AU2023367084A1 - Small molecular weight chemical compound to antagonize eph-ephrin tetramerization and inhibit bidirectional signaling - Google Patents

Abstract

Compositions and methods for inhibiting EPH-EPHRIN tetramerization are provided. The compounds and methods herein further comprise methods for administering novel EPH-EPHRIN tetramerization inhibitors to a subject in need thereof to treat conditions caused or worsened by abnormal, defective, or excessive EPH-EPHRIN signaling.

Description

SMALL MOLECULAR WEIGHT CHEMICAL COMPOUND TO ANTAGONIZE EPH- EPHRIN TETRAM ERIZATION AND INHIBIT BIDIRECTIONAL SIGNALING

CROSS-REFERENCE TO RELATED APPLICATIONS

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

ACKNOWLEDGMENT OF GOVERNMENT SUPPORT

[0002] This invention was made with Government support under grant number DK128819 awarded by the National Institutes of Health and grant numbers W81XWH-20- CPMRP-IIRA, CP200270 and DAMD 11115013 awarded by the United States Department of Defense. The government has certain rights in this invention.

INCORPORATION BY REFERENCE OF SEQUENCE LISTING

[0003] This application contains a Sequence Listing that has been submitted in computer readable format (XML) on Patent Center and is hereby incorporated 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 about 8000 bytes in size.

BACKGROUND

[0004] 1. Field

[0005] The present inventive concept is directed to compounds capable of inhibiting EPH- EPHRIN tetramerization and their use in treatments for conditions caused or worsened by disrupted, abnormal, defective, or excessive EPH-EPHRIN interactions and/or cellular signaling.

[0006] 2. Discussion of Related Art

[0007] Chronic pain is a major problem that is poorly treated using drugs with efficacy, safety, and addiction issues. A further drawback of the current painkillers available today is that they do not target the key synaptic machinery in the spinal cord that responds to peripheral nerve damage and directly causes the pathological long-lasting changes in neurons that leads to central sensitization and chronic pain. Specifically, central sensitization is due to the strengthening of synaptic connections formed by the pain-sensing peripheral nociceptor neurons, the C-fibers, which terminate superficially onto the spinal cord dorsal horn (DH) neurons. The plasticity of these synapses leads to enhanced transmission of pain impulses from the spinal cord to produce a heightened sensitivity to heat and touch stimuli (hyperalgesia and allodynia). The highly conserved EphB1 receptor tyrosine kinase interacting with its cognate transmembrane EphrinB2 (EB2) ligand are key trans-synaptic players that generate this plasticity and cause central sensitization.

[0008] The present disclosure is based on, in part, the development of a highly sensitive biochemical assay that can be run in small volumes and used for high-throughput screening (HTS) in a search for chemicals that will disrupt the protein-protein interaction of EphB1 with one of its ligands, EphrinB2. Exemplary examples herein describe how EphrinB2 reverse signaling may also contribute to chronic pain and show that compounds disclosed herein will block both forward and reverse EPH-EPHRIN signaling, and target both EphB1 and EphB2. Accordingly, the present disclosure provides for novel therapeutics aimed at decreasing the ability of an Eph receptor (e.g., EphB2) to bind with an associated Ephrin ligand (e.g., EphrinB2 ligand).

BRIEF SUMMARY

[0009] Various aspects of the present disclosure are directed to a compound comprising a structure of Formula (I) (Formula I) or pharmaceutically suitable salt thereof, wherein: A is an -

O-, -SO2, or -N(CH₂)_nRi; B is CH₂, SO₂, or CO; X is hydrogen, a halogen, an alkyenyl, an alkyl, -NO₂, or -NH₂; each R is independently hydrogen, a substituted or unsubstituted alkyl, an alkoxy, heterocycloalykl, or a carbonyl, R1 is a substituted or unsubstituted aryl, a substituted or unsubstituted heteroaryl, a substituted or unsubstituted cycloalkyl, or a substituted or unsubstituted heterocycloalkyl; and n = 0-3; with the provisos that (1) X is not hydrogen, chloro, -NO₂, or bromo when A is -O- , B is CH₂ and R is hydrogen; and (2) X is not bromine when A is NR1 and R1 is methyl

[0010] In various aspects, n is 0 or 1 . In various aspects, X is hydrogen, bromo, iodo, chloro, -NO₂, or -NH₂, methyl, or propenyl. various aspects, each R is independently hydrogen, methyl, V or For example, in some aspects, each R is hydrogen.

[0012] In various aspects, Ri 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. For example, in some aspects, Ri is a substituted or unsubstituted phenyl, a substituted or unsubstituted pyridinyl, a substituted or unsubstituted thiazole, a substituted or unsubstituted thiophenyl, a substituted or unsubstituted piperazine, or an unsubstituted C4-C10 cycloalkyl or an unsubstituted C4-C10 heterocycloalkyl. In further aspects, Ri is selected from the group consisting of wherein each Y is independently -CH2- or -O-; each R2 is independently hydrogen, -NO₂, an alkoxy, an alkyl ether of -R3OR4, -NHOR5, -NH₂, halo, a haloalkyl, or an alkyl; and R₃ and R4 are each independently C1-C4 alkyl and R5 is an alkyl or a benzyl. In various aspects, each R2 is independently hydrogen, -NO2, -OCH3, - H

[0014] In any of the foregoing or related aspects, the compound is not:

[0015] In any of the foregoing or related aspects, the compound is selected from the group

thereof.

[0016] In any of the foregoing or related aspect, the compound is a pharmaceutically equivalent salt selected from the group consisting of:

[0017] For example, in some aspects, the compound is selected from the group consisting and any pharmaceutically appropriate salt thereof. [0018] In further aspects, the compound may be a pharmaceutically appropriate salt

[0019] In various aspects, the compound of Formula I can specifically inhibit EPHB1- EPHRIN and/or EPHB2-EPHRIN tetramerization. For example, the compound that can specifically inhibit EPHB1-EPHRIN and/or EPHB2-EPHRIN tetramerization may be selected from the group consisting of: [0020] In various aspects, the compound of Formula I can specifically inhibit EPHB2-

EPHRIN tetramerization. For example, the compound that can specifically inhibit EPHB2- EPHRIN tetramerization may be selected from the group consisting of: any pharmaceutically appropriate salt thereof.

[0021] In various aspects, the compound of Formula I can specifically inhibit EPHB1-

EPHRIN tetramerization. For example, the compound that can specifically inhibit EPHB1- EPHRIN tetramerization may be selected from the group consisting of any pharmaceutically appropriate salt thereof.

[0022] In any of the foregoing or related aspects, the compound of Formula I that specifically inhibits EPHB1-EPHRIN and/or EPHB2-EPHRIN tetramerization may not inhibit EPHB4-EPHRIN tetramerization.

[0023] In any of the foregoing or related aspects, the compound of Formula I inhibits EPH- EPHRIN tetramerization with an IC50 of less than 2 pM, less than 1.6 pM, less than 1 pM or less than 0.5 pM.

[0024] Further aspects of the present disclosure provide for pharmaceutical compositions comprising any compound of Formula I provided herein and a pharmaceutically appropriate carrier or excipient.

[0025] Further aspects of the present disclosure relate to a method of inhibiting formation of an EPH-EPHRIN tetramer, the method comprising: contacting an EPH or EPHRIN with a compound of Formula I, provided herein, or a compound selected from

formation of an EPH-EPHRIN tetramer.

[0026] In various aspects, the EPH-EPHRIN tetramer may comprise EPHB1. In various aspects, the EPH-EPHRIN tetramer may comprise EPHB2. In various aspects, formation of the EPH-EPHRIN tetramer is inhibited in vivo.

[0027] Further aspects of the present disclosure relate to a method of alleviating or relieving pain in a subject in need thereof, the method comprising administering an affective amount of an EPH-EPHRIN tetramerization inhibitor to the subject. In some aspects, the pain comprises chronic neuropathic pain.

[0028] Further aspects of the present disclosure relate to a method of treating a synaptopathy in a subject in need thereof, the method comprising administering an affective amount of an EPH-EPHRIN tetramerization inhibitor to the subject. In some aspects, the synaptopathy comprises abnormal, defective, or excessive EPH-EPHRIN signaling and, optionally, comprises disrupted NMDA signaling. In various aspects, the synaptopathy is associated with anxiety or epilepsy.

[0029] Further aspects of the present disclosure relate to a method of treating addiction or opioid dependency in a subject in need thereof, the method comprising administering an affective amount of an EPH-EPHRIN tetramerization inhibitor to the subject.

[0030] Further aspects of the present disclosure relate to a method of treating a fibrotic and/or inflammatory disease or condition in a subject in need thereof, the method comprising administering an effective amount of an EPH-EPHRIN tetramerization inhibitor to the subject. In various aspects, the fibrotic and/or inflammatory disease or condition comprises abnormal, defective, or excessive EPH-EPHRIN signaling and optionally comprises NASH liver fibrosis, chronic kidney disease, scleroderma (skin fibrosis), heart fibrosis, lung fibrosis, fibrosis of another organ, and/or abnormal wound healing optionally selected from keloids and/or hypertrophic scarring.

[0031] Further aspects of the present disclosure relate to a method of treating cancer in a subject in need thereof, the method comprising administering an effective amount of an EPH- EPHRIN tetramerization inhibitor to the subject. In various aspects, the cancer comprises abnormal, defective, or excessive EPH-EPHRIN signaling and optionally comprises GBM (glioblastoma), pancreatic cancer, and/or colon cancer.

[0032] Further aspects of the present disclosure relate to a method of treating a viral infection in a subject in need thereof, the method comprising administering an effective amount of an EPH-EPHRIN tetramerization inhibitor to the subject. In various aspects, the viral infection comprises abnormal, defective, or excessive EPH-EPHRIN signaling and is optionally comprises an infection by henipavirus and/or human immunodeficiency virus (HIV).

[0033] In any of the foregoing or related methods, the EPH-EPHRIN tetramerization inhibitor may be administered as part of a pharmaceutical composition (e.g., a pharmaceutical composition comprising the EPH-EPHRIN tetramerization inhibitor). In various aspects, the pharmaceutical composition can be administered intravenously, subcutaneously, or oral dosing, IP injections, and a topical application as cream or ointment.

[0034] In any of the foregoing or related methods, the EPH-EPHRIN tetramerization pharmaceutically acceptable salt thereof. [0035] In any of the foregoing or related methods, the subject may be a human, a livestock animal, a companion animal, a lab animal, or a zoological animal.

[0036] Further aspects of the present disclosure provide for 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 as provided herein.

BRIEF DESCRIPTION OF THE FIGURES

[0037] FIG. 1 depicts a schematic of the actions of a ligand antagonist (LA) versus kinase inhibitor. An EphB KI (blue star) would disrupt only forward signaling by the intracellular catalytic domain, whereas a LA that interferes with EphB binding to EphrinB (EB) (red star) would interfere with forward and reverse signaling.

[0038] FIG. 2 shows that trans-synaptic EphrinB2-EphB1 interactions drive the plasticity of dorsal horn neurons and formation of chronic pain. Cartoon of a dorsal horn neuron excitatory synapse in the spinal cord with post-synaptic EphB1 receptor binding to pre-synaptic EphrinB2 (EB2) ligand. This induces an association of EphB1 with the NR1 subunit of the NMDAR and tyrosine phosphorylation of the NR2 subunit, which helps open the ion channel to allow calcium influx, neuron activation, and LTP to bring about central sensitization and chronic pain. The red star indicates a hypothetical drug compound that binds/docks into the ligand binding structure of the EphB1 ectodomain to disrupt its ability to bind EphrinB2 and thus act as a ligand antagonist (LA).

[0039] FIG. 3 depicts targeting the EphB1-EphrinB2 interaction in a high throughput screen (HTS). Approach to target the protein-protein interaction of EphB1 receptor binding to EphrinB2 (EB2) ligand using soluble epitope tagged ectodomain proteins in a proximity- driven chemiluminescent Alpha assay. A library of small molecular weight drug-like chemicals was screened in high throughput with this assay to identify compounds that reduce the chemiluminescent signal. The red star indicates a hypothetical compound from the library that binds/docks into the ligand binding structure of the EphB1 ectodomain to disrupt its ability to bind the EphrinB2 (EB2) ectodomain and thus act as a ligand antagonist (LA).

[0040] FIGs. 4A-4C depict the output of an alpha assay to measure the EphB1-EphrinB2 (EB2) protein-protein interaction.

[0041] FIG. 5A depicts pulldown experiments that show A20 reduces the ability of soluble EB2-His to bind to immobilized EphB1-Fc (red asterisk).

[0042] FIG. 5B depicts results from pulldown experiments that show A20 exhibits only weak/poor ability to antagonize soluble EB2-His binding to immobilized EphB4-Fc (red asterisk). [0043] FIG. 5C depicts results from pulldown experiments showing that A20 strongly reduced EB2-His binding to immobilized EphB2-His-Fc in a dose-dependent fashion and with an IC50 of ~10pM (see FIG. 5D).

[0044] FIG. 5D depicts results from an alpha assay using EphB2-Fc or EphB4-Fc with EB2- His and increasing A20 concentrations to calculate IC50 determination (~10pM, similar to that found in FIG. 5C).

[0045] FIG. 6 depicts the chemical structure of A20 (SW056428) and related A19 (QM) chemical lacking the bromine.

[0046] FIG. 7 depicts A20 and related available analog chemicals.

[0047] FIG. 8 depicts ELISA and pulldown provides IC50 data for A20 and available analog chemicals.

[0048] FIGs. 9A-9B depict Octet RED384 kinetic analysis sensorgram data of 30 nM soluble mouse EphrinB2 (mEB2-His) protein binding to sensorchip immobilized rat EphB1- Fc (FIG. 9A) or mouse EphB4-Fc (FIG. 9B) protein and effect of increased concentration of A20. Association time was for 400 sec and dissociation time was for 500 sec. Binding assays were conducted in PBS containing 0.05% Tween-20 and 1% DMSO.

[0049] FIG. 10 depict crystal structures of the EphB2-EphrinB2 dimer, the circular tetramer, and tetramer clusters.

[0050] FIG. 11 depicts Octet RED384 kinetic analysis sensorgram data showing that A20 is a reversible antagonist of EphrinB2 binding to EphB1. Immobilized rat EphB1-Fc protein pre-exposed to 100 pM A20 did not affect its subsequent ability to bind 30 nM soluble mEB2- His (red sensorgram) and interacted with similar kinetics as the sample not pre-exposed to the compound (green sensorgram). Addition of A20 during the association step did compete with EB2 binding to the immobilized EphB1 protein (purple sensorgram). Binding assays were conducted in PBS containing 0.05% Tween-20, association time was for 400 sec and dissociation time was for 500 sec.

[0051] FIGs. 12A-12F show using Octet RED384 kinetic analysis that A20 is a competitive antagonist of EphrinB2 binding to EphB1. FIG. 12A-12C shows sensorgram data that in the absence of A20, immobilized EphB1 protein can bind EB2 protein when presented at 1 , 10, 100, and 1 ,000 nM concentrations (FIG. 12A), with 10 pM A20 present the binding of EB2 is reduced and detected at only 10, 100, and 1 ,000 nM (FIG. 12B), and with 100 pM A20 the binding of EB2 can only be detected using 100 and 1 ,000 nM (FIG. 12C). FIG. 12D-12F shows that grouping the sensorgrams based on the concentration of EB2 ligand used (0.1 or 1 nM in FIG. 12D, 10 and 100 nM in FIG. 12E and 100 nM in FIG. 12F) shows that addition of A20 competes with and reduces EB2 binding to the immobilized EphB1 protein. Binding assays were conducted in PBS containing 0.05% Tween-20, association time was for 400 sec and dissociation time was for 500 sec.

[0052] FIG. 13 is an illustrative immunoblot showing that A20 antagonizes EB2 stimulation of EphB2 catalytic activity in Cos1 cells that endogenously express this receptor.

[0053] FIG. 14 depicts representative fluorescent images and quantification showing that Cos1 cells exposed to pre-clustered EB2-Fc binds to EphB2 forming spots on the cell (green) that also contain phosphotyrosine (red, arrows). Quantification of the number of spots shows A20 (40 pM) reduced the number of ligand:receptor clusters per cell and fewer of the spots contained a detectable pTyr signal (n = >60 cells per condition, a particle/spot/signal size between 1-5 pm was considered as a cluster).

[0054] FIG. 15 is a plot showing that A20 free base distributes to plasma, brain, and spinal cord following a single IP injection of 20 mg/ kg into mice.

[0055] FIG. 16 depicts a schematic describing the generation of the A20 2xHCI salt.

[0056] FIG. 17 depicts Octet RED384 kinetic analysis of immobilized rat EphB1 binding to soluble mouse EB2 and effect of A20.2xHCI salt form of compound. Binding assays were conducted in PBS containing 0.05% Tween-20, association time was for 400 sec and dissociation time was for 500 sec.

[0057] FIG. 18 is a plot showing A20.HCI salt compound exhibits greatly improved PK dynamics.

[0058] FIGs. 19A-19F depict Octet RED384 kinetic analysis of immobilized human EphrinBI (FIG. 19A), human Ephrin B2 (Fig. 19B), and human EphrinB3 (FIG. 19C) ectodomain proteins binding to soluble human EphB1 , EphB2, and EphB4 ectodomains, and the same results grouped to show how each human EphB1 (FIG. 19D), human EphB2 (FIG. 19E) and human EphB4 (FIG. 19F) bound to the three different EphrinB proteins.

[0059] FIG. 20A-20C depicts plots showing immobilized human EphrinBI (FIG. 20A), EphrinB2 (FIG. 20B) and EphrinB3 (FIG. 20C) proteins binding to human EphB1 , EphB2, and EphB4 - curve fits (thin red lines).

[0060] FIG. 21A depicts plots of immobilized human EphrinB2 binding to human EphB2 and human EphB4 in the presence or absence of different concentrations of A20.

[0061] FIG. 21 B depicts plots of immobilized human EphrinB2 binding to human EphB2 and human EphB4 in the presence or absence of 1 pM A20.

[0062] FIG. 21 C depicts plots of immobilized human EphrinB2 binding to human EphB2 and human EphB4 where various regions corresponding to dimerization and tetramerization are shaded. [0063] FIG. 21 D is a bar graph quantifying the effect of 1 pM A20 on the dimerization and tetramerization of immobilized human EphrinB2 binding to human EphB2 and human EphB4.

[0064] FIG. 22 depicts dimertetramer ratios of immobilized human EphrinB proteins binding to 50 nM human EphB proteins (estimated based off the bend in curve) and effect of A20 salt.

[0065] FIG. 23 depicts a plot of a shortened 80 sec association using immobilized rat EphB1-Fc binding to 100 nM mEB2-His (note in this experiment the baseline showed drift).

[0066] FIG. 24 depicts plots showing a shortened association 80-40-20-15-10-5 sec of immobilized rat EphB1-Fc binding to 100 nM mEB2-His (note in this experiment the baseline showed drift).

[0067] FIGs. 25A-25C depict annotated plots (FIG. 25A and 25B) and analysis (FIG. 25C) showing that A20 does not affect dimer binding kinetics, it specifically affects accumulation of the super-stable tetramer.

[0068] FIGs. 26A-26D depict illustrative mass photometry plots showing A20 chemicals specifically affect the accumulation of Eph-Ephrin tetramers. FIG. 26A-26B show formation of weak homodimers of EphB2 (FIG. 26A) or EphrinB2 (FIG. 26B) in homogenous mixtures. FIG. 26C-26D show formation of tetramers between EphB2 and Ephrin B2 without the presence of an A20 chemical (FIG. 26C) and the inhibition of tetramerization in the presence of the A20 chemical (FIG. 26D).

[0069] FIG. 27 depicts an illustrative plot and biophysical analysis of A class and cross-A/B class Eph-Ephrin protein-protein interactions.

[0070] FIG. 28A-28C depicts illustrative plots and biophysical analysis showing that A20 also disrupts formation of A class (e.g., EphA3-EA1 in FIG. 28A or EphA3-EA5 in FIG. 28B) and cross-A/B class (e.g., EphB2-EA5 in FIG. 28C) Eph-Ephrin tetramers.

[0071] FIG. 29A-29C depicts illustrative biophysical data showing that pre-existing stable Eph-Ephrin tetramers, hEphrinB2+hEphB1 (FIG. 29A), hEphrinB2+hEphB2 (FIG. 29B), and mEphB+mEphrinB2 (FIG. 29C) are also targeted by A20 chemicals.

[0072] FIG. 30A-30D depict plots showing accurate calculations of the tetramer inhibitor IC50 values for exemplary compounds A19 • 2xHCI salt (FIG. 30A), A20 • 2xHCI salt (FIG. 30B), 8009-9255 • 2xHCI salt (FIG. 30C), and 3511-0013 (FIG. 30D), on the human EphrinB2-EphB2 interaction.

[0073] FIG. 31 depicts analogs based off A20 scaffold.

[0074] FIG. 32 depicts analogs based off 3511-0013 scaffold. [0075] FIGs. 33A-33B depict first generation of novel A20 analogs. The 8-hydroxyquinoline ring system is either retained (FIG. 33A) or replaced with a 5-methoxyindole ring (FIG. 33B).

[0076] FIG. 34 depicts alpha results testing LA activity of the six first-generation novel compounds on the EphB1-EB2 interaction. Conditions included 8 nM of EphB1-Fc and EB2- His proteins, 1 % DMSO, 50 ng each bead per well, and indicated concentration of compound in free base form.

[0077] FIG. 35A-35E depict tetramer inhibitor IC50 values for exemplary first-generation compounds QPB4 (FIG. 35A), QPDF (FIG. 35B), IM (FIG. 35C), IMP2 (FIG. 35D) and IPB4 (FIG. 35E) on the human EphrinB2-EphB2 interaction.

[0078] FIG. 36A depicts chemical synthesis schematic for generating compounds in series 1a, series 1 b, and a few compounds in series 6.

[0079] FIG. 36B depicts chemical synthesis schematics for generating compounds in series 2, 3, 4, as well as a few compounds in series 6.

[0080] FIG. 36C depicts a chemical synthesis schematic for generating series 5 compounds.

[0081] FIG. 37 depicts compounds synthesized and described herein.

[0082] FIG. 38A-38D depict binding of immobilized EphB2 binding to murine EphrinB2 in the presence of A20-I (FIG. 38A) or A20 (FIG. 38B), binding of immobilized murine EphB2 binding to human EphrinA5 in the presence of A20-I (FIG. 38C) and binding of immobilized human EphrinB2 to human EphB2 in the presence of A19-NO2. These data show that second-generation compound A20-I is a stronger tetramerization inhibitor compared to A20 and that replacement of the halogen of A20/A20-I with NO2 results in poor tetramerization inhibitor activity.

[0083] FIG. 39 shows A20-I exhibits increased in vitro metabolic stability compared to A20. Both free base and salt forms of compounds were analyzed by the PK Core.

[0084] FIG. 40A-40C shows biophysical analysis of an exemplary second-generation compounds derived from QTM: QTM (FIG. 40A), QTM-Br (FIG. 40B), and QTM-I (FIG. 40C) where all compounds were tested in their salt (2xHCI) form. Assays shown are immobilized human EphrinB2 binding to 50 nM human EphB2.

[0085] FIG. 41A-41 F depict biophysical analysis of exemplary first-generation compounds derived from QPB4: QPB4 (FIG. 41A), QPPh (FIG. 41 B), QPI4 (FIG. 41C), MeQPB4 (FIG. 41 D), BQPB4-Bn (FIG. 41 E), and QPCF34 (FIG. 41 F) where each compound, except MeQPB4 (FIG. 41 D), was tested in its salt (2xHCI) form. Assays shown are immobilized human EphrinB2 binding to 50 nM human EphB2. [0086] FIG. 42A depicts the structure of QPB4-Bn (free base) and QPB4-Bn salt form (2xHCI).

[0087] FIG. 42B is an illustrative plot showing QPB4-Bn salt form (2xHCI) exhibits strong PK distribution when injected into mice.

[0088] FIG. 42C depicts quantification of experiment depicted in FIG. 42B.

[0089] FIG. 43 depicts peptides SNEW (SEQ ID NO: 1) and EWLS (SEQ ID NO: 2) which are similar peptides described to bind into the EphB1 and EphB2 dimerization pocket and disrupt EphrinB binding.

[0090] FIGs. 44A-44B show biophysical studies that the SNEW peptide (FIG. 44A), but not EWLS peptide (FIG. 44B), specifically disrupts the EphrinB2-EphB2 dimer interaction. After baseline measurements, biosensor immobilized human EphrinB2 ectodomain protein was exposed to 50 nM soluble human EphB2 ectodomain without or with the indicated concentrations of SNEW (FIG. 44A) or EWLS (FIG. 44B) peptide during the association step. The upper full trace sensorgrams at relatively low peptide concentrations show only the highest 100 pM concentration of SNEW altered the binding pattern during the association step, though its effect was different and not as significant as the 2 pM concentration of the A20-I tetramerization inhibitor used for the maximum control. The lower sensorgrams focus on the association step of a second experiment where the concentration of peptides were increased up to 400 pM. Note that the initial steep 84° dimerization slope of the EphrinB2- EphB2 interaction in the absence of peptide is strongly reduced in a dose-dependent fashion by presence of the SNEW peptide. The EWLS peptide showed no effect on the EphrinB2- EphB2 protein-protein interaction.

[0091] FIG. 45 depicts tests of salt forms of A20 and A20-I for aqueous solubility. Compounds were provided to the UT Southwestern PK Core facility and tested for soluble in water. While both are quite soluble, A20 salt was especially so and can be made into a >100% solution, whereas A20-I salt can be dissolved into >15% solution.

[0092] FIG. 46 depicts tests of A20 and A20-I free base and salt forms for in vitro plasma stability. Compounds were provided to the UT Southwestern PK Core facility and tested for stability when incubated with mouse plasma. While both compounds appear quite stable, A20-I showed superior stability whether the free base or salt form was assessed.

[0093] FIG. 47A-47D depicts data and quantification showing that A20 salt form is orally bioavailable. A20.2xHCI compound was provided to the UT Southwestern PK Core facility and tested for bioavailability after a single dose when either injected intravenous (IV) at 3 mg/kg or provided by oral gavage (PO) at 30 mg/kg using PBS as vehicle. The data on oral gavage is quite impressive and indicates significant levels of A20 persist, especially in the liver, after a single dose.

[0094] FIG. 48A-48D depicts biophysical analysis of exemplary second-generation compounds A20-I (FIG. 48A), A19-L (FIG. 48B), 2OH-A19 (FIG. 48C), and QPPh (FIG. 48D). All compounds were tested as free bases, except A20-I (tested as 2xHCI salt). Assays shown are immobilized human EphrinB2 binding to 50 nM human EphB2. Note that compound A19- L shows a slight ability to disrupt the unsurmountable tetramer at 3.2 pM concentration (bold) as its sensorgram is below the maximum control.

[0095] FIG. 49A-49E depict biophysical analysis of additional exemplary second- generation compounds A19-NO2 (FIG. 49A), QTM-NO2 (FIG. 49B), 2OH-A20 (FIG. 49C), 2- MeA20 (FIG. 49D) and QPB-NO4 (FIG. 49E). All compounds were tested as free bases, except QPB-NO4 (tested as 2xHCI salt). Assays shown are immobilized human EphrinB2 binding to 50 nM human EphB2. Note that compound QPB-NO4 shows a slight ability to disrupt the unsurmountable tetramer at 3.2 pM concentration (bold) as its sensorgram is below the maximum control.

[0096] FIG. 50A-50D depicts biophysical analysis of an exemplary second-generation compounds derived from QPA: QPA (FIG. 50A), QPA-Ac (FIG. 50B), QPA-PAc (FIG. 50C), and BQPA-PAc (FIG. 50D). Assays shown are immobilized human EphrinB2 binding to 50 nM human EphB2.

[0097] FIG. 51A-51 E depict biophysical analysis of exemplary second-generation compounds derived from 3511 : 3511-1 (FIG. 51A), 3511-0013-BF (FIG. 51 B), 3511-4OMe- BF (FIG. 51C), 3511-4OHMe-l (FIG. 51 D) and IQPB4-BN (FIG. 51 E). Assays shown are immobilized human EphrinB2 binding to 50 nM human EphB2. Note that compound 3511-1 shows fairly strong ability to disrupt some of the unsurmountable tetramer at 0.8 and 1 .6 pM concentrations tested, and compound 3511-4OMe-BF also has ability to disrupt some of the unsurmountable tetramer at 3.2 uM concentration.

[0098] FIG. 52 depicts biophysical analysis of second-generation compound BQPB4 shows it breaks through to inhibit 100% of the unsurmountable tetramer at low micromolar concentrations. Assays shown are immobilized human EphrinB2 binding to 50 nM human EphB2. Note that BQPB4 exhibits a strong, concentration-dependent ability to disrupt formation of the super-stable circular tetramer with an IC50 = -300 nM and that at 0.8, 1.6, and 3.2 pM concentrations tested the unsurmountable tetramer is disrupted with complete loss of this structure at 3.2 pM.

[0099] FIG. 53 depicts compounds that inhibit the unsurmountable EphrinB2-EphB2 tetramer. [0100] FIG. 54 depicts a schematic of medicinal chemistry efforts around the A20 lead compound to indicate changes that can enhance or decrease its Eph-Ephrin tetramerization inhibitor activity.

[0101] FIG. 55 depicts potential site modifications on A20 derivatives. (Red) Piperazine replacement, (Yellow) Hydrophobic ring extension, (Green) Carbon replacement, (Blue) Halogen replacement, (Purple) Alcohol protection.

[0102] FIG. 56 depicts potential site modifications on 3511-0013, BQPB4, and BQPB4-bn. (Green) n = number of -CHr extensions, (Yellow) Terminal substituent on extended phenyl/benzyl ring, (Purple) Substituent at meta position to R1 , (Blue) Halogen replacements.

[0103] FIGs. 57A-57B show that expression of EphB1 receptor and EphrinB2 ligand are increased in the spinal cord following chronic pain generating nerve insults. (FIG. 57A) Within 25 hr following chronic constriction injury (CCI) of the rat sciatic nerve, a strong and sustained increase in expression of EphB1 was detected by immunoblot in the ipsilateral (I), injured side of the spinal cord (red arrows) compared to the uninjured contralateral (C) side and sham treated mice. FIG. 57B, in bone cancer pain, tumor cell implantation (TCI) of Walker- 256 carcinoma cells into the intramedullary space of the rat tibia resulted in a striking increase of EphB1 and EphrinB2 expression in the ipsilateral, tumor cell implanted side as detected by immunofluorescence of sectioned spinal cords (green signal). Note that the increased expression of EphB1 and EphrinB2 is mainly restricted to the dorsal, superficial region of the ipsilateral spinal cord where DH neurons are located and where the C-fiber:DH neuron synapses are localized. Little if any expression can be detected in the contralateral control side of the spinal cord or in the sham treated rats.

[0104] FIG. 58 shows that 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 thermal heat (hyperalgesia) as indicated by a quickened response to an infrared heat source. EphB1-/- KO mice do not develop hyperalgesia post-CCI (diamonds). Even the EphB1+/- heterozygous mice do not exhibit hyperalgesia following CCI (triangles).

[0105] FIG. 59 shows that EphB1 is required for dorsal horn neuron LTP. Electrophysiological recordings from the superficial spinal cord of live mice assessed the plasticity of synapses formed between peripheral nociceptive C-fibers and DH neurons. Following a brief high frequency stimulation (HFS) train of the sciatic nerve, EphB1+/+ (WT) mice exhibited a strong and sustained increase in field potentials. Recordings from spinal cords of EphB1-/- KO mice indicated no LTP following the HFS train.

[0106] FIG. 60 is a schematic of showing the localization of EphrinB2, EphB1 and the NMDA receptor at the nociceptor nerve C-fiber - dorsal horn neuron synapse (see FIG. 2). [0107] FIG. 61 shows that A20 (red dotted line) strongly reduced inflammatory pain compared to V controls (black dotted line) when injected into mice. A total of five wildtype (WT) male mice were injected IP with A20 free base (3 mice at 10 mg/kg and 2 mice at 20 mg/kg in 6% DMSO I 94% sunflower seed oil, data is pooled) and three WT males with vehicle (V) only as control. As indicated, injections of A20 or V started two days prior to the 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 examination of withdrawal latency, both left and right hind paws were subjected to 6-12 thermal pain measurements to obtain an average response time for each mouse. Mice were at 14 weeks age when experiment started.

[0108] FIG. 62 shows that A20 appears more effective than MCD at reducing inflammatory pain. Wildtype (WT) female mice were injected IP with either A20 free base (20 mg/kg in 6% DMSO I 94% sunflower seed oil), with MCD kinase inhibitor (10 mg/kg in PBS), or vehicle only (6% DMSO I 94% sunflower seed oil). Injections of A20, MCD, or V started two days prior to 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 examination of withdrawal latency, both left and right hind paws were subjected to 6-12 thermal pain measurements to obtain an average response time, and for each mouse the average of the uninjured hind paw response time was divided by the average of the injured side response time to obtain a pain response ratio for each mouse which was then plotted and subjected to indicated statistical analysis. Data shown were those obtained at day +3 post-CFA. Mice were at 20 weeks age when experiment started.

[0109] FIG. 63 depicts tests of salt versions of compounds A20 and QPB4-Bn showing both are effective at reducing inflammatory pain when injected after the pain-generating insult and when using PBS as the vehicle. Wild-type (WT) male and female mice were injected IP with either A20.2xHCI salt (20 mg/kg in PBS), QPB4-Bn.2xHCI salt (20 mg/kg in PBS), or vehicle only (PBS). Here, the first injection of A20, QPB4-Bn, or V started 15 minutes after CFA was injected 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 examination of withdrawal latency, both left and right hind paws were subjected to 6-12 thermal pain measurements to obtain an average response time, and for each mouse the average of the uninjured hind paw response time was divided by the average of the injured side response time to obtain a pain response ratio for each mouse which was then plotted and subjected to indicated statistical analysis. Data shown were those obtained at day +3 post-CFA. Mice were at 8 weeks age when experiment started. [0110] FIG. 64 depicts tests of two more A20 analogs, salt forms of QPB4 and QPP(QPP127) show both are effective at reducing inflammatory pain when injected after the pain-generating insult and when using PBS as the vehicle. Wild-type (WT) female mice were injected IP with QPB4«2xHCI salt (20 mg/kg in PBS), QPP(QPP127) «2xHCI salt (20 mg/kg in PBS), or vehicle only (PBS). Here, the first injection of QPB4, QPP(QPP127), or vehicle started 15 minutes after CFA was injected 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 examination of withdrawal latency, both left and right hind paws were subjected to 6-12 thermal pain measurements to obtain an average response time, and for each mouse the average of the uninjured hind paw response time was divided by the average of the injured side response time to obtain a pain response ratio for each mouse which was then plotted and subjected to indicated statistical analysis. Data shown were those obtained at day +3 post-CFA. Mice were at 12 weeks age when experiment started. A topical test of an MCD cream was also assessed in this experiment and also showed efficacy.

[0111] FIG. 65 depicts development of a quantitative fluorescent method to assess the activation of DH neurons in the spinal cord following a chronic, neuropathic insult to the peripheral nerves. The left hind paws of EphB1+/+ wild-type, EphB1+/- heterozygous, and EphB1-/- homozygous mutant mice that also contained the Trap2/CreERT2 driver and the Ai9/tdTomato reporter were injected once with CFA to induce inflammatory pain. Four hours later, mice were then injected IP with 4-hydroxy-tamoxifen (4- OHT, 40 mg/kg in sunflower seed oil) to activate Cre recombinase and permanently label with red fluorescence the neurons that were activated during the short window of time (-8 hr) until the 4-OHT is degraded. Fourteen days later, animals were perfused with 4% paraformaldehyde dissolved in PBS and then 50 pM thick vibratome serial sections of the lumbar spinal cord were obtained, mounted, and viewed under fluorescence microscopy to identify red labeled neurons in the superficial dorsal horn of the spinal cords that were activated between -4-12 hr after the CFA induction of inflammatory pain. Superficial red neurons were counted on the left (injured) and right (uninjured) side dorsal horns (boxed areas) from 8-12 serial sections along the span of lumbar cord of each animal. The ratio of T rap2+ 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 the WT mice exhibited almost 3 times more red labeled neurons in the injured dorsal horn compared to uninjured, indicative of a high level of neuronal activation following inflammatory pain generating insult to the left hind paw. In contrast, the EphB1-/- knockout showed very little left-right difference (ratio -1) and the EphB1+/- heterozygotes all showed an intermediate ratio of -2, indicating gene dosedependent effect on neuronal activation that parallels the behavioral observation that heterozygotes are also refractory to chronic pain generating insults. Females at 20 weeks age were used in this experiment.

[0112] FIG. 66 shows that A20 reduces the activation of DH neurons in WT mice following CFA inflammatory insult. Shown are representative fluorescent images of the dorsal lumbar spinal cord from vehicle and A20 injected mice. Superficial DH neurons in the left, CFA injected, side of the vehicle treated mice showed ~3 times more Trap2/tdTomato red fluorescent labeled neurons compared to the uninjured right side. A20 treated mice showed significantly fewer labeled neurons in the injured side. The plotted ratio of left/ right red neurons per mouse was determined by averaging the value obtained from 8-12 serial sections along the span of lumbar cord of each animal. Note that while the small n value prevented one-way ANOVA analysis, unpaired t-tests indicate significant differences between the A20 and vehicle treated animals, but not for the MCD kinase inhibitor treatment group.

[0113] FIG. 67 shows that expression of EphB1 receptor protein is increased in the spinal cord following escalating doses of morphine.

[0114] FIG. 68 shows that EphB1-/- knockout mice lacking expression of this receptor protein exhibit greatly reduced morphine withdrawal behaviors.

[0115] FIG. 69 shows EphB1-/- knockout mice lacking expression of this receptor protein exhibit greatly reduced morphine withdrawal behaviors. This withdrawal study confirmed the involvement of EphB1.

[0116] FIGs. 70A-70B show A20 strongly reduced morphine withdrawal behaviors in mice. Pooled results from 2 different experiments using CD1 mice (3 mice V and 3 mice A20) and C57BL/6 mice (3 mice V and 4 mice A20)(FIG. 70A) or C57BL/6 (FIG. 70B). The two mice that scored zero for jumps, especially the V mouse, may have received a poor naloxone injection.

[0117] FIGs. 71A-71X depict reaction schematics and representative NMR spectra for exemplary compounds of the present disclosure.

[0118] FIGs. 72A-72C depict Octet biophysical data using sensorchip immobilized human EphrinB2-Fc ectodomain binding to 25 nM soluble human EphB1-His (FIG. 72A), EphB2-His (FIG. 72B), and EphB4-His (FIG. 72C) ectodomains (80” association and 500” dissociation) with 10 pM A20-I as max control.

[0119] FIGs. 73A-73C depict percent inhibition of immobilized human EphrinB2-Fc binding to 50 nM soluble human EphB1-His (FIG. 73A), EphB2-His (FIG. 73B), and EphB4-His (FIG. 73C) proteins (80” association and 500” dissociation) with 10 pM of either A20-1 as max control or BQPB4. [0120] FIGs. 74A-74C depict representative Octet sensorgrams showing effect of increasing concentrations of A20-I salt on immobilized human EphrinB2-Fc binding to 50 nM soluble human EphB1-His (FIG. 74A), human EphB2-His (FIG. 74B), or human EphB4-His (FIG. 74C) proteins.

[0121] FIGs. 75A-75C depict representative Octet sensorgrams showing effect of increasing concentrations of BQPB4 salt on immobilized human EphrinB2-Fc binding to 50 nM soluble human EphB1-His (FIG. 74A), human EphB2-His (FIG. 74B), or human EphB4- His (FIG. 74C) proteins.

[0122] FIGs. 76A-76F depict representative Octet sensorgrams (FIG. 76A-76C) and enlarged inserts (FIG. 76D-76F) showing maximal inhibition of different compounds (3511-1, 8009-9255, QTM-Br, A19, A19-NO2, and IM) on EphB1-EphrinB2 (FIG. 76A, enlarged in FIG. 76D), EphB2-EphrinB2 (FIG. 76B, enlarged in FIG. 76E), or EphB4-EphrinB2 (FIG. 76C, enlarged in FIG. 76F) tetramerization.

[0123] FIGs. 77A-77F depict representative Octet sensorgrams showing maximal inhibition of EphB1-EphrinB2 (FIG. 77A, FIG. 77D), EphB2-EphrinB2 (FIG. 77B, FIG. 77E) and EphB4- EphrinB2 (FIG. 77C, FIG. 77F) tetramerization using A19 and A19-NO2 (salt or free base forms) compared to maximum control, A20-I (FIGs. 77A-77C) or A19, A19-NO2 (salt or free base), QPB4-Bn, and BQPB4 compared to maximum control, A20-I (FIGs. 77D-77F).

[0124] FIG. 78 depicts structures of A20 chemicals that exhibit less inhibitory activity towards EphrinB2-EphB4 interaction and higher inhibitor activity towards EphrinB2-EphB1 and/or EphrinB2-EphB2 interaction.

[0125] FIGs. 79A-79C depict representative immunoblots (FIG. 79A) and quantification (FIG. 17B-17C) showing expression of EphB2, phospho-EphB1/B2 (pEphB1/B2), and EphrinB2 in brain protein lysates after intraperitoneal (IP) injection of A20, A20-I and 3511-1 and immunoprecipitation of EphB2, using anti-EphB2 antibodies. FIG. 79B and 79C show quantification of band intensities (number of photons detected) to obtain ratios of pEphB/EphB2 as a measure of EphB2 receptor activation levels (FIG. 79B) and ratios of EphrinB2/EphB2 as a measure of how much EphrinB2 is co-immunoprecipitated with the EphB2 receptor (FIG. 79C).

[0126] FIGs. 80A-80E depict representative immunoblots (FIGs. 80A-80C) and quantification (FIG. 80D-80E) thereof following EphB2 immunoprecipitation from mouse brain protein lysates. FIG. 80A-80C show expression of EphB2 (FIG. 80A), phospho-TyrlOOO (pTyr1000)(FIG. 80B) and the 4G10 phosphotyrosine-specific antibody (FIG. 80C) in mouse brains following oral dosing (PO) or injection (IP) for 4 days with vehicle (PBS) or A20 or A20-I. FIG. 80D show ratios of pTyr1000/EphB2 and 4G10/EphB2 in mice treated with vehicle or A20 compounds (both intraperitoneal (IP) and oral gavage (PO) administration are pooled) and FIG. 80E shows ratios of pTyr1000/EphB2 and 4G10/EphB2 in said mice (with IP and PO administration separated).

[0127] FIGs. 81A-81 D depict representative immunoblots (FIGs. 81A-81C) and quantification (FIG. 81 D) showing EphB2 (FIG. 81 A), pEphB1/B2 (FIG. 81 B), and pTyrlOOO (FIG. 81C) following EphB2 immunoprecipitated from brain lysates from WT or EphB1/EphB2 double homozygous knockout (E1 N1), EphB2-K661 R (homozygous kinase-dead point mutant), EphB2-F620D (homozygous kinase-overactive point mutant) and EphB2-lacZ (a mutation producing an intracellular truncated EphB2-beta-gal fusion protein which migrates at 220 kDa (heterozygous N2/+)) mice. FIG. 81 D shows phospho-EphB2/pan-EphB2 ratios in the mice, as indicated.

[0128] FIGs. 82A-82B show a darker representation of the EphB2 immunoblot shown in FIG. 81A (FIG. 82A) and tabulated chemiluminescent signal intensity (FIG. 82B).

[0129] FIGs. 83A-83B show a Coomassie blue stained SDS-PAGE gel of liver and brain protein lysates after immunoprecipitation with goat anti-EphB2 antibodies (FIG. 83A) and number of proteins identified in different brain tissues by mass spectrometry (FIG. 83B).

[0130] FIGs. 84A-84D depict representative immunoblots of whole liver protein lysates subjected to goat anti-EphB2 immunoprecipitation and immunoblot analysis with anti-EphB2 (FIG. 84A), anti-phospho-EphB1/B2 (FIG. 84B), anti-4G10 (FIG. 84C) and anti-pTry1000 (FIG. 84D) antibodies.

[0131] FIG. 85 is a schematic of an experimental protocol to assess the ability of oral dosing of A20 chemicals to blunt chronic long-term inflammatory pain induced by injection of complete Freund’s adjuvant (CFA) into the mouse hind paw.

[0132] FIGs. 86A-86B depict average mechanical pain thresholds (determined via von Frey filaments) sorted by treatment group (FIG. 86A) and by treatment time (FIG. 86B). In both figures, the left panels represent data from the left (CFA-injured) hind paw and the right represent data from the right (uninjured) hind paw. Animals were treated with vehicle or IP or PO (oral) administration of A20 (20 mg/kg).

[0133] FIGs. 87A-87B depict paw withdrawal time after contact with a thermal pain stimulus in mice following treatment with vehicle, A20 (IP, blue) or A20 (PO, yellow). The left hind paws were injured with CFA (same animals as examined in FIG. 86). FIG. 87A presents the data sorted by treatment group. FIG. 87B depicts the data sorted by treatment time.

[0134] FIGs. 88A-88B depict pain ratios calculated from data in FIGs. 86A-86B and 87A- 87B, by dividing the uninjured side pain responses by the injured side responses. [0135] FIGs. 89A-89D depict mechanical (FIG. 89A) and thermal (FIG. 89B) pain measurements following Zymosin treatment as well as mechanical pain ratio (FIG. 89C) and thermal pain ratio (FIG. 89D) in EphB2 WT (green), null (blue) or heterozygous (yellow) mice.

[0136] FIGs. 90A-90D depict mechanical pain (FIG. 90A) and thermal pain (FIG. 90B) ratios following Zymosin treatment and oral dosing (PO) of vehicle (green) or 5 mg/kg (orange), 10 mg/kg (yellow) or 20 mg/kg (blue) A20, with pooled A20 data depicted for mechanical pain in FIG. 90C and thermal pain in FIG. 90D.

[0137] FIGs. 91A-91B depict mechanical pain (FIG. 91A) or thermal pain (91 B) measurements and ratios in animals treated with vehicle (green), 3511-1 (blue), or BQPB4 (yellow) after exposure to Zymposin pain stimulus.

[0138] FIGs. 92A-92B depict thermal pain measurements (FIG. 92A) and ratios (FIG. 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) of BQPB4 (oral administration).

[0139] FIGs. 93A-93C show images and analysis of activated dorsal horn neurons following an inflammatory pain insult with FIG. 93A showing 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 is an example plotting the uninjured number of red Tom+ DH neurons counted from the left and right sides of the 12 different imaged sections from the same animal, and FIG. 93C showing a histogram plotting of the data as a function of the number of sections with X number of Tom+ DH neurons.

[0140] FIGs. 94A-94B are plots showing the ratio of Tom+ DH neurons (injured side I uninjured side) for all sections analyzed (FIG. 94A), and the average ratio when all sections of an individual spinal cord are grouped (FIG. 94B).

[0141] FIGs. 95A-95B are plots showing Tom+ DH neurons (injured side I uninjured side) for all sections analyzed (12 sections per mouse) of animals treated with vehicle or different A20 compounds after Zymosin administration (FIG. 94A), and the average ratio when all sections of an individual spinal cord are grouped (FIG. 94B).

DETAILED DESCRIPTION

[0142] In some aspects, the current 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 may be used to inhibit Eph forward signaling and Ephrin reverse signaling (bidirectional signaling) and have potential use in various therapeutics. Accordingly, various EPH-EPHRIN tetramerization inhibitors are described herein along with methods of treatment thereof.

I. Definitions

[0143] Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this invention belongs. The following references provide one of skill with a general definition of many of the terms used in this invention: Singleton et al., Dictionary of Microbiology and Molecular Biology (2^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). As used herein, the following terms have the meanings ascribed to them unless specified otherwise.

[0144] When introducing elements of the present disclosure or the preferred aspects(s) thereof, the articles “a”, “an”, “the” and “said” are intended to mean that there are one or more of the elements. The terms “comprising”, “including” and “having” are intended to be inclusive and mean that there may be additional elements other than the listed elements. Wherever the terms “comprising” or “including” are used, it should be understood the disclosure also expressly contemplates and encompasses additional aspects “consisting of” the disclosed elements, in which additional elements other than the listed elements are not included.

[0145] “Pharmaceutical composition” means a mixture of substances suitable for administering to an individual that includes a pharmaceutical agent. As used herein a pharmaceutical composition comprises one or more of receptors, vectors, cells disclosed herein compounded with suitable pharmaceuticals carriers or excipients.

[0146] “Treatment” or “therapy” of a subject refers to any type of intervention or process performed on, or the administration of an active agent to, the subject with the objective of reversing, alleviating, ameliorating, inhibiting, slowing down or preventing the onset, progression, development, severity or recurrence of a symptom, complication, condition or biochemical indicia associated with a disease.

[0147] As used herein, the term “patient”, “subject”, or “test subject” refers to any organism to which provided compound or compounds described herein are 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 farm animals), non-human primates, humans). In an aspect, a subject is a human. In some aspects, a subject may be suffering from, and/or susceptible to a disease, disorder, and/or condition (e.g., a condition caused or worsened by abnormal, defective, or excessive EPH-EPHRIN signaling).

[0148] The term “effective amount” as used herein is defined as the amount of the molecules of the present invention that are necessary to result in the desired physiological change in the cell or tissue to which it is administered. The term “therapeutically effective amount” as used herein is defined as the amount of the molecules of the present invention that achieves a desired effect with respect to whatever condition is being treated. For example, a desired effect in a method for treating pain could be a reduction or amelioration of pain in the subject while a desired effect in a method of treating cancer could be a reduction in tumor size or an arrest in tumor growth. A skilled artisan readily recognizes that in many cases the molecules may not provide a cure but may provide a partial benefit, such as alleviation or improvement of at least one symptom or parameter. In some embodiments, a physiological change having some benefit is also considered therapeutically beneficial. Thus, in some embodiments, an amount of molecules that provides a physiological change is considered an “effective amount” or a “therapeutically effective amount.”

[0149] As used herein, the term “alkyl” to saturated hydrocarbons 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 groups and cycloalkyl-substituted alkyl groups). The terms “alkenyl” and “alkynyl” refer to unsaturated aliphatic groups analogous to alkyls, but which contain at least one double or triple carbon-carbon bond respectively.

[0150] As used herein, the term “alkoxy” refers to alkyl groups linked to the remainder 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. The alkoxy groups can be straight-chain or branched.

[0151] As used herein, the term “Amino” refers to unsubstituted or substituted moiety of the formula — NraRb, in which Ra and Rb are each independently hydrogen, alkyl, aryl, or heterocyclyl, or Ra and Rb, taken together with the nitrogen atom to which they are attached, form a cyclic moiety having from 3 to 8 atoms in the ring. Thus, the term amino includes cyclic amino moieties such as piperidinyl or pyrrolidinyl groups, unless otherwise stated.

[0152] As used herein, the term “heterocyclic group” refers to closed ring structures analogous to carbocyclic groups in which one or more of the carbon atoms in the ring is an element other than carbon, for example, nitrogen, sulfur, or oxygen. Heterocyclic groups may be saturated or unsaturated. Additionally, heterocyclic groups (such as pyrrolyl, pyridyl, isoquinolyl, quinolyl, purinyl, and furyl) may have aromatic character, in which case they may be referred to as “heteroaryl” or “heteroaromatic” groups. Exemplary heterocyclic groups include, but are not limited to pyrrole, furan, thiophene, thiazole, isothiaozole, imidazole, triazole, tetrazole, pyrazole, oxazole, quinoline, piperazine, pyridine, pyrazine, pyridazine, pyrimidine, benzoxazole, benzodioxazole, benzothiazole, benzoimidazole, benzothiophene, methylenedioxyphenyl, quinoline, isoquinoline, napthridine, indole, benzofuran, purine, benzofuran, deazapurine, or indolizine.

[0153] As used herein, the term “acceptable salt” refers to salts of the compounds of the invention which are acceptable for the methods of the invention.

[0154] As used herein, the term “haloalkyl” refers to an alkyl group, as defined herein, substituted with one or more halogen. For example, trifluoromethyl is a haloalkyl group.

[0155] As used herein, the term “cyano” refers to a moiety comprising a carbon connected to a nitrogen with a triple bond (e.g., -CN).

[0156] As used herein, the term “azido” refers a linear, polyatomic anion with the formula N-3 and structure -N=N +=N - three nitrogens (e.g., -N3).

[0157] As used herein, the term “amido” refers to a functional group comprising a primary, secondary, or tertiary amide. The “amido” group may further comprise additional R groups (e.g., further alkyls, cycloalkyls, aryls etc).

[0158] As used herein, the term “carbonyl” refers to a functional group comprising at least one -CO moiety. It may further comprise additional R groups (e.g., further alkyls, cycloalkyls, aryls etc).

[0159] As used herein, the term C4-C10 aryl refers to an aromatic ring comprising 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 wherein at least one carbon has been replaced with a heteroatom (e.g., N, O or S). As used herein, a C4-C10 cycloalkyl refers to a non-aromatic cyclic moiety having 4 to 10 carbons. As used herein, a C4-C10 heterocycloalkyl refers to a non-aromatic cyclic moiety having 4 to 6 carbon atoms, wherein at least one carbon atom has been replaced with a heteroatom (e.g, N, O or S). Any of the C4-C10 aryls, heteroaryls, cycloalkyls or heterocycloalkyls used herein may optionally be substituted, unless otherwise stated. As used herein, the term C4-C10 when associated with a ring (e.g., a cycloalkyl, an aryl, a heterocycloalkyl or a heteroaryl as described above) explicitly and implicitly contemplates any intervening ring size (e.g., a 4-member ring, a 5-member ring, a 6- member ring, a 7- member ring, a 8- member ring, a 9 member ring, or a 10 member ring). [0160] As used herein, the term “may form a fused ring with”, means that the referenced functional groups (e.g., R groups) connected to a first ring may be connected to form a second ring fused to the first ring. A fused ring may be a fused aryl ring.

[0161] As used herein, the term EPH-EPHRIN tetramerization refers to the formation of an Eph receptor - Ephrin ligand macromolecular complex, between any of the known 14 EPH receptors and 8 Ephrin ligands, A and B class. Eph-Ephrin dimers first associate/bind together and then two of these dimers associate/bind to form the EPH-EPHRIN tetramer, which is a very stable circular structure that brings together the four components to activate the receptors and ligands to transduce their forward and reverse signals into the cells they are expressed on to mediate bidirectional signaling. EPH-EPHRIN tetramers once formed then assemble into much larger tetramer clusters which leads to greatly enhanced bidirectional signaling. Without tetramers, the formation of these larger tetramer clusters also cannot occur, and so compounds that affect tetramerization will also be envisioned to affect the formation of these larger tetramer clusters. EPH-EPHRIN tetramerization and tetramer clustering is distinct from dimerization which may or may not occur earlier. Rates of EPH- EPHRIN tetramerization and tetramer clustering may be determined according to methods in the art and as described in the Examples herein below.

[0162] As used herein, the 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,

I .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 should be understood as encompassing the specific quantities, amounts, dimensions, measurements and so on, and also as encompassing such instances modified by the term “about.” Accordingly, unless indicated to the contrary, the numerical descriptions set forth herein may vary while remaining well within the teachings of the present disclosure. At the very least, each numerical value should be construed in view of the number of significant digits and by applying routine rounding techniques. As various changes could be made in the above-described cells and methods without departing from the scope of the invention, it is intended that all matter contained in the above description and in the examples given below, shall be interpreted as illustrative and not in a limiting sense. As various changes could be made in the above-described cells and methods without departing from the scope of the invention, it is intended that all matter contained in the above description and in the examples given below, shall be interpreted as illustrative and not in a limiting sense.

II. Compounds

[0163] In various aspects of the present disclosure, a compound of Formula I is provided: (Formula I), or a pharmaceutically suitable salt thereof, wherein: A is an -O-

, -SO2, CH2, or -N(CH2)_nRi; B is CH2, SO2, or CO; X is hydrogen, a halogen, an alkyenyl, an alkyl, -NO2, or -NH2, ; each R is independently hydrogen, alkyl, an alkoxy, heterocycloalykl, or a carbonyl, Ri is a substituted or unsubstituted aryl, a substituted or unsubstituted heteroaryl, a substituted or unsubstituted cycloalkyl, or a substituted or unsubstituted heterocycloalkyl; and n

= 0-3.

[0164] In certain aspects, n may be 0 or 1. Additionally, in further aspects, X may be hydrogen, bromo, iodo, chloro, -NO2, or -NH2, methyl, or propenyl. In further embodiments, each

R may be independently selected from hydrogen, methyl, For example, each R may be independently hydrogen or methyl. In certain aspects, each R is hydrogen.

[0165] In any of the compounds provided herein, Ri may be a substituted or unsubstituted C4-C10 aryl (e.g., a C4-C8 or a C4-C6 aryl), a substituted or unsubstituted C4-C10 heteroaryl (e.g., a C4-C8 or a C4-C6 heteroaryl), a substituted or unsubstituted C4-C10 cycloalkyl (e.g., a C4-C8 or a C4-C6 cycloaryl) or a substituted or unsubstituted C4-C10 heterocycloalkyl (e.g., a C4-C8 or a C4-C6 heterocycloalkyl). For example, in various embodiments, Ri may be a substituted or unsubstituted phenyl, a substituted or unsubstituted pyridinyl, a substituted or unsubstituted thiazole, a substituted or unsubstituted thiophenyl, a substituted or unsubstituted piperazine, an unsubstituted C4-C10 cycloalkyl or an unsubstituted C4-C10 heterocycloalkyl. For example, Ri may be selected from the group consisting of: independently hydrogen, -NO2, an alkoxy, an alkyl ether of -R3OR4, -OCH3, -NHOR5, -NH2, halo, a haloalkyl, or an alkyl; R3 and R4 are each independently C1-C4 alkyl and R5 is an alkyl or a benzyl. [0166] In any of the compounds of the current disclosure, each R2 may be independently hydrogen, -NO2, -OCH3, -CH2CH2OCH3, -NHOCH3, -CF3, -NH2, bromo, chloro, fluoro, iodo,

[0168] In certain aspects, the compound disclosed herein is a compound of Formula 1 , wherein (1) X is not hydrogen, chloro, -NO2, or bromo when A is -O-, B is CH2 and R is hydrogen and (2) X is not bromine when A is NR1 and R1 is methyl

[0169] In certain aspects, the compound of Formula I is not a compound selected from:

[0170] In various aspects, compound of Formula I is selected from the group consisting of. any pharmaceutically appropriate salt thereof. [0171] In further aspects, the compound provided herein may be selected from the group [0172] As described in more detail in the Examples below, it was surprisingly found that some pharmaceutically appropriate salt versions of the compounds of Formula I showed improved properties (e.g., improved ability to inhibit Eph/Ephrin tetramerization) compared to their non-salt (e.g., free base) forms. Accordingly, in some aspects, the compound of Formula I is a pharmaceutically appropriate salt (e.g., an HCI salt).

[0173] In some aspects, the pharmaceutically appropriate salt may be selected from the group consisting of: [0174] In further aspects, the pharmaceutical salts may be selected from the group

[0175] For ease of reference, suitable compounds encompassed by Formula I as well as related compounds used in the Examples below, are provided in the following Table 1.

Table 1 :

[0176] As noted, any compound of Formula I described herein may inhibit EPH-EPHRIN tetramerization. As discussed further in the Examples, there are 14 different Eph receptors and 8 different Ephrin ligands in two classes (A and B). In some aspects, the compounds herein may be a general inhibitor of EPH-EPHRIN tetramerization - resulting in inhibition of any combination of Eph/Ephrin. In some aspects, the compounds may specifically inhibit a class of Eph/Ephrin (e.g., EphB/EphrinB). In some aspects, the compounds may specifically inhibit (e.g., preferentially inhibit) tetramerization of one or more of EphB1 , EphB2 or EphB4 to their respective Ephrins. In some aspects, the compound may specifically inhibit tetramerization of EphB1/Ephrin and/or EphB2/Ephrin over EphB4/Ephrin tetramerization. In some aspects, the compound may specifically inhibit tetramerization of EphB1/Ephrin over EphB2/Ephrin and/or EphB4/Ephrin tetramerization. In some aspects, the compound may specifically inhibit tetramerization of EphB2/Ephrin over EphB1/Ephrin and/or EphB4/Ephrin tetramerization.

[0177] As used herein, the term “specifically inhibit” does not necessarily mean that a reference compound has no ability to inhibit tetramerization of a non-preferred Eph/Ephrin combination. As shown in more detail in the illustrative examples below, many of the strong tetramerization inhibitors disclosed herein (e.g., A20-I or BQPB4) generally show about 60% ability to inhibit/antagonize EphB2-EphrinB2 binding, about 30% to inhibit EphB1-EphrinB2 binding and about 10% to inhibit EphB4-EphrinB2 binding during the association step (e.g., have a 60:30:10 ratio of inhibiting EphB2:EphB1 :EphB4 binding to EphrinB2). However, some compounds, especially those considered “specific inhibitors” herein, show a preferential ability to inhibit, for example, EphB2 or EphB1 over EphB4 tetramerization. For example, as shown in the Examples below, compounds A19-NO2 and QTM-NO2 remain potent inhibitors of EphB1-EphrinB2 and EphB2-EphrinB2 binding but show very little, if any, inhibition of EphB4-EphrinB2 binding. These compounds, for example, show a 40:25:0 ratio of inhibiting EphB2:EphB1 :EphB4 binding to EphrinB2, indicating that they have a greatly reduced activity toward the EphB4-EphrinB2 interaction. Therefore, as used herein, when described herein, compounds that specifically inhibit tetramerization of EphB1-Ephrin and/or EphB2-Ephrin over EphB4-Ephrin may, in some aspects, show less than 10% inhibition of binding to EphB4. In some aspects, the compounds may have no detectable inhibition of EphB4.

[0178] In some aspects, a compound provided herein that specifically inhibits EphB1/Ephrin and/or EphB2/Ephrin tetramerization can be selected from the group consisting of: pharmaceutically appropriate salt thereof. In various aspects, these compounds that specifically inhibit EphB1-Ephrin and/or EphB2-Ephrin tetramerization do not inhibit (or do not significantly inhibit) EphB4-Ephrin tetramerization.

[0179] In some aspects, a compound provided herein that specifically inhibits EphB1-Ephrin tetramerization can be selected from the group consisting of: or a pharmaceutically appropriate salt thereof. In various aspects, these compounds that specifically inhibit EphB1- Ephrin tetramerization do not inhibit (or do not significantly inhibit) EphB4-Ephrin tetramerization. In various aspects, these compounds that specifically inhibit EphB1-Ephrin tetramerization do not inhibit (or do not significantly inhibit) EphB2-Ephrin tetramerization. In various aspects, these compounds that specifically inhibit EphB1-Ephrin tetramerization do not inhibit (or do not significantly inhibit) EphB4-Ephrin and EphB2-Ephrin tetramerization.

[0180] In some aspects, a compound provided herein that specifically inhibits EphB2-Ephrin tetramerization can be selected from the group consisting of: , or a pharmaceutically appropriate salt thereof. In various aspects, these compounds that specifically inhibit EphB2-Ephrin tetramerization do not inhibit (or do not significantly inhibit) EphB4-Ephrin tetramerization. In various aspects, these compounds that specifically inhibit EphB2-Ephrin tetramerization do not inhibit (or do not significantly inhibit) EphB1-Ephrin tetramerization. In various aspects, these compounds that specifically inhibit EphB2-Ephrin tetramerization do not inhibit (or do not significantly inhibit) EphB4-Ephrin and EphB1-Ephrin tetramerization.

[0181] In any of the foregoing aspects, the compound may have an IC50 of less than 2 pM, less than 1.6 pM, less than 1 pM or less than 0.5 pM. In some aspects, the compound may have an IC50 greater than about 1.6 pM. In other aspects, the compound may have an IC50 of about 1.0 to 1.6 pM. In still other aspects, the compound may have an IC50 of about 0.4 to about 1.0 pM. In still other aspects, the IC50 may be less than 0.4 pM. IC50 and EPH- EPHRIN inhibition may be measured according to known methods in the art, including those described in the Examples herein.

III. Pharmaceutical Formulations and Treatment Regimens

[0182] The compounds disclosed herein for use according to the methods herein described may be provided per se or as part of a pharmaceutical composition, where the compounds can be mixed with suitable carriers or excipients.

[0183] As used herein a “pharmaceutical composition” refers to a preparation of one or more of the 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.

[0184] Herein the term “active ingredient” refers to a compound, such as those described herein, that inhibits EPH-EPHRIN tetramerization.

Pharmaceutically acceptable carriers and excipients

[0185] Hereinafter, the phrases “physiologically acceptable carrier” and “pharmaceutically acceptable carrier” which may be interchangeably used refer to a carrier or a diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered compound. An adjuvant is included under these phrases. [0186] In various embodiments, compositions disclosed herein may further compromise one or more pharmaceutically acceptable diluent(s), excipient(s), or carrier(s). As used herein, a pharmaceutically acceptable diluent, excipient, or carrier, refers to a material suitable for administration to a subject without causing undesirable biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained. Pharmaceutically acceptable diluents, carriers, and excipients can include, but are not limited to, physiological saline, Ringer’s solution, phosphate solution or buffer, buffered saline, and other carriers known in the art. Pharmaceutical compositions may also include stabilizers, anti- oxidants, colorants, other medicinal or pharmaceutical agents, carriers, adjuvants, preserving agents, stabilizing agents, wetting agents, emulsifying agents, solution promoters, salts, solubilizers, antifoaming agents, antioxidants, dispersing agents, surfactants, and combinations thereof. Herein the term “excipient” refers to an inert substance added to a pharmaceutical composition to further facilitate administration of an active ingredient. Examples, without limitation, of excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols. Techniques for formulation and administration of drugs may be found in “Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa., latest edition, which is incorporated herein by reference.

[0187] In various embodiments, pharmaceutical compositions described herein may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries to facilitate processing of genetically modified endothelial progenitor cells into preparations which can be used pharmaceutically. In other embodiments, any of the well-known techniques, carriers, and excipients may be used as suitable and as understood in the art.

[0188] In various embodiments, pharmaceutical compositions described herein may be an aqueous suspension comprising one or more polymers as suspending agents. In some aspects, polymers that may comprise pharmaceutical compositions described herein include: water- soluble polymers such as cellulosic polymers, e.g., hydroxypropyl methylcellulose; water- insoluble polymers such as cross-linked carboxyl-containing polymers; mucoadhesive polymers, selected from, for example, carboxymethylcellulose, carbomer (acrylic acid polymer), poly(methylmethacrylate), polyacrylamide, polycarbophil, acrylic acid/butyl acrylate copolymer, sodium alginate, and dextran; or a combination thereof. In other aspects, compositions disclosed herein may comprise at least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% total amount of polymers as suspending agent(s) by total weight of the composition. [0189] In various embodiments, pharmaceutical compositions disclosed herein may comprise a viscous formulation. In some aspects, viscosity of the composition may be increased by the addition of one or more gelling or thickening agents. In other aspects, compositions disclosed herein may comprise one or more gelling or thickening agents in an amount to provide a sufficiently viscous formulation to remain on treated tissue. In still other aspects, compositions disclosed herein may comprise at least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% total amount of gelling or thickening agent(s) by total weight of the composition. In yet other aspects, suitable thickening agents can be hydroxypropyl methylcellulose, hydroxyethyl cellulose, polyvinylpyrrolidone, carboxymethyl cellulose, polyvinyl alcohol, sodium chondroitin sulfate, sodium hyaluronate. In other aspects, viscosity enhancing agents can be acacia (gum arabic), agar, aluminum magnesium silicate, sodium alginate, sodium stearate, bladderwrack, bentonite, carbomer, carrageenan, Carbopol, xanthan, cellulose, microcrystalline cellulose (MCC), ceratonia, chitin, carboxymethylated chitosan, chondrus, dextrose, furcellaran, gelatin, Ghatti gum, guar gum, hectorite, lactose, sucrose, maltodextrin, mannitol, sorbitol, honey, maize starch, wheat starch, rice starch, potato starch, gelatin, sterculia gum, xanthum gum, gum tragacanth, ethyl cellulose, ethylhydroxyethyl cellulose, ethylmethyl cellulose, methyl cellulose, hydroxyethyl cellulose, hydroxyethylmethyl cellulose, hydroxypropyl cellulose, poly(hydroxyethyl methacrylate), oxypolygelatin, pectin, polygeline, povidone, propylene carbonate, methyl vinyl ether/maleic anhydride copolymer (PVM/MA), poly(methoxyethyl methacrylate), poly(methoxyethoxyethyl methacrylate), hydroxypropyl cellulose, hydroxypropylmethyl-cellulose (HPMC), sodium carboxymethylcellulose (CMC), silicon dioxide, polyvinylpyrrolidone (PVP: povidone), Splenda® (dextrose, maltodextrin and sucralose), or combinations thereof. In some embodiments, suitable thickening agent may be carboxymethylcellulose.

[0190] In various embodiments, pharmaceutical compositions disclosed herein may comprise additional agents or additives selected from a group including surface-active agents, detergents, solvents, acidifying agents, alkalizing agents, buffering agents, tonicity modifying agents, ionic additives effective to increase the ionic strength of the solution, antimicrobial agents, antibiotic agents, antifungal agents, antioxidants, preservatives, electrolytes, antifoaming agents, oils, stabilizers, enhancing agents, and the like. In some aspects, pharmaceutical compositions disclosed herein may comprise at least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% total amount of one or more agents by total weight of the composition. In other aspects, one or more of these agents may be added to improve the performance, efficacy, safety, shelf-life and/or other property of the muscarinic antagonist composition of the present disclosure. In s aspects, additives will be biocompatible, and will not be harsh, abrasive, or allergenic.

[0191] In various embodiments, pharmaceutical compositions disclosed herein may comprise one or more acidifying agents. As used herein, “acidifying agents” refers to compounds used to provide an acidic medium. Such compounds include, by way of example and without limitation, acetic acid, amino acid, citric acid, fumaric acid and other alpha hydroxy acids, such as hydrochloric acid, ascorbic acid, and nitric acid and others known to those of ordinary skill in the art. In some aspects, any pharmaceutically acceptable organic or inorganic acid may be used. In other aspects, compositions disclosed herein may comprise at least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% total amount of one or more acidifying agents by total weight of the composition.

[0192] In various embodiments, pharmaceutical compositions disclosed herein may comprise one or more alkalizing agents. As used herein, “alkalizing agents” are compounds used to provide alkaline medium. Such compounds include, by way of example and without limitation, ammonia solution, ammonium carbonate, diethanolamine, monoethanolamine, potassium hydroxide, sodium borate, sodium carbonate, sodium bicarbonate, sodium hydroxide, triethanolamine, and trolamine and others known to those of ordinary skill in the art. In some aspects, any pharmaceutically acceptable organic or inorganic base can be used. In other aspects, compositions disclosed herein may comprise at least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% total amount of one or more alkalizing agents by total weight of the composition.

[0193] In various embodiments, pharmaceutical compositions disclosed herein may comprise one or more antioxidants. As used herein, “antioxidants” are agents that inhibit oxidation and thus can be used to prevent the deterioration of preparations by the oxidative process. Such compounds include, by way of example and without limitation, ascorbic acid, ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, hypophophorous acid, monothioglycerol, propyl gallate, sodium ascorbate, sodium bisulfite, sodium formaldehyde sulfoxylate and sodium metabisulfite and other materials known to one of ordinary skill in the art. In some aspects, compositions disclosed herein may comprise at least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% total amount of one or more antioxidants by total weight of the composition.

[0194] In other embodiments, pharmaceutical compositions disclosed herein may comprise a buffer system. As used herein, a “buffer system” is a composition comprised of one or more buffering agents wherein “buffering agents” are compounds used to resist change in pH upon dilution or addition of acid or alkali. Buffering agents include, by way of example and without limitation, potassium metaphosphate, potassium phosphate, monobasic sodium acetate and sodium citrate anhydrous and dihydrate and other materials known to one of ordinary skill in the art. In some aspects, any pharmaceutically acceptable organic or inorganic buffer can be used. In another aspect, compositions disclosed herein may comprise at least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% total amount of one or more buffering agents by total weight of the composition. In other aspects, the amount of one or more buffering agents may depend on the desired pH level of a composition. In some embodiments, pharmaceutical compositions disclosed herein may have a pH of about 6 to about 9. In other embodiments, 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 a preferred embodiment, compositions disclosed herein may have a pH greater than about 6.8.

[0195] In various embodiments, pharmaceutical compositions disclosed herein may comprise one or more preservatives. As used herein, “preservatives” refers to agents or combination of agents that inhibits, reduces, or eliminates bacterial growth in a pharmaceutical dosage form. Non-limiting examples of preservatives include Nipagin, Nipasol, isopropyl alcohol and a combination thereof. In some aspects, any pharmaceutically acceptable preservative can be used. In other aspects, pharmaceutical compositions disclosed herein may comprise at least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% total amount of one or more preservatives by total weight of the composition.

[0196] In other embodiments, pharmaceutical compositions disclosed herein may comprise one or more surface-acting reagents or detergents. In some aspects, surface-acting reagents or detergents may be synthetic, natural, or semi-synthetic. In other aspects, compositions disclosed herein may comprise anionic detergents, cationic detergents, zwitterionic detergents, ampholytic detergents, amphoteric detergents, nonionic detergents having a steroid skeleton, or a combination thereof. In still other aspects, pharmaceutical compositions disclosed herein may comprise at least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% total amount of one or more surface-acting reagents or detergents by total weight of the composition.

[0197] In various embodiments, pharmaceutical compositions disclosed herein may comprise one or more stabilizers. As used herein, a “stabilizer” refers to a compound used to stabilize an active agent against physical, chemical, or biochemical process that would otherwise reduce the therapeutic activity of the agent. Suitable stabilizers include, by way of example and without limitation, succinic anhydride, albumin, sialic acid, creatinine, glycine and other amino acids, niacinamide, sodium acetyltryptophonate, zinc oxide, sucrose, glucose, lactose, sorbitol, mannitol, glycerol, polyethylene glycols, sodium caprylate and sodium saccharin and others known to those of ordinary skill in the art. In some aspects, pharmaceutical compositions disclosed herein may comprise at least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% total amount of one or more stabilizers by total weight of the composition.

[0198] In other embodiments, pharmaceutical compositions disclosed herein may comprise one or more tonicity agents. As used herein, a “tonicity agents” refers to a compound that can be used to adjust the tonicity of the 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 or ordinary skill in the art. Osmolarity in a composition may be expressed in milliosmoles per liter (mOsm/L). Osmolarity may be measured using methods commonly known in the art. In preferred embodiments, a vapor pressure depression method is used to calculate the osmolarity of the compositions disclosed herein. In some aspects, the amount of one or more tonicity agents comprising a pharmaceutical composition disclosed herein may result in a composition osmolarity 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 aspects, a composition 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, a pharmaceutical composition described herein has an osmolarity 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 aspects, pharmaceutical compositions disclosed herein may comprise at least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% total amount of one or more tonicity modifiers by total weight of the composition.

Dosage formulations

[0199] Suitable routes of administration may, for example, include oral, rectal, transmucosal, especially trans-nasal, intestinal or parenteral delivery, including intramuscular, subcutaneous and intramedullary injections as well as, intravenous, intraperitoneal, intranasal injections, intraocular (e.g., via eye drops) or topical (e.g., creams or ointments). [0200] One may administer the pharmaceutical composition in a local or systemic manner, for example, via local injection of the pharmaceutical composition directly into a tissue region of a patient. In some embodiments, a pharmaceutical composition disclosed herein can be administered parenterally, e.g., by intravenous injection, intracerebroventricular injection, intra- cisterna magna injection, intra-parenchymal injection, or a combination thereof. In some embodiments, a pharmaceutical composition disclosed herein can administered to the human patient via at least two administration routes. In some examples, the combination of administration routes by be intracerebroventricular injection and intravenous injection; intrathecal injection and intravenous injection; intra-cisterna magna injection and intravenous injection; and intra-parenchymal injection and intravenous injection.

[0201] Pharmaceutical compositions of the present disclosure may be manufactured by processes well known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.

[0202] Pharmaceutical compositions for use in accordance with the present disclosure thus may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active ingredients into preparations which, can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.

[0203] 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 salt buffer.

[0204] The pharmaceutical composition described herein may be formulated for parenteral administration, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multidose containers with optionally, an added preservative. The compositions may be suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.

[0205] Pharmaceutical compositions for parenteral administration include aqueous solutions of the active preparation in water-soluble form. Additionally, suspensions of the active ingredients may be prepared as appropriate oily or water-based injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acids esters such as ethyl oleate, triglycerides or liposomes. Aqueous injection suspensions may contain substances, which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the active ingredients to allow for the preparation of highly concentrated solutions.

[0206] Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water-based solution, before use.

[0207] Pharmaceutical compositions suitable for use in context of the present disclosure include compositions wherein the active ingredients are contained in an amount effective to achieve the intended purpose. In some embodiments, a therapeutically effective amount means an amount of active ingredients (i.e., a compound disclosed herein) effective to prevent, slow, alleviate or ameliorate symptoms of a disorder (e.g., a pain disorder, a psychiatric disorder, or other neurological/cognitive disorder brought about by disrupted synaptic activity) or prolong the survival of the subject being treated.

[0208] Determination of a therapeutically effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.

[0209] For any preparation used in the methods of the present disclosure, the therapeutically effective amount or dose can be estimated initially from in vitro and cell culture assays and or screening platforms disclosed herein. For example, a dose can be formulated in animal models to achieve a desired concentration or titer. Such information can be used to more accurately determine useful doses in humans.

[0210] Toxicity and therapeutic efficacy of the active ingredients described herein can be determined by standard pharmaceutical procedures in vitro, in cell cultures or experimental animals. The data obtained from these in vitro and cell culture assays and animal studies can be used in formulating a range of dosage for use in human. The dosage may vary depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. (See e.g., Fingl, et al., 1975, in “The Pharmacological Basis of Therapeutics”, Ch. 1 p. 1).

[0211] Dosage amount and interval may be adjusted individually to brain or blood levels of the active ingredient are sufficient to induce or suppress the biological effect (minimal effective concentration, MEC). The MEC will vary for each preparation, but can be estimated from in vitro data. Dosages necessary to achieve the MEC will depend on individual characteristics and route of administration. Detection assays can be used to determine plasma concentrations.

[0212] Depending on the severity and responsiveness of the condition to be treated, dosing can be of a single or a plurality of administrations, with course of treatment lasting from several days to several weeks or until cure is effected or diminution of the disease state is achieved. The amount of a composition to be 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- responsive curves derived from in vitro or in vivo test systems.

IV. Methods of Treatment

[0213] Provided herein are methods for inhibiting formation of an EPH-EPHRIN tetramer, the method comprising contacting an EPH or EPHRIN with a compound of Formula I provided herein, or a compound selected from:

[0214] In some aspects, the EPH-EPHRIN tetramer comprises EPHB1. In some aspects, the EPH-EPHRIN tetramer comprises EPHB2. As discussed further herein below, EPH- EPHRIN signaling contributes to many physiological conditions. Accordingly, the present disclosure provides for methods of treating, attenuating, and preventing a condition related to EPH-EPHRIN signaling in a subject in need thereof. In various aspects, disrupted Eph- Ephrin signaling leads to, exacerbates or directly contributes to the condition to be treated. For example, Eph-Ephrin tetramerization contributes to pain signaling as well as synaptic plasticity (e.g., see FIG. 1 and FIG. 2). Accordingly, the present disclosure provides for methods of treating, attenuating, and preventing pain in a subject in need thereof. In certain aspects, the pain may be chronic, neuropathic pain. In other aspects, the present disclosure provides for methods of treating, attenuating, and preventing a synaptopathy in a subject in need thereof. In some aspects, the synaptopathy is caused by disrupted NMDA receptor signaling. In various aspects, the condition may be anxiety or epilepsy. In still further aspects, a method for treating, attenuating, and/or preventing an addiction (e.g., an opioid addiction) in a subject in need thereof is provided.

[0215] In further aspects, methods of treating, attenuating, and preventing a condition selected from the group consisting of pain (e.g., chronic, neuropathic pain), addiction and dependency (e.g., opioid addiction and dependency), neurological disorders (e.g., anxiety, epilepsy, seizures), fibrotic and inflammatory diseases (e.g., NASH liver fibrosis, chronic kidney disease, scleroderma (skin fibrosis), fibrosis (e.g., lung fibrosis or heart fibrosis), and abnormal wound healing (e.g., keloids and hypertrophic scarring)), metabolic disorders (e.g., diabetes, obesity), cancer (e.g GBM (glioblastoma), pancreatic cancer, colon cancer), viral infections (e.g. henipavirus and HIV infections) is provided. In any of the methods provided herein, the condition to be treated (e.g., a cancer, a fibrotic or inflammatory disease, a viral infection, a metabolic disorder) may comprise abnormal, defective or excessive EPH- EPHRIN signaling. That is, any of the methods herein may involve treating a condition caused or worsened by abnormal, defective, or excessive EPH-EPHRIN signaling.

[0216] In any of the above or foregoing therapeutic methods, an effective amount of one or more compounds disclosed herein (e.g., compounds of Formula I) or one or more of the following compounds: pharmaceutically acceptable salt thereof, may be administered to the subject. In some aspects, the compound may be administered in a pharmaceutical composition or formulation as described herein, alone or alongside another suitable therapy for the condition.

[0217] In various embodiments, a subject in need thereof can be having, suspected of having, or at risk of being in pain, or having a synaptic disorder. For example, the subject in need thereof can be having, suspected of having, or at risk of having neuropathic pain. For example, the subject in need thereof can be having, suspected of having, or at risk of having a synaptic disorder.

[0218] A suitable subject includes a human, a livestock animal, a companion animal, a lab animal, or a zoological animal. In one embodiment, the subject may be a rodent, e.g., a mouse, a rat, a 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, a “zoological animal” refers to an animal that may be found in a zoo. Such animals may include non-human primates, large cats, wolves, and bears. In a specific embodiment, the animal is a laboratory animal. Non-limiting examples of a laboratory animal may include rodents, canines, felines, 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 preferred embodiments, the subject is a human.

V. Kits

[0219] 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 in accordance with any of the methods described herein. The included instructions can comprise a description of administration of a composition containing a compound (e.g., EPH-EPHRIN tetramerization inhibitor such as a compound of Formula I) disclosed herein and to treat, delay the onset, or alleviate a target disease as those described herein. The kit may further include a description of selecting an individual suitable for treatment based on identifying whether that individual has the target disease. In still other embodiments, the instructions can include a description of administering a compound to an individual at risk of the target disease.

[0220] The instructions relating to the use of a composition containing a compound inhibiting EPH-EPHRIN tetramerization (e.g., a compound of Formula I) generally include information as to dosage, dosing schedule, and route of administration for the intended treatment. The containers may be unit doses, bulk packages (e.g., multi-dose packages) or sub-unit doses. Instructions supplied in the kits of the invention are typically written instructions on a label or package insert (e.g., a paper sheet included in the kit), but machine- readable instructions (e.g., instructions carried on a magnetic or optical storage disk) are also acceptable. [0221] The label or package insert indicates that the composition is used for treating, delaying the onset and/or alleviating the disease. Instructions may be provided for practicing any of the methods described herein.

[0222] The kits of this invention are in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags), and the like. Also contemplated are packages for use in combination with a specific device, such as an inhaler, nasal administration device (e.g., an atomizer) or an infusion device such as a minipump. A kit may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). The container may also have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). In some examples, at least one active agent in the composition can be a compound (e.g., a CDK8 inhibitor) as described herein.

[0223] Kits may optionally provide additional components such as buffers and interpretive information. Normally, the kit includes a container and a label or package insert(s) on or associated with the container. In some embodiments, the invention provides articles of manufacture comprising contents of the kits described above.

EXAMPLES

[0224] All patents and publications mentioned in the specification are indicative of the levels of those skilled in the art to which the present 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.

[0225] The publications discussed throughout are provided solely for their disclosure before the filing date of the present application. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.

[0226] 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 persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims.

[0227] Unless specified, reagents employed in the examples are commercially available or can be prepared using commercially available instrumentation, methods, or reagents known in the art. The examples illustrate various aspects of the invention and 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, although the invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, those of ordinary skill in the art will realize readily that many changes and modifications can be made thereto without departing from the spirit or scope of the appended claims.

Example 1 : Study design

[0228] The drug discovery efforts were directed toward the interaction of EphB1 receptor with its ligand, EphrinB2, mediated by extracellular segments of these two proteins. The focus was on ligand antagonists because a ligand antagonist (LA) compound would interfere with both EphB forward signaling and EphrinB reverse signaling and be in contrast to the more traditional approach of searching for a tyrosine kinase inhibitor (KI) that would only target the catalytic domain of the receptor (FIG. 1). A focus on KI might also lead to compounds with a greater possibility for off-target effects. This is because there are -500 different kinase domain-containing proteins encoded in the mouse and human genomes that share an evolutionary related amino acid sequence and three-dimensional structure, so most all Ki’s identified cross-react to inhibit other unintended kinase domains with potential for undesirable side effects. The LA compounds which exhibit greater specificity for on-target effects and less potential for off-target effects were selected because they will disrupt a protein-protein association involving ectodomain structures specific to the Eph receptors and Ephrin ligands (FIG. 2). It is therefore, concluded that a compound which can disrupt Eph- Ephrin binding to block both forward and reverse signaling might be the most effective way to target these molecules to treat chronic pain and many other diseases.

[0229] A robust and highly sensitive chemiluminescent Alpha assay was developed for use in 384 well plates that measures the protein-protein interaction formed between the ectodomains of the EphB1 receptor tyrosine kinase and one of its interacting ligands, EphrinB2 (EB2). This assay was used to screen the -240,000 complex chemical library available in the UT Southwestern Medical Center high-throughput screening (HTS) core facility and ultimately identified a single small molecular weight compound, termed A20 (SW056428)(7-Bromo-5-(4-morpholinylmethyl)-8-quinolinol), that acts to significantly reduce the EphB1-EB2 protein-protein interaction at low micromolar concentration using multiple biochemical measurements. It was proposed that A20 is a lead compound for a new class of chemicals that act as ligand antagonists (LA) to prevent Eph receptors from binding to their Ephrin ligands. It was further proposed that use of such LA compounds will be much a better way to pharmacologically target Eph-Ephrin in disease versus the classic/typical use of a kinase inhibitor (KI), because an LA compound will interfere with both forward and reverse signaling, whereas a KI will only target forward signaling (FIG. 1). Though, HTS and initial screening was focused on EphB1-EB2 interaction because of the specific role these molecules play in chronic pain, as described below the LA compounds can be used to target the whole family of 14 different Eph receptors and 8 different Ephrin ligands and could be utilized to treat a wide swath of potential unmet medical conditions.

[0230] A series of novel A20-related analogs (termed A20 chemicals) were designed and synthesized. This includes both free base and salt versions of compounds, the latter of which are much more aqueous soluble and preferable for biochemical analysis and for administration into animals. Ongoing biochemical and biophysical kinetic analysis of this growing collection of A20 chemicals conducted in the Henkemeyer laboratory is providing key IC50 values which informs structure activity relationship (SAR) information. Importantly, the disclosure includes the identification of various novel A20 analogs, (e.g., A20-I and others described below), which exhibits improved submicromolar IC50 activity.

[0231] Detailed biophysical analysis was conducted to provide crucial and valuable insight into the mechanism by which the A20 chemicals act to disrupt/inhibit/antagonize Eph-Ephrin receptor-ligand protein-protein interactions. To understand this, one needs to understand that the interactions of Eph receptors and Ephrin ligands is extremely complex, much more complex in fact than most any other receptor-ligand system. This is because: (i) there are 14 different Eph receptors and 8 different Ephrin ligands grouped into two classes, A and B, that show promiscuous interactions, (ii) the receptors and ligands are both membrane anchored and expressed on the cell surface, (iii) this means the ligands are not typically soluble and do not work at a distance like most all other ligand-based systems, so rather Ephrins physically interact with their cognate Ephs only upon cell-cell contact, (iv) because the Ephrins are also membrane-anchored and they too can function as receptors to transduce reverse signals, one needs to understand that the Eph is not really only a receptor it is also a ligand and the Ephrin is not just a ligand it is also a receptor, these proteins signal bidirectionally, and finally (v) upon interaction, the Ephs and Ephrins first form a dimer, and then two Eph-Ephrin dimers can assemble into a circular tetramer structure, and then circular tetramers can further aggregate into higher-order clusters through lateral migration in the plasma membrane. Formation of the circular tetramer is crucial as this generates the active signaling complex necessary for transduction of forward signals (into the Eph-expressing cell) and reverse signals (into the Ephrin-expressing cell).

[0232] Biophysical kinetic binding data was also examined to better visualize and describe these complex interactions and show that A20 chemicals act as reversible competitive inhibitors that specifically target the Eph-Ephrin circular tetramer. By selectively targeting the circular tetramer, the A20 compounds exhibit a unique mode of action that centers in on the key signaling complex formed by these molecules and helps explain the observed strong/robust biological activities of these compounds when tested on cells and in vivo in preclinical studies. In the absence of A20 chemical, biophysical kinetic analysis shows that an Eph molecule and Ephrin molecule first assemble during association step into a relatively weak Eph-Ephrin dimer with a very fast on-off rate (KD = -40 nM), and then with time and a somewhat slower on rate two Eph-Ephrin dimers assemble into a circular tetramer. The circular tetramer is a super stable structure with a very slow or non-existent off rate in the dissociation step depending on the interaction under study (KD = low-nanomolar to sub- picomolar). Key data is obtained when an active A20 chemical is added to the binding assay. A striking dose-dependent loss in formation of the tetramer structure is observed during the association step that results in a steep or complete loss of stable tetramer structure in the dissociation step. A20 chemicals remarkably have no effect on Eph-Ephrin dimer dynamics, even at very high concentrations. The data disclosed further shows A20 chemicals act as pan-Eph-Ephrin tetramerization inhibitors as they can disrupt A class, B class, and A-B crossclass tetramer interactions. The biophysical kinetic analysis of the protein interactions provides a important new insight into the diverse and complicated interactions all these Ephs and Ephrins make. While some interactions are tetramer-driven and are more targeted by the A20 chemicals (examples include EphB1-EB2, EphB2-EB2, EB2-EA5, and EphA3-EA1 interactions), other interactions are more dimer-driven and are less targeted (examples include EphB4-EB2 and EphA3-EA5 interactions). The above biophysical studies were set up to assess how a compound may affect Eph-Ephrin interactions during the association step and to obtain IC50 information. The biophysical data also show that A20 chemicals can also disrupt pre-existing stable Eph-Ephrin circular tetramer structures in a dose-dependent fashion. These results show that the disclosed compounds can act on pre-existing Eph- Ephrin signaling tetramers upon administration.

[0233] A pharmacokinetic (PK) study is disclosed using a formulation of highly aqueous soluble salt (HCI) forms of the compounds injected with PBS as vehicle into mice and is compared to results obtained using the free base form which needs to be solubilized in a DMSO/s unflower seed vehicle prior to injection. The results show that the A20.HCI salt form exhibits -50 times greater PK dynamics than the free base, distributing into the plasma, liver, and brain.

[0234] It has been found that the circular tetramer forms the active Eph-Ephrin signaling complex which leads to transduction of bidirectional signals into both the Eph-expressing cell and the Ephrin-expressing cell. Without the circular tetramer, signalling does not occur. The current disclosure describes a family of novel A20 chemicals that do not affect Eph-Ephrin dimerization dynamics, but rather selectively target the circular tetramer formed by all types of Eph-Ephrin interactions, those formed by A class receptors-ligands, B class receptors- ligands, as well as cross-class A-B hetero-tetramers. The A20 chemicals, described herein, target the circular tetramer structure essential for signal transduction by these molecules and appear to target all types of Eph-Ephrin tetramers at IC50 concentrations below 1 pM. These compounds thus serve as pan-tetramerization inhibitors of Eph-Ephrin bidirectional signaling and will have great potential as pharmaceutical agent to treat individuals suffering from numerous devastating and unmet conditions that involve overexpression and excessive signaling by these molecules.

Example 2: Screening of

[0235] A chemiluminescent AlphaScreenTM bead-based proximity assay suitable for HTS was developed to measure the receptor-ligand interaction formed between EphB1 and EphrinB2 (EB2) ectodomains (FIG. 3). Here, when singlet oxygen molecules generated by high energy irradiation of donor beads travel over a constrained distance to excite acceptor beads, a chemiluminescent signal is generated and detected. If an analyte interferes with the EphB1-EB2 protein-protein interaction, close proximity of the donor and acceptor beads will be disrupted resulting in diminished or lost energy transfer and reduced acceptor bead emission. It was hypothesized that an effective inhibitor compound could either dock to the EphB1 extracellular domain (shown as a red star in FIG. 1 , FIG. 2 and FIG. 3), the EB2 protein, or perhaps both, to disrupt the EphB1-EB2 interaction.

[0236] Protein interaction assays were developed using soluble EphB ectodomains conjugated to Fc from R&D Systems (e.g., rat EphB1-Fc residues 18-538, which is 99.4% identical to human) and EphrinB ectodomains conjugated to His from Sino Biologicals (e.g., mouse EB2-His residues 18-232, which is 97.7% identical to human). Titrating concentrations of the EphB1-Fc ectodomain (or unconjugated Fc protein as negative control) was added with titrating amounts of the three different EphrinB ectodomains (EB1-His, EB2- His, or EB3-His) with 125 ng each of the Protein A donor, anti-6x-His acceptor beads, 2% DMSO, and 1X HiBlock buffer (all from PerkinElmer) in 384 well plates, and after 3 hr incubation at RT were read in an Envision Multilabel Reader 2102. Consistent with the expected high-affinity EphB1-EB interactions being able to bring the donor and acceptor beads into close proximity, very high chemiluminescent signals were observed even at 3.2 nM of each protein (>600,000) that were concentration-dependent and strong even as low as 0.4 nM (-100,000). No signal above background (<1 ,500) was detected for wells that contained an Fc control protein and the EB-His proteins or just the EphB1-Fc protein and no EB-His protein.

[0237] The Alpha assay was then tested in competition experiments using 12-mer peptides designated SNEW and EWLS previously identified to bind into the dimerization pocket formed on the surface of the EphB1/EphB2 receptors and antagonize their ability to bind EphrinB ligands (see Koolpe M, Burgess R, Dail 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 the EphB-EB interaction in vitro, they are not useful in vivo as their half-life is only a few minutes. Both SNEW and EWLS antagonized the EphB1-EB2 interaction in the Alpha assay in a concentration-dependent manner with observed IC50 of -0.8 pM (FIG. 4A). Test was conducted in the Alpha assay, to see if purified soluble Reelin (previously shown to bind EphB1 ectodomain) can interfere with EphB1-EB2 binding. A a strong dose-dependent reduction in signal was observed with an IC50 of -50 nM and almost complete inhibition at 200 nM (FIG. 4B).

[0238] The EphB1-EB2 screen was conducted using 0.8 nM of each protein, 50 ng beads per well, and assessed at 5 pM a library of -240,000 small, drug-like chemicals. An example of a plate run with a potential hit well circled in green is shown (FIG. 40). The average Z’ for the 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 the signal >10% were identified. 887 of the 1 ,055 compounds were selected from master plates, avoiding ones with “pan-assay interference” (PAINS) properties that tend to nonspecifically react with numerous biological targets and are often false positives. The 887 compounds were retested in triplicate in the EphB1-EB2 Alpha assay. They were also tested in a counter screen designed to identify nonspecific compounds. Here, a single-protein Alpha assay was set up using a dual-tagged EphB2-His-Fc ectodomain protein that can bind both donor and acceptor beads to bring them into proximity. If a selected compound simply absorbs the chemiluminescent signal or interferes with bead function/binding or protein folding, it was hypothesized that it would also reduce signals in the single protein assay, and would consider it a false positive. Together, the Alpha confirmation and counter screens allowed us to eliminate a large number of the hits, leaving 32 compounds to advance forward that showed repeated ability to reduce the EphB1-EB2 Alpha assay chemiluminescent signal >10% and had no effect on signal in the single-protein assay.

[0239] The 32 hits were then subjected to ELISA in which EphB1-Fc was first immobilized in 96 well Protein A plates, then soluble EB2-His was added with hit compound in triplicate, again at 5 pM. After 2 hr at RT to allow EB2-His binding to EphB1-Fc, wells were washed, 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 in a luminometer. A single-protein counter ELISA using the dual-tagged EphB2-His-Fc ectodomain protein was also run. This allowed us to quickly narrow down to one compound from well 268 G15 (SW056428) that reduced signal in the two-protein ELISA but had no effect on signal in the single-protein ELISA. For simplicity, we call this compound A20. Another compound, termed E13, was also noted to possibly reduce signal in the ELISA (not shown). [0240] A20, E13, and other compounds from the top 32 hits were then characterized in pulldown assays. EphB1-Fc was immobilized onto Protein A agarose beads, then mixed with compounds and the soluble EB2-His ectodomain for 1 hr at RT, after which protein complexes were washed and then bound EB2 was detected by immunoblot using anti-His antibodies. A20 (50 and 250 pM) strongly reduced EB2-His binding to EphB1-Fc as shown in FIG. 5A with red asterisks), but other compounds were inactive, including E13. Studies with other EphB receptors showed that A20 exhibited only a weak ability to prevent EphB4- Fc from binding to EB2-His in the pulldown even at 250 pM as shown in FIG. 5B with red asterisks, though it did show a strong dose-dependent ability to antagonize EphB2-His-Fc protein binding to EB2-His as shown in FIG. 5C with red asterisk, results of which were confirmed by ELISA and Alpha assays (FIG. 5D).

[0241] In summary, A20 was found to exhibit potent ability to antagonize EphrinB2 ligand binding to the two closely related neural-expressed EphB1 and EphB2 sister receptors both with IC50 -10 pM and was only weakly able to interfere with the ability of EphrinB2 to bind the more distant and cardiovascular specific EphB4 counterpart with IC50 >150 pM. The chemical structure of A20 is formed by the joining of morpholine and 7-bromo-8- hydroxyquinoline rings as shown in FIG. 6.

[0242] Two other compounds present in the UT Southwestern Medical Center library, SW022101 and SW022102, exhibited similarity to A20 (FIG. 7). While SW022102 was not identified in the HTS, SW022101 was one of the initial 887 selected primary hits, though it did not pass through the Alpha confirmation and counter screens. This early SAR information indicates the morpholine ring of A20 is important for the ability of this compound to inhibit the EphB1-EB2 protein interaction. In addition to A20, SW022101 , and SW022102, five other similar analogs not in the library were identified as being commercially available (FIG. 7). These compounds were obtained and subjected to Alpha, ELISA, and pulldown experiments. The results indicate compounds 3511 and 8009 may be as effective as A20 at antagonizing EphB1-EB2 and EphB2-EB2 interactions, with very little if any effect on the EphB4-EB2 interaction. The compounds D014, F235, and 8002 did not exhibit any activity in our assays (FIG. 8). Additional experiments indicate that A20 can also antagonize EphB1/EphB2 proteins from binding to EphrinBI (EB1) and EphrinB3 (EB3) proteins.

Example 3: Biophysical studies of compound A20

[0243] The experiments described above involve measurements following long-term incubation of proteins with compound and thus represent information after interaction homeostasis has been achieved (FIG. 5A, FIG. 5B, FIG. 5C, and FIG. 5D) or after complexes are extensively washed (ELISAs and pulldowns, FIG. 8). To further analyze biophysical activity of A20 in real-time, kinetic studies were performed using an Octet® RED384 system to visualize the effect of compound on EphB1-EB2 protein-protein binding dynamics. In these experiments, EphB1-Fc ectodomain protein was first sparsely immobilized onto a set of Octet biosensors, which after baseline measurements were then exposed to 30 nM soluble EB2- His protein that also contained 0, 1 , 10, or 100 pM A20 for a 400 sec association step followed by exposure to buffer only for a 500 sec dissociation step. In the absence of A20 compound (0 pM), soluble EB2-His ectodomain showed the expected high affinity interaction of rapid binding to biosensor immobilized EphB1-Fc in the association step followed by an off phase in the dissociation step with a response value that reached a steady-state plateau/flatline significantly above the baseline level, indicative of very strong and stable binding of EB2-His remaining at the end of the run as shown in FIG. 9A with red trace). The sensorgram provides various data of the binding kinetics, such as on rates (kon), off rates (koff), and equilibrium dissociation constant (KD). As expected, software curve fitting of the Octet data showed the EphB1-EB2 interaction was not a classic 1 :1 dimer binding interaction, but more complex/heterologous (2:1) as these molecules can form dimers, circular tetramers, and higher order clusters (FIG. 10). Eph and Ephrin ectodomains exhibit complex receptor-ligand binding interactions. Specifically, EphB2 forms a dimer with EphrinB2, and that two dimers then assemble together into a circular tetramer structure (see FIG. 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).

[0244] The distinct dimerization and tetramerization interfaces are indicated. Circular tetramers further cluster together to pack in the crystal via additional ligand-ligand (red arrow) and receptor-receptor (blue arrow) interfaces. Many studies by multiple groups show the circular tetramer and tetramer clusters are observed in vivo and that they are needed in order to form the active complex necessary for the transduction of Eph-Ephrin bidirectional signals. Calculations of KD for the interaction showed a very high affinity interaction of approximately 2 nM (with 1 :1 calculations) or 0.5 nM (with 2:1 calculations). Addition of A20 resulted in a greatly blunted, dose-dependent reduction in binding of EB2-His to biosensor immobilized EphB1-Fc during the association step, which was obvious even at 1 pM and with very little binding during association step at the 100 pM concentration (FIG. 9A). A variety of methods can be used to compare the different sensorgrams obtained from a dose-dependent run of a drug compound to quantify its LA activity and obtain IC50 information. Single time points in the assay can be picked and response signals obtained at the different compound doses can be compared, such as at 380 sec near the end of the association step or at 100 sec after start of the dissociation step. The entire sensorgram traces was taken into account for analyzing areas under the curve (AUG), which for EphB1-EB2 interaction resulted in IC50 of 9.33 pM, consistent to that calculated using Alpha, ELISA, or pulldown experiments. [0245] Biophysical analysis using immobilized EphB4-Fc protein in Octet system also showed a very high affinity interaction with EB2 in the absence of compound, however addition of A20 resulted in only a mild effect on formation of the EphB4-EB2 protein-protein interaction, with an area under the curve IC50 of >100 pM (FIG. 9B). This is consistent with the results from Alpha, ELISA, and pulldown studies that showed A20 exhibited only weak ability to disrupt the EphB4-EB2 interaction.

[0246] In summary, the disclosed new biophysical kinetic analysis is a powerful addition to methods to assess EphB receptors interacting with their EphrinB ligands. Further, the results indicate that A20 and the new A20 analog chemicals disclosed herein effectively target EphB1-EphrinB and EphB2-EphrinB binding but show little effect on EphB4-EphrinB2 binding. Further experiments were conducted using Octet® RED384 to understand the mechanism of action of the A20 chemicals.

Example 4: Compound A20 is a reversible, competitive antagonist of Eph-Ephrin receptor-ligand binding

[0247] Antagonists are classified as either competitive/reversible or irreversible, depending on how the particular inhibitor interacts with its protein targets. A reversible antagonist will bind receptor protein via non-covalent intermolecular forces, and it will eventually dissociate from the receptor, freeing the protein to bind either another antagonist chemical in the solution or its bona-fide ligand protein. Irreversible antagonists on the other hand bind via covalent forces to form a permanent receptor-antagonist complex that does not dissociate. A competitive antagonist is thought to bind to receptor protein at the same binding site/interface as the endogenous ligand (aka Ephrin), but without activating the receptor. Ligand and antagonist will thus compete for the same binding site on the receptor; when antagonist is bound to receptor it will prevent ligand binding and increasing concentrations of an antagonist will displace in a dose-dependent fashion the ability of ligand from binding the receptor. Since, most receptor antagonists participate in a reversible, non-covalent, competitive type of interaction, two Octet experiments were conducted to determine if this was indeed the mode of action of A20. To determine if A20 is a reversible inhibitor/antagonist, rEphB1-Fc was first immobilized onto sensor chips, then pre-exposed to 100 pM A20, then washed to remove A20, and after baseline measurements then tested for binding to 30 nM soluble mEB2-His protein. The results showed pretreatment with A20 did not affect subsequent ability of immobilized EphB1 protein from binding soluble EB2 in the association step, indicating compound acts as a reversible inhibitor (FIG. 11). Specifically, immobilized rEphB1-Fc protein pre-exposed to 100 pM A20 did not affect its subsequent ability to bind 30 nM soluble EB2 (red sensorgram) and interacted with similar kinetics as the sample not pre-exposed to the compound (green sensorgram). Addition of A20 during the association step did compete with EB2 binding to the immobilized EphB1 protein (purple sensorgram). Binding assays were conducted in PBS containing 0.05% Tween-20, association time was for 400 sec and dissociation time was for 500 sec. To determine if A20 is a competitive inhibitor, increasing concentrations of A20 (0, 10, 100 pM) was 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 in the association step. The data plotted in FIG. 12A- 12C groups the sensorgram results by the amount of A20 included in the binding reactions (0, 10, 100 pM), whereas the plots in FIG. 12D-12F groups them by the concentration of EB2 in the binding reaction. The data showed that increasing concentrations of A20 reduced in a dose-dependent fashion the ability of EB2 to bind EphB1 , indicating compound is a competitive inhibitor of EphB1-EB2 binding. As shown in the FIG. 12A-12C, in the absence of the compound, the super high concentration of soluble EB2 protein leads to a rapid and saturated binding response to immobilize EphB1 within seconds because of high amounts of available ligand protein. Addition of A20 to the association step strongly blunted this saturated binding response, which was especially displayed in the dissociation part of the curves where compound dramatically increased the speed of the off rate (koff) during the first 100 seconds of dissociation, resulting in a greatly reduced level of EB2 bound at the end of the run. The data shows that, as little as 10x more A20 (10 pM) in the association step can compete away some of the saturating 1 pM of the high affinity EB2 ligand from binding to EphB1 , and that 100x compound (100 pM) almost totally eliminates the super stable EphB1-EB2 tetramer at the end of the run (purple sensorgram). Additionally, at saturating 1 pM concentrations of EB2, addition of A20 had a clear effect on stable tetramers in the dissociation step, while A20 had absolutely no effect on the rapid initial binding of EB2 to the immobilized EphB1 that takes place within seconds at the beginning of the association step (kon).

[0248] These results provide insights into how A20 chemicals disrupt the Eph-Ephrin interaction, and that they do not target formation of the Eph-Ephrin dimer, but rather they specifically target formation and/or stability of the Eph-Ephrin tetramer which is extremely stable and long-lasting macromolecular complex which activates the bidirectional signaling.

Example 5: Cell-based studies of compound A20

[0249] To assess if A20 would reduce the ability of EphrinB2 to stimulate activation of the EphB tyrosine kinase domain in live cells, Cos1 cells that express endogenous EphB2 were exposed to pre-clustered EB2-Fc ectodomains in the presence or absence of A20 for 30 min. Protein lysates were immunoblotted with rabbit anti-phospho-EphB specific antibodies, showing A20 caused a dose-dependent reduction in EphB2 kinase activity (FIG. 13). Stimulated cells were fixed on the coverslip and subjected to immunofluorescence (IF) with anti-Fc antibodies to detect ligand protein bound to receptor, which leads to formation of circular Eph-Ephrin tetramers that then cluster into large obvious spots on the cell surface by lateral migration through the plasma membrane and also contain anti-phosphotyrosine signal indicating activation of the receptor’s kinase domain. Results showed that A20 significantly reduced the ability of pre-clustered EB2-Fc to bind/cluster the EphB2 receptor to spots on the cell and to activate its tyrosine kinase domain (FIG. 14). Additionally, the cellbased analysis showed that the Cos1 cells exposed to EB2-Fc flattened out on the dish as a typical response to stimulation of EphB2 forward signaling, whereas the cells exposed to both EB2-Fc and A20 showed much less flattening and generally appeared more like the unstimulated control cells. These results indicates that A20 is able to impact cellular responses induced by EphB-EphrinB interaction and signaling.

Example 6: Cytotoxicity, metabolic stability, and an early pharmacokinetic (PK) study of compound A20

[0250] Potential cytotoxicity of A20 was evaluated using MTT assay performed after culturing normal human dermal fibroblast (NHDF) cells with increasing concentrations of A20 for 48 hr. The results revealed prolonged exposure of up to 10 pM A20 was not toxic (data not shown). We next worked with the UT Southwestern Medical Center Preclinical Pharmacology Core (PPC) to assess for in vitro metabolic stability using a murine microsomal assay, initially finding that A20 has a half-life of 224 min, 3511 half-life is 136 min, and A19 was only 22 min (data not shown).

[0251] Further studies were conducted as in vivo experiments to evaluate the effect of A20 when injected into the mouse. To formulate the compound for intraperitoneal (IP) injection, A20 was tested for solubility in various buffers, pH conditions, and with cyclodextrins (e.g. Captisol). The formulation was prepared by first dissolving A20 powder in DMSO and followed by mixing with sunflower seed oil (SSO) prior to administration (in 6% DMSO/94% SSO). IP injected A20 was well tolerated in multi-day dosing spaced 12 hr apart at up to 40 mg/kg. PK profiling experiments were then conducted with A20 to determine its absorption, distribution, metabolism, and excretion (ADME) properties. Mice were given a single IP injection of 20 mg/kg A20 and after 10, 30, 90, 180, 360, 960, 1440 min, three animals per time point were sacrificed and tissue was collected and subjected to mass spectrometry. The results showed A20 quickly distributed to the plasma and passed the BBB to reach the brain and spinal cord (FIG. 15). Plasma Cmax (maximum concentration) was observed at 10 min time point with a concentration of A20 at 214 ng/ml, which calculates to be 0.665 pM, and the area under the concentration time curve (ALICO-t), a measure of systemic exposure to the compound, was equal to 14,636 ng/ml over a 24 hr period. For brain, Cmax of A20 was at 10 min and was 104 ng/ml which calculates to be 0.323 pM, and the ALICO-t was equal to 6,684 ng/ml, and for spinal cord Cmax was 88 ng/ml which calculates to be 0.273 pM, and the ALICO-t was equal to 4,928 ng/ml.

Example 7; ..Generation of compound A20 salt

[0252] A20 was synthesized to obtain compounds with 98-100% purity and the resulting free-base chemicals were subjected to salting procedures as shown for A20 (FIG. 16). The synthesized salt forms of A20 chemicals were more aqueous soluble and more active, as shown in an Octet run using the same conditions as in FIG. 9A with the newly synthesized A20.2xHCI salt (FIG. 17). The IC50 values for the synthesized compounds were calculated to be 3.09 (response at 380 sec after association), 2.40 (response at 100 sec after dissociation step), and 3.63 pM (AUG), indicating that the salt form of A20 exhibits 2-3-fold better and/or stronger antagonist activity than the free-base.

Example 8: ...Pharmacpkjnetic-study-pf-newjy

[0253] The salt forms of newly synthesized A20 compounds were extremely soluble in water or PBS (>50 mg/ml for A20.2xHCI) and allowed for the injection of aqueous formulations of compounds into animals. A study was conducted with A20 salt dissolved solely in PBS and injected at 20 mg/kg to match the previously described study of the free-base. The resulting data shows that the salt version of A20 exhibits a much more impressive PK profile (FIG. 18). The plasma Cmax was at the 10 min time point with a concentration of A20 salt at 2,880 ng/ml which is >13-fold increase over the free-base and calculates to be 7.27 pM and the ALICO-t was equal to 81 ,039 ng/ml over a 24 hr period, which is >5.5-fold increase over the free-base. For brain, Cmax of A20 was also at 10 min and was 2,787 ng/ml which is >26-fold increase over the free-base and calculates to be 7.03 pM, and the ALICO-t was equal to 136,933 ng/ml (which is >20-fold increase over the free-base), and for spinal cord Cmax was 3,310 ng/ml (which is >37-fold increase over the free-base and calculates to be 8.35 pM), and the ALICO-t was equal to 86,044 ng/ml (which is >17-fold increase over the free-base). The liver was also analyzed and the Cmax was a 22,577 ng/ml which calculates to be 57 pM, and the ALICO-t was 1 ,363,490 ng/ml. The results from the brain, spinal cord, and liver tissues are particularly outstanding and indicate that significant levels of A20 salt remain even after the final collection at 24 hr time point. These results provide strong evidence that injection with A20 salt dissolved in PBS exhibits much better PK dynamics compared to the free-base dissolved in DMSO/SSO.

Example 9; Binding ..kinetics of human EphB-Ephr

[0254] Studies were conducted to confirm that A20 chemicals can effectively target the human Eph/ Ephrin proteins. Octet system was used to first characterize the biophysical interactions of the human B class Eph and Ephrin proteins, and then was tested to assess how A20 may disrupt the interactions. Since the human and rodent proteins are highly conserved, and essentially identical, with EphB2 sharing 99.5% identity between human and mouse, with only 5 of -1 ,000 amino acids different, any species-specific differences were not anticipated. Therefore, commercially available human EphrinB1-Fc (EB1), EphrinB2-Fc (EB2), and EphrinB3-Fc (EB3) ectodomain proteins were sparsely immobilized onto biosensors and then exposed to 50 nM of soluble human EphB1-His, EphB2-His, and EphB4- His ectodomains for 800 sec association and 1 ,000 sec dissociation steps with a total of nine different interactions analyzed. In the absence of A20 chemical, the immobilized human EphrinB proteins bound to their appropriate human EphB target proteins; with EphrinBI only bound EphB1 and EphB2, EphrinB2 bound all three EphB1 , EphB2, and EphB4, and EphrinB3 only bound EphB1 and EphB2 (Fig. 19A-19B).

[0255] In summary, the analysis of human proteins clearly shows EphB1 and EphB2 receptors promiscuously bind to all three EphrinB proteins, whereas EphB4, which only functions in the vasculature system, interacts strictly and very strongly with only EphrinB2. EphB4 shows no interaction whatsoever with EphrinBI or EphrinB3. Secondly, it is obvious that all three human EphB receptors tested exhibited the strongest binding to EphrinB2 ligand, with EphB4 clearly showing the fastest initial binding with the steepest slope of the three different sensorgrams at the beginning of the association step at 85° and the strongest response at end of association (0.48). The data also shows that the steep EphB4-EB2 sensorgram curve quickly bends starting about 60 sec into the association step with the slope then flattening dramatically to only 5°. Further, the EphB1-EB2 and EphB2-EB2 sensorgrams are very different. For EphB1-EB2, the initial slope of binding is only 70° (not nearly as steep as EphB4) and then the response quickly bends to form a near straight 25° slope throughout the remaining association step. The EphB2-EB2 interaction appears hybrid of EphB4/EphB1 , as it shows a very strong initial slope of 85° at start of association (like EphB4) but then within 20 sec the response bends to form a near straight 20° slope throughout the remaining association (like EphB1). All three EphB-EB2 interactions exhibited similar dissociation profiles with a fair amount of EphB protein quickly coming off the EphrinB-bound sensorchips, though -50% or more of the respective receptor protein remained bound at the end of the 1 ,000 sec dissociation step, indicating formation of very stable and long-lasting macromolecular protein complexes. The results from sensorgrams for EphB1 and EphB2 receptors interacting with EB1 are very different compared to those with EB2. The EphB1- EB1 sensorgram did not show an initial higher slope, instead starting right from the beginning of the association step the response was a straight near 22° sloped line throughout, and remarkably in the dissociation step no EphB1 protein was lost from the sensorchip, indicating formation of a super-stable complex that basically has no off rate. The EphB2-EB1 sensorgram showed there was an extremely fast initial on rate similar to the EphB2-EB2 interaction with a slope of 85°, however this was only for a very brief <5 sec period, because then the binding curve quickly flattened to a 22° slope to run parallel with EphB1-EB1 binding kinetics. Further, in the EphB2-EB1 dissociation step there was an initial rapid loss of a small amount of bound EphB2 protein, but then the remaining bound protein was very stable similar to EphB1-EB1. The initial fast loss of some sensorchip-bound EphB2 protein at the start of dissociation step was basically reverse of the fast initial on observed during start of association.

[0256] Further, the sensorgrams for EphB1 and EphB2 receptors interacting with EB3 are basically identical to that for EphB1-EB1 , in that both EphB1-EB3 and EphB2-EB3 sensorgrams indicated a delayed, very slow steady straight-lined accumulation of the EphB1/EphB2 proteins to the immobilized EB3 protein during the association step. The slope of binding was found to be even less than the other interactions being 15° for EphB1 and only 11° for EphB2. Further, like the EphB1-EB1 interaction, the EphB1-EB3 and EphB2-EB3 complexes did not melt away at all in the dissociation step, indicating formation of a superstable complex that basically has no off rate.

[0257] While the binding kinetics described above for the various human EphB-EphrinB interactions are complicated and show clear differences, 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 the sensorgram data and first attempts to fit the information to equations that follow rather standard binding characteristics, most typified by either a classic 1 :1 binding type interaction (like formation of a simple receptor-ligand dimer) or something more complex/heterologous involving 2:1 binding type interactions (like Eph- Ephrin dimer and tetramer). Based on the information contained in the sensorgram run under analysis, the Octet software will attempt to draw curves to fit the data to 1 :1 and 2:1 binding equations. The quality of any one curve fit and thus confidence of the results the equation puts out is also provided as the R2 value, and the closer to 1 the better, this data is provided in parentheses next to the KD values in Table 2. The 1 :1 and 2:1 curve that fits for all the above human protein data is shown in FIG. 20A-20C. Focusing first on the EphB-EB2 interactions, it is clear they do not fit the 1 :1 binding curve, but rather more closely resemble a 2:1 heterologous-type interaction. The resulting curve fits are not always perfect, especially for such complex interactions as the Eph/Ephrin system. As such, while the EphB2-EB2 and EphB4-EB2 data fit a 2:1 curve fairly well, the EphB1-EB2 interaction fits neither 1 :1 or 2:1 well and the R2 values of this interaction reflects this. The interaction of EB1 with its two target proteins is more divergent. The EphB1-EB1 data indicates a clear 1 :1 curve fit and interaction with an extremely high 11.4 pM affinity in a complex that forms very slowly during association but does not melt during dissociation. On the other hand, EphB2-EB1 with its initial very fast on rate during association and fast initial off rate in the dissociation step shows a clear 2:1 curve fit interaction. The EphB1-EB3 and EphB2-EB3 interactions are like that for EphB1-EB1 , clear 1 :1 curve fits with a slow gradual on rate of association followed by no off rate in dissociation.

[0258] Further, in none of the nine sensorgrams analyzed did the binding reaction reach homeostasis where on/off rates equilibrate as would be indicated by the trace eventually bending during association step into a straight line with no slope (0°). Thus, whether the binding reactions start out fast with slopes >70° or slow and steady with slopes <30°, homeostasis was not achieved. This failure to reach homeostasis was even observed when the standard association and dissociation step lengths were doubled to 800 and 1 ,000 sec, as shown in Figs. 19 and 20. Additionally, the competitive binding studies in Fig. 12 show that binding homeostasis can be achieved if saturating amounts (somewhere between 100- 1 ,000 nM) of the soluble binding partner are used. In the above studies with human proteins, the binding reaction for EphB4-EB2 was closest to reaching homeostasis with slope 5° at the end of the association step. This indicates that the EphB4-EB2 interaction is more “dimer- driven”, whereas all the other interactions are what we term “tetramer-driven” (see more below). In the previous Octet studies presented above using biosensor immobilized rodent EphB1-Fc and EphB4-Fc ectodomain proteins binding to soluble rodent EB2 ectodomain, the sensorgram traces looked highly similar to those using human proteins where the EphrinB2- Fc protein was immobilized. Thus, whether the Eph protein or the Ephrin protein is immobilized on the biosensor, or whether human proteins or rodent proteins are used, the Octet produces highly similar binding interactions, sensorgram curves, KD information, and other biophysical kinetic values for a particular interaction under analysis. As such, the rodent EphB1-EB2 binding data shows that homeostasis is not reached and the sensorgram pattern again indicates this is a “tetramer-driven” interaction (FIG. 9A, FIG. 11 , FIG. 17), whereas the binding data shown for rodent EphB4-EB2 reached homeostasis within about 200 sec of association and can be characterized as a “dimer-driven” interaction (FIG. 9B). In conclusion, the data indicates that Eph-Ephrin interactions are very complex due to formation of dimers and tetramers, show very high affinity associations, and once formed the Eph-Ephrin tetramer is a super-stable macromolecular complex.

Table 2: KD data of biophysical studies of immobilized human EphrinB binding to human EphB.

■Example 10 : A20 ' disrupts the EphB-EphrinB tetrame

[0259] The Octet studies shown in FIG. 19A-19F, and FIG. 20A-20C, and Table 2 using human proteins involved runs in which 1 , 10, and 100 pM concentrations of the A20.2xHCI salt were included to determine how compound would affect binding kinetics. The sensorgrams for immobilized human EphrinB2 binding to EphB2 and EphB4 with 0, 1 , 10, and 100 pM concentrations of A20 salt provide good examples of how the compound can alter Eph-Ephrin protein-protein interaction dynamics. For both interactions, the addition of A20 resulted in a reduced level of binding with compound having a much stronger overall inhibitory effect on the EB2-EphB2 interaction, especially noted in the association step (FIG. 21A). Interesting, the inhibitory effect of A20 salt on the interactions of immobilized human EphrinB2 ectodomain with soluble human EphB ectodomains maxed out at the 1 pM concentration, as 10-fold (10 pM) and 100-fold (100 pM) more compound did not provide any enhanced inhibition. This indicates the effective/relative IC50 for A20 is well below 1 pM (more accurate IC50 calculations are provided below). Notwithstanding, if just the sensorgram responses for 0 and 1 pM A20 are analyzed for absolute IC50 calculations (FIG. 21 B), it is clear the compound is a much stronger inhibitor of the EB2-EphB2 interaction whether assessed near the end of association step (blue star), shortly after start of dissociation step (gold star), or by AUG, as all provide absolute IC50 values -1 pM. The calculations for EB2-EphB4 indicate absence of an absolute IC50 because compound does not inhibit any of the three response values assessed >50%, even at 100 pM. Consistent with these divergent absolute IC50 values, particular scrutiny of the association step of the sensorgrams shows 1 pM of A20 reduced EB2-EphB2 association response -50%, but only slightly reduced the EB2-EphB4 binding response. Furthermore, it is clear that addition of A20 had no effect on the initial rapid binding 85° slope observed at the start of association step for either EphB2-EB2 or EphB4-EB2 interactions, this shows compound has no effect on formation of the initial Eph-Ephrin dimer. However, past this early fast dimer phase, A20 was observed to reduce the slope of the remaining part of the association. For EphB2-EB2, the slope went from 20° to 10° in the presence of A20 and for EphB4-EB2 from 5° to 3°. The reduced slope later in the association step indicates the A20 compound is specifically affecting accumulation of the stable tetramer.

[0260] Addition of compound also altered dissociation kinetics. EphB2 response showed a very rapid loss from the immobilized EphrinB2 protein at the start of the dissociation step which then quickly stabilized to reach a plateau level significantly above the baseline that did not change for the remaining duration of the dissociation step. This observation of a low level of stable bound EphB2 protein remaining at the end of the dissociation when compound is included at 1 , 10, or even 100 pM reveals that there is a gradual accumulation of an extra- super-stable Eph-Ephrin complex that is not targeted by the A20 chemical. This is referred to as the “unsurmountable” component of the Eph-Ephrin interaction (FIG. 21 C). Additionally, AUG data was graphed for these runs (FIG. 21 D). The results indicate that in the absence of A20 compound the EB2-EphB2 dimertetramer ratio was 32:68 (tetramer-driven) and the EB2-EphB4 dimertetramer ratio was 83:17 (dimer-driven). Further, while runs in the presence of A20 showed no effect on dimer formation for either interaction, the EB2-EphB2 dimertetramer ratio was now inverted 32:21 such that it shifted to a more dimer-driven interaction that accumulated many fewer tetramers, the ones that A20 will not counter, the “unsurmountable” component. For EB2-EphB4, the dimertetramer ratio was 83:8 in the presence of A20 and was not much different from the no compound control run because it is already a strong dimer-driven interaction. Overall, the data indicates that addition of A20 compound strongly reduced the accumulation of the EB2-EphB2 tetramer and shifted it more towards a dimer-driven type of interaction that with time will form at a much slower rate an “unsurmountable” extra-super-stable complex. A similar though more muted effect of A20 on tetramer accumulation was also observed for the other interactions of immobilized human EphrinB binding to soluble human EphB1 and EphB2 ectodomains (FIG. 22). The inhibitory effect of A20 is strongest on the EB2-EphB2 interaction with absolute IC50 reaching 1 pM, followed by the EB2-EphB1 interaction with absolute IC50 -10 pM. It was found that with EB2-EphB1 , in the absence of A20 the dimertetramer ratio was 14:86, even more tetramer- driven than EB2-EphB2, and that the concentration of A20 needed to maximize the inhibitory response on EB2-EphB1 is 10 pM. The “unsurmountable” component of the EB2-EphB1 interaction was -50% of the tetramer species (14:41) whereas that for EB2-EphB2 interaction is -30%. The interactions of soluble human EphB1 and EphB2 ectodomains with immobilized human EB1 and EB3 ectodomains in the absence of any A20 are somewhat different and seem to bypass dimer formation to instead slowly accumulate as 100% tetramers that do not melt off the biosensor-bound EphrinB protein during dissociation (FIG. 19A-19F and FIG. 20A-20C). Addition of A20 to these interactions clearly reduced the level of tetramers formed during the association step with 1 pM producing the maximum inhibition for EB1-EphB1 , EB1- EphB2, and EB3-EphB2 interactions while 10 pM was needed to maximize inhibition of EB3- EphB1. A20 appeared more effective towards EphB2, having max effect at 1 pM and reducing level of tetramers with both EB1 or EB3 -45%, while reducing EphB1 tetramer formation 15% and 40%, respectively.

[0261] Together, these results show that A20 can affect the association/dissociation dynamics and tetramer accumulation of all the EphB-EphrinB complexes examined, with strongest activity towards EphB2 interacting with EphrinB2. The fact that the tetramer inhibitory effect of A20 is maximized at 1 pM in most cases and increasing amount of compound to 100 pM has no additional effect to eliminate the “unsurmountable” component, it is determined that there are at least two different tetramer configurations, one effectively targeted by A20 chemicals and the other not. While the precise nature of this particular extra- super-stable “unsurmountable” complex is unknown, it is possibly caused by the clustering of two or more circular tetramers into a conformation unable to be breached by A20 (FIG. 10). Thus, while A20 chemicals inhibit the ability of two Eph-Ephrin dimers to complex into a super-stable circular tetramer, some tetramers may still form in the presence of the drug, albeit at a much slower rate (slope of curve), and perhaps if two or more of these tetramers manage to complex together, then it is predicted this may form the extra-super-stable “unsurmountable” Eph-Ephrin complex. The results disclosed here show that Eph-Ephrin interactions form dimers, and A20 surmountable tetramers, as well as some amount of unsurmountable tetramers (see more on this below).

Example 11 : The EphB-EphrinB dimer is fast-on/fast-off, while the tetramer is slow- or no-off

[0262] In the above disclosed Octet interaction studies using human proteins the association and dissociation times were doubled to 800 and 1 ,000 sec, respectively, resulting in a greater accumulation of the super-stable surmountable and extra-super-stable unsurmountable forms of the Eph-Ephrin tetramers. To better assess early binding activities in the absence of A20, biophysical studies were conducted in which the association times were shortened to 80, 40, 20, 15, 10, and 5 sec, followed by a standard 500 sec dissociation, and where biosensor immobilized rEphB1-Fc ectodomain was exposed to 100 nM soluble mEB2-His. Focusing first on the run with 80 sec association step and for illustrative purpose (FIG. 23), the biosensor response indicates the expected rapid formation of EphB1-EB2 dimers with extremely high 87° slope for the first 10-15 sec of the association, which then quickly bends again as expected this time to a -60° slope as tetramers begin to accumulate. When placed into the dissociation step, the response showed an initial rapid 15-20 sec off phase with a high reverse slope representing some of the bound EB2 protein coming off immobilized EphB1 (dimer melting). The dissociation response then quickly bent to level off and plateau at a signal much higher than baseline to represent the species of stable EphB1- EB2 tetramers that remained complexed throughout the remaining dissociation step. Analysis of response levels showed that even with the shortened 80 sec association time, 54% of the binding can be attributed to formation of tetramers, and when the association time was reduced to 40 sec, a slightly lower 43% contribution of the response/binding is due to tetramer formation as shown in FIG. 24, upper sensorgrams. Interestingly, if the association time was reduced to 5, 10, or 15 sec, only the fast-on and fast-off responses were evident, no stable tetramers were present at end of the dissociation, but if the association was extended to 20 sec, then a very low -5% level of stable tetramer is generated as shown in FIG. 24, lower sensorgrams). This suggests a critical period of 15-20 sec association is needed to allow for two fast-on/fast-off dimers to form before they can associate into a stable tetramer complex.

[0263] To assess how A20 chemicals alter early binding activities, 80 sec Octet runs were then conducted using 0, 10, and 100 pM concentrations of the A20 salt compound (Fig. 25A 25B). In the absence of A20, the association response of immobilized EphB1 binding to soluble EB2 is typical to that shown in FIG. 24 and indicates rapid early formation of dimers with very high 87° slope that quickly bends to the tetramerization accumulation slope of -60°, and then once in the dissociation step the slope abruptly reverses to show fast-off of the dimer species with a signal that then leveled off in this experiment at 67% of the maximum response and represents accumulation of the stable tetramers. In runs containing A20, even though there was no effect on the early and fast formation of dimers during the 80 sec association step, there was a clear and very strong dose-dependent reduction in accumulation of the tetramer. The tetramer accumulation slope changed from -60° to 16° with 10 pM A20 as shown in FIG. 25 (purple sensorgram) and was a flat line with no slope with 100 pM A20 as shown in FIG. 25 (dark green sensorgram), indicating very little if any stable tetramer was able to form. Analysis of the dissociation step confirms that addition of A20 in both concentrations resulted in a fast loss of nearly all of the response signal to approach baseline levels, indicating very few super-stable and extra-super-stable unsurmountable tetramers formed.

[0264] The shortened association studies clearly show that the presence of A20 has a major effect on Eph-Ephrin binding dynamics, changing them from a 2:1 heterologous/complex- type highly stable interaction to a simple 1 :1 dimer-type fast-on/fast-off interaction. This can be especially seen in the analysis of the kinetic data where the parameters were set to calculate information based off of a 1 :1 interaction (FIG. 25C). Here, while the run with no A20 and 10 pM A20 did not fit 1 :1 interaction dynamics with R2 of 0.3279 and 0.6296, respectively, the run with 100 pM A20 gave an R2 value of 0.9623 indicating movement towards a 1 :1 dimer-type interaction. Further, the KD for the EphB1-EB2 interaction in the absence of A20 calculated to be an extremely high affinity of 0.137 nM (and even higher affinity < 1 pM if calculated using more appropriate 2:1 interaction dynamics). In the presence of A20, the KD indicates a much lower affinity interaction, 76-fold lower with 10 pM (10.47 nM) and 337-fold lower with 100 pM (46.2 nM). Furthermore, addition of A20 did not significantly change the rate of association (Ka), which is mainly driven by the formation of the fast-dimer. However, compound did cause a striking 90-fold and 275-fold increase in rate of dissociation (Kdis), respectively. This increased rate of dissociation reflects the near absence in formation of stable tetramers and provides a fairly accurate representation of the Kdis that is specifically attributed to the fast-off melting of dimers. Summarizing these biophysical studies, in the presence of A20 only the fast-on/fast-off dimers form and are visualized in the association step, quickly reaching a homeostasis level of dimers forming and melting in equilibrium (fast-on/ fast-off), and not being able to associate into tetramers. Then, when placed into the dissociation step, all the existing dimers quickly melt off leaving behind the few tetramers that were able to form. Furthermore, because the 100 pM A20 concentration essentially leads to only association of EphB1-EphrinB2 dimers, we can use that KD value of 46.2 nM and other kinetic information gained by these experiments to characterize the fast-on/fast-off dimer. Thus, the very strong sub-nanomolar/picomolar affinity of the EphB1-EphrinB2 interaction observed in the absence of A20 compound is mainly due to the assembly of two relatively weak/unstable dimers into a highly stable circular tetramer complex. Based on the results, it is concluded that A20 chemicals can be classified as specific/select inhibitors of Eph-Ephrin tetramerization.

Example 12: Mass photometry confirms A20 chemicals target the Eph-Ephrin tetramer

[0265] To verify the kinetic studies using the Octet system, mass photometry (MP) that uses optical detection to measure the mass of individual proteins and protein complexes in solution was employed. MP was used to determine the mass of EphB2 and EphrinB2 ectodomain proteins individually or after being combined and allowed to associate into tetramers for a 24 hr period either without or with 10 or 50 pM concentrations of the A20 salt (FIG. 26). For these experiments His-tagged proteins, human hEphB2-His and murine mEB2-His were used. The hEphB2-His protein when measured on its own exhibited an average mass of 130 kDa, and the mEB2-His protein measured in at an average 83 kDa, with both proteins exhibiting fairly sharp poisson distribution patterns indicating each has a relatively uniform mass (FIGs. 26A-26B). The values obtained for EphB2 and EB2 are significantly higher than the predicted monomeric masses of these two ectodomain proteins, 59.7 and 23.5 kDa, respectively. While a portion of this discrepancy is likely due to heavy glycosylation, especially for the Ephrin, and both Eph and Ephrin ectodomain proteins have been shown to have weak homo-dimerization/ oligomerization tendencies when in solution and in crystal structures. During MP experiments EphB2 and EB2 when analyzed alone would each measure as a homodimer, and when the EphB2 and EphrinB2 proteins are combined, the average mass of the protein species shifted to a larger 216 kDa species (FIG. 26C). The size was as expected if two EphB2 and two EphrinB2 molecules complexed together into the stable circular tetramer (130 + 83 = 213 kDa). Importantly, consistent with the bringing together of the two different molecules into a single tetramer complex, the total number of protein species measured in the MP did not increase when the EphB2 and EB2 proteins were combined. This indicates that the proteins combined into a single measurable unit of higher mass. Moreover, when A20 was added, a dose-dependent loss of the 216 kDa sized species into intermediate average masses of 158 kDa with 10 uM A20 and 150 kDa with 50 pM A20 was observed (FIG. 26D). The intermediate mass of 158/150 kDa would be consistent with loss of either one EphB2 or one EphrinB molecule from the tetramer, turning it into a weak Eph-Ephrin dimer with a weak homo-oligomerized Eph or Ephrin molecule attached as a trimolecular species. Consistent with breakdown of the tetramer, the total number of protein species increased in the presence of A20. It is important to understand that the MP experiments described here are powerful, but also temporally limited because we can really only assess the protein species after interaction homeostasis has been achieved and for this experiment the proteins and A20 were allowed to incubate together for 24 hr before masses were determined.

[0266] In contrast to Octet kinetic studies, MP studies focuses on measuring the protein interactions that would be present at the end of association step than using an Octet. Thus, when Eph-Ephrin proteins are combined they lose their weak homo-dimerization ability and instead prefer to assemble into a stable very high affinity tetramer structure, and when an A20 chemical is present the tetramer is inhibited leaving only the relatively weaker Eph- Ephrin dimer to form that can also weakly homodimerize with an Eph or Ephrin molecule on the side. It was hypothesized that these Eph-Ephrin:Ephrin or Eph:Eph-Ephrin trimolecular species represents a primed complex, that stands ready to tetramerize but is strongly inhibited by A20 chemicals.

Example 13: A20 chemicals also target EphA-EphrinA tetramers and EphB-EphrinA cross class tetramers

[0267] The Octet system was used to probe the interactions of epitope-tagged human A class proteins, focusing on immobilized EphA3 binding to two of its cognate ligands, EphrinAI and EphrinA5, as well as EphB2 binding to EphrinA5 (FIG. 27). The binding kinetics indicate EphA3 exhibits a tetramerization-driven interaction with EphrinAI , but a dimerization-driven interaction with EphrinA5. Focusing on the dimerization-driven EphA3- EA5 interaction first (green sensorgram trace), which mimics what is disclosed above for EphB4-EB2, the initial binding of soluble EphrinA5 to the immobilized EphA3 is very fast and strong with a long and steep 84° slope of response that then quickly bends around 35 sec into the association step to form a more gradual tetramerization accumulation slope. The overall response of EphA3-EA5 binding during the association step is much stronger than the other two A class interactions analyzed, suggesting a very high affinity interaction. However, again like the EphB4-EB2 interaction, once placed into dissociation, a significant fraction of the bound EA5 protein melts quickly and the off phase continues downward with a slow gradual decline in bound protein. Although the dissociation was for only 500 sec, the continued downward slope of the response indicates most of the bound EA5 protein would eventually melt off of the immobilized EphA3 protein. This is referred to as a classic dimerization-driven interaction, fast and strong initial binding in the association step but with most of the bound protein coming off in the dissociation due to lack of formation of stable tetramers. EphA3 interacts with EphrinAI with totally different kinetics/dynamics (orange sensorgram trace). Here, the initial high 84° dimerization slope phase is extremely brief (5 sec) with the binding quickly snapping into a linear tetramerization accumulation phase with a 25° slope, and that once placed into dissociation step almost no bound protein melts, just the small amount that would represent dimer interactions falling off in the first few seconds after being placed into dissociation. Thus, while the EphA3-EA5 interaction is strong initially because of the relatively high dimerization affinity but then falls apart because of weaker tetramerization affinity, the EphA3-EA1 interaction is more like a turtle, slow and methodical, building strong and stable high-affinity tetramers that do not melt and that will eventually overtake its faster-on but faster-off counterpart. EphB2 interacting with EA5 is similar to the EphA3-EA1 interaction, in that the association step is characterized by a very brief (5 sec) high 84° slope phase that quickly bends into a more gradual tetramer accumulation phase (blue sensorgram trace). Interestingly, once placed into dissociation, a large fraction of bound EA5 protein quickly melts off the immobilized EphB2 but then towards the end of dissociation the response levels off above baseline indicating formation of stable long-lasting tetramers. The strong initial off phase of the EphB2-EA5 complexes in the dissociation represents both the melting of weak dimers and of the relatively weaker affinity tetramers formed by the cross A/B class EphB2-EA5 molecules.

[0268] Similar to results obtained with the B class molecules, addition of A20 effectively reduced formation of A class and cross A/B class tetramers (FIGs. 28A-28C). For the EphA3- EA1 tetramerization-driven interaction (FIG. 28A), A20 produced a strong dose-dependent reduction in binding, with -50% of the tetramers lost at 1 pM and near complete loss of tetramer formation at 100 pM. While there is no effect of A20 on the initial 5 sec fast EphA3- EA1 dimer phase, there is a very strong effect of compound on tetramer accumulation. It was estimated that >70% of the EphA3-EA1 binding interaction can be attributed to the tetramer which is effectively disrupted by compound, with very little binding activity attributed to the dimer and more accurately the dimerizatiomtetramerization ratio of this interaction is 10:90 not 30:70. Regarding the EphA3-EA5 interaction (FIG. 28B), addition of A20 also had no effect on the initial fast high slope dimer phase observed at the beginning of the association step, however at even the lowest concentration of A20 tested (1 pM) there was a striking maximized loss of tetramer accumulation. Since this is a dimerization-driven type interaction (dimerizatiomtetramerization ratio of here is 72:28), the overall effect of compound on this interaction is muted. The cross A/B class interaction, EphB2-EA5 (FIG. 28C), can also be described as a tetramerization-driven interaction that is strongly inhibited by A20 (dimerizatiomtetramerization ratio here is 23:77). As this is one of the weakest of the Eph- Ephrin interactions, addition of A20 here even at 1 pM completely inhibited the formation of stable tetramers as the response level reached baseline quickly into the dissociation step (asterisk), unlike the EphA3-EA1 or EphA3-EA5 interactions which showed some low level of unsurmountable tetramers. In summary, the results indicate that the A20 compounds effectively target the formation of all classes of Eph-Ephrin tetramers, A class, B class, and A/B cross class tetramers.

Example 14: Pre-existing stable Eph-Ephrin tetramers are also disrupted by A20 ■Chemicals

[0269] Biophysical studies were conducted to explore if A20 is able to disrupt pre-existing stable tetramers, focusing on interactions of immobilized human EphrinB2 binding to soluble human EphB1 and EphB2, and immobilized mouse EphB2 binding to mouse EphrinB2 (FIGs. 29A-29C). In these experiments, sensor-chip immobilized proteins were first exposed to 50 nM of their soluble target protein for an 800 sec association step and 1 ,000 sec dissociation step to generate pre-formed stable tetramers, which were then subjected to a 1 ,000 sec competition step that included either 0, 2, 10, or 50 pM concentrations of A20 salt compound. In all three cases, it was observed that A20 was able to compete away in a dose-dependent manner a significant amount of the stable pre-formed tetramer complexes. The ability of A20 to disrupt pre-existing tetramers was most pronounced for human EB2-EphB2 and cognate murine EphB2-EB2 experiments and indicated a gradual ongoing dose-dependent decline in tetramers throughout the 1 ,000 sec competition step (and beyond if allowed) and irrespective of whether the human EphrinB2 or murine EphB2 was the immobilized protein. A20 also clearly disrupted in a dose-dependent fashion some of the pre-existing EB2-EphB1 tetramers, however, here the effect was complete within 200 sec into the competition step as the response levels then flatted out. This indicates that some of the pre-existing EB2- EphB1 tetramers are readily targeted by A20, but others are not, the latter of which may be related to the unsurmountable type of tetramers discussed previously. In summary, the results indicate pre-existing Eph-Ephrin tetramers can be disrupted by A20 chemicals and that perhaps the tetramers formed between EphB2 and EphrinB2 are more surmountable and easier to compete away than those formed between EphB1 and EphrinB2. Example 15: Method to determine accurate compound IC50 values for inhibiting the Eph-Ephrin tetramer.

[0270] To identify additional novel chemical entities which exhibit improved tetramerization inhibitor activity and display enhanced pharmacological properties, a refined method to obtain accurate IC50 information that reflects the specific effect of compound on inhibiting the Eph-Ephrin surmountable tetramer was developed. Low concentration dose-response Octet studies of a compound (e.g., 0, 0.05, 0.1 , 0.2, 0.4, 0.8, and 1.6 pM concentrations) was ran and included a “maximum control” biosensor condition, which is the necessary concentration of A20 chemical needed to eliminate all of the surmountable tetramers. The response of the maximum control is subtracted from the other responses to eliminate the contribution of binding to dimer formation and to the unsurmountable component of the tetramer, leaving behind for analysis the amount of binding that can be attributed to the surmountable tetramer. The subtracted response of the no compound control biosensor is set to 100% and the maximum control biosensor is by definition set to 0%. The subtracted and analyzed part of the run can be at a single point in time, we select 380 sec into the standard 400 sec association step and 50 sec after beginning of 500 sec dissociation step as two key time points to analyze, or the AUC of each sensorgram under analysis can be determined. The Octet runs shown in Fig. 30A-30D highlight this new method assessing immobilized human EphrinB2 binding to 50 nM soluble human EphB2, and using 2 pM A20 for the maximum control to provide accurate tetramerization inhibitor IC50’s for A19, A20, 8009, and 3511. Just glancing at the data it is clear that compared to results with A20, 8009, and 3511 , compound A19 exhibits only weak ability to inhibit formation of the EB2-EphB2 tetramer. If we focus on IC50 values 380 sec into the association step, after first subtracting the max control response from the different dose runs of a compound, a blue asterisk was placed to indicate the 50% point of the corrected data. For compound A19 the blue asterisk is below the highest concentration tested here and so the relative association IC50 here is >1.6 pM, while for A20 and 8009 the blue asterisks are just below the 0.4 concentration and so the IC50’s for these two compounds are ~0.5 uM, and for 3511 the IC50 is right on top of the 0.2 pM sensorgram run. These very accurate IC50 calculations indicate that the lead compounds exhibit IC50 values in the 200-600 nM range, which make for very potent leads as inhibitors of very high affinity protein-protein interactions.

Example 16: Making ( and test jng of analogs of

[0271] Through ongoing medicinal chemistry efforts, a number of novel analog compounds similar to A20 were designed, synthesized, salted, and purified. Compound 8009 is very similar to A20, with a chloride substituting for the bromine halogen on the 8-hydroxyquinoline, whereas in 3511 the A20 morpholine ring is replaced with a more complex piperazine extended structure. Given that changes can be made in these two regions of A20, an array of compounds that alter these regions were synthesized. Compounds were then subjected to kinetic Octet studies to determine their biophysical activities and build our understanding of SAR and inform the chemical space that defines what makes for a good Eph-Ephrin tetramerization inhibitor.

[0272] The general strategy for design of A20 analogs aimed to satisfy (a) novelty, (b) effect of different synthetic building blocks other than quinoline, (c) effect of modifications of the morpholine/piperazine extensions, and (d) pharmacokinetic parameters. Synthetic schemes for multiple different compounds based off A20 have been validated (FIG. 31) including 3511 (FIG. 32). A20 is synthesized via 3 steps, starting with quinoline-8-ol and 37% formaldehyde in the presence of HCI and catalytic ZnCI2, followed by nucleophilic substitution along with morpholine in basic conditions to yield QM, followed by an electrophilic substitution reaction using N-bromosuccinamide (NBS) in the presence of dichloromethane to yield A20. Compounds are purified using silica gel column chromatography and mobile phase gradient elution using an ethyl acetate/hexane mixture and are structurally elucidated using 1 HNMR and C13NMR spectroscopy. Different structural modifications of A20 encompass one or more combinations as those shown in FIG. 31 , including (x) replacing the Br (A20) and Cl (8009) with other halogens I or F, (y) morpholine variation to piperidine and piperazine, and (z) heteroaryl variation changing the quinoline ring system into quinazoline or indole. Analogs based off of 3511 shown in FIG. 32 will adopt structural modification via; (x) replacing Br with H, Cl, F, or I, (y) changing the methoxy group with different electron donating or electron withdrawing groups at ortho, meta, or para positions, (z) changing quinolone ring into quinazoline, and (r.o.) ring opening for piperazine to have diamino ethane as a linker. Salt forms of compounds are also generated which aid aqueous solubility and provide more favorable PK properties.

[0273] Initially, two quinoline-8-ol based morpholine variants were synthesized by extending the hydrophobic moieties through nucleophilic substitution along with different piperazine derivatives to generate novel compounds QPB4 and QPDF (FIG. 33A). Synthesis of QPB4 and QPDF also involved generating an A20-derivative in which the Br is replaced by H, leading to A19/QM (compound 1). Changes in the quinoline ring system were also synthesized in application of classical bio-isosterism via ring replacement as this is a known strategy for drug discovery and substituting with either a quinazoline or indole ring system is a viable approach. Here, a different ring system was introduced, 5-methoxyindole, in a one- step synthetic pathway reaction. This involved coupling 5-methoxy-1 H-indole-3-carboxylic acid with morpholine or substituted piperazines in the presence of 1-Ethyl-3-(3-dimethyl- aminopropyl) carbodiimide and hydroxybenzotriazole in dichloromethane to synthesize the novel indole-based analogs IM, IPM2, and IPB4 (FIG. 33B). [0274] These early first-generation novel compounds in Fig. 33 were initially tested in Alpha assays and compared to A20, 3511 , and 8009. Compound QPB4 initially appeared interesting as it exhibited strong dose-dependent antagonist activity that seemed much better than A20 or 3511 (FIG. 34). QPDF and A19/QM also exhibited some inhibitory activity though it was not apparent until higher concentrations. The 5-methoxyindole analogs failed to exhibit any activity even at 0.5 mM concentrations, indicating this ring substitution is not viable and thus informing SAR as to the importance of the quinoline ring system. To more accurately determine IC50 values, all of the compounds were subjected to refined Octet studies as described above using 0, 0.1 , 0.2, 0.4, 0.8, and 1.6 pM concentrations of compounds with immobilized human EphrinB2 binding to 50 nM soluble human EphB2 and including the subtraction method of the maximum control (Fig. 35). The tetramerization inhibitor activity of QPB4 was more muted with a relative association IC50 of 1.2 pM, and thus this compound is not as active as initial Alpha assay results indicated.

[0275] With successful making and testing of the first-generation A20-related chemicals, additional novel second-generation compounds designed and synthesized using the general synthetic schemes displayed (FIG. 36A, FIG. 36B, and FIG. 36C). The structures of all analogs made are shown in FIG. 37, and detailed methods of synthesis are described in Example 27, below. Additional information on all compounds we have studied including structures, molecular weights, and naming information are provided in Table 1 above. Tables A-G (end) include IC50s and other biophysical data measured for various compounds.

[0276] Most of these chemicals have been subjected to refined biophysical testing using immobilized human EphrinB2 binding to 50 nM soluble human EphB2 to determine accurate IC50 tetramerization inhibitor activities (see FIG. 30A - FIG. 30D and FIG. 35A- FIG. 35E), with specific numerical values shown in Tables A-G, appearing at the end of the Examples. The data collected to date are very promising and summarized below.

[0277] The novel compound A20-I is a halogen replacement that changes the 7-bromine in A20 for iodine and had enhanced tetramerization inhibitor activity (FIGs. 38A-38D). This is evident when comparing results of A20-I and A20 salt compounds on the interaction of immobilized mouse EphB2 ectodomain binding to soluble mouse EphrinB2 ectodomain (FIG. 38A and FIG. 38B) or soluble human EphrinA5 (FIG. 38C). Regarding the EphB2-EphrinA5 interaction, A20-I exhibited a stronger inhibitor activity when compared to A20 (see FIG. 28). With A20-I the strong concentration-response effect shows maximum effect of compound is achieved at 0.8 pM as this concentration and 1.6 pM produced identical overlapped sensorgram traces, whereas that for A20 showed 10 pM of this compound is necessary to achieve full inhibitory activity. Likewise, for the interaction of immobilized mouse EphB2 binding to mouse EphrinB2, A20-I eliminated all tetramer species at 100 pM, including the unsurmountable component that A20 is unable to target. During these interactions of immobilized EphB ectodomains binding to soluble EphrinB2, the tetramer is much more surmountable by addition of an active compound than for the reverse experiment where EphrinB2 is immobilized and EphB2 is the soluble component in the assay (see FIG. 11 , FIG. 17, and FIG. 25A - FIG. 25C). It is believed that the more resistant unsurmountable tetramer formed by immobilized EphrinB2 binding to soluble EphB ectodomains provides for the most rigorous test for determining IC50 values and thus utilizing this EphrinB2-EphB2 assay for analysis of all compounds, provide uniformity in observed results. The results described herein with A20-I and using the human EphrinB2-EphB2 interaction indicates replacements can be made in the 7-bromine in A20 that lead to enhanced tetramerization inhibitor activity. However, not all replacements of the 7-bromine in A20 are productive, as A19-NO2 which replaces the halogen with nitrogen dioxide at the 7 position produces a weak, essentially inactive compound (FIG. 38D). Initial studies by the PK Core indicate A20-I, both free base and salt forms, also exhibit longer in vitro half-life in a murine microsomal metabolic stability assay when directly compared to A20 (FIG. 39). These results on metabolic stability are promising and indicate that this novel compound will show significantly improved biological activity over our original A20 lead.

[0278] A group of related novel analogs that replace the oxygen in the morpholine ring with sulfur dioxide and have either H (QTM), Br (QTM-Br), or I (QTM-I) at the 7-halogen position are shown in FIG. 40A-40C. While all three compounds show some ability to inhibit the tetramer, the two that contain a halogen show strong activity as both reach the maximum control response level at 1.6 pM and with relative IC50 values -0.8 pM. Results indicate that the QTM compounds are not as active as A20, which neared the maximum inhibitory response at 0.8 pM (upper left panel).

[0279] A series of second-generation compound variations related to QPB4 are shown in FIG. 41A-41 F. Among these, compound BQPB4-Bn appears promising as the 0.8 and 1.6 pM concentrations hit the maximum response level (FIG. 41 E). Changing the Br on the piperazine of BQPB4-Bn to CF3 to generate compound QPCF34 eliminated the activity. Further, QPI4 appeared less potent and more like the parental QPB4. Other compounds exhibited very weak or no activities, most notable MeQPB4, which has a methyl group attached to the quinoline 2 position, also appears to eliminate tetramerization inhibitor activity.

[0280] The salt form of compound QPB4-Bn with strong tetramerization inhibitor activity has also been tested for PK biodistribution as shown in FIG. 42A, FIG. 42B, and FIG. 42C. The results exhibit similarity to that obtained for the A20 salt and indicates significantly high levels of compound reach intended tissues (i.e. , brain, spinal cord, liver, skin) and persist at high concentrations during the 24 hr period of the test.

Example 17: The ..Eph-Ephrin ( dimer js disrupted by SNEVV

[0281] The Octet system was used to characterize the biophysical effect of the disclosed SNEW and EWLS peptides on the Eph-Ephrin protein-protein interaction. As shown in FIG. 43, “the SNEW peptide” herein refers to a peptide having an amino acid sequence of SNEWIQPRLPQH (SEQ ID NO: 1) and the “EWLS peptide” refer to a peptide having an amino acid sequence of EWLSPNLAPSVR (SEQ ID NO: 2). These peptides are further embodied in a consensus sequence: EWX1X2PX3LX4PX5 where X1 is leucine or isoleucine (L/l), 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 about these peptides is provided in Koolpe, M., et al.O (J. Biol. Chem. (2005) 280, 17301-17311), which is incorporated herein by reference in its entirety. Alpha assay data in FIG. 4A show both peptides will disrupt the EphB1-EphrinB2 interaction. Since these two peptides are very similar to each other (FIG. 43), their common ability to disrupt the EphB1-EphrinB2 interaction was attributed to the Alpha assay being more amenable to disruption.

[0282] The extensive biophysical data indicates that A20 chemicals selectively target the Eph-Ephrin tetramer. To assess this, the Octet was employed to characterize how SNEW and EWLS peptides affect binding interactions with the hypothesis they should affect the initial very high slope “fast-on” binding we attribute to formation of the Eph-Ephrin dimer. Increasing concentrations of SNEW and EWLS peptides (0.25, 0.5, 1.0, 5, 10, and 100 pM) was tested using immobilized human EphrinB2 binding to 50 nM soluble human EphB2 (FIG. 44A and FIG. 44B - upper sensorgrams). While low concentrations of the SNEW peptide up to 10 pM had no effect on the EphrinB2-EphB2 interaction, the 100 pM concentration of the SNEW peptide specifically altered the binding in the association step. The effect of the SNEW peptide can be attributed to interference with formation of the dimer as the initial fast-on dimer slope of -85° typically observed immediately at the start of the association step was significantly flattened to 65° with 100 pM peptide present in this experiment during the association step. It is also observed that while the dimer slope is dramatically flattened by addition of SNEW peptide, the stable tetramer continues to accumulate albeit more slowly. This indicates a dimerization inhibitor like SNEW may only slow the formation of tetramers, and so the effect of these peptides will be muted compared to A20 chemicals. The EWLS peptide exhibited no effect on the EphrinB2-EphB2 interaction. Thus, the biophysical analysis with Octet show that the SNEW peptide but not the EWLS peptide is selective for disrupting the EphB2-EphrinB2 interaction consistent with previous reports. However, the need to use such high concentrations of SNEW to achieve biophysical responses can be explained by the fairly weak ability of these peptides to show any biological activity. Nevertheless, to confirm these results, a second dose-response Octet study was conducted using a higher concentration range of SNEW and EWLS peptides (50, 100, 200, and 400 uM). Results show that the SNEW peptide exhibited a strong dose-response inhibition of the fast-on dimer that again started right at the beginning of the association step at all concentrations tested (FIG. 44A and FIG. 44B- lower sensorgrams). During this experiment, the fast-on dimer slope of 84° in the absence of peptide became strongly flattened to 70°, 60°, 51°, and 41° as the concentration of SNEW was increased. EWLS again had no effect on the interaction, even at the 400 pM concentration, again demonstrating the SNEW peptide is specific for the EphrinB2-EphB2 interaction. In summary, SNEW peptide disrupts formation of the EphrinB2- EphB2 dimer and provides additional evidence that the Octet system can help discriminate Eph-Ephrin dimer interactions from Eph-Ephrin tetramer interactions.

Example 18: Pharmacokinetic studies of A20 chemicals

[0283] To examine the drug-like nature of the disclosed compounds, tests for solubility was conducted. The results show salt forms of both A20 and A20-I are extremely soluble in water (FIG. 45). Additionally, in vitro plasma stability tests show that A20-I exhibits a 3-5 times longer half-life than A20 (FIG. 46). In vivo PK studies show A20 salt compound is orally bioavailable (FIGs. 47A-47D), indicating compounds could possibly be formulated as a pill form or oral liquid for administration and strengthens the drug like qualities of the tetramerization inhibitors. Oral availability is especially exhibited for the liver which was assessed in this experiment. This suggests possible routes of administration of tetramerization inhibitors orally, for conditions such as NASH liver fibrosis, CDK chronic kidney disease, diabetes/obesity, pancreatic cancer, etc.

Example 19: Biophysical studies of additional A20 chemicals

[0284] The Octet has been used to assess many more new chemicals that have been designed and synthesized using standard assay of immobilized human EphrinB2 ectodomain binding to 50 nM soluble human EphB2 ectodomain.

[0285] FIG. 48A, FIG. 48B, FIG. 48C and FIG. 48D shows results of compounds A20-I, A19-L, 2OH-A19, and QPPh. While A20-I exhibited strong activity at disrupting the surmountable tetramer as expected given the data shown above and with an IC50 - 0.6 pM (FIG. 48A), compounds 2OH-A19 and QPPh showed only weak activity with IC50’s -3 pM (FIG. 48C and FIG. 48D). The data for A19-L was mixed in that while it showed a low tetramerization inhibitor IC50 of -1 .6 pM, at the 3.2 pM concentration it was actually able to inhibit a small amount of the EphrinB2-EphB2 unsurmountable tetramer as its sensorgram trace was clearly below that of the max control (FIG. 48B). [0286] FIG. 49A, FIG. 49B, FIG. 49C, FIG. 49D, and FIG. 49E show results of compounds A19-NO, QTM-NO, 2OH-A20, 2-MeA20, and QPB-NO4, respectively. Here, A19-NO, QTM- NO, and 2-MeA20 showed only weak ability to inhibit the tetramer with IC50’s >-3 uM, while 2OH-A20 and QPB-NO4 had poor/low activity with IC50’s -1.6 uM. Interesting, like compound A19-L, QPB-NO4 at the 3.2 pM concentration was also able to inhibit a small amount of the EphrinB2-EphB2 unsurmountable tetramer as its sensorgram was clearly below that of the max control, even more so than A19-L (FIG. 49E).

[0287] FIG. 50A, FIG. 50B, FIG. 50C, and FIG. 50D show results of compounds QPA, QPA- Ac, QPA-PAc, and BQPA-PAc, respectively. Here, QPA-Ac and BQPA-PAc showed only weak activity with IC50 > 3 pM, QPA and QPA-PAc had poor/low activity with IC50 -2 pM and -1.2 pM, respectively. Interesting again, like compound A19-L and QPB-NO4, QPA-PAc at both the 1.6 and 3.2 pM concentrations were able to inhibit a small amount of the EphrinB2- EphB2 unsurmountable tetramer as these two sensorgrams, especially the 3.2 pM, was clearly below that of the max control (FIG. 50C).

[0288] FIG. 51A, FIG. 51 B, FIG. 51C, FIG. 51 D, and FIG. 51 E show results of compounds 3511-1, 3511-0013-BF, 3511-4OMe-BF, 3511-4OHMe-l, and IQPB4-BN, respectively. All but 3511-0013-BF exhibit strong IC50 values. The Octet run for 3511-1 is particularly interesting as both the 0.8 and 1.6 pM concentrations were also able to inhibit a small amount of the EphrinB2-EphB2 unsurmountable tetramer. Interesting, the amount of unsurmountable tetramer inhibited by 3511-1 reached a maximum at 0.8 pM as there was no additional tetramer loss at the 1.6 pM concentration tested (FIG. 51 A).

[0289] Finally, FIG. 52 shows results from two independent Octet runs of compound BQPB4. Remarkably, this compound exhibits both a super-strong IC50 of ~0.3 pM for inhibiting the surmountable tetramer and at the same time shows an extremely strong concentration-dependent ability to inhibit 100% of the unsurmountable tetramer. With its exceptionally strong ability to inhibit all EphrinB-EphB2 tetramers, compound BQPB4 is the most potent chemical identified. FIG. 53 shows the six different compounds, led by BQPB4 that exhibit activity towards the unsurmountable tetramer.

Example 20: Summary of medicinal chemistry efforts

[0290] FIG. 54 a scheme that summarizes the medicinal chemistry efforts around the A20 scaffold and highlight the changes that can enhance/improve tetramer inhibitor activity (green and purple) and those which result in low/poor inhibitor activity (yellow) or weak/no activity (red). [0291] Additional modifications to both the A20 and 3511 scaffold are envisioned. These modifications may stabilize the molecule or may increase potency. Proposed modifications that will be synthesized are summarized in FIG. 55 and FIG. 56.

Example 21 : EPH-EPHRIN tetramerization inhibitors as a new class of drugs to treat chronic pain

[0292] Following a chronic pain generating nerve insult or injury, the expression of EphrinB2 becomes strongly increased in the nociceptors/C-fibers (pre-synaptic) and EphB1 becomes strongly increased in the DH neurons (post-synaptic), as previously shown using the chronic constriction injury (CGI) of the sciatic nerve model and in a bone cancer pain model (FIG. 57A-57B, see 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, each of which are incorporated herein by reference in their entirety). Important early studies of gene knockout (KO) mice showed that EphBT^/_ mutant animals are viable although they exhibit greatly diminished hyperalgesia and allodynia following various experimental forms of nerve damage that normally induce chronic pain, including CCI neuropathic pain and inflammatory pain models (FIG. 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, incorporated herein by reference in its entirety). Therefore, EphB1 KO mice lacking expression of this receptor do not get chronic pain, they are immune, and is the reason why it was sought to identify a drug compound to block this protein. Furthermore, as uninjured EphBT^/_ mutant mice exhibit normal levels of acute pain, their resistance to developing chronic pain is not simply because they cannot feel typical normal pain required for day-to-day life. EphB1 therefore is not involved in normal acute pain, but rather upon a chronic pain generating nerve injury or damage its expression become strongly upregulated and this is what drives pathological DH neuron plasticity and central sensitization. A central role for EphB1 in chronic pain is strengthened by the finding that EphB1^+/_ heterozygous animals with only one copy of the wild-type gene showed no neuropathic pain (FIG. 58 - the triangles). This indicates the EphB1 receptor protein is an ideal candidate to target for chronic pain because an effective drug compound will only need to block -50% of its activity.

[0293] Electrophysiological studies in live mice provides one of the strongest pieces of information that shows EphB1 can directly affect DH neuron plasticity in response to excessive pain stimulation. While high frequency stimulation of C-fibers evoked strong and long-lasting LTP in the spinal cord of live EphB1^+/+ wild-type mice, the same experiment done to EphBT^/_ mutant mice lacking expression of the EphB1 receptor showed absolutely no response (FIG. 59, see 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, incorporated herein by reference in its entirety). This demonstrates that without EphB1 protein expression, DH neurons fail to exhibit pathological plasticity associated with excessive stimulation of the C-fiber inputs. In addition to studies of EphB1 KO mice, other methods have been employed to assess the role of this receptor and its ligands in pathological pain, most notably intrathecal injections of soluble EphB and EphrinB ectodomain blocking/activating reagents into the spinal cord. In fact, some of the strongest data come from combined use of intrathecal injections of ectodomain proteins into EphBT^/_ mutant mice, which solidifies the idea that the EphB1 receptor tyrosine kinase is needed for the development of chronic/ neuropathic pain. Other key data show EphB1 is needed for the post-synaptic localization of the NMDAR, that EphB1 can directly bind to the NR1 subunit to control the synaptic localization of the NMDAR, that EphB1 forward signaling activates Src kinase family members to induce tyrosine phosphorylation of the NR2 subunit, which opens the NMDAR ion channel to allow for calcium influx and LTP , and by gain-of-function studies that show the NMDAR inhibitor MK-801 will block EphB1-stimulated neuropathic pain. Furthermore, it is noted that many of the above cited papers provide convincing evidence that the role for EphB1 may not be just to control synaptic localization and activity of the NMDAR, but may also act through activation of other intracellular neuronal signaling pathways implicated in plasticity, such as MAP kinases, PI3K, AKT, PKA, PKCy, ERK, CaMKII, and CREB. Thus, it appears at least with regards to DH synapses and central sensitization, the EphB1 receptor is at the top of the food chain to activate numerous downstream events to produce chronic pain (FIG. 60 and FIG. 2). This role in pain, is consistent with the trans-synaptic localization and overexpression of these proteins upon nerve injury/damage, which will lead to enhanced formation of EphrinB2-EphB1 tetramers and higher order signaling clusters. This will inappropriately over-activate intracellular bidirectional signal transduction events through both the receptor (forward signaling) and the Ephrin (reverse signaling) to strengthen pre-existing synapses and build additional new ones. And while much of the above data concerning EphB1 suggests excessive forward signaling by this receptor is to blame for chronic pain, it remains possible that reverse signaling by EphrinB2 (or other EphrinB’s) also participates in the pathology, perhaps in a role similar to what was described in the hippocampus where retrograde, pre-synaptic reverse signaling can affect synaptic plasticity. Furthermore, a closely related neuronal sister receptor EphB2 may also participate in pain by stimulating excitability of nociceptor primary sensory neurons. Thus, both EphB1 and EphB2 proteins as well as their cognate EphrinB proteins are likely involved in chronic pain and so we anticipate A20 tetramerization inhibitor chemicals will effectively target these possible interactions.

Example 22: A20 compounds exh ljke activity to b

[0294] Having demonstrated A20 will pass the BBB when injected IP at 20 mg/kg, a pilot experiment was conducted to assess if such injections will counteract inflammatory pain generated in the CFA model. It is previously determined that EphB1-/- KO mice exhibit greatly reduced formation of thermal and mechanical hyperalgesia following injection of CFA into the hind paw. A20 was therefore tested in 14-week-old CD1 mice for enhanced thermal sensitivity induced by CFA. Animals received 7 x 12 hr IP dosing of A20 or vehicle with 30 pl CFA being injected into the right hind paw between the 3rd and 4th dose. All vehicle control mice showed the expected rapid and long-lasting thermal pain hypersensitivity in their right hind paws (FIG. 61 - black stippled line), whereas the pain response in the A20 treated mice was remarkably blunted throughout the course of the 19-day study (FIG. 61 - red stippled line). Statistical analysis of the data indicated significant differences between the vehicle and A20 treated mice. Importantly, the uninjured left hind paws of A20 and vehicle injected mice showed no differences between each other (FIG. 61 - solid lines). This key internal control indicates A20 does not result in a global effect on the normal sense of pain, only on pain that develops after inflammatory insult.

[0295] In addition to A20 compounds, certain tetracycline antibiotics can act as EphB kinase inhibitors (KI) and can reduce inflammatory pain, especially an equal molar amount of three tetracycline drugs, termed MCD. A comparison was done with A20 to MCD using the same strategy as shown in FIG. 61 and found that A20 appears to be a more effective agent (FIG. 62). The results indicate that A20 may be more effective because of the ability of the tetramerization inhibitors to more potently interfere with Eph forward signaling and at the same time interfere with Ephrin reverse signaling, something the kinase inhibitors will not touch.

[0296] In the previous pain testing experiments, compounds were pre-loaded into the animal starting 2 days prior to the placement of the CFA inflammatory agent into the hind paw to induce pain. Next, it was tested if compounds would be effective at treating pain when administered after the CFA was injected into the hind paw. This is significant because any effective drug compound would likely need to be delivered after a pain-generating chronic/neuropathic insult has occurred the subject typically cannot predict when they will suffer a serious injury, except perhaps before a planned surgery. Further, the formulation of A20 for previous experiments described herein utilized the free base form of the compound and because of solubility issues required complex mixture of DMSO and sunflower seed oil as the vehicle. Therefore, tests were conducted to examine if highly soluble salt forms of A20 chemicals which can be dissolved simply in PBS as the vehicle can be effective anti-pain agents. For these purposes, compounds dissolved in PBS were first injected at 20 mg/kg 15 min after the placement of CFA inflammatory agent into the hind paw. Animals were then reinjected with compound every 12 hr for an additional 3 days (8 injections total). Thermal pain responses were determined both before CFA injection (baseline) and periodically for 2 weeks post-injection. The data shows salt forms of A20, as well as three novel analog chemicals (QPB4-Bn, QPB4, and QPP-127) all showed ability to blunt the enhanced sensitivity produced by the inflammatory pain insult (FIG. 63 and FIG. 64).

[0297] In summary, the described preclinical in vivo data shows that disclosed Eph-Ephrin tetramerization inhibitor compounds when administered either before or after a paingenerating noxious insult is highly efficacious.

Example 23: A20 compounds exhibit drug-like activity to blunt the activation of dorsal horn neurons jn the spinal cord ! associated wjth chronic pa jn and cent ra

[0298] Chronic pain generating nerve injuries/insults lead to overstimulation of the NMDAR in the superficial spinal cord and this results in the activation of DH neurons and formation of LTP. This DH neuron plasticity causes central sensitization and leads to the transmission of enhanced pain signals to the brain. And while such activity-dependent neuronal plasticity is crucial for normal learning and memory in the brain, plasticity of DH neurons involved in chronic pain can be viewed as a form of pathological or bad plasticity, for which blocking or preventing by targeting the EphB1 receptor with the disclosed tetramerization inhibitor drugs were examined. The molecular events triggered by the NMDAR that result in neuronal activation and plasticity involve a cascade of intracellular pathways, most notably an increase in expression of the immediate early gene (IEG) c-Fos. Moreover, consistent with its key role in pain, EphB1 KO mice was found to exhibit significantly reduced numbers of c-Fos expressing DH neurons after an inflammatory insult. Genetic tools are now available, Trapl and improved Trap2, that express CreERT2 under control of the c-Fos promoter and when combined with a Rosa26-stop-tdTomato Cre indicator (Ai9) and an IP injection of 4-hydroxy- tamoxifen (4-OHT), will permanently label with red fluorescence only neurons that were activated for a short window of time (~8 hr) until the 4-OHT is degraded. Trapl was used to study EphB2 in the structural plasticity of learning-associated neurons in the auditory cortex. Similarly, Trap2 can be used to study the role of EphB1 in the activation of DH neurons and to use it to quantitatively assess effect of the disclosed compounds on neuronal activation after a chronic pain-generating insult. [0299] To test and develop the system, Trap2 and Ai9 containing mice were obtained and crossed to mice that contained our EphB1 KO mutation to generate EphB1^+/+ WT mice, EphB1^+/_ heterozygous mice, and EphBT^/_homozygous mice that contained one copy of the Trap2 CreERT2 driver and one copy of the Ai9 tdTomato Cre reporter. The left hind paw of these mice was first injected with CFA to provide the chronic pain insult, and then after 4 hr to allow expression of CreERT2 to accumulate under the c-Fos/Trap2 promoter, mice were injected IP with 4-OHT to activate the Cre recombinase to induce deletion of the floxed-stop- tdTomato Ai9 element. This results in permanent strong expression of the tdTomato red fluorescent protein only in neurons that were activated during the short window of time the 4-OTH was active. Two weeks later, mice were perfused with fixative and lumbar spinal cords isolated, vibratome sectioned, and viewed using a fluorescent microscope to reveal the red labeled neurons that were activated two weeks earlier when CFA/4-OHT was injected. The fluorescent images and quantification of red neurons in the dorsal horn of the spinal cord of EphB1^+/+ WT mice revealed approximately 3 times more red labeled neurons on the injured side versus the uninjured side, whereas the EphB1^+/_ heterozygous mice showed approximately 2 times more neurons, and the EphB1-/- homozygote analyzed showed a near equal number of neurons on both sides (FIG. 65). The data shows a clear correlation in EphB1 gene dose to level of DH neuron activation in the spinal cord in response to a chronic pain-generating insult. This provides strong evidence that 50% reduction of EphB1 protein expression in EphB1^+/_ mice (or homozygosity) strongly reduces (or eliminates in the case of the KO) the early activation of DH neurons in response to a chronic pain-generating insult.

[0300] To test if injection with A20 lead compound would affect neuronal activity in the dorsal horn, it was determined that compound dramatically reduced DH neuron activation following CFA insult (FIG. 66). Thus, independent of behavioral tests, this new system allows to quantitatively visualize the effect of reduced EphB1 protein expression or its activity on chronic pain.

[0301] In summary, the use of Trap2/Ai9 system to permanently label the small number of neurons in the dorsal horn that become activated in a short period of time immediately following a chronic pain-generating insult will provide a powerful and independent method outside of the standard behavioral pain testing as shown in Figs. 61-64 to quantitatively assess the in vivo efficacy of the disclosed tetramerization inhibitor drug compounds.

Example 24: EPH-EPHRIN tetramerization inhibitors as a new class of drugs to combat opjold gjjdjjjtion.

[0302] Remarkably, like in chronic pain, it has previously been shown that expression of the EphB1 receptor is strongly upregulated in the dorsal horn of the spinal cord following escalating doses of morphine (FIG. 67), and EphBT^/_ KO mice exhibit greatly reduced intense withdrawal behaviors observed in morphine addicted mice (FIG. 68) (e.g., see 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 are incorporated herein by reference in their entirety). Targeted mutation of EphB1 receptor prevents development of neuropathic hyperalgesia and physical dependence on morphine in mice. The disclosed experiments further showed that EphB1 is also important for the development of tolerance to opioids as the KO mice exhibit a much longer analgesic responsiveness to morphine, and so tolerance builds slower in the absence of the receptor. Because EphB1 receptor is linked to the NMDAR, pharmacological targeting of the ability of EphB1 to interact with its EphrinB ligands will have great utility for countering the adverse effects of opioid use and abuse. Experiments were conducted to show that administration of tetramerization inhibitor A20 is very effective at reducing the adverse withdrawal behaviors in morphine addicted mice and will have great utility for countering the adverse effects experienced by those addicted to opioids.

Example 25; Reyjsjtjng the role of ' EphBl behaviors

[0303] It was recently reproduced and verified that EphBT^/_ KO mice exhibit greatly reduced morphine withdrawal behaviors in studies. Eight month old male EphB1^+/+ WT mice EphBT^/_ KO mice from the Henkemeyer mouse colony were injected intraperitoneally (IP) with increasing morphine doses in normal saline every 12 h for 4 consecutive days (day 1 : 20 mg/kg, day 2: 40 mg/kg, day 3: 60 mg/kg, day 4: 80 mg/kg) and on the fifth day in the morning they were injected with a final 100 mg/kg morphine dose which was staggered by one mouse every 30 minutes. Exactly 3 h after the last morphine injection, each mouse was weighed, and withdrawal was precipitated using naloxone hydrochloride that was subcutaneously injected at 1 mg/kg. Withdrawal signs were then monitored by an examiner who was blinded to animal genotypes for 30 min after naloxone administration to identify the number of jumps, wet dog shakes, and diarrhea events observed in the 30 min period, and % change in weight from before naloxone administration to 30 min post withdrawal. For tremor and ptosis, the presence of tremor and/or a drooping upper eyelid is monitored at the beginning of each 5 min interval during the 30 min monitoring period. Counting of the number of jumps a mouse makes can be accounted as one of the most robust method to score for morphine withdrawal as this behavior is very easy to identify and normal mice in a cage simply do not jump around, and here it was determined that the WT mice exhibited ~100 jumps during their 30 min period, whereas the KO mice exhibited ~50 jumps (FIG. 69). This demonstrates reproducibility of the data that connects EphB1 receptor to morphine withdrawal behaviors. [0304] Having confirmed the role for EphB1 in morphine withdrawal and having demonstrated that A20 will pass the BBB when injected IP, two pilot experiments were conducted to assess if such injections will minimize withdrawal behaviors. WT CD1 mice was used in one experiment and WT C57BL/6 mice was used in the other. All mice were 4-5- month-old males. In both cases, 30 mg/kg A20 free base dissolved in 6% DMSO I 94% sunflower seed oil was administered in 4 x 12 hr IP dosings to mice that were at an entering dose 6 of a 9 x 12 hr IP schedule of escalating morphine (2x20, 2x40, 2x60, 2x80, and1 x 100 mg/kg). Naloxone was given exactly 3 hr after the last A20/morphine dose and withdrawal behaviors were monitored for 30 min. Both groups of mice were scored for jumping (FIG. 70A) and the C57BL/6 mice were additionally scored at 5 min intervals for diarrhea, tremor, and ptosis (FIG. 70B). The A20 treated animals exhibited significantly fewer jumps and did not show signs of diarrhea or ptosis, and their tremors were reduced 50%. In summary, A20 tetramerization inhibitor compounds serve to help reduce the adverse effects of opioid use and abuse, and could possibly be used to help wean people off of terribly addictive narcotic drugs.

Example 26: Synthesis of Exemplary Compounds

[0305] The following compounds were synthesized according to schematics depicted in FIG. 36A, FIG. 36B and FIG. 36C and in FIGs. 71A-71X. Additional related compounds could be envisioned by slight modifications of the methods described herein. A method for generating a salt of one of these compounds is depicted in FIG. 16 (A20-2HCI) and is described further below. Salts of other compounds synthetized below can be easily envisioned based on the methods of FIG. 16 described herein.

Compound 1 (5-(morpholinomethyl)quinolin-8-ol, A19)

[0306] Synthetic Procedure 1 : Referring to FIG. 36A and FIG. 71 A, to a round bottom flask was added 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 equivalence of base. This solution was added dropwise to the reaction mixture, which was then refluxed at 80 °C for 12 hours. Following completion, the organic solvent was stripped from the crude reaction mixture under reduced pressure. The crude solid was taken up in dichloromethane and washed with water, NH4CI (aq) and brine, organic fractions were collected and concentrated. Resulting crude material was purified via column chromatography (EtoAc:Hex, 1 :1 , v:v) to yield target compound. NMR spectra is shown in FIG. 71A.

Compound 5 (7-bromo-5-(morpholinomethyl)quinolin-8-ol, A20)

[0307] Referring to FIG. 36A and FIG. 71 B, synthetic procedure 1 was followed to prepare Compound 1. Then, In a round bottom flask, 5-(morpholinomethyl)quinolin-8-ol (Compound 1) was dissolved in chloroform and cooled to 0 °C on an ice bath. N-bromosuccinimide was suspended in chloroform in a separate vial and added portion-wise to the reaction mixture in the flask over 5-10 minutes. The resulting mixture was stirred at 0 °C for 1 hr. Then the solvent was removed under vacuum at room temperature. The recovered solid/semisolid was purified using silica column chromatography (EtOAc:Hex, 1 :1 , v:v) to yield an off-white solid product. NMR spectra is shown in FIG. 71 B.

Compound 6 (4-((7-bromo-8-hydroxyquinolin-1-ium-5-yl)methyl)morpholin-4-ium chloride, A20-2HCI)

[0308] Referring to FIG. 36A and FIG. 71C, the free base of Compound 5 was generated as described above. Then, referring to FIG. 16, to a round bottom flask equipped with a stir bar, free-base Compound 5 (A20, 0.200 g, 0.62 mmol) was added and subsequently dissolved in CHCI3 or chloroform (5 mL). While stirring the solution at 0 °C, concentrated (12.1 M) HCI (106 uL, 1.27 mmol) diluted in methanol (2 mL) was added dropwise over 5 min. Continue to stir over ice for 30 minutes as yellow precipitate forms in solution then allow the reaction to warm to room temperature for 2.5 hours. The resulting precipitate was filtered in ambient conditions over a Buchner funnel, washed with CHCI3 or chloroform and collected as a pale-yellow solid (yield = 215 mg, 88 %). NMR spectra is shown in FIG. 71 C.

Compound 7 (7-iodo-5-(morpholinomethyl)quinolin-8-ol, A20-I)

[0309] Referring to FIG. 36A and FIG. 71 D, synthetic procedure 1 was followed to prepare Compound 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 hr. Then the solvent was removed under a vacuum. The recovered solid/semisolid was purified using silica column chromatography (EtOAc:Hex, 1 :1 , v:v) to yield an off-white solid product. NMR spectra is shown in FIG. 71 D.

Compound 9 (5-(morpholinomethyl)-7-nitroquinolin-8-ol, AI9-NO2)

[0310] Referring to FIG. 36A and FIG. 71 E, synthetic procedure 1 was followed to prepare Compound 1. In a round bottom flask, 5-(morpholinomethyl)quinolin-8-ol was dissolved in glacial acetic acid and cooled to 5-10 °C on an ice-water bath. Then the nitration mixture (H2SO4:HNO3 = 1 :1) was cooled and added drop-wise to the reaction mixture in the flask over 5-10 minutes. The resulting mixture was stirred and allowed to reach to room temperature over 3.5 hrs. Then the resulting suspension was added on to ice cold water (10-20 mL) and neutralized (pH 6-7) with a cold saturated NaHCOs until precipitation was obtained. The resulting solid was filtered under suction and washed with cold DI water and cold ether (a minimum volume) to obtain a yellow solid. NMR spectra is shown in FIG. 71 E.

Compound 14 (4-((8-hydroxyquinolin-5-yl)methyl)thiomorpholine 1,1-dioxide, QTM)

[0311] Synthetic Procedure 2: Referring to FIG. 36A and FIG. 71 F, to a round bottom flask was added thiomorpholine 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 equivalence of base. This solution was added dropwise to the reaction mixture, which was then refluxed at 80 °C for 12 hours. Following completion, the organic solvent was stripped from the crude reaction mixture under reduced pressure. The crude solid was taken up in dichloromethane and washed with water, NH₄CI (aq) and brine, organic fractions were collected and concentrated. Resulting crude material was purified via column chromatography (EtoAc:Hex, 1 :1 , v:v) to yield target compound. NMR spectra is shown in FIG. 71 F.

Compound 16 (4-((7-bromo-8-hydroxyquinolin-5-yl)methyl)thiomorpholine 1,1-dioxide, QTM-Br)

[0312] Referring to FIG. 36A and FIG. 71 G, synthetic Procedure 2 was followed to prepare Compound 14. 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 on an ice bath. N-bromosuccinimide was suspended in chloroform in a separate vial and added portion-wise to the reaction mixture in the flask over 5-10 minutes. The resulting mixture was stirred at 0 °C for 1 hr. Then the solvent was removed under vacuum at room temperature. The recovered solid/semisolid was purified using silica column chromatography (EtOAc:Hex, 1 :1 , v:v) to yield an off-white solid product. NMR spectra is shown in FIG. 71G.

Compound 18 (4-((8-hydroxy-7-iodoquinolin-5-yl)methyl)thiomorpholine 1,1-dioxide, QTM-I)

[0313] Referring to FIG. 36A and FIG. 71 H, synthetic Procedure 2 was followed to prepare Compound 14. 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 once to the reaction mixture in the flask. The resulting mixture was stirred at 50 °C for 12 hr. Then the solvent was removed under a vacuum. The recovered solid/semisolid was purified using silica column chromatography (EtOAc:Hex, 1 :1 , v:v) to yield an off-white solid product. NMR spectra is shown in FIG. 71 H.

Compound 20 (4-((8-hydroxy-7-nitroquinolin-5-yl)methyl)thiomorpholine 1,1-dioxide, QTM-NO2)

[0314] Referring to FIG. 36A and FIG. 711, Synthetic Procedure 2 was followed to prepare Compound 14. 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 on an ice-water bath. Then the nitration mixture (H2SO4:HNO3 = 1 :1) was cooled and added drop-wise to the reaction mixture in the flask over 5-10 minutes. The resulting mixture was then stirred at room temperature for over 6 hrs. The resulting solid suspension was kept in a refrigerator overnight for the completion of the reaction. Then the resulting suspension was added on to ice cold water (10-20 mL) and neutralized (pH 6-7) with a cold saturated NaHCOs until precipitation was obtained. The resulting solid was filtered under suction and washed with cold DI water and cold ether (minimum volume) to obtain a yellow solid. NMR spectra is shown in FIG. 711.

Compound 24 (7-bromo-5-((4-phenylpiperazin-1-yl)methyl)quinolin-8-ol, BQPPh)

[0315] Bromination procedure. Referring to FIG. 36B and FIG. 71 J, in a round bottom flask, 5-(chloromethyl)quinolin-8-ol was dissolved in chloroform and cooled to 0 °C on an ice bath. N-bromosuccinimide was suspended in chloroform in a separate vial and added portion-wise to the reaction mixture in the flask over 5-10 minutes. The resulting mixture was stirred at 0 °C for 1 hr. Then the solvent was removed under vacuum at room temperature. The recovered solid/semisolid was purified using silica column chromatography (EtOAc:Hex, 1 :1 , v:v) to yield an off-white solid product.

[0316] Continuing to refer to FIG. 36B and FIG. 71 J, to a round bottom flask was added 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 equivalence of base. This solution was added dropwise to the reaction mixture, which was then refluxed at 80 °C for 12 hours. Following completion, the organic solvent was stripped from the crude reaction mixture under reduced pressure. The crude solid was taken up in dichloromethane and washed with water, NH₄CI (aq) and brine, organic fractions were collected and concentrated. Resulting crude material was purified via column chromatography (EtoAc:Hex, 1 :1 , v:v) to yield target compound.

Compound 26 (5-((4-(4-bromophenyl)piperazin-1-yl)methyl)quinolin-8-ol, QPB4)

[0317] Referring to FIG. 36B and FIG. 71 K, to a round bottom flask was added 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 equivalence of base. This solution was added dropwise to the reaction mixture, which was then refluxed at 80 °C for 12 hours. Following completion, the organic solvent was stripped from the crude reaction mixture under reduced pressure. The crude solid was taken up in dichloromethane and washed with water, NH4CI (aq) and brine, organic fractions were collected and concentrated. Resulting crude material was purified via column chromatography (EtoAc:Hex, 1 :1 , v:v) to yield target compound. NMR spectra is shown in FIG. 71 K.

Compound 28 (7-bromo-5-((4-(4-bromophenyl)piperazin-1 -yl)methyl)quinolin-8-ol, BQPB4)

[0318] Referring to FIG. 36B and FIG. 71 L, the Bromination Procedure as described for Compound 24, above, was followed. Then, to a round bottom flask was added 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 equivalence of base. This solution was added dropwise to the reaction mixture, which was then refluxed at 80 °C for 12 hours. Following completion, the organic solvent was stripped from the crude reaction mixture under reduced pressure. The crude solid was taken up in dichloromethane and washed with water, NH₄CI (aq) and brine, organic fractions were collected and concentrated. Resulting crude material was purified via column chromatography (EtoAc:Hex, 1 :1 , v:v) to yield target compound. NMR spectra is shown in FIG. 71 L.

Compound 35 (7-bromo-5-((4-(5-bromopyridin-2-yl)piperazin-1-yl)methyl)quinolin-8-ol, BQPP) [0319] Referring to FIG. 36B and FIG. 71M, the Bromination Procedure as described for Compound 24 above was followed. Then to a round bottom flask was added 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 equivalence of base. This solution was added dropwise to the reaction mixture, which was then refluxed at 80 °C for 12 hours. Following completion, the organic solvent was stripped from the crude reaction mixture under reduced pressure. The crude solid was taken up in dichloromethane and washed with water, NH₄CI (aq) and brine, organic fractions were collected and concentrated. Resulting crude material was purified via column chromatography (EtoAc:Hex, 1 :1 , v:v) to yield target compound. NMR spectra is shown in FIG. 71 M.

Compound 41 (5-((4-benzylpiperazin-1-yl)methyl)quinolin-8-ol, QP-Bn)

[0320] Referring to FIG. 36B and FIG. 71 N, to a round bottom flask was added 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 equivalence of base. This solution was added dropwise to the reaction mixture, which was then refluxed at 80 °C for 12 hours. Following completion, the organic solvent was stripped from the crude reaction mixture under reduced pressure. The crude solid was taken up in dichloromethane and washed with water, NH4CI (aq) and brine, organic fractions were collected and concentrated. Resulting crude material was purified via column chromatography (EtoAc:Hex, 1 :1 , v:v) to yield target compound. NMR spectra is shown in FIG. 71 N.

Compound 42 (5-((4-(4-bromobenzyl)piperazin-1-yl)methyl)quinolin-8-ol, QPB4-Bn)

[0321] Referring to FIG. 36B and FIG. 710, to a round bottom flask was added 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 equivalence of base. This solution was added dropwise to the reaction mixture, which was then refluxed at 80 °C for 12 hours. Following completion, the organic solvent was stripped from the crude reaction mixture under reduced pressure. The crude solid was taken up in dichloromethane and washed with water, NH4CI (aq) and brine, organic fractions were collected and concentrated. Resulting crude material was purified via column chromatography (EtoAc:Hex, 1 :1 , v:v) to yield target compound. NMR spectra is shown in FIG. 710.

Compound 44 (7-bromo-5-((4-(4-bromobenzyl)piperazin-1 -yl)methyl)quinolin-8-ol, BQPB4-Bn)

[0322] Referring to FIG. 36B and FIG. 71 P, the Bromination Procedure followed as described for Compound 24 was followed. Then, to a round bottom flask was added 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 equivalence of base. This solution was added dropwise to the reaction mixture, which was then refluxed at 80 °C for 12 hours. Following completion, the organic solvent was stripped from the crude reaction mixture under reduced pressure. The crude solid was taken up in dichloromethane and washed with water, NH4CI (aq) and brine, organic fractions were collected and concentrated. Resulting crude material was purified via column chromatography (EtoAc:Hex, 1 :1 , v:v) to yield target compound. NMR spectra is shown in FIG. 71 P.

Compound 48 (5-((4-(4-(trifluoromethyl)benzyl)piperazin-1 -yl)methyl)quinolin-8-ol, QPCF34-Bn)

[0323] Referring to FIG. 36B and FIG. 71 Q, to a round bottom flask was added 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 equivalence of base. This solution was added dropwise to the reaction mixture, which was then refluxed at 80 °C for 12 hours. Following completion, the organic solvent was stripped from the crude reaction mixture under reduced pressure. The crude solid was taken up in dichloromethane and washed with water, NH4CI (aq) and brine, organic fractions were collected and concentrated. Resulting crude material was purified via column chromatography (EtoAc:Hex, 1 :1 , v:v) to yield target compound. NMR spectra is shown in FIG. 71Q.

Compound 50 (N-(4-(4-((8-hydroxyquinolin-5-yl)methyl)piperazin-1- yl)phenyl)acetamide, QPA-Ac)

[0324] Referring to FIG. 36B and FIG. 71 R, top, the amido substituted amidepiperazine derivative was prepared in a two-step procedure. First, A round bottom flask was charged with aminopiperazine derivative and stir bar was purged under vacuum then backfilled with N₂ gas (3x). To the sealed vessel was added dichloromethane and triethylamine and allowed to stir at 0 °C for 5 minutes, followed by dropwise addition of the acetyl chloride to to the reaction mixture, which was allowed to stir at room temperature for 12 hours. Following completion, the organic solvent was stripped from the crude reaction mixture under reduced pressure. The crude solid was taken up in dichloromethane and washed with water, NH₄CI (aq) and brine, organic fractions were collected and concentrated. Resulting crude material was purified via column chromatography (EtoAc:Hex, 1 :1 , v:v) to yield target compound. Then, the resulting amide product was added to a round bottom flask and dissolved in dichloromethane. To this solution was added trifluoroacetic acid dropwise, and the reaction was allowed to stir at room temperature for 12 hours. After completion, the crude mixture was stripped of organic solvent under reduced pressure to yield a pink oil. The crude material was washed with ether and resulting solid was dried under high vacuum and subjected directly to the next reaction.

[0325] Then, continuing as shown in FIG. 36B, bottom, to a round bottom flask was added the amidepiperazine 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 equivalence of base. This solution was added dropwise to the reaction mixture, which was then refluxed at 80 °C for 12 hours. Following completion, the organic solvent was stripped from the crude reaction mixture under reduced pressure. The crude solid was taken up in dichloromethane and washed with water, NH4CI (aq) and brine, organic fractions were collected and concentrated. Resulting crude material was purified via column chromatography (EtoAc:Hex, 1 :1 , v:v) to yield target compound. NMR spectra is shown in FIG. 71 R.

Compound 56 (2-methyl-5-(morpholinomethyl)quinolin-8-ol, 2-MeA19)

[0326] Synthetic Procedure 3: Referring to FIG. 36A and FIG. 71 S, to a round bottom flask was added the morpholine 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 equivalence of base. This solution was added dropwise to the reaction mixture, which was then refluxed at 80 °C for 12 hours. Following completion, the organic solvent was stripped from the crude reaction mixture under reduced pressure. The crude solid was taken up in dichloromethane and washed with water, NH4CI (aq) and brine, organic fractions were collected and concentrated. Resulting crude material was purified via column chromatography (EtoAc:Hex, 1 :1 , v:v) to yield target compound. NMR spectra is shown in FIG. 71S.

Compound 57 (7-bromo-2-methyl-5-(morpholinomethyl)quinolin-8-ol, 2-MeA20)

[0327] Referring to FIG. 36A and FIG. 71T, synthetic procedure 3 was performed 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 on an ice bath. N-bromosuccinimide was suspended in chloroform in a separate vial and added portion-wise to the reaction mixture in the flask for 5-10 minutes. The resulting mixture was stirred at 0 °C for 1 hr. Then the solvent was removed under vacuum at room temperature. The recovered solid/semisolid was purified using silica column chromatography (EtOAc:Hex, 1 :1 , v:v) to yield an off-white solid product. NMR spectra is shown in FIG. 71T.

Compound 60 (5-((4-(4-bromophenyl)piperazin-1-yl)methyl)-2-methylquinolin-8-ol, 2- MeQPB4)

[0328] Referring to FIG. 36B and FIG. 71 II, to a round bottom flask was added 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 equivalence of base. This solution was added dropwise to the reaction mixture, which was then refluxed at 80 °C for 12 hours. Following completion, the organic solvent was stripped from the crude reaction mixture under reduced pressure. The crude solid was taken up in dichloromethane and washed with water, NH4CI (aq) and brine, organic fractions were collected and concentrated. Resulting crude material was purified via column chromatography (EtoAc:Hex, 1 :1 , v:v) to yield target compound. NMR spectra is shown in FIG. 71 II.

Compound 69 (5-((4-(2-methoxyphenyl)piperazin-1-yl)methyl)quinolin-8-ol, 3511-0013- BF)

[0329] Referring to FIG. 36B and FIG. 71V, to a round bottom flask was added 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 equivalence of base. This solution was added dropwise to the reaction mixture, which was then refluxed at 80 °C for 12 hours. Following completion, the organic solvent was stripped from the crude reaction mixture under reduced pressure. The crude solid was taken up in dichloromethane and washed with water, NH4CI (aq) and brine, organic fractions were collected and concentrated. Resulting crude material was purified via column chromatography (EtoAc:Hex, 1 :1 , v:v) to yield target compound. NMR spectra is shown in FIG. 71V.

Compound 74 (7-iodo-5-((4-(4-methoxyphenyl)piperazin-1-yl)methyl)quinolin-8-ol, 3511-4OMe-l)

[0330] Referring to FIG. 36B and FIG. 71W, to a round bottom flask was added 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 equivalence of base. This solution was added dropwise to the reaction mixture, which was then refluxed at 80 °C for 12 hours. Following completion, the organic solvent was stripped from the crude reaction mixture under reduced pressure. The crude solid was taken up in dichloromethane and washed with water, NH4CI (aq) and brine, organic fractions were collected and concentrated. Resulting crude material was purified via column chromatography (EtoAc:Hex, 1 :1 , v:v) to yield 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 once to the reaction mixture in the flask. The resulting mixture was stirred at 50 °C for 12 hr. Then the solvent was removed under a vacuum. The recovered solid/semisolid was purified using silica column chromatography (25-50% EtOAc in Petroleum Ether) to yield an off-white solid product. NMR spectra is shown in FIG. 71W.

Compound 72 (5-((4-(4-methoxyphenyl)piperazin-1-yl)methyl)quinolin-8-ol, 3511- 4OMe-BF)

[0331] Referring to FIG. 36B and FIG. 71X, to a round bottom flask was added 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 equivalence of base. This solution was added dropwise to the reaction mixture, which was then refluxed at 80 °C for 12 hours. Following completion, the organic solvent was stripped from the crude reaction mixture under reduced pressure. The crude solid was taken up in dichloromethane and washed with water, NH4CI (aq) and brine, organic fractions were collected and concentrated. Resulting crude material was purified via column chromatography (EtoAc:Hex, 1 :1 , v:v) to yield target compound. NMR spectra is shown in FIG. 71X.

Compound 91 (7-bromo-5-((4-(4-bromothiazol-2-yl)piperazin-1-yl)methyl)quinolin-8-ol, BQPT)

[0332] Referring to FIG. 36B, to a round bottom flask was added 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 equivalence of organic base. This solution was added dropwise to the reaction mixture, which was then refluxed at 80 °C for 12 hours. Following completion, the organic solvent was stripped from the crude reaction mixture under reduced pressure. The crude solid was taken up in dichloromethane and washed with water, NH4CI (aq) and brine, organic fractions were collected and concentrated. Resulting crude material was purified via column chromatography (EtoAc:Hex, 1 :1 , v:v) to yield 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. N-bromosuccinimide suspended in chloroform in a separate vial and added portion- wise to the reaction mixture in the flask for 5-10 minutes. The resulting mixture was stirred at 0 °C for 1 hr. Then the solvent was removed under a vacuum. The recovered solid/semisolid was purified using silica column chromatography (EtOAc:Hex, 2:1 , v:v) to yield an 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)

[0333] Referring to FIG. 36B, to a round bottom flask was added 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 equivalence of organic base. This solution was added dropwise to the reaction mixture, which was then refluxed at 80 °C for 12 hours. Following completion, the organic solvent was stripped from the crude reaction mixture under reduced pressure. The crude solid was taken up in dichloromethane and washed with water, NH₄CI (aq) and brine, organic fractions were collected and concentrated. Resulting crude material was purified via column chromatography (EtoAc:Hex, 2:1 , v:v) to yield 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. N-bromosuccinimide suspended in chloroform in a separate vial and added portionwise to the reaction mixture in the flask for 5-10 minutes. The resulting mixture was stirred at 0 °C for 1 hr. Then the solvent was removed under a vacuum. The recovered solid/semisolid was purified using silica column chromatography (EtOAc:Hex, 2:1 , v:v) to yield an 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, BQP2CI) Referring to FIG. 36B, to a round bottom flask was added 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 equivalence of organic base. This solution was added dropwise to the reaction mixture, which was then refluxed at 80 °C for 12 hours. Following completion, the organic solvent was stripped from the crude reaction mixture under reduced pressure. The crude solid was taken up in dichloromethane and washed with water, NH₄CI (aq) and brine, organic fractions were collected and concentrated. Resulting crude material was purified via column chromatography (EtoAc:Hex, 1 :1 , v:v) to yield 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. N-bromosuccinimide suspended in chloroform in a separate vial and added portionwise to the reaction mixture in the flask for 5-10 minutes. The resulting mixture was stirred at 0 °C for 1 hr. Then the solvent was removed under a vacuum. The recovered solid/semisolid was purified using silica column chromatography (EtOAc:Hex, 2:1 , v:v) to yield an 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)

[0334] Referring to FIG. 36B, to a round bottom flask was added 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 equivalence of organic base. This solution was added dropwise to the reaction mixture, which was then refluxed at 80 °C for 12 hours. Following completion, the organic solvent was stripped from the crude reaction mixture under reduced pressure. The crude solid was taken up in dichloromethane and washed with water, NH₄CI (aq) and brine, organic fractions were collected and concentrated. Resulting crude material was purified via column chromatography (CHC^MeOH, 95:5 v:v) to yield 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. N-bromosuccinimide suspended in chloroform in a separate vial and added portion-wise to the reaction mixture in the flask for 5-10 minutes. The resulting mixture was stirred at 0 °C for 1 hr. Then the solvent was removed under a vacuum. The recovered solid/semisolid was purified using silica column chromatography (CHC MeOH, 95:5 v:v) to yield an 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)

[0335] Referring to FIG. 36B, to a round bottom flask was added 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 equivalence of organic base. This solution was added dropwise to the reaction mixture, which was then refluxed at 80 °C for 12 hours. Following completion, the organic solvent was stripped from the crude reaction mixture under reduced pressure. The crude solid was taken up in dichloromethane and washed with water, NH₄CI (aq) and brine, organic fractions were collected and concentrated. Resulting crude material was purified via column chromatography (EtoAc:Hex, 1 :1 , v:v) to yield 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)

[0336] Referring to FIG. 36B, to a round bottom flask was added 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 equivalence of organic base. This solution was added dropwise to the reaction mixture, which was then refluxed at 80 °C for 12 hours. Following completion, the organic solvent was stripped from the crude reaction mixture under reduced pressure. The crude solid was taken up in dichloromethane and washed with water, NH4CI (aq) and brine, organic fractions were collected and concentrated. Resulting crude material was purified via column chromatography (CHCl3:MeOH, 95:5 v:v) to yield 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. N-bromosuccinimide suspended in chloroform in a separate vial and added portion-wise to the reaction mixture in the flask for 5-10 minutes. The resulting mixture was stirred at 0 °C for 1 hr. Then the solvent was removed under a vacuum. The recovered solid/semisolid was purified using silica column chromatography (CHC MeOH, 95:5 v:v) to yield an off-white solid product 7-bromo-5-((4-((4-bromothiophen-2-yl)methyl)piperazin-1- yl)methyl)quinolin-8-ol.

[0337] As mentioned above, various biophysical parameters for certain synthesized compounds were determined and are described in Tables A-G at end of the Examples.

Example 27: Selective inhibition of A20-I on EphB2-EphB2 receptor interaction.

[0338] In another experiment, whether certain A20 analogs might have specificity for different Eph receptors was tested. An Octet RED384 system was used to obtain biophysical ON-OFF binding data (association-dissociation) for the three different human EphrinB2- EphB1 , EphrinB2-EphB2, and EphrinB2-EphB4 protein-protein interactions and to assess the effect of A20 chemicals on the binding kinetics.

[0339] As described above, the interactions of Ephrin-Eph ectodomains probed by this biophysical analysis allow for the investigation into the complex interplay of protein interactions that occur with these ligand-receptor partners. The association step is initially marked by quick formation of Ephrin-Eph dimers within the first 15” of the association step that exhibit fast-ON/fast-OFF dynamics, which is then followed by slower formation of the circular tetramer species when two Ephrin-Eph dimers meet and complex. Tetramers continue to slowly accumulate during the association step and form an extremely stable macromolecular complex that persists through to the end of the dissociation step. Importantly, the circular tetramer complex is the key macromolecular structure these receptors and ligands need to assemble into to activate both Eph-forward and Ephrin-reverse signaling into the cells they are expressed on.

[0340] FIGs. 72A-72C show three example Octet sensorgram response traces for the immobilized human EphrinB2 ectodomain protein binding to soluble forms of the human EphB1 , EphB2, and EphB4 ectodomain proteins, in the absence or presence of an A20 chemical (buffer conditions are always PBSTD: PBS with 0.05% Tween-20, 1 % DMSO, and +/- indicated chemical). After loading EphrinB2-Fc protein to Octet AHC biosensors, washing, and obtaining stable baseline measurements in the appropriate PBSTD (either with or without indicated chemical), the sensors are placed in its buffer that also contains the indicated soluble EphB protein for the association step (0-80”), and then returned to its buffer for the dissociation step (81”-580”). In each figure (FIGs. 72A-72C), a sensorgram run using 10 uM of the 2xHCI salt form of A20-I is compared to the no compound control run to exemplify the maximum level of reduced response signal typically obtained with a potent A20 chemical.

[0341] Based on the effect of the compound during the 80” association step, it is shown that compound A20-I, like other strong A20 tetramerization inhibitors, exhibits greatest ability to reduce the EphrinB2-EphB2 interaction (61.8%), followed by EphrinB2-EphB1 (36.6%), and then by EphrinB2-EphB4 (13.9%) as calculated by comparing the AUG of association steps without and with compound (red asterisks). In general, during the 80” association step, strong tetramerization inhibitor chemicals like A20-I exhibit approximately a 60%, 30%, and 15% ability to disrupt binding of the EphrinB2 ligand ectodomain to its cognate EphB2, EphB1 , and EphB4 receptor ectodomains, respectively.

[0342] In the 500” dissociation step, compound A20-I exhibited striking ability within 40” to rapidly reduce the amount of bound EphB2 (representing the dimer component melting), leaving a steady, but very low level of super-stable unsurmountable tetramers that do not dissociate even by the end of the full 580” run. The effect of A20-I on EphB1 dissociation is similar to that observed for EphB2, with a rapid loss of some bound EphB1 protein within the first 60-80” (the dimer melting) that then leaves behind a stabilized response level of superstable unsurmountable tetramers that remain through to the end of the full 580” run. The dissociation kinetics of EphB4 are different, here only a slow gradual loss of bound EphB4 protein is observed with a response level that continues its downward slope through the end of the 580” run.

[0343] Because the response levels during the dissociation step plateau for the EphrinB2- EphB2 and EphrinB2-EphB1 interactions by the first 80”, ALICs were also calculated for run data encompassing the first 160” of each experiment to account for both the 80” association data (red asterisks) and the first 80” of dissociation data (marked by green line and asterisks). This revealed A20-I inhibited 76.5% of the EphrinB2-EphB2 interaction, 48.5% the EphrinB2- EphB1 interaction, and only 33.9% the EphrinB2-EphB4 interaction as summarized in Table 3, below.

Table 3: % Inhibited (AUC) Example 28: Selective inhibition of A20-I and BQPB4 on EphB2-EphB2 receptor interaction.

[0344] The selectivity of another A20 analog (a 3xHCL salt form of compound BQPB4) in comparison to previously tested compound, A20-I, was tested as described in Example 27 above. FIGs. 73A-73C are representative sensorgrams showing that BQPB4, like A20-I and other strong A20 tetramerization inhibitors, exhibits greatest ability to reduce the EphrinB2- EphB2 interaction, followed by EphrinB2-EphB1 , and then EphrinB2-EphB4 as calculated by comparing the AUG of association steps with and without compounds.

[0345] Specifically, during the 80” association step, BQPB4 like A20-I exhibits a similar - 60%, -30%, and -15% ratio in ability to disrupt binding of the EphrinB2 ectodomain to its cognate EphB2, EphB1 , and EphB4 ectodomains, respectively. In the 500” dissociation step, compound BQPB4, like A20-I, exhibited striking ability to within 40” rapidly reduce the amount of bound EphB2 to a very low level of tetramers. However, unlike A20-I, with BQPB4 the dissociation response did not plateau, but rather continued to gradually decrease and reached the baseline by the end of the full 580” run (response = 0). The effect of BQPB4 on EphB1 dissociation is similar to that observed for EphB2, with a rapid loss of some bound EphB1 protein within the first 60-80”, however that was followed by only a slight gradual loss of response signal through the remaining dissociation step. This indicates BQPB4 exhibits less ability to disrupt the EphrinB2-EphB1 tetramers compared to its stronger effect on the EphrinB2-EphB2 unsurmountable tetramers. A summary of the association (area under the curve) determined for association only (0-80”) and for association + dissociation (0-160”) is provided in Table 4, below.

Table 4: % Inhibited (AUC).

Example 29: Dose-Response of A20-I at inhibiting different Eph-EphR interactions

[0346] Further to the experiments listed above, a series of dose-response experiments were conducted to determine the IC50 of a selected A20 chemical (A20-I) on the kinetics of human EphrinB2 ligand ectodomain protein binding to its cognate human EphB1 , EphB2, and EphB4 receptor ectodomain proteins. FIG. 74A-74C are representative sensorgrams showing the effect of 0, 0.1 , 0.2, 0.4, 0.8, 1.6, and 3.2 pM, concentrations of the 2xHCI salt of A20-I along with a 10 pM max standard (also A20-I) on three different Ephrin-EphB interactions (e.g., EphB1 in FIG. 74A, EphB2 in FIG. 74B and EphB4 in FIG. 74C). In addition to using AUG calculations of the sensorgrams to obtain % inhibition information for the highest dose of A20 chemical tested for the IC50 (typically 3.2 pM, see Table 5 below), the ALICs for the various dose-responses are used to calculate IC50 values for the association only (0-80”) or for the combined association+dissociation full run (0-580”). Additional IC50 values were determined at specific timepoints, at 75” into the association step and at 55” into the dissociation step (135”).

[0347] All absolute and relative IC50 values are provided in the Table 6 below, where absolute are based off the full binding sensorgram traces, and relative are based off of the max control traces. As they remove the portion of the EphrinB2-EphB binding that is due to dimer dynamics not affected by the A20 chemicals, the relative IC50 values are most useful as they provide description of a compound’s concentration needed to specifically disrupt 50% of only the tetramer species being analyzed.

Table 5: % Inhibited (AUC)

Table 6: IC₅o Values

Example 29: Dose-Response of BQP4 at inhibiting different Eph-EphR interactions [0348] Experiments described in Example 28 were repeated using BQP4. FIGs. 75A-75C depicts representative sensorgrams showing the effect of 0, 0.1 , 0.2, 0.4, 0.8, 1.6, and 3.2 pM concentrations of BQP4 along with a 10 pM max standard (also A20-I) on three different Ephrin-EphB interactions (e.g., EphB1 in FIG. 75A, EphB2 in FIG. 75B and EphB4 in FIG. 75C). It was found that BQPB4 strongly reduced sensorgram response to the max control level at only 0.4 pM.

[0349] AUG values derived from the sensorgrams are described 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.

Table 7: % Inhibited (AUC)

Table 8: IC₅o Values

Example 30: Determining the % inhibition for various A20 analog chemicals against the three different EphrinB2-EphB protein interactions

[0350] In another experiment, the % inhibition for various A20 analog chemicals against the three different EphrinB2-EphB protein interactions was determined. Specifically, the percent inhibition for immobilized human EphrinB2-Fc binding to 25 nM soluble human EphB1-His (FIG. 76A, enlarged in FIG. 76D), EphB2-His (FIG. 76B, enlarged in FIG. 76E), and EphB4- His (FIG. 76C, enlarged in FIG. 76F) with 3511-1, 8009-9255, QTM-Br, A19, A19-NO2, and IM (all at 3.2 pM) was determined. While the majority of A20 related chemicals analyzed exhibit the typical -60-30-15% ratio of inhibition of EphB2-EphB1-EphB4 binding during the 80” association step (exemplified by the A20-I max control), this analysis identified analogs that show much less activity towards the EphrinB2-EphB4 interaction. Specifically, the data shown in FIGs. 76A-76E and summarized in Tables 9-10 below, show that compound A19- NO2 exhibits approximately 20-fold less % inhibition activity toward the EphrinB2-EphB4 interaction (bolded values in Table 9 and 10 and FIGs. 76D-76E which are enlarged images of the first 160 sec of sensorgram traces in FIGs. 76A-76C) while retaining nearly all of its inhibitory activity towards EphrinB2-EphB2 and EphrinB2-EphB1 interactions. Note that compounds 3511-1, 8009-9255, and QTM-Br exhibit activity similar to A20-I max control, whereas A19 was generally less active towards all three interactions, and the indole ring compound IM showed no activity across the board.

Table 9: % Inhibition: Association ONLY (80 sec)

" nhibition percent was less than 0, indicating no inhibition of protein-protein interactions

Table 10: % Inhibition: Association +Dissociation (160 sec)

""Inhibition percent was less than 0, indicating no inhibition of protein-protein interactions

Example 31 : Determining the % inhibition for additional A20 analog chemicals against the three different EphrinB2-EphB protein interactions

[0351] A similar experiment as described in Example 30 was performed with a different set of A20 analog chemicals. Specifically, BQPB4-BN, A19, A19-NO2 (A-free base), A19-NO2 (B - 2XHCI salt), IPM2 and QPB4-BN were all tested at 3.2 pM to determine the percent inhibition of each of these compounds on immobilized human EphrinB2-Fc binding to 25 nM soluble human EphB1-His (FIG. 77A, FIG. 77D), EphB2-His (FIG. 77B, FIG. 77E), and EphB4-His (FIG. 77C, FIG. 77F). FIG. 77A-77C show close-ups of the first 160” of another Octet run testing both free base (FB) and 2xHCI salt forms of compound A19-NO2, indicating again a differential ability of this compound to selectively inhibit EphrinB2-EphB2 and EphrinB2-EphB1 interactions, while sparing most of the EphrinB2-EphB4 interaction. FIGs. 77D-77F show all eight traces from this experiment, indicating that QPB4-Bn, like A19-NO2, also exhibits ability to selectively inhibit EphrinB2-EphB2 and EphrinB2-EphB1 interactions, while sparing the EphrinB2-EphB4 interaction (see bolded values in summary Tables 11 and 12 below).

Table 11 : % Inhibition: Association ONLY (80 sec)

" nhibition percent was less than 0, indicating no inhibition of protein-protein interactions

Table 12: % Inhibition: Association +Dissociation (160 sec)

""Inhibition percent was less than 0, indicating no inhibition of protein-protein interactions

Example 32: Determining the % inhibition for additional A20 analog chemicals against the three different EphrinB2-EphB protein interactions

[0352] In another experiment, the percent inhibition of immobilized human EphrinB2-Fc binding to 25 nM soluble human EphB1-His, EphB2-His, and EphB4-His proteins with 3.2 pM of 12 compounds (2OH-A20, A20-1.2xMSA, QPCF34-BN, A20, QPB4, QPA-Pac, QTM-NO2, QPP, QP-Bn, 3511-0013) and A20-1 as max control. Data is summarized in Tables 11-14 below. Compounds QPCF34-Bn, QPB4, QTM-NO2, and QP-Bn also exhibit ability to selectively inhibit EphrinB2-EphB2 and EphrinB2-EphB1 interactions, while sparing the EphrinB2-EphB4 interaction (bolded in Tables 13-16). QPCF34-Bn appears more selective at inhibiting EphB2 interaction with little activity towards EphB1 (bolded in Tables 13 and 14), whereas QPBN appears more selective for EphB1 (bolded in Tables 15 and 16). 8- Hydroxyquinoline (8-HQ), part of the building block of A20 chemicals and a chemical with known metal chelation activity, was unable to disrupt any of the EphrinB2-EphB interactions (See Tables 13 and 14 below). This indicates metal chelator action is likely not the mode of action of A20 chemicals. Table 13: % Inhibition: Association ONLY (80 sec)

" nhibition percent was less than 0, indicating no inhibition of protein-protein interactions

Table 14: % Inhibition: Association +Dissociation (160 sec) inhibition percent was less than 0, indicating no inhibition of protein-protein interactions Table 15: % Inhibition: Association ONLY (80 sec) inhibition percent was less than 0, indicating no inhibition of protein-protein interactions

Table 16: % Inhibition: Association +Dissociation (160 sec) inhibition percent was less than 0, indicating no inhibition of protein-protein interactions

Example 33: Identification of A20 Analogs with specificity for EphB1 , EphB2, or EphB1 and EphB2

[0353] Results in the previous experiments was analyzed and used to identify different chemical structures that have different specificity for different EphrinB-EphB interactions. The results are summarized in FIG. 78 identify A19-NO2 (free base or HCL salt), QTM-NO2, and QPB4 as having less action towards EphB4 but high specificity for EphB1 and EphB2; QPA and QPCF34-Bn as having less action toward EphB4 and EphB1 but high specificity for EphB2; and QP-Bn, 3511-0113 BF and BQPA-Pac as having less action toward EphB4 and EphB2 but high specificity for EphB1.

Example 34: Intraperitoneal injection (IP) of A20, A20-I, and 3511-1 leads to reduced levels of EphB2 activation and association with EphrinB2 in the brain

[0354] Wild-type mice aged 10-12 weeks were IP injected every 12 hr for four days with vehicle (PBS) or 10 mg/kg of 2x.HCI salt forms of A20 (in PBS), A20-I (in PBS), or 3511-1, in 94% PBS/6% DMSO) for a total of 8 injections. Exactly 2 hr after final injection of A20 chemical, brains were dissected and frozen at -80 °C. Whole brain protein lysates were then prepared in a Dounce homogenizer using 5 ml PLC lysis buffer, and 1 ml of lysate was used for immunoprecipitation (IP) with 1 pg goat anti-EphB2 antibody, followed by immunoblot (IB) analysis with goat anti-EphB2, rabbit anti-phosphoEphB1/B2 (pEphB1/B2), and mouse anti- EphrinB2 antibodies using appropriate HRP conjugated secondary antibodies and chemiluminescent detection methods.

[0355] FIG. 79A depicts an illustrative immunoblot showing expression of EphB2, phospho- EphB1/B2, and EphrinB2 (co-IP) in the mouse brains. The EphB2 antibody immunoblot showed equal expression of the receptor in all the mouse brains as expected. The phospho- EphB specific antibody immunoblot, which recognizes the tyrosine phosphorylated juxtamembrane segment of EphB2 and is a readout for the activated tyrosine kinase catalytic domain, showed strong tyrosine phosphorylation of the receptor in the two vehicle (V) control lanes. The level of phospho-EphB signal in the brains of A20 chemical injected animals showed generally lower levels of activation as indicated by fainter bands in their corresponding lanes. The EphrinB2 specific antibody immunoblot shows a strong band in the vehicle (V) lane indicating EphrinB2 co-immunoprecipitates with EphB2 in the brain. The level of co-immunoprecipitated EphrinB2 from the brains of A20 chemical injected animals was generally lower as indicated by fainter bands. FIG. 79B and 79C show quantification of band intensities (number of photons detected) to obtain ratios of pEphB/EphB2 as a measure of EphB2 receptor activation levels (FIG. 79B) and ratios of EphrinB2/EphB2 as a measure of how much EphrinB2 is co-immunoprecipitated with the EphB2 receptor (FIG. 79C). The data indicates that animals IP injected with A20, A20-I, and 3511-1 show a generally lower level of activated phosphorylated EphB2 in the brain and that this is associated with a reduced level of association with EphrinB2.

Example 35: Oral dosing (PO) of A20 and A20-I leads to reduced levels of EphB2 activation in the brain

[0356] Whole brain protein lysates were prepared from wild-type mice either oral dosed (PO) or injected (IP) for 4 days with vehicle (PBS) or 20 mg/kg of 2x.HCI salt drugs of A20 or A20-I every 12 hr, with last (7th) dose staggered such that brain dissections happened 2 hr after final drug dosing. EphB2 was immunoprecipitated from 1 ml of protein lysate using 1 pg of goat anti-EphB2 antibodies and then precipitated proteins immunoblotted with goat anti-EphB2, rabbit anti-pTyr1000, and mouse 4G10 antibodies using appropriate HRP conjugated secondary antibodies and chemiluminescent detection methods.

[0357] The EphB2 antibody immunoblot (FIG. 80A) shows equal expression of the receptor in all the mouse brains as expected. The pTrylOOO and 4G10 immunoblots (FIG. 80B and FIG. 80C, respectively), which both recognize tyrosine phosphorylated proteins and are readouts for the activated EphB2 tyrosine kinase catalytic domain, both show strong tyrosine phosphorylation of the receptor in 3 of 4 vehicle (V) control lanes. The level of pTyrlOOO and 4G10 signal in the brains of A20 and A20-I chemical dosed animals showed generally lower levels of activation as indicated by fainter bands in their corresponding lanes. Quantification of band intensities to obtain ratios of pTyr1000/EphB2 and 4G10/EphB2 as measures of EphB2 receptor activation levels indicate that animals PO oral dosed (shown in both FIG. 80D and FIG. 80E) or IP injected (shown only in FIG. 80E) with A20 or A20-I show lower levels of activated phosphorylated EphB2 in the brain. The results shown here also highlight our finding that when EphB2 is tyrosine phosphorylated and catalytically activate in the intact brain, the protein migrates much slower in the SDS-PAGE gels with apparent migration in distinct bands -220-260 kDa (green arrows in FIGs. 80B and 80C), as opposed to the typical migration of unphosphorylated EphB2 protein at 125 kDa (black arrow in FIG. 80A). It is well known that phosphoproteins can exhibit significant differences in their migration in SDS- PAGE gels compare to the unphosphorylated molecules, and with EphB2 the change is quite significant.

Example 36: Biochemical studies of EphB2 in the brain indicate when the receptor protein is activated and tyrosine phosphorylated it exhibits a greatly retarded mobility in SDS-PAGE gels.

[0358] Whole brain protein lysates were prepared from two different wild-type (WT) mice and one each of the following mutant mice: EphB1/EphB2 double homozygous knockout (El N1), EphB2-K661 R (homozygous kinase-dead point mutant), EphB2-F620D (homozygous kinase-overactive point mutant), and EphB2-lacZ a mutation that produces an intracellular truncated EphB2-beta-gal fusion protein which migrates at 220 kDa (heterozygous N2/+). EphB2 was immunoprecipitated from 1 ml of each protein lysate using 1 pg of goat anti- EphB2 antibodies and then the precipitated proteins were immunoblotted with goat anti- EphB2, rabbit anti-phospho-EphB1 /B2, and rabbit anti-pTyr1000 antibodies using appropriate HRP conjugated secondary antibodies and chemiluminescent detection methods. [0359] The EphB2 antibody immunoblot (FIG. 81A) shows relatively equal expression of the 125 kDa sized EphB2 receptor in the WT, K661 R, F620D mouse brains as expected (black arrow), with somewhat lower levels of WT protein in the N2/+ brain as it only has one copy of the WT gene and also produces the 220 kDa EphB2-beta-gal fusion protein which is also recognized by the ectodomain-specific goat anti-EphB2 antibody (thin black arrow). The pEphBI /B2 (FIG. 81B) and pTyrlOOO (FIG. 81C) immunoblots both show tyrosine phosphorylation of the receptor in the two WT brains, and much stronger phosphorylation in the F620D kinase-overactive mutant brain, and that these bands detect activated EphB2 receptor migrating much slower in the SDS-PAGE gel at and just below the 250 kDa standard protein marker (green arrows in FIGs. 81 B and 81 C). The El N1 knockout and K661 R kinase- dead mutant brains showed greatly reduced tyrosine phosphorylation as expected because these brain either express no EphB1 or EphB2 protein (the knockout) or express a catalytically inactive full-length protein (the K661 R).

[0360] FIG. 81 D shows quantification of band intensities to obtain ratios of pEphB/EphB2 and pTyr1000/EphB2 as measures of EphB2 receptor activation levels and indicate that when compared to the WT brains, the F620D brain exhibits much more phospho-EphB2 whereas in the K661 R brain it is greatly reduced. Further, close inspection of the EphB2 immunoblot shows that the high MW bands of the phosphorylated EphB2 protein in the WT and F620D brains can also be detected as this antibody is a particularly strong and specific antibody with little background and thus gives a high signal-to-noise ratio (thin green arrows in FIG. 81A).

Example 37: Biochemical analysis show only about 3-4% of the total EphB2 protein is activated in WT brains

[0361] FIG. 82A shows a darker exposure of the EphB2 immunoblot from the EphB2 immunoprecipitation in FIG. 81A, with the bright unphosphorylated 125 kDa sized EphB2 band and the dim slower migrating 220-250 kDa phospho-EphB2 bands boxed for quantification of chemiluminescent signal intensity (and with a third area of each lane also boxed for subtraction of background). Here, ratios of pEphB2/EphB2 indicate only a small fraction of the total EphB2 protein in the WT brain is actually in the catalytically active, tyrosine phosphorylated state (3-4%), whereas in the kinase-overactive F620D mutant there is 2-3 times more active pEphB2 (9.9%), and in the kinase-dead K661 R there is much less (0.6%) (as tabulated in FIG. 82B).

Example 38 - Mass spectrophotometry independently identifies slow migrating higher sized EphB2 protein in the brain.

[0362] FIG. 83A shows a Coomassie blue stained SDS-PAGE gel of liver and brain protein lysates after immunoprecipitation with goat anti-EphB2 antibodies. The boxed gel pieces were cut and proteins eluted and subjected to mass spectrophotometry methods to obtain peptide sequences contained in each slice (FIG. 83B). This revealed that the gel slice with the most EphB2 protein, as expected, was the K1 (rank 2 - 2nd most abundant protein in the slice) and F1 (rank 6 - 6th most abundant protein in the slice) cutouts just below the 130 kDa protein marker. Importantly, the three higher sized gel slices from both samples (K2-K4 and F2-F4) also contained EphB2 protein sequences, confirming that the higher sized bands labeled for phospho-EphB2 in the immunoblots shown above do indeed identify the presence of bona fide EphB2 receptor protein. It is also noted that the K1 and F1 gel slices also contained some sequences for the related EphB1 and EphA4 receptors, indicating that the goat anti-EphB2 antibody either (1) somewhat cross reacts with these other two receptors and can immunoprecipitate them as well, or (2) that when EphB2 is precipitated the EphB1/EphA4 receptors can also be co-precipitated because they are complexed with EphB2.

Example 39: Slowly migrating, higher sized, tyrosine phosphorylated EphB2 protein is also detected in the liver

[0363] Repeated IP dosing of carbon tetrachloride was used to injure the liver, which has been shown induces overexpression of the EphB2 receptor. Whole liver protein lysates were prepared and subjected to goat anti-EphB2 immunoprecipitation followed by immunoblot analysis with EphB2 (FIG. 84A), phospho-EphB1/B2 (FIG. 84B), 4G10 (FIG. 84C), and pTrylOOO (FIG. 84D) antibodies. The EphB2 immunoblot (FIG. 84A) shows relatively low levels of the 125 kDa non-phosphorylated EphB2 in the livers from uninjured control mice (lanes 1 and 2) that increases in the CCI4 injured livers (remaining lanes). The EphB2 immunoblot also shows significant bands for the higher, slower migrating species of 220 and 260 kDa, that again are lower in intensity/abundance in the uninjured livers compared to the injured livers. The various phosphotyrosine-specific immunoblots (FIGs. 84B-84D) show quite clearly that the 220 kDa band in particular is phospho-EphB2, with low levels in uninjured and high levels in injured.

Example 40: Experimental protocol used to assess how well oral dosing of A20 chemicals blunt chronic long-term inflammatory pain induced by injection of complete Freund’s adjuvant (CFA) into the mouse hind paw

[0364] FIG. 85 shows a schematic of an experimental protocol to assess how well oral dosing of A20 chemicals blunt chronic long-term inflammatory pain induced by injection of complete Freund’s adjuvant (CFA) into the mouse hind paw. The week prior to injection of CFA, adult mice (>10 weeks age, male or female) were first acclimated to the chambers used in pain assessments 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 repeatably testing hind paws for their sensitivity to von Frey (VF) filaments of different stiffnesses with the mechanical withdrawal threshold for each mouse hind paw defined as the minimum gauge filament needed 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 device) that is connected to a timer to measure how long the animal takes to notice the heat and lift the paw. In these tests, both hind paws of mice were subjected to multiple measurements over a 2-3 hr period to (1) either narrow down the VF filament stiffness threshold to plot the touch pain or (2) to obtain six good thermal response measurements which are averaged to provide the data points for heat pain. A chronic pain experiment was initiated by subcutaneously injecting 30 pL of CFA (F5881 , Sigma-Aldrich) into one of the two hind paws (it doesn’t matter, left or right) to induce inflammation and pain, followed 15 min later with initial oral gavage (PO) or intraperitoneal (IP) dose of A20 chemical (or vehicle only in control mice), and then a second A20 chemical dose given 6-8 hr later in the evening. In subsequent days mice were then subjected to additional dosings of A20 chemical (orange arrows) and tested for either thermal (red arrows) or mechanical (green arrows) pain responses as indicated over the next 15 days. In all of the experiments, the person doing the actual pain testing of mice was kept blinded to the mouse genotypes (e.g. wild-type or EphB1 knockout) and/or treatment conditions (e.g. vehicle only or dosed with an A20 chemical). A total of 12 mice were assessed in any one experiment. Results from these experiments are discussed further in the following Examples.

Example 41 : Oral dosing of A20 lead compound strongly blunts mechanical touch pain in the CFA model

[0365] In this example, 12 wild-type adult male mice (11 weeks age, 129x1/SvJ background) were separated into 3 groups of 4, vehicle controls (vehicle is 97%PBS/3% DMSO), 20 mg/kg A20 IP (2x.HCI salt), and 20 mg/kg A20 PO (2x.HCI salt), and subjected to the CFA inflammatory pain model (as described in Example 40). Only the vehicle treated mice exhibited significant increases in mechanical pain as noted by their increased sensitivity to VF filaments in the left (injured) hind paw after the injection of CFA (left panels in FIG. 86A and FIG. 86B). The CFA model leads to long-term mechanical pain as indicated by the vehicle mice exhibiting a significant increase in average sensitivity to low stiffness VF filaments even 10 days post injury compared to the baseline measurements, with pain responses returning to normal at day 15. Injection or oral gavage of A20 resulted in mice that exhibited no increased sensitivity to the VF filaments on average, indicating strong ability of A20 compound to blunt mechanical pain. The right uninjured hind paws of all mice exhibited no significant differences between treatment groups throughout the study (right panels in FIG. 86A and FIG. 86B). This indicates administration of A20 has no effect on the normal sense of touch pain, and that the compound only targets pain produced by a chronic paingenerating insult.

Example 42: Oral dosing of A20 lead compound also effectively blunts thermal pain in the CFA model

[0366] The vehicle treated group (described in Examples 40-41) exhibited greatly increased sensitivity to thermal pain in the left (injured) hind paw following CFA, as noted by their strong decrease in response times to the infrared heat source (FIGs. 87A-87B). The CFA model leads to long-term thermal pain as indicated by the vehicle mice exhibiting significant increased sensitivity to the infrared heat source even 11 days post injury compared to the baseline measurements, with pain responses returning to near baseline at day 14 (green bars in left panels in FIGs. 87A and 87B). Injection or oral gavage of the 2x.HCI salt form of A20 resulted in mice that exhibited reduced sensitivity to the infrared heat source compared to the vehicle treated mice, indicating strong ability of compound to blunt pain (blue and yellow bars in left panels in FIGs. 87A and 87B). The right uninjured hind paws of all mice exhibited no significant differences between treatment groups pre/post baseline (right panels in FIGs. 87A and 87B). This indicates A20 exhibits no effect on the normal sense of heat pain, and that it only targets pain produced by a chronic pain-generating insult.

Example 43: Pain ratio analysis shows oral dosing of A20 lead compound effectively blunts pain as well as IP dosing in the CFA model

[0367] Pain ratios were calculated by dividing the uninjured side pain responses by the injured side responses (right hind paw I left hind paw) for each animal using the data shown in FIGs. 86A, 86B, 87A, and 87B). This analysis clearly shows for mechanical pain the vehicle control mice exhibited strong elevated pain ratios of 2-3 for the ten days following CFA injury, whereas orally dosed or IP injected A20 treated mice exhibited essentially no increase in pain as their pain ratios were 1 throughout the study (FIG. 88A). Similarly, assessment of thermal pain showed vehicle treated mice exhibited strong pain ratios -4 or above, whereas orally dosed or IP injected A20 treated mice exhibited greatly blunted pain as their pain ratios were <2 throughout the study (FIG. 88B).

Example 44: Use of the inflammatory agent Zymosan to induce pain, a more rapid model to study pain and get results in one day

[0368] An alternative and potentially faster pain model using Zymosin (an inflammatory agent like CFA) to induce inflammatory pain was tested. To initially test the Zymosan inflammatory pain model, we studied EphB1 -/- knockout, EphB1 +/- heterozygous, and EphB1 +/+ wild-type adult mice (a large set of 4-6” Month 'old males and females, 129/CD1 mixed background), as we have previously shown the -/- and even +/- mice exhibit reduced injury-induced pain responses due to the key role of the EphB1 receptor in chronic, neuropathic pain. Mice were first acclimated to the pain testing set ups and then subjected to baseline mechanical and thermal pain measurements. Early in the morning the following day, the left hind paws were injected subcutaneously with 30 pL of Zymosan (5 mg/ml in PBS; Z4250, Sigma-Aldrich). Two hours after the Zymosan injection, the left hind paws were assessed for thermal pain using Hargreaves device (2-4 hr), and then left paws tested for mechanical pain using VF filaments (4-6 hr), and then again for thermal pain (6-8 hr). Data from two independent experiments were pooled to increase the n values for the three different genotypes. Focusing on mechanical pain, Zymosan elicited a strong, significant increase in pain in the +/+ and +/- mice with average filament response going from greater than 6 to less than 3, whereas the response in the -/- knockout mice was not significantly lower than the baseline measurements (FIG. 89A). Calculation of pain ratios confirms that the +/+ and +/- mice exhibit a strong increase in mechanical pain with ratios of -3, whereas the pain ratio of -/- mice was close to 1 and significantly less than the wild-type, indicating essentially no increase in mechanical pain in the knockout mice following injury with Zymosan (FIG. 89C). Thermal pain increased in all group following Zymosan injury as indicated by a decreased average response time to the infrared heat source in all animals assessed, and the level of pain intensified from the 2-4 hr testing period to the 6-8 hr period. Nevertheless, the -/- and +/- animals exhibited blunted thermal pain responses compared to the +/+ mice, especially during the 6-8 hr testing session where pain ratios were significantly lower by -50% (FIGs. 89B and 89D). These data indicate Zymosan can be used as a rapid model to assess ability of A20 chemicals to reduce EphrinB2-EphB1 tetramerization/ signaling and blunt inflammatory pain.

Example 45: Oral dosing of A20 lead compound blunts mechanical and thermal pain in the Zymosan model

[0369] Inflammatory pain was induced by subcutaneous injection of 30 pl of 5 mg/ml Zymosan into the right hind paws of 12 wild-type adult male mice (5-6 months age, 129x1/SvJ background) that in previous days were acclimated to the testing chambers and assessed to obtain baseline pain measurements. After 15 min, mice were orally dosed with either vehicle only (97%PBS/3%DMSO) or with 20, 10, or 5 mg/kg of A20 (2x.HCI salt in vehicle), 3 mice for each condition. Two hours after Zymosan injection, the right right hind paws were assessed for thermal pain using Hargreaves device (2-4 hr), then mice were orally dosed again with vehicle only or A20 chemicals, and then right paws tested for mechanical pain using VF filaments (4-6 hr), and then again for thermal pain (6-8 hr).

[0370] In the vehicle treated mice, Zymosan elicited a strong increase in the mechanical pain ratio with an average approaching 3, whereas the A20 treated mice exhibited mechanical pain ratios close to 1 , which indicates no enhanced pain (FIG. 90A). Likewise, in the two rounds of thermal pain testing, the vehicle treated mice exhibited an average thermal pain ratio close to 6 indicating extreme pain levels, whereas the A20 treated mice showed thermal pain ratios around 3 (FIG. 90B). With only three mice assessed for each condition, statistical analysis is difficult because animal behavior studies are well known to show quite significant variability. As it was noted that all three A20 doses tested (20, 10, 5 mg/kg) resulted in similar blunted pain responses, these data were pooled and compared to the vehicle only for statistical analysis, indicating the A20 treated mice showed significant reductions in both mechanical and thermal pain (FIGs. 90C-90D).

Example 46: Oral dosing of 3511-1 and BQPB4 compound blunts mechanical and thermal pain in the Zymosan model

[0371] Inflammatory pain was induced by subcutaneous injection of 30 pl of 5 mg/ml Zymosan into the left hind paws of 9 wild-type adult males (5-6 months age, 129/CD1 mixed background) that in previous days were acclimated to the testing chambers and assessed to obtain baseline pain measurements. After 15 min, mice were orally dosed with either vehicle only (97%PBS/3%DMSO) or with 20 mg/kg of 3511-1 (2x.HCI salt in vehicle), or 20 mg/kg BQPB4 (3x.HCI salt in 97% sunflower seed oil/3% DMSO), 3 mice for each condition. Two hours after Zymosan injection, the left right hind paws were assessed for mechanical pain using VF filaments (2-4 hr), then mice were orally dosed again with vehicle only or A20 chemicals, and then left hind paws tested for thermal pain using Hargreaves device (4-6 hr), and then again for mechanical pain (6-8 hr).

[0372] In the vehicle treated mice, Zymosan elicited a strong significant increase in mechanical pain as the average VF filament stiffness response went from -6 in baseline to less than 4 during the 2-4 hr testing session to almost 2 during the 6-8 hr testing session, leading to mechanical pain ratios of 2 and 3, respectively (green bars in FIG. 91 A). The 3511- I treated mice exhibited a somewhat blunted mechanical pain response, especially during the 6-8 hr testing session, whereas the BQPB4 treated mice exhibited essentially no enhanced mechanical pain sensitivity with VF filament averages similar to baseline and pain ratio averages near 1 (blue and yellow bars in FIG. 91A). Similarly, the vehicle treated mice exhibited a very strong sensitivity to thermal pain as their average response to the infrared heat source went from 12 sec in baseline to below 2 sec post-Zymosan (green bars FIG. 91 B). Compared to the vehicle controls, 3511-1 and BQPB4 compound treated mice exhibited on average significantly less thermal pain (longer withdrawal times), and their average pain ratios were significantly lowered to near 3 for 3511-1 to less than 2 for BQPB4 (blue and yellow bars, FIG. 91 B). Example 47: Oral dosing of BQPB4 compound shows a dose-dependent blunting of thermal pain in the Zymosan model

[0373] Inflammatory pain was induced by subcutaneous injection of Zymosan into left hind paws, followed 15 min later with an oral dose of either vehicle or 20, 10, or 5 mg/kg of BQPB4 (3x.HCI salt in 97% sunflower seed oil/3% DMSO). Two hours after Zymosan injection, the left hind paws were assessed for mechanical pain using VF filaments (2-4 hr), then mice were orally dosed again with vehicle or BQPB4 chemical, and then hind paws tested for thermal pain using Hargreaves device (4-6 hr), and then tested again for mechanical pain (6- 8 hr). Data is pooled from two different experiments, with thermal pain results shown in FIGs. 92A-92B. The vehicle treated mice exhibited a very strong sensitivity to thermal pain as their average response to the infrared heat source went from 11.5 sec in baseline to <2 sec postZymosan. Compared to the vehicle controls, mice treated with any of the three doses of BQPB4 tested exhibited on average longer thermal withdrawal times, indicating less thermal pain. Indeed, the effect of BQPB4 was dose-dependent as the 20 mg/kg dose produced the strongest and most significant reduction in pain, followed by the 10 mg/kg dose, and then the 5 mg/kg dose (FIG. 92A). Calculation of pain ratios confirmed the significant dose-dependent effects of BQPB4 at blunting thermal pain (FIG. 92B).

Example 48: Additional studies of dorsal horn (PH) neuron activation in the spinal cord after a chronic pain generating insult

[0374] In this example, an in-house method to count and quantify/analyze Tom+ neurons (see Example 23) in the dorsal horn (DH) of the spinal cord is used to track the effect of an inflammatory insult (subcutaneous injection of CFA into the left hind paw). As described above in Example 23, we initially dissected perfusion-fixed spinal cords and then cut in the transverse plane 50 urn thick serial sections throughout the entire length of the lumbar cord of each animal using a vibratome aiming to capture all sections. As serial sections are cut, they are placed into 10 section bins (500 pm spinal cord per bin) and a total of 12 bins are collected spanning 6 mm total spinal cord length. In the earlier Examples, above, one section per each bin was mounted onto a slide and imaged to quantify red Tom+ neurons, though it was observed, especially in wild-type and vehicle-treated mice, that a consecutive group of 4 of the 12 bins usually contained the majority of sections that exhibited strong differences in ratios of left (injured) to right (uninjured) Tom+ DH neurons. Thus, it was assumed that these bins represent the key segment of spinal cord that is innervated by the sciatic nerve. Focusing in on this “sweet spot” of 4 bins, the additional sections were mounted and analyzed (aiming for at least 12 sections from these 4 bins, 3 per bin) to increase total data to analyze. FIGs. 93A-93C shows a representative example of the data collected and analyzed, with FIG. 93A showing a typical fluorescent image taken using a Zeiss Axioscan to rapidly capture images of mounted sections from all animals under analysis, FIG. 93B plotting the uninjured number of red Tom+ DH neurons counted from the left and right sides of the 12 different imaged “sweet spot” sections, and FIG. 93C showing a histogram plotting of the data as a function of the number of sections with X number of Tom+ DH neurons, and dividing the mean of the number of Tom+ neurons on injured side by the mean of the number of Tom+ neurons on the uninjured side, with the example shown giving a ratio of Tom+ neurons of 3.67.

Example 49: Dorsal horn (DH) neuron activation in the spinal cord after a chronic pain generating insult in EphB1 wildtype, heterozygous or knockout animals.

[0375] A plurality of Axioscan imaged lumbar spinal cord sections from “the sweet spots” of EphB1 +/+ wild-type, +/- heterozygous, and -/- knockout mutants subjected to CFA pain model were analyzed as described in Example 48. FIG. 94A shows the ratio of Tom+ DH neurons (injured side I uninjured side) for each individual section, and FIG. 94B shows the average ratio for all sections of an individual spinal cord. Data for two other +/+ wild-type animals that were not subjected to CFA pain model were also included in this analysis. Note that while the +/+ wild-type injured animals exhibited strong increase in Tom+ neuron ratios >3, the +/- heterozygotes and -/- homozygotes both exhibited highly significant reductions in their Tom+ neuron ratios to near 1 that more resembled the +/+ animals that were not injured (no CFA).

Example 50: Dorsal horn (DH) neuron activation in the spinal cord after a chronic pain generating insult in animals treated with A20 compounds

[0376] In another example, 307 Axioscan imaged lumbar spinal cord sections from the “sweet spots” of eight +/+ wild-type mice subjected to Zymosan pain model and oral dosing of either vehicle (n=3), 3511-1 (2X HCI, n=3), or BQPB4 (3X HCI, n=2) were analyzed. Data is plotted in FIG. 95A as a ratio of Tom+ DH neurons (injured side I uninjured side) for each individual section, and in FIG. 95B as the average ratio for all sections of an individual spinal cord. Note that on average the 3511-1 or BQPB4 treated mice exhibited a significantly lower ratio of Tom+ neurons compared to the vehicle treated animals when all sections were plotted. There was also a trend towards lower overall Tom+ ratios in the 3511-1 or BQPB4 treated mice, though the numbers of mice analyzed here were too low to reveal significance. Table A: IC50 for immobilized human EphrinB2 binding to 50 nM soluble human EphB2 (EphrinB2-EphB2)

Table B: IC50 for immobilized human EphrinB2 binding to 50 nM soluble human EphB1 (EphrinB2-EphB1) Table C: IC50 for immobilized human EphrinB2 binding to 50 nM soluble human EphB4 (EphrinB2-EphB4)

Table D: IC50 for immobilized human EphA3 binding to 50 nM soluble human EphrinAI and EphrinA5

Table E: IC50 for immobilized mouse EphB2 binding to 50 nM soluble human EphrinA5 and mouse EphrinB2

Table F: IC50 for immobilized rat EphB1 or mouse EphB4 binding to 50 nM soluble mouse EphrinB2

Table G: Percent Inhibition of immobilized human EphrinB2 binding to 25 nM soluble human EphB [0377] Having described several embodiments, it will be recognized by those skilled in the art that various modifications, alternative constructions, and equivalents may be used without departing from the spirit of the present inventive concept. Additionally, a number of well- known processes and elements have not been described in order to avoid unnecessarily obscuring the present inventive concept. Accordingly, this description should not be taken as limiting the scope of the present inventive concept.

[0378] Those skilled in the art will appreciate that the presently disclosed embodiments teach by way of example and not by limitation. Therefore, the matter contained in this description or shown in the accompanying drawings should be interpreted as illustrative and not in a limiting sense. The following claims are intended to cover all generic and specific features described herein, as well as all statements of the scope of the method and assemblies, which, as a matter of language, might be said to fall there between.

Claims

1 . A compound comprising a structure of Formula (I) (Formula I) or pharmaceutically suitable salt thereof, wherein: A is an -O-, -SO2, or -N(CH2)_nRi; B is CH2, SO2, or CO; X is hydrogen, a halogen, an alkyenyl, an alkyl, -NO2, or-NH2; each R is independently hydrogen, a substituted or unsubstituted alkyl, an alkoxy, heterocycloalykl, or a carbonyl, R1 is a substituted or unsubstituted aryl, a substituted or unsubstituted heteroaryl, a substituted or unsubstituted cycloalkyl, or a substituted or unsubstituted heterocycloalkyl; and n = 0-3; with the provisos that (1) X is not hydrogen, chloro, -NO2, or bromo when A is -O- , B is CH2 and R is hydrogen; and (2) X is not bromine when A is

NRi and Ri is methyl

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

3. The compound of claim 1 , wherein X is hydrogen, bromo, iodo, chloro, -NO2, or -NH2, methyl, or propenyl.

4. The compound of claim 1 , wherein each R is independently hydrogen, methyl,

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

6. The compound of claim 1 , wherein R1 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. The compound of claim 6, wherein R1 is a substituted or unsubstituted phenyl, a substituted or unsubstituted pyridinyl, a substituted or unsubstituted thiazole, a substituted or unsubstituted thiophenyl, a substituted or unsubstituted piperazine, or an unsubstituted C4-C10 cycloalkyl or an unsubstituted C4-C10 heterocycloalkyl.

8. The compound of claim 7, wherein Ri is selected from the group consisting of

O-; each R2 is independently hydrogen, -NO2, an alkoxy, an alkyl ether of -R3OR4, -NHOR5, - NH2, halo, a haloalkyl, or an alkyl; and R3 and R4 are each independently C1-C4 alkyl and R5 is an alkyl or a benzyl.

9. The compound of claim 7, wherein each R2 is independently hydrogen, -NO2, -OCH3, -

CH2CH2OCH3, -NHOCH3, -CF3, -NH2, bromo, fluoro, chloro, iodo, methyl, 11. The compound of any one of claims 1 to 10, wherein the compound is not:

12. The compound of claim 1, wherein the compound is selected from the group consisting of: any pharmaceutically appropriate salt thereof. 13. The compound of claim 12, wherein the compound is a pharmaceutically equivalent salt

, and any pharmaceutically appropriate salt thereof.

15. The compound of claim 14, wherein the compound is a pharmaceutically appropriate salt

16. The compound of any one of claims 1 to 15, wherein the compound inhibits EPH-EPHRIN tetramerization.

17. The compound of claim 16, wherein the compound specifically inhibits EPHB1-EPHRIN and/or EPHB2-EPHRIN tetramerization.

18. The compound of claim 17, wherein the compound is selected from the group consisting appropriate salt thereof.

19. The compound of claim 17, wherein the compound specifically inhibits EPHB2-EPHRIN tetramerization.

20. The compound of claim 19, wherein the compound is selected from the group consisting or a pharmaceutically appropriate salt thereof.

21. The compound of claim 17, wherein the compound specifically inhibits EPHB1-EPHRIN tetramerization.

22. The compound of claim 21 , wherein the compound is selected from the group consisting a pharmaceutically appropriate salt thereof.

23. The compound of claim 17, wherein the compound does not inhibit EPHB4-EPHRIN tetramerization.

24. The compound of claim 16, wherein the compound inhibits EPH-EPHRIN tetramerization with an IC50 of less than 2 pM, less than 1.6 pM, less than 1 pM or less than 0.5 pM.

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

26. A method of inhibiting formation of an EPH-EPHRIN tetramer, the method comprising: contacting an EPH or EPHRIN with a compound of any one of claims 1 to 24 or a compound inhibiting formation of an EPH-EPHRIN tetramer.

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

30. A method of alleviating or relieving pain in a subject in need thereof, the method comprising administering an affective amount of an EPH-EPHRIN tetramerization inhibitor to the subject.

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

32. A method of treating a synaptopathy in a subject in need thereof, the method comprising administering an affective amount of an EPH-EPHRIN tetramerization inhibitor to the subject.

33. The method of claim 32, wherein the synaptopathy comprises abnormal, defective, or excessive EPH-EPHRIN signaling and optionally comprises disrupted NMDA signaling.

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

35. A method of treating addiction or opioid dependency in a subject in need thereof, the method comprising administering an affective amount of an EPH-EPHRIN tetramerization inhibitor to the subject.

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

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

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

39. The method of claim 38, wherein the cancer comprises abnormal, 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, the method comprising administering an effective amount of an EPH-EPHRIN tetramerization inhibitor to the subject.

41 . The method of claim 40, wherein the viral infection comprises abnormal, defective, or excessive EPH-EPHRIN signaling and is optionally comprises an infection by henipavirus and/or human immunodeficiency virus (HIV).

42. The method of any one of claims 30 to 41 , wherein the EPH-EPHRIN tetramerization a compound of any one of claims 1 to 28, or any pharmaceutically acceptable salt thereof.

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

44. The method of claim 43, wherein the pharmaceutical composition is administered intravenously, subcutaneously, or oral dosing, IP injections, and a topical application as cream or ointment.

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

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

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