2-(piperidin-3-YL)isoindole-1,3-dione analogs and uses thereof

Abstract

2-(Piperidin-3-yl)isoindole-1,3-dione analogs and uses thereof are disclosed. The compounds have a structure according to Formula I, or a stereoisomer or pharmaceutically acceptable salt, solvate, or hydrate thereof. The compounds may be useful for inhibiting TNF-α activity, TNF-α synthesis, interleukin-6 (IL-6) level, inflammation, or SARS-COV-2 virus. In some aspects, the compounds, or pharmaceutical compositions including the compounds, are administered to a subject a traumatic brain injury (TBI), an inflammatory disorder, a neurodegenerative disease, cancer, a viral infection, or any combination thereof.

Description

2-(PIPERIDIN-3-YL)ISOINDOLE-1,3-DIONE ANALOGS AND USES THEREOF CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No.63 / 664,442 filed June 26, 2024, which is incorporated by reference herein in its entirety. ACKNOWLEDGMENT OF GOVERNMENT SUPPORT

[0002] This invention was made with government support under Z01 AG 000994 awarded by the National Institutes of Health, National Institute on Aging. The government has certain rights in the invention. FIELD

[0003] 2-(Piperidin-3-yl)isoindoline-1,3-dione analogs are disclosed, along with methods of using the compounds. BACKGROUND

[0004] The pharmacology of phthalimidoglutarimide-based anti-inflammatory and neuroprotective agents, such as thalidomide and pomalidomide and derivatives thereof, has been examined. For example, studies have demonstrated that 3,6-dithiopomalidomide mitigates microgliosis, astrogliosis and hippocampal neurodegeneration in rats impacted by moderate traumatic brain injury (Huang et al., Intl J of Molecular Sciences 2021, 22(15):8276). In a separate study, pomalidomide and 3,6-dithiopomalidomide were shown to attenuate Alzheimer’s disease (AD) amyloid-β peptide-mediated inflammation, neuronal cell loss, and neurite network loss in primary cortical cultures and in AD mice bearing human transgenes (Lin et al., eLife 2020, 9:354726; Lecca et al., Alzheimers Dement.2022, 18(11):2327-2340). Additionally, some fluorinated analogs demonstrated complementary anti-inflammatory activity in LPS-challenged RAW cells concurrent with elevated metabolic stability relative to non-halogenated counterparts (Scerba et al., Bioorganic & Medicinal Chemistry Letters 2022, 76:128972).

[0005] However, thalidomides do not typically concede simple direct routes to optically pure and biologically-stable stereoisomers, as in vivo racemization, epimerization, and disproportionation all contribute to their complex pharmacology (Nishimura et al., Chemical & Pharmaceutical Bulletin 1994, 42(5):1157-1159; Wnendt et al., Chirality 1996, 8(5):390- 396; Vargesson, Birth Defects Research. Part C, Embryo Today : Reviews 2015, 105(2):140- 156; Reist et al., Chemical Research in Toxicology 1998, 11(12):1521-1528; Lepper et al., Current Drug Metabolism 2006, 7(76):677-685). Compounding the issue is a markedly limited water solubility which often complicates compound delivery, bioavailability, and subsequent evaluations. Thus, a need exists for compounds that exhibit suitable properties of solubility and resistance to in vivo racemization, epimerization, and disproportionation, while retaining desirable biological activity. SUMMARY

[0006] This disclosure concerns aspects of 2-(piperidin-3-yl)isoindoline-1,3-dione analogs, as well as stereoisomers, pharmaceutically acceptable salts, solvates, and hydrates of the compounds. Pharmaceutical compositions comprising the compounds and methods of using the compounds also are disclosed.

[0007] The disclosed compounds have a structure according to Formula I, or a stereoisomer or pharmaceutically acceptable salt, solvate, or hydrate thereof:

[0008] where R1is -H, -N(R′)(R′′), -NO2, -OH, -ORb, -SH, -SRb, -CN, C2-C6 alkenyl, C2-C6 alkynyl, or halo , wherein R′ and R′′ independently are -H, C1-C3 alkyl, C3-C6 cycloalkyl, or - C(O)Rb, such as -N(R′)(R′′), -NO2, -OH, or halo, wherein R′ and R′′ independently are -H or C1-C3 alkyl. R2is -H, Ra, -C(O)Rb, -C(O)ORb, -S(O)2Rb, C2-C6 alkenyl, C2-C6 alkynyl, or -C(O)N(Rc)(Rd), such as -H, Ra, -C(O)Rb, -C(O)ORb, -S(O)2Rb, or -C(O)N(Rc)(Rd). R3is - H, halo, C1-C4 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, -C(O)ORb, or -C(O)N(Rc)(Rd), such as - H, halo, C1-C4 alkyl, -C(O)ORb, or -C(O)N(Rc)(Rd). Each R4independently is halo, -OH, Rb, -ORb, -SH, SRb, C2-C6 alkenyl, C2-C6 alkynyl, or -C(O)ORb, such as halo, -OH, Rb, -ORb, or-C(O)ORb, and n is 0, 1, 2, or 3. R5-R8are each independently -H, halo, Rb, C2-C6alkenyl, C2-C6 alkynyl, or -C(O)ORb, such as -H, halo, Rb, or -C(O)ORb. Rais alkyl, haloalkyl, cycloalkyl, heterocycloalkyl, or monocyclic heteroaryl. Rbis alkyl, haloalkyl, or cycloalkyl. Rcand Rdindependently are -H or C1-C4 alkyl or Rcand Rdtogether with the N atom form a 5- or 6-membered heterocycloalkyl. Z1and Z2independently are C(H)2, C(O), or C(S), wherein at least one of Z1and Z2is C(O) or C(S).

[0009] In any of the foregoing or following aspects, R1may be -NH2. In any of the foregoing or following aspects, R2may be -H, methyl, ethyl, isopropyl, trifluoromethyl, - C(O)CH3, -following aspects, R3may be -H, halo, methyl, or -C(O)ORb. In some aspects, Z1and Z2are C(O); or Z1and Z2are C(S); or one of Z1and Z2is C(O) and the other of Z1and Z2is C(S).

[0010] In any of the foregoing or following aspects, the compound may be a pharmaceutically acceptable salt, such as a hydrochloride salt. In some aspects, the compound is an enantiomer, a rotamer, or an enantiomer and a rotamer of the compound according to Formula I. In one implementation, an amount of the compound is a racemic mixture of (R)- and (S)-enantiomers. In an independent implementation, an amount of the compound has an enantiomeric excess of at least 20% of the (R)-enantiomer or (S)- enantiomer.

[0011] A pharmaceutical composition includes a compound, or a stereoisomer, pharmaceutically acceptable salt, solvate, or a hydrate thereof, according to Formula I, and a pharmaceutically acceptable carrier.

[0012] In some aspects, a method for inhibiting TNF-α activity, TNF-α synthesis, interleukin-6 (IL-6) level, inflammation, or SARS-COV-2 virus includes contacting a cell with an effective amount of a compound as disclosed herein, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some implementations, contacting the cell includes administering to a subject a therapeutically effective amount of the compound or pharmaceutically acceptable salt, solvate, or hydrate thereof, or a therapeutically effectiveamount of a pharmaceutical composition comprising the compound or pharmaceutically acceptable salt, solvate, or hydrate thereof.

[0013] In any of the foregoing or following aspects, the subject may have a disease or condition characterized at least in part by inflammation. In any of the foregoing aspects, the subject may have a traumatic brain injury (TBI), an inflammatory disorder, a neurodegenerative disease, cancer, a SARS CoV-2 virus infection, or any combination thereof.

[0014] The foregoing and other objects, features, and advantages of the disclosure will become more apparent from the following detailed description, which proceeds with reference to the accompanying figures. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] FIG.1 is a general synthetic scheme for making some compounds as disclosed by (a) secondary amine route, (b) N-alkyl route, (c) sulfonamide route, and (d) N-acyl route.

[0016] FIGS.2A-2E show chiral HPLC traces of compound (rac)-9 (FIG.2A) and traces of compound (R)-10 at time 0 (FIG.2B), after 1 hour (FIG.2C), after 2.5 hours (FIG.2D), and after 5 hours (FIG.2E) demonstrating optical stability of compound (R)-10 under biologically-relevant conditions known to racemize thalidomide.

[0017] FIGS.3A-3D show effects of several disclosed compounds on lipopolysaccharide (LPS)-induced changes in cell viability and inflammation markers in mouse RAW 264.7 cells. The percentage change from the normalized drug vehicle control + LPS (10 ng / ml, control = 100 %) for each biological variable are shown (Y-axis) for the corresponding compound concentration (µM, X-axis). Cell viability (1); media nitrite (2) and media TNF-α (3) and in a subset of assessments media IL-6 (4). Values are presented as mean ± S.E.M. of n observations (n=4). Significance markers are * P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001.

[0018] FIGS.4A-4E summarize National Institute of Mental Health (NIMH) Psychoactive Drug Screening Program binding events. The graphs show binding of compounds 6, 9, or 19 to sigma 2 receptor (FIG.4A), sigma 1 receptor (FIG.4B), 5-HT2A receptor (FIG.4C), 5- HT2Breceptor (FIG.4D), and 5-HT3receptor (FIG.4E). The corresponding reference compound is also indicated.

[0019] FIG.5 shows interaction of compounds 6, 9, 18, or 19 with a human Cereblon / / DDB1 (damaged DNA-binding protein 1) complex. Thalidomide was used as a reference compound.

[0020] FIG.6 provides graphs of percentage of change from control versus treatment group. Illustrating the anti-inflammatory action of compounds 7 (top) and 19 (bottom) in mouse microglial cells (IMG cells). DETAILED DESCRIPTION

[0021] This disclosure concerns aspects of 2-(piperidin-3-yl)isoindoline-1,3-dione analogs, as well as stereoisomers, pharmaceutically acceptable salts, solvates, and hydrates of the compounds. Pharmaceutical compositions comprising the compounds and methods of using the compounds also are disclosed. In some aspects, the compounds are useful for inhibiting TNF-α activity, TNF-α synthesis, interleukin-6 (IL-6) level, inflammation, or viral infections, such as coronavirus infections. I. Definitions and Abbreviations

[0022] The following explanations of terms and abbreviations are provided to better describe the present disclosure and to guide those of ordinary skill in the art in the practice of the present disclosure. As used herein, “comprising” means “including” and the singular forms “a” or “an” or “the” include plural references unless the context clearly dictates otherwise. The term “or” refers to a single element of stated alternative elements or a combination of two or more elements, unless the context clearly indicates otherwise.

[0023] Unless explained otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. The materials, methods, and examples are illustrative only and not intended to be limiting. Other features of the disclosure are apparent from the following detailed description and the claims.

[0024] The disclosure of numerical ranges should be understood as referring to each discrete point within the range, inclusive of endpoints, unless otherwise noted. Unless otherwise indicated, all numbers expressing quantities of components, percentages, and so forth, as used in the specification or claims are to be understood as being modified by the term “about.”Accordingly, unless otherwise implicitly or explicitly indicated, or unless the context is properly understood by a person of ordinary skill in the art to have a more definitive construction, the numerical parameters set forth are approximations that may depend on the desired properties sought and / or limits of detection under standard test conditions / methods as known to those of ordinary skill in the art. When directly and explicitly distinguishing embodiments from discussed prior art, the embodiment numbers are not approximates unless the word “about” is recited.

[0025] Definitions of common terms in chemistry may be found in Richard J. Lewis, Sr. (ed.), Hawley’s Condensed Chemical Dictionary, published by John Wiley & Sons, Inc., 2016 (ISBN 978-1-118-13515-0). Definitions of common terms in molecular biology may be found in Benjamin Lewin, Genes VII, published by Oxford University Press, 2000 (ISBN 019879276X); Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Publishers, 1994 (ISBN 0632021829); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by Wiley, John & Sons, Inc., 1995 (ISBN 0471186341); and other similar references.

[0026] In order to facilitate review of the various embodiments of the disclosure, the following explanations of specific terms are provided:

[0027] Administer: To provide a compound or a pharmaceutical composition as described herein. The compound or composition can be administered by another person to a subject (e.g., intravenously) or it can be self-administered by the subject (e.g., tablets).

[0028] Alkyl: A hydrocarbon group having a saturated carbon chain, i.e., including at least one sp3-hybridized carbon, having from 1 to 25 (C1-25) or more carbon atoms, more typically 1 to 10 (C1-10) carbon atoms such as 1 to 6 (C1-6) carbon atoms or 1 to 4 (C1-4) carbon atoms, unless otherwise specified. The chain may be branched or unbranched. Examples, without limitation, of alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, t-butyl, pentyl, hexyl, heptyl, octyl, nonyl and decyl.

[0029] Alkenyl: A hydrocarbon group having an unsaturated carbon chain with at least one double bond, i.e., including at least two sp2-hybridized carbons (C=C), and having from 2 to 25 (C2-25) or more carbon atoms, more typically 2 to 10 (C2-10) carbon atoms such as 2 to 6 (C2-6) carbon atoms or 2 to 4 (C2-4) carbon atoms, unless otherwise specified. The chain may be branched or unbranched.

[0030] Alkynyl: A hydrocarbon group having an unsaturated carbon chain with at least one triple bond, i.e., including at least two sp-hybridized carbons, and having from 2 to 25 (C2-25) or more carbon atoms, more typically 2 to 10 (C2-10) carbon atoms such as 2 to 6 (C2-6) carbon atoms or 2 to 4 (C2-4) carbon atoms, unless otherwise specified. The chain may be branched or unbranched.

[0031] Cycloalkyl: A saturated monovalent cyclic hydrocarbon radical of three to seven ring carbons, e.g., cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl.

[0032] Effective amount: An amount of a compound or composition sufficient to: achieve a particular desired result, such as to inhibit a protein or enzyme; elicit a desired biological or medical response in a tissue, system, subject or patient; treat a specified disorder or disease; ameliorate or eradicate one or more of its symptoms; and / or prevent the occurrence of the disease or disorder. The amount of a compound which constitutes an “effective amount” may vary depending on the compound, the desired result, the disease state and its severity, the age of the patient to be treated, and the like.

[0033] Enantiomeric excess: A measurement of purity for chiral substances, reflecting the degree to which one enantiomer is present in greater amount than the other enantiomer. Enantiomeric excess (ee) is calculated as the difference between the two enantiomers as the percentage of a mixture. A racemic mixture with 50% of each enantiomer has an ee of zero. A pure enantiomer has an ee of 100%. If a mixture has, for example, 70% of one enantiomer and 30% of the other, the ee is 40% (70% - 30%).

[0034] Haloalkyl: An alkyl group substituted with one or more same or different halo atoms, e.g., -CH2Cl, -CHF2, -CF3, -CH2CF3, -CF2CF3, -CH2CCl3, and the like.

[0035] Heteroaryl: An aromatic compound or group having at least one heteroatom, i.e., one or more carbon atoms in the ring has been replaced with an atom having at least one lone pair of electrons, typically nitrogen, oxygen, phosphorus, silicon, or sulfur.

[0036] Heterocycloalkyl: A cycloalkyl group having at least one heteroatom, i.e., one or more carbon atoms in the ring has been replaced with an atom having at least one lone pair of electrons, typically nitrogen, oxygen, phosphorus, silicon, or sulfur.

[0037] Pharmaceutically acceptable: A substance that can be taken into a subject without significant adverse toxicological effects on the subject. The term "pharmaceutically acceptable form" means any pharmaceutically acceptable derivative or variation, such asstereoisomers, stereoisomer mixtures, enantiomers, solvates, hydrates, isomorphs, polymorphs, pseudomorphs, neutral forms, salt forms, and prodrug agents.

[0038] Pharmaceutically acceptable carrier: The pharmaceutically acceptable carriers (vehicles) useful in this disclosure are conventional. Remington: The Science and Practice of Pharmacy, The University of the Sciences in Philadelphia, Editor, Lippincott, Williams, & Wilkins, Philadelphia, PA, 21stEdition (2005), describes compositions and formulations suitable for pharmaceutical delivery of one or more therapeutic compositions and additional pharmaceutical agents. In general, the nature of the carrier will depend on the particular mode of administration being employed. For instance, parenteral formulations usually comprise injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle. In some examples, the pharmaceutically acceptable carrier may be sterile to be suitable for administration to a subject (for example, by parenteral, intramuscular, or subcutaneous injection). In addition to biologically-neutral carriers, pharmaceutical compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate. In some examples, the pharmaceutically acceptable carrier is a non-naturally occurring or synthetic carrier. The carrier also can be formulated in a unit-dosage form that carries a preselected therapeutic dosage of the active agent, for example in a pill, vial, bottle, or syringe.

[0039] Pharmaceutically acceptable salt: A biologically compatible salt of a disclosed compound, which salts are derived from a variety of organic and inorganic counter ions well known in the art and include, by way of example only, sodium, potassium, calcium, magnesium, ammonium, tetraalkylammonium, and the like; and when the molecule contains a basic functionality, salts of organic or inorganic acids, such as hydrochloride, hydrobromide, tartrate, mesylate, acetate, maleate, oxalate, and the like. Pharmaceutically acceptable acid addition salts are those salts that retain the biological effectiveness of the free bases while formed by acid partners that are not biologically or otherwise undesirable, e.g., inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like, as well as organic acids such as acetic acid, trifluoroacetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid and the like.Pharmaceutically acceptable base addition salts include those derived from inorganic bases such as sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts and the like. Exemplary salts are the ammonium, potassium, sodium, calcium, and magnesium salts. Salts derived from pharmaceutically acceptable organic non-toxic bases include, but are not limited to, salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, ethylenediamine, glucosamine, methylglucamine, theobromine, purines, piperazine, piperidine, N-ethylpiperidine, polyamine resins, and the like. Exemplary organic bases are isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline, and caffeine. (See, for example, S. M. Berge, et al., “Pharmaceutical Salts,” J. Pharm. Sci., 1977; 66:1-19, which is incorporated herein by reference.) For therapeutic use, salts of the compounds are those wherein the counter-ion is pharmaceutically acceptable. However, salts of acids and bases which are non-pharmaceutically acceptable may also find use, for example, in the preparation or purification of a pharmaceutically acceptable compound.

[0040] Pharmaceutical composition: A composition that includes an amount (for example, a unit dosage) of one or more of the disclosed compounds together with one or more nontoxic pharmaceutically acceptable additives, including carriers, diluents, and / or adjuvants, and optionally other biologically active ingredients. Such pharmaceutical compositions can be prepared by standard pharmaceutical formulation techniques such as those disclosed in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA (19th Edition).

[0041] Solvate: A complex formed by combination of solvent molecules with molecules or ions of a solute. The solvent can be an organic solvent, an inorganic solvent, or a mixture of both. Exemplary solvents include, but are not limited to, alcohols, such as methanol, ethanol, propanol; amides such as N,N-dialiphatic amides, such as N,N-dimethylformamide; tetrahydrofuran; alkylsulfoxides, such as dimethylsulfoxide; water; and combinations thereof. The compounds described herein can exist in un-solvated as well as solvated forms when combined with solvents, pharmaceutically acceptable or not, such as water, ethanol, and the like. Solvated forms of the presently disclosed compounds are within the scope of the embodiments disclosed herein.

[0042] Stereoisomers: Isomers that differ in the arrangement of their atoms in space are termed “stereoisomers”. Stereoisomers that are not mirror images of one another are termed “diastereomers” and those that are non-superimposable mirror images of each other are termed “enantiomers.” When a compound has an asymmetric center, for example, if a carbon atom is bonded to four different groups, a pair of enantiomers is possible. An enantiomer can be characterized by the absolute configuration of its asymmetric center and is described by the R- and S-sequencing rules of Cahn and Prelog, or by the manner in which the molecule rotates the plane of polarized light and designated as dextrorotatory or levorotatory (i.e., as (+) or (-) isomers respectively). A chiral compound can exist as either individual enantiomer or as a mixture thereof. A mixture containing equal proportions of the enantiomers is called a “racemic mixture.” When the enantiomer proportions are not equal, an enantiomeric excess (ee) of one enantiomer is present. When spatial orientation is not indicated in a chemical formula, the formula includes all possible spatial orientations. Rotamers (also known as conformational isomers, rotational isomers, and conformers) are stereoisomers that are produced by twisting or rotating about sigma bonds, creating different spatial arrangements of carbon atoms. Rotamers often rapidly interconvert at ambient and in vivo temperatures.

[0043] Subject: An animal (human or non-human) subjected to a treatment, observation or experiment. Includes both human and veterinary subjects, including human and non-human mammals, such as rats, mice, cats, dogs, pigs, horses, cows, and non-human primates.

[0044] Therapeutically effective amount or dose: An amount sufficient to provide a beneficial, or therapeutic, effect to a subject or a given percentage of subjects.

[0045] Treating or treatment: With respect to disease, either term includes (1) preventing the disease, e.g., causing the clinical symptoms of the disease not to develop in an animal that may be exposed to or predisposed to the disease but does not yet experience or display symptoms of the disease, (2) inhibiting the disease, e.g., arresting the development of the disease or its clinical symptoms, or (3) relieving the disease, e.g., causing regression of the disease or its clinical symptoms. As used herein, the terms “disease” and “condition” can be used interchangeably or can be different in that the particular malady or condition may not have a known causative agent (so that etiology has not yet been determined) and it is therefore not yet recognized as a disease but only as an undesirable condition or syndrome, where a more or less specific set of symptoms have been identified by clinicians.II. 2-(Piperidin-3-yl)isoindoline-1,3-dione Analogs Compounds disclosed herein have a structure according to Formula I, or a stereoisomer or pharmaceutically acceptable salt, solvate, or hydrate thereof:

[0046] With respect to Formula I, R1is -H, -N(R′)(R′′), -NO2, -OH, -ORb, -SH, -SRb, -CN, C2-C6alkenyl, C2-C6alkynyl, or halo, such as -N(R′)(R′′), -NO2, -OH, or halo, wherein R′ and R′′ independently are -H, C1-C3 alkyl, C3-C6 cycloalkyl, or -C(O)Rb, such as -H or C1-C3 alkyl.

[0047] R2is -H, Ra, -C(O)Rb, -C(O)ORb, -S(O)2Rb, C2-C6 alkenyl, C2-C6 alkynyl, or -C(O)N(Rc)(Rd), such as -H, Ra, -C(O)Rb, -C(O)ORb, -S(O)2Rb, or -C(O)N(Rc)(Rd).

[0048] R3is -H, halo, C1-C4alkyl, C2-C6alkenyl, C2-C6alkynyl, -C(O)ORb, or - C(O)N(Rc)(Rd), such as -H, halo, C1-C4 alkyl, -C(O)ORb, or -C(O)N(Rc)(Rd).

[0049] Each R4independently is halo, -OH, Rb, -ORb, -SH, SRb, C2-C6 alkenyl, C2-C6 alkynyl, or -C(O)ORb, such as halo, -OH, Rb, -ORb, or -C(O)ORb, where n is 0, 1, 2, or 3.

[0050] R5-R8are each independently -H, halo, Rb, C2-C6 alkenyl, C2-C6 alkynyl, or -C(O)ORb, such as -H, halo, Rb, or -C(O)ORb.

[0051] Rais alkyl, haloalkyl, cycloalkyl, heterocycloalkyl, or monocyclic heteroaryl.

[0052] Rbis alkyl, haloalkyl, or cycloalkyl.

[0053] Rcand Rdindependently are -H or C1-C4alkyl or Rcand Rdtogether with the N atom form a 5- or 6-membered heterocycloalkyl.

[0054] Z1and Z2independently are C(H)2, C(O), or C(S), wherein at least one of Z1and Z2is C(O) or C(S).

[0055] In some aspects, the following provisos apply: (i) if R1is -NH2, then one of Z1and Z2is other than C(O), or n is not 0, or R2is not H, methyl, or ethyl, or R3is not methyl, or one of R5-R8is not -H; (ii) if R1is -NO2, then one of Z1and Z2is other than C(O), or n is not 0, or R2is not ethyl, or R3is not H, or one of R5-R8is not -H; iii) if R1is halo, then one of Z1and Z2is other than C(O), or R2is not H, or R3is not H, or one of R5-R8is not -H, or n is 0 or 2 when R4is halo.

[0056] In some aspects, R1is -N(R′)(R′′), -NO2, -OH, or halo, wherein R′ and R′′ independently are -H or C1-C3 alkyl. In some aspects, R1is -NH2, -N(CH3)2, -NO2, -OH, -F, or -Cl. In certain aspects, R1is -NH2.

[0057] R2is -H, Ra, -C(O)Rb, -C(O)ORb, -S(O)2Rb, or -C(O)N(Rc)(Rd) where Ra-Rdare as previously described. In some aspects, R2is -H, alkyl, haloalkyl, heterocycloalkyl, monocyclic heteroaryl, -C(O)Rbor -C(O)ORbwhere Rbis alkyl or cycloalkyl, -S(O)2Rbwhere Rbis alkyl, or -C(O)N(Rc)(Rd) where Rcand Rdare H or Rcand Rdtogether with the N atom form a 5- or 6-membered heterocycloalkyl. In some implementations, the alkyl, cycloalkyl, heterocycloalkyl or monocyclic heteroaryl group is unsubstituted. In certain aspects, R2is -H, methyl, ethyl, isopropyl, trifluoromethyl, -C(O)CH3, -C(O)CF3, , -C

[0058] R3is -H, halo, C1-C4alkyl, -C(O)ORb, or -C(O)N(Rc)(Rd) where Rb-Rdare as previously described. In some aspects, R3is -H, halo, C1-C4 alkyl, or -C(O)ORbwhere Rbis alkyl or haloalkyl. In certain aspects, R3is -H, -F, methyl, or -C(O)OCH3.

[0059] Each R4independently is halo, -OH, Rb, -ORb, or -C(O)ORb, and n is 0, 1, 2, or 3. In some aspects, n is 0. In some implementations, n is 1, 2, or 3, and R4is halo, -OH, -Rb, or -C(O)ORb. In some aspects, R4is -F, -Cl, -Br, -OH, -CH3, -CF3, -OCH3, -C(O)OCH3, or cyclopropyl. In certain implementations, n is 1.

[0060] R5-R8independently are -H, halo, Rb, or -C(O)ORb. In some aspects, R5-R8are -H. In an independent aspect, R5and R6are both halo, and R7and R8are both -H. In another independent aspect, R5and R6are both -H, and R7and R8are both halo. In some implementations, the halo atoms are fluorine. In still another independent aspect, one of R5- R8is Rbor -C(O)ORband the others of R5-R8are -H. In yet another independent aspect, one of R5-R8is Rband the others of R5-R8are -H. In some implementations, Rbis -CH3 or -CF3.

[0061] Z1and Z2independently are C(H)2, C(O), or C(S), wherein at least one of Z1and Z2is C(O) or C(S). In one aspect, Z1and Z2are C(O). In an independent aspect, Z1is C(O) and Z2is C(S). In another independent aspect, Z1is C(S) and Z2is C(O). In still anotherindependent aspect, Z1and Z2are C(S). In yet another independent aspect, Z1is C(H)2and Z2is C(O) or C(S). In yet another independent aspect, Z1is C(O) or C(S) and Z2is C(H)2.

[0062] Compounds according to Formula I include a chiral center as indicated by the asterisk (*). Accordingly, the compounds include (R) and (S) enantiomers. In some aspects, an amount of a compound according to Formula I is a racemic mixture including equal proportions of the (R) and (S) enantiomers. In other aspects, an amount of a compound according to Formula I has an enantiomeric excess (ee) of either the (R) or (S) enantiomer. In some implementations, the predominant stereoisomer is present in an enantiomeric excess of at least 20%. In certain implementations, the ee is at least 40%, at least 60%, at least 80%, from 20% to 100%, from 40% to 100%, from 60% to 100%, from 80% to 100%, or from 90% to 100%. Rotamers of compounds according to Formula I also are encompassed by this disclosure.

[0063] Several exemplary non-limiting examples of compounds according to Formula I are shown in Table 1. It is understood that the exemplary compounds may be in the form of a rotamer and / or a pharmaceutically acceptable salt, solvate, or hydrate unless otherwise indicated. In some aspects, the exemplary compounds are in the form of pharmaceutically acceptable salts. In certain aspects, the pharmaceutically acceptable salt is a hydrochloride salt. Table 1 – Exemplary CompoundsIn some aspects, the compound iseutically acceptable salt, solvate, or hydrate thereof. In certain aspects, the compound isthe stereoisomer has an ee of at least 20%, at least 40%, at least 60%, or at least 80%, such as 20-100%, 40-100%, 60-100%, or 80-100%.

[0064] In some aspects, the compound is notoplementations, the compound is not a racemic mixture, an enantiomer, or a rotamer of the foregoing nine compounds. Additionally, or alternatively, the compound is not a pharmaceutically acceptable salt, solvate, or hydrate of the foregoing nine compounds. III. Pharmaceutical Compositions

[0065] Embodiments of a pharmaceutical composition include one or more compounds according to Formula I as disclosed herein, or a stereoisomer, pharmaceutically acceptable salt, solvate, or a hydrate thereof, and a pharmaceutically acceptable carrier. The disclosed compounds can be further combined with excipients, and optionally sustained release matrices, such as biodegradable polymers. The composition may comprise a unit dosage form of the composition, and may further comprise instructions for administering the composition to a subject. Such pharmaceutical compositions may be used in methods for inhibiting TNF-α activity, TNF-α synthesis, interleukin-6 (IL-6) level, inflammation, or SARS-COV-2 virus, or any combination thereof, as well as diseases and disorders characterized by abnormal levels of TNF-α activity, TNF-α synthesis, IL-6 level, inflammation, or any combination thereof, as discussed further in section IV below.

[0066] The disclosed pharmaceutical compositions can be in the form of tablets, capsules, powders, granules, lozenges, liquid or gel preparations, such as oral, topical, or sterile parenteral solutions or suspensions (e.g., eye or ear drops, throat or nasal sprays, etc.), transdermal patches, and other forms known in the art.

[0067] Pharmaceutical compositions can be administered systemically or locally in any manner appropriate to the treatment of a given condition, including orally, parenterally, rectally, nasally, buccally, vaginally, topically, optically, by inhalation spray, or via an implanted reservoir. The term "parenterally" as used herein includes, but is not limited to subcutaneous, intravenous, intramuscular, intrasternal, intrasynovial, intrathecal, intrahepatic, intralesional, and intracranial administration, for example, by injection or infusion. For treatment of the central nervous system, the pharmaceutical compositions may readily penetrate the blood brain barrier when peripherally or intraventricularly administered.

[0068] Pharmaceutically acceptable carriers include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins (such as human serum albumin), buffers (such as phosphates), glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene polyoxypropylene block polymers, polyethylene glycol, and wool fat.

[0069] Tablets and capsules for oral administration can be in a form suitable for unit dose presentation and can contain conventional pharmaceutically acceptable excipients. Examples of these include binding agents such as syrup, acacia, gelatin, sorbitol, tragacanth, and polyvinylpyrrolidone; fillers such as lactose, sugar, corn starch, calcium phosphate, sorbitol, or glycine; tableting lubricants, such as magnesium stearate, talc, polyethylene glycol, or silica; disintegrants, such as potato starch; and dispersing or wetting agents, such as sodium lauryl sulfate. Oral liquid preparations can be in the form of, for example, aqueous or oily suspensions, solutions, emulsions, syrups or elixirs, or can be presented as a dry product for reconstitution with water or other suitable vehicle before use.

[0070] The pharmaceutical compositions can also be administered parenterally in a sterile aqueous or oleaginous medium. The composition can be dissolved or suspended in a nontoxic parenterally acceptable diluent or solvent, e.g., as a solution in 1,3-butanediol. Commonly used vehicles and solvents include water, physiological saline, Hank's solution, Ringer's solution, and sterile, fixed oils, including synthetic mono- or diglycerides, etc. For topical application, the drug may be made up into a solution, suspension, cream, lotion, or ointment in a suitable aqueous or non-aqueous vehicle. Additives may also be included, for example, buffers such as sodium metabisulfite or disodium edetate; preservatives such as bactericidal and fungicidal agents, including phenyl mercuric acetate or nitrate, benzalkonium chloride or chlorhexidine, and thickening agents, such as hypromellose.

[0071] The compounds can be used in the form of pharmaceutically acceptable salts derived from inorganic or organic acids and bases, including, but not limited to: acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate,methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate, pamoate, pectinate, persulfate, 3-phenylpropionate, picrate, pivalate, propionate, succinate, tartrate, thiocyanate, tosylate, and undecanoate. Base salts include, but are not limited to, ammonium salts, alkali metal salts (such as sodium and potassium salts), alkaline earth metal salts (such as calcium and magnesium salts), salts with organic bases (such as dicyclohexylamine salts), N-methyl-D- glucamine, and salts with amino acids (such as arginine, lysine, etc.). Basic nitrogen containing groups can be quaternized, for example, with such agents as C1-8alkyl halides (such as methyl, ethyl, propyl, and butyl chlorides, bromides, and iodides), dialkyl sulfates (such as dimethyl, diethyl, dibutyl, and diamyl sulfates), long-chain halides (such as decyl, lauryl, myristyl, and stearyl chlorides, bromides, and iodides), aralkyl halides (such as benzyl and phenethyl bromides), etc. Water- or oil-soluble or dispersible products are produced thereby. In certain aspects, the compound is a hydrochloride salt of a compound according to Formula I or a stereoisomer thereof. IV. Methods of Use

[0072] The compounds disclosed herein, and stereoisomers, pharmaceutically acceptable salts, solvates, or hydrates thereof, may be used for inhibiting TNF-α activity, TNF-α synthesis, interleukin-6 (IL-6) level, inflammation, SARS CoV 2 virus, or any combination thereof. In some aspects, a cell is contacted with an effective amount of a compound as disclosed herein, or a pharmaceutically acceptable salt, solvate, or hydrate thereof, to inhibit TNF-α activity, TNF-α synthesis, interleukin-6 (IL-6) level, inflammation, or SARS-COV-2 virus or any combination thereof. The cell may be contacted in vitro, in vivo, or ex vivo. In one implementation, the cell is contacted with a compound as disclosed in Table 1.

[0073] In any of the foregoing aspects, contacting the cell with an effective amount of the compound may comprise administering to a subject a therapeutically effective amount of the compound, or stereoisomer, pharmaceutically acceptable salt, solvate, or hydrate thereof, or a therapeutically effective amount of a pharmaceutical composition comprising the compound or stereoisomer, pharmaceutically acceptable salt, solvate, or hydrate thereof. Administration may be performed by any suitable route, including orally, parenterally, rectally, nasally, buccally, vaginally, topically, optically, by inhalation spray, or via an implanted reservoir.

[0074] In some implementations, a subject is administered a therapeutically effective amount of compound according to general Formula I, or a stereoisomer, a pharmaceutically acceptable salt, solvate, or hydrate thereof, or a pharmaceutical composition comprising thecompound. In certain implementations, a subject is administered a therapeutically effective amount of a compound as disclosed in Table 1, or a stereoisomer, a pharmaceutically acceptable salt, solvate, or hydrate thereof, or a pharmaceutical composition comprising the compound.

[0075] In some aspects, the subject has an inflammatory disorder, an autoimmune disorder, a neurodegenerative disease, cancer, a viral infection, or any combination thereof. In some implementations, the subject has a viral infection, particularly a coronavirus infection, such as a SARS CoV 2 infection. Exemplary inflammatory and / or autoimmune disorders that may be ameliorated with aspects of the disclosed compounds include, but are not limited to, neuroinflammation, rheumatoid arthritis, immune arthritis, degenerative arthritis, celiac disease, glomerulonephritis, lupus nephritis, prostatitis, inflammatory bowel disease (e.g., Crohn’s disease), pelvic inflammatory disease, graft versus host disease, interstitial cystitis, autoimmune thyroiditis, Graves’ disease; autoimmune pancreatitis, Sjogren’s syndrome, myocarditis, autoimmune hepatitis, primary biliary cirrhosis, autoimmune angioedema, bullous pemphigoid, discoid lupus erythematosus, erythema nodosum leprosum, sarcoidosis, pemphigus vulgaris psoriasis, POEMS syndrome, polymyositis, human immune deficiency virus / acquired immune deficiency syndrome, vasculitis, sarcopenia, primary myelofibrosis, myelodysplastic syndrome, white blood cell cancers (e.g., multiple myeloma, acute myeloid leukemia, chronic lymphocytic leukemia, hairy cell leukemia, large granular lymphocytic leukemia, non-Hodgkin’s lymphoma), and Kaposi sarcoma,. In some implementations, the subject has a traumatic brain injury (TBI) and / or neuroinflammation following a TBI. In some aspects, the neurodegenerative disease is Alzheimer’s disease (AD), Parkinson’s disease (PD), multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), or Huntington’s disease (HD). Subject to neurotoxicity considerations (e.g., whether the compound is well- tolerated by nervous tissue), certain aspects of the compounds disclosed herein may be used to reduce neuroinflammation as a treatment strategy for neurodegenerative disorders. Advantageously, a compound used to reduce neuroinflammation may be non-neurotoxic at a therapeutically effective dose. Examples of neurodegenerative and / or neuroinflammatory disorders that may be ameliorated with embodiments of the disclosed compounds include, but are not limited to, neurodegeneration resulting from head trauma (e.g., traumatic brain injury), spinal cord injuries, stroke, Alzheimer’s disease, Parkinson’s disease, ALS (amyotrophic lateral sclerosis), HIV (human immunodeficiency virus) dementia, Huntington’s disease, multiple sclerosis, cerebral amyloid angiopathy, tauopathies, peripheralneuropathies, macular degeneration, hearing loss, cochlear injury, epilepsy, a non-epileptic seizure disorder (e.g., due to head injury, dementia, prenatal brain injury, meningitis, lupus, encephalitis, among others), and major depressive disorder (also known as clinical depression, unipolar depression). Aspects of the disclosed compounds may be used to reduce chronic systemic and CNS inflammation and / or as immunomodulatory agents. Certain aspects of the disclosed compounds are small-molecular-weight lipophilic compounds with physicochemical properties (see, e.g., Lipinski et al., Adv Drug Deliv Rev.200146(1-3):3- 26) that may allow them to pass through the blood-brain barrier.

[0076] In some aspects, the disclosed compounds are useful for treating or preventing degenerative and / or inflammatory conditions of the eye (such as, but not limited to, macular degeneration, retinitis pigmentosa, diabetic retinopathy, uvetitis / scleritis / keratitis, or Graves’ eye disease). In some such aspects, the compound or a formulation thereof is applied topically (i.e., locally).

[0077] In some aspects, the disclosed compounds are useful for treating or preventing meningeal TB (tuberculosis), optionally in combination with an antibacterial to mitigate neuroinflammation.

[0078] In some aspects, the disclosed compounds are useful for treating or preventing noise- induced sensory hair cell loss and hearing loss, such as disorders / diseases of the cochlear (inner ear) which are associated with inflammation. In some such aspects, the compound or a formulation thereof is applied locally (for example, topically).

[0079] In any of the foregoing or following aspects, the subject may have a disease or condition characterized at least in part by inflammation. In certain aspects, the subject has a TBI, an inflammatory disorder, a neurodegenerative disease, cancer, or any combination thereof.

[0080] TNF-α serves as a regulator in acute stages of neuroinflammation, triggering signaling cascades of pro-inflammatory cytokines. Increased TNF-α is associated with several neurodegenerative disorders, including TBI, AD, PD, MS, ALS, and HD, among others. Advantageously, some implementations of the disclosed compounds inhibit TNF-α and / or ameliorate inflammation without binding to cereblon (the primary target of thalidomide teratogenicity). Reduced inflammation may be evidenced by reduced levels of pro-inflammatory cytokines and / or chemokines (e.g., TNF-α, IL-6, and others) in plasma and / or brain tissue.

[0081] TBI is a leading cause of death and disability in children and adults. TBI has been identified as a major risk factor for several neurodegenerative disorders, including PD and AD. Neuroinflammation is considered the cause of later secondary cell death following TBI, and has the potential to chronically aggravate the first impact. Within minutes to hours after TIB, mRNA and protein expression of TNF-α is elevated. Advantageously, some aspects of the disclosed compounds mitigate lipopolysaccharide induced inflammation and TNF-α levels, and decrease neuroinflammation induced by controlled cortical impact. In some implementations, the disclosed compounds decrease lesion size / volume following TBI, compared to lesion size / volume in the absence of treatment with the disclosed compounds. In certain implementations, the disclosed compounds further mitigate microglial cell activation, neuronal loss, and / or behavioral deficits when administered after TBI. For example, following administration of an effective amount of a disclosed compound, a subject may demonstrate reduced impairment in fine motor coordination and / or balance, following TBI, compared to a subject that has not been treated with a disclosed compound. The compounds further may be useful for treating longer term neurodegenerative disorders, such as those disorders mediated by neuroinflammation and / or induced by TBI (e.g., AD, PD, MS, and / or ALS).

[0082] In one aspect, the subject is administered a therapeutically effective amount of a compound or a pharmaceutically acceptable salt, solvate, or hydrate thereof, or a pharmaceutical composition comprising the compound or pharmaceutically acceptable salt, solvate, or hydrate thereof, wherein the compound issubject is administeredstereoisomer has an enantiomeric excess of at least 20%. In certain implementations, the eeis at least 40%, at least 60%, at least 80%, from 20% to 100%, from 40% to 100%, from 60% to 100%, from 80% to 100%, or from 90% to 100%.

[0083] Exemplary Aspects

[0084] The following numbered paragraphs illustrate exemplary aspects of the disclosed technology.

[0085] Paragraph 1. A compound according to Formula I, or a stereoisomer or pharmaceutically acceptable salt, solvate, or hydrate thereof:

[0086] where

[0087] R1is -N(R′)(R′′), -NO2, -OH, or halo, wherein R′ and R′′ independently are -H or C1-C3 alkyl;

[0088] R2is -H, Ra, -C(O)Rb, -C(O)ORb, -S(O)2Rb, or -C(O)N(Rc)(Rd);

[0089] R3is -H, halo, C1-C4 alkyl, -C(O)ORb, or -C(O)N(Rc)(Rd);

[0090] each R4independently is halo, -OH, Rb, -ORb, or -C(O)ORb;

[0091] R5-R8independently are -H, halo, Rb, or -C(O)ORb;

[0092] Rais alkyl, haloalkyl, cycloalkyl, heterocycloalkyl, or monocyclic heteroaryl;

[0093] Rbis alkyl, haloalkyl, or cycloalkyl;

[0094] Rcand Rdindependently are -H or C1-C4 alkyl or Rcand Rdtogether with the N atom form a 5- or 6-membered heterocycloalkyl;

[0095] Z1and Z2independently are C(H)2, C(O), or C(S), wherein at least one of Z1and Z2is C(O) or C(S); and

[0096] n is 0, 1, 2, or 3,

[0097] wherein

[0098] (i) if R1is -NH2, then one of Z1and Z2is other than C(O), or n is not 0, or R2is not H, methyl, or ethyl, or R3is not methyl, or one of R5-R8is not -H, or

[0099] (ii) if R1is -NO2, then one of Z1and Z2is other than C(O), or n is not 0, or R2is not ethyl, or R3is not H, or one of R5-R8is not -H, or

[0100] (iii) if R1is halo, then one of Z1and Z2is other than C(O), or R2is not H, or R3is not H, or one of R5-R8is not -H, or n is 0 or 2 when R4is halo.

[0101] Paragraph 2. The compound according to paragraph 1, where R1is -NH2.

[0102] Paragraph 3. The compound according to paragraph 1 or paragraph 2, wherein R2is -H, methyl, ethyl, isopropyl, trifluoromethyl, -C(O)CH3, -C(O)CF3, , -C(O)O, C

[0103] Paragraph 4. The compound according to any one of paragraphs 1-3, wherein R3is - H, halo, methyl, or -C(O)ORb.

[0104] Paragraph 5. The compound according to any one of paragraphs 1-4, wherein n is 0.

[0105] Paragraph 6. The compound according to any one of paragraphs 1-4, wherein n is 1 and R4is halo, -OH, -Rb, or -C(O)ORb.

[0106] Paragraph 7. The compound according to any one of paragraphs 1-6, wherein:

[0107] Z1and Z2are C(O); or

[0108] Z1and Z2are C(S); or

[0109] one of Z1and Z2is C(O) and the other of Z1and Z2is C(S).

[0110] Paragraph 8. The compound according to any one of paragraphs 1-7, wherein:

[0111] R5-R8are -H; or

[0112] R5and R6are halo, and R7and R8are -H; or

[0113] R5and R6are -H and R7and R8are halo; or

[0114] one of R5-R8is Rbor -C(O)ORb, and the others of R5-R8are -H.

[0115] Paragraph 9. The compound according to any one of paragraphs 4 or 6-8, where Rbis -CH3.

[0116] Paragraph 10. The compound according to any one of paragraphs 1-9, wherein the compound is a pharmaceutically acceptable salt.

[0117] Paragraph 11. The compound according to paragraph 10, wherein the pharmaceutically acceptable salt is a hydrochloride salt.

[0118] Paragraph 12. The compound according to any one of paragraphs 1-11, wherein the stereoisomer is an enantiomer, a rotamer, or an enantiomer and a rotamer of the compound according to Formula I.

[0119] Paragraph 13. The compound according to any one of paragraphs 1-12, wherein an amount of the compound is a racemic mixture of (R)- and (S)-enantiomers.

[0120] Paragraph 14. The compound according to any one of paragraphs 1-12, wherein an amount of the compound has an enantiomeric excess of at least 20% of the (R)-enantiomer or (S)-enantiomer.

[0121] Paragraph 15. The compound of paragraph 1, wherein the compound is:wherein enantiomers are present in an enantiomeric excess of at least 20% in an amount of the compound.

[0122] Paragraph 16. The compound of paragraph 1, wherein the compound is:

[0123] pharmaceutically acceptable salt, solvate, or hydrate thereof.

[0124] Paragraph 17. The compound of paragraph 1, wherein the compound is

[0125]

[0126] wherein the stereoisomer has an enantiomeric excess of at least 20% in an amount of the compound.

[0127] Paragraph 18. A pharmaceutical composition, comprising

[0128] a compound, or a stereoisomer, pharmaceutically acceptable salt, solvate, or a hydrate thereof, according to any one of paragraphs 1-17; and

[0129] a pharmaceutically acceptable carrier.

[0130] Paragraph 19. A method for inhibiting TNF-α activity, TNF-α synthesis, interleukin-6 (IL-6) level, inflammation, or SARS-COV-2 virus, comprising contacting a cell with an effective amount of a compound or a pharmaceutically acceptable salt, solvate, or hydrate thereof, according to any one of paragraphs 1-17.

[0131] Paragraph 20. The method of paragraph 19, wherein contacting the cell with an effective amount of the compound or pharmaceutically acceptable salt, solvate, or hydrate thereof, comprises administering to a subject a therapeutically effective amount of the compound or pharmaceutically acceptable salt, solvate, or hydrate thereof, or a therapeutically effective amount of a pharmaceutical composition comprising the compound or pharmaceutically acceptable salt, solvate, or hydrate thereof.

[0132] Paragraph 21. The method of paragraph 20, wherein the subject has a disease or condition characterized at least in part by inflammation.

[0133] Paragraph 22. The method of paragraph 20 or paragraph 21, wherein the subject has a traumatic brain injury (TBI), an inflammatory disorder, a neurodegenerative disease, cancer, a SARS CoV-2 virus infection, or any combination thereof.

[0134] Paragraph 23. The method of paragraph 20, wherein the subject has a TBI, neuroinflammation, a SARS CoV 2 virus infection, Alzheimer’s Disease, Parkinson’s Disease, multiple sclerosis, amyotrophic lateral sclerosis, Huntington’s Disease, a spinal cord injury, a stroke, human immunodeficiency virus dementia, cerebral amyloid angiopathy, tauopathy, peripheral neuropathy, macular degeneration, hearing loss, cochlear injury, epilepsy, a non-epileptic seizure disorder, depression, rheumatoid arthritis, immune arthritis, degenerative arthritis, celiac disease, glomerulonephritis, lupus nephritis, prostatitis, inflammatory bowel disease, pelvic inflammatory disease, graft versus host disease, interstitial cystitis, autoimmune thyroiditis, Graves’ disease; autoimmune pancreatitis,Sjogren’s syndrome, myocarditis, autoimmune hepatitis, primary biliary cirrhosis, autoimmune angioedema, bullous pemphigoid, discoid lupus erythematosus, erythema nodosum leprosum, sarcoidosis, pemphigus vulgaris psoriasis, POEMS syndrome, polymyositis, human immune deficiency virus / acquired immune deficiency syndrome, vasculitis, sarcopenia, multiple myeloma, primary myelofibrosis, myelodysplastic syndrome, acute myeloid leukemia, chronic lymphocytic leukemia, hairy cell leukemia, large granular lymphocytic leukemia, non-Hodgkin’s lymphoma, Kaposi sarcoma, or any combination thereof.

[0135] Paragraph 24. A compound for use in a method of treating aberrantly high TNF-α activity, aberrantly high level of IL-6, inflammation, SARS-CoV-2 viral infection, or any combination thereof, the method comprising administering to a subject having aberrantly high TNF-α activity, aberrantly high level of IL-6, inflammation, SARS-CoV-2 viral infection, or any combination thereof a therapeutically effective amount of a compound or a pharmaceutically acceptable salt, solvate, or hydrate thereof, according to any one of paragraphs 1-17 or a therapeutically effective amount of a pharmaceutical composition comprising the compound or pharmaceutically acceptable salt, solvate, or hydrate thereof.

[0136] Paragraph 25. The compound for use of paragraph 24, wherein the subject has a disease or condition characterized at least in part by inflammation.

[0137] Paragraph 26. The compound for use of paragraph 24, wherein the subject has a TBI, an inflammatory disorder, a neurodegenerative disease, cancer, or any combination thereof.

[0138] Paragraph 27. The compound for use of paragraph 24, wherein the subject has a TBI, neuroinflammation, a SARS CoV 2 virus infection, Alzheimer’s Disease, Parkinson’s Disease, multiple sclerosis, amyotrophic lateral sclerosis, Huntington’s Disease, a spinal cord injury, a stroke, human immunodeficiency virus dementia, cerebral amyloid angiopathy, tauopathy, peripheral neuropathy, macular degeneration, hearing loss, cochlear injury, epilepsy, a non-epileptic seizure disorder, depression, rheumatoid arthritis, immune arthritis, degenerative arthritis, celiac disease, glomerulonephritis, lupus nephritis, prostatitis, inflammatory bowel disease, pelvic inflammatory disease, graft versus host disease, interstitial cystitis, autoimmune thyroiditis, Graves’ disease; autoimmune pancreatitis, Sjogren’s syndrome, myocarditis, autoimmune hepatitis, primary biliary cirrhosis, autoimmune angioedema, bullous pemphigoid, discoid lupus erythematosus, erythema nodosum leprosum, sarcoidosis, pemphigus vulgaris psoriasis, POEMS syndrome, polymyositis, human immune deficiency virus / acquired immune deficiency syndrome, vasculitis, sarcopenia, multiple myeloma, primary myelofibrosis, myelodysplastic syndrome,acute myeloid leukemia, chronic lymphocytic leukemia, hairy cell leukemia, large granular lymphocytic leukemia, non-Hodgkin’s lymphoma, Kaposi sarcoma, or any combination thereof. V. Examples

[0139] Reagents and Instrumentation

[0140] All reagents were purchased from commercial sources and used as received unless otherwise noted. Solvents utilized were of the highest grade available given their intended use (LC / MS grade, HPLC grade, anhydrous, etc.). Reactions were carried out with anhydrous solvents under inert atmosphere unless otherwise noted. All compounds are racemic unless otherwise noted. Reactions are not optimized.1H-NMR,19F-NMR, and13C- NMR spectra were recorded on a Varian Mercury Plus 400 spectrometer or a JEOL 400YH spectrometer, and at 400 MHz, 377 MHz, and 101 MHz, respectively, and at room temperature (25°C) unless otherwise noted. The chemical shifts are given in parts per million (δ) with respect to internal tetramethylsilane, fluorotrichloromethane, or residual solvent peaks. NMR data is reported in the standard format: chemical shift, multiplicity (singlet, s; doublet, d; triplet, t; multiplet, m; broad singlet, bs; broad doublet, bd; broad multiplet, bm), integration, coupling constant (Hz). Purity analyses by qNMR were conducted under conditions ensuring complete relaxation of the nuclei in question and using appropriately certified quantitative standards as indicated. Materials were weighed into a tared vial, and then subsequently dissolved completely in DMSO-d6. Aliquots were then injected into the NMR tube and the experiments were conducted immediately after. The qNMR experiments were performed with a 90° pulse sequence, at least 8 scans, and delay of 60s to ensure complete relaxation, all maintained at 25°C. Prior to signal integration, the spectrum was manually phased, and the baseline corrected using a polynomial fit. Care was taken to integrate entire signals, and a key distinct proton was set to a corresponding value for area comparison. The purity was then determined using standard qNMR calculations, factoring in the supplied TraceCERT purity profile, ultimately demonstrating a purity of >95% (+ / - the indicated TraceCERT range) of the sample utilized. TraceCERT materials were purchased from Sigma Aldrich [1,2,4,5-tetrachloro-3-nitrobenzene, “TCNB”, lot# BCCD1553, 99.79 + / - 0.19%] and [ethylene carbonate, “EC” lot# BCCC5614, 99.92 + / - 0.23%]. Materials used for biological studies were analyzed and documented at >95% purity using elemental analysis or qNMR as noted. LC / MS spectra were obtained on an Agilent®6125-LC-MSSingle Quad system (Agilent Technologies, Inc., Santa Clara, CA) using an InfinityLab®Poroshell®120 EC-C18 (4.6 x 50mm, 2.7-μm) column (Agilent Technologies, Inc.), and a gradient elution in acetonitrile / water supplemented to 0.1% formic acid. If indicated, preparatory HPLC was carried out on an AccqPrep 125 system (Teledyne Labs) fitted with a Gemini 5-μm NX-C18110A LC column (150 x 30mm; Phenomenex, Torrance, CA) and using a gradient of acetonitrile / water supplemented to 0.1% trifluoroacetic acid. Chiral HPLC were obtained on a Shimadzu (Kyoto, Japan) LC-20 system and using Chiralpak®HPLC columns (Daicel Corporation, Osaka, Japan) as indicated and using isocratic conditions. For HRMS (MALDI) analyses, samples were dissolved in acetonitrile and diluted in an alpha-cyano-4-hydroxycinnamic acid (CHCA) matrix solution. The matrix solution was at a concentration of 10 mg / mL in 1:1 (v / v) I:H2O (0.1%TFA).1 µL of the sample / matrix mixture was added to the sample plate. An Orbitrap®XL mass spectrometer (Thermo Fisher, Waltham, MA) with a MALDI source was used for mass analysis. Samples were analyzed in positive ion mode with a mass resolution setting of 100,000. For ESI HRMS, an Orbitrap®Velos mass spectrometer (Thermo Fisher) with a HESI source was used for mass analysis coupled with an auto sampler (Ultimate 3000 HPLC, Thermo Fisher). The analysis was conducted in positive ion mode with the HESI ion source in FTMS mode with a mass resolution of 100K.

[0141] Solubility Analysis

[0142] Between 1-5mg of thalidomide, pomalidomide, or (rac)-9 was weighed into a tared vial and then the appropriate amount of 1X phosphate-buffered saline (PBS, pH 7.4) was added to bring the final titer of each to a theoretical concentration of 850mM. All of the mixtures were allowed to stir and equilibrate briefly at room temperature (RT), after which the vials were visibly inspected and subsequently examined under a microscope. From a simple visual inspection, it was apparent that the thalidomide and pomalidomide samples contained a significant amount of precipitate. The thalidomide sample also contained material that would not settle to the bottom of the vial and instead remained floating at the interface of the solvent. In the cases of (rac)-9, the vials were essentially free of undissolved material, and the liquors were very clearly homogenous yellow. The structural integrity of (rac)-9 immediately upon dissolution was also confirmed by LC / MS comparison to the parent material. Images were obtained using an Olympus CK2 microscope with a 10X eyepiece and a 4X objective with a standard digital camera.

[0143] Racemization Evaluation

[0144] A portion of (R)-10 was dissolved in dimethylsulfoxide (DMSO) to make a 10mg / 100mL stock solution. Then, 10mL of the stock was diluted into 990mL of 37oC 1X PBS (pH 7.4) to give a final concentration of 1mg / mL in a manner similar to racemization studies known for thalidomide (Cossy et al., Comprehensive Chirality 2012, Vol.1, 1-7). The mixture was monitored for 5hr (twice the t1 / 2of thalidomide racemization) to assess for evidence of racemization. Chiral HPLC were obtained on a Shimadzu LC-20 system, and using Chiralpak®HPLC columns as indicated and using isocratic conditions.

[0145] Biological Methods

[0146] Growth and maintenance of RAW 264.7 cells: RAW 264.7 cells were purchased from ATCC (Manassas, VA, USA). The cells were grown in Dulbecco’s modified eagle medium (DMEM) media (Invitrogen, DMEM, high glucose, GlutaMAX®Supplement (Life Technologies Corporation, Carlsbad, CA), pyruvate, #10569-010) supplemented with 10% FBS (Invitrogen, Fetal Bovine Serum, qualified, heat inactivated, #16140-071) with penicillin and streptomycin (Invitrogen, Penicillin-Streptomycin (10, 000 U / mL), #15140-122) and were maintained at 37˚C and 5% CO2. The cells were grown to 75% - 85% confluence on 10 cm plates (Corning Falcon, #353003) following the protocol described by ATTC.

[0147] Drug assessment of anti-inflammatory actions in LPS activated RAW 264.7 cells: Cells were seeded into black 24 well plates (PerkinElmer Black VisiplateTMTC # 1450- 605 at a density of 250 x 103cells per well. Twenty-four hours after seeding the plate the seeding media was replaced with fresh media. Two hours later the cells were exposed to a range of concentrations of drug test-compound(s) dissolved in 100% tissue culture grade dimethyl sulfoxide (DMSO, SIGMA #D2650). The effects of each concentration of drug were assessed in 4 wells per concentration in the 24 well plate (n = 4). On each plate one set of wells were assigned as drug-vehicle control (i.e. DMSO+LPS), one test compound was assessed on one 24-well plate. The drug concentration used for each test agent were 1, 10, or 30 μM. One hour after the drugs were added the cells were challenged with lipopolysaccharide (LPS, SIGMA, serotype 055:B5) at a final concentration (10 ng / ml). Twenty to twenty-four hours after the cells were challenged with LPS the culture media was collected and utilized for the measurement of markers of inflammation. Fresh media was added to the cells and then the cells underwent the assessment of cell viability.

[0148] Cellular viability assessment of drug treated RAW 264.7 cells: RAW 264.7 cell viability was determined by use of a fluorescent cell viability assay, the CellTiter-Blue Cell Viability assay following the protocol recommended by the manufacturer, (Cat # G8081, Promega, Madison. WI). In brief, the assay mixture was added to the cell culture wells containing the RAW 264.7 cells. The cells were returned to the incubator and after 25 to 30 minutes the cell viability was assessed by reading the fluorescence signals. The plate was read with an excitation at 560 nm and the emission was read at 590 nm using an Infinite M200 PRO plate reader (TECAN, USA). The relative fluorescence unit (RFU) levels from the different drug treatments were compared to those of the control wells (DMSO+LPS controls) and the RFUs were then converted to relative percentage change of control levels for each compound.

[0149] Nitrite ion detection in drug treated RAW 267.4 cell culture media: To measure the levels of nitrite ion (NO2-) we utilized the Nitrate / Nitrite Fluorometric Assay Kit (Abnova, Cat # KA1344). The protocol followed was slightly different to that recommended by the manufacturer; in brief a NO2- ion standard curve was prepared in culture media of the same composition to that used for the cell culture study. The concentration range of the standard curve was from 0.156 µM to 10 µM. Equal volumes of the standards and unknown samples were added to black 96 well plates in duplicate (Corning # 3925). Then 2,3- diaminonapthalene (DAN, 50 µg / ml in 0.62 M HCl) was added to each well, the plate was covered and incubated in the dark for 10 min. After this NaOH (2.8 N) was added to each well and the RFUs / NO2- levels were assessed. The plate was read with an excitation at 362 nm and an emission at 430 nm (Infinite M200 PRO plate reader, TECAN, USA). The raw RFU data for the standards and unknowns were used to calculate the NO2- levels (µM) using linear regression analysis in GraphPad Prism 8.3.1 (GraphPad Prism). NO2- levels were then converted to relative percentage change of control levels for each set of drug treatments (DMSO+LPS controls).

[0150] Enzyme-linked immunosorbent assay for TNF-α protein: Media TNF-α and IL-6 levels were measured by use of the Biolegend ELISA MAX Set Delux ELISA kits (TNF-α #430904; IL-6 # 431315). The protocol used was that recommended by the manufacturer. In brief, a day prior to performing the assay, a 96 well plate was coated with a capture antibody directed against TNF-α / IL-6. The following day, the assay plate was washed and blocked with the kit blocking agent for 1 h while being mixed on a plate shaker at 200 rpm. A TNF- α / IL-6 protein standard curve was prepared in the assay diluent following the kit instructions,and unknown media samples were likewise diluted in the same assay diluent. After blocking, the plate was washed and then both the standards and unknown media samples were added to the plate in duplicate. The standards and unknown samples were incubated for 2h. After the incubation, the plate was washed, and then the biotin labeled detection antibody was added to each well and incubated for a further 1h. After this incubation, the plate was washed and the avidin-HRP conjugate complex was added to each well for 30 min. The plate was washed and the chromogenic substrate 3,3’,5,5’-tetramethylbenzidine solution was added. At this point the plate was covered and incubated in the dark for 15 min, with no mixing. After 15 min, the chromogenic reaction was stopped by the addition of 2 N H2SO4and the absorbance was read at 450 nm and for background subtractions at 570 nm, on a SPECTRAmax PLUS plate reader. The absorbances were used to generate a TNF-α / IL-6 protein standard curve and the protein levels in the unknown samples were determined using SoftMax Pro V5, Molecular Devices. The TNF-α / IL-6 levels were then converted to relative percentage change of control levels for each set of drug treatments (DMSO+LPS controls).

[0151] Statistical analyses for RAW 264.7 cell assays: Data are expressed as a percentage change from the DMSO-vehicle control measurements. Measurements are expressed as mean ± standard error, where the n number represents the number of wells in a 24 well plate. Statistical comparisons were undertaken by use GraphPad Prim 8.3.1. Initially data were assessed for normality by use of the Shapiro-Wilk test, then significant outliers were assessed by use of the ROUT test, if any outliers were detected they were removed. Significantly different changes in biological variables induced by t test compounds were assessed by use of ANOVA (Ordinary one-way ANOVA or the Kruskal-Wallis test).

[0152] PDSP Studies: Samples were submitted to the NIMH PDSP as powdered samples in the provided vials. The materials were then prepared at the PDSP and subjected to multiple assay screens and processed as per the PDSP Assay Protocol Book (Version 3, March 2018). Compounds that exceeded an initial 10uM binding threshold were then delegated for secondary dose-response analysis.

[0153] Cereblon Binding Studies: Cereblon Binding studies were performed at the Reaction Biology Company using their Cereblon Binding Assay Service (Human Cereblon / DDB1 complex) with Bodipy- or Cy5-labeled thalidomide probes (https: / / www.reactionbiology.com / datasheet / cereblon_protac_malvern / ).

[0154] Example 1 – Chemical Syntheses

[0155] The classical thalidomide / pomalidomide skeleton was altered by eliminating the two carbonyl groups from the glutarimide portion. The resulting piperidine moiety retained a 6- membered formation and the relative positioning of the nitrogen atom while potentially providing attenuated acidity of the epimerizable proton.

[0156] The syntheses began by condensing 3-nitrophthalic anhydride (5) with a variety of 3- aminopiperidines to yield their corresponding nitrophthalimide intermediates (FIG.1). Sixteen (piperidin-3-yl)isoindoline materials through four distinct branches of the synthetic tree were prepared and described in detail below. In the secondary amine route (a), the use of the carboxybenzyl (CBz) protecting group facilitated a one-pot simultaneous deprotection and nitro reduction, providing the archetypical free piperidine materials in moderate yield. The (R)-7 and (S)-8 isomers were obtained from the analogous optically pure starting materials. N-alkylated materials were obtained from the appropriate Boc-protected alkyl piperidine as shown in pathway (b); the acidic conditions and elevated temperature utilized for the condensation seemed to promote Boc removal, and the resulting nitrophthalimide products were obtained in a convenient one-pot process. Subsequent reduction provided N- methyl, N-ethyl, and N-isopropyl compounds 9-17 which included racemic and enantiopure forms. Sulfonamide 18 was readily accessible while tertiary amides 19-21 were generated in a similar fashion and stemming from the reciprocal acylations and reduction using pathways (c) and (d).

[0157]

[0158] 4-amino-2-(piperidin-3-yl)isoindoline-1,3-dione (6). The racemic form of this material can be prepared by combining equivalent quantities of the corresponding pure (R)-7 and (S)-8 isomeric forms. A chiral HPLC analysis confirmed the racemate (48:52); ChiralHPLC; 12 / 88 isopropanol / hexanes (iPrOH / Hex), Chiralpak®AD, 4.6mmx250mm, 10 μm, 1mg / mL, 1ml / min. HRMS (ESI) calc for [C13H15N3O2+ H]+246.1237, found 246.1232.

[0159]

[0160] 4-amino-2-(piperidin-3-yl)isoindoline-1,3-dione (7) A 250ml round bottom flask was charged with sodium acetate (5.30g, 64.5mmol, 5.0eq), 3-nitrophthalic anhydride (2.50g, 12.9mmol, 1.0eq) and (R)-benzyl 3-aminopiperidine-1-carboxylate (3.03g, 12.9 mmol, 1.0eq). 50mL of acetic acid was added, and a reflux condenser was affixed. The mixture was stirred at 130°C overnight. Then, the reaction was cooled to RT and the solvents were removed in vacuo. The residue was combined with toluene and shaken briefly before being evaporated again to help remove traces of acetic acid. The residue was then combined with ~10mL of 10% Na2CO3 and extracted repeatedly with ethyl acetate (EtOAc). The combined organic layers were washed with brine, dried over Na2SO4and evaporated to give a crude product that was used directly in the next step and without further purification. The material was transferred to a Parr flask and combined with methanol. Under a stream of argon, a small scoop of 10% Pd / C was added, and the mixture was hydrogenated at 50psi until consumption ceased. The catalyst was subsequently filtered off, and the solvents were evaporated. The resulting residue was combined with a few mL of triethylamine and extracted with EtOAc against a minimal volume of water. The combined organics were washed with brine, dried over Na2SO4 and evaporated. The residue was purified by flash chromatography using 10% methanol (MeOH) / 1% triethylamine (TEA) / EtOAc. Isolated 184 mg of a yellow solid, 6% yield for all steps.1H-NMR (400 MHz, DMSO-D6) δ 7.37 (t, J = 7.7 Hz, 1H), 6.92-6.87 (m, 2H), 6.40 (s, 2H), 3.93-3.87 (m, 1H), 3.01 (t, J = 11.5 Hz, 1H), 2.77 (dd, J = 21.5, 12.1 Hz, 2H), 2.32 (t, J = 12.4 Hz, 1H), 2.23 (bs, 1H, exchanges with D2O), 2.16 (m, 1H), 1.70-1.62 (m, 2H), 1.38 (d, J = 12.6 Hz, 1H).13C-NMR (101 MHz, DMSO-D6) δ 169.4, 167.8, 146.2, 135.0, 132.1, 121.2, 110.5, 108.6, 48.6, 48.3, 45.4, 28.0, 26.7.1H-qNMR (400 MHz, DMSO-D6) prepared with 8.17mg analyte and 10.34mg 1,2,4,5- tetrachloro-3-nitrobenzene (TCNB) TraceCERT std; 95.76 + / - 0.19%. HRMS (ESI) calc for [C13H15N3O2+ H]+246.1237, found 246.1232 Chiral HPLC; 12 / 88 iPrOH / Hex, Chiralpak®AD, 4.6mmx250mm, 10μm, 1mg / mL, 1mL / min; >99% enantiomeric excess (ee).

[0161]

[0162] (S)-4-amino-2-(piperidin-3-yl)isoindoline-1,3-dione (8) A 250ml round bottom flask was charged with sodium acetate (6.35g, 77.4mmol, 6.0eq), 3-nitrophthalic anhydride (2.50g, 12.9mmol, 1.0eq) and (S)-benzyl 3-aminopiperidine-1-carboxylate hydrochloride (3.50g, 12.9mmol, 1.0eq). 50mL of acetic acid was added, and a reflux condenser was affixed. The mixture was stirred at 130°C overnight. Then, the reaction was cooled to RT and the solvents were removed in vacuo. The residue was combined with toluene and shaken briefly before being evaporated again to help remove traces of acetic acid. The residue was then combined with ~10mL of 10% Na2CO3 and extracted repeatedly with EtOAc. The combined organic layers were washed with brine, dried over Na2SO4and evaporated to give a crude product that was used directly in the next step and without further purification. The material was transferred to a Parr flask and combined with methanol. Under a stream of argon, a small scoop of 10% Pd / C was added, and the mixture was hydrogenated at 50psi until consumption ceased. The catalyst was subsequently filtered off, and the solvents were evaporated. The resulting residue was combined with a few mL of triethylamine and extracted with EtOAc against a minimal volume of water. The combined organics were washed with brine, dried over Na2SO4 and evaporated. The residue was purified by flash chromatography using 10% MeOH / 1% TEA / EtOAc. Isolated 146mg of a yellow solid, 5% yield, total, for all steps.1H-NMR (400 MHz, DMSO-D6) δ 7.43-7.39 (m, 1H), 6.97-6.92 (m, 2H), 6.44 (s, 2H), 3.97-3.92 (m, 1H), 3.06 (t, J = 11.5 Hz, 1H), 2.82 (dd, J = 21.2, 12.3 Hz, 2H), 2.39-2.17 (m, 3H, with 1H exchanging with D2O), 1.71 (t, J = 16.7 Hz, 2H), 1.43 (d, J = 12.1 Hz, 1H).13C-NMR (101 MHz, DMSO-D6) δ 169.5, 168.1, 146.3, 135.1, 132.1, 121.3, 110.5, 108.8, 48.6, 48.3, 45.4, 28.1, 26.7.1H-qNMR (400 MHz, DMSO-D6) prepared with 4.66mg analyte and 11.08mg TCNB TraceCERT std; 95.33 + / - 0.19%. HRMS (ESI) calc for [C13H15N3O2+ H]+246.1237, found 246.1232 Chiral HPLC; 12 / 88 iPrOH / Hex, Chiralpak®AD, 4.6mmx250mm, 10μm, 1mg / mL, 1mL / min; >99% ee.

[0163]

[0164] 4-amino-2-(1-methylpiperidin-3-yl)isoindoline-1,3-dione (9) The racemic form of this material can be prepared by combining equivalent quantities of the corresponding pure (R)-10 and (S)-11 isomeric forms. A chiral HPLC analysis confirmed the racemate (50:50); Chiral HPLC; 20 / 80 iPrOH / Hex, Chiralpak AD, 4.6mmx250mm, 10um, 1mg / mL, 1ml / min. HRMS (ESI) calc for [C14H17N3O2+ H]+260.1394, found 260.1389.

[0165]

[0166] (R)-4-amino-2-(1-methylpiperidin-3-yl)isoindoline-1,3-dione (10) A 100 ml round bottom flask was charged with 3-nitrophthalic anhydride (1.0g, 4.66mmol, 1.0eq) and tert- butyl (R)-(1-methylpiperidin-3-yl)carbamate (1.0g, 4.66mmol, 1.0eq). 20mL of acetic acid was added, and a reflux condenser was affixed. The mixture was stirred at 130°C overnight. Then, the reaction was cooled to RT and the solvents were removed in vacuo. The residue was then combined with water and extracted repeatedly with EtOAc. The combined organic layers were washed with saturated NaHCO3, water, brine, then dried over Na2SO4 and evaporated to give a crude product that was used directly in the next step and without further purification. The material was transferred to a Parr flask and combined with methanol and a few drops of sulfuric acid. Under a stream of argon, a small scoop of 10% Pd / C was added, and the mixture was hydrogenated at 50psi until consumption ceased. The catalyst was subsequently filtered off, and the solvents were evaporated. The resulting residue was combined with a few mL of triethylamine and extracted with EtOAc against a minimal volume (~5mL) of water. The organic layer was isolated, then washed once with saturated NaHCO3, water, brine, and then was finally dried over Na2SO4 and evaporated to give a yellow solid which was purified by repeated recrystallization from acetone, providing 115mg of a bright yellow powder, 10% yield, total, for all steps.1H-NMR (400 MHz, DMSO-D6) δ 7.42 (t, J = 7.7 Hz, 1H), 6.97-6.92 (m, 2H), 6.46 (s, 2H), 4.11-4.04 (m, 1H), 2.76-2.67 (m, 2H), 2.47 (m, 1H, DMSO overlap), 2.19 (s, 3H), 2.09-1.99 (m, 1H), 1.84-1.66 (m, 3H), 1.57- 1.48 (m, 1H).13C-NMR (101 MHz, DMSO-D6) δ 169.3, 167.9, 146.3, 135.0, 132.0, 121.2, 110.5, 1087.6, 57.1, 54.6, 47.3, 45.8, 26.4, 24.6.1H-qNMR (400 MHz, DMSO-D6) prepared with 4.40mg analyte and 10.43mg TCNB TraceCERT std; 95.6 + / - 0.19%. HRMS (ESI) calc for [C14H17N3O2+ H]+260.1394, found 260.1389. Chiral HPLC; 20 / 80 iPrOH / Hex, Chiralpak AD, 4.6mmx250mm, 10μm, 1mg / mL, 1ml / min; >99% ee.

[0167]

[0168] (S)-4-amino-2-(1-methylpiperidin-3-yl)isoindoline-1,3-dione (11) A 100 ml round bottom flask was charged with 3-nitrophthalic anhydride (2.5g, 12.9mmol, 1.1eq) and tert- butyl (S)-(1-methylpiperidin-3-yl)carbamate (2.5g, 11.6 mmol, 1.0eq). 50mL of acetic acid was added, and a reflux condenser was affixed. The mixture was stirred at 130°C overnight. Then, the reaction was cooled to RT and the solvents were removed in vacuo. The residue was then combined with a few mL of triethylamine and water, then extracted repeatedly with EtOAc. The combined organic layers were washed with 10% Na2CO3and brine, dried over Na2SO4 and evaporated to give a crude product that was used directly in the next step and without further purification. 1.0g of the material was transferred to a Parr flask and combined with methanol. Under a stream of argon, a small scoop of 10% Pd / C was added, and the mixture was hydrogenated at 50psi until consumption ceased. The catalyst was subsequently filtered off, and the solvents were evaporated. The resulting residue was combined with a few mL of triethylamine and extracted with EtOAc against a minimal volume of water. The combined organics were washed with saturated NaHCO3, brine, dried over Na2SO4and evaporated. Repeated recrystallizations from acetone gave 126mg of a bright yellow solid, 14% yield (based on 1.0g starting material).1H-NMR (400 MHz, DMSO-D6) δ 7.42 (t, J = 7.7 Hz, 1H), 6.98-6.92 (m, 2H), 6.45 (s, 2H), 4.07 (m, 1H), 2.75- 2.67 (m, 2H), 2.45 (m, J = 10.8 Hz, 1H), 2.19 (s, 3H), 2.04 (m, 1H), 1.81-1.67 (m, 3H), 1.53 (m, 1H).13C-NMR (101 MHz, DMSO-D6) δ 169.3, 167.8, 146.3, 135.0, 132.0, 121.2, 110.4, 108.6, 57.1, 54.6, 47.3, 45.8, 26.4, 24.5.1H-qNMR (400 MHz, DMSO-D6) prepared with 4.75mg analyte and 2.07mg EC TraceCERT std; 99.20 + / - 0.46%. HRMS (ESI) calc for [C14H17N3O2+ H]+260.1394, found 260.1389. Chiral HPLC; 20 / 80 iPrOH / Hex, Chiralpak AD, 4.6mmx250mm, 10μm, 1mg / mL, 1ml / min; >99% ee.

[0169]

[0170] 4-amino-2-(1-methylpiperidin-3-yl)isoindoline-1,3-dione (12) The racemic form of this material can be prepared by combining equivalent quantities of the corresponding pure (R)-13 and (S)-14 isomeric forms. A chiral HPLC analysis confirmed the racemate (51:49);Chiral HPLC; 8 / 92 iPrOH / Hex, Chiralpak OD, 4.6mmx250mm, 10μm, 1mg / mL, 1ml / min. HRMS (ESI) calc for [C15H19N3O2+ H]+274.1550, found 274.1545.

[0171]

[0172] (R)-4-amino-2-(1-ethylpiperidin-3-yl)isoindoline-1,3-dione (13) A 100 ml round bottom flask was charged with benzyl (R)-piperidin-3-ylcarbamate (2.5g, 10.6mmol, 1.0eq) and potassium carbonate (2.19g, 15.9mmol, 1.5eq). Acetonitrile (25mL) was added, and the mixture was cooled to 0°C. A solution of Iodoethane (0.94mL, 11.7mmol, 1.1eq) in 2.5mL acetonitrile was added dropwise, and the mixture was allowed to stir and gradually warm to RT overnight (similar processes are described in WO 2022 / 036204 A1). Then, the solids were filtered off and washed with acetonitrile. The liquor was isolated and evaporated. The resulting residue was taken up in 20mL of 6M HCl and heated to 100°C overnight. After cooling, the solvent was removed under reduced pressure to give a tan residue which was further suspended in 30mL acetic acid and combined with 3-nitrophthalic anhydride (2.04g, 10.6mmol, 1.0eq) in a 100mL round bottom flask affixed with a reflux condenser. The mixture was heated at 130°C overnight. After cooling, the acetic acid was removed under reduced pressure, and the residue was resuspended in methanol in a Parr flask. Under a stream of argon, a small scoop of 10% Pd / C was added along with a few drops of sulfuric acid. The resulting mixture was hydrogenated at 50psi until consumption ceased. After filtering off the catalyst, the liquor was evaporated, and the resulting residue was extracted into ethyl acetate, washed with 10% Na2CO3and brine, dried over Na2SO4and evaporated to give a bright yellow foamy solid. The material was further purified by flash chromatography over silica using 5% MeOH / 1% TEA / EtOAc to give 356mg of a yellow solid, 13% total, for all steps.1H-NMR (400 MHz, DMSO-D6) δ 7.36 (dd, J = 8.2, 7.1 Hz, 1H), 6.93-6.87 (m, 2H), 6.41 (s, 2H), 4.05-3.97 (m, 1H), 2.80-2.71 (m, 2H), 2.47-2.23 (m, 3H), 2.09-1.98 (m, 1H), 1.80-1.63 (m, 3H), 1.51-1.40 (m, 1H), 0.93 (t, J = 7.1 Hz, 3H).13C-NMR (101 MHz, DMSO-D6) δ 170.0, 168.5, 147.0, 135.6, 132.6, 121.8, 111.1, 109.3, 55.4, 52.9, 52.2, 48.1, 27.8, 25.2, 12.6.1H-qNMR (400 MHz, DMSO-D6) prepared with 8.48mg analyte and 11.03mg EC TraceCERT std; 96.8 + / - 0.23%. HRMS (ESI) calc for [C15H19N3O2+ H]+274.1550, found 274.1545. Chiral HPLC; 8 / 92 iPrOH / Hex, Chiralpak OD, 4.6mmx250mm, 10μm, 1mg / mL, 1ml / min; >99% ee.

[0173]

[0174] (S)-4-amino-2-(1-ethylpiperidin-3-yl)isoindoline-1,3-dione (14) A 100 ml round bottom flask was charged with benzyl (S)-piperidin-3-ylcarbamate (2.5g, 10.6mmol, 1.0eq) and potassium carbonate (2.19g, 15.9mmol, 1.5eq). Acetonitrile (25mL) was added, and the mixture was cooled to 0°C. A solution of Iodoethane (0.94mL, 11.7mmol, 1.1eq) in 2.5mL acetonitrile was added dropwise, and the mixture was allowed to stir and gradually warm to RT overnight. Then, the solids were filtered off and washed with acetonitrile. The liquor was isolated and evaporated. The resulting residue was taken up in 20mL of 6M HCl and heated to 100°C overnight. After cooling, the solvent was removed under reduced pressure to give a tan residue which was further suspended in 30mL acetic acid and combined with 3- nitrophthalic anhydride (2.04g, 10.6mmol, 1.0eq) in a 100mL round bottom flask affixed with a reflux condenser. The mixture was heated at 130°C overnight. After cooling, the acetic acid was removed under reduced pressure, and the residue was resuspended in methanol in a Parr flask. Under a stream of argon, a small scoop of 10% Pd / C was added along with a few drops of sulfuric acid. The resulting mixture was hydrogenated at 50psi until consumption ceased. After filtering off the catalyst, the liquor was evaporated, and the resulting residue was extracted into ethyl acetate, washed with 10% Na2CO3 and brine, dried over Na2SO4and evaporated to give a bright yellow foamy solid. The material was further purified by flash chromatography over silica using 5% MeOH / 1% TEA / EtOAc to give 141mg of a yellow solid, 5% total, for all steps.1H-NMR (400 MHz, DMSO-D6) δ 7.42 (t, J = 7.7 Hz, 1H), 6.97-6.92 (m, 2H), 6.46 (s, 2H), 4.09-4.03 (m, 1H), 2.86-2.78 (m, 2H), 2.48- 2.31 (m, 3H), 2.12-2.03 (m, 1H), 1.83-1.68 (m, 3H), 1.55-1.49 (m, 1H), 0.99 (t, J = 7.2 Hz, 3H).13C-NMR (101 MHz, DMSO-D6) δ 170.0, 168.5, 147.0, 135.6, 132.6, 121.8, 111.1, 109.2, 55.3, 52.9, 52.2, 48.1, 27.7, 25.2, 12.5.1H-qNMR (400 MHz, DMSO-D6) prepared with 9.45mg analyte and 10.97mg EC TraceCERT std; 97.3 + / - 0.23%. HRMS (ESI) calc for [C15H19N3O2+ H]+274.1550, found 274.1545. Chiral HPLC; 8 / 92 iPrOH / Hex, Chiralpak OD, 4.6mmx250mm, 10μm, 1mg / mL, 1ml / min; >97% ee.

[0175]

[0176] 4-amino-2-(1-isopropylpiperidin-3-yl)isoindoline-1,3-dione (15) The racemic form of this material can be prepared by combining equivalent quantities of the corresponding pure (R)-16 and (S)-17 isomeric forms. A chiral HPLC analysis confirmed the racemate (47:53); Chiral HPLC; 5 / 95 iPrOH / Hex, Chiralpak OD, 4.6mmx250mm, 10μm, 1mg / mL, 1ml / min. HRMS (ESI) calc for [C16H21N3O2+ H]+288.1707, found 288.1702.

[0177]

[0178] (R)-4-amino-2-(1-isopropylpiperidin-3-yl)isoindoline-1,3-dione (16) A Parr flask was charged tert-butyl (R)-piperidin-3-ylcarbamate (2.5g, 12.5mmol, 1.0eq). Under a stream of argon, a small scoop of platinum oxide (PtO2) was added, followed by 100mL of acetone and a few drops of acetic acid. The system was hydrogenated at 50psi until consumption ceased. The catalyst was filtered off, and the liquor was evaporated to give a light clear- yellow oil. The oil was resuspended in 50mL of acetic acid and combined with 3- nitrophthalic anhydride (2.41g, 12.5mmol, 1.0eq) in a 250mL round bottom flask affixed with a reflux condenser. The mixture was heated at 130°C overnight. After cooling, the acetic acid was removed under reduced pressure, and the residue was combined with ethyl acetate and extracted with saturated NaHCO3, brine, then dried over Na2SO4 and finally evaporated to give a crude residue. Under a stream of argon, the material was transferred to a Parr flask and combined with a small scoop of 10% Pd / C, methanol, and a few drops of sulfuric acid. The mixture was hydrogenated at 50psi until consumption ceased. The catalyst was subsequently filtered off, and the solvents were evaporated. The resulting residue was combined with EtOAc and extracted against saturated NaHCO3, brine, and then dried over Na2SO4and evaporated. The residue was purified by flash chromatography over silica gel, using 5% MeOH / 1% TEA / EtOAc to give 348mg of a yellow solid, 10% yield, total, for all steps).1H-NMR (400 MHz, DMSO-D6) δ 7.41 (dd, J = 8.2, 7.1 Hz, 1H), 6.98-6.92 (m, 2H), 6.46 (s, 2H), 4.07-4.01 (m, 1H), 2.73-2.67 (m, 4H), 2.11-2.04 (m, 2H), 1.74-1.68 (m, 2H), 1.48-1.45 (m, 1H), 0.95 (d, J = 6.6 Hz, 6H).13C-NMR (101 MHz, DMSO-D6) δ 170.0,168.5, 146.9, 135.5, 132.6, 121.7, 111.0, 109.2, 54.3, 51.0, 48.8, 48.4, 28.0, 25.6, 18.6, 18.2.1H-qNMR (400 MHz, DMSO-D6) prepared with 13.31mg analyte and 16.08mg EC TraceCERT std; 98.5 + / - 0.23%. HRMS (ESI) calc for [C16H21N3O2+ H]+288.1707, found 288.1702. Chiral HPLC; 5 / 95 iPrOH / Hex, Chiralpak OD, 4.6mmx250mm, 10μm, 1mg / mL, 1ml / min; >98% ee.

[0179]

[0180] (S)-4-amino-2-(1-isopropylpiperidin-3-yl)isoindoline-1,3-dione (17) A Parr flask was charged tert-butyl (S)-piperidin-3-ylcarbamate (2.5g, 12.5mmol, 1.0eq). Under a stream of argon, a small scoop of platinum oxide (PtO2) was added, followed by 100mL of acetone and a few drops of acetic acid. The system was hydrogenated at 50psi until consumption ceased. The catalyst was filtered off, and the liquor was evaporated to give a light clear- yellow oil. The oil was resuspended in 50mL of acetic acid and combined with 3- nitrophthalic anhydride (2.41g, 12.5mmol, 1.0eq) in a 250mL round bottom flask affixed with a reflux condenser. The mixture was heated at 130°C overnight. After cooling, the acetic acid was removed under reduced pressure, and the residue was combined with ethyl acetate and extracted with saturated NaHCO3, brine, then dried over Na2SO4and finally evaporated to give a crude residue. Under a stream of argon, the material was transferred to a Parr flask and combined with a small scoop of 10% Pd / C, methanol, and a few drops of sulfuric acid. The mixture was hydrogenated at 50psi until consumption ceased. The catalyst was subsequently filtered off, and the solvents were evaporated. The resulting residue was combined with EtOAc and extracted against saturated NaHCO3, brine, and then dried over Na2SO4and evaporated. The residue was purified by flash chromatography over silica gel, using 5% MeOH / 1% TEA / EtOAc to give 489mg of a yellow solid, 14% yield, total, for all steps.1H-NMR (400 MHz, DMSO-D6) δ 7.40 (dd, J = 8.1, 7.2 Hz, 1H), 6.96-6.90 (m, 2H), 6.44 (s, 2H), 4.02 (tt, J = 11.5, 3.8 Hz, 1H), 2.72-2.65 (m, 4H), 2.11-2.03 (m, 2H), 1.73-1.66 (m, 2H), 1.50-1.43 (m, 1H), 0.93 (d, J = 6.6 Hz, 6H).13C-NMR (101 MHz, DMSO-D6) δ 170.0, 168.5, 146.9, 135.5, 132.6, 121.7, 111.0, 109.2, 54.3, 51.0, 48.8, 48.4, 28.0, 25.6, 18.6, 18.2.1H-qNMR (400 MHz, DMSO-D6) prepared with 12.60mg analyte and 10.64mg EC TraceCERT std; 96.4 + / - 0.23%. HRMS (ESI) calc for [C16H21N3O2+ H]+288.1707, found288.1702. Chiral HPLC; 5 / 95 iPrOH / Hex, Chiralpak OD, 4.6mmx250mm, 10μm, 1mg / mL, 1ml / min; 99% ee.

[0181]

[0182] 4-amino-2-(1-(methylsulfonyl)piperidin-3-yl)isoindoline-1,3-dione (18) A 50 ml round bottom flask was charged with 3-nitrophthalic anhydride (0.295g, 1.53 mmol, 1.1eq), sodium acetate (0.345g, 4.21 mmol, 3.0eq), and 1-(methylsulfonyl)piperidin-3-amine (0.250g, 1.40 mmol, 1.0eq). 10mL of acetic acid was added, and a reflux condenser was affixed. The mixture was stirred at 130°C overnight. Then, the reaction was cooled to RT and the solvents were removed in vacuo. The residue was transferred to a Parr flask and, under a stream of argon, combined with a small scoop of 10% Pd / C, methanol, and a few drops of sulfuric acid. The mixture was hydrogenated at 50psi until consumption ceased. The catalyst was subsequently filtered off, and the solvents were evaporated. The resulting residue was combined with EtOAc and extracted against saturated NaHCO3, brine, and then dried over Na2SO4and evaporated. The residue was purified by flash chromatography over silica gel, using 5% MeOH / 1% TEA / EtOAc to give 116 mg of a yellow powder, 26% yield, total, for all steps.1H-NMR (400 MHz, DMSO-D6) δ 7.39 (d, J = 7.1 Hz, 1H), 6.95-6.91 (m, 2H), 6.44 (s, 2H), 4.04 (m, 1H), 3.55 (d, J = 10.3 Hz, 2H), 3.16 (t, J = 11.0 Hz, 1H), 2.87 (s, 3H), 2.64 (t, J = 11.9 Hz, 1H), 2.19 (m, 1H), 1.87-1.78 (m, 2H), 1.56 (m, 1H).13C-NMR (101 MHz, DMSO-D6) δ 169.9, 168.2, 147.2, 135.7, 132.3, 121.7, 111.2, 108.9, 47.7, 47.5, 45.8, 35.1, 27.1, 24.9.1H-qNMR (400 MHz, DMSO-D6) prepared with 4.06mg analyte and 10.09mg TCNB TraceCERT std; 98.65 + / - 0.19%. HRMS (ESI) calc for [C14H17N3O4S + H]+324.1013, found 324.1007.

[0183]

[0184] 4-nitro-2-(piperidin-3-yl)isoindoline-1,3-dione (S-1) A 250mL round bottom flask was fitted with a reflux condenser and charged with 3-nitrophthalic anhydride (2.41g, 12.5mmol, 1.0eq), tert-butyl 3-aminopiperidine-1-carboxylate (2.5g, 12.5mmol, 1.0eq), and 50mL of toluene. The mixture was refluxed overnight. After cooling to RT, the solvent wasremoved under reduced pressure, and the resulting off-white solid was used without further purification. Crude 1H-NMR revealed a mixture of rotamers that resolved upon heating. In general, a portion (~2g) of the crude solid was suspended in 10mL of dichloromethane, cooled to 0°C, and then treated with trifluoracetic acid (5ml) dropwise. The mixture was stirred and allowed to warm to RT. When the reaction was complete, the solvents were carefully removed under reduced pressure, and the residue was briefly extracted into ethyl acetate, washed with 10% Na2CO3and brine, dried over Na2SO4and evaporated to give a crude pale-yellow solid (S-1) that was used immediately in subsequent reactions and without further purification.

[0185]

[0186] 2-(1-acetylpiperidin-3-yl)-4-aminoisoindoline-1,3-dione (19) A 100mL round bottom flask was charged with 4-nitro-2-(piperidin-3-yl)isoindoline-1,3-dione (S-1) (1.0g, 3.63 mmol, 1.0eq) and 50mL of dichloromethane. Then, acetic anhydride (0.38mL, 3.99mmol, 1.1eq) was added, followed by triethylamine (0.76mL, 5.45 mmol, 1.5eq). The mixture was stirred for 1hr and then extracted twice with saturated NaHCO3, then once each with water then brine. The organics were dried over Na2SO4 and evaporated to give a crude white solid. Under a stream of argon, the material was transferred to a Parr flask and combined with a small scoop of 10% Pd / C, 100 mL methanol, and a few drops of sulfuric acid. The mixture was hydrogenated at 50psi until consumption ceased. The catalyst was subsequently filtered off, and the solvents were evaporated. The resulting residue was combined with a few mL of triethylamine and extracted with EtOAc against a minimal volume (~5mL) of water. The organic layer was isolated, then washed once with 5mL of 10% Na2CO3, an additional 5mL of water, brine, and then was finally dried over MgSO4 and evaporated. The residue was purified by flash chromatography over silica gel, using 5% MeOH / 1% TEA / EtOAc to give 483mg, 46% yield of a bright yellow powder.1H-NMR (400 MHz, DMSO-D6) δ 7.43 (t, J = 7.7 Hz, 1H), 6.98-6.94 (m, 2H), 6.45 (s, 2H), 4.40 (m, 1H), 3.85 (m, 2H), 3.59 (m, 0.4H), 3.03 (m, 1H), 2.42 (m, 0.12H, obscured by DMSO), 2.26 (d, J = 12.4 Hz, 1H), 2.03 (s, 1.7H), 1.97 (s, 1.3H), 1.79 (m, 2H), 1.53-1.36 (m, 1H).13C-NMR (101 MHz, DMSO-D6) δ 169.1 , 168.1, 168.0, 167.7, 146.3, 146.1, 135.0, 132.0, 121.1, 110.5, 108.4, 48.0, 47.3, 46.5, 45.6, 42.9, 40.7, 27.5, 25.1, 24.2, 21.2. The material appearsto exist as a mixture of rotamers. The DMSO sample was subsequently heated to 80°C at which point many of the peaks in question coalesced.1H-qNMR (400 MHz, DMSO-D6) prepared with 4.75mg analyte and 10.12mg TCNB TraceCERT std; 99.63 + / - 0.19%. HRMS (ESI) calc for [C15H17N3O3+ H]+288.1343, found 288.1337.

[0187]

[0188] 4-amino-2-(1-(2,2,2-trifluoroacetyl)piperidin-3-yl)isoindoline-1,3-dione (20) A 25mL round bottom flask was charged with 4-nitro-2-(piperidin-3-yl)isoindoline-1,3-dione (S-1) (0.250g, 0.91 mmol, 1.0eq) and 5mL of dichloromethane. The mixture was cooled to 0°C. Triethylamine was added (0.15mL, 1.09mmol, 1.2eq) followed by trifluoroacetic anhydride (0.14mL, 1.0mmol, 1.1eq), dropwise. The mix was stirred to room temperature over the course of 1hr, at which time the solvents were removed in vacuo. The residue was extracted into ethyl acetate and washed briefly with saturated NaHCO3 and then brine. After drying over Na2SO4, the solvents were evaporated and the residue was combined in a Parr flask with methanol, a few drops of sulfuric acid, and a small scoop of 10% Pd / C (under a stream of argon) and the mix was hydrogenated at 50psi until consumption ceased. The catalyst was subsequently filtered off, and the solvents were evaporated. The resulting residue was combined with EtOAc and extracted against saturated NaHCO3, brine, and then dried over Na2SO4and evaporated. The residue was purified by recrystallization from acetone to give 109mg of a pale yellow powder, 35% yield, total, for all steps.1H-NMR (400 MHz, DMSO-D6) δ 7.46-7.42 (m, 1H), 7.00-6.95 (m, 2H), 6.48 (s, 2H), 4.36-4.33 (m, 1H), 4.07-3.75 (m, 2.5H), 3.45 (t, J = 12.0 Hz, 0.5H), 3.28-3.21 (m, 0.5H), 2.86 (s, 0.5H), 2.37 (td, J = 12.9, 3.7 Hz, 1H), 1.94-1.87 (m, 2H), 1.60-1.54 (m, 1H).13C-NMR (101 MHz, DMSO- D6) δ 169.2, 169.1, 167.8, 167.7, 146.6, 146.5, 135.2, 135.1, 132.2, 132.1, 121.4, 121.3, 120.7, 120.6, 117.9, 117.8, 115.0, 114.9, 110.7, 110.7, 108.8, 108.7, 47.2, 47.2, 47.1, 46.2, 45.5, 45.5, 45.0, 43.3, 27.2, 27.1, 25.0, 23.9.19F-NMR (377 MHz, DMSO-D6) δ -67.5, -67.7. These spectra are complicated by extensive fluorine coupling, and additionally, the material appears to exist as a mixture of rotamers. The DMSO sample was heated to 100°C, and the 19F spectrum was monitored at which point the peaks in question coalesced.1H-qNMR (400MHz, DMSO-D6) prepared with 7.95mg analyte and 12.77mg TCNB TraceCERT std; 96.2 + / - 0.19%. HRMS (ESI) calc for [C15H14F3N3O3+ H]+342.1060, found 342.1054.

[0189]

[0190] 4-amino-2-(1-(cyclopropanecarbonyl)piperidin-3-yl)isoindoline-1,3-dione (21) A 250mL round bottom flask was charged with 4-nitro-2-(piperidin-3-yl)isoindoline-1,3-dione (S-1) (1.0g, 3.63 mmol, 1.0eq) and 100mL of ethyl acetate. Then, cyclopropanecarbonyl chloride (0.33mL, 3.63mmol, 1.0eq) was added, followed by triethylamine (0.56mL, 3.99 mmol, 1.1eq). The mixture was stirred for 30min at RT and then extracted with saturated NaHCO3, then once each with water then brine. The organics were dried over Na2SO4 and evaporated to give a crude off-white solid. Under a stream of argon, the material was transferred to a Parr flask and combined with a small scoop of 10% Pd / C, 100 mL of 50 / 50 methanol / ethyl acetate, and one drop of sulfuric acid. The mixture was hydrogenated at 50psi until consumption ceased. The catalyst was subsequently filtered off, and the solvents were evaporated. The resulting residue was combined with a few mL of triethylamine and extracted with EtOAc against a minimal volume (~5mL) of water. The combined organics were washed with brine and was finally dried over Na2SO4and evaporated to give a yellow oily reside. The material was recrystallized from acetone to give 127mg of a yellow solid, 11% yield, total, for all steps.1H-NMR (400 MHz, DMSO-D6) δ 7.43 (t, J = 7.7 Hz, 1H), 6.99-6.94 (m, 2H), 6.48 (s, 2H), 4.38-4.28 (m, 2H), 3.90 (m, 1H), 3.64 (t, J = 12.3 Hz, 0.5H), 3.18-2.90 (m, 1H), 2.31 (qd, J = 12.6, 3.8 Hz, 1H), 2.01-1.75 (m, 3H), 1.52-1.39 (m, 1H), 0.72 (m, 4H). The material appears to exist as a mixture of rotamers. The DMSO sample was subsequently heated to 90°C at which point many of the peaks in question coalesced.13C-NMR (101 MHz, DMSO-D6) δ 171.1, 169.3, 167.8, 146.5, 135.1, 132.1, 121.4, 110.6, 108.7, 47.8, 47.2, 46.6, 45.0, 43.9, 41.8, 27.7, 25.6, 24.4, 10.4, 6.9.1H-qNMR (400 MHz, DMSO-D6) prepared with 7.17mg analyte and 10.62mg TCNB TraceCERT std; 97.6 + / - 0.19%. HRMS (ESI) calc for [C17H19N3O3+ H]+314.1499, found 314.1496.

[0191] Example 2 – Compound Characterization

[0192] N-methylated compounds (rac)- 9 and (R)-10 were evaluated in initial solubility and configurational stability analyses. These compounds were selected as offering a mix of polarity and alkyl character. Initially, a simple cheminformatic analysis (Chemicalize, January 2023 – March 2024, developed by ChemAxon, Budapest, Hungary) of 9 revealed a desirable CNS MPO (central nervous system multiparameter optimization desirability) score (Table 2), one built upon seemingly favorable ClogP and ClogD parameters, yet perhaps slightly perturbed from the classical, archetypical phthalimidoglutarimide. Table 2

[0193] A set of experiments was subsequently devised to quickly gauge the pertinent and biologically-relevant aqueous solubility of 9 relative to thalidomide and pomalidomide. Samples of these agents were weighed into tared vials, and an appropriate amount of PBS (pH 7.4) was added to bring the final titer of each to a theoretical concentration of 850µΜ, a value 10 times the established solubility of pomalidomide (85µΜ) and roughly 14-fold that of thalidomide (62µΜ) (Min et al., Angew. Chemie Int. Ed.2021, 60(51):26663-26670). The (rac)-9 was visibly more soluble, with the corresponding mixture yielding a bright yellow homogenous solution after just a few minutes of stirring. Conversely, the thalidomide and pomalidomide samples remained largely precipitated, a result consistent with their known aqueous solubilities.

[0194] To probe the configurational stability, a solution of (R)-10 was subjected to biologically-relevant conditions known to racemize optically pure thalidomide (Cossy, Comprehensive Chirality 2012, Vol.1, 1-7). Briefly, (R)-10 was dissolved in PBS buffer (pH 7.4) at a concentration of 1 mg / mL. The ensuing process was monitored by chiral HPLC, and over the course of a few analogous thalidomide half-lives (a 5-hour timeline), no discernable racemization to (S)-isomer 11 was observed (FIGS.2A-2E), thereby suggesting the material to be more robust under these conditions and timepoints.

[0195] Example 3 – Effects on Cellular Inflammation

[0196] To evaluate activity against classical markers of cellular inflammation, the LPS- activated RAW 264.7 cellular system was selected, an assay platform well-suited for identifying small-molecule regulators of nitrite (a stable and widely accepted marker of nitric oxide related inflammation) and pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α,) and interleukin-6 (IL-6) (Tweedie et al., The open biochemistry journal 2011, 5:37- 44). Compounds were evaluated 1 μM, 10 μM, and 30 μM. The biological and anti- inflammatory activities are shown in Table 3 and FIGS.3A-3D. Table 3 summarizes drug effects on classical markers of cellular inflammation and viability, with the numbers in parentheses referring to the numbered bars in FIGS.3A-3D. Where appropriate, values are presented as the mean % change of the indicated parameter (relative to the control) ± the standard error of the mean. Values are presented as mean ± S.E.M. of n observations (n=4). Significance markers are * P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001. From these initial observations, it was clear that the compounds were well tolerated, with all entries maintaining or exceeding 90% viability. In fact, cellular viability seemed largely unperturbed, approaching 100% in most cases; the only materials that appeared to harbor even slight toxicity (between 90% and 95% viability) were higher-order alkyl materials 14, 15, and 16, suggesting that an increase in steric bulk at the piperidine nitrogen might be somewhat detrimental to cell survival.Table 3

[0197] Attention then turned to the key pro-inflammatory markers of interest, of which modest decreases of the various reporters were observed for multiple compounds. In general, the compounds were broadly and moderately active against nitrite, with 11 of the 16 candidates demonstrating some degree of nitrite-lowering ability over the concentrations screened. Compounds 13 and 19 reduced nitrite in a dose-dependent manner (and up to 20% of basal amounts) while 11 appeared to effect roughly 30% reductions, seemingly independent of concentration. Of particular note, the secondary amino cohort including rac- 6, (R)-7, and (S)-8 all demonstrated a pronounced dose-dependent trend in nitrite reduction, with (R)-7 showing the most significant effect. Interestingly, it appears the stereochemistry may play a key role, as the racemate 6 exhibited an intermediate activity between (R)-7, and (S)-8.

[0198] While nitrite appeared adequately responsive to the compounds, TNF-α did not seem as susceptible, as the only notable decreases were observed for 19 which abated levels by roughly 15% relative to basal mounts at both 10 µM, and 30µM. Smaller reductions were noted for a few materials, with (R)-7 effecting decreases of approximately 5% and 9% at 10 µM, and 30µM, respectively. While TNF-α is a valuable gauge of LPS-induced cellular inflammation, it has a propensity to be somewhat less responsive in the cellular assay than the other markers typically screened. Therefore, the cytokine exploration was expanded, and the compounds were subjected to IL-6 evaluation. This target proved to be much more susceptible, with rac-6 and (R)-7 demonstrating very apparent and dose-dependent reductions in secreted IL-6. Interestingly, the stereochemical effects mirrored those seen for nitrite.Meanwhile, 9, 18, and 19 notably increased IL-6 relative to basal amounts at the majority of concentrations screened; the only attenuation here was observed for 19 which demonstrated an approximate 10% decrease at 30µM only.

[0199] Example 4 – NIMH Psychoactive Drug Screening Program Evaluation

[0200] Several compounds were selected for additional screening at the National Institute of Mental Health (NIMH) Psychoactive Drug Screening Program (PDSP) (Besnard et al., Nature 2012, 492(7428):215-220). Compounds 6, 9, 18, and 19 were selected as offering the best combination of anti-inflammatory properties and structurally-diverse chemical architecture, thereby casting an appropriately wide net for any potential compounds. As seen in FIGS.4A-4E, these compounds demonstrated an interesting and focused scope of binding with regards to the numerous CNS targets screened at the PDSP. Although 18 did not elicit any appreciable interactions (all Ki >10 µM), compounds 6, 9, and 19 provided some notable results. All three compounds showed similar low micromolar binding to the sigma-2 (σ-2) receptor, with Ki of 2.2µM, 6.3µM, and 5.2µM, respectively (FIG.4A). While 19 displayed additional binding at the related sigma-1 (σ-1) receptor (Ki=2.4 µM, FIG.4B), compounds 6 and 9 appeared selective for σ-2 in this respect. The remainder of the notable hits converged on serotonin receptors, specifically 5-HT2A, 5-HT2B, and 5-HT3 (FIGS.4C-4E). Compound 6 appeared to be the most potent, with Ki of 561nM at 5-HT2Band 535nM at 5-HT3. Compound 9 effected a 1.1µM interaction at 5-HT2B while 19 demonstrated 5.2µM Ki for 5- HT2A.

[0201] Example 5 – Cereblon Binding Evaluation

[0202] A human Cereblon (CRBN) binding screen (Reaction Biology Corporation, Cereblon Binding Assay Service) of compounds 6, 9, 18, and 19 revealed no compelling or dose- dependent binding within the concentrations investigated (FIG.5). In general, a bound Cy5- tagged thalidomide-like probe was displaced from the Cereblon-DDB1 complex with the indicated ligand. Any ligand interaction was monitored through a subsequent change in fluorescence polarization activity. The classical CRBN ligand thalidomide clearly effects significant activity decreases while our compounds 6, 9, 18, and 19 maintained elevated levels of bound probe activity across all concentrations.

[0203] Discussion

[0204] RAW cell assays do not illuminate the specific mode of anti-inflammatory action. However, as a classical phenotypic assay, the platform offers an expedient depiction of general inflammation that is agnostic to underlying mechanisms. It cannot, for instance, differentiate between a NOS inhibitor and a simple nitrite-sequestering agent. In this overly- simplified regard, both avenues could present as nitrite reductions concurrent with uniformly high cell viability. When considering the target-based PDSP binding results in the context of the screened markers, consideration should be given to whether the receptors translate in RAW cells, and if so, to what extent they are implicated in RAW cell inflammatory responses.

[0205] Multiple examples are found of the sigma and serotonin receptors having direct influence on all of the classical markers of cellular inflammation, and in a wide array of analysis platforms. For instance, sigma receptor binding has been implicated in the regulation of both pro- and anti-inflammatory cytokines including TNF-α, IL-2, IL-6, and IL- 10, and has been similarly associated with attenuation of NOS and COX activity as well as processes linked to transcription factors NF-κB and NFAT, and additionally in general cellular degeneration (Lizama et al., Intl J of Molecular Sciences 2023, 24(7):6251; Yi et al., J of Neurochemistry 2017, 140(4):561-575; Iñguez et al., Frontiers in Pharmacology 2013, 4:23; Malar et al., CNS Drugs 2023, 37(5):399-440; wang et al., Scientific Reports 2022, 12(1):20753). Such effects have been observed in human neuroblastomas, leukemic T-type lymphocytes, and retinal ganglion cells. Other studies have noted the neuroprotective properties of σ-2 receptor modulators observed after controlled cortical impact TBI in rodent models (Vázquez-Rosa et al., ACS Chemical Neuroscience 2019, 10(3):1595-1602). Another pertinent study revealed that nitric oxide synthesis was reduced in LPS-stimulated RAW murine macrophages in response to small molecule ligation with sigma receptors (Gannon et al., Surgical Infections 2001, 2(4):267-273). In regards to serotonin receptors, there has been ample evidence demonstrating the roles that 5-HT2A and 5-HT2B play in inflammatory processes; the small-molecule modulation of such receptors has been linked to reductions in TNF-α mediated inflammation in lung cells as well as IL-6 decreases in vascular smooth muscle, and ROS and IL-6 attenuations in epithelial cells (Yang et al., Intl Immunopharmacology 2020, 81:106036; Nau et al., PLoS One 2013, 8(10):e75426; Robinson et al., Current Issues in Molecular Biology 2023, 45(8):6743-6774; Ito et al., Circulation 2000, 102(20:2522-2527). -HT2A activation specifically has been shown to inhibit iNOS inglioma cells (Miller et al., Annals of the New York Academy of Sciences 1998, 861:169-173), while its blockade has been linked to increased nitric oxide production in canine models of ischemic heart dysfunction (Fujita et al., J of Molecular and Cellular Cardiology 2004, 37(6):1219-1223). 5-HT2B has also been directly tied to oxidative stress, with its blockade preventing ROS-induced cardiac hypertrophy in mice; conversely, its activation has been directly coupled to constitutive NOS and iNOS activity concurrent with increased nitric oxide production (Manivet et al., The J of Biological Chemistry 2000, 275(13):9324-9331; Monassier et al., Hypertesion (Dallas, Tex. : 1979) 2008, 52(2):301-307). In a similar fashion, small molecule antagonists of 5-HT3have been shown to inhibit production of TNF- α, IL-6, and iNOS-derived nitric oxide (Graeff et al., Central Nervous System Agents in Medicinal Chemistry 2010, 10(3):207-217; Kato et al., Biological & Pharmaceutical Bulletin 2013; 36(9):1406-1409; Molderings et al., Naunyn-Schmiedeberg’s Archives of Pharmacology 1996, 354(3):245-252).

[0206] The synthesis, characterization, and biological evaluation of a family of new 4- amino-2-(piperidin-3-yl)isoindoline-1,3-diones is disclosed. The compounds were well tolerated in the biological assays, with all compounds maintaining >90% cellular viability across all concentration ranges tested, with most compounds greatly exceeding a 95% threshold. The few instances of attenuated viability seemed to coincide, roughly, with expanded alkyl bulk at the piperidine nitrogen (14, 15, and 16) and were coincident with elevated levels of secreted TNF-α and nitrite at 30µM dosing. The secondary amino cohort including rac-6, (R)-7, and (S)-8 rendered the most pronounced dose-dependent reductions in nitrite and IL-6. When dosed at 30µM, (R)-7 demonstrated the most compelling effects, with decreases of 32% and 40% for nitrite and IL-6, respectively. Interestingly, the racemate 6 exhibited an intermediate activity between (R)-7, and (S)-8, suggesting that the stereochemistry may play a key role in the observed biological activities, possibly involving a piperidine N-H hydrogen bond. Four representative compounds were further evaluated against numerous CNS receptors, channels, and transporters, with 6, 9, and 19 demonstrating varying degrees of nanomolar-to-low-micromolar binding to the σ-1and σ-2 receptors and also to serotonin receptors 5HT2A, 5HT2Band 5HT3. In this regard, 6 displayed perhaps the strongest affinities, with binding at σ-2 (Ki = 2.2µM), 5HT2B (Ki = 561nM) and 5HT3 (Ki = 536nM). Further short-term studies in rats will be performed. These studies will evaluate the degree to which the cell-based anti-inflammatory activities described above translate in vivowithin animals challenged with a systemic dose of LPS (Lecca et al., Alzheimers Dement 2022, 18(11):2327-2340).

[0207] Example 6 – Mouse microglial cells (IMG cells)

[0208] About 250,000 mouse microglial cells (IMG cells) were treated with the test compound and then one hour later were challenged with LPS (10 ng / mL). The assays were performed 24 hours after the challenge. FIG.6 provides the results from treatment with concentrations of 1 μM, 10 μM, 30 μM, and 60 μM of compound 7 in the top graph, and compound 19 in the lower graph, with CNT indicating the untreated control group in each case.

[0209] The cell viability assay (MTS assay) demonstrated that all cells were alive. That is, both compound 7 and compound 19 were well tolerated and did not cause observable cell loss.

[0210] The other assays (nitrate, TNF-α, and IL-6) demonstrated a concentration-dependent drug-induced decline in proinflammatory cytokines. This indicated that both compounds exhibited anti-inflammatory action.

[0211] In view of the many possible embodiments to which the principles of the disclosure may be applied, it should be recognized that the illustrated embodiments are only preferred examples of the disclosure and should not be taken as limiting the scope of the invention. Rather, the scope of the invention is defined by the following claims. We therefore claim as our invention all that comes within the scope and spirit of these claims.

Claims

We claim:

1. A compound according to Formula I, or a stereoisomer or pharmaceutically acceptable salt, solvate, or hydrate thereof:where R1is -H, -N(R′)(R′′), -NO2, -OH, -ORb, -SH, -SRb, -CN, C2-C6 alkenyl, C2-C6 alkynyl, or halo , wherein R′ and R′′ independently are -H, C1-C3alkyl, C3-C6cycloalkyl, or - C(O)Rb; R2is -H, Ra, -C(O)Rb, -C(O)ORb, -S(O)2Rb, C2-C6alkenyl, C2-C6alkynyl, or -C(O)N(Rc)(Rd); R3is -H, halo, C1-C4alkyl, C2-C6alkenyl, C2-C6alkynyl, -C(O)ORb, or - C(O)N(Rc)(Rd); each R4independently is halo, -OH, Rb, -ORb, -SH, SRb, C2-C6alkenyl, C2-C6alkynyl, or -C(O)ORb; R5-R8are each independently -H, halo, Rb, C2-C6alkenyl, C2-C6alkynyl, or -C(O)ORb; Rais alkyl, haloalkyl, cycloalkyl, heterocycloalkyl, or monocyclic heteroaryl; Rbis alkyl, haloalkyl, or cycloalkyl; Rcand Rdindependently are -H or C1-C4alkyl or Rcand Rdtogether with the N atom form a 5- or 6-membered heterocycloalkyl; Z1and Z2independently are C(H)2, C(O), or C(S), wherein at least one of Z1and Z2is C(O) or C(S); and n is 0, 1, 2, or 3, wherein (i) if R1is -NH2, then one of Z1and Z2is other than C(O), or n is not 0, or R2is not H, methyl, or ethyl, or R3is not methyl, or one of R5-R8is not -H, or (ii) if R1is -NO2, then one of Z1and Z2is other than C(O), or n is not 0, or R2is not ethyl, or R3is not H, or one of R5-R8is not -H, or(iii) if R1is halo, then one of Z1and Z2is other than C(O), or R2is not H, or R3is not H, or one of R5-R8is not -H, or n is 0 or 2 when R4is halo.

2. The compound of claim 1, wherein: R1is -N(R′)(R′′), -NO2, -OH, or halo, wherein R′ and R′′ independently are -H or C1-C3 alkyl; R2is -H, Ra, -C(O)Rb, -C(O)ORb, -S(O)2Rb, or -C(O)N(Rc)(Rd); R3is -H, halo, C1-C4 alkyl, -C(O)ORb, or -C(O)N(Rc)(Rd); each R4independently is halo, -OH, Rb, -ORb, or -C(O)ORb; and R5-R8independently are -H, halo, Rb, or -C(O)ORb.

3. The compound according to claim 1 or claim 2, where R1is -NH2.

4. The compound according to any one of claims 1-3, wherein R2is -H, methyl, e C5. The compound according to any one of claims 1-4, wherein R3is -H, halo, methyl, or -C(O)ORb.

6. The compound according to any one of claims 1-5, wherein n is 0.

7. The compound according to any one of claims 1-5, wherein n is 1 and R4is halo, -OH, -Rb, or -C(O)ORb.

8. The compound according to any one of claims 1-7, wherein: Z1and Z2are C(O); or Z1and Z2are C(S); or one of Z1and Z2is C(O) and the other of Z1and Z2is C(S).

9. The compound according to any one of claims 1-8, wherein: R5-R8are -H; or R5and R6are halo, and R7and R8are -H; or R5and R6are -H and R7and R8are halo; or one of R5-R8is Rbor -C(O)ORb, and the others of R5-R8are -H.

10. The compound according to any one of claims 5 or 7-9, where Rbis -CH3.

11. The compound according to any one of claims 1-10, wherein the compound is a pharmaceutically acceptable salt.

12. The compound according to claim 11, wherein the pharmaceutically acceptable salt is a hydrochloride salt.

13. The compound according to any one of claims 1-12, wherein the stereoisomer is an enantiomer, a rotamer, or an enantiomer and a rotamer of the compound according to Formula I.

14. The compound according to any one of claims 1-13, wherein an amount of the compound is a racemic mixture of (R)- and (S)-enantiomers.

15. The compound according to any one of claims 1-13, wherein an amount of the compound has an enantiomeric excess of at least 20% of the (R)-enantiomer or (S)- enantiomer.

16. The compound of claim 1, wherein the compound is:wherein enantiomers are present in an enantiomeric excess of at least 20% in an amount of the compound.

17. The compound of claim 1, wherein the compound is:pharmaceutically acceptable salt, solvate, or hydrate thereof.

18. The compound of claim 1, wherein the compound iswherein the stereoisomer has an enantiomeric excess of at least 20% in an amount of the compound.

19. A pharmaceutical composition, comprising a compound, or a stereoisomer, pharmaceutically acceptable salt, solvate, or a hydrate thereof, according to any one of claims 1-18; and a pharmaceutically acceptable carrier.

20. A method for inhibiting TNF-α activity, TNF-α synthesis, interleukin-6 (IL-6) level, inflammation, or SARS-COV-2 virus, comprising contacting a cell with an effective amount of a compound or a pharmaceutically acceptable salt, solvate, or hydrate thereof, according to any one of claims 1-18.

21. The method of claim 20, wherein contacting the cell with an effective amount of the compound or pharmaceutically acceptable salt, solvate, or hydrate thereof, comprises administering to a subject a therapeutically effective amount of the compound or pharmaceutically acceptable salt, solvate, or hydrate thereof, or a therapeutically effective amount of a pharmaceutical composition comprising the compound or pharmaceutically acceptable salt, solvate, or hydrate thereof.

22. The method of claim 21, wherein the subject has a disease or condition characterized at least in part by inflammation.

23. The method of claim 21 or claim 22, wherein the subject has a traumatic brain injury (TBI), an inflammatory disorder, a neurodegenerative disease, cancer, a SARS CoV-2 virus infection, or any combination thereof.

24. The method of claim 21, wherein the subject has a TBI, neuroinflammation, a SARS CoV 2 virus infection, Alzheimer’s Disease, Parkinson’s Disease, multiple sclerosis,amyotrophic lateral sclerosis, Huntington’s Disease, a spinal cord injury, a stroke, human immunodeficiency virus dementia, cerebral amyloid angiopathy, tauopathy, peripheral neuropathy, macular degeneration, hearing loss, cochlear injury, epilepsy, a non-epileptic seizure disorder, depression, rheumatoid arthritis, immune arthritis, degenerative arthritis, celiac disease, glomerulonephritis, lupus nephritis, prostatitis, inflammatory bowel disease, pelvic inflammatory disease, graft versus host disease, interstitial cystitis, autoimmune thyroiditis, Graves’ disease; autoimmune pancreatitis, Sjogren’s syndrome, myocarditis, autoimmune hepatitis, primary biliary cirrhosis, autoimmune angioedema, bullous pemphigoid, discoid lupus erythematosus, erythema nodosum leprosum, sarcoidosis, pemphigus vulgaris psoriasis, POEMS syndrome, polymyositis, human immune deficiency virus / acquired immune deficiency syndrome, vasculitis, sarcopenia, multiple myeloma, primary myelofibrosis, myelodysplastic syndrome, acute myeloid leukemia, chronic lymphocytic leukemia, hairy cell leukemia, large granular lymphocytic leukemia, non- Hodgkin’s lymphoma, Kaposi sarcoma, or any combination thereof.

25. The method of claim 21, wherein: the subject has a degenerative and / or inflammatory conditions of the eye, optionally selected from macular degeneration, retinitis pigmentosa, diabetic retinopathy, uvetitis / scleritis / keratitis, or Graves’ eye disease; or the subject has meningeal TB (tuberculosis); or the subject has noise-induced sensory hair cell loss and hearing loss.

26. A compound for use in a method of treating aberrantly high TNF-α activity, aberrantly high level of IL-6, inflammation, SARS-CoV-2 viral infection, or any combination thereof, the method comprising administering to a subject having aberrantly high TNF-α activity, aberrantly high level of IL-6, inflammation, SARS-CoV-2 viral infection, or any combination thereof a therapeutically effective amount of a compound or a pharmaceutically acceptable salt, solvate, or hydrate thereof, according to any one of claims 1-18 or a therapeutically effective amount of a pharmaceutical composition comprising the compound or pharmaceutically acceptable salt, solvate, or hydrate thereof.

27. The compound for use of claim 26, wherein the subject has a disease or condition characterized at least in part by inflammation.

28. The compound for use of claim 26, wherein the subject has a TBI, an inflammatory disorder, a neurodegenerative disease, cancer, or any combination thereof.

29. The compound for use of claim 26, wherein the subject has a TBI, neuroinflammation, a SARS CoV 2 virus infection, Alzheimer’s Disease, Parkinson’s Disease, multiple sclerosis, amyotrophic lateral sclerosis, Huntington’s Disease, a spinal cord injury, a stroke, human immunodeficiency virus dementia, cerebral amyloid angiopathy, tauopathy, peripheral neuropathy, macular degeneration, hearing loss, cochlear injury, epilepsy, a non-epileptic seizure disorder, depression, rheumatoid arthritis, immune arthritis, degenerative arthritis, celiac disease, glomerulonephritis, lupus nephritis, prostatitis, inflammatory bowel disease, pelvic inflammatory disease, graft versus host disease, interstitial cystitis, autoimmune thyroiditis, Graves’ disease; autoimmune pancreatitis, Sjogren’s syndrome, myocarditis, autoimmune hepatitis, primary biliary cirrhosis, autoimmune angioedema, bullous pemphigoid, discoid lupus erythematosus, erythema nodosum leprosum, sarcoidosis, pemphigus vulgaris psoriasis, POEMS syndrome, polymyositis, human immune deficiency virus / acquired immune deficiency syndrome, vasculitis, sarcopenia, multiple myeloma, primary myelofibrosis, myelodysplastic syndrome, acute myeloid leukemia, chronic lymphocytic leukemia, hairy cell leukemia, large granular lymphocytic leukemia, non-Hodgkin’s lymphoma, Kaposi sarcoma, or any combination thereof.

30. The compound for use of claim 26, wherein: the subject has a degenerative and / or inflammatory conditions of the eye, optionally selected from macular degeneration, retinitis pigmentosa, diabetic retinopathy, uvetitis / scleritis / keratitis, or Graves’ eye disease; or the subject has meningeal TB (tuberculosis); or the subject has noise-induced sensory hair cell loss and hearing loss.

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