EPHA2 targeting agents and uses thereof

Abstract

The EphA2 targeting agent developed herein is a potent peptidomimetic with high affinity (Kd 8-20 nanomolar) for the ligand-binding domain called targefurin. While the monomeric version of targefurin acts as an antagonist, the dimeric version of the agent (targefurin-dimer) triggers receptor internalization and degradation via the lysosomal pathway. Therefore, targefurin-dimer agents are effective in reducing pro-tumorigenic EphA2 levels in cancer cells when used as a single agent or in combination with standard of care. Targefurin-dimers can also sensitize cancer cells that have developed resistance to EGFR or BRAF inhibitors, and potentially other anticancer drugs. Furthermore, dimeric agents can be conjugated to chemotherapeutic agents, such as paclitaxel, to selectively deliver cytotoxic agents to EphA2-expressing cancer cells. The monomeric agent is linked to the chemotherapeutic agent via a stable, cleavable linker, allowing the cytotoxic agent to accumulate in tumors and subsequently enter the tumor. Examples of novel compositions and their applications are reported.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 416,890, filed October 17, 2022.

[0002] The entire contents of the above-referenced application are hereby incorporated by reference. [Background technology]

[0003] EphA2 is a receptor tyrosine kinase whose unbound form is pro-tumorigenic in various solid tumors. Its tumor-promoting activities include enhancing cell motility and migration, enhancing endothelial cell migration, enhancing angiogenesis, and suppressing the immune system. Furthermore, EphA2 expression confers resistance to drugs targeting EGRF inhibitors and to Her2 inhibitors in various solid tumors and to BRAF inhibitors in melanoma. Therefore, EphA2 could be used to develop effective cancer therapeutics.

[0004] Currently, there is a need for agents that target EphA2, which would be useful as cancer therapeutics, either alone or in combination with other anti-cancer agents. Summary of the Invention

[0005] Agents capable of targeting EphA2 have been identified. These EphA2 targeting agents are potent peptidomimetics with high affinity (Kds 8-20 nanomolar) for the ligand binding domain. Monomeric versions of these compounds (compounds of formula (I), where R 12 where R is H) have been found to be antagonists, while the dimeric form of the drug (compounds of formula (I) where R 12(wherein is other than H) causes receptor internalization and degradation via the lysosomal pathway. Thus, the dimeric agents are effective in reducing pro-tumorigenic EphA2 levels in cancer cells. Furthermore, the agents can be conjugated to other chemotherapeutic agents (e.g., paclitaxel) to selectively deliver cytotoxic agents to EphA2-expressing cancer cells.

[0006] Certain compounds of the invention are more soluble and 10-fold more active in causing EphA2 internalization and degradation than previously reported agents such as 135H12 (EphA2 Agonists and Uses Thereof, inventors Pellecchia et al., assigned to University of California, US20210221843A1; PCTWO2019237075A1; also published in Gambini et al. ACS Chem Biol. 2018 Sep 21;13(9):2633-2644. doi: 10.1021 / acschembio.8b00556). Thus, these agents can be used: i) to reduce pro-oncogenic EphA2 levels in solid tumors; ii) to conjugate with other chemotherapeutic agents to more selectively deliver cytotoxic agents to EphA2-expressing cancer cells; and iii) to degrade other surface receptors by linking them to molecules potent against these target receptors.

[0007] Thus, in one embodiment, the present invention provides a compound of formula (I): [ka] or a salt thereof, wherein: each R is independently selected from the group consisting of morpholino, piperidino, and piperazine, which is optionally substituted with (C-C) alkyl; Each R 1is benzyl, 3-indolylmethyl, 4-pyridinylmethyl, 1-naphthylmethyl, or 2-naphthylmethyl, wherein benzyl, 3-indolylmethyl, 4-pyridinylmethyl, 1-naphthylmethyl, and 2-naphthylmethyl are optionally substituted with one or more groups independently selected from hydroxy, amino, nitro, (C-C)alkoxy, and (C-C)alkyl; Each R 2 is independently selected from the group consisting of (C1-C6)alkyl optionally substituted with hydroxy; Each R 4 are independently selected from the group consisting of biphenyl and phenoxyphenyl, wherein the biphenyl and phenoxyphenyl are optionally substituted with one or more groups independently selected from halo, hydroxy, (C1-C6)alkyl, and (C1-C6)alkoxy, wherein each (C1-C6)alkyl and (C1-C6)alkoxy is optionally substituted with one or more groups independently selected from the group consisting of halo; Each R 7 is (C1-C6) alkyl optionally substituted with hydroxy; Each R 8 is independently selected from the group consisting of isopropyl and (C3-C6)cycloalkyl; Each R 9 is independently selected from the group consisting of benzyl optionally substituted with one or more halo; R 12 is H or is selected from the group consisting of: [ka] R 100 is H, (C3-C6)cycloalkyl optionally substituted with hydroxy, or (C1-C6)alkyl; R 101 is H, (C3-C6)cycloalkyl optionally substituted with hydroxy, or (C1-C6)alkyl; R 102is H, (C3-C6)cycloalkyl optionally substituted with hydroxy, or (C1-C6)alkyl; R 103 -L 1 -D; D is a residue of a drug or a residue of a targeting agent; p is 1, 2, or 3; m is 1, 2, or 3; n is 1, 2, or 3; R 104 is the following: [ka] and R 11 is C(=NH)NH2; L 1 is a linking group; L 2 is a linking group.

[0008] The present invention also provides a pharmaceutical composition comprising a compound of Formula I, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.

[0009] The present invention also provides a method for treating or preventing cancer in an animal (e.g., a mammal such as a human), comprising administering to the animal a compound of Formula I or a pharmaceutically acceptable salt thereof. In certain embodiments, the cancer is pancreatic cancer, prostate cancer, breast cancer, esophageal cancer, melanoma, bladder cancer, brain cancer, lung cancer, ovarian cancer, gastric cancer, or leukemia. In certain embodiments, the cancer is pancreatic cancer, prostate cancer, breast cancer, or melanoma. In certain embodiments, the cancer is metastatic cancer.

[0010] The present invention also provides a compound of formula I, or a pharmaceutically acceptable salt thereof, for use in medical therapy.

[0011] The present invention also provides a compound of formula I or a pharmaceutically acceptable salt thereof for the prophylactic or therapeutic treatment of cancer.

[0012] The invention also provides the use of a compound of Formula I, or a pharmaceutically acceptable salt thereof, to prepare a medicament for treating cancer in an animal (eg, a mammal such as a human).

[0013] The present invention also provides processes and intermediates disclosed herein that are useful for preparing compounds of formula I or salts thereof. [Brief explanation of the drawings]

[0014] [Figure 1A] 1 shows modeling and binding data for targefrin. Molecular model of targefrin in complex with EphA2-LBD based on the X-ray structure of the complex with peptide (PDB ID 6B9L). [Figure 1B] 1 shows modeling and binding data for targefrin. Chemical structure of targefrin. [Figure 1C] 1 shows modeling and binding data for targefrin. Isothermal titration calorimetry (ITC) curve for the binding between targefrin and EphA2-LBD (Kd=21.7±1.2 nM; ΔH=-20.2±0.4 kcal / mol; -TΔS=9.7±0.4 kcal / mol). [Figure 1D]

[0023] Figure 1 shows modeling and binding data for targefrin. ITC curves for the binding between targefrin and EphA4-LBD and EphA3-LBD, the two Eph receptors most similar to EphA2. The data showed no appreciable binding under these experimental conditions. [Figure 2]Data from Example 9 are shown. Targefrin acts as an antagonist. A. Western blot of BxPC3 cells starved for 1 hour, pretreated with various concentrations of targefrin for 20 minutes, and then co-treated with 2 μg / mL ephrinA1-Fc for 3 hours. B. Quantification of EphA2 levels. The EphA2 / β-actin ratio was normalized by setting the EphA2 expression from the DMSO condition without ephrinA1-Fc as 1. The EC50 value was calculated to be 1.6 ± 0.1 μM and is shown as the mean ± standard error (SE) of two independent experiments. [Figure 3A] Data from Example 11 are shown. Targefrin-dimer and its variants cause EphA2 degradation at nanomolar concentrations in pancreatic cancer cell lines. Western blot images of BxPC3 cells, in which cells were starved for 1 hour and treated with 2 μg / mL ephrinA1-Fc or the indicated doses of targefrin, targefrin-dimer, and its variants with different linkers (Table 4) for 3 hours. The previous dimerizer 135H121 is also shown as a reference. EphA2 / β-actin ratios were normalized to EphA2 expression from the DMSO control condition as 100%. [Figure 3B] Data from Example 11 are shown. Targefrin-dimer and its variants cause EphA2 degradation at nanomolar concentrations in pancreatic cancer cell lines. Western blot images of PANC-1 cells, in which cells were starved for 1 hour and treated with 2 μg / mL ephrinA1-Fc or the indicated doses of targefrin, targefrin-dimer, and its variants with different linkers (Table 4) for 3 hours. The previous dimerizer 135H121 is also shown as a reference. EphA2 / β-actin ratios were normalized to EphA2 expression from the DMSO control condition as 100%. [Figure 3C]Data from Example 11 are shown. Targefrin-dimer and its variations induce EphA2 degradation at nanomolar concentrations in pancreatic cancer cell lines. Western blot images of MIAPaCa-2 cells were starved for 1 hour and then treated with 2 μg / mL ephrinA1-Fc or the indicated doses of targefrin, targefrin-dimer, and its variations with different linkers (Table 4) for 3 hours. The previous dimerizer 135H121 is also shown as a reference. EphA2 / β-actin ratios were normalized to EphA2 expression from the DMSO control condition, which was set at 100%. [Figure 3D] Data from Example 11 are shown. Targefurin dimer and its variants cause EphA2 degradation at nanomolar concentrations in pancreatic cancer cell lines. Densitometry analysis of the data shown in A. EphA2 / β-actin ratios were normalized to EphA2 expression from the DMSO control condition as 1. ***p<0.001, ****p<0.0001, determined by one-way ANOVA with Dunnett's post-hoc analysis. [Figure 3E] Data from Example 11 are shown. Targefurin dimer and its variants cause EphA2 degradation at nanomolar concentrations in pancreatic cancer cell lines. B Densitometry analysis of the data shown. EphA2 / β-actin ratios were normalized to EphA2 expression from the DMSO control condition, with 1. ***p<0.001, ****p<0.0001, determined by one-way ANOVA with Dunnett's post-hoc analysis. [Figure 3F] Data from Example 11 are shown. Targefurin dimer and its variants cause EphA2 degradation at nanomolar concentrations in pancreatic cancer cell lines. Densitometry analysis of the data shown in C. EphA2 / β-actin ratios were normalized to EphA2 expression from the DMSO control condition as 1. ***p<0.001, ****p<0.0001, determined by one-way ANOVA with Dunnett's post-hoc analysis. [Figure 4A]1 shows data from Example 12. Chemical structure and biochemical activity of targefrin-conjugate agents. Chemical structure of targefrin conjugated to paclitaxel (targefrin-PTX). [Figure 4B] 1 shows data from Example 12. Chemical structure and biochemical activity of targefrin-conjugate agents. Chemical structure of the dimeric version of targefrin conjugated to paclitaxel (targefrin-dimer-PTX). [Figure 4C] 1 shows data from Example 12. Chemical structure and biochemical activity of targefurin-conjugate agents. Chemical structure of the dimeric version of targefurin conjugated to 5-carboxytetramethylrhodamine-azide dye (targefurin-dimer-TAMRA). [Figure 4D] 1 shows data from Example 12. Chemical structure and biochemical activity of targefurin-conjugate agents. DELFIA displacement dose-response curves comparing targefurin-PTX, targefurin-dimer-PTX, and targefurin-dimer-TAMRA by their respective IC50 values. [Figure 5] Data from Example 13 are shown. Targefurin-dimer-TAMRA is internalized in EphA2-expressing cells. BxPC3 cells were treated with 100 nM targefurin-dimer-TAMRA for 0, 30, and 60 minutes. Upon drug binding, EphA2 was internalized and targeted to lysosomes, as indicated by colocalization of 5-TAMRA and LAMP1 (arrows). Nuclei are labeled blue. Scale bar = 10 μm. [Figure 6A] Data from Example 14 are shown. Targefurin-dimer significantly inhibits the migration of pancreatic cancer cells. Cell migration assay of BxPC3 cells treated with 2 μg / mL ephrinA1-Fc and 10 μM targefurin, or the indicated doses of targefurin-dimer. Plates were imaged every 3 hours for 24 hours. The yellow line indicates the initial scratch made at 0 hours, while the black line indicates the position where the cells migrated after 24 hours. Scale bar = 250 μm. [Figure 6B]Data from Example 14 are shown. Targefurin-dimer significantly inhibits the migration of pancreatic cancer cells. Targefurin-dimer significantly inhibited cell migration after 24 hours in a dose-dependent manner, as indicated by a decrease in relative wound density. Data compared with the 12-hour time point are reported in Figure 10. ***p<0.001, ****p<0.0001, determined by one-way ANOVA with Dunnett's post-hoc analysis. [Figure 6C]

[0033] Figure 1 shows data from Example 14. Targefrin-dimer significantly inhibits the migration of pancreatic cancer cells. Time-response curves showed the effect of the drug on wound closure over 24 hours. [Figure 7A] Data from Example 15 are shown. PTX-conjugates suppressed tumor growth in tumor xenografts bearing MIA-PaCa-2 cells. Five groups of five mice bearing pre-established MIA-PaCa-2 tumors were treated for 22 days with vehicle control alone, paclitaxel (PTX, 2.5 mg / kg), targefurin-PTX (10 mg / kg, equivalent to 2.5 mg / kg of PTX), targefurin-dimer-PTX (17 mg / kg, equivalent to 2.5 mg / kg of PTX), and low-dose targefurin-dimer-PTX (10 mg / kg, equivalent to 1.5 mg / kg of PTX). Tumor volumes are reported as mean ± SE. [Figure 7B] 1 shows data from Example 15. PTX-conjugates inhibited tumor growth in tumor xenografts bearing MIA-PaCa-2 cells. Mean tumor volumes measured by treatment group on days 0, 8, 15, and 22. *p=0.03, **p<0.01, ***p=0.0001, ****p<0.0001, determined by two-way ANOVA with Tukey's post-hoc analysis. [Figure 7C]Data from Example 15 are shown. PTX-conjugates suppressed tumor growth in tumor xenografts bearing MIA-PaCa-2 cells. For each of the five treatment groups, mean body weights ± SE are reported on days 0, 8, 15, and 22. For all graphs, vehicle is reported in black, PTX in green, low-dose targefrin-dimer-PTX in light blue, targefrin-dimer-PTX in blue, and targefrin-PTX in red. [Figure 8A] Figure 1 shows that targefrin-dimer-PTX retains the ability to cause EphA2 degradation in pancreatic cancer cell lines. Western blot images of BxPC3 cells, in which cells were starved for 1 hour and treated with 2 μg / mL ephrinA1-Fc or the indicated doses of targefrin, targefrin-PTX, targefrin-dimer, and targefrin-dimer-PTX for 3 hours, show that targefrin-dimer-PTX retains the ability to cause EphA2 degradation in pancreatic cancer cell lines. EphA2 / β-actin ratios were normalized to 100% EphA2 expression from the DMSO control condition. [Figure 8B] Figure 1 shows that targefrin-dimer-PTX retains the ability to cause EphA2 degradation in pancreatic cancer cell lines. Western blot images of PANC-1 cells, in which cells were starved for 1 hour and treated with 2 μg / mL ephrinA1-Fc or the indicated doses of targefrin, targefrin-PTX, targefrin-dimer, and targefrin-dimer-PTX for 3 hours, show that targefrin-dimer-PTX retains the ability to cause EphA2 degradation in pancreatic cancer cell lines. EphA2 / β-actin ratios were normalized to 100% EphA2 expression from the DMSO control condition. [Figure 8C] Figure 1 shows that targefrin-dimer-PTX retains the ability to cause EphA2 degradation in pancreatic cancer cell lines. A Western blot image of MIAPaCa-2 cells, in which cells were starved for 1 hour and treated with 2 μg / mL ephrinA1-Fc or the indicated doses of targefrin, targefrin-PTX, targefrin-dimer, and targefrin-dimer-PTX for 3 hours. EphA2 / β-actin ratios were normalized to EphA2 expression from the DMSO control condition (100%). [Figure 8D] Densitometry analysis of Figure 8A shows that targefurin-dimer-PTX retains the ability to cause EphA2 degradation in pancreatic cancer cell lines. EphA2 / β-actin ratios were normalized to EphA2 expression from the DMSO control condition, with 1. ***p<0.001, ****p<0.0001, determined by one-way ANOVA with Dunnett's post-hoc analysis. [Figure 8E] Densitometry analysis of Figure 8B shows that targefurin-dimer-PTX retains the ability to cause EphA2 degradation in pancreatic cancer cell lines. EphA2 / β-actin ratios were normalized to EphA2 expression from the DMSO control condition, with 1. ***p<0.001, ****p<0.0001, determined by one-way ANOVA with Dunnett's post-hoc analysis. [Figure 8F] Densitometry analysis of Figure 8C shows that targefurin-dimer-PTX retains the ability to cause EphA2 degradation in pancreatic cancer cell lines. EphA2 / β-actin ratios were normalized to EphA2 expression from the DMSO control condition, with 1. ***p<0.001, ****p<0.0001, determined by one-way ANOVA with Dunnett's post-hoc analysis. [Figure 9] Pharmacokinetic Studies. Preliminary pharmacokinetic (PK) studies using targefrin-dimer. The drug was injected IV via the tail vein at a concentration of 50 mg / kg in a formulation of 80% PBS, 10% Tween 80, and 10% ethanol. Note that the formulation resulted in a clear solution containing 20 mg / ml of targefrin-dimer. Two hours after injection, Cmax was approximately 650 ng / mL. The estimated t1 / 2 was approximately 15 hours. [Figure 10A] Figure 12 shows cell migration assay of BxPC3 from Figure 9 after 12 hours. Cell migration assay of BxPC3 treated with 2 μg / mL ephrinA1-Fc and 10 μM targefurin, or the indicated doses of targefurin-dimer. The yellow line indicates the initial scratch made at 0 hours, while the black line indicates the position to which the cells migrated after 12 hours. [Figure 10B]Figure 10 shows the cell migration assay after 12 hours for BxPC3 from Figure 9. Targefurin-dimer significantly inhibited cell migration after 12 hours in a dose-dependent manner, as indicated by a decrease in relative wound density. ***p<0.01, ****p<0.0001, determined by one-way ANOVA with Dunnett's post-hoc analysis. [Figure 11] Structural details relevant to the optimization process. A. Structure of EphA2-LBD complexed with a drug previously developed in our laboratory (1). Showing the receptor surface and the DE, GH, and JK loops. B. Detail of tyrosine residue 4 of the EphA2-binding agent, which protrudes into a large hydrophobic pocket located between the DE and JK loops. C. Detail of position 1 of the EphA2-binding agent, which replaces tyrosine 1 and the N-terminal amide of the YSA peptide, located between the GH and JK loops. D. Detail of a pair of serine residues in the EphA2-binding agent, which form intramolecular hydrogen bonds (see arrows), constraining the peptide into a tight conformation in the EphA2-bound form. [Figure 12A] MIA PaCa-2 cell viability assay at 72 hours. MIA PaCa-2 cells were treated with 1 μg / mL ephrinA1-Fc, different doses of targefrin, or targefrin-dimer for 72 hours. Percent confluency was monitored using an IncuCyte S3 live cell analysis system, and percent cell viability was calculated by normalizing treatment confluency to the DMSO control confluency. Cell viability was not significantly affected by any treatment, as determined by two-way ANOVA with Bonferroni post-hoc analysis. [Figure 12B] Cell viability assay of MIA PaCa-2 at 72 hours. Time-response curves of percent confluency of MIA PaCa-2 cells after the indicated treatments. [Figure 13]In (A), targefurin-dimer mimics the natural ephrinA1 ligand, triggering EphA2 degradation and suppressing metastasis. In (B), targefurin-PTX was able to deliver PTX to cancer cells by accumulating the toxin on EphA2-expressing metastatic pancreatic cancer cells. Here, release of the toxin requires specific linker cleavage by an extracellular protein, followed by passive diffusion of the toxin within the tumor cells. In (C), however, conjugation of PTX to targefurin-dimer suppresses EphA2 degradation and prometastatic signaling. Simultaneously, the toxin is actively transported within EphA2-expressing tumor cells, actively killing primary and metastatic cancer cells. DETAILED DESCRIPTION OF THE INVENTION

[0015] Unless otherwise stated, the following definitions are used: halo or halogen is fluoro, chloro, bromo, or iodo; alkyl, alkoxy, etc. refer to both straight and branched chain groups, although when referring to individual radicals such as propyl, only straight chain radicals are included (branched chain isomers such as isopropyl are specifically mentioned).

[0016] The term "alkyl," by itself or as part of another substituent, unless otherwise stated, has the indicated number of carbon atoms (i.e., C 1-8 means a straight or branched chain hydrocarbon radical (meaning 1 to 8 carbons). Examples include (C1-C8) alkyl, (C2-C8) alkyl, C1-C6) alkyl, (C2-C6) alkyl, and (C3-C6) alkyl. Examples of alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, n-pentyl, n-hexyl, n-heptyl, n-octyl, and the higher homologs and isomers.

[0017] The term "alkoxy" refers to an alkyl group attached to the remainder of the molecule through an oxygen atom ("oxy").

[0018] The term "cycloalkyl" or "carbocycle" refers to any saturated or partially unsaturated (non-aromatic) carbocyclic ring (i.e., (C3-C8)carbocycle) having 3 to 8 carbon atoms. The term also includes saturated, all-carbon, multiple condensed ring systems (e.g., ring systems containing two, three, or four carbocycles). Thus, carbocycle includes 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 having up to about 20 carbon atoms). Rings in multiple condensed ring systems can be connected to each other through fused, spiro, and bridged bonds, where valence requirements permit. For example, polycyclic carbocycles can be connected to each other through a single carbon atom to form a spiro connection (e.g., spiropentane, spiro[4,5]decane, etc.), through two adjacent carbon atoms to form a fused connection (e.g., carbocycles such as decahydronaphthalene, norsabinane, norcarane, etc.), or through two non-adjacent carbon atoms to form a bridged connection (e.g., norbornane, bicyclo[2.2.2]octane, etc.) Non-limiting examples of cycloalkyl include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, bicyclo[2.2.1]heptane, pinane, and adamantane.

[0019] The term "aryl," as used herein, refers to an all-carbon aromatic monocyclic ring system or an all-carbon multiple condensed ring system in which at least one ring is aromatic. For example, in certain embodiments, aryl groups have 6 to 20 carbon atoms, 6 to 14 carbon atoms, 6 to 12 carbon atoms, or 6 to 10 carbon atoms. Aryl includes the phenyl radical. Aryl also includes multiple condensed carbocyclic ring systems (e.g., ring systems containing two, three, or four rings) having about 9 to 20 carbon atoms, in which at least one ring is aromatic and the remaining rings may or may not be aromatic (i.e., cycloalkyl). Rings in multiple condensed ring systems can be connected to each other via fused, spiro, and bridged bonds, where valence requirements allow. As defined above, it should be understood that the point of attachment in multiple condensed ring systems may be at any position on the ring system, including the aromatic or carbocyclic portions 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.

[0020] The term "heterocycle" refers to a saturated or partially unsaturated monocyclic ring having at least one atom other than carbon in the ring, where the atom is selected from the group consisting of oxygen, nitrogen, and sulfur; the term also includes multiple condensed ring systems having at least one such saturated or partially unsaturated ring, which are further described below. Thus, the term includes a single saturated or partially unsaturated ring (e.g., a 3-, 4-, 5-, 6-, or 7-membered ring) of about 1 to 6 carbon atoms and about 1 to 3 heteroatoms selected from the group consisting of oxygen, nitrogen, and sulfur in the ring. The sulfur and nitrogen atoms may be present in oxidized form. Exemplary heterocycles include, but are not limited to, azetidinyl, tetrahydrofuranyl, and piperidinyl. The term "heterocycle" also includes multiple fused ring systems (e.g., ring systems containing 2, 3, or 4 rings), where a heterocyclic monocycle (as defined above) can be fused to one or more groups selected from cycloalkyl, aryl, and heterocycle to form the multiple fused ring system. The rings of a multiple fused ring system can be connected to each other via fused, spiro, and bridged bonds, where permitted by valence requirements. It should be understood that the individual rings of a multiple fused ring system can be connected to each other in any order. It should also be understood that the point of attachment of a multiple fused ring system (as defined above for heterocycle) can be at any position of the multiple fused ring system, including the heterocyclic, aryl, and carbocyclic portions of the ring. In one embodiment, the term heterocycle includes a 3- to 15-membered heterocycle. In one embodiment, the term heterocycle includes a 3- to 10-membered heterocycle. In one embodiment, the term heterocycle includes a 3- to 8-membered heterocycle. In one embodiment, the term heterocycle includes a 3- to 7-membered heterocycle. In one embodiment, the term heterocycle includes a 3- to 6-membered heterocycle. In one embodiment, the term heterocycle includes a 4- to 6-membered heterocycle. In one embodiment, the term heterocycle includes a 3- to 10-membered monocyclic or bicyclic heterocycle containing 1-4 heteroatoms. In one embodiment, the term heterocycle includes a 3- to 8-membered monocyclic or bicyclic heterocycle containing 1-3 heteroatoms. In one embodiment, the term heterocycle includes a 3- to 6-membered monocyclic heterocycle containing 1-2 heteroatoms.In one embodiment, the term heterocycle includes 4-6 membered monocyclic heterocycles containing 1-2 heteroatoms. Exemplary heterocycles include 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-1,1'-isoindolinyl]-3'-one, isoindolinyl-1-one, 2-oxa-6-azaspiro[3.3]heptanyl, imidazolidin-2-one, imidazolidine, pyrazolidine, butyrolactam, valerolactam, imidazolidinone, hydantoin, dioxolane, phthalimide, and 1,4-dioxane.

[0021] The term "heteroaryl," as used herein, refers to a monocyclic aromatic ring having at least one atom other than carbon in the ring, the atom being selected from the group consisting of oxygen, nitrogen, and sulfur. "Heteroaryl" also includes multiple fused ring systems having at least one such aromatic ring, which are further described below. Thus, "heteroaryl" includes a monocyclic aromatic ring of about 1 to 6 carbon atoms and about 1 to 4 heteroatoms selected from the group consisting of oxygen, nitrogen, and sulfur. The sulfur and nitrogen atoms may also be present in 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 fused ring systems (e.g., ring systems containing 2, 3, or 4 rings) in which a heteroaryl group, as defined above, is fused to one or more rings selected from cycloalkyl, aryl, heterocycle, and heteroaryl. It should be understood that the point of attachment of the heteroaryl or heteroaryl multiple condensed ring system may be any suitable atom of the heteroaryl or heteroaryl multiple condensed ring system, including carbon atoms and heteroatoms (e.g., 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.

[0022] As used herein, the term "protecting group" refers to a substituent commonly used to block or protect a particular functional group on a compound. For example, an "amino-protecting group" is a substituent attached to an amino group that blocks or protects the amino functionality in the compound. Suitable amino-protecting groups include acetyl, trifluoroacetyl, t-butoxycarbonyl (BOC), benzyloxycarbonyl (CBZ), and 9-fluorenylmethyleneoxycarbonyl (Fmoc). Similarly, a "hydroxy-protecting group" refers to a substituent of a hydroxy group that blocks or protects the hydroxy functionality. Suitable protecting groups include acetyl and silyl. A "carboxy-protecting group" refers to a substituent of a carboxy group that blocks or protects the carboxy functionality. Common carboxy-protecting groups include phenylsulfonylethyl, cyanoethyl, 2-(trimethylsilyl)ethyl, 2-(trimethylsilyl)ethoxymethyl, 2-(p-toluenesulfonyl)ethyl, 2-(p-nitrophenylsulfenyl)ethyl, 2-(diphenylphosphino)-ethyl, nitroethyl, and the like. For a general description of protecting groups and their uses, see PGM Wuts and TW Greene, Greene's Protective Groups in Organic Synthesis 4th edition, Wiley-Interscience, New York, 2006.

[0023] As used herein, a wavy line crossing a bond in a chemical structure [ka] indicates the point of attachment of the bond that the wavy bond meets in the chemical structure to the rest of the molecule.

[0024] The terms "treat," "treatment," or "treating," insofar as it relates to a disease or condition, include inhibiting a disease or condition, eliminating a disease or condition, and / or alleviating one or more symptoms of a disease or condition. The terms "treat," "treatment," or "treating" also refer to both therapeutic treatment and / or prophylactic treatment or measures, where the objective is to prevent or slow (alleviate) the onset or spread of an undesirable physiological change or disorder, such as cancer. For example, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, whether detectable or undetectable, attenuation of the extent of the disease or disorder, stabilization of the disease or disorder state (i.e., not worsening), delay or slowing of disease progression, palliation or palliative care of the disease state or disorder, and remission (whether partial or complete). "Treat," "treatment," or "treating" can also mean prolonging survival as compared to expected survival if not receiving treatment. Those in need of treatment include those already with the disease or disorder as well as those prone to have the disease or disorder or those in whom the disease or disorder is to be prevented. In one embodiment, "treat", "treatment", or "treating" does not include prevent or prophylaxis,

[0025] The phrase "therapeutically effective amount" or "effective amount" includes, but is not limited to, an amount of a compound that (i) treats or prevents a particular disease, condition, or disorder, (ii) attenuates, ameliorate, or eliminates one or more symptoms of a particular disease, condition, or disorder, or (iii) prevents or delays the onset of one or more symptoms of a particular disease, condition, or disorder described herein.

[0026] The term "mammal", as used herein, refers to humans, higher non-human primates, rodents, livestock, cattle, horses, pigs, sheep, dogs, and cats. In one embodiment, a mammal is a human. The term "patient", as used herein, refers to any animal, including mammals. In one embodiment, the patient is a mammalian patient. In one embodiment, the patient is a human patient.

[0027] Those skilled in the art will appreciate that the present invention provides isotope ratios of, but not limited to, deuterium ( 2 It will be understood that this also includes any compound as set forth in the claims that may be enriched with one or more isotopes, such as H or D. As a non-limiting example, a -CH3 group may be replaced with -CD3.

[0028] The pharmaceutical composition of the present invention may contain one or more excipients. When used in conjunction with the pharmaceutical composition of the present invention, the term "excipient" generally refers to an additional component that is combined with the compound of formula (I) or its pharmaceutically acceptable salt to provide the corresponding composition. For example, when used in conjunction with the pharmaceutical composition of the present invention, the term "excipient" includes, but is not limited to: carriers, binders, disintegrants, lubricants, sweeteners, flavoring agents, coatings, preservatives, and dyes.

[0029] The stereochemical definitions and conventions used herein generally follow those of S.P. Parker, Ed., McGraw-Hill Dictionary of Chemical Terms (1984), McGraw-Hill Book Company, New York, and Eliel, E. and Wilen, S., "Stereochemistry of Organic Compounds," John Wiley & Sons, Inc., New York, 1994. The compounds of the present invention may contain asymmetric or chiral centers and therefore exist in various stereoisomeric forms. All stereoisomers of the compounds of the present invention (including, but not limited to, diastereomers, enantiomers, and atropisomers), as well as mixtures thereof (such as racemic mixtures), are intended to form part of the present invention. Many organic compounds exist in optically active forms, i.e., have the ability to rotate the plane of plane-polarized light. In describing an optically active compound, the prefixes D and L, or R and S, are used to denote the absolute configuration of the molecule about its chiral center(s). The prefixes d and l or (+) and (-) are used to denote the sign of rotation of plane-polarized light by a compound, where (-) or l means the compound is levorotatory. Compounds with a (+) or d prefix are dextrorotatory. For a given chemical structure, these stereoisomers are identical except that they are mirror images of each other. A specific stereoisomer can also be called an enantiomer, and a mixture of such isomers is often called an enantiomeric mixture. A 50:50 mixture of enantiomers is called a racemic mixture or racemate and can occur when there is no stereoselectivity or stereospecificity in a chemical reaction or process. The terms "racemic mixture" and "racemate" refer to an equimolar mixture of two enantiomeric species that lacks optical activity.

[0030] It will be apparent to those skilled in the art that compounds of the present invention having chiral centers can exist in and be isolated in optically active and racemic forms. Some compounds may exhibit crystalline polymorphism. It should be understood that the present invention includes any racemic, optically active, crystalline polymorphic, or stereoisomer of the compounds of the present invention, or mixtures thereof, that possess the useful properties described herein, and methods for preparing optically active forms (e.g., by recrystallization techniques, by resolution of racemates, by synthesis from optically active starting materials, by chiral synthesis, or by chromatographic separation using chiral stationary phases) are well known in the art.

[0031] In the formulas of compounds herein, when a bond is drawn in a non-stereochemical form (e.g., planar), all stereochemical possibilities are included for the atom to which the bond is attached. In the formulas of compounds herein, when a bond is drawn in a defined stereochemical form (e.g., as a bold line, a bold wedge, a dashed line, or a dashed wedge), it is understood that the atom to which the stereochemical bond is attached is enriched for the absolute stereoisomer shown, unless otherwise noted. In one embodiment, the compound may be at least 51% of the absolute stereoisomer shown. In another embodiment, the compound may be at least 60% of the absolute stereoisomer shown. In another embodiment, the compound may be at least 80% of the absolute stereoisomer shown. In another embodiment, the compound may be at least 90% of the absolute stereoisomer shown. In another embodiment, the compound may be at least 95% of the absolute stereoisomer shown. In another embodiment, the compound may be at least 99% of the absolute stereoisomer shown.

[0032] The term "residue" as applied to a residue of a compound refers to a compound that has been modified in some way that results in the creation of an open valence, where the site of the open valence becomes an open valence. An open valence can be created by removing one or more atoms from the compound (e.g., removing a single atom such as hydrogen, or removing two or more atoms such as a group of atoms including, but not limited to, an amine, hydroxyl, methyl, amide (e.g., -C(=O)NH2), or acetyl group). An open valence can also be created by chemically converting a first functional group of the compound into a second functional group of the compound (e.g., reducing a carbonyl group, replacing the carbonyl group with an amine, etc.), followed by removing one or more atoms from the second functional group to create the open valence.

[0033] In one embodiment, the present invention provides a compound of formula (I): [ka] or a salt thereof, wherein: each R is independently selected from the group consisting of morpholino, piperidino, and piperazine, which is optionally substituted with (C-C) alkyl; Each R 1 is benzyl, 1-naphthylmethyl, or 2-naphthylmethyl, wherein the benzyl, 1-naphthylmethyl, and 2-naphthylmethyl are optionally substituted with one or more groups independently selected from hydroxy, amino, nitro, and (C1-C6)alkyl; Each R 2 is independently selected from the group consisting of (C1-C6)alkyl optionally substituted with hydroxy; Each R 4 are independently selected from the group consisting of biphenyl and phenoxyphenyl, wherein the biphenyl and phenoxyphenyl are optionally substituted with one or more groups independently selected from halo, hydroxy, (C1-C6)alkyl, and (C1-C6)alkoxy, wherein each (C1-C6)alkyl and (C1-C6)alkoxy is optionally substituted with one or more groups independently selected from the group consisting of halo; Each R7 is (C1-C6) alkyl optionally substituted with hydroxy; Each R 8 is independently selected from the group consisting of isopropyl and (C3-C6)cycloalkyl; Each R 9 is independently selected from the group consisting of benzyl optionally substituted with one or more halo; R 12 is H or is selected from the group consisting of: [ka] R 100 is H, (C1-C2) alkyl optionally substituted with hydroxy, or (C3-C6) cycloalkyl; R 101 is H, (C1-C2) alkyl optionally substituted with hydroxy, or (C3-C6) cycloalkyl; R 102 is H, (C1-C2) alkyl optionally substituted with hydroxy, or (C3-C6) cycloalkyl; R 103 -L 1 -D; D is a residue of a drug or a residue of a targeting agent; p is 1, 2, or 3; R 104 is the following: [ka] and R 11 is C(=NH)NH2; L 1 is a linking group; L 2 is a linking group.

[0034] The specific values ​​listed below for radicals, substituents, and ranges are for illustrative purposes only; they do not exclude other defined values ​​or other values ​​within defined ranges for radicals and substituents. It is understood that two or more values ​​can be combined. It is also understood that the values ​​listed below (or any subset thereof) can be excluded.

[0035] Specifically, (C1-C6)alkyl can be methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, pentyl, 3-pentyl, or hexyl; (C3-C6)cycloalkyl can be cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl; (C3-C6)cycloalkyl(C1-C6)alkyl can be cyclopropylmethyl, cyclobutylmethyl, cyclopentylmethyl, cyclohexylmethyl, 2-cyclopropylethyl, 2-cyclobutylethyl, 2-cyclopentylethyl, or 2-cyclohexylethyl; (C1-C6)alkoxy can be methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, sec-butoxy, pentoxy, 3-pentoxy, or hexyloxy; and aryl can be phenyl, indenyl, or naphthyl.

[0036] A specific value for R is morpholino.

[0037] A specific value for R is piperidino.

[0038] A specific value for R is piperazinyl (e.g., 1-piperazinyl), which is optionally substituted with (C1-C6) alkyl.

[0039] A specific value for R is 1-piperazyl.

[0040] R 1 A specific value for is benzyl optionally substituted with amino.

[0041] R 1A specific value for is benzyl optionally substituted with nitro.

[0042] R 1 A specific value for is benzyl optionally substituted with (C1-C6) alkyl.

[0043] R 1 A specific value for is benzyl optionally substituted with hydroxyl.

[0044] R 1 A specific value for is benzyl optionally substituted with (C1-C6)alkoxyl.

[0045] R 1 A specific value for is 2-nitrobenzyl, 4-methylbenzyl, or 4-aminobenzyl.

[0046] R 1 A specific value for is 4-hydroxybenzyl, or 4-methoxybenzyl.

[0047] R 1 A specific value for is 3-indolylmethyl, 4-pyridinylmethyl, 1-naphthylmethyl, or 2-naphthylmethyl.

[0048] R 1 A specific value for is 3-indolylmethyl optionally substituted with hydroxyl.

[0049] R 2 A specific value for is selected from the group consisting of (C1-C4) alkyl optionally substituted with hydroxy.

[0050] R 2 A specific value for is isobutyl or hydroxymethyl.

[0051] R 2 A specific value for is hydroxyethyl (eg, 1-hydroxyethyl).

[0052] R 4 A specific value for is biphenyl optionally substituted with one or more groups independently selected from halo, hydroxy, (C-C) alkyl, and (C-C) alkoxy, wherein each (C-C) alkyl and (C-C) alkoxy is optionally substituted with one or more groups independently selected from the group consisting of halo.

[0053] R 4 A specific value for is phenoxyphenyl optionally substituted with one or more groups independently selected from the group consisting of halo, hydroxy, (C-C) alkyl, and (C-C) alkoxy, wherein each (C-C) alkyl and (C-C) alkoxy is optionally substituted with one or more groups independently selected from the group consisting of halo.

[0054] R 4 Specific values ​​of are selected from the group consisting of biphenyl, 2'-trifluoromethylbiphenyl, 2'-methylbiphenyl, 4'-chlorobiphenyl, 2'-methoxybiphenyl, 3'-methylbiphenyl, 2'-methyl-4'-methoxybiphenyl, phenoxyphenyl, and 4-(4-hydroxyphenyloxy)phenyl.

[0055] R 7 A specific value for is methyl optionally substituted with hydroxyl.

[0056] R 7 A specific value for is methyl.

[0057] R 8 Specific values ​​of are independently selected from the group consisting of isopropyl and (C3-C6)cycloalkyl.

[0058] R 8 A specific value for is cyclohexyl.

[0059] R 9 A specific value for is benzyl.

[0060] R 12 The specific value of is H.

[0061] R 12 Specific values ​​for are: [ka] is.

[0062] R 12 Specific values ​​for are: [ka] is.

[0063] A specific value of m is 1. A specific value of m is 2. A specific value of m is 3.

[0064] A specific value of n is 1. A specific value of n is 2. A specific value of n is 3.

[0065] R 100 A specific value for is H, (C3-C6)cycloalkyl, or (C1-C3)alkyl.

[0066] R 101 A specific value for is H, (C3-C6)cycloalkyl, or (C1-C3)alkyl.

[0067] R 102 A specific value for is H, (C3-C6)cycloalkyl, or (C1-C3)alkyl.

[0068] R 100 A specific value for is H, (C3-C6)cycloalkyl, or (C1-C2)alkyl.

[0069] R 101 A specific value for is H, (C3-C6)cycloalkyl, or (C1-C2)alkyl.

[0070] R102 A specific value for is H, (C3-C6)cycloalkyl, or (C1-C2)alkyl.

[0071] R 100 A specific value for is H, methyl, ethyl, isopropyl, cyclohexyl, or hydroxymethyl.

[0072] R 101 A specific value for is H, methyl, ethyl, isopropyl, cyclohexyl, or hydroxymethyl.

[0073] R 102 A specific value for is H, methyl, ethyl, isopropyl, cyclohexyl, or hydroxymethyl.

[0074] R 12 Specific values ​​for are: [ka] is.

[0075] R 12 Specific values ​​for are: [ka] is.

[0076] A specific value of m is 1. A specific value of m is 2. A specific value of m is 3.

[0077] A specific value of n is 1. A specific value of n is 2. A specific value of n is 3.

[0078] L 1 A specific value for is a branched or unbranched, saturated or unsaturated hydrocarbon chain having about 1 to 100 carbon atoms, where one or more carbon atoms are selected from the group consisting of -O-, -S, -N(R a)-, a 3- to 7-membered heterocycle, a 5- to 6-membered heteroaryl, or a carbocycle, wherein each chain, 3- to 7-membered heterocycle, 5- to 6-membered heteroaryl, or carbocycle is optionally independently substituted with (C-C)alkyl, (C-C)alkoxy, (C-C)cycloalkyl, (C-C)alkanoyl, (C-C)alkanoyloxy, (C-C)alkoxycarbonyl, (C-C)alkylthio, azido, cyano, nitro, halo, -N(R a )2, hydroxy, oxo (=O), carboxy, aryl, aryloxy, heteroaryl, and heteroaryloxy, wherein each R a are independently H or (C1-C6) alkyl.

[0079] L 1 A specific value for is a branched or unbranched, saturated or unsaturated hydrocarbon chain having about 1 to 20 carbon atoms, where one or more carbon atoms are selected from the group consisting of -O-, -S, -N(R a )-, a 3- to 7-membered heterocycle, a 5- to 6-membered heteroaryl, or a carbocycle, wherein each carbon atom, 3- to 7-membered heterocycle, 5- to 6-membered heteroaryl, or carbocycle is optionally independently replaced by (C-C)alkyl, (C-C)alkoxy, (C-C)cycloalkyl, (C-C)alkanoyl, (C-C)alkanoyloxy, (C-C)alkoxycarbonyl, (C-C)alkylthio, azido, cyano, nitro, halo, -N(R a )2, hydroxy, oxo (=O), and carboxy; a are independently H or (C1-C6) alkyl.

[0080] L 1A specific value for is a branched or unbranched saturated hydrocarbon chain having about 5 to 15 carbon atoms, wherein one or more carbon atoms are optionally independently replaced with O, NH, or a divalent triazine ring, wherein each carbon atom is optionally independently replaced with one or more (e.g., 1, 2, 3, 4, 5, or more) substituents selected from halo and oxo (=O).

[0081] L 1 Specific values ​​for are: [ka] is.

[0082] A specific value for D is the residue of a drug.

[0083] Specific values ​​for D are residues of anti-cancer drugs, including classical chemotherapeutic agents such as antimetabolites (e.g., gemcitabine), antimitotics (e.g., taxanes, or DM1, or MMAE), alkylating agents (e.g., chlorambucil), DNA damaging agents (e.g., doxorubicin), and targeted therapeutics (e.g., kinase inhibitors such as erlotinib). In one embodiment, D is a residue of an EGRF inhibitor (e.g., cetuximab, gefitinib, erlotinib), a Her2 inhibitor (e.g., trastuzumab), a BRAF inhibitor (e.g., vemurafenib, dabrafenib), gemcitabine, 5FU, or another antimetabolite, taxane, alkylating agent, or DNA damaging agent.

[0084] In certain embodiments, the antimetabolite is gemcitabine. In certain embodiments, the mitotic inhibitor is a taxane, DM1, or MMAE. In certain embodiments, the alkylating agent is chlorambucil. In certain embodiments, the DNA damaging agent is doxorubicin. In certain embodiments, the targeted therapeutic agent is a kinase inhibitor such as erlotinib. In one embodiment, D is a residue of an EGRF inhibitor, and the EGRF inhibitor is cetuximab, gefitinib, or erlotinib. In one embodiment, D is a residue of a Her2 inhibitor, and the Her2 inhibitor is trastuzumab. In one embodiment, D is a residue of a BRAF inhibitor, and the BRAF inhibitor is vemurafenib or dabrafenib. In one embodiment, D is a residue of gemcitabine, 5FU, or another antimetabolite, taxane, alkylating agent, or DNA damaging agent.

[0085] Specific values ​​for D are residues of taxanes, including paclitaxel, docetaxel, and cabazitaxel.

[0086] A specific value for D is the residue of paclitaxel.

[0087] A specific value for D is a residue of a targeting agent.

[0088] R 12 Specific values ​​for are: [ka] is.

[0089] R 12 Specific values ​​for are: [ka] is.

[0090] A specific value of p is 1. A specific value of p is 2. A specific value of p is 3.

[0091] L 2A specific value for is a branched or unbranched, saturated or unsaturated hydrocarbon chain having about 1 to 100 carbon atoms, where one or more carbon atoms are selected from the group consisting of -O-, -S, -N(R a )-, a 3- to 7-membered heterocycle, a 5- to 6-membered heteroaryl, or a carbocycle, wherein each chain, 3- to 7-membered heterocycle, 5- to 6-membered heteroaryl, or carbocycle is optionally independently substituted with (C-C)alkyl, (C-C)alkoxy, (C-C)cycloalkyl, (C-C)alkanoyl, (C-C)alkanoyloxy, (C-C)alkoxycarbonyl, (C-C)alkylthio, azido, cyano, nitro, halo, -N(R a )2, hydroxy, oxo (=O), carboxy, aryl, aryloxy, heteroaryl, and heteroaryloxy, wherein each R a are independently H or (C1-C6) alkyl.

[0092] L 2 A specific value for is a branched or unbranched, saturated or unsaturated hydrocarbon chain having about 1 to 20 carbon atoms, where one or more carbon atoms are selected from the group consisting of -O-, -S, -N(R a )-, a 3- to 7-membered heterocycle, a 5- to 6-membered heteroaryl, or a carbocycle, wherein each carbon atom, 3- to 7-membered heterocycle, 5- to 6-membered heteroaryl, or carbocycle is optionally independently replaced by (C-C)alkyl, (C-C)alkoxy, (C-C)cycloalkyl, (C-C)alkanoyl, (C-C)alkanoyloxy, (C-C)alkoxycarbonyl, (C-C)alkylthio, azido, cyano, nitro, halo, -N(R a )2, hydroxy, oxo (=O), and carboxy; a are independently H or (C1-C6) alkyl.

[0093] L2 A specific value for is a branched or unbranched saturated hydrocarbon chain having about 3 to 110 carbon atoms (e.g., 3 to 10 carbon atoms), where each carbon atom is optionally independently substituted with one or more (e.g., 1, 2, 3, 4, 5 or more) substituents selected from halo and oxo (=O).

[0094] L 2 Specific values ​​for are -CH2C(=O)-, -CH2CH2C(=O)-, -CH2CH2CH2C(=O)-, -CH2CH2CH2CH2C(=O)-, or -CH2CH2CH2CH2CH2C(=O)-.

[0095] A specific compound or salt is targefrin (Figure 1), or targefrin conjugated with paclitaxel, or targefrin-dimer, or targefrin-dimer conjugated to paclitaxel: [ka] [ka] [ka] and salts thereof.

[0096] In certain embodiments, a compound of Formula (I) or specific compounds described herein is a homodimeric compound (eg, a targefurin-dimer).

[0097] Processes for preparing compounds of formula I are provided as further embodiments of the present invention.

[0098] If the compound is sufficiently basic or acidic, salts of the compound of Formula I may be useful as intermediates for isolating or purifying the compound of Formula I. Furthermore, it may be preferable to administer the compound of Formula I as a pharmaceutically acceptable acid or base salt. Examples of pharmaceutically acceptable salts are organic acid addition salts formed with acids that form physiologically acceptable anions, such as tosylate, methanesulfonate, acetate, citrate, malonate, tartrate, succinate, benzoate, ascorbate, α-ketoglutarate, and α-glycerophosphate. Suitable inorganic salts may also be formed, including hydrochloride, sulfate, nitrate, bicarbonate, and carbonate salts.

[0099] Salts can be obtained using standard procedures well known in the art, for example, by reacting a sufficiently basic compound, such as an amine, with a suitable acid to provide a physiologically acceptable anion. Alkali metal (e.g., sodium, potassium, or lithium) or alkaline earth metal (e.g., calcium) salts of carboxylic acids can also be prepared.

[0100] The compounds of formula I can be formulated as pharmaceutical compositions and administered to a mammalian host, such as a human patient, in a variety of forms adapted to the selected route of administration, i.e., orally or parenterally, intravenously, intramuscularly, topically, or subcutaneously.

[0101] That is, the compounds of the present invention can be administered systemically, e.g., orally, in combination with a pharmaceutically acceptable vehicle, such as an inert diluent or an assimilable edible carrier. They can be enclosed in hard or soft shell gelatin capsules, compressed into tablets, or incorporated directly into the patient's diet. For oral therapeutic administration, the active compound can be combined with one or more excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Such compositions and preparations should contain at least 0.1% of the active compound. The percentage of the compositions and preparations may, of course, vary and may conveniently be about 2 to about 60% of the weight of a given unit dosage form. The amount of active compound in such therapeutically useful compositions is such that an effective dosage level will be obtained.

[0102] Tablets, troches, pills, capsules, and the like may contain the following: binders such as tragacanth gum, acacia, cornstarch, or gelatin; excipients such as dicalcium phosphate; disintegrating agents such as cornstarch, potato starch, alginic acid, and the like; lubricants such as magnesium stearate; and sweeteners such as sucrose, fructose, lactose, or aspartame, or flavorings such as peppermint, wintergreen oil, or cherry flavoring. When the unit dosage form is a capsule, in addition to the above types of materials, it may contain a liquid carrier such as vegetable oil or polyethylene glycol. Various other materials may be present as coatings or otherwise to modify the physical form of the solid unit dosage form. For example, tablets, pills, or capsules may be coated with gelatin, wax, shellac, sugar, or the like. Syrup or elixir may contain the active compound, sucrose or fructose as a sweetener, methylparaben and propylparaben as preservatives, a dye, and a flavoring such as cherry flavor or orange flavor. Of course, any material used to prepare any unit dosage form must be pharmaceutically acceptable and substantially non-toxic in the amounts used. In addition, the active compound may be incorporated into sustained-release preparations and sustained-release devices.

[0103] The active compound can also be administered intravenously or intraperitoneally by infusion or injection.The solution of the active compound or its salt can be prepared in water, optionally mixed with a non-toxic surfactant.Dispersions can also be prepared in glycerol, liquid polyethylene glycol, triacetin and their mixtures, and in oil.These preparations contain preservatives to prevent the growth of microorganisms under normal storage and use conditions.

[0104] Pharmaceutical dosage forms suitable for injection or infusion include sterile aqueous solutions or dispersions containing the active ingredient, or sterile powders containing the active ingredient adapted for the extemporaneous preparation of sterile solutions or dispersions for injection or infusion, optionally encapsulated in liposomes. In either case, the final dosage form must be sterile, fluid, and stable under the conditions of manufacture and storage. Liquid carriers or liquid vehicles can be solvents or liquid dispersion media containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), vegetable oils, non-toxic glyceryl esters, and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the formation of liposomes, by maintaining the required particle size in the case of dispersions, or by the use of surfactants. Prevention of microbial action can be achieved by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, such as 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.

[0105] Sterile injectable solutions are prepared by incorporating the active compound in the required amount in a suitable solvent with various other ingredients as enumerated above, as needed, and then filtering and sterilizing. In the case of sterile powders for preparing sterile injectable solutions, the preferred preparation method is vacuum drying and freeze-drying, which yields a powder of the active ingredient and any additional desired ingredients present in a previously sterile-filtered solution.

[0106] For topical or subcutaneous administration, the compounds may be administered as a composition or formulation in combination with a dermatologically acceptable carrier.

[0107] Useful liquid carriers include water, alcohols, or glycols, or water-alcohol / glycol blends, in which the compounds of the present invention can be dissolved or dispersed at effective levels, optionally with the aid of non-toxic surfactants. Adjuvants such as fragrances and additional antimicrobial agents can be added to optimize the properties for a given application. The resulting liquid compositions can be applied from absorbent pads, used to impregnate bandages and other dressings, or sprayed onto the affected area using pump-type or aerosol sprayers.

[0108] Additionally, thickening agents such as synthetic polymers, fatty acids, fatty acid salts and esters, fatty acid alcohols, modified cellulose, or modified mineral substances can be used with liquid carriers to form spreadable pastes, gels, ointments, soaps, and the like for direct application to the user's skin.

[0109] Useful dosages of the compounds of Formula I can be determined by comparing their in vitro activity, and in vivo activity in animal models. Methods for the extrapolation of effective dosages in mice, and other animals, to humans are known to the art; see, for example, U.S. Patent No. 4,938,949.

[0110] The amount of a compound of the invention, or an active salt or derivative thereof, required for therapeutic use will vary not only with the particular salt chosen, but also with the route of administration, the nature of the condition being treated, and the age and condition of the patient, and will ultimately be at the discretion of the attending physician or clinician.

[0111] The desired dose may conveniently be presented in a single dose or as divided doses administered at appropriate intervals, for example, as two, three, four or more sub-doses per day. The sub-dose itself may be further divided, into a number of discrete loosely spaced administrations, such as, for example, multiple inhalations from an insufflator or multiple applications by eye drops.

[0112] The compounds of the present invention can also be administered in combination with other therapeutic agents, such as other agents useful in the treatment of cancer. Examples of such agents include EGRF inhibitors (i.e., cetuximab, gefitinib, erlotinib), Her2 inhibitors (i.e., trastuzumab), or BRAF inhibitors (vemurafenib, dabrafenib), gemcitabine, 5FU, and other classical chemotherapeutic agents, such as other antimetabolites, taxanes, alkylating agents, and DNA-damaging agents. Thus, in one embodiment, the present invention provides a composition comprising a compound of Formula I, or a pharmaceutically acceptable salt thereof, at least one other therapeutic agent, and a pharmaceutically acceptable diluent or carrier. The present invention also provides a kit comprising a compound of Formula I, or a pharmaceutically acceptable salt thereof, at least one other therapeutic agent, packaging materials, and instructions for administering a compound of Formula I, or a pharmaceutically acceptable salt thereof, and one or more other therapeutic agents to an animal to treat cancer.

[0113] Linking group L 1 and L 2 The compounds of formula (I) contain a linking group L 1 and L 2 In one embodiment, the linking group is absent. The linking group may vary in length and atomic composition, for example, branched, unbranched, cyclic, or a combination thereof. The linking group may also adjust the properties of the final compound of formula (I), for example, solubility, stability, or aggregation.

[0114] In one embodiment, the linker comprises about 3 to 100 atoms. In one embodiment, the linker comprises about 3 to 90 atoms. In one embodiment, the linker comprises about 3 to 80 atoms. In one embodiment, the linker comprises about 3 to 70 atoms. In one embodiment, the linker comprises about 3 to 60 atoms. In one embodiment, the linker comprises about 3 to 50 atoms. In one embodiment, the linker comprises about 3 to 400 atoms. In one embodiment, the linker comprises about 3 to 30 atoms. In one embodiment, the linker comprises about 3 to 20 atoms. In one embodiment, the linker comprises about 3 to 10 atoms.

[0115] In one embodiment, the linker comprises atoms selected from H, C, N, S, and O.

[0116] In one embodiment, the linker comprises an atom selected from H, C, N, and O.

[0117] In one embodiment, the linker is a branched or unbranched, saturated or unsaturated hydrocarbon chain having about 1 to 100 (1 to 90, 1 to 80, 1 to 70, 1 to 60, 1 to 50, 1 to 40, 1 to 30, 1 to 20, or 1 to 10) carbon atoms, wherein one or more carbon atoms are selected from the group consisting of -O-, -S, -N(R a )-, a 3- to 7-membered heterocycle, a 5- to 6-membered heteroaryl, or a carbocycle, wherein each chain, 3- to 7-membered heterocycle, 5- to 6-membered heteroaryl, or carbocycle is optionally independently substituted with (C-C)alkyl, (C-C)alkoxy, (C-C)cycloalkyl, (C-C)alkanoyl, (C-C)alkanoyloxy, (C-C)alkoxycarbonyl, (C-C)alkylthio, azido, cyano, nitro, halo, -N(R a )2, hydroxy, oxo (=O), carboxy, aryl, aryloxy, heteroaryl, and heteroaryloxy, wherein each R a are independently H or (C1-C6) alkyl.

[0118] In one embodiment, the linker is a branched or unbranched, saturated or unsaturated hydrocarbon chain having about 1 to 20 carbon atoms, wherein one or more carbon atoms are —O—, —S, —N(R a )-, a 3- to 7-membered heterocycle, a 5- to 6-membered heteroaryl, or a carbocycle, wherein each carbon atom, 3- to 7-membered heterocycle, 5- to 6-membered heteroaryl, or carbocycle is optionally independently replaced by (C-C)alkyl, (C-C)alkoxy, (C-C)cycloalkyl, (C-C)alkanoyl, (C-C)alkanoyloxy, (C-C)alkoxycarbonyl, (C-C)alkylthio, azido, cyano, nitro, halo, -N(R a )2, hydroxy, oxo (=O), and carboxy; a are independently H or (C1-C6) alkyl.

[0119] The invention will now be illustrated by the following non-limiting examples. [Example]

[0120] Example 1. Chemical composition and mass spectrometry data for representative compounds. The compounds shown in Table 1 below were prepared using procedures similar to those described herein or using known and available methods and materials. [Table 1-1] [Table 1-2]

[0121] All compounds were analyzed using an Agilent 6545 QTOF LC / MS instrument.

[0122] Example 2. The chemical structures and dissociation constants of EphA2 binding agents are shown. For each compound, the Kd values ​​obtained by ITC and the IC values ​​obtained from DELFIA displacement measurements against the EphA2 ligand binding domain (LBD) are shown. 50 Report the value.

[0123] [Table 2]

[0124] Example 3. Synthetic scheme for the synthesis of targefurin. Conditions: (a) Rink Amide resin + 3 equivalents of Fmoc-Pro-OH, 3 equivalents of DIC, 1 equivalent of OximaPure in 4.5 mL of DMF. Reaction in a microwave-assisted Liberty Blue peptide synthesizer at 90 °C for 5 minutes. (b) Two Fmoc deprotections with 20% N-methylpiperidine in DMF at 90 °C for 3 minutes in a microwave-assisted Liberty Blue peptide synthesizer; (c) Peptide propagation using the previous conditions in the Liberty Blue system; (d) TFA / TIS / water / phenol (94:2:2:2) at room temperature for 5 hours. [ka]

[0125] Example 4. Synthetic scheme for the synthesis of targefurin-dimer. Conditions: (a) Rink Amide resin + 3 equivalents of Fmoc-Lys(Fmoc)-OH, 3 equivalents of DIC, 1 equivalent of OximaPure in 4.5 mL of DMF. Reaction in a microwave-assisted Liberty Blue peptide synthesizer at 90 °C for 5 minutes; (b) Two Fmoc deprotections with 20% N-methylpiperidine in DMF at 90 °C for 3 minutes in a microwave-assisted Liberty Blue peptide synthesizer; (c) Peptide propagation using the previous conditions but with twice the equivalents for dimer propagation: 6 equivalents of Fmoc-amino acid, 6 equivalents of DIC, 2 equivalents of OximaPure in 4.5 mL of DMF. Reaction in a microwave-assisted Liberty Blue peptide synthesizer at 90 °C for 5 minutes; (d) TFA / TIS / water / phenol (94:2:2:2) at room temperature for 5 hours. [ka]

[0126] Example 5. Synthetic scheme for the synthesis of the compound targefrin motif (an intermediate for the synthesis of targefrin-paclitaxel). Conditions: (a) Rink Amide resin + 3 equivalents of Fmoc-Lys(ivDde)-OH, 3 equivalents of Fmoc-Lys(ivDde)-OH HATU, 3 equivalents of OximaPure, and 5 equivalents of DIPEA in 1 mL of DMF at room temperature for 1 hour; (b) Fmoc deprotection twice with 20% piperidine in DMF; (c) 3 equivalents of Fmoc-Gly-OH, 3 equivalents of Fmoc-Gly-OH HATU, 3 equivalents of OximaPure, and 5 equivalents of DIPEA in 1 mL of DMF at room temperature for 1 hour; (d) either the above conditions or Liberty Peptide amplification using Blue; (e) ivDde deprotection using 4% N2H2 in DMF (3 x 5 mL) at room temperature; (f) 3 equivalents of 5-hexynoic acid, 3 equivalents of HATU, 3 equivalents of OximaPure, and 5 equivalents of DIPEA in 1 mL of DMF at room temperature for 1 hour; (g) TFA / TIS / water / phenol (94:2:2:2) at room temperature for 5 hours. [ka]

[0127] Example 6. Synthetic scheme for the synthesis of the compound targefurin-dimer motif (an intermediate for the synthesis of targefurin-dimer-paclitaxel). Conditions: (a) Rink Amide resin + 3 equivalents of Fmoc-Lys(ivDde)-OH, 3 equivalents of Fmoc-Lys(ivDde)-OH HATU, 3 equivalents of OximaPure, and 5 equivalents of DIPEA in 1 mL of DMF at room temperature for 1 hour. (b) Fmoc deprotection twice with 20% N-methylpiperidine in DMF. (c) 3 equivalents of Fmoc-Gly-OH, 3 equivalents of Fmoc-Gly-OH HATU, 3 equivalents of OximaPure, and 5 equivalents of DIPEA in 1 mL of DMF at room temperature for 1 hour. (d) 3 equivalents of Fmoc-Lys(Fmoc)-OH, 3 equivalents of HATU, 3 equivalents of OximaPure, and 5 equivalents of DIPEA in 1 mL of DMF at room temperature for 1 hour. (e) Peptide amplification was performed using twice the equivalents for dimer amplification: 6 equivalents of Fmoc-amino acid, 6 equivalents of DIC, and 2 equivalents of OximaPure in 4.5 mL of DMF. The reaction was carried out at 90 °C for 5 minutes in a microwave-assisted Liberty Blue peptide synthesizer. (f) ivD deprotection was carried out at room temperature using 4% N2H2 in DMF (3 x 5 mL); (g) 3 equivalents of 5-hexanoic acid, 3 equivalents of HATU, 3 equivalents of OximaPure, and 5 equivalents of DIPEA in 1 mL of DMF at room temperature for 1 hour; (h) TFA / TIS / water / phenol (94:2:2:2) at room temperature for 5 hours. [ka]

[0128] Example 7. Synthetic scheme for the synthesis of targefurin-PTX. Conditions: (a) Targefurin motif crude, 1 equivalent of PTX-azide in 4 mL of 4:1 DMSO:water solution. Add 50 uL of CuSO4 1 M and 50 uL of sodium ascorbate 1 M. Mix at room temperature for 48 hours. [ka]

[0129] Example 8. Synthetic scheme for targefurin-dimer-PTX synthesis. Conditions: (a) Targefurin-dimer motif crude, 1 equivalent of PTX-azide in 4 mL of 4:1 DMSO:water solution. Add 50 uL of CuSO4 1M and 50 uL of sodium ascorbate 1M. Mix at room temperature for 48 hours. [ka]

[0130] Example 9. Antagonistic function of targefurin monomer. Figure 2A shows a Western blot of BxPC3 cells starved for 1 hour, pretreated with various concentrations of targefrin for 20 minutes, and then co-treated with 2 μg / mL ephrinA1-Fc for 3 hours. Figure 2B shows quantification of EphA2 levels. The EphA2 / β-actin ratio was normalized to 1, which is the EphA2 expression from the DMSO condition without ephrinA1-Fc. EC 50 The value was calculated to be 1.6±0.1 μM and is presented as the mean±standard error (SE) of two independent experiments.

[0131] Example 10. Chemical structures of dimeric EphA2 binding agents. I C 50 Values ​​were obtained by repeated DELFIA measurements for EphA2-LBD. [Table 3] Table 3. Chemical structures of dimeric EphA2-binding agents. IC 50 Values ​​were obtained by repeated DELFIA measurements.

[0132] Example 11. Targefurin-dimer and its variations cause degradation of EphA2 at nanomolar concentrations in pancreatic cancer cell lines. Figures 3A-3C show Western blot images of BxPC3, PANC-1, and MIAPaCa-2 cells, respectively. Cells were starved for 1 hour and then treated with 2 μg / mL ephrinA1-Fc or the indicated doses of targefurin, targefurin-dimer, and its variants with different linkers for 3 hours. The dimerizer 135H12 (see US20210221843A1; PCTWO2019237075A1, and Gambini et al. ACS Chem Biol. 2018, 13(9), 2633-2644) is shown as a reference. Figures 3D-3F show densitometric analysis of the data shown in Figures 3A-3C, respectively. EphA2 / β-actin ratios were normalized to EphA2 expression from the DMSO control condition as 100% in Figures 3A-3C or 1 in Figures 3D-3F. ***p<0.001, ****p<0.0001, determined by one-way ANOVA with Dunnett's post-hoc analysis.

[0133] Example 12. Chemical structure and biochemical activity of targefurin-conjugate agents. Figure 4A shows the chemical structure of targefurin conjugated to paclitaxel (targefurin-PTX). Figure 4B shows the dimeric version of targefurin conjugated to paclitaxel (targefurin-dimer-PTX). Figure 4C shows the dimeric version of targefurin conjugated to 5-carboxytetramethylrhodamine-azide dye (targefurin-dimer-TAMRA). Figure 4D shows the chemical structure of targefurin-PTX, targefurin-dimer-PTX, and targefurin-dimer-TAMRA conjugated to their respective IC 50 DELFIA displacement dose-response curves are shown, comparing the values.

[0134] Example 13. Targefurin-dimer-TAMRA is internalized into EphA2-expressing cells. BxPC3 cells were treated with 100 nM targefurin-dimer-TAMRA for 0, 30, and 60 minutes. Upon drug binding, EphA2 was internalized and targeted to lysosomes, as indicated by colocalization of 5-TAMRA with LAMP1 (arrows). See Figure 5. Scale bar = 10 μm.

[0135] Example 14. Targefurin-dimer significantly inhibits the migration of pancreatic cancer cells. Figure 6A shows a cell migration assay of BxPC3 cells treated with 2 μg / mL ephrinA1-Fc and 10 μM targefurin, or the indicated doses of targefurin-dimer. Plates were imaged every 3 hours for 24 hours. Figure 6B shows that targefurin-dimer significantly inhibited cell migration after 24 hours in a dose-dependent manner, as indicated by a decrease in relative wound density. Figure 6C shows time-response curves illustrating the effect of the drugs on wound closure over 24 hours. ***p<0.001, ****p<0.0001, determined by one-way ANOVA with Dunnett's post-hoc analysis. Scale bar = 250 μm.

[0136] Example 15. PTX conjugates suppress tumor growth in tumor xenografts bearing MIA-PaCa-2 cells. Figure 7A shows five groups of five mice bearing pre-established MIA-PaCa-2 tumors treated for 22 days with vehicle control alone, paclitaxel (PTX, 2.5 mg / kg), targefurin-PTX (10 mg / kg, equivalent to 2.5 mg / kg of PTX), targefurin-dimer-PTX (17 mg / kg, equivalent to 2.5 mg / kg of PTX), and low-dose targefurin-dimer-PTX (10 mg / kg, equivalent to 1.5 mg / kg of PTX). Tumor volumes are reported as mean ± SE. Figure 7B shows the mean tumor volumes per treatment group measured on days 0, 8, 15, and 22. *p=0.03, **p<0.01, ***p=0.0001, ****p<0.0001, as determined by two-way ANOVA with Tukey's post-hoc analysis. FIG. 7C shows that mean body weights±SE are reported on days 0, 8, 15, and 22 for each of the five treatment groups.

[0137] Example 16. Representative Pharmaceutical Dosage Forms The following illustrates a representative pharmaceutical dosage form containing a compound of Formula I ("Compound X") for therapeutic or prophylactic use in humans: An IV injectable formulation consisting of 80% PBS, 10% Tween 80, 10% ethanol, with the drug dissolved at up to 20 mg / ml. This formulation, and several variations of this formulation, can be obtained by conventional procedures well known in the pharmaceutical industry.

[0138] Example 17. Synthetic scheme for the synthesis of targefurin-dimer-TAMRA. Conditions: (a) Targefurin-dimer motif crude, 1 equivalent of 5-TAMRA-azide in 4 mL of 4:1 DMSO:water solution. Add 50 uL of CuSO4 1 M and 50 uL of sodium ascorbate 1 M. Mix at room temperature for 48 hours. [ka]

[0139] Example 18. Targefrin: a potent drug targeting the ligand-binding domain of EphA2 Overexpression of the receptor tyrosine kinase EphA2 is associated with poor prognosis and the development of aggressive metastatic cancer. In this example, using the recently elucidated X-ray structure of the complex between an agonist peptide and EphA2-LBD as a guide, we demonstrate that EphA2-LBD binds to EphA2-LBD with a dissociation constant of 21 nM as determined by isothermal titration calorimetry and an IC50 of 10.8 nM in a biochemical assay. 50 We describe a novel drug, targefrin, that demonstrates therapeutic value. In cell-based assays, the dimeric version of the drug is as effective as the natural dimeric ligand (ephrinA1-Fc) in inducing cellular receptor internalization and degradation in several pancreatic cancer cell lines. When conjugated with a chemotherapeutic drug, the drug can effectively deliver paclitaxel to pancreatic cancer in mouse xenograft studies. Given the pivotal role of EphA2 in tumor progression, the drug reported herein has the potential to be further developed as an innovative EphA2-targeted therapeutic.

[0140] Introduction The receptor tyrosine kinase EphA2 functions as a tumor suppressor in its ephrin-binding form, preventing cancer cell migration, tumor growth, and angiogenesis. On the other hand, when the receptor is in an unbound state, for example, when the receptor is abnormally overexpressed, the receptor can cause cancer cells to develop tumors, such as pancreatic cancer, 2-4 prostate cancer, 5-7 breast cancer, 8-10 esophageal cancer, 11 12 melanoma, 13 bladder cancer, 14 brain tumors, 15-17 lung cancer, 18 ovarian cancer, 19 stomach cancer, 20 and some types of leukemia 21-24 It confers pro-oncogenic properties that induce metastatic behavior in some solid tumors, including tumours with tumour suppressor activity. Therefore, targeting EphA2 has been a target for the development of various potential therapeutic strategies, including the intracellular kinase domain, 25-28 or a ligand-binding domain, 29、30The unbound EphA2 receptor functions as a potent oncogene, but its tumorigenic effects can be suppressed and possibly reversed by synthetic drugs that mimic its ligand, membrane-anchored ephrinA1. 31

[0141] In cellular assays, binding of ephrinA1-Fc, a chimeric protein consisting of ephrinA1 and the Fc region of an antibody, to the EphA2 ligand-binding domain (LBD) triggers receptor dimerization, followed by clustering, internalization, and receptor degradation via the lysosomal pathway. 32 Therefore, ephrinA1-Fc could, in principle, reverse the oncogenic EphA2 into a tumor suppressor, and the design of potent and effective ephrinA1-Fc mimetics could potentially lead to the development of novel anti-metastatic therapeutics. Because such agents may induce receptor internalization, they could further be utilized as transport molecules for selective targeted delivery of chemotherapeutic drugs to EphA2-expressing cancers. In this regard, we recently demonstrated that ephrinA1-Fc, like ephrinA1-Fc, could suppress tumor metastasis in an orthotopic model of prostate cancer. 33 Suppression of cell migration in pancreatic cancer cell lines 34 We have developed an EphA2 dimer agonist peptidomimetic. 35 or paclitaxel 8、36-37 When conjugated with, these have been shown to be effective in treating pancreatic cancer, 35 prostate cancer, 36-37 breast cancer, 8、38 and delivered their cargo to EphA2-expressing tumors, including melanoma. 38 More recently, we have solved the first X-ray structure of an agonistic ephrin peptidomimetic in complex with EphA2-LBD. 1 Previous structure-activity relationship studies of peptide binders previously obtained in our laboratory 1、37-38 and high-resolution X-ray structure 1Taking advantage of this, we sought to further derive drugs that could rival the affinity and activity of ephrinA1-Fc, which targets the EphA2-LBD. As shown herein, novel drugs with low nanomolar affinity for the EphA2-LBD were identified, which exhibit affinity for the receptor comparable to that of ephrinA1. In cellular assays, a dimeric version of our most potent drug (which we named targefrin) induces receptor degradation at nanomolar concentrations similar to the effects of ephrinA1-Fc, as assessed by Western blot analysis in the pancreatic cancer cell lines BxPC3, PANC-1, and MIAPaCa2, representing KRAS wild-type (BxPC3) and KRAS mutant (PANC-1 and MIAPaCa2) tumors. In phenotypic assays, the drug was also effective in inhibiting cell migration in the BxPC3 pancreatic cancer cell line. When conjugated with paclitaxel, the drug is effective in suppressing tumor growth in the MIA PaCa2 xenograft model of pancreatic cancer. The exceptional affinity of targefurin for the ligand-binding domain of EphA2 makes this drug a novel pharmacological tool for studying this receptor tyrosine kinase and for the development of novel therapeutics and / or targeted delivery strategies.

[0142] result Design, synthesis, and characterization of targefrin To rapidly and repeatedly characterize the binding properties of novel EphA2-binding ligands (Table 4), we performed binding studies by isothermal titration calorimetry using recombinant EphA2 ligand-binding domains (LBDs). Figure 11A shows a prior drug complexed with EphA2-LBD (PDB ID 6B9L). 1 Phage display-derived YSA peptide 39 The sequence YSAYPDSVPFRP(K) was merged with the sequence of the natural ephrin ligand. d Using a ligand of 1230 nM, ITC; Table 4, compounds), 1We began investigating optimization strategies (Figure 11). First, we investigated substitutions that could protrude into the large hydrophobic pocket located near Tyr4 of the peptide (Figures 11A-11B, Table 4). Here, replacing the Tyr residue at position 4 with a bulkier aromatic group significantly enhanced affinity (Table 4). Therefore, we subsequently fixed phenyl-Phe at position 4 of the peptide and explored modifications at other positions. [Table 4] Table 4. Structure-activity relationship studies at position 4 for reported EphA2 binding agents. Chemical structures and dissociation constants are reported. d Values ​​were obtained by reverse isothermal titration calorimetry.

[0143] These include modifications of the N-terminal amide (Figures 11A, 11C, Table 5), a position that is not only susceptible to aminopeptidases in plasma but also involved in ligand recognition. 1、37-38 Replacement of the amino group with piperazine or morpholino increased the binding affinity to EphA2-LBD (Table 5). Furthermore, we investigated additional modifications, including modifications of the Tyr and Ser residue pair at position 1 (Table 5). We found that Tyr1 can be replaced with various substituents, thereby eliminating the potential pharmacological defect represented by the phenolic hydroxyl group (Table 5). In its bound state, the peptide adopts a closed conformation, with the two Ser residues forming intramolecular hydrogen bonds within the first peptide (Figures 11A and 11D). We further investigated additional modifications of this amino acid pair and assessed their effect on binding affinity to EphA2-LBD via ITC measurements (Table 5).

[0144] Finally, a set of peptides containing optimal substituents from the drugs reported in Tables 4 and 5 were synthesized to yield the final drugs listed in Table 2. These compounds were also evaluated for binding properties using an orthogonal biochemical displacement assay based on the DELFIA platform, as previously described. 1 [Table 5-1] [Table 5-2] Table 5. Chemical structures and dissociation constants of EphA2 binding agents. K d Values ​​were obtained by reverse isothermal titration calorimetry.

[0145] These studies demonstrated that EphA2-LBD IC 50 This culminated in the selection of drug 27, herein named targefurin, with a value of 10.8 nM (Table 2). In Figure 1 we report a molecular model of targefurin in complex with EphA2-LBD, based on the X-ray structure of its complex with one of our prior peptidomimetics (PDB ID 6B9L). 1 To obtain a preliminary but significant snapshot of the selectivity of targefurin for EphA2-LBD relative to other members of this protein family, we tested targefurin against the ligand-binding domains of EphA3 and EphA4, the two Eph receptors with the highest similarity to EphA2 (58% homology with EphA3-LBD and 57% homology with EphA4-LBD). When tested under similar experimental conditions, the ligand was inactive against both domains (Figure 1D).

[0146] Monomeric peptides induce agonist activity only at very high concentrations and may actually act as antagonists at physiologically achievable concentrations. 8、34、40 Consistent with its high affinity for EphA2, pretreatment of BxPC3 pancreatic cancer cells with targefrin effectively antagonized EphA2 degradation induced by the potent ephrinA1-Fc ligand, achieving approximately an EC 50 was approximately 1.6 μM (Figure 2).

[0147] This agent alone did not induce appreciable EphA2 degradation in BxPC3 pancreatic cancer cells (FIG. 3).

[0148] However, dimerization of EphA2 targeting agents may result in compounds with increased agonist activity within cells, whereas monomeric peptides act as antagonists similar to ephrin A1. 1、8、33-34、41-42 This is likely due to the fact that enhancing dimerization can promote subsequent receptor clustering and internalization. 8、42 We prepared dimeric versions of targefurin (Table 3) using Lys residues separated by Gly, β-Ala, or γ-aminobutyric acid at the C-terminus of targefurin as dimerization linkers (Table 3), which also reports our previously identified dimeric agent 135H12. 1

[0149] Targefrin-dimers and targefrin-drug conjugates An interesting property of agonist drugs is that they induce the internalization of the EphA2 receptor via the lysosomal pathway, which leads to its degradation. Therefore, potent agonist drugs can induce EphA2 degradation, thereby eliminating its pro-oncogenic effects. EphA2 internalization induced by agonist drugs does not necessarily affect cell proliferation, as shown in Figure 12. However, due to the lysosomal internalization event, EphA2 agonists may be used for the targeted delivery of cytotoxic chemotherapeutic drugs by synthesizing suitable peptide-drug conjugates (PDCs). Therefore, to evaluate the EphA2 internalization and degradation properties of our drugs, we tested them in various pancreatic cancer cell lines in parallel with dimerized ephrinA1-Fc as a positive control. As reported above, the monomeric version of targefrin was not active in causing EphA2 degradation when tested at nanomolar concentrations, consistent with our previous observation that the monomeric peptide was agonistic only at higher micromolar concentrations, which appeared to be true for all three cell lines tested: BxPC3, PANC-1, and MIA PaCa2 (Figure 3).

[0150] However, dimeric versions of targefrin, particularly dimers with a Gly-Lys linker (Table 3), showed significantly increased receptor activation and caused receptor degradation at submicromolar concentrations in all pancreatic cancer cell lines tested (Figure 3). Furthermore, our new agent is significantly more effective than our previously reported dimeric agent, 135H12 (Table 3, Figure 3). 1

[0151] To assess the utility of targefrin and targefrin-dimers as carriers for targeted delivery, we synthesized and tested drug conjugates containing the chemotherapeutic agent paclitaxel and the fluorescent dye TAMRA (Figure 4). The synthesis of these agents followed our previously described "click chemistry" linkers, which allow efficient incorporation of drugs or imaging reagents into dimeric or monomeric agents (Examples 7, 8, and 17). 1、8 Conjugation of TAMRA or paclitaxel to the dimeric agents did not significantly alter their binding properties to the isolated EphA2-LBD (Fig. 4D ), although a more significant loss of binding affinity was observed with targefurin-monomer-PTX, likely due to the shorter linker selected.

[0152] Immunofluorescence microscopy data using BxPC3 cells showed intermittent cytoplasmic fluorescence that colocalized with the lysosomal marker LAMP-1 in targefurin-dimer-TAMRA-treated cells (Figure 5), confirming EphA2-specific lysosomal internalization events triggered by the agonist drug. Indeed, targefurin-dimer-PTX retained the ability to induce EphA2 degradation in all three pancreatic cancer cell lines tested (Figure 8).

[0153] In contrast, targefurin-monomer-PTX alone did not induce receptor internalization. These data clearly identify targefurin as a potent EphA2-LBD binder with antagonistic activity, whereas targefurin-dimer exhibited similar strong affinity for isolated EphA2-LBD but also potent EphA2 degradation activity in pancreatic cancer cells.

[0154] Finally, to determine whether our EphA2 agonist drugs interfere with the cell migration of pancreatic cancer cells, we performed a cell migration assay using the scratch wound method and detected the cell migration using time-lapse live cell analysis (IncuCyte S3, Sartorius) in the pancreatic cancer cell line BxPC3. We previously reported that knockout of EphA2 alone significantly reduced cell migration in BxPC3. 34 Similarly, treatment of BxPC3 cells with increasing concentrations of targefurin-dimer significantly inhibited cell migration (Fig. 6). From these data, we conclude that targefurin and targefurin-dimer are potent antagonistic and agonistic EphA2 agents, respectively.

[0155] In vivo pharmacology and mouse xenograft studies A preliminary pharmacokinetic study was conducted with targefrin-dimer, in which plasma drug concentrations were measured over time after administration of a single dose of 50 mg / kg of the drug via the tail vein (Figure 9). The data showed a C of 100–200 nM, far exceeding the C required for the drug to induce EphA2 degradation in cells. max Reaching t 1 / 2 The study showed that the onset of vasoconstriction was estimated to be approximately 15 hours, suggesting that lower drug concentrations could be used in subsequent in vivo efficacy studies. Blood chemistry analysis after this high dose of targefrin-dimer showed a significant improvement in the blood chemistry panel (e.g., albumin, ALP, ALT, amylase, bilirubin, Ca, P, Na). + , K. +No significant changes in blood glucose, total protein, globulin, creatinine, urea nitrogen, or glucose were evident. In an additional preliminary in vivo toxicity study, Balb / C mice were administered repeated doses of PTX (8 mg / kg), targefurin-dimer (50 mg / kg), or targefurin-dimer-PTX (50 mg / kg) daily for 5 days; thus, each group received an equal dose of PTX. Two of the three mice receiving PTX were found dead after the second dose, while the remaining mice appeared lethargic and were found dead by day 5. On the other hand, no adverse signs of toxicity were observed in the targefurin-dimer-treated and targefurin-dimer-PTX-treated groups (mice in the latter group appeared lethargic after day 1 but recovered). Body weight was monitored throughout the experiment (Table 6). These preliminary data suggest that targefrin is well tolerated and can selectively deliver PTX to EphA2-expressing tumor cells. [Table 6] Table 6. Repeated-dose toxicity study of targefrin-dimer-PTX versus PTX alone. Balc / c mice were given equimolar doses of PTX or targefrin-dimer-PTX (IV) daily and weighed daily. FD = found dead. By day 5, all three mice in the PTX-treated group were found dead. Mice treated with targefrin-dimer-PTX were lethargic after the first dose but recovered. No signs of toxicity were observed in mice treated with targefrin-dimer.

[0156] Therefore, to further evaluate the ability of the drug conjugates to deliver chemotherapeutic agents to pancreatic cancer in vivo, we evaluated the ability of the drugs to suppress tumor growth in tumor xenografts using MIA PaCa-2 cells. First, MIA PaCa-2 cells (1.0 × 10) were cultured in 100 μL of PBS. 7 100 cells / mouse) were injected into the right flank of five nu / nu mice to obtain tumor stock fragments. 3MIA PaCa-2 tumor fragments were implanted into the right flank of each of 25 mice. Tumor growth was measured with a caliper on day 18 after tumor implantation. Mice were divided into groups and treated on days 1, 4, 8, 11, 15, and 18. Drugs were dissolved in a mixture of 80% PBS, 10% Tween 80, and 10% ethanol. Five groups received vehicle control alone, paclitaxel (PTX; 2.5 mg / kg), targefurin-PTX (10 mg / kg, equivalent to 2.5 mg / kg of PTX), targefurin-dimer-PTX (17 mg / kg, equivalent to 2.5 mg / kg of PTX), and low-dose targefurin-dimer-PTX (10 mg / kg, equivalent to 1.5 mg / kg of PTX). Both targefrin-PTX and targefrin-dimer-PTX showed significant antitumor effects compared with both the untreated and PTX-treated groups (Figure 7). Furthermore, although the standard deviation of the PTX-treated group was too large to assess significance, the group treated with a substoichiometric dose of PTX was also more effective than the PTX-treated group (Figure 7). Taken together, these data suggest that the agents can deliver drugs to EphA2-expressing tumors.

[0157] Discussion and Conclusion In recent years, the present inventors have seen an increase in efforts to target EphA2 with various strategies for the development of novel therapeutic agents. 43 These include computational docking strategies; 31、44-46 NMR screening, 40、47-48 High-throughput screening, 49 phage display screening, 39 These efforts include the development of potential small molecule compounds, 31、44、8 or EphA2 / ephrin antagonists 45-46、49-51、52 However, none of these cited drugs are mature enough to be used as potential therapeutics. Conversely, mAbs have been proposed to target EphA2, but have not performed well in the clinic due to reduced selectivity or prolonged half-life, which leads to drug accumulation in unwanted tissues. 53Indeed, a very recent phase I clinical study aimed to evaluate the biodistribution of DS-8895a, an anti-EphA2 antibody, in patients with advanced EphA2-positive cancer. 54 Encouragingly, no treatment-related toxicity was reported, but low tumor uptake was observed, suggesting that the therapeutic efficacy of DS-8895a was limited, leading to the discontinuation of further development of DS-8895a. 54

[0158] More recently, Bicycle Therapeutics reported on a peptide antagonist that binds to EphA2-LBD with a dissociation constant in the low nanomolar range. 55 The antagonistic drug was conjugated with monomethyl auristatin linked by a cathepsin-cleavable linker, which is currently in phase I clinical trials (clinicaltrials.gov / ct2 / splay / NCT04180371). While this drug shows great promise as the first to translate an EphA2-targeting agent into a potential therapeutic, targefurin and targefurin-dimer offer a viable alternative strategy to Bicycle Therapeutics' drug. First, targefurin has a similar affinity for EphA2 as Bicycle Therapeutics' compound, but its lower molecular weight likely enhances its tissue penetration. Second, targefurin-dimer induces active internalization of receptor function as an effective EphA2 degrader; therefore, it could be exploited as an effective EphA2-based therapeutic agent for inhibiting cell migration as an alternative to agonistic antibodies (Figure 6). Therefore, we envision that targefurin dimers may be exploited as EphA2 degraders to suppress the metastatic behavior of cancer cells, as we have recently demonstrated with the lead drug 135H12 in an orthotopic model of prostate cancer (Figure 13). 33

[0159] Furthermore, the active internalization of the drug conjugate induced by the targefurin dimer potentially increases the distribution of chemotherapeutic agents to EphA2-expressing tumor cells without the need for cleavage of the extracellular linker and passive diffusion of the cargo. We observed that the preceding dimeric EphA2 targeting agent conjugated with paclitaxel induced a significant reduction in circulating tumor cells in tumor-bearing mice. 8 Here, we observed that subtherapeutic doses of paclitaxel were effective in reducing tumor volume when conjugated to both the monomeric and dimeric versions of targefurin (Figure 7).

[0160] In conjunction with the reported preliminary toxicity and pharmacokinetic studies, the inventors suggest that this dimer can be utilized as a single agent or in combination with standard therapies to suppress EphA2 in cancer cells. Furthermore, the preliminary studies on drug conjugates should encourage further evaluation of such agents, particularly conjugation to targefurin-dimers, to take advantage of the active internalization that this agent offers to EphA2-overexpressing tumors.

[0161] In conclusion, the agents reported herein open the way to a wide range of opportunities, ranging from developing more effective PDC to diagnostic agents, to developing EphA2-targeted therapeutics, or devising more effective combination therapies targeting tumor metastasis.

[0162] Experimental Section chemistry General. All reagents and solvents, including Fmoc-protected amino acids and resins for solid-phase synthesis, were obtained from commercial sources. All peptides were synthesized in-house using a Liberty Blue peptide synthesizer (CEM) on Rink amide resin using a standard microwave-assisted Fmoc peptide synthesis protocol. For each coupling reaction, 3 equivalents of Fmoc-AA, 3 equivalents of DIC, and 1 equivalent of OximaPure in 4.5 mL of DMF were used. Coupling reactions proceeded for 5 minutes at 90 °C in a microwave reactor. Fmoc deprotection was performed by treating the resin-bound peptide with 20% N-methylpiperidine in DMF (2 × 3 mL) at 90 °C for 3 minutes. The peptide was cleaved from the resin using a cleavage cocktail containing TFA / TIS / HO / phenol (94:2:2:2) for 5 hours (see Example 3). The cleavage solution was filtered from the resin, and the peptide was precipitated in EtO, centrifuged, and dried under ultra-vacuum. Solution 1 Concentrations were confirmed using H NMR, and spectra were recorded on a Bruker Avance III 700 MHz instrument. High-resolution mass spectral data were acquired on an Agilent LC-TOF instrument. RP-HPLC purification was performed on an XTerra C18 10μ 10 × 250 mm (Waters) column with a PDA detector and fraction collector controlled by a ChromNAV system (JASCO). Purity of test compounds was assessed by HPLC using an Atlantis T3 3μm 4.6 × 150 mm column (HO / ACN gradient, 5% to 100% in 45 min). All compounds were >95% pure.

[0163] Preparation of dimeric agents and targefurin-dimers. Dimeric agents were prepared according to the procedure described above, but with double the equivalents for each coupling and with Fmoc-Lys(Fmoc)-OH incorporated as the first amino acid in the sequence, as shown in Example 4.

[0164] Preparation of the Targefurin-motif and Targefurin-dimer-motif. To prepare the Targefurin-motif and Targefurin-dimer-motif, we introduced the Fmoc-Lys(ivDde)-OH amino acid as the first amino acid coupled to Rink Amide resin. The peptide was then grown according to a solid-phase synthesis scheme similar to that previously described by the inventors. Upon completion of synthesis, the fully protected peptide on the Rink Amide resin was treated with a 4% solution of hydrazine in DMF (3 × 5 mL, 30 min each) to remove the ivDde protecting group, followed by washing with DMF (3 × 5 mL). This was followed by coupling with 3 equivalents of 5-hexynoic acid in the presence of 3 equivalents of HATU, 3 equivalents of OximaPure, and 5 equivalents of DIPEA in 1 mL of DMF at room temperature for 1 hour. The resin was then washed with DMF (3 × 5 mL) and DCM (3 × 5 mL), dried under vacuum, and cleaved with a cleavage cocktail containing TFA / TIS / HO / phenol (94:2:2:2) for 5 h. The synthetic schemes for the targefurin motif and targefurin dimer motif are reported in Examples 5 and 6, respectively.

[0165] Preparation of targefurin-PTX, targefurin-dimer-PTX, and targefurin-dimer-TAMRA. Crude targefurin motif was dissolved in 4 mL of 4:1 DMSO:water in the presence of 50 μL of 1 M CuSO4 and 50 μL of 1 M sodium ascorbate together with 1 equivalent of PTX-azide and stirred at room temperature for 48 hours (Example 7). Targefurin-dimer-PTX was obtained as described above, but using crude targefurin-dimer-motif as the starting point (Example 8). Targefurin-dimer-TAMRA was obtained as previously described, but using 5-TAMRA-azide instead of 1 equivalent of PTX-azide (Example 17). Mass spectrometry data for representative peptides synthesized are reported in Table 1.

[0166] Isothermal titration calorimetry (ITC) We have determined the dissociation constants (K dTo obtain information about the binding and thermodynamics of compounds, compounds were tested by isothermal titration calorimetry (ITC) performed against EphA2-LBD using a TA Instruments (New Castle, DE) Affinity ITC autosampler. Titrations were performed in the inverse manner by titrating the protein into the ligand solution. All titrations were performed at 25°C, with both the drug and targeting protein dissolved in 25 mM Tris (pH 7.5), 150 mM NaCl, and a final concentration of 1% DMSO. Syringes were filled with 200 μM solutions of EphA2-LBD, EphA3-LBD chimera, or EphA4-LBD, and 20 injections of 2.5 μL each were made into cells containing 10 μM solutions of the compounds. Injections were performed at 200-second intervals with a stirring speed of 75 rpm. The solutions were maintained at 4°C in the autosampler. Data analysis was performed with NanoAnalyze software (TA Instruments, New Castle, DE) and then exported to Microsoft Excel.

[0167] DELFIA displacement assay To test the activity of dimeric and monomeric agents, 1 μM 123B9-biotin 1100 μL of a solution of either 100 nM of the drug PiperazineAcAcid-YSA-(2MeBip)-PDS-Chg-PFRP-GK (biotin LC) or 100 nM of the drug was added to each well of a 96-well streptavidin-coated plate and incubated for 2 hours. The plate was then washed three times. Subsequently, a mixture containing 11 μL of EphA2 protein and serial dilutions of test compounds was added to each well and incubated with a solution containing 89 μL of Eu-N1-labeled anti-6x-His antibody (PerkinElmer) for 1 hour. At the end of the incubation period, the plate was washed three times and incubated with DELFIA enhancement solution (PerkinElmer) for 10 minutes. The final concentrations of EphA2 protein used to test the activity of dimeric and monomeric agents were 71.2 nM and 10 nM, respectively. The antibody concentrations in 89 μL of solution used to test the dimeric and monomeric agents were 4.17 nM and 3.13 nM, respectively. EphA2 protein, biotinylated peptide, and antibody were prepared in DELFIA assay buffer (PerkinElmer). Fluorescence measurements were performed using a VICTOR X5 microplate reader (ex / em 340 / 615 nm), normalized to DMSO wells, and reported as percent inhibition. IC was calculated using Prism9 (GraphPad). 50 The value was calculated.

[0168] Cell lines, cell cultures, and antibodies BxPC3, MIA PaCa-2, and PANC-1 cell lines were purchased from the American Type Culture Collection (ATCC). BxPC3 and PANC-1 cells were cultured in RPMI-1640 medium and DMEM medium, respectively, supplemented with 10% fetal bovine serum (FBS). MIA PaCa-2 cells were cultured in DMEM medium supplemented with 10% FBS and 2.5% horse serum. Cells were maintained at 37°C in a humidified incubator containing 5% CO2. Anti-EphA2 antibody (#374400), HRP-conjugated goat anti-mouse secondary antibody (#31432), and Alexa Fluor 488-conjugated goat anti-rabbit secondary antibody (#A-11034) were purchased from ThermoFisher Scientific. Anti-β-actin antibody (#sc-69879) was purchased from Santa Cruz Biotechnology, and anti-LAMP1 antibody (#9091) was purchased from Cell Signaling Technology.

[0169] Immunofluorescence BxPC3 cells were plated on glass coverslips overnight. Cells were serum-starved for 1 hour and treated with 100 nM targefurin-dimer-TAMRA for 0, 30, and 60 minutes. Cells were then fixed with 4% paraformaldehyde for 20 minutes, permeabilized with 0.2% Triton X-100 for 5 minutes, blocked with 10% goat serum for 1 hour, and incubated with anti-LAMP1 antibody at 4°C overnight, followed by incubation with anti-rabbit secondary antibody conjugated with Alexa Fluor™ 488 for 1 hour at room temperature. VECTASHIELD antifade mounting medium containing DAPI (Vector Laboratories) was added to the coverslips to stain nuclei. Images were then acquired using a Zeiss Axicovert 200M fluorescence deconvolution microscope and processed with SlideBook software version 6 (Intelligent Imaging Innovations).

[0170] Immunoblotting After treatment, cells were lysed on ice using lysis buffer (20 mM Tris, pH 7.4, 120 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 1% IGEPAL, and 5 mM EDTA, supplemented with protease inhibitor cocktail and PhosSTOP (Sigma-Aldrich)). Lysates were then centrifuged at 16,000 x g for 20 minutes at 4°C, and the supernatant was collected. Protein measurements were performed using the Pierce BCA Protein Assay Kit (ThermoFisher Scientific) according to the manufacturer's protocol. Samples were prepared and loaded onto 4-12% NuPAGE Bis-Tris precast gels, then transferred to PVDF membranes. Blots were blocked with 5% nonfat milk for 1 hour at room temperature, incubated overnight at 4°C with monoclonal EphA2 or actin antibodies, followed by incubation with anti-mouse HRP-conjugated antibodies for 1 hour at room temperature. Clarity Western ECL kit (BIO-RAD) was added to the blots, and images were captured with a ChemiDoc imaging system (BIO-RAD) and analyzed using ImageJ software.

[0171] Cell migration assay BxPC3 cells were seeded into IncuCyte® ImageLock 96-well plates (Sartorius) to achieve approximately 95-100% confluency at the time of treatment. Next, wounds were created on the cell monolayer using a WoundMAker™ (Sartorius) followed by two washes with PBS. Cells were then treated with 2 μg / mL ephrinA1-Fc (R&D Systems) or test agents, and the plates were imaged every 3 hours using an IncuCyte® S3 Live Cell Analysis System (Sartorius). Relative wound density percentages were quantified using the IncuCyte® Cell Migration Software Module.

[0172] In vivo pharmacokinetics, toxicity, and xenograft studies In vivo efficacy experiments were conducted at AntiCancer, Inc. (San Diego). Thirty-five male nu / nu mice, 8–10 weeks old (AntiCancer Inc., San Diego), were used in the xenograft study, consisting of 25 mice for randomization and 10 reserve mice. All mice were housed in a barrier facility on high-efficiency particulate air (HEPA)-filtered racks under standard conditions with a 12-h light / dark cycle. Animal studies were conducted in accordance with the principles and procedures outlined in the National Institutes of Health Guide for the Care and Use of Laboratory Animals, using an AntiCancer Institutional Animal Care and Use Committee (IACUC) protocol specifically approved for this study under assurance number A3873-1. All animals were given ad libitum access to autoclaved acidified water (pH 2.5–3). Frozen vials containing MIA-PaCa-2 pancreatic cancer cells were thawed from liquid nitrogen storage and expanded for in vitro cell culture. Subcutaneous stock tumors were prepared for subsequent flank tumor fragment implantation. MIA-PaCa-2 cells were maintained in DMEM supplemented with 10% heat-inactivated fetal bovine serum and 1% penicillin and cultured at 37°C in a 5% CO2 incubator. Thus, MIA-PaCa-2 cells (1.0 × 107 cells / mouse) in 100 μL of PBS were injected into the right flank of five male nu / nu mice. After placing the mice under anesthesia using ketamine solution, an approximately 5 mm incision was made on the back of the nude mice. After creating a space under the skin of the right flank, a 1 mm incision prepared from the stock was inserted. 3MIA PaCa-2 tumor fragments were inserted into the incision. The incision was closed with 5-0 PDS-II sutures. 18 days after tumor implantation (day 0), tumors were measured with a vernier caliper using the formula: (tumor volume) = (length) × (width) × (width) × ½. Twenty-five of the 35 mice were randomized into five treatment groups of five mice, with no significant differences in tumor volume between groups. All treatments (dissolved in 100 μl of a formulation containing 80% PBS, 10% Tween 80, and 10% ethanol) were administered via tail vein injection twice weekly for three weeks for a total of six injections. Treatment began the day after randomization (day 1), and mice received drug or vehicle control on days 1, 4, 8, 11, 15, and 18. Tumor volume and body weight were measured weekly. The study was terminated 22 days after treatment initiation.

[0173] Molecular Modeling Molecular models were analyzed using MOE2022.02 (Chemical Computing Group). A model of targefrin complexed with EphA2-LBD was obtained by modifying and appropriately minimizing our previous crystal structure of the drug with EphA2-LBD (PDB-ID 6B9L).

[0174] The contents of C. Baggio, et al., J. Med. Chem. 2022, 65, 22, 15443-15456 are incorporated herein by reference.

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K., Emerging Strategies for Epha2 Receptor Targeting for Cancer Therapeutics. Expert Opin Ther Targets 2011, 15 (1), 31-51. 53. Annunziata, C. M.;Kohn, E. C.;LoRusso, P.;Houston, N. D.;Coleman, R. L.;Buzoianu, M.;Robbie, G.;Lechleider, R., Phase 1, Open-Label Study of Medi-547 in Patients with Relapsed or Refractory Solid Tumors. Invest New Drugs 2013, 31 (1), 77-84. 54. Gan, H. K.; Parakh, S.; Lee, F. T.; Tebbutt, N. C.; Ameratunga, M.; Lee, S. T.; O’Keefe, G. J.; Gong, S. J.; Vanrenen, C.; Caine, J.; Giovannetti, M.; Murone, C.; Scott, F. E.; Guo, N.; Burvenich, I. J. G.; Paine, C.; Macri, M. J.; Kotsuma, M.; Senaldi, G.; Venhaus, R.; Scott, A. M., A Phase 1 Safety and Bioimaging Trial of Antibody Ds-8895a against Epha2 in Patients with Advanced or Metastatic Epha2 Positive Cancers. Invest New Drugs 2022, 40 (4), 747-755. 55. Mudd, G. E.; Brown, A.; Chen, L.; van Rietschoten, K.; Watcham, S.; Teufel, D. P.; Pavan, S.; Lani, R.; Huxley, P.; Bennett, G. S., Identification and Optimization of Epha2-Selective Bicycles for the Delivery of Cytotoxic Payloads. J Med Chem 2020, 63 (8), 4107-4116.

[0176] Example 19. [Table 7] [Table 8]

[0177] All publications, patents, and patent applications are incorporated herein by reference, as if individually incorporated by reference. The invention has been described with reference to various specific and preferred embodiments and techniques. However, it should be understood that many variations and modifications may be made while remaining within the spirit and scope of the invention.

Claims

1. Compounds of formula (I): 【Chemistry 1】 or a salt thereof (wherein Each R is independently selected from the group consisting of morpholino, piperidino, and piperazine, which is １ -C ６ ) optionally substituted with alkyl; Each R １ is benzyl, 3-indolylmethyl, 4-pyridinylmethyl, 1-naphthylmethyl, or 2-naphthylmethyl, and benzyl, 3-indolylmethyl, 4-pyridinylmethyl, 1-naphthylmethyl, and 2-naphthylmethyl are hydroxy, amino, nitro, (C １ -C ６ ) alkoxy, and (C １ -C ６ ) optionally substituted with one or more groups independently selected from alkyl; Each R ２ is optionally substituted with hydroxy (C １ -C ６ ) alkyl; Each R ４ is independently selected from the group consisting of biphenyl and phenoxyphenyl, wherein the biphenyl and phenoxyphenyl are selected from the group consisting of halo, hydroxy, (C １ -C ６ ) alkyl, and (C １ -C ６ ) alkoxy, wherein each (C １ -C ６ ) alkyl and (C １ -C ６ ) the alkoxy is optionally substituted with one or more groups independently selected from the group consisting of halo; Each R ７ is optionally substituted with hydroxy (C １ -C ６ ) alkyl; Each R ８ isopropyl and (C ３ -C ６ ) cycloalkyl; Each R ９ is independently selected from the group consisting of benzyl optionally substituted with one or more halo; R １２ is H or is selected from the group consisting of: 【Chemistry 2】 R １００ is optionally substituted with H, hydroxy (C ３ -C ６ ) cycloalkyl, or (C １ -C ６ ) alkyl; R １０１ is optionally substituted with H, hydroxy (C ３ -C ６ ) cycloalkyl, or (C １ -C ６ ) alkyl; R １０２ is optionally substituted with H, hydroxy (C ３ -C ６ ) cycloalkyl, or (C １ -C ６ ) alkyl; R １０３ Is, -L １ -D; D is a residue of a drug or a residue of a targeting agent; p is 1, 2, or 3; m is 1, 2, or 3; n is 1, 2, or 3; R １０４ Below: 【Transformation 3】 and R １１ is C(=NH)NH ２ and L １ is a linking group; L ２ is a linking group).

2. 2. A compound or salt according to claim 1, Each R １ is benzyl, 1-naphthylmethyl, or 2-naphthylmethyl, and the benzyl, 1-naphthylmethyl, and 2-naphthylmethyl are hydroxy, amino, nitro, and (C １ -C ６ ) optionally substituted with one or more groups independently selected from alkyl; R １２ is H or is selected from the group consisting of: 【Chemistry 4】 R １００ is H, (C ３ -C ６ ) cycloalkyl, or (C １ -C ２ ) alkyl; R １０１ is H, (C ３ -C ６ ) cycloalkyl, or (C １ -C ２ ) alkyl; R １０２ is H, (C ３ -C ６ ) cycloalkyl, or (C １ -C ２ ) alkyl.

3. 3. The compound or salt of claim 1, wherein each R is morpholino or piperidino.

4. Each R is (C １ -C ６ 3. The compound or salt according to any one of claims 1 to 2, which is piperazine optionally substituted with alkyl.

5. 3. The compound or salt of any one of claims 1 to 2, wherein each R is 1-piperazinyl.

6. Each R １ 6. The compound or salt of any one of claims 1 to 5, wherein is benzyl optionally substituted with amino.

7. Each R １ 6. The compound or salt of any one of claims 1 to 5, wherein is benzyl optionally substituted with hydroxy.

8. Each R １ However, (C １ -C ６ 6. The compound or salt of claim 1, wherein R is 1 or 2. 7.) benzyl optionally substituted with alkyl.

9. Each R １ The compound or salt according to any one of claims 1 to 5, wherein is 2-nitrobenzyl, 4-methylbenzyl, 4-hydroxybenzyl, or 4-aminobenzyl.

10. Each R ２ is optionally substituted with hydroxy (C １ -C ４ 10. The compound or salt of any one of claims 1 to 9, wherein the aryl group is independently selected from the group consisting of: aryl, aryl(s), ...

11. Each R ２ The compound or salt according to any one of claims 1 to 9, wherein is isobutyl or hydroxymethyl.

12. Each R ４ However, halo, hydroxy, (C １ -C ６ ) alkyl, and (C １ -C ６ ) alkoxy, wherein each (C １ -C ６ ) alkyl and (C １ -C ６ 12. The compound or salt of any one of claims 1 to 11, wherein the alkoxy is optionally substituted with one or more groups independently selected from the group consisting of halo.

13. Each R ４ are independently halo, hydroxy, (C １ -C ６ ) alkyl, and (C １ -C ６ phenoxyphenyl optionally substituted with one or more groups independently selected from the group consisting of (C)alkoxy, １ -C ６ ) alkyl and (C １ -C ６ 12. The compound or salt of any one of claims 1 to 11, wherein the alkoxy is optionally substituted with one or more groups independently selected from the group consisting of halo.

14. Each R ４ are independently selected from the group consisting of biphenyl, 2'-trifluoromethylbiphenyl, 2'-methylbiphenyl, 4'-chlorobiphenyl, 2'-methoxybiphenyl, 3'-methylbiphenyl, 2'-methyl-4'-methoxybiphenyl, phenoxyphenyl, and 4-(4-hydroxyphenyloxy)phenyl.

15. Each R ８ isopropyl and (C ３ -C ６ 15. The compound or salt of any one of claims 1 to 14, wherein the aryl group is independently selected from the group consisting of: aryl, aryl(s), ...

16. Each R ８ The compound or salt according to any one of claims 1 to 14, wherein is cyclohexyl.

17. R １２ The compound or salt of any one of claims 1 to 16, wherein is H.

18. R １２ but the following: 【Transformation 5】 The compound or salt according to any one of claims 1 to 16,

19. R １００ is H, -CH3, -C ２ H ５ , i-pr, cyclohexyl, or —CH ２ 19. The compound or salt of claim 18, wherein:

20. R １０１ is H, -CH3, -C ２ H ５ , i-pr, cyclohexyl, or —CH ２ 19. The compound or salt of claim 18, wherein:

21. R １０２ is H, -CH3, -C ２ H ５ , i-pr, cyclohexyl, or —CH ２ 19. The compound or salt of claim 18, wherein:

22. R １２ but the following: 【Transformation 6】 The compound or salt according to any one of claims 1 to 16,

23. A compound or salt according to any one of claims 1 to 22, wherein L １ is a branched or unbranched, saturated or unsaturated hydrocarbon chain having about 1 to 100 carbon atoms, wherein one or more of said carbon atoms is selected from the group consisting of —O—, —S, —N(R ａ )-, a 3- to 7-membered heterocycle, a 5- to 6-membered heteroaryl, or a carbocycle, wherein each chain, 3- to 7-membered heterocycle, 5- to 6-membered heteroaryl, or carbocycle is optionally independently replaced by (C １ -C ６ ) alkyl, (C １ -C ６ ) alkoxy, (C ３ -C ６ ) cycloalkyl, (C １ -C ６ ) alkanoyl, (C １ -C ６ ) alkanoyloxy, (C １ -C ６ ) alkoxycarbonyl, (C １ -C ６ ) alkylthio, azido, cyano, nitro, halo, -N(R ａ ) ２ , hydroxy, oxo (═O), carboxy, aryl, aryloxy, heteroaryl, and heteroaryloxy, wherein each R ａ are independently H or (C １ -C ６ ) alkyl.

24. A compound or salt according to any one of claims 1 to 22, wherein L １ is a branched or unbranched, saturated or unsaturated hydrocarbon chain having about 1 to 20 carbon atoms, wherein one or more of said carbon atoms is selected from the group consisting of —O—, —S, —N(R ａ )-, a 3- to 7-membered heterocycle, a 5- to 6-membered heteroaryl, or a carbocycle, wherein each carbon atom, 3- to 7-membered heterocycle, 5- to 6-membered heteroaryl, or carbocycle is optionally independently replaced by (C １ -C ６ ) alkyl, (C １ -C ６ ) alkoxy, (C ３ -C ６ ) cycloalkyl, (C １ -C ６ ) alkanoyl, (C １ -C ６ ) alkanoyloxy, (C １ -C ６ ) alkoxycarbonyl, (C １ -C ６ ) alkylthio, azido, cyano, nitro, halo, -N(R ａ ) ２ , hydroxy, oxo (═O), and carboxy; ａ are independently H or (C １ -C ６ ) alkyl.

25. A compound or salt according to any one of claims 1 to 22, wherein L １ is a branched or unbranched saturated hydrocarbon chain having from about 5 to 15 carbon atoms, wherein one or more of said carbon atoms are optionally independently replaced by O, NH, or a divalent triazine ring, wherein each carbon atom is optionally independently substituted with one or more (e.g., 1, 2, 3, 4, 5 or more) substituents selected from halo and oxo (=O).

26. L １ but the following: 【Transformation 7】 The compound or salt according to any one of claims 1 to 22,

27. 27. The compound or salt of any one of claims 1 to 26, wherein D is a residue of a drug.

28. The compound or salt according to any one of claims 1 to 26, wherein D is a residue of an anticancer drug.

29. 27. The compound or salt of any one of claims 1 to 26, wherein D is a residue of a taxane, including paclitaxel, docetaxel, or cabazitaxel.

30. 27. The compound or salt of any one of claims 1 to 26, wherein D is a residue of gemcitabine.

31. 27. The compound or salt of any one of claims 1 to 26, wherein D is a residue of a targeting agent.

32. R １２ but the following: 【Transformation 8】 The compound or salt according to any one of claims 1 to 16,

33. 33. The compound or salt of claim 32, wherein p is 2.

34. A compound or salt according to any one of claims 1 to 17 and 22 to 33, wherein L ２ is a branched or unbranched, saturated or unsaturated hydrocarbon chain having about 1 to 100 carbon atoms, wherein one or more of said carbon atoms is selected from the group consisting of —O—, —S, —N(R ａ )-, a 3- to 7-membered heterocycle, a 5- to 6-membered heteroaryl, or a carbocycle, wherein each chain, 3- to 7-membered heterocycle, 5- to 6-membered heteroaryl, or carbocycle is optionally independently replaced by (C １ -C ６ ) alkyl, (C １ -C ６ ) alkoxy, (C ３ -C ６ ) cycloalkyl, (C １ -C ６ ) alkanoyl, (C １ -C ６ ) alkanoyloxy, (C １ -C ６ ) alkoxycarbonyl, (C １ -C ６ ) alkylthio, azido, cyano, nitro, halo, -N(R ａ ) ２ , hydroxy, oxo (═O), carboxy, aryl, aryloxy, heteroaryl, and heteroaryloxy, wherein each R ａ are independently H or (C １ -C ６ ) alkyl.

35. A compound or salt according to any one of claims 1 to 17 and 22 to 33, wherein L ２ is a branched or unbranched, saturated or unsaturated hydrocarbon chain having about 1 to 20 carbon atoms, wherein one or more of said carbon atoms is selected from the group consisting of —O—, —S, —N(R ａ )-, a 3- to 7-membered heterocycle, a 5- to 6-membered heteroaryl, or a carbocycle, wherein each carbon atom, 3- to 7-membered heterocycle, 5- to 6-membered heteroaryl, or carbocycle is optionally independently replaced by (C １ -C ６ ) alkyl, (C １ -C ６ ) alkoxy, (C ３ -C ６ ) cycloalkyl, (C １ -C ６ ) alkanoyl, (C １ -C ６ ) alkanoyloxy, (C １ -C ６ ) alkoxycarbonyl, (C １ -C ６ ) alkylthio, azido, cyano, nitro, halo, -N(R ａ ) ２ , hydroxy, oxo (═O), and carboxy; ａ are independently H or (C １ -C ６ ) alkyl.

36. A compound or salt according to any one of claims 1 to 17 and 22 to 33, wherein L ２ is a branched or unbranched saturated hydrocarbon chain having from about 3 to 110 carbon atoms, wherein each carbon atom is optionally independently substituted with one or more (e.g., 1, 2, 3, 4, 5 or more) substituents selected from halo and oxo (=O).

37. A compound or salt according to any one of claims 1 to 17 and 22 to 33, wherein L ２ But -CH ２ C(=O)-, -CH ２ CH ２ C(=O)-, -CH ２ CH ２ CH ２ C(=O)-, -CH ２ CH ２ CH ２ CH ２ C(=O)- or -CH ２ CH ２ CH ２ CH ２ CH ２ The compound or salt thereof, wherein C(=O)-.

38. A compound selected from the group consisting of: 【Chemistry 9-1】 【Chemistry 9-2】 【Chemistry 9-3】 Or its salt.

39. A pharmaceutical composition comprising a compound or salt according to any one of claims 1 to 38 and a pharmaceutically acceptable excipient.

40. 40. A method of treating cancer in an animal, comprising administering to the animal a compound of formula I as defined in any one of claims 1 to 38, or a pharmaceutically acceptable salt thereof.

41. A compound of formula I or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 38 for use in medical therapy.

42. A compound of formula I according to any one of claims 1 to 38 or a pharmaceutically acceptable salt thereof for the preventive or therapeutic treatment of cancer.

43. 40. Use of a compound of formula I, or a pharmaceutically acceptable salt thereof, according to any one of claims 1 to 38, for the preparation of a medicament for treating cancer in an animal.

44. 44. The method, compound or use of any one of claims 40 to 43 in combination with an EGRF inhibitor, a Her2 inhibitor, or a BRAF inhibitor.

45. 44. The method, compound or use of any one of claims 40 to 43 in combination with any chemotherapeutic agent or other anti-cancer targeted therapeutic agent.

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