Prodrugs for Granzyme B-Specific Compounds and Their Use

Abstract

Compounds that bind to granzyme B and can contain a radioactive moiety, such as the compound of formula (I), and pharmaceutical compositions comprising the same. Uses of the compound and pharmaceutical composition in cancer treatment, as well as kits and methods for cancer treatment, are also provided herein.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims the benefits of U.S. Provisional Patent Application No. 63 / 506,732, filed on June 7, 2023, the contents of which are incorporated herein by reference in their entirety.

[0002] Technical field This disclosure relates to prodrug radiocompounds that can be converted into an active form useful as a therapeutic agent in vivo, and more particularly to prodrug radiocompounds that can be converted into a granzyme B-specific active compound that can eliminate cancer cells containing it. [Background technology]

[0003] Granzyme B is the most commonly found serine protease in the granules of natural killer cells and cytotoxic T cells. Granzyme B is released along with the membrane pore-forming protein perforin at immunological synapses formed between T cells and their targets. Subsequently, a portion of the released granzyme B enters cancer cells, primarily via perforin pores, where it 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 responding to immunotherapy. [Overview of the Initiative] [Problems that the invention aims to solve]

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

[0005] This disclosure is based, at least in part, on the development of prodrug compounds that can be converted, for example, in vivo to active granzyme B (GZB) conjugate compounds. Such prodrug compounds (i.e., pro-forms of GZB conjugate compounds) exhibit excellent features such as the generation of a single isomer, synthesis with reliable stereochemical results, easy release of an active GZB conjugate compound in vivo, or a combination thereof. Such pro-forms of granzyme B (GZB) conjugate compounds can be used, for example, when targeting GZB for therapeutic purposes.

[0006] 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.

[0007] In some embodiments, compounds having the structure of formula (I) or their stereoisomers, tautomers, or salts are provided herein.

[0008] [ka]

[0009] In formula (I), M represents the radioactive portion; A is the chelater that chelates the radioactive portion of M; B is selected from the group consisting of aryl, heteroaryl, cycloalkyl, and heterocyclyl; B may also be a 6-membered ring; X is -CH2C(NH)-, -CH2C(O)-, -CH2C(S)-, -NHC(NH)-, -NHC(O)-, -NHC(S)-, -OC(NH)-, -OC(O)-, and -OC(S)-, and X may also be -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)-; Z may be -CH2- or -CH2C(O)-; L is a peptide linker having 1 to 6 amino acid residues including the values at both ends; R 1 is H or C 1~6 alkyl, and R 1 may be H or methyl; R 2 is C 1~6 alkyl or C 3~6 cycloalkyl; R 3 is C 1~6 alkyl.

[0010] In some embodiments, R 2 is C 1~6 alkyl. In some examples, R 2 is C4 alkyl. In a specific example, R 2 is sec-butyl (-CH(CH3)CH2CH3).

[0011] In some embodiments, the compound is of formula (Ia):

[0012]

Chemical formula

[0013] For any of the compounds of formula (I), for example, in the compound of formula (Ia), X may be -CH2C(O)-.

[0014] In some examples, the compound is of formula (Ib):

[0015]

Chemical formula

[0016] In any of the compounds of formula (I), for example, the compounds of formula (Ia) or formula (Ib) provided herein, R 3 It is a C1 alkyl group. For example, R 3 R is methyl (-CH3). In any of the compounds of formula (I), for example, in the compounds of formula (Ia) or formula (Ib) provided herein, R 3 It is a C2 alkyl group. For example, R 3 It is ethyl (-CH2CH3). In some examples, the compound is of formula (Ic):

[0017] [ka] That is the case.

[0018] In any of the compounds of formula (I), for example, the compounds of formula (Ia), formula (Ib), or formula (Ic), B can be a six-membered ring.

[0019] Alternatively, or in addition, in any of the compounds of formula (I), for example, the compounds of formula (Ia), formula (Ib), or formula (Ic) provided herein, Z may be -CH2- or -CH2C(O)-.

[0020] In some examples, the compound has the structure of formula (Ic-A), (Ic-B), or (Ic-C):

[0021] [ka] It has one of the following.

[0022] In some examples, the compounds have the structures of formula (Ic-Aa), (Ic-Ab), (Ic-Ba), (Ic-Bb), (Ic-Ca), and (Ic-Cb):

[0023] [ka]

[0024] [ka] It has one of the following.

[0025] In some examples, the compound is given by formula (Id):

[0026] [ka] It has.

[0027] In any of the compounds of formula (I), for example, the compounds of formula (Ia), formula (Ib), formula (Ic), or formula (Id) provided herein, the chelate A may be 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), a restricted complexing agent (RESCA), or MACROPA.

[0028] In any of the compounds of formula (I), for example, the compounds of formula (Ia), formula (Ib), formula (Ic), or formula (Id) provided herein, L may be a peptide having 1 to 5 amino acid residues (1, 2, 3, 4, or 5 including the values ​​at both ends).

[0029] In any of the compounds of formula (I), for example, in the compounds of formula (Ia), formula (Ib), formula (Ic), or formula (Id) provided herein, L may be a peptide having 1 to 3 amino acid residues (1, 2, or 3 including the values ​​at both ends). In any of the compounds of formula (I), for example, in the compounds of formula (Ia), formula (Ib), formula (Ic), or formula (Id) provided herein, L may be a peptide having 3 to 6 amino acid residues (3, 4, 5, or 6 including the values ​​at both ends).

[0030] In any of the compounds of formula (I), for example, the compounds of formula (Ia), formula (Ib), formula (Ic), or formula (Id) provided herein, L may have one or more non-naturally occurring amino acid residues. Exemplary peptides of L are listed below: Gly, Gly-Gly, Gln-Gly, Glu, Glu-Gly, Glu-Gly-Gly, Glu-βAla-βAla, D Glu, D Glu-βAla-βAla, D Glu-Gly-Gly, D Glu-AEA, D Glu-AEEA-AEEA, D Glu- D Glu-AEA, D Glu- D Glu-βAla-βAla, γGlu, γGlu-βAla, D γGlu, Lys-Gly, Arg-Gly, N-acid-βAla-βAla, βAla-N-acid-βAla, βAla-Glu-Gly-Gly, βAla- D Glu-βAla, and diacid-βAla-βAla.

[0031] In any of the compounds of formula (I), for example, the compounds of formula (Ia), formula (Ib), formula (Ic), or formula (Id) provided herein, the radioactive moiety of M may be a therapeutic radioisotope. Examples include, but are not limited to, 67 Cu, 90 Y, 177 Lu, 225 Ac, 47 Sc,131 I, 161 Tb, 153 Sm, 211 At, 212 Pb, 212 Bi, 223 Ra, or 227Th This includes. In some specific examples, therapeutic radioisotopes are 90 It is Y.

[0032] In some embodiments, the chelated portion is NOTA or DOTA in the compounds provided herein, and the therapeutic radioisotope is 90 Y, 177 Lu, or 225 It is Ac. In other examples, the chelated portion is NODA, and the therapeutic radioisotope is 47 Sc or 67 It is Cu.

[0033] In some cases, the compound is represented by formula (Id-A):

[0034] [ka] It has.

[0035] In some cases, the compound has the structure of the following formula (Id-Aa) or (Id-Ab):

[0036] [ka] It has one of the following.

[0037] In some examples, the compounds disclosed herein have the following structure:

[0038] [ka] [wherein M is, 177 Lu, 90 Y, 225 Ac, or213 [It is Bi]

[0039] In other examples, the compounds disclosed herein have the following structure:

[0040] [ka] [wherein M is, 177 Lu, 90 Y, 225 Ac, or 213 [It is Bi]

[0041] In other examples, the compounds disclosed herein have the following structure:

[0042] [ka] [wherein M is, 177 Lu, 90 Y, 225 Ac, or 213 [It is Bi]

[0043] Pharmaceutical compositions comprising one or more compounds of formula (I) described above are also provided, and this is within the scope of the disclosure.

[0044] In other embodiments, the present disclosure is characterized by a method for treating cancer in a subject, comprising the step of administering to a subject in need of such treatment a pharmaceutical composition comprising any of the formula (I) compounds disclosed herein, or any other such compound.

[0045] In some cases, an immunotherapy drug may be administered to the subject before receiving the compound of formula (I). Exemplary immunotherapy drugs include, but are not limited to, immune checkpoint inhibitors (e.g., PD1 inhibitors such as anti-PD1 or anti-PD-L1 antibodies) or genetically engineered T cells that express chimeric antigen receptors (CARs).

[0046] Alternatively, or in addition, imaging agents can be administered to the subject to image granzyme B.

[0047] In some examples, any of the methods disclosed herein may further include treating the subject with one or more additional therapeutic agents, such as anti-inflammatory agents, steroids, immunotherapies, and / or chemotherapies.

[0048] The scope of this disclosure also includes pharmaceutical compositions containing any of the formula (I) compounds disclosed herein (e.g., formula (Ia), formula (Ib), formula (Ic), and formula (Id) compounds) for use in cancer treatment, as well as the use of any of the formula (I) compounds for manufacturing pharmaceuticals for cancer treatment.

[0049] Various aspects and embodiments will be described more fully later in this specification. Such aspects and embodiments may take many different forms, and the examples disclosed herein should not be construed as limiting; rather, the disclosure will be complete and its scope fully conveyed to those skilled in the art if these embodiments are provided. [Modes for carrying out the invention]

[0050] Cancer immunotherapy has shown remarkable progress in cancer treatment in recent years. Antibodies against immune checkpoints such as programmed cell death protein 1 (PD-1) and cytotoxic T lymphocyte-associated protein 4 (CTLA-4) have been approved, and outcomes have been positive in some patients. Research in the field of immuno-oncology continues with strategies including CAR-T cells, vaccines, small molecules, and antibodies under development. Despite the potential of these therapies, they are not panaceas. These immunotherapies can be associated with serious adverse events with a heavy cost, and response rates are typically 20-50%, meaning that the majority of patients do not respond to treatment. Furthermore, determining an individual patient's response to treatment can be difficult using conventional methods, as the response is often thought to be associated with immune cell infiltration, which may enlarge responsive tumors on anatomical imaging (e.g., CT, MRI) and show increased avidity with FDG-PET imaging due to the influx of metabolically active immune cells. Given the limitations of current imaging technologies, clinical studies in cancer immunotherapy typically use overall survival, rather than progression-free survival, as their research endpoint.

[0051] Theranostics refers to a combination of imaging and therapeutic approaches for diagnosing and treating target diseases. Generally, the agents used in theranostics therapy contain imaging compounds and radiotherapeutic compounds. In some cases, the imaging and therapeutic compounds may have the same molecule, except for different radiolabelings, one being a nuclide for imaging purposes and the other a nuclide for therapeutic purposes. In some cases, the imaging and therapeutic compounds may have the same pharmacohore or different elements bound to the molecule, each having different radioisotopes for imaging and therapeutic purposes.

[0052] Granzyme B, a downstream marker of cytotoxic T cell activity, may serve as a novel biomarker for evaluating the efficacy of cancer immunotherapy. Granzyme B expression within tumors can be evaluated not only for the presence or absence of CTLs, but also as an effector protein released by active T cells, integrating the measurement of CTL activity and thus addressing the problem of T cell exhaustion that makes it difficult to assess the presence of CTLs.

[0053] This disclosure provides certain specific compounds that can bind to granzyme B (GZB), such as compounds of formula (I), including compounds of formula (Ia) and formula (Ib), which exhibit high binding affinity to granzyme B. Such compounds may carry a radioactive moiety, such as a radioisotope for therapeutic use. Such compounds exhibit excellent features, including a good metabolic profile with little to no gut intake, and complete renal clearance without the formation of metabolites. For further details, see International Patent Application PCT / US2022 / 081098, which is incorporated by reference with respect to the subject matter and objectives of the invention referred herein.

[0054] Accordingly, granzyme B-targeted theranostic therapy for treating cancer in subjects requiring treatment is provided herein. The theranostic therapy comprises a granzyme B-targeted therapeutic compound containing a therapeutic radioisotope as disclosed herein, and may also comprise a granzyme B-targeted imaging compound. In some examples, the granzyme B-targeted therapeutic compound and the granzyme B-targeted imaging compound may be the same molecule carrying different types of radioactive moieties, e.g., therapeutic radioisotope versus imaging radioisotope. In some embodiments, the subject to be treated by the granzyme B-targeted theranostic therapy may have received or be receiving immunotherapy. In some embodiments, the theranostic therapy disclosed herein may be administered to the subject concurrently with immunotherapy.

[0055] definition It should be understood that the technical terms used herein are intended to describe specific embodiments and are not intended to be limiting. Furthermore, any methods, devices, and materials similar to or equivalent to those described herein may be used in the implementation and testing of the present invention, but preferred methods, devices, and materials are described herein. In addition to the foregoing, as used herein and in the appended claims, unless otherwise specified, the following terms have the given meanings:

[0056] "Amino" refers to the -NH2 radical.

[0057] "Cyano" refers to the -CN radical.

[0058] "Hydroxyl" refers to the -OH radical.

[0059] "Imino" refers to the =NH substituent.

[0060] "Nitro" refers to the -NO2 radical.

[0061] "Oxo" refers to an =O substituent.

[0062] "Thioxo" refers to the =S substituent.

[0063] "Trifluoromethyl" refers to the -CF3 radical.

[0064] "Alkyl" refers to a linear saturated acyclic monovalent hydrocarbon radical or a branched saturated acyclic monovalent hydrocarbon radical having 1 to 6 carbon atoms bonded to the rest of the molecule by a single bond, such as methyl, ethyl, n-propyl, 1-methylethyl (isopropyl), n-butyl, n-pentyl, 1,1-dimethylethyl (t-butyl), 3-methylpentyl-1,2-methylpentyl, and similar. The alkyl portion may be unsubstituted. Alternatively, the alkyl portion may be substituted. Optionally, alkyl radicals may be halo, cyano, nitro, aryl, cycloalkyl, heterocyclyl, heteroaryl, oxo, trimethylsilanyl, -OR, if their valency allows. 3 -OC(O)-R 3 , -N(R 3 )2, -C(O)R 4 , -C(O)OR 3 ,-C(O)N(R 3 )2, -N(R 3 )C(O)OR 5 , -N(R 3 )C(O)R 5 , -N(R 3 )S(O) t R 5 (where t is 1 or 2), -S(O) t Ure 5 (where t is 1 or 2), -S(O) p R 5 (where p is 0, 1, or 2) and -S(O) t N(R 3 An alkyl radical which may be substituted with 1, 2, 3, 4, or 5 substituents independently selected from the group consisting of )2 (where t is 1 or 2), and each R 3 R is independently hydrogen, alkyl, haloalkyl, cycloalkyl, cycloalkylalkyl, aryl, aralkyl, heterocyclyl, or heteroaryl; each R 4 R is independently hydrogen, cycloalkyl, aryl, heterocyclyl, or heteroaryl; each R 5 These are independently alkyl, haloalkyl, cycloalkyl, aryl, heterocyclyl, or heteroaryl.

[0065] "Cycloalkyl" refers to a stable non-aromatic monocyclic or polycyclic hydrocarbon radical that is saturated or unsaturated and has 3 to 15 carbon atoms, preferably 3 to 10 carbon atoms, and is bonded to the rest 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. Monocyclic cycloalkyl radicals include, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. Polycyclic cycloalkyl radicals include, for example, adamantyl, norbornyl, decalinyl, and the like. The cycloalkyl moiety may be unsubstituted. Alternatively, the cycloalkyl moiety may be substituted. The optionally substituted cycloalkyl is 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 [[ID=3​​​​​​​​​​​​​​​​​​​R 5 (where p is 0, 1, or 2) and -R 4 -S(O) t N(R 3 A cycloalkyl radical which may be substituted with 1, 2, 3, 4, or 5 substituents independently selected from the group consisting of )2 (where t is 1 or 2), and each R 3 R is independently hydrogen, alkyl, haloalkyl, cycloalkyl, aryl, heterocyclyl, or heteroaryl; each R 4 Each R is independently a directly linked, linear, or branched alkylene or alkenylene chain; 5 These are independently alkyl, haloalkyl, cycloalkyl, cycloalkylalkyl, aryl, aralkyl, heterocyclyl, or heteroaryl.

[0066] The "chelated moiety" is a molecule or ion that can act as a polydentate ligand for a metal ion. For example, a molecule having multiple atoms (including, but not limited to, nitrogen and oxygen) with available lone pairs of electrons can act as a chelated moiety. The chelated moiety may be linear (e.g., EDTA) or cyclic (macro-ring, including DOTA, porphyrin), and may include macro-rings generally known in the art. The chelated 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 with the metal.

[0067] In some embodiments, the preparation of the compound may require the addition of an acid or base, for example, to affect the catalytic action of the desired reaction or the formation of a salt form such as an acid addition salt.

[0068] Exemplary acids may be inorganic or organic acids and include, but are not limited to, strong and weak acids. 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, but are not limited to, acetic acid, propionic acid, butanoic acid, benzoic acid, tartaric acid, pentanoic acid, hexanoic acid, heptanoic acid, octanoic acid, nonaneoic acid, and decanoic acid.

[0069] Exemplary bases include lithium hydroxide, sodium hydroxide, potassium hydroxide, lithium carbonate, sodium carbonate, potassium carbonate, and sodium bicarbonate. Examples of some strong bases, but not limited to, include hydroxides, alkoxides, metal amides, metal hydrides, metal dialkylamides, and arylamines. Alkoxides include lithium, sodium, and potassium salts of methyl, ethyl, and t-butyl oxides; metal amides include sodium amides, potassium amides, and lithium amides; metal hydrides include sodium hydride, potassium hydride, and lithium hydride; and metal dialkylamides include lithium, sodium, and potassium salts of methyl, ethyl, n-propyl, iso-propyl, n-butyl, tert-butyl, trimethylsilyl, and cyclohexyl-substituted amides.

[0070] As used herein, the term “pharmaceutically acceptable salt” refers to a derivative of a disclosed compound in which the parent compound is modified by converting an existing acidic or base moiety to its salt form. Examples of pharmaceutically acceptable salts include, but are not limited to, inorganic or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as carboxylic acids; and similar. The pharmaceutically acceptable salts of this application include, for example, conventional non-toxic salts of parent compounds formed from non-toxic inorganic or organic acids. The pharmaceutically acceptable salts of this application can be synthesized from parent compounds containing basic or acidic moieties by conventional chemical methods. Generally, such salts can be prepared by reacting the free acidic or base form of these compounds with a stoichiometric amount of a suitable base or acid in water or an organic solvent, or a mixture thereof (generally, non-aqueous media such as ether, ethyl acetate, alcohol (e.g., methanol, ethanol, iso-propanol, or butanol) or acetonitrile (MeCN) are preferred). A list of suitable 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.

[0071] 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 enriched. Substantial isolation may include a composition containing at least about 50% by weight, at least about 60% by weight, at least about 70% by weight, at least about 80% by weight, at least about 90% by weight, at least about 95% by weight, at least about 97% by weight, or at least about 99% by weight of the compounds or salts thereof provided herein. Methods for isolating compounds and their salts are routine in the art.

[0072] As used herein, the terms “ambient temperature” and “room temperature” or “rt” refer to the temperature that is understood in the art and generally refers to the approximate temperature of the room in which the reaction is carried out, for example, the reaction temperature, for example, a temperature of about 20°C to about 30°C.

[0073] I. Granzyme B Target Compounds In some embodiments, granzyme B target compounds, such as compounds of formula (I), are provided herein. The compounds disclosed herein include the compounds themselves, their pharmaceutically acceptable salts, and their stereoisomers.

[0074] The compounds described herein may contain one or more chiral centers and therefore may exist in various isomeric forms, such as enantiomers and / or diastereomers. For example, the compounds described herein may be in the form of individual enantiomers, diastereomers or geometric isomers, or in the form of a mixture of stereoisomers, including racemic mixtures and mixtures enriched with 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 (ed. E.L. Eliel, Univ. of Notre Dame Press, Notre Dame, IN 1972). This disclosure also includes the compounds described herein as individual isomers substantially free from other isomers, and, by alternative means, as mixtures of various isomers.

[0075] A. Compound of formula (I) The GZB target compounds provided herein have the structure of formula (I):

[0076] [ka] or it may have stereoisomers, tautomers, or salts thereof. In formula (I), M is the radioactive moiety; A is the chelate moiety that chelates the radioactive moiety; B is an aryl, heteroaryl, cycloalkyl, or heterocyclyl; X is -CH2C(NH)-, -CH2C(O)-, -CH2C(S)-, -NHC(NH)-, -NHC(O)-, -NHC(S)-, -OC(NH)-, -OC(O)-, or -OC(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 the values ​​at both ends; R 1 is H or C 1~6 It is alkyl; R 2 C 1~6 Alkyl or C 3~6 It is cycloalkyl; R 3 C 1~6 It is alkyl. In some embodiments, B is a six-membered ring, for example, as provided herein. Alternatively, or in addition, X may be -CH2C(O)- or -NHC(S)-.

[0077] In some embodiments, R 1 In other embodiments, R 1 C 1~6 It is alkyl. For example, R 1 It is a C1 alkyl group. Alternatively, R 1 It is a C2 alkyl group. In other examples, R 1 It is a C3 alkyl group. In yet another example, R 1 This is a C4 alkyl, C5 alkyl, or C6 alkyl. A specific example is R 1 It is methyl (-CH3).

[0078] In some embodiments, R 2 C 1~6 It is alkyl. For example, R 1 It is a C1 alkyl group. Alternatively, R 1It is a C2 alkyl group. In other examples, R 1 It is a C3 alkyl group. In yet another example, R 1 It is a C4 alkyl group. A specific example is R 2 This is sec-butyl (-CH(CH3)CH2CH3). In yet another example, R 1 It is a C5 alkyl group. In yet another example, R 1 is a C6 alkyl group. In some specific embodiments, R 2 C 3~6 It is alkyl. For example, R 2 C 3~6 Alkyl is branched or unbranched substituted or unsubstituted C 3~6 It is alkyl.

[0079] In other embodiments, R 2 C 3~6 It is a cycloalkyl. For example, R 2 This may be cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl.

[0080] In one embodiment, the compound is given by formula (Ia):

[0081] [ka] It has.

[0082] In some embodiments, X is -CH2C(NH)-. In some embodiments, X is -CH2C(O)-. In some embodiments, X is -CH2C(S)-. In some embodiments, X is -NHC(NH)-. In some embodiments, X is -NHC(O)-. In some embodiments, X is -NHC(S)-. In some embodiments, X is -OC(NH)-. In some embodiments, X is -OC(O)-. In some embodiments, X is -OC(S)-. In some specific embodiments, X is -CH2C(O)-.

[0083] In one embodiment, the compound is of formula (Ib):

[0084] [ka] It has.

[0085] Any of the compounds of formula (I) provided herein, R 3 In some embodiments, C 1~6 It may be alkyl. In some embodiments, R 3 is a C1 alkyl group. In some embodiments, R 3 is a C2 alkyl group. In some embodiments, R 3 is a C3 alkyl group. In some embodiments, R 3 is a C4 alkyl group. In some embodiments, R 3 is a C5 alkyl group. In some embodiments, R 3 is a C6 alkyl group. In some specific embodiments, R 3 It is methyl (-CH3).

[0086] In one embodiment, the compound is of formula (Ic):

[0087] [ka] It has.

[0088] In any of the formula (I) compounds provided herein, B may be a six-membered ring, and in some embodiments, it may be, for example, an aryl, heteroaryl, cycloalkyl, or heterocyclyl. In some examples, B is an aryl. In some examples, B is a heteroaryl. In some examples, B is a cycloalkyl. In other examples, B is a heterocyclyl.

[0089] In any of the compounds of formula (I) provided herein, Z may be -CH2- in some embodiments. In some embodiments, Z is -CH2C(NH)-. In some embodiments, Z is -CH2C(O)-. In some embodiments, Z is -CH2C(S)-. In some embodiments, Z is -NHC(NH)-. In some embodiments, Z is -NHC(O)-. In some embodiments, Z is -NHC(S)-. In some embodiments, Z is -OC(NH)-. In some embodiments, Z is -OC(O)-. In some embodiments, Z is -OC(S)-. In some examples, the compound has the structure (Ic-A):

[0090] [ka] It has.

[0091] For example, the compound of formula (Ic-A) is the diastereomer of formula (Ic-Aa) shown below:

[0092] [ka] It is possible.

[0093] In some examples, the compound has the structure (Ic-B):

[0094] [ka] It has.

[0095] For example, the compound of formula (Ic-B) is a diastereomer of formula (Ic-Ba) or (Ic-Bb) shown below:

[0096] [ka] It is possible.

[0097] In some examples, the compound has the structure (Ic-C):

[0098] [ka] It has.

[0099] For example, the compound of formula (Ic-C) is a diastereomer of formula (Ic-Ca) or (Ic-Cb) shown below: It is possible.

[0100] In some embodiments, the compound has the structure of (Ic-Ca):

[0101] [ka] It has.

[0102] In some embodiments, the compound has the structure of (Id):

[0103] [ka] It has.

[0104] In any of the formula (I) compounds provided herein, L is a peptide linker having 1 to 6 amino acid residues, including the values ​​at both ends. In some embodiments, L is 1 to 5 amino acid residues, including the values ​​at both ends. In some embodiments, L is 2 to 4 amino acid residues, including the values ​​at both ends. In some embodiments, L is 1 amino acid residue. In some embodiments, L is 2 amino acids. In some embodiments, L is 3 amino acid residues. In some embodiments, L is 4 amino acid residues. In some embodiments, L is 5 amino acid residues. In some embodiments, L is 6 amino acid residues.

[0105] In some embodiments, the amino acid residues may be standard proteolytic amino acids (i.e., the 20 naturally occurring amino acid residues found in naturally occurring proteins), or non-natural amino acids that may be derivatives of naturally occurring proteins or isomers of naturally occurring amino acid residues. As used herein, proteolytic amino acid residues refer to the 20 naturally occurring amino acid residues that serve as building blocks for protein synthesis. The amino acid residues may form chains by standard peptide bonds or by forming amide bonds with compatible side chains (e.g., glutamic acid (e.g., D-Glu), aspartic acid). The structures of exemplary non-natural amino acid residues that may be included in the L-linker are shown in Table 1 below.

[0106] [Table 1]

[0107] Examples of amino acid sequences include Gly, Gly-Gly, Gln-Gly, Glu, Glu-Gly, Glu-Gly-Gly, Glu-βAla-βAla, D Glu, D Glu-βAla-βAla, D Glu-Gly-Gly, D Glu-AEA, D Glu-AEEA-AEEA, D Glu- D Glu-AEA, D Glu- D Glu-βAla-βAla, γGlu, γGlu-βAla, D γGlu, Lys-Gly, Arg-Gly, N-acid-βAla-βAla, βAla-N-acid-βAla, βAla-Glu-Gly-Gly, βAla- DThis includes Glu-βAla and diacid-βAla-βAla. In some embodiments, L has a Gly sequence. In some embodiments, L has a Gly-Gly sequence. In some embodiments, L has a Gln-Gly sequence. In some embodiments, L has a Glu sequence. In some embodiments, L has a Glu-Gly sequence. In some embodiments, L has a Glu-Gly-Gly sequence. In some embodiments, L has a Glu-βAla-βAla sequence. In some embodiments, L is D Glu, D It has a Glu-βAla-βAla sequence. In some embodiments, L is D It has a Glu-Gly-Gly 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 has a γGlu sequence. In some embodiments, L has a γGlu-βAla sequence. In some embodiments, L is D It has a γGlu sequence. In some embodiments, L has a Lys-Gly sequence. In some embodiments, L has an Arg-Gly sequence. In some embodiments, L has an N-acid-βAla-βAla sequence. In some embodiments, L has a βAla-N-acid-βAla sequence. In some embodiments, L has a βAla-Glu-Gly-Gly sequence. In some embodiments, L has a βAla- D It has a Glu-βAla sequence. In some embodiments, L has a diacid-βAla-βAla sequence. See Table 2 for the structures of these exemplary L linkers.

[0108] [Table 2-1]

[0109] Table 2-2

[0110] Table 2-3

[0111] In formula (I), A is the chelate moiety. The chelate moiety is a molecule or ion that can act as a polydentate ligand for a metal ion. For example, a molecule having multiple atoms (including, but not limited to, nitrogen and oxygen) with available lone pairs of electrons can act as a chelate moiety. The chelate moiety may be linear (e.g., EDTA) or cyclic (macro-ring, including DOTA, porphyrin), and may include macro-rings generally known in the art. The chelate moiety may have 2, 3, 4, 5, or 6 functional groups (e.g., amines, amides, hydroxyls, carboxylic acids, etc.) with lone pairs of electrons available to coordinate with the metal. Exemplary chelated moieties for use in the granzyme B target 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), cyclohexyl-l,2-diaminetetraacetic acid (CDTA), and This product contains 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-l-glutaric acid-4,7-diacetic acid (NODAGA), a restricting 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).

[0112] In one embodiment, the chelated moiety 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 chelated moiety 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:

[0113] [ka] It has.

[0114] In some embodiments, the chelated moiety 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:

[0115] [ka] It has the following structure. In some embodiments, the chelated moiety A is 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), which has the following structure:

[0116] [ka] It has the following structure. In some embodiments, the chelated moiety A is 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid (DO3A), which has the following structure:

[0117] [ka] It has.

[0118] The selection of a suitable chelating agent in combination 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 content of each of these is incorporated by reference with respect to the subject matter and objectives of the invention referred herein) or the guidance provided herein. In some examples, the compound of formula (I) may have a pair of chelating agent and therapeutic radioisotope listed in Table 3 below. Such a pair of chelating agent and therapeutic radioisotope may result in high loading efficiency of the therapeutic radioisotope.

[0119] [Table 3]

[0120] In some embodiments, the radioactive portion of M is a therapeutic radioisotope. In some embodiments, the therapeutic radioisotope of M is 90 It is Y. In some embodiments, the therapeutic radioactive isotope of M is 177 It is Lu. In some embodiments, the therapeutic radioactive isotope of M is 225 It is Ac. In some embodiments, the therapeutic radioactive isotope of M is 47It is Sc. In some embodiments, the therapeutic radioactive isotope of M is 67 It is Cu. 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 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 227Th In some specific embodiments, the chelated portion is NOTA or DOTA, and the therapeutic radioisotope is 90 Y, 177 Lu, or 225 It is Ac. In some other embodiments, the chelated portion is NODA, and the therapeutic radioisotope is 47 Sc or 67 It is Cu.

[0121] A compound of exemplary formula (Ic-A) is disclosed in Table 4 below.

[0122] [Table 4-1]

[0123] [Table 4-2]

[0124] [Table 4-3]

[0125] [Table 4-4]

[0126] In some embodiments, the compound has the structure of formula (Ic-B):

[0127] [ka] It has.

[0128] Exemplary compounds of formula (Ic-B) are disclosed in Table 5.

[0129] [Table 5-1]

[0130] [Table 5-2]

[0131] [Table 5-3]

[0132] In some embodiments, the compound has the structure of formula (Ic-C):

[0133] [ka] It has.

[0134] Exemplary compounds of formula (Ic-C) are disclosed in Table 6.

[0135] [Table 6-1]

[0136] [Table 6-2]

[0137] [Table 6-3]

[0138] [Table 6-4]

[0139] In some embodiments, the compound has the structure of formula (Id):

[0140] [Chemical Structure] and has.

[0141] Exemplary compounds of formula (Id) are disclosed in Table 7.

[0142] [Table 7]

[0143] As shown in the examples below, the GZB-binding compounds disclosed herein that include either a piperidine or a piperazine linking a peptide linker and a chelating moiety, and further include a specific peptide linker structure, exhibited better in vivo binding to GZB and clearance profiles compared to other GZB-binding compounds. See International Applications PCT / US2022 / \\081125 and WO2021 / 252\\644, which are incorporated by reference herein with respect to the subject matter and objectives of the invention referred to herein.

[0144] Exemplary improved properties include improved pharmacokinetics (e.g., renal clearance), pharmacodynamics, and efficacy. In particular, improved pharmacokinetics can be observed in the absence of gastrointestinal uptake, the absence of radiometabolites in urine, and / or dominant renal clearance. See international application PCT / US2022 / 081125.

[0145] The compounds described herein, when containing radioisotopes such as therapeutic radioisotopes, are useful as theranostic drugs 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 refer to compounds, materials, compositions, and / or dosage forms that, within the bounds of good medical judgment, are suitable for use in contact with human and animal tissues in proportion to a reasonable benefit-to-risk ratio without excessive toxicity, irritation, allergic response, or other problems or complications.

[0146] B. Chemical synthesis of granzyme B target compounds As will be acknowledged, the compounds provided herein, including their stereoisomers and salts, can be prepared using known organic synthesis techniques and can be synthesized by any of a number of possible synthetic routes.

[0147] The compounds disclosed herein, or pharmaceutically acceptable salts thereof, can be prepared by following the exemplary protocols described below. Suitable protecting groups for use in such synthesis are known in the art. See, for example, McOmie, Protective Groups in Organic Chemistry (1973):98.

[0148] A general synthetic procedure for preparing the peptide linker L, Fmoc-Haic(2S,5S)-OH tricyclic moiety, and examples thereof, as well as appropriate metal complexing at the chelated moiety, can be found in International Application PCT / US2021 / 036661, filed 9 June 2021, which is incorporated by reference with respect to the subject matter and objectives of the invention referred herein.

[0149] Many suitable imaging agents (e.g., radioisotopes) are known in the art (the contents of each of these are incorporated herein by reference in whole; see, for example, U.S. Patents 5,021,236; 4,938,948; and 4,472,509). Radiolabeled compounds, or pharmaceutically acceptable salts thereof, provided herein may be prepared by 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.

[0150] 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 potential use in the synthesis of the compounds provided herein. Those skilled in the art will know how to select and carry out appropriate synthetic routes. Preferred synthetic methods for starting materials, intermediates, and products are found in Advances in Heterocyclic Chemistry, Vols. 1-107 (Elsevier, 1963-2012); Journal of Heterocyclic Chemistry Vols. 1-49 (Journal of Heterocyclic Chemistry, 1964-2012); Carreira et al. (eds.), 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. (eds.), Comprehensive Organic Functional Group Transformations (Pergamon Press, 1996); Katritzky et al. (eds.), Comprehensive Organic Functional Group Transformations II (Elsevier, 2001-2012). nd Edition, 2004);Katritzky et al. (eds.), Comprehensive Heterocyclic Chemistry (Pergamon Press, 1984);Katritzky et al., Comprehensive Heterocyclic Chemistry II, (Pergamon Press, 1996);Smith et al., March's Advanced Organic Chemistry:Reactions, Mechanisms, and Structure, 6 thIt can be identified by referring to literature including references such as Ed. (Wiley, 2007); Trost et al. (eds.), Comprehensive Organic Synthesis (Pergamon Press, 1991).

[0151] The reactions for preparing the compounds described herein can be carried out in suitable solvents that can be readily selected by those skilled in the art of organic synthesis. Suitable solvents can be substantially inactive with the starting materials (reactants), intermediates, or products at the temperature in which the reaction is carried out (for example, a temperature that may 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 more than one solvent. Depending on the specific reaction step, suitable solvents for a particular reaction step can be selected by those skilled in the art.

[0152] The preparation of the compounds described herein may require 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 action of protecting groups is described, for example, in TWGreene and PGMWuts, Protective Groups in Organic Synthesis, 3 rd This can be found in Ed., Wiley & Sons, Inc., New York (1999).

[0153] The reaction can be monitored by any suitable method known in the art. For example, the formation of the product 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., visible UV), by mass spectrometry, or by chromatographic methods such as high-performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LCMS), or thin-layer chromatography (TLC). Compounds can be purified by various methods, including high-performance liquid chromatography (HPLC) and normal-phase silica chromatography, according to those skilled in the art.

[0154] II. Pharmaceutical Compositions Any compound of formula (I), for example, a compound of formula (Ia), a compound of formula (Ib), a compound of formula (Ic) such as formula (Ic-A), formula (Ic-B), formula (Ic-D), formula (Ic-Aa), formula (Ic-Ab), formula (Ic-Ba), formula (Ic-Bb), formula (Ic-Ca), and formula (Ic-Cb), a compound of formula (Id) such as formula (Id-Aa) and formula (Id-Ab), or a pharmaceutically acceptable salt thereof, can be mixed with a pharmaceutically acceptable carrier to form a pharmaceutical composition for therapeutic purposes, for example, 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 (additives) are provided herein. "Acceptable" means that the carrier must be compatible with the active ingredient of the composition (preferably able to stabilize the active ingredient) and not harmful to the object being treated. Suitable carriers include microcrystalline cellulose, mannitol, glucose, skim milk powder, polyvinylpyrrolidone, and starch, or combinations thereof.

[0155] Some examples of suitable additives 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 also include, 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; flavorings; or combinations thereof. For further information on acceptable pharmaceutical compositions, see Remington's Pharmaceutical Sciences, 17 th See ed., Mack Publishing Company, Easton, Pa., 1985, p. 1418.

[0156] A 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 by other conventional routes, for example, orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally, or via an implanted reservoir. The term "parenteral," as used herein, includes subcutaneous, intradermal, intravenous, intramuscular, intra-articular, intra-arterial, intra-bursal, intrasternal, intrathecal, intralesional, and intracranial injection or infusion techniques. Parenteral administration may take 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 injection depot route of administration, such as using 1, 3, or 6-month depot injection or biodegradable materials and methods.

[0157] 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, and similar). For intravenous injection, water-soluble antibodies can be administered by drip infusion, thereby injecting a pharmaceutical preparation containing the antibody and physiologically acceptable additives. Physiologically acceptable additives may include, for example, 5% dextrose, 0.9% physiological saline, Ringer's solution, or other suitable additives. Intramuscular preparations of preferred soluble salt forms of the antibody, such as sterile preparations, can be dissolved and administered in pharmaceutical additives such as sterile water for injection, 0.9% physiological saline, or 5% glucose solution.

[0158] For oral administration, the composition is prepared by conventional means using acceptable additives such as binders (e.g., pregelatinized corn starch, polyvinylpyrrolidone or hydroxypropylmethylcellulose); diluents (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); or wetting agents (e.g., sodium lauryl sulfate), and may take the form of, for example, tablets or capsules. Tablets can be coated by methods well known in the art.

[0159] In some embodiments, the compounds provided herein, or pharmaceutically acceptable salts thereof, are suitable for parenteral administration. In some embodiments, the compounds provided herein, or pharmaceutically acceptable salts thereof, are suitable for intravenous administration.

[0160] 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, powder or oily bases, thickeners and the like may be necessary or desirable.

[0161] In making the pharmaceutical compositions provided herein, the active ingredient is typically admixed with, diluted by, or enclosed within a carrier in the form of, for example, capsules, sachets, papers, or other containers. When the additive serves as a diluent, it can be a solid, semi-solid, or liquid material that acts as a vehicle, carrier or medium for the active ingredient.

[0162] Thus, the pharmaceutical composition may 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.

[0163] 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 treatment.

[0164] To carry out the methods disclosed herein, an effective amount of a pharmaceutical composition comprising a compound of formula (I) as disclosed herein, or a pharmaceutically acceptable salt thereof, can be administered via a route suitable for the subject requiring treatment. In some embodiments, such methods may further include administering to the subject an effective amount of a granzyme B-targeted drug, e.g., a compound of formula (I) as disclosed herein, or a pharmaceutically acceptable salt thereof, which may be done prior to the administration of the therapeutic agent. In some embodiments, the subject may or may have previously received an immunotherapy, e.g., one disclosed herein.

[0165] 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.

[0166] In some embodiments, the subjects are human patients with cancer. In some cases, the cancer is a solid tumor. Examples include, but are not limited to, brain cancer, breast cancer (e.g., HER2+, ER+ / PR+ / HER2-, or tri-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, 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's sarcoma, liposarcoma, fibrosarcoma, leiomyosarcoma, peripheral neuroectodermal tumors, squamous cell carcinoma of the vagina, and squamous cell carcinoma of the vulva. In some cases, the cancer is colorectal cancer.

[0167] In other embodiments, cancer is hematological cancer (e.g., leukemia, lymphoma, and similar). 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, T-cell prelymphocytic leukemia, classical Hodgkin lymphoma, B-cell non-Hodgkin lymphoma, chronic lymphocytic leukemia, acute myeloid leukemia, myelodysplastic syndrome, primary myelofibrosis, myelofibrosis secondary to essential thrombocythemia, or myelofibrosis secondary to polycythemia vera.

[0168] As used herein, “effective dose” refers to the amount of each active agent required to impart a therapeutic effect to a target, either alone or in combination with one or more other active agents. Determining whether an antibody dose achieves 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 specific condition being treated, the severity of the condition, individual patient parameters including age, physical condition, build, sex, and weight, the duration of treatment, the nature of any concurrent treatments, the specific route of administration, and similar factors within the scope of the healthcare professional’s understanding and expertise. These factors are well known to those skilled in the art and can be addressed through routine experimentation or less. It is generally preferable to use the maximum dose of the individual components or combinations thereof, i.e., the maximum safe dose determined by appropriate medical judgment.

[0169] In some cases, the maximum permissible dose of a checkpoint inhibitor can be used in the methods disclosed herein. Alternatively, or in addition, the minimum effective dose of any of the therapeutic granzyme B target molecules of formula (I) disclosed herein can be used in such methods.

[0170] Empirical considerations, such as half-life, generally contribute to determining the dosage. For example, antibodies 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 protect it from being attacked by the recipient's immune system. The frequency of administration can be determined and adjusted during the course of treatment, generally, but not necessarily, based on the treatment and / or suppression and / or improvement 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.

[0171] In some embodiments, the dosage 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 g, approximately 100 μg to approximately 2 g, for example, approximately 100 μg to approximately 2 g, approximately 100 μg to approximately 1000 mg, approximately 100 μg to approximately 500 mg, approximately 100 μg to approximately 100 mg, approximately 100 μg to approximately 50 mg, approximately 100 μg to approximately 1 mg, approximately 100 μg to approximately 500 μg, approximately 500 μg to approximately 2 g, for example, approximately 500 μg to approximately 2 g, approximately 50 The dosage ranges are 0 μg to approximately 1000 mg, approximately 500 μg to approximately 500 mg, approximately 500 μg to approximately 100 mg, approximately 500 μg to approximately 50 mg, approximately 500 μg to approximately 1 mg, approximately 1 mg to approximately 2 g, approximately 1 mg to approximately 1000 mg, approximately 1 mg to approximately 500 mg, approximately 1 mg to approximately 100 mg, approximately 1 mg to approximately 50 mg, or approximately 50 mg to approximately 500 mg.

[0172] As used herein, the terms “to treat” or “treatment” mean one or more of the following: (1) inhibiting cancer; for example, inhibiting cancer in an individual experiencing or presenting with cancerous disease or overall symptoms (i.e., preventing further progression of disease and / or overall symptoms); and (2) alleviating cancer; for example, reducing the severity of cancer or reducing or alleviating one or more of the symptoms of cancer, or alleviating cancer in an individual experiencing or presenting with cancerous disease or overall symptoms (i.e., improving disease and / or overall symptoms).

[0173] In some embodiments, subjects for treatment can be identified and / or diagnosed as having cancer prior to administration of the therapeutic compound of formula (I). In some examples, subjects having the target cancer can be identified by routine medical examinations, e.g., clinical 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 previously received or are receiving anti-cancer treatment, e.g., chemotherapy, radiation therapy, immunotherapy, or surgery.

[0174] Human patient subjects for any of the cancer treatments disclosed herein (e.g., theranostic therapy as disclosed herein) may have prior treatment with an immunotherapy, or may be subjects of immunotherapy simultaneously. Immunotherapy generally targets cells (e.g., cancer cells) and induces immune effector cells and molecules that destroy them. Immune effectors may be, for example, antibodies specific to markers on the surface of cells (e.g., tumor cells). Antibodies may act as therapeutic effectors alone, or they may mobilize other cells that carry out cell death. Various effector cells include, but are not limited to, cytotoxic T cells and NK cells.

[0175] Examples of immunotherapies 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 (e.g., Ravindranath & Morton, International reviews of See immunology, 7.4(1991):303-329), hormonal therapy, corticosteroids, progestins (e.g., hydroxyprogesterone caproate, medroxyprogesterone acetate, and megestrol acetate), estrogens (e.g., diethylstilbestrol and ethinylestradiol), antiestrogens (e.g., testosterone propionate and fluoxymesterone), antiandrogens (e.g., flutamide), and gonadotropin-releasing hormone analogs (e.g., leuprolide). Additional immunotherapies are known in the art and can be found, for example, in Rosenberg et al., New England Journal of Medicine, 319.25 (1988):1676-1680; and Rosenberg et al., Annals of surgery, 210.4(1989):474).

[0176] In some embodiments, the immunotherapy agent is an immune checkpoint inhibitor, such as a PD1 inhibitor (e.g., an anti-PD-1 antibody such as nivolumab, pembrolizumab, or semiprimab; 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 relatrimab). In other embodiments, the immunotherapy agent may be CAR-T cells, such as axicaptagen silolucel or brexcabutagen oatlucel.

[0177] The therapeutic agents provided herein may be effective across a wide range of dosages and are generally administered in effective doses. However, it will be understood that the actual amount of therapeutic agent administered will usually be determined by a physician based on the imagined condition, the chosen route of administration, the actual compound administered, the age, weight, and response of the individual subject, the severity of the subject's symptoms, and other relevant circumstances.

[0178] In some embodiments, any of the granzyme B-targeted theranostic compounds can be used concurrently with anticancer therapies, such as those disclosed herein, for example, immunotherapy and chemotherapy.

[0179] 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.

[0180] Any granzyme B imaging agent known in the art can be used in the methods disclosed herein. Examples include those disclosed in U.S. Patent No. 11,559,590, WO2021 / 252664, PCT / US2022 / 081098, and PCT / US2022 / 081125, the relevant content of which is incorporated by reference with respect to the subject matter and objectives of the invention referred herein.

[0181] In some embodiments, the granzyme B imaging compound is a compound of formula (II) shown below:

[0182] [ka] or its stereoisomers, tautomers, or salts. In formula (II), M is an imaging agent; A is a chelate 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)- (e.g., -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 the values ​​at both ends; R 1 is H or C 1~6 It is alkyl, R 1 R may be H or methyl; 2 C 1~6 Alkyl or C 3~6 It is cycloalkyl; R 3 C 1~6 It is alkyl.

[0183] In some cases, granzyme B-imaging compounds have the structure shown in formula (II-A) below:

[0184] [ka] It may have, where M, A, X, L, R 1 , and R 2 This is as defined above.

[0185] In some cases, granzyme B-imaging compounds have the structure shown in formula (II-B) below:

[0186] [ka] It may have, where M, Z, A, X, L, R 1 and R 2 As defined above, Y is either CH or N.

[0187] In any of the imaging agents disclosed herein, M may be a metal or a metal bound to a radioisotope suitable for imaging. Suitable metals for use in this disclosure include those useful in imaging granzyme B, e.g., metals that are suitable radioimaging agents, and metals that can bind to nonmetallic radioisotopes that are suitable radioimaging agents. Exemplary metal radioisotopes are 68 It is Ga. An example of a nonmetallic radioactive isotope is... 18 F is present, which can be conjugated with Al to support the granzyme B-binding compounds disclosed herein.

[0188] IV. Kits for Granzyme B-Targeted Theranostics Therapy Kits for granzyme B-targeted cancer therapy (e.g., pharmaceutical packs) are also included in this disclosure. The kits provided may include containers (e.g., vials, ampoules, bottles, syringes, and / or dispenser packaging, or other suitable containers) capable of holding the pharmaceutical compositions as disclosed herein. In some embodiments, the kits provided may further include a second container containing pharmaceutical additives for diluting or suspending the pharmaceutical compositions. In some embodiments, the pharmaceutical compositions provided in the first and second containers are combined to form a single unit dosage form. In some embodiments, the kits may include additional containers containing one or more additional therapeutic agents, e.g., immunotherapeutic agents, as disclosed herein. In some embodiments, the kits may include imaging compounds or their pharmaceutical compositions, and therapeutic compounds or their pharmaceutical compositions.

[0189] 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 as required by regulatory authorities 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 for treating cancer in subjects requiring it and / or for reducing the risk of cancer. The kit described herein may include one or more additional pharmacoactive substances described herein as separate compositions.

[0190] Without further details, those skilled in the art will be able to make the most of the present invention based on the above description. Therefore, the following specific embodiments should be construed as illustrative only and not in any way limit the remainder of this disclosure. All publications cited herein are incorporated by reference with respect to the purposes or subject matter referred to herein. [Examples]

[0191] Example 1: Synthesis of an exemplary compound of formula (I') This example presents an exemplary synthetic method for preparing a precursor compound of formula (I') that does not contain the radioactive moiety from the corresponding compound of formula (I).

[0192] Preparation of chelated partial coupling partners

[0193] [ka] 2-(4-((4,7-bis(2-(tert-butoxy)-2-oxoethyl)-1,4,7-triazonan-1-yl)methyl)piperidine-1-yl)acetic acid

[0194] [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 ratio 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). Benzyl 2-(4-(hydroxymethyl)piperidine-1-yl)acetate (92.0 g, 349 mmol) was obtained as a yellow oil. 1H NMR: (400 MHz, CDCl3) δ 7.35-7.37 (m, 5H), 5.16 (s, 2H), 3.49 (d, J = 6.4 Hz, 2H), 3.26 (s, 2H), 2.95 (d, J = 11.2 Hz, 2H), 2.16-2.22 (m, 2H), 1.72 (d, J = 13.2 Hz, 2H), 1.59 (s, 1H), 1.49-1.55 (m, 1H), 1.30-1.40 (m, 2H).

[0195] [ka]

[0196] 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 at -65°C for a further 30 minutes. TLC (dichloromethane:methanol = 10:1, R f A reading of 0.33 indicated that the starting material had been completely consumed. The reaction mixture was quenched by adding NaHCO3 (500 mL) at 0°C and then 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. 1H NMR: 400 MHz, CDCl3δ 9.65 (d, J = 1.2 Hz, 1H), 7.33-7.38 (m, 5H), 5.17 (s, 2H), 3.29 (s, 2H), 2.86-2.91 (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).

[0197] [ka]

[0198] Step 3: Di-tert-butyl 2,2'-(1,4,7-triazonan-1,4-diyl) diacetate (32.0 g, 89.5 mmol) and AcOH (4.30 g, 71.6 mmol, 4.10 mL) were added at 0°C to a mixture of benzyl 2-(4-formylpiperidine-1-yl) 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 equivalents) 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 from 30.0 g and 32.0 g of di-tert-butyl 2,2'-(1,4,7-triazonan-1,4-diyl)diacetate were combined and further purified. The combined crude product was ground with SiO2 (120 mL) for 12 hours to obtain 51.0 g of di-tert-butyl 2,2'-(7-((1-(2-(benzyloxy)-2-oxoethyl)piperidine-4-yl)methyl)-1,4,7-triazonan-1,4-diyl)diacetate as a white solid. ES / MS m / z 603.5 (M+H)+ .

[0199] [ka]

[0200] Step 4: To a solution of di-tert-butyl 2,2'-(7-((1-(2-(benzyloxy)-2-oxoethyl)piperidine-4-yl)methyl)-1,4,7-triazonan-1,4-diyl) diacetate (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 ground in 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 / z 513.4(M+H) + .

[0201] General peptide synthesis procedure: Peptides were synthesized using H-Asp(OtBu)-H resin according to a standard Fmoc solid-phase peptide synthesis procedure. The final peptide was deprotected and cleaved from the resin according to a two-step procedure: 1) treatment with trifluoroacetic acid (TFA) for 2 hours at room temperature or with TFA / dichloromethane (DCM) overnight at room temperature, followed by concentration; and 2) treatment with 0.1% TFA in acetonitrile / water (60:40) at 60°C for 1 hour (https: / / www.emdmillipore.com / US / en / product / H-AspOtBu-H-NovaSyn-TG-resin,MDA_CHEM-856072#documentation). The crude peptide was concentrated or lyophilized and then subjected to preparative HPLC purification (0.1% formic acid or 0.1% TFA in water / acetonitrile mobile phase). The product-containing fraction was collected and freeze-dried to obtain the peptide as a white, fluffy solid.

[0202] Compound 1 is synthesized according to a general peptide synthesis procedure.

[0203] [ka]

[0204] Compound 2 is synthesized according to a general peptide synthesis procedure.

[0205] [ka]

[0206] Compound 3 is synthesized according to a general peptide synthesis procedure.

[0207] [ka]

[0208] Compound 4 is synthesized according to a general peptide synthesis procedure.

[0209] [ka]

[0210] Compound 5 is synthesized according to a general peptide synthesis procedure.

[0211] [ka]

[0212] Compound 6 is synthesized according to a general peptide synthesis procedure.

[0213] [ka]

[0214] Compound 7 is synthesized according to a general peptide synthesis procedure.

[0215] [ka]

[0216] Compound 8 is synthesized according to a general peptide synthesis procedure.

[0217] [ka]

[0218] Compound 9 is synthesized according to a general peptide synthesis procedure.

[0219] [ka]

[0220] Compound 10 is synthesized according to a general peptide synthesis procedure.

[0221] [ka]

[0222] Compound 11 is synthesized according to a general peptide synthesis procedure.

[0223] [ka]

[0224] Compound 12 is synthesized according to a general peptide synthesis procedure.

[0225] [ka]

[0226] Compound 13 is synthesized according to a general peptide synthesis procedure.

[0227] [ka]

[0228] Compound 14 is synthesized according to a general peptide synthesis procedure.

[0229] [ka]

[0230] Compound 15 is synthesized according to a general peptide synthesis procedure.

[0231] [ka]

[0232] Compound 16 is synthesized according to a general peptide synthesis procedure.

[0233] [ka]

[0234] Compound 17 is synthesized according to a general peptide synthesis procedure.

[0235] [ka]

[0236] Compound 18 is synthesized according to a general peptide synthesis procedure.

[0237] [ka]

[0238] Compound 19 is synthesized according to a general peptide synthesis procedure.

[0239] [ka]

[0240] Compound 20 is synthesized according to a general peptide synthesis procedure.

[0241] [ka]

[0242] Compound 21 is synthesized according to a general peptide synthesis procedure.

[0243] [ka]

[0244] Compound 22 is synthesized according to a general peptide synthesis procedure.

[0245] [ka]

[0246] Compound 23 is synthesized according to a general peptide synthesis procedure.

[0247] [ka]

[0248] Compound 34 is synthesized according to a general peptide synthesis procedure.

[0249] [ka]

[0250] Compound 25 is synthesized according to a general peptide synthesis procedure.

[0251] [ka]

[0252] Compound 26 is synthesized according to a general peptide synthesis procedure.

[0253] [ka]

[0254] Compound 27 is synthesized according to a general peptide synthesis procedure.

[0255] [ka]

[0256] Compound 28 is synthesized according to a general peptide synthesis procedure.

[0257] [ka]

[0258] Compound 29 is synthesized according to a general peptide synthesis procedure.

[0259] [ka]

[0260] Compound 30 is synthesized according to a general peptide synthesis procedure.

[0261] [ka]

[0262] Compound 31 is synthesized according to a general peptide synthesis procedure.

[0263] [ka]

[0264] Compound 32 is synthesized according to a general peptide synthesis procedure.

[0265] [ka]

[0266] Compound 33 is synthesized according to a general peptide synthesis procedure.

[0267] [ka]

[0268] Compound 34 is synthesized according to a general peptide synthesis procedure.

[0269] [ka]

[0270] Compound 35 is synthesized according to a general peptide synthesis procedure.

[0271] [ka]

[0272] Compound 36 is synthesized according to a general peptide synthesis procedure.

[0273] [ka]

[0274] Compound 37 is synthesized according to a general peptide synthesis procedure.

[0275] [ka]

[0276] Compound 38 is synthesized according to a general peptide synthesis procedure.

[0277] [ka]

[0278] Compound 39 is synthesized according to a general peptide synthesis procedure.

[0279] [ka]

[0280] Compound 40 is synthesized according to a general peptide synthesis procedure.

[0281] [ka]

[0282] Compound 41 is synthesized according to a general peptide synthesis procedure.

[0283] [ka]

[0284] Compound 42 is synthesized according to a general peptide synthesis procedure.

[0285] [ka]

[0286] Compound 43 is synthesized according to a general peptide synthesis procedure.

[0287] [ka]

[0288] Compound 44 is synthesized according to a general peptide synthesis procedure.

[0289] [ka]

[0290] Compound 45 is synthesized according to a general peptide synthesis procedure.

[0291] [ka]

[0292] Compound 46 is synthesized according to a general peptide synthesis procedure.

[0293] [ka]

[0294] Compound 47 is synthesized according to a general peptide synthesis procedure.

[0295] [ka]

[0296] Compound 48 is synthesized according to a general peptide synthesis procedure.

[0297] [ka]

[0298] Compound 49 is synthesized according to a general peptide synthesis procedure.

[0299] [ka]

[0300] Example 2: Synthesis of an exemplary compound of formula (I) General procedure for complexing therapeutic isotopes A reaction vial containing a peptide precursor and a stirring rod is filled with a suitable amount of therapeutic radioisotope (e.g., an equal equivalent amount of solvent (1.5 to 3.0 equivalents relative to the peptide) (e.g., 90 Add Y) and stir. This will result in therapeutic isotope complexing.

[0301] Compound 1-M was synthesized according to the general procedure for complexing therapeutic isotopes.

[0302] [ka]

[0303] Compound 2-M was synthesized according to the general procedure for complexing therapeutic isotopes.

[0304] [ka]

[0305] Compound 3-M was synthesized according to the general procedure for complexing therapeutic isotopes.

[0306] [ka]

[0307] Compound 4-M was synthesized according to the general procedure for complexing therapeutic isotopes.

[0308] [ka]

[0309] Compound 5-M was synthesized according to the general procedure for complexing therapeutic isotopes.

[0310] [ka]

[0311] Compound 6-M was synthesized according to the general procedure for complexing therapeutic isotopes.

[0312] [ka]

[0313] Compound 7-M was synthesized according to the general procedure for complexing therapeutic isotopes.

[0314] [ka]

[0315] Compound 8-M was synthesized according to the general procedure for complexing therapeutic isotopes.

[0316] [ka]

[0317] Compound 9-M was synthesized according to the general procedure for complexing therapeutic isotopes.

[0318] [ka]

[0319] Compound 10-M was synthesized according to the general procedure for complexing therapeutic isotopes.

[0320] [ka]

[0321] Compound 11-M was synthesized according to the general procedure for complexing therapeutic isotopes.

[0322] [ka]

[0323] Compound 12-M was synthesized according to the general procedure for complexing therapeutic isotopes.

[0324] [ka]

[0325] Compound 13-M was synthesized according to the general procedure for complexing therapeutic isotopes.

[0326] [ka]

[0327] Compound 14-M was synthesized according to the general procedure for complexing therapeutic isotopes.

[0328] [ka]

[0329] Compound 15-M was synthesized according to the general procedure for complexing therapeutic isotopes.

[0330] [ka]

[0331] Compound 16-M was synthesized according to the general procedure for complexing therapeutic isotopes.

[0332] [ka]

[0333] Compound 17-M was synthesized according to the general procedure for complexing therapeutic isotopes.

[0334] [ka]

[0335] Compound 18-M was synthesized according to the general procedure for complexing therapeutic isotopes.

[0336] [ka]

[0337] Compound 19-M was synthesized according to the general procedure for complexing therapeutic isotopes.

[0338] [ka]

[0339] Compound 20-M was synthesized according to the general procedure for complexing therapeutic isotopes.

[0340] [ka]

[0341] Compound 21-M was synthesized according to the general procedure for complexing therapeutic isotopes.

[0342] [ka]

[0343] Compound 22-M was synthesized according to the general procedure for complexing therapeutic isotopes.

[0344] [ka]

[0345] Compound 23-M was synthesized according to the general procedure for complexing therapeutic isotopes.

[0346] [ka]

[0347] Compound 24-M was synthesized according to the general procedure for complexing therapeutic isotopes.

[0348] [ka]

[0349] Compound 25-M was synthesized according to the general procedure for complexing therapeutic isotopes.

[0350] [ka]

[0351] Compound 26-M was synthesized according to the general procedure for complexing therapeutic isotopes.

[0352] [ka]

[0353] Compound 27-M was synthesized according to the general procedure for complexing therapeutic isotopes.

[0354] [ka]

[0355] Compound 28-M was synthesized according to the general procedure for complexing therapeutic isotopes.

[0356] [ka]

[0357] Compound 29-M was synthesized according to the general procedure for complexing therapeutic isotopes.

[0358] [ka]

[0359] Compound 30-M was synthesized according to the general procedure for complexing therapeutic isotopes.

[0360] [ka]

[0361] Compound 31-M was synthesized according to the general procedure for complexing therapeutic isotopes.

[0362] [ka]

[0363] Compound 32-M was synthesized according to the general procedure for complexing therapeutic isotopes.

[0364] [ka]

[0365] Compound 33-M was synthesized according to the general procedure for complexing therapeutic isotopes.

[0366] [ka]

[0367] Compound 34-M was synthesized according to the general procedure for complexing therapeutic isotopes.

[0368] [ka]

[0369] Compound 35-M was synthesized according to the general procedure for complexing therapeutic isotopes.

[0370] [ka]

[0371] Compound 36-M was synthesized according to the general procedure for complexing therapeutic isotopes.

[0372] [ka]

[0373] Compound 37-M was synthesized according to the general procedure for complexing therapeutic isotopes.

[0374] [ka]

[0375] Compound 38-M was synthesized according to the general procedure for complexing therapeutic isotopes.

[0376] [ka]

[0377] Compound 39-M was synthesized according to the general procedure for complexing therapeutic isotopes.

[0378] [ka]

[0379] Compound 40-M was synthesized according to the general procedure for complexing therapeutic isotopes.

[0380] [ka]

[0381] Compound 41-M was synthesized according to the general procedure for complexing therapeutic isotopes.

[0382] [ka]

[0383] Compound 42-M was synthesized according to the general procedure for complexing therapeutic isotopes.

[0384] [ka]

[0385] Compound 43-M was synthesized according to the general procedure for complexing therapeutic isotopes.

[0386] [ka]

[0387] Compound 44-M was synthesized according to the general procedure for complexing therapeutic isotopes.

[0388] [ka]

[0389] Compound 45-M was synthesized according to the general procedure for complexing therapeutic isotopes.

[0390] [ka]

[0391] Compound 46-M was synthesized according to the general procedure for complexing therapeutic isotopes.

[0392] [ka]

[0393] Compound 47-M was synthesized according to the general procedure for complexing therapeutic isotopes.

[0394] [ka]

[0395] Compound 48-M was synthesized according to the general procedure for complexing therapeutic isotopes.

[0396] [ka]

[0397] Compound 49-M was synthesized following a general procedure for complexing therapeutic isotopes.

[0398] [ka]

[0399] Example 3: 90 Radioactive synthesis of Y-conjugated compounds

[0400] [ka] Typical compound 47- synthesized with ORA Neptis Perform radio synthesizer 90Y RCY was produced in the range of 14–20% (n=7) using a starting activity of 29–55 GBq. The synthesis time was 68 ± 5 minutes with a product radiochemical purity >93% and specific activity in the range of 167–1320 GBq / μmol (3.9–30.9 mCi / μg).

[0401] [ka]

[0402] Compound 48- 90 Synthesize Y according to the same method described above.

[0403] [ka]

[0404] Compound 49- 90 Synthesize Y according to the same method described above.

[0405] Example 4: Competitive binding assay of human granzyme B Purpose: Compound 1- 90 A competitive binding assay with human granzyme B was performed using Y, and the IC of a promising granzyme B ligand was determined. 50 To determine a value.

[0406] Preparation of the initial plate (including fully bound and non-specific bound): 1. Add 78 μL of reaction buffer as described above to each well in the 96-well plate. 2. In the wells used to examine complete binding (defined as binding of the radioactive ligand in the absence of competing factors), 2 μL of DMSO is added to obtain a final 1% DMSO concentration in all wells. 3. In the wells used to examine nonspecific binding (defined as the binding of the radioactive ligand in the presence of 10 μM 21), 2 μL of 1 mM compound 1 is added to obtain a final concentration of 10 μM.

[0407] Enzyme preparation: 1. Dilute Granzyme B (GZB, human lymphocytes) to 0.5 μg / mL using the reaction buffer described above. 2. Add 100 μL of 0.5 μg / mL diluted human GzB to each well to obtain final concentrations of 50 ng and 0.25 μg / mL per well.

[0408] 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 compound stock to 40 μL of DMSO, mix by moving the pipette up and down, and transfer 18.5 μL of the mixture to 40 μL of DMSO, thereby serially diluting the 1 mM compound stock solution logarithmically (3.16 times). Repeat this process to create 10 dilution points for the test compound. 3. For the wells used to examine ligand binding competition, 2 μL of each diluent is dispensed into a 96-well plate prepared as described above to obtain a further 1 / 100 dilution.

[0409] Preparation of radioactive ligands: 1. Aim for approximately 2 million CPM per 20 μL of solution (input), using a 200 nM compound 1- 90 Prepare Y (10× radioactive ligand stock) in reaction buffer. 2. Dispense 20 μL of radioactive ligand stock into each well to obtain a final concentration of 20 nM per well.

[0410] Incubation conditions, post-incubation sample processing, and data analysis: 1. All 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 the assay plate are pre-soaked in PBS buffer, pH 7.4, and placed in a MultiScreen. HTSThe sample was transferred to an FB plate and filtered using a vacuum manifold. An additional 150 μL of PBS buffer was added to each well in the assay plate, ensuring that all remaining sample was transferred. 3. MultiScreen HTS Wash the FB plate three times with 150 μL of PBS buffer. 4. Remove all filters and transfer them to individual tubes using Multiscreen punch tips and a Millipore multiple punch apparatus. 5. The sample set was analyzed using a Wizard 2480 automatic gamma-counter [Perkin Elmer]. Values ​​are reported as decay-corrected counts per minute (CPM). 6. Normalize the obtained CPM values ​​and convert them to inhibition percentages using the following formula:

[0411]

number

[0412] * The fully bound fraction is typically less than 10% of the added radioligand under such assay conditions.

[0413] Using GraphPad Prism 8.4.3 software, One site - Fit logIC 50 Using the formula, plot the obtained inhibition % values ​​and the IC at each ligand. 50 The value was determined. Y = A + (BA) / (1 + 10) (x-LogIC50) ) Y = Inhibition % X = Logarithmic concentration of the cold competing ligand (M) A=Minimum Y(0%) B=Max Y(100%) LogIC 50 = Logarithmic concentration of cold competing ligand (M) midway between minimum and maximum Y

[0414] Other Embodiments All features disclosed herein may be combined in any combination. Each feature disclosed herein may be replaced by an alternative feature that serves the same, equivalent, or similar purpose. Thus, unless otherwise explicitly stated, each disclosed feature is merely an example of a general series of equivalent or similar features.

[0415] Furthermore, from the above description, those skilled in the art can easily identify the essential features of this disclosure and adapt it in various ways to suit various uses and conditions without departing from its intent and scope. Therefore, other embodiments are also within the scope of the claims.

[0416] Equal parts While several embodiments of the present invention have been described and illustrated herein, those skilled in the art will readily conceive of various other means and / or structures to perform the functions described herein and / or to obtain one or more of the results and / or benefits, and each of such variations and / or modifications will be considered within the scope of the embodiments of the present invention described herein. More generally, those skilled in the art will readily understand that all parameters, dimensions, materials and configurations described herein are intended to be illustrative, and that actual parameters, dimensions, materials and / or configurations will depend on the particular use or use in which the teachings of the present invention are used. Those skilled in the art will be able to recognize or confirm many equivalents to the particular embodiments of the present invention described herein by means of routine experimentation. Therefore, it should be understood that the embodiments described above are presented merely as examples, and within the scope of the appended claims and their equivalents, embodiments of the present invention may be carried out in ways other than those specifically described and claimed. The embodiments of the present invention in this disclosure cover each individual feature, system, article, material, kit and / or method 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 provided that they are not mutually inconsistent.

[0417] All definitions, when 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 being defined.

[0418] All references, patents, and patent applications disclosed herein are incorporated by reference with respect to the subject matter of the invention, each cited for that purpose, and in some cases encompassing the entire document.

[0419] Where used herein and in the claims, the indefinite articles "a" and "an" should be understood to mean "at least one" unless explicitly stated otherwise.

[0420] When used herein and in the claims, the phrase “and / or” should be understood to mean “either or both” of the elements thus combined, i.e., elements that exist in some cases conjugately and in others separately. Any multiple elements listed in “and / or” should similarly be interpreted as “one or more” of the elements thus combined. Other elements other than those specifically identified by the “and / or” clause may exist, whether related to or unrelated to those specifically identified elements. Therefore, as a non-restrictive example, a reference to “A and / or B,” when used in combination with an open-ended word such as “including,” may, in one embodiment, refer to A only (which may include elements other than B); in another embodiment, refer to B only (which may include elements other than A); and in yet another embodiment, refer to both A and B (which may include other elements), and so on.

[0421] 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 an enumeration, “or” or “and / or” should be interpreted as inclusive, that is, including at least one of several elements or enumerations of elements and additional unenumerated items, but more than one. In contrast, the only explicitly stated terms such as “one of” or “exactly one of” or, as used in the claims, “consisting of” refer to including exactly one element of several elements or enumerations. In general, where used herein, the term “or” should be interpreted only as indicating an exclusive substitute (i.e., “one or the other, but not both”) when preceded by terms of exclusivity such as “either one,” “one of,” “one of,” or “exactly one of.” Where used in the claims, “essentially consisting of” should have the ordinary meaning as used in the field of patent law.

[0422] As used herein and in the claims, the phrase “at least one” relating to an enumeration of one or more elements should be understood to mean at least one element selected from any one or more of the elements in the enumeration of elements, but not necessarily including each element and at least one of any elements specifically enumerated in the enumeration of elements, and not excluding any combination of elements in the enumeration of elements. This definition also allows for the presence of elements other than those specifically identified in the enumeration of elements referred to by the phrase “at least one,” whether related to those specifically identified elements or not. 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”) could mean, in one embodiment, that B is absent and there may be at least one or more A's (and other elements); in another embodiment, that A is absent and there may be at least one or more B's (and other elements); and in yet another embodiment, that may be at least one or more A's and at least one or more B's (and other elements), and so on.

[0423] In contrast, unless explicitly stated otherwise, any method claimed herein, including more than one step or action, should be understood that 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】 or its stereoisomers, tautomers, or salts [In the formula, M is the radioactive part; A is a chelate portion that chelates the radioactive portion; B is selected from the group consisting of aryl, heteroaryl, cycloalkyl, and heterocyclyl; B may also be a six-membered ring; X is -CH ２ C(NH)-, -CH ２ C(O)-, -CH ２ X is selected from the group consisting of C(S)-, -NHC(NH)-, -NHC(O)-, -NHC(S)-, -OC(NH)-, -OC(O)-, and -OC(S)-, where X is -CH ２ It may also be C(O)- or -NHC(S)-; Z is -CH ２ -ien-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)-, or -OC(S)-; L is a peptide linker having 1 to 6 amino acid residues, including the values ​​at both ends; R １ is H or C １～６ alkyl, and R １ may be H or methyl; R ２ C １～６ Alkyl or C ３～６ It is a cycloalkyl; R ３ C １～６ It is alkyl.

2. R ２ However, C １～６ The compound according to claim 1, wherein it is alkyl.

3. R ２ However, C ４ The compound according to claim 1 or 2, wherein it is alkyl.

4. R ２ However, sec-butyl(-CH(CH ３ )CH ２ CH ３ The compound according to claim 3, which is the compound described in claim 3.

5. Equation (Ia): 【Chemistry 2】 A compound according to claim 4, having the structure of the compound, its stereoisomer, tautomer, or salt thereof.

6. X is CH ２ A compound according to any one of claims 1 to 5, wherein the compound is C(O)- or -NHC(S)-.

7. Formula (Ib): 【Transformation 3】 A compound according to claim 6, having the structure of the compound, its stereoisomer, tautomer, or salt thereof.

8. R ３ However, C １ A compound according to any one of claims 1 to 7, wherein it is alkyl.

9. R ３ However, methyl (-CH ３ The compound according to claim 8, which is the compound described in claim 8.

10. Formula (Ic): 【Chemistry 4】 A compound according to claim 9, having the structure of the compound, its stereoisomer, tautomer, or salt thereof.

11. The compound according to any one of claims 1 to 10, wherein B is a six-membered ring.

12. Z is -CH ２ - or -CH ２ The compound according to any one of claims 1 to 11, wherein it is C(O)-.

13. Equations (Ic-A), (Ic-B), and (Ic-C): 【Transformation 5】 A compound according to claim 12, having one of the structures of the compound, or a stereoisomer, tautomer, or salt thereof.

14. Formulas (Ic-Aa), (Ic-Ab), (Ic-Ba), (Ic-Bb), (Ic-Ca), and (Ic-Cb): 【Chemistry 6-1】 【Chemistry 6-2】 A compound according to any one of claims 1 to 13, having one of the structures of the compound or a stereoisomer, tautomer, or salt thereof.

15. The following formula (Id): 【Transformation 7】 A compound according to any one of claims 1 to 11 having the structure, or a stereoisomer, tautomer, or salt thereof.

16. The compound according to any one of claims 1 to 15, wherein the chelated 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), a restricting complexing agent (RESCA), or MACROPA.

17. The compound according to any one of claims 1 to 16, wherein A is NODA.

18. The compound according to any one of claims 1 to 17, wherein L has 1 to 3 amino acid residues, including the values ​​at both ends.

19. The compound according to any one of claims 1 to 17, wherein L has 3 to 6 amino acid residues, including the values ​​at both ends.

20. L, Gly, Gly-Gly, Gln-Gly, Glu, Glu-Gly, Glu-Gly-Gly, Glu-βAla-βAla, Ｄ Glu、 Ｄ Glu-βAla-βAla、 Ｄ Glu-Gly-Gly、 Ｄ Glu-AEA, Ｄ ---AEEA-AEEA、 Ｄ Glu- Ｄ Glu-AEA, Ｄ Glu- Ｄ Glu-βAla-βAla、 γGlu, γGlu-βAla, Ｄ γGlu, Lys-Gly, Arg-Gly, N-acid-βAla-βAla, βAla-N-acid-βAla, βAla-Glu-Gly-Gly, βAla- Ｄ Glu-βAla, and Diacid-βAla-βAla A compound according to any one of claims 1 to 19, having an amino acid sequence selected from the group consisting of the following.

21. The compound according to any one of claims 1 to 20, wherein the radioactive portion of M is a therapeutic radioisotope.

22. 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 The therapeutic radioactive isotope is ９０ The compound according to claim 21, which may also be Y.

23. The chelated portion is NOTA or DOTA, and the therapeutic radioisotope is ９０ Y, １７７ Lu, or ２２５ A compound according to any one of claims 1 to 22, wherein the compound is Ac.

24. The chelated portion is NODA, and the therapeutic radioactive isotope is ４７ Sc or ６７ A compound according to any one of claims 1 to 22, wherein the compound is Cu.

25. The following equation (Id - A): 【Transformation 8】 A compound according to claim 24 having the structure, or a stereoisomer, tautomer, or salt thereof. [In the formula, M is, １７７ Lu, ９０ Y, ２２５ Ac, or ２１３ It is Bi.

26. The following formula (Id-Aa) or (Id-Ab) structure: 【Chemistry 9】 A compound according to claim 25 having one of the above, or a stereoisomer, tautomer, or salt thereof. [In the formula, M is, １７７ Lu, ９０ Y, ２２５ Ac, or ２１３ It is Bi.

27. Next structure: 【Chemistry 10】 The compound according to claim 1, having one of the following [In the formula, M is, １７７ Lu, ９０ Y, ２２５ Ac, or ２１３ It is Bi.

28. A pharmaceutical composition comprising a compound according to any one of claims 1 to 27 and a pharmaceutically acceptable carrier.

29. A method for treating cancer in a subject, (a) The step of administering an effective amount of the compound according to any one of claims 1 to 27 or a pharmaceutical composition containing the same to a subject that requires it. A method that includes this.

30. Before step (a), The method according to claim 29, further comprising the step of administering an immunotherapy drug to the subject.

31. The method according to claim 29 or 30, wherein the immunotherapy drug is an immune checkpoint inhibitor which may be a PD1 inhibitor, or a genetically modified T cell expressing a chimeric antigen receptor (CAR).

32. The method according to any one of claims 29 to 31, further comprising the step of administering an imaging agent for imaging granzyme B to the subject before step (a).

33. The method according to any one of claims 29 to 32, further treating the subject with one or more additional therapeutic agents.

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

35. A kit for cancer treatment comprising a first container containing a compound according to any one of claims 1 to 27 or a pharmaceutical composition containing the same.

36. The kit according to claim 35, comprising a pharmaceutical additive.

37. The kit according to claim 36, comprising a second container, wherein the pharmaceutical additive is contained within the second container.

38. The kit according to claim 36 or 37, wherein the compound or pharmaceutical composition and the pharmaceutical additive are combined to form a single unit dosage form.

39. A kit according to any one of claims 35 to 38, comprising an imaging agent suitable for imaging granzyme B.

40. A kit according to any one of claims 35 to 39, comprising an additional therapeutic agent.

41. The kit according to claim 40, wherein the additional therapeutic agent includes an immunotherapy drug.

42. The kit according to claim 40 or 41, wherein the additional therapeutic agent is contained in the first container.

43. The kit according to claim 41 or 42, wherein the immunotherapy drug is an immune checkpoint inhibitor which may be a PD1 inhibitor, or genetically modified T cells which express a chimeric antigen receptor (CAR).

44. A kit according to any one of claims 35 to 43, comprising instructions for using one or more of the compounds, pharmaceutical compositions, imaging agents, and additional therapeutic agents according to any of the preceding claims.

45. A kit according to any one of claims 35 to 44, comprising instructions for imaging granzyme B in a subject and / or treating cancer and / or reducing the risk of cancer.

46. The kit according to any one of claims 35 to 45, wherein the first container comprises a vial, ampoule, bottle, syringe, or dispenser packaging.

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