BENZOFURAN DERIVATIVES FOR TARGETING AUTOPHAGY

Provided herein are compounds of formula (I), (II), and (III), or pharmaceutically acceptable salts, stereoisomers, or deuterated forms thereof, wherein X1, X2, X3, R1, R2, a, b, L0, L1, L2, and A are defined herein. Also provided herein are pharmaceutical compositions comprising a compound of formula (I), (II), or (III), or pharmaceutically acceptable salt, a stereoisomer, or deuterated form thereof, and methods of using a compound of formula (I), (II), or (III), or pharmaceutically acceptable salt, a stereoisomer, or deuterated form thereof, e.g., in the treatment of a disease or disorder by modulating autophagic degradation and/or p62 activity.

BACKGROUND

Autophagy is a major degradation pathway for maintaining cellular homeostasis. Autophagy eliminates unnecessary, aged, dysfunctional, or damaged intracellular components through lysosome-mediated degradation. The autophagy process can degrade bulky cellular cargoes (e.g., proteins aggregates, intracellular pathogens) that cannot be degraded by other processes, such as the ubiquitin-proteasome system (UPS).

During autophagy, cytoplasmic contents are delivered into the lysosomal system by double-membraned organelles called autophagosomes. Specifically, cytoplasmic material is sequestered into autophagosomes, which subsequently fuse with lysosomes where degradation occurs via the action of acidic lysosomal hydrolases. Autophagy helps to keep cells healthy, and dysregulation of this process can contribute to a wide range of diseases, including cancer, inflammation, neurodegeneration, and infectious diseases.

Sequestosome-1, also known as ubiquitin-binding protein p62 (SQSTM1 or p62, hereinafter “p62”), is a key autophagy receptor for targeted degradation. p62 operates as an autophagy adaptor that brings ubiquitinated substrates (e.g., damaged proteins) into contact with autophagosomes in preparation for autophagy. In addition to autophagic degradation, p62 also plays an important role in the UPS, cellular signaling, metabolism, and apoptosis.

There is a need to develop therapeutics that target p62 and modulate autophagy.

SUMMARY

or a pharmaceutically acceptable salt, stereoisomer, or deuterated form thereof, wherein:

In another aspect, the present disclosure provides a compound of formula (II)

or a pharmaceutically acceptable salt, stereoisomer, or deuterated form thereof, wherein:

In a further aspect, the present disclosure provides a compound of formula (III)

or a pharmaceutically acceptable salt, stereoisomer, or deuterated form thereof, wherein:

DETAILED DESCRIPTION

Targeted Protein degradation (TPD) has emerged as a promising modality in drug discovery, typically involving heterobifunctional molecules that combine a target binder with a degradation-inducing moiety (Békés, Miklós, David R. Langley, and Craig M. Crews. 2022. “PROTAC Targeted Protein Degraders: The Past Is Prologue.” Nature Reviews. Drug Discovery 21 (3): 181-200, incorporated herein by reference in its entirety). This approach is particularly attractive for targeting proteins previously considered undruggable. However, current TPD technologies, such as PROteolysis-TArgeting Chimeras (PROTACs), are primarily limited to inducing ubiquitination of target substrates for degradation. Despite its potential, PROTAC technology faces limitations due to challenges in forming substrate-PROTAC-E3 ligase complexes, restricting its application to a limited set of targets and E3 ligases.

Autophagy is a vital cellular process that manages the degradation and recycling of cellular components (Lamark, Trond, and Terje Johansen. 2021. “Mechanisms of Selective Autophagy.” Annual Review of Cell and Developmental Biology 37 (October): 143-69; Aman, Yahyah, Tomas Schmauck-Medina, Malene Hansen, Richard I. Morimoto, Anna Katharina Simon, Ivana Bjedov, Konstantinos Palikaras, et al. 2021. “Autophagy in Healthy Aging and Disease.” Nature Aging 1 (8): 634-50; each incorporated herein by reference in their entirety). It plays a crucial role in maintaining cellular homeostasis by removing damaged or dysfunctional organelles and proteins. The process of autophagy involves the formation of autophagosomes, which engulf cellular debris and fuse with lysosomes for degradation. This mechanism is essential for cellular health, and its dysfunction is associated with various diseases, including neurodegenerative disorders, cancers, and metabolic diseases. Autophagy can be broadly classified into macroautophagy, microautophagy, and chaperone-mediated autophagy, with each type serving specific functions in the cell.

Autophagy plays a dual role in disease pathology. While it can prevent the accumulation of toxic protein aggregates and damaged organelles, its dysregulation can also contribute to disease progression. In neurodegenerative diseases such as Alzheimer's, Parkinson's, and Huntington's disease, impaired autophagy leads to the accumulation of misfolded proteins, exacerbating neurodegeneration. In cancer, autophagy can have both tumor-suppressive and tumor-promoting roles, depending on the context. Enhancing or inhibiting autophagy has been explored as a therapeutic strategy in various diseases. For instance, autophagy inducers are being investigated for their potential to clear protein aggregates in neurodegenerative diseases, while autophagy inhibitors are being explored in certain cancers (Maiuri, Maria Chiara, and Guido Kroemer. 2019. “Therapeutic Modulation of Autophagy: Which Disease Comes First?” Cell Death and Differentiation 26 (4): 680-89, incorporated herein by reference in its entirety).

p62 is a multifunctional protein that plays a significant role in autophagy, particularly in selective autophagy (Kumar, Anita V., Joslyn Mills, and Louis R. Lapierre. 2022. “Selective Autophagy Receptor P62/SQSTM1, a Pivotal Player in Stress and Aging.” Frontiers in Cell and Developmental Biology 10 (February): 793328, incorporated herein by reference in its entirety). It acts as a link between LC3 (a protein associated with autophagosomes) and ubiquitinated substrates. p62 binds to ubiquitin-tagged proteins and aggregates, delivering them to autophagosomes for degradation. It is also involved in the formation of protein aggregates known as aggresomes, which are targeted for autophagic degradation. The regulation of p62 and its interaction with other autophagy-related proteins are critical for the efficient execution of selective autophagy.

p62 is a key autophagy adaptor involved in the autophagic degradation of ubiquitinated substrates. p62 also interacts with ATG8 proteins that are found on the surface of developing autophagosomes. As such, p62 enables selective degradation by directing ubiquitinated substrates to the growing autophagosomes. Oligomerization of individual p62 units (i.e., p62 oligomer) has been shown to provide a stronger interaction with autophagosome. In addition to autophagic degradation, p62 influences other cellular pathways and is associated with pathological conditions including neurodegenerative diseases and cancer.

The N-degron pathway recognizes specific N-terminal amino acids (N-degrons) of proteins for degradation (Varshavsky, Alexander. 2019. “N-Degron and C-Degron Pathways of Protein Degradation.” Proceedings of the National Academy of Sciences of the United States of America 116 (2): 358-66, incorporated herein by reference in its entirety). The Arginylation branch of this pathway utilizes Arg, Lys, His (type 1), and Phe, Tyr, Trp, Leu, Ile (type 2) as N-degrons. Recent discoveries have shown that the Arg/N-degron pathway mediates not only ubiquitylation-dependent proteasomal clearance but also macroautophagic protein degradation. In this process, p62/SQSTM1 acts as an N-recognin, binding type-1 and type-2 N-degrons via its ZZ domain, activating p62 into an autophagy-compatible form for efficient autophagosome biogenesis (Kwon, Do Hoon, Ok Hyun Park, Leehyeon Kim, Yang Ouk Jung, Yeonkyoung Park, Hyeongseop Jeong, Jaekyung Hyun, Yoon Ki Kim, and Hyun Kyu Song. 2018. “Insights into Degradation Mechanism of N-End Rule Substrates by P62/SQSTM1 Autophagy Adapter.” Nature Communications 9 (1): 3291; Cha-Molstad, Hyunjoo, Ji Eun Yu, Zhiwei Feng, Su Hyun Lee, Jung Gi Kim, Peng Yang, Bitnara Han, et al. 2017. “P62/SQSTM1/Sequestosome-1 Is an N-Recognin of the N-End Rule Pathway Which Modulates Autophagosome Biogenesis.” Nature Communications 8 (1): 1-17; each incorporated herein by reference in their entirety).

p62 (SQSTM1) and NBR1 are key autophagy receptors that mediate the selective degradation of ubiquitinated proteins by bridging cargo to the autophagic machinery. Both proteins share several conserved domains critical to their function, including the ZZ-type zinc finger domain (ZZ domain), a PB1 domain for oligomerization, a UBA domain for binding polyubiquitinated substrates, and an LC3-interacting region (LIR) essential for autophagosome recruitment.

A defining feature of p62 and NBR1 is their ability to bind LC3, a core autophagy component embedded in the autophagosome membrane. This interaction, driven by their LIR motifs, is crucial for targeting cargo to autophagosomes for subsequent lysosomal degradation. Oligomerization through the PB1 domain promotes the clustering of cargo, enhancing both LC3 binding and autophagosome formation. The ZZ domain further stabilizes this process by indirectly facilitating interactions with ubiquitinated substrates and upstream signalling proteins involved in autophagy initiation.

Given the high degree of conservation between p62 and NBR1, the designed warheads could potentially interact with both receptors, offering a broad mechanism for modulating selective autophagy.

This disclosure presents compositions and methods for the manipulation of the intrinsic autophagic pathway for the selective degradation of pathogenic proteins. The disclosure comprises novel heterobifunctional compounds designed to engage the autophagy adaptor protein p62/SQSTM1, thereby triggering its activation and subsequent assembly of autophagosomes. These chimeric molecules are composed of a targeting ligand (also referred to as a “protein binding component” or “PBC”), which exhibits high-affinity binding to designated pathogenic proteins, conjoined via a designed, flexible linker to a p62 activating moiety (also referred to as a “warhead”). This bifunctional architecture enables the precise orchestration of p62 oligomerization and spatial localization, thereby enhancing the sequestration of the targeted proteins within nascent autophagosomes. The utility of this inventive approach lies in its capacity to harness the cell's autophagic machinery, thereby offering a therapeutic modality with broad-spectrum applicability in the attenuation of diseases characterized by aberrant protein accumulation or defective protein clearance.

Throughout this disclosure, various patents, patent applications and publications are referenced. The disclosures of these patents, patent applications and publications in their entireties are incorporated into this disclosure by reference for all purposes in order to more fully describe the state of the art as known to those skilled therein as of the date of this disclosure. This disclosure will govern in the instance that there is any inconsistency between the patents, patent applications and publications cited and this disclosure.

Definitions

Listed below are definitions of various terms used in the specification and claims to describe the present disclosure.

Unless defined otherwise, all technical and scientific terms used in this disclosure have the same meanings as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

The term “about” when immediately preceding a numerical value means a range encompassing said numerical value plus or minus an acceptable amount of variation in the art (e.g., plus or minus 10% of that value). For example, “about 50” can mean 45 to 55, “about 25,000” can mean 22,500 to 27,500, etc., unless the context of the disclosure indicates otherwise, or is inconsistent with such an interpretation. For example in a list of numerical values such as “about 49, about 50, about 55, . . . ”, “about 50” means a range extending to less than half the interval(s) between the preceding and subsequent values, e.g., more than 49.5 to less than 50.5. Furthermore, the phrases “less than about” a value or “greater than about” a value should be understood in view of the definition of the term “about” provided herein. Similarly, the term “about” when preceding a series of numerical values or a range of values (e.g., “about 10, 20, 30” or “about 10-30”) refers, respectively to all values in the series, or the endpoints of the range.

The terms below, as used herein, have the following meanings, unless indicated otherwise:

“Cyano” refers to the —CN radical.

“Hydroxy” or “hydroxyl” refers to the —OH radical.

“Oxo” refers to the ═O substituent.

“Alkyl” or “alkyl group” refers to a fully saturated, straight or branched hydrocarbon chain radical having from one to twelve carbon atoms, and which is attached to the rest of the molecule by a single bond. Alkyls comprising any number of carbon atoms from 1 to 50 are included. An alkyl comprising up to 50 carbon atoms is a C1-C50 alkyl, an alkyl comprising up to 24 carbon atoms is a C1-C24 alkyl, an alkyl comprising up to 12 carbon atoms is a C1-C12 alkyl, an alkyl comprising up to 10 carbon atoms is a C1-C10 alkyl, an alkyl comprising up to 6 carbon atoms is a C1-C6 alkyl and an alkyl comprising up to 5 carbon atoms is a C1-C5 alkyl. A C1-C5 alkyl includes C5 alkyls, C4 alkyls, C3 alkyls, C2 alkyls and C1 alkyl (i.e., methyl). A C1-C6 alkyl includes all moieties described above for C1-C5 alkyls but also includes C6 alkyls. A C1-C10 alkyl includes all moieties described above for C1-C5 alkyls and C1-C6 alkyls, but also includes C7, C8, C9 and C10 alkyls. Similarly, a C1-C12 alkyl includes all the foregoing moieties, but also includes C11 and C12 alkyls. Non-limiting examples of C1-C12 alkyl include methyl, ethyl, n-propyl, i-propyl, sec-propyl, n-butyl, i-butyl, sec-butyl, t-butyl, n-pentyl, t-amyl, n-hexyl, n-heptyl, n-octyl, n-nonyl, n-decyl, n-undecyl, and n-dodecyl. Unless stated otherwise specifically in the specification, an alkyl group can be optionally substituted.

“Alkylene” or “alkylene chain” refers to a fully saturated, straight or branched divalent hydrocarbon chain radical, and having from 1 to 50 carbon atoms. Non-limiting examples of C2-C50 alkylene include ethylene, propylene, n-butylene, ethenylene, propenylene, n-butenylene, propynylene, n-butynylene, and the like. The alkylene chain is attached to the rest of the molecule through a single bond and to the radical group through a single bond. The points of attachment of the alkylene chain to the rest of the molecule and to the radical group can be through one carbon or any two carbons within the chain. Unless stated otherwise specifically in the specification, an alkylene chain can be optionally substituted. Non-limiting examples of substituted alkylene include —CH(CH3)—, —CH2—CH(CH3)—, and the like.

“Alkenylene” or “alkenylene chain” refers to a straight or branched divalent hydrocarbon chain radical, having from 2 to 25 carbon atoms, and having one or more carbon-carbon double bonds. Non-limiting examples of C2-C25 alkenylene include ethene, propene, butene, and the like. The alkenylene chain is attached to the rest of the molecule through a single bond and to the radical group through a single bond. The points of attachment of the alkenylene chain to the rest of the molecule and to the radical group can be through one carbon or any two carbons within the chain. Unless stated otherwise specifically in the specification, an alkenylene chain can be optionally substituted.

“Alkynyl” or “alkynyl group” refers to a straight or branched hydrocarbon chain radical having from 2 to 25 carbon atoms, and having one or more carbon-carbon triple bonds. Each alkynyl group is attached to the rest of the molecule by a single bond. Alkynyl group comprising any number of carbon atoms from 2 to 25 are included. An alkynyl group comprising up to 25 carbon atoms is a C2-C25 alkynyl, an alkynyl comprising up to 10 carbon atoms is a C2-C10 alkynyl, an alkynyl group comprising up to 6 carbon atoms is a C2-C6 alkynyl and an alkynyl comprising up to 5 carbon atoms is a C2-C5 alkynyl. A C2-C5 alkynyl includes C5 alkynyls, C4 alkynyls, C3 alkynyls, and C2 alkynyls. A C2-C6 alkynyl includes all moieties described above for C2-C5 alkynyls but also includes C6 alkynyls. A C2-C10 alkynyl includes all moieties described above for C2-C5 alkynyls and C2-C6 alkynyls, but also includes C7, C8, C9 and C10 alkynyls. Similarly, a C2-C12 alkynyl includes all the foregoing moieties, but also includes C11 and C12 alkynyls. Non-limiting examples of C2-C25 alkynyl include ethynyl, propynyl, butynyl, pentynyl and the like. Unless stated otherwise specifically in the specification, an alkynyl group can be optionally substituted.

“Alkynylene” or “alkynylene chain” refers to a straight or branched divalent hydrocarbon chain radical, having from 2 to 25 carbon atoms, and having one or more carbon-carbon triple bonds. Non-limiting examples of C2-C25 alkynylene include ethynylene, propargylene and the like. The alkynylene chain is attached to the rest of the molecule through a single bond and to the radical group through a single bond. The points of attachment of the alkynylene chain to the rest of the molecule and to the radical group can be through one carbon or any two carbons within the chain. Unless stated otherwise specifically in the specification, an alkynylene chain can be optionally substituted.

“Alkoxy” refers to a radical of the formula —ORa where Ra is an alkyl, alkenyl or alkynyl radical as defined above containing one to twelve carbon atoms. Unless stated otherwise specifically in the specification, an alkoxy group can be optionally substituted.

“Hydroxyalkyl” refers to an alkyl radical, as defined above, that is substituted by one or more hydroxy groups. As used herein, the term “hydroxyalkyl” encompasses alkyls having a primary (terminal) hydroxy group, such as —CH2OH, —CH2CH2OH, —CH2CH2CH2OH, —CH2CH(CH3)CH2OH, and —CH2CH2CH2CH2OH, those having branched (non-terminal) hydroxy groups, such as —CH(OH)CH3, —CH2CH(CH3)OH, and those having both primary and branched hydroxy groups, such as —CH2CH(OH)CH2CH2OH.

“Alkylamino” refers to a radical of the formula —NHRa or —NRaRa where each Ra is, independently, an alkyl, alkenyl or alkynyl radical as defined above containing one to twelve carbon atoms. Unless stated otherwise specifically in the specification, an alkylamino group can be optionally substituted.

“Aryl” refers to a hydrocarbon ring system radical comprising hydrogen, 6 to 18 carbon ring atoms and at least one aromatic ring. For purposes of this disclosure, the aryl radical can be a monocyclic, bicyclic, tricyclic or tetracyclic ring system, which can include fused, bridged, or spiro ring systems. Aryl radicals include, but are not limited to, aryl radicals derived from aceanthrylene, acenaphthylene, acephenanthrylene, anthracene, azulene, benzene, chrysene, fluoranthene, fluorene, as-indacene, s-indacene, indane, indene, naphthalene, phenalene, phenanthrene, pleiadene, pyrene, and triphenylene. Unless stated otherwise specifically in the specification, the term “aryl” is meant to include aryl radicals that are optionally substituted.

“Aralkyl” or “arylalkyl” refers to a radical of the formula —Rb—Rc where Rb is an alkylene group as defined above and Rc is one or more aryl radicals as defined above, for example, benzyl, diphenylmethyl and the like. Unless stated otherwise specifically in the specification, an arylalkyl group can be optionally substituted.

“Carbocycle,” “carbocyclyl,” or “carbocyclic ring” or refers to a ring structure, wherein the atoms which form the ring are each carbon. Carbocyclic rings can comprise from 3 to 20 carbon atoms in the ring. Carbocyclic rings include cycloalkyl, cycloalkenyl and cycloalkynyl as defined herein. Unless stated otherwise specifically in the specification, a carbocyclyl group can be optionally substituted.

“Cycloalkyl” refers to a stable non-aromatic monocyclic or polycyclic fully saturated hydrocarbon radical consisting solely of carbon and hydrogen atoms, which can include fused, bridged, or spiro ring systems, having from three to twenty carbon atoms, e.g., having from three to ten carbon atoms, and which is attached to the rest of the molecule by a single bond. Monocyclic cycloalkyl radicals include, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. Polycyclic cycloalkyl radicals include, for example, adamantyl, norbornyl, decalinyl, 7,7-dimethyl-bicyclo[2.2.1]heptanyl, and the like. Unless otherwise stated specifically in the specification, a cycloalkyl group can be optionally substituted.

“Cycloalkylene” refers to a divalent, non-aromatic, and fully saturated monocyclic or polycyclic hydrocarbon ring having 3 to 20 carbon atoms, or 3 to 8 carbon atoms. Non-limiting examples of C3-8 cycloalkylene include

“Cycloalkenyl” refers to a stable non-aromatic monocyclic or polycyclic hydrocarbon radical consisting solely of carbon and hydrogen atoms, having one or more carbon-carbon double bonds, which can include fused, bridged, or spiro ring systems, having from three to twenty carbon atoms, e.g., having from three to ten carbon atoms, and which is attached to the rest of the molecule by a single bond. Monocyclic cycloalkenyl radicals include, for example, cyclopentenyl, cyclohexenyl, cycloheptenyl, cycloctenyl, and the like. Polycyclic cycloalkenyl radicals include, for example, bicyclo[2.2.1]hept-2-enyl and the like. Unless otherwise stated specifically in the specification, a cycloalkenyl group can be optionally substituted.

“Cycloalkynyl” refers to a stable non-aromatic monocyclic or polycyclic hydrocarbon radical consisting solely of carbon and hydrogen atoms, having one or more carbon-carbon triple bonds, which can include fused, bridged, or spiro ring systems, having from three to twenty carbon atoms, e.g., having from three to ten carbon atoms, and which is attached to the rest of the molecule by a single bond. Monocyclic cycloalkynyl radicals include, for example, cycloheptynyl, cyclooctynyl, and the like. Unless otherwise stated specifically in the specification, a cycloalkynyl group can be optionally substituted.

“Cycloalkylalkyl” refers to a radical of the formula —Rb—Rd where Rb is an alkylene, alkenylene, or alkynylene group as defined above and Rd is a cycloalkyl, cycloalkenyl, cycloalkynyl radical as defined above. Unless stated otherwise specifically in the specification, a cycloalkylalkyl group can be optionally substituted.

“Haloalkyl” refers to an alkyl radical, as defined above, that is substituted by one or more halo radicals, as defined above, e.g., difluoromethyl, trifluoromethyl, trichloromethyl, 2,2,2-trifluoroethyl, 1,2-difluoroethyl, 3-bromo-2-fluoropropyl, 1,2-dibromoethyl, and the like. The haloalkyl group of the present disclosure can be e.g., a C1-10 haloalkyl group, a C1-6 haloalkyl group, or a C1-3 haloalkyl group. Unless stated otherwise specifically in the specification, a haloalkyl group can be optionally substituted.

“Heterocyclyl” “heterocyclic ring” or “heterocycle” refers to a stable 3- to 20-membered non-aromatic, saturated or partially unsaturated ring radical which consists of two to twelve carbon ring atoms and from one to six heteroatoms as ring atoms selected from nitrogen, oxygen or sulfur, at least one non-aromatic, saturated or partially unsaturated ring containing at least one heteroatom as a ring atom. Unless stated otherwise specifically in the specification, the heterocyclyl radical can be a monocyclic, bicyclic, tricyclic or tetracyclic ring system, which can include fused, bridged, or spiro ring systems; and the nitrogen, carbon or sulfur atoms in the heterocyclyl radical can be optionally oxidized; the nitrogen atom can be optionally quaternized; and the heterocyclyl radical can be partially or fully saturated. Examples of such heterocyclyl radicals include, but are not limited to, dioxolanyl, thienyl[1,3]dithianyl, decahydroisoquinolyl, imidazolinyl, imidazolidinyl, isothiazolidinyl, isoxazolidinyl, morpholinyl, octahydroindolyl, octahydroisoindolyl, 2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolidinyl, oxazolidinyl, piperidinyl, piperazinyl, 4-piperidonyl, pyrrolidinyl, pyrazolidinyl, quinuclidinyl, thiazolidinyl, tetrahydrofuryl, trithianyl, tetrahydropyranyl, thiomorpholinyl, thiamorpholinyl, 1-oxo-thiomorpholinyl, and 1,1-dioxo-thiomorpholinyl. In embodiments where “L” is heterocyclyl, the heterocyclyl radical is a diradical. Unless stated otherwise specifically in the specification, a heterocyclyl group can be optionally substituted.

“Protecting group” refers to a moiety that, when attached to a chemically reactive group in a molecule, masks or reduces chemical reactivity of the group. Protecting groups are well known in the art and include those described in detail in Protective Groups in Organic Synthesis, T. W. Greene, et al., 3rd edition, John Wiley & Sons, 1999, incorporated by reference herein. Non-limiting examples of an amino protecting group (also referred to as a nitrogen protecting group) include those forming carbamates, such as tert-Butyloxycarbonyl (BOC) group, Carbobenzyloxy (Cbz) group, p-Methoxybenzyl carbonyl (Moz or MeOZ) group, Troc, 9-Fluorenylmethyloxycarbonyl (Fmoc) group, etc., those forming an amide, such as acetyl, trifluoroacetyl, benzoyl, etc., those forming a benzylic amine, such as benzyl, p-methoxybenzyl, 3,4-dimethoxybenzyl, etc., and others such as p-methoxyphenyl. Non-limiting examples of a hydroxy protecting group (also referred to as an oxygen protecting group) include those forming alkyl ethers or substituted alkyl ethers, such as methyl, allyl, benzyl, substituted benzyls such as 4-methoxybenzyl, methoxylmethyl (MOM), benzyloxymethyl (BOM), 2-methoxyethoxymethyl (MEM), etc., those forming silyl ethers, such as trymethylsilyl (TMS), triethylsilyl (TES), triisopropylsilyl (TIPS), t-butyldimethylsilyl (TBDMS), etc., those forming acetals or ketals, such as tetrahydropyranyl (THP), and those forming esters such as formate, acetate, chloroacetate, dichloroacetate, trichloroacetate, trifluoroacetate, methoxyacetate, etc.

The term “substituted” used herein means any of the above groups (i.e., alkyl, alkylene, alkenyl, alkenylene, alkynyl, alkynylene, alkoxy, alkylamino, thioalkyl, aryl, aralkyl, carbocyclyl, cycloalkyl, cycloalkenyl, cycloalkynyl, cycloalkylalkyl, haloalkyl, heterocyclyl, N-heterocyclyl, heterocyclylalkyl, heteroaryl, N-heteroaryl and/or heteroarylalkyl) wherein at least one hydrogen atom is replaced by a bond to a non-hydrogen atoms such as, but not limited to: a halogen atom such as F, Cl, Br, and I; an oxygen atom in groups such as hydroxyl groups, alkoxy groups, and ester groups; a sulfur atom in groups such as thiol groups, thioalkyl groups, sulfone groups, sulfonyl groups, and sulfoxide groups; a nitrogen atom in groups such as amines, amides, alkylamines, dialkylamines, arylamines, alkylarylamines, diarylamines, N-oxides, imides, and enamines; a silicon atom in groups such as trialkylsilyl groups, dialkylarylsilyl groups, alkyldiarylsilyl groups, and triarylsilyl groups; and other heteroatoms in various other groups.

“Substituted” also means any of the above groups in which one or more hydrogen atoms are replaced by a higher-order bond (e.g., a double- or triple-bond) to a heteroatom such as oxygen in oxo, carbonyl, carboxyl, and ester groups; and nitrogen in groups such as imines, oximes, hydrazones, and nitriles. For example, “substituted” includes any of the above groups in which one or more hydrogen atoms are replaced with —NRgRh, —NRgC(═O)Rh, —NRgC(═O)NRgRh, —NRgC(═O)ORh, —NRgSO2Rh, —OC(═O)NRgRh, —ORg, —SRg, —SORg, —SO2Rg, —OSO2Rg, —SO2ORg, ═NSO2Rg, and —SO2NRgRh. “Substituted also means any of the above groups in which one or more hydrogen atoms are replaced with —C(═O)Rg, —C(═O)ORg, —C(═O)NRgRh, —CH2SO2Rg, —CH2SO2NRgRh. In the foregoing, Rg and Rh are the same or different and independently hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, thioalkyl, aryl, aralkyl, cycloalkyl, cycloalkenyl, cycloalkynyl, cycloalkylalkyl, haloalkyl, haloalkenyl, haloalkynyl, heterocyclyl, N-heterocyclyl, heterocyclylalkyl, heteroaryl, N-heteroaryl and/or heteroarylalkyl. “Substituted” further includes any of the above groups in which one or more hydrogen atoms are replaced by a bond to an amino, cyano, hydroxyl, imino, nitro, oxo, thioxo, halo, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, thioalkyl, aryl, aralkyl, cycloalkyl, cycloalkenyl, cycloalkynyl, cycloalkylalkyl, haloalkyl, haloalkenyl, haloalkynyl, heterocyclyl, N-heterocyclyl, heterocyclylalkyl, heteroaryl, N-heteroaryl and/or heteroarylalkyl group. In addition, each of the foregoing substituents can also be optionally substituted with one or more of the above substituents.

As used herein, the symbol

(hereinafter can be referred to as “a point of attachment bond”) denotes a bond that is a point of attachment between two chemical entities, one of which is depicted as being attached to the point of attachment bond and the other of which is not depicted as being attached to the point of attachment bond. For example,

indicates that the chemical entity “XY” is bonded to another chemical entity via the point of attachment bond. Furthermore, the specific point of attachment to the non-depicted chemical entity can be specified by inference.

In this specification, unless stated otherwise, the term “pharmaceutically acceptable” is used to characterize a moiety (e.g., a salt, dosage form, or excipient) as being appropriate for use in accordance with sound medical judgment. In general, a pharmaceutically acceptable moiety has one or more benefits that outweigh any deleterious effect that the moiety may have. Deleterious effects may include, for example, excessive toxicity, irritation, allergic response, and other problems and complications.

The term “pharmaceutically acceptable salt” includes both acid and base addition salts. Pharmaceutically acceptable salts include those obtained by reacting the active compound functioning as a base, with an inorganic or organic acid to form a salt, for example, salts of hydrochloric acid, sulfuric acid, phosphoric acid, methanesulfonic acid, camphorsulfonic acid, oxalic acid, maleic acid, succinic acid, citric acid, formic acid, hydrobromic acid, benzoic acid, tartaric acid, fumaric acid, salicylic acid, mandelic acid, carbonic acid, etc. Those skilled in the art will further recognize that acid addition salts may be prepared by reaction of the compounds with the appropriate inorganic or organic acid via any of a number of known methods.

The present disclosure is intended to encompass deuterated forms of the compounds described herein, which include isotopes of atoms occurring in the compounds. Isotopes include those atoms having the same atomic number but different mass numbers. By way of general example, and without limitation, isotopes of hydrogen include deuterium and tritium, and isotopes of carbon include 13C and 14C. Isotopically labeled compounds of the present disclosure can generally be prepared by conventional techniques known to those skilled in the art or by processes and methods analogous to those described herein, using an appropriate isotopically labeled reagent in place of the non-labeled reagent otherwise employed.

A “derivative” refers to a chemically or biologically modified version of a chemical compound that is structurally similar to a parent compound and derivable from that parent compound. Derivatization (i.e., modification) may involve substitution of one or more moieties within the parent compound (e.g., a change in functional group). For example, when ligand A of the present disclosure is a derivative of a compound, ligand A can have a structure in which part of the structure of the compound is modified by binding to linker L2. Exemplary modifications include replacement of a substituent (e.g., H, halogen, etc.) for subsequent formation of a bond via chemical process such as amidation, amination, acylation, alkylation, esterification, or dehydration.

The terms “subject,” “individual,” and “patient” are used interchangeably herein to refer to a vertebrate, such as a mammal. The mammal may be, for example, a mouse, a rat, a rabbit, a cat, a dog, a pig, a sheep, a horse, a non-human primate (e.g., cynomolgus monkey, chimpanzee), or a human.

The term “treating” as used herein with regard to a patient, refers to improving at least one symptom of the patient's disorder. Treating can be improving, or at least partially ameliorating a disorder or an associated symptom of a disorder.

An “effective amount” means the amount compound or pharmaceutical formulation, that when administered to a patient for treating a state, disorder or condition is sufficient to affect such treatment.

The term “therapeutically effective” applied to dose or amount refers to that quantity of a compound or pharmaceutical formulation that is sufficient to result in a desired clinical benefit after administration to a patient in need thereof. A “therapeutically effective amount”, in some embodiments, is a dose or amount of a compound or pharmaceutical formulation that is sufficient to result in prophylaxis after administration to a patient in need thereof.

Compounds

This disclosure presents compositions and methods for the manipulation of the intrinsic autophagic pathway for the selective degradation of pathogenic proteins. The disclosure comprises novel heterobifunctional compounds designed to engage the autophagy adaptor protein p62/SQSTM1, thereby triggering its activation and subsequent assembly of autophagosomes. These chimeric molecules are composed of a targeting ligand (also referred to as a “protein binding component” or “PBC”), which exhibits high-affinity binding to designated pathogenic proteins, conjoined via a designed, flexible linker to a p62 activating moiety (also referred to as a “warhead”). This bifunctional architecture enables the precise orchestration of p62 oligomerization and spatial localization, thereby enhancing the sequestration of the targeted proteins within nascent autophagosomes. The utility of this inventive approach lies in its capacity to harness the cell's autophagic machinery, thereby offering a therapeutic modality with broad-spectrum applicability in the attenuation of diseases characterized by aberrant protein accumulation or defective protein clearance.

In embodiments, the compounds of the present disclosure can be useful for targeted protein degradation, including for inducing targeted autophagy. In embodiments, the compounds of the present disclosure can be also useful for modulating activity of p62.

As illustrated above, these bifunctional compounds contain: (1) a first component (“warhead”) that targets and recruits an autophagy adaptor such as p62, and (2) a second component (“protein binding component” or “PBC”) that binds to a protein target to be degraded. In some embodiments, ligand A in the formulas described herein is a PBC. In embodiments, bifunctional compounds contain (3) a linker that covalently couples the warhead to the protein binding component. As such, the compounds disclosed herein can be applied for therapeutically degrading any specific targets, including, but not limited to, proteins, protein aggregates, protein complexes, lipids, lipid droplets, or pathogens (e.g., viruses) within the cell. Additional autophagy adaptor proteins include, but are not limited to, LC3, Optineurin, TAX1BP1, NBR1, NDP52, NUFIP1, WDFY3, RETREG1, Nix, and TOLLIP.

In embodiments, the present disclosure provides a compound of formula (X-I):

or a pharmaceutically acceptable salt, stereoisomer, or deuterated form thereof, wherein:

In embodiments, the present disclosure provides a compound of formula (X-I):

or a pharmaceutically acceptable salt, stereoisomer, or deuterated form thereof, wherein:

In embodiments, the present disclosure provides a compound of formula (I):

or a pharmaceutically acceptable salt, stereoisomer, or deuterated form thereof, wherein:

In embodiments, the present disclosure provides a compound of formula (I):

or a pharmaceutically acceptable salt, stereoisomer, or deuterated form thereof, wherein:

In embodiments, the present disclosure provides a compound of formula (I-A):

or a pharmaceutically acceptable salt, a stereoisomer, or a deuterated form thereof, wherein:

In embodiments, the present disclosure provides a compound of formula (I-A):

or a pharmaceutically acceptable salt, a stereoisomer, or a deuterated form thereof, wherein:

In embodiments, the present disclosure provides a compound of of formula (I-A-1), (I-A-2), (I-A-3), or (I-A-4):

or a pharmaceutically acceptable salt, a stereoisomer, or a deuterated form thereof, wherein:

In embodiments, the present disclosure provides a compound of of formula (I-A-1), (I-A-2), (I-A-3), or (I-A-4):

or a pharmaceutically acceptable salt, a stereoisomer, or a deuterated form thereof, wherein:

In embodiments, the present disclosure provides a compound of formula (I-B):

or a pharmaceutically acceptable salt, a stereoisomer, or a deuterated form thereof, wherein:

In embodiments, the present disclosure provides a compound of formula (I-B):

or a pharmaceutically acceptable salt, a stereoisomer, or a deuterated form thereof, wherein:

In embodiments, the present disclosure provides a compound of formula (I-B-1), (I-B-2), (I-B-3), or (I-B-4):

or a pharmaceutically acceptable salt, a stereoisomer, or a deuterated form thereof, wherein:

In embodiments, the present disclosure provides a compound of formula (I-B-1), (I-B-2), (I-B-3), or (I-B-4):

or a pharmaceutically acceptable salt, a stereoisomer, or a deuterated form thereof, wherein:

In embodiments, the present disclosure provides a compound of formula (I-C) or (I-D):

or a pharmaceutically acceptable salt, a stereoisomer, or a deuterated form thereof, wherein:

In embodiments, the present disclosure provides a compound of formula (I-C) or (I-D):

or a pharmaceutically acceptable salt, a stereoisomer, or a deuterated form thereof, wherein:

In embodiments, the present disclosure provides a compound of formula (I-C-1), (I-C-2), (I-C-3), (I-C-4), (I-D-1), (I-D-2), (I-D-3), or (I-D-4):

or a pharmaceutically acceptable salt, a stereoisomer, or a deuterated form thereof, wherein:

In embodiments, the present disclosure provides a compound of formula (I-C-1), (I-C-2), (I-C-3), (I-C-4), (I-D-1), (I-D-2), (I-D-3), or (I-D-4):

or a pharmaceutically acceptable salt, a stereoisomer, or a deuterated form thereof,

In some embodiments, L0 is —CH2NHCH2CH2OH, —CH2NHCH2CH(CH3)OH, or

In some embodiments, L0 is —CH2NHCH2CH2OH.

In some embodiments, L0 is or

In some embodiments, the C3-8 cycloalkylene is

In embodiments, the present disclosure provides a compound of formula (I-A-1-a), (I-A-2-a), or (I-A-3-a):

or a pharmaceutically acceptable salt, a stereoisomer, or a deuterated form thereof, wherein:

In embodiments, the present disclosure provides a compound of formula (I-B-1-a), (I-B-2-a), or (I-B-3-a):

or a pharmaceutically acceptable salt, a stereoisomer, or a deuterated form thereof,

In embodiments, the present disclosure provides a compound of formula (I-C-1-a) or (I-D-1-a):

or a pharmaceutically acceptable salt, a stereoisomer, or a deuterated form thereof,

In embodiments, the present disclosure provides for components in which a linker is covalently attached to the compounds of (X-I), (I), (I-A), (I-B), (I-C), (I-D), (I-A-1), (I-A-2), (I-A-3), (I-A-4), (I-B-1), (I-B-2), (I-B-3), (I-B-4), (I-C-1), (I-C-2), (I-C-3), (I-C-4), (I-D-1), (I-D-2), (I-D-3), (I-D-4), (I-A-1-a), (I-A-2-a), (I-A-3-a), (I-B-1-a), (I-B-2-a), (I-B-3-a), (I-C-1-a). The linker (e.g., L1 disclosed herein) may be any moiety that is capable of covalently binding to the warhead and to the protein binding component (PBC). In embodiments, the present disclosure provides a compound of formula (X-II):

or a pharmaceutically acceptable salt, stereoisomer, or deuterated form thereof,

In embodiments, the present disclosure provides a compound of formula (II)

or a pharmaceutically acceptable salt, stereoisomer, or deuterated form thereof,

In embodiments, the present disclosure provides a compound of formula (II-A):

or a pharmaceutically acceptable salt, a stereoisomer, or a deuterated form thereof,

In embodiments, the present disclosure provides a compound of formula (II-A-1), (II-A-2), (II-A-3), or (II-A-4):

or a pharmaceutically acceptable salt, a stereoisomer, or a deuterated form thereof,

In embodiments, the present disclosure provides a compound of formula (II-B):

or a pharmaceutically acceptable salt, a stereoisomer, or a deuterated form thereof, wherein:

In embodiments, the present disclosure provides a compound of formula (II-B-1), (II-B-2), (II-B-3), or (II-B-4):

or a pharmaceutically acceptable salt, a stereoisomer, or a deuterated form thereof,

In embodiments, the present disclosure provides a compound of formula (II-C) or (II-D):

or a pharmaceutically acceptable salt, a stereoisomer, or a deuterated form thereof, wherein:

In embodiments, the present disclosure provides a compound of formula (II-C-1), (II-C-2), (II-C-3), (II-C-4), (II-D-1), (II-D-2), (II-D-3), or (II-D-4):

or a pharmaceutically acceptable salt, a stereoisomer, or a deuterated form thereof,

In embodiments of the compound of formula (X-II), (II), (II-A), (II-B), (II-C), (II-D), (II-A-1), (II-A-2), (II-A-3), (II-A-4), (II-B3-1), (II-B3-2), (II-B-3), (II-B-4), (II-C-1), (II-C-2), (II-C-3), (II-C-4), (II-D-1), (II-D-2), (II-D-3), or (II-D-4), or a pharmaceutically acceptable salt, stereoisomer, or deuterated form thereof, L1 comprises —NH(CH2CH2O)m—(CH2)n—, wherein m is an integer of 1-12, and n is an integer of 0-12. In embodiments, m is an integer of 1-12, 1-6, or 1-4. In embodiments, n is an integer of 0-12, 0-8, or 0-3.

In embodiments of the compound of formula (II), (II-A), (II-B), (II-C), (II-D), (II-A-1), (II-A-2), (II-A-3), (II-A-4), (II-B3-1), (II-B3-2), (II-B-3), (II-B-4), (II-C-1), (II-C-2), (II-C-3), (II-C-4), (II-D-1), (II-D-2), (II-D-3), or (II-D-4), or a pharmaceutically acceptable salt, stereoisomer, or deuterated form thereof, L1 is —(C1-6 alkylene)-NH(CH2CH2O)m—(CH2)n—C≡CH. In some embodiments, L1 is —(C1-3 alkylene)-NH—(CH2CH2O)m—(CH2)n—C≡CH. In some embodiments, the alkylene is optionally substituted with 1 or 2 RZ, wherein each RZ is independently —OH, C1-6 alkyl, or halogen. In some embodiments, m is an integer of 1-6. In some embodiments, m is an integer of 1-4. In some embodiments, n is an integer of 0-12. In some embodiments, n is an integer of 0-8. In some embodiments, L1 is —CH2NH—(CH2CH2O)—(CH2)2—C≡CH, —CH2NH—(CH2CH2O)—(CH2)5—C≡CH, —CH2NH—(CH2CH2O)—(CH2)6—C≡CH, —CH2NH—(CH2CH2O)—(CH2)8—C≡CH, —CH2NH—(CH2CH2O)2—(CH2)—C≡CH, —CH2NH—(CH2CH2O)2—(CH2)2—C≡CH, —CH2NH—(CH2CH2O)3—(CH2)—C≡CH, or —CH2NH—(CH2CH2O)3—(CH2)2—C≡CH.

In embodiments of the compound of formula (II), (II-A), (II-B), (II-C), (II-D), (II-A-1), (II-A-2), (II-A-3), (II-A-4), (II-B3-1), (II-B3-2), (II-B-3), (II-B-4), (II-C-1), (II-C-2), (II-C-3), (II-C-4), (II-D-1), (II-D-2), (II-D-3), or (II-D-4), or a pharmaceutically acceptable salt, stereoisomer, or deuterated form thereof, L1 is —(C1-6 alkylene)-NH(CH2CH2O)m—(CH2)n—C≡CH. In some embodiments, L1 is —(C1-3 alkylene)-NH—(CH2CH2O)m—(CH2)n—C≡CH. In some embodiments, the alkylene is optionally substituted with 1 or 2 RZ, wherein each RZ is independently —OH, C1-6 alkyl, or halogen. In some embodiments, m is an integer of 1-6. In some embodiments, m is an integer of 1-4. In some embodiments, n is an integer of 0-3. In some embodiments, L1 is —CH2NH—(CH2CH2O)2—(CH2)2—C≡CH or —CH2NH—(CH2CH2O)3—(CH2)2—C≡CH.

In embodiments of the compound of formula (II), (II-A), (II-B), (II-C), (II-D), (II-A-1), (II-A-2), (II-A-3), (II-A-4), (II-B3-1), (II-B3-2), (II-B-3), (II-B-4), (II-C-1), (II-C-2), (II-C-3), (II-C-4), (II-D-1), (II-D-2), (II-D-3), or (II-D-4), or a pharmaceutically acceptable salt, stereoisomer, or deuterated form thereof, L1 is —(C1-6 alkylene)-NH(CH2CH2O)m—(CH2)n-arylene-C≡CH. In some embodiments, L1 is —(C1-3 alkylene)-NH(CH2CH2O)m—(CH2)n-arylene-C≡CH. In some embodiments, the arylene is optionally substituted phenylene. In some embodiments, the alkylene is optionally substituted with 1 or 2 RZ, wherein each RZ is independently —OH, C1-6 alkyl, or halogen. In some embodiments, m is an integer of 1-6. In some embodiments, m is an integer of 1-4. In some embodiments, n is an integer of 0-3. In some embodiments, L is

In embodiments of the compound of formula (II), (II-A), (II-B), (II-C), (II-D), (II-A-1), (II-A-2), (II-A-3), (II-A-4), (II-B3-1), (II-B3-2), (II-B-3), (II-B-4), (II-C-1), (II-C-2), (II-C-3), (II-C-4), (II-D-1), (II-D-2), (II-D-3), or (II-D-4), or a pharmaceutically acceptable salt, stereoisomer, or deuterated form thereof, L1 is —O—(C1-6 alkylene)-CH(OH)—(C1-6 alkylene)-NH(CH2CH2O)m—(CH2)n—C≡CH. In some embodiments, L1 is —O—(C1-3 alkylene)-CH(OH)—(C1-3 alkylene)-NH(CH2CH2O)m—(CH2)n—C≡CH. In some embodiments, the alkylene is optionally substituted with 1 or 2 RZ, wherein each RZ is independently —OH, C1-6 alkyl, or halogen. In some embodiments, m is an integer of 1-6. In some embodiments, m is an integer of 1-4. In some embodiments, n is an integer of 0-4. In some embodiments, n is an integer of 1-3. In some embodiments, L1 is —O—CH2—CH(OH)—CH2—NH—(CH2CH2O)2—(CH2)2—C≡CH.

In embodiments of the compound of formula (II), (II-A), (II-B), (II-C), (II-D), (II-A-1), (II-A-2), (II-A-3), (II-A-4), (II-B3-1), (II-B3-2), (II-B-3), (II-B-4), (II-C-1), (II-C-2), (II-C-3), (II-C-4), (II-D-1), (II-D-2), (II-D-3), or (II-D-4), or a pharmaceutically acceptable salt, stereoisomer, or deuterated form thereof, L1 is —(C1-6 alkylene)-NH(CH2CH2O)m—(CH2)n—NH—C(O)O—(C1-6 alkyl). In some embodiments, the alkylene or alkyl is optionally and independently substituted with 1, 2, or 3 RZ, wherein each RZ is independently —OH, C1-6 alkyl, or halogen. In some embodiments, m is an integer of 1-6. In some embodiments, m is an integer of 1-4. In some embodiments, n is an integer of 0-12. In some embodiments, n is an integer of 0-8. In some embodiments, L1 is —CH2NH—(CH2CH2O)—(CH2)2—NHC(O)OC(CH3)3, —CH2NH—(CH2CH2O)—(CH2)3—NHC(O)OC(CH3)3, —CH2NH—(CH2CH2O)—(CH2)5—NHC(O)OC(CH3)3, —CH2NH—(CH2CH2O)—(CH2)6—NHC(O)OC(CH3)3, —CH2NH—(CH2CH2O)—(CH2)8—NHC(O)OC(CH3)3, —CH2NH—(CH2CH2O)2—(CH2)2—NHC(O)OC(CH3)3, or —CH2NH—(CH2CH2O)3—(CH2)2—NHC(O)OC(CH3)3.

In some embodiments, m is an integer of 1-6. In some embodiments, m is an integer of 1-4. In some embodiments, n is an integer of 0-12. In some embodiments, n is an integer of 0-8. In some embodiments, L1 is

In embodiments of the compound of formula (II), (II-A), (II-B), (II-C), (II-D), (II-A-1), (II-A-2), (II-A-3), (II-A-4), (II-B3-1), (II-B3-2), (II-B-3), (II-B-4), (II-C-1), (II-C-2), (II-C-3), (II-C-4), (II-D-1), (II-D-2), (II-D-3), or (II-D-4), or a pharmaceutically acceptable salt, stereoisomer, or deuterated form thereof, L1 is —(C1-6 alkylene)-NH(CH2CH2O)m—(CH2)n-arylene-NH—C(O)O—(C1-6 alkyl). In some embodiments, the alkylene, arylene, or alkyl is optionally and independently substituted with 1, 2, or 3 RZ, wherein each RZ is independently —OH, C1-6 alkyl, or halogen. In some embodiments, the arylene is phenylene. In embodiments, the phenylene is p-phenylene, m-phenylene o-phenylene. In some embodiments, m is an integer of 1-6. In some embodiments, m is an integer of 1-4. In some embodiments, n is an integer of 0-12. In some embodiments, n is an integer of 0-6. In some embodiments, L1 is

In some embodiments L1 is

In some embodiments, L1 is

In some embodiments, L1 is

In some embodiments, HETA is azetidinyl, pyrrolidinyl, piperidinyl, piperazinyl, or oxetanyl optionally substituted with —(C1-6 alkylene)-C≡CH. In some embodiments, HETA is

In some embodiments, L1 is

In some embodiments, L1 is

In some embodiments, HETA is azetidinyl, pyrrolidinyl, piperidinyl, piperazinyl, or oxetanyl optionally substituted with -arylene-C(O)O—(C1-6 alkyl). In some embodiments, the arylene is optionally substituted phenylene. In some embodiments, HETA is

In some embodiments, L1 is

In some embodiments, L1 is

In some embodiments, L1 is

In embodiments of the compound of formula (II), (II-A), (II-B), (II-C), (II-D), (II-A-1), (II-A-2), (II-A-3), (II-A-4), (II-B3-1), (II-B3-2), (II-B-3), (II-B-4), (II-C-1), (II-C-2), (II-C-3), (II-C-4), (II-D-1), (II-D-2), (II-D-3), or (II-D-4), or a pharmaceutically acceptable salt, stereoisomer, or deuterated form thereof, L1 is —(C1-6 alkylene)-NH(CH2CH2O)m—(CH2)n—NH—(C1-6 alkyl). In some embodiments, the alkylene or alkyl is optionally and independently substituted with 1, 2, or 3 RZ, wherein each RZ is independently —OH, C1-6 alkyl, or halogen. In some embodiments, m is an integer of 1-6. In some embodiments, m is an integer of 1-4. In some embodiments, n is an integer of 0-12. In some embodiments, n is an integer of 0-6. In some embodiments, L1 is —(C1-6 alkylene)-NH(CH2CH2O)m—(CH2)n—NHCH3. In some embodiments, L1 is —CH2NH—[CH2CH2O]2—[CH2]2—NHCH3, —CH2NH—(CH2CH2O)2—(CH2)3—NHCH3, or —CH2NH—[CH2CH2O]3—[CH2]2—NHCH3.

In embodiments of the compound of formula (II), (II-A), (II-B), (II-C), (II-D), (II-A-1), (II-A-2), (II-A-3), (II-A-4), (II-B3-1), (II-B3-2), (II-B-3), (II-B-4), (II-C-1), (II-C-2), (II-C-3), (II-C-4), (II-D-1), (II-D-2), (II-D-3), or (II-D-4), or a pharmaceutically acceptable salt, stereoisomer, or deuterated form thereof, L1 is —(C1-6 alkylene)-NH(CH2CH2O)m—(CH2)n—C(O)NH—(C1-6 alkyl). In some embodiments, the alkylene or alkyl is optionally and independently substituted with 1, 2, or 3 RZ, wherein each RZ is independently —OH, C1-6 alkyl, or halogen. In some embodiments, m is an integer of 1-6. In some embodiments, m is an integer of 1-4. In some embodiments, n is an integer of 0-12. In some embodiments, n is an integer of 0-6. In embodiments, L1 is —(C1-3 alkylene)-NH(CH2CH2O)m—(CH2)n—C(O)NH—CH3. In some embodiments, L1 is —CH2NH—(CH2CH2O)2—(CH2)2—C(O)NH—CH3.

In embodiments of the compound of formula (II), (II-A), (II-B), (II-C), (II-D), (II-A-1), (II-A-2), (II-A-3), (II-A-4), (II-B3-1), (II-B3-2), (II-B-3), (II-B-4), (II-C-1), (II-C-2), (II-C-3), (II-C-4), (II-D-1), (II-D-2), (II-D-3), or (II-D-4), or a pharmaceutically acceptable salt, stereoisomer, or deuterated form thereof, L1 is —(C1-6 alkylene)-NH(CH2CH2O)m—(CH2)n—C(O)NH—(CH2)n—C(O)O—(C1-6 alkyl). In some embodiments, the alkylene or alkyl is optionally and independently substituted with 1, 2, or 3 RZ, wherein each RZ is independently —OH, C1-6 alkyl, or halogen. In some embodiments, m is an integer of 1-6. In some embodiments, m is an integer of 1-4. In some embodiments, n is an integer of 0-12. In embodiments, L1 is —(C1-6 alkylene)-NH(CH2CH2O)m—(CH2)n—C(O)NH—(CH2)n—C(O)OCH2CH3. In embodiments, L1 is —CH2NH—(CH2CH2O)—(CH2)—C(O)NH—(CH2)—C(O)OCH2CH3.

In embodiments of the compound of formula (II), (II-A), (II-B), (II-C), (II-D), (II-A-1), (II-A-2), (II-A-3), (II-A-4), (II-B3-1), (II-B3-2), (II-B-3), (II-B-4), (II-C-1), (II-C-2), (II-C-3), (II-C-4), (II-D-1), (II-D-2), (II-D-3), or (II-D-4), or a pharmaceutically acceptable salt, stereoisomer, or deuterated form thereof, L1 is —(C1-6 alkylene)-NH(CH2CH2O)m—(CH2)n—C(O)O—(C1-6 alkyl). In some embodiments, L1 is —(C1-6 alkylene)-NH(CH2CH2O)m—(CH2)n—C(O)O—(C1-4 alkyl). In some embodiments, the alkylene or alkyl is optionally and independently substituted with 1, 2, or 3 RZ, wherein each RZ is independently —OH, C1-6 alkyl, or halogen. In some embodiments, L1 is —(C1-6 alkylene)-NH(CH2CH2O)m—(CH2)n—C(O)OCH3 or —(C1-6 alkylene)-NH(CH2CH2O)m—(CH2)n—C(O)OC(CH3)3. In some embodiments, m is an integer of 1-6. In some embodiments, m is an integer of 1-4. In some embodiments, n is an integer of 0-12. In some embodiments, n is an integer of 0-8. In some embodiments, L1 is —CH2NH—[CH2CH2O]2—[CH2]2—C(O)OCH3, —CH2NH—(CH2CH2O)3—(CH2)2—C(O)OCH3, —CH2NH(CH2CH2O)—(CH2)—C(O)OC(CH3)3, —CH2NH(CH2CH2O)—(CH2)2—C(O)OC(CH3)3, —CH2NH(CH2CH2O)—(CH2)3—C(O)OC(CH3)3, —CH2NH(CH2CH2O)—(CH2)4—C(O)OC(CH3)3, —CH2NH(CH2CH2O)—(CH2)5—C(O)OC(CH3)3, —CH2NH(CH2CH2O)—(CH2)8—C(O)OC(CH3)3, or —CH2NH(CH2CH2O)2—(CH2)2—C(O)OC(CH3)3.

In embodiments of the compound of formula (II), (II-A), (II-B), (II-C), (II-D), (II-A-1), (II-A-2), (II-A-3), (II-A-4), (II-B3-1), (II-B3-2), (II-B-3), (II-B-4), (II-C-1), (II-C-2), (II-C-3), (II-C-4), (II-D-1), (II-D-2), (II-D-3), or (II-D-4), or a pharmaceutically acceptable salt, stereoisomer, or deuterated form thereof, L1 is —O—(C1-6 alkylene)-CH(OH)—(C1-6 alkylene)-NH(CH2CH2O)m—(CH2)n—C(O)O—(C1-6 alkyl). In some embodiments, L1 is —O—(C1-3 alkylene)-CH(OH)—(C1-3 alkylene)-NH(CH2CH2O)m—(CH2)n—C(O)O—(C1-3 alkyl). In some embodiments, the alkylene is optionally substituted with 1 or 2 RZ, wherein each RZ is independently —OH, C1-6 alkyl, or halogen. In some embodiments, m is an integer of 1-6. In some embodiments, m is an integer of 1-4. In some embodiments, n is an integer of 0-4. In some embodiments, n is an integer of 1-3. In some embodiments, L1 is —O—CH2—CH(OH)—CH2NH—(CH2CH2O)—(CH2)2—C(O)OCH3.

In embodiments of the compound of formula (II), (II-A), (II-B), (II-C), (II-D), (II-A-1), (II-A-2), (II-A-3), (II-A-4), (II-B3-1), (II-B3-2), (II-B-3), (II-B-4), (II-C-1), (II-C-2), (II-C-3), (II-C-4), (II-D-1), (II-D-2), (II-D-3), or (II-D-4), or a pharmaceutically acceptable salt, stereoisomer, or deuterated form thereof, L1 is —(C1-6 alkylene)-NH(CH2CH2O)m—(CH2)n—O—C6-10 aryl. In some embodiments, m is an integer of 1-6. In some embodiments, m is an integer of 1-4. In some embodiments, n is an integer of 0-12. In some embodiments, n is an integer of 0-6. In some embodiments, the alkylene or aryl is optionally and independently substituted with 1, 2, or 3 RZ, wherein each RZ is independently —OH, C1-6 alkyl, or halogen. In some embodiments, L1 is

In embodiments of the compound of formula (II), (II-A), (II-B), (II-C), (II-D), (II-A-1), (II-A-2), (II-A-3), (II-A-4), (II-B3-1), (II-B3-2), (II-B-3), (II-B-4), (II-C-1), (II-C-2), (II-C-3), (II-C-4), (II-D-1), (II-D-2), (II-D-3), or (II-D-4), or a pharmaceutically acceptable salt, stereoisomer, or deuterated form thereof, L1 is —(C1-6 alkylene)-NH(CH2CH2O)m—(CH2)n-arylene-C(O)O—(C1-6 alkyl). In some embodiments, the alkylene, arylene or alkyl is optionally and independently substituted with 1, 2, or 3 RZ, wherein each RZ is independently —OH, C1-6 alkyl, or halogen. In some embodiments, the arylene is phenylene. In embodiments, the phenylene is p-phenylene, m-phenylene o-phenylene. In some embodiments, m is an integer of 1-6. In some embodiments, m is an integer of 1-4. In some embodiments, n is an integer of 0-12. In some embodiments, n is an integer of 0-6. In some embodiments, L1 is

In embodiments of the compound of formula (II), (II-A), (II-B), (II-C), (II-D), (II-A-1), (II-A-2), (II-A-3), (II-A-4), (II-B3-1), (II-B3-2), (II-B-3), (II-B-4), (II-C-1), (II-C-2), (II-C-3), (II-C-4), (II-D-1), (II-D-2), (II-D-3), or (II-D-4), or a pharmaceutically acceptable salt, stereoisomer, or deuterated form thereof, L1 is —(C1-6 alkylene)-NH(CH2CH2O)m—(CH2)n-heteroarylene-C(O)O—(C1-6 alkyl). In some embodiments, the alkylene, heteroarylene or alkyl is optionally and independently substituted with 1, 2, or 3 RZ, wherein each RZ is independently —OH, C1-6 alkyl, or halogen. In some embodiments, the heteroarylene is pyridinylene. In some embodiments, m is an integer of 1-6. In some embodiments, m is an integer of 1-4. In some embodiments, n is an integer of 0-12. In some embodiments, n is an integer of 0-6. In some embodiments, L1 is

In embodiments of the compound of formula (II), (II-A), (II-B), (II-C), (II-D), (II-A-1), (II-A-2), (II-A-3), (II-A-4), (II-B3-1), (II-B3-2), (II-B-3), (II-B-4), (II-C-1), (II-C-2), (II-C-3), (II-C-4), (II-D-1), (II-D-2), (II-D-3), or (II-D-4), or a pharmaceutically acceptable salt, stereoisomer, or deuterated form thereof, L1 is —(C1-6 alkylene)-NH(CH2CH2O)m—(CH2)n—XH, wherein XH is halogen. In some embodiments, XH is chloro or bromo. In some embodiments, m is an integer of 1-6. In some embodiments, m is an integer of 1-4. In some embodiments, n is an integer of 0-12. In some embodiments, n is an integer of 0-6. In some embodiments, L1 is —CH2NH—(CH2CH2O)2—(CH2)3—C1.

In embodiments of the compound of formula (II), (II-A), (II-B), (II-C), (II-D), (II-A-1), (II-A-2), (II-A-3), (II-A-4), (II-B3-1), (II-B3-2), (II-B-3), (II-B-4), (II-C-1), (II-C-2), (II-C-3), (II-C-4), (II-D-1), (II-D-2), (II-D-3), or (II-D-4), or a pharmaceutically acceptable salt, stereoisomer, or deuterated form thereof, L1 is —O—CH2CH(OH)—(C1-6 alkylene)-NH(CH2CH2O)m—(CH2)n—C≡CH. In some embodiments, m is an integer of 1-6. In some embodiments, m is an integer of 1-4. In some embodiments, n is an integer of 0-12. In some embodiments, n is an integer of 0-6. In some embodiments, L1 is —O—CH2CH(OH)—CH2—NH(CH2CH2O)2—(CH2)2—C≡CH.

In embodiments of the compound of formula (II), (II-A), (II-B), (II-C), (II-D), (II-A-1), (II-A-2), (II-A-3), (II-A-4), (II-B3-1), (II-B3-2), (II-B-3), (II-B-4), (II-C-1), (II-C-2), (II-C-3), (II-C-4), (II-D-1), (II-D-2), (II-D-3), or (II-D-4), or a pharmaceutically acceptable salt, stereoisomer, or deuterated form thereof, L1 is —O—CH2CH(OH)—(C1-6 alkylene)-NH(CH2CH2O)m—(CH2)n—C(O)O—(C1-6 alkyl). In some embodiments, m is an integer of 1-6. In some embodiments, m is an integer of 1-4. In some embodiments, n is an integer of 0-12. In some embodiments, n is an integer of 0-6. In some embodiments, L1 is —O—CH2CH(OH)—CH2—NH(CH2CH2O)—(CH2)2—C(O)OCH3.

In embodiments of the compound of formula (II), (II-A), (II-B), (II-C), (II-D), (II-A-1), (II-A-2), (II-A-3), (II-A-4), (II-B3-1), (II-B3-2), (II-B-3), (II-B-4), (II-C-1), (II-C-2), (II-C-3), (II-C-4), (II-D-1), (II-D-2), (II-D-3), or (II-D-4), or a pharmaceutically acceptable salt, stereoisomer, or deuterated form thereof, L1 is —(C1-6 alkylene)-NH(CH2CH2O)m—(CH2)n—(CH2CH2O)m—(CH2)n—C(O)O—(C1-6 alkyl). In some embodiments, each m is independently an integer of 1-12; and each n is independently an integer of 0-12. In some embodiments, each m is an integer of 1-6, 1-4, or 2-3. In some embodiments, each n is an integer of 0-6, 1-5, or 2-3. In some embodiments, L1 is —CH2NH(CH2CH2O)—(CH2)—(CH2CH2O)—(CH2)—C(O)OC(CH3)3.

In embodiments, a ligand (“A”) that binds to a protein, a protein aggregate, a protein complex, or a lipid that is targeted for degradation is covalently attached to the compounds of formula (X-II), (II), (II-A), (II-B3), (II-C), (II-D), (II-A-1), (II-A-2), (II-A-3), (II-A-4), (II-B3-1), (II-B3-2), (II-B3-3), (II-B3-4), (II-C-1), (II-C-2), (II-C-3), (II-C-4), (II-D-1), (II-D-2), (II-D-3), or (II-D-4). In embodiments, the present disclosure provides a compound of formula (X-III):

or a pharmaceutically acceptable salt, stereoisomer, or deuterated form thereof,

In embodiments, the present disclosure provides a compound of formula (III)

or a pharmaceutically acceptable salt, stereoisomer, or deuterated form thereof,

In embodiments, the present disclosure provides a compound of formula (III-A): (III-A):

or a pharmaceutically acceptable salt, a stereoisomer, or a deuterated form thereof,

In embodiments, the present disclosure provides a compound of formula (III-A-1), (III-A-2), (III-A-3), or (III-A-4):

or a pharmaceutically acceptable salt, a stereoisomer, or a deuterated form thereof,

In embodiments, the present disclosure provides a compound of formula (III-B):

or a pharmaceutically acceptable salt, a stereoisomer, or a deuterated form thereof,

In embodiments, the present disclosure provides a compound of formula (III-B-1), (III-B-2), (III-B-3), or (III-B-4):

or a pharmaceutically acceptable salt, a stereoisomer, or a deuterated form thereof,

In embodiments, the present disclosure provides a compound of formula (III-C) or (III-D):

or a pharmaceutically acceptable salt, a stereoisomer, or a deuterated form thereof,

In embodiments, the present disclosure provides a compound of formula (III-A) formula (III-C-1), (III-C-2), (III-C-3), (III-C-4), (III-D-1), (III-D-2), (III-D-3), or (III-D-4):

or a pharmaceutically acceptable salt, a stereoisomer, or a deuterated form thereof,

In some embodiments of the compound of formula (X-III), (III), (III-A), (III-B), (III-pharmaceutically acceptable salt, a stereoisomer, or a deuterated form thereof, L2 is:

In some embodiments of the compound of formula (III), (III-A), (III-B), (III-C), (III-D), (III-A-1), (III-A-2), (III-A-3), (III-A-4), (III-B-1), (III-B-2), (III-B-3), (III-B-4), (III-C-1) (III-C-2), (III-C-3), (III-C-4), (III-D-1), (III-D-2), (III-D-3), or (III-D-4), or a pharmaceutically acceptable salt, a stereoisomer, or a deuterated form thereof, other linker moieties can be used as L2 to covalently link A with ring W. Non-limiting examples of additional linker moiety as L2 include the following:

Ligand A (Protein Binding Component “PBC”)

In some embodiments of the compound of formula (X-III), (III), (III-A), (III-B3), (III-C), (III-D), (III-A-1), (III-A-2), (III-A-3), (III-A-4), (III-B3-1), (III-B3-2), (III-B3-3), (III-B-4), (III-C-1), (III-C-2), (III-C-3), (III-C-4), (III-D-1), (III-D-2), (III-D-3), or (III-D-4), or a pharmaceutically acceptable salt, a stereoisomer, or a deuterated form thereof, A is a ligand (e.g., a PBC) that binds to a protein. Once a protein targeted for degradation is identified, any ligand that binds to said target protein may be used in the compounds of (X-III), (III), (III-A), (III-B), (III-C), (III-D), (III-A-1), (III-A-2), (III-A-3), (III-A-4), (III-B-1), (III-B-2), (III-B-3), (III-B-4), (III-C-1), (III-C-2), (III-C-3), (III-C-4), (III-D-1), (III-D-2), (III-D-3), or (III-D-4) described herein. Such ligands may be covalently bound to the linker using a functional group found on the ligand, or the ligand may be modified to include an appropriate functional group to facilitate conjugation to the linker.

In some embodiments of the compound of (X-III), (III), (III-A), (III-B), (III-C), (III-D), (III-A-1), (III-A-2), (III-A-3), (III-A-4), (III-B3-1), (III-B-2), (III-B-3), (III-B-4), (III-C-1), (III-C-2), (III-C-3), (III-C-4), (III-D-1), (III-D-2), (III-D-3), or (III-D-4), or a pharmaceutically acceptable salt, a stereoisomer, or a deuterated form thereof, the protein is a protein that is associated with cancer. In some embodiments, the protein associated with cancer comprises a mutation or a fusion. In some embodiments, the protein associated with cancer is BRD4. In some embodiments, the protein associated with cancer is BRD4, and A is

In some embodiments, Riv is methyl.

Assays for assessing the binding of BRD4 with compounds having a corresponding ligand (e.g., the compound of formula (III), (III-A), (III-B), (III-C), (III-D), (III-A-1), (III-A-2), (III-A-3), (III-A-4), (III-B3-1), (III-B-2), (III-B-3), (III-B-4), (III-C-1), (III-C-2), (III-C-3), (III-C-4), (III-D-1), (III-D-2), (III-D-3), or (III-D-4), or a pharmaceutically acceptable salt, a stereoisomer, or a deuterated form thereof having the aforementioned A group) are generally known to a person of ordinary skill in the art. Exemplary BRD4 binding assays include, but are not limited to, surface plasmon resonance (SPR) assay, fluorescence polarization (FP), and thermal shift assay (TSA). See e.g., ACS Chem. Biol. 2019, 14, 3, 361-368; J Chem Inf Model. 2023, 63(17), 5408-5432; Structure 2023, 31(8), 912-923; SLAS Discovery 2015, 20(2), 180-189; Current Protocols in Pharmacology 2018, 80, 3.16.1-3.16.14, which are incorporated by reference herein in their entirety.

In some embodiments of the compound of formula (III), (III-A), (III-B), (III-C), (III-D), (III-A-1), (III-A-2), (III-A-3), (III-A-4), (III-B3-1), (III-B-2), (III-B-3), (III-B-4), (III-C-1), (III-C-2), (III-C-3), (III-C-4), (III-D-1), (III-D-2), (III-D-3), or (III-D-4), or a pharmaceutically acceptable salt, a stereoisomer, or a deuterated form thereof, the protein is a protein that is associated with a metabolic disease.

In some embodiments of the compound of formula (III), (III-A), (III-B), (III-C), (III-D), (III-A-1), (III-A-2), (III-A-3), (III-A-4), (III-B3-1), (III-B-2), (III-B-3), (III-B-4), (III-C-1), (III-C-2), (III-C-3), (III-C-4), (III-D-1), (III-D-2), (III-D-3), or (III-D-4), or a pharmaceutically acceptable salt, a stereoisomer, or a deuterated form thereof, the protein is a protein that is associated with inflammation.

In some embodiments of the compound of formula (III), (III-A), (III-B), (III-C), (III-D), (III-A-1), (III-A-2), (III-A-3), (III-A-4), (III-B3-1), (III-B-2), (III-B-3), (III-B-4), (III-C-1), (III-C-2), (III-C-3), (III-C-4), (III-D-1), (III-D-2), (III-D-3), or (III-D-4), or a pharmaceutically acceptable salt, a stereoisomer, or a deuterated form thereof, A is a ligand that binds to a protein aggregate. In some embodiments of the compound of formula (III), (III-A), (III-B), (III-C), (III-D), (III-A-1), (III-A-2), (III-A-3), (III-A-4), (III-B-1), (III-B-2), (III-B-3), (III-B-4), (III-C-1), (III-C-2), (III-C-3), (III-C-4), (III-D-1), (III-D-2), (III-D-3), or (III-D-4), or a pharmaceutically acceptable salt, a stereoisomer, or a deuterated form thereof, the protein aggregate is a Tau protein aggregate, an alpha-synuclein protein aggregate, a mutant Huntingtin protein aggregate, a β-sheet aggregate, a mitochondrial protein aggregate, an amyloid protein aggregate, or a TDP-43 protein aggregate. In some embodiments of the compound of formula (III), (III-A), (III-B), (III-C), (III-D), (III-A-1), (III-A-2), (III-A-3), (III-A-4), (III-B3-1), (III-B-2), (III-B-3), (III-B-4), (III-C-1), (III-C-2), (III-C-3), (III-C-4), (III-D-1), (III-D-2), (III-D-3), or (III-D-4), or a pharmaceutically acceptable salt, a stereoisomer, or a deuterated form thereof, the protein aggregate is an alpha-synuclein protein aggregate, a mutant Huntingtin protein aggregate, a β-sheet aggregate, an amyloid protein aggregate, or a TDP-43 protein aggregate.

In some embodiments, the protein aggregate is a Tau protein aggregate, and A is

wherein: each Ri is independently H or C1-6 alkyl; and M is CH or N. In some embodiments, each Ri is independently H or methyl.

Assays for assessing the binding of Tau with compounds having a corresponding ligand (e.g., the compound of formula (III), (III-A), (III-B), (III-C), (III-D), (III-A-1), (III-A-2), (III-A-3), (III-A-4), (III-B3-1), (III-B-2), (III-B-3), (III-B-4), (III-C-1), (III-C-2), (III-C-3), (III-C-4), (III-D-1), (III-D-2), (III-D-3), or (III-D-4), or a pharmaceutically acceptable salt, a stereoisomer, or a deuterated form thereof having the aforementioned A group) are generally known to a person of ordinary skill in the art. Exemplary Tau binding assays include, but are not limited to, radioligand binding assay, surface plasmon resonance (SPR) assay, differential scanning fluorimetry (DSF) and nuclear magnetic resonance (NMR) titration. See e.g., Chembiochem. 2023 May 16; 24(10):e202300163; J Chem Inf Model. 2023, 63(17), 5408-5432; Alzheimer's Res Therapy 2017, 9, 96; Eur J Nucl Med Mol Imaging 2024, 51, 3960-3977; bioRxiv 2024.03.15.585148, which are incorporated by reference herein in their entirety.

In some embodiments, the protein aggregate is an alpha-synuclein protein aggregate, and A is

wherein: Ri is H or C1-6 alkyl; and M is CH or N. In some embodiments, Ri is H or methyl.

Assays for assessing the binding of alpha-synuclein with compounds having a corresponding ligand (e.g., the compound of formula (III), (III-A), (III-B), (III-C), (III-D), (III-A-1), (III-A-2), (III-A-3), (III-A-4), (III-B3-1), (III-B-2), (III-B-3), (III-B-4), (III-C-1), (III-C-2), (III-C-3), (III-C-4), (III-D-1), (III-D-2), (III-D-3), or (III-D-4), or a pharmaceutically acceptable salt, a stereoisomer, or a deuterated form thereof having the aforementioned A group) are generally known to a person of ordinary skill in the art. Exemplary alpha-synuclein binding assays include, but are not limited to, nuclear magnetic resonance (NMR) spectroscopy, surface plasmon resonance (SPR) assay, and radioligand binding assay. See e.g., Chembiochem. 2023 May 16; 24(10):e202300163; J Chem Inf Model. 2023, 63(17), 5408-5432; Commun Biol 2018, 1, 44; J Neurochem. 2008, 105(4), 1428-37; Eur J Nucl Med Mol Imaging 2024, 51, 3960-3977, which are incorporated by reference herein in their entirety.

In some embodiments, the protein aggregate is a mutant Huntingtin protein aggregate (mHTT), and A is

Assays for assessing the binding of mutant Huntingtin protein with compounds having a corresponding ligand (e.g., the compound of formula (III), (III-A), (III-B), (III-C), (III-D), (III-E), (III-F), (III-G), (III-A-1), (III-A-2), (III-A-3), (III-B-1), (III-C-1), (III-D-1), (III-E-1), (III-F-1), or (III-G-1), or a pharmaceutically acceptable salt, a stereoisomer, or a deuterated form thereof having the aforementioned A group) are generally known to a person of ordinary skill in the art. Exemplary mutant Huntingtin protein binding assays include, but are not limited to, surface plasmon resonance (SPR) assay, differential scanning fluorimetry (DSF), and radioligand binding assay. See e.g., Chembiochem. 2023 May 16; 24(10):e202300163; J Chem Inf Model. 2023, 63(17), 5408-5432; Structure 2023, 31(9), 1121-1131.e6; Sci Rep 2021, 11, 17977, which are incorporated by reference herein in their entirety.

In some embodiments, the protein aggregate is a p-sheet aggregate, and A is

Assays for assessing the binding of p-sheet aggregate with compounds having a corresponding ligand (e.g., the compound of formula (III), (III-A), (III-B), (III-C), (III-D), (III-E), (III-F), (III-G), (III-A-1), (III-A-2), (III-A-3), (III-B3-1), (III-C-1), (III-D-1), (III-E-1), (III-F-1), or (III-G-1), or a pharmaceutically acceptable salt, a stereoisomer, or a deuterated form thereof having the aforementioned A group) are generally known to a person of ordinary skill in the art. Exemplary β-sheet aggregate binding assays include, but are not limited to, fluorescence spectroscopy, surface plasmon resonance (SPR) assay, differential scanning fluorimetry (DSF), nanoscale differential scanning fluorimetry (nanoDSF), and thermal shift assay (TSA). See e.g., Chembiochem. 2023 May 16; 24(10):e202300163; J Chem Inf Model. 2023, 63(17), 5408-5432; Anal Biochem. 2022, 654, 114828; Eur J Nucl Med Mol Imaging 2024, 51, 3960-3977, which are incorporated by reference herein in their entirety.

In some embodiments, the protein is a mitochondrial protein, and A is

wherein: Ri is H or C1-6 alkyl, and Rii and Riii are each independently a halogen or an alkyl. In some embodiments, Ri is H or methyl, and Rii and Riii are each independently F, Cl, or C1-6 alkyl.

Assays for assessing the binding of mitochondrial protein with compounds having a corresponding ligand (e.g., the compound of formula (III), (III-A), (III-B), (III-C), (III-D), (III-E), (III-F), (III-G), (III-A-1), (III-A-2), (III-A-3), (III-B3-1), (III-C-1), (III-D-1), (III-E-1), (III-F-1), or (III-G-1), or a pharmaceutically acceptable salt, a stereoisomer, or a deuterated form thereof having the aforementioned A group) are generally known to a person of ordinary skill in the art. Exemplary mitochondrial protein binding assays include, but are not limited to, radioligand binding assay, surface plasmon resonance (SPR) assay, thermal shift assay (TSA), and X-ray crystallography. See e.g., Chembiochem. 2023 May 16; 24(10):e202300163; J Chem Inf Model. 2023, 63(17), 5408-5432; Journal of Med. Chem. 2004, 47(7), 1852-1855; Biochemistry 2023, 62, 7, 1262-1273, which are incorporated by reference herein in their entirety.

Riv is

In some embodiments of the compound of formula (III), (III-A), (III-B), (III-C), (III-D), (III-A-1), (III-A-2), (III-A-3), (III-A-4), (III-B3-1), (III-B-2), (III-B-3), (III-B-4), (III-C-1), (III-C-2), (III-C-3), (III-C-4), (III-D-1), (III-D-2), (III-D-3), or (III-D-4), or a pharmaceutically acceptable salt, a stereoisomer, or a deuterated form thereof, other protein binding components (PBCs) that binds to a specific target protein, protein aggregates, protein complexes, lipids, or lipids can be used as ligand A. Non-limiting examples of the protein binding component include the following:

In some embodiments, the protein binding component is a derivative (e.g., a monovalent derivative that covalently bonded to linker L2) of one of the following compounds:

As a non-limiting example, a monovalent derivative of compound

may be:

In some embodiments, the protein binding component is an androgen receptor (AR) binding ligand. Non-limiting examples of androgen receptor binding ligand as protein binding component include:

In embodiments, R3 is

each optionally substituted with one R4. In some embodiments R3 is

In some embodiments R3 is

each optionally substituted with one or two R4. In some embodiments, R4 is halogen. In some embodiments, each R4 is fluoro. In some embodiments, R3 is

In some embodiments, R3 is

In some embodiments, R3 is

each optionally substituted with one R4. In some embodiments, R3 is

each optionally substituted with 1 or 2 R4. In some embodiments, R3 is

In some embodiments, R3 is

In some embodiments, R3 is

or —CH2CH2—NH—CH(CH3)2.

In some embodiments, -Q-R3 is

Another embodiment is a product obtainable by any of the processes or examples disclosed herein.

The compound disclosed herein is not

In embodiments, provided herein is a compound in Table 1, 2, or 3, or a pharmaceutically acceptable salt thereof, racemic form thereof, or stereoisomer thereof.

In embodiments, provided herein is a compound in Table 1, 2, or 3, or a pharmaceutically acceptable salt thereof, or stereoisomer thereof.

In embodiments, provided herein is a compound in Table 1, 2, or 3, or a pharmaceutically acceptable salt thereof.

In one embodiment, provided herein is a compound set forth in Table 1, 2, or 3.

In some embodiments, provided herein is a pharmaceutically acceptable salt of a compound in Table 1, 2, or 3.

Various compounds of the disclosure (series I)

ID
Structure

Various compounds of the disclosure (series II)

ID
Structure

Various compounds of the disclosure (series III)

ID
Structure

Compositions

In some embodiments, the present disclosure provides pharmaceutical composition(s) comprising one or more compounds of Table 1, 2, or 3, or a pharmaceutically acceptable salt, stereoisomer, or deuterated form thereof, and pharmaceutically acceptable adjuvant(s), diluent(s) or carrier(s).

The pharmaceutically acceptable excipients and adjuvants are added to the composition or formulation for a variety of purposes. In some embodiments, a pharmaceutical composition comprising one or more compounds disclosed herein, or a pharmaceutically acceptable salt, stereoisomer, or deuterated form thereof, further comprise a pharmaceutically acceptable carrier. In some embodiments, a pharmaceutically acceptable carrier includes a pharmaceutically acceptable excipient, binder, and/or diluent. In some embodiments, suitable pharmaceutically acceptable carriers include, but are not limited to, inert solid fillers or diluents and sterile aqueous or organic solutions. In some embodiments, suitable pharmaceutically acceptable excipients include, but are not limited to, water, salt solutions, alcohol, polyethylene glycols, gelatin, lactose, amylase, magnesium stearate, talc, silicic acid, viscous paraffin, and the like.

For the purposes of this disclosure, the compounds of the present disclosure can be formulated for administration by a variety of means including orally, parenterally, by inhalation spray, topically, transdermally, buccally, sublingually, or rectally in formulations containing pharmaceutically acceptable carriers, adjuvants, and vehicles. The term parenteral as used here includes subcutaneous, intravenous, intramuscular, and intraarterial injections with a variety of infusion techniques. Intraarterial and intravenous injection as used herein includes administration through catheters.

In some embodiments, the pharmaceutical composition can be formulated for oral administration. The oral formulations can be presented in discrete units, such as capsules, pills, cachets, lozenges, or tablets, each containing a predetermined amount of the active compound; as a powder or granules; as a solution or a suspension in an aqueous or non-aqueous liquid; or as an oil-in-water or water-in-oil emulsion.

In some embodiments, the pharmaceutical composition is formulated for parenteral administration (such as intravenous injection or infusion, subcutaneous or intramuscular injection). The parenteral formulations can be, for example, an aqueous solution, a suspension, or an emulsion.

In some embodiments, the pharmaceutical composition is formulated for inhalation. The inhalable formulations can be, for example, formulated as a nasal spray, dry powder, or an aerosol administrable through a metered-dose inhaler.

Therapeutic Use

In embodiments, the compounds of the present disclosure are p62 modulators, and thus may be used in any disease area where p62 plays a role. In embodiments, the compounds of the present disclosure are NBR1 modulators, and thus may be used in any disease area where NBR1 plays a role. As such, in one aspect of the disclosure, a method of treatment is provided. The method of treatment, in one embodiment, comprises, administering to a subject in need thereof, a composition comprising an effective amount of a compound of formula (I), (I-A), (I-B), (I-C), (I-D), (I-A-1), (I-A-2), (I-A-3), (I-A-4), (I-B-1), (I-B-2), (I-B-3), (I-B-4), (I-C-1), (I-C-2), (I-C-3), (I-C-4), (I-D-1), (I-D-2), (I-D-3), (I-D-4), (I-A-1-a), (I-A-2-a), (I-A-3-a), (I-B-1-a), (I-B-2-a), (I-B-3-a), (I-C-1-a), (I-D-1-a), (II), (II-A), (II-B), (II-C), (II-D), (II-A-1), (II-A-2), (II-A-3), (II-A-4), (II-B3-1), (II-B3-2), (II-B3-3), (II-B3-4), (II-C-1), (II-C-2), (II-C-3), (II-C-4), (II-D-1), (II-D-2), (II-D-3), (II-D-4), (III), (III-A), (III-B), (III-C), (III-D), (III-A-1), (III-A-2), (III-A-3), (III-A-4), (III-B-1), (III-B-2), (III-B-3), (III-B-4), (III-C-1), (III-C-2), (III-C-3), (III-C-4), (III-D-1), (III-D-2), (III-D-3), or (III-D-4), or Table 1, 2, or 3, or a pharmaceutically acceptable salt, stereoisomer, or deuterated form thereof. In embodiments, the composition is administered to the patient for an administration period.

In embodiments, a compound or composition of the present disclosure is administered to a subject in need thereof in a method for inducing autophagy. In some embodiments, the compound contacts p62, thereby inducing autophagy.

In embodiments, a compound or composition of the present disclosure is administered to a subject in need thereof in a method for inducing autophagy. In some embodiments, the compound contacts NBR1, thereby inducing autophagy.

In embodiments, a compound or composition of the present disclosure is administered to a subject in need thereof in a method for degrading target proteins, protein aggregates, protein complexes, lipids (e.g., lipid droplets), bacteria, or viruses. In some embodiments, a compound or composition of the present disclosure is administered to a subject in need thereof in a method for reducing the quantity of target proteins, protein aggregates, protein complexes, lipids, bacteria, or viruses.

In embodiments, a compound or composition of the present disclosure is administered to a subject in need thereof in a method for degrading a target protein. In some embodiments, a compound or composition of the present disclosure is administered to a subject in need thereof in a method for reducing the quantity of target proteins.

In embodiments, a compound or composition of the present disclosure is administered to a patient in a method for treating cancer (e.g., cancer metastasis), metabolic diseases, inflammation, neurodegenerative disorders, and infectious diseases.

In some embodiments, the cancer is breast cancer (e.g., ER negative breast cancer, triple negative breast cancer, basal-like breast cancers, HER2-positive breast cancers), kidney cancer (e.g., renal cell carcinoma (RCC)), prostate cancer, glioblastoma, or leukemia (e.g., chronic myelogenous leukemia, acute myelogenous leukemia, acute lymphoblastic leukemia).

In some embodiments, the infectious disease is a bacterial infection and/or a virus infection.

NUMBERED EMBODIMENTS OF THE DISCLOSURE

In addition to the disclosure above, the Examples below, and the appended claims, the disclosure sets forth the following numbered embodiments.

1. A compound of formula (I)

or a pharmaceutically acceptable salt, stereoisomer, or deuterated form thereof, wherein:

or a pharmaceutically acceptable salt, a stereoisomer, or a deuterated form thereof.

3. The compound of embodiment 1, having a structure of formula (I-B):

or a pharmaceutically acceptable salt, a stereoisomer, or a deuterated form thereof.

4. The compound of embodiment 1, having a structure of formula (I-C) or (I-D):

or a pharmaceutically acceptable salt, a stereoisomer, or a deuterated form thereof.

5. The compound of embodiment 1 or 2, having a structure of formula (I-A-1), (I-A-2), (I-A-3), or (I-A-4):

or a pharmaceutically acceptable salt, a stereoisomer, or a deuterated form thereof.

6. The compound of embodiment 1 or 3, having a structure of formula (I-B-1), (I-B-2), (I-B-3), or (I-B-4):

or a pharmaceutically acceptable salt, a stereoisomer, or a deuterated form thereof.

or a pharmaceutically acceptable salt, a stereoisomer, or a deuterated form thereof.

11. The compound of any one of embodiments 1, 2, 5, and 10, having a structure of formula (I-A-1-a), (I-A-2-a), or (I-A-3-a):

or a pharmaceutically acceptable salt, a stereoisomer, or a deuterated form thereof.

12. The compound of any one of embodiments 1, 3, 6, and 10, having a structure of formula (I-B-1-a), (I-B-2-a), or (I-B-3-a):

or a pharmaceutically acceptable salt, a stereoisomer, or a deuterated form thereof.

13. The compound of any one of embodiments 1, 4, 7, and 10, having a structure of formula (I-C-1-a) or (I-D-1-a):

or a pharmaceutically acceptable salt, a stereoisomer, or a deuterated form thereof.

14. The compound of any one of the preceding embodiments, wherein at least one H in L0 is replaced by conjugate comprising a ligand that binds to a protein, a protein aggregate, a protein complex, or a lipid.

15. A compound of formula (II)

or a pharmaceutically acceptable salt, stereoisomer, or deuterated form thereof,

or a pharmaceutically acceptable salt, a stereoisomer, or a deuterated form thereof.

17. The compound of embodiment 15, or a pharmaceutically acceptable salt, a stereoisomer, or a deuterated form thereof, wherein the compound has a structure of formula (II-B):

or a pharmaceutically acceptable salt, a stereoisomer, or a deuterated form thereof.

18. The compound of embodiment 15, or a pharmaceutically acceptable salt, a stereoisomer, or a deuterated form thereof, wherein the compound has a structure of formula (II-C) or (II-D):

or a pharmaceutically acceptable salt, a stereoisomer, or a deuterated form thereof.

19. The compound of embodiment 16, or a pharmaceutically acceptable salt, a stereoisomer, or a deuterated form thereof, wherein the compound has a structure of formula (II-A-1), (II-A-2), (II-A-3), or (II-A-4):

or a pharmaceutically acceptable salt, a stereoisomer, or a deuterated form thereof.

20. The compound of embodiment 17, or a pharmaceutically acceptable salt, a stereoisomer, or a deuterated form thereof, wherein the compound has a structure of formula (II-B-1), (II-B-2), (II-B-3), or (II-B-4):

or a pharmaceutically acceptable salt, a stereoisomer, or a deuterated form thereof.

21. The compound of embodiment 18, or a pharmaceutically acceptable salt, a stereoisomer, or a deuterated form thereof, wherein the compound has a structure of formula (II-C-1), (II-C-2), (II-C-3), (II-C-4), (II-D-1), (II-D-2), (II-D-3), or (II-D-4):

or a pharmaceutically acceptable salt, a stereoisomer, or a deuterated form thereof.

23. The compound of any one of embodiments 15-22, or a pharmaceutically acceptable salt, a stereoisomer, or a deuterated form thereof, wherein L1 is:

29. The compound of any one of embodiments 15-22, or a pharmaceutically acceptable salt, a stereoisomer, or a deuterated form thereof, wherein L1 comprises a bivalent moiety selected from:

30. A compound of formula (III)

or a pharmaceutically acceptable salt, stereoisomer, or deuterated form thereof,

or a pharmaceutically acceptable salt, a stereoisomer, or a deuterated form thereof.

32. The compound of embodiment 30, or a pharmaceutically acceptable salt, a stereoisomer, or a deuterated form thereof, wherein the compound has a structure of formula (III-B):

or a pharmaceutically acceptable salt, a stereoisomer, or a deuterated form thereof.

33. The compound of embodiment 30, or a pharmaceutically acceptable salt, a stereoisomer, or a deuterated form thereof, wherein the compound has a structure of formula (III-C) or (III-D3):

or a pharmaceutically acceptable salt, a stereoisomer, or a deuterated form thereof.

34. The compound of embodiment 31, or a pharmaceutically acceptable salt, a stereoisomer, or a deuterated form thereof, wherein the compound has a structure of formula (III-A-1), (III-A-2), (III-A-3), or (III-A-4):

or a pharmaceutically acceptable salt, a stereoisomer, or a deuterated form thereof.

35. The compound of embodiment 32, or a pharmaceutically acceptable salt, a stereoisomer, or a deuterated form thereof, wherein the compound has a structure of formula (III-B-1), (III-B-2), (III-B-3), or (III-B-4):

or a pharmaceutically acceptable salt, a stereoisomer, or a deuterated form thereof.

36. The compound of embodiment 33, or a pharmaceutically acceptable salt, a stereoisomer, or a deuterated form thereof, wherein the compound has a structure of formula (III-C-1), (III-C-2), (III-C-3), (III-C-4), (III-D-1), (III-D-2), (III-D-3), or (III-D-4):

or a pharmaceutically acceptable salt, a stereoisomer, or a deuterated form thereof.

38. The compound of any one of embodiments 30-37, or a pharmaceutically acceptable salt, a stereoisomer, or a deuterated form thereof, wherein L2 is:

40. The compound of any one of embodiments 30-37, or a pharmaceutically acceptable salt, a stereoisomer, or a deuterated form thereof, wherein L2 is:

41. The compound of any one of embodiment 30-40, or a pharmaceutically acceptable salt, a stereoisomer, or a deuterated form thereof, wherein the protein is a protein that is associated with cancer.

42. The compound of embodiment 41, or a pharmaceutically acceptable salt, a stereoisomer, or a deuterated form thereof, wherein the protein associated with cancer comprises a mutation or a fusion.

43. The compound of embodiment 41 or 42, or a pharmaceutically acceptable salt, a stereoisomer, or a deuterated form thereof, wherein the protein associated with cancer is BRD4.

44. The compound of any one of embodiments 30-40, or a pharmaceutically acceptable salt, a stereoisomer, or a deuterated form thereof, wherein the protein is a protein associated with a metabolic disease.

45. The compound of any one of embodiments 30-40, or a pharmaceutically acceptable salt, a stereoisomer, or a deuterated form thereof, wherein the protein is a protein associated with inflammation.

46. The compound of any one of embodiments 30-40, or a pharmaceutically acceptable salt, a stereoisomer, or a deuterated form thereof, wherein the protein is present in bacteria.

47. The compound of any one of embodiments 30-40, or a pharmaceutically acceptable salt, a stereoisomer, or a deuterated form thereof, wherein the protein is present in a virus particle.

48. The compound of any one of embodiments 30-40, or a pharmaceutically acceptable salt, a stereoisomer, or a deuterated form thereof, wherein the protein aggregate is an alpha-synuclein protein aggregate, a mutant Huntingtin protein aggregate, an amyloid protein aggregate, or a TDP-43 protein aggregate.

49. The compound of any one of embodiments 30-40, or a pharmaceutically acceptable salt, a stereoisomer, or a deuterated form thereof, wherein the protein is an intracellular protein.

51. The compound of any one of embodiments 30-40 and 50, wherein A is

70. The compound of embodiment 62, or a pharmaceutically acceptable salt, a stereoisomer, or a deuterated form thereof, wherein Q is —CH2C(O)—.

71. The compound of embodiment 62, or a pharmaceutically acceptable salt, a stereoisomer, or a deuterated form thereof, wherein Q is

73. The compound of embodiment 72, or a pharmaceutically acceptable salt, a stereoisomer, or a deuterated form thereof, wherein R3 is C6-10 aryl optionally substituted with 1 or 2 R4, and wherein each R4 is independently halogen, C1-6 alkyl, C1-6 alkoxy, or —C(O)—NRXRY.

74. The compound of embodiment 73, or a pharmaceutically acceptable salt, a stereoisomer, or a deuterated form thereof, wherein R3 is C6-10 aryl optionally substituted with —F, —C1, C1-3 alkoxy, or —C(O)—NRXRY, wherein RX is H or C1-6 alkyl, and RY is C1-6 alkyl.

75. The compound of embodiment 73 or 74, or a pharmaceutically acceptable salt, a stereoisomer, or a deuterated form thereof, wherein R3 is

76. The compound of embodiment 72, or a pharmaceutically acceptable salt, a stereoisomer, or a deuterated form thereof, wherein R3 is 5-10 membered heteroaryl optionally substituted with 1 or 2 R4; and wherein the heteroaryl contains 1 or 2 heteroatoms selected from N, S, or O.

77. The compound of embodiment 76, or a pharmaceutically acceptable salt, a stereoisomer, or a deuterated form thereof, wherein R3 is

each optionally substituted with one R4.

78. The compound of embodiment 76 or 77, or a pharmaceutically acceptable salt, a stereoisomer, or a deuterated form thereof, wherein R3 is

79. The compound of embodiment 72, or a pharmaceutically acceptable salt, a stereoisomer, or a deuterated form thereof, wherein R3 is C3-8 carbocycle optionally substituted with 1 or 2 R4, and wherein each R4 is independently halogen, —OH, C1-6 alkyl, or C1-6 alkoxy.

80. The compound of embodiment 79, or a pharmaceutically acceptable salt, a stereoisomer, or a deuterated form thereof, wherein R3 is

or each optionally substituted with one R4.

81. The compound of embodiment 72, or a pharmaceutically acceptable salt, a stereoisomer, or a deuterated form thereof, wherein R3 is 3-10 membered heterocycle containing 1 or 2 heteroatoms selected from N, O, or S, and the heterocycle is optionally substituted with 1 or 2 R4, and wherein each R4 is independently —OH, C1-6 alkyl, or —C(O)O—(C1-6 alkyl).

82. The compound of embodiment 81, or a pharmaceutically acceptable salt, a stereoisomer, or a deuterated form thereof, wherein each R4 is independently —OH, C1-3 alkyl, or —C(O)O—(C1-5 alkyl).

83. The compound of embodiment 81 or 82, or a pharmaceutically acceptable salt, a stereoisomer, or a deuterated form thereof, wherein R3 is

each optionally substituted with 1 or 2 R4.

84. The compound of any one of embodiments 81-83, or a pharmaceutically acceptable salt, a stereoisomer, or a deuterated form thereof, wherein R3 is

86. The compound of embodiment 85, or a pharmaceutically acceptable salt, a stereoisomer, or a deuterated form thereof, wherein RX is H, or methyl, and R, is methyl, —CH(CH3)2

87. The compound of embodiment 85 or 86, or a pharmaceutically acceptable salt, a stereoisomer, or a deuterated form thereof, wherein R3 is

88. The compound of any one of embodiments 1-63 and 72-87, or a pharmaceutically acceptable salt, a stereoisomer, or a deuterated form thereof, wherein Q is absent, and R3 is

89. The compound of any one of embodiments 1-72, or a pharmaceutically acceptable salt, a stereoisomer, or a deuterated form thereof, wherein -Q-R3 is

or —CH2CH2—NH—CH(CH3)2.

90. The compound of any one of embodiments 1-72, or a pharmaceutically acceptable salt, a stereoisomer, or a deuterated form thereof, wherein -Q-R3 is

91. The compound of any one of embodiments 1-72 and 89-90, or a pharmaceutically acceptable salt, a stereoisomer, or a deuterated form thereof, wherein -Q-R3 is

92. The compound of any one of embodiments 1, 2, and 5, wherein the compound of formula (I) is:

or a Pharmaceutically acceptable salt, a stereoisomer, or deuterated form thereof.

93. The compound of any one of embodiments 1, 3, and 6, wherein the compound of formula (I) is:

or a pharmaceutically acceptable salt or deuterated form thereof.

94. The compound of any one of embodiments 1, 4, and 7, wherein the compound of formula (I) is:

or a pharmaceutically acceptable salt or deuterated form thereof.

95. The compound of embodiment 15, wherein the compound of formula (II) is

or a pharmaceutically acceptable salt, a stereoisomer, or deuterated form thereof.

96. The compound of embodiment 30, wherein the compound of formula (III) is

or a pharmaceutically acceptable salt, or deuterated form thereof.

97. A method of modulating autophagy in a subject, comprising administering to the subject a compound of any one of the preceding claims.

98. The method of embodiment 97, wherein the compound contacts p62.

99. The method of embodiment 98, wherein the compound increases activity of p62, thereby causing autophagy.

100. The method of embodiment 98, wherein the compound decreases activity of p62, thereby reducing autophagy.

101. A method of degrading a target protein in a subject in need thereof, comprising administering a compound of any one of the preceding embodiments.

102. A method of inducing autophagy in a subject in need thereof, comprising administering a compound of any one of the preceding embodiments.

103. The method of embodiment 102, wherein the compound contacts p62, thereby inducing autophagy.

EXAMPLES

The present disclosure is further illustrated by reference to the following Examples. However, it should be noted that these Examples, like the embodiments described above, are illustrative and are not to be construed as restricting the scope of the disclosure in any way.

In embodiments, compounds of the present disclosure can be synthesized using the following methods. General reaction conditions are given, and reaction products can be purified by generally known methods including silica gel chromatography using various organic solvents such as hexane, dichloromethane, ethyl acetate, methanol and the like or preparative reverse phase high pressure liquid chromatography.

Abbreviations

min
minutes

RT
room temperature

TLC
thin layer chromatography

FA
formic acid

Bn
Benzyl

Example 1: Synthesis of Synthesis of Compound 2 Via Synthesis Route A

Synthesis of methyl 2-phenylbenzofuran-6-carboxylate (A-3)

Synthesis of 2-(((2-phenylbenzofuran-6-yl)methyl)amino)ethan-1-ol as Formic Acid Salt (Compound 2)

Example 2: Synthesis of Compound 6 Via Synthesis Route A

Synthesis of methyl 2-benzylbenzofuran-6-carboxylate (A-7)

Synthesis of 2-benzylbenzofuran-6-carboxylic acid (A-8)

Example 3: Synthesis of Compound 7 Via Synthesis Route A

Example 4: Synthesis of Compound 36 Via Synthesis Route A

Synthesis of methyl 2-fluoro-5-hydroxy-4-iodobenzoate (A-14)

Synthesis of methyl 2-benzyl-5-fluorobenzofuran-6-carboxylate (A-15)

Compound A-17 was obtained following the alcohol oxidation procedure similar to that for the synthesis of compound A-5, utilizing DMP (2.0 equiv.) and reaction conditions of 0° C.

Synthesis of 2-(((2-benzyl-5-fluorobenzofuran-6-yl)methyl)amino)ethan-1-ol as Formic Acid Salt (Compound 36)

Example 5: Synthesis of Compound 9 Via Synthesis Route B

Synthesis of 1-((2-benzylbenzofuran-6-yl)oxy)-3-(isopropylamino)propan-2-ol as Formic Acid Salt (Compound 9)

Example 6: Synthesis of Compound 11 Via Synthesis Route C

Example 7: Synthesis of Compound 13 Via Synthesis Route D

To a solution of carboxylate D-1 (6.70 g, 32.8 mmol, 1.0 equiv.) in CCl4 (140 mL) at RT, was added NBS (7.58 g, 42.6 mmol, 1.3 equiv.) and benzoic peroxyanhydride (795 mg, 3.28 mmol, 0.1 equiv.). The resultant mixture was stirred at 90° C. for 16 h and upon the completion of reaction by TLC, the mixture was diluted with H2O and extracted with CH2Cl2 (2×100 mL). The combined organic layers were dried over Na2SO4 and evaporated to afford D-2 as a brown solid (9.20 g, crude). TLC system: EtOAc/hexane (20:80), Rf value=0.4; LCMS (ESI) m/z 283.0 [Cl2H11BrO3+H]+. The crude material was used in the next step without purification.

Synthesis of 2-(((2-(4-chlorobenzyl)benzofuran-6-yl)methyl)amino)ethan-1-ol as Formic Acid Salt (Compound 13)

Example 8: Synthesis of Compound 33 Via Synthesis Route D

Compound D-8 was obtained following the reductive amination procedure similar to that for the synthesis of compound 2, utilizing 3-aminocyclohexan-1-ol (1.5 equiv.) with NaBH4 (2.0 equiv.) and reaction conditions of 70° C. for 16 h for imine formation, and 0° C. to RT for 1 h for imine reduction. Compound D-8 was obtained in 45% yield as a brown liquid following reverse phase column chromatography (Grace column, gradient elution with 20-30% MeCN/0.1% FA in H2O). TLC system: MeOH/CH2Cl2 (10:90), Rf value=0.2; LCMS (ESI) m/z 419.1 [C23H34N2O5+H]+.

Synthesis of 3-(((6-(((2-hydroxyethyl)amino)methyl)benzofuran-2-yl)methyl)amino)cyclohexan-1-ol as bis-formic acid salt (Compound 33)

Example 9: Synthesis of Compound 49 Via Synthesis Route D

To a stirred solution of (4-fluorobenzyl)triphenylphosphonium bromide (1.17 g, 2.60 mmol, 2.5 equiv.) in dry toluene (4.5 mL) at 0° C., was added NaH (60% in mineral oil) (104 mg, 2.60 mmol, 2.5 equiv.). The reaction mixture was warmed to RT and stirred at RT for 45 min before the reaction mixture was cooled back to 0° C., and D-6 (450 mg, 1.04 mmol, 1.0 equiv.) was added. The resultant mixture was heated to 80° C. and stirred at 80° C. for 3 h. Upon the completion of reaction by TLC, the reaction mixture was diluted with H2O and extracted with EtOAc (2×50 mL). The combined organic layers were washed with brine, dried over Na2SO4, filtered, and concentrated. The crude obtained was purified by silica gel (60-120 mesh) column chromatography (elution with 10% EtOAc/hexane) to afford D-9 (mixture of E and Z isomers in 1:1 ratio) as a colourless liquid (380 mg, yield: 69%). TLC system: EtOAc/hexane (10:90), Rf value=0.3; LCMS (ESI) m/z 526.4 [C30H40FNO4Si+H]+.

To a stirred solution of D-9 (mixture of E and Z isomers) (300 mg, 0.57 mmol, 1.0 equiv.) in MeOH (3 mL) at 0° C., was added NiCl2·6H2O (13.6 mg, 0.06 mmol, 0.1 equiv.) and NaBH4 (64.7 mg, 1.71 mmol, 3.0 equiv.). The resultant mixture was warmed to RT and stirred at RT for 1 h. Upon the completion of reaction by TLC, the reaction mixture was quenched with ice-cold water (30 mL) and extracted with EtOAc (2×40 mL). The combined organic layers were dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to afford D-10 as a colourless liquid (280 mg, crude). TLC system: EtOAc/hexane (10:90), Rf value=0.3; LCMS (ESI) m/z 528.2 [C30H42FNO4Si+H]+. The crude material was taken forward to the next step without further purification.

Synthesis of 2-(((2-(4-fluorophenethyl)benzofuran-6-yl)methyl)amino)ethan-1-ol as Formic Acid Salt (Compound 49)

Example 10: Synthesis of Compound 34 Via Synthesis Route D

Synthesis of 6-(((tert-butoxycarbonyl)(2-((tert-butyldimethylsilyl)oxy)ethyl)amino)methyl)benzofuran-2-carboxylic acid (D-11)

To a stirred solution of D-4 (300 mg, 0.63 mmol, 1.0 equiv.) in a mixture of 2:1:1 THF/MeOH/H2O (2 mL) at 0° C., was added LiOH·H2O (31.9 mg, 0.76 mmol, 1.2 equiv.). The resultant mixture was allowed to warm to RT and stirred at RT for 2 h. Upon the completion of reaction by TLC, the reaction mixture was neutralized with aq. citric acid solution and extracted with CH2Cl2 (3×30 mL). The combined organic layers were dried over Na2SO4 and concentrated under reduced pressure to afford D-11 as a pale-yellow solid (267 mg) in its crude. TLC system: MeOH/CH2Cl2 (10:90), Rf value=0.4; LCMS (ESI) m/z 450.2 [C23H35NO6Si+H]+. The crude compound was used in the next step without further purification.

Synthesis of (6-(((2-hydroxyethyl)amino)methyl)benzofuran-2-yl)(piperidin-1-yl)methanone as formic acid salt (Compound 34)

Example 11: Synthesis of Compound 16 Via Synthesis Route E

Example 12: Synthesis of Compound 18 Via Synthesis Route F

Synthesis of (E)-3-(2-hydroxy-4-methylphenyl)acrylic acid (F-3)

Compound F-5 was obtained following the bromination procedure similar to that for the synthesis of compound D-2, utilizing NBS (1.7 equiv.) with benzoic peroxyanhydride (0.1 equiv.) and reaction conditions of 90° C. for 16 h. Compound F-5 was obtained as a brown solid following silica gel (60-120 mesh) column chromatography (elution with 20% EtOAc/hexane) as a semi-pure material. TLC system: EtOAc/hexane (20:80), Rf value=0.4. The semi-pure material was used in the next step without further purification.

Synthesis of 2-(((2-(phenylsulfonyl)benzofuran-6-yl)methyl)amino)ethan-1-ol as Formic Acid Salt (Compound 18)

Example 13: Synthesis of Compound 19 Via Synthesis Route G

Compound G-4 was obtained following the bromination procedure similar to that for the synthesis of compound D-2, utilizing NBS (1.5 equiv.) with benzoic peroxyanhydride (0.1 equiv.) and reaction conditions of 90° C. for 8 h. Compound G-4 was obtained as a brown solid as a crude material. TLC system: MeOH/CH2Cl2 (10:90), Rf value=0.7; 1H NMR (400 MHz, CDCl3) δ ppm: 7.50-7.43 (m, 2H), 7.29-7.27 (m, 1H), 6.72 (s, 1H), 4.61 (s, 2H). The crude material was used in the next step without further purification.

To a stirred solution of G-7 (1.00 g, 1.99 mmol, 1.0 equiv.) in CH2Cl2 (10 mL) at 0° C., was added AcOH (2.5 mL), H2O (7.5 mL) and DCDMH (1.96 g, 9.95 mmol, 5 equiv.). The resultant reaction mixture was stirred at 0° C. for 1 h. Upon the completion of reaction by TLC, the reaction mixture was diluted with H2O (20 mL) and extracted with CH2Cl2 (2×40 mL). The combined organic layers were dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to afford G-8 as a brown gum (2 g, crude). TLC system: EtOAc/hexane (10:90), Rf value=0.6. The crude material was used in the next step immediately.

To a stirred solution of G-8 (2 g crude, 4.17 mmol, 1.0 equiv.) in CH2Cl2 (20 mL) at 0° C., was added pyridine (0.67 mL, 8.34 mmol, 2 equiv.) and aniline (0.76 mL, 8.34 mmol, 2 equiv.). The resultant mixture was warmed to RT and stirred at RT for 16 h. Upon the completion of reaction by TLC, the reaction mixture was diluted with H2O (20 mL) and extracted with CH2Cl2 (2×40 mL). The combined organic layers were dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The crude compound obtained was purified by silica gel (60-120 mesh) column chromatography (elution with 20% EtOAc/hexane) to afford G-9 as a pale-brown gum (260 mg, 64% pure by LCMS) as a semi-pure material. TLC system: EtOAc/hexane (30:70), Rf value=0.4; LCMS (ESI) m/z 535.5 [C29H32N2O6S−H]−. The semi-pure material was used in the next step without further purification.

Example 14: Synthesis of Compound 29 Via Synthesis Route H

Synthesis of methyl 2-(2-hydroxyethyl)benzofuran-6-carboxylate (H-2)

Synthesis of 2-(6-(methoxycarbonyl)benzofuran-2-yl)acetic acid (H-3)

Synthesis of methyl 2-(2-(dimethylamino)-2-oxoethyl)benzofuran-6-carboxylate (H-4)

Synthesis of 2-(2-(dimethylamino)-2-oxoethyl)benzofuran-6-carboxylic acid (H-5)

Example 15: Synthesis of Compound 38 Via Synthesis Route I

Synthesis of methyl 2-benzyl-7-fluorobenzofuran-6-carboxylate (I-4)

Example 16: Synthesis of Compound 42 Via Synthesis Route I

Synthesis of methyl 2-benzyl-3-methylbenzofuran-6-carboxylate (I-10)

Synthesis of 2-(((2-benzyl-3-methylbenzofuran-6-yl)methyl)amino)ethan-1-ol as Formic Acid Salt (Compound 42)

Example 17: Synthesis of Compound 43 Via Synthesis Route J

Synthesis of methyl 3-(cinnamyloxy)-4-iodobenzoate (J-2)

Synthesis of methyl 3-benzylbenzofuran-6-carboxylate (J-3)

Example 18: Synthesis of Compound 47 Via Synthesis Route K

Synthesis of 2-(((2-benzylbenzo[d]oxazol-6-yl)methyl)amino)ethan-1-ol as Formic Acid Salt (Compound 47)

Example 19: Synthesis of Compound 48 Via Synthesis Route K

Compound K-9 was obtained following the ester reduction procedure similar to that for the synthesis of compound A-4, utilizing DIBAL-H (2.5 equiv.) and reaction conditions of −78° C. -RT for 2 h. Compound K-9 was obtained as a light yellow gummy liquid in its crude. TLC system: EtOAc/hexane (40:60), Rf value=0.3; LCMS (ESI) m/z 256.1 [C15H13NOS+H]+. The crude material was used in the next step without further purification.

Example 20: Synthesis of Compound 50 Via Synthesis Route L

Synthesis of methyl 2-(hydroxymethyl)benzofuran-6-carboxylate (L-2)

Synthesis of methyl 2-formylbenzofuran-6-carboxylate (L-3)

Synthesis of methyl (E/Z)-2-(4-fluorostyryl)benzofuran-6-carboxylate (L-4)

Synthesis of methyl 2-(4-fluorophenethyl)benzofuran-6-carboxylate (L-5)

Synthesis of 2-(4-fluorophenethyl)benzofuran-6-carboxylic acid (L-6)

Example 21: Synthesis of Compound 81 Via Synthesis Route A

Synthesis of methyl 2-phenethylbenzofuran-6-carboxylate (A-18)

Example 22: Synthesis of Compound 82 Via Synthesis Route A

Example 23: Synthesis of Compound 83 Via Synthesis Route A

Example 24: Synthesis of Compound 95 Via Synthesis Route A

Example 25: Synthesis of Compound 98 Via Synthesis Route B

Example 26: Synthesis of Compound 100 Via Synthesis Route M

To a stirred solution of M-1 (400 mg, 0.54 mmol, 1 equiv.) in CH2Cl2 (4 mL) at 0° C., was added TFA (0.4 mL). The reaction mixture was warmed to RT and stirred at RT for 2 h. Upon the completion of reaction by TLC, the volatiles were removed under reduced pressure and the residue was triturated with n-pentane (2×10 mL) to afford M-2 (TFA salt) as a pink gummy liquid (450 mg, crude). TLC system: MeOH/CH2Cl2 (10:90), Rf value=0.1; LCMS (ESI) m/z 635.4 [C39H42N2O6+H]+. The crude compound was used in the next step without further purification.

Example 27: Synthesis of Compound 104 Via Synthesis Route M

Where absolute stereochemistry has not been indicated, compounds herein are racemic mixtures with relative stereochemistry as drawn.

Summary of compounds (series I) prepared according to the Examples above

and characterizations thereof

Cpd
Synthesis

NMR and Mass

No.
Route
Structure
Spectrometry

Mixture of isomers

observed in NMR.

isomers observed in NMR.

105
A

107
A

of isomers observed in

108
A

109
A

110
A

111
A

112
A

118
A

Summary of compounds (series II) prepared according to the examples above and characterizations thereof

Cpd
Synthesis

NMR and Mass

No.
Route
Structure
Spectrometry

120
A

121
A

122
A

123
A

124
A

125
A

126
A

127
A

128
A

129
A

130
A

131
A

132
A

133
A

134
A

135
A

136
A

137
A

138
A

139
A

140
A

141
A

142
A

143
A

144
A

145
A

146
A

147
A

148
A

149
A

150
A

151
A

152
A

153
A

154
A

155
A

156
A

157
A

158
A

159
A

160
A

161
A

162
A

163
A

164
A

165
A

166
A

167
A

168
A

Summary of compounds (series III) prepared according to the examples above and characterizations thereof

Cpd
thesis

Cpd No.
NMR and Mass Spectrometry

Example 28: Surface Plasmon Resonance (SPR) Analysis

Protein Preparation

Recombinant p62 protein was buffer exchanged into Biacore SPR running buffer (Cytiva 100671) using the Slide-A-Lyzer Mini Dialysis system 7K MWCO (Thermo Fisher Scientific, Cat no. 69560). The final protein concentration was determined using the Nanodrop One spectrophotometer (Thermo Fisher Scientific, cat no. ND-ONE-W), with calibration for the dialysis buffer.

Protein Immobilisation

The prepared protein was diluted in sodium acetate buffer (NaAc, pH 5.5, Cytiva) to concentrations ranging from approximately 0.25-40 μg/mL. Post-immobilization, the protein required approximately 16 hours of equilibration in running buffer supplemented with 1% DMSO at 15° C. to ensure adequate surface equilibration and protein reorganization.

SPR Running Conditions

All SPR experiments were conducted at a constant temperature of 15° C. The SPR chip surface was activated using EDC/NHS for a contact time of 6-10 min at a flow rate of 10 μL/min. This was followed by protein immobilization for 2100 seconds and ethanolamine capping for 420 seconds, both at the same flow rate. Immediately after immobilization, the NH2—REEE peptide was analyzed in Single Cycle Kinetics mode (SCK) to verify surface activity, ensuring it binds the sample with the published affinity (−20 μM by SPR).

Compound Binding Experiments

For general compound binding studies, experiments were performed at 15° C. using a running buffer composed of 10 mM HEPES, 150 mM NaCl, 1 mM DTT, 0.05% Tween 20, and 1% DMSO at pH 7.4. The test compound was initially prepared at a 20 mM concentration in DMSO and subsequently titrated in 2-fold dilutions. For each assay, 2 μL of the compound was mixed with 198 μL of the running buffer, and the mixture was thoroughly pipetted approximately 15 times for homogenization.

A series of blank injections (0 μM concentration) were included for double referencing, with at least 8 or 16 per plate depending on the format. Typical injection parameters were 120 seconds for association and 180 seconds for dissociation at a flow rate of 50 L/min. A four-point solvent correction was prepared with DMSO concentrations ranging from 2% to 0.5% in the buffer. Post each compound injection, the system's needles were washed with 50% DMSO.

Data Acquisition and Analysis

Analysis of the SPR data was conducted using the Cytiva Evaluation software. The binding levels at the late time-point of each interaction were corrected for reference, then normalized to percentage bound values to account for variations in immobilization levels, protein molecular weight, and compound molecular weight.

Example 29: Western Blot Protocol

HeLa cells were seeded in 6-well plates (700,000 cells) and allowed to adhere overnight. Media was replaced one hour before administering either vehicle (DMSO) or test compounds. After 24 hours of treatment, the media was discarded, and cells were rinsed once with ice-cold 1×PBS before detached from plates with ice-cold RIPA Lysis and Extraction Buffer (Thermo Fisher Scientific) which was supplemented with protease and phosphatase inhibitors. The resulting cell suspensions were transferred to pre-chilled microcentrifuge tubes, incubated on ice for 20 min with intermittent mixing and then centrifuged at 12,000 rpm for 20 min at 4° C. The supernatants were then aspirated slowly and transferred into new tubes kept on ice. The protein concentration in the samples was determined using BCA kit (Thermo Fisher Scientific) as per manufacturer's instructions.

For the p62 oligomerization and LC3 lipidation assays, cell lysates were prepared using RIPA buffer supplemented with 2% SDS and benzonase (Sigma). The lysates were then combined with 4× Laemmli protein sample buffer (Bio-Rad), heated at 70° C. for 10 min and subjected to a brief centrifugation of 10-15 seconds.

For the BRD4 degradation assay, cell lysates were prepared with RIPA buffer containing 0.25% SDS. These were mixed with 4× Laemmli protein sample buffer (Bio-Rad) supplemented with beta-mercaptoethaol (355 mM). The lysates were then heated at 95° C. for 5 min, and briefly centrifuged for 10-15 seconds.

For the FKBP12(F36V) degradation assay, cell lysates were prepared with a Triton-X-100 buffer supplemented with 2% SDS. The lysates were mixed with 4× Laemmli protein sample buffer (Bio-Rad) supplemented with beta-mercaptoethaol (355 mM). The lysates were then heated at 95° C. for 5 min, and briefly centrifuged for 10-15 seconds.

For p62 oligomerization and LC3 lipidation assays, lysates containing 30 μg of protein and for BRD4 and FKBP12(F36V) degradation assays, lysates containing 20 μg of protein, were loaded slowly into the wells of 4-20% gradient Tris-glycine gels (Bio-Rad). The gels were run at 100V until completion and transferred onto PVDF membranes. Membranes were blocked using 5% milk in TBS-Tween (0.1%) buffer at room temperature for 1 hour. The membranes were incubated with primary antibody in 5% milk-TBST with shaking, overnight at 4° C. The primary antibodies used were: p62 (Novus Biologicals 2C11, H00008878-MO1), LC3A/B (Cell Signalling Technology D3U4C, 12741S), FKBP12 (Abcam ab24373), BRD4 (Cell Signalling Technology E2A7X, 13440S), GAPDH (Cell Signalling Technology D4C6R, 97166S), and β-Actin (Proteintech 66009-1-Ig). Following primary antibody incubation, membranes were washed thrice with TBST 10 min before and after incubation with secondary-HRP conjugated antibody in 5% milk-TBST for 1 hour at room temperature. The secondary antibodies used were: Anti-Mouse IgG HRP Conjugate (Promega W4021B) and Anti-Rabbit IgG HRP Conjugate (Promega W4011B). For the p62 oligomerization assay, the total protein levels were assessed using Ponceau staining (Thermo Fisher Scientific) of PVDF membrane as per recommendation by manufacturer's guidelines.

Imaging was acquired using SuperSignal™ West Pico PLUS Chemiluminescent Substrate (Thermo Fisher Scientific) with iBright Invitrogen FL100 imaging system. Band signal intensity, reflective of protein concentrations, were quantified using Invitrogen™ iBright™ Analysis and Image J software.

Example 30: Immunocytochemistry and Imaging Protocol

HeLa cells were seeded in coated 96-well plates (10,000 cells) and allowed to adhere overnight. After 24 hours of treatment with either vehicle (DMSO) or test compounds, the media was discarded, and cells were rinsed with ice-cold 1×PBS before fixation by 4% formaldehyde or paraformaldehyde for 20 min. Cells were then washed with 1×PBS and permeabilized with 0.1% Triton X-100 in PBS for 20 min and blocked with Blocking Buffer in PBS for 1 hour. The cells were incubated thereafter for 1 hour with anti-p62 primary antibody in Blocking Buffer, followed by incubation with secondary antibody for 1 hour. DAPI dye was used to visualize nuclei of fixed cells. The blocking buffer used were 5% Fetal Bovine Serum (FBS, ThermoFIsher Scientific, 10270106) and Li-cor Intercept (PBS) Blocking Buffer (Li-cor, 927-70001). The primary antibody used was mouse anti-p62 primary antibody (Novus Biologicals, H00008878-MO1). The secondary antibodies used were goat anti-mouse Alexa Fluor Plus 488 secondary antibody (Invitrogen, GOXMS) and goat anti-mouse Alexa Fluor 488 secondary antibody (Invitrogen, A-11029). The DAPI dyes used were DAPI dye (Invitrogen, 62248) and DAPI dye (Invitrogen, D1306).

For the EVOS M7000 System, cells were imaged on the EVOS M7000 microscope using a 20× air objective (Nikon, N.A 0.45). DAPI and p62 channels were illuminated with LED light cubes using integrated hard-coated filter sets for DAPI (357/447 nm) and GFP (470/525 nm) respectively. Images were analysed for p62 puncta count on the Celeste 6.0 software.

For the Operetta CLS System, cells were imaged on the Operetta CLS using a 63× water objective on the DAPI and Alexa 488 channels. Images were analysed for p62 puncta count on the Harmony 5.1 software.

Example 31: Studies on Compound Binding to p62

Compound binding studies were carried out according to experimental procedures above and SPR % occupancy was determined and summarized in Table 7.

Example 32: Oligomerization Activity of p62 Protein

HeLa cells were treated with compounds for 24 hours to evaluate the effect of these compounds on p62 oligomerisation activity. The resulting changes in levels of p62 oligomers were assessed by Western blotting, as described in experimental procedures. The fold change of p62 oligomer levels induced by compound treatment relative to vehicle control (DMSO) was determined and summarized in Table 8. Representative Western blot images are shown in FIGS. 2A-C. Specifically, FIGS. 2A-C show Western blot images and their corresponding quantification showing the levels of high molecular weight (100-200 kDa) p62 oligomers by 24 hours compound treatments at listed concentrations, relative to vehicle control (DMSO) in HeLa cells. Protein loading consistencies was assessed by Ponceau staining (not shown).

Compound effect on p62 oligomerization

++: > 2-fold change relative to DMSO control

+: < 2-fold change relative to DMSO control

Example 33: Lipidation Activity of LC3 Protein

HeLa cells were treated with compounds for 24 hours to evaluate the effect of these compounds on LC3-II/LC3-I levels, a measure of autophagy activation. The changes in levels of LC3-I, LC3-II and β-Actin (loading control) were assessed by Western blotting, as described in experimental procedures. The fold increase of LC3-II/LC3-I levels induced by compound treatment relative to vehicle control (DMSO) was determined and summarized in Table 9. Representative Western blot images are shown in FIGS. 3A-C. Specifically, FIGS. 3A-C show Western blot images and their corresponding quantification showing the lipidation levels of LC3 protein (LC3-II/LC3-I) by 24 hours compound treatments at listed concentrations, relative to vehicle control (DMSO) in HeLa cells.

Compound effect on LC3 lipidation

++: > 2-fold change in LC3-II/LC3-I relative to DMSO control

+: < 2-fold change in LC3-II/LC3-I relative to DMSO control

Example 34: Puncta Activity of p62 Protein

HeLa cells were treated with compounds for 24 hours to evaluate the effect of these compounds on p62 puncta activity. The resulting changes in levels of p62 puncta were assessed by imaging, either on the EVOS M7000 or Operetta CLS systems as described in experimental procedures. The fold change of p62 puncta levels induced by compound treatment relative to vehicle control (DMSO) was determined and summarized in Table 10.

++: > 2-fold change in p62 puncta levels relative to DMSO control

+: < 2-fold change in p62 puncta levels relative to DMSO control

Example 35: Degradation of BRD4

To evaluate levels of protein degradation by compounds 100-103, HeLa cells were treated with the corresponding compounds at doses up to 10 μM for 24 hours. The changes in levels of BRD4 and GAPDH (loading control) were assessed by Western blotting, as described in experimental procedures. Results are summarized in Table 11 below, measured by percentage change in BRD4 levels by compound treatment relative to vehicle (DMSO) control.

To demonstrate p62 dependency of the compounds, wild type (WT) and p62 knockout (p62-KO) HeLa cells were treated with respective compounds for 24 hours. Compound-mediated BRD4 degradation was not observed in p62-KO cells in contrast to WT HeLa cells. Representative Western blot images are shown in FIGS. 4A-B. Specifically, FIGS. 4A-B show Western blot images and their corresponding quantifications showing the degradation levels of BRD4 by 24 hours compound treatments at listed concentrations, relative to vehicle control (DMSO) in wild type (WT) and/or p62 knockout (p62-KO) HeLa cells.

++: > 2-fold change in BRD4 levels relative to DMSO control

+: < 2-fold change in BRD4 levels relative to DMSO control

Example 36: Degradation of FKBP12(F36V)

HeLa cells expressing FKBP12-F36V were treated with compound 104 for 24 hours to evaluate its ability to degrade the target protein. The changes in levels of FKBP12 and 0-Actin (loading control) were assessed by Western blotting, as described in experimental procedures. Representative Western blots images are shown in FIG. 5. Specifically, FIG. 5 shows Western blot images and their corresponding quantification showing the degradation levels of FKBP12 by 24 hours compound treatment at listed concentrations, relative to vehicle control (DMSO) in HeLa cells expressing FKBP12(F36V).