general procedure for therapeutic isotope complexing.

[0159] [ka] Compound 4-M Compound 4-M is synthesized according to the general procedure for therapeutic isotope complexing.

[0160] [ka] Compound 5-M Compound 5-M was synthesized according to the general procedure for therapeutic isotope complexing.

[0161] [ka] Compound 6-M Compound 6-M was synthesized according to the general procedure for therapeutic isotope complexing.

[0162] [ka] Compound 7-M Compound 7-M was synthesized according to the general procedure for therapeutic isotope complexing.

[0163] [ka] Compound 8-M Compound 8-M was synthesized following the general procedure for therapeutic isotope complexing.

[0164] [ka] Compound 9-M Compound 9-M was synthesized following the general procedure for therapeutic isotope complexing.

[0165] [ka] Compound 10-M Compound 10-M is synthesized according to the general procedure for therapeutic isotope complexing.

[0166] [ka] Compound 11-M Compound 11-M was synthesized according to the general procedure for therapeutic isotope complexing.

[0167] [ka] Compound 12-M Compound 12-M was synthesized according to the general procedure for therapeutic isotope complexing.

[0168] [ka] Compound 13-M Compound 13-M was synthesized according to the general procedure for therapeutic isotope complexing.

[0169] [ka] Compound 14-M Compound 14-M was synthesized according to the general procedure for therapeutic isotope complexing.

[0170] [ka] Compound 15-M Compound 15-M was synthesized according to the general procedure for therapeutic isotope complexing.

[0171] [ka] Compound 16-M Compound 16-M was synthesized according to the general procedure for therapeutic isotope complexing.

[0172] [ka] Compound 17-M Compound 17-M was synthesized according to the general procedure for therapeutic isotope complexing.

[0173] [ka] Compound 18-M Compound 18-M was synthesized according to a general procedure for therapeutic isotope complexing.

[0174] Example 3: 90 Radioactive Synthesis of Y-Conjugate Compounds [ka] Typical compound 1- synthesized in ORA Neptis Perform radiosynthesizer 90 The RCY of Y is in the range of 14–20% (n=7) using a 29–55 GBq initiation activity. The synthesis time is 68 ± 5 minutes, the radiochemical purity of the product is over 93%, and the specific activity is in the range of 167–1320 GBq / μmol (3.9–30.9 mCi / μg).

[0175] [ka] Compound 18- 90 Y is synthesized according to the same method as described above.

[0176] Example 4: Competitive binding assay of human granzyme B Purpose: Compound 1- 90 By performing a competitive binding assay with human granzyme B using Y, the IC of potential granzyme B ligands was determined. 50 Determining the value

[0177] Preparation of the initial plate (including fully bound and non-specific bound plates): 1. As described above, add 78 μL of reaction buffer to each well of the 96-well plate. 2. In the wells used to examine complete binding (defined as binding of the radioactive ligand in the absence of competitors), 2 μL of DMSO is added to give a final DMSO concentration of 1% in all wells. 3. In the wells used to examine nonspecific binding (defined as the binding of a radioactive ligand in the presence of 10 μM 21), 2 μL of 1 mM compound 1 is added to give a final concentration of 10 μM.

[0178] Enzyme preparation: 1. Dilute granzyme B (GZB, human lymphocytes) to 0.5 μg / mL using the reaction buffer described above. 2.100 μL of 0.5 μg / mL diluted human GzB was added to each well to obtain final concentrations of 50 ng and 0.25 ug / mL per well.

[0179] Preparation of compounds: 1. Add DMSO or water to each compound vial to prepare a 1 mM compound stock solution. 2. Add 18.5 μL of the compound stock solution to 40 μL of DMSO and mix by pipetting up and down. Transfer 18.5 μL of the mixture to 40 μL of DMSO to perform a 1 / 2 log (3.16-fold) serial dilution of the 1 mM compound stock solution. Repeat this process to prepare 10 dilution points of the test compound. 3. For the wells used to examine ligand binding competition, dispense 2 μL of each dilution into a 96-well plate prepared as described above to obtain a further 1 / 100 dilution.

[0180] Preparation of radioactive ligands: Aiming for approximately 2 million CPM per 1.20 μL of solution (input volume), a 200 nM compound 1- 90 Prepare Y (10× radioactive ligand stock) in the reaction buffer. Dispense 2.20 μL of radioactive ligand stock into each well to obtain a final concentration of 20 nM per well.

[0181] Incubation conditions, post-incubation sample processing, and data analysis: All 1.96 wells should have a final volume of 200 μL, and the assay plate should be incubated at 37°C for 90 minutes. 2. All samples from assay plates are pre-immersed in PBS buffer, pH 7.4, and then processed using MultiScreen. HTS The samples are transferred to an FB plate and filtered using a vacuum manifold. An additional 150 μL of PBS buffer is added to each well of the assay plate and combined to ensure that any remaining samples are transferred. 3. MultiScreenHTS Wash the FB plate three times with 150 μL of PBS buffer. 4. Separate all filters and transfer them to individual tubes using Multiscreen punch tips and Millipore multi-punch equipment. 5. Analyze the sample set using the Wizard 2480 automated gamma counter [Perkin Elmer]. Values ​​are reported as attenuated counts per minute (CPM). 6. Normalize the obtained CPM value and convert it to % inhibition using the following formula:

number

[0182] The obtained % inhibition values ​​are One site-Fit logIC 50 The plots were generated using the software GraphPad Prism 8.4.3, which uses the formula, and the IC of each ligand. 50 The value has been determined. Y = A + (BA) / (1 + 10) (x-LogIC50) ) Y = % inhibition X = Log concentration (M) of the cold competing ligand A = Minimum Y (0%) B=Max Y(100%) LogIC 50 = Log concentration of cold competing ligand (M) midway between minimum Y and maximum Y

[0183] Other Embodiments All features disclosed herein can be combined in any combination. Each feature disclosed herein may be replaced by an alternative feature that serves the same, equivalent, or similar purpose. Therefore, unless expressly otherwise specified, each disclosed feature is merely an example of a general set of equivalent or similar features.

[0184] Furthermore, from the above description, those skilled in the art will readily be able to identify the essential features of this disclosure and adapt various modifications and forms of this disclosure to various uses and conditions without departing from the spirit and scope of this disclosure. For this reason, other embodiments are also within the scope of the claims.

[0185] Equal parts While several embodiments of the present invention have been described and illustrated herein, various other means and / or structures for performing the functions described herein and / or obtaining the results and / or one or more advantages will be readily foreseeable to those skilled in the art. Furthermore, each of such variations and / or modifications will be considered to fall within the scope of the embodiments of the present invention described herein. More generally, all parameters, dimensions, materials and configurations described herein are intended to be exemplary, and it will be readily apparent to those skilled in the art that actual parameters, dimensions, materials and / or configurations will depend on the specific one or more applications in which the teachings of the present invention are used. Those skilled in the art will be able to identify, or verify, many equivalent forms to the specific embodiments of the present invention described herein without going beyond ordinary experimentation. Thus, the above embodiments are presented merely as examples, and it should be understood that embodiments of the present invention can be carried out in ways other than those specifically described and claimed, within the scope of the appended claims and their equivalents. The embodiments of the present invention in this disclosure relate to each of the individual features, systems, articles, materials, kits and / or methods described herein. In addition, any combination of two or more such features, systems, articles, materials, kits, and / or methods is included within the scope of the present invention as long as such features, systems, articles, materials, kits, and / or methods do not contradict each other.

[0186] All definitions defined and used herein should be understood to take precedence over dictionary definitions, definitions in literature incorporated by reference, and / or the ordinary meanings of the terms defined.

[0187] All references, patents, and patent applications disclosed herein are incorporated by reference with respect to the subject matter from which they are cited, and in some cases may encompass the entirety of the references.

[0188] As used herein and in the claims, the indefinite articles “a” and “an” should be understood to mean “at least one” unless explicitly stated otherwise.

[0189] When the phrase “and / or” is used herein and in the claims, it should be understood to mean “either one or both” of the elements that are thus coordinately connected, i.e., elements that exist in some cases conjunctively and in other cases disjunctively. Multiple elements enumerated by “and / or” should be interpreted in the same manner, i.e., “one or more” of the elements that are thus coordinately connected. Other elements not specifically identified by the phrase “and / or” may be optionally present, whether or not they relate to the specifically identified elements. For this reason, as a non-restrictive example, when used in combination with open-ended language such as “includes,” a reference to “A and / or B” may, in one embodiment, refer to A only (optionally including elements other than B), in another embodiment, refer to B only (optionally including elements other than A), in yet another embodiment, refer to both A and B (optionally including other elements), and so on.

[0190] Where used herein and in the claims, “or” should be understood to have the same meaning as “and / or” as defined above. For example, when separating items in a list, “or” or “and / or” shall be interpreted as inclusive; that is, it includes at least one, but also several elements or two or more of the enumerated elements, and optionally additional unenumerated items. Only terms that are clearly contradictory, such as “only one of” or “exactly one of” or, as used in the claims, “consisting of,” would refer to the inclusion of several elements or exactly one of the enumerated elements. In general, where used herein, the term “or” shall be interpreted only as indicating an exclusive choice (i.e., “one or the other, but not both”) when preceded by an exclusive term such as “either,” “one of,” “only one of,” or “exactly one of.” Where used in the claims, “consisting of” shall have its usual meaning as used in the field of patent law.

[0191] As used herein and in the claims, the phrase “at least one” in relation to a list of one or more elements means at least one element selected from any one or more elements in the list of elements, but not necessarily including at least one of every element specifically enumerated in the list of elements, nor excluding any combination of elements in the list of elements. Furthermore, this definition allows for the optional presence of elements other than those specifically identified in the list of elements referred to by the phrase “at least one,” whether or not they are related to the specifically identified elements. Therefore, as a non-restrictive example, "at least one of A and B" (or equivalently "at least one of A or B" or equivalently "at least one of A and / or B") may refer to, in one embodiment, at least one A including two or more A's, where B is absent (and other elements are optionally included); in another embodiment, at least one B including two or more B's, where A is absent (and other elements are optionally included); in yet another embodiment, at least one A including two or more A's, and at least one B including two or more B's, where other elements are optionally included; and so on.

[0192] Unless otherwise clearly indicated, it should be understood that in any method claimed herein that includes two or more steps or actions, the order of the steps or actions of the Method is not necessarily limited to the order in which the steps or actions of the Method are listed.

Claims

1. Compound of formula (I): 【Chemistry 1】 (In the formula, M is the radioactive part; A is a chelating portion that chelates the radioactive portion; X is -CH ２ C(NH)-, -CH ２ C(O)-, -CH ２ Selected from the group consisting of C(S)-, -NHC(NH)-, -NHC(O)-, -NHC(S)-, -OC(NH)-, -OC(O)-, and -OC(S)-, X is arbitrarily selected as -CH ２ It is C(O)- or -NHC(S)-; Y is either CH or N; Z is -CH ２ -, -CH ２ C(NH)-, -CH ２ C(O)-, -CH ２ C(S)-, -NHC(NH)-, -NHC(O)-, -NHC(S)-, -OC(NH)-, -OC(O)-, or -OC(S)-; optionally, Z is -CH ２ - or -CH ２ C(O)-; L is a peptide linker having 1 to 6 amino acid residues (including both ends); R １ is H or C １～６ It is alkyl, and optionally R １ is H or methyl; and R ２ C １～６ Alkyl or C ３～６ (It is cycloalkyl.) , or its stereoisomers, tautomers, or salts.

2. The aforementioned compound is of formula (Ia): 【Chemistry 2】 The compound according to claim 1, wherein the compound is as described above.

3. X is -CH ２ The compound according to claim 1 or 2, wherein it is C(O)-.

4. The aforementioned compound is of formula (Ib): 【Transformation 3】 The compound according to claim 3, wherein the compound is as described above.

5. The compound according to any one of claims 1 to 4, wherein L has 1 to 5 amino acid residues (including both ends); optionally, L has 1 or more non-natural amino acid residues.

6. L, Glu-Gly-Gly, Glu-βAla-βAla, γGlu, Ｄ γGlu, γGlu-βAla, Ｄ Glu-βAla-βAla、 ｄ Glu-AEA, ｄ -+-AEEA-AEEA、 Ｄ Glu-DGlu-AEA、 Ｄ Glu-DGlu-βAla-βAla、 βAla-DGlu-βAla, Diacid-βAl-βAl, N-acid-βAla-βAla, and βAla-N-acid-βAla A compound according to any one of claims 1 to 5, having an amino acid sequence selected from the group consisting of the following.

7. The compound according to any one of claims 1 to 6, wherein the chelating portion A is 1,4,7-triazacyclononane-N,N',N''-triacetic acid (NOTA), 1,4,7-triazacyclononane-4,7-diyldiacetic acid (NODA), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid (DO3A), 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid (DO3A), a restraining complexing agent (RESCA), or MACROPA.

8. The compound according to any one of claims 1 to 7, wherein the radioactive portion of M is a therapeutic radioisotope.

9. The aforementioned therapeutic radioactive isotope, ６７ Cd, ９０ Y, １７７ Lu, ２２５ Ac, ４７ Sc, １３１ I, １５３ Sm, １６１ Tb, ２１１ At, ２１２ Pb, ２１２ Bi, ２２３ Ra, or ２２７Ｔｈ Selected from the group consisting of; optionally, the therapeutic radioisotope ９０ The method according to claim 8, wherein Y.

10. The chelating portion is NOTA or DOTA, and the therapeutic radioactive isotope is ９０ Y, １７７ Lu, or ２２５ A compound according to any one of claims 1 to 9, wherein the compound is Ac.

11. The chelating portion is NODA, and the therapeutic radioactive isotope is ４７ Sc or ６７ A compound according to any one of claims 1 to 10, wherein the compound is Cu.

12. The aforementioned compound has the following structure: 【Chemistry 4】 (In the formula, M is １７７ Lu, ９０ Y, ２２５ Ac, or ２１３ Bi is), or 【Transformation 5】 (In the formula, M is １７７ Lu, ９０ Y, ２２５ Ac, or ２１３ (It is Bi) The compound according to claim 1, which is one of the compounds.

13. A pharmaceutical composition comprising a compound according to any one of claims 1 to 12 and a pharmaceutically acceptable excipient.

14. A method for treating cancer in a subject, (a) Administer an effective amount of the compound described in any one of claims 1 to 12 or a pharmaceutical composition containing the same to the target subject. A method that includes this.

15. Before step (a), Administering an immunotherapy agent to the aforementioned subject. The method according to claim 14, further comprising:

16. The method according to claim 14 or 15, wherein the immunotherapy agent is an immune checkpoint inhibitor, which is optionally a PD1 inhibitor, or a genetically engineered T cell expressing a chimeric antigen receptor (CAR).

17. The method according to any one of claims 14 to 16, further comprising administering an imaging agent to the subject in order to perform imaging of granzyme B prior to step (a).

18. The method according to any one of claims 14 to 17, wherein the subject is further treated with one or more additional therapeutic agents.

19. The method according to claim 18, wherein the one or more additional therapeutic agents are selected from the group consisting of anti-inflammatory agents, steroids, immunotherapy agents, and chemotherapeutic agents.

20. A kit for cancer therapy comprising a first container having a compound according to any one of claims 1 to 12 or a pharmaceutical composition containing the same.

21. The kit according to claim 20, comprising a pharmaceutical excipient.

22. The kit according to claim 21, comprising a second container, wherein the second container contains the pharmaceutical excipient.

23. The kit according to claim 21 or 23, wherein the compound or pharmaceutical composition and the pharmaceutical excipient are combined to form a single unit dosage form.

24. A kit according to any one of claims 20 to 23, comprising an imaging agent suitable for imaging granzyme B.

25. A kit according to any one of claims 20 to 24, comprising an additional therapeutic agent.

26. The kit according to claim 25, wherein the additional therapeutic agent comprises an immunotherapeutic agent.

27. The kit according to claim 25 or 26, wherein the additional therapeutic agent is contained in the first container.

28. The kit according to claim 26 or 27, wherein the immunotherapy agent is an immune checkpoint inhibitor, which is optionally a PD1 inhibitor, or a genetically engineered T cell expressing a chimeric antigen receptor (CAR).

29. A kit according to any one of claims 20 to 28, comprising instructions for using one or more of the compounds, pharmaceutical compositions, imaging agents, and additional therapeutic agents according to any one of claims 1 to 28.

30. A kit according to any one of claims 20 to 29, comprising instructions for imaging and / or treating cancer and / or reducing the risk of cancer in a subject.

31. The kit according to any one of claims 20 to 30, wherein the first container comprises a vial, ampoule, bottle, syringe, or dispenser package.

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