BIFUNCTIONAL DEGRADERS COMPRISING ELECTROPHILIC PROTACS

THAT ENGAGE DCAF1 AND PHARMACEUTICAL COMPOSITIONS

COMPRISING THE SAME

CROSS REFERENCE TO RELATED APPLICATION

[0001] This application claims priority to U.S. provisional patent application No. 63/343,583, which was filed on May 19, 2022, and which is hereby incorporated by reference in its entirety.

GOVERNMENT SUPPORT

[0002] This invention was made with government support under grant numbers CA231881 and CA248715 awarded by the National Institute of Health. The government has certain rights in the invention.

FIELD OF INVENTION

[0003] This invention relates to bifunctional degraders comprising electrophilic PROTACs that engage DCAF1.

BACKGROUND OF THE DISCLOSURE

[0004] Most small molecules affect the functions of proteins through binding-mediated agonism or antagonism. Targeted protein degradation has recently emerged as a distinct type of pharmacology wherein small molecules induce the formation of complexes between a substrate protein and a component of the ubiquitin-proteasome system (typically, an E3 ligase) to promote the physical turnover of the substrate protein ¹ ' ³ . Targeted protein degradation is often enacted by compounds termed PROTACs (proteolysis-targeting chimeras), which are heterobifunctional molecules that contain two independent recognition units - one that binds the substrate protein and the other that binds an E3 ligase - connected by a linker to form a ternary complex that brings the substrate protein into proximity of the E3 ligase, resulting in substrate ubiquitination and proteasome-mediated degradation ^{2, 4} . PROTACs have the potential to address gaps in chemical probe and drug development by, for instance, providing a means to i) eliminate multidomain or multifunctional proteins for which small-molecule antagonism proves insufficient to block the full scope of protein activities, and ii) convert silent ligand-protein interactions into functional (degradation) outcomes ² . The

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SUBSTITUTE SHEET (RULE 26) successful design of PROTACs depends on identifying ligands for E3 ligases. Even though human cells express hundreds of E3 ligases, small-molecule ligands have been discovered so far for only a small number of these proteins ^{2, 8} , and most PROTACs utilize one of two E3 ligases - cereblon (CRBN) or VHL ^{2, 9} ' ¹⁰ . Recent work indicates that CRBN and VHL display distinct and restricted substrate specificities for executing targeted protein degradation ¹¹ ' ¹⁴ [0005] There is therefore a need to identify ligands for additional E3 ligases for realizing the full potential of targeted protein degradation.

SUMMARY OF THE DISCLOSURE

[0006] Some embodiments described herein provide a bifunctional degrader of Formula

(I)

A-B-C

Formula (I) wherein, A is a ligand to a protein of interest, B is a linker that is a bond or a molecular linker that is chemically linked to A and C, and C is a ligand to the E3 ligase substrate receptor DCAF1, wherein: the protein of interest is any protein having a ligand that can form a covalent bond with the linker B; and

C comprises an azetidinyl acrylamide that forms a covalent bond with Cl 113 of DCAF1 through a Michael addition reaction.

[0007] Some embodiments described herein also provide a DCAF1 protein-probe adduct, wherein the probe binds to cysteine residue Cl 113 of DCAF1, and wherein the probe comprises an azetidinyl acrylamide moiety.

[0008] Some embodiments described herein also provide a compound of Formula (I)

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SUBSTITUTE SHEET (RULE 26) or a pharmaceutically acceptable salt thereof, wherein:

BRIEF DESCRIPTION OF THE FIGURES

[0009] Figure 1 shows the chemical proteomic discovery of a stereo- and site-selective covalent ligand for DCAF1. (A) Structures of a set of stereochemically defined azetidine acrylamides MY-1A (1), MY-1B (2), MY-3A (3), and MY-3B (4). (B) Heat map showing cysteines that were substantially engaged by azetidine acrylamides (> 75% by at least one compound) in human T cells (20 pM compound, 3 h) as determined by MS-ABPP using an iodoacetamide desthiobiotin (IA-DTB) probe following previously described methods ²¹ . Red arrow marks DCAF1 C1113. (C) MS-ABPP quantification of IA-DTB labeling of DCAF1 C1113 from T cells treated with the indicated azetidine acrylamides (20 pM, 3 h) relative to control T cells treated with DMSO. (D) MS-ABPP quantification of IA-DTB labeling of the indicated cysteines in DCAF1 from T cells treated with MY-1B (20 pM, 3 h) relative to control T cells treated with DMSO. For (B-D), data represent average values (for C and D, average values ± SEM) from three independent experiments each performed with two technical replicates, where cysteines were required to have been quantified in at least two experiments for interpretation.

[0010] Figure 2 that azetidine acrylamides stereo- and site-selectively engage recombinant DCAF1 C1113. (A) Domain map of DCAF1 with the region used for recombinant protein studies marked in a bracket (a.a.’s 1046-1396) and Cl 113 highlighted in red. (B) Crystal structure of a DCAFl-Vpx complex (pdb: 5AJA). DCAF1 (a.a.’s 1046-1396) is shown in cyan, Vpx is shown in yellow, and Cl 113, which is located at the DCAFl-Vpx interface is shown in red. (C) Structures of alkyne-modified azetidine acrylamides MY-11 A (5) and MY-1 IB (6). (D) Gel-ABPP showing the concentration-dependent reactivity of MY- 11 A and MY-1 IB (1 h) reactivity with recombinant, purified DCAF1-WT or the DCAF1- C1113A mutant (0.06 pg/pL of DCAF1 protein per sample) doped into HEK293T cell proteome (1 pg/uL). MY-11A/B labeling of proteins was visualized by CuAAC conjugation

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SUBSTITUTE SHEET (RULE 26) to an azide-rhodamine tag followed by SDS-PAGE and in-gel fluorescence scanning. Results are from a single experiment representative of two independent experiments. (E) Gel-ABPP showing the concentration-dependent effects of MY-1 A and MY-1B on MY-1 IB reactivity with DCAF1-WT. Samples were treated with MY-1A/B for 2 h, followed by MY-1 IB (25 pM, 1 h) and analyzed as described in (D). Left, quantification of data presented as mean values ± SEM from two independent experiments. Right, representative gel-ABPP results. (D, E) Lower images are Coomassie blue-stained gels corresponding to ABPP gels shown in upper images.

[0011] Figure 3 shows that azetidine acrylamide-based heterobifunctional compounds stereoselectively engage DCAF1. (A) Structures of candidate electrophilic PROTACs YT41R (7), YT41S (8), YT47R (9), and YT47S (10). (B) Gel-ABPP showing the concentrationdependent effects of YT41R and YT47R on MY-1 IB reactivity with DCAF1-WT protein (0.06 pg/pL of DCAF1 protein per sample) doped into HEK293T cell proteome (1 pg/uL). Also shown are the effects of YT41S and YT47S tested at a single concentration (100 pM). Samples were treated with heterobifunctional compounds for 2 h, followed by MY-1 IB (25 pM, 1 h) and analyzed as described in Figure 2D. Left, representative gel-ABPP results. Lower image is Coomassie blue-stained gel corresponding to ABPP gel shown in upper image. Right, quantification of data presented as mean values ± SEM from two independent experiments.

[0012] Figure 4 shows that DCAF1 -directed electrophilic PROTACs promote FKBP12 degradation in a stereo- and site-selective manner. (A) Left, western blots showing concentration-dependent effects of YT47R (upper) and YT41R (lower) on FKBP12 abundance in HEK293T cells expressing HA-FBKP12 with or without co-expression of FLAG-DCAF1. Right, quantification of HA-FKBP12 abundance in the indicated experimental groups. (B) Left, western blots showing effects of YT47R versus YT47S (upper) and YT41R versus YT41S (lower) on FKBP12 abundance in HEK293T cells coexpressing HA-FBKP12 and FLAG-DCAF1. Right, quantification of HA-FKBP12 abundance in the indicated experimental groups. (C) Western blots showing effects of YT47R (left blots) and YT41R (right blots) on FKBP12 abundance in HEK293T cells coexpressing HA-FBKP12 and either FLAG-DCAF1-WT or FLAG-DCAF1-C1113A mutant. Quantification of HA-FKBP12 abundance in the indicated experimental groups is shown in the adjacent bar graphs. For (A-C), FKBP12 abundance was determined at 24 h posttreatment with compounds, and data are mean values ± SEM for three independent

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SUBSTITUTE SHEET (RULE 26) experiments. Statistical significance was calculated with unpaired two-tailed Student’s t-tests comparing YT41R/YT47R- and DMSO-treated cells. ***P < 0.001, ****p < 0.0001.

[0013] Figure 5 shows that FKBP12 degradation induced by DCAF1 -directed electrophilic PROTACs is dependent on the ubiquitin-proteasome system. (A) Left, western blots showing effects of proteasome (MG132) and Neddylation (MLN4924) inhibitors (1 h pre-treatment) on YT47R- and YT41R-dep endent degradation of FKBP12 in HEK293T cells co-expressing HA-FBKP12 and FLAG-DCAF1. FKBP12 abundance was determined at 24 h post-treatment with YT47R or YT41R. Right, quantification of HA-FKBP12 abundance in the indicated experimental groups. (B) Left, western blots showing effects of the FKBP12 ligand SLF (20 pM) or DCAF1 ligand MY-1 IB (1 h pre-treatment with 20 pM SLF or 5 pM MY-1 IB) on YT47R- and YT41R-dependent degradation of FKBP12 in HEK293T cells coexpressing HA-FBKP12 and FLAG-DCAF1. FKBP12 abundance was determined at 24 h post-treatment with YT47R or YT41R. Right, quantification of HA-FKBP12 abundance in the indicated experimental groups. (C) Left, western blots showing effects of MY-11 A or MY-1 IB (5 pM, 1 h pre-treatment) on YT47R- and YT41R-dependent degradation of FKBP12 in HEK293T cells co-expressing HA-FBKP12 and FLAG-DCAF1. FKBP12 abundance was determined at 24 h post-treatment with YT47R or YT41R. Right, quantification of HA-FKBP12 abundance in the indicated experimental groups. For (A-C), data are mean values ± SEM for three-five independent experiments. Statistical significance was calculated with unpaired two-tailed Student’s t-tests comparing YT41R/YT47R- and DMSO-treated cells. ****p < 0.0001.

[0014] Figure 6 shows the evidence for ternary complex formation and ubiquitination of HA-FKBP12 by DCAFl-directed electrophilic PROTACs. (A) Left, western blots showing effects of YT47R and YT47S on the immunoprecipitation (IP) of FLAG-DCAF1-WT or FLAG-DCAF1-C1113A with HA-FKBP12 in HEK293T cells coexpressing these proteins. Cells were treated with YT47R or YT47S (5 pM) for 2 h in the presence of MG132 (10 pM) prior to lysis and IP with an anti-HA antibody and western blotting for the indicated proteins (DCAF1, FKBP12, and ubiquitinated protein). Right, bar graphs quantifying DCAF1 (top) and ubiquitination (Ub) (bottom) in the indicated IP groups. (B) Left, western blots showing effects of YT47R and YT47S on the IP of endogenous DCAF1 with HA-FKBP12 in HEK293T cells expressing HA-FKBP12. Cells were treated with YT47R or YT47S (5 pM) for 2 h in the presence of MG132 (10 pM) prior to lysis and IP with an anti-HA antibody and western blotting for the indicated proteins (DCAF1,

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SUBSTITUTE SHEET (RULE 26) FKBP12). Right, bar graph quantifying DCAF1 in the indicated IP groups. For (A, B), data are mean values ± SEM for three independent experiments. Statistical significance was calculated with unpaired two-tailed Student’s t-tests comparing YT41R/YT47R- and DMSO- treated cells. **P < 0.01, ****p < 0.0001. (C) MS-ABPP quantification of IA-DTB labeling of the indicated cysteines in DCAF1 from FLAG-DCAFl-WT-expressing HEK293T cells treated with the indicated concentrations of YT47R (3 h) relative to cells treated with DMSO. Data represent average values ± SEM from four independent experiments. Statistical significance was calculated with unpaired two-tailed Student’s t-tests comparing compound to DMSO treated cells. *P < 0.1.

[0015] Figure SI. A computational model recapitulates the stereoselective engagement of DCAF1 C1113 by MY-1B. (A) Predicted binding pose of MY-1B. Apical (left) and lateral (right) views are shown, with the latter being clipped for clarity.

DCAF1 C1113 is shown in yellow and a dashed line connects Cl 113 sulfhydryl and acrylamide terminal carbon to illustrate a simplified nucleophile trajectory. (B) Diagram illustrating predicted interactions between MY-1B and DCAF1 residues located within 4 A of the ligand. Green contour: hydrophobic residues, blue contour: polar residues; green lines: TI- n stacking interactions, brown arrow: halogen bond. (C) Predicted binding energies of MY- 1B and its stereoisomers (Schrodinger Prime MM-GBSA, best of 3 poses) and respective distances between Cl 113 sulfhydryl and acrylamide terminal carbon.

[0016] Figure S2. A computational model supports conjugation strategies of MY-1B core scaffold. (A) Conjugation was considered at the phenyl 4-position (solid arrow) and the acrylamide position (dashed arrow). The former was prioritized to avoid affecting the cysteine-reactivity of the parent compound. (B) Constrained docking poses of MY-1B- derived conjugate S.10 (apical view, 3 poses shown). (C) Left: Diagram illustrating predicted interactions between MY-1B conjugate S.10 and DCAF1 residues located within 4 A of the ligand. Green contour: hydrophobic residues, blue contour: polar residues; green lines: TT-TC stacking interactions, brown arrow: halogen bond; grey shading denotes solvent exposure. Right: corresponding docking pose.

[0017] Figure S3. Concentration-dependent degradation of FKBP12 by DCAF1- directed electrophilic PROTACs. Top, western blots showing concentration-dependent effects of YT47R and YT41R on FKBP12 abundance in HEK293T cells co-expressing HA- FBKP12 and FLAG-DCAF1. Data are mean values ± SEM for four independent experiments. Statistical significance was calculated with unpaired two-tailed Student’s t-tests comparing

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SUBSTITUTE SHEET (RULE 26) YT41R/YT47R- and DMSO-treated cells. *P < 0.1, **P < 0.01, ***P < 0.001, ****P 0.0001.

[0018] Figure S4. Time-dependent degradation of FKBP12 by DCAFl-directed electrophilic PROTACs. Top, western blots showing time-dependent effects of YT47R and YT41R (2 pM each) on FKBP12 abundance in HEK293T cells co-expressing HA-FBKP12 and FLAG-DCAF1. Data are mean values ± SEM for four independent experiments.

Statistical significance was calculated with unpaired two-tailed Student’s t-tests comparing YT41R/YT47R- and DMSO-treated cells. ****P < 0.0001.

[0019] Figure S5. A reversible small molecule CYCA-117-70 binds DCAF1 near Cl 113. Apical and lateral views are shown. Cl 113 is indicated in yellow for visual reference. X-ray diffraction data generated by Kimani, S. et al. (1.62-A resolution, PDB: 7SSE).

DETAILED DESCRIPTION OF THE DISCLOSURE

[0020] Unless otherwise stated, the following terms used in the specification and claims are defined for the purposes of this Application and have the following meaning. All undefined technical and scientific terms used in this Application have the meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0021] As used herein, “a” or “an” entity refers to one or more of that entity; for example, a compound refers to one or more compounds or at least one compound unless stated otherwise. As such, the terms “a” (or “an”), “one or more”, and “at least one” can be used interchangeably herein.

[0022] When a range of values is listed, it is intended to encompass each value and subrange within the range. For example, “Ci-6 alkyl” is intended to encompass, Ci, C2, C3, C4, C ₅ , C ₆ , C1-6, Ci-5, C1-4, C1-3, C1-2, C2-6, C2-5, C2-4, C2-3, Csv, C3-5, C3-4, C v, C4-5, and C _5-6 alkyl.

[0023] “Alkyl” refers to a radical of a straight-chain or branched saturated hydrocarbon group having from 1 to 20 carbon atoms (“C1-20 alkyl”). In some embodiments, an alkyl group has 1 to 15 carbon atoms (“C1-15 alkyl”). In some embodiments, an alkyl group has 1 to 14 carbon atoms (“C1-14 alkyl”). In some embodiments, an alkyl group has 1 to 13 carbon atoms (“Ci-13 alkyl”). In some embodiments, an alkyl group has 1 to 12 carbon atoms (“C1-12 alkyl”). In some embodiments, an alkyl group has 1 to 11 carbon atoms (“Ci-u alkyl”). In some embodiments, an alkyl group has 1 to 10 carbon atoms (“C1-10 alkyl”). In some embodiments, an alkyl group has 1 to 9 carbon atoms (“C1-9 alkyl”). In some embodiments,

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SUBSTITUTE SHEET (RULE 26) an alkyl group has 1 to 8 carbon atoms (“Ci-8 alkyl”). In some embodiments, an alkyl group has 1 to 7 carbon atoms (“C1-7 alkyl”). In some embodiments, an alkyl group has 1 to 6 carbon atoms (“C1-6 alkyl”). In some embodiments, an alkyl group has 1 to 5 carbon atoms (“C1-5 alkyl”). In some embodiments, an alkyl group has 1 to 4 carbon atoms (“C1-4 alkyl”). In some embodiments, an alkyl group has 1 to 3 carbon atoms (“C1-3 alkyl”). In some embodiments, an alkyl group has 1 to 2 carbon atoms (“C1-2 alkyl”). In some embodiments, an alkyl group has 1 carbon atom (“Ci alkyl”). In some embodiments, an alkyl group has 2 to 6 carbon atoms (“C2-6 alkyl”). Examples of C1-6 alkyl groups include methyl (Ci), ethyl (C2), n-propyl (C3), isopropyl (C3), n-butyl (C4), tert-butyl (C4), sec-butyl (C4), iso-butyl (C4), n- pentyl (C5), 3-pentanyl (C5), amyl (C5), neopentyl (C5), 3-methyl-2-butanyl (C5), tertiary amyl (C5), and n-hexyl (Ce). Additional examples of alkyl groups include n-heptyl (C7), n- octyl (Cs) and the like.

[0024] “Alkenyl” refers to a radical of a straight-chain or branched hydrocarbon group having from 2 to 10 carbon atoms and 1, 2, 3, or 4 carbon-carbon double bonds (“C2-10 alkenyl”). In some embodiments, an alkenyl group has 2 to 9 carbon atoms (“C2-9 alkenyl”). In some embodiments, an alkenyl group has 2 to 8 carbon atoms (“C2-8 alkenyl”). In some embodiments, an alkenyl group has 2 to 7 carbon atoms (“C2-7 alkenyl”). In some embodiments, an alkenyl group has 2 to 6 carbon atoms (“C2-6 alkenyl”). In some embodiments, an alkenyl group has 2 to 5 carbon atoms (“C2-5 alkenyl”). In some embodiments, an alkenyl group has 2 to 4 carbon atoms (“C2-4 alkenyl”). In some embodiments, an alkenyl group has 2 to 3 carbon atoms (“C2-3 alkenyl”). In some embodiments, an alkenyl group has 2 carbon atoms (“C2 alkenyl”). The one or more carboncarbon double bonds can be internal (such as in 2-butenyl) or terminal (such as in 1-butenyl). Examples of C2-4 alkenyl groups include ethenyl (C2), 1-propenyl (C3), 2-propenyl (C3), 1- butenyl (C4), 2-butenyl (C4), butadienyl (C4), and the like. Examples of C2-6 alkenyl groups include the aforementioned C2-4 alkenyl groups as well as pentenyl (C5), pentadienyl (C5), hexenyl (Ce), and the like. Additional examples of alkenyl include heptenyl (C7), octenyl (Cs), octatrienyl (Cs), and the like.

[0025] “Alkynyl” refers to a radical of a straight-chain or branched hydrocarbon group having from 2 to 10 carbon atoms and one or more carbon-carbon triple bonds (e.g., 1, 2, 3, or 4 triple bonds) (“C2-10 alkynyl”). In some embodiments, an alkynyl group has 2 to 9 carbon atoms (“C2-9 alkynyl”). In some embodiments, an alkynyl group has 2 to 8 carbon atoms (“C2-8 alkynyl”). In some embodiments, an alkynyl group has 2 to 7 carbon atoms

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SUBSTITUTE SHEET (RULE 26) (“C2-7 alkynyl”). In some embodiments, an alkynyl group has 2 to 6 carbon atoms (“C2-6 alkynyl”). In some embodiments, an alkynyl group has 2 to 5 carbon atoms (“C2-5 alkynyl”). In some embodiments, an alkynyl group has 2 to 4 carbon atoms (“C2-4 alkynyl”). In some embodiments, an alkynyl group has 2 to 3 carbon atoms (“C2-3 alkynyl”). In some embodiments, an alkynyl group has 2 carbon atoms (“C2 alkynyl”). The one or more carboncarbon triple bonds can be internal (such as in 2-butynyl) or terminal (such as in 1-butynyl). Examples of C2-4 alkynyl groups include, without limitation, ethynyl (C2), 1-propynyl (C3), 2-propynyl (C3), 1-butynyl (C4), 2-butynyl (C4), and the like. Examples of C2-6 alkenyl groups include the aforementioned C2-4 alkynyl groups as well as pentynyl (C5), hexynyl (Ce), and the like. Additional examples of alkynyl include heptynyl (C7), octynyl (Cs), and the like.

[0026] “Carbocyclyl” or “carbocyclic” refers to a radical of a non-aromatic cyclic hydrocarbon group having from 3 to 14 ring carbon atoms (“C3-14 carbocyclyl”) and zero heteroatoms in the non-aromatic ring system. In some embodiments, a carbocyclyl group has 3 to 10 ring carbon atoms (“C3-10 carbocyclyl”). In some embodiments, a carbocyclyl group has 3 to 8 ring carbon atoms (“C3-8 carbocyclyl”). In some embodiments, a carbocyclyl group has 3 to 7 ring carbon atoms (“C3-7 carbocyclyl”). In some embodiments, a carbocyclyl group has 3 to 6 ring carbon atoms (“C3-6 carbocyclyl”). In some embodiments, a carbocyclyl group has 4 to 6 ring carbon atoms (“C4-6 carbocyclyl”). In some embodiments, a carbocyclyl group has 5 to 6 ring carbon atoms (“C5-6 carbocyclyl”). In some embodiments, a carbocyclyl group has 5 to 10 ring carbon atoms (“C5-10 carbocyclyl”). Exemplary C3-6 carbocyclyl groups include, without limitation, cyclopropyl (C3), cyclopropenyl (C3), cyclobutyl (C4), cyclobutenyl (C4), cyclopentyl (C5), cyclopentenyl (C5), cyclohexyl (Ce), cyclohexenyl (Ce), cyclohexadienyl (Ce), and the like. Exemplary C3-8 carbocyclyl groups include, without limitation, the aforementioned C3-6 carbocyclyl groups as well as cycloheptyl (C7), cycloheptenyl (C7), cycloheptadienyl (C7), cycloheptatrienyl (C7), cyclooctyl (Cs), cyclooctenyl (Cs), bicyclo[2.2.1]heptanyl (C7), bicyclo[2.2.2]octanyl (Cs), and the like. Exemplary C3-10 carbocyclyl groups include, without limitation, the aforementioned C3-8 carbocyclyl groups as well as cyclononyl (C9), cyclononenyl (C9), cyclodecyl (C10), cyclodecenyl (C10), octahydro- 177-indenyl (C9), decahydronaphthalenyl (C10), spiro[4.5]decanyl (C10), and the like. As the foregoing examples illustrate, in certain embodiments, the carbocyclyl group is either monocyclic (“monocyclic carbocyclyl”) or polycyclic (e.g., containing a fused, bridged or spiro ring system such as a bicyclic system

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SUBSTITUTE SHEET (RULE 26) (“bicyclic carbocyclyl”) or tricyclic system (“tricyclic carbocyclyl”)) and can be saturated or can contain one or more carbon-carbon double or triple bonds. “Carbocyclyl” also includes ring systems wherein the carbocyclyl ring, as defined above, is fused with one or more aryl or heteroaryl groups wherein the point of attachment is on the carbocyclyl ring, and in such instances, the number of carbons continue to designate the number of carbons in the carbocyclic ring system.

[0027] In some embodiments, “carbocyclyl” is a monocyclic, saturated carbocyclyl group having from 3 to 14 ring carbon atoms (“C3-14 cycloalkyl”). In some embodiments, “carbocyclyl” is a monocyclic, saturated carbocyclyl group having from 3 to 10 ring carbon atoms (“C3-10 cycloalkyl”). In some embodiments, a cycloalkyl group has 3 to 8 ring carbon atoms (“C3-8 cycloalkyl”). In some embodiments, a cycloalkyl group has 3 to 6 ring carbon atoms (“C3-6 cycloalkyl”). In some embodiments, a cycloalkyl group has 4 to 6 ring carbon atoms (“C4-6 cycloalkyl”). In some embodiments, a cycloalkyl group has 5 to 6 ring carbon atoms (“C5-6 cycloalkyl”). In some embodiments, a cycloalkyl group has 5 to 10 ring carbon atoms (“C5-10 cycloalkyl”). Examples of C5-6 cycloalkyl groups include cyclopentyl (C5) and cyclohexyl (C5). Examples of C3-6 cycloalkyl groups include the aforementioned C5-6 cycloalkyl groups as well as cyclopropyl (C3) and cyclobutyl (C4). Examples of C3-8 cycloalkyl groups include the aforementioned C3-6 cycloalkyl groups as well as cycloheptyl (C7) and cyclooctyl (Cs).

[0028] “Heterocyclyl” or “heterocyclic” refers to a group or radical of a 3- to 14- membered non-aromatic ring system having ring carbon atoms and 1 to 4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“3-14 membered heterocyclyl”). In heterocyclyl groups that contain one or more nitrogen atoms, the point of attachment can be a carbon or nitrogen atom, as valency permits. A heterocyclyl group can either be monocyclic (“monocyclic heterocyclyl”) or polycyclic (e.g., a fused, bridged or spiro ring system such as a bicyclic system (“bicyclic heterocyclyl”) or tricyclic system (“tricyclic heterocyclyl”)), and can be saturated or can contain one or more carboncarbon double or triple bonds. Heterocyclyl polycyclic ring systems can include one or more heteroatoms in one or both rings. “Heterocyclyl” also includes ring systems wherein the heterocyclyl ring, as defined above, is fused with one or more carbocyclyl groups wherein the point of attachment is either on the carbocyclyl or heterocyclyl ring, or ring systems wherein the heterocyclyl ring, as defined above, is fused with one or more aryl or heteroaryl groups, wherein the point of attachment is on the heterocyclyl ring, and in such instances, the number

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SUBSTITUTE SHEET (RULE 26) of ring members continue to designate the number of ring members in the heterocyclyl ring system.

[0029] In some embodiments, a heterocyclyl group is a 5-10 membered non-aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5-10 membered heterocyclyl”). In some embodiments, a heterocyclyl group is a 5-8 membered non-aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5-8 membered heterocyclyl”). In some embodiments, a heterocyclyl group is a 5-6 membered non-aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5-6 membered heterocyclyl”). In some embodiments, the 5-6 membered heterocyclyl has 1-3 ring heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, the 5-6 membered heterocyclyl has 1-2 ring heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, the 5-6 membered heterocyclyl has 1 ring heteroatom selected from nitrogen, oxygen, and sulfur. [0030] Exemplary 3-membered heterocyclyl groups containing 1 heteroatom include, without limitation, azirdinyl, oxiranyl, and thiiranyl. Exemplary 4-membered heterocyclyl groups containing 1 heteroatom include, without limitation, azetidinyl, oxetanyl and thietanyl. Exemplary 5-membered heterocyclyl groups containing 1 heteroatom include, without limitation, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothiophenyl, dihydrothiophenyl, pyrrolidinyl, dihydropyrrolyl, and pyrrolyl-2, 5-dione. Exemplary 5- membered heterocyclyl groups containing 2 heteroatoms include, without limitation, dioxolanyl, oxathiolanyl and dithiolanyl. Exemplary 5-membered heterocyclyl groups containing 3 heteroatoms include, without limitation, triazolinyl, oxadiazolinyl, and thiadiazolinyl. Exemplary 6-membered heterocyclyl groups containing 1 heteroatom include, without limitation, piperidinyl, tetrahydropyranyl, dihydropyridinyl, and thianyl. Exemplary 6-membered heterocyclyl groups containing 2 heteroatoms include, without limitation, piperazinyl, morpholinyl, dithianyl, and dioxanyl. Exemplary 6-membered heterocyclyl groups containing 3 heteroatoms include, without limitation, triazinanyl. Exemplary 7-membered heterocyclyl groups containing 1 heteroatom include, without limitation, azepanyl, oxepanyl and thiepanyl. Exemplary 8-membered heterocyclyl groups containing 1 heteroatom include, without limitation, azocanyl, oxecanyl and thiocanyl. Exemplary bicyclic heterocyclyl groups include, without limitation, indolinyl, isoindolinyl,

11

SUBSTITUTE SHEET (RULE 26) dihydrobenzofuranyl, dihydrobenzothienyl, tetrahydrobenzothienyl, tetrahydrobenzofuranyl, tetrahydroindolyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl, decahydroisoquinolinyl, octahydrochromenyl, octahydroisochromenyl, decahydronaphthyridinyl, decahydro-1, 8-naphthyridinyl, octahydropyrrolo[3,2-b]pyrrole, indolinyl, phthalimidyl, naphthalimidyl, chromanyl, chromenyl, lH-benzo[e][l,4]diazepinyl, l,4,5,7-tetrahydropyrano[3,4-b]pyrrolyl, 5,6-dihydro-4H-furo[3,2-b]pyrrolyl, 6,7-dihydro- 5H-furo[3,2-b]pyranyl, 5,7-dihydro-4H-thieno[2,3-c]pyranyl, 2,3-dihydro-lH- pyrrolo[2,3-b]pyridinyl, 2,3-dihydrofuro[2,3-b]pyridinyl, 4,5,6,7-tetrahydro-lH-pyrrolo- [2,3-b]pyridinyl, 4,5,6,7-tetrahydrofuro[3,2-c]pyridinyl, 4,5,6,7-tetrahydrothieno[3,2- b]pyridinyl, l,2,3,4-tetrahydro-l,6-naphthyridinyl, and the like.

[0031] “Aryl” refers to a radical of a monocyclic or polycyclic (e.g., bicyclic or tricyclic) 4n+2 aromatic ring system (e.g., having 6, 10, or 14 pi electrons shared in a cyclic array) having 6-14 ring carbon atoms and zero heteroatoms provided in the aromatic ring system (“Ce-i4 aryl”). In some embodiments, an aryl group has 6 ring carbon atoms (“Ce aryl”; e.g., phenyl). In some embodiments, an aryl group has 10 ring carbon atoms (“Cio aryl”; e.g., naphthyl such as 1-naphthyl (a-naphthyl) and 2-naphthyl (P-naphthyl)). In some embodiments, an aryl group has 14 ring carbon atoms (“Cu aryl”; e.g., anthracyl). “Aryl” also includes ring systems wherein the aryl ring, as defined above, is fused with one or more carbocyclyl or heterocyclyl groups wherein the radical or point of attachment is on the aryl ring, and in such instances, the number of carbon atoms continue to designate the number of carbon atoms in the aryl ring system.

[0032] “Heteroaryl” refers to a radical of a 5-14 membered monocyclic or polycyclic e.g., bicyclic, tricyclic) 4n+2 aromatic ring system (e.g., having 6, 10, or 14 pi electrons shared in a cyclic array) having ring carbon atoms and 1-4 ring heteroatoms provided in the aromatic ring system, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5-14 membered heteroaryl”). In heteroaryl groups that contain one or more nitrogen atoms, the point of attachment can be a carbon or nitrogen atom, as valency permits. Heteroaryl polycyclic ring systems can include one or more heteroatoms in one or both rings. “Heteroaryl” includes ring systems wherein the heteroaryl ring, as defined above, is fused with one or more carbocyclyl or heterocyclyl groups wherein the point of attachment is on the heteroaryl ring, and in such instances, the number of ring members continue to designate the number of ring members in the heteroaryl ring system. “Heteroaryl” also includes ring systems wherein the heteroaryl ring, as defined above, is fused with one or

12

SUBSTITUTE SHEET (RULE 26) more aryl groups wherein the point of attachment is either on the aryl or heteroaryl ring, and in such instances, the number of ring members designates the number of ring members in the fused polycyclic (aryl/heteroaryl) ring system. Polycyclic heteroaryl groups wherein one ring does not contain a heteroatom (e.g., indolyl, quinolinyl, carbazolyl, and the like) the point of attachment can be on either ring, i.e., either the ring bearing a heteroatom (e.g., 2-indolyl) or the ring that does not contain a heteroatom (e.g., 5-indolyl).

[0033] In some embodiments, a heteroaryl group is a 5-10 membered aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms provided in the aromatic ring system, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5-10 membered heteroaryl”). In some embodiments, a heteroaryl group is a 5-8 membered aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms provided in the aromatic ring system, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5-8 membered heteroaryl”). In some embodiments, a heteroaryl group is a 5-6 membered aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms provided in the aromatic ring system, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5-6 membered heteroaryl”). In some embodiments, the 5-6 membered heteroaryl has 1-3 ring heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, the 5-6 membered heteroaryl has 1-2 ring heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, the 5-6 membered heteroaryl has 1 ring heteroatom selected from nitrogen, oxygen, and sulfur.

[0034] Exemplary 5-membered heteroaryl groups containing 1 heteroatom include, without limitation, pyrrolyl, furanyl and thiophenyl. Exemplary 5-membered heteroaryl groups containing 2 heteroatoms include, without limitation, imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, and isothiazolyl. Exemplary 5-membered heteroaryl groups containing 3 heteroatoms include, without limitation, triazolyl, oxadiazolyl, and thiadiazolyl. Exemplary

5-membered heteroaryl groups containing 4 heteroatoms include, without limitation, tetrazolyl. Exemplary 6-membered heteroaryl groups containing 1 heteroatom include, without limitation, pyridinyl. Exemplary 6-membered heteroaryl groups containing 2 heteroatoms include, without limitation, pyridazinyl, pyrimidinyl, and pyrazinyl. Exemplary

6-membered heteroaryl groups containing 3 or 4 heteroatoms include, without limitation, triazinyl and tetrazinyl, respectively. Exemplary 7-membered heteroaryl groups containing 1 heteroatom include, without limitation, azepinyl, oxepinyl, and thiepinyl. Exemplary 5,6- bicyclic heteroaryl groups include, without limitation, indolyl, isoindolyl, indazolyl,

13

SUBSTITUTE SHEET (RULE 26) benzotri azolyl, benzothiophenyl, isobenzothiophenyl, benzofuranyl, benzoisofuranyl, benzimidazolyl, benzoxazolyl, benzisoxazolyl, benzoxadi azolyl, benzthiazolyl, benzisothi azolyl, benzthiadi azolyl, indolizinyl, and purinyl. Exemplary 6,6-bicyclic heteroaryl groups include, without limitation, naphthyridinyl, pteridinyl, quinolinyl, isoquinolinyl, cinnolinyl, quinoxalinyl, phthalazinyl, and quinazolinyl. Exemplary tricyclic heteroaryl groups include, without limitation, phenanthridinyl, dibenzofuranyl, carbazolyl, acridinyl, phenothiazinyl, phenoxazinyl and phenazinyl.

[0035] Saturated” refers to a ring moiety that does not contain a double or triple bond, z.e., the ring contains all single bonds.

[0036] Alkyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl groups may be optionally substituted. Optionally substituted refers to a group which may be substituted or unsubstituted. In general, the term “substituted” means that at least one hydrogen present on a group is replaced with a non-hydrogen substituent, and which upon substitution results in a stable compound, e.g., a compound which does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, or other reaction. Heteroatoms such as nitrogen, oxygen, and sulfur may have hydrogen substituents and/or non-hydrogen substituents which satisfy the valencies of the heteroatoms and results in the formation of a stable compound.

[0037] Exemplary non-hydrogen substituents may be selected from the group consisting of halogen, -CN, -NO ₂ , -N ₃ , -SO ₂ H, -SO ₃ H, -OH, -OR ^aa , -N(R ^bb ) ₂ , -N(OR ^cc )R ^bb , -SH, - S(=O)R ^aa , -OS(=O)R ^aa , -B(OR ^CC ) ₂ , Ci-io alkyl, C ₂ -io alkenyl, C ₂ -io alkynyl, C ₃ -i ₄ carbocyclyl, 3- to 14- membered heterocyclyl, Ce-14 aryl, and 5- to 14- membered heteroaryl, wherein each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 R ^dd groups, or two geminal hydrogens on a carbon atom are replaced with the group =0;

[0038] each instance of R ^aa is, independently, selected from the group consisting of Ci-io alkyl, Ci-io perhaloalkyl, C ₂ -io alkenyl, C ₂ -io alkynyl, C ₃ -i ₄ carbocyclyl, 3- to 14- membered heterocyclyl, Ce-14 aryl, and 5- to 14- membered heteroaryl, or two R ^aa groups are joined to form a 3- to 14- membered heterocyclyl or 5- to 14- membered heteroaryl ring, wherein each

14

SUBSTITUTE SHEET (RULE 26) alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 R ^dd groups;

[0039] each instance of R ^bb is, independently, selected from the group consisting of hydrogen, -OH, -OR ^aa , -N(R ^CC ) ₂ , -CN, -C(=O)R ^aa , -C(=O)N(R ^CC ) ₂ , -CO ₂ R ^aa , -SO ₂ R ^aa , - SO ₂ N(R ^CC ) ₂ , -SOR ^aa , Ci-io alkyl, Ci-io perhaloalkyl, C2-10 alkenyl, C2-10 alkynyl, C3-14 carbocyclyl, 3- to 14- membered heterocyclyl, Ce-14 aryl, and 5- to 14- membered heteroaryl, or two R ^bb groups are joined to form a 3- to 14- membered heterocyclyl or 5- to 14- membered heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 R ^dd groups;

[0040] each instance of R ^cc is, independently, selected from the group consisting of hydrogen, C1-10 alkyl, C1-10 perhaloalkyl, C2-10 alkenyl, C2-10 alkynyl, C3-14 carbocyclyl, 3- to 14- membered heterocyclyl, Ce-14 aryl, and 5- to 14- membered heteroaryl, or two R ^cc groups are joined to form a 3- to 14- membered heterocyclyl or 5- to 14- membered heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 R ^dd groups; and [0041] each instance of R ^dd is, independently, selected from the group consisting of halogen, -CN, -NO ₂ , -N3, -SO ₂ H, -SO3H, -OH, -OCi- ₆ alkyl, -ON(CI- ₆ alkyl) ₂ , -N(CI- ₆ alkyl) ₂ , -N(OCI- ₆ alkyl)(Ci- ₆ alkyl), -N(OH)(CI- ₆ alkyl), -NH(OH), -SH, -SCi^ alkyl, - C(=O)(Ci-6 alkyl), -CO ₂ H, -CO ₂ (Ci- ₆ alkyl), -OC(=O)(Ci- ₆ alkyl), -OCO ₂ (Ci- ₆ alkyl), - C(=O)NH ₂ , -C(=O)N(CI-6 alkyl) ₂ , -OC(=O)NH(CI- ₆ alkyl), -NHC(=O)( Ci- ₆ alkyl), -N(Ci- 6 alkyl)C(=O)( Ci- ₆ alkyl), -NHCO ₂ (CI- ₆ alkyl), -NHC(=O)N(CI- ₆ alkyl) ₂ , - NHC(=O)NH(CI- ₆ alkyl), -NHC(=0)NH ₂ , -C(=NH)O(CI- ₆ alkyl),-OC(=NH)(Ci- ₆ alkyl), - OC(=NH)OCI- ₆ alkyl, -C(=NH)N(CI- ₆ alkyl) ₂ , -C(=NH)NH(CI- ₆ alkyl), -C(=NH)NH ₂ , - OC(=NH)N(CI-6 alkyl) ₂ , -OC(NH)NH(CI- ₆ alkyl), -0C(NH)NH ₂ , -NHC(NH)N(CI- ₆ alkyl) ₂ , -NHC(=NH)NH ₂ , -NHSO ₂ (CI- ₆ alkyl), -SO ₂ N(CI- ₆ alkyl) ₂ , -SO ₂ NH(CI- ₆ alkyl), - SO ₂ NH ₂ ,-SO ₂ CI-6 alkyl, -B(OH) ₂ , -B(OCI-6 alkyl) ₂ ,Ci-6 alkyl, Ci-6 perhaloalkyl, C ₂ -6 alkenyl, C ₂ -6 alkynyl, C3-10 carbocyclyl, Ce-io aryl, 3-to 10- membered heterocyclyl, and 5- to 10- membered heteroaryl; or two geminal R ^dd substituents on a carbon atom may be joined to form =0.

[0042] “Halo” or “halogen” refers to fluorine (fluoro, -F), chlorine (chloro, -Cl), bromine (bromo, -Br), or iodine (iodo, -I).

15

SUBSTITUTE SHEET (RULE 26) [0043] It should be noted that in hetero-atom containing ring systems described herein, there are no hydroxyl groups on carbon atoms adjacent to a N, O or S, as well as there are no N or S groups on carbon adjacent to another heteroatom. Thus, for example, in the ring: there is no -OH attached directly to carbons marked 2 and 5.

[0044] It should also be noted that tautomeric forms such as, for example, the moieties: are considered equivalent unless otherwise specified.

[0045] As used herein, the term “Michael addition reaction” is well known to those of ordinary skill in the art of organic chemistry. In this reaction, a new covalent bond is formed between a portion of the Michael acceptor moiety (for example, an alpha, beta unsaturated functionality, such as an acrylamide) and a donor moiety. The Michael acceptor moiety is an electrophile and the “donor moiety” is a nucleophile (such as the sulfur atom in the SH group of cysteine)

[0046] As used herein, the term “composition” is intended to encompass a product comprising the specified ingredients in the specified amounts, as well as any product which results, directly or indirectly, from combination of the specified ingredients in the specified amounts.

[0047] “Effective amount” or “therapeutically effective amount” is meant to describe an amount of compound or a composition described herein that is effective in inhibiting the recited diseases or conditions, and thus producing the desired therapeutic, ameliorative, inhibitory and/or preventative effect.

[0048] Salt” includes any and all salts. “Pharmaceutically acceptable salt” refers to those salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit/risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, Berge et al., describes pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences (1977) 66: 1-19.

Pharmaceutically acceptable salts include those derived from inorganic and organic acids and

16

SUBSTITUTE SHEET (RULE 26) bases. Examples of pharmaceutically acceptable, nontoxic acid addition salts are salts of an amino group formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid or with organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid or by using other methods used in the art such as ion exchange. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2- naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate salts, and the like. Pharmaceutically acceptable salts derived from appropriate bases include alkali metal, alkaline earth metal, ammonium and N ⁺ (Ci-4alkyl)4 salts. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. Further pharmaceutically acceptable salts include, when appropriate, nontoxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, lower alkyl sulfonate, and aryl sulfonate. [0049] Unless otherwise indicated, compounds described herein can comprise one or more asymmetric centers, and thus can exist in various stereoisomeric forms, e.g., enantiomers and/or diastereomers. For example, the compounds described herein can be in the form of an individual enantiomer, diastereomer or geometric isomer, or can be in the form of a mixture of stereoisomers, including racemic mixtures and mixtures enriched in one or more stereoisomer. Isomers can be isolated from mixtures by methods known to those skilled in the art, including chiral high pressure liquid chromatography (HPLC). Compounds described herein can be in the form of individual isomers substantially free of other isomers, and alternatively, as mixtures of various isomers.

[0050] Unless otherwise stated, structures depicted herein are also meant to include compounds that differ only in the presence of one or more isotopically enriched atoms. For example, compounds having the present structures except for the replacement of hydrogen by deuterium or tritium, replacement of ¹⁹ F with ¹⁸ F, replacement of a carbon by a ¹³ C- or ¹⁴ C- enriched carbon, and/or replacement of an oxygen atom with ¹⁸ O, are within the scope of the

17

SUBSTITUTE SHEET (RULE 26) disclosure. Other examples of isotopes include ¹⁵ N, ¹⁸ O, ¹⁷ 0, ³¹ P, ³² P, ³⁵ S, ¹⁸ F, ³⁶ C1 and ¹²³ I. Compounds with such isotopically enriched atoms are useful, for example, as analytical tools or probes in biological assays.

[0051] Certain isotopically-labelled compounds (e.g., those labeled with ³ H and ¹⁴ C) are useful in compound and/or substrate tissue distribution assays. Tritiated (i.e., ³ H) and carbon-14 (i.e., ¹⁴ C) isotopes are particularly preferred for their ease of preparation and detectability.

[0052] Certain isotopically-labelled compounds of Formula (I) can be useful for medical imaging purposes, for example, those labeled with positron-emitting isotopes like ⁿ C or ¹⁸ F can be useful for application in Positron Emission Tomography (PET) and those labeled with gamma ray emitting isotopes like ¹²³ I can be useful for application in Single Photon Emission Computed Tomography (SPECT). Further, substitution with heavier isotopes such as deuterium (i.e., ² H) may afford certain therapeutic advantages resulting from greater metabolic stability (e.g., increased in vivo half-life or reduced dosage requirements) and hence may be preferred in some circumstances. Further, substitution with heavier isotopes such as deuterium (i.e., ² H) may afford certain therapeutic advantages resulting from greater metabolic stability (e.g., increased in vivo half-life or reduced dosage requirements), and hence, may be preferred in some circumstances. Additionally, isotopic substitution at a site where epimerization occurs may slow or reduce the epimerization process and thereby retain the more active or efficacious form of the compound for a longer period of time. Isotopically labeled compounds of Formula (I), in particular those containing isotopes with longer halflives (ti/2 >1 day), can generally be prepared by following procedures analogous to those disclosed in the Schemes and/or in the Examples herein below, by substituting an appropriate isotopically labeled reagent for a non-isotopically labeled reagent.

[0053] The compounds described herein can also be used in combination with one or more additional therapeutic and/or prophylactic agents.

[0054] It is also possible to combine any compound of the invention with one or more additional active therapeutic agents in a unitary dosage form for simultaneous or sequential administration to a patient. The combination therapy may be administered as a simultaneous or sequential regimen. When administered sequentially, the combination may be administered in two or more administrations.

[0055] Co-administration of a compound of the invention with one or more other active therapeutic agents generally refers to simultaneous or sequential administration of a

18

SUBSTITUTE SHEET (RULE 26) compound of the invention and one or more other active therapeutic agents, such that therapeutically effective amounts of the compound of the invention and one or more other active therapeutic agents are both present in the body of the patient.

[0056] Co-administration includes administration of unit dosages of the compounds of the invention before or after administration of unit dosages of one or more other active therapeutic agents, for example, administration of the compounds of the invention within seconds, minutes, or hours of the administration of one or more other active therapeutic agents. For example, a unit dose of a compound of the invention can be administered first, followed within seconds or minutes by administration of a unit dose of one or more other active therapeutic agents. Alternatively, a unit dose of one or more other therapeutic agents can be administered first, followed by administration of a unit dose of a compound of the invention within seconds or minutes. In some cases, it may be desirable to administer a unit dose of a compound of the invention first, followed, after a period of hours (e.g., 1-12 hours), by administration of a unit dose of one or more other active therapeutic agents. In other cases, it may be desirable to administer a unit dose of one or more other active therapeutic agents first, followed, after a period of hours ( e.g., 1-12 hours), by administration of a unit dose of a compound of the invention.

[0057] The combination therapy may provide "synergy" and "synergistic", i.e. the effect achieved when the active ingredients used together is greater than the sum of the effects that results from using the compounds separately. A synergistic effect may be attained when the active ingredients are: (1) co-formulated and administered or delivered simultaneously in a combined formulation; (2) delivered by alternation or in parallel as separate formulations; or (3) by some other regimen. When delivered in alternation therapy, a synergistic effect may be attained when the compounds are administered or delivered sequentially, e.g. in separate tablets, pills or capsules, or by different injections in separate syringes. In general, during alternation therapy, an effective dosage of each active ingredient is administered sequentially, i.e. serially, whereas in combination therapy, effective dosages of two or more active ingredients are administered together. A synergistic anti-viral effect denotes an antiviral effect which is greater than the predicted purely additive effects of the individual compounds of the combination.

Embodiments

[0058] Examples of embodiments of the present application include the following:

19 SUBSTITUTE SHEET (RULE 26) Embodiment 1

[0059] a bifunctional degrader of Formula (I)

A-B-C

Formula (I) wherein, A is a ligand to a protein of interest, B is a linker that is a bond or a chemical linker that is chemically linked to A and C, and C is a ligand to the E3 ligase substrate receptor DCAF1, wherein:

[0060] the protein of interest is any protein having a ligand that can form a covalent bond with the linker B; and

[0061] C comprises an azetidinyl acrylamide that forms a covalent bond with Cl 113 wherein the amino acid numbering is based on DCAF1 Isoform 1 (Accession No. Q9Y4B6- 1), Cl 112 of DCAF1, wherein the amino acid numbering is based on DCAF1 Isoform 2 (Accession No. Q9Y4B6-2), or C664 of DCAF1, wherein the amino acid numbering is based on DCAF1 Isoform 3 (Accession No. Q9Y4B6-3), through a Michael addition reaction. Embodiment 2

[0062] The bifunctional degrader of Embodiment 1, wherein the protein of interest is selected from the group consisting of FKBP12, BRD4, androgen receptor, estrogen receptor, IRAK4, a JAK protein, BCL-XL, BCL-2, and Stat3.

Embodiment 3

[0063] The bifunctional degrader of Embodiment 1 or 2, wherein azetidinyl acrylamide of C forms a covalent bond with Cl 113 of DCAF1 through a Michal addition reaction. Embodiment 4

[0064] The bifunctional degrader of any one of Embodiments 1-3, wherein C has the structural Formula (C-l): wherein:

[0065] Ar is a Ce-Cio aryl, optionally substituted with 1-3 moi eties selected from the group consisting of: halo, hydroxy, cyano, optionally substituted Ci-Ce alkyl, -O-(Ci-Ce

20

SUBSTITUTE SHEET (RULE 26) alkyl), optionally substituted Ce-Cio aryl, optionally substituted C3-C8 cycloalkyl, -C(=O)- (Ci-Ce alkyl), optionally substituted -(Ci-C3)n-heterocyclylphenyl, Ce-Cio aryl, -(Ci-C3)n- linked optionally substituted five- to six-membered heterocyclyl, -(Ci-C3)n-linked optionally substituted five- to six-membered heterocyclyl fused to an optionally substituted Ce-Cio aryl, and -(Ci-C3)n-linked optionally substituted five- to six-membered heteroaryl;

[0066] each R ¹ independently is an optional substituent selected from the group consisting of halo, cyano, optionally substituted Ci-Ce alkyl, -O- Ci-Ce alkyl, optionally substituted Ce-Cio aryl, optionally substituted C3-C8 cycloalkyl, -C(=O)(Ci-Ce alkyl), optionally substituted five- to six-membered heterocyclyl, and optionally substituted five- to six-membered heteroaryl; n is 0, 1, or 2; p is 0, 1 or 2; ring A is a five- or six-membered heteroaryl; each R ² independently is an optional substituent selected from the group consisting of halo, cyano, optionally substituted Ci-Ce alkyl, -O- Ci-Ce alkyl, optionally substituted Ce-Cio aryl, optionally substituted C3-C8 cycloalkyl, -C(=O)(Ci-Ce alkyl), optionally substituted five- to six-membered heterocyclyl, and optionally substituted five- to six-membered heteroaryl; q is 0, 1, or 2; and indicates the point of attachment to the linker B.

Embodiment 5

[0067] The bifunctional degrader of Embodiment 4 wherein:

[0068] Ar is substituted with 1-2 substituents selected from the group consisting of halo, hydroxy, cyano, -O-Ci-Cealkyl, -alkynylphenyl, flurophenoxy, methoxyphenyl, -((Ci-C3)n)-4- (4-methoxyphenyl)piperidine and -(Ci-C3)n-linked optionally substituted five- to sixmembered heterocyclyl fused to an optionally substituted Ce-Cio aryl.

Embodiment 6

[0069] The bifunctional degrader of Embodiment 5, wherein: the -(Ci-C3)n-linked optionally substituted five- to six-membered heterocyclyl fused to an optionally substituted Ce-Cio aryl is selected from the group consisting of -((Ci-C3)n)- benzo[ ][l,3]dioxolyl, -((Ci-C3)n)-l,2,3,4-tetrahydroquinoline-l-yl, -((Ci-C3)n)-1, 2,3,4- tetrahydroisoquinoline-2-yl, -((Ci-C3)n)-6-methoxy-l,2,3,4-tetrahydroisoquinoline-2-yl, and - ((Ci-C3)n)-indoline-l-yl; and

21

SUBSTITUTE SHEET (RULE 26) n is 0 or 1.

Embodiment 7

[0070] The bifunctional degrader of any one of Embodiments 1-6, wherein Formula (C-l) has the formula (C-l a):

[0071] The bifunctional degrader of any one of Embodiments 1-7, wherein

22

SUBSTITUTE SHEET (RULE 26)

Embodiment 9

[0072] The bifunctional degrader of any one of Embodiments 1-8, wherein linker B comprises a moiety having ethylene repeat units, the moiety having the formula (B-l) wherein r is an integer from 1 to 10; or a moiety having ethylene glycol repeat units, the moiety having the formula (B-2) wherein s is an integer from 1 to 10.

Embodiment 10

[0073] The bifunctional degrader of any one of Embodiments 1-9, wherein linker B

Embodiment 10a

[0074] The bifunctional degrader of any one of Embodiments 1-9, wherein linker B is selected from the group consisting of:

23

SUBSTITUTE SHEET (RULE 26)

SUBSTITUTE SHEET (RULE 26)

SUBSTITUTE SHEET (RULE 26)

SUBSTITUTE SHEET (RULE 26)

SUBSTITUTE SHEET (RULE 26)

SUBSTITUTE SHEET (RULE 26)

SUBSTITUTE SHEET (RULE 26)

SUBSTITUTE SHEET (RULE 26)

SUBSTITUTE SHEET (RULE 26)

SUBSTITUTE SHEET (RULE 26)

SUBSTITUTE SHEET (RULE 26)

SUBSTITUTE SHEET (RULE 26)

SUBSTITUTE SHEET (RULE 26)

SUBSTITUTE SHEET (RULE 26)

SUBSTITUTE SHEET (RULE 26)

SUBSTITUTE SHEET (RULE 26)

SUBSTITUTE SHEET (RULE 26)

SUBSTITUTE SHEET (RULE 26)

SUBSTITUTE SHEET (RULE 26)

SUBSTITUTE SHEET (RULE 26)

SUBSTITUTE SHEET (RULE 26)

SUBSTITUTE SHEET (RULE 26) [0075] Descriptions of most of the linkers above are disclosed in published US patent application US 2019/0192668 Al.

Embodiment 11

[0076] The bifunctional degrader of any one of Embodiments l-10a, wherein the protein of interest is FKBP12.

Embodiment 12

[0077] The bifunctional degrader of Embodiment 11, wherein ligand A is SLF, has the structure (A-l):

Embodiment 13

[0078] The bifunctional degrader of either Embodiment 11 or 12, wherein the linker B is

Embodiment 14

[0079] The bifunctional degrader of any one of Embodiments 11-13, having the structure: or a pharmaceutically acceptable salt thereof.

Embodiment 15

[0080] The bifunctional degrader of any one of Embodiments 1-10, wherein the protein of interest is the androgen receptor.

Embodiment 16

[0081] The bifunctional degrader of Embodiment 15, wherein ligand A comprises at least one chemical moiety selected from the group consisting of:

46

SUBSTITUTE SHEET (RULE 26)

47

SUBSTITUTE SHEET (RULE 26)

; wherein in each instance, _O r * can be a point of attachment for the linker B.

[0082] Various androgen receptor ligands are disclosed in:

Xin Han et al., J. Med. Chem. 2019, 62, 941-964; DOI: 10.1021/acs.jmedchem.8b01631; and

Michael L. Mohler et al., Int. J. Mol. Sci. 2021, 22, 2124. https://doi.org/10.3390/ijms22042124;

Embodiment 17

[0083] The bifunctional degrader of any one of Embodiments 1-10, wherein the protein of interest is the estrogen receptor.

Embodiment 18

[0084] The bifunctional degrader of Embodiment 17, wherein ligand A comprises at least one chemical moiety selected from the group consisting of:

48

SUBSTITUTE SHEET (RULE 26)

wherein _can be a point of attachment for the linker B.

[0085] Various ligands for the estrogen receptor are disclosed for example in the following references: Hui Qin et al., Curr Med Chem. 2021 Nov 9. doi: 10.2174/0929867328666211110101018; and US Patent 10,071,164.

Embodiment 19

[0086] The bifunctional degrader of any one of Embodiments 1-10, wherein the protein of interest is IRAK4.

Embodiment 20

49

SUBSTITUTE SHEET (RULE 26) [0087] The bifunctional degrader of Embodiment 19, wherein ligand A comprises at least one chemical moiety selected from the group consisting of: ; wherein ^vww can be a point of attachment for the linker B.

[0088] Various ligands for IRAK are disclosed for example, in published US patent application US 2019/0192668 Al.

Embodiment 21

50

SUBSTITUTE SHEET (RULE 26) [0089] The bifunctional degrader of any one of Embodiments 1-10, wherein the protein of interest is a JAK protein.

Embodiment 22

[0090] The bifunctional degrader of Embodiment 21, wherein the JAK protein is JAK1, JAK2, or JAK3.

Embodiment 23

[0091] The bifunctional degrader of Embodiment 21 or 22, wherein ligand A comprises at least one chemical moiety selected from the group consisting of: point of attachment for the linker B.

51

SUBSTITUTE SHEET (RULE 26) [0092] Ligands for various JAK proteins are disclosed in Rishi R. Shah et al., Bioorganic & Medicinal Chemistry, Volume 28, Issue 5, 1 March 2020, 115326 (including the supplemental supporting information) ; https://doi.Org/10.1016/j.bmc.2020.115326.

Embodiment 24

[0093] The bifunctional degrader of any one of Embodiments 1-10, wherein the protein of interest is BCL-XL or BCL-2.

Embodiment 25

[0094] The bifunctional degrader of Embodiment 24, wherein ligand A comprises at least one chemical moiety selected from the group consisting of: wherein > ZWW can be a point of attachment for the linker B.

[0095] Various ligands for BCL-XL and BCL-2 are disclosed in Sajid Khan et.al., Nature Medicine, VOL 25, December 2019, 1938-1947, and www.nature.com/articles/s41591-Q19- 0668-z.pdf; doi: 10.1038/s41591-019-0668-z.

Embodiment 26

[0096] The bifunctional degrader of any one of Embodiments 1-10, wherein the protein of interest is Stat3.

Embodiment 27

[0097] The bifunctional degrader of Embodiment 26, wherein ligand A comprises at least one chemical moiety of Formula (A-2):

52

SUBSTITUTE SHEET (RULE 26)

wherein * is the point of attachment for R and R ¹ , and _can b _e a point of attachment for the linker B.

[0098] Various ligands for Stat3 protein are disclosed, for example, in the following references:

Longchuan Bai et al., Cancer Cell 36, 498-511, November 11, 2019; https://doi.Org/10.1016/j.ccell.2019.10.002;

Haibin Zhou et al., ACS Medicinal Chemistry Letters, 2021 May 10;12(6):996-1004; https ://doi . org/ 10.1021 / acsmedchemlett.1 cOO 155 ; and

Jianyong Chen et al., ACS Med Chem Lett., 2010 May 13 ; 1 (2): 85-89. doi:

10.1021/mll00010j.

Embodiment 28

[0099] The bifunctional degrader of any one of Embodiments 1-10, wherein the protein of interest is BRD4.

Embodiment 29

53

SUBSTITUTE SHEET (RULE 26) [0100] The bifunctional degrader of Embodiment 28, wherein ligand A comprises at least one chemical moiety selected from the group consisting of: be a point of attachment for the linker B.

[0101] Various ligands for BRD4 are disclosed for example, in the following references: Germano Heinzelmann & Michael K. Gilson, Scientifc Reports, (2021) 11 : 1116; doi:

10.1038/s41598-020-80769- 1 ;

Bryce K. Allen et al., ACS Omega 2017, 2, 4760-4771, doi: 10.1021/acsomega.7b00553;

Tamara Minko, Trends Pharmacol Sci. 2020 Oct; 41(10):684-686. doi:

10.1016/j.tips.2020.08.008;

54

SUBSTITUTE SHEET (RULE 26) Serena G. Piticchio et al., J. Med. Chem. 2021, 64, 17887-17900; doi: 10.1021/acs.jmedchem. lc01108m; including supporting information at https://pubs.acs.org/doi/10.1021/acs.jmedchem.lc01108;

Fred L. Ciske and Thomas G. Brock, Ph.D., Cayman Chemical. Bromodomain Targeting with PROTACs. Article from 2017-04-05; https://www.caymanchem.com/news/bromodomain-targeting-with-p rotacs; and

US Patent 11,179,373 B2

Embodiment 30

[0102] The bifunctional degrader of Embodiment 28 or 29, wherein ligand A is

Embodiment 31

[0103] The bifunctional degrader of Embodiment 30, having the structure:

Embodiment 32

[0104] A pharmaceutical composition comprising the bifunctional degrader of any one of

Embodiments 1-31 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

Embodiment 33

[0105] The pharmaceutical composition of Embodiment 32, further comprising an additional therapeutic agent.

Embodiment 34

[0106] A method of degrading a protein selected from the group consisting of FKBP12,

BRD4, androgen receptor, estrogen receptor, IRAK4, a JAK protein, BCL-XL, BCL-2, and

55

SUBSTITUTE SHEET (RULE 26) Stat3 in a patient or biological sample comprising administering to said patient, or contacting said biological sample with the bifunctional degrader of any one of Embodiments 1-31. Embodiment 35

[0107] A method of treating a disorder, disease or condition mediated by a protein selected from the group consisting of FKBP12, BRD4, androgen receptor, estrogen receptor, IRAK4, a JAK protein, BCL-XL, BCL-2, and Stat3, in a patient, comprising administering to the patient a therapeutically effective amount of the degrader of any of Embodiments 1-31 or a pharmaceutically acceptable salt thereof.

Embodiment 36

[0108] The method of Embodiment 35, wherein the disorder, disease or condition is a cancer, a neurodegenerative disease, a viral disease, an autoimmune disease, an inflammatory disorder, a hereditary disorder, a hormone-related disease, a hematopoietic disorder, a metabolic disorder, a condition associated with organ transplantation, an immunodeficiency disorder, a destructive bone disorder, a proliferative disorder, an infectious disease, a condition associated with cell death, thrombin-induced platelet aggregation, liver disease, a pathologic immune condition involving T cell activation, a cardiovascular disorder, and a CNS disorder.

Embodiment 37

[0109] The method of either Embodiment 35 or 36, further comprising administering an additional therapeutic agent.

Embodiment 38

[0110] A DCAF1 protein-probe adduct, wherein the probe binds to cysteine residue Cl 113 of DCAF1, wherein the amino acid numbering is based on DCAF1 Isoform 1 (Accession No. Q9Y4B6-1), cysteine residue Cl 112 of DCAF1, wherein the amino acid numbering is based on DCAF1 Isoform 2 (Accession No. Q9Y4B6-2), or cysteine residue C664 of DCAF1, wherein the amino acid numbering is based on DCAF1 Isoform 3 (Accession No. Q9Y4B6-3) and wherein the probe comprises an azetidinyl acrylamide moiety.

Embodiment 39

[OHl] The DCAF1 protein-probe adduct of Embodiment 38, wherein the probe is a compound of Formula (I):

56

SUBSTITUTE SHEET (RULE 26) wherein:

X is selected from the group consisting of halo,

Embodiment 40

[0112] The DCAF1 protein-probe adduct of Embodiment 39, having the structure of

Formula (II): wherein:

[0113] S represents the sulfur atom of a cysteine residue Cl 113 of DCAF1, wherein the amino acid numbering is based on DCAF1 Isoform 1 (Accession No. Q9Y4B6-1), cysteine residue Cl 112 of DCAF1, wherein the amino acid numbering is based on DCAF1 Isoform 2 (Accession No. Q9Y4B6-2), or cysteine residue C664 of DCAF1, wherein the amino acid numbering is based on DCAF1 Isoform 3 (Accession No. Q9Y4B6-3); and

[0114] DP represents the DCAF1 polypeptide wherein the amino acid numbering is based on DCAFl Isoform 1 (Accession No. Q9Y4B6-1), DCAF1 Isoform 2 (Accession No.

Q9Y4B6-2), or DCAFl Isoform 3 (Accession No. Q9Y4B6-3).

Embodiment 41

57

SUBSTITUTE SHEET (RULE 26) [0115] The DCAF1 protein-probe adduct of any of Embodiments 38-40, wherein the probe is selected from the group consisting of:

Embodiment 42

[0116] A compound of Formula (I) or a pharmaceutically acceptable salt thereof, wherein:

58

SUBSTITUTE SHEET (RULE 26) X is selected from the group consisting of halo,

Embodiment 43

[0117] The compound of Embodiment 42, selected from the group consisting of: pharmaceutically acceptable salt thereof.

Embodiment 44

[0118] A method of agonizing or antagonizing DCAF1 protein, wherein the amino acid numbering is based on DCAF1 Isoform 1 (Accession No. Q9Y4B6-1), in a patient in need of such agonization or antagonization, or in a biological sample, comprising administering to the patient, or contacting the biological sample with the compound of Embodiment 43 or 43, or a pharmaceutically acceptable salt thereof.

59

SUBSTITUTE SHEET (RULE 26) EXAMPLES

[0119] Three isoforms of DCAF1 protein sequences.

Isoform 1 : ( identifier : Q9Y4B 6-1 )

10 20 30 40 50

MTTVWHVDS KAELTTLLEQ WEKEHGSGQD MVPILTRMSQ LIEKETEEYR

60 70 80 90 100

KGDPDPFDDR HPGRADPECM LGHLLRILFK NDDFMNALVN AYVMTSREPP

110 120 130 140 150

LNTAACRLLL DIMPGLETAV VFQEKEGIVE NLFKWAREAD QPLRTYSTGL

160 170 180 190 200

LGGAMENQDI AANYRDENSQ LVAIVLRRLR ELQLQEVALR QENKRPSPRK

210 220 230 240 250

LSSEPLLPLD EEAVDMDYGD MAVDWDGDQ EEASGDMEI S FHLDSGHKTS

260 270 280 290 300

SRVNSTTKPE DGGLKKNKSA KQGDRENFRK AKQKLGFSSS DPDRMFVELS

310 320 330 340 350

NSSWSEMSPW VIGTNYTLYP MTPAIEQRLI LQYLTPLGEY QELLPIFMQL

360 370 380 390 400

GSRELMMFYI DLKQTNDVLL TFEALKHLAS LLLHNKFATE FVAHGGVQKL

60

SUBSTITUTE SHEET (RULE 26) 410 420 430 440 450

LEIPRPSMAA TGVSMCLYYL SYNQDAMERV CMHPHNVLSD WNYTLWLME

460 470 480 490 500

CSHASGCCHA TMFFSICFSF RAVLELFDRY DGLRRLVNLI STLEILNLED

510 520 530 540 550

QGALLSDDEI FASRQTGKHT CMALRKYFEA HLAIKLEQVK QSLQRTEGGI

560 570 580 590 600

LVHPQPPYKA CSYTHEQIVE MMEFLIEYGP AQLYWEPAEV FLKLSCVQLL

610 620 630 640 650

LQLISIACNW KTYYARNDTV RFALDVLAIL TWPKIQLQL AESVDVLDEA

660 670 680 690 700

GSTVSTVGIS IILGVAEGEF FIHDAEIQKS ALQIIINCVC GPDNRISSIG

710 720 730 740 750

KFISGTPRRK LPQNPKSSEH TLAKMWNWQ SNNGIKVLLS LLSIKMPITD

760 770 780 790 800

ADQIRALACK ALVGLSRSST VRQIISKLPL FSSCQIQQLM KEPVLQDKRS

810 820 830 840 850

DHVKFCKYAA ELIERVSGKP LLIGTDVSLA RLQKADVVAQ SRISFPEKEL

860 870 880 890 900

61

SUBSTITUTE SHEET (RULE 26) LLLIRNHLI S KGLGETATVL TKEADLPMTA ASHS SAFTPV TAAASPVSLP

910 920 930 940 950

RT PRIANGIA TRLGSHAAVG ASAPSAPTAH PQPRPPQGPL ALPGPSYAGN

960 970 980 990 1000

SPLIGRI SFI RERPSPCNGR KIRVLRQKSD HGAYSQS PAI KKQLDRHLPS

1010 1020 1030 1040 1050

PPTLDS I ITE YLREQHARCK NPVATCPPFS LFTPHQCPEP KQRRQAPINF

1060 1070 1080 1090 1100

TSRLNRRASF PKYGGVDGGC FDRHLI FSRF RPI SVFREAN EDESGFTCCA

1110 1120 1130 1140 1150

FSARERFLML GTCTGQLKLY NVFSGQEEAS YNCHNSAITH LEPSRDGSLL

1160 1170 1180 1190 1200

LT SATWSQPL SALWGMKSVF DMKHSFTEDH YVEFSKHSQD RVIGTKGDIA

1210 1220 1230 1240 1250

HI YDIQTGNK LLTLFNPDLA NNYKRNCATF NPTDDLVLND GVLWDVRSAQ

1260 1270 1280 1290 1300

AIHKFDKFNM NI SGVFHPNG LEVI INTEIW DLRT FHLLHT VPALDQCRW

1310 1320 1330 1340 1350

FNHTGTVMYG AMLQADDEDD LMEERMKSPF GSSFRTFNAT DYKP IAT IDV

62

SUBSTITUTE SHEET (RULE 26) 1360 1370 1380 1390 1400

KRNIFDLCTD TKDCYLAVIE NQGSMDALNM DTVCRLYEVG RQRLAEDEDE

1410 1420 1430 1440 1450

EEDQEEEEQE EEDDDEDDDD TDDLDELDTD QLLEAELEED DNNENAGEDG

1460 1470 1480 1490 1500

DNDFSPSDEE LANLLEEGED GEDEDSDADE EVELILGDTD SSDNSDLEDD

IILSLNE

Isoform 2: (identifier: Q9Y4B6-2)

The sequence of this isoform differs from the canonical sequence as follows :

87-87: Missing.

63

SUBSTITUTE SHEET (RULE 26) 460 470 480 490 500

SHASGCCHAT MFFSICFSFR AVLELFDRYD GLRRLVNLIS TLEILNLEDQ

510 520 530 540 550

GALLSDDEIF ASRQTGKHTC MALRKYFEAH LAIKLEQVKQ SLQRTEGGIL

560 570 580 590 600

VHPQPPYKAC SYTHEQIVEM MEFLIEYGPA QLYWEPAEVF LKLSCVQLLL

610 620 630 640 650

QLISIACNWK TYYARNDTVR FALDVLAILT WPKIQLQLA ESVDVLDEAG

660 670 680 690 700

STVSTVGISI ILGVAEGEFF IHDAEIQKSA LQIIINCVCG PDNRISSIGK

710 720 730 740 750

FISGTPRRKL PQNPKSSEHT LAKMWNVVQS NNGIKVLLSL LSIKMPITDA

760 770 780 790 800

DQIRALACKA LVGLSRSSTV RQIISKLPLF SSCQIQQLMK EPVLQDKRSD

810 820 830 840 850

HVKFCKYAAE LIERVSGKPL LIGTDVSLAR LQKADWAQS RISFPEKELL

860 870 880 890 900

LLIRNHLISK GLGETATVLT KEADLPMTAA SHSSAFTPVT AAASPVSLPR

910 920 930 940 950

TPRIANGIAT RLGSHAAVGA SAPSAPTAHP QPRPPQGPLA LPGPSYAGNS

960 970 980 990 1000

PLIGRISFIR ERPSPCNGRK IRVLRQKSDH GAYSQSPAIK KQLDRHLPSP

1010 1020 1030 1040 1050

PTLDSIITEY LREQHARCKN PVATCPPFSL FTPHQCPEPK QRRQAPINFT

1060 1070 1080 1090 1100

SRLNRRASFP KYGGVDGGCF DRHLIFSRFR PISVFREANE DESGFTCCAF

1110 1120 1130 1140 1150

SARERFLMLG TCTGQLKLYN VFSGQEEASY NCHNSAITHL EPSRDGSLLL

1160 1170 1180 1190 1200

TSATWSQPLS ALWGMKSVFD MKHSFTEDHY VEFSKHSQDR VIGTKGDIAH

1210 1220 1230 1240 1250

IYDIQTGNKL LTLFNPDLAN NYKRNCATFN PTDDLVLNDG VLWDVRSAQA

1260 1270 1280 1290 1300

IHKFDKFNMN ISGVFHPNGL EVIINTEIWD LRTFHLLHTV PALDQCRVVF

1310 1320 1330 1340 1350

NHTGTVMYGA MLQADDEDDL MEERMKSPFG SSFRTFNATD YKPIATIDVK

1360 1370 1380 1390 1400

RNIFDLCTDT KDCYLAVIEN QGSMDALNMD TVCRLYEVGR QRLAEDEDEE

64

SUBSTITUTE SHEET (RULE 26)

65

SUBSTITUTE SHEET (RULE 26) Isoform 3 (identifier: Q9Y4B6-3)

The sequence of this isoform differs from the canonical sequence as follows :

225-673: Missing.

66

SUBSTITUTE SHEET (RULE 26)

General Examples for the Processes and Compounds of the Invention

[0120] So far, efforts to expand the scope of E3 ligases addressable with small-molecule ligands have largely relied on phenotypic screening ¹⁵ ' ¹⁷ or focused studies of purified E3 ligases ¹⁸ ' ¹⁹ . This work has uncovered the potential to target a range of E3 ligases with covalent chemistry ²⁰ , including DCAF16, DCAF11, RNF4, FEM1B, and RNF114 ¹⁵ ' ¹⁹ , which has enabled the design of electrophilic PROTACs that can promote targeted protein degradation at remarkably low stoichiometric engagement of the E3 ligase ¹⁵ ' ¹⁶ (reflecting the high catalytic potential of electrophilic PROTACs ⁹ ). Nonetheless, the E3 ligase-targeting components of most of these PROTACs represent simple fragments bearing highly reactive cysteine-directed electrophiles such as a-chloroacetamides, and it therefore remains unclear whether such small molecule-E3 ligase interactions can be progressed to more advanced, selective chemical probes. Indeed, in multiple cases (e.g., DCAF11 and DCAF16), initial data suggest that the electrophilic PROTACs may have the capacity to engage more than one cysteine on the E3 ligase itself, underscoring the persistent challenges facing the discovery of selective electrophilic ligands for E3 ligases.

[0121] We recently introduced an activity-based protein profiling (ABPP) strategy for the chemical proteomic discovery of small molecule-protein interactions in human cells that leverages sets of stereoisomeric electrophilic compounds, wherein the interaction are prioritized based on a combination of cellular potency, stereochemical selectivity, and sitespecificity ²¹ . Using these criteria, we discovered a striking number of cysteines on

67 SUBSTITUTE SHEET (RULE 26) structurally and functionally diverse proteins that were stereo selectively engaged in primary human T cells by a focused set of tryptoline acrylamides. Here, we screened a distinct set of stereoisomeric azetidine acrylamides in human T cells using cysteine-directed mass spectrometry (MS)-ABPP ²¹ These experiments identified a stereoselectively engaged cysteine (Cl 113) in the substrate binding domain of the Cullin4-RING E3 ligase (CRL4) substrate receptor DCAF1 (or VPRBP). We show that azetidine acrylamide-derived PROTACs promote the degradation of a target protein FBKP12 in a stereoselective manner that depends on DCAF1 and is blocked by mutation of Cl 113. We further demonstrate that the azetidine acrylamide PROTACs form a ternary complex with FKBP12 and DCAF1 and promote FBKP12 degradation at low fractional engagement (-20%) of DCAF1 C1113. These findings, taken together, designate CRL4 ^DCAF1 as an E3 ligase capable of supporting targeted protein degradation mediated by electrophilic PROTACS that act in a stereo- and site-selective manner.

Discovery of a stereo- and site-selective electrophilic compound-cysteine interaction in DCAF1

[0122] Guided by the principles of diversity-oriented synthesis ²² , specifically the design of compounds with densely functionalized and entropically constrained sp ³ -rich cores bearing one or more stereocenters, we generated a set of stereoisomeric azetidine acrylamides (Figure 1A) and screened these compounds by cysteine-directed MS-ABPP ²³ ' ²⁶ in primary human T cells. Across more than 10000 quantified cysteines, these experiments uncovered several azetidine acrylamide-cysteine interactions (Figure IB and Dataset SI), including the stereo- (Figure IB, C) and site- (Figure ID) selective engagement of Cl 113 on the E3 ligase substrate adaptor DCAF1. Among the azetidine acrylamides, MY-1B (2) showed the strongest reactivity with DCAF1 C1113, while the enantiomer MY-1 A (1) was inactive (Figure IB, C). A weaker enantioselective interaction was also observed for DCAF1 C1113 with MY-3B (4) compared to MY-3A (3) (Figure IB, C). Other quantified cysteines in DCAF1 were unaffected by MY-1B (Figure ID).

[0123] DCAF1 is a multi-domain substrate receptor for CRL4 ligases (Figure 2A) that has diverse physiological and disease functions, including being co-opted by the HIV-2 virus to promote degradation of the antiviral host protein SAMHD1 ²⁷ ' ²⁸ . This outcome is achieved by interactions between DCAF1 and the viral protein Vpx, and, interestingly, Cl 113, which is located in the WD40 domain of DCAF1, resides in close proximity to the Vpx interface ²⁹ ' ³⁰

68

SUBSTITUTE SHEET (RULE 26) (Figure 2B). We verified the stereo- and site-selective engagement of DCAF1 C1113 by azetidine acrylamides using alkynylated analogues of MY-1 A and MY-1B (MY-11 A (5) and MY-1 IB (6), respectively; Figure 2C). For these experiments, we doped into HEK293T cell lysates a recombinantly expressed and purified form of DCAF1 containing the WD40 domain (amino acids (a.a.’s) 1046-1396), in complex with the CRL4 adaptor protein DDB I ³¹ , and exposed these samples to varying concentrations of MY-11 A or MY-1 IB. After 1 h, we assessed MY-11 A and MY-1 IB engagement of DCAF1 by copper-catalyzed azide-alkyne cycloaddition (CuAAC) ³² with an azide-rhodamine reporter tag followed by SDS-PAGE and in-gel fluorescence scanning ³³ . This gel-ABPP experiment revealed much greater concentration-dependent labeling of recombinant DCAF1 by MY-1 IB versus MY-11 A, and this labeling was blocked by mutation of Cl 113 to alanine (DCAF1-C1113 A mutant) (Figure 2D) or by pre-treatment with MY-1B, but not MY-1 A (6.25 - 100 pM, 2 h pre-treatment; Figure 2E, right). We used gel-ABPP to quantify an in vitro target engagement value (TE50) of approximately 25 pM (95% C.I. = 13 - 38 pM) for MY-1B labeling of DCAF1 (Figure 2E, left). Modeling studies also supported a preferred interaction of DCAF1 C1113 with MY-1B over the other stereoisomers MY-1 A, MY-3A, and MY-3B (Figure SI). These data, combined with our original MS-ABPP studies in T cells, indicate that MY-1B serves as a stereo- and site-selective covalent ligand for Cl 113 of DCAF1.

Design and characterization of DCAFl-directed electrophilic PROTACs.

[0124] Considering the location of Cl 113 at the interface of DCAF1 binding to the viral Vpx protein, which in turn recruits the host antiviral protein SAMHD1 for ubiquitination and degradation ³⁰ , we hypothesized that covalent ligands targeting this cysteine might serve as the basis for PROTAC-mediated targeted protein degradation. The structural model for the MY-1B-DCAF1 C1113 complex suggested multiple potential exit vectors for PROTAC construction, including the para position of the phenyl ring and the acrylamide electrophile (Figure S2). For our initial studies, we elected to focus on constructing PROTACs that extended from the phenyl ring so that the acrylamide electrophile was left intact. We synthesized two heterobifunctional compounds - YT41R (7) and YT47R (9) - that connected MY-1B to the small molecule SLF (Figure 3A), which is a high-affinity ligand for FKBP12 that is frequently used for assessing PROTAC performance ¹⁵ ' ^{16, 34} ' ³⁷ . YT41R and YT47R differed in the length of the PEG (polyethylene glycol) linker connecting the MY-1B and

69

SUBSTITUTE SHEET (RULE 26) SLF units, and we also prepared two analogous control probes based on the inactive enantiomer MY- 1 A (YT41S (8) and YT47S (10); Figure 3A).

[0125] Gel-ABPP experiments revealed that both YT41R and YT47R produced a concentration-dependent inhibition of MY-1 IB probe reactivity with recombinant DCAF1 (Figure 3B). The blockade of MY-1 IB labeling plateaued at -75% at 20 pM test concentrations of YT41R and YT47R, which could reflect the limited solubility of these candidate PROTACs at higher concentrations. In contrast, YT41S and YT47S did not impair the MY-11B-DCAF1 interaction (Figure 3B). Interestingly, we also observed an apparent gel-shift for recombinant DCAF1 by Coomassie blue staining in the presence of YT41R or YT47R (but not YT41S or YT47S), likely reflecting the substantial molecular weight change in the protein caused by covalent reaction with these compounds, which have MWs > 1000 Da (Figure 3B, lower image).

[0126] We next treated HEK293T cells co-transfected with FLAG epitope-tagged full length DCAF1 (a.a.’s 1 - 1507) and HA (hemagglutinin) epitope-tagged FKBP12 with a concentration range of YT41R or YT47R and, after 24 h, measured HA-FKBP12 abundance by Western blotting. Robust concentration-dependent degradation of HA-FKBP12 was observed for both YT41R and YT47R in co-transfected cells, but not HEK293T cells transfected only with HA-FKBP12 (Figure 4A). Evidence of YT41R/YT47R-induced degradation of FKBP12 was observed at concentrations of YT41R and YT47R as low at 0.25 pM (Figure S3) with a possible modest hook effect emerging at 5 pM (Figure 4A). Timecourse studies revealed limited evidence of HA-FKBP12 degradation until -24 h after treatment of FLAG-DCAF1 -transfected cells with YT41R and YT47R (Figure S4), which could reflect the time required for PROTAC -mediated degradation to overcome the robust expression of HA-FKBP12 in a transient co-transfection system.

[0127] YT41R and YT47R acted in a stereoselective manner, as neither of the control enantiomeric probes YT41S and YT47S supported HA-FKBP12 degradation (Figure 4B). Additionally, we found that YT41R/YT47R did not promote the degradation of FKBP12 in cells expressing a DCAF1-C1113 A mutant (Figure 4C).

Taken together, these data indicate YT41R and YT47R act as electrophilic PROTACs that promote targeted protein degradation through the stereoselective engagement of Cl 113 of DCAF1.

Mechanistic studies of DCAFl-directed electrophilic PROTACs.

70

SUBSTITUTE SHEET (RULE 26) [0128] YT41R/YT47R-mediated degradation of HA-FKBP12 was blocked by pretreatment with the proteasome inhibitor MG132 (5 pM, 1 h pre-treatment followed by 24 h co-treatment with YT41R/YT47R) (Figure 5A); however, interpretation of these experiments was complicated by the increased quantities of both HA-FKBP12 and FLAG-DCAF1 observed in MG132-treated cells (Figure 5A). On the other hand, treatment with the NAE inhibitor MLN4924, which blocks the Neddylation and activity of cullin-RING E3 ligases ³⁸ , also impaired YT41R/YT47R-mediated degradation of HA-FKBP12 without causing alterations in the quantity of HA-FKBP12 or FLAG-DCAF1 in control cells (Figure 5A). These data support that the targeted protein degradation activity of YT41R and YT47R is proteasome-and cullin-RING E3 ligase-dependent.

[0129] PROTAC -mediated protein degradation requires ternary complex formation between the target protein, the heterobifunctional compound, and the E3 ligase, and this process can often be competitively disrupted by monovalent ligands targeting individual components of the complex. Consistent with this model for YT41R/YT47R action, we found that pre-treatment of HA-FKBP12 and FLAG-DCAF1 co-transfected HEK293T cells with either the FKBP12 ligand SLF (20 pM) or the DCAF1 ligand MY-1 IB (5 pM) blocked YT41R and YT47R-mediated degradation of HA-FKBP12 (Figure 5B). The control enantiomer MY-11 A, which does not engage DCAF1 (Figure 2D), did not block YT41R/YT47R-mediated HA-FKBP12 degradation (Figure 5C).

[0130] Finally, we investigated whether electrophilic PROTACs induce a stable ternary complex of FKBP12 with DCAF1 and promote FKBP12 ubiquitination. These coimmunoprecipitation experiments were performed in HEK293T cells co-transfected with HA-FKBP12 and FLAG-DCAF1 and treated with YT47R (or the control enantiomer YT47S) in the presence of MG132 (to block FKBP12 degradation) and revealed clear evidence of a ternary complex of FKBP12 and DCAF1 with YT47R, but not YT47S (Figure 6A). A ternary complex was not observed in cells expressing the DCAF1-C1113 A mutant (Figure 6A), supporting that YT47R induces this complex through site-specifically reacting with Cl 113. We also detected robust polyubiquitination of the immunoprecipitated HA-FKBP12 in YT47R-treated, DCAFl-WT-expressing cells compared to YT47S- or DMSO-treated, DCAFl-WT-expressing cells or YT47R-treated, DCAF1-C1113A-expressing cells (Figure 6A). Interestingly, we also observed increased FKBP12 ubiquitination in YT47R- versus YT47S-treated HEK293T cells that did not express exogenous DCAF1 (Figure 6A). We wondered whether this outcome might be mediated by endogenous DCAF1, and, consistent

71

SUBSTITUTE SHEET (RULE 26) with this possibility, we found that endogenous DCAF1 formed a more robust ternary complex with HA-FKBP12 in HEK293T cells treated with YT47R compared to YT47S or DMSO (Figure 6B). In considering possible reasons why this ternary complex formation with endogenous DCAF1 was insufficient to support the degradation of recombinantly expressed FKBP12 (Figure 4A), we speculate that our current PROTACs (YT41R, YT47R) may require further improvements in cellular properties to enable robust targeted protein degradation mediated by physiological concentrations of DCAF1. Possibly supporting this hypothesis, MS-ABPP experiments revealed that YT47R (5 pM) only engaged -20% of Cl 113 of recombinantly expressed DCAF1 (Figure 6C and Data set S2). This minor engagement, which is consistent with previous studies of other electrophilic PROTACs that also act sub-stoichiometrically (and presumably catalytically) to degrade proteins ¹⁵ ' ¹⁶ , was both stereo- and site-selective (Figure 6C) and suggests more optimized PROTACs that engage a greater fraction of DCAF1 C1113 in cells may enable targeted protein degradation by endogenous DC AF 1.

[0131] Here, we have described the chemical proteomic discovery of a ligandable cysteine (Cl 113) in the E3 ligase substrate receptor protein DCAF1 and the conversion of azetidine acrylamides targeting this cysteine into electrophilic PROTACs. These findings are significant because, to our knowledge, they describe the first electrophilic PROTACs that have been shown to act in a stereo- and site-selective manner. The properties of stereoselectivity and site-specificity provide convenient ways to establish controls to verify on-target activity for electrophilic PROTACs, as we have shown herein for DCAF1 and others have demonstrated for reversibly binding PROTACs that engage VHL in a stereoselective manner ³⁹ . We further interpret these features to indicate the presence of a high-quality druggable pocket in proximity to DCAF1 C1113. Also supportive of this conclusion is the recent report of a noncovalent DCAF1 ligand that binds a pocket near Cl 113 (PDB code: 7SSE, Figure S5). While we have taken advantage of the druggability of DCAF1 C1113 to create electrophilic PROTACs, we also imagine that more advanced covalent ligands might serve as molecular glues ⁵ ' ⁷ or antagonists of the various physiological and pathological functions of DCAF1 ⁴⁰ ' ⁴⁴ . Indeed, small-molecule antagonists of DCAF1 could counteract the pathogenesis of viruses like HIV-1 and HIV-2 that co-opt this E3 ligase substrate receptor to suppress immune cell responses ^{30, 44} ' ⁴⁷ . More generally, the rich dataset of stereoselective interactions reported herein between azetidine acrylamides and cysteines in the human T-cell proteome (Figure IB and Dataset SI) should offer attractive starting points

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SUBSTITUTE SHEET (RULE 26) for chemical probe discovery for additional proteins from structurally and functionally diverse classes.

[0132] As has been described previously ¹⁵ ' ¹⁹ , electrophilic PROTACs have the potential to maximally leverage the catalytic potential of targeted protein degradation by creating “neo”-E3 ligases that are permanently modified (until physical turnover) with a substratebinding compound. However, key variables can impact the success of such endeavors, including the half-life and substrate compatibility of the E3 ligase ¹¹ ' ¹⁴ , as well as the quality of the chemical probes that target it. On the latter point, we view the azetidine acrylamide reactive group found herein to stereoselectively engage DCAF1 C1113 as an encouraging starting point for probe optimization, especially in comparison to electrophilic PROTACs reported to target other E3 ligases, which mostly use high-reactivity groups such as a- chloroacetamides ¹⁵ ' ^{16, 18} ' ¹⁹ . Nonetheless, we have, so far, only observed targeted protein degradation for electrophilic PROTACs in cells expressing recombinant DCAF1, and it will be important in future studies to determine if this activity can be extended to endogenous DCAF1. That we observed evidence of electrophilic PROTACs promoting a ternary complex with endogenous DCAF1 and FBKP12, as well as inducing FKBP12 ubiquitination, is encouraging, even though these effects were not apparently robust enough to lead to substantial FKBP12 degradation in cells expressing endogenous DCAF1 (possibly due to counteracting cellular deubiquitinases ⁹ ). Further improvements in electrophilic PROTAC performance may require greater levels of cellular engagement of DCAF1, as our first- generation compounds only appear to modify < 20% of recombinant DCAF1 at functional concentrations in cells. A recent study has also described an autoinhibitory oligomerization mechanism for DCAF1 that may not be shared by other CRL4 substrate receptors ⁴⁸ . If a substantial proportion of endogenous DCAF1 is in an autoinhibited tetrameric state, then a greater quantity of total DCAF1 may need to be modified by electrophilic PROTACs to support targeted protein degradation. On the other hand, we wonder whether this autoregulatory feature of DCAF1 might also be exploited to create electrophilic PROTACs and/or molecular glues that carry out context-dependent protein degradation only in those cell types where DCAF1 is in an activated state.

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SUBSTITUTE SHEET (RULE 26) References

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SUBSTITUTE SHEET (RULE 26) EXAMPLES

General Information

[0133] Compounds of the invention can be made by a variety of methods depicted in the illustrative synthetic reactions described below in this Examples section.

The starting materials and reagents used in preparing these compounds generally are either available from commercial suppliers, such as Aldrich Chemical Co., or are prepared by methods known to those skilled in the art following procedures set forth in references such as Fieser and Fieser's Reagents for Organic Synthesis,' Wiley & Sons: New York, 1991, Volumes 1-15; Rodd's Chemistry of Carbon Compounds, Elsevier Science Publishers, 1989, Volumes 1-5 and Suppiementals; and Organic Reactions, Wiley & Sons: New York, 1991, Volumes 1-40. It should be appreciated that the synthetic reaction schemes shown in the Examples section are merely illustrative of some methods by which the compounds of the invention can be synthesized, and various modifications to these synthetic reaction schemes can be made and will be suggested to one skilled in the art having referred to the disclosure contained in this application.

[0134] The starting materials and the intermediates of the synthetic reaction schemes can be isolated and purified if desired using conventional techniques, including but not limited to, filtration, distillation, crystallization, chromatography, and the like. Such materials can be characterized using conventional means, including physical constants and spectral data. [0135] Unless specified to the contrary, the reactions described herein are typically conducted under an inert atmosphere at atmospheric pressure at a reaction temperature range of from about -78 °C to about 150 °C, often from about 0 °C to about 125 °C, and more often and conveniently at about room (or ambient) temperature, e.g., about 20 °C.

[0136] Various substituents on the compounds of the invention can be present in the starting compounds, added to any one of the intermediates or added after formation of the final products by known methods of substitution or conversion reactions. If the substituents themselves are reactive, then the substituents can themselves be protected according to the techniques known in the art. A variety of protecting groups are known in the art, and can be employed. Examples of many of the possible groups can be found in “Protective Groups in Organic Synthesis" by Green et al., John Wiley and Sons, 1999. For example, nitro groups can be added by nitration and the nitro group can be converted to other groups, such as amino by reduction, and halogen by diazotization of the amino group and replacement of the diazo group with halogen. Acyl groups can be added by Friedel-Crafts acylation. The acyl groups

79

SUBSTITUTE SHEET (RULE 26) can then be transformed to the corresponding alkyl groups by various methods, including the Wolff-Kishner reduction and Clemmenson reduction. Amino groups can be alkylated to form mono- and di-alkylamino groups; and mercapto and hydroxy groups can be alkylated to form corresponding ethers. Primary alcohols can be oxidized by oxidizing agents known in the art to form carboxylic acids or aldehydes, and secondary alcohols can be oxidized to form ketones. Thus, substitution or alteration reactions can be employed to provide a variety of substituents throughout the molecule of the starting material, intermediates, or the final product, including isolated products.

[0137] All chemicals, including anhydrous solvents, were obtained from commercial suppliers and used without further purification. Merck silica gel TLC plates (0.25 mm, 60 F254) were used to monitor reaction progress and were visualized under UV light (254 nm) or by staining with potassium permanganate (KmnCh). Flash chromatography was performed using SiliaFlash® F60 silica gel (SiCh, 40-60 pM, 60 A), loaded with dichloromethane unless otherwise noted. NMR spectra were recorded on a Bruker 600 or 500 MHz instrument. All ¹ H NMR chemical shifts (5) are reported in parts per million (ppm) relative to residual signals for CHCh (7.26 ppm) or CH3OH (3.31 ppm) in the deuterated solvent. Reactions were monitored by LCMS and TLC. High-resolution mass spectra (HRMS) were obtained on an Agilent LC/MSD TOF mass spectrometer by electrospray ionization-time-of-flight (ESI- TOF). Analytical supercritical fluid chromatography (SFC) was performed on a Shimadzu LC system (flow rate: 3 mL/min, back pressure: 100 Bar, column temperature: 35 °C) equipped with a polydiode array detector unless otherwise noted. Tandem liquid chromatography/mass spectrometry (LC-MS) was performed on an Agilent 1200 series LC/MSD system equipped with an Agilent G6110A mass detector, alternatively a Shimadzu LC-20AD or AB series LC-MS system equipped with Shimadzu SPD-M20A or SPDM40 mass detectors, alternatively a Waters H-Class LC with equipped with diode array and Qda mass detector.

Materials and methods

[0138] All reactions were carried out under positive pressure of argon unless otherwise noted. Glassware was oven-dried at 120 °C for a minimum of 12 hours, or flame-dried with a propane torch under vacuum (< 1 torr). Anhydrous dichloromethane (CH2Q2) was distilled from calcium hydride (5% w/v) under positive pressure of nitrogen. Anhydrous tetrahydrofuran (THF) containing 250 ppm BHT (peroxide inhibitor) was purchased from MilliporeSigma / SigmaAldrich. Anhydrous toluene was obtained by passing the previously

80

SUBSTITUTE SHEET (RULE 26) degassed solvent through an activated alumina column. Other commercially available solvents or reagents were used without further purification unless otherwise noted. Reactions were monitored by thin layer chromatography (TLC) using precoated silica gel plates from EMD Chemicals (TLC Silica gel 60 F254, 250 pm thickness). Flash column chromatography was performed over Silica gel 60 (particle size 0.04- 0.063 mm) from EMD Chemicals and activated neutral alumina (Brockmann I, 150 mesh) from Sigma- Aldrich. Room temperature or ambient temperature in Beckman Building, Lab 420 is 22 °C. Organic solvent from crude reaction mixtures and solutions of pure compounds was evaporated on a Buchi Rotavapor R3 (rotavap or rotovap, referred to in the experimental procedures).

[0139] Hexanes (ACS grade), ethyl acetate (ACS grade), diethyl ether (anhydrous ACS grade), dichloromethane (ACS grade), chloroform (ACS grade), and isopropanol (ACS grade) were purchased from Fisher Chemical and used without further purification. Anhydrous tetrahydrofuran, DMF, and acetonitrile were purchased from Sigma-Aldrich. Anhydrous DMSO was purchased from Acros Organics. Anhydrous ethanol was obtained from Pharmco-Aaper. [Ir{dF(CF3)ppy}2(dtbbpy)]PFe was prepared according to the procedure of Lowry et al. ¹ Photocatalysts 3CzCHPN and 4CzIPN were prepared according to the procedure of Zeitler et al ¹ NiCL’glyme and IrCh’nHiO were purchased from Strem Chemicals. Commercially available substrates were used without further purification unless otherwise noted. The reactions were monitored by thin layer chromatography (TLC) using precoated silica gel plates from EMD Chemicals (TLC Silica gel 60 F254) or by LC/MS on an Agilent 6120 Quadrupole system with an ESI probe. Flash column chromatography was performed over Silica gel 60 (particle size 0.04-0.063 mm) from Fischer Scientific or Florsil® from Sigma Aldrich or Acros Organics. ¹ H NMR and ¹³ C NMR spectra were recorded on Bruker DPX-400, a Bruker DPX-500 or Bruker DPX-600 equipped with cry oprobe, and the residual solvent peaks were used as internal standard (CDCL: 7.26 ppm 'H NMR, 77.16 ppm, DMSO-t/ ₆ : 2.50 ppm 'H NMR, 39.52 ppm, MeOH-t/ ₄ : 3.31 ppm 'H NMR, 49.00 ppm ¹³ C NMR. NMR data is denoted with apparent multiplicities: s = singlet, d = doublet, t = triplet, q = quartet, and combinations thereof.

Abbreviations

[0140] DCAF1 = DDBl-Cul4-associated factor 1; DDB1 = DNA damage-binding protein 1; Cul4=Cullin-4A; FKBP12 = FK506-binding protein 12; BRD4 =Bromodomain- containing protein 4; IRAK4 = interleukin- 1 receptor-associated kinase 4; JAK = Janus

81 SUBSTITUTE SHEET (RULE 26) kinase; BCL-XL = B-cell lymphoma-extra large; BCL-2 = B-cell lymphoma 2; Stat3 = Signal transducer and activator of transcription 3.

[0141] Commonly used abbreviations include: acetyl (Ac), azo-Zh -isobutyrylnitrile (AIBN), atmospheres (Atm), 9-borabicyclo[3.3.1]nonane (9-BBN or BBN), tertbutoxycarbonyl (Boc), di-tert-butyl pyrocarbonate or boc anhydride (BOC2O), benzyl (Bn), butyl (Bu), Chemical Abstracts Registration Number (CASRN), benzyloxycarbonyl (CBZ or Z), carbonyl diimidazole (CDI), l,4-diazabicyclo[2.2.2]octane (DABCO), di ethylaminosulfur trifluoride (DAST), dibenzylideneacetone (dba), l,5-diazabicyclo[4.3.0]non-5-ene (DBN), l,8-diazabicyclo[5.4.0]undec-7-ene (DBU), N,N'-dicyclohexylcarbodiimide (DCC), 1,2- di chloroethane (DCE), dichloromethane (DCM), diethyl azodi carb oxy late (DEAD), di-z.w- propylazodicarboxylate (DIAD), di-Ao-butylaluminumhydride (DIBAL or DIBAL-H), di- iso-propylethylamine (DIPEA), N,N-dimethyl acetamide (DMA), 4-N,N- dimethylaminopyridine (DMAP), N,N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), l,l'-Z>A-(diphenylphosphino)ethane (dppe), l,l'-Z>z5-(diphenylphosphino)ferrocene (dppf), 1 -(3 -dimethylaminopropyl)-3 -ethylcarbodiimide hydrochloride (EDCI), ethyl (Et), ethyl acetate (EtOAc), ethanol (EtOH), 2-ethoxy-2J/-quinoline-l -carboxylic acid ethyl ester (EEDQ), diethyl ether (Et2O), O-(7-azabenzotriazole-l-yl)-N, N,N’N’-tetramethyluronium hexafluorophosphate acetic acid (HATU), acetic acid (HO Ac), 1-N-hydroxybenzotriazole (HOBt), high pressure liquid chromatography (HPLC), z.w-propanol (IP A), lithium hexamethyl disilazane (LiHMDS), methanol (MeOH), melting point (mp), MeSO2- (mesyl or Ms), , methyl (Me), acetonitrile (MeCN), zzz-chloroperbenzoic acid (MCPBA), mass spectrum (ms), methyl /-butyl ether (MTBE), N-bromosuccinimide (NBS), N-carboxyanhydride (NCA), N-chlorosuccinimide (NCS), N-methylmorpholine (NMM), N-methylpyrrolidone (NMP), pyridinium chlorochromate (PCC), pyridinium dichromate (PDC), phenyl (Ph), propyl (Pr), z.w-propyl (z-Pr), pounds per square inch (psi), pyridine (pyr), room temperature (rt or RT), tez7-butyldimethylsilyl or Z-BuMe2Si (TBDMS), triethylamine (TEA or EtsN), 2,2,6,6-tetramethylpiperidine 1-oxyl (TEMPO), triflate or CF3SO2- (Tf), tri fluoroacetic acid (TFA), l,l'-Z>z5-2,2,6,6-tetramethylheptane-2,6-dione (TMHD), O-benzotriazol-l-yl- N,N,N',N'-tetramethyluronium tetrafluoroborate (TBTU), thin layer chromatography (TLC), tetrahydrofuran (THF), trimethyl silyl or MesSi (TMS), -toluenesulfonic acid monohydrate (TsOH or pTsOH), 4-Me-CeH4SO2- or tosyl (Ts), N-urethane-N-carboxyanhydride (UNCA),. Conventional nomenclature including the prefixes normal (n), iso (i-), secondary (sec-), tertiary tert-) and neo have their customary meaning when used with an alkyl moiety. (J.

82

SUBSTITUTE SHEET (RULE 26) Rigaudy and D. P. Klesney, Nomenclature in Organic Chemistry, IUPAC 1979 Pergamon Press, Oxford.).

83

SUBSTITUTE SHEET (RULE 26) Synthesis of MY- 1 A

To a precooled (0 °C) solution of SI (50.0 mg, 131 pmol) ⁴ in dichloromethane (1 mL) were added triethylamine (26.5 mg, 262 pmol) and acryloyl chloride (14.2 mg, 157 pmol). The mixture was stirred at 0 °C for 0.5 hours. Upon completion, the reaction mixture was concentrated under reduced pressure to obtain a residue, which was purified by prep-TLC (SiCh, petroleum ether/EtOAc = 2:1) to give MY-1A (51.0 mg, 89% yield) as a white solid. HRMS ESI-TOF m/z calculated for C22Hi ₉ BrN ₃ O2 [M+H] ⁺ 436.0655. Found 436.0646. ⁴ H NMR (400 MHz, CDCh): 5 10.47 (br s, 1H), 8.81 (d, J= 3.6 Hz, 1H), 8.43 (d, J= 4.0 Hz, 1H), 8.16 (d, J= 8.0 Hz, 1H), 7.54-7.42 (m, 3H), 7.29-7.25 (m, 4H + CHCh), 6.53 (d, J = 16.3 Hz, 1H), 6.48-6.18 (m, 1H), 5.98-5.62 (m, 1H), 5.38 (d, J= 8.0 Hz, 1H), 4.64 (t, J= 9.3 Hz, 1H), 4.59-4.40 (m, 1H), 4.33-4.24 (m, 1H).

Synthesis of MY- IB

Prepared in analogous fashion from ewt-Sl ⁴ .

(2A,35)-l-acryloyl-3-(4-bromophenyl)-A-(84uinoline-8-yl)a zetidine-2-carboxamide (MY- 1B)

84

SUBSTITUTE SHEET (RULE 26)

HRMS ESI-TOF m/z calculated for C22Hi ₉ BrN ₃ O2 [M+H] ⁺ 436.0655. Found 436.0649.

^X H NMR (400 MHz, CDCh): 5 10.47 (s, 1H), 8.80 (d, J= 4.0 Hz, 1H), 8.42 (d, J= 4.0 Hz, 1H), 8.15 (d, J= 8.0 Hz, 1H), 7.52-7.40 (m, 3H), 7.30-7.25 (m, 4H + CHCh), 6.64-6.15 (m, 2H), 5.90-5.64 (m, 1H), 5.37 (d, J= 8.0 Hz, 1H), 4.63 (t, J= 9.3 Hz, 1H), 4.57-4.38 (m, 1H), 4.33-4.22 (m, 1H).

Synthesis of MY-3A

(25,35)-3-(4-bromophenyl)-l-(terLbutoxycarbonyl)azetidine -2-carboxylic acid (S3)

To a solution of c -S I (450 mg, 1.18 mmol) in acetonitrile (3 mL) was added BOC2O (308 mg, 1.41 mmol), and the resulting mixture was stirred at 50 °C for 15 min. Upon completion, the reaction mixture was filtered over Celite and concentrated under reduced pressure to give S2 (600 mg, crude) as a yellow oil. To a solution of S2 (450 mg, 933 pmol) in ethanol (3 mL) was added sodium hydroxide (373 mg, 9.33 mmol), and the resulting mixture was stirred at 110 °C for 15 min. Upon completion, the mixture was diluted with water and washed with

85

SUBSTITUTE SHEET (RULE 26) di chloromethane (2x 100 mL). The resulting aqueous solution was acidified with HC1 (1 M) to adjust pH to 5~6, exhaustively extracted with z-PrOH/CHCL (3:7, 5x60 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give S3 (330 mg, 99% yield over two steps) as a white solid.

LC-MS m/z calculated for CisHwBrNCU [M+H] ⁺ 356.0. Found 356.0.

(25,35)-3-(4-bromophenyl)-7V-(86uinoline-8-yl)azetidine-2 -carboxamide (S5) To a solution of S3 (330 mg, 926 pmol) in dichloromethane (4 mL) were added diisopropylethylamine (239 mg, 1.85 mmol) and 8-aminoquinoline (23.7 mg, 262 pmol), followed by HATU (705 mg, 1.85 mmol). The resulting mixture was stirred at 25 °C for 2 hours. Upon completion, the reaction mixture was concentrated under reduced pressure to give a residue, which was purified by prep-TLC (SiCh, petroleum ether/EtOAc = 2: 1) to give S4 (200 mg) as a yellow solid used directly in the next step. To a solution of S4 (100 mg) in dichloromethane (1 mL) was added HCl/dioxane (4 M, 1 mL). and the resulting mixture was stirred at 25 °C for 1 hour. Upon completion, the reaction mixture was partitioned between ethyl acetate (40 mL) and brine (30 mL). The water layer was extracted with z-PrOH/CHCL (3:7, 5x20 mL), then the organic phase was dried over sodium sulfate, filtered, and concentrated under reduced pressure to give S5 (90.0 mg, 51% yield over two steps) as an off-white solid.

LC-MS m/z calculated for Ci9Hi ₇ BrN ₃ O [M+H] ⁺ 382.1. Found 382.1.

(25,35)- l-acryloyl-3-(4-bromophenyl)-A-(86uinoline-8-yl)azetidine-2- carboxamide (MY-3A)

86

SUBSTITUTE SHEET (RULE 26) To a solution of S5 (80.0 mg, 209 pmol) in dichloromethane (1 mL) were added triethylamine (42.4 mg, 419 pmol) and acryloyl chloride (37.9 mg, 419 pmol). The mixture was stirred at 25 °C for 2 hours. Upon completion, the reaction mixture was concentrated under reduced pressure. The residue was purified by preparative TLC (SiCh, petroleum ether/EtOAc = 2: 1) to give MY-3 A (34.0 mg, 37% yield) as a white solid.

HRMS ESI-TOF m/z calculated for C ₂₂ Hi ₉ BrN ₃ O2 [M+H] ⁺ 436.0655. Found 436.0649.

^X H NMR (400 MHz, CDCh): 5 11.13-10.67 (m, 1H), 8.95-8.84 (m, 1H), 8.83-8.75 (m, 1H), 8.16 (d, J= 8.0 Hz, 1H), 7.60-7.50 (m, 4H), 7.45 (dd, J= 8.2, 4.2 Hz, 1H), 7.33-7.28 (m, 2H), 6.54 (br d, J= 16.9 Hz, 1H), 6.42-6.25 (m, 1H), 5.91-5.75 (m, 1H), 5.18-4.95 (m, 1H), 4.76-4.55 (m, 1H), 4.43-4.14 (m, 2H).

Synthesis of MY-3B

Prepared in analogous fashion from SI.

(25,35)-l-acryloyl-3-(4-bromophenyl)-A-(87uinoline-8-yl)a zetidine-2-carboxamide (MY-3B) HRMS ESI-TOF m/z calculated for C ₂₂ Hi ₉ BrN ₃ O2 [M+H] ⁺ 436.0655. Found 436.0643.

^X H NMR (400 MHz, CDCh): 5 10.95 (br s, 1H), 8.97-8.69 (m, 2H), 8.15 (d, J= 8.2 Hz, 1H), 7.55 (d, J= 4.7 Hz, 2H), 7.52 (d, J= 8.1 Hz, 2H), 7.45 (dd, J= 8.3, 4.2 Hz, 1H), 7.28 (d, J= 8.2 Hz, 2H), 6.53 (d, J= 16.9 Hz, 1H), 6.41-6.27 (m, 1H), 5.90-5.75 (m, 1H), 5.15-5.02 (m, 1H), 4.73-4.59 (m, 1H), 4.37-4.21 (m, 2H).

87

SUBSTITUTE SHEET (RULE 26)

(2R,3S)-3-(4-formylphenyl)-N-(88uinoline-8-yl)-l-(2,2,2-t rifluoroacetyl)azetidine-2- carboxamide (S7)

In a dry flask, S6 (2.42 g, 7.49 mmol, 1.0 equiv.) was dissolved in dry DCE (7.5 mL). To the flask were added AgOAc (2.50 g, 15.0 mmol, 2.0 equiv.), (BnO^PChH (625 mg, 2.25 mmol, 0.3 equiv.), Pd(Oac)2 (252 mg, 1.12 mmol, 0.15 equiv.), and 4-iodobenzaldehyde (5.20 g, 22.4 mmol, 3.0 equiv.). The reaction was heated to 110 °C under nitrogen atmosphere for 36 h. The mixture was then cooled to ambient temperature, concentrated and directly purified by flash chromatography (SiCh, Hexanes/EtOAc = 3 : 1 to 1 : 1) to yield S7 as a yellow oil (1.28 g, 40%).

^X H NMR (600 MHz, CDCh) 5 10.12 (s, 1H), 9.83 (s, 1H), 9.81 (s, 0.5H), 9.74 (s, 0.5H), 8.78 (d, J= 3.3 Hz, 1H), 8.76 (d, J= 3.6 Hz, 0.5H), 8.37 (d, J= 7.5 Hz, 1.5H), 8.15 (dd, J= 8.2, 1.3 Hz, 1.5H), 7.68 (d, J= 8.1 Hz, 2H), 7.60 (d, J= 8.0 Hz, 10H), 7.56 (d, J= 8.1 Hz, 3H), 7.52 (d, J= 8.0 Hz, 1H), 7.51 - 7.45 (m, 4H), 7.42 (q, J= 8.5, 8.1 Hz, 2H), 7.38 (d, J= 3.9 Hz, 1H), 5.70 (d, J= 8.2 Hz, 0.5H), 5.51 (d, J= 9.8 Hz, 1H), 5.12 - 5.03 (m, 0.4H), 4.99 - 4.92 (m, 1H), 4.86 (t, J= 92 Hz, 1H), 4.74 (d, J= 7.5 Hz, 0.5H), 4.64 - 4.52 (m, 2H), 4.28 - 4.07 (m, 0.5H).

HRMS (ESI+) m/z calculated for C22H17F3N3O3 [M+H] ⁺ 428.1217. Found 428.1219.

(2R,3S)-l-acryloyl-3-(4-ethynylphenyl)-N-(88uinoline-8-yl )azetidine-2-carboxamide (MY- 11B)

In a 1-dram vial, S7 (160 mg, 0.37 mmol, 1.0 equiv.) was dissolved in methanol (3 mL). K2CO3 (77.0 mg, 0.56 mmol, 1.5 equiv.) was then added, followed by Ohira-Bestmann reagent (83.5 pL, 0.56 mmol, 1.5 equiv.). The reaction was allowed to stir at 23 °C for 24 h, then quenched with saturated NaHCOs (aq), extracted with ethyl acetate (6 times). The combined organic phase was then washed with brine, dried with Na2SO4, concentrated, and used directly in the next step.

88

SUBSTITUTE SHEET (RULE 26) The crude mixture was dissolved in dichloromethane (1 mL), then triethylamine (0.10 mL, 0.75 mmol, 2.0 equiv.) and acryloyl chloride (40.3 mg, 0.44 mmol, 1.2 equiv.) were added at 0 °C. The mixture was stirred at 0 °C for 0.5 hours. Upon completion, the reaction mixture was concentrated under reduced pressure and purified by prep-TLC to give MY-1 IB (27.0 mg, 22% yield) as a white solid.

'H NMR (600 MHz, CDCh) 5 10.47 (s, 1H), 8.89 - 8.68 (m, 1H), 8.40 (d, J= 13 Hz, 1H), 8.14 (d, J= 7.9 Hz, 1H), 7.54 - 7.39 (m, 3H), 7.34 (d, J= 7.9 Hz, 2H), 7.27 - 7.20 (m, 2H), 6.52 (m, 1H), 6.30 ( m, 1H), 5.79 (m, 1H), 5.37 (d, J= 9.8 Hz, 1H), 4.64 (t, J= 9.3 Hz, 1H), 4.51 (s, 1H), 4.31 (s, 1H), 2.97 (s, 1H).

¹³ C NMR (151 MHz, CDCh) 8 167.87, 165.59, 148.44, 138.36, 136.51, 137.12, 132.78, 132.21, 129.37, 128.07, 127.89, 127.26, 125.71, 122.41, 121.64, 121.51, 117.02, 83.19, 77.55, 69.01, 67.24, 53.46, 51.87, 38.18.

Note: Certain peaks in NMR are broad and split due to the presence of rotamers. HRMS (ESI+) m/z calcd for C24H ₂ oN ₃ 02 ⁺ [M+H] ⁺ : 382.1550, found: 382.1564.

The enantiomer MY-11 A was prepared in the same way from ent- .

General procedure for the synthesis of heterobifunctional compounds

[0142] S7 or erit- n (1.0 equiv.) was dissolved in THF, into which sulfamic acid (2.0 equiv.) was added. The mixture was cooled to 0 °C, and an aqueous solution of Na ₂ CCh (4.0 equiv.) was added dropwise with vigorous stirring over 15 min. The reaction was further stirred for 1 h at 23 °C until complete consumption of the limiting starting material by TLC. The mixture was then extracted with ethyl acetate (3 times), washed with Na2S20s (aq),

89

SUBSTITUTE SHEET (RULE 26) brine, and dried over Na2SO4. It was then concentrated to give a yellow oil which was used in the next step without further purification.

[0143] The crude acid was then dissolved in DCM, COMU (1.5 equiv.), DIPEA (3 equiv.) and the linker amine (1.2 equiv.) were added. The reaction was stirred at 23 °C overnight. The mixture was then concentrated and purified by flash column (SiCh, Hexanes/EtOAc = 1 : 1 to 0: 1) to give the coupling product (confirmed by LC-MS) which was then used without further characterization. The crude product was then dissolved in methanolic ammonia (7N) and stirred at 23 °C for 3 h until complete conversion by LC-MS. The mixture was concentrated under reduced pressure and dried under high vacuum, then dissolved in dichloromethane. The resulting solution was cooled to 0 °C, following which, followed by the addition of tri ethylamine (3.0 equiv.) and acryloyl chloride (2.0 equiv.) at 0 °C. The mixture was stirred at 0 °C for 0.5 hour. Upon completion, the reaction mixture was concentrated under reduced pressure and purified by prep-TLC to give S8a-S8d.

[0144] S8a-S8d was dissolved in DCM/TFA (3: 1) and stirred at 23 °C for 3 h until LC- MS showed complete conversion of the starting material. The mixture was then concentrated and dried under high vacuum overnight, then dissolved in dichloromethane, followed by the addition of DIPEA (3 equiv.), DMAP (0.1 equiv.), COMU (1.5 equiv.), and S9 (1.1 equiv.). The reaction was stirred at 23 °C overnight. The mixture was concentrated and first purified by prep-TLC, then prep-HPLC to give the final product as a white residue.

'H NMR (500 MHz, CDCh) 5 10.53 (s, 1H), 8.91 - 8.74 (m, 1H), 8.39 (d, J= 7.6 Hz, 1H), 8.24 - 8.09 (m, 1H), 7.75 - 7.55 (m, 2H), 7.44 (dt, J= 30.4, 7.9 Hz, 5H), 6.63 - 6.48 (m, 2H), 6.36 (br, 1H), 5.79 (br, 1H), 5.42 (d, J= 9.8 Hz, 1H), 5.00 (s, 1H), 4.67 (t, J= 92 Hz, 1H), 4.56 (s, 1H), 4.36 (d, J= 11.0 Hz, 1H), 3.67 - 3.54 (m, 8H), 3.49 (s, 2H), 3.26 (q, J= 5.6 Hz, 2H), 1.43 (s, 9H).

¹³ C NMR (126 MHz, CDCh) 5 167.81, 166.83, 165.58, 155.98, 148.61, 140.20, 138.49, 136.36, 136.34, 133.79, 129.43, 128.22, 127.88, 127.21, 127.13, 125.70, 122.32, 121.70, 116.82, 79.33, 70.22, 70.18, 70.08, 69.77, 54.03, 51.83, 40.21, 39.66, 38.10, 28.35.

90

SUBSTITUTE SHEET (RULE 26) HRMS (ESI+) m/z calcd for C34H ₄ 2N ₅ O ₇ ⁺ [M+H] ⁺ : 632.3079, found: 632.3091.

The enantiomer S8b was prepared in the same way from ent- 7.

'H NMR (600 MHz, CD ₃ OD) 5 8.84 (dd, J= 4.2, 1.6 Hz, 1H), 8.29 - 8.14 (m, 2H), 7.63 (d, J= 7.9 Hz, 2H), 7.58 - 7.47 (m, 4H), 7.39 (t, J= 8.0 Hz, 1H), 6.65 (dd, J= 17.2, 10.5 Hz, 0.5H), 6.48 - 6.30 (m, 1.2H), 5.93 (d, J= 10.5 Hz, 0.5H), 5.82 - 5.68 (m, 0.6H), 5.50 (d, J= 9.8 Hz, 0.5H), 4.75 (dd, J= 31.2, 8.3 Hz, 1.1H), 4.59 - 4.42 (m, 1.6H), 3.61 - 3.54 (m, 8H), 3.50 (s, 2H), 3.49 - 3.45 (m, 2H), 3.45 - 3.41 (m, 2H), 3.34 - 3.32 (m, 1H), 3.18 (t, J= 5.6 Hz, 2H), 1.43 (s, 9H). (Some peaks are broad and split due to the presence of rotamers.)

¹³ C NMR (151 MHz, CD3OD) 5 168.15, 167.64, 166.26, 157.00, 148.71, 140.26, 138.52, 136.20, 133.28, 133.03, 128.11, 128.06, 127.32, 126.93, 126.43, 125.97, 122.64, 122.37, 121.73, 117.14, 116.53, 78.66, 70.11, 70.09, 69.80, 69.75, 69.59, 69.05, 67.13, 53.62, 39.85, 39.44, 37.43, 27.40.

HRMS (ESI+) m/z calcd for C36H ₄ 6N ₅ O ₈ ⁺ [M+H] ⁺ : 676.3341, found: 676.3352.

The enantiomer S8d was prepared in the same way from ent- 7.

^X H NMR (500 MHz, CDCh) 5 10.53 (s, 1H), 8.82 (s, 1H), 8.39 (d, J= 7.1 Hz, 1H), 8.14 (d, J = 8.0 Hz, 1H), 7.59 (d, J= 13 Hz, 2H), 7.48 (t, J= 10.4 Hz, 4H), 7.40 (t, J= 7.8 Hz, 1H), 7.32 - 7.30 (m, 1H), 7.01 (t, J= 7.9 Hz, 2H), 6.93 (s, 1H), 6.86 - 6.77 (m, 2H), 6.70 (d, J=

8.7 Hz, 2H), 6.63 - 6.48 (m, 2H), 5.85 - 5.72 (m, 2H), 5.42 (d, J= 9.8 Hz, 1H), 5.32 (d, J=

4.8 Hz, 1H), 4.67 (t, J= 9.2 Hz, 1H), 4.48 (s, 3H), 4.36 (s, 1H), 3.91 - 3.84 (m, 6H), 3.61 - 3.53 (m, 8H), 3.52 - 3.48 (m, 2H), 3.39 (d, J= 12.9 Hz, 1H), 3.19 (t, J= 12.2 Hz, 1H), 2.60 (ddt, J= 29.7, 15.2, 6.9 Hz, 2H), 2.38 (d, J= 13.4 Hz, 1H), 2.25 (dt, J= 9.0, 4.6 Hz, 1H),

91

SUBSTITUTE SHEET (RULE 26) 2.06 (dt, J= 14.3, 7.3 Hz, 1H), 1.87 - 1.61 (m, 6H), 1.44 (dq, J= 60.5, 12.8 Hz, 3H), 1.23 (d, J= 7.7 Hz, 6H), 1.14 (s, 1H), 0.90 (t, J= 7.3 Hz, 3H).

¹³ C NMR (126 MHz, CDCh) 5207.86, 169.71, 168.20, 168.13, 167.93, 167.32, 165.57, 157.38, 148.92, 148.71, 147.41, 141.90, 138.61, 136.24, 133.76, 133.34, 131.21, 130.06, 129.49, 128.23, 127.87, 127.21, 127.07, 125.68, 121.74, 120.16, 120.06, 117.21, 116.70, 114.15, 113.37, 111.75, 111.34, 76.51, 70.22, 70.19, 69.71, 69.62, 67.38, 55.94, 55.86, 51.28, 46.71, 44.16, 39.64, 39.53, 38.73, 38.61, 38.20, 32.48, 31.27, 26.43, 24.94, 23.56, 23.47, 23.17, 21.20, 21.03, 8.82, 8.77.

HRMS (ESI+) m/z calcd for C ₆ IH ₇₃ N ₆ 0I ₃ ⁺ [M+H] ⁺ : 1097.5230, found: 1097.5238.

'H NMR (600 MHz, CD ₃ OD) 5 8.83 (s, 1H), 8.24 (d, J= 7.9 Hz, 2H), 7.65 - 7.46 (m, 7H), 7.38 (t, J= 7.8 Hz, 1H), 7.29 (t, J= 7.8 Hz, 1H), 7.04 - 6.95 (m, 2H), 6.90 (d, J= 7.8 Hz, 1H), 6.85 (d, J= 8.0 Hz, 1H), 6.79 (d, J= 12.5 Hz, 1H), 6.71 (d, J= 7.9 Hz, 1H), 6.48 - 6.31 (m, 0.6H), 5.98 - 5.65 (m, 1.4H), 5.55 - 5.43 (m, 2H), 4.55 - 4.41 (m, 4H), 3.80 (s, 3H), 3.79 (s, 3H), 3.52 (d, J= 17.0 Hz, 8H), 3.42 (s, 3H), 3.41 (s, 3H), 3.21 (t, J= 12.9 Hz, 1H), 2.59 (m, 2H), 2.40 - 2.18 (m, 2H), 2.11 - 1.99 (m, 1H), 1.70 (m, 5H), 1.53 - 1.27 (m, 4H), 1.22 (d, J= 6.0 Hz, 6H), 1.12 (s, 1H), 0.89 (t, J= 7.3 Hz, 3H).

¹³ C NMR (151 MHz, CD3OD) 5 207.65, 169.62, 169.60, 169.58, 167.70, 157.87, 149.01, 148.70, 147.43, 146.45, 141.96, 136.19, 133.80, 133.26, 133.01, 129.62, 128.11, 128.05, 126.89, 126.42, 125.91, 121.71, 120.30, 119.68, 119.58, 118.92, 114.17, 112.92, 112.17, 111.83, 76.56, 69.85, 69.07, 69.03, 66.88, 56.87, 55.14, 55.04, 53.41, 51.42, 46.30, 44.30, 39.38, 38.48, 37.86, 32.26, 32.18, 30.85, 27.20, 25.95, 25.67, 24.49, 24.27, 22.68, 22.42, 22.36, 22.25, 20.75, 20.66, 7.72.

HRMS (ESI+) m/z calcd for C6iH ₇ 3N ₆ 0i3 ⁺ [M+H] ⁺ : 1097.5230, found: 1097.5237.

92

SUBSTITUTE SHEET (RULE 26)

(Mixture of rotamers)

'H NMR (600 MHz, CD ₃ OD) 5 8.83 (dd, J= 4.2, 1.6 Hz, 1H), 8.30 - 8.12 (m, 2H), 7.61 (d, J = 7.6 Hz, 2H), 7.57 - 7.46 (m, 4H), 7.38 (t, J= 8.0 Hz, 1H), 7.29 (t, J= 7.9 Hz, 1H), 7.03 - 6.95 (m, 2H), 6.91 (dd, J= 8.3, 2.3 Hz, 1H), 6.85 (dd, J= 8.2, 2.2 Hz, 1H), 6.81 - 6.75 (m, 1H), 6.73 - 6.67 (m, 1H), 6.67 - 6.29 (m, 2H), 6.00 - 5.67 (m, 2H), 5.54 - 5.19 (m, 1H), 4.73 (d, J= 30.1 Hz, 1H), 4.51 (m, 4H), 3.80 (s, 3H), 3.79 (s, 3H), 3.61 - 3.46 (m, 13H), 3.46 - 3.37 (m, 5H), 3.21 (td, J= 13.2, 3.0 Hz, 1H), 2.68 - 2.53 (m, 2H), 2.38 - 2.20 (m, 2H), 2.11 - 2.01 (m, 1H), 1.82 - 1.59 (m, 5H), 1.54 - 1.43 (m, 1H), 1.42 - 1.27 (m, 2H), 1.22 (d, J= 6.3 Hz, 6H), 1.12 (d, J= 1.5 Hz, 1H), 0.88 (t, J= 7.5 Hz, 2.5H), 0.80 (t, J= 7.5 Hz, 0.5H).

¹³ C NMR (151 MHz, CD3OD) 5 207.64, 207.59, 169.58, 169.57, 168.11, 167.69, 166.99, 166.22, 157.86, 149.02, 148.71, 147.44, 141.97, 141.79, 140.28, 138.50, 136.20, 134.95, 134.42, 133.84, 133.79, 133.27, 133.04, 129.62, 128.06, 126.92, 126.43, 125.96, 121.72, 120.30, 119.67, 119.56, 114.15, 113.00, 112.96, 112.18, 111.83, 76.76, 76.54, 70.11, 70.08, 69.81, 69.79, 69.03, 68.96, 67.10, 66.90, 56.86, 55.14, 55.05, 51.41, 46.30, 44.30, 39.44, 38.75, 38.55, 37.88, 37.74, 37.42, 32.26, 32.18, 30.86, 27.21, 25.95, 24.49, 24.08, 22.69, 22.43, 22.36, 22.26, 20.75, 20.66, 7.79, 7.73.

HRMS (ESI+) m/z calcd for C63H77N6O1 [M+H] ⁺ : 1141.5492, found: 1141.5519.

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SUBSTITUTE SHEET (RULE 26)

^X H NMR (600 MHz, CDCh) 8 10.52 (s, 1H), 8.81 (s, 1H), 8.39 (d, J= 6.7 Hz, 1H), 8.15 (d, J = 7.9 Hz, 1H), 7.60 (d, J= 6.8 Hz, 2H), 7.52 - 7.38 (m, 4H), 7.29 (d, J= 6.2 Hz, 1H), 7.06 (d, J= 5.4 Hz, 1H), 6.99 (dd, J= 13.0, 7.8 Hz, 1H), 6.95 - 6.89 (m, 1H), 6.85 (d, J= 8.2 Hz, 1H), 6.80 (dd, J= 8.4, 4.5 Hz, 1H), 6.75 - 6.65 (m, 3H), 6.53 (s, 1H), 5.77 (q, J= 5.8 Hz, 1H), 5.42 (d, J= 9.8 Hz, 1H), 4.66 (t, J= 9.2 Hz, 1H), 4.49 (s, 2H), 4.37 (s, 1H), 3.87 (d, J= 6.9 Hz, 6H), 3.62 - 3.51 (m, 17H), 3.39 (d, J= 12.9 Hz, 1H), 3.19 (td, J= 13.1, 2.6 Hz, 1H), 2.58 (m, 2H), 2.38 (d, J= 13.7 Hz, 1H), 2.29 - 2.20 (m, 1H), 2.10 - 2.04 (m, 1H), 1.83 - 1.61 (m, 6H), 1.57 - 1.43 (m, 1H), 1.40 - 1.30 (m, 1H), 1.23 (d, J= 10.1 Hz, 6H), 1.14 (d, J= 4.6 Hz, 1H), 0.90 (t, J= 7.4 Hz, 3H).

¹³ C NMR (151 MHz, CDCh) 5207.87, 169.69, 168.13, 167.31, 166.80, 166.53, 165.58, 157.41, 148.90, 148.60, 147.38, 141.86, 138.47, 136.33, 133.78, 133.34, 130.04, 129.43, 128.20, 127.89, 127.26, 127.12, 125.68, 121.72, 120.15, 120.06, 116.54, 116.39, 114.25, 113.30, 111.72, 111.32, 76.51, 70.47, 70.23, 70.20, 69.75, 69.64, 67.39, 56.69, 55.93, 55.85, 53.46, 51.27, 46.71, 44.17, 39.69, 38.85, 38.78, 38.20, 38.03, 32.58, 32.48, 31.27, 26.44, 24.94, 23.55, 23.47, 23.15, 21.22, 8.83, 8.78, 1.92.

HRMS (ESI+) m/z calcd for C63H77N6O1 [M+H] ⁺ : 1141.5492, found: 1141.5482.

References

1. Zhang, X.; Luukkonen, L. M.; Eissler, C. L.; Crowley, V. M.; Yamashita, Y .; Schafroth, M. A.; Kikuchi, S.; Weinstein, D. S.; Symons, K. T.; Nordin, B. E.; Rodriguez, J.

L.; Wucherpfennig, T. G.; Bauer, L. G.; Dix, M. M.; Stamos, D.; Kinsella, T. M.; Simon, G.

M.; Baltgalvis, K. A.; Cravatt, B. F., DCAF11 Supports Targeted Protein Degradation by Electrophilic Proteolysis-Targeting Chimeras. J. Am. Chem. Soc. 2021, 143 (13), 5141-5149.

2. Banchenko, S.; Krupp, F.; Gotthold, C.; Burger, J.; Graziadei, A.; O’Reilly, F. J.;

Sinn, L.; Ruda, O.; Rappsilber, J.; Spahn, C. M., Structural insights into Cullin4-RING

94

SUBSTITUTE SHEET (RULE 26) ubiquitin ligase remodelling by Vpr from simian immunodeficiency viruses. PLoS Pathog. 2021, 77 (8), el009775.

3. Vinogradova, E. V.; Zhang, X.; Remillard, D.; Lazar, D. C.; Suciu, R. M.; Wang, Y.; Bianco, G.; Yamashita, Y.; Crowley, V. M.; Schafroth, M. A.; Yokoyama, M.; Konrad, D. B.; Lum, K. M.; Simon, G. M.; Kemper, E. K.; Lazear, M. R.; Yin, S.; Blewett, M. M.; Dix, M. M.; Nguyen, N.; Shokhirev, M. N.; Chin, E. N.; Lairson, L. L.; Melillo, B.; Schreiber, S. L.; Forli, S.; Teijaro, J. R.; Cravatt, B. F., An Activity-Guided Map of Electrophile-Cysteine Interactions in Primary Human T Cells. Cell 2020, 182 (4), 1009-1026 e29.

4. Maetani, M.; Zoller, J.; Melillo, B.; Verho, O.; Kato, N.; Pu, J.; Comer, E.; Schreiber,

5. L., Synthesis of a Bicyclic Azetidine with In Vivo Antimalarial Activity Enabled by Stereospecific, Directed C(sp(3))-H Arylation. J Am Chem Soc 2017, 139 (32), 11300-11306.

95

SUBSTITUTE SHEET (RULE 26) Scheme for synthesis of additional compounds:

[0145] The foregoing disclosure has been described in some detail by way of illustration and example, for purposes of clarity and understanding. It will be obvious to one of skill in the art that changes and modifications may be practiced within the scope of the appended claims. Therefore, it is to be understood that the above description is intended to be illustrative and not restrictive. The scope of the disclosure should, therefore, be determined not with reference to the above description, but should instead be determined with reference

96

SUBSTITUTE SHEET (RULE 26) to the following appended claims, along with the full scope of equivalents to which such claims are entitled.

[0146] This application refers to various issued patents, published patent applications, journal articles, and other publications, each of which are incorporated herein by reference.

97

SUBSTITUTE SHEET (RULE 26)