A small molecule compound that antagonizes EPH-ephrin tetramerization and inhibits bidirectional signaling

Abstract

Compositions and methods for inhibiting EPH-ephrin tetramerization are provided. The compounds and methods herein further include methods of administering novel EPH-ephrin tetramerization inhibitors to a subject in need thereof to treat conditions caused by or exacerbated by aberrant, defective, or excessive EPH-ephrin signaling.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 381,090, filed October 26, 2022, which is incorporated herein by reference in its entirety.

[0002] Acknowledgments of government support This invention was made with government support under Grant No. DK128819 awarded by the National Institutes of Health and Grant Nos. W81XWH-20-CPMRP-IIRA, CP200270, and DAMD 11115013 awarded by the United States Department of Defense. The government has certain rights in this invention.

[0003] Incorporation by reference of sequence listing This application contains a Sequence Listing that has been submitted to the Patent Center in computer readable format (XML) and is incorporated herein by reference in its entirety. The computer readable file, created on October 23, 2023, is named 106546-769841_UTSD_4148-PCT_SequenceListing.xml and is approximately 8000 bytes in size.

[0004] 1. Field The present invention is directed to compounds capable of inhibiting EPH-ephrin tetramerization and their use in the treatment of conditions caused by or exacerbated by disturbed, aberrant, defective, or excessive EPH-ephrin interactions and / or cell signaling. [Background technology]

[0005] 2. Discussion of related technologies Chronic pain is a major problem that is inadequately treated using medications with efficacy, safety, and addiction challenges. A further drawback of current pain medications available today is that they do not target the critical synaptic mechanisms in the spinal cord that directly trigger the pathological, long-lasting changes in neurons that respond to peripheral nerve injury, resulting in central sensitization and chronic pain. Specifically, central sensitization results from strengthening of synaptic connections formed by pain-sensing peripheral nociceptive neurons, C-fibers, that terminate superficially on dorsal horn (DH) neurons. Plasticity at these synapses leads to enhanced transmission of pain impulses from the spinal cord and enhanced sensitivity to heat and touch stimuli (hyperalgesia and allodynia). The highly conserved EphB1 receptor tyrosine kinase, which interacts with its cognate transmembrane ephrinB2 (EB2) ligand, is a key transsynaptic player in mediating this plasticity and causing central sensitization.

[0006] The present disclosure is based, in part, on the development of an extremely sensitive biochemical assay that can be performed in small quantities and used for high-throughput screening (HTS) in the search for chemicals that disrupt protein-protein interactions between EphB1 and one of its ligands, ephrinB2. The illustrative examples herein illustrate how ephrinB2 retrograde signaling can also contribute to chronic pain, and demonstrate that compounds disclosed herein block both anterograde and retrograde EPH-ephrin signaling and target both EphB1 and EphB2. Thus, the present disclosure provides novel therapeutic agents aimed at reducing the ability of Eph receptors (e.g., EphB2) to bind to their cognate ephrin ligands (e.g., ephrinB2 ligands). Summary of the Invention

[0007] Various aspects of the present disclosure include a compound comprising the structure of formula (I): [ka] or a pharmaceutically acceptable salt thereof, wherein A is -O-, -SO2, or -N(CH2) n R1; B is CH2, SO2, or CO; X is hydrogen, halogen, alkenyl, alkyl, -NO2, or -NH2; each R is independently hydrogen, substituted or unsubstituted alkyl, alkoxy, heterocycloalkyl, or carbonyl, and R1 is substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, or substituted or unsubstituted heterocycloalkyl; n=0-3; with the proviso that (1) when A is -O-, B is CH2, and R is hydrogen, then X is not hydrogen, chloro, -NO2, or bromo; (2) A is NR1, and R1 is methyl or [ka] (X is not bromine when The target is.

[0008] In various embodiments, n is 0 or 1. In various embodiments, X is hydrogen, bromo, iodo, chloro, —NO 2 , or —NH 2 , methyl, or propenyl.

[0009] In various embodiments, each R is independently hydrogen, methyl, [ka] For example, in some embodiments, each R is hydrogen.

[0010] In various embodiments, R1 is substituted or unsubstituted C4-C10 aryl, substituted or unsubstituted C4-C10 heteroaryl, substituted or unsubstituted C4-C10 cycloalkyl, or substituted or unsubstituted C4-C10 heterocycloalkyl. For example, in some embodiments, R1 is substituted or unsubstituted phenyl, substituted or unsubstituted pyridinyl, substituted or unsubstituted thiazole, substituted or unsubstituted thiophenyl, substituted or unsubstituted piperazine, or unsubstituted C4-C10 cycloalkyl, or unsubstituted C4-C10 heterocycloalkyl. In further embodiments, R1 is [ka] wherein each Y is independently -CH2- or -O-; each R2 is independently hydrogen, -NO2, alkoxy, an alkyl ether of -R3OR4, -NHOR5, -NH2, halo, haloalkyl, or alkyl; R3 and R4 are each independently C1-C4 alkyl, and R5 is alkyl or benzyl. In various embodiments, each R2 is independently hydrogen, -NO2, -OCH 3、 -CH2CH2OCH3, -NHOCH3, -CF 3、 -NH2, bromo, fluoro, chloro, iodo, methyl, or [ka] is.

[0011] In any of the foregoing embodiments, R1 is [ka] may be selected from:

[0012] In any of the above or related embodiments, the compound is: [ka] isn't it.

[0013] In any of the above or related embodiments, the compound is: [ka] [ka] [ka] and any pharmaceutically suitable salts thereof.

[0014] In any of the above or related embodiments, the compound is: [ka] and a pharmaceutically equivalent salt selected from the group consisting of:

[0015] For example, in some embodiments, the compound is: [ka] and any pharmaceutically suitable salts thereof.

[0016] In a further aspect, the compound is [ka] The compound may be a pharmaceutically suitable salt selected from the group consisting of:

[0017] In various embodiments, compounds of Formula I can specifically inhibit EPHB1-ephrin and / or EPHB2-ephrin tetramerization. For example, compounds that can specifically inhibit EPHB1-ephrin and / or EPHB2-ephrin tetramerization include: [ka] and any pharmaceutically suitable salt thereof.

[0018] In various embodiments, compounds of Formula I can specifically inhibit EPHB2-ephrin tetramerization. For example, compounds that can specifically inhibit EPHB2-ephrin tetramerization include: [ka] and any pharmaceutically suitable salts thereof.

[0019] In various embodiments, the compounds of Formula I can specifically inhibit EPHB1-ephrin tetramerization. For example, compounds that can specifically inhibit EPHB1-ephrin tetramerization include: [ka] and any pharmaceutically suitable salts thereof.

[0020] In any of the above or related embodiments, compounds of Formula I that specifically inhibit EPHB1-ephrin and / or EPHB2-ephrin tetramerization are unable to inhibit EPHB4-ephrin tetramerization.

[0021] In any of the above or related embodiments, compounds of Formula I inhibit EPH-ephrin tetramerization with an IC50 of less than 2 μM, less than 1.6 μM, less than 1 μM, or less than 0.5 μM.

[0022] A further aspect of the present disclosure provides pharmaceutical compositions comprising any compound of Formula I provided herein and a pharmaceutically suitable carrier or excipient.

[0023] A further aspect of the present disclosure is a method of inhibiting the formation of EPH-ephrin tetramers, comprising administering EPH or an ephrin to a compound of formula I provided herein, or [ka] with a compound selected from the group consisting of:

[0024] In various embodiments, the EPH-ephrin tetramer can comprise EPHB 1. In various embodiments, the EPH-ephrin tetramer can comprise EPHB 2. In various embodiments, formation of the EPH-ephrin tetramer is inhibited in vivo.

[0025] A further aspect of the present disclosure relates to a method of alleviating or reducing pain in a subject in need thereof, comprising administering to the subject an effective amount of an EPH-ephrin tetramerization inhibitor, hi some aspects, the pain comprises chronic neuropathic pain.

[0026] A further aspect of the present disclosure relates to a method for treating synaptopathy in a subject in need thereof, comprising administering to the subject an effective amount of an EPH-ephrin tetramerization inhibitor. In some aspects, the synaptopathy comprises abnormal, defective, or excessive EPH-ephrin signaling, and optionally disturbed NMDA signaling. In various aspects, the synaptopathy is associated with anxiety or epilepsy.

[0027] A further aspect of the present disclosure relates to a method of treating addiction or opioid dependence in a subject in need thereof, comprising administering to the subject an effective amount of an EPH-ephrin tetramerization inhibitor.

[0028] A further aspect of the present disclosure relates to a method of treating a fibrotic and / or inflammatory disease or condition in a subject in need thereof, the method comprising administering to the subject an effective amount of an EPH-ephrin tetramerization inhibitor. In various aspects, the fibrotic and / or inflammatory disease or condition comprises aberrant, defective, or excessive EPH-ephrin signaling, and optionally comprises NASH liver fibrosis, chronic kidney disease, scleroderma (skin fibrosis), cardiac fibrosis, pulmonary fibrosis, fibrosis of another organ, and / or abnormal wound healing, optionally selected from keloids and / or hypertrophic scars.

[0029] A further aspect of the present disclosure relates to a method of treating cancer in a subject in need thereof, comprising administering to the subject an effective amount of an EPH-ephrin tetramerization inhibitor. In various aspects, the cancer comprises aberrant, defective, or excessive EPH-ephrin signaling, and optionally comprises GBM (glioblastoma), pancreatic cancer, and / or colon cancer.

[0030] A further aspect of the present disclosure relates to a method of treating a viral infection in a subject in need thereof, comprising administering to the subject an effective amount of an EPH-ephrin tetramerization inhibitor. In various aspects, the viral infection comprises aberrant, defective, or excessive EPH-ephrin signaling, and optionally comprises infection with Henipavirus and / or Human Immunodeficiency Virus (HIV).

[0031] In any of the foregoing or related methods, the EPH-ephrin tetramerization inhibitor can be administered as part of a pharmaceutical composition (e.g., a pharmaceutical composition comprising an EPH-ephrin tetramerization inhibitor). In various embodiments, the pharmaceutical composition can be administered intravenously, subcutaneously, or orally, by IP injection, and topically as a cream or ointment.

[0032] In any of the aforementioned or related methods, the EPH-ephrin tetramerization inhibitor comprises: [ka] , a compound of Formula I, or any pharmaceutically acceptable salt thereof.

[0033] In any of the foregoing or related methods, the subject may be a human, a livestock animal, a companion animal, a laboratory animal, or a zoological animal.

[0034] A further aspect of the present disclosure provides a kit comprising: (a) an EPH-ephrin tetramerization inhibitor; and (b) a container. In various aspects, the EPH-ephrin tetramerization inhibitor can be a compound of Formula I provided herein. [Brief explanation of the drawings]

[0035] [Figure 1] Schematic of the effects of ligand antagonists (LA) on kinase inhibitors: EphB KIs (blue asterisk) only prevent anterograde signaling through the intracellular catalytic domain, whereas LAs (red asterisk), which interfere with EphB binding to ephrin B (EB), disrupt both anterograde and retrograde signaling. [Figure 2] This figure shows that transsynaptic ephrinB2-EphB1 interactions drive dorsal horn neuron plasticity and the formation of chronic pain. A cartoon illustration of a dorsal horn neuron excitatory synapse in the spinal cord, where postsynaptic EphB1 receptor binding to presynaptic ephrinB2 (EB2) ligand induces EphB1 binding to the NR1 subunit of NMDAR and tyrosine phosphorylation of the NR2 subunit, opening ion channels to allow calcium influx, neuronal activation, and LTP, leading to central sensitization and chronic pain. The red asterisk indicates a hypothetical drug compound that binds / docks to the ligand-binding structure of the EphB1 ectodomain, preventing its ability to bind ephrinB2 and thus acting as a ligand antagonist (LA). [Figure 3] Figure 1. Targeting the EphB1-EphrinB2 interaction in high-throughput screening (HTS). A soluble epitope-tagged ectodomain protein is used in a proximity-driven chemiluminescent Alpha assay to target the protein-protein interaction of EphB1 receptor binding to the EphrinB2 (EB2) ligand. A library of low-molecular-weight drug-like chemicals was screened in a high-throughput manner using this assay to identify compounds that reduced the chemiluminescent signal. The red star indicates a hypothetical compound from the library that binds / docks to the ligand-bound conformation of the EphB1 ectodomain, preventing its ability to bind to the EphrinB2 (EB2) ectodomain and thus acts as a ligand antagonist (LA). [Figure 4A]FIG. 1 shows the output of the Alpha assay, which measures EphB1-EphrinB2 (EB2) protein-protein interactions. [Figure 4B] FIG. 1 shows the output of the Alpha assay, which measures EphB1-EphrinB2 (EB2) protein-protein interactions. [Figure 4C] FIG. 1 shows the output of the Alpha assay, which measures EphB1-EphrinB2 (EB2) protein-protein interactions. [Figure 5A] FIG. 1 shows a pull-down experiment demonstrating that A20 reduces the ability of soluble EB2-His to bind to immobilized EphB1-Fc (red asterisk). [Figure 5B] FIG. 1 shows the results of a pull-down experiment demonstrating that A20 exhibits only a weak / poor ability to antagonize the binding of soluble EB2-His to immobilized EphB4-Fc (red asterisk). [Figure 5C] FIG. 5D shows the results of a pull-down experiment demonstrating that A20 potently reduced the binding of EB2-His to immobilized EphB2-His-Fc in a dose-dependent manner and with an IC50 of approximately 10 μM (see FIG. 5D). [Figure 5D] FIG. 5C shows the results of an Alpha assay using EphB2-Fc or EphB4-Fc with EB2-His and increasing A20 concentrations to calculate IC50 determinations (approximately 10 μM, similar to those seen in FIG. 5C). [Figure 6] FIG. 1 shows the chemical structures of A20 (SW056428) and the related A19 (QM) chemical lacking bromine. [Figure 7] FIG. 1 shows A20 and related available analogue chemicals. [Figure 8] FIG. 1 shows that ELISA and pull-downs provide IC50 data for A20 and available analogue chemistries. [Figure 9]Octet RED384 kinetic analysis sensorgram data of the binding of 30 nM soluble mouse ephrin B2 (mEB2-His) protein to sensor chip-immobilized rat EphB1-Fc (FIG. 9A) or mouse EphB4-Fc (FIG. 9B) protein and the effect of increasing concentrations of A20. The association time was 400 s and the dissociation time was 500 s. Binding assays were performed in PBS containing 0.05% Tween-20 and 1% DMSO. [Figure 10] FIG. 1 shows the crystal structures of an EphB2-ephrinB2 dimer, a cyclic tetramer, and a tetramer cluster. [Figure 11] Figure 1 shows Octet RED384 kinetic analysis sensorgram data demonstrating that A20 is a reversible antagonist of ephrin B2 binding to EphB1. Pre-exposure of immobilized rat EphB1-Fc protein to 100 μM A20 did not affect its subsequent ability to bind 30 nM soluble mEB2-His (red sensorgram), and the interaction kinetics were similar to that of samples not pre-exposed to the compound (green sensorgram). Addition of A20 during the binding phase competed with EB2 binding to the immobilized EphB1 protein (purple sensorgram). Binding assays were performed in PBS containing 0.05% Tween-20, with an association time of 400 seconds and a dissociation time of 500 seconds. [Figure 12]Using Octet RED384 kinetic analysis, A20 is a competitive antagonist of ephrin-B2 binding to EphB1. Figures 12A-12C show sensorgram data showing that in the absence of A20, immobilized EphB1 protein can bind to EB2 protein when provided at concentrations of 1, 10, 100, and 1,000 nM (Figure 12A). In the presence of 10 μM A20, EB2 binding is reduced and is only detected at 10, 100, and 1,000 nM (Figure 12B). At 100 μM A20, EB2 binding can only be detected using 100 and 1,000 nM (Figure 12C). Figures 12D-12F show that grouping of sensorgrams based on the concentration of EB2 ligand used (0.1 or 1 nM in Figure 12D, 10 and 100 nM in Figure 12E, and 100 nM in Figure 12F) indicates that addition of A20 competes with and reduces the binding of EB2 to immobilized EphB1 protein. Binding assays were performed in PBS containing 0.05% Tween-20, with association times of 400 s and dissociation times of 500 s. [Figure 13] FIG. 1 shows an exemplary immunoblot demonstrating that A20 antagonizes EB2 stimulation of EphB2 catalytic activity in Cos1 cells that endogenously express this receptor. [Figure 14] Representative fluorescence images and quantification showing that Cos1 cells exposed to pre-clustered EB2-Fc bind EphB2, forming spots (green) in cells that also contain phosphotyrosine (red, arrows). Quantification of the number of spots shows that A20 (40 μM) reduced the number of ligand:receptor clusters per cell, and most of the spots contained no detectable pTyr signal (n = >60 cells per condition; particles / spots / signal sizes between 1 and 5 μm were considered clusters). [Figure 15] 1 is a plot showing that A20 free base is distributed to plasma, brain, and spinal cord after a single IP injection of 20 mg / kg in mice. [Figure 16] A20 Schematic diagram illustrating the production of 2×HCl salt. [Figure 17] Figure 1 shows Octet RED384 kinetic analysis of the binding of immobilized rat EphB1 to soluble mouse EB2 and the effect of the A20.2x HCl salt form of the compound. Binding assays were performed in PBS containing 0.05% Tween-20, with an association time of 400 seconds and a dissociation time of 500 seconds. [Figure 18] A20. Plot showing that the HCl salt compound exhibits greatly improved PK kinetics. [Figure 19A] FIG. 1 shows Octet RED384 kinetic analysis of binding of immobilized human ephrinB1 ectodomain protein to soluble human EphB1, EphB2, and EphB4 ectodomains. [Figure 19B] FIG. 1 shows Octet RED384 kinetic analysis of binding of immobilized human ephrin B2 ectodomain protein to soluble human EphB1, EphB2, and EphB4 ectodomains. [Figure 19C] FIG. 1 shows Octet RED384 kinetic analysis of binding of immobilized human ephrin B3 ectodomain protein to soluble human EphB1, EphB2, and EphB4 ectodomains. [Figure 19D] FIG. 1 shows the same results grouped to show how human EphB1 bound to three different ephrin B proteins. [Figure 19E] FIG. 1 shows the same results grouped to show how human EphB2 bound to three different ephrin B proteins. [Figure 19F] FIG. 1 shows the same results grouped to show how human EphB4 bound to three different ephrin B proteins. [Figure 20A] FIG. 1 shows plots—curve fits (thin red lines) showing binding of immobilized human ephrinB1 protein to human EphB1, EphB2, and EphB4. [Figure 20B]FIG. 1 shows plots—curve fits (thin red lines) showing binding of immobilized human ephrin B2 protein to human EphB1, EphB2, and EphB4. [Figure 20C] FIG. 1 shows plots—curve fits (thin red lines) showing binding of immobilized human ephrinB3 protein to human EphB1, EphB2, and EphB4. [Figure 21A] FIG. 1 shows plots of binding of immobilized human ephrin B2 to human EphB2 and human EphB4 in the presence or absence of different concentrations of A20. [Figure 21B] FIG. 1 shows plots of binding of immobilized human ephrinB2 to human EphB2 and human EphB4 in the presence or absence of 1 μM A20. [Figure 21C] FIG. 1 shows plots of binding of immobilized human ephrinB2 to human EphB2 and human EphB4, with the various regions corresponding to dimerization and tetramerization shaded. [Figure 21D] 1 is a bar graph quantifying the effect of 1 μM A20 on the dimerization and tetramerization of immobilized human ephrinB2 binding to human EphB2 and human EphB4. [Figure 22] FIG. 1 shows the effect of dimer:tetramer ratio (estimated based on the curvature of the curve) and A20 salt on the binding of immobilized human ephrin B protein to 50 nM human EphB protein. [Figure 23] FIG. 1 shows a plot of an abbreviated 80 second binding using binding of immobilized rat EphB1-Fc to 100 nM mEB2-His (note that the baseline showed drift in this experiment). [Figure 24] Figure 10 shows a plot showing the shortened 80-40-20-15-10-5 sec binding of immobilized rat EphB1-Fc to 100 nM mEB2-His (note that the baseline exhibited drift in this experiment). [Figure 25A]FIG. 10 shows an annotated plot showing that A20 does not affect dimer binding kinetics but specifically affects the accumulation of ultrastable tetramers. [Figure 25B] FIG. 10 shows an annotated plot showing that A20 does not affect dimer binding kinetics but specifically affects the accumulation of ultrastable tetramers. [Figure 25C] FIG. 1 shows an analysis demonstrating that A20 does not affect dimer binding kinetics but specifically affects the accumulation of ultrastable tetramers. [Figure 26A] Figure 26A shows exemplary mass photometry plots demonstrating that A20 chemicals specifically affect the accumulation of EPH-ephrin tetramers. Figure 26A shows the formation of weak homodimers of EphB2 in a homogenous mixture. [Figure 26B] Figure 26B shows exemplary mass spectrometry plots demonstrating that A20 chemicals specifically affect the accumulation of EPH-ephrin tetramers. Figure 26B shows the formation of weak homodimers of ephrin B2 in a homogenous mixture. [Figure 26C] Figure 26C shows exemplary mass spectrometry plots demonstrating that the A20 chemical specifically affects the accumulation of EPH-ephrin tetramers. Figure 26C shows the formation of tetramers between EphB2 and ephrinB2 in the absence of the A20 chemical. [Figure 26D] Figure 26D shows exemplary mass spectrometry plots demonstrating that the A20 chemicals specifically affect the accumulation of EPH-ephrin tetramers. Figure 26C shows the inhibition of tetramerization in the presence of the A20 chemicals. [Figure 27] FIG. 1 shows exemplary plots and biophysical analysis of A-class and A / B-class cross-sectional EPH-ephrin protein-protein interactions. [Figure 28A] FIG. 1 shows exemplary plots and biophysical analyses demonstrating that A20 also prevents the formation of A-class (eg, EphA3-EA1) EPH-ephrin tetramers. [Figure 28B]FIG. 1 shows exemplary plots and biophysical analyses demonstrating that A20 also prevents the formation of A-class (eg, EphA3-EA5) EPH-ephrin tetramers. [Figure 28C] FIG. 1 shows exemplary plots and biophysical analyses demonstrating that A20 also prevents the formation of A / B cross-class (eg, EphB2-EA5) EPH-ephrin tetramers. [Figure 29A] FIG. 1 shows exemplary biophysical data demonstrating that the pre-existing stable Eph-ephrin tetramer, hEphrinB2+hEphB1, is also targeted by the A20 chemistry. [Figure 29B] FIG. 1 shows exemplary biophysical data demonstrating that the pre-existing stable Eph-ephrin tetramer, hEphrinB2+hEphB2, is also targeted by the A20 chemistry. [Figure 29C] FIG. 1 shows exemplary biophysical data demonstrating that the pre-existing stable Eph-Ephrin tetramer, mEphB+mEphrinB2, is also targeted by the A20 chemistry. [Figure 30A] FIG. 10 shows plots illustrating accurate calculation of tetrameric inhibitor IC50 values ​​for exemplary compound A19·2×HCl salt against human ephrinB2-EphB2 interaction. [Figure 30B] FIG. 1 shows a plot illustrating accurate calculation of the tetrameric inhibitor IC50 value for exemplary compound A20·2×HCl salt against the human ephrinB2-EphB2 interaction. [Figure 30C] FIG. 1 shows a plot illustrating accurate calculation of tetrameric inhibitor IC50 values ​​for exemplary compound 8009-9255·2×HCl salt against human ephrinB2-EphB2 interaction. [Figure 30D] FIG. 10 shows a plot showing accurate calculation of tetrameric inhibitor IC50 values ​​for exemplary compound 3511-0013 against human ephrinB2-EphB2 interaction. [Figure 31] FIG. 1 shows analogs based on the A20 scaffold. [Figure 32]FIG. 1 shows analogs based on the 3511-0013 scaffold. [Figure 33] Figure 33 shows first generation novel A20 analogs, in which the 8-hydroxyquinoline ring system is retained (Figure 33A) or replaced with a 5-methoxyindole ring (Figure 33B). [Figure 34] Figure 1 shows the results of Alpha, which tested the LA activity of six first-generation novel compounds against the EphB1-EB2 interaction. Conditions included 8 nM EphB1-Fc and EB2-His proteins, 1% DMSO, 50 ng of beads each per well, and the free base form of the compounds at the indicated concentrations. [Figure 35A] FIG. 1 shows tetrameric inhibitor IC50 values ​​for an exemplary first generation compound, QPB4, against the human ephrinB2-EphB2 interaction. [Figure 35B] FIG. 1 shows tetrameric inhibitor IC50 values ​​for an exemplary first generation compound, QPDF, against the human ephrinB2-EphB2 interaction. [Figure 35C] FIG. 1 shows tetrameric inhibitor IC50 values ​​for exemplary first generation compounds, IM, against human ephrinB2-EphB2 interaction. [Figure 35D] FIG. 1 shows tetrameric inhibitor IC50 values ​​for an exemplary first generation compound, IMP2, against the human ephrinB2-EphB2 interaction. [Figure 35E] FIG. 1 shows tetrameric inhibitor IC50 values ​​for an exemplary first generation compound, IPB4, against the human ephrinB2-EphB2 interaction. [Figure 36A] Schematic of chemical synthesis for producing compounds in series 1a, series 1b, and a few compounds in series 6. [Figure 36B] Schematic of chemical synthesis for producing compounds in series 2, 3, 4, and a few compounds in series 6. [Figure 36C] FIG. 1 is a schematic diagram of the chemical synthesis for producing compounds in series 5. [Figure 37]FIG. 1 shows compounds synthesized and described herein. [Figure 38A] Figure 1 shows binding of immobilized EphB2 to mouse ephrinB2 in the presence of A20-I. These data demonstrate that the second-generation compound A20-I is a potent tetramerization inhibitor compared to A20, and that replacement of the halogen in A20 / A20-I with NO2 results in poor tetramerization inhibitor activity. [Figure 38B] Figure 1 shows binding of immobilized EphB2 to mouse ephrinB2 in the presence of A20. These data demonstrate that the second-generation compound, A20-I, is a more potent tetramerization inhibitor than A20, and that replacement of the halogen in A20 / A20-I with NO2 results in poor tetramerization inhibitor activity. [Figure 38C] Figure 1 shows binding of immobilized mouse EphB2 to human ephrin A5 in the presence of A20-I. These data demonstrate that the second-generation compound A20-I is a more potent tetramerization inhibitor than A20, and that replacement of the halogen in A20 / A20-I with NO2 results in poor tetramerization inhibitor activity. [Figure 38D] Figure 1 shows binding of immobilized human ephrinB2 to human EphB2 in the presence of A19-NO2. These data demonstrate that the second-generation compound, A20-I, is a more potent tetramerization inhibitor than A20, and that replacement of the halogen in A20 / A20-I with NO2 results in poor tetramerization inhibitor activity. [Figure 39] 1 shows that A20-I exhibits increased in vitro metabolic stability compared to A20. Both the free base and salt forms of the compound were analyzed by PK Core. [Figure 40A] FIG. 1 shows biophysical analysis of exemplary second-generation compounds derived from QTM:QTM (where all compounds were tested in their salt (2×HCl) form). The assay shown is the binding of immobilized human ephrinB2 to 50 nM human EphB2. [Figure 40B]

[0023] Figure 1 shows biophysical analysis of exemplary second generation compounds derived from QTM-Br (where all compounds were tested in their salt (2xHCl) form). The assay shown is the binding of immobilized human ephrin B2 to 50 nM human EphB2. [Figure 40C]

[0023] Figure 1 shows biophysical analysis of exemplary second generation compounds derived from QTM-I (where all compounds were tested in their salt (2xHCl) form). The assay shown is the binding of immobilized human ephrinB2 to 50 nM human EphB2. [Figure 41A] QPB4: Biophysical analysis of exemplary first generation compounds derived from QPB4 (where compounds were tested in their salt (2xHCl) form). The assay shown is the binding of immobilized human ephrinB2 to 50 nM human EphB2. [Figure 41B]

[0023] Figure 1 shows biophysical analysis of exemplary first generation compounds derived from QPPh, where the compounds were tested in their salt (2xHCl) form. The assay shown is the binding of immobilized human ephrinB2 to 50 nM human EphB2. [Figure 41C]

[0023] Figure 1 shows biophysical analysis of exemplary first generation compounds derived from QPI4, where the compounds were tested in their salt (2xHCl) form. The assay shown is the binding of immobilized human ephrinB2 to 50 nM human EphB2. [Figure 41D] 1 shows biophysical analysis of exemplary first generation compounds derived from MeQPB4. The assay shown is binding of immobilized human ephrinB2 to 50 nM human EphB2. [Figure 41E]

[0023] Figure 1 shows biophysical analysis of exemplary first generation compounds derived from BQPB4-Bn, where the compounds were tested in their salt (2xHCl) form. The assay shown is the binding of immobilized human ephrin B2 to 50 nM human EphB2. [Figure 41F]1 shows biophysical analysis of exemplary first generation compounds derived from QPCF34 (where compounds were tested in their salt (2×HCl) form). The assay shown is binding of immobilized human ephrinB2 to 50 nM human EphB2. [Figure 42A] FIG. 1 shows the structures of QPB4-Bn (free base) and QPB4-Bn salt form (2×HCl). [Figure 42B] FIG. 1 shows exemplary plots demonstrating that QPB4-Bn salt form (2×HCl) exhibits a potent PK profile when injected into mice. [Figure 42C] FIG. 42B shows quantification of the experiment shown in FIG. 42B. [Figure 43] FIG. 1 shows peptides SNEW (SEQ ID NO: 1) and EWLS (SEQ ID NO: 2), which are similar peptides that have been described to bind to the EphB1 and EphB2 dimerization pockets and prevent ephrin B binding. [Figure 44] Figure 44 shows biophysical experiments demonstrating that the SNEW peptide (Figure 44A), but not the EWLS peptide (Figure 44B), specifically disrupts ephrinB2-EphB2 dimer interactions. After baseline measurements, biosensor-immobilized human ephrinB2 ectodomain protein was exposed to 50 nM soluble human EphB2 ectodomain during the binding phase, without or with the indicated concentrations of SNEW (Figure 44A) or EWLS (Figure 44B) peptides. The upper full-trace sensorgram at a relatively low peptide concentration shows that only the highest 100 μM concentration of SNEW altered the binding pattern during the binding phase, but the effect was not as dramatic as that of the 2 μM concentration of the A20-I tetramerization inhibitor used as the maximum control. The lower sensorgram focuses on the binding phase of a second experiment, in which the peptide concentration was increased to 400 μM. Note that the initial steep 84° dimerization slope of ephrinB2-EphB2 interaction in the absence of peptide was strongly reduced in a dose-dependent manner by the presence of the SNEW peptide. The EWLS peptide showed no effect on ephrinB2-EphB2 protein-protein interaction. [Figure 45]Figure 1 shows testing of the aqueous solubility of salt forms of A20 and A20-I. The compounds were sent to the UT Southwestern PK Core facility and tested for aqueous solubility. While both are highly soluble, the A20 salt is particularly soluble and can be made into a greater than 100% solution, while the A20-I salt can be dissolved into a greater than 15% solution. [Figure 46] Figure 1 shows in vitro plasma stability studies of the free base and salt forms of A20 and A20-I. The compounds were sent to the UT Southwestern PK Core facility and tested for stability when incubated with mouse plasma. Both compounds appear to be quite stable, but A20-I exhibited superior stability when evaluated in either the free base or salt form. [Figure 47A]

[0023] Figure 1 shows data and quantification demonstrating that the A20 salt form is orally bioavailable. The A20.2xHCl compound was sent to the UT Southwestern PK Core facility and tested for bioavailability after a single dose when injected intravenously (IV) at 3 mg / kg or given by oral gavage (PO) at 30 mg / kg using PBS as the vehicle. The data for oral gavage are quite impressive, showing that significant levels of A20 persist after a single dose, particularly in the liver. [Figure 47B]

[0023] Figure 1 shows data and quantification demonstrating that the A20 salt form is orally bioavailable. The A20.2xHCl compound was sent to the UT Southwestern PK Core facility and tested for bioavailability after a single dose when injected intravenously (IV) at 3 mg / kg or given by oral gavage (PO) at 30 mg / kg using PBS as the vehicle. The data for oral gavage are quite impressive, showing that significant levels of A20 persist after a single dose, particularly in the liver. [Figure 47C]

[0023] Figure 1 shows data and quantification demonstrating that the A20 salt form is orally bioavailable. The A20.2xHCl compound was sent to the UT Southwestern PK Core facility and tested for bioavailability after a single dose when injected intravenously (IV) at 3 mg / kg or given by oral gavage (PO) at 30 mg / kg using PBS as the vehicle. The data for oral gavage are quite impressive, showing that significant levels of A20 persist after a single dose, particularly in the liver. [Figure 47D]

[0023] Figure 1 shows data and quantification demonstrating that the A20 salt form is orally bioavailable. The A20.2xHCl compound was sent to the UT Southwestern PK Core facility and tested for bioavailability after a single dose when injected intravenously (IV) at 3 mg / kg or given by oral gavage (PO) at 30 mg / kg using PBS as the vehicle. The data for oral gavage are quite impressive, showing that significant levels of A20 persist after a single dose, particularly in the liver. [Figure 48A]

[0023] Figure 1 shows biophysical analysis of an exemplary second-generation compound, A20-I. All compounds were tested as the free base except for A20-I (tested as the 2xHCl salt). The assay shown is the binding of immobilized human ephrinB2 to 50 nM human EphB2. [Figure 48B] Figure 1 shows biophysical analysis of an exemplary second-generation compound, A19-L. All compounds were tested as free bases except A20-I (tested as the 2xHCl salt). The assay shown is binding of immobilized human ephrinB2 to 50 nM human EphB2. Note that compound A19-L exhibits little ability to prevent insurmountable tetramers at 3.2 μM concentration (bold), as its sensorgram falls below the maximum control. [Figure 48C]Figure 1 shows biophysical analysis of an exemplary second-generation compound, 2OH-A19. All compounds were tested as the free base except for A20-I (tested as the 2xHCl salt). The assay shown is the binding of immobilized human ephrin B2 to 50 nM human EphB2. [Figure 48D]

[0023] Figure 1 shows biophysical analysis of an exemplary second-generation compound, QPPh. All compounds were tested as the free base except for A20-I (tested as the 2xHCl salt). The assay shown is the binding of immobilized human ephrinB2 to 50 nM human EphB2. [Figure 49A]

[0023] Figure 1 shows biophysical analysis of an additional exemplary second-generation compound, A19-NO2. All compounds were tested as the free base, except for QPB-NO4 (tested as the 2xHCl salt). The assay shown is the binding of immobilized human ephrin B2 to 50 nM human EphB2. [Figure 49B]

[0023] Figure 1 shows biophysical analysis of an additional exemplary second-generation compound, QTM-NO2. All compounds were tested as the free base, except for QPB-NO4 (tested as the 2xHCl salt). The assay shown is binding of immobilized human ephrin B2 to 50 nM human EphB2. [Figure 49C]

[0023] Figure 1 shows biophysical analysis of an additional exemplary second-generation compound, 2OH-A20. All compounds were tested as the free base, except for QPB-NO4 (tested as the 2xHCl salt). The assay shown is binding of immobilized human ephrin B2 to 50 nM human EphB2. [Figure 49D]

[0023] Figure 1 shows biophysical analysis of an additional exemplary second-generation compound, 2-MeA20. All compounds were tested as the free base, except for QPB-NO4 (tested as the 2xHCl salt). The assay shown is binding of immobilized human ephrinB2 to 50 nM human EphB2. [Figure 49E]Figure 1 shows biophysical analysis of an additional exemplary second-generation compound, QPB-NO4. All compounds were tested as free bases except for QPB-NO4 (tested as the 2xHCl salt). The assay shown is binding of immobilized human ephrinB2 to 50 nM human EphB2. Note that compound QPB-NO4 exhibits little ability to prevent insurmountable tetramers at 3.2 μM concentration (bold), as its sensorgram falls below the maximum control. [Figure 50A] QPA: Biophysical analysis of exemplary second generation compounds derived from QPA. The assay shown is binding of immobilized human ephrinB2 to 50 nM human EphB2. [Figure 50B] 1 shows biophysical analysis of exemplary second generation compounds derived from QPA-Ac. The assay shown is binding of immobilized human ephrinB2 to 50 nM human EphB2. [Figure 50C] 1 shows biophysical analysis of exemplary second generation compounds derived from QPA-PAc. The assay shown is binding of immobilized human ephrinB2 to 50 nM human EphB2. [Figure 50D] 1 shows biophysical analysis of exemplary second generation compounds derived from BQPA-PAc. The assay shown is binding of immobilized human ephrinB2 to 50 nM human EphB2. [Figure 51A] 3511: Biophysical analysis of exemplary second-generation compounds derived from 3511-I. The assay shown is the binding of immobilized human ephrin B2 to 50 nM human EphB2. Note that compound 3511-I exhibits a fairly strong ability to block a portion of the insurmountable tetramer at the 0.8 and 1.6 μM concentrations tested. [Figure 51B] 1 shows biophysical analysis of exemplary second generation compounds derived from 3511-0013-BF. The assay shown is binding of immobilized human ephrinB2 to 50 nM human EphB2. [Figure 51C]

[0023] Figure 1 shows biophysical analysis of exemplary second-generation compounds derived from 3511-4OMe-BF. The assay shown is the binding of immobilized human ephrin B2 to 50 nM human EphB2. Note that compound 3511-4OMe-BF also has the ability to block a portion of the insurmountable tetramer at a concentration of 3.2 μM. [Figure 51D] 1 shows biophysical analysis of exemplary second generation compounds derived from 3511-4OHMe-I. The assay shown is binding of immobilized human ephrin B2 to 50 nM human EphB2. [Figure 51E] 1 shows biophysical analysis of exemplary second generation compounds derived from IQPB4-BN. The assay shown is binding of immobilized human ephrinB2 to 50 nM human EphB2. [Figure 52]

[0023] Figure 1 shows biophysical analysis of the second-generation compound BQPB4, demonstrating that it breaks through and inhibits 100% of insurmountable tetramers at low micromolar concentrations. The assay shown is the binding of immobilized human ephrinB2 to 50 nM human EphB2. Note that BQPB4 exhibits a strong concentration-dependent ability to prevent the formation of ultrastable cyclic tetramers with an IC50 of approximately 300 nM, and that at the 0.8, 1.6, and 3.2 μM concentrations tested, insurmountable tetramers are prevented by complete loss of this structure at 3.2 μM. [Figure 53] FIG. 1 shows compounds that inhibit insurmountable ephrinB2-EphB2 tetramers. [Figure 54] FIG. 1 is a schematic representation of medicinal chemistry efforts toward A20 lead compounds that exhibit alterations that can enhance or decrease EPH-ephrin tetramerization inhibitor activity. [Figure 55] Potential site modifications of A20 derivatives: (red) piperazine substitution, (yellow) hydrophobic ring expansion, (green) carbon substitution, (blue) halogen substitution, and (purple) alcohol protection. [Figure 56]Potential site modifications of 3511-0013, BQPB4, and BQPB4-bn: (green) number of n = -CHr extensions, (yellow) terminal substituents on extended phenyl / benzyl rings, (purple) substituents meta to R1, (blue) halogen substitutions. [Figure 57] Figure 57A shows that EphB1 receptor and ephrinB2 ligand expression increases in the spinal cord after nerve injury producing chronic pain. (Figure 57A) Within 25 hours after chronic constriction injury (CCI) of the rat sciatic nerve, a strong and sustained increase in EphB1 expression was detected by immunoblotting in the ipsilateral (I), injured side of the spinal cord compared with the uninjured (C) side and sham-treated mice (red arrow). (Figure 57B) In bone cancer pain, tumor cell implantation (TCI) of Walker-256 cancer cells into the intramedullary space of rat tibiae resulted in a significant increase in EphB1 and ephrinB2 expression in the ipsilateral, tumor cell-implanted side, as detected by immunofluorescence (green signal) of dissected spinal cords. Note that increased EphB1 and ephrinB2 expression was primarily restricted to the dorsal, superficial region of the ipsilateral spinal cord, where DH neurons are located and C fiber:DH neuron synapses are localized. Little, if any, expression could be detected in the contralateral control side of the spinal cord or in sham-treated rats. [Figure 58] Figure 1. EphB1 in nerve injury pain. In EphB1+ / + wild-type mice (filled circles), CCI of the sciatic nerve results in a sustained increase in sensitivity to heat (hyperalgesia), as indicated by a rapid response to an infrared heat source. EphB1- / - KO mice do not develop hyperalgesia after CCI (diamonds). Even EphB1+ / - heterozygous mice do not exhibit hyperalgesia after CCI (triangles). [Figure 59]This figure shows that EphB1 is required for dorsal horn neuron LTP. Electrophysiological recordings from the superficial spinal cord of live mice were used to assess the plasticity of synapses formed between peripheral nociceptive C fibers and DH neurons. After brief high-frequency stimulation (HFS) training of the sciatic nerve, EphB1+ / + (WT) mice showed robust and sustained increases in field potentials. Recordings from the spinal cord of EphB1- / - KO mice did not demonstrate LTP after HFS training. [Figure 60] Schematic diagram showing the localization of ephrinB2, EphB1, and NMDA receptors at the nociceptor C fiber-dorsal horn neuron synapse (see FIG. 2). [Figure 61] This figure shows that when injected into mice, A20 (red dotted line) strongly reduced inflammatory pain compared to the V control (black dotted line). A total of five wild-type (WT) male mice were IP injected with A20 free base (in 6% DMSO / 94% sunflower seed oil; three mice at 10 mg / kg and two at 20 mg / kg; data pooled), and three WT male mice were IP injected with vehicle (V) alone as controls. As indicated, injections of A20 or V began two days before a single injection of CFA into one of the two hind paws to initiate inflammatory pain, and injections continued for two additional days. For each day's withdrawal latency experiment, both the left and right hind paws were subjected to 6–12 thermal pain measurements to obtain the average response time for each mouse. Mice were 14 weeks old at the start of the experiment. [Figure 62]This figure shows that A20 appears more effective than MCD in reducing inflammatory pain. Wild-type (WT) female mice were IP injected with either A20 free base (20 mg / kg in 6% DMSO / 94% sunflower seed oil), MCD kinase inhibitor (10 mg / kg in PBS), or vehicle alone (6% DMSO / 94% sunflower seed oil). Injections of A20, MCD, or V were initiated two days before a single CFA injection into one of the two hind paws to initiate inflammatory pain, and injections continued for two additional days. For each day's withdrawal latency experiment, both the left and right hind paws were subjected to 6–12 thermal pain measurements to obtain the mean response time. For each mouse, the mean uninjured hind paw response time was divided by the mean injured side response time to obtain the pain response ratio for each mouse, which was then plotted and subjected to the statistical analysis shown. Data shown were obtained on day +3 after CFA. Mice were 20 weeks old at the start of the experiment. [Figure 63] Figure 1 shows the effectiveness of salt versions of compounds A20 and QPB4-Bn in reducing inflammatory pain when injected after pain-producing injury and when PBS was used as the vehicle. Wild-type (WT) male and female mice were IP injected with either A20 2xHCl salt (20 mg / kg in PBS), QPB4-Bn 2xHCl salt (20 mg / kg in PBS), or vehicle alone (PBS). Here, the first injection of A20, QPB4-Bn, or V began 15 minutes after CFA injection into one of the two hind paws to initiate inflammatory pain, and injections continued every 12 hours for three additional days. For each day's withdrawal latency experiment, both the left and right hind paws were subjected to 6–12 thermal pain measurements to obtain the average response time. For each mouse, the average non-injured hind paw response time was divided by the average injured side response time to obtain the pain response ratio for each mouse, which was then plotted and subjected to the statistical analysis shown. Data shown were obtained on day +3 after CFA. Mice were 8 weeks old at the start of the experiment. [Figure 64]This figure shows that the salt forms of two additional A20 analogs, QPB4 and QPP (QPP127), were tested and shown to be effective in reducing inflammatory pain when injected after pain-producing injury and when PBS was used as the vehicle. Wild-type (WT) female mice were IP injected with QPB4 2×HCl salt (20 mg / kg in PBS), QPP (QPP127) 2×HCl salt (20 mg / kg in PBS), or vehicle alone (PBS). Here, the first injection of QPB4, QPP (QPP127), or vehicle began 15 minutes after CFA injection into one of the two hind paws to initiate inflammatory pain, and injections continued every 12 hours for an additional 3 days. For each day's withdrawal latency experiment, both the left and right hind paws were subjected to 6-12 thermal pain measurements to obtain the average response time. For each mouse, the average non-injured hind paw response time was divided by the average injured side response time to obtain the pain response ratio for each mouse, which was then plotted and subjected to the statistical analysis shown. Data shown were obtained on day +3 after CFA. Mice were 12 weeks old at the start of the experiment. Topical MCD cream was also evaluated in this experiment and showed similar efficacy. [Figure 65]Development of a quantitative fluorescent method for assessing DH neuron activation in the spinal cord after chronic neuropathic injury to peripheral nerves. EphB1+ / + wild-type, EphB1+ / - heterozygous, and EphB1- / - homozygous mutant mice, also containing the Trap2 / CreERT2 driver and Ai9 / tdTomato reporter, received a single CFA injection into the left hind paw to induce inflammatory pain. Four hours later, the mice were IP injected with 4-hydroxytamoxifen (4-OHT, 40 mg / kg in sunflower seed oil) to activate Cre recombinase, permanently labeling activated neurons with red fluorescence for a short period (approximately 8 hours) until 4-OHT was degraded. After 14 days, animals were perfused with 4% paraformaldehyde in PBS. 50 μM-thick serial vibratome sections of the lumbar spinal cord were then obtained, mounted, and observed under a fluorescent microscope to identify red-labeled neurons in the superficial dorsal horn of the spinal cord that were activated approximately 4–12 h after CFA induction of inflammatory pain. Superficial red neurons were counted in the left (injured) and right (uninjured) dorsal horns (boxed areas) from 8–12 serial sections along the length of the lumbar spinal cord of each animal. The ratio of Trap2+ activated neurons for each animal was then determined by dividing the average number of red neurons from the injured side by the number obtained from the uninjured side. Note that WT mice showed nearly three times more red-labeled neurons in the injured dorsal horn compared to the uninjured side, indicating high levels of neuronal activation after injury that generates inflammatory pain to the left hindpaw. In contrast, EphB1- / - knockouts showed only a very small left-right difference (ratio of approximately 1), and all EphB1+ / - heterozygotes showed an intermediate ratio of approximately 2, indicating a gene dose-dependent effect on neuronal activation, consistent with the behavioral observation that heterozygotes are also refractory to injury that produces chronic pain. Twenty-week-old females were used in this experiment. [Figure 66]This figure shows that A20 reduces DH neuron activation in WT mice after CFA inflammatory insult. Representative fluorescent images of the dorsal lumbar spinal cord of vehicle- and A20-injected mice are shown. Superficial DH neurons on the left, CFA-injected side of vehicle-treated mice exhibited approximately three times more Trap2 / tdTomato red fluorescently labeled neurons compared to the uninjured right side. A20-treated mice exhibited significantly fewer labeled neurons on the injured side. The plot ratio of left / right red neurons per mouse was determined by averaging values ​​obtained from 8–12 serial sections along the length of the lumbar spinal cord for each animal. Note that while the small n value precluded one-way ANOVA analysis, unpaired t-tests demonstrate significant differences between A20- and vehicle-treated animals, but not the MCD kinase inhibitor-treated group. [Figure 67] FIG. 1 shows that EphB1 receptor protein expression increases in the spinal cord with increasing doses of morphine. [Figure 68] FIG. 1 shows that EphB1 − / − knockout mice, which lack expression of this receptor protein, exhibit greatly reduced morphine withdrawal behavior. [Figure 69] Figure 1 shows that EphB1- / - knockout mice, which lack expression of this receptor protein, exhibit greatly reduced morphine withdrawal behavior. This withdrawal study confirmed the involvement of EphB1. [Figure 70A] Figure 1 shows that A20 strongly reduced morphine withdrawal behavior in mice. Results are pooled from experiments using CD1 mice (3 mice V and 3 mice A20) and C57BL / 6 mice (3 mice V and 4 mice A20). The two mice that received zero points for jumping, especially the V mouse, may have received an insufficient naloxone injection. [Figure 70B] A20 potently reduced morphine withdrawal behavior in mice. Pooled results from experiments using C57BL / 6. [Figure 71A] FIG. 1 shows reaction schematics and representative NMR spectra for exemplary compounds of the present disclosure. [Figure 71B] FIG. 1 shows reaction schematics and representative NMR spectra for exemplary compounds of the present disclosure. [Figure 71C] FIG. 1 shows reaction schematics and representative NMR spectra for exemplary compounds of the present disclosure. [Figure 71D] FIG. 1 shows reaction schematics and representative NMR spectra for exemplary compounds of the present disclosure. [Figure 71E] FIG. 1 shows reaction schematics and representative NMR spectra for exemplary compounds of the present disclosure. [Figure 71F] FIG. 1 shows reaction schematics and representative NMR spectra for exemplary compounds of the present disclosure. [Figure 71G] FIG. 1 shows reaction schematics and representative NMR spectra for exemplary compounds of the present disclosure. [Figure 71H] FIG. 1 shows reaction schematics and representative NMR spectra for exemplary compounds of the present disclosure. [Figure 71I] FIG. 1 shows reaction schematics and representative NMR spectra for exemplary compounds of the present disclosure. [Figure 71J] FIG. 1 shows reaction schematics and representative NMR spectra for exemplary compounds of the present disclosure. [Figure 71K] FIG. 1 shows reaction schematics and representative NMR spectra for exemplary compounds of the present disclosure. [Figure 71L] FIG. 1 shows reaction schematics and representative NMR spectra for exemplary compounds of the present disclosure. [Figure 71M] FIG. 1 shows reaction schematics and representative NMR spectra for exemplary compounds of the present disclosure. [Figure 71N] FIG. 1 shows reaction schematics and representative NMR spectra for exemplary compounds of the present disclosure. [Figure 71O] FIG. 1 shows reaction schematics and representative NMR spectra for exemplary compounds of the present disclosure. [Figure 71P]FIG. 1 shows reaction schematics and representative NMR spectra for exemplary compounds of the present disclosure. [Figure 71Q] FIG. 1 shows reaction schematics and representative NMR spectra for exemplary compounds of the present disclosure. [Figure 71R] FIG. 1 shows reaction schematics and representative NMR spectra for exemplary compounds of the present disclosure. [Figure 71S] FIG. 1 shows reaction schematics and representative NMR spectra for exemplary compounds of the present disclosure. [Figure 71T] FIG. 1 shows reaction schematics and representative NMR spectra for exemplary compounds of the present disclosure. [Figure 71U] FIG. 1 shows reaction schematics and representative NMR spectra for exemplary compounds of the present disclosure. [Figure 71V] FIG. 1 shows reaction schematics and representative NMR spectra for exemplary compounds of the present disclosure. [Figure 71W] FIG. 1 shows reaction schematics and representative NMR spectra for exemplary compounds of the present disclosure. [Figure 71X] FIG. 1 shows reaction schematics and representative NMR spectra for exemplary compounds of the present disclosure. [Figure 72A] FIG. 1 shows Octet biophysical data (80 sec association and 500 sec dissociation) using binding of sensorchip-immobilized human ephrinB2-Fc ectodomain to 25 nM soluble human EphB1-His ectodomain, with 10 μM A20-I as the maximum control. [Figure 72B] FIG. 1 shows Octet biophysical data (80 sec association and 500 sec dissociation) using binding of sensor chip-immobilized human ephrin B2-Fc ectodomain to 25 nM soluble human EphB2-His ectodomain, with 10 μM A20-I as the maximum control. [Figure 72C]FIG. 1 shows Octet biophysical data (80 sec association and 500 sec dissociation) using binding of sensorchip-immobilized human ephrin B2-Fc ectodomain to 25 nM soluble human EphB4-His ectodomain, with 10 μM A20-I as a maximum control. [Figure 73A] Figure 1 shows the percent inhibition of binding of immobilized human ephrinB2-Fc to 50 nM soluble human EphB1-His protein (80 sec association and 500 sec dissociation) with A20-1 or BQPB4 as 10 μM maximum control. [Figure 73B] Figure 1 shows the percent inhibition of binding of immobilized human ephrinB2-Fc to 50 nM soluble human EphB2-His protein (80 sec association and 500 sec dissociation) with A20-1 or BQPB4 as 10 μM maximum control. [Figure 73C] Figure 1 shows the percent inhibition of binding of immobilized human ephrinB2-Fc to 50 nM soluble human EphB4-His protein (80 sec association and 500 sec dissociation) with A20-1 or BQPB4 as 10 μM maximum control. [Figure 74A] FIG. 1 shows representative Octet sensorgrams showing the effect of increasing concentrations of A20-I salt on the binding of immobilized human ephrinB2-Fc to 50 nM soluble human EphB1-His protein. [Figure 74B] FIG. 1 shows representative Octet sensorgrams showing the effect of increasing concentrations of A20-I salt on the binding of immobilized human ephrinB2-Fc to 50 nM soluble human EphB2-His protein. [Figure 74C] FIG. 1 shows representative Octet sensorgrams showing the effect of increasing concentrations of A20-I salt on the binding of immobilized human ephrinB2-Fc to 50 nM soluble human EphB4-His protein. [Figure 75A] FIG. 1 shows representative Octet sensorgrams showing the effect of increasing concentrations of BQPB4 salt on the binding of immobilized human ephrinB2-Fc to 50 nM soluble human EphB1-His protein. [Figure 75B]FIG. 1 shows representative Octet sensorgrams showing the effect of increasing concentrations of BQPB4 salt on the binding of immobilized human ephrinB2-Fc to 50 nM soluble human EphB2-His protein. [Figure 75C] FIG. 1 shows representative Octet sensorgrams showing the effect of increasing concentrations of BQPB4 salt on the binding of immobilized human ephrinB2-Fc to 50 nM soluble human EphB4-His protein. [Figure 76] Representative Octet sensorgrams (Figures 76A-C) and enlarged insets (Figures 76D-F) showing the maximum inhibition of different compounds (3511-I, 8009-9255, QTM-Br, A19, A19-NO2, and IM) on EphB1-EphrinB2 (Figure 76A, enlarged in Figure 76D), EphB2-EphrinB2 (Figure 76B, enlarged in Figure 76E), or EphB4-EphrinB2 (Figure 76C, enlarged in Figure 76F) tetramerization. [Figure 77A] FIG. 1 shows representative Octet sensorgrams demonstrating maximal inhibition of EphB1-EphrinB2 tetramerization using A19 and A19-NO2 (salt or free base form) compared to the maximal control, A20-I. [Figure 77B] FIG. 1 shows representative Octet sensorgrams demonstrating maximal inhibition of EphB2-EphrinB2 tetramerization using A19 and A19-NO2 (salt or free base form) compared to the maximal control, A20-I. [Figure 77C] FIG. 1 shows representative Octet sensorgrams demonstrating maximal inhibition of EphB4-EphrinB2 tetramerization using A19 and A19-NO2 (salt or free base form) compared to the maximal control, A20-I. [Figure 77D] FIG. 1 shows representative Octet sensorgrams demonstrating maximal inhibition of EphB1-EphrinB2 tetramerization using A19, A19-NO2 (salt or free base), QPB4-Bn, and BQPB4 compared to the maximal control, A20-I. [Figure 77E]FIG. 1 shows representative Octet sensorgrams demonstrating maximal inhibition of EphB2-ephrinB2 tetramerization using A19, A19-NO2 (salt or free base), QPB4-Bn, and BQPB4 compared to the maximal control, A20-I. [Figure 77F] FIG. 1 shows representative Octet sensorgrams demonstrating maximal inhibition of EphB4-ephrinB2 tetramerization using A19, A19-NO2 (salt or free base), QPB4-Bn, and BQPB4 compared to the maximal control, A20-I. [Figure 78] FIG. 1 shows the structure of A20 chemical entity that exhibits lower inhibitory activity against ephrinB2-EphB4 interaction and higher inhibitory activity against ephrinB2-EphB1 and / or ephrinB2-EphB2 interactions. [Figure 79] Representative immunoblots (Figure 79A) and quantification (Figures 17B-17C) of EphB2, phospho-EphB1 / B2 (pEphB1 / B2), and ephrin-B2 expression in brain protein lysates after intraperitoneal (IP) injection of A20, A20-I, and 3511-I and immunoprecipitation of EphB2 using an anti-EphB2 antibody. Figures 79B and 79C show quantification of band intensity (number of photons detected) to obtain the ratio of pEphB / EphB2 (Figure 79B) as a measure of EphB2 receptor activation level and the ratio of ephrin-B2 / EphB2 (Figure 79C) as a measure of how much ephrin-B2 is co-immunoprecipitated with the EphB2 receptor. [Figure 80A] Representative immunoblots following EphB2 immunoprecipitation from mouse brain protein lysates. Figure 80A shows EphB2 expression in mouse brain after 4 days of oral administration (PO) or injection (IP) with vehicle (PBS) or A20 or A20-I. [Figure 80B]Figure 80B shows a representative immunoblot after EphB2 immunoprecipitation from mouse brain protein lysate. Figure 80B shows the expression of phospho-Tyr1000 (pTyr1000) in mouse brain after 4 days of oral administration (PO) or injection (IP) with vehicle (PBS) or A20 or A20-I. [Figure 80C] Figure 80C shows a representative immunoblot following EphB2 immunoprecipitation from mouse brain protein lysates. Figure 80D shows the expression of 4G10 phosphotyrosine-specific antibody in mouse brain after 4 days of oral administration (PO) or injection (IP) with vehicle (PBS) or A20 or A20-I. [Figure 80D] Figure 80D shows the quantification of representative immunoblots after EphB2 immunoprecipitation from mouse brain protein lysates. Figure 80D shows the ratios of pTyr1000 / EphB2 and 4G10 / EphB2 (pooled for both intraperitoneal (IP) and oral gavage (PO) administration) in mice treated with vehicle or A20 compound. [Figure 80E] Figure 80E shows the quantification of representative immunoblots following EphB2 immunoprecipitation from mouse brain protein lysates. Figure 80E shows the ratios of pTyr1000 / EphB2 and 4G10 / EphB2 (IP and PO administrations separated) in the mice. [Figure 81] Representative immunoblots (Figures 81A-81C) and quantification (Figure 81D) of EphB2 (Figure 81A), pEphB1 / B2 (Figure 81B), and pTyr1000 (Figure 81C) after immunoprecipitation of EphB2 from brain lysates of WT, or EphB1 / EphB2 double homozygous knockout (E1N1), EphB2-K661R (homozygous kinase-dead point mutant), EphB2-F620D (homozygous kinase-hyperactive point mutant), and EphB2-lacZ (mutation resulting in an intracellular truncated EphB2-β-gal fusion protein migrating at 220 kDa (heterozygous N2 / +)) mice. Figure 81D shows the phospho-EphB2 / pan-EphB2 ratio in the indicated mice. [Figure 82A]FIG. 81B shows a darker representation of the EphB2 immunoblot shown in FIG. 81A. [Figure 82B] FIG. 81B shows tabulated chemiluminescent signal intensities of the EphB2 immunoblot shown in FIG. 81A. [Figure 83] Coomassie blue stained SDS-PAGE gel of liver and brain protein lysates after immunoprecipitation with goat anti-EphB2 antibody (Figure 83A) and the number of proteins identified in different brain tissues by mass spectrometry (Figure 83B). [Figure 84] Representative immunoblots of total liver protein lysates subjected to goat anti-EphB2 immunoprecipitation and immunoblot analysis using anti-EphB2 (Figure 84A), anti-phospho-EphB1 / B2 (Figure 84B), anti-4G10 (Figure 84C), and anti-pTry1000 (Figure 84D) antibodies. [Figure 85] A20 Schematic representation of the experimental protocol to evaluate the ability of oral administration of chemicals to blunt chronic long-term inflammatory pain induced by injection of complete Freund's adjuvant (CFA) into the mouse hind paw. [Figure 86A] Figure 1 shows mean mechanical pain thresholds (determined via von Frey filaments) categorized by treatment group. In the figure, the left panel represents data for the left (CFA-injured) hind paw, and the right represents data for the right (uninjured) hind paw. Animals were treated with vehicle or A20 (20 mg / kg) administered IP or PO (orally). [Figure 86B] Figure 1 shows mean mechanical pain thresholds (determined via von Frey filaments) categorized by treatment time. In the figure, the left panel represents data for the left (CFA-injured) hind paw, and the right represents data for the right (uninjured) hind paw. Animals were treated with vehicle or A20 (20 mg / kg) administered IP or PO (orally). [Figure 87A]Figure 87A shows paw withdrawal latency following contact with a thermal painful stimulus in mice treated with vehicle, A20 (IP, blue), or A20 (PO, yellow). The left hind paw was injured with CFA (same animals tested in Figure 86). Figure 87A provides data categorized by treatment group. [Figure 87B] Figure 87B shows paw withdrawal latency following contact with a thermal painful stimulus in mice treated with vehicle, A20 (IP, blue), or A20 (PO, yellow). The left hind paw was injured with CFA (same animals tested in Figure 86). Figure 87B shows data sorted by treatment time. [Figure 88] FIG. 87A shows the pain ratio calculated from the data in FIGS. 86A-B and 87A-B by dividing the uninjured side pain response by the injured side response. [Figure 89A] FIG. 1 shows mechanical pain measurements after Zymosin treatment in EphB2WT (green), null (blue), or heterozygous (yellow) mice. [Figure 89B] FIG. 1 shows heat pain measurements after Zymosin treatment in EphB2WT (green), null (blue), or heterozygous (yellow) mice. [Figure 89C] FIG. 1 shows mechanical pain ratios after Zymosin treatment in EphB2WT (green), null (blue), or heterozygous (yellow) mice. [Figure 89D] FIG. 1 shows heat pain ratios after Zymosin treatment in EphB2WT (green), null (blue), or heterozygous (yellow) mice. [Figure 90A] FIG. 1 shows mechanical pain ratio after Zymosin treatment and oral administration (PO) of vehicle (green), or 5 mg / kg (orange), 10 mg / kg (yellow), or 20 mg / kg (blue) A20. [Figure 90B] FIG. 1 shows heat pain ratio after Zymosin treatment and oral administration (PO) of vehicle (green), or 5 mg / kg (orange), 10 mg / kg (yellow), or 20 mg / kg (blue) A20. [Figure 90C]FIG. 1 shows pooled A20 data for mechanical pain. [Figure 90D] FIG. 1 shows pooled A20 data for heat pain. [Figure 91A] FIG. 1 shows mechanical pain measurements and ratios in animals treated with vehicle (green), 3511-1 (blue), or BQPB4 (yellow) after exposure to Zymposin pain stimuli. [Figure 91B] FIG. 1 shows thermal pain measurements and ratios in animals treated with vehicle (green), 3511-1 (blue), or BQPB4 (yellow) after exposure to Zymposin pain stimuli. [Figure 92] Figure 92A shows thermal pain measurements (Figure 92A) and ratios (Figure 92B) in animals exposed to Zymosin and treated with vehicle (green), or 20 mg / kg (blue), 10 mg / kg (yellow), or 5 mg / kg (orange) BQPB4 (oral administration). [Figure 93] 93A-93C show imaging and analysis of activated dorsal horn neurons after inflammatory pain insult. FIG. 93A shows a typical fluorescent image taken using a Zeiss Axioscan to rapidly capture images of mounted sections from the lumbar spinal cord of all animals under analysis. FIG. 93B shows an example plot of the number of uninjured red Tom+ DH neurons counted from the left and right sides of 12 different imaged sections from the same animal. FIG. 93C shows a histogram plot of the data as a function of the number of sections, with the number of Tom+ DH neurons x. [Figure 94] Figure 94A shows plots showing the ratio of Tom+ DH neurons (lesioned / unlesioned) for all sections analyzed (Figure 94A) and the average ratio when all sections of an individual spinal cord are grouped together (Figure 94B). [Figure 95] Figure 94A shows plots showing Tom+ DH neurons (injured / uninjured side) for all sections analyzed (12 sections per mouse) from animals treated with vehicle or different A20 compounds after Zymosin administration (Figure 94A), and the average ratio when all sections from individual spinal cords are grouped together (Figure 94B). DETAILED DESCRIPTION OF THE INVENTION

[0036] In some aspects, the present disclosure is based on the surprising discovery and identification of novel low molecular weight compounds that selectively inhibit EPH-ephrin receptor-ligand tetramerization.These compounds can be used to inhibit Eph anterograde signaling and ephrin retrograde signaling (bidirectional signaling), and can have potential applications in various therapeutics.Therefore, various EPH-ephrin tetramerization inhibitors are described herein together with their treatment methods.

[0037] I. Definition Unless otherwise defined, all technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this invention belongs. nd ed.1994);The Cambridge Dictionary of Science and Technology(Walker ed.,1988);The Glossary of Genetics,5 th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991), provide those skilled in the art with general definitions of many of the terms used in this invention. As used herein, the following terms have the meanings ascribed to them unless otherwise specified.

[0038] When introducing elements of the present disclosure or preferred embodiments thereof, the articles "a," "an," "the," and "said" are intended to mean that there are one or more elements. The terms "comprising," "including," and "having" are intended to be inclusive and mean that additional elements may be present other than the listed elements. Whenever the terms "comprising" or "including" are used, it should be understood that the present disclosure also expressly contemplates and encompasses additional embodiments "consisting of" the disclosed elements that do not include additional elements other than the listed elements.

[0039] "Pharmaceutical composition" means a mixture of substances, including pharmaceutical agents, suitable for administration to an individual. As used herein, a pharmaceutical composition comprises one or more receptors, vectors, cells disclosed herein formulated with a suitable pharmaceutical carrier or excipient.

[0040] "Treatment" or "treatment" of a subject refers to any type of intervention or process performed on a subject, or administration of an active agent to a subject, with the intent of ameliorating, alleviating, ameliorating, inhibiting, slowing, or preventing the onset, progression, development, severity, or recurrence of symptoms, complications, conditions, or biochemical manifestations associated with a disease.

[0041] As used herein, the terms "patient," "subject," or "test subject" refer to any organism to which a provided compound or a compound described herein is administered in accordance with the present invention, e.g., for experimental, diagnostic, prophylactic, and / or therapeutic purposes. Typical subjects include animals (e.g., mammals such as mice, rats, rabbits, dogs, cats, horses (and other domestic animals), non-human primates, and humans). In certain embodiments, the subject is a human. In some embodiments, the subject may be suffering from and / or susceptible to a disease, disorder, and / or condition (e.g., a condition caused or exacerbated by aberrant, defective, or excessive EPH-ephrin signaling).

[0042] The term "effective amount," as used herein, is defined as the amount of a molecule of the present invention necessary to produce a desired physiological change in a cell or tissue to which it is administered. The term "therapeutically effective amount," as used herein, is defined as the amount of a molecule of the present invention that achieves a desired effect for whatever condition is being treated. For example, the desired effect in a method for treating pain may be a reduction or amelioration of pain in the subject, while the desired effect in a method for treating cancer may be a reduction in tumor size or cessation of tumor growth. Those skilled in the art will readily recognize that in many cases, a molecule may not provide a cure, but may provide a partial benefit, such as a reduction or improvement of at least one symptom or parameter. In some embodiments, a physiological change that has a benefit is also considered therapeutically beneficial. Thus, in some embodiments, the amount of a molecule that provides a physiological change is considered an "effective amount" or a "therapeutically effective amount."

[0043] As used herein, the term "alkyl" refers to a saturated hydrocarbon having one or more carbon atoms, including straight-chain alkyl groups (e.g., methyl, ethyl, propyl, butyl, pentyl, hexyl, etc.), cyclic alkyl groups (or "cycloalkyl" or "alicyclic" or "carbocyclic" groups) (e.g., cyclopropyl, cyclopentyl, cyclohexyl, etc.), branched-chain alkyl groups (isopropyl, tert-butyl, sec-butyl, isobutyl, etc.), and alkyl-substituted alkyl groups (e.g., alkyl-substituted cycloalkyl and cycloalkyl-substituted alkyl groups). The terms "alkenyl" and "alkynyl" refer to unsaturated aliphatic groups analogous to alkyl, but which contain at least one double or triple carbon-carbon bond, respectively.

[0044] As used herein, the term "alkoxy" refers to an alkyl group attached to the rest of the molecule through an oxygen atom. Examples of alkoxy groups include, but are not limited to, methoxy, ethoxy, isopropyloxy, propoxy, butoxy, and pentoxy groups. Alkoxy groups may be straight-chain or branched.

[0045] As used herein, the term "amino" refers to an unsubstituted or substituted moiety of the formula -NraRb, where Ra and Rb are each independently hydrogen, alkyl, aryl, or heterocyclyl, or Ra and Rb, together with the nitrogen atom to which they are attached, form a cyclic moiety having 3 to 8 atoms in the ring. Thus, unless otherwise specified, the term amino includes cyclic amino moieties such as piperidinyl or pyrrolidinyl groups.

[0046] As used herein, the term "heterocyclic group" refers to a closed ring structure similar to a carbocyclic group in which one or more of the carbon atoms in the ring is an element other than carbon, such as nitrogen, sulfur, or oxygen. Heterocyclic groups can be saturated or unsaturated. Furthermore, heterocyclic groups (such as pyrrolyl, pyridyl, isoquinolyl, quinolyl, purinyl, and furyl) can have aromatic character, in which case they can be referred to as "heteroaryl" or "heteroaromatic" groups. Exemplary heterocyclic groups include, but are not limited to, pyrrole, furan, thiophene, thiazole, isothiazole, imidazole, triazole, tetrazole, pyrazole, oxazole, quinoline, piperazine, pyridine, pyrazine, pyridazine, pyrimidine, benzoxazole, benzodioxazole, benzothiazole, benzimidazole, benzothiophene, methylenedioxyphenyl, quinoline, isoquinoline, naphthyridine, indole, benzofuran, purine, benzofuran, deazapurine, or indolizine.

[0047] As used herein, the term "acceptable salt" refers to a salt of a compound of the present invention that is acceptable for the methods of the present invention.

[0048] As used herein, the term "haloalkyl" refers to an alkyl group, as defined herein, that is substituted with one or more halogens. For example, trifluoromethyl is a haloalkyl group.

[0049] As used herein, the term "cyano" refers to a moiety containing a carbon connected to a nitrogen with a triple bond (eg, --CN).

[0050] As used herein, the term "azide" refers to a linear polyatomic anion having three nitrogens (e.g., -N3) of the formula N-3 and the structure -N=N+=N-.

[0051] As used herein, the term "amide" refers to a functional group that includes a primary, secondary, or tertiary amide. The "amide" group may further include additional R groups (e.g., additional alkyl, cycloalkyl, aryl, etc.).

[0052] As used herein, the term "carbonyl" refers to a functional group that includes at least one -CO moiety. The carbonyl may further include additional R groups (e.g., additional alkyl, cycloalkyl, aryl, etc.).

[0053] As used herein, the term C4-C10 aryl refers to an aromatic ring containing 4 to 10 carbons and no heteroatoms. As used herein, the term C4-C10 heteroaryl refers to an aromatic ring of 4 to 10 carbons in which at least one carbon is replaced with a heteroatom (e.g., N, O, or S). As used herein, C4-C10 cycloalkyl refers to a non-aromatic cyclic moiety having 4 to 10 carbons. As used herein, C4-C10 heterocycloalkyl refers to a non-aromatic cyclic moiety having 4 to 6 carbon atoms in which at least one carbon atom is replaced with a heteroatom (e.g., N, O, or S). Any of the C4-C10 aryl, heteroaryl, cycloalkyl, or heterocycloalkyl used herein may be optionally substituted unless otherwise specified. As used herein, the term C4-C10, when referring to a ring (e.g., a cycloalkyl, aryl, heterocycloalkyl, or heteroaryl as described above), explicitly and implicitly contemplates any intervening ring size (e.g., 4-, 5-, 6-, 7-, 8-, 9-, or 10-membered ring).

[0054] As used herein, the term "capable of forming a fused ring with" means that a referenced functional group (e.g., an R group) attached to a first ring can be connected to form a second ring fused to the first ring. The fused ring can be a fused aryl ring.

[0055] As used herein, the term EPH-ephrin tetramerization refers to the formation of an Eph receptor-ephrin ligand macromolecular complex between any of the 14 known EPH receptors and any of the eight ephrin ligands, A and B classes. EPH-ephrin dimers first associate / bind, and then two of these dimers associate / bind to form an EPH-ephrin tetramer, a highly stable ring-shaped structure that brings together the four components, activates the receptor and ligand, and transmits anterograde and retrograde signals to cells in which they are expressed, mediating bidirectional signaling. Once formed, the EPH-ephrin tetramers then assemble into much larger tetrameric clusters, resulting in greatly enhanced bidirectional signaling. Because the formation of these larger tetrameric clusters cannot occur without tetramers, it is important to remember that compounds that affect tetramerization also affect the formation of these larger tetrameric clusters. EPH-ephrin tetramerization and tetrameric clustering are distinct from dimerization, which may or may not occur early. The rates of EPH-ephrin tetramerization and tetramer clustering can be determined according to methods in the art and as described in the Examples herein below.

[0056] As used herein, disclosure of numerical ranges by numerical endpoints includes all numbers encompassed by that range (e.g., "1 to 5" includes, but is not limited to, 1, 1.25, 1.5, 1.75, 2, 2.3, 2.5, 2.8, 3, 3.1, 3.3, 3.8, 3.9, 4, 4.25, 4.5, 4.75, and 5). Unless otherwise indicated, all numbers used herein to express quantities, amounts, dimensions, measurements, and the like are to be understood as encompassing the specific quantity, amount, dimension, measurement, and the like, as well as examples of such modified by the term "about." Accordingly, unless otherwise indicated, the numerical descriptions set forth herein can vary within the teachings of the present disclosure. At the very least, each numerical value should be construed in light of the number of significant digits and by applying routine rounding techniques. It is intended that everything contained in the above description and in the examples given below be interpreted as illustrative and not limiting, as various changes can be made in the cells and methods without departing from the scope of the present invention.It is intended that everything contained in the above description and in the examples given below be interpreted as illustrative and not limiting, as various changes can be made in the cells and methods without departing from the scope of the present invention.

[0057] II. Compounds In various embodiments of the present disclosure, a compound of Formula I: [ka] or a pharmaceutically suitable salt thereof, wherein A is -O-, -SO2, CH2, or -N(CH2) n R1; B is CH2, SO2, or CO; X is hydrogen, halogen, alkenyl, alkyl, -NO2, or -NH2; each R is independently hydrogen, alkyl, alkoxy, heterocycloalkyl, or carbonyl, and R1 is substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, or substituted or unsubstituted heterocycloalkyl; and n=0-3).

[0058] In certain aspects, n can be 0 or 1. In yet further aspects, X can be hydrogen, bromo, iodo, chloro, -NO2, or -NH2, methyl, or propenyl. In further embodiments, each R can be hydrogen, methyl, [ka] and R may be independently selected from the group consisting of: For example, each R may independently be hydrogen or methyl. In certain embodiments, each R is hydrogen.

[0059] In any of the compounds provided herein, R1 can be a substituted or unsubstituted C4-C10 aryl (e.g., C4-C8 or C4-C6 aryl), a substituted or unsubstituted C4-C10 heteroaryl (e.g., C4-C8 or C4-C6 heteroaryl), a substituted or unsubstituted C4-C10 cycloalkyl (e.g., C4-C8 or C4-C6 cycloaryl), or a substituted or unsubstituted C4-C10 heterocycloalkyl (e.g., C4-C8 or C4-C6 heterocycloalkyl). For example, in various embodiments, R1 can be a substituted or unsubstituted phenyl, substituted or unsubstituted pyridinyl, substituted or unsubstituted thiazole, substituted or unsubstituted thiophenyl, substituted or unsubstituted piperazine, an unsubstituted C4-C10 cycloalkyl, or an unsubstituted C4-C10 heterocycloalkyl. For example, R1 can be: [ka] wherein each Y is independently -CH- or -O-; each R is independently hydrogen, -NO, alkoxy, an alkyl ether of -ROR, -OCH, -NHOR, -NH, halo, haloalkyl, or alkyl; R and R are each independently C-C alkyl, and R is alkyl or benzyl. may be selected from the group consisting of:

[0060] In any of the compounds of the present disclosure, each R2 is independently hydrogen, -NO2, -OCH3, -CH2CH2OCH3, -NHOCH3, -CF3, -NH2, bromo, chloro, fluoro, iodo, methyl, or [ka] It could be.

[0061] Thus, in various embodiments, R1 is [ka] It could be.

[0062] In certain embodiments, the compounds disclosed herein are: (1) when A is -O-, B is CH2, and R is hydrogen, then X is not hydrogen, chloro, -NO2, or bromo; (2) when A is NR1, and R1 is methyl or [ka] where X is not bromine, then the compound is of formula 1.

[0063] In certain embodiments, the compound of formula I is [ka] is not a compound selected from

[0064] In various embodiments, the compound of formula I is [ka] [ka] [ka] and any pharmaceutically suitable salts thereof.

[0065] In a further aspect, the compounds provided herein are [ka] or any pharmaceutically suitable salt thereof.

[0066] As described in more detail in the Examples below, it has surprisingly been found that some pharmaceutically suitable salt versions of compounds of Formula I exhibit improved properties (e.g., improved ability to inhibit Eph / ephrin tetramerization) compared to their non-salt (e.g., free base) forms. Accordingly, in some embodiments, the compound of Formula I is a pharmaceutically suitable salt (e.g., HCl salt).

[0067] In some embodiments, the pharmaceutically suitable salt is [ka] may be selected from the group consisting of:

[0068] In a further aspect, the pharmaceutical salt is [ka] may be selected from the group consisting of:

[0069] For ease of reference, suitable compounds encompassed by Formula I and related compounds used in the examples below are provided in Table 1 below.

[0070] [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] Table 1-6 Table 1-7 Table 1-8 Table 1-9 Table 1-10 Table 1-11 Table 1-12 Table 1-13 Table 1-14 Table 1-15 Table 1-16 Table 1-17 Table 1-18 Table 1-19 Table 1-20 Table 1-21 Table 1-22

[0071] As mentioned, any compound of Formula I described herein can inhibit EPH-ephrin tetramerization. As further discussed in the Examples, there are 14 different Eph receptors and 8 different ephrin ligands in two classes (A and B). In some embodiments, the compounds herein can be general inhibitors of EPH-ephrin tetramerization, resulting in the inhibition of any combination of Eph / ephrins. In some embodiments, the compounds can specifically inhibit a class of Eph / ephrins (e.g., EphB / ephrinB). In some embodiments, the compounds can specifically inhibit (e.g., preferentially inhibit) the tetramerization of EphB1, EphB2, or EphB4 with one or more respective ephrins. In some embodiments, the compounds can specifically inhibit EphB1 / ephrin and / or EphB2 / ephrin tetramerization over EphB4 / ephrin tetramerization. In some embodiments, the compounds may specifically inhibit EphB1 / ephrin tetramerization over EphB2 / ephrin and / or EphB4 / ephrin tetramerization, hi some embodiments, the compounds may specifically inhibit EphB2 / ephrin tetramerization over EphB1 / ephrin and / or EphB4 / ephrin tetramerization.

[0072] As used herein, the term "specifically inhibits" does not necessarily mean that the reference compound does not have the ability to inhibit the tetramerization of undesired Eph / ephrin combinations. As shown in more detail in the illustrative examples below, many of the potent tetramerization inhibitors disclosed herein (e.g., A20-I or BQPB4) generally exhibit the ability to inhibit / antagonize EphB2-ephrinB2 binding by about 60%, EphB1-ephrinB2 binding by about 30%, and EphB4-ephrinB2 binding by about 10% during the binding phase (e.g., having a 60:30:10 ratio of inhibition of EphB2:EphB1:EphB4 binding to ephrinB2). However, some compounds, particularly those considered "specific inhibitors" herein, exhibit preferential ability to inhibit, for example, EphB2 or EphB1 tetramerization over EphB4 tetramerization. For example, as shown in the following examples, compounds A19-NO2 and QTM-NO2 remain potent inhibitors of EphB1-ephrinB2 and EphB2-ephrinB2 binding, but show only slight, if any, inhibition of EphB4-ephrinB2 binding.These compounds show, for example, a 40:25:0 ratio of inhibition of EphB2:EphB1:EphB4 binding to ephrinB2, and show significantly reduced activity against EphB4-ephrinB2 interaction.Thus, as used herein, and as described herein, a compound that specifically inhibits the tetramerization of EphB1-ephrin and / or EphB2-ephrin rather than EphB4-ephrin may, in some embodiments, show less than 10% inhibition of EphB4 binding.In some embodiments, the compound cannot detectably inhibit EphB4.

[0073] In some aspects, compounds provided herein that specifically inhibit EphB1 / ephrin and / or EphB2 / ephrin tetramerization are [ka] or a pharmaceutically suitable salt thereof. In various embodiments, these compounds that specifically inhibit EphB1-ephrin and / or EphB2-ephrin tetramerization do not inhibit (or do not significantly inhibit) EphB4-ephrin tetramerization.

[0074] In some aspects, the compounds provided herein that specifically inhibit EphB1-ephrin tetramerization are [ka] or a pharmaceutically suitable salt thereof. In various embodiments, these compounds that specifically inhibit EphB1-ephrin tetramerization do not inhibit (or do not significantly inhibit) EphB4-ephrin tetramerization. In various embodiments, these compounds that specifically inhibit EphB1-ephrin tetramerization do not inhibit (or do not significantly inhibit) EphB2-ephrin tetramerization. In various embodiments, these compounds that specifically inhibit EphB1-ephrin tetramerization do not inhibit (or do not significantly inhibit) EphB4-ephrin and EphB2-ephrin tetramerization.

[0075] In some aspects, compounds provided herein that specifically inhibit EphB2-ephrin tetramerization are [ka] or a pharmaceutically suitable salt thereof. In various embodiments, these compounds that specifically inhibit EphB2-ephrin tetramerization do not inhibit (or do not significantly inhibit) EphB4-ephrin tetramerization. In various embodiments, these compounds that specifically inhibit EphB2-ephrin tetramerization do not inhibit (or do not significantly inhibit) EphB1-ephrin tetramerization. In various embodiments, these compounds that specifically inhibit EphB2-ephrin tetramerization do not inhibit (or do not significantly inhibit) EphB4-ephrin and EphB1-ephrin tetramerization.

[0076] In any of the above embodiments, the compound may have an IC50 of less than 2 μM, less than 1.6 μM, less than 1 μM, or less than 0.5 μM. In some embodiments, the compound may have an IC50 of greater than about 1.6 μM. In other embodiments, the compound may have an IC50 of about 1.0 to 1.6 μM. In still other embodiments, the compound may have an IC50 of about 0.4 to about 1.0 μM. In still other embodiments, the IC50 may be less than 0.4 μM. IC50 and EPH-ephrin inhibition may be measured according to methods known in the art, including those described in the Examples herein.

[0077] III. Pharmaceutical Formulations and Treatment Regimens The compounds disclosed herein for use in accordance with the methods described herein can be provided by themselves or as part of a pharmaceutical composition in which the compound can be mixed with a suitable carrier or excipient.

[0078] As used herein, "pharmaceutical composition" refers to a preparation of one or more active ingredients described herein with other chemical components, such as physiologically suitable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration of a compound to an organism.

[0079] As used herein, the term "active ingredient" refers to a compound, such as the compounds described herein, that inhibits EPH-ephrin tetramerization.

[0080] Pharmaceutically Acceptable Carriers and Excipients Hereinafter, the phrases "physiologically acceptable carrier" and "pharmaceutically acceptable carrier," which may be used interchangeably, refer to a carrier or diluent that does not cause significant irritation to an organism and does not interfere with the biological activity and properties of the administered compound. Adjuvants are included in these phrases.

[0081] In various embodiments, the compositions disclosed herein may further comprise one or more pharmaceutically acceptable diluents, excipients, or carriers. As used herein, a pharmaceutically acceptable diluent, excipient, or carrier refers to a material that is suitable for administration to a subject without causing any undesired biological effects or adversely interacting with any of the components of the composition in which it is contained. Pharmaceutically acceptable diluents, carriers, and excipients include, but are not limited to, saline, Ringer's solution, phosphate solution or buffer, buffered saline, and other carriers known in the art. Pharmaceutical compositions may also include stabilizers, antioxidants, colorants, other medicinal or pharmaceutical agents, carriers, adjuvants, preservatives, stabilizers, wetting agents, emulsifiers, solubility enhancers, salts, solubilizers, antifoaming agents, antioxidants, dispersing agents, surfactants, and combinations thereof. As used herein, the term "excipient" refers to an inert substance added to a pharmaceutical composition to further facilitate administration of an active ingredient. Examples of excipients include, but are not limited to, calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils, and polyethylene glycols. Techniques for drug formulation and administration may be found in "Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton, Pa., latest edition, which is incorporated herein by reference.

[0082] In various embodiments, the pharmaceutical compositions described herein can be formulated in a conventional manner using one or more physiologically acceptable carriers, including excipients and auxiliary agents that facilitate the processing of recombinant endothelial progenitor cells into pharmaceutically usable preparations. In other embodiments, any of the well-known techniques, carriers, and excipients can be used as appropriate and as understood in the art.

[0083] In various embodiments, the pharmaceutical compositions described herein can be aqueous suspensions containing one or more polymers as suspending agents.In some aspects, the polymers that can be contained in the pharmaceutical compositions described herein include water-soluble polymers such as cellulose polymers, for example, hydroxypropylmethylcellulose; water-insoluble polymers such as cross-linked carboxyl-containing polymers; mucoadhesive polymers selected from, for example, carboxymethylcellulose, carbomer (acrylic acid polymer), poly(methyl methacrylate), polyacrylamide, polycarbophil, acrylic acid / butyl acrylate copolymer, sodium alginate, and dextran; or combinations thereof.In other aspects, the compositions disclosed herein can contain at least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50% of the total amount of polymer as suspending agent based on the total weight of the composition.

[0084] In various embodiments, the pharmaceutical compositions disclosed herein may comprise a viscous formulation. In some aspects, the viscosity of the composition may be increased by adding one or more gelling agents or thickening agents. In other aspects, the compositions disclosed herein may comprise one or more gelling agents or thickening agents in an amount that provides the formulation with sufficient viscosity to remain on the treated tissue. In still other aspects, the compositions disclosed herein may comprise a total amount of gelling agents or thickening agents of at least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50% by weight of the total composition. In still other aspects, suitable thickening agents may be hydroxypropylmethylcellulose, hydroxyethylcellulose, polyvinylpyrrolidone, carboxymethylcellulose, polyvinyl alcohol, sodium chondroitin sulfate, or sodium hyaluronate.In other embodiments, the thickening agent is selected from the group consisting of acacia (gum arabic), agar, magnesium aluminum silicate, sodium alginate, sodium stearate, fucus, bentonite, carbomer, carrageenan, Carbopol, xanthan, cellulose, microcrystalline cellulose (MCC), ceratonia, chitin, carboxymethylated chitosan, hornwort, dextrose, furcellaran, gelatin, ghatti gum, guar gum, hectorite, lactose, sucrose, maltodextrin, mannitol, sorbitol, honey, corn starch, wheat starch, rice starch, potato starch, gelatin, sterculia gum, xanthan gum, tragacanth gum, ethyl cellulose, ethyl hydroxyethyl cellulose, Can be ethyl methylcellulose, methylcellulose, hydroxyethylcellulose, hydroxyethylmethylcellulose, hydroxypropylcellulose, poly(hydroxyethyl methacrylate), oxypolygelatin, pectin, polygelatin, povidone, propylene carbonate, methyl vinyl ether / maleic anhydride copolymer (PVM / MA), poly(methoxyethyl methacrylate), poly(methoxyethoxyethyl methacrylate), hydroxypropylcellulose, hydroxypropylmethylcellulose (HPMC), sodium carboxymethylcellulose (CMC), silicon dioxide, polyvinylpyrrolidone (PVP: povidone), Splenda® (dextrose, maltodextrin, and sucralose), or combinations thereof.In some embodiments, suitable thickener can be carboxymethylcellulose.

[0085] In various embodiments, the pharmaceutical compositions disclosed herein may contain additional agents or additives selected from the group including surfactants, detergents, solvents, acidifying agents, alkalizing agents, buffers, tonicity agents, ionic additives effective to increase the ionic strength of a solution, antimicrobial agents, antibiotics, antifungals, antioxidants, preservatives, electrolytes, antifoaming agents, oils, stabilizers, enhancers, and the like. In some aspects, the pharmaceutical compositions disclosed herein may contain one or more agents in a total amount of at least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50% by weight of the total composition. In other aspects, one or more of these agents may be added to improve the performance, efficacy, safety, shelf life, and / or other properties of the muscarinic antagonist compositions of the present disclosure. In some aspects, the additives are biocompatible and not harsh, unpleasant, or allergenic.

[0086] In various embodiments, the pharmaceutical compositions disclosed herein may contain one or more acidifying agents. As used herein, the term "acidifying agent" refers to a compound used to provide an acidic medium. Examples of such compounds include, but are not limited to, acetic acid, amino acids, citric acid, fumaric acid, and other alphahydroxy acids, such as hydrochloric acid, ascorbic acid, and nitric acid, as well as other acids known to those skilled in the art. In some embodiments, any pharmaceutically acceptable organic or inorganic acid may be used. In other embodiments, the compositions disclosed herein may contain one or more acidifying agents in a total amount of at least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50% by weight of the total composition.

[0087] In various embodiments, the pharmaceutical compositions disclosed herein may contain one or more alkalizing agents. As used herein, an "alkalinizing agent" refers to a compound used to provide an alkaline medium. Examples of such compounds include, but are not limited to, ammonia solution, ammonium carbonate, diethanolamine, monoethanolamine, potassium hydroxide, sodium borate, sodium carbonate, sodium bicarbonate, sodium hydroxide, triethanolamine, and trolamine, as well as others known to those skilled in the art. In some aspects, any pharmaceutically acceptable organic or inorganic base may be used. In other aspects, the compositions disclosed herein may contain one or more alkalizing agents in a total amount of at least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50% by weight of the total composition.

[0088] In various embodiments, the pharmaceutical compositions disclosed herein may contain one or more antioxidants. As used herein, an "antioxidant" refers to an agent that inhibits oxidation and can therefore be used to prevent preparations from deteriorating due to the oxidative process. Examples of such compounds include, but are not limited to, ascorbic acid, ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, hypophosphorous acid, monothioglycerol, propyl gallate, sodium ascorbate, sodium bisulfite, sodium formaldehyde sulfoxylate, and sodium metabisulfite, as well as other substances known to those skilled in the art. In some aspects, the compositions disclosed herein may contain one or more antioxidants in a total amount of at least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50% by weight of the total composition.

[0089] In other embodiments, the pharmaceutical compositions disclosed herein may include a buffer system. As used herein, a "buffer system" refers to a composition composed of one or more buffering agents, and a "buffering agent" refers to a compound used to resist pH changes upon dilution or the addition of acid or alkali. Examples of buffering agents include, but are not limited to, potassium metaphosphate, potassium phosphate, monosodium acetate, and sodium citrate anhydrous and dihydrate, as well as others known to those of skill in the art. In some embodiments, any pharmaceutically acceptable organic or inorganic buffering agent may be used. In other embodiments, the compositions disclosed herein may include one or more buffering agents in a total amount of at least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50% by weight of the composition. In other embodiments, the amount of one or more buffering agents may depend on the desired pH level of the composition. In some embodiments, the pharmaceutical compositions disclosed herein may have a pH of about 6 to about 9. In other embodiments, the pharmaceutical compositions disclosed herein may have a pH greater than about 8, greater than about 7.5, greater than about 7, greater than about 6.5, or greater than about 6. In preferred embodiments, the compositions disclosed herein may have a pH greater than about 6.8.

[0090] In various embodiments, the pharmaceutical compositions disclosed herein may contain one or more preservatives. As used herein, "preservative" refers to an agent or combination of agents that inhibit, reduce, or eliminate bacterial growth in a pharmaceutical dosage form. Non-limiting examples of preservatives include Nipagin, Nipasol, isopropyl alcohol, and combinations thereof. In some aspects, any pharmaceutically acceptable preservative may be used. In other aspects, the pharmaceutical compositions disclosed herein may contain one or more preservatives in a total amount of at least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50% by weight of the total composition.

[0091] In other embodiments, the pharmaceutical compositions disclosed herein may contain one or more surfactants or detergents. In some aspects, the surfactants or detergents may be synthetic, natural, or semi-synthetic. In other aspects, the compositions disclosed herein may contain an anionic detergent, a cationic detergent, a zwitterionic detergent, an amphoteric electrolytic detergent, an amphoteric detergent, a nonionic detergent having a steroid skeleton, or a combination thereof. In still other aspects, the pharmaceutical compositions disclosed herein may contain one or more surfactants or detergents in a total amount of at least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50% by weight of the composition.

[0092] In various embodiments, the pharmaceutical compositions disclosed herein may contain one or more stabilizers. As used herein, the term "stabilizer" refers to a compound used to stabilize an active agent against physical, chemical, or biochemical processes that would otherwise reduce the therapeutic activity of the agent. Suitable stabilizers include, by way of example and not limitation, succinic anhydride, albumin, sialic acid, creatinine, glycine and other amino acids, niacinamide, sodium acetyltryptophan, zinc oxide, sucrose, glucose, lactose, sorbitol, mannitol, glycerol, polyethylene glycol, sodium caprate, and sodium saccharin, as well as others known to those skilled in the art. In some aspects, the pharmaceutical compositions disclosed herein may contain one or more stabilizers in a total amount of at least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50% by weight of the composition.

[0093] In other embodiments, the pharmaceutical compositions disclosed herein may contain one or more tonicity agents. As used herein, the term "tonicity agent" refers to a compound that can be used to adjust the tonicity of a liquid formulation. Suitable tonicity agents include, but are not limited to, glycerin, lactose, mannitol, dextrose, sodium chloride, sodium sulfate, sorbitol, trehalose, and others known to those skilled in the art. The osmolality of a composition can be expressed in milliosmoles per liter (mOsm / L). Osmolality can be measured using methods commonly known in the art. In a preferred embodiment, the vapor pressure depression method is used to calculate the osmolality of a composition disclosed herein. In some embodiments, the amount of one or more tonicity agents comprising the pharmaceutical compositions disclosed herein may result in an osmolality of the composition of about 150 mOsm / L to about 500 mOsm / L, about 250 mOsm / L to about 500 mOsm / L, about 250 mOsm / L to about 350 mOsm / L, about 280 mOsm / L to about 370 mOsm / L, or about 250 mOsm / L to about 320 mOsm / L. In other embodiments, the compositions herein may have an osmolality ranging from about 100 mOsm / kg to about 1000 mOsm / kg, from about 200 mOsm / kg to about 800 mOsm / kg, from about 250 mOsm / kg to about 500 mOsm / kg, or from about 250 mOsm / kg to about 320 mOsm / kg, or from about 250 mOsm / kg to about 350 mOsm / kg, or from about 280 mOsm / kg to about 320 mOsm / kg. In some embodiments, the pharmaceutical compositions described herein have an osmolality of about 100 mOsm / L to about 1000 mOsm / L, about 200 mOsm / L to about 800 mOsm / L, about 250 mOsm / L to about 500 mOsm / L, about 250 mOsm / L to about 350 mOsm / L, about 250 mOsm / L to about 320 mOsm / L, or about 280 mOsm / L to about 320 mOsm / L.In still other embodiments, the pharmaceutical compositions disclosed herein may comprise one or more tonicity agents in a total amount of at least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50% by total weight of the composition.

[0094] Dosage Formulation Suitable routes of administration may include, for example, oral, rectal, transmucosal, particularly nasal, intestinal, or parenteral delivery, including intramuscular, subcutaneous, and intramedullary injection, as well as intravenous, intraperitoneal, intranasal injection, intraocular (e.g., via eye drops) or topical (e.g., cream or ointment).

[0095] For example, the pharmaceutical composition can be administered locally or systemically, for example, by directly injecting the pharmaceutical composition into a tissue region of a patient. In some embodiments, the pharmaceutical composition disclosed herein can be administered parenterally, for example, by intravenous injection, intraventricular injection, intracisternal injection, intraparenchymal injection, or a combination thereof. In some embodiments, the pharmaceutical composition disclosed herein can be administered to a human patient via at least two administration routes. In some examples, the combination of administration routes can be intraventricular injection and intravenous injection; intrathecal injection and intravenous injection; intracisternal injection and intravenous injection; and intraparenchymal injection and intravenous injection.

[0096] The pharmaceutical compositions of the present disclosure may be manufactured by processes well known in the art, for example, by conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping, or lyophilizing processes.

[0097] Therefore, pharmaceutical compositions for use according to the present disclosure can be formulated in a conventional manner using one or more physiologically acceptable carriers, including excipients and adjuvants that facilitate the processing of the active ingredient into a pharmaceutically usable preparation. The appropriate formulation depends on the selected route of administration.

[0098] For injection, the active ingredients of the pharmaceutical composition may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological saline buffer.

[0099] The pharmaceutical compositions described herein can be formulated for parenteral administration, for example, by bolus injection or continuous infusion.The preparation for injection can be provided in unit dosage form, for example, in ampoules, or in multi-dose containers, optionally containing preservatives.The composition can be a suspension, solution, or emulsion in oily or aqueous vehicles, and can contain formulating agents such as suspending agents, stabilizing agents, and / or dispersing agents.

[0100] Pharmaceutical compositions for parenteral administration include aqueous solutions of active preparations in water-soluble form.In addition, suspensions of active ingredients can be prepared as suitable oily or aqueous injection suspensions.Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters such as ethyl oleate, triglycerides, or liposomes.Aqueous injection suspensions can contain substances that increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, or dextran.Optionally, suspensions can also contain suitable stabilizers or agents that increase the solubility of active ingredients, allowing the preparation of highly concentrated solutions.

[0101] Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, eg, sterile pyrogen-free aqueous solution, before use.

[0102] Pharmaceutical compositions suitable for use in the context of the present disclosure include compositions containing an active ingredient in an amount effective to achieve its intended purpose. In some embodiments, a therapeutically effective amount refers to an amount of active ingredient (i.e., a compound disclosed herein) effective to prevent, slow, alleviate, or ameliorate the symptoms of a disorder (e.g., a pain disorder, psychiatric disorder, or other neurological / cognitive disorder caused by disrupted synaptic activity), or to prolong the survival of the subject being treated.

[0103] Determination of a therapeutically effective amount is well within the capabilities of those skilled in the art, especially in light of the detailed disclosure provided herein.

[0104] For any preparation used in the method of the present disclosure, therapeutically effective amount or dosage can be estimated first from the in vitro and cell culture assay and / or screening platform disclosed herein.For example, a dosage can be formulated to achieve desired concentration or titer in animal model.This information can be used to more accurately determine the dosage that is useful in humans.

[0105] The toxicity and therapeutic efficacy of the active ingredients described herein can be determined by standard pharmaceutical procedures in vitro, in cell cultures, or in experimental animals. Data obtained from these in vitro and cell culture assays and animal tests can be used to formulate a dosage range for use in humans. The dosage can vary depending on the dosage form used and the route of administration utilized. The exact formulation, route of administration, and dosage can be chosen by the individual physician in consideration of the patient's condition (see, for example, Fingl, et al., 1975, in "The Pharmacological Basis of Therapeutics", Ch. 1, p. 1).

[0106] Dosage and interval can be individually adjusted to achieve brain or blood levels of the active ingredient sufficient to induce or suppress the biological effect (minimum effective concentration, MEC). The MEC varies for each preparation but can be estimated from in vitro data. The dosage required to achieve the MEC depends on individual characteristics and the route of administration. Plasma concentrations can be determined using detection assays.

[0107] Depending on the severity and responsiveness of the condition to be treated, administration may be single or multiple, with the course of treatment lasting from several days to several weeks, or until a cure is effected or a diminution of the disease state is achieved. The amount of composition administered will, of course, be dependent on the subject being treated, the severity of the affliction, the manner of administration, the judgment of the prescribing physician, etc. Effective doses may be extrapolated from dose-response curves derived from in vitro or in vivo test systems.

[0108] IV. Treatment Methods A method for inhibiting EPH-ephrin tetramer formation, comprising administering EPH or an ephrin to a patient receiving a compound of formula I provided herein, or [ka] Provided herein are methods comprising contacting a compound selected from:

[0109] In some embodiments, the EPH-ephrin tetramer comprises EPHB1. In some embodiments, the EPH-ephrin tetramer comprises EPHB2. As discussed further herein below, EPH-ephrin signaling contributes to many physiological conditions. Accordingly, the present disclosure provides methods for treating, attenuating, and preventing conditions associated with EPH-ephrin signaling in a subject in need thereof. In various embodiments, disrupted Eph-ephrin signaling causes, worsens, or directly contributes to the condition being treated. For example, Eph-ephrin tetramerization contributes to pain signaling and synaptic plasticity (see, e.g., Figures 1 and 2). Accordingly, the present disclosure provides methods for treating, attenuating, and preventing pain in a subject in need thereof. In certain embodiments, the pain can be chronic neuropathic pain. In other embodiments, the present disclosure provides methods for treating, attenuating, and preventing synaptopathy in a subject in need thereof. In some embodiments, synaptopathy is caused by disrupted NMDA receptor signaling. In various embodiments, the condition can be anxiety or epilepsy. In yet a further embodiment, a method is provided for treating, attenuating, and / or preventing addiction (e.g., opioid addiction) in a subject in need thereof.

[0110] In a further aspect, methods are provided for treating, attenuating, and preventing a condition selected from the group consisting of pain (e.g., chronic neuropathic pain), addiction and dependence (e.g., opioid addiction and dependence), neuropathy (e.g., anxiety, epilepsy, seizures), fibrotic and inflammatory diseases (e.g., NASH liver fibrosis, chronic kidney disease, scleroderma (skin fibrosis), fibrosis (e.g., pulmonary fibrosis or cardiac fibrosis), and abnormal wound healing (e.g., keloids and hypertrophic scars)), metabolic disorders (e.g., diabetes, obesity), cancer (e.g., GBM (glioblastoma), pancreatic cancer, colon cancer), viral infection (e.g., henipavirus and HIV infection). In any of the methods provided herein, the condition being treated (e.g., cancer, fibrotic or inflammatory disease, viral infection, metabolic disorder) may involve aberrant, defective, or excessive EPH-ephrin signaling. That is, any of the methods herein can involve treating a condition caused or exacerbated by aberrant, defective, or excessive EPH-ephrin signaling.

[0111] In any of the above or preceding methods of treatment, an effective amount of one or more compounds disclosed herein (e.g., compounds of Formula I) or one or more of the following compounds: [ka] or any pharmaceutically acceptable salt thereof may be administered to the subject. In some aspects, the compound may be administered in a pharmaceutical composition or formulation described herein, alone or in combination with another appropriate treatment for the condition.

[0112] In various embodiments, the subject in need thereof may have pain, be suspected of having pain, or be at risk of suffering from pain, or have synaptopathy.For example, the subject in need thereof may have neuropathic pain, be suspected of having neuropathic pain, or be at risk of having neuropathic pain.For example, the subject in need thereof may have synaptopathy, be suspected of having synaptopathy, or be at risk of having synaptopathy.

[0113] Suitable subjects include humans, livestock animals, companion animals, laboratory animals, or zoological animals. In one embodiment, the subject may be a rodent, such as a mouse, rat, guinea pig, etc. In another embodiment, the subject may be a livestock animal. Non-limiting examples of suitable livestock animals may include pigs, cows, horses, goats, sheep, llamas, and alpacas. In yet another embodiment, the subject may be a companion animal. Non-limiting examples of companion animals may include pets such as dogs, cats, rabbits, and birds. In yet another embodiment, the subject may be a zoological animal. As used herein, "zoological animal" refers to animals found in zoos. Such animals may include non-human primates, big cats, wolves, and bears. In certain embodiments, the animal is a laboratory animal. Non-limiting examples of laboratory animals may include rodents, dogs, cats, and non-human primates. In certain embodiments, the animal is a rodent. Non-limiting examples of rodents may include mice, rats, guinea pigs, etc. In a preferred embodiment, the subject is a human.

[0114] V. Kit The present disclosure provides kits for use in treating or alleviating a target condition, such as neuropathic pain, as described herein. In some embodiments, the kit can include instructions for use according to any of the methods described herein. The included instructions can include instructions for administering a composition containing a compound disclosed herein (e.g., an EPH-ephrin tetramerization inhibitor, such as a compound of Formula I) to treat, delay the onset of, or alleviate a target disease as described herein. The kit can further include instructions for selecting an individual suitable for treatment based on identifying whether the individual has the target disease. In yet other embodiments, the instructions can include instructions for administering the compound to an individual at risk for the target disease.

[0115] Instructions for use of a composition containing a compound that inhibits EPH-ephrin tetramerization (e.g., a compound of Formula I) generally include information about the dosage, administration schedule, and route of administration for the intended treatment. The container may be a unit dose, bulk package (e.g., multi-dose package), or sub-unit dose. Instructions provided with kits of the invention are typically written instructions on a label or package insert (e.g., a paper sheet included with the kit), although machine-readable instructions (e.g., instructions recorded on a magnetic or optical storage disk) are also acceptable.

[0116] The label or package insert indicates that the composition is used for treating, delaying the onset of, and / or alleviating the disease. Instructions can be provided for practicing any of the methods described herein.

[0117] The kit of the present invention is in suitable packaging.Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (for example, sealed Mylar or plastic bags), etc.Packaging is also intended for use in combination with specific devices, such as inhalers, nasal administration devices (for example, atomizers), or infusion devices, such as minipumps.The kit can have a sterile access port (for example, the container can be an infusion bag or vial with a stopper that can be punctured by a hypodermic needle).The container can also have a sterile access port (for example, the container can be an infusion bag or vial with a stopper that can be punctured by a hypodermic needle).In some cases, at least one active agent in the composition can be a compound described herein (for example, a CDK8 inhibitor).

[0118] The kit may optionally provide additional components such as buffers and interpretive information. Typically, the kit comprises a container and a label or package insert on or associated with the container. In some embodiments, the invention provides an article of manufacture comprising the contents of the above-described kit. [Example]

[0119] All patents and publications mentioned in this specification are indicative of the levels of those skilled in the art to which this disclosure pertains. All patents and publications are herein incorporated by reference to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference.

[0120] The publications discussed throughout are provided solely for their disclosure prior to the filing date of the present application, and nothing herein should be construed as an admission that the present invention is not entitled to antedate such disclosure by virtue of prior invention.

[0121] It is understood that the examples and embodiments described herein are for illustrative purposes only, and that various modifications or changes in light thereof will be suggested to those skilled in the art and are to be included within the spirit and scope of this application and the appended claims.

[0122] Unless specified, reagents used in the examples are commercially available or can be prepared using commercially available equipment, methods, or reagents known in the art. The examples illustrate various aspects of the invention and the practice of the methods of the invention. The examples are not intended to provide an exhaustive description of the many different embodiments of the invention. Thus, while the invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, those skilled in the art will readily appreciate that many changes and modifications can be made therein without departing from the spirit or scope of the appended claims.

[0123] [Example 1] Test Design We focused our drug discovery efforts on the interaction between the EphB1 receptor and its ligand, ephrinB2, mediated by the extracellular segments of these two proteins. We focused on ligand antagonist (LA) compounds because they disrupt both EphB anterograde signaling and ephrinB retrograde signaling, in contrast to the more traditional approach of searching for tyrosine kinase inhibitors (KIs) that target only the catalytic domain of the receptor (Figure 1). Furthermore, focusing on KIs can lead to compounds with a greater potential for off-target effects. This is because there are 500 different kinase domain-containing proteins encoded in the mouse and human genomes that share evolutionarily related amino acid sequences and three-dimensional structures. Therefore, almost all identified KIs cross-react and inhibit other unintended kinase domains, potentially resulting in unwanted side effects. We selected LA compounds that exhibit great specificity for on-target effects and a low potential for off-target effects because they interfere with protein-protein binding with ectodomain structures specific to Eph receptors and ephrin ligands (Figure 2). Therefore, we conclude that compounds that can interfere with Eph-ephrin binding and block both anterograde and retrograde signaling may be the most effective way to target these molecules to treat chronic pain and many other diseases.

[0124] A robust and highly sensitive chemiluminescent Alpha assay for measuring protein-protein interactions formed between the EphB1 receptor tyrosine kinase and the ectodomain of one of its interacting ligands, ephrin B2 (EB2), was developed for use in 384-well plates. Using this assay, we screened a 240,000-complex chemical library available at the UT Southwestern Medical Center High-Throughput Screening (HTS) Core Facility. Finally, using multiple biochemical measurements, we identified a single low-molecular-weight compound, designated A20 (SW056428) (7-bromo-5-(4-morpholinylmethyl)-8-quinolinol), that acts to significantly reduce the EphB1-EB2 protein-protein interaction at low micromolar concentrations. A20 is proposed to be a lead compound for a novel class of chemicals that act as ligand antagonists (LAs), preventing Eph receptors from binding to their ephrin ligands. Because LA compounds disrupt both anterograde and retrograde signaling, whereas KIs only target anterograde signaling (Figure 1), it was further proposed that the use of such LA compounds represents a much better method for pharmacologically targeting Eph-ephrins in disease than the classical / typical use of kinase inhibitors (KIs). While the HTS and initial screening focused on the EphB1-EB2 interaction due to the specific role these molecules play in chronic pain, as described below, LA compounds can be used to target the entire family of 14 different Eph receptors and 8 different ephrin ligands, potentially treating a wide range of unmet medical conditions.

[0125] A series of novel A20-related analogs (referred to as A20 chemicals) has been designed and synthesized. This includes both free base and salt versions of the compounds, the latter of which are much more water-soluble and preferred for biochemical analysis and animal administration. Ongoing biochemical and biophysical kinetic analysis of this ever-growing collection of A20 chemicals conducted in the Henkemeyer laboratory has provided important IC50 values ​​that yield structure-activity relationship (SAR) information. Importantly, this disclosure includes the identification of various novel A20 analogs (e.g., A20-I and others described below) that exhibit improved submicromolar IC50 activity.

[0126] Detailed biophysical analyses have been performed to provide important and valuable insights into the mechanism by which A20 chemicals act to prevent / inhibit / antagonize Eph-ephrin receptor-ligand protein-protein interactions. To understand this, it is necessary to understand that the interaction of Eph receptors with ephrin ligands is extremely complex, in fact, far more complex than most any other receptor-ligand system. This is because (i) there are 14 different Eph receptors and 8 different ephrin ligands divided into two classes, A and B, that exhibit promiscuous interactions; (ii) both receptors and ligands are membrane-anchored and expressed on the cell surface; (iii) this means that ligands are typically not soluble and do not act at a distance as in almost all other ligand-based systems; rather, ephrins physically interact with their cognate Ephs only upon cell-cell contact; (iv) ephrins are also membrane-anchored and can function as receptors and transmit retrograde signals, so it is necessary to understand that Ephs are not just receptors but also ligands, and ephrins are not just ligands but also receptors, and that these proteins signal bidirectionally; and finally (v) upon interaction, Ephs and ephrins first form dimers, and then two Eph-ephrin dimers assemble into a cyclic tetrameric structure, which can then further aggregate into higher-order clusters through lateral movement at the plasma membrane. Formation of the circular tetramer is important because it generates the active signaling complex required for the transmission of anterograde (to Eph-expressing cells) and retrograde (to ephrin-expressing cells) signals.

[0127] Biophysical kinetic binding data were also examined to better visualize and describe these complex interactions, demonstrating that the A20 chemicals act as reversible competitive inhibitors that specifically target the Eph-ephrin cyclic tetramer. By selectively targeting the cyclic tetramer, the A20 compounds focus on the critical signaling complex formed by these molecules, demonstrating a unique mode of action that helps explain the potent and robust biological activity of these compounds observed when tested in cells and in vivo in preclinical studies. In the absence of the A20 chemical, biophysical kinetic analysis shows that Eph and ephrin molecules initially assemble into relatively weak Eph-ephrin dimers during the binding phase with very fast association-dissociation rates (KD = 40 nM). Then, over time, the two Eph-ephrin dimers assemble into a cyclic tetramer with somewhat slower association rates. The cyclic tetramer is an ultrastable structure with very slow or negligible dissociation rates during the dissociation phase (KD = low nanomolar to sub-picomolar concentrations), depending on the interaction under study. Addition of active A20 chemicals to binding assays yields important data. A significant dose-dependent loss of tetrameric structure formation is observed during the binding phase, leading to a rapid or complete loss of stable tetrameric structure during the dissociation phase. Remarkably, A20 chemicals have no effect on Eph-ephrin dimer dynamics, even at very high concentrations. The disclosed data further demonstrate that A20 chemicals act as pan-Eph-ephrin tetramerization inhibitors, as they can disrupt A-class, B-class, and AB-class cross-tetramer interactions. Biophysical kinetic analysis of protein interactions provides important new insights into the diverse and complex interactions of all these Ephs and ephrins. Some interactions are tetramer-driven and are better targeted by A20 chemistry (examples include EphB1-EB2, EphB2-EB2, EB2-EA5, and EphA3-EA1 interactions), while other interactions are more dimer-driven and less well targeted (examples include EphB4-EB2 and EphA3-EA5 interactions).The biophysical tests were designed to assess how compounds can affect Eph-ephrin interactions during the binding phase, providing IC50 information. Biophysical data also show that A20 chemicals can disrupt pre-existing stable Eph-ephrin cyclic tetramer structures in a dose-dependent manner. These results indicate that the disclosed compounds can act on pre-existing Eph-ephrin signaling tetramers upon administration.

[0128] A pharmacokinetic (PK) study is disclosed using a formulation of the highly water-soluble salt (HCl) form of the compound injected into mice using PBS as a vehicle, and compared to results obtained using the free base form, which must be solubilized in a DMSO / sunflower seed vehicle prior to injection. The results show that the A20.HCl salt form exhibits 50-fold greater PK kinetics than the free base, distributing to plasma, liver, and brain.

[0129] It has been found that cyclic tetramers form active Eph-ephrin signaling complexes that result in bidirectional signal transmission to both Eph- and ephrin-expressing cells. Without the cyclic tetramer, signaling does not occur. This disclosure describes a family of novel A20 chemical compounds that selectively target all types of Eph-ephrin interactions, including A-class receptor-ligand tetramers, B-class receptor-ligand tetramers, and cross-class AB heterotetramers, without affecting Eph-ephrin dimerization kinetics. The A20 chemical compounds described herein target the cyclic tetramer structure essential for signaling by these molecules and appear to target all types of Eph-ephrin tetramers with IC50 concentrations below 1 μM. Thus, these compounds function as pan-tetramerization inhibitors of Eph-ephrin bidirectional signaling and have great potential as pharmaceuticals to treat individuals suffering from a number of devastating and untreatable conditions involving overexpression and excessive signaling by these molecules.

[0130] [Example 2] Compound screening To measure the receptor-ligand interaction formed between the EphB1 ectodomain and the ephrin B2 (EB2) ectodomain, we developed a chemiluminescent AlphaScreen™ bead-based proximity assay suitable for HTS (Figure 3). Here, singlet oxygen molecules generated by high-energy irradiation of donor beads travel a limited distance to excite acceptor beads, generating and detecting a chemiluminescent signal. If the analyte disrupts the EphB1-EB2 protein-protein interaction, the proximity of the donor and acceptor beads is prevented, reducing or eliminating energy transfer and decreasing the luminescence of the acceptor beads. We hypothesized that effective inhibitor compounds could dock to the EphB1 ectodomain (indicated as a red star in Figures 1, 2, and 3), the EB2 protein, or possibly both, thereby disrupting the EphB1-EB2 interaction.

[0131] We developed a protein interaction assay using soluble EphB ectodomains conjugated to Fc (e.g., rat EphB1-Fc residues 18–538, 99.4% identical to human EphB1) from R&D Systems and His-conjugated ephrin B ectodomains (e.g., mouse EB2-His residues 18–232, 97.7% identical to human EphB1) from Sino Biologicals. Titrated concentrations of EphB1-Fc ectodomains (or unconjugated Fc protein as a negative control) were added to titrated amounts of three different ephrin B ectodomains (EB1-His, EB2-His, or EB3-His) in a 384-well plate containing 125 ng each of Protein A donor, anti-6x-His acceptor beads, 2% DMSO, and 1x HiBlock buffer (all from PerkinElmer). After 3 hours of incubation at RT, the assay was read on an Envision Multilabel Reader 2102. Consistent with the expected high-affinity EphB1-EB interaction, which brings donor and acceptor beads into close proximity, very high chemiluminescent signals were observed even at 3.2 nM of each protein (>600,000), which were concentration-dependent and strong even at as low as 0.4 nM (100,000). No signal above background (<1,500) was detected for wells containing the Fc control protein and EB-His protein, or for wells containing only EphB1-Fc protein but no EB-His protein.

[0132] The Alpha assay was then tested in competition experiments using 12-mer peptides, termed SNEW and EWLS, previously identified to bind to the dimerization pocket formed on the surface of the EphB1 / EphB2 receptors and antagonize their ability to bind ephrin-B ligands (see Koolpe M, Burgess R, Daile M, and Pasquale EB. (2005). EphB Receptor-binding Peptides Identified by Phage Display Enable Design of an Antagonist with Ephrin-like Affinity. J. Biol. Chem. 280:17301-17311, incorporated herein by reference in its entirety). While these peptides antagonize EphB-EB interactions in vitro, their half-lives of only a few minutes make them unuseful in vivo. Both SNEW and EWLS concentration-dependently antagonized the EphB1-EB2 interaction in the Alpha assay with an observed IC50 of 0.8 μM (Figure 4A). To determine whether purified soluble Reelin (previously shown to bind to the EphB1 ectodomain) could disrupt EphB1-EB2 binding, it was tested in the Alpha assay. A strong dose-dependent decrease in signal was observed with an IC50 of 50 nM, with near-complete inhibition observed at 200 nM (Figure 4B).

[0133] The EphB1-EB2 screen was performed using 0.8 nM of each protein and 50 ng beads / well to evaluate a library of 240,000 small, drug-like chemicals at 5 μM. An example plate is shown (Figure 4C), with potential hit wells circled in green. The average Z for HTS was 0.92, indicating a very high-quality screen with little well-to-well or plate-to-plate variability. A total of 1,055 potential hits that reduced signal by more than 10% were identified. 887 of the 1,055 compounds were selected from the master plate, avoiding compounds with "pan-assay interference" (PAINS) properties, which tend to react nonspecifically with multiple biological targets and typically result in false positives. The 887 compounds were retested in triplicate in the EphB1-EB2 Alpha assay. These compounds were also tested in a counterscreen designed to identify nonspecific compounds. Here, we set up a single-protein Alpha assay using a dual-tagged EphB2-His-Fc ectodomain protein that can bind to both donor and acceptor beads, bringing them into close proximity. It was hypothesized that if selected compounds simply absorbed the chemiluminescent signal or interfered with bead function / binding or protein folding, they would also reduce the signal in the single-protein assay and be considered false positives. Collectively, Alpha confirmation and counterscreening eliminated numerous hits, leaving 32 compounds that repeatedly demonstrated the ability to reduce the EphB1-EB2 Alpha assay chemiluminescent signal by more than 10% and had no effect on the signal in the single-protein assay.

[0134] The 32 hits were then subjected to ELISA, in which EphB1-Fc was first immobilized on a 96-well Protein A plate, and then hit compounds were added to soluble EB2-His in triplicate, again at 5 μM. After 2 h at RT to allow binding of EB2-His to EphB1-Fc, the wells were washed and incubated with nickel-activated horseradish peroxidase (HisProbe-HRP) to detect bound EB2-His protein, washed, and then incubated with ELISA Pico Chemiluminescent Substrate (both from Thermo-Fisher) before being read on a luminometer. A single-protein counter ELISA was also performed using dual-tagged EphB2-His-Fc ectodomain protein. This allowed us to rapidly narrow down the search to one compound (SW056428) from well 268 G15, which reduced the signal in the two-protein ELISA but had no effect on the signal in the single-protein ELISA. For simplicity, we refer to this compound as A20. Another compound called E13 was also found to be capable of reducing the signal in ELISA (not shown).

[0135] A20, E13, and other compounds from the top 32 hits were then characterized in a pull-down assay. EphB1-Fc was immobilized on protein A agarose beads and then mixed with the compounds and soluble EB2-His ectodomain at RT for 1 hour. The protein complexes were then washed, and bound EB2 was detected by immunoblotting using an anti-His antibody. A20 (50 and 250 μM) strongly reduced the binding of EB2-His to EphB1-Fc, as shown by the red asterisk in Figure 5A, whereas other compounds, including E13, were inactive. Tests with other EphB receptors showed that A20 exhibited only a weak ability to prevent EphB4-Fc from binding to EB2-His in pull-downs, even at 250 μM, as shown in Figure 5B with a red asterisk, but exhibited a strong, dose-dependent ability to antagonize binding of EphB2-His-Fc protein to EB2-His, as shown in Figure 5C with a red asterisk, and these results were confirmed by ELISA and Alpha assay (Figure 5D).

[0136] In summary, A20 was found to exhibit a potent ability to antagonize the binding of ephrinB2 ligands to two closely related neuronally expressed sister receptors, EphB1 and EphB2, both with IC50s of 10 μM, and was only weakly able to interfere with the ability of ephrinB2 to bind to its more distant, cardiovascular-specific EphB4 counterpart with an IC50 of >150 μM. The chemical structure of A20 is formed by the combination of a morpholine and a 7-bromo-8-hydroxyquinoline ring, as shown in Figure 6.

[0137] Two other compounds present in the UT Southwestern Medical Center library, SW022101 and SW022102, showed similarity to A20 (Figure 7). While SW022102 was not identified by HTS, SW022101 was one of the initial 887 selected primary hits but did not pass Alpha validation and counterscreening. This initial SAR information indicates that the morpholine ring of A20 is important for this compound's ability to inhibit EphB1-EB2 protein interaction. In addition to A20, SW022101, and SW022102, five other similar analogs not present in the library were identified as commercially available (Figure 7). These compounds were obtained and subjected to Alpha, ELISA, and pull-down experiments. The results indicate that compounds 3511 and 8009 may be as effective as A20 in antagonizing EphB1-EB2 and EphB2-EB2 interactions, with minimal, if any, effect on the EphB4-EB2 interaction. Compounds D014, F235, and 8002 showed no activity in our assay (Figure 8). Additional experiments show that A20 can also antagonize EphB1 / EphB2 proteins from binding to ephrinB1 (EB1) and ephrinB3 (EB3) proteins.

[0138] [Example 3] Biophysical testing of compound A20 The above experiments involved measurements after prolonged incubation of the protein with the compound and therefore represent information after interaction constancy had been achieved (Figures 5A, 5B, 5C, and 5D) or after the complex had been extensively washed (ELISA and pull-down, Figure 8). To further analyze the biophysical activity of A20 in real time, kinetic studies were performed using the Octet® RED384 system to visualize the effect of compounds on EphB1-EB2 protein-protein binding kinetics. In these experiments, EphB1-Fc ectodomain proteins were first immobilized at low density on a set of Octet biosensors. After baseline measurements, they were then exposed to 30 nM soluble EB2-His protein containing 0, 1, 10, or 100 μM A20 for a 400-second association phase, followed by a 500-second dissociation phase with buffer alone. In the absence of A20 compound (0 μM), soluble EB2-His ectodomain exhibited the expected high-affinity interaction of rapid binding to biosensor-immobilized EphB1-Fc during the binding phase, followed by a dissociation phase with a steady-state plateau / flatline response significantly above baseline levels, indicating very strong and stable binding of EB2-His remaining at the end of the experiment, as shown in Figure 9A by the red trace. The sensorgram provides various data on binding kinetics, such as the binding rate (k), dissociation rate (k), and equilibrium dissociation constant (K). As expected, software curve fits of the Octet data indicated that the EphB1-EB2 interaction is not a classical 1:1 dimeric binding interaction, but is more complex / heterogeneous (2:1), as these molecules can form dimers, cyclic tetramers, and higher-order clusters (Figure 10). Eph ectodomains and ephrin ectodomains exhibit complex receptor-ligand binding interactions.Specifically, EphB2 dimerizes with ephrinB2, and the two dimers then assemble into a ring-shaped tetrameric structure (see Figure 10 and Himanen JP, Rajashankar KR, Lackmann M, Cowan CA, Henkemeyer M, and Nikolov DB. (2001). Crystal structure of an Eph receptor-ephrin complex. Nature 414:933-8, which is incorporated herein by reference in its entirety).

[0139] Distinct dimerization and tetramerization interfaces are shown. The cyclic tetramers further cluster and pack into crystals through additional ligand-ligand (red arrow) and receptor-receptor (blue arrow) interfaces. Numerous studies by multiple groups have demonstrated that cyclic tetramers and tetramer clusters have been observed in vivo and are required to form the active complexes necessary for Eph-ephrin bidirectional signaling. KD calculations for the interaction indicated a very high affinity interaction of approximately 2 nM (1:1 calculation) or 0.5 nM (2:1 calculation). Addition of A20 resulted in a significantly blunted, dose-dependent decrease in the binding of EB2-His to biosensor-immobilized EphB1-Fc during the binding phase, which was evident even at 1 μM, with only negligible binding at 100 μM (Figure 9A). Using various methods, different sensorgrams obtained from dose-dependent experiments of drug compounds can be compared to quantify their LA activity and obtain IC50 information. A single time point during the assay can be chosen, and the response signals obtained with different compound doses can be compared, for example, at 380 s near the end of the association phase or 100 s after the start of the dissociation phase. When the entire sensorgram trace is taken into account for analysis of the area under the curve (AUC), an IC50 of 9.33 μM was obtained for the EphB1-EB2 interaction, consistent with that calculated using Alpha, ELISA, or pull-down experiments.

[0140] Biophysical analysis using immobilized EphB4-Fc protein in the Octet system also demonstrated a very high affinity interaction with EB2 in the absence of compound, whereas the addition of A20 had only a moderate effect on the formation of the EphB4-EB2 protein-protein interaction, with an area under the curve (IC50) of >100 μM (Figure 9B). This is consistent with the results of Alpha, ELISA, and pull-down studies, which showed that A20 exhibited only a weak ability to disrupt EphB4-EB2 interaction.

[0141] In summary, the new biophysical kinetic analysis disclosed is a powerful addition to methods for evaluating EphB receptor interactions with EphrinB ligands. Furthermore, the results show that A20 and the new A20 analog chemistries disclosed herein effectively target EphB1-EphrinB and EphB2-EphrinB binding, but show little effect on EphB4-EphrinB2 binding. To understand the mechanism of action of the A20 chemistries, further experiments were performed using Octet® RED384.

[0142] [Example 4] Compound A20 is a reversible, competitive antagonist of Eph-ephrin receptor-ligand binding Antagonists are classified as either competitive / reversible or irreversible, depending on how a particular inhibitor interacts with its protein target. Reversible antagonists bind to receptor proteins via noncovalent intermolecular forces and eventually dissociate from the receptor, freeing the protein to bind to another antagonist chemical or its true ligand protein in solution. Irreversible antagonists, on the other hand, bind via covalent forces to form a permanent receptor-antagonist complex that does not dissociate. Competitive antagonists are thought to bind to receptor proteins at the same binding site / interface as the endogenous ligand (also known as ephrins), but do not activate the receptor. Thus, the ligand and antagonist compete for the same binding site on the receptor; when the antagonist is bound to the receptor, it prevents ligand binding, and increasing concentrations of the antagonist dose-dependently negate the ligand's ability to bind to the receptor. Because most receptor antagonists involve reversible, noncovalent, competitive interactions, two Octet experiments were performed to determine whether this was indeed A20's mode of action. To determine whether A20 is a reversible inhibitor / antagonist, rEphB1-Fc was first immobilized on a sensor chip and then pre-exposed to 100 μM A20, then washed to remove A20, and then tested for binding to 30 nM soluble mEB2-His protein after a baseline measurement. The results showed that pre-treatment with A20 did not affect the ability of the immobilized EphB1 protein to bind to soluble EB2 in the subsequent binding step, indicating that the compound acts as a reversible inhibitor (Figure 11). Specifically, pre-exposure of immobilized rEphB1-Fc protein to 100 μM A20 did not affect its ability to subsequently bind to 30 nM soluble EB2 (red sensorgram), and the interaction kinetics were similar to that of samples not pre-exposed to the compound (green sensorgram). Addition of A20 during the binding step competed with EB2 binding to immobilized EphB1 protein (purple sensorgram). Binding assays were performed in PBS containing 0.05% Tween-20, with an association time of 400 s and a dissociation time of 500 s.To determine whether A20 is a competitive inhibitor, increasing concentrations of A20 (0, 10, 100 μM) were combined with different concentrations of soluble mEB2-His protein (0.1, 1, 10, 100, 1000 nM) and tested for binding to sensorchip-immobilized rEphB1-Fc during the binding step. The data plotted in Figures 12A-C group the sensorgram results by the amount of A20 included in the binding reaction (0, 10, 100 μM), while the plots in Figures 12D-F group these by the concentration of EB2 in the binding reaction. The data show that increasing concentrations of A20 dose-dependently reduce the ability of EB2 to bind to EphB1, indicating that the compound is a competitive inhibitor of EphB1-EB2 binding. As shown in Figures 12A-C, in the absence of compound, ultrahigh concentrations of soluble EB2 protein immobilize EphB1 within seconds, resulting in a rapid, saturable binding response due to the large amount of available ligand protein. Addition of A20 to the binding phase strongly blunted this saturable binding response, particularly in the dissociation portion of the curve, where the compound dramatically increased the dissociation rate (koff) during the first 100 seconds of dissociation and significantly reduced the level of bound EB2 at the end of the experiment. The data show that just 10-fold more A20 (10 μM) during the binding phase can compete with a portion of the saturating 1 μM high-affinity EB2 ligand for binding to EphB1, and that at the end of the experiment, 100-fold more compound (100 μM) almost completely eliminated the ultrastable EphB1-EB2 tetramer (purple sensorgram). Furthermore, at a saturating 1 μM concentration of EB2, the addition of A20 had a clear effect on the stable tetramer during the dissociation phase, whereas A20 had no effect on the rapid initial binding of EB2 to immobilized EphB1, which occurred within the first few seconds of the association phase (k).

[0143] These results provide insight into how A20 chemicals interfere with Eph-ephrin interactions and that they do not target the formation of Eph-ephrin dimers but specifically target the formation and / or stability of Eph-ephrin tetramers, highly stable and long-lasting macromolecular complexes that activate bidirectional signaling.

[0144] [Example 5] Cell-based testing of compound A20 To assess whether A20 reduces the ability of ephrinB2 to stimulate activation of the EphB tyrosine kinase domain in live cells, Cos1 cells expressing endogenous EphB2 were exposed to pre-clustered EB2-Fc ectodomains for 30 minutes in the presence or absence of A20. Immunoblotting of protein lysates with a rabbit anti-phospho-EphB-specific antibody demonstrated that A20 caused a dose-dependent reduction in EphB2 kinase activity (Figure 13). Stimulated cells were fixed onto coverslips and subjected to immunofluorescence (IF) with an anti-Fc antibody to detect receptor-bound ligand proteins, which subsequently clustered through the plasma membrane into a large, distinct spot on the cell surface and contained an anti-phosphotyrosine signal, indicating activation of the receptor's kinase domain. The results showed that A20 significantly reduced the ability of pre-clustered EB2-Fc to bind / cluster to EphB2 receptors, resulting in spots on cells and activating their tyrosine kinase domains (Figure 14). Furthermore, cell-based analysis showed that Cos1 cells exposed to EB2-Fc flattened on the dish, a typical response to stimulation of EphB2 anterograde signaling, whereas cells exposed to both EB2-Fc and A20 exhibited much less flattening and generally resembled unstimulated control cells. These results indicate that A20 can affect cellular responses induced by EphB-ephrinB interactions and signaling.

[0145] [Example 6] Cytotoxicity, metabolic stability, and initial pharmacokinetic (PK) studies of compound A20 The potential cytotoxicity of A20 was evaluated using an MTT assay performed after culturing normal human dermal fibroblast (NHDF) cells with increasing concentrations of A20 for 48 hours. Results revealed that long-term exposure to A20 up to 10 μM was not toxic (data not shown). Working with the UT Southwestern Medical Center Preclinical Pharmacology Core (PPC), we next evaluated in vitro metabolic stability using a mouse microsome assay and initially found that A20 had a half-life of 224 minutes, A19 had a half-life of 136 minutes, and A20 had a half-life of only 22 minutes (data not shown).

[0146] Further studies were performed in vivo to evaluate the effects of A20 when injected into mice. To formulate the compound for intraperitoneal (IP) injection, A20 was tested for solubility in various buffers, pH conditions, and with cyclodextrins (e.g., Captisol). Formulations were prepared by first dissolving A20 powder in DMSO and subsequently mixing with sunflower seed oil (SSO) (in 6% DMSO / 94% SSO) prior to administration. IP-injected A20 was well tolerated at doses up to 40 mg / kg, administered 12 hours apart on multiple days. PK profiling experiments were then performed on A20 to determine its absorption, distribution, metabolism, and excretion (ADME) properties. Mice were given a single IP injection of 20 mg / kg A20, and three animals per time point were sacrificed at 10, 30, 90, 180, 360, 960, and 1440 minutes, and tissues were collected and subjected to mass spectrometry. Results showed that A20 rapidly distributed into plasma and crossed the BBB to reach the brain and spinal cord (Figure 15). The plasma Cmax (maximum concentration) was observed at 10 minutes, with an A20 concentration of 214 ng / ml calculated to be 0.665 μM, and the area under the concentration-time curve (AUC0-t), a measure of systemic exposure to the compound, was equal to 14,636 ng / ml over 24 hours. In the brain, the Cmax of A20 was 104 ng / ml calculated to be 10 min and 0.323 μM, with an AUC0-t equal to 6,684 ng / ml, and in the spinal cord, the Cmax was 88 ng / ml calculated to be 0.273 μM, with an AUC0-t equal to 4,928 ng / ml.

[0147] [Example 7] Formation of Compound A20 Salt A20 was synthesized to yield a compound with 98-100% purity, and the resulting free base chemical was subjected to the salting procedure shown for A20 (Figure 16). The synthesized salt form of the A20 chemical was more water-soluble and more active, as shown in an Octet experiment using the same conditions as in Figure 9A, but with the newly synthesized A20.2×HCl salt (Figure 17). The IC50 values ​​for the synthesized compound were calculated to be 3.09 (response at 380 s after binding), 2.40 (response at 100 s after the dissociation phase), and 3.63 μM (AUC), indicating that the salt form of A20 exhibits 2-3 times better and / or more potent antagonist activity than the free base.

[0148] [Example 8] Pharmacokinetic study of newly synthesized A20 salts The salt forms of the newly synthesized A20 compound were highly soluble in water or PBS (greater than 50 mg / ml for A20.2×HCl), allowing for the injection of aqueous formulations of the compound into animals. Studies were performed with the A20 salt dissolved in PBS alone and injected at 20 mg / kg, consistent with the free base studies previously described. The data obtained demonstrate that the salt version of A20 exhibits a much more impressive PK profile (Figure 18). Plasma Cmax was greater than 13-fold increased relative to the free base at 10 minutes, at a concentration of 2,880 ng / ml of A20 salt calculated to be 7.27 μM, and AUC0-t was equal to 81,039 ng / ml over 24 hours, a greater than 5.5-fold increase relative to the free base. In the brain, the Cmax of A20 was 2,787 ng / ml, calculated to be 7.03 μM, a more than 26-fold increase over the free base, and the AUC0-t was equal to 136,933 ng / ml (a more than 20-fold increase over the free base). In the spinal cord, the Cmax was 3,310 ng / ml (a more than 37-fold increase over the free base, calculated to be 8.35 μM), and the AUC0-t was equal to 86,044 ng / ml (a more than 17-fold increase over the free base). Liver analysis also showed a Cmax of 22,577 ng / ml, calculated to be 57 μM, and an AUC0-t of 1,363,490 ng / ml. The results for brain, spinal cord, and liver tissues were particularly striking, demonstrating that significant levels of A20 salt remained even after the final collection at 24 hours. These results provide strong evidence that injection with A20 salt dissolved in PBS exhibits significantly better PK kinetics compared to the free base dissolved in DMSO / SSO.

[0149] [Example 9] Binding kinetics of human EphB-ephrin B proteins Tests were conducted to confirm that the A20 chemical could effectively target human Eph / ephrin proteins. Using the Octet system, we first characterized the biophysical interactions between human B-class Eph and ephrin proteins, and then tested A20 to assess how it might disrupt the interaction. Because human and rodent proteins are highly conserved and essentially identical, with EphB2 sharing 99.5% identity between humans and mice and differing by only five of its approximately 1,000 amino acids, species-specific differences were not expected. Therefore, commercially available human ephrinB1-Fc (EB1), ephrinB2-Fc (EB2), and ephrinB3-Fc (EB3) ectodomain proteins were immobilized at low density on a biosensor and then exposed to 50 nM soluble human EphB1-His, EphB2-His, and EphB4-His ectodomains for an 800-second association phase and a 1,000-second dissociation phase, analyzing a total of nine distinct interactions. In the absence of A20 chemical, immobilized human ephrinB proteins bound to their appropriate human EphB target proteins; ephrinB1 bound only to EphB1 and EphB2, ephrinB2 bound to all three, and ephrinB3 bound only to EphB1 and EphB2 (Figures 19A-B).

[0150] In summary, analysis of the human proteins clearly demonstrates that EphB1 and EphB2 receptors bind promiscuously to all three ephrin B proteins, whereas EphB4, which functions exclusively in the vasculature, interacts strictly and very strongly with ephrin B2 only. EphB4 does not show any interaction with ephrin B1 or ephrin B3. Second, it is clear that all three human EphB receptors tested exhibited the strongest binding to ephrin B2 ligands, with EphB4 clearly demonstrating the fastest initial binding at the beginning of the binding phase, with the steepest slope of 85° among the three different sensorgrams, and the strongest response at the end of binding (0.48). The data also show that the steep EphB4-EB2 sensorgram curve rapidly bends starting approximately 60 seconds into the binding phase, after which the slope dramatically flattens to only 5°. Furthermore, the sensorgrams for EphB1-EB2 and EphB2-EB2 are very different. In the case of EphB1-EB2, the initial slope of binding was only 70° (not at all steep compared to EphB4), and then the response curved rapidly to form a nearly straight 25° slope throughout the remaining binding phase. The EphB2-EB2 interaction exhibited a very strong initial slope of 85° at the onset of binding (like EphB4), but then within 20 seconds, the response curved to form a nearly straight 20° slope throughout the remaining binding phase (like EphB1), resembling an EphB4 / EphB1 hybrid. All three EphB-EB2 interactions exhibited similar dissociation profiles, with significant amounts of EphB protein rapidly detaching from the ephrinB-bound sensor chip. However, approximately 50% or more of each receptor protein remained bound at the end of the 1000-second dissociation phase, indicating the formation of a highly stable, long-lasting macromolecular protein complex. The sensorgram results for EphB1 and EphB2 receptors interacting with EB1 were very different compared to those for EB2.The EphB1-EB1 sensorgram did not show an initial higher slope. Instead, starting from the beginning of the binding phase, the response was a straight, approximately 22° slope throughout. Notably, no EphB1 protein was lost from the sensor chip during the dissociation phase, indicating the formation of an ultrastable complex with essentially no dissociation rate. The EphB2-EB1 sensorgram showed an extremely fast initial binding rate, similar to the EphB2-EB2 interaction, with a slope of 85°. However, this lasted only for a very short period of time (less than 5 seconds), as the binding curve then rapidly flattened to a 22° slope and ran parallel to the EphB1-EB1 binding kinetics. Furthermore, during the EphB2-EB1 dissociation phase, there was an initial rapid loss of a small amount of bound EphB2 protein, but thereafter, the remaining bound protein remained very stable, similar to EphB1-EB1. The initial rapid loss of some sensor chip-bound EphB2 protein at the beginning of the dissociation phase was essentially the opposite of the fast initial binding observed during the onset of binding.

[0151] Furthermore, the sensorgrams for EphB1 and EphB2 receptors interacting with EB3 are essentially identical to the sensorgram for EphB1-EB1 in that both the EphB1-EB3 and EphB2-EB3 sensorgrams showed a delayed, very slow, and stable linear accumulation of EphB1 / EphB2 proteins to immobilized EB3 protein during the binding phase. The binding slopes were found to be even lower than those of the other interactions, at 15° for EphB1 and only 11° for EphB2. Furthermore, like the EphB1-EB1 interaction, the EphB1-EB3 and EphB2-EB3 complexes did not melt at all during the dissociation phase, indicating the formation of ultrastable complexes with essentially no dissociation rate.

[0152] The binding kinetics described above for various human EphB-ephrinB interactions are complex and show clear differences, but all are extremely high-affinity interactions with KD values ​​in the low nanomolar to picomolar range (Table 2). Octet software allows for detailed biophysical analysis of sensorgram data, first attempting to fit the information to equations that follow fairly standard binding characteristics, mostly representative of either classical 1:1 binding interactions (such as simple receptor-ligand dimer formation) or more complex / heterogeneous interactions involving 2:1 binding interactions (such as Eph-ephrin dimers and tetramers). Based on the information contained in the sensorgram experiment being analyzed, Octet software attempts to draw curves to fit the data to 1:1 and 2:1 binding equations. The quality of any one curve fit, and therefore the confidence in the equation's results, is also provided as an R2 value; the closer to 1, the better. This data is provided in parentheses next to the KD value in Table 2. 1:1 and 2:1 curve fits for all of the human protein data are shown in Figures 20A-C. Focusing first on the EphB-EB2 interactions, it is clear that they do not fit a 1:1 binding curve but rather closely resemble a 2:1 heterotypic interaction. The resulting curve fits are not always perfect, especially for complex interactions such as the Eph / ephrin system. Thus, while the EphB2-EB2 and EphB4-EB2 data fit fairly well to 2:1 curves, the EphB1-EB2 interaction fits poorly to either 1:1 or 2:1 curves, as reflected by the R2 values ​​for this interaction. The interaction between EB1 and its two target proteins is more diverse. The EphB1-EB1 data show a clear 1:1 curve fit and an extremely high affinity interaction of 11.4 pM in a complex that forms very slowly during binding and does not melt during dissociation. On the other hand, EphB2-EB1, with its initial very fast on-rate during binding and a fast initial off-rate during dissociation, shows a clear 2:1 curve fit interaction. EphB1-EB3 and EphB2-EB3 interactions are similar to EphB1-EB1, with clear 1:1 curve fits, without a slow stepwise on-rate during binding followed by a fast off-rate during dissociation.

[0153] Furthermore, in none of the nine sensorgrams analyzed did the binding reaction reach homeostasis, where the association / dissociation rates equilibrate, as indicated by the traces that eventually curved during the binding phase to a straight line with no slope (0°). Thus, whether the binding reaction started fast with a slope greater than 70° or slow and steady with a slope less than 30°, homeostasis was not achieved. This inability to reach homeostasis was observed even when the length of the standard association and dissociation phases was doubled to 800 and 1,000 seconds, as shown in Figures 19 and 20. Furthermore, the competitive binding studies in Figure 12 demonstrate that binding homeostasis can be achieved when using saturating amounts (anywhere between 100 and 1,000 nM) of soluble binding partner. In the above studies using human proteins, the binding reaction for EphB4-EB2 came closest to reaching homeostasis with a slope of 5° at the end of the binding phase. This indicates that the EphB4-EB2 interaction is more "dimer-driven," while all other interactions are what are referred to as "tetramer-driven" (see further below). In the previous Octet study provided above, which used biosensor-immobilized rodent EphB1-Fc and EphB4-Fc ectodomain proteins binding to soluble rodent EB2 ectodomain, the sensorgram traces appeared very similar to those using human protein-immobilized ephrinB2-Fc protein. Thus, whether an Eph or ephrin protein is immobilized on the biosensor, or whether a human or rodent protein is used, Octet provides very similar binding interactions, sensorgram curves, KD information, and other biophysical kinetic values ​​for the specific interaction being analyzed. Thus, while the rodent EphB1-EB2 binding data shows that it does not reach homeostasis, and the sensorgram patterns also indicate that this is a "tetramer-driven" interaction (Figure 9A, Figure 11, Figure 17), the binding data shown for rodent EphB4-EB2 reaches homeostasis within approximately 200 seconds of binding and can be characterized as a "dimer-driven" interaction (Figure 9B).In conclusion, the data show that Eph-ephrin interactions are highly complex due to the formation of dimers and tetramers, exhibit very high affinity binding, and that Eph-ephrin tetramers, once formed, are ultrastable macromolecular complexes.

[0154] [Table 2]

[0155] [Example 10] A20 interferes with EphB-ephrinB tetramerization The Octet studies using human proteins, shown in Figures 19A-F and 20A-C and in Table 2, were accompanied by experiments to determine how compounds, including 1, 10, and 100 μM concentrations of A20.2×HCl salt, affected binding kinetics. Sensorgrams for binding of immobilized human ephrinB2 to EphB2 and EphB4 using 0, 1, 10, and 100 μM concentrations of A20 salt provide good examples of how compounds can alter Eph-ephrin protein-protein interaction kinetics. For both interactions, the addition of A20 resulted in reduced binding levels by the compounds, which had a much stronger overall inhibitory effect on EphB2-EphB2 interactions, particularly at the binding stage (Figure 21A). Interestingly, the inhibitory effect of A20 salt on the interaction between immobilized human ephrinB2 ectodomain and soluble human EphB ectodomain reached a maximum at a concentration of 1 μM, as 10-fold (10 μM) and 100-fold (100 μM) more compound did not result in enhanced inhibition. This indicates that the effective / relative IC50 of A20 is well below 1 μM (more precise IC50 calculations are provided below). Nevertheless, simply analyzing the sensorgram responses for 0 and 1 μM A20 for absolute IC50 calculations (Figure 21B) reveals that the compound is a much more potent inhibitor of EB2-EphB2 interaction, as all evaluations, whether near the end of the association phase (blue star), immediately after the beginning of the dissociation phase (gold star), or AUC, provide absolute IC50 values ​​of 1 μM. Calculations for EB2-EphB4 indicate the absence of an absolute IC50, as the compound, even at 100 μM, does not inhibit any of the three estimated response values ​​by more than 50%. Consistent with these diverse absolute IC50 values, specific inspection of the binding phase of the sensorgrams shows that 1 μM A20 reduced the EB2-EphB2 binding response by 50%, but only slightly reduced the EB2-EphB4 binding response.Furthermore, it was clear that the addition of A20 had no effect on the initial, rapid 85° slope of association observed at the beginning of the association phase for EphB2-EB2 or EphB4-EB2 interactions, indicating that the compound had no effect on the formation of early Eph-ephrin dimers. However, once this initial, rapid dimerization phase had passed, A20 was observed to reduce the slope of the remaining portion of association. In the case of EphB2-EB2, the slope went from 20° to 10° in the presence of A20, and in the case of EphB4-EB2, it went from 5° to 3°. The reduced slope at the later stage of the association phase indicates that the A20 compound specifically affects the accumulation of stable tetramers.

[0156] Addition of compound also altered dissociation kinetics. The EphB2 response showed a very rapid loss from immobilized ephrinB2 protein at the beginning of the dissociation phase, which then rapidly stabilized to a plateau level well above baseline and remained unchanged for the remainder of the dissociation phase. This finding of low levels of stably bound EphB2 protein remaining at the end of dissociation when compound was included at 1, 10, or even 100 μM reveals a gradual accumulation of ultra-ultrastable Eph-ephrin complexes that are not targeted by the A20 chemistry. This is referred to as the "insurmountable" component of Eph-ephrin interactions (Figure 21C). Furthermore, AUC data were graphed for these experiments (Figure 21D). The results show that in the absence of A20 compound, the EB2-EphB2 dimer:tetramer ratio was 32:68 (tetramer-driven) and the EB2-EphB4 dimer:tetramer ratio was 83:17 (dimer-driven). Furthermore, experiments in the presence of A20 showed no effect on dimer formation for either interaction, but reversed the EB2-EphB2 dimer:tetramer ratio, now to 32:21, resulting in a shift toward a dimer-driven interaction and the accumulation of much fewer tetramers, an "insurmountable" component that A20 does not counter. In the case of EB2-EphB4, the dimer:tetramer ratio was 83:8 in the presence of A20, already a strong dimer-driven interaction, and was not significantly different from the control experiment without compound. Overall, the data indicate that addition of the A20 compound strongly reduced the accumulation of EB2-EphB2 tetramers, shifting this over time to a more dimer-driven interaction that forms "insurmountable" ultra-hyperstable complexes at a much slower rate. A similar but weaker effect of A20 on tetramer accumulation was also observed for other interactions: binding of immobilized human ephrinBs to soluble human EphB1 and EphB2 ectodomains (Figure 22). The inhibitory effect of A20 was strongest on the EB2-EphB2 interaction, with an absolute IC50 reaching 1 μM, followed by the EB2-EphB1 interaction with an absolute IC50 of 10 μM.For EB2-EphB1, in the absence of A20, the dimer:tetramer ratio was found to be 14:86, making it even more tetramer-driven than EB2-EphB2, and the concentration of A20 required to maximize the inhibitory response to EB2-EphB1 was 10 μM. The "insurmountable" component of the EB2-EphB1 interaction was 50% of the tetrameric species (14:41), compared with 30% for the EB2-EphB2 interaction. The interaction of soluble human EphB1 and EphB2 ectodomains with immobilized human EB1 and EB3 ectodomains in the absence of any A20 was somewhat different, bypassing dimer formation and instead appearing to slowly accumulate as 100% tetramers that do not dissolve into the biosensor-bound ephrin B protein during dissociation (Figures 19A-F and 20A-C). Addition of A20 to these interactions clearly reduced the level of tetramers formed during the binding step; 1 μM resulted in maximal inhibition for the EB1-EphB1, EB1-EphB2, and EB3-EphB2 interactions, whereas 10 μM was required for maximal inhibition of EB3-EphB1. A20 appeared to be more effective against EphB2, with a maximal effect at 1 μM, reducing tetramer levels by 45% for both EB1 and EB3, but reducing EphB1 tetramer formation by 15% and 40%, respectively.

[0157] Taken together, these results demonstrate that A20 can affect the association / dissociation kinetics and tetramer accumulation of all EphB-ephrinB complexes tested, with the strongest activity against EphB2 interacting with ephrinB2. The tetramer-inhibitory effect of A20 was maximized at 1 μM in most cases, and increasing the compound's concentration up to 100 μM had no additional effect on eliminating the "insurmountable" component, confirming the existence of at least two distinct tetramer configurations, one effectively targeted by the A20 chemistry and the other not. While the exact nature of this particular ultra-ultrastable "insurmountable" complex is unknown, it is likely caused by the clustering of two or more cyclic tetramers into a conformation that cannot be broken by A20 (Figure 10). Thus, although the A20 chemical inhibits the ability of two Eph-ephrin dimers to complex into ultrastable circular tetramers, it is expected that some tetramers can still form in the presence of the drug, albeit at a much slower rate (slope of the curve), and perhaps if two or more of these tetramers manage to complex together, this can form an ultra-ultrastable "insurmountable" Eph-ephrin complex. The results disclosed here show that Eph-ephrin interactions form dimers and tetramers that can be surmounted by A20, as well as a certain amount of insurmountable tetramers (see below for more on this).

[0158] [Example 11] EphB-ephrinB dimers bind / dissociate rapidly, whereas tetramers bind / dissociate slowly or not at all In the Octet interaction studies disclosed above using human proteins, the association and dissociation times were doubled to 800 and 1,000 seconds, respectively, resulting in greater accumulation of the ultrastable surmountable and ultra-ultrastable insurmountable forms of Eph-ephrin tetramers. To better assess initial binding activity in the absence of A20, we performed biophysical experiments in which the association time was shortened to 80, 40, 20, 15, 10, and 5 seconds, followed by a standard 500-second dissociation, exposing biosensor-immobilized rEphB1-Fc ectodomain to 100 nM soluble mEB2-His. Focusing first on the 80-second association phase for illustrative purposes (Figure 23), the biosensor response shows the expected rapid formation of EphB1-EB2 dimers with a very high 87° slope during the first 10–15 seconds of association, followed by a rapid bend again, this time to a 60° slope, as expected, as the tetramers begin to accumulate. Upon dissociation, the response showed an initial, rapid 15-20 s dissociation phase with a high inverse slope representing the dissociation of a portion of the bound EB2 protein from immobilized EphB1 (dimer melting). The dissociation response then rapidly curved and leveled off, reaching a plateau with a signal much higher than baseline, representing stable EphB1-EB2 tetrameric species that remained complexed throughout the remainder of the dissociation phase. Analysis of the response levels indicated that even with a shortened 80 s binding time, 54% of the binding could be attributed to tetramer formation. When the binding time was reduced to 40 s, a slightly lower 43% contribution of the response / binding was attributed to tetramer formation, as shown in the top sensorgram of Figure 24. Interestingly, when the binding time was reduced to 5, 10, or 15 seconds, only fast binding and fast dissociation responses were evident, with no stable tetramers present at the end of dissociation, whereas when binding was extended to 20 seconds, a very low 5% level of stable tetramers was produced, as shown in the bottom sensorgram of Figure 24. This suggests that a critical period of 15-20 seconds of binding is required to allow two fast-binding / fast-dissociating dimers to form before they can bind into a stable tetrameric complex.

[0159] To assess how A20 chemicals alter initial binding activity, we next performed 80-s Octet experiments using 0, 10, and 100 μM concentrations of the A20 salt compound (Figure 25A, Figure 25B). In the absence of A20, the binding response of immobilized EphB1 binding to soluble EB2 was typical of that shown in Figure 24, showing rapid early formation of dimers with a very high 87° slope, which rapidly curved into a 60° tetramerization accumulation slope, followed by a sudden reversal of the slope once the dissociation phase began, indicating rapid dissociation of the dimeric species, which then plateaued at 67% of the maximum response in this experiment, with a signal representing stable tetramer accumulation. In experiments with A20, there was no effect on the early and rapid formation of dimers during the 80-s binding phase, but there was a clear and very strong dose-dependent decrease in tetramer accumulation. The tetramer accumulation gradient changed from 60° to 16° at 10 μM A20, as shown in Figure 25 (purple sensorgram), and was a flat line with no gradient at 100 μM A20, as shown in Figure 25 (dark green sensorgram), indicating that very few, if any, stable tetramers could be formed. Analysis of the dissociation phase confirmed that addition of both concentrations of A20 rapidly lost nearly all response signal, approaching baseline levels, indicating that very few ultrastable and ultra-ultra-ultrastable insurmountable tetramers were formed.

[0160] The shortened binding experiments clearly demonstrate that the presence of A20 has a profound effect on Eph-ephrin binding kinetics, shifting them from a highly stable 2:1 heterogeneous / complex interaction to a simple 1:1 dimeric, fast-on / fast-off interaction. This is particularly evident in the analysis of kinetic data where parameters were set to calculate information based on the 1:1 interaction (Figure 25C). Here, experiments without A20 and with 10 μM A20 failed to fit the 1:1 interaction kinetics with R2 values ​​of 0.3279 and 0.6296, respectively, whereas experiments with 100 μM A20 yielded an R2 value of 0.9623, indicating a shift toward a 1:1 dimeric interaction. Furthermore, the KD for the EphB1-EB2 interaction in the absence of A20 was calculated to be an extremely high affinity of 0.137 nM (and an even higher affinity of less than 1 pM when calculated using the more appropriate 2:1 interaction kinetics). In the presence of A20, the KD is much lower, 76-fold lower at 10 μM (10.47 nM) and 337-fold lower at 100 μM (46.2 nM). Furthermore, the addition of A20 did not significantly alter the association rate (Ka), which is primarily driven by fast dimer formation. However, the compound caused a significant 90-fold and 275-fold increase in the dissociation rate (Kdis), respectively. This increased dissociation rate reflects the near-absence of stable tetramer formation and provides a fairly accurate representation of Kdis, which is specifically attributed to the fast-dissociating melting of dimers. Summarizing these biophysical tests, in the presence of A20, only fast-binding / fast-dissociating dimers are formed and visualized during the binding phase, rapidly reaching a homeostatic level where dimers form and melt at equilibrium (fast-binding / fast-dissociating), and are unable to bind to form tetramers. When subjected to the dissociation step, all pre-existing dimers rapidly melted, leaving only the few tetramers that had been able to form. Furthermore, because a 100 μM A20 concentration resulted in essentially only EphB1-ephrinB2 dimer binding, its K value of 46.2 nM and other kinetic information obtained from these experiments could be used to characterize fast-binding / fast-dissociating dimers.Thus, the very strong subnanomolar / picomolar affinity of the EphB1-ephrinB2 interaction observed in the absence of A20 compound is primarily due to the assembly of two relatively weak / unstable dimers into a highly stable cyclic tetrameric complex. Based on these results, we conclude that A20 chemical compounds can be classified as specific / selective inhibitors of Eph-ephrin tetramerization.

[0161] [Example 12] Mass photometry confirms that A20 chemicals target Eph-ephrin tetramers To validate kinetic studies using the Octet system, we used mass photometry (MP), which uses optical detection to measure the mass of individual proteins and protein complexes in solution. Using MP, we determined the masses of EphB2 and ephrin B2 ectodomain proteins, individually and in combination, after allowing them to associate and form tetramers for 24 hours with or without 10 or 50 μM A20 salt (Figure 26). For these experiments, we used His-tagged proteins, human hEphB2-His and mouse mEB2-His. The hEphB2-His protein, when measured alone, exhibited an average mass of 130 kDa, while the mEB2-His protein measured an average of 83 kDa. Both proteins displayed a fairly steep Poisson distribution pattern, indicating a relatively uniform mass within each (Figure 26A-B). The values ​​obtained for EphB2 and EB2 are significantly higher than the predicted monomer masses of these two ectodomain proteins, 59.7 kDa and 23.5 kDa, respectively. While part of this discrepancy may be due to heavy glycosylation, particularly in the case of ephrins, both Eph and ephrin ectodomain proteins have been shown to have weak homodimerization / oligomerization tendencies in solution and in crystal structures. During MP experiments, EphB2 and EB2 were measured as homodimers, respectively, when analyzed alone. When EphB2 and ephrinB2 proteins were combined, the average mass of the protein species shifted to a larger 216 kDa species (Figure 26C). When two EphB2 and two ephrinB2 molecules complexed together into a stable cyclic tetramer, the size was as expected (130 + 83 = 213 kDa). Importantly, consistent with assembling two distinct molecules into a single tetrameric complex, the total number of protein species measured in the MP did not increase when EphB2 and EB2 proteins were combined, indicating that the proteins had combined into a single measurable entity of higher mass.Furthermore, upon addition of A20, a dose-dependent loss of a 216 kDa-sized species was observed to an intermediate average mass of 158 kDa at 10 μM A20 and 150 kDa at 50 μM A20 (Figure 26D). The intermediate mass of 158 / 150 kDa would be consistent with the loss of one EphB2 or one ephrinB molecule from the tetramer, converting it into a weak Eph-ephrin dimer with a weakly homo-oligomerized Eph or ephrin molecule bound as a trimer. In the presence of A20, the total number of protein species increased, consistent with tetramer disassembly. Indeed, it is important to understand that the MP experiments described here are powerful but also time-limited, since protein species could only be assessed after interaction homeostasis was achieved; in this experiment, the protein and A20 were allowed to incubate together for 24 h before mass determination.

[0162] In contrast to the Octet kinetic assay, the MP assay focuses on measuring protein interactions present at the end of the binding phase, rather than using Octet. Thus, when Eph-ephrin proteins are combined, they lose their weak homodimerization ability and instead prefer to combine into stable, very high-affinity tetrameric structures. In the presence of A20 chemistry, tetramerization is inhibited, leaving only relatively weak Eph-ephrin dimers that can also weakly homodimerize with other Eph or ephrin molecules. These Eph-ephrin:ephrin or Eph:Eph-ephrin trimer species are hypothesized to represent primed complexes that are ready to tetramerize but are strongly inhibited by A20 chemistry.

[0163] [Example 13] A20 chemistry also targets EphA-ephrinA tetramers and EphB-ephrinA cross-class tetramers We used the Octet system to probe the interactions of epitope-tagged human A-class proteins, focusing on the binding of immobilized EphA3 to its two cognate ligands, ephrinA1 and ephrinA5, and EphB2 to ephrinA5 (Figure 27). The binding kinetics indicate that EphA3 exhibits a tetramerization-driven interaction with ephrinA1 but a dimerization-driven interaction with ephrinA5. Focusing first on the dimerization-driven EphA3-EA5 interaction (green sensorgram trace), which mimics that disclosed above for EphB4-EB2, we observed that the initial binding of soluble ephrinA5 to immobilized EphA3 was very fast and strong, with a long, steep 84° slope response, which then rapidly curved around 35 seconds into the binding phase, forming a more gradual tetramerization accumulation gradient. The overall response of EphA3-EA5 binding during the binding phase was much stronger than the other two Class A interactions analyzed, suggesting a very high affinity interaction. However, also like the EphB4-EB2 interaction, once subjected to dissociation, a significant proportion of the bound EA5 protein rapidly dissolved, and the dissociation phase continued downward with a slow, gradual decrease in bound protein. Although dissociation only took 500 seconds, the continued downward slope of the response indicates that the majority of bound EA5 protein eventually dissolved from the immobilized EphA3 protein. This is referred to as a classical dimerization-driven interaction, in which there is a fast and strong initial binding during the binding phase, but the lack of stable tetramer formation results in the release of most of the bound protein during dissociation. EphA3 interacts with ephrin-A1 with completely different kinetics / dynamics (orange sensorgram trace). Here, the initial high 84° dimerization gradient phase is extremely short (5 seconds), and the binding transitions rapidly to a linear tetramerization accumulation phase with a 25° gradient; once subjected to the dissociation step, the bound protein barely melts, and only a small amount, representing the dimeric interaction, leaves in the first few seconds after being subjected to dissociation.Thus, whereas the EphA3-EA5 interaction is initially strong due to its relatively high dimerization affinity but then collapses due to its weak tetramerization affinity, the EphA3-EA1 interaction is more like a turtle, slow and orderly, building strong, stable, high-affinity tetramers that do not melt and eventually catch up with their fast-binding, fast-dissociating counterparts. EphB2 interacting with EA5 resembles the EphA3-EA1 interaction in that the binding phase is characterized by a very short (5 s) high 84° gradient phase (blue sensorgram trace) that rapidly curves into a more gradual tetramer accumulation phase. Interestingly, once subjected to dissociation, the majority of bound EA5 protein dissolves from immobilized EphB2, but then, toward the end of dissociation, the response levels off above baseline, indicating the formation of a stable, long-lasting tetramer. The strong initial dissociation phase of the EphB2-EA5 complex represents both the melting of a weak dimer and the melting of a relatively weak affinity tetramer formed by A / B cross-class EphB2-EA5 molecules.

[0164] Similar to the results obtained with B-class molecules, the addition of A20 effectively reduced the formation of A-class and A / B-class cross-tetramers (Figures 28A-C). In the case of the EphA3-EA1 tetramerization-driven interaction (Figure 28A), A20 caused a strong dose-dependent decrease in binding, with approximately 50% loss of tetramers at 1 μM and almost complete loss of tetramer formation at 100 μM. While there was no effect of A20 on the rapid EphA3-EA1 dimerization phase during the first 5 s, there was a very strong effect of the compound on tetramer accumulation. Over 70% of the EphA3-EA1 binding interaction was attributed to tetramers, which were effectively disrupted by the compound, and negligible binding activity was attributed to dimers. More precisely, the dimerization:tetramerization ratio for this interaction was estimated to be 10:90, not 30:70. For the EphA3-EA5 interaction (Figure 28B), the addition of A20 also had no effect on the initial rapid, high-slope dimerization phase observed at the beginning of the binding phase, although there was a significant maximal loss of tetramer accumulation even at the lowest concentration of A20 tested (1 μM). Because this is a dimerization-driven interaction (the dimerization:tetramerization ratio here is 72:28), the overall effect of the compound on this interaction is attenuated. The A / B cross-class interaction, EphB2-EA5 (Figure 28C), can also be described as a tetramerization-driven interaction that is strongly inhibited by A20 (the dimerization:tetramerization ratio here is 23:77). Because this is one of the weakest Eph-ephrin interactions, addition of A20 here completely inhibited stable tetramer formation even at 1 μM. Unlike the EphA3-EA1 or EphA3-EA5 interactions, which showed low levels of insurmountable tetramers, the response level rapidly reached baseline during the dissociation phase (asterisk). In summary, the results demonstrate that the A20 compound effectively targets the formation of all classes of Eph-ephrin tetramers: A-class, B-class, and cross-A / B-class tetramers.

[0165] [Example 14] Pre-existing stable Eph-ephrin tetramers are also disrupted by the A20 chemical Biophysical studies were performed to investigate whether A20 could disrupt pre-existing stable tetramers, focusing on the binding of immobilized human ephrinB2 to soluble human EphB1 and EphB2, and the binding of immobilized mouse EphB2 to mouse ephrinB2 (Figures 29A-C). In these experiments, sensor chip-immobilized proteins were first exposed to 50 nM of their soluble target protein for an 800-second association phase and a 1000-second dissociation phase to generate pre-formed stable tetramers, which were then subjected to a 1000-second competition phase containing either 0, 2, 10, or 50 μM concentrations of A20 salt compounds. In all three cases, we observed that A20 could compete with a significant amount of the stable pre-formed tetramer complex in a dose-dependent manner. The ability of A20 to disrupt pre-existing tetramers was most evident in human EB2-EphB2 and cognate mouse EphB2-EB2 experiments, which showed a progressive, dose-dependent reduction in tetramer formation throughout (and possibly beyond) the 1,000-second competition phase, regardless of whether human ephrinB2 or mouse EphB2 was the immobilized protein. A20 also clearly disrupted a portion of pre-existing EB2-EphB1 tetramers in a dose-dependent manner, but the response level then plateaued, indicating that the effect was complete within 200 seconds of the competition phase. This suggests that some pre-existing EB2-EphB1 tetramers are readily targeted by A20, while others are not, potentially forming the insurmountable tetramers discussed previously. In summary, the results indicate that pre-existing Eph-ephrin tetramers can be disrupted by the A20 chemical and that the tetramer formed between EphB2 and ephrinB2 is probably easier to overcome and compete with than the tetramer formed between EphB1 and ephrinB2.

[0166] [Example 15] Methods for determining accurate compound IC50 values ​​for inhibition of Eph-ephrin tetramers. To identify additional novel chemical entities that exhibit improved tetramerization inhibitor activity and enhanced pharmacological properties, we developed a precise method to obtain accurate IC50 information reflecting the specific effect of compounds on inhibiting Eph-ephrin surmountable tetramers. Low-concentration dose-response Octet studies of compounds (e.g., 0, 0.05, 0.1, 0.2, 0.4, 0.8, and 1.6 μM concentrations) were performed, including a "maximum control" biosensor condition, which represents the concentration of A20 chemical required to eliminate all surmountable tetramers. The maximum control response was subtracted from the other responses to eliminate the contributions of dimer formation and binding to the insurmountable components of the tetramer, leaving the amount of binding attributable to surmountable tetramers for analysis. The subtracted response of the no-compound control biosensor was set to 100%, and the maximum control biosensor was set to 0% by definition. The subtracted and analyzed portion of the experiment can be a single time point, such as selecting two critical time points for analysis: 380 s during the standard 400 s association phase and 50 s after the start of the 500 s dissociation phase, or analyzing the AUC of each sensorgram during the analysis. The Octet experiment shown in Figures 30A-30D highlights this novel approach, evaluating the binding of immobilized human ephrinB2 to 50 nM soluble human EphB2 and providing accurate tetramerization inhibitor IC50s for A19, A20, 8009, and 3511, using 2 μM A20 as a maximum control. A quick glance at the data reveals that compound A19 exhibits only a weak ability to inhibit EB2-EphB2 tetramer formation compared to the results with A20, 8009, and 3511. Focusing on the IC50 values ​​at 380 seconds during the binding phase, we first subtracted the maximum control response from the compound at different doses, and then placed a blue asterisk to indicate the 50% point of the corrected data. For compound A19, the blue asterisk is below the highest concentration tested here, so the relative binding IC50 here is greater than 1.6 μM. For A20 and 8009, the blue asterisk is just below the 0.4 concentration, so the IC50 for these two compounds is approximately 0.5 μM. For 3511, the IC50 is just above the 0.2 μM sensorgram experiment.These highly accurate IC50 calculations show that the lead compounds exhibit IC50 values ​​in the 200-600 nM range, generating highly potent leads as inhibitors of very high affinity protein-protein interactions.

[0167] [Example 16] Preparation and testing of analogs of the A20 compound Through ongoing medicinal chemistry efforts, we have designed, synthesized, salted, and purified several novel analog compounds similar to A20. Compound 8009 is very similar to A20, with a chloride replacing the bromine halogen on the 8-hydroxyquinoline, while compound 3511 replaces the A20 morpholine ring with a more complex piperazine-extended structure. Given the potential for modifications in these two regions of A20, we synthesized a series of compounds that varied these regions. The compounds were then subjected to kinetic Octet studies to determine their biophysical activity, build an understanding of SAR, and provide information about the chemical space that defines what makes a good Eph-ephrin tetramerization inhibitor.

[0168] A general strategy for designing A20 analogs was developed, focusing on (a) novelty, (b) the effect of different synthetic building blocks other than quinoline, (c) the effect of modifications of the morpholine / piperazine extension, and (d) satisfying pharmacokinetic parameters. The synthetic schemes for several different compounds based on A20 were examined (Figure 31), including 3511 (Figure 32). A20 was synthesized via three steps, starting with quinolin-8-ol and 37% formaldehyde in the presence of HCl and catalytic ZnCl, followed by nucleophilic substitution with morpholine under basic conditions to give QM, followed by electrophilic substitution using N-bromosuccinamide (NBS) in the presence of dichloromethane to give A20. Compounds were purified using silica gel column chromatography and mobile phase gradient elution using an ethyl acetate / hexane mixture and structurally elucidated using H NMR and C NMR spectroscopy. Different structural modifications of A20 encompass one or more combinations as shown in Figure 31, including (x) substitution of Br (A20) and Cl (8009) with other halogens I or F, (y) modification of morpholine to piperidine and piperazine, and (z) heteroaryl modification changing the quinoline ring system to quinazoline or indole. Analogs based on 3511, shown in Figure 32, incorporate structural modifications via (x) substitution of Br with H, Cl, F, or I, (y) modification of the methoxy group with different electron-donating or electron-withdrawing groups at the ortho, meta, or para positions, (z) modification of the quinolone ring to quinazoline, and (ro) ring-opening of the piperazine to have diaminoethane as a linker. Salt forms of the compound are also produced, which aid in aqueous solubility and provide more favorable PK properties.

[0169] First, two quinolin-8-ol-based morpholine variants were synthesized by expanding the hydrophobic moiety through nucleophilic substitution with different piperazine derivatives to generate the novel compounds QPB4 and QPDF (Figure 33A). The synthesis of QPB4 and QPDF also involved generating the A20 derivative in which Br was replaced by H, resulting in A19 / QM (compound 1). Because ring substitution is a known strategy for drug discovery, and substitution with quinazoline or indole ring systems is a viable approach, modifications of the quinoline ring system were also synthesized in the application of classical bioisosterism via ring substitution. Here, a different ring system, 5-methoxyindole, was introduced in a one-step synthetic pathway. This involved the synthesis of novel indole-based analogs IM, IPM2, and IPB4 by coupling 5-methoxy-1H-indole-3-carboxylic acid with morpholine or substituted piperazine in the presence of 1-ethyl-3-(3-dimethyl-aminopropyl)carbodiimide and hydroxybenzotriazole in dichloromethane (Figure 33B).

[0170] These initial first-generation novel compounds in Figure 33 were initially tested in the Alpha assay and compared with A20, 3511, and 8009. Compound QPB4 initially appeared interesting because it exhibited potent dose-dependent antagonist activity that appeared far superior to A20 or 3511 (Figure 34). QPDF and A19 / QM also exhibited some inhibitory activity, but this was not evident until higher concentrations. The 5-methoxyindole analog failed to demonstrate activity even at 0.5 mM, indicating that this ring substitution was not viable and thus providing information about the SAR regarding the importance of the quinoline ring system. To more accurately determine IC50 values, all compounds were subjected to the refined Octet assay described above, including the maximum control subtraction method, using compound concentrations of 0, 0.1, 0.2, 0.4, 0.8, and 1.6 μM at binding of immobilized human ephrinB2 to 50 nM soluble human EphB2 (Figure 35). The tetramerization inhibitor activity of QPB4 was weaker with a relative binding IC50 of 1.2 μM, suggesting that this compound is not as active as the initial Alpha assay results indicated.

[0171] Given the successful preparation and testing of first-generation A20-related chemicals, additional novel second-generation compounds were designed and synthesized using the general synthetic scheme shown (Figures 36A, 36B, and 36C). Structures of all analogs prepared are shown in Figure 37, and detailed synthetic methods are described in Example 27 below. Additional information on all tested compounds, including structure, molecular weight, and nomenclature information, is provided in Table 1 above. Tables A-G (last) contain the IC50 and other biophysical data measured for the various compounds.

[0172] Most of these chemicals were subjected to sophisticated biophysical testing using binding of immobilized human ephrinB2 to 50 nM soluble human EphB2 to determine precise IC50 tetramerization inhibitor activity (see Figures 30A-30D and 35A-35E), with specific values ​​shown in Tables A-G at the end of the Examples. The data collected to date are very encouraging and are summarized below.

[0173] The novel compound A20-I, which is a halogen substitution that changes the 7-bromine of A20 to an iodine, possessed enhanced tetramerization inhibitory activity (Figures 38A-38D). This is evident when comparing the results of A20-I with those of the A20 salt compound for the binding of immobilized mouse EphB2 ectodomain to soluble mouse ephrinB2 ectodomain (Figures 38A and 38B) or soluble human ephrinA5 (Figure 38C). For the EphB2-ephrinA5 interaction, A20-I exhibited potent inhibitory activity compared to A20 (see Figure 28). For A20-I, the potent concentration-response effect was observed, as 0.8 μM and 1.6 μM yielded identical, overlapping sensorgram traces, indicating that the compound's maximum effect was achieved at 0.8 μM, whereas for A20, 10 μM of this compound was required to achieve full inhibitory activity. Similarly, for the binding interaction of immobilized mouse EphB2 to mouse ephrinB2, A20-I at 100 μM eliminated all tetrameric species containing an insurmountable component that A20 cannot target. During these interactions of immobilized EphB ectodomain binding to soluble ephrinB2, the tetramer was much more easily overcome by the addition of active compound than in the reverse experiment, in which ephrinB2 was immobilized and EphB2 was the soluble component in the assay (see Figures 11, 17, and 25A-25C). The more resistant insurmountable tetramer formed by binding of immobilized ephrinB2 to soluble EphB ectodomain provided the most rigorous test for determining IC50 values, and therefore, utilizing this ephrinB2-EphB2 assay for analysis of all compounds is believed to provide uniformity in the results observed. The results described herein with A20-I and with human ephrinB2-EphB2 interactions indicate that substitutions can be made at the 7-bromine of A20 that result in enhanced tetramerization inhibitor activity. However, not all substitutions at the 7-bromine of A20 are productive, as A19-NO2, which replaces the halogen at the 7 position with nitrogen dioxide, results in a weak, essentially inactive compound (Figure 38D).Initial studies by PK Core indicate that both the free base and salt forms of A20-I also exhibit a long in vitro half-life in a mouse microsomal metabolic stability assay in direct comparison to A20 (Figure 39). These metabolic stability results are encouraging and indicate that this novel compound exhibits significantly improved biological activity compared to our initial A20 lead.

[0174] A group of related novel analogs, in which the oxygen in the morpholine ring is replaced with sulfur dioxide and the 7-halogen position contains either H (QTM), Br (QTM-Br), or I (QTM-I), are shown in Figures 40A-C. All three compounds show some ability to inhibit the tetramer, but the two containing halogens exhibit potent activity, both reaching a maximum control response level at 1.6 μM and with a relative IC value of 0.8 μM. The results show that the QTM compound is not as active as A20, which approached a maximum inhibitory response at 0.8 μM (upper left panel).

[0175] A series of second-generation compound variants related to QPB4 are shown in Figures 41A-41F. Among these, compound BQPB4-Bn appears promising, as concentrations of 0.8 and 1.6 μM correspond to maximum response levels (Figure 41E). Changing the Br on the piperazine of BQPB4-Bn to CF3, generating compound QPCF34, eliminated activity. Furthermore, QPI4 appeared less potent and closer to the parent QPB4. Other compounds showed very weak or no activity, most notably MeQPB4, which has a methyl group attached to the quinoline 2-position, also appearing to eliminate tetramerization inhibitor activity.

[0176] The salt form of compound QPB4-Bn, which has potent tetramerization inhibitor activity, was also tested for PK biodistribution, as shown in Figures 42A, 42B, and 42C. The results show similarity to those obtained with the A20 salt, indicating that significantly higher levels of the compound reach the intended tissues (i.e., brain, spinal cord, liver, skin) and persist at high concentrations throughout the 24-hour study period.

[0177] [Example 17] Eph-ephrin dimerization is disrupted by SNEW and EWLS peptides The Octet system was used to characterize the biophysical effects of the disclosed SNEW and EWLS peptides on Eph-ephrin protein-protein interactions. As shown in Figure 43, "SNEW peptide" herein refers to a peptide having the amino acid sequence SNEWIQPRLPQH (SEQ ID NO: 1), and "EWLS peptide" refers to a peptide having the amino acid sequence EWLSPNLAPSVR (SEQ ID NO: 2). These peptides are further embodied in the consensus sequence EWX1X2PX3LX4PX5 (wherein X1 is leucine or isoleucine (L / I), X2 and X5 are each independently glutamine (Q) or serine (S), X3 is any amino acid, and X4 is alanine or absent) (SEQ ID NO: 3). Further information regarding these peptides is provided in Koolpe, M., et al. (J. Biol. Chem. (2005) 280, 17301-17311), which is incorporated herein by reference in its entirety. The Alpha assay data in Figure 4A show that both peptides interfere with EphB1-ephrinB2 interaction. Because these two peptides are very similar to each other (Figure 4C), their general ability to interfere with EphB1-ephrinB2 interaction was attributed to the Alpha assay, which is more suitable for interference.

[0178] Extensive biophysical data indicate that the A20 chemical selectively targets Eph-ephrin tetramers. To assess this, we used Octet to characterize how the SNEW and EWLS peptides affect the binding interaction, hypothesizing that they affect the initial, very high gradient "fast-on" binding, which is attributed to the formation of Eph-ephrin dimers. Using the binding of immobilized human ephrinB2 to 50 nM soluble human EphB2, we tested increasing concentrations of the SNEW and EWLS peptides (0.25, 0.5, 1.0, 5, 10, and 100 μM) (Figures 44A and 44B—top sensorgrams). While concentrations of the SNEW peptide up to 10 μM had no effect on the ephrinB2-EphB2 interaction, a 100 μM concentration of the SNEW peptide specifically altered the binding phase. The effect of the SNEW peptide can be attributed to disruption of dimer formation, as the initial fast-binding dimer gradient of 85° typically observed immediately after the start of the binding phase significantly flattened to 65° during the binding phase in the presence of 100 μM peptide in this experiment. Although the dimer gradient dramatically flattens with the addition of the SNEW peptide, stable tetramers continue to accumulate, albeit more slowly. This indicates that dimerization inhibitors such as SNEW may only slow tetramer formation, thereby weakening the effect of these peptides compared to the A20 chemical. The EWLS peptide showed no effect on ephrinB2-EphB2 interactions. Thus, consistent with previous reports, biophysical analysis by Octet indicates that the SNEW peptide, but not the EWLS peptide, is selective in disrupting EphB2-ephrinB2 interactions. However, the need to use such high concentrations of SNEW to achieve a biophysical response can be explained by the rather weak ability of these peptides to exhibit biological activity. Nevertheless, to confirm these results, a second dose-response Octet study was performed using a higher concentration range of SNEW and EWLS peptides (50, 100, 200, and 400 μM).The results show that the SNEW peptide exhibited potent dose-response inhibition of fast-binding dimers, beginning again just at the beginning of the binding phase, at all concentrations tested (Figures 44A and 44B—bottom sensorgrams). During this experiment, the fast-binding dimer gradient of 84° in the absence of peptide strongly flattened to 70°, 60°, 51°, and 41° as the concentration of SNEW increased. EWLS again had no effect on the interaction, even at a 400 μM concentration, again demonstrating that the SNEW peptide is specific for ephrinB2-EphB2 interactions. In summary, the SNEW peptide prevented the formation of ephrinB2-EphB2 dimers, providing additional evidence that the Octet system can help distinguish Eph-ephrin dimer interactions from Eph-ephrin tetramer interactions.

[0179] [Example 18] Pharmacokinetic studies of A20 chemicals To investigate the drug-like properties of the disclosed compounds, solubility studies were performed. Results indicate that both salt forms of A20 and A20-I are highly water-soluble (Figure 45). Furthermore, in vitro plasma stability studies indicate that A20-I exhibits a half-life 3-5 times longer than A20 (Figure 46). In vivo PK studies demonstrate that the A20 salt compound is orally bioavailable (Figures 47A-47D), suggesting that the compound could potentially be formulated as a pill or oral liquid for administration, enhancing the drug-like qualities of the tetramerization inhibitors. The oral bioavailability evaluated in this study is particularly evident in the liver. This suggests a possible route of administration for oral tetramerization inhibitors for conditions such as NASH liver fibrosis, CDK chronic kidney disease, diabetes / obesity, pancreatic cancer, etc.

[0180] [Example 19] Biophysical testing of additional A20 chemicals The Octet was used to evaluate a number of newer chemical entities that had been designed and synthesized using a standard assay of binding of immobilized human ephrin B2 ectodomain to 50 nM soluble human EphB2 ectodomain.

[0181] Figures 48A, 48B, 48C, and 48D show the results for compounds A20-I, A19-L, 2OH-A19, and QPPh. A20-I exhibited strong activity in preventing surmountable tetramers with an IC50 of 0.6 μM (Figure 48A), as expected given the data presented above. However, compounds 2OH-A19 and QPPh exhibited only weak activity with IC50s of 3 μM (Figures 48C and 48D). Data for A19-L were mixed in that it showed a low tetramerization inhibitor IC50 of 1.6 μM at a 3.2 μM concentration, but its sensorgram trace was clearly below that of the maximum control, so it was actually able to inhibit a small amount of ephrinB2-EphB2 insurmountable tetramers (Figure 48B).

[0182] Figures 49A, 49B, 49C, 49D, and 49E show the results for compounds A19-NO, QTM-NO, 2OH-A20, 2-MeA20, and QPB-NO4, respectively. Here, A19-NO, QTM-NO, and 2-MeA20 only showed weak ability to inhibit the tetramer with IC50s above 3 μM, whereas 2OH-A20 and QPB-NO4 had poor / low activity with IC50s of 1.6 μM. Interestingly, like compound A19-L, QPB-NO4 was also able to inhibit a small amount of the insurmountable ephrinB2-EphB2 tetramer, as its sensorgram at 3.2 μM was clearly below that of the maximum control and even lower than that of A19-L (Figure 49E).

[0183] Figures 50A, 50B, 50C, and 50D show the results for compounds QPA, QPA-Ac, QPA-PAc, and BQPA-PAc, respectively. Here, QPA-Ac and BQPA-PAc showed only weak activity with IC50s above 3 μM, while QPA and QPA-PAc had poor / low activity with IC50s of 2 μM and 1.2 μM, respectively. Interestingly, like compounds A19-L and QPB-NO4, QPA-PAc was able to inhibit a small amount of the insurmountable ephrinB2-EphB2 tetramer at both 1.6 μM and 3.2 μM concentrations, as these two sensorgrams, especially at 3.2 μM, were clearly below those of the maximum control (Figure 50C).

[0184] Figures 51A, 51B, 51C, 51D, and 51E show the results for compounds 3511-I, 3511-0013-BF, 3511-4OMe-BF, 3511-4OHMe-I, and IQPB4-BN, respectively. All except 3511-0013-BF exhibit strong IC50 values. The Octet experiment for 3511-I was particularly interesting because both the 0.8 μM and 1.6 μM concentrations were able to inhibit small amounts of the ephrinB2-EphB2 insurmountable tetramer. Interestingly, the amount of insurmountable tetramer inhibited by 3511-I reached a maximum at 0.8 μM, as there was no additional tetramer loss at the 1.6 μM concentration tested (Figure 51A).

[0185] Finally, Figure 52 shows results from two independent Octet experiments for compound BQPB4. Remarkably, this compound exhibits an ultrapotent IC50 of approximately 0.3 μM for inhibition of surmountable tetramers, while simultaneously exhibiting an extremely potent, concentration-dependent ability to inhibit 100% of insurmountable tetramers. Due to its exceptional ability to inhibit all ephrinB-EphB2 tetramers, compound BQPB4 is the most potent chemical entity identified. Figure 53 shows six different compounds derived from BQPB4 that exhibit activity against insurmountable tetramers.

[0186] [Example 20] Overview of Medicinal Chemistry Initiatives Figure 54 is a scheme summarizing medicinal chemistry efforts around the A20 scaffold, highlighting changes that can enhance / improve tetrameric inhibitor activity (green and purple) and changes that result in low / poor inhibitor activity (yellow) or weak / no activity (red).

[0187] Additional modifications to both the A20 and 3511 scaffolds are also envisioned. These modifications may stabilize the molecule or increase potency. Proposed modifications to be synthesized are summarized in Figures 55 and 56.

[0188] [Example 21] EPH-ephrin tetramerization inhibitors as a new class of drugs for the treatment of chronic pain As previously shown using chronic constriction injury (CCI) in a sciatic nerve model and in a bone cancer pain model, following nerve injury or damage that produces chronic pain, ephrinB2 expression is strongly increased in nociceptors / C fibers (presynaptic) and EphB1 is strongly increased in DH neurons (postsynaptic) (Figures 57A-57B, each of which is incorporated by reference in its entirety; Song XJ, Cao JL, Li HC, Zheng JH, Song XS, and Xiong LZ. (2008). Upregulation and redistribution of ephrinB and EphB receptor in dorsal root ganglion and spinal dorsal horn neurons after peripheral nerve injury and dorsal rhizotomy. Eur J Pain 12:1031-9 and Liu S, Liu WT, Liu YP, Dong HL, Henkemeyer M, Xiong LZ, and Song XJ. (2011). Blocking EphB1 Receptor Forward Signaling in Spinal Cord Relieves Bone Cancer Pain and Rescues Analgesic Effect of Morphine Treatment in Rodents. Cancer Res 71:4392-4402. - / -We have shown that mutant animals are viable but exhibit greatly reduced hyperalgesia and allodynia after various experimental forms of nerve injury that normally induce chronic pain, including CCI neuropathic and inflammatory pain models (Figure 58, see Han Y, Song XS, Liu WT, Henkemeyer M, and Song XJ. (2008). Targeted mutation of EphB1 receptor prevents development of neuropathic hyperalgesia and physical dependence on morphine in mice. Mol Pain 4:60, which is incorporated herein by reference in its entirety). Thus, EphB1 KO mice lacking expression of this receptor do not experience chronic pain and are immune, which is why we sought to identify drug compounds that block this protein. Furthermore, uninjured EphB1 - / - Because mutant mice exhibit normal levels of acute pain, their resistance to chronic pain development is not simply due to their inability to sense typical, normal pain required for daily life. Thus, EphB1 is not involved in normal acute pain, but its expression is strongly upregulated upon nerve injury or damage that generates chronic pain, which drives pathological DH neuron plasticity and central sensitization. The central role of EphB1 in chronic pain is due to the fact that EphB1 possesses only one copy of the wild-type gene. + / - This is strengthened by the finding that heterozygous animals did not exhibit neuropathic pain (Figure 58 - triangles), indicating that the EphB1 receptor protein is an ideal candidate to target for chronic pain, as an effective drug compound would only need to block approximately 50% of its activity.

[0189] Electrophysiological studies in live mice provide one of the most compelling evidences that EphB1 can directly influence DH neuron plasticity in response to excessive pain stimuli. High-frequency stimulation of C-fibers induces EphB1 in live mice. + / +EphB1 induced robust and long-lasting LTP in the spinal cord of wild-type mice but lacked expression of the EphB1 receptor. - / - The same experiments performed on mutant mice showed no response at all (see Figure 59, incorporated herein by reference in its entirety, Liu WT, Han Y, Li HC, Adams B, Zheng JH, Wu YP, Henkemeyer M, and Song XJ. (2009). An in vivo mouse model of long-term potentiation at synapses between primary afferent C-fibers and spinal dorsal horn neurons: essential role of EphB1 receptor. Mol Pain 5:29). This demonstrates that without EphB1 protein expression, DH neurons fail to exhibit the pathological plasticity associated with excessive stimulation of C-fiber inputs. In addition to studying EphB1 KO mice, other methods were used to evaluate the role of this receptor and its ligands in pathological pain, most notably intrathecal injection of soluble EphB and ephrinB ectodomain-blocking / activating reagents into the spinal cord. Indeed, some of the strongest data solidifying the idea that the EphB1 receptor tyrosine kinase is required for the development of chronic / neuropathic pain come from the ectodomain protein EphB1. - / -This work stems from the combined use of intrathecal injections into mutant mice. Other important data include the following: EphB1 is required for the postsynaptic localization of NMDARs; EphB1 can directly bind to the NR1 subunit and regulate the synaptic localization of NMDARs; EphB1 anterograde signaling activates Src kinase family members to induce tyrosine phosphorylation of the NR2 subunit, thereby opening the NMDAR ion channel and enabling calcium influx and LTP; and gain-of-function studies show that the NMDAR inhibitor MK-801 blocks EphB1-stimulated neuropathic pain. Furthermore, many of the above-cited papers provide compelling evidence that EphB1's role is not simply to regulate the synaptic localization and activity of NMDARs, but may also act through the activation of other intracellular neuronal signaling pathways implicated in plasticity, such as MAP kinase, PI3K, AKT, PKA, PKCγ, ERK, CaMKII, and CREB. Thus, at least with respect to DH synapses and central sensitization, the EphB1 receptor appears to be at the top of the food chain, activating numerous downstream events that result in chronic pain (Figure 6 and Figure 2). This role in pain is consistent with the transsynaptic localization and overexpression of these proteins upon nerve injury / injury, leading to enhanced formation of ephrinB2-EphB1 tetramers and higher-order signaling clusters. This inappropriately overactivates intracellular bidirectional signaling events through both the receptor (anterograde signaling) and ephrins (retrograde signaling) to strengthen existing synapses and build additional new synapses. Furthermore, while much of the above data on EphB1 suggests that excessive anterograde signaling by this receptor is responsible for chronic pain, it remains possible that retrograde signaling by ephrinB2 (or other ephrinBs) may also be involved in pathology, perhaps in a role similar to that described in the hippocampus, where retrograde, presynaptic retrograde signaling can affect synaptic plasticity. Furthermore, the closely related neuronal sister receptor EphB2 may also be involved in pain by stimulating the excitability of nociceptor primary sensory neurons.Thus, because both EphB1 and EphB2 proteins and their cognate ephrin B proteins may be involved in chronic pain, we anticipate that A20 tetramerization inhibitor chemicals will effectively target these possible interactions.

[0190] [Example 22] A20 compound exhibits drug-like activity in dampening inflammatory pain Having demonstrated that A20 crosses the BBB when injected IP at 20 mg / kg, pilot experiments were performed to assess whether such injections attenuate inflammatory pain generated in the CFA model. It has previously been determined that EphB1- / -KO mice exhibit greatly reduced thermal and mechanical hyperalgesia following injection of CFA into the hindpaw. Therefore, A20 was tested for CFA-induced thermal sensitivity enhancement in 14-week-old CD1 mice. Animals received 7 x 12-h IP administrations of A20 or vehicle, with 30 μl of CFA injected into the right hindpaw between the third and fourth administrations. While all vehicle-control mice exhibited the expected rapid and long-lasting thermal hyperalgesia in the right hindpaw (Figure 61 - black stippled line), pain responses in A20-treated mice were significantly blunted throughout the course of the 19-day study (Figure 61 - red stippled line). Statistical analysis of the data revealed significant differences between vehicle- and A20-treated mice. Importantly, the uninjured left hind paws of A20- and vehicle-injected mice showed no difference from each other (Figure 61 - solid line). This important internal control indicates that A20 does not have an overall effect on normal pain sensation, but only on the pain that develops after inflammatory insult.

[0191] In addition to the A20 compound, certain tetracycline antibiotics, particularly equimolar amounts of three tetracycline drugs referred to as MCD, can act as EphB kinase inhibitors (KIs) and reduce inflammatory pain. A comparison of A20 and MCD was performed using the same strategy as shown in Figure 61, and found that A20 appears to be the more effective drug (Figure 62). The results indicate that A20 may be more effective due to its ability as a tetramerization inhibitor to more potently disrupt Eph anterograde signaling and, at the same time, to disrupt ephrin retrograde signaling, something that kinase inhibitors do not do.

[0192] In previous pain-testing experiments, animals were preloaded with the compound starting two days before CFA inflammatory agent placement in the hind paw to induce pain. Next, we tested whether the compound was effective in treating pain when administered after CFA injection into the hind paw. Any effective drug compound likely needs to be delivered after the pain-producing chronic / neuropathic injury has occurred, and subjects typically cannot predict when severe injury will occur, except perhaps before planned surgery. Furthermore, the formulation of A20 in the previous experiments described herein utilized the free base form of the compound and, due to solubility issues, required a complex mixture of DMSO and sunflower seed oil as a vehicle. Therefore, we conducted studies to investigate whether a highly soluble salt form of the A20 chemical, which can be easily dissolved in PBS as a vehicle, could be an effective pain reliever. For these purposes, the compound dissolved in PBS was first injected at 20 mg / kg 15 minutes after CFA inflammatory agent placement in the hind paw. The animals were then injected with the compound every 12 hours for three additional days (a total of eight injections). Thermal pain responses were determined both before CFA injection (baseline) and periodically over a two-week period following injection. Data show that the salt form of A20 and three novel analogue chemicals (QPB4-Bn, QPB4, and QPP-127) all demonstrated the ability to blunt the increased sensitivity induced by the inflammatory pain insult (Figures 63 and 64).

[0193] In summary, the preclinical in vivo data described demonstrate that the disclosed Eph-ephrin tetramerization inhibitor compounds are highly effective when administered either before or after a pain-producing noxious injury.

[0194] [Example 23] A20 compound exhibits drug-like activity in blunting activation of dorsal horn neurons in the spinal cord associated with chronic pain and central sensitization Nerve injury / injury that generates chronic pain leads to overstimulation of NMDARs in the superficial spinal cord, which activates DH neurons and forms LTP. This DH neuron plasticity causes central sensitization, resulting in enhanced pain signal transmission to the brain. Although such activity-dependent neuronal plasticity is important for normal learning and memory in the brain, DH neuron plasticity involved in chronic pain may be considered a pathological or adverse form of plasticity. Therefore, we investigated its blockade or prevention by targeting the EphB1 receptor with the disclosed tetramerization inhibitor drug. The molecular events triggered by NMDARs that lead to neuronal activation and plasticity involve a cascade of intracellular pathways, most notably increased expression of the immediate-early gene (IEG) c-Fos. Furthermore, consistent with its important role in pain, EphB1 KO mice were found to exhibit significantly reduced numbers of c-Fos-expressing DH neurons after inflammatory injury. Genetic tools, Trap1 and improved Trap2, are now available that express CreERT2 under the control of the c-Fos promoter and, when combined with the Rosa26-stop-tdTomato Cre indicator (Ai9) and IP injection of 4-hydroxy-tamoxifen (4-OHT), permanently label only activated neurons with red fluorescence for a short time period (approximately 8 hours) until 4-OHT is degraded. Trap1 was used to examine EphB2 in the structural plasticity of learning-related neurons in the auditory cortex. Similarly, Trap2 was used to examine the role of EphB1 in DH neuron activation, which can be used to quantitatively assess the effects of the disclosed compounds on neuronal activation after injury that produces chronic pain.

[0195] To test and develop the system, Trap2 and Ai9-containing mice were obtained and crossed with mice containing the EphB1 KO mutation to express one copy of the Trap2 CreERT2 driver and one copy of the Ai9 tdTomato Cre reporter. + / + WT mice, EphB1 + / -Heterozygous mice, and EphB1 - / - Homozygous mice were generated. First, the left hind limbs of these mice were injected with CFA to induce a chronic pain injury. Four hours later, CreERT2 expression was accumulated under the c-Fos / Trap2 promoter. The mice were then IP injected with 4-OHT to activate Cre recombinase and induce deletion of the floxed-stop-tdTomato Ai9 element. This results in persistent and robust expression of tdTomato red fluorescent protein only in activated neurons during the brief window in which 4-OHT is active. Two weeks later, the mice were perfused with fixative, and the lumbar spinal cord was isolated, vibratome sectioned, and viewed under a fluorescent microscope to reveal red-labeled neurons that had been activated 2 weeks prior to the CFA / 4-OHT injection. EphB1 + / + Fluorescence imaging and quantification of red neurons in the dorsal horn of the spinal cord of WT mice revealed approximately three times more red-labeled neurons in the injured side compared with the uninjured side, but EphB1 + / - Heterozygous mice showed approximately twice as many neurons, and the EphB1- / - homozygotes analyzed showed approximately equal numbers of neurons on each side (Figure 65). The data show a clear correlation between EphB1 gene dosage and the level of DH neuron activation in the spinal cord in response to injury producing chronic pain. This is due to the EphB1 + / - We provide strong evidence that a 50% reduction in EphB1 protein expression in mice (or homozygous) strongly reduces (or, in the case of KO, eliminates) the early activation of DH neurons in response to injury that generates chronic pain.

[0196] To test whether injection of the A20 lead compound affects neuronal activity in the dorsal horn, it was determined that the compound dramatically reduced DH neuron activation after CFA injury (Figure 66). Thus, independent of behavioral testing, this new system allows for quantitative visualization of the effects of reduced EphB1 protein expression or activity on chronic pain.

[0197] In summary, the use of the Trap2 / Ai9 system to permanently label a small number of neurons in the dorsal horn that are activated for a short period immediately after injury to produce chronic pain provides a powerful and independent method for quantitatively assessing the in vivo efficacy of the disclosed tetramerization inhibitor drug compounds, in addition to the standard behavioral pain tests shown in Figures 61-64.

[0198] [Example 24] EPH-ephrin tetramerization inhibitors as a new class of drugs to combat opioid addiction. Notably, as in chronic pain, EphB1 receptor expression was previously shown to be strongly upregulated in the dorsal horn of the spinal cord after increasing doses of morphine (Fig. 67), and EphB1 - / -KO mice exhibit a significant reduction in the severe withdrawal behavior observed in morphine-addicted mice (Figure 68) (see, e.g., Liu WT, Li HC, Song XS, Huang ZJ, and Song XJ. (2009). EphB receptor signaling in mouse spinal cord contributes to physical dependence on morphine. FASEB J 23:90-8 and Han Y, Song XS, Liu WT, Henkemeyer M, and Song XJ. (2008), each of which is incorporated herein by reference in its entirety). Targeted mutation of the EphB1 receptor prevents the development of neuropathic hyperalgesia and physical dependence on morphine in mice. The disclosed experiments further demonstrated that EphB1 is also important for the development of tolerance to opioids, as KO mice exhibited a much longer analgesic response to morphine, and thus, in the absence of the receptor, tolerance builds slowly. Because the EphB1 receptor is associated with NMDARs, pharmacological targeting of EphB1's ability to interact with its ephrin B ligands has great utility for countering the adverse effects of opioid use and abuse. Experiments were performed to demonstrate that administration of the tetramerization inhibitor A20 was highly effective in reducing adverse withdrawal behaviors in morphine-addicted mice, making it potentially useful for countering the adverse effects experienced by mice addicted to opioids.

[0199] [Example 25] Revisiting the role of EphB1 receptors in adverse opioid withdrawal behaviors EphB1 - / - Recently, it has been replicated and verified that 8-month-old male EphB1 KO mice from the Henkemeyer mouse colony show a significant reduction in morphine withdrawal behavior in the test. + / + WT mouse EphB1 - / -KO mice were intraperitoneally (IP) injected with increasing doses of morphine in saline every 12 hours for 4 consecutive days (Day 1: 20 mg / kg, Day 2: 40 mg / kg, Day 3: 60 mg / kg, Day 4: 80 mg / kg). On the morning of Day 5, a final 100 mg / kg morphine dose was injected every 30 minutes until one mouse became staggered. Exactly 3 hours after the final morphine injection, each mouse was weighed and withdrawal was facilitated using naloxone hydrochloride subcutaneously injected at 1 mg / kg. An investigator blinded to the animals' genotype then monitored withdrawal signs for 30 minutes after naloxone administration to determine the number of jumping, violent shaking, and diarrheal events observed during the 30 minutes, as well as the % body weight change from before naloxone administration to 30 minutes after withdrawal. For tremor and ptosis, the presence of tremor and / or ptosis of the upper eyelid is monitored at the beginning of each 5-minute interval during the 30-minute monitoring period. Counting the number of jumps performed by mice can be described as one of the most robust methods for scoring morphine withdrawal, as this behavior is very easy to identify and normal mice in cages do not simply jump around. Here, it was determined that WT mice exhibited approximately 100 jumps over 30 minutes, while KO mice exhibited approximately 50 jumps (Figure 69). This demonstrates the reproducibility of data linking the EphB1 receptor to morphine withdrawal behavior.

[0200] Having confirmed the role of EphB1 in morphine withdrawal and demonstrated that A20 crosses the BBB when injected IP, two pilot experiments were performed to evaluate whether such injections minimize withdrawal behavior. One experiment used wild-type CD1 mice, while the other used wild-type C57BL / 6 mice. All mice were 4-5 months old and male. In both cases, 30 mg / kg of A20 free base dissolved in 6% DMSO / 94% sunflower seed oil was administered IP in 4 × 12-h doses to mice entering a 9 × 12-h IP schedule of increasing doses of morphine (2 × 20, 2 × 40, 2 × 60, 2 × 80, and 1 × 100 mg / kg). Naloxone was administered exactly 3 h after the final A20 / morphine dose, and withdrawal behavior was monitored for 30 min. Both groups of mice were scored for jumping (Figure 70A), and C57BL / 6 mice were additionally scored at 5-minute intervals for diarrhea, tremors, and ptosis (Figure 70B). A20-treated animals showed significantly fewer jumps, no signs of diarrhea or ptosis, and a 50% reduction in tremors. In summary, A20 tetramerization inhibitor compounds act to help reduce the harmful effects of opioid use and abuse and could potentially be used to help people wean themselves off highly addictive narcotics.

[0201] [Example 26] Synthesis of exemplary compounds The following compounds were synthesized according to the schematics shown in Figures 36A, 36B, and 36C, and Figures 71A-71X. Additional related compounds can be envisioned by slight modifications of the methods described herein. A method for producing a salt of one of these compounds is shown in Figure 16 (A20-2HCl) and is further described below. Salts of the other compounds synthesized below can be readily envisioned based on the method of Figure 16 described herein. Compound 1 (5-(morpholinomethyl)quinolin-8-ol, A19)

[0202] [ka]

[0203] Synthetic Procedure 1: Referring to Figure 36A and Figure 71A, a round-bottom flask was charged with piperazine derivative and diisopropylethylamine in dichloromethane and stirred at room temperature for 5 minutes. In a separate vial, 5-chloromethyl-2-methyl-8-quinolinol hydrochloride was dissolved in dichloromethane and 1 equivalent of base. This solution was added dropwise to the reaction mixture, which was then refluxed at 80 °C for 12 hours. Upon completion, the organic solvent was removed from the crude reaction mixture under reduced pressure. The crude solid was dissolved in dichloromethane and washed with water, NHCl (aq), and brine, and the organic fraction was collected and concentrated. The resulting crude material was purified via column chromatography (EtoAc:Hex, 1:1, v:v) to afford the target compound. The NMR spectrum is shown in Figure 71A. Compound 5 (7-bromo-5-(morpholinomethyl)quinolin-8-ol, A20)

[0204] [ka]

[0205] Referring to Figure 36A and Figure 71B, compound 1 was prepared according to synthetic procedure 1. Then, in a round-bottom flask, 5-(morpholinomethyl)quinolin-8-ol (compound 1) was dissolved in chloroform and cooled to 0 °C in an ice bath. In a separate vial, N-bromosuccinimide was suspended in chloroform and added portionwise to the reaction mixture in the flask over a period of 5-10 minutes. The resulting mixture was stirred at 0 °C for 1 hour. The solvent was then removed under vacuum at room temperature. The recovered solid / semi-solid was purified using silica column chromatography (EtOAc:Hex, 1:1, v:v) to afford an off-white solid product. The NMR spectrum is shown in Figure 71B. Compound 6 (4-((7-bromo-8-hydroxyquinolin-1-ium-5-yl)methyl)morpholin-4-ium chloride, A20-2HCl)

[0206] [ka]

[0207] Referring to Figures 36A and 71C, the free base of compound 5 was prepared as described above. Referring to Figure 16, the free base compound 5 (A20, 0.200 g, 0.62 mmol) was then added to a round-bottom flask equipped with a stir bar and subsequently dissolved in CHCl or chloroform (5 mL). While stirring the solution at 0°C, concentrated (12.1 M) HCl (106 μL, 1.27 mmol) diluted in methanol (2 mL) was added dropwise over 5 minutes. A yellow precipitate formed in the solution, so stirring was continued on ice for 30 minutes, and then the reaction was allowed to warm to room temperature for 2.5 hours. The resulting precipitate was filtered through a Buchner funnel at ambient conditions, washed with CHCl or chloroform, and recovered as a pale yellow solid (yield = 215 mg, 88%). The NMR spectrum is shown in Figure 71C. Compound 7 (7-iodo-5-(morpholinomethyl)quinolin-8-ol, A20-I)

[0208] [ka]

[0209] Referring to Figure 36A and Figure 71D, Compound 1 was prepared according to Synthetic Procedure 1. Then, in a round-bottom flask, 5-(morpholinomethyl)quinolin-8-ol (Compound 1) was dissolved in chloroform at room temperature. N-iodosuccinimide was added once to the reaction mixture in the flask. The resulting mixture was stirred at 50°C for 12 hours. The solvent was then removed under vacuum. The recovered solid / semi-solid was purified using silica column chromatography (EtOAc:Hex, 1:1, v:v) to give an off-white solid product. The NMR spectrum is shown in Figure 71D. Compound 9 (5-(morpholinomethyl)-7-nitroquinolin-8-ol, A19-NO2)

[0210] [ka]

[0211] Referring to Figure 36A and Figure 71E, compound 1 was prepared according to synthetic procedure 1. In a round-bottom flask, 5-(morpholinomethyl)quinolin-8-ol was dissolved in glacial acetic acid and cooled to 5-10 °C in an ice-water bath. The nitration mixture (H2SO4:HNO3 = 1:1) was then cooled and added dropwise to the reaction mixture in the flask over 5-10 minutes. The resulting mixture was stirred and allowed to reach room temperature over 3.5 hours. The resulting suspension was then added to ice-cold water (10-20 mL) and neutralized (pH 6-7) with cold saturated NaHCO3 until a precipitate was obtained. The resulting solid was filtered under suction and washed with cold DI water and a minimal amount of cold ether to yield a yellow solid. The NMR spectrum is shown in Figure 71E. Compound 14 (4-((8-hydroxyquinolin-5-yl)methyl)thiomorpholine 1,1-dioxide, QTM)

[0212] [ka]

[0213] Synthetic Procedure 2: Referring to Figure 36A and Figure 71F, thiomorpholine and diisopropylethylamine in dichloromethane were added to a round-bottom flask and allowed to stir at room temperature for 5 minutes. In a separate vial, 5-chloromethyl-8-quinolinol hydrochloride was dissolved in dichloromethane and 1 equivalent of base. This solution was added dropwise to the reaction mixture, which was then refluxed at 80 °C for 12 hours. Upon completion, the organic solvent was removed from the crude reaction mixture under reduced pressure. The crude solid was dissolved in dichloromethane and washed with water, NHCl (aq), and brine, and the organic fraction was collected and concentrated. The resulting crude material was purified via column chromatography (EtoAc:Hex, 1:1, v:v) to afford the target compound. The NMR spectrum is shown in Figure 71F. Compound 16 (4-((7-bromo-8-hydroxyquinolin-5-yl)methyl)thiomorpholine 1,1-dioxide, QTM-Br)

[0214] [ka]

[0215] Referring to Figure 36A and Figure 71G, compound 14 was prepared according to synthetic procedure 2. Then, in a round-bottom flask, 4-((8-hydroxyquinolin-5-yl)methyl)thiomorpholine 1,1-dioxide was dissolved in chloroform and cooled to 0 °C in an ice bath. In a separate vial, N-bromosuccinimide was suspended in chloroform and added portionwise to the reaction mixture in the flask over a period of 5-10 minutes. The resulting mixture was stirred at 0 °C for 1 hour. The solvent was then removed under vacuum at room temperature. The recovered solid / semi-solid was purified using silica column chromatography (EtOAc:Hex, 1:1, v:v) to afford an off-white solid product. The NMR spectrum is shown in Figure 71G. Compound 18 (4-((8-hydroxy-7-iodoquinolin-5-yl)methyl)thiomorpholine 1,1-dioxide, QTM-I)

[0216] [ka]

[0217] Referring to Figure 36A and Figure 71H, compound 14 was prepared according to synthetic procedure 2. Then, in a round-bottom flask, 4-((8-hydroxyquinolin-5-yl)methyl)thiomorpholine 1,1-dioxide was dissolved in chloroform at room temperature. N-iodosuccinimide was added in one portion to the reaction mixture in the flask. The resulting mixture was stirred at 50°C for 12 hours. The solvent was then removed under vacuum. The recovered solid / semi-solid was purified using silica column chromatography (EtOAc:Hex, 1:1, v:v) to give an off-white solid product. The NMR spectrum is shown in Figure 71H. Compound 20 (4-((8-hydroxy-7-nitroquinolin-5-yl)methyl)thiomorpholine 1,1-dioxide, QTM-NO2)

[0218] [ka]

[0219] Referring to Figure 36A and Figure 71I, compound 14 was prepared according to synthetic procedure 2. Then, in a round-bottom flask, 4-((8-hydroxyquinolin-5-yl)methyl)thiomorpholine 1,1-dioxide was dissolved in glacial acetic acid and cooled to 5-10 °C in an ice-water bath. The nitration mixture (H2SO4:HNO3 = 1:1) was then cooled and added dropwise to the reaction mixture in the flask over a period of 5-10 minutes. The resulting mixture was then stirred at room temperature for over 6 hours. To complete the reaction, the resulting solid suspension was stored overnight in a refrigerator. The resulting suspension was then added to ice-cold water (10-20 mL) and neutralized (pH 6-7) with cold saturated NaHCO3 until a precipitate was obtained. The resulting solid was filtered under suction and washed with cold DI water and a minimal amount of cold ether to obtain a yellow solid. The NMR spectrum is shown in Figure 71I. Compound 24 (7-bromo-5-((4-phenylpiperazin-1-yl)methyl)quinolin-8-ol, BQPPh)

[0220] [ka]

[0221] Bromination Procedure. Referring to Figure 36B and Figure 71J, in a round-bottom flask, 5-(chloromethyl)quinolin-8-ol was dissolved in chloroform and cooled to 0 °C in an ice bath. In a separate vial, N-bromosuccinimide was suspended in chloroform and added portionwise to the reaction mixture in the flask over a period of 5-10 minutes. The resulting mixture was stirred at 0 °C for 1 hour. The solvent was then removed under vacuum at room temperature. The recovered solid / semi-solid was purified using silica column chromatography (EtOAc:Hex, 1:1, v:v) to afford an off-white solid product.

[0222] Continuing with reference to Figure 36B and Figure 71J, a round-bottom flask was charged with the piperazine derivative and diisopropylethylamine in dichloromethane and allowed to stir at room temperature for 5 minutes. In a separate vial, 5-chloromethyl-8-quinolinol hydrochloride was dissolved in dichloromethane and 1 equivalent of base. This solution was added dropwise to the reaction mixture, which was then refluxed at 80 °C for 12 hours. Upon completion, the organic solvent was removed from the crude reaction mixture under reduced pressure. The crude solid was dissolved in dichloromethane and washed with water, NH4Cl (aq), and brine, and the organic fraction was collected and concentrated. The resulting crude material was purified via column chromatography (EtoAc:Hex, 1:1, v:v) to afford the target compound. Compound 26 (5-((4-(4-bromophenyl)piperazin-1-yl)methyl)quinolin-8-ol, QPB4)

[0223] [ka]

[0224] Referring to Figure 36B and Figure 71K, a round-bottom flask was charged with the piperazine derivative and diisopropylethylamine in dichloromethane and allowed to stir at room temperature for 5 minutes. In a separate vial, 5-chloromethyl-8-quinolinol hydrochloride was dissolved in dichloromethane and 1 equivalent of base. This solution was added dropwise to the reaction mixture, which was then refluxed at 80 °C for 12 hours. Upon completion, the organic solvent was removed from the crude reaction mixture under reduced pressure. The crude solid was dissolved in dichloromethane and washed with water, NHCl (aq), and brine, and the organic fraction was collected and concentrated. The resulting crude material was purified via column chromatography (EtoAc:Hex, 1:1, v:v) to afford the target compound. The NMR spectrum is shown in Figure 71K. Compound 28 (7-bromo-5-((4-(4-bromophenyl)piperazin-1-yl)methyl)quinolin-8-ol, BQPB4)

[0225] [ka]

[0226] Referring to Figure 36B and Figure 71L, the bromination procedure described for compound 24 above was followed. Then, the piperazine derivative and diisopropylethylamine in dichloromethane were added to a round-bottom flask and allowed to stir at room temperature for 5 minutes. In a separate vial, 5-chloromethyl-8-quinolinol hydrochloride was dissolved in dichloromethane and 1 equivalent of base. This solution was added dropwise to the reaction mixture, which was then refluxed at 80 °C for 12 hours. Upon completion, the organic solvent was removed from the crude reaction mixture under reduced pressure. The crude solid was dissolved in dichloromethane and washed with water, NHCl (aq), and brine, and the organic fraction was collected and concentrated. The resulting crude material was purified via column chromatography (EtoAc:Hex, 1:1, v:v) to afford the target compound. The NMR spectrum is shown in Figure 71L. Compound 35 (7-bromo-5-((4-(5-bromopyridin-2-yl)piperazin-1-yl)methyl)quinolin-8-ol, BQPP)

[0227] [ka]

[0228] Referring to Figure 36B and Figure 71M, the bromination procedure described for compound 24 above was followed. Then, the piperazine derivative and diisopropylethylamine in dichloromethane were added to a round-bottom flask and allowed to stir at room temperature for 5 minutes. In a separate vial, 5-chloromethyl-8-quinolinol hydrochloride was dissolved in dichloromethane and 1 equivalent of base. This solution was added dropwise to the reaction mixture, which was then refluxed at 80 °C for 12 hours. Upon completion, the organic solvent was removed from the crude reaction mixture under reduced pressure. The crude solid was dissolved in dichloromethane and washed with water, NHCl (aq), and brine, and the organic fraction was collected and concentrated. The resulting crude material was purified via column chromatography (EtoAc:Hex, 1:1, v:v) to afford the target compound. The NMR spectrum is shown in Figure 71M. Compound 41 (5-((4-benzylpiperazin-1-yl)methyl)quinolin-8-ol, QP-Bn)

[0229] [ka]

[0230] Referring to Figure 36B and Figure 71N, a round-bottom flask was charged with the benzylpiperazine derivative and diisopropylethylamine in dichloromethane and allowed to stir at room temperature for 5 minutes. In a separate vial, 5-chloromethyl-8-quinolinol hydrochloride was dissolved in dichloromethane and 1 equivalent of base. This solution was added dropwise to the reaction mixture, which was then refluxed at 80 °C for 12 hours. Upon completion, the organic solvent was removed from the crude reaction mixture under reduced pressure. The crude solid was dissolved in dichloromethane and washed with water, NH4Cl (aq), and brine, and the organic fraction was collected and concentrated. The resulting crude material was purified via column chromatography (EtoAc:Hex, 1:1, v:v) to afford the target compound. The NMR spectrum is shown in Figure 71N. Compound 42 (5-((4-(4-bromobenzyl)piperazin-1-yl)methyl)quinolin-8-ol, QPB4-Bn)

[0231] [ka]

[0232] Referring to Figure 36B and Figure 71O, a round-bottom flask was charged with the benzylpiperazine derivative and diisopropylethylamine in dichloromethane and allowed to stir at room temperature for 5 minutes. In a separate vial, 5-chloromethyl-8-quinolinol hydrochloride was dissolved in dichloromethane and 1 equivalent of base. This solution was added dropwise to the reaction mixture, which was then refluxed at 80 °C for 12 hours. Upon completion, the organic solvent was removed from the crude reaction mixture under reduced pressure. The crude solid was dissolved in dichloromethane and washed with water, NH4Cl (aq), and brine, and the organic fraction was collected and concentrated. The resulting crude material was purified via column chromatography (EtoAc:Hex, 1:1, v:v) to afford the target compound. The NMR spectrum is shown in Figure 71O. Compound 44 (7-bromo-5-((4-(4-bromobenzyl)piperazin-1-yl)methyl)quinolin-8-ol, BQPB4-Bn)

[0233] [ka]

[0234] The bromination procedure described for compound 24 was followed, with reference to Figure 36B and Figure 71P. Then, the benzylpiperazine derivative and diisopropylethylamine in dichloromethane were added to a round-bottom flask and allowed to stir at room temperature for 5 minutes. In a separate vial, 5-chloromethyl-8-quinolinol hydrochloride was dissolved in dichloromethane and 1 equivalent of base. This solution was added dropwise to the reaction mixture, which was then refluxed at 80 °C for 12 hours. Upon completion, the organic solvent was removed from the crude reaction mixture under reduced pressure. The crude solid was dissolved in dichloromethane and washed with water, NHCl (aq), and brine, and the organic fraction was collected and concentrated. The resulting crude material was purified via column chromatography (EtoAc:Hex, 1:1, v:v) to afford the target compound. The NMR spectrum is shown in Figure 71P. Compound 48 (5-((4-(4-(trifluoromethyl)benzyl)piperazin-1-yl)methyl)quinolin-8-ol, QPCF34-Bn)

[0235] [ka]

[0236] Referring to Figure 36B and Figure 71Q, a round-bottom flask was charged with the benzylpiperazine derivative and diisopropylethylamine in dichloromethane and allowed to stir at room temperature for 5 minutes. In a separate vial, 5-chloromethyl-8-quinolinol hydrochloride was dissolved in dichloromethane and 1 equivalent of base. This solution was added dropwise to the reaction mixture, which was then refluxed at 80 °C for 12 hours. Upon completion, the organic solvent was removed from the crude reaction mixture under reduced pressure. The crude solid was dissolved in dichloromethane and washed with water, NHCl (aq), and brine, and the organic fraction was collected and concentrated. The resulting crude material was purified via column chromatography (EtoAc:Hex, 1:1, v:v) to afford the target compound. The NMR spectrum is shown in Figure 71Q. Compound 50 (N-(4-(4-((8-hydroxyquinolin-5-yl)methyl)piperazin-1-yl)phenyl)acetamide, QPA-Ac)

[0237] [ka]

[0238] Referring to Figure 36B and Figure 71R, above, the amide-substituted amide piperazine derivative was prepared in a two-step procedure. First, a round-bottom flask was charged with the aminopiperazine derivative, and the stir bar was purged under vacuum and then backfilled with N2 gas (3x). Dichloromethane and triethylamine were added to a sealed vessel and allowed to stir at 0 °C for 5 minutes. Subsequently, acetyl chloride was added dropwise to the reaction mixture, which was allowed to stir at room temperature for 12 hours. Upon completion, the organic solvent was removed from the crude reaction mixture under reduced pressure. The crude solid was dissolved in dichloromethane and washed with water, NHCl (aq), and brine, and the organic fraction was collected and concentrated. The resulting crude material was purified via column chromatography (EtoAc:Hex, 1:1, v:v) to afford the target compound. The resulting amide product was then added to a round-bottom flask and dissolved in dichloromethane. Trifluoroacetic acid was added dropwise to this solution, and the reaction was allowed to stir at room temperature for 12 hours. After completion, the organic solvent was removed from the crude mixture under reduced pressure to give a pink oil. The crude material was washed with ether and the resulting solid was dried under high vacuum and used directly in the next reaction.

[0239] Next, as shown in Figure 36B, bottom, the amidopiperazine derivative and diisopropylethylamine in dichloromethane were added to a round-bottom flask and allowed to stir at room temperature for 5 minutes. In a separate vial, 5-chloromethyl-8-quinolinol hydrochloride was dissolved in dichloromethane and 1 equivalent of base. This solution was added dropwise to the reaction mixture, which was then refluxed at 80 °C for 12 hours. Upon completion, the organic solvent was removed from the crude reaction mixture under reduced pressure. The crude solid was dissolved in dichloromethane and washed with water, NH4Cl (aq), and brine, and the organic fraction was collected and concentrated. The resulting crude material was purified via column chromatography (EtoAc:Hex, 1:1, v:v) to afford the target compound. The NMR spectrum is shown in Figure 71R. Compound 56 (2-methyl-5-(morpholinomethyl)quinolin-8-ol, 2-MeA19)

[0240] [ka]

[0241] Synthetic Procedure 3: Referring to Figure 36A and Figure 71S, morpholine and diisopropylethylamine in dichloromethane were added to a round-bottom flask and allowed to stir at room temperature for 5 minutes. In a separate vial, 5-chloromethyl-2-methyl-8-quinolinol hydrochloride was dissolved in dichloromethane and 1 equivalent of base. This solution was added dropwise to the reaction mixture, which was then refluxed at 80 °C for 12 hours. Upon completion, the organic solvent was removed from the crude reaction mixture under reduced pressure. The crude solid was dissolved in dichloromethane and washed with water, NHCl (aq), and brine, and the organic fraction was collected and concentrated. The resulting crude material was purified via column chromatography (EtoAc:Hex, 1:1, v:v) to afford the target compound. The NMR spectrum is shown in Figure 71S. Compound 57 (7-bromo-2-methyl-5-(morpholinomethyl)quinolin-8-ol, 2-MeA20)

[0242] [ka]

[0243] Referring to Figure 36A and Figure 71T, synthetic procedure 3 was carried out as described for compound 55. Then, in a round-bottom flask, 2-methyl-5-(morpholinomethyl)quinolin-8-ol was dissolved in chloroform and cooled to 0 °C in an ice bath. In a separate vial, N-bromosuccinimide was suspended in chloroform and added portionwise to the reaction mixture in the flask over 5-10 minutes. The resulting mixture was stirred at 0 °C for 1 hour. The solvent was then removed under vacuum at room temperature. The recovered solid / semi-solid was purified using silica column chromatography (EtOAc:Hex, 1:1, v:v) to afford an off-white solid product. The NMR spectrum is shown in Figure 71T. Compound 60 (5-((4-(4-bromophenyl)piperazin-1-yl)methyl)-2-methylquinolin-8-ol, 2-MeQPB4)

[0244] [ka]

[0245] Referring to Figure 36B and Figure 71U, a round-bottom flask was charged with the piperazine derivative and diisopropylethylamine in dichloromethane and allowed to stir at room temperature for 5 minutes. In a separate vial, 5-chloromethyl-2-methyl-8-quinolinol hydrochloride was dissolved in dichloromethane and 1 equivalent of base. This solution was added dropwise to the reaction mixture, which was then refluxed at 80 °C for 12 hours. Upon completion, the organic solvent was removed from the crude reaction mixture under reduced pressure. The crude solid was dissolved in dichloromethane and washed with water, NHCl (aq), and brine, and the organic fraction was collected and concentrated. The resulting crude material was purified via column chromatography (EtoAc:Hex, 1:1, v:v) to afford the target compound. The NMR spectrum is shown in Figure 71U. Compound 69 (5-((4-(2-methoxyphenyl)piperazin-1-yl)methyl)quinolin-8-ol, 3511-0013-BF)

[0246] [ka]

[0247] Referring to Figure 36B and Figure 71V, the piperazine derivative and diisopropylethylamine in dichloromethane were added to a round-bottom flask and allowed to stir at room temperature for 5 minutes. In a separate vial, 5-chloromethyl-2-methyl-8-quinolinol hydrochloride was dissolved in dichloromethane and 1 equivalent of base. This solution was added dropwise to the reaction mixture, which was then refluxed at 80 °C for 12 hours. Upon completion, the organic solvent was removed from the crude reaction mixture under reduced pressure. The crude solid was dissolved in dichloromethane and washed with water, NHCl (aq), and brine, and the organic fraction was collected and concentrated. The resulting crude material was purified via column chromatography (EtoAc:Hex, 1:1, v:v) to afford the target compound. The NMR spectrum is shown in Figure 71V. Compound 74 (7-iodo-5-((4-(4-methoxyphenyl)piperazin-1-yl)methyl)quinolin-8-ol, 3511-4OMe-I)

[0248] [ka]

[0249] Referring to Figure 36B and Figure 71W, a round-bottom flask was charged with the piperazine derivative and diisopropylethylamine in dichloromethane and allowed to stir at room temperature for 5 minutes. In a separate vial, 5-chloromethyl-2-methyl-8-quinolinol hydrochloride was dissolved in dichloromethane and 1 equivalent of base. This solution was added dropwise to the reaction mixture, which was then refluxed at 80 °C for 12 hours. Upon completion, the organic solvent was removed from the crude reaction mixture under reduced pressure. The crude solid was dissolved in dichloromethane and washed with water, NHCl (aq), and brine, and the organic fraction was collected and concentrated. The resulting crude material was purified via column chromatography (EtoAc:Hex, 1:1, v:v) to afford the target compound (5-((4-(4-methoxyphenyl)piperazin-1-yl)methyl)quinolin-8-ol). Then, in a round-bottom flask, 5-((4-(4-methoxyphenyl)piperazin-1-yl)methyl)quinolin-8-ol was dissolved in chloroform at room temperature. N-iodosuccinimide was added in one portion to the reaction mixture in the flask. The resulting mixture was stirred at 50 °C for 12 h. The solvent was then removed under vacuum. The recovered solid / semi-solid was purified using silica column chromatography (25-50% EtOAc in petroleum ether) to give an off-white solid product. The NMR spectrum is shown in Figure 71W. Compound 72 (5-((4-(4-methoxyphenyl)piperazin-1-yl)methyl)quinolin-8-ol, 3511-4OMe-BF)

[0250] [ka]

[0251] Referring to Figure 36B and Figure 71X, a round-bottom flask was charged with the piperazine derivative and diisopropylethylamine in dichloromethane and allowed to stir at room temperature for 5 minutes. In a separate vial, 5-chloromethyl-2-methyl-8-quinolinol hydrochloride was dissolved in dichloromethane and 1 equivalent of base. This solution was added dropwise to the reaction mixture, which was then refluxed at 80 °C for 12 hours. Upon completion, the organic solvent was removed from the crude reaction mixture under reduced pressure. The crude solid was dissolved in dichloromethane and washed with water, NH4Cl (aq), and brine, and the organic fraction was collected and concentrated. The resulting crude material was purified via column chromatography (EtoAc:Hex, 1:1, v:v) to afford the target compound. The NMR spectrum is shown in Figure 71X. Compound 91 (7-bromo-5-((4-(4-bromothiazol-2-yl)piperazin-1-yl)methyl)quinolin-8-ol, BQPT)

[0252] [ka]

[0253] Referring to Figure 36B, the piperazine derivative and diisopropylethylamine in dichloromethane were added to a round-bottom flask and allowed to stir at room temperature for 5 minutes. In a separate vial, 5-chloromethyl-2-methyl-8-quinolinol hydrochloride was dissolved in dichloromethane and 1 equivalent of organic base. This solution was added dropwise to the reaction mixture, which was then refluxed at 80°C for 12 hours. Upon completion, the organic solvent was removed from the crude reaction mixture under reduced pressure. The crude solid was dissolved in dichloromethane and washed with water, NHCl (aq), and brine, and the organic fraction was collected and concentrated. The resulting crude material was purified via column chromatography (EtoAc:Hex, 1:1, v:v) to afford the target compound 5-((4-(4-bromothiazol-2-yl)piperazin-1-yl)methyl)quinolin-8-ol, which was then dissolved in chloroform in a round-bottom flask at room temperature and then cooled to 0°C in an ice bath. In a separate vial, N-bromosuccinimide was suspended in chloroform and added portionwise to the reaction mixture in the flask over 5-10 minutes. The resulting mixture was stirred at 0 °C for 1 hour. The solvent was then removed under vacuum. The recovered solid / semi-solid was purified using silica column chromatography (EtOAc:Hex, 2:1, v:v) to afford the off-white solid product, 7-bromo-5-((4-(4-bromothiazol-2-yl)piperazin-1-yl)methyl)quinolin-8-ol. Compound 93 (7-bromo-5-((4-(2,3,4-trimethoxybenzyl)piperazin-1-yl)methyl)quinolin-8-ol, BQP3M)

[0254] [ka]

[0255] Referring to Figure 36B, the piperazine derivative and diisopropylethylamine in dichloromethane were added to a round-bottom flask and allowed to stir at room temperature for 5 minutes. In a separate vial, 5-chloromethyl-2-methyl-8-quinolinol hydrochloride was dissolved in dichloromethane and 1 equivalent of organic base. This solution was added dropwise to the reaction mixture, which was then refluxed at 80°C for 12 hours. Upon completion, the organic solvent was removed from the crude reaction mixture under reduced pressure. The crude solid was dissolved in dichloromethane and washed with water, NHCl (aq), and brine, and the organic fraction was collected and concentrated. The resulting crude material was purified via column chromatography (EtoAc:Hex, 2:1, v:v) to afford the target compound 5-((4-(2,3,4-trimethoxybenzyl)piperazin-1-yl)methyl)quinolin-8-ol, which was then dissolved in chloroform in a round-bottom flask at room temperature and then cooled to 0°C in an ice bath. In a separate vial, N-bromosuccinimide was suspended in chloroform and added portionwise to the reaction mixture in the flask over 5-10 minutes. The resulting mixture was stirred at 0 °C for 1 hour. The solvent was then removed under vacuum. The recovered solid / semi-solid was purified using silica column chromatography (EtOAc:Hex, 2:1, v:v) to afford the off-white solid product, 7-bromo-5-((4-(2,3,4-trimethoxybenzyl)piperazin-1-yl)methyl)quinolin-8-ol. Compound 95 (7-bromo-5-((4-(2,3-dichlorophenyl)piperazin-1-yl)methyl)quinolin-8-ol, BQP2Cl)

[0256] [ka]

[0257] Referring to Figure 36B, the piperazine derivative and diisopropylethylamine in dichloromethane were added to a round-bottom flask and allowed to stir at room temperature for 5 minutes. In a separate vial, 5-chloromethyl-2-methyl-8-quinolinol hydrochloride was dissolved in dichloromethane and 1 equivalent of organic base. This solution was added dropwise to the reaction mixture, which was then refluxed at 80°C for 12 hours. Upon completion, the organic solvent was removed from the crude reaction mixture under reduced pressure. The crude solid was dissolved in dichloromethane and washed with water, NHCl (aq), and brine, and the organic fraction was collected and concentrated. The resulting crude material was purified via column chromatography (EtoAc:Hex, 1:1, v:v) to afford the target compound 5-((4-(2,3-dichlorophenyl)piperazin-1-yl)methyl)quinolin-8-ol, which was then dissolved in chloroform in a round-bottom flask at room temperature and then cooled to 0°C in an ice bath. In a separate vial, N-bromosuccinimide was suspended in chloroform and added portionwise to the reaction mixture in the flask over 5-10 minutes. The resulting mixture was stirred at 0 °C for 1 hour. The solvent was then removed under vacuum. The recovered solid / semi-solid was purified using silica column chromatography (EtOAc:Hex, 2:1, v:v) to afford the off-white solid product, 7-bromo-5-((4-(2,3-dichlorophenyl)piperazin-1-yl)methyl)quinolin-8-ol. Compound 97 (1-((4-((7-bromo-8-hydroxyquinolin-5-yl)methyl)piperazin-1-yl)methyl)-4-(2-methoxyethyl)piperazine-2,3-dione, BQPD2)

[0258] [ka]

[0259] Referring to Figure 36B, a round-bottom flask was charged with the piperazine derivative and diisopropylethylamine in dichloromethane and allowed to stir at room temperature for 5 minutes. In a separate vial, 5-chloromethyl-2-methyl-8-quinolinol hydrochloride was dissolved in dichloromethane and 1 equivalent of organic base. This solution was added dropwise to the reaction mixture, which was then refluxed at 80°C for 12 hours. Upon completion, the organic solvent was removed from the crude reaction mixture under reduced pressure. The crude solid was dissolved in dichloromethane and washed with water, NH4Cl (aq), and brine, and the organic fraction was collected and concentrated. The resulting crude material was purified via column chromatography (CHCl3:MeOH, 95:5 v:v) to give the target compound 1-((4-((8-hydroxyquinolin-5-yl)methyl)piperazin-1-yl)methyl)-4-(2-methoxyethyl)piperazine-2,3-dione, which was then dissolved in chloroform in a round-bottom flask at room temperature and then cooled to 0°C in an ice bath. In a separate vial, N-bromosuccinimide was suspended in chloroform and added portionwise to the reaction mixture in the flask over 5-10 minutes. The resulting mixture was stirred at 0°C for 1 hour. The solvent was then removed under vacuum. The recovered solid / semi-solid was purified using silica column chromatography (CHCl3:MeOH, 95:5 v:v) to give the off-white solid product 1-((4-((7-bromo-8-hydroxyquinolin-5-yl)methyl)piperazin-1-yl)methyl)-4-(2-methoxyethyl)piperazine-2,3-dione. Compound 98 (5-((4-(1,4-dioxaspiro[4.5]decan-8-yl)piperazin-1-yl)methyl)quinolin-8-ol, QPO2)

[0260] [ka]

[0261] Referring to Figure 36B, a round-bottom flask was charged with the piperazine derivative and diisopropylethylamine in dichloromethane and allowed to stir at room temperature for 5 minutes. In a separate vial, 5-chloromethyl-2-methyl-8-quinolinol hydrochloride was dissolved in dichloromethane and 1 equivalent of organic base. This solution was added dropwise to the reaction mixture, which was then refluxed at 80°C for 12 hours. Upon completion, the organic solvent was removed from the crude reaction mixture under reduced pressure. The crude solid was dissolved in dichloromethane and washed with water, NHCl (aq), and brine, and the organic fraction was collected and concentrated. The resulting crude material was purified via column chromatography (EtoAc:Hex, 1:1, v:v) to afford the target compound 5-((4-(1,4-dioxaspiro[4.5]decan-8-yl)piperazin-1-yl)methyl)quinolin-8-ol. Compound 100 (7-bromo-5-((4-((4-bromothiophen-2-yl)methyl)piperazin-1-yl)methyl)quinolin-8-ol, BQPT-Bn)

[0262] [ka]

[0263] Referring to Figure 36B, the piperazine derivative and diisopropylethylamine in dichloromethane were added to a round-bottom flask and allowed to stir at room temperature for 5 minutes. In a separate vial, 5-chloromethyl-2-methyl-8-quinolinol hydrochloride was dissolved in dichloromethane and 1 equivalent of organic base. This solution was added dropwise to the reaction mixture, which was then refluxed at 80°C for 12 hours. Upon completion, the organic solvent was removed from the crude reaction mixture under reduced pressure. The crude solid was dissolved in dichloromethane and washed with water, NHCl (aq), and brine, and the organic fraction was collected and concentrated. The resulting crude material was purified via column chromatography (CHCl:MeOH, 95:5 v:v) to afford the target compound 5-((4-((4-bromothiophen-2-yl)methyl)piperazin-1-yl)methyl)quinolin-8-ol, which was then dissolved in chloroform in a round-bottom flask at room temperature and then cooled to 0°C in an ice bath. In a separate vial, N-bromosuccinimide was suspended in chloroform and added portionwise to the reaction mixture in the flask over 5-10 minutes. The resulting mixture was stirred at 0 °C for 1 hour. The solvent was then removed under vacuum. The recovered solid / semi-solid was purified using silica column chromatography (CHCl3:MeOH, 95:5 v:v) to afford the off-white solid product 7-bromo-5-((4-((4-bromothiophen-2-yl)methyl)piperazin-1-yl)methyl)quinolin-8-ol.

[0264] As noted above, various biophysical parameters were determined for certain synthesized compounds and are listed in Tables A-G at the end of the Examples.

[0265] [Example 27] Selective inhibition of EphB2-EphB2 receptor interaction by A20-I. In separate experiments, we tested whether certain A20 analogs have specificity for different Eph receptors. Using the Octet RED384 system, we obtained biophysical on-off binding data (association-dissociation) for three different human ephrinB2-EphB1, ephrinB2-EphB2, and ephrinB2-EphB4 protein-protein interactions and assessed the effect of A20 chemistry on binding kinetics.

[0266] As described above, the ephrin-Eph ectodomain interactions investigated by this biophysical analysis allow for investigation of the complex interrelationships of protein interactions occurring with these ligand-receptor partners. The binding phase initially shows the rapid formation of ephrin-Eph dimers within the first 15 seconds of the binding phase, exhibiting fast on / fast off kinetics. This is then followed by the slower formation of a cyclic tetrameric species, in which two ephrin-Eph dimers meet and complex. The tetramers continue to accumulate slowly during the binding phase, forming a highly stable macromolecular complex that persists until the end of the dissociation phase. Importantly, the cyclic tetrameric complex is the key macromolecular structure into which these receptors and ligands must assemble to activate both Eph-anterograde and ephrin-retrograde signaling in the cells in which they are expressed.

[0267] Figures 72A-72C show three exemplary Octet sensorgram response traces for binding of immobilized human ephrinB2 ectodomain protein to soluble forms of human EphB1, EphB2, and EphB4 ectodomain proteins in the absence or presence of A20 chemicals (buffer conditions were always PBSTD: PBS containing 0.05% Tween-20, 1% DMSO, and + / - the indicated chemicals). After loading the ephrinB2-Fc protein onto an Octet AHC biosensor, washing, and obtaining a stable baseline measurement in the appropriate PBSTD (with or without the indicated chemicals), the sensor was placed in that buffer also containing the indicated soluble EphB protein for the association phase (0-80 seconds), and then returned to the buffer for the dissociation phase (81-580 seconds). In each figure (Figures 72A-C), a sensorgram experiment using 10 μM of the 2×HCl salt form of A20-I is compared to a no-compound control experiment to illustrate the reduction in the maximum level of response signal typically obtained with potent A20 chemistry.

[0268] Based on the compound's effect during the 80-second binding phase, as calculated by comparing the AUC (red asterisks) for the binding phase without and with compound, compound A20-I, like other potent A20 tetramerization inhibitors, exhibits the greatest ability to reduce ephrinB2-EphB2 interactions (61.8%), followed by ephrinB2-EphB1 (36.6%), and then ephrinB2-EphB4 (13.9%). In general, during the 80-second binding phase, potent tetramerization inhibitor chemicals such as A20-I exhibit approximately 60%, 30%, and 15% ability to prevent binding of ephrinB2 ligand ectodomains to their cognate EphB2, EphB1, and EphB4 receptor ectodomains, respectively.

[0269] During the 500-second dissociation phase, compound A20-I demonstrated a remarkable ability to rapidly reduce the amount of bound EphB2 within 40 seconds (representing dimer component melting), leaving a stable but very low level of ultrastable, insurmountable tetramers that did not dissociate by the end of the entire 580-second experiment. The effect of A20-I on EphB1 dissociation was similar to that observed for EphB2, with a rapid loss of some bound EphB1 protein (dimer melting) within the first 60–80 seconds, followed by a stabilized response level of ultrastable, insurmountable tetramers that remained until the end of the entire 580-second experiment. The dissociation kinetics of EphB4 were different; here, only a slow, gradual loss of bound EphB4 protein was observed, with the response level continuing its downward slope until the end of the 580-second experiment.

[0270] For ephrinB2-EphB2 and ephrinB2-EphB1 interactions, because the response level during the dissociation phase plateaus by the first 80 seconds, we also calculated the AUC for the experimental data encompassing the first 160 seconds of each experiment, accounting for both the 80 seconds of association data (red asterisk) and the first 80 seconds of dissociation data (green line and asterisk). This revealed that A20-I inhibited 76.5% of the ephrinB2-EphB2 interaction, 48.5% of the ephrinB2-EphB1 interaction, but only 33.9% of the ephrinB2-EphB4 interaction, as summarized in Table 3 below.

[0271] [Table 3]

[0272] [Example 28] Selective inhibition of EphB2-EphB2 receptor interaction by A20-I and BQPB4. The selectivity of another A20 analog (the 3×HCl salt form of compound BQPB4) relative to a previously tested compound, A20-I, was tested as described above in Example 27. Figures 73A-C are representative sensorgrams showing that BQPB4, like A20-I and other potent A20 tetramerization inhibitors, exhibits the greatest ability to reduce ephrinB2-EphB2 interactions, followed by ephrinB2-EphB1 and then ephrinB2-EphB4, as calculated by comparing the AUC of the binding phase with and without compound.

[0273] Specifically, during the 80-second association phase, BQPB4, like A20-I, exhibited similar ratios of 60%, 30%, and 15% of the ability to block ephrin-B2 ectodomain binding to its cognate EphB2, EphB1, and EphB4 ectodomains, respectively. During the 500-second dissociation phase, compound BQPB4, like A20-I, demonstrated a remarkable ability to rapidly reduce the amount of bound EphB2 to very low levels of tetramers within 40 seconds. However, unlike A20-I, with BQPB4, the dissociation response did not plateau but continued to decrease gradually, reaching baseline (response = 0) by the end of the entire 580-second experiment. The effect of BQPB4 on EphB1 dissociation was similar to that observed for EphB2, with a rapid loss of some bound EphB1 protein within the first 60–80 seconds, followed by only a slight gradual loss of response signal throughout the remainder of the dissociation phase. This indicates that BQPB4 exhibits a weak ability to impede ephrinB2-EphB1 tetramers compared to its strong effect on the insurmountable ephrinB2-EphB2 tetramers. A summary of the binding (area under the curve) determined for binding only (0-80 s) and binding + dissociation (0-160 s) is provided in Table 4 below.

[0274] [Table 4]

[0275] [Example 29] Dose-response of A20-I in inhibiting different Eph-EphR interactions In addition to the experiments listed above, a series of dose-response experiments were performed to determine the IC50 of selected A20 chemical entities (A20-I) on the binding kinetics of human ephrin B2 ligand ectodomain proteins to their cognate human EphB1, EphB2, and EphB4 receptor ectodomain proteins. Figures 74A-74C are representative sensorgrams showing the effect of 0, 0.1, 0.2, 0.4, 0.8, 1.6, and 3.2 μM concentrations of A20-I 2×HCl salt, along with the effect of the 10 μM maximum standard (also A20-I), on three different ephrin-EphB interactions (e.g., Figure 74A for EphB1, Figure 74B for EphB2, and Figure 74C for EphB4). In addition to using the AUC calculation of the sensorgram to obtain % inhibition information for the highest dose of A20 chemical tested for IC50 (typically 3.2 μM, see Table 5 below), the AUC for the various dose-responses is used to calculate IC50 values ​​for binding only (0-80 s) or for the entire experiment (0-580 s) for binding + dissociation combined. Additional IC50 values ​​were determined at specific time points: 75 s of the association phase and 55 s of the dissociation phase (135 s).

[0276] All absolute and relative IC50 values ​​are provided below in Table 6, where absolute values ​​are based on the full binding sensorgram trace and relative values ​​are based on the maximum control trace. Because the A20 removes the portion of ephrinB2-EphB binding due to dimer kinetics that is unaffected by the chemical, the relative IC50 values ​​are most useful as they provide a description of the concentration of compound required to specifically inhibit 50% of only the tetrameric species analyzed.

[0277] [Table 5]

[0278] [Table 6]

[0279] [Example 29] Dose-response of BQP4 in inhibiting different Eph-EphR interactions The experiment described in Example 28 was repeated using BQP4. Figures 75A-75C show representative sensorgrams showing the effects of BQP4 at concentrations of 0, 0.1, 0.2, 0.4, 0.8, 1.6, and 3.2 μM, along with the effect of a 10 μM maximum standard (also A20-I) on three different ephrin-EphB interactions (e.g., Figure 75A for EphB1, Figure 75B for EphB2, and Figure 75C for EphB4). BQPB4 was found to strongly reduce the sensorgram response relative to maximum control levels at as little as 0.4 μM.

[0280] The AUC values ​​derived from the sensorgrams are set forth in Table 7 below, and the absolute and relative IC50 values ​​for BQP4 (derived as discussed in Example 28) are shown in Table 8 below.

[0281] [Table 7]

[0282] [Table 8]

[0283] [Example 30] Determination of % inhibition for various A20 analogue chemicals on three different ephrinB2-EphB protein interactions In a separate experiment, the percent inhibition of three different ephrinB2-EphB protein interactions was determined for various A20 analog chemicals. Specifically, the percent inhibition of binding of immobilized human ephrinB2-Fc to 25 nM soluble human EphB1-His (Figure 76A, expanded in Figure 76D), EphB2-His (Figure 76B, expanded in Figure 76E), and EphB4-His (Figure 76C, expanded in Figure 76F) by 3511-I, 8009-9255, QTM-Br, A19, A19-NO2, and IM (all at 3.2 μM) was determined. While most of the A20-related chemicals analyzed exhibited typical 60-30-15% inhibition of EphB2-EphB1-EphB4 binding during the 80-second binding phase (exemplified by the A20-I maximum control), this analysis identified analogs that exhibited much lower activity against ephrinB2-EphB4 interactions. Specifically, the data shown in Figures 76A-76E and summarized in Tables 9-10 below indicate that compound A19-NO2 exhibits approximately 20-fold lower % inhibitory activity against ephrinB2-EphB4 interactions (bold values ​​in Tables 9 and 10 and Figures 76D-76E, which are enlarged images of the first 160 seconds of the sensorgram traces in Figures 76A-76C), while retaining nearly all of its inhibitory activity against ephrinB2-EphB2 and ephrinB2-EphB1 interactions. Note that compounds 3511-I, 8009-9255, and QTM-Br exhibited activity similar to the A20-I maximum control, whereas A19 was generally less active for all three interactions, and the indole ring compound IM showed no activity overall.

[0284] [Table 9]

[0285] [Table 10]

[0286] [Example 31] Determination of % inhibition for additional A20 analog chemicals on three different ephrinB2-EphB protein interactions Similar experiments were performed with different sets of A20 analog chemistries, similar to those described in Example 30. Specifically, BQPB4-BN, A19, A19-NO2 (A-free base), A19-NO2 (B-2×HCl salt), IPM2, and QPB4-BN were all tested at 3.2 μM to determine the percent inhibition of each of these compounds on the binding of immobilized human ephrin B2-Fc to 25 nM soluble human EphB1-His (Figures 77A, 77D), EphB2-His (Figures 77B, 77E), and EphB4-His (Figures 77C, 77F). Figures 77A-77C show a close-up of the first 160 seconds of another Octet experiment testing both the free base (FB) and 2xHCl salt forms of compound A19-NO2, again demonstrating the distinct abilities of these compounds to selectively inhibit ephrinB2-EphB2 and ephrinB2-EphB1 interactions while sparing the majority of ephrinB2-EphB4 interactions. Figures 77D-77F show all eight traces from this experiment, demonstrating that QPB4-Bn, like A19-NO2, also exhibits the ability to selectively inhibit ephrinB2-EphB2 and ephrinB2-EphB1 interactions while sparing ephrinB2-EphB4 interactions (see bolded values ​​in the summaries in Tables 11 and 12 below).

[0287] [Table 11]

[0288] [Table 12]

[0289] [Example 32] Determination of % inhibition for additional A20 analog chemicals on three different ephrinB2-EphB protein interactions In a separate experiment, percent inhibition of binding of immobilized human ephrinB2-Fc to 25 nM soluble human EphB1-His, EphB2-His, and EphB4-His proteins by 3.2 μM of 12 compounds (2OH-A20, A20-1.2×MSA, QPCF34-BN, A20, QPB4, QPA-Pac, QTM-NO2, QPP, QP-Bn, 3511-0013) and A20-1 as the maximum control was measured. The data are summarized in Tables 11-14 below. Compounds QPCF34-Bn, QPB4, QTM-NO2, and QP-Bn also demonstrate the ability to selectively inhibit ephrinB2-EphB2 and ephrinB2-EphB1 interactions, while sparing the ephrinB2-EphB4 interaction (bold in Tables 13-16). QPCF34-Bn appears to have little activity against EphB1 and is more selective in inhibiting EphB2 interactions (bold in Tables 13 and 14), whereas QPBN appears to be more selective for EphB1 (bold in Tables 15 and 16). 8-Hydroxyquinoline (8-HQ), part of the building blocks of the A20 chemistry and a chemical with known metal chelating activity, was unable to interfere with any of the ephrinB2-EphB interactions (see Tables 13 and 14 below), indicating that metal chelating activity is likely not the mode of action of the A20 chemistry.

[0290] [Table 13]

[0291] [Table 14]

[0292] [Table 15]

[0293] [Table 16]

[0294] [Example 33] Identification of A20 analogs with specificity for EphB1, EphB2, or EphB1 and EphB2 The results of previous experiments were analyzed and used to identify different chemical structures with different specificities for different ephrin B-EphB interactions. The results, summarized in Figure 78, identify A19-NO2 (free base or HCl salt), QTM-NO2, and QPB4 as having low activity against EphB4 but high specificity for EphB1 and EphB2; QPA and QPCF34-Bn as having low activity against EphB4 and EphB1 but high specificity for EphB2; and QP-Bn, 3511-0113 BF, and BQPA-Pac as having low activity against EphB4 and EphB2 but high specificity for EphB1.

[0295] [Example 34] Intraperitoneal (IP) injection of A20, A20-I, and 3511-I results in reduced EphB2 activation levels and binding to ephrinB2 in the brain Ten- to 12-week-old wild-type mice were IP injected every 12 hours for 4 days with vehicle (PBS) or 10 mg / kg of the 2x HCl salt form of A20 (in PBS), A20-I (in PBS), or 3511-I (in 94% PBS / 6% DMSO) for a total of eight injections. Just 2 hours after the last injection of A20, brains were dissected and frozen at -80°C. Whole-brain protein lysates were then prepared using a Dounce homogenizer with 5 ml of PLC lysis buffer. 1 ml of the lysate was used for immunoprecipitation (IP) with 1 μg of goat anti-EphB2 antibody, followed by immunoblot (IB) analysis with goat anti-EphB2, rabbit anti-phospho-EphB1 / B2 (pEphB1 / B2), and mouse anti-ephrin B2 antibodies using the appropriate HRP-conjugated secondary antibody and chemiluminescence detection.

[0296] Figure 79A shows an exemplary immunoblot showing the expression of EphB2, phospho-EphB1 / B2, and ephrinB2 (co-IP) in mouse brain. EphB2 antibody immunoblots showed equal receptor expression in all mouse brains, as expected. Phospho-EphB-specific antibody immunoblots, which recognize the tyrosine-phosphorylated juxtamembrane portion of EphB2 and are a readout for the activated tyrosine kinase catalytic domain, showed strong tyrosine phosphorylation of the receptor in the two vehicle (V) control lanes. The levels of phospho-EphB signal in the brains of animals injected with the A20 chemical generally showed low activation levels, as indicated by the faint bands in the corresponding lanes. EphrinB2-specific antibody immunoblots showed a strong band in the vehicle (V) lane, indicating that ephrinB2 co-immunoprecipitates with EphB2 in the brain. The levels of co-immunoprecipitated ephrinB2 in the brains of animals injected with the A20 chemical were generally low, as indicated by the faint bands. Figures 79B and 79C show quantification of band intensity (number of detected photons) to obtain the ratio of pEphB / EphB2 (Figure 79B) as a measure of the level of EphB2 receptor activation and the ratio of ephrinB2 / EphB2 (Figure 79C) as a measure of how much ephrinB2 is co-immunoprecipitated with the EphB2 receptor. The data show that animals IP-injected with A20, A20-I, and 3511-I generally exhibit low levels of activated, phosphorylated EphB2 in the brain, and this correlates with reduced levels of binding to ephrinB2.

[0297] [Example 35] Oral administration (PO) of A20 and A20-I reduces EphB2 activation levels in the brain Whole-brain protein lysates were prepared from wild-type mice orally (PO) or intravenously (IP) injected with vehicle (PBS) or 20 mg / kg of A20 or A20-I 2x HCl salt every 12 h for 4 days, with the final (7th) dose staggered so that brain dissection was performed 2 h after the last drug administration. EphB2 was immunoprecipitated from 1 ml of protein lysate using 1 μg of goat anti-EphB2 antibody, and the precipitated proteins were then immunoblotted with goat anti-EphB2, rabbit anti-pTyr1000, and mouse 4G10 antibodies using the appropriate HRP-conjugated secondary antibody and chemiluminescent detection.

[0298] EphB2 antibody immunoblots (Figure 80A) show equal expression of the receptor in all mouse brains, as expected. pTry1000 and 4G10 immunoblots (Figure 80B and Figure 80C, respectively), which both recognize tyrosine-phosphorylated proteins and are readouts of the activated EphB2 tyrosine kinase catalytic domain, both show strong tyrosine phosphorylation of the receptor in three of the four vehicle (V) control lanes. The levels of pTyr1000 and 4G10 signal in the brains of animals administered the A20 and A20-I chemicals generally showed low levels of activation, as indicated by faint bands in the corresponding lanes. Quantification of band intensities to obtain the ratios of pTyr1000 / EphB2 and 4G10 / EphB2 as a measure of EphB2 receptor activation levels indicates that animals administered A20 or A20-I PO (shown in both Figures 80D and 80E) or IP (shown in Figure 80E only) showed low levels of activated, phosphorylated EphB2 in the brain. The results shown here also highlight our finding that in intact brain, when EphB2 is tyrosine phosphorylated and catalytically active, the protein migrates much slower in SDS-PAGE gels, with an apparent migration in a distinct band of 220-260 kDa (green arrows in Figures 80B and 80C), in contrast to the typical migration of unphosphorylated EphB2 protein at 125 kDa (black arrow in Figure 80A). It is well known that phosphoproteins can exhibit significant differences in migration in SDS-PAGE gels compared to non-phosphorylated molecules, and for EphB2 the variation is quite large.

[0299] [Example 36] Biochemical studies of EphB2 in the brain show that when the receptor protein is activated and tyrosine phosphorylated, it exhibits a significantly retarded mobility in SDS-PAGE gels. Whole-brain protein lysates were prepared from two wild-type (WT) mice and one each of the following mutant mice: an EphB1 / EphB2 double homozygous knockout (El N1), an EphB2-K661R (homozygous kinase-dead point mutant), an EphB2-F620D (homozygous kinase-hyperactive point mutant), and an EphB2-lacZ (heterozygous N2 / +) mutant, which produces an intracellular truncated EphB2-β-gal fusion protein migrating at 220 kDa. EphB2 was immunoprecipitated from 1 ml of each protein lysate using 1 μg of goat anti-EphB2 antibody, and the precipitated proteins were then immunoblotted with goat anti-EphB2, rabbit anti-phospho-EphB1 / B2, and rabbit anti-pTyr1000 antibodies using the appropriate HRP-conjugated secondary antibody and chemiluminescent detection.

[0300] EphB2 antibody immunoblots (Figure 81A) show, as expected, relatively equal expression of the 125 kDa EphB2 receptor in WT, K661R, and F620D mouse brains (black arrows). N2 / + brains, which have only one copy of the WT gene and therefore have somewhat lower levels of WT protein, also produce a 220 kDa EphB2-β-gal fusion protein that is recognized by a goat anti-EphB2 antibody specific for the ectodomain (thin black arrow). pEphB1 / B2 (Figure 81B) and pTyr1000 (Figure 81C) immunoblots both show tyrosine phosphorylation of the receptor in the two WT brains and much stronger phosphorylation in the F620D kinase hyperactive mutant brain. These bands detect the activated EphB2 receptor, which migrates much slower on SDS-PAGE gels, at and just below the 250 kDa standard protein marker (green arrows in Figures 81B and 81C). El N1 knockout and K661R kinase-dead mutant brains expressed neither EphB1 nor EphB2 protein (knockout) or catalytically inactive full-length protein (K661R), and as expected, showed greatly reduced tyrosine phosphorylation.

[0301] Figure 81D shows quantification of band intensity to obtain the ratios of pEphB / EphB2 and pTyr1000 / EphB2 as a measure of EphB2 receptor activation levels, showing that compared to WT brains, F620D brains exhibit much more phospho-EphB2, whereas K661R brains exhibit a large reduction in this. Furthermore, inspection of EphB2 immunoblots shows that this antibody is particularly strong and specific with little background, thus providing a high signal-to-noise ratio, and is therefore able to detect the high MW band of phosphorylated EphB2 protein in WT and F620D brains (light green arrows in Figure 81A).

[0302] [Example 37] Biochemical analysis indicates that only about 3–4% of total EphB2 protein is activated in WT brain. Figure 82A shows a darker exposure of the EphB2 immunoblot from the EphB2 immunoprecipitation in Figure 81A, with the bright, non-phosphorylated 125 kDa-sized EphB2 band and the dim, slower-migrating 220-250 kDa phospho-EphB2 band boxed for quantification of chemiluminescent signal intensity (a third region of each lane is also boxed for background subtraction). Here, the ratio of pEphB2 / EphB2 indicates that while only a small fraction of total EphB2 protein in WT brain is actually in the catalytically active, tyrosine-phosphorylated state (3-4%), there is 2-3-fold more active pEphB2 in the kinase-hyperactive F620D mutant (9.9%) and much less in the kinase-dead K661R mutant (0.6%) (as tabulated in Figure 82B).

[0303] [Example 38] Mass spectrophotometry independently identifies a slower migrating, larger sized EphB2 protein in the brain. Figure 83A shows a Coomassie blue-stained SDS-PAGE gel of liver and brain protein lysates after immunoprecipitation with goat anti-EphB2 antibody. The boxed gel slices were cut, and proteins were eluted and subjected to mass spectrometry to obtain the peptide sequences contained in each slice (Figure 83B). This revealed that the gel slices containing the most abundant EphB2 protein were, as expected, the K1 (rank 2—the second most abundant protein in the slice) and F1 (rank 6—the sixth most abundant protein in the slice) cutouts just below the 130 kDa protein marker. Importantly, the three larger-sized gel slices (K2–K4 and F2–F4) from both samples also contained EphB2 protein sequences, confirming that the larger-sized band labeled for phospho-EphB2 in the immunoblot shown above indeed identifies the presence of authentic EphB2 receptor protein. It is also noteworthy that the K1 and F1 gel sections also contain some sequences for the related EphB1 and EphA4 receptors, indicating that the goat anti-EphB2 antibody either (1) cross-reacts somewhat with these other two receptors and can also immunoprecipitate them, or (2) when EphB2 is precipitated, the EphB1 / EphA4 receptors can also be co-precipitated because they are complexed with EphB2.

[0304] [Example 39] A slower-migrating, larger-sized, tyrosine-phosphorylated EphB2 protein is also detected in the liver Livers were injured using repeated IP administration of carbon tetrachloride, which has been shown to induce overexpression of the EphB2 receptor. Total liver protein lysates were prepared and subjected to goat anti-EphB2 immunoprecipitation followed by immunoblot analysis using EphB2 (Figure 84A), phospho-EphB1 / B2 (Figure 84B), 4G10 (Figure 84C), and pTry1000 (Figure 84D) antibodies. The EphB2 immunoblot (Figure 84A) showed relatively low levels of 125 kDa non-phosphorylated EphB2 (lanes 1 and 2) in livers from uninjured control mice, which increased in CCI4-injured livers (remaining lanes). The EphB2 immunoblot also showed prominent bands for higher and slower migrating species at 220 and 260 kDa, which were also less intense / abundant in uninjured livers compared to injured livers. Various phosphotyrosine-specific immunoblots (Figures 84B-84D) very clearly show that the 220 kDa band is specifically phospho-EphB2, present at low levels in uninjured and at high levels in injured.

[0305] [Example 40] Experimental protocol used to evaluate how well oral administration of A20 chemicals blunts chronic long-term inflammatory pain induced by injection of complete Freund's adjuvant (CFA) into the hind paw of mice Figure 85 shows a schematic diagram of the experimental protocol for assessing how well oral administration of A20 chemical blunts chronic long-term inflammatory pain induced by injection of complete Freund's adjuvant (CFA) into the mouse hind paw. The week before CFA injection, adult mice (over 10 weeks old, male or female) were first acclimated to the chamber used in pain assessment, and then their left and right hind paws were subjected to thermal (heat) and mechanical (touch) pain measurements to obtain baseline data. Mechanical pain was assessed by placing mice on a wire grid and repeatedly testing the hind paws for their sensitivity to von Frey (VF) filaments of different stiffness, using the mechanical withdrawal response threshold for each mouse hind paw, defined as the minimum gauge filament required to elicit a reflex. Thermal pain was assessed by placing mice on a glass surface and subjecting their hind paws to an infrared heat source (Hargreaves apparatus) connected to a timer to measure how long it took the animals to notice the heat and lift their paws. In these studies, both hind paws of mice were subjected to multiple measurements over a 2-3 h period to either (1) refine the VF filament stiffness threshold and plot contact pain, or (2) obtain six successful thermal response measurements, which were averaged to obtain a data point for thermal pain. Chronic pain experiments were initiated by subcutaneously injecting 30 μL of CFA (F5881, Sigma-Aldrich) into one of the two hind paws (left or right is not important) to induce inflammation and pain, followed 15 min later by a first oral gavage (PO) or intraperitoneal (IP) administration of A20 chemical (or vehicle alone in control mice), followed by a second A20 chemical administration 6-8 h later in the evening. Over the following days, mice were then subjected to additional administrations of A20 chemical (orange arrows) and tested for the indicated thermal (red arrows) or mechanical (green arrows) pain responses over the next 15 days. In all experiments, the person performing the actual pain testing on the mice remained blinded to the mouse genotype (e.g., wild-type or EphB1 knockout) and / or treatment condition (e.g., vehicle only or administration of A20 chemical). A total of 12 mice were evaluated per experiment. Results from these experiments are further discussed in the Examples below.

[0306] [Example 41] Oral administration of the A20 lead compound potently attenuates mechanical touch pain in the CFA model In this example, 12 wild-type adult male mice (11 weeks old, 129x1 / SvJ background) were divided into three groups of four: vehicle control (vehicle is 97% PBS / 3% DMSO), 20 mg / kg A20 IP (2x HCl salt), and 20 mg / kg A20 PO (2x HCl salt), and subjected to the CFA inflammatory pain model (as described in Example 40). Only vehicle-treated mice showed a significant increase in mechanical pain, as indicated by increased sensitivity to the VF filament in the left (injured) hindpaw after CFA injection (left panels of Figures 86A and 86B). The CFA model results in long-term mechanical pain, as indicated by vehicle mice showing a significant increase in mean sensitivity to the low-stiffness VF filament even 10 days after injury compared to baseline measurements, with pain responses returning to normal by day 15. Injection or oral gavage of A20 resulted in mice that, on average, showed no increased sensitivity to VF filaments, demonstrating the potent ability of the A20 compound to blunt mechanical pain. The right uninjured hind paws of all mice showed no significant differences between treatment groups throughout the study (right panels of Figures 86A and 86B). This indicates that administration of A20 has no effect on normal tactile pain sensation, and that the compound only targets pain caused by injury, which produces chronic pain.

[0307] [Example 42] Oral administration of the A20 lead compound also effectively blunts heat pain in the CFA model The vehicle-treated group (described in Examples 40-41) showed a significant increase in sensitivity to thermal pain in the left (injured) hind paw after CFA, as indicated by a significant decrease in response time to the infrared heat source (Figures 87A-87B). The CFA model results in prolonged thermal pain, as shown by vehicle mice showing a significant increase in sensitivity to the infrared heat source even 11 days after injury compared to baseline measurements, with pain responses returning to near baseline by day 14 (green bars in the left panels of Figures 87A and 87B). Injection or gavage of the 2xHCI salt form of A20 resulted in mice showing decreased sensitivity to the infrared heat source compared to vehicle-treated mice, demonstrating the compound's potent ability to blunt pain (blue and yellow bars in the left panels of Figures 87A and 87B). The right, non-injured hind paw of all mice showed no significant differences between treatment groups before and after baseline (right panels of Figures 87A and 87B). This indicates that A20 has no effect on normal thermal pain sensation, but only targets pain caused by injury that produces chronic pain.

[0308] [Example 43] Pain ratio analysis shows that oral administration of the A20 lead compound effectively blunts pain as well as IP administration in the CFA model Using the data shown in Figures 86A, 86B, 87A, and 87B, pain ratios were calculated for each animal by dividing the non-injured side pain response by the injured side response (right hindpaw / left hindpaw). This analysis clearly shows that for mechanical pain, vehicle-controlled mice exhibited a significantly increased pain ratio of 2-3 for 10 days after CFA injury, whereas orally or IP-injected A20-treated mice exhibited essentially no increase in pain, as their pain ratio remained at 1 throughout the study (Figure 88A). Similarly, assessment of thermal pain indicated that vehicle-treated mice exhibited a strong pain ratio of 4 or greater, whereas orally or IP-injected A20-treated mice exhibited significantly blunted pain, as their pain ratio remained below 2 throughout the study (Figure 88B).

[0309] [Example 44] Use of the inflammatory agent Zymosan to induce pain, a more rapid model for testing pain and obtaining results in one day We tested an alternative, potentially more rapid, pain model using Zymosin (an inflammatory agent similar to CFA) to induce inflammatory pain. To initially test the Zymosan inflammatory pain model, we tested EphB1 knockout, EphB1 heterozygote, and EphB1 wild-type adult mice (a large set of males and females, 4–6 months old, on a 129 / CD1 mixed background), because we previously showed that - / - and even + / - mice exhibit reduced pain responses induced by injury due to the critical role of the EphB1 receptor in chronic neuropathic pain. Mice were first acclimated to the pain testing setup and then subjected to baseline mechanical and thermal pain measurements. Early the next morning, 30 μL of Zymosan (5 mg / ml in PBS; Z4250, Sigma-Aldrich) was injected subcutaneously into the left hind paw. Two hours after Zymosan injection, the left hind paw was assessed for thermal pain using a Hargreaves apparatus (2–4 h). The left paw was then tested for mechanical pain using a VF filament (4–6 h), and then again for thermal pain (6–8 h). Data from two independent experiments were pooled to increase the n value for the three different genotypes. Focusing on mechanical pain, Zymosan induced a strong and significant increase in pain in + / + and + / − mice, with the mean filament response going from >6 to <3, whereas responses in − / − knockout mice were not significantly lower than baseline measurements (Figure 89A). Calculation of the pain ratio confirmed that + / + and + / − mice showed a strong increase in mechanical pain, with a ratio of 3, whereas the pain ratio in − / − mice was close to 1, significantly lower than that of wild-type mice, indicating essentially no increase in mechanical pain in knockout mice after Zymosan injury (Figure 89C). Heat pain increased in all groups after Zymosan injury, as indicated by a decrease in mean response time to the infrared heat source in all animals evaluated, with pain levels increasing from the 2-4 hour test period to the 6-8 hour period. Nevertheless, - / - and + / - animals showed a blunted heat pain response compared to + / + mice, particularly during the 6-8 hour test session, where the pain ratio was significantly reduced by 50% (Figures 89B and 89D).These data indicate that Zymosan A20 can be used as a rapid model to assess the ability of chemicals to decrease ephrinB2-EphB1 tetramerization / signaling and blunt inflammatory pain.

[0310] [Example 45] Oral administration of the A20 lead compound blunts mechanical and thermal pain in the Zymosan model Inflammatory pain was induced by subcutaneous injection of 30 μl of 5 mg / ml Zymosan into the right hind paw of 12 wild-type adult male mice (5-6 months old, 129x1 / SvJ background) that had been acclimated to the test chambers over the past few days, and assessed to obtain baseline pain measurements. Fifteen minutes later, mice were orally administered either vehicle alone (97% PBS / 3% DMSO) or 20, 10, or 5 mg / kg A20 (2x HCl salt in vehicle), with three mice per condition. Two hours after Zymosan injection, the right hind paw was assessed for thermal pain using a Hargreaves apparatus (2-4 hours). Mice were then again orally administered vehicle alone or A20 chemical. The right paw was then tested for mechanical pain using a VF filament (4-6 hours) and then again for thermal pain (6-8 hours).

[0311] In vehicle-treated mice, Zymosan induced a strong increase in the mechanical pain ratio, approaching an average of 3, whereas A20-treated mice exhibited a mechanical pain ratio close to 1, indicating no increase in pain (Figure 90A). Similarly, in two heat pain tests, vehicle-treated mice exhibited an av...

Claims

1. Compounds containing the structure of formula (I) 【Chemistry 1】 or a pharmaceutically suitable salt thereof (Wherein, A is —O—, —SO ２ , or -N(CH ２ ) ｎ R １ and B is CH ２ , S.O. ２ or CO; X is hydrogen, halogen, alkenyl, alkyl, —NO ２ , or —NH ２ each R is independently hydrogen, substituted or unsubstituted alkyl, alkoxy, heterocycloalkyl, or carbonyl; R １ is a substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, or substituted or unsubstituted heterocycloalkyl; n=0-3; provided that (1) A is —O— and B is CH ２ and when R is hydrogen, X is hydrogen, chloro, or —NO ２ But not bromo; (2) A is NR １ and R １ is methyl or 【Chemistry 2】 where X is not bromine).

2. 2. The compound of claim 1, wherein n is 0 or 1.

3. X is hydrogen, bromo, iodo, chloro, -NO ２ , or —NH ２ , methyl, or propenyl.

4. Each R is independently hydrogen, methyl, 【Transformation 3】 2. The compound of claim 1, wherein:

5. 5. The compound of claim 4, wherein each R is hydrogen.

6. R １ is a substituted or unsubstituted C4-C10 aryl, a substituted or unsubstituted C4-C10 heteroaryl, a substituted or unsubstituted C4-C10 cycloalkyl, or a substituted or unsubstituted C4-C10 heterocycloalkyl.

7. R １ is substituted or unsubstituted phenyl, substituted or unsubstituted pyridinyl, substituted or unsubstituted thiazole, substituted or unsubstituted thiophenyl, substituted or unsubstituted piperazine, or unsubstituted C4-C10 cycloalkyl, or unsubstituted C4-C10 heterocycloalkyl.

8. R １ but, 【Chemistry 4】 wherein each Y is independently —CH ２ - or -O-; each R ２ are independently hydrogen, —NO ２ , alkoxy, -R ３ OR ４ alkyl ether of -NHOR ５ , -NH ２ , halo, haloalkyl, or alkyl; R ３ and R ４ are each independently C1-C4 alkyl, and R ５ is alkyl or benzyl) 8. The compound of claim 7 selected from the group consisting of:

9. Each R ２ are independently hydrogen, —NO ２ , -OCH ３、 -CH ２ CH ２ OCH ３ , -NHOCH ３ , -CF ３、 -NH ２ , bromo, fluoro, chloro, iodo, methyl, or 【Transformation 5】 8. The compound of claim 7, wherein:

10. R １ but, 【Transformation 6】 2. The compound of claim 1, wherein: 【Request Item 11】 【Chemistry 7】 11. The compound of any one of claims 1 to 10, which is not 【Request Item 12】 【Chemistry 8-1】 【Chemistry 8-2】 【Chemistry 8-3】 10. The compound of claim 1, selected from the group consisting of: and any pharmaceutically suitable salt thereof. 【Request Item 13】 【Chemistry 9】 13. The compound of claim 12, which is a pharmaceutically equivalent salt selected from the group consisting of: 【Request Item 14】 【Chemistry 10】 10. The compound of claim 1, selected from the group consisting of: and any pharmaceutically suitable salt thereof. 【Request Item 15】 【Chemistry 11】 15. The compound of claim 14, which is a pharmaceutically suitable salt selected from the group consisting of:

16. 16. A compound according to any one of claims 1 to 15, which inhibits EPH-ephrin tetramerization.

17. The compound of claim 16, which specifically inhibits EPHB1-ephrin and / or EPHB2-ephrin tetramerization. 【Request Item 18】 【Chemistry 12】 18. The compound of claim 17, selected from the group consisting of: or a pharmaceutically suitable salt thereof.

19. The compound of claim 17, which specifically inhibits EPHB2-ephrin tetramerization. 【Request Item 20】 【Chemistry 13】 20. The compound of claim 19, selected from the group consisting of: or a pharmaceutically suitable salt thereof.

21. The compound of claim 17, which specifically inhibits EPHB1-ephrin tetramerization. 【Request Item 22】 【Chemistry 14】 22. The compound of claim 21, selected from the group consisting of: or a pharmaceutically suitable salt thereof.

23. The compound of claim 17, which does not inhibit EPHB4-ephrin tetramerization.

24. 17. The compound of claim 16, which inhibits EPH-ephrin tetramerization with an IC50 of less than 2 μM, less than 1.6 μM, less than 1 μM, or less than 0.5 μM.

25. 25. A pharmaceutical composition comprising a compound according to any one of claims 1 to 24 and a pharmaceutically suitable carrier or excipient.

26. A method for inhibiting the formation of EPH-ephrin tetramers, comprising administering to EPH or ephrin a compound according to any one of claims 1 to 24 or 【Chemistry 15】 with a compound selected from the group consisting of:

27. The method of claim 26, wherein the EPH-ephrin tetramer comprises EPHB1.

28. The method of claim 26, wherein the EPH-ephrin tetramer comprises EPHB2.

29. 27. The method of claim 26, wherein the formation of the EPH-ephrin tetramer is inhibited in vivo.

30. A method of alleviating or reducing pain in a subject in need thereof, comprising administering to said subject an effective amount of an EPH-ephrin tetramerization inhibitor.

31. 31. The method of claim 30, wherein the pain comprises chronic neuropathic pain.

32. A method of treating synaptopathy in a subject in need thereof, comprising administering to said subject an effective amount of an EPH-ephrin tetramerization inhibitor.

33. 33. The method of claim 32, wherein the synaptopathy comprises aberrant, defective, or excessive EPH-ephrin signaling, and optionally disturbed NMDA signaling.

34. 33. The method of claim 32, wherein the synaptopathy is associated with anxiety or epilepsy.

35. A method of treating addiction or opioid dependence in a subject in need thereof, comprising administering to said subject an effective amount of an EPH-ephrin tetramerization inhibitor.

36. A method for treating a fibrotic and / or inflammatory disease or condition in a subject in need thereof, comprising administering to said subject an effective amount of an EPH-ephrin tetramerization inhibitor.

37. 37. The method of claim 36, wherein the fibrotic and / or inflammatory disease or condition comprises aberrant, defective, or excessive EPH-ephrin signaling, and optionally comprises NASH liver fibrosis, chronic kidney disease, scleroderma (skin fibrosis), cardiac fibrosis, pulmonary fibrosis, fibrosis of another organ, and / or abnormal wound healing, optionally selected from keloids and / or hypertrophic scars.

38. A method of treating cancer in a subject in need thereof, comprising administering to said subject an effective amount of an EPH-ephrin tetramerization inhibitor.

39. 39. The method of claim 38, wherein the cancer comprises aberrant, defective, or excessive EPH-ephrin signaling, and optionally comprises GBM (glioblastoma), pancreatic cancer, and / or colon cancer.

40. A method of treating a viral infection in a subject in need thereof, comprising administering to said subject an effective amount of an EPH-ephrin tetramerization inhibitor.

41. 41. The method of claim 40, wherein the viral infection involves aberrant, defective, or excessive EPH-ephrin signaling, optionally including infection with Henipavirus and / or human immunodeficiency virus (HIV).

42. The EPH-ephrin tetramerization inhibitor is 【Chemistry 16】 42. The method of any one of claims 30 to 41, comprising a compound of any one of claims 1 to 28, or any pharmaceutically acceptable salt thereof.

43. 43. The method of any one of claims 30 to 42, further comprising administering to the subject a pharmaceutical composition comprising the EPH-ephrin tetramerization inhibitor.

44. 44. The method of claim 43, wherein the pharmaceutical composition is administered intravenously, subcutaneously, or orally, by IP injection, and topically as a cream or ointment.

45. 45. The method of any one of claims 30 to 44, wherein the subject is a human, a livestock animal, a companion animal, a laboratory animal, or a zoological animal.

46. A kit comprising: (a) an EPH-ephrin tetramerization inhibitor; and (b) a container.

47. 47. The kit of claim 46, wherein the EPH-ephrin tetramerization inhibitor comprises a compound according to any one of claims 1 to 24.

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