Azole compounds useful as inhibitors of voltage-dependent anion channel oligomerization

Abstract

A method of inhibiting voltage-dependent anion channel oligomerization in a subject in need thereof includes administering an effective amount of a compound of Formula I, a solvate or salt thereof to the subject:

Description

[0001] NIH0164PCT AZOLE COMPOUNDS USEFUL AS INHIBITORS OF VOLTAGE-DEPENDENT ANION CHANNEL OLIGOMERIZATION CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application Ser. No. 63 / 663,336 filed June 24, 2024, which is incorporated by reference herein in its entirety. STATEMENT OF GOVERNMENT SUPPORT [1] This invention was made in part with government support from the US Department of Health and Human Services, National Institutes of Health. The Government has certain rights in this invention. BACKGROUND OF THE INVENTION 1. Field of the Invention [2] This invention relates generally to inhibitors of voltage-dependent anion channel (VDAC) oligomerization, and more particularly to azole compounds, pharmaceutical compositions containing these compounds, and methods for the treatment of a disease, disorder, or medical condition associated with mitochondrial dysfunction and / or stress. 2. Brief Description of the Art [3] Many diseases are associated with mitochondrial stress. They include rare diseases as well as common diseases such as covid-19, long covid, cancers, atherosclerosis, autoimmunity, hepatitis, diabetes, and T-cell exhaustion. Prior findings suggest that oligomerization of mitochondrial outer membrane protein VDAC and production of reactive oxygen species (ROS) contribute to mitochondrial dysfunction (Kim et al. Science, 2019, vol. 366, Issue 6472, pp 1531- 1536). The best known VDAC inhibitors such as VBIT-4 can be toxic in some cells. Accordingly, there is a continuing need for new compounds that can reduce VDAC oligomerization and ROS production with improved safety and efficacy. SUMMARY OF THE INVENTION [4] In one aspect, the invention is directed to a compound of Formula I 1 NIH0164PCT R10R11R9A R5R6Formula I or a pharmaceut alt thereof, wherein: R1 is halogen; R2 is halogen or hydrogen; R3is hydroxyl, or -N(R17)2, wherein each R17is independently hydrogen, or C1-C6alkyl; R4, R5, R6, R7, and R8are independently chosen from hydrogen, halogen, C1-C6alkyl, and C1-C6alkoxy, C1-C2haloalkyl, and C1-C2haloalkoxy; R9, R10, R11, R12, and R13 are independently chosen from hydrogen, C1-C6alkyl, and C1-C6alkoxy, C1-C2haloalkyl, and C1-C4haloalkoxy; R3 is halogen or C1-C6alkyl; A ring is NNR16N NRN 15N or , wherein R15 is C1-4 alkyl, and R16 is hydrogen or C1-4 alkyl; B ring is 6-membered heteroaryl ring having one or two nitrogen atoms, which B ring is optionally substituted with one or more substituents independently chosen from halogen and C1- C6alkyl, with the proviso that the compound is not 2-(2,4-difluorophenyl)-1,1-difluoro-3-(2H- tetrazol-2-yl)-1-(5-(4-(2,2,2-trifluoroethoxy) phenyl)pyridin-2-yl)propan-2-ol. [5] A method of inhibiting voltage-dependent anion channel oligomerization in a subject in need thereof, comprising administering an effective amount of a compound of Formula I, a solvate or salt thereof to the subject: R10R11R9a I or a pharmaceut ca y accep a e so va e, or sa ereo , w erein: R1 is halogen; R2 is halogen or hydrogen; R3is hydroxyl, or -N(R17)2, wherein each R17is independently hydrogen, or C1-C6alkyl; R4, R5, R6, R7, and R8are independently chosen from hydrogen, halogen, C1-C6alkyl, and 2 NIH0164PCT C1-C6alkoxy, C1-C2haloalkyl, and C1-C2haloalkoxy; R9, R10, R11, R12, and R13are independently chosen from hydrogen, C1-C6alkyl, and C1-C6alkoxy, C1-C2haloalkyl, and C1-C4haloalkoxy; R3 is halogen or C1-C6 alkyl; A ring is NNR16N N R N N , wherein R15 is hydrogen or C1-4 alkyl, and R16 is hydrogen bered heteroaryl ring having one or two nitrogen atoms, which B ring is optionally substituted with one or more substituents independently chosen from halogen and C1-C6alkyl. In another aspect, the present invention is directed to a pharmaceutical composition comprising at least one of a compound of Formula I or a pharmaceutically acceptable solvate, or salt thereof, together with a pharmaceutically acceptable carrier. In another aspect, the present invention is directed to a method of inhibiting voltage- dependent anion channel oligomerization in a subject in need thereof, comprising administering an effective amount of at least one of a compound of Formula I, a solvate or salt thereof to the subject. BRIEF DESCRIPTION OF THE DRAWING [6] FIG. 1A shows that IVO-21 blocks the formation of VDAC1 dimer in stressed HeLa cells, and FIG. 1B shows that IVO-21 blocks the formation of 8-12 mer (300-350 kDa) in stressed fibroblasts; [7] FIG. 2A shows that IVO-21 does not disrupt VDAC1-HK1 interaction but disrupts VDAC1-HK2 interaction, FIG. 2B shows that IVOs decrease thrombosis that is stimulated by immunostimulant oligonucleotide, without decreasing platelet aggregation below the baseline (Control); [8] FIG. 3 shows that IVOs decrease ROS produced in response to endoplasmic reticulum stressor Tunicamycin; [9] FIG. 4A shows that IVO-21 increases AMPK activity (p-AMPK) and suppresses mTORC1 (p-S6K1), and FIG. 4B shows that IVO-21 increases autophagy as evidenced by increased p62 levels and LC3 II;

[0010] FIG. 5A shows the mitochondrial DNA (mtDNA) released into the cytoplasm (cmtDNA), 3 NIH0164PCT FIG. 5B shows the mtDNA released into the extracellular medium, and FIG. 5C shows the inflammatory cytokine IL-1β released into the extracellular medium;

[0011] FIG. 6 shows that tumor growth factor-b (TGF-b) promotes fibrosis by increasing production of collagen (COL1A1), but IVOs (17-21) mitigate it;

[0012] FIG. 7 shows that expression levels of IP3R is mitigated by IVO-21;

[0013] FIG. 8 shows the frequency of live CD8 T cells expressing exhaustion markers (TIGIT+ / CD39+) after chronic stimulation is decreased by IVO-21;

[0014] FIG. 9 shows the measurement of NETosis in the presence of NETosis inducer A23187 and / or IVO-21;

[0015] FIG. 10A shows the effects of azole-based drugs on ROS-induced VDAC1 oligomerization. FIG. 10B shows recombinant VDAC1 that binds more strongly to over- oxidized C127 than to native C127. FIG. 10C shows that IVO-21 selectively binds to over- oxidized C127, while oteseconazole and VBIT-4 do not. FIG. 10D is a diagram illustrating how IVO-21 blocks VDAC1-VDAC1 interaction (i.e. oligomerization) by binding to over-oxidized C127. FIG. 10E shows an IC50curve of IVO-21 on CpG-ODN-induced platelet aggregation (+ADP). FIG. 10F shows that IVO-21 inhibits IL-1β-induced platelet aggregation (+ADP). FIG. 10G shows that IVO-21 inhibits LPS-induced platelet aggregation (+ADP);

[0016] FIG. 11A shows a co-immunoprecipitation of VDAC1 and HK2 in vdac1+ / +and vdac1C127A / +platelets after treatment with CpG-ODN. Inputs are shown below. FIG. 11B: IVO- 21 does not disrupt VDAC1-HK1 interaction. FIG. 11C: IVO-21 disrupts VDAC1-HK2 interaction, but VBIT does not. Inputs are shown below. FIG. 11D: Recombinant HK2 binds more strongly to over-oxidized C127 than to native C127. FIGS. 11E-F: Glucose flux analysis showing the incorporation of13C glucose into metabolites of glycolysis, pentose phosphate pathway (PPP), and TCA cycle 15 min (FIG. 11E) and 30 min (FIG. 11F) after the addition of13C glucose. Platelets were preincubated with CpG-ODN for 1 hour (-IVO-21, left; +IVO-21, right), and their metabolites were compared to untreated platelets. FIG. 11G:13C glucose flux into the TCA cycle is enhanced by IVO-2115 min (top) but not 30 min (bottom) after13C glucose addition;

[0017] FIG. 12A shows that IVO-21, mito-TEMPO, and VAS2870 inhibit CpG-ODN-induced ROS production. FIG. 12B: ROS levels in vdac1+ / +and vdac1C127A / +platelets. FIG. 12C: Aggregation of vdac1+ / +and vdac1C127A / +platelets (+ ADP). FIG. 12D: CpG-ODN-induced 4 NIH0164PCT oligomerization of VDAC1 (total VDAC1, left; over-oxidized C127, right) in the presence of IVO-21. FIG. 12E: LPS-induced VDAC1 oligomerization in the presence of IVO-21. FIG. 12F: IL-1β-induced VDAC1 oligomerization in the presence of IVO-21. FIG. 12G: Cytosolic mtDNA after treatment with CpG-ODN in the presence of IVO-21. FIGS. 12H-J: Extracellular mtDNA after treatment with CpG-ODN (FIG. 12H), LPS (FIG. 12I) and IL-1β (FIG. 12J). FIG. 12K: Extracellular mtDNA after CpG-ODN treatment in the presence of VAS2870 or mito-TEMPO. FIG. 12L: Extracellular mtDNA released from vdac1+ / +and vdac1C127A / +platelets. FIG. 12M: Effect of IVO-21 on aPL antibody-induced platelet aggregation (two donors). FIG. 12N: Effect of IVO-21 on the levels of extracellular mtDNA in aPL antibody-induced platelets. FIG. 12O: The effect of IVO-21 on the levels of IL-1β released after CpG-ODN treatment. FIG. 12P: Diagram showing how over-oxidation of C127 is a critical mediator of the thromboinflammation loop;

[0018] FIG. 13A show plasma platelet levels of mice injected with LPS with or without pre- treatment with IVO-21. FIG. 13B: Plasma lactate levels from the same mice. FIG. 13C: Plasma IL-1β levels from the same mice. FIG. 13D: Plasma IL-6 levels from the same mice. FIG. 13E: Plasma eosinophil levels from the same mice. FIG. 13F: Kaplan-Meir curve showing the effect of IVO-21 injected after CLP on mouse survival. FIG. 13G: Diagram illustrating how VDAC activation loop 1 (VAL1) and VAL2 work together to promote thromboinflammation;

[0019] Fig. 14 shows that IVO-21 protects against respiratory distress caused by tracheal infusion of LPS;

[0020] Fig. 15A shows that IVO-21 protects against deep vein thrombus. Clotting was induced by electrolysis (left). Prior treatment with IVO-21 reduced the thrombus size (center and right); Fig. 15B shows that IVO-21 binds specifically to oxidized Cys127 in VDAC1.

[0021] FIGS. 16A and 17B show an analysis of sorted naïve and memory T cell populations (CM: CD62L+ CD44+; EM: CD62L- CD44+) using mass spectrometry to detect thiol modifications caused by ROS during T cell activation. FIG. 16A: Expression intensity of Vdac1 peptide via mass spectrometry. FIG. 16B: Identification of sulfinic acid modification in VDAC1 C127 residue. FIG. 16C: assessment of oxidative stress in activated T cells from WT and Vdac1C127A / wtmice using flow cytometry;

[0022] FIGS. 17A-C show the vitiligo response scored based on the greying pattern observed on the skin / fur of these animals at ~10 months of age. FIG. 17A: Representative images of Pmel 5 NIH0164PCT transgenic animals with or without Vdac1 expression at approximately 12 months of age. FIG. 17B: Average rank score metric of graded vitiligo response in Pmel transgenic animals expressing varying degrees of Vdac1; the genetic identities were blinded during scoring, and the average scores from two independent investigators are shown. FIG. 17C: Bar plots showing the frequencies of Tim3-aPD1+ exhausted cells in circulation obtained from these animals at approximately 10 months of age;

[0023] FIG. 18A: Bar graph shows mean fluorescent intensity of TOX expression under different treatment conditions across 2 healthy donors. TOX is a transcription factor that is key to the expression of several exhaustion related genes in T cells. FIG. 18B: Representative bar graph showing the expression of exhaustion proteins Lag3, Tim3, CD39, and TIGIT in a healthy donor T cells treated with various VDAC1 inhibitors. The data is representative of response from 2 healthy donors;

[0024] FIG. 19A: C57BL6 mice were fed HFD and simultaneous treated with different doses of oral IVO-21 or saline. FIG. 19B: Body weight of IVO-treated mice. FIG. 19C: Glucose tolerance test with IVO-21. FIG. 19D: Insulin tolerance test with IVO-21. FIG. 19E: Insulin tolerance test of vdac1C127A / + and vdac1+ / + mice on HFD. FIG. 19F: Glucose tolerance test of same mice;

[0025] FIGS. 20A-C show that IVO-21 mitigates myocardial infarction-induced heart failure. FIG. 20A shows the effects of IVO21 on global heart volume. FIG. 20B shows the effects of IVO21 on mid papillary of heart. FIG. 20C shows the effects of IVO21 on the apex of the heart;

[0026] FIG. 21A shows that IVO-21 protects against renal atrophy. Fig. 21B: PSR staining shows that IVO-21 reduces renal fibrosis. Fig. 21C: IVO-21 preserves kidney function (BUN, creatinine);

[0027] FIG. 22A shows the structures of IVO-18 enantiomers (IVO-18.2 and IVO-18.4). FIG. 22B: platelet aggregation was blocked (green shade) by both IVO-18 enantiomers at <0.5 nM; and

[0028] FIGS. 23A-B: Azole compounds activate AMPK. Itraconazole (10 mg / kg) activates AMPK in skeletal muscle (FIG. 23A) and white adipose tissue (FIG. 23B) in wild-type (WT) mice but not in VDAC1 KO mice. FIG. 23C: Itraconazole activates AMPK in C2C12 myotubes. FIG. 23D: Measurement of Extracellular Acidification Rate (ECAR), G6P and F1,6BP levels in C2C12 myotubes after treatment with itraconazole. FIG. 23E: Activation of AMPK, inhibition of mTORC1 and activation of TFEB after treatment with IVO-18.2 and IVO-18.4. 6 NIH0164PCT

[0029] FIG. 24 shows CpG-ODN (oligo)-induced coagulation in the presence of varying concentrations of IVO-18, IVO-21 and 1:1 mixture of IVO-18 and IVO-21. Platelets were preincubated with IVOs for 30 minutes prior to oligo addition, and coagulation was measured 30 minutes after oligo addition. For 1:1 mixture, the concentration reflects the total IVO concentration. Both IVO-18 and IVO-21 are racemic mixtures. DETAILED DESCRIPTION OF THE INVENTION TERMINOLOGY

[0030] Compounds of the present disclosure are generally described using standard nomenclature.

[0031] The terms “a” and “an” do not denote a limitation of quantity, but rather denote the presence of at least one of the referenced items. The term “or” means “and / or.” The open-ended transitional phrase “comprising” encompasses the intermediate transitional phrase “consisting essentially of” and the close-ended phrase “consisting of.” Claims reciting one of these three transitional phrases, or with an alternate transitional phrase such as “containing” or “including” can be written with any other transitional phrase unless clearly precluded by the context or art. Recitation of ranges of values are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. The endpoints of all ranges are included within the range and independently combinable. All methods described herein can be performed in a suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”), is intended merely to better illustrate the disclosure and does not pose a limitation on its scope unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention as used herein. Unless defined otherwise, technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which this disclosure belongs.

[0032] Formula I includes all subformulae such as Formula I-A, Formula I-B, and Formula I-C.

[0033] The compounds described herein include tautomers and polymorphs.

[0034] In certain situations, the compounds described herein may contain one or more 7 NIH0164PCT asymmetric elements such as stereogenic centers, stereogenic axes and the like, e.g. asymmetric carbon atoms, so that the compounds can exist in different stereoisomeric forms. The disclosed compounds include all stereoisomeric forms, including racemates, optically enriched, and optically pure forms. All isomeric forms of the compounds are included in the present disclosure. In these situations, the single enantiomers, i.e., optically active forms can be obtained by asymmetric synthesis, synthesis from optically pure precursors, or by resolution of the racemates. Resolution of the racemates can also be accomplished, for example, by conventional methods such as crystallization in the presence of a resolving agent, or chromatography, using, for example, a chiral HPLC column.

[0035] The disclosure of compounds includes all isotopes of atoms occurring in the present compounds. Isotopes include those atoms having the same atomic number but different mass numbers. By way of general example, and without limitation, isotopes of hydrogen include tritium and deuterium and isotopes of carbon include11C,13C, and14C and isotopes of fluorine including19F.

[0036] Certain compounds are described herein using a general formula that includes variables, e.g., R1-R17, A ring, and B ring. Unless otherwise specified, each variable within such a formula is defined independently of other variables. Thus, if a group is said to be substituted, e.g., with 0-2 R*, then said group may be substituted with up to two R* groups and R* at each occurrence is selected independently from the definition of R*.

[0037] Combinations of substituents and / or variables are permissible only if such combinations result in stable compounds. A stable compound or stable structure is meant to imply a compound that is sufficiently robust to survive isolation from a reaction mixture, and subsequent formulation into an effective therapeutic agent.

[0038] The term “substituted” means that any one or more hydrogen atoms bound to the designated atom or group is replaced with a selection from the indicated group, provided that the designated atom's normal valence is not exceeded. Combinations of substituents and / or variables are permissible only if such combinations result in stable compounds or useful synthetic intermediates.

[0039] Substituents are named into the ring unless otherwise indicated. A dash ("-") that is not between two letters or symbols indicates the point of attachment for a substitu . For example, -CONH2is attached through the carbon atom. 8 NIH0164PCT

[0040] An “active agent” means a compound (including a compound disclosed herein), element, or mixture that when administered to a patient, alone or in combination with another compound, element, or mixture, confers, directly or indirectly, a physiological effect on the subject. The indirect physiological effect may occur via a metabolite or other indirect mechanism. The “active agent” may also potentiate or make more active another active agent. For example, the compounds of Formula I potentiate the activity of other active agents when given in combination with another active agent, for example by lowering the effective dose of the other active agent.

[0041] “Alkyl” includes both branched and straight-chain saturated aliphatic hydrocarbon groups, having the specified number of carbon atoms. Thus, the term C1- C6alkyl includes alkyl groups having from 1 to about 6 carbon atoms. Examples of alkyl include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, n-pentyl, and sec-pentyl. C1-C4alkyl includes alkyl groups having 1, 2, 3, or 4 carbon atoms.

[0042] “Alkoxy” is an alkyl group as defined above with the indicated number of carbon atoms attached through an oxygen bridge. Examples of alkoxy include, but are not limited to, methoxy, ethoxy, n-propoxy, i-propoxy, n-butoxy, 2-butoxy, t-butoxy, n-pentoxy, 2-pentoxy, 3-pentoxy, isopentoxy, neopentoxy, n-hexoxy, 2-hexoxy, 3-hexoxy, and 3- methylpentoxy.

[0043] “Haloalkyl” includes both branched and straight-chain saturated aliphatic hydrocarbon groups, having the specified number of carbon atoms, substituted with 1 or more halogen atoms, generally up to the maximum allowable number of halogen atoms. Thus, the term C1- C6haloalkyl includes haloalkyl groups having from 1 to about 6 carbon atoms. Examples of haloalkyl include, but are not limited to, trifluoromethyl, difluoromethyl, 2-fluoroethyl, chloromethyl, chloroethyl, and penta-fluoroethyl. C1-C2alkyl includes alkyl groups having 1or 2 carbon atoms, substituted with 1 or more halogen atoms, generally up to the maximum allowable number of halogen atoms.

[0044] “Haloalkoxy” is an haloalkyl group as defined above with the indicated number of carbon atoms attached through an oxygen bridge. Examples of haloalkoxy include, but are not limited to, fluoromethoxy, trifluoromethoxy, fluoroethoxy, trifluoroethoxy, chloromethoxy, chloroethoxy, bromo-n-propoxy, bromo-i-propoxy, iodo-n-butoxy, iodo-2-butoxy, or chloro-n- pentoxy. C1-C2haloalkoxy includes alkoxy groups having 1or 2 carbon atoms, substituted with 1 or more halogen atoms, generally up to the maximum allowable number of halogen atoms.

[0045] “Halo” or “halogen” as used herein refers to fluoro, chloro, bromo, or iodo. 9 NIH0164PCT

[0046] “IVO” as used herein refers to inhibitor of voltage-dependent anion channel (VDAC) oligomerization.

[0047] “Pharmaceutical compositions” are compositions comprising at least one active agent, such as a compound of Formula I or Formula II, or a pharmaceutically acceptable salt, derivative, or solvate thereof, and at least one other excipient, such as a carrier. “Carriers” are any inactive materials, including excipients and diluents, which may be added to the pharmaceutical compositions including carriers and diluents. Pharmaceutical compositions meet the U.S. FDA’s GMP (good manufacturing practice) standards for human or non-human drugs. Also included are any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, and isotonic and absorption delaying agents. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions. A “pharmaceutically acceptable carrier” includes both one and more than one such carrier.

[0048] “Pharmaceutically acceptable salt” includes derivatives of the disclosed compounds wherein the parent compound is modified by making non-toxic acid or base salts thereof, and further refers to pharmaceutically acceptable hydrates or solvates of such compounds and such salts. Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as carboxylic acids; and the like. The pharmaceutically acceptable salts include the conventional non-toxic salts and the quaternary ammonium salts of the parent compound formed, for example, from non-toxic inorganic or organic acids. For example, conventional non-toxic acid salts include those derived from inorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, nitric and the like; and the salts prepared from organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, pamoic, maleic, hydroxylmaleic, phenylacetic, glutamic, benzoic, salicylic, mesylic, esylic, besylic, sulfanilic, 2- acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, isethionic, HOOC-(CH2)n-COOH where n is 0-4, and the like. Lists of additional suitable salts may be found, e.g., in G. Steffen Paulekuhn, et al., Journal of Medicinal Chemistry 2007, 50, 6665 and Handbook of Pharmaceutically Acceptable Salts: Properties, Selection and Use, P. Heinrich 10 NIH0164PCT Stahl and Camille G. Wermuth Editors, Wiley-VCH, 2002.

[0049] The term “carrier” applied to pharmaceutical compositions / combinations of the disclosure refers to a diluent, excipient, or vehicle with which an active compound is provided. A “pharmaceutically acceptable carrier” means a substance, e.g., excipient, diluent, or vehicle, that is useful in preparing a pharmaceutical composition that is generally safe, non-toxic and neither biologically nor otherwise undesirable, and includes a carrier that is acceptable for veterinary use as well as human pharmaceutical use. A “pharmaceutically acceptable carrier” includes both one and more than one such carrier.

[0050] A “patient” or a “subject” is a human or non-human animal in need of medical treatment. Medical treatment can include treatment of an existing condition, such as a disease or disorder or diagnostic treatment. In some embodiments the patient is a human patient.

[0051] “Providing” means giving, administering, selling, distributing, transferring (for profit or not), manufacturing, compounding, or dispensing.

[0052] “Treatment” or “treating” means providing an active compound to a patient in an amount sufficient to measurably reduce any existing condition or slow existing condition progression.

[0053] The term "therapeutically effective amount" of a compound of Formula I, or a pharmaceutical composition, means an amount effective, when administered to a patient or subject, to provide a therapeutic benefit such as an amelioration of symptoms, decrease disease progression, or cause disease regression. Thus, a therapeutically effective amount of a compound or composition is also an amount sufficient to significantly reduce the indicia of the disease or condition being treated. A significant reduction is any detectable negative change that is statistically significant in a standard parametric test of statistical significance, such as Student’s t-test, in which p < 0.05.

[0054] “Administering” means giving, providing, applying, or dispensing by any suitable route. Administration of a combination of active agents includes administration of the combination in a single formulation or unit dosage form, administration of the individual active agents of the combination concurrently but separately, or administration of the individual active agents of the combination sequentially by any suitable route. The dosage of the individual active agents of the combination may require more frequent administration of one of the active agent(s) as compared to the other active agent(s) in the combination. Therefore, to permit appropriate dosing, packaged pharmaceutical products may contain one or more dosage forms that contain the 11 NIH0164PCT combination of active agents, and one or more dosage forms that contain one of the combination of active agents, but not the other active agent(s) of the combination. CHEMICALDESCRIPTION

[0055] The present disclosure provides compounds having VDAC oligomerization inhibitory activity. Certain compounds of Formula I can decrease mitochondrial stress, oxidative stress and ultimately inflammation.

[0056] In addition to compounds of Formula I shown above in the SUMMARY section, the disclosure includes the following particular embodiments of Formula I R10R11R9A R6 or a pharmaceutically acceptable solvate, or salt thereof, in which the variables may carry any of the values disclosed herein.

[0057] Formulae I, I-A, I-B, and I-C include embodiments in which the variables, e.g. R1, R2, R3, R4, R5, R6, R7, R8, R9, R10, R11, R12, R13, R15, R16, R17, A, and B carry the definitions set forth below. The variable definitions can be combined in any combination that results in a stable compound.

[0058] (a) The A ring is NNN R15N , and R15is hydrogen or methyl.

[0059] (b) The A ring is 12 NIH0164PCT R16N N N is hydrogen or methyl.

[0060] dyl.

[0061] (d) R1and R2are both halogen, preferably fluoro.

[0062] (e) R3 is hydroxyl.

[0063] (f) R3is -NH2.

[0064] (g) R4and R6are halogen, preferably fluoro, and R5, R7, and R8are all hydrogen.

[0065] (h) R9, R10, R12, and R13 are all hydrogen, and R11 is C1-C2haloalkoxy such as -OCF3 or - OCH2CF3.

[0066] The disclosure includes the following compounds of Formula I shown below, and their pharmaceutically acceptable solvate or salt thereof: , , , 13 NIH0164PCT 18 19 or 14 NIH0164PCT 28. In alternative embodiments, ring A may be an imidazole moiety and have one or more of the following structures: PHARMACEUTICALPREPARATIONS

[0067] Compounds disclosed herein can be administered as the neat chemical, but are preferably administered as a pharmaceutical composition. Accordingly, the disclosure provides pharmaceutical compositions comprising a compound of Formula I, or a pharmaceutically acceptable solvate, or salt thereof, (also referred to as “VDAC oligomerization inhibitor”) together with at least one pharmaceutically acceptable carrier. The pharmaceutical composition / combination may contain a compound of Formula I, or a pharmaceutically acceptable solvate or salt thereof as the only active agent or may be combined with one or more additional active agents. In certain embodiments the pharmaceutical composition is in a dosage form that contains from about 0.1 milligrams (mg) to about 4000 mg, from 0.1 mg to 3000 mg, from about 10 mg to about 2000 mg, from about 25 mg to about 1200 mg, or from about 50 mg to about 1000 mg of a compound of Formula I or a pharmaceutically acceptable salt or solvate thereof.

[0068] Compounds disclosed herein may be administered orally, topically, parenterally, by inhalation or spray, sublingually, intrathecally, intracranially, transdermally, via buccal administration, or by other means routine in the art for administering pharmaceutical compositions. The pharmaceutical composition may be formulated as any pharmaceutically useful form, e.g., as an aerosol, a cream, a gel, a pill, a capsule, a tablet, a syrup, a transdermal 15 NIH0164PCT patch, or an ophthalmic solution. Some dosage forms, such as tablets and capsules, are subdivided into suitably sized unit doses containing appropriate quantities of the active components, e.g., an effective amount to achieve the desired purpose.

[0069] Carriers include excipients and diluents and must be of sufficiently high purity and sufficiently low toxicity to render them suitable for administration to the patient being treated. The carrier can be inert, or it can possess pharmaceutical benefits of its own. The amount of carrier employed in conjunction with the compound is sufficient to provide a practical quantity of material for administration per unit dose of the compound.

[0070] Classes of carriers include, but are not limited to binders, buffering agents, coloring agents, diluents, disintegrants, emulsifiers, flavorants, glidants, lubricants, preservatives, stabilizers, surfactants, tableting agents, and wetting agents. Some carriers may be listed in more than one class, for example vegetable oil may be used as a lubricant in some formulations and a diluent in others. Exemplary pharmaceutically acceptable carriers include sugars, starches, celluloses, powdered tragacanth, malt, gelatin, talc, and vegetable oils. Optional active agents may be included in a pharmaceutical composition, which do not substantially interfere with the activity of the compound of the present invention.

[0071] The pharmaceutical compositions / combinations can be formulated for oral administration. These compositions contain between 0.1 and 99 weight % (wt.%) of a compound of Formula I or a pharmaceutically acceptable solvate or salt thereof and usually at least about 5 wt.% of a compound of Formula I or a pharmaceutically acceptable solvate or salt thereof. Some embodiments contain from about 25 wt.% to about 50 wt. % or from about 5 wt.% to about 75 wt.% of a compound of Formula I or a pharmaceutically acceptable solvate or salt thereof. METHODS OF TREATMENT

[0072] The disclosure also provides methods of inhibiting VDAC oligomerization in a subject in need thereof, wherein the method comprises administering to the subject a compound of Formula I, or a pharmaceutically acceptable solvate or salt thereof in an amount effective to inhibit VDAC oligomerization in the subject.

[0073] The subject can have an autoimmune disease, blood disorder, bone disease, brain disease, a cancer, a cardiovascular disease, a dermatological disease, an eye and ear disease, a gastrointestinal disorder, a gynecological disorder, an inflammatory disease, a kidney disease, a 16 NIH0164PCT liver disease, a lysosomal storage disease, a metabolic disorder, a mitochondrial disease, a muscle disorder, a nerve disease, a neurodegenerative disease, an oral or throat disease, a radiation / chemotherapy / immunotherapy-induced condition, a post-infection disease, a post- trauma tissue damage, a respiratory disease, or a testicular or prostate disease.

[0074] Examples of autoimmune diseases that can respond to the beneficial effects of the VDAC oligomerization inhibitors as disclosed herein can include acromegaly, acquired aplastic anemia, acquired hemophilia, agammaglobulinemia, primary Alopecia areata, ankylosing spondylitis (AS), anti-NMDA receptor encephalitis, antiphospholipid syndrome (APS) | catastrophic antiphospholipid syndrome (CAPS) / Asherson's syndrome, arteriosclerosis, autoimmune Addison’s disease (AAD), autoimmune autonomic ganglionopathy (AAG) / autoimmune dysautonomia | autoimmune gastrointestinal dysmotility (AGID), autoimmune encephalitis | acute disseminated encephalomyelitis (ADEM), autoimmune gastritis, autoimmune hemolytic anemia (AIHA), autoimmune hepatitis (AIH), autoimmune hyperlipidemia, autoimmune hypophysitis / lymphocytic hypophysitis, autoimmune inner ear disease (AIED), autoimmune lymphoproliferative syndrome (ALPS), autoimmune myelofibrosis, autoimmune myocarditis, autoimmune oophoritis, autoimmune pancreatitis (AIP), autoimmune polyglandular syndromes, types I, II, & III (APS type 1, APS type 2, APS type 3, APECED), autoimmune progesterone dermatitis, autoimmune retinopathy (AIR), autoimmune sudden sensorineural hearing loss (SNHL), Balo disease, Behçet’s disease, birdshot chorioretinopathy / birdshot uveitis, bullous pemphigoid, Castleman disease, celiac disease, chagas disease, chronic inflammatory demyelinating polyneuropathy (CIDP), chronic autoimmune urticaria, Churg-Strauss syndrome / eosinophilic granulomatosis with polyangiitis (EGPA), Cogan’s syndrome, cold agglutinin disease, CREST syndrome | limited cutaneous systemic sclerosis, Crohn’s disease (CD), Cronkhite-Canada syndrome (CSS), cryptogenic organizing pneumonia (COP), dermatitis herpetiformis, dermatomyositis, diabetes, type 1, Discoid lupus, Dressler’s syndrome / postmyocardial infarction / postpericardiotomy syndrome, eczema / atopic dermatitis, endometriosis, eosinophilic esophagitis / eosinophilic gastroenteritis, eosinophilic fasciitis, erythema nodosum, essential mixed cryoglobulinemia, Evans syndrome, fibrosing alveolitis / idiopathic pulmonary fibrosis (IPF), giant cell arteritis / temporal arteritis / Horton’s disease, giant cell myocarditis, glomerulonephritis, Goodpasture’s syndrome / anti-GBM / anti- TBM disease, granulomatosis with polyangiitis (GPA) / Wegener’s granulomatosis, graves' 17 NIH0164PCT disease / thyroid eye disease, Guillain-Barré syndrome (GBS), Hashimoto’s thyroiditis / chronic lymphocytic thyroiditis / autoimmune thyroiditis, Henoch-Schönlein purpura / IgA vasculitis, hidradenitis suppurativa, hurst’s disease / acute hemorrhagic leukoencephalitis (AHLE), hypogammaglobulinemia IgA nephropathy / Berger's disease, immune-mediated necrotizing myopathy (IMNM), immune thrombocytopenia (ITP) / autoimmune thrombocytopenic purpura / autoimmune thrombocytopenia, Inclusion body myositis, IgG4-related sclerosing disease (ISD), interstitial cystitis, juvenile idiopathic arthritis / adult-onset Still's disease, juvenile polymyositis | juvenile dermatomyositis | juvenile myositis, Kawasaki disease, Lambert-Eaton myasthenic syndrome (LEMS), leukocytoclastic vasculitis, lichen planus | lichen planopilaris, lichen sclerosus, ligneous conjunctivitis, linear IgA disease (LAD) | linear IgA bullous dermatosis (LABD), lupus nephritis, Lyme disease / chronic Lyme disease / post-treatment Lyme disease syndrome (PTLDS), lymphocytic colitis / microscopic colitis, lymphocytic hypophystitis / autoimmune hypophystitis, mast cell activation syndrome, Ménière’s disease, microscopic polyangiitis (MPA) / ANCA-associated vasculitis, mixed connective tissue disease (MCTD), Mooren’s ulcer, Mucha-Habermann disease, multifocal motor neuropathy, multiple sclerosis (MS), Myalgic encephalomyelitis (ME) / chronic fatigue syndrome (CFS), myasthenia gravis (MG), narcolepsy, neuromyelitis Optica / Devic's disease, ocular cicatricial pemphigoid, opsoclonus-myoclonus syndrome (OMS), palindromic rheumatism, paraneoplastic cerebellar degeneration, paraneoplastic pemphigus, Parry-Romberg syndrome (PRS) / hemifacial atrophy (HFA) / progressive facial hemiatrophy, paroxysmal nocturnal hemoglobinuria (PNH), peripheral uveitis / pars planitis, PANS / PANDAS, parsonage-turner syndrome, pemphigus gestationis / herpes gestationis, pemphigus foliaceus, pemphigus vulgaris, pernicious anemia, POEMS syndrome, polyarteritis nodosa, polymyalgia rheumatica, polymyositis, postural orthostatic tachycardia syndrome (POTS), primary biliary cirrhosis (PBC) / primary biliary cholangitis, primary sclerosing cholangitis (PSC), psoriasis, palmoplantar pustulosis, psoriatic arthritis, pulmonary fibrosis, idiopathic (IPF), pure red cell aplasia (PRCA), pyoderma gangrenosum, Rasmussen's encephalitis, Raynaud’s syndrome / phenomenon, reactive arthritis / Reiter’s syndrome, reflex sympathetic dystrophy syndrome (RSD) / complex regional pain syndrome (CRPS), relapsing polychondritis, restless leg syndrome (RLS) / Willis-Ekbom disease, rheumatic fever, rheumatoid arthritis, sarcoidosis, Schmidt syndrome / autoimmune polyendocrine syndrome type II / polyglandular autoimmune syndrome, type 2, scleritis, 18 NIH0164PCT scleroderma, sclerosing mesenteritis / mesenteric panniculitis, serpiginous choroidopathy, Sjögren’s syndrome, stiff person syndrome (SPS), small fiber sensory neuropathy, systemic lupus erythematosus (SLE), subacute bacterial endocarditis (SBE), subacute cutaneous lupus, susac syndrome, sydenham's chorea, sympathetic ophthalmia, Takayasu’s arteritis (vasculitis), testicular autoimmunity (vasculitis, orchitis), Tolosa-Hunt syndrome, transverse myelitis (TM), tubulointerstitial nephritis uveitis syndrome (TINU), ulcerative colitis (UC), undifferentiated connective tissue disease (UCTD), uveitis | anterior / intermediate / posterior, vasculitis, VEXAS syndrome, vitiligo, and Vogt-Koyanagi-Harada syndrome (VKH).

[0075] Examples of blood disorders that can respond to the beneficial effects of the VDAC oligomerization inhibitors as disclosed herein can include iron-deficiency anemia, vitamin B12 deficiency anemia (pernicious anemia), folate deficiency anemia, anemia of chronic disease, aplastic anemia, hemolytic anemia (autoimmune, drug-induced), sideroblastic anemia, sickle cell disease, thalassemia (alpha and beta), hereditary spherocytosis, hereditary elliptocytosis, G6PD deficiency, pyruvate kinase deficiency, paroxysmal nocturnal hemoglobinuria (PNH), acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), Hodgkin lymphoma, non-Hodgkin lymphoma, neutropenia (congenital, autoimmune, cyclic), leukocytosis, leukemoid reaction, eosinophilia, monocytosis, myelodysplastic syndromes (MDS), myeloproliferative neoplasms (e.g., polycythemia vera, essential thrombocythemia), polycythemia vera, essential thrombocythemia, myelofibrosis, immune thrombocytopenic purpura (ITP), heparin-induced thrombocytopenia (HIT), thrombocytopenia, thrombocytosis, Glanzmann thrombasthenia, Bernard-Soulier syndrome, platelet storage pool disease, hemophilia A (Factor VIII deficiency), hemophilia B (Factor IX deficiency), von Willebrand disease, Factor XI deficiency, disseminated intravascular coagulation (DIC), antiphospholipid antibody syndrome, protein C deficiency, protein S deficiency, antithrombin III deficiency, Factor V Leiden mutation, prothrombin G20210A mutation, multiple myeloma, Waldenström macroglobulinemia, monoclonal gammopathy of undetermined significance (MGUS), light chain (AL) amyloidosis, POEMS syndrome, Fanconi anemia, Diamond-Blackfan anemia, Shwachman-Diamond syndrome, Evans syndrome, and hemophagocytic lymphohistiocytosis (HLH).

[0076] Osteoporosis, osteomalacia, rickets, osteogenesis imperfecta, Paget’s disease of bone, osteopetrosis, fibrous dysplasia, osteitis fibrosa cystica (secondary to hyperparathyroidism), bone 19 NIH0164PCT metastases, primary bone cancers (such as osteosarcoma, Ewing sarcoma, and chondrosarcoma), avascular necrosis (osteonecrosis), osteomyelitis, rheumatoid arthritis-related bone erosion, ankylosing spondylitis, hypertrophic osteoarthropathy, multiple myeloma-associated bone lesions, bone cysts (simple and aneurysmal), hyperostosis, melorheostosis, and skeletal dysplasias (such as achondroplasia and thanatophoric dysplasia)..

[0077] Examples of brain diseases (non-degeneration) that can respond to the beneficial effects of the VDAC oligomerization inhibitors can include epilepsy, anxiety, depression, bipolar disease, fragile X syndrome, schizophrenia, obsessive-compulsive disorder (OCD), post- traumatic stress disorder (PTSD), traumatic brain injury, chronic traumatic encephalopathy, panic disorder, autism spectrum disorder (ASD), borderline personality disorder (BPD), eating disorders (anorexia nervosa, bulimia nervosa, binge-eating disorder), substance use disorders (substance abuse and dependence), dissociative disorders (dissociative identity disorder, dissociative amnesia), phobias, leaky blood-brain barrier, addiction, headaches, including migraine and cluster headache, essential tremor, ADD and ADHD, and insomnia.

[0078] Examples of cancers that can respond to the beneficial effects of the VDAC oligomerization inhibitors can include carcinomas, sarcomas, leukemias, lymphomas, myelomas, melanomas, germ cell tumors, blastomas, and mixed-type cancers. VDAC1 oligomerization inhibitors will also improve immune response to all cancers. It will also enhance the efficacy of immune therapies, including immune checkpoint inhibitors (PD-1, PD-L1, CTLA-4, LAG-3, TIGIT, TIM-3), adoptive cell therapies (CAR-T, TCR-T, TILs, CAR-NK), cancer vaccines (preventive HPV / HBV and therapeutic like sipuleucel-T and neoantigen vaccines), monoclonal antibodies (naked, ADCs, BiTEs), cytokine therapies (IL-2, IFN-α, IL-15, IL-12), oncolytic viruses (like T-VEC), immune agonists (TLR, STING, CD40, OX40, 4-1BB, GITR), and combination therapies involving immunotherapy with chemo, radiation, or other immuno- modulators.

[0079] Examples of cardiovascular diseases that can respond to the beneficial effects of the VDAC oligomerization inhibitors as disclosed herein can include coronary artery disease (CAD), myocardial infarction (heart attack), heart failure, arrhythmias (irregular heartbeats), including atrial fibrillation, hypertensive heart disease, stroke, cardiomyopathy (heart muscle disease), peripheral artery disease (PAD), endocarditis, pulmonary embolism, deep vein thrombosis (DVT), stroke (though primarily neurological, often related to cardiovascular issues), stent (IVO- 20 NIH0164PCT eluting), post-stent recovery, atherosclerosis, blood vessel thickening, aortic dissection, aneurysm, disseminated intravascular coagulation (DIC), pericarditis, hypertension, progeria and dysautonomia.

[0080] Examples of dermatological diseases that can respond to the beneficial effects of the VDAC oligomerization inhibitors as disclosed herein can include psoriasis, dermatitis (including eczema), cellulitis, shingles, herpes simplex, urticaria, rosacea, rhytids and wrinkles, lentigo, and dermatoporosis.

[0081] Examples of eye and ear diseases that can respond to the beneficial effects of the VDAC oligomerization inhibitors as disclosed herein can include conjunctivitis, uveitis, blepharitis, keratitis, scleritis, episcleritis, retinitis, iritis, endophthalmitis, optic neuritis, choroiditis, orbital cellulitis, macular degeneration, retinal detachment, retinitis pigmentosa, dry eye syndrome, hearing impairment, ear infection, vestibular neuritis, cataracts, tinnitus, Meniere’s disease, vertigo, glaucoma, and diabetic retinopathy.

[0082] Examples of gastrointestinal disorders that can respond to the beneficial effects of the VDAC oligomerization inhibitors as disclosed herein can include chronic pancreatitis, microbiome change, gastroesophageal reflux disease (GERD), peptic ulcer disease, gastritis, irritable bowel syndrome (IBS), diverticulitis, celiac disease, gall bladder inflammation, pancreatitis, gastroenteritis (Stomach flu), hemorrhoids, small intestine inflammation, intestinal infarct, intestinal torsion, inflammatory bowel disease, leaky gut syndrome, and appendicitis.

[0083] Examples of gynecological disorders that can respond to the beneficial effects of the VDAC oligomerization inhibitors as disclosed herein can include pelvic inflammatory disease, endometriosis, polycystic ovarian disease, dyspareunia, dryness, fibroids, vaginitis, vulva infection and damage and dysmenorrhia.

[0084] Examples of inflammatory diseases (in addition to autoimmune diseases listed above) that can respond to the beneficial effects of the VDAC oligomerization inhibitors as disclosed herein can include ME / CFS (chronic fatigue syndrome), long-covid (PASC), fibromyalgia, microbiome change, post-infection syndrome, chronic obstructive pulmonary disease (COPD), gulf war syndrome, sepsis, meningitis, encephalitis, alopecia areata and other forms of hair loss, cellular senescence (SASP), cytokine storm, anti-NMDAR encephalitis, and tissue fibrosis.

[0085] Examples of kidney diseases that can respond to the beneficial effects of the VDAC oligomerization inhibitors as disclosed herein can include chronic kidney disease (CKD), acute 21 NIH0164PCT kidney injury (AKI), glomerulonephritis, kidney stones (nephrolithiasis), nephrotic syndrome, urinary tract infections (UTIs), renal artery stenosis, Alport syndrome, IgA nephropathy, lupus nephritis, diabetic nephropathy, hemolytic uremic syndrome (HUS), and cystitis.

[0086] Examples of liver diseases that can respond to the beneficial effects of the VDAC oligomerization inhibitors as disclosed herein can include hepatitis (including hepatitis A, hepatitis B, and hepatitis C), drug-induced hepatitis, cirrhosis, non-alcoholic fatty liver disease (NAFLD), alcoholic liver disease, liver cancer (hepatocellular carcinoma), Wilson's disease, hemochromatosis, primary biliary cholangitis (formerly known as primary biliary cirrhosis), primary sclerosing cholangitis, autoimmune hepatitis, Gilbert's syndrome, Budd-Chiari syndrome, alpha-1 antitrypsin deficiency, liver abscess, liver fibrosis, non-alcoholic fatty liver disease (NAFLD), alcoholic liver disease (ALD), viral hepatitis, and liver cirrhosis.

[0087] Examples of lysosomal storage diseases that can respond to the beneficial effects of the VDAC oligomerization inhibitors as disclosed herein can include Gaucher disease, Fabry disease, Pompe disease (Glycogen storage disease type II), Niemann-Pick disease, Tay-Sachs disease, Krabbe disease (Globoid cell leukodystrophy), metachromatic leukodystrophy, hurler syndrome (Mucopolysaccharidosis type I), hunter syndrome (mucopolysaccharidosis type II), Sanfilippo syndrome (mucopolysaccharidosis type III), Maroteaux-Lamy syndrome (Mucopolysaccharidosis type VI), sly syndrome (Mucopolysaccharidosis type VII), Anderson- Fabry disease, Wolman disease, and Farber disease.

[0088] Examples of metabolic disorders that can respond to the beneficial effects of the VDAC oligomerization inhibitors as disclosed herein can include diabetes mellitus (Type 1 and Type 2), obesity, metabolic syndrome, hyperthyroidism, hypothyroidism, acidosis, alkalosis, Addison's disease, Wilson's disease, gout, hyperlipidemia (high cholesterol or triglycerides), maple syrup urine disease (MSUD), glycogen storage diseases, Fabry disease, obesity, and metabolic syndrome.

[0089] Examples of mitochondrial diseases that can respond to the beneficial effects of the VDAC oligomerization inhibitors as disclosed herein can include neurological disorders, metabolic disorders, arrhythmias, liver disorders, and skeletal muscle disorders. Neurological disorders can include mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes (MELAS), leigh syndrome, mitochondrial myopathy, and amyotrophic lateral sclerosis (ALS). Metabolic disorders can include mitochondrial DNA depletion syndromes, leigh syndrome, 22 NIH0164PCT Barth syndrome, Pearson syndrome, mitochondrial diabetes, mitochondrial myopathy, Kearns- Sayre syndrome, Leber's hereditary optic neuropathy (LHON), mitochondrial neurogastrointestinal encephalopathy (MNGIE) syndrome, cardiac disorders such as dilated cardiomyopathy and hypertrophic cardiomyopathy. Arrhythmias can include mitochondrial myopathy, liver disorders can include mitochondrial hepatopathy. Skeletal muscle disorders can include mitochondrial myopathy, myoclonic epilepsy with ragged-red fibers (MERRF), Kearns- Sayre syndrome, ophthalmologic disorders such as Leber's hereditary optic neuropathy (LHON), Kearns-Sayre syndrome, and chronic progressive external ophthalmoplegia (CPEO).

[0090] Examples of muscle disorders that can respond to the beneficial effects of the VDAC oligomerization inhibitors as disclosed herein can include muscular dystrophy (including Duchenne muscular dystrophy, Becker muscular dystrophy, and others), myasthenia gravis, polymyositis, dermatomyositis, inclusion body myositis, facioscapulohumeral muscular dystrophy (FSHD), limb-girdle muscular dystrophy, myotonic dystrophy (including type 1 and type 2), McArdle disease (Glycogen storage disease type V), Pompe disease (Glycogen storage disease type II), Charcot-Marie-Tooth disease, spinal muscular atrophy (SMA), metabolic myopathies (e.g., mitochondrial myopathies), polymyalgia rheumatica, rhabdomyolysis, sarcopenia, tendinitis, ligament tear, and Pompe disease.

[0091] Examples of nerve diseases that can respond to the beneficial effects of the VDAC oligomerization inhibitors as disclosed herein can include peripheral neuropathy, Guillain-Barré syndrome, neuropathic pain, non-neuropathic pain, trigeminal neuralgia, diabetic neuropathy, carpal tunnel syndrome, Bell's palsy, and myasthenia gravis.

[0092] Examples of neurodegenerative diseases that can respond to the beneficial effects of the VDAC oligomerization inhibitors as disclosed herein can include Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis (ALS), multiple sclerosis (MS), frontotemporal dementia (FTD), progressive supranuclear palsy (PSP), lewy body dementia, spinocerebellar ataxia, Friedreich's ataxia, Prion diseases (such as Creutzfeldt-Jakob disease), corticobasal degeneration (CBD), Wilson's disease, spinal muscular atrophy (SMA), batten disease, ataxia telangiectasia, and Bell’s palsy.

[0093] Examples of oral / throat diseases that can respond to the beneficial effects of the VDAC oligomerization inhibitors as disclosed herein can include periodontal disease (Gingivitis and Periodontitis), temporomandibular joint disorder (TMJ) canker sores (Aphthous ulcers), salivary 23 NIH0164PCT gland disorders, efflux (LPR, a condition where stomach acid flows back into the throat, causing irritation and inflammation), tonsillitis, esophagitis, vocal cord nodules / polyps (noncancerous growths on the vocal cords, often caused by vocal strain or misuse), mononucleosis (Mono, a viral infection caused by the Epstein-Barr virus, characterized by sore throat, fever, and fatigue), GERD (gastroesophageal reflux disease), chronic acid reflux, pharyngitis and laryngitis, and sinusitis.

[0094] Examples of post-cancer treatment that can respond to the beneficial effects of the VDAC oligomerization inhibitors as disclosed herein can include radiation / chemotherapy / immunotherapy-induced peripheral neuropathy, radiation / chemotherapy / immunotherapy-induced hair loss (alopecia), radiation / chemotherapy / immunotherapy-induced skin changes (radiation dermatitis), radiation / chemotherapy / immunotherapy-induced fatigue, radiation / chemotherapy / immunotherapy-induced gastrointestinal symptoms (diarrhea, nausea, and vomiting), radiation / chemotherapy / immunotherapy-induced organ damage (e.g., lung damage from thoracic radiation), radiation / chemotherapy / Immunotherapy-induced secondary cancers, radiation / chemotherapy / immunotherapy / surgery-related complications (infection, bleeding, scarring, organ damage), radiation / chemotherapy / immunotherapy / surgery-related lymphedema (swelling due to lymphatic system damage), radiation / chemotherapy / immunotherapy-related cardiovascular toxicities (e.g., hypertension, heart failure), radiation / chemotherapy / immunotherapy-related adverse events (e.g., colitis, hepatitis), radiation / chemotherapy / immunotherapy-related endocrine disorders (e.g., thyroid dysfunction), radiation / chemotherapy / immunotherapy-related skin reactions (e.g., rash, pruritus), and radiation / chemotherapy / immunotherapy long-term effects on fertility and reproductive health.

[0095] Examples of infections or post-infection diseases that can respond to the beneficial effects of the VDAC inhibitors as disclosed herein can include those caused by any type of microbes (bacteria, archaea, fungi, protozoa, algae, viruses, prions, helminths, and viroid) and post-streptococcal glomerulonephritis, post-infectious irritable bowel syndrome (PI-IBS), post- infectious arthritis, post-infectious reactive airway disease, post-infectious encephalitis, post- infectious myocarditis, post-infectious neuropathies, post-infectious glomerulonephritis, post- infectious cough, post-Lyme syndrome, HIV AIDS, malaria, dengue fever, typhoid fever, 24 NIH0164PCT infection-associated pain, Ebola virus, rabies, parasitic diseases, yellow fever, cholera, tetanus, and polio.

[0096] Examples of post-trauma tissue damage that can respond to the beneficial effects of the VDAC inhibitors as disclosed herein can include post-trauma tissue damage to all organs, including the brain (e.g CTE), post-surgical damage, infection-associated tissue damage, and chemical and radiation-associated tissue damage.

[0097] Examples of testicular / prostate diseases that can respond to the beneficial effects of the VDAC inhibitors as disclosed herein can include testicular torsion, epididymitis, orchitis, testicular trauma or injury, testicular infarction, prostatitis, and erectile dysfunction.

[0098] Examples of respiratory diseases that can respond to the beneficial effects of the VDAC inhibitors as disclosed herein can include acute respiratory distress syndrome (ARDS), bronchiolitis, sarcoidosis, bronchiectasis, cystic fibrosis, allergy (including food, chemicals, environmental, and infectious), tuberculosis, asthma, pulmonary hypertension, pulmonary fibrosis, pneumonia, laryngitis, and bronchitis.

[0099] Preferably, a method of inhibiting voltage-dependent anion channel oligomerization in a subject in need thereof comprises administering an effective amount of a compound of Formula I, a solvate or salt thereof to the subject, wherein the subject has covid-19, long covid, atherosclerosis, autoimmunity, hepatitis, fibrosis, thrombosis, diabetes, Alzheimer's disease, Parkinson's disease, or T-cell exhaustion.

[0100] An inhibitor for VDAC oligomerization as described herein may be the only active agent administered (monotherapy) or may be combined with one or more other active agents, including other IVO compounds (combination, adjunct, or augmentation therapy).

[0101] Methods of treatment include providing certain dosage amounts of a compound of Formula I or a pharmaceutically acceptable solvate or salt thereof to a patient. Dosage levels of each compound of from about 1 ng to about 250 mg per kilogram of body weight per day are useful in the treatment of the above-indicated conditions (about 0.1 µg to about 15 g per patient per day). The amount of compound that may be combined with the carrier materials to produce a single dosage form will vary depending upon the patient treated and the particular mode of administration. Dosage unit forms will generally contain between from about 0.1 µg to about 1000 mg of each active compound. In certain embodiments 0.1 µg to 1000 mg, or 0.1 µg to 400 25 NIH0164PCT mg of a soluble epoxide hydrolase inhibitor is provided daily to a patient. Frequency of dosage may also vary depending on the compound.

[0102] In an embodiment, the method further comprises administering to the patient in need thereof at least one additional therapeutic agent.

[0103] Such administration encompasses co-administration of these therapeutic agents in a substantially simultaneous manner, such as in a single dosage form having a fixed ratio of active ingredients or in separate dosage forms for each active ingredient. In addition, such administration also encompasses administration of each therapeutic agent in a sequential manner, either at approximately the same time or at different times. In either case, the treatment regimen will provide the beneficial effects of each therapeutic agent in the drug combination in treating the conditions or disorders described herein.

[0104] It will be understood, however, that the specific dose level for any particular patient will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, route of administration, and rate of excretion, drug combination and the severity of the particular disease undergoing therapy.

[0105] Methods of treatment provided herein are also useful for treatment of mammals other than humans, including for veterinary applications such as to treat horses and livestock e.g. cattle, chickens, sheep, cattles, goats, swine and the like, and pets (companion animals) such as dogs and cats. EXAMPLES GENERAL METHODS FOR CHEMISTRY

[0106] All reagents were used as received from the following suppliers: NIH Chemistry and Synthesis Center. The1H and13C NMR spectra were recorded on Bruker 400 MHz and Varian 400 MHz spectrometer. Chemical shifts are reported in parts per million and were referenced to residual proton solvent signals. Example 1. Synthesis of IVO-14 26 NIH0164PCT N F C N N N3O N N O O F [10 tetrazol-1-yl)propan-2-ol (140 mg, 0.324 mmol) in tetrahydrofuran (THF, 2 ml) was degassed by bubbling argon (Ar) gas through it for 5 minutes (min). Then to this solution were added (4- (trifluoromethoxy)phenyl)boronic acid (103 mg, 0.356 mmol, 1.1 eq.), sodium carbonate (86 mg, 0.81 mmol, 2.5 eq.), and water (250 ml) at room temperature (rt) while continuing degassing with Ar. Finally PdCl2dppf•CH2Cl2 catalyst (26 mg, 32 mmol, 10 mol%) was added, and the solution was degassed with Ar for an additional 10 min. The reaction mixture was heated at 65oC for 4 hours (h), then left at room temperature overnight. The crude product was filtered through celite. All the volatiles were removed, and residue was chromatographed over silica gel, 12 G gold SiO2 column using 10 to 80 % ethyl acetate (EtOAc) in hexane 20 column volume (CV) to give the product (IVO-14) as off white solid 110 mg (66 %). Example 2. Synthesis of IVO-15 N F C N N3O N HO OH F

[0108] A solution of 1-(5-bromopyridin-2-yl)-2-(2,4-difluorophenyl)-1,1-difluoro-3-(5-methyl- 1H-tetrazol-2-yl)propan-2-ol (64 mg, 0.14 mmol) in THF (2 ml) was degassed by bubbling Ar gas through it for 5 min. Then to this solution were added (4-(trifluoromethoxy)phenyl)boronic acid (35 mg, 0.17 mmol, 1.2 eq.), sodium carbonate (38 mg, 0.36 mmol, 2.5 eq.), and water (250 ml) at room temperature while continuing degassing with Ar. Finally PdCl2dppf•CH2Cl2catalyst 27 NIH0164PCT (12 mg, 0.14 mmol, 10 mol%) was added, and the solution was degassed with Ar for an additional 10 min. The reaction mixture was then heated at 65oC for 4 h, and then left at room temperature overnight. The crude product was filtered through celite. All the volatiles were removed, and residue was chromatographed over silica gel, 12 G gold SiO2 column using 0 to 60 % EtOAc in hexane 25 CV to give the product (IVO-15) as off white solid 50 mg (66 %). Example 3. Synthesis of IVO-16 NNF3CONNF [ py y , p y , ethyl- 2H-tetrazol-2-yl)propan-2-ol (82 mg, 0.18 mmol) in THF (2 ml) was degassed by bubbling Ar gas through it for 5 min. Then to this solution were added (4-(trifluoromethoxy)phenyl)boronic acid (45 mg, 0.22 mmol, 1.2 eq.), sodium carbonate (49 mg, 0.46 mmol, 2.5 eq.), and water (250 ml) at room temperature while continuing degassing with Ar. Finally PdCl2dppf•CH2Cl2 catalyst (15 mg, 0.18 mmol, 10 mol%) was added, and the solution was degassed with Ar for an additional 10 min. The reaction mixture was heated at 65oC for 4 h, then left at room temperature overnight. The crude product was filtered through celite. All the volatiles were removed, and residue was chromatographed over silica gel, 12 G gold SiO2 column using 0 to 40 % EtOAc in hexane 20 CV to give the product (IVO-16) as off white solid 67 mg (69 %). Example 4. Synthesis of IVO-17 28 NIH0164PCT N O N N N HO O Br N H N B CF N N F [1 tetrazol-1-yl)propan-2-ol (55 mg, 0.13 mmol) in THF (1.25 ml) was degassed by bubbling Ar gas through it for 5 min. To this solution were added (4-(trifluoromethoxy)phenyl)boronic acid (31 mg, 0.15 mmol, 1.2 eq.), sodium carbonate (34 mg, 0.32 mmol, 2.5 eq.), and water (250 ml) at room temperature while continuing degassing with Ar. Finally PdCl2dppf•CH2Cl2 catalyst (10 mg, 13 mmol, 10 mol%) was added, and the solution was degassed with Ar for an additional 10 min. The reaction mixture was heated to 65oC for 4 h, and then left at room temperature overnight. The crude product was filtered through celite. All the volatiles were removed, and residue was chromatographed over silica gel, 12 G gold SiO2 column using 0 to 40 % EtOAc in hexane 25 CV. to give the product (IVO-17) as off white solid 42 mg (82 %). Example 5. Synthesis of IVO-18 NNF3CO HO ONNN H F [1 py y , p y , tetrazol-2-yl)propan-2-ol (42 mg, 97 mmol) in THF (1.25 ml) was degassed by bubbling Ar gas through it for 5 min. To this solution were added (4-(2,2,2-trifluoroethoxy)phenyl)boronic acid (26 mg, 0.12 mmol, 1.2 eq.), sodium carbonate (26 mg, 0.24 mmol, 2.5 eq.), and water (250 ml) at room temperature while continuing degassing with Ar. Finally PdCl2dppf•CH2Cl2 catalyst (7.9 mg, 9.7 mmol, 10 mol%) was added, and the solution was degassed with Ar for an additional 10 min. The reaction was heated at 65oC for 4 h, then left at room temperature 29 NIH0164PCT overnight. The crude product was filtered through celite. All the volatiles were removed, and residue was chromatographed over silica gel, 12 G gold SiO2 column using 5 to 70 % EtOAc in hexane 25 CV. to give the product (IVO-18) as off white solid 56 mg (86 %). IVO-18 is comprised of two enantiomers, IVO-18.2 and IVO-18.4: o a sou o o e - - , - uoop e y o a --y uoo e y - - - , , - trifluoroethoxy)phenyl)pyridine (80 mg, 0.17 mmol) in 1.5 ml anhydrous dimethylformamide (DMF) was added sodium 5-methyltetrazol-1-ide dihydrate (25 mg.0.17 mmol, 1 eq.) at room temperature. The resulting reaction mixture was heated at 80oC for 12 h. After this time the reaction was quenched by addition of water and extracted with Ethyl Acetate (3 times). The organic layers were combined, dried over anhydrous sodium sulfate, concentrated and 30 NIH0164PCT chromatographed over silica gel (12 G gold column, 5 to 75 % Ethyl acetate in hexane to give two isomeric products the non-polar compound being 2,5 isomer [III] (IVO-19, 29 mg, 31 %) and polar compound being 1,5-isomer [IV] (IVO-20, 25 mg, 26 %). Example 7. Synthesis of IVO-17 and IVO-21 O ONNO N N CF3Na F CF3NNCF3NNO N HO F HO F F trifluoroethoxy)phenyl)pyridine (110 mg, 0.24 mmol) in 1 ml anhydrous DMF was added sodium tetrazol-1-ide hydrate (29 mg. 0.26 mmol, 1.1 eq.) at room temperature. The resulting reaction mixture was heated at 80oC for 12 h. After this time the reaction was quenched by addition of water and extracted with Ethyl Acetate (3 times). The organic layers were combined, dried over anhydrous sodium sulfate, concentrated and chromatographed over silica gel (12 G gold column, 5 to 75 % Ethyl acetate in hexane to give two isomeric products the non-polar compound being 2,5 isomer [III] (IVO-21, 25 mg, 20 %) and polar compound being 1,5-isomer [IV] (IVO-17, 18 mg, 14 %). IVO-21 is comprised of two enantiomers, IVO-21.2 and IVO-21.4: BIOLOGICAL RESULTS AND METHODS

[0114] The methods used to generate the results in the figures are described.

[0115] Cell culture and reagents. Hela cells were maintained in DMEM supplemented with 10% fetal bovine serum. Cells were treated with Ketoconazole, Posaconazole, Miconazole (Cayman chem), VBIT-4 (Selleckchem), Otesaconazole, IVO10 - 21 (KareBay Biochem), Tunicamycin (Sigma-Aldrich), EGS (Thermo Scientific) or DMSO for the indicated time and doses. 31 NIH0164PCT

[0116] Immunoblotting. Cells were lysed in RIPA buffer and subjected to immunoblotting. The following antibodies were used: AMPK, p-AMPK (T172), p-S6k1 (T389), S6K1, p62, LC3B, GAPDH (Cell Signaling Technology), VDAC1 (Abcam), HK1, HK2 (ProteinTech). VDAC1 interaction with Hexokinases were visualized by immunoprecipitating Hexokinases (ProteinTech) from the extract (300 μg) and immunoblotting with VDAC1 antibody (Abcam).

[0117] VDAC1 cross-linking assay. Hela cells were rinsed 3 times with PBS and harvested by scraping. Cell pellets were incubated with 0.25 mM EGS in PBS, pH 8.3 at 30 °C for 15 minutes. The pellets were lysed in NP-40 lysis buffer by sonication on ice. The protein content was measured using the Pierce BCA Protein Assay. The samples (50 µg) were then subjected to SDS‒PAGE and immunoblotting using VDAC1 antibody.

[0118] Semi-denaturing detergent agarose gel electrophoresis (SDD–AGE). LX-2 cells were seeded in 6-well culture plates (ThermoFisher Scientific, USA) at a density of 2 x 105cells per well and cultured in DMEM supplemented with additives for 24 hours. Following pre-treatment with IVO 21 at a concentration of 1 µM in complete growth medium for 1 hour, cells were subsequently exposed to TGF-beta (InvivoGen, San Diego, USA) at a concentration of 5 ng / ml for 48 hours. Whole cell lysates were prepared and suspended in 1× sample buffer (0.5× TBE, 10% glycerol, 2% SDS, and 0.0025% bromophenol blue), then subjected to semi-denaturing detergent agarose gel electrophoresis (SDD-AGE). For SDD-AGE, samples were loaded onto a 1.5% vertical agarose gel (1× TBE and 0.1% SDS) and electrophoresed in running buffer (1× TBE and 0.1% SDS) for 50 minutes at 100 V and 4°C. Subsequently, proteins were transferred onto PVDF membranes (Millipore, USA) for immunoblotting.

[0119] Cells were lysed in RIPA buffer and subjected to immunoblotting. The following antibodies were used: AMPK, p-AMPK (T172), p-S6k1 (T389), S6K1, p62, LC3B, GAPDH (Cell Signaling Technology), VDAC1 (Abcam), HK1, HK2 (ProteinTech). VDAC1 interaction with Hexokinases were visualized by immunoprecipitating Hexokinases (ProteinTech) from the extract (300 μg) and immunoblotting with VDAC1 antibody (Abcam).

[0120] 96-well aggregometry. As described by Chan et al. in Platelets, 2018 Nov; 29(7):650-655 and by Vinholt et al. in PLos One, 2017 Oct 12; 12(10):e0185675, fresh platelets from the blood bank were adjusted to 5 x 108cells / ml. Platelets were pretreated with IVOs (5 μM) for 1 hour, followed by mtDNA (oligo) at 0.5 μM for 24 hours. A 45 μL aliquot of the cell suspension was added to wells containing agonists, adenosine diphosphate (ADP, 5.1 μM), resulting in a final 32 NIH0164PCT assay volume of 50 μL. The ADP used was derived from Bio / Data Corporation (Horsham, PA). The plate was shaken (807 cycles per minute, orbital shaking) for 2 minutes at 37°C, and absorbance was measured using a BioTek Cytation 5 (Santa Clara, CA, USA) at 595 nm. Platelet-poor plasma (PPP), collected by centrifugation at 1,000 x g for 10 minutes, was used as the blank. % Platelet aggregation was calculated from the optical density (OD) in wells with agonists, referenced to fresh platelets (set to 0% aggregation). Platelet aggregation = (OD platelets - OD sample) / (OD platelets – OD PPP) x 100 %

[0121] ROS measurements. Hela cells were first treated with tunicamycin (5 µg / ml) for 6 hr and then CM-H2DCFDA (Thermo Scientific) dissolved in PBS was added to achieve 2 μM final concentration and incubated at 37°C for 1hr in a 96-well plate. Then, the cells were washed 3 times with PBS followed by prewarmed growth medium (DMEM supplemented with 10% fetal bovine serum). The change in fluorescence intensity was monitored at two time points (0 and 20 min) by using a microplate fluorescence reader (Bio-Tek Instruments), at excitation 485 nm / emission 530 nm.

[0122] Quantification of mtDNA release by reverse-transcription quantitative PCR (RT-qPCR). Washed platelets (107cells) were resuspended in RPMI and stimulated with IVO21 (1 μM) for 1 hour, followed by oligo (0.5 μM) stimulation for 24 hours. Cytoplasmic mtDNA (cmtDNA) quantification was performed as previously described by Kim et al. in Science, 2019 Dec 20;366(6472):1531-1536 with slight modifications. Briefly, cells were resuspended in digitonin buffer containing 150 mM NaCl, 50 mM HEPES pH 7.4, and 25 µg / ml digitonin (EMD Millipore Corp), and incubated on a rotator for 10 min at room temperature. The homogenates were then centrifuged at 800 xg for 3 min and at 16,000 xg for another 15 min at 4°C. The supernatant was used for qPCR. The pellet was resuspended in lysis buffer containing 5 mM EDTA and proteinase K (Invitrogen), and incubated at 55°C overnight.

[0123] For the extracellular release of mtDNA, medium was purified using the QIAquick Nucleotide Removal Kit (Qiagen) following the manufacturer’s instructions. The pellet was resuspended in 50 µM NaOH and then boiled at 100°C for 30 minutes. Next, 50 µL of 1 M Tris- HCl pH 8.0 was added, and the mixture was centrifuged at 13,000 rpm for 10 minutes at 4°C. The resulting whole-cell extraction was used for qPCR. RT-qPCR was then performed using the 33 NIH0164PCT LightCycler 96 system (Roche Life Science) with SYBR Green master mix (Roche) and human MT-ND6 primers (see Blood, 2021 Jun 3;137(22):3116-3126).

[0124] Enzyme-linked immunosorbent assay (ELISA). Cytokine production was determined by ELISA. Washed platelets (107cells) were resuspended in RPMI and stimulated with IVO21 (1 μM) for 1 hour, followed by oligo (0.5 μM) stimulation for 24 hours. The culture media were collected, and protein levels were measured using an ELISA kit specific for Human IL-1β (R&D Systems, Minneapolis, MN, USA), following the manufacturer’s instructions.

[0125] Cell culture. LX-2 cells were cultured in Dulbecco’s modified Eagle medium (DMEM, ThermoFisher Scientific, USA) supplemented with 2% fetal bovine serum (Sigma-Aldrich, USA), 100 units / mL penicillin, 100 µg / mL streptomycin (ThermoFisher Scientific, USA), and 4 mM L-glutamine (ThermoFisher Scientific, USA) at 37°C in a 5% CO2 humidified chamber. When the cells reached approximately 80% confluency, they were detached from the culture flask using 0.25% trypsin-EDTA solution (ThermoFisher Scientific, USA) and subsequently re- seeded at a split ratio of 1:3.

[0126] Western Blotting. LX-2 cells were seeded in 6-well culture plates (ThermoFisher Scientific, USA) at a density of 2 x 105cells per well and cultured in DMEM supplemented with additives for 24 hours. After pre-treatment with IVO 14-21 and VBIT-4 at a concentration of 5 µM in complete growth medium for 1 hour, cells were subsequently exposed to TGF-beta (InvivoGen, San Diego, USA) at a concentration of 2 ng / ml for 24 hours. The cells were rinsed with phosphate-buffered saline (PBS, ThermoFisher Scientific, USA) and lysed using RIPA lysis buffer (Thermo Scientific, USA) supplemented with 1% protease inhibitor cocktail (ThermoFisher Scientific, USA). Protein concentration was determined using the bicinchoninic acid colorimetric assay (BCA, ThermoFisher Scientific, USA). Equal amounts of protein (20-30 μg per well) were separated by SDS-PAGE gels (10–12%) and transferred onto PVDF membranes. The membranes were blocked for 1 hour in a blocking buffer containing 5% bovine serum albumin (BSA) in 1X Tris-buffered saline (TBS) with 0.1% Tween-20 (1X TBS-T) and then incubated overnight at 4°C with specific primary antibodies: COL1A1 (1:1000, Cell Signaling Technology, MA, USA), pSmad2 (1:1000, Cell Signaling Technology, MA, USA), Smad2 (1:2000, Cell Signaling Technology, MA, USA), α-SMA (1:2000, Abcam, Cambridge, 34 NIH0164PCT UK), GAPDH (1:10000, Cell Signaling Technology, MA, USA), HK2 (1:2000, Cell Signaling Technology, MA, USA), VDAC1 (1:2000, Abcam, Cambridge, UK), IP3R (1:2000, Abcam, Cambridge, UK). Following washing with 1X TBS-T six times for 5 minutes each, the membranes were then incubated with corresponding secondary antibodies for 1 hour at room temperature and subsequently visualized using the ECL Western blotting substrate (ThermoFisher Scientific, USA).

[0127] Human T cell exhaustion. Healthy donor buffy coats were obtained from the NIH Blood Bank following protocol. Peripheral blood mononuclear cells (PBMCs) were separated using human lymphocyte separation medium (MP Biochemicals) and density gradient centrifugation. Following the manufacturer's instructions, CD8 T cells were isolated using the untouched human CD8+T cell isolation kit (Stem cell Technology). The method for inducing T-cell exhaustion was adapted from Dunsford et al. (Immuno-Oncology: 640 Cellular and Translational Approaches. New York, NY: Springer US; 2020. p. 89-101). Essentially, T cells were cultured in human serum-containing RPMI Aim550:50 media supplemented with 30IU IL2 and stimulated with anti-CD3 / anti-CD28 Dynabeads at a ratio of 1 bead to every 10 cells. Every 3 days, the cells were collected and stripped of the magnetic beads. They were then washed, counted, and restimulated with fresh beads spanning 9 days. On day 9, cells were collected, washed, and stripped of the beads. Subsequently, the cells were restimulated either in the presence or absence of IVO21 (5µM). Three days after this last stimulation, the cells were collected, and FACS analyzed for the expression of exhaustion markers CD39 and TIGIT after co-staining with viability and the CD8+ T cell marker.

[0128] Visualization and quantification of NETs. NETs were induced in NDGs by incubating human granulocytes with the calcium ionophore A23187 (25 μM) (Thermo Fisher) in RPMI 1640 medium for 2 h, and NETs were quantified as previously described in Nat Med 22, 146-153 (2016) using SYTOX fluorescent dye at 485 / 520 nm to quantify extracellular DNA. The fluorescence of PicoGreen (Life Technologies) at t = 0 min was measured at 485 / 520 nm (emission / extinction) to quantify the total DNA. The fluorescence was quantified using a microplate reader (Synergy HTX; BIOTEK). NETs were also quantified by fluorescence microscopy as previously described (Lood et al., Nat Med 22, 146-153 (2016)). In brief, the cells 35 NIH0164PCT were attached to coverslip chambers, stimulated for 90 min at 37 °C with A23187 (25 μM), fixed with 4% paraformaldehyde overnight at 4 °C, and permeabilized with 0.2% Triton X-100 for 10 min, followed by 0.5% gelatin for 20 min. The cells were stained with antibodies against human neutrophil elastase (ab21595, abcam) for 2 h at room temperature, washed in PBS, and stained with Hoechst 33342 (Life Technologies) and Alexa Fluor 488 secondary antibody (A31570, Life Technologies) for 2 h at room temperature. After mounting, the cells were visualized with a LSM880 confocal microscope (Zeiss). VDAC1 Oligomerization Inhibitory Activity

[0129] IVOs inhibit VDAC1 oligomerization which causes mitochondrial rupture and release of mtDNA and mitochondrial RNA (mtRNA). As a result, they will also protect against diseases that conventional VDAC1 oligomerization inhibitors VBIT-4 or VBIT-12 protect.

[0130] FIG. 1 shows that IVO-21 is a stronger VDAC1 oligomerization inhibitor than the conventional inhibitor VBIT-4 and antifungals ketoconazole, oteseconazole and miconazole. It blocks the formation of dimer in stressed HeLa cells (FIG. 1A) and 8-12 mer (300-350 kDa) in stressed fibroblasts (FIG. 1B). VDAC1-HK2 Interaction Inhibitory Activity

[0131] IVOs inhibit VDAC1-HK2 interaction, decreasing glycolysis and shifting metabolism to mitochondrial oxidative phosphorylation. As glycolysis promotes inflammation, decreasing glycolysis and the resulting lactic production may decreases inflammation. One example is immunothrombosis wherein inflammation signal enhances thrombosis.

[0132] FIG. 2 shows that IVO-21 does not disrupt VDAC1-HK1 interaction but disrupts VDAC1-HK2 interaction. VDAC1-HK interaction enhances production of glucose-6 phosphate, the first step in glycolysis. (B). IVOs decrease thrombosis that is stimulated by immunostimulant oligonucleotide, without decreasing platelet aggregation below the baseline (Control). This property decreases immunothrombosis without the bleeding risk which is present in the existing anti-coagulation drugs. Effects on Mitochondrial Stress and Reactive Oxygen Species Production 36 NIH0164PCT

[0133] IVOs decrease mitochondrial stress and reactive oxygen species (ROS) production. As a result, they protect against mitochondrial dysfunction and oxidative damage.

[0134] FIG. 3 shows that IVOs decrease ROS produced in response to endoplasmic reticulum stressor Tunicamycin. Effects on AMP-dependent kinase (AMPK)

[0135] IVOs activate AMPK, which promotes autophagy and lysosomal activation. As a result, they eliminate damaged organelles such as mitochondria as well as protein aggregations and protect against diseases caused by fibrils such as neurodegenerative diseases and lysosomal diseases.

[0136] FIG. 4A shows that IVO-21 increases AMPK activity (p-AMPK) and suppresses mTORC1 (p-S6K1); and FIG. 4B shows that IVO-21 increases autophagy as evidenced by increased p62 levels and LC3 II. Effects on the Release of Immune Stimulators

[0137] IVOs prevent the release of immune stimulators such as cytokines and DNA. As a result, they protect against tissue damage after trauma and mitigate autoimmune and other inflammatory diseases.

[0138] FIG. 5 shows that immune stimulation of platelets with CpG-ODN (oligo) releases more immunostimulants, but this is mitigated by IVO-21. Left, mtDNA released into the cytoplasm; Center, mtDNA released into the extracellular medium; Right, inflammatory cytokine IL-1β released into the extracellular medium. Effects on Fibrosis

[0139] IVOs decrease fibrosis, which often occur after chronic tissue damage or inflammation and can lead to organ failure. As a result, they protect against fibrosis of important organs such as liver, lung, heart, and kidney.

[0140] FIG. 6 shows that tumor growth factor-b (TGF-b) promotes fibrosis by increasing production of collagen (COL1A1), but IVOs (17-21) mitigate it. Effects on IP3R 37 NIH0164PCT

[0141] IVOs decrease IP3R, which release Ca2+from endoplasmic reticulum. Therefore, they can decrease mitochondrial influx of Ca2+, which when excessive, can cause mitochondrial hyperactivity, mitochondrial damage, and cell death. Excessive mitochondrial activity in neurons can potentially cause abnormal brain activity, including neuronal hyperactivity.

[0142] FIG. 7 shows that expression levels of IP3R, which release Ca2+from endoplasmic reticulum, is increased by (TGF-b), but this is mitigated by IVO-21. This effect will prevent mitochondrial Ca2+overload. Effects on T Cell Exhaustion

[0143] IVOs decrease T cell exhaustion. T cells become exhausted after chronic stimulation, resulting in weakened immune response against infection and cancer. Preventing exhaustion will enhance the ability of the immune system to eliminate infection and cancer.

[0144] FIG. 8 shows that frequency of live CD8 T cells expressing exhaustion markers (TIGIT+ / CD39+) after chronic stimulation is decreased by IVO-21. Effects on NETosis

[0145] IVOs suppress NETosis. Neutrophils rupture and release DNA into the extracellular space in an inflammatory process called NETosis. Inhibiting NETosis will decrease inflammation and thrombosis in various disease states.

[0146] FIG. 9 shows the measurement of NETosis in the presence of NETosis inducer A23187 and / or IVO-21. The results show that IVO-21 significantly decreases DNA released into the extracellular space.

[0147] Inhibitor of VDAC oxidation inhibitor mitigates thromboinflammation by stabilizing mitochondria and decreasing glycolysis.

[0148] Thromboinflammation is driven by a self-perpetuating cycle in which damage-associated molecular patterns (DAMPs), pathogen-associated molecular patterns (PAMPs) and pro- inflammatory cytokines promote thrombosis, which in turn leads to the release of prothrombotic and inflammatory mediators such as mitochondrial DNA (mtDNA) and RNA (mtRNA). Most inflammatory mediators increase ROS production and glycolysis both of which promote platelet activation. 38 NIH0164PCT

[0149] All inflammatory processes require a metabolic shift to glycolysis, and inhibition of glycolysis may mitigate inflammation. Currently there is no FDA-approved glycolysis inhibitor, but several experimental compounds are known to inhibit glycolysis by inhibiting the activity of hexokinase (HK), typically Hexokinase 2 (HK2), which catalyzes the first step of glycolysis: ATP-dependent phosphorylation of glucose to glucose 6-phosphate (G6P). HK2 exists in both soluble and VDAC1-bound states, with the VDAC1-bound HK2 being more active because the local concentration of ATP near VDAC1 is likely to be highest in the cytoplasm. The VDAC1- HK2 interaction is also crucial for preventing VDAC1 oligomerization. Therefore, inactivation of HK2 or disruption of VDAC-HK2 interaction, which translocate HK2 from mitochondria to the cytoplasm, induces VDAC1 oligomerization and mitochondrial rupture. This process leads to apoptosis and the release of pro-inflammatory cytokines such as IL-1β. Therefore, these inhibitors are primarily designed for tumoricidal therapy.

[0150] A promising strategy for developing a safe glycolysis inhibitor involves designing an HK2 inhibitor that also prevents VDAC1 oligomerization. The commonly used VDAC1 oligomerization inhibitor, VBIT-4, has been reported to strengthen, rather than weaken, the VDAC1-HK2 interaction. Furthermore, there is evidence suggesting that VBIT-4 may induce dose-dependent mitochondrial toxicity. To overcome these challenges, it is crucial to develop a compound capable of inhibiting both VDAC1-HK2 interaction and VDAC1 oligomerization.

[0151] To design a small molecule inhibitor of VDAC1-HK2 and VDAC1-VDAC1 (i.e. oligomerization) interactions, previous observations were considered that azole-containing antifungal compounds itraconazole binds to VDAC1, while other azole-containing antifungals ketoconazole and posaconazole inhibit HK activity. These three azole-containing antifungals with very different structures (ketoconazole, miconazole and oteseconazole) were tested whether inhibited ROS (H2O2)-induced VDAC1 oligomerization by using ethylene glycol bis(succinimidyl succinate) (EGS) to cross-link and stabilize VDAC1 oligomers. Ketoconazole and oteseconazole inhibited VDAC1 oligomerization better than miconazole (FIG. 10A).

[0152] Given the propensity of over-oxidized VDAC1 to form large VDAC1 oligomers, whether IVO-21 inhibits VDAC1 oligomerization by binding to over-oxidized C127 was tested. Recombinant VDAC1 with biotin-tagged native (N)-VDAC1 peptide (a.a. 121-132) or sulfonic acid-modified C127 (Ox)-peptide was incubated and a pull-down experiment with streptavidin 39 NIH0164PCT beads with or without IVO-21 (FIG. 10B) was performed. VDAC1 did not bind to the N-peptide, but bound to the Ox-peptide, and this interaction was inhibited by IVO-21. To determine if IVO- 21 bound preferentially to the Ox-peptide compared to the N-peptide, oteseconazole, VBIT-4 and IVO-21 was incubated with the two peptides (FIG. 10C). After washing unbound compounds, the levels of bound compounds were measured with mass spectrometry (MS). Among the three drugs, only IVO-21 had preferential affinity for the Ox-peptide. Therefore, it was concluded that the VDAC1-VDAC1 interaction, which is enhanced by over-oxidation of C127, but IVO-21, by binding to over-oxidized C127, prevents the hydrogen-bond formation between over-oxidized C127 and an adjacent VDAC1 (FIG. 10D).

[0153]

[0154] IVO-21 inhibits VDAC1-HK2 interaction and decreases glycolysis.

[0155] Activated immune cells increase glycolysis and ROS production partly by inducing expression of several proteins: 1. glucose transporters, which take up glucose; 2. Pentose phosphate pathway (PP) enzyme glucose-6-phosphate dehydrogenase, which generates NADPH in converting G6P to 6-phosphoglucanolactone; 3. malic enzyme, which generates NADPH in converting malate to pyruvate; 4. NADPH oxidase (NOX), which generates superoxide anion. However, these events don’t occur in cells without a nucleus. Therefore, it is not clear how pro- inflammatory cytokines, DAMPs and PAMPs increase glycolysis and superoxide anion production in platelets.

[0156] VDAC1-HK2 interaction has been well characterized, but the VDAC1 residue(s) that is essential for ROS-induced binding to VDAC1 has not been identified. Even though IVO-21 inhibits platelet aggregation induced by CpG-ODN, IL-1β and LPS (FIG. 10E-G), CpG-ODN was focused on because it induced platelet aggregation more strongly than the others, and released mtDNA, which like CpG-ODN, activates TLR9 and triggers potent systemic pro- inflammatory response. Since disruption of VDAC1-HK2 interaction leads to VDAC1 oligomerization, it was hypothesized that HK2 also binds to over-oxidized C127. To test this, platelets from vdac1C127A / +and vdac1+ / +were stimulated with CpG-ODN and performed co- immunoprecipitation with HK2. It was found that CpG-ODN increased VDAC1-HK2 interaction in vdac1+ / +platelets but not in vdac1C127A / +platelets (FIG. 11A). To determine if IVO-21 inhibited VDAC1 interaction with HK2 is specific, co-immunoprecipitation was performed between VDAC1 and either HK1 or HK2. It was found that IVO-21 did not inhibit VDAC1- 40 NIH0164PCT HK1 interaction (FIG. 11B) but inhibited VDAC1-HK2 interaction in less than 30 min (FIG. 11C); VBIT-4 inhibited neither interactions. A pull-down experiment with recombinant HK2 with the N- and Ox-peptides revealed that like VDAC1, HK2 bound preferentially to the Ox- peptide (FIG. 11D). These findings suggest that IVO-21 binds to hyperoxidized C127 to disrupt VDAC1-VDAC1 interaction as well as VDAC1-HK2 interaction.

[0157] To confirm the inhibitory effect of IVO-21 on glycolysis, a glucose-flux analysis was conducted by treating platelets with CpG-ODN followed by incubation with12C-glucose and13C- glucose for 15 and 30 min before analyzing the metabolome. Compared to untreated platelets (- CpG-ODN), IVO-21 did not affect glycolysis, Pentose Phosphate Pathway (PPP) or the tricyclic acid (TCA) cycle after 15 min (FIG. 11E). However, after 30 min, IVO-21 decreased glycolysis, PPP and the TCA cycle and increased the accumulation of unmetabolized glucose (FIG. 11F). A closer examination of the glucose-flux in the TCA cycle after CpG-ODN treatment showed by IVO-21 increased the flux in the TCA cycle after 15 min (FIG. 11G, top). This difference was no longer visible after 30 min (FIG. 11G, bottom). Taken together, these findings indicate that IVO- 21 causes a relative shift in energy production from HK2-mediated glycolysis and PPP to oxidative phosphorylation (OXPHOS) which is ∼18 fold more efficient than glycolysis.

[0158] IVO-21 attenuates the thromboinflammation loop.

[0159] Nearly all immune activators increase ROS which is required for their full inflammatory action, including in thromboinflammation. NADPH oxidase, which is essential for platelet aggregation, generates the potent ROS superoxide anion by using NADPH as cofactor. Two molecules of NADPH are generated for every molecule of G6P that enters PPP. If IVO-21 attenuates PPP as shown in FIG. 11F, then it should also reduce ROS production. Indeed, IVO- 21, NOX inhibitor VAS2870, and mitochondrial antioxidant mito-TEMPO mitigated CpG-ODN- induced ROS production (FIG. 12A). Consistent with the hypothesis that IVO-21 binds to over- oxidized C127, platelets from vdac1C127A / +platelets exhibited lower ROS production (FIG. 12B) and platelet aggregation (FIG. 12C) compared to vdac1+ / +platelets.

[0160] VDAC1 oligomer visualization has relied on cross-linking with EGS, followed by SDS- PAGE electrophoresis. However, cross-linking can irreversibly bind transiently-interacting proteins, potentially introducing artifacts. To address this limitation, immunoblotting of VDAC1 was performed using platelet lysates electrophoresed on a modified native gel without prior 41 NIH0164PCT cross-linking. As shown in FIG. 12D (left), stimulation with CpG-ODN enhanced VDAC1 oligomerization, an effect that was reversed by IVO-21. Furthermore, IVO-21 reduced levels of oxidized VDAC1 (Ox-VDAC1), as detected by an antibody specific for VDAC1 with sulfonic acid-modified C127 (FIG. 12D, right). Notably, the Ox-VDAC1 antibody did not stain VDAC1 monomers, indicating that oxidation at C127 occurs exclusively within oligomeric VDAC1 complexes (FIG. 12D). Additionally, IVO-21 mitigated VDAC1 oligomerization induced by LPS (FIG. 12E) and IL-1β (FIG. 12F). These findings indicate that over-oxidation of C127 not only leads to VDAC1 oligomerization but exacerbates ROS production.

[0161] VDAC1 oligomerization facilitates the release of mtDNA into the cytosol and extracellular space.

[0162] Treatment with IVO-21 significantly reduced both cytosolic (FIG. 12G) and extracellular (FIG. 12H) mtDNA levels following CpG-ODN stimulation. Similarly, IVO-21 attenuated extracellular mtDNA release in response to LPS (FIG. 12I) and IL-1β (FIG. 12J).

[0163] To further validate the hypothesis that excessive oxidation at C127 triggers mtDNA release into the extracellular space, it was demonstrated that quenching reactive oxygen species (ROS) with VAS2870 or mito-TEMPO reduced extracellular mtDNA levels (FIG. 12K). Moreover, vdac1C127A / +platelets released less extracellular mtDNA than their vdac1+ / +counterparts (Fig. 12L), supporting the role of C127 oxidation in this process.

[0164] In autoimmune diseases such as systemic lupus erythematosus (SLE), high levels of prothrombotic antibodies such as anti-phospholipid (aPL) antibody are often present in the sera. Incubation of sera from two SLE patients with high aPL antibody titre stimulated aggregation but IVO-21 prevented this (FIG. 12M). The levels of extracellular mtDNA released by aPL antibody were also reduced by IVO-21 (FIG. 12N). The feed-forward nature of thromboinflammation is also disrupted by IVO-21 as IL-1β released from platelets that have been stimulated with CpG- ODN was attenuated by IVO-21 (FIG. 12O). These findings indicate that over-oxidation of C127 is a critical mediator of the thromboinflammation loop (FIG. 12P).

[0165] IVO-21 protects against sepsis.

[0166] As a bacterial endotoxin, Lipopolysaccharide (LPS) is a potent trigger of aggregation and septic shock. If IVO-21 inhibits the thromboinflammatory cycle, it should suppress disseminated intravascular aggregation (DIC) and the associated thrombocytopenia following LPS 42 NIH0164PCT administration. Indeed, IVO-21 treatment prevented LPS-induced thrombocytopenia (FIG. 13A) and, consistent with its anti-glycolytic activity, reduced lactic acidemia in C57BL / 6 mice (FIG. 13B). Additionally, IVO-21 mitigated the LPS-induced surge in IL-1β (FIG. 13C) and IL-6 (FIG. 13D), as well as eosinophilia (FIG. 13E).

[0167] Next, whether IVO-21 provides protective effects against sepsis was investigated. The cecal ligation and puncture (CLP) model, which releases fecal content into the peritoneum, is widely regarded as the gold standard for studying sepsis in animals due to its close resemblance to human sepsis progression. IVO-21 was administered immediately after CLP and monitored survival for approximately six days. The results showed that IVO-21 reduced mortality by approximately 60% (FIG. 13F). Moreover, deaths from tracheal infusion of LPS, which causes respiratory failure, is reduced by IVO-21 (FIG. 14). In order to confirm that IVO-21 mitigates thromboinflammation, clotting was induced by running electricity to an electrode inserted into inferior vena cava. The thrombus was removed, and its mass was measured. As shown in FIG. 15A, the size of the thrombus was reduced in IVO-21 treated mice, providing in vivo evidence that IVO-21 mitigates thromboinflammation.

[0168] In addition, it was shown that IVO-21 binds specifically to oxidized Cys127 in VDAC1 (Fig 15B) using a streptavidin-pull down experiment.

[0169] In conclusion, ROS production and the resulting C127 over-oxidation drive two VDAC1 amplification loops of (VALs): VAL1, which promotes VDAC1 oligomerization, and VAL2, which recruits HK2 to the mitochondria to enhance glycolysis (FIG. 13G). This distinctive feature enables IVO-21 to selectively target both VALs by binding specifically to over-oxidized C127. Therefore, IVO-21 holds promise as a therapeutic candidate for treating thromboinflammation, sepsis and possibly other inflammation-related diseases.

[0170] The role of VDAC1 and IVO-21 in T cell function.

[0171] The link between autoimmunity, cancer progression, and inflammatory diseases like chronic fatigue syndrome (CFS) and long COVID highlights how immune system dysregulation, especially involving T cells, leads to a range of clinical effects. T cells are essential players in the regulation of tissue homeostasis and preventing diseases. However, when T cell function is impaired, it can lead to immune disturbances associated with aging, autoimmune disorders, and chronic inflammatory diseases. A decline in T cell functionality, including T cell exhaustion, 43 NIH0164PCT occurs when T cells progressively lose their ability to proliferate, secrete cytotoxic cytokines, and destroy infected or malignant cells. This weakened immune surveillance diminishes the body’s ability to combat infections and tumors, potentially worsening disease progression or an improvement to their immune functions can alternatively promote autoimmunity.

[0172] Mitochondria are vital hubs that dictate T cell fate and function by regulating cellular metabolism and key signaling pathways. They are pivotal intracellular sites for generating intracellular reactive oxygen species (ROS), and the levels of ROS are critical for modulating T cell activity. Optimal levels of ROS, ranging from low to moderate, are essential for activating CD8 T cells, facilitating their proliferation, and enabling effective cytokine secretion, including IL2 and IL4, which are indispensable for sustaining T cell activation. Furthermore, mitochondrial ROS (mROS) is pivotal for activating transcription factors that stimulate CD8 T cells to produce more IFNg against tumor cells.

[0173] In conditions of chronic antigen stimulation, such as cancer and chronic viral infections, T cells exhibit compromised metabolic processes, marked by the continual loss of mitochondrial content and function, as well as reduced oxidative metabolism. In tumor-infiltrating lymphocytes (TIL), dysfunctional mitochondria lead to impaired ATP production and excessive accumulation of mROS, which activates NFAT signaling, contributing to T cell exhaustion. Moreover, higher levels of ROS in aging T cells show accelerated T cell senescence and telomere shortening. In summary, elevated ROS originating from stressed mitochondria hastens the functional decline of CD8+ T cells, thereby weakening their effectiveness.

[0174] Genetic Loss of VDAC-1 In T-cells Uncouples Cell Proliferation and Effector Differentiation.

[0175] To gain deeper insights into the role of Vdac1 in T-cells, a Vdac1 genetic knockout model was employed, in which exons 2-5 of Vdac1 were deleted. Splenocytes from both Vdac1+ / + and Vdac1- / - mice were isolated and activated in vitro using αCD3 / αCD28 stimulation in the presence of IL-2. The cells were then expanded until day 10 by restimulating them with αCD3 / αCD28 on day 5. T-cell phenotypic analysis was conducted at day 10 using flow cytometry and RNA sequencing.

[0176] It was revealed that the genetic loss of Vdac1 in T-cells significantly enhanced T-cell proliferation while preserving the expression of memory markers CD62L, CD25, and CD27. 44 NIH0164PCT Additionally, Vdac1- / - splenocytes expanded in vitro exhibited a significantly reduced frequency of Klrg1+ cells, a marker of T-cell senescence. Furthermore, global RNA sequencing analysis aligned with the protein data, showing that Vdac1- / - T-cells had a notable upregulation of memory-associated genes, including Tcf7, Lef1, IL7R, Myb, and Sell, while genes linked to T- cell inhibition and exhaustion, such as Pdcd1, Lag3, and Tim3, were downregulated. Collectively, these findings highlight the crucial role of Vdac1 in restricting T-cell proliferation while promoting effector differentiation, and its loss decouples cellular expansion from effector differentiation.

[0177] Role of VDAC1 C127 Residue in driving T Cell Exhaustion.

[0178] The primary aim was to investigate how VDAC oligomerization occurs under oxidative stress and explore methods to target this process using small molecule inhibitors. Mitochondrial oxidative stress (mtROS) can induce reversible and irreversible modifications of cysteine residues. It was hypothesized that under oxidative stress, cysteine residues within the transmembrane region of VDAC1 are modified to sulfinic and, under prolonged stress, to sulfonic forms, enhancing the adhesive properties among neighboring VDACs and promoting multimer formation. Using mass spectrometric analysis, potential sulfinic and sulfonic acid modifications in T cells that were activated and sorted into specific subsets were examined. FIG. 16A and FIG. 16B depict the results of mass spectrometry analysis for VDAC1 levels and C127- CysOx modification in the Cys127 residues of VDAC1. The data suggest a probable role of this residue in facilitating VDAC1 multimer formation. To further validate the hypothesis, a new mouse model carrying a single point mutation in the VDAC1 where the Cysteine was altered to Alanine, was developed, referred to as CysC127A. Splenocytes were collected from these mice, and activated T cells were analyzed. FIG. 16C demonstrates the levels of oxidative stress measured by DCFDA staining in activated T cells. Notably, the oxidative stress levels were significantly reduced in VDAC1(C127A / wt)T cells compared to the WT, VDAC1(wt / wt)counterparts. Importantly, a striking reduction in oxidative stress levels in Effector memory T cells from VDAC1(C127A / wt)T cells was comparable to that observed in the CM population of WT T cells. This finding supports the hypothesis that VDAC1 C127 is crucial in sensing oxidative stress, promoting VDAC1 multimer formation. Due to decreased oligomer formation in VDAC1 C127A mutants, there is a potential reduction in oxidative stress. Furthermore, in day 7 activated 45 NIH0164PCT T cells, the frequency of exhausted T cells was notably diminished in VDAC1(C127A / wt)T cells compared to WT counterparts. Utilizing this new mouse model, the significance of VDAC1 oligomerization in regulating T cell exhaustion and antitumor immunity is studied.

[0179] Loss of VDAC1 drives autoimmune response in a T Cell Model of Vitiligo.

[0180] To explore the role of VDAC1 in a CD8 T cell-driven vitiligo model, Pmel-1 CD8+ TCR transgenic mice—whose CD8+ T cells specifically target the GP100 antigen expressed by melanocytes—with VDAC1 knockout mice were bred, generating Pmel VDAC1+ / +and Pmel- VDAC1- / -mice. These animals were aged for approximately one year to allow for the natural progression of the disease, and their vitiligo response was assessed after 10 months of age. FIGS. 17A-C show the vitiligo response scored based on the greying pattern observed on the skin / fur of these animals at ~10 months of age. Current ongoing experiment involves assessing vitiligo response in Pmel transgenic mice carrying the VDAC1 C127A transgene, referred to as Pmel-VDAC1C127A / wtand Pmel-VDAC1wt / wt.

[0181] Vdac1 oligomerization and T cell exhaustion in human T cells.

[0182] To investigate the role of VDAC1 oligomerization in promoting T cell exhaustion in human T cells, an in vitro chronic activation driven T cell exhaustion model was used. CD8 T cells were isolated from healthy donors, activated using CD3 / CD28 beads, and performed sequential washing, counting, and restimulation with fresh activation beads every 48 hours. After two stimulations, the cells were counted and restimulated either in the presence or absence of VDAC1 oligomerization inhibitors E, VBIT4, and IVO21 (FIGS. 18A-B). Targeting VDAC1 oligomerization using VDAC1 oxidation inhibitors like IVO21 limits T cell exhaustion. FIG. 18A illustrates the TOX expression levels under different treatment conditions across2 healthy donors, while FIG. 18B presents the expression of exhaustion markers Lag3, Tim3, CD39, and TIGIT in one donor. The data shown represents data from 2 donors.

[0183] Effect of IVO-21 on obesity and insulin sensitivity.

[0184] Glucagon-like peptide-1 (GLP-1) agonists are receiving widespread attention for their ability to improve glucose tolerance and suppress appetite. However, despite these benefits, more than 70% of patients discontinue GLP-1 agonists within two years. There may be a number of 46 NIH0164PCT reasons for this (e.g., cost and side-effects), but one reason may be that food and social interactions involving food play a crucial role in mental well-being.

[0185] Given the intricate crosstalk between metabolism, ROS, and inflammation, reducing ROS and inflammation with IVO-21 may provide an alternative approach to promoting weight loss and improving glucose control—without compromising the psychological and social aspects of eating.

[0186] C57BL6J mice were fed on 45% HFD and treated half of them with IVO-21 and the other half with saline. IVO-21 did not affect food intake (FIG. 19A) but decreased weight gain at higher doses (FIG. 19B) and increased glucose tolerance (FIG. 19C) and glucose tolerance (FIG. 19D). Since IVO-21 acts by binding to over-oxidized C127 and interfering with VDAC1 oligomerization and VDAC1-HK2 interaction, C127A mutation should have similar phenotype as IVO-21-treated mice. Indeed, vdac1C127A / +mice had higher insulin sensitivity (FIG. 19E) and glucose tolerance (FIG. 19F) than vdac1+ / +mice.

[0187] IVO-21 mitigates myocardial infarction-induced heart failure.

[0188] Myocardial infarction (MI) is a leading cause of heart failure, with mitochondrial dysfunction playing a central role in post-infarction cell death and maladaptive cardiac remodeling. To evaluate the cardioprotective potential of IVO-21, the compound was administered to mice prior to inducing MI via ligation of the left anterior descending (LAD) coronary artery. Cardiac function was assessed one-month post-MI using echocardiography.

[0189] IVO-21 treatment significantly improved left ventricular ejection fraction (EF) compared to untreated MI controls, indicating partial restoration of systolic function (FIGS. 20A-C). Additionally, IVO-21 attenuated cardiac hypertrophy, as evidenced by reduced global heart volume. MI-induced wall thinning was also mitigated by IVO-21, with treated mice exhibiting preserved thickness of the left ventricular anterior wall during systole (LVAW;s) and diastole (LVAW;d), as well as the left ventricular posterior wall during systole (LVPW;s) and diastole (LVPW;d).

[0190] The definition of % EF: ejection fraction is the percentage of blood ejected from the ventricle during systole (contraction). The meaning of % EF: ejection fraction represents the pumping efficiency of the heart. In healthy mice, EF typically ranges from 60–80%. The definition of % FS: fractional shortening is the percentage change in the diameter of the left 47 NIH0164PCT ventricle between diastole and systole. The meaning of % FS: fractional shortening is a simple index of left ventricular contractility. The definition of Volume;d (End-Diastolic Volume, EDV) is that the volume of blood in the ventricle at the end of diastole (before contraction). The meaning of Volume;d (End-Diastolic Volume, EDV) is that it indicates how much blood the ventricle fills before contracting. The definition of Volume;s (End-Systolic Volume, ESV) is that the volume of blood remaining in the ventricle at the end of systole (after contraction). The meaning of Volume;s (End-Systolic Volume, ESV) is that it reflects the amount of blood not ejected during contraction—used to assess systolic function. LVEDD (Left Ventricular End- Diastolic Diameter) is defined as an internal diameter of the left ventricle when the heart is relaxed and filled with blood (Indicates ventricular filling and chamber size). LVAW;d: Left Ventricular Anterior Wall thickness – diastole is defined as a thickness of the anterior wall of the left ventricle during diastole (Used to assess structural changes (e.g., hypertrophy)). LVAW;s: Left Ventricular Anterior Wall thickness – systole is defined as a thickness of the anterior wall during ventricular contraction (Reflects wall thickening and contractility). LVPW;d: Left Ventricular Posterior Wall thickness – diastole is defined as a thickness of the posterior wall of the LV during diastole (Indicates baseline wall structure). LVPW;s: Left Ventricular Posterior Wall thickness – systole is defined as a posterior wall thickness during contraction (Helps assess contractile function of the ventricular wall).

[0191] These findings demonstrate that IVO-21 confers cardioprotection following MI by preserving cardiac structure and function, thereby mitigating the progression to heart failure.

[0192] IVO-21 mitigates renal fibrosis.

[0193] Fibrosis commonly arises as a consequence of chronic inflammation and follows a conserved pathological cascade across different organs. It was demonstrated that the C127A mutation effectively prevents MASH-induced liver fibrosis. In the present study, whether the small molecule IVO-21 can confer protection against high-dose folate-induced renal fibrosis is evaluated.

[0194] Mice received a single injection of high-dose folate, and beginning approximately 9 days post-injection, were treated with IVO-21 two to three times per week. Treatment continued until day 28, at which point kidneys were harvested for analysis. IVO-21 treatment significantly attenuated renal atrophy (FIG. 21A) and reduced fibrotic deposition, as visualized by Picrosirius 48 NIH0164PCT Red (PSR) staining (FIG. 21B). Notably, IVO-21 also prevented the elevation of blood urea nitrogen (BUN) and serum creatinine levels, suggesting preservation of renal function (FIG. 21C).

[0195] Potent activity of IVO-18 enantiomers.

[0196] Racemic mixtures can exhibit varying activities depending on the amount of each enantiomer in each production batch. In order to mitigate this variability, the two enantiomers of IVO-18 (FIG. 22A) were separated and examined the effect of each enantiomer on preventing oligo-induced platelet aggregation (FIG. 22B). The two enantiomers are IVO-18.2 and IVO-18.4. IVO-18.4 (Isomer 4), was more effective in reducing thromboinflammation and had a wider therapeutic window than IVO-18.2 (Isomer 2) at <1 nM.

[0197]

[0198] IVOs mimic calorie restriction and increase lysosomal function.

[0199] Calorie restriction (CR) has been widely shown to delay aging and improve metabolic health. However, long-term adherence to a CR lifestyle is challenging for most individuals. One well-established mechanism through which CR exerts its anti-aging effects is the activation of AMP-activated protein kinase (AMPK), an essential cellular energy sensor. AMPK is activated 49 NIH0164PCT in response to energy stress, typically via an increased AMP / ATP ratio and a reduction in glycolytic intermediates such as fructose 1,6-bisphosphate (F1,6BP).

[0200] Given prior observations that IVO inhibits glycolysis, it was hypothesized that IVO, or a structurally related azole compound such as itraconazole, would reduce F1,6BP levels and subsequently activate AMPK. Indeed, administration of itraconazole significantly increased phospho-AMPK levels—a marker of AMPK activation—in skeletal muscle (FIG. 23A) and white adipose tissue (WAT) (FIG. 23B) of wild-type (WT) mice. Notably, this effect was absent in VDAC1 knockout mice, suggesting that VDAC1 is necessary for AMPK activation in this context. Similarly, itraconazole induced AMPK phosphorylation in C2C12 myotubes (FIG. 23C).

[0201] Consistent with a glycolysis-inhibitory effect, itraconazole treatment led to reduced extracellular acidification rate (ECAR), glucose-6-phosphate, and F1,6BP levels in C2C12 cells (FIG. 23D). Furthermore, both enantiomers of IVO-18 and IVO-21 activated AMPK (FIG. 23E), inhibited mTORC1 signaling—as evidenced by decreased phosphorylation of S6 kinase (p- S6K)—and reduced mTORC1-mediated phosphorylation of TFEB, collectively indicating enhanced lysosomal activity.

[0202] CpG-ODN (oligo)-induced coagulation in the presence of varying concentrations of IVO- 18, IVO-21 and 1:1 (equimolar) mixture of IVO-18 and IVO-21. As shown in FIG. 24, even though the total concentration of IVO is equal in all three groups, the IVO-18 / IVO-21 mixture reduced oligo-induced coagulation at a lower concentration than IVO-18 or IVO-21 alone, indicating that the mixture has more potent anti-inflammatory activity than either one alone. Unexpectedly, at high concentrations (for all three groups), there is a tendency the anti- inflammatory effect to decrease or wear-off. This limits the therapeutic window of IVO-18 and less so of IVO-21. Considering the mix has higher potency compared to IVO-18 or IVO-21 alone and a wider therapeutic window compared to IVO-18, it is possible that the 1:1 mix is a superior anti-inflammatory drug than either IVO-18 or IVO-21 alone. It will be appreciated, however, that other ratios of enantiomers, such as 2:1, 3:1, 4:1, may be implemented in the present invention.

[0203] The present inventive concept has been described in terms of exemplary principles and embodiments, but those skilled in the art will recognize that variations, such as the ratio of each 50 NIH0164PCT enantiomer may be made and equivalents substituted for what is described without departing from the scope and spirit of the disclosure as defined by the following claims. 51

Claims

NIH0164PCT CLAIMS What is claimed is:

1. A compound of Formula I R1011 9A I or a phareof, wherein: R1is halogen; R2is halogen or hydrogen; R3 is hydroxyl, or -N(R17)2, wherein each R17 is independently hydrogen, or C1-C6 alkyl; R4, R5, R6, R7, and R8 are independently chosen from hydrogen, halogen, C1-C6alkyl, and C1-C6alkoxy, C1-C2haloalkyl, and C1-C2haloalkoxy; R9, R10, R11, R12, and R13 are independently chosen from hydrogen, C1-C6alkyl, and C1- C6alkoxy, C1-C2haloalkyl, and C1-C4haloalkoxy; R3is halogen or C1-C6alkyl; A ring is NNR16N wherein R15is C1-4alkyl, and R16is hydrogen or C1-4alkyl;B ring is 6-membered heteroaryl ring having one or two nitrogen atoms, which B ring is optionally substituted with one or more substituents independently chosen from halogen and C1- C6alkyl, with the proviso that the compound is not 2-(2,4-difluorophenyl)-1,1-difluoro-3-(2H- tetrazol-2-yl)-1-(5-(4-(2,2,2-trifluoroethoxy) phenyl)pyridin-2-yl)propan-2-ol.

2. The compound of claim 1, wherein the A ring is 52NIH0164PCT NNN R15N d3. The compound of claim 1, wherein the A ring is R16N N N N , and R16 is hydrogen or methyl.

4. The compound of any one of claims 1 to 3, wherein R3 is hydroxyl.

5. The compound of any one of claims 1 to 3, wherein R3is -NH2.

6. The compound of any one of claims 1 to 5, wherein R4 and R6 are halogen; and R5, R7, and R8are all hydrogen.

7. The compound of any one of claims 1 to 6, wherein R9, R10, R12, and R13 are allhydrogen; and R11 isC1-C2haloalkoxy.

8. The compound of any one of claims 1 to 7, wherein the compound of Formula I is a compound of Formula I-B: R11A 6 Formula I-B.

9. The compound of claim 8, wherein R1 and R2 are fluoro.

10. The compound of any one of claims 1 to 9, wherein the compound is 53NIH0164PCT , , F , ,54NIH0164PCT F3CO N F3CONNN N N N N F .

11. A pharmaceutical composition comprising at least one of a compound of any one of claims 1 to 10, or a pharmaceutically acceptable solvate, or salt thereof, together with a pharmaceutically acceptable carrier.

12. A method of inhibiting voltage-dependent anion channel oligomerization in a subject in need thereof, comprising administering an effective amount of at least one of a compound of Formula I, a solvate or salt thereof to the subject: R10R11R9I or a phar, eof, wherein: R1 is halogen; R2 is halogen or hydrogen; R3is hydroxyl, or -N(R17)2, wherein each R17is independently hydrogen, or C1-C6alkyl; R4, R5, R6, R7, and R8are independently chosen from hydrogen, halogen, C1-C6alkyl, and C1-C6alkoxy, C1-C2haloalkyl, and C1-C2haloalkoxy; R9, R10, R11, R12, and R13are independently chosen from hydrogen, C1-C6alkyl, and C1- C6alkoxy, C1-C2haloalkyl, and C1-C4haloalkoxy; R3 is halogen or C1-C6 alkyl; A ring is 55NIH0164PCT NNR16N N R1N N N , wherein R15 is hydrogen or C1-4 alkyl, and R16 is hydrogB ring is 6-membered heteroaryl ring having one or two nitrogen atoms, which B ring is optionally substituted with one or more substituents independently chosen from halogen and C1- C6alkyl.

13. The method of claim 12, wherein the subject has an autoimmune disease, blood disorder, bone disease, brain disease, a cancer, a cardiovascular disease, a dermatological disease, an eye and ear disease, a gastrointestinal disorder, a gynecological disorder, an inflammatory disease, a kidney disease, a liver disease, a lysosomal storage disease, a metabolic disorder, a mitochondrial disease, a muscle disorder, a nerve disease, a neurodegenerative disease, an oral or throat disease, a radiation / chemotherapy / immunotherapy-induced condition, a post-infection disease, a post-trauma tissue damage, a respiratory disease, or a testicular or prostate disease.

14. The method of claim 12 or claim 13, wherein the subject has covid-19, long covid, atherosclerosis, autoimmunity, hepatitis, fibrosis, thrombosis, diabetes, Alzheimer's disease, Parkinson's disease, or T-cell exhaustion.

15. The method of any one of claims 12 to 14, further comprising administering to the subject in need thereof at least one additional therapeutic agent.

16. The method of any one of claims 12 to 15, wherein the A ring is NNN d.

17. The method of any one of claims 12 to 15, wherein the A ring is 56NIH0164PCT R16N N N d n or methyl.

18. The method of any one of claims 12 to 17, wherein R3 is hydroxyl.

19. The method of any one of claims 12 to 18, wherein R3is -NH2.

20. The method of any one of claims 12 to 19, wherein R4 and R6 are halogen, and R5, R7, and R8are all hydrogen.

21. The method of any one of claims 12 to 20, wherein R9, R10, R12, and R13 are all hydrogen, and R11is C1-C2haloalkoxy.

22. The method of any one of claims 12 to 21, wherein the compound of Formula I is a compound of Formula I-B: R11A 6 Formula I-B.

23. The method of claim 22, wherein R1and R2are fluoro.

24. The method of any one of claims 12 to 23, wherein the compound is ,57NIH0164PCTNIH0164PCT F3CO N F3CONNN N N N N or25. The method of any one of claims 12 to 24, wherein the compound is .

26. The method of any one of claims 12 to 24, wherein the compound is .59NIH0164PCT 27. The compound of any one of claims 1 to 9, wherein the compound is IVO-18.2 or IVO- 18.4: .

28. The method of claim 26, wherein the compound is IVO-18.2 or IVO-18.4: 60NIH0164PCT .

29. A pharmaceutical composition comprising an equimolar mixture of IVO-18 and IVO-21.

30. The method of claim 12, wherein the compound of Formula I comprises an equimolar mixture of IVO-18 and IVO-21.

31. The compound of claim 1, wherein the compound is selected from 61NIH0164PCT62

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