EPHA2 targeting radioligand compounds and methods thereof

Abstract

Disclosed herein are EphA2 targeting compounds or pharmaceutically acceptable salt or radioactive metal complex thereof, and methods of their use in the treatment of EphA2 positive cancer. In certain embodiments, the compound is a Targefrin monomer or dimer compound complexed with a radiometal, such as lutetium-177. In certain embodiments, the compound is an irreversible covalent binder of EphA2. In certain embodiments, the compound is a cyclic compound.

Description

[0001] EPHA2 TARGETING RADIOLIGAND COMPOUNDS AND METHODS THEREOF 1. CROSS REFERENCE TO RELATED APPLICATION

[0002] This application claims priority to United States Provisional Application Number 63 / 744,735 filed on January 13, 2025. The entire content of the application referenced above is hereby incorporated by reference herein.

[0003] 2. GOVERNMENT FUNDING

[0004] This invention was made with government support under NS107479 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0005] 3. BACKGROUND OF THE INVENTION

[0006] Systemic and localized radiation are often used to reduce tumor burden or as prevention of tumor spread or recurrence in patients. However, while this approach is effective in killing rapidly dividing cancer cells, similar to chemotherapy, systemic toxicity is a major limitation of the approach. Improved approach for delivering radioligand to cancer cells are needed.

[0007] 4. SUMMARY OF THE INVENTION

[0008] Certain embodiments of the invention provide an EphA2 targeting compound, or pharmaceutically acceptable salt, or metal complex thereof as described herein (e.g., a reversible binder of EphA2, an irreversible covalent binder of EphA2, and / or a cyclic compound, or a radioactive metal complex thereof).

[0009] Certain embodiments of the invention provide a compound of Formula (I)

[0010] X'-R. (I)

[0011] or a pharmaceutically acceptable salt or metal complex thereof, wherein:

[0012] R is -L°-R5, -L'-R6, or

[0013]

[0014] L° is a linking group (e.g., a solubilizing group);

[0015] R5is H, (C2-Ce)alkanoyl, a chelating group, or -L4-D;

[0016] L4is absent or a linking group;

[0017] D is the residue of a drug;

[0018] L1is absent or a linking group (e.g., a solubilizing group) of the formula

[0019]

[0020] each n is independently 1 to 100 (e.g., 2 to 100, 2 to 30, 2 to 10, 1 to 30, or 1 to 10); R6is -NH2 or -(CH2)XNHC(=O)CH3;

[0021] R7is H or (Ci-C3)alkyl (e.g. methyl);

[0022] Y is -L°-R5, or -L'-R6;

[0023] each of a, g, and x is independently 1, 2, or 3;

[0024] each of X1and X2is of the formula

[0025]

[0026] each R° is independently CH2, NH, N(Ra), or O, wherein each Rais independently (Ci- Ce)alkyl or (C2-Ce)alkanoyl;

[0027] each R1and R2is independently (C2-Ce)alkynyl or optionally substituted (Ci-Ce)alkyl, which optional substitution is with azido, hydroxy, or thiol; or

[0028] each R1and R2are taken together to form a linking group L3;

[0029] each R3is independently benzyl optionally substituted with one or more halo or is of the formula

[0030]

[0031] each Z is independently CH2, NH, or O;

[0032] each R3ais independently H, -SO2F, -OSO2F, -SO2-(C5-Ce)heteroaryl (e.g., SO2-tetrazole), (C3-C7)heterocycle (e.g., oxiranyl, or cyclic imine such as 1 -pyrroline), acrylamide (-NHC(=O)CH=CH2), halo substituted (C2-Ce)alkanoyl (e.g., halo substituted on the a carbon next to the oxo(=O) group), or isothiocyanate (-N=C=S);

[0033] eachR3bis independently (Ce-Cio)aryl, (Ci-Ce)alkyl, (Ci-Ce)alkoxy, (C3-Ce)cycloalkyl, -CH2O(Ci-Ce)alkyl, -CN, -C(=O)NH2, (C5-Ce)heteroaryl, (C3-C7)heterocycle (e.g., aziridinyl, or oxiranyl), hydroxy, nitro, -0(Ce-Cio)aryl, -O(C3-C6)cycloalkyl, -O(C5-Ce)heteroaryl, or -O(C3-C7)heterocycle; andeach of d, e, and f is independently 0, 1, 2, or 3.

[0034] In certain embodiment, the compound is not 152E2 or 152E3.

[0035] In certain embodiment, at least one (e.g., one or both) R3is not benzyl optionally substituted with one or more halo.

[0036] In certain embodiment, the invention provides a compound having structure of Formula (I), (e.g., an irreversible, covalent binder of EphA2), wherein in at least one of (e.g., one or both) of X1and X2, R3is independently

[0037]

[0038] In certain embodiment, d and e are not simultaneously 0 when Z is CEE.

[0039] In certain embodiment, each of d, e, and f is independently 1, 2, or 3.

[0040] In certain embodiment, each Z is independently NH, or O.

[0041] In certain embodiment, R3ais not H.

[0042] In certain embodiment, the invention provides a compound having structure of Formula (I), wherein, in at least one (e.g., one or both) of X1and X2, each R1and R2is independently (C2-Ce)alkynyl or substituted (Ci-Ce)alkyl, which substitution is with azido, or thiol; or in at least one (e.g., one or both) of X1and X2, R1and R2are taken together to form a linking group L3.

[0043] In certain embodiment, the invention provides a compound having structure of Formula (I), (e.g., a cyclic compound), wherein, in at least one (e.g., one or both) of X1and X2, R1 —R2are taken together to form a linking group L3.

[0044] In certain embodiment, the invention provides a compound having structure of Formula (I), (e.g., a compound having a PEG or poly-sarcosine tail), wherein R is -L'-R6, or Y is -LkR6.

[0045] Certain embodiments of the invention provide a method of treating cancer, which comprises administering a therapeutically effective amount of a compound or pharmaceutically acceptable salt or metal complex thereof described herein to a patient in need thereof.

[0046] Certain embodiments of the invention provide a pharmaceutical composition comprising a compound as described herein or a pharmaceutically acceptable salt or metal complex thereof, and a pharmaceutically acceptable carrier.

[0047] Certain embodiments of the invention provide a compound as described herein or a pharmaceutically acceptable salt or metal complex thereof for the prophylactic or therapeutic treatment of cancer, or for use in medical imaging (e.g., magnetic resonance imaging (MRI), or Positron Emission Tomography (PET)).Certain embodiments of the invention provide the use of a compound as described herein or a pharmaceutically acceptable salt or metal complex thereof to prepare a medicament for treating cancer or to prepare an imaging agent for medical imaging.

[0048] Certain embodiments of the invention provide a compound as described herein or a pharmaceutically acceptable salt or metal complex thereof for use in medical therapy or medical imaging.

[0049] Certain embodiments of the invention provide a method (e.g., an imaging and / or a treatment method), which comprises 1) administering a first compound or pharmaceutically acceptable salt or metal complex thereof as described herein to a patient in need thereof, 2) generating one or more medical image of the patient from a MRI or PET machine, and 3) optionally diagnosing the patient as having cancer (e.g., EphA2 positive cancer) and / or optionally administering an anti-cancer drug (e.g., a second compound or pharmaceutically acceptable salt or metal complex thereof as described herein) to the patient.

[0050] 5. BRIEF DESCRIPTION OF THE FIGURES

[0051] Figure 1. Mode of Action of Targefrin-monomer-radioisotope conjugate.

[0052] Figure 2. Mode of Action of Targefrin-dimer-radioisotope conjugate.

[0053] Figure 3. DELFIA displacement assay curves for a Targefrin-monomer-DOTA reversible compound (152G5), and an irreversible agent Targefrin-monomer-covalent-DOTA (152F11). The covalency to Tyr 48 residue of EphA2 increased the affinity of the agent for EphA2 down to the limit of detection of the assay.

[0054] Figure 4. Thermal denaturation curve of EphA2 (left), EpHA2 in presence of Targefrin-monomer (middle at +3.91°C), or EphA2 in presence of Targefrin-monomer-covalent (152F11) (right, at + 10.82°C). The covalent agent increases thermal stability significantly.

[0055] Figure 5. SDS-Gel of EphA2 collected in absence and presence of Targefrin-monomer-covalent (152F11). The MW band shift observed in the presence of the covalent agent indicates the formation of a stable covalent adduct between Targefrin-monomer-covalent and EphA2.

[0056] Figure 6. DELFIA displacement curves for Targefrin-dimer (152E3) and the covalent agent Targefrin-dimer-covalent (152F10). The covalency to Tyr 48 increased the affinity of the agent for EphA2 down to the limit of detection of the assay.

[0057] Figure 7. SDS-Gel of EphA2 collected in presence of DMSO control only, in presence of Targefrin-dimer-covalent (152F10), or in presence of Targefrin-dimer non-covalent (152E3). The two bands observed with Targefrin-dimer indicate the single covalent adduct and the ternary covalent adduct (at around 45 kDa), both indicating the formation of a stable complex.Figure 8. Targefrin-monomer-DOTA (152G5), results in a Kd of 12.5 nM by ITC, which is comparable to Targefrin-monomer.

[0058] Figure 9. DELFIA displacement curves for Targefrin-dimer-DOTA (152G12).

[0059] Figure 10. Targefrin-monomer-covalent (152F11) Time Course. lOpM EphA2 in 25 mM TRIS pH 75, 150 mM NaCl has been incubated at the reported time points with lOOpM of Targefrin-monomer-covalent. EphA2 molecular weight (MW) is 23,373 Da. The higher molecular weight band is due to the covalent reaction with the covalent irreversible agent 152F11 (MW = 1,812 Da), giving a final adduct of a MW of 25,165 Da.

[0060] Figure 11. Targefrin-monomer-covalent-DOTA (152G8) Time Course. lOpM EphA2 in 25 mM TRIS pH 75, 150 mM NaCl has been incubated at the reported time points with lOOpM of Targefrin-monomer-covalent. EphA2 MW is 23,373 Da. The higher molecular weight band is due to the covalent reaction with the covalent irreversible agent 152G8 (MW = 2,483 Da), giving a final adduct of a MW of 25,836 Da.

[0061] Figure 12. Kinact / Ki calculation done by DELFIA displacement assay, of Targefrin-monomer-covalent-DOTA (152G8). Kinact / Ki is an indication of the efficacy of the covalent binding. 152G8 has a Kinact / Ki in the same order of magnitude as FDA-approved irreversible agents, such as Sotorasib.

[0062] Figure 13. Protein Thermal Shift (PTS) denaturation curves of EphA2 in the presence of Targefrin-monomer-DOTA (152G5), Targefrin-monomer-covalent-DOTA (152G8), and a nonactive scrambled version of Targefrin-monomer-DOTA. lOpM of EphA2 in 25 mM TRIS pH 7.5, 150 mM NaCl, was incubated for 2 h @ RT with lOOpM of each agent. 10X SYPRO Orange dye was used.

[0063] Figure 14. Targefrin-monomer-DOTA (152G5) selectivity for EphA2, as tested by ITC, over two closely related proteins, EphA4 and EphA3.

[0064] Figure 15. Targefrin-monomer-DOTA (152G5) and Targefrin-monomer-covalent-DOTA (152G8) selectivity were tested by Protein Thermal Shift (PTS) denaturation curve, against two closely related proteins, EphA4 and EphA3. lOpM of EphA2, EphA4, and EphA3 in 25 mM TRIS pH 7.5, 150 mM NaCl, was incubated for 2 h at room temperature (RT) with lOOpM of each agent. 10X SYPRO Orange dye was used.

[0065] Figure 16. Targefrin-monomer-covalent-DOTA (152G8) selectivity for EphA2 was tested by SDS-Gel, over two closely related proteins, EphA4 and EphA3. A higher Molecular weight band is visible only in the presence of EphA2, due to the covalent adduct formation.

[0066] Figure 17. Comparison of the affinity and thermodynamic features of Targefrin-Cyclic (152H2), its linear analog (152H3), and Targefrin-monomer (152E2). Targefrin-Cyclic, given its constrained nature, has a bigger entropic contribution compared to the other agents.Figure 18. Targefrin-Cyclic (152H2) shows a similar melting temperature compared to Targefrin-monomer (152E2), and its non-cyclic analog (152H3). ATmwas tested by Protein Thermal Shift (PTS) denaturation curve with lOpM of EphA2 in 25 mM TRIS pH 7.5, 150 mM NaCl, with lOOpM of each agent. 10X SYPRO Orange dye was used.

[0067] Figure 19. Radiolabeling with 177Lu of Targefrin-monomer-DOTA (152G5), Targefrin-monomer-covalent-DOTA (152G8), and Targefrin-dimer-DOTA (152G12). Radio-HPLC QC Results: Radiochemical purity > 99%. Molar activity of all radiolabeled peptides ranged from 75 - 99 mCi / pmol (2.7 - 3.6 GBq / pmol).

[0068] Figure 20. Radiolabeling with 177Lu of Targefrin-monomer-DOTA (152G5), Targefrin-monomer-covalent-DOTA (152G8), and Targefrin-dimer-DOTA (152G12). iTLC-HPLC QC Results: Radiochemical purity > 99%. Molar activity of all radiolabeled peptides ranged from 75 - 99 mCi / pmol (2.7 - 3.6 GBq / pmol)

[0069] 6. DETAILED DESCRIPTION

[0070] Certain embodiments of the invention provide a compound, or a salt or a metal complex thereof as described herein, or a method of use as described herein, e.g., which comprises administering a compound, or a salt or a metal complex thereof, as described herein to a patient in need of (e.g., for imaging or treating a cancer).

[0071] Systemic and localized radiation are often used to reduce tumor burden or as prevention of tumor spread or recurrence in patients. However, while this approach is effective in killing rapidly dividing cancer cells, similar to chemotherapy, systemic toxicity is a major limitation of the approach. Novel strategies aimed at delivering radioligands directly to cancer cells involve a tumor homing agent that can be conjugated with radioligands. For example, the FDA has recently approved the PSMA-targeted radioligand lutetium-177-PSMA-617, for patients with PSMA-positive metastatic castration-resistant prostate cancer that has failed androgen receptor pathway¬ inhibitors and taxane chemotherapy. Genetic and cellular studies identified the EphA2 receptor as a possible target for targeted delivery of chemotherapy, by conjugating EphA2 targeting agents such as Targefrin, with cytotoxic drugs, such as a taxane. As described herein, Targefrin and analog compounds are designed herein to deliver radioligands to EphA2 expressing tumors.

[0072] Targeted radioligands can be devised by new compounds in which Targefrin is synthesized with a metal chelating agent. In a first implementation of this strategy, we prepared Targefrin-monomer-DOTA, where DOTA can be used to tightly chelate any radioisotope, including Lutenium-177, or other elements that can be used for imaging purposes, such as Gadolinium (e.g., for MRI) or Gallium (e.g., for PET). In a second application, to potentially further increase the residence time of the homing agent to EphA2 expressing tumor cells, we alsodevised an irreversible Targefrin ligand. This was accomplished by modifying the side chain of the Phe residue of Targefrin to reach a Tyr residue in the binding site of EphA2. For the reaction to take place we introduced a mild electrophile such as a substituted sulfonyl fluoride of fluorosulfate.

[0073] In addition, Targefrin-dimer can also actively deliver its cargo to EphA2 expressing cancer cells. Hence, we also derivatized Targefrin-dimer with radioligand chelating groups such as DOTA or DTPA to actively deliver radioisotopes to tumor cells. Hence, Targefrin-dimer-DOTA, for example, can be prepared and conjugated with radioligands such as Lutenium-177. Our data with those agents (e.g., see Example 1) suggest that Targefrin-monomer and Targefrin-monomer-covalent could be deployed for targeted delivery of radioisotopes. The dimers could also be deployed for targeted delivery of radioisotopes, although the reversible targefrin-dimer seems to be potentially more advantageous since it appears that the targefrin-dimer-covalent does not induce EphA2 degradation as effectively as reversible targefrin-dimer.

[0074] Accordingly, disclosed herein are EphA2 targeting compounds or pharmaceutically acceptable salt or metal complex thereof, and methods of their use in the imaging and / or treatment of EphA2 positive cancer. In certain embodiments, the compound is a Targefrin monomer or dimer compound complexed with a radiometal, such as lutetium- 177. In certain embodiments, the compound is an irreversible covalent binder of EphA2, a cyclic compound, and / or with improved solubility (e.g., with a PEG or sarcosine tail).

[0075] 6.1. COMPOUNDS

[0076] Certain embodiments of the invention provide a compound of Formula (I)

[0077] Xx-R (I)

[0078] or a pharmaceutically acceptable salt or metal complex thereof, wherein:

[0079] R is -L°-R5, -L'-R6, or

[0080]

[0081] L° is a linking group;

[0082] R5is H, (C2-Ce)alkanoyl, a chelating group, or -L4-D;

[0083] L4is absent or a linking group;

[0084] D is the residue of a drug;

[0085] L1is absent or a linking group of the formula

[0086]

[0087] each n is independently 1 to 100 (e.g., n is 2 to 100, 2 to 30, 2 to 20, 2 to 10, or 1 to 30); R6is -NH2or -(CH2)XNHC(=O)CH3;

[0088] R7is H or (Ci-C3)alkyl (e.g., methyl);

[0089] Y is -L°-R5, or -L'-R6;

[0090] each of a, g, and x is independently 1, 2, or 3;

[0091] each of X1and X2is of the formula

[0092]

[0093] each R° is independently CH2, NH, N(Ra), or O, wherein each Rais independently (Ci-C6)alkyl, or (C2-C6)alkanoyl (e.g., C(=O)CH3);

[0094] each R1and R2is independently (C2-Ce)alkynyl or optionally substituted (Ci-Ce)alkyl, which optional substitution is with azido, hydroxy, or thiol; or

[0095] each R1and R2are taken together to form a linking group L3;

[0096] each R3is independently benzyl optionally substituted with one or more halo or is of the formula

[0097]

[0098] each Z is independently CH2, NH, or O;

[0099] each R3ais independently H, -SO2F, -OSO2F, -SO2-(C5-Ce)heteroaryl (e.g., SO2-tetrazole), (C3-C7)heterocycle (e.g., oxiranyl, or cyclic imine such as 1 -pyrroline), acrylamide (-NHC(=O)CH=CH2), halo substituted (C2-Ce)alkanoyl (e.g., halo substituted on the a carbon next to the oxo(=O) group), or isothiocyanate (-N=C=S);

[0100] eachR3bis independently (Ce-Cio)aryl, (Ci-Ce)alkyl, (Ci-Ce)alkoxy, (C3-C6)cycloalkyl, -CH2O(Ci-Ce)alkyl, -CN, -C(=O)NH2, (C5-Ce)heteroaryl, (C3-C7)heterocycle (e.g., aziridinyl, or oxiranyl), hydroxy, nitro, -0(Ce-Cio)aryl, -O(C3-C6)cycloalkyl, -O(C5-Ce)heteroaryl, or -O(C3-C7)heterocycle; andeach of d, e, and f is independently 0, 1, 2, or 3.

[0101] In certain embodiment, the compound is not 152E2 or 152E3.

[0102] In certain embodiment, at least one (e.g., one or both) R3is not benzyl optionally substituted with one or more halo.

[0103] In certain embodiments, the compound comprises a chelating group, wherein R is -L°-R5and / or Y is -L°-R5.

[0104] In certain embodiments, L°is

[0105]

[0106] , each of b and c is independently 0, 1, 2, or 3. In certain embodiments, R is

[0107]

[0108] , wherein L2is a linking group and each of b and c is independently 0, 1, 2, or 3.

[0109] Metal chelating agents or groups are known in the art and also described herein. In certain embodiments, the metal chelating agent or group comprises a plurality of carboxy groups (e.g., 2, 3, 4, 5, or more) and a plurality e.g., 2, 3, 4, or more) of tertiary amine groups. In certain embodiments, the metal chelating agent or group comprises 3 or 4 carboxy groups and 2, 3, or 4 tertiary amine groups.

[0110] In certain embodiments, the metal chelating agent or group is DOTA (1,4,7,10-tetraazacyclododecane- 1,4,7, 10-tetraacetic acid), DTPA (diethylenetriaminepentaacetic acid); EDTA (ethylenediaminetetraacetic acid), NOTA (l,4,7-triazacyclononane-l,4,7-triacetic acid), or a residue thereof (e.g., wherein an -OH of one carboxy group has been replaced by an amide bond with -NH-).

[0111] In certain embodiments, the compound forms a metal complex wherein a metal (e.g., a radioactive metal) is complexed with the R5chelating group (e.g., DOTA).

[0112] In certain embodiments, the metal complex comprises Lutetium- 177, Gadolinium, Gallium-68, Terbium-161, Holmium-166, Samarium-153, Promethium- 149, Terbium-149, Terbium-152, Terbium-155, Techneti um-99, Actinium-225, Indium-Ill, Copper-64, Copper-67, Yttrium-90, Lead-212, Scandium-47, Scandium-44, or Bismuth-213 that is complexed with the R5chelating group.In certain embodiments, the metal complex comprises a radiolanthanide. In certain embodiments, the metal complex comprises Holmium-166, Samarium-153, Promethium- 149, Terbium-149, Terbium-152, Terbium-155, or Technetium-99 that is complexed with the R5chelating group.

[0113] In certain embodiments, the metal complex comprises Lutetium- 177, Gadolinium, Gallium-68, or Copper-64 that is complexed with the R5chelating group. In certain embodiments, the metal complex comprises Lutetium- 177.

[0114] In certain embodiments, R5is a chelating group of the formula

[0115]

[0116] In certain embodiments, the compound is a monomer compound, wherein R is -L°-R5or - L^R6.

[0117] In certain embodiments, L° is a hydrocarbon chain (e.g., branched or unbranched, saturated or unsaturated) comprising from 1 to 340 carbon atoms (e.g., 1 to 320, 1 to 300, 1 to 260, 1 to 220, 1 to 200, 1 to 150, 1 to 120, 1 to 110, 1 to 100, 1 to 90, 1 to 80, 1 to 60, 1 to 40, 1 to 30, 1 to 20, 1 to 10, 1 to 9, 1 to 8, 1 to 6, 1 to 5, 1 to 3, 2 to 220, 2 to 200, 2 to 150, 2 to 100, 2 to 80, 2 to 60, 2 to 40, 2 to 30, 2 to 20, 2 to 10, 2 to 9, 2 to 8, 2 to 6, 2 to 5, or 2 to 3), wherein one or more carbon atoms of the chain is optionally replaced with O, S, N(RS), or an optionally substituted divalent (Ce-C2o)aryl, (Cs-C2o)heteroaryl, (C3-Cio)heterocycle, or (C5-Ce)carbocycle, which optional substitution is with one or more of C(=0)NH2, halo, hydroxy, or oxo (=0); and Rsis H or (Ci-Ce)alkyl.

[0118] In certain embodiments, L° is

[0119]

[0120] In certain embodiments, R is

[0121]

[0122] In certain embodiments, L2is a hydrocarbon chain (e.g., branched or unbranched, saturated or unsaturated) comprising from 1 to 320 carbon atoms (e.g., 1 to 300, 1 to 260, 1 to 220, 1 to 200, 1 to 150, 1 to 100, 1 to 90, 1 to 80, 1 to 60, 1 to 40, 1 to 30, 1 to 20, 1 to 10, 1 to 9, 1 to 8, 1 to 6, 1 to 5, 1 to 3, 2 to 220, 2 to 200, 2 to 150, 2 to 100, 2 to 80, 2 to 60, 2 to 40, 2 to 30, 2 to 20, 2 to 10, 2 to 9, 2 to 8, 2 to 6, 2 to 5, or 2 to 3), wherein one or more carbon atoms of the chain is optionally replaced with O, S, N(RS), or an optionally substituted divalent (Ce-C2o)aryl, (C5-C2o)heteroaryl, (C3-Cio)heterocycle, or (C5-Ce)carbocycle, which optional substitution is with one or more of C(=0)NH2, halo, hydroxy, or oxo (=0); and Rsis H or (Ci-Ce)alkyl.

[0123] In certain embodiments, L2is -NH(CH2)hC(=O)-,

[0124]

[0125] , ; each n is independently is 1 to 100; h is 1, 2, or 3; and R7is H or (Ci-C3)alkyl (e.g., methyl).

[0126] In certain embodiments, each n is independently 1 to 90, 1 to 80, 1 to 70, 1 to 60, 1 to 50, 1 to 40, 1 to 30, 1 to 20, 1 to 15, 1 to 10, or 1 to 5, or 1 to 3. In certain embodiments, each n is independently about 100, 90, 80, 70, 60, 50, 40, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1.

[0127] In certain embodiments, the compound comprises a PEG or sarcosine tail and / or may have improved solubility. In certain embodiments, R is -L^R6or Y is -L^R6.

[0128] In certain embodiments, L1is a linking group (e.g., a solubilizing group) of the formula

[0129]

[0130] In certain embodiments, L1is

[0131]

[0132] 10, and R7is H or (Ci-C3)alkyl (e.g. methyl).

[0133] In certain embodiments, R6is -NH2. In certain embodiments, R6is -(CH2)2NHC(=O)CH3. In certain embodiments, the compound is

[0134]

[0135] Targefrin-monomer-SarlO, or a pharmaceutically acceptable salt thereof.

[0136] In certain embodiments, the compound is

[0137]

[0138] Targefrin-monomer-PEG28, or a pharmaceutically acceptable salt thereof.

[0139] In certain embodiments, L1is absent.

[0140] In certain embodiments, the compound is a drug conjugate wherein R5is -L4-D.

[0141] In certain embodiments, L4is a hydrocarbon chain (e.g., branched or unbranched, saturated or unsaturated) comprising from 1 to 30 carbon atoms (e.g., 5 to 15, 1 to 20, 1 to 10, 1 to 9, 1 to 8, 1 to 6, 1 to 5, 1 to 3, 2 to 9, 2 to 8, 2 to 6, 2 to 5, or 2 to 3), wherein one or more carbon atoms of the chain is optionally replaced with O, S, N(RS), or an optionally substituted divalent (Ce-C2o)aryl, (Cs-C2o)heteroaryl, (C3-Cio)heterocycle, or (Cs-Cejcarbocycle, which optional substitution is with one or more of C(=O)NH2, halo, hydroxy, or oxo (=0); and Rsis H or (Ci-Ce)alkyl.

[0142] In certain embodiments, L4is:

[0143]

[0144] In certain embodiments, D is a residue of an anti-cancer agent, such as a residue of a taxane, including paclitaxel (PTX), docetaxel, or cabazitaxel. In certain embodiments, D is a residue of Monomethyl auristatin E (MMAE), Mertansine (DM1), or SN38 (7-ethyl-10-hydroxy-camptothecin). In certain embodiments, D is a residue of gemcitabine. In certain embodiments, -L4-D is -L4-PTX of the formula

[0145]

[0146] In certain embodiments, at least one or both R° is independently CH2, NH, NRa, or O, wherein each Rais independently (Ci-C3)alkyl (e.g., methyl or ethyl) or (C2-Ce)alkanoyl (e.g., C(=O)CH3). In certain embodiments, at least one or both R° is independently NH or NRa. In certain embodiments, at least one or both R° is NH or O.

[0147] In certain embodiments, at least one or both R3is independently benzyl that is optionally substituted with one or more halo. In certain embodiments, at least one or both R3is benzyl.

[0148] In certain embodiments, the compound is an irreversible covalent binder of EphA2. Without wanting to be bound by theory, in certain embodiments, the compound may form a covalent bond with a Tyrosine residue (Tyr 48) of the EphA2 protein (e.g., see NCBI accession number P29317). In certain embodiments, in at least one (one or both) of X1and X2, R3is independently

[0149]

[0150] each R3ais independently H, -SO2F, -OSO2F, -SO2-(C5-Ce)heteroaryl (e.g., SO2-tetrazole), (C3-C?)heterocycle (e.g., oxiranyl, or cyclic imine such as 1 -pyrroline), acrylamide (-NHC(=O)CH=CH2), halo substituted (C2-Ce)alkanoyl (e.g., halo substituted on the a carbon next to the oxo(=O) group), or isothiocyanate (-N=C=S);

[0151] eachR3bis independently (Ce-Cio)aryl, (Ci-Ce)alkyl, (Ci-Ce)alkoxy, (C3-Ce)cycloalkyl, -CH2O(Ci-Ce)alkyl, -CN, -C(=O)NH2, (C5-Ce)heteroaryl, (C3-C7)heterocycle (e.g., aziridinyl, or oxiranyl), hydroxy, nitro, -0(Ce-Cio)aryl, -O(C3-C6)cycloalkyl, -O(C5-Ce)heteroaryl, or -O(C3-C7)heterocycle;

[0152] each d, e, and f is independently 0, 1, 2, or 3.

[0153] In certain embodiments, each d and e are independently 1.

[0154] In certain embodiments, each f is 0.

[0155] In certain embodiments, each R3ais in the para position.

[0156] In certain embodiments, each R3ais -SO2F. In certain embodiments, each R3ais -OSO2F. In certain embodiments, each R3bis independently aziridinyl, (Ce-Cio)aryl, (C3-C6)cycloalkyl, -CH3, -C2H5, -CH2OCH3, -CN, -CONH2, -NO2, oxiranyl, -OCH3, -OC2H5, -OCH2CH2CH3, -O(C3-C6)cycloalkyl, -OH, -0(Ce-Cio)aryl, or -O(C5-C6)heteroaryl.

[0157] In certain embodiments, each R3bis independently aziridinyl, -CH3, -C2H5, -CH2OCH3, -CN, -CONH2, -NO2, -OCH3, -OC2H5, -OCH2CH2CH3, or -OH.

[0158] In certain embodiments, the compound is a monomer wherein R is

[0159]

[0160] In certain embodiments, the compound is a compound of formula (la):

[0161]

[0162] In certain embodiments, the compound is a compound of formula (lb):

[0163]

[0164] In certain embodiments, the compound is a compound of formula (Ic):

[0165]

[0166] In certain embodiments, the compound is a compound of formula:

[0167]

[0168] In certain embodiments, the compound is 152G5.

[0169] In certain embodiments, the compound is 152G8.

[0170] In certain embodiments, the compound is a compound of formula:

[0171]

[0172] In certain embodiments, the compound is a dimer (homodimer, or heterodimer).

[0173] In certain embodiments, the compound is a dimer wherein R is

[0174]

[0175] In certain embodiments, the compound is a dimer wherein R is

[0176]

[0177] In certain embodiments, a is 1. In certain embodiments, h is 1. In certain embodiments, b certain embodiments, g is 3. In certain embodiments, c is 3.

[0178] In certain embodiments, Y is -I^-R6.

[0179] In certain embodiments, the compound is a compound of formula

[0180]

[0181] In certain embodiments, L1is a poly-sarcosine (e.g., n is 1 to 100, or 1 to 10, wherein R7l) segment.

[0182] In certain embodiments, Y is -L°-R5.In certain embodiments, the compound is a compound of formula (Id)

[0183]

[0184] In certain embodiments, the compound is a compound of formula

[0185]

[0186] In certain embodiments, the compound is 152G12, 152G12-13, or 152G12-15.

[0187] In certain embodiments, at least one or both R1is independently (Ci-Ce)alkyl (e.g., isopropyl) or (C2-Ce)alkynyl, wherein the (Ci-Ce)alkyl is optionally substituted with hydroxy, thiol, or azido.

[0188] In certain embodiments, at least one or both R2is independently (Ci-Ce)alkyl (e.g., methyl) or (C2-Ce)alkynyl, wherein the (Ci-Ce)alkyl is optionally substituted with hydroxy, thiol, or azido.

[0189] In certain embodiments, at least one or both R1is isopropyl.

[0190] In certain embodiments, at least one or both R2is methyl.

[0191] In certain embodiments, each R1is isopropyl and each R2is methyl.

[0192] In certain embodiments, in at least one or both of X1and X2, R1and R2are capable of forming, a disulfide bond (-S-S-) or a triazole moiety via click chemistry, between each other.

[0193] In certain embodiments, in at least one or both of X1and X2, R1 —R2together forms a linking group L3.

[0194] In certain embodiments, L3is a hydrocarbon chain (branched or unbranched, saturated or unsaturated) comprising from 1 to 12 carbon atoms, wherein one or more carbon atoms of the chain is optionally replaced with O, S, N(RS), or an optionally substituted divalent (Ce-C2o)aryl,(C5-C2o)heteroaryl, (C3-Cio)heterocycle, or (Cs-Cejcarbocycle, which optional substitution is with one or more of C(=O)NH2, halo, hydroxy, or oxo (=0); and Rsis H or (Ci-Ce)alkyl.

[0195] In certain embodiments, L3is -CH2-, -O-, -C(=0)NH-, -NHC(=0)-, -NHS(=O)2-, -S(=O)2NH-, -NH-, -OC(=O)-, -C(=O)O-, (CH2)P-S-S-(CH2)q or (CH2)p-triazolyl-(CH2)q; p is 0, 1, 2, or 3; and q is 0, 1, 2, or 3.

[0196] In certain embodiments, in at least one or both of X1and X2, R1 —R2together is L3, which is a branched or unbranched, saturated or unsaturated, hydrocarbon chain having from about 3 to 10 carbon atoms, wherein one or more of the carbon atoms of the chain is replaced with -S-S- or triazolyl.

[0197] In certain embodiments, in at least one or both of X1and X2, R1 —R2together is L3, which is (CH2)P-S-S-(CH2)q or (CH2)P-triazolyl-(CH2)q; p is 0, 1, 2, or 3; and q is 0, 1, 2, or 3.

[0198] In certain embodiments, in at least one or both of X1and X2, R1 —R2together is L3, which is

[0199]

[0200] In certain embodiments, in at least one or both of X1and X2, each R1and R2are independently (Ci-C3)alkyl that is substituted with thiol. In certain embodiments, in at least one or both of X1and X2, each R1and R2are independently -CH2SH or -(CH2)2SH.

[0201] In certain embodiments, in at least one or both of X1and X2, each R1is -CH2SH and each R2is -(CH2)2SH.

[0202] In certain embodiments, in at least one or both of X1and X2, each R2is -CH2SH and each R1is -(CH2)2SH.

[0203] In certain embodiments, in at least one or both of X1and X2, each R1is independently (C2-Ce)alkynyl and each R2is independently (Ci-Ce)alkyl that is substituted with azido. In certain embodiments, in at least one or both of X1and X2, each R1is independently (C2-C4)alkynyl, and each R2is independently (Ci-C3)alkyl is substituted with azido.

[0204] In certain embodiments, in at least one or both of X1and X2, each R2is independently (C2-Ce)alkynyl and each R1is independently (Ci-Ce)alkyl that is substituted with azido. In certain embodiments, in at least one or both of X1and X2, each R2is independently (C2-C4)alkynyl, and each R1is independently (Ci-C3)alkyl is substituted with azido.

[0205] In certain embodiments, in at least one or both of X1and X2, each R1and R2are independently the side chain of the amino acid residue of cysteine, homocysteine, 2-Amino-4-pentynoic Acid, 2-Amino-3-butynoic Acid, 2-Amino-5-hexynoic Acid, 2-amino-3-azidopropanoic acid, or 2-amino-3 -azidobutanoic acid (e.g., wherein R1and R2are capable of forming, a triazole moiety via click chemistry or a disulfide bond (-S-S-), between each other).

[0206] In certain embodiments, in at least one or both of X1and X2, each R1is ethynyl, 2-propynyl, or 3-butynyl.

[0207] In certain embodiments, in at least one or both of X1and X2, each R2is ethynyl, 2-propynyl, or 3-butynyl.

[0208] In certain embodiments, in at least one or both of X1and X2, each R1is ethynyl, 2-propynyl, or 3-butynyl, and each R2is (Ci-C3)alkyl substituted with azido.

[0209] In certain embodiments, in at least one or both of X1and X2, each R2is ethynyl, 2-propynyl, or 3-butynyl, and each R1is (Ci-C3)alkyl substituted with azido.

[0210] In certain embodiments, the compound is of Formula (Ig)

[0211]

[0212] In certain embodiments, the compound is of formula:

[0213]

[0214] In certain embodiments, the compound is of formula:

[0215]

[0216] In certain embodiments, the compound is a cyclic compound of formula

[0217]

[0218] In certain embodiments, the compound is

[0219]

[0220] In certain embodiments, the compound is

[0221]

[0222] In certain embodiments, the compound is

[0223]

[0224] TABLE A. Certain exemplary EphA2 targeting compounds

[0225]

[0226]

[0227] In certain embodiments, the compound (e.g., a monomer) is an antagonist of EphA2. In certain embodiments, the compound (e.g., a dimer) may cause internalization and degradation of EphA2. In certain embodiments, the compound (e.g., a dimer) is an agonist of EphA2.

[0228] The synthetic schemes for certain targefrin compounds are known in the art (e.g. , International Patent Application Number WO2024086161; and Carlo Baggio, el al., J. Med. Chem. 2022, 65, 22, 15443-15456) and also described herein (e.g., Example 1)6.2. CERTAIN METHODS

[0229] Certain embodiments of the invention provide a method of treating cancer, which comprises administering a therapeutically effective amount of a compound or pharmaceutically acceptable salt or metal complex thereof described herein to a patient in need thereof.

[0230] In certain embodiments, the cancer is pancreatic cancer, prostate cancer, breast cancer, esophageal cancer, melanoma, urinary bladder, brain cancer, lung cancer, ovarian cancer, stomach cancer, or leukemia. In certain embodiments, the cancer is a metastatic cancer. In certain embodiments, the cancer is an EphA2 positive cancer.

[0231] In certain embodiments, the method further comprises administering a second anti-cancer agent (e.g., chemotherapeutic agent) to the patient. In certain embodiments, the method further comprises administering an EGRF inhibitor, a HER2 inhibitor, or a BRAF inhibitor to the patient.

[0232] Certain embodiments of the invention provide the use of a compound as described herein or a pharmaceutically acceptable salt or metal complex thereof to prepare a medicament for treating cancer or to prepare an imaging agent for medical imaging.

[0233] Certain embodiments of the invention provide a compound as described herein or a pharmaceutically acceptable salt or metal complex thereof for the prophylactic or therapeutic treatment of cancer, or for use in medical imaging (e.g., magnetic resonance imaging (MRI), or Positron Emission Tomography (PET)).

[0234] Certain embodiments of the invention provide a compound as described herein or a pharmaceutically acceptable salt or metal complex thereof for use in medical therapy or medical imaging.

[0235] Certain embodiments of the invention provide a method (e.g., an imaging and / or a treatment method), which comprises 1) administering a first compound or pharmaceutically acceptable salt or metal complex thereof described herein to a patient in need thereof, 2) generating one or more medical image of the patient from a MRI or PET machine, and 3) optionally diagnosing the patient as having cancer (e.g., EphA2 positive cancer) and / or optionally administering an anti -cancer drug (e.g., a second compound or pharmaceutically acceptable salt or metal complex thereof described herein) to the patient.

[0236] 6.3. COMPOSITION AND ADMINISTRATION

[0237] Certain embodiments of the invention also provide a composition comprising a compound as described, or a salt (e.g., pharmaceutically acceptable) or metal complex thereof, and a pharmaceutically acceptable carrier. Compounds described herein (including salt, or metal complex thereof) can be formulated as pharmaceutical compositions and administered to amammalian host, such as a human patient in a variety of forms adapted to the chosen route of administration, i.e., orally or parenterally, by intravenous, intramuscular, intrathecal, intracerebroventricular, intraperitoneal, intradermal or subcutaneous routes.

[0238] Compounds disclosed herein may be systemically (e.g., orally or intravenously) administered in combination with a pharmaceutically acceptable vehicle such as an inert diluent or an assimilable, edible carrier. They may be in the form of single unit dosage forms (e.g., enclosed in hard or soft shell gelatin capsules or compressed into tablets). For oral therapeutic administration, an active compound may be combined with one or more excipients in the form of ingestible tablets, buccal tablets, capsules, caplets, troches, elixirs, suspensions, syrups, and wafers.

[0239] Compounds may also be administered intravenously, intrathecally, intracerebroventricularly, or intraperitoneally by infusion or injection. Solutions of an active compound or its salts may be prepared in water, optionally mixed with a nontoxic surfactant. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, triacetin, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

[0240] Pharmaceutical dosage forms suitable for injection or infusion may include sterile aqueous solutions or dispersions or sterile powders comprising the active ingredient which are adapted for the extemporaneous preparation of sterile injectable or infusible solutions or dispersions, optionally encapsulated in liposomes. In all cases, the ultimate dosage form should be sterile, fluid and stable under the conditions of manufacture and storage. The liquid carrier or vehicle can be a solvent or liquid dispersion medium comprising, for example, water, ethanol, a polyol (for example, glycerol, propylene glycol, liquid polyethylene glycols, and the like), vegetable oils, nontoxic glyceryl esters, and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the formation of liposomes, by the maintenance of the required particle size in the case of dispersions or by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, buffers or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0241] Sterile injectable solutions are prepared by incorporating the active compound in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filter sterilization. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuumdrying and the freeze drying techniques, which yield a powder of the active ingredient plus any additional desired ingredient present in the previously sterile-filtered solutions.

[0242] Lyophilized formulations may also contain carrier such as bulking agent (e.g., mannitol or glycine) and cryoprotectant / lyoprotectant (e.g., trehalose or sucrose). Lyophilized formulation can be reconstituted into a liquid dosage form using saline, 5% dextrose solution or sterile water before administration.

[0243] 6.4. CERTAIN DEFINITIONS

[0244] The following definitions are used, unless otherwise described: halo or halogen is fluoro, chloro, bromo, or iodo. Alkyl, alkoxy, alkenyl, alkynyl, etc. denote both straight and branched groups; but reference to an individual radical such as propyl embraces only the straight chain radical, a branched chain isomer such as isopropyl being specifically referred to.

[0245] The term "alkyl", by itself or as part of another substituent, means, unless otherwise stated, a straight or branched chain hydrocarbon radical, having the number of carbon atoms designated (i.e., Ci-8 means one to eight carbons). Examples include (Ci-Cs)alkyl, (C2-Cs)alkyl, (Ci-Ce)alkyl, (C2-Ce)alkyl, (Ci-C3)alkyl, and (C3-Ce)alkyl. Examples of alkyl groups include methyl, ethyl, n-propyl, iso-propyl, n-butyl, t-butyl, iso-butyl, sec-butyl, n-pentyl, n-hexyl, n-heptyl, n-octyl, and higher homologs and isomers.

[0246] The term "alkenyl" refers to an unsaturated alkyl radical having one or more double bonds. Examples of such unsaturated alkyl groups include vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl), 2,4-pentadienyl, 3-(l,4-pentadienyl) and the higher homologs and isomers.

[0247] The term "alkynyl" refers to an unsaturated alkyl radical having one or more triple bonds. Examples of such unsaturated alkyl groups ethynyl, 1- and 3-propynyl, 3-butynyl, and higher homologs and isomers.

[0248] The term “alkoxy” refers to the formula -OR or radical thereof, where R is an alkyl as defined.

[0249] The term “halo” or “halogen” refers to bromo, chloro, fluoro or iodo. In some embodiments, halogen refers to chloro or fluoro.

[0250] The term “cycloalkyl” or “carbocycle” refers to a saturated or partially unsaturated (nonaromatic) all carbon ring having 3 to 8 carbon atoms (i.e., (C3-Cs)carbocycle). The term also includes multiple condensed, saturated all carbon ring systems (e.g., ring systems comprising 2, 3 or 4 carbocyclic rings). Accordingly, carbocycle includes multicyclic carbocyles such as a bicyclic carbocycles (e.g., bicyclic carbocycles having about 3 to 15 carbon atoms, about 6 to 15 carbon atoms, or 6 to 12 carbon atoms such as bicyclo[3.1.0]hexane and bicyclo[2.1.1]hexane),and polycyclic carbocycles (e.g tricyclic and tetracyclic carbocycles with up to about 20 carbon atoms). The rings of the multiple condensed ring system can be connected to each other via fused, spiro and bridged bonds when allowed by valency requirements. For example, multicyclic carbocyles can be connected to each other via a single carbon atom to form a spiro connection (e.g., spiropentane, spiro[4,5]decane, etc), via two adjacent carbon atoms to form a fused connection (e.g., carbocycles such as decahydronaphthalene, norsabinane, norcarane) or via two non-adjacent carbon atoms to form a bridged connection (e.g., norbornane, bicyclo[2.2.2]octane, etc). Non-limiting examples of cycloalkyls include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, bicyclo[2.2.1]heptane, pinane, and adamantane. In certain embodiments, the carbocycle is a Ce carbocycle.

[0251] The term “aryl” as used herein refers to a single all carbon aromatic ring or a multiple condensed all carbon ring system wherein at least one of the rings is aromatic. For example, in certain embodiments, an aryl group has 6 to 20 carbon atoms, 6 to 14 carbon atoms, 6 to 12 carbon atoms, or 6 to 10 carbon atoms. Aryl includes a phenyl radical. Aryl also includes multiple condensed carbon ring systems (e.g., ring systems comprising 2, 3 or 4 rings) having about 9 to 20 carbon atoms in which at least one ring is aromatic and wherein the other rings may be aromatic or not aromatic (i.e., cycloalkyl). The rings of the multiple condensed ring system can be connected to each other via fused, spiro and bridged bonds when allowed by valency requirements. It is to be understood that the point of attachment of a multiple condensed ring system, as defined above, can be at any position of the ring system including an aromatic or a carbocycle portion of the ring. Non-limiting examples of aryl groups include, but are not limited to, phenyl, indenyl, indanyl, naphthyl, 1, 2, 3, 4-tetrahydronaphthyl, anthracenyl, and the like.

[0252] The term “heterocycle” or “heterocycloalkyl” refers to a single saturated or partially unsaturated ring that has at least one atom other than carbon in the ring, wherein the atom is selected from the group consisting of oxygen, nitrogen and sulfur; the term also includes multiple condensed ring systems that have at least one such saturated or partially unsaturated ring, which multiple condensed ring systems are further described below. Thus, the term includes single saturated or partially unsaturated rings (e.g., 3, 4, 5, 6 or 7-membered rings) from about 1 to 6 carbon atoms and from about 1 to 3 heteroatoms selected from the group consisting of oxygen, nitrogen and sulfur in the ring. The sulfur and nitrogen atoms may also be present in their oxidized forms. Exemplary heterocycles include but are not limited to azetidinyl, tetrahydrofuranyl and piperidinyl. The term “heterocycle” or “heterocycloalkyl” also includes multiple condensed ring systems (e.g., ring systems comprising 2, 3 or 4 rings) wherein a single heterocycle ring (as defined above) can be condensed with one or more groups selected from cycloalkyl, aryl, and heterocycle to form the multiple condensed ring system. The rings of themultiple condensed ring system can be connected to each other via fused, spiro and bridged bonds when allowed by valency requirements. It is to be understood that the individual rings of the multiple condensed ring system may be connected in any order relative to one another. It is also to be understood that the point of attachment of a multiple condensed ring system (as defined above for a heterocycle) can be at any position of the multiple condensed ring system including a heterocycle, aryl and carbocycle portion of the ring. In one embodiment the term heterocycle includes a 3-15 membered heterocycle. In one embodiment the term heterocycle includes a 3-10 membered heterocycle. In one embodiment the term heterocycle includes a 3-8 membered heterocycle. In one embodiment the term heterocycle includes a 3-7 membered heterocycle. In one embodiment the term heterocycle includes a 3-6 membered heterocycle. In one embodiment the term heterocycle includes a 4-6 membered heterocycle. In one embodiment the term heterocycle includes a 3-10 membered monocyclic or bicyclic heterocycle comprising 1 to 4 heteroatoms. In one embodiment the term heterocycle includes a 3-8 membered monocyclic or bicyclic heterocycle heterocycle comprising 1 to 3 heteroatoms. In one embodiment the term heterocycle includes a 3-6 membered monocyclic heterocycle comprising 1 to 2 heteroatoms. In one embodiment the term heterocycle includes a 4-6 membered monocyclic heterocycle comprising 1 to 2 heteroatoms. Exemplary heterocycles include, but are not limited to aziridinyl, azetidinyl, pyrrolidinyl, piperidinyl, homopiperidinyl, morpholinyl, thiomorpholinyl, piperazinyl, tetrahydrofuranyl, dihydrooxazolyl, tetrahydropyranyl, tetrahydrothiopyranyl, 1, 2,3,4-tetrahydroquinolyl, benzoxazinyl, dihydrooxazolyl, chromanyl, 1,2-dihydropyridinyl, 2,3-dihydrobenzofuranyl, 1,3-benzodioxolyl, 1,4-benzodioxanyl, spiro[cyclopropane-l,l'-isoindolinyl]-3'-one, isoindolinyl-l-one, 2-oxa-6-azaspiro[3.3]heptanyl, imidazolidin-2-one imidazolidine, pyrazolidine, butyrolactam, valerolactam, imidazolidinone, hydantoin, dioxolane, phthalimide, and 1,4-di oxane.

[0253] The term “heteroaryl” as used herein refers to a single aromatic ring that has at least one atom other than carbon in the ring, wherein the atom is selected from the group consisting of oxygen, nitrogen and sulfur; “heteroaryl” also includes multiple condensed ring systems that have at least one such aromatic ring, which multiple condensed ring systems are further described below. Thus, “heteroaryl” includes single aromatic rings of from about 1 to 6 carbon atoms and about 1-4 heteroatoms selected from the group consisting of oxygen, nitrogen and sulfur. The sulfur and nitrogen atoms may also be present in an oxidized form provided the ring is aromatic. Exemplary heteroaryl ring systems include but are not limited to pyridyl, pyrimidinyl, oxazolyl or furyl. “Heteroaryl” also includes multiple condensed ring systems (e.g., ring systems comprising 2, 3 or 4 rings) wherein a heteroaryl group, as defined above, is condensed with one or more rings selected from cycloalkyl, aryl, heterocycle, and heteroaryl. Itis to be understood that the point of attachment for a heteroaryl or heteroaryl multiple condensed ring system can be at any suitable atom of the heteroaryl or heteroaryl multiple condensed ring system including a carbon atom and a heteroatom (e.g., a nitrogen). Exemplary heteroaryls include but are not limited to pyridyl, pyrrolyl, pyrazinyl, pyrimidinyl, pyridazinyl, pyrazolyl, thienyl, indolyl, imidazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, thiazolyl, furyl, oxadiazolyl, thiadiazolyl, quinolyl, isoquinolyl, benzothiazolyl, benzoxazolyl, indazolyl, quinoxalyl, and quinazolyl.

[0254] The terms "treat" and "treatment" refer to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) an undesired physiological change or disorder. For example, the onset of a disorder or disease is prevented or delayed. The progression of a disease is slowed or stopped.

[0255] The phrase “therapeutically effective amount” means an amount of a compound of the present invention that (i) treats the particular disease, condition, or disorder, (ii) attenuates, ameliorates, or eliminates one or more symptoms of the particular disease, condition, or disorder, or (iii) prevents or delays the onset of one or more symptoms of the particular disease, condition, or disorder described herein.

[0256] The term “mammal” as used herein refers to, e.g., humans, higher non-human primates, rodents, cows, horses, pigs, sheep, dogs and cats. In one embodiment, the mammal is a human.

[0257] Unless otherwise indicated, an adjective before a string of nouns should be construed to apply to each. For example, the phrase “optionally substituted pyridyl, pyrazyl, or furanyl” means the same as “optionally substituted pyridyl, optionally substituted pyrazyl, or optionally substituted furanyl”.

[0258] A dashed line “ — ” used to depict a bond in a chemical structure indicates that the bond may be delocalized (e.g., as in a tautomer) or may or may not exist (e.g., as in the case where a solid and a dashed line are used together to depict a bond that may exist as a single bond or a double bond).

[0259] A wavy line “ ” that intersects a bond in a chemical structure indicates the point of attachment of the bond that the wavy bond intersects in the chemical structure to the remainder of a molecule.

[0260] Compounds disclosed herein may exist as tautomeric isomers. Although only one delocalized resonance structure may be depicted, all such forms are contemplated within the scope of the invention.

[0261] Compounds disclosed herein may exist as zwitterions (e.g., at pharmacological pH). Unless otherwise indicated, it should be understood that a chemical drawing depicting the structure of such a compound encompasses all of its zwitterionic forms.Some compounds may exist as stereoisomers. When a stereoisomer of compound is defined by its name (e.g., with the use of R or 5) or is depicted in a drawn structure (e.g., using a bold, bold- wedge, dashed, or dashed- wedge to depict the relevant chemical bond), the enantiomeric excess (ee) of that compound, unless otherwise indicated, is to be understood to be at least 60, 70, 80, 90, 95, or 99%. A compound or composition enriched with one stereoisomer of the compound has that one stereoisomer in an amount measurably greater than the compound’s other stereoisomer(s). For example, a compound enriched with an R enantiomer will have an enantiomeric excess of that enantiomer versus the S enantiomer.

[0262] Unless explicitly noted, chemical structures named or shown herein encompass derivatives wherein one or more of their constituent atoms is / are replaced or enriched (i.e., present in a quantity measurably greater than its natural abundance) with an isotope thereof. Thus, unless noted otherwise, a chemical structure that contains hydrogen atoms encompasses deuterated forms of the represented molecule or moiety.

[0263] 6.5. EXAMPLES

[0264] 6.5.1. EXAMPLE 1.

[0265] Systemic and localized radiation are often used to reduce tumor burden or as prevention of tumor spread or recurrence in patients. However, while this approach is effective in killing rapidly dividing cancer cells, similar to chemotherapy, systemic toxicity is a major limitation of the approach. Novel strategies aimed at delivering radioligands directly to cancer cells are being expl ored. These strategies include a tumor homing agent that targets and accumulates preferentially to cancer cells, conjugated with radioligands. For example, the FDA has recently approved the PSMA-targeted radioligand lutetium-177-PSMA-617, for patients with PSMA-positive metastatic castration-resistant prostate cancer that has failed androgen receptor pathway inhibitors and taxane chemotherapy. Genetic and cellular studies identified the EphA2 receptor as a possible target for targeted delivery of chemotherapy, by conjugating EphA2 targeting agents such as Targefrin, with cytotoxic drugs, such as a taxane. Here we report innovative aspects that exploits Targefrin (Figure 1) to deliver radioligands to EphA2 expressing aggressive tumors.

[0266] In a first implementation of this strategy, we prepared an exemplary Targefrin-monomer-DOTA (152G5, PiperazAcAcid-(4-NH2-Phe)-L-A-(2-CF3-Bip)-PD-A-Chg-PFRPG-K(Ava)-DOTA), where DOTA can be used to tightly chelate any radioisotope, such as Lutetium- 177, or other elements that can be used for imaging purposes, such as Gadolinium (MRI) or Gallium (PET).

[0267]

[0268] Targefrin-monomer-DOTA (152G5, PiperazAcAcid-(4-NH2-Phe)-L-A-(2-CF3-Bip)-PD- A-Chg-PFRPG-K(Ava)-DOTA)

[0269] In a second application, to further increase the residence time of the homing agent to EphA2 expressing tumor cells, we also devised an irreversible Targefrin ligand. This was accomplished by modifying the side chain of the Phe residue of Targefrin to reach a Tyr residue in the binding site of EphA2. For the reaction to take place we introduced a mild electrophile such as a substituted sulfonyl fluoride of fluoro-sulfate in this Example.

[0270]

[0271] Targefrin-monomer-covalent-DOTA (152G8, PiperazAcAcid-(4-NH2-Phe)-L-A-(2-CF3- Bip)-PD-A-Chg-P-Dap(benzylSF)-RPG-K(Ava)-DOTA)

[0272] Taregfrin-dimer can actively deliver its cargo to EphA2 expressing cancer cells. We also propose to derivatize Targefrin-dimer with radioligand chelating groups such as DOTA or DTPA to more actively deliver radioisotopes to tumor cells (Figure 2). Hence, Targefrin-dimer-DOTA, for example, can be prepared and conjugated with radioligands such as Lutetium- 177.

[0273]

[0274] Targefrin-dimer-SarlO-DOTA (152G12-15, [Ac-PiperazAcAcid-(4-NH2-Phe)-L-A-(2- CF3-Bip)-PD-A-Chg-PFRPG]2-K(Sar)ioK(Ava)-DOTA)

[0275] Data described in this Example suggest that Targefrin-monomer and targefrin-monomer-covalent could be deployed for targeted delivery of radioisotopes. Among the dimers, the reversible targefrin-dimer seems to be more suitable as compared to the targefrin-dimer-covalent version, which does not appear to induce EphA2 degradation.

[0276] Complex formation of the agents with the selected radioisotope is obtained by combining one or more equivalents of the desired radioisotope with the DOTA or DTPA conjugate, followed by subsequent purification by reverse phase HPLC.

[0277] DELFIA displacement curves were obtained for Targefrin-monomer-DOTA reversible binder (152G5, IC50 of 8.5nM), and the irreversible binder agent Targefrin-monomer-covalent-DOTA (152F11, IC50 of 5.3nM). The covalency to EphA2 Tyr48 residue increased the affinity of the agent for EphA2 down to the limit of detection of the assay (Figure 3).

[0278] Thermal denaturation curve of EphA2 was obtained, along with EphA2 in the presence of Targefrin-monomer (middle at +3.91°C), or EphA2 in presence of Targefrin-monomer-covalent (152F11) (right, at + 10.82°C). It is shown that the covalent agent increases thermal stability of EphA2 significantly (Figure 4).SDS-Gel of EphA2 was collected in absence and presence of Targefrin-monomer-covalent (152F11). The MW band shift observed in the presence of the covalent agent indicates the formation of a stable covalent adduct between Targefrin-monomer-covalent and EphA2 (Figure 5).

[0279] DELFIA displacement curves were for Targefrin-dimer (152E3, IC50 of 6.5nM) and the covalent agent Targefrin-dimer-covalent (152F10, IC50 of 2.3nM). The covalency to EphA2 Tyr48 residue increased the affinity of the agent for EphA2 down to the limit of detection of the assay (Figure 6).

[0280] SDS-Gel of EphA2 were collected in presence of DMSO control only, in presence of Targefrin-dimer-covalent (152F10), or in presence of Targefrin-dimer non-covalent (152E3). The two bands observed with Targefrin-dimer indicate the single covalent adduct and the ternary covalent adduct (at around 45 kDa), both indicating the formation of a stable complex (Figure 7).

[0281] Targefrin-monomer-DOTA (152G5), results in a Kd of 12.5 nM by ITC, that is comparable to Targefrin-monomer without DOTA (Figure 8).

[0282] DELFIA displacement curves was obtained for Targefrin-dimer-DOTA (152G12), showing an IC 50 of 1.2 nM (Figure 9).

[0283] Targefrin-monomer-covalent (152F11) was incubated with EphA2 for various durations. lOpM EphA2 in 25 mM TRIS pH 75, 150 mM NaCl has been incubated at the reported time points with lOOpM of Targefrin-monomer-covalent. EphA2 MW is 23,373 Da. The higher molecular weight band is due to the covalent reaction with the covalent irreversible agent 152F11 (MW = 1,812 Da), giving a final adduct of a MW of 25,165 Da (Figure 10).

[0284] Targefrin-monomer-covalent-DOTA (152G8) was incubated with EphA2 for various durations. lOpM EphA2 in 25 mM TRIS pH 75, 150 mM NaCl has been incubated at the reported time points with lOOpM of Targefrin-monomer-covalent. EphA2 MW is 23,373 Da. The higher molecular weight band is due to the covalent reaction with the covalent irreversible agent 152G8 (MW = 2,483 Da), giving a final adduct of a MW of 25,836 Da (Figure 11).

[0285] Kinact / Ki calculation done by DELFIA displacement assay was used to further determine the covalent binding of Targefrin-monomer-covalent-DOTA (152G8). Kinact / Ki is an indication of the efficacy of the covalent binding. 152G8 has a Kinact / Ki in the same order of magnitude as FDA-approved irreversible agents, such as Sotorasib (Figure 12).

[0286] Protein Thermal Shift (PTS) denaturation curves of EphA2 was also determined in the presence of Targefrin-monomer-DOTA (152G5), Targefrin-monomer-covalent-DOTA (152G8), and a non-active scrambled version of Targefrin-monomer-DOTA. It is shown that covalent binder (+ 9.35°C) has higher thermal shift than the non-covalent binder (+ 5.15°C) (Figure 13).It is also demonstrated that Targefrin-monomer-DOTA (152G5) has excellent selectivity for EphA2, as tested by ITC, over two closely related proteins, EphA4 and EphA3 (Figure 14).

[0287] Similarly, Targefrin-monomer-DOTA (152G5) (+ 5.15°C) and Targefrin-monomer-covalent-DOTA (152G8) (+ 9.35°C) selectivity for EphA2 were also demonstrated by Protein Thermal Shift (PTS) denaturation assays, over two closely related proteins, EphA4 and EphA3 (Figure 15).

[0288] In addition, Targefrin-monomer-covalent-DOTA (152G8) selectivity for EphA2 was further tested by SDS-Gel, over two closely related proteins, EphA4 and EphA3. A higher Molecular weight band is visible only in the presence of EphA2, due to the covalent adduct formation (Figure 16).

[0289] Furthermore, cyclic versions of targefrin, by introducing appropriate residues in position 3 and position 8 were made. Cyclization occurs through the formation of either a disulfide bond or a click chemistry reaction between an azido and an alkyne group. For example, Targefrin-Cyclic (152G9) was made from a linear compound with 2-Amino-4-pentynoic Acid residue in position 3, and 2-amino-3-azidopropanoic acid residue in position 8.

[0290] Synthesis of agents with the linking / solubilizing moiety poly-sarcosine

[0291]

[0292] Amino acid coupling is done in solid phase using a Liberty Blue peptide synthesizer (CEM). 6 equiv. of Fmoc-AA, 3 equiv. of DIC, and 1 equiv. of Oxima Pure in 4.5 mL of DMF were used. For some special amino acids, standard coupling has been done, using 3 equiv. of Fmoc-AA, 3 equiv. of HATU, 3 equiv. of Oxima Pure, and 5 equiv. of DIPEA in 1 mL of DMF, for Ih at RT. a) Rink Amide resin LL + Fmoc-Lys(ivDde)-OH, standard coupling, b) Fmoc deprotection with 20% piperidine in DMF twice, c) Fmoc-Sar-OH on Liberty Blue Steps c and b have been repeated another 9 times to get a total of ten sarcosine residues, d) Peptides growth using standard conditions or Liberty Blue for peptide growth, e) ivDde deprotection using 4% N2H2 in DMF (3 x 5 mL), rt. f) Fmoc-5-Aminovaleric acid, standard coupling, g) DOTA-tris(tert-butyl ester), standard coupling, h) Peptides were cleaved from Rink amide resin with a cleavage cocktail containing TFA / TIS / water / phenol (94:2:2:2) for 3 h.

[0293]

[0294] a) 400 mM 12 in DMF (4 equiv.)

[0295] with 0.5 equiv. TEMPO, 30 min, rt

[0296] b) Cleavage Cocktail

[0297]

[0298]

[0299] Additionally, Targefrin-cyclic (152H2) was made from a linear compound with L-Cysteine in position 3, and L-HomoCysteine in position 8.

[0300]

[0301] The majority of amino acid coupling is done in solid phase using a Liberty Blue peptide synthesizer (CEM). 6 equiv. of Fmoc-AA, 3 equiv. of DIC, and 1 equiv. A solution of Oxima Pure in 4.5 mL of DMF was used. For some special amino acids, standard coupling has been done, using 3 equiv. of Fmoc-AA, 3 equiv. of HATU, 3 equiv. of Oxima Pure, and 5 equiv. of DIPEA in 1 mL of DMF, for 1 h at RT.

[0302] Example of synthesis of Targefrin-dimer-Sar 10-DOTA

[0303]

[0304] Amino acid coupling is done in solid phase using a Liberty Blue peptide synthesizer (CEM). 6 equiv. of Fmoc-AA, 3 equiv. of DIC, and 1 equiv. of Oxima Pure in 4.5 mL of DMF were used. For some special amino acids, standard coupling has been done, using 3 equiv. of Fmoc-AA, 3 equiv. of HATU, 3 equiv. of Oxima Pure, and 5 equiv. of DIPEA in 1 mL of DMF, for Ih at RT. a) Rink Amide resin LL + Fmoc-Lys(ivDde)-OH, standard coupling, b) Fmoc deprotection with 20% piperidine in DMF twice, c) Fmoc-Sar-OH on Liberty Blue Steps c and b have been repeated another 9 times to get a total of ten sarcosine residues, d) Fmoc-Lys(Fmoc)-OH, on Liberty Blue e) Fmoc-Gly-OH, on Liberty Blue, at double scale synthesis for dimer growth, f) Peptides growth using standard conditions or Liberty Blue using double scale synthesis for dimer growth, g) ivDde deprotection using 4% N2H2 in DMF (3 x 5 mL), rt. h) Fmoc-5-Aminovaleric acid, standard coupling, i) DOTA-tris(tert-butyl ester), standard coupling, j) Peptides were cleaved from Rink amide resin with a cleavage cocktail containing TFA / TIS / water / phenol (94:2:2:2) for 3 h.

[0305] Targefrin-Cyclic (152H2) results in having a slightly lower affinity compared to Targefrin-monomer (152E2) but a higher affinity compared to its non-cyclic analog (152H3) as determined by Isothermal Titration Calorimetry (ITC). It shows a higher entropic contribution given the more constrained nature of the peptides. It also shows a similar melting temperature to what is observed for 152E2 by the Protein Thermal Shift denaturation curve (Figure 17).

[0306] It is shown that Targefrin-Cyclic (152H2) (+ 5.7°C) has a similar melting temperature compared to Targefrin-monomer (152E2) (+ 6.5°C), and its non-cyclic analog (152H3) (+ 5.4°C) (Figure 18). Moreover, Targefrin-Cyclic compounds, can also be conjugated with chelating agents (e.g., DOTA), for the chelation of a radioisotope. Targefrin-cyclic can also be modified to be covalent binder of EphA2 as described herein.

[0307] We successfully radiolabeled with 177Lu, for Targefrin-monomer-DOTA (152G5), Targefrin-monomer-covalent-DOTA (152G8), and Targefrin-dimer-DOTA (152G12) with reaction conditions of: Lu-177 (0.4-2 pL, 284-870 pCi in 0.04M HC1), compound (10.6 nmol, 8.8 - 9 pL), ammonium acetate IM (50- 100 pL) at 95°C, for 15min, 400rpm.

[0308] The agents resulted in a Radiochemical purity of > 99% by both radio-HPLC and radio-ITLC. Radiolabeling with 177Lu of Targefrin-monomer-DOTA (152G5), Targefrin-monomer-covalent-DOTA (152G8), and Targefrin-dimer-DOTA (152G12) resulted in Radiochemical purity > 99% as determined by Radio-HPLC. Molar activity of all radiolabeled peptide compounds ranged from 75 to 99 mCi / pmol (2.7 - 3.6 GBq / pmol) (Figure 19). iTLC-HPLC shows the similar results of a Radiochemical purity > 99%. Molar activity of all radiolabeled peptide compounds ranged from 75 to 99 mCi / pmol (2.7 - 3.6 GBq / pmol) (Figure 20).Furthermore, the compounds may be modified with a PEG or sarcosine tail. It is shown herein that the modification with such linking group increase agent’s solubility significantly (Tables B, C, and D).

[0309] Table B. exemplary R5= H, or a residue of DOTA, DTP A, EDTA, NOTA, or a drug (in certain embodiments of a structure described herein, wherein R5= a residue of DOTA, DTP A, EDTA, NOTA, or a drug; in certain embodiments of a structure described herein, wherein R5= a residue of DOTA)

[0310]

[0311] Table C. exemplary R5= H, or residue of DOTA, DTP A, EDTA, NOTA, or a drug (in certain embodiments of a structure described herein, wherein R5= a residue of DOTA, DTP A, EDTA, NOTA, or a drug; in certain embodiments of a structure described herein, wherein R5= a residue

[0312]

[0313]

[0314] Table D. exemplary R5= H, or residue of DOTA, DTP A, EDTA, NOTA, or a drug (in certain embodiments of a structure described herein, wherein R5= a residue of DOTA, DTP A, EDTA, NOTA, or a drug; in certain embodiments of a structure described herein, wherein R5= a residue of DOT A)

[0315]

[0316] All publications, patents, and patent documents are incorporated by reference herein, as though individually incorporated by reference.

Claims

CLAIMSWhat is claimed is:A compound of Formula (I):XkR (I)or a pharmaceutically acceptable salt or metal complex thereof, wherein:R is -L°-R5, -LkR6, orL° is a linking group (e.g., a solubilizing group);R5is H, (C2-Ce)alkanoyl, a chelating group, or -L4-D;L4is absent or a linking group;D is the residue of a drug;L1is absent or a linking group (e.g., a solubilizing group) of the formulaeach of a, g, and x is independently 1, 2, or 3;each of X1and X2is of the formulaeach R° is independently CH2, NH, N(Ra), or O, wherein each Rais independently (Ci- Ce)alkyl or (C2-Ce)alkanoyl;each R1and R2is independently (C2-Ce)alkynyl or optionally substituted (Ci-Ce)alkyl (e.g., isopropyl, methyl), which optional substitution is with azido, hydroxy, or thiol; or each R1and R2are taken together to form a linking group L3;each R3is independently benzyl optionally substituted with one or more halo or is of the formulaeach Z is independently CH2, NH, or O;each R3ais independently H, -SO2F, -OSO2F, -SO2-(C5-Ce)heteroaryl (e.g., SO2- tetrazole), (C3-C7)heterocycle (e.g., oxiranyl, or cyclic imine such as 1 -pyrroline), acrylamide (-NHC(=O)CH=CH2), halo substituted (C2-Ce)alkanoyl (e.g, halo substituted on the a carbon next to the oxo(=O) group), or isothiocyanate (-N=C=S);eachR3bis independently (Ce-Cio)aryl, (Ci-Ce)alkyl, (Ci-Ce)alkoxy, (C3-Ce)cycloalkyl, - CH2O(Ci-Ce)alkyl, -CN, -C(=0)NH2, (C5-Ce)heteroaryl, (C3-C7)heterocycle (e.g., aziridinyl, or oxiranyl), hydroxy, nitro, -0(Ce-Cio)aryl, -O(C3-C6)cycloalkyl, -O(C5-Ce)heteroaryl, or -O(C3- C7)heterocycle; andeach of d, e, and f is independently 0, 1, 2, or 3.

2. The compound of claim 1, which is:wherein L2is a linking group, and each of b and c is independently 0, 1, 2, or 3.

3. The compound of claim 1, which is:wherein each of b and c is independently 0, 1, 2, or 3.

4. The compound of claim 1, which is:wherein each of b and c is independently 0, 1, 2, or 3.

5. The compound of claim 1, which is:wherein L2is a linking group and each of b and c is independently 0, 1, 2, or 3.

6. The compound of claim 1, which is:

7. The compound of any of the previous claims, wherein R5is a chelating group.

8. The compound of any of the previous claims, wherein R5is a residue of DOTA (l,4,7,10-tetraazacyclododecane-l,4,7,10-tetraacetic acid), DTPA (di ethylenetriaminepentaacetic acid), EDTA (ethylenediaminetetraacetic acid), or NOTA (l,4,7-triazacyclononane-l,4,7- tri acetic acid).

9. The compound of any of the previous claims, which is a metal complex that comprises Lutetium-177, Gadolinium, Gallium-68, Terbium-161, Holmium-166, Samarium-153, Promethium- 149, Terbium-149, Terbium-152, Terbium-155, Technetium-99, Actinium-225, Indium-Ill, Copper-64, Copper-67, Yttrium-90, Lead-212, Scandium-47, Scandium-44, or Bismuth-213, wherein the metal is complexed with the R5chelating group.

10. The compound of any of the previous claims, wherein at least one R3is not benzyl optionally substituted with one or more halo.

11. The compound of any of the previous claims, wherein, in at least one of X1and X2, R1and R2are taken together to form a linking group L3.

12. The compound of any of the previous claims, wherein, in at least one of X1and X2, each of R1and R2is independently (C2-Ce)alkynyl or substituted (Ci-Ce)alkyl, which substitution is with azido, or thiol.

13. The compound of any of the previous claims, wherein R is -L°-R5.

14. The compound of any of the previous claims, wherein L° is a hydrocarbon chain (e.g., branched or unbranched, saturated or unsaturated) comprising from 1 to 340 carbon atoms, wherein one or more carbon atoms of the chain is optionally replaced with O, S, N(RS), or an optionally substituted divalent (Ce-C2o)aryl, (Cs-C2o)heteroaryl, (C3-Cio)heterocycle, or (Cs-Ce)carbocycle, which optional substitution is with one or more of C(=O)NH2, halo, hydroxy, or oxo (=0); and Rsis H or (Ci-Ce)alkyl.

15. The compound of claim 14, wherein L° comprises from 1 to 110 carbon atoms.

16. The compound of any of the previous claims, wherein R is", wherein L2is a linking group, and each of b and c is independently 0, 1, 2, or 3.

17. The compound of any of the previous claims, wherein R is, wherein each of b and c is independently 0, 1, 2, or 3.

18. The compound of any of the previous claims, wherein L2is a hydrocarbon chain (e.g., branched or unbranched, saturated or unsaturated) comprising from 1 to 320 carbon atoms,wherein one or more carbon atoms of the chain is optionally replaced with O, S, N(RS), or an optionally substituted divalent (Ce-C2o)aryl, (Cs-C2o)heteroaryl, (C3-Cio)heterocycle, or (Cs-Ce)carbocycle, which optional substitution is with one or more of C(=0)NH2, halo, hydroxy, or oxo (=0); and Rsis H or (Ci-Ce)alkyl.

19. The compound of claim 18, wherein L2comprises from 1 to 90 carbon atoms.

20. The compound of claim 18, wherein L2is -NH(CH2)hC(=0)-, or is of the structureeach n is independently 1 to 100 (e.g., 2 to 100); h is 1, 2, or 3; and R7is H or methyl.

21. The compound of claim 18, wherein L2is, and R7is H or methyl.

22. The compound of any of claims 20-21, wherein each n is independently 1-30 (e.g., 2 to 30, or 1 to 10).

23. The compound of any of the previous claims, wherein R is24. The compound of claim 23, wherein Y is -L°-R5.

25. The compound of claim 23, wherein Y is26. The compound of any of the previous claims, wherein at least one R° is NH.

27. The compound of any of the previous claims, wherein at least one R1is isopropyl.

28. The compound of any of the previous claims, wherein at least one R2is methyl.

29. The compound of any of the previous claims, wherein at least one R3is benzyl.

30. The compound of any of the previous claims, wherein, in at least one of X1and X2, R3is31. The compound of any of the previous claims, wherein each R1and R2is independently (C2-Ce)alkynyl or substituted (Ci-Ce)alkyl (e.g., substituted methyl or ethyl), which substitution is with thiol or azido.

32. The compound of any of the previous claims, wherein, in at least one of X1and X2, R1and R2are taken together to form a linking group L3.

33. The compound of any of the previous claims, wherein L3is a hydrocarbon chain (e.g., branched or unbranched, saturated or unsaturated) comprising from 1 to 12 carbon atoms, wherein one or more carbon atoms of the chain is optionally replaced with O, S, N(RS), or an optionally substituted divalent (Ce-C2o)aryl, (Cs-C2o)heteroaryl, (C3-Cio)heterocycle, or (C5-Ce)carbocycle, which optional substitution is with one or more of C(=O)NH2, halo, hydroxy, or oxo (=0); and Rsis H or (Ci-Ce)alkyl.

34. The compound of claim 33, wherein L3is -CH2-, -O-, -C(=0)NH-, -NHC(=0)-, -NHS(=O)2-, -S(=O)2NH-, -NH-, -OC(=O)-, -C(=O)O-, (CH2)P-S-S-(CH2)q or (CH2)P-triazolyl-(CH2)q; p is 0, 1, 2, or 3; and q is 0, 1, 2, or 3.

35. The compound of claim 33, wherein L3is>36. The compound of claim 1, which is 152G5, 152G5-1, 152G5-3, 152G5-5, 152G8, 152G8-6, 152G8-8, 152G8-10, 152G12, 152G12-11, 152-13, or 152-15.or a pharmaceutically acceptable salt or metal complex thereof.

37. The compound of claim 1, wherein R is -L^R6.

38. The compound of claim 37, wherein R6is -NH2 or (CH2)2NHC(=O)CH3.

39. The compound of claim 37, wherein L1isr methyl.

40. The compound of claim 1, which isor a pharmaceutically acceptable salt thereof.

41. The compound of claim 1, which isTargefrin-monomer-PEG28, or a pharmaceutically acceptable salt thereof.

42. The compound of claim 1, wherein, in at least one (e.g., one or both) of X1and X2, R3is of the formula43. The compound of claim 42, wherein L1is absent and R6is -NH2.

44. The compound of claim 42, which is 152F11.

45. The compound of claim 42 or 43, wherein R is46. The compound of claim 42 or 43 or 45, wherein R5is H.

47. The compound of claim 42 or 43, which is 152F10.

48. The compound of claim 42 or 43 or 45, wherein L2is a hydrocarbon chain (branched or unbranched, saturated or unsaturated) comprising from 1 to 60 carbon atoms, wherein one or more carbon atoms of the chain is optionally replaced with O, S, N(RS), or an optionally substituted divalent (Ce-C2o)aryl, (Cs-C2o)heteroaryl, (C3-Cio)heterocycle, or (C5-Ce)carbocycle, which optional substitution is with one or more of C(=O)NH2, halo, hydroxy, or oxo (=0); and Rsis H or (Ci-Ce)alkyl.

49. The compound of claim 42, wherein D is a residue of paclitaxel.

50. The compound of claim 1, wherein, in at least one of X1and X2, R1and R2is independently (C2-Ce)alkynyl or substituted (Ci-Ce)alkyl, which substitution is with azido, or thiol; or in at least one of X1and X2, R1and R2are taken together to form a linking group L3.

51. The compound of claim 50, wherein L1is absent and R6is -NH2.

52. The compound of claim 50 or 51, which isor a pharmaceutically acceptable salt.

53. The compound of claim 50 or 51, which is:

54. The compound of claim 50 or 51 or 53, wherein L3is -CH2-, -O-, -C(=O)NH-, -NHC(=O)-, -NHS(=O)2-, -S(=O)2NH-, -NH-, -OC(=O)-, -C(=O)O-, (CH2)P-S-S-(CH2)q or (CH2)P-triazolyl-(CH2)q; p is 0, 1, 2, or 3; and q is 0, 1, 2, or 3.

55. The compound of claim 50 or 51 or 53, wherein L3is3.

56. The compound of claim 50 or 51 or 53, which isor a pharmaceutically acceptable salt.

57. A pharmaceutical composition comprising a compound or salt or metal complex of any one of claims 1-56 and a pharmaceutically acceptable carrier.

58. A method for treating cancer, which comprises administering a compound of formula I of any one of claims 1-56 or a pharmaceutically acceptable salt or metal complex thereof to a patient in need of.

59. The method of claim 56, which further comprises administering a second anticancer agent (e.g., chemotherapeutic agent) to the patient.

60. The method of claim 56, which further comprises administering an EGRF inhibitor, a HER2 inhibitor, or a BRAF inhibitor to the patient.

61. A compound of formula I of any one of claims 1-56 or a pharmaceutically acceptable salt or metal complex thereof for the prophylactic or therapeutic treatment of cancer.

62. Use of a compound of formula I of any oen of claims 1-56 or a pharmaceutically acceptable salt or metal complex thereof to prepare a medicament for treating cancer in an animal.

63. Use of any one of claims 61-62, in combination with an EGRF inhibitor, a HER2 inhibitor, or a BRAF inhibitor.

64. A method (e.g., an imaging and / or a treatment method), which comprises 1) administering a first compound of formula I of any one of claims 1-56 or a pharmaceutically acceptable salt or metal complex thereof to a patient in need of, 2) generating one or more medical image of the patient from a MRI or PET machine, and 3) optionally diagnosing thepatient as having cancer (e.g., EphA2 positive cancer) and / or optionally administering an anticancer drug e.g., a second compound of formula I of any one of claims 1-56 or a pharmaceutically acceptable salt or metal complex thereof) to the patient.

Images