Seranostics compounds targeting granzyme B

Abstract

A seranostic agent targeting granzyme B for use in diagnosing and treating cancer. Exemplary therapeutic compounds targeting granzyme B can have the structure of formula (I) disclosed herein. Also provided herein is a method for treating cancer, the method comprising administering to a subject in need of treatment a granzyme B-targeted therapeutic compound disclosed herein.

Description

Technical Field

[0001] Related Applications This application claims the benefit of the filing date of U.S. Provisional Patent Application No. 63 / 391,905, filed Jul. 25, 2022, the entire contents of which are incorporated herein by reference.

[0002] The present disclosure relates to compounds useful for seranostics therapy, and more particularly, to radiolabeled compounds that are useful for identifying granzyme B and eliminating cancer cells containing granzyme B.

Background Art

[0003] Granzyme B is a serine protease most commonly found in the granules of natural killer cells and cytotoxic T cells. Granzyme B is released together with the pore-forming protein perforin at the immunological synapse formed between a T cell and its target. Subsequently, a portion of the released granzyme B enters the cancer cell mainly through the perforin pores, where granzyme B activates multiple substrates, leading 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 that respond to immunotherapy.

[0004] There is a need to develop novel compounds that function as effective granzyme B imaging agents and therapies for treating immune dysregulation such as cancer.

Summary of the Invention

[0005] This application provides compounds capable of targeting granzyme B and their use as seranostics agents for treating cancer.

[0006] In one aspect, the present disclosure is a method of treating cancer in a subject, the method comprising a compound of formula (I):

Chemical Formula

[0007] In another aspect, the present disclosure features a method of treating cancer in a subject, the method comprising: (i) administering an immunotherapeutic agent to a subject in need of treating cancer; and (ii) after step (i), administering to the subject an effective amount of a pharmaceutical composition comprising a compound of formula (I) provided herein.

[0008] In formula (I), M comprises a therapeutic radioisotope, A is a chelating moiety that chelates M, X is -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 inclusively from 1 to 6 amino acid residues, R 1 is H or C 1-6 alkyl, R 2 is C 1-6 alkyl or C 3-6 cycloalkyl,

[0009] In some examples, X is -CH2C(NH)-. In other examples, X is -NHC(S)-. In still other examples, X is -CH2C(O)-. Alternatively or additionally, R 1 can be H. In other examples, R 1 can be methyl.

[0010] In some embodiments, the compound of formula (I) is of formula (Ia).

Chemical formula

[0011] In some examples, L is a peptide linker having inclusively 1 to 3 (1, 2, or 3) amino acid residues. In one example, L is a peptide linker having 3 amino acid residues in length.

[0012] In some cases, the compound of formula (Ia) is of formula (Ia-A).

Chemical formula

[0013] Alternatively, the compound of formula (Ia) is of formula (Ia-B).

Chemical formula

[0014] For any of the compounds of formula (I) disclosed herein, the chelating moiety A can be 1,4,7-triazacyclononane-4,7-diyl diacetic acid (NODA), 1,4,7-triazacyclononane-N,N’,N”-triacetic acid (NOTA), 2-S-(4-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid (p-SCN-Bn-NOTA), or 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA). Moiety M in any of the compounds of formula (I) disclosed herein is 90 Y, 177 Lu, 225 Ac, 47 Sc, 67 Cu, 131 I, 153 Sm, 211 At, 212 Pb, 212 Bi, 223 Ra, or 227 Th and can include therapeutic radioisotopes.

[0015] In some examples, the chelating moiety is NOTA or DOTA and the therapeutic radioisotope is 90 Y, 177 Lu, or225 It is Ac. In other examples, the chelating moiety is NOTA and the therapeutic radioisotope is 90 Y, 177 Lu, or 225 Ac. In yet other examples, the chelating moiety is DOTA and the therapeutic radioisotope is 90 Y, 177 Lu, or 225 Ac. In another example, the chelating moiety is NODA and the therapeutic radioisotope is 47 Sc or 67 Cu.

[0016] In some specific examples, the compound of formula (I) is

Chemical formula

[0017] In any of the methods disclosed herein, a subject (e.g., a human patient) may have received or is receiving a treatment comprising an immunotherapeutic agent. In some embodiments, the immunotherapeutic agent is an immune checkpoint inhibitor. In some examples, the checkpoint inhibitor is a PD-1 inhibitor (e.g., an antagonist of PD-1 such as an anti-PD-1 antibody, or an antagonist of PD-L1 such as an anti-PD-L1 antibody). In other examples, the checkpoint inhibitor can be a CTLA-4 inhibitor or a LAG-3 inhibitor. In other embodiments, the immunotherapeutic agent can be a genetically engineered T cell expressing a chimeric antigen receptor (CAR-T cell therapy).

[0018] Alternatively or additionally, the methods disclosed herein may further comprise administering an imaging agent to a subject to image granzyme B prior to administration of a pharmaceutical composition comprising a compound of formula (I). In some cases, the imaging agent is a compound of formula (II) or a pharmaceutically acceptable salt thereof. [Chemical formula]

[0019] In formula (II), M contains a contrast agent, A is a chelating moiety that chelates 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 optionally, X is -CH2C(O)- or -NHC(S)-, L is a peptide linker that inclusively has 1 to 6 amino acid residues, R 1 0]is H or C 1-6 alkyl, and optionally, R 1 is H or methyl, R 2 is C 1-6 alkyl or C 3-6 cycloalkyl.

[0020] In some examples, the imaging agent can be any of the compounds listed in Table 2.

[0021] In some embodiments, a subject being treated by any of the methods disclosed herein may be further treated with one or more additional therapeutic agents. Examples of one or more additional therapeutic agents include, but are not limited to, anti-inflammatory agents, steroids, immunotherapeutic agents, chemotherapeutic agents, and therapeutic antibodies.

[0022] In yet another aspect, the disclosure features (i) a diagnostic compound for imaging granzyme B (e.g., a compound of formula (II) disclosed herein such as those listed in Table 2), and (ii) a therapeutic compound, which can be a compound of formula (I) disclosed herein or a pharmaceutically acceptable salt thereof, in a theranostic agent.

[0023] A therapeutic compound of formula (I), or a pharmaceutically acceptable salt thereof, and any of the pharmaceutical compositions containing such are within the scope of the present disclosure. Further, the present disclosure features any of the use of a therapeutic compound of formula (I) or a pharmaceutically acceptable salt thereof, and a pharmaceutical composition containing such, for use in treating cancer, and the use of such a compound, pharmaceutically acceptable salt, or pharmaceutical composition for manufacturing a medicament for cancer treatment.

[0024] Details of one or more embodiments of the present invention are described in the following specification. Other features or advantages of the present invention will become apparent from the following drawings and detailed description of several embodiments, and also from the appended claims.

Mode for Carrying Out the Invention

[0025] Cancer immunotherapy represents a significant advance in recent cancer therapy. Antibodies targeting immune checkpoints such as programmed cell death protein 1 (PD-1) and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) have been approved to have a positive outcome for some patients. Research in the field of immuno-oncology continues, and strategies including CAR-T cells, vaccines, small molecules, and antibodies are under development. However, these immunotherapies can be associated with significant adverse events, and the response rate is typically 20 - 50%, which means that most patients do not respond to the therapy. Further, the response is frequently associated with immune cell infiltration, which can make it difficult to determine the response of an individual patient to the therapy using conventional methods, as the responding tumors may grow on anatomical imaging (e.g., CT, MRI) and appear to demonstrate increased avidity by FDG-PET imaging due to the influx of metabolically active immune cells.

[0026] Seranostics refers to a combination of imaging diagnostics and therapies for diagnosing and treating a target disease. Generally, the agents used in seranostics therapies contain an imaging diagnostic compound and a radiotherapeutic compound. In some cases, the imaging diagnostic and therapeutic compounds can have the same molecule, one radionuclide for imaging purposes, and one radionuclide for therapeutic purposes, except for different radioactive labels. In some cases, the imaging diagnostic and therapeutic compounds can have different elements bound to the same pharmacophore or molecule with different radioisotopes for imaging and therapeutic purposes.

[0027] Granzyme B, a downstream marker of cytotoxic T cell activity, can function as a novel biomarker for evaluating the efficacy of cancer immunotherapy. Granzyme B expression in tumors can be evaluated not only in terms of the presence or absence of CTLs but also as an effector protein released by activated T cells that integrates a measure of CTL activity, thus explaining the problem of T cell exhaustion that makes it difficult to achieve an assessment of the presence of CTLs.

[0028] Accordingly, provided herein is a granzyme B-targeted seranostics therapy for treating cancer in a subject in need thereof. The seranostics therapy comprises a granzyme B-targeted therapeutic compound comprising a therapeutic radionuclide 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 can be the same molecule loaded with a therapeutic radionuclide relative to an imaging radionuclide, for example, different types of radioactive moieties. In some embodiments, the subject for treatment by the granzyme B-targeted seranostics therapy may have received or be receiving immunotherapy. In some embodiments, the seranostics therapy disclosed herein may be administered to the subject concurrently with immunotherapy.

[0029] I. Granzyme B-Targeted Seranostics Agents In some embodiments, provided herein is a granzyme B-targeted therapeutic agent comprising a granzyme B-targeting compound for treatment, and optionally a granzyme B-targeting compound for imaging. Compounds disclosed herein include the compound itself, its pharmaceutically acceptable salts, and its stereoisomers.

[0030] The compounds described herein can contain one or more asymmetric centers and, thus, can exist in various isomeric forms, such as enantiomers and / or diastereomers. For example, the compounds described herein can 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 enriched in one or more stereoisomers. Isomers can 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 the preferred isomers can 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 (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 that are substantially free of other isomers and, alternatively, as mixtures of various isomers.

[0031] A. Granzyme B-Targeting Compounds for Treatment In some embodiments, the granzyme B-targeting compounds for treatment disclosed herein are compounds of formula (I). [Chemical formula]

[0032] In formula (I), M includes a therapeutic radioisotope. Any radioisotope suitable for therapeutic use (e.g., suitable for cancer treatment) can be used in the compound of formula (I). Examples include 90 Y, 177 Lu, 225 Ac, 47 Sc, 131 I, 153 Sm, 211 At, 212 Pb, 212 Bi, 223 Ra, or 227 Th, but are not limited thereto.

[0033] In formula (I), A is a chelating moiety. The chelating moiety is a molecule or ion that can function as a polydentate ligand for a metal ion. For example, a molecule having a plurality of atoms with available lone pairs (including, but not limited to, nitrogen and oxygen) can function as a chelating moiety. The chelating moiety can be linear (e.g., EDTA), cyclic (including macrocyclic molecules such as DOTA, porphyrin), and can include macrocyas generally known in the art. The chelating moiety can have 2, 3, 4, 5, or 6 functional groups (e.g., amine, amide, hydroxyl, carboxylic acid, etc.) having lone pairs available for coordination with a metal. Exemplary chelating moieties for use in the granzyme B targeting compounds disclosed herein include 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-1-glutaric acid-4,7-diacetic acid (NODAGA), ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), cyclohexyl-1,2-diaminetetraacetic acid (CDTA), ethylene glycol-0,0'-bis(2-aminoethyl)-N,N,N',N'-tetraacetic acid (EGTA), N,N-bis(hydroxybenzyl)-ethylenediamine-N,N'-diacetic acid (HBED), triethylenetetraminehexaacetic 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-diyl diacetic acid (NODA), and deferoxamine B (DFO), but are not limited thereto.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-diyl diacetic acid (NODA), and 1,4,7-triazacyclononane-1-glutaric acid-4,7-diacetic acid (NODAGA). In some embodiments, the chelating agent is 1,4,7-triazacyclononane triacetic acid (NOTA). In other embodiments, the chelating agent is 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA).

[0034] In some examples, the chelating agent can be 1,4,7-triazacyclononane-4,7-diyl diacetic acid (NODA). In other examples, the chelating agent can be 1,4,7-triazacyclononane-N,N’,N”-triacetic acid (NOTA). In some examples, the chelating agent can be 2-S-(4-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid (p-SCN-Bn-NOTA). In still other examples, the chelating agent can be 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA).

[0035] The selection of a suitable chelating agent to pair with a therapeutic radioisotope may be according to 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 of which are incorporated herein by reference for the subject matter and purposes related hereto), or according to the guidance provided herein. In some cases, the compound of formula (I) may have a pair of a chelating agent listed in Table 1 below and a therapeutic radioisotope. Such pairs of chelating agents and therapeutic radioisotopes can result in high loading efficiencies of the therapeutic radioisotope [Table 1]

[0036] X can be -CH2C(NH)-, -CH2C(O)-, -CH2C(S)-, -NHC(NH)-, -NHC(O)-, -NHC(S)-, -OC(NH)-, -OC(O)-, or -OC(S)-. In some examples, X is -CH2C(NH)-. In other examples, X is -NHC(S)-. In other examples, X is -CH2C(O)-.

[0037] L can be a peptide linker having inclusively 1 to 6 amino acid residues. In some examples, L inclusively contains 1 to 3 amino acid residues. In other examples, L inclusively contains 4 to 6 amino acid residues. In one example, L contains 1 amino acid residue. In another example, L contains 2 amino acids. In yet another example, L contains 3 amino acid residues. Alternatively, L contains 4 amino acid residues. In still another example, L contains 5 amino acid residues. Alternatively, L contains 6 amino acid residues.

[0038] Suitable amino acid residues in the peptide linker L can include natural and non-natural amino acid residues (including β-amino acid residues and D-amino acids), but are not limited to prothienogenic amino acid residues. The amino acid residues can form a chain via standard peptide bonds or by forming amide bonds with computable side chains (such as glutamic acid (such as D-Glu), aspartic acid). Exemplary peptide linkers include, but are not limited to, Glu-Gly-Gly, D-Glu-β-Ala-β-Ala, Gly-Gly, Gly, Glu-Gly, Glu, D-Glu, Arg-Gly, Lys-Gly.

[0039] In some embodiments, R 1 is H. In other embodiments, R 1 is C 1-6 alkyl. For example, R 1 can be methyl.

[0040] In some embodiments, R2 may be C 1-6 alkyl. Alternatively, R 2 may be C 3-6 cycloalkyl (e.g., branched or unbranched, substituted or unsubstituted) or C 3-6 cycloalkyl (e.g., cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl).

[0041] In some cases, the hemiacetal unit in the compound of formula (I) may be in the open-chain aldehyde form. As a result, the compound of formula (I) can have the following structure.

Chemical formula

[0042] In any of formulas (I-1) to (I-6), each of M, A, X, L, R 1 , and R 2 is as described herein.

[0043] In some embodiments, R 1 is H and R 2 is C4 alkyl such as in the compound of formula (Ia).

Chemical formula

[0044] In some specific examples, as in the compound of formula (Ia-A), X is -CH2C(O)-.

Chemical formula

[0045] In other specific examples, as in the compound of formula (Ia-B), X is -NHC(S)-.

Chemical formula

[0046] In specific examples, compounds of formula (I) provided herein have the following structure: [ka] where R* is a therapeutic isotope, e.g., 177 Lu, 90 Y, 225 Ac, or 213 I'm Bi.

[0047] The present disclosure also includes stereoisomers of compounds of Formula (I), such as the stereoisomers of compound 21 provided herein. The stereoisomers arise from two chiral centers (closed ring) or one chiral center (open chain) of the hemiacetal unit.

[0048] B. Granzyme B-Targeted Imaging Compounds Any imaging compound known in the art that targets Granzyme B can be used in the theranostic therapy disclosed herein.

[0049] In some embodiments, the granzyme B targeted imaging compound can be a compound of formula (II) shown below. [ka]

[0050] In formula (II), M is a metal or a metal linked to a radioisotope suitable for imaging. Suitable metals for use in the present disclosure include those useful for imaging granzyme B, such as metals that are suitable radioactive imaging agents, as well as metals that can be linked to non-metallic radioisotopes that are suitable radioactive imaging agents. Exemplary metal radioisotopes include: 68 Exemplary non-metallic radioisotopes are: 18 F, which can be conjugated with Al for loading into the granzyme B binding compounds disclosed herein.

[0051] A, X, L, R 1 , and R2 and each of the following is as defined herein. See, e.g., the section above entitled Granzyme B Targeted Therapeutic Compounds.

[0052] In some cases, the hemiacetal unit in the compound of formula (II) may be in the open-chain aldehyde form. As a result, the compound of formula (II) may have the following structure: [ka]

[0053] In any of formulas (II-1) to (II-6), M, A, X, L, R 1 , and R 2 each of which is as described herein.

[0054] In some embodiments, as in compounds of formula (IIa), R 1 is H and R 2 is a C4 alkyl. [ka]

[0055] In some embodiments, such as in compounds of formula (IIa-A), X is —CH 2 C(O)—. [ka]

[0056] In another embodiment, X is -NHC(S)-, as in the compound of formula (IIa-B). [ka]

[0057] Exemplary granzyme B-targeted imaging compounds of formula (II) are provided in Table 2. [Table 2-1]

Table 2-2

Table 2-3

Table 2-4

Table 2-5

[0058] In one example, the compound of formula (II) is compound 7-Al, 18 It can be filled with F. Also, reference is made to WO2021 / 252664, the relevant disclosure of which is incorporated by reference for the subject matter and purposes referred to herein.

[0059] Also within the scope of the present disclosure are variants of the compounds of formula (I) and formula (II) disclosed herein, and the lactone ring in the compounds of formula (I) and formula (II) can be substituted with other moieties, such as an aryl ring, a heteroaryl ring, etc. An example of a variant of formula (II) is provided below.

Chemical formula

[0060] The compounds of formula (II) provided herein contain radioisotopes useful as imaging agents. For example, the radioisotope can be 18 F, which can be useful as an imaging agent (e.g., as a non-toxic and / or non-therapeutic radioisotope) when administered to a subject at a low concentration (e.g., 5 mCi).

[0061] As pointed out above, this application also includes pharmaceutically acceptable salts of the compounds described herein. As used herein, the term "pharmaceutically acceptable" means within the scope of sound medical judgment of these compounds, materials, compositions, and / or dosage forms that are suitable for use in contact with the tissues of humans and animals without undue toxicity, irritation, allergic response, or other problems or complications commensurate with a reasonable benefit / risk ratio.

[0062] This disclosure also includes stereoisomers of the compounds of formula (II), such as the stereoisomers of compound 7-Al and 7-Al described herein. See the following examples.

Chemical formula

[0063] C. Chemical Synthesis of Compounds for Granzyme B Targeted Therapy and Imaging Compounds As will be appreciated, the compounds provided herein, including stereoisomers and their salts, can be prepared using known organic synthesis techniques and can be synthesized according to any of a number of possible synthetic routes.

[0064] The compounds disclosed herein, or pharmaceutically acceptable salts thereof, can be prepared by following the exemplary protocols described below. Suitable protecting groups for use in such syntheses are known in the art. See, for example, McOmie, Protective Groups in Organic Chemistry, (1973):98. Briefly, the synthesis of the granzyme B targeting compounds disclosed herein can involve the following processes: A) attaching the desired tricyclic ring (such as in the precursor) to a resin (e.g., peptide coupling) and deprotecting the tricyclic ring; B) attaching either of the L peptide linkers (which can be formed by repeatedly coupling amino acid residues to the desired length) to the resin-bound tricyclic ring to form a tricyclic-peptide linker moiety; C) attaching the tricyclic-peptide linker moiety to a chelating site to form a precursor compound, cleaving this to release the resin, and subsequently optionally, D) chelating a metal with the precursor compound to form a compound of formula (I).

[0065] For example, in step A, the tricyclic ring (e.g., Fmoc-Haic(2S,5S)-OH) found in the precursor and the compound of formula (I) can be attached to a resin (e.g., H-Asp(OtBu)-H) and deprotected. Then, in step B, the free amine of the tricyclic ring can be coupled with an Fmoc-protected amino acid (e.g., isoleucine), and then the Fmoc may be removed. The resulting free amine can be coupled to a desired number of amino acids using conventional peptide synthesis methods to reach the tricyclic-peptide linker moiety. Fmoc protection and deprotection can be carried out in each round of amino acid residue addition. In step C, a chelating moiety having a terminal coupling group (e.g., carboxylic acid, carbamothio O-acid, isothiocyanate, or thiocyanate) can be coupled to the last free amine of the amino acid chain in the tricyclic-peptide linker moiety to produce a precursor compound, which can be liberated from the resin and purified if necessary. Then, the precursor thus prepared can be chelated with a metal (e.g., Ga, Al) disclosed herein to produce a compound of formula (I). Exemplary examples for synthesizing the exemplary precursor and the compound of formula (I) are provided in the following examples. In an alternative embodiment of step C, the compound can be cleaved from the resin before being attached to the chelating moiety.

[0066] Many suitable imaging agents (e.g., radioisotopes) are known in the art (see, e.g., U.S. Pat. Nos. 5,021,236, 4,938,948, and 4,472,509, the disclosures of each of which are hereby incorporated by reference in their entirety). The radiolabeled compounds, or pharmaceutically acceptable salts thereof, provided herein can be prepared according to methods well known in the art. Synthetic methods for incorporating radioisotopes into organic compounds are well known in the art, and those skilled in the art will readily recognize other methods applicable to the compounds provided herein.

[0067] The processes described herein are not the exclusive means by which the compounds provided herein can be synthesized, and it will be understood by those skilled in the art that a wide repertoire of synthetic organic reactions are available for potential use in the synthesis of the compounds provided herein. Those skilled in the art know how to select and implement appropriate synthetic routes. Suitable synthetic methods for starting materials, intermediates, and products are described in Advances in Heterocyclic Chemistry, Vols. 1 - 107 (Elsevier, 1963 - 2012), Journal of Heterocyclic Chemistry Vols. 1 - 49 (Journal of Heterocyclic Chemistry, 1964 - 2012), Carreira, et al. (Ed.) Science of Synthesis, Vols. 1 - 48 (2001 - 2010) and Knowledge Updates KU2010 / 1 - 4; 2011 / 1 - 4; 2012 / 1 - 2 (Thieme, 2001 - 2012), Katritzky, et al. (Ed.) Comprehensive Organic Functional Group Transformations, (Pergamon Press, 1996), Katritzky et al. (Ed.); Comprehensive Organic Functional Group Transformations II (Elsevier, 2 nd nd Edition, 2004), Katritzky et al. (Ed.), Comprehensive Heterocyclic Chemistry (Pergamon Press, 1984), Katritzky et al., Comprehensive Heterocyclic Chemistry II, (Pergamon Press, 1996), Smith et al., March’s Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, 6 thIt can be identified by referring to documents including the sources of documents such as Ed. (Wiley, 2007), Trost et al. (Ed.), Comprehensive Organic Synthesis (Pergamon Press, 1991).

[0068] The reactions for preparing the compounds described herein can be carried out in a suitable solvent that can be easily selected by those skilled in the art of organic synthesis. A suitable solvent can be substantially non-reactive with the starting materials (reactants), intermediates, or products at the temperature at which the reaction is carried out (e.g., a temperature in the 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 the specific reaction step can be selected by those skilled in the art.

[0069] The preparation of the compounds described herein can involve the protection and deprotection of various chemical groups. The need for protection and deprotection, as well as the selection of appropriate protecting groups, can be readily determined by those skilled in the art. The chemical properties of the protecting groups can be found, for example, in T.W. Greene and P.G.M. Wuts, Protective Groups in Organic Synthesis, 3 rd Ed., Wiley & Sons, Inc., New York (1999).

[0070] The reaction can be monitored according to any suitable method known in the art. For example, the formation of the product can be monitored by spectroscopic means such as nuclear magnetic resonance spectroscopy (e.g., 1 H or 13 C), infrared spectroscopy, spectrophotometry (e.g., UV-visible), mass spectrometry, or by chromatographic means such as high performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LCMS), or thin layer chromatography (TLC). The compounds can be purified by those skilled in the art by various methods including high performance liquid chromatography (HPLC) and normal phase silica chromatography.

[0071] In some embodiments, a precursor compound having the structure of formula (III) shown below may be provided, and a suitable radioisotope (e.g., a therapeutic radioisotope for preparing a compound of formula (I) or an imaging radioisotope for preparing a compound of formula (II)) may be loaded into the precursor compound via the chelate moiety A therein, whereby a compound of formula (I) (for therapeutic use) or a compound of formula (II) (for imaging use) is produced.

[0072]

Chemical formula

[0073] The hemiacetal unit in the compound of formula (III) may be in the open-chain aldehyde form. As a result, the compound of formula (III) may have the following structure.

Chemical formula

[0074] In any of formulas (III-1) to (III-6), each of A, X, L, R 1 , and R 2 is as described herein.

[0075] In some embodiments, as in the compound of formula (IIIa), R 1 is H and R 2 is C4 alkyl.

Chemical formula

[0076] In some specific examples, as in the compound of formula (IIIa-A), X is -CH2C(O)-.

Chemical formula

[0077] In another specific example, as in the compound of formula (IIIa-B), X is -NHC(S)-.

Chemical formula

[0078] Exemplary precursor compounds of formula (III) include those listed in Table 3.

Table 3-1

Table 3-2

Table 3-3

Table 3-4

[0079] Also, reference is made to WO2021 / 252664, the relevant disclosure of which is incorporated by reference for the subject matter and purposes referred to herein.

[0080] II. Pharmaceutical Compositions A compound of formula (I) and / or formula (II), or a pharmaceutically acceptable salt thereof, can be mixed with a pharmaceutically acceptable carrier to form a pharmaceutical composition for use in the granzyme B-targeted seranostics therapy disclosed herein. "Acceptable" means that the carrier must be compatible with the active ingredient of the composition (and preferably can stabilize the active ingredient) and not be harmful to the subject being treated. Suitable carriers include microcrystalline cellulose, mannitol, glucose, skim milk powder, polyvinylpyrrolidone, and starch, or combinations thereof.

[0081] 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 further include, but are not limited to, lubricants such as talc, magnesium stearate, and mineral oil, wetting agents, emulsifying agents and suspending agents, preservatives such as methyl and propyl hydroxybenzoates, sweetening agents, flavoring agents, or combinations thereof. For more information on acceptable pharmaceutical compositions, see Remington’s Pharmaceutical Sciences, 17 th th ed., Mack Publishing Company, Easton, Pa., 1985, p. 1418.

[0082] The pharmaceutical compositions disclosed herein can be formulated for a suitable route of administration, such as oral administration, parenteral administration, inhalation, topical administration, rectal administration, nasal administration, or buccal administration. As used herein, the term “parenteral” includes subcutaneous, intradermal, intravenous, intramuscular, intra-articular, intra-arterial, intrasynovial, intrasternal, intrathecal, intralesional, and intracranial injection or infusion techniques.

[0083] Injectable compositions can contain various carriers such as vegetable oils, dimethylactamide, dimethylformamide, ethyl lactate, ethyl carbonate, isopropyl myristate, ethanol, and polyols (such as glycerol, propylene glycol, liquid polyethylene glycol, etc.). In the case of intravenous injection, water-soluble antibodies can be administered by the drip method, whereby a pharmaceutical preparation containing the antibody and a physiologically acceptable excipient is infused. Physiologically acceptable excipients can include, for example, 5% dextrose, 0.9% saline, Ringer's solution, or other suitable excipients. Intramuscular preparations, for example, sterile preparations of suitable soluble salt forms of antibodies can be dissolved and administered to pharmaceutical excipients such as water for injection, 0.9% saline, or 5% glucose solution.

[0084] For oral administration, the composition can be in the form of tablets or capsules prepared by conventional means with acceptable excipients such as binders (e.g., pre-gelatinized corn starch, polyvinylpyrrolidone, or hydroxypropylmethylcellulose); fillers (e.g., lactose, microcrystalline cellulose, or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc, or silica); disintegrants (e.g., potato starch or sodium glycolate); or wetting agents (e.g., sodium lauryl sulfate). Tablets can be coated by methods well known in the art.

[0085] In some embodiments, the above compounds provided by the present invention, 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.

[0086] 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, thickening agents, etc. may be required or desirable.

[0087] In the preparation of the pharmaceutical compositions provided herein, the active ingredient is typically mixed with, diluted by, or enclosed within a carrier such as, for example, in the form of capsules, sachets, paper, or other containers. When the excipient functions as a diluent, it can be a solid, semi-solid, or liquid material that functions as a vehicle, carrier, or medium for the active ingredient.

[0088] Thus, the pharmaceutical compositions can be in the form of tablets, pills, powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols (as a solid or in a liquid medium), ointments, soft and hard gelatin capsules, suppositories, sterile injectable solutions, and sterile packaged powders.

[0089] III. Celanostics Therapy Any of the granzyme B-targeted therapeutic compounds of formula (I) disclosed herein can be used in celanostics therapy in cancer therapy, for example, together with a granzyme B-targeted imaging compound such as a compound of formula (II) disclosed herein.

[0090] To carry out the methods disclosed herein, an effective amount of a pharmaceutical composition comprising a compound of formula (I) disclosed herein or a pharmaceutically acceptable salt thereof can be administered to a subject in need of treatment via a suitable route. In some embodiments, such methods can further comprise administering to the subject an effective amount of a granzyme B-targeted imaging agent, for example, any of the compounds of formula (I) disclosed herein, or a pharmaceutically acceptable salt thereof, which can be carried out prior to the administration of the therapeutic agent. In some embodiments, the subject may have received or be receiving immunotherapy, for example, as disclosed herein.

[0091] As used herein, the term "subject" refers to any mammal, such as a mouse, rat, other rodent, rabbit, dog, cat, pig, cow, sheep, horse, non-human primate, or human. In some embodiments, the subject is a human.

[0092] In some embodiments, the subject is a human patient having cancer. In some cases, the cancer is a solid tumor. Examples include, but are not limited to, brain, breast cancer (e.g., HER2+, ER+ / 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 cancer, multiple myeloma, pancreatic cancer, prostate cancer, glioblastoma, hepatocellular carcinoma, urothelial carcinoma, esophageal cancer, gastroesophageal cancer, gastric cancer, squamous cell carcinoma of the head and neck, epithelial ovarian cancer (EOC), primary peritoneal cancer, fallopian tube cancer, Merkel cell carcinoma, nasopharyngeal cancer, adrenocortical carcinoma, meningioma, neuroblastoma, retinoblastoma, osteosarcoma, rhabdomyosarcoma, Ewing sarcoma, liposarcoma, fibrosarcoma, leiomyosarcoma, undifferentiated neuroectodermal tumor, squamous cell carcinoma of the vagina, and squamous cell carcinoma of the vulva. In some examples, the cancer is colon cancer.

[0093] In other embodiments, the cancer is a hematological cancer (e.g., leukemia, lymphoma, etc.). Examples include, but are not limited to, hairy cell leukemia, Kaposi sarcoma, follicular lymphoma, chronic myeloid leukemia, cutaneous T cell lymphoma, peripheral T cell lymphoma, T cell prolymphocytic leukemia, classical Hodgkin lymphoma, B cell non-Hodgkin lymphoma, chronic lymphocytic leukemia, acute myeloid leukemia, myelodysplastic syndrome, primary myelofibrosis, myelofibrosis transformed from essential thrombocythemia, and myelofibrosis transformed from polycythemia vera.

[0094] As used herein, "effective amount" refers to the amount of each active agent required to confer a therapeutic effect on a subject, alone or in combination with one or more other active agents. It will be apparent to those skilled in the art how to determine whether a given amount of an antibody has achieved a therapeutic effect. The effective amount will vary depending on individual patient parameters, including the particular condition being treated, the severity of the condition, age, health status, size, gender, and weight, the duration of treatment, the nature of any combination therapy (if any), the particular route of administration, and similar factors within the knowledge and expertise of the medical practitioner. These factors are well known to those skilled in the art and can be addressed by routine experimentation only. Generally, the maximum dosage of the individual components or combinations thereof, i.e., the highest safe dosage in accordance with sound medical judgment, is preferably used.

[0095] In some instances, the maximum allowable dosage of a checkpoint inhibitor can be used in the methods disclosed herein. Alternatively or additionally, the minimum effective dosage of any of the therapeutic granzyme B-targeting molecules of formula (I) disclosed herein can be used in such methods.

[0096] Generally, empirical considerations such as half-life will contribute to the determination of dosage. For example, antibodies that are compatible with the human immune system, such as humanized or fully human antibodies, can be used to extend the half-life of the antibody and prevent the antibody from being attacked by the host immune system. The dosing frequency can be determined and adjusted over the course of therapy and generally need not necessarily be based on the treatment and / or suppression and / or improvement and / or delay of the target disease / disorder. Alternatively, a sustained continuous release formulation of the antibody may be appropriate. A variety of formulations and devices for achieving sustained release are known in the art.

[0097] In some embodiments, the dosage of one of the compounds or a pharmaceutically acceptable salt thereof to be administered to a subject or individual is from about 1 μg to about 2 g, such as, for example, from about 1 μg to about 2 g, from about 1 μg to about 1000 mg, from about 1 μg to about 500 mg, from about 1 μg to about 100 mg, from about 1 μg to about 50 mg, from about 1 μg to about 1 mg, from about 1 μg to about 500 μg, from about 1 μg to about 100 μg, from about 1 μg to about 10 μg, from about 10 μg to about 2 g, such as, for example, from about 10 μg to about 2 g, from about 10 μg to about 1000 mg, from about 10 μg to about 500 mg, from about 10 μg to about 100 mg, from about 10 μg to about 50 mg, from about 10 μg to about 1 mg, from about 10 μg to about 500 μg, from about 10 μg to about 100 μg, from about 100 μg to about 2 g, such as, for example, from about 100 μg to about 2 g, from about 100 μg to about 1000 mg, from about 100 μg to about 500 mg, from about 100 μg to about 100 mg, from about 100 μg to about 50 mg, from about 100 μg to about 1 mg, from about 100 μg to about 500 μg, from about 500 μg to about 2 g, such as, for example, from about 500 μg to about 2 g, from about 500 μg to about 1000 mg, from about 500 μg to about 500 mg, from about 500 μg to about 100 mg, from about 500 μg to about 50 mg, from about 500 μg to about 1 mg, from about 1 mg to about 2 g, from about 1 mg to about 1000 mg, from about 1 mg to about 500 mg, from about 1 mg to about 100 mg, from about 1 mg to 50 mg, or from about 50 mg to about 500 mg.

[0098] As used herein, the terms “treating” or “treatment” refer to one or more of (1) inhibiting cancer, e.g., inhibiting cancer in an individual who is experiencing or showing the pathology or symptoms of cancer (i.e., arresting further development of the pathology and / or symptoms), and (2) improving cancer, e.g., improving cancer in an individual who is experiencing or showing the pathology or symptoms of cancer (i.e., reversing the pathology and / or symptoms), such as reducing the severity of cancer or reducing or alleviating one or more symptoms of cancer.

[0099] In some embodiments, the subject to be treated can be identified and / or diagnosed as having cancer prior to administration of the therapeutic compound of formula (I). In some cases, a subject having a target cancer can be identified by routine medical tests such as clinical tests, organ function tests, CT scans, or ultrasound tests. In some embodiments, the subject to be treated by the methods described herein can be a human cancer patient who has received or is receiving anti-cancer therapy such as chemotherapy, radiation therapy, immunotherapy, or surgery.

[0100] In some cases, the subject is treated with a granzyme B-targeted imaging agent such as any of the imaging compounds of formula (II) disclosed herein for cancer imaging prior to treatment with the therapeutic compound of formula (I).

[0101] A human patient to be administered any of the cancer therapies disclosed herein (e.g., the seranostics therapy disclosed herein) may have received pre-treatment with an immunotherapeutic agent or may be administered immunotherapy concurrently. Immunotherapeutic agents generally elicit immune effector cells and molecules that 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 function as an effector of the therapy or can recruit other cells to kill the cell. Exemplary effector cells include, but are not limited to, cytotoxic T cells and NK cells.

[0102] Exemplary 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 such as MIP-1, MIP-1β, MCP-1, RANTES, IL-8; or growth factors such as FLT3 ligand), antigenic peptides, polypeptides, or proteins, or autologous or allogeneic tumor cell compositions (see, e.g., Ravindranath & Morton, International reviews of immunology, 7.4(1991):303-329), hormonal therapy, corticosteroids, progestins (e.g., hydroxyprogesterone caproate, medroxyprogesterone acetate, and megestrol acetate), estrogens (e.g., diethylstilbestrol and ethinyl estradiol), anti-estrogens (e.g., testosterone propionate and fluoxymesterone), anti-androgens (e.g., flutamide), and gonadotropin-releasing hormone analogs (e.g., leuprolide). Additional immunotherapeutic agents 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).

[0103] In some embodiments, the immunotherapeutic agent is an immune checkpoint inhibitor, such as a PD1 inhibitor (e.g., an anti-PD-1 antibody such as nivolumab, pembrolizumab, or cemiplimab; or an anti-PD-L1 antibody such as atezolizumab, avelumab, or durvalumab), a CTLA-4 inhibitor (e.g., an anti-CTLA-4 antibody such as ipilimumab), or a LAG-3 inhibitor (e.g., an anti-LAG-3 antibody such as relatlimab). In other embodiments, the immunotherapeutic agent can be CAR-T cells, such as axicabtagene ciloleucel or brexucabtagene autoleucel.

[0104] The therapeutic agents provided herein can be effective over a wide range of dosages and are generally administered in an effective amount. However, it will be understood that the amount of the therapeutic agent actually administered will typically be determined by a physician according to relevant circumstances including the condition being imaged, the route of administration selected, the compound actually being administered, the age, weight, and response of the individual subject, the severity of the subject's symptoms, and the like.

[0105] IV. Kits for Granzyme B-Targeted Theranostics Also included by the present disclosure are kits (e.g., pharmaceutical packs) for Granzyme B-targeted cancer therapy and optionally for Granzyme B-targeted cancer imaging. The kits provided may include a container (e.g., a vial, ampule, bottle, syringe, and / or dispenser package, or other suitable container) in which a pharmaceutical composition disclosed herein may be disposed. In some embodiments, the kits provided may further include a second container optionally containing a pharmaceutical excipient for dilution or suspension of the pharmaceutical composition. In some embodiments, combining the pharmaceutical compositions provided in the first and second containers forms a single unit dosage form. In some embodiments, the kit may include additional containers containing one or more additional therapeutic agents disclosed herein, such as immunotherapeutic agents.

[0106] In certain embodiments, the kits described herein further include instructions for using the compounds or compositions included in the kit. The kits described herein may include information required by regulatory agencies such as the U.S. Food and Drug Administration (FDA). In certain embodiments, the information included in the kit is prescription information. In certain embodiments, the kit and instructions are provided for imaging granzyme B and / or treating and / or reducing the risk in a subject in need thereof. The kits described herein may include one or more additional pharmaceutical agents described herein as separate compositions.

[0107] Definitions It is understood that the terms used herein are for the purpose of describing particular embodiments and are not intended to be limiting. Further, any methods, devices, and materials similar or equivalent to those described herein may be used in the practice or testing of the present invention, but the preferred methods, devices, and materials are described herein. In addition to the above, when used in this specification and the appended claims, the following terms have the indicated meanings unless otherwise specified. "Amino" refers to the -NH2 radical. "Cyano" refers to the -CN radical. "Hydroxyl" refers to the -OH radical. "Imino" refers to the =NH substituent. "Nitro" refers to the -NO2 radical. "Oxo" refers to the =O substituent. "Thioxo" refers to the =S substituent. "Trifluoromethyl" refers to the -CF3 radical.

[0108] "Alkyl" refers to a straight-chain, saturated, acyclic, monovalent hydrocarbon radical having 1 to 6 carbon atoms, such as methyl, ethyl, n-propyl, 1-methylethyl (iso-propyl), n-butyl, n-pentyl, 1,1-dimethylethyl (t-butyl), 3-methylpentyl, 2-methylpentyl, etc., or a branched, saturated, acyclic, monovalent hydrocarbon radical, which is bonded to the rest of the molecule by a single bond. The alkyl moiety may be unsubstituted. Alternatively, the alkyl moiety may be optionally substituted. An optionally substituted alkyl radical is independently selected from the group consisting of halo, cyano, nitro, aryl, cycloalkyl, heterocyclyl, heteroaryl, oxo, 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 (wherein t is 1 or 2), -S(O) t OR 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), and is an alkyl radical optionally substituted by 1, 2, 3, 4, or 5 substituents as allowed by valence, 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, each R 5is independently alkyl, haloalkyl, cycloalkyl, aryl, or heteroaryl.

[0109] "Cycloalkyl" refers to a stable non-aromatic monocyclic or polycyclic hydrocarbon radical having 3 to 15 carbon atoms, preferably 3 to 10 carbon atoms, which is saturated or unsaturated and is bonded to the remainder of the molecule by a single bond. The polycyclic hydrocarbon radical is a bicyclic, tricyclic, or tetracyclic ring system. The unsaturated cycloalkyl contains 1, 2, or 3 carbon-carbon double bonds and / or 1 carbon-carbon triple bond. Examples of monocyclic cycloalkyl radicals include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. Examples of polycyclic cycloalkyl radicals include adamantyl, norbornyl, decalinyl, etc. The cycloalkyl moiety may be unsubstituted. Alternatively, the cycloalkyl moiety may be optionally substituted. Optionally substituted cycloalkyl is independently 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) t R 5 (wherein t is 1 or 2), -R 4 -S(O) t OR 5(wherein t is 1 or 2), -R 4 -S(O) p R 5 (wherein p is 0, 1, or 2), and -R 4 -S(O) t N(R 3 )2 (wherein t is 1 or 2), and is a cycloalkyl radical optionally substituted by 1, 2, 3, 4, or 5 substituents selected from the group consisting of, and each R 3 is independently hydrogen, alkyl, haloalkyl, cycloalkyl, aryl, heterocyclyl, or heteroaryl, and each R 4 is independently a direct bond, or a straight-chain or branched alkylene or alkenylene chain, and each R 5 is independently alkyl, haloalkyl, cycloalkylalkyl, aryl, aralkyl, heterocyclyl, or heteroaryl.

[0110] In some embodiments, the preparation of the compounds can involve, for example, the addition of an acid or base that affects the catalysis of the desired reaction or the formation of a salt form such as an acid addition salt.

[0111] Exemplary acids can be inorganic or organic acids, including but not limited to strong and weak acids. Some exemplary acids include hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, p-toluenesulfonic acid, 4-nitrobenzoic acid, methanesulfonic acid, benzenesulfonic acid, trifluoroacetic acid, and nitric acid. Some weak acids include acetic acid, propionic acid, butanoic acid, benzoic acid, tartaric acid, pentanoic acid, hexanoic acid, heptanoic acid, octanoic acid, nonanoic acid, and decanoic acid, but are not limited thereto.

[0112] Exemplary bases include lithium hydroxide, sodium hydroxide, potassium hydroxide, lithium carbonate, sodium carbonate, potassium carbonate, and sodium bicarbonate. Some exemplary strong bases include, but are not limited to, hydroxides, alkoxides, metal amides, metal hydrides, metal dialkylamides, and arylamines. Examples of alkoxides include lithium, sodium, and potassium salts of methyl, ethyl, and t-butoxide. Examples of metal amides include sodium amide, potassium amide, and lithium amide. Examples of metal hydrides include sodium hydride, potassium hydride, and lithium hydride. Examples of metal dialkylamides include lithium, sodium, and potassium salts of methyl, ethyl, n-propyl, iso-propyl, n-butyl, tert-butyl, trimethylsilyl, and cyclohexyl-substituted amides.

[0113] As used herein, the phrase "pharmaceutically acceptable salt" refers to derivatives of the disclosed compounds in which the parent compound is modified by converting an existing acidic or basic 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, and alkali or organic salts of acidic residues such as carboxylic acids. Pharmaceutically acceptable salts of the present application include, for example, conventional non-toxic salts of the parent compound formed from non-toxic inorganic or organic acids. Pharmaceutically acceptable salts of the present application can be synthesized from parent compounds containing basic or acidic moieties by conventional chemical methods. Generally, such salts can be prepared by reacting the free acid or base form of these compounds with a stoichiometric amount of an appropriate base or acid in water or an organic solvent, or a mixture of the two, and generally, non-aqueous media such as ether, ethyl acetate, alcohols (e.g., methanol, ethanol, isopropanol, or butanol), or acetonitrile (MeCN) are preferred. A list of suitable salts is provided in Remington’s Pharmaceutical Sciences,17 thIt can be found in the 1985 edition, Mack Publishing Company, Easton, Pa., 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.

[0114] In some embodiments, the compounds provided by the present invention, or salts thereof, 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 separation can include, for example, a composition concentrated in the compounds provided herein. Substantial separation can include compositions containing at least about 50 wt%, at least about 60 wt%, at least about 70 wt%, at least about 80 wt%, at least about 90 wt%, at least about 95 wt%, at least about 97 wt%, or at least about 99 wt% of the compounds provided herein, or salts thereof. Methods for isolating compounds and their salts are conventional in the art.

[0115] As used herein, the expressions "ambient temperature" and "room temperature" or "rt" are understood in the art and generally refer to, for example, the reaction temperature, i.e., a temperature of about room temperature at which the reaction is carried out, for example, about 20°C to about 30°C.

[0116] Without further elaboration, it is believed that those skilled in the art can make maximum use of the present invention based on the above description. Accordingly, the following specific embodiments should be construed as merely illustrative and not limiting the remainder of the disclosure in any way. All publications cited herein are incorporated by reference for the purposes or subject matter referred to herein.

[0117] Example 1: Synthesis of an exemplary compound of formula (I) This example illustrates an exemplary synthetic process for producing the compound of formula (I) disclosed herein. Additional information for preparing the compound of formula (I) can be found, for example, in WO2021 / 252664, the relevant disclosure of which is incorporated by reference for the subject matter and purposes herein.

[0118] (i) Synthesis of the peptide linker Using H-Asp(OtBu)-H resin, peptides can be synthesized according to standard Fmoc solid-phase peptide synthesis procedures. The final peptide is deprotected and cleaved from the resin according to a two-step procedure: 1) treatment with either trifluoroacetic acid (TFA) at room temperature for 2 hours or TFA / dichloromethane (DCM) at room temperature overnight, followed by concentration, and 2) treatment with acetonitrile / water (60:40) containing 0.1% TFA at 60 °C for 1 hour. The crude peptide can be either concentrated or lyophilized and then subjected to preparative HPLC purification (using 0.1% formic acid or 0.1% TFA in a water / acetonitrile mobile phase). The product-containing fractions are collected and lyophilized to obtain the peptide as a white fluffy solid.

[0119] (ii) Synthesis of the granzyme B target compound of formula (I) Using the following general procedure, the compounds of formula (I) disclosed herein can be formed. To a reaction vial containing the peptide precursor and a stir bar, an equivalent of 20 mM AlCl3 in 0.1 M NaOAc (pH ~4.5) and 100 mM NaF in H2O (1.5 - 3.0 equivalents relative to the peptide) was added. Then, acetonitrile (0 - 34% of the total reaction volume) was added. The mixture was heated to 100 °C for 15 - 30 minutes. The acetonitrile was removed under reduced pressure and the aqueous solution was purified by either a C18 ISCO column or a C18 HPLC preparative column (using water and acetonitrile containing 0.1% formic acid as the eluent). The appropriate fractions were collected and lyophilized to obtain the peptide AlF complex as a white fluffy solid.

[0120] (iii) Radiosynthesis of the target compound Typical * For the R-GZB compound RCY, using a starting activity of 0.5 - 2.0 Ci, the synthesis time is 75 ± 10 minutes and it ranges from 5.6 - 63%. A precursor (e.g., 0.2 - 0.6 mg) in an acetic acid / sodium acetate aqueous buffer (e.g., 200 - 400 μL, 1 mol / L, pH 3.0 - 5.0), AlCl3·6H2O (e.g., 34 - 82 μg, 100 - 240 nmol), and acetonitrile (e.g., 25 - 50% of the total reaction mixture volume) is added to a reaction vial. * The R activity is retained on an anion exchange resin (e.g., Sep-Pak Accell Plus QMA Carbonate Plus Light Cartridge, 46 mg of adsorbent per cartridge, 40 μm particle size, Waters part number 186004540) conditioned with [5 mL of 0.9% saline, followed by 5 mL of WFI (Water for Injection)]. The retained * R is eluted from the cartridge into the reaction vial with 0.9% saline (e.g., 0.5 - 0.8 mL). The resulting mixture is heated for a certain time (e.g., 15 minutes) (e.g., 105 °C), then cooled (e.g., 60 °C), and then diluted with water (e.g., 1.0 - 5.0 mL, HPLC grade). The resulting crude product is loaded onto a semi-preparative reverse phase HPLC column (e.g., Agilent ZORBAX Eclipse XDB-C18, 5 μm, 9.4 mm × 250 mm, part number 990967-202) (e.g., a mobile phase containing an aqueous acetonitrile solution (8 - 20%), pH 1 - 8) for purification. The purified * The HPLC fraction containing the R-GZB compound is diluted with a 0.5% w / v aqueous sodium ascorbate solution (e.g., 30 - 50 mL), and then passed through a reverse phase cartridge (e.g., Sep-Pak® Light C18 Cartridge, 130 mg of adsorbent per cartridge, particle size 55 - 105 μm, Waters part number WAT0523501) conditioned with [5 mL of ethanol (USP grade), followed by 5 mL of water (HPLC grade)]. The retained *The R-GZB was washed with an aqueous solution of sodium ascorbate at 0.5% w / v (e.g., 5 - 15 mL), and eluted from the cartridge using ethanol (e.g., 1.0 - 1.5 mL) into a formulation vial containing sodium ascorbate at 0.5% w / v in 0.9% saline (e.g., 6 - 10 mL). Subsequently, the C18 cartridge was rinsed with additional sodium ascorbate at 0.5% w / v in 0.9% saline (e.g., 3.0 - 3.5 mL), and the rinse solution was collected into the formulation vial. A certain amount of diluent (90% v / v 0.9% saline containing 10% v / v ethanol and 0.5% w / v sodium ascorbate) may be added to adjust the strength of the product.

[0121] To prepare a sterile product, the resulting product (in 90% v / v 0.9% saline containing 10% v / v ethanol and 0.5% w / v sodium ascorbate) of * R-GZB) was sterile filtered through a 0.22 μm filter (e.g., Millex® GV Sterile Filter, Millipore part number SLGV033RS; Millex GV 25 mm Sterile Filter, Millipore part number SLGVV255F; and Millex LG 25 mm Sterile Filter, Millipore part number SLLG025SS) into a bulk product vial.

[0122] Other embodiments All features disclosed in this specification may be combined in any combination. Each feature disclosed in this specification may be replaced by an alternative feature that serves the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is only an example of a general series of equivalent or similar features.

[0123] From the above specification, those skilled in the art can easily identify the essential features of the present invention and make various changes and modifications to the present invention to adapt to various applications and conditions without departing from the spirit and scope thereof. Accordingly, other embodiments are also within the scope of the claims.

[0124] Equivalents While several embodiments of the present invention have been described and illustrated herein, those skilled in the art will readily envision various other means and / or structures for performing the function and / or obtaining one or more of the results and / or advantages described herein, and each such variation and / or modification is to be regarded as within the scope of the embodiments of the invention described herein. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are exemplary, and that actual parameters, dimensions, materials, and / or configurations will depend upon the particular application for which the teachings of the invention are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Accordingly, the foregoing embodiments are presented by way of example only, and it is to be understood that the embodiments of the invention may be practiced otherwise than as specifically described and claimed within the scope of the appended claims and their equivalents. Embodiments of the inventions disclosed herein are directed to each and every individual feature, system, article, material, kit, and / or method described herein. Additionally, any combination of two or more such features, systems, articles, materials, kits, and / or methods is included within the scope of the inventions disclosed herein so long as such features, systems, articles, materials, kits, and / or methods are not mutually inconsistent.

[0125] All definitions, as defined and used herein, are to be understood to control the dictionary definitions, definitions in the documents incorporated by reference, and / or the ordinary meaning of the defined terms.

[0126] All references, patents, and patent applications disclosed herein are hereby incorporated by reference in their entirety for the subject matter for which each is cited, and in some cases they may encompass the entire disclosure of this document.

[0127] As used in this specification and the claims, the indefinite articles "a" and "an" should be understood to mean "at least one" unless the contrary is clearly indicated.

[0128] As used herein in this specification and the claims, the phrase "and / or" should be understood to mean "either or both" of the elements so joined together, i.e., elements that in some cases exist conjunctively and in other cases exist disjunctively. A plurality of elements listed with "and / or" should be construed in the same manner, i.e., as "one or more" of the elements so joined together. Other elements other than those specifically identified by the "and / or" clause may optionally exist, whether or not they are related to those specifically identified elements. Thus, by way of non-limiting example, a reference to "A and / or B", when used in conjunction with open-ended language such as "comprising", can in one embodiment refer to only A (optionally including elements other than B), in another embodiment can refer to only B (optionally including elements other than A), and in yet another embodiment can refer to both A and B (optionally including other elements), and so on.

[0129] As used herein in the specification and 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" should be construed as inclusive, i.e., including at least one of several elements or a list of elements, but including more than one, and optionally including additional unlisted items. Only terms such as "only one of" or "only one of", or "consisting of" when used in the claims, refer to the inclusion of only one element of several elements or a list of elements. Generally, as used herein, the term "or" is to be construed as indicating an exclusive alternative (i.e., "one or the other, but not both") only when followed by exclusive terms such as "either", "only one of", "only one of", or "only one of". When used within the claims, "consisting essentially of" shall have the ordinary meaning as used in the field of patent law.

[0130] As used in this specification and the claims, the phrase "at least one" with respect to a list of one or more elements means at least one element selected from any one or more of the elements in the list of elements, but does not necessarily include at least one of every element specifically recited in the list of elements, and is to be understood not to exclude any combinations of elements in the list of elements. This definition also permits that elements other than those specifically identified in the list of elements referred to by the phrase "at least one" may optionally be present, whether or not they are related to those specifically identified elements. Thus, by way of non-limiting 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") can, in one embodiment, refer to at least one A, optionally including two or more, without including B (and optionally including elements other than B), in another embodiment, can refer to at least one B, optionally including two or more, without including A (and optionally including elements other than A), and in yet another embodiment, can refer to at least one A, optionally including two or more, and at least one B, optionally including two or more (and optionally including other elements), and so on.

[0131] Also, it should be understood that, unless explicitly indicated to the contrary, in any method claimed in this specification that includes two or more steps or acts, the order of the method steps or acts is not necessarily limited to the order in which the method steps or acts are recited.

Claims

1. A method for treating cancer in a subject, said method comprising a compound of formula (I): 【Chemical 1】 、 or a pharmaceutically acceptable salt thereof, wherein M comprises a therapeutic radioisotope, A is a chelating moiety that chelates M, X is -CH ２ C(NH)-, -CH ２ C(O)-, -CH ２ is selected from the group consisting of C(S)-, -NHC(NH)-, -NHC(O)-, -NHCS)-, -OC(NH)-, -OC(O)-, and -OC(S)-, and optionally, X is -CH ２ C(O)- or -NHCS)-, L is a peptide linker having inclusively from 1 to 6 amino acid residues, R １ is H or C １－６ is alkyl, and optionally, R １ is H or methyl R ２ is C １－６ alkyl or C ３－６ administering to a subject in need of treating cancer an effective amount of a pharmaceutical composition comprising a compound, or a pharmaceutically acceptable salt thereof, wherein R is C said subject having received or being receiving a treatment comprising an immunotherapeutic agent.

2. A method for treating cancer in a subject, comprising (i) administering an immunotherapeutic agent to a subject in need of treating cancer, and (ii) after step (i), a compound of formula (I): 【Chemical 2】 、 or a pharmaceutically acceptable salt thereof, wherein M comprises a therapeutic radioisotope, A is a chelating moiety that chelates M, X is -CH ２ C(NH)-, -CH ２ C(O)-, -CH ２ C(S)-, -NHC(NH)-, -NHC(O)-, -NHCS)-, -OC(NH)-, -OC(O)-, and -OC(S)-, and optionally, X is -CH ２ C(O)- or -NHCS)-, L is a peptide linker having inclusively from 1 to 6 amino acid residues. R １ is H or C １－６ is alkyl, and optionally, R １ is H or methyl R ２ is C １－６ alkyl or C ３－６ administering to the subject an effective amount of a pharmaceutical composition comprising a compound, or a pharmaceutically acceptable salt thereof, wherein R is C

3. The method according to claim 1 or 2, wherein said compound is a compound of formula (Ia). 【Chemical 3】

4. The method according to any one of claims 1 to 3, wherein L has inclusively from 1 to 3 amino acid residues.

5. The method according to claim 4, wherein L has 3 amino acid residues.

6. The method according to any one of claims 3 to 5, wherein said compound is a compound of formula (Ia-A): 【Chemical Formula 4】 、 or a compound of formula (Ia-B): [Chemical Formula 5] .

7. The method according to any one of claims 1 to 6, wherein said chelating moiety A is 1,4,7-triazacyclononane-4,7-diyl diacetic acid (NODA), 1,4,7-triazacyclononane-N,N',N''-triacetic acid (NOTA), 2-S-(4-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid (p-SCN-Bn-NOTA), or 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA).

8. The therapeutic radioisotope is ９０ Y, １７７ Lu, ２２５ Ac, ４７ Sc, １３１ I, １５３ Sm, ２１１ At, ２１２ Pb, ２１２ Bi, ２２３ Ra, or ２２７ Th, the method according to any one of claims 1 to 7.

9. wherein the chelate moiety is NOTA or DOTA, and the therapeutic radioisotope is ９０ Y, １７７ Lu, or ２２５ Ac, the method according to any one of claims 1 to 7.

10. wherein the chelate moiety is NODA and the therapeutic radioisotope is ４７ Sc or ６７ Cu, the method according to any one of claims 1 to 7.

11. The compound of formula (I) is [Chemical Formula 6] wherein R* is １７７ Lu, ９０ Y, ２２５ Ac, or ２１３ Bi, the method according to claim 1.

12. The method according to any one of claims 1 to 11, wherein said immunotherapeutic agent is optionally a PD1 inhibitor, an immune checkpoint inhibitor, or a genetically engineered T cell expressing a chimeric antigen receptor (CAR).

13. The method according to any one of claims 1 to 12, further comprising administering an imaging agent to said subject to image granzyme B prior to administering said pharmaceutical composition comprising the compound of formula (I).

14. The imaging agent is a compound of formula (I): 【Chemical Formula 7】 or a pharmaceutically acceptable salt thereof, wherein M includes a contrast agent, A is a chelating moiety that chelates M, X is -CH ２ C(NH)-, -CH ２ C(O)-, -CH ２ selected from the group consisting of C(S)-, -NHC(NH)-, -NHC(O)-, -NH C(S)-, -OC(NH)-, -OC(O)-, and -OC(S)-, and optionally, X is -CH ２ C(O)- or -NHC(S)-, L is a peptide linker having inclusively 1 to 6 amino acid residues, R １ is H or C １－６ is alkyl, and optionally, R １ is H or methyl, R ２ is C １－６ alkyl or C ３－６ cycloalkyl, the method according to claim 13.

15. The method according to claim 14, wherein the imaging agent is selected from those listed in Table 2.

16. The method according to claim 15, wherein the imaging agent is a compound selected from the group consisting of the following. 【Chemical 8】

17. The method according to any one of claims 1 to 16, wherein the subject is further treated with one or more additional therapeutic agents.

18. The method according to claim 17, wherein the one or more additional therapeutic agents are selected from the group consisting of anti-inflammatory agents, steroids, immunotherapeutic agents, chemotherapeutic agents, and therapeutic antibodies.

19. A seranostics agent, comprising: (i) a diagnostic compound for imaging granzyme B; and (ii) a therapeutic compound having the structure of formula (I) according to any one of claims 1 to 11.

20. The seranostics agent according to claim 19, wherein the diagnostic compound has the structure according to any one of claims 14 to 16.

21. A pharmaceutical composition comprising a therapeutic compound and a pharmaceutically acceptable carrier, wherein the therapeutic compound is as described in any one of claims 1 to 11.

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