TRIM21 binding molecules

Abstract

This invention relates to binding molecules comprising a TRIM21 binding portion and a protein binding portion for the selective degradation of target proteins. The invention also relates to compositions comprising these molecules and their use in therapies. Furthermore, the invention relates to TRIM21 binding molecules and their use.

Description

[Technical Field]

[0001] This invention relates to binding molecules comprising a TRIM21 binding portion and a protein binding portion for the selective degradation of target proteins. The invention also relates to compositions comprising these molecules and their use in therapies. Furthermore, the invention relates to TRIM21 binding molecules and their use. [Background technology]

[0002] Small molecules known as "protein degradation targeting chimeras" (PROTACs) can be used to deplete target proteins. PROTAC molecules typically contain ligands, such as small molecule inhibitors of the target protein, which are covalently bound to a ligand of an E3 ubiquitin ligase. When the small molecule inhibitor binds to the target protein, the PROTAC molecule recruits the E3 ubiquitin ligase, which can ubiquitinate the target protein, thereby targeting it for subsequent degradation. PROTAC molecules are considered a potential therapy in the treatment of several diseases, allowing clinicians to specifically target and degrade certain proteins. [Overview of the project] [Problems that the invention aims to solve]

[0003] However, while PROTAC molecules are selective for target proteins, they may not distinguish between different forms of the same protein, and therefore could lead to the degradation of non-pathogenic forms of the protein.

[0004] Therefore, there is a need for molecules that can target the pathogenic form of proteins. Such molecules would enable more targeted therapies. The use of more targeted proteolysis as a therapeutic strategy can minimize off-target effects of drugs and avoid or reduce systemic drug exposure. [Means for solving the problem]

[0005] The present invention relates to a bifunctional molecule comprising a TRIM21 binding moiety and a protein targeting moiety. Due to their ability to cluster TRIM21 in close proximity to the target protein, the compounds of the present invention can enable the selective degradation of specific forms of the target protein.

[0006] Therefore, a first aspect of the present invention relates to a compound of formula (I). ALB (I) [In the formula, A is the part that binds to TRIM21, and the molecular weight of A is 1000 Da or less. L is a linker, B is the part that binds to the target protein. Or provide a pharmaceutically acceptable salt thereof.

[0007] The target protein is a protein that may include a first form (e.g., a pathogenic form) that provides multiple binding sites for B. The target protein may be a protein capable of forming oligomeric species. In the oligomeric form, multiple binding sites for B are provided.

[0008] A second aspect of the present invention provides a pharmaceutical composition comprising a compound of the first aspect of the present invention or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

[0009] A third aspect of the present invention provides an in vitro method for selectively degrading an oligomeric or pathogenic form of a target protein, comprising the step of contacting the target protein with TRIM21 and a compound according to the first aspect of the present invention or a pharmaceutically acceptable salt thereof.

[0010] A fourth aspect of the present invention provides a compound having the structure of formula (IV).

[0011] A fifth aspect of the present invention relates to a compound having the structure of formula (V).

[0012] Further aspects and embodiments of the present invention are described below and in the accompanying drawings.

[0013] All preferred features of the second and subsequent embodiments of the present invention are the same as those relating to the first embodiment, with necessary modifications. [Brief explanation of the drawing]

[0014] [Figure 1] Figure 1 shows the thermal stabilization of Trim21 PRYSPRY (10 μM) in the presence of compounds 36, 37, and 38 (100 μM) compared to a control with DMSO alone. [Figure 2] Figure 2 shows the quenching of the intrinsic fluorescence of Trim21 PRYSPRY upon ligand binding - compounds 36 (black circles), 37 (dark gray squares), and 38 (gray triangles). The data were fitted using a four-parameter binding model. [Figure 3] Figure 3 shows the fluorescence polarization substitution assay. White circles indicate the titration of human IgG-Fc to Trim21 PRYSPRY labeled with Alexa-488. For the substitution experiment, 0.5 μM IgG-Fc was used along with the titrations of competing compounds 36 (black circles), 37 (dark gray squares), and 38 (gray triangles). [Figure 4] Figure 4 shows lane views (a) and quantifications (b) of Hsp60 protein levels and TRIM21 protein levels normalized to DMSO conditions after RPE-1 cells were treated with DMSO or 2.5 μM compound 37 for 18 hours and whole cell extracts were collected for capillary-based immunoblotting. Significance is based on Student's t-test (two-sided). [Figure 5]Figure 5 shows a schematic diagram of the assay for TRIM21-mediated degradation. Expression of the NbGFP-Fc antibody construct leads to TRIM21 recruitment to H2B-mEGFP, causing H2B-mEGFP proteasomal degradation. Compound 37 competes with NbGFP-Fc for binding to TRIM21 PRYSPRY and therefore inhibits TRIM21 recruitment and H2B-mEGFP degradation. [Figure 6] Figure 6 shows the normalized H2B-mEGFP fluorescence values ​​for each condition (DMSO or compound 37) against NbGFP-FcH433A (an Fc variant that cannot bind to TRIM21 PRYSPRY; a control with no degradation). A value of 1 is equivalent to no degradation. (A) shows the time course of degradation in cells treated with DMSO or 50 μM and 100 μM of compound 37. (B) shows the dose-response curve of H2B-mEGFP degradation in cells treated with titration of compound 37 or the corresponding DMSO concentration. [Figure 7] Figure 7 shows a schematic diagram of the inhibition of the viral neutralization assay. Cells are infected with adenovirus 5 (Adv5) in the presence of an anti-Adv5 antibody. Normally, TRIM21 neutralizes the antibody-bound virus, resulting in a reduction of infection as measured by a fluorescent reporter gene (EGFP). The compound competes with the anti-Adv5 antibody for binding to TRIM21 PRYSPRY and therefore inhibits TRIM21-mediated neutralization. [Figure 8] Figure 8 shows the results of the experiment shown in Figure 7. The TRIM21 compounds dose-dependently rescued the infection (increased EGFP), demonstrating that they inhibit TRIM21 in cells. [Figure 9] Figure 9 shows the confluence of cells after compound treatment. [Figure 10] Figure 10 shows the thermal stabilization of Trim21 PRYSPRY in the presence of compound 51 (compound 37 as a mixed stereoisomer) and compound 71 (compound 37 bonded to a short PEG linker and chloroalkane). This figure shows that the bond is only slightly affected, as significant thermal stabilization is still observed. [Figure 11] Figure 11 shows the AUC velocity experiment; the c(s) distribution shows that both TRIM21 PRYSPRY (solid black line) and HaloTag (solid gray line) alone are monomers that settle at 2.3S (T, Sw,20=2.5S) and 2.9S (H, Sw,20=3.2S), respectively, with calculated masses of 22.0kDa and 34.4kDa, and friction ratios of 1.143 and 1.142, respectively. The mixture alone (dotted black line) yields a single broad distribution, which may simply be a cumulative Gaussian distribution of two individual unbound species (T - H). Addition of 10 μM ligand (dashed black line) reduces the concentration of T21 PRYSPRY and significantly reduces HaloTag, resulting in the appearance of a species that settles at 3.7S (T-71-H, Sw,20=4.1S). This species has an approximate predicted mass of 57.3 kDa for a three-component composite, assuming a friction ratio of 1.286 (characteristic of extended non-spherical composites). [Figure 12] Figure 12 shows the AUC equilibrium experiment. In the absence of the compound, the average mass of 30,316 Da is close to the average of the two proteins at equimolar concentrations. As the concentration of the compound increases, the average mass reaches a maximum at 5 μM, where the average mass is 50,482 ± 82 Da, which is close to the value expected for a 1:1 complex. Beyond this point, the average mass decreases as the compound binds to individual components in competition with complex formation. [Figure 13]Figure 13(A) shows a schematic diagram of the cellular three-component complex assay. Cells co-expressing mCherry-TRIM21 and H2B-mEGFP-Halo. The presence of a large mCherry tag on TRIM21 hinders transport to the nucleus. During cell division, transient nuclear membrane collapse allows mCherry-TRIM21 to access chromatin. Nuclear localization of mCherry-TRIM21 after cell division indicates the formation of a three-component complex between mCherry-TRIM21, TRIMTAC, and H2B-mEGFP-Halo. (B) and (C) show representative cells undergoing cell division. RPE-1 TRIM21 KO cells co-expressing mCherry-TRIM21 and H2B-mEGFP-Halo were treated with 1 μM compound 37(B) or 1 μM compound 71(C) and imaged using the IncuCyte system. [Figure 14] RPE-1 TRIM21 KO cells co-expressing H2B-mEGFP-Halo and one of the following: mCherry-TRIM21(A), mCherry-TRIM21ΔRB((B); RING-Box deletion), or mCherry-TRIM21ΔPS((C); PRYSPRY deletion) were treated with titration of compound 37 or compound 71 and imaged using the IncuCyte system. 40 hours after compound addition, the tricomponent complex was quantified by normalizing the red / green overlap area (mCherry-TRIM21-positive nuclei) relative to the total green area (total nuclei). Tricomponent complex formation requires the PRYSPRY domain of TRIM21. Compound 71 exhibits a bell-shaped concentration dependence (Hook effect) on tricomponent complex formation. [Figure 15](A) RPE-1 TRIM21 KO cells co-expressing mCherryTRIM21 and H2B-mEGFP-Halo were treated with titration of the indicated compounds, and tricomponent complex formation was quantified at 48 hours by normalizing the red / green overlap area (mCherry-TRIM21-positive nuclei) to the total green area (total nuclei). (B) RPE-1 cells were seeded at approximately 15% confluence, treated with titration of the indicated compounds or with corresponding DMSO controls, and imaged using the IncuCyte system. Cellular confluence was quantified at 72 hours post-treatment by normalizing the total cell area per image for each compound concentration to the corresponding DMSO control. Cells treated with 40 μM of compound 71 or compound 109 did not reach 100% confluence, indicating toxicity. No toxicity was observed for any of the compounds at concentrations of 20 μM or less. [Figure 16] Stable cell lines of RPE-1 mEGFP-Halo, RPE-1 CAV1-mEGFP-Halo, and RPE-1 Cavin1-mEGFP-Halo were treated with VHL-mobilized PROTAC (HaloPROTAC1) or compounds 37, 71, 109, 110, and 111 at the indicated concentrations and imaged for 48 hours using the IncuCyte system. For each image, GFP fluorescence was quantified by normalizing the GFP integrated density (and therefore the product of area and mean intensity) against the total cell area (phase), and then normalizing it against the corresponding DMSO condition such that a value of 1 (gray dashed line) represents no degradation. HaloPROTAC1 degrades all three proteins, while TRIMTAC (compounds 71, 109, 110, and 111) degrades oligomeric CAV1- and Cavin1-mEGFP-Halo but not monomeric mEGFP-Halo. [Figure 17]Stable cell lines of RPE-1 mEGFP-Halo, RPE-1 CAV1-mEGFP-Halo, and RPE-1 Cavin1-mEGFP-Halo were treated with titration of the indicated compounds for 48 hours, and GFP fluorescence was quantified using the IncuCyte system as described for Figure 16. At all concentrations tested, TRIMTAC compounds (71, 109, 110, and 111) selectively degraded oligomeric CAV1- and Cavin1-mEGFP-Halo more selectively than monomeric mEGFP-Halo, exhibiting a characteristic "PROTAC hook effect" concentration dependence for degradation. [Figure 18] WT or TRIM21 KO cells stably expressing Cavin1-mEGFP-Halo (A) or CAV1-mEGFP-Halo (B) were treated with titration of compound 71 for 48 hours, and GFP fluorescence was quantified using the IncuCyte system as described for Figure 16. The degradation of the oligomeric proteins CAV1- and Cavin1-mEGFP-Halo by compound 71 is TRIM21-dependent. [Figure 19](A) - RPE-1 Cavin1-mEGFP-Halo cells were treated with DMSO, 2.5 μM HaloPROTAC1, or compound 37 for 24 hours, and whole cell extracts were collected for capillary-based immunoblotting. Anti-GFP immunoblotting showed the presence of a smaller species (*-mEGFP-Halo) in addition to full-length Cavin1-mEGFP-Halo, which may correspond to cleavage products lacking the Cavin1 sequence. Treatment with HaloPROTAC1 resulted in a slight reduction of the Cavin1-mEGFP-Halo band and complete loss of the mEGFP-Halo band. Conversely, treatment with compound 71 resulted in a substantial reduction of the Cavin1-mEGFP-Halo band, while the *-mEGFP-Halo band remained unaffected. (B) - Cavin1-mEGFP-Halo and *-mEGFP-Halo bands were normalized to the anti-actin band and expressed as a percentage of the total anti-GFP band intensity. Treatment with compound 71 resulted in a significant reduction in Cavin1-mEGFP-Halo protein levels, but not a significant reduction in *-mEGFP-Halo protein levels. Significance is based on Student's t-test (two-sided). [Figure 20] (A) RPE-1 Cavin1-mEGFP-Halo cells were treated with DMSO or 1.25 μM of the indicated compound (or 10 μM for compound 110) for 48 hours and imaged using the IncuCyte system. Cavin1-mEGFP-Halo cells showed a bright signal at the cell membrane and some diffused cytoplasmic fluorescence under control (DMSO) conditions. (B) RPE-1 Cavin1-mEGFP-Halo cells were seeded on polymer coverslips (Ibidi) in an 8-well chamber and imaged before and after treatment with DMSO or 2.5 μM HaloPROTAC1 or compound 71 using an Etaluma Lumascope LS720 wide-field microscope equipped with a 40× apochromatic 0.95NA air objective lens housed in a 37C 5% CO2 incubator. [Figure 21](A) and (B) Representative examples of individual cells from Figure 20(B) before and 24 hours after treatment with 2.5 μM HaloPROTAC1 or compound 71. The graph shows pixel intensity along the yellow line in the image. [Figure 22] RPE-1 Cavin1-mEGFP-Halo cells were seeded and imaged 2.5 hours after treatment with DMSO, 2.5 μM compound 37, or 2.5 μM compound 37 + 25 μM MG132, as shown in Figure 20(B). The graph shows the pixel intensity along the yellow line in the image. The dashed line indicates the peak maximum cytosolic pixel intensity, and peaks above this line represent membrane-bound fluorescence. [Figure 23] The crystal structures of Trim21-PRYSPRY in complex with compounds 36(A), 37(B), and 38(C) are shown. Trim21 PRYSPRY is shown as a surface representation with several residues forming the binding site, indicated and labeled as rods (white). Key residues are underlined. The ligand itself is shown as a rod (black). (B) also includes an arrow (black) indicating a potential exit for developing a heterobifunctional molecule. This is also shown in (D) using the skeletal representation of compound 37. [Figure 24] The image shows a superposition of the complex between TRIM21 and compound 37, and the complex between TRIM21 and IgG Fc (PDB: 2IWG). Antibody residues that form a native epitope for TRIM21 are shown in white (HNHY, labeled), and compound 37 is shown in black. [Figure 25]Stable cell lines of RPE-1 mEGFP-FKBP(F36V) and RPE-1 Cavin-1-mEGFP-FKBP(F36V) were treated with compound 414 (Cmp 414) (5 μM) or CRBN mobilization (dTAG13) or VHL mobilization (dTAGv1) PROTAC (80 nM), and GFP fluorescence was quantified using the IncuCyte system as shown in Figure 16. dTAG13 and dTAGv1 degrade both FKBP(F36V) fusion proteins, preferentially degrading the monomer mEGFP-FKBP(F36V), while Cmp 414 degrades the oligomeric Cavin1-mEGFP-FKBP(F36V) but not the monomer mEGFP-FKBP(F36V). [Figure 26] Stable cell lines of RPE-1 mEGFP-FKBP(F36V) and RPE-1 Cavin-1-mEGFP-FKBP(F36V) were treated with titration with Cmp 414, dTAG13, or dTAGv1 for 8 hours, and GFP fluorescence was quantified using the IncuCyte system as shown in Figure 16. Cmp 414 degrades the oligomeric Cavin-1-mEGFP-FKBP(F36V) in a concentration-dependent manner, but does not degrade the monomer mEGFP-FKBP(F36V). dTAG13 and dTAGv1 degrade both FKBP(F36V) fusion proteins, preferentially degrading the monomer mEGFP-FKBP(F36V). [Figure 27] WT or TRIM21 KO cells stably expressing Cavin-1-mEGFP-FKBP(F36V) were treated with Cmp 414 titration and imaged for 8 hours using the IncuCyte system. GFP fluorescence was quantified as shown in Figure 16. (A) At 5 μM, Cmp414 rapidly degrades Cavin-1-mEGFP-FKBP(F36V) in WT cells but not in TRIM21 KO cells. (B) Concentration-dependent degradation of Cavin-1-mEGFP-FKBP(F36V) is TRIM21-dependent. [Figure 28](A) The expression construct contains full-length Myd88 fused at the c-terminus to the GyrB domain of Eschericia coli DNA gyrase. The GyrB domain dimerizes upon binding to the bivalent antibiotic coumermycin. Coumermycin-induced GyrB dimerization induces oligomerization of Myd88 and assembly of active midosomes. The mEGFP tag is included for visualization. The FKBP(F36V) domain enables recruitment of TRIM21 via Cmp 414. (B) U2OS cells expressing Myd88-GyrB-mEGFP-FKBP(F36V) were treated with coumermycin (100 nM) for 5 hours to induce midosome assembly or mock-treated (control). Five hours after cumermycin / mock treatment (time 0, dotted line), cells were treated with DMSO, Cmp 414 (5 μM), or compound 37 (Cmp 37) (5 μM), and GFP fluorescence was imaged and quantified using the IncuCyte system as shown in Figure 16. Cmp 414 did not degrade Myd88 in control-treated cells, but rapidly degraded Myd88 in cumermycin-treated cells. The addition of DMSO or Cmp 37 did not affect Myd88 levels. (B) Representative image from A. In control-treated cells, Myd88 is monomeric and shows a diffused cytosolic distribution. Cumermycin treatment induces midsome assembly, as observed by the accumulation of cytosolic Myd88 spots (time -17 min). Cmp 414 degrades Myd88 spots but does not degrade diffused Myd88, resulting in a partial reduction of Myd88 fluorescence in cumermycin-treated cells, as quantified in A. [Figure 29](A) The expression construct contains full-length RIPK3 fused at the N-terminus to mEGFP for visualization, and FKBP(F36V) for TRIM21 mobilization via Cmp 414. T / S / Z (TNFα / Smac mimetic / ZVAD-FMK) treatment induces RIPK3 oligomerization (necrosome) and promotes necroptosis. (B, C) RPE-1 cells stably expressing FKBP(F36V)-mEGFP-RIPK3 were treated with T / S / Z or mocked (control) for 8 hours in the presence of Cmp 414 titration to induce RIPK3 oligomerization, and GFP fluorescence was imaged and quantified using the IncuCyte system as shown in Figure 16. Cmp 414 degrades oligomeric RIPK3 (T / S / Z) in a concentration-dependent manner but does not degrade monomeric RIPK3 (control). TNFα was used at 20 ng / μl. Smac mimetic agent (AZD5582) was used at 100 nM. ZVAD-FMK was used at 25 μM. [Figure 30]RPE-1 cells expressing FKBP(F36V)-mEGFP-RIPK3 were treated with T / S / Z or mock (control) in the presence of DMSO, Cmp 414 (2.5 μM), or the RIPK3 kinase inhibitor GSK'872 (0.3 μM) to induce RIPK3 oligomerization and necroptosis, and GFP fluorescence and cell area were quantified using the IncuCyte system. (A) Cmp 414 does not degrade monomeric RIPK3 in control-treated cells, but rapidly degrades oligomeric RIPK3 in T / S / Z-treated cells. (B) Representative image from A. In control-treated cells, RIPK3 is monomeric and shows a diffused cytosolic distribution. T / S / Z treatment induces RIPK3 oligomerization, as observed by the accumulation of cytosolic RIPK3 spots. Cmp 414 degrades T / S / Z-induced RIPK3 oligomers during assembly, leading to a reduction in RIPK3 signaling. GSK'872 inhibits RIPK3 kinase activity but does not affect RIPK3 oligomerization. (C~E) Necroptosis cell death is quantified by a reduction in total cell area. (C) Cmp 414 (2.5 μM) partially rescues the viability of T / S / Z-treated cells. (D) Representative phase images (top panel) and segmented cell regions (white, bottom panel) 48 hours post-treatment. (E) Quantification of cell area normalized to control / DMSO-treated cells 48 hours post-treatment. Significance is based on one-way ANOVA (*P<0.05, ****<0.0001). [Figure 31](A) RPE-1 TRIM21 KO cells co-expressing Cavin-1-mEGFP-FKBP(F36V) and SmBiT-TRIM21 with either LgBiT-TRIM21 or LgBiT-TUBES were seeded in a white 96-well plate. The medium was changed to CO2-independent medium containing Vivazine live cell luciferase substrate, and incubated at 37C for 30 minutes. Luminescence was measured using a Promega GloMax Discover plate reader with an integration time of 2 seconds every 2 minutes for 30 minutes before the addition of compound 414 (5 μM) or DMSO, and then for a further 90 minutes thereafter. Luminescence values ​​were normalized within each well to the time immediately before compound / DMSO addition, and then the values ​​for compound 414-treated wells were normalized to the DMSO-treated control. (B) RPE-1 TRIM21 KO cells co-expressing Cavin-1-mEGFP-FKBP(F36V) and SmBiT-TRIM21 with either LgBiT-TRIM21 or LgBiT-TUBES were seeded in 96-well plates, the culture medium was changed to Fluorobrite medium containing either compound 414 (5 μM) or DMSO, and GFP fluorescence was quantified using the IncuCyte system as shown in Figure 16. [Modes for carrying out the invention]

[0015] This disclosure describes a bifunctional compound that binds to a target protein and recruits the E3 ligase TRIM21 to promote the degradation of the target protein. This disclosure provides a compound comprising a TRIM21-binding moiety that is a small molecule ligand (i.e., having a molecular weight of 1000 Da or less, e.g., 100 Da to 1000 Da or 100 Da to 600 Da) and a moiety capable of binding to the target protein (also called the "protein-targeting moiety"). The compounds of the present invention induce molecular clustering of multiple TRIM21 molecules, thereby inducing a process of ubiquitination that results in efficient degradation of the target protein. This allows the bifunctional compound to selectively degrade the target protein bound to multiple copies of the compound. This is therapeutically very beneficial as it provides superior potency while avoiding potential toxicity. One of these positive outcomes is improved therapeutic metrics.

[0016] The compounds of the present invention have two criteria. First, the compounds of the present invention enable the formation of a three-component complex between the compound of the present invention, a target protein, and TRIM21. Second, these three-component complexes form part of a higher-order structure in which multiple TRIM21 molecules are placed in close proximity. The compounds of the present invention are capable of activating the enzymatic activity of TRIM21, resulting in the ubiquitination and degradation of the target protein. This makes it possible to selectively degrade the oligomeric form of the protein because protein monomers can satisfy the first criterion but not the second. In other words, the present invention relates to compounds that are capable of forming a three-component complex with a target protein and TRIM21, and that this three-component complex can be part of a higher-order structure that results in the activation of TRIM21.

[0017] In one embodiment, the target protein may be a protein having a first form containing two or more binding sites to the protein targeting moiety, and a second form having a single binding site to the protein targeting moiety. The protein targeting moiety can bind to both the first and second forms of the protein. However, since multiple copies of TRIM21 are required to activate the proteolytic pathway, the compound selectively degrades the first form of the protein. The multiple binding sites allow for the recruitment of multiple copies of TRIM21 to the target protein, resulting in the clustering of TRIM21 required for TRIM21 activation.

[0018] In one embodiment, the target protein may have both a pathogenic and a non-pathogenic form. The pathogenic form of the target protein may include a repeat domain or be an oligomeric form of the protein, for example, a form of the protein that provides two or more binding sites for the compound of the present invention. The protein targeting moiety may bind to both the pathogenic and non-pathogenic forms of the protein. However, the compound selectively degrades the pathogenic form of the protein so that multiple copies of TRIM21 are recruited to the target protein, enabling activation of the degradation pathway. The multiple copies of TRIM21 recruited to the target protein result in the clustering of TRIM21 required for TRIM21 activation.

[0019] In one embodiment, the compound recruits TRIM21 to enable TRIM21-mediated degradation of the oligomeric form of the target protein, in a manner selective to its monomeric form.

[0020] "Selective degradation" or similar terms mean that a compound degrades one form of a protein more than another. For example, "selective degradation" or similar terms mean that the pathogenic form (e.g., the oligomeric form) is degraded, while the non-pathogenic form of the target protein (e.g., the monomeric form) is not. While we do not wish to be bound by theory, this is thought to be because TRIM21 activation is induced by the clustering of multiple adjacent TRIM21 molecules. Multiple compounds of the present invention can bind to the oligomeric form of a protein, thereby enabling the clustering of multiple TRIM21 molecules and activation of the TRIM21 proteolytic pathway, resulting in ubiquitination and degradation of the pathogenic form (e.g., the oligomeric form) of the protein.

[0021] Therefore, the molecules of the present invention have a different mechanism of action from known PROTACs in that they require multiple compounds of the present invention to bind to a target protein and activate a proteolytic pathway. Consequently, the molecules of the present invention can selectively degrade the oligomeric form of the same protein, but not the monomeric form. This is in contrast to known PROTACs that can degrade both the oligomeric and monomeric forms of proteins. In the present invention, efficient degradation is not induced simply by the molecules binding to the target protein unless the protein is, for example, in an oligomeric form, so that multiple TRIM21 molecules can be recruited in close proximity to induce a proteolytic pathway. The molecules of the present invention can degrade oligomers more efficiently, in contrast to known PROTACs.

[0022] Therefore, the present invention relates to the compound of formula (I). ALB (I) [In the formula, A is the part that connects to TRIM21. L is a linker, B is the part that binds to the target protein. Or provide a pharmaceutically acceptable salt thereof.

[0023] The target protein is one to which binding of the compound of formula (I) can lead to TRIM21 clustering and enable TRIM21 activation. TRIM21 activation can lead to the degradation of the target protein.

[0024] In one embodiment, the target protein is a protein that may be in a first form (e.g., a pathogenic form) that provides multiple binding sites for B. In one embodiment, the target protein may be capable of forming an oligomeric species. In the oligomeric form, multiple binding sites for B are provided.

[0025] The bifunctional compound of the present invention binds to TRIM21 via "A". TRIM21 is a member of the tripartite motif-containing protein family, and the sequence of human TRIM21 can be accessed as P19474 in the UniProt database. The sequence of human TRIM21 is shown in Sequence ID No. 1. References to TRIM21 herein usually refer to human TRIM21. TRIM21 has ubiquitin ligase activity and can initiate proteolysis via the ubiquitin-proteasome system (UPS).

[0026] In the compound of the present invention having the structure of formula (I), "A" is the TRIM21 binding site. In one embodiment, "A" binds to the PRYSPRY domain of TRIM21 or a variant thereof. The PRYSPRY domain consists of the PRY region and SPRY region at positions 286-337 and 339-465 of the human TRIM21 amino acid sequence shown in Sequence ID No. 1.

[0027] In one embodiment, "A" binds to one or more residues selected from W381, W383, D355, F369, L370, L371, F450, Y328, H368, S447, M330, D452, R364, Q395, Y393, E389, and K387 of human TRIM21 shown in SEQ ID NO: 1. In one embodiment, "A" binds to one or more residues selected from W381, W383, D355, F369, L370, L371, F450, Y328, H368, S447, and M330. In one embodiment, "A" binds to one or more residues selected from D452, R364, Q395, Y393, E389, and K387. In one embodiment, "A" binds to at least residue W381. In one embodiment, "A" binds to residues W381, W383, and D355 of TRIM21.

[0028] In some embodiments, "A" binds to residue D355 of TRIM21. In some embodiments, "A" includes a portion capable of forming a hydrogen bond (e.g., a charged hydrogen bond) with residue D355 of TRIM21. In some embodiments, "A" binds to residue W381 of TRIM21. In some embodiments, "A" includes a portion capable of hydrophobic stacking with residue W381 of TRIM21. In some embodiments, "A" binds to residue W383 of TRIM21. In some embodiments, "A" includes a portion capable of hydrophobic stacking with residue W383 of TRIM21.

[0029] "A" may compete with IgG binding to TRIM21. In one embodiment, "A" may compete with Fc binding to TRIM21.

[0030] The binding affinity of the compounds described herein to TRIM21 is determined using a method known in the art (e.g., fluorescent quenching) by the dissociation constant (K) of the adduct between the compound and TRIM21. d) may be measured by the value. In certain embodiments, the adduct comprises a compound and TRIM21 PRYSPRY, which are bonded to each other (e.g., non-covalently). In certain embodiments, the compound of formula (I) is K to TRIM21 in the range of 0.1 nM to 1000 μM, for example 1 nM to 1000 μM, preferably 10 nM to 100 μM. d It has a value.

[0031] Binding to specific residues can be determined by comparing the binding affinity to the wild type versus the binding affinity to the mutant (for example, comparing the binding affinity of wild-type TRIM21 with that of TRIM21 mutated at the target residue, e.g., D355, W381, or W383) using a suitable biophysical technique such as differential scanning fluorescence (DSF), isothermal titration calorimetry (ITC), or surface plasmon resonance (SPR). The contribution of the specific residue can then be directly quantified by the difference in binding energy. Furthermore, binding to specific residues can be demonstrated by elucidating the structure of the TRIM21 PRYSPRY domain complexed with the ligand to show its interaction with the residue.

[0032] As described herein, the TRIM21 binding moiety (e.g., "A") targets the C-terminal PRYSPRY domain and binds within the "SPRY" element. Specifically, it targets the concave binding surface surrounded by six flexible loops within "SPRY". The TRIM21 binding moiety described herein is capable of replacing the intrinsic ligand IgG and competing with it for binding. This moiety functionally replaces the linear sequence of "HNHY" found at the C-terminus of IgG. This moiety may do so by performing interactions similar to those of the "HNHY" motif, such as hydrophobic stacking interactions (e.g., with W381 and W383) and charged hydrogen bonding (e.g., with D355A). TRIM21 binding agents can be obtained by selecting molecules whose complex formation with the PRYSPRY domain causes an increase in the PRYSPRY melting point (Tm), alters the intrinsic tryptophan fluorescence of residues W381 or W383, and / or replaces IgG Fc. Such methods are described in the examples.

[0033] In some embodiments, A has the structure of formula (II).

[0034] [ka]

[0035] [In the formula, R 1 and R 2 These are, independently, hydrogen and C 1~2 Selected from alkyl groups, or R 1 and R 2 One of them is hydrogen, and the other is selected from an amino acid residue and a covalent bonding site to L. R 3is selected from hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted heteroalkyl, optionally substituted heteroalkenyl, optionally substituted heteroalkynyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted aryl and optionally substituted heteroaryl, R 3 optionally contains a site of a covalent bond to L, R 4 is hydrogen, halo or C 1~2 alkyl, R 5 is hydrogen or C 1~2 alkyl, R 6 is optionally substituted alkyl, or R 5 and R 6 combine to form an optionally substituted heterocyclic ring, R 7 is

[0036]

Chemical formula

[0037] a site of a covalent bond to L, or

[0038]

Chemical formula

[0039] and R 8 is hydrogen or C 1~2 alkyl, R 9 is optionally substituted alkyl, R 14 and R 15 are each independently selected from hydrogen and C 1~2 alkyl, R 7 in the structure of

[0040] [ka]

[0041] This indicates the site of covalent bonding to L. However, this is conditional on A containing a covalent bond site to L. It holds.

[0042] In some embodiments, A has the following structure:

[0043] [ka]

[0044] [In the formula, R 1 and R 2 These are, independently, hydrogen and C 1~2 Selected from alkyl groups, or R 1 and R 2 One of them is hydrogen, and the other is selected from L-amino acid residues and the site of covalent bonding to L. R 7 teeth,

[0045] [ka]

[0046] , the site of covalent bonding to L, or

[0047] [ka]

[0048] is It holds.

[0049] In some embodiments, A has the following structure:

[0050] [ka]

[0051] [In the formula, R 1 and R 2 These are, independently, hydrogen and C 1~2 Selected from alkyl groups, or R 1 and R 2 One of them is hydrogen, and the other is selected from a D-amino acid residue and a covalent bonding site to L. R 7 teeth,

[0052] [ka]

[0053] , the site of covalent bonding to L, or

[0054] [ka]

[0055] is It holds.

[0056] If the structure of part A is based on D-amino acids, this may be beneficial in preventing cleavage by proteolytic enzymes.

[0057] In some embodiments, A has the structure of formula (IIa), (IIb), or (IIc):

[0058] [ka]

[0059] [In the formula, each

[0060] [ka]

[0061] Independently,

[0062] [ka]

[0063] Selected from, R 1 and R 2 These are, independently, hydrogen and C 1~2 Selected from alkyl groups, or R 1 and R 2 One is hydrogen, and the other is an amino acid residue. R 3 This is selected from hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted heteroalkyl, optionally substituted heteroalkenyl, optionally substituted heteroalkynyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted aryl, and optionally substituted heteroaryl. R 4 is hydrogen, halo, or C 1~2 It is alkyl, Each R 5 These are, independently, hydrogen or C 1~2 It is alkyl, R 6 is an alkyl that may be substituted, Or, R 5 and R 6 If R is bonded to an adjacent atom, 5 and R 6 These combine to form a complex ring, which may be substituted. R 8 is hydrogen or C 1~2 It is alkyl, R 9 is an alkyl group which may be substituted, R 18 and R 19 These are, independently, hydrogen and C 1~2 Selected from alkyl groups, R 20[This is an alkyl group that may be substituted.] It holds.

[0064] In some embodiments, R 1 , R 2 and R 4 It is hydrogen.

[0065] In some embodiments, R 3 R was selected from optionally substituted aryls and optionally substituted heteroaryls. For example, R 3 This can be selected from furan and optionally substituted phenyl.

[0066] In some embodiments, R 7 (If it exists),

[0067] [ka]

[0068] [In the formula, R 9 is selected from aryls which may be substituted with -alkylene- and heteroaryls which may be substituted with -alkylene-, and optionally R 9 [These are selected from aryls which may be substituted with -CH2- and heteroaryls which may be substituted with -CH2-] In some embodiments, R 9 is selected from aryls which may be substituted with -alkylene- and heteroaryls which may be substituted with -alkylene-, and optionally R 9 This is selected from aryls which may be substituted with -CH2- and heteroaryls which may be substituted with -CH2-.

[0069] In some embodiments, R 7 (If it exists),

[0070] [ka]

[0071] [wherein, R 9 is

[0072]

Chem.

[0073] is selected from, and R 9 in the structure of

[0074]

Chem.

[0075] is the site of the covalent bond to the remainder of R 7 . In some embodiments, R is 9 is

[0076]

Chem.

[0077] selected from.

[0078] In some embodiments, R 9 is selected from a histidine side chain, a phenylalanine side chain, a tryptophan side chain, a tyrosine side chain, a cysteine side chain, a serine side chain, a threonine side chain, a methionine side chain, an asparagine side chain, and a glutamine side chain.

[0079] In some embodiments, R 5 and R 6 are combined to be hydroxy, nitro, cyano, halo, -NR 10 R 11、which may be substituted by an optionally substituted alkyl, an optionally substituted alkoxy, an optionally substituted cycloalkyl, an optionally substituted heterocyclyl, an optionally substituted aryl, an optionally substituted heteroaryl, an optionally substituted -O-cycloalkyl, an optionally substituted -O-heterocyclyl, an optionally substituted -O-aryl, or an optionally substituted -O-heteroaryl, to form a 5-membered heterocyclic ring, R 10 and R 11 are each independently H, an optionally substituted alkyl, or an optionally substituted cycloalkyl.

[0080] In some embodiments, A has the structure of formula (III)

[0081]

Chemical formula

[0082] [wherein, each of rings E, F, C and D is independently selected from an optionally substituted cycloalkyl, an optionally substituted heterocyclyl, an optionally substituted aryl, and an optionally substituted heteroaryl, in the structure of formula (III)

[0083]

Chemical formula

[0084] indicates the site of the covalent bond to L] has.

[0085] In some embodiments, ring C is selected from aryl and heteroaryl, and the aryl or heteroaryl is substituted by an aryl or heteroaryl.

[0086] In some embodiments, A is

[0087] [ka] TIFF2026525346000022.tif241164TIFF2026525346000023.tif232159

[0088] [In the formula, in the structure of A

[0089] [ka]

[0090] [This indicates the site of the covalent bond to L.] Selected from.

[0091] In some embodiments, A is

[0092] [ka]

[0093] [In the formula, in the structure of A

[0094] [ka]

[0095] [This indicates the site of the covalent bond to L.] Selected from.

[0096] In the structure of formula (I), A includes a covalent bond site to L. Preferably, A includes one covalent bond site to L. If A has the structure of formula (II), R 1 / R 2 , R 3 and R 7 One or more of these may include a covalent bonding site to L. In one embodiment, R 1 and R 2 One of them may be a covalent bond site to L, and the other may be a hydrogen atom. In another embodiment, R7 is the site of the covalent bond to L. In another embodiment, R 7 is

[0097] [Chemical formula]

[0098] [wherein,

[0099] [Chemical formula]

[0100] indicates the site of the covalent bond to L, and R 8 and R 9 are as defined in formula (II)] is. When R 3 is said to be the "optionally substituted" moiety, this includes embodiments where the substituent is the site of the covalent bond to L. Thus, in one embodiment, R 3 includes the site of the covalent bond to L. For example, R 3 is

[0101] [Chemical formula]

[0102] [wherein,

[0103] [Chemical formula]

[0104] indicates the site of the covalent bond to L, and * indicates the site of the covalent bond to the remainder of A] may be.

[0105] In one embodiment, the molecular weight of A is 1000 Da or less. In another embodiment, the molecular weight of A is in the range of 100 Da to 1000 Da. The molecular weight can be calculated based on the standard atomic weights of chemical elements determined and published by the International Union of Pure and Applied Chemistry (IUPAC) Committee on Isotope Abundances and Atomic Weights (CIAAW).

[0106] In the compound of the present invention having the structure of formula (I), B is a portion that binds to the target protein and is also referred herein as the "target protein binding portion" or "protein targeting portion" or similar names. B is a small molecule that binds to the target protein and positions / presents that protein in close proximity to TRIM21, so that protein degradation by ubiquitin ligase can occur when multiple TRIM21 molecules are present.

[0107] The target protein may be a protein having a morphology that includes multiple binding sites to B, which, when bound by the compound of the present invention, can result in the clustering and activation of TRIM21.

[0108] TRIM21 activation can be measured by methods discussed in the examples and known in the art. For example, intracellular TRIM21 clustering can be directly measured using an in vitro ubiquitination assay with purified TRIM21 and target proteins, or by using a fluorescence resonance energy transfer assay (FRET), a bioluminescence resonance energy transfer assay (BRET), or a protein fragment complementation assay (PCA).

[0109] In one embodiment, TRIM21 activation can be determined by a NanoBiT split luciferase assay. In the NanoBiT split luciferase assay, luciferase enzyme activity is reconstituted only when the two protein fragments, LgBiT and SmBiT, are placed in close proximity. Specifically, the NanoBiT split luciferase assay may involve co-expressing two N-terminally tagged versions of TRIM21, LgBiT-TRIM21 and SmBiT-TRIM21, in TRIM21 KO cells. The compound to be tested is added to the cells. Luciferase enzyme activity is measured. The presence of luciferase activity indicates TRIM21 activation.

[0110] In the absence of the compounds of the present invention, TRIM21 is not clustered, and therefore the LgBiT tag and SmBiT tag do not interact, resulting in no luciferase activity. For example, when the compounds of the present invention having the structure of formula (I) as defined herein are present, they bind to a target protein containing multiple binding sites for B. This results in TRIM21 clustering, which can be measured by the luciferase enzyme activity reconstituted by the interaction of the LgBiT tag and SmBiT tag at the N-terminus of the clustered TRIM21 molecule.

[0111] In one embodiment, the target protein may have a first and second form, which are at least two different forms, the first protein form providing multiple binding sites for "B". The second protein form may provide only a single binding site for "B". B can bind to both the first and second forms of the target protein. However, the first form of the target protein allows TRIM21 to cluster around the target protein when a compound having the structure of formula (I) is bound, whereas the second form does not allow for the clustering of TRIM21.

[0112] "Protein morphology" includes, but is not limited to, the monomeric, oligomeric, and multimeric forms of proteins, as well as multicomponent complexes. The oligomeric, multimeric, and multicomponent forms of proteins contain two or more monomeric molecules of the bound protein.

[0113] The oligomeric forms of proteins include, but are not limited to, multimeric forms and aggregates of proteins. Oligomers include, but are not limited to, dimers, trimers, tetramers, pentamers, and hexamers, or aggregates having larger molecules, such as 2, 3, 4, 5, 6 or more units. Oligomers may be homooligomers or heterooligomers. If an oligomer is heterooligomer, it contains at least two units of target protein to provide multiple binding sites for "B".

[0114] "Multiple binding sites" means that the protein morphology has two or more binding sites to a protein binding portion, for example, the protein binding portion "B" has 2, 3, 4, 5, 6 or more binding sites to the compound, that is, multiple compounds of the present invention (i.e., compounds of formula (I)) can bind to a target protein morphology via their respective protein binding portions "B". The protein morphology may be a monomeric protein, or a heteromeric oligomer or homomeric oligomer of a protein.

[0115] In one embodiment, the target protein may be a protein having pathogenic and non-pathogenic forms, the pathogenic form containing two or more binding sites to the compound of the present invention. The pathogenic form may be an oligomeric form of the protein, and the non-pathogenic form may be a monomeric form of the protein. The compound of the present invention enables selective degradation of the oligomeric form of the protein relative to the monomeric form.

[0116] In some embodiments, the target protein is a protein having a pathogenic form that includes multiple binding sites for the compound of the present invention. For example, a mutant form of the protein having multiple binding sites for the protein binding portion compared to the wild-type non-pathogenic form of the protein. The compound of the present invention enables selective degradation of the pathogenic form having multiple binding sites for the compound compared to the non-pathogenic form of the protein that does not contain multiple binding sites.

[0117] In some embodiments, the target protein is a pathogenic form of a protein that is part of a multicomponent complex. The multicomponent complex includes multiple subunits of the target protein, as well as other proteins and / or compounds that form the complex.

[0118] As used herein, the term “pathogenic form” or similar terms may mean a form or conformation of a protein that causes or is associated with a disease, and therefore is present in the presence of a disease that involves aggregates or complexes of multiple copies of the protein. The pathogenic form of a protein has multiple binding sites to the protein targeting moiety, e.g., two or more binding sites. The pathogenic form allows for the clustering of TRIM21 when a compound of formula (I) is bound to it. The term “non-pathogenic form” or similar terms are used to refer to a normal form of a protein whose presence is not associated with a disease. The non-pathogenic form may have a single binding site to the protein targeting moiety. The non-pathogenic form does not allow for the clustering of TRIM21 when a compound of formula (I) is bound to it.

[0119] In some embodiments, the target protein is a protein having monomeric and oligomeric forms, where the oligomeric form is associated with the presence of disease. Because TRIM21 does not cluster around the monomeric form of the target protein, the compounds of the present invention enable selective degradation of the oligomeric form of the protein relative to the monomeric form.

[0120] In some embodiments, target proteins can form aggregates. For example, target proteins may include, but are not limited to, tau, synuclein (including α-synuclein), amyloid-beta, islet amyloid polypeptide (IAPP), serum amyloid-A (SAA), prion proteins, misfolded transthyretin protein (TTR), TAR DNA-binding protein 43 (TDP-43), NOTCH3 receptor, mutant cystatin C, medin, dipeptide repeat proteins, and fused-in-sarcoma. For example, the target protein may be a protein selected from tau, synuclein (including α-synuclein), amyloid-beta, prion proteins, TAR DNA-binding protein 43 (TDP-43), dipeptide repeat proteins, and fused-in-sarcoma. These proteins can form aggregates or oligomers associated with neurodegenerative diseases of impairment. Since multiple compounds of the present invention can bind to aggregates and lead to clustering of TRIM21, the compounds of the present invention cause selective degradation of aggregates.

[0121] In some embodiments, the target protein has an elongated repeat element in the case of the pathogenic form. For example, mutant huntingtin may be the target protein, but is not limited to it. Mutant huntingtin may contain an elongated polyglutamine repeat and is associated with Huntington's disease. The polyglutamine repeat provides multiple binding sites to the protein targeting moiety (B), which can result in clustering of TRIM21. Mutant huntingtin provides enough binding sites to the compound of the present invention to enable clustering of TRIM21.

[0122] In some embodiments, the target protein is involved in signaling pathways in inflammation and / or cancer, and preferably the protein is selected from Myd88, IRAK4, IRAK2, IRAK1, TRAF6, NLRP3, RIPK3, and ASC. For example, the protein may be selected from Myd88, IRAK4, IRAK2, IRAK1, TRAF6, NLRP3, and ASC. These proteins are part of a multicomponent complex containing multiple (e.g., two or more) units of the target protein. The multiple units of the protein in the complex provide multiple binding sites for the protein targeting portion (B), which can result in clustering of TRIM21.

[0123] In some embodiments, the target protein is a viral protein that forms an oligomer, preferably selected from the nucleoproteins of CoV2 and influenza virus. These proteins form multimers, providing multiple binding sites to the protein targeting region (B), which can enable clustering of TRIM21.

[0124] In some embodiments, the target protein is selected from bromodomain-containing protein 4 (BRD4), mutant gelsolin, misfolded rhodopsin, and atrial natriuretic peptide. These proteins provide multiple binding sites to the protein targeting region (B), which can enable clustering of TRIM21.

[0125] In some embodiments, the target protein is an intracellular protein.

[0126] "B" may be selected from, or based on, known ligands of the target protein.

[0127] "B" includes a covalent bonding site to L. In some embodiments, B is derivatized to include a covalent bonding site to L before conjugation with the remainder of the compound of formula (I).

[0128] As those skilled in the art will understand, if B is a known ligand for the target protein, it is present in the compound of formula (I) in a modified form that allows for covalent bonding to L. For example, if B is methylene blue:

[0129] [ka]

[0130] If so, it is in the following form in the compound of formula (I)

[0131] [ka]

[0132] [In the formula,

[0133] [ka]

[0134] [This indicates the site of the covalent bond to L.] It can exist in that form.

[0135] In some embodiments, the target protein is tau. Tau protein is a protein found primarily in nerve cells in the central nervous system. In healthy neurons, tau binds to microtubules and regulates microtubule stability, which is important for axonal elongation and neuronal plasticity. However, tau can also form insoluble aggregates in cells. The accumulation of abnormal tau aggregates in neurons is a significant pathological feature in several neurodegenerative disorders, including Alzheimer's disease. As used herein, reference to “tau” refers to all forms of the tau protein, including normal cellular tau, as well as tau aggregates, fibrils, aggregates, and other abnormal conformations. This includes post-translational modified versions of tau, including phosphorylated, acetylated, glycosylated, ubiquitinated, and variants modified in other ways. This also includes variant forms of tau, particularly those associated with genetically transmitted predispositions to neurodegenerative diseases. Thus, in pathogenic forms, tau aggregates into aggregates, also called oligomeric or multimeric forms. Tau aggregates, oligomers, and multimers are used to represent the same thing and are interchangeable for the purposes of this invention.

[0136] Compounds that bind to tau are known in the art. Small molecule ligands for tau are well known, for example, in Leuzy, A et al (2019) and Silva et al (2019). Silva et al (2019) proposed the use of tau-binding small molecules previously used for tau PET imaging to promote tau degradation. Such small molecules, such as F-AV-1451, may be useful in the context of the present invention. Various other tau PET tracers and other tau-binding small molecules are available. Therefore, if the target protein is tau, B may be based on compounds including, but not limited to, PI-2014, FDDNP, AV680(T808), GTP-1, THK523, THK5105, PBB3, AV1451(T807), THK5117, THK5351, N-methyllansoprazole, (E)-6-iodo-2-styryl-1H-benzo[d]imidazole, RO6958948, PI-2620, JNJ-067, APN-1607(PM-PBB3), MK-6240, and methylene blue.

[0137] In some embodiments, B is

[0138] [ka]

[0139] [In the formula, in the structure of B

[0140] [ka]

[0141] [This indicates the site of the covalent bond to L.] These are selected from. These parts can be used when the target protein is tau.

[0142] In one embodiment, the target protein is BRD4. In such an embodiment, B may be selected from, for example, JQ1 and apabetalon.

[0143] In the compound of the present invention, L is a linker that covalently connects the target protein binding portion (B) and the TRIM21 binding portion (A).

[0144] In some embodiments, the linker is a bond. In some embodiments, the linker is a chain of 2 to 20 carbon atoms, one or more of which are optionally replaced by 1 to 12 ethylene glycol units and / or heteroatoms selected from O, N, S, and P. In some embodiments, the linker includes poly(ethylene glycol) (PEG), unsubstituted alkylene, substituted alkylene, or a combination thereof.

[0145] In some embodiments, the linker is

[0146] [ka] TIFF2026525346000037.tif102166

[0147] [In the formula, in the structure of the linker

[0148] [ka]

[0149] Each of these cases indicates a covalent bond to A or B. Selected from.

[0150] A further aspect of the present invention is the structure of formula (IV):

[0151] [ka]

[0152] [In the formula, R 1 and R 2 These are, independently, hydrogen and C1~2 is selected from alkyl, or R 1 and R 2 one of which is hydrogen and the other is an amino acid residue, R 3 is selected from hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted heteroalkyl, optionally substituted heteroalkenyl, optionally substituted heteroalkynyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted aryl and optionally substituted heteroaryl, R 4 is hydrogen, halo or C 1~2 alkyl, R 5 is hydrogen or C 1~2 alkyl, R 6 ]>is optionally substituted alkyl, or R 5 and R 6 are combined to form an optionally substituted heterocyclic ring, R 9 is optionally substituted alkyl, R 12 and R 13 are each independently selected from hydrogen and C 1~2 alkyl, or one of R 12 and R 13 is hydrogen and the other is optionally substituted C 1~2 alkyl and -CHR 9 C(O)NR 14 R 15 selected from R 14 and R 15 are each independently selected from hydrogen and C 1~2 alkyl selected] relates to a compound having

[0153] A further aspect of the present invention is a structure of formula (IVa), (IVb) or (IVc):

[0154] [ka]

[0155] [In the formula, each

[0156] [ka]

[0157] Independently,

[0158] [ka]

[0159] Selected from, R 1 and R 2 These are, independently, hydrogen and C 1~2 Selected from alkyl groups, or R 1 and R 2 One is hydrogen, and the other is an amino acid residue. R 3 R is selected from hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted heteroalkyl, optionally substituted heteroalkenyl, optionally substituted heteroalkynyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted aryl, and optionally substituted heteroaryl. 3 This optionally includes a covalent bond site to L. R 4 is hydrogen, halo, or C 1~2 It is alkyl, Each R 5 These are, independently, hydrogen or C 1~2 It is alkyl, R 6 is an alkyl that may be substituted, Or, R 5 and R 6 If R is bonded to an adjacent atom,5 and R 6 These combine to form a complex ring, which may be substituted. R 8 is hydrogen or C 1~2 It is alkyl, R 9 is an alkyl group which may be substituted, R 12 and R 13 These are, independently, hydrogen and C 1~2 Selected from alkyl groups, R 14 and R 15 These are, independently, hydrogen and C 1~2 Selected from alkyl groups, R 18 and R 19 These are, independently, hydrogen and C 1~2 Selected from alkyl groups, R 20 [This is an alkyl group that may be substituted.] This relates to compounds having the following properties.

[0160] A further aspect of the present invention is the structure of formula (V):

[0161] [ka]

[0162] [In the formula, Each of rings E, F, C, and D is independently selected from an optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted aryl, or optionally substituted heteroaryl. R 16 and R 17 These are, independently, hydrogen and C 1~2 Selected from alkyl groups] This relates to compounds having the following properties.

[0163] The compounds of formulas (IV), (IVa), (IVb), (IVc), and (V) have the ability to bind to TRIM21.

[0164] The compound of the present invention is

[0165] [ka] TIFF2026525346000045.tif225164TIFF2026525346000046.tif108164

[0166] and their pharmaceutically acceptable salts can be selected. Such compounds have the ability to bind to TRIM21.

[0167] "Alkyl" refers to a radical of a linear or branched saturated hydrocarbon group having 1 to 20 carbon atoms ("C 1~20 (alkyl). In some embodiments, the alkyl group has 1 to 12 carbon atoms ("C"). 1~12 "Alkyl"). In some embodiments, the alkyl group has 1 to 10 carbon atoms, 1 to 6 carbon atoms, 1 to 3 carbon atoms, or 1 to 2 carbon atoms. "Alkylene" refers to an alkyl group from which two hydrogen atoms have been removed to provide a divalent radical.

[0168] "Alkenyl" refers to a radical of a linear or branched hydrocarbon group having 2 to 20 carbon atoms, one or more carbon-carbon double bonds, and optionally one or more carbon-carbon triple bonds ("C 2~20 (Alkenyl). In certain embodiments, the alkenyl does not contain a triple bond. In some embodiments, the alkenyl group has 2 to 10 carbon atoms, 2 to 5 carbon atoms, 2 to 3 carbon atoms, or 2 carbon atoms.

[0169] "Alkynyl" refers to a radical of a linear or branched hydrocarbon group having 2 to 20 carbon atoms, one or more carbon-carbon triple bonds, and optionally one or more carbon-carbon double bonds. In certain embodiments, the alkynyl does not contain double bonds. In some embodiments, the alkynyl group has 2 to 10 carbon atoms, 2 to 5 carbon atoms, 2 to 3 carbon atoms, or 2 carbon atoms.

[0170] "Aryl" refers to a radical of a monocyclic or polycyclic 4n+2 aromatic ring system having 6 to 14 ring carbon atoms and 0 heteroatoms provided in the aromatic ring system. In some embodiments, the aryl group has 6 ring carbon atoms or 10 ring carbon atoms. "Aryl" also includes a ring system in which the aryl ring as defined above is fused with one or more cycloalkyl or heterocyclyl groups, with the bonding site located on the aryl ring. In certain embodiments, the aryl group is substituted with one or more groups selected from halo, C1-C8 alkyl, C1-C8 haloalkyl, cyano, hydroxy, C1-C8 alkoxy, and amino.

[0171] When "hetero" is used to describe a compound or a group present on a compound, it means that one or more carbon atoms in the compound or group are replaced by nitrogen, oxygen, or sulfur heteroatoms. Hetero can be applied to groups such as alkyl, e.g., heteroalkyl; cycloalkyl, e.g., heterocyclyl; and aryl, e.g., heteroaryl.

[0172] "Heteroaryl" refers to a 5-10 membered monocyclic or bicyclic 4n+2 aromatic ring radical having a ring carbon atom and 1-4 ring heteroatoms in the aromatic ring system, where each heteroatom is independently selected from nitrogen, oxygen, and sulfur ("5-10 membered heteroaryl"). A heteroaryl bicyclic ring system may contain one or more heteroatoms in one or both rings. "Heteroaryl" includes ring systems in which a heteroaryl ring is fused with one or more cycloalkyl or heterocyclyl groups, where the bonding site is on the heteroaryl ring. "Heteroaryl" also includes ring systems in which the heteroaryl ring defined above is fused with one or more aryl groups, where the bonding site is on either the aryl ring or the heteroaryl ring.

[0173] In some embodiments, the heteroaryl group is a 5-6 membered aromatic ring system having a ring carbon atom and 1-4 ring heteroatoms provided in the aromatic ring system, where each heteroatom is independently selected from nitrogen, oxygen, and sulfur ("5-6 membered heteroaryl").

[0174] Examples of five-membered heteroaryl groups containing one heteroatom include, but are not limited to, pyrrolyl, furanyl, and thiophenyl. Examples of five-membered heteroaryl groups containing two heteroatoms include, but are not limited to, imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, and isothiazolyl. Examples of five-membered heteroaryl groups containing three heteroatoms include, but are not limited to, triazolyl, oxadiazolyl, and thiadiazolyl. Examples of five-membered heteroaryl groups containing four heteroatoms include, but are not limited to, tetrazolyl. Examples of six-membered heteroaryl groups containing one heteroatom include, but are not limited to, pyridinyl. Examples of six-membered heteroaryl groups containing two heteroatoms include, but are not limited to, pyridazinyl, pyrimidinyl, and pyrazinyl. Examples of six-membered heteroaryl groups containing three or four heteroatoms include, but are not limited to, triazinyl and tetradinyl, respectively.

[0175] In certain embodiments, the heteroaryl group is substituted with one or more groups selected from halo, C1-C8 alkyl, C1-C8 haloalkyl, cyano, hydroxy, C1-C8 alkoxy, and amino.

[0176] Non-exclusive examples of heteroaryl groups include pyridinyl, pyrimidinyl, thiophenyl, thienyl, furanyl, indolyl, benzoxadiazolyl, benzodioxolyl, benzodioxanyl, thianaphthalyl, pyrrolopyridinyl, indazolyl, quinolinyl, quinoxalinyl, pyridopyradinyl, quinazolinonyl, benzoisoxazolyl, imidazopyridinyl, benzofuranyl, benzothienyl, benzothiophenyl, phenyl, naphthyl, biphenyl, and pyro Examples include lyl, pyrazolyl, imidazolyl, pyrazinyl, oxazolyl, isoxazolyl, thiazolyl, furylthienyl, pyridyl, pyrimidyl, benzothiazolyl, prinyl, benzimidazolyl, isoquinolyl, thiadiazolyl, oxadiazolyl, pyrrolyl, diazolyl, triazolyl, tetrazolyl, benzothiadiazolyl, isothiazolyl, pyrazolopyrimidinyl, pyroropyrimidinyl, benzotriazolyl, benzoxazolyl, or quinolyl. The above examples may be substituted or unsubstituted, and the divalent radicals of each of the above heteroaryl examples are non-limiting examples of heteroarylenes.

[0177] "Cycloalkyl" refers to a radical of a saturated cyclic hydrocarbon group having 3 to 10 ring carbon atoms and 0 heteroatoms in a non-aromatic ring system. In some embodiments, the cycloalkyl group has 3 to 8 ring carbon atoms. In some embodiments, the cycloalkyl group has 3 to 6 ring carbon atoms. In some embodiments, the cycloalkyl group has 5 to 10 ring carbon atoms. Examples of cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

[0178] A "heterocyclyl" or "heterocyclic" radical is a 3-10 membered non-aromatic ring system having a ring carbon atom and 1-4 ring heteroatoms, where each heteroatom is independently selected from nitrogen, oxygen, sulfur, boron, phosphorus, and silicon. The heterocyclyl group may be monocyclic, fused, bridged, or spirocyclic, such as a bicyclic system, and may be saturated or partially unsaturated. A "heterocyclyl" also includes a ring system in which the heterocyclyl ring as defined above is fused with one or more cycloalkyl groups, with the bond site located either on the cycloalkyl ring or the heterocyclyl ring, or a ring system in which the heterocyclyl ring as defined above is fused with one or more aryl or heteroaryl groups, with the bond site located on the heterocyclyl ring. In some embodiments, the heterocyclyl group is a 5-6 membered non-aromatic ring system having a ring carbon atom and 1-4 ring heteroatoms, where each heteroatom is independently selected from nitrogen, oxygen, and sulfur.

[0179] Examples of three-membered heterocyclyl groups containing one heteroatom include, but are not limited to, azilidinyl, oxylanil, and thiorenyl. Examples of four-membered heterocyclyl groups containing one heteroatom include, but are not limited to, azetidinyl, oxetanil, and thietanil. Examples of five-membered heterocyclyl groups containing one heteroatom include, but are not limited to, tetrahydrofuranil, dihydrofuranil, tetrahydrothiophenyl, dihydrothiophenyl, pyrrolidinyl, dihydropyrrolyl, and pyrrolyl-2,5-dione. Examples of five-membered heterocyclyl groups containing two heteroatoms include, but are not limited to, dioxolanil, oxasulfuranil, disulfuranil, and oxazolidine-2-one. Examples of five-membered heterocyclyl groups containing three heteroatoms include, but are not limited to, triazolinil, oxadiazolinil, and thiadiazolinil. Examples of six-membered heterocyclyl groups containing one heteroatom include, but are not limited to, piperidinyl, tetrahydropyranil, dihydropyridinyl, and thianil. Examples of six-membered heterocyclyl groups containing two heteroatoms include, but are not limited to, piperazinyl, morpholinil, dithianil, and dioxanil. Examples of six-membered heterocyclyl groups containing two heteroatoms include, but are not limited to, triazinyl. Examples of seven-membered heterocyclyl groups containing one heteroatom include, but are not limited to, azepanil, oxepanil, and thiepanil. Examples of eight-membered heterocyclyl groups containing one heteroatom include, but are not limited to, azocanil, oxecanil, and thiocanil. Examples of five-membered heterocyclyl groups fused to a C6 aryl ring (also referred to herein as 5,6-bicyclic heterocyclic rings) include, but are not limited to, indolinyl, isoindolinyl, dihydrobenzofuranyl, dihydrobenzothienyl, and benzoxazolinonyl. Examples of six-membered heterocyclyl groups fused to an aryl ring (also referred to herein as 6,6-bicyclic heterocyclic rings) include, but are not limited to, tetrahydroquinolinyl and tetrahydroisoquinolinyl.

[0180] Examples of nitrogen-containing heterocyclyl groups include morpholine, piperidine (e.g., 2-piperidinyl, 3-piperidinyl, and 4-piperidinyl), pyrrolidine (e.g., 2-pyrrolidinyl and 3-pyrrolidinyl), azetidine, pyrrolidone, imidazoline, imidazolidinone, 2-pyrazoline, pyrazolidine, piperazine, and N-alkylpiperazine, such as N-methylpiperazine.

[0181] "Alkoxy" refers to the -O-alkyl group. In some embodiments, alkoxy is -OC 1~6 It is alkyl. In some embodiments, the alkoxy is -OC 1~2 It is alkyl.

[0182] "Halo" or "halogen" refers to fluoro(F), chloro(Cl), bromo(Br), and iodine(I). In certain embodiments, the halo group is either fluoro or chloro.

[0183] "Amino acid" refers to a compound having both an amino functional group and an acid functional group. In some embodiments, amino acids are naturally occurring amino acids found in proteins. In some embodiments, amino acids may be selected from glycine, alanine, valine, cysteine, leucine, isoleucine, serine, threonine, methionine, glutamic acid, aspartic acid, glutamine, asparagine, lysine, arginine, proline, histidine, phenylalanine, tyrosine, and tryptophan.

[0184] An "amino acid residue" refers to the portion of an amino acid after removing a hydrogen atom from the N-terminus (-NH2) and / or a hydroxyl group from the C-terminus (-COOH).

[0185] Exemplary carbon atom substituents include, but are not limited to, halogens, -CN, -NO2, -N3, -SO2H, -SO3H, -OH, and -OR aa , -N(R bb )2, -SH, -SR aa -C(=O)Raa -CO2H, -CHO, -CO2R aa , -OCO2R aa , -SO2R aa , C 1~10 Alkyl, C 1~10 Haloalkyl, C 2~10 Alkenil, C 2~10 Alkinyl, C 3~10 Cycloalkyl, 3-14 member heterocyclyl, C 6~14 Examples include aryls and 5- to 14-membered heteroaryls. R aa In each of these cases, independently, C 1~10 Alkyl, C 1~10 Haloalkyl, C 2~10 Alkenil, C 2~10 Alkinyl, C 3~10 Cycloalkyl, 3-14 member heterocyclyl, C 6~14 Selected from aryls and 5-14 member heteroaryls, or two R aa The groups come together to form a 3-14 member heterocyclyl or a 5-14 member heteroaryl ring. R bb In each case, independently, hydrogen, -OH, -OR aa , -N(R cc )2, -CN, -C(=O)R aa , -CO2R aa , -SO2R aa , -SO2R cc , C 1~10 Alkyl, C 1~10 Haloalkyl, C 2~10 Alkenil, C 2~10 Alkinyl, C 3~10 Cycloalkyl, 3-14 member heterocyclyl, C 6~14 Selected from aryls and 5-14 member heteroaryls, or two R bb The groups come together to form a 3-14 member heterocyclyl or a 5-14 member heteroaryl ring. R cc In each case, independently, hydrogen and C 1~10 Alkyl, C 1~10 Haloalkyl, C 2~10 Alkenil, C 2~10 Alkinyl, C3~10 Cycloalkyl, 3-14 member heterocyclyl, C 6~14 Selected from aryls and 5-14 member heteroaryls, or two R cc The groups combine to form a 3- to 14-membered heterocycline or a 5- to 14-membered heteroaryl ring.

[0186] The nitrogen atom may be substituted or unsubstituted, depending on its valence, and may include primary, secondary, tertiary, and quaternary nitrogen atoms. Exemplary nitrogen atom substituents include, but are not limited to, hydrogen, -OH, and -OR. aa , -N(R cc )2, -CN, -C(=O)R aa -C(=O)N(R cc )2, -CO2R aa , -SO2R aa , -SO2R cc -SOR aa , C 1~10 Alkyl, C 1~10 Haloalkyl, C 2~10 Alkenil, C 2~10 Alkinyl, C 3~10 Cycloalkyl, 3-14 member heterocyclyl, C 6~14 Examples include aryls and 5- to 14-membered heteroaryls, or two Rs bonded to a nitrogen atom. cc The groups combine to form a 3- to 14-membered heterocycline or a 5- to 14-membered heteroaryl ring.

[0187] As used herein, the term “may be substituted” is used to refer to a moiety that may be unsubstituted or substituted by one or more groups. Possible substituents include acyl, acylamino, acyloxy, alkoxy, alkoxycarbonyl, alkoxycarbonylamino, amino, substituted amino, aminocarbonyl (e.g., amide), aminocarbonylamino, aminosulfonyl, sulfonylamino, heteroaryl, aryl, aryloxy, azide, carboxyl, cyano, heterocyclyl, cycloalkyl, halogen, hydroxy, keto, nitro, thiol, -S-alkyl, -S-aryl, -S(O)-alkyl, -S(O)-aryl, -S(O)2-alkyl, and -S(O)2-aryl.

[0188] In one embodiment, A is not an anti-TRIM21 antibody or its antigen-binding fragment. In one embodiment, B does not bind to TRIM21, for example, the target protein is not TRIM21. In one embodiment, B is not an anti-TRIM21 antibody or its antigen-binding fragment. In one embodiment, A is not an anti-TRIM21 antibody or its antigen-binding fragment, and B is not an anti-TRIM21 antibody or its antigen-binding fragment. In particular, the compounds of the present invention do not contain Fc or an Fc region.

[0189] In one embodiment, B is not an anti-tau antigen or its antigen-binding fragment. In one embodiment, A is not an anti-TRIM21 antibody or its antigen-binding fragment, and B is not an anti-tau antibody or its antigen-binding fragment.

[0190] As used herein, “antibodies” include, but are not limited to, polyclonal, monoclonal, recombinant, chimeric, complementarity-determining region (CDR) grafted, single-chain, bispecific, Fab fragments, and fragments generated by Fab expression libraries. Such fragments include fragments of whole antibodies, Fv, F(ab'), F(ab')2 fragments, and F(v), or V, that retain their binding activity to a desired antigen. H Contains antibody fragments.

[0191] As used herein, the term “antibody fragment” refers to a portion of an intact, full-length antibody, such as the antigen-binding region or variable region of an intact antibody. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv); multispecific antibody fragments, such as bispecific, tripspecific, and multispecific antibodies (e.g., diabodies, triabodies, tetrabodies); binding domain immunoglobulin fusion proteins; camelized antibodies; minibodies; chelated recombinant antibodies; tribodies or bibodies; intrabodies; nanobodies; small modular immunosorbents (SMIPs), V HH This includes antibodies containing antibodies and any other polypeptides formed from antibody fragments.

[0192] Antibodies and their fragments also include antibody variants and their fragments. Variants include peptides and polypeptides having one or more amino acid sequence substitutions, deletions, and / or additions that have the same or substantially the same epitope-binding affinity and specificity as the antigen-specific antibody or its fragment.

[0193] "Fc" or "Fc region," as used herein, refers to the constant region of an antibody excluding the first constant region immunoglobulin domain. Thus, Fc refers to the last two constant region immunoglobulin domains of IgA, IgD, and IgG, as well as the last three constant region immunoglobulin domains of IgE and IgM, and the flexible hinge at the N-terminus of these domains. In the case of IgA and IgM, Fc may include the J chain. In the case of IgG, Fc refers to the immunoglobulin domain C H 2 and C H 3, and C H 1 and C HIt includes a hinge between 2 and 3. The boundary of the Fc region may vary, but the human IgG heavy chain Fc region is typically defined to include residues C226 or P230 at its carboxyl terminus. TRIM21 recognizes the Fc domain of the antibody. However, the compounds of the present invention do not contain Fc or an Fc region as "A", that is, the TRIM21 binding portion of the compounds of the present invention does not contain or consist of Fc or an Fc region.

[0194] The term "pharmaceutically acceptable salt" refers to a salt that, within reasonable medical judgment, is suitable for use in contact with human and lower animal tissues, without excessive toxicity, irritation, or allergic reactions, and with a reasonable benefit-to-risk ratio. pharmaceutically acceptable salts are well known in the art.

[0195] The present invention also provides compositions comprising the compounds of the present invention. In one embodiment, a pharmaceutical composition is provided comprising the compound according to the present invention or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

[0196] The compositions of this disclosure may be formulated in a conventional manner using one or more pharmaceutically acceptable carriers. Examples of pharmaceutically acceptable carriers that may be used in these pharmaceutical compositions include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, e.g., human serum albumin, buffers, e.g., phosphates, glycine, sorbic acid, potassium sorbate, partially glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, e.g., prolamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulosic substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol, and wool fat.

[0197] In any aspect or embodiment described herein, the therapeutic composition comprising the compound described herein may be in any preferred dosage form, such as a solid or a liquid, and may be configured to be delivered by any preferred route, such as orally, parenterally, intravenously, intraperitoneally, subcutaneously, or intramuscularly.

[0198] The compounds of the present invention may be used therapeutically as pharmaceuticals. In some embodiments, compounds having the structure of formula (I) ALB as described herein are provided for use in the treatment of a subject. In some embodiments, the use of compounds having the structure of formula (I) ALB is provided for use in the manufacture of a pharmaceutical for the treatment of a subject. In some embodiments, a method for treating a subject is provided, comprising the step of administering a compound having the structure of formula (I) ALB to the subject. In some embodiments, the present invention also provides the use of compounds having the structure of formula (I) ALB in the manufacture of a pharmaceutical for use in the treatment of a subject.

[0199] The disease to be treated depends on the target protein. For example, in one embodiment, the present invention provides a compound having the structure of formula (I) ALB for use in the treatment of neurodegenerative diseases. Neurodegenerative diseases may be related to the accumulation and aggregation of tau, such as Alzheimer's disease. In such embodiments, the target protein may be tau, and for example, B may be a tau protein binding site.

[0200] The terms “to treat,” “to treat,” or “treatment” (or equivalent terms) mean that the severity of an individual’s condition is reduced or at least partially improved or cured, and / or some reduction, alleviation or decrease of at least one clinical symptom is achieved, and / or the progression of the condition is inhibited or delayed, and / or the onset of a disease or illness is prevented or delayed.

[0201] The terms “patient,” “individual,” or “subject” include human and other mammalian subjects receiving prophylactic or therapeutic treatment with the compounds described herein. Mammalian subjects include primates, e.g., non-human primates. Mammalian subjects also include laboratory animals commonly used in research, e.g., rabbits and rodents, e.g., rats and mice.

[0202] In another embodiment, the Disclosure provides a method for ubiquitinating / degrading a target protein (e.g., tau) in cells. The method comprises administering a compound comprising the structure of formula (I) ALB described herein such that degradation of the target protein occurs when the target protein is positioned in close proximity to a ubiquitin ligase, thus resulting in degradation / inhibition of the effect of the target protein and control of protein levels. The control of protein levels resulting from the Disclosure provides treatment for a disease state or condition, which is modulated via the target protein by reducing the level of that protein in the patient's cells.

[0203] The present invention also relates to a method for selectively degrading an oligomeric or pathogenic form of a target protein, comprising the step of contacting the target protein with TRIM21 and a compound having the structure of formula (I) ALB or a pharmaceutically acceptable salt thereof.

[0204] The method may be an in vivo method. Alternatively, the method may be an in vitro method in which the oligomeric form of the target protein is present in the sample. The method may include the step of introducing the compound of the present invention into the sample.

[0205] Wherever it exists in this specification, the phrase "selected from the group containing ~" may be replaced with the phrase "selected from the group consisting of ~", and vice versa.

[0206] Similarly, the singular forms "a," "an," and "the" refer to multiple objects unless explicitly indicated otherwise in the context.

[0207] The contents of all publications referenced herein are incorporated in their entirety by reference to provide a more complete description of the latest technology to which the present invention relates. [Examples]

[0208] The present invention is described herein with reference to the following non-limiting embodiments.

[0209] Example 1: Screening to identify compounds that bind to Trim21 PRYSPRY Details of the construct: The Trim21 PRYSPRY protein was expressed as an N-terminal fusion with a hexahistidine tag, which added the residue MAHHHHHHM to the hsTrim21 PRYSPRY sequence (residues 287-475). Expression and purification were carried out as previously described (James et al., 2007).

[0210] [Table 1]

[0211] Capture of T21 PRYSPRY on beads. To ensure that the screening would function, it was necessary to capture the T21 PRYSPRY protein on beads and ensure that it remained active. The activity of T21 PRYSPRY can be monitored by its ability to maintain binding to IgG-Fc, which is only possible when T21 PRYSPRY is in its native fold. T21 PRYSPRY was covalently bound to magnetic carboxyl-derived beads using a two-step EDC-NHS protocol, as detailed below.

[0212] Covalent capture of Trim21 PRYSPRY to magnetic beads 1) Dilute 10 μl of MagnaBind carboxyl beads (catalog number: 21353) in 500 μl of MPQ in Eppendorf and mix well.

[0213] 2) Wash the beads in 100 μL of MPQ (using a magnetic tube rack, collect the beads on the side of the Eppendorf tube and remove the current buffer with a pipette. Immediately add the solution described and mix by short vortexing).

[0214] 3) Prepare the coupling reaction product. 1. Thaw the aliquots of EDC (0.4M) and NHS (0.1M). These were prepared in advance in MPQ and stored at -20°C. 2. Mix 20 μL of 0.5 M MES buffer pH 6 with 100 μL each of EDC and NHS in an Eppendorf container, and mix thoroughly by pipetting. 3. Transfer 100 μL of this reaction mixture to the washed beads (discard the previous solution), vortex immediately to prevent bead aggregation, and place on a shaker (20°C, 1400 RPM). 4. Incubate for 30 minutes.

[0215] 4) Addition of protein to activation beads 1. Prepare the protein dilution: Dilute His-Trim21-PRYSPRY (15 mg / ml) in HEPES buffer (20 mM HEPES (pH 7.8), 150 mM NaCl, and 1 mM TCEP) to a final concentration of 10 μg (0.1 mg / ml) per 100 μL and store on ice. 100 μL is required per reaction. 2. Wash the beads with HEPES buffer (100 μL). 3. Add 100 μL of protein solution (discard the previous solution) to the beads, vortex immediately, and place on a shaker. 4. Incubate for 15 minutes while shaking (20°C, 1400 RPM).

[0216] 5) Quenching and blocking 1. Add 10 μL of 1 M ethanolamine-HCl pH 8.5 to the reactants (do not discard the solution!). 2. Incubate for 10 minutes while shaking. 3. Collect the flow-through and change the pH to 50 mM ethanolamine-HCl (100 μL) at pH 8.5 for 15 minutes. 4. Change to any necessary buffer solution.

[0217] Abbreviations. EDC: 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. NHS: N-hydroxysuccinimide. MPQ: Milli-Q filtered water.

[0218] IgG pulldown by immobilized T21 PRYSPRY. T21 PRYSPRY, protein-free, or control protein were covalently bound to magnetic carboxyl-derived beads. Binding experiments were performed in 20 mM HEPES pH 7.8, 150 mM NaCl, and 1 mM TCEP. Beads were incubated with 2 μM IgG for 15 minutes, washed, and bound beads were collected. Beads were incubated at 95°C for 15 minutes and analyzed by gel electrophoresis and Coomassie staining. For competitive experiments, protein AG was added at 0.1, 1, or 10 μM during binding incubation, otherwise processed as before.

[0219] Results: PRYSPRY-conjugated beads were able to efficiently capture IgG. To demonstrate that the capture was due to a specific interaction, incubation was repeated in the presence of increasing concentrations of protein AG, which binds to the same epitope on IgG Fc as TRIM21. The ability of protein AG to dose-dependently inhibit the capture of IgG by PRYSPRY-conjugated beads confirmed that the interaction in this form is specific. Subsequently, specific ligands were captured using PRYSPRY-conjugated beads. Enrichment was then evaluated and specific compounds were selected.

[0220] Examples of compounds recovered from the screening process are detailed in Table 1 below.

[0221] [Table 2]

[0222] Example 2: Synthesis of N-boc protected compound 37

[0223] [ka]

[0224] To a stirred solution of trans-4-cyclohexyl-L-proline (100 mg, 0.507 mmol, 1.00 equivalent) in ethanol (2.5 mL, 0.20 M) at 0°C, thionyl chloride (0.111 mL, 1.52 mmol, 3.00 equivalent) was added dropwise. The resulting reaction mixture was stirred at room temperature for 18 hours. The reaction mixture was concentrated under vacuum (toxic Buchi) to obtain the desired product 2 as a white solid (130 mg, 98%). δ H (400 MHz, CDCl3) 0.84-1.38 (9H, m), 1.56-1.78 (5H, m), 1.94-2.10 (2H, m), 2.24-2.38 (1H, m), 3.06 (1H, br s), 3.75 (1H, br s), 4.28 (2H, q, J 7.1 Hz), 4.48 (1H, br s), 8.88 (1H, br s), 11.11 (1H, br s); δ C (101 MHz, CDCl3) 14.2 (CH3), 25.9 (2 × CH2), 26.2 (CH2), 31.6 (CH2), 32.0 (CH2), 33.3 (CH2), 41.1 (CH3), 43.1 (CH3), 49.4 (CH2), 59.4 (CH3), 63.2 (CH2), 169.1 (C); m / z (ESI) 226.1806 (MH + .C 13 H 24 NO2 requires 226.1802.

[0225] [ka]

[0226] To a stirred solution of 3-(4-pyridyl)-L-alanine (100 mg, 0.602 mmol, 1.00 equivalent) in methanol (2.5 mL, 0.25 M) at 0°C, thionyl chloride (0.132 mL, 1.81 mmol, 3.00 equivalent) was added dropwise. The resulting reaction mixture was stirred at 60°C for 18 hours. The reaction mixture was concentrated under vacuum (toxic Buchi) to obtain the desired product 7 as a white solid (155 mg, 100%). δ H (400 MHz, CD3OD) 3.55 (1H, dd, J 14.5, 6.5 Hz, 2-HH), 3.63 (1H, dd, J 14.5, 7.9 Hz, 2-HH), 3.83 (3H, s, CH3), 4.67 (1H, dd, J 7.9, 6.5 Hz, 1-H), 8.10-8.16 (2H, m, 2 × ArH), 8.85-8.90 (2H, m, 2 × ArH); δ C (101 MHz, CD3OD) 37.0 (CH2), 53.5 (CH), 54.0 (CH3), 129.7 (2 × CH), 142.9 (2 × CH), 158.3 (C), 169.4 (C); m / z (ESI) 181.0973 (MH + .C9H 12 (N2O2 requires 181.0972 units)

[0227] [ka]

[0228] (S)-3-amino-3-(2-methoxyphenyl)propionic acid (100 mg, 0.512 mmol, 1.00 equivalent) was dissolved in tetrahydrofuran (1.3 mL) and water (1.3 mL), and the resulting solution was cooled to 0°C. Sodium bicarbonate (129 mg, 1.54 mmol, 3.00 equivalent) was added all at once, followed by di-tert-butyl dicarbonate. The resulting reaction mixture was stirred at room temperature for 18 hours. Tetrahydrofuran was removed under reduced pressure, and the reaction mixture was acidified to pH 1 with 1 M hydrochloric acid aqueous solution. The aqueous layer was extracted with dichloromethane (3 × 20 mL). The combined organic layers were dried over MgSO4, filtered, and concentrated under vacuum to obtain the desired product 4 as a white solid (131 mg, 87%);δ H (400 MHz, CD3OD) 1.42 (9H, s, 3 × CH3), 2.63 (1H, dd, J 15.5, 8.8 Hz, 2-HH), 2.75 (1H, dd, J 15.5, 5.1 Hz, 2-HH), 3.87 (3H, s, OCH3), 5.30 (1H, br s, 3-H), 6.90 (1H, t, J 7.5 Hz, ArH), 6.96 (1H, d, J 8.2 Hz, ArH), 7.19-7.29 (2 H, m, 2 × ArH); δ C (101 MHz, CD3OD) 28.7 (3 × CH3), 40.7 (CH2), 54.8 (C), 55.8 (CH), 80.2 (C), 111.8 (CH), 121.5 (CH), 128.0 (CH), 129.6 (CH), 131.2 (C), 157.4 (C), 158.0 (C), 174.8 (C); m / z (ESI) 318.1319 (MNa + .C 15 H 21 NNaO5 requires 318.1312.

[0229] [ka]

[0230] To a stirred solution of (S)-3-((tert-butoxycarbonyl)amino)-3-(2-methoxyphenyl)propanoic acid (38 mg, 0.13 mmol, 1.0 equivalent) in dichloromethane (0.4 mL), N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (30 mg, 0.15 mmol, 1.2 equivalents) and 1-hydroxybenzotriazole hydrate (24 mg, 0.15 mmol, 1.2 equivalents) were added, and the resulting solution was stirred at room temperature for 10 minutes. To the reaction mixture, a stirred solution of (2S,4S)-ethyl4-cyclohexylpyrrolidine-2-carboxylate hydrochloride (34 mg, 0.13 mmol, 1.0 equivalent) and N,N-diisopropylethylamine (0.067 mL, 0.39 mmol, 3.0 equivalents) in dichloromethane was added, and the resulting reaction mixture was stirred at room temperature for 18 hours. The reaction mixture was concentrated under vacuum and purified by flash column chromatography (petroleum ether / ethyl acetate 7:3) to obtain the desired product 5 as a white solid (55 mg, 85%). δ H (400 MHz, CD3OD) 0.78-1.46 (19H, m), 1.54-1.91 (6H, m), 1.94-2.11 (2H, m), 2.71 (1H, dd, J 15.0, 5.0 Hz, 2-HH), 2.80 (1H, dd, J 15.0, 7.9 Hz, 2-HH), 2.88-3.03 (1H, m), 3.64-3.79 (1H, m), 3.88 (3H, s, OCH3), 4.09-4.25 (2H, m), 4.41 (1H, d, J 9.3, CH), 5.34 (1H, br s, 3-H), 6.92 (1H, td, J 7.4, 1.1 Hz, ArH), 6.95-7.00 (1H, m, ArH), 7.20-7.29 (2H, m, 2 × ArH); δ C(101 MHz, CD3OD) 14.4 (3 × CH3), 27.1 (CH), 27.1 (CH), 27.4 (CH), 28.8 (CH2), 32.3 (CH), 32.9 (CH), 34.2 (CH), 36.0 (C), 40.1 (CH), 42.9 (CH), 44.9 (CH), 51.7 (CH2), 52.5 (CH2), 55.9 (CH3), 60.5 (CH), 61.4 (CH), 62.3 (CH2), 111.7 (CH), 121.6 (CH), 127.6 (C), 129.5 (CH), 129.7 (CH), 157.9 (C), 171.9 (C), 173.7 (C); m / z (LC-MS, ESI + ) 503 (MH + , 37%); (HR-MS, ESI) 525.2943 (MNa + .C 28 H 42 N2NaO6 requires 525.2935 units.

[0231] [ka]

[0232] Lithium hydroxide hydrate (15.8 mg, 0.376 mmol, 1.50 equivalent) was added to a stirred solution of 5 (126 mg, 0.251 mmol, 1.00 equivalent) in tetrahydrofuran (2.4 mL) and water (2.4 mL). The resulting reaction mixture was stirred at room temperature for 18 hours. Tetrahydrofuran was removed under vacuum, and the reaction mixture was acidified to pH 1 with 1 M hydrochloric acid aqueous solution. The aqueous layer was extracted with dichloromethane (3 × 30 mL). The combined organic layers were dried over MgSO4, filtered, and concentrated under vacuum to obtain the desired product 6 as a white solid (115 mg, 97%). δ H(400 MHz, CD3OD) 0.81 - 1.33 (7H, m), 1.41 (9H, s, 3 × CH3), 1.53 - 1.93 (7H, m), 1.95 - 2.52 (2H, m), 2.63 - 3.02 (3H, m), 3.57 - 3.74 (1H, m), 3.87 (3H, s, OCH3), 4.42 (1H, d, J 9.2 Hz, CH), 5.17 - 5.39 (1H, m, 3 - H), 6.91 (1H, br t, J 7.4 Hz, ArH), 6.96 (1H, br d, J 8.5 Hz, ArH), 7.19 - 7.28 (2H, m, 2 × ArH); δ C (101 MHz, MeOD) 27.1, 27.1, 27.4, 28.8, 30.7, 32.3, 32.4, 32.9, 34.3, 36.0, 40.0, 40.9, 42.9, 43.0, 43.1, 44.8, 48.3, 51.7, 52.5, 54.8, 55.8, 55.9, 60.3, 61.3, 80.3, 111.6, 111.7, 121.6, 121.7, 127.6, 128.0, 129.5, 129.7, 130.9, 131.3, 157.1, 157.3, 157.8, 171.9, 171.9, 175.4; m / z (LC - MS, ESI - ) 473 (MH - , 100%); (HR - MS, ESI + ) 475.2807 (MH + [[ID=IO]].C[[ID=II]] 26 H 39 N2O6 requires 475.2803).

[0233]

Chemical Structure

[0234] It should be noted that there seems to be a typo in the original text where "IO" should probably be "10". This has been corrected in the translation where appropriate.To a stirred solution of 6 (35 mg, 0.074 mmol, 1.0 equivalent) in dichloromethane (1.0 mL), N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (17 mg, 0.089 mmol, 1.2 equivalents) and 1-hydroxybenzotriazole hydrate (14 mg, 0.089 mmol, 1.2 equivalents) were added, and the resulting solution was stirred at room temperature for 10 minutes. To the reaction mixture, a stirred solution of 7 (16 mg, 0.074 mmol, 1.0 equivalent) in dichloromethane and N,N-diisopropylethylamine (0.039 mL, 0.22 mmol, 3.0 equivalents) was added, and the resulting reaction mixture was stirred at room temperature for 18 hours. The reaction mixture was concentrated under vacuum and purified by flash column chromatography (dichloromethane / methanol 49:1) to obtain the desired product 8 as a white solid (32 mg, 79%). δ H (400 MHz, CDCl3) 0.66-1.22 (7H, m), 1.41 (9H, s, 3 × CH3), 1.54-1.81 (5H, m), 2.18-2.36 (2H, m, 2-H2), 2.59 (1H, dd, J 14.5, 5.2 Hz), 2.78-2.96 (2H, m), 3.08-3.23 (2H, m), 3.74 (3H, s, OCH3), 3.87 (3H, s, OCH3), 4.42 (1 H, d, J 7.9 Hz, CH), 4.82 (1H, td, J 8.3, 5.4 Hz), 5.36 (1H, br s), 6.33-6.45 (1H, m, NH), 6.80-6.94 (2H, m, ArH), 6.98-7.08 (2H, m, 2 × ArH), 7.17-7.29 (2H, m, 2 × ArH), 7.61 (1H, br d, J 8.2 Hz, NH), 8.43-8.59 (2H, m, 2 × ArH); δ C(101 MHz, CDCl3) 26.0 (CH2), 26.1 (CH2), 26.3 (CH2), 28.5 (3 × CH3), 30.9 (CH2), 31.3 (CH2), 31.9 (CH2), 37.4 (CH2), 38.3 (CH2), 41.9 (CH), 43.8 (CH), 47.3 (CH), 51.7 (CH2), 52.3 (CH), 52.6 (CH3), 55.5 (CH3), 59.8 (CH), 77.4 (C), 79.5 (C), 110.4 (CH), 110.6 (C), 120.8 (CH), 124.7 (2 × CH), 127.3 (CH), 128.6 (CH), 145.7 (C), 149.7 (2 × CH), 155.1 (C), 156.4 (C), 170.9 (C), 171.4 (C); m / z (LC-MS, ESI + ) 637 (MH + , 100%); (ESI) 659.3419 (MNa + .C 35 H 48 N4NaO7 requires 659.3415.

[0235] [ka]

[0236] Lithium hydroxide hydrate (3.0 mg, 0.071 mmol, 1.5 equivalents) was added to a stirred solution of 8 (30 mg, 0.047 mmol, 1.0 equivalent) in tetrahydrofuran (0.5 mL) and water (0.5 mL). The resulting reaction mixture was stirred at room temperature for 4 hours. Tetrahydrofuran was removed under vacuum, and the reaction mixture was acidified to pH 1 with 1 M hydrochloric acid aqueous solution. The aqueous layer was extracted with dichloromethane (3 × 20 mL). The combined organic layers were dried over MgSO4, filtered, and concentrated under vacuum to obtain the desired product 9 as a white solid (20 mg, 69%). δ H(400 MHz, CD3OD) 0.71-2.10 (25H, m), 2.29-2.71 (2H, m), 2.74-3.15 (3H, m), 3.36-3.77 (2H, m), 3.85 (3H, s, OCH3), 4.37 (1H, d, J 8.8 Hz), 4.69 (1H, dd, J 8.1, 5.0 Hz), 5.25-5.42 (1H, m), 6.84-7.01 (2H, m), 7.17-7.33 (3H, m), 7.39 (1H, d, J 5.1 Hz), 8.26-8.52 (2H, m); δ C (101 MHz, CD3OD) 25.7, 25.7, 26.0, 27.4, 31.0, 31.6, 33.0, 35.4, 36.4, 38.5, 41.6, 43.3, 50.7, 51.4, 54.6, 60.0, 61.3, m / z (LC-MS, ESI + ) 637 (MH + , 100%); (APCI) 623.3439 (MH + .C 34 H 47 N4O7 requires 623.3439.

[0237] To obtain compound 37, the N-boc group of 9 may be removed under acidic conditions. Alternatively, 9 may be used in further synthesis to obtain a bifunctional molecule.

[0238] Example 3: Structural and biophysical characterization of TRIM21 ligand The binding of compounds 36-38 to the TRIM21 PRYSPRY protein was tested using a combination of nanoDSF (Figure 1), intrinsic tryptophan fluorescence (Figure 2), and competitive fluorescence polarization (Figure 3). The methods are described below.

[0239] NanoDSF (thermal shift assay). The thermal shift assay was performed using the nanoDSF Prometheus instrument. The assay was carried out using 10 μM T21 PRYSPRY in 150 mM NaCl, 50 mM Tris pH 8, 1 mM DTT, and 1% DMSO. The compound was included at 100 μM. Denaturation was performed over an 80°C range from 15 to 95°C with a gradient of 2°C / s. The data were analyzed using Nano Temper software.

[0240] Intrinsic tryptophan fluorescence quenching. Measurements were performed using a nanoDSF Prometheus instrument at 15°C for compound 36, or using a PHERAstar plate reader at 25°C for the other two compounds (37 and 38). The assays were carried out in 150 mM NaCl, 50 mM Tris pH 8, 1 mM DTT, and 1% DMSO. Compound dilutions were prepared as needed (usually 2-fold dilutions), starting from 100 μM. For final protein concentrations of 8 μM for the Prometheus experiment and 0.15 μM for the PHERAstar experiment, proteins were added from 10× stock solutions prepared in the same buffer. Samples were prepared in 384-well format (final volume of 50 μL in each well) or in microtubes (40 μL of reaction prepared), equilibrated for 15 minutes, and then fluorescence was measured. Data were normalized and analyzed by PRISM.

[0241] Fluorescence polarization substitution assay. The experiment was performed in a 384-well low-binding plate in 150 mM NaCl, 50 mM Tris pH 8, 1 mM DTT, 0.01% v / v tween20, and 1% DMSO at 25°C using a PHERAstar plate reader. Trim21-PRYSPRY-Alexa488 (50 nM) was pre-mixed with human IgG Fc (5 μM) and equilibrated. 5 μL of this master mix was dispensed into a 384-well plate. Compound dilutions were prepared in a low-binding 96-well plate, typically starting from 10–100 μM and progressing to 2-fold dilutions. Next, 45 μL of the compound solution was transferred to each well in the 384-well plate. The plate was centrifuged at 700 g for 1 minute and equilibrated in the dark for 30 minutes. Finally, the polarization was read using a PHERAstar plate reader. Data were analyzed using PRISM.

[0242] Crystallization and data acquisition. To produce diffraction-quality crystals, a solution of Trim21-PRYSPRY (15 mg / ml) was mixed with a ligand stock solution (100 mM in DMSO) at a 50:1 ratio and equilibrated for 15–30 minutes before preparing the crystallization plate. Using a Mosquito (TTP Labtech) instrument, a sparse matrix screening was prepared with a 100 nL protein droplet size and 200 nL of precipitant. Crystals were obtained by sitting vapor diffusion at 17°C. Diffraction-quality crystals for compounds 37 and 38 were obtained in 30% PEG10000, 0.1 M TRIS HCl pH 8.5, and flash-frozen in liquid nitrogen without cryoprotection. For compound 36, crystals were obtained in 20% PEG4000, 5% isopropanol, 0.1 M sodium citrate, cryoprotected by replenishing the reservoir with 20% glycerol, and frozen in liquid nitrogen. Data was collected at Diamond Light Source (Didcot, UK) at beamline I-24 (for compounds 36 and 37) and at beamline I-04 (for compound 38).

[0243] Structural analysis and model construction. Data were processed using the CCP4i package as follows: Data were indexed, scaled, and integrated using the FASTDP (Winter et al., 2011) and Xia2dials (Winter et al., 2022) pipelines. Molecular substitution was performed using PHASER with the TRIM21 PRYSPRY model 2IWG. Models were iteratively refined using REFMAC5 and COOT for model construction. Constraints to ligands were generated using AceDRG.

[0244] Results. In nanoDSF experiments, the ability of the compounds to bind to TRIM21 was confirmed by an increase in PRYSPRY thermal stability (Tm) in the range of 2–7°C (Figure 1). Intrinsic tryptophan fluorescence experiments were able to utilize the presence of two tryptophan residues (W381 and W383) at the TRIM21 PRYSPRY binding site. Upon antibody binding, the intrinsic fluorescence of these tryptophan residues at approximately 340 nm was quenched. Similar behavior was observed for all three compounds (Figure 2), suggesting that these compounds not only reach the same binding site as the native TRIM21 ligand (IgG Fc) but also allow for the determination of affinity. Therefore, a series of competitive anisotropy experiments were performed using Alexa488-labeled PRYSPRY to test whether the compounds compete with IgG. Incubation of increasing concentrations of IgG Fc with labeled PRYSPRY resulted in the formation of a complex, where a larger mass induced a change in polarization (Figure 3). To test the inhibition of the compounds, increasing concentrations of ligands were incubated with pre-formed IgG:PRYSPRY complexes. Titration of each ligand resulted in a decrease in polarization, indicating that they compete with IgG for binding to labeled PRYSPRY. Three different mutants were tested by fluorescence quenching experiments to provide a detailed assessment of which residues drive the binding of the compounds to TRIM21. For compound 36, any mutation at D355, W381, or W383 was sufficient to invalidate measurable binding. For compounds 37 and 38, mutant W381A showed the weakest affinity, suggesting that this residue is the primary driver of binding (Figure 23A). TRIM21: To obtain atomic-resolution information about compound binding and to evaluate ligand complementarity, the X-ray structures of TRIM21 complexed with ligands 36, 37, and 38 were elucidated (Figures 23A-C). These structures confirm that all three ligands access the same pocket in the PRYSPRY domain, explaining why the W381 mutation is particularly important for interaction with ligands 37 and 38.The selected small molecule ligands also replicate the features of the native IgG Fc epitope in unexpected ways, such as forming a bidentate motif that penetrates deeply into the PRYSPRY pocket (Figure 24). This structure also allowed us to evaluate possible exit vectors for constructing additional groups on the TRIM21 ligand. Three different vectors were observed that could be used to extend the TRIM21 ligand, such as binding to other parts capable of binding to other proteins (Figures 23B and D).

[0245] Table 2 below shows the K values ​​obtained from fluorescence quenching experiments using Trim21 PRYSPRY and its related mutants. D Show the value.

[0246] [Table 3]

[0247] Example 4: TRIM21 compound inhibits TRIM21 cell activity To evaluate the cell penetration and in situ engagement of TRIM21 compounds with endogenous proteins, RPE-1 cells were treated with 2.5 μM compound 37 for 18 hours and then blotting for TRIM21 protein levels. The treatment resulted in an increase in TRIM21 levels (Figure 4), which was consistent with the compound stabilization of the protein as shown by previous biophysical data (Figures 1 and 2). To further confirm that TRIM21 compounds are cell-permeable, non-toxic, and capable of functionally interacting with TRIM21, two assays were performed. The first assay monitored targeted proteolysis by TRIM21, while the second assay measured TRIM21's ability to neutralize viral infection in the presence of antibodies.

[0248] In the first assay, mRNA encoding a protein consisting of nanobodies (NbGFP) fused to IgG Fc-bound EGFP is electroporated into cells stably expressing H2B-mEGFP. Once the nanobodies-Fc are generated, they bind to H2B-mEGFP and recruit endogenous TRIM21. The resulting degradation of the H2B-mEGFP protein by TRIM21 is then monitored by recording cell fluorescence (Figure 5).

[0249] In RPE-1 cells that stably express H2B-mEGFP, NbGFP-Fc or NbGFPFc H433A mRNA encoding EGFP-H2B was electroporated and immediately seeded in a medium containing DMSO or compound 37 at the concentrations shown in Figure 6. Cells were imaged using the IncuCyte system, and H2B-mEGFP fluorescence was quantified by normalizing the integrated density of GFP (and therefore the product of area and mean intensity) for each image against the total cell area (phase). The H2B-mEGFP fluorescence values ​​were then normalized for each condition against NbGFPFcH433A (an Fc variant that cannot bind to TRIM21 PRYSPRY; a control with no degradation), i.e., a value of 1 is equivalent to no degradation. Treatment of cells with compound 37 was sufficient to dose-dependently inhibit the degradation of EGFP-H2B by TRIM21, and this inhibition was almost complete even after 15 hours (Figure 6).

[0250] In the second assay, cells were infected with adenovirus 5 (Adv5) in the presence of anti-adenovirus antibodies. Endogenous TRIM21 can detect and degrade the incoming antibody-coated virus, resulting in potent inhibition of infection as measured by the expression of the virus-encoded GFP gene (Figure 7). Treatment with compounds 37 and 38 dose-dependently rescued the infection, which was consistent with inhibition of TRIM21 activity by competing with IgG for binding (Figure 8).

[0251] The confluence of cells treated for 48 hours was evaluated to determine the toxicity of the TRIM21 compound. The confluence was largely unaffected by the compound treatment (Figure 9).

[0252] Example 5: Biophysical characterization of TrimTac and in vitro evidence of three-component complex formation. To investigate the possibility that the TRIM21 compound interacts with a second protein in addition to PRYSPRY, a short linker and a chloroalkane were added to compound 37 to form compound 71.

[0253] [ka]

[0254] Expression and Purification. The HaloTag protein was expressed as an N-terminal fusion with a hexahistidine tag, which adds the residue MAHHHHHHM to the N-terminus of the protein. This protein was expressed in Escherichia coli (C41 strain) using self-inducing medium (ZYP-5052, Studier et al). Cells were lysed by sonication in 1M NaCl, 50mM Tris pH8, 2mM DTT, 10mM imidazole, 10% v / v BugBuster, and cOmplete protease inhibitor. The lysates were clarified by centrifugation at 18000 RPM for 45 minutes using a JA25.50 fixed-angle rotor. The clarified lysates were subjected to an IMAC affinity step using a gravity-flow column packed with NiNTA agarose. After washing and elution, the peak fraction was further purified using size exclusion chromatography (Superdex S75 16 / 60) equilibrated in 50 mM Tris pH 8, 150 mM NaCl, and 1 mM DTT.

[0255] AUC velocity experiment. The c(s) distribution shows that both TRIM21 PRYSPRY (solid black line) and HaloTag (dashed black line) alone are monomers that settle at 2.3S (Sw,20=2.5S) and 2.9S (Sw,20=3.2S), respectively, with calculated masses of 22.0kDa and 34.4kDa, and friction ratios of 1.143 and 1.142, respectively. The mixture alone (dashed orange line) yields a single broad distribution, which may simply be a cumulative Gaussian distribution of two individual unbound species. Addition of 10 μM ligand (solid orange line) reduces the concentration of T21 PRYSPRY and significantly reduces HaloTag, resulting in the appearance of a species that settles at 3.7S (Sw,20=4.1S). If the friction coefficient is the same as for T alone (1.143), the calculated mass would be 48.1kDa. This is lower than the 57.3 kDa mass expected for a 1:1 ratio. If this species represents a composite, it is expected to have a friction ratio of 1.286, which suggests a slightly more expanded / non-spherical conformation.

[0256] AUC equilibrium experiment. 5 μM TRIM PRYSPRY (22,490 Da) and Halo Tag (34,765 Da) were subjected to AUC precipitation equilibrium at 20°C using both absorbance and interference optics at 280 nm in 50 mM Tris HCl pH 8.0, 150 mM NaCl, 1 mM DTT, and 0.08% (v / v) DMSO, both in the absence and in the presence of the compound. The data were analyzed using SEDPHAT with a single exponential function to obtain the average mass. In the absence of the compound, the average mass of 30,316 Da is close to the average of the two proteins at equimolar concentrations. As the concentration of the compound increases, the average mass reaches a maximum at 5 μM, with an average mass of 50,482 ± 82 Da, which is close to the expected value for a 1:1 complex. Beyond this point, increasing the concentration further decreases the average mass because the compound binds to individual components in competition with complex formation.

[0257] Crystallization, data acquisition, and structural elucidation. To generate diffraction-quality crystals, a solution of HaloTag protein (13 mg / ml) was mixed 50:1 with a stock solution of compound 71 (50 mM in DMSO) and equilibrated for 30 minutes before preparing the crystallization plate. Using a Mosquito (TTP Labtech) instrument, a sparse matrix screening was prepared with a 100 nL protein droplet size along with 100 nL of precipitant. Crystals were obtained by sitting vapor diffusion at 17°C. Diffraction-quality crystals were obtained in 25% PEG4000, 0.1 M Na MES pH 6.5, and 0.2 M magnesium chloride. Crystals were cryoprotected with 20-30% glycerol and frozen in liquid nitrogen. Data were collected at beamline I-04 at Diamond Light Source (Didcot, UK). The data was processed using the CCP4i package as follows: The data was indexed, scaled, and integrated using the Xia2dials (Winter et al., 2022) pipeline. Molecular substitution was performed using PHASER with a HaloTag model from 5UY1. The model was iteratively refined using REFMAC5 and COOT for model construction. Constraints to the ligand were generated using AceDRG.

[0258] Model construction. Using HADDOCK software, docked models of Trim21 PRYSPRY and HaloTag were generated. These were checked for proper orientation. COOT was used to construct models with fitted ligands. PyMOL was used to visualize the resulting structures and models.

[0259] Results. Conjugation of a short linker and chloroalkane to compound 37 did not alter the engagement efficiency with PRYSPRY, as assessed by nanoDSF (Figure 10). The ability of compound 71 to simultaneously bind to PRYSPRY and the second HaloTag protein was tested by kinetic ultracentrifugation (AUC). Binding of compound 71 did not significantly alter the mass of either PRYSPRY or HaloTag protein alone (Figure 11). Furthermore, the two proteins were unable to interact with each other in the absence of the compound, as indicated by a broadened peak with a mass between the masses of both proteins. Importantly, however, the addition of compound 71 to a solution containing both PRYSPRY and HaloTag resulted in the formation of a complex with a predicted mass of 57 kDa (Figure 11). This result indicates that a triplicate complex can be formed in vitro. Extending these experiments, the mass of the triplicate complex was measured at a range of compound concentrations. The mass of the triplicate complex peaked near the expected mass (approximately 50 kDa) and then decreased as the compound concentration increased further (Figure 12). This behavior is consistent with the "Hook effect," a well-known phenomenon in triplicate complex formation that occurs when an excess of the mediator is added, forming separate 1:1 complexes with each of the other two partners. To understand how compound 71 mediates triplicate complex formation, the X-ray structure of the complex with HaloTag was elucidated. A clear density of the chloroalkane component of compound 71 was observed, which, as expected, was located at the HaloTag active site. This structure was then used together with the complex of compound 37 bound to PRYSPRY to construct a triplicate complex model. This model shows that there are no steric collisions during triplicate complex formation, and the linker between the chloroalkane and the TRIM21 warhead is sufficient to allow simultaneous engagement of both protein partners.

[0260] These formed compounds, comprising a TRIM21 binding site and a warhead directed at a target protein, are referred to herein as TrimTac.

[0261] Example 6. TrimTac forms a three-component complex between TRIM21 and the target in cells. To investigate whether the formation of a triplicate complex between two protein targets can be mediated by the TRIM21 ligand in living cells, we developed a fluorescence assay for colocalizing ternary structures ("FACTS"). In this assay, one protein (in this case, TRIM21) is labeled with a fluorescent protein tag (in this case, mCherry) and expressed in the cytosol, while a second protein (in this case, HaloTag) is bound to chromatin and therefore (in this case, by fusing to H2B) to the nucleus and expressed together with a fluorescent protein tag of a different color (in this case, mEGFP). Normally, the two proteins do not mix and cannot form a complex. During cell division, the nuclear membrane undergoes transient collapse, allowing mixing of the cytosol and chromatin, but unless the two proteins form a complex, they reseparate into the cytosol and nucleus during nuclear membrane reformation (Figure 13A). However, in the presence of a compound capable of mediating triplicate complex formation, the two proteins remain colocalized in the nucleus. This can be easily detected by the co-localization of mEGFP and mCherry signals. Using this assay, compounds 37 and 71 were compared (compound 37 is capable of binding only to TRIM21, while compound 71 is capable of binding to both TRIM21 and HaloTag). Co-localization of TRIM21 (mCherry-TRIM21) and HaloTag (H2B-mEGFP-HaloTag) was not observed for either compound before cell division. After cell division, the two proteins remained in separate compartments in the presence of compound 37 (Figure 13B). In contrast, in cells treated with compound 71, co-localization of mEGFP and mCherry fluorescence indicated that TRIM21 remained in the nucleus as part of a triplicate complex with HaloTag (Figure 13C). This experiment was repeated at a range of compound concentrations, and no evidence of triplicate complex with compound 37 was found at any dose. For compound 71, a dose-dependent increase in the co-localization of EGFP and mCherry was observed up to a concentration exceeding approximately 2 μM, indicating the formation of more triplicate complexes.The efficiency of tricomponent complex formation decreased at the highest compound concentration, consistent with the hook effect. To confirm these results, colocalization behavior was compared in cells expressing either mCherry-TRIM21 (ΔRB) lacking the RING and B Box domains, or mCherry-TRIM21 (ΔPS) lacking the PRYSPRY domain. Removal of the PRYSPRY domain invalidated tricomponent complex formation, but removal of the RING-B Box domain did not, consistent with the former being required for compound binding (Figure 14). Finally, a series of different TrimTac compounds were prepared and their ability to form tricomponent complexes and their toxicity were compared.

[0262] Compound 71 - 11-atom linker

[0263] [ka]

[0264] Compound 109 - 14-atom linker

[0265] [ka]

[0266] Compound 110 - 8-atom linker

[0267] [ka]

[0268] Compound 111 - 18-atom linker

[0269] [ka]

[0270] RPE-1 TRIM21 KO cells co-expressing mCherryTRIM21 and H2B-mEGFP-Halo were treated with titration of compounds 37, 71, 109, 110, and 111, and tricomponent complex formation was quantified at 48 hours by normalizing the red / green overlap area (mCherry-TRIM21-positive nuclei) to the total green area (total nuclei). In each case, the compounds were able to mediate tricomponent complex formation and showed a hook effect at similar concentrations (Figure 15A).

[0271] RPE-1 cells were seeded at approximately 15% confluence and treated with titration of compounds 37, 71, 109, 110, and 111 or with corresponding DMSO controls, and imaged using the IncuCyte system. Cellular confluence was quantified 72 hours after treatment by normalizing the total cell area per image for each compound concentration relative to the corresponding DMSO control. Cells treated with 40 μM of compound 71 or compound 109 did not reach 100% confluence, indicating toxicity. No toxicity was observed for any of the compounds at concentrations below 20 μM (Figure 15B).

[0272] Example 7: TrimTac selectively degrades oligomer targets but does not degrade monomer targets. Next, we investigated TrimTac's ability to degrade different cellular targets, namely the monomer mEGFP-Halo and the oligomers CAV1-mEGFP-Halo and Cavin1-mEGFP-Halo. TrimTac's degradation activity was compared to that of a standard VHL-based PROTAC (HaloPROTAC1), and protein levels were monitored by measuring mEGFP fluorescence. Specifically, stable RPE-1 mEGFP-Halo, RPE-1 CAV1-mEGFP-Halo, and RPE-1 Cavin1-mEGFP-Halo cell lines were treated with VHL-mobilized PROTAC (HaloPROTAC1) or compounds 37, 71, 109, 110, and 111 at concentrations shown in Figure 16, and imaged for 48 hours using the IncuCyte system. GFP fluorescence was quantified by normalizing the GFP integrated density (and therefore the product of area and mean intensity) for each image against the total cell area (phase), and then normalizing it against the corresponding DMSO condition such that a value of 1 (gray dashed line in Figure 16) represents no degradation. As expected, HaloPROTAC1 efficiently degraded all three targets within 24 hours, whereas compound 37, which binds to TRIM21 but not to protein targets, did not (Figure 16). TrimTac71, 109, 110, and 111 were all able to efficiently degrade CAV1-mEGFP-Halo and Cavin1-mEGFP-Halo. Unexpectedly, however, none of the TrimTacs showed any activity against mEGFP-Halo.

[0273] To further explore this, both HaloPROTAC1 and TrimTac were titrated over a wide range of concentrations, and their substrate degradation profiles were compared. Stable cell lines of RPE-1 mEGFP-Halo, RPE-1 CAV1-mEGFP-Halo, and RPE-1 Cavin1-mEGFP-Halo were treated with titrations of HaloPROTAC1 and compounds 37, 71, 109, 110, and 111 for 48 hours, and GFP fluorescence was quantified using the IncuCyte system as described for Figure 16. HaloPROTAC1 dose-dependently degraded all three substrates, and there was evidence of a hook effect at the highest concentrations for CAV1-mEGFP-Halo and Cavin1-mEGFP-Halo (Figure 17). TrimTac also dose-dependently degraded CAV1-mEGFP-Halo and Cavin1-mEGFP-Halo, similarly with a hook effect, but did not exhibit degrading activity against mEGFP-Halo at any concentration, as previously observed.

[0274] WT or TRIM21 knockout cells stably expressing Cavin1-mEGFP-Halo or CAV1-mEGFP-Halo were treated with titration of compound 71 for 48 hours, and GFP fluorescence was quantified using the IncuCyte system as described for Figure 16. The results showed that TrimTac activity is TRIM21-dependent, as no substrate degradation was observed in TRIM21 knockout cells (Figure 18).

[0275] To confirm that the selective degradation of the oligomeric substrate is maintained by non-covalent TRIMTAC, a short linker and AP1867 (a synthetic ligand that non-covalently binds to FKBP(F36V)) were added to compound 37 to form compound 414:

[0276] [ka]

[0277] Next, we investigated the ability of compound 414 to degrade different cellular targets, namely the monomer mEGFP-FKBP(F36V) and the oligomer Cavin1-mEGFP-FKBP(F36V). The degradation activity of compound 414 was compared to standard VHL-based (dTAGv1) and CRBN-based (dTAG13) PROTACs, and protein levels were monitored by measuring mEGFP fluorescence. Specifically, RPE-1 mEGFP-FKBP(F36V) and RPE-1 Cavin1-mEGFP-FKBP(F36V) stable cell lines were treated with compound 414, dTAG13, and dTAGv1 at the concentrations shown in Figure 25, and imaged for 8 hours using the IncuCyte system. GFP fluorescence was quantified by normalizing the GFP integrated density (and therefore the product of area and mean intensity) for each image against the total cell area (phase), and then normalizing it against the corresponding DMSO condition such that a value of 1 (dashed line in Figure 25) represents no degradation. As expected, dTAG13 and dTAGv1 efficiently degraded both targets, but monomer mEGFP-FKBP(F36V) was preferred (Figure 25). In contrast, compound 414 efficiently degraded oligomer Cavin-1-mEGFP-FKBP(F36V) but showed no activity against monomer mEGFP-FKBP(F36V).

[0278] To further explore this, compounds 414, dTAG13, and dTAGv1 were titrated over a wide range of concentrations, and their substrate degradation profiles were compared. Specifically, stable RPE-1 mEGFP-FKBP(F36V) and Cavin1-mEGFP-FKBP(F36V) cell lines were treated with titration of compound 414, dTAG13, or dTAGv1 for 8 hours, and GFP fluorescence was quantified using the IncuCyte system as described for Figure 25. Both dTAG13 and dTAGv1 dose-dependently degraded both substrates, with a strong preference for monomer substrates over oligomeric substrates (Figure 26). In contrast, compound 414 dose-dependently degraded the oligomeric Cavin1-mEGFP-FKBP(F36V), but, as previously observed, showed no degradation activity against monomeric mEGFP-FKBP(F36V) at any concentration.

[0279] Finally, WT or TRIM21 KO cells stably expressing Cavin1-mEGFP-FKBP(F36V) were treated with titration of compound 414 for 8 hours, and GFP fluorescence was quantified using the IncuCyte system as described for Figure 25. The results showed that compound 414 activity is TRIM21-dependent, as no substrate degradation was observed in TRIM21 knockout cells (Figure 27).

[0280] In summary, the data indicate that TrimTac can mediate highly efficient TRIM21-dependent substrate degradation. However, unlike VHL-mobilized and CRBN-mobilized protacs, TrimTac exhibits target selectivity, being able to degrade oligomeric substrates but not monomeric substrates.

[0281] Example 8: TRIMTAC, upon binding to an oligomeric target, induces activation of TRIM21 clustering and ubiquitination. To investigate the mechanism by which TRIMTAC selectively degrades oligomeric proteins, we developed an assay to detect TRIM21 clustering and activation in living cells. This assay uses a NanoBiT split luciferase system, which reconstitutes luciferase activity only when two protein fragments, LgBiT and SmBiT, are placed in proximity. Specifically, two N-terminally tagged versions of TRIM21, SmBiT-TRIM21 and LgBiT-TRIM21, were co-expressed in TRIM21 KO Cavin-1-mEGFP-FKBP(F36V) cells. Addition of compound 414 induced an immediate and rapid increase in luciferase activity, suggesting that compound 414 placed the two versions of TRIM21 in proximity, indicating TRIM21 clustering when TRIMTAC bound to the oligomeric target (Figure 31A, B). This luciferase activity peaked 8 minutes after the addition of compound 414 and then decreased over the next 90 minutes, which occurred simultaneously with the degradation of Cavin-1-mEGFP-FKBP(F36V) and may indicate that TRIM21 is co-degraded along with its target (Figure 31A, B).

[0282] Next, the inventors adapted this assay to detect TRIMTAC-induced ubiquitination by co-expressing SmBiT-TRIM21 together with LgBiT-TUBEs (tandem ubiquitin-binding elements) that bind to polyubiquitin chains. The addition of compound 414 induced an immediate and rapid increase in luciferase activity, suggesting that TRIMTAC, upon binding to the oligomeric target, induces rapid assembly of ubiquitin chains in proximity to TRIM21 (Figure 31A, B). Luciferase activity for SmBiT-TRIM21 + LgBiT-TUBEs started slightly later than for SmBiT-TRIM21 + LgBiT-TRIM21, peaking 12–16 minutes after compound 414 addition, which was consistent with ubiquitin chain formation occurring after TRIM21 activation. This luciferase activity decreased over the next 90 minutes, occurring concurrently with the degradation of Cavin-1-mEGFP-FKBP(F36V), which may represent the degradation of polyubiquitinated species (Figure 31A, B).

[0283] Example 9: Synthesis of TrimTac based on Compound 37 and AP1867

[0284] [ka]

[0285] A stirred solution of ortho-AP1867(42) (15 mg, 0.022 mmol, 1.0 equivalent) and 43 (6.9 mg, 0.022 mmol, 1.0 equivalent) in N,N-dimethylformamide (0.1 mL) was to be mixed with a stirred solution of 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (8.2 mg, 0.022 mmol, 1.0 equivalent) and N,N-diisopropylethylamine (0.011 mL, 0.065 mmol, 3.0 equivalents) in N,N-dimethylformamide (0.1 mL). The resulting reaction mixture was stirred at room temperature in the dark for 18 hours. Due to the presence of residual 1 as determined by TLC and NMR, a further portion of 43 (2.0 mg) and 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (2.0 mg) was added to the reaction mixture and stirred for a further 24 hours. The reaction mixture was washed with 5% aqueous lithium chloride solution and extracted with ethyl acetate (3 × 20 mL). The combined organic layer was washed with 5% aqueous lithium chloride solution (2 × 20 mL), dried over MgSO4, filtered, and concentrated under vacuum. The residue was purified by flash column chromatography (0-5% methanol in dichloromethane) to obtain the desired product 44 as a yellow oil (16 mg, 74%). δ H (400 MHz, CD3OD) 0.88 (3H, t, J 7.3 Hz, 1-H3), 1.16-1.48 (11H, m), 1.55-1.83 (8H, m), 1.87-2.15 (3H, m), 2.27 (1H, d, J 13.2 Hz), 2.40-2.68 (3H, m), 3.06-3.29 (4H, m), 3.35-3.63 (13H, m), 3.64-3.84 (15H, m), 3.87 (1H, t, J 7.3 Hz), 4.13 (1H, br d, J 13.7 Hz), 4.38-4.68 (2H, m), 5.38-5.48 (1H, m), 6.13 (1H, dd, J 8.2, 5.8 Hz), 6.32-7.50 (10 H, m); δ C(101 MHz, CD3OD) 12.6, 21.9, 26.4, 27.6, 28.8, 29.3, 30.3, 30.9, 32.2, 37.6, 38.9, 45.0, 51.0, 53.4, 56.5, 56.5, 56.6, 56.7, 61.1, 61.1, 68.3, 69.8, 69.9, 70.8, 71.2, 71.5, 79.8, 106.6, 113.0, 113.1, 113.5, 113.6, 121.7, 121.8, 123.2, 128.3, 130.0, 130.5, 135.0, 136.9, 138.0, 148.8, 150.4, 154.6, 155.5, 170.6, 172.5, 174.8; + .C 53 H 77 N3NaO 15 (This requires 1018.5247).

[0286] [ka]

[0287] To a stirred solution of 44 (16 mg, 0.016 mmol, 1.00 equivalent) in 1,4-dioxane (0.20 mL) at 0°C, 4M hydrochloric acid in 1,4-dioxane (0.20 mL) was added. The resulting reaction mixture was stirred at room temperature for 4 hours. The reaction mixture was concentrated under vacuum to obtain the desired product 45 as a colorless oil (14 mg, 93%). The product was proceeded to the next step without further purification or characterization.

[0288] [ka]

[0289] To a stirred solution of 9 (4.0 mg, 0.0064 mmol, 1.0 equivalent) in 0.50 mL of dichloromethane, N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (1.5 mg, 0.0077 mmol, 1.2 equivalents) and 1-hydroxybenzotriazole hydrate (1.2 mg, 0.0077 mmol, 1.2 equivalents) were added, and the resulting solution was stirred at room temperature for 10 minutes. To the reaction mixture, a stirred solution of 45 (6.0 mg, 0.0064 mmol, 1.0 equivalent) and N,N-diisopropylethylamine (0.0025 mL, 0.019 mmol, 3.0 equivalents) in 0.50 mL of dichloromethane was added, and the resulting reaction mixture was stirred at room temperature for 20 hours. The reaction mixture was concentrated under vacuum and purified by flash column chromatography (0-5% methanol in dichloromethane) to obtain the desired product 46 as a colorless oil (4 mg, 41%). δ H (400 MHz, CD3OD) 0.68-3.08 (51H, m), 3.10-3.26 (3H, m), 3.35-3.93 (31H, m), 3.95-4.74 (6H, m), 5.27-5.47 (2H, m), 6.13 (1H, t, J 7.0 Hz), 6.32-7.13 (11H, m), 7.14-7.87 (6H, m), 8.42 (2H, d, J 4.6 Hz); + ) 701.1 ([M-Boc+2H] 2+ , 38%).

[0290] [ka]

[0291] To a stirred solution of 46 (4.0 mg, 0.0027 mmol, 1.0 equivalent) in 0.10 mL of dichloromethane at 0°C, trifluoroacetic acid (0.0020 mL, 0.027 mmol, 10 equivalents) was added as a stock solution in dichloromethane. The resulting reaction mixture was stirred at room temperature for 2 hours. The reaction mixture was concentrated under vacuum to obtain the desired product 47 as a colorless oil (4.0 mg, quant.). To prepare the compound for biological evaluation, the product was further purified by preparative HPLC (30-95% acetonitrile (+0.1% formic acid) in water (+0.1% formic acid) - the desired product was eluted as digate at tR=13 min (48% acetonitrile)), and lyophilized to obtain the product as a white solid (2.0 mg). δ H (400 MHz, CD3OD) 0.69-0.97 (6H, m), 0.99-1.31 (7H, m), 1.42-1.83 (16H, m), 1.86-2.12 (5H, m), 2.26 (1H, d, J 12.5 Hz), 2.39-2.70 (4H, m), 2.78-3.15 (6H, m), 3.13 (1H, p, J 1.6 Hz), 3.21-3.28 (4H, m), 3.36-3.42 (2H, m), 3.43-3.61 (11H, m), 3.64-3.71 (7H, m), 3.75 (1H, d, J 7.0), 3.77-3.82 (7H, m), 3.82-3.91 (4H, m), 4.14 (1H, br d, J 13.5 Hz), 4.37-4.50 (2H, m), 4.52-4.77 (3H, m), 5.42 (1H, s), 6.13 (1H, dd, J 8.2, 5.7 Hz), 6.54-6.65 (2H, m), 6.67 (1H, dd, J 8.2, 2.0 Hz), 6.73-6.80 (1H, m), 6.81-6.93 (2H, m), 6.94-7.11 (2H, m), 7.20-7.50 (4H, m), 8.37-8.49 (2H, m), 8.54 (1H, br s); m / z (LC-MS, ESI + ) 701.1 ([M+2H] 2+ , 100%).

[0292] Example 10: TrimTac selectively degrades oligomeric proteins in the same cell, but does not degrade monomer proteins. To obtain further evidence regarding TrimTac selectivity, microscopy was used to determine where the substrate was degraded within the cell. RPE-1 Cavin1-mEGFP-Halo cells were treated with DMSO, 1.25 μM HaloPROTAC1, 1.25 μM compounds 37, 71, 109, 111, or 10 μM compound 110 for 48 hours and imaged using the IncuCyte system. Cavin1-mEGFP-Halo cells showed bright fluorescence at the cell membrane and some diffused cytoplasmic fluorescence under control (DMSO) conditions. Treatment with HaloPROTAC1 reduced overall fluorescence, but residual fluorescence remained concentrated at the cell membrane. Incubation with HaloPROTAC1 resulted in protein degradation from both the membrane and the cytosol pool, but significant levels of membrane-bound substrate remained even after 48 hours. Treatment with TRIMTAC (compounds 71, 109, 110, and 111) caused a complete loss of bright fluorescence from the cell membrane, but diffused cytosolic fluorescence remained. Therefore, the membrane-bound form of Cavin1-mEGFP-Halo was degraded by TRIMTAC, but the cytosolic form was unaffected.

[0293] This was investigated further. RPE-1 Cavin1-mEGFP-Halo cells were seeded on polymer coverslips (Ibidi) in an 8-well chamber, and images were taken before and after treatment with DMSO, 2.5 μM HaloPROTAC1, or compound 71 using an Etaluma Lumascope LS720 wide-field microscope equipped with a 40× apochromatic 0.95NA air objective lens housed in a 37C 5% CO2 incubator. Treatment with HaloPROTAC1 induced a gradual loss of both cytosolic and membrane fluorescence for more than 24 hours. Treatment with compound 71 induced a rapid loss of membrane-bound fluorescence within 1 hour. After 1 hour, almost all membrane-bound Cavin1-mEGFP-Halo was found to be degraded in TrimTac-treated cells, whereas the cytosolic pool remained unaffected even after 24 hours (Figure 20A). In contrast, cytosolic Cavin1-mEGFP-Halo was rapidly degraded in HaloPROTAC1-treated cells, but a significant membrane-bound protein remained after 24 hours (Figure 20B).

[0294] This was further supported by quantifying fluorescence intensity across cells. HaloPROTAC1-treated cells showed a decrease in protein levels across cells (Figure 21A), while TrimTac-treated cells showed degradation only of oligomeric membrane-bound proteins (Figure 21B).

[0295] Finally, we tested whether TrimTac could selectively degrade oligomeric protein species when both oligomeric and monomeric versions were expressed in the same cells. RPE-1 Cavin1-mEGFP-Halo cells were treated with DMSO, 2.5 μM HaloPROTAC1, or compound 37 for 24 hours, and whole cell extracts were collected for capillary-based immunoblotting. Anti-GFP immunoblotting showed the presence of a smaller species (*-mEGFP-Halo) in addition to full-length Cavin1-mEGFP-Halo, which may correspond to cleavage products lacking the Cavin1 sequence. Treatment with HaloPROTAC1 resulted in a slight reduction of the Cavin1-mEGFP-Halo band and complete loss of the mEGFP-Halo band. Conversely, treatment with compound 71 resulted in a substantial reduction of the Cavin1-mEGFP-Halo band, while the *-mEGFP-Halo band remained unaffected (Figure 19(A)). Cavin1-mEGFP-Halo and *-mEGFP-Halo bands were normalized relative to the anti-actin band and expressed as a percentage of the total anti-GFP band intensity. Treatment with compound 71 caused a significant reduction in Cavin1-mEGFP-Halo protein levels, but not a significant reduction in *-mEGFP-Halo protein levels (Figure 19(B)). Therefore, in cells expressing both mEGFP-Halo and Cavin1-mEGFP-Halo and treated with HaloPROTAC1, there was efficient degradation of monomeric mEGFP-Halo and inefficient degradation of oligomeric Cavin1-mEGFP-Halo. In contrast, TrimTac treatment left monomeric mEGFP-Halo unaffected, while only oligomeric Cavin1-mEGFP-Halo was degraded. As discussed herein, TrimTac's activity is TRIM21-dependent.

[0296] RPE-1 Cavin1-mEGFP-Halo cells were seeded on polymer coverslips (Ibidi) in an 8-well chamber, and images were taken 2.5 hours after treatment with DMSO, 2.5 μM compound 37, or 2.5 μM compound 37 + 25 μM MG132. Treatment with MG132 was sufficient to rescue TrimTac-mediated substrate degradation, indicating that proteasomes are also required (Figure 22).

[0297] Example 11: TRIMTAC selectively degrades Myd88, which assembles the midosome. To obtain further evidence regarding TRIMTAC selectivity, the degradation activity of compound 414 was tested against Myd88, either in its resting monomer form or when Myd88 oligomerizes and assembles the midsome, a pro-inflammatory signaling complex. Briefly, we established an assay in which Myd88 fused c-terminus to GyrB-mEGFP-FKBP(F36V) is induced to oligomerize and assemble the midsome upon addition of the antibiotic cummermycin (Figure 28A). U2OS cells stably expressing Myd88-GyrB-mEGFP-FKBP(F36V) were treated with cummermycin (100 nM) to induce midsome assembly or mock-treated (control), and then treated with DMSO, Cmp 414 (5 μM), or Cmp 37 (5 μM), and Myd88 protein levels were quantified by GFP fluorescence. Cmp 414 does not degrade Myd88 in control-treated cells, but rapidly degrades Myd88 in Kumermycin-treated cells. Addition of DMSO or Cmp 37 does not affect Myd88 levels (Figure 28B). Microscopic images show that compound 414 rapidly degrades oligomeric Myd88 splatters (midosomes) but tolerates residual diffused Myd88 signaling.

[0298] Example 12: TRIMTAC selectively degrades RIPK3, which assembles necrosomes. To obtain further evidence of TRIMTAC selectivity, the degradation activity of compound 414 was tested against RIPK3, either in its inactive resting monomer state or when RIPK3 oligomerizes to assemble active necrosomes that induce cellular necroptosis. An assay was established in which treatment of cells expressing FKBP(F36V)-mEGFP-RIPK3 with a combination of (T)TNFα, (S)Smac mimetic, and (Z) caspase inhibitor ZVAD-FMK (T / S / Z) induced RIPK3 oligomerization and necroptosis (Figure 29A). RPE-1 cells stably expressing FKBP(F36V)-mEGFP-RIPK3 were treated with T / S / Z or a mock for 8 hours in the presence of DMSO or titration of compound 414 (control). Microscopic images show that in control-treated cells, RIPK3 is present as a monomer and exhibits a diffused cytosolic distribution, while T / S / Z treatment induces RIPK3 oligomerization, as observed by the accumulation of cytosolic RIPK3 spots (Figure 29B, DMSO). Compound 414 dose-dependently degraded oligomeric RIPK3 (T / S / Z) fluorescent spots, but showed no degrading activity against monomer-diffused RIPK3 (control) at any concentration.

[0299] To test whether TRIMTAC-selective degradation of oligomeric RIPK3 inhibits downstream necroptosis, the activity of compound 414 was compared to that of the RIPK3 kinase inhibitor GSK'872. RPE-1 cells expressing FKBP(F36V)-mEGFP-RIPK3 were treated with T / S / Z or mock (control) in the presence of DMSO, Cmp 414 (2.5 μM), or GSK'872 (0.3 μM) to induce RIPK3 oligomerization and necroptosis, and GFP fluorescence (RIPK3 protein level) and cell area (necroptosis cell death) were quantified using the IncuCyte system. Compound 414 rapidly degrades T / S / Z-induced RIPK3 oligomers upon assembly but does not affect monomer-diffusing RIPK3 protein levels. The RIPK3 kinase inhibitor GSK'872, like DMSO, did not affect RIPK3 oligomerization (Figure 30A, B). T / S / Z treatment induced necroptosis, which could be quantified by measuring cell area from phase-contrast microscopy images. As expected, the RIPK3 kinase inhibitor GSK'872 prevented necroptosis, as the cell area was similar to that of control cells at 48 hours. Compound 414 also partially rescued cell area in T / S / Z-treated cells, suggesting that selective degradation of oligomeric RIPK3 mediated by TRIMTAC may inhibit necroptosis.

[0300] In summary, the data indicate that TrimTac possesses unexpected target specificity, degrading oligomeric substrates but not monomeric substrates. This is due to its TRIM21-recruiting property, as its activity is lost in TRIM21 knockout cells. Therefore, compounds that recruit TRIM21 access a unique degradation mechanism rather than PROTAC.

[0301] This data demonstrates that the compound of the present invention (also known herein as TRIMTAC) can selectively degrade oligomeric proteins rather than their monomeric forms.

[0302] Further embodiments of the present invention are provided below:

[0303] 1. Compound of formula (I) ALB (I) [In the formula, A is the part that connects to TRIM21. L is a linker, B is the part that binds to the target protein. or a pharmaceutically acceptable salt thereof.

[0304] 2. The compound according to claim 1, wherein the target protein may include a first form that provides multiple binding sites to B, and / or the target protein may be capable of forming an oligomeric species.

[0305] 3. The compound according to claim 1 or 2, wherein the compound is configured to recruit TRIM21 to enable selective degradation by TRIM21 of one or more oligomeric forms of a target protein.

[0306] 4. A compound according to any one of items 1 to 3, wherein A binds to the PRYSPRY domain of TRIM21 (SEQ ID NO: 1).

[0307] 5. A is bound to one or more residues selected from W381, W383, D355, F369, L370, L371, F450, Y328, H368, S447, M330, D452, R364, Q395, Y393, E389, and K387 of TRIM21 (SEQ ID NO: 1), preferably one or more of the residues being selected from W381, W383, and D355, as described in any one of items 1 to 4.

[0308] 6. A compound described in any one of items 1 to 5, wherein A is bound to residue D355 of TRIM21 (SEQ ID NO: 1).

[0309] 7. A compound according to any one of items 1 to 6, wherein A includes a portion capable of forming a hydrogen bond with residue D355 of TRIM21 (SEQ ID NO: 1).

[0310] 8. The compounds described in item 7, wherein the hydrogen bonds are charged hydrogen bonds.

[0311] 9. A compound described in any one of items 1 to 8, wherein A is bound to residue W381 of TRIM21 (SEQ ID NO: 1).

[0312] 10. A compound according to any one of items 1 to 9, wherein A contains a portion that can hydrophobically stack with residue W381 of TRIM21 (SEQ ID NO: 1).

[0313] 11. A compound described in any one of items 1 to 10, wherein A is bound to residue W383 of TRIM21 (SEQ ID NO: 1).

[0314] 12. A compound according to any one of items 1 to 11, wherein A contains a portion that can hydrophobically stack with residue W383 of TRIM21 (SEQ ID NO: 1).

[0315] 13. A compound according to any one of items 1 to 12, wherein A is bound to residues W381, W383 and D355 of TRIM21 (SEQ ID NO: 1).

[0316] 14. A compound according to any one of items 1 to 13, wherein A competes with IgG for binding to TRIM21.

[0317] 15. A compound according to any one of items 1 to 14, wherein A is not an Fc region or a fragment thereof.

[0318] 16. A compound according to any one of items 1 to 15, wherein A is not an anti-TRIM21 antibody or its antigen-binding fragment.

[0319] 17. A compound according to any one of items 1 to 16, wherein the target protein has a pathogenic form and a non-pathogenic form, and the pathogenic form of the protein includes two or more binding sites to B.

[0320] 18. A compound according to any one of items 1 to 17, wherein B is not an anti-tau antibody or an antibody-conjugated fragment thereof.

[0321] 19. A compound according to any one of items 1 to 18, wherein A is not an anti-TRIM21 antibody or its antigen-binding fragment, and B is not an anti-tau antibody or its antigen-binding fragment.

[0322] 20. A compound described in any one of items 1 to 19, wherein the molecular weight of A is 1000 Da or less.

[0323] 21. A compound described in any one of items 1 to 20, wherein the molecular weight of A is in the range of 100 Da to 1000 Da.

[0324] 22. The compound's K in relation to TRIM21 in the range of 0.1 nM to 1000 μM. d A compound according to any one of items 1 to 21, having a value.

[0325] 23. The compound has a K2 concentration in TRIM21 in the range of 1 nM to 1000 μM. d A compound according to any one of items 1 to 22, having a value.

[0326] 24. The compound has a K2 concentration in the range of 10 nM to 100 μM relative to TRIM21. d A compound according to any one of items 1 to 23, having a value.

[0327] 25. A is the structure of equation (II).

[0328] [ka]

[0329] [In the formula, R 1and R 2 These are, independently, hydrogen and C 1~2 Selected from alkyl groups, or R 1 and R 2 One of them is hydrogen, and the other is selected from an amino acid residue and a covalent bonding site to L. R 3 R is selected from hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted heteroalkyl, optionally substituted heteroalkenyl, optionally substituted heteroalkynyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted aryl, and optionally substituted heteroaryl. 3 This optionally includes a covalent bond site to L. R 4 is hydrogen, halo, or C 1~2 It is alkyl, R 5 is hydrogen or C 1~2 It is alkyl, R 6 is an alkyl which may be substituted, or R 5 and R 6 These combine to form a complex ring, which may be substituted. R 7 teeth,

[0330] [ka]

[0331] , the site of covalent bonding to L, or

[0332] [ka]

[0333] And, R 8 is hydrogen or C 1~2 It is alkyl, R 9is an alkyl group which may be substituted, R 14 and R 15 These are, independently, hydrogen and C 1~2 Selected from alkyl groups, R 7 In the structure

[0334] [ka]

[0335] This indicates the site of covalent bonding to L. However, this is conditional on A containing a covalent bond site to L. A compound according to any one of items 1 to 24, having the properties of:

[0336] 26. A has the following structure:

[0337] [ka]

[0338] [In the formula, R 1 and R 2 These are, independently, hydrogen and C 1~2 Selected from alkyl groups, or R 1 and R 2 One of them is hydrogen, and the other is selected from L-amino acid residues and the site of covalent bonding to L. R 7 teeth,

[0339] [ka]

[0340] , the site of covalent bonding to L, or

[0341] [ka]

[0342] is The compound described in item 25, having the properties of:

[0343] 27. A has the following structure:

[0344] [ka]

[0345] [In the formula, R 1 and R 2 These are, independently, hydrogen and C 1~2 Selected from alkyl groups, or R 1 and R 2 One of them is hydrogen, and the other is selected from a D-amino acid residue and a covalent bonding site to L. R 7 teeth,

[0346] [ka]

[0347] , the site of covalent bonding to L, or

[0348] [ka]

[0349] is The compound described in item 25, having the properties of:

[0350] 28. A has the structure of formula (IIa), (IIb), or (IIc):

[0351] [ka]

[0352] [In the formula, each

[0353] [ka]

[0354] Independently,

[0355] [ka]

[0356] Selected from, R 1 and R 2 These are, independently, hydrogen and C 1~2 Selected from alkyl groups, or R 1 and R 2 One is hydrogen, and the other is an amino acid residue. R 3 This is selected from hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted heteroalkyl, optionally substituted heteroalkenyl, optionally substituted heteroalkynyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted aryl, and optionally substituted heteroaryl. R 4 is hydrogen, halo, or C 1~2 It is alkyl, Each R 5 These are, independently, hydrogen or C 1~2 It is alkyl, R 6 is an alkyl that may be substituted, Or, R 5 and R 6 If R is bonded to an adjacent atom, 5 and R 6 These combine to form a complex ring, which may be substituted. R 8 is hydrogen or C 1~2 It is alkyl, R 9 is an alkyl group which may be substituted, R 18 and R 19 These are, independently, hydrogen and C 1~2 Selected from alkyl groups, R 20 [This is an alkyl group that may be substituted.] A compound according to any one of items 1 to 27, having the properties of:

[0357] 29. R 1 and R 2 The compounds described in sections 25-28, wherein one of the atoms is hydrogen and the other is selected from an amino acid residue, and optionally the amino acid residue is selected from a histidine residue, a phenylalanine residue, a tryptophan residue, and a tyrosine residue.

[0358] 30. The compounds described in item 29, wherein the amino acid residue is a histidine residue.

[0359] 31. R 1 , R 2 and R 4 Compounds described in sections 25-28, wherein the hydrogen atom is hydrogen.

[0360] 32. R 3 The compound according to any one of claims 25 to 31, selected from optionally substituted aryls and optionally substituted heteroaryls.

[0361] 33. R 3 The compound according to any one of claims 25 to 32, wherein the compound is selected from aryl and heteroaryl, and the aryl or heteroaryl may be substituted with one or more substituents selected from halo, C1-C8 alkyl, C1-C8 haloalkyl, cyano, hydroxy, C1-C8 alkoxy, and amino.

[0362] 34. R 3 However, the compounds according to any one of claims 25 to 33, selected from furan and optionally substituted phenyl.

[0363] 35. R 3However, unsubstituted phenyl, C 1~2 Phenyl, C substituted with alkyl 1~2 A compound according to any one of claims 25 to 34, selected from phenyls substituted with alkoxy and unsubstituted 5-6 membered heteroaryls.

[0364] 36. R 7 but,

[0365] [ka]

[0366] [In the formula, (a)R 9 is selected from aryls which may be substituted with -alkylene- and heteroaryls which may be substituted with -alkylene-, and optionally R 9 This is selected from aryls which may be substituted with -CH2- and heteroaryls which may be substituted with -CH2-, or (b)R 9 teeth,

[0367] [ka]

[0368] Selected from, R 9 In the structure

[0369] [ka]

[0370] R 7 This indicates the site of covalent bonding to the remainder, or (c)R 9 [The side chain is selected from histidine, phenylalanine, tryptophan, tyrosine, cysteine, serine, threonine, methionine, asparagine, and glutamine side chains.] The compound described in any one of the items 25-27 or 29-35.

[0371] 37. (a)R 9 However, selected from aryls which may be substituted with -alkylene- and heteroaryls which may be substituted with -alkylene-, and optionally R 9 However, they may be selected from aryls that may be substituted with -CH2- and heteroaryls that may be substituted with -CH2-, or (b)R 9 but,

[0372] [ka]

[0373] Select from, or (c)R 9 The compound described in any one of items 25 to 36, wherein the side chain is selected from histidine, phenylalanine, tryptophan, tyrosine, cysteine, serine, threonine, methionine, asparagine, and glutamine.

[0374] 38. R 5 and R 6 These are combined to form hydroxy, nitro, cyano, halo, and -NR. 10 R 11 , forming a 5-membered heterocyclic ring which may be substituted by an optionally substituted alkyl, optionally substituted alkoxy, optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted -O-cycloalkyl, optionally substituted -O-heterocyclyl, optionally substituted -O-aryl, or optionally substituted -O-heteroaryl, R 10 and R 11The compound according to any one of items 25 to 37, wherein each is independently H, an optionally substituted alkyl group, or an optionally substituted cycloalkyl group.

[0375] 39. R 7 but,

[0376] [ka]

[0377] And R 3 but,

[0378] [ka]

[0379] [In the formula,

[0380] [ka]

[0381] * indicates the site of covalent bonding to L, and * indicates the site of covalent bonding to the remainder of A. The compound described in any one of items 25-27 or 29-38.

[0382] 40. R 7 but,

[0383] [ka]

[0384] And R 3 However, it is selected from 5-6 member heteroaryls and phenyl, where phenyl is hydroxy, C 1~2 Alkoxy or C 1~2 The compounds described in any one of items 25-27 or 29-38, which may be substituted with alkyl.

[0385] 41. R 3 However, it is selected from 5-6 member heteroaryls and phenyl, where phenyl is hydroxy, C 1~2 Alkoxy or C 1~2 The compounds described in any one of items 25 to 40, which may be substituted with alkyl groups.

[0386] 42. A is structure:

[0387] [ka]

[0388] [In the formula, R x , R y and R z Each of these is independently selected from optionally substituted cycloalkyls, optionally substituted heterocyclines, optionally substituted aryls, and optionally substituted heteroaryls. A compound according to any one of items 1 to 41, having the properties of:

[0389] 43. R x However, R is selected from substituted phenyl and substituted 5-6 member heteroaryls. y However, R is selected from substituted or substituted 5-6 member cycloalkyls and substituted or substituted 5-6 member heterocyclines. z The compound according to item 42, selected from optionally substituted phenyl and optionally substituted 5-6 membered heteroaryls.

[0390] 44. A is the structure of equation (III).

[0391] [ka]

[0392] [In the formula, Each of rings E, F, C, and D is independently selected from an optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted aryl, and optionally substituted heteroaryl. In the structure of equation (III)

[0393] [ka]

[0394] [This indicates the site of the covalent bond to L.] A compound according to any one of items 1 to 24, having the properties of:

[0395] 45. The compound according to item 44, wherein ring C is selected from aryl and heteroaryl, and the aryl or heteroaryl is substituted with aryl or heteroaryl.

[0396] 46. ​​A is structure:

[0397] [ka]

[0398] [In the formula, R a R is selected from 5-membered heteroaryls, 6-membered heteroaryls, and phenyls. b is hydrogen, C 1~2 alkyl, halo, and C 1~2 Selected from alkoxy, R c is hydrogen, C 1~2 alkyl, halo, and C 1~2 [Selected from alkoxy] A compound according to any one of claims 1 to 34, 44, or 45, having the properties of:

[0399] 47. R a The compound according to item 46, wherein is a 5-membered heteroaryl.

[0400] 48. R aThe compound according to item 46 or 47, wherein is a nitrogen-containing heteroaryl compound.

[0401] 49. A,

[0402] [ka] TIFF2026525346000093.tif172150

[0403] [In the formula, in the structure of A

[0404] [ka]

[0405] [This indicates the site of the covalent bond to L.] A compound selected from any one of items 1 to 48.

[0406] 50. A,

[0407] [ka] TIFF2026525346000096.tif56153

[0408] [In the formula, in the structure of A

[0409] [ka]

[0410] [This indicates the site of the covalent bond to L.] A compound selected from the compounds listed in item 49.

[0411] 51. A compound according to any one of items 1 to 50, wherein the target protein is selected from proteins that form aggregates.

[0412] 52. A compound according to any one of items 1 to 51, wherein the target protein is selected from tau, synuclein, amyloid-beta, islet amyloid polypeptide (IAPP), serum amyloid-a (SAA), prion protein, misfolded transthyretin protein (TTR), TAR DNA-binding protein 43 (TDP-43), NOTCH3 receptor, mutant cystatin C, mezin, dipeptide repeat protein, and sarcoma fusion.

[0413] 53. A compound according to any one of items 1 to 52, wherein the target protein is selected from tau, synuclein, amyloid-beta, prion protein, TAR DNA-binding protein 43 (TDP-43), dipeptide repeat protein, and sarcoma fusion.

[0414] 54. A compound described in any one of items 1 to 53, wherein the target protein is tau.

[0415] 55. A compound according to any one of items 1 to 54, wherein the target protein is selected from proteins having extended repeat elements.

[0416] 56. A compound described in any one of items 1 to 55, wherein the target protein is mutant huntingtin.

[0417] 57. A compound according to any one of items 1 to 56, wherein the target protein is selected from proteins involved in signaling pathways in inflammation and / or cancer.

[0418] 58. A compound according to any one of items 1 to 57, wherein the target protein is selected from Myd88, IRAK4, IRAK2, IRAK1, TRAF6, NLRP3, RIPK3, and ASC.

[0419] 59. A compound according to any one of items 1 to 58, wherein the target protein is selected from Myd88, IRAK4, IRAK2, IRAK1, TRAF6, NLRP3, and ASC.

[0420] 60. A compound according to any one of items 1 to 59, wherein the target protein is selected from viral proteins that form oligomers.

[0421] 61. A compound according to any one of items 1 to 60, wherein the target protein is selected from the nucleoproteins of CoV2 and influenza virus.

[0422] 62. A compound according to any one of items 1 to 61, wherein the target protein is selected from bromodomain-containing protein 4 (BRD4), mutant gelsolin, misfolded rhodopsin, and atrial natriuretic peptide.

[0423] 63. A compound according to any one of items 1 to 62, wherein B is selected from PI-2014, FDDNP, AV680(T808), GTP-1, THK523, THK5105, PBB3, AV1451(T807), THK5117, THK5351, N-methyllansoprazole, (E)-6-iodo-2-styryl-1H-benzo[d]imidazole, RO6958948, PI-2620, JNJ-067, APN-1607(PM-PBB3), MK-6240, and methylene blue.

[0424] 64. B,

[0425] [ka]

[0426] [In the formula, in the structure of B

[0427] [ka]

[0428] [This indicates the site of the covalent bond to L.] A compound selected from any one of items 1 to 63.

[0429] 65. Linker (L) (a) is a combination, or (b) A chain of 2 to 20 carbon atoms, wherein one or more carbon atoms are optionally replaced by 1 to 12 ethylene glycol units and / or heteroatoms selected from O, N, S, and P, and / or (c) comprising poly(ethylene glycol) (PEG), unsubstituted alkylene, substituted alkylene, or a combination thereof, and / or (d)

[0430] [ka] TIFF2026525346000101.tif173165

[0431] [In the formula, in the structure of the linker

[0432] [ka]

[0433] Each of these cases indicates a covalent bond to A or B. A compound selected from any one of items 1 to 64.

[0434] 66. The compound,

[0435] [ka] TIFF2026525346000104.tif28163

[0436] A compound selected from any one of items 1 to 65.

[0437] 67. The compound,

[0438] [ka]

[0439] A compound selected from any one of items 1 to 66.

[0440] 68. Compounds of formula (I) ALB (I) [In the formula, A is the part that connects to TRIM21. L is a linker, B is a portion that binds to the target protein, and the target protein has a first form containing multiple binding sites to B and a second form containing a single binding site to B. or a pharmaceutically acceptable salt thereof.

[0441] 69. The compound according to item 68, wherein A, L, B and the target protein are as defined in any one of items 1 to 67.

[0442] 70. Compounds capable of simultaneously binding to TRIM21 and target proteins.

[0443] 71. The compound according to item 70, wherein the target protein has a first form in which it provides multiple binding sites for the compound.

[0444] 72. The compound according to item 70 or 71, wherein the target protein has a first form comprising multiple binding sites to the compound and a second form comprising a single binding site to the compound.

[0445] 73. A compound according to any one of items 70 to 72, wherein the target protein is capable of forming an oligomeric species.

[0446] 74. A compound according to any one of items 70-73, wherein the binding allows for the clustering of multiple TRIM21 molecules.

[0447] 75. A compound according to any one of items 70 to 74, wherein the binding enables ubiquitination and degradation of a target protein.

[0448] 76. A compound according to any one of items 70 to 75, wherein the target protein has multiple binding sites for the compound, and the binding of the compound enables the clustering of multiple TRIM21 molecules.

[0449] 77. The compound according to item 74 or 76, wherein clustering enables ubiquitination and degradation of the target protein.

[0450] 78. A compound according to any one of the items 70-77, wherein the target protein is as defined in any one of the items 2, 3, 17, or 51-62.

[0451] 79. A compound according to any one of items 70 to 78, wherein the compound binds to the PRYSPRY domain of TRIM21 (SEQ ID NO: 1).

[0452] 80. A compound according to any one of items 70 to 79, wherein the compound binds to one or more residues selected from W381, W383, D355, F369, L370, L371, F450, Y328, H368, S447, M330, D452, R364, Q395, Y393, E389, and K387 of TRIM21 (SEQ ID NO: 1).

[0453] 81. The compound described in item 80, wherein one or more residues are selected from W381, W383, and D355.

[0454] 82. A compound according to any one of items 70 to 81, wherein the compound binds to residue D355 of TRIM21 (SEQ ID NO: 1).

[0455] 83. A compound according to any one of items 70 to 82, wherein the compound includes a portion capable of forming a hydrogen bond with residue D355 of TRIM21 (SEQ ID NO: 1).

[0456] 84. The compounds described in item 83, wherein the hydrogen bonds are charged hydrogen bonds.

[0457] 85. A compound described in any one of items 70 to 84, wherein the compound binds to residue W381 of TRIM21 (SEQ ID NO: 1).

[0458] 86. A compound according to any one of items 70 to 85, wherein the compound includes a portion that can hydrophobically stack with residue W381 of TRIM21 (SEQ ID NO: 1).

[0459] 87. A compound according to any one of items 70 to 86, wherein the compound binds to residue W383 of TRIM21 (SEQ ID NO: 1).

[0460] 88. A compound according to any one of items 70 to 87, wherein the compound includes a portion that can hydrophobically stack with residue W383 of TRIM21 (SEQ ID NO: 1).

[0461] 89. A compound according to any one of items 70 to 88, wherein the compound is bound to residues W381, W383 and D355 of TRIM21 (SEQ ID NO: 1).

[0462] 90. A compound described in any one of sections 70-89, wherein the compound competes with IgG for binding to TRIM21.

[0463] 91. A compound according to any one of the items 70 to 90, wherein the compound is not an Fc region or a fragment thereof.

[0464] 92. A compound according to any one of items 70 to 91, wherein the compound is not an anti-TRIM21 antibody or an antigen-binding fragment thereof.

[0465] 93. A compound according to any one of items 70 to 92, wherein the compound is as defined in any one of items 1 to 69.

[0466] 94. A pharmaceutical composition comprising a compound described in any one of sub-sub

[0467] 95. A compound or a pharmaceutically acceptable salt thereof as described in any one of sections 1 to 93, or a pharmaceutical composition as described in section 94, for use in therapy.

[0468] 96. A compound or a pharmaceutically acceptable salt thereof as described in any one of items 1 to 93, or a pharmaceutical composition as described in item 94, for use in the treatment of neurodegenerative diseases.

[0469] 97. A compound or composition for use as described in item 96, wherein the neurodegenerative disease is selected from Alzheimer's disease, Parkinson's disease, and dementia.

[0470] 98. A method for treating a subject having a disease or disorder, comprising the step of administering a compound or a pharmaceutically acceptable salt thereof as described in any one of the items 1 to 93, or a pharmaceutical composition as described in item 94.

[0471] 99. The method of paragraph 98, wherein the disease or disorder is a neurodegenerative disease.

[0472] 100. Use of any compound or pharmaceutically acceptable salt thereof described in any one of subheadings 1 to 93 for the manufacture of a medicine for the treatment of a disease or disorder.

[0473] 101. Use as described in paragraph 100, where the disease or disorder is a neurodegenerative disease.

[0474] 102. An in vitro method for selectively degrading an oligomeric or pathogenic form of a target protein, comprising the step of contacting the target protein with TRIM21 and a compound described in any one of sub-sub

[0475] 103. Structure of Equation (IV)

[0476] [ka]

[0477] [In the formula, R 1 and R 2 These are, independently, hydrogen and C 1~2 Selected from alkyl groups, or R 1 and R 2 One is hydrogen, and the other is an amino acid residue. R 3 This is selected from hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted heteroalkyl, optionally substituted heteroalkenyl, optionally substituted heteroalkynyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted aryl, and optionally substituted heteroaryl. R 4 is hydrogen, halo, or C 1~2 It is alkyl, R 5 is hydrogen or C 1~2 It is alkyl, R 6 is an alkyl which may be substituted, or R 5 and R 6 These combine to form a complex ring, which may be substituted. R 9 is an alkyl group which may be substituted, R 12 and R 13 These are, independently, hydrogen and C 1~2 Selected from alkyl groups, or R 12 and R 13 One of them is hydrogen, and the other is a carbon atom which may be substituted. 1~2 Alkyl and -CHR 9 C(O)NR 14 R 15 Selected from, R 14 and R 15 These are, independently, hydrogen and C 1~2 Selected from alkyl groups] A compound having the following properties.

[0478] 103a. The compound has the following structure:

[0479] [ka]

[0480] [In the formula, R 1 and R 2 These are, independently, hydrogen and C 1~2 Selected from alkyl groups, or R 1 and R 2 One of them is hydrogen, and the other is an L-amino acid residue. R 12 and R 13 These are, independently, hydrogen and C 1~2 Selected from alkyl groups, or R 12 and R 13 One of them is hydrogen, and the other is a carbon atom which may be substituted. 1~2 Alkyl and

[0481] [ka]

[0482] [Selected from] A compound according to item 103, having the properties of:

[0483] 103b. The compound has the following structure:

[0484] [ka]

[0485] [In the formula, R 1 and R 2 These are, independently, hydrogen and C 1~2 Selected from alkyl groups, or R 1 and R 2 One of them is hydrogen, and the other is a D-amino acid residue. R 12 and R13 These are, independently, hydrogen and C 1~2 Selected from alkyl groups, or R 12 and R 13 One of them is hydrogen, and the other is a carbon atom which may be substituted. 1~2 Alkyl and

[0486] [ka]

[0487] [Selected from] A compound according to item 103, having the properties of:

[0488] 104. Structure of formula (IVa), (IVb), or (IVc):

[0489] [ka]

[0490] [In the formula, each

[0491] [ka]

[0492] Independently,

[0493] [ka]

[0494] Selected from, R 1 and R 2 These are, independently, hydrogen and C 1~2 Selected from alkyl groups, or R 1 and R 2 One is hydrogen, and the other is an amino acid residue. R 3R is selected from hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted heteroalkyl, optionally substituted heteroalkenyl, optionally substituted heteroalkynyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted aryl, and optionally substituted heteroaryl. 3 This optionally includes a covalent bond site to L. R 4 is hydrogen, halo, or C 1~2 It is alkyl, Each R 5 These are, independently, hydrogen or C 1~2 It is alkyl, R 6 is an alkyl that may be substituted, Or, R 5 and R 6 If R is bonded to an adjacent atom, 5 and R 6 These combine to form a complex ring, which may be substituted. R 8 is hydrogen or C 1~2 It is alkyl, R 9 is an alkyl group which may be substituted, R 12 and R 13 These are, independently, hydrogen and C 1~2 Selected from alkyl groups, R 14 and R 15 These are, independently, hydrogen and C 1~2 Selected from alkyl groups, R 18 and R 19 These are, independently, hydrogen and C 1~2 Selected from alkyl groups, R 20 [This is an alkyl group that may be substituted.] A compound having the following properties.

[0495] 105. R 1 and R 2The compound according to item 103, 103a, 103b, or 104, wherein one of the atoms is hydrogen and the other is selected from an amino acid residue, optionally, the amino acid residue is selected from a histidine residue, a phenylalanine residue, a tryptophan residue, and a tyrosine residue.

[0496] 106. The compounds described in item 105, wherein the amino acid residue is a histidine residue.

[0497] 107. R 1 , R 2 and R 4 A compound according to item 103, 103a, 103b, or 104, wherein the compound is hydrogen.

[0498] 108. R 3 However, selected from substituted or substituted aryls and substituted or substituted heteroaryls, optionally R 3 A compound according to any one of claims 103 to 107, selected from furan and optionally substituted phenyl.

[0499] 109. R 12 and R 13 One of them is hydrogen, and the other is -CHR 9 C(O)NR 14 R 15 And, (a)R 9 However, selected from aryls which may be substituted with -alkylene- and heteroaryls which may be substituted with -alkylene-, and optionally R 9 However, they may be selected from aryls that may be substituted with -CH2- and heteroaryls that may be substituted with -CH2-, or (b)R 9 but,

[0500] [ka]

[0501] A compound selected from any one of the items 103, 103a, 103b, or 105-108.

[0502] 110. (a)R 9 However, selected from aryls which may be substituted with -alkylene- and heteroaryls which may be substituted with -alkylene-, and optionally R 9 However, they may be selected from aryls that may be substituted with -CH2- and heteroaryls that may be substituted with -CH2-, or (b)R 9 but,

[0503] [ka]

[0504] A compound selected from any one of items 103 to 109.

[0505] 111. R 5 and R 6 These are combined to form hydroxy, nitro, cyano, halo, and -NR. 10 R 11 , forming a 5-membered heterocyclic ring which may be substituted by an optionally substituted alkyl, optionally substituted alkoxy, optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted -O-cycloalkyl, optionally substituted -O-heterocyclyl, optionally substituted -O-aryl, or optionally substituted -O-heteroaryl, R 10 and R 11 The compound according to any one of claims 103 to 110, wherein each is independently H, an optionally substituted alkyl group, or an optionally substituted cycloalkyl group.

[0506] 112. The compound has the following structure:

[0507] [ka]

[0508] [In the formula, R x , R y and R z Each of these is independently selected from optionally substituted cycloalkyls, optionally substituted heterocyclines, optionally substituted aryls, and optionally substituted heteroaryls. A compound having any one of the items 103 to 111.

[0509] 113. R x However, R is selected from substituted phenyl and substituted 5-6 member heteroaryls. y However, R is selected from substituted or substituted 5-6 member cycloalkyls and substituted or substituted 5-6 member heterocyclines. z The compound according to item 112, selected from optionally substituted phenyl and optionally substituted 5-6 membered heteroaryls.

[0510] 114. Structure of equation (V)

[0511] [ka]

[0512] [In the formula, Each of rings E, F, C, and D is independently selected from an optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted aryl, or optionally substituted heteroaryl. R 16 and R 17 These are, independently, hydrogen and C 1~2 Selected from alkyl groups] A compound having the following properties.

[0513] 115. The compound according to item 114, wherein ring C is selected from aryl and heteroaryl, and the aryl or heteroaryl is substituted with aryl or heteroaryl.

[0514] 116. The compound has the following structure:

[0515] [ka]

[0516] [In the formula, R a R is selected from 5-membered heteroaryls, 6-membered heteroaryls, and phenyls. b is hydrogen, C 1~2 alkyl, halo, and C 1~2 Selected from alkoxy, R c is hydrogen, C 1~2 alkyl, halo, and C 1~2 [Selected from alkoxy] A compound according to item 114 or 115, having the properties of:

[0517] 117. R a The compound according to item 116, wherein is a 5-membered heteroaryl.

[0518] 118. R a The compound according to item 116 or 117, wherein is a nitrogen-containing heteroaryl compound.

[0519] 119. The compound,

[0520] [ka] TIFF2026525346000120.tif166163

[0521] A compound selected from any one of items 103 to 118.

[0522] 120. The compound,

[0523] [ka] TIFF2026525346000122.tif60158

[0524] A compound selected from the compounds listed in item 119.

[0525] 121. A method for inhibiting the interaction between TRIM21 and an antibody, comprising the step of contacting TRIM21 with a compound described in any one of the items 103 to 120.

[0526] 122. The method according to item 121, wherein the method is an in vitro method.

[0527] one two three.

[0528] [ka] TIFF2026525346000124.tif106168

[0529] and compounds selected from pharmaceutically acceptable salts thereof.

[0530] 124. A pharmaceutical composition comprising a compound described in item 123 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

[0531] 125. A compound or a pharmaceutically acceptable salt thereof as described in item 123, or a pharmaceutical composition as described in item 124, for use in therapy.

[0532] 126. A compound or a pharmaceutically acceptable salt thereof as described in item 123, or a pharmaceutical composition as described in item 124, for use in the treatment of neurodegenerative diseases.

[0533] 127. A compound or composition for use as described in item 126, wherein the neurodegenerative disease is selected from Alzheimer's disease, Parkinson's disease, and dementia.

[0534] Sequences referred to herein:

[0535] [Table 4]

[0536] References: James LC et al, (2007) Proc Natl Acad Sci US A. 2007104(15):6200-6205 Leuzy A et al, (2019) Mol Psychiatry 24, 1112-113 Silva MC et al eLife v. 8 (2019) e45457 Studier FW et al, (2005). Protein Expr Purif, 41, 207-234 Winter G et al, (2011) Methods. 2011; 55:81-93 Winter G et al, (2022) Protein Sci. 2022 Jan;31(1):232-250

Claims

1. Compound of formula (I) ALB (I) [In the formula, A is the part that binds to TRIM21, and the molecular weight of A is 1000 Da or less. L is a linker, B is a portion that binds to the target protein, and the target protein may have a first form that provides multiple binding sites for B. or a pharmaceutically acceptable salt thereof.

2. The compound according to claim 1, wherein the target protein is capable of forming an oligomeric species.

3. The compound according to claim 1 or 2, wherein the compound is configured to recruit TRIM21 to enable selective degradation by TRIM21 of one or more oligomeric forms of a target protein.

4. A (a) the PRYSPRY domain of TRIM21 (SEQ ID NO: 1), and / or (b) One or more residues selected from W381, W383, D355 and F369, L370, L371, F450, Y328, H368, S447, M330, D452, R364, Q395, Y393, E389 and K387 of TRIM21, preferably one or more residues selected from W381, W383 and D355. A compound according to any one of claims 1 to 3, which is bonded to the compound.

5. A is a compound according to any one of claims 1 to 4, wherein A competes with IgG for binding to TRIM21.

6. A is a compound according to any one of claims 1 to 5, wherein A is not an antibody.

7. The compound has a K content in TRIM21 ranging from 0.1 nM to 1000 μM, preferably from 10 nM to 100 μM. d A compound according to any one of claims 1 to 6, having a value.

8. A is the structure of equation (II) 【Chemistry 1】 [In the formula, R 1 and R 2 These are, independently, hydrogen and C 1～2 Selected from alkyl groups, or R 1 and R 2 One of them is hydrogen, and the other is selected from an amino acid residue and a covalent bonding site to L. R 3 R is selected from hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted heteroalkyl, optionally substituted heteroalkenyl, optionally substituted heteroalkynyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted aryl, and optionally substituted heteroaryl. 3 This optionally includes a covalent bond site to L. R 4 is hydrogen, halo or C 1～2 alkyl, and R 5 is hydrogen or C 1～2 It is alkyl, R 6 is an alkyl which may be substituted, or R 5 and R 6 These combine to form a complex ring, which may be substituted. R 7 teeth, 【Chemistry 2】 , the site of covalent bonding to L, or 【Transformation 3】 And, R 8 is hydrogen or C 1～2 It is alkyl, R 9 is an alkyl group which may be substituted, R 14 and R 15 These are, independently, hydrogen and C 1～2 Selected from alkyl groups, R 7 In the structure 【Chemistry 4】 This indicates the site of covalent bonding to L. However, this is conditional on A containing a covalent bond site to L. A compound according to any one of claims 1 to 7, having the following characteristics.

9. A has the structure of formula (IIa), (IIb), or (IIc): 【Transformation 5】 [In the formula, each 【Transformation 6】 Independently, 【Transformation 7】 Selected from, R 1 and R 2 These are, independently, hydrogen and C 1～2 Selected from alkyl groups, or R 1 and R 2 One is hydrogen, and the other is an amino acid residue. R 3 This is selected from hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted heteroalkyl, optionally substituted heteroalkenyl, optionally substituted heteroalkynyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted aryl, and optionally substituted heteroaryl. R 4 is hydrogen, halo, or C 1～2 It is alkyl, Each R 5 These are, independently, hydrogen or C 1～2 It is alkyl, R 6 is an alkyl that may be substituted, Or, R 5 and R 6 If R is bonded to an adjacent atom, 5 and R 6 These combine to form a complex ring, which may be substituted. R 8 is hydrogen or C 1～2 It is alkyl, R 9 is an alkyl group which may be substituted, R 18 and R 19 These are, independently, hydrogen and C 1～2 Selected from alkyl groups, R 20 [This is an alkyl group that may be substituted.] A compound according to any one of claims 1 to 8, having the following characteristics.

10. R 1 , R 2 and R 4 The compound according to claim 8 or claim 9, wherein the compound is hydrogen.

11. R 3 However, selected from substituted or substituted aryls and substituted or substituted heteroaryls, optionally R 3 The compound according to any one of claims 8 to 10, selected from furan and optionally substituted phenyl.

12. R 7 but, 【Transformation 8】 [In the formula, (a)R 9 is selected from aryls which may be substituted with -alkylene- and heteroaryls which may be substituted with -alkylene-, and optionally R 9 is, -CH 2 Aryl and -CH may be substituted with -. 2 Selected from heteroaryls which may be substituted with -, (b)R 9 teeth, 【Chemistry 9】 Selected from, R 9 In the structure 【Chemistry 10】 R 7 [Indicates the site of covalent bonding to the remainder] The compound according to any one of claims 8, 10, and 11.

13. (a)R 9 However, selected from aryls which may be substituted with -alkylene- and heteroaryls which may be substituted with -alkylene-, and optionally R 9 However, -CH 2 Aryl and -CH may be substituted with -. 2 Selected from heteroaryls which may be substituted with -, (b)R 9 but, 【Chemistry 11】 A compound according to any one of claims 9 to 11, selected from the above.

14. R 5 and R 6 These are combined to form hydroxy, nitro, cyano, halo, and -NR. 10 R 11 , forming a 5-membered heterocyclic ring which may be substituted by an optionally substituted alkyl, optionally substituted alkoxy, optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted -O-cycloalkyl, optionally substituted -O-heterocyclyl, optionally substituted -O-aryl, or optionally substituted -O-heteroaryl, R 10 and R 11 The compound according to any one of claims 8 to 13, wherein each is independently H, an optionally substituted alkyl, or an optionally substituted cycloalkyl.

15. A is the structure of equation (III) 【Chemistry 12】 [In the formula, Each of rings E, F, C, and D is independently selected from an optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted aryl, and optionally substituted heteroaryl. In the structure of equation (III) 【Chemistry 13】 [This indicates the site of covalent bonding to L.] A compound according to any one of claims 1 to 14, having the following characteristics.

16. The compound according to claim 15, wherein ring C is selected from aryl and heteroaryl, and the aryl or heteroaryl is substituted with aryl or heteroaryl.

17. A 【Chemistry 14】 【change】 【change】 [In the formula, in the structure of A 【Chemistry 15】 [This indicates the site of the covalent bond to L.] A compound according to any one of claims 1 to 16, selected from the above.

18. A 【Chemistry 16】 [In the formula, in the structure of A 【Chemistry 17】 [This indicates the site of covalent bonding to L.] A compound according to claim 17, selected from the above.

19. The target protein is (a) A protein that forms aggregates involved in neurodegeneration, preferably selected from tau, synuclein, amyloid-beta, islet amyloid polypeptide (IAPP), serum amyloid-A (SAA), prion protein, misfolded transthyretin protein (TTR), TAR DNA-binding protein 43 (TDP-43), NOTCH3 receptor, mutant cystatin C, mezin, dipeptide repeat protein, and fused-in-sarcoma, for example, selected from tau, synuclein, amyloid-beta, prion protein, TAR DNA-binding protein 43 (TDP-43), dipeptide repeat protein, and fused-in-sarcoma, preferably tau, (b) A protein having an extended repeat element, preferably mutant huntingtin, (c) Proteins involved in signaling pathways in inflammation and / or cancer, preferably selected from Myd88, IRAK4, IRAK1, IRAK2, TRAF6, NLRP3, RIPK3 and ASC, (d) A viral protein that forms an oligomer, preferably selected from the nucleoproteins of CoV2 and influenza virus, or (e) A protein selected from bromodomain-containing protein 4 (BRD4), mutant gelsolin, misfolded rhodopsin, and atrial natriuretic peptide. A compound according to any one of claims 1 to 18, selected from the above.

20. The compound according to any one of claims 1 to 19, wherein B is selected from PI-2014, FDDNP, AV680(T808), GTP-1, THK523, THK5105, PBB3, AV1451(T807), THK5117, THK5351, N-methyllansoprazole, (E)-6-iodo-2-styryl-1H-benzo[d]imidazole, RO6958948, PI-2620, JNJ-067, APN-1607(PM-PBB3), MK-6240, and methylene blue.

21. B, [Chemistry 18] [In the formula, in the structure of B 【Chemistry 19】 [This indicates the site of covalent bonding to L.] A compound according to any one of claims 1 to 20, selected from the above.

22. Linker (L) (a) is a combination, or (b) A chain of 2 to 20 carbon atoms, wherein one or more carbon atoms are optionally replaced by 1 to 12 ethylene glycol units and / or heteroatoms selected from O, N, S, and P, and / or (c) comprising poly(ethylene glycol) (PEG), unsubstituted alkylene, substituted alkylene, or a combination thereof, and / or (d) 【Chemistry 20】 【change】 [In the formula, in the structure of the linker 【Chemistry 21】 Each of these cases indicates a covalent bond to A or B. A compound according to any one of claims 1 to 21, selected from the above.

23. The compound, 【Chemistry 22】 A compound according to any one of claims 1 to 22, selected from the above.

24. A pharmaceutical composition comprising a compound according to any one of claims 1 to 23 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

25. A compound according to any one of claims 1 to 23 or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to claim 24, for use in therapeutic purposes.

26. A compound according to any one of claims 1 to 23 or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to claim 24, for use in the treatment of neurodegenerative diseases.

27. An in vitro method for selectively degrading the oligomeric form of a target protein, comprising the step of contacting the target protein with TRIM21 and a compound according to any one of claims 1 to 23 or a pharmaceutically acceptable salt thereof.

28. Structure of equation (IV) 【Chemistry 23】 [In the formula, R 1 and R 2 These are, independently, hydrogen and C 1～2 Selected from alkyl groups, or R 1 and R 2 One is hydrogen, and the other is an amino acid residue. R 3 This is selected from hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted heteroalkyl, optionally substituted heteroalkenyl, optionally substituted heteroalkynyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted aryl, and optionally substituted heteroaryl. R 4 is hydrogen, halo, or C 1～2 It is alkyl, R 5 is hydrogen or C 1～2 It is alkyl, R 6 is an alkyl which may be substituted, or R 5 and R 6 These combine to form a complex ring, which may be substituted. R 9 is an alkyl group which may be substituted, R 12 and R 13 are each independently selected from hydrogen and C 1～2 alkyl, or one of R 12 and R 13 is hydrogen and the other is optionally substituted C 1～2 alkyl and -CHR 9 C(O)NR 14 R 15 selected from, R 14 and R 15 These are, independently, hydrogen and C 1～2 Selected from alkyl groups] A compound having the following properties.

29. Structure of formula (IVa), (IVb), or (IVc): 【Chemistry 24】 [In the formula, each 【Chemistry 25】 Independently, 【Chemistry 26】 Selected from, R 1 and R 2 each independently is selected from hydrogen and C 1～2 alkyl, or one of R 1 and R 2 is hydrogen and the other is an amino acid residue, R 3 R is selected from hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted heteroalkyl, optionally substituted heteroalkenyl, optionally substituted heteroalkynyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted aryl, and optionally substituted heteroaryl. 3 This optionally includes a covalent bond site to L. R 4 is hydrogen, halo, or C 1～2 It is alkyl, Each R 5 These are, independently, hydrogen or C 1～2 It is alkyl, R 6 is an alkyl that may be substituted, Or, R 5 and R 6 If R is bonded to an adjacent atom, 5 and R 6 These combine to form a complex ring, which may be substituted. R 8 is hydrogen or C 1～2 It is alkyl, R 9 is an alkyl group which may be substituted, R 12 and R 13 These are, independently, hydrogen and C 1～2 Selected from alkyl groups, R 14 and R 15 These are, independently, hydrogen and C 1～2 Selected from alkyl groups, R 18 and R 19 These are, independently, hydrogen and C 1～2 Selected from alkyl groups, R 20 [This is an alkyl group that may be substituted.] A compound having the following properties.

30. Structure of equation (V) 【Chemistry 27】 [In the formula, Each of rings E, F, C, and D is independently selected from an optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted aryl, or optionally substituted heteroaryl. R 16 and R 17 These are, independently, hydrogen and C 1～2 Selected from alkyl groups] A compound having the following properties.

31. The compound, 【Chemistry 28】 【change】 A compound according to any one of claims 28 to 30, selected from the above.

32. The compound, 【Chemistry 29】 【change】 A compound according to claim 31, selected from the above.

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