DCAF16-based covalent handle for the rational design of monovalent degraders

Abstract

Compositions and methods deploy a vinylsulfonyl covalent handle in monovalent degraders that act through targeting a cysteine within DCAF16 to enable the degradation of a target protein.

Description

DCAF16-Based Covalent Handle for the Rational Design of Monovalent Degraders

[0001] Cross-references to related application

[0002] This application claims priority to U.S. Provisional Application No. 63 / 623,304; filed: Jan 21, 2024, the disclosure of which is hereby incorporated by reference in its entirety for all purposes.

[0003] Government Support Clause

[0004] This invention was made with government support under grant numbers CA263814 and CA240981 awarded by the National Institutes of Health, and grant number 2127788 awarded by the National Science Foundation. The government has certain rights in the invention.

[0005] Reference to a Sequence Listing

[0006] A Sequence Listing in XML format is incorporated by reference into the specification.The name of the XML file containing the Sequence Listing is B24-080-2WO.xml. The XML file is l,827bytes and was created and submitted electronically via EFS-Web on Dec 3, 2024.

[0007] Introduction

[0008] Monovalent molecular glue degraders have arisen as a powerful therapeutic modality for degrading therapeutic targets of interest through inducing the proximity of an E3 ubiquitin ligase with a neo-substrate protein to ubiquitinate and degrade the target through the proteasome ’ . Molecular glue degraders are potentially more promising compared to heterobifunctional Proteolysis Targeting Chimeras (PROTACs) because of their lower molecular weights and associated drug-like properties, as well as their potential to exploit shallow protein-protein interfaces between an E3 ligase and less tractable therapeutic proteins that may not possess deep binding pockets '. However, most molecular glue degraders have either been discovered fortuitously or through phenotypic screens. Rational chemical design of molecular glue or monovalent degraders in a target-based manner remains challenging.

[0009] Many recent studies have reported how subtle chemical alterations to otherwise non- degradative small-molecule inhibitors converted them into molecular glue degraders of their respective targets6“9. These studies gave rise to the exciting possibility of transplantable chemical handles that could be appended onto the exit vector of protein-targeting ligands to convert these compounds into molecular glue degraders of their targets. E3 ligases have been shown to be ligandable with covalent small-molecules and chemoproteomic approaches10“16. Covalent handles have also been successfully used in heterobifunctional PROTACs to identifypermissive chemical handle and ligandable E3 ligase pairs that can be exploited for targeted protein degradation applications. These studies have identified various covalent handles targeting cysteines in E3 ligase substrate receptors DCAF16 and DCAF1113,17,18. Covalent ligand screens against specific ubiquitin proteasome system components have also yielded new E3 ligase, E2 ubiquitin conjugating enzyme, or Cullin adaptor recruiters against RNF114, RNF4, FEM1B, UBE2D, DDB1, and SKP1 that can be used for PROTACs12’19"24.

[0010] Recent studies have also revealed that covalent chemistry can be used to identify potential chemical handles that enable the rational design of monovalent or molecular glue degraders. We previously discovered a covalent chemical handle that targets a cysteine in the quality control E3 ligase RNF126 that could be appended to the exit vector of a diverse range of protein-targeting ligands without the necessity for a linker to enable degradation of their respective targets . Covalent molecular glue degraders have also been discovered that enhance weak existing interactions between DCAF16 and BRD4 to degrade BRD4 in a template-assisted covalent modification approach26,27.[Oil] Summary of the Invention

[0012] We sought to identify additional transplantable covalent chemical handles that can convert non-degradative inhibitors into molecular glue or monovalent degraders of their respective targets. We have identified, inter alia, a vinylsulfonyl piperazine handle that acts through targeting a cysteine within DCAF16 to not only enable the degradation of BRD4, but also several additional neo-substrates.

[0013] The invention provides attaching a vinylsulfonyl covalent handle on protein targeting ligands to degrade a target protein, and related compositions. The invention provides compositions and methods for the rational design of monovalent degraders that act through targeting a cysteine within DCAF16 to enable the degradation of a target protein.

[0014] In aspects and embodiments the invention provides:

[0015] 1. A vinylsulfonyl piperazine covalent handle for the rational design of monovalent degraders that act through targeting a cysteine within DCAF16 to enable the degradation of a target protein, of structure:

[0016] 2. The vinylsulfonyl piperazine covalent handle of a claim herein, attached to a proteintargeting ligand, R, as of structure:

[0017] 3. The vinylsulfonyl piperazine covalent handle of a claim herein, wherein the target protein is selected from BRD4, CDK4 / 6, SMARCA2 / 4, AR, BTK, and BCR-ABL / c-ABL.

[0018] 4. The vinylsulfonyl piperazine covalent handle of a claim herein, wherein the proteintargeting ligand is selected from:

[0019] BET bromodomain inhibitors (e.g. et al. Molecules. 2023 Mar 29;28(7): 3043), such as JQ1 (ML 1 -50),

[0020] CDK4 / 6 inhibitors such as ribociclib (ML 1-71),

[0021] AR inhibitors, such as derived from the ARV- 110 PROTAC (ML 2-9)

[0022] SMARCA2 inhibitors, such as in ML 1-96,

[0023] BCR-ABL / c-ABL inhibitors, such as dasatinib derivative, as in ML 2-5.

[0024] 5. A vinylsulfonyl piperidine covalent handle that acts through targeting a cysteine within DCAF16 to enable the degradation of a target protein, attached to a protein-targeting ligand, R, as of structure:R / li _JO

[0025] 6. A vinylsulfonyl derivative of a vinyl ketone protein-targeting ligand.

[0026] 7. A vinylsulfonyl derivative of a vinyl ketone protein-targeting ligand, wherein the derivative effects the degradation of the target protein, whereas the ligand does not.

[0027] 8. A vinylsulfonyl derivative of the BTK inhibitor ibrutinib (TH 1-9).

[0028] 9. A pharmaceutical composition comprising a compound herein, or a pharmaceutically acceptable salt, a hydrate or a stereoisomer thereof, and a pharmaceutically acceptable carrier or excipient, preferably in a pharmaceutically acceptable unit dosage.

[0029] 10. A method to treat a disease or physiological condition comprising administering to a person in need thereof a compound herein.

[0030] 11. A method herein, further comprising the antecedent step of detecting or diagnosing a disease or condition indicating the need thereof, and / or the subsequent step of detecting a resultant improvement or delay of progression of the disease or condition.

[0031] 12. Use of a compound herein in the manufacture of a medicament for treating a disease or condition, including all embodiments and examples thereof.

[0032] 13. A compound herein for use in treating a disease or condition, including all embodiments and examples thereof.

[0033] The invention encompasses all combinations of the particular embodiments recited herein, as if each combination had been laboriously recited.

[0034] Brief Description of the Drawings

[0035] Figs. 1A-1C. Identifying Covalent Handles that Enable the Degradation of BRD4.Fig. 1A. Series of analogs of the BET family inhibitor JQ1 bearing various electrophilic handles. Fig. IB. Testing for BRD4 degradation with covalent JQ1 derivatives. HEK293T cells were treated with DMSO vehicle or covalent JQ1 derivatives (5 pM) for 24 h and BRD4 long and short isoforms and GAPDH loading control levels were assessed by Western blotting. Shown are gels that are representative of n=3 biologically independent replicates per group. Fig. 1C. Quantitation of BRD4 long and short isoforms from experiment described in Fig. IB showing individual replicate values and average ± sem. Significance is expressed as *p<0.001 compared to vehicle-treated controls.

[0036] Figs. 2A-2I. Characterization of the Monovalent and Covalent BRD4 Degrader ML 1-50. Fig. 2A. Structure of ML 1-50 with the vinylsulfonyl piperazine covalent chemical handle in red. Figs. 2B-2C. Dose-response of BRD4 degradation. HEK293T cells were treated with DMSO vehicle or ML 1-50 for 24 h. BRD4 and actin loading control levels were assessed by Western blotting and quantified in Fig. 2C. Figs. 2D-2E. Proteasome inhibitor attenuation of BRD4 degradation. HEK293T cells were pre-treated with DMSO vehicle or bortezomib (1 pM) 1 h prior to DMSO vehicle or ML 1-50 (1 pM) treatment for 24 h. BRD4 and actin loading control levels were assessed by Western blotting and quantified in Fig. 2E. Figs. 2F-2G.NEDD8 activating enzyme inhibitor attenuation of BRD4 degradation. HEK293T cells were pre-treated with DMSO vehicle or MLN4924 (1 pM) 1 h prior to DMSO vehicle or ML 1-50 (1 pM) treatment for 24 h. BRD4 and actin loading control levels were assessed by Western blotting and quantified in Fig. 2G. Fig. 2H. BRD4 degradation in MDA-MB-231 cells. MDA- MB-231 cells were treated with DMSO vehicle or ML 1-50 for 24 h and BRD4 and actin loading control levels were assessed by Western blotting. Fig. 21. Tandem mass tagging (TMT)- based quantitative proteomic profiling of ML 1-50 in MDA-MB-231 cells. MDA-MB-231 cellswere treated with DMSO vehicle or ML 1-50 (IpM) for 24 h. Proteins that were lowered in levels by >2-fold with pcO.OOl are highlighted in red with BRD4 specifically labeled. Data are from n-3 biologically independent replicates per group. Blots shown in Figs. B, D, F, H are representative of n=3 biologically independent replicates per group. Bar graphs in Figs. C, E, G show average ± sem. Significance is expressed as *p<0.05 compared to vehicle-treated controls and #p<0.05 compared to ML 1-50 treatment alone.

[0037] Figs. 3A-3F. Identifying the E3 Ligase Responsible for ML 1-50-Mediated BRD4 Degradation. Fig. 3A. Structure of alkyne-functionalized probe of the vinylsulfonyl piperazine handle (highlighted in red). Fig. 3B. ML 1-50-outcompeted targets enriched by ML 2-33. HEK293T cells were pre-treated with DMSO vehicle or ML 1-50 (50 pM) 1 h prior to treatment of cells with the ML 2-33 probe (10 pM). Probe-modified proteins were subjected to copper- catalyzed azide alkyne cycloaddition (CuAAC) with an azide-functionalized biotin enrichment handle. Probe-modified proteins were avidin-enriched, tryptically digested, and analyzed by TMT-based proteomics. Among the significantly outcompeted targets, DCAF16 highlighted in red was the only Cullin E3 ligase substrate receptor identified. Fig. 3C. Gel-based ABPP of ML 1-50 against pure DCAF16. Pure DCAF16 protein was pre-incubated with DMSO vehicle or ML 1-50 for 30 min prior to addition of a rhodamine-functionalized cysteine-reactive iodoacetamide probe (lA-rhodamine) (100 nM) for 1 h. Proteins were resolved by SDS / PAGE and assessed by in-gel fluorescence and protein loading was assessed by silver staining. Fig. 3D. TMT-based quantitative proteomic analysis of DCAF16 wild-type (WT) versus knockout (KO) cells. Because there was no commercial DCAF16 antibody, proteomic methods were used to confirm DCAF16 knockout. DCAF16 is labeled in red. Figs. 3E-3F. BRD4 degradation in DCAF16 WT and KO cells. DCAF16 WT and KO cells were treated with DMSO vehicle or ML 1-50 (1 pM) for 24 h and BRD4 and actin loading control levels were assessed by Western blotting and quantified in Fig. 3F. Proteomics experiments and blots in Figs. 3B, 3C, 3D, 3E are from n=3 biologically independent replicates per group and blots are representative. Bar graph in Fig. 3F shows individual replicate values average ± sem. Significance is expressed as *p<0.05 compared to vehicle-treated controls and #p<0.05 compared to ML 1-50 treated DCAF16 WT cells.

[0038] Figs. 4A-4D. Testing the Dependence of Covalent Monovalent BRD4 Degraders on DCAF16 versus RNF126. Fig. 4A. Structure of our previously published covalent monovalent BRD4 degrader JP-2-197 bearing the covalent “fumarate” handle shown in red. Fig. 4B. BRD4 degradation in DCAF16 WT and KO cells. DCAF16 WT and KO HEK293T cells were treated with DMSO vehicle or JP-2-197 for 24 h and BRD4 and actin loading control levels were assessed by Western blotting. Figs. 4C-4D. BRD4 degradation in RNF126 WT and KO cells.RNF126 WT and KO HEK293T cells were treated with JP-2-197 for 10 h and BRD4, RNF126, and GAPDH loading control levels were assessed by Western blotting and quantified in Fig. 4D. Blots in Figs. 4B-4C are representative of n=3 biologically independent replicates per group. Bar graph in Fig. 4D shows individual replicate values and average ± sem. Significance is expressed as *p<0.05 compared to vehicle-treated controls and #p<0.05 compared to JP-2-197 treated RNF126 WT cells.

[0039] Figs. 5A-5F. Characterization of CDK4 Monovalent Degrader. Fig. 5A. Structure of ML 1-71, a CDK4 inhibitor ribociclib bearing a vinylsulfonyl piperazine handle highlighted in red. Figs. 5B-5C. CDK4 degradation in C33A cervical cancer cells. C33A cells were treated with DMSO vehicle or ML 1-71 for 24 h and CDK4 and actin loading control levels were assessed by Western blotting and quantified in Fig. 5C. Figs. 5D-5E. CDK4 degradation in DCAF16 WT and KO cells. DCAF16 WT and KO HEK293T cells were treated with DMSO vehicle or ML 1-71 for 24 h and CDK4 and actin loading control levels were assessed by Western blotting and quantified in Fig. 5E. Fig. 5F. TMT-based quantitative proteomic profiling of ML 1 -71 in C33A cells. C33A cells were treated with DMSO vehicle or ML 1-71 (10 pM) for 24 h. Proteins that were reduced in levels by >2-fold with p<0.05 are designated in red with CDK4 labeled. Data are from n=3 biologically independent replicates per group. Blots in Figs. 5B, 5D are representative of n=3 biologically independent replicates per group. Bar graph in Fig. 5E shows individual replicate values and average ± sem. Significance is expressed as *p<0.05 compared to vehicle-treated controls and #p<0.05 compared to ML 1-71 treated DCAF16 WT cells.

[0040] Figs. 6A-6H. Characterization of AR and BTK Monovalent Degraders. Fig. 6A.Structure of AR monovalent degrader ML 2-9 with AR- targeting ligand derived from the ARV- 110 PROTAC bearing the covalent vinylsulfonyl piperazine handle highlighted in red. Figs. 6B- 6C. AR degradation in LNCaP prostate cancer cells. LNCaP cells were treated with DMSO vehicle or ML 2-9 for 24 h and AR and actin loading control levels were assessed by Western blotting and quantified in Fig. 6C. Fig. 6D. TMT-based quantitative proteomic profiling of ML 2-9 in LNCaP cells. LNCaP cells were treated with DMSO vehicle or ML 2-9 (1 pM) for 24 h. Proteins that were reduced in levels by >4-fold with p<0.01 are designated in red with AR labeled. Data are from n=3 biologically independent replicates per group. Fig. 6E. Structure of BTK monovalent degrader TH 1-9 with BTK inhibitor derived from ibrutinib bearing the covalent vinylsulfonyl piperazine handle highlighted in red. Figs. 6F-6G. BTK degradation in MINO lymphoma cancer cells. MINO cells were treated with DMSO vehicle or TH 1-9 for 24 h and BTK and GAPDH loading control levels were assessed by Western blotting and quantified in Fig. 6G. Fig. 6H. TMT-based quantitative proteomic profiling of TH 1-9 in MINO cells.MINO cells were treated with DMSO vehicle or TH 1-9 (5 pM) for 24 h. Proteins that were reduced in levels by >8-fold with p<0.001 are designated in red with BTK labeled. Data are from n=3 biologically independent replicates per group. Blots in Figs. 6B, 6F are representative of n=3 biologically independent replicates per group. Bar graphs in Figs. 6C, 6G show individual replicate values and average ± sem. Significance is expressed as *p<0.05 compared to vehicle-treated controls.

[0041] Figs. 7A-7D. Characterization of ML 1-50. Fig. 7A. Cell viability of HEK293T cells. HEK293T cells were treated with DMSO vehicle or ML 1-50 for 24 h and cell viability was assessed by CellTiter-Glo. Fig. 7B. Quantification of BRD4 degradation in MDA-MB-231 cells from experiment described in Fig. 2H. Fig. 7C. IsoDTB-ABPP profiling of ML 1-50. HEK293T cells were treated with DMSO vehicle or ML 1-50 (10 pM) for 2h. Subsequent lysates were labeled with an alkyne-functionalized iodoacetamide probe (200 pM) for 1 h, followed by CuAAC-mediated attachment of an isotopically light (for control) or heavy (for treated) azide- functionalized desthiobiotin handle, after which probe-modified proteins were streptavidin- enriched, tryptically digested, eluted from beads, and analyzed by LC-MS / MS. The light versus heavy probe-modified peptide ratio for Cl 19 of DCAF16 is noted with associated p-value. Each blue dot corresponds to the average light / heavy ratio of each probe-modified peptide. Data are from n=3 biologically independent replicates per group. Fig. 7D. Site of modification analysis of ML 1-50 on pure DCAF16 protein (SEQ ID NO: 1). Pure DCAF16 protein was labeled with ML 1-50 (50 pM) for 1 h. Protein was tryptically digested and analyzed for the ML 1-50 adduct by LC-MS / MS.

[0042] Figs. 8A-8E. Characterization of SMARCA2 Monovalent Degrader. Fig. 8A. Structure of ML 1 -96 with the vinylsulfonyl piperazine covalent chemical handle in red. Figs. 8B-8C. SMARCA2 degradation in MV-4-11 leukemia cancer cells. MV-4-11 cells were treated with DMSO vehicle or ML 1-96 for 24 h and SMARCA2 / 4 and actin loading control levels were assessed by Western blotting and quantified in Fig. 8C. Figs. 8D-8E. BRD4 degradation in DCAF16 WT and KO cells. DCAF16 WT and KO HEK293 cells were treated with ML 1-96 for 24 h and SMARCA2, SMARCA4, and actin loading control levels were assessed by Western blotting and quantified in Fig. 8D. Blots in Figs. 8B, 8D are representative of n=3 biologically independent replicates per group. Bar graphs in Figs. 8B, 8D show individual replicate values and average ± sem. Significance is expressed as *p<0.05 compared to vehicle-treated controls and #p<0.05 compared to ML 1-96 treated DCAF16 WT cells.

[0043] Figs. 9A-9C. Characterization of a BCR-ABL / c-ABL Monovalent Degrader. Fig. 9A.Structure of ML 2-5 with a dasatinib derivative bearing a vinylsulfonyl piperazine covalent chemical handle in red. Figs. 9B-9C. BCR-ABL and c-ABL degradation in K562 leukemiacancer cells. K562 cells were treated with DMSO vehicle or ML 2-5 for 24 h and SMARCA2 / 4 and actin loading control levels were assessed by Western blotting and quantified in Fig. 9C. Blot is representative is n=3 biologically independent replicates per group. Bar graph in Fig. 9C shows individual replicate values and average ± sem. Significance is expressed as *p<0.05 compared to vehicle- treated controls.

[0044] Description of Particular Embodiments of the Invention

[0045] Unless contraindicated or noted otherwise, in these descriptions and throughout this specification, the terms “a” and “an” mean one or more, the term “or” means and / or. It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein, including citations therein, are hereby incorporated by reference in their entirety for all purposes.

[0046] The term hydrocarbyl refers to hydrocarbon radical, including alkyl, akenyl, alkynyl and aryl.

[0047] The term "alkyl" refers to a hydrocarbon group selected from linear and branched saturated hydrocarbon groups of 1-18, or 1-12, or 1-6 carbon atoms. Examples of the alkyl group include methyl, ethyl, 1 -propyl or n-propyl ("n-Pr"), 2-propyl or isopropyl ("i-Pr"), 1 -butyl or n-butyl ("n-Bu"), 2-methyl-l -propyl or isobutyl ("i-Bu"), 1 -methylpropyl or s-butyl ("s-Bu"), and 1,1 -dimethylethyl or t-butyl ("t-Bu"). Other examples of the alkyl group include 1-pentyl, 2-pentyl, 3 -pentyl, 2-methyl-2-butyl, 3-methyl-2-butyl, 3 -methyl- 1 -butyl, 2-methyl-l -butyl, 1- hexyl, 2-hexyl, 3-hexyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl, 4-methyl-2-pentyl, 3-methyl-3- pentyl, 2-methyl-3-pentyl, 2,3-dimethyl-2-butyl and 3,3-dimethyl-2-butyl groups.

[0048] Lower alkyl means 1-8, preferably 1-6, more preferably 1-4 carbon atoms; lower alkenyl or alkynyl means 2-8, 2-6 or 2-4 carbon atoms.

[0049] The term "alkenyl" refers to a hydrocarbon group selected from linear and branched hydrocarbon groups comprising at least one C=C double bond and of 2-18, or 2-12, or 2-6 carbon atoms. Examples of the alkenyl group may be selected from ethenyl or vinyl, prop-1- enyl, prop-2-enyl, 2-methylprop- 1 -enyl, but-l-enyl, but- 2-enyl, but-3-enyl, buta- 1,3-dienyl, 2- methylbuta-l,3-diene, hex-l-enyl, hex-2-enyl, hex-3-enyl, hex-4-enyl, and hexa-1, 3-dienyl groups.

[0050] The term "alkynyl" refers to a hydrocarbon group selected from linear and branched hydrocarbon group, comprising at least one C=C triple bond and of 2-18, or 2-12, or 2-6 carbonatoms. Examples of the alkynyl group include ethynyl, 1-propynyl, 2-propynyl (propargyl), 1- butynyl, 2-butynyl, and 3-butynyl groups.

[0051] The term "cycloalkyl" refers to a hydrocarbon group selected from saturated and partially unsaturated cyclic hydrocarbon groups, comprising monocyclic and polycyclic (e.g., bicyclic and tricyclic) groups. For example, the cycloalkyl group may be of 3-12, or 3-8, or 3-6 carbon atoms. Even further for example, the cycloalkyl group may be a monocyclic group of 3-12, or 3- 8, or 3-6 carbon atoms. Examples of the monocyclic cycloalkyl group include cyclopropyl, cyclobutyl, cyclopentyl, 1 -cyclopent- 1-enyl, l-cyclopent-2-enyl, l-cyclopent-3-enyl, cyclohexyl, 1 -cyclohex- 1-enyl, l-cyclohex-2-enyl, 1 -cyclohex-3 -enyl, cyclohexadienyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl, cycloundecyl, and cyclododecyl groups. Examples of the bicyclic cycloalkyl groups include those having 7-12 ring atoms arranged as a bicycle ring selected from [4,4], [4,5], [5,5], [5,6] and [6,6] ring systems, or as a bridged bicyclic ring selected from bicyclo [2.2. l]heptane, bicyclo[2.2.2]octane, and bicyclo[3.2.2]nonane. The ring may be saturated or have at least one double bond (i.e. partially unsaturated), but is not fully conjugated, and is not aromatic, as aromatic is defined herein.

[0052] The term “aryl” herein refers to a group selected from:5- and 6-membered carbocyclic aromatic rings, for example, phenyl; bicyclic ring systems such as 7-12 membered bicyclic ring systems wherein at least one ring is carbocyclic and aromatic, selected, for example, from naphthalene, indane, and 1,2,3,4-tetrahydroquinoline; and tricyclic ring systems such as 10-15 membered tricyclic ring systems wherein at least one ring is carbocyclic and aromatic, for example, fluorene.

[0053] For example, the aryl group is selected from 5- and 6-membered carbocyclic aromatic rings fused to a 5- to 7-membered cycloalkyl or heterocyclic ring optionally comprising at least one heteroatom selected from N, O, and S, provided that the point of attachment is at the carbocyclic aromatic ring when the carbocyclic aromatic ring is fused with a heterocyclic ring, and the point of attachment can be at the carbocyclic aromatic ring or at the cycloalkyl group when the carbocyclic aromatic ring is fused with a cycloalkyl group. Bivalent radicals formed from substituted benzene derivatives and having the free valences at ring atoms are named as substituted phenylene radicals. Bivalent radicals derived from univalent polycyclic hydrocarbon radicals whose names end in "-yl" by removal of one hydrogen atom from the carbon atom with the free valence are named by adding "-idene" to the name of the corresponding univalent radical, e.g., a naphthyl group with two points of attachment is termed naphthylidene. Aryl, however, does not encompass or overlap with heteroaryl, separately defined below. Hence, if one or more carbocyclic aromatic rings are fused with a heterocyclic aromatic ring, the resulting ring system is heteroaryl, not aryl, as defined herein.

[0054] The term "halogen" or “halo” refers to F, Cl, Br or I.

[0055] The term "heteroalkyl" refers to alkyl comprising at least one heteroatom.

[0056] The term "heteroaryl" refers to a group selected from:

[0057] 5 - to 7-membered aromatic, monocyclic rings comprising 1, 2, 3 or 4 heteroatoms selected from N, O, and S, with the remaining ring atoms being carbon;

[0058] 8- to 12-membered bicyclic rings comprising 1, 2, 3 or 4 heteroatoms, selected from N, O, and S, with the remaining ring atoms being carbon and wherein at least one ring is aromatic and at least one heteroatom is present in the aromatic ring; and

[0059] 11 - to 14-membered tricyclic rings comprising 1, 2, 3 or 4 heteroatoms, selected from N, O, and S, with the remaining ring atoms being carbon and wherein at least one ring is aromatic and at least one heteroatom is present in an aromatic ring.

[0060] For example, the heteroaryl group includes a 5- to 7-membered heterocyclic aromatic ring fused to a 5- to 7-membered cycloalkyl ring. For such fused, bicyclic heteroaryl ring systems wherein only one of the rings comprises at least one heteroatom, the point of attachment may be at the heteroaromatic ring or at the cycloalkyl ring.

[0061] When the total number of S and O atoms in the heteroaryl group exceeds 1 , those heteroatoms are not adjacent to one another. In some embodiments, the total number of S and O atoms in the heteroaryl group is not more than 2. In some embodiments, the total number of S and O atoms in the aromatic heterocycle is not more than 1.

[0062] Examples of the heteroaryl group include, but are not limited to, (as numbered from the linkage position assigned priority 1) pyridyl (such as 2-pyridyl, 3-pyridyl, or 4-pyridyl), cinnolinyl, pyrazinyl, 2,4-pyrimidinyl, 3,5-pyrimidinyl, 2,4-imidazolyl, imidazopyridinyl, isoxazolyl, oxazolyl, thiazolyl, isothiazolyl, thiadiazolyl, tetrazolyl, thienyl, triazinyl, benzothienyl, furyl, benzofuryl, benzoimidazolyl, indolyl, isoindolyl, indolinyl, phthalazinyl, pyrazinyl, pyridazinyl, pyrrolyl, triazolyl, quinolinyl, isoquinolinyl, pyrazolyl, pyrrolopyridinyl (such as lH-pyrrolo[2,3-b]pyridin-5-yl), pyrazolopyridinyl (such aslH- pyrazolo[3,4-b]pyridin-5-yl), benzoxazolyl (such as benzo[d]oxazol-6-yl), pteridinyl, purinyl, 1- oxa-2,3-diazolyl, l-oxa-2,4-diazolyl, l-oxa-2,5-diazolyl, l-oxa-3,4-diazolyl, l-thia-2,3-diazolyl, l-thia-2,4-diazolyl, l-thia-2,5-diazolyl, l-thia-3,4-diazolyl, furazanyl, benzofurazanyl, benzothiophenyl, benzothiazolyl, benzoxazolyl, quinazolinyl, quinoxalinyl, naphthyridinyl, furopyridinyl, benzothiazolyl (such as benzo[d]thiazol-6-yl), indazolyl (such as lH-indazol-5-yl) and 5,6,7,8-tetrahydroisoquinoline.

[0063] The term "heterocyclic" or "heterocycle" or "heterocyclyl" refers to a ring selected from 4- to 12-membered monocyclic, bicyclic and tricyclic, saturated and partially unsaturated rings comprising at least one carbon atoms in addition to 1, 2, 3 or 4 heteroatoms, selected fromoxygen, sulfur, and nitrogen. “Heterocycle” also refers to a 5- to 7-membered heterocyclic ring comprising at least one heteroatom selected from N, O, and S fused with 5-, 6-, and / or 7- membered cycloalkyl, carbocyclic aromatic or heteroaromatic ring, provided that the point of attachment is at the heterocyclic ring when the heterocyclic ring is fused with a carbocyclic aromatic or a heteroaromatic ring, and that the point of attachment can be at the cycloalkyl or heterocyclic ring when the heterocyclic ring is fused with cycloalkyl.

[0064] “Heterocycle” also refers to an aliphatic spirocyclic ring comprising at least one heteroatom selected from N, O, and S, provided that the point of attachment is at the heterocyclic ring. The rings may be saturated or have at least one double bond (i.e. partially unsaturated). The heterocycle may be substituted with oxo. The point of the attachment may be carbon or heteroatom in the heterocyclic ring. A heterocyle is not a heteroaryl as defined herein.

[0065] Examples of the heterocycle include, but not limited to, (as numbered from the linkage position assigned priority 1) 1-pyrrolidinyl, 2-pyrrolidinyl, 2,4-imidazolidinyl, 2,3-pyrazolidinyl, 1 -piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-piperidinyl, 2,5-piperazinyl, pyranyl, 2- morpholinyl, 3-morpholinyl, oxiranyl, aziridinyl, thiiranyl, azetidinyl, oxetanyl, thietanyl, 1 ,2- dithietanyl, 1,3-dithietanyl, dihydropyridinyl, tetrahydropyridinyl, thiomorpholinyl, thioxanyl, piperazinyl, homopiperazinyl, homopiperidinyl, azepanyl, oxepanyl, thiepanyl, 1,4-oxathianyl, 1 ,4-dioxepanyl, 1 ,4-oxathiepanyl, 1,4-oxaazepanyl, 1 ,4-dithiepanyl, 1 ,4-thiazepanyl and 1,4- diazepane 1 ,4-dithianyl, 1 ,4-azathianyl, oxazepinyl, diazepinyl, thiazepinyl, dihydrothienyl, dihydropyranyl, dihydrofuranyl, tetrahydrofuranyl, tetrahydro thienyl, tetrahydropyranyl, tetrahydrothiopyranyl, 1-pyrrolinyl, 2-pyrrolinyl, 3-pyrrolinyl, indolinyl, 2H-pyranyl, 4H- pyranyl, 1 ,4-dioxanyl, 1,3-dioxolanyl, pyrazolinyl, pyrazolidinyl, dithianyl, dithiolanyl, pyrazolidinylimidazolinyl, pyrimidinonyl, 1,1-dioxo-thiomorpholinyl, 3- azabicyco[3.1.0]hexanyl, 3-azabicyclo[4. 1.0]heptanyl and azabicyclo[2.2.2]hexanyl.Substituted heterocycle also includes ring systems substituted with one or more oxo moieties, such as piperidinyl N-oxide, morpholinyl-N-oxide, 1 -oxo- 1 -thiomorpholinyl and 1, 1 -dioxo- 1- thiomorpholinyl.

[0066] Substituents are selected from: halogen, -R', -OR', =0, =NR', =N-OR’, -NR'R", -SR', - SiR'R"R"', -OC(O)R’, -C(O)R', -CO2R', -CONR'R", -OC(O)NR'R", -NR"C(O)R', -NR'- C(O)NR"R"', -NR’-SO2NR'", -NR 'CO2R', -NH-C(NH2)=NH, -NR'C(NH2)=NH, -NH- C(NH2)=NR’, -S(O)R’, -SO2R’, -SO2NR R", -NR"SO2R, -CN and -NO2, -N3, -CH(Ph)2, perfluoro(Cl-C4)alkoxy and perfluoro(Cl-C4)alkyl, in a number ranging from zero to three, with those groups having zero, one or two substituents being particularly preferred. R, R', R" and R'" each independently refer to hydrogen, unsubstituted (Cl-C8)alkyl and heteroalkyl, unsubstituted aryl, aryl substituted with one to three halogens, unsubstituted alkyl, alkoxy orthioalkoxy groups, or aryl-(Cl-C4)alkyl groups. When R' and R" are attached to the same nitrogen atom, they can be combined with the nitrogen atom to form a 5-, 6- or 7 -membered ring. Hence, -NR'R" includes 1-pyrrolidinyl and 4-morpholinyl, "alkyl” includes groups such as trihaloalkyl (e.g., -CF3 and -CH2CF3), and when the aryl group is 1,2,3,4-tetrahydronaphthalene, it may be substituted with a substituted or unsubstituted (C3-C7)spirocycloalkyl group. The (C3- C7)spirocycloalkyl group may be substituted in the same manner as defined herein for "cycloalkyl".

[0067] Preferred substituents are selected from: halogen, -R', -OR', =0. -NR’R", -SR', - SiR'R' R’ ", -OC(O)R’, -C(0)R’, -C02R', -CONR'R", -0C(0)NR'R", -NR"C(0)R’, -NR"CO2R’, - NR'-S02NR"R ", -S(O)R', -SO2R', -SO2NR’R", -NR"SO2R, -CN and -NO2, perfhioro(Cl- C4)alkoxy and perfluoro(Cl-C4)alkyl, where R' and R" are as defined above.

[0068] The term "fused ring" refers to a polycyclic ring system, e.g., a bicyclic or tricyclic ring system, in whcih two rings share only two ring atoms and one bond in common. Examples of fused rings may comprise a fused bicyclic cycloalkyl ring such as those having from 7 to 12 ring atoms arranged as a bicyclic ring selected from [4,4], [4,5], [5,5], [5,6] and [6,6] ring systems as mentioned above; a fused bicylclic aryl ring such as 7 to 12 membered bicyclic aryl ring systems as mentioned above, a fused tricyclic aryl ring such as 10 to 15 membered tricyclic aryl ring systems mentioned above; a fused bicyclic heteroaryl ring such as 8- to 12- membered bicyclic heteroaryl rings as mentioned above, a fused tricyclic heteroaryl ring such as 11- to 14- membered tricyclic heteroaryl rings as mentioned above; and a fused bicyclic or tricyclic heterocyclyl ring as mentioned above.

[0069] The compounds may contain an asymmetric center and may thus exist as enantiomers. Where the compounds possess two or more asymmetric centers, they may additionally exist as diastereomers. Enantiomers and diastereomers fall within the broader class of stereoisomers.All such possible stereoisomers as substantially pure resolved enantiomers, racemic mixtures thereof, as well as mixtures of diastereomers are intended to be included. All stereoisomers of the compounds and / or pharmaceutically acceptable salts thereof are intended to be included. Unless specifically mentioned otherwise, reference to one isomer applies to any of the possible isomers. Whenever the isomeric composition is unspecified, all possible isomers are included.

[0070] The compounds of the invention may also contain unnatural proportions of atomic isotopes at one or more of the atoms that constitute such compounds, such as deuterium, e.g. - CD3, CD2H or CDH2in place of methyl. For example, the compounds may be radiolabeled with radioactive isotopes, such as for example tritium (3H), iodine- 125 (125I) or carbon- 14 (14C). All isotopic variations of the compounds of the invention, whether radioactive or not, are intended to be encompassed within the scope of the invention.

[0071] Synthetic Methods and Characterization

[0072] All chemical reactions were carried out under a nitrogen atmosphere with dry solvents under anhydrous conditions, unless otherwise noted. Reagents were purchased at the highest commercial quality and used without further purification, unless otherwise stated. Room temperature is defined as between 21-25 °C. Reactions were stirred magnetically and monitored by thin layer chromatography (TLC) using TLC plates precoated with silica gel 60 F254 on aluminium (Merck KGaA). Detection was by UV (254 nm and 365 nm) or chemical stain (KMnO4, ninhydrin, iodine). Solvents were removed in vacuo using either a Buchi R-300 Rotavapor (equipped with an 1-300 Pro Interface, B-300 Base Heating Bath, Welch 2037B-01 DryFast pump, and VWR AD15R-40-V 1 IB Circulating Bath). Solvents for silica gel chromatography were used as supplied by Sigma- Aldrich. Automated flash chromatography was performed on a Biotage Isolera instrument, equipped with a UV detector. Chromatograms were recorded at 254 and 280 nm. If additional purification is needed, compound was further purified using Thermo Scientific’s semi-prep reversed phase high-performance liquid chromatography equipped (RP-HPLC: Ultimate 3000 HPLC) equipped with C18 column (Luna® 10 pm c 18(2), 100 A, Serial #:5293-0084). Elution of the sample was monitored using DIONEX UltiMate 3000. Eluting buffer A: 100% distilled water + 0.1% trifluoracetic acid (TFA). Eluting buffer B: 95% acetonitrile + 5% distilled water + 0.1% TFA. High-resolution mass spectra (HRMS) were obtained using Q Exactive™ Plus Hybrid Quadrupole-Orbitrap™ Mass Spectrometer.1H and13C Nuclear Magnetic Resonance (NMR) spectra were recorded on BRUKER AV (600 MHz and 700 MHz), AVB (400 MHz), AVQ (400 MHz) and NEO (500 MHz) spectrometers. Measurements were carried out at ambient temperature. Chemical shifts (5) are reported in ppm with the residual solvent signal as internal standard (chloroform at 7.26 and 77.2 ppm for1H NMR and13C NMR, respectively, methanol at 3.31 and 49.0, respectively and DMSO at 2.50 and 39.5, respectively). Multiplicity is reported as follows: singlet (s), doublet (d), doublet of doublet (dd) doublet of triplet (dt), triplet (t), triplet of doublet (td), quartet (q), and multiplet (m). Coupling constants (I) are reported in Hertz (Hz).13C NMR spectra were recorded with broadband 1 H decoupling.

[0073] General Procedures

[0074] Amide Couplings

[0075] General Procedure A

[0076] The corresponding carboxylic acid (1.0 equiv.) was added to a vessel and purged with N2 for 5 minutes. The acid was dissolved in N,N-dimethylformamide (DMF) (0.1 M) and N,N- diisopropylethylamine (DIPEA) (3 equiv.) was added. A >50% wt. solution of propylphosphonic anhydride (T3P) in EtOAc (1.5 equiv.) was added dropwise, and the reactionmixture was stirred at ambient temperature for 30 minutes. The corresponding amine (1.2 equiv.) was dissolved in DMF (0.1 M) then added dropwise and the reaction mixture was stirred at ambient temperature overnight. The reaction was quenched with 5 times the reaction volume of 5% LiCl(aq) and extracted 3 times with ethyl acetate (EtOAc). The organic extracts were washed once with brine, dried over Na2SC>4, vacuum filtered, and concentrated in vacuo. The resultant residue was purified by silica gel flash chromatography to afford the title compound.

[0077] General Procedure B

[0078] A mixture of the corresponding carboxylic acid (1.1 equiv.) and HATU (1.2 equiv.) was purged with N2for 5 minutes. The mixture was dissolved in DMF (0.1 M), DIPEA (3 equiv.) was added and the reaction mixture was allowed to stir at ambient temperature for 30 minutes. The corresponding amine (1 equiv.) was dissolved in DMF (0.1 M) then added dropwise and the reaction mixture was stirred at ambient temperature overnight. The reaction was quenched with 5 times the reaction volume of 5% LiCl(aq) and extracted 3 times with EtOAc. The organic extracts were dried over Na2SO4, vacuum filtered, and concentrated in vacuo. The resultant residue was purified by silica gel flash chromatography to afford the title compound.

[0079] Ter t-butyloxy carbonyl Deprotection

[0080] General Procedure C

[0081] The corresponding tert-butyloxycarbonyl protected amine (1 equiv.) was dissolved in DCM (0. 1 M). Trifluoroacetic acid (32 equiv.) was added, and the reaction mixture was stirred at ambient temperature for 30 minutes to overnight. The volatiles were removed in vacuo and the crude residue was used without further purification, unless otherwise noted.

[0082] Synthesis of Vinyl Sulfonamides

[0083] General Procedure D

[0084] The corresponding amine (1.0 equiv.) was dissolved in dichloromethane (DCM) (0.1 M) and triethylamine (3.0 equiv.) was added at 0 °C. 2-chloroethanesulfonyl chloride (0.8-1.2 equiv.) in DCM (0.1 M) was added dropwise and the resulting reaction mixture was stirred at ambient temperature overnight. The reaction was quenched with 5 times the reaction volume of water and extracted 3 times with DCM. The organic extracts were washed once with brine, dried over Na2SC>4, vacuum filtered, and concentrated in vacuo. The resultant residue was purified by silica gel flash chromatography to afford the title compound.

[0085] (S)-4-(2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][l,2,4]triazolo[4,3- a][l,4]diazepin-6-yl)acetamido)benzenesulfonyl fluoride (ML1-10)

[0087] General Procedure A was followed with (S)-2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H- thieno [3,2-f][l,2,4]triazolo[4,3-a][l,4]diazepin-6-yl)acetic acid (JQl-Acid) (50.0 mg, 0.12 mmol), T3P (0.1 mL, 0.19 mmol), DIPEA (0.07 mL, 0.37 mmol), and 4-aminobenzenesulfonyl fluoride (24.0 mg, 0.14 mmol). The crude residue was purified by silica gel chromatography (0- 7% MeOH in DCM) to afford 16.6 mg (24%) of the title compound as a yellow-white powder.

[0088] XH NMR (600 MHz, CDC13) 5 10.54 (s, 1H), 7.76 (d, J= 9.0 Hz, 2H), 7.72 (d, J = 9.0 Hz, 2H), 7.40 (d, J = 8.5 Hz, 2H), 7.31 (d, J = 8.8 Hz, 2H), 4.75 (dd, J = 9.7, 4.5 Hz, 1H), 3.99 (dd, J = 15.0, 9.7 Hz, 1H), 3.59 (dd, 7 = 15.0, 4.5 Hz, 1H), 2.72 (s, 3H), 2.44 (s, 2H), 1.71 (s, 2H).

[0089] X3C NMR (151 MHz, CDC13) 5 169.72, 164.43, 155.93, 150.35, 145.15, 137.14, 136.24, 131.89, 131.42, 131.03, 130.60, 129.90, 129.56, 128.78, 126.48, 126.32, 119.48, 54.24, 40.36, 14.42, 13.14, 11.86.

[0090] HRMS (ESI) m / z calcd for C25H21ClFN5NaO3S2+[M+Na]+: 580.0651; found: 580.0651

[0091] (S)-tert-butyl (4-(2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2- f][l,2,4]triazolo[4,3-a] [l,4]diazepin-6-yl)acetamido)phenyl) carbonate (ML1-13)

[0093] General Procedure A was followed with (S)-2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H- thieno [3,2-f][l,2,4]triazolo[4,3-a][l,4]diazepin-6-yl)acetic acid (JQl-Acid) (30.0 mg, 0.07 mmol), T3P (0.07 mL, 0.11 mmol), DIPEA (0.04 mL, 0.22 mmol), and 4-aminophenyl tert-butyl carbonate (17.0 mg, 0.08 mmol). The crude residue was purified by silica gel chromatography (0-7% MeOH in DCM) to afford 30.0 mg (68%) of the title compound as a yellow-white powder.

[0094] XH NMR (600 MHz, CDCI3) 8 9.36 (s, 1H), 7.57 (d, J = 9.0 Hz, 2H), 7.39 (d, J = 8.3 Hz, 2H), 7.30 (d, J = 8.8 Hz, 2H), 7.05 (d, J = 9.0 Hz, 2H), 4.68 (dd, J = 8.1, 5.9 Hz, 1H), 3.81 (dd, J = 14.4, 8.2 Hz, 1H), 3.58 (dd, 7 = 14.3, 5.9 Hz, 1H), 2.67 (s, 3H), 2.39 (s, 3H), 1.67 (s, 3H), 1.53 (s, 9H).

[0095] 13C NMR (151 MHz, CDC13) 6 168.91, 164.05, 155.79, 151.92, 149.99, 147.08, 136.86, 136.51, 136.00, 132.08, 130.99, 130.96, 130.52, 129.88, 128.72, 121.46, 120.78, 83.35, 54.59, 40.36, 27.71, 14.38, 13.09, 11.82.

[0096] HRMS (ESI) m / z calcd for C3oH3oClN5Na04S+[M+Na]+: 614. 1599; found:614. 1600

[0097] (S)-2,4-dichloro-5-(2-(4-(4-chIorophenyl)-2,3,9-trimethyl-6H-thieno[3,2- f][l,2,4]triazolo[4,3-a][l,4]diazepin-6-yl)acetamido)phenyl ethyl carbonate (ML1-14)

[0099] General Procedure A was followed with (S)-2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H- thieno [3,2-f][l,2,4]triazolo[4,3-a][l,4]diazepin-6-yl)acetic acid (JQl-Acid) (30.0 mg, 0.07 mmol), T3P (0.07 mL, 0.11 mmol), DIPEA (0.04 mL, 0.22 mmol), and 5-amino-2,4- dichlorophenyl ethyl carbonate (20.6 mg, 0.08 mmol). The crude residue was purified by silica gel chromatography (0-8% MeOH in DCM) to afford 5.0 mg (10.6%) of the title compound as a yellow-white powder.

[0100] 'H NMR (600 MHz, CDC13) 5 8.98 (s, 1H), 8.50 (s, 1H), 7.44 (s, 1H), 7.43 (d, 7 = 8.5 Hz, 2H), 7.34 (d, 7 = 8.8 Hz, 2H), 4.60 (t, 7 = 6.5 Hz, 1H), 4.33 (q, 7 = 7.2 Hz, 2H), 3.77 (dd, 7 = 14.6, 6.9 Hz, 1H), 3.58 (dd, 7 = 14.5, 6.1 Hz, 1H), 2.68 (s, 3H), 2.41 (d, 7 = 0.8 Hz, 3H), 1.71 - 1.68 (m, 3H), 1.38 (1, 7 = 7.1 Hz, 3H).

[0101] 13C NMR (151 MHZ, CDC13) 5 169.09, 164.38, 152.30, 150.09, 146.02, 137.08, 134.61, 132.26, 130.26, 129.94, 129.68, 128.76, 121.58, 120.49, 116.07, 65.52, 54.22, 40.80, 14.43,14.16, 13.11, 11.84.

[0102] HRMS (ESI) m / z calcd for C28H24Cl3N5NaO4S+[M+Na]+: 654.0507; found: 654.0512

[0103] (S)-2-(4-(4-chlorophenyI)-2,3,9-trimethyI-6H-thieno[3,2-f][l,2,4]triazolo[4,3- a][l,4]diazepin-6-yl)-N-(2,4-dioxo-l,4-dihydro-2H-benzo[d][l,3]oxazin-7-yl)acetamide

[0105] General Procedure A was followed with (S)-2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H- thieno [3,2-f][l,2,4]triazolo[4,3-a][l,4]diazepin-6-yl)acetic acid (JQl-Acid) (30.0 mg, 0.07 mmol), T3P (0.07 mL, 0.11 mmol), DIPEA (0.04 mL, 0.22 mmol), and 7-amino-2H-benzo[d][l,3]oxazine-2,4 (IH)-dione (14.7 mg, 0.08 mmol). The crude residue was purified by silica gel chromatography (0-8% MeOH in DCM) to afford 8.6 mg (20.5%) of the title compound as a yellow-white powder.

[0106] XH NMR (600 MHz, CDC13) 6 11.23 (s, 1H), 10.42 (s, 1H), 7.47 (s, 1H), 7.38 (d, J = 8.2 Hz, 2H), 7.21 (d, J = 8.2 Hz, 2H), 5.03 - 5.00 (m, 1H), 4.24 - 4.21 (m, 1H), 3.76 - 3.73 (m, 1H), 2.72 (s, 3H), 2.38 (s, 3H), 1.72 (s, 3H).

[0107] 13C NMR (151 MHz, CDC13) 5 164.37, 156.26, 155.10, 153.41, 150.58, 136.64, 136.56, 131.85, 131.03, 130.95, 130.43, 130.00, 129.78, 129.04, 128.53, 115.66, 54.18, 40.37, 14.38, 13.11, 11.93.

[0108] HRMS (ESI) m / z calcd for C27H21ClN6NaO4S+[M+Na]+: 583.0926; found: 583.0934

[0109] (S)-2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][l,2,4]triazolo[4,3- a][l,4]diazepin-6-yl)-N-(2-methoxy-3,4-dioxocyclobut-l-en-l-yl)acetamide (ML1-16)

[0111] General Procedure A was followed with (S)-2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H- thieno [3,2-f][l,2,4]triazolo[4,3-a][l,4]diazepin-6-yl)acetic acid (JQl-Acid) (30.0 mg, 0.07 mmol), T3P (0.07 mL, 0.11 mmol), DIPEA (0.04 mL, 0.22 mmol), and 3-amino-4- methoxycyclobut-3-ene- 1,2-dione (10.5 mg, 0.08 mmol). The crude residue was purified by silica gel chromatography (0-7% MeOH in DCM) to afford 8.0 mg (21.0%) of the title compound as a yellow-white powder.

[0112] 'H NMR (600 MHz, CDC13) 5 10.97 (s, 1H), 7.42 (d, J= 8.3 Hz, 2H), 7.35 (d, J = 8.8 Hz, 2H), 4.65 (t, 7 = 6.5 Hz, 1H), 4.50 (s, 3H), 3.81 (dd, 7 = 8.0, 6.5 Hz, 2H), 2.72 (s, 3H), 2.42 (s, 3H), 1.70 (s, 3H).

[0113] 13C NMR (151 MHZ, CDC13) 5 168.01 , 150.62, 150.36, 136.05, 135.32, 132.58, 132.13, 132.12, 131.30, 131.03, 129.94, 128.87, 61.21, 53.67, 39.03, 14.47, 13.13, 11.73.

[0114] HRMS (ESI) m / z calcd for C24H2oClN5Na04S+[M+Na]+: 532.0817; found: 532.0819

[0115] (S)-2-(4-(4-chlorophenyI)-2,3,9-trimethyI-6H-thieno[3,2-f][l,2,4]triazolo[4,3- a][l,4]diazepin-6-yl)-N-(6-formylpyridin-3-yl)acetamide (ML1-17)

[0117] General Procedure A was followed with (S)-2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H- thieno [3,2-f][l,2,4]triazolo[4,3-a][l,4]diazepin-6-yl)acetic acid (JQl-Acid) (30.0 mg, 0.07 mmol), T3P (0.07 mL, 0.11 mmol), DIPEA (0.04 mL, 0.22 mmol), and 5-aminopicolinaldehyde (10.5 mg, 0.08 mmol). The crude residue was purified by silica gel chromatography (0-7% MeOH in DCM) to afford 4.0 mg (10.5%) of the title compound as a yellow-white powder. NMR (600 MHz, CDC13) 5 10.45 (s, 1 H), 9.91 (s, 1H), 8.75 (d, J = 2.4 Hz, 1H), 8.22 (dd, J = 8.5, 2.4 Hz, 1H), 7.78 (d, J = 8.5 Hz, 1H), 7.41 (d, J = 8.4 Hz, 2H), 7.32 (d, J = 8.7 Hz, 2H), 4.76 (dd, 9.2, 4.6 Hz, 1H), 3.97 (dd, 7 = 14.9, 9.3 Hz, 1H), 3.63 (dd, 7 = 14.9, 4.7 Hz, 1H), 2.72 (s, 3H), 2.44 (s, 3H), 1.71 (s, 3H).

[0119] 13C NMR (151 MHZ, CDC13) 6 192.15, 169.82, 164.47, 155.82, 150.34, 147.97, 141.08, 139.10, 137.17, 136.22, 131.94, 131.43, 131.02, 130.56, 129.89, 128.81, 126.17, 122.50, 54.27, 40.19, 14.43, 13.16, 11.88.

[0120] HRMS (ESI) m / z calcd for C25H2]ClN6NaO2S+[M+Na]+: 527. 1027; found: 527.1034

[0121] (S)-2-(4-(4-chlorophenyI)-2,3,9-trimethyI-6H-thieno[3,2-f][l,2,4]triazolo[4,3- a][l,4]diazepin-6-yl)-N-(6-formylpyridin-2-yl)acetamide (ML1-25)

[0123] General Procedure A was followed with (S)-2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H- thieno [3,2-f][l,2,4]triazolo[4,3-a][l,4]diazepin-6-yl)acetic acid (JQl-Acid) (25.0 mg, 0.06 mmol), T3P (0.06 mL, 0.09 mmol), DIPEA (0.03 mL, 0.19 mmol), and 6-aminopicolinaldehyde (9.1 mg, 0.07 mmol). The crude residue was purified by silica gel chromatography (0-7% MeOH in DCM) to afford 6.0 mg (19.0%) of the title compound as a yellow-white powder.

[0124] 'H NMR (600 MHz, CDC13) 5 9.96 (s, 1H), 9.42 (s, 1H), 8.46 (d, 7 = 8.3 Hz, 1H), 7.87 (t, 7 = 7.9 Hz, 1H), 7.69 (d, 7 = 7.4 Hz, 1H), 7.52 (d, 7 = 8.1 Hz, 2H), 7.36 (d, 7 = 8.7 Hz, 2H), 4.66 (t, 7 = 6.8 Hz, 1H), 3.70 (d, 7 = 6.9 Hz, 2H), 2.69 (s, 3H), 2.41 (s, 3H), 1.69 (s, 3H).

[0125] 13C NMR (151 MHz, CDC13) 8 192.68, 169.71, 164.55, 155.29, 151.79, 150.99, 150.14, 139.25, 137.16, 136.39, 132.32, 131.06, 130.98, 130.29, 129.98, 128.82, 118.66, 117.83, 54.13, 41.03, 40.35, 14.47, 13.12, 11.88.

[0126] HRMS (ESI) m / z calcd for C25H21ClN6NaO2S+[M+Na]+: 527.1027; found: 527.1024

[0127] tert-butyl (S)-4-(2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2- f][l,2,4]triazolo[4,3-a] [l,4]diazepin-6-yl)acetyl)piperazine-l-carboxylate (ML1-26)

[0129] General Procedure A was followed with (S)-2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H- thieno [3,2-f][l,2,4]triazolo[4,3-a][l,4]diazepin-6-yl)acetic acid (JQl-Acid) (322.0 mg, 0.80 mmol), T3P (0.53 mL, 0.88 mmol), DIPEA (0.56 mL, 3.21 mmol), and 1 -boc-piperazine (179.5 mg, 0.96 mmol). The crude residue was purified by silica gel chromatography (0-5% MeOH in DCM) to afford 338.9 mg (74.1%) of the title compound as a yellow oil.

[0130] XH NMR (600 MHz, CDC13) 5 7.41 (d, J = 8.6 Hz, 2H), 7.34 (d, J = 8.7 Hz, 2H), 4.84 - 4.79 (m, 1H), 3.84 (ddd, 7 = 13.3, 6.3, 3.5 Hz, 1H), 3.75 (dd, 7 = 15.9, 6.2 Hz, 2H), 3.72 - 3.65 (m, 1H), 3.58 (ddp, 7 = 15.4, 7.6, 4.0 Hz, 4H), 3.52 (s, 1H), 3.41 (ddd, 7 = 13.2, 7.5, 3.6 Hz, 1H), 2.68 (s, 3H), 2.41 (d, 7 = 0.9 Hz, 3H), 1.69 (s, 3H), 1.50 (s, 9H).

[0131] X3C NMR (151 MHz, CDC13) 5 169.22, 163.71, 155.82, 154.61, 149.83, 136.82, 136.67, 132.24, 130.91, 130.65, 130.52, 129.80, 128.69, 80.25, 54.47, 45.74, 41.70, 35.35, 28.41, 14.35, 13.06, 11.83.

[0132] HRMS (ESI) m / z calcd for C28H33ClN6NaO3S+[M+Na]+: 591.1916; found: 691.1922

[0133] (S)-4-(4-(2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][l,2,4]triazolo[4,3- a][l,4]diazepin-6-yl)acetyl)piperazine-l-carbonyl)-2-hydroxybenzaldehyde (ML1-29)

[0135] General Procedure C was followed with ML1-26 (35.0 mg, 0.06 mmol), and TFA(0.15 mL, 1.97 mmol) for 2 hours. The crude material was used without further purification.

[0136] General Procedure A was followed with 4-formyl-3 -hydroxybenzoic acid (12.8 mg, 0.08 mmol), T3P (0.06 mL, 0.10 mmol), DIPEA (0.06 mL, 0.32 mmol), and the amine from above (30.0 mg, 0.06 mmol). The crude residue was purified by silica gel chromatography (0-7% MeOH in DCM) to afford 10.0 mg (25.3%) of the title compound as a yellow-white powder.

[0137] XH NMR (600 MHz, CDC13) 8 11.12 (s, 1H), 9.95 (s, 1H), 7.67 (d, 7 = 7.8 Hz, 1H), 7.39 (d, 7 = 8.2 Hz, 2H), 7.33 (d, 7 = 8.7 Hz, 2H), 7.06 (d, 7 = 6.4 Hz, OH), 7.02 (s, 1H), 4.80 (t, 7 = 6.8 Hz, 1H), 4.02 - 3.97 (m, 2H), 3.91 - 3.84 (m, 1H), 3.81 - 3.78 (m, 2H), 3.63 - 3.56 (m, 2H), 3.51 - 3.45 (m, 1H), 3.44 - 3.39 (m, 2H), 2.67 (s, 3H), 2.40 (s, 3H), 1.67 (s, 1H).

[0138] 13C NMR (151 MHZ, CDC13) 5 196.18, 168.70, 163.96, 161.66, 149.97, 143.44, 136.81, 136.70, 134.37, 132.20, 130.94, 130.45, 129.79, 128.77, 121.11, 118.20, 116.10, 54.36, 46.34, 41.96, 14.42, 13.14, 11.88.

[0139] HRMS (ESI) m / z calcd for C3iH29ClN6NaO4S+[M+Na]+: 639. 1552; found: 639.1562

[0140] (S)-5-(4-(2-(4-(4-chlorophenyI)-2,3,9-trimethyI-6H-thieno[3,2- / ][l,2,4]triazolo[4,3- o][l, 4]diazepin-6-yl)acetyl)piperazine-l -carbonyl)-! 7 / -pyrrole-3-sulfonyl fluoride (ML1-30)

[0142] General Procedure C was followed with ML1-26 (35.0 mg, 0.06 mmol), and TFA (0.15 mL, 1.97 mmol) for 2 hours. The crude material was used without further purification.

[0143] General Procedure A was followed with 4-(fluorosulfonyl)-lH-pyrrole-2-carboxylic acid (14.8 mg, 0.08 mmol), T3P (0.06 mL, 0.10 mmol), DIPEA (0.06 mL, 0.32 mmol), and the amine from above (30.0 mg, 0.06 mmol). The crude residue was purified by silica gel chromatography (0-7% MeOH in DCM) to afford 24.8 mg (60.2%) of the title compound as a yellow- white powder.

[0144] 'H NMR (600 MHz, CDC13) 5 11.71 (s, 1H), 7.66 (s, 1H), 7.40 (d, J = 8.3 Hz, 2H), 7.33 (d, 7 = 8.3 Hz, 2H), 6.93 (d, 7 = 1.5 Hz, 1H), 4.82 (t, 7 = 6.9 Hz, 1H), 4.10 - 4.01 (m, 2H), 3.96 (tt, 7 = 15.2, 6.8 Hz, 3H), 3.89 - 3.75 (m, 3H), 3.67 - 3.60 (m, 1H), 3.52 (dd, 7 = 16.3, 6.4 Hz, 1H), 2.67 (s, 3H), 2.40 (s, 3H), 1.68 (s, 3H).

[0145] 13C NMR (151 MHz, CDC13) 5 169.57, 164.00, 160.46, 155.72, 149.99, 136.82, 136.71, 132.16, 130.94, 130.90, 130.54, 129.81, 128.75, 127.05, 126.70, 116.16, 115.96, 111.68, 54.57, 45.44, 41.37, 35.36, 31.92, 22.68, 14.36, 13.09, 11.81.

[0146] HRMS (ESI) m / z calcd for C28H27ClFN7NaO4S2+[M+Na]+: 666.1131; found: 666.1127

[0147] (S)-4-(4-(2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2- / ][l,2,4]triazolo[4,3- a][l,4]diazepin-6-yl)acetyl)piperazine-l-carbonyl)benzenesulfonyl fluoride (ML1-31)

[0149] General Procedure C was followed with ML1-26 (35.0 mg, 0.06 mmol), and TFA(0.15 mL, 1.97 mmol) for 2 hours. The crude material was used without further purification.

[0150] General Procedure A was followed with 4-(fluorosulfonyl)benzoic acid (15.7 mg, 0.08 mmol), T3P (0.06 mL, 0.10 mmol), DIPEA (0.06 mL, 0.32 mmol), and the amine from above (30.0 mg, 0.06 mmol). The crude residue was purified by silica gel chromatography (0-7% MeOH in DCM) to afford 22.7 mg (54.2%) of the title compound as a yellow-white powder.

[0151] 'H NMR (600 MHz, CDC13) 5 8.10 (d, 7 = 8.2 Hz, 2H), 7.68 (d, 7 = 8.0 Hz, 2H), 7.39 (d, 7 = 8.2 Hz, 2H), 7.33 (d, 7 = 8.6 Hz, 2H), 4.80 (dd, 7 = 7.4, 6.4 Hz, 1 H), 4.09 - 4.00 (m, 3H), 3.88 - 3.81 (m, 2H), 3.74 - 3.30 (m, 6H), 2.67 (s, 3H), 2.40 (s, 3H), 1.67 (s, 3H).

[0152] 13C NMR (151 MHz, CDC13) 8 155.77, 150.10, 142.56, 136.96, 136.83, 134.54, 134.37, 132.31, 131.06, 131.00, 130.61, 129.91, 129.12, 128.88, 128.54, 128.46, 32.04, 30.44, 29.47, 22.81, 14.48, 13.21, 11.92.

[0153] HRMS (ESI) m / z calcd for C30H28ClFN6NaO4S2+[M+Na]+: 677.1178; found: 677.1175

[0154] (S)-3-(4-(2-(4-(4-chlorophenyI)-2,3,9-trimethyI-6H-thieno[3,2- / ][l,2,4]triazolo[4,3- a][l,4]diazepin-6-yl)acetyl)piperazine-l-carbonyl)benzenesulfonyI fluoride (ML1-32)

[0156] General Procedure C was followed with ML1-26 (35.0 mg, 0.06 mmol), and TFA(0.15 mL, 1.97 mmol) for 2 hours. The crude material was used without further purification.

[0157] General Procedure A was followed with 3-(fluorosulfonyl)benzoic acid (15.7 mg, 0.08 mmol), T3P (0.06 mL, 0.10 mmol), DIPEA (0.06 mL, 0.32 mmol), and the amine from above(30.0 mg, 0.06 mmol). The crude residue was purified by silica gel chromatography (0-7% MeOH in DCM) to afford 10.0 mg (23.9%) of the title compound as a yellow-white powder.

[0158] 'H NMR (600 MHz, CDC13) 3 8.10 (dq, 7 = 3.3, 1.7 Hz, 2H), 7.85 (dt, 7 = 7.7, 1.4 Hz, 1H), 7.74 (t, 7 = 8.1 Hz, 1H), 7.40 (d, 7 = 8.3 Hz, 2H), 7.34 (d, 7 = 8.5 Hz, 2H), 4.82 (t, 7 = 6.8 Hz, 1H), 4.11 - 3.95 (m, 2H), 3.84 (dd, 7 = 15.9, 7.1 Hz, 2H), 3.78 - 3.30 (m, 5H), 2.69 (s, 3H), 2.40 (s, 3H), 1.68 (s, 3H).

[0159] 13C NMR (151 MHZ, CDC13) 6 169.41, 167.64, 164.04, 155.64, 150.02, 137.17, 136.89, 136.64, 134.05, 132.04, 130.99, 130.62, 130.25, 129.79, 129.72, 128.77, 127.31, 35.26, 31.92,30.32, 22.68, 14.36, 13.09, 11.75.

[0160] HRMS (ESI) m / z calcd for C30H28ClFN6NaO4S2+[M+Na]+: 677.1178; found: 677.1183

[0161] (S)-butyl (4-(4-(2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2- / ][l,2,4]triazolo[4,3-a][l, 4]diazepin-6-yl)acetyl)piperazine-l-carbonyI)phenyl) carbonate (ML1-33)7 = 8.6 Hz, 2H), 7.27 (d, 7 = 4.3 Hz, 2H), 4.80 (t, 7 = 6.8 Hz, 1H), 4.27 (t, 7 = 6.7 Hz, 2H), 3.93 (s, 2H), 3.80 (dd, 7 = 15.8, 6.9 Hz, 2H), 3.54 (s, 6H), 2.66 (s, 3H), 2.39 (s, 3H), 1.78 - 1.70 (m, 2H), 1.67 (s, 3H), 1.51 - 1.44 (m, 2H), 0.97 (t, 7 = 7.4 Hz, 3H).

[0166] 13C NMR (151 MHZ, CDC13) 8 169.74, 169.35, 163.87, 155.72, 153.30, 152.30, 149.89, 136.76, 136.75, 132.83, 132.23, 130.92, 130.74, 130.49, 129.79, 128.74, 128.72, 121.37, 68.97, 54.55, 35.30, 30.57, 29.68, 18.91, 14.35, 13.63, 13.07, 11.81.

[0167] HRMS (ESI) m / z calcd for C35H37ClN6NaO5S+[M+Na]+: 711.2127; found: 711.2125

[0168] (S)-2-(2-(4-(2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2- / ][l,2,4]triazolo[4,3-a][l,4]diazepin-6-yl)acetyI)piperazin-l-yl)-2-oxoethoxy)isoindoline-l,3- dione (ML1-43)

[0170] General Procedure C was followed with ML1-26 (35.0 mg, 0.06 mmol), and TFA(0. 15 mL, 1.97 mmol) for 2 hours. The crude material was used without further purification.

[0171] General Procedure B was followed with 2-((l,3-dioxoisoindolin-2-yl)oxy)acetic acid (15.6 mg, 0.07 mmol), HATU (29.3 mg, 0.08 mmol), DIPEA (0.05 mL, 0.26 mmol), and the amine from above (30.0 mg, 0.06 mmol). The crude residue was purified by silica gel chromatography (0-7% MeOH in DCM) to afford 5.0 mg (11.6%) of the title compound as a yellow-white powder.

[0172] 'H NMR (500 MHz, CDC13) 5 7.88 (dd, 7 = 5.4, 3.0 Hz, 2H), 7.76 (dd, 7 = 5.5, 3.1 Hz, 2H), 7.35 (d, 7 = 8.5 Hz, 2H), 7.26 (d, 7 = 8.7 Hz, 2H), 4.74 (dd, 7 = 7.2, 5.9 Hz, 1H), 4.69 (s, 2H), 3.74 (q, 7 = 5.3 Hz, 2H), 3.71 - 3.62 (m, 2H), 3.63 - 3.51 (m, 2H), 2.77 (ddd, 7 = 6.4, 4.0, 2.2 Hz, 2H), 2.70 - 2.65 (m, 2H), 2.63 (s, 3H), 2.37 (s, 3H), 1.64 (s, 3H).

[0173] 13C NMR (151 MHz, CDC13) 5 167.76, 162.97, 149.94, 136.76, 136.72, 134.76, 132.47, 132.06, 131.00, 130.92, 130.88, 130.64, 129.85, 128.85, 128.81 , 128.74, 123.81 , 77.24, 77.03,76.82, 68.17, 38.75, 30.37, 28.93, 23.76, 22.98, 14.39, 14.04, 13.10, 11.79, 10.96.

[0174] HRMS (ESI) m / z calcd for C33H30ClN7NaO5S+[M+Na]+: 694. 1610; found: 694.1589

[0175] (S)-4-((4-(2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2- / ][l,2,4]triazolo[4,3- a][l,4]diazepin-6-yl)acetyl)piperazin-l-yl)methyl)benzenesulfonyl fluoride (ML1-45)

[0177] General Procedure C was followed with ML1-26 (35.0 mg, 0.06 mmol), and TFA (0.15 mL, 1.97 mmol) for 2 hours. The crude material was used without further purification.

[0178] To a mixture of the above product (30.0 mg, 0.06 mmol) and 4- (bromomethyl)benzenesulfonyl fluoride (17.8 mg, 0.07 mmol) in DMF (ImL) was added DIPEA (0.06 mL, 0.35 mmol). The reaction was stirred at ambient temperature for 20 min and concentrated in vacuo before it was redissolved in EtOAc (20 mL) and saturated aqueous sodium bicarbonate (10 mL). The aqueous phase was extracted 3 times with EtOAc, and the organic extracts were washed with brine (10 mL), dried over Na2SO4, vacuum filtered, and concentrated in vacuo. The resultant residue was purified by silica gel chromatography (0-5% MeOH in DCM) to afford 19.9 mg (48.5 %) of the title compound as a yellow-white powder.

[0179] 'H NMR (600 MHz, CDC13) 5 7.98 (d, J = 8.4 Hz, 2H), 7.64 (d, J = 8.2 Hz, 2H), 7.40 (d, 7 = 8.5 Hz, 2H), 7.32 (d, 7 = 8.4 Hz, 2H), 4.83 - 4.77 (m, 1H), 3.90 - 3.84 (m, 1H), 3.80 (ddd, 7 = 13.2, 6.4, 3.3 Hz, 1H), 3.71 (dt, 7 = 15.8, 4.8 Hz, 2H), 3.66 (s, 2H), 3.59 (dtd, 7 = 19.9, 15.2, 9.3 Hz, 2H), 2.66 (s, 3H), 2.63 - 2.54 (m, 2H), 2.50 (ddd, 7 = 10.0, 6.4, 3.4 Hz, 1H), 2.43 (ddd, 7 = 11.1, 7.5, 3.3 Hz, 1H), 2.39 (s, 3H), 1.67 (s, 3H).

[0180] 13C NMR (151 MHz, CDC13) 5 168.99, 163.67, 155.89, 149.83, 147.10, 136.85, 136.66, 132.23, 131.90, 131.74, 130.92, 130.66, 130.56, 129.83, 129.80, 128.68, 128.60, 62.03, 54.44, 53.30, 52.86, 45.78, 41.81, 35.23, 14.35, 13.06, 11.83.

[0181] HRMS (ESI) m / z calcd for C3oH30ClFN6Na03S2+[M+Na]+: 663.1386; found: 663.1387

[0182] (S)-2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-i / ][ l,2,4]triazolo[4,3- a][l,4]diazepin-6-yl)-l-(4-(vinyIsulfonyl)piperazin-l-yl)ethan-l-one (ML1-50)

[0184] General Procedure C was followed with ML1-26 (200.0 mg, 0.35 mmol), and TFA (0.86 mL, 11.25 mmol) for 2 hours. The crude material was used without further purification.

[0185] General Procedure D was followed with the above product (164.8 mg, 0.06 mmol), triethylamine (0.25 mL, 1.76 mmol), and 2-chloroethanesulfonyl chloride (0.04 mL, 0.39 mmol). The crude residue was purified by silica gel chromatography (0-5% MeOH in DCM) to afford 103.5mg (52.7%) of the title compound as a yellow-white powder.

[0186] 'H NMR (600 MHz, CDC13) 6 7.38 (d, J = 8.5 Hz, 2H), 7.32 (d, J = 8.7 Hz, 2H), 6.44 (dd, J = 16.6, 10.0 Hz, 1H), 6.28 (d, J = 16.6 Hz, 1H), 6.09 (d, J = 10.0 Hz, 1H), 4.77 (t, J = 6.8 Hz, 1H), 4.03 - 3.94 (m, 2H), 3.79 - 3.71 (m, 2H), 3.59 - 3.46 (m, 2H), 3.38 (ddd, 7 = 11.8, 5.7, 3.2 Hz, 1H), 3.27 (dddt, J = 22.7, 11.6, 8.1, 3.4 Hz, 2H), 3.05 (ddd, 7 = 11.7, 8.2, 3.3 Hz, 1H), 2.65 (s, 3H), 2.39 (s, 3H), 1.67 (s, 3H).

[0187] 13C NMR (151 MHz, CDCI3) 6 177.74, 172.47, 164.27, 158.48, 145.34, 145.32, 140.79, 140.77, 139.50, 139.33, 139.04, 138.36, 138.06, 137.30, 85.81, 85.60, 85.38, 62.98, 54.25, 54.15, 53.99, 50.01, 43.86, 22.94, 21.66, 20.41.

[0188] HRMS (ESI) m / z calcd for C25H27ClN6NaO3S2+[M+Na]+: 581.1167; found: 581.1171

[0189] (S)-5-(4-(2-(4-(4-chlorophenyI)-2,3,9-trimethyI-6H-thieno[3,2- / ][ l,2,4]triazolo[4,3- a][l,4]diazepin-6-yl)acetyl)piperazine-l-carbonyl)picolinaldehyde (ML1-52)

[0191] General Procedure C was followed with ML1-26 (35.0 mg, 0.06 mmol), and TFA (0.15 mL, 1.97 mmol) for 2 hours. The crude material was used without further purification.

[0192] General Procedure B was followed with 6-formylnicotinic acid (10.2 mg, 0.07 mmol), HATU (28.1 mg, 0.07 mmol), DIPEA (0.04 mL, 0.25 mmol), and the amine from above (28.8 mg, 0.06 mmol). The crude residue was purified by silica gel chromatography (0-10% MeOH in DCM) to afford 15.0 mg (40.5%) of the title compound as a yellow- white powder.

[0193] 'H NMR (600 MHz, CDC13) 5 10.12 (s, 1H), 8.85 (s, 1H), 8.05 (d, 7 = 7.9 Hz, 1H), 7.95 (dd, 7 = 8.0, 2.0 Hz, 1H), 7.40 (d, 7 = 8.3 Hz, 2H), 7.33 (d, 2H), 4.80 (t, 7 = 6.9 Hz, 1H), 4.10 -4.01 (m, 2H), 3.88 - 3.75 (m, 2H), 3.76 - 3.64 (m, 1H), 3.62 - 3.34 (m, 1H), 2.66 (s, 3H), 2.40 (s, 3H), 1.68 (s, 3H).

[0194] 13C NMR (151 MHz, CDC13) 5 192.43, 171.12, 166.92, 153.35, 148.43, 136.82, 136.74, 136.16, 134.90, 132.26, 130.92, 130.44, 129.77, 128.75, 121.49, 60.38, 29.69, 21.03, 14.36,13.08, 11.83.

[0195] HRMS (ESI) m / z calcd for C30H2SaN7NaO3S+[M+Na]+: 624.1555; found: 624.1563

[0196] (S)-3-(4-(2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2- / ][l,2,4]triazolo[4,3- a][l,4]diazepin-6-yl)acetyl)piperazine-l-carbonyl)-4-hydroxybenzenesulfonyl fluoride

[0198] General Procedure C was followed with ML1-26 (30.0 mg, 0.05 mmol), and TFA (0. 13 mL, 1.69 mmol) for 2 hours. The crude material was used without further purification.

[0199] General Procedure A was followed with 5-(fluorosulfonyl)-2-hydroxybenzoic acid (13.9 mg, 0.06 mmol), T3P (0.04 mL, 0.06 mmol), DIPEA (0.05 mL, 0.26 mmol), and the amine from above (24.7 mg, 0.05 mmol). The crude residue was purified by silica gel chromatography (0-8% MeOH in DCM) to afford 5.5 mg (15.6%) of the title compound as a yellow-white powder.

[0200] 3H NMR (600 MHz, CDC13) 5 7.97 - 7.91 (m, 2H), 7.41 (d, J = 8.4 Hz, 2H), 7.34 (d, J = 8.7 Hz, 2H), 7.25 (d, 7 = 8.7 Hz, 1H), 4.82 (dd, 7= 7.7, 6.1 Hz, 1H), 4.09 - 4.04 (m, 2H), 3.97 - 3.89 (m, 2H), 3.87 (dd, 7 = 15.8, 7.9 Hz, 1H), 3.77 (dd, 7 = 13.0, 5.7 Hz, 2H), 3.68 - 3.63 (m, 1H), 3.61 - 3.53 (m, 1H), 3.45 (dd, 7 = 15.8, 6.1 Hz, 1H), 2.68 (s, 3H), 2.42 (s, 3H), 1.69 (s, 3H).

[0201] 13C NMR (151 MHZ, CDC13) 6 169.32, 168.37, 155.72, 149.97, 136.93, 136.59, 132.57, 132.02, 131.03, 130.98, 130.64, 129.87, 129.79, 129.59, 128.79, 119.25, 118.87, 54.58, 41.51,35.35, 14.37, 13.10, 11.81.

[0202] HRMS (ESI) m / z calcd for C30H28ClFN6NaO5S2+[M+Na]+: 693.1127; found: 693.1132

[0203] (S)-2,4-di-te / 7-butyl-5-(2-(4-(4-chIorophenyl)-2,3,9-trimethyl-6H-thieno[3,2- / ][l,2,4]triazolo[4,3-«][l,4]diazepin-6-yl)acetamido)phenyl methyl carbonate (ML1-55)

[0205] General Procedure A was followed with (S)-2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H- thieno [3,2-f][l,2,4]triazolo[4,3-a][l,4]diazepin-6-yl)acetic acid (JQl-Acid) (25.0 mg, 0.06 mmol), T3P (0.05 mL, 0.07 mmol), DIPEA (0.04 mL, 0.25 mmol), and 5-amino-2,4-di-tert- butylphenyl methyl carbonate (19.2 mg, 0.07 mmol). The crude residue was purified by silica gel chromatography (0-8% MeOH in DCM) to afford 5.8 mg (14.0%) of the title compound as a yellow-white powder.

[0206] 'H NMR (400 MHz, CDC13) 3 8.03 (s, 1H), 7.49 (s, 1H), 7.42 (s, 1H), 7.39 (d, J= 5.1 Hz, 2H), 7.32 (d, J = 8.5 Hz, 2H), 4.72 (t, J = 6.7 Hz, 1H), 3.87 (s, 3H), 3.71 (dd, J = 14.6, 6.2 Hz, 1H), 3.59 (dd, 7 = 14.6, 7.2 Hz, 1H), 2.68 (s, 3H), 2.41 (s, 3H), 1.68 (s, 3H), 1.46 (s, 9H), 1.34 (s, 9H).

[0207] 13C NMR (151 MHz, CDC13) 6 168.68, 164.01, 155.70, 154.24, 149.99, 147.53, 139.50, 137.85, 136.82, 136.56, 133.56, 132.14, 131.00, 130.85, 130.53, 129.93, 128.68, 125.25, 121.85, 55.31, 54.23, 40.46, 34.78, 34.72, 30.79, 30.22, 14.40, 13.09, 11.81.

[0208] HRMS (ESI) m / z calcd for C35H4oClN5Na04S+[M+Na]+: 684.2382; found: 684.2385

[0209] 7-cyclopentyl- / V, / V-dimethyl-2-((5-(4-(vinylsulfonyl)piperazin-l-yl)pyridin-2- yl)amino)-7 / / -pyrrolo[2,3-d]pyrimidine-6-carboxamide (ML1-71)

[0211] General Procedure D was followed with 7-cyclopentyl-N,N-dimethyl-2-((5-(piperazin- 1-yl) pyridin-2-yl)amino)-7H-pyrrolo[2,3-d]pyrimidine-6-carboxamide (ribociclib) (50.0 mg, 0.12 mmol), triethylamine (0.05 mL, 0.35 mmol), and 2-chloroethanesulfonyl chloride (0.01 mL, 0.13 mmol). The crude residue was purified by silica gel chromatography (0-8% MeOH in DCM) to afford 21.7 mg (36.0%) of the title compound as a yellow-white powder.

[0212] XH NMR (600 MHz, CDCI3) 8 8.69 (s, 1H), 8.38 (d, J = 8.3 Hz, 1H), 8.00 (d, J = 2.3 Hz, 1H), 7.87 (s, 1H), 7.32 (dd, J = 9.1, 2.9 Hz, 1H), 6.48 (dd, J = 16.6, 10.0 Hz, 1H), 6.44 (s, 1H), 6.30 (d, J = 16.6 Hz, 1H), 6.10 (d, J = 10.0 Hz, 1H), 4.80 (p, J = 8.9 Hz, 1H), 3.38 - 3.34 (m, 4H), 3.25 - 3.20 (m, 5H), 2.58 (qd, J = 10.1, 5.0 Hz, 2H), 2.12 - 2.00 (m, 5H), 1.75 - 1.68 (m, 2H).

[0213] 13C NMR (151 MHz, CDCI3) 6 164.06, 154.47, 151.96, 151.77, 147.78, 141.85, 132.32, 132.18, 129.16, 128.34, 127.52, 112.80, 112.37, 100.94, 57.90, 50.22, 45.57, 30.20, 24.73.

[0214] HRMS (ESI) m / z calcd for C25H33N8O3S+[M+Na]+: 525.2391; found: 525.2382

[0215] 2-(6-amino-5-(4-(vinylsulfonyl)piperazin-l-yl)pyridazin-3-yl)phenol (ML1-96)

[0217] General Procedure D was followed with 2-(6-amino-5-(piperazin-l-yl)pyridazin-3- yl)phenol (50.0 mg, 0.18 mmol), triethylamine (0.08 mL, 0.55 mmol), and 2- chloroethanesulfonyl chloride (0.02 mL, 0.15 mmol). The crude residue was purified by silica gel chromatography (0-8% MeOH in DCM) and another round of purification using HPLC (buffer B : buffer A = 25:75 to 60:40) to afford 12.4 mg (18.6%) of the title compound as a yellow-white powder.

[0218] 1H NMR (600 MHz, CDC13) 3 7.61 (dd, J = 8.0, 1.6 Hz, 1H), 7.38 (s, 1H), 7.33 (ddd, J = 8.5, 7.3, 1.6 Hz, 1H), 7.09 (dd, J = 8.2, 1.2 Hz, 1H), 6.99 - 6.92 (m, 1H), 6.52 (dd, 7 = 16.6, 9.9 Hz, 1H), 6.37 (d, J = 16.6 Hz, 1H), 6. 16 (d, J = 9.9 Hz, 1H), 4.81 (s, 2H), 3.43 (t, J = 4.9 Hz, 4H), 3.28 (t, J = 4.9 Hz, 4H).

[0219] 13C NMR (151 MHz, CDC13) 8 159.23, 155.52, 153.88, 140.25, 132.32, 131.13, 129.58, 125.18, 118.82, 118.62, 117.35, 111.46, 48.91, 45.44.

[0220] HRMS (ESI) m / z calcd for Ci6H2oN503S+[M+H]+: 362.1281; found: 362.1291

[0221] ( / ?)-3-(4-phenoxyphenyl)-l-(l-(vinylsuIfonyl)piperidin-3-yl)-LH-pyrazolo[3,4- d]pyrimidin-4-amine (TH1-9)

[0223] General Procedure C was followed with tert-butyl (R)-3-(4-amino-3-(4- phenoxyphenyl)-lH- pyrazolo[3,4-d]pyrimidin-l-yl)piperidine-l -carboxylate (lOO.Omg, 0.21mmol), and TFA (0.5 mL, 6.58 mmol) overnight. The crude material was used without further purification.

[0224] General Procedure D was followed with the above product (50.0mg, 0.13mmol), triethylamine (0.08 mL, 0.58 mmol), and 2-chloroethanesulfonyl chloride (0.02 mL, 0.19 mmol).The crude residue was purified by silica gel chromatography (0-10% MeOH in DCM) to afford 18.2 mg (29.5%) of the title compound as a yellow-white powder.

[0225] 'H NMR (600 MHz, CDC13) 3 8.37 (s, 1H), 7.62 (d, J = 8.6 Hz, 2H), 7.38 (dd, J = 8.6,7.4 Hz, 2H), 7.17 (t, 1H), 7.15 (d, 2H), 7.07 (d, J = 7.6 Hz, 1H), 6.46 (dd, J = 16.6, 10.0 Hz, 1H), 6.24 (d, J = 16.6 Hz, 1H), 6.02 (d, 7= 10.0 Hz, 1H), 5.50 (s, 2H), 5.05 - 4.97 (m, 1H), 4.01 -3.94 (m, 1H), 3.81 (dt, J = 12.2, 2.3 Hz, 1H), 3.28 (t, J = 11.2 Hz, 1H), 2.72 (td, J = 12.2, 2.9 Hz, 1H), 2.24 - 2.18 (m, 2H), 2.02 - 1.95 (m, 1H), 1.92 - 1.83 (m, 1H).

[0226] 13C NMR (151 MHz, CDC13) 5 158.64, 157.76, 156.32, 156.00, 154.38, 144.04, 132.98, 129.99, 129.96, 128.57, 127.65, 124.11, 119.58, 119.13, 98.62, 52.89, 49.19, 45.52, 29.57, 24.37.

[0227] HRMS (ESI) m / z calcd for C24H24N6NaO3S+[M+Na]+: 499.1523; found: 499.1523

[0228] / V-(2-chloro-6-methyIphenyl)-2-((2-methyl-6-(4-(vinylsulfonyI)piperazin-l- yl)pyrimidin-4-yl)amino)thiazole-5-carboxamide (ML2-5)

[0230] General Procedure D was followed with N-(2-chloro-6-methylphenyl)-2-((2-methyl-6- (piperazin- l-yl)pyrimidin-4-yl)amino)thiazole-5-carboxamide (100.0 mg, 0.23 mmol), triethylamine (0.09 mL, 0.68 mmol), and 2-chloroethanesulfonyl chloride (0.02 mL, 0.23 mmol). The crude residue was purified by silica gel chromatography (50-100% EtOAc in Hexanes) to afford 35.6 mg (29.6%) of the title compound as a yellow-white powder.

[0231] XH NMR (600 MHz, DMSO) 6 11.52 (s, 1H), 9.87 (s, 1H), 8.23 (s, 1H), 7.40 (dd, J = 7.8, 1.7 Hz, 1H), 7.28 (dt, 7 = 15.4, 7.5 Hz, 2H), 6.83 (dd, 7= 16.5, 10.0 Hz, 1H), 6.20 (d, 7 = 10.0 Hz, 1H), 6.16 (d, 7 = 16.5 Hz, 1H), 6.11 (s, 1H), 3.66 (t, 7 = 5.1 Hz, 4H), 3.31 (s, 3H), 3.14 (t, 7 = 5.1 Hz, 4H), 2.43 (s, 3H), 2.25 (s, 3H).

[0232] X3C NMR (151 MHZ, DMSO) 5 170.21, 165.22, 162.37, 162.08, 159.78, 157.01, 140.71, 138.71, 133.41, 132.42, 132.33, 129.64, 128.92, 128.07, 126.90, 125.73, 82.99, 59.63, 44.75, 43.02, 25.44, 20.65, 18.18, 13.98.

[0233] HRMS (ESI) m / z calcd for C22H24ClN7NaO3S2+[M+Na]+: 556.0963; found: 556.0966

[0234] tert-butyl 4-(6-(((lr,4r)-4-(3-chloro-4- cyanophenoxy)cyclohexyl)carbamoyl)pyridazin-3-yl) piperazine- 1-carboxylate (ML2-8)

[0236] To 6-chloro-N-((lr,4r)-4-(3-chloro-4-cyanophenoxy)cyclohexyl)pyridazine-3- carboxamide (200.0 mg, 0.51 mmol) in DMF (4 mL) was added 1 -hoc -piperazine (142.8 mg, 0.77 mmol), followed by triethylamine (0.21 mL, 1.53 mmol). The resultant mixture was stirred at 80 °C overnight. It was then cooled to RT and the precipitate was filtered off and dried to afford 156.6mg (56.6%) of the title compound as a white powder.

[0237] XH NMR (400 MHz, CDC13) 5 8.01 (d, 7 = 9.5 Hz, 1H), 7.84 (d, 7 = 8.5 Hz, 1H), 7.54 (d, 7 = 8.6 Hz, 1H), 7.01 - 6.94 (m, 2H), 6.84 (dd, 7 = 8.8, 2.5 Hz, 1H), 4.31 (tt, 7 = 9.9, 3.6 Hz,1H), 4.04 (dtt, 7 = 10.8, 7.4, 3.9 Hz, 1H), 3.78 - 3.71 (m, 4H), 3.62 - 3.54 (m, 4H), 2.16 (tt, 7 = 10.8, 4.2 Hz, 5H), 1.81 - 1.59 (m, 2H), 1.48 (s, 9H), 1.45 - 1.31 (m, 2H).

[0238] 13C NMR (151 MHz, CDCI3) 5 162.60, 161.63, 160.20, 154.64, 144.83, 138.35, 135.08, 126.98, 116.94, 116.40, 114.66, 112.28, 104.86, 80.44, 75.68, 47.12, 30.03, 29.67, 28.41.

[0239] HRMS (ESI) m / z calcd for C27H33ClN6NaO4+[M+Na]+: 563.2144; found: 563.2146

[0240] N-((lr,4r)-4-(3-chloro-4-cyanophenoxy)cycIohexyl)-6-(4-(vinylsulfonyl)piperazin-l- yl)pyridazine-3-carboxamide (ML2-9)

[0242] General Procedure C was followed with ML2-8 (70.0 mg, 0.13 mmol), and TFA (0.32 mL, 4. 14 mmol) for 2 hours. The crude material was used without further purification.

[0243] General Procedure D was followed with the above product (57.1 mg, 0.13 mmol), triethylamine (0.08 mL, 0.58 mmol), and 2-chloroethanesulfonyl chloride (0.02 mL, 0.16 mmol). The crude residue was purified by silica gel chromatography (0-100% EtOAc in Hexanes) to afford 27.4mg (39.9%) of the title compound as a yellow-white powder.

[0244] 'H NMR (600 MHz, CDC13) 5 8.05 (d, J = 9.4 Hz, 1H), 7.84 (d, J = 8.2 Hz, 1H), 7.55 (d, J = 8.7 Hz, 1H), 7.02 (s, 1H), 7.02 - 6.97 (m, 1H), 6.85 (dd, J = 8.7, 2.4 Hz, 1H), 6.43 (dd, J = 16.6, 9.9 Hz, 1H), 6.30 (d, 7 = 16.6 Hz, 1H), 6.09 (d, 7 = 9.9 Hz, 1H), 4.32 (td, 7 = 10.3, 5.2 Hz, 1H), 4.06 (did, 7 = 10.8, 7.4, 4.0 Hz, 1H), 3.89 (t, 7 = 5.1 Hz, 4H), 3.32 (t, 7 = 5.1 Hz, 4H), 2.23 - 2.13 (m, 4H), 1.74 - 1.66 (m, 2H), 1.52 - 1.42 (m, 2H).

[0245] 13C NMR (151 MHZ, CDCI3) 5 162.38, 161.60, 159.99, 145.30, 138.34, 135.09, 132.10, 129.68, 127.20, 116.91, 116.40, 114.66, 1 12.65, 104.84, 75.62, 47.18, 45.04, 44.65, 30.02, 29.70, 29.66.

[0246] HRMS (ESI) m / z calcd for Ci^ClNeNaCLS* [M+Na]+: 553. 1395; found: 553.1387

[0247]

[0248] tert-butyl 4-(prop-2-yn-l-yl)piperazine-l -carboxylate (ML2-32)Boe

[0249]

[0250] A combination of 1-boc -piperazine (1.50 g, 8.05 mmol) and potassium carbonate (1.67 g, 12.08 mmol) was suspended in acetonitrile (30 mL) and stirred at ambient temperature for 10 minutes. Propargyl bromide (0.84 mL, 8.86 mmol) was added, and the reaction mixture was stirred at 60°C for 2 hours. The reaction mixture was concentrated in vacuo, and the cruderesidue was purified by silica gel chromatography (0-10% MeOH in EtOAc) to afford 1.50 g (82.9%) of the title compound as a yellow oil.

[0251] 'H NMR (600 MHz, CDC13) 5 3.38 (t, J = 5.1 Hz, 4H), 3.22 (d, J = 2.5 Hz, 2H), 2.42 (t, J = 5.1 Hz, 4H), 2.19 (t, J = 2.4 Hz, 1H), 1.37 (s, 9H).

[0252] 13C NMR (151 MHz, CDCI3) 6 154.55, 79.54, 78.35, 73.41, 51.54, 46.90, 28.35.

[0253]

[0254] l-(prop-2-yn-l-yl)-4-(vinylsulfonyl)piperazine (ML2-33)

[0256] General Procedure C was followed with ML2-32 (500.0 mg, 2.23 mmol), and TFA (5.5 mL, 73.3 mmol) for 2 hours. The crude material was used without further purification.

[0257] General Procedure D was followed with the above product (28 mg, 0.23 mmol), triethylamine (0.14 mL, 1.01 mmol), and 2-chloroethanesulfonyl chloride (0.03 mL, 0.27 mmol). The reaction mixture was concentrated in vacuo, and the crude residue was purified by silica gel chromatography (0-10% MeOH in EtOAc) to afford 13.0 mg (26.9%) of the title compound as a yellow oil.

[0258] 'H NMR (600 MHz, CDC13) 5 6.42 (dd, J = 16.6, 10.0 Hz, 1H), 6.24 (d, J = 16.6 Hz, 1H), 6.04 (d, J = 10.0 Hz, 1H), 3.33 (d, J = 2.4 Hz, 2H), 3.22 (t, J = 5.0 Hz, 4H), 2.68 - 2.63 (m, 4H), 2.28 (t, J = 2.5 Hz, 1H).

[0259] 13C NMR (151 MHZ, CDCI3) 6 132.32, 128.81, 77.98, 73.72, 51.10, 46.68, 45.45.

[0260] Examples

[0261] Abstract

[0262] In this example we sought to identify a transplantable and linker-less covalent handle that could be appended onto the exit vector of various protein-targeting ligands to induce the degradation of their respective targets. Using the BET family inhibitor JQ1 as a testbed, we synthesized and screened a series of covalent JQ1 analogs and identified a vinylsulfonyl piperazine handle that led to the potent and selective degradation of BRD4 in cells. Through chemoproteomic profiling, we identified DCAF16 as the E3 ligase responsible for BRD4 degradation — an E3 ligase substrate receptor that has been previously covalently targeted for molecular glue-based degradation of BRD4. Interestingly, we demonstrated that this covalent handle can be transplanted across a diverse array of protein- targeting ligands spanning many different protein classes to induce the degradation of CDK4, the androgen receptor, BTK, SMARCA2 / 4, and BCR-ABL / c-ABL. Our study reveals a DCAF16-based covalent degradativeand linker- less chemical handle that can be attached to protein-targeting ligands to induce the degradation of several different classes of protein targets.

[0263] Results

[0264] Identifying Covalent Handles that Enable the Degradation of BRD4

[0265] To identify covalent chemical handles that could convert non-degradative inhibitors into molecular glue or monovalent degraders of their targets, we used the BET family inhibitor JQ1 as a testbed to generate a series of covalent JQ1 analogs bearing various electrophilic handles that could react with cysteines, lysines, or other nucleophilic amino acids on E3 ligases to degrade BRD4 (Fig. 1A). Among the 18 derivatives tested, we only identified one compound, ML 1-50, that led to the loss of both the long and short isoforms of BRD4 in HEK293T cells (Figs. 1B-1C; Fig. 2A). ML 1-50 reduced BRD4 levels in a dose-dependent manner with preferential degradation of the short BRD4 isoform with nanomolar potency in HEK293T cells (Figs. 2B-2C). We observed hook effects with degradation of the long BRD4 isoform. ML 1-50 only showed modest cell viability impairments in HEK293T cells at the highest concentration of 10 uM tested (Fig. 7A). This BRD4 degradation was attenuated by pre -treatment with either proteasome inhibitor or NEDD8-activating enzyme inhibitor MLN4924 (Figs. 2D-2G). We had previously observed preference for degradation of the long versus short isoforms of BRD4 with covalent PROTACs that appeared be specific to HEK293T cells compared to other cell lines23,24Similarly, we found that ML 1-50 potently degraded both the long and short BRD4 isoforms in the MDA-MB-231 breast cancer cell line, with no hook effects observed (Fig. 2H, Fig. 7B). Quantitative proteomic profiling of ML 1-50 in MDA-MB-231 cells showed relatively selective BRD4 degradation with only 11 other proteins that were significantly reduced in levels by greater than 2-fold (Fig. 21).

[0266] Mapping the E3 Ligase Responsible for BRD4 Degradation

[0267] We next sought to identify the E3 ligase responsible for the BRD4 degradation observed with ML 1-50. We synthesized an alkyne-functionalized probe based on the vinylsulfonyl piperazine handle, ML 2-33 (Fig. 3A). Given the simplicity of this handle without a more elaborated ligand attached, we surmised that the handle would likely be more promiscuous compared to ML 1-50. We thus performed a competitive pulldown chemoproteomic experiment from ML 2-33-treated cells searching for proteins that were significantly outcompeted by ML 1- 50 pre-treatment. Among these outcompeted targets, there was only one E3 ligase that was part of the Cullin E3 ligase family that could be regulated by NEDD8 — DCAF16 (Fig. 3B). Using competitive activity-based protein profiling (ABPP), we showed significant engagement of Cl 19 of DCAF16 in cells by ~28 % (Fig. 7C). This modest degree of E3 ligase engagement is consistent with previous reports with covalent PROTACs showing that only a small fraction ofthe E3 ligase needs to be engaged to enable degradation of target proteins12 13 17. Site of modification analysis by liquid chromatography-mass spectrometry (LC-MS / MS) on tryptic digests of pure DCAF16 labeled with ML 1-50 also showed a single labeled site on Cl 19 (Fig. 7D).

[0268] To further confirm direct interaction of ML 1-50 with DCAF16, we showed that ML 1- 50 displaced cysteine-reactive probe labeling of pure DCAF16 protein by gel-based ABPP approaches without causing any precipitation of the protein (Fig. 3C). Consistent with the role of DCAF16 in our observed effects, we showed that the BRD4 degradation from ML 1-50 treatment was significantly attenuated in DCAF16 knockout cells compared to wild-type cells (Figs. 3D-3F).

[0269] Given that several previous studies have identified DCAF16 as the E3 ligase substrate receptor responsible for the degradation of covalent BRD4 degraders bearing various different types of electrophilic handles ' , we next determined whether our previously discovered covalent monovalent BRD4 degrader, JP-2-197, bearing a but-2-ene, 1,4-dione “fumarate” covalent degrader handle that targets RNF126 instead acts through DCAF1625. We showed that the BRD4 degradation observed by JP-2-197 was not attenuated at all in DCAF16 knockout cells (Figs. 4A-4B). In contrast, we observed complete and significant mitigation of BRD4 degradation in RNF126 knockout cells (Figs. 4C-4D). Our data thus indicate that different electrophilic degraders against the same target can act through distinct E3 ligases.

[0270]

[0271] Exploring the Applicability of the Covalent Handle Against Other Target Proteins

[0272] While we identified a covalent handle that can convert the non-degradative JQ1 into a degrader of BRD4, BRD4 is one of the easiest proteins to degrade and thus demonstrating proof- of-concept of a covalent handle that can degrade BRD4 does not speak to the broader applicability of this handle for other targets. Furthermore, previous studies have already demonstrated covalent and noncovalent BRD4 molecular glue degraders that act through DCAF16, through strengthening already existing weak interactions between DCAF16 and BRD48’9,26’28. Previous studies have also used covalent DCAF16 recruiters to generate heterobifunctional PROTACs against other targets beyond BRD4n. Whether a DCAF16- targeting covalent handle could be broadly transplanted across other protein-targeting ligands to generate linker-less monovalent degraders of neo-substrate proteins beyond BRD4 is unknown. We first appended the vinylsulfonyl piperazine handle onto the clinically approved CDK4 / 6 inhibitor ribociclib to generate ML 1-71 (Fig. 5A). ML 1-71 significantly degraded CDK4 in cells in a dose-dependent manner, albeit less potently compared to ML 1-50 and BRD4 (Figs.5B-5C). Despite the modest potency of this degrader, we still observed significant attenuation ofCDK4 degradation in DC AF 16 knockout cells (Figs. 5D-5E). Quantitative proteomic profiling of ML 1-71 in C33A cervical cancer cells also demonstrated relatively selective CDK4 degradation with only 16 other targets reduced in levels that may arise from transcriptional effects downstream of CDK4 inhibition or off-target effects (Fig. 5F). We next generated a monovalent degrader for SMARCA2 / 4 bearing the vinylsulfonyl piperazine handle — ML 1-96 (Fig. 8A)29. ML 1 -96 dose-responsively and significantly degraded both SMARCA2 and SMARCA4 in MV-4-11 leukemia cancer cells (Figs. 8B-8C). This SMARCA2 / 4 loss was once again attenuated in DCAF16 knockout cells (Figs. 8D-8E).

[0273] To further explore the substrate scope of our covalent degradative handle, we next generated an androgen receptor (AR) monovalent degrader consisting of the AR-targeting ligand from the AR PROTAC ARV-110 and the vinylsulfonyl piperazine handle — ML 2-9 (Fig. 6A)30ML 2-9 significantly degraded AR in LNCaP prostate cancer cells (Fig. 6B-6C). Interestingly, a hook effect was observed with this degrader. Proteomic data showed selective AR degradation with only 7 other proteins reduced in levels (Fig. 6D). We also generated a vinylsulfonyl derivative of the BTK inhibitor ibrutinib, TH 1-9, and showed that this compound also degrades BTK in dose-dependent manner in MINO lymphoma cancer cells (Figs. 6E-6G). Proteomic analyses revealed a higher number of proteins beyond BTK that were lowered in levels compared to the other degraders (Fig. 6H). This may be due to other off-targets of ibrutinib that are also being degraded or downstream transcriptional changes resulting from BTK inhibition and degradation. We also made a vinylsulfonyl piperazine bearing derivative of the BCR-ABL and c-ABL kinase inhibitor dasatinib, ML 2-5, and demonstrated the degradation of both the fusion oncogene and the parent kinase in K562 leukemia cancer cells (Figs. 9A-9C). This degradative covalent handle thus enabled the degradation of not only BRD4, but also several other proteins, including CDK4, SMARCA2 / 4, AR, BTK, and BCR-ABL / c-ABL.

[0274] Methods

[0275] Bortezomib or MLN4924 Rescue Studies

[0276] 2E6 of HEK293T cells per 3 mL of media were plated in 6-cm plates and left overnight to adhere. Cells were pretreated for 1 h with either Bortezomib (Cayman, C835F70) or MLN4924 (Tocris Bioscience, 649910) at a final concentration of 1 pM. Cells were then treated with ML 1-50 until desired time point. Cells from both the supernatant and on the plate were harvested and assessed via western blot.

[0277] Cell Viability Assay

[0278] HEK293T cells were seeded at a density of 20,000 / well (100 pL) in 96-well white plates overnight. Cells were then treated with DMSO vehicle control or ML1-50 and incubated at 37 °C for 24 h. Cell viability assay was performed using CellTiter-Glo® 2.0 reagent (Promega,G9241) according to manufacturer’s protocol. Luminescent signals were measured using the Tecan Spark Plate reader (30086376).

[0279] Isotopic desthiobiotin (isoDTB)-ABPP Cysteine Chemoproteomic Profiling of ML1- 50

[0280] HEK293T cells were treated with either ML1-50 (10 pM) or DMSO for 2 h before cell collection and lysis. The proteome concentrations were determined using BCA assay and adjusted to 2 mg / mL. For each biological replicate, 2 aliquots of 1 mb of 2 mg / mL were used (i.e. 4 mg per condition). Each aliquot was treated with 20 pL of lA-alkyne (26.6 mg / mL in DMSO, 200 pM final concentration) for 1 h at RT. Two master mixes of the click reagents were prepared in the meanwhile, each containing 510 pL TBTA (0.9 mg / mL in 4: 1 tBuOH / DMSO), 165 pL CuSO4 (12.5 mg / mL in H2O), 165 pL TCEP (14.0 mg / mL in H2O) and 160 pL of either heavy or light isoDTB tags (4 mg in DMSO, Click Chemistry Tools, 1565). The samples were then treated with 120 pL of the heavy (DMSO treated) or light (compound treated) master mix for 1 h at RT. After incubation, one light and one heavy labeled samples were combined and acetone-precipitated overnight at -20 °C. The samples were then centrifuged at 3,500 rpm for 10 min, acetone was removed, and the protein pellets resuspended in cold MeOH by sonication. The samples were centrifuged at 3,500 rpm for 10 min and MeOH was removed (repeated 3x in total). The pellets were dissolved in 600 pL urea (8 M in 0. 1 M TEAB) by sonication and the urea concentration was then adjusted to 2 M by adding 1800 pL of TEAB (0. 1 M). Two tubes containing solubilized proteins were combined, further diluted with 2400 pL 0.2% NP40 in PBS, and bound to high-capacity streptavidin agarose beads (200 pL / sample, ThermoFisher, 20357) for 1 h at RT with mixing. The beads were then centrifuged for 1 min at 1 ,000 g, the supernatant was removed, and the beads were washed 3 times with 0.1% NP40 in PBS, 3 times with PBS and 3 times with H2O. The samples were then resuspended in 8 M urea (600 pL in 0.1 M TEAB) and treated with DTT (30 pL, 31 mg / mL in H2O) for 45 min at 37 °C. They were then reacted with iodoacetamide (30 pL, 74 mg / mL in H2O) for 30 min at RT, followed by DTT (30 pL, 31 mg / mL in H2O) for 30 min at RT. The samples were diluted with 1800 pL TEAB (0.1 M), centrifuged for 1 min at 1 ,000 g, and the supernatant was removed. The beads were resuspended in 400 pL urea (2M in 0.1 M TEAB), and trypsin (8 pL, 0.5 mg / mL) was added and incubated for 20 h at 37 °C. The samples were then diluted with 800 pL 0.1% NP40 in PBS and the beads were washed 3 times with 0.1% NP40 in PBS, 3 times with PBS, and 3 times with H2O.Peptides were then eluted with 0.1% formic acid in 50% acetonitrile (3 x 400 pL). The samples were then dried using a vacuum concentrator at 30 °C, resuspended in 300 pL 0. 1% TFA in H2O, and fractionated using high pH reversed-phase peptide fractionation kits (ThermoFisher, 84868) according to the manufacturer’s protocol.

[0281] ML1-50-Competed Targets from ML2-33 Probe Pulldown Proteomics

[0282] HEK293T cells were harvested, lysed, and the proteome concentration was adjusted to 5 mg / mL in 500 pL of PBS using the BCA assay. HEK293T cell lysate were pre-treated with DMSO vehicle or ML1-50 (200 pM) for 1 h at room temperature prior to ML2-33 probe labeling (20 pM) at room temperature for 1 h. To each tube containing cell lysate, the following reagents were added: 10 pL of 10 mM biotin picolyl azide (Sigma Aldrich, 900912) in DMSO, 10 pL of 50 mM TCEP in H2O, 10 pL of 50 mM CuSO4in H2O, and 30 pL of TBTA ligand (1.7 mM in 1:4 DMSO / tBuOH, Cayman Chemical, 18816). The reaction mixture was incubated at room temperature for 60 minutes, and the reaction was quenched by protein precipitation. Precipitated pellets were washed using 500 pL of MeOH and centrifuged again to yield white pellets. Samples were resuspended in 1.2% SDS-PBS (1 mL), completely dissolved, and heated to 90 °C for 5 minutes. The soluble proteome was then diluted with 5 mL of PBS and further incubated with high-capacity streptavidin-agarose beads (100 pL / sample, ThermoFisher Scientific, 20357). Beads and lysates were incubated overnight at 4 °C with rotation. On the following day, beads were suspended and washed three times with 0.1 % SDS-PBS, PBS, and H2O. Washed beads were resuspended in 6 M Urea / PBS (500 pL), and the samples were further treated with DTT and iodoacetamide. After removing the supernatant, beads were resuspended in 100 pL of 50 mM TEAB and enzymatically digested overnight using sequencing-grade trypsin (Promega, V51 11). Digested peptides were eluted through centrifugation and labeled using commercially available TMTsixplex tags (ThermoFisher, P / N 90061). After labeling, 35 pg of each labeled sample was combined and dried using a vacufuge. Dried samples were redissolved with 300 pL of 0.1% TFA in H2O and further fractionated using high-pH re versed- phase peptide fractionation kits (ThermoFisher, P / N 84868) following the manufacturer’s protocol. Dried fractions were then resuspended in 25 pL of 0.1 % Formic acid / H2O (w / v) to be analyzed by LC-MS / MS.

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Claims

CLAIMS1. A compound comprising a vinylsulfonyl piperazine covalent handle for the rational design of monovalent degraders that act through targeting a cysteine within DCAF16 to enable the degradation of a target protein, of structure:

2. The compound of claim 1, wherein the vinylsulfonyl piperazine covalent handle is attached to a protein-targeting ligand, R, as of structure:

3. The compound of claim 2, wherein the target protein is selected from BRD4, CDK4 / 6, SMARCA2 / 4, AR, BTK, and BCR-ABL / c-ABL.

4. The compound of claim 2, wherein the protein-targeting ligand is selected from:BET bromodomain inhibitors ,CDK4 / 6 inhibitors,AR inhibitors,SMARCA2 inhibitors, andBCR-ABL / c-ABL inhibitors.

5. The compound of claim 2, wherein the protein-targeting ligand is selected from:BET bromodomain inhibitors, that is JQ1 (ML 1-50),CDK4 / 6 inhibitors that is ribociclib (ML 1-71),AR inhibitors, that is derived from the ARV- 110 PROTAC (ML 2-9),SMARCA2 inhibitors, that is ML 1-96, andBCR-ABL / c-ABL inhibitors, that is dasatinib derivative, that is ML 2-5.

6. A compound comprising a vinylsulfonyl piperidine covalent handle that acts through targeting a cysteine within DCAF16 to enable the degradation of a target protein, attached to a proteintargeting ligand, R, as of structure:

7. The compound of claim 2, 3, 4, 5 or 6, wherein the protein- targeting ligand is a vinylsulfonyl derivative of a vinyl ketone protein-targeting ligand.

8. The compound of claim 2, 3, 4, 5 or 6, wherein the protein- targeting ligand is a vinylsulfonyl derivative of a vinyl ketone protein-targeting ligand, wherein the derivative effects the degradation of the target protein, whereas the ligand does not.

9. A compound comprising a vinylsulfonyl derivative of the BTK inhibitor ibrutinib (TH 1-9).

10. A pharmaceutical composition comprising a compound of claim 2, 3, 4, 5, 6 or 9, or a pharmaceutically acceptable salt, a hydrate or a stereoisomer thereof, and a pharmaceutically acceptable carrier or excipient, preferably in a pharmaceutically acceptable unit dosage.

11. A method of using a compound of claim 2, 3, 4, 5, 6 or 9 to treat a disease or physiological condition comprising administering to a person in need thereof a compound herein.

12. A method of using a compound of claim 2, 3, 4, 5 6 or 9, further comprising the antecedent step of detecting or diagnosing a disease or condition indicating the need thereof, and the subsequent step of detecting a resultant improvement or delay of progression of the disease or condition.

13. Use of a compound of claim 2, 3, 4, 5, 6 or 9 in the manufacture of a medicament for treating a disease or condition.

14. A compound of claim 2, 3, 4, 5, 6 or 9 for use in treating a disease or condition.

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