USP28-based deubiquitinase-targeting chimeras for cancer treatment

Abstract

Deubiquitinase-targeting chimeras (DUBTACs) are an emerging class of therapeutics that can stabilize tumor suppressors by hijacking a deubiquitinase (DUB), thereby offering a strategic pivot from conventional approaches to target tumor suppressors. However, only OTUB1 and USP7 have been harnessed for DUBTAC development to date. Here, we show for the first time that USP28 can be leveraged for developing DUBTACs. Utilizing a USP28 non-covalent ligand, we crafted USP28-recruiting DUBTACs that effectively stabilized the ΔF508-CFTR mutant protein, with comparable effectiveness to the previously reported OTUB1- and USP7-recruiting CFTR DUBTACs. Furthermore, we developed USP28-recruiting cGAS DUBTACs that effectively stabilized cGAS, elevated the cGAS-STING signaling pathway, and elicited an anti-proliferative effect. We also developed first-in-class PPARγ DUBTACs to intervene with cancer metabolism pathways. Our lead PPARγ DUBTACs effectively stabilized PPARγ and suppressed the proliferation in cancer cells, thus providing a new potential anti-cancer therapeutic approach. Hence, this work advances the targeted protein stabilization field.

Description

Attorney Docket No.: 27527-0235 WO 1USP28-BASED DEUBIQUITINASE- TARGETING CHIMERAS FOR CANCER TREATMENT CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims priority to U. S. Provisional Patent Application No.63 / 743,452, filed on January 9, 2025, the contents of which are incorporated herein by reference in their entirety.STATEMENT OF FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

[0002] This work was supported in part by the National Institutes of Health (NIH) grant no. R35CA253027. The government has certain rights in the invention.TECHNICAL FIELD

[0003] This invention relates to deubiquitinase-targeting chimeras, and more particularly to deubiquitinase-targeting chimeras including a ubiquitin-specific protease 28 (USP28) recruiting ligand conjugated to a target protein ligand by a linker.BACKGROUND OF THE INVENTION

[0004] Deubiquitinase-targeting chimeras (DUBTACs)1'4are an emerging class of therapeutic modalities that leverage cellular deubiquitination mechanisms to stabilize proteins of interest (POI) such as tumor suppressors. In contrast to targeted protein degradation (TPD) strategies to eliminate aberrant proteins, such as proteolysis-targeting chimeras (PROTACs)5'9, molecular glues7, 10, n, lysosome-targeting chimeras (LYTACs)12 15, autophagosometethering compound (ATTEC)16, autophagy-targeting chimera (AUTAC)17, and nucleotide-based degradation system18'23, DUBTACs stabilize the target proteins by hijacking a deubiquitinase (DUB)1'3, offering a potential new approach to targeting tumor suppressors such as p5324. While the drug discovery community has focused primarily on targeting oncoproteins than tumor suppressors to date, likely due to the lack of viable strategies to target tumor suppressors, the DUBTAC technology could provide a much-needed new approach to effectively target tumor suppressors. However, despite the initial successes in stabilizing critical proteins such as Cystic Fibrosis Transmembrane Conductance Regulator (CTFR)1, 3’4, Wee I1, FOXO3A2, p532, IRF32, AMPK3, and cGAS4by DUBTACs, the current DUBTAC technology has several limitations. First, OTUB1 and USP7 are the only DUBsAttorney Docket No.: 27527-0235 WO 1that have been harnessed for developing DUBTAC to date1'3. Hence, exploration of additional DUBs for DUBTAC development is desirable. Second, there are concerns over the potential toxicity of the 0TUB1 covalent ligand utilized for DUBTAC development1, 2’25, 26. Although we previously showed that noncovalent inhibitors of USP7 can be utilized to generate DUBTACs3, USP7 is the only successful example thus far. Additional examples for utilizing noncovalent ligands of DUBs for DUBTAC development are desirable. To this end, we investigated noncovalently harnessing USP28 (ubiquitin-specific protease 28) for DUBTAC development (Figure 1A and IB). USP28 is a crucial DUB in the ubiquitin-proteasome system and is highly expressed in many types of human cancer27'29. Moreover, USP28 has been shown to regulate the stability of several critical proteins involved in the DNA damage response, such as 53BP1 and Claspin, thereby influencing the ability of the cell to repair DNA damage and maintain genomic integrity30, 31. The aberrant high expression or activity of USP28 is linked to various types of cancer, including breast, colorectal, and lung cancers, making USP28 a potential cancer drug taiget27'29. Several noncovalent smallmolecule inhibitors of USP28, including CT1073 and KL9, which are very close analogs, have been reported (Figure 1C)32and the co-crystal structure of USP28 in complex with KL9 has been solved (PDB code: 7TUO)33(Figure ID). Interestingly, in this co-crystal structure, we observed that one molecule of KL9 binds to an allosteric site on the surface of USP28 (site 1), and another KU9 binds to the active site of USP28 (site 2). In site 1, the bridged piperidinyl moiety is solvent-exposed. In contrast, the whole KL9 is buried in the binding pocket in site 2. Therefore, we hypothesized that the linker extension from the bridged piperidinyl group would likely maintain KU9 binding in site 1, but disrupt the binding in site 2, thus likely making DUBTACs derived from this linking strategy still capable of binding USP28, but not inhibiting the deubiquitinase activity of USP28. In this study, we utilized these USP28 noncovalent inhibitors and this linking strategy to hijack USP28 and successfully engineered effective DUBTACs for CFTR, cGAS, and PPARy, respectively. This work demonstrated for the first time that USP28 can be harnessed for DUBTAC development, thus expanding the limited toolbox for targeted protein stabilization (TPS).Attorney Docket No.: 27527-0235WO1SUMMARY OF THE INVENTION

[0005] The present disclosure is based in part, on the unexpected discovery of a deubiquitinase-targeting chimera (DUBTAC), including a ubiquitin-specific protease 28 (USP28) recruiting ligand conjugated to a target protein ligand by a linker.

[0006] In an embodiment, the deubiquitinase-targeting chimera, includes ubiquitinspecific protease 28 (USP28) recruiting ligand CT1073 or KL9 conjugated to cystic fibrosis transmembrane conductance regulator (CTFR) ligand, lumacaftor, by a linker.

[0007] In an embodiment, the deubiquitinase-targeting chimera has the followings structure: # & s M &COTTS COT?? OT-gA S', ■COT M. n a C8U82; - 1. C8U7S, n « A COWS n ®

[0008] In an embodiment, the deubiquitinase-targeting chimera, includes ubiquitinspecific protease 28 (USP28) recruiting ligand CT1073 or KL9, conjugated to cyclic GMP-AMP synthase (cGAS) antagonist GMO, by a linker.Attorney Docket No.: 27527-0235WO1

[0009] In an embodiment, the deubiquitinase-targeting chimera has the following structure:

[0010] In an embodiment, the deubiquitinase-targeting chimera includes ubiquitinspecific protease 28 (USP28) recruiting ligand CT1073 or KL9, conjugated to peroxisome proliferator-activated receptor gamma (PPARy) ligand, lobeglitazone, by a linker.

[0011] In an embodiment, the deubiquitinase-targeting chimera has the following structure:W.1 n sa 2- IW-Sm «n * 24^282?) PDTqgiass a; n ® 4;POT-25: PTO& m « 5f^S2O3O}

[0012] In an embodiment, the present disclosure is directed to a method of treating cystic fibrosis, including administering to a subject in need thereof, a therapeutically effective amount of a deubiquitinase-targeting chimera including a CTFR ligand.

[0013] In an embodiment, the present disclosure is directed to a method of treating cancer including administering to a subject in need thereof, a therapeutically effective amount of a deubiquitinase-targeting chimera including a cGAS antagonist.Attomey Docket No.: 27527-0235 WO 1

[0014] In an embodiment, the present disclosure is directed to a method of treating cancer, including administering to a subject in need thereof, a therapeutically effective amount of a deubiquitinase-targeting chimera including a PPARy ligand.

[0015] In an embodiment, the present disclosure is directed to a pharmaceutical composition, including a deubiquitinase-targeting chimera as disclosed herein, and a pharmaceutically acceptable carrier.

[0016] The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials are described herein for use in the present invention; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

[0017] Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.BRIEF DESCRIPTION OF DRAWINGS

[0018] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0019] FIG 1. is the schematic model of USP28 -recruiting DUBTACs to stabilize target proteins. (A) Schematic illustration of the protein degradation mediated by the ubiquitin-proteasome system. Ubiquitinases attach ubiquitin molecules to the protein-of-interest (POI) until it is sufficiently polyubiquitinated and recognized by the proteasome, leading to the subsequent degradation of the POI. This process can be reversed by deubiquitinases, which remove ubiquitin chains. (B) Schematic illustration for the mechanism of the POI stabilization induced by a USP28-recruiting DUBTAC. The DUBTAC recruits USP28 to the polyubiquitinated POI, leading to the removal of ubiquitin from the POI, thereby rescuing it from proteasomal degradation. (C) Chemical structures of USP28 noncovalent small-Attorney Docket No.: 27527-0235 WO 1molecule inhibitors, CT1073 and KL9. (D) The co-crystal structure of USP28 in complex with KL9 (PDB ID: 7TU0). (Top) Overview of two KL9 molecules binding with USP28; (Bottom) detailed views of the two different binding sites.

[0020] FIG. 2. USP28-recruiting CFTR DUBTACs stabilize AF508-CFTR mutant protein in cells. (A) Workflow for screening USP28-recruiting DUBTACs that stabilize CFTR. (B) Chemical structures of the synthesized USP28-based CFTR DUBTACs, CDT-72 to CDT-84. (C-D) Western blot (WB) analysis of compounds CDT-72 to CDT-82 (C) and CDT-82 to CDT-84 (D) in CF41o-AF508 human cystic fibrosis bronchial epithelial cells, which were treated with DMSO, positive control NJH-2-057 or the indicated compound at 10 pM for 24 h. MS9279 (CDT-079) was identified as the lead compound from this screen. (E) WB analysis to assess the effect of MS9279 on increasing the CFTR protein level at multiple concentrations. CF41o-AF508 human cystic fibrosis bronchial epithelial cells were treated with increasing concentrations of MS9279, ranging from 0 to 20 pM, for 24 h. (F) WB analysis investigating the time-dependent effect of MS9279 on the protein levels of CFTR. CF41o-AF508 human cystic fibrosis bronchial epithelial cells were treated with MS9279 (10 pM) for 0, 3, 6, 12, 18, or 24 h. Numbers showing below WB are relative protein levels by normalizing to DMSO controls. The brackets indicate the bands for CFTR protein.

[0021] FIG. 3. USP28-recruiting cGAS DUBTACs stabilize cGAS, and suppress the proliferation and colony formation in cancer cells. (A) Schematic representation for increasing cGAS / STING protein level via stabilizing cGAS by USP28 -recruiting cGAS DUBTACs. (B-C) Chemical structures of the synthesized USP28-recruiting cGAS DUBTACs. (D-G) WB analysis of the cGAS protein level in HeEa cells treated with GDT-51 to -61 (D), GDT-86 to -97 (E), and GDT-62 to -65 and -98 to -100 (F). HeLa cells were treated with DMSO or the indicated compound at 10 pM for 24 h. The quantification results shown are representative of three independent experiments (G). (H-I) WB confirming the screening results of D-F. MS2099 and MS2100 were identified as the lead compounds. The quantification results shown are representative of three independent experiments (I). (J) WB analysis to assess the effect of MS2100 on increasing the cGAS protein level at multiple concentrations. HeLa cells were treated with the indicated compound at the indicated concentration for 24 h. (K) WB analysis assessing the kinetics of cGAS stabilization and STING up-regulation induced by MS2100. HeLa cells were treated with MS2100 at 10 pM for the indicated times. (L) WB analysis of cGAS and STING protein levels upon theAttorney Docket No.: 27527-0235 WO 1treatment of the parent cGAS ligand (GMO, 30 pM), the parent USP28 ligand (CT1073, 10 pM), MS2099 (10 pM), and MS2100 (10 pM) in combination with CT1073 or GMO. HeLa cells were treated with these compound(s) as indicated at 10 or 30 pM for 24 h. (M) The immunoprecipitation results for cGAS and USP28. HEK293 cells were transfected with HA-cGAS and Flag-USP28 plasmids as indicated. 24 h post-transfection, cells were treated with the DUBTAC, MS2099 or MS2100 (10 pM), for 24 h. To stablize the ternary complex, cells were co-treated with MG132 (10 pM) for the final 12 h before harvesting for immunoprecipitation analysis. (N) Immunoprecipitaion analysis of the ubiquitination level of cGAS. IB analysis of HA-IP and WCL derived from HEK293 cells transfected with HA-cGAS and Myc-ubi quitin (Ub) expression plasmids. Cells were treated with 10 pM MS2100 (with or without) for 24 h and 30 pM proteasome inhibitor MG132 for 8 h before harvesting. (O) The evaluation of cGAMP level after the treatment of cGAS-DUBTAC. The level of cGAMP was evaluated in HeLa cells following treatment with 10 pM MS2099 or MS2100 for 24 hours, with an additional 12-hour treatment in the presence or absence of HT-DNA ( 1 pg / mL). (P) The downstream activation of STING and IRF3 following DUBTAC-induced stabilization of cGAS. HeLa cells were treated with 10 pM MS2099 or MS2100 for 24 h, and further incubated for 12 h either in the presence or absence of high molecular weight DNA (HT-DNA) (1 pg / mL). Additionally, cells treated with MS2100 were co-treated with the STING inhibitor SN-011 (1 pM) as indicated. Error bars represent the SD from the mean of triplicate experiments. The quantification of the data was analyzed by one-way or two-way ANOVA with Tukey’s correction using GraphPad Prism software and is presented as mean ± SD. Differences were considered statistically significant at p < 0.05. ns, *, **, ***, ****: no significance, p < 0.05, p < 0.01, p < 0.001, p < 0.0001.

[0022] FIG. 4. USP28-recruiting PPARy DUBTACs stabilize PPARy. (A) Schematic illustration of PPARy stabilization induced by PPARy-targeting DUBTACs, resulting in cancer cell death via apoptosis. (B) The co-crystal structure of PPARy in complex with LGZ (PDB ID: 5YCN). Top: The structure of Lobeglitazone (LGZ) molecule; Bottom: LGZ in complex with PPARy ligand binding domain (PDB ID: 5YCN). The red dashed circle indicates the solvent exposure part. (C) The chemical structures of the synthesized PPARy DUBTACs (PDTs). (D-E) WB analysis of the PPARy protein level in HeLa cells treated with DMSO or the indicated DUBTAC candidate compounds (PDT-16 to -23 (D) and PDT-24 to -30 (E)) at 10 pM for 24 h. Numbers showing below WB are relative protein levels by normalizing to DMSO controls. MS 1727 to MS 1730 were identified as lead compounds inAttorney Docket No.: 27527-0235 WO 1this screening. (F-G) WB analysis of the PPARy protein level in cells treated with lead compounds, MS 1727 to MS 1730, at 10 pM for 24 h in HeLa (F) and MDA-MB-231 (G) cells. The quantification results shown are representative of three independent experiments. (H-I) A time (H) and dose-dependent (I) analysis of MS 1728 in HeLa cells at different time points and concentrations. The error bars represent the SD from the mean of triplicate experiments. (J) WB analysis of PPARy protein level upon the treatment of the parental PPARy ligand, LGZ, the parental USP28 ligand, CT 1073, MS 1728, in combination with CT1073 or LGZ, as indicated, at 10 pM for 24 h. The quantification results shown are representative of three independent experiments. (K) The immunoprecipitation results for PPARy and USP28. HEK293 cells were transfected with HA-PPARy and Flag-USP28 plasmids as indicated in Figure 4A. At 24 h post-transfection, cells were treated with the DUBTAC compounds MS1728 to MS1730 (5 pM) for an additional 12 h, with each compound applied separately and subsequently co-treated with MG132 (10 pM) for another 12 h before harvesting for the immunoprecipitation analysis. (L) The drug specificity was analyzed with shRNA-mediated knockdown of USP28 in MDA-MB-231 cells. Resulting cells were treated with DUBTAC, and the cell lysate were collected to analyze PPARy protein level. The quantification of the data was analyzed by one-way or two-way ANOVA with Tukey’s correction using GraphPad Prism software and is presented as mean ± SD. Differences were considered statistically significant at p < 0.05. ns, *, ***, ****: no significance, p < 0.05, p < 0.001, p < 0.0001.

[0023] FIG. 5. PPARy DUBTACs exhibit anti-proliferative effects in cancer cells. (A) Structure of the compounds for function analysis. (B and C) Colony formation analysis of HeLa cells treated with DMSO, LGZ, PDT-16, PDT-17, MS 1727, MS 1728, MS 1729, or MS 1730 at 5 pM for two weeks. The quantified results shown are representative of three independent experiments. (D and E) Cell imaging analysis of HeLa cells treated with DMSO, LGZ, MS1727N, and MS1727 at 10 pM for 24 h. The quantified results shown are representative of three independent experiments. (F) The effects of MS 1727 on the proliferation of HeLa cells. Cells were treated with DMSO or the indicated compound at 10 pM over three days. Error bars represent the SD from the mean of triplicate experiments. (G) Concentration-response curves evaluating the effect of MS 1727 on the viability of HeLa cells treated with DMSO or the indicated compound at the indicated concentration for three days. The error bars represent the SD from the mean of triplicate experiments. The quantification of the data was analyzed by one-way or two-way ANOVA with Tukey’s correction usingAttorney Docket No.: 27527-0235 WO 1GraphPad Prism software and is presented as mean ± SD. Differences were considered statistically significant at p < 0.05. ns, *, ***, ****: no significance, p < 0.05, p < 0.001, p < 0.0001.

[0024] FIG. 6. The full membrane images of CFTR western blot data for Figure 2C - 2F.

[0025] FIG. 7. The mechanism study of cGAS DUBTAC. (A) The screening of cGAS DUBTACs. (B) The Time course analysis for the stabilization of cGAS by MS2099. (C) WB analysis of cGAS stabilization and STING elevation in HeLa cells treated with DMSO, the parent cGAS ligand (G140), MS2099, or MS2100 at 10 pM for 24 h. The quantification results shown are representative of three independent experiments. (D) The hook effect analysis for MS2099 and MS2100 in a series of dosages, including 10, 20, 30, 40, and 50 pM for 24 h. (E) The drug specificity was analyzed with shRNA-mediated knockdown of USP28 in HeLa cells. Resulting cells were treated with DUBTAC, and the cell lysate were collected to analyze the cGAS and STING protein levels.

[0026] FIG. 8. The anti-proliferative effects of cGAS DUBTACs in cancer cells. (A) The structures of cGAS DUBTACs and ligand control for the downstream functional studies, including the cGAS ligand (G140), USP28-linker version (QC251-34), MS2099, and MS2100. (B) Colony formation analysis of HeLa cells treated with DMSO, G140, QC251-34, MS2099, or MS2100 at 10 pM for two weeks. The quantification results shown are representative of three independent experiments. (C) The effect of MS2099 and MS2100 on the proliferation of HeLa cells over four days. HeLa cells were treated with DMSO or the indicated compound at 10 pM. Error bars represent the standard deviation (SD) from the mean of triplicate experiments. (D) Concentration-response curves evaluating the effect of G140, GDT-87, MS2099, and MS2100 on the viability of HeLa cells. HeLa cells were treated with DMSO or the indicated compound at the indicated concentration for four days. (E) Cell imaging analysis of HeLa cells treated with DMSO, GMO, GDT-87, MS2099, or MS2100 at 10 pM for 24 h. The quantification results shown in the blue color graph are representative of three independent experiments.

[0027] FIG. 9. The anti-proliferative effects of PPARy DUBTACs in cancer cells.(A)WB analysis of cGAS stabilization in HeLa cells treated with DMSO, the parent cGAS ligand, GMO, the parent USP28 ligand, CT1073, MS2099, or MS2100 at 10 pM for 24 h. (B) Cell imaging analysis of HeLa cells treated with DMSO, LGZ, PDT-16, MS1727, MS1728, MS1729, or MS1730 at 10 pM for 24 h. The quantification results shown are representativeAttorney Docket No.: 27527-0235 WO 1of three independent experiments. (C) Colony formation analysis of HeLa cells treated with DMSO, LGZ, PDT-16, PDT-17, MS1727, MS1728, MS1729, or MS1730 at 5 pM for two weeks. The quantification results shown are representative of three independent experiments. (D) The effect of MS1727 to MS1730 on the proliferation of HeLa cells over three days. HeLa cells were treated with DMSO or the indicated compound at 10 pM. Error bars represent the SD from the mean of triplicate experiments. (E) Concentration-response curves evaluating the effect of MS1727 to MS1730 on the viability of HeLa cells. HeLa cells were treated with DMSO or the indicated compound at the indicated concentration for three days. The error bars represent the SD from the mean of triplicate experiments. The quantification of the data was analyzed by one-way or two-way ANOVA with Tukey’s correction using GraphPad Prism software and is presented as mean ± SD. Differences were considered statistically significant at p < 0.05. ns, *, ***, ****: no significance, p < 0.05, p < 0.001, p < 0.0001.

[0028] FIG. 10. Schematic illustration of DUBTAC mechanism of action.

[0029] FIGS. 11 - 22 are LC-MS and NMR spectra of compounds according to the present disclosure.

[0030] Like reference symbols in the various drawings indicate like elements.DETAILED DESCRIPTIONPharmaceutically acceptable isotopic variations of the compounds disclosed herein are contemplated and can be synthesized using conventional methods known in the art or methods corresponding to those described in the Examples (substituting appropriate reagents with appropriate isotopic variations of those reagents). Specifically, an isotopic variation is a compound in which at least one atom is replaced by an atom having the same atomic number, but an atomic mass different from the atomic mass usually found in nature. Useful isotopes are known in the art and include, for example, isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, and chlorine. Exemplary isotopes thus include, e. g-,2H,3H,13C,14C,15N,170,18O,32P,35S,18F, and36C1.

[0001] Isotopic variations (e.g, isotopic variations containing2H) can provide therapeutic advantages resulting from greater metabolic stability, e.g., increased in vivo half-life or reduced dosage requirements. In addition, certain isotopic variations (particularly those containing a radioactive isotope) can be used in drug or substrate tissue distribution studies.Attorney Docket No.: 27527-0235 WO 1The radioactive isotopes tritium (3H) and carbon-14 (14C) are particularly useful for this purpose in view of their ease of incorporation and ready means of detection.

[0002] Pharmaceutically acceptable solvates of the compounds disclosed herein are contemplated. A solvate can be generated, e.g., by substituting a solvent used to crystallize a compound disclosed herein with an isotopic variation (e.g., D2O in place of H2O, e-acetone in place of acetone, or de-DMSO in place of DMSO).

[0003] Pharmaceutically acceptable fluorinated variations of the compounds disclosed herein are contemplated and can be synthesized using conventional methods known in the art or methods corresponding to those described in the Examples (substituting appropriate reagents with appropriate fluorinated variations of those reagents). Specifically, a fluorinated variation is a compound in which at least one hydrogen atom is replaced by a fluoro atom. Fluorinated variations can provide therapeutic advantages resulting from greater metabolic stability, e.g., increased in vivo half-life or reduced dosage requirements.Definition of Terms

[0004] As used herein, the terms “comprising” and “including” are used in their open, non-limiting sense.

[0005] " Pharmaceutically acceptable salt" includes both acid and base addition salts. A pharmaceutically acceptable salt of any one of the compounds described herein is intended to encompass any and all pharmaceutically suitable salt forms. Preferred pharmaceutically acceptable salts of the compounds described herein are pharmaceutically acceptable acid addition salts and pharmaceutically acceptable base addition salts.

[0006] " Pharmaceutically acceptable acid addition salt" refers to those salts which retain the biological effectiveness and properties of the free bases, which are not biologically or otherwise undesirable, and which are formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, hydroiodic acid, hydrofluoric acid, phosphorous acid, and the like. Also included are salts that are formed with organic acids such as aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxy alkanoic acids, alkanedioic acids, aromatic acids, aliphatic and. aromatic sulfonic acids, etc. and include, for example, acetic acid, trifluoroacetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid,Attorney Docket No.: 27527-0235 WO 1salicylic acid, and the like. Exemplary salts thus include sulfates, pyrosulfates, bisulfates, sulfites, bisulfites, nitrates, phosphates, monohydrogenphosphates, dihydrogenphosphates, metaphosphates, pyrophosphates, chlorides, bromides, iodides, acetates, trifluoroacetates, propionates, caprylates, isobutyrates, oxalates, malonates, succinate suberates, sebacates, fumarates, maleates, mandelates, benzoates, chlorobenzoates, methylbenzoates, dinitrobenzoates, phthalates, benzenesulfonates, toluenesulfonates, phenylacetates, citrates, lactates, malates, tartrates, methanesulfonates, and the like. Also contemplated are salts of amino acids, such as arginates, gluconates, and galacturonates (see, for example, Berge S. M. et al., " Pharmaceutical Salts," Journal of Pharmaceutical Science, 66: 1-19 (1997), which is hereby incorporated by reference in its entirety). Acid addition salts of basic compounds may be prepared by contacting the free base forms with a sufficient amount of the desired acid to produce the salt according to methods and techniques with which a skilled artisan is familiar.

[0007] " Pharmaceutically acceptable base addition salt" refers to those salts that retain the biological effectiveness and properties of the free acids, which are not biologically or otherwise undesirable. These salts are prepared from addition of an inorganic base or an organic base to the free acid. Pharmaceutically acceptable base addition salts may be formed with metals or amines, such as alkali and alkaline earth metals or organic amines. Salts derived from inorganic bases include, but are not limited to, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts and the like. Salts derived from organic bases include, but are not limited to, salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, for example, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, diethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, N, N-dibenzylethylenediamine, chloroprocaine, hydrabamine, choline, betaine, ethylenediamine, ethylenedianiline, N-methylglucamine. glucosamine, methylglucamine, theobromine, purines, piperazine, piperidine, N-ethylpiperidine, polyamine resins and the like. See Berge et al., supra.Pharmaceutical Compositions

[0008] In some aspects, the compositions and methods described herein include the manufacture and use of pharmaceutical compositions and medicaments that include one or more compounds as disclosed herein. Also included are the pharmaceutical compositions themselves.Attorney Docket No.: 27527-0235 WO 1

[0009] In some aspects, the compositions disclosed herein can include other compounds, drugs, or agents used for the treatment. For example, in some instances, pharmaceutical compositions disclosed herein can be combined with one or more (e.g, one, two, three, four, five, or less than ten) compounds.

[0010] In some aspects, the pH of the compositions disclosed herein can be adjusted with pharmaceutically acceptable acids, bases, or buffers to enhance the stability of the compounds or its delivery form.

[0011] Pharmaceutical compositions typically include a pharmaceutically acceptable carrier, adjuvant, or vehicle. As used herein, the phrase “pharmaceutically acceptable” refers to molecular entities and compositions that are generally believed to be physiologically tolerable and do not typically produce an allergic or similar untoward reaction, such as gastric upset, dizziness and the like, when administered to a human. A pharmaceutically acceptable carrier, adjuvant, or vehicle is a composition that can be administered to a patient, together with a compound of the invention, and which does not destroy the pharmacological activity thereof and is nontoxic when administered in doses sufficient to deliver a therapeutic amount of the compound. Exemplary conventional nontoxic pharmaceutically acceptable carriers, adjuvants, and vehicles include saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration.

[0012] In particular, pharmaceutically acceptable carriers, adjuvants, and vehicles that can be used in the pharmaceutical compositions of this invention include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, self-emulsifying drug delivery systems (SEDDS) such as d-a-tocopherol polyethylene glycol 1000 succinate, surfactants used in pharmaceutical dosage forms such as Tweens or other similar polymeric delivery matrices, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol and wool fat. Cyclodextrins such asAttorney Docket No.: 27527-0235 WO 1a-, P-, and y-cyclodextrin, may also be advantageously used to enhance delivery of compounds of the formulae described herein.

[0013] As used herein, the compounds disclosed herein are defined to include pharmaceutically acceptable derivatives or prodrugs thereof. A “pharmaceutically acceptable derivative” means any pharmaceutically acceptable salt, solvate, or prodrug, e.g, carbamate, ester, phosphate ester, salt of an ester, or other derivative of a compound or agent disclosed herein, which upon administration to a recipient is capable of providing (directly or indirectly) a compound described herein, or an active metabolite or residue thereof.Particularly favored derivatives and prodrugs are those that increase the bioavailability of the compounds disclosed herein when such compounds are administered to a mammal (e.g., by allowing an orally administered compound to be more readily absorbed into the blood) or which enhance delivery of the parent compound to a biological compartment (e.g., the brain or lymphatic system) relative to the parent species. Preferred prodrugs include derivatives where a group that enhances aqueous solubility or active transport through the gut membrane is appended to the structure of formulae described herein. Such derivatives are recognizable to those skilled in the art without undue experimentation. Nevertheless, reference is made to the teaching of Burger’s Medicinal Chemistry and Drug Discovery, 5thEdition, Vol. 1:Principles and Practice, which is incorporated herein by reference to the extent of teaching such derivatives.

[0014] The compounds disclosed herein include pure enantiomers, mixtures of enantiomers, pure diastereoisomers, mixtures of diastereoisomers, diastereoisomeric racemates, mixtures of diastereoisomeric racemates and the meso-form and pharmaceutically acceptable salts, solvent complexes, morphological forms, or deuterated derivative thereof.

[0015] In particular, pharmaceutically acceptable salts of the compounds disclosed herein include, e. g., those derived from pharmaceutically acceptable inorganic and organic acids and bases. Examples of suitable acid salts include acetate, adipate, benzoate, benzenesulfonate, butyrate, citrate, digluconate, dodecylsulfate, formate, fumarate, glycolate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, lactate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, palmoate, phosphate, picrate, pivalate, propionate, salicylate, succinate, sulfate, tartrate, tosylate, trifluoromethylsulfonate, and undecanoate. Salts derived from appropriate bases include, e.g., alkali metal (e.g., sodium), alkaline earth metal (e.g., magnesium), ammonium salts. TheAttorney Docket No.: 27527-0235 WO 1invention also envisions the quatemization of any basic nitrogen-containing groups of the compounds disclosed herein. Water or oil-soluble or dispersible products can be obtained by such quatemization.

[0016] In some aspects, the pharmaceutical compositions disclosed herein can include an effective amount of one or more compounds. The terms “effective amount” and “effective to treat,” as used herein, refer to an amount or a concentration of one or more compounds or a pharmaceutical composition described herein utilized for a period of time (including acute or chronic administration and periodic or continuous administration) that is effective within the context of its administration for causing an intended effect or physiological outcome. In some aspects, pharmaceutical compositions can further include one or more additional compounds, drugs, or agents used for the treatment in amounts effective for causing an intended effect or physiological outcome.

[0017] In some aspects, the pharmaceutical compositions disclosed herein can be formulated for sale in the United States, import into the United States, or export from the United States.Administration of Pharmaceutical Compositions

[0018] The pharmaceutical compositions disclosed herein can be formulated or adapted for administration to a subject via any route, e.g., any route approved by the Food and Drug Administration (FDA). Exemplary methods are described in the FDA Data Standards Manual (DSM) (available at http: / / www.fda.gov / Drugs / DevelopmentApprovalProcess / FormsSubmissionRequirements / ElectronicSubmissions / DataStandardsManuahnonographs). In particular, the pharmaceutical compositions can be formulated for and administered via oral, parenteral, or transdermal delivery. The term “parenteral” as used herein includes subcutaneous, intracutaneous, intravenous, intramuscular, intraperitoneal, intra-articular, intra-arterial, intrasynovial, intrastemal, intrathecal, intralesional, and intracranial injection or infusion techniques.

[0019] For example, the pharmaceutical compositions disclosed herein can be administered, e.g., topically, rectally, nasally (e.g, by inhalation spray or nebulizer), buccally, vaginally, subdermally (e.g, by injection or via an implanted reservoir), or ophthalmically.Attomey Docket No.: 27527-0235 WO 1

[0020] For example, pharmaceutical compositions of this invention can be orally administered in any orally acceptable dosage form including, but not limited to, capsules, tablets, emulsions and aqueous suspensions, dispersions and solutions. In the case of tablets for oral use, carriers which are commonly used include lactose and com starch. Lubricating agents, such as magnesium stearate, are also typically added. For oral administration in a capsule form, useful diluents include lactose and dried com starch. When aqueous suspensions or emulsions are administered orally, the active ingredient may be suspended or dissolved in an oily phase is combined with emulsifying or suspending agents. If desired, certain sweetening, flavoring, or coloring agents can be added.

[0021] For example, the pharmaceutical compositions of this invention can be administered in the form of suppositories for rectal administration. These compositions can be prepared by mixing a compound of this invention with a suitable non-irritating excipient which is solid at room temperature but liquid at the rectal temperature and therefore will melt in the rectum to release the active components. Such materials include, but are not limited to, cocoa butter, beeswax, and polyethylene glycols.

[0022] For example, the pharmaceutical compositions of this invention can be administered by nasal aerosol or inhalation. Such compositions are prepared according to techniques well-known in the art of pharmaceutical formulation and can be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, or other solubilizing or dispersing agents known in the art.

[0023] For example, the pharmaceutical compositions of this invention can be administered by injection (e.g., as a solution or powder). Such compositions can be formulated according to techniques known in the art using suitable dispersing or wetting agents (such as, for example, Tween 80) and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, e.g., as a solution in 1,3 -butanediol. Among the acceptable vehicles and solvents that may be employed are mannitol, water, Ringer’s solution, and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil can be employed, including synthetic mono- or diglycerides. Fatty acids, such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically-Attorney Docket No.: 27527-0235 WO 1acceptable oils, e.g, olive oil or castor oil, especially in their polyoxyethylated versions. These oil solutions or suspensions can also contain a long-chain alcohol diluent or dispersant, or carboxymethyl cellulose or similar dispersing agents which are commonly used in the formulation of pharmaceutically acceptable dosage forms such as emulsions and or suspensions. Other commonly used surfactants such as Tweens, Spans, or other similar emulsifying agents or bioavailability enhancers which are commonly used in the manufacture of pharmaceutically acceptable solid, liquid, or other dosage forms can also be used for the purposes of formulation.

[0024] In some aspects, an effective dose of a pharmaceutical composition of this invention can include, but is not limited to, e.g., about 0.00001, 0.0001, 0.001, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, 1, 1.25, 1.5, 1.75, 2, 2.5, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2500, 5000, or 10000 mg / kg / day, or according to the requirements of the particular pharmaceutical composition.

[0025] When the pharmaceutical compositions disclosed herein include a combination of a compound of the formulae described herein and one or more additional compounds, both the compound and the additional compound should be present at dosage levels of between about 1 to 100%, and more preferably between about 5 to 95% of the dosage normally administered in a monotherapy regimen. The additional agents can be administered separately, as part of a multiple dose regimen, from the compounds of this invention.Alternatively, those agents can be part of a single dosage form, mixed together with the compounds of this invention in a single composition.

[0026] In some aspects, the pharmaceutical compositions disclosed herein can be included in a container, pack, or dispenser together with instructions for administration. Methods of Treatment

[0027] The methods disclosed herein contemplate administration of an effective amount of a compound or composition to achieve the desired or stated effect. Typically, the compounds or compositions of the invention will be administered from about 1 to about 6 times per day or, alternately or in addition, as a continuous infusion. Such administration can be used as a chronic or acute therapy. The amount of active ingredient that can be combined with the carrier materials to produce a single dosage form will vary depending upon the hostAttorney Docket No.: 27527-0235 WO 1treated and the particular mode of administration. A typical preparation will contain from about 5% to about 95% active compound (w / w). Alternatively, such preparations can contain from about 20% to about 80% active compound.

[0028] In some aspects, the present disclosure provides methods for using a composition comprising a compound, including pharmaceutical compositions (indicated below as ‘X’) disclosed herein in the following methods: Substance X for use as a medicament in the treatment of one or more diseases or conditions disclosed herein. Use of substance X for the manufacture of a medicament for the treatment of Y; and substance X for use in the treatment ofY.

[0029] In some aspects, the methods disclosed include the administration of a therapeutically effective amount of one or more of the compounds or compositions described herein to a subject (e.g, a mammalian subject, e.g., a human subject) who is in need of, or who has been determined to be in need of, such treatment. In some aspects, the methods disclosed include selecting a subject and administering to the subject an effective amount of one or more of the compounds or compositions described herein, and optionally repeating administration as required.

[0030] In some aspects, subject selection can include obtaining a sample from a subject (e.g, a candidate subject) and testing the sample for an indication that the subject is suitable for selection. In some aspects, the subject can be confirmed or identified, e.g, by a health care professional, as having had or having a condition or disease. In some aspects, suitable subjects include, for example, subjects who have or had a condition or disease but that resolved the disease or an aspect thereof, present reduced symptoms of disease (e.g., relative to other subjects (e.g., the majority of subjects) with the same condition or disease), or that survive for extended periods of time with the condition or disease (e.g., relative to other subjects (e.g, the majority of subjects) with the same condition or disease), e.g, in an asymptomatic state (e.g., relative to other subjects (e.g., the majority of subjects) with the same condition or disease). In some aspects, exhibition of a positive immune response towards a condition or disease can be made from patient records, family history, or detecting an indication of a positive immune response. In some aspects, multiple parties can be included in subject selection. For example, a first party can obtain a sample from a candidate subject and a second party can test the sample. In some aspects, subjects can be selected or referred by a medical practitioner (e.g, a general practitioner). In some aspects, subjectAttorney Docket No.: 27527-0235 WO 1selection can include obtaining a sample from a selected subject and storing the sample or using the in the methods disclosed herein. Samples can include, e.g., cells or populations of cells.

[0031] In some aspects, methods of treatment can include a single administration, multiple administrations, and repeating administration of one or more compounds disclosed herein as required for the prevention or treatment of the disease or condition from which the subject is suffering. In some aspects, methods of treatment can include assessing a level of disease in the subject prior to treatment, during treatment, or after treatment. In some aspects, treatment can continue until a decrease in the level of disease in the subject is detected.

[0032] The term “subject,” as used herein, refers to any animal. In some instances, the subject is a mammal. In some instances, the term “subject,” as used herein, refers to a human (e.g., a man, a woman, or a child).

[0033] The terms “administer,” “administering,” or “administration,” as used herein, refer to implanting, ingesting, injecting, inhaling, or otherwise absorbing a compound or composition, regardless of form. For example, the methods disclosed herein include administration of an effective amount of a compound or composition to achieve the desired or stated effect.

[0034] The terms “treat”, “treating,” or “treatment,” as used herein, refer to partially or completely alleviating, inhibiting, ameliorating, or relieving the disease or condition from which the subject is suffering. This means any manner in which one or more of the symptoms of a disease or disorder are ameliorated or otherwise beneficially altered. As used herein, amelioration of the symptoms of a particular disorder refers to any lessening, whether permanent or temporary, lasting or transient that can be attributed to or associated with treatment by the compositions and methods of the present invention.

[0035] The terms “prevent,” “preventing,” and “prevention,” as used herein, shall refer to a decrease in the occurrence of a disease or decrease in the risk of acquiring a disease or its associated symptoms in a subject. The prevention may be complete, e.g., the total absence of disease or pathological cells in a subject. The prevention may also be partial, such that the occurrence of the disease or pathological cells in a subject is less than, occurs later than, or develops more slowly than that which would have occurred without the present invention.

[0036] As used herein, the term “preventing a disease” in a subject means for example, to stop the development of one or more symptoms of a disease in a subject before they occur orAttorney Docket No.: 27527-0235 WO 1are detectable, e.g., by the patient or the patient’s doctor. Preferably, the disease does not develop at all, i.e., no symptoms of the disease are detectable. However, it can also mean delaying or slowing of the development of one or more symptoms of the disease.Alternatively, or in addition, it can mean decreasing the severity of one or more subsequently developed symptoms.

[0037] Specific dosage and treatment regimens for any particular patient will depend upon a variety of factors, including the activity of the specific compound employed, the age, body weight, general health status, sex, diet, time of administration, rate of excretion, drug combination, the severity and course of the disease, condition or symptoms, the patient’s disposition to the disease, condition or symptoms, and the judgment of the treating physician.

[0038] An effective amount can be administered in one or more administrations, applications or dosages. A therapeutically effective amount of a therapeutic compound (i.e., an effective dosage) depends on the therapeutic compounds selected. Moreover, treatment of a subject with a therapeutically effective amount of the compounds or compositions described herein can include a single treatment or a series of treatments. For example, effective amounts can be administered at least once. The compositions can be administered one from one or more times per day to one or more times per week; including once every other day. The skilled artisan will appreciate that certain factors can influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health or age of the subject, and other diseases present.

[0039] Following administration, the subject can be evaluated to detect, assess, or determine their level of disease. In some instances, treatment can continue until a change (e.g, reduction) in the level of disease in the subject is detected. Upon improvement of a patient’s condition (e.g., a change (e.g, decrease) in the level of disease in the subject), a maintenance dose of a compound, or composition disclosed herein can be administered, if necessary. Subsequently, the dosage or frequency of administration, or both, can be reduced, e.g., as a function of the symptoms, to a level at which the improved condition is retained. Patients may, however, require intermittent treatment on a long-term basis upon any recurrence of disease symptoms.Attorney Docket No.: 27527-0235 WO 1RESULTS AND DISCUSSION

[0031] Proof-of-concept studies of USP-28-based DUBTAC platform to stabilize CFTR. To demonstrate that USP28 can be effectively harnessed for DUBTAC development and protein stabilization, we synthesized several USP28-based CFTR DUBTACs (CDTs) and examined their ability to stabilize CFTR, the benchmark protein used in DUBTAC studies (Figure 2A). The USP28 non-covalent ligand, CT1073, was linked with the CFTR ligand, Lumacaftor1, by various linkers to generate the USP28-based CDTs (Figure 2B). Notably, screening of these CDTs in CFBE41o-4.7 AF508-CFTR (CF41o-AF508) human cystic fibrosis bronchial epithelial cells identified CDT-79 (MS9279) as the lead compound for enhancing the stability of the AF508-CFTR mutant protein, which showed similar potency as the reported OTUBl-based CFTR DUBTAC, NJH-2-0571(Figure 2C and 2D). Further characterization studies revealed that MS9279 stabilized CFTR protein in a concentrationfigure 2E) and time-dependent manner, with CFTR protein level increasing drastically after 18h (Figure 2F). Overall, we have demonstrated that USP28 can be successfully recruited in DUBTAC for targeted protein stabilization, as our USP28-based CFTR DUBTAC effectively stabilized CFTR with an efficacy comparable to OTUBl-based CFTR DUBTAC, NJH-2-0571, and USP7-based CFTR DUBTAC3, as previously reported.

[0032] Developing USP28-based cGAS DUBTACs (GDTs) and analyzing their effects on cancer cell proliferation. Following the successful discovery of effective USP28-recruiting CFTR DUBTACs, we extended this approach to stabilize cancer-suppressive proteins, such as cGAS, a key mediator of the cGAS / STING pathway that regulates innate immunity and tumor suppression, for potential anti-cancer therapy34. Briefly, we propose that USP28-based GDTs will stabilize cGAS by recruiting the USP28 deubiquitinase to polyubiquitinated cGAS to facilitate the cleavage of its ubiquitin molecules. The removal of these ubiquitin chains prevents the proteasomal degradation of cGAS, thereby enhancing its stability and activating downstream STING signaling35(Figure 3A). Hence, we synthesized a series of USP28 -recruiting cGAS DUBTACs (GDT compounds) (Figures 3B-C) by conjugating the cGAS antagonist, G14036, to CT1073, the USP28 non-covalent ligand, via reported linkage strategy4and evaluated their cGAS stabilization efficacy in HeLa cells. Treatment of these GDT compounds in HeLa cells identified GDT-99 (MS2099) and GDT-100 (MS2100) as the most potent compounds in this series that effectively increased cGAS protein level as revealed by WB analysis (Figures 3D-I). Furthermore, we showed that both MS2099 and MS2100 enhanced cGAS stabilization in a concentration-dependent mannerAttorney Docket No.: 27527-0235 WO 1(Figure 3J and S2B), and in the time course study, MS2100 potently increased cGAS protein level and its downstream effector, STING, as early as 3 h (Figure 3K). Additionally, we confirmed that cGAS stabilization required the engagement of both cGAS and USP28, as treatment with the cGAS antagonist, G140, or the USP28 ligand, CT1073, alone showed minimal effect on cGAS protein level (Figure 3L). The stabilization of the cGAS protein induced by MS2099 or MS2100 treatment was laigely abolished by competition with a single ligand specific to either cGAS (GMO) or USP28 (CT1073) (Figure 3L). To determine whether DUBTAC treatment could facilitate ternary complex formation, we conducted an immunoprecipitation assay using HA-cGAS and Flag-USP28 plasmids. HA-cGAS was immunoprecipitated only in the presence of MS2099 or MS2100 (Figure 3M), confirming that DUBTAC treatment induced the formation of a ternary complex. Additionally, the analysis of cGAS ubiquitination levels, with or without MS2100, showed that the treatment with MS2100 reduced the ubiquitination level of cGAS in the presence of the proteasome inhibitor MG 132 (Figure 3N). Further evaluation of downstream activation from cGAS stabilization was conducted. Stabilization of cGAS led to a direct increase in cGAMP levels (Figure 30), which subsequently promoted p-STING activation in the presence of high molecular weght DNA (HT-DNA) resulting in elevated levels of p-IRF3 protein (Figure 3P). To confirm pathway specificity, the STING inhibitor SN-01137was included to block STING activation. SN-011 treatment effectively disrupted the activation of both p-STING and downstream p-IRF3, even in the presence of DUBTAC MS2100 (Figure 30). Since activation of the cGAS-STTNG pathway has been shown to inhibit cancer cell growth34, we evaluated the antiproliferative effects of MS2099 and MS2100 through cell imaging and colony formation assays (Figure 8). The parent cGAS antagonist, GMO, and GDT-87 (Figure 3C and 3E) were included as a negative control due to its minimal stabilization efficacy, and we also included a linker version of negative control of MS2099, termed MS2099N (Figure 8C and 8D). Our results showed that both MS2099 and MS2100 drastically reduced the number of HeUa cells to a greater extent than the cGAS antagonist, GMO, following a 10 pM treatment for one day in cell imaging and for two weeks in colony formation assays (Figure 8B). In addition, we monitored cell growth in response to MS2099, MS2100, GMO, MS2099N, or GDT-87 treatment at 10 pM over four days by counting the cell number in each condition (Figure 8C). We found that MS2099 and MS2100 significantly suppressed HeUa cell growth, and even led to a reduction in cell number that is lower than the starting cell number, indicating cell death. Dose-response evaluation of MS2099, MS2100, GMO andAttorney Docket No.: 27527-0235 WO 1GDT-87 over one day also demonstrated that MS2099 suppressed HeLa cell viability potently with an IC50 of 0.53 ± 0.21 pM, compared to GMO and USP28 linker control (MS2099N), which have an IC50 of 13.5 ± 2.3 pM and 4.9 ± 0.6 pM, respectively (Figure 8D), further supporting the potential of USP28 -based cGAS DUBTACs as effective agents to inhibit the cancer cell proliferation. In addition, imaging of HeLa cells treated with DMSO, G140, GDT-87, MS2099, or MS2100 at 10 pM for 24 h demonstrated that MS2099 and MS2100 significantly inhibited the HeLa cell proliferation (Figure 8E). Altogether, MS2099 and MS2100 emerged as lead compounds that effectively stabilized cGAS protein in a concentration- and time-dependent manner, leading to the activation of the STING pathway. The activation of the cGAS-STING pathway by MS2099 and MS2100 subsequently suppressed cancer cell growth in HeLa cells, as demonstrated through the cell viability and colony formation assays. These results underscore the potential of targeted protein stabilization via DUBTAC for therapeutic purposes.

[0033] Developing USP28-based PPARy DUBTACs (PDTs). Inspired by the success of USP28-based cGAS DUBTACs, we aimed to develop USP28-based PPARy DUBTACs. Notably, PPARy plays a pivotal role in cellular processes such as differentiation, proliferation, and apoptosis38. It has been identified as a promising taiget in cancer therapy39'41, particularly due to its ability to alter the tumor microenvironment and suppress tumor progression and metastasis. Moreover, previous studies have shown that activation of PPARy suppressed cancer cell growth and tumor invasiveness in multiple human cancers, such as breast, lung, and colon cancer42, 43. Here, we propose a novel therapeutic strategy to inhibit cancer cell growth through the stabilization of PPARy by USP28-recruiting PPARy DUBTACs (PDT compounds) (Figure 4A). To synthesize these DUBTACs, we first analyzed the co-crystal structure of PPARy in complex with its ligand, Lobeglitazone (LGZ) (PDB ID: 5YCN)44, and found that the methoxyl group in the phenyl ring is solvent-exposed (Figure 4B). Therefore, from this methoxy group of LGZ, we linked LGZ to the USP28 ligand, CT1073, via various linkers (Figure 4C). Subsequent screening using western blot analysis in HeLa cells identified PDT-27 to -30 (MS1727 to MS1730) as the most effective compounds in stabilizing PPARy (Figures 4D and 4E). Further analysis confirmed that these four compounds (MS1727 to MS1730) significantly stabilized PPARy in both HeLa and MDA-MB-231 cells (Figure 4F and 4G). Additionally, time-course and dosage studies of MS1728-mediated PPARy stabilization were performed by treating HeLa cells with MS 1728 for 0 - 24 h and at concentrations ranging from 0 to 20 pM, respectively. Our results revealed HeLa’sAttorney Docket No.: 27527-0235 WO 1clear time- and concentration-dependent response to MS1728 (Figure 4H and 41). Notably, competitive rescue experiments with the USP28 ligand, CT1073, or the parental PPARy ligand, LGZ, significantly reduced the PPARy stabilizing effect of MS 1728, suggesting that binding to both USP28 and PPARy is required for MS1728-mediated PPARy stabilization (Figure 4J). Subsequently, we attempted to validate the ternary complex formation by immunoprecipitaion assay using 293 cells transfected with HA -PPARy and Flag-USP28. The HA -PPARy was immunoprecipitated only in the presence of DUBTACs, confirming that DUBTAC treatment successfully induced the formation of a ternary complex (Figure 4K). In addition, the USP28 konckdown experiment indicated that the observed PPARy upregulation is largely dependent on USP28 (Figure 4L).

[0034] The validation of PPARy DUBTACs (PDTs) as a novel therapy for inhibiting cancer cell proliferation. To investigate the anti-proliferation effects of compounds MS 1727 to MS 1730 (Figure 5 A and Figure 9), we treated He La cells with these compounds, or the USP28 ligand with linker (MS1727N), or the PPARy ligand (LGZ), and analyzed their effect on cell numberthrough colony formation assays (Figure 5B and 5C) and cell imaging (Figure 5D and 5E). We also included PDT-16 and PDT-17 as negative controls, as these compounds did not stabilize PPARy, but encompassed both USP28 and PPARy binding arms. As shown in Figures 5F and 9D, compounds MS1727 to MS1730, which potently stabilized PPARy, greatly reduced cell number, whereas LGZ, PDT-16, and PDT-17 had minimal effect on HeLa cell number. Additionally, a cell proliferation assay conducted over four days further demonstrated that MS 1727 to MS 1730 significantly suppressed cell growth (Figure 5G and 9E). Dose-response evaluation of MS1727 to MS1730, and LGZ over three days also showed that MS1727 suppressed HeLa cell viability most potently with an IC50 of 0.33 ± 0.05 pM, exhibited a concentration-dependent anti-proliferative effect of MS 1727 to MS 1730 in HeLa cells (Figure 5G). In contrast, LGZ and PDT-16, which did not or weakly increase PPARy protein level, were less potent in suppressing the proliferation of HeLa cells with an IC50 of 32.5 ± 9.3 pM and >100 pM (Figure 9E), respectively, indicating that the superior antigrowth effect of MS 1727 to MS 1730 is likely due to the stabilization of PPARy. Taken together, we have provided another example supporting that USP28 can be successfully harnessed in DUBTACs for targeted protein stabilization. In particular, we discovered several first-in-class USP28-based PPARy DUBTACs that potently stabilize PPARy in both HeLa and MDA-MB-231 cells, in a concentration- and time-dependent manner that requires binding to both USP28 and PPARy and significantly suppressed growth of HeLa cancer cells.Attorney Docket No.: 27527-0235 WO 1

[0035] In this study, we have demonstrated for the first time that USP28 can be harnessed for DUBTAC development. USP28 is the third DUB, only after OTUB1 and USP7, which has been successfully hijacked to generate effective DUBTACs. By utilizing a noncovalent small-molecule inhibitor of USP28 as a USP28 binder, we developed CFTR, cGAS, and PPARy DUBTACs that effectively stabilized these target proteins, respectively. The lead USP28-recruiting cGAS DUBTACs we developed effectively stabilized cGAS, activated cGAS-STING signaling, and exhibited anti-proliferative effects in cancer cells. Notably, the PPARy-targeting DUBTACs we developed are first-in-class. Our engineered USP28-based DUBTACs not only effectively stabilized PPARy, but also suppressed the proliferation in cancer cells. While PPARy has controversial roles in cancer cells27'30, our results suggest that the PPARy stabilization induced by DUBTACs could provide a new potential therapeutic approach for the treatment of cancer. Future studies will focus on evaluating the in vivo pharmacokinetics and anti-tumor efficacy of these compounds. cGAS-targeting DUBTACs hold the potential to modulate and activate the cGAS-STING-IRF3-mediated immune pathway, thereby offering opportunities to enhance immunotherapy approaches. Moreover, further studies are needed to evaluate the in vivo therapeutic potential of PPARy-targeting DUBTACs. In conclusion, the novel CFTR, cGAS, and PPARy-targeting DUBTACs developed in this study could serve as potentially useful chemical biology tools to further explore CFTR, cGAS, and PPARy physiological and pathophysiological functions.

[0036] A number of embodiments of the invention have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.EXPERIMENTAL METHODSCell culture

[0037] CFBE41o-4.7 AF508-CFTR (CF41o-AF508) human cystic fibrosis bronchial epithelial cells, commercially available from Millipore Sigma (SCC159), are a derivative of the parental CFBE41o- cells modified to ectopically express the AF508-CFTR construct. These cells were cultured in a-MEM media (Sigma, #M2279), supplemented with 10% FBS, 2 mM L-glutamine, 300 pg / mL Hygromycin B, 10,000 units / mL penicillin, and 10,000 pg / mL streptomycin. For experiments, cells were seeded in 6-well plates coated with a mixture of 10 pg / mL Fibronectin (Sigma, #F2006), 30 pg / mL PureCol Collagen (Sigma,Attorney Docket No.: 27527-0235 WO 1#5006), and 100 pg / mL BSA (Sigma, #126575), and treated with the indicated drugs at approximately 60-70% confluency. HeLa and MDA-MB-231 cells were maintained in Dulbecco’s Modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U / mL penicillin, and 100 pg / mL streptomycin. Lentiviral packaging and cell line infections were carried out as previously described32. Following viral infection, CF41o-AF508 cells were selected using 1 pg / mL puromycin for at least 72 h to eliminate noninfected cells.Western blot assays

[0038] Cells were lysed using the EBC lysis buffer (50 mM Tris pH 7.5, 120 mM NaCl, 0.5% NP-40), supplemented with protease inhibitors from Pierce and phosphatase inhibitors from Calbiochem. Protein concentrations were determined. Approximately 50 pg of whole cell lysates (WCL) were separated using 8% SDS-PAGE at 130 V for 120 min for CFTR protein analysis and 10% SDS-PAGE at 130 V for 90 min for cGAS and PPARy. Proteins were then transferred to PVDF membranes and probed with the indicated specific primary antibodies at 4°C overnight. After five washes with Tris-buffered saline containing 0.1% Tween-20 (TBST), the membranes were incubated with the corresponding secondary antibodies for 1 h at room temperature and washed four times with the TBST buffer.Antibodies used included CFTR (#78335, 1:1,000), cGAS (#15102, 1:1,000), STING (#13647, 1:1,000), PPARy (#2435, 1:1,000), USP28 (#4217, 1:1000), p-STING (#50907, 1:1,000), and IRF3 (11904), from Cell Signaling Technologies; anti-p-IRF3 (ab76493, 1:1000) antibody from Abeam; anti-Actin (A2228, 1:50,000), anti-Vinculin (V-4505, 1:50,000), anti-mouse secondary (A-4416, 1:3,000), and anti-rabbit secondary (A -4914, 1:3,000) from Sigma.Cell imaging

[0039] Cells were first cultured in 24-well plates until they reached approximately 50% confluence. The growth medium was removed and replaced with indicated DUBTACs or the control in fresh growth medium. Following a 24-hour incubation at 37°C, the cells were washed with PBS. Cells transfected with GFP were imaged using a LEICA DMi8 fluorescence microscope. Images were processed using ImageJ software.Attorney Docket No.: 27527-0235 WO 1Cell proliferation assay

[0040] Cells (30,000 cells / well) in a 12-well plate were treated with individual compounds (10 pM). The cells were trypsinized and the cell numbers were counted using a hemocytometer at the indicated time.Cell viability assay

[0041] Cells were treated with different compounds at different concentrations for 72 h. CCK-based viability test was then conducted to determine cell viability via optical absorbance measurement (96-well plate reader). Briefly, cells were treated with lx solution of CCK-8 (Dojindo, CK04) and re-incubated at 37 °C for 2-4 h until the absorbance at 450 nm reads 1.0 or greater for the control group. Assays were performed in triplicates for each concentration point. The ICso was calculated from the cell survival (%) vs. log (drug concentration) curves using a non-linear regression method in GraphPad Prism.Clonal formation assays

[0042] HeLa cells were treated with the indicated DUBTACs for two weeks. After that cells were fixed with a fixation buffer (acetic acid: methanol=l:7, v:v) and stained with 0.4% crystal violet in 20% ethanol. The number of cell colonies was quantified using Image J (NIH).Immunoprecipitation assays

[0043] HEK293 cells were transfected with HA-cGAS or HA-PPARy plasmids alongside Flag-USP28 for 24 hours. After transfection, cells were treated individually with DUBTAC compounds (5 or 10 pM) for an additional 12 hours. Following this, cells were co-treated with MG132 (10 pM) for another 12 hours, after which they were harvested for immunoprecipitation analysis. The cell lysates were collected with EBC buffer (as mentioned above) for Flag-immunoprecipitation. Lysates (around 8 mg) were incubated with 8 pL of anti -Flag antibody-conjugated bead (Sigma- Aldrich) for 4 h at 4°C with gentle rocking. Then we washed the beads four times with 1 mL of NP-40 washing buffer (20 mM Tris, pH 8.0, 100 mM NaCl, 1 mM EDTA, and 0.5% NP-40). The washed anti-Flag beads were resuspended in 50 pL IxSDS sample buffer and heated at 95°C. Spined down the beads and collected the supernatant for western blot analysis using the indicated antibodies.Attorney Docket No.: 27527-0235 WO 1In cells ubiquitination assay

[0044] HA-cGAS and Myc -ubiquitin (Ub) expression plasmids were transfected with polyethylenimine (PEI) into HEK293 cells, as indicated. At 24 h after transfection, cells were treated with 10 pM MS2100 for 24 h and the proteasome inhibitor MG132 (30 pM) for the final 8 h before harvesting. Cells were lysed in PBS containing 1% SDS. The cell lysates were sonicated and diluted to 0.1% SDS with EBC buffer containing protease inhibitors for HA-immunoprecipitation. Lysates (1.5 mg) were incubated with 8 pL of anti-HA antibody-conjugated bead slurry (Sigma- Aldrich) for 4 h at 4°C with gentle rocking. The beads were washed five times with 1 mL of NP-40 washing buffer (20 mM Tris, pH 8.0, 100 mM NaCl, 1 mM EDTA, and 0.5% NP-40). The washed anti-HA or anti-Myc-IP beads were resuspended in 50 pL 2xSDS sample buffer and heated at 95°C for 5 min. Eluted samples and whole-cell lysates were separated by SDS-PAGE, followed by western blot analysis using the indicated antibodies.Lentivirus knockdown protocol

[0045] HEK293T cells were cultured to 70-80% confluency in DMEM supplemented with 10% FBS. The medium was replaced with fresh DMEM containing 15% FBS before transfection. Cells were transfected with the carrier plasmid, containing the shUSP28 (Obtained from Sigma Mission), along with lentiviral helper plasmids using a standard lipofectamine protocol (ThermoFisher Scientific). The next day, the medium was replaced with fresh DMEM containing 15% FBS. After 36 hours, the supernatant containing lentiviral particles was harvested and centrifuged at 15,000g for 10 minutes. The to be infected cells (MDA-MB-231 and HeLa) were seeded and grown to 50-70% confluency before transduction. The lentiviral supernatant was diluted at a ratio of 1:4 in the appropriate growth medium and added to the target cells. After overnight incubation, the medium was replaced with fresh growth medium, and the cells were cultured for additional three days. The acquired scramble control cells (shScramble) and USP28 knockdown cells (shUSP28) were then treated with or without DUBTACs. Finally, cell lysates were collected and analyzed for western blot.General chemistry methods

[0046] Flash column chromatography was conducted using a Teledyne ISCO CombiFlash Rf+ equipped with a UV detector (220 / 254 / 280 nm) and a fraction collector. Additionally, normal phase column chromatography was carried out with silica gel columns, employingAttorney Docket No.: 27527-0235 WO 1either hexane / ethyl acetate or dichloromethane / methanol as eluents. Reverse-phase column chromatography utilized Cl 8 RediSep Rf columns with a gradient starting at 10% acetonitrile in H2O containing 0.1% TFA, gradually increasing to 100% acetonitrile. All final compounds were purified using preparative high-performance liquid chromatography (HPLC) on an Agilent Prep 1200 series system, maintaining a flow rate of 40 mL / min and utilizing a UV detector set at 220 / 254 nm. Samples were injected onto a Phenomenex Luna 750 x 30 mm, 5 pm Cl 8 column with a similar gradient as mentioned earlier. The purity of respective compounds tested for biological activity was confirmed to be > 95% through chromatography analysis using an Agilent 1200 series system with a DAD detector and a Zorbax 300SB-C18 2.1 mm x 150 mm, 5 pm column. High-resolution mass spectra (HRMS) were acquired in positive ion mode on an Agilent G6230B A Accurate Mass TOF with an electrospray ionization (ESI) source. In another configuration, 0.8 pL samples were injected at a flow rate of 0.8 mL / min at room temperature onto a C18 column using water containing 0.1% formic acid as solvent A and acetonitrile containing 0.1% formic acid as solvent B. In addition, analyses were conducted on an Agilent G1969AAPI-TOF using the same electrospray ionization (ESI) method, where two microliters of samples were injected onto a Cl 8 column with a flow rate of 0.4 mL / min, using the same solvents. No further purification was performed on the commercial chemicals and solvents used in the experiments.

[0047] Scheme SI. Synthesis of CFTR-USP28 DUBTAC linkers CL-072“.MCL-072

[0048] “Reaction and conditions: a) HOAt, EDCI, NMM, DMF, rt, 2h; b)TFA, DCM, rt, Ih, 61% yield in 2 steps.

[0049] (3-(6-(l-(2,2-difluorobenzo[< / ][l,3]dioxol-5-yl)cyclopropane-l-carboxamido)-3-methylpyridin-2-yl)benzoyl)glycine (CL-072) Compound 1-1 (commercial available) (100 mg, 0.22 mmol, 1.0 eq), tert-butyl glycinate (31 mg, 0.24 mmol, 1.1 eq), EDC HC1 (63 mg, 0.33 mmol, 1.5 eq), HOAt (45 mg, 0.33 mmol, 1.5 eq), NMM (67 mg, 0.66 mmol, 3.0 eq) were stirred in DMF (1.5 mL) at room temperature for 2 h. Then the mixture was purified via reverse-ISCO to get related intermediate. Then the intermediate was dissolved in DCM / TFA (1:1, 2 mL), the mixture was stirred at room temperature for 1 h. After removing all the volatiles and resulted residue was purified via reverse-ISCO to yield title compoundAttorney Docket No.: 27527-0235 WO 1as a white solid (70 mg, 61% yield over two steps). 'HNMR (400 MHz, CD3OD) 5 8.08 (d, J = 8.5 Hz, 1H), 7.96 (d, J= 6.6 Hz, 1H), 7.92 (s, 1H), 7.84 (d, J= 8.5 Hz, 1H), 7.64 - 7.57 (dt,.7= 6.4, 1.6 Hz, 2H), 7.39 (s, 1H), 7.34 (d, J = 8.3 Hz, 1H), 7.23 (d, J = 8.3 Hz, 1H), 4.14 -4.06 (m, 2H), 2.32 - 2.26 (s, 3H), 1.75 -1.68 (m, 2H), 1.32 - 1.23 (m, 2H).CL-073: n = 2; CL-077: n = 6; CL-080: m = 1;CL-074: n = 3; CL-078: n = 7; CL-081: m =2;CL-075: n = 4; CL-079: n = 8; CL-082: m = 3;CL-076: n = 5; CL-083: m = 4;CL-084: m = 5;

[0050] Linkers CL-073 - CL-084 were synthesized following the same procedure for preparing Linker CL-072.

[0051] 3-(3-(6-(l-(2,2-difhiorobenzo[< / | [l,3]dioxol-5-yl)cyclopropane-l-carboxamido)-3-methylpyridin-2-yl)benzamido)propanoic acid (CL-073) White solid, 51% yield.1HNMR(400 MHz, CD3OD) 58.09 - 7.90 (m, 4H), 7.69 - 7.60 (m, 2H), 7.40 (s, 1H), 7.35 (d, J= 8.4 Hz, 1H), 7.25 (d, J= 8.3 Hz, 1H), 3.71 - 3.63 (m, 2H), 2.66 (t, J= 7.3 Hz, 2H), 2.33 (s, 3H), 1.76 - 1.69 (m, 2H), 1.36 - 1.27 (m, 2H).

[0052] 4-(3-(6-(l-(2,2-difluorobenzo[t / ][l,3]dioxol-5-yl)cyclopropane-l-carboxamido)-3-methylpyridin-2-yl)benzamido)butanoic acid (CL-074) White solid, 63% yield. 'HNMR (400 MHz, CD3OD) 57.96 (d, J= 8.5 Hz, 1H), 7.83 - 7.76 (m, 3H), 7.54 - 7.43 (m, 2H), 7.27 (s, 1H), 7.22 (d, J= 8.3 Hz, 1H), 7.11 (d, J= 8.3 Hz, 1H), 3.33 (t, J = 6.9 Hz, 2H), 2.28 (t, J= 7.3 Hz, 2H), 2.18 (s, 3H), 1.85 - 1.79 (m, 2H), 1.63 - 1.57 (m, 2H), 1.20 - 1.12(m, 2H).

[0053] 5-(3-(6-(l-(2,2-difluorobenzo[t / ][l,3]dioxol-5-yl)cyclopropane-l-carboxamido)-3-methylpyridin-2-yl)benzamido)pentanoic acid (CL-075) White solid, 65% yield. 'HNMR (400 MHz, CD3OD) 58.08 (d, J= 8.6 Hz, 1H), 7.94 - 7.84 (m, 3H), 7.62 - 7.57 (m, 2H), 7.40 (s, 1H), 7.34 (d, J= 10.4 Hz, 1H), 7.24 (d, J= 8.4 Hz, 1H), 3.47 -3.35 (m, 2H), 2.39 - 2.32 (m, 2H), 2.29 (s, 3H), 1.73 - 1.63 (m, 6H), 1.33 - 1.26 (m, 2H).

[0054] 6-(3-(6-(l-(2,2-difluorobenzo[d][l,3]dioxol-5-yl)cyclopropane-l-carboxamido)-3-methylpyridin-2-yl)benzamido)hexanoic acid (CL-076) White solid, 61%Attorney Docket No.: 27527-0235 WO 1yield. 'H NMR (400 MHz, CD3OD) 58.06 (d, J= 8.5 Hz, 1H), 7.88 (d, J= 6.4 Hz, 1H), 7.85 - 7.80 (m, 2H), 7.56 (d, J= 5.2 Hz, 2H), 7.37 (s, 1H), 7.31 (d, J= 8.3 Hz, 1H), 7.21 (d, J= 8.3 Hz, 1H), 3.38 (t, J= 6.3 Hz, 2H), 2.31 (t, J= 8.4 Hz, 2H), 2.27 (s, 3H), 1.71 - 1.58 (m, 6H), 1.48 - 1.37 (m, 2H), 1.30 - 1.24 (m, 2H).

[0055] 7-(3-(6-(l-(2,2-difluorobenzo[t / ][l,3]dioxol-5-yl)cyclopropane-l-carboxamido)-3-methylpyridin-2-yl)benzamido)heptanoic acid (CL-077) White solid, 50% yield. *HNMR (400 MHz, CD3OD) 58.05 (d, J= 8.5 Hz, 1H), 7.87 (d, J= 5.1 Hz, 1H), 7.85 -7.79 (m, 2H), 7.59 - 7.51 (m, 2H), 7.35 (s, 1H), 7.30 (d, J= 8.3 Hz, 1H), 7.19 (d, J = 8.3 Hz, 1H), 3.38 - 3.33 (m, 2H), 2.31 - 2.24 (m, 5H), 1.71 - 1.64 (m, 2H), 1.64 - 1.56 (m, 4H), 1.44 - 1.36 (m, 4H), 1.29 - 1.22 (m, 2H).

[0056] 8-(3-(6-(l-(2,2-difluorobenzo[t / ][l,3]dioxol-5-yl)cyclopropane-l-carboxamido)-3-methylpyridin-2-yl)benzamido)octanoic acid (CL-078) White solid, 63% yield. 'H NMR (400 MHz, CD3OD) 58.04 (d, J= 8.5 Hz, 1H), 7.86 (d, J= 6.0 Hz, 1H), 7.83 (s, 1H), 7.79 (d, J= 8.5 Hz, 1H), 7.54 (d, J= 5.0 Hz, 2H), 7.34 (s, 1H), 7.29 (d, J= 8.1 Hz, 1H), 7.18 (d, J= 8.3 Hz, 1H), 3.38 - 3.33 (m, 2H), 2.30 - 2.22 (m, 5H), 1.70 - 1.64 (m, 2H), 1.62 - 1.53 (m, 4H), 1.42 - 1.30 (m, 6H), 1.27 - 1.20 (m, 2H).

[0057] 9-(3-(6-(l-(2,2-difluorobenzo[t / ][l,3]dioxol-5-yl)cyclopropane-l-carboxamido)-3-methylpyridin-2-yl)benzamido)nonanoic acid (CL-079) White solid, 56%. 'HNMR (400 MHz, Methanol-oh) 58.05 (d, J= 7.6 Hz, 1H), 7.89 - 7.76 (m, 3H), 7.54 (d, J= 5.0 Hz, 2H), 7.34 (s, 1H), 7.29 (d, J= 8.4 Hz, 1H), 7.19 (d, J= 8.4 Hz, 1H), 3.40 -3.33 (m, 2H), 2.30 - 2.22 (m, 5H), 1.68 - 1.53 (m, 6H), 1.42 - 1.31 (m, 8H), 1.25 - 1.20 (m, 2H).

[0058] 3-(2-(3-(6-(l-(2,2-difluorobenzo[t / ][l,3]dioxol-5-yl)cyclopropane-l-carboxamido)-3-methylpyridin-2-yl)benzamido)ethoxy)propanoic acid (CL-080) White solid, 52% yield. 'H NMR (400 MHz, CD3OD) 58.03 (d, J= 8.6 Hz, 1H), 7.88 (d, J= 6.8 Hz, 1H), 7.83 (s, 1H), 7.78 (d, J= 8.6 Hz, 1H), 7.57 - 7.49 (m, 2H), 7.32 (s, 1H), 7.28 (d, J= 8.3 Hz, 1H), 7.17 (d, J= 8.3 Hz, 1H), 3.71 (t, J= 6.1 Hz, 2H), 3.64 - 3.57 (m, 2H), 3.56 -3.50 (m, 2H), 2.52 (t, J= 6.1 Hz, 2H), 2.24 (s, 3H), 1.67 - 1.64 (m, 2H), 1.25 - 1.20 (m, 2H).

[0059] 3-(2-(2-(3-(6-(l-(2,2-difluorobenzo[t / |[l,3]dioxol-5-yl)cyclopropane-l-carboxamido)-3-methylpyridin-2-yl)benzamido)ethoxy)ethoxy)propanoic acid (CL-081) White solid, 58% yield. 'HNMR (400 MHz, CD3OD) 58.05 (d, J= 8.8 Hz, 1H), 7.93 - 7.89 (m, 1H), 7.86 (s, 1H), 7.83 (d, J = 7.9 Hz, 1H), 7.60 - 7.53 (m, 2H), 7.36 (s, 1H), 7.31 (d, J=Attorney Docket No.: 27527-0235 WO 18.2 Hz, 1H), 7.21 (d, J= 8.3 Hz, 1H), 3.71 (d, J= 5.5 Hz, 2H), 3.67 - 3.53 (m, 8H), 2.48 (t, J = 5.8 Hz, 2H), 2.26 (s, 3H), 1.70 - 1.62 (m, 2H), 1.28 - 1.22 (m, 2H).

[0060] l-(3-(6-(l-(2,2-difluorobenzo[t / |[l,3]dioxol-5-yl)cyclopropane-l-carboxamido)-3-methylpyridin-2-yl)phenyl)-l-oxo-5,8,ll-trioxa-2-azatetradecan-14-oic acid (CL-082) White solid, 60% yield. ' H NMR (400 MHz, CD30D) 58.04 (d, J= 7.8 Hz, 1H), 7.95 - 7.85 (m, 3H), 7.63 - 7.54 (m, 2H), 7.36 (s, 1H), 7.30 (d, J= 9.0 Hz, 1H), 7.20 (d, J= 7.4 Hz, 1H), 3.67 - 3.50 (m, 14H), 2.47 (t, J= 6.1 Hz, 2H), 2.27 (s, 3H), 1.72 - 1.66 (m, 2H), 1.30 - 1.24 (m, 2H).

[0061] l-(3-(6-(l-(2,2-difluorobenzo[t / ][l,3]dioxol-5-yl)cyclopropane-l-carboxamido)-3-methylpyridin-2-yl)phenyl)-l-oxo-5, 8, 11,14-tetraoxa- 2- azaheptadecan-17-oic acid (CL-083) White solid, 57% yield. ' H NMR (400 MHz, CD3OD) 58.04 - 7.97 (m, 2H), 7.97 - 7.93 (m, 1H), 7.91 (s, 1H), 7.65 - 7.56 (m, 2H), 7.36 (s, 1H), 7.30 (d, J= 7.9 Hz, 1H), 7.20 (d, J= 8.1 Hz, 1H), 3.68 - 3.60 (m, 8H), 3.59 - 3.50 (m, 10H), 2.48 (t, J= 6.1 Hz, 2H), 2.29 (s, 3H), 1.74 - 1.67 (m, 2H), 1.30 - 1.25 (m, 2H).

[0062] l-(3-(6-(l-(2,2-difluorobenzo[t / ][l,3]dioxol-5-yl)cyclopropane-l-carboxamido)-3-methylpyridin-2-yl)phenyl)-l-oxo-5,8,ll,14,17-pentaoxa-2-azaicosan-20-oic acid (CL-084) White solid, 55% yield. 'HNMR (400 MHz, CD3OD) 57.97 (s, 2H), 7.91 (d, J= 6.9 Hz, 1H), 7.87 (s, 1H), 7.63 - 7.53 (m, 2H), 7.31 (s, 1H), 7.25 (d, J= 8.2 Hz, 1H), 7.16 (d, J= 8.3 Hz, 1H), 3.65 - 3.56 (m, 8H), 3.55 - 3.47 (m, 14H), 2.45 (t, J= 6.0 Hz, 2H), 2.25 (s, 3H),1.69 - 1.63 (m, 2H), 1.27 - 1.21 (m, 2H).

[0063] Scheme 2. Synthesis of CDT series compounds""reaction and conditions: a) HOAt, EDCI, NMM, DMF rt, 3h, 40 - XX yield.

[0064] JV-(4-(8-((3-(6-(l-(2,2-difluorobenzo[< / ][l,3]dioxol-5-yl)cyclopropane-l-carboxamido)-3-methylpyridin-2-yl)benzoyl)glycyl)-3,8-diazabicyclo[3.2.1]octan-3-yl)phenethyl)-3-(methylamino)thieno[2,3-Z>] pyridine-2-carboxamide (CDT-072) CL-072 (5.1 mg, 0.01 mmol, 1.0 eq), CT1073 (4.4 mg, 0.01 mmol, 1.0 eq), EDCI (2.9 mg, 0.015Attomey Docket No.: 27527-0235 WO 1mmol, 1.5 eq), HOAt (2.0 mg, 0.015 mmol, 1.5 eq), NMM (3.0 mg, 0.3 mmol, 3.0 eq) were stirred in DMF (1.0 mL) at room temperature for 3 h. Then, the mixture was purified via prep-HPLC to yield titled compound as a yellow solid (3.8 mg, 40%).1H NMR (400 MHz, Methanol-A) 5 8.55 - 8.44 (m, 2H), 8.05 - 7.77 (m, 4H), 7.61 - 7.51 (m, 2H), 7.39 - 7.03 (m, 6H), 6.79 (d, J= 8.6 Hz, 2H), 4.70 - 4.66 (m, 1H), 4.50 - 4.46 (m, 1H), 4.22 (s, 2H), 3.56 - 3.41 (m, 4H), 3.17 (s, 3H), 2.96 - 2.79 (m, 2H), 2.74 (t, J= 7.4 Hz, 2H), 2.21 (s, 3H), 2.08 - 1.84 (m, 4H), 1.63 (q, J= 4.0 Hz, 2H), 1.21 (q, J= 4.0 Hz, 2H). HRMS (m / z): ESI [M] calculated for C49H47F2N8O6S+[M+H]+: 913.3302, Found [M+H]+: 913.3304.

[0065] CDT-073 - CDT-084 were synthesized following the same procedure for preparing CDT-072 with related linkers.

[0066] A-(4-(8-(3-(3-(6-(l-(2,2-difhiorobenzo[< / ][l,3]dioxol-5-yl)cyclopropane-l-carboxamido)-3-methylpyridin-2-yl)benzamido)propanoyl)-3,8-diazabicyclo[3.2.1]octan-3-yl)phenethyl)-3-(methylamino)thieno[2,3-Z>]pyridine-2-carboxamide (CDT-073) Yellow solid, 37% yield. 'HNMR (400 MHz, Mcthanol-tfi) 5 8.54 - 8.48 (m, 2H), 7.98 (d, J= 8.5 Hz, 1H), 7.83 - 7.74 (m, 3H), 7.53 - 7.42 (m, 2H), 7.37 -7.29 (m, 2H), 7.24 (dd, J= 8.3, 1.8 Hz, 1H), 7.14 (d, J= 8.3 Hz, 1H), 7.02 (d, J= 8.5 Hz, 2H), 6.70 (d, J= 8.4 Hz, 2H), 4.70 - 4.64 (m, 1H), 4.42 - 4.38 (m, 1H), 3.62 (t, J= 6.7 Hz, 2H), 3.48 - 3.39 (m, 4H), 3.16 (s, 3H), 2.79 - 2.66 (m, 6H), 2.14 (s, 3H), 1.97 - 1.78 (m, 4H), 1.62 (q, J= 3.9 Hz, 2H), 1.20 (q, J= 4.0 Hz, 2H). HRMS (m / z): ESI [M] calculated for C5OH49F2N806S+[M+H]+: 927.3458, Found [M+H]+: 927.3465.

[0067] A-(4-(8-(4-(3-(6-(l-(2,2-difluorobenzo[< / ][l,3]dioxol-5-yl)cyclopropane-l-carboxamido)-3-methylpyridin-2-yl)benzamido)butanoyl)-3,8-diazabicyclo[3.2.1]octan-3-yl)phenethyl)-3-(methylamino)thieno[2,3-Z>]pyridine-2-carboxamide (CDT-074) Yellow solid, 24% yield. ' H NMR (400 MHz, Methanol-A) 58.54 - 8.48 (m, 2H), 8.01 (d, J = 8.5 Hz, 1H), 7.84 - 7.73 (m, 3H), 7.53 - 7.45 (m, 2H), 7.37 - 7.30 (m, 2H), 7.23 (dd, J = 8.3, 1.8 Hz, 1H), 7.13 (d, J= 8.3 Hz, 1H), 7.03 (d, J= 8.3 Hz, 2H), 6.72 (d, J= 8.4 Hz, 2H), 4.65 - 4.60 (m, 1H), 4.38 - 4.33 (m, 1H), 3.48 - 3.35 (m, 6H), 3.16 (s, 3H), 2.82 - 2.62 (m, 4H), 2.55 - 2.37 (m, 2H), 2.18 (s, 3H), 1.97 - 1.71 (m, 6H), 1.62 (q, J= 3.9 Hz, 2H), 1.19 (q, J= 4.0 Hz, 2H). HRMS (m / z): ESI [M] calculated for C5iH5iF2N8O6S+[M+H]+: 941.3615, Found [M+H]+: 941.3611.

[0068] A-(4-(8-(5-(3-(6-(l-(2,2-difluorobenzo[< / ][l,3]dioxol-5-yl)cyclopropane-l-carboxamido)-3-methylpyridin-2-yl)benzamido)pentanoyl)-3,8-diazabicyclo[3.2.1]octan-Attorney Docket No.: 27527-0235 WO 13-yl)phenethyl)-3-(methylamino)thieno[2,3-Z>]pyridine-2-carb oxamide (CDT-075) Yellow solid, 25% yield. 'H NMR (400 MHz, Methanol-4) 5 8.53 - 8.49 (m, 2H), 7.99 (d, J = 8.5 Hz, 1H), 7.86 - 7.74 (m, 3H), 7.55 - 7.46 (m, 2H), 7.36 - 7.28 (m, 2H), 7.22 (dd, J = 8.2, 1.8 Hz, 1H), 7.13 (d, J= 8.3 Hz, 1H), 7.02 (d, J= 8.5 Hz, 2H), 6.71 (d, J= 8.5 Hz, 2H), 4.67 - 4.62 (m, 1H), 4.42 - 4.37 (m, 1H), 3.47 - 3.31 (m, 6H), 3.16 (s, 3H), 2.76 - 2.68 (m, 4H), 2.46 - 2.37 (m, 2H), 2.18 (s, 3H), 1.96 - 1.75 (m, 4H), 1.68 - 1.56 (m, 6H), 1.19 (q, J = 3.9 Hz, 2H). HRMS (m / z): ESI [M] calculated for C52H53F2N8O6S+[M+H]+: 955.3771, Found [M+H]+: 955.3780.

[0069] A-(4-(8-(6-(3-(6-(l-(2,2-difluorobenzo[< / ][l,3]dioxol-5-yl)cyclopropane-l-carboxamido)-3-methylpyridin-2-yl)benzamido)hexanoyl)-3,8-diazabicyclo[3.2.1]octan-3-yl)phenethyl)-3-(methylamino)thieno[2,3-Z>]pyridine-2-carboxamide (CDT-076) Yellow solid’ 22% yield. 'H NMR (400 MHz, Methanol^) 5 8.55 - 8.48 (m, 2H), 8.00 (d, J = 8.6 Hz, 1H), 7.84 - 7.78 (m, 3H), 7.55 - 7.45 (m, 2H), 7.37 - 7.28 (m, 2H), 7.23 (dd, J = 8.2, 1.7 Hz, 1H), 7.13 (d, J= 8.2 Hz, 1H), 7.04 (d, J= 8.3 Hz, 2H), 6.74 (d, J= 8.6 Hz, 2H), 4.64 - 4.54 (m, 1H), 4.39 - 4.34 (m, 1H), 3.50 - 3.30 (m, 6H), 3.16 (s, 3H), 2.79 - 2.65 (m, 4H), 2.47 - 2.27 (m, 2H), 2.20 (s, 3H), 1.94 - 1.72 (m, 4H), 1.67 - 1.55 (m, 6H), 1.43 - 1.32 (m, 2H), 1.19 (q, J= 4.0 Hz, 2H). HRMS (m / z): ESI [M] calculated for C53H55F2N8O6S+[M+H]+: 969.3928, Found [M+H]+: 969.3919.

[0070] A-(4-(8-(7-(3-(6-(l-(2,2-difluorobenzo[< / ][l,3]dioxol-5-yl)cyclopropane-l-carboxamido)-3-methylpyridin-2-yl)benzamido)heptanoyl)-3,8-diazabicyclo[3.2.1]octan-3-yl)phenethyl)-3-(methylamino)thieno[2,3-Z>]pyridine-2-carboxamide (CDT-077) Yellow solid, 22% yield. 'H NMR (400 MHz, Methanol^) 5 8.53 - 8.49 (m, 2H), 8.00 (d, J = 8.5 Hz, 1H), 7.84 - 7.72 (m, 3H), 7.53 - 7.47 (m, 2H), 7.37 - 7.29 (m, 2H), 7.23 (dd, J = 8.2, 1.8 Hz, 1H), 7.14 (d, J= 8.3 Hz, 1H), 7.04 (d, J= 8.5 Hz, 2H), 6.74 (d, J= 8.3 Hz, 2H), 4.68 - 4.61 (m, 1H), 4.39 - 4.33 (s, 1H), 3.48 - 3.28 (m, 6H), 3.16 (s, 3H), 2.76 - 2.68 (m, 4H), 2.41 - 2.29 (m, 2H), 2.19 (s, 3H), 1.94 - 1.78 (m, 4H), 1.64 - 1.54 (m, 6H), 1.40 - 1.32 (m, 4H), 1.19 (q, J= 4.0 Hz, 2H). HRMS (m / z): ESI [M] calculated for C54H57F2N8O6S+[M+H]+: 983.4084, Found [M+H]+: 983.4093.

[0071] A-(4-(8-(8-(3-(6-(l-(2,2-difluorobenzo[< / ][l,3]dioxol-5-yl)cyclopropane-l-carboxamido)-3-methylpyridin-2-yl)benzamido)octanoyl)-3,8-diazabicyclo[3.2.1]octan-3-yl)phenethyl)-3-(methylamino)thieno[2,3-Z>]pyridine-2-carboxamide (CDT-078) Yellow solid, 23% yield. 'HNMR (400 MHz, Methanol-^) 5 8.53 - 8.49 (m, 2H), 8.00 (d, JAttorney Docket No.: 27527-0235 WO 1= 8.5 Hz, 1H), 7.85 - 7.70 (m, 3H), 7.54 - 7.46 (m, 2H), 7.36 - 7.29 (m, 2H), 7.24 (dd, J = 8.2, 1.8 Hz, 1H), 7.14 (d, J= 8.3 Hz, 1H), 7.04 (d, J= 8.4 Hz, 2H), 6.75 (d, J= 8.4 Hz, 2H), 4.67 - 4.63 (m, 1H), 4.40 - 4.35 (m, 1H), 3.50 - 3.27 (m, 6H), 3.16 (s, 3H), 2.76 - 2.67 (m, 4H), 2.43 - 2.27 (m, 2H), 2.19 (s, 3H), 1.95 - 1.78 (m, 4H), 1.65 - 1.51 (m, 6H), 1.35 - 1.29 (m, 6H), 1.19 (q, J= 4.0 Hz, 2H). HRMS (m / z): ESI [M] calculated for C55H59F2N8O6S+[M+H]+: 997.4241, Found [M+H]+: 997.4255.

[0072] A-(4-(8-(9-(3-(6-(l-(2,2-difluorobenzo[< / ][l,3]dioxol-5-yl)cyclopropane-l-carboxamido)-3-methylpyridin-2-yl)benzamido)nonanoyl)-3,8-diazabicyclo[3.2.1]octan-3-yl)phenethyl)-3-(methylamino)thieno[2,3-Z>]pyridine-2-carboxamide (CDT-079, MS9279) Yellow solid, 21% yield. ' H NMR (400 MHz, Methanol-^) 5 8.54 - 8.47 (m, 2H), 7.99 (d, J= 8.5 Hz, 1H), 7.85 - 7.72 (m, 3H), 7.52 - 7.48 (m, 2H), 7.37 - 7.29 (m, 2H), 7.24 (dd, J= 8.3, 1.7 Hz, 1H), 7.14 (d, J= 8.2 Hz, 1H), 7.04 (d, J= 8.6 Hz, 2H), 6.75 (d,. / = 8.6 Hz, 2H), 4.68 - 4.62 (m, 1H), 4.40 - 4.34 (m, 1H), 3.50 - 3.27 (m, 6H), 3.16 (s, 3H), 2.79 - 2.68 (m, 4H), 2.39 - 2.29 (m, 2H), 2.19 (s, 3H), 1.95 - 1.75 (m, 4H), 1.64 - 1.49 (m, 6H), 1.33 - 1.26(m, 8H), 1.19 (q, J= 4.1 Hz, 2H).13CNMR (101 MHz, Methanol-4) 5 173.88, 170.58, 170.50, 168.15, 161.22, 153.42, 151.75, 151.06, 137.86, 137.26, 135.72, 135.12, 132.14, 131.70, 130.02, 127.38, 126.26, 125.30, 123.76, 123.69, 121.50, 117.24, 116.71, 109.64, 108.14, 72.62 - 72.38 (m), 71.56, 69.58, 62.51, 57.74, 57.18, 56.51, 56.07, 54.13, 45.83, 43.71, 41.72, 39.25, 37.08, 36.36, 35.25, 30.42, 28.84, 25.35. HRMS (m / z): ESI [M] calculated for C56H6IF2N8O6S+[M+H]+: 1011.4397, Found [M+H]+: 1011.4371.

[0073] A-(4-(8-(3-(2-(3-(6-(l-(2,2-difluorobenzo[< / ][l,3]dioxol-5-yl)cyclopropane-l-carboxamido)-3-methylpyridin-2-yl)benzamido)ethoxy)propanoyl)-3,8-diazabicyclo[3.2.1]octan-3-yl)phenethyl)-3-(methylamino)thieno[2,3-Z>]pyridine-2-carboxamide (CDT-080) Yellow solid, 26% yield.1H NMR (400 MHz, Methanol-<74) 5 8.55 - 8.44 (m, 2H), 8.00 (d, J= 8.6 Hz, 1H), 7.87 - 7.83 (m, 2H), 7.77 (d, J= 8.6 Hz, 1H), 7.54 -7.46 (m, 2H), 7.36 - 7.31 (m, 1H), 7.24 (d, J= 1.7 Hz, 1H), 7.18 (dd, J= 8.3, 1.8 Hz, 1H), 7.09 (d, J= 8.2 Hz, 1H), 7.01 (d, J= 8.4 Hz, 2H), 6.67 (d, J= 8.3 Hz, 2H), 4.44 - 4.34 (m, 2H), 3.72 (t, J= 5.8 Hz, 2H), 3.60 - 3.44 (m, 4H), 3.44 - 3.33 (m, 3H), 3.23 - 3.19 (m, 1H), 3.15 (s, 3H), 2.78 - 2.53 (m, 6H), 2.19 (s, 3H), 1.92 - 1.57 (m, 6H), 1.16 (q, J= 3.1 Hz, 2H). HRMS (m / z): ESI [M] calculated for C52H53F2N8O7S+[M+H]+: 971.3720, Found [M+H]+: 971.3737.Attorney Docket No.: 27527-0235 WO 1

[0074] A-(4-(8-(3-(2-(2-(3-(6-(l-(2,2-difluorobenzo[< / |[l,3]dioxol-5-yl)cyclopropane-l-carboxamido)-3-methylpyridin-2-yl)benzamido)ethoxy)ethoxy)propanoyl)-3,8-diazabicyclo[3.2.1]octan-3-yl)phenethyl)-3-(methylamino)thieno[2,3-Z>]pyridine-2-carboxamide (CDT-081) Yellow solid, 27% yield.1H NMR (400 MHz, Mcthanol-A) 58.53 - 8.48 (m, 2H), 7.94 (d, J= 8.6 Hz, 1H), 7.87 - 7.82 (m, 2H), 7.77 (d, J= 8.6 Hz, 1H), 7.53 -7.46 (m, 2H), 7.34 (dd, J= 8.2, 4.8 Hz, 1H), 7.29 (d, J= 1.7 Hz, 1H), 7.22 (dd, J= 8.3, 1.8 Hz, 1H), 7.12 (d, J= 8.3 Hz, 1H), 7.01 (d, J= 8.4 Hz, 2H), 6.69 (d, J= 8.6 Hz, 2H), 4.63 -4.57 (m, 1H), 4.36 - 4.30 (m, 1H), 3.72 - 3.64 (m, 2H), 3.55 - 3.31 (m, 12H), 3.16 (s, 3H), 2.82 - 2.66 (m, 4H), 2.55 - 2.48 (m, 2H), 2.17 (s, 3H), 1.96 - 1.73 (m, 4H), 1.61 (q, J= 4.0 Hz, 2H), 1.19 (q, J= 4.0 Hz, 2H). HRMS (m / z): ESI [M] calculated for C54H57F2N8O8S+[M+H]+: 1015.3983, Found [M+H]+: 1015.3992.

[0075] JV-(4-(8-(l-(3-(6-(l-(2,2-difluorobenzo[< / ][l,3]dioxol-5-yl)cyclopropane-l-carboxamido)-3-methylpyridin-2-yl)phenyl)-l-oxo-5, 8, 11-trioxa- 2- azatetradecan-14-oyl)-3,8-diazabicyclo[3.2.1]octan-3-yl)phenethyl)-3-(methylamino)thieno[2,3-Z>]pyridine-2-carboxamide (CDT-082) Yellow solid, 22% yield. 'HNMR (400 MHz, Methanol-t / 4) 5 8.53 - 8.48 (m, 2H), 7.95 (d, J= 8.6 Hz, 1H), 7.89 - 7.77 (m, 3H), 7.55 - 7.47 (m, 2H), 7.37 - 7.28 (m, 2H), 7.23 (dd, J= 8.2, 1.8 Hz, 1H), 7.13 (d, J= 8.3 Hz, 1H), 7.02 (d, J= 8.5 Hz, 2H), 6.72 (d, J= 8.6 Hz, 2H), 4.65 - 4.59 (m, 1H), 4.40 - 4.32 (m, 1H), 3.69 - 3.56 (m, 2H), 3.54 - 3.37 (m, 16H), 3.15 (s, 3H), 2.86 - 2.66 (m, 4H), 2.60 - 2.43 (m, 2H), 2.18 (s, 3H), 1.96 - 1.75 (m, 4H), 1.62 (q, J= 3.9 Hz, 2H), 1.20 (q, J= 4.0 Hz, 2H). HRMS (m / z): ESI [M] calculated for C56H6IF2N8O9S+[M+H]+: 1059.4245, Found [M+H]+: 1059.4258.

[0076] A-(4-(8-(l-(3-(6-(l-(2,2-difluorobenzo[< / ][l,3]dioxol-5-yl)cyclopropane-l-carboxamido)-3-methylpyridin-2-yl)phenyl)-l-oxo-5,8,ll,14-tetraoxa-2-azaheptadecan-17-oyl)-3,8-diazabicyclo[3.2.1]octan-3-yl)phenethyl)-3-(methylamino)thieno[2,3-Z>] pyridine- 2-carboxamide (CDT-083) Yellow solid, 22% yield.1H NMR (400 MHz, Methanol-t / 4) 58.54 - 8.44 (m, 2H), 7.98 (d, J= 8.6 Hz, 1H), 7.87 - 7.75 (m, 3H), 7.55 -7.46 (m, 2H), 7.37 - 7.28 (m, 2H), 7.23 (dd, J= 8.3, 1.8 Hz, 1H), 7.14 (d, J= 8.3 Hz, 1H), 7.03 (d, J= 8.3 Hz, 2H), 6.73 (d, J= 8.7 Hz, 2H), 4.67 - 4.60 (m, 1H), 4.43 - 4.35 (m, 1H), 3.68 - 3.35 (m, 22H), 3.15 (s, 3H), 2.88 - 2.45 (m, 6H), 2.18 (s, 3H), 1.99 - 1.78 (m, 4H), 1.62 (q, J = 3.9 Hz, 2H), 1.20 (q, J = 4.0 Hz, 2H). HRMS (m / z): ESI [M] calculated for C58H65F2N8OIOS+[M+H]+: 1103.4507, Found [M+H]+: 1103.4522.Attorney Docket No.: 27527-0235 WO 1

[0077] A-(4-(8-(l-(3-(6-(l-(2,2-difluorobenzo[< / ][l,3]dioxol-5-yl)cyclopropane-l-carboxamido)-3-methylpyridin-2-yl)phenyl)-l-oxo-5,8,ll,14,17-pentaoxa-2-azaicosan-20-oyl)-3,8-diazabicyclo[3.2.1]octan-3-yl)phenethyl)-3-(methylamino)thieno[2,3-Z>] pyridine- 2-carboxamide (CDT-084) Yellow solid, 23% yield. 'H NMR (400 MHz, Methanol-A) 5 8.53 - 8.48 (m, 2H), 7.99 (d, J= 8.5 Hz, 1H), 7.87 - 7.78 (m, 2H), 7.73 (d, J = 8.6 Hz, 1H), 7.54 - 7.45 (m, 2H), 7.36 - 7.29 (m, 2H), 7.24 (dd, J= 8.3, 1.8 Hz, 1H), 7.15 (d, J= 8.3 Hz, 1H), 7.04 (d, J= 8.3 Hz, 2H), 6.75 (d, J= 8.4 Hz, 2H), 4.69 - 4.63 (m, 1H), 4.45 - 4.37 (m, 1H), 3.74 - 3.61 (m, 2H), 3.59 - 3.38 (m, 24H), 3.16 (s, 3H), 2.88 - 2.52 (m, 6H), 2.18 (s, 3H), 1.97 - 1.78 (m, 4H), 1.62 (q, J= 3.9 Hz, 2H), 1.19 (q, J= 4.0 Hz, 2H).HRMS (m / z): ESI [M] calculated for C6oH69F2N8OnS+[M+H]+: 1147.4769, Found [M+H]+: 1147.4782.Scheme 3. Synthesis of cGAS-USP28 DUBTAC linkers GL-51“

[0078] “reaction and conditions: a) K2COs, CH3CN, 80 °C, 6 h, 80%; b) Pd(dppf)Cl2, KO Ac, cpd 1-4, dioxane, 100 °C, 6 h, 65%; c) LiOH, MeOH / H2O, rt, 10 min; d) TFA, DCM, rt, 1 h, 45% yield in 2 steps; f) Malonic acid, HO At, EDCI, NMM, DMF, O°C-rt, 12 h, 50%.Attorney Docket No.: 27527-0235 WO 1

[0079] 2-(9-(l-(2-(( / e / 7-butoxycarbonyl)amino)ethyl)-l / / -pyrazol-3-yl)-6,7-dichloro-l,3,4,5-tetrahydro-2 / f-pyrido[4,3-Z>]indol-2-yl)-2-oxoethyl acetate (1-5) To a solution of 3-(4, 4, 5, 5 -tetramethyl- 1,3,2-dioxaborolan-2-yl)-lH-pyrazole (7.3 g, 37.6 mmol, 1.0 eq) in MeCN (30ml) was added K2CO3 (15.6 g, 112.9 mmol, 3.0 eq) and tert-butyl (2-bromoethyl)carbamate (10.1 g, 45.1 mmol, 1.2 eq). The mixture was stirred at 80 °C for 6 h. After that, water was added and the mixture was extracted with ethyl acetate. The organic layer was collected, and excess solvent was removed to get intermediate 1-3 (10.1 g, 80% yield) and used for the next step directly without further purification. To a solution of compound 1-4 (prepared following previous reported lit1) (1.05 g, 2.5 mmol, 1 eq) and intermediate 1-3 (3.2 g, 9.5 mmol, 3.8 eq) in dioxane (40 ml) were added KOAc (981 mg, 10.0 mmol, 4.0 eq) and Pd(dppf)Ch (408 mg, 0.5 mmol, 0.2 eq). The mixture was stirred at 100 °C for 6 h under N2 followed concentrated under reduced pressure. The resulted residue was purified by ISCO to give title compound as a brown oil, yield (894 mg, 65% yield).1H NMR (400 MHz, DMSO-6) 57.88 (s, 1H), 7.32 (d, J= 9.7 Hz, 1H), 6.70 (dd, J= 27.9, 2.3 Hz, 1H), 4.82 (d, J= 61.8 Hz, 2H), 4.68 - 4.36 (m, 4H), 3.76 (dt, J= 45.2, 5.9 Hz, 2H), 2.89 (dt, J= 46.7, 5.8 Hz, 2H), 2.07 (d, J= 1.9 Hz, 5H), 1.11 (s, 9H).

[0080] l-(9-(l-(2-aminoethyl)-l / 7-pyrazol-3-yl)-6,7-dichloro-l,3,4,5-tetrahydro-2 / f-pyrido[4,3-Z>]indol-2-yl)-2-hydroxyethan-l-one (1-6) To a solution of intermediate 1-5 (420 mg, 0.76 mmol, 1 eq) in MeOH / H2O (10: 1, 4.4 ml) was added LiOH. H2O (91 mg, 2.3 mmol, 3 eq). The mixture was stirred at rt for 10 min followed by concentrated and diluted with H2O (4 mL). After that, the pH value of the solution was adjusted to 6 with HC1 aq. solution (IM) followed by concentration. The residue was re-dissolved in DCM / TFA (2:1, 7.5 mL). After stirring at rt for 1 h, excess solvent was removed and the residue was purified by prep-HPLC to give titled compound as a yellow solid, yield (178.6 mg, 45% yield).1H NMR (400 MHz, CD3OD) 57.77 (d, J= 13.4 Hz, 1H), 7.23 (dd, J= 9.9, 3.0 Hz, 1H), 6.56 (d, J= 9.3 Hz, 1H), 4.73 > 4.47 (m, 4H), 4.40 - 4.13 (m, 2H), 3.97 - 3.46 (m, 4H), 3.07 - 2.81 (m, 2H).

[0081] 3-((2-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5-tetrahydro-LH-pyrido[4,3-Z>]indol-9-yl)-LH-pyrazol-l-yl)ethyl)amino)-3-oxopropanoic acid (GL-051) To a solution of Malonic acid (35.4mg, 0.3 mmol, 2.0 eq) inDMF (0.75 mL) and DCM (18 mL) was added NMM (75.9mg, 0.75 mmol, 5.0 eq), intermediate 1-6 (78.3mg, 0.15 mmol, 1.0 eq), HOAt (24.5mg, 0.18 mmol, 1.2 eq), and EDCI (34.5mg, 0.18 mmol, 12eq) at 0°C. The resulted reaction solution was stirred at 0 °C for 6 h before being stirred at room temperature overnight followed by concentrated. The resulted residue was purified by reverse-phaseAttorney Docket No.: 27527-0235 WO 1chromatography to yield the titled product (38.1 mg, 50% yield). 'H NMR (400 MHz, CD3OD) 57.74 (d, J= 13.4 Hz, 1H), 7.19 (d, J= 17.1 Hz, 1H), 6.45 (d, J = 24.9 Hz, 1H), 4.64 - 4.51 (m, 2H), 4.43 -4.17 (m, 4H), 3.96 - 3.62 (m, 6H), 2.95 - 2.88 (m, 2H).Mr*- GL-52: n =2; GL-57: n = 7; GL-61; m s 1GL-53: n =3; GL-58: n = 8; GL-62: m = 2GL-54: n =4; GL-59: n = 9; GL-63: m = 3GL-55: n =5; GL-60: n = 10; GL-64: m = 4GL-56: n =6; GL-65: m = 5

[0082] Linkers GL-52 - GL-65 were synthesized following the same procedure for preparing linkers GL-51.

[0083] 4-((2-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5-tetrahydro-LH-pyrido[4,3- Z»]indol-9-yl)-LH-pyrazol-l-yl)ethyl)amino)-4-oxobutanoic acid (GL-52) Brown solid, 50% yield. *HNMR (400 MHz, CD3OD) 57.78 - 7.65 (m, 1H), 7.21 (d, J= 18.1 Hz, 1H), 6.49 (dd, J= 23.8, 2.3 Hz, 1H), 4.55 (d, J= 7.0 Hz, 2H), 4.42 - 4.16 (m, 4H), 3.99 - 3.61 (m, 4H), 2.94 (dt, J= 22.2, 5.8 Hz, 2H), 2.59 (t, J= 6.8 Hz, 2H), 2.46 (dt, J= 10.8, 6.7 Hz, 2H).

[0084] 5-((2-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5-tetrahydro-l / / -pyrido[4,3- Z»]indol-9-yl)-LH-pyrazol-l-yl)ethyl)amino)-5-oxopentanoic acid (GL-53). *HNMR (400 MHz, CD3OD) 57.67 (dd, J= 16.3, 2.3 Hz, 1H), 7.21 (d, J= 19.1 Hz, 1H), 6.48 (dd, J= 26.0, 2.3 Hz, 1H), 4.68 - 4.14 (m, 6H), 3.99 - 3.63 (m, 4H), 3.06 - 2.84 (m, 2H), 2.42 - 2.13 (m, 4H), 1.85 (d, J= 29.4 Hz, 2H).

[0085] 6-((2-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5-tetrahydro-LH-pyrido[4,3-Z»]indol-9-yl)-LH-pyrazol-l-yl)ethyl)amino)-6-oxohexanoic acid (GL-54). 'H NMR (400 MHz, CD3OD) 57.69 (dd, J= 17.0, 2.2 Hz, 1H), 7.23 (d, J= 18.8 Hz, 1H), 6.51 (dd, J= 26.7, 2.3 Hz, 1H), 4.72 - 4.19 (m, 6H), 4.00 - 3.59 (m, 4H), 3.05 - 2.86 (m, 2H), 2.21 (d, J = 53.8 Hz, 4H), 1.58 (d, J= 63.0 Hz, 4H).

[0086] 7-((2-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5-tetrahydro-lH-pyrido[4,3-b]indol-9-yl)-lH-pyrazol-l-yl)ethyl)amino)-7-oxoheptanoic acid (GL-55).XH NMR (400 MHz, CD3OD) 57.66 (dd, J= 17.2, 2.3 Hz, 1H), 7.20 (d, J= 18.0 Hz, 1H), 6.49 (dd, J= 25.4, 2.3 Hz, 1H), 4.65 - 4.17 (m, 6H), 3.79 - 3.56 (m, 4H), 3.02 - 2.74 (m, 2H), 2.40 - 2.03 (m, 4H), 1.40 (s, 6H).Atorney Docket No.: 27527-0235 WO 1

[0087] 8-((2-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5-tetrahydro-LH-pyrido[4,3- / >|indol-9-yl)-l / / -pyrazol-l-yl)ethyl)amino)-8-oxooctanoic acid (GL-56). 'HNMR (400 MHz, CD3OD) 57.98 - 7.47 (m, 1H), 7.20 (d, J= 14.3 Hz, 1H), 6.50 (dd, J= 23.5, 2.3 Hz, 1H), 4.70 - 4.18 (m, 6H), 3.99 - 3.88 (m, 1H), 3.78 - 3.57 (m, 3H), 3.01 - 2.74 (m, 4H), 2.34 - 2.05 (m, 2H), 1.73 - 1.13 (m, 8H).

[0088] 9-((2-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5-tetrahydro-LH-pyrido[4,3-Z>]indol-9-yl)-LH-pyrazol-l-yl)ethyl)amino)-9-oxononanoic acid (GL-57).1HNMR (400 MHz, CD3OD) 57.73 - 7.56 (m, 1H), 7.20 (d, J= 14.1 Hz, 1H), 6.49 (dd, J= 24.0, 2.3 Hz, 1H), 4.68 - 4.19 (m, 6H), 4.00 - 3.61 (m, 4H), 3.06 - 2.76 (m, 4H), 2.35 - 2.06 (m, 2H), 1.73 - 1.03 (m, 10H).

[0089] 10-((2-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5-tetrahydro-LH-pyrido[4,3-Z>]indol-9-yl)-LH-pyrazol-l-yl)ethyl)amino)-10-oxodecanoic acid (GL-58). 'HNMR (400 MHz, CD3OD) 57.65 (dd, J= 20.7, 2.3 Hz, 1H), 7.20 (d, J= 14.7 Hz, 1H), 6.48 (dd, J= 24.8, 2.3 Hz, 1H), 4.65 - 4.17 (m, 6H), 3.97 - 3.56 (m, 4H), 2.92 (d, J = 27.6 Hz, 2H), 2.18 (t, J= 59.4 Hz, 4H), 1.67 - 1.04 (m, 12H).

[0090] ll-((2-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5-tetrahydro-LH-pyrido[4,3-Z>]indol-9-yl)-LH-pyrazol-l-yl)ethyl)amino)-ll-oxoundecanoic acid (GL-59). 'H NMR (400 MHz, CD3OD) 57.65 (dd, J= 20.6, 2.3 Hz, 1H), 7.21 (d, J= 13.3 Hz, 1H), 6.49 (dd, J = 24.3, 2.3 Hz, 1H), 4.70 - 4.10 (m, 6H), 3.97 - 3.59 (m, 1H), 3.05 - 2.80 (m, 2H), 2.30 - 2.03 (m, 4H), 1.64 - 0.92 (m, 14H).

[0091] 12-((2-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5-tetrahydro-LH-pyrido[4,3-Z>]indol-9-yl)-LH-pyrazol-l-yl)ethyl)amino)-12-oxododecanoic acid (GL-60). 'H NMR (400 MHz, CD3OD) 57.65 (dd, J= 21.4, 2.0 Hz, 1H), 7.21 (d, J= 13.7 Hz, 1H), 6.48 (dd, J = 25.4, 2.2 Hz, 1H), 4.70 - 4.18 (m, 6H), 4.00 - 3.56 (m, 4H), 3.09 - 2.81 (m, 2H), 2.38 - 2.02 (m, 4H), 1.64 - 0.93 (m, 16H).

[0092] 3-(3-((2-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5-tetrahydro-LH-pyrido[4,3-Z>]indol-9-yl)-LH-pyrazol-l-yl)ethyl)amino)-3-oxopropoxy)propanoic acid (GL-61). 'HNMR (400 MHz, CD3OD) 57.69 (dd, J= 15.3, 2.3 Hz, 1H), 7.20 (d, J= 17.8 Hz, 1H), 6.49 (dd, J= 23.4, 2.3 Hz, 1H), 4.62 - 4.17 (m, 6H), 3.97 - 3.56 (m, 8H), 3.02 -2.82 (m, 2H), 2.57 - 2.31 (m, 4H).

[0093] 3-(2-(3-((2-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5-tetrahydro-LH-pyrido[4,3-Z>]indol-9-yl)-LH-pyrazol-l-yl)ethyl)amino)-3-oxopropoxy)ethoxy)propanoicAtorney Docket No.: 27527-0235 WO 1acid (GL-62) 'HNMR (400 MHz, CD3OD) 57.70 (dd, J= 17.0, 2.2 Hz, 1H), 7.20 (d, J= 16.8 Hz, 1H), 6.48 (dd, J= 24.2, 2.3 Hz, 1H), 4.62 - 4.15 (m, 6H), 3.97 - 3.42 (m, 12H), 3.01 - 2.82 (m, 2H), 2.54 - 2.32 (m, 4H).

[0094] l-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5-tetrahydro-l / 7-pyrido[4,3-Z>]indol-9-yl)-LH-pyrazol-l-yl)-4-oxo-7,10,13-trioxa-3-azahexadecan-16-oic acid (GL-63) 'HNMR (400 MHz, CD3OD) 57.81 - 7.62 (m, 1H), 7.30 - 7.15 (m, 1H), 6.57 - 6.40 (m, 1H), 4.64 - 4.19 (m, 6H), 3.98 - 3.45 (m, 16H), 3.04 - 2.84 (m, 2H), 2.58 - 2.31 (m, 4H).

[0095] (E)-A^-(2-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5-tetrahydro-l / 7-pyrido[4,3-Z>]indol-9-yl)-LH-pyrazol-l-yl)ethyl)-JV -(2-(5-(4-(4-(dimethylamino)but-2-enoyl)-2-oxopiperazin-l-yl)thiophen-2-yl)ethyl)-4,7,10,13-tetraoxahexadecanediamide (GL-64). 'HNMR (400 MHz, CD3OD) 57.80 - 7.66 (m, 1H), 7.23 (d, J= 17.3 Hz, 1H), 6.52 (dd, J= 24.5, 2.3 Hz, 1H), 4.63 - 4.17 (m, 6H), 3.79 - 3.47 (m, 20H), 3.01 - 2.86 (m, 2H), 2.63 - 2.28 (m, 4H).

[0096] (£')-N'-(2-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5-tetrahydro-l / / -pyrido[4,3- / >|indol-9-yl)-l / / -pyrazol-l-yl)ethyl)- / V' -(2-(5-(4-(4-(dimethylamino)but-2-enoyl)-2-oxopiperazin-l-yl)thiophen-2-yl)ethyl)-4,7,10,13,16-pentaoxanonadecanediamide (GL-65)1H NMR (400 MHz, CD3OD) 57.79 - 7.49 (m, 1H), 7.11 (d, J= 17.3 Hz, 1H), 6.40 (dd, J= 24.3, 2.3 Hz, 1H), 4.54 - 4.10 (m, 6H), 3.73 - 3.38 (m, 24H), 2.92 - 2.76 (m, 2H), 2.51 - 2.14 (m, 4H).

[0097] Scheme 4. Synthesis of cGAS-USP28 DUBTAC linkers GL-86aAttorney Docket No.: 27527-0235 WO 1

[0098] “reaction and conditions: a) K2CO3, CH3CN, 80°C, 6h, 80%; b) Pd(dppf)Cl2, KOAc, Cpd 1-4, dioxane, 100°C, 6h; c) LiOH, MeOHTLO, rt, lOmin, 33% in 2 steps; d) tert-Butyl glycinate, HOAt, EDCI, NMM, DMF, rt, 12h; e) TFA / DCM, rt, 30min, 63% over 2 steps.

[0099] 2-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5-tetrahydro-l / / -pyrido[4,3-Z>]indol-9-yl)-LH-pyrazol-l-yl)acetic acid (1-8) To a solution of 1-2 (7.3 g, 37.6 mmol, 1.0 eq) in MeCN (30 ml) was added K2CO3 (15.6 g, 113 mmol, 3.0 eq) and ethyl bromoacetate (7.5 g, 45.1 mmol, 1.2 eq). After stirred at 80 °C for 6h, water was added and the mixture was extracted with ethyl acetate. The organic layer was collected, and excess solvent was removed to get compound 1-6 (8.4 g, 80%) and used for the next step directly without further purification. To a solution of 1-6 (2.7 g, 9.5 mmol, 3.8 eq) and 1-4 (1.1 g, 2.5 mmol, 1 eq) in dioxane (40 ml) was added KOAc (981mg, 10.0 mmol, 4.0 eq) and Pd(dppf)Cl2CH2C12(408 mg, 0.5 mmol, 0.2 eq). The mixture was stirred at 100 °C for 6 h under N2. After cooling down to room temperature, the reaction mixture was concentrated under reduced pressure and purified by ISCO to yield intermediate as a brown oil (840 mg). To a solution of got intermediate (840 mg, 1.7 mmol, 1 eq) in MeOH (20 ml) and H2O (5 ml) was added LiOH (214 mg, 5.1 mmol, 3 eq). After stirred at rt for 10 min, excess MeOH was removed, resulted residue was diluted with H2O (2 mL) and the pH value was adjusted to 6 with HC1 aq. (IM) followed by purified by prep-HPLC to yield title compound as a yellow solid (456 mg, 33% in 2 steps). ' H NMR (400 MHz, CD3OD) 57.72 (d, J= 2.3 Hz, 1H), 7.24 (s, 1H), 6.53 (d, J = 2.3 Hz, 1H), 4.89 - 4.84 (m, 2H), 4.63 - 4.49 (m, 2H), 4.37 - 4.26 (m, 2H), 4.01 - 3.70 (m, 2H), 3.01 - 2.85 (m, 2H).

[0100] A-(2-aminoethyl)-2-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5-tetrahydro-l / / -pyrido[4,3- / >|indol-9-yl)-l / / -pyrazol-l-yl)acetamide (GL-86) To a solution of 1-9 (65 mg, 0.12 mmol) in DMF (0.5 mL) were added / -butyl glycinate (15.7 mg, 0.12 mmol, 1.0 equiv), l-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI, 34.3 mg, 0.18 mmol, 1.5 equiv), 1 -hydroxy-7 -azabenzo-triazole (HOAt, 24.5 mg, 0.18 mmol, 1.5 equiv), and N-methyhnorpholine (NMM, 36.4 mg, 0.36 mmol, 3.0 equiv). After being stirred overnight at rt, the resulting mixture was purified by prep-HPLC to afford crude product. The crude product was dissolved in DCM / TFA (1:1, 2 mL) followed by stirred at rt for 30 min. The resulting mixture was purified by prep-HPLC to afford title compound as white solid (18.4 mg, 32% yield). ' H NMR (400 MHz, Methanol-4) 57.83 (d, J= 10.2 Hz, 1H), 7.27 (s, 1H),Atorney Docket No.: 27527-0235 WO 16.60 (s, 1H), 5.08 - 5.04 (m, 2H), 4.50 (s, 1H), 4.23 (s, 1H), 4.00 - 3.91 (m, 2H), 3.81 - 3.66 (m, 4H), 3.04 - 2.87 (m, 2H).GL-96: m = 1;GL-97: m = 2;GL-89: n =4; GL-94: n = 9; GL-98: m = 3;GL-90: n =5; GL-95: n = 10; GL-99: m = 4;GL-91: n =6; GL-100: m = 5;

[0101] Linkers GL-87 - GL-100 were synthesized following the same procedure for preparing linker GL-86.

[0102] 3-(2-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5-tetrahydro-l / 7-pyrido[4,3- / >|indol-9-yl)-l / / -pyrazol-l-yl)acetamido)propanoic acid (GL-87) White solid, 49%. 'H NMR (400 MHz, CD3OD) 57.75 (s, 1H), 7.23 (s, 1H), 6.55 (s, 1H), 4.94 (s, 2H), 4.69 - 4.59 (m, 1H), 4.49 - 4.39 (m, 1H), 4.36 - 4.26 (m, 1H), 4.26 - 4.17 (m, 1H), 4.03 - 3.87 (m, 1H), 3.83 - 3.64 (m, 1H), 3.58 - 3.40 (m, 2H), 3.02 - 2.76 (m, 2H), 2.60 - 2.45 (m, 2H).

[0103] 4-(2-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5-tetrahydro-l / 7-pyrido[4,3-Z»]indol-9-yl)-LH-pyrazol-l-yl)acetamido)butanoic acid (GL-88) White solid, 70%. 'H NMR (400 MHz, CD3OD) 57.76 (s, 1H), 7.22 (s, 1H), 6.55 (s, 1H), 4.97 - 4.93 (m, 2H), 4.69 - 4.59 (m, 1H), 4.49 - 4.39 (m, 1H), 4.36 - 4.26 (m, 1H), 4.25 -4.15 (m, 1H), 4.00 -3.88 (m, 1H), 3.79 - 3.69 (m, 1H), 3.23 - 3.17 (m, 2H), 3.01 - 2.75 (m, 2H), 2.41 - 2.25 (m, 2H), 1.69 - 1.43 (m, 2H).

[0104] 5-(2-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5-tetrahydro-l / 7-pyrido[4,3- / >|indol-9-yl)-l / / -pyrazol-l-yl)acetamido)pentanoic acid (GL-89) White solid, 47%. 'H NMR (400 MHz, CD30D) 57.75 (s, 1H), 7.22 (s, 1H), 6.55 (s, 1H), 5.01 - 4.90 (m, 2H), 4.73 -4.61 (m, 1H), 4.48 -4.38 (m, 1H), 4.37 - 4.28 (m, 1H), 4.26 -4.14 (m, 1H), 4.00-3.88 (m, 1H), 3.81 - 3.69 (m, 1H), 3.27 - 3.19 (m, 2H), 3.01 - 2.78 (m, 2H), 2.41 - 2.24 (m, 2H), 1.72 - 1.45 (m, 4H).

[0105] 6-(2-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5-tetrahydro-l / 7-pyrido[4,3-Z»]indol-9-yl)-LH-pyrazol-l-yl)acetamido)hexanoic acid (GL-90) Yellow solid, 25%. 'HAttorney Docket No.: 27527-0235 WO 1NMR (400 MHz, CD3OD) 57.74 (s, 1H), 7.21 (s, 1H), 6.55 (s, 1H), 4.99 - 4.91 (m, 2H), 4.71 - 4.60 (m, 1H), 4.50 - 4.37 (m, 1H), 4.37 - 4.26 (m, 1H), 4.27 - 4.14 (m, 1H), 4.02 -3.85 (m, 1H), 3.79 - 3.64 (m, 1H), 3.28 - 3.15 (m, 2H), 3.03 - 2.79 (m, 2H), 2.33 - 2.14 (m, 2H), 1.69 - 1.43 (m, 4H), 1.43 - 1.24 (m, 2H).

[0106] 7-(2-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5-tetrahydro-l / 7-pyrido[4,3-Z>]indol-9-yl)-LH-pyrazol-l-yl)acetamido)heptanoic acid (GL-91) Yellow solid, 27%. 'H NMR (400 MHz, CD3OD) 57.74 (s, 1H), 7.21 (s, 1H), 6.54 (s, 1H), 4.99 - 4.90 (m, 2H), 4.72 - 4.62 (m, 1H), 4.49 - 4.39 (m, 1H), 4.37 - 4.27 (m, 1H),4.26 - 4.12 (m, 1H), 4.03 -3.89 (m, 1H), 3.82 - 3.65 (m, 1H), 3.27 - 3.14 (m, 2H), 3.01 - 2.81 (m, 2H), 2.33 - 2.15 (m, 2H), 1.66 - 1.42 (m, 4H), 1.41 - 1.22 (m, 4H).

[0107] 8-(2-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5-tetrahydro-l / 7-pyrido[4,3-Z>]indol-9-yl)-LH-pyrazol-l-yl)acetamido)octanoic acid (GL-92) White solid, 67%. 'H NMR (400 MHz, CD3OD) 57.75 (s, 1H), 7.23 (s, 1H), 6.55 (s, 1H), 4.99 - 4.89 (m, 2H), 4.71 - 4.59 (m, 1H), 4.48 - 4.36 (m, 1H), 4.35 - 4.26 (m, 1H), 4.24 -4.15 (m, 1H), 4.00- 3.87 (m, 1H), 3.82 - 3.65 (m, 1H), 3.26 - 3.16 (m, 2H), 3.01 - 2.77 (m, 2H), 2.36 - 2.18 (m, 2H), 1.67 - 1.42 (m, 4H), 1.39 - 1.26 (m, 6H).

[0108] 9-(2-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5-tetrahydro-l / 7-pyrido[4,3-Z>]indol-9-yl)-LH-pyrazol-l-yl)acetamido)nonanoic acid (GL-93) White solid, 54%. 'H NMR (400 MHz, CD3OD) 57.74 (s, 1H), 7.21 (s, 1H), 6.54 (s, 1H), 5.03 - 4.90 (m, 2H), 4.71 -4.53 (m, 1H), 4.49 - 4.38 (m, 1H), 4.37 - 4.26 (m, 1H), 4.26 -4.12 (m, 1H), 4.02- 3.87 (m, 1H), 3.78 - 3.63 (m, 1H), 3.27 - 3.15 (m, 2H), 2.99 - 2.81 (m, 2H), 2.26 - 2.16 (m, 2H), 1.62 - 1.43 (m, 4H), 1.42 - 1.19 (m, 8H).

[0109] 10-(2-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5-tetrahydro-lH-pyrido[4,3-Z>]indol-9-yl)-LH-pyrazol-l-yl)acetamido)decanoic acid (GL-94) Yellow solid, 50%. 'H NMR (400 MHz, CD3OD) 57.77 (s, 1H), 7.24 (s, 1H), 6.57 (s, 1H), 5.06 - 4.93 (m, 2H), 4.74 - 4.6O (m, 1H), 4.48 -4.38 (m, 1H), 4.39 - 4.30 (m, 1H), 4.28 - 4.14 (m, 1H), 3.99 - 3.87 (m, 1H), 3.81 - 3.69 (m, 1H), 3.28 - 3.14 (m, 2H), 3.08 - 2.78 (m, 2H), 2.38 - 2.09 (m, 2H), 1.72 - 1.40 (m, 4H), 1.39 - 1.07 (m, 10H).

[0110] ll-(2-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5-tetrahydro-l / 7-pyrido[4,3-Z>]indol-9-yl)-LH-pyrazol-l-yl)acetamido)undecanoic acid (GL-95) Yellow solid, 52%.1H NMR (400 MHz, CD3OD) 57.76 (s, 1H), 7.23 (s, 1H), 6.56 (s, 1H), 5.05 - 4.92 (m, 2H), 4.75 - 4.60 (m, 1H), 4.47 - 4.37 (m, 1H), 4.35 - 4.25 (m, 1H), 4.24 - 4.10 (m, 1H), 4.00 -Atorney Docket No.: 27527-0235 WO 14.87 (m, 1H), 3.79 - 3.70 (m, 1H), 3.27 - 3.15 (m, 2H), 2.92 (m, 2H), 2.26 (m, 2H), 1.53 (m, 4H), 1.35 - 1.14 (m, 12H).

[0111] 3-(2-(2-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5-tetrahydro-lH-pyrido[4,3- / ’|indol-9-yl)-l / / -pyrazol-l-yl)acetamido)ethoxy)propanoic acid (GL-96) White solid, 35%.1HNMR(400 MHz, CD30D) 57.75 (s, 1H), 7.21 (s, 1H), 6.55 (s, 1H), 5.01 - 4.92 (m, 2H), 4.68 - 4.58 (m, 1H), 4.46 - 4.39 (m, 1H), 4.34 - 4.27 (m, 1H), 4.26 -4.11 (m, 1H), 3.96 - 3.87 (m, 1H), 3.77 - 3.56 (m, 3H), 3.59 - 3.47 (m, 2H), 3.46 - 3.36 (m, 2H), 3.01 - 2.79 (m, 2H), 2.63 - 2.38 (m, 2H).

[0112] 3-(2-(2-(2-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5-tetrahydro-l / f-pyrido[4,3- / >|indol-9-yl)-l / / -pyrazol-l-yl)acetamido)ethoxy)ethoxy)propanoic acid (GL- 97) Yellow solid, 47%. 'H NMR (400 MHz, CD3OD) 57.75 (s, 1H), 7.22 (s, 1H), 6.55 (s, 1H), 5.04 - 4.93 (m, 2H), 4.70 - 4.56 (m, 1H), 4.50 - 4.40 (m, 1H), 4.37 - 4.26 (m, 1H), 4.24 - 4.16 (m, 1H), 4.00 - 3.89 (m, 1H), 3.76 - 3.60 (m, 3H), 3.60 - 3.45 (m, 6H), 3.45 - 3.36 (m, 2H), 3.01 - 2.80 (m, 2H), 2.62 - 2.40 (m, 2H).

[0113] l-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5-tetrahydro-l / 7-pyrido[4,3-Z>]indol-9-yl)-LH-pyrazol-l-yl)-2-oxo-6,9,12-trioxa-3-azapentadecan-15-oic acid (GL- 98). Yellow solid, 71%. *HNMR (400 MHz, CD3OD) 57.74 (s, 1H), 7.20 (s, 1H), 6.54 (s, 1H), 5.03 - 4.95 (m, 2H), 4.79 - 4.58 (m, 1H), 4.53 - 4.36 (m, 1H), 4.35 - 4.24 (m, 1H), 4.24 - 4.11 (m, 1H), 4.01 -3.83 (m, 1H), 3.75 - 3.60 (m, 3H), 3.60 - 3.33 (m, 12H), 3.00- 2.72 (m, 2H), 2.60 -2.44 (m, 2H).

[0114] l-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5-tetrahydro-l / 7-pyrido[4,3-Z>]indol-9-yl)-LH-pyrazol-l-yl)-2-oxo-6,9,12,15-tetraoxa-3-azaoctadecan-18-oic acid (GL-99). Yellow solid, 61%. ‘HNMR (400 MHz, CD3OD) 57.74 (s, 1H), 7.21 (s, 1H), 6.55 (s, 1H), 4.98 - 4.91 (m, 2H), 4.72 - 4.60 (m, 1H), 4.52 - 4.40 (m, 1H), 4.38 - 4.27 (m, 1H), 4.25 - 4.17 (m, 1H), 3.99 - 3.86 (m, 1H), 3.79 - 3.61 (m, 3H), 3.61 - 3.31 (m, 16H), 2.97 - 2.82 (m, 2H), 2.64 - 2.38 (m, 2H).

[0115] l-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5-tetrahydro-l / 7-pyrido[4,3-Z>]indol-9-yl)-LH-pyrazol-l-yl)-2-oxo-6,9,12,15,18-pentaoxa-3-azahenicosan-21-oic acid (GL-100) Yellow solid, 63%.XH NMR (400 MHz, CD3OD) 57.75 (s, 1H), 7.21 (s, 1H), 6.55 (s, 1H), 5.01 - 4.93 (m, 2H), 4.70 - 4.61 (m, 1H), 4.52 - 4.42 (m, 1H), 4.38 - 4.26 (m, 1H), 4.26 - 4.13 (m, 1H), 4.00 - 3.85 (m, 1H), 3.78 - 3.64 (m, 3H), 3.62 - 3.35 (m, 20H), 2.98 -2.77 (m, 2H), 2.65 - 2.37 (m, 2H).Attorney Docket No.: 27527-0235 WO 1

[0116] Scheme 5. Synthesis of cGAS-USP28 DUBTAC GDT-5 la

[0117] “reaction and conditions: a) HOAt, EDCI, NMM, DMSO, rt.

[0118] A-(4-(8-(3-((2-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5-tetrahydro-l / 7-pyrido[4,3-Z>]indol-9-yl)-EH-pyrazol-l-yl)ethyl)amino)-3-oxopropanoyl)-3,8-diazabicyclo[3.2.1]octan-3-yl)phenethyl)-3-(methylamino)thieno[2,3-Z>]pyridine-2-carboxamide (GDT-51) Yellow solid (1.3 mg, 15%).1HNMR(400 MHz, Methanol-< A) 5 8.61 - 8.58 (m, 2H), 7.43 (q, J= 7.3 Hz, 1H), 7.25 (d, J= 9.2 Hz, 1H), 7.16 (d, J= 8.2 Hz, 1H), 7.10 - 7.04 (m, 2H), 6.77 - 6.71 (m, 2H), 6.55 - 6.44 (m, 1H), 4.54 - 4.42 (m, 3H), 3.87 - 3.62 (m, 5H), 3.55 - 3.30 (m, 8H), 3.25 (s, 3H), 3.02 - 2.95 (m, 3H), 2.86 - 2.72 (m, 5H), 1.93 - 1.87 (m, 4H). HRMS (m / z): ESI [M] calculated for C44H47Cl2Ni0O5S+[M+H]+:897.2823, Found [M+H]+: 897.2835.

[0119] cGAS-USP28 DUBTACs GDT-52 - GDT-65 and GDT-86 - GDT-100 were synthesized following the same procedure for preparing GDT-51.Attorney Docket No.: 27527-0235 WO 1

[0120] A-(4-(8-(4-((2-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5-tetrahydro-l / / -pyrido[4,3- / ’|indol-9-yl)-l / / -pyrazol-l-yl)ethyl)amino)-4-oxobutanoyl)-3,8-diazabicyclo[3.2.1]octan-3-yl)phenethyl)-3-(methylamino)thieno[2,3-Z>]pyridine-2-carboxamide (GDT-52) Yellow solid (1.7 mg, 19%).1HNMR(400 MHz, Methanol-A) 5 8.65 - 8.53 (m, 2H), 7.72 (d, J= 12.5 Hz, 1H), 7.42 (dd, J= 8.4, 4.7 Hz, 1H), 7.23 (d, J= 18.0 Hz, 1H), 7.14 - 7.08 (m, 2H), 6.84 - 6.72 (m, 2H), 6.49 (d, J= 19.2 Hz, 1H), 4.73 - 4.56 (m, 3H), 4.44 - 4.19 (m, 5H), 3.75 - 3.65 (m, 3H), 3.58 - 3.30 (m, 5H), 3.23 (s, 3H), 2.99 -2.76 (m, 6H), 2.70 - 2.43 (m, 4H), 1.98 - 1.83 (m, 4H). HRMS (m / z): ESI [M] calculated for C45H49C12NIO05S+[M+H]+: 911.2980, Found [M+H]+: 911.2989.

[0121] JV-(4-(8-(5-((2-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5-tetrahydro-l / 7-pyrido[4,3-Z>]indol-9-yl)-LH-pyrazol-l-yl)ethyl)amino)-5-oxopentanoyl)-3,8-diazabicyclo[3.2.1]octan-3-yl)phenethyl)-3-(methylamino)thieno[2,3-Z>]pyridine-2-carboxamide (GDT-53) Yellow solid (2.0 mg, 22%).1HNMR(400 MHz, Methanol-A) 5 8.62 - 8.51 (m, 2H), 7.69 (d, J= 14.9 Hz, 1H), 7.42 (dd, J= 8.4, 4.7 Hz, 1H), 7.23 (d, J= 21.2 Hz, 1H), 7.14 - 7.05 (m, 2H), 6.76 - 6.66 (m, 2H), 6.48 (d, J= 17.4 Hz, 1H), 4.72 -4.53 (m, 3H), 4.45 - 4.20 (m, 5H), 3.77 - 3.67 (m, 3H), 3.57 - 3.30 (m, 5H), 3.23 (s, 3H), 3.00 - 2.63 (m, 6H), 2.40 - 2.20 (m, 4H), 1.97 - 1.77 (m, 6H). HRMS (m / z): ESI [M] calculated for CreHsiChNioOsS [M+H]+: 925.3136, Found [M+H]+: 925.3128.

[0122] JV-(4-(8-(6-((2-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5-tetrahydro-l / 7-pyrido[4,3-Z>]indol-9-yl)-LH-pyrazol-l-yl)ethyl)amino)-6-oxohexanoyl)-3,8-diazabicyclo[3.2.1]octan-3-yl)phenethyl)-3-(methylamino)thieno[2,3-Z>]pyridine-2-carboxamide (GDT-54) Yellow solid (2.0 mg, 21%).1HNMR(400 MHz, Methanol-tA) 5 8.64 - 8.54 (m, 2H), 7.66 (d, J= 18.7 Hz, 1H), 7.41 (dd, J= 8.5, 4.7 Hz, 1H), 7.23 (d, J= 17.4 Hz, 1H), 7.15 - 7.07 (m, 2H), 6.84 - 6.74 (m, 2H), 6.48 (d, J= 26.3 Hz, 1H), 4.73 -4.58 (m, 3H), 4.45 - 4.24 (m, 5H), 3.75 - 3.64 (m, 3H), 3.56 - 3.30 (m, 5H), 3.22 (s, 3H), 3.01 - 2.62 (m, 6H), 2.38 - 2.17 (m, 4H), 2.04 - 1.76 (m, 4H), 1.66 - 1.54 (m, 4H). HRMS (m / z): ESI [M] calculated for CrrHssChNioOsS [M+H]+: 939.3293, Found [M+H]+:939.3281.

[0123] JV-(4-(8-(7-((2-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5-tetrahydro-LH-pyrido[4,3-Z>]indol-9-yl)-LH-pyrazol-l-yl)ethyl)amino)-7-oxoheptanoyl)-3,8-diazabicyclo[3.2.1]octan-3-yl)phenethyl)-3-(methylamino)thieno[2,3-Z>]pyridine-2-carboxamide (GDT-55) Yellow solid (1.8 mg, 19%).1H NMR (400 MHz, Methanol-t / 4) 5Attorney Docket No.: 27527-0235 WO 18.62 - 8.54 (m, 2H), 7.68 (d, J= 16.1 Hz, 1H), 7.41 (dd, J= 8.5, 4.4 Hz, 1H), 7.24 (d, J= 15.6 Hz, 1H), 7.16 - 7.08 (m, 2H), 6.84 - 6.74 (m, 2H), 6.51 (d, J = 21.8 Hz, 1H), 4.73 -4.54 (m, 3H), 4.46 - 4.11 (m, 5H), 3.78 - 3.62 (m, 3H), 3.57 - 3.35 (m, 5H), 3.23 (s, 3H), 3.03 - 2.64 (m, 6H), 2.37 - 2.26 (m, 4H), 1.97 - 1.80 (m, 4H), 1.63 - 1.55 (m, 4H), 1.28 (p, J = 7.7 Hz, 2H). HRMS (m / z): ESI [M] calculated for CrsHssChNioOsS [M+H]+: 953.3449, Found [M+H]+: 953.3443.

[0124] A-(4-(8-(8-((2-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5-tetrahydro-l / 7-pyrido[4,3-Z>]indol-9-yl)-EH-pyrazol-l-yl)ethyl)amino)-8-oxooctanoyl)-3,8-diazabicyclo[3.2.1]octan-3-yl)phenethyl)-3-(methylamino)thieno[2,3-Z>]pyridine-2-carboxamide (GDT-56) Yellow solid (1.7 mg, 18%). 'H NMR (400 MHz, Mcthanol-t / 4) 5 8.61 - 8.53 (m, 2H), 7.67 (d, J= 19.2 Hz, 1H), 7.41 (dd, J= 8.2, 4.7 Hz, 1H), 7.23 (d, J= 14.5 Hz, 1H), 7.17 - 7.07 (m, 2H), 6.85 - 6.75 (m, 2H), 6.50 (d, J= 24.0 Hz, 1H), 4.75 - 4.55 (m, 3H), 4.44 - 4.21 (m, 5H), 3.76 - 3.65 (m, 3H), 3.56 - 3.30 (m, 5H), 3.23 (s, 3H), 3.00 -2.74 (m, 6H), 2.40 - 2.10 (m, 4H), 2.03 - 1.81 (m, 4H), 1.65 - 1.52 (m, 4H), 1.35 - 1.24 (m, 4H). HRMS (m / z): ESI [M] calculated for C49H57C12NI0O5S+[M+H]+: 967.3606, Found [M+H]+: 967.3630.

[0125] JV-(4-(8-(9-((2-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5-tetrahydro-l / 7-pyrido[4,3-Z>]indol-9-yl)-LH-pyrazol-l-yl)ethyl)amino)-9-oxononanoyl)-3,8-diazabicyclo[3.2.1]octan-3-yl)phenethyl)-3-(methylamino)thieno[2,3-Z>]pyridine-2-carboxamide (GDT-57) Yellow solid (1.7 mg, 17%).XH NMR (400 MHz, Methanol-t / 4) 5 8.62 - 8.52 (m, 2H), 7.66 (d, J= 21.6 Hz, 1H), 7.45 - 7.37 (m, 1H), 7.23 (d, J= 12.5 Hz, 1H), 7.19 - 7.07 (m, 2H), 6.87 - 6.77 (m, 2H), 6.51 (d, J= 25.0 Hz, 1H), 4.76 - 4.54 (m, 3H), 4.46 - 4.20 (m, 5H), 3.78 - 3.64 (m, 3H), 3.58 - 3.30 (m, 5H), 3.23 (s, 3H), 3.01 - 2.73 (m, 6H), 2.42 - 2.10 (m, 4H), 2.03 - 1.81 (m, 4H), 1.65 - 1.45 (m, 4H), 1.29 - 1.19 (m, 6H). HRMS (m / z): ESI [M] calculated for CsoHsgChNioOsS [M+H]+: 981.3762, Found [M+H]+: 981.3778.

[0126] A-(4-(8-(10-((2-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5-tetrahydro-lH-pyrido[4,3- / ?]indol-9-yl)-lH-pyrazol-l-yl)ethyl)amino)-10-oxodecanoyl)-3,8-diazabicyclo[3.2.1]octan-3-yl)phenethyl)-3-(methylamino)thieno[2,3- / j]pyridine-2-carboxamide (GDT-58) Yellow solid (1.8 mg, 18%).1H NMR (400 MHz, Methanol-t / 4) 5 8.62 - 8.50 (m, 2H), 7.67 (d, J= 21.2 Hz, 1H), 7.41 (dd, J= 8.4, 4.7 Hz, 1H), 7.23 (d, J= 12.9 Hz, 1H), 7.18 - 7.09 (m, 2H), 6.86 - 6.78 (m, 2H), 6.51 (d, J= 23.1 Hz, 1H), 4.77 -Attorney Docket No.: 27527-0235 WO 14.54 (m, 3H), 4.45 - 4.21 (m, 5H), 3.79 - 3.65 (m, 3H), 3.58 - 3.35 (m, 5H), 3.23 (s, 3H), 3.02 -2.76 (m, 6H), 2.45 -2.08 (m, 4H), 2.03 - 1.82 (m, 4H), 1.66 - 1.46 (m, 4H), 1.32-1.16 (m, 8H). HRMS (m / z): ESI [M] calculated for C5iH6iCl2Nio05S+[M+H]+: 995.3919, Found [M+H]+: 995.3908.

[0127] JV-(4-(8-(l l-((2-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5-tetrahydro-EH-pyrido[4,3-Z>]indol-9-yl)-FH-pyrazol-l-yl)ethyl)amino)-ll-oxoundecanoyl)-3,8-diazabicyclo[3.2.1]octan-3-yl)phenethyl)-3-(methylamino)thieno[2,3-Z>]pyridine-2-carboxamide (GDT-59) Yellow solid (1.9 mg, 19%). 'H NMR (400 MHz, Methanol-A) 5 8.63 - 8.52 (m, 2H), 7.67 (d, J= 22.5 Hz, 1H), 7.41 (dd, J= 8.4, 4.7 Hz, 1H), 7.23 (d, J= 12.6 Hz, 1H), 7.19 - 7.09 (m, 2H), 6.86 - 6.78 (m, 2H), 6.51 (d, J = 25.0 Hz, 1H), 4.78 -4.53 (m, 3H), 4.48 - 4.21 (m, 5H), 3.78 - 3. 68 (m, 3H), 3.59 - 3.33 (m, 5H), 3.23 (s, 3H), 3.02 -2.78 (m, 6H), 2.46 -2.09 (m, 4H), 2.04- 1.83 (m, 4H), 1.69 - 1.47 (m, 4H), 1.33 -1.15 (m, I OH). HRMS (m / z): ESI [M] calculated for C52H63C12NIO05S+[M+H]+: 1009.4075, Found [M+H]+: 1009.4071.

[0128] JV-(4-(8-(12-((2-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5-tetrahydro-LH-pyrido[4,3-Z>]indol-9-yl)-LH-pyrazol-l-yl)ethyl)amino)-12-oxododecanoyl)-3,8-diazabicyclo[3.2.1]octan-3-yl)phenethyl)-3-(methylamino)thieno[2,3-Z>]pyridine-2-carboxamide (GDT-60) Yellow solid (1.7 mg, 17%).XH NMR (400 MHz, Methanol-t / 4) 5 8.62 - 8.52 (m, 2H), 7.68 (d, J= 22.0 Hz, 1H), 7.41 (dd, J= 8.4, 4.7 Hz, 1H), 7.23 (d, J= 12.8 Hz, 1H), 7.18 - 7.12 (m, 2H), 6.88 - 6.80 (m, 2H), 6.51 (d, J = 24.9 Hz, 1H), 4.79 -4.56 (m, 3H), 4.50 - 4.24 (m, 5H), 3.80 - 3.63 (m, 3H), 3.60 - 3.37 (m, 5H), 3.24 (s, 3H), 3.04 -2.77 (m, 6H), 2.50 -2.11 (m, 4H), 2.06 - 1.84 (m, 4H), 1.65 - 1.48 (m, 4H), 1.32-1.13 (m, 12H). HRMS (m / z): ESI [M] calculated for C53H65C12NI0O5S+[M+H]+: 1023.4232, Found [M+H]+: 1023.4218.

[0129] JV-(4-(8-(3-(3-((2-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5-tetrahydro-l / 7-pyrido[4,3-Z>]indol-9-yl)-LH-pyrazol-l-yl)ethyl)amino)-3-oxopropoxy)propanoyl)-3,8-diazabicyclo[3.2.1]octan-3-yl)phenethyl)-3-(methylamino)thieno[2,3-Z>]pyridine-2-carboxamide (GDT-61) Yellow solid (1.4 mg, 15%).1H NMR (400 MHz, Methanol-t / 4) 5 8.62 - 8.54 (m, 2H), 7.68 (d, J= 15.0 Hz, 1H), 7.41 (dd, J= 8.4, 4.7 Hz, 1H), 7.30 - 7.22 (m, 1H), 7.14 - 7.06 (m, 2H), 6.77 - 6.67 (m, 2H), 6.52 (d, J= 20.1 Hz, 1H), 4.74 - 4.58 (m, 3H), 4.44 - 4.22 (m, 5H), 3.78 - 3.64 (m, 7H), 3.56 - 3.33 (m, 5H), 3.23 (s, 3H), 2.99 - 2.90Attomey Docket No.: 27527-0235 WO 1(m, 2H), 2.83 - 2.68 (m, 4H), 2.58 - 2.40 (m, 4H), 1.90 - 1.77 (m, 4H). HRMS (m / z): ESI [M] calculated for C47H53C12NIO06S+[M+H]+: 955.3242, Found [M+H]+: 955.3263.

[0130] A-(4-(8-(3-(2-(3-((2-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5-tetrahydro- LH-pyrido[4,3-Z>]indol-9-yl)-l / f-pyrazol-l-yl)ethyl)amino)-3-oxopropoxy)ethoxy)propanoyl)-3,8-diazabicyclo[3.2.1]octan-3-yl)phenethyl)-3-(methylamino)thieno[2,3-Z>]pyridine-2-carboxamide (GDT-62) Yellow solid(1.7 mg, 17%). ' H NMR (400 MHz, Methanol-d4) 58.63 - 8.50 (m, 2H), 7.71 (d, J= 18.6 Hz, 1H), 7.41 (dd, J= 8.4, 4.6 Hz, 1H), 7.23 (d, J= 16.4 Hz, 1H), 7.14- 7.08 (m, 2H), 6.79 - 6.70 (m, 2H), 6.51 (d, J= 24.0 Hz, 1H), 4.77 - 4.54 (m, 3H), 4.45 - 4.23 (m, 5H), 3.76 - 3.39 (m, 16H), 3.23 (s, 3H), 3.00 -2.54 (m, 8H), 2.42- 2.31(m, 2H), 1.95 - 1.75 (m, 4H). HRMS (m / z): ESI [M] calculated for C49H57C12NI0O7S+[M+H]+: 999.3504, Found [M+H]+:999.3529.

[0131] JV-(4-(8-(l-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5-tetrahydro-LH-pyrido[4,3-Z>]indol-9-yl)-LH-pyrazol-l-yl)-4-oxo-7,10,13-trioxa-3-azahexadecan-16-oyl)-3,8-diazabicyclo[3.2.1]octan-3-yl)phenethyl)-3-(methylamino)thieno[2,3-Z>]pyridine-2-carboxamide (GDT-63) Yellow solid (1.4 mg, 13%). 'HNMR (400 MHz, Methanol- r) 5 8.62 - 8.53 (m, 2H), 7.71 (d, J= 15.4 Hz, 1H), 7.41 (dd, J= 8.4, 4.6 Hz, 1H), 7.24 (d, J= 14.8 Hz, 1H), 7.14 - 7.08 (m, 2H), 6.79 - 6.72 (m, 2H), 6.51 (d, J = 20.5 Hz, 1H), 4.75 -4.53 (m, 3H), 4.45 - 4.23 (m, 5H), 3.77 - 3.64 (m, 7H), 3.55 - 3.38 (m, 13H), 3.23 (s, 3H), 2.96 - 2.76 (m, 6H), 2.67 - 2.35 (m, 4H), 1.98 - 1.81 (m, 4H). HRMS (m / z): ESI [M] calculated for C5iH6iCl2Nio08S+[M+H]+: 1043.3766, Found [M+H]+: 1043.3748.

[0132] A-(4-(8-(l-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5-tetrahydro-lH-pyrido[4,3-Z>]indol-9-yl)-EH-pyrazol-l-yl)-4-oxo-7,10,13,16-tetraoxa-3-azanonadecan-19-oyl)-3,8-diazabicyclo[3.2.1]octan-3-yl)phenethyl)-3-(methylamino)thieno[2,3-Z>]pyridine-2-carboxamide (GDT-64) Yellow solid (1.6mg, 15%).1H NMR (400 MHz, Methanol-dr) 5 8.62 - 8.54 (m, 2H), 7.72 (d, J= 16.9 Hz, 1H), 7.44 - 7.38 (m, 1H), 7.25 (d, J= 17.2 Hz, 1H), 7.12 (d, J= 8.1 Hz, 2H), 6.90 - 6.75 (m, 2H), 6.52 (d, J= 24.7 Hz, 1H), 4.77 - 4.56 (m, 3H), 4.45 - 4.20 (m, 5H), 3.81 - 3.67 (m, 7H), 3.55 - 3.37 (m, 17H), 3.24 (s, 3H), 2.98 - 2.44 (m, 10H), 1.97 - 1.84 (m, 4H). HRMS (m / z): ESI [M] calculated for CssHssChNioOgS [M+H]+: 1087.4028, Found [M+H]+: 1087.4035.

[0133] JV-(4-(8-(l-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5-tetrahydro-lH-pyrido[4,3-Z>]indol-9-yl)-l / f-pyrazol-l-yl)-4-oxo-7,10,13,16,19-pentaoxa-3-azadocosan-Attorney Docket No.: 27527-0235 WO 122-oyl)-3,8-diazabicyclo[3.2.1]octan-3-yl)phenethyl)-3-(methylamino)thieno[2,3- Z>] pyridine- 2-carboxamide (GDT-85) Yellow solid(1.4 mg, 12%). 'H NMR (400 MHz, Methanol-^) 58.64 - 8.53 (m, 2H), 7.73 (d, J= 17.5 Hz, 1H), 7.41 (dd, J= 8.2, 4.7 Hz, 1H), 7.25 (d, J= 17.0 Hz, 1H), 7.15 - 7.08 (m, 2H), 6.88 - 6.78 (m, 2H), 6.52 (d, J= 24.1 Hz, 1H), 4.77 - 4.58 (m, 3H), 4.48 - 4.18 (m, 5H), 3.79 - 3.67 (m, 7H), 3.60 - 3.38 (m, 21H), 3.24 (s, 3H), 2.97 - 2.38 (m, 10H), 2.00 - 1.85 (m, 4H). HRMS (m / z): ESI [M] calculated for C55H69C12NIOOIOS+[M+H]+: 1131.4290, Found [M+H]+: 1131.4278.

[0134] A-(4-(8-((2-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5-tetrahydro-lH-pyrido[4,3-Z>]indol-9-yl)-LH-pyrazol-l-yl)acetyl)glycyl)-3,8-diazabicyclo[3.2.1]octan-3-yl)phenethyl)-3-(methylamino)thieno[2,3-Z>]pyridine-2-carboxamide (GDT-86) Yellow solid (1.0 mg, 11%). 'HNMR (400 MHz, Methanol-4) 58.62 - 8.57 (m, 2H), 7.83 (d, J= 13.4 Hz, 1H), 7.42 - 7.38 (m, 1H), 7.28 - 7.24 (m, 1H), 7.16 - 7.08 (m, 2H), 6.88 - 6.79 (m, 2H), 6.60 (s, 1H), 5.11 - 5.05 (m, 2H), 4.76 - 4.68 (m, 2H), 4.50 - 4.17 (m, 4H), 3.98 - 3.90 (m, 1H), 3.76 - 3.68 (m, 1H), 3.58 - 3.40 (m, 6H), 3.24 (s, 3H), 2.99 - 2.81 (m, 6H), 2.09 -1.88 (m, 4H). HRMS (m / z): ESI [M] calculated for C43H45C12NIO05S+[M+H]+: 883.2667, Found [M+H]+: 883.2682.

[0135] A-(4-(8-(3-(2-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5-tetrahydro-l / 7-pyrido[4,3-Z>]indol-9-yl)-LH-pyrazol-l-yl)acetamido)propanoyl)-3,8-diazabicyclo[3.2.1]octan-3-yl)phenethyl)-3-(methylamino)thieno[2,3-Z>]pyridine-2-carboxamide (GDT-87) Yellow solid (1.4 mg, 16%).XH NMR (400 MHz, Methanol-r / 4) 5 8.63 - 8.49 (m, 2H), 7.79 - 7.67 (m, 1H), 7.39 (dd, J= 8.4, 4.7 Hz, 1H), 7.24 (s, 1H), 7.16 -7.08 (m, 2H), 6.83 - 6.73 (m, 2H), 6.51 (dd, J= 15.2, 2.3 Hz, 1H), 4.97 - 4.97 (m, 2H), 4.70 - 4.63 (m, 2H), 4.50 - 4.22 (m, 4H), 4.02 - 3.93 (m, 1H), 3.75 - 3.70 (m, 1H), 3.61 - 3.40 (m, 6H), 3.25 - 3.18 (m, 3H), 2.99 - 2.64 (m, 8H), 2.06 - 1.78 (m, 4H). HRMS (m / z): ESI [M] calculated for C44H47C12NIO05S+[M+H]+: 897.2823, Found [M+H]+: 897.2810.

[0136] JV-(4-(8-(4-(2-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5-tetrahydro-l / 7-pyrido[4,3-Z>]indol-9-yl)-LH-pyrazol-l-yl)acetamido)butanoyl)-3,8-diazabicyclo[3.2.1]octan-3-yl)phenethyl)-3-(methylamino)thieno[2,3-Z>]pyridine-2-carboxamide (GDT-88) Yellow solid (1.7 mg, 19%).XH NMR (400 MHz, Methanol-r / 4) 5 8.67 - 8.50 (m, 2H), 7.80 - 7.78 (m, 1H), 7.42 - 7.38 (m, 1H), 7.25 (d, J= 3.1 Hz, 1H), 7.16 - 7.04 (m, 2H), 6.76 - 6.54 (m, 3H), 5.03 - 4.95 (m, 2H), 4.82 - 4.60 (m, 2H), 4.51 - 4.09 (m, 4H), 3.96 - 3.90 (m, 1H), 3.75 - 3.69 (m, 1H), 3.59 - 3.35 (m, 6H), 3.25 - 3.15 (m, 3H),Attorney Docket No.: 27527-0235 WO 13.07 - 2.68 (m, 6H), 2.55 - 2.25 (m, 2H), 1.93 - 1.74 (m, 6H). HRMS (m / z): ESI [M] calculated for C45H49C12NI0O5S+[M+H]+:911.2980, Found [M+H]+: 911.2999.

[0137] JV-(4-(8-(5-(2-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5-tetrahydro-l / 7-pyrido[4,3-Z>]indol-9-yl)-LH-pyrazol-l-yl)acetamido)pentanoyl)-3,8-diazabicyclo[3.2.1]octan-3-yl)phenethyl)-3-(methylamino)thieno[2,3-Z>]pyridine-2-carboxamide (GDT-89) Yellow solid (2.0 mg, 22%).1H NMR (400 MHz, Methanol-<74) 5 8.60 - 8.52 (m, 2H), 7.81 - 7.69 (m, 1H), 7.40 (dd, J= 8.4, 4.7 Hz, 1H), 7.28 - 7.08 (m, 3H), 6.84 - 6.76 (m, 2H), 6.58 - 6.53 (m, 1H), 4.97 - 4.93 (m, 2H), 4.74 - 4.66 (m, 2H), 4.49 - 4.17 (m, 4H), 3.96 - 3.92 (m, 1H), 3.75 - 3.72 (m, 1H), 3.57 - 3.36 (m, 6H), 3.24 - 3.16 (m, 3H), 2.99 - 2.75 (m, 6H), 2.46 - 2.38 (m, 2H), 2.06 - 1.82 (m, 4H), 1.75 - 1.55 (m, 4H). HRMS (m / z): ESI [M] calculated for C46H5iCl2Ni0O5S+[M+H]+:925.3136, Found [M+H]+: 925.3145.

[0138] JV-(4-(8-(6-(2-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5-tetrahydro-l / 7-pyrido[4,3-Z>]indol-9-yl)-LH-pyrazol-l-yl)acetamido)hexanoyl)-3,8-diazabicyclo[3.2.1]octan-3-yl)phenethyl)-3-(methylamino)thieno[2,3-Z>]pyridine-2-carboxamide (GDT-90)

[0139] Yellow solid (1.8 mg, 19%). *HNMR (400 MHz, Methanol -t / 4) 58.62 - 8.54 (m, 2H), 7.78 - 7.74 (m, J = 3.2Hz, 1H), 7.41 (dd, J = 8.3, 4.6 Hz, 1H), 7.27 - 7.19 (m, 1H), 7.17 - 7.09 (m, 2H), 6.83 - 6.77 (m, 2H), 6.60 - 6.56 (m, 1H), 4.98 - 4.90 (m, 2H), 4.74 -4.68 (m, 2H), 4.50 - 4.18 (m, 4H), 3.97 - 3.92 (m, 1H), 3.76 - 3.70 (m, 1H), 3.59 - 3.35 (m, 6H), 3.24 - 3.18 (m, 3H), 3.02 - 2.66 (m, 6H), 2.39 - 2.22 (m, 2H), 2.06 - 1.78 (m, 4H), 1.73 - 1.51 (m, 4H), 1.45 - 1.30 (m, 2H). HRMS (m / z): ESI [M] calculated for G HssChNioOsS [M+H]+:939.3293, Found [M+H]+: 939.3279.

[0140] A-(4-(8-(7-(2-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5-tetrahydro-l / 7-pyrido[4,3-Z>]indol-9-yl)-LH-pyrazol-l-yl)acetamido)heptanoyl)-3,8-diazabicyclo[3.2.1]octan-3-yl)phenethyl)-3-(methylamino)thieno[2,3-Z>]pyridine-2-carboxamide (GDT-91) Yellow solid (1.4 mg, 15%).XH NMR (400 MHz, Methanol-<74) 5 8.62 - 8.53 (m, 2H), 7.80 - 7.76 (m, 1H), 7.41 (dd, J= 8.5, 4.8 Hz, 1H), 7.26 - 7.22 (m, 1H), 7.18 - 7.06 (m, 2H), 6.86 - 6.78 (m, 2H), 6.61 - 6.55 (m, 1H), 4.96 - 4.93 (m, 2H), 4.76 -4.66 (m, 2H), 4.48 - 4.18 (m, 4H), 3.98 - 3.94 (m, 1H), 3.75 - 3.71 (m, 1H), 3.60- 3.37 (m, 6H), 3.23 (s, 3H), 3.00- 2.77 (m, 6H), 2.47 - 2.32 (m, 2H), 2.05 - 1.85 (m, 4H), 1.68 - 1.50Attorney Docket No.: 27527-0235 WO 1(m, 4H), 1.42 -1.33 (m, 4H). HRMS (m / z): ESI [M] calculated for C48H55C12NIO05S+[M+H]+:953.3449, Found [M+H]+: 953.3476.

[0141] JV-(4-(8-(8-(2-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5-tetrahydro-l / 7-pyrido[4,3-Z>]indol-9-yl)-lH-pyrazol-l-yl)acetamido)octanoyl)-3,8-diazabicyclo[3.2.1]octan-3-yl)phenethyl)-3-(methylamino)thieno[2,3-Z>]pyridine-2-carboxamide (GDT-92) Yellow solid (1.5 mg, 16%).1HNMR(400 MHz, Methanol-<74) 5 8.62 - 8.54 (m, 2H), 7.80 - 7.76 (m, 1H), 7.41 (dd, J= 8.4, 4.7 Hz, 1H), 7.25 (s, 1H), 7.18 -7.10 (m, 2H), 6.87 - 6.80 (m, 2H), 6.62 - 6.58 (m, 1H), 4.97 - 4.90 (m, 2H), 4.76 - 4.69 (m, 2H), 4.49 - 4.18 (m, 4H), 3.98 - 3.94 (m, 1H), 3.75 - 3.71 (m, 1H), 3.57 - 3.36 (m, 4H), 3.26 - 3.19 (m, 5H), 3.00 - 2.77 (m, 6H), 2.46 - 2.29 (m, 2H), 2.04 - 1.83 (m, 4H), 1.68 -1.47 (m, 4H), 1.37 - 1.25 (m, 6H). HRMS (m / z): ESI [M] calculated for C49H57C12NI0O5S+[M+H]+:967.3606, Found [M+H]+: 967.3605.

[0142] JV-(4-(8-(9-(2-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5-tetrahydro-l / 7-pyrido[4,3-Z>]indol-9-yl)-LH-pyrazol-l-yl)acetamido)nonanoyl)-3,8-diazabicyclo[3.2.1]octan-3-yl)phenethyl)-3-(methylamino)thieno[2,3-Z>]pyridine-2-carboxamide (GDT-93) Yellow solid (1.7 mg, 17%).1HNMR(400 MHz, Methanol-r / 4) 5 8.62 - 8.56 (m, 2H), 7.80 - 7.76 (m, 1H), 7.41 (dd, J= 8.3, 4.8 Hz, 1H), 7.25 (s, 1H), 7.18 -7.09 (m, 2H), 6.87 - 6.80 (m, 2H), 6.60 - 6.56 (m, 1H), 4.97 - 4.93 (m, 2H), 4.77 - 4.68 (m, 2H), 4.48 - 4.19 (m, 4H), 3.98 -3.94 (m, 1H), 3.75 - 3.71 (m, 1H), 3.58 - 3.46 (m, 4H), 3.27 - 3.19 (m, 5H), 2.98 - 2.79 (m, 6H), 2.44 - 2.35 (m, 2H), 2.02 - 1.86 (m, 4H), 1.69 -1.47 (m, 4H), 1.36- 1.25 (m, 8H). HRMS (m / z): ESI [M] calculated for CsoHsgChNioOsS [M+H]+: 981.3762, Found [M+H]+: 981.3777.

[0143] A-(4-(8-(10-(2-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5-tetrahydro-lH-pyrido[4,3-Z>]indol-9-yl)-LH-pyrazol-l-yl)acetamido)decanoyl)-3,8-diazabicyclo[3.2.1]octan-3-yl)phenethyl)-3-(methylamino)thieno[2,3-Z>]pyridine-2-carboxamide (GDT-94)

[0144] Yellow solid (1.5 mg, 15%). 'H NMR (400 MHz, Mcthanol-A) 58.62 - 8.56 (m, 2H), 7.80 - 7.76 (m, 1H), 7.42 (dd, J= 8.4, 4.7 Hz, 1H), 7.25 (s, 1H), 7.18 - 7.09 (m, 2H), 6.91 - 6.78 (m, 2H), 6.60 - 6.55 (m, 1H), 4.97 - 4.93 (m, 2H), 4.77 - 4.69 (m, 2H), 4.50 -4.16 (m, 4H), 3.98 - 3.94 (m, 1H), 3.75 - 3.71 (m, 1H), 3.55 - 3.48 (m, 4H), 3.27 - 3.19 (m, 5H), 3.03 - 2.78 (m, 6H), 2.45 -2.38 (m, 2H), 2.09- 1.85 (m, 4H), 1.71 - 1.44 (m, 4H), 1.35Attorney Docket No.: 27527-0235 WO 1- 1.23 (m, 10H). HRMS (m / z): ESI [M] calculated for CsiHsiChNioOsS [M+H]+: 995.3919, Found [M+H]+: 995.3939.

[0145] A-(4-(8-(ll-(2-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5-tetrahydro-lH-pyrido[4,3-Z>]indol-9-yl)-lH-pyrazol-l-yl)acetamido)undecanoyl)-3,8-diazabicyclo[3.2.1]octan-3-yl)phenethyl)-3-(methylamino)thieno[2,3-Z>]pyridine-2-carboxamide (GDT-95)

[0146] Yellow solid (1.6 mg, 16%). 'HNMR (400 MHz, Methanol-^) 58.62 - 8.56 (m, 2H), 7.80 - 7.76 (m, 1H), 7.42 (dd, J= 8.4, 4.7 Hz, 1H), 7.25 (s, 1H), 7.18 - 7.11 (m, 2H), 6.91 - 6.82 (m, 2H), 6.60 - 6.55 (m, 1H), 4.97 - 4.93 (m, 2H), 4.77 - 4.68 (m, 2H), 4.54 -4.17 (m, 4H), 3.98 - 3.94 (m, 1H), 3.75 - 3.71 (m, 1H), 3.58 - 3.47 (m, 4H), 3.28 - 3.21(m, 5H), 3.00 - 2.79 (m, 6H), 2.44 - 2.38 (m, 2H), 2.05 - 1.85 (m, 4H), 1.73 - 1.48 (m, 4H), 1.38 - 1.20 (m, 12H). HRMS (m / z): ESI [M] calculated for C52H63C12NI0O5S+[M+H]+:1009.4075, Found [M+H]+: 1009.4061.

[0147] JV-(4-(8-(3-(2-(2-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5-tetrahydro-lH-pyrido[4,3-Z>]indol-9-yl)-LH-pyrazol-l-yl)acetamido)ethoxy)propanoyl)-3,8-diazabicyclo[3.2.1]octan-3-yl)phenethyl)-3-(methylamino)thieno[2,3-Z>]pyridine-2-carboxamide (GDT-96) Yellow solid (1.7 mg, 18%).1HNMR(400 MHz, Mcthanol-t / 4) 5 8.66 - 8.52 (m, 2H), 7.83 - 7.72 (m, 1H), 7.45 - 7.38 (m, 1H), 7.28 (d, J= 7.4 Hz, 1H), 7.15 - 7.07 (m, 2H), 6.78 - 6.70 (m, 2H), 6.63 - 6.58 (m, 1H), 4.97 - 4.93 (m, 2H), 4.75 - 4.66 (m, 2H), 4.56 - 4.20 (m, 4H), 4.03 - 3.88 (m, 2H), 3.75 - 3.66 (m, 3H), 3.60 - 3.37 (m, 8H), 3.24 - 3.20 (m, 3H), 3.00 - 2.39 (m, 9H), 1.96 - 1.76 (m, 4H). HRMS (m / z): ESI [M] calculated for C46H5iCl2Nio06S+[M+H]+: 941.3085, Found [M+H]+: 941.3069.

[0148] A-(4-(8-(3-(2-(2-(2-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5-tetrahydro- LH-pyrido[4,3-Z>]indol-9-yl)-l / f-pyrazol-l-yl)acetamido)ethoxy)ethoxy)propanoyl)-3,8-diazabicyclo[3.2.1]octan-3-yl)phenethyl)-3-(methylamino)thieno[2,3-Z>]pyridine-2-carboxamide (GDT-97) Yellow solid (1.3 mg, 13%).1HNMR(400 MHz, Methanol-t / 4) 5 8.62 - 8.52 (m, 2H), 7.82 - 7.79 (m, 1H), 7.41 (dd, J= 8.3, 4.7 Hz, 1H), 7.26 (s, 1H), 7.14 -7.08 (m, 2H), 6.79 - 6.76 (m, 2H), 6.62 - 6.58 (m, 1H), 5.01 - 4.97 (m, 2H), 4.76 - 4.67 (m, 2H), 4.53 - 4.32 (m, 3H), 4.24 (s, 1H), 3.99 - 3.89 (m, 2H), 3.78 - 3.68 (m, 3H), 3.56 - 3.38 (m, 12H), 3.25 - 3.21 (m, 3H), 3.00 - 2.76 (m, 7H), 2.65 - 2.48 (m, 2H), 1.97- 1.84 (m, 4H). HRMS (m / z): ESI [M] calculated for C48H55C12NI0O7S+[M+H]+: 985.3347, Found [M+H]+: 985.3357.Attorney Docket No.: 27527-0235 WO 1

[0149] A-(4-(8-(l-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5-tetrahydro-l / f-pyrido[4, 3- / >|indol-9-yl)-l / / -pyrazol-l-yl)-2-oxo-6, 9,12-trioxa-3-azapentadecan-l 5-oyl)-3,8-diazabicyclo[3.2.1]octan-3-yl)phenethyl)-3-(methylamino)thieno[2,3-Z>]pyridine-2-carboxamide (GDT-98) Yellow solid (1.5 mg, 15%).1HNMR (400 MHz, Methanol-r / 4) 5 8.60 -8.53(m, 2H), 7.81 - 7.75 (m, 1H), 7.44 - 7.38 (m, 1H), 7.25 (s, 1H), 7.14 - 7.08 (m, 2H), 6.80 - 6.74 (m, 2H), 6.60 - 6.56 (m, 1H), 5.03 - 4.97 (m, 2H), 4.76 - 4.67 (m, 2H), 4.52 - 4.33 (m, 3H), 4.23 (s, 1H), 3.98 - 3.90 (m, 2H), 3.78 - 3.68 (m, 3H), 3.54 - 3.39 (m, 16H), 3.26 - 3.20 (m, 3H), 3.02 - 2.54 (m, 9H), 2.01 - 1.83 (m, 4H). HRMS (m / z): ESI [M] calculated for CsoHsgChNioOsS [M+H]+: 1029.3610, Found [M+H]+: 1029.3633.

[0150] JV-(4-(8-(l-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5-tetrahydro-LH-pyrido[4,3-Z>]indol-9-yl)-LH-pyrazol-l-yl)-2-oxo-6,9,12,15-tetraoxa-3-azaoctadecan-18-oyl)-3,8-diazabicyclo[3.2.1]octan-3-yl)phenethyl)-3-(methylamino)thieno[2,3-Z>]pyridine-2-carboxamide (GDT-99, MS2099) Yellow solid (1.7 mg, 16%). 'HNMR (400 MHz, Methanol-^) 5 8.64 - 8.52 (m, 2H), 7.80 - 7.75 (m, 1H), 7.41 (dd, J= 8.4, 4.7 Hz, 1H), 7.25 - 7.22 (m, 1H), 7.14 - 7.08 (m, 2H), 6.83 - 7.67 (m, 2H), 6.59 - 6.56 (m, 1H), 5.03 - 4.97 (m, 2H), 4.74 - 4.67 (m, 2H), 4.52 - 4.33 (m, 3H), 4.23 (s, 1H), 3.98 - 3.90 (m, 2H), 3.78 - 3.68 (m, 3H), 3.57 - 3.43 (m, 20H), 3.26 - 3.20 (m, 3H), 2.96 - 2.56 (m, 9H), 1.99 - 1.82 (m, 4H).13C NMR (101 MHz, Methanol-A) 5 171.43, 168.12, 168.05, 165.71, 158.78, 150.94, 149.30, 148.61, 135.40, 134.79, 133.23, 132.66, 129.65, 129.22, 127.53, 124.94, 123.77, 122.85, 121.27, 121.21, 119.01, 114.75, 114.23, 107.19, 105.66, 70.22 - 69.88 (m), 69.07, 67.11, 60.04, 55.26, 54.69, 54.04, 53.58, 51.68, 43.36, 41.23, 39.25, 34.60, 33.88, 32.73, 27.93, 26.35, 22.87. HRMS (m / z): ESI [M] calculated for C52H63C12NIO09S+[M+H]+: 1073.3872, Found [M+H]+: 1073.3881.

[0151] A-(4-(8-(l-(3-(6,7-dichloro-2-(2-hydroxyacetyl)-2,3,4,5-tetrahydro-lH-pyrido[4,3-Z>]indol-9-yl)-EH-pyrazol-l-yl)-2-oxo-6,9,12,15,18-pentaoxa-3-azahenicosan-21-oyl)-3,8-diazabicyclo[3.2.1]octan-3-yl)phenethyl)-3-(methylamino)thieno[2,3- Z>] pyridine- 2-carboxamide (GDT-100, MS2100) Yellow solid (2.2 mg, 20%).XH NMR (400 MHz, Methanol-A) 58.61 - 8.52 (m, 2H), 7.80 - 7.75 (m, 1H), 7.40 (dd, J= 8.4, 4.7 Hz, 1H), 7.25 (s, 1H), 7.14 - 7.08 (m, 2H), 6.83 - 7.67 (m, 2H), 6.62 - 6.56 (m, 1H), 5.03 - 4.97 (m, 2H), 4.74 - 4.67 (m, 2H), 4.53 - 4.32 (m, 3H), 4.24 (s, 1H), 3.98 - 3.90 (m, 2H), 3.82 -3.70 (m, 3H), 3.58 - 3.45 (m, 24H), 3.26 - 3.19 (m, 3H), 2.98 - 2.59 (m, 9H), 2.03 - 1.87 (m, 4H).13C NMR (101 MHZ, Methanol-4) 5 173.88, 170.58, 170.50, 168.15, 161.22, 153.42, 151.75, 151.06, 137.86, 137.26, 135.72, 135.12, 132.14, 131.70, 130.02, 127.38, 126.26,Attorney Docket No.: 27527-0235 WO 1125.30, 123.76, 123.69, 121.50, 117.24, 116.71, 109.64, 108.14, 72.62 - 72.38 (m), 71.56, 69.58, 62.51, 57.74, 57.18, 56.51, 56.07, 54.13, 45.83, 43.71, 41.72, 39.25, 37.08, 36.36, 35.25, 30.42, 28.84, 25.35. HRMS (m / z): ESI [M] calculated for CsrHe ChNioOioS [M+H]+: 1117.4134, Found [M+H]+: 1117.4156.

[0152] Scheme 6. Synthesis of PPARy-USP28 DUBTAC linker PL-16““reaction and conditions: a) PPhs, DIAD, THF, 0°C - rt, 0.5 h, 73%; b) HC1 in dioxane, 40°C, 1 h; c) 1-12, 1-13, KF, DMF, 80 - 90°C, 56%; d) AcOH, cone. HC1, reflux, 30 min, 83%; e) HO At, EDCI, NMM, DCM, rt; f) TFA, DCM, rt.

[0153] 5-(4-(2-(methylamino)ethoxy)benzyl)thiazolidine-2, 4-dione (1-11) 1-10 (1.05 g, 4.71 mmol, 1.1 eq), tert-butyl (2-hydroxyethyl)(methyl)carbamate (750 mg, 4.28 mmol, 1.0 eq), PPI13 (1.68 g, 6.42 mmol, 1.5 eq) in 30 mL THF were added DIAD (1.3 g, 6.42 mmol, 1.5 eq) dropwise at 0°C. The mixture was stirred at room temperature for 0.5 h, then purified with silica gel to yield a white solid (1.2 g, 73%). Then the product was dissolved in MeOH (3 mL) and HC1 (4M in dioxane, 3 mL, 12 mmol), and stirred at 40°C for 1 h. After removal of all the volatiles, white solid was obtained without further purification.1H NMR (400 MHz, Methanol-t / 4) 57.08 (d, J= 7.8 Hz, 2H), 6.73 (d, J= 8.5 Hz, 2H), 4.76 - 4.68 (m, 1H), 3.88 -3.83 (m, 2H), 3.46 - 3.39 (m, 1H), 3.23 - 3.04 (m, 3H), 2.71 (s, 3H), 1.28 -1.25 (m, 1H).

[0154] Ethyl 2-(4-((6-((2-(4-((2,4-dioxothiazolidin-5-yl)methyl)phenoxy)ethyl)(methyl)amino)pyrimidin-4-yl)oxy)phenoxy)acetate (1-14) 1-12 (470 mg, 3.15 mmol, 1.0 eq), 1-13 (618 mg, 3.15 mmol, 1.0 eq), KF (700 mg, 12.6 mmol, 4.0 eq) in DMF (5 mL) were stirred at 80°C for 2h. Then 1-11 (1.0 g, 3.15 mmol, 1.0 eq) wasAtorney Docket No.: 27527-0235WO1added and the mixture was stirred at 90°C for 2h. 10 mL water was added and the mixture was extracted with ethyl acetate for 3 times and the organic layer was purified with silica gel (hexane / Ethyl acetate = 3 / 7). The product was obtained as a white solid (1.0 g, 56%). 'H NMR (400 MHz, Methanol-^) 58.10 (s, 1H), 7.94 (s, 1H), 7.00 - 6.90 (m, 6H), 6.68 - 6.64 (m, 2H), 5.71 (s, 1H), 4.67 (s, 2H), 4.45 - 4.40 (m, 1H), 4.21 (q, J= 7.2, 2H), 2.95 (s, 3H), 2.90 - 2.86 (m, 2H), 2.84 - 2.80 (m, 4H), 1.25 (t, J= 7.2 Hz, 3H).

[0155] 2-(4-((6-((2-(4-((2,4-dioxothiazolidin-5-yl)methyl)phenoxy)ethyl)(methyl)amino)pyrimidin-4-yl)oxy)phenoxy)acetic acid (1-15) 1-14 (1.0 g, 1.8 mmol) in AcOH / conc. HC1 (2: 1, 3.9 mL) was stirred under reflux for 30 min. Then purified via reverse-ISCO to give a white solid (780 mg, 83%).1H NMR (400 MHz, Methanol-^) 5 8.25 (s, 1H), 7.12 - 6.95 (m, 6H), 6.69 (d, J= 7.6 Hz, 2H), 5.72 (s, 1H), 4.68 (s, 2H), 4.55 - 4.47 (m, 1H), 3.91 - 3.52 (m, 3H), 3.33 - 3.25 (m, 3H), 2.98 - 2.88 (m, 3H).

[0156] (2-(4-((6-((2-(4-((2,4-dioxothiazolidin-5-yl)methyl)phenoxy)ethyl)(methyl)amino)pyrimidin-4-yl)oxy)phenoxy)acetyl)glycine (PL-16) 1-15 (52.4 mg, 0.1 mmol, 1.0 eq), tert-butyl glycinate (14.4 mg, 0.11 mmol, 1.1 eq), EDCI (29 mg, 0.15 mmol, 1.5 eq), HOAt (20 mg, 0.15 mmol, 1.5 eq), NMM (30 mg, 0.3 mmol, 3.0 eq) were stirred in DCM (2.0 mL) at room temperature for 1 h. After stirred at rt for 1 h, TFA (1 mL) was added, the mixture was stirred for 2another 20 mins and purified via reverse-ISCO to give a white solid (26.2 mg, 45%). 'H NMR (400 MHz, Methanol-oh) 5 8.28 (s, 1H), 7.12 - 6.98 (m, 4H), 6.89 (d, J= 7.8 Hz, 2H), 6.58 (d, J= 8.3 Hz, 2H), 5.59 (s, 1H), 4.57 - 4.39 (m, 3H), 3.90 (s, 2H), 3.77 - 3.53 (m, 3H), 3.23 - 3.15 (m, 2H), 2.93 - 2.50 (m, 4H).ooL=PL-17: n = 2; PL-22: n = 7; PL-26: m = 1;PL-18: n = 3; PL-23: n = 8; PL-27: m =2;PL-19: n s 4; PL-24: ns 9; PL-28: m =3;PL-20: n = 5; PL-25: n = 10; PL-29: m =4;PL-21: n s 6; PL-30: m =5;

[0157] PPARy-USP28 DUBTAC linkers PL-17 - PL-30 were synthesized following the same method for preparing PL-16.Attomey Docket No.: 27527-0235 WO 1

[0158] 3-(2-(4-((6-((2-(4-((2,4-dioxothiazolidin-5-yl)methyl)phenoxy)ethyl)(methyl)amino)pyrimidin-4-yl)oxy)phenoxy)acetamido)propanoic acid (PL-17) White solid (26.2 mg, 44%). *HNMR (400 MHz, Methanol-^) 5 8.22 (s, 1H), 7.06 - 6.80 (m, 6H), 6.55 (d, J= 7.6 Hz, 2H), 5.56 (s, 1H), 4.40 (s, 3H), 3.76 - 3.47 (m, 3H), 3.41 - 3.35 (m, 2H), 3.18 - 3.10 (m, 2H), 2.91 -2.34 (m, 6H).

[0159] 4-(2-(4-((6-((2-(4-((2,4-dioxothiazolidin-5-yl)methyl)phenoxy)ethyl)(methyl)amino)pyrimidin-4-yl)oxy)phenoxy)acetamido)butanoic acid (PL-18) White solid (28.1 mg, 46%). 'HNMR (400 MHz, Methanol-^) 5 8.24 (s, 1H), 7.12 - 6.84 (m, 6H), 6.57 (d, J= 7.4 Hz, 2H), 5.58 (s, 1H), 4.42 (s, 3H), 3.78 - 3.53 (m, 3H), 3.23 - 3.16 (m, 4H), 2.92 - 2.44 (m, 4H), 2.24 - 2.14 (m, 2H), 1.75 - 1.63 (m, 2H).

[0160] 5-(2-(4-((6-((2-(4-((2,4-dioxothiazolidin-5-yl)methyl)phenoxy)ethyl)(methyl)amino)pyrimidin-4-yl)oxy)phenoxy)acetamido)pentanoic acid (PL-19) White solid (26.8 mg, 43%). 'HNMR (400 MHz, Methanol-^) 5 8.24 (s, 1H), 7.11 - 6.85 (m, 6H), 6.59 (d, J= 8.3 Hz, 2H), 5.60 (s, 1H), 4.44 (s, 3H), 3.81 - 3.51 (m, 3H), 3.22 - 3.16 (m, 4H), 2.96 - 2.40 (m, 4H), 2.26 - 2.15 (m, 2H), 1.53 - 1.42 (m, 4H).

[0161] 6-(2-(4-((6-((2-(4-((2,4-dioxothiazolidin-5-yl)methyl)phenoxy)ethyl)(methyl)amino)pyrimidin-4-yl)oxy)phenoxy)acetamido)hexanoic acid (PL-20) White solid (28.7 mg, 45%). 'HNMR (400 MHz, Methanol-^) 5 8.25 (s, 1H), 7.13 - 6.87 (m, 6H), 6.60 (d, J= 8.2 Hz, 2H), 5.62 (s, 1H), 4.45 (s, 3H), 3.83 - 3.55 (m, 3H), 3.23 - 3.17 (m, 4H), 2.96 - 2.50 (m, 4H), 2.19 (t, J = 7.2 Hz, 2H), 1.55 - 1.40 (m, 4H), 1.30 - 1.20 (m, 2H).

[0162] 7-(2-(4-((6-((2-(4-((2,4-dioxothiazolidin-5-yl)methyl)phenoxy)ethyl)(methyl)amino)pyrimidin-4-yl)oxy)phenoxy)acetamido)heptanoic acid (PL-21) White solid (30.6 mg, 47%). 'HNMR (400 MHz, Methanol-A) 5 8.17 (s, 1H), 7.03 - 6.81 (m, 6H), 6.56 (d, J= 7.7 Hz, 2H), 5.58 (s, 1H), 4.40 (s, 3H), 3.76 - 3.43 (m, 3H), 3.18 - 3.10 (m, 4H), 2.90 - 2.36 (m, 4H), 2.13 (t, J = 7.2 Hz, 2H), 1.48 - 1.36 (m, 4H), 1.26 - 1.13 (m, 4H).

[0163] 8-(2-(4-((6-((2-(4-((2,4-dioxothiazolidin-5-yl)methyl)phenoxy)ethyl)(methyl)amino)pyrimidin-4-Attorney Docket No.: 27527-0235 WO 1yl)oxy)phenoxy)acetamido)octanoic acid (PL-22) White solid (31.6 mg, 47%). 'HNMR (400 MHz, Methanol-^) 5 8.13 (s, 1H), 7.02 - 6.85 (m, 6H), 6.61 (d, J= 7.4 Hz, 2H), 5.65 (s, 1H), 4.43 (s, 3H), 3.81 - 3.43 (m, 3H), 3.23 - 3.17 (m, 4H), 2.90 - 2.30 (m, 4H), 2.18 (t, J = 7.3 Hz, 2H), 1.55 - 1.37 (m, 4H), 1.30 - 1.16 (m, 6H).

[0164] 9-(2-(4-((6-((2-(4-((2,4-dioxothiazolidin-5-yl)methyl)phenoxy)ethyl)(methyl)amino)pyrimidin-4-yl)oxy)phenoxy)acetamido)nonanoic acid (PL-23) White solid (28.8 mg, 41%). 'HNMR (400 MHz, Methanol-^) 5 8.12 (s, 1H), 7.03 - 6.85 (m, 6H), 6.60 (d, J= 8.4 Hz, 2H), 5.65 (s, 1H), 4.42 (s, 3H), 3.79 - 3.38 (m, 3H), 3.23 - 3.14 (m, 4H), 2.94 - 2.29 (m, 4H), 2.17 (t, J = 7.1 Hz, 2H), 1.55 - 1.37 (m, 4H), 1.25 - 1.15 (m, 8H).

[0165] 10-(2-(4-((6-((2-(4-((2,4-dioxothiazolidin-5-yl)methyl)phenoxy)ethyl)(methyl)amino)pyrimidin-4-yl)oxy)phenoxy)acetamido)decanoic acid (PL-24) White solid (33.3 mg, 48%). 'HNMR (400 MHz, Methanol-^) 5 8.17 (s, 1H), 7.09 - 6.85 (m, 6H), 6.58 (d, J= 6.8 Hz, 2H), 5.62 (s, 1H), 4.42 (s, 3H), 3.82 - 3.48 (m, 3H), 3.22 - 3.10 (m, 4H), 2.90 - 2.40 (m, 4H), 2.22 - 2.10 (m, 2H), 1.55 - 1.40 (m, 4H), 1.30 - 1.16 (m, 10H).

[0166] ll-(2-(4-((6-((2-(4-((2,4-dioxothiazolidin-5-yl)methyl)phenoxy)ethyl)(methyl)amino)pyrimidin-4-yl)oxy)phenoxy)acetamido)undecanoic acid (PL-25) White solid (33.3 mg, 46%). 'HNMR (400 MHz, Methanol-^) 5 8.20 (s, 1H), 7.06 - 6.83 (m, 6H), 6.57 (d, J= 7.6 Hz, 2H), 5.59 (s, 1H), 4.41 (s, 3H), 3.75 - 3.43 (m, 3H), 3.18 - 3.10 (m, 4H), 2.86 - 2.43 (m, 4H), 2.16 - 2.11 (m, 2H), 1.48 - 1.38 (m, 4H), 1.29 - 1.13 (m, 12H).

[0167] 3-(2-(2-(4-((6-((2-(4-((2,4-dioxothiazolidin-5-yl)methyl)phenoxy)ethyl)(methyl)amino)pyrimidin-4-yl)oxy)phenoxy)acetamido)ethoxy)propanoic acid (PL-26) White solid (29.4 mg, 46%). 'H NMR (400 MHz, Methanol-4) 58.19 (s, 1H), 7.06 - 6.84 (m, 6H), 6.61 (d, J= 8.4 Hz, 2H), 5.64 (s, 1H), 4.45 (s, 3H), 3.79 - 3.52 (m, 5H), 3.48 - 3.35 (m, 4H), 3.24 - 3.16 (m, 2H), 2.95 - 2.70 (m, 3H), 2.52 - 2.40 (m, 3H). LCMS m / z = 640.2.

[0168] 3-(2-(2-(2-(4-((6-((2-(4-((2,4-dioxothiazolidin-5-yl)methyl)phenoxy)ethyl)(methyl)amino)pyrimidin-4-yl)oxy)phenoxy)acetamido)ethoxy)ethoxy)propanoic acid (PL-27) White solid (29.5 mg, 43%). 'HNMR (400 MHz, Methanol-4) 5 8.22 (s, 1H), 7.06 - 6.88 (m, 6H), 6.60 (d, J= 8.5Attorney Docket No.: 27527-0235 WO 1Hz, 2H), 5.63 (s, 1H), 4.46 (s, 3H), 3.78 - 3.57 (m, 5H), 3.51 - 3.45 (m, 6H), 3.38 - 3.34 (m, 2H), 3.23 - 3.16 (m, 2H), 2.98 - 273 (m, 3H), 2.56 - 2.40 (m, 3H).

[0169] l-(4-((6-((2-(4-((2,4-dioxothiazolidin-5-yl)methyl)phenoxy)ethyl)(methyl)amino)pyrimidin-4-yl)oxy)phenoxy)-2-oxo-6,9,12-trioxa-3-azapentadecan-15-oic acid (PL-28) White solid (30.6 mg, 42%). 'HNMR (400 MHz, Methanol-^) 58.22 (s, 1H), 7.06 - 6.89 (m, 6H), 6.62 (d, J= 8.4 Hz, 2H), 5.65 (s, 1H), 4.48 (s, 3H), 3.81 - 3.59 (m, 5H), 3.55 - 3.46 (m, 10H), 3.39 (t, J= 5.5 Hz, 2H), 3.25 -3.18 (m, 2H), 2.95 - 2.75(m, 3H), 2.57 - 2.36 (m, 3H).

[0170] l-(4-((6-((2-(4-((2,4-dioxothiazolidin-5-yl)methyl)phenoxy)ethyl)(methyl)amino)pyrimidin-4-yl)oxy)phenoxy)-2-oxo-6,9,12,15-tetraoxa-3-azaoctadecan-18-oic acid (PL-29) White solid (34.7 mg, 45%).1HNMR(400 MHz, Methanol-^) 58.24 (s, 1H), 7.07 - 6.87 (m, 6H), 6.59 (d, J= 8.4 Hz, 2H), 5.61 (s, 1H), 4.46 (s, 3H), 3.80 - 3.56 (m, 5H), 3.53 - 3.44 (m, 14H), 3.38 - 3.32 (m, 2H), 3.24 - 3.17 (m, 2H), 2.95 - 2.76 (m, 3H), 2.60 - 2.37 (m, 3H).

[0171] l-(4-((6-((2-(4-((2,4-dioxothiazolidin-5-yl)methyl)phenoxy)ethyl)(methyl)amino)pyrimidin-4-yl)oxy)phenoxy)-2-oxo-6,9,12,15,18-pentaoxa-3-azahenicosan-21-oic acid (PL-30) White solid (34.7 mg, 45%). 'H NMR (400 MHz, Methanol^) 58.21 (s, 1H), 7.04 - 6.83 (m, 6H), 6.54 (d, J= 8.0 Hz, 2H), 5.56 (s, 1H), 4.41 (s, 3H), 3.72 - 3.51 (m, 5H), 3.47 - 3.40 (m, 18H), 3.35 - 3.30 (m, 2H), 3.17 - 3.10 (m, 2H), 2.92 - 2.67 (m, 3H), 2.58 - 2.33 (m, 3H). LCMS m / z = 816.3.

[0172] Scheme 8. Synthesis of PPARy-USP28 DUBTACs PDT-16aCT1073Attorney Docket No.: 27527-0235 WO 1“reaction and conditions: a) HOAt, EDCI, NMM, DMSO, rt, 30%.

[0173] A-(4-(8-((2-(4-((6-((2-(4-((2,4-dioxothiazolidin-5-yl)methyl)phenoxy)ethyl)(methyl)amino)pyrimidin-4-yl)oxy)phenoxy)acetyl)glycyl)-3,8-diazabicyclo[3.2.1]octan-3-yl)phenethyl)-3-(methylamino)thieno[2,3-Z>]pyridine-2-carboxamide (PDT-16) Yellow solid (2.9 mg, 30%).1H NMR (400 MHz, Methanol-oh) 5 8.61 - 8.48 (m, 2H), 8.35 (s, 1H), 7.46 - 7.36 (m, 1H), 7.16 - 7.05 (m, 6H), 6.95 (d, J= 8.0 Hz, 2H), 6.79 (d, J= 8.0 Hz, 2H), 6.68 - 6.60 (m, 2H), 5.66 (s, 1H), 4.72 - 4.37 (m, 5H), 4.14 (s, 2H), 3.83 - 3.41 (m, 7H), 3.25 - 3.20 (m, 2H), 3.19 (s, 3H), 3.02- 2.54 (m, 8H), 2.09 - 1.82 (m, 4H). HRMS (m / z): ESI [M] calculated for C5OH53NIO08S2+[M+H]+: 985.3484, Found [M+H]+: 985.3487.*° ifo

[0174] PPARy-USP28 DUBTACs PDT-17 - PDT-30 were synthesized following the same procedure for preparing PDT-16.

[0175] A-(4-(8-(3-(2-(4-((6-((2-(4-((2,4-dioxothiazolidin-5-yl)methyl)phenoxy)ethyl)(methyl)amino)pyrimidin-4-yl)oxy)phenoxy)acetamido)propanoyl)-3,8-diazabicyclo[3.2.1]octan-3-yl)phenethyl)-3-(methylamino)thieno[2,3-Z>]pyridine-2-carboxamide (PDT-17) Yellow solid (2.9 mg, 29%). 'H NMR (400 MHz, Methanol-^) 58.60 - 8.49 (m, 2H), 8.33 (s, 1H), 7.46 - 7.36 (m, 1H), 7.09 - 6.89 (m, 8H), 6.75 (d, J= 8.6 Hz, 2H), 6.64 (d, J= 8.4 Hz, 2H), 5.64 (s, 1H), 4.71 - 4.33 (m, 5H), 3.84 - 3.38 (m, 9H), 3.26 - 3.15 (m, 5H), 3.00 - 2.57 (m, 10H), 2.00 -1.80 (m, 4H). HRMS (m / z): ESI [M] calculated for CsiHssNioOsS [M+H]+: 999.3640, Found [M+H]+: 999.3631.

[0176] A-(4-(8-(4-(2-(4-((6-((2-(4-((2,4-dioxothiazolidin-5-yl)methyl)phenoxy)ethyl)(methyl)amino)pyrimidin-4-yl)oxy)phenoxy)acetamido)butanoyl)-3,8-diazabicyclo[3.2.1]octan-3-yl)phenethyl)-3-(methylamino)thieno[2,3-Z>]pyridine-2-carboxamide (PDT-18) Yellow solid (3.1 mg,Attorney Docket No.: 27527-0235 WO 130%). 'H NMR (400 MHz, Methanol-4) 58.58 - 8.48 (m, 2H), 8.30 (s, 1H), 7.42 - 7.33 (m, 1H), 7.13 - 6.90 (m, 8H), 6.76 (d, J= 8.6 Hz, 2H), 6.64 (d, J= 8.2 Hz, 2H), 5.65 (s, 1H), 4.72 - 4.27 (m, 5H), 3.84 - 3.28 (m, 11H), 3.18 (s, 3H), 3.06 - 2.25 (m, 10H), 1.99 - 1.77 (m, 6H). HRMS (m / z): ESI [M] calculated for C52H57NIO08S2+[M+H]+: 1013.3797, Found [M+H]+: 1013.3790.

[0177] A-(4-(8-(5-(2-(4-((6-((2-(4-((2,4-dioxothiazolidin-5-yl)methyl)phenoxy)ethyl)(methyl)amino)pyrimidin-4-yl)oxy)phenoxy)acetamido)pentanoyl)-3,8-diazabicyclo[3.2.1]octan-3-yl)phenethyl)-3-(methylamino)thieno[2,3-Z>]pyridine-2-carboxamide (PDT-19) Yellow solid (3.0 mg, 29%). 'H NMR (400 MHz, Methanol-4) 58.60 - 8.47 (m, 2H), 8.32 (s, 1H), 7.43 - 7.33 (m, 1H), 7.13 - 6.91 (m, 8H), 6.77 (d, J = 8.6Hz, 2H), 6.64 (d, J= 8.3 Hz, 2H), 5.64 (s, 1H), 4.72 - 4.37 (m, 5H), 3.86 - 3.27 (m, 11H), 3.18 (s, 3H), 3.03 - 2.35 (m, 10H), 1.98 - 1.79 (m, 4H), 1.59 (d, J= 10.2 Hz, 4H). HRMS (m / z): ESI [M] calculated for C53H59NIO08S2+[M+H]+: 1027.3953, Found [M+H]+: 1027.3955.

[0178] JV-(4-(8-(6-(2-(4-((6-((2-(4-((2,4-dioxothiazolidin-5-yl)methyl)phenoxy)ethyl)(methyl)amino)pyrimidin-4-yl)oxy)phenoxy)acetamido)hexanoyl)-3,8-diazabicyclo[3.2.1]octan-3-yl)phenethyl)-3-(methylamino)thieno[2,3-Z>]pyridine-2-carboxamide (PDT-20) Yellow solid (2.8 mg, 27%). 'HNMR (400 MHz, Methanol-4) 58.58 - 8.49 (m, 2H), 8.32 (s, 1H), 7.41 - 7.35 (m, 1H), 7.14 - 6.93 (m, 8H), 6.78 (d, J= 8.8 Hz, 2H), 6.65 (d, J= 8.4 Hz, 2H), 5.65 (s, 1H), 4.71 - 4.32 (m, 5H), 3.82 - 3.39 (m, 7H), 3.29 - 3.14 (m, 7H), 2.95 - 2.28 (m, 10H), 2.00 -1.79 (m, 4H), 1.66 - 1.47 (m, 4H), 1.37 - 1.29 (m, 2H). HRMS (m / z): ESI [M] calculated for C54H6INIO08S2+[M+H]+: 1041.4110, Found [M+H]+: 1041.4122.

[0179] N-(4-(8-(7-(2-(4-((6-((2-(4-((2,4-dioxothiazolidin-5-yl)methyl)phenoxy)ethyl)(methyl)amino)pyrimidin-4-yl)oxy)phenoxy)acetamido)heptanoyl)-3,8-diazabicyclo[3.2.1]octan-3-yl)phenethyl)-3-(methylamino)thieno[2,3-Z>]pyridine-2-carboxamide (PDT-21) Yellow solid (3.3 mg, 32%). ‘HNMR (400 MHz, Methanol-4) 58.60 - 8.49 (m, 2H), 8.30 (s, 1H), 7.42 - 7.34 (m, 1H), 7.16 - 6.93 (m, 8H), 6.78 (d, J= 7.8 Hz, 2H), 6.65 (d, J= 8.1 Hz, 2H), 5.66 (s, 1H), 4.72 - 4.35 (m, 5H), 3.84 - 3.41 (m, 7H), 3.26 - 3.17 (m, 7H), 3.03 - 2.28 (m, 10H), 2.02 -1.78 (m, 4H), 1.65 - 1.26 (m, 8H). HRMS (m / z): ESI [M] calculated for CssIfeNioOsS [M+H]+: 1055.4266, Found [M+H]+: 1055.4250.Attorney Docket No.: 27527-0235 WO 1

[0180] JV-(4-(8-(8-(2-(4-((6-((2-(4-((2,4-dioxothiazolidin-5-yl)methyl)phenoxy)ethyl)(methyl)amino)pyrimidin-4-yl)oxy)phenoxy)acetamido)octanoyl)-3,8-diazabicyclo[3.2.1]octan-3-yl)phenethyl)-3-(methylamino)thieno[2,3-Z>]pyridine-2-carboxamide (PDT-22) Yellow solid (3.2 mg, 30%). ' H NMR (400 MHz, Methanol^) 58.59 - 8.49 (m, 2H), 8.31 (s, 1H), 7.42 - 7.34 (m, 1H), 7.16 - 6.92 (m, 8H), 6.78 (d, J= 8.0 Hz, 2H), 6.65 (d, J= 8.4 Hz, 2H), 5.66 (s, 1H), 4.71 - 4.36 (m, 5H), 3.83 - 3.37 (m, 7H), 3.26 - 3.16 (m, 7H), 3.00 - 2.29 (m, 10H), 1.99 -1.76 (m, 4H), 1.61 - 1.43 (m, 4H), 1.35 - 1.22 (m, 6H). HRMS (m / z): ESI [M] calculated for C56H65NIO08S2+[M+H]+: 1069.4423, Found [M+H]+: 1069.4450.

[0181] JV-(4-(8-(9-(2-(4-((6-((2-(4-((2,4-dioxothiazolidin-5-yl)methyl)phenoxy)ethyl)(methyl)amino)pyrimidin-4-yl)oxy)phenoxy)acetamido)nonanoyl)-3,8-diazabicyclo[3.2.1]octan-3-yl)phenethyl)-3-(methylamino)thieno[2,3-Z>]pyridine-2-carboxamide (PDT-23) Yellow solid (3.3 mg, 31%). 'H NMR (400 MHz, Methanol-4) 58.58 - 8.49 (m, 2H), 8.32 (s, 1H), 7.41 - 7.34 (m, 1H), 7.13 - 6.92 (m, 8H), 6.78 (d, J= 8.0 Hz, 2H), 6.65 (d, J= 8.5 Hz, 2H), 5.66 (s, 1H), 4.72 - 4.38 (m, 5H), 3.83 - 3.41 (m, 7H), 3.26 - 3.16 (m, 7H), 3.02 - 2.26 (m, 10H), 1.98 - 1.80 (m, 4H), 1.66 - 1.21 (m, 12H). HRMS (m / z): ESI [M] calculated for C57H67NI0O8S2+[M+H]+: 1083.4579, Found [M+H]+: 1083.4577.

[0182] JV-(4-(8-(10-(2-(4-((6-((2-(4-((2,4-dioxothiazolidin-5-yl)methyl)phenoxy)ethyl)(methyl)amino)pyrimidin-4-yl)oxy)phenoxy)acetamido)decanoyl)-3,8-diazabicyclo[3.2.1]octan-3-yl)phenethyl)-3-(methylamino)thieno[2,3-Z>]pyridine-2-carboxamide (PDT-24) Yellow solid (3.1 mg, 28%). ‘HNMR (400 MHz, Methanol-4) 58.60 - 8.51 (m, 2H), 8.31 (s, 1H), 7.41 - 7.35 (m, 1H), 7.14 - 6.93 (m, 8H), 6.79 (d, J= 7.7 Hz, 2H), 6.65 (d, J= 8.4 Hz, 2H), 5.66 (s, 1H), 4.74 > 4.36 (m, 5H), 3.81 - 3.44 (m, 7H), 3.26 - 3.17 (m, 7H), 3.07 - 2.26 (m, 10H), 2.02 - 1.81 (m, 4H), 1.64 - 1.23 (m, 14H). HRMS (m / z): ESI [M] calculated for C58H69NIO08S2+[M+H]+: 1097.4736, Found [M+H]+: 1097.4743.

[0183] N-(4-(8-(ll-(2-(4-((6-((2-(4-((2,4-dioxothiazolidin-5-yl)methyl)phenoxy)ethyl)(methyl)amino)pyrimidin-4-yl)oxy)phenoxy)acetamido)undecanoyl)-3,8-diazabicyclo[3.2.1]octan-3-yl)phenethyl)-3-(methylamino)thieno[2,3-Z>]pyridine-2-carboxamide (PDT-25) Yellow solid (3.3 mg, 30%). ‘HNMR (400 MHz, Methanol-4) 58.64 - 8.48 (m, 2H), 8.32 (s, 1H), 7.44 - 7.34 (m,Attorney Docket No.: 27527-0235 WO 11H), 7.16 - 6.92 (m, 8H), 6.79 (d, J= 8.6 Hz, 2H), 6.66 (d, J= 8.4 Hz, 2H), 5.66 (s, 1H), 4.73 - 4.36 (m, 5H), 3.86 - 3.42 (m, 7H), 3.26 - 3.15 (m, 7H), 3.00 - 2.30 (m, 10H), 1.99 -1.76 (m, 4H), 1.62 - 1.21 (m, 16H). HRMS (m / z): ESI [M] calculated for C59H71N10O8S [M+H]+: 1111.4892, Found [M+H]+: 1111.4911.

[0184] A-(4-(8-(3-(2-(2-(4-((6-((2-(4-((2,4-dioxothiazolidin-5-yl)methyl)phenoxy)ethyl)(methyl)amino)pyrimidin-4-yl)oxy)phenoxy)acetamido)ethoxy)propanoyl)-3,8-diazabicyclo[3.2.1]octan-3-yl)phenethyl)-3-(methylamino)thieno[2,3-Z>] pyridine-2-carboxamide (PDT-26) Y ellow solid (3.3 mg, 32%). ' H NMR (400 MHz, Methanol-^) 58.57 - 8.50 (m, 2H), 8.32 (s, 1H), 7.40 - 7.34 (m, 1H), 7.12 - 6.91 (m, 8H), 6.72 (d, J= 8.6 Hz, 2H), 6.64 (d, J= 8.4 Hz, 2H), 5.61 (s, 1H), 4.72 - 4.29 (m, 5H), 3.77 - 3.36 (m, 13H), 3.26 - 3.17 (m, 5H), 2.96 - 2.52 (m, 10H), 1.97 - 1.78 (m, 4H). HRMS (m / z): ESI [M] calculated for CssHsgNioCW [M+H]+: 1043.3902, Found [M+H]+: 1043.3925.

[0185] A-(4-(8-(3-(2-(2-(2-(4-((6-((2-(4-((2,4-dioxothiazolidin-5-yl)methyl)phenoxy)ethyl)(methyl)amino)pyrimidin-4-yl)oxy)phenoxy)acetamido)ethoxy)ethoxy)propanoyl)-3,8-diazabicyclo[3.2.1]octan-3-yl)phenethyl)-3-(methylamino)thieno[2,3-Z>]pyridine-2-carboxamide (PDT-27, MS1727) Yellow solid (2.9 mg, 27%).XH NMR (400 MHz, Methanol-^) 5 8.61 - 8.55 (m, 2H), 8.24 (s, 1H), 7.40 - 7.34 (m, 1H), 7.13 - 6.97 (m, 8H), 6.81 (d, J = 8.0 Hz, 2H), 6.71 (d, J= 8.7 Hz, 2H), 5.73 (s, 1H), 4.76 - 4.45 (m, 5H), 3.83 - 3.40 (m, 19H), 3.25 (s, 3H), 2.98 - 2.49 (m, 10H), 1.99 - 1.86 (m, 4H). HRMS (m / z): ESI [M] calculated for C55H63NIOOIOS2+[M+H]+: 1087.4165, Found [M+H]+: 1087.4160.

[0186] JV-(4-(8-(l-(4-((6-((2-(4-((2,4-dioxothiazolidin-5-yl)methyl)phenoxy)ethyl)(methyl)amino)pyrimidin-4-yl)oxy)phenoxy)-2-oxo-6,9,12-trioxa-3-azapentadecan-15-oyl)-3,8-diazabicyclo[3.2.1]octan-3-yl)phenethyl)-3- (methylamino)thieno[2,3-Z>]pyridine-2-carboxamide (PDT-28, MS1728) Yellow solid (2.8 mg, 26%). 'HNMR (400 MHz, Methanol-^) 58.57 - 8.51 (m, 2H), 8.23 (s, 1H), 7.41 - 7.35 (m, 1H), 7.12 - 6.94 (m, 8H), 6.78 (d, J= 8.0 Hz, 2H), 6.66 (d, J= 8.4 Hz, 2H), 5.68 (s, 1H), 4.77 - 4.36 (m, 5H), 3.81 - 3.39 (m, 23H), 3.20 (s, 3H), 2.97 - 2.43 (m, 10H), 1.98 - 1.83 (m, 4H).13CNMR (101 MHZ, Methanol-4) 5 174.08, 171.60, 169.29, 168.19, 165.60, 158.72, 156.53, 155.99, 153.52, 149.34, 148.67, 146.95, 145.96, 133.29, 130.18, 129.74,Attomey Docket No.: 27527-0235 WO 1129.22, 127.54, 126.61, 122.06, 119.11, 116.21, 115.11, 114.79, 84.02, 70.20, 70.18, 70.15, 69.89, 69.07, 67.29, 67.16, 55.32, 54.78, 53.62, 51.71, 51.69, 46.85, 41.23, 38.69, 36.91, 35.50, 34.58, 33.90, 32.71, 27.93, 26.34. HRMS (m / z): ESI [M] calculated for C57H67NIOOIIS2+[M+H]+: 1131.4427, Found [M+H]+: 1131.4417.

[0187] A-(4-(8-(l-(4-((6-((2-(4-((2,4-dioxothiazolidin-5-yl)methyl)phenoxy)ethyl)(methyl)amino)pyrimidin-4-yl)oxy)phenoxy)-2-oxo-6,9,12,15-tetraoxa-3-azaoctadecan-18-oyl)-3,8-diazabicyclo[3.2.1]octan-3-yl)phenethyl)-3-(methylamino)thieno[2,3-Z>]pyridine-2-carboxamide (PDT-29, MS1729) Yellow solid (2.9 mg, 25%). 'HNMR (400 MHz, Methanol-^) 58.59 - 8.50 (m, 2H), 8.24 (s, 1H), 7.38 (d, J= 7.6 Hz, 1H), 7.12 - 6.94 (m, 8H), 6.79 (d, J= 8.1 Hz, 2H), 6.66 (d, J= 8.6 Hz, 2H), 5.69 (s, 1H), 4.74 - 4.42 (m, 5H), 3.79 - 3.41 (m, 27H), 3.20 (s, 3H), 2.97 - 2.50 (m, 10H), 2.00 -1.85 (m, 4H). HRMS (m / z): ESI [M] calculated for C59H7iNioOi2S2+[M+H]+: 1175.4689, Found [M+H]+: 1175.4678.

[0188] A-(4-(8-(l-(4-((6-((2-(4-((2,4-dioxothiazolidin-5-yl)methyl)phenoxy)ethyl)(methyl)amino)pyrimidin-4-yl)oxy)phenoxy)-2-oxo-6,9,12,15,18-pentaoxa-3-azahenicosan-21-oyl)-3,8-diazabicyclo[3.2.1]octan-3-yl)phenethyl)-3-(methylamino)thieno[2,3-Z>]pyridine-2-carboxamide (PDT-30, MS1730) Yellow solid (3.1 mg, 25%).1HNMR(400 MHz, Methanol-4) 5 8.57 - 8.50 (m, 2H), 8.24 (s, 1H), 7.41 - 7.34 (m, 1H), 7.10 - 6.95 (m, 8H), 6.79 (d, J= 8.3 Hz, 2H), 6.67 (d, J= 8.4 Hz, 2H), 5.69 (s, 1H), 4.72 - 4.44 (m, 5H), 3.83 - 3.43 (m, 31H), 3.20 (s, 3H), 2.94 - 2.54 (m, 10H), 2.00 - 1.86 (m, 4H). HRMS (m / z): ESI [M] calculated for C61H75N10O13S [M+H]+: 1219.4951, Found [M+H]+: 1219.4944.Scheme 9. Synthesis of Linker 14-(3-(4-(2-(3-(methylamino)thieno[2,3-h]pyridine-2-carboxamido)ethyl)phenyl)-3,8-diazabicyclo[3.2.1]octan-8-yl)-4-oxobutanoic acid (Linker l)jV-(4-(3,8-diazabicyclo[3.2.1]octan-3-yl)phenethyl)-3-(methylamino)thieno[2,3- / >]pyridine-2-carboxamide (30 mg, 0.07 mmol, 1.0 eq), 4-butoxy-4-Atorney Docket No.: 27527-0235 WO 1oxobutanoic acid (13.3 mg, 0.077 mmol, 1.1 eq), HATU (29 mg, 0.077 mmol, 1.5 eq), DIPEA (18 mg, 0.138 mmol, 1.5 eq), were stirred in DCM (2 mL) at room temperature for 1 h. Then TFA (1 mL) was added and the mixture was stirred for addtional 10 mins. After removal of all the volatiles, the resulted residue was purified via reverse-ISCO to yield title compound as a orange solid (28 mg, 75% yield over two steps). *H NMR (400 MHz, MeOD) 58.62 - 8.53 (m, 2H), 7.43 (dd, J = 8.4, 4.7 Hz, 1H), 7.13 (d, J = 8.5 Hz, 2H), 6.85 (d, J = 8.6 Hz, 2H), 4.73 -4.67 (m, 1H), 4.48 (d, J= 6.5 Hz, 1H), 3.56 - 3.45 (m, 4H), 3.22 (s, 3H), 3.00 -2.91 (m, 1H), 2.89 -2.75 (m, 3H), 2.70 - 2.60 (m, 4H), 2.09 - 1.85 (m, 4H). LCMS m / z = 522.2.]Linkers 2- 13 were synthesized following the same procedure for preparing Linker 1.o o5-(3-(4-(2-(3-(methylamino)thieno[2,3-h]pyridine-2-carboxamido)ethyl)phenyl)-3,8-diazabicyclo[3.2.1]octan-8-yl)-5-oxopentanoic acid (Linker 2) Orange solid (32 mg, 83% yield ). *H NMR (400 MHz, MeOD) 58.62 - 8.54 (m, 2H), 7.43 (dd, J = 8.4, 4.7 Hz, 1H), 7.12 (d, J = 8.5 Hz, 2H), 6.83 (d, J = 8.6 Hz, 2H), 4.72 (d, J= 5.8 Hz, 1H), 4.45 (d, J= 5.9 Hz, 1H), 3.56 - 3.46 (m, 4H), 3.22 (s, 3H), 2.88 - 2.75 (m, 4H), 2.57 - 2.35 (m, 4H), 2.03 - 1.85 (m, 6H). LCMS m / z = 536.2.6-(3-(4-(2-(3-(methylamino)thieno[2,3-b]pyridine-2-carboxamido)ethyl)phenyl)-3,8-diazabicyclo[3.2.1]octan-8-yl)-6-oxohexanoic acid (Linker 3) Orange solid(34mg, 86% yield ). 'HNMR (400 MHz, MeOD) 58.65 - 8.55 (m, 2H), 7.45 (dd, J = 8.4, 4.7 Hz, 1H), 7.13 (d, J = 8.6 Hz, 2H), 6.84 (d, J = 8.6 Hz, 2H), 4.78 - 4.67 (m, 1H), 4.49 - 4.40 (m, 1H), 3.59 - 3.45 (m, 4H), 3.22 (s, 3H), 2.90 - 2.77 (m, 4H), 2.54 -2.29 (m, 4H), 2.09 - 1.83 (m, 4H), 1.67 (p, J= 4.3 Hz, 4H). LCMS m / z =550.2.7-(3-(4-(2-(3-(methylamino)thieno[2,3-b]pyridine-2-carboxamido)ethyl)phenyl)-3,8-diazabicyclo[3.2.1]octan-8-yl)-7-oxoheptanoic acid (Linker 4) Orange solid (36 mg, 89% yield ). *H NMR (400 MHz, MeOD) 58.63 - 8.53 (m, 2H), 7.43 (dd, J = 8.4, 4.7 Hz, 1H), 7.12 (d, J = 8.6 Hz, 2H), 6.83 (d, J =Atorney Docket No.: 27527-0235 WO 18.7 Hz, 2H), 4.77 - 4.66 (m, 1H), 4.49 - 4.39 (m, 1H), 3.57 - 3.44 (m, 4H), 3.22 (s, 3H), 2.87 - 2.74 (m, 4H), 2.49 -2.26 (m, 4H), 2.06 - 1.84 (m, 4H), 1.70 - 1.56 (m, 4H), 1.45 - 1.35 (m, 2H). LCMS m / z = 564.3.8-(3-(4-(2-(3-(methylamino)thieno[2,3-h]pyridine-2-carboxamido)ethyl)phenyl)-3,8-diazabicyclo[3.2.1]octan-8-yl)-8-oxooctanoic acid (Linker 5) Orange solid (38 mg, 92% yield ). *H NMR (400 MHz, MeOD) 58.42 - 8.34 (m, 2H), 7.22 (dd, J = 8.3, 4.6 Hz, 1H), 6.93 (d, J = 8.4 Hz, 2H), 6.63 (d, J = 8.2 Hz, 2H), 4.57 - 4.49 (m, 1H), 4.29 - 4.20 (m, 1H), 3.37 - 3.26 (m, 4H), 3.02 (s, 3H), 2.65 - 2.57 (m, 4H), 2.28 - 2.06 (m, 4H), 1.84 - 1.64 (m, 4H), 1.47 - 1.37 (m, 4H), 1.22 - 1.14 (m, 4H). LCMS m / z = 578.3.9-(3-(4-(2-(3-(methylamino)thieno[2,3-b]pyridine-2-carboxamido)ethyl)phenyl)-3,8-diazabicyclo[3.2.1]octan-8-yl)-9-oxononanoic acid (Linker 6) Orange solid (29 mg, 68% yield ). *H NMR (400 MHz, MeOD) 58.44 - 8.35 (m, 2H), 7.25 (dd, J = 8.4, 4.6 Hz, 1H), 6.93 (d, J = 8.3 Hz, 2H), 6.64 (d, J = 8.3 Hz, 2H), 4.57 - 4.50 (m, 1H), 4.28 - 4.21 (m, 1H), 3.38 - 3.25 (m, 4H), 3.03 (s, 3H), 2.68 - 2.57 (m, 4H), 2.25 -2.07 (m, 4H), 1.85 - 1.67 (m, 4H), 1.48 - 1.36 (m, 4H), 1.19 - 1.12 (m, 6H). LCMS m / z = 592.3.o o10-(3-(4-(2-(3-(methylamino)thieno|2,3- / >|pyridine-2-carboxamido)ethyl)phenyl)-3,8-diazabicyclo[3.2.1]octan-8-yl)-10-oxodecanoic acid (Linker 7) Orange solid (29 mg, 67% yield ). *H NMR (400 MHz, MeOD) 58.43 - 8.35 (m, 2H), 7.23 (dd, J = 8.4, 4.7 Hz, 1H), 6.93 (d, J = 8.4 Hz, 2H), 6.64 (d, J = 8.6 Hz, 2H), 4.57 - 4.49 (m, 1H), 4.29 - 4.19 (m, 1H), 3.37 - 3.25 (m, 4H), 3.03 (s, 3H), 2.67 - 2.58 (m, 4H), 2.27 -2.05 (m, 4H), 1.83 - 1.66 (m, 4H), 1.45 - 1.35 (m, 4H), 1.18 - 1.12 (m, 8H). LCMS m / z = 606.3.Atorney Docket No.: 27527-0235 WO 1 o ll-(3-(4-(2-(3-(methylamino)thieno[2,3-b]pyridine-2-carboxamido)ethyl)phenyl)-3,8- diazabicyclo[3.2.1]octan-8-yl)-ll-oxoundecanoic acid (Linker 8) Orange solid (33 mg, 74% yield ). *H NMR (400 MHz, MeOD) 58.42 - 8.35 (m, 2H), 7.22 (dd, J = 8.3, 4.7 Hz, 1H), 6.93 (d, J = 8.6 Hz, 2H), 6.64 (d, J = 8.7 Hz, 2H), 4.57 - 4.52 (m, 1H), 4.29 - 4.22 (m, 1H), 3.37 - 3.27 (m, 4H), 3.03 (s, 3H), 2.67 - 2.58 (m, 4H), 2.27-2.05 (m, 4H), 1.85 - 1.65 (m,4H), 1.48 - 1.34 (m, 4H), 1.16 - 1.10 (m, 10H). LCMS m / z = 620.3.o o 3-(3-(3-(4-(2-(3-(methylamino)thieno[2,3-h]pyridine-2-carboxamido)ethyl)phenyl)-3,8- diazabicyclo[3.2.1]octan-8-yl)-3-oxopropoxy)propanoic acid (Linker 9) Orange solid (29 mg, 75% yield ). *H NMR (400 MHz, MeOD) 58.64 - 8.52 (m, 2H), 7.44 (dd, J= 8.3, 4.7 Hz, 1H), 7.13 (d, J= 8.5 Hz, 2H), 6.84 (d, J = 8.7 Hz, 2H), 4.74 (d, J = 5.5 Hz, 1H), 4.49 (d, J = 6.4 Hz, 1H), 3.75 (t, J = 6.1 Hz, 2H), 3.69 (t, J = 6.1 Hz, 2H), 3.55 - 3.47 (m, 4H), 3.22 (s, 3H), 2.93 - 2.46 (m, 8H), 2.07 - 1.85 (m, 4H). LCMS m / z = 566.2.3-(2-(3-(3-(4-(2-(3-(methylamino)thieno|2,3- / >|pyridine-2-carboxamido)ethyl)phenyl)-3,8- diazabicyclo[3.2.1]octan-8-yl)-3-oxopropoxy)ethoxy)propanoic acid (Linker 10) Orange solid (32 mg, 73% yield ). > H NMR (400 MHz, MeOD) 58.47 - 8.32 (m, 2H), 7.24 (dd, J = 8.4, 4.7 Hz, 1H), 6.93 (d, J = 8.4 Hz, 2H), 6.65 (d, J= 8.2 Hz, 2H), 4.60 -4.49 (m, 1H), 4.33 - 4.28 (m, 1H), 3.60 - 3.52 (m, 2H), 3.44 - 3.28 (m, 10H), 3.03 (s, 3H), 2.80 - 2.59 (m, 4H), 2.49 - 2.42 (m, 2H), 2.25 (t, J= 6.3 Hz, 2H), 1.92 - 1.65 (m, 4H). LCMS m / z = 610.3.Atorney Docket No.: 27527-0235 WO 13-(2-(2-(3-(3-(4-(2-(3-(methylamino)thieno[2,3-b]pyridine-2-carboxamido)ethyl)phenyl)-3,8-diazabicydo[3.2.1]octan-8-yl)-3-oxopropoxy)ethoxy)ethoxy)propanoic acid (Linker 11) Orange solid (40 mg, 86% yield ). II NMR (400 MHz, MeOD) 58.47 - 8.38 (m, 2H), 7.28 (dd, J= 8.4, 4.7 Hz, 1H), 6.96 (d, J= 8.3 Hz, 2H), 6.68 (d, J = 8.7 Hz, 2H), 4.57 (d, J = 5.5 Hz, 1H), 4.34 (d, J = 6.4 Hz, 1H), 3.65 - 3.54 (m, 2H), 3.49 (t, J= 6.3 Hz, 2H), 3.39 - 3.29 (m, 12H), 3.05 (s, 3H), 2.84 - 2.78 (m, 1H), 2.73 - 2.60 (m, 3H), 2.54 -2.42 (m, 2H), 2.34 (1,7 = 6.3 Hz,2H), 1.90 - 1.68 (m, 4H). LCMS m / z = 654.3.o o16-(3-(4-(2-(3-(methylamino)thieno[2,3-b]pyridine-2-carboxamido)ethyl)phenyl)-3,8-diazabicyclo[3.2.1]octan-8-yl)-16-oxo-4,7,10,13-tetraoxahexadecanoic acid (Linker 12) Orange solid (42 mg, 84% yield ). II NMR (400 MHz, MeOD) 58.45 - 8.34 (m, 2H), 7.26 (dd, 7= 8.3, 4.7 Hz, 1H), 6.95 (d,7 = 8.6 Hz, 2H), 6.67 (d, 7 = 8.7 Hz, 2H), 4.56 (d, 7 = 5.5 Hz, 1H), 4.32 (d, 7 = 6.4 Hz, 1H), 3.62 - 3.47 (m, 4H), 3.38 - 3.29 (m, 16H), 3.03 (s, 3H), 2.83 - 2.76 (m, 1H), 2.70 - 2.58 (m, 3H), 2.55 - 2.40 (m, 2H), 2.33 (t, 7 = 6.3 Hz, 2H), 1.89 - 1.69 (m, 4H). LCMS m / z = 698.3.19-(3-(4-(2-(3-(methylamino)thieno[2,3-b]pyridine-2-carboxamido)ethyl)phenyl)-3,8-diazabicyclo[3.2.1]octan-8-yl)-19-oxo-4,7,10,13,16-pentaoxanonadecanoic acid (Linker 13) Orange solid (45 mg, 85% yield ). 'HNMR (400 MHz, MeOD) 58.53 - 8.46 (m, 2H), 7.34 (dd, 7= 8.4, 4.7 Hz, 1H), 7.04 (d, J= 8.5 Hz, 2H), 6.79 - 6.73 (m, 2H), 4.70 - 4.61 (m, 1H), 4.42 (d, 7= 6.4 Hz, 1H), 3.70 - 3.60 (m, 4H), 3.49 -3.39 (m, 20H), 3.13 (s, 3H), 2.92 - 2.85 (m, 1H), 2.79 - 2.69 (m, 3H), 2.62 - 2.50 (m, 2H), 2.43 (t, 7 = 6.3 Hz, 2H), 1.97 - 1.78 (m, 4H). LCMS m / z = 742.3.Ethyl-3-((2-(4-(3-(N-methyl-4-(3-(4-(2-(3-(methylamino)thieno[2,3-b]pyridine-2-carboxamido)ethyl)phenyl)-3,8-diazabicyclo[3.2.1]octan-8-yl)-4-oxobutanamido)propyl)pyridin-2-yl)-6-(l,2,4,5-tetrahydro-3H-benzo[d]azepin-3-yl)pyrimidin-4-yl)amino)propanoate (QC272-062) Ethyl-3-((2- (4-(3-(methylamino)propyl)pyridin-2-yl)-6-(l,2,4,5-tetrahydro-3H-benzo[d]azepin-3-yl)py-rimidin-4-yl)amino)propanoate (3.0 mg, 0.006 mmol, 1.0 eq), 4-(3-(4-(2-(3-(methylamino)thieno[2,3-b]pyridine-2-Atorney Docket No.: 27527-0235 WO 1carboxamido)ethyl)phenyl)-3,8-diazabicyclo[3.2.1]octan-8-yl)-4-oxobutanoic acid (Linker 1) (4.0 mg, 0.006 mmol, 1.0 eq), HATU (2.5 mg, 0.0066 mmol, 1.1 eq), DIPEA (2.6 mg, 0.018 mmol, 3.0 eq) were stirred in DMF (1.0 mL) at room temperature for 2 h. the mixture was purified via prep-HPLC to yield titled compound as a yellow solid (1.8 mg, 30%). 'HNMR (400 MHz, MeOD) 58.69 - 8.53 (m, 3H), 8.48 - 8.37 (m, 1H), 7.70 - 7.55 (m, 1H), 7.46 - 7.34 (m, 1H), 7.24 - 7.01 (m, 6H), 6.84 - 6.73 (m, 2H), 5.96 - 5.93 (m, 1H), 4.70 - 4.65 (m, 1H), 4.53 - 4.46 (m, 1H), 4.22 - 4.16 (m, 2H), 3.73 - 3.68 (m, 2H), 3.55 - 3.45 (m, 7H), 3.16 - 3.07 (m, 8H), 2.85 - 2.60 (m, 13H), 2.00 - 1.86 (m, 8H), 1.34 - 1.24 (m, 5H). HRMS (m / z): ESI [M] calculated for C55H<56NIIO5S+[M+H]+: 992.4964, Found [M+H]+: 992.4968.Ethyl-3-((2-(4-(3-(N-methyl-5-(3-(4-(2-(3-(methylamino)thieno[2,3-b]pyridine-2- carboxamido)ethyl)phenyl)-3,8-diazabicyclo[3.2.1]octan-8-yl)-5-oxopentanamido)propyl)pyridin-2-yl)-6- (l,2,4,5-tetrahydro-3H-benzo[d]azepin-3-yl)pyrimidin-4-yl)amino)propanoate (QC272-063) Yellow solid (2.0 mg, 33%). > H NMR (400 MHz, MeOD) 58.68 - 8.31 (m, 4H), 7.63 - 7.34 (m, 2H), 7.21 - 7.05 (m, 6H), 6.81 - 6.71 (m, 2H), 5.96 - 5.93 (m, 1H), 4.72 - 4.68 (m, 1H), 4.48 - 4.44 (m, 1H), 4.19 (q, J = 7.1 Hz, 2H), 3.70 (t, J= 6.2 Hz, 2H), 3.56 - 3.46 (m, 7H), 3.24 (s, 3H), 3.11 - 3.05 (m, 5H), 2.90 - 2.70 (m, 9H), 2.53 - 2.43 (m, 4H), 2.02 - 1.86 (m, 10H), 1.35 - 1.22 (m, 5H). HRMS (m / z): ESI [M] calculated for C56H68NIIO5S+[M+H]+: 1006.5120, Found [M+H]+: 1006.5130.Ethyl-3-((2-(4-(3-(N-methyl-6-(3-(4-(2-(3-(methylamino)thieno[2,3-b]pyridine-2- carboxamido)ethyl)phenyl)-3,8-diazabicyclo[3.2.1]octan-8-yl)-6-oxohexanamido)propyl)pyridin-2-yl)-6- (l,2,4,5-tetrahydro-3H-benzo[d]azepin-3-yl)pyrimidin-4-yl)amino)propanoate (QC272-064) Yellow solid (1.6 mg, 26%). *H NMR (400 MHz, MeOD) 58.67 - 8.30 (m, 4H), 7.62 - 7.34 (m, 2H), 7.20 - 7.04 (m, 6H), 6.82 - 6.71 (m, 2H), 5.97 - 5.93 (m, 1H), 4.71 - 4.68 (m, 1H), 4.48 - 4.43 (m, 1H), 4.20 (q, J = 7.1 Hz, 2H), 3.72 (t, J= 6.2 Hz, 2H), 3.52 - 3.47 (m, 7H), 3.23 (s, 3H), 3.11 - 3.05 (m, 5H), 2.92 - 2.71 (m, 9H), 2.53 - 2.42 (m, 4H), 2.01 - 1.84 (m, 12H), 1.35 - 1.22 (m, 5H). HRMS (m / z): ESI [M] calculated for C57H7oNn05S+[M+H]+: 1020.5277, Found [M+H]+: 1020.5268.Atorney Docket No.: 27527-0235 WO 1Ethyl-3-((2-(4-(3-(N-methyl-7-(3-(4-(2-(3-(methylamino)thieno[2,3-b]pyridine-2-carboxamido)ethyl)phenyl)-3,8-diazabicydo[3.2.1]octan-8-yl)-7-oxoheptanamido)propyl)pyridin-2-yl)-6-(l,2,4,5-tetrahydro-3H-benzo[d]azepin-3-yl)pyrimidin-4-yl)amino)propanoate (QC272-065) Yellow solid (2.0 mg, 32%). *H NMR (400 MHz, MeOD) 58.55 - 8.26 (m, 4H), 7.50 (s, 1H), 7.28 (dd, J = 8.1, 4.8 Hz, 1H), 7.10 - 6.95 (m, 6H), 6.71 - 6.65 (m, 2H), 5.87 - 5.81 (m, 1H), 4.62- 4.58 (m, 1H), 4.34 - 4.28 (m, 1H), 4.11 -4.05 (m, 2H), 3.60 - 3.54 (m, 2H), 3.42 - 3.28 (m, 7H), 3.11 (s, 3H), 3.01 - 2.92 (m, 5H), 2.75 - 2.58 (m, 8H), 2.35 - 2.22 (m, 4H), 1.92 - 1.75 (m, 7H), 1.58 - 1.45 (m, 5H), 1.33 - 1.12 (m, 8H). HRMS (m / z): ESI [M] calculated for C58H72N11O5S+[M+H]+: 1034.5433, Found [M+H]+: 1034.5422.o oEthyl-3-((2-(4-(3-(N-methyl-8-(3-(4-(2-(3-(methylamino)thieno[2,3-b]pyridine-2-carboxamido)ethyl)phenyl)-3,8-diazabicyclo[3.2.1]octan-8-yl)-8-oxooctanamido)propyl)pyridin-2-yl)-6-(l,2,4,5-tetrahydro-3H-benzo[d]azepin-3-yl)pyrimidin-4-yl)amino)propanoate (QC272-066) Yellow solid (1.7 mg, 27%). *H NMR (400 MHz, MeOD) 58.60 - 8.26 (m, 4H), 7.50 (s, 1H), 7.32 - 7.24 (m, 1H), 7.10 -6.93 (m, 6H), 6.70 (d, J = 8.0 Hz, 2H), 5.90 - 5.80 (m, 1H), 4.63 - 4.59 (m, 1H), 4.34 - 4.28 (m, 1H), 4.12 -4.05 (m, 2H), 3.60 - 3.54 (m, 2H), 3.45 - 3.33 (m, 7H), 3.11 (s, 3H), 3.00 - 2.90 (m, 5H), 2.76 - 2.59 (m, 8H), 2.35 - 2.22 (m, 4H), 1.95 - 1.75 (m, 7H), 1.58 - 1.44 (m, 5H), 1.30 - 1.13 (m, 10H). HRMS (m / z): ESI [M] calculated for C59H74NIIO5S+[M+H]+: 1048.5590, Found [M+H]+: 1048.5596.Ethyl-3-((2-(4-(3-(N-methyl-9-(3-(4-(2-(3-(methylamino)thieno[2,3-b]pyridine-2-carboxamido)ethyl)phenyl)-3,8-diazabicyclo[3.2.1]octan-8-yl)-9-oxononanamido)propyl)pyridin-2-yl)-6- (l,2,4,5-tetrahydro-3H-benzo[d]azepin-3-yl)pyrimidin-4-yl)amino)propanoate (QC272-067) Yellow solidAtorney Docket No.: 27527-0235 WO 1(1.8 mg, 28%). > H NMR (400 MHz, MeOD) 58.61 - 8.26 (m, 4H), 7.51 (s, 1H), 7.39 - 7.20 (m, 1H), 7.10 -6.98 (m, 6H), 6.71 (d, J= 8.5 Hz, 2H), 5.84 - 5.80 (m, 1H), 4.64 - 4.58 (m, 1H), 4.34 - 4.28 (m, 1H), 4.11 -4.06 (m, 2H), 3.63 - 3.55 (m, 2H), 3.44 - 3.33 (m, 7H), 3.11 (s, 3H), 3.00 - 2.90 (m, 5H), 2.75 -2.60 (m, 8H), 2.30 -2.12 (m, 4H), 1.95 - 1.75 (m, 7H), 1.49 (s, 5H), 1.28 - 1.16 (m, 12H). HRMS (m / z): ESI [M] calculated for C6OH76NII05S+[M+H]+: 1062.5746, Found [M+H]+: 1062.5747.o oEthyl-3-((2-(4-(3-(N-methyl-10-(3-(4-(2-(3-(methylamino)thieno[2,3-b]pyridine-2-carboxamido)ethyl)phenyl)-3,8-diazabicyclo[3.2.1]octan-8-yl)-10-oxodecanamido)propyl)pyridin-2-yl)-6-(l,2,4,5-tetrahydro-3H-benzo[d]azepin-3-yl)pyrimidin-4-yl)amino)propanoate (QC272-068) Yellow solid (1.8 mg, 26%). > H NMR (400 MHz, MeOD) 58.53 - 8.26 (m, 4H), 7.51 (s, 1H), 7.29 - 7.25 (m, 1H), 7.10 -6.98 (m, 6H), 6.71 (d, J= 8.6 Hz, 2H), 5.84 - 5.80 (m, 1H), 4.64 - 4.60 (m, 1H), 4.35 - 4.31 (m, 1H), 4.11 -4.03 (m, 2H), 3.64 - 3.51 (m, 2H), 3.48 - 3.30 (m, 7H), 3.11 (s, 3H), 3.00 - 2.92 (m, 5H), 2.77 -2.59 (m, 8H), 2.29 -2.13 (m, 4H), 1.95 - 1.71 (m, 7H), 1.59 - 1.40 (m, 5H), 1.27- 1.14 (m, 14H). HRMS (m / z): ESI [M] calculated for C6iH7sNiiO5S+[M+H]+: 1076.5903, Found [M+H]+: 1076.5912.Ethyl-3-((2-(4-(3-(N-methyl-ll-(3-(4-(2-(3-(methylamino)thieno[2,3-b]pyridine-2-carboxamido)ethyl)phenyl)-3,8-diazabicyclo[3.2.1]octan-8-yl)-ll-oxoundecanamido)propyl)pyridin-2-yl)-6-(l,2,4,5-tetrahydro-3H-benzo[d]azepin-3-yl)pyrimidin-4-yl)amino)propanoate (QC272-069) Yellow solid (2.2 mg, 34%). > H NMR (400 MHz, MeOD) 58.60 - 8.27 (m, 4H), 7.51 (s, 1H), 7.28 (dd, J = 8.3, 4.7 Hz, 1H), 7.11 -6.98 (m, 6H), 6.72 (d, J= 8.3 Hz, 2H), 5.85 - 5.81 (m, 1H), 4.65 - 4.61 (m, 1H), 4.35 - 4.31 (m, 1H), 4.08 (q, J = 7.1 Hz, 2H), 3.59 (t, J= 6.2 Hz, 2H), 3.48 - 3.29 (m, 7H), 3.11 (s, 3H), 3.00 - 2.94 (m, 5H), 2.77 -2.60 (m, 8H), 2.33 -2.12 (m, 4H), 1.98 - 1.71 (m, 7H), 1.55 - 1.35 (m, 5H), 1.25 - 1.09 (m, 16H). HRMS (m / z): ESI [M] calculated for C62HSONII05S+[M+H]+: 1090.6059, Found [M+H]+: 1090.6070.Atorney Docket No.: 27527-0235 WO 1Ethyl-3-((2-(4-(3-(N-methyl-3-(3-(3-(4-(2-(3-(methylamino)thieno[2,3-b]pyridine-2- carboxamido)ethyl)phenyl)-3,8-diazabicyclo[3.2.1]octan-8-yl)-3- oxopropoxy)propanamido)propyl)pyridin-2-yl)-6-(l,2,4,5-tetrahydro-3H-benzo[d]azepin-3-yl)pyrimidin- 4-yl)amino)propanoate (QC272-070) Yellow solid (1.6 mg, 26%). > H NMR (400 MHz, MeOD) 58.56 - 8.18 (m, 4H), 7.47 (s, 1H), 7.28 (dd, J= 8.4, 4.6 Hz, 1H), 7.10 - 6.92 (m, 6H), 6.70 - 6.57 (m, 2H), 5.79 - 5.76 (m, 1H), 4.62 - 4.58 (m, 1H), 4.36 - 4.32 (m, 1H), 4.07 (q, J = 7.2 Hz, 2H), 3.69 - 3.50 (m, 7H), 3.40 - 3.24 (m, 6H), 3.10 (s, 3H), 2.95 (br, 4H), 2.80 - 2.74 (m, 4H), 2.70 -2.58 (m, 8H), 2.50 - 2.35 (m, 3H), 1.91 - 1.67 (m, 7H), 1.24 - 1.08 (m, 5H). HRMS (m / z): ESI [M] calculated for C57H7ONII06S+[M+H]+: 1036.5226, Found [M+H]+: 1036.5211.Ethyl-3-((2-(4-(3-(N-methyl-3-(2-(3-(3-(4-(2-(3-(methylamino)thieno[2,3-b]pyridine-2- carboxamido)ethyl)phenyl)-3,8-diazabicyclo[3.2.1]octan-8-yl)-3- oxopropoxy)ethoxy)propanamido)propyl)pyridin-2-yl)-6-(l,2,4,5-tetrahydro-3H-benzo[d]azepin-3- yl)pyrimidin-4-yl)amino)propanoate (QC272-071) Yellow solid (1.8 mg, 27%). *H NMR (400 MHz, MeOD) 88.56 - 8.25 (m, 4H), 7.49 (s, 1H), 7.29 - 7.23 (m, 1H), 7.10 - 6.92 (m, 6H), 6.68 (d, J= 9.4 Hz, 2H), 5.81 - 5.77 (m, 1H), 4.63 - 4.59 (m, 1H), 4.37 - 4.33 (m, 1H), 4.08 (q, J = 7.3 Hz, 2H), 3.70 - 3.32 (m, 17H), 3.10 (s, 3H), 2.98 -2.90 (m, 4H), 2.84 -2.54 (m, 12H), 2.46 -2.33 (m, 3H), 1.95 - 1.71 (m, 7H), 1.25 - 1.12 (m, 5H). HRMS (m / z): ESI [M] calculated for C59H74NIIO7S+[M+H]+: 1080.5488, Found [M+H]+: 1080.5487.Ethyl-3-((2-(4-(14-methyl-l-(3-(4-(2-(3-(methylamino)thieno[2,3-b]pyridine-2-carboxamido)ethyl)phenyl)- 3,8-diazabicyclo[3.2.1]octan-8-yl)-l,13-dioxo-4,7,10-trioxa-14-azaheptadecan-17-yl)pyridin-2-yl)-6- (l,2,4,5-tetrahydro-3H-benzo[d]azepin-3-yl)pyrimidin-4-yl)amino)propanoate (QC272-072) Yellow solidAtorney Docket No.: 27527-0235 WO 1(1.9 mg, 27%). > H NMR (400 MHz, MeOD) 58.59 - 8.21 (m, 4H), 7.49 (s, 1H), 7.27 (dd, J = 8.4, 4.7 Hz, 1H), 7.09 - 6.95 (m, 6H), 6.69 (d, J= 8.1 Hz,2H), 5.81 - 5.78 (m, 1H), 4.63 - 4.59 (m, 1H), 4.39 - 4.35 (m, 1H),4.11 - 4.05 (m, 2H), 3.69 - 3.55 (m, 7H), 3.41 - 3.32 (m, 14H), 3.10 (s, 3H), 2.98 -2.49 (m, 19H), 1.95 - 1.71 (m, 7H), 1.25 - 1.10 (m, 5H). HRMS (m / z): ESI [M] calculated for C6IH78NIIO8S+[M+H]+: 1124.5750, Found [M+H]+: 1124.5747.Ethyl-3-((2-(4-(17-methyl-l-(3-(4-(2-(3-(methylamino)thieno[2,3-b]pyridine-2-carboxamido)ethyl)phenyl)- 3,8-diazabicyclo[3.2.1]octan-8-yl)-l,16-dioxo-4,7,10,13-tetraoxa-17-azaicosan-20-yl)pyridin-2-yl)-6- (l,2,4,5-tetrahydro-3H-benzo[d]azepin-3-yl)pyrimidin-4-yl)amino)propanoate (QC272-073) Yellow solid (2.0 mg, 28%). *H NMR (400 MHz, MeOD) 58.59 - 8.25 (m, 4H), 7.49 (s, 1H), 7.27 (dd, J = 8.5, 4.7 Hz, 1H), 7.12 - 6.93 (m, 6H), 6.72 (dd, J = 19.7, 8.1 Hz, 2H), 5.83- 5.79 (m, 1H), 4.64 - 4.60 (m, 1H), 4.40 - 4.36 (m, 1H), 4.08 (q, J= 7.2 Hz, 2H), 3.70 - 3.53 (m, 7H), 3.45 - 3.32 (m, 18H), 3.11 (s, 3H), 2.98 -2.48 (m, 19H), 1.86 (q, J= 22.5 Hz, 7H), 1.30 - 1.07 (m, 5H). HRMS (m / z): ESI [M] calculated for C63H82NIIO9S+[M+H]+: 1168.6012, Found [M+H]+: 1168.6023.Ethyl-3-((2-(4-(20-methyl-l-(3-(4-(2-(3-(methylamino)thieno[2,3-b]pyridine-2-carboxamido)ethyl)phenyl)- 3,8-diazabicyclo[3.2.1]octan-8-yl)-l,19-dioxo-4,7,10,13,16-pentaoxa-20-azatricosan-23-yl)pyridin-2-yl)-6- (l,2,4,5-tetrahydro-3H-benzo[d]azepin-3-yl)pyrimidin-4-yl)amino)propanoate (QC272-074) Yellow solid (2.2 mg, 30%). > H NMR (400 MHz, MeOD) 58.59 - 8.24 (m, 4H), 7.47 (s, 1H), 7.27 (dd, J = 8.3, 4.8 Hz, 1H), 7.11 - 6.97 (m, 6H), 6.79 - 6.64 (m, 2H), 5.90 - 5.79 (m, 1H), 4.64 - 4.60 (m, 1H), 4.40 - 4.36 (m, 1H), 4.08 (q, J= 7.2 Hz, 2H), 3.68 - 3.57 (m, 7H), 3.47- 3.35 (m, 22H), 3.11 (s, 3H), 2.98 -2.45 (m, 19H), 1.95 - 1.71 (m, 7H), 1.24 - 1.08 (m, 5H). HRMS (m / z): ESI [M] calculated for C65H86NnOioS+[M+H]+: 1212.6274, Found [M+H]+: 1212.6277.TABLE 1Atorney Docket No.: 27527-0235 WO 1Compo structure Nameund IDQC272 Ethyl-3-((2-(4-(3-(N-methyl-4-(3-(4-(2-(3- (methylamino)thieno[2,3-b]pyridine-2- -062 V > o o carboxamido)ethyl)phenyl)-3,8- diazabicyclo[3.2.1]octan-8-yl)-4- r r r r oxobutanamido)propyl)pyridin -2-yl)-6-(l, 2,4,5- tetrahydro-3H-benzo[d]azepin-3-yl)pyrimidin- 4-yl)amino)propanoateQC272 A A Ethyl-3-((2-(4-(3-(N-methyl-5-(3-(4-(2-(3- (methylamino)thieno[2,3-b]pyridine-2- -063 carboxamido)ethyl)phenyl)-3,8-:1 I A diazabicyclo[3.2.1]octan-8-yl)-5- ■ i d:•>u°. Xl A oxopentanamido)propyl)pyridin-2-yl)-6- O N^N AA A°(l,2,4,5-tetrahydro-3H-benzo[d]azepin-3- <y A^ N AJL N \H x A A°°°Hs J \A 7} — v N= / yl)pyrimidin-4-yl)amino)propanoate AA QC272 O 0 0 0 0A Ethyl-3-((2-(4-(3-(N-methyl-6-(3-(4-(2-(3- (methylamino)thieno[2,3-b]pyridine-2- -064 carboxamido)ethyl)phenyl)-3,8- O.. diazabicyclo[3.2.1]octan-8-yl)-6- 1 \ Il?HN.... oxohexanamido)propyl)pyridin-2-yl)-6-(l, 2,4,5- O N^Ntetrahydro-3H-benzo[d]azepin-3-yl)pyrimidin- ZVVNAJV>H4-yl)amino)propanoateEthyl-3-((2-(4-(3-(N-methyl-7-(3-(4-(2-(3- QC272 (methylamino)thieno[2,3-b]pyridine-2- -065 carboxamido)ethyl)phenyl)-3,8- diazabicyclo[3.2.1]octan-8-yl)-7- oxoheptanamido)propyl)pyridin-2-yl)-6- (l,2,4,5-tetrahydro-3H-benzo[d]azepin-3- yl)pyrimidin-4-yl)amino)propanoate Ethyl-3-((2-(4-(3-(N-methyl-8-(3-(4-(2-(3- QC272(methylamino)thieno[2,3-b]pyridine-2- -066 carboxamido)ethyl)phenyl)-3,8- diazabicyclo[3.2.1]octan-8-yl)-8- oxooctanamido)propyl)pyridin-2-yl)-6-(l, 2,4,5- tetrahydro-3H-benzo[d]azepin-3-yl)pyrimidin- 4-yl)amino)propanoateO 0 Ethyl-3-((2-(4-(3-(N-methyl-9-(3-(4-(2-(3- QC272 (methylamino)thieno[2,3-b]pyridine-2- rr^AA -067 carboxamido)ethyl)phenyl)-3,8- NA1A.i'k^diazabicyclo[3.2.1]octan-8-yl)-9- T Al 1 A oxononanamido)propyl)pyridin-2-yl)-6- o N^N(l,2,4,5-tetrahydro-3H-benzo[d]azepin-3- A / - AAH®AAyl)pyrimidin-4-yl)amino)propanoate H 1 \. N= AAAO 0 Ethyl-3-((2-(4-(3-(N-methyl-10-(3-(4-(2-(3- QC272(methylamino)thieno[2,3-b]pyridine-2- -068 rA^A^A carboxamido)ethyl)phenyl)-3,8- diazabicyclo[3.2.1]octan-8-yl)-10- A Xl A oxodecanamido)propyl)pyridin -2-yl) -6-(l, 2,4,5- O N^Ntetrahydro-3H-benzo[d]azepin-3-yl)pyrimidin- A / - AAHSA A <y N A 4-yl)amino)propanoate H 1 } —. N= / AA^AEthyl-3-((2-(4-(3-(N-methyl-ll-(3-(4-(2-(3- QC272 (methylamino)thieno[2,3-b]pyridine-2- -069 carboxamido)ethyl)phenyl)-3,8- diazabicyclo[3.2. l]octan-8-yl)-l 1- oxoundecanamido)propyl)pyridin-2-yl)-6- (l,2,4,5-tetrahydro-3H-benzo[d]azepin-3- yl)pyrimidin-4-yl)amino)propanoateAtorney Docket No.: 27527-0235 WO 10 oQC272 Ethyl-3-((2-(4-(3-(N-methyl-3-(3-(3-(4-(2-(3- N A^o-A N ''V| (methylamino)thieno[2,3-b]pyridine-2- -070 A 1?hn^ carboxamido)ethyl)phenyl)-3,8- diazabicyclo[3.2.1]octan-8-yl)-3- O N N X AT «A oxopropoxy)propanamido)propyl)pyridin-2- H I yl)-6-(l,2,4,5-tetrahydro-3H-benzo[d]azepin-3- yl)pyrimidin-4-yl)amino)propanoate O O Ethyl-3-((2-(4-(3-(N-methyl-3-(2-(3-(3-(4-(2-(3- QC272 rII I ' '2 1 / 1 (methylamino)thieno[2,3-b]pyridine-2- -0711Y / N.i T1?HN^ carboxamido)ethyl)phenyl)-3,8- diazabicyclo[3.2.1]octan-8-yl)-3- / -x <y Jk / -x oxopropoxy)ethoxy)propanamido)propyl)pyrid hr AT / ~~\ V-, A 7 in- i ATN^ 2-yl)-6-(l,2,4,5-tetrahydro-3H- A |o benzo[d]azepin-3-yl)pyrimidin-4- yl)amino)propanoateEthyl-3-((2-(4-(14-methyl-l-(3-(4-(2-(3- QC272 o (methylamino)thieno[2,3-b]pyridine-2- -072 carboxamido)ethyl)phenyl)-3,8- diazabicyclo[3.2.1]octan-8-yl)-l,13-dioxo- 74,7,10-trioxa-14-azaheptadecan-17-yl)pyridin- 2-yl)-6-(l,2,4,5-tetrahydro-3H-benzo[d]azepin- 3-yl)pyrimidin-4-yl)amino)propanoate 0 0 Ethyl-3-((2-(4-(17-methyl-l-(3-(4-(2-(3- QC272 (methylamino)thieno[2,3-b]pyridine-2- 1v 4I / 1 -073 carboxamido)ethyl)phenyl)-3,8- xz 1 1 11?HN^ diazabicyclo[3.2.1]octan-8-yl)-l,16-dioxo- T° 0 N^N4,7,10J3-tetraoxa-17-azaicosan-20-yl)pyridin- / \ JL Ax A / — \ 2-yl)-6-(l,2,4,5-tetrahydro-3H-benzo[d]azepin- ( ATN=T 3-yl)pyrimidin-4-yl)amino)propanoate 0 0 Ethyl-3-((2-(4-(20-methyl-l-(3-(4-(2-(3- QC272(methylamino)thieno[2,3-b]pyridine-2- YA A II 1 ' » 1 / 1 -074 carboxamido)ethyl)phenyl)-3,8- Al i?HN^ diazabicyclo[3.2.1]octan-8-yl)-l,19-dioxo- o A AA4,7,10J3,16-pentaoxa-20-azatricosan-23- XX A XX Jxjk X-x,Hs- / l> yl)pyridin-2-yl)-6-(l,2,4,5-tetrahydro-3H- benzo[d]azepin-3-yl)pyrimidin-4- yl)amino)propanoateAttomey Docket No.: 27527-0235 WO 1REFERENCES:(1) Henning, N. J.; Boike, L.; Spradlin, J. N.; Ward, C. C.; Liu, G.; Zhang, E.; Belcher, B. P.; Brittain, S. M.; Hesse, M. J.; Dovala, D., Deubiquitinase-targeting chimeras fortargeted protein stabilization. Nat. Chem. Biol. 2022, 18 (4), 412-421.(2) Liu, J.; Yu, X.; Chen, H.; Kaniskan, H. U. m.; Xie, L.; Chen, X.; Jin, J.; Wei, W, TF-DUBTACs stabilize tumor suppressor transcription factors. J. Am. Chem. Soc. 2022, 144 (28), 12934-12941.(3) Liu, J.; Hu, X.; Luo, K.; Xiong, Y; Chen, L.; Wang, Z.; Inuzuka, H.; Qian, C.; Yu, X.; Xie, L.; Muneer, A.; Zhang, D.; Paulo, J. A.; Chen, X.; Jin, J.; Wei, W, USP7-based deubiquitinase-targeting chimeras stabilize AMPK. J. Am. Chem. Soc. 2024, 146 (16), 11507— 11514.(4) Deng, Z.; Chen, L.; Qian, C.; Liu, J.; Wu, Q.; Song, X.; Xiong, Y; Wang, Z.; Hu, X.; Inuzuka, H.; Zhong, Y; Xiang, Y; Lin, Y; Pham, N. D.; Shi, Y; Wei, W; Jin, J., The First-in-class Deubiquitinase-targeting Chimera Stabilizes and Activates cGAS. Angew. Chem. Int. Ed. 2024, e202415168.(5) Sakamoto, K. M.; Kim, K. B.; Kumagai, A.; Mercurio, F.; Crews, C. M.; Deshaies, R., Protacs: Chimeric molecules that target proteins to the Skpl-Cullin-F box complex for ubiquitination and degradation. Proceedings of the National Academy of Sciences 2001, 98 (15), 8554-8559.(6) Schneekloth, A. R.; Pucheault, M.; Tae, H. S.; Crews, C. M., Targeted intracellular protein degradation induced by a small molecule: En route to chemical proteomics. Bioorg. Med. Chem. Lett. 2008, 18 (22), 5904-5908.(7) Liu, J.; Ma, J.; Liu, Y; Xia, J.; Li, Y; Wang, Z. P.; Wei, W. In PROTACs: A novel strategy for cancer therapy, Semin. Cancer Biol., Elsevier: 2020; pp 171-179.(8) Dale, B.; Cheng, M.; Park, K. S.; Kaniskan, H.; Xiong, Y; Jin, J., Advancing targeted protein degradation for cancer therapy. Nat. Rev. Cancer 2021, 21 (10), 638-654.(9) Schneekloth, A. R.; Pucheault, M.; Tae, H. S.; Crews, C. M., Targeted intracellular protein degradation induced by a small molecule: En route to chemical proteomics. Bioorganic & medicinal chemistry letters 2008, 18 (22), 5904-5908.(10) Schreiber, S. L., The Rise of Molecular Glues. Cell 2021, 184 (1), 3-9.(11) Tsai, J. M.; Nowak, R. P.; Ebert, B. L.; Fischer, E. S., Targeted protein degradation: from mechanisms to clinic. Ata. Rev. Mol. CellBiol. 2024, https: / / doi.org / 10.1038 / s41580-024-Attorney Docket No.: 27527-0235 WO 100729-9.(12) Marei H; Tsai WK; Kee YS; Ruiz K; He J; CoxC; Sun T; Penikalapati S; Dwivedi P; Choi M; Kan D; Saenz-Lopez P; Dorighi K; Zhang P; KschonsakYT; KljavinN; Amin D, K. I.; Mancini AG; Nguyen T; Wang C; Janezic E; Doan A; Mai E; Xi H; Gu C; Heinlein M; Biehs B; WuJ; LehouxI; Harris S; Comps-Agrar L; Seshasayee D; de Sauvage FJ; Grimmer M, L. J.; Agard NJ; F., d. S. E. M., Antibody targeting of E3 ubiquitin ligases for receptor degradation. Nature 2022, 610 (7930), 182-189.(13) Pance, K.; Gramespacher, J. A.; Byrnes, J. R.; Salangsang, F.; Serrano, J.-A. C.; Cotton, A. D.; Steri, V; Wells, J. A., Modular cytokine receptor-targeting chimeras for targeted degradation of cell surface and extracellular proteins. Nat. Biotech. 2023, 41 (2), 273-281. (14) Zhang, D., Duque-Jimenez, J., Facchinetti, F., Brixi, G., Rhee, K., Feng, W. W., Janne, P. A. and Zhou, X., Transferrin receptor targeting chimeras for membrane protein degradation. Nature 2024, pp.1-9.(15) Banik, S. M.; Pedram, K.; Wisnovsky, S.; Ahn, G.; Riley, N. M.; Bertozzi, C. R., Lysosome-targeting chimaeras for degradation of extracellular proteins. Nature 2020, 584 (7820), 291-297.(16) Li, Z.; Wang, C.; Wang, Z.; Zhu, C.; Li, J.; Sha, T; Ma, L.; Gao, C.; Yang, Y; Sun, Y, Allele-selective lowering of mutant HTT protein by HTT-LC3 linker compounds. Nature 2019, 575 (7781), 203-209.(17) Takahashi, D.; Moriyama, J.; Nakamura, T; Miki, E.; Takahashi, E.; Sato, A.; Akaike, T; Itto-Nakama, K.; Arimoto, H., AUTACs: cargo -specific degraders using selective autophagy. Mol. Cell 2019, 76 (5), 797-810. elO.(18) Wang, Z.; Liu, J.; Chen, H.; Qiu, X.; Xie, L.; Kaniskan, H. U. m.; Chen, X.; Jin, J.; Wei, W, Telomere targeting chimera enables targeted destruction of telomeric repeat-binding factor proteins. J. Am. Chem. Soc. 2023, 145 (19), 10872-10879.(19) Wang, Z.; Liu, J.; Qiu, X.; Zhang, D.; Inuzuka, H.; Chen, L.; Chen, H.; Xie, L.; Kaniskan, H. U. m.; Chen, X.; Jin, J.; Wei, W, Methylated nucleotide-based proteolysistargeting chimera enables targeted degradation of methyl-CpG-binding protein 2. J. Am. Chem. Soc. 2023, 145 (40), 21871-21878.(20) Wang, Z.; Zhang, D.; Qiu, X.; Inuzuka, H.; Xiong, Y; Liu, J.; Chen, L.; Chen, H.; Xie, L.; Kaniskan, H. U.; Chen, X.; Jin, J.; Wei, W, Structurally specific Z-DNA proteolysis targeting chimera enables targeted degradation of adenosine deaminase acting on RNA 1. J. Am. Chem. Soc. 2024, 146 (11), 7584-7593.Attorney Docket No.: 27527-0235 WO 1(21) Samarasinghe, K. T. G.; Jaime-Figueroa, S.; Burgess, M.; Nalawansha, D. A.; Dai, K.; Hu, Z.; Bebenek, A.; Holley, S. A.; Crews, C. M., Targeted degradation of transcription factors by TRAFTACs: TRAnscription Factor TAigeting Chimeras. Cell Chem. Biol. 2021, 28 (5), 648-661.e5.(22) Liu, J.; Chen, H.; Kaniskan, H. U.; Xie, L.; Chen, X.; Jin, J.; Wei, W., TF-PROTACs enable targeted degradation of transcription factors. J. Am. Chem. Soc. 2021, 143 (23), 8902-8910.(23) Shao, J.; Yan, Y; Ding, D.; Wang, D.; He, Y; Pan, Y; Yan, W; Kharbanda, A.; Li, H. Y; Huang, H., Destruction of DNA-b inding proteins by programmable oligonucleotide PROTAC (O'PROTAC): Effective targeting of LEF1 and ERG. Adv. Sci. (Weinh) 2021, 8 (20), e2102555.(24) Oren, M., Regulation of the p53 tumor suppressor protein. J. Biol. Chem. 1999, 274 (51), 36031-36034.(25) Chen, Y; Xue, H.; Jin, J. J. J. o. B. C., Applications of protein ubiquitylation and deubiquitylation in drug discovery. J. Biol. Chem 2024, 300 (5), 107264.(26) Jani, C.; Singh, H.; Walker, A.; Rupal, A.; Perrino, C. M.; Caughey, T. D.; Lam, P, Pomalidomide induced lupus type hypersensitivity reaction. Current Problems in Cancer: Case Reports 2021, 3, 100065.(27) Harrigan, J. A.; Jacq, X.; Martin, N. M.; Jackson, S. P., Deubiquitylating enzymes and drug discovery: emerging opportunities. Nat. Rev. Drug Discov. 2018, 17 (1), 57-78.(28) Sacco, J. J.; Coulson, J. M.; Clague, M. J.; Urbe, S., Emerging roles of deubiquitinases in cancer-associated pathways. IUBMB life 2010, 62 (2), 140-157.(29) Hussain, S.; Zhang, Y; Galardy, P, DUBs and cancer: the role of deubiquitinating enzymes as oncogenes, non-oncogenes and tumor suppressors. Cell Cycle. 2009, 8 (11), 1688-1697.(30) Meitinger, F.; Anzola, J. V.; Kaulich, M.; Richardson, A.; Stender, J. D.; Benner, C.; Glass, C. K.; Dowdy, S. F.; Desai, A.; Shiau, A. K., 53BP1 andUSP28 mediate p53 activation and G1 arrest after centrosome loss or extended mitotic duration. J. Cell Biol. 2016, 214 (2), 155-166.(31) Wrigley, J. D.; Gavory, G.; Simpson,!.; Preston, M.; Plant, H.; Bradley, J.; Goeppert, A. U.; Rozycka, E.; Davies, G.; Walsh, J., Identification and characterization of dual inhibitors of the USP25 / 28 deubiquitinating enzyme subfamily. ACS Chem. Biol. 2017, 12 (12), 3113-3125.Attorney Docket No.: 27527-0235 WO 1(32) Peng, J.; Jiang, K.; Sun, X.; Wu, L.; Wang, J.; Xi, X.; Tan, X.; Liang, T; Tan, C.; Zhang, P, Identification of a class of potent USP25 / 28 inhibitors with broad-spectrum anticancer activity. Signal Transduct. Target. Ther. 2022, 7(1), 393.(33) Seo, H.-S.; Dhe-Paganon, S., Crystal Structure Analysis of human USP28 complex with a compound. 2023, https: / / doi.org / 10.2210 / pdb7TUO / pdb.(34) Kwon, J.; Bakhoum, S. F., The cytosolic DNA-sensing cGAS-STING pathway in cancer. Cancer Discov. 2020, 10 (1), 26-39.(35) Samson, N.; Ablasser, A., The cGAS-STING pathway and cancer. Nat. Cancer 2022, 3 (12), 1452-1463.(36) Lama, L.; Adura, C.; Xie, W; Tomita, D.; Kamei, T; Kuryavyi, V.; Gogakos, T; Steinberg, J. L; Miller, M.; Ramos-Espiritu, L.; Asano, Y; Hashizume, S.; Aida, J.; Imaeda, T; Okamoto, R.; Jennings, A. J.; Michino, M.; Kuroita, T; Stamford, A.; Gao, P.; Meinke, P.; Glickman, J. F.; Patel, D. J.; Tuschl, T. Development of human cGAS-specific small-molecule inhibitors for repression of dsDNA -triggered interferon expression. Nat. Commun. 2019, 10 (1), 2261.(37) Hong, Z.; Mei, J.; Li, C., Bai, G.; Maimaiti, M.; Hu, H.; Yu, W.; Sun, L. I.; Zhang, L.; Cheng, D.; Liao, Y, STING inhibitors target the cyclic dinucleotide binding pocket. Proc. Natl. Acad. Sci. U. S. A. 2021, 775(24), p.e2105465118.(38) Rosen, E. D.; Spiegelman, B. M., PPARy: a nuclear regulator of metabolism, differentiation, and cell growth. J. Biol. Chem. 2001, 276 (41), 37731-37734.(39) Elrod, H. A.; Sun, S.-Y, PPARy and apoptosis in cancer. PPAR Res. 2008, 2008, 704165.(40) Farrow, B.; Evers, B. M., Activation of PPARy increases PTEN expression in pancreatic cancer cells. Biochem. Biophys. Res. Commun. 2003, 301 (1), 50-53.(41) Campbell, M. J.; Carlberg, C.; Koeffler, H. P, Arole forthe PPARy in cancer therapy. PPAR Res. 2008, 2008, 314974.(42) Chen, L.; Yuan, Y; Kar, S.; Kanchi, M. M.; Arora, S.; Kim, J. E.; Koh, P. F.; Yousef, E.; Samy, R. P; Shanmugam, M. K.; Tan, T. Z.; Shin, S. W.; Arfuso, F.; Shen, H. M.; Yang, H.; Goh, B. C.; Park, J. L; Gaboury, L.; Lobie, P. E.; Sethi, G.; Lim, L. H. K.; Kumar, A. P, PPARgamma ligand-induced annexin Al expression determines chemotherapy response via deubiquitination of death domain kinase RIP in triple-negative breast cancers. Mol. Cancer Ther. 2017, 76 (11), 2528-2542.(43) Xu, X.; Liu, M.; Yang, Y; Wei, C.; Zhang, X.; Song, H.; Wang, Y; Duan, X., VSP-17Attorney Docket No.: 27527-0235 WO 1suppresses the migration and invasion of triple-negative breast cancer cells through inhibition of the EMT process via the PPARgamma / AMPK signaling pathway. Oncol. Rep. 2021, 45 (3), 975-986.(44) Jang, J. Y.; Bae, H.; Lee, Y. J.; Choi, Y. I.; Kim, H. J.; Park, S. B.; Suh, S. W.; Kim, S. W.; Han, B. W., Structural basis for the enhanced anti-Diabetic efficacy of Lobeglitazone on PPARy. Sci. Rep. 2018, 8 (1), 31.

Claims

Attorney Docket No.: 27527-0235WO1WHAT IS CLAIMED IS:

1. A deubiquitinase-targeting chimera, comprising a ubiquitin-specific protease 28 (USP28) recruiting ligand conjugated to a target protein ligand by a linker.

2. A deubiquitinase-targeting chimera, comprising ubiquitin-specific protease 28 (USP28) recruiting ligand CT1073 or KL9 conjugated to cystic fibrosis transmembrane conductance regulator (CTFR) ligand, lumacaftor, by a linker.

3. A deubiquitinase-targeting chimera having the following structure:L:CO'MA w 4; CSH3: m a 4:and pharmaceutically acceptable salts and enantiomers thereof.

4. Deubiquitinase-targeting chimera MS9279.

5. A deubiquitinase-targeting chimera, comprising ubiquitin-specific protease 28 (USP28) recruiting ligand CT1073 or KL9, conjugated to cyclic GMP-AMP synthase (cGAS) antagonist GMO, by a linker.Attorney Docket No.: 27527-0235WO16. A deubiquitinase-targeting chimera having the following structure:O''?* " O cur s; - ' ' b«U<? -s x?"???* rare**ami: « 4and pharmaceutically acceptable salts and enantiomers thereof.

7. Deubiquitinase-targeting chimera MS2099.

8. Deubiquitinase-targeting chimera MS2100.

9. A deubiquitinase-targeting chimera, comprising ubiquitin-specific protease 28 (USP28) recruiting ligand CT1073 or KL9, conjugated to peroxisome proliferator-activated receptor gamma (PPARy) ligand, lobeglitazone, by a linker.

10. A deubiquitinase-targeting chimera having the following structure:PPT Sg; m » 1c m « 2$O») 1W2& ra ®W2§; H)W: m *and pharmaceutically acceptable salts and enantiomers thereof.Attorney Docket No.: 27527-0235 WO 111. Deubiquitinase-targeting chimera selected from any one of MS2027 -MS2030.

12. A method of treating cystic fibrosis, comprising administering to a subject in need thereof, a therapeutically effective amount of a deubiquitinase-targeting chimera according to any one of claims 2 - 4.

13. A method of treating cancer, comprising administering to a subject in need thereof, a therapeutically effective amount of a deubiquitinase-targeting chimera according to any one of claims 5 - 11.

14. A pharmaceutical composition, comprising a deubiquitinase-targeting chimera according to any one of claims 1 - 11 and a pharmaceutically acceptable carrier.

15. Deubiquitinase-targeting chimera selected from the group consisting of QC272-062, QC272-063, QC272-064, QC272-065, QC272-066, QC272-067, QC272-068, QC272-069, QC272-070, QC272-071, QC272-072, QC272-0732 and QC272-074 and pharmaceutically acceptable salts and enantiomers thereof.

16. A method of treating cystic fibrosis, comprising administering to a subject in need thereof, a therapeutically effective amount of a deubiquitinase-targeting chimera according to claim 15.

17. A method of treating cancer, comprising administering to a subject in need thereof, a therapeutically effective amount of a deubiquitinase-targeting chimera according to claim 15.

18. A pharmaceutical composition, comprising a deubiquitinase-targeting chimera according to claim 15 and a pharmaceutically acceptable carrier.

Images