Prodrugs for compounds specific for granzyme B and uses thereof

Abstract

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

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 287,473, filed December 8, 2021, the contents of which are incorporated herein by reference in their entirety.

[0002] The present disclosure relates to prodrug compounds that can be converted to active forms useful for in vivo imaging techniques, and more specifically, to prodrug compounds that are specific for Granzyme B and that can be converted to active compounds useful for imaging Granzyme B using medical imaging methods including positron emission tomography. [Background technology]

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

[0004] There is a need for developing novel compounds that function as effective granzyme B imaging agents and therapies for treating immune dysregulation, such as cancer. Summary of the Invention

[0005] The present disclosure is based at least in part on the development of prodrug compounds that can be converted into active granzyme B (GZB) binding compounds, for example, in vivo. Such prodrug compounds (i.e., pro-type GZB binding compounds) exhibit excellent features, such as the generation of single isomers, synthesis with reliable stereochemical results, easy release of active GZB binding compounds in vivo, or combinations thereof. Such pro-type granzyme B (GZB) binding compounds can be used for GZB imaging (e.g., in vivo), the results of which can be relied upon for therapeutic and diagnostic purposes, for example, to identify patients suitable for treatment and / or to monitor treatment effectiveness.

[0006] Accordingly, in one aspect, the disclosure features a compound of formula (I): [ka]

[0007] In formula (I), A is a chelating moiety (e.g., those disclosed herein); B is selected from the group consisting of aryl, heteroaryl, cycloalkyl, and heterocyclyl; X is selected from the group consisting of -CHC(NH)-, -CHC(O)-, -CHC(S)-, -NHC(NH)-, -NHC(O)-, -NHC(S)-, -OC(NH)-, -OC(O)-, and -OC(S)-; Z is -CH-, -CHC(NH)-, -CHC(O)-, -CHC(S)-, -NHC(NH)-, -NHC(O)-, -NHC(S)-, -OC(NH)-, -OC(O)-, and -OC(S)-; L is a peptide linker having 1 to 6 amino acid residues, inclusive; R 1 But H or C 1-6 alkyl, and optionally R 1 is H or methyl, R 2 But, C1-6 Alkyl or C 3-6 is cycloalkyl, R 3 But, C 1-6 It is an alkyl.

[0008] In some examples, B can be a 6-membered ring. In certain examples, B can be a 6-membered aryl ring. In other examples, B can be a 6-membered heterocyclyl.

[0009] In some examples, X can be -CHC(O)-. In other examples, X can be -NHC(S)-.

[0010] In some examples, Z can be -CH- (e.g., when B is a piperidyl ring). Alternatively, Z can be -CHC(O)- (e.g., when B is a piperazyl ring).

[0011] In some embodiments, R 1 is H and R 2 is a C4 alkyl; [ka] where the variables A, B, X, Z, L, and R 3 is as defined herein.

[0012] In a further embodiment, X is -CH2C(O)-, such as, for example, in compounds of formula (Ib): [ka] In the formula, A, B, Z, L, and R 3 is as defined herein.

[0013] In some of the above embodiments, for example, in formula (Ic), R 3 is methyl, [ka] wherein the variables A, B, Z, and L are each as defined herein.

[0014] In some of the above embodiments, B is piperidyl and Z is -CH-, e.g., as in formula (Ic-A): [ka] wherein the variables A and L are each as defined herein.

[0015] In some embodiments, the compound has a defined stereochemistry, such as formula (Ic-Aa): [ka] wherein the variables A and L are each as defined herein.

[0016] In certain instances, the compound has a defined stereochemistry, such as formula (Ic-Ab): [ka] wherein the variables A and L are each as defined herein.

[0017] Exemplary compounds of formula (Ic-A) include compounds 1-17.

[0018] In some of the above embodiments, B is piperazyl and Z is -CH2C(O)-, for example, as in formula (Ic-B): [ka] wherein the variables A and L are each as defined herein.

[0019] In some embodiments, the compound has a defined stereochemistry, such as formula (Ic-Ba): [ka] wherein the variables A and L are each as defined herein.

[0020] In certain instances, the compound has a defined stereochemistry, such as formula (Ic-Bb): [ka] wherein the variables A and L are each as defined herein.

[0021] Exemplary compounds of formula (Ic-B) include compounds 18-28.

[0022] In some embodiments, B is phenyl and Z is -CH-, e.g., as in Formula (Ic-C): [ka] wherein the variables A and L are each as defined herein.

[0023] In some embodiments, the compound has a defined stereochemistry, such as formula (Ic-Ca): [ka] wherein the variables A and L are each as defined herein.

[0024] In certain examples, the compounds have a defined stereochemistry, such as formula (Ic-Cb): [ka] wherein the variables A and L are each as defined herein.

[0025] Exemplary compounds of formula (Ic-C) include compounds 29-46.

[0026] In some embodiments, as in formula (Id), B is phenyl and R 1is H and R 2 is C4 alkyl, R 3 is methyl, [ka] wherein the variables A, Z, X, and L are each as defined herein.

[0027] In some embodiments, A is NOTA and Z is -CH2-, as in formula (Id-A): [ka] wherein the variables X and L are each as defined herein.

[0028] In some embodiments, the compound has a defined stereochemistry, such as formula (Id-Aa): [ka] wherein the variables X and L are as defined herein.

[0029] In certain instances, the compound has a defined stereochemistry, such as formula (Id-Ab): [ka] wherein the variables X and L are as defined herein.

[0030] Exemplary compounds of formula (Id-A) include compounds 47-49.

[0031] In another aspect, the disclosure features a compound of formula (II), or a pharma- ceutically acceptable salt thereof: [ka]

[0032] The variables of formula (II) may be written as follows: M is a metal or a metal linked to a radioisotope; A is a chelating moiety that chelates a metal; B is selected from the group consisting of aryl, heteroaryl, cycloalkyl, and heterocyclyl, optionally B is a 6-membered ring; X is selected from the group consisting of -CHC(NH)-, -CHC(O)-, -CHC(S)-, -NHC(NH)-, -NHC(O)-, -NHC(S)-, -OC(NH)-, -OC(O)-, and -OC(S)-; optionally X is -CHC(O)- or -NHC(S)-; Z is -CH-, -CHC(NH)-, -CHC(O)-, -CHC(S)-, -NHC(NH)-, -NHC(O)-, -NHC(S)-, -OC(NH)-, -OC(O)-, and -OC(S)-, optionally Z is -CH- or -CHC(O)-; L is a peptide linker having 1 to 6 amino acid residues, inclusive; R 1 But H or C 1-6 alkyl, and optionally R 1 is H or methyl, R 2 But, C 1-6 Alkyl or C 3-6 is cycloalkyl, R 3 But, C 1-6 It is an alkyl.

[0033] In some examples, B is a 6-membered ring. In certain examples, B is a 6-membered aryl. In other examples, B can be a 6-membered heterocyclyl.

[0034] In some examples, X can be -CHC(O)-. In other examples, X can be -NHC(S)-.

[0035] In some examples, Z can be -CH- (e.g., when B is a piperidyl ring). Alternatively, Z can be -CHC(O)- (e.g., when B is a piperazyl ring).

[0036] In some embodiments, R 1 is H and R 2 is a C4 alkyl; [ka] where the variables M, A, B, X, Z, and R 3 and L is as defined herein.

[0037] In a further embodiment, X is -CH2C(O)-, such as, for example, in compounds of formula (IIb): [ka] where the variables M, A, B, Z, and R 3 and L is as defined herein.

[0038] In some embodiments, R 3 is methyl, [ka] wherein the variables M, A, B, Z, and L are each as defined herein.

[0039] In some embodiments, B is piperidyl and Z is -CH-, as in Formula (IIc-A), [ka] wherein the variables M, A, and L are each as defined herein.

[0040] In some embodiments, the compound has a defined stereochemistry, such as formula (IIc-Aa): [ka] wherein the variables M, A, and L are each as defined herein.

[0041] In certain instances, the compound has a defined stereochemistry, such as formula (IIc-Ab): [ka] wherein the variables M, A, and L are each as defined herein.

[0042] Exemplary compounds of formula (IIc-Bb) include compounds 1-Al through 17-Al, as described herein.

[0043] In some embodiments, B is phenyl and Z is -CH-, as in Formula (IIc-B): [ka] wherein the variables A and L are each as defined herein.

[0044] In some embodiments, the compound has a defined stereochemistry, such as formula (IIc-Ba): [ka] wherein the variables A and L are each as defined herein.

[0045] In certain instances, the compound has a defined stereochemistry, such as formula (IIc-Bb): [ka] wherein the variables A and L are each as defined herein.

[0046] Exemplary compounds of formula (IIc-B) include compounds 18-Al through 28-Al, as described herein.

[0047] In some embodiments, B is phenyl and R 3 is methyl, [ka] wherein the variables M, A, Z, X, and L are each as defined herein.

[0048] In some embodiments, A is NOTA, -CH2-, as in formula (IId-A): [ka] wherein the variables M, X, and L are each as defined herein.

[0049] In some embodiments, the compound has a defined stereochemistry, such as formula (IId-Aa): [ka] wherein the variables M, A, X, and L are each as defined herein.

[0050] In certain instances, the compound has a defined stereochemistry, such as formula (IId-Ab): [ka] wherein the variables M, A, X, and L are each as defined herein.

[0051] Exemplary compounds of formula (IId) include compounds 47-Al through 49-Al, as described herein.

[0052] In any of the GZB binding compounds disclosed herein, L can have 1-6 amino acid residues, e.g., 1-5 amino acid residues. The amino acid residues can be standard proteinogenic amino acids (the 20 amino acid residues found in naturally occurring proteins) or non-natural amino acids (e.g., derivatives or isomers of any of the 20 naturally occurring amino acid residues). Exemplary structures of non-naturally occurring amino acid residues that can be included in the L linker are provided in Table 1 below. Exemplary 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- D Glu-βAla, and diacid-βAla-βAla. See Table 2 for the structures of these exemplary L linkers.

[0053] In any of the compounds of formula (I) or (II) described herein, A can be a chelating moiety known in the art to be useful as described herein, for example, for binding to metals. In some examples, the chelating moiety is 1,4,7-triazacyclononane-N,N',N''-triacetic acid (NOTA) or 1,4,7-triazacyclononane-4,7-diyldiacetic acid (NODA).

[0054] In any of the compounds of formula (II) disclosed herein, M can be a metal known in the art to be useful as described herein, for example, for imaging purposes. In some examples, the metal (either alone or in combination with a radioisotope) can be toxic. In other examples, the metal (either alone or in conjugation with a radioisotope) can be non-toxic. Exemplary metals include Ga (e.g., 68 Ga) and Al( 18 F).

[0055] As shown in the exemplary compounds herein, the stereochemistry of the two stereocenters of the 5-oxotetrahydrofuranyl moiety can affect the properties of the compounds disclosed herein. In some instances, the relationship between the amide and the alkoxy is cis (i.e., syn). In certain instances, the stereocenter of the amide-bearing carbon is assigned (S). In other instances, the stereocenter of the alkoxy-bearing carbon is assigned (R).

[0056] In another aspect, the disclosure features a pharmaceutical composition including any of the GZB binding compounds disclosed herein, their stereoisomers, or their pharma- ceutically acceptable salts, and a pharma- ceutically acceptable carrier.

[0057] In another aspect, the disclosure features a method of imaging granzyme B in a tissue, the method comprising: (i) contacting any of the compounds of formula (II) disclosed herein, or a pharma- ceutically acceptable salt thereof, with tissue suspected of containing granzyme B; (ii) imaging the tissue based on a radioisotope signal emitted from the compound or a pharma- ceutically acceptable salt thereof.

[0058] In some embodiments, the imaging methods disclosed herein are performed in vitro, for example, tissue for imaging by the methods is in a biological sample that can be obtained from a subject (e.g., a human patient) as disclosed herein.

[0059] In other embodiments, the imaging methods disclosed herein may be performed in vivo, and an effective amount of the compound or a pharma- ceutically acceptable salt thereof may be administered to a subject (e.g., a human patient) in need thereof.

[0060] In some embodiments, the subject is receiving treatment (e.g., anti-inflammatory agents, steroids, immunotherapeutic agents, chemotherapeutic agents, and therapeutic antibodies) for an immune dysregulation (e.g., autoimmune disorders, inflammatory disorders, skin disorders, cancer, and cardiovascular disorders).

[0061] In some embodiments, the immune response in a subject is monitored based on imaging of granzyme B.

[0062] Also provided herein is a compound of formula (II), a pharma- ceutically acceptable salt thereof, or a pharmaceutical composition comprising same, for use in imaging granzyme B for diagnostic purposes or to monitor the effect of treatment.

[0063] The details of one or more embodiments of the invention are set forth in the description below. Other features or advantages of the invention will become apparent from the following drawings and detailed description of certain embodiments, and from the appended claims. [Brief description of the drawings]

[0064] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure, which may be better understood by reference to the drawings in combination with the detailed description of specific embodiments presented herein.

[0065] [Figure 1A-1B] Included is a diagram showing the synthesis of compound 18F-4-Al. [Figure 1A] Semi-preparative HPLC radiotrace for the preparation of compound 18F-4-Al. [Figure 1B] Analytical HPLC radiotrace of compound 18F-4-Al. [Diagram 2] 1 includes diagrams showing the structures of exemplary pro- and active forms of GZB binding compounds. 29-Al and 4-Al (prodrug form) are shown in their active forms. [Diagram 3] Includes diagrams showing chemical structures of exemplary prodrugs. [Figure 4A-4D] FIG. 1 includes a diagram showing the in vivo imaging activity of an exemplary prodrug compound 18F-4-Al (cis form). [Figure 4A] The structure of the compound 18F-4-Al. [Figure 4B] Chart showing the in vivo imaging activity of compound 18F-4-Al. Left panel: %ID / g. Right panel: TBR. [Figure 4C] FIG. 1 shows the presence of active compound in mouse plasma 5 min after dosing. [Figure 4D] Photographs showing the in vivo imaging activity of compound 18F-4-Al. [Figure 5A-5D] FIG. 1 includes a diagram showing the in vivo imaging activity of an exemplary prodrug compound 18F-3-Al (trans form). [Figure 5A] The structure of the compound 18F-3-Al. [Figure 5B] Chart showing the in vivo imaging activity of compound 18F-3-Al. Left panel: %ID / g. Right panel: TBR. [Figure 5C] FIG. 1 shows the presence of the indicated compounds in mouse plasma 5 min after administration. [Figure 5D] Photographs showing the in vivo imaging activity of compound 18F-3-Al. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0066] Cancer immunotherapy represents a significant advancement in cancer therapy in recent years. Antibodies targeting immune checkpoints such as programmed cell death protein 1 (PD-1) and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) have been approved with positive outcomes for several patients. Research in the field of immuno-oncology continues with strategies including CAR-T cells, vaccines, small molecules, and antibodies under development. Despite the promise of these therapies, they are not a panacea. These immunotherapies can be associated with significant adverse events, are expensive, and response rates are typically 20-50%, meaning that a large proportion of patients do not respond to the therapy. Furthermore, it can be difficult to determine an individual patient's response to therapy using traditional methods, as the response may be seen as growing on anatomical imaging (e.g., CT, MRI) and is frequently associated with immune cell infiltration that demonstrates increased avidity on FDG-PET imaging due to the influx of metabolically active immune cells. Given the limitations of current imaging technology, clinical studies of cancer immunotherapy typically use overall survival as a study endpoint, as opposed to progression-free survival.

[0067] Granzyme B, a downstream marker of cytotoxic T cell activity, can serve as a novel biomarker for evaluating the efficacy of cancer immunotherapy. Granzyme B expression in tumors can be evaluated not only for the presence or absence of CTLs, but also as an effector protein released by activated T cells that integrates a measure of CTL activity, thus accounting for the problem of T cell exhaustion that makes the assessment of the presence of CTLs difficult to achieve.

[0068] The present disclosure provides prodrug compounds (also known as pro-forms), such as compounds of formula (I) and formula (II), which can encompass in vivo active granzyme B (GZB) binding compounds. Such compounds are chemically stable compared to their active counterparts. In addition, the prodrug strategy used herein allows reliable stereochemical stability during the synthesis of compounds of formula (I) and (II), while providing easy release of active drugs in vitro. Thus, the pro-forms of GZB binding compounds disclosed herein can function as granzyme B imaging agents through rapid in vivo conversion. As reported herein, certain stereochemical isomers (e.g., cis isomers) have shown superior in vivo conversion and imaging activity. The prodrug forms of GZB binding compounds disclosed herein can be used to identify patients who are responsive to immunotherapeutic agents or monitor the therapeutic efficacy of immunotherapeutic agents based on the level and / or location of granzyme B determined by the imaging assays disclosed herein.

[0069] definition It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. Furthermore, although any methods, devices, and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods, devices, and materials are described herein. In addition to the above, as used in the specification and appended claims, unless specified to the contrary, the following terms have the meanings indicated. "Amino" refers to the -NH2 radical. "Cyano" refers to the -CN radical. "Hydroxyl" refers to the -OH radical. "Imino" refers to the =NH substituent. "Nitro" refers to the -NO2 radical. "Oxo" refers to the =O substituent. "Thioxo" refers to the =S substituent. "Trifluoromethyl" refers to the -CF3 radical.

[0070] "Alkyl" refers to a linear, saturated, acyclic, monovalent hydrocarbon radical or a branched, saturated, acyclic, monovalent hydrocarbon radical having from 1 to 6 carbon atoms and attached to the remainder of the molecule by a single bond, e.g., methyl, ethyl, n-propyl, 1-methylethyl (iso-propyl), n-butyl, n-pentyl, 1,1-dimethylethyl (t-butyl), 3-methylpentyl-1,2-methylpentyl, and the like. The alkyl portion can be unsubstituted. Alternatively, the alkyl portion can be optionally substituted. Optionally substituted alkyl radicals can include, depending on the valence, independently, halo, cyano, nitro, aryl, cycloalkyl, heterocyclyl, heteroaryl, oxo, trimethylsilanyl, -OR, -O- ... 3 , -OC(O)-R 3 , -N(R 3 )2, -C(O)R 4 , -C(O)OR 3 , -C(O)N(R 3 )2, -N(R 3 )C(O)OR 5 , -N(R 3 )C(O)R 5 , -N(R 3 )S(O) t R 5 (wherein t is 1 or 2), -S(O) t OR 5 (wherein t is 1 or 2), -S(O) p R 5 (wherein p is 0, 1, or 2) and -S(O) t N(R 3 t is 1 or 2; and each R 3 is independently hydrogen, alkyl, haloalkyl, cycloalkyl, cycloalkylalkyl, aryl, aralkyl, heterocyclyl, or heteroaryl; 4 is independently hydrogen, cycloalkyl, aryl, heterocyclyl, or heteroaryl; each R 5is independently alkyl, haloalkyl, cycloalkyl, aryl, or heteroaryl.

[0071] "Cycloalkyl" refers to a stable non-aromatic monocyclic or polycyclic hydrocarbon radical having 3 to 15 carbon atoms, preferably 3 to 10 carbon atoms, saturated or unsaturated, and attached to the remainder of the molecule by a single bond. Polycyclic hydrocarbon radicals are bicyclic, tricyclic, or tetracyclic ring systems. Unsaturated cycloalkyls contain one, two, or three carbon-carbon double bonds and / or one 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 can be unsubstituted. Alternatively, the cycloalkyl moiety can be optionally substituted. Optionally substituted cycloalkyl is independently selected from alkyl, alkenyl, halo, haloalkyl, haloalkenyl, cyano, nitro, oxo, aryl, aralkyl, cycloalkyl, heterocyclyl, heteroaryl, -R 4 -OR 3 , -R 4 -OC(O)-R 3 , -R 4 -N(R 3 )2, -R 4 -C(O)R 3 , R 4 -C(O)OR 3 , -R 4 -C(O)N(R 3 )2, -R 4 -N(R 3 )C(O)OR 5 , -R 4 -N(R 3 )C(O)R 5 , -R 4 -N(R 3 )S(O) t R 5 (wherein t is 1 or 2), -R 4 -S(O) t OR 5(wherein t is 1 or 2), -R 4 -S(O) p R 5 (wherein p is 0, 1, or 2), and -R 4 -S(O) t N(R 3 )2, where t is 1 or 2; and each R 3 is independently hydrogen, alkyl, haloalkyl, cycloalkyl, aryl, heterocyclyl, or heteroaryl; each R 4 is independently a direct bond or a linear or branched alkylene or alkenylene chain; 5 is independently alkyl, haloalkyl, cycloalkylalkyl, aryl, aralkyl, heterocyclyl, or heteroaryl.

[0072] A "chelating moiety" is a molecule or ion that can function as a multidentate ligand for a metal ion. For example, molecules with multiple atoms with available loan pairs (including, but not limited to, nitrogen and oxygen) can function as chelating moieties. Chelating moieties can be linear (e.g., EDTA), cyclic (including macrocycles, e.g., DOTA, porphyrins), and can include macrocycles that are commonly known in the art. Chelating moieties can have 2, 3, 4, 5, or 6 functional groups (e.g., amines, amides, hydroxyls, carboxylic acids, etc.) with available loan pairs for coordinating with a metal.

[0073] In some embodiments, preparation of compounds may involve the addition of acids or bases, for example to affect catalysis of a desired reaction or the formation of salt forms, such as acid addition salts.

[0074] Exemplary acids may be inorganic or organic, including, but not limited to, strong and weak acids. Some exemplary acids include hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, p-toluenesulfonic acid, 4-nitrobenzoic acid, methanesulfonic acid, benzenesulfonic acid, trifluoroacetic acid, and nitric acid. Some weak acids include, but are not limited to, acetic acid, propionic acid, butanoic acid, benzoic acid, tartaric acid, pentanoic acid, hexanoic acid, heptanoic acid, octanoic acid, nonanoic acid, and decanoic acid.

[0075] Exemplary bases include lithium hydroxide, sodium hydroxide, potassium hydroxide, lithium carbonate, sodium carbonate, potassium carbonate, and sodium bicarbonate. Some examples of strong bases include, but are not limited to, hydroxides, alkoxides, metal amides, metal hydrides, metal dialkylamides, and arylamines, where alkoxides include the lithium, sodium, and potassium salts of methyl, ethyl, and t-butyl oxides, metal amides include sodium amide, potassium amide, and lithium amide, metal hydrides include sodium hydride, potassium hydride, and lithium hydride, and metal dialkylamides include the lithium, sodium, and potassium salts of methyl, ethyl, n-propyl, iso-propyl, n-butyl, tert-butyl, trimethylsilyl, and cyclohexyl substituted amides.

[0076] As used herein, the phrase "pharmaceutically acceptable salts" refers to derivatives of the disclosed compounds, in which the parent compound is modified by converting an existing acid or base moiety into its salt form. Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines, alkali or organic salts of acidic residues such as carboxylic acids, and the like. The pharmaceutically acceptable salts of the present application include conventional non-toxic salts of the parent compound formed, for example, from non-toxic inorganic or organic acids. The pharmaceutically acceptable salts of the present application can be synthesized from the parent compound containing a basic or acidic moiety by conventional chemical methods. In general, such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or an organic solvent, or in a mixture of the two, generally with non-aqueous media such as ether, ethyl acetate, alcohol (e.g., methanol, ethanol, isopropanol, or butanol), or acetonitrile (MeCN) being preferred. A list of suitable salts can be found in Remington's Pharmaceutical Sciences, 17 th 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.

[0077] In some embodiments, the compounds provided herein, or salts thereof, are substantially isolated. "Substantially isolated" means that the compounds are at least partially or substantially separated from the environment in which they were formed or detected. Partial separation can include, for example, compositions enriched in the compounds provided herein. Substantial separation can include compositions containing at least about 50% by weight, at least about 60% by weight, at least about 70% by weight, at least about 80% by weight, at least about 90% by weight, at least about 95% by weight, at least about 97% by weight, or at least about 99% by weight of the compounds provided herein, or salts thereof. Methods for isolating compounds and their salts are routine in the art.

[0078] As used herein, the expressions "ambient temperature" and "room temperature" or "rt" are understood in the art and generally refer to, for example, the reaction temperature, i.e., room temperature, at which a reaction is carried out, for example, a temperature of about 20° C. to about 30° C.

[0079] I. Prodrugs of Granzyme B Targeting Compounds In some aspects, provided herein are granzyme B targeting compounds disclosed herein, such as compounds of formula (I) or formula (II). The compounds disclosed herein include the compounds themselves, pharma- ceutically acceptable salts thereof, and stereoisomers thereof.

[0080] The compounds described herein may contain one or more asymmetric 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 may be in the form of a mixture of stereoisomers, including racemic mixtures and mixtures enriched in one or more stereoisomers. Isomers may be isolated from mixtures 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 (EL Eliel, Ed., Univ. of Notre Dame Press, Notre Dame, IN 1972). The present disclosure further encompasses the compounds described herein as individual isomers substantially free of other isomers, or alternatively, as mixtures of various isomers.

[0081] A. Compounds of Formula (I) In some embodiments, the present disclosure provides compounds of formula (I), as shown below, that are prodrugs of compounds capable of binding to Granzyme B with high binding affinity and specificity. [ka]

[0082] In formula (I), A is a chelating moiety. Exemplary chelating moieties for use in the granzyme B targeting compounds disclosed herein include 1,4,7-triazycyclonan triacetic acid (NOTA), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), 1,4,7-triazycyclonan-1-glutaric acid-4,7-diacetic acid (NODAGA), ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), cyclohexyl-1,2-diaminetetraacetic acid (CDTA), ethyleneglycol-0,0'-bis(2-aminoethyl)-N,N,N',N'-tetraacetic acid (E GTA), N,N-bis(hydroxybenzyl)-ethylenediamine-N,N'-diacetic acid (HBED), triethylenetetraminehexaacetic acid (TTHA), hydroxydiaminetriacetic acid (HEDTA), 1,4,8,11-tetraazacyclotetradecane-N,N',N'',N'''-tetraacetic acid (TETA), 1,4,7,10-tetraaza-1,4,7,10-tetra-(2-carbamoylmethyl)-cyclododecane (TCMC), 1,4,7-triazacyclononane-4,7-diyldiacetic acid (NODA), and desferrioxamine B (DFO). In some embodiments, the chelating agent is selected from the group consisting of 1,4,7-triazacyclononane triacetic acid (NOTA), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), 1,4,7-triazacyclononane-4,7-diyl diacetic acid (NODA) and 1,4,7-triazacyclononane-1-glutaric acid-4,7-diacetic acid (NODAGA). In some embodiments, the chelating agent is 1,4,7-triazacyclononane triacetic acid (NOTA). In other embodiments, the chelating agent is 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA). In some embodiments, the chelating agent is 1,4,7-triazacyclononane-4,7-diyl diacetic acid (NODA).

[0083] B can be aryl, heteroaryl, cycloalkyl, or heterocyclyl. In some examples, B is a 6-membered ring. In one example, B is a 6-membered aryl ring, such as phenyl. In another example, B is a 6-membered heterocyclyl, such as a piperazine ring or a piperazine ring.

[0084] X can be -CHC(NH)-, -CHC(O)-, -CHC(S)-, -NHC(NH)-, -NHC(O)-, -NHC(S)-, -OC(NH)-, -OC(O)-, or -OC(S)-. In one example, X is -CHC(O)-. In another example, X is -NHC(S)-.

[0085] X can be -CH-, -CHC(NH)-, -CHC(O)-, -CHC(S)-, -NHC(NH)-, -NHC(O)-, -NHC(S)-, -OC(NH)-, -OC(O)-, and -OC(S)-. In some embodiments, Z is -CH- (e.g., when attached to a piperazine ring). In other embodiments, Z is -CHC(O)- (e.g., when attached to a piperazine ring).

[0086] L can be a peptide linker having 1-6 amino acid residues, inclusive. In some examples, L includes 1-5 amino acid residues, inclusive. In other examples, L includes 2-4 amino acid residues, inclusive. In one example, L includes 1 amino acid residue. In another example, L includes 2 amino acids. In yet another example, L includes 3 amino acid residues. Alternatively, L includes 4 amino acid residues. In yet another example, L includes 5 amino acid residues. Alternatively, L includes 6 amino acid residues.

[0087] Compatible amino acid residues in the peptide linker L can include natural and non-natural amino acid residues, including β-amino acid residues and D-amino acids, but are not limited to proteogenic amino acid residues. As used herein, proteinogenic amino acid residues refer to the 20 amino acid residues that occur naturally as building blocks for synthesizing proteins. The amino acid residues can form chains through standard peptide bonds or by forming amide bonds with calculable side chains (e.g., glutamic acid (e.g., D-Glu), aspartic acid). Table 1 provides some exemplary non-proteinogenic (non-naturally occurring) amino acids that can be used in any of the peptide linkers L disclosed herein, including their chemical structures. [Table 1]

[0088] Exemplary peptide linkers 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- D These include, but are not limited to, Glu-βAla, and diacid-βAla-βAla. [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4]

[0089] In some embodiments, R 1 is H. In other embodiments, R 1 is C 1-6 For example, R 1 can be methyl.

[0090] In some embodiments, R 2 is C 1-6 Alternatively, R 2 is C 3-6 Alkyl (e.g., branched or unbranched, substituted or unsubstituted) or C 3-6 It can be cycloalkyl (eg, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl).

[0091] In some embodiments, R 1 is H and R 2 is a C4 alkyl such as in the compound of formula (Ia). [ka]

[0092] In some embodiments, X is -CH2C(O)-, such as in the compound of formula (Ib) below: [ka]

[0093] In some instances, such as in compounds of formula (Ic), R 3 is methyl. [ka]

[0094] In some examples of formula (Ic), B is piperidyl and Z is -CH-, such as compounds of formula (Ic-A). [ka]

[0095] Exemplary compounds of formula (Ic-A) include those listed in Table 3. [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4]

[0096] In some examples, the compound of formula (I) is compound 1, 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, 15, 16, or 17. In one embodiment, the compound of formula (I) is compound 4. In another embodiment, the compound of formula (I) is compound 7. As described herein, the above examples benefit from the piperidine ring, which exhibits improved properties over other related moieties.

[0097] In some examples of formula (Ic), B is piperazyl and Z is -CHC(O)-, such as compounds of formula (Ic-B). [ka]

[0098] Exemplary compounds of formula (Ic-B) include those listed in Table 4. [Table 4-1] [Table 4-2] [Table 4-3]

[0099] In some examples, the compound of formula (I) is compound 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28. As described herein, the above examples benefit from a piperazine ring that exhibits improved properties over several other related moieties.

[0100] In some examples of formula (Ic), B is phenyl and Z is -CH-, such as compounds of formula (Ic-C): [ka]

[0101] Exemplary compounds of formula (Ic-C) include those listed in Table 5. [Table 5-1] [Table 5-2] [Table 5-3] [Table 5-4]

[0102] In some examples, the compound of formula (I) is compound 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, or 46. In other examples, the compound of formula (I) is compound 29.

[0103] In some examples of formula (Ia), A is NOTA, B is phenyl, Z is -CH-, and R is 1, such as the compound of formula (Id). 3 is methyl [ka]

[0104] Exemplary compounds of formula (Id) include those listed in Table 6. [Table 6]

[0105] In some examples, the compound of formula (I) is compound 47, 48, or 49.

[0106] B. Compound of Formula (II) In some embodiments, the present disclosure provides a compound of formula (II) as shown below: In comparison to the compounds of formula (I) disclosed herein, the compounds of formula (II) further contain a metal that can be conjugated to a radioisotope via a chelating moiety A. [ka]

[0107] In formula (II), M is a metal or a metal bound to a radioisotope. Suitable metals for use in the present disclosure include those useful for imaging granzyme B, such as metals that are suitable radioimaging agents, as well as metals that can be bound to non-metallic radioisotopes that are suitable radioimaging agents. Exemplary metallic radioisotopes include: 68 Exemplary non-metallic radioisotopes are: 18 F, which can be conjugated with Al for loading into the granzyme B binding compounds disclosed herein.

[0108] A, X, Y, Z, L, R 1 , R 2 , and R 3are as defined herein. See, for example, the section entitled Compounds of Formula (I) above.

[0109] In some embodiments, as in the compound of formula (Ia), R 1 is H and R 2 is a C4 alkyl. [ka]

[0110] In some embodiments, X is -CH2C(O)-, such as in the compound of formula (IIb). [ka]

[0111] In some instances, such as in compounds of formula (IIc), R 3 is methyl [ka]

[0112] In some examples of formula (IIc), B is piperidyl and Z is -CH-, such as compounds of formula (Ic-A). [ka]

[0113] Exemplary compounds of formula (IIc-A) include those listed in Table 7. [Table 7-1] [Table 7-2] [Table 7-3] [Table 7-4]

[0114] In some examples, the compound of formula (I) is compound 1-Al, 2-Al, 3-Al, 4-Al, 5-Al, 6-Al, 7-Al, 8-Al, 9-Al, 10-Al, 11-Al, 12-Al, 13-Al, 14-Al, 15-Al, 16-Al, or 17-Al. In one embodiment, the compound of formula (I) is compound 4-Al. In another embodiment, the compound of formula (I) is compound 7-Al. As described herein, the above examples benefit from the piperidine ring, which exhibits improved properties over other related moieties.

[0115] In some examples of formula (IIc), B is piperazyl and Z is -CHC(O)-, such as compounds of formula (IIc-B). [ka]

[0116] Exemplary compounds of formula (IIc-B) include those listed in Table 8. [Table 8-1] [Table 8-2] [Table 8-3]

[0117] In some examples, the compound of formula (I) is the compound 18-Al, 19-Al, 20-Al, 21-Al, 22-Al, 23-Al, 24-Al, 25-Al, 26-Al, 27-Al, or 28-Al. As described herein, the above examples benefit from a piperazine ring that exhibits improved properties over some other related moieties.

[0118] In some examples of formula (IIc), B is phenyl and Z is -CH-, such as compounds of formula (IIc-C). [ka]

[0119] Exemplary compounds of formula (IIc-C) include those listed in Table 9. [Table 9-1] [Table 9-2] [Table 9-3] [Table 9-4]

[0120] In some examples, the compound of formula (I) is compound 29-Al, 30-Al, 31-Al, 32-Al, 33-Al, 34-Al, 35-Al, 36-Al, 37-Al, 38-Al, 39-Al, 40-Al, 41-Al, 42-Al, 43-Al, 44-Al, 45-Al, or 46-Al. In another embodiment, the compound of formula (I) is compound 29.

[0121] In some examples of formula (IIa), A is NOTA, B is phenyl, Z is -CH-, and R is 1, such as the compound of formula (IId). 3 is methyl [ka]

[0122] Exemplary compounds of formula (IId) include those listed in Table 10. [Table 10]

[0123] In some examples, the compound of formula (I) is compound 47-Al, 48-Al, or 49-Al.

[0124] As shown in the examples below, prodrugs of the GZB binding compounds disclosed herein, including methyl or ethyl ethers, as well as certain peptide linker structures, exhibit one or more of the following favorable features: production of a single isomer, synthesis with reliable stereochemical outcomes, and facile release of active drug in vivo.

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

[0126] C. Chemical Synthesis of Granzyme B Targeting Compounds As will be understood, the compounds provided herein, including stereoisomers and salts thereof, may be prepared using known organic synthesis techniques and may be synthesized according to any of a number of possible synthetic routes.

[0127] The compounds disclosed herein, or pharma- ceutically acceptable salts thereof, can be prepared by following the exemplary protocols described below. Suitable protecting groups for use in such syntheses are known in the art. See, for example, McOmie, Protective Groups in Organic Chemistry, (1973):98.

[0128] General synthetic procedures and examples thereof for the preparation of peptide linkers L, Fmoc-Haic(2S,5S)-OH tricyclic moieties, and suitable metal complexes with chelating moieties can be found in International Application No. PCT / US2021 / 036661, filed June 9, 2021, the relevant disclosures of which are incorporated by reference for the subject matter and purposes referenced herein.

[0129] The chelating moiety may be prepared and attached to the peptide linker, L, by any suitable means known in the art.

[0130] The prodrug moiety can be prepared using synthetic means known in the art. In a preferred embodiment, the enantiopure α-amino alcohol of reduced aspartic acid is oxidized to an aldehyde and formed into a selected acetal. The γ-carboxylate can then be condensed onto the acetal to form a ring. The resulting diastereomers can then be separated. The use of natural or unnatural aspartic acid provides access to all four diastereomers.

[0131] Many suitable imaging agents (e.g., ratio isotopes) are known in the art (see, e.g., U.S. Pat. Nos. 5,021,236, 4,938,948, and 4,472,509, the disclosures of each of which are incorporated herein by reference in their entirety). The radiolabeled compounds provided herein, or pharma- ceutically acceptable salts thereof, can be prepared according to methods well known in the art. Synthetic methods for incorporating radioisotopes into organic compounds are well known in the art, and those skilled in the art will readily recognize other methods that are applicable to the compounds provided herein.

[0132] It will be understood by those of skill in the art that the processes described herein are not the exclusive means for synthesizing the compounds provided herein, and that a wide repertoire of synthetic organic reactions is available for potential use in synthesizing the compounds provided herein, and those of skill in the art will know how to select and carry out appropriate synthetic routes. Suitable methods for synthesizing the starting materials, intermediates, and products are described in Advances in Heterocyclic Chemistry, Vols. 1-107 (Elsevier, 1963-2012); Journal of Heterocyclic Chemistry Vols. 1-49 (Journal of Heterocyclic Chemistry, 1964-2012), Carreira, et al. (Ed.) Science of Synthesis, Vols. 1-48 (2001-2010) and Knowledge Updates KU2010 / 1-4; 2011 / 1-4; 2012 / 1-2 (Thieme, 2001-2012), Katritzky, et al. (Ed.) Comprehensive Organic Functional Group Transformations, (Pergamon Press, 1996), Katritzky et al. (Ed.); Comprehensive Organic Functional Group Transformations II (Elsevier, 2003), and others. ndEdition, 2004), Katritzky et al. (Ed.), Comprehensive Heterocyclic Chemistry (Pergamon Press, 1984), Katritzky et al., Comprehensive Heterocyclic Chemistry II, (Pergamon Press, 1996), Smith et al., March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, 6 th Ed. (Wiley, 2007), Trost et al. (Ed.), Comprehensive Organic Synthesis (Pergamon Press, 1991), and other literature sources.

[0133] The reactions for preparing the compounds described herein can be carried out in a suitable solvent that can be easily selected by those skilled in the art of organic synthesis. A suitable solvent can be substantially non-reactive with the starting materials (reactants), intermediates, or products at the temperature at which the reaction is carried out (e.g., temperatures ranging from the freezing temperature of the solvent to the boiling temperature of the solvent). A given reaction can be carried out in one solvent or a mixture of two or more solvents. Depending on the particular reaction step, a suitable solvent for a particular reaction step can be selected by those skilled in the art.

[0134] Preparation of the compounds described herein can involve the protection and deprotection of various chemical groups. The need for protection and deprotection, and the selection of appropriate protecting groups, can be readily determined by one of ordinary skill in the art. The chemistry of protecting groups can be found, for example, in TW Greene and PG M Huts, Protective Groups in Organic Synthesis, 3 rd Ed., Wiley & Sons, Inc., New York (1999).

[0135] The reaction can be monitored according to any suitable method known in the art. For example, the formation of the product can be monitored by nuclear magnetic resonance spectroscopy (e.g., 1 H or 13 C), infrared spectroscopy, spectrophotometry (e.g., UV-visible), mass spectrometry, or chromatographic methods such as high performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LCMS), or thin layer chromatography (TLC). Compounds can be purified by one of skill in the art by a variety of methods, including high performance liquid chromatography (HPLC) and normal phase silica chromatography.

[0136] II. Pharmaceutical Compositions Any of the compounds of formula (I) and formula (II), or a pharma- ceutically acceptable salt thereof, may be mixed with a pharma- ceutically acceptable carrier to form a pharmaceutical composition for use in granzyme B imaging and / or for therapeutic purposes as disclosed herein. In some embodiments, provided herein is a pharmaceutical composition comprising, as an active ingredient, a metal-bearing compound provided by the present invention (a compound of formula (II)), or a pharma- ceutically acceptable salt thereof, in combination with one or more pharma- ceutical acceptable carriers (excipients). By "acceptable," it is meant that the carrier must be compatible with the active ingredient of the composition (preferably capable of stabilizing the active ingredient) and not harmful to the subject being treated. Suitable carriers include microcrystalline cellulose, mannitol, glucose, skim milk powder, polyvinylpyrrolidone, and starch, or a combination thereof.

[0137] Some examples of suitable excipients include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum acacia, calcium phosphate, alginate, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, and methylcellulose. Pharmaceutical preparations may further include, but are not limited to, lubricants such as talc, magnesium stearate, and mineral oil, wetting agents, emulsifying and suspending agents, preserving agents such as methyl and propylhydroxybenzoates, sweeteners, flavoring agents, or combinations thereof. See Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa., 1985, p.1418, which describes the details of acceptable pharmaceutical compositions.

[0138] Conventional methods known to those skilled in the medical field can be used to administer the pharmaceutical composition to a subject depending on the type of disease or site of disease to be treated. The composition can also be administered by other conventional routes, such as orally, parenterally, by inhalation spray, topically, rectally, nasally, bucally, vaginally, or via an implanted reservoir. As used herein, the term "parenteral" includes subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal, intralesional, and intracranial injection or infusion techniques. Parenteral administration can be in the form of a single bolus dose or can be, for example, by a continuous perfusion pump. In addition, it can be administered to a subject via injectable depot route of administration, such as using 1, 3, or 6 month depot injectable or biodegradable materials and methods.

[0139] Injectable compositions may contain a variety of carriers, such as vegetable oils, dimethylactamide, dimethylformamide, ethyl lactate, ethyl carbonate, isopropyl myristate, ethanol, and polyols (such as glycerol, propylene glycol, and liquid polyethylene glycol). For intravenous injection, water-soluble antibodies may be administered by drip method, whereby a pharmaceutical formulation containing the antibody and a physiologically acceptable excipient is infused. Physiologically acceptable excipients may include, for example, 5% dextrose, 0.9% saline, Ringer's solution, or other suitable excipients. For intramuscular preparations, for example, a sterile formulation of a suitable soluble salt form of the antibody may be dissolved and administered in a pharmaceutical excipient such as water for injection, 0.9% saline, or 5% glucose solution.

[0140] For oral administration, the compositions can take the form of tablets or capsules prepared by conventional means using acceptable excipients such as binding agents (e.g., pregelatinized corn starch, polyvinylpyrrolidone, or hydroxypropylmethylcellulose), fillers (e.g., lactose, microcrystalline cellulose, or calcium hydrogen phosphate), lubricants (e.g., magnesium stearate, talc, or silica), disintegrants (e.g., potato starch or sodium glycolate), or wetting agents (e.g., sodium lauryl sulfate). Tablets can be coated by methods well known in the art.

[0141] In some embodiments, the compounds provided herein, or pharma- ceutically acceptable salts thereof, are suitable for parenteral administration, hi some embodiments, the compounds provided herein, or pharma- ceutically acceptable salts thereof, are suitable for intravenous administration.

[0142] 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.

[0143] In making the pharmaceutical compositions provided herein, the active ingredient is typically mixed with an excipient, diluted by an excipient, or enclosed within such a carrier, for example, in the form of a capsule, sachet, paper, or other container. When the excipient 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.

[0144] Thus, the pharmaceutical compositions can be in the form of tablets, pills, powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols (as a solid or in a liquid medium), ointments, soft and hard gelatin capsules, suppositories, sterile injectable solutions, and sterile packaged powders.

[0145] III.How to use The present application further provides a method for imaging Granzyme B using one of the above-mentioned prodrug compounds, or a pharma- ceutically acceptable salt thereof. In some embodiments, the method is an in vitro method. In some embodiments, the method is an in vivo method.

[0146] Alternatively, the imaging method disclosed herein may be an in vivo imaging method comprising administering to a subject in need thereof a GZB-binding compound disclosed herein or a pharmaceutical composition comprising the same via a suitable route, for example, intravenous injection or local injection.

[0147] As used herein, the term "subject" refers to any animal, including mammals and invertebrates. For example, mice, rats, other rodents, rabbits, dogs, cats, pigs, cows, sheep, horses, primates, fish, and humans. In some embodiments, the subject is a human. In some embodiments, the subject is a mouse. In some embodiments, the subject is a fish (e.g., a zebrafish).

[0148] The present application further provides a method for imaging granzyme B in a cell or tissue, comprising: i) contacting a cell or tissue with an effective amount of one of the compounds described above, or a pharma- ceutically acceptable salt thereof; ii) imaging the cell or tissue with a suitable imaging technique, thereby imaging granzyme B within the cell or tissue.

[0149] The present application further provides a method for imaging granzyme B in a sample, a cell sample, or a tissue sample, comprising: i) contacting the sample, cell sample or tissue sample with an effective amount of one of the compounds described above, or a pharma- ceutically acceptable salt thereof; ii) imaging the sample, cell sample or tissue sample with a suitable imaging technique, thereby imaging Granzyme B within the sample, cell sample or tissue sample.

[0150] As used herein in the context of imaging, the term "sample" refers to a biological sample other than a cell or tissue sample obtained from a subject. For example, samples include, but are not limited to, saliva, blood, and andrin.

[0151] The present application further provides a method for imaging granzyme B in a subject, comprising: i) administering to a subject an effective amount of one of the compounds described above, or a pharma- ceutically acceptable salt thereof; ii) imaging the subject with a suitable imaging technique, thereby imaging granzyme B in the subject.

[0152] The present application further provides a method for imaging an immune response in a cell or tissue sample, comprising: i) contacting a cell or tissue sample with an effective amount of one of the compounds described above, or a pharma- ceutically acceptable salt thereof; ii) imaging the cell or tissue sample with a suitable imaging technique, thereby imaging the immune response within the cell or tissue sample.

[0153] The present application further provides a method for imaging an immune response in a subject, comprising: i) administering to a subject an effective amount of one of the compounds described above, or a pharma- ceutically acceptable salt thereof; ii) imaging the subject with a suitable imaging technique, thereby imaging the immune response in the subject.

[0154] The present application further provides a method for monitoring treatment of a disease in a subject, comprising: i) administering to a subject an effective amount of one of the compounds described above, or a pharma- ceutically acceptable salt thereof; ii) imaging the object with a suitable imaging technique.

[0155] The present application further provides a method for monitoring an immune response in the treatment of a disease in a subject, comprising: i) administering to a subject an effective amount of one of the compounds described above, or a pharma- ceutically acceptable salt thereof; ii) imaging the object with a suitable imaging technique.

[0156] In some embodiments, the methods provided herein further include waiting a sufficient amount of time prior to imaging to allow the compound, or a pharma- ceutically acceptable salt thereof, to accumulate in a cell or tissue site associated with a disease (e.g., a cell or tissue site in a subject).

[0157] In some embodiments, the methods provided herein further include waiting a sufficient amount of time prior to imaging to allow the compound, or a pharma- ceutically acceptable salt thereof, to bind to Granzyme B at a cell or tissue site associated with the disease (e.g., a cell or tissue site in a subject).

[0158] In some embodiments, a sufficient time is from about 30 seconds to about 24 hours, e.g., from about 30 seconds to about 24 hours, from about 30 seconds to about 12 hours, from about 30 seconds to about 6 hours, from about 30 seconds to about 2 hours, from about 30 seconds to about 1 hour, from about 30 seconds to about 30 minutes, from about 30 seconds to about 10 minutes, from about 10 minutes to about 24 hours, from about 10 minutes to about 12 hours, from about 10 minutes to about 6 hours, from about 10 minutes to about 2 hours, from about 10 minutes to about 1 hour, from about 10 minutes to about 30 minutes, about 30 minutes to about 24 hours, about 30 minutes to about 12 hours, about 30 minutes to about 6 hours, about 30 minutes to about 2 hours, about 30 minutes to about 1 hour, about 1 hour to about 24 hours, about 1 hour to about 12 hours, about 1 hour to about 6 hours, about 1 hour to about 2 hours, about 2 hours to about 24 hours, about 2 hours to about 12 hours, about 2 hours to about 6 hours, about 6 hours to about 24 hours, about 6 hours to about 12 hours, or about 12 hours to about 24 hours.

[0159] In some embodiments, the preferred imaging technique is a non-invasive imaging technique. In some embodiments, the preferred imaging technique is a minimally invasive imaging technique. As used herein, the term "minimally invasive imaging technique" includes imaging techniques that use an internal probe or injection of one of the above compounds, or a pharma-ceutically acceptable salt thereof, or the use of a radioactive tracer via a syringe.

[0160] Exemplary imaging techniques include, but are not limited to, fluoroscopic imaging, x-ray imaging, magnetic resonance imaging (MRI), ultrasound imaging, photoacoustic imaging, thermography imaging, tomography imaging, echocardiography imaging, positron emission tomography (PET) imaging, PET with computed tomography (CT) imaging, PET-MRI, single photon emission computed tomography (SPECT), and ultrasound imaging. In some embodiments, the preferred imaging technique is selected from the group consisting of PET imaging, PET-CT, PET-MRI, and SPECT.

[0161] In some embodiments, the preferred imaging technique is selected from the group consisting of PET imaging, PET with computed tomography imaging, and PET with magnetic resonance imaging (MRI). In some embodiments, the preferred imaging technique is selected PET imaging.

[0162] The results from any of the granzyme B imaging methods disclosed herein may be relied upon for diagnostic and / or prognostic purposes. In some embodiments, the results may be relied upon to identify patients suitable for treatment of immune dysregulation (diseases) with a therapeutic agent (e.g., an anti-inflammatory agent, a steroid, an immunotherapeutic agent, a chemotherapeutic agent, or a therapeutic antibody). In other embodiments, the results may be relied upon to monitor the effectiveness of a therapeutic agent, such as those provided herein. For example, the presence of granzyme B or an increase in GZB levels may indicate that the patient is suitable and / or responsive to a therapeutic agent. In that case, the methods disclosed herein may further include administering a therapeutic agent to the patient to treat the target disease.

[0163] In some embodiments, the disease described herein is selected from the group consisting of an autoimmune disorder, an inflammatory disorder, a skin disorder, cancer, and a cardiovascular disorder. As used herein, the term "disease" is used interchangeably with the term "immune dysregulation."

[0164] In some embodiments, the disease is cancer. In some embodiments, the cancer comprises a solid tumor. In some embodiments, the cancer is a hematological cancer (e.g., leukemia, lymphoma, etc.). Exemplary solid cancers include, but are not limited to, brain, breast, cervical, colorectal, lung, lymphoma, melanoma, bladder, renal cell, multiple myeloma, pancreatic, and prostate cancer. Exemplary hematological cancers include, but are not limited to, hairy cell leukemia, Kaposi's sarcoma, follicular lymphoma, chronic myelogenous leukemia, cutaneous T-cell lymphoma, peripheral T-cell lymphoma, T-cell prolymphocytic leukemia, classical Hodgkin's lymphoma, B-cell non-Hodgkin's lymphoma, chronic lymphocytic leukemia, acute myelogenous leukemia, myelodysplastic syndrome, primary myelofibrosis, myelofibrosis transitioning from essential thrombocythemia, and myelofibrosis transitioning from polycythemia vera. Other examples include melanoma, renal cell carcinoma, prostate cancer, non-small cell lung cancer, small cell lung cancer, glioblastoma, hepatocellular carcinoma, urothelial carcinoma, esophageal cancer, gastroesophageal cancer, gastric cancer, multiple myeloma, colon cancer, rectal cancer, squamous cell carcinoma of the head and neck, epithelial ovarian cancer (EOC), primary peritoneal cancer, fallopian tube cancer, HER2+ breast cancer, ER+ / PR+ / HER2- breast cancer, triple-negative breast cancer, gastric cancer, pancreatic cancer, bladder cancer, Merkel cell carcinoma, nasopharyngeal carcinoma, adrenal cortical carcinoma, meningioma, neuroblastoma, retinomas, osteosarcoma, Labeohem sarcoma, Ewing's sarcoma, liposarcoma, fibrous sarcoma, leiomyosarcoma, peripheral primary neurotomy skin tumor, squamous cell carcinoma of the cervix, squamous cell carcinoma of the vagina, and squamous cell carcinoma of the vulva. In some examples, the cancer is colon cancer.

[0165] Additional exemplary immune dysregulation disorders include graft versus host disease, rheumatoid arthritis, systemic lupus erythematosus, Hashimoto's thyroiditis, multiple sclerosis, myasthenia gravis, type I diabetes mellitus, uveitis, posterior uveitis, allergic encephalomyelitis, glomerulonephritis, rheumatic fever, post-infectious glomerulonephritis, psoriasis, atopic dermatitis, contact dermatitis, eczematous dermatitis, seborrheic dermatitis, lichen planus, pemphigus, bullous pemphigus, epidermolysis bullosa, urticaria, angioedema, vasculitis, erythema, cutaneous eosinophilia, lupus erythematosus, acne, alopecia areata, keratoconjunctivitis, vernal keratoconjunctivitis, uveitis associated with Behcet's disease, keratitis, herpetic keratitis, keratoconus, corneal epithelial atrophy, corneal leukoplakia, ocular pemphigus, Mooren's ulcer, scleritis, Graves' ophthalmopathy, V OGT-Koyanagi-Harada syndrome, sarcoidosis, pollen allergy, reversible obstructive airway disease, bronchial asthma, allergic asthma, intrinsic asthma, extrinsic asthma, dust asthma, chronic or reflux asthma, late stage asthma and airway hyperresponsive asthma, bronchitis, gastric ulcer, vascular damage due to ischemic disease and thrombosis, ischemic bowel disease, inflammatory bowel disease, necrotizing enterocolitis, intestinal lesions associated with burns, colon disease, proctitis, eosinophilic gastroenteritis, masticatory cell disease, Crohn's disease, ulcerative colitis, migraine, rhinitis, dermatitis, interstitial nephritis, Goodpasture's syndrome, hemolytic uremic syndrome, diabetic nephropathy, polymyositis, Guillain-Barre syndrome, Meniere's disease, polyneuritis, multiple neuropathy neuritis, mononeuritis, radiculopathy, hyperthyroidism, Bassdau disease, pure red cell aplasia, aplastic anemia, hypoplastic anemia, idiopathic thrombocytopenic purpura, autoimmune hemolytic anemia, agranulocytosis, pernicious anemia, megaloblastic anemia, red blood cell hypoplasia, osteoporosis, sarcoidosis, fibrotic lung, idiopathic interstitial pneumonia, dermatomyositis, albinism, ichthyosis vulgaris, photoallergic sensitivity, cutaneous T-cell lymphoma, arteriosclerosis, atherosclerosis, aortitis syndrome, polyarteritis nodosa, cardiomyopathy, scleroderma, Wegener's granulomatosis, Sjogren's syndrome, adiposity, eosinophilic fasciitisfascitis), lesions of the gums, periodontium, alveolar bone, dental cementum, glomerulonephritis, male pattern baldness, senile alopecia by preventing hair loss, senile alopecia by providing hair germination and / or promoting hair production and hair growth, muscular dystrophy, pyoderma, Sezary syndrome, Addison's disease, organ ischemia-reperfusion injury, transplant disease, ischemic disease, endotoxic shock, pseudomembranous colitis, drug or radiation colitis, ischemic acute renal failure, chronic renal failure, pulmonary oxygen or drug induced toxicosis, lung cancer, emphysema, cataracts, siderosis, retinitis pigmentosa, senile macular degeneration, vitreous scar, corneal alkali burn, erythema multiforme, linear IgA bulbar dermatitis and cement dermatitis, gingivitis, periodontitis, sepsis, pancreas These include, but are not limited to, inflammation, aging, cancer development, metastasis of carcinoma and hypobaropathy, histamine or leukotriene-C4 release associated diseases, Behcet's disease, autoimmune hepatitis, primary biliary cirrhosis, sclerosing cholangitis, partial liver resection, acute liver necrosis, necrosis caused by toxins, viral hepatitis, shock, anoxia, B-viral hepatitis, non-A / non-B hepatitis, liver cirrhosis, alcoholic liver cirrhosis, liver failure, fulminant liver failure, delayed liver failure, acute exacerbation of chronic liver failure, cytomegalovirus infection, HCMV infection, AIDS, senile dementia, trauma, chronic bacterial infection, malignant tumors of lymphatic origin, acute lymphocytic leukemia, chronic lymphocytic leukemia, acute lymphocytic lymphoma, and chronic lymphocytic lymphoma.

[0166] Exemplary autoimmune diseases include, but are not limited to, systemic lupus erythematosus, rheumatoid arthritis, type I diabetes, inflammatory bowel disease, biliary cirrhosis, uveitis, multiple sclerosis, Crohn's disease, ulcerative colitis, bullous pemphigus, sarcoidosis, psoriasis, autoimmune myositis, Wegener's granulomatosis, ichthyosis, Graves' ophthalmopathy, asthma, Schleroeder's disease, and Sjogren's syndrome.

[0167] In some embodiments, the disease can be bone marrow rejection, organ transplant rejection, and graft-versus-host disease.

[0168] When used in the method for treating disease, the compound of formula (II) provided herein above or its pharma- ceutically acceptable salt can be administered in combination with one or more additional therapeutic agents.In some cases, the additional therapeutic agent induces an immune response in the subject undergoing treatment.The compound of formula (II) can be used to monitor such an immune response based on the presence / absence of GZB or the change in GZB level in the subject.

[0169] In some embodiments, the compound of formula (II) may be administered to a patient after the patient has received at least one dose of an additional therapeutic agent. Based on the GZB imaging results resulting from the compound of formula (II), the patient may continue treatment with the additional therapeutic agent. In some examples, the dosage and / or dosage schedule of the additional therapeutic agent may be adjusted.

[0170] Examples of additional therapeutic agents include, but are not limited to, anti-inflammatory agents, steroids, immunotherapeutic agents, chemotherapeutic agents, and therapeutic antibodies.

[0171] In some embodiments, the therapeutic agent is an antibody. Exemplary antibodies for use in combination therapy include, but are not limited to, trastuzumab (e.g., anti-HER2), ranibizumab (e.g., anti-VEGF-A), bevacizumab (e.g., anti-VEGF), panitumumab (e.g., anti-EGFR), cetuximab (e.g., anti-EGFR), Rituxan (anti-CD20), antibodies against c-MET, and antibody inhibitors of granzyme B (e.g., clone GB11, clone GrB-7, and NCL-L-gran-B), ipilimumab (anti-CTLA-4), nivolumab (anti-PD-1), pembrolizumab (anti-PD-1), atezolizumab (anti-PD-1), elotuzumab (anti-SLAM7), and daratumumab (anti-CD38).

[0172] In some embodiments, the additional therapeutic agent is a steroid. Exemplary steroids include corticosteroids, such as cortisone, dexamethasone, hydrocortisone, methylprednisolone, prednisolone, and prednisone. In some embodiments, the additional therapeutic agent is a corticosteroid.

[0173] In some embodiments, the additional therapeutic agent is an anti-inflammatory compound. Exemplary anti-inflammatory compounds include aspirin, choline salicylate, celecoxib, diclofenac potassium, diclofenac sodium, diclofenac sodium with misoprostol, diflunisal, etodolac, fenoprofen, flurbiprofen, ibuprofen, ketoprofen, meclofenamate sodium, mefenamic acid, nabumetone, naproxen, naproxen sodium, oxaprozin, piroxican, rofecoxib, salsalate, sodium salicylate, sulindac, tolmetin sodium, and valdecoxib.

[0174] In some embodiments, the additional therapeutic agent is a chemotherapeutic agent. Exemplary chemotherapeutic agents include cytostatic agents, cisplatin, doxorubicin, taxol, etoposide, irinotecan, topotecan, paclitaxel, docetaxel, epothilone, tamoxifen, 5-fluorouracil, methotrexate, temozolomide, cyclophosphamide, SCH66336, R115777, L778,123, BMS214662, gefitinib, erlotinib hydrochloride, antibodies against EGFR, imatinib mesylate, intron, ara-C, gemcitabine, uracil mustard, chlormethine, ifosfamide, melphalan, chlorambucil, pipobroman, triethylenemelamine, triethylenethiophosphoramine, busulfan, carmustine, lomustine, streptozocin, dacarbazine, floxuridine, cytarabine, 6-mercaptopurine, 6-thioguanine, fludarabine phosphate, oxaliplatin, folinic acid, penicillin tostatin, vinblastine, vincristine, vindesine, bleomycin, dactinomycin, daunorubicin, doxorubicin, epirubicin, idarubicin, mithramycin, deoxycoformycin, mitomycin C, L-asparaginase, teniposide, 17α-ethynyl estradiol, diethylstilbestrol, testosterone, prednisone, fluoxymesterone, dromostanolone propionate, testolactone, megestrol acetate, methylprednisolone, methyltestosterone, prednisolone, Liamcinolone, chlorotrianisene, hydroxyprogesterone, aminoglutethimide, estramustine, medroxyprogesterone acetate, leuprolide, flutamide, toremifene, goserelin, carboplatin, hydroxyurea, amsacrine, procarbazine, mitotane, mitoxantrone, levamisole, vinorelbine, anastrozole, letrozole, capecitabine, reloxafine, hexamethylmelamine, bevacizumab, Bexar, Velcade, Zevalin, Trisenox, Xeroda, vinorelbine, porfima These include, but are not limited to, erbitux, liposomal, thiotepa, altretamine, melphalan, trastuzumab, fulvestrant, exemestane, ifosfamide, rituximab, C225, alemtuzumab, clofarabine, cladribine, aphidicolin, sunitinib, dasatinib, tezacitabine, Sml1, triapine, didox, trimidox, amidox, 3-AP, MDL-101.731, bendamustine, ofatumumab, and GS-1101 (also known as CAL-101).

[0175] In some embodiments, the chemotherapeutic agent is an alkylating agent (e.g., busulfan, chlorambucil, cisplatin, cyclophosphamide (Cytoxan), dacarbazine, ifosfamide, mechlorethamine (Masterzin), and melphalan), a nitrosourea (e.g., carmustine, lomustine, semustine, and streptozocin), a triazine (e.g., dacarbazine), antimetabolites (e.g., 5-fluorouracil (5-FU), cytarabine (Ara-C), fludarabine, gemcitabine, and methotrexate), a purine analog (e.g., 6-mercaptopurine, 6-thiourea, guanine, and pentosteoxycoccin (2-deoxycoccin), mitotic inhibitors (e.g., docetaxel, etoposide (VP16), tenocide, paclitaxel, taxol, vinblastine, vincristine, and vinorelbine), antitumor antibiotics (e.g., bleomycin, dactinomycin, daunorubicin, doxorubicin, mitomycin, plicamycin, and idarubicin), platinum chemotherapeutic agents (e.g., cisplatin and carboplatin), anthracenediones (e.g., mitoxantrone), toxins (e.g., ricin A chain (Burbage, Leukemia research, 21.7(1997):681-690), diphtheria toxin A (Massuda et al., Proceedings of the National Academy of Sciences, 94.26(1997):14701-14706; Lidor, American journal of obstetrics and gynecology, 177.3(1997):579-585), pertussis toxin A subunit, Escherichia coli enterotoxin A subunit, cholera toxin A subunit and Pseudomonas toxin c-terminus), and gene therapy vectors (e.g., signal transduction proteins (e.g., Src, Abl, and Ras), Jun, Fos, and Myc).

[0176] In some embodiments, the additional therapeutic agent is an immunotherapeutic agent. Immunotherapeutic agents generally trigger immune effector cells and molecules to target and destroy cells (e.g., cancer cells). The immune effector can be, for example, an antibody specific to a marker on the surface of a cell (e.g., a tumor cell). The antibody alone can function as an effector of therapy or can recruit other cells to kill the cell. Various effector cells include, but are not limited to, cytotoxic T cells and NK cells.

[0177] Exemplary immunotherapeutic agents include azathioprine, chlorambucil, cyclophosphamide, cyclosporine, daclizumab, infliximab, methotrexate, tacrolimus, immune stimulants (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 Immunology, 7.4 (1991): 303-329), hormone therapy, corticosteroids, progestins (e.g., hydroxyprogesterone caproate, medroxyprogesterone acetate, and megestrol acetate), estrogens (e.g., diethylstilbestrol and ethinyl estradiol), antiestrogens (e.g., testosterone propionate and fluoxymesterone), antiandrogens (e.g., flutamide), and gonadotropin stimulating hormone analogs (e.g., leuprolide). Additional immunotherapeutic agents are known in the art and can be found, for example, in Rosenberg et al, New England Journal of Medicine, 319.25 (1988): 1676-1680, and Rosenberg et al, Annals of surgery, 210.4 (1989): 474).

[0178] The therapeutic agents provided herein may be effective over a wide range of doses and are generally administered in effective amounts. However, it will be understood that the amount of therapeutic agent actually administered will usually be determined by a physician depending on the relevant circumstances, including the condition being imaged, the route of administration selected, the compound actually administered, the age, weight, and response of the individual subject, the severity of the subject's symptoms, and the like.

[0179] Additional information may be found in International Application No. PCT / US2021 / 036661, filed June 9, 2021, the relevant disclosure of which is incorporated by reference with respect to the subject matter and purposes referenced herein.

[0180] IV. Granzyme B Imaging Kit Also included in the present disclosure is a kit (e.g., pharmaceutical pack) for granzyme B imaging using any of the GZB-binding compounds disclosed herein. The provided kit may include a container (e.g., a vial, an ampoule, a bottle, a syringe, and / or a dispenser package, or other suitable container) into which the pharmaceutical composition disclosed herein may be placed. In some embodiments, the provided kit may optionally further include a second container that includes a pharmaceutical excipient for diluting or suspending the pharmaceutical composition. In some embodiments, the pharmaceutical composition provided in the first container and the second container are combined to form a single unit dosage form. In some embodiments, the kit may include an additional container that includes one or more additional therapeutic agents disclosed herein, such as anti-inflammatory agents, steroids, immunotherapeutic agents, chemotherapeutic agents, and therapeutic antibodies described herein.

[0181] In certain embodiments, the kits described herein further comprise instructions for using the compounds or compositions contained in the kit. The kits described herein may comprise information required by regulatory agencies such as the U.S. Food and Drug Administration (FDA). In certain embodiments, the information contained in the kit is prescription information. In certain embodiments, the kit and instructions provide for imaging granzyme B and evaluating the efficacy of treatment with any of the therapeutic agents disclosed herein in a subject in need thereof. The kits described herein may comprise one or more additional pharmaceutical agents described herein as separate compositions.

[0182] Common techniques The practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are within the skill of those in the art. For such techniques, see, for example, Molecular Cloning: A Laboratory Manual, Second edition (Sambrook, et al., 1989) Cold Spring Harbor Press, Oligonucleotide Synthesis (MJ Gait, ed. 1984), Methods in Molecular Biology, Humana Press, Cell Biology: A Laboratory Notebook (JECellis, ed., 1989) Academic Press, Animal Cell. Culture (RIFreshney, ed. 1987), Introduction to Cell and Tissue Culture (JP Mather and PE Roberts, 1998) Plenum Press, Cell and Tissue Culture: Laboratory Procedures (A. Doyle, JBGriffiths, and DG Newell, eds. 1993-8) J. Wiley and Sons, Methods in Enzymology (Academic Press, Inc.), Handbook of Experimental Immunology (DM Weir and CC Blackwell, eds.): Gene Transfer Vectors for Mammalian Cells (JMMiller and MPCalos, eds., 1987), Current Protocols in Molecular Biology (FMAusubel, et al. eds. 1987), PCR: The Polymerase Chain Reaction, (Mullis, et al., eds. 1994), Current Protocols in Immunology (JEColigan et al., eds., 1991), Short Protocols in Molecular Biology (Wiley and Sons, 1999), Immunobiology (C.A. Janeway and P. Travers, 1997), Antibodies (P. Finch, 1997), Antibodies: a practice approach (D. Catty., ed., IRL Press, 1988 - 1989), Monoclonal antibodies: a practical approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000), Using antibodies: a laboratory manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999), The Antibodies (M. Zanetti and J.D. Capra, eds. Harwood Academic Publishers, 1995), DNA Cloning: A practical Approach, Volumes I and II (D.N. Glover ed. 1985), Nucleic Acid Hybridization (B.D. Hames & S.J. Higgins eds. (1985.

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[0183] Without further elaboration, it is believed that one skilled in the art can, based on the above specification, utilize the present invention to its fullest extent. The following specific embodiments are therefore to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way. All publications cited herein are incorporated by reference for the purposes or subject matter referenced herein.

[0184] Example 1: 18 F-Granzyme B( 18 Radiation synthesis of F-GZB Typical 18 The F-GZB compound RCY ranges from 10 to 54% with a synthesis time of 75 ± 10 min using a starting activity of 0.4 to 1.2 Ci. The reaction vessel was pre-loaded with the reaction mixture containing the precursor (e.g., 0.2-0.4 mg), AlCl3·6H2O (e.g., 34-82 μg), acetic acid / sodium acetate aqueous buffer (e.g., 0.1-0.2 mL, 1 M, pH 3-5), water (e.g., 0.1-0.3 mL), and acetonitrile (e.g., 25-50% of the total reaction mixture volume). [ 18[F]fluoride activity was retained on a conditioned anion exchange resin (e.g., Sep-Pak Accell Plus QMA Carbonate Plus Light cartridges, 46 mg sorbent per cartridge, particle size 40 μm, Waters part number 186004540) and then eluted into a reaction vessel using 0.9% saline (e.g., 0.5-0.8 mL). The resulting mixture was heated (e.g., 105 °C) for a period of time (e.g., 15 min) and then cooled (e.g., 60 °C) before diluting with water (e.g., 4-5 mL). The resulting crude was loaded onto a semi-preparative reversed-phase HPLC column (e.g., Agilent ZORBAX Eclipse XDB-C18, 5 μm, 9.4 × 250 mm, part number 990967-202) for purification (e.g., mobile phase with aqueous acetonitrile (10-20%), pH 1-8). The purified 18 The HPLC fractions containing the F-GZB compounds were diluted with 0.5% w / v aqueous sodium ascorbate (e.g., 30-50 mL) and then passed through a conditioned reverse-phase cartridge (e.g., Sep-Pak® Light C18 cartridges, 130 mg of sorbent per cartridge, particle size 55-105 μm, Waters part number WAT0523501). 18 F-GZB was washed with 0.5% w / v aqueous sodium ascorbate (e.g., 5-15 mL) and eluted from the cartridge using ethanol (e.g., 1-1.5 mL) into a formulation vial containing 0.5% w / v sodium ascorbate in 0.9% saline (e.g., 6-10 mL). The C18 cartridge was then rinsed with additional 0.5% w / v sodium ascorbate in 0.9% saline (e.g., 3-3.5 mL) and the rinse was collected into a formulation vial. A volume of diluent (10% v / v ethanol, and 90% v / v 0.9% saline containing 0.5% w / v sodium ascorbate) can be added to adjust the strength of the product.

[0185] To prepare a sterile product, the resulting product (in 90% v / v 0.9% saline solution containing 10% v / v ethanol and 0.5% w / v sodium ascorbate) was diluted with 10% ethanol and 0.5% w / v sodium ascorbate. 18 F-GZB) was sterile filtered through a 0.22 μm filter (e.g., Millex® GV sterile filter, Millipore part number SLGV033RS, Millex GV 25 mm sterile filter, Millipore part number SLGV255F, and Millex LG 25 mm sterile filter, Millipore part number SLLG025SS) into a bulk product vial.

[0186] Example 2: Synthesis of intermediates ((2R,3S)-2-Methoxy-5-oxotetrahydrofuran-3-yl)carbamate and Preparation of benzyl ((2S,3S)-2-methoxy-5-oxotetrahydrofuran-3-yl)carbamate [ka] Step 1: [ka] To a solution of tert-butyl (S)-3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-hydroxybutanoate (11.5 g, 28.9 mmol) in dichloromethane (120 mL) was added Dess-Martin periodinane (18.6 g, 43.4 mmol). The mixture was stirred at 20 °C for 3 h under N2. The reaction mixture was quenched with saturated sodium bicarbonate solution (200 mL) and extracted with dichloromethane (200 mL x 3). The combined organic layers were washed with brine (300 mL), dried over sodium sulfate, filtered, and concentrated under reduced pressure. The residue was combined with another reaction of the same scale and purified by flash silica gel chromatography (ISCO; 220 g, 0-40% ethyl acetate / petroleum ether 100 mL / min). tert-Butyl (S)-3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-oxobutanoate (18 g, 45.52 mmol) was obtained as a yellow oil and used in the next step.

[0187] Step 2: To a solution of tert-butyl (S)-3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-oxobutanoate (12 g, 30.35 mmol) in methanol (60 mL) was added trimethoxymethane (13.7 g, 129 mmol) and p-toluenesulfonic acid (227 mg, 1.3 mmol). The reaction mixture was stirred at 20° C. for 16 h. The reaction mixture was concentrated under reduced pressure to remove the solvent to give the crude product. The crude product was combined with another reaction starting with 6 g of tert-butyl (S)-3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-oxobutanoate. The combined mixture was dissolved in dichloromethane (300 mL). The dichloromethane solution was washed with saturated NaHCO3 (200 mL) and brine (200 mL), dried over Na2SO4, filtered, and concentrated. The residue was purified by flash silica gel chromatography (ISCO; 220 g, 0-25% ethyl acetate / petroleum ether, 100 mL / min) to give tert-butyl (S)-3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4,4-dimethoxybutanoate (13.6 g, 29.9 mmol) as a colorless oil.

[0188] ES / MS m / z 464.2(M+Na) + .

[0189] Step 3: [ka] To a solution of tert-butyl (S)-3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4,4-dimethoxybutanoate (13.6 g, 29.9 mmol) in tetrahydrofuran (80 mL) was added diethylamine (80 mL, 760 mmol). The reaction mixture was stirred at 20° C. for 5 h. The reaction mixture was concentrated under reduced pressure to remove the solvent to give crude tert-butyl (S)-3-amino-4,4-dimethoxybutanoate (14 g) as a colorless oil, which was used in the next step without further purification.

[0190] Step 4: To a solution of tert-butyl (S)-3-amino-4,4-dimethoxybutanoate (14 g) and sodium carbonate (7.2 g, 68 mmol) in tetrahydrofuran (80 mL) and water (4 mL) was added a solution of benzyl chloroformate (8.6 g, 50 mmol) in tetrahydrofuran (20 mL) slowly at 0° C. The reaction mixture was stirred at room temperature until TLC showed the reaction was complete. Water (80 mL) was added and the mixture was extracted with ethyl acetate (100 mL×3). The combined organic layers were washed with brine (150 mL), dried over sodium sulfate, filtered and concentrated. The residue was purified by flash silica gel chromatography (ISCO; 220 g silica gel flash column, 0-40% ethyl acetate / petroleum ether, 100 mL / min) to give tert-butyl (S)-3-(((benzyloxy)carbonyl)amino)-4,4-dimethoxybutanoate (11.7 g, 32.82 mmol) as a pale yellow oil.

[0191] 1 H NMR(400 MHz,CDCl3) δ:7.39-7.29(m,5H),5.33(d,J=8.8 Hz,1H),5.17-5.05(m,2H),4.35(d,J=3.6 Hz,1H),4.25-4.16(m,1H),3.45-3.38(s,6H),2.57-2.41(m,2H),1.43(s,9H).

[0192] Step 5: [ka] To a solution of tert-butyl (S)-3-(((benzyloxy)carbonyl)amino)-4,4-dimethoxybutanoate (10.2 g, 27.4 mmol) in dichloromethane (100 mL) was added TFA (10 mL, 129 mmol) and anisole (1.0 mL, 9.1 mmol) at 0° C. The reaction mixture was warmed to 20° C. and stirred for 1 h. The reaction mixture was concentrated under reduced pressure to remove the solvent. The residue was combined with another reaction starting with 1.5 g of tert-butyl (S)-3-(((benzyloxy)carbonyl)amino)-4,4-dimethoxybutanoate. The residue was purified several times by flash silica gel chromatography (ISCO, 220 g, 0-1% MeOH / DCM, 80 mL / min) to give two compounds: [ka] Benzyl ((2R,3S)-2-methoxy-5-oxotetrahydrofuran-3-yl)carbamate (3.85 g, 12.2 mmol) was obtained as a white solid.

[0193] 1H NMR (400 MHz, DMSO): δ 7.58(d,J=7.9 Hz,1H),7.43-7.29(m,5H),5.43(d,J=5.0 Hz,1H),5.07(s,2H),4.47-4.39(m,1H),3.42(s,3H),2.71(dd,J=17.2,8.9 Hz,1H),2.52(dd,J=17.2,9.9 Hz,1H).

[0194] [ka] Benzyl ((2S,3S)-2-methoxy-5-oxotetrahydrofuran-3-yl)carbamate (1.93 g, 6.40 mmol) was obtained as a colorless oil.

[0195] 1H NMR(400 MHz,DMSO): δ 7.88(s,1H),7.43-7.29(m,5H),5.29(d,J=1.5 Hz,1H),5.07(s,2H),4.06-4.00(m,1H),3.42(s,3H),2.98(dd,J=18.1,8.3 Hz,1H),2.41(dd,J=18.1,2.9 Hz,1H).

[0196] Preparation of benzyl ((2S,3R)-2-methoxy-5-oxotetrahydrofuran-3-yl)carbamate and benzyl ((2R,3R)-2-methoxy-5-oxotetrahydrofuran-3-yl)carbamate [ka] Step 1: The intermediate tert-butyl (R)-3-(((benzyloxy)carbonyl)amino)-4,4-dimethoxybutanoate was prepared following a similar synthetic procedure described above.

[0197] Step 2: [ka] To a solution of tert-butyl (R)-3-(((benzyloxy)carbonyl)amino)-4,4-dimethoxybutanoate (4 g, 10.87 mmol) in dichloromethane (40 mL) was added TFA (4 mL, 51.4 mmol) and anisole (0.4 mL, 4 mmol). The reaction mixture was stirred at 20° C. for 1 h. The reaction mixture was concentrated under reduced pressure to remove the solvent. The residue was combined with two other batch reactions each starting with 5 g of tert-butyl (R)-3-(((benzyloxy)carbonyl)amino)-4,4-dimethoxybutanoate. It was purified by flash silica gel chromatography (ISCO; 220 g, 0-1% MeOH / dichloromethane, 100 mL / min).

[0198] [ka] Benzyl ((2S,3R)-2-methoxy-5-oxotetrahydrofuran-3-yl)carbamate (2.2 g, 8.0 mmol) was obtained as a white solid.

[0199] 1H NMR (400 MHz, CDCl3) δ:7.41-7.33(m,5 H),5.38-5.29(m,2 H),5.15-5.12(s,2 H),4.64-4.52(m,1 H),3.54(s,3 H),2.86(dd,J=8.4,17.3 Hz,1 H),2.45(dd,J=10.4,17.3 Hz,1 H).

[0200] [ka] Benzyl ((2R,3R)-2-methoxy-5-oxotetrahydrofuran-3-yl)carbamate (1.5 g, 5.3 mmol) was obtained as a colorless oil.

[0201] 1H NMR (400 MHz, CDCl3) δ:7.42-7.31(m,5 H),5.31(s,1 H),5.17-5.10(s,2 H),5.06-4.94(m,1 H),4.22(t,J=6.6 Hz,1 H),3.51(s,3 H),3.00(dd,J=7.8,18.0 Hz,1 H),2.40(d,J=18.0 Hz,1 H).

[0202] Preparation of (R)-2-(2-(4-(((9H-fluoren-9-yl)methoxy)carbonyl)piperazin-1-yl)acetamido)-5-(benzyloxy)-5-oxopentanoic acid [ka] Step 1: [ka] To a solution of (R)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-(benzyloxy)-5-oxopentanoic acid (3.3 g, 7.2 mmol) in dimethylformamide (30 mL) was added potassium carbonate (1.82 g, 13.0 mmol) and allyl bromide (1.58 g, 13.1 mmol). The reaction mixture was stirred at 20° C. for 12 h. Water (80 mL) was added. The mixture was extracted with ethyl acetate (100 mL). The combined organic layers were washed with brine (3×60 mL), dried over sodium sulfate, and concentrated in vacuo to give crude 1-allyl 5-benzyl(((9H-fluoren-9-yl)methoxy)carbonyl)-D-glutamic acid (4.1 g, 6.6 mmol) as a yellow oil.

[0203] ES / MS m / z 500.1(M+H) + .

[0204] Step 2: [ka] To a solution of 1-allyl 5-benzyl(((9H-fluoren-9-yl)methoxy)carbonyl)-D-glutamic acid (4.1 g, 6.6 mmol) in tetrahydrofuran (30 mL) was added diethylamine (1.82 g, 24.8 mmol). The reaction mixture was stirred at 20° C. for 3 h. The solvent was removed under reduced pressure to give crude 1-allyl 5-benzyl-D-glutamic acid (1.9 g) as a yellow oil, which was then used directly. To a solution of 2-(4-(((9H-fluoren-9-yl)methoxy)carbonyl)piperazin-1-yl)acetic acid (2.5 g, 6.8 mmol) and ethyl cyanohydroxyiminoacetate (1.2 g, 8.2 mmol) in dimethylformamide (20 mL) was added N,N-diisopropylcarbodiimide (1.1 g, 8.5 mmol) at 0° C. The reaction mixture was stirred at 0° C. for 0.5 h under N2. Then, crude 1-allyl 5-benzyl D-glutamic acid (1.9 g) in dimethylformamide (5 mL) was added. The reaction mixture was stirred at 20° C. for 16 h under N2. H2O (50 mL) was added and the reaction mixture was extracted with ethyl acetate (8×80 mL). The combined organic layers were washed with brine (3×100 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by flash silica gel chromatography (ISCO, 40 g, 0-5% MeOH / DCM with 0.3% NH3.H2O as additive). 1-allyl 5-benzyl(2-(4-(((9H-fluoren-9-yl)methoxy)carbonyl)piperazin-1-yl)acetyl)-D-glutamic acid (2.5 g, 2.9 mmol) was obtained as a yellow oil.

[0205] ES / MS m / z 626.3(M+H) + .

[0206] Step 3: [ka] To a solution of 1-allyl 5-benzyl(2-(4-(((9H-fluoren-9-yl)methoxy)carbonyl)piperazin-1-yl)acetyl)-D-glutamic acid (2.5 g, 2.9 mmol) and morpholine (700 mg, 7.9 mmol) in tetrahydrofuran (40 mL) was added tetrakis(triphenylphosphine)palladium(0) (140 mg, 0.12 mmol). The mixture was stirred at room temperature for 12 h. The solvent was removed under reduced pressure to give the crude product. The crude product was purified by C18 prep-HPLC (0.225% formic acid in H2O-CH3CN). The fractions were combined and lyophilized to give (R)-2-(2-(4-(((9H-fluoren-9-yl)methoxy)carbonyl)piperazin-1-yl)acetamido)-5-(benzyloxy)-5-oxopentanoic acid (510 mg, 0.84 mmol) as a brown oil.

[0207] ES / MS m / z 586.6(M+H) + .

[0208] Preparation of 2-(4,7-bis(2-(benzyloxy)-2-oxoethyl)-1,4,7-triazonan-1-yl)acetic acid [ka] Step 1: [ka] To a solution of 1,4,7-triazonane hydrochloride (15 g, 61.0 mmol) in toluene (180 mL) was added sodium hydroxide (7.4 g, 180 mmol) in water (12 mL). The reaction mixture was stirred at 135 °C for 12 h using a Dean-Stark condenser. Additional sodium hydroxide (3.9 g, 97 mmol) was added and the mixture was stirred at 130 °C for 2 h. The mixture was filtered while hot and the cake was washed with ethyl acetate (3 × 15 mL). The filtrate was concentrated to give 1,4,7-triazonane (7.0 g, 54 mmol) as a white solid.

[0209] To a solution of 1,4,7-triazonane (4 g, 31.0 mmol) in chloroform (120 mL) was added benzyl 2-bromoacetate (13.5 g, 58.9 mmol) in chloroform (120 mL) dropwise at -10 °C for 1.5 h. The reaction mixture was allowed to warm to room temperature and stirred for 12 h. The reaction mixture was concentrated under reduced pressure to remove the solvent. The residue was purified by flash silica gel chromatography (ISCO, 220 g, 0-8% MeOH (2% 7M NH3 in methanol) / DCM gradient). Dibenzyl 2,2'-(1,4,7-triazonane-1,4-diyl) diacetate (4.6 g, 10 mmol) was obtained as a yellow oil.

[0210] ES / MS m / z 426.1(M+H) + .

[0211] Step 2: [ka] To a solution of dibenzyl 2,2'-(1,4,7-triazonane-1,4-diyl)diacetate (6.1 g, 13 mmol) in acetonitrile (150 mL) was added potassium carbonate (2.97 g, 21.3 mmol) and tert-butyl 2-bromoacetate (2.66 g, 13.6 mmol). The reaction mixture was stirred at room temperature for 12 h. The reaction mixture was concentrated under reduced pressure to remove the solvent. The crude product was purified by C18 preparative HPLC (0.225% formic acid in H2O / CH3CN). The fractions were combined and lyophilized to give dibenzyl 2,2'-(7-(2-(tert-butoxy)-2-oxoethyl)-1,4,7-triazonane-1,4-diyl)diacetate (5.7 g, 9.4 mmol) as a colorless oil.

[0212] ES / MS m / z 540.2(M+H) + .

[0213] Step 3: [ka] To a solution of dibenzyl 2,2'-(7-(2-(2-(tert-butoxy)-2-oxoethyl)-1,4,7-triazonane-1,4-diyl)diacetate (5.7 g, 11 mmol) in dichloromethane (30 mL) was added trifluoroacetic acid (30 mL). The reaction mixture was stirred at room temperature for 20 h. The reaction mixture was concentrated under reduced pressure to remove the solvent. Saturated NaHCO3 was added to adjust the pH of the mixture to 8. The mixture was then lyophilized to give crude 2-(4,7-bis(2-(benzyloxy)-2-oxoethyl)-1,4,7-triazonan-1-yl)acetic acid (4.7 g, 9.7 mmol) as a yellow oil. The crude product was used in the next step without further purification.

[0214] ES / MS m / z 540.2(M+H) + .

[0215] Preparation of dibenzyl 2,2'-(7-((1-(2-((2,5-dioxopyrrolidin-1-yl)oxy)-2-oxoethyl)piperidin-4-yl)methyl)-1,4,7-triazonane-1,4-diyl) diacetate [ka] Step 1: [ka] To a solution of dibenzyl 2,2'-(1,4,7-triazonane-1,4-diyl)diacetate (28.0 g, 65.8 mmol) and tert-butyl 2-(4-formylpiperidin-1-yl)acetate (17.9 g, 79.0 mmol) in DCE (245 mL) was added AcOH (3.16 g, 52.6 mmol, 3.01 mL, 0.80 equiv) at 0°C and the mixture was stirred at 0°C for 1 h. Then NaBH(OAc)3 (20.9 g, 98.7 mmol, 1.50 equiv) was added and the resulting mixture was stirred at 25°C for 2 h. The reaction mixture was quenched by adding NaHCO3 (500 mL) and then extracted with EA (500 mL x 2). The combined organic layers were dried over Na2SO4, filtered and concentrated under reduced pressure to give dibenzyl 2,2'-(7-((1-(2-(tert-butoxy)-2-oxoethyl)piperidin-4-yl)methyl)-1,4,7-triazonane-1,4-diyl)diacetate (50.0 g, crude) as a brown oil. The crude product was used directly in the next step without further purification.

[0216] ES / MS m / z 637.4(M+H) + .

[0217] Step 2: [ka] To a solution of dibenzyl 2,2'-(7-((1-(2-(tert-butoxy)-2-oxoethyl)piperidin-4-yl)methyl)-1,4,7-triazonane-1,4-diyl)diacetate (50.0 g, 78.5 mmol) in DCM (350 mL) was added TFA (231 g, 2.03 mol, 150 mL). The mixture was stirred at 20° C. for 12 h. The reaction mixture was concentrated under reduced pressure to remove the solvent. The residue was purified by preparative HPLC (TFA conditions) and the TFA salt was dissolved in deionized water and cleaved with chloride ion resin to form the HCl salt to give 2-(4-((4,7-bis(2-(benzyloxy)-2-oxoethyl)-1,4,7-triazonan-1-yl)methyl)piperidin-1-yl)acetic acid, HCl salt (20.0 g, 31.0 mmol) as a light brown foam. ES / MS m / z 581.2 (M+H). + .

[0218] Step 3: [ka] To a solution of 2-(4-((4,7-bis(2-(benzyloxy)-2-oxoethyl)-1,4,7-triazonan-1-yl)methyl)piperidin-1-yl)acetic acid (0.4 g, 2.4 mmol) and N,N-diisopropylethylamine (470 mg, 3.6 mmol) in DMF (20 mL) was added TSTU (tetramethyl-O-(N-succinimidyl)uronium tetrafluoroborate, 820 mg, 2.6 mmol) at 0° C. under N2. The mixture was then stirred at 20° C. for 1 h. The reaction mixture of dibenzyl 2,2'-(7-((1-(2-((2,5-dioxopyrrolidin-1-yl)oxy)-2-oxoethyl)piperidin-4-yl)methyl)-1,4,7-triazonane-1,4-diyl)diacetate (2.4 mmol) was used directly in the next step without further purification.

[0219] Example 3: Synthesis of peptide acid intermediates: General peptide synthesis procedure A: Peptides were synthesized according to standard Fmoc solid phase peptide synthesis procedures using 2-CTC (2-chlorotrityl chloride) resin. The final peptide was deprotected and cleaved from the resin via 20% 1,1,1,3,3,3-hexafluoro-2-propanol in dichloromethane treatment. The resin was treated with 20% 1,1,1,3,3,3-hexafluoro-2-propanol in dichloromethane (15-20 mL of solution per gram of resin) and shaken for 10 min. The solution was drained into a round bottom flask. The treatment was repeated twice with fresh aliquots of 20% 1,1,1,3,3,3-hexafluoro-2-propanol in dichloromethane (15-20 mL of solution per gram of resin), shaken for 30 min each. The solutions from all treatments were drained and combined. The crude peptide was concentrated and then subjected to preparative HPLC or reverse-phase C18 column purification (water with or without appropriate modifier / acetonitrile mobile phase). Product-containing fractions were collected and lyophilized to give the peptide as a solid. [ka]

[0220] [ka] Following general procedure A, (3S,6S)-3-((2S,13R)-13-(2-(4-((4,7-bis(2-(tert-butoxy)-2-oxoethyl)-1,4,7-triazonan-1-yl)methyl)piperidin-1-yl)acetamido)-2-((S)-sec-butyl)-18,18-dimethyl-4,8,12,16-tetraoxo-17-oxa-3,7,11-triazanonadecanamido)-4-oxo-1,2,3,4,6,7-hexahydroazepino[3,2,1-hi]indole-6-carboxylic acid (1005.2 mg) was synthesized using solid phase and isolated as a grey powder.

[0221] ES / MS m / z 1181.8(M+H) + .

[0222] [ka] Following procedure A, (3S,6S)-3-((6R,17S)-6-(2-(4-((4,7-bis(2-(benzyloxy)-2-oxoethyl)-1,4,7-triazonan-1-yl)methyl)piperidin-1-yl)acetamido)-17-((S)-sec-butyl)-3,7,11,15-tetraoxo-1-phenyl-2-oxa-8,12,16-triazaoctadecane-18-amido)-4-oxo-1,2,3,4,6,7-hexahydroazepino[3,2,1-hi]indole-6-carboxylic acid (354 mg) was synthesized as a white solid using solid phase.

[0223] ES / MS m / z 1284.1(M+H) + .

[0224] In this embodiment, the solid phase synthesis protocol was modified. [ka]

[0225] To a fritted glass funnel containing (3S,6S)-3-(((5R,16S)-5-(3-((benzyloxy)-3-oxopropyl)-16-((S)-sec-butyl)-1-(9H-fluoren-9-yl)-3,6,10,14-tetraoxo-2-oxa-4,7,11,15-tetraazaheptadecan-17-amido)-4-oxo-1,2,3,4,6,7-hexahydroazepino[3,2,1-hi]indole-6-carboxylic acid on 2-CTC resin (2 mmol), DBU (25 mL, 2% in DMF) was added and stirred for 2 min. The mixture was filtered and DBU (25 mL, 2% in DMF) was added and stirred for 2 min. The solution was then removed from the resin and the resin was washed with DMF (10 mL x 8).

[0226] A mixture of the resulting resin and crude dibenzyl 2,2'-(7-((1-(2-((2,5-dioxopyrrolidin-1-yl)oxy)-2-oxoethyl)piperidin-4-yl)methyl)-1,4,7-triazonane-1,4-diyl)diacetate (2.4 mmol) in DMF (20 mL) in a fritted glass funnel was shaken at room temperature for 12 hours. The solution was then removed from the resin and the resin was washed with DMF (30 mL*2), then DCM (30 mL*2), then DMF (30 mL*2) and finally DCM (30 mL*2).

[0227] Resin cleavage was performed according to general procedure A.

[0228] [ka] Following general procedure A, (3S,6S)-3-((2S,11R,14R)-14-(2-(4-((4,7-bis(2-(tert-butoxy)-2-oxoethyl)-1,4,7-triazonan-1-yl)methyl)piperidin-1-yl)acetamido)-11-(3-(tert-butoxy)-3-oxopropyl)-2-((S)-sec-butyl)-19,19-dimethyl-4,10,13,17-tetraoxo-6,18-dioxa-3,9,12-triazaicosanamido)-4-oxo-1,2,3,4,6,7-hexahydroazepino[3,2,1-hi]indole-6-carboxylic acid (555.8 mg) was synthesized using solid phase and isolated as an off-white powder.

[0229] ES / MS m / z 1325.6(M+H) + .

[0230] [ka] Following general procedure A, (3S,6S)-3-((S)-6-(2-(4-((4,7-bis(2-(tert-butoxy)-2-oxoethyl)-1,4,7-triazonan-1-yl)methyl)piperidin-1-yl)acetyl)-18-((S)-sec-butyl)-2,2-dimethyl-4,8,12,16-tetraoxo-3-oxa-6,9,13,17-tetraazanonadecane-19-amido)-4-oxo-1,2,3,4,6,7-hexahydroazepino[3,2,1-hi]indole-6-carboxylic acid (340.9 mg) was synthesized using solid phase and isolated as an off-white powder.

[0231] ES / MS m / z 1167.8(M+H) + .

[0232] [ka] Following procedure A, (3S,6S)-3-((2S,3S)-2-(3-((S)-4-(2-(4-((4,7-bis(2-(tert-butoxy)-2-oxoethyl)-1,4,7-triazonan-1-yl)methyl)piperidin-1-yl)acetamido)-5-(tert-butoxy)-5-oxopentanamido)propanamido)-3-methylpentanamido)-4-oxo-1,2,3,4,6,7-hexahydroazepino[3,2,1-hi]indole-6-carboxylic acid (325.0 mg) was synthesized using solid phase and isolated as an off-white powder.

[0233] ES / MS m / z 1110.6(M+H) + .

[0234] [ka] Following procedure A, (3S,6S)-3-((2S,11S)-11-(2-(4-((4,7-bis(2-(tert-butoxy)-2-oxoethyl)-1,4,7-triazonan-1-yl)methyl)phenyl)acetamido)-2-((S)-sec-butyl)-16,16-dimethyl-4,7,10,14-tetraoxo-15-oxa-3,6,9-triazaheptadecanamido)-4-oxo-1,2,3,4,6,7-hexahydroazepino[3,2,1-hi]indole-6-carboxylic acid (1 g) was synthesized using solid phase and isolated as a white fluffy solid.

[0235] ES / MS m / z 1146.7(M+H) + .

[0236] [ka] Following procedure A, (3S,6S)-3-((6R,17S)-6-(2-(4-(2-(4,7-bis(2-(benzyloxy)-2-oxoethyl)-1,4,7-triazonan-1-yl)acetyl)piperazin-1-yl)acetamido)-17-((S)-sec-butyl)-3,7,11,15-tetraoxo-1-phenyl-2-oxa-8,12,16-triazaoctadecane-18-amido)-4-oxo-1,2,3,4,6,7-hexahydroazepino[3,2,1-hi]indole-6-carboxylic acid (184 mg) was synthesized using solid phase. In this specific example, one dipeptide intermediate, (R)-2-(2-(4-(((9H-fluoren-9-yl)methoxy)carbonyl)piperazin-1-yl)acetamido)-5-(benzyloxy)-5-oxopentanoic acid, was used in the solid-phase synthesis. It was isolated as a pale yellow solid.

[0237] ES / MS m / z 1313.1(M+H) + .

[0238] General procedure for precursor synthesis: General Procedure B: Active Ester Synthesis [ka] The peptide acid was dissolved in DCM (0.1 M) in a 20 mL vial equipped with a stir bar, followed by the addition of 2,3,5,6-tetrafluorophenyl trifluoroacetate (TFPTFA, 2 eq.). The resulting light yellow solution was cooled to 0° C. and triethylamine (2 eq.) was added. The yellow solution was allowed to warm to ambient temperature and monitored by LCMS. TFA (same volume) was then added slowly to the reaction mixture and the reaction was stirred at ambient temperature and monitored by LCMS. Upon completion, the yellow reaction mixture was concentrated and purified by gold aq-C18 ISCO column using water and 0.1% formic acid in acetonitrile as eluents. The appropriate fractions were collected and lyophilized to give the active ester as a white fluffy solid.

[0239] General Procedure C: Amide Bonding [ka] The Cbz-protected amine partner (>3 equiv.) was hydrogenated in ethyl acetate using H2 and 20% Pd(OH)2 / C at ambient temperature; 1 The reaction was monitored by H NMR analysis. The amine solution was concentrated and redissolved in DMF (1 mL). The pale yellow solution was transferred to a vial containing the active ester (1 eq.) and additional acetonitrile (2 mL) was added to aid transfer. The reaction was then mixed with a drop of TEA and monitored by LCMS. The clear pale yellow reaction mixture was then concentrated and purified by Phenomenex Gemini C18 RP-HPLC preparatory column using 0.1% formic acid (or 20 mM ammonium acetate) in water and acetonitrile as eluents. The appropriate fractions were collected and lyophilized to give the prodrug precursor as an off-white fluffy solid (if purified with 0.1% FA in water) or a pale yellow solid (if purified with 20 mM ammonium acetate in water).

[0240] [ka] According to general procedure B, 2,2'-(7-((1-(4R,15S,16S)-4-(2-carboxyethyl)-16-methyl-2,5,9,13-tetraoxo-15-(((3S,6S)-4-oxo-6-((2,3,5,6-tetrafluorophenoxy)carbonyl)-1,2,3,4,6,7-hexahydroazepino[3,2,1-hi]indol-3-yl)carbamoyl)-3,6,10,14-tetraazaoctadecyl)piperidin-4-yl)methyl)-1,4,7-triazonane-1,4-diyl)diacetic acid (72.3 mg) was added to (3S,6S)- Synthesized from 3-((2S,13R)-13-(2-(4-((4,7-bis(2-(tert-butoxy)-2-oxoethyl)-1,4,7-triazonan-1-yl)methyl)piperidin-1-yl)acetamido)-2-((S)-sec-butyl)-18,18-dimethyl-4,8,12,16-tetraoxo-17-oxa-3,7,11-triazanonadecanamido)-4-oxo-1,2,3,4,6,7-hexahydroazepino[3,2,1-hi]indole-6-carboxylic acid (100.0 mg) and isolated as an off-white fluffy solid.

[0241] ES / MS m / z 1161.4(M+H) + .

[0242] [ka] Following general procedure C, 2,2'-(7-((1-(4R,15S,16S)-4-(2-carboxyethyl)-15-((3S,6S)-6-(((2R,3S)-2-methoxy-5-oxotetrahydrofuran-3-yl)carbamoyl)-4-oxo-1,2,3,4,6,7-hexahydroazepino[3,2,1-hi]indol-3-yl)carbamoyl)-16-methyl-2,5,9,13-tetraoxo-3,6,10,14-tetraazaoctadecyl)piperidin-4-yl)methyl)-1,4,7-triazonane-1,4-diyl)diacetic acid (25.0 mg) was synthesized from the corresponding active ester (40.0 mg) and isolated as an off-white fluffy solid.

[0243] ES / MS m / z 1126.5(M+H) + .

[0244] [ka] Following general procedure C, 2,2'-(7-((1-(4R,15S,16S)-4-(2-carboxyethyl)-15-((3S,6S)-6-(((2S,3S)-2-methoxy-5-oxotetrahydrofuran-3-yl)carbamoyl)-4-oxo-1,2,3,4,6,7-hexahydroazepino[3,2,1-hi]indol-3-yl)carbamoyl)-16-methyl-2,5,9,13-tetraoxo-3,6,10,14-tetraazaoctadecyl)piperidin-4-yl)methyl)-1,4,7-triazonane-1,4-diyl)diacetic acid (24.6 mg) was synthesized from the corresponding active ester (36.9 mg) and isolated as an off-white fluffy solid.

[0245] ES / MS m / z 1126.6(M+H) + .

[0246] [ka] (3S,6S)-3-((6R,17S)-6-(2-(4-((4,7-bis(2-(benzyloxy)-2-oxoethyl)-1,4,7-triazonan-1-yl)methyl)piperidin-1-yl)acetamide)-17-((S)-sec-butyl)-3,7,11,15-tetraoxo-1-phenyl-2-oxa-8,12,1 To a solution of 6-triazaoctadecane-18-amido)-4-oxo-1,2,3,4,6,7-hexahydroazepino[3,2,1-hi]indole-6-carboxylic acid (180 mg, 0.12 mmol), ethyl cyano(hydroxyimino)acetate (50 mg, 0.34 mmol) and DIC (45 mg, 0.35 mmol) were added at 0° C. under N2. The mixture was stirred at 0° C. for 0.5 h. (4R,5S)-4-amino-5-methoxydihydrofuran-2(3H)-one (34 mg, 0.26 mmol, prepared from the corresponding Cbz intermediate by catalytic hydrogenation) was added. The mixture was stirred at 20° C. for 12 h. The mixture was filtered and the filtrate was purified by C18 preparative HPLC (formic acid in water-CH3CN). The obtained fractions were combined and lyophilized. Dibenzyl 2,2'-(7-((1-(4R,15S,16S)-4-(3-(benzyloxy)-3-oxopropyl)-15-(((3S,6S)-6-(((2S,3R)-2-methoxy-5-oxotetrahydrofuran-3-yl)carbamoyl)-4-oxo-1,2,3,4,6,7-hexahydroazepino[3,2,1-hi]indol-3-yl)carbamoyl)-16-methyl-2,5,9,13-tetraoxo-3,6,10,14-tetraazaoctadecyl)piperidin-4-yl)methyl)-1,4,7-triazonane-1,4-diyl)diacetate (132 mg, 0.05 mmol) was obtained as a white solid.

[0247] ES / MS m / z 1397.6(M+H) + .

[0248] Dibenzyl 2,2'-(7-((1-((4R,15S,16S)-4-(3-(benzyloxy)-3-oxopropyl)-15-(((3S,6S)-6-(((2S,3R)-2-methoxy-5-oxotetrahydrofuran-3-yl)carbamoyl)-4-oxo-1,2,3,4,6,7-hexafluorophenyl)-2,4'-dihydro-2,5'-dimethyl-2,6'-dihydro-1,7'-dihydro-2,7'-dihydro-2,5 ... To a solution of xahydroazepino[3,2,1-hi]indol-3-yl)carbamoyl)-16-methyl-2,5,9,13-tetraoxo-3,6,10,14-tetraazaoctadecyl)piperidin-4-yl)methyl)-1,4,7-triazonane-1,4-diyl)diacetate (130 mg, 0.05 mmol) was added 10% Pd / C (50 mg). The reaction mixture was degassed and purged with H2 several times. The reaction mixture was stirred under H2 (15 Psi) at 25°C for 5 h. The mixture was filtered and the filtrate was purified by C18 preparative HPLC (formic acid in water-CH3CN). The resulting fractions were combined and lyophilized. 2,2'-(7-((1-((4R,15S,16S)-4-(2-carboxyethyl)-15-(((3S,6S)-6-(((2S,3R)-2-methoxy-5-oxotetrahydrofuran-3-yl)carbamoyl)-4-oxo-1,2,3,4,6,7-hexahydroazepino[3,2,1-hi]indol-3-yl)carbamoyl)-16-methyl-2,5,9,13-tetraoxo-3,6,10,14-tetraazaoctadecyl)piperidin-4-yl)methyl)-1,4,7-triazonane-1,4-diyl)diacetic acid (40 mg, 0.036 mmol) was obtained as a white solid.

[0249] ES / MS m / z 1126.9(M+H) + .

[0250] [ka] Compound (3S,6S)-3-((6R,17S)-6-(2-(4-((4,7-bis(2-(benzyloxy)-2-oxoethyl)-1,4,7-triazonan-1-yl)methyl)piperidin-1-yl)acetamide)-17-((S)-sec-butyl)-3,7,11,15-tetraoxo-1-phenyl-2-oxa-8,12 To a solution of (4R,5R)-4-oxo-1,2,3,4,6,7-hexahydroazepino[3,2,1-hi]indole-6-carboxylic acid (210 mg, 0.13 mmol) was added ethyl cyano(hydroxyimino)acetate (48 mg, 0.33 mmol) and DIC (43 mg, 0.33 mmol) at 0° C. under N2. The mixture was stirred at 0° C. for 0.5 h. (4R,5R)-4-amino-5-methoxydihydrofuran-2(3H)-one (34 mg, 0.26 mmol, prepared from the corresponding Cbz intermediate by catalytic hydrogenation) was added. The mixture was stirred at 20° C. for 12 h. The crude product was purified by C18 preparative HPLC (formic acid in water-CH3CN). The obtained fractions were combined and lyophilized. Dibenzyl 2,2'-(7-((1-(4R,15S,16S)-4-(3-(benzyloxy)-3-oxopropyl)-15-(((3S,6S)-6-(((2R,3R)-2-methoxy-5-oxotetrahydrofuran-3-yl)carbamoyl)-4-oxo-1,2,3,4,6,7-hexahydroazepino[3,2,1-hi]indol-3-yl)carbamoyl)-16-methyl-2,5,9,13-tetraoxo-3,6,10,14-tetraazaoctadecyl)piperidin-4-yl)methyl)-1,4,7-triazonane-1,4-diyl)diacetate (112 mg, 0.07 mmol) was obtained as a white solid.

[0251] ES / MS m / z 1397.1(M+H) + .

[0252] Dibenzyl 2,2'-(7-((1-((4R,15S,16S)-4-(3-(benzyloxy)-3-oxopropyl)-15-((3S,6S)-6-(((2R,3R)-2-methoxy-5-oxotetrahydrofuran-3-yl)carbamoyl)-4-oxo-1,2,3,4,6,7-hexafluorophenyl)-2,4'-dihydro-2,5'-dimethyl-2,6'-dihydro-1,7'-dihydro-2,7'-dihydro-2,5 ... To a solution of xahydroazepino[3,2,1-hi]indol-3-yl)carbamoyl)-16-methyl-2,5,9,13-tetraoxo-3,6,10,14-tetraazaoctadecyl)piperidin-4-yl)methyl)-1,4,7-triazonane-1,4-diyl)diacetate (110 mg, 0.07 mmol) was added 10% Pd / C (50 mg). The reaction mixture was degassed and purged with H2 several times. The reaction mixture was stirred under H2 (15 Psi) at 20°C for 2 h. The mixture was filtered and the cake was washed with DCM (10 mL*3). The combined filtrate was concentrated. The residue was purified by C18 preparative HPLC (formic acid in water-CH3CN). The obtained fractions were combined and lyophilized. 2,2'-(7-((1-((4R,15S,16S)-4-(2-carboxyethyl)-15-(((3S,6S)-6-(((2R,3R)-2-methoxy-5-oxotetrahydrofuran-3-yl)carbamoyl)-4-oxo-1,2,3,4,6,7-hexahydroazepino[3,2,1-hi]indol-3-yl)carbamoyl)-16-methyl-2,5,9,13-tetraoxo-3,6,10,14-tetraazaoctadecyl)piperidin-4-yl)methyl)-1,4,7-triazonane-1,4-diyl)diacetic acid (26 mg, 0.02 mmol) was obtained as a white solid.

[0253] ES / MS m / z 1126.9(M+H) + .

[0254] [ka] Following general procedure C, 2,2'-(7-((1-((4R,15S,16S)-4-(2-carboxyethyl)-15-(((3S,6S)-6-(((2R,3S)-2-ethoxy-5-oxotetrahydrofuran-3-yl)carbamoyl)-4-oxo-1,2,3,4,6,7-hexahydroazepino[3,2,1-hi]indol-3-yl)carbamoyl)-16-methyl-2,5,9,13-tetraoxo-3,6,10,14-tetraazaoctadecyl)piperidin-4-yl)methyl)-1,4,7-triazonane-1,4-diyl)diacetic acid (21.9 mg) was synthesized from the corresponding active ester (33.0 mg) and isolated as an off-white fluffy solid.

[0255] ES / MS m / z 1140.6(M+H) + .

[0256] [ka] According to general procedure B, 2,2'-(7-((1-((4R,7R,16S,17S)-4,7-bis(2-carboxyethyl)-17-methyl-2,5,8,14-tetraoxo-16-(((3S,6S)-4-oxo-6-((2,3,5,6-tetrafluorophenoxy)carbonyl)-1,2,3,4,6,7-hexahydroazepino[3,2,1-hi]indol-3-yl)carbamoyl)-12-oxa-3,6,9,15-tetraazanonadecyl)piperidin-4-yl)methyl)-1,4,7-triazonane-1,4-diyl)diacetic acid (85.1 mg) was added to (3S,6S)-3-((2S,11 Synthesized from (R,14R)-14-(2-(4-(4,7-bis(2-(tert-butoxy)-2-oxoethyl)-1,4,7-triazonan-1-yl)methyl)piperidin-1-yl)acetamido-11-(tert-butoxy)-3-oxopropyl)-2-((S)-sec-butyl)-19,19-dimethyl-4,10,13,17-tetraoxo-6,18-dioxa-3,9,12-triazaicosanamido)-4-oxo-1,2,3,4,6,7-hexahydroazepino[3,2,1-hi]indole-6-carboxylic acid (191.2 mg) and isolated as an off-white fluffy solid.

[0257] ES / MS m / z 1249.6(M+H) + .

[0258] [ka] Following general procedure C, 2,2'-(7-((1-((4R,7R,16S,17S)-4,7-bis(2-carboxyethyl)-16-(((3S,6S)-6-(((2R,3S)-2-methoxy-5-oxotetrahydrofuran-3-yl)carbamoyl)-4-oxo-1,2,3,4,6,7-hexahydroazepino[3,2,1-hi]indo (3-yl)carbamoyl)-17-methyl-2,5,8,14-tetraoxo-12-oxa-3,6,9,15-tetraazanonadecyl)piperidin-4-yl)methyl)-1,4,7-triaza-1,4-diyl)diacetic acid (61.9 mg) was synthesized from the corresponding active ester (65.0 mg) and isolated as an off-white fluffy solid.

[0259] ES / MS m / z 1214.8(M+H) + .

[0260] [ka] Following general procedure C, 2,2'-(7-((1-((4R,7R,16S,17S)-4,7-bis(2-carboxyethyl)-16-(((3S,6S)-6-(((2S,3S)-2-methoxy-5-oxotetrahydrofuran-3-yl)carbamoyl)-4-oxo-1,2,3,4,6,7-hexahydroazepino[3,2,1-hi]indo (3-yl)carbamoyl)-17-methyl-2,5,8,14-tetraoxo-12-oxa-3,6,9,15-tetraazanonadecyl)piperidin-4-yl)methyl)-1,4,7-triaza-1,4-diyl)diacetic acid (14.1 mg) was synthesized from the corresponding active ester (25.3 mg) and isolated as an off-white fluffy solid.

[0261] ES / MS m / z 1214.8(M+H) + .

[0262] [ka] According to general procedure C, 2,2'-(7-((1-((4R,7R,16S,17S)-4,7-bis(2-carboxyethyl)-16-(((3S,6S)-6-(((2R,3S)-2-ethoxy-5-oxotetrahydrofuran-3-yl)carbamoyl)-4-oxo-1,2,3,4,6,7-hexahydroazepino[3,2,1-hi]indoline was prepared. (3-yl)carbamoyl)-17-methyl-2,5,8,14-tetraoxo-12-oxa-3,6,9,15-tetraazanonadecyl)piperidin-4-yl)methyl)-1,4,7-triazonane-1,4-diyl)diacetic acid (26.5 mg) was synthesized from the corresponding active ester (43.0 mg) and isolated as an off-white fluffy solid.

[0263] ES / MS m / z 1228.9(M+H) + .

[0264] [ka] According to general procedure B, 2,2'-(7-((1-((15S,16S)-3-(carboxymethyl)-16-methyl-2,5,9,13-tetraoxo-15-(((3S,6S)-4-oxo-6-((2,3,5,6-tetrafluorophenoxy)carbonyl)-1,2,3,4,6,7-hexahydroazepino[3,2,1-hi]indol-3-yl)carbamoyl)-3,6,10,14-tetraazaoctadecyl)piperidin-4-yl)methyl)-1,4,7-triazonane-1,4-diyl)diacetic acid (91.8 mg) was added to (3S,6S)-3 -((S)-6-(2-(4-((4,7-bis(2-(tert-butoxy)-2-oxoethyl)-1,4,7-triazonan-1-yl)methyl)piperidin-1-yl)acetyl)-18-((S)-sec-butyl)-2,2-dimethyl-4,8,12,16-tetraoxo-3-oxa-6,9,13,17-tetraazanonadecane-19-amido)-4-oxo-1,2,3,4,6,7-hexahydroazepino[3,2,1-hi]indole-6-carboxylic acid (180.0 mg) and isolated as an off-white fluffy solid.

[0265] ES / MS m / z 1147.5(M+H) + .

[0266] [ka] Following general procedure C, 2,2'-(7-((1-((15S,16S)-3-(carboxymethyl)-15-(((3S,6S)-6-(((2R,3S)-2-methoxy-5-oxotetrahydrofuran-3-yl)carbamoyl)-4-oxo-1,2,3,4,6,7-hexahydroazepino[3,2,1-hi]indol-3-yl)carbamoyl)-16-methyl-2,5,9,13-tetraoxo-3,6,10,14-tetraazaoctadecyl)piperidin-4-yl)methyl)-1,4,7-triazonane-1,4-diyl)diacetic acid (43.2 mg) was synthesized from the corresponding active ester (50.0 mg) and isolated as an off-white fluffy solid.

[0267] ES / MS m / z 1112.5(M+H) + .

[0268] [ka] According to procedure B, 2,2'-(7-((1-(2-(((S)-1-carboxy-4-((3-(((2S,3S)-3-methyl-1-oxo-1-(((3S,6S)-4-oxo-6-((2,3,5,6-tetrafluorophenoxy)carbonyl)-1,2,3,4,6,7-hexahydroazepino[3,2,1-hi]indol-3-yl)amino)pentan-2-yl)amino)-3-oxopropyl)amino)-4-oxobutyl)amino)-2-oxoethyl)piperidin-4-yl)methyl)-1,4,7-triazonane-1,4-diyl)diacetic acid (8 3.0 mg) was synthesized from (3S,6S)-3-((2S,3S)-2-(3-((S)-4-(2-(4-((4,7-bis(2-(tert-butoxy)-2-oxoethyl)-1,4,7-triazonan-1-yl)methyl)piperidin-1-yl)acetamido-5-(tert-butoxy)-5-oxopentanamido)propanamido)-3-methylpentanamido)-4-oxo-1,2,3,4,6,7-hexahydroazepino[3,2,1-hi]indole-6-carboxylic acid (100.0 mg) and isolated as an off-white fluffy solid.

[0269] ES / MS m / z 1090.5(M+H) + .

[0270] [ka] According to procedure C, 2,2'-(7-((1-(2-(((S)-1-carboxy-4-((3-(((2S,3S)-1-(((3S,6S)-6-(((2R,3S)-2-methoxy-5-oxotetrahydrofuran-3-yl)carbamoyl)-4-oxo-1,2,3,4,6,7-hexahydroazepino[3,2,1-hi]indol-3-yl)amino )-3-Methyl-1-oxopentan-2-yl)amino)-3-oxopropyl)amino)-4-oxobutyl)amino)-2-oxoethyl)piperidin-4-yl)methyl)-1,4,7-triazonane-1,4-diyl)diacetic acid (24.8 mg) was synthesized from the corresponding active ester (83.0 mg) and isolated as an off-white fluffy solid.

[0271] ES / MS m / z 1055.5(M+H) + .

[0272] [ka] According to procedure B, 2,2'-(7-(4-((4S,13S,14S)-4-(2-carboxyethyl)-14-methyl-2,5,8,11-tetraoxo-13-(((3S,6S)-4-oxo-6-((2,3,5,6-tetrafluorophenoxy)carbonyl)-1,2,3,4,6,7-hexahydroazepino[3,2,1-hi]indol-3-yl)carbamoyl)-3,6,9,12-tetraazahexadecyl)benzyl)-1,4,7-triazonane-1,4-diyl)diacetic acid (20.0 mg) was added to (3S,6S)-3-(( Synthesized from 2S,11S)-11-(2-(4-((4,7-bis(2-(tert-butoxy)-2-oxaethyl)-1,4,7-triazonan-1-yl)methyl)phenyl)acetamido)-2-((S)-sec-butyl)-16,16-dimethyl-4,7,10,14-tetraoxo-15-oxa-3,6,9-triazaheptadecanamido)-4-oxo-1,2,3,4,6,7-hexahydroazepino[3,2,1-hi]indole-6-carboxylic acid (30.6 mg) and isolated as an off-white fluffy solid.

[0273] ES / MS m / z 1126.4(M+H) + .

[0274] [ka] Following general procedure C, 2,2'-(7-(4-(4S,13S,14S)-4-(2-carboxyethyl)-13-(((3S,6S)-6-(((2R,3S)-2-methoxy-5-oxotetrahydrofuran-3-yl)carbamoyl)-4-oxo-1,2,3,4,6,7-hexahydroazepino[3,2,1-hi]indol-3-yl)carbamoyl)-14-methyl-2,5,8,11-tetraoxo-3,6,9,12-tetraazahexadecyl)benzyl)-1,4,7-triazonane-1,4-diyl)diacetic acid (12.0 mg) was synthesized from the corresponding active ester (19.0 mg) and isolated as an off-white fluffy solid.

[0275] ES / MS m / z 1091.5(M+H) + .

[0276] [ka] A stirred solution of 2,2'-(7-(4-((4S,13S,14S)-4-(2-carboxyethyl)-13-((3S,6S)-6-((2-hydroxy-5-oxotetrahydrofuran-3-yl)carbamoyl)-4-oxo-1,2,3,4,6,7-hexahydroazepino[3,2,1-hi]indol-3-yl)carbamoyl)-14-methyl-2,5,8,11-tetraoxo-3,6,9,12-tetraazahexadecyl)benzyl)-1,4,7-triazonane-1,4-diyl)diacetic acid (15.0 mg) in a mixture of 3.0 mL of ethanol and 0.75 mL of ethyl orthoacetate with approximately 20 μL of TFA was heated to 65° C. overnight. The solution was concentrated and the residue was redissolved in 10% DMSO in H2O. The mixture was purified by HPLC (HO to 0.1% FA in CHCN, 0-60%) to give 2,2'-(7-(4-((4S,13S,14S)-4-(2-carboxyethyl)-13-(((3S,6S)-6-((2-ethoxy-5-oxotetrahydrofuran-3-yl)carbamoyl)-4-oxo-1,2,3,4,6,7-hexahydroazepino[3,2,1-hi]indol-3-yl)carbamoyl)-14-methoxy-2,5,8,11-tetraoxo-3,6,9,12-tetraazahexadecyl)benzyl)-1,4,7-triazonane-1,4-diyl)diacetic acid (2.5 mg) as an off-white solid.

[0277] ES / MS m / z 1105.5(M+H) + .

[0278] [ka] Compound (3S,6S)-3-((6R,17S)-6-(2-(4-(2-(4,7-bis(2-(benzyloxy)-2-oxoethyl)-1,4,7-triazonan-1-yl)acetyl)pipiperazin-1-yl)acetamide)-17-((S)-sec-butyl)-3,7,11,15-tetraoxo-1-phenyl-2-oxa-8,12, To a solution of 16-triazaoctadecane-18-amido)-4-oxo-1,2,3,4,6,7-hexahydroazepino[3,2,1-hi]indole-6-carboxylic acid (100 mg, 0.069 mmol) and ethyl cyanohydroxyiminoacetate (28 mg, 0.19 mmol) was added N,N-diisopropylcarbodiimide (25 mg, 0.19 mmol) at 0° C. The reaction mixture was stirred at 0° C. for 0.5 h under N2. Then, (4S,5R)-4-amino-5-methoxydihydrofuran-2(3H)-one (25 mg, 0.19 mmol) in DMF (2 mL) was added. The reaction mixture was stirred at 20° C. for 16 h under N2. The mixture was filtered and the filtrate was purified by preparative HPLC (formic acid in water-CH3CN). The resulting fractions were combined and lyophilized to give dibenzyl 2,2'-(7-(2-(4-((4R,15S,16S)-4-(3-(benzyloxy)-3-oxopropyl)-15-(((3S,6S)-6-(((2R,3S)-2-methoxy-5-oxotetrahydrofuran-3-yl)carbamoyl)-4-oxo-1,2,3,4,6,7-hexafluoropropanediol. Sahydroazepino[3,2,1-hi]indol-3-yl)carbamoyl)-16-methyl-2,5,9,13-tetraoxo-3,6,10,14-tetraazaoctadecyl)piperazin-1-yl)-2-oxoethyl)-1,4,7-triazonane-1,4-diyl)diacetate (41 mg, 0.025 mmol) was obtained as a white solid. ES / MS m / z 1426.7 (M+H) + .

[0279] Dibenzyl 2,2'-(7-(2-(4-((4R,15S,16S)-4-(3-(benzyloxy)-3-oxopropyl)-15-(((3S,6S)-6-(((2R,3S)-2-methoxy-5-oxotetrahydrofuran-3-yl)carbamoyl)-4-oxo-1,2,3,4,6,7-hexafluorophenyl)-2,4'-dihydro-2,5'-dimethyl-2,6'-dihydro-1,7'-dihydro-2,7'-dihydro-2,5'-dihydro-2,6'-dihydro-1 ... To a solution of xahydroazepino[3,2,1-hi]indol-3-yl)carbamoyl)-16-methyl-2,5,9,13-tetraoxo-3,6,10,14-tetraazaoctadecyl)piperazin-1-yl)-2-oxoethyl)-1,4,7-triazonane-1,4-diyl)diacetate (41 mg, 0.025 mmol) was added Pd / C (15 mg). The reaction mixture was degassed and purged with H2 several times. The reaction mixture was stirred under H2 atmosphere at 20° C. for 3 h under N2. The mixture was filtered and the filtrate was concentrated under reduced pressure. The crude product was purified by C18 preparative HPLC (formic acid in water-CH3CN). The resulting fractions were combined and lyophilized to give 2,2'-(7-(2-(4-((4R,15S,16S)-4-(2-carboxyethyl)-15-(((3S,6S)-6-(((2R,3S)-2-methoxy-5-oxotetrahydrofuran-3-yl)carbamoyl)-4-oxo-1,2,3,4,6,7-hexahydroazepino[3,2,1-hi]indol-3-yl)carbamoyl)-16-methyl-2,5,9,13-tetraoxo-3,6,10,14-tetraazaoctadecyl)piperazin-1-yl)-2-oxoethyl)-1,4,7-triazonane-1,4-diyl)diacetic acid (12.4 mg, 0.01 mmol).

[0280] ES / MS m / z 1156.0(M+H) + .

[0281] AlF complex General Procedure D: To a reaction vial containing the peptide precursor and a stir bar were added equal equivalents (1.5-3.0 equivalents relative to peptide) of 20 mM AlCl3 in 0.1 M NaOAc (pH approx. 4.5) and 100 mM NaF in HO. Acetonitrile (0-34% of the total reaction volume) was then added. The mixture was heated to 100°C for 15-30 min. Acetonitrile was removed under reduced pressure and the aqueous solution was purified by either a gold aq-C18 ISCO column or a Phenomenex Gemini C18 RP-HPLC preparative column (20 mM ammonium acetate in water and acetonitrile as eluents). Appropriate fractions were collected and lyophilized to give the peptide AlF complex as a white fluffy solid (purified by 0.1% FA in water) or a pale yellow solid (purified by 20 mM ammonium acetate in water).

[0282] General procedure E: To a 4 mL vial containing 100 μL of peptide precursor solution (approximately 200 nmol, 2 mg / mL in 1 N acetate buffer, pH 3.5), 60 μL of AlCl3 solution [144 nmol, 2.4 mM in WIC (water for ion chromatography)], 10 μL of NaF (100 nmol, 10 mM in WIC), and 230 μL of acetonitrile (HPLC grade) were added. The mixture was heated at 105° C. for 15 min in a heating block and then diluted with 1.6 mL of WIC. The reaction mixture was then analyzed by LCMS.

[0283] [ka] According to the general procedure D, 2,2'-(7-((1-((4R,15S,16S)-4-(2-carboxyethyl)-15-(((3S,6S)-6-(((2R,3S)-2-methoxy-5-oxotetrahydrofuran-3-yl)carbamoyl)-4-oxo-1,2,3,4,6,7-hexahydroazepino[3,2,1-hi]indol-3-yl)carbamoyl)-16-methyl-2,5,9,13-tetraoxo-3,6,10,14-tetraazaoctadecyl)piperidin-4-yl)methyl)-1,4,7-triazonane-1,4-diyl)diacetic acid, aluminum fluoride complex (1:1) (14.6 mg) was obtained. ) was synthesized from 2,2'-(7-((1-((4R,15S,16S)-4-(2-carboxyethyl)-15-(((3S,6S)-6-(((2R,3S)-2-methoxy-5-oxotetrahydrofuran-3-yl)carbamoyl)-4-oxo-1,2,3,4,6,7-hexahydroazepino[3,2,1-hi]indol-3-yl)carbamoyl)-16-methyl-2,5,9,13-tetraoxo-3,6,10,14-tetraazaoctadecyl)piperidin-4-yl)methyl)-1,4,7-triazonane-1,4-diyl)diacetic acid (20.4 mg) and isolated as a white fluffy solid.

[0284] ES / MS m / z 1170.7(M+H) + .

[0285] [ka] According to the general procedure E, 2,2'-(7-((1-(4R,15S,16S)-4-(2-carboxyethyl)-15-(((3S,6S)-6-(((2S,3S)-2-methoxy-5-oxotetrahydrofuran-3-yl)carbamoyl)-4-oxo-1,2,3,4,6,7-hexahydroazepino[3,2,1-hi]indol-3-yl)carbamoyl)-16-methyl-2,5,9,13-tetraoxo-3,6,10,14-tetraazaoctadecyl)piperidin-4-yl)methyl)-1,4,7-triazonane-1,4-diyl)diacetic acid, aluminum fluoride complex (1:1) was detected by HPLC.

[0286] ES / MS m / z 1192.6(M+Na) + .

[0287] [ka] According to the general procedure E, 2,2'-(7-((1-((4R,15S,16S)-4-(2-carboxyethyl)-15-(((3S,6S)-6-(((2S,3R)-2-methoxy-5-oxotetrahydrofuran-3-yl)carbamoyl)-4-oxo-1,2,3,4,6,7-hexahydroazepino[3,2,1-hi]indol-3-yl)carbamoyl)-16-methyl-2,5,9,13-tetraoxo-3,6,10,14-tetraazaoctadecyl)piperidin-4-yl)methyl)-1,4,7-triazonane-1,4-diyl)diacetic acid, aluminum fluoride complex (1:1) was detected by HPLC.

[0288] ES / MS m / z 1170.6(M+H) + .

[0289] [ka] According to the general procedure E, 2,2'-(7-((1-((4R,15S,16S)-4-(2-carboxyethyl)-15-(((3S,6S)-6-(((2R,3R)-2-methoxy-5-oxotetrahydrofuran-3-yl)carbamoyl)-4-oxo-1,2,3,4,6,7-hexahydroazepino[3,2,1-hi]indol-3-yl)carbamoyl)-16-methyl-2,5,9,13-tetraoxo-3,6,10,14-tetraazaoctadecyl)piperidin-4-yl)methyl)-1,4,7-triazonane-1,4-diyl)diacetic acid, aluminum fluoride complex (1:1) was detected by HPLC.

[0290] ES / MS m / z 1170.6(M+H) + .

[0291] [ka] According to the general procedure D, 2,2'-(7-((1-((4R,15S,16S)-4-(2-carboxyethyl)-15-(((3S,6S)-6-((2R,3S)-2-ethoxy-5-oxotetrahydrofuran-3-yl)carbamoyl)-4-oxo-1,2,3,4,6,7-hexahydroazepino[3,2,1-hi]indol-3-yl)carbamoyl)-16-methyl-2,5,9,13-tetraoxo-3,6,10,14-tetraazaoctadecyl)piperidin-4-yl)methyl)-1,4,7-triazonane-1,4-diyl)diacetic acid, aluminum fluoride complex (1:1) (5.0 mg) was synthesized from 2,2'-(7-((1-((4R,15S,16S)-4-(2-carboxyethyl)-15-(((3S,6S)-6-(((2R,3S)-2-ethoxy-5-oxotetrahydrofuran-3-yl)carbamoyl)-4-oxo-1,2,3,4,6,7-hexahydroazepino[3,2,1-hi]indol-3-yl)carbamoyl)-16-methyl-2,5,9,13-tetraoxo-3,6,10,14-tetraazaoctadecyl)piperidin-4-yl)methyl)-1,4,7-triazonane-1,4-diyl)diacetic acid (10.4 mg) and isolated as a white fluffy solid.

[0292] ES / MS m / z 1184.6(M+H) + .

[0293] [ka] According to the general procedure D, 2,2'-(7-((1-((4R,7R,16S,17S)-4,7-bis(2-carboxyethyl)-16-(((3S,6S)-6-(((2R,3S)-2-methoxy-5-oxotetrahydrofuran-3-yl)carbamoyl)-4-oxo-1,2,3,4,6,7-hexahydroazepino[3,2,1-hi]indol-3-yl)carbamoyl)-17-methyl-2,5,8,14-tetraoxo-12-oxa-3,6,9,15-tetraazanonadecyl)piperidin-4-yl)methyl)-1,4,7-triazonane-1,4-diyl)diacetic acid, aluminum fluoride complex (1:1) (10.6 mg) was obtained. ) was synthesized from 2,2'-(7-((1-((4R,7R,16S,17S)-4,7-bis(2-carboxyethyl)-16-(((3S,6S)-6-(((2R,3S)-2-methoxy-5-oxotetrahydrofuran-3-yl)carbamoyl)-4-oxo-1,2,3,4,6,7-hexahydroazepino[3,2,1-hi]indol-3-yl)carbamoyl)-17-methyl-2,5,8,14-tetraoxo-12-oxa-3,6,9,15-tetraazanonadecyl)piperidin-4-yl)methyl)-1,4,7-triazonane-1,4-diyl)diacetic acid (20.0 mg) and isolated as a white fluffy solid.

[0294] ES / MS m / z 1258.6(M+H) + .

[0295] [ka] According to the general procedure D, 2,2'-(7-((1-((4R,7R,16S,17S)-4,7-bis(2-carboxyethyl)-16-(((3S,6S)-6-(((2S,3S)-2-methoxy-5-oxotetrahydrofuran-3-yl)carbamoyl)-4-oxo-1,2,3,4,6,7-hexahydroazepino[3,2,1-hi]indol-3-yl)carbamoyl)-17-methyl-2,5,8,14-tetraoxo-12-oxa-3,6,9,15-tetraazanonadecyl)piperidin-4-yl)methyl)-1,4,7-triazonane-1,4-diyl)diacetic acid, aluminum fluoride complex (1:1) (1.1 mg) was prepared. ) was synthesized from 2,2'-(7-((1-((4R,7R,16S,17S)-4,7-bis(2-carboxyethyl)-16-(((3S,6S)-6-(((2S,3S)-2-methoxy-5-oxotetrahydrofuran-3-yl)carbamoyl)-4-oxo-1,2,3,4,6,7-hexahydroazepino[3,2,1-hi]indol-3-yl)carbamoyl)-17-methyl-2,5,8,14-tetraoxo-12-oxa-3,6,9,15-tetraazanonadecyl)piperidin-4-yl)methyl)-1,4,7-triazonane-1,4-diyl)diacetic acid (4.8 mg) and isolated as a white fluffy solid.

[0296] ES / MS m / z 1258.6(M+H) + .

[0297] [ka] According to the general procedure D, 2,2'-(7-((1-(4R,7R,16S,17S)-4,7-bis(2-carboxyethyl)-16-((3S,6S)-6-(((2R,3S)-2-ethoxy-5-oxotetrahydrofuran-3-yl)carbamoyl)-4-oxo-1,2,3,4,6,7-hexahydroazepino[3,2,1-hi]indol-3-yl)carbamoyl)-17-methyl-2,5,8,14-tetraoxo-12-oxa-3,6,9,15-tetraazanonadecyl)piperidin-4-yl)methyl)-1,4,7-triazonane-1,4-diyl)diacetic acid, aluminum fluoride complex (1:1) (3.3 mg) was synthesized from 2,2'-(7-((1-((4R,7R,16S,17S)-4,7-bis(2-carboxyethyl)-16-(((3S,6S)-6-(((2R,3S)-2-ethoxy-5-oxotetrahydrofuran-3-yl)carbamoyl)-4-oxo-1,2,3,4,6,7-hexahydroazepino[3,2,1-hi]indol-3-yl)carbamoyl)-17-methyl-2,5,8,14-tetraoxo-12-oxa-3,6,9,15-tetraazanonadecyl)piperidin-4-yl)methyl)-1,4,7-triazonane-1,4-diyl)diacetic acid (10.5 mg) and isolated as a white fluffy solid.

[0298] ES / MS m / z 1272.6(M+H) + .

[0299] [ka] According to the general procedure D, 2,2'-(7-((1-((15S,16S)-3-(carboxymethyl)-15-(((3S,6S)-6-(((2R,3S)-2-methoxy-5-oxotetrahydrofuran-3-yl)carbamoyl)-4-oxo-1,2,3,4,6,7-hexahydroazepino[3,2,1-hi]indol-3-yl)carbamoyl)-16-methyl-2,5,9,13-tetraoxo-3,6,10,14-tetraazaoctadecyl)piperidin-4-yl)methyl)-1,4,7-triazonane-1,4-diyl)diacetic acid, aluminum fluoride complex (1:1) (7.5 mg) was prepared. ) was synthesized from 2,2'-(7-((1-((15S,16S)-3-(carboxyethyl)-15-(((3S,6S)-6-(((2R,3S)-2-methoxy-5-oxotetrahydrofuran-3-yl)carbamoyl)-4-oxo-1,2,3,4,6,7-hexahydroazepino[3,2,1-hi]indol-3-yl)carbamoyl)-16-methyl-2,5,9,13-tetraoxo-3,6,10,14-tetraazaoctadecyl)piperidin-4-yl)methyl)-1,4,7-triazonane-1,4-diyl)diacetic acid (10.2 mg) and isolated as a white fluffy solid.

[0300] ES / MS m / z 1156.6(M+H) + .

[0301] [ka] General procedure E was followed and 2,2'-(7-((1-(2-(((S)-1-carboxy-4-((3-(((2S,3S)-1-(((3S,6S)-6-(((2R,3S)-2-methoxy-5-oxotetrahydrofuran-3-yl)carbamoyl)-4-oxo-1,2,3,4,6,7-hexahydroazepino[3,2,1-hi]indol-3-yl)amino)-3-methyl-1-oxopentan-2-yl)amino)-3-oxopropyl)amino)-4-oxobutyl)amino)-2-oxoethyl)piperidin-4-yl)methyl)-1,4,7-triazonane-1,4-diyl)diacetic acid, aluminum fluoride complex (1:1) was detected by HPLC.

[0302] ES / MS m / z 1099.5(M+H) + .

[0303] [ka] General procedure E was followed to detect 2,2'-(7-(2-(4-((4R,15S,16S)-4-(2-carboxyethyl)-15-(((3S,6S)-6-(((2R,3S)-2-methoxy-5-oxotetrahydrofuran-3-yl)carbamoyl)-4-oxo-1,2,3,4,6,7-hexahydroazepino[3,2,1-hi]indol-3-yl)carbamoyl)-16-methyl-2,5,9,13-tetraoxo-3,6,10,14-tetraazaoctadecyl)piperazin-1-yl)-2-oxoethyl)-1,4,7-triazonane-1,4-diyl)diacetic acid, aluminum fluoride complex (1:1) by HPLC.

[0304] ES / MS m / z 1199.6(M+H) + .

[0305] [ka] According to the general procedure E, 2,2'-(7-(4-((4S,13S,14S)-4-(2-carboxyethyl)-13-(((3S,6S)-6-(((2R,3S)-2-methoxy-5-oxotetrahydrofuran-3-yl)carbamoyl)-4-oxo-1,2,3,4,6,7-hexahydroazepino[3,2,1-hi]indol-3-yl)carbamoyl)-14-methyl-2,5,8,11-tetraoxo-3,6,9,12-tetraazahexadecyl)benzyl)-1,4,7-triazonane-1,4-diyl)diacetic acid, aluminum fluoride complex (1:1) was detected by HPLC.

[0306] ES / MS m / z 1135.4(M+H) + .

[0307] [ka] A stirred solution of 2,2'-(7-(4-((4S,13S,14S)-4-(2-carboxyethyl)-13-(((3S,6S)-6-((2-hydroxy-5-oxotetrahydrofuran-3-yl)carbamoyl)-4-oxo-1,2,3,4,6,7-hexahydroazepino[3,2,1-hi]indol-3-yl)carbamoyl)-14-methyl-2,5,8,11-tetraoxo-3,6,9,12-tetraazahexadecyl)benzyl)-1,4,7-triazonane-1,4-diyl)diacetic acid, aluminum fluoride complex (1:1) (15.0 mg) in a mixture of 3.0 mL of ethanol and 0.75 mL of ethyl orthoacetate with approximately 20 μL of TFA was heated to 65° C. overnight. The solution was concentrated and the residue was redissolved in 10% DMSO in H2O. The mixture was purified by HPLC (HO to 0.1% FA in CHCN, 0-60%) to give 2,2'-(7-(4-((4S,13S,14S)-4-(2-carboxyethyl)-13-(((3S,6S)-6-((2-ethoxy-5-oxotetrahydrofuran-3-yl)carbamoyl)-4-oxo-1,2,3,4,6,7-hexahydroazepino[3,2,1-hi]indol-3-yl)carbamoyl)-14-methoxy-2,5,8,11-tetraoxo-3,6,9,12-tetraazahexadecyl)benzyl)-1,4,7-triazonane-1,4-diyl)diacetic acid, aluminum fluoride complex (1:1) (5.5 mg) as an off-white solid.

[0308] ES / MS m / z 1149.5(M+H) + .

[0309] Example 4: Compounds 18 Radiation synthesis of F-4-Al Representative compounds synthesized with the ORA Neptis Perform radiosynthesizer 18 The F-4-Al RCY ranges from 42 to 45% using starting activities of 20 to 26 GBq. The synthesis time is 62 ± 5 min, the radiochemical purity of the products is greater than 97%, and the specific activities range from 744 to 894 GBq / μmol (17 to 21 mCi / μg).

[0310] A reaction vessel was pre-charged with the precursor (0.22 mg), AlCl3·6H2O (38.6 μg), acetic acid / sodium acetate buffer (0.2 mL, 1 M, pH = 3.5), water (0.19 mL), and acetonitrile (0.8 mL). 18 [F]Fluoride was retained on a preconditioned Waters Sep-Pak Accell Plus QMA Carbonate Plus Light cartridge (46 mg sorbent per cartridge, particle size 40 μm, Waters part number 186004540) and then eluted into the reaction vessel using 0.9% saline (0.8 mL). The resulting mixture was held at 105 °C for 15 min and then cooled to 60 °C. 4.5 mL of water was added to the reaction vessel. The diluted reaction mixture was then loaded onto a semi-preparative HPLC column (Agilent ZORBAX Eclipse XDB-C18, 5 μm, 9.4 × 250 mm, part number 990967-202) and purified using a mobile phase containing 82% 20 mM ammonium acetate in water (pH 7) and 18% acetonitrile at a flow rate of 4 mL / min. Typical peak collection ranged from 23 to 25 min. (Figure 1A) The collected fractions were then diluted with 0.5% (w / v) aqueous sodium ascorbate (approximately 30 mL) and passed through a preconditioned Waters Sep-Pak C18Plus Light cartridge (130 mg sorbent per cartridge, 55-105 μm, part number: 023501). The product retained on the cartridge was then washed with aqueous sodium ascorbate (0.5%, w / v, approximately 15 mL) and then eluted with 1 mL of ethanol into a final product vial containing 6 mL of 0.9% saline and sodium ascorbate (0.5% w / v). The C18 cartridge was then rinsed with an additional 3 mL of 0.9% saline and sodium ascorbate (0.5% w / v) to obtain 10 mL of formulated product as 10% v / v ethanol, and 90% v / v 0.9% saline containing 0.5% w / v sodium ascorbate. A sample was removed from the product vial for HPLC analysis (Figure 1B).

[0311] Example 5: Active and pro-granzyme B in a Matrigel mouse model 18 In vivo imaging of F-granzyme B tracer This example explores the in vivo imaging activity of exemplary prodrug compounds disclosed herein, which are capable of generating Granzyme B binding compounds in vivo.

[0312] Female nude athymic (5-6 weeks, 15-30 g) mice were purchased from Jackson Laboratories. Both Granzyme B (human lymphocytes, Enzo Life Sciences) and inactivated human pro-Granzyme B (R&D Systems) were also purchased commercially. On the day of the imaging diagnostic study, each mouse had a cannula (SAI 27 g butterfly with 12 cm tubing, #BF27-01) inserted into the lateral tail vein to allow for intravenous radiotracer administration. Each Granzyme B enzyme was then achieved to a final concentration of 0.05 μg / ul using phosphate buffered saline (GE, Hyclone). Matrigel (65 μl, Corning) was mixed with 0.75 μg of each Granzyme B enzyme (Granzyme B and pro-Granzyme B) in each Eppendorf tube and 15 μl. Mice were then anesthetized with 2.5-3% isoflurane mixed with oxygen. Approximately 60-80 μl of Granzyme B / Matrigel or pro-Granzyme B / Matrigel was then injected using a 28 gauge x 0.5 inch insulin syringe (Terumo) to form implants in the right and left shoulder flanks of the mice. Approximately 5 minutes later, the mice were administered 100 μl of Granzyme B / Matrigel via bolus intravenous injection (approximately 150 μCi in a total volume of 100 μl saline, plus an additional approximately 25 μl saline to flush the catheter line). 18F radiotracer was administered. After radiotracer injection, the catheter was removed and residual radioactivity was measured. Mice were then returned to their cages for recovery. A nanoScan® PET / CT (Mediso, Hungary) was used for microPET / CT imaging, and a 15-minute static PET scan was performed 75 minutes after injection of the radiotracer. Tera-Tomo™ 3D PET iterative reconstruction was then performed post-acquisition with scatter correction. A short high-resolution computed tomography (CT) scan was also performed immediately after to allow anatomical registration. PET signal in the granzyme B implants was quantified by manually drawing regions of interest (ROIs) on the Matrigel implants based on the fused PET / CT images, and the corresponding activity values ​​were determined with VivoQuant (Invicro, Massachusetts). All values ​​were expressed as % injected dose per gram (%ID / g). Target-to-background ratio (TBR) was then calculated by dividing the %ID / g value of granzyme B by the %ID / g value of pro-granzyme B. Figure 2 shows the chemical structures of active compounds 29-Al and 4-Al (exemplary prodrugs). Figure 3 shows the chemical structures of compounds 29-Al (R = methyl, MeO prodrug) and 30-Al (R = ethyl, EtO prodrug). The pro-compounds showed lower %ID / g and TBR compared to the corresponding active compounds.

[0313] Two exemplary cis / trans pairs of prodrug compounds, compounds 18 F-4-Al(cis-) / compound 18 F-3-Al(trans-) and compounds 18 F-6-Al(cis-) / compound 18 F-7-Al(trans-) was investigated in this example. 18 The cis isomer of F-4-Al showed rapid conversion to the active form and good differentiation in Matrigel. 18 The trans isomer of F-3-Al was slow to convert to the active form and resulted in poor differentiation in Matrigel (Figures 5A-5D).

[0314] Example 6: Metabolism of Exemplary Compounds In vivo conversion of the prodrug was examined in male CD-1 mice [ 18 F] granzyme B tracer was injected and monitored by radiometabolite screening studies performed by subsequent analysis of extracted blood samples by radio-HPLC. Results are reported as the percent of active form present in the blood (Table 11).

[0315] Briefly, male CD-1 mice were administered approximately 250 μCi of [ 18 Mice were anesthetized (n=3 mice / time point) prior to dosing with [F] granzyme B tracer. For blood collection, mice were sacrificed after cardiac exsanguination with a syringe pre-rinsed with sodium heparin 5 and 15 min after injection, and samples were prepared for analysis via wireless HPLC.

[0316] Blood samples were centrifuged at 1500 RCF for 5 minutes to allow separation of plasma. Plasma samples were mixed with 2 volumes of methanol, vortexed for 30 seconds, and then centrifuged at 1500 RCF for 2 minutes. The supernatant was separated from the pellet and diluted 1:4 with PBS buffer pH 7.4 or water before analysis by HPLC.

[0317] The samples were then analyzed on an Agilent 1290 Series UPLC UV coupled with a BGO coincident detector. The HPLC method utilized: 2) Phenomenex Monolithic C18 (100 x 4.6 mm); injection volume of 100-200 μL; flow rate: 1.2 mL / min; solvent for A: 20 mM ammonium acetate in water; solvent for B: 100% methanol; gradient: initial hold at 5% B, hold for 1 min, 5% to 40% in 7 min, hold 40% B for 2 min, increase to 40% to 95% B in 1 min, hold at 95% B for 2 min, then re-equilibrate back to 5% B.

[0318] Table 11 provides the results from this example, reported as the percent active present. [Table 11]

[0319] Other embodiments All features disclosed herein may be combined in any combination. Each feature disclosed herein may be replaced by an alternative feature serving the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is only an example of a generic series of equivalent or similar features.

[0320] Moreover, from the above specification, those skilled in the art can easily ascertain the essential features of the present disclosure, and can make various changes and modifications to the present disclosure to adapt it to various applications and conditions without departing from the spirit and scope thereof. Accordingly, other embodiments are within the scope of the appended claims.

[0321] equivalent While several embodiments of the invention have been described and illustrated herein, those of ordinary skill in the art will readily envision various other means and / or structures for performing the functions and / or obtaining one or more of the results and / or advantages described herein, and each such variation and / or modification is deemed to be within the scope of the embodiments of the invention described herein. More generally, those of ordinary skill in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary, and that the actual parameters, dimensions, materials, and / or configurations will depend on the particular application for which the teachings of the invention are used. Those of ordinary skill in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Thus, the foregoing embodiments are presented by way of example only, and it will be understood that within the scope of the appended claims and equivalents thereof, embodiments of the invention may be practiced other than as specifically described and claimed. The inventive embodiments of the present disclosure relate to 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 inventive scope of the present disclosure, if such features, systems, articles, materials, kits, and / or methods are not mutually inconsistent.

[0322] All definitions as defined and used herein should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and / or ordinary meaning of the defined terms.

[0323] All references, patents, and patent applications disclosed herein are incorporated by reference with respect to the subject matter for which each is cited, which in some cases may include the entirety of this specification.

[0324] The indefinite articles "a" and "an," as used in the specification and claims, unless expressly indicated to the contrary, should be understood to mean "at least one."

[0325] As used in the specification and claims, the phrase "and / or" should be understood to mean "either or both" of the elements so conjoined, i.e., elements that are conjunctively present in some cases and alternatively present in other cases. Multiple elements listed with "and / or" should be construed in the same manner, i.e., "one or more" of the elements so conjoined. Other elements, whether related or unrelated to those specifically identified elements, other than the elements specifically identified by the "and / or" clause may optionally be present. Thus, as a non-limiting example, a reference to "A and / or B," when used in conjunction with open-ended language such as "comprising," may in one embodiment refer to only A (optionally including elements other than B), in another embodiment refer to only B (optionally including elements other than A), in yet another embodiment refer to both A and B (optionally including other elements), and so forth.

[0326] As used herein in the specification and claims, "or" should be understood to have the same meaning as "and / or" as defined above. For example, when separating items in a list, "or" or "and / or" should be interpreted as inclusive, i.e., including at least one of, but including more than one, and optionally including additional unlisted items, of an element or list of elements. Only terms clearly indicated to the contrary, such as "only one of" or "only one of," or "consisting of," when used in the claims, refer to the inclusion of only one element of an element or list of elements. In general, as used herein, the term "or" shall only be interpreted as indicating exclusive alternatives (i.e., "one or the other, but not both") when followed by a term of exclusivity, such as "either," "one of," "only one of," or "only one of." When used in the claims, "consisting essentially of" shall have its ordinary meaning as used in the field of patent law.

[0327] As used herein and in the claims, the phrase "at least one" in reference to a list 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 list of elements, but not necessarily including at least one of each and every element specifically listed in the list of elements, and not excluding any combination of elements in the list of elements. This definition also allows that elements other than the elements specifically identified in the list of elements to which the phrase "at least one" refers may optionally be present, whether related or unrelated to those specifically identified elements. Thus, as a non-limiting example, "at least one of A and B" (or, equivalently, "at least one of A or B" or, equivalently, "at least one of A and / or B") can refer in one embodiment to at least one, optionally including more than one, A, but not to B (and optionally including elements other than B); in another embodiment to at least one, optionally including more than one, B, but not to A (and optionally including elements other than A); in yet another embodiment to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); and so forth.

[0328] It should also be understood that, unless expressly stated to the contrary, in any method claimed herein that includes two or more steps or actions, the order of the method steps or actions is not necessarily limited to the order in which the method steps or actions are recited.

Claims

1. A compound, or a pharmaceutically acceptable salt thereof, wherein said compound is of formula (I): 【Chemistry 1】 During the ceremony, A is a chelating moiety; B is selected from the group consisting of aryl, heteroaryl, cycloalkyl, and heterocyclyl, optionally B is a 6-membered ring; X is -CH ２ C(NH)-, -CH ２ C(O)-, -CH ２ and optionally X is selected from the group consisting of -C(S)-, -NHC(NH)-, -NHC(O)-, -NHC(S)-, -OC(NH)-, -OC(O)-, and -OC(S)-; ２ -C(O)- or -NHC(S)-; Z is -CH ２ -, -CH ２ C(NH)-, -CH ２ C(O)-, -CH ２ and optionally Z is -CH ２ - or -CH ２ C(O)—, L is a peptide linker having 1 to 6 amino acid residues, inclusive; R １ is H or C １－６ alkyl, and optionally R １ is H or methyl, R ２ But C １－６ Alkyl or C ３－６ is cycloalkyl, R ３ But C １－６ alkyl, or a pharmaceutically acceptable salt thereof.

2. The compound is of formula (Ia) 【Chemistry 2】 wherein A, B, X, Z, L, and R ３ 2. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein:

3. the compound is of formula (Ib) 【Transformation 3】 wherein A, B, Z, L, and R ３ 2. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein:

4. 2. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein the chelating moiety A is 1,4,7-triazacyclononane-N,N',N''-triacetic acid (NOTA) or 1,4,7-triazacyclononane-4,7-diyldiacetic acid (NODA).

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

6. 2. The compound according to claim 1, or a pharmaceutically acceptable salt thereof, wherein L has 3 to 6 amino acid residues inclusive.

7. 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 2. The compound of claim 1, or a pharmaceutically acceptable salt thereof, having an amino acid sequence selected from the group consisting of: diacid-βAla-βAla.

8. the compound is of formula (Ic) 【Chemistry 4】 2. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein A, B, Z, and L are each defined as in claim 1.

9. the compound is of formula (Ic-A), 【Transformation 5】 9. The compound of claim 8, or a pharmaceutically acceptable salt thereof, wherein A and L are each defined as in claim 8.

10. the compound is of formula (Ic-Aa), 【Transformation 6】 Optionally, the compound is of formula (Ic-Ab): 【Transformation 7】 10. The compound of claim 9, or a pharmaceutically acceptable salt thereof, wherein A and L are each defined as in claim 9.

11. 10. The compound of claim 9, or a pharmaceutically acceptable salt thereof, wherein the compound is selected from the group consisting of compounds 1 to 17.

12. the compound is of formula (Ic-B), 【Transformation 8】 9. The compound of claim 8, or a pharmaceutically acceptable salt thereof, wherein A and L are each defined as in claim 8.

13. the compound is of formula (Ic-Ba), 【Chemistry 9】 Optionally, the compound is of formula (Ic-Bb): 【Chemistry 10】 13. The compound of claim 12, or a pharmaceutically acceptable salt thereof, wherein A and L are each defined as in claim 12.

14. 13. The compound of claim 12, or a pharmaceutically acceptable salt thereof, wherein A is NODA, and optionally the compound is selected from the group consisting of compounds 18-28.

15. the compound is of formula (Ic-C), 【Chemistry 11】 9. The compound of claim 8, or a pharmaceutically acceptable salt thereof, wherein L is as defined in claim 8.

16. the compound is of formula (Ic-Ca), 【Chemistry 12】 Optionally, the compound is of formula (Ic-Cb): 【Chemistry 13】 16. The compound of claim 15, or a pharmaceutically acceptable salt thereof, wherein L is as defined in claim 15.

17. 16. The compound of claim 15, or a pharmaceutically acceptable salt thereof, wherein A is NODA, and optionally the compound is selected from the group consisting of compounds 29-46.

18. the compound is of formula (Id) 【Chemistry 14】 2. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein A, Z, X, and L are each defined as in claim 1.

19. the compound is of formula (Id-A), 【Chemistry 15】 19. The compound of claim 18, or a pharmaceutically acceptable salt thereof, wherein X and L are as defined in claim 18.

20. the compound is of formula (Id-Aa), 【Chemistry 16】 Optionally, the compound is of formula (Id-Ab): 【Chemistry 17】 20. The compound of claim 19, or a pharmaceutically acceptable salt thereof, wherein X and L are as defined in claim 19.

21. 20. The compound of claim 19, or a pharmaceutically acceptable salt thereof, wherein the compound is selected from the group consisting of compounds 47 to 49.

22. A compound, or a pharmaceutically acceptable salt thereof, wherein the compound is of formula (II): [Chemistry 18] During the ceremony, M is a metal or a metal linked to a radioisotope; A is a chelating moiety that chelates the metal; B is selected from the group consisting of aryl, heteroaryl, cycloalkyl, and heterocyclyl, optionally B is a 6-membered ring; X is -CH ２ C(NH)-, -CH ２ C(O)-, -CH ２ and optionally X is selected from the group consisting of -C(S)-, -NHC(NH)-, -NHC(O)-, -NHC(S)-, -OC(NH)-, -OC(O)-, and -OC(S)-; ２ -C(O)- or -NHC(S)-; Z is -CH ２ -, -CH ２ C(NH)-, -CH ２ C(O)-, -CH ２ and optionally Z is -CH ２ - or -CH ２ C(O)—, L is a peptide linker having 1 to 6 amino acid residues, inclusive; R １ is H or C １－６ alkyl, and optionally R １ is H or methyl, R ２ But C １－６ Alkyl or C ３－６ is cycloalkyl, R ３ But C １－６ alkyl, or a pharmaceutically acceptable salt thereof.

23. The compound is of formula (IIa) 【Chemistry 19】 wherein M, A, B, X, Z, L, and R ３ 23. The compound of claim 22, or a pharmaceutically acceptable salt thereof, wherein:

24. the compound is of formula (IIb) 【Chemistry 20】 wherein M, A, B, Z, L, and R ３ 23. The compound of claim 22, or a pharmaceutically acceptable salt thereof, wherein:

25. 23. The compound of claim 22, or a pharmaceutically acceptable salt thereof, wherein the chelating moiety A is 1,4,7-triazacyclononane-N,N',N''-triacetic acid (NOTA) or 1,4,7-triazacyclononane-4,7-diyldiacetic acid (NODA).

26. 23. The compound of claim 22, or a pharmaceutically acceptable salt thereof, wherein L has 1 to 3 amino acid residues inclusive.

27. 23. The compound of claim 22, or a pharmaceutically acceptable salt thereof, wherein L has 3 to 6 amino acid residues inclusive.

28. 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 23. The compound of claim 22, or a pharmaceutically acceptable salt thereof, having an amino acid sequence selected from the group consisting of: diacid-βAla-βAla.

29. The metal linked to a radioisotope is Al, and optionally the radioisotope is １８ 23. The compound of claim 22, wherein R is H, or a pharmaceutically acceptable salt thereof.

30. the compound is of formula (IIc) 【Chemistry 21】 23. The compound of claim 22, or a pharmaceutically acceptable salt thereof, wherein M, A, B, Z, and L are each as defined in claim 22.

31. the compound is of formula (IIc-A), 【Chemistry 22】 31. The compound of claim 30, or a pharmaceutically acceptable salt thereof, wherein M, A, and L are each defined as in claim 30.

32. the compound is of formula (IIc-Aa), 【Chemistry 23】 Optionally, the compound is of formula (IIc-Ab): 【Chemistry 24】 32. The compound of claim 31, or a pharmaceutically acceptable salt thereof, wherein M, A, and L are each as defined in claim 31.

33. 32. The compound of claim 31, or a pharmaceutically acceptable salt thereof, wherein A is NODA.

34. 34. The compound of claim 33, or a pharmaceutically acceptable salt thereof, wherein the compound is selected from the group consisting of compounds 1-Al to 17-Al.

35. the compound is of formula (IIc-B), 【Chemistry 25】 31. The compound of claim 30, or a pharmaceutically acceptable salt thereof, wherein M, A, and L are each defined as in claim 30.

36. the compound is of formula (IIc-Ba), 【Chemistry 26】 Optionally, the compound is of formula (Ic-Bb): 【Chemistry 27】 36. The compound of claim 35, or a pharmaceutically acceptable salt thereof, wherein M, A, and L are each defined as in claim 35.

37. the compound is of formula (IIc-C), 【Chemistry 28】 23. The compound of claim 22, or a pharmaceutically acceptable salt thereof, wherein A and L are as defined in claim 22.

38. the compound is of formula (IIc-Ca), 【Chemistry 29】 Optionally, the compound is of formula (IIc-Cb): 【Transformation 30】 38. The compound of claim 37, or a pharmaceutically acceptable salt thereof, wherein A and L are each defined as in claim 37.

39. 39. The compound of claim 38, or a pharmaceutically acceptable salt thereof, wherein A is NODA, and optionally the compound is selected from the group consisting of compounds 18-28.

40. The metal is Ga, which optionally comprises: ６８ 23. The compound of claim 22, or a pharmaceutically acceptable salt thereof, wherein:

41. the compound is of formula (IId) 【Chemistry 31】 23. The compound of claim 22, or a pharmaceutically acceptable salt thereof, wherein M, A, Z, X, and L are each defined as in claim 22.

42. the compound is of formula (IId-A), 【Chemistry 32】 42. The compound of claim 41, or a pharmaceutically acceptable salt thereof, wherein M, X, and L are each defined as in claim 41.

43. the compound is of formula (IId-Aa), 【Transformation 33】 Optionally, the compound is of formula (IId-Ab): 【Transformation 34】 43. The compound of claim 42, or a pharmaceutically acceptable salt thereof, wherein M, X, and L are each defined as in claim 42.

44. 43. The compound of claim 42, or a pharmaceutically acceptable salt thereof, wherein the compound is selected from the group consisting of compounds 47 to 49.

45. A pharmaceutical composition comprising the compound of any one of claims 1 to 44, its stereoisomer, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

46. ​​A pharmaceutical composition for use in a method for treating an immune dysregulation in a subject, comprising an effective amount of a compound described in any one of claims 22 to 44, said method comprising administering to a subject in need thereof said pharmaceutical composition and, optionally, one or more additional therapeutic agents for treating said immune dysregulation.

47. 47. The pharmaceutical composition of claim 46, wherein the one or more additional therapeutic agents are selected from the group consisting of anti-inflammatory agents, steroids, immunotherapeutic agents, chemotherapeutic agents, and therapeutic antibodies.

48. 48. The pharmaceutical composition of claim 47, wherein the immune dysregulation is selected from the group consisting of an autoimmune disorder, an inflammatory disorder, a skin disorder, cancer, and a cardiovascular disorder.

49. 49. The pharmaceutical composition of claim 48, wherein the immune dysregulation is cancer.

50. 1. A method for imaging granzyme B in a tissue, said method comprising: (i) contacting a compound according to any one of claims 22 to 44 or a pharmaceutically acceptable salt thereof with tissue suspected of containing granzyme B; (ii) imaging the tissue based on a radioisotope signal emitted from the compound or the pharmaceutically acceptable salt thereof; (iii) optionally, the radioisotope contained in the compound or the pharmaceutically acceptable salt thereof is １８ F or ６８ The method is Ga.

51. 51. The method of claim 50, wherein said contacting step is carried out by administering said compound or said pharmaceutically acceptable salt thereof to a subject in need thereof.

52. 52. The method of claim 51, wherein the subject is undergoing treatment for an immune dysregulation.

53. 53. The method of claim 52, wherein the immune dysregulation is selected from the group consisting of an autoimmune disorder, an inflammatory disorder, a skin disorder, cancer, and a cardiovascular disorder, and optionally, the immune dysregulation is cancer.

54. 54. The method of claim 53, wherein the treatment comprises one or more additional therapeutic agents selected from the group consisting of anti-inflammatory agents, steroids, immunotherapeutic agents, chemotherapeutic agents, and therapeutic antibodies.

55. 51. The method of claim 50, further comprising monitoring an immune response in the subject based on imaging of the granzyme B.

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