Granzyme b pet imaging agents

Abstract

The invention is directed to compounds of Formula (I) and their pharmaceutically acceptable salts, which may be suitable for imaging Granzyme B activity and hence are useful in binding and imaging Granzyme B in patients with cancer or inflammatory diseases.

Description

25927 GRANZYME B PET IMAGING AGENTS CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Patent Application Serial No. 63 / 637,424 filed April 23, 2024, the entire contents of which are incorporated by reference herein. BACKGROUND

[0002] Granzyme B (GzmB) is a serine protease secreted by cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells. GzmB-induced cell death has been traditionally viewed as a primary mechanism used by CTLs and NK cells to eliminate harmful target cells including allogeneic, virally infected, and tumor cells. See WO2020 / 167989, Willoughby, C. A. et al., Bioorganic & Med. Chem Ltrs 2002, 12(16), 2197-200; US2019 / 0224348; Larimer, B.; et al. Cancer Res 77, 2017, 2318-2327 and Larimer, B.; et al. U. Clin Cancer Res 25, 2019, 1196-1205. Measurement of GzmB can serve as a readout for the functional status of CTLs and NKs and is of interest in cancer immunotherapy and autoimmunity. Presently disclosed are compounds that bind human granzyme B with high affinity and may be labelled with [18F]fluoride for use in granzyme B positron emission tomography. SUMMARY OF THE DISCLOSURE

[0003] Provided are novel pyrrolidine derivatives, their salts, pharmaceutical compositions comprising them, diagnostic and therapeutic uses and processes for making such compounds, which may be suitable for imaging granzyme B. Further provided are compounds useful as a radiotracer for positron emission tomography (PET) imaging. This disclosure further relates to use of the compounds as imaging agents of granzyme B.

[0004] The summary of the disclosure described above is non-limiting and other features and advantages of the technology will be apparent from the following detailed description, and from the claims. BRIEF DESCRIPTION OF THE FIGURES

[0005] Figure 1:Representative PET / CT images of mice injected with18F-example Y in hPMC and NOG control mice. - 1 -25927 DETAILED DESCRIPTION OF THE DISCLOSURE

[0006] Described are aminoproline derivatives of Formula I or a pharmaceutically acceptable salt thereof, which are capable of binding granzyme B. In some embodiments, the compound of Formula I, or a pharmaceutically acceptable salt thereof is an irreversible binder of granzyme B. In some embodiments, the compound of Formula I, or a pharmaceutically acceptable salt thereof is an inhibitor of granzyme B. Still, in some embodiments, the compounds of Formula I, or a pharmaceutically acceptable salt thereof, contain one or more imaging agents. In some embodiments, the compound of Formula I, or a pharmaceutically acceptable salt thereof is a compound that binds granzyme B.

[0007] Provided are compounds of Formula I:and pharmaceutically acceptable salts thereof, wherein, R1is C1-6 alkyl; R2is -NHC(O)R4, or (CH2)nheteroaryl, wherein said heteroaryl is optionally substituted with 1 to 3 Rxsubstituents; R3is hydrogen, or =O; R4is selected from C1-6alkyl, -C1-3haloalkyl, and –(CH2NHC(O))1-2(CH2)qNH-R4b; R4bis selected from:p p achment; - 2 -25927 R7and R8are independently selected from hydrogen and C1-6alkyl, wherein said alkyl is optionally substituted with 1 to 3 groups of Rx, or together R7and R8form =O, provided that when R7and R8form =O, R3is hydrogen; R5is selected from –O(CH2)naryl, –O(CH2)nheteroaryl, heteroaryl, -NHC(O)(CH2)nC3- 6cycloalkyl, -NHC(O)(CH2)naryl, -NHC(O)(CH2)nheteroaryl, -NHC(O)(CH2)nheterocyclyl, - OC(O)NH(CH2)naryl, -OC(O)(CH2)naryl, -NHCHO, -NHC(O)C1-6alkyl, and NH2, wherein said alkyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl are optionally substituted with 1 to 3 Rxsubstituents; R6is -C(O)NHCH(CH2COOH)CH=O, or -C(O)NHCH2heteroaryl, wherein said heteroaryl is optionally substituted with 1 to 3 Rxsubstituents; Rxis selected from C1-6 alkyl, halogen, OH, OC1-6 alkyl, -CO2H, -C1-3 haloalkyl, -(CH2)naryl, - (CH2)nheteroaryl, and (CH2)nheterocyclyl, wherein said alkyl, aryl, heteroaryl, and heterocyclyl are optionally substituted with 1 to 3 Ry; Ryis selected from C1-6alkyl, OC1-6alkyl, halogen, CN, and -C1-3haloalkyl; q is 1, 2, or 3; and n is 0, 1, 2, or 3.

[0008] An embodiment of Formula I is realized when n is 0. Another embodiment of Formula I is realized when n is 1. Another embodiment of Formula I is realized when n is 2. Another embodiment of Formula I is realized when n is 3.

[0009] An embodiment of the disclosure is realized when R1is -CH(CH3)2, or - C(CH3)CH2CH3. Another embodiment of the disclosure is realized when R1is -CH(CH3)2. Another embodiment of the disclosure is realized when R1is -C(CH3)CH2CH3.

[0010] An embodiment of the disclosure is realized when R2is -NHC(O)R4. A subembodiment of this aspect of the disclosure is realized when R4is C1-6 alkyl. Another subembodiment of this aspect of the disclosure is realized when R4is C1-6alkyl selected from -(CH2)nCH3, -CH(CH3)2,and -C(CH3)CH2CH3. Another subembodiment of this aspect of the disclosure is realized when R4is -C1-3haloalkyl. Another subembodiment of this aspect of the disclosure is realized when R4is -C1-3 haloalkyl selected from CF3, -CH2F, or -(CH2)2CH2F.

[0011] An embodiment of the disclosure is realized when R2is (CH2)nheteroaryl selected from pyridyl, pyrimidinyl, pyrazolyl, pyrrolyl, imidazolyl, triazolyl and tetrazolyl, said pyridyl, pyrimidinyl, pyrazolyl, pyrrolyl, imidazolyl, triazolyl and tetrazolyl, optionally substituted with 1 to 3 Rxsubstituents. Another embodiment of this disclosure is realized when R2is an optionally substituted pyridyl. - 3 -25927

[0012] An embodiment of the disclosure is realized when R3is hydrogen and together R7and R8form =O. An embodiment of the disclosure is realized when R3is =O.

[0013] An embodiment of the disclosure is realized when R4is C1-6alkyl. Another embodiment of the disclosure is realized when R4is -C1-3 haloalkyl. Another embodiment of the disclosure is realized when R4is –(CH2NHC(O))1-2(CH2)qNH-R4b, wherein 1 of –(CH2NHC(O))- is present. Another embodiment of the disclosure is realized when R4is –(CH2NHC(O))1-2(CH2)qNH-R4b, wherein 2 of –(CH2NHC(O))- is present.

[0014] Another embodiment of the disclosure is realized when R8is C1-6alkyl, said alkyl optionally substituted with 1 to 3 Rxsubstituents. A subembodiment of this aspect of the disclosure is realized when R8is -CH2or CH(CH2)2CO2H.

[0015] Another embodiment of the disclosure is realized when R7is H, R8is -CH2 or CH(CH2)2CO2H and R3is =O.

[0016] Another embodiment of the disclosure is realized when R5is –O(CH2)naryl, said aryl optionally substituted with 1 to 3 Rxsubstituents. Another embodiment of the disclosure is realized when R5is –O(CH2)nheteroaryl, said heteroaryl optionally substituted with 1 to 3 Rxsubstituents. Another embodiment of the disclosure is realized when R5is heteroaryl, said heteroaryl optionally substituted with 1 to 3 Rxsubstituents. Another embodiment of the disclosure is realized when R5is-NHC(O)(CH2)nC3-6cycloalkyl, said cycloalkyl optionally substituted with 1 to 3 Rxsubstituents. Another embodiment of the disclosure is realized when R5is -NHC(O)(CH2)naryl, wherein said aryl is optionally substituted with 1 to 3 Rxsubstituents. Another embodiment of the disclosure is realized when R5is -NHC(O)(CH2)nheteroaryl, said heteroaryl optionally substituted with 1 to 3 Rxsubstituents. Another embodiment of the disclosure is realized when R5is -NHC(O)(CH2)nheterocyclyl, said heterocyclyl optionally substituted with 1 to 3 Rxsubstituents. Another embodiment of the disclosure is realized when R5is -OC(O)(CH2)naryl, said aryl optionally substituted with 1 to 3 Rxsubstituents. Another embodiment of the disclosure is realized when R5is -OC(O)NH(CH2)naryl, said aryl optionally substituted with 1 to 3 Rxsubstituents. Another embodiment of the disclosure is realized when R5is -NHCHO. Another embodiment of the disclosure is realized when R5is -NHC(O)C1-6 alkyl, said alkyl optionally substituted with 1 to 3 Rxsubstituents. Another embodiment of the disclosure is realized when R5is NH2. Another embodiment of the disclosure is realized when the R5aryl is selected from optionally substituted phenyl and naphthyl. Another embodiment of the disclosure is realized when the R5heteroaryl is selected from optionally substituted indolyl, thiophene, thienyl, benzofuranyl, tetrahydrofuranyl, furanyl, pyridyl and triazolyl. Another - 4 -25927 embodiment of the disclosure is realized when the R5cycloalkyl is selected from optionally substituted cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl.

[0017] Another embodiment of this disclosure is realized when R6is - C(O)NHCH(CH2COOH)CH=O. Another embodiment of this disclosure is realized when R6is - C(O)NHCH2heteroaryl, said heteroaryl optionally substituted with 1 to 3 Rxsubstituents. Another embodiment of this disclosure is realized when the heteroaryl in C(O)NHCH2heteroaryl is selected from optionally substituted triazolyl, tetrazolyl, imidazolyl, and pyrazolyl. Another embodiment of this disclosure is realized when R6is -C(O)NHCH2triazolyl. Another embodiment of this disclosure is realized when R6is -C(O)NHCH2tetrazolyl. Another embodiment of this disclosure is realized when R6is -C(O)NHCH2imidazolyl. Another embodiment of this disclosure is realized when R6is -C(O)NHCH2pyrazolyl.

[0018] Another embodiment of this disclosure is realized when Rxis selected from C1-6alkyl, halogen, OH, OC1-6 alkyl, - CF3, -(CH2)nphenyl, - (CH2)noxadiazolyl, (CH2)nquinolinyl, (CH2)nthienyl, (CH2)npyrimidinyl, (CH2)npyridyl, (CH2)npyrrolyl, (CH2)nisoxazolyl, (CH2)nbenzodioxolyl, (CH2)noxoimidazolidinyl, and (CH2)nbenzoxazolyl, wherein said alkyl, phenyl, oxadiazolyl, quinolinyl, thienyl, pyrimidinyl, pyridyl, pyrrolyl isoxazolyl, benzodioxolyl, (CH2)noxoimidazolidinyl, and benzoxazolyl are optionally substituted with 1 to 3 Ry.

[0019] An embodiment of the disclosure is realized when Ryis selected from methyl, ethyl, propyl, butyl, pentyl, hexyl, OH, OCH3, OCH2CH3, CF3, halogen, and CN.

[0020] An embodiment of the disclosure is realized when n is 0. An embodiment of the disclosure is realized when n is 1. An embodiment of the disclosure is realized when n is 2. An embodiment of the disclosure is realized when n is 3.

[0021] An embodiment of the disclosure is realized by the compounds structural Formula II: and pharmaceutically accep, , , , R5, R7and R8are as described herein. An embodiment of the compounds of Formula II is realized when R7is H, R8is -CH2, or - CH(CH2)2CO2H; and R3is =O. Another embodiment of the compounds of Formula II is realized when R7is H; R8is CH2; and R3is =O. Another embodiment of the compounds of Formula II is - 5 -25927 realized when R7is H; R8is - CH(CH2)2CO2H; and R3is =O. Another embodiment of the compounds of Formula II is realized when R7is H; R8is -CH2, or -CH(CH2)2CO2H; R3is =O, and R1is -CH(CH3)2, or -C(CH3)CH2CH3. Another embodiment of the compounds of Formula II is realized when R7is H; R8is -CH2, or -CH(CH2)2CO2H; R3is =O; and R2is -NHC(O)R4.

[0022] A subembodiment of this aspect of the disclosure is realized when R4is selected from - (CH2)nCH3, CH(CH3)2, -C(CH3)CH2CH3, CF3, -CH2F, and -(CH2)2CH2F. Another embodiment of the compounds of Formula II is realized when R7is H; R8is -CH2, or -CH(CH2)2CO2H; R3is =O; and R2is (CH2)nheteroaryl wherein the heteroaryl moiety is selected from pyridyl, pyrimidinyl, pyrazolyl, pyrrolyl, imidazolyl, triazolyl and tetrazolyl, and wherein said pyridyl, pyrimidinyl, pyrazolyl, pyrrolyl, imidazolyl, triazolyl and tetrazolyl, are optionally substituted with 1 to 3 Rxsubstituents.

[0023] Another embodiment of the compounds of Formula II is realized when R8is -CH2, or - CH(CH2)2CO2H; R3is =O; and R1is -CH(CH3)2, or -C(CH3)CH2CH3; R2is selected from - NHC(O)(CH2)nCH3, -NHC(O)CH(CH3)2, -NHC(O)C(CH3)CH2CH3, -NHC(O)CF3, - NHC(O)CH2F, -NHC(O)(CH2)2CH2F, and heteroaryl. Another embodiment of the compounds of Formula II is realized when R5is selected from –O(CH2)naryl, -NHC(O)(CH2)nC3-6cycloalkyl, - NHC(O)(CH2)naryl, -NHC(O)(CH2)nheteroaryl, -NHCHO, and -NHC(O)C1-6 alkyl, wherein said alkyl, cycloalkyl, aryl, and heteroaryl, are optionally substituted with 1 to 3 Rxsubstituents.

[0024] Another embodiment of the compounds of Formula II is realized when R5is selected from –O(CH2)naryl, -NHC(O)(CH2)naryl, -NHC(O)(CH2)nheteroaryl, - NHC(O)(CH2)nheterocyclyl, -NHCHO, and -NHC(O)C1-6 alkyl, wherein said alkyl, aryl, heteroaryl and heterocyclyl moieties are optionally substituted with 1 to 3 Rxsubstituents. Another embodiment of the compounds of Formula II is realized when the R5aryl moiety is selected from optionally substituted phenyl and naphthyl; the R5heteroaryl moiety is selected from optionally substituted indolyl, thiophene, thienyl, benzofuranyl, furanyl, pyridyl and triazolyl; the R5heteroaryl moiety is tetrahydrofuranyl; and the R5cycloalkyl moiety is selected from optionally substituted cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl.

[0025] Another embodiment of the compounds of Formula II is realized when R4is C1-6 alkyl. Another embodiment of the compounds of Formula II is realized when R4is -C1-3haloalkyl. Another embodiment of the compounds of Formula II is realized when R4is –(CH2NHC(O))1-2(CH2)qNH-R4b, wherein 1 of –(CH2NHC(O))- is present. Another embodiment of the compounds of Formula II is realized when R4is –(CH2NHC(O))1-2(CH2)qNH-R4b, wherein 2 of – (CH2NHC(O))- is present.

[0026] An embodiment of the disclosure is realized by compounds of structural Formula III: - 6 -25927and pharmaceutically acceptable salts thereof, wherein R , R , R3R5, R7and R8are as described herein and R6ais heteroaryl, said heteroaryl optionally substituted by 1 to 3 Rxsubstituents. An embodiment of the compounds of Formula III is realized when R6ais selected from optionally substituted triazolyl, tetrazolyl, imidazolyl, and pyrazolyl.

[0027] An embodiment of the compounds of Formula III is realized when R7is H, R8is -CH2, or -CH(CH2)2CO2H; R3is =O; and R6ais selected from optionally substituted triazolyl, tetrazolyl, imidazolyl, and pyrazolyl. Another embodiment of the compounds of Formula III is realized when R7is H; R8is CH2; R3is =O; and R6ais selected from optionally substituted triazolyl, tetrazolyl, imidazolyl, and pyrazolyl. Another embodiment of the compounds of Formula III is realized when R7is H; R8is - CH(CH2)2CO2H; R3is =O; and R6ais selected from optionally substituted triazolyl, tetrazolyl, imidazolyl, and pyrazolyl. Another embodiment of the compounds of Formula III is realized when R7is H; R8is -CH2, or -CH(CH2)2CO2H; R3is =O; R1is -CH(CH3)2, or -C(CH3)CH2CH3; and R6ais selected from optionally substituted triazolyl, tetrazolyl, imidazolyl, and pyrazolyl. Another embodiment of the compounds of Formula III is realized when R7is H,; R8is -CH2, or -CH(CH2)2CO2H; R3is =O; and R2is -NHC(O)R4; and R6ais selected from optionally substituted triazolyl, tetrazolyl, imidazolyl, and pyrazolyl.

[0028] A subembodiment of Formula III is realized when R4is selected from -(CH2)nCH3, CH(CH3)2, -C(CH3)CH2CH3, CF3, -CH2F, and -(CH2)2CH2F.

[0029] Another embodiment of the compounds of Formula III is realized when R7is H; R8is - CH2, or -CH(CH2)2CO2H; R3is =O; and R2is (CH2)nheteroaryl, wherein the heteroaryl moiety is selected from pyridyl, pyrimidinyl, pyrazolyl, pyrrolyl, imidazolyl, triazolyl and tetrazolyl, and wherein said pyridyl, pyrimidinyl, pyrazolyl, pyrrolyl, imidazolyl, triazolyl and tetrazolyl, are optionally substituted with 1 to 3 Rxsubstituents; and R6ais selected from optionally substituted triazolyl, tetrazolyl, imidazolyl, and pyrazolyl.

[0030] Another embodiment of the compounds of Formula III is realized when R7is H; R8is - CH2, or -CH(CH2)2CO2H; R3is =O; and R1is -CH(CH3)2, or -C(CH3)CH2CH3; R2is selected from -NHC(O)(CH2)nCH3, -NHC(O)CH(CH3)2, -NHC(O)C(CH3)CH2CH3, -NHC(O)CF3, - - 7 -25927 NHC(O)CH2F, -NHC(O) (CH2)2CH2F, and heteroaryl; and R6ais selected from optionally substituted triazolyl, tetrazolyl, imidazolyl, and pyrazolyl. An aspect of Formula III is realized when n is 0. Another aspect of Formula III is realized when n is 1. Another aspect of Formula III is realized when n is 2. Another aspect of Formula III is realized when n is 3.

[0031] Another embodiment of the compounds of Formula III is realized when R5is selected from –O(CH2)naryl, -NHC(O)(CH2)nC3-6cycloalkyl, -NHC(O)(CH2)naryl, - NHC(O)(CH2)nheteroaryl, -NHC(O)(CH2)nheterocyclyl, -NHCHO, and -NHC(O)C1-6 alkyl, wherein said alkyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl are optionally substituted with 1 to 3 Rxsubstituents. Another embodiment of the compounds of Formula III is realized when R5is selected from –O(CH2)naryl, -NHC(O)(CH2)naryl, -NHC(O)(CH2)nheteroaryl, -NHCHO, and - NHC(O)C1-6 alkyl, wherein said alkyl, aryl, and heteroaryl are optionally substituted with 1 to 3 Rxsubstituents. Another embodiment of the compounds of Formula III is realized when the R5aryl moiety is selected from optionally substituted phenyl and naphthyl; the R5heteroaryl moiety is selected from optionally substituted indolyl, thiophene, thienyl, benzofuranyl, furanyl, pyridyl and triazolyl; the R5heterocyclyl moiety is tetrahydrofuranyl; and the R5cycloalkyl moiety is selected from optionally substituted cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl.

[0032] Another embodiment of the compounds of Formula III is realized when R4is C1-6 alkyl. Another embodiment of the compounds of Formula III is realized when R4is -C1-3haloalkyl. Another embodiment of the compounds of Formula III is realized when R4is –(CH2NHC(O))1-2(CH2)qNH-R4b, wherein 1 of –(CH2NHC(O))- is present. Another embodiment of the compounds of Formula III is realized when R4is –(CH2NHC(O))1-2(CH2)qNH-R4b, wherein 2 of –(CH2NHC(O))- is present.

[0033] An embodiment of the disclosure is realized by compounds of structural Formula IV:and pharmaceutically acceptable salts thereof, wherein R1, R2, R5and R6are as described herein.

[0034] An embodiment of the compounds of Formula II is realized R1is -CH(CH3)2or - C(CH3)CH2CH3. Another embodiment of the compounds of Formula IV is realized when R2is - - 8 -25927 NHC(O)R4. A subembodiment of this aspect of the disclosure is realized when R4is selected from -(CH2)nCH3, CH(CH3)2, -C(CH3)CH2CH3, CF3, -CH2F, and -(CH2)2CH2F.

[0035] Another embodiment of the compounds of Formula IV is realized when R2is (CH2)nheteroaryl wherein the heteroaryl moiety is selected from pyridyl, pyrimidinyl, pyrazolyl, pyrrolyl, imidazolyl, triazolyl and tetrazolyl, and wherein said pyridyl, pyrimidinyl, pyrazolyl, pyrrolyl, imidazolyl, triazolyl and tetrazolyl are optionally substituted with 1 to 3 Rxsubstituents.

[0036] Another embodiment of the compounds of Formula IV is realized when R1is - CH(CH3)2, or -C(CH3)CH2CH3; and R2is selected from -NHC(O)(CH2)nCH3, - NHC(O)CH(CH3)2, -NHC(O)C(CH3)CH2CH3, -NHC(O)CF3, -NHC(O)CH2F, -NHC(O) (CH2)2CH2F, and heteroaryl.

[0037] Another embodiment of the compounds of Formula IV is realized when R6is C(O)NHCH(CH2COOH)CH=O. Another embodiment of the compounds of Formula IV is realized when R6is -C(O)NHCH2heteroaryl, wherein said heteroaryl is optionally substituted with 1 to 3 Rxsubstituents. Another embodiment of this disclosure is realized when the heteroaryl in -C(O)NHCH2heteroaryl is selected from optionally substituted triazolyl, tetrazolyl, imidazolyl, and pyrazolyl.

[0038] Another embodiment of the compounds of Formula IV is realized when R5is selected from –O(CH2)naryl, -NHC(O)(CH2)nC3-6cycloalkyl, -NHC(O)(CH2)naryl, - NHC(O)(CH2)nheteroaryl, -NHCHO, and -NHC(O)C1-6 alkyl, wherein said alkyl, cycloalkyl, aryl, and heteroaryl are optionally substituted with 1 to 3 Rxsubstituents. Another embodiment of the compounds of Formula IV is realized when R5is selected from –O(CH2)naryl, - NHC(O)(CH2)naryl, -NHC(O)(CH2)nheteroaryl, -NHCHO, and -NHC(O)C1-6 alkyl, wherein said alkyl, aryl, and heteroaryl are optionally substituted with 1 to 3 Rxsubstituents. Another embodiment of the compounds of Formula IV is realized when the R5aryl is selected from optionally substituted phenyl and naphthyl; the R5heteroaryl is selected from optionally substituted indolyl, thiophene, thienyl, benzofuranyl, tetrahydrofuranyl, furanyl, pyridyl and triazolyl; and the R5cycloalkyl is selected from optionally substituted cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl.

[0039] Another embodiment of the compounds of Formula IV is realized when R4is C1-6alkyl. Another embodiment of the compounds of Formula IV is realized when R4is -C1-3 haloalkyl. Another embodiment of the compounds of Formula IV is realized when R4is –(CH2NHC(O)) (CH2)qNH-R4b. Another embodiment of the compounds of Formula IV is realized when R4is – (CH2NHC(O))2(CH2)qNH-R4b. - 9 -25927

[0040] Non-limiting examples of the compounds, or pharmaceutically acceptable salts thereof of the disclosure are found in Table 1 below. Table 1 COMPOUND Chemical Name MS [M+H]- 10 -25927 COMPOUND Chemical Name MS [M+H]- 11 -25927 COMPOUND Chemical Name MS [M+H]- 12 -25927 COMPOUND Chemical Name MS [M+H]- 13 -25927 COMPOUND Chemical Name MS [M+H]- 14 -25927 COMPOUND Chemical Name MS [M+H]- 15 -25927 COMPOUND Chemical Name MS [M+H]- 16 -25927 COMPOUND Chemical Name MS [M+H]- 17 -25927 COMPOUND Chemical Name MS [M+H]- 18 -25927 COMPOUND Chemical Name MS [M+H]- 19 -25927 COMPOUND Chemical Name MS [M+H]- 20 -25927 COMPOUND Chemical Name MS [M+H]- 21 -25927 COMPOUND Chemical Name MS [M+H]- 22 -25927 COMPOUND Chemical Name MS [M+H]- 23 -25927 COMPOUND Chemical Name MS [M+H] H] 5- 24 -25927 COMPOUND Chemical Name MS [M+H] H] 5selected from the group consisting of a paramagnetic ion, an x-ray imaging agent, a fluorophore, and a radioisotope. Many appropriate imaging agents are known in the art, as are methods for their attachment to antibodies (see, e.g., U.S. Pat. Nos.5,021,236; 4,938,948; and 4,472,509, the disclosure of each of which is incorporated herein by reference in its entirety). Radioactive labeled compounds of Formula I, II, III and IV or a pharmaceutically acceptable salt thereof provided herein may be prepared according to well-known methods in the art. In a further example, compounds of Formula I, II, III and IV or a pharmaceutically acceptable salt thereof, provided herein may be labeled with18F-fluoride by nucleophilic substitution, or18F-labelled prosthetic groups (Wester at al, Nuclear Med Biol 1996, 365-372), or by radiometalation of a bifunctional chelator (e.g., 1, 4, 7 - triazacyclononane - N, N ', N "- triacetic acid (NOTA), 1, 4, 7, 10-tetraacetic acid (DOTA), or 1,4,7-triazacyclononane,1-glutaric acid-4,7-acetic acid (NODAGA), or a similar derivative thereof. Synthetic methods for incorporating radioisotopes into organic compounds are well known in the art, and one of ordinary skill in the art will readily recognize other methods applicable for the compounds provided herein.

[0042] In another embodiment, the imaging agent comprises 1, 2, or 3 imaging agents selected from the group consisting of a paramagnetic ion, an x-ray imaging agent, a fluorophore, and a radioisotope. In another embodiment, Formula I, II, III and IV comprise one imaging agent. In another embodiment Formula I, II, III and IV comprise two imaging agents. In another embodiment, Formula I, II, III and IV comprise comprises three imaging agents. In another embodiment the compound of Formula I, II, III and IV comprise comprises one or more imaging agents which can include one or more independently selected paramagnetic ions.

[0043] A subembodiment of this aspect of the disclosure is realized when each of the paramagnetic ions are independently selected from the group consisting of fluoride, chromium (III), manganese (II), iron (III), iron (II), cobalt (II), nickel (II), copper (II), neodymium (III), samarium (III), ytterbium (III), gado-linium (III), vanadium (II), terbium (III), dysprosium (III), - 25 -25927 holmium (III), and erbium (III). Another subembodiment of this aspect of the disclosure is realized when the compound of Formula I, II, III and IV comprise 1, 2, or 3 independently selected paramagnetic ions. Another subembodiment of this aspect of the disclosure is realized when the one or more independently paramagnetic ions are independently directly or indirectly (e.g., through a chelator) bound to the compounds provided herein.

[0044] Another embodiment of this aspect of the disclosure is realized when the compound of Formula I, II, III and IV comprises one or more imaging agents which are independently selected x-ray imaging agents. A subembodiment of this aspect of the disclosure is realized when each of the x-ray imaging agents are independently selected from the group consisting of lanthanum (III), gold (III), lead (II), bismuth(III), and iodinated x-ray imaging agents (e.g., diatrizoate, ioxaglate, metrizoate, iopamidol, iohexol, ioxilan, iopromide, iodixanol, and ioversol).

[0045] Another embodiment of this disclosure is realized when the imaging agent is a fluorophore. A subembodiment of this aspect of the disclosure is realized when the is fluorophore selected from the group consisting of Alexa 350, Alexa 430, AMCA, BODIPY 630 / 650, BODIPY 650 / 665, BODIPY-FL, BODPY-R6G, 13BODLPY-TMR, BODLPY-TRX, cascade blue, Cy3, Cy5, 6-FAM, fluorescein isothiocyanate, HEX, 6-JOE, Oregon green 488, Oregon green 500, Oregon green 514, a quantum dot, pacific blue, REG, rhodamine green, rhod- amine red, renographin, ROX, TAMRA, TET, tetramethyl- rhodamine, Texas Red, AF 350, 405, AF532, AF488, AF647, AF680, AF750, Cy5, Cy5.5, Cy7, indocyanine green (ICG), green fluorescent protein (GFP), red fluorescent protein (RFP), dsRED, and IRdye 800.

[0046] Another embodiment of this aspect of the disclosure is realized when the compound of Formula I, II, III and IV comprise one or more imaging agents which include one or more independently selected radioisotopes. A subembodiment of this aspect of the disclosure is realized when the radioisotopes provided herein are useful as imaging agents in one or more of the methods provided herein. A subembodiment of this aspect of the disclosure is realized when one or more of the radioistopes provided herein may also be useful in one or more therapeutic applications, (e.g., when administered to a subject in a therapeutically effective amount).131I and64Cu, for example, may be useful as imaging agents (e.g., as non-toxic and / or non-therapeutic radioisotopes) when administered to the subject at low concentrations (e.g., 5 mCi) and may also be useful as therapeutic agents (i.e., as toxic radioisotopes and / or therapeutic radioisotopes) when administered to the subject at a higher concentration. A subembodiment of this aspect of the disclosure is realized when each of the radioisotopes are independently selected from the group consisting of3H,11C,14C,18F,32P,35S,36C1,51Cr52Fe,57Co,58Co,59Fe,64Cu,67Cu,67Ga,68Ga,75Se,76Br,77Br,89Zr,90Y,99mTc111In123I124I125I131I152Eu153Sm166Ho,177Lu,186Re,188Re,201Tl, - 26 -25927203Pb,212Pb,210At,211At,212Bi,213Bi, and225Ac. A subembodiment of this aspect of the disclosure is realized when one or more independently radioisotopes are independently directly or indirectly (e.g., through a chelator) bound to the compounds of Formula Formula I, II, III and IV.

[0047] Another embodiment of this disclosure is realized when the compound of Formula I, II, III and IV comprises one or more imaging agents selected from PET (positron emission tomography), SPECT (single-photon emission computed tomography), and computed tomography imaging agent. A subembodiment of this aspect of the disclosure is realized when the imaging agent is PET. Another subembodiment of this aspect of the disclosure is realized when the imaging agent is SPECT. Another subembodiment of this aspect of the disclosure is realized when the imaging agent is computed tomography imaging agent. Another subembodiment of this aspect of the disclosure is realized when the imaging agent is a radioisotopic computed tomography imaging agent. Another subembodiment of the disclosure is realized when the imaging agent is PET or SPECT comprising one or more radioisotopes selected from11C,18F,64Cu,68Ga,76Br,77Br,89Zr,111In,123I,124I,186Re,188Re, and201T1. In further aspects of the disclosure the imaging agent is a PET or SPECT comprising68Ga or18F. In further aspects, non-limiting radioisotopes that form stable complexes with a chelating moiety and have physical half-lives suitable for PET imaging purposes are selected from89Zr,68Ga,64Cu,44Sc, and86Y. In further aspects, non-limiting radioisotopes that directly bond with peptide, including, but not limited to,76Br and124I. Still in further aspects, non-limiting radioisotopes that are introduced via prosthetic group are, for example,18F.

[0048] Another embodiment of the disclosure is realized when the compound of Formula Formula I, II, III and IV or a pharmaceutically acceptable salt thereof is linked to one or more imaging agents through a linking group. See, for example, linking groups disclosed in Grazytrace, J. Cin. Invest.2022. A subembodiment of this aspect of the disclosure is realized when the linking group comprises one or more amino acid residues. A subembodiment of this aspect of the disclosure is realized when there are about 1 to about 100, about 1 to about 80, about 1 to about 60, about 1 to about 40, about 1 to about 20, about 1 to about 10, about 1 to about 5, about 5 to about 100, about 5 to about 80, about 5 to about 60, about 5 to about 40, about 5 to about 20, about 5 to about 10, about 10 to about 100, about 10 to about 80, about 10 to about 60, about 10 to about 40, about 10 to about 20, about 20 to about 100, about 20 to about 80, about 20 to about 60, about 20 to about 40, about 40 to about 100, about 40 to about 80, about 40 to about 60, about 60 to about 100, about 60 to about 80, or about 80 to about 100 amino acid residues. - 27 -25927

[0049] Another embodiment of this aspect of the disclosure is realized when the linking group comprises one or more alkylene groups, one or more amine groups, one or more amide groups, one or more alkyleneoxy groups, one or more thiol groups, one or more carbohydrate groups, or any combination thereof. A subembodiment of this aspect of the disclosure is realized when the linking group comprises one or more C1-50 alkylene groups, one or more amine groups, one or more amide groups, one or more C1-50alkyleneoxy groups, one or more C1-50thiol groups, or any combination thereof. Another subembodiment of this aspect of the disclosure is realized when the linking group comprises one or more —(OCH2CH2)p— groups wherein p is an integer, for example, from about 1 to about 100, about 1 to about 80, about 1 to about 60, about 1 to about 40, about 1 to about 20, about 1 to about 10, about 1 to about 5, about 5 to about 100, about 5 to about 80, about 5 to about 60, about 5 to about 40, about 5 to about 20, about 5 to about 10, about 10 to about 100, about 10 to about 80, about 10 to about 60, about 10 to about 40, about 10 to about 20, about 20 to about 100, about 20 to about 80, about 20 to about 60, about 20 to about 40, about 40 to about 100, about 40 to about 80, about 40 to about 60, about 60 to about 100, about 60 to about 80, or about 80 to about 100 amino acid residues. In some embodiments, p is an integer from about 10 to about 40. In some embodiments, p is an integer from about 20 to about 40. In some embodiments, p is an integer from about 25 to about 35.

[0050] Another embodiment of the disclosure is realized when the compounds of Formula I, II and III or a pharmaceutically acceptable salt thereof is linked to a chelating moiety containing one or more of the imaging agents described herein.

[0051] Listed below are definitions of various terms used herein. These definitions apply to the terms as they are used throughout this specification and claims, unless otherwise limited in specific instances, either individually or as part of a larger group.

[0052] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Generally, the nomenclature used herein and the laboratory procedures in cell culture, molecular genetics, organic chemistry, and peptide chemistry are those well-known and commonly employed in the art.

[0053] As used herein, the articles “a” and “an” refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element. Furthermore, use of the term “including” as well as other forms, such as “include,” “includes,” and “included,” is not limiting.

[0054] As used herein, the term “about” in quantitative terms refers to plus or minus 10% of the value it modifies (rounded up to the nearest whole number if the value is not sub-dividable, such as a number of molecules or nucleotides). - 28 -25927

[0055] All ranges disclosed herein are inclusive of the recited endpoint and independently combinable (for example, the range of “from 50 mg to 500 mg” is inclusive of the endpoints, 50 mg and 500 mg, and all the intermediate values). The endpoints of the ranges and any values disclosed herein are not limited to the precise range or value; they are sufficiently imprecise to include values approximating these ranges and / or values.

[0056] As used herein, the term “comprising” may include the embodiments “consisting of” and “consisting essentially of.” The terms “comprise(s),” “include(s),” “having,” “has,” “may,” “contain(s),” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that require the presence of the named ingredients / steps and permit the presence of other ingredients / steps. However, such description should be construed as also describing compositions or processes as “consisting of” and “consisting essentially of” the enumerated components, which allows the presence of only the named components or compounds, along with any acceptable carriers or fluids, and excludes other components or compounds.

[0057] As used herein, the term “optionally substituted” means unsubstituted or substituted with the named substituents or just unsubstituted or substituted.

[0058] The compounds of the disclosure may contain one or more asymmetric centers and can thus occur as racemates and racemic mixtures, single enantiomers, diastereomeric mixtures and individual diastereomers. Additional asymmetric centers may be present depending upon the nature of the various substituents on the molecule. Each such asymmetric center will independently produce two optical isomers and it is intended that all the possible optical isomers and diastereomers in mixtures and as pure or partially purified compounds are included within the ambit of this disclosure. Unless a specific stereochemistry is indicated, the present disclosure is meant to encompass all such isomeric forms of these compounds.

[0059] The independent syntheses of these diastereomers or their chromatographic separations may be achieved as known in the art by appropriate modification of the methodology disclosed herein. Their absolute stereochemistry may be determined, amongst other methods, by the x-ray crystallography of crystalline products or crystalline intermediates which are derivatized, if necessary, with a reagent containing an asymmetric center of known absolute configuration.

[0060] If desired, racemic mixtures of the compounds may be separated so that the individual enantiomers are isolated. The separation can be carried out by methods well known in the art, such as the coupling of a racemic mixture of compounds to an enantiomerically pure compound to form a diastereomeric mixture, followed by separation of the individual diastereomers by standard methods, such as fractional crystallization or chromatography. The coupling reaction is - 29 -25927 often the formation of salts using an enantiomerically pure acid or base. The diastereomeric derivatives may then be converted to the pure enantiomers by cleavage of the added chiral residue. The racemic mixture of the compounds can also be separated directly by chromatographic methods utilizing chiral stationary phases, which methods are well known in the art.

[0061] Alternatively, any enantiomer of a compound may be obtained by stereoselective synthesis using optically pure starting materials or reagents of known configuration by methods well known in the art.

[0062] In the compounds of Formula I, II, III and IV the atoms may exhibit their natural isotopic abundances, or one or more of the atoms may be artificially enriched in a particular isotope having the same atomic number, but an atomic mass or mass number different from the atomic mass or mass number predominantly found in nature. The present disclosure may include all suitable isotopic variations of the compounds of generic Formula I, II, III and IV. For example, different isotopic forms of hydrogen (H) include protium (1H) and deuterium (2H). Protium is the predominant hydrogen isotope found in nature. Enriching for deuterium may afford certain therapeutic advantages, such as increasing in vivo half-life or reducing dosage requirements, or may provide a compound useful as a standard for characterization of biological samples. For purposes of this disclosure when a compound is said to be “not deuterated” it means not enriched in deuterium beyond the background state. Isotopically-enriched compounds within generic Formula I, II, III and IV can be prepared without undue experimentation by conventional techniques well known to those skilled in the art or by processes analogous to those described in the Schemes and Examples herein using appropriate isotopically-enriched reagents and / or intermediates.

[0063] When any variable (e.g., R1, etc.) occurs more than one time in any constituent, its definition on each occurrence is independent at every other occurrence. Also, combinations of substituents and variables are permissible only if such combinations result in stable compounds. Lines drawn into the ring systems from substituents represent that the indicated bond may be attached to any of the substitutable ring atoms. If the ring system is bicyclic, it is intended that the bond be attached to any of the suitable atoms on either ring of the bicyclic moiety.

[0064] It is understood that one or more silicon (Si) atoms can be incorporated into the compounds of the instant disclosure in place of one or more carbon atoms by one of ordinary skill in the art to provide compounds that are chemically stable and that can be readily synthesized by techniques known in the art from readily available starting materials. Carbon and silicon differ in their covalent radius leading to differences in bond distance and the steric - 30 -25927 arrangement when comparing analogous C-element and Si-element bonds. These differences lead to subtle changes in the size and shape of silicon-containing compounds when compared to carbon. One of ordinary skill in the art would understand that size and shape differences can lead to subtle or dramatic changes in potency, solubility, lack of off-target activity, packaging properties, and so on. (Diass, J. O. et al. Organometallics (2006) 5:1188-1198; Showell, G.A. et al. Bioorganic & Medicinal Chemistry Letters (2006) 16:2555-2558).

[0065] It is understood that substituents and substitution patterns on the compounds of the instant disclosure can be selected by one of ordinary skill in the art to provide compounds that are chemically stable and that can be readily synthesized by techniques known in the art, as well as those methods set forth below, from readily available starting materials. If a substituent is itself substituted with more than one group, it is understood that these multiple groups may be on the same carbon or on different carbons, so long as a stable structure results. The phrase “optionally substituted with one or more substituents” should be understood as meaning that the group in question is either unsubstituted or may be substituted with one or more substituents.

[0066] Absolute stereochemistry is illustrated by the use of hashed and solid wedge bonds. As shown in Illus-I and Illus-II. Accordingly, the methyl group of Illus-I is emerging from the page of the paper and the ethyl group in Illus-II is descending into the page, where the cyclohexene ring resides within the plane of the paper. It is assumed that the hydrogen on the same carbon as the methyl group of Illus-I descends into the page and the hydrogen on the same carbon as the ethyl group of Illus-II emerges from the page. The convention is the same where both a hashed and solid rectangle are appended to the same carbon as in Illus-III, the methyl group is emerging from the plane of the paper and the ethyl group is descending into the plane of the paper with the cyclohexene ring in the plane of the paper. s-3

[0067] As is conventional, unless otherwise noted in accompanying text, ordinary "stick" bonds or "wavy" bonds indicate that all possible stereochemistry is represented, including, pure compounds, mixtures of isomers, and racemic mixtures.

[0068] As used herein, unless otherwise specified, the following terms have the following meanings:

[0069] The phrase “at least one” used in reference to the number of components comprising a composition, for example, "at least one pharmaceutical excipient" means that one member of the - 31 -25927 specified group is present in the composition, and more than one may additionally be present. Components of a composition are typically aliquots of isolated pure material added to the composition, where the purity level of the isolated material added into the composition is the normally accepted purity level for a reagent of the type.

[0070] Whether used in reference to a substituent on a compound or a component of a pharmaceutical composition the phrase "one or more", means the same as "at least one";

[0071] “Effective amount” or “therapeutically effective amount” is meant to describe the provision of an amount of at least one active compound or pharmaceutical agent of the disclosure or of a composition comprising at least one compound or pharmaceutical agent of the disclosure that elicits the biological or medicinal response that is being sought in a tissue, system, animal, individual or human, or which is effective in treating or inhibiting a disease or condition described herein, and thus produce the desired therapeutic, ameliorative, inhibitory or preventative effect. For example, in treating central nervous system diseases or disorders with one or more of the compounds described herein “effective amount” (or “therapeutically effective amount”) means, for example, providing the amount of at least one compound of Formula I, II, III, IV or a pharmaceutically acceptable salt thereof that results in a therapeutic response in a patient afflicted with a central nervous system disease or disorder ("condition"), including a response suitable to manage, alleviate, ameliorate, or treat the condition or alleviate, ameliorate, reduce, or eradicate one or more symptoms attributed to the condition and / or long-term stabilization of the condition, for example, as may be determined by the analysis of pharmacodynamic markers or clinical evaluation of patients afflicted with the condition;

[0072] “Patient” and "subject" means an animal, such as a mammal (e.g., a human being) and is preferably a human being;

[0073] The term “substituted” means that one or more of the enumerated substituents can occupy one or more of the bonding positions on the substrate typically occupied by "–H", provided that such substitution does not exceed the normal valency rules for the atom in the bonding configuration presented in the substrate, and that the substitution ultimately provides a stable compound, which is to say that such substitution does not provide compounds with mutually reactive substituents located geminal or vicinal to each other; and wherein the substitution provides a compound sufficiently robust to survive isolation to a useful degree of purity from a reaction mixture.

[0074] Where optional substitution of a moiety is described (e.g., "optionally substituted") the term means that if substituents are present, one or more of the enumerated substituents for the specified substrate can be present on the substrate in a bonding position normally occupied by the - 32 -25927 default substituent normally occupying that position. For example, a default substituent on the carbon atoms of an alkyl moiety is a hydrogen atom, an optional substituent can replace the default substituent.

[0075] As used herein, unless otherwise specified, the following terms used to describe moieties, whether comprising the entire definition of a variable portion of a structural representation of a compound of the disclosure or a substituent appended to a variable portion of a structural representation of a group of compounds of the disclosure have the following meanings, and unless otherwise specified, the definitions of each term (i.e., moiety or substituent) apply when that term is used individually or as a component of another term (e.g., the definition of aryl is the same for aryl and for the aryl portion of arylalkyl, alkylaryl, arylalkynyl moieties, and the like); moieties are equivalently described herein by structure, typographical representation or chemical terminology without intending any differentiation in meaning, for example, an "acyl" substituent may be equivalently described herein by the term “acyl”, by typographical representations "R' (C=O)-" or "R'-C(O)-", or by a structural representation:, equally, with no differentiation implied using any or all of these representations;

[0076] The term “alkyl” (including the alkyl portions of other moieties, such as trifluoromethyl-alkyl- and alkoxy-) means a straight or branched aliphatic hydrocarbon moiety comprising up to about 20 carbon atoms (for example, a designation of "C1-20alkyl" indicates an aliphatic hydrocarbon moiety of from 1 to 20 carbon atoms). In some embodiments, alkyls preferably comprise up to about 10 carbon atoms, unless the term is modified by an indication that a shorter chain is contemplated, for example, an alkyl moiety of from 1 up to 8 carbon atoms is designated herein "C1-8-alkyl". Where the term "alkyl" is indicated with two hyphens (i.e., " alkyl " it indicates that the alkyl moiety is bonded in a manner that the alkyl moiety connects the substituents on either side of it, for example, " alkyl-OH" indicates an alkyl moiety connecting a hydroxyl moiety to a substrate.

[0077] "Dose", "dosage", "unit dose", "unit dosage", "effective dose" and related terms refer to physically discrete units that contain a predetermined quantity of active ingredient (e.g., peptidomimetic macrocycle) calculated to produce a desired therapeutic effect (e.g., death of cancer cells). These terms are synonymous with the therapeutically effective amounts and amounts sufficient to achieve the stated goals of the methods disclosed herein.

[0078] As used herein, “cycloalkyl” intended to include saturated or unsaturated nonaromatic ring groups, such as cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl. For example, the term “C3-8cycloalkyl” is intended to include C3, C4, C5, C6, C7, and C8cycloalkyl groups. Cycloalkyls may include multiple spiro- or fused or bridged rings. For example, cycloalkyl can include, but is - 33 -25927 not limited to, spiro butyl, pentyl, hexyl, heptyl, octyl, nonyl, or decyl groups, bicyclo butyl, pentyl, hexyl, heptyl, octyl, nonyl, or decyl groups, adamantyl groups, and norbornyl groups Examples of cycloalkyl moieties include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl. The term “cycloalkyl” also includes non-aromatic, fused multicyclic ring system comprising up to 20 carbon. Suitable multicyclic cycloalkyls are, for example, but are not limited to: 1-decalin; norbornyl; adamantyl; and the like. Cycloalkyl also includes fused multicyclic ring systems comprising a non-aromatic ring fused to an aromatic ring, such as benzoannulenyl.

[0079] As used herein, the term “alkylene” refers to a saturated linear or branched aliphatic hydrocarbon group having two residues derived from the removal of two hydrogen atoms from the same carbon atom or two different carbon atoms of the parent alkane. The alkylene is a linear or branched group having 1 to 20 carbon atoms, preferably 1 to 12 carbon atoms, and more preferably 1 to 6 carbon atoms. Non-limiting examples are methylene, ethylene, propylene, butylene, pentylene, and the like.

[0080] Where a structural formula represents bonding between a moiety and a substrate using a bonding line that terminates in the middle of the structure, for example the following representations: hered to the substrate through any of available ring atom, for example, the numbered atoms of the example moieties.

[0081] The term "aryl" refers to a 6 to 14 membered all-carbon monocyclic ring or polycyclic fused ring (i.e., each ring in the system shares an adjacent pair of carbon atoms with another ring in the system) having a conjugated electron system, preferably a 6 to 10 membered aryl, for example, phenyl and naphthyl, and preferably phenyl.

[0082] The term "heteroaryl" refers to an aromatic 5-8 membered monocyclic, 8-12 membered bicyclic, or 11-14 membered tricyclic ring system having 1-3 heteroatoms for monocyclic, 1-6 heteroatoms for bicyclic, or 1-9 heteroatoms for tricyclic, said heteroatoms selected from O, N, or S (e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of N, O, or S for monocyclic, bicyclic, - 34 -25927 or tricyclic, respectively). Non-limiting examples of heteroaryls are imidazolyl, pyridyl, pyrazolyl, pyrimidinyl, furanyl, oxazolyl, triazolyl, oxadiazolyl, quinolinyl, pyrrolyl, and thiophenyl. The heteroaryl groups herein described may also contain fused rings that share a common carbon-carbon bond, such as indolyl and benzofuranyl.

[0083] The term "heterocyclyl” (or heterocycloalkyl) means a non-aromatic saturated or unsaturated monocyclic or multicyclic ring system comprising 3 to 10 ring atoms, preferably 5 to 10 ring atoms, in which one or more of the atoms in the ring system is an element other than carbon, for example nitrogen (e.g., azetidinyl, piperidyl, pyrrolidinyl, tetrahydroisoquinolinyl,), oxygen (e.g., tetrahydrofuranyl and tetrahydropyranyl) or sulfur (e.g., tetrahydrothiophenyl and tetrahydrothiopyranyl); and wherein the heteroatoms can be alone or in combination provided that the moiety does not contain adjacent oxygen and / or sulfur atoms present in the ring system.

[0084] The nitrogen or sulfur atom of the heterocyclyl can be optionally oxidized to the corresponding N-oxide, S-oxide or S,S-dioxide (SO2). Non-limiting examples of suitable monocyclic heterocyclyl rings include azetidinyl, piperidyl, pyrrolidinyl, piperazinyl, morpholinyl - (where unless otherwise noted the moiety is bonded to the substrate through any on atoms C2, C3, C5, or C6), thiomorpholinyl, thiazolidinyl, 1,3- dioxolanyl, 1,4-dioxanyl, tetrahydrofuranyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, and the like; and bicyclic rings such as tetrahydroisoquinolinyl

[0085] The term “halogen” means fluorine, chlorine, bromine, or iodine; preferred halogens, unless specified otherwise where the term is used, are fluorine, chlorine and bromine, a substituent which is a halogen atom means –F, -Cl, -Br, or –I, and “halo” means fluoro, chloro, bromo, or iodo substituents bonded to the moiety defined, for example, "haloalkyl” means an alkyl, as defined above, wherein one or more of the bonding positions on the alkyl moiety typically occupied by hydrogen atoms are instead occupied by a halo group, perhaloalkyl (or “fully halogenated” alkyl) means that all bonding positions not participating in bonding the alkyl substituent to a substrate are occupied by a halogen, for example, where the alkyl is selected to be methyl, the term perfluoroalkyl means -CF3;

[0086] The term "hydroxyl" and "hydroxy" means an HO- group, “hydroxyalkyl” means a substituent of the formula: "HO-alkyl-",wherein the alkyl group is bonded to the substrate and may be substituted or unsubstituted as defined above; preferred hydroxyalkyl moieties comprise - 35 -25927 a lower alkyl; Non-limiting examples of suitable hydroxyalkyl groups include hydroxymethyl and 2-hydroxyethyl; and

[0087] The bonding sequence is indicated by hyphens where moieties are represented in text, for example –alkyl, indicates a single bond between a substrate and an alkyl moiety, -alkyl-X, indicates that an alkyl group bonds an "X" substituent to a substrate, and in structural representation, bonding sequence is indicated by a wavy line terminating a bond representation, for example , indicates that the methylphenyl moiety is bonded to a substrate through a catho to the methyl substituent, while a bond representation terminated with a wavy line and drawn into a structure without any particular indication of an atom to which it is bonded indicates that the moiety may be bonded to a substrate via any of the atoms in the moiety which are available for bonding as described in the examples above.

[0088] The line —, as a bond generally indicates a mixture of, or either of, the possible isomers, e.g., containing (R)- and (S)- stereochemical configuration.

[0089] Furthermore, unwedged-bolded or unwedged-hashed lines are used in structures containing multiple stereocenters in order to depict relative configuration where it is known. For example: ., p p y d to capture each of the stereochemical permutations that are possible for a given structural isomer based on the synthetic operations employed in its preparation. Lists of discrete stereoisomers that are conjoined using or indicate that the presented compound (e.g., ‘Example number’) was isolated as a single stereoisomer, and that the identity of that stereoisomer corresponds to one of - 36 -25927 the possible configurations listed. Lists of discrete stereoisomers that are conjoined using and indicate that the presented compound was isolated as a racemic mixture or diastereomeric mixture.

[0091] A specific absolute configuration is indicated by use of a wedged-bolded or wedged- hashed line. Unless a specific absolute configuration is indicated, the present disclosure is meant to encompass all such stereoisomeric forms of these compounds.

[0092] In this specification, where there are multiple oxygen and / or sulfur atoms in a ring system, there cannot be any adjacent oxygen and / or sulfur present in said ring system.

[0093] As well known in the art, a bond drawn from a particular atom wherein no moiety is depicted at the terminal end of the bond indicates a methyl group bound through that bond to the atom, unless stated otherwise. For example: s, structural formulae, and any Tablesherein is assumed to have a hydrogen atom or atoms of sufficient number to satisfy the valences.

[0095] One or more compounds of the disclosure may also exist as, or optionally be converted to, a solvate. Preparation of solvates is generally known. Thus, for example, M. Caira et al., J. Pharmaceutical Sci., 93(3), 601-611 (2004) describe the preparation of the solvates of the antifungal fluconazole in ethyl acetate as well as from water. Similar preparations of solvates, and hemisolvate, including hydrates (where the solvent is water or aqueous-based) and the like are described by E. C. van Tonder et al., AAPS PharmSciTech., 5(1), article 12 (2004); and A. L. Bingham et al., Chem. Commun., 603-604 (2001). A typical, non-limiting, process involves dissolving the inventive compound in desired amounts of the desired solvent (for example, an organic solvent, an aqueous solvent, water or mixtures of two or more thereof) at a higher than ambient temperature, and cooling the solution, with or without an antisolvent present, at a rate sufficient to form crystals which are then isolated by standard methods. Analytical techniques such as, for example I.R. spectroscopy, show the presence of the solvent (including water) in the crystals as a solvate (or hydrate in the case where water is incorporated into the crystalline form).

[0096] This disclosure also includes the compounds of this disclosure in isolated and purified form obtained by routine techniques. Polymorphic forms of the compounds of Formula I, II, III and IV and of the salts, solvates and prodrugs of the compounds of Formula I, II, III and IV are intended to be included in the present disclosure. Certain compounds of the disclosure may exist - 37 -25927 in different isomeric forms (e.g., enantiomers, diastereoisomers, atropisomers). The inventive compounds include all isomeric forms thereof, both in pure form and admixtures of two or more, including racemic mixtures.

[0097] In the same manner, unless indicated otherwise, presenting a structural representation of any tautomeric form of a compound which exhibits tautomerism is meant to include all such tautomeric forms of the compound. Accordingly, where compounds of the disclosure, their salts, and solvates and prodrugs thereof, may exist in different tautomeric forms or in equilibrium among such forms, all such forms of the compound are embraced by, and included within the scope of the disclosure. Examples of such tautomers include, but are not limited to, ketone / enol tautomeric forms, imine-enamine tautomeric forms, and for example heteroaromatic forms such as the following moieties: .p p cally acceptable" is employed herein to refer to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0099] As used herein, "pharmaceutically acceptable salts" refer to derivatives wherein the parent compound is modified by making acid or base salts thereof. Salts in the solid form may exist in more than one crystal structure and may also be in the form of hydrates. 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 include the conventional non-toxic salts or the quaternary ammonium salts of the parent compound formed, for example, from non-toxic inorganic or organic acids. For example, such conventional non-toxic salts include those derived from inorganic acids such as formic, hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, nitric and the like; and the salts prepared from organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, pamoic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic, sulfanilic, 2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, isethionic, and the like. Salts derived from inorganic - 38 -25927 bases include aluminum, ammonium, calcium, copper, ferric, ferrous, lithium, magnesium, manganic salts, manganous, potassium, sodium, zinc, and the like.

[0100] When the compound of the present disclosure is basic, salts may be prepared from pharmaceutically acceptable non-toxic acids, including inorganic and organic acids. Such acids include acetic, benzenesulfonic, benzoic, camphorsulfonic, citric, ethanesulfonic, fumaric, gluconic, glutamic, hydrobromic, hydrochloric, isethionic, lactic, maleic, malic, mandelic, methanesulfonic, mucic, nitric, pamoic, pantothenic, phosphoric, succinic, sulfuric, tartaric, p- toluenesulfonic acid, and the like. In one aspect of the disclosure the salts are citric, hydrobromic, hydrochloric, maleic, phosphoric, sulfuric, fumaric, and tartaric acids. Similarly, the salts of the acidic compounds are formed by reactions with the appropriate inorganic or organic base.

[0101] The terms “treating” or “treatment” (of, e.g., a disease, disorder, or conditions or associated symptoms, which together or individually may be referred to as “indications”) as used herein include: inhibiting the disease, disorder or condition, i.e., arresting or reducing the development of the disease or its biological processes or progression or clinical symptoms thereof; or relieving the disease, i.e., causing regression of the disease or its biological processes or progression and / or clinical symptoms thereof. “Treatment” as used herein also refers to control, amelioration, or reduction of risks to the subject afflicted with a disease, disorder or condition in which a tumor is involved. The terms “preventing” or “prevention” or “prophylaxis” of a disease, disorder or condition as used herein includes: impeding the development or progression of clinical symptoms of the disease, disorder, or condition in a mammal that may be exposed to or predisposed to the disease, disorder or condition but does not yet experience or display symptoms of the disease, and the like.

[0102] As would be evident to those skilled in the art, subjects treated by the methods described herein are generally mammals, including humans and non-human animals (e.g., laboratory animals and companion animals). The term "therapeutically effective amount" means the amount of the subject compound that will elicit the biological or medical response of a tissue, system, animal or human that is being sought by the researcher, veterinarian, medical doctor or other clinician.

[0103] The term "composition" as used herein is intended to encompass a product comprising a compound of the disclosure or a pharmaceutically acceptable salt thereof, together with one or more additional specified ingredients in the specified amounts, as well as any product which results, directly or indirectly, from combination of the specified ingredients in the specified amounts. Such term in relation to a pharmaceutical composition, is intended to encompass a product comprising the active ingredient(s), which include a compound of the disclosure or a - 39 -25927 pharmaceutically acceptable salt thereof, optionally together with one or more additional active ingredients, and the inert ingredient(s) that make up the carrier, as well as any product which results, directly or indirectly, from combination, complexation or aggregation of any two or more of the ingredients, or from dissociation of one or more of the ingredients, or from other types of reactions or interactions of one or more of the ingredients. Accordingly, the pharmaceutical compositions of the present disclosure encompass any composition made by admixing a compound of the present disclosure, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier. By "pharmaceutically acceptable" it is meant the carrier, diluent or excipient must be compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.

[0104] As noted above, additional embodiments of the present disclosure are each directed to a method for the treatment a disease, disorder, or condition, or one or more symptoms thereof (“indications”) which method comprises administering to a subject in need of such treatment a therapeutically effective amount of a compound of the disclosure, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising said compound or salt thereof. Thus, an embodiment of this disclosure is a method of treating a disease, disorder, or condition, or one or more symptoms thereof (“indications”) where granzyme B is implicated.

[0105] Another embodiment of this aspect of the disclosure is realized when the disease is selected from an autoimmune disorder, inflammatory disorder, skin disorder, cancer and cardiovascular disorder. A subembodiment of this aspect of the disclosure relates to a disease that is cancer selected from breast cancer, ovarian cancer, cervical cancer, uterine cancer, prostate cancer, kidney cancer, urethral cancer, bladder cancer, liver cancer, stomach cancer, endometrial cancer, salivary gland cancer, esophageal cancer, melanoma, glioma, neuroblastoma, sarcoma, lung cancer (for example, small cell lung cancer and non-small cell lung cancer) colon cancer, rectal cancer, colorectal cancer, leukemia (for example, acute lymphocytic leukemia, acute myeloid leukemia, acute promyelocytic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia), bone cancer, skin cancer, thyroid cancer, pancreatic cancer, and lymphoma (for example, Hodgkin’s lymphoma, non-Hodgkin’s lymphoma, or recurrent anaplastic large cell lymphoma). Another subembodiment of this aspect of the disclosure relates to a method of treating or preventing cancer, in a subject in need thereof, said method comprising administering to a subject in need of such treatment a therapeutically effective amount of a compound of Formula I, II, III, IV, or a pharmaceutically acceptable salt or solvate thereof, or a pharmaceutical composition comprising said compound, salt or solvate thereof. In one such embodiment, the subject is a human. - 40 -25927

[0106] Another embodiment of the disclosure relates to methods of imaging granzyme B. A subembodiment of this aspect of the disclosure relates to a method wherein imaging is performed in a cell, a tissue, a cell sample, a tissue sample, or a subject. As used herein, the term “subject,” refers to any animal, including mammals (e.g., humans, domestic animals, farm animals, etc.) and invertebrates (e.g., fish). Another embodiment of the disclosure relates to a method of imaging granzyme B in a cell or tissue, comprising contacting the cell or tissue with a compound of Formula I, II, III, IV or a pharmaceutically acceptable salt thereof and imaging the cell or tissue with a suitable imaging technique to image the granzyme B in the cell or tissue. A subembodiment of this aspect of the disclosure is realized when the compound of Formula I, II, III, IV or a pharmaceutically acceptable salt thereof comprising an imaging agent.

[0107] Another embodiment of the disclosure relates to a method of imaging an immune response in a subject, comprising administering to the subject a compound of Formula I, II, III, IV or a pharmaceutically acceptable salt thereof, and imaging the subject with a suitable imaging technique to image the granzyme B in the cell or tissue. A subembodiment of this aspect of the disclosure is realized when the compound of Formula I, II, III, IV, or a pharmaceutically acceptable salt thereof comprising an imaging agent.

[0108] Another embodiment of the disclosure relates to a method of monitoring an immune response in the treatment of a disease in a subject, comprising administering to the subject a compound of Formula I, II, III, IV or a pharmaceutically acceptable salt thereof, and imaging the subject with a suitable imaging technique to image the granzyme B in the cell or tissue. A subembodiment of this aspect of the disclosure is realized when the compound of Formula I, II, III, IV or a pharmaceutically acceptable salt thereof comprising an imaging agent.

[0109] Another embodiment of the disclosure is realized when the compounds are used as imaging agents in diseases selected from graft-versus-host disease, rheumatoid arthritis, systemic lupus erythematosus, Hashimoto’s thyroiditis, multiple sclerosis, myasthenia gravis, type I diabetes, uveitis, posterior uveitis, allergic encephalomyelitis, glomerulonephritis, rheumatic fever, post-infectious glomerulonephritis, psoriasis, atopic dermatitis, contact dermatitis, eczematous dermatitis, seborrhoeic dermatitis, lichen planus, pemphigus, bullous pemphigoid, epidermolysis bullosa, urticaria, angioedemas, vasculitis, erythema, cutaneous eosinophilia, lupus erythematosus, acne, alopecia areata, keratoconjunctivitis, vernal conjunctivitis, uveitis associated with Behcet’s disease, keratitis, herpetic keratitis, conical cornea, dystrophia epithelialis corneae, corneal leukoma, ocular pemphigus, Mooren’s ulcer, scleritis, Graves’ opthalmopathy, Vogt-Koyanagi-Harada syndrome, sarcoidosis, pollen allergies, reversible obstructive airway disease, bronchial asthma, allergic asthma, intrinsic asthma, extrinsic asthma, - 41 -25927 dust asthma, chronic or inveterate asthma, late asthma and airway hyper-responsiveness, bronchitis, gastric ulcers, vascular damage caused by ischemic diseases and thrombosis, ischemic bowel diseases, inflammatory bowel diseases, necrotizing enterocolitis, intestinal lesions associated with thermal burns, coeliac diseases, proctitis, eosinophilic gastroenteritis, mastocytosis, Crohn’s disease, ulcerative colitis, migraine, rhinitis, eczema, interstitial nephritis, Goodpasture’s syndrome, hemolyticuremic syndrome, diabetic nephropathy, multiple myositis, Guillain-Barre syndrome, Meniere’s disease, polyneuritis, multiple neuritis, mononeuritis, radiculopathy, hyperthyroidism, Basedow’s disease, pure red cell aplasia, aplastic anemia, hypoplastic anemia, idiopathic thrombocytopenic purpura, autoimmune hemolytic anemia, agranulocytosis, pernicious anemia, megaloblastic anemia, anerythroplasia, osteoporosis, sarcoidosis, fibroid lung, idiopathic interstitial pneumonia, der-matomyositis, leukoderma vulgaris, ichthyosis vulgaris, photoallergic sensitivity, cutaneous T cell lymphoma, arteriosclerosis, atherosclerosis, aortitis syndrome, polyarteritis nodosa, myocardosis, scleroderma, Wegener’s granuloma, Sjogren’s syndrome, adiposis, eosinophilic fascitis, lesions of gingiva, periodontium, alveolar bone, substantia ossea dentis, glomerulonephritis, male pattern alopecia, alopecia senilis by preventing epilation, alopecia senilis by providing hair germination and / or promoting hair generation and hair growth, muscular dystrophy, pyoderma, Sezary’s syndrome, Addison’s disease, ischemia-reperfusion injury of organs, transplantation disease, ischemic disease, endotoxin-shock, pseudomembranous colitis, colitis caused by drug or radiation, ischemic acute renal insufficiency, chronic renal insufficiency, toxinosis caused by lung-oxygen or drugs, lung cancer, pulmonary emphysema, cataracta, siderosis, retinitis pigmentosa, senile macular degeneration, vitreal scarring, corneal alkali bum, dermatitis erythema multiforme, linear IgA ballous dermatitis and cement dermatitis, gingivitis, periodontitis, sepsis, pancreatitis, aging, carcinogenesis, 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 toxin, viral hepatitis, shock, anoxia, B-virus hepatitis, non-A / non-B hepatitis, cirrhosis, alcoholic cirrhosis, hepatic failure, fulminant hepatic failure, late-onset hepatic failure, acute-on-chronic liver failure, cyto-megalovirus infection, HCMV infection, AIDS, senile dementia, trauma, chronic bacterial infection, malignancy of lymphoid origin, acute lymphocytic leukemia, chronic lymphocytic leukemia, acute lymphocytic lymphoma, and chronic lymphocytic lymphoma.

[0110] Another embodiment of the disclosure is realized when the disease is selected from systemic lupus erythematosis, chronic rheumatoid arthritis, type I diabetes mellitus, - 42 -25927 inflammatory bowel disease, biliary cirrhosis, uveitis, multiple sclerosis, Crohn’s disease, ulcerative colitis, bullous pemphigoid, sarcoidosis, psoriasis, autoimmune myositis, Wegener’s granulomatosis, ichthyosis, Graves ophthalmopathy, asthma, schleroderma and Sjogren’s syndrome. Another embodiment of the disclosure is realized when the disease is selected from bone marrow rejection, organ transplant rejection, and graft-versus-host disease.

[0111] Another aspect of the disclosure relates to uses and processes for making such compounds, which may be suitable for imaging granzyme B.

[0112] The present disclosure includes within its scope prodrugs of the compounds of this disclosure. In general, such prodrugs will be functional derivatives of the compounds of this disclosure which are readily convertible in vivo into the required compound. Thus, in the methods of treatment of the present disclosure, the terms "administration of" or "administering a" compound shall encompass the treatment of the various conditions described with the compound specifically disclosed or with a compound which may not be specifically disclosed, but which converts to the specified compound in vivo after administration to the patient. Conventional procedures for the selection and preparation of suitable prodrug derivatives are described, for example, in "Design of Prodrugs," ed. H. Bundgaard, Elsevier, 1985. Metabolites of these compounds include active species produced upon introduction of compounds of this disclosure into the biological milieu.

[0113] The compounds described herein, or pharmaceutically acceptable salts and / or solvates thereof, may be administered singly, in combination with other compounds of the disclosure, and / or in cocktails combined with other therapeutic agents. The choice of therapeutic agents that can be co-administered with the compounds of the disclosure will depend, in part, on the condition being treated.

[0114] The compounds of the present disclosure may be administered by oral, parenteral (e.g., intramuscular, intraperitoneal, intravenous, ICV, intracisternal injection or infusion, subcutaneous injection, or implant), by inhalation spray, nasal, vaginal, rectal, sublingual, buccal or topical routes of administration and may be formulated, alone or together, in suitable dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants and vehicles appropriate for each route of administration. In addition to the treatment of warm-blooded animals the compounds of the disclosure are effective for use in humans.

[0115] The pharmaceutical compositions for the administration of the compounds of this disclosure may conveniently be presented in dosage unit form and may be prepared by any of the methods well known in the art of pharmacy. All methods include the step of bringing the active ingredient into association with the carrier which constitutes one or more accessory ingredients. - 43 -25927 In general, the pharmaceutical compositions are prepared by uniformly and intimately bringing the active ingredient into association with a liquid carrier or a finely divided solid carrier or both, and then, if necessary, shaping the product into the desired formulation. In the pharmaceutical composition the active compound is included in an amount sufficient to produce the desired effect upon the process or condition of diseases. As used herein, the term "composition" is intended to encompass a product comprising the specified ingredients in the specified amounts, as well as any product which results, directly or indirectly, from combination of the specified ingredients in the specified amounts.

[0116] The pharmaceutical compositions containing the active ingredient may be in a form suitable for oral use, for example, as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions, solutions, hard or soft capsules, or syrups or elixirs. Compositions intended for oral use may be prepared according to any method known to the art for the manufacture of pharmaceutical compositions and such compositions may contain one or more agents selected from the group consisting of sweetening agents, flavoring agents, coloring agents and preserving agents in order to provide pharmaceutically elegant and palatable preparations. Tablets contain the active ingredient in admixture with non-toxic pharmaceutically acceptable excipients which are suitable for the manufacture of tablets. These excipients may be for example, inert diluents, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, for example, corn starch, or alginic acid; binding agents, for example starch, gelatin or acacia; and lubricating agents, for example magnesium stearate, stearic acid or talc. The tablets may be uncoated, or they may be coated by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as glyceryl monostearate or glyceryl distearate may be employed. They may also be coated by the techniques described in the U.S. Patents 4,256,108; 4,166,452; and 4,265,874 to form osmotic therapeutic tablets for control release. Oral tablets may also be formulated for immediate release, such as fast melt tablets or wafers, rapid dissolve tablets or fast dissolve films.

[0117] Formulations for oral use may also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, for example peanut oil, liquid paraffin, or olive oil.

[0118] Aqueous suspensions contain the active materials in admixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients are suspending agents, for example sodium carboxymethylcellulose, methylcellulose, hydroxy-propylmethylcellulose, sodium - 44 -25927 alginate, polyvinyl-pyrrolidone, gum tragacanth and gum acacia; dispersing or wetting agents may be a naturally-occurring phosphatide, for example lecithin, or condensation products of an alkylene oxide with fatty acids, for example polyoxyethylene stearate, or condensation products of ethylene oxide with long chain aliphatic alcohols, for example heptadecaethyleneoxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, for example polyethylene sorbitan monooleate. The aqueous suspensions may also contain one or more preservatives, for example ethyl, or n-propyl, p-hydroxybenzoate, one or more coloring agents, one or more flavoring agents, and one or more sweetening agents, such as sucrose or saccharin.

[0119] Oily suspensions may be formulated by suspending the active ingredient in a vegetable oil, for example arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin. The oily suspensions may contain a thickening agent, for example beeswax, hard paraffin or acetyl alcohol. Sweetening agents such as those set forth above, and flavoring agents may be added to provide a palatable oral preparation. These compositions may be preserved by the addition of an antioxidant such as ascorbic acid.

[0120] Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the active ingredient in admixture with a dispersing or wetting agent, suspending agent and one or more preservatives. Suitable dispersing or wetting agents and suspending agents are exemplified by those already mentioned above. Additional excipients, for example sweetening, flavoring and coloring agents, may also be present.

[0121] The pharmaceutical compositions of the disclosure may also be in the form of oil-in- water emulsions. The oily phase may be a vegetable oil, for example olive oil or arachis oil, or a mineral oil, for example liquid paraffin or mixtures of these. Suitable emulsifying agents may be naturally- occurring gums, for example gum acacia or gum tragacanth, naturally-occurring phosphatides, for example soy bean, lecithin, and esters or partial esters derived from fatty acids and hexitol anhydrides, for example sorbitan monooleate, and condensation products of the said partial esters with ethylene oxide, for example polyoxyethylene sorbitan monooleate. The emulsions may also contain sweetening and flavoring agents.

[0122] Syrups and elixirs may be formulated with sweetening agents, for example glycerol, propylene glycol, sorbitol or sucrose. Such formulations may also contain a demulcent, a preservative and flavoring and coloring agents.

[0123] The pharmaceutical compositions may be in the form of a sterile injectable aqueous or oleagenous suspension. This suspension may be formulated according to the known art using - 45 -25927 those suitable dispersing or wetting agents and suspending agents which have been mentioned above. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example as a solution in 1,3-butane diol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil may be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables.

[0124] The compounds of the present disclosure may also be administered in the form of suppositories for rectal administration of the drug. These compositions can be prepared by mixing the drug with a suitable non-irritating excipient which is solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum to release the drug. Such materials are cocoa butter and polyethylene glycols.

[0125] For topical use, creams, ointments, jellies, solutions or suspensions and the like, containing the compounds of the present disclosure are employed. Similarly, transdermal patches may also be used for topical administration.

[0126] The pharmaceutical composition and method of the present disclosure may further comprise other therapeutically active compounds as noted herein which are usually applied in the treatment of the above-mentioned pathological conditions.

[0127] In the treatment, prevention, control, amelioration, or reduction of risk of the conditions disclosed herein the phrase “therapeutically effective amount” refers to the amount of active compound or pharmaceutical agent that elicits the biological or medicinal response that is being sought in a tissue, system, animal, individual or human. In some embodiments, the dosage of the compound, or a pharmaceutically acceptable salt thereof, administered to a subject or individual is about 1 µg to about 2 g, about 1 µg to about 1000 mg, about 1 µg to about 500 mg, about 1 µg to about 100 mg, about 1 µg to about 50 mg, about 1 µg to about 1 mg, about 1 µg to about 500 µg, about 1 µg to about 100 µg, about 1 µg to about 10 µg, about 10 µg to about 2 g and the like. The compounds may be administered on a regimen of 1 to 4 times per day or may be administered once or twice per day.

[0128] Another embodiment of the disclosure is realized wherein the compounds of Formula I, II, III, IV or a pharmaceutically acceptable salt thereof can be administered in combination with one or more of the additional therapeutic agents provided herein. A subembodiment of this aspect of the disclosure is realized when the additional therapeutic agents include, but are not limited to, - 46 -25927 anti-inflammatory agents, steroids, immuno-therapy agents, chemotherapeutic agents, and therapeutic antibodies.

[0129] Another embodiment of the disclosure is realized when administration of the therapeutic agent induces an immune response cell or tissue sample or subject. A subembodiment of this aspect of the disclosure is realized when the therapeutic agent is a compound of Formula I, II, III, IV or a pharmaceutically acceptable salt thereof. A subembodiment of this aspect of the disclosure is realized when the therapeutic agent is a compound of Formula I, II, III, IV or a pharmaceutically acceptable salt thereof, comprising a radioisotope (e.g., a therapeutic radioisotope). Another subembodiment of this aspect of the disclosure is realized when the therapeutic agent is a compound of Formula I, II, III, IV or a pharmaceutically acceptable salt, comprising a toxic radioisotope. Another subembodiment of this aspect of the disclosure is realized when the toxic radioisotope is selected from alpha emitters (211At,212Pb,212Bi,213Bi,225Ac,227Th) and beta emitters (e.g.,90Y,131I and177Lu,161Tb).

[0130] It will be understood, however, that the specific dose level and frequency of dosage for any particular patient may be varied and will depend upon a variety of factors including the activity of the specific compound employed, the metabolic stability and length of action of that compound, the age, body weight, general health, sex, diet, mode and time of administration, rate of excretion, drug combination, the severity of the particular condition, and the host undergoing therapy.

[0131] Methods for preparing the compounds of this disclosure are illustrated in the following Schemes and Examples. Starting materials are made according to procedures known in the art or as illustrated herein.

[0132] It will be appreciated by one skilled in the art that the processes described are not the exclusive means by which compounds provided herein may be synthesized and that a broad repertoire of synthetic organic reactions is available to be potentially employed in synthesizing compounds provided herein. The compounds of the present disclosure can be prepared according to the following illustrative schemes and specific examples, or modifications thereof, using readily available starting materials, reagents and conventional synthesis procedures. It is also possible to make use of variants which are themselves known to those of ordinary skill in this art but are not mentioned in detail. The general procedures for making the compounds claimed in this disclosure can be readily understood by one skilled in the art from viewing the following schemes and descriptions. Abbreviations used in the experimentals may include, but are not limited to the following: - 47 -25927 AcOH Acetic acid aq Aqueous All Alll b l25927 Thr Threonine Trp Tryptophan T T i

[0133] All reagents and solvents were purchased from commercial sources and used as is unless otherwise noted. Reaction progress and synthetic intermediate analysis were assessed by LCMS (UV detection with electrospray ionization (ESI), atmosphermic pressure chemical ionization (APCI), or other mass detection) when applicable using a MeCN / water gradient with either TFA, formic acid, or NH4HCO3modifier. Silica gel and reverse-phase flash column chromatography were conducted with commercially available pre-packed columns. Reverse- phase preparative HPLC purification was performed on preparative HPLC instruments with UV and MS detection using a MeCN / water gradient with either TFA, formic acid, or NH4OH modifier. Unless otherwise noted, all KDdata presented in tables refers to the surface plasmon resonance assay that is described in the Biological Assay section.

[0134] Peptides were synthesized using standard solid phase synthesis using Fmoc / tert-butyl chemistry as exemplified in Chan, W. C.; White, P. D. “Fmoc Solid-Phase Synthesis: a Practical Approach”, Oxford University Press, Oxford, 2000; Steward, J.; Young, J. “Solid Phase Peptide Synthesis”, Pierce Chemical Company, Rockford, 1984.; N. L. Benoiton, “Chemistry of Peptide Synthesis”, CRC Press, New York, 2006; and Lloyd-Williams, P.; Albericio, F. “Chemical Approaches to the Synthesis of Peptides and Proteins”, CRC Press, New York, 1997.

[0135] The following examples are meant to be illustrative and should not be construed as further limiting. The contents of the figures and all references, patents, and published patent applications cited throughout this application are expressly incorporated herein by reference. Example 1:- -25927 1 Preparation of compound 1 -2-yl)acetic acid (1.75 g, 9.40 mmol)was suspended in toluene (47 mL) and oxalyl chloride (2.42 mL, 28.2 mmol) was added, and the reaction stirred for 2.5 hours. The mixture was filtered to remove particles, and the filtrate was concentrated to give 1.85 g of an oil that crystallized to give a solid.n dichloromethane (4.4 mL) and treated with trifluoroacetic acid (4.42 mL, 57.7 mmol). After stirring for 1 hour, the reaction was concentrated to dryness under reduced pressure, with heptane azeotrope to remove residual TFA (3 x 20 mL), to give 1d. MS: m / z 353.3 = [M+H].

[0138] Step C – Synthesis of compound 1e. Compound 1d (TFA salt, 2.35 g, 5.04 mmol) was dissolved in dichloromethane (25 mL) and treated with the acid chloride and DIEA (2.63 mL, 15.1 mmol). The reaction was allowed to stir for 30 minutes, then the reaction was quenched by the addition of water (25 mL), and the layers were separated. The aqueous layer was extracted with CH2Cl2(2 x 25 mL), and the combined organic layers were dried over MgSO4, filtered, concentrated, and purified by silica gel flash column chromatography, eluting with 0-5%MeOH / CH2Cl2+ 0.1% AcOH over 33 minutes (120-gram cartridge) to give 1e. MS: m / z 521.4 =[M+H]. - 50 -25927 ertyBlue synthesizer (CEM, Matthews, NC). The synthesis was started using 100 µmol H- Asp(OtBu)-H NovaSyn TG resin (Novabiochem), 0.21 mmol / g (CEM). Each amino acid coupling cycle used: 1) Amino acid or carboxylic acid as 0.2 M solution in DMF (0.5 mmol, 2.5 mL). (S)-2- ((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-(tert-butoxy)-5-oxopentanoic acid (Fmoc-Glu-OH), (((9H-fluoren-9-yl)methoxy)carbonyl)-L-isoleucine (Fmoc-Ile-OH), and 2-fluoroacetic acid were commercially available. 2) 0.45 M 1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate (HATU) in DMF (0.45 mmol, 1 mL) 3) N,N’-diisopropylethylamine (DIEA) in NMP (1 mmol, 0.5 mL)

[0140] Fmoc-deprotection steps were performed with 20% pyrrolidine in DMF for 60 sec at 90oC. Following solid-phase peptide synthesis, the resin was dried under vacuum for 2 hours. The dry resin was treated with 2 x 3 mL of TFA (10 min cycles), filtered, and washed with CH2Cl2(2 x 10 mL). The resin was suspended in 4 mL of AcOH / H2O / DCM / MeOH (10: 5: 63: 22) for 30 min, then filtered; this process was repeated an additional two times. The resulting solution was concentrated under reduced pressure and dried under high vacuum to give an oil. The crude material was purified using preparative HPLC using a Phenomenex Luna C185u 100A 250*21.2mm column and 5-95% gradient of acetonitrile in water, both buffered with 0.1% TFA. The HPLC fractions containing a pure peptide product were pooled and lyophilized to give compound 1 as a solid. MS: m / z 348.9 = [M-2H] / 2. - 51 -25927 Example 2 Compound 2 was made using the method described in Example 1 and substituting the appropriate reactants and / or reagents: Compound# Structure Observed m / z 2)Compound 3

[0141] Step A – Synthesis of compound 3a. The peptide sequence was assembled by solid phase synthesis. H-Asp(OtBu)-H NovaSyn TG resin (2.50 g, 0.50 mmol, 0.20 mmol / g) was transferred to a reaction vessel and swelled with 10 mL of DMF for 1 hour. - 52 -25927

[0142] The resin was washed with 20 mL of DMF (5 x 0.1 min) and followed by addition of Fmoc-Pro(4NHAlloc)-OH (1.00 mmol, 436.5 mg, 2.00 equiv.), HATU (0.95 mmol, 361 mg, 1.90 equiv.) and DIEA (2.00 mmol, 0.331 mL, 4.00 equiv.) in DMF (15 mL). The coupling reaction was mixed for 1h, filtered. After completing the coupling reaction, the resin was washed with 25 mL of DMF (3 x 0.1 min) prior to starting the next deprotection / coupling cycle.

[0143] Coupling of Fmoc-Pro(4NHAlloc)-OH: The resin was washed with 20 mL of DMF (5 x 0.1 min) and followed by addition of Fmoc-Pro(4NHAlloc)-OH (1.00 mmol, 436.5 mg, 2.00 equiv.), HATU (0.95 mmol, 361 mg, 1.90 equiv.) and DIEA (2.00 mmol, 0.331 mL, 4.00 equiv.) in DMF (15 mL). The coupling reaction was mixed for 1h, filtered. After completing the coupling reaction, the resin was washed with 25 mL of DMF (3 x 0.1 min). Deprotection of the Fmoc group was accomplished with 20 mL of 20% piperidine in DMF to the swollen resin for 30 min. After deprotection the resin was washed with 20 mL of DMF (5 x 0.1 min). The other Fmoc amino acids were incorporated using the same procedure outlined above. After incorporation of the last amino acid, the resin was washed with 20 mL of DMF (5 x 0.1 min), and 10%Ac2O / 5%NMM / 85%DMF (30 mL) was added, the reaction mixed for 30 mins, and then filtered.

[0144] Step B – Synthesis of compound 3b The resin was swollen with 20 mL DCM for 1 min then filtered, and this process repeated two additional times. The resin was treated with [Ph3P]4Pd (0.10 equiv.) and phenylsilane (10 equiv.) in DCM (20 mL), agitated at room temperature for 30 minutes, and filtered; this process was repeated an additional three times. The resin was washed with 0.5% DIEA / DMF and 0.5% Sodiumdiethyldithiocarbamatetrihydrate 5 times. Finally, the resin was washed with DMF (20 mL) 5 times.

[0145] Step C – Synthesis of compound 3c 4-fluorobenzoic acid was coupled using the same procedure as step A. After last step, the resin was washed with MeOH (50 mL) for 3 times, and dried under N2.

[0146] Step D – Synthesis of compound 3 The resin was treated with anhydrous TFA (10 mL) for 30 min, then filtered. The resin was then treated with 10% AcOH / 5%H2O / 63% DCM / 21% MeOH (50 mL) solution for 30 minutes and filtered; this process was repeated an additional two times. The reaction mixture was concentrated under reduced pressure. Semi-Preparative reverse phase HPLC was performed on a YMC-Actus 10 μm C18 column (30 mm x 250 mm) (GLX- 281). Separations were achieved using linear gradients of buffer B in A (Mobile phase A: water containing 0.075% TFA, mobile phase B: Acetonitrile (ACN)), at a flow rate of 20 mL / min (preparative). The Gradient was 10-40% B over 40 minutes. Fractions containing desired product were combined and lyophilized to provide compound 3. MS: m / z 636.5 = [M+H]. - 53 -25927 Example 4 Intermediate compound X was made using the method described in Example 3 Compound# Structure Observed m / z H)Compound Y

[0147] Compound Y is the1ve s o o o pou . - luoride was produced with a Siemens RDS-111 cyclotron. A target containing18O-H2O was irradiated with an 11-MeV proton beam, generating18F-fluoride. The18F-fluoride was trapped on an ion exchange resin. PET tracer identity, radiochemical purity, and specific activity were determined by evaluation on a Agilent 1100 LC system (Agilent Technologies) equipped with a FlowCount photodiode radiodetector (Bioscan Inc.). The product was purified using a Gemini C18150 × 10 mm, 5 μm HPLC column (Phenomex) at a flow rate of 5 mL / min. The mobile phase started at 15% acetonitrile / 85% 0.1% TFA to 60 % CH3CN / 40 % 0.1% TFA in 20 min. The final preparation was tested for chemical and radiochemical purity by means of an analytical HPLC system (Agilent) using an Gemini C18 150 × 4.6 mm, 5 μ HPLC column (Phenomex) at a flow rate of 1.0 mL / min. The mobile phase was 10% acetonitrile / 90% 0.1% TFA to 65% acetonitrile / 45% 0.1% TFA in 10 min. Compound 5 concentration was determined by means of an ultraviolet detector (254 nm). Confirmation of the identity of the product was determined by coinjection of a sample of - 54 -25927 Compound 5, and radiochemical purity was determined using a sodium iodide detector (Bioscan). Radiochemical purity was calculated from the percentage radioactivity attributed to isolated peak in the radioactive HPLC trace after integration of all radioactive peaks. The molar activity (GBq / μmol) of the radioligand was determined by withdrawing an aliquot (0.1 mL) of the preparation, determining the amount of radioactivity in a dose-calibrator, and correcting for decay from end of synthesis. The aliquot was evaluated by HPLC, and the ultraviolet response was compared against a calibration curve that was prepared with the unlabeled reference standard to determine the mass associated with the decay-corrected radioactivity of the injected aliquot.

[0148] [18F]N-Succinimidyl 4-fluorobenzoate ([18F]SFB) was prepared using the triflate salt of p-trimethylammonium benzoate (3 mg) purified by solid-phase extraction (LiChrolut EN – Millipore-Sigma) according to previous literature.(Wester, H.-J.; Hamacher, K.; Stöcklin, G., A comparative study of N.C.A. Fluorine-18 labeling of proteins via acylation and photochemical conjugation. Nuclear Medicine and Biology 1996, 23 (3), 365-372.) 2 ml of [18F]SFB in acetonitrile was concentrated at 70 ˚C under argon gas stream to ~0.5 ml. [18F]SFB in acetonitrile was cooled and Example 4 (3.7 mg, 5.90 µmol) in DMF (250 μl) and 0.2 M Na2HPO4 (250 μl) was added. The reaction mixture was heated at 65 °C for 15 min, diluted with water (1.5 ml) and loaded onto the high-performance liquid chromatography (HPLC) purification loop.prior to injection onto a semipreparation scale HPLC column. The fraction containing Example [18F]-5 (~9 min) was collected in a heated pear-shaped flask, evaporated under negative pressure, diluted with 0.9% sterile saline solution (6 mL), and transferred into a sterile vial. - 55 -25927 Example 6 Fmoc N H2N O O O NH tBu O HN Alloc tBuStep 1, Synthesis of 1b: 2.50 g of H-Asp(OtBu)-H NovaSyn TG resin (0.50 mmol, 0.20 mmol / g loading) was swollen with 100 mL of DMF (2 h). The resin was treated with (4S)-1-Fmoc-4-(Allyloxycarbonylamino)- L-proline (3.00 equiv.), HATU (2.85 equiv.) and DIEA (6.00 equiv.) dissolved in 20 mL DMF. The sample was filtered, and rinsed with DMF. Step 2, Synthesis of 1c: The resin 1b was swollen with 100 mL DMF and treated with PhSiH3 (10.00 equiv.) and Pd(PPh3)4(0.10 equiv.) for 15 min, then filtered. This process was repeated two additional times. The resin was treated with 4-fluorobenzoicacid (3.00 equiv.), HATU (2.85 equiv.), and DIEA (6.00 equiv.) dissolved in 20 mL DMF. Step 3, Synthesis of 76: The resin 1c was treated with 40 ml of 20% piperidine in DMF for 30 min, then washed with 50 mL of DMF (5 x 0.1 min). The resin was treated with Fmoc-Glu(OtBu)-OH, HATU (2.85 equiv.) - 56 -25927 and DIEA (6.00 equiv.) in 20 mL DMF for one hour. The sample was filtered, treated with 40 ml of 20% piperidine in DMF for 30 min, then washed with 50 mL of DMF (5 x 0.1 min). The resin was then treated with Fmoc-Ile-OH, HATU (2.85 equiv.) and DIEA (6.00 equiv.) in 20 mL DMF for one hour. The sample was filtered, treated with 40 ml of 20% piperidine in DMF for 30 min, then washed with 50 mL of DMF (5 x 0.1 min). The resin was then treated with Fmoc-Gly-Gly- OH, HATU (2.85 equiv.) and DIEA (6.00 equiv.) in 20 mL DMF for one hour. The sample was filtered, treated with 40 ml of 20% piperidine in DMF for 30 min, then washed with 50 mL of DMF (5 x 0.1 min). The resin was then treated with Fmoc-beta-Ala-OH, HATU (2.85 equiv.) and DIEA (6.00 equiv.) in 20 mL DMF for one hour. The sample was filtered, treated with 40 ml of 20% piperidine in DMF for 30 min, then washed with 50 mL of DMF (5 x 0.1 min). The resin was then treated with 2,2',2''-(2-(4-isothiocyanatobenzyl)-1,4,7-triazonane-1,4,7-triyl)triacetic acid (3.00 equiv.) and DIEA (6.00 equiv.) in DMF (15 mL) at 35-40℃ for 3 hours. The coupling reaction was monitored by ninhydrin color reaction. The resin was washed with 50 mL of DMF (5 x 0.1 min) and 50 mL of MeOH (3 x 0.1 min), then dried under vacuum. The resin was then treated with 25 mL TFA for 0.5 hr and drained. Then the resin was treated with 25 mL 10%ACOH / 5%H2O / 63%DCM / 21%MeOH (3 x 30 mins), and the filtrate was collected and spin-dried and lyophilized. The sample was purified using reversed-phase HPLC on a YMC-Actus 5 μm C18 column (30 mm x 250 mm) (GX-281) and eluting with a gradient of water containing 0.075% TFA and acetonitrile at a flow rate of 20 mL / min. Fractions containing desired material were combined and lyophilized to provide compound 76. MS: m / z = 615.5 [M+2H] / 2 Example 7 Compound 77 was prepared using the method described in Example 6 and substituting the appropriate reactants and / or reagents:- 57 -25927 Example 8 Synthesis of 78 - 81:[68Ga]-Gallium chloride was eluted from a IGG100 Gallium-68 generator (Eckert & Ziegler) using 5 ml of 0.1 M HCl and passed through a strong cation exchange cartridge (Phenomenex Strata-XC or Agilent Bond elute SCX). [68Ga]GaCl3 was then eluted from the cartridge with NaCl 1N / HCl 0.1N (0.8 mL) onto a solution containing 76 (40 μg, 40 μL, 813.47 μmolar, 1 Eq, 0.033 μmol) in ammonium acetate 0.2 M - pH 5.5 (1.5 mL), and heated at 70 °C for 15 min . Radiochemical purity was monitored by radio-HPLC (Onyx Monolithic C18 column (100 mm × 3 mm,) eluted with a gradient from 5 % acetonitrile in aqueous (0.1 % HCO2H) to 45 % acetonitrile at a flow rate of 1 ml / min over 7 min). Synthesis of 79:[111In]-Indium chloride (Jubilant Radiopharmacies, Folcroft, PA) was added to a solution containing 76 (40 μg, 40 μL, 813.47 μmolar, 1 Eq, 0.033 μmol) in ammonium acetate 0.2 M - pH 5.5 (1.5 mL). The mixture was heated at 70 °C for 15 min. Radiochemical purity was monitored by radio-HPLC (Onyx Monolithic C18 column (100 mm × 3 mm,) eluted with a gradient from 5 - 58 -25927 % acetonitrile in aqueous (0.1 % HCO2H) to 45 % acetonitrile at a flow rate of 1 ml / min over 7 min). Synthesis of 80: Compound 80 was prappropriate reactants and / or reagents. Synthesis of 81:containing 77(40 μg, 40 μL, 784.07 μmolar, 1 Eq, 0.031 μmol) in ammonium acetate 0.2 M - pH 5.5 (1.5 mL). The mixture was heated at 70 °C for 15 min. Radiochemical purity was monitored by radio-HPLC (Onyx Monolithic C18 column (100 mm × 3 mm,) eluted with a gradient from 5 % acetonitrile in aqueous (0.1 % HCO2H) to 45 % acetonitrile at a flow rate of 1 ml / min over 7 min).

[0149] As readily understood by the ordinary skilled artisan, Compounds 4 through 81 in Table 1 were made using the method described in Examples 1 through 8 and substituting the appropriate reactants and / or reagents. - 59 -25927 Granzyme B biochemical inhibition assay

[0150] The tested compounds were manually prepared in a 2-fold serial dilutions in DMSO, spanning 200 μM to 0.39 μM in a Labcyte Echo qualified low dead volume 384-well plate. Then an ECHO acoustic liquid handler was used to dispense 125 nL of each compound from 384-well plate into a PerkinElmer Proxiplate-384 Plus F assay plate. For the positive control of inhibitor, (S)-3-((3S,6S)-3-((2S,3S)-2-acetamido-3-methylpentanamido)-4-oxo-1,2,3,4,6,7- hexahydroazepino[3,2,1-hi]indole-6-carboxamido)-4-oxobutanoic acid (see C. A. Willoughby et al. / Bioorg. Med. Chem. Lett.12 (2002) 2197–22002199) was used at 2 μM assay concentration. For no inhibition control, DMSO was used. Each compound was run in duplicate.

[0151] Once the compounds were dispensed in the plates, enzyme mix and substrate mix were prepared. Enzyme mix was prepared by preparing a mixture of 300 μL of a granzyme B (final concentration 8 nM) and 10.8 mL of assay buffer (from Sigma kit). Substrate mix was prepared by combining 500 μL substrate (from Sigma kit) and 3 mL of assay buffer (from Sigma kit). Enzyme mixture was loaded on BioRaptr and 9.25 μL were added to each well of the assay plate containing compounds. The plate was incubated at room temperature under a metal gasket cover for 30 minutes. Subsequently, 3.125 μL of substrate mix were added to each well using the BioRaptr, reaching a final assay volume of 12.5 μL, and the plate was monitored using a Pherastar FS by 1 read per minute for 30 minutes in fluorescence mode, excitation wavelength is 400 nm and emission wavelength is 510 nm.

[0152] The data analysis was performed first in Microsoft Excel®, where the slopes of the measured fluorescence from each sample were determined using MARS software from BMG LABTECH. These rates were then multiplied by 10,000 for visualization purposes. The rates, multiplied by 10,000, were normalized to 0% and 100% activity based on the average values of the samples with either 2 μM inhibitor (S)-3-((3S,6S)-3-((2S,3S)-2-acetamido-3- methylpentanamido)-4-oxo-1,2,3,4,6,7-hexahydroazepino[3,2,1-hi]indole-6-carboxamido)-4- oxobutanoic acid, or no inhibitor respectively. All samples were then normalized against these values using the formula = {100 - [(sample - 100%inhib) / 0%inhib]}*100 to achieve a normalized percent inhibition. Normalized percent inhibition versus concentration inhibitor was then plotted in Graphpad Prism and a variable slope (four parameters) fit was calculated for each inhibitor. Table 2 lists inhibition data (IC50 values) for representative compounds of this disclosure. In-vivo preclinical imaging:

[0153] Diseased model: Graft versus host disease (GvHD) model was selected to evaluate radiolabeled granzyme-B peptides due to presence of human granzyme-B after onset of disease. - 60 -25927 Ten million human peripheral blood mononuclear cell (PBMC) were engrafted in immunocompromised NOG (NOD / Shi-scid / IL-2Rγ) mice. Body weight was monitored routinely. Once the mice started to lose weight (indication of onset of disease), PET / CT scans were performed. To determine target specificity, NOG mice without human PBMC were used as control.

[0154] Mice were intravenously injected with approximately 150 uCi (35 Ci / mmol) of18F- labeled Compound Y and 45-minute whole body static PET / CT scans were performed 1 h after injection. Attenuation correction was performed using low-dose CT scan. Regions of interests (ROI) were manually drawn over tissues of interest to determine mean standardized uptake value (SUVmean).

[0155] PET imaging performed with18F-labeled anti-granzyme-B (Compound Y) from a hPBMC donor revealed significantly higher uptake in lung, liver, and bone in GvHD model than NOG control 3 week after hPBMC engraftment and onset of GvHD as observed by body weight loss (See Figure 1). Table 2 COMPOUND# Granzyme B Ki (Human, v2)[nM] 1 14.025927 COMPOUND# Granzyme B Ki (Human, v2)[nM] 29 33.2 30 364 ND=Not determined- 62 -

Claims

25927 WHAT IS CLAIMED IS: 1.or a pharmaceutically acceptable salt thereof, wherein, R1is C1-6alkyl; R2is -NHC(O)R4, or (CH2)nheteroaryl, wherein said heteroaryl is optionally substituted with 1 to 3 Rxsubstituents; R3is hydrogen, or =O; R4is selected from C1-6alkyl, -C1-3haloalkyl, and –(CH2NHC(O))1-2(CH2)qNH-R4b; R4bis selected from:chment; R7and R8are independently selected from hydrogen and C1-6 alkyl, wherein said alkyl is optionally substituted with 1 to 3 groups of Rx, or together R7and R8form =O, provided that when R7and R8form =O, R3is hydrogen; R5is selected from –O(CH2)naryl, –O(CH2)nheteroaryl, heteroaryl, -NHC(O)(CH2)nC3-6cycloalkyl, -NHC(O)(CH2)naryl, -NHC(O)(CH2)nheteroaryl, -NHC(O)(CH2)nheterocyclyl, - OC(O)NH(CH2)naryl, -OC(O)(CH2)naryl, -NHCHO, -NHC(O)C1-6alkyl, and NH2, wherein said alkyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl are optionally substituted with 1 to 3 Rxsubstituents; - 63 -25927 R6is -C(O)NHCH(CH2COOH)CH=O, or -C(O)NHCH2heteroaryl, wherein said heteroaryl is optionally substituted with 1 to 3 Rxsubstituents; Rxis selected from C1-6alkyl, halogen, OH, OC1-6alkyl, -CO2H, -C1-3haloalkyl, -(CH2)naryl, - (CH2)nheteroaryl, and (CH2)nheterocyclyl, wherein said alkyl, aryl, heteroaryl, and heterocyclyl are optionally substituted with 1 to 3 Ry; Ryis selected from C1-6alkyl, OC1-6alkyl, halogen, CN, and -C1-3haloalkyl; q is 1, 2, or 3; and n is 0, 1, 2, or 3.

2. The compound according to claim 1, or a pharmaceutically acceptable salt thereof, wherein R1is -CH(CH3)2 or C(CH3)CH2CH3; and R3is hydrogen or =O.

3. The compound according to any of claims 1 and 2, or a pharmaceutically acceptable salt thereof, wherein R2is -NHC(O)R4.

4. The compound according to any of claims 1-3, or a pharmaceutically acceptable salt thereof, wherein R4is selected from -(CH2)nCH3, CH(CH3)2, -C(CH3)CH2CH3, CF3, -CH2F, and - (CH2)2CH2F.

5. The compound according to any of claims 1-4, or a pharmaceutically acceptable salt thereof, wherein R2is optionally substituted (CH2)nheteroaryl.

6. The compound according to any of claims 1-3, or a pharmaceutically acceptable salt thereof, wherein R3is hydrogen; and R7and R8together form =O.

7. The compound according to any of claims 1-3, or a pharmaceutically acceptable salt thereof, wherein R3is =O.

8. The compound according to any of claims 1-5 and 7, or a pharmaceutically acceptable salt thereof, wherein R7is H; and R8is C1-6 alkyl, wherein said alkyl is optionally substituted with 1 to 3 Rxsubstituents.

9. The compound according to any of claims 1-5, 7 and 8, or a pharmaceutically acceptable salt thereof, wherein R7is H; and R8is -CH2 or CH(CH2)2CO2H. - 64 -25927 10. The compound according to any of claims 1-9, or a pharmaceutically acceptable salt thereof, wherein R5is selected from –O(CH2)naryl, -NHC(O)(CH2)nC3-6cycloalkyl, - NHC(O)(CH2)naryl, -NHC(O)(CH2)nheteroaryl, -NHCHO, and -NHC(O)C1-6 alkyl, wherein said alkyl, cycloalkyl, aryl, and heteroaryl are optionally substituted with 1 to 3 Rxsubstituents.

11. The compound according to any of claims 1-10, or a pharmaceutically acceptable salt thereof, wherein R6is -C(O)NHCH(CH2COOH)CH=O.

12. The compound according to any of claims 1-10, or a pharmaceutically acceptable salt thereof, wherein R6is C(O)NHCH2heteroaryl.

13. The compound according to claim 1 of Formula II: or a pharmaceutically acce14. The compound according to claim 13, or a pharmaceutically acceptable salt thereof, wherein R7is H; R8is -CH2, or -CH(CH2)2CO2H; R3is =O; R1is -CH(CH3)2or - C(CH3)CH2CH3; and R2is -NHC(O)R4or -(CH2)nheteroaryl.

15. The compound according to claim 1 of Formula III:- 65 -25927 III or a pharmaceutically acceptable salt thereof, wherein R6ais selected from heteroaryl optionally substituted with 1 to 3 Rxsubstituents.

16. The compound according to claim 15, or a pharmaceutically acceptable salt thereof, wherein R7is H; R8is -CH2, or -CH(CH2)2CO2H; R3is =O; R1is -CH(CH3)2or - C(CH3)CH2CH3; R2is -NHC(O)R4or -(CH2)nheteroaryl; and R6ais selected from optionally substituted triazolyl, tetrazolyl, imidazolyl, and pyrazolyl.

17. The compound according to claim 1 of Formula IV:or a pharmaceutically acceptable salt thereof.

18. A compound selected from: ,- 66 -25927 O OH H O N O O N H O N OH ,- 67 -25927 ,- 68 -25927- 69 -25927 ,- 70 -25927 ,- 71 -2592719. The compound of any one of Claims 1-18, or a pharmaceutically acceptable salt thereof, which is a binder of granzyme B.

20. The compound of any one of Claims 1-18, or a pharmaceutically acceptable salt thereof, which is an inhibitor of granzyme B.

21. A method of imaging granzyme B in a cell or tissue comprising contacting the cell or tissue with a compound any one of Claims 1-18, or a pharmaceutically acceptable salt thereof, and imaging the cell or tissue with a suitable imaging technique, thereby imaging granzyme B in the cell or tissue.

22. A method for treating a disease where granzyme B is implicated, comprising administering to a subject in need thereof a compound of any one of Claims 1-18, or a pharmaceutically acceptable salt thereof.

23. Use of any one of the compounds of Claims 1-18, or a pharmaceutically acceptable salt thereof, for treating a disease where granzyme B is implicated.

24. Use of any one of the compounds of Claims 1-18, or a pharmaceutically acceptable salt thereof, for imaging granzyme B in the cell or tissue.

25. A pharmaceutical composition comprising a compound of any one of claims 1-18, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or excipient. - 72 -

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