sumoylation targeting chimera (sutaC)

Abstract

The present disclosure relates to SUMOylated targeting chimera (SUTACs), and to their use for treating diseases, SUMOylated targeting chimera (SUTACs) comprising a SUMOylase binding moiety attached to a target protein binding moiety via a linker.

Description

[0001] sequence list This application includes a sequence list, which has been submitted electronically in XML file format and incorporated herein by reference in its entirety. The XML copy created on January 2, 2025, is named “P-629947-PC_SL.xml” and has a size of 8,263 bytes.

[0002] The scope of this disclosure This disclosure relates to SUMOylation-targeting chimeras (SUTAC) and their use in treating diseases, wherein the SUMOylation-targeting chimera includes a SUMOylase-binding group attached to a target protein-binding group via a linker.

[0003] background Post-translational modifications via small ubiquitin-like modifiers (SUMOs) constitute key molecular regulators of many proteins involved in countless cellular processes (Gareau, JR & Lima, CD The SUMO pathway: Emerging mechanisms that shape specificity, conjugation and recognition. NatRev Mol Cell Biol 11, 861-871, 2010).

[0004] SUMO reversibly conjugates to substrate proteins to regulate a variety of fundamental cellular processes, such as proteolysis, signal transduction, cell division, gene expression, and differentiation (Hay, RT SUMO: A history of modification. Molecular Cell vol. 18 1-12, 2005). SUMO conjugation to substrates is facilitated through a multi-step enzymatic cascade involving E1 activators, E2 conjugators, and E3 ligases. SUMOylation has been shown to be a robust protein modification capable of modulating protein stability, activity, solubility, localization, and interactions with other proteins in both environment- and substrate-specific ways. Previous studies have identified antagonistic interactions between ubiquitination and SUMOylation targeting specific proteins by competing for the same receptor lysine residues. In other cases, SUMOylation has been shown to have an agonistic interaction with ubiquitination, leading to proteasome degradation of the protein. Beyond protein stability, SUMOylation has been shown to be a key regulator of the activity and function of critical proteins by promoting or eliminating the binding of critical proteins to their protein chaperones.

[0005] With the advent of heterobifunctional chimeras, the concept of altering the modification state of proteins of interest (POIs) to regulate their appropriate levels or functions has been recognized as a promising therapeutic strategy (Modell, AE, Lai, S., Nguyen, TM & Choudhary, A. Bifunctional modalities for repurposing protein function. Cell Chemical Biology vol. 28, 2021), including proteolysis-targeted chimeras (PROTACs) (Békés, M., Langley, DR & Crews, CM PROTAC targeted protein degraders: the past is prologue. Nature Reviews Drug Discovery vol. 21, 2022). To date, no modalities have been established for selectively triggering protein SUMOylation. Unlike PROTACs, which are designed to cause protein degradation, SUTAC modalities can be used to alter protein function. Therefore, it is desirable to develop SUMOylation-targeted chimeras (SUTACs) to induce protein SUMOylation.

[0006] Overview of this disclosure In one aspect, this paper discloses a SUMOylation-targeting chimera (SUTAC) comprising a SUMOylation enzyme-binding group attached to a target protein-binding group via a linker.

[0007] In some embodiments, the SUMOylase-binding group is capable of binding SUMO E3 ligase. In some embodiments, the SUMO E3 ligase is selected from the group consisting of: PIAS1, PIAS2, PIAS3, PIAS4, NSMCE2, TOPORS, Trim19, Trim28, PML, ZNF451, and RANBP2.

[0008] In some implementations, SUTAC is represented by a structure of formula I or a salt thereof: (I) in, E is H, amide, acetylamide, or an electrophilic group; Z represents O, S, and NH; Each of R6 and R7 is independently selected from the group consisting of: H, halogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, haloaryl, heteroaryl, heterocyclic, hydroxyl, alkoxy, aryloxy, thioalkoxy, thioalkyl, thioalkenyl, thiohaloalkyl, cyano, nitro, azide, amino, -COOH, -C(O)NHR', -NHCOR''-; wherein R' is H, alkyl, alkenyl, aryl, heterocyclic alkyl, or heteroaryl; and R'' is hydroxyl, alkoxy, aryloxy, alkyl, alkenyl, aryl, heterocyclic alkyl, or heteroaryl; or R6 and R7 together form a 5-8 elemental ring, wherein the ring is either substituted or not substituted; R 11 It is H, substituted or unsubstituted alkyl, alkenyl, aryl, heterocyclic or heteroaryl; L1 is a bond, -(CH2CH2O) q -(CH2CH2O) q (CH2CH2)NH(CO)-, -(CH2CH2O) q (CH2CH2)-、-(CH2CH2O) q (CH2CH2)NH-, -NHCH2(CO)NHCH2Ph-, -N(alkyl)CH2(CO)NHCH2Ph-, substituted or unsubstituted straight-chain or branched alkylene group, substituted or unsubstituted straight-chain or branched alkenyl group, substituted or unsubstituted straight-chain or branched alkyne group, substituted or unsubstituted cycloalkyl group, substituted or unsubstituted heterocyclic group, substituted or unsubstituted aryl group, substituted or unsubstituted heteroaryl group, ether group, ester group, amine group, amide group or any combination thereof, wherein q is an integer from 2 to 10; L2 is a bond, -(CH2CH2O) q -(CH2CH2O) q (CH2CH2)NH(CO)-, -(CH2CH2O) q (CH2CH2)-、-(CH2CH2O) q (CH2CH2)NH-, -NHCH2(CO)NHCH2Ph-, -N(alkyl)CH2(CO)NHCH2Ph-, substituted or unsubstituted straight-chain or branched alkylene groups, substituted or unsubstituted straight-chain or branched alkenyl groups, substituted or unsubstituted straight-chain or branched alkyne groups, substituted or unsubstituted cycloalkyl groups, substituted or unsubstituted heterocyclic groups, substituted or unsubstituted aryl groups, substituted or unsubstituted heteroaryl groups, ether groups, ester groups, amine groups, amide groups, or any combination thereof, wherein q is an integer from 2 to 10; and W is the target protein binding group.

[0009] In some implementations, Z is S.

[0010] In some implementations, SUTAC is represented by a structure of formula I' or a salt thereof: Among them, R6, R7, R 11 L1, L2, Z and W are defined as for Equation I.

[0011] In some implementations, SUTAC is represented by the structure of formula IB or a salt thereof: (IB) in: Represents saturated or unsaturated bonds; If If the bond is saturated, then X is C, N, O, or S; where if If the bond is unsaturated, then X is C or N; R8, R9, R 10 Each of the following is independently selected from the group consisting of: H, halogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, haloaryl, heteroaryl, heterocyclic, hydroxyl, alkoxy, aryloxy, thioalkoxy, thioalkyl, thioalkenyl, thiohaloalkyl, cyano, nitro, azide, amino, -COOH, -C(O)NHR', -NHCOR''-; wherein R' is H, alkyl, alkenyl, aryl, heterocyclic alkyl, heteroaryl; and R'' is hydroxyl, alkoxy, aryloxy, alkyl, alkenyl, aryl, heterocyclic alkyl, or heteroaryl; R 11 It is H, substituted or unsubstituted alkyl, alkenyl, aryl, heterocyclic or heteroaryl; n is an integer, which is 1, 2, or 3; L1 is a bond, -(CH2CH2O) q -(CH2CH2O) q (CH2CH2)NH(CO)-, -(CH2CH2O) q (CH2CH2)-、-(CH2CH2O) q(CH2CH2)NH-, -NHCH2(CO)NHCH2Ph-, -N(alkyl)CH2(CO)NHCH2Ph-, substituted or unsubstituted straight-chain or branched alkylene group, substituted or unsubstituted straight-chain or branched alkenyl group, substituted or unsubstituted straight-chain or branched alkyne group, substituted or unsubstituted cycloalkyl group, substituted or unsubstituted heterocyclic group, substituted or unsubstituted aryl group, substituted or unsubstituted heteroaryl group, ether group, ester group, amine group, amide group or any combination thereof, wherein q is an integer from 2 to 10; L2 is a bond, -(CH2CH2O) q -(CH2CH2O) q (CH2CH2)NH(CO)-, -(CH2CH2O) q (CH2CH2)-、-(CH2CH2O) q (CH2CH2)NH-, -NHCH2(CO)NHCH2Ph-, -N(alkyl)CH2(CO)NHCH2Ph-, substituted or unsubstituted straight-chain or branched alkylene groups, substituted or unsubstituted straight-chain or branched alkenyl groups, substituted or unsubstituted straight-chain or branched alkyne groups, substituted or unsubstituted cycloalkyl groups, substituted or unsubstituted heterocyclic groups, substituted or unsubstituted aryl groups, substituted or unsubstituted heteroaryl groups, ether groups, ester groups, amine groups, amide groups, or any combination thereof, wherein q is an integer from 2 to 10; and W is the target protein binding group.

[0012] In some embodiments, the target protein binding group comprises a protein 4 (BRD4) targeting binder containing a bromo domain to form a BRD4-targeting SUTAC.

[0013] In some embodiments, the BRD4 targeting binder comprises JQ1 or its acid or ester.

[0014] In some implementations, BRD4 targeting SUTAC is represented by one of the following structures: In some implementations, the target protein binding group contains a P300 targeting binder to form a P300-targeting SUTAC.

[0015] In some embodiments, the P300 targeting binder comprises GNE-207 or a derivative thereof.

[0016] In some implementations, the P300-targeted SUTAC is represented by one of the following structures: In some implementations, the target protein binding group contains an androgen receptor (AR) targeting binder to form an AR-targeting SUTAC.

[0017] In some implementations, the AR-targeted SUTAC is represented by one of the following structures: In another aspect, this disclosure provides a pharmaceutical composition comprising SUTAC as described herein and a suitable, acceptable carrier.

[0018] In another aspect, the SUTAC described herein is used to treat subjects suffering from cancer, improve the condition of said subjects, and inhibit the decline of said subjects. In some embodiments, the cancer is selected from the group consisting of: kidney cancer, lung cancer, endometrial / uterine cancer, esophageal cancer, breast cancer, cervical cancer, liver cancer, gastric cancer, esophageal cancer, head and neck cancer, ovarian cancer, skin cancer, bile duct cancer, leukemia, lymphoma, rhabdoid tumor, brain cancer, colon / colorectal cancer, pancreatic cancer, myeloma, prostate cancer, neuroblastoma, gastric cancer, sarcoma, thyroid cancer, bladder cancer, bone cancer, or eye cancer.

[0019] In another aspect, the SUTAC described herein is used to treat subjects suffering from inflammation, neuroinflammation, allergies, aging, autoimmune diseases, viral infections, bacterial infections, obesity, neurodegenerative diseases, fibrosis, cardiovascular diseases, diabetes, Crohn's disease, colitis, osteoporosis, multiple sclerosis (MS), SLE, or non-alcoholic fatty liver disease, to improve the condition of said subjects, and to inhibit the decline of said subjects.

[0020] In some implementations, diabetes is type 2 diabetes.

[0021] In some embodiments, administration of the SUTAC to a subject reduces the degradation of the target protein compared to an untreated subject. In some embodiments, administration of the SUTAC to a subject increases the degradation of the target protein compared to an untreated subject.

[0022] In some embodiments, administration of the SUTAC to a subject reduces the stabilization of the target protein compared to an untreated subject. In some embodiments, administration of the SUTAC to a subject increases the stabilization of the target protein compared to an untreated subject.

[0023] In some embodiments, administration of the SUTAC to a subject reduces the activation of the target protein compared to an untreated subject. In some embodiments, administration of the SUTAC to a subject increases the activation of the target protein compared to an untreated subject.

[0024] In some embodiments, administration of the SUTAC to a subject reduces repression of the target protein compared to an untreated subject. In some embodiments, administration of the SUTAC to a subject increases repression of the target protein compared to an untreated subject.

[0025] In some embodiments, administration of the SUTAC to a subject increases the binding of the target protein to other proteins or DNA compared to an untreated subject. In some embodiments, administration of the SUTAC to a subject decreases the binding of the target protein to other proteins or DNA compared to an untreated subject.

[0026] In some embodiments, administration of the SUTAC to a subject reduces the solubility of the target protein compared to an untreated subject. In some embodiments, administration of the SUTAC to a subject increases the solubility of the target protein compared to an untreated subject. Brief description of the attached diagram Figures 1A-1C Figure 1A A schematic overview highlighting the differences between PROTAC and SUTAC modes is shown; Figure 1B Electrophile screening for recombinant domains derived from SUMO E3 ligase is illustrated schematically. Figure 1C The initial hits from the screening of PIAS1 binders are shown.

[0028] Figure 2 The binding efficiency (PIAS1 binding IC50 value) and thiol reactivity characterization of the derivatives of molecules 2 and 3 are shown.

[0029] Figures 3A-3C The binding efficiency (PIAS1 binding IC50 value) and thiol reactivity characterization of the derivatives of molecule 1 are shown. Figure 3A Derivatives at the amide position; Figure 3B Thiophene ring substituents; Figure 3C Acetamide derivatives of chloroacetamide aminosulfonate.

[0030] Figures 4A-4C The cells of 1-12 with PIAS protein are shown. Figure 4A Structures 1-34 for proteomics characterization of cellular targets; Figure 4BGel-based ABPP experiments were performed in which Daudi cells were incubated with 1-34 for 6 hours, with 1-12 for 6 hours, or pre-incubated with 1-12 for 2 hours followed by incubation with 1-34 for 6 hours. Cells were lysed, and the lysates were clicked onto TAMRA azide and imaged on the gel. Figure 4C Target proteomics characterization involved treating Daudi cells with 1 µM 1-34 for 6 hours, with or without pre-incubation with 10 µM 1-12 for 2 hours. Cells were lysed, clicked onto biotin-functionalized, trypsin-cleavable azide-containing peptides, and the labeled proteins were enriched on streptavidin beads, trypsinized, and analyzed using LC-MS / MS.

[0031] Figures 5A-5D The study showed that BRD4 targeting SUTAC reduces the protein levels of BRD4 and c-MYC. Figure 5A The structure of BRD4 targeting SUTAC (left) and its IC50 binding with PIAS1, as well as the structure JQ1 (right), are shown. Figure 5B Western blotting of Daudi cells with the indicated antibody, the Daudi cells being treated with an increased concentration of the indicated SUTAC molecule for 20 hours; Figure 5C The structures of 1-5, 1 PIAS1 binders and PEG linkers are shown (left), as well as the structure of JQ1-ester (right), which is cell-permeable; Figure 5D Western blotting of Daudi cells with the indicated antibody, the Daudi cells being treated with an increased concentration of the indicated molecule for 20 hours.

[0032] Figures 6A-6B It was shown that BRD4 targeting SUTAC reduces BRD4 levels in a SUMOylation and PIAS-dependent manner. Figure 6A Western blotting of Daudi cells with the indicated antibody, the Daudi cells having been treated with an increased concentration of the indicated SUTAC molecule for 20 hours. Cells were pre-incubated with 50 µM 2D08 (Sigma, SML1052) or DMSO as a control for 1 hour; Figure 6B Western blotting of TK6 cells with the indicated genetic background using the indicated antibody, the TK6 cells being treated with the indicated SUTAC for 20 hours.

[0033] Figure 7This demonstrates the modification of recombinant BRD4 with SUTAC under SUMOylation-promoting conditions. Recombinant (Glu49-Glu460)-FLAG-BRD4 was incubated with increased concentrations of SUTAC or DMSO controls in an active protein extract derived from A549 cells for 1 hour at 30°C under either SUMOylation-promoting retention (SUMO1-aldehyde and SUMO2-aldehyde) or control conditions. This was followed by flag-tag pull-down and Western blotting of the eluent using the indicated antibody.

[0034] Figures 8A-8B The optimized binder is shown to enhance the effect of SUTAC. Figure 8A The structure of BRD4 targeting SUTAC and its IC50 binding to PIAS1; Figure 8B Western blotting of Daudi cells with the indicated antibody, the Daudi cells being treated with an increased concentration of the indicated SUTAC molecule for 20 hours.

[0035] Figures 9A-9C This study demonstrates that BRD4 targeting SUTAC is effective in reducing the proliferation of blood cancer cells. Figure 9A Daudi cells were treated with increased doses of BRD4-targeted SUTAC or JQ1, and relative proliferation was determined by Cell TiterGlow assay after 72 hours of treatment. Figure 9B OCI-AML2 cells were treated with increased doses of 1-26 BRD4-targeted SUTAC or JQ1 or 1-12 PIAS binding agent, and relative proliferation was determined by Cell Titer Glow assay after 72 hours of treatment. Figure 9C Daudi cells were treated with increased doses of 1-26 BRD4-targeted SUTAC or JQ1 or 1-12 PIAS binding agent, and relative proliferation was determined by Cell Titer Glow assay after 72 hours of treatment.

[0036] Figures 10A-10C It was shown that targeting SUTAC with AR can reduce AR levels. Figure 10A The structure of AR-targeted SUTAC is shown. Figure 10B LNCaP cells were treated with an increased concentration of the indicated SUTAC molecule for 20 hours and then subjected to Western blotting using the indicated antibody. Figure 10C After 48 hours, the 293T cells transiently transfected with the indicated plasmid were treated with an increased concentration of the indicated SUTAC molecule for an additional 20 hours and then subjected to Western blotting using the indicated antibody.

[0037] Figures 11A-11CEnhanced SUMOylation of BRD4 via ID-1 (=1-26) SUTAC and chromatin expulsion of BRD4. Figure 11A Pre-incubate PIAS4 or the medium with 1-26 SUTAC or DMSO controls at 25°C for 4 hours. Then, incubate them together with SUMO1,2 and SUMO-E1 (SAE1 / 2) with or without SUMO-E2 enzyme (UBC 9) and with or without recombinant BRD4 at 30°C for 1 hour. The samples are then subjected to Western blotting using the indicated antibody. Figure 11B OCI-AML2 was treated with an increased concentration of ID-1 for approximately 20 hours and then harvested and analyzed using Western blotting with the indicated antibody. Figure 11C OCI-AML2 cells were treated with 1 μM ID-1SUTAC, JQ1-ester, and MZ1 (PROTAC) for approximately 20 hours. Cells were then fractionated and analyzed using Western blotting with antibodies targeting His-tag, BRD4-tag, UBC9-tag, and MYC-tag.

[0038] Figure 12 Library screening for chloroacetamide fragments of PIAS1 and PIAS4. Thiophene-based hits containing amide (left), ester (middle), and nitrile (right) are shown.

[0039] Figures 13A-13B The medicinal chemical activities used to optimize the PIAS4 binder are shown. Figure 13A Compounds 1-10, 1-11, 1-12, 1-16, 1-17, 1-18, 1-19, and 1-40, which are extended around thiophene; compounds 1-36, 1-37, 1-38, and 1-39, which are optimized with amide substituents; and the combination of compounds 1-40 and 1-39 (compound 1-41). Figure 13B Add adapters and recruiters for the target protein.

[0040] Figures 14A-14B Identification of PIAS4 binding sites. Figure 14A MS / MS spectra of modified peptides from PIAS4 (322-330). Y ions are indicated in blue and B ions in magenta: modified by urea methylation (top) and modified by compounds 1-41 (bottom); Figure 14BLeft: Superposition of the α-sheet structures of unbound PIAS4 (gray) and bound PIAS (protein: salmon, compounds 1-41: blue), and right: Model of compounds 1-41 bound to PIAS4.

[0041] Figure 15 Compound 1-43 binds to the cellular binding of the PIAS protein. For proteomic characterization of the target, Daudi cells were pre-incubated with 1 μM of compound 1-43 (or DMSO) for 2 hours, followed by incubation with 0.1 μM of compound 1-42 (or DMSO) for 1 hour. Cells were lysed, clicked onto biotin-functionalized, trypsin-cleavable azide-containing peptides, and the labeled proteins were enriched on streptavidin beads, trypsinized, and analyzed using LC-MS / MS.

[0042] Figures 16A-16E BRD4 targets SUTAC (ID-2) to enhance the SUMOylation of recombinant BRD4 and its downstream target c-MYC. Figure 16A Pre-incubate PIAS4 or the medium with ID-2 (SUTAC) or DMSO control at 25°C for 1.5 hours. Then, incubate them together with SUMO1,2 and SUMO-E1 (SAE1 / 2) with or without SUMO-E2 enzyme (UBC 9), with or without recombinant BRD4, at 30°C for 1 hour. The samples are then subjected to Western blotting using an anti-BRD4 antibody. Figure 16B Cancer cell lines Daudi, OCI-AML2, RAMOS, and RPMI-8226 were treated with increased concentrations of ID-2 for 20 hours. Cells were lysed and subjected to Western blotting using antibodies against BRD4, cMYC, and actin. Figure 16C Daudi cells were treated with increased concentrations of ID-2 or JQ1-ester for 24 hours. Cells were then lysed and subjected to Western blot analysis using antibodies against BRD4, cMYC, and actin. Figure 16D Daudi cells were pretreated with 10 µM MG132 or DMSO and then treated with ID-2 for 4 hours. Cells were then lysed and subjected to Western blot analysis using antibodies against BRD4, ubiquitin, and actin. Figure 16E Double knockout (DKO) TK6 cells of PIAS1 and PIAS4 were treated with either (left) 1 µM ID-2 for 4 hours or (right) 0.1 µM ID-2 overnight. Cells were then lysed and subjected to Western blot analysis using antibodies against BRD4, PIAS1, PIAS2, PIAS3, PIAS4, and actin.

[0043] Figures 17A-17B The SUMOylation of recombinant BRD4 was enhanced by compound 7 (SUTAC) in the in vitro SUMOylation reaction. Figure 17A The structures of ID-2 and ID-3; Figure 17B Pre-incubate PIAS4 or the mediator with ID-2 (SUTAC) or ID-3 (SUTAC) or DMSO controls at 25°C for 1.5 hours. Then, incubate them together with SUMO1,2 and SUMO-E1 (SAE1 / 2) with or without SUMO-E2 enzyme (UBC 9) and with or without recombinant BRD4 at 30°C for 1 hour. The samples are then subjected to Western blotting using an anti-BRD4 antibody (abcam).

[0044] Figures 18A-18E P300 targets SUTAC to enhance the SUMOylation of P-300 and its downstream target c-MYC. Figure 18A P300 targets the structure of SUTAC (ID-4); Figure 18B Daudi (right) or OCI-AML2 (left) cancer cell lines were treated with increased concentrations of ID-4 for 20 hours. Cells were lysed and subjected to Western blotting using antibodies against P300, H2B, cMYC, and actin. Figure 18C OCI-AML2 cells were treated with increased doses of ID-4 or 1-41, and relative proliferation was determined by Cell Titer Glow assay after 72 hours of treatment. Figure 18D The structures of ID-4, ID-5 and the P300 inhibitor (compound 10); Figure 18E The OCI-AML2 cell line was treated for 20 hours with an increased dose of ID-4, ID-5, P300 inhibitor (compound 10), or a DMSO control. Cells were lysed and subjected to Western blot analysis using an antibody against cMYC.

[0045] Detailed Explanation The subject matter of the invention can be more readily understood by referring to the following detailed description, which forms part of this disclosure. It should be understood that this disclosure is not limited to the specific products, methods, conditions, or parameters described and / or illustrated herein, and that the terminology used herein is for the purpose of describing particular embodiments by way of example only and is not intended to limit the claimed disclosure.

[0046] This disclosure illustrates the development of SUTAC as a novel drug model in the form of a modular heterobifunctional molecule containing two functional groups: a first part (part 1) is a novel chemical moiety that selectively binds to a specific SUMO E3 ligase (a SUMOylating enzyme binding group). A second part (part 2) is a chemical moiety (a target protein binding group) that specifically binds to a protein of interest (POI) (target protein). These two parts are conjugated by a short chemical linker (part 3) to produce a chimeric molecule. The SUTAC molecule induces selective SUMOylation of any target POI as needed. The SUTAC approach can utilize other SUMO E3 ligases or SUMO E2 conjugates to induce SUMOylation of any POI.

[0047] To establish the SUTAC model, a dedicated electrophile screening was performed targeting the recombinant domain derived from the well-defined SUMO E3 ligase (PIAS1). Three binding agents capable of labeling PIAS1 at single-digit μM concentrations were identified. The binding efficiency of these molecules was optimized, and binding to PIAS1 and two other PIAS family members (PIAS4 and PIAS2) in cells was verified. Three of the identified binding agents were used to generate first-generation SUTACs.

[0048] Using a short PEG linker, the binder was conjugated to JQ1, a potent binder targeting the transcriptional activator BRD4. BRD4 is a transcriptional activator known to promote the expression of key oncogenes in several cancers and other pathologies. BRD4-targeting SUTACs are cell-permeable and induce SUMOylation of recombinant BRD4 in cell extracts at ~200 nM, and SUMOylation of recombinant BRD4 is induced in an in vitro SUMOylation assay using purified SUMOylase. Furthermore, these SUTAC molecules reduce BRD4 activity and / or levels in a SUMOylation and PIAS-dependent manner. These SUTAC molecules can lead to the evection of BRD4 from chromatin fractions. In addition, SUTAC treatment significantly reduces the level of the oncoprotein cMYC, which is characteristically transactivated by BRD4. Notably, these SUTACs are effective in attenuating the proliferation of hematologic cancer cell lines. Furthermore, the androgen receptor (AR) was generated to target SUTAC, and its efficacy in reducing AR protein levels in prostate cancer cell lines and in an AR overexpression environment was demonstrated.

[0049] In addition, a more extensive electrophile screening was conducted on recombinant PIAS1 and PIAS4, identifying an amide-containing thiophene scaffold as a dual binder. Through optimization, the binding of this amide-containing thiophene scaffold to PIAS4 was enhanced by more than 100-fold, and its co-structure with PIAS4 was resolved. The binding of these compounds to PIAS was validated in cells, and it was demonstrated that BRD4-targeting SUTACs utilizing these binders could induce BRD4 SUMOylation. Furthermore, these BRD4-targeting SUTACs were shown to affect BRD4 activity and / or levels and significantly attenuate cancer cell growth. Notably, when these binders were conjugated with a P300 / CBP binder, the resulting SUTACs effectively downregulated P300 activity and / or levels and significantly attenuated cancer cell growth.

[0050] In some embodiments, this document provides a SUMOylation-targeting chimera (SUTAC) comprising a SUMOylating enzyme-binding group attached to a target protein-binding group via a linker (e.g., L1, L2). In some embodiments, the linker comprises one or more polyethylene glycol (PEG) groups. In some embodiments, the linker comprises L1 as described in detail herein. In some embodiments, the linker comprises L2 as described in detail herein.

[0051] In some embodiments, this document provides a SUMOylation-targeting chimera (SUTAC) comprising a SUMOylation enzyme-binding group attached to a target protein-binding group via a linker. In some embodiments of the SUTAC described herein, the SUMOylation enzyme-binding group is capable of binding SUMO E3 ligase. In some embodiments of the SUTAC described herein, the SUMOylation enzyme-binding group is capable of binding SUMO E2 conjugate enzyme.

[0052] In some embodiments, the term "SUMOylation" may encompass the binding of a SUMO protein to a target protein, which may affect the activity of the target protein. In some embodiments, "SUMOylation enzyme binding group" may encompass a compound or molecule that binds to or at least interacts with a SUMOylation enzyme. In some embodiments, the SUMOylation enzyme comprises SUMOase E1. In some embodiments, the SUMOylation enzyme comprises SUMO conjugate E2. In some embodiments, the SUMOylation enzyme comprises a SUMO E3 ligase. In some embodiments, the SUMO E3 ligase is selected from the group consisting of protein inhibitors of activated STAT1 (PIAS1), PIAS2, PIAS3, PIAS4, NSMCE2, TOPORS, Trim19, Trim28, PML, ZNF451, and Ran-binding protein 2 (RanBP2).

[0053] In some embodiments, the SUMO E3 ligase is selected from the group consisting of: PIAS1, PIAS2, PIAS3, PIAS4, NSMCE2, TOPORS, Trim19, Trim28, PML, ZNF451, and RANBP2. In some embodiments, the SUMO E3 ligase comprises PIAS1. In some embodiments, the SUMO E3 ligase comprises PIAS2. In some embodiments, the SUMO E3 ligase comprises PIAS3. In some embodiments, the SUMO E3 ligase comprises PIAS4. In some embodiments, the SUMO E3 ligase comprises NSMCE2. In some embodiments, the SUMO E3 ligase comprises TOPORS. In some embodiments, the SUMO E3 ligase comprises Trim19. In some embodiments, the SUMO E3 ligase comprises Trim28. In some embodiments, the SUMO E3 ligase comprises PML. In some embodiments, the SUMO E3 ligase comprises ZNF451. In some embodiments, the SUMO E3 ligase comprises RANBP2.

[0054] In some embodiments, the "target protein binding group" may encompass a polypeptide or protein that binds to or at least interacts with the target protein. In some embodiments, the target protein binding group comprises an inhibitor of the target protein. In some embodiments, the target protein binding group comprises JQ1 or an acid or ester thereof. In some embodiments, the target protein binding group comprises GNE-207 or a derivative thereof. In some embodiments, the target protein binding group comprises A-485 or a derivative thereof. In some embodiments, the target protein binding group comprises a kinase inhibitor. In some embodiments, the target protein binding group comprises gefitinib. In some embodiments, the target protein binding group comprises W as described in detail herein.

[0055] In some embodiments, the "target protein" may encompass a polypeptide or a protein of interest (POI). In some embodiments, the protein of interest includes any protein that can undergo SUMOylation, i.e., covalently modified by a SUMO protein. In some embodiments, the protein of interest includes any protein requiring increased degradation. In some embodiments, the protein of interest includes any protein requiring reduced degradation. In some embodiments, the protein of interest includes any protein requiring increased stabilization. In some embodiments, the protein of interest includes any protein requiring reduced stabilization. In some embodiments, the protein of interest includes any protein requiring increased activation. In some embodiments, the protein of interest includes any protein requiring reduced activation. In some embodiments, the protein of interest includes any protein requiring increased repression. In some embodiments, the protein of interest includes any protein requiring reduced repression. In some embodiments, the protein of interest includes any protein requiring increased solubility. In some embodiments, the protein of interest includes any protein requiring reduced solubility.

[0056] In some embodiments, the protein of interest is selected from proteases, cellulases, amylases, glycoases, lipases, isomerases, transferases, kinases, and phosphatases. In some embodiments, the protein of interest is selected from the group consisting of antibodies, hormones, and growth factors.

[0057] In some implementations, the proteins of interest are selected from the group consisting of: DNA-binding proteins, transcription factors, RNA-binding proteins, scaffold proteins, GTPases, solute carriers, kinases, phosphatases, chromatin remodellers, transcription activators, transcription repressors, proteins containing bromo domains and cromo domains, and G protein-coupled receptors.

[0058] In some embodiments, the protein of interest is selected from the group consisting of integrases, recombinases, transcription factors, proteases, kinases, and growth factors. In some embodiments, the protein of interest includes integrases. In some embodiments, the protein of interest includes recombinases. In some embodiments, the protein of interest includes proteases. In some embodiments, the protein of interest includes kinases. In some embodiments, the protein of interest includes growth factors.

[0059] In some embodiments, the target protein includes protein 4 (BRD4), which contains a bromo domain. In some embodiments, the target protein includes the androgen receptor (AR). In some embodiments, the target protein includes the estrogen receptor (ER). In some embodiments, the target protein includes the P300 / CBP protein.

[0060] In some implementations, this document provides SUMO-targeted chimeras (SUTACs) represented by the structure of Formula I or a salt thereof: (I) in, E is H, amide, acetylamide, or an electrophilic group; Z represents O, S, and NH; Each of R6 and R7 is independently selected from the group consisting of: H, halogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, haloaryl, heteroaryl, heterocyclic, hydroxyl, alkoxy, aryloxy, thioalkoxy, thioalkyl, thioalkenyl, thiohaloalkyl, cyano, nitro, azide, amino, -COOH, -C(O)NHR', -NHCOR''-; wherein R' is H, alkyl, alkenyl, aryl, heterocyclic alkyl, or heteroaryl; and R'' is hydroxyl, alkoxy, aryloxy, alkyl, alkenyl, aryl, heterocyclic alkyl, or heteroaryl; or R6 and R7 together form a 5-8 elemental ring, wherein the ring is either substituted or not substituted; R 11 It is H, substituted or unsubstituted alkyl, alkenyl, aryl, heterocyclic or heteroaryl; L1 is a bond, -(CH2CH2O) q -(CH2CH2O) q (CH2CH2)NH(CO)-, -(CH2CH2O) q (CH2CH2)-、-(CH2CH2O) q (CH2CH2)NH-, -NHCH2(CO)NHCH2Ph-, -N(alkyl)CH2(CO)NHCH2Ph-, substituted or unsubstituted straight-chain or branched alkylene group, substituted or unsubstituted straight-chain or branched alkenyl group, substituted or unsubstituted straight-chain or branched alkyne group, substituted or unsubstituted cycloalkyl group, substituted or unsubstituted heterocyclic group, substituted or unsubstituted aryl group, substituted or unsubstituted heteroaryl group, ether group, ester group, amine group, amide group or any combination thereof, wherein q is an integer from 2 to 10; L2 is a bond, -(CH2CH2O) q -(CH2CH2O) q(CH2CH2)NH(CO)-, -(CH2CH2O) q (CH2CH2)-、-(CH2CH2O) q (CH2CH2)NH-, -NHCH2(CO)NHCH2Ph-, -N(alkyl)CH2(CO)NHCH2Ph-, substituted or unsubstituted straight-chain or branched alkylene groups, substituted or unsubstituted straight-chain or branched alkenyl groups, substituted or unsubstituted straight-chain or branched alkyne groups, substituted or unsubstituted cycloalkyl groups, substituted or unsubstituted heterocyclic groups, substituted or unsubstituted aryl groups, substituted or unsubstituted heteroaryl groups, ether groups, ester groups, amine groups, amide groups, or any combination thereof, wherein q is an integer from 2 to 10; and W is the target protein binding group.

[0061] In some embodiments, the electrophilic group E of Formula I includes any known electrophilic group. In some embodiments, the electrophilic group includes chloroacetamide, acrylamide, methacrylamide, aminosulfonate, propynamide, butyramide, or vinyl sulfone. In some embodiments, the electrophilic group includes chloroacetamide. In some embodiments, the electrophilic group includes acrylamide. In some embodiments, the electrophilic group includes methacrylamide. In some embodiments, the electrophilic group includes aminosulfonate. In some embodiments, the electrophilic group includes propynamide. In some embodiments, the electrophilic group includes butyramide. In some embodiments, the electrophilic group includes vinyl sulfone.

[0062] In some implementations, the electrophilic group is represented by the following structure: .

[0063] In some implementations, this document provides SUMO-targeted chimeras (SUTACs) represented by structures of formula I' or salts thereof: (I') in, Z represents O, S, and NH; Each of R6 and R7 is independently selected from the group consisting of: H, halogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, haloaryl, heteroaryl, heterocyclic, hydroxyl, alkoxy, aryloxy, thioalkoxy, thioalkyl, thioalkenyl, thiohaloalkyl, cyano, nitro, azide, amino, -COOH, -C(O)NHR', -NHCOR''-; wherein R' is H, alkyl, alkenyl, aryl, heterocyclic alkyl, or heteroaryl; and R'' is hydroxyl, alkoxy, aryloxy, alkyl, alkenyl, aryl, heterocyclic alkyl, or heteroaryl; or R6 and R7 together form a 5-8 elemental ring, wherein the ring is either substituted or not substituted; R 11 It is H, substituted or unsubstituted alkyl, alkenyl, aryl, heterocyclic or heteroaryl; L1 is a bond, -(CH2CH2O) q -(CH2CH2O) q (CH2CH2)NH(CO)-, -(CH2CH2O) q (CH2CH2)-、-(CH2CH2O) q (CH2CH2)NH-, -NHCH2(CO)NHCH2Ph-, -N(alkyl)CH2(CO)NHCH2Ph-, substituted or unsubstituted straight-chain or branched alkylene group, substituted or unsubstituted straight-chain or branched alkenyl group, substituted or unsubstituted straight-chain or branched alkyne group, substituted or unsubstituted cycloalkyl group, substituted or unsubstituted heterocyclic group, substituted or unsubstituted aryl group, substituted or unsubstituted heteroaryl group, ether group, ester group, amine group, amide group or any combination thereof, wherein q is an integer from 2 to 10; L2 is a bond, -(CH2CH2O) q -(CH2CH2O) q (CH2CH2)NH(CO)-, -(CH2CH2O) q (CH2CH2)-、-(CH2CH2O) q (CH2CH2)NH-, -NHCH2(CO)NHCH2Ph-, -N(alkyl)CH2(CO)NHCH2Ph-, substituted or unsubstituted straight-chain or branched alkylene groups, substituted or unsubstituted straight-chain or branched alkenyl groups, substituted or unsubstituted straight-chain or branched alkyne groups, substituted or unsubstituted cycloalkyl groups, substituted or unsubstituted heterocyclic groups, substituted or unsubstituted aryl groups, substituted or unsubstituted heteroaryl groups, ether groups, ester groups, amine groups, amide groups, or any combination thereof, wherein q is an integer from 2 to 10; and W is the target protein binding group.

[0064] In some implementations, this document provides SUMO-targeted chimeras (SUTACs) represented by structures of formula IA or salts thereof: (IA) in, Each of R6 and R7 is independently selected from the group consisting of: H, halogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, haloaryl, heteroaryl, heterocyclic, hydroxyl, alkoxy, aryloxy, thioalkoxy, thioalkyl, thioalkenyl, thiohaloalkyl, cyano, nitro, azide, amino, -COOH, -C(O)NHR', -NHCOR''-; wherein R' is H, alkyl, alkenyl, aryl, heterocyclic alkyl, or heteroaryl; and R'' is hydroxyl, alkoxy, aryloxy, alkyl, alkenyl, aryl, heterocyclic alkyl, or heteroaryl; or R6 and R7 together form a 5-8 elemental ring, wherein the ring is either substituted or not substituted; R 11 It is H, substituted or unsubstituted alkyl, alkenyl, aryl, heterocyclic or heteroaryl; L1 is a bond, -(CH2CH2O) q -(CH2CH2O) q (CH2CH2)NH(CO)-, -(CH2CH2O) q (CH2CH2)-、-(CH2CH2O) q (CH2CH2)NH-, -NHCH2(CO)NHCH2Ph-, -N(alkyl)CH2(CO)NHCH2Ph-, substituted or unsubstituted straight-chain or branched alkylene group, substituted or unsubstituted straight-chain or branched alkenyl group, substituted or unsubstituted straight-chain or branched alkyne group, substituted or unsubstituted cycloalkyl group, substituted or unsubstituted heterocyclic group, substituted or unsubstituted aryl group, substituted or unsubstituted heteroaryl group, ether group, ester group, amine group, amide group or any combination thereof, wherein q is an integer from 2 to 10; L2 is a bond, -(CH2CH2O) q -(CH2CH2O) q (CH2CH2)NH(CO)-, -(CH2CH2O) q (CH2CH2)-、-(CH2CH2O) q (CH2CH2)NH-, -NHCH2(CO)NHCH2Ph-, -N(alkyl)CH2(CO)NHCH2Ph-, substituted or unsubstituted straight-chain or branched alkylene groups, substituted or unsubstituted straight-chain or branched alkenyl groups, substituted or unsubstituted straight-chain or branched alkyne groups, substituted or unsubstituted cycloalkyl groups, substituted or unsubstituted heterocyclic groups, substituted or unsubstituted aryl groups, substituted or unsubstituted heteroaryl groups, ether groups, ester groups, amine groups, amide groups, or any combination thereof, wherein q is an integer from 2 to 10; and W is the target protein binding group.

[0065] In some implementations, this document provides SUMO-targeted chimeras (SUTACs) represented by the structure of formula IB or a salt thereof: (IB) in: Represents saturated or unsaturated bonds; If If the bond is saturated, then X is C, N, O, or S; where if If the bond is unsaturated, then X is C or N; R8, R9, R 10 Each of the following is independently selected from the group consisting of: H, halogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, haloaryl, heteroaryl, heterocyclic, hydroxyl, alkoxy, aryloxy, thioalkoxy, thioalkyl, thioalkenyl, thiohaloalkyl, cyano, nitro, azide, amino, -COOH, -C(O)NHR', -NHCOR''-; wherein R' is H, alkyl, alkenyl, aryl, heterocyclic alkyl, heteroaryl; and R'' is hydroxyl, alkoxy, aryloxy, alkyl, alkenyl, aryl, heterocyclic alkyl, or heteroaryl; R 11 It is H, substituted or unsubstituted alkyl, alkenyl, aryl, heterocyclic or heteroaryl; n is an integer, which is 1, 2, or 3; L1 is a bond, -(CH2CH2O) q -(CH2CH2O) q (CH2CH2)NH(CO)-, -(CH2CH2O) q (CH2CH2)-、-(CH2CH2O) q (CH2CH2)NH-, -NHCH2(CO)NHCH2Ph-, -N(alkyl)CH2(CO)NHCH2Ph-, substituted or unsubstituted straight-chain or branched alkylene group, substituted or unsubstituted straight-chain or branched alkenyl group, substituted or unsubstituted straight-chain or branched alkyne group, substituted or unsubstituted cycloalkyl group, substituted or unsubstituted heterocyclic group, substituted or unsubstituted aryl group, substituted or unsubstituted heteroaryl group, ether group, ester group, amine group, amide group or any combination thereof, wherein q is an integer from 2 to 10; L2 is a bond, -(CH2CH2O) q -(CH2CH2O) q (CH2CH2)NH(CO)-, -(CH2CH2O) q (CH2CH2)-、-(CH2CH2O)q (CH2CH2)NH-, -NHCH2(CO)NHCH2Ph-, -N(alkyl)CH2(CO)NHCH2Ph-, substituted or unsubstituted straight-chain or branched alkylene groups, substituted or unsubstituted straight-chain or branched alkenyl groups, substituted or unsubstituted straight-chain or branched alkyne groups, substituted or unsubstituted cycloalkyl groups, substituted or unsubstituted heterocyclic groups, substituted or unsubstituted aryl groups, substituted or unsubstituted heteroaryl groups, ether groups, ester groups, amine groups, amide groups, or any combination thereof, wherein q is an integer from 2 to 10; and W is the target protein binding group.

[0066] In some implementations, this document provides SUMO-targeted chimeras (SUTACs) represented by the structure of formula IC or a salt thereof: (IC) in, R8, R9, R 10 Each of the following is independently selected from the group consisting of: H, halogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, haloaryl, heteroaryl, heterocyclic, hydroxyl, alkoxy, aryloxy, thioalkoxy, thioalkyl, thioalkenyl, thiohaloalkyl, cyano, nitro, azide, amino, -COOH, -C(O)NHR', -NHCOR''-; wherein R' is H, alkyl, alkenyl, aryl, heterocyclic alkyl, heteroaryl; and R'' is hydroxyl, alkoxy, aryloxy, alkyl, alkenyl, aryl, heterocyclic alkyl, or heteroaryl; R 11 It is H, substituted or unsubstituted alkyl, alkenyl, aryl, heterocyclic or heteroaryl; n is an integer, which is 1, 2, or 3; L1 is a bond, -(CH2CH2O) q -(CH2CH2O) q (CH2CH2)NH(CO)-, -(CH2CH2O) q (CH2CH2)-、-(CH2CH2O) q (CH2CH2)NH-, -NHCH2(CO)NHCH2Ph-, -N(alkyl)CH2(CO)NHCH2Ph-, substituted or unsubstituted straight-chain or branched alkylene group, substituted or unsubstituted straight-chain or branched alkenyl group, substituted or unsubstituted straight-chain or branched alkyne group, substituted or unsubstituted cycloalkyl group, substituted or unsubstituted heterocyclic group, substituted or unsubstituted aryl group, substituted or unsubstituted heteroaryl group, ether group, ester group, amine group, amide group or any combination thereof, wherein q is an integer from 2 to 10; L2 is a bond, -(CH2CH2O) q -(CH2CH2O) q (CH2CH2)NH(CO)-, -(CH2CH2O) q (CH2CH2)-、-(CH2CH2O) q (CH2CH2)NH-, -NHCH2(CO)NHCH2Ph-, -N(alkyl)CH2(CO)NHCH2Ph-, substituted or unsubstituted straight-chain or branched alkylene groups, substituted or unsubstituted straight-chain or branched alkenyl groups, substituted or unsubstituted straight-chain or branched alkyne groups, substituted or unsubstituted cycloalkyl groups, substituted or unsubstituted heterocyclic groups, substituted or unsubstituted aryl groups, substituted or unsubstituted heteroaryl groups, ether groups, ester groups, amine groups, amide groups, or any combination thereof, wherein q is an integer from 2 to 10; and W is the target protein binding group.

[0067] In some implementations, this paper provides SUMO-targeted chimeras (SUTACs) represented by a structure of formula ID or a salt thereof: (ID) in, R8, R9, R 10 Each of the following is independently selected from the group consisting of: H, halogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, haloaryl, heteroaryl, heterocyclic, hydroxyl, alkoxy, aryloxy, thioalkoxy, thioalkyl, thioalkenyl, thiohaloalkyl, cyano, nitro, azide, amino, -COOH, -C(O)NHR', -NHCOR''-; wherein R' is H, alkyl, alkenyl, aryl, heterocyclic alkyl, heteroaryl; and R'' is hydroxyl, alkoxy, aryloxy, alkyl, alkenyl, aryl, heterocyclic alkyl, or heteroaryl; R 11 It is H, substituted or unsubstituted alkyl, alkenyl, aryl, heterocyclic or heteroaryl; n is an integer, which is 1, 2, or 3; L1 is a bond, -(CH2CH2O) q -(CH2CH2O) q (CH2CH2)NH(CO)-, -(CH2CH2O) q (CH2CH2)-、-(CH2CH2O) q(CH2CH2)NH-, -NHCH2(CO)NHCH2Ph-, -N(alkyl)CH2(CO)NHCH2Ph-, substituted or unsubstituted straight-chain or branched alkylene group, substituted or unsubstituted straight-chain or branched alkenyl group, substituted or unsubstituted straight-chain or branched alkyne group, substituted or unsubstituted cycloalkyl group, substituted or unsubstituted heterocyclic group, substituted or unsubstituted aryl group, substituted or unsubstituted heteroaryl group, ether group, ester group, amine group, amide group or any combination thereof, wherein q is an integer from 2 to 10; L2 is a bond, -(CH2CH2O) q -(CH2CH2O) q (CH2CH2)NH(CO)-, -(CH2CH2O) q (CH2CH2)-、-(CH2CH2O) q (CH2CH2)NH-, -NHCH2(CO)NHCH2Ph-, -N(alkyl)CH2(CO)NHCH2Ph-, substituted or unsubstituted straight-chain or branched alkylene groups, substituted or unsubstituted straight-chain or branched alkenyl groups, substituted or unsubstituted straight-chain or branched alkyne groups, substituted or unsubstituted cycloalkyl groups, substituted or unsubstituted heterocyclic groups, substituted or unsubstituted aryl groups, substituted or unsubstituted heteroaryl groups, ether groups, ester groups, amine groups, amide groups, or any combination thereof, wherein q is an integer from 2 to 10; and W is a target protein binding group. In some implementations, this document provides SUMO-targeted chimeras (SUTACs) represented by the structure of Formula II or a salt thereof: (II); in, R1 is H, -S-CF3, S-CHF2, S-CH2F, -O-CF3, -O-CHF2, -O-CH2F, thioalkyl, thioalkenyl, thiohaloalkyl, halogen, alkyl, alkenyl, alkynyl, aryl, haloaryl, heteroaryl, heterocyclic, hydroxyl, alkoxy, aryloxy, thioalkoxy, cyano, nitro, azide, amino, -COOH, -C(O)NHR', -NHCOR''-; wherein R' is H, alkyl, alkenyl, aryl, heterocyclic alkyl, heteroaryl; and R'' is hydroxyl, alkoxy, aryloxy, alkyl, alkenyl, aryl, heterocyclic alkyl, or heteroaryl; R2 is H, -S-CF3, S-CHF2, S-CH2F, -O-CF3, -O-CHF2, -O-CH2F, thioalkyl, thioalkenyl, thiohaloalkyl, halogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocyclic, hydroxyl, alkoxy, aryloxy, thioalkoxy, cyano, nitro, azide, amino, -COOH, -C(O)NHR', -NHCOR''-; wherein R' is H, alkyl, alkenyl, aryl, heterocyclic alkyl, heteroaryl; and R'' is hydroxyl, alkoxy, aryloxy, alkyl, alkenyl, aryl, heterocyclic alkyl, or heteroaryl; n is an integer from 1 to 3; L1 is a bond, -(CH2CH2O) q -(CH2CH2O) q (CH2CH2)NH(CO)-, -(CH2CH2O) q (CH2CH2)-、-(CH2CH2O) q (CH2CH2)NH-, -NHCH2(CO)NHCH2Ph-, -N(alkyl)CH2(CO)NHCH2Ph-, substituted or unsubstituted straight-chain or branched alkylene group, substituted or unsubstituted straight-chain or branched alkenyl group, substituted or unsubstituted straight-chain or branched alkyne group, substituted or unsubstituted cycloalkyl group, substituted or unsubstituted heterocyclic group, substituted or unsubstituted aryl group, substituted or unsubstituted heteroaryl group, ether group, ester group, amine group, amide group or any combination thereof, wherein q is an integer from 2 to 10; L2 is a bond, -(CH2CH2O) q -(CH2CH2O) q (CH2CH2)NH(CO)-, -(CH2CH2O) q (CH2CH2)-、-(CH2CH2O) q (CH2CH2)NH-, -NHCH2(CO)NHCH2Ph-, -N(alkyl)CH2(CO)NHCH2Ph-, substituted or unsubstituted straight-chain or branched alkylene groups, substituted or unsubstituted straight-chain or branched alkenyl groups, substituted or unsubstituted straight-chain or branched alkyne groups, substituted or unsubstituted cycloalkyl groups, substituted or unsubstituted heterocyclic groups, substituted or unsubstituted aryl groups, substituted or unsubstituted heteroaryl groups, ether groups, ester groups, amine groups, amide groups, or any combination thereof, wherein q is an integer from 2 to 10; and W is the target protein binding group.

[0068] In some implementations, this document provides SUMO-targeted chimeras (SUTACs) represented by structures of formula IIA or salts thereof: (IIA) in, R1 is H, -S-CF3, -S-CHF2, -S-CH2F, -O-CF3, -O-CHF2, -O-CH2F, thioalkyl, thioalkenyl, thiohaloalkyl, halogen, alkyl, alkenyl, alkynyl, aryl, haloaryl, heteroaryl, heterocyclic, hydroxyl, alkoxy, aryloxy, thioalkoxy, cyano, nitro, azide, amino, -COOH, -C(O)NHR' or -NHCOR''-; wherein R' is H, alkyl, alkenyl, aryl, heterocyclic alkyl, heteroaryl; and R'' is hydroxyl, alkoxy, aryloxy, alkyl, alkenyl, aryl, heterocyclic alkyl or heteroaryl; R2 is H, -S-CF3, -S-CHF2, -S-CH2F, -O-CF3, -O-CHF2, -O-CH2F, thioalkyl, thioalkenyl, thiohaloalkyl, halogen, alkyl, alkenyl, alkynyl, aryl, haloaryl, heteroaryl, heterocyclic, hydroxyl, alkoxy, aryloxy, thioalkoxy, cyano, nitro, azide, amino, -COOH, -C(O)NHR' and -NHCOR''-; wherein R' is H, alkyl, alkenyl, aryl, heterocyclic alkyl, heteroaryl; and R'' is hydroxyl, alkoxy, aryloxy, alkyl, alkenyl, aryl, heterocyclic alkyl or heteroaryl; n is an integer from 1 to 3; L1 is a bond, -(CH2CH2O) q -(CH2CH2O) q (CH2CH2)NH(CO)-, -(CH2CH2O) q (CH2CH2)-、-(CH2CH2O) q (CH2CH2)NH-, -NHCH2(CO)NHCH2Ph-, -N(alkyl)CH2(CO)NHCH2Ph-, substituted or unsubstituted straight-chain or branched alkylene group, substituted or unsubstituted straight-chain or branched alkenyl group, substituted or unsubstituted straight-chain or branched alkyne group, substituted or unsubstituted cycloalkyl group, substituted or unsubstituted heterocyclic group, substituted or unsubstituted aryl group, substituted or unsubstituted heteroaryl group, ether group, ester group, amine group, amide group or any combination thereof, wherein q is an integer from 2 to 10; L2 is a bond, -(CH2CH2O) q -(CH2CH2O) q (CH2CH2)NH(CO)-, -(CH2CH2O)q (CH2CH2)-、-(CH2CH2O) q (CH2CH2)NH-, -NHCH2(CO)NHCH2Ph-, -N(alkyl)CH2(CO)NHCH2Ph-, substituted or unsubstituted straight-chain or branched alkylene groups, substituted or unsubstituted straight-chain or branched alkenyl groups, substituted or unsubstituted straight-chain or branched alkyne groups, substituted or unsubstituted cycloalkyl groups, substituted or unsubstituted heterocyclic groups, substituted or unsubstituted aryl groups, substituted or unsubstituted heteroaryl groups, ether groups, ester groups, amine groups, amide groups, or any combination thereof, wherein q is an integer from 2 to 10; and W is the target protein binding group.

[0069] In some implementations, this document provides SUMO-targeted chimeras (SUTACs) represented by structures of formula IIB or salts thereof: (IIB) in, Each of Q1, Q2, and Q3 is independently selected from the group consisting of: H, halogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclic. L1 is a bond, -(CH2CH2O) q -(CH2CH2O) q (CH2CH2)NH(CO)-, -(CH2CH2O) q (CH2CH2)-、-(CH2CH2O) q (CH2CH2)NH-, -NHCH2(CO)NHCH2Ph-, -N(alkyl)CH2(CO)NHCH2Ph-, substituted or unsubstituted straight-chain or branched alkylene group, substituted or unsubstituted straight-chain or branched alkenyl group, substituted or unsubstituted straight-chain or branched alkyne group, substituted or unsubstituted cycloalkyl group, substituted or unsubstituted heterocyclic group, substituted or unsubstituted aryl group, substituted or unsubstituted heteroaryl group, ether group, ester group, amine group, amide group or any combination thereof, wherein q is an integer from 2 to 10; L2 is a bond, -(CH2CH2O) q -(CH2CH2O) q (CH2CH2)NH(CO)-, -(CH2CH2O) q (CH2CH2)-、-(CH2CH2O) q(CH2CH2)NH-, -NHCH2(CO)NHCH2Ph-, -N(alkyl)CH2(CO)NHCH2Ph-, substituted or unsubstituted straight-chain or branched alkylene groups, substituted or unsubstituted straight-chain or branched alkenyl groups, substituted or unsubstituted straight-chain or branched alkyne groups, substituted or unsubstituted cycloalkyl groups, substituted or unsubstituted heterocyclic groups, substituted or unsubstituted aryl groups, substituted or unsubstituted heteroaryl groups, ether groups, ester groups, amine groups, amide groups, or any combination thereof, wherein q is an integer from 2 to 10; and W is the target protein binding group.

[0070] In some embodiments, Z in Formula I or Formula I' is O, S, and NH. In another embodiment, Z in Formula I is O. In another embodiment, Z in Formula I is S. In yet another embodiment, Z in Formula I is NH.

[0071] In some embodiments, R1 of formula II or IIA is H, -S-CF3, S-CHF2, S-CH2F, -O-CF3, -O-CHF2, -O-CH2F, thioalkyl, thioalkenyl, thiohaloalkyl, halogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, haloaryl, heteroaryl, heterocyclic, hydroxyl, alkoxy, aryloxy, thioalkoxy, cyano, nitro, azide, amino, -COOH, -C(O)NHR', -NHCOR''-; wherein R' is H, alkyl, alkenyl, aryl, heterocyclic alkyl, heteroaryl; and R'' is hydroxyl, alkoxy, aryloxy, alkyl, alkenyl, aryl, heterocyclic alkyl, or heteroaryl. In another embodiment, R1 is H. In another embodiment, R1 is -S-CF3. In another embodiment, R1 is S-CHF2. In another embodiment, R1 is S-CH2F. In another embodiment, R1 is -O-CF3. In another embodiment, R1 is -O-CHF2. In another embodiment, R1 is -O-CH2F. In another embodiment, R1 is a thioalkyl. In another embodiment, R1 is a thioalkenyl. In another embodiment, R1 is a thiohaloalkyl. In another embodiment, R1 is a halogen. In another embodiment, R1 is an alkyl. In another embodiment, R1 is an alkenyl. In another embodiment, R1 is an alkane. In another embodiment, R1 is an aryl. In another embodiment, R1 is a haloaryl. In another embodiment, R1 is a heteroaryl. In another embodiment, R1 is a heterocyclic. In another embodiment, R1 is a hydroxyl. In another embodiment, R1 is an alkoxy. In another embodiment, R1 is an aryloxy. In another embodiment, R1 is a thioalkoxy. In another embodiment, R1 is a cyano. In another embodiment, R1 is a nitro. In another embodiment, R1 is an azide. In another embodiment, R1 is an amino group. In another embodiment, R1 is -COOH. In another embodiment, R1 is -C(O)NHR'. In another embodiment, R1 is -NHCOR''-. In another embodiment, R' is H, alkyl, alkenyl, aryl, heterocyclic alkyl, or heteroaryl. In another embodiment, R'' is hydroxyl, alkoxy, aryloxy, alkyl, alkenyl, aryl, heterocyclic alkyl, or heteroaryl.

[0072] In some embodiments, R2 of formula II or IIA is H, -S-CF3, S-CHF2, S-CH2F, -O-CF3, O-CHF2, O-CH2F, thioalkyl, thioalkenyl, thiohaloalkyl, halogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, heterocyclic, hydroxyl, alkoxy, aryloxy, thioalkoxy, cyano, nitro, azide, amino, -COOH, -C(O)NHR', -NHCOR''-; wherein R' is H, alkyl, alkenyl, aryl, heterocyclic alkyl, heteroaryl; and R'' is hydroxyl, alkoxy, aryloxy, alkyl, alkenyl, aryl, heterocyclic alkyl, or heteroaryl. In another embodiment, R2 is H. In another embodiment, R2 is -S-CF3. In another embodiment, R2 is S-CHF2. In another embodiment, R2 is S-CH2F. In another embodiment, R2 is -O-CF3. In another embodiment, R2 is O-CHF2. In another embodiment, R2 is O-CH2F. In another embodiment, R2 is a thioalkyl. In another embodiment, R2 is a thioalkenyl. In another embodiment, R2 is a thiohaloalkyl. In another embodiment, R2 is a halogen. In another embodiment, R2 is an alkyl. In another embodiment, R2 is an alkenyl. In another embodiment, R2 is an alkane. In another embodiment, R2 is an aryl. In another embodiment, R2 is a heteroaryl. In another embodiment, R2 is a heterocyclic. In another embodiment, R2 is a hydroxyl. In another embodiment, R2 is an alkoxy. In another embodiment, R2 is an aryloxy. In another embodiment, R2 is a thioalkoxy. In another embodiment, R2 is a cyano. In another embodiment, R2 is a nitro. In another embodiment, R2 is an azide. In another embodiment, R2 is an amino. In another embodiment, R2 is -COOH. In another embodiment, R2 is -C(O)NHR'. In another embodiment, R2 is -NHCOR''-. In another embodiment, R' is H, alkyl, alkenyl, aryl, heterocyclic alkyl, or heteroaryl. In another embodiment, R'' is hydroxyl, alkoxy, aryloxy, alkyl, alkenyl, aryl, heterocyclic alkyl, or heteroaryl.

[0073] In some embodiments, each of R6 and R7 of Formula I, Formula I', or Formula IA is independently selected from the group consisting of: H, halogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, haloaryl, heteroaryl, heterocyclic, hydroxyl, alkoxy, aryloxy, thioalkoxy, thioalkyl, thioalkenyl, thiohaloalkyl, cyano, nitro, azide, amino, -COOH, -C(O)NHR', -NHCOR''-; wherein R' is H, alkyl, alkenyl, aryl, heterocyclic alkyl, or heteroaryl; and R'' is hydroxyl, alkoxy, aryloxy, alkyl, alkenyl, aryl, heterocyclic alkyl, or heteroaryl; or R6 and R7 together form a 5-8 membered ring, wherein the ring is substituted or unsubstituted. In another embodiment, each of R6 and R7 of Formula I, Formula I', or Formula IA is independently H. In another embodiment, each of R6 and R7 of Formula I, Formula I', or Formula IA is independently a halogen. In another embodiment, each of R6 and R7 of Formula I, Formula I', or Formula IA is independently an alkyl group. In another embodiment, each of R6 and R7 of Formula I, Formula I', or Formula IA is independently an alkenyl group. In another embodiment, each of R6 and R7 of Formula I, Formula I', or Formula IA is independently an alkynyl group. In another embodiment, each of R6 and R7 of Formula I, Formula I', or Formula IA is independently a cycloalkyl group. In another embodiment, each of R6 and R7 of Formula I, Formula I', or Formula IA is independently an aryl group. In another embodiment, each of R6 and R7 of Formula I, Formula I', or Formula IA is independently a haloaryl group. In another embodiment, each of R6 and R7 of Formula I, Formula I', or Formula IA is independently a heteroaryl group. In another embodiment, each of R6 and R7 of Formula I, Formula I', or Formula IA is independently a heterocyclic group. In another embodiment, R6 and R7 of Formula I, I', or IA are independently hydroxyl groups. In another embodiment, each of R6 and R7 of Formula I, I', or IA is independently alkoxy. In another embodiment, each of R6 and R7 of Formula I, I', or IA is independently aryloxy. In another embodiment, each of R6 and R7 of Formula I, I', or IA is independently thioalkoxy. In another embodiment, each of R6 and R7 of Formula I, I', or IA is independently thioalkyl. In another embodiment, each of R6 and R7 of Formula I, I', or IA is independently thioalkenyl. In another embodiment, each of R6 and R7 of Formula I, I', or IA is independently thiohaloalkyl. In another embodiment, each of R6 and R7 of Formula I, I', or IA is independently cyano. In another embodiment, each of R6 and R7 in Formula I, Formula I', or Formula IA is independently a nitro group.In another embodiment, each of R6 and R7 of Formula I, Formula I', or Formula IA is independently an azide. In another embodiment, each of R6 and R7 of Formula I, Formula I', or Formula IA is independently an amino group. In another embodiment, each of R6 and R7 of Formula I, Formula I', or Formula IA is independently -COOH. In another embodiment, each of R6 and R7 of Formula I, Formula I', or Formula IA is independently -C(O)NHR'. In another embodiment, each of R6 and R7 of Formula I, Formula I', or Formula IA is independently -NHCOR''-. In other embodiments, R' is H, alkyl, alkenyl, aryl, heterocyclic alkyl, or heteroaryl. In other embodiments, R'' is hydroxyl, alkoxy, aryloxy, alkyl, alkenyl, aryl, heterocyclic alkyl, or heteroaryl. In some embodiments, R6 and R7 of Formula I, Formula I', or Formula IA together form a 5-8 membered ring, wherein the ring is substituted or unsubstituted.

[0074] In some implementations, R8, R9, R of form IB, form IC, or form ID 10 Each of the following is independently selected from the group consisting of: H, halogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, haloaryl, heteroaryl, heterocyclic, hydroxyl, alkoxy, aryloxy, thioalkoxy, thioalkyl, thioalkenyl, thiohaloalkyl, cyano, nitro, azide, amino, -COOH, -C(O)NHR', -NHCOR''-; wherein R' is H, alkyl, alkenyl, aryl, heterocyclic alkyl, heteroaryl; and R'' is hydroxyl, alkoxy, aryloxy, alkyl, alkenyl, aryl, heterocyclic alkyl, or heteroaryl. In another embodiment, R8, R9, R 10 Each of them is independently H. In another implementation, R8, R9, R 10 Each of them is independently a halogen. In another embodiment, R8, R9, R 10 Each of them is independently an alkyl group. In another embodiment, R8, R9, R 10 Each of them is an alkenyl group independently. In another embodiment, R8, R9, R 10 Each of these groups is independently an alkynyl group. In another embodiment, R8, R9, R... 10 Each of them is independently a cyclic hydrocarbon group. In another embodiment, R8, R9, R 10 Each of R8, R9, and R is an aryl group. In another embodiment, R8, R9, and R are... 10 Each of them is independently a haloaryl group. In another embodiment, R8, R9, R 10 Each of them is independently a heteroaryl group. In another embodiment, R8, R9, R 10Each of these is independently a heterocyclic ring. In another embodiment, R8, R9, and R... 10 Each of these is independently a hydroxyl group. In another embodiment, R8, R9, R... 10 Each of them is independently an alkoxy group. In another embodiment, R8, R9, R 10 Each of these is independently an aryloxy group. In another embodiment, R8, R9, R... 10 Each of them is independently a thioalkoxy group. In another embodiment, R8, R9, R 10 Each of them is independently a thioalkyl group. In another embodiment, R8, R9, R 10 Each of them is independently a thioalkenyl group. In another embodiment, R8, R9, R 10 Each of them is independently a thiohaloalkyl group. In another embodiment, R8, R9, R 10 Each of them is independently a cyano group. In another embodiment, R8, R9, R 10 Each of them is independently a nitro group. In another embodiment, R8, R9, R 10 Each of them is independently an azide. In another embodiment, R8, R9, R 10 Each of them is independently an amino group. In another embodiment, R8, R9, R 10 Each of them is independently -COOH. In another embodiment, R8, R9, R 10 Each of them is independently -C(O)NHR'. In another implementation, R8, R9, R 10 Each of these is independently -NHCOR''-. In another embodiment, R' is H, alkyl, alkenyl, aryl, heterocyclic alkyl, or heteroaryl. In another embodiment, R'' is hydroxyl, alkoxy, aryloxy, alkyl, alkenyl, aryl, heterocyclic alkyl, or heteroaryl.

[0075] In some embodiments, n in formula IB, formula IC, formula ID, formula II, or formula IIA is an integer 1, 2, or 3. In another embodiment, n is 1. In another embodiment, n is 2. In another embodiment, n is 3.

[0076] In some embodiments, each of Q1, Q2, and Q3 of Formula IIB is independently selected from the group consisting of: H, halogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclic. In another embodiment, each of Q1, Q2, and Q3 is independently H. In another embodiment, each of Q1, Q2, and Q3 is independently halogen. In another embodiment, each of Q1, Q2, and Q3 is independently alkyl. In another embodiment, each of Q1, Q2, and Q3 is independently alkenyl. In another embodiment, each of Q1, Q2, and Q3 is independently alkynyl. In another embodiment, each of Q1, Q2, and Q3 is independently cycloalkyl. In another embodiment, each of Q1, Q2, and Q3 is independently aryl. In another embodiment, each of Q1, Q2, and Q3 is independently heteroaryl. In another embodiment, each of Q1, Q2, and Q3 is independently heterocyclic.

[0077] In some implementations, L1 of formulas I, I', IA, IB, IC, ID, II, IIA, or IIB is a bond, -(CH2CH2O). q The following groups may be substituted or unsubstituted straight-chain or branched alkylene groups, substituted or unsubstituted straight-chain or branched alkenyl groups, substituted or unsubstituted straight-chain or branched alkyne groups, substituted or unsubstituted cyclic hydrocarbon groups, substituted or unsubstituted heterocyclic groups, substituted or unsubstituted aryl groups, substituted or unsubstituted heteroaryl groups, ether groups, ester groups, amine groups, amide groups, or any combination thereof, wherein q is an integer from 2 to 10. In another embodiment, L1 is a bond. In another embodiment, L1 is -(CH2CH2O). q Where q is an integer from 2 to 10. In another embodiment, L1 is a substituted or unsubstituted straight-chain or branched alkylene group. In another embodiment, L1 is a substituted or unsubstituted straight-chain or branched alkenyl group. In another embodiment, L1 is a substituted or unsubstituted straight-chain or branched alkyne group. In another embodiment, L1 is a substituted or unsubstituted cyclic hydrocarbon group. In another embodiment, L1 is a substituted or unsubstituted heterocyclic ring. In another embodiment, L1 is a substituted or unsubstituted aryl group. In another embodiment, L1 is a substituted or unsubstituted heteroaryl group. In another embodiment, L1 is an ether group. In another embodiment, L1 is an ester. In another embodiment, L1 is an amine group. In another embodiment, L1 is an amide group. In another embodiment, L1 is an amide group. In another embodiment, L1 is -(CH2CH2O). qIn another implementation, L1 is -(CH2CH2O). q (CH2CH2)NH(CO)-. In another embodiment, L1 is -(CH2CH2O). q (CH2CH2)-. In another embodiment, L1 is -(CH2CH2O). q (CH2CH2)NH-. In another embodiment, L1 is -NHCH2(CO)NHCH2Ph-. In another embodiment, L1 is -N(alkyl)CH2(CO)NHCH2Ph-.

[0078] In some implementations, L2 of formulas I, I', IA, IB, IC, ID, II, IIA, or IIB is a bond, -(CH2CH2O). q The following groups may be substituted or unsubstituted straight-chain or branched alkylene groups, substituted or unsubstituted straight-chain or branched alkenyl groups, substituted or unsubstituted straight-chain or branched alkyne groups, substituted or unsubstituted cyclic hydrocarbon groups, substituted or unsubstituted heterocyclic groups, substituted or unsubstituted aryl groups, substituted or unsubstituted heteroaryl groups, ether groups, ester groups, amine groups, amide groups, or any combination thereof, wherein q is an integer from 2 to 10. In another embodiment, L2 is a bond. In another embodiment, L2 is -(CH2CH2O). q Where q is an integer from 2 to 10. In another embodiment, L2 is a substituted or unsubstituted straight-chain or branched alkylene group. In another embodiment, L2 is a substituted or unsubstituted straight-chain or branched alkenyl group. In another embodiment, L2 is a substituted or unsubstituted straight-chain or branched ynylene group. In another embodiment, L2 is a substituted or unsubstituted cyclic hydrocarbon group. In another embodiment, L2 is a substituted or unsubstituted heterocyclic ring. In another embodiment, L2 is a substituted or unsubstituted aryl group. In another embodiment, L2 is a substituted or unsubstituted heteroaryl group. In another embodiment, L2 is an ether group. In another embodiment, L2 is an ester. In another embodiment, L2 is an amine group. In another embodiment, L2 is an amide group. In another embodiment, L2 is -(CH2CH2O). q In another implementation, L2 is -(CH2CH2O). q (CH2CH2)NH(CO)-. In another embodiment, L2 is -(CH2CH2O). q (CH2CH2)-. In another embodiment, L2 is -(CH2CH2O). q(CH2CH2)NH-. In another embodiment, L2 is -NHCH2(CO)NHCH2Ph-. In another embodiment, L2 is -N(alkyl)CH2(CO)NHCH2Ph-.

[0079] In some implementations, R of formulas I, I', IA, IB, IC, and ID 11 It is H, a substituted or unsubstituted alkyl, alkenyl, aryl, heterocyclic, or heteroaryl group. In another embodiment, R of formula I... 11 It is H. In another implementation, R of formula I, formula I', formula IA, formula IB, formula IC, and formula ID. 11 It is a substituted or unsubstituted alkyl group. In another embodiment, R of formula I, formula I', formula IA, formula IB, formula IC, formula ID 11 It is a substituted or unsubstituted alkenyl group. In another embodiment, R of formula I, formula I', formula IA, formula IB, formula IC, formula ID 11 It is a substituted or unsubstituted aryl group. In another embodiment, R of formula I, formula I', formula IA, formula IB, formula IC, formula ID 11 It is a substituted or unsubstituted heterocyclic ring. In another embodiment, R of formula I, formula I', formula IA, formula IB, formula IC, and formula ID 11 It is a heteroaryl group that has been substituted or not substituted.

[0080] In some implementations, W of Formula I, Formula I', Formula IA, Formula IB, Formula IC, Formula ID, Formula II, Formula IIA, or Formula IIB is a target protein binding group. The target protein binding group is defined based on the target protein. For example, for BRD4, the target protein binding group is JQ1 (structure [a] below), and for kinases, the target protein binding group is a kinase inhibitor, such as gefitinib (structure [b] below). In some embodiments, the target protein binding group includes JQ1. In some embodiments, the target protein binding group includes a kinase inhibitor. In some embodiments, the target protein binding group includes gefitinib. In some embodiments, the target protein binding group includes GNE-207 or a derivative thereof. In some embodiments, the target protein binding group includes A-485 or a derivative thereof.

[0081] This invention includes SUTAC salts, which can be produced by reacting the SUTACs of this invention with an acid or base. Certain SUTACs, particularly those having acidic or basic groups, may also be in the form of salts, optionally pharmaceutically acceptable salts. The term "pharmaceutically acceptable salt" refers to those salts that retain the bioavailability and properties of a free base or free acid, and which are not biologically or otherwise undesirable. The salts are formed from inorganic and organic acids, such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and similar inorganic acids, and such as acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, etc. N - Acetylcysteine ​​and similar organic acids. Other salts are known to those skilled in the art and can be readily adapted for use according to the present invention.

[0082] Suitable and acceptable salts of the amines of the SUTAC of the present invention can be prepared from inorganic or organic acids. In various embodiments, examples of inorganic salts of the amines are hydrogen sulfates, borates, bromides, chlorides, hemisulfates, hydrobroms, hydrochlorides, 2-hydroxyethyl sulfonates (hydroxyethane sulfonates), iodates, iodides, hydroxyethyl sulfonates, nitrates, persulfates, phosphates, sulfates, aminosulfonates, sulfanamides, sulfonic acids (alkyl sulfonates, aryl sulfonates, halogenated alkyl sulfonates, halogenated aryl sulfonates), sulfonates, and thiocyanates.

[0083] In various embodiments, examples of organic salts of amines can be selected from organic acids of the aliphatic, alicyclic, aromatic, araliphatic, heterocyclic, carboxylic, and sulfonic acid classes, including acetates, arginine salts, aspartate salts, ascorbic acid salts, adipic acid salts, anthranilates, alginates, alkane carboxylates, substituted alkane carboxylates, alginates, benzenesulfonates, benzoates, hydrogen sulfates, butyrates, bicarbonates, hydrogen tartrates, citrates, camphorates, camphor sulfonates, cyclohexylaminosulfonate, cyclopentane propionate, and calcium edetate. edetate), dextrorotatory camphor sulfonate, camsylate, carbonate, clavulanate, cinnamate, dicarboxylate, digluconate, dodecyl sulfonate, dihydrochloride, decanoate, enanthate, ethanesulfonate, edetate, ethanedisulfonate, propionate, estolate, esylate, fumarate, formate, fluoride, galacturonate, gluconate, glutamate, glycolate, glucorate, gluconate ucoheptanoate, glycerol phosphate, gluceptate, glycolyllarsanilate, glutamate, glutamate, heptanate, hexanoate, hydroxymaleate, hydroxycarboxylic acid, hexylresorcinol, hydroxybenzoate, hydroxynaphthyl carboxate, hydrofluoric acid, lactate, lactobionate, laurate, malate, maleate, methylenebis(beta-oxynaphthoate), malonate, mandelate, mesylate, methanesulfonatesulfonate), methyl bromide, methyl nitrate, methanesulfonate, monopotassium maleate, mucate, monocarboxylate, naphthalenesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, napsylate, N-methylglucosamine, oxalate, octanoate, oleate, dihydroxynaphthalate, phenylacetate, picrate, phenylbenzoate, pivalate, propionate, phthalate, styrene Salts, pectates, phenylpropionates, palmitates, pantothenates, polygalactosates, pyruvates, quinates, salicylates, succinates, stearates, sulfaniates, basic acetates, tartrates, theophylline acetates, p-toluenesulfonates (toluenesulfonates), trifluoroacetates, terephthalates, tannates, theophylline, trihaloacetates, triethyliodide, tricarboxylates, undecanoates, and valerates.

[0084] In various embodiments, examples of inorganic salts of carboxylic acids or hydroxyl groups may be selected from ammonium, alkali metals (including lithium, sodium, potassium, cesium); alkaline earth metals (including calcium, magnesium, aluminum; zinc, barium, choline, quaternary ammonium).

[0085] In some embodiments, examples of organic salts of carboxylic acids or hydroxyl groups may be selected from arginine; organic amines, including aliphatic organic amines, alicyclic organic amines, and aromatic organic amines; benzathine penicillin, tert-butylamine, phenethylbenzylamine (… N (-Benzylphenethylamine), dicyclohexylamine, dimethylamine, diethanolamine, ethanolamine, ethylenediamine, hydrabamine, imidazole, lysine, methylamine, meglumine, N -methyl- D -glucosamine, N , N' - Dibenzylethylenediamine, nicotinamide, organic amines, ornithine, pyridine, picolides, piperazine, procaine, tri(hydroxymethyl)methylamine, triethylamine, triethanolamine, trimethylamine, tromethamine, and urea.

[0086] In various embodiments, the salt can be formed by conventional means, such as by reacting the product in the form of a free base or free acid with one or more equivalents of a suitable acid or base in a solvent or medium in which the salt is insoluble, or in a solvent such as water, wherein the solvent is removed in a vacuum or by freeze-drying or by exchanging the ions of the existing salt for another ion or a suitable ion exchange resin.

[0087] The alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclic, aryl, heteroaryl, and rings formed with R6 and R7 mentioned in this article may have one or more substituents, wherein each substituent group may be, independently, for example, cycloalkyl, aryl, heteroaryl, heteroalicyclic, halogen, hydroxyl, alkoxy, aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, sulfinyl, sulfonyl, sulfonate, sulfate, cyano, nitro, azide, phosphonyl, oxophosphonyl, oxo, carbonyl, thiocarbonyl, urea group, thiourea group, O-carbamoyl, N-carbamoyl, O-thiocarbamoyl, N-thiocarbamoyl, C-amide, N-amide The substituents include: alkyl, C-carboxyl, O-carboxyl, sulfonamido, amidine, guanidine, hydrazine, acylhydrazine, thioacylhydrazine, aminohalogen, alkylaryloxy, heteroaryloxy, oxo, cycloalkyl, phenyl, heteroaryl, heterocyclic, naphthyl, amino, alkylamino, arylamino, heteroarylamino, dialkylamino, diarylamino, alkylarylamino, alkylheteroarylamino, arylheteroarylamino, acyl, acyloxy, nitro, carboxyl, carbamoyl, carboxamide, cyano, sulfonyl, sulfonylamino, sulfoneamido, sulfinyl, sulfinylamino, thiol, alkylthio, arylthio, or alkylsulfonyl groups. Any substituent may be unsubstituted or further substituted by any of the substituents mentioned above.

[0088] In this document, the term "alkyl" describes saturated aliphatic hydrocarbons, including straight-chain and branched groups. Optionally, the alkyl group has 2 to 20 carbon atoms. More optionally, the alkyl group is a medium-sized alkyl group having 2 to 10 carbon atoms. Most optionally, the alkyl group is a lower alkyl group having 2 to 4 carbon atoms or 1 to 6 carbon atoms. The alkyl group may be substituted or unsubstituted.

[0089] In some embodiments, unless otherwise stated, the substituted alkyl or alkylene group may be substituted by one or more (e.g., one, two, three or more, as long as the valence allows) groups independently selected from halogens, hydroxyl groups, alkoxy groups, cyano groups, and oxo groups. In some embodiments, the substituted alkyl group may be substituted by one or more (e.g., one, two, three or more, as long as the valence allows) groups independently selected from halogens and cyano groups.

[0090] In this document, the term "alkenyl" describes an unsaturated aliphatic hydrocarbon containing at least one carbon-carbon double bond, including straight-chain and branched groups. Optionally, the alkenyl group has 2 to 20 carbon atoms. More optionally, the alkenyl group is a medium-sized alkenyl group having 2 to 10 carbon atoms or 2 to 6 carbon atoms. Most optionally, unless otherwise specified, the alkenyl group is a lower alkenyl group having 2 to 4 carbon atoms. The alkenyl group may be substituted or unsubstituted.

[0091] The substituted alkenyl or alkenyl group may have one or more substituents, wherein each substituent group may be, independently, for example, alkynyl, cycloalkyl, alkynyl, aryl, heteroaryl, heteroalicyclic, halogen, hydroxyl, alkoxy, aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, sulfinyl, sulfonyl, sulfonate, sulfonamide, sulfate, cyano, nitro, azide, phosphonyl, oxophosphonyl, oxo, carbonyl, thiocarbonyl, urea, thiourea, O-carbamoyl, N-carbamoyl, O-thiocarbamoyl, N-thiocarbamoyl, C-amide, N-amide, C-carboxyl, O-carboxyl, sulfonylamide, amido, guanidine, hydrazine, acylhydrazine, thioacylhydrazine, and amino.

[0092] In some embodiments, unless otherwise stated, the substituted alkenyl or alkenyl group may be substituted by one or more (e.g., one, two, three or more, provided the valence allows) groups independently selected from halogens, hydroxyl groups, alkoxy groups, cyano groups, and oxo groups. In some embodiments, the substituted alkenyl group may be substituted by one or more (e.g., one, two, three or more, provided the valence allows) groups independently selected from halogens and cyano groups.

[0093] In this document, the term "alkynyl" describes an unsaturated aliphatic hydrocarbon containing at least one carbon-carbon triple bond, including straight-chain and branched groups. Optionally, the alkynyl group has 2 to 20 carbon atoms. More optionally, the alkynyl group is a medium-sized alkynyl group having 2 to 10 carbon atoms. Most optionally, unless otherwise stated, the alkynyl group is a lower alkynyl group having 2 to 4 carbon atoms. The alkynyl group may be substituted or unsubstituted.

[0094] The substituted alkynyl or alkynyl group may have one or more substituents, wherein each substituent group may be, independently, for example, cycloalkyl, alkenyl, aryl, heteroaryl, heteroalicyclic, halogen, hydroxyl, alkoxy, aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, sulfinyl, sulfonyl, sulfonate, sulfate, cyano, nitro, azide, phosphonyl, oxophosphonyl, oxo, carbonyl, thiocarbonyl, urea, thiourea, O-carbamoyl, N-carbamoyl, O-thiocarbamoyl, N-thiocarbamoyl, C-amide, N-amide, C-carboxyl, O-carboxyl, sulfonylamide, amido, guanidine, hydrazine, acylhydrazine, thioacylhydrazine, and amino.

[0095] In some embodiments, unless otherwise stated, the substituted alkynyl or ynylene group may be substituted by one or more (e.g., one, two, three or more, provided the valence allows) groups independently selected from halogens, hydroxyl groups, alkoxy groups, cyano groups, and oxo groups. In some embodiments, the substituted alkynyl group may be substituted by one or more (e.g., one, two, three or more, provided the valence allows) groups independently selected from halogens and cyano groups.

[0096] As used herein, "alkylene" refers to a straight-chain, branched, or cyclic divalent aliphatic hydrocarbon group, in some embodiments of which is straight-chain or branched, having from one to about 20 carbon atoms in one embodiment and from one to 12 carbon atoms in another embodiment. In further embodiments, alkylene includes lower alkylene groups. Optionally, one or more oxygen, sulfur (including S(=O) groups and S(=O)2 groups), or substituted or unsubstituted nitrogen atoms (including -NR- groups and -N groups) may be inserted along the alkylene group. + The alkylene group (RR-) is a nitrogen-substituent that is alkyl, aryl, aralkyl, heteroaryl, heteroaryl, or COR, wherein each R is independently selected from alkyl, aryl, aralkyl, heteroaryl, heteroaryl, -OY, or -NYY, and each Y is independently selected from hydrogen, alkyl, aryl, heteroaryl, cycloalkyl, or heterocyclic. The alkylene group includes, but is not limited to, methylene (-CH2), ethylene (-CH2CH2-), propylene (-(CH2)3), methylenedioxy (-O-CH2-O-), and ethylenedioxy (-O-(CH2)2-O-). The term "lower alkylene" refers to an alkylene group having 1 to 6 carbon atoms. In some embodiments, the alkylene group is a lower alkylene group, which includes alkylene groups having 1 to 3 carbon atoms.

[0097] As used herein, "alkenyl" refers to a straight-chain, branched, or cyclic divalent aliphatic hydrocarbon group, in one embodiment of which is straight-chain or branched, having from 2 to about 20 carbon atoms and at least one double bond in some embodiments, and having 1 to 12 carbon atoms in other embodiments. In further embodiments, the alkenyl group comprises a lower alkenyl group. One or more oxygen, sulfur, or substituted or unsubstituted nitrogen atoms may optionally be inserted along the alkenyl group, wherein the nitrogen substituent is an alkyl group. Alkenyl groups include, but are not limited to, -CH=CH-CH=CH- and -H=CH-CH2. The term "lower alkenyl" refers to an alkenyl group having 2 to 6 carbon atoms. In some embodiments, the alkenyl group is a lower alkenyl group, which includes alkenyl groups having 3 to 4 carbon atoms.

[0098] In some embodiments, unless otherwise stated, the substituted alkenyl group may be substituted by one or more (e.g., one, two, three or more, provided the valence allows) groups independently selected from halogens, hydroxyl groups, alkoxy groups, cyano groups, and oxo groups. In some embodiments, the substituted alkenyl group may be substituted by one or more (e.g., one, two, three or more, provided the valence allows) groups independently selected from halogens and cyano groups.

[0099] As used herein, "ynynyl" refers to a straight-chain, branched, or cyclic divalent aliphatic hydrocarbon group, in some embodiments of which is straight-chain or branched, having from 2 to about 20 carbon atoms and at least one triple bond in one embodiment, and having 1 to 12 carbon atoms in another embodiment. In further embodiments, the ynynyl group includes a lower ynynyl group. One or more oxygen, sulfur, or substituted or unsubstituted nitrogen atoms may optionally be inserted along the ynynyl group, wherein the nitrogen substituent is an alkyl group. The ynynyl group includes, but is not limited to, -C≡CC≡C, -C≡C-, and -C≡C-CH2-. The term "lower ynynyl" refers to an ynynyl group having 2 to 6 carbon atoms. In some embodiments, the ynynyl group is a lower ynynyl group, which includes an ynynyl group having 3 to 4 carbon atoms.

[0100] In some embodiments, unless otherwise stated, the substituted ynylene group may be replaced by one or more (e.g., one, two, three or more, provided the valence allows) groups independently selected from halogens, hydroxyl groups, alkoxy groups, cyano groups, and oxo groups. In some embodiments, the substituted ynylene group may be replaced by one or more (e.g., one, two, three or more, provided the valence allows) groups independently selected from halogens and cyano groups.

[0101] A "cyclic hydrocarbon group" refers to a saturated or unsaturated monocyclic or fused-ring (i.e., rings sharing adjacent carbon atom pairs) group, wherein one or more of the rings do not have a fully conjugated π-electron system. Examples of cyclic hydrocarbon groups are, without limitation, cyclopropane, cyclobutane, cyclopentane, cyclopentene, cyclohexane, cyclohexadiene, cycloheptane, cycloheptanetriene, and adamantane. Cyclic hydrocarbon groups can be substituted or unsubstituted. When substituted, the substituent group can be, for example, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, heteroalicyclic, halogen, hydroxyl, alkoxy, aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, sulfinyl, sulfonyl, sulfonate, sulfate, cyano, nitro, azide, phosphonyl, oxophosphonyl, oxo, carbonyl, thiocarbonyl, urea, thiourea, O-carbamoyl, N-carbamoyl, O-thiocarbamoyl, N-thiocarbamoyl, C-amide, N-amide, C-carboxyl, O-carboxyl, sulfonylamide, amidine, guanidine, hydrazine, acylhydrazine, thioacylhydrazine, and amino, as these terms are defined herein. When the cycloalkyl group is unsaturated, it can contain at least one carbon-carbon double bond and / or at least one carbon-carbon triple bond. The cyclic hydrocarbon group can be an end group as defined herein, wherein the cyclic hydrocarbon group is attached to a single adjacent atom; or it can be a linking group as defined herein, connecting two or more parts.

[0102] In some embodiments, unless otherwise stated, the substituted cyclic hydrocarbon group may be replaced by one or more (e.g., one, two, three or more, as long as the valence allows) groups independently selected from halogens, hydroxyl groups, alkoxy groups, cyano groups, and oxo groups. In some embodiments, the substituted cyclic hydrocarbon group may be replaced by one or more (e.g., one, two, three or more, as long as the valence allows) groups independently selected from halogens and cyano groups.

[0103] An "aryl" group refers to an all-carbon monocyclic or fused-ring polycyclic (i.e., rings sharing adjacent carbon atom pairs) terminal group that has a fully conjugated π-electron system. Examples of aryl groups are not limited to phenyl, naphthyl, and anthracene. Aryl groups can be substituted or unsubstituted. When substituted, the substituent group can be, for example, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, heteroalicyclic, halogen, hydroxyl, alkoxy, aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, sulfinyl, sulfonyl, sulfonate, sulfate, cyano, nitro, azide, phosphonyl, oxophosphonyl, oxo, carbonyl, thiocarbonyl, urea, thiourea, O-carbamoyl, N-carbamoyl, O-thiocarbamoyl, N-thiocarbamoyl, C-amide, N-amide, C-carboxyl, O-carboxyl, sulfonylamide, amidine, guanidine, hydrazine, acylhydrazine, thioacylhydrazine, and amino, as these terms are defined herein. An aryl group can be an end group as defined herein, wherein the aryl group is attached to a single adjacent atom; or it can be a linking group as defined herein, connecting two or more parts.

[0104] In some embodiments, unless otherwise stated, the substituted aryl group may be substituted by one or more (e.g., one, two, three or more, provided the valence allows) groups independently selected from halogens, hydroxyl groups, alkoxy groups, cyano groups, and oxo groups. In some embodiments, the substituted aryl group may be substituted by one or more (e.g., one, two, three or more, provided the valence allows) groups independently selected from halogens and cyano groups.

[0105] A "heteroaryl" group refers to a monocyclic or fused-ring (i.e., a ring sharing adjacent atom pairs) end group that has one or more atoms in the ring, such as nitrogen, oxygen, and sulfur, and in addition has a fully conjugated π-electron system. Examples of heteroaryl groups are not limited to pyrrole, furan, thiophene, imidazole, oxazole, thiazole, pyrazole, pyridine, pyrimidine, quinoline, isoquinoline, and purine. Heteroaryl groups can be substituted or unsubstituted. When substituted, the substituent group can be, for example, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, heteroalicyclic, halogen, hydroxyl, alkoxy, aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, sulfinyl, sulfonyl, sulfonate, sulfate, cyano, nitro, azide, phosphonyl, oxophosphonyl, oxo, carbonyl, thiocarbonyl, urea, thiourea, O-carbamoyl, N-carbamoyl, O-thiocarbamoyl, N-thiocarbamoyl, C-acylamino, N-acylamino, C-carboxyl, O-carboxyl, sulfonylamino, amido, guanidinyl, hydrazine, acylhydrazine, thioacylhydrazine, and amino, as these terms are defined herein.

[0106] In some embodiments, unless otherwise stated, the substituted heteroaryl group may be substituted by one or more (e.g., one, two, three or more, provided the valence allows) groups independently selected from halogens, hydroxyl groups, alkoxy groups, cyano groups, and oxo groups. In some embodiments, the substituted heteroaryl group may be substituted by one or more (e.g., one, two, three or more, provided the valence allows) groups independently selected from halogens and cyano groups.

[0107] A "heterocyclic" group is a monocyclic or fused-ring group having one or more atoms, such as nitrogen, oxygen, and sulfur, in the ring. The ring may also have one or more double bonds. However, the ring does not have a fully conjugated π-electron system. Heterocyclic rings can be substituted or unsubstituted. When substituted, the substituted group can be, for example, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, heteroalicyclic, halogen, hydroxyl, alkoxy, aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, sulfinyl, sulfonyl, sulfonate, sulfate, cyano, nitro, azide, phosphonyl, oxophosphonyl, oxo, carbonyl, thiocarbonyl, urea, thiourea, O-carbamoyl, N-carbamoyl, O-thiocarbamoyl, N-thiocarbamoyl, C-amide, N-amide, C-carboxyl, O-carboxyl, sulfonylamide, amidine, guanidine, hydrazine, acylhydrazine, thioacylhydrazine, and amino, as these terms are defined herein. Representative examples are piperidine, piperazine, tetrahydrofuran, tetrahydropyran, morpholine, etc. The heterocyclic group can be an end group as defined herein, wherein the heterocyclic group is attached to a single adjacent atom; or it can be a linking group as defined herein, connecting two or more parts.

[0108] In some embodiments, unless otherwise stated, the substituted heterocycle may be substituted by one or more (e.g., one, two, three or more, provided the valence allows) groups independently selected from halogens, hydroxyl groups, alkoxy groups, cyano groups, and oxo groups. In some embodiments, the substituted heterocycle may be substituted by one or more (e.g., one, two, three or more, provided the valence allows) groups independently selected from halogens and cyano groups.

[0109] In this article, the terms "amine" and "amino" each refer to -NR x R y end base, -N + R x R y R z end base, -NR x -Linking group or -N + R x R y - Linking group, where R x Ry and R z Each is hydrogen or a substituted or unsubstituted alkyl, alkenyl, alkynyl, cycloalkyl, heteroalicyclic (linked to an amine nitrogen via its ring carbon), aryl, or heteroaryl (linked to an amine nitrogen via its ring carbon), as defined herein. Optionally, R x R y and R z It is hydrogen or an alkyl group containing one to four carbon atoms. Optionally, R x and R y (and R) z (if present) is hydrogen. When substituted, R is bonded to the nitrogen atom of the amine. x R y Or R z The carbon atoms in the hydrocarbon moiety are optionally not substituted with oxygen, making R x R y and R z Not, for example, carbonyl, C-carboxyl, or amide groups as defined herein, unless otherwise stated.

[0110] The term "alkoxy" or "alkoxyl" refers to both an -O-alkyl terminal group and an -O-cyclic hydrocarbon terminal group as defined herein, or an -O-alkylene-linking group or an -O-cyclic hydrocarbon-linking group as defined herein.

[0111] "Aryloxy" group refers to both -O-aryl terminal group and -O-heteroaryl terminal group as defined herein, or -O-arylene-linking group as defined herein.

[0112] The "azide" group refers to -N=N + =N - Group.

[0113] The "hydroxyl" group refers to the -OH group.

[0114] The "nitro" group refers to the -NO2 group.

[0115] The "cyano" group refers to -C N-group.

[0116] In some embodiments, the chimera is represented by the structures of formulas IA-1, IA-2, IA-3, IA-4, IA-5, IA-6, IA-7 (1-24), IC-1 (1-27), ID-1 (1-26), ID-2, ID-3, ID-4, and ID-5: In some embodiments, the SUMOylase-binding group is selected from the groups presented in Table 1. In some embodiments, the SUMOylase-binding group binds to SUMO E3 ligase. In some embodiments, the SUMOylase-binding group binds to PIAS1. In some embodiments, the SUMOylase-binding group binds to PIAS4. In some embodiments, the SUMOylase-binding groups in Table 1 bind to PIAS1. In some embodiments, the SUMOylase-binding groups in Table 1 bind to PIAS4.

[0117] Table 1: SUMOylating compounds used as SUMOylating enzyme binding groups in the chimeras of the present invention. In some embodiments, the SUMOylase-binding group in the chimera of the present invention comprises NC(O)-CH2Cl or NC(O)-CH=CH2 (acrylamide), wherein, for example, the chimera of Formula I (as disclosed above); or an acrylamide derivative thereof, as presented below: .

[0118] Similarly, the SUMOylase-binding groups presented in Table 1 are part of the SUTAC chimera of the present invention.

[0119] In some embodiments, this document provides a pharmaceutical composition comprising a SUMOylation-targeting chimera (SUTAC) and a suitable, acceptable carrier, wherein the SUTAC comprises a SUMOylation enzyme-binding group attached to a target protein-binding group via a linker.

[0120] In some embodiments, this document provides a pharmaceutical composition comprising SUTAC as described in detail herein, and a suitable, acceptable carrier.

[0121] Compounds / compositions used In some embodiments, this document provides a SUMOylation-targeting chimera (SUTAC) comprising a SUMOylase-binding group attached to a target protein-binding group via a linker for treating a subject with cancer, improving the condition of the subject, or inhibiting the decline of the subject. In some embodiments, this document provides a SUMOylation-targeting chimera (SUTAC) as described in detail herein for treating a subject with cancer, improving the condition of the subject, or inhibiting the decline of the subject.

[0122] In some embodiments, this document provides methods for treating a subject with cancer, improving the condition of the subject, or inhibiting the decline of the subject, the method comprising administering to the subject a SUMOylation-targeting chimera (SUTAC), the SUTAC comprising a SUMOylase-binding group attached to a target protein-binding group via a linker. In some embodiments, this document provides methods for treating a subject with cancer, improving the condition of the subject, or inhibiting the decline of the subject, the method comprising administering to the subject a SUMOylation-targeting chimera (SUTAC) as described in detail herein.

[0123] In some embodiments, the target protein comprises protein 4 (BRD4) containing a bromo domain. In some embodiments, the target protein comprises an androgen receptor (AR). In some embodiments, the target protein comprises the P300 / CBP protein.

[0124] In some implementations, the target protein includes the estrogen receptor (ER).

[0125] In some implementations, treatment may include directly affecting or curing, inhibiting, suppressing, preventing, or reducing the severity of a disease, disorder, or condition; delaying the onset of a disease, disorder, or condition; reducing symptoms associated with a disease, disorder, or condition; or a combination thereof. Therefore, in some implementations, "treatment" has the same meaning as "ameliorating" and "alleviating," and specifically refers to delaying progression, accelerating remission, inducing remission, enhancing remission, accelerating recovery, reducing the severity of symptoms, reducing the severity of acute exacerbations, reducing the number of symptoms, reducing the incidence of disease-related symptoms, reducing the latency of symptoms, improving symptoms, or a combination thereof.

[0126] As used herein, the terms “administering,” “administer,” or “administration” refer to the delivery of the SUTAC or composition described herein to a subject. In some embodiments, the compositions described herein may be administered parenterally, enterally, or topically. Illustrative examples of parenterally administration include, but are not limited to, intravenous, intramuscular, intraarterial, intrasheath, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, tracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, intraspinal, and intrasternal injections and infusions. Illustrative examples of enteral administration include, but are not limited to, sublingual and oral administration.

[0127] In some embodiments, the SUTAC or composition described herein is administered in a therapeutically effective amount. The terms “effective,” “efficacy,” or “effectiveness” are used herein to refer to the ability of a therapy to achieve a beneficial or desired outcome, such as a clinical result, and therefore, an “effective amount” depends on the context in which it is applied. For example, in the context of administering a SUTAC or composition for treating cancer, an effective amount of the SUTAC or composition is, for example, an amount sufficient to achieve a treatment as defined herein, compared to a response obtained without the administration of the SUTAC or composition. In some embodiments, a therapeutically effective amount is the amount of SUTAC or composition to be delivered that, when administered to a subject with cancer, is sufficient to treat the subject with cancer, improve the subject's condition, or inhibit the subject's decline.

[0128] In some implementations, cancer or tumor includes non-solid tumors. In some implementations, non-solid cancer or tumor includes hematopoietic malignancies, blood cell carcinomas, leukemia, myelodysplastic syndromes, lymphoma, multiple myeloma (plasma cell myeloma), acute promyelocytic leukemia, acute lymphoblastic leukemia, acute myeloid leukemia, chronic myeloid leukemia, Hodgkin lymphoma, non-Hodgkin lymphoma, Burkitt lymphoma (BL), or plasma cell leukemia.

[0129] In some implementations, cancer or tumor includes solid tumors. In some implementations, cancer is selected from the group consisting of: kidney cancer, lung cancer, endometrial / uterine cancer, esophageal cancer, breast cancer, cervical cancer, liver cancer, stomach cancer, esophageal cancer, head and neck cancer, ovarian cancer, skin cancer, bile duct cancer, rhabdomyosarcoma, brain cancer, colon / colorectal cancer, pancreatic cancer, myeloma, neuroblastoma, gastric cancer, sarcoma, thyroid cancer, bladder cancer, bone cancer, or eye cancer.

[0130] In some embodiments, the use of a pharmaceutical composition comprising SUTAC as described herein reduces the number of cancer cells, the size of the tumor, or the amount of cancer in the subject, or any combination thereof, compared to a subject who has not received a pharmaceutical composition comprising SUTAC as described herein. In some embodiments, treating cancer, improving the condition of cancer, or inhibiting the decline of cancer includes reducing tumor size.

[0131] In one implementation, the term "reduction in tumor size," as used herein, is assessed using the Response Evaluation Criteria for Solid Tumors (RECIST). In one implementation, RECIST measures tumor size reduction by measuring the longest dimension of the target lesion. In one implementation, target lesions are selected based on their size (the lesion with the longest diameter) and their suitability for accurate repeat measurements (by imaging techniques or clinically). In one implementation, all other lesions (or disease sites) are identified as non-target lesions and are also recorded at baseline. Measurement of these lesions is not required, but the presence or absence of each lesion is noted throughout follow-up.

[0132] In some embodiments, treating cancer, improving the condition of cancer, or inhibiting the decline of cancer includes reducing metastasis. In some embodiments, treating cancer, improving the condition of cancer, or inhibiting the decline of cancer includes eliminating metastasis. In some embodiments, treating cancer, improving the condition of cancer, or inhibiting the decline of cancer includes increased survival compared to subjects who did not receive a pharmaceutical composition containing SUTAC as described herein.

[0133] In some embodiments of the methods disclosed herein, subjects who received a pharmaceutical composition containing SUTAC as described herein maintained a longer period of disease-free duration than subjects who did not receive a pharmaceutical composition containing SUTAC.

[0134] Skilled professionals will understand that, as used herein, the term “disease-free” can refer to a subject remaining alive for a defined period of time without a recurrence of cancer or tumor, such as approximately 1 year, 2 years, 3 years, 4 years, 5 years, 10 years, or longer from the start of treatment or from the initial diagnosis.

[0135] In some implementations, the subject remains disease-free for at least 1, 2, 3, 4, 5, 6, 7, 8, or 10 years. In some implementations, the subject remains disease-free for at least 1 year. In some implementations, the subject remains disease-free for at least 5 years. In some implementations, the subject remains disease-free for at least 10 years.

[0136] In some embodiments, the methods disclosed herein reduce the tumor burden of cancer or tumors or decrease the incidence of cancer or tumors in subjects who have not received a pharmaceutical composition containing SUTAC. In some embodiments, the methods disclosed herein reduce minimal residual disease, increase remission, increase duration of remission, decrease tumor recurrence rate, prevent metastasis of the tumor or cancer, or decrease the metastasis rate of the tumor or cancer, or any combination thereof.

[0137] In some embodiments, this document provides a SUMOylation-targeting chimera (SUTAC) comprising a SUMOylation enzyme-binding group attached to a target protein-binding group via a linker for treating subjects suffering from obesity, neurodegenerative diseases (e.g., Parkinson's disease, Alzheimer's disease), fibrosis, cardiovascular disease, diabetes, Crohn's disease, osteoporosis, multiple sclerosis (MS), SLE, or non-alcoholic fatty liver disease, improving the condition of said subjects, and inhibiting the decline of said subjects.

[0138] In some implementations, the neurodegenerative disease includes Parkinson's disease. In some implementations, the neurodegenerative disease includes Alzheimer's disease. In some implementations, the diabetes is type II diabetes.

[0139] In some embodiments, administration of SUTAC to subjects reduces target protein degradation compared to untreated subjects. In some embodiments, administration of SUTAC to subjects increases target protein degradation compared to untreated subjects.

[0140] In some implementations, administration of SUTAC to subjects reduces the stabilization of the target protein compared to untreated subjects. In other implementations, administration of SUTAC to subjects increases the stabilization of the target protein compared to untreated subjects.

[0141] In some implementations, administration of SUTAC to a subject reduces target protein activation compared to untreated subjects. In some implementations, administration of SUTAC to a subject increases target protein activation compared to untreated subjects.

[0142] In some implementations, administration of SUTAC to a subject reduces repression of the target protein compared to untreated subjects. In some implementations, administration of SUTAC to a subject increases repression of the target protein compared to untreated subjects.

[0143] In some embodiments, administration of SUTAC to a subject increases the binding of the target protein to other proteins or DNA compared to an untreated subject. In some embodiments, administration of the SUTAC to a subject decreases the binding of the target protein to other proteins or DNA compared to an untreated subject.

[0144] In some implementations, administration of SUTAC to a subject reduces the solubility of the target protein compared to an untreated subject. In some implementations, administration of SUTAC to a subject increases the solubility of the target protein compared to an untreated subject.

[0145] In some embodiments, the terms “increase” or “decrease” used to describe protein state can encompass the state of the protein relative to different times (in some embodiments, this could be a pre-administration time point), different tissues or cells, or in other subjects (such as untreated subjects), and are presented, for example, as a fold change. Those skilled in the art will recognize the tools and methods that can be used to determine protein levels (amounts) and / or their state (i.e., the level of protein activation or stabilization).

[0146] Unless otherwise defined herein, scientific and technical terms used in connection with this application shall have the meanings commonly understood by one of ordinary skill in the art. Furthermore, unless the context requires otherwise, singular terms shall include plural terms, and plural terms shall include singular terms.

[0147] In this disclosure, the singular forms “a,” “an,” and “the” include plural indicators, and references to a particular numerical value include at least that particular value, unless the context clearly indicates otherwise. As used herein, the term “more than one” means more than one. When indicating a range of values, another embodiment includes from one particular value and / or to another particular value. Similarly, when a value is expressed as an approximation using the antecedent “about,” it should be understood that the particular value forms another embodiment. All ranges are inclusive and composable. In some embodiments, the term “about” means a deviation from the indicated number or range of numbers between 0.0001% and 5%. In some embodiments, the term “about” means a deviation from the indicated number or range of numbers between 1% and 10%. In some embodiments, the term “about” means a deviation from the indicated number or range of numbers up to 25%. The term "comprises" means that it encompasses all the listed elements, but may also include additional unnamed elements, and it is used interchangeably with the terms "encompasses," "includes," or "contains," which have all the same nature and meaning. The term "consisting of" means that it consists of the listed elements or steps, and it is used interchangeably with the term "composed of," which has all the same nature and meaning.

[0148] It should be understood that the disclosure presented herein is not limited to the specific methods, schemes, reagents, and embodiments described herein. The terminology and embodiments used herein are for the purpose of describing particular implementations and providing guidance to those skilled in the art, and are not intended to limit the scope of the disclosure presented herein.

[0149] Example Example 1: Synthesis of SUTAC (AR-targeted SUTAC) of Formula IA-1 Scheme 1: Synthesis scheme: (a) Cs2CO3 / Pd(OAc)2 / BINAP / Toluene, 110℃ (b) HCl / Dioxane / DCM / RT (c) EDCI / HOBt / TEA / DMF / 25℃ (d) NaOH / MeOH / 25℃ (e) EDCI / HOBt / TEA / DMF / 25℃ (f) NaHCO3 / THF / H2O / 0℃ 4-[2-[2-[2-(tert-butoxycarbonylamino)ethoxy]ethoxy]ethylamino]methyl benzoate: A mixture of N-[2-[2-(2-aminoethoxy)ethoxy]ethyl]carbamate tert-butyl ester (2 g, 1 equivalent), methyl 4-iodobenzoate (2.53 g, 1.2 equivalent), Cs₂CO₃ (7.87 g, 3 equivalent), Pd(OAc)₂ (180.82 mg, 0.1 equivalent), and BINAP (1.00 g, 0.2 equivalent) in toluene (40 mL) was degassed and purged three times with N₂. The mixture was then stirred at 110 °C under N₂ atmosphere for 6 h. The residue was purified by preparative HPLC (NaHCO₃ conditions) to provide the compound. The product was given as a yellow oil (1.3 g, 42.20% yield). LC-MS (m / z): Calculated: 382.46; Found: 381.1 [MH] + .

[0150] 1 H NMR (400 MHz, DMSO) δ 7.68 (d, J = 8.8 Hz, 2H), 6.77 (br t, J =5.5 Hz, 1H), 6.62 (d, J = 8.9 Hz, 2H), 6.53 (t, J = 5.6 Hz, 1H), 3.74 (s,3H), 3.58 - 3.49 (m, 6H), 3.39 - 3.39 (m, 2H), 3.29 - 3.23 (m, 2H), 3.09 -3.02 (m, 2H), 1.37 (s, 9H).

[0151] 4-((2-(2-(2-aminoethoxy)ethoxy)ethyl)amino)benzoate methyl ester: A solution of methyl 4-[2-[2-[2-(tert-butoxycarbonylamino)ethoxy]ethoxy]ethylamino]benzoate (1.3 g, 1 equivalent) was added to a solution of HCl / dioxane (2 M, 1.70 mL). The mixture was stirred at 25 °C for 1 h. The reaction mixture was concentrated under reduced pressure to remove the solvent. The product was given as a brown solid (1.2 g, crude, HCl). LC-MS (m / z): Calculated: 282.34; Found: 283.1 [M+H] + .

[0152] 4-[2-[2-[2-[(2-aminothiophene-3-carbonyl)amino]ethoxy]ethoxy]ethylamino]benzoic acid ester: HOBt (635.78 mg, 1.5 equivalent) and EDCI (901.99 mg, 1.5 equivalent), TEA (1.27 g, 1.75 mL, 4 equivalent) were added to a solution of 2-aminothiophene-3-carboxylic acid (449.08 mg, 1 equivalent) in DMF (10 mL). Then, methyl 4-[2-[2-(2-aminoethoxy)ethoxy]ethylamino]benzoate (1 g, 1 equivalent, HCl) was added at 25 °C. The mixture was stirred at 25 °C for 2 h. The residue was purified by preparative HPLC (FA conditions) to provide the compound. The product was given as a yellow oil (560 mg, 43.81% yield). LC-MS (m / z): Calculated: 407.49; Found: 408.1 [M+H] + .

[0153] 1 H NMR (400 MHz, DMSO) δ 7.78 (d, J = 8.9 Hz, 2H), 6.96 (d, J = 5.9Hz, 1H), 6.62 (d, J = 8.9 Hz, 2H), 6.23 (d, J = 5.9 Hz, 1H), 4.60 (br s, 1H), 3.83 (s, 3H), 3.72 - 3.59 (m, 8H), 3.53 - 3.47 (m, 2H), 3.36 - 3.32 (m, 5H).

[0154] 4-[2-[2-[2-[(2-aminothiophene-3-carbonyl)amino]ethoxy]ethoxy]ethylamino]benzoic acid: NaOH (215.94 mg, 4 equivalents) was added to a solution of methyl 4-[2-[2-[2-[(2-aminothiophene-3-carbonyl)amino]ethoxy]ethoxy]ethylamino]benzoate (550 mg, 1 equivalent) in THF (1 mL), MeOH (1 mL), and H₂O (1 mL). The mixture was stirred at 25 °C for 2 h. The residue was purified by preparative HPLC (NaHCO₃ conditions) to provide a yellow solid (133 mg, 25.04% yield). LC-MS (m / z): Calculated: 393.46; Found: 394.0 [M+H] + .

[0155] 1 H NMR (400 MHz, DMSO) δ 7.76 (t,J = 5.6 Hz, 1H), 7.68 - 7.63 (m,3H), 7.20 (s, 2H), 7.08 (d, J = 5.9 Hz, 1H), 6.55 (d, J = 8.8 Hz, 3H), 6.25 (d, J = 5.9 Hz, 1H), 6.19 (br t, J = 5.4 Hz, 2H), 3.53 - 3.46 (m, 4H), 3.36 -3.20 (m, 6H).

[0156] 2-Amino-N-[2-[2-[2-[4-[[3-(3-chloro-4-cyano-phenoxy)-2,2,4,4-tetramethyl-cyclobutyl] [Carbamoyl]Aniline]Ethoxy]Ethoxy]Ethio]Thiophene-3-carboxamide: To a solution of 4-[2-[2-[2-[(2-aminothiophene-3-carbonyl)amino]ethoxy]ethoxy]ethylamino]benzoic acid (133 mg, 1 equivalent) in DMF (2 mL), EDCI (97.20 mg, 1.5 equivalent) and TEA (102.61 mg, 141.15 μL, 3 equivalent), HOBt (68.51 mg, 1.5 equivalent) were added. Then, 4-(3-amino-2,2,4,4-tetramethyl-cyclobutoxy)-2-chlorobenzonitrile (94.23 mg, 1 equivalent) was added. The mixture was stirred at 25 °C for 2 h. The residue was purified by preparative HPLC (FA conditions) to provide a yellow solid (150 mg, 67.83% yield). LC-MS (m / z): Calculated: 654.22; Found: 655.1 [M+H] + .

[0157] 1 H NMR (400 MHz, DMSO) δ 7.95 (d, J = 8.8 Hz, 1H), 7.78 (br t, J =5.7 Hz, 1H), 7.69 (d, J = 8.8 Hz, 2H), 7.39 (d, J = 9.1 Hz, 1H), 7.28 - 7.20(m, 3H), 7.11 (d, J = 5.9 Hz, 1H), 7.04 (dd, J = 2.4, 8.8 Hz, 1H), 6.65 (d, J= 8.8 Hz, 2H), 6.29 (d, J = 5.8 Hz, 1H), 6.22 (s, 1H), 4.35 (s, 1H), 4.08 (d, J = 9.1 Hz, 1H), 3.63 - 3.57 (m, 6H), 3.55 - 3.50 (m, 2H), 3.36 (br d, J =6.0 Hz, 2H), 3.28 (q, J = 5.7 Hz, 2H), 1.25 (s, 6H), 1.16 (s, 6H).

[0158] 2-[(2-chloroacetyl)amino]-N-[2-[2-[2-[4-[[3-(3-chloro-4-cyano-phenoxy)-2,2,4,4- Tetramethyl-cyclobutyl]carbamoyl]aniline]ethoxy]ethoxy]ethyl]thiophene-3-carboxamide: TEA (13.92 mg, 19.15 μL, 3 equivalents) was added to a solution of 2-amino-N-[2-[2-[2-[4-[[3-(3-chloro-4-cyano-phenoxy)-2,2,4,4-tetramethyl-cyclobutyl]carbamoyl]aniline]ethoxy]ethoxy]ethyl]thiophene-3-carboxamide (30 mg, 1 equivalent) in DCM (1 mL). Then, 2-chloroacetyl chloride (5.18 mg, 3.65 μL, 1 equivalent) was added at 0 °C. The mixture was stirred at 0 °C for 1 h. The reaction mixture was concentrated under reduced pressure to remove the solvent. The residue was purified by preparative HPLC (TFA conditions) to provide the compound. The product was given as a blue solid (2.52 mg, 7.37% yield). LC-MS (m / z): Calculated: 730.7; Found: 731.1 [M+H] + .

[0159] 1 H NMR (400 MHz, CDCl3) δ 12.82 (br s, 1H), 7.59 (dd, J = 8.6, 16.2Hz, 3H), 7.02 (d, J = 5.8 Hz, 1H), 6.98 (d, J = 2.4 Hz, 1H), 6.82 (dd, J =2.4, 8.8 Hz, 1H), 6.77 (d, J = 5.9 Hz, 1H), 6.70 - 6.63 (m, 1H), 6.60 (d, J=8.8 Hz, 2H), 6.12 (br d, J = 8.3 Hz, 1H), 4.25 (s, 2H), 4.15 (d, J = 8.3 Hz,1H), 4.06 (s, 1H), 3.76 - 3.71 (m, 2H), 3.70 - 3.65 (m, 8H), 3.35 (t, J = 5.1Hz, 2H), 1.27 (s, 6H), 1.22 (s, 6H).

[0160] Example 2: Synthesis of SUTAC (AR-targeted SUTAC) of Formula IA-2 Scheme 2: Synthesis Scheme: (a) DMSO / DIEA / 120℃ (b) HCl / Dioxane / DCM / RT (c) EDCI / HOBt / TEA / DMF / 25℃ (d) NaOH / MeOH / THF / H2O / 25℃ (e) EDCI / HOBt / TEA / DMF / 25℃ (f) DCM / TEA / 0℃ 4-[8-(tert-butoxycarbonylamino)octylamino]methyl benzoate: DIEA (1.59 g, 12.28 mmol) was added to a solution of N-(8-aminooctyl)carbamate tert-butyl ester (1 g, 4.09 mmol) and methyl 4-fluorobenzoate (630.75 mg, 4.09 mmol) in DMSO (2 mL). The mixture was stirred at 120 °C for 18 h. The residue was purified by preparative HPLC (NH4HCO3 conditions) to provide methyl 4-[8-(tert-butoxycarbonylamino)octylamino]benzoate (500 mg, 32.28% yield) as a white solid. LC-MS (m / z): Calculated: 378.51; Found: 379.5 [M+H] + .

[0161] 1H NMR (400 MHz, DMSO) δ 7.67 (d, J = 8.8 Hz, 2H), 6.74 (br t, J =5.0 Hz, 1H), 6.56 (d, J = 8.8 Hz, 2H), 6.48 (br t, J = 5.2 Hz, 1H), 3.73 (s,3H), 3.07-3.01 (m, 2H), 2.88 (q, J = 6.5 Hz, 2H), 1.53 (quin, J = 7.0 Hz,2H), 1.39-1.31 (m, 14H), 1.25 (br s, 6H).

[0162] 4-(8-aminooctylamino)benzoate methyl benzoate: DCM (5 mL) was added to a solution of methyl 4-[8-(tert-butoxycarbonylamino)octylamino]benzoate (500 mg, 1.32 mmol) in HCl / dioxane (4 M, 6.06 mL). The mixture was stirred at 15 °C for 1 h. The reaction mixture was concentrated under reduced pressure to remove the solvent. The product was given as a yellow solid (415 mg, 99.78% yield, HCl). LC-MS (m / z): Calculated: 278.40; Found: 279.3 [M+H] + .

[0163] 4-[8-[(2-aminothiophene-3-carbonyl)amino]octylamino]methyl benzoate: HOBt (291.23 mg, 2.16 mmol), EDCI (413.17 mg, 2.16 mmol), and TEA (436.18 mg, 4.31 mmol) were added to a solution of methyl 4-(8-aminooctylamino)benzoate (400 mg, 1.44 mmol), 2-aminothiophene-3-carboxylic acid (205.70 mg, 1.44 mmol), and DMSO (4 mL). The mixture was stirred at 25 °C for 1 h. The product was obtained as a white solid (265 mg, 45.70% yield). LC-MS (m / z): Calculated: 403.54; Found: 404.3 [M+H] + .

[0164] 4-[2-[2-[2-[(2-aminothiophene-3-carbonyl)amino]ethoxy]ethoxy]ethylamino]benzoic acid: NaOH (103.08 mg, 2.58 mmol) was added to a solution of methyl 4-[8-[(2-aminothiophene-3-carbonyl)amino]octylamino]benzoate (260 mg, 644.30 μmol) in MeOH (2 mL), H₂O (1 mL), and THF (2 mL). The mixture was stirred at 25 °C for 1 h. The residue was purified by preparative HPLC (NH₄HCO₃ conditions) to provide 4-[8-[(2-aminothiophene-3-carbonyl)amino]octylamino]benzoic acid (45 mg, 17.93% yield) as a yellow solid. LC-MS (m / z): Calculated: 389.51; Found: 389.6 [M+H] + .

[0165] 2-Amino-N-[8-[4-[[3-(3-chloro-4-cyano-phenoxy)-2,2,4,4-tetramethyl-cyclobutyl]aminomethyl Acyl]Aniline]Octyl]Thiophene-3-carboxamide: Add EDCI (29.53 mg, 154.04 μmol), HOBt (20.81 mg, 154.04 μmol), and TEA (41.57 mg, 410.77 μmol) to a solution of 4-[8-[(2-aminothiophene-3-carbonyl)amino]octylamino]benzoic acid (40 mg, 102.69 μmol), 4-(3-amino-2,2,4,4-tetramethyl-cyclobutoxy)-2-chlorobenzonitrile (28.63 mg, 102.69 μmol) in DCM (1 mL). Stir the mixture at 25 °C for 1 h. The residue was purified by preparative HPLC (FA conditions) to provide 2-amino-N-[8-[4-[[3-(3-chloro-4-cyano-phenoxy)-2,2,4,4-tetramethyl-cyclobutyl]carbamoyl]anilino]octyl]thiophene-3-carboxamide as a brown solid (40 mg, 59.90% yield). LC-MS (m / z): Calculated: 650.28; Found: 651.1 [M+H] + .

[0166] 2-[(2-chloroacetyl)amino]-N-[8-[4-[[3-(3-chloro-4-cyano-phenoxy)-2,2,4,4-tetramethyl- Cyclobutyl]carbamoyl]aniline]octyl]thiophene-3-carboxamide: TEA (7.00 mg, 69.20 μmol) was added to a solution of 2-amino-N-[8-[4-[[3-(3-chloro-4-cyano-phenoxy)-2,2,4,4-tetramethyl-cyclobutyl]carbamoyl]anilino]octyl]thiophene-3-carboxamide (15 mg, 23.07 μmol) and 2-chloroacetyl chloride (2.61 mg, 23.07 μmol) in DCM (1 mL). The mixture was stirred at 0 °C for 1 h. The reaction mixture was concentrated under reduced pressure to remove the solvent. The residue was purified by preparative HPLC (FA conditions) to provide 2-[(2-chloroacetyl)amino]-N-[8-[4-[[3-(3-chloro-4-cyano-phenoxy)-2,2,4,4-tetramethyl-cyclobutyl]carbamoyl]anilino]octyl]thiophene-3-carboxamide (3.23 mg) as a white solid. LC-MS (m / z): Calculated value: 726.76; Measured value: 727.5 [M+H] + .

[0167] 1 H NMR (400 MHz, CDCl3) δ 12.86 (br s, 1H), 7.63 (br d J = 8.6 Hz, 2H), 7.57 (d, J = 8.6 Hz, 1H), 7.00-6.96 (m, 2H), 6.86 (d, J = 5.9 Hz, 1H), 6.81 (dd, J = 2.3, 8.7 Hz, 1H), 6.59 (br d, J = 8.8 Hz, 2H), 6.08 (br d, J =8.3 Hz, 1H), 5.98 (br s, 1H), 4.26 (s, 2H), 4.15 (d, J = 8.0 Hz, 1H), 4.05(s, 1H), 3.49 - 3.41 (m, 2H), 3.17 (t, J = 6.9 Hz, 2H), 1.69-1.65 (m, 2H), 1.44-1.33 (m, 10H), 1.27 (s, 6H), 1.22 (s, 6H).

[0168] Example 3: Synthesis of SUTAC (AR-targeted SUTAC) of Formula IA-3 Scheme 3: Synthesis Scheme: (a) DMSO / DIEA / 110℃ (b) HCl / Dioxane / DCM / RT (c) EDCI / HOBt / TEA / DMF / 25℃ (d) NaOH, MeOH / THF / H2O / 25℃ (e) EDCI / HOBt / TEA / DMF / 25℃ (f) DCM / TEA / 0℃ 4-[2-[2-[2-[2-[2-[2-(tert-butoxycarbonylamino)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]eth Methyl benzoate [oxy]ethylamino]benzoate : To N-[2-[2-[2-[2-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethyl]carbamate tert-butyl ester (1.4 g, 1 equivalent) 、 Methyl 4-iodobenzoate (1.16 g, 1.2 equivalents) was added to a solution of toluene (1 mL) with Cs₂CO₃ (3.60 g, 3 equivalents), Pd(OAc)₂ (82.61 mg, 0.1 equivalents), and BINAP (458.24 mg, 0.2 equivalents). The mixture was stirred at 110 °C for 6 h. The residue was purified by preparative HPLC (NH₄HCO₃ conditions) to provide a yellow oil (900 mg, 1.75 mmol, 47.53% yield). LC-MS (m / z): Calculated: 514.29; Found: 515.1 [M+H] + .

[0169] 4-[2-[2-[2-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethylamino] Methyl benzoate: To a solution of methyl 4-[2-[2-[2-[2-[2-[2-(tert-butoxycarbonylamino)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethylamino]benzoate (700 mg, 1 equivalent) in DCM (5 mL), HCl / dioxane (2 M, 14 mL, 20.58 equivalents) was added. The mixture was stirred at 25 °C for 1 h. The reaction mixture was concentrated under reduced pressure to give a yellow oil (600 mg, 97.81% yield, HCl). LC-MS (m / z): Calculated: 414.24; Found: 415.3 [M+H] + .

[0170] 1 H NMR (400 MHz, DMSO) δ 8.11 - 7.79 (m, 3H), 7.68 (d, J= 8.9 Hz,2H), 6.68 - 6.56 (m, 2H), 3.74 (s, 3H), 3.55 - 3.47 (m, 19H), 3.26 (t, J =5.7 Hz, 2H), 2.95 (qd, J = 5.3, 10.8 Hz, 2H).

[0171] 4-[2-[2-[2-[2-[2-[2-[(2-aminothiophene-3-carbonyl)amino]ethoxy]ethoxy]ethoxy]eth Methyl benzoate: [oxy]ethoxy]ethylamino]benzoate HOBt (269.68 mg, 1.5 equivalents) and EDCI (382.59 mg, 1.5 equivalents) and TEA (538.53 mg, 740.76 μL, 4 equivalents) were added to a solution of methyl 4-[2-[2-[2-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethylamino]benzoate (600 mg, 1 equivalent, HCl), 2-aminothiophene-3-carboxylic acid (190.48 mg, 1 equivalent) in DMF (1.5 mL). The mixture was stirred at 25 °C for 2 h. LCMS (EW42073-456-P1A1) showed a peak of 27% of the desired compound. The residue was purified by preparative HPLC (NH4HCO3 conditions) to provide a yellow oil (400 mg, 55.71% yield). LC-MS (m / z): Calculated value: 539.23; Measured value: 540.0 [M+H] + .

[0172] 1 H NMR (400 MHz, DMSO) δ 7.75 - 7.65 (m, 3H), 7.19 (s, 2H), 7.07 (d, J = 5.9 Hz, 1H), 6.62 (d, J = 8.9 Hz, 2H), 6.53 (t, J = 5.6 Hz, 1H), 6.25 (d, J = 5.8 Hz, 1H), 3.74 (s, 3H), 3.58 - 3.42 (m, 23H).

[0173] 4-[2-[2-[2-[2-[2-[2-[(2-aminothiophene-3-carbonyl)amino]ethoxy]ethoxy]ethoxy]eth [Oxygen group] [Ethoxy group] [Ethylamino group] [Benzoic acid] Methyl benzoate (400 mg, 1 equivalent) was added to a solution of 4-[2-[2-[2-[2-[2-[(2-aminothiophene-3-carbonyl)amino]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethylamino]benzoate (118.59 mg, 4 equivalents) of NaOH in MeOH (0.5 mL), THF (0.5 mL), and H₂O (0.5 mL). The residue was purified by preparative HPLC (NH₄HCO₃ conditions) to provide a red oil (210 mg, 53.90% yield). LC-MS (m / z): Calculated: 525.21; Found: 526.1 [M+H] + .

[0174] 1 H NMR (400 MHz, DMSO) δ 7.81 - 7.74 (m, 1H), 7.69 (d, J = 8.8 Hz,2H), 7.22 (s, 2H), 7.11 (d, J = 5.9 Hz, 1H), 6.62 (d, J = 8.8 Hz, 2H), 6.38(br t, J = 5.5 Hz, 1H), 6.28 (d, J = 5.8 Hz, 1H), 3.62 - 3.56 (m, 10H), 3.56 - 3.53 (m, 14H).

[0175] 2-Amino-N-[2-[2-[2-[2-[2-[2-[4-[[3-(3-chloro-4-cyano-phenoxy)-2,2,4,4-tetramethyl [[-cyclobutyl]carbamoyl]aniline]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethyl]thiophene-3-methyl Amide: Add (3-amino-2,2,4,4-tetramethyl-cyclobutoxy)-2-chlorobenzonitrile (106.08 mg, 1 equivalent) and TEA (115.51 mg, 158.89 μL, 3 equivalents) and HOBt (77.12 mg, 1.5 μL, 1.5 μL) to a solution of 4-[2-[2-[2-[2-[2-[2-[2-[2-[(2-aminothiophene-3-carbonyl)amino]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethylamino]benzoic acid (200 mg, 1 equivalent) in DMF (1 mL) to a solution of 4-[2-[2-[2-[2-[2-[2-[2-[(2-aminothiophene-3-carbonyl)amino]ethoxy]ethoxy]ethoxy]ethoxy]ethylamino]benzoic acid (200 mg, 1 equivalent) in DMF (1 mL) to a solution of 4 ... eq), EDCI (109.42 mg, 1.5 equivalents). The mixture was stirred at 25 °C for 2 h. The residue was purified by preparative HPLC (NH4HCO3 conditions) to provide a brown solid (190 mg, 63.50% yield). LC-MS (m / z): Calculated: 785.32; Found: 786.2 [M+H] + .

[0176] 1 H NMR (400 MHz, DMSO) δ 7.90 (d, J = 8.6 Hz, 1H), 7.72 (br t, J =5.4 Hz, 1H), 7.65 (br d, J = 8.5 Hz, 2H), 7.34 (br d, J = 9.3 Hz, 1H), 7.25 -7.11 (m, 3H), 7.06 (d, J = 5.9 Hz, 1H), 7.00 (dd, J = 1.9, 8.8 Hz, 1H), 6.61(br d, J = 8.5 Hz, 2H), 6.24 (d, J = 5.9 Hz, 1H), 6.17 (br t, J = 5.3 Hz,1H), 4.31 (s, 1H), 4.04 (br d, J = 9.1 Hz, 1H), 3.63 - 3.40 (m, 22H), 3.24(br d, J = 5.5 Hz, 2H), 1.21 (s, 6H), 1.11 (s, 6H).

[0177] 2-[(2-chloroacetyl)amino]-N-[2-[2-[2-[2-[2-[2-[4-[[3-(3-chloro-4-cyano-phenoxy)- 2,2,4,4-Tetramethyl-cyclobutyl]carbamoyl]aniline]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]eth [By]thiophene-3-carboxamide: TEA (7.72 mg, 0.62 μL, 3 equivalents) was added to a solution of 2-amino-N-[2-[2-[2-[2-[2-[4-[[3-(3-chloro-4-cyano-phenoxy)-2,2,4,4-tetramethylcyclobutyl]carbamoyl]aniline]ethoxy]ethoxy]ethoxy]ethoxy]ethyl]thiophene-3-carboxamide (20 mg, 1 equivalent) and 2-chloroacetyl chloride (2.87 mg, 2.03 μL, 1 equivalent) in DCM (2 mL). The mixture was stirred at 0 °C for 1 h. The residue was purified by preparative HPLC (TFA conditions) to provide a white solid (4.7 mg, 20.69% yield). LC-MS (m / z): Calculated: 861.29; Found: 862.1 [M+H] + .

[0178] 1 H NMR (400 MHz, DMSO) δ 12.93 (s, 1H), 7.62 (d, J = 8.5 Hz, 2H), 7.57 (d, J = 8.6 Hz, 1H), 7.54 - 7.46 (m, 1H), 7.29 (d, J = 5.9 Hz, 1H), 6.97 (d, J = 2.4 Hz, 1H), 6.83 - 6.79 (m, 2H), 6.67 (br d, J = 8.1 Hz, 2H), 6.13(br d, J = 8.3 Hz, 1H), 4.25 (s, 2H), 4.15 (d, J = 8.3 Hz, 1H), 4.05 (s, 1H), 3.69 - 3.62 (m, 22H), 3.32 (t, J = 5.0 Hz, 2H), 1.27 (s, 6H), 1.22 (s, 6H).

[0179] Example 4: Synthesis of SUTAC of Formula IA-4 (BRD4-targeted SUTAC) Scheme 4: Synthesis Scheme: (a) HOBt / EDCI / DIEA / DMF / RT (b) TEA / ETOH / 68℃ (c) LiOH / 25℃ (d) HATU / DIEA / DMF / 25℃ (e) DCM / TEA / 0℃ 2-[2-[2-[(2-cyanoacetyl)amino]ethoxy]ethoxy]tert-butyl acetate: EDCI (2.19 g, 2.5 equivalents), HOBt (924.33 mg, 1.5 equivalents), and DIEA (1.77 g, 2.38 mL, 3 equivalents) were added to a solution of 2-[2-(2-aminoethoxy)ethoxy]tert-butyl acetate (1 g, 1 equivalent), 2-cyanoacetic acid (504.29 mg, 1.3 equivalents) in DMF (10 mL). The mixture was stirred at 20 °C for 16 h. The residue was purified by preparative HPLC (NH4HCO3 conditions) to provide a yellow oil (500 mg, 38.29% yield). LC-MS (m / z): Calculated: 286.15; Found: 287.1 [M+H] + .

[0180] 2-[2-[2-[(2-aminothiophene-3-carbonyl)amino]ethoxy]ethoxy]tert-butyl acetate: 2-[2-[2-[(2-cyanoacetyl)amino]ethoxy]ethoxy]tert-butyl acetate (500 mg, 1 equivalent) 、 1,4-Dithiane-2,5-diol (265.84 mg, 1 equivalent) was added to a solution of EtOH (3 mL) with TEA (353.41 mg, 486.12 μL, 2 equivalents). The mixture was stirred at 68 °C for 10 h. The reaction mixture was concentrated under reduced pressure to remove the solvent. The residue was purified by preparative HPLC (NH4HCO3 conditions) to provide a yellow oil (300 mg, 49.88% yield). LC-MS (m / z): Calculated: 342.16; Found: 341.1 [MH] + .

[0181] 2-[2-[2-[(2-aminothiophene-3-carbonyl)amino]ethoxy]ethoxy]acetic acid: LiOH·H₂O (36.55 mg, 3 equivalents) was added to a solution of 2-[2-[2-[(2-aminothiophene-3-carbonyl)amino]ethoxy]ethoxy]tert-butyl acetate (100 mg, 1 equivalent) in THF (1 mL), MeOH (1 mL), and H₂O (1 mL). The mixture was stirred at 25 °C for 3 h. The reaction mixture was concentrated under reduced pressure to remove the solvent. The residue was purified by preparative HPLC (NH₄HCO₃ conditions) to provide a yellow oil (60 mg, 71.68% yield). LC-MS (m / z): Calculated: 288.08; Found: 289.0 [M+H] + .

[0182] 1 H NMR (400 MHz, DMSO) δ 7.88 (br t, J = 5.6 Hz, 1H), 7.17 (br s,2H), 7.12 (d, J = 5.9 Hz, 1H), 6.23 (d, J= 5.9 Hz, 1H), 3.67 (s, 3H), 3.36 -3.28 (m, 7H).

[0183] 2-Amino-N-[2-[2-[2-[2-[4-[(Z)-1-(4-hydroxyphenyl)-2-phenyl-but-1-enyl]phenoxy] [Ethyl-methyl-amino]-2-oxo-ethoxy]ethoxy]ethyl]thiophene-3-carboxamide: HATU (98.91 mg, 1.5 equivalent) and DIEA (67.24 mg, 90.62 μL, 3 equivalent) were added to a solution of 2-[2-[2-[(2-aminothiophene-3-carbonyl)amino]ethoxy]ethoxy]acetic acid (50 mg, 1 equivalent), 4-[(Z)-1-[4-[2-(methylamino)ethoxy]phenyl]-2-phenyl-but-1-enyl]phenol (64.77 mg, 1 equivalent) in DMF (1 mL). The mixture was stirred at 25 °C for 2 h. The residue was purified by preparative HPLC (NH4HCO3 conditions) to provide a yellow solid (13 mg, 11.64% yield). LC-MS (m / z): Calculated: 643.27; Found: 644.1 [M+H] + .

[0184] 2-[(2-chloroacetyl)amino]-N-[2-[2-[2-[2-[4-[(Z)-1-(4-hydroxyphenyl)-2-phenyl-but- 1-Alkenyl]phenoxy]ethyl-methyl-amino]-2-oxo-ethoxy]ethoxy]ethyl]thiophene-3-carboxamide: TEA (6.13 mg, 8.43 μL, 3 equivalents) was added to a solution of 2-amino-N-[2-[2-[2-[2-[4-[(Z)-1-(4-hydroxyphenyl)-2-phenyl-but-1-enyl]phenoxy]ethyl-methyl-amino]-2-oxo-ethoxy]ethoxy]ethyl]thiophene-3-carboxamide (13 mg, 1 equivalent) and 2-chloroacetyl chloride (2.28 mg, 1.61 μL, 1 equivalent) in DCM (1 mL). The mixture was stirred at 0 °C for 1 h. The reaction mixture was concentrated under reduced pressure to remove the solvent. The residue was purified by preparative HPLC (FA conditions) to provide an off-white solid (4.47 mg, 30.73% yield). LC-MS (m / z): Calculated: 719.24; Found: 720.2 [M+H] + .

[0185] 1 H NMR (400 MHz, DMSO) δ 7.34 (dd, J = 3.3, 5.8 Hz, 1H), 7.17 - 7.05(m, 5H), 7.01 (d, J = 8.4 Hz, 2H), 6.89 (d, J = 5.6 Hz, 1H), 6.80 - 6.70 (m,4H), 6.53 (d, J = 8.5 Hz, 2H), 4.34 (d, J = 19.3 Hz, 2H), 4.29 - 4.17 (m,2H), 3.98 (br d, J = 5.4 Hz, 2H), 3.68 - 3.51 (m, 10H), 3.04 - 2.84 (m, 3H), 2.48 (q, J = 7.3 Hz, 2H), 0.90 (t, J = 7.4 Hz, 3H).

[0186] Example 5: Synthesis of SUTAC of Formula IA-5 (BRD4-targeted SUTAC) Scheme 5: Synthesis scheme: (a) EDCI / HOBt / DIEA / DMF, RT (b) TEA / EtOH, 70℃ (c) LiOH / MeOH / THF / H2O (d) EDCI / HOBt / DIEA / DMF, RT (e) NaHCO3,THF / H2O, 0℃ 8-[(2-cyanoacetyl)amino]tert-butyl octanoate: EDCI (2.23 g, 2.5 equivalence) and HOBt (941.27 mg, 1.5 equivalence), DIEA (1.80 g, 2.43 mL, 3 equivalence) were added to a solution of 2-cyanoacetic acid (513.53 mg, 1.3 equivalence) in DMF (10 mL), followed by the addition of tert-butyl 8-aminooctanoate (1 g, 1 equivalence). The mixture was stirred at 25 °C for 18 h. The residue was purified by preparative HPLC (FA conditions) to provide a yellow oil (500 mg, 38.13% yield). LC-MS (m / z): Calculated: 282.19; Found: 283.0 [M+H] + .

[0187] 8-[(2-aminothiophene-3-carbonyl)amino]tert-butyl octanoate: TEA (358.35 mg, 492.91 μL, 2 equivalents) was added to a solution of tert-butyl 8-[(2-cyanoacetyl)amino]octanoate (500 mg, 1 equivalent) and 1,4-dithiaran-2,5-diol (269.56 mg, 1 equivalent) in EtOH (5 mL). The mixture was stirred at 68 °C for 10 h. The reaction mixture was concentrated under reduced pressure to remove the solvent. The residue was purified by preparative HPLC (NH4HCO3 conditions) to provide a yellow oil (300 mg, 49.76% yield). LC-MS (m / z): Calculated: 340.18; Found: 341.0 [M+H] + .

[0188] 8-[(2-aminothiophene-3-carbonyl)amino]octanoic acid: LiOH·H₂O (73.95 mg, 3 equivalents) was added to a solution of tert-butyl 8-[(2-aminothiophene-3-carbonyl)amino]octanoate (200 mg, 1 equivalent) in THF (1.5 mL), MeOH (1.5 mL), and H₂O (1.5 mL). The mixture was stirred at 25 °C for 3 h. The reaction mixture was concentrated under reduced pressure to remove the solvent. The residue was purified by preparative HPLC (NH₄HCO₃ conditions) to provide a yellow oil (95 mg, 56.87% yield). LC-MS (m / z): Calculated: 284.12; Found: 307.1 [M+Na] + .

[0189] 2-Amino-N-[8-[2-[4-[(Z)-1-(4-hydroxyphenyl)-2-phenyl-but-1-enyl]phenoxy]ethyl-methyl [[-amino]-8-oxo-octyl]thiophene-3-carboxamide: Add HATU (190.53 mg, 1.5 equivalents) and DIEA (129.53 mg, 174.57 μL, 3 equivalents) to a solution of 8-[(2-aminothiophene-3-carbonyl)amino]octanoic acid (95 mg, 1 equivalent) in DMF (1 mL). ， Then 4-[(Z)-1-[4-[2-(methylamino)ethoxy]phenyl]-2-phenyl-but-1-enyl]phenol (124.77 mg, 1 equivalent) was added. The mixture was stirred at 25 °C for 2 h. The residue was purified by preparative HPLC (NH4HCO3 conditions) to provide a yellow solid (80 mg, 37.43% yield). LC-MS (m / z): Calculated: 639.31; Found: 638.3 [MH] + .

[0190] 2-[(2-chloroacetyl)amino]-N-[8-[2-[4-[(Z)-1-(4-hydroxyphenyl)-2-phenyl-but-1-enyl] [Phenoxy]ethyl-methyl-amino]-8-oxo-octyl]thiophene-3-carboxamide: TEA (5.22 mg, 7.18 μL, 3 equivalents) was added to a solution of 2-amino-N-[8-[2-[4-[(Z)-1-(4-hydroxyphenyl)-2-phenyl-but-1-enyl]phenoxy]ethyl-methyl-amino]-8-oxo-octyl]thiophene-3-carboxamide (11 mg, 1 equivalent) in DCM (1 mL), followed by the addition of 2-chloroacetyl chloride (1.94 mg, 1.37 μL, 1 equivalent) at 0 °C. The mixture was stirred at 0 °C for 1 h. The reaction mixture was concentrated under reduced pressure to remove the solvent. The residue was purified by preparative HPLC (FA conditions) to provide the product as a grayish-white solid (5.16 mg, 41.06% yield). LC-MS (m / z): Calculated: 715.28; Found: 716.2 [M+H] + .

[0191] 1 H NMR (400 MHz, DMSO-d6) δ 7.33 (dd, J = 2.5, 5.9 Hz, 1H), 7.17 -6.97 (m, 7H), 6.94 (dd, J = 3.5, 5.8 Hz, 1H), 6.81 - 6.68 (m, 4H), 6.53 (brd, J = 7.4 Hz, 2H), 4.36 (d,J = 4.5 Hz, 2H), 4.00 (td, J = 5.1, 10.2 Hz,2H), 3.73 - 3.59 (m, 2H), 3.37 - 3.32 (m, 2H), 3.10 - 2.88 (m, 3H), 2.52 -2.28 (m, 4H), 1.66 - 1.52 (m, 4H), 1.38 - 1.29 (m, 6H), 0.89 (t, J = 7.4 Hz, 3H).

[0192] Example 6: Synthesis of SUTAC of Formula IA-6 (BRD4-targeted SUTAC) Scheme 6: Synthesis scheme: (a) DIEA / HOBt / EDCI / DMF, 25℃ (b) TEA, ETOH, 68℃ (c) LiOH / THF / H2O / MeOH, 25℃ (d) HATU / DIEA / DMF / 25℃ (e) TEA, DCM, 0℃.

[0193] 3-[2-[2-[2-[2-[(2-cyanoacetyl)amino]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy [By] tert-butyl propionate: To a solution of tert-butyl propionate (500 mg, 1 equivalent) and 2-cyanoacetic acid (116.38 mg, 1 equivalent) in DMF (5 mL), EDCI (655.68 mg, 2.5 equivalent) and HOBt (277.30 mg, 1.5 equivalent), DIEA (353.64 mg, 476.61 μL, 2 equivalent) were added. The mixture was stirred at 25 °C for 18 h. The residue was purified by preparative HPLC (FA conditions) to give a yellow oil (300 mg, 50.70% yield). LC-MS (m / z): calculated 432.25; found: 431.0 [MH] + .

[0194] 3-[2-[2-[2-[2-[2-[(2-aminothiophene-3-carbonyl)amino]ethoxy]ethoxy]ethoxy]ethoxy [B-] ethoxy[2-] tert-butyl propionate: tert-butyl propionate (231 mg, 1 equivalent) 、1,4-Dithiane-2,5-diol (81.31 mg, 1 equivalent) was added to a solution of EtOH (2.5 mL) with TEA (108.09 mg, 148.68 μL, 2 equivalents). The mixture was stirred at 68 °C for 10 h. The reaction mixture was poured into water (20 mL) and extracted with ethyl acetate (20 mL × 2). The combined organic layers were washed with brine (40 mL), dried over Na₂SO₄, filtered, and concentrated under reduced pressure to give the residue. The residue was purified by preparative HPLC (NH₄HCO₃ conditions) to give a yellow oil (150 mg, 57.24% yield). LC-MS (m / z): Calculated 490.23; Found: 491.0 [M+H] + .

[0195] 3-[2-[2-[2-[2-[2-[(2-aminothiophene-3-carbonyl)amino]ethoxy]ethoxy]ethoxy]ethoxy [By]ethoxy]propionic acid: LiOH·H₂O (30.79 mg, 3 equivalents) was added to a solution of tert-butyl propionate (120 mg, 1 equivalent) in THF (1.5 mL), H₂O (1.5 mL), and MeOH (1.5 mL). The mixture was stirred at 25 °C for 1 h. The residue was purified by preparative HPLC (NH₄HCO₃ conditions) to give a yellow oil (60 mg, 56.46% yield). LC-MS (m / z): Calculated 434.17; Found 435.0 [M+H] + .

[0196] 1 H NMR (400 MHz, MeOH) δ = 7.00 (d, J = 5.9 Hz, 1H), 6.27 (d, J = 5.9Hz, 1H), 3.74 (t, J = 6.8 Hz, 3H), 3.69 - 3.60 (m, 21H), 3.52 - 3.48 (m, 2H), 2.50 (t, J = 6.7 Hz, 2H).

[0197] 2-Amino-N-[2-[2-[2-[2-[2-[3-[2-[4-[(Z)-1-(4-hydroxyphenyl)-2-phenyl-but-1-ene] [By]phenoxy]ethyl-methyl-amino]-3-oxo-propoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethyl]thi Fen-3-formamide: To a solution of 3-[2-[2-[2-[2-[2-[(2-aminothiophene-3-carbonyl)amino]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propionic acid (50 mg, 1 equivalent) in DMF (1 mL), HATU (65.63 mg, 1.5 equivalent) and DIEA (44.62 mg, 60.13 μL, 3 equivalent), and 4-[(Z)-1-[4-[2-(methylamino)ethoxy]phenyl]-2-phenyl-but-1-enyl]phenol (42.98 mg, 1 equivalent) were added. The mixture was stirred at 25 °C for 2 h. The residue was purified by preparative HPLC (FA conditions) to give a brown solid (40 mg, 44.00% yield). LC-MS (m / z): Calculated 789.37; Found: 790.4 [M+H] + .

[0198] 2-[(2-chloroacetyl)amino]-N-[2-[2-[2-[2-[2-[3-[2-[4-[(Z)-1-(4-hydroxyphenyl)-2- [Phenyl-but-1-enyl]phenoxy]ethyl-methyl-amino]-3-oxo-propoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy] [By]ethyl]thiophene-3-carboxamide: TEA (7.69 mg, 10.57 μL, 3 equivalents) was added to a solution of 2-amino-N-[2-[2-[2-[2-[3-[2-[2-[4-[(Z)-1-(4-hydroxyphenyl)-2-phenyl-but-1-enyl]phenoxy]ethyl-methyl-amino]-3-oxo-propoxy]ethoxy]ethoxy]ethoxy]ethyl]thiophene-3-carboxamide (20 mg, 1 equivalent) in DCM (1 mL). Then, 2-chloroacetyl chloride (2.86 mg, 2.02 μL, 1 equivalent) was added at 0 °C. The mixture was stirred at 0 °C for 1 h. The reaction mixture was concentrated under reduced pressure to remove the solvent. The residue was purified by preparative HPLC (FA conditions) to provide a brown solid (13.66 mg, 62.27% yield). LC-MS (m / z): Calculated value 789.37; Measured value: 790.4 [M+H] + .

[0199] 1 H NMR (400 MHz, CDCl3) δ12.93 (br s, 1H), 7.44 (br s, 1H), 7.27 -7.25 (m, 1H), 7.21 - 7.01 (m, 7H), 6.94 - 6.62 (m, 6H), 6.50 (br d, J= 8.5Hz, 2H), 4.30 - 4.20 (m, 2H), 4.04 - 3.90 (m, 2H), 3.78 - 3.51 (m, 24H), 3.15- 2.89 (m, 3H), 2.67 - 2.44 (m, 4H), 0.92 (br t, J = 7.4 Hz, 3H).

[0200] Example 7: Synthesis of SUTAC of Formula IA-7 (BRD4-targeted SUTAC) Use 3 in the reaction mol of 1-5 (30 (100 mM stock solution in DMSO). Weigh 2 mg of JQ1 acid (5 mol) and dissolved in 10 In DMF. Dissolve EDC and HOBT in DMF to a concentration of 0.4 M, and then add 12.5... The solution contains 1 ml of this solution + 3.5 ml. l DIPEA (20 (mol) was added to a solution of JQ1 acid. After 5 minutes, it was added to samples 1–5 under gentle vortexing. LCMS indicated complete reaction after 1 hour. The samples were diluted with 15% acetonitrile aqueous solution + 0.1% TFA and purified by reversed-phase HPLC based on a 12%–65% acetonitrile gradient. The product was lyophilized from 30% acetic acid to remove TFA. The overall yield obtained was 0.94 mg (43%). The quality was confirmed by LCMS.

[0201] Example 8: Synthesis of SUTAC of Formula ID-1 (BRD4-targeted SUTAC 1-26) Scheme 8 presents the synthesis of SUTAC with the structure of conjugate ID-1. Weigh 6.2 1-30 and 10.7 mol mol of JQ1 acid dissolved in 400 In dichloromethane, l. Then, while stirring on ice, add 10.7 mol of EDC was added to the mixture. LCMS indicated 20% conversion after 3 hours on ice, and the reaction was then transferred to room temperature. After two hours, 10 mol of EDC was added. The reaction was stopped by adding TFA. The sample was evaporated and dissolved in 80% acetonitrile:water, then diluted with 0.1% TFA aqueous solution to approximately 30% acetonitrile. The product was separated using a 20%–70% gradient by reversed-phase HPLC. The product was lyophilized in 30% acetic acid to remove TFA. The yield was 3.4 mg (4.3 mg / L). (mol, 70%). Mass was confirmed by LCMS.

[0202] Example 9: Synthesis of SUTAC of Formula IC-1 (BRD4-targeted SUTAC) Scheme 9 presents the synthesis of SUTAC having the structure of the conjugate IC-1. Weigh 6.2 5-1 and 10.7 mol mol of JQ1 acid dissolved in 400 In dichloromethane, l. Then, while stirring on ice, add 10.7 mol of EDC was added to the mixture. LCMS indicated 20% conversion after 3 hours on ice, and the reaction was then transferred to room temperature. After two hours, 10 mol of EDC was added. The reaction was stopped by adding TFA. The sample was evaporated and dissolved in 80% acetonitrile:water, then diluted with 0.1% TFA aqueous solution to approximately 30% acetonitrile. The product was separated using a 20%–70% gradient by reversed-phase HPLC. The product was lyophilized in 30% acetic acid to remove TFA. The yield was 3.88 mg (4.9 mg). (mol, 79%). Mass was confirmed by LCMS.

[0203] Example 10: Synthesis of compound of formula I-34 (PIAS4 binder) Option 10 presents a structure with I-34 ( Figure 4A Synthesis of PIAS4 binder. Weigh 7.7 1-30 mol and dissolved in methanol. They were then mixed with 0.8 equivalents of 4-pentynealdehyde and 10 3 M sodium acetate at pH = 5.2 (4 equivalents) was mixed. After 5 minutes, 2 equivalents of sodium cyanoborohydride were added. LCMS indicated 10%–20% conversion after 2 hours, with no further progress after another 4 hours. An additional 1.6 equivalents of the aldehyde and an additional 16 equivalents of sodium cyanoborohydride were added. The product was purified by LCMS, and its mass was confirmed by LCMS (0.56 mg, 16% yield).

[0204] Example 11: Materials and methods of Examples 12-25.

[0205] Protein expression and purification PIAS1(123-419) The sequence of PIAS1 (123-419, SEQ ID NO: 2) was cloned into a modified pET41 plasmid encoding an N-terminal GST tag, a His tag, and a subsequent TEV cleavage site. The plasmid was transformed into BL21(DE3) bacteria. The bacteria were inoculated with 5 L of a solution of 2YT + 1 mM MgSO4 + 1% glucose + NPS (25 mM ammonium sulfate, 50 mM disodium hydrogen phosphate, 50 mM potassium dihydrogen phosphate) + 100 µg / ml kanamycin and grown at 37°C until a density of 0.5 was reached. The bacteria were then induced overnight at 15°C with 0.2 mM IPTG.

[0206] Cells were lysed in 200 ml of Tris 50 mM pH 7.5, 0.5 M NaCl containing a mixture of lysozyme and protease inhibitors, followed by probe sonication (55% amplitude, on for 5 seconds, off for 10 seconds, total sonication time 2 minutes, on ice). After sonication, the sample was centrifuged at 20,000 rpm for 20 minutes, and the supernatant was filtered. Imidazole was added to a final concentration of 20 mM, and the sample was loaded onto a 5 ml Ni-NTA column. The column was washed with 30 ml of Tris 50 mM pH 7.5, 0.5 M NaCl, and 20 mM imidazole, followed by elution with Tris 50 mM pH 7.5, 0.5 M NaCl, and 0.5 M imidazole. The sample was lysed with TEV protease (molar ratio 1:50) and dialyzed into Tris 25 mM pH 7.5, 50 mM NaCl. The sample was replenished with 25 mM imidazole and passed through the Ni-NTA column again. PIAS1 (123-419) maintained a weak binding to the column and did not elute in the flowthrough. The column was washed with buffer containing 25 mM imidazole, and then PIAS1 (123-419) was eluted with 50 mM imidazole. The fractions containing PIAS1 (123-419) were pooled, concentrated, and injected into a Superdex 75 120 mL column equilibrated with Tris 25 mM pH = 7.5, 50 mM NaCl, and 2 mM DTT. The fractions containing PIAS1 (123-419) were combined and concentrated to 36 µM, then aliquoted, rapidly frozen in liquid nitrogen, and then frozen at -80 °C.

[0207] PIAS4(126-411) The synthetic gene encoding PIAS4 (126-411, SEQ ID NO: 4) was cloned into the pET28 plasmid, which contains a His-tagged yeast SUMO fusion protein (Integrated DNA Technologies) preceding the PIAS4 construct. BL21(DE3) bacteria transformed with the plasmid were grown in 2 YT + 1 mM MgSO4 + 1% glucose + NPS + 100 µg / ml kanamycin and induced overnight at 16°C with 0.1 mM IPTG. Bacteria were lysed in buffer A (HEPES 25 mM pH = 7.5, 20 mM imidazole, 0.5 M NaCl) supplemented with lysozyme and protease inhibitors, using sonication as described for PIAS1. The samples were centrifuged, and the supernatant was sonicated again and filtered 0.45 μm before being loaded onto a NiNTA column. The column was washed with buffer A, followed by washing with buffer A + 48 mM imidazole. Proteins were eluted with 300 mM imidazole. The tag was lysed overnight at 4°C using His6-Ulp1, while simultaneously dialyzing against buffer A. The sample was then reloaded onto a Ni-NTA column. The flow-through was collected, concentrated, and loaded onto a Superdex 75 120 mL column equilibrated with 25 mM HEPES, pH = 7.5, and 0.5 M NaCl. The fraction containing pure PIAS4 (126-411) (final concentration 38 μM) was pooled, rapidly frozen in liquid nitrogen, and stored at -80°C.

[0208] Electrophilic screening LC-MS was performed on a Waters ACQUITY UPLC class H instrument in positive ion mode using electrospray ionization. UPLC separation was performed using a C4-BEH column (300 Å, 1.7 μm, 21 mm × 100 mm). The column was maintained at 40 °C and the autosampler at 10 °C. Mobile phase A was 0.1% aqueous formic acid, and mobile phase B was 0.1% formic acid in acetonitrile. The running flow rate was 0.4 mL / min. The gradient used was 1% B for 2 min, linearly increased to 80% B over 2.5 min, held at 80% B for 0.5 min, changed to 20% B over 0.2 min, and held at 1% B for 0.8 min. MS data were collected in the range of 600 m / z–1900 m / z on a Waters SQD2 detector with an m / z range of 2–3071.98. The desolvation temperature was 500 °C, and the flow rate was 800 L / h. The voltage used for the capillary was 1.00 kV, and the voltage used for the cone was 24 V. MassLynx version 4.2 was used to operate LC-MS and analyze the data. The raw data was processed using openLYNX, and deconvolution was performed using MaxEnt at a resolution of 1 Da / channel and a range of 30000 Da–36000 Da.

[0209] 384-well polypropylene plates were prepared using Echo, designed to contain pools of five compounds with a mass difference of at least 20 Da in each well. The compounds were added to the plates as DMSO stock solutions. To initiate screening, proteins were diluted to 1 μM in 25 mM HEPES pH = 7.5, 50 mM NaCl, and the protein solution was added to the plates while they were on ice. The plates were mixed (1400 rpm for 6 seconds), briefly centrifuged, and incubated overnight at 4°C for incubation. The reactions were quenched by adding formic acid to 0.4%, and the samples were analyzed by LC-MS. PIAS4 was incubated with 15 μM of the compound, and PIAS1 was incubated with 20 μM of the compound.

[0210] Dose response of new compounds and further analysis The identified hits were further characterized in dose-response experiments, where each compound was tested at concentrations within a defined range without mixing with other compounds. Different amounts of the compound were added to the plates using Echo dispensing, with DMSO added to maintain a uniform DMSO concentration across all samples. PIAS1 was added to these samples as in the screening, and the label was tested using LCMS. Additional derivatives of the confirmed hits were synthesized and tested using a similar protocol.

[0211] The reactivity of compounds was measured using DTNB assay. The intrinsic reactivity of the compounds was measured using the thiol reactivity assay described by Resnick et al. (pubmed 31060360). This assay was performed in 25 mM NaPi at pH = 7.4 and 150 mM NaCl. 0.5 20 mM of the compound was added to each well of a black 384-well plate with a transparent bottom. 5,5′-Dithiobis(2-nitrobenzoic acid) (DTNB) was added to a concentration of 50 μL. The concentration of M was increased, and TCEP was added to 200. The concentration of M causes DTNB to be reduced to the yellow TNB anion. After several minutes, 50... A solution of l was added to each well. Compounds were measured in triplicate, and for each compound, in addition to the triplicate measured in the presence of TCEP and DTNB, an additional triplicate was measured in the presence of TCEP, solely for correcting the compound's intrinsic absorbance (if any). The plate was covered with a transparent strip and incubated at 37°C inside a plate reader with shaking, wherein absorbance at 492 nm was measured every 15 minutes over a 14-hour period.

[0212] The DTNB measurement data analysis is as follows: 1. Average the absorbance of three copies of each sample and calculate the 95% confidence interval. The same applies to the blank sample (excluding DTNB).

[0213] 2. Subtract the blank from the average signal.

[0214] 3. Normalize the data by dividing each data point by the data at time 0. Propagate the error from the confidence interval using a standard formula.

[0215] When fitting the data, assume that TNB is initially 100. M and the compound is 200 M, and they only react with each other, calculate the actual concentrations of the two substances (compound and TNB). The signal comes from the absorbance of TNB, therefore [TNB] = 0.0001M. (Normalized absorbance) [compound] = [TNB] + 0.0001M.

[0216] The following fitting of the analytical solution to the second-order rate equation enables a simple linear fit to obtain the rate constant. Due to the time required for the plate to equilibrate to 37°C, the first few data points were discarded when fitting the sample, and for highly reactive compounds, linearity was often lost when TNB concentrations were low; therefore, only data from the first 4 hours were used for fitting. In cases where the compound had low solubility in the aqueous buffer, adding 20% ​​acetonitrile to the buffer slightly reduced the observed reactivity, while the relative observation rate remained consistent with measurements performed in the buffer.

[0217] Time-process combined experiments for optimizing medicinal chemistry The compound was diluted 50-fold from a 100 μM stock solution in DMSO using 1 μM protein dissolved in 25 mM HEPES, pH = 7.5, and 50 mM NaCl. Incubation was performed at room temperature, and the reaction was stopped at a specified time by mixing the sample with an equal volume of 40% acetonitrile aqueous solution + 0.5% trifluoroacetic acid.

[0218] The intrinsic reactivity of electrophiles was assessed using N-acetylcysteine ​​methyl ester. For each compound, 1 µl of a 20 mM sample in DMSO was diluted to 50 µl with reaction buffer (NaPi 25 mM pH = 7.5, 50% acetonitrile). In parallel, NAC was dissolved in fresh water to 100 mM and diluted to 2 mM in reaction buffer. The solutions of the compound and NAC were mixed in equal volumes to initiate the reaction, which was then carried out at room temperature. At determined time points, 10 µl of the sample was mixed with 35 µl of a 0.1% trifluoroacetic acid aqueous solution and injected into LC-MS.

[0219] LC-MS runs for NAC determination were performed using the same instrument with a C18-CSH column (300 Å, 1.7 μm, 21 mm × 100 mm) and a gradient of 1% B for 1 minute, increasing to 95% B over 4.5 minutes, holding at 95% B for 0.75 minutes, then decreasing to 1% B over 0.75 minutes and holding at 1% B for 1 minute. MS data were collected in the range of 80 m / z–2500 m / z using the same ionization conditions as for proteins.

[0220] LC-MS / MS analysis of the compound-bound PIAS4 complex PIAS4 (38 μM) was incubated with DMSO or 1.5 equivalents of compound 1-41 at room temperature in 25 mM HEPES, pH 7.5, and 0.5 M NaCl for 30 min. After confirming complete protein labeling by LC-MS, 40 µl of each sample buffer was exchanged for 100 mM triethylammonium bicarbonate using a BioSpin column. Following buffer exchange, 40 µl of each sample was incubated overnight at 37 °C with 1.25 µg of sequencing-grade trypsin (Promega). After trypsinization, 1 µl of 200 mM DTT was added to each sample, followed by incubation at 37 °C for 30 min. At this point, 1 µl of freshly dissolved 0.8 M iodoacetamide was added to each sample, and the sample was incubated at room temperature in the dark for 30 min. Then, 40 µl of 0.2% TFA aqueous solution was added, and the samples were desalted using an Oasis column (Waters) and dried using a speedvac. The sample was dissolved in 50 µl of 3% acetonitrile + 0.1% formic acid, and 0.5 µl was injected into LC-MSMS.

[0221] Samples were analyzed using an EASY-nLC 1200 nanoflow UPLC system with a PepMap RSLC C18 column (2 μm particle size, 100 Å pore size, 75 μm diameter × 50 cm length). The PepMap RSLC C18 column was mounted on an Exploris 240 mass spectrometer operating with Xcalibur version 4.4.16.14 using an EASY-Spray source. UPLC / MS grade solvent was used for all chromatographic steps at 300 nL / min. The mobile phases were: (A) H₂O + 0.1% formic acid and (B) 80% acetonitrile + 0.1% formic acid. Peptides were eluted from the column to the mass spectrometer using the following gradients: 1%–40% B over 60 min, 40%–100% B over 5 min, held at 100% for 20 min, 100% to 1% over 10 min, and a final 1% for 5 min. Ionization was achieved using a spray voltage of 2100 V, with the ion transfer tube temperature set at 275 °C. Initially, data were acquired in data-dependent acquisition (DDA) mode. MS1 resolution was set to 120,000 (at 200 m / z), with a mass range of 375 m / z–1650 m / z, a normalized AGC of 300%, and a maximum injection time of 20 ms. MS2 resolution was set to 15,000, with a quadrupole isolation of 1.4 m / z, a normalized AGC of 100%, a maximum injection time of 22 ms, and an HCD collision energy of 30%. Three 0.5 µl injections were performed on each sample. DDA data were analyzed using Fragpipe version 22.0. The database contained sequences of the PIAS4 constructs used in the study and included contaminants. Methionine oxidation and N-terminal acetylation were variable modifications, and carbamidomethyl and modifications via compounds 1–41 were set as variable modifications in the analysis, with up to 3 modifications per peptide. Digestion was defined as trypsin / P with up to 2 missed cleavages. Data were imported into Skyline (version 22.2.0.351), and precursors from 16 peptides were selected for parallel reaction monitoring (PRM). A full MS spectrum was acquired at each acquisition cycle with the following parameters: 350 Da–1400 Da range, 300% AGC target, maximum injection time 20 ms, and resolution 120,000. Data for each precursor were measured during a 4–5 min window around the retention time measured in the DDA run, with Q1 resolution of 2 Da, orbital trap resolution of 15,000, 300% AGC target, and maximum injection time of 160 ms. The acquired data were then analyzed in Skyline using a spectral library generated from the DDA run. The five strongest product ions were used for quantification relative to the DMSO control.

[0222] PIAS4's DSF PIAS4 (WT or mutant) was incubated at 20 μM in 25 mM HEPES pH = 7.5, 150 mM NaCl containing 1% DMSO for 10 minutes, with or without compounds 1-41. At this point, 19 µl of the sample was mixed with 1 µl of SYPRO Orange (Sigma) X100 (the final dilution of SYPROOrange X5) in 25 mM HEPES pH = 7.5, 50 mM NaCl. The samples were measured in triplicate on a StepOne Plus instrument using FAM as the target and ROX as the passive reference.

[0223] Crystallography of the PIAS4(126-411) PROSS mutant with compounds 1-41 The PROSS mutant of PIAS4(126-411) (SEQ ID NO: 5) was expressed and purified using the same protocol as the wild-type protein and concentrated to 724 μM before freezing. The free protein was first subjected to crystallographic screening. The protein was desalted using Biospin (Bio-Rad) to 25 mM HEPES pH = 7.5 and 100 mM NaCl. Crystallization was performed using a 96-well iQ plate and Salt-Rx, PEG-Rx, and PEG-ion sieves from Hampton via the sitting drop method. 150 nL of protein and 100 nL of buffer were placed in each spot and incubated at 20 °C. After one week, small crystals appeared under several conditions, with 1.3 M diammonium hydrogen tartrate and 0.1 M BIS-TRIS propane at pH 7.0 selected for seed crystal preparation. The crystals were crushed, vortexed with stainless steel beads, and serially diluted in 1.4 M diammonium hydrogen tartrate and 0.1 M Tris at pH 8.5.

[0224] To crystallize the complex containing compounds 1-41, 90 µl of protein was mixed with 2 µl of 50 mM compound in DMSO (1.5 equivalents). LCMS confirmed complete labeling after 20 minutes. The sample was centrifuged, and the supernatant was desalted to HEPES 25 mM pH = 7.5, 100 mM NaCl. The sample was screened using a Salt-Rx screen by mixing 150 nml of the complex with 50 nml of seed stock and 100 nml of buffer. Large crystals appeared after 3 days in 4.0 M sodium nitrate, Tris 0.1 M pH = 8.5. The crystals were collected, immersed in cryo-oil, and measured.

[0225] Gel-based characterization of proteomic selectivity of molecules 1-34 Daudi cells were treated with 1–34 for 6 hours under various pre-incubation (2 hours) conditions. Cells were then incubated at 100... The protein was lysed and centrifuged in RIPA + protease inhibitor, and the protein concentration was measured by BCA. The lysate was then diluted to 2 mg / ml with RIPA and the following additives were used to adjust the concentration of 50 mg / ml protein. The sample underwent a click reaction: - 1.8 0.1 M CuSO4 / THPTA - 1 5 mM TAMRA-azide - 1.5 150 mM sodium ascorbate freshly dissolved in water All samples were precipitated with methanol-chloroform (150). 1 water, 200 Methanol and 50 Chloroform, rotate, remove the top, and use 200... (Repeat twice with methanol), then dissolve in 120... In 1X LDS buffer containing 5 mM DTT, heat to 70°C for 10 minutes, and add 50 ml of the solution. The l was loaded onto the gel. The gel was run, fixed with 45% methanol, 45% water, and 10% acetic acid, and imaged on Typhoon.

[0226] cell lines Daudi, OCI-AML2, Ramos, and RPMI-8226 cells were grown in RPMI medium supplemented with 10% FBS (GIBCO), 2 mM glutamine (Invitrogen), 1% penicillin-streptomycin (Invitrogen), and 1% sodium pyruvate (Invitrogen). LNCaP cells were grown in RPMI medium supplemented with 10% FBS (GIBCO), 2 mM glutamine (Invitrogen), 1% penicillin-streptomycin (Invitrogen), and 1% sodium pyruvate (Invitrogen). WT, PIAS1 KO, and PIAS1 / 4 DKO TK6- cells (Mohiuddin, M. et al. SUMOylation of PCNA by PIAS1 and PIAS4 promotes template switch in the chicken and human B cell lines. ProcNatl Acad Sci USA 115, 2018) were grown in RPMI medium supplemented with 5% horse serum (H1138 Sigma-Aldrich), 2 mM glutamine (Invitrogen), 1% penicillin-streptomycin (Invitrogen), and 1% sodium pyruvate (Invitrogen). 293T and A549 cells were grown in DMEM medium supplemented with 10% FBS (GIBCO), 2 mM glutamine (Invitrogen), 1% penicillin-streptomycin (Invitrogen), and 1% sodium pyruvate (Invitrogen). All cell lines were grown at 37°C and 5% CO2, and mycoplasma contamination was routinely tested.

[0227] plasmid overexpression According to the manufacturer's instructions, 293T cells were transfected with JetPEI (PolyPlus) using pCMV-mCherry or pCMV-FLAG-hAR (Plasmid addgene #89080).

[0228] Characterization of proteomic selectivity of molecules 1-34 Prepare the following cell samples in triplicate (40 million cells in each sample): -3 replicates: pre-incubate with DMSO (2 hours) and then with DMSO (6 hours), labeled (1, 2, 3).

[0229] -3 replicates: DMSO (2 hours) pre-incubation and then 1 M 1-34 (6 hours), marked (4, 5, 6).

[0230] -3 repetitions: 10 M 1-12 (2 hours) pre-incubate and then 1 M 1-34 (6 hours), marked (7, 8, 9).

[0231] Cells were lysed in RIPA and BCA was tested, yielding lysate concentrations of 8-10 mg / ml. The lysate was reacted with a biotin-containing peptide (Biotin-GGGGGGRK(az)-NH2) that is cleavable by trypsin. The sample was diluted to 2 mg / ml in 15 ml polypropylene tubes of RIPA, resulting in a total volume of 250 ml. l. Add 1 to each sample 40 mM peptide azide, 2.5 l of CuSO4:THPTA 100 mM and 2.5 Freshly dissolved 200 mM sodium ascorbate was added. The reaction was carried out at room temperature in the dark for 1 hour, followed by methanol-chloroform precipitation as follows: 750 μL of freshly dissolved sodium ascorbate was added. 1 ml water, 1 ml methanol and 250 ml Mix the sample with 1 ml of chloroform, then mix vigorously and centrifuge at 3200 x g for 10 minutes. Aspirate the top layer and add 1 ml of methanol, then mix the sample and centrifuge again. Remove the supernatant and allow the sample to dry completely before storing at -80°C.

[0232] The sample was dispersed at 180°C using ultrasonic treatment (16-second, 2-second pulse). l was placed in 2.5% SDS in PBS. The sample was diluted X20 with PBS and washed with streptavidin beads (from Cytiva-20) at room temperature. Incubate the beads (1 bead / sample) together by tumbling for 3 hours. Then transfer the beads to a rotating column and wash as follows: Use 300 Wash three times with 1% SDS / PBS. Add 3 ml of [unspecified solution] before draining the third wash. Add 1 MDTT solution and incubate the beads in the solution at room temperature for 40 minutes, mixing occasionally, then add 15 MDTT solution. Add 1 liter of fresh 0.8 M iodoacetamide, followed by 30 minutes of incubation at room temperature, with occasional mixing. At this point, drain the solution and continue washing.

[0233] Use 350 The sample was washed three times with freshly dissolved 6 M urea in PBS.

[0234] Use 400 The sample was washed three times with 20% methanol in PBS.

[0235] Use 400 Wash once with PBS.

[0236] Use 400 Wash twice with water.

[0237] At this point, transfer the beads to two portions of 50. 50 mM TEAB in Eppendorf, and add 2 to each sample. 0.5 of l g / I. Trypsin, then incubate at 37°C with shaking at 1150 rpm for 6 hours. Centrifuge the beads and aspirate the supernatant (90 g / L). l), and further use 100 beads l was washed with 2M NaCl in 50 mM TEAB, and then with a first 90 Combine the supernatants. Then combine the combined supernatants with 190... The sample was mixed with a 0.2% TFA aqueous solution and then desalted using an Oasis (Waters) column according to the manufacturer's instructions. The eluted sample was then evaporated under vacuum.

[0238] The dried peptides were dissolved in 3% acetonitrile + 0.1% formic acid (25 µl) and injected in 2 µl. Samples were analyzed using an EASY-nLC 1200 nanofluid UPLC system with a PepMap RSLC C18 column (2 μm particle size, 100 Å pore size, 75 μm diameter × 50 cm length), mounted on an Exploris 240 mass spectrometer using an EASY-Spray source. UPLC / MS grade solvent was used at 300 nL / min for all chromatographic steps. The mobile phases were: (A) H₂O + 0.1% formic acid and (B) 80% acetonitrile + 0.1% formic acid. The peptides were eluted from the column to the mass spectrometer using the following gradient: 1%–40% B over 160 min, 40%–100% B over 5 min, held at 100% for 20 min, 100% to 1% over 10 min, and a final 1% for 5 min. Ionization was achieved using a 2100 V spray voltage, with the ion transfer tube temperature set at 275 °C. Initially, data was acquired in data-dependent acquisition (DDA) mode. MS1 resolution was set to 120,000 (at 200 m / z), with a mass range of 375 m / z–1650 m / z, a normalized AGC of 300%, and a maximum injection time of 20 ms. MS2 resolution was set to 15,000, with quadrupole isolation of 1.4 m / z, a normalized AGC of 50%, an automatic maximum injection time, and an HCD collision energy of 30%. Three samples were analyzed under each condition.

[0239] Data analysis was performed using Fragpipe (version 19.1), the Msfragger search engine (version 3.8), IonQuant 1.8.10, and Philosopher 4.8.1. Analysis was conducted using the Human Proteome Database (Uniprot) from December 2022, with contaminants added and streptavidin manually added as a contaminant. Msfragger analysis was performed using trypsin as the cleavage enzyme following Arg and Lys, with up to two missed cleavages, peptide lengths of 7–50, and N-terminal methionine removed. N-terminal acetylation and methionine oxidation were defined as variable modifications, and carbamoylmethylation was defined as a fixed modification. A false discovery rate of 0.01 was used at both the peptide and protein levels. Label-free quantification was performed using IonQuant with a minimum peptide count, and a 1-minute tolerance enabled matching between runs. After analysis, the merged protein files were analyzed using Perseus.

[0240] Intensities were converted to Log2 values. One sample from the dataset with pre-incubation of 1-12 was contaminated with SDS and removed from the analysis. Triplets of the first two groups (DMSO-treated; 1-34-treated) and duplicates of the last group (1-34-treated with 1-12 pre-incubation) were grouped, and all proteins with at least two valid values ​​in one group were retained in the analysis. Missing values ​​were replaced by imputation from a normal distribution (shifted down by 2.7, width 0.3), and Student's t-tests were used to calculate differences and p-values ​​between the two pairs of groups: cells treated with 1-34 versus cells treated with DMSO; and cells treated with 1-34 versus cells treated with 1-34-treated with 1-12 pre-incubation. Results were filtered based on both tests and selected proteins that gave significant differences for both (proteins effectively pulled down by 1-34 but effectively competed with 1-12).

[0241] Characterization of the proteomic selectivity of compounds 1-43 Daudi cells (20 million per sample) were incubated with 1 μM compound 1-43 (or DMSO) for 2 hours, followed by incubation with 0.1 μM compound 1-42 (or DMSO) for 1 hour. Four replicates were measured for each sample. After incubation, cells were washed with PBS, harvested, and lysed in 200 µl RIPA buffer (Sigma) supplemented with protease inhibitors. Lysis was performed by dispersing cells in the buffer and incubating the sample on ice for 15 minutes with occasional vortexing. Samples were centrifuged at 21,000 × g for 10 minutes at 4 °C, and the protein concentration in the supernatant was estimated using BCA determination. At this point, a sample of 200 µl / 2.5 mg / ml protein was prepared from each replicate by diluting with RIPA buffer.

[0242] For each sample, a pre-click mixture (total volume 40 µl) containing 0.5 µl of 50 mM biotin azide in DMSO, 4.5 µl of DMSO, 22.5 µl of RIPA buffer, and 12.5 µl of 100 mM THPTA:20 mM CuSO4 aqueous solution was added. Then, 10 µl of freshly dissolved 200 mM sodium ascorbate was added, and the sample was vortexed and incubated in the dark at room temperature for 1.5 h. After the reaction, each sample was precipitated by adding 750 µl of water, 1 ml of methanol, and 250 µl of chloroform, followed by vigorous vortexing and centrifugation (3200 × g, 10 min, 4 °C). The supernatant was aspirated, and the precipitate was washed twice with cold 1 ml of methanol and air-dried.

[0243] The precipitate was dispersed in 250 µl of 2.5% SDS in PBS and then sonicated (20%, 8 pulses for 2 seconds, 2-second intervals). The sample was then diluted 20-fold with PBS and 10 µl of streptavidin beads (cytiva) was added. The sample was incubated at room temperature for 3 hours using a small rotating column in a vacuum manifold. The beads were washed three times with 1% SDS (400 µl) in PBS, and then 400 µl of 1% SDS in PBS was added, followed by 4 µl of 1 M DTT with intermittent mixing. After 30 minutes, 25 µl of freshly dissolved 0.8 M iodoacetamide was added, and the sample was incubated for another 30 minutes in the dark. The buffer was then pumped out, and the sample was washed as follows: three times with fresh 6 M urea in PBS, four times with 20% methanol in PBS, twice with PBS, and twice with water.

[0244] The beads were then transferred to Eppendorf tubes using two 100 µl aliquots of 100 mM TEAB, and 0.5 μg of trypsin was added. The tubes were then incubated overnight at 37°C with shaking (1300 rpm). The samples were then centrifuged, the supernatant was removed, and the beads were washed once with TEAB and once with TEAB + 2 M NaCl. The washes were combined with the supernatant, and TFA was added to a final concentration of 0.1%. The samples were then desalted using an Oasis desalting column (Waters). They were dissolved in 3% ACN + 0.1% formic acid and injected (5 µl) into the LCMS / MS instrument.

[0245] Samples were analyzed using an EASY-nLC 1200 nanoflow UPLC system with a PepMap RSLC C18 column (2 μm particle size, 100 Å pore size, 75 μm diameter × 50 cm length), mounted on an Exploris 240 mass spectrometer using an EASY-Spray source. uLC / MS grade solvent was used at 300 nL / min for all chromatographic steps. The mobile phase was (A) H₂O + 0.1% formic acid and (B) 80% acetonitrile + 0.1% formic acid. Peptides were eluted from the column to the mass spectrometer using the following gradients: 1%–40% B over 160 min, 40%–100% B over 5 min, held at 100% for 20 min, 100% to 1% over 10 min, and a final 1% for 5 min. Ionization was achieved using a 2100 V spray voltage with the ion transfer tube temperature at 275 °C. Initially, data were acquired using data-independent acquisition (DIA) mode. MS1 resolution was set to 60,000 (at 200 m / z), with a mass range of 370 m / z–1450 m / z, a normalized AGC of 300%, and a maximum injection time of 20 ms. MS2 resolution was set to 30,000, with 31 isolation windows of 19 Da, 1 Da overlap, a maximum injection time of 50 ms, and an HCD collision energy of 27%. Four samples were analyzed under each condition.

[0246] Data analysis was performed using Fragpipe (version 22.0) with the Msfragger search engine (version 4.1, PMID 28394336) and DIANN 1.9 (PMID 31768060). Analysis was performed using the Human Proteome Database (Uniprot) from December 2022, with contaminants added and streptavidin manually added as a contaminant. Msfragger analysis was performed using trypsin as the cleavage enzyme following Arg and Lys, with up to two missed cleavages, peptide lengths of 7–50, and N-terminal methionine removed. N-terminal acetylation and methionine oxidation were defined as variable modifications, and carbamoylmethylation was defined as a fixed modification. A false discovery rate of 0.01 was used at both the peptide and protein levels. Label-free quantification was performed using DIANN. Following analysis, the quantified protein files were analyzed using Perseus (PMID 27348712). The intensity was converted to Log2 values, each type was grouped into quadruplets, and missing values ​​were replaced with 13. Differences and p-values ​​were calculated using a two-tailed Student's t-test. The difference between 2 and Log2 and the difference between 1 (2-fold) was used as the significance limit.

[0247] 1-26 In vitro SUMOylation assay of BRD4 by SUTAC The full-length MYC-DDK-PIAS4 (TP306748, Origene) was recombined with 3.5 M 1-26 SUTAC or DMSO were pre-incubated at 25°C for 4 hours. Afterwards, they were incubated with the following recombinant proteins in reaction buffer: 10 M SUMO1 (UL-712, Boston Biochem), 10 M SUMO2 (UL-752, Boston Biochem), 1 MSAE1 / SAE2 (E315 Boston Biochem), ± 1 MUBC9 (E2-645 Boston Biochem), ± 350 nM BRD4 (SP-600, R&D systems), the reaction buffer consisted of: 50 mM HEPES, 4 mM ATP, 15 mM creatine phosphate, 0.7 / ml creatine phosphokinase, 10 mM MgCl2, 10 mM MgOAC, 50 mM KOAc and 0.5 mM DTT. The mixture was then allowed to incubate at 30°C for 60 minutes, after which it was subjected to Western blot analysis.

[0248] In vitro SUMOylation assay of BRD4 by ID-2 200 nM of recombinant full-length MYC-DDK-PIAS4 (TP306748, Origene) was pre-incubated with 200 nM ID-2 or DMSO at 25°C for 90 minutes. Afterwards, they were incubated with the following recombinant proteins in reaction buffer: 4 M SUMO1 (UL-712, Boston Biochem), 4 M SUMO2 (UL-752, Boston Biochem), 0.5 M SAE1 / SAE2 (E315 Boston Biochem), ± 0.2 MUBC9 (E2-645 Boston Biochem), ± 200 nM BRD4 (SP-600, R&D systems), the reaction buffer consisted of: 50 mM HEPES, 4 mM ATP, 15 mM creatine phosphate, 0.7 / ml creatine phosphokinase, 10 mM MgCl2, 10 mM MgOAC, 50 mM KOAc and 0.5 mM DTT. The mixture was then allowed to incubate at 30°C for 60 minutes, after which it was subjected to Western blot analysis.

[0249] Denaturing lysis buffer for whole-cell lysates Harvest cells, wash and count them. Take an equal number of cells for lysis. Lyse cells using 5× lysis buffer (2% SDS, 50 mM Tris pH 7.5, 10 mM NaCl, protease inhibitor mixture 1:250 (Merck), 20 mM NEM (Merck)). Incubate the lysates on ice for 10 minutes, then add 4 volumes of supplemental protease inhibitor mixture, 20 mM NEM, and 1 ml of [unspecified solution] in 1000 ml. 1 L of Benzonase (71205-3, Merck) was added to PBS+ / +. The lysate was then incubated on ice for 5 min, followed by incubation at 30 °C for 10 min. Finally, the lysate was centrifuged (15000 rcf, 15 min, 4 °C) and the supernatant was collected for further analysis.

[0250] Chromatin grading Harvest cells, wash and count. Resuspend equal numbers of cells in lysis buffer (10 mM HEPES pH 7.4, 10 mM KCl, 0.05% NP-40) and incubate on ice for 20 min. Then centrifuge the lysate at 14,000 RPM for 10 min at 4 °C to separate the cytoplasmic and nucleoplasmic fractions. Resuspend the precipitate in low-salt buffer (10 mM Tris-HCl pH 7.4, 0.2 mM MgCl2, 1% Triton X-100) and incubate on ice for 15 min. After centrifugation under the same conditions, separate the chromatin and nucleoplasmic fractions. Resuspend the chromatin-containing precipitate in 0.2 N HCl, incubate on ice for 30 min, and centrifuge. Collect the resulting supernatant and neutralize it by mixing with 1 M Tris-HCl pH 8.0 at a 1:1 ratio. All buffers were replenished with 25 mM NEM, a mixture of protease inhibitors (Sigma-Aldrich), and 1 mM MPMSF.

[0251] Active extracts Cells were collected, washed, and then lysed using a swelling buffer containing 25 mM HEPES (pH 7.5), 1.5 mM MgCl2, 5 mM KCl, 1 mM DTT, and a mixture of protease inhibitors. The lysate was further homogenized using a freeze-thaw process and passed through a needle. To remove impurities, the extract underwent a series of centrifugation steps, with the first centrifugation at 5,000 rpm for 5 minutes and the second centrifugation at 14,000 rpm for 60 minutes. A portion of the resulting extract (20 µL, 25 mg / mL) was combined with an energy mixture containing 150 mM creatine phosphate, 20 mM ATP, 2 mM EGTA, and 20 mM MgCl2 (pH 7.6). 1–5 M SUMO1-aldehyde and SUMO2-aldehyde (R&D system) are included in some downstream assays.

[0252] Immunoprecipitation Flag magnetic beads (M2, Sigma) were used for immunoprecipitation. Lysates were incubated at 4°C for 2 hours while being rotated with the beads. After this incubation, the beads were used to remove unbound proteins, and the elution buffer was then incubated with 150 mM Tris-HCl (pH 7.4), 1.5 mM MgCl2, 150 mM NaCl, and 0.5 mg / ml X3flag peptide (Sigma). All buffers were supplemented with 25 mM NEM, a 1:250 protease inhibitor mixture, and 1 mM MPMSF. The samples were then subjected to analysis by Western blot, as described in further detail below.

[0253] Protein blot Protein concentration was assessed using the BCA kit (Thermo Scientific). Proteins were separated by SDS-PAGE on 4%–15% Criterion™ TGX unstained gels and transferred to nitrocellulose membranes using the iBlot 2 gel transfer device (Thermo Fisher Scientific). The membrane was sealed in 5% emulsion prepared in TBS-0.1% Tween and incubated overnight at 4°C with primary antibodies. The primary antibodies were: anti-SUMO1 (Cell Signaling Technology Cat# 4930S, 1:1000), anti-UBC9 (Cell Signaling Technology Cat# 4918S, 1:1000), anti-ubiquitinated protein (FK2) (Sigma-Aldrich #04-263, 1:1000), anti-ubiquitin (P4D1) (Cell Signaling Technology #3936, 1:1000), anti-BRD4 (Cell Signaling Technology #13440, 1:1000), anti-BRD4 (abcamab128874, 1:1000), anti-H3K9me3 (abcamab8898, 1:1000), anti-PIAS1 (abcamab32219, 1:1000), and anti-PIAS2 (abcamab155556). (1:1000), Anti-PIAS3 Cell Signaling Technology Cat#4164, Anti-PIAS4 Cell Signaling Technology Cat#4392, Anti-P300 and anti-cMYC Cell Signaling Technology #9402 (1:1000), Anti-AR Cell Signaling Technology #5153 (1:1000), Anti-P300 C20 Santa Cruz SC-585 (1000), Anti-actin Cell Signaling Technology Cat#4970, Anti-HSP90 abcam ab13495 (1:20000), Anti-fouling protein (E1E9V) Cell Signaling Technology #13901 (1:1000), Anti-γ tubulin (GTU-88) abcam ab11316 (1:10000), Anti-H2B abcam ab1790.For secondary antibody detection, peroxide-conjugated AffiniPure goat anti-rabbit (1:5,000 dilution, product number 111-035-003, from Jackson Immuno Research), goat anti-mouse (1:5,000, Jackson Immuno Research), and donkey anti-sheep (1:5,000, Jackson Immuno Research) were used. Finally, the blots were examined using SuperSignal West Pico chemiluminescent substrates from Thermo Scientific and the ChemiDoc XRS+ system from BIO-RAD.

[0254] Proliferation assay To evaluate the effect of the compound on cell proliferation, Cell Titer Glo (Promega, #G7572) was used according to the manufacturer's instructions. Briefly, cells were plated at a density of ~5,000 cells per well with 100 µl of the compound in 96-well tissue culture plates. After a 72-hour incubation period, 100 µl of reconstituted Cell-Titer-Glo reagent was added to each well, and luminescence was measured using a plate reader. Relative cell growth was determined by comparing the measurement readings of the treated cells with those of control cells treated with DMSO.

[0255] Example 12: Electrophilic screening and initial dose response for PIAS1 Objective: To identify candidate molecules that bind to SUMO-specific E3 ligases.

[0256] Internal electrophilic fragment screening (Resnick, E. et al. Rapid Covalent-Probe Discovery by Electrophile-Fragment Screening. J Am Chem Soc 141, 2019) was used to screen for recombinant domains (sequences 123-419) of the human protein PIAS1. Figure 1B The screening of molecules pooled at 20 μM was performed using OpenLynx analysis, and the output was parsed to reveal the initial hits. Some molecules revealed by the screening were also frequent hits in other protein screenings and were filtered out. After this step, six candidate hits remained. As a secondary screening, dose-response measurements of the proteins versus the candidates were performed (overnight at 4°C). Three of the molecules were found to be unlabeled proteins, leaving three confirmed hits from two different scaffolds. Figure 1CMolecular 1 is based on a thiophene scaffold with a proximal amide group, while molecules 2 and 3 are based on phenyl groups with electron-rich groups at the para position relative to chloroacetamide. Examination of data from the original screening revealed some structure-activity relationships (SARs), although the quantitative values ​​of this data are limited due to the pooling of compounds.

[0257] To optimize binding efficiency, three hit modified versions were created. Parallel to dose-response experiments on the derivatives, the intrinsic thiol reactivity of the compounds was also measured for a subset of molecules to reveal how intrinsic reactivity contributes to their binding to proteins. Derivatives of molecules 2 and 3 were prepared by modification of a bulky substituent at the para position and other modifications on the benzene ring. Figure 2 Several substitutions on the ring enhance the binding, particularly for molecules 3-3, 3-5, and 3-7, but this is accompanied by enhanced inherent reactivity, especially for the meta position, indicating that these changes primarily improve binding by enhancing the reactivity of the electrophile rather than by better recognition. The only change made to the para substituent that results in slightly better performance is the addition of a methyl group to the difluorine group on the sulfur, but this may also be due to slightly enhanced reactivity.

[0258] The thiophene scaffold of molecule 1 was tested using several derivatization methods. First, the amide substituents on the ring were examined. Several amides were tested, and it was found that the molecule is tolerant to a relatively wide range of amide substituents, but not to secondary amides. Figure 3A This result is also consistent with observations from the initial screening, namely that molecules similar to molecule 1 but with ester groups instead of amides showed poor binding to PIAS1. Various modifications to the ring structure on molecule 1 were also tested. Figure 3B The most effective derivatives comprise a cyclohexane or phenyl ring fused with thiophene, exhibiting relatively limited extension directions, as seen through the differential effect of chlorination on the ring (molecules 1-12 to 1-19). The replacement of the chloroacetamide warhead with an aminosulfonate acetamide warhead was also tested (Lu, J. et al. Hijacking the E3 Ubiquitin Ligase Cereblon to Efficiently TargetBRD4. Chem Biol 22, 2015).

[0259] Example 13: Characterization of cellular targets of molecules 1-12 Objective: To validate PIAS1 as a true cellular target of the molecule.

[0260] The most effective binding agent compounds 1-12 were selected, and alkyne-modified derivative compounds 1-34 were prepared. Figure 4AThis molecule exhibits efficient binding to PIAS1 in vitro, but also displays high intrinsic reactivity, a property observed with several other positively charged 1-12 derivatives. When cells were incubated with 1-34 and lysates were labeled with TAMRA azide using copper-catalyzed azide-alkyne coupling (CuAAC), extensive labeling of many proteins was observed. Figure 4B Almost all proteins were not competitively removed by the less confounded 1-12. For proteomics experiments, cells were incubated with 1-34 and CuAAC was used to attach biotin to the labeled proteins, followed by pull-down, trypsinization, and LC-MS / MS. Targets labeled with 1-34 were identified relative to a DMSO-treated control, and targets labeled with 1-12 were identified by pre-incubating lysates before incubation with 1-34 and comparing the proteins obtained. As expected, 1-12 was much more selective than 1-34. PIAS1 was identified by a single peptide and could be pulled down and competed for, but not statistically significant, likely due to its low abundance. However, two other homologs, PIAS2 and PIAS4, were identified more robustly and were clearly bound by 1-34 and effectively competed for by 1-12. Figure 4C Importantly, preliminary LC-MS / MS findings revealed that 1–5 PIAS binders covalently bind Cys315 of PIAS1 (data not shown), which is conserved across the PIAS family (PIAS1 / 2 / 3 / 4) and may be the basis for non-selective labeling of the PIAS family.

[0261] Example 14: BRD4-targeted SUTAC reduces the levels of BRD4 and its downstream target c-MYC. Objective: To test the feasibility of using the SUTAC pattern to modify and change Points of Interest (POIs).

[0262] The transcriptional activator BRD4 was chosen as the first target. This protein is crucial for the expression of c-MYC-driven oncogenic programs and the proliferation of various cancer types, including acute myeloid leukemia (AML) and Burkitt lymphoma (BL). Furthermore, BRD4 has been widely used as a model substrate for PROTAC development due to its potent small-molecule binding agents (e.g., the BET inhibitor JQ1), and it is hypothesized that SUMOylation of BRD4 by SUTAC might disrupt its binding to chromatin and inhibit its activity. Conjugates of PIAS binding agent 1 and +JQ1 were prepared, linked by linkers having 2 or 5 polyethylene glycol (PEG) groups, thereby forming 1-24 (IA-7) BRD4 targeting SUTAC and 1-25 BRD4 targeting SUTAC, respectively. Figure 5AInvariably, conjugation reduced the binding efficiency of the molecule to PIAS1. However, treatment of the B-cell lymphoma cell line Daudi with 1–24 BRD4 targeting SUTAC instead of 1–25 BRD4 targeting SUTAC for 20 hours resulted in a significant decrease in BRD4 levels. Figure 5B ).

[0263] This effect is achieved at 100 nM and is accompanied by a decrease in c-MYC levels. Importantly, the building blocks of SUTAC, namely JQ1 and PEG-modified PIAS binder 1.5 (… Figure 5C The conjugation between these two molecules is crucial for mediating their effect and for achieving a 100-fold improvement in reducing c-MYC levels compared to free JQ1. Figure 5D In summary, these results support the proximity-induced mechanism of SUTAC.

[0264] To examine whether the effect of BRD4 targeting SUTAC is indeed promoted by a SUMOylation mechanism, the same reaction was performed with pre-incubation with the SUMO E2 conjugate inhibitor 2D08. It was found that SUMOylation inhibition partially eliminated the SUTAC-induced decrease in BRD4 levels. Figure 6A Furthermore, BRD4 levels were less effectively reduced in cells where both PIAS1 and PIAS4 were double knocked out (DKO), rather than in cells where only PIAS1 was knocked out. Figure 6B This observation is consistent with the fact that the identified PIAS1 binder interacts not only with PIAS1, but also with PIAS2 and PIAS4. Figures 4A-4C ).

[0265] Example 15: SUTAC modifies recombinant BRD4 only under conditions that promote SUMOylation Objective: To test the ability of 1-24 (IA-7) BRD4 targeting SUTAC to induce SUMOylation of recombinant BRD4 (short form: (Glu49-Glu460)-FLAG-BRD4) under controlled cell-free conditions.

[0266] Significantly, only under SUMOylation retention conditions (SUMO-aldehyde supplementation), SUTAC treatment induces high molecular weight BRD4 bands, indicating that BRD4 is SUMOylated. Figure 7It is noteworthy that the anti-SUMO1 antibody used failed to detect the BRD4 band, most likely due to the low efficiency of anti-SUMO antibodies in detecting all SUMOized proteins (Garvin, AJ, Lanz, AJ & Morris, JR SUMO monoclonal antibodies vary in sensitivity, specificity, and ability to detect types of SUMO conjugate. Sci Rep 12, 2022).

[0267] Example 16: Optimization of the PIAS binder enhances the effect of SUTAC against BRD4. Objective: To test the enhanced binders 1-9 and 1-12 ( Figures 3A-3C Whether this could lead to an enhanced effect of BRD4 targeting SUTAC.

[0268] The conjugation of JQ1 with binders 1-9 and 1-12 forms 1-26 SUTAC and 1-27 SUTAC, respectively, which significantly reduces the binding efficiency of SUTAC with recombinant PIAS1. Figure 8A Treatment of Daudi cells with 1-26 SUTAC and 1-27 SUTAC for 20 hours resulted in a stronger reduction in BRD4 and cMYC levels compared to the original 1-24 SUTAC treatment. Figure 8B It is noteworthy that a decrease in BRD4 levels is readily observed at concentrations of 20 nM in the range of 1–26.

[0269] Example 17: BRD4-targeted SUTAC inhibits the growth of hematologic malignancies in vitro. The significant reduction in c-MYC levels induced by SUTAC treatment compared to JQ1 indicates that SUTAC can induce a high killing effect on lymphoma cells. After 72 hours of treatment, the SUTAC variant showed a more significant inhibition (up to 120-fold) of Daudi cell proliferation compared to JQ1. Figure 9A Consistently, these effects were also more pronounced than those observed with JQ1 in the OCI-AML2 AML cell line. Figure 9B ), with IC50 values ​​of 3.8 nM-10 nM. Importantly, treatment with the binder alone showed significantly lower toxicity than SUTAC ( Figures 9B-9C ).

[0270] BRD4 targeting SUTAC can alter the ability of BRD4 to activate c-MYC-dependent malignant pro-malignant programs in hematological cancer cells, leading to cell death.

[0271] Example 18: AR-targeted SUTAC reduces AR protein levels in prostate cancer cells To establish SUTAC as a widely applicable modality against other protein targets, an androgen receptor (AR)-targeting SUTAC was developed. SUTAC is used to induce SUMOylation of AR, aiming to alleviate excessive AR activity associated with promoting cancer progression. To this end, an AR-targeting SUTAC molecule based on the PIAS1 binding agent was developed. Figure 10A The conjugation with the AR binder significantly inhibited binding to PIAS1, as no binding to PIAS1 was observed at 20 μM, and no reaction with the DTNB compound was detected even at 200 μM. The activity of this SUTAC was evaluated because it may still be manipulated by other PIAS proteins. AR-targeted SUTAC treatment reduced the protein level of AR in the LNCaP prostate cancer cell line. Figure 10B ) and the level of overexpressed flag-labeled AR ( Figure 10C ).

[0272] AR-targeting SUTAC promotes AR degradation in prostate cancer cells, demonstrating the broad utility and versatility of the SUTAC approach.

[0273] Example 19: SUMOylation of recombinant BRD4 is enhanced and drives chromatin expulsion of BRD4 via ID-1 (1-26) SUTAC. Objective: To further verify that ID-1 SUTAC can enhance the SUMOylation of recombinant BRD4 by PIAS4 in a purified reaction.

[0274] Pre-incubate PIAS4 or the mediator with ID-1 SUTAC or DMSO controls. Then, incubate them with SUMO1,2 and SUMO-E1 (SAE1 / 2) with or without SUMO-E2 enzyme (UBC9), with or without recombinant BRD4. The samples are then subjected to Western blotting. It was found that BRD4 is SUMOylated by ID-1 SUTAC only when all the required SUMOylating enzymes are present. Figure 11A ).

[0275] When examining how ID-1 SUTAC altered BRD4 and cMYC in OCI-AML2 cells (which exhibited significant growth retardation after 72 hours of ID-1 SUTAC treatment), it was found that while cMYC levels decreased, BRD4 levels did not change significantly. This suggests that the downregulation of cMYC may be driven by the dissociation of BRD4 from chromatin. Indeed, ID-1 treatment led to an increase in cytoplasmic BRD4 levels, accompanied by a decrease in its chromatin levels. Figure 11B and Figure 11C This differs from the way classic PROTACs work for BRD4 (e.g., MZ1), highlighting a different mechanism of action than PROTACs.

[0276] Example 20: Electrophilic screening for discovering PIAS4 and PIAS1 binders and medicinal chemistry activities for binder optimization. Objective: To identify common binding agents for PIAS family proteins.

[0277] Two PIAS isoforms, PIAS1 and PIAS4, were screened from a library of approximately 1500 chloroacetamides. The binding (…) was estimated using liquid chromatography-mass spectrometry (LC-MS). Figure 12 Generally, PIAS4 is considerably more reactive than PIAS1 and exhibits a much higher hit rate. However, a small group of compounds showed broad labeling for both proteins. The vast majority of these hits were based on aminothiazole-based chloroacetamides, which are known from previous screenings to be inherently reactive compounds for labeling most proteins. However, a large group of thiophene-based chloroacetamide fragments appeared as hits. Thiophene compounds were classified into three classes based on the substituents adjacent to the chloroacetamide: amides, esters, and nitriles. While nitriles and esters primarily labeled PIAS4, amide-containing thiophenes showed significant binding to both isoforms. Therefore, starting with compound 1, an amide-containing thiophene scaffold was selected.

[0278] Due to the high reactivity of PIAS4, this protein was used for optimization activities. The optimization activities were conducted in two phases: A. Extension of the aromatic system surrounding thiophene: extending the ring system and adding substituents at multiple positions ( Figure 13A Several substituents were experimented with to obtain compounds 1-12, which bound PIAS4 10 times more effectively than compound 1. Chlorinated substituents were then systematically added to the fused phenyl groups of compounds 1-12 (compounds 1-16-1-19), with only one permissible position shown (compound 1-18). Further improvement was obtained by replacing the chlorination with a methyl group (compound 1-40).

[0279] B. Extension from amides – First, test whether the amide position can be modified. The corresponding acids are almost unreactive, and methylation of the amide is permitted, but dimethylation is not. Therefore, it is deduced that several primary amines can be coupled with compounds 1-36 to produce active binders ( Figure 13AA high-throughput experiment was established in which 1-36 was coupled with a µl-scale primary amine library and the crude reaction for binding to PIAS4 was tested. Since acid 1-36 is virtually inactive, any labeling was expected to be generated by the resulting amide and was normalized to the measured yield of the coupling reaction. From the highest-scoring amine, a benzylglycine derivative was chosen because it would serve as a scaffold easily modified with linkers and recruiters targeting the target protein. This yielded compound 1-39, which was 6-fold more effective than compound 1.

[0280] Finally, the features from compounds 1-40 and 1-39 were combined to obtain compound 1-41. Figure 13A Compound 1-41 was 100 times more effective than compound 1. Introducing an alkyne at the para position of the benzyl group (compound 1-42) resulted in a slight improvement in efficacy. Finally, the effects of introducing a PEG handle (1-44) and the BRD4 binder JQ1 (ID-2) on binding to PIAS4 were tested. Figure 13B Compared to compounds 1-41, the conjugates exhibited weakened binding to PIAS4, but still labeled PIAS4 on a minute timescale, making them potential candidates for targeting PIAS4 recruiters.

[0281] The effect of changes in the intrinsic reactivity of the electrophile on the binding of compounds to PIAS4 was determined. To this end, reactivity assays were performed against N-acetylcysteine ​​methyl ester (NAC). Compounds 1-41 were found to be 6-7 times more reactive to NAC than compound 1, while showing a >100-fold difference in binding rate to PIAS4. These results indicate that enhanced recognition of PIAS4 plays a significant role in the increased efficacy from compound 1 to compounds 1-41. On the other hand, ID-2 was only 33% more reactive than compound 1, indicating that enhanced reactivity only slightly contributes to the improved binding observed along this series.

[0282] Example 21: Identification of PIAS4 binding sites Objective: To identify the binding sites of compounds in PIAS4.

[0283] To identify the binding sites of compounds in PIAS4, the protein was incubated with compounds 1-41 and digested with trypsin, and the binding sites were identified using LC-MS / MS. In the unmodified peptides, peptides containing residues 322-330 were specifically depleted in the compound-treated samples, pointing to Cys326 as the modification site. Furthermore, peptides modified by compounds 1-41 were clearly identified, thus confirming Cys326 as the target site. Figure 14A ).

[0284] PIAS4 was crystallized with compounds 1-41 to elucidate the structure of the complex and confirm the binding site. Wild-type PIAS4 was not sufficiently soluble to allow for crystallography. Therefore, PROSS(1) was used to design stable mutants of PIAS4 (126-411). After inputting the protein sequence and α-sheet-derived structures, focus was placed on mutant number 7, which contained five mutations: N140D / C405R / M141V / S388N / I291C. The mutations were visually examined on the α-sheet structure and found to be plausible except for I291C, which replaced the surface isoleucine with a nonpolar cysteine. To consider alternatives, the sequence of PIAS4 was compared with sequences of other PIAS isotypes. It was found that they all contained lysine at that position. Therefore, the N140D / C405R / M141V / S388N / I291K mutant was selected. Following the cloning of the mutant gene, it was expressed in bacteria, yielding approximately 10-fold higher yields compared to the wild type. Differential scanning fluorescence (DSF) experiments revealed that the mutant was 10°C more stable than the wild type, and for both proteins, binding to compound 1-41 destabilized the proteins by 3-4°C.

[0285] The structures of the mutated PIAS4 and compounds 1-41 were determined at a resolution of 2.2 Å. Figure 14B As predicted by proteomics data, this compound binds to Cys326. To bind to cysteine, the compound substitutes the ring containing residues 324-333. Figure 14B (Left). The displacement of the ring exposed the hydrophobic bag ( Figure 14B (Right), chloroacetamide and thiophene scaffolds are inserted into a hydrophobic bag and form hydrophobic interactions with the ring and the α-helix containing residues 350-359. This perturbation of protein folding can explain the destabilization observed in the DSF. Although hydrogen in the amide group is essential for binding, the crystal structure does not indicate hydrogen bonding in the complex. Furthermore, besides crystal contact, the significantly enhanced benzylglycine does not form any direct interactions. It is possible that these two features contribute to binding either by enabling the compound to adopt the conformation necessary for binding or by participating in the 324-333 ring kinetics prior to covalent bond formation.

[0286] Example 22: Derivatives of compounds 1-41 (compounds 1-43) bind to PIAS protein in cells Objective: To verify that the PIAS protein is indeed the cellular target of the optimized binding compound 1-41.

[0287] To verify that the PIAS protein is indeed the cellular target of the optimized binding compound 1-41, an alkyne-modified derivative compound 1-42 was prepared. Figure 13ACompound 1-42 allows for the identification of its interacting proteins via proteomics, with compound 1-43 used as a competitive agent. Specifically, Daudi cells were incubated with compound 1-42, and biotin was attached to the labeled proteins using CuAAC, followed by pull-down, trypsinization, and LC-MS / MS. Targets labeled with 1-42 were identified relative to a DMSO-treated control, and the proteins obtained were compared by pre-incubating the lysates before incubation with 1-42. Figure 15 The targets labeled 1-43 (chlorine competitive agents) were identified. Figure 13A In fact, PIAS4 and PIAS2 (PIAS1 was also identified, but the difference was not significant) were identified in a large pool of proteins pulled down (1032 proteins). As expected, the competition with 1-43 pre-incubated proteins was an indicator of how many of these proteins were labeled, and only 17 proteins were identified in the competing data, one of which was PIAS4. Many off-target effects were observed in common reactive proteins, such as GSTO1 and selenoproteins, in many experiments.

[0288] Example 23: BRD4-targeted SUTAC (ID-2) enhances the SUMOylation of recombinant BRD4 and its downstream target c-MYC. Objective: To test the ability of compounds 1-41 to function in a SUTAC environment.

[0289] Compound 1-41 is conjugated with JQ1 targeting BRD4 to obtain ID-2 ( Figure 13B To assess its ability to promote BRD4 SUMOylation, an in vitro SUMOylation assay was performed. Using a purified reaction containing recombinant BRD4, SUMO, and the essential enzymes of the SUMOylation pathway, ID-2 (SUTAC) was observed to significantly enhance BRD4 SUMOylation only in the presence of all essential SUMOylating enzymes (including PIAS4). Figure 16A This confirms that compound 1-41 is a potential agent for inducing its binding with PIAS4, which is adjacent to POI.

[0290] To examine the ability of ID-2 to affect the cellular levels and function of BRD4, different cancer cell lines (Daudi, OCI-AML2, RAMOS, and RPMI-8226) were treated overnight with increased concentrations of ID-2. Indeed, ID-2 treatment resulted in a significant reduction in cMYC levels across all tested lines. Figure 16B It is noteworthy that the decrease in cMYC was significantly more pronounced than the decrease induced by JQ1-1 alone. Figure 16C ).

[0291] Importantly, no decrease in BRD4 levels was observed in any cell line, suggesting that the underlying mechanism of cMYC downregulation is not solely driven by degradation, at least not in OCI-AML2 cells. Notably, in Daudi cells, where a decrease in cMYC levels was associated with a decrease in BRD4 levels, proteasome inhibition completely eliminated the ID-2-induced decrease in BRD4 levels. Figure 16D ).

[0292] Furthermore, in PIAS1 and PIAS4 dual knockout (DKO) cells, the ID-2-induced reduction in BRD4 levels was partially but consistently reversed. This validates the mechanism of action of SUTAC and highlights the importance of PIAS in mediating its effects in cells. Figure 16E ).

[0293] Example 24: BRD4-targeted SUTAC (ID-3)-enhanced SUMOylation of Recombinant BRD4 Although the hydrogen in the amide group of the binder is crucial for the covalent bonding with recombinant PIAS4 (126-411) Figures 13A-13B However, the crystal structure did not show evidence of hydrogen bonding in the complex. Figure 14B Therefore, it is assumed that binders with methyl groups can still covalently or non-covalently bind full-length PIAS4. Results reveal that JQ1-based SUTAC (ID-3, ...) incorporating such binders... Figure 17A It enhances the SUMOylation of BRD4 by full-length PIAS4 even more effectively than ID-2. Figure 17B It is worth noting that ID-3 also induces the poly-SUMOylation of BRD4. Figure 17B This demonstrates the utility of methyl derivatives of compounds 1-41 (i.e., 1-46) in the SUTAC method.

[0294] Example 25: P300-targeted SUTAC reduces cMYC levels and cancer cell proliferation. Objective: To test the versatility of compounds 1-41 and their methyl derivatives in altering other targets.

[0295] To further test the versatility of compound 1-41 and its methyl derivative 1-46 in altering other targets, the target of P300 / CBP was chosen. P300 is a histone acetyltransferase that drives cancer progression, and its activity is known to be negatively regulated by SUMOylation. SUTAC (ID-4, based on compound 1-41 and the P300 / CBP inhibitor GNE-207) was developed. Figure 18A In fact, it was found that P300 was targeted and altered in SUTAC in two cancer cell lines, Daudi and OCI-AML2. Figure 18B Importantly, this also led to a decrease in cMYC levels, a downstream gene activated by P300, indicating that P300 activity was altered. Importantly, treatment of OCI-AML2 cells with ID-4 for 72 hours... Figure 18C This leads to reduced growth, while 1-41 has no significant effect on their growth.

[0296] Finally, when comparing ID-4 with the methylamine derivative (compound 10) of the P300 inhibitor GNE-207, Figure 18D ) and P300 targeting SUTAC(ID-5) based on methyl derivatives of compounds 1-41. Figure 18D When considering the effects of ID-5, we observed that it was most effective in reducing cMYC levels. Figure 18E ).

[0297] Example 26: Synthesis of ID-2-based SUTAC (BRD4-targeted SUTAC) Scheme 11 presents the synthesis of SUTAC with the structure of the conjugate ID-2. 10 mg of molecule 1-44 was dissolved in 600 µl of dichloromethane and 140 µl of trifluoroacetic acid. Complete deprotection was confirmed by LC-MS after tumbling at room temperature for 40 minutes. The solution was evaporated under an argon atmosphere, and dichloromethane was added and evaporated three times to thoroughly remove trace amounts of TFA. 2.5 mg (4.12 μmol) of the product was used for coupling.

[0298] For coupling, 6 mg of JQ1 acid (14.9 μmol) was dissolved in dichloromethane and mixed with an amine. 50 µl of 0.2 M EDC / 0.6 M DIPEA (10 µmol EDC, 30 µmol DIPEA) in DCM was added to the mixture. The reaction was carried out at room temperature for 24 hours, the DCM was evaporated, and the product was purified by HPLC. 1.32 mg of pure RG104 was obtained (yield 32%).

[0299] Synthesis of 1-44: Synthesis schemes: (a) KOH, p-xylene, 25℃, 12 h (b) PtO2, MeOH, 25℃, 16 h (c) EDCI, Py, 30℃, 2 h.

[0300] N-[2-[2-[2-[2-[2-(4-cyanophenyl)ethoxy]ethoxy]ethoxy]ethyl]tert-butyl carbamate: A mixture of N-[2-[2-(2-bromoethoxy)ethoxy]ethyl]carbamate tert-butyl ester (1.59 g, 1.5 equivalents), 4-(2-hydroxyethyl)benzonitrile (500 mg, 1 equivalent), KOH (953.05 mg, 5 equivalents), and tetrabutylammonium bromide (547.60 mg, 0.5 equivalents) in p-xylene (10 mL) was degassed and purged three times with N2, and then stirred at 25 °C under N2 atmosphere for 12 h. The reaction mixture was quenched by NH4Cl (100 mL) and extracted with ethyl acetate (100 mL × 2). The combined organic layers were washed with brine (50 mL × 2), dried over Na2SO4, filtered, and concentrated under reduced pressure to give the residue. The residue was purified by preparative HPLC to give a yellow oil (430 mg, 33.44% yield).

[0301] LC-MS (m / z): Calculated value: 378.22; Measured value: 379.1 [M+H] + .

[0302] N-[2-[2-[2-[2-[4-(aminomethyl)phenyl]ethoxy]ethoxy]ethoxy]ethyl]carbamate tert-ethyl Butyl ester: PtO2 (144.00 mg, 1.20 equivalent) was added to a solution of N-[2-[2-[2-[2-(4-cyanophenyl)ethoxy]ethoxy]ethoxy]ethyl]carbamate tert-butyl ester (200 mg, 1 equivalent) in MeOH (5 mL). The mixture was degassed and purged three times with H2, and then stirred at 25 °C under an H2 (30 Psi) atmosphere for 16 h. The reaction mixture was filtered and the mother liquor was concentrated. The residue was purified by preparative HPLC to provide a white solid (150 mg, 74.21% yield).

[0303] LC-MS (m / z): Calculated value: 382.25; Measured value: 383.1 [M+H] + .

[0304] 1H NMR (400 MHz, DMSO) δ 7.36 - 7.31 (m, 2H), 7.28 - 7.22 (m, 2H), 6.78 (br t, J = 5.2 Hz, 1H), 3.88 (s, 2H), 3.52 - 3.49 (m, 10H), 3.38 (br t, J = 6.0 Hz, 3H), 3.07 (q, J = 6.0 Hz, 2H), 2.81 (t, J = 7.0 Hz, 2H), 1.38 (s, 9H).

[0305] N-[2-[2-[2-[2-[4-[[[2-[[2-[(2-chloroacetyl)amino]-5-methyl-benzothiophene-3-carbonyl]] [Amino]acetyl]amino]methyl]phenyl]ethoxy]ethoxy]ethoxy]ethyl]tert-butyl carbamate: Add EDCI (75.18 mg, 1.5 equivalents) to a solution of N-[2-[2-[2-[2-[4-(aminomethyl)phenyl]ethoxy]ethoxy]ethoxy]ethyl]carbamate (100 mg, 1 equivalent) and 2-[[2-[(2-chloroacetyl)amino]-5-methyl-benzothiophene-3-carbonyl]amino]acetic acid (89.09 mg, 1 equivalent) in Py (2.5 mL). Stir the mixture at 30 °C for 2 h. Concentrate the reaction mixture under reduced pressure to remove the solvent. Purify the residue by preparative HPLC to provide a brown solid (41.69 mg, 22.61% yield).

[0306] LC-MS (m / z): Calculated value: 704.26; Measured value: 705.1 [M+H] + .

[0307] 1H NMR (400 MHz, CDCl3) δ 12.91 (s, 1H), 7.81 (s, 1H), 7.71 (d, J =8.2 Hz, 1H), 7.27 - 7.17 (m, 6H), 6.50 (br d, J = 4.2 Hz, 1H), 5.05 (br s,1H), 4.51 (d, J = 5.6 Hz, 2H), 4.30 - 4.25 (m, 4H), 3.69 (t, J = 7.0 Hz, 2H), 3.65 - 3.59 (m, 8H), 3.50 (t, J = 5.1 Hz, 2H), 3.23 (q, J = 5.4 Hz, 2H), 2.90(t, J = 7.0 Hz, 2H), 2.54 (s, 3H), 1.44 (s, 9H).

[0308] Example 27: Synthesis of ID-3-based SUTAC (BRD4-targeted SUTAC) Scheme 12 presents the synthesis of SUTAC with the structure of the conjugate ID-3. The amine derivative (a) from Scheme 12 and the JQ1 acid were dissolved in DMSO to a final volume of 200 mM. Oxyma pure was dissolved in DMS to a final volume of 0.4 M, and EDC was dissolved in DMSO to a final volume of 0.4 M. 18 µl of the EDC solution and 36 µl of the Oxyma pure solution were premixed and added to 30 µl of the JQ1 acid (6 µmol). The mixture was incubated at room temperature for 5 min, followed by the addition of 57 µl of DMF, 3 µl of DIPEA, and 20 µl of the amine derivative (a) (4 µmol). The reaction was carried out at room temperature for 3 h, after which the product was purified by HPLC. Yield: 2.65 mg (66%).

[0309] Example 28: Synthesis of ID-4-targeted SUTAC (P300 / CBP-targeted SUTAC) Scheme 13 presents the synthesis of SUTAC with the structure of the conjugate ID-4. 2.3 mg of compound (b) (4.62 µmol) from Scheme 13 was dissolved in 50 µl of DMF. A mixture containing 13.9 µl of 0.4 M EDC (DMSO) and 27.8 µl of 0.4 M oxyma pure (DMF) was added, followed by incubation at room temperature for 5 min. The sample was diluted to 55 mL with 10 mM DIPEA in DMF, and 3.6 mg of the deprotected amine (a) was added. The reaction was carried out at 37 °C for 48 h. HCl was then added to 20 mM, and the sample was evaporated and purified by HPLC. 1.39 mg of the pure product was obtained (yield 28%).

[0310] Example 29: Synthesis of ID-5-targeted SUTAC (P300 / CBP-targeted SUTAC) Scheme 14 presents the synthesis of SUTAC with the structure of the conjugate ID-5. Acid (b) from Scheme 14 was dissolved in DMSO to a final concentration of 200 mM. 30 µl of 0.2 M EDC / DMSO and 30 µl of 0.4 M Oxyma / DMF were premixed and added to 25 µl of the acid (b) solution from Scheme 14 (5 μmol). After 5 minutes at room temperature, 4.3 µl of DIPEA and 30 µl of amine (a) from Scheme 14 (200 mM in DMSO) were added. The reaction was carried out at room temperature for 24 hours, after which the sample was evaporated and purified by HPLC. Yield: 1.02 mg (19%).

[0311] Example 30: Synthesis of Compound 10 (P300 / CBP Inhibitor) Schemes 15A and 15B present the synthesis of compound 10.

[0312] Option 15A) Synthesis schemes: (a) Py, 0~25℃, 2 h; (b) Pd(dppf)Cl2, K2CO3, THF, H2O, 80℃, 3 h; (c) Pd2(dba)3, KOAc, dioxane, 120℃, 8 h; (d) XPhos Pd G2, XPhos, CsCO3, dioxane, H2O, 90℃, 1 h. (8-Chloro-3-isoquinolinyl)trifluoromethanesulfonate: The 8-chloroisoquinoline-3-ol (2 g, 1 equivalent) in Py (80 mL) was cooled to 0 °C and carefully treated at 0 °C with trifluoromethanesulfonyl trifluoromethanesulfonate (5.03 g, 1.6 equivalent). The mixture was allowed to be heated to 25 °C and stirred for 2 h. The reaction mixture was concentrated under reduced pressure to remove the solvent. The residue was purified by column chromatography to provide a yellow solid (2.91 g, 83.85% yield).

[0313] LC-MS (m / z): Calculated value: 310.96; Measured value: 312.1 [M+H] + .

[0314] methyl 5-(8-chloro-3-isoquinolinyl)pyridine-2-carboxylate: A mixture of (8-chloro-3-isoquinolinyl)trifluoromethanesulfonate (2.91 g, 1 equivalent), methyl 5-(4,4,5,5-tetramethyl-1,3,2-dioxaborhecyclopentan-2-yl)pyridine-2-carboxylate (3.68 g, 1.5 equivalent), Pd(dppf)Cl2 (683.19 mg, 0.1 equivalent), and K2CO3 (3.87 g, 3 equivalent) in THF (35 mL) and H2O (7 mL) was stirred at 80 °C for 3 h. The reaction mixture was poured into 10 mL of H2O, filtered, and collected. The collected solid was washed with 15 mL of THF and dried under high vacuum. The residue was purified by filtration to provide a gray solid (2.1 g, 75.29% yield).

[0315] LC-MS (m / z): Calculated value: 298.72; Measured value: 299.1 [M+H] + .

[0316] 5-[8-(4,4,5,5-tetramethyl-1,3,2-dioxaborhecyclopentan-2-yl)-3-isoquinolinyl]pyridine-2- Methyl formate: To a mixture of methyl 5-(8-chloro-3-isoquinolinyl)pyridine-2-carboxylate (1 g, 1 equivalent), 4,4,5,5-tetramethyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborhexacyclopentan-2-yl)-1,3,2-dioxaborhexacyclopentane (1.28 g, 1.5 equivalent) in dioxane (30 mL), tricyclohexylphosphine (375.50 mg, 0.4 equivalent), KOAc (985.62 mg, 10.04 mmol, 3 equivalent), and Pd2(dba)3 (306.54 mg, 0.1 equivalent) was added. The mixture was degassed and purged three times with N2, and then stirred at 120 °C under N2 atmosphere for 8 h. The reaction mixture was poured into 20 mL of dioxane, collected by filtration, and dried under high vacuum. The residue was purified by filtration to provide a yellow solid (600 mg, 45.93% yield).

[0317] LC-MS (m / z): Calculated value: 390.18; Measured value: 391.1 [M+H] + .

[0318] 5-[8-(5-acetyl-1-tetrahydropyran-4-yl-6,7-dihydro-4H-pyrazolo[4,3-c]pyridin-3-yl)-3- [Isoquinolinyl]pyridine-2-carboxylic acid: Add methyl 5-[8-(4,4,5,5-tetramethyl-1,3,2-dioxaborhexylcyclopentan-2-yl)-3-isoquinolinyl]pyridine-2-carboxylate (500 mg, 1 equivalent) and 1-(3-iodo-1-tetrahydropyran-4-yl-6,7-dihydro-4H-pyrazolo[4,3-c]pyridin-5-yl)acetone (480.74 mg) to a mixture of dicyclohexyl-[2-(2,4,6-triisopropylphenyl)phenyl]phosphine (61.08 mg, 0.1 equivalent), [2-(2-aminophenyl)phenyl]-chloro-palladium; dicyclohexyl-[3-(2,4,6-triisopropylphenyl)phenyl]phosphine (100.81 mg, 0.1 equivalent) and Cs2CO3 (1.25 g, 3 equivalent) to a mixture of dicyclohexyl-[2 ...61.08 mg, 0.1 equivalent), [2-(2-aminophenyl)phenyl]-chloro-palladium; dicyclohexyl-[3-(2,4,6-triisopropylphenyl)phenyl]phosphine (100.81 mg, 0.1 equivalent) and Cs2CO3 (1.25 g, 3 equivalent) and methyl 1-(3-iodo-1-te A solution of 1 mg (1 equivalent) of dioxane (20 mL) and H₂O (10 mL) was prepared. The reaction mixture was heated to 90 °C for 2 h. The reaction mixture was quenched with H₂O (20 mL), extracted with ethyl acetate (20 mL × 2), the aqueous solution was adjusted to pH 6 with saturated FA, extracted with ethyl acetate (20 mL × 2), dried over Na₂SO₄, filtered, and concentrated under reduced pressure to give the residue. The residue was purified by preparative HPLC to give an off-white solid (36.77 mg, 5.25% yield).

[0319] LC-MS (m / z): Calculated value: 497.21; Measured value: 498.1 [M+H] + .

[0320] 1 H NMR (400 MHz, DMSO) δ 10.01 (s, 1H), 9.55 (br s, 1H), 8.83 - 8.66(m, 2H), 8.19 (br d, J = 8.0 Hz, 1H), 8.09 (br d, J = 8.0 Hz, 1H), 7.93 (brs, 1H), 7.80 - 7.67 (m, 1H), 4.59(br s, 2H), 4.55 - 4.43 (m, 1H), 4.08 - 4.00(m, 2H), 3.90 - 3.80 (m, 2H), 3.58 - 3.51 (m, 2H), 2.99 (br t, J = 5.2 Hz,1H), 2.87 (br d, J = 4.8 Hz, 1H), 2.21 - 2.10 (m, 4H), 2.05 - 1.94 (m, 3H).

[0321] Option 15B) The product of Scheme 15A (structure (a) in Scheme 15B) was dissolved in DMSO to a final concentration of 200 mM. 60 µl of 0.2 M EDC / DMSO and 60 µl of 0.4 M Oxyma / DMF were premixed and added to 50 µl of a 10 µmol solution of compound (a) from Scheme 15A. After 5 minutes at room temperature, 17.2 µl of DIPEA and 50 µl of 1 M methylamine hydrochloride in DMSO were added. The reaction was carried out at room temperature for 24 hours, after which the sample was evaporated and purified by HPLC. Yield: 1.21 mg (24%).

[0322] Example 31: Synthesis of SUMOylation compound used as a SUMOylation enzyme binding group in the chimera of the present invention.

[0323] Synthesis of 1-2 Synthesis schemes: (a) TEA / EtOH (b) NaHCO3 / THF / H2O / 0℃ 2-Amino-N-methylthiophene-3-carboxamide: 2-cyano-N,N-dimethylacetamide (368.28 mg, 3.28 mmol) and TEA (664.69 mg, 6.57 mmol, 914.29 μL) were added to a solution of 1,4-dithiaran-2,5-diol (500 mg, 3.28 mmol) in EtOH (15 mL). The mixture was stirred at 70 °C for 16 h. After cooling to room temperature, the mixture was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 3 / 1) to give a yellow solid (320 mg).

[0324] LC-MS (m / z): Calculated value: 156.04; Measured value: 157.01 [M+H] + .

[0325] 1 H NMR (400 MHz, DMSO) δ 6.75 (d, J = 5.6 Hz, 1H), 6.48 (s, 2H), 6.32(d, J = 5.6 Hz, 1H), 2.96 (s, 6H).

[0326] 2-(2-Chloroacetamido)-N,N-Dimethylthiophene-3-carboxamide: At 0 °C, 2-cyano-N,N-dimethylacetamide (79.62 mg, 704.92 μmol, 56.07 μL) was added to a solution of 2-amino-N-methylthiophene-3-carboxamide (100 mg, 587.43 μmol), NaHCO3 (98.70 mg, 1.17 mmol, 45.69 μL) in THF (1 mL) and H2O (1 mL). The mixture was stirred at 20 °C for 0.5 h. The mixture was concentrated under vacuum to give a residue. The residue was purified by preparative HPLC (FA conditions) to give a white solid (62.49 mg).

[0327] LC-MS (m / z): Calculated value: 246.02; Measured value: 247.1 [M+H] + .

[0328] 1 H NMR (400 MHz, DMSO) δ 11.36 (s, 1H), 7.15-7.03 (m, 2H), 4.49 (s,2H), 3.01 (s, 6H).

[0329] Synthesis of 1-18. Synthesis schemes: (a) NaH / DMSO / 15~100℃ (b) NaOH / DMSO / 100℃ (c) H2SO4 / 60℃ (d) / NaHCO3 / THF / H2O / 0℃ N-(5-chloro-3-cyano-benzothiophene-2-yl)carbamate: A mixture of 2-(4-chloro-2-fluoro-phenyl)acetonitrile (5 g, 29.48 mmol, 1 equivalent) and NaH (2.83 g, 35.38 mmol, 30% purity, 1.2 equivalent) in DMSO (50 mL) was degassed and purged with N2, and then stirred at 15 °C under N2 atmosphere for 0.5 h. Ethyl N-(thiomethylene)carbamate (3.87 g, 29.48 mmol, 1 equivalent) was added. ， The mixture was then stirred at 100°C under a nitrogen atmosphere for 2.5 h. The reaction was not post-treated and was used directly in the next step.

[0330] LC-MS (m / z): Calculated value: 280.01; Measured value: 278.9 [MH].

[0331] 2-Amino-5-chloro-benzothiophene-3-carboxynitrile: A mixture of N-(5-chloro-3-cyano-benzothiophene-2-yl)carbamate (5 g, 17.81 mmol, 1 equivalent) and NaOH (2.14 g, 53.43 mmol, 3 equivalents) in DMSO (50 mL) was degassed and purged three times with N2. The mixture was then stirred at 100 °C under N2 atmosphere for 2 h. The reaction liquid was filtered, and the solid was the crude product. The product was given as a yellow solid (3.7 g, 17.73 mmol, 99.56% yield).

[0332] LC-MS (m / z): Calculated value: 207.99; Measured value: 206.81 [MH].

[0333] 1 H NMR (400 MHz, DMSO) δ 7.95 (br s, 2H), 7.64 (d, J = 7.9 Hz, 1H), 7.29 (d, J= 7.9 Hz, 1H), 7.08 (t, J = 7.9 Hz, 1H).

[0334] 2-Amino-5-chloro-benzothiophene-3-carboxamide: H₂SO₄ (3 mL) was added to a solution of 2-amino-5-chloro-benzothiophene-3-carboxynitrile (500 mg, 2.40 mmol, 1 equivalent). The mixture was stirred at 60 °C for 1 h. The reaction solution was poured into a NaHCO₃ solution (100 mL) and then extracted with ethyl acetate (20 mL × 2). The combined organic layers were washed with brine (40 mL), dried over Na₂SO₄, filtered, and concentrated under reduced pressure to give the residue. The residue was analyzed by preparative HPLC (column: Waters Xbridge Prep OBD C18150). 40mm 10 μm; mobile phase: [water (NH4HCO3)-ACN]; gradient: 6%-36% over 15 min. B) Purification. The product was obtained as a white solid (180 mg, 794.06 μmol, 33.14% yield).

[0335] LC-MS (m / z): Calculated value: 226; Measured value: 226.9 [M+H] + .

[0336] 1 H NMR (400 MHz, DMSO) δ 7.59 (dd, J = 0.8, 7.8 Hz, 1H), 7.43 - 7.27(m, 2H), 7.22 (dd, J = 0.8, 7.8 Hz, 1H), 7.00 (t, J = 7.8 Hz, 1H), 6.58 (s, 2H).

[0337] 5-Chloro-2-[(2-chloroacetyl)amino]benzothiophene-3-carboxamide: NaHCO3 (148.24 mg, 1.76 mmol, 68.66 μL, 4 equivalents) was added to a solution of 2-amino-5-chloro-benzothiophene-3-carboxamide (100 mg, 441.15 μmol, 1 equivalent) and 2-chloroacetyl chloride (59.79 mg, 529.38 μmol, 42.16 μL, 1.2 equivalents) in THF (1 mL) and H2O (1 mL). The mixture was stirred at 0 °C for 1 h. The reaction mixture was concentrated under reduced pressure to remove the solvent. The product was given as a white solid (12.46 mg, 39.46 μmol, 8.94% yield).

[0338] LC-MS (m / z): Calculated value: 301.97; Measured value: 300.9 [MH] + .

[0339] 1 H NMR (400 MHz, DMSO) δ 11.13 (br s, 1H), 8.03 (s, 1H), 7.92 - 7.89(m, 1H), 7.70 (br s, 1H), 7.42 (dd, J = 0.9, 7.8 Hz, 1H), 7.31 - 7.26 (m,1H), 4.54 (s, 2H).

[0340] Synthesis of 1-19. Synthesis schemes: (a) NaH / DMSO / 15~100℃ (b) NaOH / DMSO / 100℃ (c) H2SO4 / 60℃ (d) / NaHCO3 / THF / H2O / 0℃ N-(4-chloro-3-cyano-benzothiophene-2-yl)carbamate: A mixture of 2-(2-chloro-6-fluoro-phenyl)acetonitrile (1 g, 5.90 mmol, 1 equivalent), N-(thiomethylene)carbamate (773.38 mg, 5.90 mmol, 1 equivalent), and NaH (283.02 mg, 7.08 mmol, 60% purity, 1.2 equivalent) in DMSO (10 mL) was degassed and purged with N2. The mixture was then stirred at 15 °C under N2 atmosphere for 0.5 h. N-(thiomethylene)carbamate (773.38 mg, 5.90 mmol, 1 equivalent) was added. ，The mixture was then stirred at 100°C under a nitrogen atmosphere for 2.5 h. The reaction was not post-treated and was used directly in the next step.

[0341] LC-MS (m / z): Calculated value: 280.01; Measured value: 279.0 [MH] + .

[0342] 2-Amino-4-chloro-benzothiophene-3-carboxynitrile: A mixture of N-(4-chloro-3-cyano-benzothiophene-2-yl)carbamate (1 g, 3.56 mmol, 1 equivalent) and NaOH (5 M, 15 mL, 21.05 equivalent) in DMSO (10 mL) was degassed and purged three times with N2. The mixture was then stirred at 100 °C under N2 atmosphere for 3 h. 50 mL of water was added to the reaction solution, the mixture was cooled to 15 °C, filtered, and the residue was the crude product (coarse product). The product was given as a yellow solid (720 mg, 3.45 mmol, 96.86% yield).

[0343] LC-MS (m / z): Calculated value: 207.99; Measured value: 206.81 [MH] + .

[0344] 1 H NMR (400 MHz, DMSO) δ 7.95 (br s, 2H), 7.65 (br d, J = 7.9 Hz, 1H), 7.29 (br d, J = 7.9 Hz, 1H), 7.08 (br t, J M = 7.8 Hz, 1H).

[0345] 2-Amino-4-chloro-benzothiophene-3-carboxamide: Add 3 mL of H₂SO₄ to a solution of 2-amino-4-chloro-benzothiophene-3-carboxynitrile (300 mg, 1.44 mmol, 1 equivalent). Stir the mixture at 60 °C for 0.5 h. Pour the reaction solution into a container with EA (10 3) The residue was separated into 30 mL of NaHCO3 solution. The residue was analyzed by preparative HPLC (column: Waters Xbridge Prep OBD C18 150). 40mm 10 μm; mobile phase: [water (NH4HCO3)-ACN]; gradient: 5%-35% over 15 min. B) Purification. The product was obtained as a white solid (25 mg, 110.29 μmol, 7.67% yield).

[0346] LC-MS (m / z): Calculated value: 226; Measured value: 225.1 [MH] + .

[0347] 1 H NMR (400 MHz, DMSO) δ 7.58 (dd, J = 0.8, 7.8 Hz, 1H), 7.42 - 7.28(m, 2H), 7.22 (dd, J = 0.9, 7.8 Hz, 1H), 6.99 (t, J = 7.8 Hz, 1H), 6.58 (s, 2H).

[0348] 4-Chloro-2-[(2-chloroacetyl)amino]benzothiophene-3-carboxamide: NaHCO3 (37.06 mg, 441.15 μmol, 17.16 μL, 4 equivalents) was added to a solution of 2-amino-4-chloro-benzothiophene-3-carboxamide (25 mg, 110.29 μmol, 1 equivalent) in THF (1 mL) and H2O (1 mL). The mixture was stirred at 0 °C for 1 h. The reaction mixture was concentrated under reduced pressure to remove the solvent. The product was given as a white solid (8.67 mg, 27.74 μmol, 25.15% yield).

[0349] LC-MS (m / z): Calculated value: 301.97; Measured value: 300.9 [MH] + .

[0350] 1 H NMR (400 MHz, DMSO) δ 11.14 (br s, 1H), 8.04 (s, 1H), 7.91 (d, J =7.9 Hz, 1H), 7.71 (br s, 1H), 7.43 (d, J = 7.8 Hz, 1H), 7.32 - 7.27 (m, 1H), 4.55 (s, 2H).

[0351] Synthesis of 1-36. 143 mmol of chloroacetic acid (1.5 mmol) was dissolved in dichloromethane and mixed with 173 mg of EDC (0.9 mmol), 138 mg of HOBT hydrate (0.9 mmol), and 695 µl of DIPEA (4 mmol). After 30 minutes, 80 mg of 2-aminothiophene-3-carboxylic acid (0.56 mmol) was added, and the reaction was carried out at room temperature for 1 hour. The reaction mixture was washed twice with 1N HCl, followed by washing with brine and evaporation. The crude product was purified by HPLC to obtain 1-36 as a bright brown solid, with a total weight of 11.71 mg (yield 9.5%).

[0352] Synthesis of 1-39 Synthesis schemes: (a) NaOH, MeOH, H2O, 80℃, 2 h (b) HOBt, EDCI, TEA, DMF, 25℃, 12 h (c) TEA, DCM, 0℃, 1 h.

[0353] 2-Aminothiophene-3-carboxylic acid: A mixture of methyl 2-aminothiophene-3-carboxylate (1 g, 1 equivalent), NaOH (763.35 mg, 3 equivalents) in MeOH (10 mL), H₂O (2 mL) was degassed and purged three times with N₂, and then stirred at 80 °C for 2 h. The reaction mixture was concentrated under reduced pressure to remove the solvent. The reaction mixture was filtered and the mother liquor was concentrated. The residue was purified by preparative HPLC (NH₄HCO₃ conditions) to provide a yellow solid (800 mg, 87.84% yield).

[0354] LC-MS (m / z): Calculated value: 143; Measured value: 141.9 [MH] + .

[0355] 1 H NMR (400 MHz, DMSO) δ 7.31 - 6.33 (m, 3H), 6.04 (d, J = 5.6 Hz, 1H).

[0356] 2-Amino-N-[2-(benzylamino)-2-oxo-ethyl]thiophene-3-carboxamide: HOBt (424.73 mg, 1.5 equivalent) and EDCI (602.57 mg, 1.5 equivalent), TEA (636.13 mg, 875.00 μL, 3 equivalent), and 2-amino-N-benzyl-acetamide (344.09 mg, 1 equivalent) were added to a solution of 2-aminothiophene-3-carboxylic acid (300 mg, 875.00 μL, 3 equivalent) in DMF (5 mL). The mixture was stirred at 25 °C for 12 h. The reaction mixture was poured into water (50 mL) and extracted with ethyl acetate (50 mL × 2). The combined organic layers were washed with brine (40 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure to give the residue. The residue was purified by column chromatography to give a pink oil (120 mg, 19.79% yield).

[0357] LC-MS (m / z): Calculated value: 289.09; Measured value: 290.1 ​​[M+H] + .

[0358] N-[2-(benzylamino)-2-oxo-ethyl]-2-[(2-chloroacetyl)amino]thiophene-3-carboxamide: At 0 °C, TEA (62.95 mg, 3 equivalents) and 2-chloroacetyl chloride (46.84 mg, 2 equivalents) were added to a solution of 2-amino-N-[2-(benzylamino)-2-oxo-ethyl]thiophene-3-carboxamide (60 mg, 1 equivalent) in DCM (1.5 mL). The mixture was stirred at 0 °C for 1 h. The reaction mixture was concentrated under reduced pressure to remove the solvent. The residue was purified by preparative HPLC (NH4HCO3 conditions) to provide a yellow solid (27.06 mg, 35.67% yield).

[0359] LC-MS (m / z): Calculated value: 365.06; Measured value: 366.1 [M+H] + .

[0360] 1 H NMR (400 MHz, DMSO) δ 12.73 (s, 1H), 8.82 (t, J = 6.0 Hz, 1H), 8.51 (t, J = 6.0 Hz, 1H), 7.51 (d, J = 6.0 Hz, 1H), 7.34 - 7.26 (m, 4H), 7.25- 7.19 (m, 1H), 7.09 (d, J= 5.7 Hz, 1H), 4.58 (s, 2H), 4.30 (d, J = 6.0 Hz, 2H), 3.91 (d, J = 6.0 Hz, 2H).

[0361] Synthesis of 1-40. Synthesis schemes: (a) NH4Ac, AcOH, toluene, 60℃, 12 h (b) S, NaHCO3, EtOH, 80℃, 2 h (c) NaHCO3, THF / H2O, 0℃, 1 h.

[0362] (2Z)-2-cyano-2-(3-methylcyclohex-2-en-1-yl)acetamide: CH3COONH4 (8.92 g, 2.55 equivalents) and CH3COOH (8.15 g, 2.99 equivalents) were added to a solution of 3-methylcyclohexyl-2-en-1-one (5 g, 1 equivalent), 2-cyanoacetamide (10.88 g, 2.85 equivalents) in toluene (70 mL). The mixture was stirred at 60 °C for 12 h. The reaction mixture was poured into water (200 mL) and extracted with ethyl acetate (200 mL × 2). The combined organic layers were washed with brine (100 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure to give the residue. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 1 / 1, Rf = 0.2) to provide a yellow solid (3.5 g, 47.36% yield).

[0363] LC-MS (m / z): Calculated value: 176.09; Measured value: 175.1 [MH] + .

[0364] 2-Amino-5-methyl-benzothiophene-3-carboxamide: S (0.72 g, 7.91 equivalents) and NaHCO3 (2.4 g, 10.07 equivalents) were added to a solution of (2Z)-2-cyano-2-(3-methylcyclohexyl-2-en-1-yl)acetamide (500 mg, 1 equivalent) in EtOH (16 mL). The mixture was stirred at 80 °C for 2 h. The reaction mixture was filtered and the mother liquor was concentrated. The residue was purified by preparative HPLC (FA conditions) to provide a yellow solid (250 mg, 42.72% yield).

[0365] LC-MS (m / z): Calculated value: 206.05; Measured value: 207.1 [M+H] + .

[0366] 1 H NMR (400 MHz, DMSO) δ 7.75 - 7.67 (m, 2H), 7.59 - 7.49 (m, 2H),7.02 (s, 2H), 6.90 (d, J = 8.0 Hz, 1H), 2.40 - 2.37 (m, 3H).

[0367] 2-[(2-chloroacetyl)amino]-5-methyl-benzothiophene-3-carboxamide: NaHCO3 (61.09 mg, 3 equivalents) was added to a solution of 2-amino-5-methyl-benzothiophene-3-carboxamide (50 mg, 1 equivalent) and 2-chloroacetyl chloride (27.38 mg, 19.31 μL, 1 equivalent) in THF (0.5 mL) and H2O (0.5 mL). The mixture was stirred at 0 °C for 1 h. The residue was purified by filtration to provide a yellow solid (24.22 mg, 34.63% yield).

[0368] LC-MS (m / z): Calculated value: 282.02; Measured value: 283.1 [M+H]+.

[0369] 1 H NMR (400 MHz, DMSO) δ 12.55 (s, 1H), 7.89 - 7.76 (m, 4H), 7.17 (d, J = 8.0 Hz, 1H), 4.58 (s, 2H), 2.43 (s, 3H).

[0370] Synthesis of 1-41. Synthesis schemes: (a) EDCI, HOBt, TEA, DMF, 25℃, 1 h (b) NaHCO3, THF, H2O, 0℃, 1 h.

[0371] 2-Amino-N-[2-(benzylamino)-2-oxo-ethyl]-5-methyl-benzothiophene-3-carboxamide: HOBt (215.16 mg, 1.5 equivalent) and EDCI (305.24 mg, 1.5 equivalent), TEA (322.24 mg, 3 equivalent), and 2-amino-N-benzyl-acetamide (174.31 mg, 1 equivalent) were added to a solution of 2-amino-5-methyl-benzothiophene-3-carboxylic acid (220 mg, 1 equivalent) in DMF (2 mL). The mixture was stirred at 25 °C for 1 h. The reaction mixture was filtered and the mother liquor was concentrated. The residue was purified by preparative HPLC to provide a yellow solid (50 mg, 13.33% yield).

[0372] LC-MS (m / z): Calculated value: 353.12; Measured value: 354.1 [M+H] + .

[0373] 1 H NMR (400 MHz, DMSO) δ 8.43 (br t, J = 6.0 Hz, 1H), 7.60 - 7.52 (m,4H), 7.48 (d, J = 8.0 Hz, 1H), 7.38 - 7.15 (m, 5H), 6.87 (d, J = 8.2 Hz, 1H), 4.32 (d, J = 6.0 Hz, 2H), 3.95 (d, J = 5.6 Hz, 2H), 2.35 (s, 3H).

[0374] N-[2-(benzylamino)-2-oxo-ethyl]-2-[(2-chloroacetyl)amino]-5-methyl-benzothiophene-3- Formamide: At 0 °C, NaHCO3 (17.83 mg, 3 equivalents) and 2-chloroacetyl chloride (15.98 mg, 2 equivalents) were added to a solution of 2-amino-N-[2-(benzylamino)-2-oxo-ethyl]-5-methyl-benzothiophene-3-carboxamide (25 mg, 1 equivalent) in THF (2 mL) and H2O (1 mL). The mixture was stirred at 0 °C for 1 h. The residue was purified by filtration to provide an off-white solid (14.88 mg, 48.93% yield).

[0375] LC-MS (m / z): Calculated value: 429.09; Measured value: 430.1 [M+H] + .

[0376] 1H NMR (400 MHz, DMSO) δ 12.30 (s, 1H), 8.67 (t, J = 6.0 Hz, 1H), 8.49 (t, J = 6.0 Hz, 1H), 7.89 - 7.76 (m, 2H), 7.37 - 7.28 (m, 4H), 7.28 -7.22 (m, 1H), 7.21 - 7.14 (m, 1H), 4.56 (s, 2H), 4.36 (d, J = 6.0 Hz, 2H), 4.07 (d, J = 6.0 Hz, 2H), 2.43 (s, 3H).

[0377] Synthesis of 1-43 Synthesis scheme: (a) EDCI, Py, 30℃, 2 h 2-[(2-chloroacetyl)amino]-N-[2-[(4-chlorophenyl)methylamino]-2-oxo-ethyl]-5-methyl-benzene Thiophene-3-carboxamide: EDCI (33.75 mg, 1.5 equivalents) was added to a solution of 2-[[2-[(2-chloroacetyl)amino]-5-methyl-benzothiophene-3-carbonyl]amino]acetic acid (40 mg, 1 equivalent) and (4-chlorophenyl)methylamine (33.24 mg, 2 equivalents) in Py (1 mL). The mixture was stirred at 40 °C for 1 h. The reaction mixture was concentrated under reduced pressure to remove the solvent. The residue was purified by preparative HPLC to provide a white solid (8.68 mg, 15.92% yield).

[0378] LC-MS (m / z): Calculated value: 463.05; Measured value: 464.1 [M+H] + .

[0379] 1 H NMR (400 MHz, DMSO) δ 12.28 (s, 1H), 8.68 (br t, J = 6.0 Hz, 1H), 8.48 (br t, J = 6.0 Hz, 1H), 7.86 - 7.79 (m, 2H), 7.41 - 7.36 (m, 2H), 7.36 -7.31 (m, 2H), 7.18 (d, J = 7.6 Hz, 1H), 4.55 (s, 2H), 4.34 (d, J = 6.0 Hz, 2H), 4.06 (d, J = 5.8 Hz, 2H), 2.43 (s, 3H).

[0380] Synthesis of 1-45 Synthetic schemes: (a) CH3COOH, CH3COONH4, toluene, 60℃, 12 h; (b) S, NaHCO3, EtOH, 80℃, 2 h; (c) NaOH, MeOH, THF, H2O, 80℃, 12 h; (d) HOBt, EDCI, TEA, DMF, 25℃, 12 h; (e) DCM, TEA, 0℃, 1 h; (f) TFA, DCM, 25℃, 2 h; (g) EDCI, Py, 30℃, 2 h. (2Z)-2-cyano-2-(3-methylcyclohexyl-2-en-1-yl)acetate: CH3COOH (16.30 g, 2.99 equivalents) and CH3COONH4 (17.84 g, 2.55 equivalents) were added to a solution of 3-methylcyclohexyl-2-en-1-one (10 g, 1 equivalent), methyl 2-cyanoacetate (25.64 g, 2.85 equivalents) in toluene (100 mL). The mixture was stirred at 60 °C for 12 h. The reaction mixture was poured into water (200 mL) and extracted with ethyl acetate (100 mL × 3). The combined organic layers were washed with brine (100 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure to give the residue. The residue was purified by preparative HPLC (FA conditions) to give a yellow oil (12 g, 69.12% yield).

[0381] LC-MS (m / z): Calculated value: 191.09; Measured value: 192.0 [M+H] + .

[0382] 1 H NMR (400 MHz, DMSO) δ 7.65 - 6.47 (m, 1H), 3.85 - 3.72 (m, 3H), 3.06 - 2.67 (m, 2H), 2.32 (d, J = 7.0 Hz, 2H), 2.05 (d, J = 12.6 Hz, 3H),1.89 - 1.70 (m, 2H).

[0383] 2-Amino-5-methyl-benzothiophene-3-carboxylic acid methyl ester: A mixture of (2Z)-2-cyano-2-(3-methylcyclohexyl-2-en-1-yl)acetate (7 g, 1 equivalent) in EtOH (100 mL), NaHCO3 (30.75 g, 10 equivalent), and S (7.01 g, 5.98 equivalent) was degassed and purged three times with N2. The mixture was then stirred at 80 °C under N2 atmosphere for 2 h. The reaction mixture was filtered and the mother liquor was concentrated. The residue was purified by column chromatography to provide a yellow solid (3.4 g, 41.98% yield).

[0384] 1 H NMR (400 MHz, DMSO) δ 7.94 (s, 2H), 7.79 (d, J = 0.6 Hz, 1H), 7.47(d, J = 8.0 Hz, 1H), 6.90 (dd, J = 1.1, 8.0 Hz, 1H), 3.82 (s, 3H), 2.34 (s, 3H).

[0385] 2-Amino-5-methyl-benzothiophene-3-carboxylic acid: NaOH (1.54 g, 2.5 equivalents) was added to a solution of methyl 2-amino-5-methyl-benzothiophene-3-carboxylate (3.4 g, 1 equivalent) in THF (30 mL), MeOH (30 mL), and H₂O (30 mL). The mixture was stirred at 80 °C for 12 h. The reaction mixture was adjusted to pH 7 with HCl (1 mol / L), and the filtered solid was the crude product. The residue was purified by preparative HPLC (NH₄HCO₃ conditions) to provide a red solid (6 g, crude).

[0386] LC-MS (m / z): Calculated value: 207.04; Measured value: 208.1 [M+H].

[0387] 1 H NMR (400 MHz, DMSO) δ 12.74 - 11.82 (m, 1H), 7.99 - 7.78 (m, 3H),7.47 (d, J = 7.9 Hz, 1H), 6.95 - 6.85 (m, 1H), 2.35 (s, 3H).

[0388] 2-[(2-amino-5-methyl-benzothiophene-3-carbonyl)amino]tert-butyl acetate: HOBt (1.66 g, 1.5 equivalent) and EDCI (2.36 g, 1.5 equivalent), TEA (2.49 g, 3 equivalent), and tert-butyl 2-aminoacetate (1.08 g, 1 equivalent) were added to a solution of 2-amino-5-methyl-benzothiophene-3-carboxylic acid (2 g, 1 equivalent, HCl) in DMF (20 mL). The mixture was stirred at 25 °C for 2 h. The residue was purified by preparative HPLC (NH4HCO3 conditions) to provide a yellow solid (491 mg, 18.67% yield).

[0389] LC-MS (m / z): Calculated value: 320.12; Measured value: 319.2 [MH] + .

[0390] 1 H NMR (400 MHz, DMSO) δ 7.65 (br t, J = 5.8 Hz, 1H), 7.55 (s, 3H), 7.49 (d, J = 7.9 Hz, 1H), 6.88 (d, J = 8.0 Hz, 1H), 3.91 (d, J = 6.0 Hz, 2H), 2.36 (s, 3H), 1.44 (s, 9H).

[0391] 2-[[2-[(2-chloroacetyl)amino]-5-methyl-benzothiophene-3-carbonyl]amino]tert-butyl acetate: At 0 °C, TEA (739.01 mg, 3 equivalents) and 2-chloroacetyl chloride (549.90 mg, 2 equivalents) were added to a solution of 2-[(2-amino-5-methyl-benzothiophene-3-carbonyl)amino]tert-butyl acetate (780 mg, 1 equivalent) in DCM (10 mL). The mixture was stirred at 0 °C for 1 h. The reaction mixture was concentrated under reduced pressure to remove the solvent, providing a yellow solid (700 mg, 72.45% yield).

[0392] LC-MS (m / z): Calculated value: 396.09; Measured value: 395.1 [MH] + .

[0393] 2-[[2-[(2-chloroacetyl)amino]-5-methyl-benzothiophene-3-carbonyl]amino]acetic acid: TFA (6.14 g, 26.71 equivalents) was added to a solution of tert-butyl 2-[[2-[(2-chloroacetyl)amino]-5-methyl-benzothiophene-3-carbonyl]amino]acetate (800 mg, 1 equivalent) in DCM (16 mL). The mixture was stirred at 25 °C for 2 h. The reaction mixture was concentrated under reduced pressure to remove the solvent. The reaction mixture was poured into 20 mL of petroleum ether and collected by filtration and dried under high vacuum to provide a yellow solid (550 mg, 80.07% yield).

[0394] LC-MS (m / z): Calculated value: 340.03; Measured value: 339.1 [MH] + .

[0395] 1 H NMR (400 MHz, DMSO) δ 13.32 - 12.38 (m, 1H), 12.17 (br s, 1H),8.54 (s, 1H), 7.88 (br d, J = 8.8 Hz, 2H), 7.22 (d, J = 8.1 Hz, 1H), 4.60 (s,2H), 4.07 (d, J = 5.8 Hz, 2H), 2.47 (s, 3H).

[0396] 2-[(2-chloroacetyl)amino]-N-[2-[(4-ethynylphenyl)methylamino]-2-oxo-ethyl]-5-methyl Benzothiophene-3-carboxamide: EDCI (25.31 mg, 1.5 equivalent) was added to a solution of 2-[[2-[(2-chloroacetyl)amino]-5-methyl-benzothiophene-3-carbonyl]amino]acetic acid (30 mg, 1 equivalent) and (4-ethynylphenyl)methylamine (23.10 mg, 2 equivalent) in Py (1 mL). The mixture was stirred at 30 °C for 2 h. The reaction mixture was concentrated under reduced pressure to remove the solvent. The residue was purified by preparative HPLC (FA conditions) to provide a white solid (3.31 mg, 8.28% yield).

[0397] LC-MS (m / z): Calculated value: 453.09; Measured value: 454.4 [M+H] + .

[0398] 1H NMR (400 MHz, DMSO) δ 12.29 (s, 1H), 8.75 - 8.64 (m, 1H), 8.49 (s,1H), 7.89 - 7.78 (m, 2H), 7.44 (d, J = 8.0 Hz, 2H), 7.32 (d, J = 8.0 Hz, 2H),7.18 (d, J = 8.1 Hz, 1H), 4.55 (s, 2H), 4.37 (d, J = 5.8 Hz, 2H), 4.14 (s,1H), 4.07 (d, J = 6.0 Hz, 2H), 2.43 (s, 3H)。

Claims

1. A SUMOylation-targeting chimera (SUTAC) comprising a SUMOylation enzyme-binding group attached to a target protein-binding group via a linker.

2. The SUTAC according to claim 1, wherein the SUMOylase-binding group is capable of binding SUMO E3 ligase.

3. The SUTAC according to claim 1, wherein the SUMO E3 ligase is selected from the group consisting of: PIAS1, PIAS2, PIAS3, PIAS4, NSMCE2, TOPORS, Trim19, Trim28, PML, ZNF451 and RANBP2.

4. The SUTAC according to claim 1, represented by the structure of formula I or a salt thereof: in, E is H, amide, acetylamide, or an electrophilic group; Z represents O, S, and NH; Each of R6 and R7 is independently selected from the group consisting of: H, halogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, haloaryl, heteroaryl, heterocyclic, hydroxyl, alkoxy, aryloxy, thioalkoxy, thioalkyl, thioalkenyl, thiohaloalkyl, cyano, nitro, azide, amino, -COOH, -C(O)NHR', -NHCOR''-; wherein R' is H, alkyl, alkenyl, aryl, heterocyclic alkyl, or heteroaryl; and R'' is hydroxyl, alkoxy, aryloxy, alkyl, alkenyl, aryl, heterocyclic alkyl, or heteroaryl; or R6 and R7 together form a 5-8 elemental ring, wherein the ring is either substituted or not substituted; R 11 It is H, substituted or unsubstituted alkyl, alkenyl, aryl, heterocyclic or heteroaryl; L1 is a bond, -(CH2CH2O) q -(CH2CH2O) q (CH2CH2)NH(CO)-, -(CH2CH2O) q (CH2CH2)-、-(CH2CH2O) q (CH2CH2)NH-, -NHCH2(CO)NHCH2Ph-, -N(alkyl)CH2(CO)NHCH2Ph-, substituted or unsubstituted straight-chain or branched alkylene group, substituted or unsubstituted straight-chain or branched alkenyl group, substituted or unsubstituted straight-chain or branched alkyne group, substituted or unsubstituted cycloalkyl group, substituted or unsubstituted heterocyclic group, substituted or unsubstituted aryl group, substituted or unsubstituted heteroaryl group, ether group, ester group, amine group, amide group or any combination thereof, wherein q is an integer from 2 to 10; L2 is a bond, -(CH2CH2O) q -(CH2CH2O) q (CH2CH2)NH(CO)-, -(CH2CH2O) q (CH2CH2)-、-(CH2CH2O) q (CH2CH2)NH-, -NHCH2(CO)NHCH2Ph-, -N(alkyl)CH2(CO)NHCH2Ph-, substituted or unsubstituted straight-chain or branched alkylene groups, substituted or unsubstituted straight-chain or branched alkenyl groups, substituted or unsubstituted straight-chain or branched alkyne groups, substituted or unsubstituted cycloalkyl groups, substituted or unsubstituted heterocyclic groups, substituted or unsubstituted aryl groups, substituted or unsubstituted heteroaryl groups, ether groups, ester groups, amine groups, amide groups, or any combination thereof, wherein q is an integer from 2 to 10; and W is the target protein binding group.

5. The SUTAC according to claim 4, represented by the structure of formula I' or a salt thereof: (I’)。 6. The SUTAC according to claim 4 or claim 5, wherein Z is S.

7. The SUTAC according to any one of claims 4-6, represented by the structure of formula IB or a salt thereof: (ONE) in: Represents saturated or unsaturated bonds; If If the bond is saturated, then X is C, N, O, or S; where if If the bond is unsaturated, then X is C or N; R8, R9, R 10 Each of the following is independently selected from the group consisting of: H, halogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, haloaryl, heteroaryl, heterocyclic, hydroxyl, alkoxy, aryloxy, thioalkoxy, thioalkyl, thioalkenyl, thiohaloalkyl, cyano, nitro, azide, amino, -COOH, -C(O)NHR', -NHCOR''-; wherein R' is H, alkyl, alkenyl, aryl, heterocyclic alkyl, heteroaryl; and R'' is hydroxyl, alkoxy, aryloxy, alkyl, alkenyl, aryl, heterocyclic alkyl, or heteroaryl; R 11 It is H, substituted or unsubstituted alkyl, alkenyl, aryl, heterocyclic or heteroaryl; n is an integer, which is 1, 2, or 3; L1 is a bond, -(CH2CH2O) q -(CH2CH2O) q (CH2CH2)NH(CO)-, -(CH2CH2O) q (CH2CH2)-、-(CH2CH2O) q (CH2CH2)NH-, -NHCH2(CO)NHCH2Ph-, -N(alkyl)CH2(CO)NHCH2Ph-, substituted or unsubstituted straight-chain or branched alkylene group, substituted or unsubstituted straight-chain or branched alkenyl group, substituted or unsubstituted straight-chain or branched alkyne group, substituted or unsubstituted cycloalkyl group, substituted or unsubstituted heterocyclic group, substituted or unsubstituted aryl group, substituted or unsubstituted heteroaryl group, ether group, ester group, amine group, amide group or any combination thereof, wherein q is an integer from 2 to 10; L2 is a bond, -(CH2CH2O) q -(CH2CH2O) q (CH2CH2)NH(CO)-, -(CH2CH2O) q (CH2CH2)-、-(CH2CH2O) q (CH2CH2)NH-, -NHCH2(CO)NHCH2Ph-, -N(alkyl)CH2(CO)NHCH2Ph-, substituted or unsubstituted straight-chain or branched alkylene groups, substituted or unsubstituted straight-chain or branched alkenyl groups, substituted or unsubstituted straight-chain or branched alkyne groups, substituted or unsubstituted cycloalkyl groups, substituted or unsubstituted heterocyclic groups, substituted or unsubstituted aryl groups, substituted or unsubstituted heteroaryl groups, ether groups, ester groups, amine groups, amide groups, or any combination thereof, wherein q is an integer from 2 to 10; and W is the target protein binding group.

8. The SUTAC according to any one of claims 1-7, wherein the target protein binding group comprises a protein 4 (BRD4) targeting binder containing a bromo domain to form a BRD4-targeting SUTAC.

9. The SUTAC of claim 8, wherein the BRD4 targeting binder comprises JQ1 or its acid or ester.

10. The SUTAC of claim 8, wherein the BRD4-targeted SUTAC is represented by one of the following structures: 。 11. The SUTAC according to any one of claims 1-7, wherein the target protein binding group comprises an androgen receptor (AR) targeting binder to form an AR-targeting SUTAC.

12. The SUTAC of claim 11, wherein the AR-targeted SUTAC is represented by one of the following structures: 。 13. The SUTAC according to any one of claims 1-7, wherein the target protein binding group comprises a P300 targeting binder to form a P300-targeting SUTAC.

14. The SUTAC of claim 13, wherein the P300 targeting binder comprises GNE-207 or a derivative thereof.

15. The SUTAC of claim 13, wherein the P300-targeted SUTAC is represented by one of the following structures: ID-4 ID-5.

16. A pharmaceutical composition comprising SUTAC according to any one of claims 1-15 and a suitable, acceptable carrier.

17. The SUTAC according to any one of claims 1-15, for use in treating a subject suffering from cancer, improving the condition of the subject, or inhibiting the decline of the subject.

18. The SUTAC for use according to claim 17, wherein the cancer is selected from the group consisting of: kidney cancer, lung cancer, endometrial / uterine cancer, esophageal cancer, breast cancer, cervical cancer, liver cancer, gastric cancer, esophageal cancer, head and neck cancer, ovarian cancer, skin cancer, bile duct cancer, leukemia, lymphoma, rhabdoid tumor, brain cancer, colon / colorectal cancer, pancreatic cancer, myeloma, prostate cancer, neuroblastoma, gastric cancer, sarcoma, thyroid cancer, bladder cancer, bone cancer, or eye cancer.

19. The SUTAC according to any one of claims 1-15, for use in treating subjects suffering from inflammation, neuroinflammation, allergy, aging, autoimmune diseases, viral infections, bacterial infections, obesity, neurodegenerative diseases, fibrosis, cardiovascular diseases, diabetes, Crohn's disease, osteoporosis, multiple sclerosis (MS), SLE, or non-alcoholic fatty liver disease, improving the condition of said subjects, or inhibiting the decline of said subjects.

20. The SUTAC for use according to claim 19, wherein the diabetes is type II diabetes.

21. The SUTAC for use according to any one of claims 17-20, wherein administration of the SUTAC to the subject reduces the degradation of the target protein compared to an untreated subject.

22. The SUTAC for use according to any one of claims 17-20, wherein administration of the SUTAC to a subject increases the degradation of the target protein compared to an untreated subject.

23. The SUTAC for use according to any one of claims 17-20, wherein administration of the SUTAC to a subject reduces the stabilization of the target protein compared to an untreated subject.

24. The SUTAC for use according to any one of claims 17-20, wherein administration of the SUTAC to the subject increases the stabilization of the target protein compared to an untreated subject.

25. The SUTAC for use according to any one of claims 17-20, wherein administration of the SUTAC to the subject reduces the activation of the target protein compared to an untreated subject.

26. The SUTAC for use according to any one of claims 17-20, wherein administration of the SUTAC to a subject increases the activation of the target protein compared to an untreated subject.

27. The SUTAC for use according to any one of claims 17-20, wherein administration of the SUTAC to a subject reduces repression of the target protein compared to an untreated subject.

28. The SUTAC for use according to any one of claims 17-20, wherein administration of the SUTAC to a subject increases repression of the target protein compared to an untreated subject.

29. The SUTAC for use according to any one of claims 17-20, wherein administration of the SUTAC to a subject reduces the binding of the target protein to other proteins or DNA compared to an untreated subject.

30. The SUTAC for use according to any one of claims 17-20, wherein administration of the SUTAC to a subject increases the binding of the target protein to other proteins or DNA compared to an untreated subject.

31. The SUTAC for use according to any one of claims 17-20, wherein administration of the SUTAC to the subject reduces the solubility of the target protein compared to an untreated subject.

32. The SUTAC for use according to any one of claims 17-20, wherein administration of the SUTAC to the subject increases the dissolution of the target protein compared to an untreated subject.

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