EPHA2 targeting agents and uses thereof

Abstract

The EphA2 targeting agents developed herein are potent peptidomimetics having a high affinity (Kd 8-20 nanomole) to the ligand binding domain referred to as tagfolin. Monomeric versions of tagfolin act as antagonists, while dimeric versions of said agents (tagfolin dimers) result in internalization and degradation of receptors via lysosomal pathways. Thus, when used as a single agent or in combination with a standard care, the tagfolin dimer agent is effective in reducing cancerogenic EphA2 levels in cancer cells. Tagfolin dimers may also sensitize cancer cells that are resistant to EGFR or BRAF inhibitors and potentially other anti-cancer agents. Furthermore, the dimeric agent can be conjugated to a chemotherapeutic agent, such as paclitaxel, to selectively deliver a cytotoxic agent to the EphA2-expressing cancer cells. The monomeric agent can be linked to the chemotherapeutic agent via a stable cleavable linker, causing the cytotoxic to accumulate at the tumor, which then enters the tumor. Novel compositions and examples of these applications are reported.

Description

[0001] Cross - reference to related applications

[0002] This application claims the priority of U.S. Provisional Application No. 63 / 416,890, filed on October 17, 2022. The entire content of the application cited above is hereby incorporated herein by reference. Background of the Invention

[0003] EphA2 is a receptor tyrosine kinase that has pro - oncogenic effects in a variety of solid tumors in its unbound form. Its tumor - promoting activities include increasing cell motility and cell migration, increasing trans - endothelial migration, increasing angiogenesis, and suppressing the immune system. In addition, the expression of EphA2 renders cancer cells resistant to drugs targeting EGRF inhibitors, as well as to Her2 inhibitors in various solid tumors, and to BRAF inhibitors in melanoma. Therefore, it can be converted into effective cancer therapeutics.

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

[0005] Agents that can target EphA2 have been identified. These EphA2 - targeting agents are potent peptidomimetics that have high affinity (Kd 8 - 20 nanomolar) for the ligand - binding domain. The monomeric version (version) of the compound (the compound of formula (I) where R 12 is H) has been found to be an antagonist, while the dimeric version of the agent (the compound of formula (I) where R 12 is not H) results in receptor internalization and degradation via the lysosomal pathway. Thus, the dimeric agent can effectively reduce the pro - oncogenic EphA2 levels in cancer cells. In addition, the agent can be conjugated with other chemotherapeutic agents (such as paclitaxel) to selectively deliver cytotoxic agents to EphA2 - expressing cancer cells.

[0006] Certain compounds of the present invention are more soluble than previously reported agents such as 135H12 (EphA2 agonists and uses thereof, inventors Pellecchia et al., assigned to the University of California, US20210221843A1; PCT WO2019237075A1; also published in Gambini et al., ACS Chem Biol. September 21, 2018; 13(9):2633–2644. doi:10.1021 / acschembio.8b00556), and are 10-fold more active than the previously reported agents in causing EphA2 internalization and degradation. Thus, the agent can i) be used to reduce the pro-cancerous EphA2 level in solid tumors; ii) be conjugated with other chemotherapeutic agents to more selectively deliver cytotoxic agents to EphA2-expressing cancer cells; and iii) be used to degrade other surface receptors by linking with molecules that have a strong effect on the targeted receptor.

[0007] Accordingly, in one embodiment, the present invention provides a compound of formula (I):

[0008]

[0009] or a salt thereof, wherein:

[0010] Each R is independently selected from the group consisting of morpholino, piperidino, and piperazine, optionally substituted with (C1-C6) alkyl;

[0011] Each R 1 is benzyl, 3-indolylmethyl, 4-pyridylmethyl, 1-naphthylmethyl, or 2-naphthylmethyl, and the benzyl, 3-indolylmethyl, 4-pyridylmethyl, 1-naphthylmethyl, and 2-naphthylmethyl are optionally substituted with one or more groups independently selected from hydroxy, amino, nitro, (C1-C6) alkoxy, and (C1-C6) alkyl;

[0012] Each R 2 is independently selected from the group consisting of (C1-C6) alkyl, optionally substituted with hydroxy;

[0013] Each R 4 is independently selected from the group consisting of biphenyl and phenoxyphenyl, and 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;

[0014] Each R 7 is an optionally hydroxy-substituted (C1-C6) alkyl group;

[0015] Each R 8 is independently selected from the group consisting of isopropyl and (C3-C6) cycloalkyl;

[0016] Each R 9 is independently selected from the group consisting of benzyl optionally substituted with one or more halogen groups;

[0017] R 12 is H or is selected from the group consisting of:

[0018]

[0019] R 100 is H, (C3-C6) cycloalkyl, or an optionally hydroxy-substituted (C1-C6) alkyl group;

[0020] R 101 is H, (C3-C6) cycloalkyl, or an optionally hydroxy-substituted (C1-C6) alkyl group;

[0021] R 102 is H, (C3-C6) cycloalkyl, or an optionally hydroxy-substituted (C1-C6) alkyl group;

[0022] R 103 is -L 1 -D;

[0023] D is a residue of a drug or a residue of a targeting agent;

[0024] p is 1, 2, or 3;

[0025] m is 1, 2, or 3;

[0026] n is 1, 2, or 3;

[0027] R 104 is:

[0028]

[0029] R 11 is C(=NH)NH2;

[0030] L 1 is a linking group; and

[0031] L 2 is a linking group.

[0032] 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.

[0033] The present invention also provides a method for treating or preventing cancer in an animal (e.g., a mammal such as a human), the method 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.

[0034] The present invention also provides a compound of formula I or a pharmaceutically acceptable salt thereof for use in medical therapy.

[0035] The present invention also provides a compound of formula I or a pharmaceutically acceptable salt thereof for prophylactic or therapeutic treatment of cancer.

[0036] The present invention also provides the use of a compound of formula I or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating cancer in an animal (e.g., a mammal such as a human).

[0037] The present invention also provides methods and intermediates useful for preparing the compounds of formula I or salts thereof disclosed herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figures 1A - 1D Shows the modeling and binding data of targefrin. Figure 1A ) Molecular model of targefrin complexed with EphA2-LBD based on the X-ray structure of the complex with a peptide (PDB ID 6B9L). Figure 1B ) Chemical structure of targefrin. Figure 1C ) Isothermal titration calorimetry (ITC) curve of the binding between targefrin and EphA2-LBD (K d = 21.7 ± 1.2 nM; ΔH = -20.2 ± 0.4 kcal / mol; -TΔS = 9.7 ± 0.4 kcal / mol). Figure 1D ) ITC curves of the binding between targefrin and EphA4-LBD and EphA3-LBD, which are the two Eph receptors most similar to EphA2. The data show no significant binding under these experimental conditions.

[0039] Figures 2A - 2B Shows data from Example 9. Targefrin acts as an antagonist. ( Figure 2A ) Western blot of BxPC3 cells starved for 1 h and pretreated with different concentrations of targefrin for 20 min, followed by treatment with 2 μg / mL ephrin A1-Fc for 3 h.Figure 2B )Quantification of EphA2 levels. The EphA2 / β-actin ratio was normalized by designating the EphA2 expression in DMSO without ephrin A1-Fc condition as 1. EC 50 values were calculated to be 1.6 ± 0.1 μM and presented as the mean ± standard error (SE) of two independent experiments.

[0040] Figures 3A - 3F Data from Example 11 are shown. Tagafolin dimer and its variants cause EphA2 degradation in pancreatic cancer cell lines at nanomolar concentrations. ([[]] Figures 3A - 3C )Western blot images of BxPC3, PANC-1, and MIA PaCa-2 cells, respectively, where the cells were starved for 1 h and treated with 2 μg / mL ephrin A1-Fc or indicated doses of tagafolin and tagafolin dimer and its variants using different linkers (Table 4) for 3 h. The previous dimerizer 135H121 is also shown as a reference. ([[]] Figures 3D - 3F )Respectively for the Figures 3A - 3C )data shown in. Figures 3A - 3C )The EphA2 / β-actin ratio was normalized by designating the EphA2 expression from the DMSO control condition as 100% (for Figures 3D - 3F )or 1 (for

[0041] Figures 4A - 4D Data from Example 12 are shown. Chemical structures and biochemical activities of tagafolin-conjugated agents. ([[]] Figure 4A )Tagafolin conjugated to paclitaxel (tagafolin-PTX), Figure 4B )dimeric form of tagafolin conjugated to paclitaxel (tagafolin dimer-PTX), Figure 4C )dimeric form of tagafolin conjugated to 5-carboxytetramethylrhodamine-azide dye (tagafolin dimer-TAMRA) chemical structures. ([[]] Figure 4D )DELFIA displacement dose-response curves comparing tagafolin-PTX, tagafolin dimer-PTX, and tagafolin dimer-TAMRA and their respective IC 50 values.

[0042] Figure 5Data from Example 13 are shown. Tagflorine dimer-TAMRA was internalized in cells expressing EphA2. BxPC3 cells were treated with 100 nM tagflorine dimer-TAMRA for 0, 30, and 60 min. After agent binding, EphA2 was internalized and targeted to lysosomes, as demonstrated by the co-localization of 5-TAMRA and LAMP1 (arrows). The nuclei were labeled blue. Scale bar = 10 μm.

[0043] Figures 6A - 6C Data from Example 14 are shown. Tagflorine dimer significantly inhibited pancreatic cancer cell migration. ( Figure 6A ) Cell migration assay of BxPC3 treated with 2 μg / mL ephrin A1-Fc and 10 μM tagflorine or indicated doses of tagflorine dimer. The plates were imaged every 3 h for 24 h. The yellow line shows the initial scratch made at 0 h, while the black line shows the position to which the cells migrated after 24 h. ( Figure 6B ) Tagflorine dimer significantly inhibited cell migration in a dose-dependent manner at 24 h, as demonstrated by the decrease in relative wound density. Data related to the 12 h time point were reported as 10. ( Figure 6C ) The time-response curve shows the effect of these agents on wound closure over a 24 h period. ***p < 0.001, ****p < 0.0001, determined by one-way ANOVA analyzed using Dunnett's post hoc test. Scale bar = 250 μm.

[0044] Figures 7A - 7C Data from Example 15 are shown. PTX conjugation inhibited tumor growth in tumor xenografts with MIA-PaCa-2 cells. ( Figure 7A ) Five groups of five mice bearing pre-established MIA-PaCa-2 tumors were treated with vehicle control alone, paclitaxel (PTX; 2.5 mg / kg), tagflorine-PTX (10 mg / kg, which is equivalent to 2.5 mg / kg PTX), tagflorine dimer-PTX (17 mg / kg, which is equivalent to 2.5 mg / kg PTX), and lower dose tagflorine dimer-PTX (10 mg / kg, which is equivalent to 1.5 mg / kg PTX) for 22 days. Tumor volumes were reported as mean ± SE. ( Figure 7B ) Mean tumor volumes of each treatment group measured at days 0, 8, 15, and 22. *p = 0.03, **p < 0.01, ***p = 0.0001, ****p < 0.0001, determined by two-way ANOVA analyzed using Tukey's post hoc test. ( Figure 7C) Reported the mean body weight ± SE of each of the five treatment groups at days 0, 8, 15, and 22. For all graphs, the vehicle was reported in black, PTX in green, the lower dose of tagafolin dimer-PTX in light blue, tagafolin dimer-PTX in blue, and tagafolin-PTX in red.

[0045] Figures 8A - 8F Showed that tagafolin dimer-PTX retained its ability to cause EphA2 degradation in pancreatic cancer cell lines. ( Figure 8A –8C) Western blot images of BxPC3, PANC-1, and MIA PaCa-2 cells, respectively, in which the cells were starved for 1 h and treated with 2 μg / mL ephrin A1-Fc or indicated doses of tagafolin, tagafolin-PTX, tagafolin dimer, and tagafolin dimer-PTX for 3 h. Figures 8D - 8F ) Respectively for ( Figures 8A - 8C ) Densitometric analysis. By designating the EphA2 expression from the DMSO control condition as 100% (for Figures 8A - 8C ) or 1 (for Figures 8D - 8F ), the EphA2 / β-actin ratio was normalized. ***p < 0.001, ****p < 0.0001, as determined by one-way ANOVA analyzed using Dunnett's post hoc test.

[0046] Figure 9 . Pharmacokinetic study. A preliminary pharmacokinetic (PK) study using tagafolin dimer. The agent was injected via the tail vein IV at a concentration of 50 mg / Kg in a formulation with 80% PBS, 10% Tween 80, and 10% ethanol. Note that this formulation produced a clear solution containing 20 mg / ml tagafolin dimer. At 2 h after injection, C max was approximately 650 ng / mL. The estimated t 1 / 2 was approximately 15 h.

[0047] Figures 10A - 10B Showed the cell migration assay of BxPC3 at 12 h from Figure 9 . Figure 10A ) Cell migration assay of BxPC3 treated with 2 μg / mL ephrin A1-Fc and 10 μM tagafolin or indicated doses of tagafolin dimer. The yellow line shows the initial scratch made at 0 h, while the black line shows the position to which the cells migrated after 12 h. Figure 10B)Tagfurin dimer significantly inhibited cell migration in a dose-dependent manner at 12 h, as demonstrated by the decrease in relative wound density. ***p < 0.01, ****p < 0.0001, determined by one-way ANOVA analyzed using Dunnett's post hoc test.

[0048] Figures 11A - 11D . Structural details related to the optimization process. ( Figure 11A ) Structure of EphA2-LBD complexed with an early agent developed in our laboratory (1). The surface of the receptor and the D–E, G–H, and J–K loops are shown. ( Figure 11B ) Details of the tyrosine residue at position 4 of the EphA2 binder, which protrudes into a large hydrophobic pocket located between the D-E and J-K loops. ( Figure 11C ) Details of position 1 of the EphA2 binder, which replaces tyrosine 1 and the N-terminal amide of the YSA peptide, and is located between the G-H and J-K loops. ( Figure 11D ) Details of the serine residue pair in the EphA2 binder, which forms an intramolecular hydrogen bond (see arrow) in its bound form, thus constraining the peptide in a tight conformation.

[0049] Figures 12A - 12B . Cell viability assay of MIA PaCa-2 at 72 h. Figure 12A ) MIA PaCa-2 cells were treated with 1 μg / mL ephrin A1-Fc, different doses of tagfurin, or tagfurin dimer for 72 h. The percentage of confluence was monitored using the IncuCyte S3 live cell analysis system, and the percentage of cell viability was calculated by normalizing the confluence of these treatments against the confluence of the DMSO control. The percentage of cell viability was not significantly affected in all treatments, as determined by two-way ANOVA analyzed using Bonferroni's post hoc test. Figure 12B ) Time-response curve of the percentage of confluence of MIAPaCa-2 cells after the indicated treatments.

[0050] Figures 13A - 13C . ( Figure 13A ) Tagfurin dimer mimics the natural ephrin A1 ligand and causes EphA2 degradation, thus inhibiting metastasis. ( Figure 13B ) Tagfurin-PTX can deliver PTX to cancer cells by accumulating the toxin on metastatic pancreatic cancer cells expressing EphA2. This requires extracellular proteases to cleave a specific linker to release the toxin, and then passive diffusion in tumor cells. ( Figure 13C)However, when PTX is conjugated to tagifolin dimer, EphA2 degradation and prometastatic signals are inhibited. Meanwhile, the toxin is actively transported in tumor cells expressing EphA2, thereby actively killing primary and metastatic cancer cells. Detailed Description

[0051] Unless otherwise described, the following definitions are used: Halo or halogen is fluorine, chlorine, bromine or iodine. Alkyl, alkoxy, etc. denote both straight-chain and branched-chain groups; but a reference to a single group such as propyl includes only the straight-chain group, and the branched-chain isomers such as isopropyl are specifically mentioned.

[0052] Unless otherwise specified, the term "alkyl" by itself or as part of another substituent means a straight-chain or branched-chain hydrocarbon group having the specified number of carbon atoms (i.e., C 1-8 means a straight-chain or branched-chain hydrocarbon group having one to eight carbons. Examples include (C1-C8) alkyl, (C2-C8) alkyl, (C1-C6) alkyl, (C2-C6) alkyl and (C3-C6) alkyl. Examples of alkyl include methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, sec-butyl, n-pentyl, n-hexyl, n-heptyl, n-octyl and higher homologues and isomers.

[0053] The term "alkoxy" refers to an alkyl group attached to the remainder of the molecule via an oxygen atom ("oxy").

[0054] The term "cycloalkyl" or "carbocyclic" refers to a saturated or partially unsaturated (non-aromatic) all-carbon ring having 3 to 8 carbon atoms (i.e., (C3-C8) carbocyclic). The term also includes polycondensed, saturated all-carbon ring systems (e.g., a ring system containing 2, 3 or 4 carbon rings). Accordingly, carbocyclics include polycyclic carbocyclics, such as bicyclic carbocyclics (e.g., having from about 3 to 15 carbon atoms, from 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 carbocyclics (e.g., tricyclic and tetracyclic carbocyclics having up to about 20 carbon atoms). When the valence requires it, the rings of a polycondensed ring system may be connected to each other via fused, spiro and bridged bonds. For example, polycyclic carbocyclics may be connected to each other by a single carbon atom to form a spiro linkage (e.g., spiropentane, spiro[4,5]decane, etc.), by two adjacent carbon atoms to form a fused linkage (e.g., carbocyclics such as decalin, norsabinane, norcarane), or by two non-adjacent carbon atoms to form a bridged linkage (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.

[0055] As used herein, the term "aryl" refers to a single fully carbon aromatic ring or a polycondensed fully carbon ring system, wherein at least one ring is aromatic. For example, in certain embodiments, aryl has 6 to 20 carbon atoms, 6 to 14 carbon atoms, 6 to 12 carbon atoms, or 6 to 10 carbon atoms. Aryl includes phenyl. Aryl also includes polycondensed carbocyclic ring systems having about 9 to 20 carbon atoms (e.g., ring systems containing 2, 3, or 4 rings), wherein at least one ring is aromatic and wherein the other rings may or may not be aromatic (i.e., cycloalkyl). When valency permits, the rings of the polycondensed ring system may be connected to each other via fused, spiro, and bridging bonds. It should be understood that, as defined above, the point of attachment of a multiple fused ring system may be at any position of the ring system, including the aromatic portion or the carbocyclic portion of the ring. Non-limiting examples of aryl include, but are not limited to, phenyl, indenyl, indanyl, naphthyl, 1,2,3,4-tetrahydronaphthyl, anthracenyl, and the like.

[0056] The term "heterocycle" refers to a single saturated or partially unsaturated 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 a polyfused ring system having at least one such saturated or partially unsaturated ring, which polyfused ring system will be further described below. Thus, the term includes a single saturated or partially unsaturated ring having from about 1 to 6 carbon atoms and from about 1 to 3 heteroatoms selected from the group consisting of oxygen, nitrogen, and sulfur (e.g., 3-, 4-, 5-, 6-, or 7-membered rings). Sulfur and nitrogen atoms may also be in their oxidized forms. Exemplary heterocycles include, but are not limited to, azetidinyl, tetrahydrofuranyl, and piperidinyl. The term "heterocycle" also includes polyfused ring systems (e.g., ring systems containing 2, 3, or 4 rings), where a single heterocycle (as defined above) may be fused with one or more groups selected from cycloalkyl, aryl, and heterocycle to form a polyfused ring system. When valency permits, the rings of a polycondensed ring system may be connected to each other via fused, spiro, and bridging bonds. It should be understood that the individual rings of a polyfused ring system may be connected to each other in any order. It should also be understood that the point of attachment of a polyfused ring system (as defined above for heterocycles) may be at any position of the polyfused ring system, which positions include the heterocyclic, aryl, and carbocyclic moieties of the ring. In one embodiment, the term heterocycle includes 3- to 15-membered heterocycles. In one embodiment, the term heterocycle includes 3- to 10-membered heterocycles. In one embodiment, the term heterocycle includes 3- to 8-membered heterocycles. In one embodiment, the term heterocycle includes 3- to 7-membered heterocycles. In one embodiment, the term heterocycle includes 3- to 6-membered heterocycles. In one embodiment, the term heterocycle includes 4- to 6-membered heterocycles. In one embodiment, the term heterocycle includes 3- to 10-membered monocyclic or bicyclic heterocycles containing 1 to 4 heteroatoms. In one embodiment, the term heterocycle includes 3- to 8-membered monocyclic or bicyclic heterocycles containing 1 to 3 heteroatoms. In one embodiment, the term heterocycle includes 3- to 6-membered monocyclic heterocycles containing 1 to 2 heteroatoms. In one embodiment, the term heterocycle includes 4- to 6-membered monocyclic heterocycles containing 1 to 2 heteroatoms. Exemplary heterocycles include, but are not limited to, aziridinyl, azetidinyl, pyrrolidinyl, piperidinyl, homopiperidinyl, morpholinyl, thiomorpholinyl, piperazinyl, tetrahydrofuranyl, dihydrooxazolyl, tetrahydropyranyl, tetrahydrothiopyranyl, 1,2,3,4-tetrahydroquinolinyl, benzoxazinyl, dihydrooxazolyl, chromanyl, 1,2-dihydropyridinyl, 2,3-dihydrobenzofuranyl, 1,3-benzodioxolyl, 1,4-benzodioxanyl, spiro[cyclopropane-1,1'-isoindolinyl]-3'-one, isoindolin-1-one, 2-oxa-6-azaspiro[3.3]heptyl, imidazolidin-2-one, imidazolidine, pyrazolidine, butyrolactam, valerolactam, imidazolidinone, hydantoin, dioxolane, phthalimide, and 1,4-dioxane.

[0057] As used herein, the term "heteroaryl" refers to a single aromatic 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; "heteroaryl" also includes a multiple fused ring system having at least one such aromatic ring, which multiple fused ring system will be further described below. Thus, "heteroaryl" includes a single aromatic ring having from about 1 to 6 carbon atoms and from about 1 - 4 heteroatoms selected from the group consisting of oxygen, nitrogen, and sulfur. The sulfur and nitrogen atoms may also be in oxidized form, provided that 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), where a heteroaryl 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 fused ring system may be at any suitable atom of the heteroaryl or heteroaryl multiple fused 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, quinolinyl, isoquinolinyl, benzothiazolyl, benzoxazolyl, indazolyl, quinoxalinyl, and quinazolinyl.

[0058] As used herein, the term "protecting group" refers to a substituent that is commonly used to block or protect a specific functional group on a compound. For example, an "amino protecting group" is a substituent that blocks or protects the attachment of the amino functional group in a compound to the amino group. Suitable amino protecting groups include acetyl, trifluoroacetyl, tert-butoxycarbonyl (BOC), benzyloxycarbonyl (CBZ), and 9-fluorenylmethyloxycarbonyl (Fmoc). Similarly, a "hydroxy protecting group" refers to a hydroxy substituent that blocks or protects the hydroxy functional group. Suitable protecting groups include acetyl and silyl. A "carboxy protecting group" is a carboxy substituent that blocks or protects the carboxy functional group. Common carboxy protecting groups include phenylsulfonylethyl, cyanoethyl, 2-(trimethylsilyl)ethyl, 2-(trimethylsilyl)ethoxymethyl, 2-(p-toluenesulfonyl)ethyl, 2-(p-nitrophenylsulfinyl)ethyl, 2-(diphenylphosphino)ethyl, nitroethyl, and the like. For a general description of protecting groups and their uses, see P.G.M. Wuts and T.W.G. Greene, Greene's Protective Groups in Organic Synthesis, 4th Edition, Wiley-Interscience, New York, 2006.

[0059] As used herein, a wavy line intersecting a bond in a chemical structure represents the point of attachment of the wavy bond to the bond that intersects the remainder of the molecule in that chemical structure.

[0060] The terms "treat / treatment / treating", when referring to a disease or disorder, include inhibiting the disease or disorder, eliminating the disease or disorder, and / or alleviating one or more symptoms of the disease or disorder. The terms "treat / treatment / treating" also refer to both therapeutic treatment and / or prophylactic treatment or preventive measures, where the goal is to prevent or slow down (mitigate) an undesired physiological change or condition, such as the development or spread of cancer. By way of example, beneficial or desired clinical outcomes include, but are not limited to, alleviation of detectable or non-detectable symptoms, diminishment of the degree of a disease or disorder, stabilization (i.e., non-worsening) of the state of a disease or disorder, delay or slowing of disease progression, improvement or alleviation of the disease state or disorder, and remission (partial or total). "Treat / treatment / treating" may also mean prolonging survival as compared to expected survival if not receiving treatment. Those in need of treatment include those who already have the disease or disorder and those who are predisposed to having the disease or disorder or are to prevent the disease or disorder. In one embodiment, "treat / treatment / treating" does not include preventing / prevention.

[0061] 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, disorder, or condition, (ii) diminishes, ameliorates, or eliminates one or more symptoms of a particular disease, disorder, or condition, or (iii) prevents or delays the onset of one or more symptoms of a particular disease, disorder, or condition described herein.

[0062] As used herein, the term "mammal" refers to a human, a higher non-human primate, a rodent, a domestic animal, cattle, horses, pigs, sheep, dogs, and cats. In one embodiment, the mammal is a human. As used herein, the term "patient" refers to any animal, including a mammal. In one embodiment, the patient is a mammalian patient. In one embodiment, the patient is a human patient.

[0063] One of ordinary skill in the art will understand that the invention also includes any claimed compound that can be enriched in one or more isotopes at a ratio higher than the naturally occurring isotope ratio at any or all atoms, such as, but not limited to, deuterium ( 2 H or D). As a non-limiting example, a -CH3 group can be replaced by a -CD3 group.

[0064] The pharmaceutical composition of the present invention may comprise one or more excipients. When used in combination with the pharmaceutical composition of the present invention, the term "excipient" generally refers to an additional ingredient that, in combination with the compound of formula (I) or its pharmaceutically acceptable salt, provides the corresponding composition. For example, when used in combination with the pharmaceutical composition of the present invention, the term "excipient" includes, but is not limited to: carriers, binders, disintegrants, lubricants, sweeteners, flavorants, coating agents, preservatives, and dyes.

[0065] The stereochemical definitions and conventions used herein generally follow those described in S.P. Parker, editor, 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 thus exist in different stereoisomeric forms. It is intended that all stereoisomeric forms of the compounds of the present invention (including, but not limited to, diastereoisomers, enantiomers, and atropisomers) and mixtures thereof (such as racemic mixtures) are part of the present invention. Many organic compounds exist in optically active forms, i.e., they have the ability to rotate the plane of plane-polarized light. When describing optically active compounds, the prefixes D and L or R and S are used to denote the absolute configuration of the molecule with respect to its chiral center. The prefixes d and l or (+) and (-) are used to specify the sign of rotation of the compound with respect to plane-polarized light, (-) or l meaning that the compound is levorotatory. Compounds with the prefix (+) or d are dextrorotatory. For a given chemical structure, these stereoisomers are identical except that they are mirror images of each other. Specific stereoisomers may also be referred to as enantiomers, and mixtures of such isomers are generally called enantiomeric mixtures. A 50:50 mixture of enantiomers is called a racemic mixture or racemate, which may occur in chemical reactions or processes without stereoselection or stereospecificity. The terms "racemic mixture" and "racemate" refer to an equimolar mixture of two enantiomeric substances that is not optically active.

[0066] Those skilled in the art will understand that the compounds of the present invention having chiral centers can exist in optically active forms and racemic forms and can be separated in optically active forms and racemic forms. Some compounds may exhibit polymorphism. It should be understood that the present invention encompasses any racemic, optically active, polymorphic or stereoisomeric forms of the compounds of the present invention or mixtures thereof, which substances have the useful properties described herein, and it is well known in the art how to prepare optically active forms (for example, by resolving the racemic form with recrystallization techniques, by synthesis from optically active starting materials, by chiral synthesis or by chromatographic separation using a chiral stationary phase).

[0067] When the bonds in the compound formulas herein are drawn in a non-stereochemical manner (e.g., flat), the atoms to which the bonds are attached include all stereochemical possibilities. Unless otherwise indicated, when the bonds in the compound formulas herein are drawn in a defined stereochemical manner (e.g., bold, bold wedge, dash or dash wedge), it should be understood that the atoms to which the stereochemical bonds are attached are enriched in the depicted absolute stereoisomer. In one embodiment, the compound can be at least 51% of the depicted absolute stereoisomer. In another embodiment, the compound can be at least 60% of the depicted absolute stereoisomer. In another embodiment, the compound can be at least 80% of the depicted absolute stereoisomer. In another embodiment, the compound can be at least 90% of the depicted absolute stereoisomer. In another embodiment, the compound can be at least 95% of the depicted absolute stereoisomer. In another embodiment, the compound can be at least 99% of the depicted absolute stereoisomer.

[0068] The term "residue" when applied to a residue of a compound refers to a compound that has been modified in any way that results in the creation of an open valence, where the site of the open valence. The 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 more than one atom such as a group of atoms, including but not limited to an amine, a hydroxyl group, a methyl group, an amide (e.g., -C(=O)NH2) or an acetyl group). The open valence can also be created by chemically converting a first functional group of the compound to a second functional group of the compound (e.g., reducing a carbonyl group, replacing a carbonyl group with an amine), and then removing one or more atoms from the second functional group to create the open valence.

[0069] In one embodiment, the present invention provides a compound of formula (I):

[0070]

[0071] or a salt thereof, wherein:

[0072] Each R is independently selected from the group consisting of morpholino, piperidino and piperazine, each optionally substituted by (C1-C6) alkyl;

[0073] Each R 1 is benzyl, 1-naphthylmethyl or 2-naphthylmethyl, where the benzyl, 1-naphthylmethyl and 2-naphthylmethyl are each optionally substituted by one or more groups independently selected from hydroxyl, amino, nitro and (C1-C6) alkyl;

[0074] Each R 2 is independently selected from the group consisting of (C1-C6) alkyl, each optionally substituted by hydroxyl;

[0075] Each R 4 is independently selected from the group consisting of biphenyl and phenoxyphenyl, where the biphenyl and phenoxyphenyl are each optionally substituted by one or more groups independently selected from halogen, hydroxyl, (C1-C6) alkyl and (C1-C6) alkoxy, where each (C1-C6) alkyl and (C1-C6) alkoxy is optionally substituted by one or more groups independently selected from the group consisting of halogen;

[0076] Each R 7 is (C1-C6) alkyl, optionally substituted by hydroxyl;

[0077] Each R 8 is independently selected from the group consisting of isopropyl and (C3-C6) cycloalkyl;

[0078] Each R 9 is independently selected from the group consisting of benzyl, each optionally substituted by one or more halogens;

[0079] R 12 is H or selected from the group consisting of:

[0080]

[0081] R 100 is H, (C1-C2) alkyl or (C3-C6) cycloalkyl, optionally substituted by hydroxyl;

[0082] R 101 is H, (C1-C2) alkyl or (C3-C6) cycloalkyl, optionally substituted by hydroxyl;

[0083] R 102 is H, (C1-C2) alkyl or (C3-C6) cycloalkyl, optionally substituted by hydroxyl;

[0084] R 103 is -L 1 -D;

[0085] D is a residue of a drug or a residue of a targeting agent;

[0086] p is 1, 2 or 3;

[0087] R 104 is:

[0088]

[0089] R 11 is C(=NH)NH2;

[0090] L 1 is a linking group; and

[0091] L 2 is a linking group.

[0092] The specific values listed below for groups, substituents and ranges are for illustration only; they do not exclude other determined values of groups and substituents or other values within the determined ranges. It should be understood that two or more values can be combined. It should also be understood that the values (or subsets thereof) listed below herein can be excluded.

[0093] 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, pentyloxy, 3-pentyloxy or hexyloxy; and aryl can be phenyl, indenyl or naphthyl.

[0094] A specific value of R is morpholino.

[0095] A specific value of R is piperidino.

[0096] A specific value of R is a piperazinyl optionally substituted by (C1-C6)alkyl (e.g., 1-piperazinyl).

[0097] A specific value of R is 1-piperazinyl.

[0098] R 1 has a specific value of benzyl optionally substituted by amino.

[0099] R 1 has a specific value of benzyl optionally substituted by nitro.

[0100] R 1The specific value of is benzyl optionally substituted by (C1-C6) alkyl.

[0101] R 1 The specific value of is benzyl optionally substituted by hydroxy.

[0102] R 1 The specific value of is benzyl optionally substituted by (C1-C6) alkoxy.

[0103] R 1 The specific value of is 2-nitrobenzyl, 4-methylbenzyl or 4-aminobenzyl.

[0104] R 1 The specific value of is 4-hydroxybenzyl or 4-methoxybenzyl.

[0105] R 1 The specific value of is 3-indolylmethyl, 4-pyridylmethyl, 1-naphthylmethyl or 2-naphthylmethyl.

[0106] R 1 The specific value of is 3-indolylmethyl optionally substituted by hydroxy.

[0107] R 2 The specific value of is selected from the group consisting of (C1-C4) alkyl optionally substituted by hydroxy.

[0108] R 2 The specific value of is isobutyl or hydroxymethyl.

[0109] R 2 The specific value of is hydroxyethyl (e.g., 1-hydroxyethyl).

[0110] R 4 The specific value of is biphenyl optionally substituted by one or more groups independently selected from halogen, hydroxy, (C1-C6) alkyl and (C1-C6) alkoxy, wherein each (C1-C6) alkyl and (C1-C6) alkoxy is optionally substituted by one or more groups independently selected from the group consisting of halo groups.

[0111] R 4 The specific value of is phenoxyphenyl optionally substituted by one or more groups independently selected from the group consisting of halo, hydroxy, (C1-C6) alkyl and (C1-C6) alkoxy, wherein each (C1-C6) alkyl and (C1-C6) alkoxy is optionally substituted by one or more groups independently selected from the group consisting of halo groups.

[0112] R 4The specific value of is 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-hydroxyphenoxy)phenyl.

[0113] R 7 The specific value of is methyl optionally substituted by a hydroxyl group.

[0114] R 7 The specific value of is methyl.

[0115] R 8 The specific value of is selected from the group consisting of isopropyl and (C3-C6) cycloalkyl.

[0116] R 8 The specific value of is cyclohexyl.

[0117] R 9 The specific value of is benzyl.

[0118] R 12 The specific value of is H.

[0119] R 12 The specific value of is:

[0120]

[0121] R 12 The specific value of is:

[0122]

[0123] The specific value of m is 1. The specific value of m is 2. The specific value of m is 3.

[0124] The specific value of n is 1. The specific value of n is 2. The specific value of n is 3.

[0125] R 100 The specific value of is H, (C3-C6) cycloalkyl, or (C1-C3) alkyl.

[0126] R 101 The specific value of is H, (C3-C6) cycloalkyl, or (C1-C3) alkyl.

[0127] R 102 The specific value of is H, (C3-C6) cycloalkyl, or (C1-C3) alkyl.

[0128] R 100 The specific value of is H, (C3-C6) cycloalkyl, or (C1-C2) alkyl.

[0129] R 101The specific value of is H, a (C3-C6) cycloalkyl group, or a (C1-C2) alkyl group.

[0130] R 102 The specific value of is H, a (C3-C6) cycloalkyl group, or a (C1-C2) alkyl group.

[0131] R 100 The specific value of is H, methyl, ethyl, isopropyl, cyclohexyl or hydroxymethyl.

[0132] R 101 The specific value of is H, methyl, ethyl, isopropyl, cyclohexyl or hydroxymethyl.

[0133] R 102 The specific value of is H, methyl, ethyl, isopropyl, cyclohexyl or hydroxymethyl.

[0134] R 12 The specific value of is:

[0135]

[0136] R 12 The specific value of is:

[0137]

[0138] The specific value of m is 1. The specific value of m is 2. The specific value of m is 3.

[0139] The specific value of n is 1. The specific value of n is 2. The specific value of n is 3.

[0140] L 1 The specific value of is a branched or unbranched, saturated or unsaturated hydrocarbon chain having from about 1 to 100 carbon atoms, wherein one or more of said carbon atoms are optionally and independently replaced by -O-, -S-, -N(R a )-, a 3-7 membered heterocycle, a 5-6 membered heteroaryl or a carbocycle, and wherein each chain, 3-7 membered heterocycle, 5-6 membered heteroaryl or carbocycle is optionally and independently substituted by one or more (e.g., 1, 2, 3, 4, 5 or more) substituents selected from: (C1-C6) alkyl, (C1-C6) alkoxy, (C3-C6) cycloalkyl, (C1-C6) alkanoyl, (C1-C6) alkanoyloxy, (C1-C6) alkoxycarbonyl, (C1-C6) alkylthio, azido, cyano, nitro, halo, -N(R a )2, hydroxy, oxo(=O), carboxy, aryl, aryloxy, heteroaryl and heteroaryloxy, wherein each R a is independently H or (C1-C6) alkyl.

[0141] L 1The specific value of is a branched or unbranched, saturated or unsaturated hydrocarbon chain having from about 1 to 20 carbon atoms, wherein one or more of said carbon atoms are optionally and independently replaced by -O-, -S-, -N(R a )-, a 3- to 7-membered heterocycle, a 5- to 6-membered heteroaryl or a carbocycle, and wherein each carbon atom, 3- to 7-membered heterocycle, 5- to 6-membered heteroaryl or carbocycle is optionally and independently substituted by one or more (e.g., 1, 2, 3, 4, 5 or more) substituents selected from: (C1-C6)alkyl, (C1-C6)alkoxy, (C3-C6)cycloalkyl, (C1-C6)alkanoyl, (C1-C6)alkanoyloxy, (C1-C6)alkoxycarbonyl, (C1-C6)alkylthio, azido, cyano, nitro, halo, -N(R a )2, hydroxy, oxo(=O) and carboxy, wherein each R a is independently H or (C1-C6)alkyl.

[0142] L 1 The specific value of 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 and independently replaced by O, NH or a divalent triazine ring. Wherein each carbon atom is optionally and independently substituted by one or more (e.g., 1, 2, 3, 4, 5 or more) substituents selected from halo and oxo(=O).

[0143] L 1 The specific value of is:

[0144]

[0145] The specific value of D is a residue of a drug.

[0146] The specific value of D is a residue of an anti-cancer agent, which anti-cancer agent includes classical chemotherapeutic agents such as antimetabolites (e.g., gemcitabine), anti-mitotic agents (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, a taxane, an alkylating agent or a DNA damaging agent.

[0147] In certain embodiments, the antimetabolite is gemcitabine. In certain embodiments, the antimitotic agent is a taxane or 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 is a kinase inhibitor, such as erlotinib. In one embodiment, D is a residue of the EGRF inhibitors cetuximab, gefitinib or erlotinib. In one embodiment, D is a residue of the Her2 inhibitor trastuzumab. In one embodiment, D is a residue of the BRAF inhibitors vemurafenib or dabrafenib. In one embodiment, D is a residue of gemcitabine, 5FU or another antimetabolite, taxane, alkylating agent or DNA damaging agent.

[0148] A specific value of D is a residue of a taxane, which taxane includes paclitaxel, docetaxel, cabazitaxel.

[0149] A specific value of D is a residue of paclitaxel.

[0150] A specific value of D is a residue of a targeting agent.

[0151] R 12 The specific value of is:

[0152]

[0153] R 12 The specific value of is:

[0154]

[0155] The specific value of p is 1. The specific value of p is 2. The specific value of p is 3.

[0156] L 2 The specific value of is a branched or unbranched, saturated or unsaturated hydrocarbon chain having from about 1 to 100 carbon atoms, wherein one or more of said carbon atoms are optionally and independently replaced by -O-, -S-, -N(Ra)-, a 3-7 membered heterocycle, a 5-6 membered heteroaryl or a carbocycle, and wherein each chain, 3-7 membered heterocycle, 5-6 membered heteroaryl or carbocycle is optionally and independently substituted with one or more (e.g., 1, 2, 3, 4, 5 or more) substituents selected from: (C1-C6)alkyl, (C1-C6)alkoxy, (C3-C6)cycloalkyl, (C1-C6)alkanoyl, (C1-C6)alkanoyloxy, (C1-C6)alkoxycarbonyl, (C1-C6)alkylthio, azido, cyano, nitro, halo, -N(R a )2, hydroxy, oxo(=O), carboxy, aryl, aryloxy, heteroaryl and heteroaryloxy, wherein each Ra independently H or (C1 - -C6)alkyl.

[0157] L 2 has a specific value of a branched or unbranched, saturated or unsaturated hydrocarbon chain having from about 1 to 20 carbon atoms, wherein one or more of said carbon atoms are optionally independently replaced by -O-, -S-, -N(R a )-, a 3- to 7-membered heterocycle, a 5- to 6-membered heteroaryl or a carbocycle, and wherein each carbon atom, 3- to 7-membered heterocycle, 5- to 6-membered heteroaryl or carbocycle is optionally and independently substituted by one or more (e.g., 1, 2, 3, 4, 5 or more) substituents selected from: (C1-C6)alkyl, (C1-C6)alkoxy, (C3-C6)cycloalkyl, (C1-C6)alkanoyl, (C1-C6)alkanoyloxy, (C1-C6)alkoxycarbonyl, (C1-C6)alkylthio, azido, cyano, nitro, halo, -N(R a )2, hydroxy, oxo(=O) and carboxy, wherein each R a is independently H or (C1-C6)alkyl.

[0158] L 2 has a specific value of a branched or unbranched, saturated hydrocarbon chain having from about 3 to 110 carbon atoms (e.g., 3 to 10 carbon atoms), wherein each carbon atom is optionally and independently substituted by one or more (e.g., 1, 2, 3, 4, 5 or more) substituents selected from halo and oxo(=O).

[0159] L 2 has a specific value of -CH2C(=O)-, -CH2CH2C(=O)-, -CH2CH2CH2C(=O)-, -CH2CH2CH2CH2C(=O)- or -CH2CH2CH2CH2CH2C(=O)-.

[0160] Specific compounds or salts are selected from the group consisting of: tagafurine (Figure 1) or tagafurine conjugated to paclitaxel, or a tagafurine dimer, or a tagafurine dimer conjugated to paclitaxel:

[0161]

[0162]

[0163] n = 1, 2, 3

[0164]

[0165] and their salts.

[0166] In certain embodiments, the compound of formula (I) or a particular compound described herein is a homodimeric compound (e.g., tagafolin dimer).

[0167] A method for preparing a compound of formula I is provided as a further embodiment of the present invention.

[0168] When the compound is sufficiently basic or acidic, salts of the compound of formula I can be used as intermediates for the separation or purification of the compound of formula I. Additionally, it may be appropriate 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 toluenesulfonate, methanesulfonate, acetate, citrate, malonate, tartrate, succinate, benzoate, ascorbate, α-ketoglutarate, and α-glycerophosphate. Suitable inorganic salts can also be formed, including hydrochloride, sulfate, nitrate, bicarbonate, and carbonate.

[0169] 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 that provides 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.

[0170] The compound of formula I can be formulated into a pharmaceutical composition and administered to a mammalian host, such as a human patient, in a variety of forms suitable for the selected route of administration (i.e., orally or parenterally, by intravenous, intramuscular, topical, or subcutaneous routes).

[0171] Thus, the compounds of the present invention can be administered systemically, for example, orally, in combination with a pharmaceutically acceptable vehicle such as an inert diluent or an absorbable edible carrier. They can be encapsulated in hard or soft shell gelatin capsules, compressed into tablets, or incorporated directly into the food of 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, lozenges, troches, capsules, elixirs, suspensions, syrups, powder tablets, etc. Such compositions and preparations should contain at least 0.1% of the active compound. Of course, the percentages of the compositions and preparations can vary and can conveniently be between about 2% and about 60% of the weight of a given unit dosage form. The amount of the active compound in such therapeutically useful compositions is an amount such that an effective dosage level will be obtained.

[0172] Tablets, lozenges, pills, capsules, etc. may also contain the following substances: binders such as tragacanth, gum arabic, corn starch or gelatin; excipients such as dibasic calcium phosphate; disintegrants such as corn starch, potato starch, alginic acid, etc.; lubricants such as magnesium stearate; and sweeteners such as sucrose, fructose, lactose or aspartame, or flavoring agents such as peppermint, wintergreen oil or cherry flavor may be added. When the unit dosage form is a capsule, in addition to materials of the above types, it may also contain a liquid carrier such as vegetable oil or polyethylene glycol. Various other substances may be present as coatings or may be present to otherwise modify the physical form of the solid unit dosage form. For example, tablets, pills or capsules may be coated with gelatin, wax, shellac or sugar. Syrups or elixirs may contain the active compound, sucrose or fructose as a sweetener, methylparaben and propylparaben as preservatives, dyes and flavoring agents (such as cherry or orange flavor). Of course, any substance used in the preparation of any unit dosage form should be pharmaceutically acceptable and substantially non-toxic at the amounts used. In addition, the active compound may be incorporated into sustained release formulations and devices.

[0173] The active compound can also be administered intravenously or intraperitoneally by infusion or injection. Solutions of the active compound or its salts can be prepared in water, optionally mixed with a non-toxic surfactant. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, triacetin and its mixtures, and in oils. Under ordinary storage and use conditions, these preparations contain preservatives to prevent the growth of microorganisms.

[0174] Pharmaceutical dosage forms suitable for injection or infusion may include sterile aqueous solutions or dispersions or sterile powders containing the active ingredient, optionally encapsulated in liposomes, which are suitable for the extemporaneous preparation of sterile injectable or infusible solutions or dispersions. In all cases, the final dosage form should be sterile, fluid and stable under the conditions of manufacture and storage. The liquid carrier or vehicle can be a solvent or a liquid dispersion medium, which includes, for example, water, ethanol, polyols (such as glycerol, propylene glycol, liquid polyethylene glycols, etc.), vegetable oils, non-toxic glycerides and suitable mixtures thereof. Adequate fluidity can be maintained, for example, by forming liposomes, by maintaining the desired particle size in the case of dispersions, or by using surfactants. The action of microorganisms can be prevented by various antibacterial and antifungal agents (such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc.). In many cases, it will be preferable to include isotonic agents such as sugars, buffers or sodium chloride. Prolonged absorption of injectable compositions can be achieved by using agents that delay absorption (such as aluminum monostearate and gelatin) in the composition.

[0175] A sterile injectable solution is prepared by incorporating the required amount of the active compound in an appropriate solvent with the various other ingredients enumerated above as required, followed by filtered sterilization. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred method of preparation is vacuum drying and freeze-drying techniques that yield a powder of the active ingredient plus any additional required ingredients present in a previously sterile filtered solution.

[0176] For topical or subcutaneous administration, the compounds of the invention may be administered as a composition or formulation in combination with a dermatologically acceptable carrier.

[0177] Useful liquid carriers include water, alcohols, or diols or water-alcohol / diol blends in which the compounds of the invention may be optionally dissolved or dispersed at effective levels, with the aid of a non-toxic surfactant. Adjuvants such as fragrances and additional antimicrobial agents may be added to optimize the properties for a given use. The resultant liquid compositions may be applied from absorbent pads, for impregnating bandages and other dressings, or sprayed onto the affected area using a pump-type or aerosol sprayer.

[0178] Thickeners such as synthetic polymers, fatty acids, fatty acid salts and esters, fatty alcohols, modified celluloses or modified minerals may also be used with the liquid carrier to form a spreadable paste, gel, ointment, soap, etc. for direct application to the skin of the user.

[0179] The appropriate dosage of the compounds of formula I can be determined by comparing their in vitro activity and in vivo activity in animal models. Methods for extrapolating effective doses in mice and other animals to humans are known in the art; see, for example, U.S. Patent No. 4,938,949.

[0180] The amount of the compound, or its active salt or derivative, required for use in therapy will vary not only with the particular salt selected, but also with the route of administration, the nature of the disorder being treated and the age and condition of the patient, and will ultimately be at the discretion of the attending physician or clinician.

[0181] The required dosage may conveniently be presented in a single dose or as divided doses administered at appropriate intervals, such as two, three, four or more sub-doses per day. The sub-doses themselves may further be divided, for example, into multiple discrete, loosely spaced administrations; such as multiple inhalations from an inhaler or by multiple drops into the eye.

[0182] The compounds of the present invention can also be used in combination with other therapeutic agents, such as other agents that can be used to treat 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 (including other antimetabolites, taxanes, alkylating agents, and DNA-damaging agents). Thus, in one embodiment, the present invention also 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 material, and instructions for administering the compound of formula I or a pharmaceutically acceptable salt thereof and one or more other therapeutic agents to an animal for treating cancer.

[0183] Linking group L 1 and L 2

[0184] The compound of formula (I) may comprise a linking group L 1 and L 2 . In one embodiment, the linking group is absent. The linking group can vary in length and atomic composition and can be, for example, branched or unbranched or cyclic or a combination thereof. The linking group can also modulate the properties of the final compound of formula (I), such as solubility, stability, or aggregation.

[0185] In one embodiment, the linker comprises from about 3 to 100 atoms. In one embodiment, the linker comprises from about 3 to 90 atoms. In one embodiment, the linker comprises from about 3 to 80 atoms. In one embodiment, the linker comprises from about 3 to 70 atoms. In one embodiment, the linker comprises from about 3 to 60 atoms. In one embodiment, the linker comprises from about 3 to 50 atoms. In one embodiment, the linker comprises from about 3 to 40 atoms. In one embodiment, the linker comprises from about 3 to 30 atoms. In one embodiment, the linker comprises from about 3 to 20 atoms. In one embodiment, the linker comprises from about 3 to 10 atoms.

[0186] In one embodiment, the linker comprises atoms selected from H, C, N, S, and O.

[0187] In one embodiment, the linker comprises atoms selected from H, C, N, and O.

[0188] In one embodiment, the linker comprises a branched or unbranched, saturated or unsaturated hydrocarbon chain having from about 1 to 100 (1 - 90, 1 - 80, 1 - 70, 1 - 60, 1 - 50, 1 - 40, 1 - 30, 1 - 20 or 1 - 10) carbon atoms, wherein one or more of said carbon atoms are optionally and independently replaced by -O-, -S-, -N(R a )-, a 3 - 7 membered heterocycle, a 5 - 6 - membered heteroaryl or a carbocycle, and wherein each chain, 3 - 7 membered heterocycle, 5 - 6 membered heteroaryl or carbocycle is optionally and independently substituted by one or more (e.g., 1, 2, 3, 4, 5 or more) substituents selected from: (C1 - C6)alkyl, (C1 - C6)alkoxy, (C3 - C6)cycloalkyl, (C1 - C6)alkanoyl, (C1 - C6)alkanoyloxy, (C1 - C6)alkoxycarbonyl, (C1 - C6)alkylthio, azido, cyano, nitro, halo, -N(R a )2, hydroxy, oxo(=O), carboxy, aryl, aryloxy, heteroaryl and heteroaryloxy, wherein each R a is independently H or (C1 - C6)alkyl.

[0189] In one embodiment, the linker comprises a branched or unbranched, saturated or unsaturated hydrocarbon chain having from about 1 to 20 carbon atoms, wherein one or more of said carbon atoms are optionally and independently replaced by -O-, -S-, -N(R a )-, a 3 - 7 membered heterocycle, a 5 - 6 - membered heteroaryl or a carbocycle, and wherein each carbon atom, 3 - 7 membered heterocycle, 5 - 6 membered heteroaryl or carbocycle is optionally and independently substituted by one or more (e.g., 1, 2, 3, 4, 5 or more) substituents selected from: (C1 - C6)alkyl, (C1 - C6)alkoxy, (C3 - C6)cycloalkyl, (C1 - C6)alkanoyl, (C1 - C6)alkanoyloxy, (C1 - C6)alkoxycarbonyl, (C1 - C6)alkylthio, azido, cyano, nitro, halo, -N(R a )2, hydroxy, oxo(=O) and carboxy, wherein each R a is independently H or (C1 - C6)alkyl.

[0190] The present invention will now be illustrated by the following non - limiting examples.

[0191] Examples

[0192] Example 1. Chemical composition and mass spectrometry data of representative compounds.

[0193] The compounds shown in Table 1 below were prepared using procedures similar to those described herein or using known and available methods and materials.

[0194] Table 1.

[0195]

[0196]

[0197] All compounds were analyzed using an Agilent 6545 QTOF LC / MS instrument.

[0198] Example 2.

[0199] The chemical structures and dissociation constants of the EphA2 binders are shown. For each compound, we report the Kd value obtained by ITC against the EphA2 ligand-binding domain (LBD) and the IC 50 value.

[0200] Table 2. Chemical structures and dissociation constants of EphA2 binders containing the best substituents from previous SAR. For each compound, we report the Kd value obtained by ITC, as well as the IC 50 value.

[0201]

[0202] Example 3. Synthetic scheme for the synthesis of tagifolin.

[0203] 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. React at 90 °C for 5 min in a microwave-assisted Liberty Blue peptide synthesizer. (b) Deprotect Fmoc twice for 3 min with 20% N-methylpiperidine in DMF at 90 °C in a microwave-assisted Liberty Blue peptide synthesizer; (c) Peptide growth using the previous conditions on the Liberty Blue system; (d) TFA / TIS / water / phenol (94:2:2:2), 5 h at room temperature.

[0204]

[0205] Example 4. Synthetic scheme for the synthesis of tagifolin.

[0206] 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. React at 90 °C for 5 minutes in a microwave-assisted Liberty Blue peptide synthesizer; (b) Perform Fmoc deprotection twice for 3 minutes each with 20% N-methylpiperidine in DMF in a microwave-assisted Liberty Blue peptide synthesizer at 90 °C; (c) Use the previous conditions for peptide growth, but use twice the equivalents for dimer growth: 6 equivalents of Fmoc-amino acid, 6 equivalents of DIC, 2 equivalents of OximaPure, in 4.5 mL of DMF. React at 90 °C for 5 minutes in a microwave-assisted Liberty Blue peptide synthesizer; (d) TFA / TIS / water / phenol (94:2:2:2), react at room temperature for 5 hours.

[0207]

[0208] Example 5. Synthetic scheme for the synthesis of the compound tagifolin motif (an intermediate for the synthesis of tagifolin-paclitaxel).

[0209] Conditions: (a) Rink amide resin + 3 equivalents of Fmoc-Lys(ivDde)-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; (b) Deprotect Fmoc twice with 20% piperidine in DMF; (c) 3 equivalents of Fmoc-Gly-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; (d) Use the previous conditions or LibertyBlue for peptide growth; (e) Deprotect ivDde with 4% N2H2 in DMF (3 × 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.

[0210]

[0211] Example 6. Synthetic scheme for the synthesis of the compound tagifolin dimer motif (an intermediate for the synthesis of tagifolin dimer-paclitaxel).

[0212] Conditions: (a) Rink amide resin + 3 equivalents of Fmoc-Lys(ivDde)-OH, 3 equivalents of HATU, 3 equivalents of OximaPure, and 5 equivalents of DIPEA in 1 mL of DMF for 1 hour at room temperature. (b) Deprotect Fmoc twice with 20% N-methylpiperidine in DMF. (c) 3 equivalents of Fmoc-Gly-OH, 3 equivalents of HATU, 3 equivalents of OximaPure, and 5 equivalents of DIPEA in 1 mL of DMF for 1 hour at room temperature. (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 and react for 1 hour at room temperature. (e) Use twice the equivalent amount of peptide growth for dimer growth: 6 equivalents of Fmoc-amino acid, 6 equivalents of DIC, 2 equivalents of OximaPure in 4.5 mL of DMF. React in a microwave-assisted Liberty Blue peptide synthesizer at 90 °C for 5 minutes. (f) Perform ivDde deprotection using 4% N2H2 in DMF (3 × 5 mL) at room temperature; (g) 3 equivalents of 5-hexynoic acid, 3 equivalents of HATU, 3 equivalents of OximaPure, and 5 equivalents of DIPEA in 1 mL of DMF for 1 hour at room temperature; (h) TFA / TIS / water / phenol (94:2:2:2) for 5 hours at room temperature.

[0213]

[0214] Example 7. Synthetic scheme for synthesizing tagifolin-PTX.

[0215] Conditions: (a) Crude tagifolin motif, 1 equivalent of PTX-azide in 4 mL of 4:1 DMSO:aqueous solution. Add 50 μL of 1 M CuSO4 and 50 μL of 1 M sodium ascorbate.

[0216] Mix at room temperature for 48 hours.

[0217]

[0218] Example 8. Synthetic scheme for synthesizing tagifolin dimer-PTX.

[0219] Conditions: (a) Crude tagifolin dimer motif, 1 equivalent of PTX-azide in 4 mL of 4:1 DMSO:aqueous solution. Add 50 μL of 1 M CuSO4 and 50 μL of 1 M sodium ascorbate. Mix at room temperature for 48 hours.

[0220]

[0221] Example 9. Tagfurin monomer functions as an antagonist.

[0222] Figure 2A A Western blot of BxPC3 cells is shown. The cells were starved for 1 hour and pretreated with different concentrations of tagfurin for 20 minutes, followed by treatment with 2 μg / mL ephrin A1-Fc conjugate for 3 hours. Figure 2B Quantification of EphA2 levels is shown. The EphA2 / β-actin ratio was normalized by designating the EphA2 expression in DMSO without ephrin A1-Fc condition as 1. EC 50 values were calculated to be 1.6 ± 0.1 μM and are presented as the mean ± standard error (SE) of 2 independent experiments.

[0223] Example 10. Chemical structure of the dimeric EphA2 binder.

[0224] IC 50 values were obtained by performing repeated DELFIA measurements on EphA2-LBD.

[0225]

[0226] Table 3. Chemical structure of the dimeric EphA2 binder. IC 50 values were obtained by performing repeated DELFIA measurements.

[0227] Example 11. Tagfurin dimer and its variants cause EphA2 degradation in pancreatic cancer cell lines at nanomolar concentrations.

[0228] Figures 3A - 3C Western blot images of BxPC3, PANC-1, and MIAPaCa-2 cells are shown, respectively, where the cells were starved for 1 hour and treated with 2 μg / mL ephrin A1-Fc or indicated doses of tagfurin, tagfurin dimer, and its variants with different linkers for 3 hours. Dimer 135H12 (see US20210221843A1; PCTWO2019237075A1; and Gambini et al. ACS Chem Biol. 2018, 13(9), 2633–2644) is shown as a reference. Figures 3D - 3F Densitometric analysis of the data shown in Figures 3A - 3C is shown, respectively. The EphA2 / β-actin ratio was normalized by designating the EphA2 expression from the DMSO control condition as 100% (for Figures 3A - 3C ) or 1 (for Figures 3D - 3F ). ***p < 0.001, ****p < 0.0001, as determined by one-way ANOVA using Dunnett's post hoc test.

[0229] Example 12. Chemical Structure and Biochemical Activity of Tagafolin Conjugates.

[0230] Figure 4A The chemical structure of tagafolin conjugated to paclitaxel (tagafolin-PTX) is shown. Figure 4B The dimeric form of tagafolin conjugated to paclitaxel (tagafolin dimer-PTX) is shown. Figure 4C The dimeric form of tagafolin conjugated to 5-carboxytetramethylrhodamine-azide dye (tagafolin dimer-TAMRA) is shown. Figure 4D DELFIA displacement dose-response curves comparing the IC 50 values of tagafolin-PTX, tagafolin dimer-PTX, and tagafolin dimer-TAMRA and their respective IC

[0231] Example 13. Internalization of Tagafolin Dimer-TAMRA in Cells Expressing EphA2.

[0232] BxPC3 cells were treated with 100 nM tagafolin dimer-TAMRA for 0, 30, and 60 minutes. Once the agent binds, EphA2 is internalized and targeted to lysosomes, as demonstrated by the co-localization of 5-TAMRA and LAMP1 (arrows). See Figure 5 . Scale bar = 10 μm.

[0233] Example 14. Tagafolin Dimer Significantly Inhibits Pancreatic Cancer Cell Migration.

[0234] Figure 6A Cell migration assays of BxPC3 treated with 2 μg / mL ephrin A1-Fc and 10 μM tagafolin or indicated doses of tagafolin dimer are shown. The plates were imaged every 3 hours for 24 hours. Figure 6B Tagafolin dimer significantly inhibits cell migration in a dose-dependent manner at 24 h, as demonstrated by the decrease in relative wound density. Figure 6C Time-response curves showing the effect of these agents on wound closure over a 24-h period are shown. ***p < 0.001, ****p < 0.0001, as determined by one-way ANOVA analyzed using Dunnett's post-test. Scale bar = 250 μm.

[0235] Example 15. PTX Conjugation Inhibited Tumor Growth in Tumor Xenografts with MIA-PaCa-2 Cells.

[0236] Figure 7AShows the situation of five groups of five mice bearing pre-established MIA-PaCa-2 tumors treated with separate vehicle control, paclitaxel (PTX; 2.5 mg / kg), tagifolin-PTX (10 mg / kg, which is equivalent to 2.5 mg / kg PTX), tagifolin dimer-PTX (17 mg / kg, which is equivalent to 2.5 mg / kg PTX), and a lower dose of tagifolin dimer-PTX (10 mg / kg, which is equivalent to 1.5 mg / kg PTX) for 22 days. Tumor volumes are reported as mean ± SE. Figure 7B Shows the mean tumor volume of each treatment group measured at days 0, 8, 15, and 22. *p = 0.03, **p < 0.01, ***p = 0.0001, ****p < 0.0001, as determined by two-way ANOVA analyzed using Tukey's post hoc test. Figure 7C Shows the mean body weight ± SE reported for each of the five treatment groups at days 0, 8, 15, and 22.

[0237] Example 16. Representative pharmaceutical dosage forms

[0238] The following illustrates representative pharmaceutical dosage forms containing the compound of formula (I) ('Compound X') for therapeutic or prophylactic use in humans. The IV injection formulation consists of 80% PBS, 10% Tween 80, and 10% ethanol containing the agent dissolved at up to 20 mg / ml. This formulation and several variants thereof can be obtained by conventional procedures known in the pharmaceutical art.

[0239] Example 17. Synthetic scheme of tagifolin dimer-TAMRA.

[0240] Conditions: (a) Crude tagifolin dimer motif, 1 equivalent of 5-TAMRA-azide, in 4 mL of 4:1 DMSO:aqueous solution. Add 50 μL of 1 M CuSO4 and 50 μL of 1 M sodium ascorbate. Mix at room temperature for 48 h.

[0241]

[0242] Example 18. Tagifolin: A potent agent targeting the ligand-binding domain of EphA2

[0243] Overexpression of the receptor tyrosine kinase EphA2 is associated with poor prognosis and development of invasive metastatic cancers. Guided by the X-ray structure of the complex between an agonist peptide and EphA2-LBD that we recently resolved, this example describes a novel agent, tagifolin, which binds to EphA2-LBD with a dissociation constant of 21 nM according to isothermal titration calorimetry and exhibits an IC of 10.8 nM in biochemical assays50 Value. In cell-based assays, the dimeric form of the agent induces receptor internalization and degradation in several pancreatic cancer cell lines as effectively as the native dimeric ligand (ephrin A1-Fc). In mouse xenograft studies, when conjugated with a chemotherapy agent, the agent can effectively deliver paclitaxel to pancreatic cancer. Given the key role of EphA2 in tumor progression, the agent reported herein can be further developed into an innovative EphA2-targeted therapeutic.

[0244] Introduction

[0245] The receptor tyrosine kinase EphA2 functions as a tumor suppressor in its ephrin-bound form, preventing cancer cell migration, tumor growth, and angiogenesis. In contrast, when the receptor is in its unbound state, such as when it is abnormally overexpressed, it confers pro-cancer properties to cancer cells, inducing metastatic behavior in several solid tumors, including pancreatic cancer, 2-4 prostate cancer, 5-7 breast cancer, 8-10 esophageal cancer, 11 12 melanoma, 13 bladder cancer, 14 brain cancer, 15-17 lung cancer, 18 ovarian cancer, 19 gastric cancer, 20 and certain types of leukemia. 2 1-24 Thus, due to the role of EphA2 as a tumor suppressor, targeting EphA2 is becoming the goal of developing various possible therapeutic strategies, including targeting its intracellular kinase domain 25-28 or its ligand-binding domain. 29,30 Although the unbound EphA2 receptor functions as a potent oncogene, its tumorigenic effects can be inhibited and even reversed by synthetic agents that mimic its ligand (membrane-anchored ephrin A1). 31

[0246] In cell assays, when the chimeric protein ephrin A1-Fc, composed of ephrin A1 and the antibody Fc region, binds to the EphA2 ligand-binding domain (LBD), it causes receptor dimerization, followed by aggregation and internalization, leading to receptor degradation via the lysosomal pathway. 32Thus, since ephrinA1-Fc can, in principle, reverse the pro-oncogenic gene EphA2 into a tumor suppressor, the design of potent and effective ephrinA1-Fc mimetics has the potential to develop novel anti-metastatic therapeutics. Since such agents may cause receptor internalization, they can additionally be deployed as carrier molecules for the selective targeted delivery of chemotherapeutic drugs to EphA2-expressing cancers. In this regard, we recently developed an EphA2 dimeric agonistic peptide mimetic, which, similar to ephrinA1-Fc, can inhibit tumor metastasis in an orthotopic model of prostate cancer, 33 and cell migration in pancreatic cancer cell lines. 34 When certain early agents were conjugated with the chemotherapeutic agents gemcitabine 35 or paclitaxel 8,36-37 they delivered their cargo to EphA2-expressing tumors, which included pancreatic cancer, 35 prostate cancer, 36-37 breast cancer, 8,38 and melanoma. 38 Recently, we solved for the first time the X-ray structure of an agonistic ephrin peptide mimetic complexed with EphA2-LBD. 1 Using our laboratory's previous structure-activity relationship studies on previous peptide binders 1,37-38 and high-resolution X-ray structures 1 here we sought to further derive agents that could approach the affinity and activity of ephrinA1-Fc in targeting EphA2-LBD. As shown herein, we identified a novel agent with low nanomolar affinity for EphA2-LBD, which has an affinity for this receptor comparable to that of ephrinA1. In cellular assays, the dimeric form of our most potent agent (which we call tagfurin) induced receptor degradation at nanomolar concentrations, similar to the effect of ephrinA1-Fc, as evaluated by Western blot analysis in the pancreatic cancer cell lines BxPC3, PANC-1, and MIAPaCa2, which represent KRAS wild-type (BxPC3) and KRAS mutant (PANC-1 and MIAPaCa2) tumors. In phenotypic assays, these agents also potently inhibited cell migration in the BxPC3 pancreatic cancer cell line. When conjugated with paclitaxel, the agent effectively inhibited tumor growth in the MIA PaCa2 xenograft model of pancreatic cancer. The extraordinary affinity of tagfurin for the ligand-binding domain of EphA2 makes this agent an unprecedented pharmacological tool for studying this receptor tyrosine kinase and for developing novel therapeutics and / or targeted delivery strategies.

[0247] Results

[0248] Design, synthesis, and characterization of tagfurin

[0249] To rapidly and repeatedly characterize the binding properties of the novel EphA2-binding ligands (Table 4), we performed isothermal titration calorimetry binding measurements using the recombinant EphA2 ligand-binding domain (LBD). Figure 11A An early agent complexed with EphA2-LBD (PDB ID 6B9L) is shown. 1 We used a ligand (K 39 with the sequence YSAYPDSVPFRP that combines the sequence of a phage-display-derived YSA peptide d with that of a native ephrin ligand (K 1 1230 nM, ITC; Table 4, compound 1) to initiate exploration of optimization strategies (Figure 11). First, we probed substitutions that could project into a large hydrophobic pocket located near Tyr 4 of the peptide ( Figures 11A - 11B , Table 4). Here, substitution of the Tyr residue at position 4 with a bulkier aromatic group significantly enhanced the affinity (Table 4). Thus, subsequently, we fixed Phe at position 4 of the peptide and explored modifications at other positions.

[0250]

[0251] Table 4. Structure-activity relationship studies at position 4 for reported EphA2 binders. Chemical structures and dissociation constants are reported. K d values were obtained by reverse isothermal titration calorimetry measurements.

[0252] These included modifications to the N-terminal amide ( Figure 11A , 11C ; Table 5), a position that may be vulnerable not only to aminopeptidases in plasma but also involved in ligand recognition. 1,37-38 Substitution of the amino group with piperazine or morpholine increased the binding affinity for EphA2-LBD (Table 5). Additionally, we explored additional modifications, including modification of the Tyr residue at position 1, and modification of the Ser residue pair (Table 5). We found that Tyr 1 could be substituted with a variety of substituents to eliminate the potential pharmacological liability represented by the phenolic hydroxyl group (Table 5). The peptide adopts a closed conformation in its bound state, in which two Ser residues form an intramolecular hydrogen bond in the initial peptide ( Figure 11A , 11D ). We further explored additional modifications of this amino acid pair and evaluated their effects on the binding affinity for EphA2-LBD via ITC measurements (Table 5).

[0253] Finally, a set of peptides was synthesized that contained the optimal substituents from the agents reported in Tables 4 and 5, leading to the final agents listed in Table 2. For these compounds, we also evaluated their binding properties using an orthogonal biochemical displacement assay based on the DELFIA platform, as previously described. 1

[0254]

[0255]

[0256] Table 5. Chemical structures and dissociation constants of EphA2 binders. K d values were obtained by measuring via reverse isothermal titration calorimetry.

[0257] These studies ultimately selected agent 27, which we herein call tagfurin, with an IC 50 value of 10.8 nM for EphA2-LBD (Table 2). Figure 1 reports a molecular model based on the X-ray structure of the complex of tagfurin with EphA2-LBD in complex with one of our earlier peptidomimetics (PDB ID 6B9L). 1 To obtain a preliminary but important snapshot of the selectivity of tagfurin for EphA2-LBD compared to other members of this protein family, we tested tagfurin against the ligand-binding domains of EphA3 and EphA4, which are the two Eph receptors with the highest similarity to EphA2 (58% identity with EphA3-LBD and 57% identity with EphA4-LBD). When tested under similar experimental conditions, the ligand was inactive against both domains ( Figure 1D ).

[0258] Monomeric peptides are only likely to elicit agonistic activity at very high concentrations and actually act as antagonists at physiologically achievable concentrations. 8,34,40 Consistent with the high affinity of tagfurin for EphA2, pretreatment of BxPC3 pancreatic cancer cells with tagfurin effectively antagonized EphA2 degradation induced by the potent ephrin A1-Fc ligand, with an EC 50 of approximately 1.6 μM under these experimental conditions (Figure 2).

[0259] The agent alone did not induce significant EphA2 degradation in BxPC3 pancreatic cancer cells (Figure 3).

[0260] However, while monomeric peptides act as antagonists, similar to ephrin A1, dimerization of EphA2-targeting agents may lead to increased agonistic activity of the compounds in cells. 1,8,33-34,41-42This may be due to the fact that enhanced dimerization may promote subsequent receptor clustering and internalization. 8,42 We prepared dimeric forms of tagafalin using Lys residues as dimerization linkers separated by Gly, β-Ala, or γ-aminobutyric acid at the C-terminus of tagafalin (Table 3). Table 3 also reports the dimerizer 135H12 that we previously identified. 1

[0261] Tagafalin dimers and tagafalin drug conjugates

[0262] An interesting property of agonists is that they induce internalization of the EphA2 receptor via the lysosomal pathway, leading to its degradation. Thus, potent agonists can induce EphA2 degradation, thereby eliminating its pro-cancer effects. Agonist-induced EphA2 internalization does not necessarily affect cell proliferation, as is the case in Figure 12. However, due to lysosomal internalization events, EphA2 agonists can be used for targeted delivery of cytotoxic chemotherapy drugs by synthesizing appropriate peptide-drug conjugates (PDCs). Therefore, to evaluate the EphA2 internalization and degradation properties of our agents, we tested them in a variety of pancreatic cancer cell lines and used dimerized ephrin A1-Fc as a positive control. As reported above, when tested at nanomolar concentrations, the monomeric form of tagafalin was inactive in causing EphA2 degradation, consistent with our previous observations (i.e., monomeric peptides are only agonistic at higher micromolar concentrations). This appears to be the case for all 3 cell lines tested (i.e., BxPC3, PANC-1, and MIA PaCa2) (Figure 3).

[0263] However, the dimeric forms of tagafalin showed significantly increased receptor activation, especially the dimer with a Gly-Lys linker (Table 3), resulting in receptor degradation at submicromolar concentrations in all pancreatic cancer cell lines tested (Figure 3). In addition, our new agents were significantly more effective than the previously reported dimerizer 135H12 (Table 3, Figure 3). 1

[0264] To evaluate the utility of tagafalin and tagafalin 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 the "click chemistry" linker that we previously described, which allows for the efficient incorporation of drugs or imaging reagents into dimeric or monomeric agents (Examples 7, 8, 17). 1,8 Conjugation of the dimerizer with TAMRA or paclitaxel did not significantly alter their binding properties to isolated EphA2-LBD Figure 4D), while a more significant loss of binding affinity was observed with tagfurine monomer-PTX, which may be due to the shorter linker selected.

[0265] Immunofluorescence microscopy data using BxPC3 cells showed punctate cytoplasmic fluorescence co-localized with the lysosomal marker LAMP-1 in cells treated with tagfurine dimer-TAMRA ( Figure 5 ), which confirmed the EphA2-specific lysosomal internalization event triggered by this agonist. Indeed, tagfurine dimer-PTX retained the ability to cause EphA2 degradation in all three tested pancreatic cancer cell lines (Figure 8).

[0266] In contrast, tagfurine monomer-PTX alone did not cause receptor internalization. These data clearly identified tagfurine as a potent EphA2-LBD binder with antagonistic activity, while tagfurine dimer showed a similar potent affinity for isolated EphA2-LBD, but it also showed potent EphA2 degradation activity in pancreatic cancer cells.

[0267] Finally, to determine whether our EphA2 agonist could block the cell motility of pancreatic cancer cells, we performed a cell migration assay using the scratch wound method as detected by time-lapse live cell analysis (IncuCyte S3, Sartorius) of the pancreatic cancer cell line BxPC3. We reported that in BxPC3, knocking out EphA2 alone led to a significant reduction in cell migration in this assay. 34 Similarly, treating BxPC3 cells with increasing concentrations of tagfurine dimer significantly inhibited cell migration (Figure 6). These data concluded that tagfurine and tagfurine dimer are potent EphA2 antagonists and agonists, respectively.

[0268] In vivo pharmacology and mouse xenograft studies

[0269] A preliminary pharmacokinetic study was performed on this agent after administering a single dose of tagfurine dimer at 50 mg / Kg via the tail vein and measuring plasma drug concentrations over time ( Figure 9 ). The data showed that the C max reached by this agent was much higher than the 100 - 200 nM required to induce EphA2 degradation in cells, and the estimated t1 / 2 was approximately 15 hours, indicating that lower drug concentrations could be used for subsequent in vivo efficacy studies. Blood chemistry analysis performed after this high dose of tagfurine dimer did not find any abnormalities in the blood chemistry panel (e.g., albumin, ALP, ALT, amylase, bilirubin, Ca, P, Na +, K + Any value of (total protein, globulin, creatinine, blood urea nitrogen, glucose) changed significantly. In another preliminary in vivo toxicity study, Balb / C mice were administered repeated doses (daily for 5 days) of PTX (8 mg / Kg), tagafolin dimer (50 mg / Kg), or tagafolin dimer-PTX (50 mg / Kg); thus, each group received an equivalent dose of PTX. Two out of three mice receiving PTX were found dead after the second dose, and the remaining mice appeared lethargic and were found dead by day 5. In contrast, no signs of adverse toxicity were observed in the groups treated with tagafolin dimer or tagafolin dimer-PTX (mice in the latter group appeared lethargic after the first day but recovered). Body weight was monitored during the experiment (Table 6). These preliminary data indicate that tagafolin is well tolerated and can selectively deliver PTX to EphA2-expressing tumor cells.

[0270]

[0271]

[0272] Table 6. Repeated-dose toxicity study of tagafolin dimer-PTX compared to PTX alone. Balc / c mice received equimolar doses of PTX or tagafolin dimer-PTX daily (IV), and body weight was measured daily. FD = found dead. By day 5, all three mice in the group treated with PTX were found dead. Mice treated with tagafolin dimer-PTX were lethargic after the first dose but recovered. No signs of toxicity were observed in mice treated with tagafolin dimer.

[0273] Therefore, to further evaluate the ability of the drug conjugate to direct chemotherapeutic agents to pancreatic cancer in vivo, we evaluated the ability of the agent to inhibit tumor growth in tumor xenografts with MIAPaCa-2 cells. First, MIAPaCa-2 cells (1.0 × 10 7 cells / mouse) in 100 μL PBS were injected into the right flank of five nu / nu mice to obtain tumor stock fragments. Subsequently, 1 mm was transplanted into the right flank of 25 mice 3MIAPaCa-2 tumor fragments, tumor growth was measured with calipers 18 days after tumor implantation, and mice were grouped for treatment on days 1, 4, 8, 11, 15, and 18. The agent was dissolved in a formulation having 80% PBS, 10% Tween 80, and 10% ethanol. Each of the 5 groups received either a separate vehicle control, paclitaxel (PTX; 2.5 mg / Kg), tagafolin-PTX (10 mg / Kg, which is equivalent to 2.5 mg / Kg of PTX), tagafolin dimer-PTX (17 mg / kg, which is equivalent to 2.5 mg / Kg of PTX), and a lower dose of tagafolin dimer-PTX (10 mg / kg, which is equivalent to 1.5 mg / kg of PTX). Both tagafolin-PTX and tagafolin dimer-PTX showed significant anti-tumor effects compared to the untreated and PTX-treated groups (Figure 7). In addition, even the group treated with a sub-stoichiometric dose of PTX became more effective than the PTX-treated group (Figure 7), although the standard deviation of the PTX-treated group was too large to assess significance. These data together suggest that the agent is capable of delivering the drug to EphA2-expressing tumors.

[0274] Discussion and conclusions

[0275] In recent years, we have witnessed continuous efforts to develop novel therapeutics by targeting EphA2 through various strategies. 43 These strategies include computational docking strategies, 31,44-46 NMR-based screening, 40,47-48 high-throughput screening, 49 phage display screening, 39 and these efforts have led to the generation of potential small molecule compounds, 31,44,8 or EphA2 / ephrin antagonists. 45-46,49-51,52 However, none of the agents listed are mature enough to be used as potential therapeutics. Instead, targeting EphA2 with mAbs has been proposed, but mAbs have performed poorly clinically, with reduced selectivity or extended half-life, leading to accumulation of the agent in undesired tissues. 53 In fact, a recent Phase I clinical study aimed to evaluate the biodistribution of the anti-EphA2 antibody DS-8895a in patients with advanced EphA2-positive cancers. 54 Although encouragingly no treatment-related toxicities were reported, the therapeutic efficacy of DS-8895a was limited, likely due to the low tumor uptake rate observed, which led to the discontinuation of further development of DS-8895a. 54

[0276] Recently, Bicycle Therapeutics reported a peptide antagonist that binds to EphA2-LBD with a dissociation constant in the low nanomolar range. 55 The antagonist is conjugated to monomethyl auristatin via a protease-cleavable linker and is currently in Phase I clinical trials (clinicaltrials.gov / ct2 / show / NCT04180371). While this agent holds great promise for being the first to translate an EphA2-targeting agent into a potential therapeutic, tagafurine and tagafurine dimer offer effective alternative strategies for Bicycle Therapeutics agents. First, tagafurine has a similar affinity for EphA2 as the Bicycle Therapeutics compound but with a reduced molecular weight, which presumably enhances its tissue penetration; second, tagafurine dimer can induce the active internalization of the receptor and act as an effective EphA2 degrader; thus, it can be deployed as an effective EphA2-based therapeutic for inhibiting cell migration as an alternative to agonist antibodies (Figure 6). Therefore, we envision that tagafurine dimer can be deployed as an EphA2 degrader for inhibiting the metastatic behavior of cancer cells (Figure 13), as we recently demonstrated using the early agent 135H12 in an orthotopic prostate cancer model. 33

[0277] Furthermore, in drug conjugates, the active internalization induced by tagafurine dimer does not require the cleavage of extracellular linkers and the passive diffusion of cargo, potentially increasing the distribution of chemotherapeutic agents to EphA2-expressing tumor cells. We observed that an early dimeric EphA2-targeting agent conjugated to 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 the monomeric and dimeric forms of tagafurine (Figure 7).

[0278] Combined with the preliminary toxicity and pharmacokinetic studies we reported, we propose to deploy the dimer as a single agent or in combination with the standard of care for inhibiting EphA2 in cancer cells. In addition, the preliminary studies with drug conjugates should encourage further evaluation of such agents, especially when the conjugation is with tagafurine dimer to take advantage of the active internalization provided by this agent to tumors overexpressing EphA2.

[0279] In summary, the agents reported herein open up a wide range of opportunities for EphA2-targeted therapeutic development ranging from more effective PDCs to diagnostic agent development, or for designing more effective combination therapies against tumor metastasis.

[0280] Experimental section.

[0281] Chemical reaction

[0282] General rules. All reagents and solvents were obtained from commercial sources, including Fmoc-protected amino acids and resins for solid-phase synthesis. All peptides were synthesized in-house using a Liberty Blue peptide synthesizer (CEM) on Rink amide resin via 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. The coupling reaction was carried out in a microwave reactor at 90 °C for 5 minutes. Fmoc deprotection was carried out by treating the resin-bound peptide with 20% N-methylpiperidine in DMF (2 × 3 mL) at 90 °C for 3 min. The peptide was cleaved from the resin using a cleavage mixture containing TFA / TIS / H2O / phenol (94:2:2:2) for 5 h (see Example 3). The cleavage solution was filtered from the resin, and the peptide was precipitated in Et2O, centrifuged, and dried under high vacuum. Solution 1 1H NMR was used to check the concentration, and spectra were recorded on a Bruker Avance III 700 MHz. High-resolution mass spectrometry data were acquired on an Agilent LC-TOF instrument. RP-HPLC purification was carried out on a JASCO preparative system equipped with a PDA detector and a fraction collector controlled by a ChromNAV system (JASCO) on an XTerra C18 10 μ 10 x 250 mm (Waters). The purity of the tested compounds was evaluated by HPLC using an Atlantis T3 3 μm 4.6 × 150 mm2 column (H2O / ACN gradient from 5% to 100% in 45 min). The purity of all compounds was >95%

[0283] Preparation of dimerizer and tagifolin dimer. The preparation of the dimerizer was carried out according to the above procedure, but the equivalents used for each coupling were doubled and Fmoc-Lys(Fmoc)-OH was introduced as the first amino acid in the sequence, as exemplified in Example 4.

[0284] Preparation of tagforin motif and tagforin dimer motif. To prepare the tagforin motif and tagforin dimer motif, we introduced the Fmoc-Lys(ivDde)-OH amino acid as the first amino acid coupled to Rink amide resin. Subsequently, the peptide was grown according to a solid-phase synthesis protocol similar to that described previously by us. At the completion of the synthesis, the fully protected peptide on the Rink amide resin was treated with a solution of 4% hydrazine in DMF (3 × 5 mL, 30 min each) to remove the ivDde protecting group and then washed with DMF (3 × 5 mL). It was then coupled with 3 equivalents of 5-hexynoic acid in 1 mL of DMF in the presence of 3 equivalents of HATU, 3 equivalents of OximaPure and 5 equivalents of DIPEA at room temperature for 1 hour. The resin was then washed with DMF (3 × 5 mL) and DCM (3 × 5 mL), dried in vacuo and cleaved with a cleavage cocktail containing TFA / TIS / H2O / phenol (94:2:2:2) for 5 h. The synthesis protocols for the tagforin motif and tagforin dimer motif are reported in Example 5 and Example 6, respectively.

[0285] Preparation of tagforin-PTX, tagforin dimer-PTX, tagforin dimer-TAMRA. The crude tagforin motif was dissolved together with 1 equivalent of PTX-azide in a stirred solution of 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 at room temperature for 48 hours (Example 7). Tagforin dimer-PTX was obtained as described above, but using the crude tagforin dimer motif as the starting point (Example 8). Tagforin dimer-TAMRA was obtained as described above, but using 1 equivalent of 5-TAMRA-azide instead of PTX-azide (Example 17). The mass spectrometry data of the representative peptides synthesized are reported in Table 1.

[0286] Isothermal titration calorimetry (ITC) measurements.

[0287] To obtain the dissociation constant (K d) and thermodynamic information, we tested the compound against EphA2-LBD by isothermal titration calorimetry (ITC) using an Affinity ITC autosampler from TA Instruments (New Castle, DE). Titration was performed in the reverse manner, i.e., titrating the protein into the ligand solution. All titrations were carried out by dissolving both the agent and the target protein at a final DMSO concentration of 1% in 25 mM Tris (pH 7.5), 150 mM NaCl at 25 °C. A 200 μM solution of EphA2-LBD, EphA3-LBD chimera, or EphA4-LBD was loaded into the syringe, and 20 injections of 2.5 μL each were made into the cell containing the 10 μM compound solution. These injections were made at 200-second intervals with a stirring speed of 75 rpm. The solution was kept at 4 °C in the autosampler. Data analysis was performed using NanoAnalyze software (TA Instruments, New Castle, DE) and then exported to Microsoft Excel.

[0288] DELFIA displacement assay

[0289] To test the activity of the dimerizer and monomeric agent, 100 μL of 1 μM 123B9-Biotin 1 solution or 100 nM solution of the agent piperazine acetic acid-YSA-(2MeBip)-PDS-Chg-PFRP-G K (Biotin LC) was added to each well of a 96-well streptavidin-coated plate and incubated for 2 h. The plate was then washed 3 times. Subsequently, a mixture containing 11 μL of EphA2 protein and serial dilutions of the test compound was added to each well and incubated for 1 h with a solution containing 89 μL of Eu-N1-labeled anti-6x-His antibody (PerkinElmer). At the end of the incubation period, the plate was washed 3 times and incubated with DELFIA enhancement solution (PerkinElmer) for 10 min. The final concentrations of EphA2 protein used to test the activity of the dimerizer and monomeric agent were 71.2 nM and 10 nM, respectively. The antibody concentrations in the 89 μL solutions used to test the dimerizer and monomeric agent 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 taken using a VICTOR X5 microplate reader (ex / em = 340 / 615 nm), normalized against DMSO wells, and reported as percentage inhibition. IC 50 values were calculated using Prism 9 (GraphPad).

[0290] Cell lines, cell cultures, and antibodies

[0291] The BxPC3, MIAPaCa-2, and PANC-1 cell lines were all purchased from the American Type Culture Collection (ATCC). The BxPC3 and PANC-1 cells were cultured in RPMI-1640 medium and DMEM medium, respectively, supplemented with 10% fetal bovine serum (FBS). The MIA PaCa-2 cells were cultured in DMEM medium containing 10% FBS and 2.5% horse serum. The cells were maintained at 37 °C in a humidified incubator with 5% CO2. The 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 all purchased from ThermoFisher Scientific. The anti-β-actin antibody (#sc-69879) was purchased from Santa Cruz Biotechnology, and the anti-LAMP1 antibody (#9091) was purchased from Cell Signaling Technology.

[0292] Immunofluorescence

[0293] The BxPC3 cells were plated on cover slips overnight. The cells were serum-starved for 1 h and treated with 100 nM tagfurin dimer TAMRA for 0, 30, and 60 min. The cells were then fixed with 4% paraformaldehyde for 20 min, permeabilized with 0.2% Triton X-100 for 5 min, blocked with 10% goat serum for 1 h, and incubated overnight at 4 °C with the anti-LAMP1 antibody, followed by incubation with the Alexa Fluor TM 488-conjugated anti-rabbit secondary antibody for 1 h at room temperature. VECTASHIELD antifade mounting medium with DAPI (Vector Laboratories) was added to the cover slips to stain the cell nuclei. Images were then acquired using a Zeiss Axiovert 200M fluorescence deconvolution microscope and processed using SlideBook software version 6 (Intelligent Imaging Innovations).

[0294] Immunoblotting

[0295] After treatment, cells were lysed on ice with 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)). The lysate was then centrifuged at 16,000 x g for 20 min at 4°C, and the supernatant was collected. Protein assays were performed using the Pierce BCA Protein Assay Kit (ThermoFisher Scientific) according to the manufacturer's protocol. Samples were prepared and loaded onto a 4-12% NuPAGE Bis-Tris precast gel and then transferred to a PVDF membrane. The blot was blocked with 5% non-fat milk for 1 h at room temperature and incubated overnight at 4°C with monoclonal EphA2 or actin antibody, followed by incubation with anti-mouse HRP-conjugated antibody for 1 h at room temperature. The Clarity Western ECL Kit (BIO-RAD) was added to the blot, and images were captured using the ChemiDoc imaging system (BIO-RAD) and analyzed using ImageJ software.

[0296] Cell migration assay

[0297] BxPC3 cells were seeded in ImageLock 96-well plates (Sartorius) such that they were approximately 95-100% confluent at the time of treatment. A wound was then made in the cell monolayer using a WoundMaker TM (Sartorius), followed by two washes with PBS. Cells were then treated with 2 μg / mL ephrin A1-Fc (R&D Systems) or the test agent and the plate was imaged every 3 h using a live cell analysis system (Sartorius). The percentage of relative wound density was quantified using the cell migration software module.

[0298] In vivo pharmacokinetics, toxicity, xenograft studies

[0299] In vivo efficacy experiments were conducted at AntiCancer, Inc. (San Diego). For xenograft studies, 35 male nu / nu mice, 8 - 10 weeks old (AntiCancer Inc, San Diego), consisting of 25 mice for randomization and 10 additional mice, were used. All mice were housed in a barrier facility on a high - efficiency particulate air (HEPA) filtered rack under standard conditions of a 12 h light / dark cycle. Animal studies were conducted in accordance with the protocol of the AntiCancer Institutional Animal Care and Use Committee (IACUC), specifically approved for this study, and conformed to the principles and procedures outlined in the National Institutes of Health Guide for the Care and Use of Animals (Assurance Number A3873 - 1). All animals had free access to autoclaved acidified water (pH 2.5 - 3). Cryovials containing MIAPaCa - 2 pancreatic cancer cells were thawed from liquid nitrogen storage and amplified for in vitro cell culture to prepare subcutaneous stock tumors for subsequent flank tumor fragment implantation. MIAPaCa - 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, MIAPaCa - 2 cells (1.0×107 cells / mouse) in 100 μL PBS were injected into the right flank of five male nu / nu mice. After anesthetizing the mice with a ketamine solution, an incision of approximately 5 mm was made on the back of the nude mice. After creating a space under the skin of the right flank, a 1 mm 3 MIA PaCa - 2 tumor fragment prepared from the stock was inserted. The incision was sutured with 5 - 0 PDS - II sutures. Eighteen days after tumor implantation (day 0), tumors were measured with calipers using the following formula: (tumor volume)=(length)×(width)×(width)×1 / 2. Twenty - five of the 35 mice were randomly divided into five treatment groups of 5 mice each, with no significant differences in tumor volume between groups. All therapeutic agents (dissolved in 100 μl of a formulation consisting of 80% PBS, 10% Tween 80, 10% ethanol) were administered by tail vein injection twice a week for 3 weeks, for a total of 6 injections. Treatment started on the day of randomization (day 1), and mice received the agent or vehicle control on days 1, 4, 8, 11, 15, 18. Tumor volume and body weight were measured weekly. The study was terminated 22 days after treatment initiation.

[0300] Molecular modeling

[0301] The molecular models were analyzed using MOE 2022.02 (Chemical Computing Group). The model of targifolin complexed with EphA2-LBD was obtained by modifying and appropriately minimizing the crystal structure of our previous agent with EphA2-LBD (PDB-ID 6B9L).

[0302] The content of C. Baggio et al., J. Med. Chem. 2022, 65, 22, 15443–15456 is incorporated herein by reference.

[0303] References in Example 18

[0304] 1. Gambini, L.; Salem, A. F.; Udompholkul, P.; Tan, X. F.; Baggio, C.; Shah, N.; Aronson, A.; Song, J.; Pellecchia, M., Structure-Based Design of Novel Epha2 Agonistic Agents with Nanomolar Affinity in Vitro and in Cell. ACS Chem Biol 2018, 13(9), 2633-2644.

[0305] 2. Duxbury, M. S.; Ito, H.; Zinner, M. J.; Ashley, S. W.; Whang, E. E., Ligation of EphA2 by Ephrin A1-Fc Inhibits Pancreatic Adenocarcinoma Cellular Invasiveness. Biochemical and biophysical research communications 2004, 320(4), 1096-1102.

[0306] 3. Duxbury, M. S.; Ito, H.; Zinner, M. J.; Ashley, S. W.; Whang, E. E, EphA2: A Determinant of Malignant Cellular Behavior and a Potential Therapeutic Tareetin Pancreatic Adenocarcinoma. Oncogene 2004, 23(7), 1448-1456

[0307] 4. Mudali, S.V.; Fu, B.; Lakkur, S.S.; Luo, M.; Embuscado, E.E.; Iacobuzio-Donahue, C.A., Patterns of Epha2 Protein Expression in Primary and Metastatic Pancreatic Carcinoma and Correlation with Genetic Status. Clinical & experimental metastasis 2006, 23(7-8), 357-365.

[0308] 5. Hess, A.R.; Seftor, E.A.; Gardner, L.M.; Carles-Kinch, K.; Schneider, G.B.; Seftor, R.E.; Kinch, M.S; Hendrix, M.J., Molecular Regulation of Tumor Cell Vasculogenic Mimicry by Tyrosine Phosphorylation: Role of Epithelial Cell Kinase (Eck / Epha2). Cancer research 2001, 61(8), 3250-3255.

[0309] 6. Walker-Daniels, J.; Coffman, K.; Azimi, M.; Rhim, J.S.; Bostwick, D.G.; Snyder, P.; Kerns, B.J.; Waters, D.J.; Kinch, M.S, Overexpression of the Epha2 Tyrosine Kinase in Prostate Cancer. The Prostate 1999, 41(4), 275-280.

[0310] 7.Zeng,G.;Hu,Z.;Kinch,M.S.;Pan,C.X.;Flockhart,D.A.;Kao,C.;Gardner,T.A.;Zhang,S.;Li,L.;Baldridge,L.A.;Koch,M.O.;Ulbright,T.M.;Eble,J.N.;Cheng,L.,High-Level Expression of Epha2 Receptor Tyrosine Kinase in ProstaticIntraepithelial Neoplasia.The American journal of pathology 2003,163(6),2271-2276.

[0311] 8.Salem,A.F.;Wang,S.;Billet,S.;Chen,J.F.;Udompholkul,P.;Gambini,L.;Baggio,C.;Tseng,H.R.;Posadas,E.M.;Bhowmick,N.A.;Pellecchia,M.,Reduction ofCirculating Cancer Cells and Metastases in Breast-Cancer Models by a PotentEpha2-Agonistic Peptide-Drug Conjugate.J Med Chem 2018,61(5),2052-2061.

[0312] 9.Zhao,P.;Jiang,D.;Huang,Y.;Chen,C.,Epha2:A Promising TherapeuticTarget in Breast Cancer.J Genet Genomics 2021,48(4),261-267

[0313] 10.Zhou,L;Lu,X.;Zhang,B.;Shi,Y.;Li,Z.,Epha2 as a New Target forBreast Cancer and Its Potential Clinical APPlication.Int J Clin Exp Pathol2021,14(4),484-492.

[0314] 11. Miyazaki, T.; Kato, H.; Fukuchi, M.; Nakajima, M.; Kuwano, H., Epha2 Overexpression Correlates with Poor Prognosis in Esophageal Squamous Cell Carcinoma. International journal of cancer. Journal international du cancer 2003, 103(5), 657 - 663.

[0315] 12. Ogawa, K.; Pasqualini, R.; Lindberg, R.A.; Kain, R.; Freeman, A.L.; Pasquale, E.B., The Ephrin-A1 Ligand and Its Receptor, Epha2, Are Expressed During Tumor Neovascularization. Oncogene 2000, 19(52), 6043 - 6052.

[0316] 13. Margaryan, N.V.; Strizzi, L.; Abbott, D.E.; Seftor, E.A.; Rao, M.S.; Hendrix, M.J.; Hess, A.R., Epha2 as a Promoter of Melanoma Tumorigenicity. Cancer biology & therapy 2009, 8(3), 279 - 288.

[0317] 14. Abraham, S.; Knapp, D.W.; Cheng, L.; Snyder, P.W.; Mittal, S.K.; Bangari, D.S.; Kinch, M.; Wu, L; Dhariwal, J.; Mohammed, S.I, Expression of Epha2 and Ephrin a-1 in Carcinoma of the Urinary Bladder. Clinical cancer research: an official journal of the American Association for Cancer Research 2006, 12(2), 353 - 360

[0318] 15. Wang, L.F.; Fokas, E.; Bieker, M.; Rose, F.; Rexin, P.; Zhu, Y.; Pagenstecher, A.; Engenhart-Cabillic, R; An, H.X., Increased Expression of Epha2 Correlates with Adverse Outcome in Primary and Recurrent Glioblastoma Multiforme Patients. Oncology reports 2008, 19(1), 151 - 156.

[0319] 16. Wykosky, J.; Gibo, D.M.; Stanton, C.; Debinski, W., Epha2 as a Novel Molecular Marker and Target in Glioblastoma Multiforme. Molecular cancer research: MCR 2005, 3(10), 541 - 551.

[0320] 17. Binda, E.; Visioli, A.; Giani, F.; Lamorte, G.; Copetti, M.; Pitter, K.L.; Huse, J.T.; Cajola, L.; Zanetti, N.; Di Meco, F.; De Filippis, L.; Mangiola, A.; Maira, G.; Anile, C.; De Bonis, P.; Reynolds, B.A.; Pasquale, E.B.; Vescovi, A.L., The Epha2 Receptor Drives Self-Renewal and Tumorigenicity in Stem-Like Tumor-Propagating Cells from Human Glioblastomas. Cancer cell 2012, 22(6), 765 - 780.

[0321] 18Faoro, L; Singleton, P.A; Cervantes, G.M; Lennon, F.E; Choong, N.W.; Kanteti, R.; Ferguson, B.D.; Husain, A.N.; Tretiakova, M.S.; Ramnath, N.; Vokes, E.E.; Salgia, R., Epha2 Mutation in Lung Squamous Cell Carcinoma Promotes Increased CellSurvival, Cell Invasion, Focal Adhesions, and Mammalian Target of RapamycinActivation. The Joumal of biological chemistry 2010, 285(24), 18575-18585.

[0322] 19.Merritt, W.M.; Thaker, P.H.; Landen, C.N., Jr.; Deavers, M.T.; Fletcher, M.S.; Lin, Y.G.; H8n, L Y.; Kamat, A.A.; Schmandt, R.; Gershenson, D.M.; Kinch, M.S.; Sood, A.K., Aaalysis of Epha2 Expression and Mutant P53 in OvarianCarcinoma. Cancer biology&therapy 2006, 5(10), 1357-1360.

[0323] 20.Yusan, W.J.; Ge, J.; Chen, Z.K.; Wu, S.B.; Shen, H.; Yang, P.; Hu, B.; Zhang, G.W.; Chen, Z.H., Over-Expression ofEpha2 and Ephrina-1in Human GastricAdenocarcinomaa and Its Prognostic Value for Postoperative Patients. Digestivediseases and sciences 2009, 54(11), 2410-2417

[0324] 21. Takahashi, Y.; Itoh, M.; Nara, N.; Tohda, S., Effect of Eph-Ephrin Signaling on the Growth of Human Leukemia Cells. Anticancer Res 2014, 34(6), 2913-2918.

[0325] 22. Trinidad, E.M.; Zapata, A.G.; Alonso-Colmenar, L.M., Eph-Ephrin Bidirectional Signaling Comes into the Context of Lymphocyte Transendothelial Migration. Cell Adh Migr 2010, 4(3), 363-367.

[0326] 23. Alonso, C.L.; Trinidad, E.M.; de Garcillan, B.; Ballesteros, M.; Castellanos, M.; Cotillo, I.; Munoz, J.J.; Zapata, A.G., Expression Profile of Eph Receptors and Ephrin Ligands in Healthy Human B Lymphocytes and Chronic Lymphocytic Leukemia B-Cells. Leuk Res 2009, 33(3), 395-406.

[0327] 24. Guan, M.; Liu, L.; Zhao, X.; Wu, Q; Yu, B.; Shao, Y.; Yang, H.; Fu, X.; Wan, J.; Zhang, W., Copy Number Variations of Epha3 Are Associated with Multiple Types of Hematologic Malignancies. Clin Lymphoma Myeloma Leuk 2011, 11(1), 50-53.

[0328] 25. Amato, K.R.; Wang, S.; Hastings, A.K.; Youngblood, V.M.; Santapuram, P.R.; Chen, H.; Cates, J.M.; Colvin, D.C; Ye, F.; Brantley-Sieders, D.M.; Cook, R.S.; Tan, L; Gray, N.S.; Chen, J., Genetic 8nd Pharmacologic Inhibition of Epha2 Promotes Apoptosis in Nsclc. J Clin Invest 2014, 124(5), 2037-2049

[0329] 26. Amato, K.R.; Wang, S.; Tan, L.; Hastings, A.K.; Song, W.; Lovly, C.M.; Meador, C.B.; Ye, F; Lu, P.; Balko, J.M; Colvin, D.C.; Cates, J.M; Pao, W; Gray, N.S; Chen, J., Epha2 Blockade Overcomes Acquired Resistance to Egfr Kinase Inhibitors in Lung Cancer. Cancer research 2016, 76(2), 305-318.

[0330] 27. Miao, B.; Ji, Z.; Tan, L.; Taylor, M.; Zhang, J.; Choi, H.G.; Frederick, D.T.; Kumar, R.; Wargo, J.A.; Flaherty, K.T.; Gray, N.S.; Tsao, H., Epha2 Is a Mediator of Vemurafenib Resistance and a Novel Therapeutic Tatget in Melanoma. Cancer Discov 2015, 5(3), 274 - 287. 28. Heinzlmeir, S.; Kudlinzki, D.; Sreeramulu, S.; Klaeger, S.; Gande, S.L; Linhard, V.; Wilhelm, M; Qiao, H.; Helm, D.; Ruprecht, B.; Saxena, K; Medard, G; Schwalbe, H.; Kuster, B., Chemical Proteomics and Structural Biology Define Epha2 Inhibition by Clinical Kinase Drugs. ACS Chem Biol 2016, 11(12), 3400 - 3411.

[0331] 29. Petty, A.; Idippily, N.; Bobba, V.; Geldenhuys, W.J.; Zhong, B.; Su, B.; Wang, B., Design and Synthesis of Small Molecule Agonists of Epha2 Receptor. Eur J Med Chem 2018, 143, 1261 - 1276.

[0332] 30. Hasegawa., J.; Sue, M.; Yamato, M; Ichikawa, J; Ishida, S.; Shibutani, T.; Kitamura,

[0333] M.; Wada, T.; Agatsuma, T., Novel Anti - Epha2 Antibody, Ds - 8895a for Cancer Treatment. Cancer biology&therapy 2016, 17(11), 1158 - 1167.

[0334] 31.Petty,A.;Myshkin,E.;Qin,H.;Guo,H.;Miao,H.;Tochtrop,G.P.;Hsieh,J.T.;Page,P.;Liu,L.;Lindner,D.J.;Acharya,C.;MacKerell,A.D.,Jr.;Ficker,E.;Song,J.;Wang,B.,A Small Molecule Agonist of Epha2 Receptor Tyrosine KinaseInhibits Tumor Cell Migration in Vitro and Prostate Cancer Metastasis inVivo.PloS one 2012,7(8),e42120.

[0335] 32Singh,D.R.;Kanvinde,P.;King,C.;Pasquale,E.B.;Hristova,K.,The Epha2Receptor Is Activated through Induction of Distinct,Ligand-DependentOligomeric Structures.Commun Biol 2018,1,15

[0336] 33.Salem,A.F.;Gambini,L.;Billet,S.;Sun,Y.;Oshiro,H.;Zhao,M.;Hoffman,R.M.;Bhowmick,N.A.;Pellecchia,M.,Prostate Cancer Metastases Are StronglyInhibited by Agonistic Epha2 Ligands in an Orthotopic Mouse Model.Cancers(Basel)2020,12(10).

[0337] 34.Salem,A.F.;Gambini,L;Udompholkul,P.;Baggio,C.;Pellecchia,M,Therapeutic Taraeting of Pancteatic Cancer Via Epha2 Dimeric AgonisticAgents.Pharmaceuticals(Basel)2020,13(5)

[0338] 35. Quiun, B.A.; Wang, S.; Barile, E.; Das, S.K.; Emdad, L; Sarkar, D.; De, S.K.; Morvaridi, S.K.; Stebbins, J.L.; Pandol, S.J.; Fisher, P.B; Pellecchia, M., Therapy of Pancreatic Cancer Via an Epha2 Receptor-Targeted Delivery of Gemcitabine. Oncotarget 2016, 7(13), 17103-17110.

[0339] 36. Wang, S.; Placzek, W.J.; Stebbins, J.L; Mitra, S.; Noberini, R.; Koolpe, M.; Zhang, Z.; Dahl, R.; Pasquale, E.B.; Pellecchia, M., Novel Targeted System to Deliver

[0340] Chemotherapeutic Drugs to Epha2-Expressing Cancer Cells. J Med Chem 2012, 55(5), 2427-2436.

[0341] 37. Wu, B.; Wang, S.; De, S.K.; Barile, E.; Quiun, B.A.; Zharkikh, I.; Purves, A.; Stebbins, J.L; Oshima, R.G.; Fisher, P.B.; Pellecchia, M., Design and Characterization of Novel Epha2 Agonists for Targeted Delivery of Chemotherapy to Cancer Cells. Chem Biol 2015, 22(7), 876-887.

[0342] 38. Barile, E.; Wang, S.; Das, S.K.; Noberini, R.; Dahl, R.; Stebbins, J.L; Pasquale, E.B.; Fisher, P.B.; Pellecchia, M., Design, Synthesis and Bioevaluation of an Epha2 Receptor-Based Targeted Delivery System. ChemMedChem 2014, 9(7), 1403-1412.

[0343] 39. Mitra, S.; Duggineni, S.; Koolpe, M.; Zhu, X.; Huang, Z.; Pasquale, E.B., Structure-Activity Relationship Analysis of Peptides Targeting the Epha2 Receptor. Biochemistry 2010, 49(31), 6687-6695.

[0344] 40. Wu, B.; Barile, E.; De, S.K.; Wei, J.; Purves, A.; Pellecchia, M., High-Throughput Screening by Nuclear Magnetic Resonance (Hts by Nmr) for the Identification of Ppis Antagonists. Curr Top Med Chem 2015, 15(20), 2032-2042.

[0345] 41. Udompholkul, P.; Baggio, C; Gambini, L.; Sun, Y.; Zhao, M.; Hoffman, R.M.; Pellecchia, M., Effective Tumor Targeting by Epha2-Agonist-Biotin-Streptavidin Conjugates. Molecules 2021, 26(12).

[0346] 42Duggineni, S.; Mitra, S; Lamberto, I.; Han, X.; Xu, Y.; An, J.; Pasquale, E.B.; Huang, Z., Design and Synthesis of Potent Bivalent Peptide Agonists Targeting the Epha2 Receptor. ACS Med Chem Lett 2013, 4(3)

[0347] 43. Lodola, A.; Giorgio, C.; Incerti, M.; Zanotti, I.; Tognolini, M., Targeting Eph / Ephrin System in Cancer Therapy. Eur J Med Chem 2017, 142, 152 - 162.

[0348] 44 Petty, A.; Idippily, N.; Bobba, V.; Geldenhuys, W.J.; Zhong, B.; Su, B.; Wang, B., Design and Synthesis of Small Molecule Agonists of Epha2 Receptor. Eur J Med Chem 2017.

[0349] 45 Hassan - Mohamed, I.; Giorgio, C.; Incerti, M.; Russo, S.; Pala, D.; Pasquale, E.B.; Zanotti, I.; Vicini, P.; Barocelli, E.; Rivara, S.; Mor, M.; Lodola, A.; Tognolini, M., Uniprl29 Is a Competitive Small Molecule Eph - Ephrin Antagonist Blocking in Vitro Angiogenesis at Low Micromolar Concentrations. Br J Pharmacol 2014, 171(23), 5195 - 5208

[0350] 46. Tognolini, M.; Incerti, M.; Pala, D.; Russo, S.; Castelli, R.; Hassan-Mohamed, I.; Giorgio, C.; Lodola, A., Target Hopping as a Useful Tool for the Identification of Novel Epha2 Protein-protein Antagonists. ChemMedChem 2014, 9(1), 67 - 72.

[0351] 47. Wu, B.; De, S.K.; Kulinich, A.; Salem, A.F.; Koeppen, J.; Wang, R.; Barile, E.; Wang, S.; Zhang, D.; Ethell, I.; Pellecchia, M., Potent and Selective Epha4 Agonists for the Treatment of Als. Cell Chem Biol 2017, 24(3), 293 - 305.

[0352] 48. Wu, B.; Zhang, Z.; Noberini, R.; Barile, E.; Giulianotti, M.; Pinilla, C.; Houghten, R.A.; Pasquale, E.B.; Pellecchia, M., Hts by Nmr of Combinatorial Libraries: A Fragment-Based Approach to Ligand Discovery. Chem Biol 2013, 20(1), 19 - 33.

[0353] 49 Giorgio, C; Incerti, M.; Corrado, M.; Rusnati, M.; Chiodelli, P.; Russo, S.; Callegari, D.; Ferlenghi, F.; Ballabeni, V.; Barocelli, E.; Lodola, A.; Tognolini, M., Pharmacological Evaluation of New Bioavailable Small Molecules Targeting Eph / Ephrin Interaction. Biochem Pharmacol 2017.

[0354] 50.Incerti,M.;Tognolini,M.;Russo,S.;Pala,D.;Giorgio,C.;Hassan-Mohamed,I.Noberini, R.;Pasquale,EB;Vicini,P;Piersanti,S.;Rivara,S.;Barocelli,E.;Mor,M.;Lodola,A.Amino Acid Conjugates ofLithocholic Acid asAntagonists ofthe Epha2 Receptor.J Med Chem 2013,56(7),2936-2947.

[0355] 51.Castelli,R.;Tognolini,M.;Vacondio,F.;Incerti,M.;Pala,D.;Callegari,D.;Bertoni,S.;Giorgio,C.Hassan-MoedZ.I. anotti,I.,Bugatti,A.;Rusnati,M.Festuccia,C.;Rivara,S.;Barocelli,E.;Mor,M.,Lodola,A,Delta(5)-Choleno-AminoAcids as Selective and Orally Available Antagonists of the Eph-EphrinSystem.Eur J Med Chem 2015,103,312-324.

[0356] 52Tandon,M;Vemula,SV;Mittal,SK,Emerging Strategies for Epha2Receptor Targeting for Cancer Therapeutics.Expert Opin Ther Targets 2011,15(1),,31-51.

[0357] 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.

[0358] 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 l 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.

[0359] 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.

[0360] Example 19.

[0361] Table 7. Agents with 4-phenyl-L-phenylalanine fixed at position 4 and relative Kd values (nM) from ITC. d Values (nM).

[0362]

[0363] Table 8. Agents with 4-(2-methylphenyl)-L-phenylalanine fixed at position 4 and relative Kd values (nM) from ITC.

[0364]

[0365] All publications, patents, and patent documents 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 is to be understood that many variations and modifications can be made while remaining within the spirit and scope of the invention.

Claims

1. A compound of formula (I): or a salt thereof, wherein: each R is independently selected from the group consisting of morpholino, piperidino, and piperazine, optionally substituted with (C1-C6) alkyl; Each R 1 is benzyl, 3-indolylmethyl, 4-pyridylmethyl, 1-naphthylmethyl or 2-naphthylmethyl, and the benzyl, 3-indolylmethyl, 4-pyridylmethyl, 1-naphthylmethyl and 2-naphthylmethyl are optionally substituted by one or more groups independently selected from hydroxy, amino, nitro, (C1-C6) alkoxy 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 is independently selected from the group consisting of biphenylyl and phenoxyphenyl, said biphenylyl and phenoxyphenyl being optionally substituted with one or more groups independently selected from the group consisting of halogen, 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 halogen; Each R 7 is an optionally hydroxy-substituted (C1-C6) alkyl group; 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 by one or more halogen groups; R 12 is H or is selected from the group consisting of: R 100 is H, (C3-C6) cycloalkyl, or (C1-C6) alkyl optionally substituted with hydroxyl; R 101 is H, (C3-C6) cycloalkyl, or (C1-C6) alkyl optionally substituted with hydroxyl; R 102 is H, (C3-C6) cycloalkyl, or (C1-C6) alkyl optionally substituted with hydroxyl; 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 is: R 11 is C(=NH)NH2; L 1 is a linking group; and L 2 is a linking group.

2. The compound or salt according to claim 1, wherein Each R 1 is benzyl, 1-naphthylmethyl or 2-naphthylmethyl, and the benzyl, 1-naphthylmethyl and 2-naphthylmethyl are optionally substituted by one or more groups independently selected from hydroxy, amino, nitro and (C1-C6) alkyl; R 12 is H or is selected from the group consisting of: R 100 is H, (C3-C6) cycloalkyl or (C1-C2) alkyl; R 101 is H, (C3-C6) cycloalkyl, or (C1-C2) alkyl; R 102 is H, (C3-C6) cycloalkyl, or (C1-C2) alkyl.

3. The compound or salt according to any one of claims 1-2, wherein each R is morpholino or piperidino.

4. The compound or salt according to any one of claims 1-2, wherein each R is piperazine optionally substituted with (C1-C6) alkyl.

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

6. The compound or salt according to any one of claims 1-5, wherein each R 1 is benzyl optionally substituted with an amino group.

7. The compound or salt according to any one of claims 1-5, wherein each R 1 is benzyl optionally substituted by hydroxy.

8. The compound or salt according to any one of claims 1-5, wherein each R 1 is benzyl optionally substituted by (C1-C6) alkyl.

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

10. The compound or salt according to any one of claims 1-9, wherein each R 2 is independently selected from the group consisting of (C1-C4) alkyl optionally substituted with a hydroxyl group.

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

12. The compound or salt according to any one of claims 1-11, wherein each R 4 is a biphenyl group optionally substituted by one or more groups independently selected from the group consisting of halo, hydroxy, (C1-C6)alkyl, and (C1-C6)alkoxy, wherein each (C1-C6)alkyl and (C1-C6)alkoxy is optionally substituted by one or more groups independently selected from the group consisting of halo.

13. The compound or salt according to any one of claims 1-11, wherein each R 4 is independently a phenoxyphenyl optionally substituted by one or more groups independently selected from the group consisting of halo, hydroxy, (C1-C6)alkyl, and (C1-C6)alkoxy, wherein each (C1-C6)alkyl and (C1-C6)alkoxy is optionally substituted by one or more groups independently selected from the group consisting of halo.

14. The compound or salt according to any one of claims 1-11, wherein each R 4 is 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-hydroxyphenoxy)phenyl.

15. The compound or salt according to any one of claims 1-14, wherein each R 8 is independently selected from the group consisting of isopropyl and (C3-C6) cycloalkyl.

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

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

18. The compound or salt according to any one of claims 1-16, wherein R 12 is:

19. The compound or salt according to claim 18, wherein R 100 is H, -CH3, -C2H5, i-pr, cyclohexyl or -CH2OH.

20. The compound or salt according to claim 18, wherein R 101 is H, -CH3, -C2H5, i-pr, cyclohexyl or -CH2OH.

21. The compound or salt according to claim 18, wherein R 102 is H, -CH3, -C2H5, i-pr, cyclohexyl or -CH2OH.

22. The compound or salt according to any one of claims 1-16, wherein R 12 is:

23. The compound or salt according to any one of claims 1-22, wherein L 1 is a branched or unbranched, saturated or unsaturated hydrocarbon chain having from about 1 to 100 carbon atoms, wherein one or more of said carbon atoms are optionally and independently replaced by -O-, -S-, -N(R a )-, a 3- to 7-membered heterocycle, a 5- to 6-membered heteroaryl or a carbocycle, and wherein each chain, 3- to 7-membered heterocycle, 5- to 6-membered heteroaryl or carbocycle is optionally and independently substituted by one or more (e.g., 1, 2, 3, 4, 5 or more) substituents selected from: (C1-C6)alkyl, (C1-C6)alkoxy, (C3-C6)cycloalkyl, (C1-C6)alkanoyl, (C1-C6)alkanoyloxy, (C1-C6)alkoxycarbonyl, (C1-C6)alkylthio, azido, cyano, nitro, halo, -N(R a )2, hydroxy, oxo(=O), carboxy, aryl, aryloxy, heteroaryl and heteroaryloxy, wherein each R a is independently H or (C1-C6)alkyl.

24. A compound or salt according to any one of claims 1-22, wherein L 1 is a branched or unbranched, saturated or unsaturated hydrocarbon chain having from about 1 to 20 carbon atoms, wherein one or more of said carbon atoms are optionally and independently replaced by -O-, -S-, -N(R a )-, a 3- to 7-membered heterocycle, a 5- to 6-membered heteroaryl or a carbocycle, and wherein each carbon atom, 3- to 7-membered heterocycle, 5- to 6-membered heteroaryl or carbocycle is optionally and independently substituted by one or more (e.g., 1, 2, 3, 4, 5 or more) substituents selected from: (C1-C6)alkyl, (C1-C6)alkoxy, (C3-C6)cycloalkyl, (C1-C6)alkanoyl, (C1-C6)alkanoyloxy, (C1-C6)alkoxycarbonyl, (C1-C6)alkylthio, azido, cyano, nitro, halo, -N(R a )2, hydroxy, oxo(=O) and carboxy, wherein each R a is independently H or (C1-C6)alkyl.

25. The compound or salt according to any one of claims 1-22, 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 and independently replaced by O, NH or a divalent triazine ring, and wherein each carbon atom is optionally and independently substituted by one or more (e.g., 1, 2, 3, 4, 5 or more) substituents selected from halo and oxo (=O).

26. The compound or salt according to any one of claims 1-22, wherein L 1 is:

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

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

29. The compound or salt according to any one of claims 1-26, wherein D is a residue of a taxane, the taxane comprising paclitaxel, docetaxel, or cabazitaxel.

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

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

32. The compound or salt according to any one of claims 1-16, wherein R 12 is:

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

34. A compound or salt according to any one of claims 1-17 and 22-33, wherein L 2 is a branched or unbranched, saturated or unsaturated hydrocarbon chain having from about 1 to 100 carbon atoms, wherein one or more of said carbon atoms are optionally and independently replaced by -O-, -S, -N(R a ), a 3- to 7-membered heterocycle, a 5- to 6-membered heteroaryl or a carbocycle, and wherein each chain, 3- to 7-membered heterocycle, 5- to 6-membered heteroaryl or carbocycle is optionally and independently substituted by one or more (e.g., 1, 2, 3, 4, 5 or more) substituents selected from: (C1-C6)alkyl, (C1-C6)alkoxy, (C3-C6)cycloalkyl, (C1-C6)alkanoyl, (C1-C6)alkanoyloxy, (C1-C6)alkoxycarbonyl, (C1-C6)alkylthio, azido, cyano, nitro, halo, -N(R a )2, hydroxy, oxo(=O), carboxy, aryl, aryloxy, heteroaryl and heteroaryloxy, wherein each R a is independently H or (C1-C6)alkyl.

35. A compound or salt according to any one of claims 1-17 and 22-33, wherein L 2 is a branched or unbranched, saturated or unsaturated hydrocarbon chain having from about 1 to 20 carbon atoms, wherein one or more of said carbon atoms are optionally and independently replaced by -O-, -S, -N(R a ), a 3- to 7-membered heterocycle, a 5- to 6-membered heteroaryl or a carbocycle, and wherein each carbon atom, 3- to 7-membered heterocycle, 5- to 6-membered heteroaryl or carbocycle is optionally and independently substituted by one or more (e.g., 1, 2, 3, 4, 5 or more) substituents selected from: (C1-C6)alkyl, (C1-C6)alkoxy, (C3-C6)cycloalkyl, (C1-C6)alkanoyl, (C1-C6)alkanoyloxy, (C1-C6)alkoxycarbonyl, (C1-C6)alkylthio, azido, cyano, nitro, halo, -N(R a )2, hydroxy, oxo(=O) and carboxy, wherein each R a is independently H or (C1-C6)alkyl.

36. The compound or salt according to any one of claims 1-17 and 22-33, 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 and independently substituted with one or more (e.g., 1, 2, 3, 4, 5 or more) substituents selected from halo and oxo (=O).

37. The compound or salt according to any one of claims 1-17 and 22-33, wherein L 2 is -CH2C(=O)-, -CH2CH2C(=O)-, -CH2CH2CH2C(=O)-, -CH2CH2CH2CH2C(=O)- or -CH2CH2CH2CH2CH2C(=O)-.

38. A compound selected from the group consisting of: or their salts.

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

40. A method for treating cancer in an animal, which comprises administering to the animal the compound of formula I or a pharmaceutically acceptable salt thereof according to any one of claims 1-38.

41. The 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. The compound of formula I or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 38, for prophylactic or therapeutic treatment of cancer.

43. Use of the compound of formula I or a pharmaceutically acceptable salt thereof according to any one of claims 1-38 in the manufacture of a drug for treating cancer in an animal.

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

45. The method, compound, or use according to any one of claims 40-43, in combination with any chemotherapeutic agent or other anticancer targeting agent.

Images