Small molecules that block proteasome-associated ubiquitin receptor RPN13 function and uses thereof

Abstract

Provided herein are bifunctional compounds with a moiety (e.g., lenalidomide, thalidomide) that is a binder of an E3 ubiquitin ligase (e.g., Cereblon) and another moiety (e.g., RA190) that is a binder of the ubiquitin receptor RPN13 to induce degradation of RPN13 and thereby inhibit proteasome function. Also provided are pharmaceutical compositions comprising the bifunctional compounds, and methods of treating and/or preventing diseases (e.g., proliferative diseases, cancers, benign neoplasms, pathological angiogenesis, inflammatory diseases, and autoimmune diseases). Provided also are methods of inducing the degradation of ubiquitin receptor RPN13 by administering a bifunctional compound or composition described herein, wherein one component of the bifunctional compound is a binder of an E3 ubiquitin ligase (e.g., lenalidomide, thalidomide) and another component of the compound is a binder of ubiquitin receptor RPN13 (e.g., RA190) in a subject.

Claims

1. A compound of Formula (I) or Formula (I′): ##STR00084## or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or stereoisomer thereof, wherein: in each pair of A's, one A is hydrogen, and the other A is one of: (i) phenyl, optionally substituted with 1-5 substituents selected from the group consisting of R.sup.1, OR.sup.1, NR.sup.1R.sup.2, S(O).sub.qR.sup.1, SO.sub.2R.sup.1R.sup.2, NR.sup.1SO.sub.2R.sup.2, C(O)R.sup.1, C(O)OR.sup.1, C(O)NR.sup.1R.sup.2, NR.sup.1C(O)R.sup.2, NR.sup.1C(O)OR.sup.2, CF.sub.3, and OCF.sub.3; (ii) naphthyl, optionally substituted with 1-5 substituents selected from the group consisting of R.sup.1, OR.sup.1, NR.sup.1R.sup.2, S(O).sub.qR.sup.1, SO.sub.2R.sup.1R.sup.2, NR.sup.1SO.sub.2R.sup.2, C(O)R.sup.1, C(O)OR.sup.1, C(O)NR.sup.1R.sup.2, NR.sup.1C(O)R.sup.2, NR.sup.1C(O)OR.sup.2, CF.sub.3, and OCF.sub.3; (iii) a 5-or 6-membered monocyclic heteroaryl group, having 1-3 heteroatoms selected from the group consisting of O, N, and S, optionally substituted with 1-3 substituents selected from the group consisting of R.sup.1, OR.sup.1, NR.sup.1R.sup.2, S(O).sub.qR.sup.1, SO.sub.2R.sup.1R.sup.2, NR.sup.1SO.sub.2R.sup.2, C(O)R.sup.1, C(O)OR.sup.1, C(O)NR.sup.1R.sup.2, NR.sup.1C(O)R.sup.2, NR.sup.1C(O)OR.sup.2, CF.sub.3, and OCF.sub.3; and (iv) an 8- to 10-membered bicyclic heteroaryl group containing 1-3 heteroatoms selected from the group consisting of O, N, and S; and the second ring is fused to the first ring using 2 carbon atoms, and the bicyclic heteroaryl group is optionally substituted with 1-3 substituents selected from the group consisting of R.sup.1, OR.sup.1, NR.sup.1R.sup.2, S(O).sub.qR.sup.1, SO.sub.2R.sup.1R.sup.2, NR.sup.1SO.sub.2R.sup.2, C(O)R.sup.1, C(O)OR.sup.1, C(O)NR.sup.1R.sup.2, NR.sup.1C(O)R.sup.2, NR.sup.1C(O)OR.sup.2, CF.sub.3, and OCF.sub.3; wherein Y is selected from the group consisting of O, S, NR.sup.1 and CR.sup.1R.sup.2, and wherein R.sup.1 and R.sup.2 are selected from the group consisting of hydrogen, nitro, hydroxyl, carboxy, amino, halogen, cyano and C.sub.1-C.sub.14 linear or branched alkyl groups, that are optionally substituted with 1-3 substituents selected from the group consisting of C.sub.1-C.sub.14 linear or branched alkyl, up to perhalo substituted C.sub.1-C.sub.14 linear or branched alkyl, C.sub.1-C.sub.14 alkoxy, hydrogen, nitro, hydroxyl, carboxy, amino, C.sub.1-C.sub.14 alkylamino, C.sub.1-C.sub.14 dialkylamino, halogen, and cyano; wherein R.sup.7 is hydrogen, C.sub.1-6 alkyl, or a nitrogen protecting group; wherein Z is selected from the group consisting of hydrogen; C.sub.1 to C.sub.14 linear, branched, or cyclic alkyl; phenyl; benzyl, 1-5 substituted benzyl, C.sub.1 to C.sub.3 alkyl-phenyl, wherein the alkyl moiety is optionally substituted with halogen up to perhalo; C.sub.1 to C.sub.14 linear or branched alkyl, wherein the alkyl moiety is optionally substituted with halogen up to perhalo; —(CH.sub.2).sub.q—K, where K is a 5 or 6 membered monocyclic heterocyclic ring, containing 1 to 4 atoms selected from oxygen, nitrogen and sulfur, which is saturated, partially saturated, or aromatic, or an 8 to 10 membered bicyclic heteroaryl having 1-4 heteroatoms selected from the group consisting of O, N, and S, and wherein the variable q is an integer ranging from 0 to 4, provided that when the compound is of Formula (I′), Z is not hydrogen, and provided that when A is substituted with S(O).sub.qR.sup.1, q is an integer ranging from 0 to 2; L is a linker; and D is an E3 ubiquitin ligase binding moiety, wherein D is of the formula IA or IB: ##STR00085## wherein: X.sup.A is C(O) or C(R.sup.3A).sub.2; each R.sup.1A is independently halogen, OH, C.sub.1-C.sub.6 alkyl, or C.sub.1-C.sub.6 alkoxy; each R.sup.3A is independently H or C.sub.1-C.sub.3 alkyl; each R.sup.3′ is independently C.sub.1-C.sub.3 alkyl; each R.sup.4A is independently H or C.sub.1-C.sub.3 alkyl; or two R.sup.4A, together with the carbon atom to which they are attached, form a C(O), C.sub.3-C.sub.6 carbocycle, or a 4-, 5-, or 6-membered heterocycle comprising 1 or 2 heteroatoms selected from N and O; R.sup.5A is H, C.sub.1-C.sub.3 alkyl, or halogen; m is 0, 1, 2 or 3; n is 0, 1, or 2; and a1 is 0 or 1; or ##STR00086## wherein: —X.sup.1—X.sup.2— is C(R.sup.3A)═N or C(R.sub.3A).sub.2—C(R.sup.3A).sub.2; each R.sup.1A is independently halogen, OH, C.sub.1-C.sub.6 alkyl, or C.sub.1-C.sub.6 alkoxy; R.sup.3A is H or C.sub.1-C.sub.3 alkyl; each R.sup.3′ is independently C.sub.1-C.sub.3 alkyl; each R.sup.4A is independently H or C.sub.1-C.sub.3 alkyl; or two R.sup.4A, together with the carbon atom to which they are attached, form a C(O), C.sub.3-C.sub.6 carbocycle, or a 4-, 5-, or 6-membered heterocycle comprising 1 or 2 heteroatoms selected from N and O; R.sup.5A is H, C.sub.1-C.sub.3 alkyl, or halogen; m is 0, 1, 2, or 3; n is 0, 1, or 2; and a1 is 0 or 1.

2. The compound of claim 1, wherein D is of the formula: ##STR00087##

3. The compound of claim 1, wherein D is of the formula: ##STR00088##

4. The compound of claim 1, wherein the compound is of formula: ##STR00089## or a pharmaceutically acceptable salt thereof.

5. The compound of claim 1, wherein the compound is of formula: ##STR00090## or a pharmaceutically acceptable salt thereof.

6. The compound of claim 1, or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or stereoisomer thereof, wherein L is a bond, a substituted or unsubstituted C.sub.1-12 hydrocarbon chain, optionally wherein one or more chain atoms of the hydrocarbon chain are independently replaced with —C(═O)—, —O, S, NR.sup.b, N═, or ═N—, substituted or unsubstituted carbocyclylene, substituted or unsubstituted heterocyclylene, substituted or unsubstituted arylene, substituted or unsubstituted heteroarylene, or substituted or unsubstituted heteroalkylene, wherein each instance of R.sup.b is independently hydrogen, substituted or unsubstituted C.sub.1-6 alkyl, or a nitrogen protecting group, or optionally two instances of R.sup.b are taken together with their intervening atoms to form a substituted or unsubstituted heterocyclic or substituted or unsubstituted heteroaryl ring.

7. The compound of claim 1, wherein in each pair of A's, one A is hydrogen and the other A is phenyl optionally substituted with R.sup.1.

8. The compound of claim 7, wherein in each pair of A's, one A is hydrogen and the other A is phenyl substituted with halogen.

9. The compound of claim 1, wherein both instances of Y are O.

10. The compound of claim 1, wherein one instance of Y is O and the other instance of Y is —CH.sub.2.

11. The compound of claim 1, wherein Z is benzyl.

12. The compound of claim 6, wherein L is an unsubstituted C.sub.1-12 hydrocarbon chain, optionally wherein one or more chain atoms of the hydrocarbon chain are independently replaced with —C(═O)—, —O—, or —NR.sup.b—.

13. The compound of claim 1, wherein L is of the formula: ##STR00091## wherein: g is 1-5; p is 2-5; and s is 1-5; ##STR00092## wherein: g is 2, 3, or 4; p is 0, 1, 2, 3, 4, or 5; and s is 1, 2, 3, 4, 5, or 6; ##STR00093## wherein: g is 1-5; and g1 is 1-5; ##STR00094## wherein: g is 1-5; and s is 2-4: or ##STR00095## wherein: g is 2, 3, or 4; wherein l.sup.A indicates the point of attachment to D, and l.sup.R indicates the point of attachment to ##STR00096##

14. The compound of claim 1, wherein: L is of the formula: ##STR00097## wherein l.sup.A indicates the point of attachment to D, and l.sup.R indicates the point of attachment to ##STR00098##

15. The compound of claim 1, wherein the compound is of the formula: ##STR00099## ##STR00100## or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or stereoisomer thereof.

16. A pharmaceutical composition comprising a therapeutically effective amount of the compound of claim 1, or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or stereoisomer thereof, and optionally a pharmaceutically acceptable excipient.

17. The compound of claim 1, or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or stereoisomer thereof, wherein R.sup.3A is H.

18. A method of treating a proliferative disease in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the compound of claim 17, or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or stereoisomer thereof.

19. The method of claim 18, wherein the proliferative disease is cancer.

20. The method of claim 19, wherein the cancer is multiple myeloma, leukemia, or lymphoma.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIGS. 1A and 1B show the design principle behind the bifunctional compounds described herein. FIG. 1A shows the use of a RPN13 small molecule inhibitor (RA190) design in conjunction with a moiety based on an immunomodulatory imide drug (iMiD), which is an ImiD-based degrader, to induce RPN13 degradation. FIG. 1B shows the use of RA190 as a covalent inhibitor of RPN13, and an ImiD-based degrader for binding an E3 ligase (e.g., cereblon (CRBN)).

(2) FIG. 2 shows MM.1S cells treated with WL40 or WL44 at 200 nM and 400 nM for 4 hours; treated with WL40 or WL44 at 200 nM, 400 nM, and 800 nM for 8 hours; and treated with WL40 or WL44 at 200 nM, 400 nM, and 800 nM for 16 hours. Protein lysates were loaded on SDS-PAGE and transferred to PVDF membrane, and then immunoblotted with anti-alpha tubulin and Rpn13 antibodies. Western blots show that exemplary bifunctional compounds WL40 and WL44 degrade Rpn13. FIG. 2 also shows the structures of exemplary bifunctional compounds

(3) ##STR00014##

(4) FIG. 3 shows a reporter cell line expressing Ub-tagged GFP that is constitutively targeted for proteasomal degradation treated with RA190 (1 μM); WL40 (0.5 μM, 1.0 μM, and 2.0 μM); and WL44 (0.5 μM, 1.0 μM, and 2.0 μM) for 16 hours. Cell lysates were loaded on SDS-PAGE, transferred to PVDF membrane, then immunoblotted with anti-GFP, and anti beta-actin antibodies. The blots show that exemplary bifunctional compounds WL40 and WL44 block proteasome function.

(5) FIGS. 4A and 4B show multiple myeloma 1S (MM.1S) cells treated with RA190, WL40, and WL44 at the indicated concentrations for 24 hours. Wst-1 assay was used to test cell viability. FIGS. 4A and 4B show that exemplary bifunctional compounds WL40 and WL44 decrease multiple myeloma 1S cell viability with lower IC.sub.50 compared to compound RA190.

(6) FIGS. 5A and 5B show PBMCs from normal healthy donor treated with WL40 and WL44 at the indicated concentrations for 48 hours. Wst-1 assay was used to test cell viability. FIGS. 5A and 5B show that exemplary bifunctional compounds WL40 (FIG. 5A) and WL44 (FIG. 5B) at the IC.sub.50 do not significantly affect the viability of normal peripheral blood mononuclear cells (PBMC's).

(7) FIGS. 6A and 6B show multiple myeloma cell lines treated with WL40 or WL44 at the indicated concentrations for 48 hours. Wst-1 assay was used to test cell viability. FIGS. 6A and 6B show the effect of exemplary bifunctional compounds WL40 and WL44 on multiple myeloma cell lines. Exemplary bifunctional compounds WL40 (FIG. 6A) and WL44 (FIG. 6B) have a high IC.sub.50 in several cell lines.

(8) FIGS. 7A and 7B show MM.1S and MM.1S CRBN KO (multiple myeloma 1S-cereblon knockout) cell lines were treated with WL40 or WL44 at the indicated concentrations for 24 hours. Wst-1 assay was used to test cell viability. FIGS. 7A and 7B show that exemplary bifunctional compounds WL40 (FIG. 7A) and WL44 (FIG. 7B) decrease cell viability in the multiple myleoma cell line 1S-cereblon knockout (MM.1S-CRBN KO).

(9) FIGS. 8A and 8B show HCT116 and HCT116-Rpn13KO cell lines that were treated with WL40 or WL44 at the indicated concentrations for 48 hours. Wst-1 assay was used to test cell viability. FIGS. 8A and 8B show that the cytotoxicity of exemplary bifunctional compounds WL40 (FIG. 8A) and WL44 (FIG. 8B) is RPN13-dependent.

(10) FIGS. 9A and 9B show CD138+MM cells from patients that were treated with WL40 or WL44 at the indicated concentrations for 48 hours. CTG assay was used to test cell viability. FIGS. 9A and 9B show the effect of exemplary bifunctional compounds WL40 (FIG. 8A) and WL44 (FIG. 8B) at certain concentrations (nM) on the viability of cells in patients.

(11) FIG. 10 shows the development of other exemplary bifunctional compounds which include degraders of RPN13.

(12) FIG. 11 shows a scheme of the development of the exemplary bifunctional compounds with the RPN13 probe molecule and degraders, including synthetic schemes.

(13) FIG. 12 shows the structure of exemplary compound dRPN13-3.

(14) FIG. 13A and FIG. 13B show that RA190 induces stronger MM cytotoxicity compared to exemplary compound JQRA, and depict the structure of exemplary compound JQRA. FIG. 13A depicts the structure of exemplary compound JQRA. In FIG. 13B, multiple myeloma cells (MM.1S) were treated with RA190 or compound JQRA at the indicated concentrations for 72 hours, and the percentage of viable cells was measured.

(15) FIG. 14 shows that RA190 induced more robust MM cytotoxicity in MM.1S, MM.1S-len resistant, and CRBN-KO cells compared with exemplary compound JQRA. The experiment was repeated twice and JQRA concentration was increased (as indicated) at the second time. MM.1S Len-resistant (MM.1S lenalidomide resistant) cells are more sensitive to exemplary compound JQRA compared to wild type MM.1S. In FIG. 14, multiple myeloma cells (MM.1SRor MM.1S) were treated with RA190 or compound JQRA at the indicated concentrations for 72 hours, and the percentage of viable cells was measured.

(16) FIG. 15 shows that treatment with JQRA does not affect the percentage of viability of HCT116 and HCT-Rpn13KO cells. In FIG. 15, HCT116 and HCT-Rpn13KO cells were treated with JQRA for 72 hours, and the percentage of viable cells was measured.

(17) FIG. 16 shows that in the different indicated multiple myeloma cell lines, RA190 induces stronger cytotoxicity than JQRA. In FIG. 16, the MM.1S cell lines were treated with JQRA or RA190 for 72 hours, and the percentage of viable cells was measured at 72 hours.

(18) FIG. 17 shows that JQRA degrades RPN13 at a certain time point. In FIG. 17, MM.1S cells were treated with RA190 or JQRA at 200 nM and 400 nM, respectively, for 48 hours; and treated with RA190 or JQRA at 200 nM and 800 nM, respectively, for 48 hours. Protein lysates were loaded on SDS-PAGE and transferred to PVDF membrane, and then immunoblotted with anti-alpha tubulin and Rpn13 antibodies. Western blots show that exemplary bifunctional compound JQRA degrades RPN13.

(19) FIG. 18 shows that JQRA does not block proteasome function as RA190 does. In FIG. 18, a reporter cell line expressing Ub-tagged GFP that is constitutively targeted for proteasomal degradation was treated with RA190 (312 μM, 625 μM, 1250 μM, 2500 μM, 5000 μM); and JQRA (312 μM, 625 μM, 1250 μM, 2500 μM, 5000 μM) for 16 hours. Cell lysates were loaded on SDS-PAGE, transferred to PVDF membrane, then immunoblotted with anti-GFP, and anti-alpha tubulin antibodies. The blots show that exemplary bifunctional compound JQRA does not block proteasome function as RA190 blocks proteasome function.

(20) FIGS. 19A-19I show the exemplary design and characterization of exemplary Rpn13 degrader WL40. FIG. 19A shows the synthesis of WL40. WL40 was created by linking the Rpn13 inhibitor RA190 to the IMiD thalidomide as a ligand for the CRBN E3 ligase via a PEG linker. FIG. 19B shows results of a Cereblon AlphaScreen assay to measure the displacement of biotinylated-Pomalidomide probe (triplicate means±SD). FIG. 19C is a schematic cartoon for the novel AlphaScreen assay to measure the binding activity of inhibitor with RPN13 proteins. FIG. 19D shows the results of a RPN13 AlphaScreen assay to measure the binding activity of inhibitor with RPN13 proteins (triplicate means±SD). FIG. 19E depicts immunoblot analysis of multiple myeloma 1S (MM.1S) cells, which were treated with WL40 or RA190 at the indicated concentrations and time periods; protein lysates were subjected to the immunoblot analysis using anti-Rpn13 or anti-tubulin Abs. FIG. 19F depicts flow cytometry analysis of MM.1S cells, which were treated with DMSO control, WL40 (400 nM), or RA190 (500 nM) for 16 hours; cells were then washed and stained with Rpn13 Ab conjugated with AlexaFluor-647, followed by the flow cytometry analysis. Isotype Ab conjugated to AlexaFluor-647 was used as control for non-specific binding. Data was quantified using FACS Diva (BD Biosciences, USA) and FlowJo (FlowJo LLC, USA). FIG. 19G depicts immunoblot analysis of MM.1S-CRBN KO cells, which were treated with WL40 (400 nM) or RA190 (500 nM) for 16 hours; protein lysates were subjected to the immunoblot analysis using anti-Rpn13 or anti β-actin Abs. Insert: Protein lysates from MM.1S.WT control and MM.1S-CRBN KO cells were subjected to immunoblot analysis using anti-CRBN or anti-β-actin Abs. FIG. 19H is an immunoblot showing the levels of Ub-GFP accumulation in a GFPu-1 reporter cell line treated with indicated concentrations of WL40 and RA190 for 16 hours. Blots shown are representative of 3 independent experiments. FIG. 19I shows an assessment for cell viability of HCT116-WT and HCT116-CRISPR Rpn13KO cells, which were treated with WL40 or RA190 at the indicated concentrations for 48 hours, followed by the assessment of cell viability using WST assay (mean±SD; p<0.001; n=3). Insert: Protein lysates from Rpn13-WT and KO cells were subjected to immunoblot analysis using anti-Rpn13 or β-actin Abs.

(21) FIGS. 20A to 20F show that WL40 blocks proteasome-mediated protein degradation without inhibiting proteasome proteolytic activities FIG. 20A is a bar graph showing percent proteasome activity after normalization using DMSO control (mean±SD; n=3). MM.1S cells, which were treated with DMSO control, bortezomib, or WL40 at indicated concentration for 3 hours; protein lysates were analyzed for different proteasome activities (CT-L, chymotrypsin-like; T-L, trypsin-like; C-L, caspase-like). FIG. 20B is a bar graph showing percent proteasome activity after normalization with DMSO control (mean±SD; n=3). Recombinant 20S proteasome was incubated with DMSO, bortezomib, or WL40 for 30 minutes, followed by assessment of the above-noted proteasome activities. FIG. 20C MM.1S cells were pretreated with exemplary proteasome inhibitor MG132 (10 μM) for 1 hour, followed by addition of WL40 (400 nM) for 8 hours. As a positive control for WL40-induced Rpn13 degradation, cells were also treated with WL40 alone for 8 hours. Total protein lysates were subjected to immunoblots for Rpn13 and α-tubulin. FIG. 20D shows immunoblot analysis of RPMI-8226 cells, which were treated with WL40 (400 nM) for 16 hours; protein lysates were subjected to the immunoblot analysis using anti-Rpn13 or anti-β-actin Abs. FIG. 20E ANBL6.BR cells were treated with WL40 (400 nM) or RA190 (500 nM) for 16 hours; protein lysates were subjected to immunoblot analysis using anti-Rpn13 or anti-β-actin Abs. FIG. 20F shows immunoblot analysis of MM.1S cells, which were treated with DMSO control, bortezomib (5 nM), RA190 (300 nM), or WL40 (200 nM) for indicated time periods; protein lysates were subjected to the immunoblot analysis using anti-polyubiquitin or anti-β-actin Abs (left panel). FIG. 20F shows that ANBL6.BR cells were treated with DMSO as a control or WL40 (1 μM) for 6 hours; protein lysates were subjected to immunoblot analysis using anti-polyubiquitin or anti-β-actin Abs (right panel).

(22) FIGS. 21A to 21E show that WL40 triggers anti-MM activity as well as overcomes the bortezomib-resistance and cytoprotective activity of the MM bone marrow (BM) microenvironment FIG. 21A shows an assessment for the cell viability of MM.1S and MM.1R cells, which were treated with DMSO control, WL40, or RA190 at indicated concentrations for 48 hours, followed by the assessment for cell viability using a standard WST (cell viability and proliferation) assay (p<0.05 for both cell lines; n=3). Table: ANBL6.WT (plasma cell myeloma; wild type), ANBL6.BR (plasma cell myeloma; bortezomib-resistant), RPMI-8226 (B-lymphocyte), or INA6 MM (plasma cell myeloma) cell lines were treated with DMSO as a control or WL40 for 48 hours, followed by assessment for cell viability. The IC.sub.50 of WL40 for cell lines is shown. FIG. 21B is a bar graph showing the percentage of viable cells in different patients (patients 1-4). Purified CD138.sup.+ patient MM cells, which were treated with DMSO control or WL40 at indicated concentrations for 48 hours, followed by assessment for cell viability using CellTiter-Glo assay (mean±SD of triplicate cultures; p<0.001). FIG. 21C is a bar graph showing the percentage of viable cells within the PBMCs. Normal PBMCs from healthy donors were treated with DMSO control or WL40 at indicated concentrations for 48 hours, and then analyzed for cell viability using CellTiter-Glo assay (mean±SD of quadruplicate cultures). FIG. 21D is a bar graph showing the percentage of viable cells within MM cells co-cultured with the patient BMSCs (bone marrow stromal cells). MM.1S cells were cultured with or without patient BMSCs in the presence or absence of WL40 for 48 hours, and cell proliferation was measured by WST assay (mean±SD; n=3; p<0.0001). FIG. 21E is a bar graph showing the percentage of viable cells within MM cells co-cultured with patient plasmacytoid dendritic cells (pDCs). MM.1S cells were cultured with or without patient pDCs in the presence or absence of WL40 for 48 hours, and cell proliferation was measured by WST assay (mean±SD; n=3; p<0.0001).

(23) FIGS. 22A to 22F show the mechanisms of WL40-induced MM cell death. FIG. 22A shows an analysis of apoptosis in view of treatment of MM.1S cells with the control DMS or WL40. MM.1S cells were treated with DMSO control or WL40 (200 nM) for 16 hours, and then analyzed for apoptosis using Annexin V/PI double staining assay (mean±SD; n=3; p<0.001). FIG. 22B is an immunoblot of MM.1S and ANBL6.BR cells, which were treated with DMSO control or WL40 (200 nM for MM.1S; 1 μM for ANBL6.BR) for 16 hours; protein lysates were then subjected to the immunoblotting using antibodies against poly ADP ribose polymerase (PARP), caspase-3, caspase-7, caspase-8, caspase-9, or β-actin. FL, full length; CF, cleaved fragment. FIG. 22C is a bar graph measuring enzymatic activity. MM.1S cells were treated with DMSO control or WL40 (200 nM) for 12 hours, followed by the measurement of caspase-3, caspase-8, or caspase-9 enzymatic activity (mean±SD; n=3; p<0.0001). FIG. 22D is an immunoblot of MM.1S and ANBL6.BR cells, which were treated with DMSO control or WL40 (200 nM for MM.1S; 1 μM for ANBL6.BR) for 16 hours; protein lysates were then subjected to the immunoblotting using specific antibodies against Cyclin-B1, Cell Division Cycle 25C (CDC25C), Cell Division Cycle 2 (CDCl.sub.2), or β-actin. FIG. 22E is an immunoblot of ANBL6.BR cells, which were treated with DMSO as a control, WL40 (1 μM), or RA190 (1 μM) for the indicated time periods; protein lysates were subjected to the immunoblotting using specific antibodies against p53, p21, BIP, PERK, p-eIF2α, calnexin, LC3A/B, and β-actin. FIG. 22F is an immunoblot of MM.1S cells, which were treated with DMSO as a control, WL40 (200 nM), or RA190 (300 nM) for the indicated time periods; protein lysates were subjected to the immunoblotting using specific antibodies against p53, p21, BIP, PERK, p-eIF2a, calnexin, LC3A/B, and β-actin. Blots shown are representative of three independent experiments.

(24) FIGS. 23A to 23D show that WL40 inhibits xenografted human MM cell growth and prolongs host survival FIG. 23A shows the tumor volume (mean tumor volume±SD in mm.sup.3, 10 mice/group) upon treatment with WL40 and RA190 at the indicated concentration versus time. Mice bearing human MM.1S MM tumors were treated with either vehicle control, WL40 (14.7 μM/kg; i.p.), or RA190 (26.8 μM/kg; i.p.) twice weekly for 18 days. FIG. 23B are Kaplan-Meier plots which show survival of mice. FIG. 23C shows immunoblot analysis of lysates of tumors harvested from control mice, and WL40- and RA190-treated mice. The lysates were subjected to the immunoblot analysis using anti-polyubiquitin, anti-cleaved-caspase-8, or anti-β-actin Abs. FIG. 23D show tumor sections from the vehicle control, and WL40-treated mice that were stained with anti-polyubiquitin, Ki67, caspase-3 (cleaved form), and CD31 antibodies. Scale bar, 10 μm.

(25) FIGS. 24A to 24C show that combinations of WL40 with pomalidomide, lenalidomide, or bortezomib trigger synergistic anti-MM activity. FIG. 24A shows MM.1S cells that were treated with WL40, pomalidomide, or WL40 plus pomalidomide for 48 hours, and then assessed for viability using WST assay. Isobologram analysis shows the synergistic cytotoxic effect of WL40 and pomalidomide. The graph is derived from the values given in the table (lower panel). Combination index (CI)<1 indicates synergy. FIG. 24B shows MM.1S cells that were treated with WL40, lenalidomide, or WL40 plus lenalidomide for 48 hours, and then assessed for viability using WST assay. Synergistic anti-MM activity was analyzed as in FIG. 24A. FIG. 24C shows MM.1S cells that were treated with WL40, bortezomib, or WL40 plus bortezomib for 48 hours, and then assessed for viability using a WST assay. Synergistic anti-MM activity was analyzed as in FIG. 24A.

(26) FIGS. 25A to 25B show NMR spectra for WL40. The compound WL40 was fully characterized using .sup.1H NMR (FIG. 25A) and .sup.13C NMR (FIG. 25B).

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS OF THE INVENTION

(27) The bifunctional compounds described herein interact with an E3 ubiquitin ligase and the ubiquitin receptor RPN13 (ADRM1). As described herein, without wishing to be bound by any particular theory, the therapeutic effect may be the result of degradation, modulation, or binding of an E3 ubiquitin ligase (e.g., Cereblon) by a compound described herein. For example, the therapeutic effect may be a result of recruitment of an E3 ubiquitin ligase (e.g., Cereblon) by modulation, targeting, binding, or modification of the E3 ubiquitin ligase, which induces the ubiquitination of a target protein, such as RPN13, and the use of a binder of the ubiquitin receptor RPN13 (e.g., RA190) which brings the E3 ubiquitin ligase in proximity to RPN13. E3 ubiquitin ligase is brought into proximity with RPN13, which leads to the ubiquitination of RPN13 and its subsequent degradation by the proteasome.

(28) A compound may be provided for use in any composition, kit, or method described herein as a pharmaceutically acceptable salt, co-crystal, tautomer, stereoisomer, solvate, hydrate, polymorph, isotopically enriched derivative, or prodrug thereof.

(29) In certain embodiments, the compounds that bind to RPN13 are compounds derived from the compounds described in U.S. patent application U.S. Ser. No. 14/889,768, filed May 6, 2014, which is incorporated herein by reference.

(30) In certain embodiments, the E3 ubiquitin ligase binding moiety of the bifunctional compounds of Formulae (I) and (I′) is derived from E3 ubiquitin ligase binding moiety (based on an immunomodulatory imide drug (e.g., derivatives of lenalidomide, thalidomide) of the bifunctional compounds described in U.S. patent application U.S. Ser. No. 15/148,253, filed May 6, 2016, U.S. Ser. No. 14/707,930, filed May 8, 2015, U.S. Ser. No. 62/096,318, filed Dec. 23, 2014, U.S. Ser. No. 62/128,457, filed Mar. 4, 2015, U.S. Ser. No. 62/149,170, filed Apr. 17, 2015, each of which is incorporated herein by reference.

(31) In certain embodiments, the E3 ubiquitin ligase binding moiety of the bifunctional compounds of Formula (I) is derived from E3 ubiquitin ligase binding moiety within the bifunctional compounds described in U.S. patent application U.S. Ser. No. 15/148,253, filed May 6, 2016, U.S. Ser. No. 14/707,930, filed May 8, 2015, U.S. Ser. No. 62/096,318, filed Dec. 23, 2014, U.S. Ser. No. 62/128,457, filed Mar. 4, 2015, U.S. Ser. No. 62/149,170, filed Apr. 17, 2015, each of which is incorporated herein by reference.

(32) In one aspect, disclosed are compounds of Formulae (I) and (I′):

(33) ##STR00015##
or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or stereoisomer thereof, wherein:

(34) in each pair of A's, one A is hydrogen, and the other A is one of:

(35) (i) phenyl, optionally substituted with 1-5 substituents selected from the group consisting of R.sup.1, OR.sup.1, NR.sup.1R.sup.2, S(O).sub.qR.sup.1, SO.sub.2R.sup.1R.sup.2, NR.sup.1R.sup.2SO.sub.2R.sub.2, C(O)R.sup.1, C(O)OR.sup.1, C(O)NR.sup.1R.sup.2, NR.sup.1C(O)R.sup.2, NR.sup.1C(O)OR.sup.2, CF.sub.3, and OCF.sub.3;

(36) (ii) naphthyl, optionally substituted with 1-5 substituents selected from the group consisting of R.sup.1, OR.sup.1, NR.sup.1R.sup.2, S(O).sub.qR.sup.1, SO.sub.2R.sup.1R.sup.2, NR.sup.1SO.sub.2R.sup.2, C(O)R.sup.1, C(O)OR.sup.1, C(O)NR.sup.1R.sup.2, NR.sup.1C(O)R.sup.2, NR.sup.1C(O)OR.sup.2, CF.sub.3, and OCF.sub.3;

(37) (iii) a 5- or 6-membered monocyclic heteroaryl group, having 1-3 heteroatoms selected from the group consisting of O, N, and S, optionally substituted with 1-3 substituents selected from the group consisting of R.sup.1, OR.sup.1, NR.sup.1R.sup.2, S(O).sub.qR.sup.1, SO.sub.2R.sup.1R.sup.2, NR.sup.1SO.sub.2R.sup.2, C(O)R.sup.1, C(O)OR.sup.1, C(O)NR.sup.1R.sup.2, NR.sup.1C(O)R.sup.2, NR.sup.1C(O)OR.sup.2, CF.sub.3, and OCF.sub.3; and

(38) (iv) an 8- to 10-membered bicyclic heteroallyl group containing 1-3 heteroatoms selected from the group consisting of O, N, and S; and the second ring is fused to the first ring using 3 to 4 carbon atoms, and the bicyclic hetero aryl group is optionally substituted with 1-3 substituents selected from the group consisting of R.sup.1, OR.sup.1, NR.sup.1R.sup.2, S(O).sub.qR.sup.1, SO.sub.2R.sup.1R.sup.2, NR.sup.1SO.sub.2R.sup.2, C(O)R.sup.1, C(O)OR.sup.1, C(O)NR.sup.1R.sup.2, NR.sup.1C(O)R.sup.2, NR.sup.1C(O)OR.sup.2, CF.sub.3, and OCF.sub.3;

(39) wherein Y is selected from the group consisting of O, S, NR.sup.1, and CR.sup.1R.sup.2, and

(40) wherein R.sup.1 and R.sup.2 are selected from the group consisting of hydrogen, nitro, hydroxyl, carboxy, amino, halogen, cyano and C.sub.1-C.sub.14 linear or branched alkyl groups, that are optionally substituted with 1-3 substituents selected from the group consisting of C.sub.1-C.sub.14 linear or branched alkyl, up to perhalo substituted C.sub.1-C.sub.14 linear or branched alkyl, C.sub.1-C.sub.14 alkoxy, hydrogen, nitro, hydroxyl, carboxy, amino, C.sub.1-C.sub.14 alkylamino, C.sub.1-C.sub.14 dialkylamino, halogen, and cyano;

(41) wherein R.sup.7 is hydrogen, C.sub.1-6 alkyl, or a nitrogen protecting group;

(42) wherein Z is selected from the group consisting of hydrogen; C.sub.1 to C.sub.14 linear, branched, or cyclic alkyls; phenyl; benzyl, 1-5 substituted benzyl, C.sub.1 to C.sub.3 alkyl-phenyl, wherein the alkyl moiety is optionally substituted with halogen up to perhalo; up to perhalo substituted C.sub.1 to C.sub.14 linear or branched alkyls; —(CH.sub.2).sub.q—K, where K is a 5 or 6 membered monocyclic heterocyclic ring, containing 1 to 4 atoms selected from oxygen, nitrogen and sulfur, which is saturated, partially saturated, or aromatic, or an 8 to 10 membered bicyclic heteroaryl having 1-4 heteroatoms selected from the group consisting of O, N, and S, wherein said alkyl moiety is optionally substituted with halogen up to perhalo, and wherein the variable q is an integer ranging from 0 to 4;

(43) L is a linker; and

(44) D is an E3 ubiquitin ligase binding moiety.

(45) Group D

(46) In certain embodiments, D is an E3 ubiquitin ligase binding moiety. D is inclusive of all moieties that bind, or can bind, any E3 ubiquitin ligase. For example, in certain embodiments, D is capable of binding an E3 ubiquitin ligase, such as Cereblon. In certain embodiments, D is capable of binding to multiple different E3 ubiquitin ligases. In certain embodiments, E binds to Cereblon. In certain embodiments, D is based on an immunomodulatory imide drug. In certain embodiments, D is derived from lenalidomide. In certain embodiments, D is derived from thalidomide.

(47) Human Cereblon (CRBN) is a protein of 442 amino acids with an apparent molecular weight of ˜51 kDa (GenBank: AAH17419). (For the CRBN protein sequence see: Higgins et al., Neurology. 2004, 63, 1927-31. For additional information related to the CRBN structure see Hartmann et al., PLoS One. 2015, 10, e0128342.) Human CRBN contains the N-terminal part (237-amino acids from 81 to 317) of ATP-dependent Lon protease domain without the conserved Walker A and Walker B motifs, 11 casein kinase II phosphorylation sites, 4 protein kinase C phosphorylation sites, 1 N-linked glycosylation site, and 2 myristoylation sites. CRBN is widely expressed in testis, spleen, prostate, liver, pancreas, placenta, kidney, lung, skeletal muscle, ovary, small intestine, peripheral blood leukocyte, colon, brain, and retina. CRBN is located in the cytoplasm, nucleus, and peripheral membrane. (Chang et al., Int. J. Biochem. Mol. Biol. 2011, 2, 287-94.)

(48) Cereblon is an E3 ubiquitin ligase, and it forms an E3 ubiquitin ligase complex with damaged DNA binding protein 1 (DDB1), Cullin-4A (CUL4A), and regulator of cullins 1 (ROC1). This complex ubiquitinates a number of other proteins. Through a mechanism which has not been completely elucidated, Cereblon ubiquitination of target proteins results in increased levels of fibroblast growth factor 8 (FGF8) and fibroblast growth factor 10 (FGF10). FGF8, in turn, regulates a number of developmental processes, such as limb and auditory vesicle formation.

(49) In certain embodiments, D is a modulator, binder, inhibitor, or ligand of Cereblon. In certain embodiments, D is a modulator of Cereblon. In certain embodiments, D is a binder of Cereblon. In certain embodiments, D is an inhibitor of Cereblon. In certain embodiments, D is a ligand of Cereblon. In certain embodiments, D is any modulator, binder, inhibitor, or ligand of Cereblon disclosed in U.S. patent application U.S. Ser. No. 14/792,414, filed Jul. 6, 2015, U.S. patent application U.S. Ser. No. 14/707,930, filed May 8, 2015, and International Patent Application, PCT/US2013/054663, filed Aug. 13, 2013, each of which is incorporated herein by reference. In certain embodiments, D is a modulator, binder, inhibitor, or ligand of a Cereblon variant. In certain embodiments, D is a modulator, binder, inhibitor, or ligand of a Cereblon isoform.

(50) In certain embodiments, D comprises a heteroaryl ring. In certain embodiments, D comprises a fused bicyclic heteroaryl ring. In certain embodiments, D comprises a fused bicyclic heteroaryl ring and a heterocyclic ring. In certain embodiments, D comprises a fused bicyclic heteroaryl ring and a heterocyclic ring, where the heterocyclic ring contains at least one nitrogen. In certain embodiments, D comprises a fused bicyclic heteroaryl ring and a heterocyclic ring, where the fused bicyclic heteroaryl ring and heterocyclic ring each contain at least one nitrogen. In certain embodiments, D comprises a fused bicyclic heteroaryl ring and a heterocyclic ring, where the fused bicyclic heteroaryl ring and heterocyclic ring each contain one nitrogen. In certain embodiments, D comprises a phthalimido group, or an analogue or derivative thereof. In certain embodiments, D comprises a phthalimido-glutarimide group, or an analogue or derivative thereof.

(51) In certain embodiments, D is of Formula (E-I):

(52) ##STR00016##
wherein:

(53) Ring A is a substituted or unsubstituted heterocyclyl, or substituted or unsubstituted heteroaryl ring;

(54) each R.sup.1A is, independently, halogen, —OH, C.sub.1-C.sub.6 alkyl, or C.sub.1-C.sub.6 alkoxy;

(55) each R.sup.3A is, independently, H or C.sub.1-C.sub.3 alkyl;

(56) each R.sup.3′ is, independently, C.sub.1-C.sub.3 alkyl;

(57) each R.sup.4A is, independently, H or C.sub.1-C.sub.3 alkyl; or two R.sup.4A, together with the carbon atom to which they are attached, form a C(O), C.sub.3-C.sub.6 carbocycle, or a 4-, 5-, or 6-membered heterocycle comprising 1 or 2 heteroatoms selected from N and O;

(58) R.sup.5A is H, C.sub.1-C.sub.3 alkyl, F, or Cl;

(59) m is 0, 1, 2, or 3; and

(60) n is 1 or 2.

(61) In certain embodiments, Formula (E-I) is derived from an immunomodulatory imide drug (e.g., derived from lenalidomide or thalidomide). In certain embodiments, Formula (E-I) is of Formula (IA) or Formula (IB).

(62) In certain embodiments, D is of Formula (IA):

(63) ##STR00017##
wherein:

(64) X.sup.A is C(O) or C(R.sup.3A).sub.2;

(65) each R.sup.1A is independently halogen, OH, C.sub.1-C.sub.6 alkyl, or C.sub.1-C.sub.6 alkoxy;

(66) R.sup.3A is H or C.sub.1-C.sub.3 alkyl;

(67) each R.sup.3′ is independently C.sub.1-C.sub.3 alkyl;

(68) each R.sup.4A is independently H or C.sub.1-C.sub.3 alkyl; or two R.sup.4A, together with the carbon atom to which they are attached, form a C(O), C.sub.3-C.sub.6 carbocycle, or a 4-, 5-, or 6-membered heterocycle comprising 1 or 2 heteroatoms selected from N and O;

(69) R.sup.5A is H, C.sub.1-C.sub.3 alkyl, or halogen;

(70) m is 0, 1, 2, or 3;

(71) n is (3, 1 or 2; and

(72) a1 is 0 or 1.

(73) In certain embodiments, D is of Formula (IA-a):

(74) ##STR00018##
wherein:

(75) X.sup.A is C(O) or C(R.sup.3A).sub.2;

(76) each R.sup.1A is, independently, halogen, OH, C.sub.1-C.sub.6 alkyl, or C.sub.1-C.sub.6 alkoxy;

(77) each R.sup.4A is, independently, H or C.sub.1-C.sub.3 alkyl; or two R.sup.4A, together with the carbon atom to which they are attached, form a C(O), C.sub.3-C.sub.6 carbocycle, or a 4-, 5-, or 6-membered heterocycle comprising 1 or 2 heteroatoms selected from N and O;

(78) R.sup.5A is H, C.sub.1-C.sub.3 alkyl, F, or Cl; and

(79) m is 0, 1, 2, or 3.

(80) In certain embodiments, D is of Formula (IA-b):

(81) ##STR00019##
wherein:

(82) X.sup.A is C(O) or C(R.sup.3A).sub.2;

(83) each R.sup.4A is, independently, H or C.sub.1-C.sub.3 alkyl; or two R.sup.4A, together with the carbon atom to which they are attached, form a C(O), C.sub.3-C.sub.6 carbocycle, or a 4-, 5-, or 6-membered heterocycle comprising 1 or 2 heteroatoms selected from N and O; and

(84) R.sup.5A is H, C.sub.1-C.sub.3 alkyl, F, or Cl.

(85) In certain embodiments, D is of Formula (IA-c):

(86) ##STR00020##
wherein:

(87) each R.sup.4A is, independently, H or C.sub.1-C.sub.3 alkyl; or two R.sup.4A, together with the carbon atom to which they are attached, form a C(O), C.sub.3-C.sub.6 carbocycle, or a 4-, 5-, or 6-membered heterocycle comprising 1 or 2 heteroatoms selected from N and O; and

(88) R.sup.5A is H, C.sub.1-C.sub.3 alkyl, F, or Cl.

(89) In certain embodiments, D is of Formula (IA-d):

(90) ##STR00021##
wherein: each R.sup.4A is, independently, H or C.sub.1-C.sub.3 alkyl; or two R.sup.4A, together with the carbon atom to which they are attached, form a C(O), C.sub.3-C.sub.6 carbocycle, or a 4-, 5-, or 6-membered heterocycle comprising 1 or 2 heteroatoms selected from N and O; and

(91) R.sup.5A is H, C.sub.1-C.sub.3 alkyl, F, or Cl.

(92) In certain embodiments, D is of Formula (IB):

(93) ##STR00022##

(94) wherein:

(95) —X.sup.1—X.sup.2— is C(R.sup.3A)═N or C(R.sub.3A).sub.2—C(R.sup.3A).sub.2;

(96) each R.sup.1A is independently halogen, OH, C.sub.1-C.sub.6 alkyl, or C.sub.1-C.sub.6 alkoxy;

(97) R.sup.3A is H or C.sub.1-C.sub.3 alkyl;

(98) each R.sup.3′ is independently C.sub.1-C.sub.3 alkyl;

(99) each R.sup.4A is independently H or C.sub.1-C.sub.3 alkyl; or two R.sup.4A, together with the carbon atom to which they are attached, form a C(O), C.sub.3-C.sub.6 carbocycle, or a 4-, 5-, or 6-membered heterocycle comprising 1 or 2 heteroatoms selected from N and O;

(100) R.sup.5A is H, C.sub.1-C.sub.3 alkyl, or halogen;

(101) m is 0, 1, 2, or 3;

(102) n is 0, 1, or 2; and

(103) a1 is 0 or 1.

(104) In certain embodiments, D is of Formula (IB-a):

(105) ##STR00023##
wherein:

(106) X.sup.1—X.sup.2 is C(R.sup.3A)═N or C(R.sup.3A).sub.2—C(R.sup.3A).sub.2;

(107) each R.sup.1A is, independently, halogen, —OH, C.sub.1-C.sub.6 alkyl, or C.sub.1-C.sub.6 alkoxy;

(108) each R.sup.3A is, independently, H or C.sub.1-C.sub.3 alkyl;

(109) each R.sup.4A is, independently, H or C.sub.1-C.sub.3 alkyl; or two R.sup.4A, together with the carbon atom to which they are attached, form a C(O), C.sub.3-C.sub.6 carbocycle, or a 4-, 5-, or 6-membered heterocycle comprising 1 or 2 heteroatoms selected from N and O;

(110) R.sup.5A is H, C.sub.1-C.sub.3 alkyl, F, or Cl; and

(111) m is 0, 1, 2, or 3.

(112) In certain embodiments, D is of Formula (IB-b):

(113) ##STR00024##
wherein:

(114) X.sup.3—X.sup.2 is C(R.sup.3A)═N or C(R.sup.3A).sub.2—C(R.sup.3A).sub.2;

(115) each R.sup.3A is, independently, H or C.sub.1-C.sub.3 alkyl;

(116) each R.sup.4A is, independently, H or C.sub.1-C.sub.3 alkyl; or two R.sup.4A, together with the carbon atom to which they are attached, form a C(O), C.sub.3-C.sub.6 carbocycle, or a 4-, 5-, or 6-membered heterocycle comprising 1 or 2 heteroatoms selected from N and O;

(117) R.sup.5A is H, C.sub.1-C.sub.3 alkyl, F, or Cl.

(118) In certain embodiments, D is of Formula (IB-c):

(119) ##STR00025##
wherein:

(120) each R.sup.4A is, independently, H or C.sub.1-C.sub.3 alkyl; or two R.sup.4A, together with the carbon atom to which they are attached, form a C(O), C.sub.3-C.sub.6 carbocycle, or a 4-, 5-, or 6-membered heterocycle comprising 1 or 2 heteroatoms selected from N and O;

(121) R.sup.5A is H, C.sub.1-C.sub.3 alkyl, F, or Cl.

(122) Formulae (IA), (IA-a), and (IA-b) include substituent X.sup.A. In certain embodiments, X.sup.A is C(O). In certain embodiments, X.sup.A is C(R.sup.3A).sub.2.

(123) Formulae (IA), (IA-a), and (IA-b) include substituents —X.sup.1—X.sup.2. In certain embodiments, —X.sup.1—X.sup.2— is C(R.sup.3A)═N. In certain embodiments, —X.sup.1—X.sup.2— is C(H)═N. In certain embodiments, —X.sup.1—X.sup.2— is C(C.sub.1-C.sub.3 alkyl)=N. In certain embodiments, —X.sup.1—X.sup.2— is C(R.sub.3A).sub.2—C(R.sup.3A).sub.2. In certain embodiments, —X.sup.1—X.sup.2— is C(H).sub.2—C(H).sub.2. In certain embodiments, —X.sup.1—X.sup.2— is C(H).sub.2—C(C.sub.1-C.sub.3 alkyl).sub.2. In certain embodiments, —X.sup.1—X.sup.2— is C(H).sub.2—C(C.sub.1-C.sub.3 alkyl).sub.2. In certain embodiments, —X.sup.1—X.sup.2— is C(H).sub.2—C(C.sub.1-C.sub.3 alkyl).sub.2. In certain embodiments, —X.sup.1—X.sup.2— is C(C.sub.1-C.sub.3 alkyl).sub.2-C(C.sub.1-C.sub.3 alkyl).sub.2.

(124) Formula (E-I) includes Ring A. In certain embodiments, Ring A is a substituted or unsubstituted heterocyclyl ring. In certain embodiments, Ring A is a substituted or unsubstituted heterocyclyl ring, which is a 3- to 10-membered non-aromatic ring system having ring carbon atoms and 1 to 4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, or sulfur. In certain embodiments, Ring A is a substituted or unsubstituted heteroaryl ring. In certain embodiments, Ring A is a substituted or unsubstituted heterocyclyl ring, which is a 5-10 membered monocyclic or bicyclic 4n+2 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 the group consisting of nitrogen, oxygen and sulfur. In certain embodiments, Ring A is a substituted or unsubstituted pyrrolidin-2-one. In certain embodiments, Ring A is a substituted or unsubstituted pyrrolidine-2,5-dione. In certain embodiments, Ring A is a substituted or unsubstituted 5,6-dihydropyrimidin-4(3H)-one. In certain embodiments, Ring A is a substituted or unsubstituted tetrahydropyrimidin-4(1H)-one.

(125) Formulae (E-I), (IA-a), (IA), (IB), and (IB-a) include substituent R.sup.1A. In certain embodiments, R.sup.1A is independently halogen, OH, C.sub.1-C.sub.6 alkyl, or C.sub.1-C.sub.6 alkoxy. In certain embodiments, at least one instance of R.sup.1A is halogen (e.g., F, Cl, Br, or I). In certain embodiments, at least one instance of R.sup.1A is OH. In certain embodiments, at least one instance of R.sup.1A is C.sub.1-C.sub.6 alkyl (e.g., methyl, ethyl). In certain embodiments, at least one instance of R.sup.1A is C.sub.1-C.sub.6 alkoxy (e.g., methoxy, ethoxy). In certain embodiments, m is 0. In certain embodiments, m is 1. In certain embodiments, m is 2. In certain embodiments, m is 3.

(126) Formulae (E-I), (IA), (IB), (IB-a), and (IB-b) include substituent R.sup.3A In certain embodiments, at least one instance of R.sup.3A is H, In certain embodiments, at least one instance of R.sup.3A is C.sub.1-C.sub.3 alkyl (e.g., methyl, ethyl). In certain embodiments, at least one instance of R.sup.3 is C.sub.1-C.sub.3 alkyl (e.g., methyl, ethyl). In certain embodiments, n is 0. In certain embodiments, n is 1. In certain embodiments, n is 2.

(127) Formulae (E-I), (IA-a), (IA-b), (IA-c), (IA-d), (IA), (IB), (IB-a), (IB-b), and (IB-c) include substituent R.sup.5A. In certain embodiments, R.sup.5A is H, In certain embodiments, R.sup.5A is deuterium. In certain embodiments, R.sup.5A is C.sub.1-C.sub.3 alkyl. In certain embodiments, R.sup.3A is halogen (e.g., F, Cl, Br, or I).

(128) In certain embodiments, a1 is 0. In certain embodiments, a1 is 1.

(129) Formulae (E-I), (IA-a), (IA-b), (IA-c), (IA-d), (IA), (IB), (IB-a), (IB-b), and (IB-c) include substituent R.sup.4A. In certain embodiments, at least one instance of R.sup.4A is H. In certain embodiments, at least one instance of R.sup.4A is C.sub.1-C.sub.3 alkyl (e.g., methyl, ethyl). In certain embodiments, two R.sup.4A, together with the carbon atom to which they are attached, form a C(O), C.sub.3-C.sub.6 carbocycle, or a. 4-, 5-, or 6-membered heterocycle comprising 1 or 2 heteroatoms selected from N and O.

(130) In certain embodiments, m and n are both 0; R.sup.3A is H; two R.sup.4A, together with the carbon atom to which they are attached, form a C(O); and R.sup.5A is H.

(131) In certain embodiments, a1 is 1; m and n are both 0; R.sup.3A is H; two R.sup.4A, together with the carbon atom to which they are attached, form a C(O); R.sup.5A is H; and X.sup.A is C(O). In certain embodiments, a1 is 1; m and n are both 0; R.sup.3A is H; two R.sup.4A, together with the carbon atom to which they are attached, form a C(O); R.sup.5A is H; and X.sup.A is C(R.sup.3A).sub.2. In certain embodiments, two R.sup.4A, together with the carbon atom to which they are attached, form a C(O); R.sup.5A is H; and X.sup.A is C(O). In certain embodiments, two R.sup.4A, together with the carbon atom to which they are attached, form a C(O); R.sup.5A is H; and X.sup.A is C(R.sup.3A).sub.2. In certain embodiments, two R.sup.4A, together with the carbon atom to which they are attached, form a C(O); and R.sup.5A is H.

(132) In certain embodiments, —X.sup.1—X.sup.2— is C(R.sup.3A)═N: R.sup.3A is H; and two R.sup.4A, together with the carbon atom to which they are attached, form a C(O). In certain embodiments, —X.sup.1—X.sup.2— is C(R.sub.3A).sub.2—C(R.sup.3A).sub.2; R.sup.3A is H; and two R.sup.4A, together with the carbon atom to which they are attached, form a C(O). In certain embodiments, —X.sup.1—X.sup.2— is C(R.sup.3A)═N; R.sup.5A is H; and two R.sup.4A, together with the carbon atom to which they are attached, form a C(O). In certain embodiments, —X.sup.1—X.sup.2— is C(R.sub.3A).sub.2—C(R.sup.3A).sub.2; R.sup.5A is H; and two R.sup.4A, together with the carbon atom to which they are attached, form a C(O).

(133) In certain embodiments, D is thalidomide, lenalidomide, pomalidomide, CC-885 (Matyskiela et al., Nature 2016, 535, 252-257), 3-(5-amino-2-methyl-4-oxoquinazolin-3(4H)-yl)piperidine-2,6-dione, or an analogue or derivative thereof. In certain embodiments, D is thalidomide. In certain embodiments, D is lenalidomide.

(134) In certain embodiments, D is:

(135) ##STR00026##

(136) In certain embodiments, D is:

(137) ##STR00027##

(138) In certain embodiments, D is

(139) ##STR00028##

(140) In certain embodiments, D is of the formula:

(141) ##STR00029##

(142) In certain embodiments, the E3 ligase binding moiety binds an E3 ubiquitin ligase with a K.sub.d of less than 50,000 nM, less than 20,000 nM, less than 10,000 nM, less than 5,000 nM, less than 2,500 nM, less than 1,000 nM, less than 900 nM, less than 800 nM, less than 700 nM, less than 600 nM, less than 500 nM, less than 400 nM, less than 300 nM, less than 200 nM, less than 100 nM, less than 90 nM, less than 80 nM, less than 70 nM, less than 60 nM, less than 50 nM, less than 40 nM, less than 30 nM, less than 20 nM, less than 10 nM, less than 5 nM, less than 4 nM, less than 3 nM, less than 2 nM, or less than 1 nM.

(143) In certain embodiments, the E3 ligase binding moiety binds Cereblon with a K.sub.d of less than 50,000 nM, less than 20,000 nM, less than 10,000 nM, less than 5,000 nM, less than 2,500 nM, less than 1,000 nM, less than 900 nM, less than 800 nM, less than 700 nM, less than 600 nM, less than 500 nM, less than 400 nM, less than 300 nM, less than 200 nM, less than 100 nM, less than 90 nM, less than 80 nM, less than 70 nM, less than 60 nM, less than 50 nM, less than 40 nM, less than 30 nM, less than 20 nM, less than 10 nM, less than 5 nM, less than 4 nM, less than 3 nM, less than 2 nM, or less than 1 nM.

(144) In certain embodiments, the E3 ligase binding moiety selectively binds an E3 ubiquitin ligase as compared to another protein. In some embodiments, the E3 ligase binding moiety selectively binds Cereblon over another protein. In some embodiments, the E3 ligase binding moiety selectively binds Cereblon over another E3 ubiquitin ligase. In certain embodiments, the selectivity is between about 2-fold and about 5-fold. In certain embodiments, the selectivity is between about 5-fold and about 10-fold. In certain embodiments, the selectivity is between about 10-fold and about 20-fold. In certain embodiments, the selectivity is between about 20-fold and about 50-fold. In certain embodiments, the selectivity is between about 50-fold and about 100-fold. In certain embodiments, the selectivity is between about 100-fold and about 200-fold. In certain embodiments, the selectivity is between about 200-fold and about 500-fold. In certain embodiments, the selectivity is between about 500-fold and about 1000-fold. In certain embodiments, the selectivity is at least about 1000-fold.

(145) Substituents A, Y, Z, R.sup.1, R.sup.2, R.sup.7

(146) Compounds of Formulae (I) and (I′) are bifunctional compounds that bind to the ubiquitin receptor RPN13 on one end and bind to E3 ligase on the other end. For compounds of Formulae (I) and (I′), the E3 ligase binding moiety includes substituents A, Y, Z, R.sup.1, and R.sup.2.

(147) In each pair of A's, one A is hydrogen, and the other A is one of:

(148) (i) phenyl, optionally substituted with 1-5 substituents selected from the group consisting of R.sup.1, OR.sup.1, NR.sup.1R.sup.2, S(O).sub.qR.sup.1, SO.sub.2R.sup.1R.sup.2, NR.sup.1SO.sub.2R.sup.2, C(O)R.sup.1, C(O)OR.sup.1, C(O)NR.sup.1R.sup.2, NR.sup.1C(O)R.sup.2, NR.sup.1C(O)OR.sup.2, CF.sub.3, and OCF.sub.3;

(149) (ii) naphthyl, optionally substituted with 1-5 substituents selected from the group consisting of R.sup.1, OR.sup.1, NR.sup.1R.sup.2, S(O).sub.qR.sup.1, SO.sub.2R.sup.1R.sup.2, NR.sup.1SO.sub.2R.sup.2, C(O)R.sup.1, C(O)OR.sup.1, C(O)NR.sup.1R.sup.2, NR.sup.1C(O)R.sup.2, NR.sup.1C(O)OR.sup.2, CF.sub.3, and OCF.sub.3;

(150) (iii) a 5 or 6 membered monocyclic heteroaryl group, having 1-3 heteroatoms selected from the group consisting of O, N, and S, optionally substituted with 1-3 substituents selected from the group consisting of R.sup.1, OR.sup.1, NR.sup.1R.sup.2, S(O).sub.qR SO.sub.2R.sup.1R.sup.2, NR.sup.1SO.sub.2R.sup.2, C(O)R.sup.1, C(O)OR.sup.1, C(O)NR.sup.1R.sup.2, NR.sup.1C(O)R.sup.2, NR.sup.1C(O)OR.sup.2, CF.sub.3, and OCF.sub.3; and

(151) (iv) an 8 to 10 membered bicyclic heteroallyl group containing 1-3 heteroatoms selected from the group consisting of O, N, and S; and the second ring is fused to the first ring using 3 to 4 carbon atoms, and the bicyclic hetero aryl group is optionally substituted with 1-3 substituents selected from the group consisting of R.sup.1, OR.sup.1, NR.sup.1R.sup.2, S(O).sub.qR.sup.1, SO.sub.2R.sup.1R.sup.2, NR.sup.1SO.sub.2R.sup.2, C(O)R.sup.1, C(O)OR.sup.1, C(O)NR.sup.1R.sup.2, NR.sup.1C(O)R.sup.2, NR.sup.1C(O)OR.sup.2, CF.sub.3, and OCF.sub.3.

(152) In each pair of A's, in some embodiments, one A is phenyl, optionally substituted with 1-5 substituents including R.sup.1, OR.sup.1, NR.sup.1R.sup.2, S(O).sub.qR.sup.1, SO.sub.2R.sup.1R.sup.2, NR.sup.1SO.sub.2R.sup.2, C(O)R.sup.1, C(O)OR.sup.1, C(O)NR.sup.1R.sup.2, NR.sup.1C(O)R.sup.2, NR.sup.1C(O)OR.sup.2, CF.sub.3, or OCF.sub.3. In some embodiments, one A is phenyl. In some embodiments, one A is phenyl substituted with halogen. In some embodiments, in each pair of A's, one A is hydrogen; and the other A is phenyl, optionally substituted with R.sup.1. In some embodiments, in each pair of A's, one A is hydrogen; and the other A is phenyl substituted with halogen (e.g., F, Cl, Br, or I).

(153) In each pair of A's, in some embodiments, one A is naphthyl, optionally substituted with 1-5 substituents including R.sup.1, OR.sup.1, NR.sup.1R.sup.2, S(O).sub.qR.sup.1, SO.sub.2R.sup.1R.sup.2, NR.sup.1SO.sub.2R.sup.2, C(O)R.sup.1, C(O)OR.sup.1, C(O)NR.sup.1R.sup.2, NR.sup.1C(O)R.sup.2, NR.sup.1C(O)OR.sup.2, CF.sub.3, or OCF.sub.3.

(154) In some embodiments, in each pair of A's, one A is hydrogen; and the other A is phenyl, optionally substituted with R.sup.1. In some embodiments, in each pair of A's, one A is hydrogen and the other A is phenyl substituted with halogen. In some embodiments, in each pair of A's, one A is hydrogen and the other A is phenyl substituted with F. In some embodiments, in each pair of A's, one A is hydrogen and the other A is phenyl substituted with Cl. In some embodiments, in each pair of A's, one A is hydrogen and the other A is phenyl substituted with Br. In some embodiments, in each pair of A's, one A is hydrogen and the other A is phenyl substituted with I. In some embodiments, in each pair of A's, both A's are phenyl, optionally substituted with 1-5 substituents including R.sup.1, OR.sup.1, NR.sup.1R.sup.2, S(O).sub.qR.sup.1, SO.sub.2R.sup.1R.sup.2, NR.sup.1SO.sub.2R.sup.2, C(O)R.sup.1, C(O)OR.sup.1, C(O)NR.sup.1R.sup.2, NR.sup.1C(O)R.sup.2, NR.sup.1C(O)OR.sup.2, CF.sub.3, or OCF.sub.3. In some embodiments, in each pair of A's, both A's are phenyl, optionally substituted with R.sup.1.

(155) In some embodiments, at least one instance of Y is selected from the group consisting of O, S, NR.sup.1, and CR.sup.2. In some embodiments, at least one instance of Y is O. In certain embodiments, both Y are O. In some embodiments, one instance of Y is O and the other instance of Y is —CH.sub.2. In some embodiments, both instances of Y are —CH.sub.2.

(156) In some embodiments, R.sup.1 and R.sup.2 are selected from the group consisting of hydrogen, nitro, hydroxyl, carboxy, amino, halogen, cyano, and C.sub.1-C.sub.14 linear or branched alkyl groups, that are optionally substituted with 1-3 substituents selected from the group consisting of C.sub.1-C.sub.14 linear or branched alkyl, up to perhalo substituted C.sub.1-C.sub.14 linear or branched alkyl, C.sub.1-C.sub.14 alkoxy, hydrogen, nitro, hydroxyl, carboxy, amino, C.sub.1-C.sub.14 alkylamino, C.sub.1-C.sub.14 dialkylamino, halogen, and cyano. In some embodiments, R.sup.1 is substituted or unsubstituted alkyl (e.g., substituted or unsubstituted C.sub.1-6 alkyl). In some embodiments, R.sup.1 is halogen (e.g., F, Br, Cl). In some embodiments, R.sup.2 is substituted or unsubstituted alkyl (e.g., substituted or unsubstituted C.sub.1-6 alkyl). In some embodiments, all the instances of R.sup.1 are the same.

(157) In some embodiments, R.sup.7 is hydrogen. In some embodiments, R.sup.7 is C.sub.1-6 alkyl (e.g., substituted or unsubstituted C.sub.1-6 alkyl) (e.g., methyl, ethyl, or propyl)). In some embodiments, R.sup.7 is a nitrogen protecting group (e.g., benzyl (Bn), t-butyl carbonate (BOC or Boc), benzyl carbamate (Cbz), 9-fluorenylmethyl carbonate (Fmoc), trifluoroacetyl, triphenylmethyl, acetyl, or p-toluenesulfonamide (Ts)).

(158) In some embodiments, Z is selected from the group consisting of hydrogen; C.sub.1 to C.sub.14 linear, branched, or cyclic alkyls; phenyl; benzyl, 1-5 substituted benzyl, C.sub.1 to C.sub.3 alkyl-phenyl, wherein the alkyl moiety is optionally substituted with halogen up to perhalo; up to perhalo substituted C.sub.1 to C.sub.14 linear or branched alkyls; and —(CH.sub.2).sub.q—K. In some embodiments, K is a 5 or 6 membered monocyclic heterocyclic ring, containing 1 to 4 atoms selected from oxygen, nitrogen, and sulfur, which is saturated, partially saturated, or aromatic, or an 8 to 10 membered bicyclic heteroaryl having 1-4 heteroatoms selected from the group consisting of O, N, and S, wherein said alkyl moiety is optionally substituted with halogen up to perhalo, and wherein the variable q is an integer ranging from 0 to 4. In certain embodiments, q is 0. In certain embodiments, q is 1. In certain embodiments, q is 2. In certain embodiments, q is 3. In certain embodiments, q is 4.

(159) In some embodiments, Z is phenyl. In some embodiments, Z is benzyl. In some embodiments, Z is unsubstituted benzyl.

(160) In certain embodiments, a compound of Formula (I) is of formula:

(161) ##STR00030##
or a pharmaceutically acceptable salt thereof.

(162) In certain embodiments, a compound of Formula (I) is of formula:

(163) ##STR00031##
or a pharmaceutically acceptable salt thereof.

(164) In certain embodiments, a compound of Formula (I) is of formula:

(165) ##STR00032##
or a pharmaceutically acceptable salt thereof.

(166) In certain embodiments, a compound of Formula (I) is of formula:

(167) ##STR00033##
or a pharmaceutically acceptable salt thereof.

(168) In certain embodiments, a compound of Formula (I) is of formula:

(169) ##STR00034##
or a pharmaceutically acceptable salt thereof.
Linker L

(170) In Formula (I), L is a divalent moiety linking the group D to the moiety of

(171) ##STR00035##
(i.e., the RPN13 binding moiety). In Formula (I), L covalently links the group D to the moiety of

(172) ##STR00036##

(173) In Formulae (I) and (I′), L is a divalent moiety. In certain embodiments, L is a bond, a substituted or unsubstituted C.sub.1-12 hydrocarbon chain, optionally wherein one or more chain atoms of the hydrocarbon chain are independently replaced with —C(═O)—, —O—, —S—, —NR.sup.b, —N═, or ═N—, substituted or unsubstituted carbocyclylene, substituted or unsubstituted heterocyclylene, substituted or unsubstituted arylene, substituted or unsubstituted heteroarylene, or substituted or unsubstituted heteroalkylene. In certain embodiments, L is any “L0” group or “Linker” group recited in U.S. patent application U.S. Ser. No. 14/707,930, filed May 8, 2015, which is incorporated herein by reference. In certain embodiments, L is any “L” group recited in U.S. patent application U.S. Ser. No. 14/792,414, filed Jul. 6, 2015, which is incorporated herein by reference.

(174) In certain embodiments, the chain of linker L comprises up to 50 atoms as the shortest path between D and the moiety of

(175) ##STR00037##
excluding hydrogen atoms. In certain embodiments, the chain of linker L comprises up to 50 atoms, excluding hydrogen atoms. In certain embodiments, L comprises up to 40 atoms, excluding hydrogen atoms. In certain embodiments, L comprises up to 30 atoms, excluding hydrogen atoms. In certain embodiments, L comprises up to 20 atoms, excluding hydrogen atoms. In certain embodiments, L comprises up to 14 atoms, excluding hydrogen atoms. In certain embodiments, L comprises up to 15 atoms, excluding hydrogen atoms. In certain embodiments, L comprises up to 12 atoms, excluding hydrogen atoms. In certain embodiments, L comprises up to 10 atoms, excluding hydrogen atoms. In certain embodiments, L comprises up to 9 atoms excluding hydrogen atoms. In certain embodiments, L comprises up to 6 atoms excluding hydrogen atoms. In certain embodiments, L comprises up to 5 atoms excluding hydrogen atoms. In certain embodiments, L comprises up to 3 atoms excluding hydrogen atoms.

(176) In certain embodiments, any of the atoms in L can be substituted. In certain embodiments, none of the atoms in the linker L are substituted. In certain embodiments, none of the carbon atoms in the linker are substituted.

(177) In certain embodiments, L is substituted or unsubstituted carbocyclylene, substituted or unsubstituted heterocyclylene, substituted or unsubstituted arylene, substituted or unsubstituted heteroarylene, or substituted or unsubstituted heteroalkylene. In certain embodiments, each instance of R.sup.b is independently hydrogen, substituted or unsubstituted C.sub.1-6 alkyl, or a nitrogen protecting group, or optionally two instances of R.sup.b are taken together with their intervening atoms to form a substituted or unsubstituted heterocyclic or substituted or unsubstituted heteroaryl ring.

(178) In certain embodiments, L is a substituted or unsubstituted C.sub.1-14 hydrocarbon chain, optimally wherein one or more chain atoms of the hydrocarbon chain are independently replaced with —O— or —NR.sup.b—. In certain embodiments, L is a substituted or unsubstituted C.sub.12-14 hydrocarbon chain, optimally wherein one or more chain atoms of the hydrocarbon chain are independently replaced with —O— or —NR.sup.b—. In certain embodiments, L is a substituted or unsubstituted C.sub.14 hydrocarbon chain, optimally wherein one or more chain atoms of the hydrocarbon chain are independently replaced with —O— or —NR.sup.b—. In certain embodiments, L is a substituted or unsubstituted C.sub.12 hydrocarbon chain, optimally wherein one or more chain atoms of the hydrocarbon chain are independently replaced with —O— or —NR.sup.b—. In certain embodiments, L is a substituted or unsubstituted C.sub.1-12 hydrocarbon chain, optimally wherein one or more chain atoms of the hydrocarbon chain are independently replaced with —O— or —NR.sup.b—. In certain embodiments, L is a substituted or unsubstituted C.sub.8-12 hydrocarbon chain, optimally wherein one or more chain atoms of the hydrocarbon chain are independently replaced with —O— or —NR.sup.b—. In certain embodiments, L is a substituted or unsubstituted C.sub.8 hydrocarbon chain, optimally wherein one or more chain atoms of the hydrocarbon chain are independently replaced with —O— or —NR.sup.b—. In certain embodiments, L is a substituted or unsubstituted C.sub.12 hydrocarbon chain, optimally wherein one or more chain atoms of the hydrocarbon chain are independently replaced with —O— or —NR.sup.b—. In certain embodiments, L is a substituted or unsubstituted C.sub.1-12 hydrocarbon chain. In certain embodiments, one or more chain atoms of the hydrocarbon chain of L are independently replaced with —C(═O)—, —O—, —S—, —NR.sup.b—, —N═, or ═N—. In certain embodiments, L is a substituted or unsubstituted C.sub.1-12 hydrocarbon chain, wherein one chain atom of the hydrocarbon chain is independently replaced with —O—. In certain embodiments, L is an unsubstituted C.sub.1-3 hydrocarbon chain.

(179) In certain embodiments, L is an all-carbon, substituted or unsubstituted C.sub.6-12 hydrocarbon chain.

(180) In certain embodiments, L is an all-carbon, substituted or unsubstituted C.sub.1-12 hydrocarbon chain. In certain embodiments, L is an all-carbon, substituted or unsubstituted C.sub.1-6 hydrocarbon chain. In certain embodiments, L is of the formula:

(181) ##STR00038##
wherein a is 0, 1, 2, 3, 4, 5, or 6. In certain embodiments, a is 0. In certain embodiments, L is a bond. In certain embodiments, a is 1. In certain embodiments, a is 2. In certain embodiments, a is 3. In certain embodiments, a is 4. In certain embodiments, a is 5. In certain embodiments, a is 6. In certain embodiments, L is of the formula:

(182) ##STR00039##
wherein l.sup.A indicates the point of attachment to D, and l.sup.R indicates the point of attachment to the moiety of formula

(183) ##STR00040##
In certain embodiments, L is of the formula:

(184) ##STR00041##

(185) In certain embodiments, L is a substituted or unsubstituted C.sub.1-12 hydrocarbon chain, optionally wherein one or more chain atoms of the hydrocarbon chain are independently replaced with —C(═O)—, —O—, —S—, —NR.sup.b—, —N═, or ═N—. In certain embodiments, L is an unsubstituted C.sub.1-12 hydrocarbon chain, wherein one or more chain atoms of the hydrocarbon chain are independently replaced with —O— or —NR.sup.b—. In certain embodiments, L is an unsubstituted C.sub.1-2 hydrocarbon chain, wherein one chain atom of the hydrocarbon chain is replaced with —O—. In certain embodiments, L is of the formula:

(186) ##STR00042##
wherein l.sup.A indicates the point of attachment to D, and l.sup.R indicates the point of attachment to the

(187) ##STR00043##
moiety. In certain embodiments, L is of the formula:

(188) ##STR00044##
In certain embodiments, L is a substituted C.sub.1-6 hydrocarbon chain, wherein one chain atom of the hydrocarbon chain is replaced with —N—. In certain embodiments, L is of the formula:

(189) ##STR00045##
In certain embodiments, L is of the formula:

(190) ##STR00046##
In certain embodiments, L is of the formula:

(191) ##STR00047##
In certain embodiments, L is of the formula:

(192) ##STR00048##
In certain embodiments, L is an unsubstituted C.sub.1-3 hydrocarbon chain, wherein one chain atom of the hydrocarbon chain is replaced with —C(═O)—. In certain embodiments, L is an unsubstituted C.sub.1-3 hydrocarbon chain, wherein one chain atom of the hydrocarbon chain is replaced with —S—. In certain embodiments, L is an unsubstituted C.sub.1-3 hydrocarbon chain, wherein one chain atom of the hydrocarbon chain is replaced with —NR.sup.b—. In certain embodiments, L is an unsubstituted C.sub.1-3 hydrocarbon chain, wherein one chain atom of the hydrocarbon chain is replaced with —N═. In certain embodiments, L is an unsubstituted C.sub.1-3 hydrocarbon chain, wherein one chain atom of the hydrocarbon chain is replaced with ═N—.

(193) In certain embodiments, L is of the formula:

(194) ##STR00049##
In certain embodiments, L is of the formula:

(195) ##STR00050##

(196) In certain embodiments, L is L is

(197) ##STR00051##
and g is 2, 3, or 4; p is 0, 1, 2, 3, 4, or 5; and s is 1, 2, 3, 4, 5, or 6. In certain embodiments, g is 2. In certain embodiments, g is 3. In certain embodiments, g is 4. In certain embodiments, p is 0. In certain embodiments, p is 1. In certain embodiments, p is 2. In certain embodiments, p is 3. In certain embodiments, p is 4. In certain embodiments, p is 5. In certain embodiments, s is 1. In certain embodiments, s is 2. In certain embodiments, s is 3. In certain embodiments, s is 4. In certain embodiments, s is 5. In certain embodiments, s is 6. In certain embodiments, L is

(198) ##STR00052##
and g is 2, 3, or 4. In certain embodiments, L is

(199) ##STR00053##
wherein p is 0 and s is 2, 3, 4, 5, or 6. In certain embodiments, L is

(200) ##STR00054##
wherein p is 0; and s is 4, 5, or 6. In certain embodiments, p is 0; and s is 2, 3, 4, 5, or 6. In certain embodiments, p is 0; and s is 3, 4, or 5. In certain embodiments, s is 3, 4, or 5.

(201) In certain embodiments, L is

(202) ##STR00055##
g is 1-5; p is 2-5; and s is 1-5. In certain embodiments, g is 1. In certain embodiments, g is 2. In certain embodiments, g is 3. In certain embodiments, g is 4. In certain embodiments, g is 5. In certain embodiments, p is 2. In certain embodiments, p is 3. In certain embodiments, p is 4. In certain embodiments, p is 5. In certain embodiments, s is 1. In certain embodiments, s is 2. In certain embodiments, s is 3. In certain embodiments, s is 4. In certain embodiments, s is 5. In certain embodiments, L is an all-carbon, substituted or unsubstituted C.sub.1-12 hydrocarbon chain. In certain embodiments, L is an all-carbon, substituted C.sub.1-12 hydrocarbon chain. In certain embodiments, L is an all-carbon, unsubstituted C.sub.1-12 hydrocarbon chain.

(203) In certain embodiments, L is of the formula:

(204) ##STR00056##
g is 1-5; and g1 is 1-5. In certain embodiments, g is 1, 2, 3, 4, or 5. In certain embodiments, g1 is 1, 2, 3, 4, or 5. In certain embodiments, L is of the formula:

(205) ##STR00057##
g is 3 or 4; and g1 is 3 or 4. In certain embodiments, L is of the formula:

(206) ##STR00058##
g is 3; and g1 is 3 or 4. In certain embodiments, g is 1. In certain embodiments, g is 2. In certain embodiments, g is 3. In certain embodiments, g is 4. In certain embodiments, g is 5. In certain embodiments, g1 is 1. In certain embodiments, g1 is 2. In certain embodiments, g1 is 3. In certain embodiments, g1 is 4. In certain embodiments, g1 is 5.

(207) In certain embodiments, L is

(208) ##STR00059##
g is 1, 2, 3, 4, or 5; and s is 2, 3, or 4. In certain embodiments, L is

(209) ##STR00060##
g is 3 or 4; and s is 2, 3, or 4. In certain embodiments, L is

(210) ##STR00061##
g is 3 or 4; and s is 2 or 3. In certain embodiments, L is

(211) ##STR00062##
g is 3 or 4; and s is 2, 3, or 4. In certain embodiments, L is

(212) ##STR00063##
g is 3 or 4; and s is 2 or 3. In certain embodiments, g is 1. In certain embodiments, g is 2. In certain embodiments, g is 3. In certain embodiments, g is 4. In certain embodiments, g is 5. In certain embodiments, s is 2. In certain embodiments, s is 3. In certain embodiments, s is 4.

(213) In certain embodiments, L is of the formula:

(214) ##STR00064##
wherein: g is 1-5; and g1 is 1-5. In certain embodiments, g is 1. In certain embodiments, g is 2. In certain embodiments, g is 3. In certain embodiments, g is 4. In certain embodiments, g is 5. In certain embodiments, g1 is 1. In certain embodiments, g1 is 2. In certain embodiments, g1 is 3. In certain embodiments, g1 is 4. In certain embodiments, g1 is 5. In certain embodiments, L is of the formula:

(215) ##STR00065##
In certain embodiments, L is of the formula:

(216) ##STR00066##
In certain embodiments, L is of the formula:

(217) ##STR00067##

(218) In certain embodiments, L is of the formula:

(219) ##STR00068##
In certain embodiments, L is of the formula:

(220) ##STR00069##
In certain embodiments, L is of the formula:

(221) ##STR00070##
In certain embodiments, L is of the formula:

(222) ##STR00071##
In certain embodiments, L is of the formula:

(223) ##STR00072##
In certain embodiments, L is of the formula:

(224) ##STR00073##

(225) In certain embodiments, a compound of Formulae (I) or (I′) is of the formula:

(226) ##STR00074## ##STR00075##
or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or stereoisomer thereof.

(227) In certain embodiments, a compound of Formulae (I) or (I′) is of the formula:

(228) ##STR00076## ##STR00077##
or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or stereoisomer thereof.

(229) In certain embodiments, a compound of Formulae (I) or (I′) is a final product compound depicted in Example 1. In certain embodiments, a compound of Formulae (I) or (I′) is a product compound depicted in Example 2. In certain embodiments, a compound of Formulae (I) or (I′) is a product compound depicted in Example 1 or Example 2. In certain embodiments, a compound of Formulae (I) or (I′) is a product compound depicted in one of the Figures.

(230) In some embodiments, the compound of Formulae (I) or (I′) selectively binds ubiquitin receptor RPN13 over another protein. In some embodiments, the compound of Formulae (I) or (I′) selectively binds ubiquitin receptor RPN13 over another ubiquitin receptor. In certain embodiments, the selectivity is between about 2-fold and about 5-fold. In certain embodiments, the selectivity is between about 5-fold and about 10-fold. In certain embodiments, the selectivity is between about 10-fold and about 20-fold. In certain embodiments, the selectivity is between about 20-fold and about 50-fold. In certain embodiments, the selectivity is between about 50-fold and about 100-fold. In certain embodiments, the selectivity is between about 100-fold and about 200-fold. In certain embodiments, the selectivity is between about 200-fold and about 500-fold. In certain embodiments, the selectivity is between about 500-fold and about 1000-fold. In certain embodiments, the selectivity is at least about 1000-fold.

(231) In certain embodiments, the compound of Formulae (I) or (I′) induces the degradation of up to 10%, up to 15%, up to 20%, up to 25%, up to 30%, up to 35%, up to 40%, up to 45%, up to 50%, up to 55%, up to 60%, up to 65%, up to 70%, up to 75%, up to 80%, up to 85%, up to 90%, up to 95%, up to 99%, or up to 100% of the ubiquitin receptor RPN13 at a concentration of 100,000 nM or less, 50,000 nM or less, 20,000 nM or less, 10,000 nM or less, 5,000 nM or less, 3,500 nM or less, 2,500 nM or less, 1,000 nM or less, 900 nM or less, 800 nM or less, 700 nM or less, 600 nM or less, 500 nM or less, 400 nM or less, 300 nM or less, 200 nM or less, 100 nM or less, 90 nM or less, 80 nM or less, 70 nM or less, 60 nM or less, 50 nM or less, 40 nM or less, 30 nM or less, 20 nM or less, 10 nM or less, 5 nM or less, 4 nM or less, 3 nM or less, 2 nM or less, or 1 nM or less.

(232) In certain embodiments, the compound of Formulae (I) or (I′) increases the rate of degradation of the ubiquitin receptor RPN13 up to 10%, up to 15%, up to 20%, up to 25%, up to 30%, up to 35%, up to 40%, up to 45%, up to 50%, up to 55%, up to 60%, up to 65%, up to 70%, up to 75%, up to 80%, up to 85%, up to 90%, up to 95%, up to 99%, or up to 100% at a concentration of 100,000 nM or less, 50,000 nM or less, 20,000 nM or less, 10,000 nM or less, 5,000 nM or less, 3,500 nM or less, 2,500 nM or less, 1,000 nM or less, 900 nM or less, 800 nM or less, 700 nM or less, 600 nM or less, 500 nM or less, 400 nM or less, 300 nM or less, 200 nM or less, 100 nM or less, 90 nM or less, 80 nM or less, 70 nM or less, 60 nM or less, 50 nM or less, 40 nM or less, 30 nM or less, 20 nM or less, 10 nM or less, 5 nM or less, 4 nM or less, 3 nM or less, 2 nM or less, or 1 nM or less.

(233) Pharmaceutical Compositions, Kits, and Administration

(234) The present disclosure provides pharmaceutical compositions comprising a compound of Formulae (I) or (I′), or a pharmaceutically acceptable salt, co-crystal, tautomer, stereoisomer, solvate, hydrate, polymorph, isotopically enriched derivative, or prodrug thereof, and optionally a pharmaceutically acceptable excipient. In certain embodiments, the pharmaceutical composition described herein comprises a compound of Formulae (I) or (I′), or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.

(235) In certain embodiments, the compound of Formulae (I) or (I′) is provided in an effective amount in the pharmaceutical composition. In certain embodiments, the effective amount is a therapeutically effective amount. In certain embodiments, the effective amount is a prophylactically effective amount. In certain embodiments, the effective amount is an amount effective for treating a proliferative disease in a subject in need thereof. In certain embodiments, the effective amount is an amount effective for preventing a proliferative disease in a subject in need thereof. In certain embodiments, the effective amount is an amount effective for treating cancer in a subject in need thereof. In certain embodiments, the effective amount is an amount effective for preventing cancer in a subject in need thereof. In certain embodiments, the effective amount is an amount effective for reducing the risk of developing a disease (e.g., proliferative disease or cancer) in a subject in need thereof.

(236) In certain embodiments, the subject is an animal. The animal may be of either sex and may be at any stage of development. In certain embodiments, the subject described herein is a human. In certain embodiments, the subject is a non-human animal. In certain embodiments, the subject is a mammal. In certain embodiments, the subject is a non-human mammal. In certain embodiments, the subject is a domesticated animal, such as a dog, cat, cow, pig, horse, sheep, or goat. In certain embodiments, the subject is a companion animal, such as a dog or cat. In certain embodiments, the subject is a livestock animal, such as a cow, pig, horse, sheep, or goat. In certain embodiments, the subject is a zoo animal. In another embodiment, the subject is a research animal, such as a rodent (e.g., mouse, rat), dog, pig, or non-human primate. In certain embodiments, the anima1 is a genetically engineered animal. In certain embodiments, the anima1 is a transgenic animal (e.g., transgenic mice and transgenic pigs). In certain embodiments, the subject is a fish or reptile.

(237) In certain embodiments, the effective amount is an amount effective for inducing the degradation of at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% of the ubiquitin receptor RPN13 in a cell. In certain embodiments, the effective amount is an amount effective for inducing the degradation of ubiquitin receptor RPN13 in a cell by a range between a percentage described in this paragraph and another percentage described in this paragraph, inclusive.

(238) The present disclosure provides pharmaceutical compositions comprising a compound that interacts with a E3 ubiquitin ligase (e.g., cereblon) and the ubiquitin receptor RPN13 for use in treating a proliferative disease in a subject in need thereof. In certain embodiments, the composition is for use in treating a neurodegenerative disease. In certain embodiments, the composition is for use in treating cancer. In certain embodiments, the composition is for use in treating multiple myeloma, leukemia, lymphoma, or a cancer resistant to a proteasome inhibitor. In certain embodiments, the composition is for use in treating cancer resistant to bortezomib. In certain embodiments, the composition is for use in treating cancer resistant to carfilzomib. In certain embodiments, the composition is for use in treating multiple myeloma.

(239) Pharmaceutical compositions can be prepared, packaged, and/or sold in bulk, as a single unit dose, and/or as a plurality of single unit doses. A “unit dose” is a discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient. The amount of the active ingredient is generally equal to the dosage of the active ingredient which would be administered to a subject and/or a convenient fraction of such a dosage, such as one-half or one-third of such a dosage.

(240) Relative amounts of the active ingredient, the pharmaceutically acceptable excipient, and/or any additional ingredients in a pharmaceutical composition described herein will vary, depending upon the identity, size, and/or condition of the subject treated and further depending upon the route by which the composition is to be administered. The composition may comprise between 0.1% and 100% (w/w) active ingredient.

(241) Pharmaceutically acceptable excipients used in the manufacture of provided pharmaceutical compositions include inert diluents, dispersing and/or granulating agents, surface active agents and/or emulsifiers, disintegrating agents, binding agents, preservatives, buffering agents, lubricating agents, and/or oils. Excipients such as cocoa butter and suppository waxes, coloring agents, coating agents, sweetening, flavoring, and perfuming agents may also be present in the composition.

(242) Exemplary diluents include calcium carbonate, sodium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, calcium hydrogen phosphate, sodium phosphate lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodium chloride, dry starch, cornstarch, powdered sugar, and mixtures thereof.

(243) Exemplary granulating and/or dispersing agents include potato starch, corn starch, tapioca starch, sodium starch glycolate, clays, alginic acid, guar gum, citrus pulp, agar, bentonite, cellulose, and wood products, natural sponge, cation-exchange resins, calcium carbonate, silicates, sodium carbonate, cross-linked poly(vinyl-pyrrolidone) (crospovidone), sodium carboxymethyl starch (sodium starch glycolate), carboxymethyl cellulose, cross-linked sodium carboxymethyl cellulose (croscarmellose), methylcellulose, pregelatinized starch (starch 1500), microcrystalline starch, water insoluble starch, calcium carboxymethyl cellulose, magnesium aluminum silicate (Veegum), sodium lauryl sulfate, quaternary ammonium compounds, and mixtures thereof.

(244) Exemplary surface active agents and/or emulsifiers include natural emulsifiers (e.g., acacia, agar, alginic acid, sodium alginate, tragacanth, chondrux, cholesterol, xanthan, pectin, gelatin, egg yolk, casein, wool fat, cholesterol, wax, and lecithin), colloidal clays (e.g., bentonite (aluminum silicate) and Veegum (magnesium aluminum silicate)), long chain amino acid derivatives, high molecular weight alcohols (e.g., stearyl alcohol, cetyl alcohol, oleyl alcohol, triacetin monostearate, ethylene glycol distearate, glyceryl monostearate, and propylene glycol monostearate, polyvinyl alcohol), carbomers (e.g., carboxy polymethylene, polyacrylic acid, acrylic acid polymer, and carboxyvinyl polymer), carrageenan, cellulosic derivatives (e.g., carboxymethylcellulose sodium, powdered cellulose, hydroxymethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, methylcellulose), sorbitan fatty acid esters (e.g., polyoxyethylene sorbitan monolaurate (Tween® 20), polyoxyethylene sorbitan (Tween® 60), polyoxyethylene sorbitan monooleate (Tween® 80), sorbitan monopalmitate (Span® 40), sorbitan monostearate (Span® 60), sorbitan tristearate (Span® 65), glyceryl monooleate, sorbitan monooleate (Span® 80), polyoxyethylene esters (e.g., polyoxyethylene monostearate (Myrj® 45), polyoxyethylene hydrogenated castor oil, polyethoxylated castor oil, polyoxymethylene stearate, and Solutol®), sucrose fatty acid esters, polyethylene glycol fatty acid esters (e.g., Cremophor®), polyoxyethylene ethers, (e.g., polyoxyethylene lauryl ether (Brij® 30)), poly(vinyl-pyrrolidone), diethylene glycol monolaurate, triethanolamine oleate, sodium oleate, potassium oleate, ethyl oleate, oleic acid, ethyl laurate, sodium lauryl sulfate, Pluronic® F-68, poloxamer P-188, cetrimonium bromide, cetylpyridinium chloride, benzalkonium chloride, docusate sodium, and/or mixtures thereof.

(245) Exemplary binding agents include starch (e.g., cornstarch and starch paste), gelatin, sugars (e.g., sucrose, glucose, dextrose, dextrin, molasses, lactose, lactitol, mannitol, etc.), natural and synthetic gums (e.g., acacia, sodium alginate, extract of Irish moss, panwar gum, ghatti gum, mucilage of isapol husks, carboxymethylcellulose, methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, microcrystalline cellulose, cellulose acetate, poly(vinyl-pyrrolidone), magnesium aluminum silicate (Veegum®), and larch arabogalactan), alginates, polyethylene oxide, polyethylene glycol, inorganic calcium salts, silicic acid, polymethacrylates, waxes, water, alcohol, and/or mixtures thereof.

(246) Exemplary preservatives include antioxidants, chelating agents, antimicrobial preservatives, antifungal preservatives, antiprotozoan preservatives, alcohol preservatives, acidic preservatives, and other preservatives. In certain embodiments, the preservative is an antioxidant. In other embodiments, the preservative is a chelating agent.

(247) Exemplary antioxidants include alpha tocopherol, ascorbic acid, acorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, monothioglycerol, potassium metabisulfite, propionic acid, propyl gallate, sodium ascorbate, sodium bisulfite, sodium metabisulfite, and sodium sulfite.

(248) Exemplary chelating agents include ethylenediaminetetraacetic acid (EDTA) and salts and hydrates thereof (e.g., sodium edetate, disodium edetate, trisodium edetate, calcium disodium edetate, dipotassium edetate, and the like), citric acid and salts and hydrates thereof (e.g., citric acid monohydrate), fumaric acid and salts and hydrates thereof, malic acid and salts and hydrates thereof, phosphoric acid and salts and hydrates thereof, and tartaric acid and salts and hydrates thereof. Exemplary antimicrobial preservatives include benzalkonium chloride, benzethonium chloride, benzyl alcohol, bronopol, cetrimide, cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethyl alcohol, glycerin, hexetidine, imidurea, phenol, phenoxyethanol, phenylethyl alcohol, phenylmercuric nitrate, propylene glycol, and thimerosal.

(249) Exemplary antifungal preservatives include butyl paraben, methyl paraben, ethyl paraben, propyl paraben, benzoic acid, hydroxybenzoic acid, potassium benzoate, potassium sorbate, sodium benzoate, sodium propionate, and sorbic acid.

(250) Exemplary alcohol preservatives include ethanol, polyethylene glycol, phenol, phenolic compounds, bisphenol, chlorobutanol, hydroxybenzoate, and phenylethyl alcohol.

(251) Exemplary acidic preservatives include vitamin A, vitamin C, vitamin E, beta-carotene, citric acid, acetic acid, dehydroacetic acid, ascorbic acid, sorbic acid, and phytic acid.

(252) Other preservatives include tocopherol, tocopherol acetate, deteroxime mesylate, cetrimide, butylated hydroxyanisol (BHA), butylated hydroxytoluened (BHT), ethylenediamine, sodium lauryl sulfate (SLS), sodium lauryl ether sulfate (SLES), sodium bisulfite, sodium metabisulfite, potassium sulfite, potassium metabisulfite, Glydant® Plus, Phenonip®, methylparaben, Germall® 115, Germaben® II, Neolone®, Kathon®, and Euxyl®.

(253) Exemplary buffering agents include citrate buffer solutions, acetate buffer solutions, phosphate buffer solutions, ammonium chloride, calcium carbonate, calcium chloride, calcium citrate, calcium glubionate, calcium gluceptate, calcium gluconate, D-gluconic acid, calcium glycerophosphate, calcium lactate, propanoic acid, calcium levulinate, pentanoic acid, dibasic calcium phosphate, phosphoric acid, tribasic calcium phosphate, calcium hydroxide phosphate, potassium acetate, potassium chloride, potassium gluconate, potassium mixtures, dibasic potassium phosphate, monobasic potassium phosphate, potassium phosphate mixtures, sodium acetate, sodium bicarbonate, sodium chloride, sodium citrate, sodium lactate, dibasic sodium phosphate, monobasic sodium phosphate, sodium phosphate mixtures, tromethamine, magnesium hydroxide, aluminum hydroxide, alginic acid, pyrogen-free water, isotonic saline, Ringer's solution, ethyl alcohol, and mixtures thereof.

(254) Exemplary lubricating agents include magnesium stearate, calcium stearate, stearic acid, silica, talc, malt, glyceryl behanate, hydrogenated vegetable oils, polyethylene glycol, sodium benzoate, sodium acetate, sodium chloride, leucine, magnesium lauryl sulfate, sodium lauryl sulfate, and mixtures thereof.

(255) Exemplary natural oils include almond, apricot kernel, avocado, babassu, bergamot, black current seed, borage, cade, camomile, canola, caraway, carnauba, castor, cinnamon, cocoa butter, coconut, cod liver, coffee, corn, cotton seed, emu, eucalyptus, evening primrose, fish, flaxseed, geraniol, gourd, grape seed, hazel nut, hyssop, isopropyl myristate, jojoba, kukui nut, lavandin, lavender, lemon, litsea cubeba, macademia nut, mallow, mango seed, meadowfoam seed, mink, nutmeg, olive, orange, orange roughy, palm, palm kernel, peach kernel, peanut, poppy seed, pumpkin seed, rapeseed, rice bran, rosemary, safflower, sandalwood, sasquana, savoury, sea buckthorn, sesame, shea butter, silicone, soybean, sunflower, tea tree, thistle, tsubaki, vetiver, walnut, and wheat germ oils. Exemplary synthetic oils include, but are not limited to, butyl stearate, caprylic triglyceride, capric triglyceride, cyclomethicone, diethyl sebacate, dimethicone 360, isopropyl myristate, mineral oil, octyldodecanol, oleyl alcohol, silicone oil, and mixtures thereof.

(256) Liquid dosage forms for oral and parenteral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active ingredients, the liquid dosage forms may comprise inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (e.g., cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. Besides inert diluents, the oral compositions can include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents. In certain embodiments for parenteral administration, the conjugates described herein are mixed with solubilizing agents such as Cremophor®, alcohols, oils, modified oils, glycols, polysorbates, cyclodextrins, polymers, and mixtures thereof.

(257) Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions can be formulated according to the known art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation can be a sterile injectable solution, suspension, or emulsion in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that can be employed are water, Ringer's solution, U.S.P., and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil can be employed including synthetic mono- or di-glycerides. In addition, fatty acids such as oleic acid are used in the preparation of injectables.

(258) The injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.

(259) In order to prolong the effect of a drug, it is often desirable to slow the absorption of the drug from subcutaneous or intramuscular injection. This can be accomplished by the use of a liquid suspension of crystalline or amorphous material with poor water solubility. The rate of absorption of the drug then depends upon its rate of dissolution, which, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered drug form may be accomplished by dissolving or suspending the drug in an oil vehicle.

(260) Compositions for rectal or vaginal administration are typically suppositories which can be prepared by mixing the conjugates described herein with suitable non-irritating excipients or carriers such as cocoa butter, polyethylene glycol, or a suppository wax which are solid at ambient temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the active ingredient.

(261) Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active ingredient is mixed with at least one inert, pharmaceutically acceptable excipient or carrier such as sodium citrate or dicalcium phosphate and/or (a) fillers or extenders such as starches, lactose, sucrose, glucose, mannitol, and silicic acid, (b) binders such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone, sucrose, and acacia, (c) humectants such as glycerol, (d) disintegrating agents such as agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate, (e) solution retarding agents such as paraffin, (f) absorption accelerators such as quaternary ammonium compounds, (g) wetting agents such as, for example, cetyl alcohol and glycerol monostearate, (h) absorbents such as kaolin and bentonite clay, and (i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof. In the case of capsules, tablets, and pills, the dosage form may include a buffering agent.

(262) Solid compositions of a similar type can be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like. The solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings and other coatings well known in the art of pharmacology. They may optionally comprise opacifying agents and can be of a composition that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner. Examples of encapsulating compositions which can be used include polymeric substances and waxes. Solid compositions of a similar type can be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polethylene glycols and the like.

(263) The active ingredient can be in a micro-encapsulated form with one or more excipients as noted above. The solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings, release controlling coatings, and other coatings well known in the pharmaceutical formulating art. In such solid dosage forms the active ingredient can be admixed with at least one inert diluent such as sucrose, lactose, or starch. Such dosage forms may comprise, as is normal practice, additional substances other than inert diluents, e.g., tableting lubricants and other tableting aids such a magnesium stearate and microcrystalline cellulose. In the case of capsules, tablets and pills, the dosage forms may comprise buffering agents. They may optionally comprise opacifying agents and can be of a composition that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner. Examples of encapsulating agents which can be used include polymeric substances and waxes.

(264) Dosage forms for topical and/or transdermal administration of a compound described herein may include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants, and/or patches. Generally, the active ingredient is admixed under sterile conditions with a pharmaceutically acceptable carrier or excipient and/or any needed preservatives and/or buffers as can be required. Additionally, the present disclosure contemplates the use of transdermal patches, which often have the added advantage of providing controlled delivery of an active ingredient to the body. Such dosage forms can be prepared, for example, by dissolving and/or dispensing the active ingredient in the proper medium. Alternatively or additionally, the rate can be controlled by either providing a rate controlling membrane and/or by dispersing the active ingredient in a polymer matrix and/or gel.

(265) Suitable devices for use in delivering intradermal pharmaceutical compositions described herein include short needle devices. Intradermal compositions can be administered by devices which limit the effective penetration length of a needle into the skin. Alternatively or additionally, conventional syringes can be used in the classical mantoux method of intradermal administration. Jet injection devices which deliver liquid formulations to the dermis via a liquid jet injector and/or via a needle which pierces the stratum corneum and produces a jet which reaches the dermis are suitable. Ballistic powder/particle delivery devices which use compressed gas to accelerate the compound in powder form through the outer layers of the skin to the dermis are suitable.

(266) Formulations suitable for topical administration include, but are not limited to, liquid and/or semi-liquid preparations such as liniments, lotions, oil-in-water and/or water-in-oil emulsions such as creams, ointments, and/or pastes, and/or solutions and/or suspensions. Topically administrable formulations may, for example, comprise from about 1% to about 10% (w/w) active ingredient, although the concentration of the active ingredient can be as high as the solubility limit of the active ingredient in the solvent. Formulations for topical administration may further comprise one or more of the additional ingredients described herein.

(267) A pharmaceutical composition described herein can be prepared, packaged, and/or sold in a formulation suitable for pulmonary administration via the buccal cavity. Such a formulation may comprise dry particles which comprise the active ingredient and which have a diameter in the range from about 0.5 to about 7 nanometers, or from about 1 to about 6 nanometers. Such compositions are conveniently in the form of dry powders for administration using a device comprising a dry powder reservoir to which a stream of propellant can be directed to disperse the powder and/or using a self-propelling solvent/powder dispensing container such as a device comprising the active ingredient dissolved and/or suspended in a low-boiling propellant in a sealed container. Such powders comprise particles wherein at least 98% of the particles by weight have a diameter greater than 0.5 nanometers and at least 95% of the particles by number have a diameter less than 7 nanometers. Alternatively, at least 95% of the particles by weight have a diameter greater than 1 nanometer and at least 90% of the particles by number have a diameter less than 6 nanometers. Dry powder compositions may include a solid fine powder diluent such as sugar and are conveniently provided in a unit dose form.

(268) Low boiling propellants generally include liquid propellants having a boiling point of below 65° F. at atmospheric pressure. Generally the propellant may constitute 50 to 99.9% (w/w) of the composition, and the active ingredient may constitute 0.1 to 20% (w/w) of the composition. The propellant may further comprise additional ingredients such as a liquid non-ionic and/or solid anionic surfactant and/or a solid diluent (which may have a particle size of the same order as particles comprising the active ingredient).

(269) Pharmaceutical compositions described herein formulated for pulmonary delivery may provide the active ingredient in the form of droplets of a solution and/or suspension. Such formulations can be prepared, packaged, and/or sold as aqueous and/or dilute alcoholic solutions and/or suspensions, optionally sterile, comprising the active ingredient, and may conveniently be administered using any nebulization and/or atomization device. Such formulations may further comprise one or more additional ingredients including, but not limited to, a flavoring agent such as saccharin sodium, a volatile oil, a buffering agent, a surface active agent, and/or a preservative such as methylhydroxybenzoate. The droplets provided by this route of administration may have an average diameter in the range from about 0.1 to about 200 nanometers.

(270) Formulations described herein as being useful for pulmonary delivery are useful for intranasal delivery of a pharmaceutical composition described herein. Another formulation suitable for intranasal administration is a coarse powder comprising the active ingredient and having an average particle from about 0.2 to 500 micrometers. Such a formulation is administered by rapid inhalation through the nasal passage from a container of the powder held close to the nares.

(271) Formulations for nasal administration may, for example, comprise from about as little as 0.1% (w/w) to as much as 100% (w/w) of the active ingredient, and may comprise one or more of the additional ingredients described herein. A pharmaceutical composition described herein can be prepared, packaged, and/or sold in a formulation for buccal administration. Such formulations may, for example, be in the form of tablets and/or lozenges made using conventional methods, and may contain, for example, 0.1 to 20% (w/w) active ingredient, the balance comprising an orally dissolvable and/or degradable composition and, optionally, one or more of the additional ingredients described herein. Alternately, formulations for buccal administration may comprise a powder and/or an aerosolized and/or atomized solution and/or suspension comprising the active ingredient. Such powdered, aerosolized, and/or aerosolized formulations, when dispersed, may have an average particle and/or droplet size in the range from about 0.1 to about 200 nanometers, and may further comprise one or more of the additional ingredients described herein.

(272) Although the descriptions of pharmaceutical compositions provided herein are principally directed to pharmaceutical compositions which are suitable for administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to animals of all sorts. Modification of pharmaceutical compositions suitable for administration to humans in order to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist can design and/or perform such modification with ordinary experimentation.

(273) Compounds provided herein are typically formulated in dosage unit form for ease of administration and uniformity of dosage. It will be understood, however, that the total daily usage of the compositions described herein will be decided by a physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular subject or organism will depend upon a variety of factors including the disease being treated and the severity of the disorder; the activity of the specific active ingredient employed; the specific composition employed; the age, body weight, general health, sex, and diet of the subject; the time of administration, route of administration, and rate of excretion of the specific active ingredient employed; the duration of the treatment; drugs used in combination or coincidental with the specific active ingredient employed; and like factors well known in the medical arts.

(274) The compounds and compositions provided herein can be administered by any route, including enteral (e.g., oral), parenteral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, subcutaneous, intraventricular, transdermal, interdermal, rectal, intravaginal, intraperitoneal, topical (as by powders, ointments, creams, and/or drops), mucosal, nasal, bucal, sublingual; by intratracheal instillation, bronchial instillation, and/or inhalation; and/or as an oral spray, nasal spray, and/or aerosol. Specifically contemplated routes are oral administration, intravenous administration (e.g., systemic intravenous injection), regional administration via blood and/or lymph supply, and/or direct administration to an affected site. In general, the most appropriate route of administration will depend upon a variety of factors including the nature of the agent (e.g., its stability in the environment of the gastrointestinal tract), and/or the condition of the subject (e.g., whether the subject is able to tolerate oral administration). In certain embodiments, the compound or pharmaceutical composition described herein is suitable for topical administration to the eye of a subject.

(275) The exact amount of a compound required to achieve an effective amount will vary from subject to subject, depending, for example, on species, age, and general condition of a subject, severity of the side effects or disorder, identity of the particular compound, mode of administration, and the like. An effective amount may be included in a single dose (e.g., single oral dose) or multiple doses (e.g., multiple oral doses). In certain embodiments, when multiple doses are administered to a subject or applied to a biological sample, tissue, or cell, any two doses of the multiple doses include different or substantially the same amounts of a compound described herein. In certain embodiments, when multiple doses are administered to a subject or applied to a biological sample, tissue, or cell, the frequency of administering the multiple doses to the subject or applying the multiple doses to the biological sample, tissue, or cell is three doses a day, two doses a day, one dose a day, one dose every other day, one dose every third day, one dose every week, one dose every two weeks, one dose every three weeks, or one dose every four weeks. In certain embodiments, the frequency of administering the multiple doses to the subject or applying the multiple doses to the biological sample, tissue, or cell is one dose per day. In certain embodiments, the frequency of administering the multiple doses to the subject or applying the multiple doses to the biological sample, tissue, or cell is two doses per day. In certain embodiments, the frequency of administering the multiple doses to the subject or applying the multiple doses to the biological sample, tissue, or cell is three doses per day. In certain embodiments, when multiple doses are administered to a subject or applied to a biological sample, tissue, or cell, the duration between the first dose and last dose of the multiple doses is one day, two days, four days, one week, two weeks, three weeks, one month, two months, three months, four months, six months, nine months, one year, two years, three years, four years, five years, seven years, ten years, fifteen years, twenty years, or the lifetime of the subject, tissue, or cell. In certain embodiments, the duration between the first dose and last dose of the multiple doses is three months, six months, or one year. In certain embodiments, the duration between the first dose and last dose of the multiple doses is the lifetime of the subject, tissue, or cell. In certain embodiments, a dose (e.g., a single dose, or any dose of multiple doses) described herein includes independently between 0.1 μg and 1 μg, between 0.001 mg and 0.01 mg, between 0.01 mg and 0.1 mg, between 0.1 mg and 1 mg, between 1 mg and 3 mg, between 3 mg and 10 mg, between 10 mg and 30 mg, between 30 mg and 100 mg, between 100 mg and 300 mg, between 300 mg and 1,000 mg, or between 1 g and 10 g, inclusive, of a compound described herein. In certain embodiments, a dose described herein includes independently between 1 mg and 3 mg, inclusive, of a compound described herein. In certain embodiments, a dose described herein includes independently between 3 mg and 10 mg, inclusive, of a compound described herein. In certain embodiments, a dose described herein includes independently between 10 mg and 30 mg, inclusive, of a compound described herein. In certain embodiments, a dose described herein includes independently between 30 mg and 100 mg, inclusive, of a compound described herein.

(276) Dose ranges as described herein provide guidance for the administration of provided pharmaceutical compositions to an adult. The amount to be administered to, for example, a child or an adolescent can be determined by a medical practitioner or person skilled in the art and can be lower or the same as that administered to an adult.

(277) A compound or composition, as described herein, can be administered in combination with one or more additional pharmaceutical agents (e.g., therapeutically and/or prophylactically active agents). The compounds or compositions can be administered in combination with additional pharmaceutical agents that improve their activity (e.g., activity (e.g., potency and/or efficacy) in treating a disease in a subject in need thereof, in preventing a disease in a subject in need thereof, in inducing the degradation of a target protein, and/or in reducing the risk to develop a disease in a subject in need thereof), improve bioavailability, improve their ability to cross the blood-brain barrier, improve safety, reduce drug resistance, reduce and/or modify metabolism, inhibit excretion, and/or modify distribution in a subject or cell. It will also be appreciated that the therapy employed may achieve a desired effect for the same disorder, and/or it may achieve different effects. In certain embodiments, a pharmaceutical composition described herein including a compound described herein and an additional pharmaceutical agent exhibit a synergistic effect that is absent in a pharmaceutical composition including one of the compound and the additional pharmaceutical agent, but not both.

(278) The compound or composition can be administered concurrently with, prior to, or subsequent to one or more additional pharmaceutical agents, which may be useful as, e.g., combination therapies. Pharmaceutical agents include therapeutically active agents. Pharmaceutical agents also include prophylactically active agents. Pharmaceutical agents include small organic molecules such as drug compounds (e.g., compounds approved for human or veterinary use by the U.S. Food and Drug Administration as provided in the Code of Federal Regulations (CFR)), peptides, proteins, carbohydrates, monosaccharides, oligosaccharides, polysaccharides, nucleoproteins, mucoproteins, lipoproteins, synthetic polypeptides or proteins, small molecules linked to proteins, glycoproteins, steroids, nucleic acids, DNAs, RNAs, nucleotides, nucleosides, oligonucleotides, antisense oligonucleotides, lipids, hormones, vitamins, and cells. In certain embodiments, the additional pharmaceutical agent is a pharmaceutical agent useful for treating and/or preventing a disease (e.g., proliferative disease, inflammatory disease, autoimmune disease, genetic disease, hematological disease, neurological disease, painful condition, psychiatric disorder, or metabolic disorder). Each additional pharmaceutical agent may be administered at a dose and/or on a time schedule determined for that pharmaceutical agent. The additional pharmaceutical agents may also be administered together with each other and/or with the compound or composition described herein in a single dose or administered separately in different doses. The particular combination to employ in a regimen will take into account compatibility of the compound described herein with the additional pharmaceutical agent(s) and/or the desired therapeutic and/or prophylactic effect to be achieved. In general, it is expected that the additional pharmaceutical agent(s) in combination be utilized at levels that do not exceed the levels at which they are utilized individually. In some embodiments, the levels utilized in combination will be lower than those utilized individually.

(279) The additional pharmaceutical agents include, but are not limited to, anti-proliferative agents, anti-cancer agents, anti-angiogenesis agents, anti-inflammatory agents, immunosuppressants, anti-bacterial agents, anti-viral agents, cardiovascular agents, cholesterol-lowering agents, anti-diabetic agents, anti-allergic agents, contraceptive agents, pain-relieving agents, and a combination thereof. In certain embodiments, the additional pharmaceutical agent is an anti-proliferative agent (e.g., anti-cancer agent). In certain embodiments, the additional pharmaceutical agent is an anti-leukemia agent. In certain embodiments, the additional pharmaceutical agent is ABITREXATE (methotrexate), ADE, Adriamycin RDF (doxorubicin hydrochloride), Ambochlorin (chlorambucil), ARRANON (nelarabine), ARZERRA (ofatumumab), BOSULIF (bosutinib), BUSULFEX (busulfan), CAMPATH (alemtuzumab), CERUBIDINE (daunorubicin hydrochloride), CLAFEN (cyclophosphamide), CLOFAREX (clofarabine), CLOLAR (clofarabine), CVP, CYTOSAR-U (cytarabine), CYTOXAN (cyclophosphamide), ERWINAZE (Asparaginase Erwinia Chrysanthemi), FLUDARA (fludarabine phosphate), FOLEX (methotrexate), FOLEX PFS (methotrexate), GAZYVA (obinutuzumab), GLEEVEC (imatinib mesylate), Hyper-CVAD, ICLUSIG (ponatinib hydrochloride), IMBRUVICA (ibrutinib), LEUKERAN (chlorambucil), LINFOLIZIN (chlorambucil), MARQIBO (vincristine sulfate liposome), METHOTREXATE LPF (methorexate), MEXATE (methotrexate), MEXATE-AQ (methotrexate), mitoxantrone hydrochloride, MUSTARGEN (mechlorethamine hydrochloride), MYLERAN (busulfan), NEOSAR (cyclophosphamide), ONCASPAR (Pegaspargase), PURINETHOL (mercaptopurine), PURIXAN (mercaptopurine), Rubidomycin (daunorubicin hydrochloride), SPRYCEL (dasatinib), SYNRIBO (omacetaxine mepesuccinate), TARABINE PFS (cytarabine), TASIGNA (nilotinib), TREANDA (bendamustine hydrochloride), TRISENOX (arsenic trioxide), VINCASAR PFS (vincristine sulfate), ZYDELIG (idelalisib), or a combination thereof. In certain embodiments, the additional pharmaceutical agent is an anti-lymphoma agent. In certain embodiments, the additional pharmaceutical agent is ABITREXATE (methotrexate), ABVD, ABVE, ABVE-PC, ADCETRIS (brentuximab vedotin), ADRIAMYCIN PFS (doxorubicin hydrochloride), ADRIAMYCIN RDF (doxorubicin hydrochloride), AMBOCHLORIN (chlorambucil), AMBOCLORIN (chlorambucil), ARRANON (nelarabine), BEACOPP, BECENUM (carmustine), BELEODAQ (belinostat), BEXXAR (tositumomab and iodine I 131 tositumomab), BICNU (carmustine), BLENOXANE (bleomycin), CARMUBRIS (carmustine), CHOP, CLAFEN (cyclophosphamide), COPP, COPP-ABV, CVP, CYTOXAN (cyclophosphamide), DEPOCYT (liposomal cytarabine), DTIC-DOME (dacarbazine), EPOCH, FOLEX (methotrexate), FOLEX PFS (methotrexate), FOLOTYN (pralatrexate), HYPER-CVAD, ICE, IMBRUVICA (ibrutinib), INTRON A (recombinant interferon alfa-2b), ISTODAX (romidepsin), LEUKERAN (chlorambucil), LINFOLIZIN (chlorambucil), Lomustine, MATULANE (procarbazine hydrochloride), METHOTREXATE LPF (methotrexate), MEXATE (methotrexate), MEXATE-AQ (methotrexate), MOPP, MOZOBIL (plerixafor), MUSTARGEN (mechlorethamine hydrochloride), NEOSAR (cyclophosphamide), OEPA, ONTAK (denileukin diftitox), OPPA, R-CHOP, REVLIMID (lenalidomide), RITUXAN (rituximab), STANFORD V, TREANDA (bendamustine hydrochloride), VAMP, VELBAN (vinblastine sulfate), VELCADE (bortezomib), VELSAR (vinblastine sulfate), VINCASAR PFS (vincristine sulfate), ZEVALIN (ibritumomab tiuxetan), ZOLINZA (vorinostat), ZYDELIG (idelalisib), or a combination thereof. In certain embodiments, the additional pharmaceutical agent is REVLIMID (lenalidomide), DACOGEN (decitabine), VIDAZA (azacitidine), CYTOSAR-U (cytarabine), IDAMYCIN (idarubicin), CERUBIDINE (daunorubicin), LEUKERAN (chlorambucil), NEOSAR (cyclophosphamide), FLUDARA (fludarabine), LEUSTATIN (cladribine), or a combination thereof. In certain embodiments, the additional pharmaceutical agent is ABITREXATE (methotrexate), ABRAXANE (paclitaxel albumin-stabilized nanoparticle formulation), AC, AC-T, ADE, ADRIAMYCIN PFS (doxorubicin hydrochloride), ADRUCIL (fluorouracil), AFINITOR (everolimus), AFINITOR DISPERZ (everolimus), ALDARA (imiquimod), ALIMTA (pemetrexed disodium), AREDIA (pamidronate disodium), ARIMIDEX (anastrozole), AROMASIN (exemestane), AVASTIN (bevacizumab), BECENUM (carmustine), BEP, BICNU (carmustine), BLENOXANE (bleomycin), CAF, CAMPTOSAR (irinotecan hydrochloride), CAPOX, CAPRELSA (vandetanib), CARBOPLATIN-TAXOL, CARMUBRIS (carmustine), CASODEX (bicalutamide), CEENU (lomustine), CERUBIDINE (daunorubicin hydrochloride), CERVARIX (recombinant HPV bivalent vaccine), CLAFEN (cyclophosphamide), CMF, COMETRIQ (cabozantinib-s-malate), COSMEGEN (dactinomycin), CYFOS (ifosfamide), CYRAMZA (ramucirumab), CYTOSAR-U (cytarabine), CYTOXAN (cyclophosphamide), DACOGEN (decitabine), DEGARELIX, DOXIL (doxorubicin hydrochloride liposome), DOXORUBICIN HYDROCHLORIDE, DOX-SL (doxorubicin hydrochloride liposome), DTIC-DOME (dacarbazine), EFUDEX (fluorouracil), ELLENCE (epirubicin hydrochloride), ELOXATIN (oxaliplatin), ERBITUX (cetuximab), ERIVEDGE (vismodegib), ETOPOPHOS (etoposide phosphate), EVACET (doxorubicin hydrochloride liposome), FARESTON (toremifene), FASLODEX (fulvestrant), FEC, FEMARA (letrozole), FLUOROPLEX (fluorouracil), FOLEX (methotrexate), FOLEX PFS (methotrexate), FOLFIRI, FOLFIRI-BEVACIZUMAB, FOLFIRI-CETUXIMAB, FOLFIRINOX, FOLFOX, FU-LV, GARDASIL (recombinant human papillomavirus (HPV) quadrivalent vaccine), GEMCITABINE-CISPLATIN, GEMCITABINE-OXALIPLATIN, GEMZAR (gemcitabine hydrochloride), GILOTRIF (afatinib dimaleate), GLEEVEC (imatinib mesylate), GLIADEL (carmustine implant), GLIADEL WAFER (carmustine implant), HERCEPTIN (trastuzumab), HYCAMTIN (topotecan hydrochloride), IFEX (ifosfamide), IFOSFAMIDUM (ifosfamide), INLYTA (axitinib), INTRON A (recombinant interferon alfa-2b), IRESSA (gefitinib), IXEMPRA (ixabepilone), JAKAFI (ruxolitinib phosphate), JEVTANA (cabazitaxel), KADCYLA (ado-trastuzumab emtansine), KEYTRUDA (pembrolizumab), KYPROLIS (carfilzomib), LIPODOX (doxorubicin hydrochloride liposome), LUPRON (leuprolide acetate), LUPRON DEPOT (leuprolide acetate), LUPRON DEPOT-3 MONTH (leuprolide acetate), LUPRON DEPOT-4 MONTH (leuprolide acetate), LUPRON DEPOT-PED (leuprolide acetate), MEG ACE (megestrol acetate), MEKINIST (trametinib), METHAZOLASTONE (temozolomide), METHOTREXATE LPF (methotrexate), MEXATE (methotrexate), MEXATE-AQ (methotrexate), MITOXANTRONE HYDROCHLORIDE, MITOZYTREX (mitomycin c), MOZOBIL (plerixafor), MUSTARGEN (mechlorethamine hydrochloride), MUTAMYCIN (mitomycin c), MYLOSAR (azacitidine), NAVELBINE (vinorelbine tartrate), NEOSAR (cyclophosphamide), NEXAVAR (sorafenib tosylate), NOLVADEX (tamoxifen citrate), NOVALDEX (tamoxifen citrate), OFF, PAD, PARAPLAT (carboplatin), PARAPLATIN (carboplatin), PEG-INTRON (peginterferon alfa-2b), PEMETREXED DISODIUM, PERJETA (pertuzumab), PLATINOL (cisplatin), PLATINOL-AQ (cisplatin), POMALYST (pomalidomide), prednisone, PROLEUKIN (aldesleukin), PROLIA (denosumab), PROVENGE (sipuleucel-t), REVLIMID (lenalidomide), RUBIDOMYCIN (daunorubicin hydrochloride), SPRYCEL (dasatinib), STIVARGA (regorafenib), SUTENT (sunitinib malate), SYLATRON (peginterferon alfa-2b), SYLVANT (siltuximab), SYNOVIR (thalidomide), TAC, TAFINLAR (dabrafenib), TARABINE PFS (cytarabine), TARCEVA (erlotinib hydrochloride), TASIGNA (nilotinib), TAXOL (paclitaxel), TAXOTERE (docetaxel), TEMODAR (temozolomide), THALOMID (thalidomide), TOPOSAR (etoposide), TORISEL (temsirolimus), TPF, TRISENOX (arsenic trioxide), TYKERB (lapatinib ditosylate), VECTIBIX (panitumumab), VEIP, VELBAN (vinblastine sulfate), VELCADE (bortezomib), VELSAR (vinblastine sulfate), VEPESID (etoposide), VIADUR (leuprolide acetate), VIDAZA (azacitidine), VINCASAR PFS (vincristine sulfate), VOTRIENT (pazopanib hydrochloride), WELLCOVORIN (leucovorin calcium), XALKORI (crizotinib), XELODA (capecitabine), XELOX, XGEVA (denosumab), XOFIGO (radium 223 dichloride), XTANDI (enzalutamide), YERVOY (ipilimumab), ZALTRAP (ziv-aflibercept), ZELBORAF (vemurafenib), ZOLADEX (goserelin acetate), ZOMETA (zoledronic acid), ZYKADIA (ceritinib), ZYTIGA (abiraterone acetate), ENMD-2076, PCI-32765, AC220, dovitinib lactate (TKI258, CHIR-258), BIBW 2992 (TOVOK™), SGX523, PF-04217903, PF-02341066, PF-299804, BMS-777607, ABT-869, MP470, BIBF 1120 (VARGATEF®), AP24534, JNJ-26483327, MGCD265, DCC-2036, BMS-690154, CEP-11981, tivozanib (AV-951), OSI-930, MM-121, XL-184, XL-647, and/or XL228), proteasome inhibitors (e.g., bortezomib (Velcade)), mTOR inhibitors (e.g., rapamycin, temsirolimus (CCI-779), everolimus (RAD-001), ridaforolimus, AP23573 (Ariad), AZD8055 (AstraZeneca), BEZ235 (Novartis), BGT226 (Norvartis), XL765 (Sanofi Aventis), PF-4691502 (Pfizer), GDC0980 (Genetech), SF1126 (Semafoe) and OSI-027 (OSI)), oblimersen, gemcitabine, carminomycin, leucovorin, pemetrexed, cyclophosphamide, dacarbazine, procarbizine, prednisolone, dexamethasone, campathecin, plicamycin, asparaginase, aminopterin, methopterin, porfiromycin, melphalan, leurosidine, leurosine, chlorambucil, trabectedin, procarbazine, discodermolide, carminomycin, aminopterin, and hexamethyl melamine, or a combination thereof. In certain embodiments, the additional pharmaceutical agent is a protein kinase inhibitor (e.g., tyrosine protein kinase inhibitor). In certain embodiments, the additional pharmaceutical agent is selected from the group consisting of epigenetic or transcriptional modulators (e.g., DNA methyltransferase inhibitors, histone deacetylase inhibitors (HDAC inhibitors), lysine methyltransferase inhibitors), antimitotic drugs (e.g., taxanes and vinca alkaloids), hormone receptor modulators (e.g., estrogen receptor modulators and androgen receptor modulators), cell signaling pathway inhibitors (e.g., tyrosine protein kinase inhibitors), modulators of protein stability (e.g., proteasome inhibitors), Hsp90 inhibitors, glucocorticoids, all-/trans retinoic acids, and other agents that promote differentiation. In certain embodiments, the compounds described herein or pharmaceutical compositions can be administered in combination with an anti-cancer therapy including, but not limited to, surgery, radiation therapy, transplantation (e.g., stem cell transplantation, bone marrow transplantation), immunotherapy, and chemotherapy.

(280) Also encompassed by the disclosure are kits (e.g., pharmaceutical packs). The kits provided may comprise a pharmaceutical composition or compound described herein and a container (e.g., a vial, ampule, bottle, syringe, and/or dispenser package, or other suitable container). In some embodiments, provided kits may optionally further include a second container comprising a pharmaceutical excipient for dilution or suspension of a pharmaceutical composition or compound described herein. In some embodiments, the pharmaceutical composition or compound described herein provided in the first container and the second container are combined to form one unit dosage form.

(281) Thus, in one aspect, provided are kits including a first container comprising a compound or pharmaceutical composition described herein. In certain embodiments, the kits are useful for treating a disease (e.g., proliferative disease, inflammatory disease, autoimmune disease, genetic disease, hematological disease, neurological disease, painful condition, psychiatric disorder, or metabolic disorder) in a subject in need thereof. In certain embodiments, the kits are useful for preventing a disease (e.g., proliferative disease, inflammatory disease, autoimmune disease, genetic disease, hematological disease, neurological disease, painful condition, psychiatric disorder, or metabolic disorder) in a subject in need thereof. In certain embodiments, the kits are useful for inducing apoptosis in a cell.

(282) In certain embodiments, a kit described herein further includes instructions for using the compound or pharmaceutical composition included in the kit. A kit described herein may also include information as required by a regulatory agency such as the U.S. Food and Drug Administration (FDA). In certain embodiments, the information included in the kits is prescribing information. In certain embodiments, the kits and instructions provide for treating a disease (e.g., proliferative disease, inflammatory disease, autoimmune disease, genetic disease, hematological disease, neurological disease, painful condition, psychiatric disorder, or metabolic disorder) in a subject in need thereof. In certain embodiments, the kits and instructions provide for preventing a disease (e.g., proliferative disease, inflammatory disease, autoimmune disease, genetic disease, hematological disease, neurological disease, painful condition, psychiatric disorder, or metabolic disorder) in a subject in need thereof. In certain embodiments, the kits and instructions provide for inducing the degradation of RPN13 in a subject, biological sample, tissue, or cell. In certain embodiments, the kits and instructions provide for inducing apoptosis in a cell. A kit described herein may include one or more additional pharmaceutical agents described herein as a separate composition.

(283) Methods of Treatment and Uses

(284) The compounds described herein are capable of binding (e.g., reversibly binding or irreversibly binding) an E3 ubiquitin ligase (e.g., Cereblon) and RPN13 and inducing the degradation of RPN13. The present disclosure thus also provides methods of inducing the degradation of RPN13 in a subject, biological sample, tissue, or cell. The present disclosure further provides methods for the treatment of a wide range of diseases, such as proliferative diseases, inflammatory diseases, autoimmune diseases, genetic diseases, hematological diseases, neurological diseases, painful conditions, psychiatric disorders, and metabolic disorders in a subject in need thereof.

(285) In certain embodiments, the application provides a method of binding an ubiquitin receptor RPN13 and promoting the degradation of RPN13. In another aspect, the present disclosure provides methods of inducing the degradation of RPN13 in a subject in need thereof, the methods comprise administering to the subject an effective amount of a compound or pharmaceutical composition described herein. In another aspect, the present disclosure provides methods of inducing the degradation of RPN13 in a biological sample, tissue, or cell, the methods comprise contacting the biological sample, tissue, or cell with an effective amount of a compound or pharmaceutical composition described herein.

(286) In certain embodiments, the application provides a method of binding an E3 ubiquitin ligase (e.g., Cereblon) and RPN13 and inducing the degradation of ubiquitin receptor RPN13. In certain embodiments, the ubiquitin receptor is RPN13. In certain embodiments, the ubiquitin receptor is RPN13. In certain embodiments, the binder of the ubiquitin receptor RPN13 is RA190 (below).

(287) ##STR00078##

(288) Use of a bifunctional compound that binds an E3 ubiquitin ligase (e.g., Cereblon) and a ubiquitin receptor RPN13 provides a strategy for treating diseases associated with RPN13 (e.g. proliferative diseases).

(289) The present disclosure also provides a compound of Formulae (I) or (I′), or a pharmaceutically acceptable salt, co-crystal, tautomer, stereoisomer, solvate, hydrate, polymorph, isotopically enriched derivative, or prodrug, or composition thereof, for use in the treatment of diseases, such as proliferative diseases, inflammatory diseases, autoimmune diseases, genetic diseases, hematological diseases, neurological diseases, painful conditions, psychiatric disorders, and metabolic disorders in a subject in need thereof.

(290) The present disclosure also provides uses of a compound of Formulae (I) or (I′), or a pharmaceutically acceptable salt, co-crystal, tautomer, stereoisomer, solvate, hydrate, polymorph, isotopically enriched derivative, or prodrug, or composition thereof, in the manufacture of a medicament for the treatment of diseases, such as proliferative diseases, inflammatory diseases, autoimmune diseases, genetic diseases, hematological diseases, neurological diseases, painful conditions, psychiatric disorders, and metabolic disorders in a subject in need thereof.

(291) In certain embodiments, the methods of the disclosure comprise administering to the subject an effective amount of a compound of Formulae (I) or (I′), or a pharmaceutically acceptable salt, co-crystal, tautomer, stereoisomer, solvate, hydrate, polymorph, isotopically enriched derivative, or prodrug, or composition thereof. In some embodiments, the effective amount is a therapeutically effective amount. In some embodiments, the effective amount is a prophylactically effective amount.

(292) In certain embodiments, the subject being treated is an animal. The animal may be of either sex and may be at any stage of development. In certain embodiments, the subject is a mammal. In certain embodiments, the subject being treated is a human. In certain embodiments, the subject is a domesticated animal, such as a dog, cat, cow, pig, horse, sheep, or goat. In certain embodiments, the subject is a companion animal, such as a dog or cat. In certain embodiments, the subject is a livestock animal, such as a cow, pig, horse, sheep, or goat. In certain embodiments, the subject is a zoo animal. In another embodiment, the subject is a research animal such as a rodent (e.g., mouse, rat), dog, pig, or non-human primate. In certain embodiments, the anima1 is a genetically engineered animal. In certain embodiments, the anima1 is a transgenic animal.

(293) Certain methods described herein may comprise administering one or more additional pharmaceutical agent(s) in combination with the compounds described herein. The additional pharmaceutical agent(s) may be administered at the same time as the compound of Formulae (I) or (I′), or at different times than the compound of Formulae (I) or (I′). For example, the compound of Formulae (I) or (I′) and any additional pharmaceutical agent(s) may be on the same dosing schedule or different dosing schedules. All or some doses of the compound of Formulae (I) or (I′) may be administered before all or some doses of an additional pharmaceutical agent, after all or some does an additional pharmaceutical agent, within a dosing schedule of an additional pharmaceutical agent, or a combination thereof. The timing of administration of the compound of Formulae (I) or (I′) and additional pharmaceutical agents may be different for different additional pharmaceutical agents.

(294) In certain embodiments, the additional pharmaceutical agent comprises an agent useful in the treatment of diseases, such as proliferative diseases, inflammatory diseases, autoimmune diseases, genetic diseases, hematological diseases, neurological diseases, painful conditions, psychiatric disorders, and metabolic disorders in a subject in need thereof. In certain embodiments, the additional pharmaceutical agent is useful in the treatment of a proliferative disease. In certain embodiments, the additional pharmaceutical agent is useful in the treatment of an inflammatory disease. In certain embodiments, the additional pharmaceutical agent is useful in the treatment of proliferative diseases, inflammatory diseases, autoimmune diseases, genetic diseases, hematological diseases, neurological diseases, painful conditions, psychiatric disorders, or metabolic disorders. In certain embodiments, the additional pharmaceutical agent is useful in the treatment of multiple myeloma. In certain embodiments, the additional pharmaceutical agent is useful in the treatment of leukemia. In certain embodiments, the additional pharmaceutical agent is useful in the treatment of lymphoma. In certain embodiments, the additional pharmaceutical agent is useful in the treatment of a non-Hodgkin's lymphoma. In certain embodiments, the additional pharmaceutical agent is useful in the treatment of cancer resistant to proteasome inhibitors (e.g., resistant to bortezomib or resistant to carfilzomib). In certain embodiments, the additional pharmaceutical agent is useful in the treatment of cancer resistant to bortezomib. In certain embodiments, the additional pharmaceutical agent is useful in the treatment of cancer resistant to carfilzomib.

(295) In another aspect, the present disclosure provides methods for inducing the degradation of RPN13, the method comprising administering to the subject a compound of Formulae (I) or (I′), or a pharmaceutically acceptable salt, co-crystal, tautomer, stereoisomer, solvate, hydrate, polymorph, isotopically enriched derivative, or prodrug, or composition thereof.

(296) In another aspect, the present disclosure provides methods for binding an E3 ubiquitin ligase and promoting the degradation of a ubiquitin receptor RPN13, the method comprising administering to the subject a compound of Formulae (I) or (I′), or a pharmaceutically acceptable salt, co-crystal, tautomer, stereoisomer, solvate, hydrate, polymorph, isotopically enriched derivative, or prodrug, or composition thereof. In one aspect, the present disclosure provides methods for inducing the ubiquitination of RPN13. In one aspect, the present disclosure provides methods for inhibiting and/or blocking proteasome function in a subject. In one aspect, the present disclosure provides methods for inhibiting and/or blocking proteasome function in a cell.

(297) In still another aspect, the present disclosure provides the pharmaceutical compositions described herein for use in binding an E3 ubiquitin ligase and RPN13 and promoting the degradation of a ubiquitin receptor RPN13; inducing the ubiquitination of RPN13 in a subject, biological sample, tissue, or cell; treating and/or preventing proliferative diseases, inflammatory diseases, autoimmune diseases, genetic diseases, hematological diseases, neurological diseases, painful conditions, psychiatric disorders, and metabolic disorders; and inhibiting and/or blocking proteasome function in a cell. The present disclosure provides methods of inducing apoptosis of a cell in a subject, the method comprising administering to the subject a therapeutically effective amount of a compound described herein.

EXAMPLES

(298) In order that the present disclosure may be more fully understood, the following examples are set forth. The synthetic and biological examples described in this application are offered to illustrate the compounds, pharmaceutical compositions, and methods provided herein and are not to be construed in any way as limiting their scope.

(299) The compounds provided herein can be prepared from readily available starting materials using the following general methods and procedures or methods known in the art. It will be appreciated that where typical or preferred process conditions (i.e., reaction temperatures, times, mole ratios of reactants, solvents, pressures, etc.) are given, other process conditions can also be used unless otherwise stated. Optimum reaction conditions may vary with the particular reactants or solvents used, but such conditions can be determined by those skilled in the art by routine optimization procedures.

(300) Compounds of Formulae (I) or (I′) may be prepared using the synthetic schemes and procedures described in detail below.

Example 1. Experimental Procedures for Synthesis of RPN13 Compounds

Synthesis of LW-RPN13-4

(301) In an exemplary synthesis, LW-RPN13-4 was synthesized using the steps shown in Scheme 1.

(302) ##STR00079##

(303) (1) A4-((S)-1-(3,5-bis((E)-3,4-dichlorobenzylidene)-4-oxopiperidin-1-yl)-1-oxo-3-phenylpropan-2-yl)-N.sup.5-(15-oxo-19-((4S)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)-4,7,10-trioxa-14-azanonadecyl)glutaramide: To a solution of N-Biotinyl-(PEG)2-COOH.DIPEA (20 atoms) (5.37 mg, 0.0077 mmol), RA190 (4.46 mg, 0.0080 mmol) and HATU (4.36 mg, 0.0115 mmol) in DMF (0.2 mL) was added DIPEA (6 mg, 0.0400 mmol) at room temperature. The reaction mixture was stirred at room temperature for 15 minutes, and purified via HPLC (0.1% TFA/MeCN) to afford LW-RPN13-4 (6.79 mg). MS: m/z (M+1).sup.+: 1103.42.

Synthesis of LW-RPN13-1

(304) In an exemplary synthesis, LW-RPN13-1 was synthesized using the steps shown in Scheme 2.

(305) ##STR00080##

(306) (2)N—((S)-1-(3,5-bis((E)-3,4-dichlorobenzylidene)-4-oxopiperidin-1-yl)-1-oxo-3-phenylpropan-2-yl)-3-(2-(2-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethoxy)ethoxy)ethoxy)propanamide: To a solution of LW-9296-034 (3.2 mg, 0.0067 mmol), RA190 (4 mg, 0.0070 mmol) and HATU (2.80 mg, 0.0074 mmol) in DMF (0.2 mL) was added DIPEA (8.6 mg, 0.067 mmol) at room temperature. The reaction mixture was stirred at room temperature for 15 minutes, and purified via HPLC (0.1% TFA/MeCN) to afford LW-RPN13-1 (4.32 mg). MS: m/z (M+1).sup.+: 1021.08.

Synthesis of LW-RPN13-2

(307) In an exemplary synthesis, LW-RPN13-2 was synthesized using the steps shown in Scheme 3.

(308) ##STR00081##

(309) (3)N-((5)-1-(3,5-bis((E)-3,4-dichlorobenzylidene)-4-oxopiperidin-1-yl)-1-oxo-3-phenylpropan-2-yl)-3-(2-(2-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)amino)ethoxy)ethoxy)ethoxy)propanamide: To a solution of LW-9296-043 (3.43 mg, 0.0072 mmol), RA190 (4.14 mg, 0.0074 mmol) and HATU (3.01 mg, 0.0080 mmol) in DMF (0.2 mL) was added DIPEA (9.3 mg, 0.072 mmol) at room temperature. The reaction mixture was stirred at room temperature for 15 minutes, and purified via HPLC (0.1% TFA/MeCN) to afford LW-RPN13-2 (2.13 mg). MS: m/z (M+1).sup.+: 1020.98.

Example 2. Exemplary RPN13 Degrader WL40

(310) Proteasome inhibition is an effective treatment for multiple myeloma (MM); however, targeting different components of the Ubiquitin-Proteasome-System (UPS) remains elusive. RNA-interference studies described herein identified proteasome-associated ubiquitin-receptor Rpn13 as a mediator of MM cell growth and survival. Here, the exemplary degrader of Rpn13, WL40, was developed using a small-molecule-induced targeted protein degradation strategy to selectively degrade this component of the UPS. WL40 was synthesized by linking the Rpn13 covalent inhibitor RA190 with the Cereblon (CRBN) binding ligand thalidomide. WL40 binds to both Rpn13 and CRBN and triggers degradation of cellular Rpn13, and is therefore beneficial in exploiting a covalent inhibitor for the development of degraders. Biochemical and cellular studies show that WL40-induced Rpn13 degradation is both CRBN E3 ligase- and Rpn13-dependent. Importantly, WL40 decreases viability in MM cell lines and patient MM cells, even those resistant to bortezomib. Mechanistically, WL40 interrupts Rpn13 function and activates caspase apoptotic-cascade, ER stress response- and p53/p21-signaling. In animal model studies, WL40 inhibits xenografted human MM cell growth and prolongs survival. Overall, the data show the development of a UbR Rpn13 degrader with potent anti-MM activity.

Materials and Methods

Chemical Synthesis of WL40

(311) All of the chemical reagents were purchased from Sigma-Aldrich with proper quality control. The compound WL40 was characterized using .sup.1H NMR (see FIG. 25A), .sup.13C NMR (see FIG. 25B), and MS. RA190 and WL40 were synthesized in the Qi laboratory and were characterized using .sup.1H NMR, .sup.13C NMR, and MS.

(312) ##STR00082##

Procedure for the Synthesis of Compound WL40

(313) Compound 1 (221 mg, 0.463 mmol, 1 eq.) and HATU (184 mg, 0.467 mmol, 1.01 eq) were added to a 25 mL round-bottom flask. DMF (2 mL) was added to generate a colorless solution, and then DIPEA (400 μL, 5 eq) was added. The resulting mixture was stirred at room temperature for 15 minutes. RA190 (260 mg, 0.463 mmol, 1 eq) was added into the reaction flask, and the reaction was continued with continuous stirring at room temperature for 30 minutes. The reaction mixture was then purified directly via HPLC (0.1% TFA, water/MeCN) to give WL40 as yellow powder. 440 mg, yield: 93.2%.

(314) ##STR00083##

(315) Compound WL40. 440 mg, yield: 93.2%; yellow powder. NMR characterization is shown in FIGS. 25A and 25B. .sup.1H NMR (500 MHz, DMSO) δ 11.09 (s, 1H), 8.27 (d, J=8.3 Hz, 1H), 7.87 (d, J=1.6 Hz, 1H), 7.81 (d, J=1.6 Hz, 1H), 7.75 (dd, 7=11.3, 8.4 Hz, 2H), 7.65 (s, 1H), 7.61-7.54 (m, 3H), 7.48 (d, J=8.4 Hz, 1H), 7.14-7.11 (m, 3H), 7.03 (d, J=7.0 Hz, 1H), 6.90 (dd, 7=7.1, 2.3 Hz, 2H), 6.58 (s, 1H), 5.05 (dd, J=12.8, 5.4 Hz, 1H), 4.87-4.67 (m, 5H), 3.59 (t, J=5.4 Hz, 2H), 3.52 (dd, J=5.8, 3.2 Hz, 2H), 3.48 (dd, J=6.1, 3.6 Hz, 9H), 2.87 (ddd, J=17.0, 13.9, 5.4 Hz, 2H), 2.74 (dd, J=13.6, 5.8 Hz, 1H), 2.67-2.52 (m, 4H), 2.17 (t, J=6.6 Hz, 2H), 2.04-1.99 (m, 1H). .sup.13C NMR (126 MHz, DMSO) δ 185.64, 173.28, 170.53, 170.30, 169.99, 169.39, 167.76, 146.85, 137.61, 136.67, 135.42, 135.08, 134.52, 134.39, 134.23, 133.96, 132.77, 132.61, 132.53, 132.49, 132.13, 131.39, 130.87, 130.71, 129.31, 128.45, 126.85, 117.88, 111.15, 109.69, 70.21, 70.15, 69.89, 69.33, 66.92, 50.16, 49.02, 46.37, 43.13, 42.15, 37.53, 36.01, 31.45, 22.62. MS (EI) calcd. for C.sub.50H.sub.47Cl.sub.4N.sub.5O.sub.10: 1019.75, Found: 1020.58.

(316) Procedures for Assays Described Herein

(317) CRBN AlphaScreen

(318) Assays were performed with minimal modifications from the manufacturer's protocol (PerkinElmer, USA). All reagents were diluted in 50 mM HEPES, 150 mM NaCl, 0.1% w/v BSA, 0.01% w/v Tween20, pH 7.5, and allowed to equilibrate to room temperature prior to addition to plates. After addition of Alpha beads to master solutions, all subsequent steps were performed under low light conditions. A 2× solution of components with final concentrations of CRBN-DDB1 at 50 nM, Ni-coated Acceptor Bead at 20 μg/ml, and 15 nM biotinylated-pomalidomide was added in 10 μL to 384-well plates (AlphaPlate-384, PerkinElmer, USA). Plates were spun down at 150×g, and 100 nL of compound in DMSO from stock plates were added by pin transfer using a Janus Workstation (PerkinElmer, USA). The streptavidin-coated donor beads (20 μg/ml final) were added as to the solution in a 2×, 10 μL volume. Following this addition, plates were sealed with foil to prevent light exposure and evaporation. The plates were spun down again at 150 g. Plates were incubated at room temperature for 1 hour, and then read on an Envision 2104 (PerkinElmer, USA), using the manufacturer's protocol.

(319) RPN13 AlphaScreen

(320) Assays were performed with minimal modifications from the manufacturer's protocol (PerkinElmer, USA). All reagents were diluted in 50 mM HEPES, 150 mM NaCl, 0.1% w/v BSA, 0.01% w/v Tween20, pH 7.5, and allowed to equilibrate to room temperature prior to addition to plates. After addition of Alpha beads to master solutions, all subsequent steps were performed under low light conditions. A 2× solution of components with final concentrations of RPN13 at 670 nM, Ni-coated Acceptor Bead at 20 μg/ml, and 5 nM biotinylated-poly-Ub K48-linked chains (UCB-230, BostonBiochem, USA) was added in 10 μL to 384-well plates (AlphaPlate-384, PerkinElmer, USA). Plates were spun down at 150×g, and 100 nL of compound in DMSO from stock plates were added by pin transfer using a Janus Workstation (PerkinElmer, USA). The streptavidin-coated donor beads (20 μg/ml final) were added, as with previous the solution, in a 2×, 10 μL volume. Following this addition, plates were sealed with foil to prevent light exposure and evaporation. The plates were spun down again at 150 g. Plates were incubated at room temperature for 1 hour, and then read on an Envision 2104 (PerkinElmer, USA), using the manufacturer's protocol.

(321) Cell Culture and Reagents

(322) Human MM cell lines MM.1S, MM.1R, RPMI-8226, ANBL6.WT, ANBL6.BR, DOX40, INA6, and normal PBMCs were cultured in RPMI1640 complete medium. Informed consent was obtained from ah patients in accordance with the Helsinki protocol. MM CD138-positive cells, bone marrow stromal cells (BMSCs), and plasmacytoid dendritic cells (pDCs) from MM patients were isolated and cultured as described previously..sup.32

(323) Immunoblotting

(324) Cellular protein extracts were prepared using RIPA lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, and 0.1% SDS). Protein lysates were subjected to immunoblotting using antibodies against poly ADP ribose polymerase (PARP, BD Bioscience Pharmingen, San Diego, Calif.), caspase 3, caspase 8, p53 (Santa Cruz Biotechnology), caspase 9, p-eIF2a (Abeam, Cambridge, Mass.), caspase 7, cyclin B1, CDC25C, CDCl.sub.2, p21, Rpn13, PERK, BIP, Calnexin, GFP, LC3A/B, α-tubulin (Cell Signaling, Beverly, Mass.), polyubiquitin (Enzo Life Sciences, Inc., Farmingdale, N.Y.), or β-actin (Sigma-Aldrich, St. Louis, Mo.).

(325) Proteasome Activity Assays

(326) MM.1S cells were treated with WL40 (1 μM or 10 μM) or Bortezomib (1 μM) for 3 hours; cells were then harvested and lysed in lysis buffer, followed by removal of debris by centrifugation. Total protein (25 μg) was analyzed for proteasome activity using 20S proteasome Assay Kit (Calbiochem), as previously described..sup.33

(327) Cell Viability and Apoptosis Analysis

(328) Cell viability was determined by WST-1/CellTiter-Glo Luminescent assays, as described previously..sup.34 Apoptosis was measured using Annexin/PI staining..sup.33 Caspase activity assay and cell cycle analysis were performed as described previously..sup.35

(329) Generation of CRISPR/Cas9-Knockout Cell Lines

(330) CRISPR-Cas9 genome editing was performed to generate Rpn13-knockout (Rpn13-KO) HCT116 and MM.1S cell lines. Cells were transfected with Rpn13-CRISPR/Cas9-knockout (KO) plasmid (Santa Cruz Biotechnology, Inc., Santa Cruz, Calif., USA) using Lipofectamine 2000 (Thermo Fisher Scientific, Waltham, Mass., USA) or the cell line Nucleofector Kit V (Amaxa Biosystems, Cologne, Germany), respectively. After 48 h incubation, green fluorescent protein (GFP)-positive cells were sorted. Rpn13 KO was confirmed by both protein expression studies and DNA sequencing.

(331) Human MM Xenograft Model

(332) Animal model studies were performed as described previously..sup.32, 33, 36 Briefly, CB17 SCID mice were subcutaneously inoculated with 5.0×10.sup.6 MM.1S cells. When tumors were measurable (100 mm.sup.3) at approximately 3 weeks after MM-cell injection, mice (10 mice/group) were treated on a twice-weekly schedule with vehicle alone, WL40, or RA190. Mice were euthanized when tumor volume reached institutional limit (2000 mm.sup.3). All animal experiments protocols were approved by and conformed to the relevant regulatory standards of the Institutional Animal Care and Use Committee at the Dana-Farber Cancer Institute.

(333) Analysis of Mice Tumors

(334) Tumors were harvested from WL40-treated and control animals. Tumor sections were fixed and paraffin embedded for immunostaining to detect growth inhibition (Ki67), apoptosis (cleaved caspase-3), poly-ubiquitination (PolyU), and angiogenesis (CD31), as described previously..sup.32, 34 Tumor protein lysates from control vehicle- and WL40-treated mice were analyzed for caspase-8, poly-Ubiquitin or β-actin level using immunoblot analyses.

(335) Statistical Analysis

(336) Statistical significance was derived using the two-tailed Student's t test. Survival of mice was analyzed by GraphPad Prism software.

(337) Results and Discussion

(338) Development of Rpn13 Degrader

(339) The main principle underlying the synthetic design of degraders is that bivalent molecules can interact with targeted proteins and E3 ligases simultaneously to induce the ubiquitination of targeted proteins. Therefore, a small-molecule Rpn13 inhibitor, RA190, that covalently binds to Cys88 of the Rpn13 Pru domain, was enlisted. The covalent binding of RA190 to Rpn13 interrupts the recognition of polyubiquitinylated proteins that signals for subsequent degradation by the proteasome. The degradation strategy utilized the E3 ligase, CRBN, and its binding molecules, immunomodulatory drugs (IMiDs), one of which is thalidomide. These IMiDs bind to the E3 ubiquitin ligase complex (CUL4-RBX-DDB1 CRBN/CRL4.sup.CRBN) and have also been used in the treatment of MM. More specifically, RA190 was linked with thalidomide via either an alkyl linker or a polyethylene glycol (PEG) linker to create a set of potential degraders. Amongst these compounds, WL40, created by linking RA190 to thalidomide with a short PEG linker, showed promising activity (FIG. 19A) as a potent degrader..sup.37

(340) Specificity and Functionality of WL40

(341) Several experiments were performed to confirm the specificity of WL40. Firstly, WL40-CRBN binding activity was assessed using in vitro AlphaScreen assays, as previously described..sup.27 Biochemical CRBN binding analysis confirmed that WL40 interacts with the E3 ubiquitin receptor CRBN using thalidomide and lenalidomide as positive controls (FIG. 19B). As expected, the Rpn13 inhibitor RA190 alone did not show any binding to CRBN (FIG. 19B). It was then examined whether the bivalent molecule, WL40, can bind to Rpn13. A novel biochemical AlphaScreen assay was designed to measure compound binding to Rpn13 (FIG. 19C). Since Rpn13 recognizes polyubiquitin (polyUb), the GST-tagged Rpn13 and biotinylated polyUb are immobilized on the acceptor and donor beads of the AlphaScreen assay (obtained from PerkinElmer). Upon excitation, the donor bead releases a singlet of oxygen, which reaches the acceptor bead and creates an emission. The binding of RA190 and WL40 interrupts this recognition event and eliminates the signal. Using RA190 as a positive control, it was further confirmed that WL40 binds to Rpn13 and interrupts Rpn13's recognition of the biotinylated polyubiquitin tail. (FIG. 19D). Thus, it was confirmed that WL40 can bind both Rpn13 and CRBN complex.

(342) Next, it was examined whether WL40 decreases cellular Rpn13 levels in MM cells. MM.1S cells were treated with various concentrations of WL40 (20 0 nM, 400 nM, or 800 nM) for 4 hours, 8 hours, and 16 hours; protein lysates were then analyzed for Rpn13 levels using immunoblot analysis. A marked decrease in Rpn13 levels was noted in WL40-treated cells in a time-dependent manner (FIG. 19E, upper panel). Decrease in Rpn13 levels was detectable as early as 8 hours after WL40 treatment and maximally (95%) reduced at 16 hours. Rpn13 protein level reduction was not observed with RA190 treatment (FIG. 19E, lower panel). There was no change in protein levels of tubulin, used as a loading control (FIG. 19E). To further corroborate the findings, MM.1S cells were treated with WL40, and then analyzed intracellular alterations in Rpn13 using flow cytometry. In concert with the data obtained using immunoblot analysis, a significant reduction in Rpn13 expression was noted in WL40-versus DMSO-, or RA190-treated cells (FIG. 19E).

(343) To confirm that CRBN presence is a prerequisite for the function of WL40 in cells, CRBN-knockout (KO) MM.1S cells.sup.27, 38 were utilized, and the effect of WL40 on Rpn13 degradation was examined. As shown in FIG. 19G, no decrease in Rpn13 levels was observed in WL40-versus DMSO control-treated CRBN-KO cells. These data demonstrate that WL40 degrades Rpn13 in a CRBN-dependent manner (FIG. 19G). Next, a Green Fluorescent Protein (GFP)u-1 reporter cell line expressing Ub-tagged GFP, which is marked for constitutive degradation by the proteasome, was utilized. GFPu-1 cells were treated with WL40, and GFP levels were then analyzed using immunoblotting. As shown in FIG. 19H, WL40 treatment increases GFP levels, indicating the blockade of proteasome-mediated GFP degradation. Similar results were observed in RA190-treated cells. These findings provide evidence for the requirement of Rpn13 engagement by WL40 for its activity. Thus, a novel degrader has been developed that can engage with both RPN13 and Cereblon (CRBN) simultaneously and induce targeted protein degradation.

(344) To eliminate the potential impact of WL40 on proteasome function, the effect of WL40 on proteasome activities was assessed. Examination of both cellular extracts from WL-40-treated MM cells and purified recombinant 20S proteasome showed that WL40 does not inhibit 20S proteasomal activities (chymotrypsin-like, trypsin-like, or caspase-like activities) (FIGS. 20A and 20B). Pretreatment of MM.1S cells with the proteasome inhibitor, MG132, blocked WL40-mediated Rpn13 degradation, suggesting that Rpn13 degradation occurs through the proteasome and that proteasome function is required for WL40-induced RPN13 degradation, consistent with other reports of targeted protein degradation (FIG. 20C). Together, these biochemical and cellular findings demonstrate that WL40 binds to CRBN and Rpn13 within cells and promotes ubiquitination of Rpn13, followed by subsequent proteasomal degradation of Rpn13.

(345) Next, the effect of WL40 on additional MM cell lines, including p53-mutated RPMI-8226 cells or bortezomib-resistant ANBL6.BR cells, was evaluated. WL40 (400 nM) treatment induced RPN13 degradation in both cell lines (FIGS. 20D and 20E). It was also examined whether WL40 triggers the accumulation of polyubiquitinated (PolyUb) protein, a hallmark event during proteasome inhibition, in these cell lines.sup.39, 40 MM.1S and ANBL6.BR cells were treated with WL40, followed by analysis of ubiquitinated protein using immunoblotting. A marked increase in PolyUb proteins was detected in WL40-versus DMSO-treated cells (FIG. 20F). PolyUb increase was also observed in bortezomib- and RA190-treated cells, albeit to a lesser extent than observed in WL40-exposed cells (FIG. 20F). The finding that WL40 triggered a more rapid and higher molecular weight PolyUb versus bortezomib suggests a distinct mechanism of action (MOA) for these agents. Indeed, bortezomib only targets the 20S proteasomal activities and therefore leads to aggregation of lower molecular weight PolyUb proteins; whereas WL40 blocks the 19S proteasome and prevents deubiquitination of substrates, thereby resulting in higher molecular weight PolyUb proteins. Taken together, these data demonstrate that WL40 blocks proteasome-mediated protein degradation upstream of the 20S proteasome and promotes RPN13 degradation selectively, without inhibiting proteasomal activities.

(346) Anti-MM Activity of WL40

(347) In order to examine whether WL40-triggered Rpn13 degradation affects the viability of MM cells, a panel of MM cell lines sensitive or resistant to conventional (dexamethasone, alkylating agents, anthracyclines) or novel (bortezomib) therapies, including lines representing cytogenetically-distinct MM subtypes, was utilized. WL40 is more cytotoxic than the parental Rpn13 inhibitor RA190 against dexamethasone-sensitive MM.1S and resistant MM.1R isogenic MM cell lines (FIG. 21A), indicating potent anti-MM activity of WL40 versus RA190 due to its ability to degrade Rpn13. IC.sub.50 values for both MM.1S and MM.1R cells correlate with the DC.sub.50 for Rpn13. Importantly, WL40 overcomes bortezomib-resistance, evidenced by similar IC.sub.50 values for both bortezomib-sensitive (ANBL6.WT) and -resistant (ANBL6.BR) cells (FIG. 21A, Table). In addition, cytotoxic activity of WL40 was observed even against p53-mutated RPMI-8226 cells and MM growth factor IL-6-dependent INA6 MM cells (FIG. 21A, Table). These data suggest that WL40 can overcome p53-mutation, a high-risk feature conferring drug resistance in MM, and triggers MM cell death even in the presence of pro-growth and -survival factor IL-6.

(348) To further evaluate the clinical potential of the novel degrader, the effect of WL40 in MM patient cells was examined next. First, primary tumor (CD138.sup.+) cells from newly diagnosed (patient #4) and MM refractory to bortezomib/lenalidomide (patient #2-4) were analyzed (FIG. 21B). Treatment with WL40 decreased viability of all CD138.sup.+ patient cells (IC.sub.50 range: 95 nM-170 nM) (FIG. 21B). Importantly, WL40 at the IC.sub.50 for MM cells does not affect viability of normal PBMCs (FIG. 21C), suggesting a favorable therapeutic index.

(349) Adhesion of MM cells to bone marrow stromal cells (BMSCs) induces MM-promoting growth factors and protects against cytotoxic activity of anti-MM drugs..sup.42, 43 Moreover, BM accessory cells such as plasmacytoid dendritic cells (pDCs), can also trigger MM cell proliferation, survival, and drug-resistance..sup.32 Therefore, the effect of WL40 was assessed next using the patient MM-BMSCs or MM-pDCs in vitro co-culture assays. Even in these co-cultures with BMSCs or pDCs, WL40 induced a dose-dependent decrease in the viability of MM cells. (FIGS. 21D and 21E). These data demonstrate that WL40 retains its anti-MM activity in the tumor-protective MM-host BM microenvironment.

(350) Rpn13 Degradation-Induced Signal Transduction

(351) Next, the downstream signaling triggered by WL40 during Rpn13 degradation was examined. WL40 treatment induces an increase in early- (Annexin V.sup.+/PI.sup.−) and late-stage (Annexin V.sup.+/PI.sup.+) apoptosis, associated with proteolytic cleavage of Poly (ADP) ribose polymerase (PARP) by immunoblotting, as well as activation of caspase-3, caspase-7, caspase-8 and caspase-9, assessed in caspase enzymatic activity assays (FIG. 22A and FIG. 22B, and FIG. 22C, respectively). Additionally, treatment of MM.1S and ANBL6.BR cells with WL40 triggers a reduction in the levels of cell-cycle regulatory proteins (cyclin-B1, CDC25C, and CDCl.sub.2), indicating growth arrest in these cells (FIG. 22D). Examination of the p53/p21 apoptotic signaling axis showed an earlier induction of this pathway in WL40-versus Rpn13 inhibitor RA190-treated ANBL6.BR cells (FIG. 22E).

(352) Elevated endoplasmic reticulum (ER)-associated protein degradation (ERAD) signaling is a hallmark of MM, which confers enhanced sensitivity to proteasome inhibitors. Since WL40, like botezomib, triggers PolyUb accumulation (FIG. 20F), it was next examined whether it increases ER stress and triggers associated unfolded protein response (UPR) signaling. Indeed, a rapid and robust induction of UPR proteins (BIP, PERK, phosphorylated eIF2α, or a lectin protein calnexin) was found in WL40-treated ANBL6.BR and MM.1S cells (FIG. 22E and FIG. 22F, respectively). Of note, Rpn13 inhibitor RA190 also triggered UPR signaling, but with delayed kinetics and to a lesser extent than Rpn13 degrader WL40. Prior studies have established that ER stress also induces an alternative lysosomal pathway (autophagy) for degradation of misfolded proteins..sup.40 Here, it was found that WL40 induces PERK, a key component in autophagic signaling. To further confirm the activation of autophagy by WL40, alterations in the autophagic molecule LC3/Atg8 were examined. During autophagy, LC3/Atg8 is processed and attached to the autophagosome membrane by conjugation with phosphatidylethanolamine. Immunoblot analysis showed a significant increase in LC3A/B in WL40-versus DMSO-treated ANBL6.BR or MM.1S cells (FIG. 22E and FIG. 22F, respectively). As for UPR signaling in ANBL6.BR cells, a more pronounced LC3 activation was noted after WL40 than RA190 treatment. Overall, these findings show that WL40-induced apoptosis is associated with activation of the caspase-cascade, p53/p21 signaling, ER stress response signaling, and autophagy. Importantly, it is shown that degradation of Rpn13 triggers more pronounced biologic sequelae in MM cells than domain-specific Rpn13 inhibition.

(353) In Vivo Anti-MM Activity of WL40

(354) To fully understand the therapeutic potential of the RPN13 degrader, the in vivo efficacy of WL40 was evaluated using the human plasmacytoma xenograft mouse model..sup.3, 34 This model has been useful in validating novel anti-MM therapies bortezomib, carfilzomib, ixazomib, lenalidomide, and pomalidomide, which have translated to clinical trials and FDA approval. Treatment of MM.1S-bearing mice with intraperitoneal (IP) injections of WL40 (14.7 μM/kg) inhibits MM growth and prolongs host survival (FIG. 23A). Rpn13 inhibitor RA190 (26.8 μM/kg) also attenuates MM tumor progression and extends mice survival (FIGS. 23A and 23B, respectively). Importantly, use of even half the equimolar dose of WL40 versus RA190 achieves similar extent of tumor growth inhibition and host survival. These findings suggest that Rpn13 degradation could be a more potent strategy in blocking tumor progression than Rpn13 inhibition. WL40 was well tolerated, with no significant weight loss in WL40-treated mice (data not shown). Analysis of tumors harvested from treated mice showed that WL40 induced increased accumulation of PolyUb proteins relative to tumors from control mice (FIGS. 23C and 23D). WL40 decreases proliferation, induces apoptosis, and blocks angiogenesis in harvested tumors, as assessed by Ki67, cleaved caspase-3, and CD31 staining, respectively (FIG. 23D). These data therefore show more potent in vivo anti-MM activity of exemplary compound WL40 versus RA190.

(355) In summary, the development of a small-molecule degrader, WL40, targeting UbR Rpn13 is described herein, and its specificity and functionality is validated using both biochemical and genetic models. Importantly, the studies using both in vitro and in vivo preclinical models of MM show potent anti-MM activity of WL40. Novel findings include: 1) the development of the covalent inhibitor-based heterobifunctional degrader molecule of Rpn13 is demonstrated; 2) using both pharmacological assays and in vivo tumor efficacy models, it is shown that the Rpn13 degrader is cell permeable and triggers potent anti-MM activity, even in the presence of cytoprotective tumor BM microenvironment, overcomes bortezomib-resistance, and is active even in the context of mutated-p53; 3) Rpn13 degradation is a more efficient inducer of MM cell death than Rpn13 inhibition, evidenced by a more rapid and robust induction of ER stress response/UPR- and p53/p21 apoptotic signaling by WL40 than RA190; 4) the MM xenograft model study showed that significant tumor growth inhibition can be achieved using half the equimolar dose of WL40 versus Rpn13 inhibitor RA190; and 5) the study strongly suggests that degradation of tumor-promoting proteins within the UPS using the degronimid strategy is a plausible therapeutic approach, especially in cancers with elevated ER stress/UPR signaling such as MM.

(356) Finally, the anti-MM activity of the IMiD, lenalidomide, occurs via CRBN complex-mediated degradation of Ikaros proteins, IKZF1 and IKZF3..sup.38 This finding supports the therapeutic potential of strategies to induce degradation of tumorigenic target proteins via chemically synthesized small-molecule degraders. Importantly, extensive preclinical research shows that degraders may: reduce the need to maintain high systemic inhibitor levels for target inhibition and efficacy in vivo; neutralize even high levels of target protein expression and function; as well as degrade substrates and thereby avoid resistance mechanisms such as gene mutation or copy number alterations. Overall, provided herein is an exemplary rationale for the development of UPS-based degrader therapies, which further indicates the potential clinical utility of novel therapeutics targeting UbR Rpn13 to improve patient outcome in MM.

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Equivalents and Scope

(358) In the claims articles such as “a,” “an,” and “the” may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that include “or” between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The disclosure includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The disclosure includes embodiments in which more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process.

(359) Furthermore, the disclosure encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, and descriptive terms from one or more of the listed claims is introduced into another claim. For example, any claim that is dependent on another claim can be modified to include one or more limitations found in any other claim that is dependent on the same base claim. Where elements are presented as lists, e.g., in Markush group format, each subgroup of the elements is also disclosed, and any element(s) can be removed from the group. It should it be understood that, in general, where the disclosure, or aspects described herein, is/are referred to as comprising particular elements and/or features, certain embodiments described herein or aspects described herein consist, or consist essentially of, such elements and/or features. For purposes of simplicity, those embodiments have not been specifically set forth in haec verba herein. It is also noted that the terms “comprising” and “containing” are intended to be open and permits the inclusion of additional elements or steps. Where ranges are given, endpoints are included. Furthermore, unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or sub-range within the stated ranges in different embodiments described herein, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise.

(360) This application refers to various issued patents, published patent applications, journal articles, and other publications, all of which are incorporated herein by reference. If there is a conflict between any of the incorporated references and the instant specification, the specification shall control. In addition, any particular embodiment of the present disclosure that falls within the prior art may be explicitly excluded from any one or more of the claims. Because such embodiments are deemed to be known to one of ordinary skill in the art, they may be excluded even if the exclusion is not set forth explicitly herein. Any particular embodiment described herein can be excluded from any claim, for any reason, whether or not related to the existence of prior art.

(361) Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation many equivalents to the specific embodiments described herein. The scope of the present embodiments described herein is not intended to be limited to the above Description, but rather is as set forth in the appended claims. Those of ordinary skill in the art will appreciate that various changes and modifications to this description may be made without departing from the spirit or scope of the present disclosure, as defined in the following claims.