A molecular scaffold for depleting HRPN13 and a select group of ken box proteins

Abstract

The invention provides scaffold compounds having anti-hRPN13 activity and pharmaceutical compositions comprising the same. The invention also provides methods of treating a disease, such as cancer, in a subject, comprising administering the scaffold compounds and pharmaceutical compositions described herein.

Description

A MOLECULAR SCAFFOLD FOR DEPLETING HRPN13 AND A SELECT GROUP OF KEN BOX PROTEINS CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 539,663, filed September 21, 2023, which is hereby incorporated by reference in its entirety. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with Government support under Grant Numbers 1 ZIA BC011490, 1 ZIA BC011627, and 1 ZIA BC011065 awarded by, and a FLEX award given from, the National Cancer Institute, National Institutes of Health. The Government has certain rights in this invention. BACKGROUND OF THE INVENTION

[0003] The 26S proteasome performs regulated protein degradation (Finley et al., Trends Biochem. Sci., 41, 77-93 (2016)). Proteasome substrates are typically marked post-translationally by ubiquitin chains which are in turn recognized by ubiquitin receptors in the proteasome 19S regulatory particle (RP) (Osei-Amponsa et al., Trends Biochem. Sci., 47(11), 950-964 (2022)). The RP caps either end of the proteasome 20S core particle (CP), which is assembled from four heptameric rings with a hollow interior where substrate proteolysis occurs (Chen et al., FEBS. J., 288, 5231-5251 (2020)).

[0004] Small molecule inhibitors of the CP are used to treat hematological cancers and an inhibitor of the CP variant immunoproteasome (iP) present in hematopoietic cells or following pro-inflammatory signaling is used against autoimmune inflammatory myopathies. Additional therapeutic applications of the ubiquitin-proteasome pathway have emerged with proteolysis targeting chimeras (PROTACs), bifunctional molecules that cause the degradation of a protein of interest by linking it to cellular machinery that catalyzes ubiquitination (Ramachandran et al., Curr. Opin. Struct. Biol., 67, 110-119 (2020)). PROTACs for antigen and estrogen receptors arein phase II clinical trials for prostate and breast cancer, respectively (Gao et al., J. Clin. Oncol., 40, 17-17 (2022) and Hamilton et al., J. Clin. Oncol., 40, TPS1120 (2022)).

[0005] RP subunit hRpn13 is a substrate receptor with a Pru domain that binds to ubiquitin (Schreiner et al., Nature, 453, 548-52 (2008) and Husnjak et al., Nature, 453, 481-8 (2008)) with preference for K48-linked ubiquitin chains (Chen et al., Structure, 24, 1257-1270 (2016)) due to interactions involving the ubiquitin linker region (Lu et al., Structure, 28, 495-506 e3 (2020)).

[0006] Adjacent and opposite to the ubiquitin-binding surface is a channel across the Pru domain where the intrinsically disordered extreme C-terminus of RP subunit hRpn2 binds (Lu et al., PLoS One, 10, e0140518 (2015), Lu et al., Nat. Commun., 8, 15540 (2017), and VanderLinden et al., J. Biol. Chem., 292, 9493-9504 (2017)). hRpn13 also has a deubiquitinase adaptor (DEUBAD) domain that binds and activates the deubiquitinase UCHL5 / Uch37 (Yao et al., Nat. Cell. Biol., 8, 994-1002 (2006), Hamazaki et al., EMBO J, 25, 4524-36 (2006), and Qiu et al., EMBO J, 25, 5742-53 (2006)).

[0007] hRpn13 has emerged as a therapeutic target, with a cysteine residue (Cys88) peripheral to the hRpn2-binding region (Lu et al., Nat. Commun., 8, 15540 (2017) and VanderLinden et al., J. Biol. Chem., 292, 9493-9504 (2017)) that is available for covalent modification by small molecules (Anchoori et al., ACS Omega, 3, 11917-11929 (2018), Anchoori et al., Cancer Cell, 24, 791-805 (2013), Lu et al., Nat. Commun., 12, 7318 (2021), and Lu et al., Nat. Commun.15: 2485 (2024)).

[0008] By linking one such molecule (XL5) to an E3 ligase-targeting compound to generate a PROTAC, a naturally existing hRpn13 fragment was discovered in multiple myeloma cells that lacks the DEUBAD domain but has an intact Pru domain (hRpn(Lu et al., Nat. Commun., 12, 7318 (2021)).

[0009] hRpn13Pruis selectively ubiquitinated and degraded by hRpn13 PROTACs that use the XL5 ligand (Lu et al., Nat. Commun., 12, 7318 (2021)). The treatment of hRpn13Pru- producing cells with these hRpn13 PROTACs induces apoptosis (Lu et al., Nat. Commun., 12, 7318 (2021)). The hRpn13 Pru and DEUBAD domains interact when free of binding partners (Chen et al., Mol. Cell, 38, 404-15 (2010)), and loss of the DEUBAD removes any regulatory activities associated with the Pru:DEUBAD interaction as well as potential deubiquitination of hRpn13 and / or its bound proteins by UCHL5 / Uch37. An independent study further supported thetherapeutic potential of hRpn13-targeting by a peptoid ligand, KDT-11, that restricts cell viability in multiple myeloma cells (Trader et al., J. Am. Chem. Soc., 137, 6312-9 (2015)).

[0010] PCNA clamp associated factor (PCLAF) is a PCNA-binding co-factor that regulates DNA repair by facilitating PCNA interaction with DNA polymerase η at stalled replisomes (Povlsen et al., Nat. Cell Biol., 14, 1089-98 (2012)) and drives cellular exit from quiescence to promote lung tumorigenesis by remodeling the DREAM (dimerization partner, retinoblastoma (RB)-like, E2F, and multi-vulval class B) complex (Kim et al., Mol. Cell, 81, 1698-1714 e6 (2021)). Ribonucleoside-diphosphate reductase subunit M2 (RRM2) is one of two subunits of ribonucleotide reductase, which catalyzes formation of deoxyribonucleotide diphosphates from ribonucleotide diphosphates, a rate-limiting step in the production of deoxyribonucleotide triphosphates. RRM2 is transcriptionally upregulated at the G1 / S phase transition (Chabes et al., J. Biol. Chem., 279, 10796-807 (2004) and DeGregori et al., Mol. Cell Biol., 15, 4215-24 (1995)) and at the end of the S phase, selected for ubiquitin-mediated degradation by the E3 ubiquitin ligase cyclin F (D’Angiolella et al., Cell, 149, 1023-34 (2012)).

[0011] Elevated RRM2 levels correlate with poor prognosis in patients with breast cancers (Shi et al., PLoS One, 17, e0265195 (2022)) and decreased overall survival in patients with neuroblastoma (Nunes et al., Sci. Adv., 8, eabn1382 (2022)) and epithelial ovarian cancer (Aird et al., Cell Cycle, 13, 199-207 (2014)), with its knockdown reported to reduce cell proliferation in epithelial ovarian cancer cell lines (Aird et al., Cell Cycle, 13, 199-207 (2014)).

[0012] The anaphase-promoting complex (APC), or cyclosome, is a multi-subunit E3 ligase that ubiquitinates cell regulators at the metaphase-anaphase and mitosis-G1 transitions. FZR1 also named Cdh1 is a substrate adaptor that binds and activates the APC in late mitosis and G1 and promotes the ubiquitination of mitotic cyclins. The E3 ligase complex APC / CFZR1promotes the degradation of PCLAF (Emanuele et al., Proc. Natl. Acad. Sci. USA, 108, 9845-50 (2011)) and RRM2 (Chabes et al., Proc. Natl. Acad. Sci. USA, 100, 3925-3929 (2003)) by recognition of KEN boxes in their protein sequences. KEN boxes are a known degron for APC / CFZR1substrates (Pfleger et al., Genes Dev., 14, 655-665 (2000)). KEN boxes are so named for their amino acid consensus sequence which is K-E-N.

[0013] Aberrant hRpn13 activity has been implicated in a number of human cancers, including, but not limited to, multiple myeloma, lymphoma, mantle cell lymphoma, acuteleukemia, cancers associated with human papillomavirus, colorectal cancer, gastric cancer, ovarian cancer, liver cancer, breast cancer, cervical cancer, prostate cancer, and pancreatic cancer. Although some hRpn13 binding compounds have been developed and have shown some efficacy, these compounds have displayed some degree of cytotoxicity or other off-target effects or have required high dosages that may lead to systemic side effects in clinical applications and / or false positives in diagnostic assays. It would be advantageous to develop new hRpn13- targeting molecules, including small molecule scaffolds, that have greater specific activity against hRpn13, in order to more effectively treat hRpn13-associated cancers. hRpn13 can regulate gene expression by interacting with histone deacetylase HDAC8 (Osei-Amponsa et al., Mol. Cell, 84: 522-537, e8 (2024); Santos-Barriopedro et al., Genes Cancer, 10: 119-133 (2019)) and arginine deiminase PADI4 (Osei-Amponsa et al., Mol. Cell, 84: 522-537, e8 (2024)).

[0014] Thus, there is an ongoing need in the art for small molecule compounds that can be used to target hRpn13 and recruit ubiquitin machinery to induce protein degradation and apoptosis, which can be used for treating cancers. The invention provides such compounds. The small molecule compounds of the invention also can be used to deplete PCLAF, RRM2, CDC25A, PTTG1, and PRR11. These and other advantages of the invention, as well as additional inventive features, will be apparent from the description of the invention provided herein. BRIEF SUMMARY OF THE INVENTION

[0015] The invention provides a compound of Formula (I):or a pharmaceutically acceptable salt thereof, wherein X1is N or CR1; X2is N or CR2; X3is N or CR3; X4is N or CR4; X5is N or CR5; each of R1, R2, R3, R4, and R5is independently selected from hydrogen, halogen, −OH, −CF3, C1-C6 alkylamino, substituted or unsubstituted C1-C6 alkyl, C1-C6 alkoxy, perhaloalkyl, perhaloalkoxy, haloalkoxy, heteroarylalkoxy, −O-Bn-tetrazolyl, aryloxy substituted with 5- or 6- membered aryl or heteroaryl, and benzyloxy substituted with 5- or 6-membered aryl or heteroaryl; provided that no more than two of X1-X5are N, and at least one of X1-X5is not CH; W is selected from substituted or unsubstituted C1-C6alkyl, C1-C6alkoxy, −S(O)2−,, −R12S(O)2NR11−, −C(O)O−, −R12C(O)O−, −C(O)OR12−, −R12C(O)OR12−, pyrazolyl, 1,2,3-triazolyl, 1,2,4-triazolyl, tetrazolyl, imidazolyl, oxadiazolonyl and isoxazolyl; Y is substituted or unsubstituted aryl or heteroaryl ring having 5-6 members, or substituted or unsubstituted bicyclic ring or two-ring system, the bicyclic ring or two-ring system having 9-11 members; and Z is selected from Z1, Z2, Z3, Z4, and Z5,wherein X6is N or CR6; X7is N or CR7; X8is N or CR8; X9is N or CR9; X10is N or CR10; each of R6, R7, R8, R9, and R10is independently selected from hydrogen, halogen, cyano, perhaloalkyl, substituted or unsubstituted C1-C6 alkyl, perhaloalkoxy, 5- or 6-membered substituted or unsubstituted aryl ring, 5- or 6-membered substituted or unsubstituted heteroaryl rprovided that no more than two of X6-X10are N; wherein R9and R10, R8and R9, R7and R8, or R6and R7together with the atoms to which they are attached optionally form a 5- or 6-membered substituted or unsubstituted aryl ring, or a5- or 6-membered substituted or unsubstituted heteroaryl ring; each R11is independently selected from hydrogen and substituted or unsubstituted C1-C6alkyl; and each R12independently is C1-C6alkyl.

[0016] The invention also provides a compound of Formula (II):Formula (II) or a pharmaceutically acceptable salt thereof, wherein Y is an aryl or heteroaryl ring having 5-6 members, or substituted or unsubstituted bicyclic ring or two-ring system, the bicyclic ring or two-ring system having 9-11 members; X6is N or CR6; X7is N or CR7; X8is N or CR8; X9is N or CR9; each of R6, R7, R8, and R9is independently selected from hydrogen, halogen, cyano, perhaloalkyl, substituted or unsubstituted C1-C6 alkyl, perhaloalkoxy, 5- or 6-membered substituted or unsubstituted aryl ring, or 5- or 6-membered substituted or unsubstituted heteroaryl ring, −R12CN, −NO2, −R12NO2, −OH, −R12OH, −C(O)OR11, −R12C(O)OR11, −NR112, −R12NR112, −C(O)NR112, −R12C(O)NR112, −B(OR11)2, −R12B(OR11)2, −S(O)2NR112, and −R12S(O)2NR112; provided that no more than two of X6-X9are N, and at least one of X6-X9is not CH; wherein R8and R9, R7and R8, or R6and R7together with the atoms to which they areattached optionally form a 5- or 6-membered substituted or unsubstituted aryl ring, or a 5- or 6- membered substituted or unsubstituted heteroaryl ring; each R11is independently selected from hydrogen and substituted or unsubstituted C1-C6 alkyl; and each R12independently is C1-C6 alkyl.

[0017] The invention further provides a pharmaceutical composition comprising the compound of Formula I or II, or a pharmaceutically acceptable salt thereof, and a pharmaceutical carrier.

[0018] The invention also provides a method for treating a disease in a subject, the method comprising administering to the subject a compound of Formula I or II, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising a compound of Formula I or II.

[0019] The invention also provides a method for modifying the activity of hRPN13, hRPN13Pru, or a variant thereof in a subject, the method comprising administering to the subject a compound of Formula I or II, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising a compound of Formula I or II.

[0020] The invention further provides a method for modifying the activity of one of the binding partners for hRPN13, hRPN13Pru, or a variant thereof in a subject, the method comprising administering to the subject a compound of Formula I or II, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising a compound of Formula I or II.

[0021] The invention also provides a method for providing a molecular glue, the method comprising administering to a subject a compound of Formula I or II, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising a compound of Formula I or II, wherein the molecular glue enhances interactions between hRPN13, hRPN13Pru, or a variant thereof, and one of the binding partners thereof in a subject.

[0022] The invention further provides a method for inducing degradation of KEN box proteins in a subject, the method comprising administering to the subject a compound of Formula I or II, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising a compound of Formula I or II.

[0023] The invention also provides a method for inducing apoptosis of cells in a subject, the method comprising administering to the subject a compound of Formula I or II, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising a compound of Formula I or II.

[0024] The invention also provides a method for disrupting cell cycle progression of cells in a subject, the method comprising administering to the subject a compound of Formula I or II, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising a compound of Formula I or II.

[0025] The invention also provides a method for restricting cell viability by degrading KEN box proteins in a subject, the method comprising administering to the subject a compound of Formula I or II, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising a compound of Formula I or II.

[0026] The invention further provides a method for restricting cell cycle progression by degrading KEN box proteins in a subject, the method comprising administering to the subject a compound of Formula I or II, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising a compound of Formula I or II. BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)

[0027] FIGs.1A-1F provide the results of a covalent docking screen conducted based on the hRpn13 Pru:XL5 structure and including the hRpn13-binding compound XL44.

[0028] FIG.1A shows a ribbon diagram of the hRpn13 Pru-XL5 structure (Protein Data Bank ID 7KXI) as used for virtual screening and the model structure of hRpn13 Pru with XL44.

[0029] FIG.1B shows the1H,15N HSQC spectra of 20 PM15N-hRpn13 Pru (black) or with equimolar XL44 (gray) in NMR buffer at 10qC. Arrows highlight the shifting of hRpn13 signals from their free state to their XL44-bound state. Residue signals that disappear (italicized text) or appear (S90, V38, V85, and V93) following XL44 addition are labeled.

[0030] FIG.1C shows an intensity graph of emission at 350 nm for 1 μM hRpn13 Pru with either addition of XL44 or XL5. The plot depicts mean ±SD from three parallel recordings against compound concentration and were fit by using the equation ‘[inhibitor vs. response – Variable slope (four parameters)’ in GraphPad Prism9.

[0031] FIG.1D shows LC-MS analysis of 2 μM purified hRpn13 Pru (MW: 17017.3 g / mol) incubated with 20 μM XL44 for 2 hours at 4qC. The resulting XL44 adduct and unmodified hRpn13 Pru are labeled along with the detected molecular weight (Da).

[0032] FIG.1E shows the chemical structure of XL44 with fluorine and a graph of the19F spectra of 50 PM XL44, 50 PM XL44 with equimolar hRpn13 Pru, 25 PM hRpn2 (940-953) added to the preincubated 50 PM XL44 with equimolar hRpn13 Pru in NMR buffer without or with DTT at 10qC.

[0033] FIG.1F shows immunoblots with antibodies against hRpn13 of input from RPMI 8226 whole cell lysates incubated with 40 μM XL5B, biotin (negative control) or XL44B (bottom panel) and the corresponding biotin pulldowns with streptavidin (top panel).

[0034] FIGs.2A-2D provide the crystal structure of hRpn13 Pru bound to ubiquitin and XL44 with the dynamic β1 – β2 hairpin resolved by NMR.

[0035] FIG.2A shows the structure of hRpn13 Pru bound to ubiquitin with XL44 (boxed ball-and-stick rendering) covalently linked to hRpn13 Cys88. An expansion of the XL44 region is included with the methoxybenzamide, benzene and indolin-2-one groups labeled (right). All protein secondary structural elements are labeled. The 2Fo − Fc electron density map is included for XL44 (gray mesh) and Cys88 (black mesh) contoured at 1.7σ. The protons at the two stereocenters of XL44 are labeled and adopt the R and S configuration for C12 and C13, respectively.

[0036] FIG.2B shows an expansion of the structure of the XL44-binding pocket of hRpn13 Pru with key interactions indicated by black lines and distances (Å).

[0037] FIG.2C shows the 2Fo − Fc electron density map (black mesh) contoured at 1.7σ illustrating that except for the backbone of Gly35 and Thr36 (ball-and-stick model), the hRpn13 E1-E2 hairpin residues Leu33 - Val38 show poor electron density, which was addressed by NMR data, examples of which are provided in FIG.3A and 3B.

[0038] FIG.2D shows the results of the hetNOE analysis recorded on apo hRpn13 Pru mapped on the structure of apo hRpn13 Pru (PDB ID 5IRS).

[0039] FIGs.3A-3C provide the structure of hRpn13 PRU-XL44 by integrated NMR and X- ray.

[0040] FIG.3A shows selected regions focusing on the XL44 methyl group (top) or hRpn13 Leu33, Val38 and Lys42 (bottom) from13C half-filtered NOESY (100 ms) experiment acquired on a sample containing 0.4 mM13C-labeled hRpn13 Pru and equimolar unlabeled XL44 dissolved in NMR buffer.

[0041] FIG.3B shows the chemical structure of XL44-13C6-CB (left panel) and selected regions from a1H,1H plane of a 3-dimensional1H,13C half-filtered NOESY (100 ms) experiment acquired without incrementing the13C dimension on a sample with 0.4 mM hRpn13 Pru mixed with equimolar of XL44-13C6-BA in NMR buffer (right panel).

[0042] FIG.3C shows an overlay of the X-ray structure (black) and integrated NMR / X-ray refined structure (gray) for the XL44-bound regions. The methyl group of XL44 as well as side chains of Leu33, Val38 and Lys42 are reoriented in the integrated structure (shown as black arrow) based on NOE interactions with XL44.

[0043] FIGs.4A-4H provide structures showing XL44 forms extensive hydrophobic interactions with hRpn13 Pru.

[0044] FIG.4A shows an overlay of the modeled (XL44 and hRpn13 Pru, black) and experimental (XL44 and hRpn13 Pru, light gray) structures not showing ubiquitin.

[0045] FIGs.4B-4C show expanded views of XL44-bound hRpn13 showing close contacts between XL44 H10 and H18 and hRpn13 Leu33, Val38, Val85 (FIG.4B) and Met31 (FIG.4C) with distances (Å) included. Interactions consistent with detected NOEs are represented with black lines, while those that do not support an R stereo-configuration at C12 are displayed with gray line.

[0046] FIGs.4D-4F show comparative structural analysis of hRpn13 Pru bound with XL44 (FIG.4D), hRpn2 peptide (944-953) (FIG.4E), or XL5 (FIG.4F) with similar interactions highlighted (black lines with distances (Å) included) for the XL44 methoxybenzamide and hRpn2 Pro944 and Pro945. Distinct interactions between hRpn13 Pru amino acid residues T39 and P40 to the XL54-methylbenzamide group are indicated.

[0047] FIGs.4G-4H show an expanded view of extensive hydrophobic interactions between the XL44 central benzene (FIG.4G) and indolin-2-one (FIG.4H) rings with hRpn13 Pru residues.

[0048] FIGs.5A-5G provide immunoblots and graphs showing XL44 induces apoptosis in an hRpn13-dependent manner and reduces the protein levels of hRpn13Pru.

[0049] FIGs.5A-5B show immunoblots of lysates from RPMI 8226 WT and trRpn13-MM2 cells treated with 20 μM (FIG.5A) or the indicated concentration of XL44 or DMSO (control) (FIG.5B) for 24 hours that were immunoprobed for cleaved caspase-9, hRpn13, or ^E-actin (as a loading control). A black asterisk indicates cleaved caspase-9 in the immunoblot for hRpn13, as hRpn13 was probed following cleaved caspase-9 and without stripping the membrane (top panel).

[0050] FIG.5C shows immunoblots of whole cell extract from RPMI 8226 WT cells treated for the indicated time with 20 PM XL44 or DMSO (0 hour, vehicle control) detecting hRpn13 or E-actin. Percentage (%) is calculated as the ratio of intensities for hRpn13Prunormalized to E- actin (divided by that of DMSO-treated cells and multiplied by 100. Experiments in 5B and 5C were performed twice. Percentage (%) (mean r SD) of hRpn13Pruwere plotted against XL44 concentration (FIG.5B, PM) or time (FIG.5C, hours) and fit by using the equation [Inhibitor] vs. normalized response - Variable slope (FIG.5B) and One phase decay (FIG.5C) in GraphPad Prism9. Half-degrading concentration (DC50, FIG.5B), half- maximal depletion concentration (DepC50, FIG.5B) and (differential protein abundance following treatment) (d1 / 2, FIG.5C) values are included.

[0051] FIG.5D shows a graph of cell viability of RPMI 8226 WT or trRpn13-MM2 cells that were treated with the indicated concentrations of XL44 for 48 hours and had cell metabolism measured by an MTT assay; data represent mean ± SD of n = 6 biological replicates. Viability is calculated as (

[0052] FIG.5E shows a graph of cell viability of MCF7, OVCAR-4, and OVCAR-5 cells that were treated with the indicated concentrations of XL44 for 48 hours and had cell metabolism measured by an MTT assay; data represent mean ± SD of n = 3 biological replicates. Viability is calculated as (

[0053] FIG.5F shows a graph of cell viability of Human Fibroblast 1634 and HS5 (BMSCs) cells that were treated with the indicated concentrations of XL44 for 48 hours and had cell metabolism measured by an MTS assay. Data represent mean r SD of n = 4 biological replicates. Viability is calculated as (l490)sample / (l490)control*100 (%). IC50values in FIGs.5D and 5F wereanalyzed by using the equation [Inhibitor] vs. normalized response] - Variable slope in GraphPad Prism9.

[0054] FIG.5G shows a graph of percent cytotoxicity of RPMI 8226 WT and trRpn13-MM2 cells that were treated with the indicated concentrations of XL44 for 24 hours and LDH activity measured by CQUANT LDH Cytotoxicity Assay Kit. Data represent mean r SD of n = 4 biological replicates. Cytotoxicity is calculated as ((LDH activity)sample-(LDH activity)spontaneous)) / ((LDH activity)Maximum-(LDH activity)spontaneous))*100 (%).

[0055] FIGs.6A-6G provide immunoblots and graphs showing that XL44 reduces protein levels of PCLAF, its downstream target PTTG1 and RRM2.

[0056] FIG.6A shows a volcano plot displaying proteomic changes caused by XL44 treatment determined by quantitative TMT proteomics analysis performed on lysates from RPMI 8226 WT cells treated for 8 hours with DMSO (control) or 20 PM XL44 in triplicate. p-value was calculated by two-tailed two-sample equal variance t-test. A horizontal and two vertical dashed lines indicate the value -log100.001, log20.5 and log22, respectively. Proteins reduced at > 0.5-fold with p-values < 0.001 are displayed by black dots.

[0057] FIGs.6B-6C show immunoblots of cell lysates from repeated experiments of FIG.5A (FIG.6B) or FIG.5B (FIG.6C) with inclusion of immunoblots for PCLAF, PTTG1, or RRM2 with mouse or rabbit primary and secondary antibodies.

[0058] FIG.6D shows an immunoblot of whole cell lysates from RPMI 8226 WT cells treated with indicated concentration of XL44 or DMSO (a vehicle control) for 24 hours that were immunoprobed for PCLAF, RRM2 (rabbit) and E-actin (as a loading control) and a percent intensity graph thereof.

[0059] FIG.6E shows immunoblots of lysates from repeated experiments of FIG.5C with inclusion of immunoblots for PCLAF, PTTG1 and RRM2 (mouse and rabbit) and a percent intensity graph thereof.

[0060] FIG.6F shows immunoblots of whole cell extract from RPMI 8226 WT cells treated for the indicated time with 20 PM XL44 or DMSO (0 hour, vehicle control) detecting PCLAF or ^E-actin and a percent intensity graph thereof. Percentage (%) in FIGs.6D-6F is calculated as the ratio of intensities for PCLAF (FIGs.6D-6F), RRM2 (FIGs.6D-6F) or hRpn13Pru(FIG.6E) normalized to E-actin (Iedivided by that of DMSO-treated cells andmultiplied by 100. Percentage (%) of PCLAF, RRM2 (FIGs.6D-6F), and hRpn13Pru(FIG.6E) plotted against XL44 concentration (FIG.6D, PM) or time (FIGs.6E-6F, hours) and fit by using the equation [Inhibitor] vs. normalized response - Variable slope (FIG.6D) and One phase decay (FIGs.6E-6F) in GraphPad Prism9. DepC50 ( 6D) and d1 / 2, (6F) values are included. The “” in FIGs.6B, 6C, and 6E indicates an unspecific band detected by the anti-RRM2 mouse antibodies.

[0061] FIG.6G shows a graphical display of the correlation (p-value <0.001) between high and low PCLAF (left graph) or RRM2 (right graph) expression levels with overall survival for patients with multiple myeloma.

[0062] FIGs.7A-7G provide immunoblots and graphs showing that XL44 causes loss of PCLAF and is less efficacious in cells following PCLAF knockdown.

[0063] FIG.7A shows an immunoblots of hRpn13 from 640 nM purified hRpn13 incubated without or with 30 nM commercial 26S proteasome at 37°C for 10 minutes.

[0064] FIG.7B shows immunoblots of hRpn13, PCLAF, and E-actin as indicated from lysates of RPMI 8226 WT cells that were pre-treated with 100 nM proteasome inhibitor carfilzomib (CZ), 1 μM UAE inhibitor MLN7243 (UAEi) or 1 μM NAE inhibitor MLN4924 (NAEi) for 2 hours and then for another 22 hours with DMSO or 20 μM XL44.

[0065] FIG.7C shows schematic representation of hRpn13 proteolysis by proteasomes to generate hRpn13Pru(displayed as a scissor) and its clearance by XL44. XL44-induced depletion of hRpn13Prurequires UAE and proteasome activity, with hRpn13Pruproteolysis followed by ubiquitin-dependent degradation. hRpn13 domains and intrinsically disordered regions are represented by grey ovals and black lines, respectively.

[0066] FIG.7D shows schematic representation of degradation of PCLAF and RRM2 by proteasomes, as accelerated by XL44.

[0067] FIG.7E shows immunoblots of lysates from RPMI 8226 WT cells treated with DMSO (vehicle control), 1 μM UAE inhibitor MLN4924 (UAEi), 20 μM XL44 or 1 μM MLN4924 (UAEi) and 20 μM XL44 (co-treatment) for 24 hours that were immunoprobed for hRpn13, PCLAF, and ^E-actin.

[0068] FIG.7F shows immunoblots of lysates from HCT116 WT cells that were transfected with 1 μg empty vector (control), HA-PCLAF, or HA-PCLAF (AA) with K78A and E79Asubstitutions and then 48 hours later, treated with 20 μM XL44 or DMSO (a control) for 24 hours, and then immunoprobed for PCLAF and β -actin (loading control).

[0069] FIG.7G shows a cell viability graph of HCT116 WT or trRpn13 cells transfected with 50 nM scramble (control, WT and trRpn13 are colored in black with circle and gray with square, respectively) or PCLAF siRNA (WT and trRpn13 are colored in dark gray with hollow square and black with hollow circle, respectively) for 48 hours and that were then treated with 10 μM or 20 μM XL44 for 24 hours, and had cell metabolism measured by an MTT assay. Data represent mean ±SD of n = 6 biological replicates. Viability is calculated as (λ570)sample / (λ570)control*100 (%).

[0070] FIGs.8A-8B provide biophysical analyses of hRpn13 binding to XL44 stereoisomers. FIG.8A shows a LC-MS UV chromatogram of XL44. FIG.8B shows1H,15N HSQC spectra of 20 PM15N-labeled hRpn13 binding Pru with addition of vehicle control DMSO (black) or 2-fold molar excess XL44 major or minor stereoisomer. Spectra were acquired at 600 MHz and 10qC.

[0071] FIGs.9A-9B provide results showing addition of a biotin handle to XL44 reduces its affinity for hRpn13 Pru. FIG.9A shows the chemical structures of biotinylated XL44 (XL44B) and XL5 (XL5B). FIG.9B shows a graph of1H,15N HSQC spectra of 20 PM15N-labeled hRpn13 Pru with addition of vehicle control DMSO (black) or 2-fold molar excess XL44B (gray). Spectra were acquired at 600 MHz and 10qC.

[0072] FIGs.10A-10F provide X-ray density maps and NMR data for XL44-bound hRpn13 Pru.

[0073] FIGs.10A-10B show the electron density map for the hRpn13 sidechain atoms of V85, C88, P89, F106, and W108 (FIG.10A) or M31, P40, K42, and W108 (FIG.10B) of the XL44 binding pocket. Black mesh indicates the 2Fo − Fc electron density map at 1.7σ for the side chain of hRpn13 residues shown with ball-and-stick rendering.

[0074] FIG.10C shows a graph of1N heteronuclear NOE results, that were acquired on 0.44 mM hRpn13 Pru at 25 qC and 850 MHz. Prolines and unassigned residues (Val38, Val85 and Ser90) are not plotted. Error bars are indicated.

[0075] FIGs.10D-10F show the chemical structure of XL44 including the ligated sulfur atom from hRpn13 Cys88. Hydrogen atoms are labeled with the numbers used in the text and figures. Chiral center C12 or C13 is labeled and indicted with a star (FIG.10D, left), and imagesof selected regions focusing on XL44 H10 and H18 (FIG.10D, middle), the XL44 methyl group (FIG.10D), or hRpn13 Leu33 and Val38 (FIGs.10E-10F) from a1H,13C half-filtered NOESY (100 ms) experiment acquired on a sample containing 0.4 mM13C-labeled hRpn13 Pru and equimolar unlabeled XL44 dissolved in NMR buffer. In FIG.10D a 1D1H trace is displayed to the right for Leu33 and Val38 methyl groups indicating the presence of two chemical shift values for XL44 proton H18.

[0076] FIGs.11A-11F provide comparative structural analyses of hRpn13 Pru alone or with XL5, XL44, or hRpn2 (940-953).

[0077] FIGs.11A-11B shows an interaction network for XL44 when bound to the A- (FIG. 11A) or B- (FIG.11B) chain of hRpn13 Pru. The definition of these two chains for the two molecules present in the asymmetric unit is arbitrary. Ligplot analyses shows similar hydrophobic interactions between XL44 and hRpn13 Pru from either chain.

[0078] FIG.11C shows the structural superimposition of the hRpn13 Pru:ubiquitin:XL44 ternary complex with hRpn13 Pru:ubiquitin:hRpn2 (black, PDB ID 5V1Y) which depicts an identical interface between hRpn13 and ubiquitin. Interface residues are represented with stick rendering.

[0079] FIG.11D shows a model of XL44-ligated hRpn13 Pru with S, S stereochemistry for the two chiral centers C12 and C13.

[0080] FIG.11E shows an overlay of hRpn13 Pru structure bound with XL5 (PDB ID 7KXI) or XL44.

[0081] FIG.11F shows a structural comparison of apo (or unbound) and holo (or bound) structure of hRpn13 Pru. Changes in the β1 - β2 hairpin upon binding to different molecules is highlighted with arrows and distances shown for the Gly35 Cα atom.

[0082] FIG.12 provides graphs of spectra from NMR analyses of compounds of Formula (I). The graphs show1H,15N HSQC spectra of 20 PM15N-labeled hRpn13 Pru with addition of DMSO (a control, black) or 2-fold molar excess of compounds XL52 - XL56 (gray). Spectra were acquired at 600 MHz and 10qC. In the first panel, arrows highlight shifting of hRpn13 Pru signals from their apo state to their XL52-bound state. Residue signals that disappear (R92, K30, F106, and R104) or appear (V38, V85, S90, and V93) after XL52 addition are labeled. In otherpanels, some XL44-binding hRpn13 residues (Met31, Leu33, Lys34, Cys88, Phe106 and Trp108) are labeled. hRpn13 Val38 is labeled when present in the spectra.

[0083] FIGs.13A-13B provide results of a MTT assay of RPMI 8226 cells treated with XL44, XL52, or XL44 stereoisomers. FIGs.13A-13B show graphs of cell viability of RPMI 8226 WT cells that were treated with varying concentrations of XL52 or XL44 (FIG.13A) or XL44 major or minor (FIG.13B) for 48 hours and cell metabolism measured by an MTT assay; data represent mean r SD of n = 6 biological replicates. Viability is calculated as (λ570)sample / (λ570)control*100 (%).

[0084] FIG.14 provides results showing an upper band detected by anti-RMM2 mouse antibodies is reduced by siRNA knockdown of RRM2. FIG.14 shows immunoblots of RRM2 (mouse) and β-actin (a loading control) from cell lysates extracted from HCT116 WT cells that were transfected with 50 nM scramble (control) or RRM2 siRNA for 48 hours. A black pound “ ” indicates an unspecific band detected by the antibody RRM2 (mouse).

[0085] FIGs.15A-15C provide results showing the correlation of expression level with patient survival for PTTG1 and hRpn13. FIGs 15A and 15C show graphical depiction of survival for multiple myeloma patients with high or low levels of PTTG1 (FIG.15A, p-value < 0.0001) or Adrm1 (FIG.15C, p-value = 0.07) expression. FIG.15B shows a graphical depiction of correlation of PCLAF and PTTG1 gene expression levels in multiple myeloma patients.

[0086] FIGs.16A-16C provides immunoblots of PCLAF, hRpn13, and β-actin (a loading control) from cell lysates of various cell types.

[0087] FIG.16A shows an immunoblot of lysate extracted from RPMI 8226 WT cells treated with DMSO (vehicle control), 1 μM UAE inhibitor MLN4924 (UAEi), 20 μM XL44 or 1 μM MLN4924 (UAEi) and 20 μM XL44 (co-treatment) for 24 hours and then immunoprobed for hRpn13, PCLAF and β-actin.

[0088] FIG.16B shows scanning of an SDS-PAGE gel that showed ubiquitination of the fluorescently labeled PCLAF by APC / CFZR1with or without XL44.

[0089] FIG.16C shows immunoblots of HCT116 WT or trRpn13 cells that were transfected with 50 nM scramble (control) or PCLAF siRNA for 48 hours and then treated for 24-hours with 20 μM XL44 or DMSO (control) and then immuoprobed for hRpn13, PCLAF and β-actin.

[0090] FIGs.17A-17B show immunoblots of hRpn13 and hRpn13Pruin xenografts of human cancer cell lines grown in the flanks of 6-8 week old NOD-scid-gamma (NSG) mice (myeloma cells) or athymic nude mice (prostate cancer cells).

[0091] FIG.17A shows immunoblots from lysates of xenografts from 5 NSG mice inoculated in the flank with 5x105cells of the myeloma cell line RPMI-8226 after 26 days. ^E-actin was used as a loading control for the immunoblots. All 5 mice were given 50% E-cyclodextrin (BCD) once / day for 5 days.

[0092] FIG.17B shows immunoblots of lysates of xenografts of the myeloma cell lines allowed to grow for 14 days in the flanks of NSG mice (lane 1) L363 (2.5x105cells inoculated) and (lane 2) LP1 (2.5x106cells inoculated) labeled with luciferase (luc), the prostate cancer lines at 1x106cells suspended in matrigel and allowed to grow until tumors reached 100mm2(lane 3) LNCaP, (lane 4) MDA Pca 2b (mouse j), and (lane 5) MDA Pca 2b (mouse e). Abcam antibody 157218 recognizes hRpn13 DEUBAD domain and Abcam antibody 157185 recognizes both hRpn13 and the cleavage product hRpn13Pru. Vinculin was used as a loading control for the immunoblots.

[0093] FIG.18 shows immunoblots with antibodies against hRpn13 (left panel) or ubiquitin (right panel) of RPMI 8226 lysates (bottom) or following a pulldown experiment with streptavidin-bound biotin or XL44B (top). For the pulldown, lysates from RPMI 8226 cells were incubated with streptavidin-bound by biotin or XL44B and washed to remove unbound proteins (top).

[0094] FIG.19 shows an immunoprobe of lysates from RPMI 8226 WT, trRpn13-MM2 or trRpn13-MM2 with reintroduced hRpn13 (trRpn13-MM2 / hRpn13) treated for 24 hours with 20 μM XL44 or DMSO (control).

[0095] FIG.20 shows immunoblots with antibodies against RRM2 (1 or 30 min) or PCLAF of streptavidin-bound XL44B or biotin (negative control) following incubation with RPMI 8226 cell lysates and subsequent wash (top panels) and corresponding input are included (lower panels).

[0096] FIG.21 provides the1H,15N HSQC spectra of 20 PM PCLAF (left panel) and with 10-fold molar excess of XL44 (right panel) in NMR buffer. The spectra were recorded at 700 MHz and 25 qC.

[0097] FIG.22 provides the LC-MS analysis of samples containing 4 μM purified PCLAF (MW: 12397.95 g / mol) incubated with DMSO (a vehicle control, left panel) or 40 μM XL44 (right panel) for 2 hours at 4 qC to detect the formation of XL44 adducts. UV spectra are displayed for each sample. Samples eluted between 1.74 and 2.01 minutes (dashed rectangle) were analyzed to generate MS spectra as indicated.

[0098] FIG.23 shows the volcano plot, extracted from FIG.6A, of proteomic changes for putative APC / C substrates.

[0099] FIG.24 shows the cell metabolism measured by an MTT assay for HCT116 WT or trRpn13 cells transfected with 50 nM scramble or PCLAF siRNA for 48 hours, followed by 24 hour treatment with 20 mM XL44 or DMSO (control). Data represent mean + SD of n = 6 biological replicates. ****, P < 0.0001; **, P < 0.004; P (WT Scramble vs. WT PCLAF) = 0.0018; P (WT Scramble vs. trRpn13 Scramble) = 0.0029; P (trRpn13 Scramble vs. trRpn13 PCLAF) = 0.0031; P (WT Scramble vs. trRpn13 PCLAF) < 0.0001; ‘one-way ANOVA’ (Tukey’s multiple comparisons test) in GraphPad Prism9. Viability is calculated as (l570)sample / (l570)control*100 (%).

[0100] FIG.25 provides the results of a MTT assay of RPMI 8226 or HS5 (BMSC) cells treated with XL44, XL69, or XL80 at varying concentration for 48 hours. Data is represented by mean and standard deviation of n=3 replicates.

[0101] FIG.26 provides the polarized light microscopy image of bulk powder XL44 at 20x magnification. Bulk powder was immersed in paraffin oil and imaged with the polarizer parallel (top) and crossed (bottom) to observe morphology and birefringence. A scale bar of 50 μm is on the bottom right.

[0102] FIG.27A provides an exemplary protocol for preparing a nanosuspension with XL44, having varied milling bead size, polymer, and surfactant stabilizers.

[0103] FIG.27B shows the average particle size of 24 screened formulations analyzed by DLS at 37 ºC in singlet. The D50 (black) and D90 (white) values were plotted, with a closer D50-to-D90 value signifying more uniform particle distribution. A black dotted line is at the 1000 nm diameter scale, and any formulations with a D90 below 1000 nm is considered a nanosuspension. Each formulation was prepared for DLS in singlet (20 acquisitions per run, n=1).

[0104] FIG.28A provides the dynamic light scattering (DLS) results for the stability analysis of XL44 screening formulations in simulated biorelevant media, where each formulation was diluted approximately ten-fold in water (control, black circle), FaSSGF (black hollow circle), FaSSIF (black triangle), or SPF (black hollow square), prepared for DLS at 37 ºC in singlet (20 acquisitions per run, n=1) and represented by the D90 value in nm.

[0105] FIG.28B provides the dynamic light scattering (DLS) results for the stability analysis of XL44 screening formulations at 4 ºC storage over 2 weeks, where each formulation was diluted approximately ten-fold in water and measured by DLS when freshly prepared (black circle), after 4 ºC storage at 1 week (black hollow square), or after 4 ºC storage at 2 weeks (black hollow circle) and represented by the D90 value in nm.

[0106] FIG.29A shows the average particle diameter of PPF nanosuspensions containing XL44, XL69, and XL80, where each formulation was prepared in singlet (n=1), and measurements for dynamic light scattering (DLS) at 37 ºC were prepared in technical triplicate (20 acquisitions per run, n=3). Each formulation was diluted approximately 10-fold in water (control), FaSSGF, FaSSIF, or SPF, and measurements are represented as the D50 (black) or D90 (white) values. For XL69 and XL80, 2-week stability at 4 ºC is shown. Formulations were diluted approximately 10-fold in water and measured by DLS. D50 and D90 values are represented as the mean ± standard deviation of the technical triplicate.

[0107] FIG.29B shows the polarized light (PLM) microscopy results of PPF nanosuspensions containing XL44 (left panel), XL69 (middle panel), and XL80 (right panel), which were visualized by PLM at 100x magnification with the polarizer parallel (top) and crossed (bottom) to observe birefringence. For each image, a scale bar of 10 μm is on the bottom right.

[0108] FIG.30 provides a scatter plot showing the blood concentration (ng / mL) of XL44, XL69, and XL80, which were dosed at 48 mg / kg in CD-1 male mice for a pharmacokinetic study (n=3 per group). Blood was sampled at 0.083, 0.25, 0.5, 1, 2, 4, 8, and 24 hours. XL69 and XL80 were formulated as nanosuspensions (NS) in PPF and dosed intraperitoneally (IP) or by oral gavage (PO). XL44 was formulated as NS and a suspension (S), where both NS and S formulations were dosed PO but only NS dosed as IP. The lower limit of quantification (dashed black line) was at 0.5 ng / mL.

[0109] FIG.31A provides a scatterplot showing myeloma tumor volumes (Day 5-Day 0) in female NSG (NOD-SCID gamma) mice dosed IP at 48 mg / kg once per day for 5 days with nanosuspensions of XL44 in PPF or PPF vehicle alone.

[0110] FIG.31B provides a scatterplot showing myeloma tumor volumes (Day 5-Day 0) in male NSG (NOD-SCID gamma) mice dosed IP at 48 mg / kg once per day for 5 days with nanosuspensions of XL44 in PPF or PPF vehicle alone.

[0111] FIG.31C provides a scatterplot showing myeloma tumor weights at day 5 in both female and male NSG (NOD-SCID gamma) mice dosed IP at 48 mg / kg once per day for 5 days with nanosuspensions of XL44 in PPF or PPF vehicle alone. DETAILED DESCRIPTION OF THE INVENTION

[0112] The invention provides a compound of Formula (I):Formula (I) or a pharmaceutically acceptable salt thereof, wherein X1is N or CR1; X2is N or CR2; X3is N or CR3; X4is N or CR4; X5is N or CR5; each of R1, R2, R3, R4, and R5is independently selected from hydrogen, halogen, −OH,−CF3, C1-C6alkylamino, substituted or unsubstituted C1-C6alkyl, C1-C6alkoxy, perhaloalkyl, perhaloalkoxy, haloalkoxy, heteroarylalkoxy, −O-Bn-tetrazolyl, aryloxy substituted with 5- or 6- membered aryl or heteroaryl, and benzyloxy substituted with 5- or 6-membered aryl or heteroaryl; provided that no more than two of X1-X5are N, and at least one of X1-X5is not CH; W is selected from substituted or unsubstituted C1-C6 alkyl, C1-C6 alkoxy, −S(O)2−, R11−,pyrazolyl, 1,2,3-triazolyl, 1,2,4-triazolyl, tetrazolyl, imidazolyl, oxadiazolonyl and isoxazolyl; Y is substituted or unsubstituted aryl or heteroaryl ring having 5-6 members, or substituted or unsubstituted bicyclic ring or two-ring system, the bicyclic ring or two-ring system having 9-11 members; andwherein X6is N or CR6; X7is N or CR7; X8is N or CR8;X9is N or CR9; X10is N or CR10; each of R6, R7, R8, R9, and R10is independently selected from hydrogen, halogen, cyano, perhaloalkyl, substituted or unsubstituted C1-C6alkyl, perhaloalkoxy, 5- or 6-membered substituted or unsubstituted aryl ring, 5- or 6-membered substituted or unsubstituted heteroarylprovided that no more than two of X6-X10are N; wherein R9and R10, R8and R9, R7and R8, or R6and R7together with the atoms to which they are attached optionally form a 5- or 6-membered substituted or unsubstituted aryl ring, or a 5- or 6-membered substituted or unsubstituted heteroaryl ring; each R11is independently selected from hydrogen and substituted or unsubstituted C1-C6 alkyl; and each R12independently is C1-C6alkyl.

[0113] In some embodiments of the compound of Formula (I), at least one of X6-X10is not CH, or if the compound of Formula (I) does not have an X10substituent, at least one of X6-X9is not CH.

[0114] In the compounds disclosed herein (e.g., in any one or more of the compounds of Formula (I)), each of R1, R2, R3, R4, and R5may be independently selected from hydrogen (H), halogen, −OH (hydroxyl), −CF3(trifluoromethyl), C1-C6alkylamino, substituted or unsubstituted C1-C6 alkyl, C1-C6 alkoxy, perhaloalkyl, perhaloalkoxy, haloalkoxy, heteroarylalkoxy, −O-Bn-tetrazolyl, aryloxy substituted with 5- or 6-membered aryl or heteroaryl, and benzyloxy substituted with 5- or 6-membered aryl or heteroaryl, provided that no more than two of X1-X5are N, and at least one of X1-X5is not CH.

[0115] In some embodiments of the compounds disclosed herein (e.g., in any one or more of the compounds of Formula (I)), X1is CR1, X2is CR2, X3is CR3, X4is CR4, and X5is CR5, and in some embodiments, each of R1, R2, R3, R4, and R5is independently selected from hydrogen, halogen, methyl, methoxy, and –O-Bn-tetrazolyl. In some embodiments, each of R1, R2, R3, R4, and R5is independently selected from hydrogen, chlorine, methyl, methoxy, and–O-Bn-tetrazolyl. In some embodiments, each of R1, R2and R4is hydrogen, R3is selected from hydrogen, halogen, methyl, methoxy and –O-Bn-tetrazolyl, and R5is selected from hydrogen and halogen, provided that at least one of R3and R5is not hydrogen. In some embodiments, each of R1, R2and R4is hydrogen, R3is selected from hydrogen, chlorine, methyl, methoxy and –O-Bn-tetrazolyl, and R5is selected from hydrogen and chlorine, provided that at least one of R3and R5is not hydrogen.

[0116] In some embodiments of the compounds disclosed herein, X1is CR1, X2is CR2, X3is CR3, X4is CR4, and X5is CR5, each of R1, R2, R4and R5is hydrogen, and R3is a C1-C6alkoxy.

[0117] In some embodiments of the compounds disclosed herein, X1is CR1, X2is CR2, X3is CR3, X4is CR4, and X5is CR5, each of R1, R2, R4and R5is hydrogen, and R3is methoxy.

[0118] In some embodiments of the compounds disclosed herein (e.g., in any one or more of the compounds of Formula (I)), each of R1, R2, R3, R4, and R5is independently selected from hydrogen, methyl, methoxy, chlorine, fluorine, and –O-Bn-tetrazolyl. In some embodiments, at least one of R1, R2, R3, R4, and R5is not hydrogen. In other embodiments, at least two of R1, R2, R3, R4, and R5are not hydrogen. In some embodiments, only one of R1, R2, R3, R4, and R5is not hydrogen. In other embodiments, only two of R1, R2, R3, R4, and R5are not hydrogen. In some embodiments of the compounds disclosed herein, each of R1, R2, R3, R4, and R5is independently selected from hydrogen and methoxy, and only one of R1, R2, R3, R4, and R5is not hydrogen. In other embodiments, each of R1, R2, R3, R4, and R5is independently selected from hydrogen, chlorine, and methoxy, and only one of R1, R2, R3, R4, and R5is not hydrogen.

[0119] In some embodiments of the compounds disclosed herein (e.g., in any one or more of the compounds of Formula (I)), W is selected from substituted or unsubstituted C1-C6 alkyl, C1-C6 alkoxy, −S(O)2−, −R12S(O)2−, −C(O)−, −R12C(O)−, −C(O)NR11−, −R12C(O)NR11−, −S(O)2NR11−, −R12S(O)2NR11−, −C(O)O−, −R12C(O)O−, −C(O)OR12−, −R12C(O)OR12−, pyrazolyl, 1,2,3-triazolyl, 1,2,4-triazolyl, tetrazolyl, imidazolyl, oxadiazolonyl and isoxazolyl, wherein each R11is independently selected from hydrogen and substituted or unsubstituted C1-C6 alkyl, and each R12independently is C1-C6 alkyl.

[0120] In some embodiments of the compounds disclosed herein, W is selected from −C(O)NR11− and −R12C(O)NR11−. In other embodiments, W is −C(O)NR11−. In some embodiments, W is selected from −R12C(O)−, –CH2C(O) –, –(CH2)2C(O) –, and –CH2−. In someembodiments, W is −C(O)NH−. In some embodiments, W is selected from pyrazolyl, −C(O)NR11−, and 1,2,3-triazolyl. In some embodiments, W is pyrazolyl. In some embodiments, W is 1,2,3-triazolyl.

[0121] In some embodiments of the compounds disclosed herein, W is selected from carboxamide compounds. For example, W can be selected from −C(O)NR11and −R12C(O)NR12.

[0122] In some embodiments, W is selected from sulfo-oxo compounds. For example, W can be selected from sulfonyl compounds, including −S(O)2− and −R12S(O)2−, where R12is C1-C6 alkyl. In other embodiments, W is selected from sulfonamide compounds, including −S(O)2NR11− and −R12S(O)2NR11−, where each R11is selected from hydrogen and substituted or unsubstituted C1-C6 alkyl, and R12is C1-C6 alkyl.

[0123] In other embodiments, W is selected from ester compounds. For example, W can be selected from −C(O)O−,12wherein each R independently is C1-C6 alkyl.

[0124] In other embodiments of the compounds disclosed herein, W is selected from heteroaryl compounds. For example, W can be selected from pyrazolyl, 1,2,3-triazolyl, 1,2,4- triazolyl, tetrazolyl, imidazolyl, oxadiazolonyl and isoxazolyol.

[0125] In some embodiments of the compounds disclosed herein, each R11is independently selected from hydrogen and substituted or unsubstituted C1-C6alkyl, and each R12independently is C1-C6 alkyl. In some embodiments, R11is hydrogen. In some embodiments, R12is methyl. In some embodiments, R11is hydrogen and R12is methyl, and in other embodiments, each of R11and R12is methyl.

[0126] In some embodiments of the compounds disclosed herein (e.g., in any one or more of the compounds of Formula (I) or Formula (II)), Y is substituted or unsubstituted aryl or heteroaryl ring having 5-6 members, or substituted or unsubstituted bicyclic ring or two-ring system, the bicyclic ring or two-ring system having 9-11 members. In some embodiments, Y is selected from:

[0127] In some embodiments of the compounds disclosed herein, Y may be any one of the above compounds, or Y may be selected from any two or more of the above compounds. For example, Y can be selected from:.

[0128] In some embodiments of the compounds disclosed herein, Y is selected from:.

[0129] In some embodiments, Y is:.

[0130] In some embodiments of the compounds disclosed herein, Y is substituted or unsubstituted aryl or heteroaryl ring having 5-6 members, or substituted or unsubstituted bicyclic ring or two-ring system, the bicyclic ring or two-ring system having 9-11 members, and is optionally substituted with C1-C6alkyl, halogen, pseudohalogen, or perhaloalkyl. In some embodiments of Y, one hydrogen is substituted with C1-C6 alkyl, halogen, pseudohalogen, or perhaloalkyl. In some embodiments, Y is substituted or unsubstituted aryl or heteroaryl ring having 5-6 members, or substituted or unsubstituted bicyclic ring or two-ring system, the bicyclic ring or two-ring system having 9-11 members, and is optionally substituted with methyl, fluorine, or trifluoromethyl. In some embodiments of Y, one hydrogen is substituted with methyl, fluorine, or trifluoromethyl. In some embodiments, Y is unsubstituted aryl or heteroaryl ring having 5-6 members, or unsubstituted bicyclic ring or two-ring system, the unsubstituted bicyclic ring or two-ring system having 9-11 members.

[0131] In some embodiments of Y, no more than three members (e.g., no more than two members) of the heteroaryl ring having 5-6 members, or the bicyclic ring or two-ring system having 9-11 members, are nitrogen. In other embodiments of Y, no more than one member of the heteroaryl ring having 5-6 members, or the bicyclic ring or two-ring system having 9-11 members, is nitrogen.

[0132] In some embodiments of the compounds disclosed herein (e.g., in any one or more of the compounds of Formula (I)), Z is selected from Z1, Z2, Z3, Z4 and Z5:wherein X6is N or CR6; X7is N or CR7; X8is N or CR8; X9is N or CR9; X10is N or CR10; each of R6, R7, R8, R9, and R10is independently selected from hydrogen, halogen, cyano, perhaloalkyl, substituted or unsubstituted C1-C6 alkyl, perhaloalkoxy, 5- or 6-membered substituted or unsubstituted aryl ring, 5- or 6-membered substituted or unsubstituted heteroaryl ring, −R12CN, −NO2, −R12NO2, −OH, −R12OH, −C(O)OR11, −R12C(O)OR11, −NR112,provided that no more than two of X6-X10are N;wherein R9and R10, R8and R9, R7and R8, or R6and R7together with the atoms to which they are attached optionally form a 5- or 6-membered substituted or unsubstituted aryl ring, or a 5- or 6-membered substituted or unsubstituted heteroaryl ring.

[0133] In some embodiments of Z, at least one of X6-X10is not CH, or if the compound of Formula (I) does not have an X10substituent, at least one of X6-X9is not CH.

[0134] In some embodiments of the compounds disclosed herein, each of R6, R7, R8, R9, and R10is independently selected from hydrogen, halogen (for example, chlorine or fluorine), cyano (for example, −CN), perhaloalkyl, substituted or unsubstituted C1-C6alkyl (for example, methyl), perhaloalkoxy, 5- or 6-membered substituted or unsubstituted aryl ring, 5- or 6- membered substituted or unsubstituted heteroaryl ring, nitrile (for example, −R12CN), nitro (for example, −NO2or −R12NO2), hydroxyl (for example, –OH or −R12OH), carboxylic acid (−C(O)OH), ester (for example, −C(O)OR11or −R12C(O)OR11), amino (for example, −NR112, −R12NR112, carboxamide (for example, −C(O)NR112, −R12C(O)NR112, or C(O)NH2), boronic esters (for example −B(OR11)2or −R12B(OR11)2), boronic acid (−B(OH)2), and sulfonamide (for example, −S(O)2NR112, −R12S(O)2NR112, or −S(O)2NH2). In some embodiments, at least one of R6, R7, R8, R9, and R10is not hydrogen.

[0135] In some embodiments of the compounds disclosed herein, each of R6, R7, R8, R9, and R10is independently selected from hydrogen, methyl, methoxy, fluorine, cyano (−CN), carboxylic acid (−C(O)OH), carboxamide (e.g., C(O)NH2), sulfonamide (for example, −S(O)2NH2), hydroxyl (for example, –OH), boronic acid (−B(OH)2). In some embodiments, at least one of R6, R7, R8, R9, and R10is not hydrogen. In other embodiments, each of R6, R7, R8, R9, and R10is hydrogen. In other embodiments, only one of R6, R7, R8, R9and R10is not hydrogen.

[0136] In some embodiments of the compounds disclosed herein, Z iswherein variables R7, R8, and R11are as described with respect to any of the embodiments disclosed herein.

[0137] In some embodiments of the compounds disclosed herein, Z is.

[0138] In some embodiments of the compounds disclosed herein, Z is, wherein variables R8, R9, R10, and R11are as described with respect to any of the embodiments disclosed herein.

[0139] In some embodiments the compound is of Formula (Ia):Formula (Ia), wherein R3is selected from hydrogen, methoxy and –O-Bn-tetrazolyl; R5is selected from hydrogen and halogen; provided that at least one of R3and R5is not hydrogen; and variable Z is as described with respect to any of the embodiments disclosed herein.

[0140] In some embodiments the compound is of Formula (Ib):Formula (Ib), wherein R3is selected from hydrogen, chlorine, methyl, methoxy and –O-Bn-tetrazolyl; R5is selected from hydrogen and chlorine; provided that at least one of R3and R5is not hydrogen; R7is selected from hydrogen and cyano; R8is selected from hydrogen, fluorine, −OH, −C(O)NH2, −C(O)OH, −S(O)2NH2, and −B(OH)2; provided that at least one of R7and R8is not hydrogen; and each R11is independently selected from hydrogen and methyl.

[0141] In some embodiments the compound is selected from:

[0142] For example, the compound may be selected from any one or more of XL44, XL45, XL46, XL47, XL48, XL92, XL93, XL94, XL95, XL96, XL52 and XL56.

[0143] In some embodiments, the compound is of Formula (Ic):Formula (Ic), wherein R3is selected from hydrogen, chlorine, and methoxy; R5is selected from hydrogen and chlorine; and provided that at least one of R3and R5is not hydrogen.

[0144] In some embodiments the compound is selected from XL49, XL50, and XL51:

[0145] In some embodiments the compound is of Formula (Id):Formula (Id), wherein R8is selected from hydrogen, fluorine, methyl, and methoxy; and R9is selected from hydrogen, methyl, and methoxy; R10is selected from hydrogen, fluorine, methyl, and methoxy; provided that at least one of R8-R10is not hydrogen; and R11is selected from hydrogen and methyl.

[0146] In some embodiments the compound is selected from:

[0147] For example, the compound may be selected from any one or more of XL34, XL35, XL36, XL37, XL38, XL39, XL40, XL41, XL42, and XL43.

[0148] In some embodiments the compound is of Formula (Ie):Formula (Ie), wherein variables X1, X2, X3, X4, X5, X6, X7, X8, X9, W, Y, and R11are as described with respect to any of the embodiments disclosed herein.

[0149] In some embodiments of the compounds of Formula (Ie), X1is CR1, and in some embodiments, X1is CH. In some embodiments, X2is N or CR2, and when X2is CR2, R2is hydrogen. In some embodiments, R3is selected from hydrogen, chlorine, methyl, methoxy, and – O-Bn-tetrazolyl. For example, R3 can be methoxy. In some embodiments, each of R4and R5is independently selected from hydrogen and chlorine.

[0150] In some embodiments of the compounds of Formula (Ie), W is a carboxamide. For example, W can be selected from −C(O)NR11− and −R12C(O)NR11−. In other embodiments, W is −C(O)NR11−. For example, W can be −C(O)NH.

[0151] In further embodiments of the compounds of Formula (Ie), Y is selected from:

[0152] In some embodiments of the compounds of Formula (Ie), Y is.

[0153] In some embodiments the compound is of Formula (If):Formula (If), wherein X2is N or CR2, and when X2is CR2, R2is hydrogen or fluorine; R3is selected from hydrogen, chlorine, methyl, methoxy, and –O-Bn-tetrazolyl; R4is selected from hydrogen and chlorine; R5is selected from hydrogen and chlorine; provided that at least one of R3-R5is not hydrogen; R7is selected from hydrogen, fluorine, and cyano; R8is selected from hydrogen, fluorine, and cyano; provided that at least one of R7and R8is not hydrogen; each R11is independently selected from hydrogen and methyl; and each R12independently is C1-C6 alkyl.

[0154] In some embodiments of the compounds of Formula (If), W is a carboxamide. For example, W can be selected from −C(O)NR11− and −R12C(O)NR11−. In other embodiments, W is −C(O)NR11−. For example, W can be −C(O)NH.

[0155] In further embodiments of the compounds of Formula (If), Y is selected from:

[0156] In some embodiments of the compounds of Formula (If), Y is.

[0157] In some embodiments the compound is selected from:

[0158] For example, the compound may be selected from any one or more of XL44, XL45, XL46, XL47, XL48, XL66, XL67, XL68, XL69, XL70, XL71, XL72, XL73, XL74, XL75, XL76, XL77, XL80, XL93, XL96, XL94, XL92, XL95, XL56, XL53, XL79, and XL52. In some embodiments, the compound is selected from XL44, XL69, and XL80.

[0159] In some embodiments the compound is of Formula (Ig):Formula (Ig), wherein R3is selected from hydrogen and methoxy; R5is selected from hydrogen and chlorine; provided that at least one of R3and R5is not hydrogen; W is selected from substituted or unsubstituted C1-C6alkyl, C1-C6alkoxy, −S(O)2−, − NR11−, −, pyrazolyl, 1,2,3-triazolyl, 1,2,4-triazolyl, tetrazolyl, imidazolyl, oxadiazolonyl and isoxazolyl; R7is selected from hydrogen, fluorine, and cyano; R8is selected from hydrogen, fluorine, and cyano; and provided that at least one of R7and R8is not hydrogen.

[0160] For example, in some embodiments of the compounds of Formula (Ig), W can be a carboxamide. For example, W can be selected from −C(O)NR11− and −R12C(O)NR11−. In other embodiments, W is −C(O)NR11−. In some embodiments, W is −C(O)NH. In other embodiments, W is selected from −C(O)NH−, −S(O)2NH−, pyrazolyl, and 1,2,3-triazolyl.

[0161] In some embodiments the compound is of Formula (Ih):Formula (Ih), wherein W is selected from −C(O)NH−, −S(O)2NH−, pyrazolyl, and 1,2,3-triazolyl.

[0162] In some embodiments, the compound is:, XL44.

[0163] In some embodiments, the compound is:

[0164] In some embodiments, the compound is:

[0165] In some embodiments, the compound is of Formula (Ii):Formula (Ii),wherein variables X1, X2, X3, X4, X5, X6, X7, X8, X9, X10, W, Y, and R11are as described with respect to any of the embodiments disclosed herein.

[0166] In some embodiments of the compounds of Formula (Ii), W is a carboxamide. For example, W can be selected from −C(O)NR11− and −R12C(O)NR11−. For example, W can be −C(O)NR11−. In some embodiments, W is −C(O)NH.

[0167] In some embodiments of the compounds of Formula (Ii), Y is selected from:

[0168] In some embodiments of the compounds of Formula (Ii), Y is.

[0169] In some embodiments, the compound is of Formula (Ij):Formula (Ij), wherein variables X1, X2, X3, X4, X5, X6, X7, X8, X9, and X10are as described with respect to any of the embodiments disclosed herein.

[0170] In some embodiments, the compound is of Formula (Ik):Formula (Ik), wherein R3is selected from hydrogen, chlorine, and methoxy; R5is selected from hydrogen and chlorine; provided that at least one of R3and R5is not hydrogen; and variables X6, X7, X8, X9, X10are as described with respect to any of the embodiments disclosed herein.

[0171] In some embodiments, the compound is of Formula (Im):. Formula (Im) wherein R3is selected from hydrogen, chlorine, and methoxy; R5is selected from hydrogen and chlorine; and provided that at least one of R3and R5is not hydrogen.

[0172] In some embodiments, the compound is selected from:

[0173] In some embodiments, the compound is selected from XL49, XL50, and XL51.

[0174] In some embodiments, the compound is of Formula (In):Formula (In), wherein variables X1, X2, X3, X4, X5, X6, X7, X8, X9, X10, W, Y, and R11are as described with respect to any of the embodiments disclosed herein.

[0175] In some embodiments, of the compounds of Formula (In), Y is selected from:

[0176] In some embodiments of the compounds of Formula (In), Y is.

[0177] In some embodiments of the compounds of Formula (In), W is a carboxamide. For example, W can be selected from −C(O)NR11− and −R12C(O)NR11−. In other embodiments, W is −C(O)NR11−. In some embodiments, W is −C(O)NH.

[0178] In some embodiments, the compound is of Formula (Io):Formula (Io), wherein variables X1, X2, X3, X4, X5, X6, X7, X8, X9, X10, and R are as described with respect to any of the embodiments disclosed herein.

[0179] In some embodiments, the compound is of Formula (Ip):Formula (Ip), wherein variables X6, X7, X8, X9, X10, and R are as described with respect to any of the embodiments disclosed herein

[0180] In some embodiments, the compound is of Formula (Iq):Formula (Iq), wherein each of R8, R9, and R10is independently selected from hydrogen, fluorine, methyl, and methoxy; provided that at least one of R8-R10is not hydrogen; and R11is selected from hydrogen and methyl.

[0181] In some embodiments, the compound is selected from:

[0182] In some embodiments, the compound is selected from any one or more of XL34, XL35, XL36, XL37, XL38, XL39, XL40, XL41, XL42, and XL43.

[0183] In some embodiments, the compound is of Formula (Ir):Formula (Ir), wherein variables X1, X2, X3, X4, X5, X6, X7, X8, X9, X10, W, Y, and R11are as described with respect to any of the embodiments disclosed herein.

[0184] In some embodiments of the compounds of Formula (Ir), W is a carboxamide. For example, W can be selected from −C(O)NR11− and −R12C(O)NR11−. For example, W can be −C(O)NR11−. In some embodiments, W is −C(O)NH.

[0185] In some embodiments, of the compounds of Formula (Ir), Y is selected from:

[0186] In some embodiments, of the compounds of Formula (Ir), Y is.

[0187] In some embodiments, the compound is:

[0188] In some embodiments, the compound is of Formula (Is):Formula (Is), wherein variables X1, X2, X3, X4, X5, X6, X7, X8, X9, W, and Y are as described with respect to any of the embodiments disclosed herein.

[0189] In some embodiments, of the compounds of Formula (Is), W is a carboxamide. For example, W can be selected from −C(O)NR11− and −R12C(O)NR11−. For example, W can be −C(O)NR11−. In some embodiments, W is −C(O)NH.

[0190] In some embodiments, of the compounds of Formula (Is), Y is selected from:

[0191] In some embodiments, of the compounds of Formula (Is), Y is.

[0192] In some embodiments, the compound is:.

[0193] The invention also provides a compound of Formula (II):or a pharmaceutically acceptable salt thereof, wherein Y is an aryl or heteroaryl ring having 5-6 members, or substituted or unsubstituted bicyclic ring or two-ring system, the bicyclic ring or two-ring system having 9-11 members; X6is N or CR6; X7is N or CR7; X8is N or CR8; X9is N or CR9; each of R6, R7, R8, and R9is independently selected from hydrogen, halogen, cyano, perhaloalkyl, substituted or unsubstituted C1-C6 alkyl, perhaloalkoxy, 5- or 6-membered substituted or unsubstituted aryl or substituted or unsubstituted heteroaryl ring, −R12CN, −NO2, ,provided that no more than two of X6-X9is N, and at least one of X6-X9is not CH; wherein R8and R9, R7and R8, or R6and R7together with the atoms to which they are attached optionally form a 5- or 6-membered substituted or unsubstituted aryl ring, or a 5- or 6- membered substituted or unsubstituted heteroaryl ring; each R11is independently selected from hydrogen and substituted or unsubstituted C1-C6alkyl; and each R12independently is C1-C6 alkyl.

[0194] In some embodiments of the compounds of Formula (II) disclosed herein, each of R6, R7, R8, and R9is independently selected from hydrogen, halogen (for example, chlorine or fluorine), cyano (for example, −CN), perhaloalkyl, substituted or unsubstituted C1-C6 alkyl (for example, methyl), perhaloalkoxy, 5- or 6-membered substituted or unsubstituted aryl ring, 5- or 6-membered substituted or unsubstituted heteroaryl ring, nitrile (for example, −R12CN), nitro (for example, −NO2 or −R12NO2), hydroxyl (for example, –OH or −R12OH), carboxylic acid (−C(O)OH), ester (for example, −C(O)OR11or −R12C(O)OR11), amino (for example, −NR112, −R12NR112, carboxamide (for example, −C(O)NR112, −R12C(O)NR112, or C(O)NH2), boronic esters (for example −B(OR11)2 or −R12B(OR11)2), boronic acid (−B(OH)2), and sulfonamide (for example, −S(O)2NR112, −R12S(O)2NR112, or −S(O)2NH2). In some embodiments, at least one of R6, R7, R8, and R9is not hydrogen.

[0195] In some embodiments of the compounds Formula (II) disclosed herein, each of R6, R7, R8and R9is independently selected from hydrogen, methyl, methoxy, fluorine, cyano (−CN), carboxylic acid (−C(O)OH), carboxamide (e.g., C(O)NH2), sulfonamide (for example, −S(O)2NH2), hydroxyl (for example, –OH), boronic acid (−B(OH)2). In some embodiments, at least one of R6, R7, R8, and R9is not hydrogen. In other embodiments, each of R6, R7, R8, and R9is hydrogen. In other embodiments, only one of R6, R7, R8, and R9is not hydrogen.

[0196] In some embodiments of the compounds of Formula (II), Y is selected from:

[0197] In some embodiments of the compounds of Formula (II) disclosed herein, Y is substituted or unsubstituted aryl or heteroaryl ring having 5-6 members, or substituted or unsubstituted bicyclic ring or two ring system, the bicyclic ring or two ring system having 9-11 members, and is optionally substituted with C1-C6alkyl, halogen, pseudohalogen, or perhaloalkyl. In some embodiments of Y, no more than two hydrogens are substituted with C1-C6 alkyl, halogen, pseudohalogen, or perhaloalkyl. In some embodiments, Y is substituted or unsubstituted aryl or heteroaryl ring having 5-6 members, or substituted or unsubstituted bicyclic ring or two ring system, the bicyclic ring or two ring system having 9-11 members, and is optionally substituted with methyl, fluorine, or trifluoromethyl. In some embodiments of Y, no more than two hydrogens are substituted with methyl, fluorine, or trifluoromethyl. In some embodiments, Y is unsubstituted aryl or heteroaryl ring having 5-6 members, or unsubstituted bicyclic ring or two ring system, the unsubstituted bicyclic ring or two ring system having 9-11 members.

[0198] In some embodiments of Y, no more than three members (e.g., no more than two members) of the heteroaryl ring having 5-6 members, or the bicyclic ring or two ring system having 9-11 members, are nitrogen. In other embodiments of Y, no more than one member of theheteroaryl ring having 5-6 members, or the bicyclic ring or two ring system having 9-11 members, is nitrogen.

[0199] For example, in some embodiments of the compounds of Formula (II), Y can be.

[0200] In some embodiments, the compound is of Formula (IIa):Formula (IIa), wherein variables X6, X7, X8, X9, and R11are as described with respect to any of the embodiments disclosed herein

[0201] In some embodiments the compound of Formula (II) is:.

[0202] Any of the disclosed compounds (e.g., the compounds of Formula (I) or Formula (II), or subgenera thereof) can be present in the form of salts (e.g., pharmaceutically acceptable salts) derived from inorganic or organic acids. As used herein, the term “pharmaceutically acceptable salt” includes salts of acidic or basic groups present in the disclosed compounds. Suitable pharmaceutically acceptable salts include base addition salts, including alkali metal salts, e.g., sodium or potassium salts; alkaline earth metal salts, e.g., calcium or magnesium salts, and salts formed with suitable organic ligands, e.g., quaternary ammonium salts, which may be similarly prepared by reacting the drug compound with a suitable pharmaceutically acceptable base. The salts can be prepared in situ during the final isolation and purification of the compounds of the present disclosure, or following final isolation by reacting a free base function, such as a secondary or tertiary amine, of a disclosed compound with a suitable inorganic or organic acid, or reacting a free acid function, such as a carboxylic acid, of a disclosed compound with a suitable inorganic or organic base.

[0203] Acidic addition salts can be prepared in situ during the final isolation and purification of a disclosed compound, or separately by reacting moieties comprising one or more nitrogen groups with a suitable acid. In various aspects, acids which may be employed to form pharmaceutically acceptable acid addition salts include such inorganic acids as hydrochloric acid, sulfuric acid and phosphoric acid and such organic acids as oxalic acid, maleic acid, succinic acid and citric acid. In a further aspect, salts further include, but are not limited, to the following: hydrochloride, hydrobromide, hydroiodide, nitrate, sulfate, bisulfate, phosphate, acidphosphate, isonicotinate, acetate, lactate, salicylate, citrate, tartrate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucaronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzensulfonate, p-toluenesulfonate, butyrate, camphorate, camphorsulfonate, digluconate, glycerophosphate, hemisulfate, heptanoate, hexanoate, fumarate, hydrochloride, 2-hydroxyethanesulfonate (isethionate), nicotinate, 2-naphthalenesulfonate, oxalate, pectinate, persulfate, 3-phenylpropionate, picrate, pivalate, propionate, succinate, tartrate, thiocyanate, phosphate, glutamate, bicarbonate, undecanoate, and pamoate (i.e., 1,1’-methylene-bis-(2-hydroxy-3-naphthoate)) salts. Also, basic nitrogen- containing groups can be quatemized with such agents as lower alkyl halides, such as methyl, ethyl, propyl, and butyl chloride, bromides, and iodides; dialkyl sulfates like dimethyl, diethyl, dibutyl, and diamyl sulfates, long chain halides such as decyl, lauryl, myristyl and stearyl chlorides, bromides and iodides, aralkyl halides like benzyl and phenethyl bromides, and others.

[0204] Basic addition salts can be prepared in situ during the final isolation and purification of a disclosed compound, or separately by reacting carboxylic acid moieties with a suitable base such as the hydroxide, carbonate or bicarbonate of a pharmaceutical acceptable metal cation or with ammonia, or an organic primary, secondary or tertiary amine. Pharmaceutically acceptable salts include, but are not limited to, cations based on the alkali and alkaline earth metals, such as sodium, lithium, potassium, calcium, magnesium, aluminum salts and the like, as well as nontoxic ammonium, quaternary ammonium, and amine cations, including, but not limited to ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, and the like. Other representative organic amines useful for the formation of base addition salts include diethylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine and the like. In further aspects, bases which may be used in the preparation of pharmaceutically acceptable salts include the following: ammonia, L- arginine, benethamine, benzathine, calcium hydroxide, choline, deanol, diethanolamine, diethylamine, 2-(diethylamino)-ethanol, ethanolamine, ethylenediamine, N-methyl-glucamine, hydrabamine, 1H-imidazole, L-lysine, magnesium hydroxide, 4-(2-hydroxyethyl)-morpholine, piperazine, potassium hydroxide, 1-(2-hydroxyethyl)-pyrrolidine, secondary amine, sodium hydroxide, triethanolamine, tromethamine and zinc hydroxide.

[0205] In some embodiments of the compounds disclosed herein (e.g., in any one or more of the compounds of Formula (I) or a subgenera thereof), the compound is not one or more of the following compounds:

[0206] In some embodiments of the compounds disclosed herein (e.g., in any one or more of the compounds of Formula (I)), the compound is not XL5:

[0207] In some embodiments of the compounds disclosed herein (e.g., in any one or more of the compounds of Formula (I)), the compound is not XL23:.

[0208] The following definitions are used herein with respect to the disclosed compounds, e.g., with respect to the compounds of Formula (I) and Formula (II).

[0209] As used herein, the term C1-C6alkyl refers to an alkyl group having from one to six carbon atoms. The term “alkyl” as used herein is a branched or unbranched saturated hydrocarbon group of 1 to 24 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, s-butyl, t-butyl, n-pentyl, isopentyl, s-pentyl, neopentyl, hexyl, heptyl, octyl, nonyl, decyl, dodecyl, tetradecyl, hexadecyl, eicosyl, tetracosyl, and the like. The alkyl group can be cyclic (i.e., cycloalkyl) or acyclic, saturated or unsaturated (e.g., alkenyl or alkynyl), or a combination thereof (e.g., cycloalkenyl). The alkyl group can be branched or unbranched. The alkyl group can also be substituted or unsubstituted. For example, the alkyl group can be substituted with one or more groups including, but not limited to, alkyl, cycloalkyl, alkoxy, amino, ether, halide, hydroxy, nitro, silyl, sulfo-oxo, or thiol, as described herein. A “lower alkyl” group is an alkyl group containing from one to six (e.g., from one to four) carbon atoms. The term alkyl group can also be a C1alkyl, C1-C2alkyl, C1-C3alkyl, C1-C4alkyl, C1-C5alkyl, C1-C6 alkyl, C1-C7 alkyl, C1-C8 alkyl, C1-C9 alkyl, C1-C10 alkyl, and the like up to and including a C1-C24alkyl.

[0210] Throughout the specification, “alkyl” is generally used to refer to both unsubstituted alkyl groups and substituted alkyl groups; however, substituted alkyl groups are also specifically referred to herein by identifying the specific substituent(s) on the alkyl group. For example, the term “halogenated alkyl” or “haloalkyl” specifically refers to an alkyl group that is substituted with one or more halides, e.g., fluorine, chlorine, bromine, or iodine. Alternatively, the term “monohaloalkyl” specifically refers to an alkyl group that is substituted with a single halide, e.g., fluorine, chlorine, bromine, or iodine. The term “polyhaloalkyl” specifically refers to an alkyl group that is independently substituted with two or more halides, i.e., each halide substituent need not be the same halide as another halide substituent, nor do the multiple instances of a halide substituent need to be on the same carbon. The term “perhaloalkyl” specifically refers to an alkyl group that has all its hydrogens substituted with halides, e.g., −CF3(trifluoromethyl). The term “alkoxyalkyl” specifically refers to an alkyl group that is substituted with one or more alkoxy groups, as described below. The term “aminoalkyl” specifically refers to an alkyl group that is substituted with one or more amino groups. The term “hydroxyalkyl” specifically refers to an alkyl group that is substituted with one or more hydroxy groups. When “alkyl” is used in oneinstance and a specific term such as “hydroxyalkyl” is used in another, it is not meant to imply that the term “alkyl” does not also refer to specific terms such as “hydroxyalkyl” and the like.

[0211] This practice is also used for other groups described herein. That is, while a term such as “cycloalkyl” refers to both unsubstituted and substituted cycloalkyl moieties, the substituted moieties can, in addition, be specifically identified herein; for example, a particular substituted cycloalkyl can be referred to as, e.g., an “alkylcycloalkyl.” Similarly, a substituted alkoxy can be specifically referred to as, e.g., a “halogenated alkoxy,” a particular substituted alkenyl can be, e.g., an “alkenylalcohol,” and the like. Again, the practice of using a general term, such as “cycloalkyl,” and a specific term, such as “alkylcycloalkyl,” is not meant to imply that the general term does not also include the specific term.

[0212] As used herein, the term “substituted” is contemplated to include all permissible substituents of organic compounds. In general, the term “substituted,” whether preceded by the term “optionally” or not, means that one or more hydrogens of the designated moiety are replaced with a suitable substituent. Unless otherwise indicated, an “optionally substituted” group may have a suitable substituent at each substitutable position of the group, and when more than one position in any given structure may be substituted with more than one substituent selected from a specified group, the substituent may be either the same or different at every position.

[0213] The term “cycloalkyl” as used herein is a non-aromatic carbon-based ring composed of at least three carbon atoms. Examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, norbornyl, and the like. The term “heterocycloalkyl” is a type of cycloalkyl group as defined above, and is included within the meaning of the term “cycloalkyl,” where at least one of the carbon atoms of the ring is replaced with a heteroatom such as, but not limited to, nitrogen, oxygen, sulfur, or phosphorus. The cycloalkyl group and heterocycloalkyl group can be substituted or unsubstituted. The cycloalkyl group and heterocycloalkyl group can be substituted with one or more groups including, but not limited to, alkyl, cycloalkyl, alkoxy, amino, ether, halide, hydroxy, nitro, silyl, sulfo-oxo, thiol, or boronic acid as described herein.

[0214] In defining various terms, “A1,” “A2,” “A3,” and “A4” are used herein as generic symbols to represent various specific substituents. These symbols can be any substituent, notlimited to those disclosed herein, and when they are defined to be certain substituents in one instance, they can, in another instance, be defined as some other substituents.

[0215] As used herein, the term C1-C6 alkoxy refers to an alkoxy group having from one to six carbon atoms. The terms “alkoxy” and “alkoxyl” as used herein to refer to an alkyl or cycloalkyl group bonded through an ether linkage; that is, an “alkoxy” group can be defined as – OA1where A1is alkyl or cycloalkyl as defined above. “Alkoxy” also includes polymers of alkoxy groups as just described; that is, an alkoxy can be a polyether such as –OA1-OA2or –OA1-(OA2)a-OA3, where “a” is an integer of from 1 to 200 and A1, A2, and A3are alkyl and / or cycloalkyl groups.

[0216] In embodiments of the compounds disclosed herein, the alkoxy group can be substituted or unsubstituted. The alkoxy group can be substituted with one or more groups including, but not limited to, alkyl, cycloalkyl, alkoxy, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, heteroaryl, aldehyde, –NH2, carboxylic acid, ester, ether, halide, hydroxy, ketone, azide, nitro, silyl, sulfo-oxo, or thiol as described herein. For example, in some embodiments of the compounds described herein, the alkoxy group can be substituted with halide or heteroaryl.

[0217] In embodiments of the compounds disclosed herein, the alkoxy group can be a haloalkoxy, perhaloalkoxy, or heteroarylalkoxy. The term “haloalkoxy” refers to an alkoxyl group that is independently substituted with one or more halides, i.e., each halide substituent need not be the same halide as another halide substituent, nor do the multiple instances of a halide substituent need to be on the same carbon. The term “perhaloalkoxy” refers to an alkoxy group that has all of its hydrogens substituted with halides. The term “heteroarylalkoxy” refers to an alkoxy group that is independently substituted with one or more heteroaryl groups.

[0218] The term “alkenyl” as used herein is a hydrocarbon group of from 2 to 24 carbon atoms with a structural formula containing at least one carbon-carbon double bond. The alkenyl group can be substituted with one or more groups including, but not limited to, alkyl, cycloalkyl, alkoxy, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxy, ketone, azide, nitro, silyl, sulfo-oxo, or thiol, as described herein.

[0219] The term “cycloalkenyl” as used herein is a non-aromatic carbon-based ring composed of at least three carbon atoms and containing at least one carbon-carbon double bound. Examples of cycloalkenyl groups include, but are not limited to, cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclopentadienyl, cyclohexenyl, cyclohexadienyl, norbornenyl, and the like. The term “heterocycloalkenyl” is a type of cycloalkenyl group as defined above, and is included within the meaning of the term “cycloalkenyl,” where at least one of the carbon atoms of the ring is replaced with a heteroatom such as, but not limited to, nitrogen, oxygen, sulfur, or phosphorus. The cycloalkenyl group and heterocycloalkenyl group can be substituted or unsubstituted. The cycloalkenyl group and heterocycloalkenyl group can be substituted with one or more groups including, but not limited to, alkyl, cycloalkyl, alkoxy, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxy, ketone, azide, nitro, silyl, sulfo-oxo, or thiol as described herein.

[0220] The term “alkynyl” as used herein is a hydrocarbon group of 2 to 24 carbon atoms with a structural formula containing at least one carbon-carbon triple bond. The alkynyl group can be unsubstituted or substituted with one or more groups including, but not limited to, alkyl, cycloalkyl, alkoxy, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxy, ketone, azide, nitro, silyl, sulfo-oxo, or thiol, as described herein. The term “cycloalkynyl” as used herein is a non-aromatic carbon- based ring composed of at least seven carbon atoms and containing at least one carbon-carbon triple bound. Examples of cycloalkynyl groups include, but are not limited to, cycloheptynyl, cyclooctynyl, cyclononynyl, and the like. The term “heterocycloalkynyl” is a type of cycloalkenyl group as defined above, and is included within the meaning of the term “cycloalkynyl,” where at least one of the carbon atoms of the ring is replaced with a heteroatom such as, but not limited to, nitrogen, oxygen, sulfur, or phosphorus. The cycloalkynyl group and heterocycloalkynyl group can be substituted or unsubstituted. The cycloalkynyl group and heterocycloalkynyl group can be substituted with one or more groups including, but not limited to, alkyl, cycloalkyl, alkoxy, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxy, ketone, azide, nitro, silyl, sulfo- oxo, or thiol as described herein.

[0221] The term “aromatic group” as used herein refers to a ring structure having cyclic clouds of delocalized TT electrons above and below the plane of the molecule, where the TT clouds contain (4n+2) TT electrons. Further description of aromaticity is disclosed in Morrison and Boyd, Organic Chemistry, (5th Ed., 1987), Chapter 13, entitled “Aromaticity,” pages 477-497, incorporated herein by reference. The term “aromatic group” is inclusive of both aryl and heteroaryl groups.

[0222] The term “aryl” or “aryl ring” as used herein is a group that contains any carbon- based aromatic group including, but not limited to, benzene, naphthalene, phenyl, biphenyl, anthracene, and the like. The aryl group can be substituted or unsubstituted. The aryl group can be substituted with one or more groups including, but not limited to, alkyl, cycloalkyl, alkoxy, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, heteroaryl, aldehyde, –NH2, carboxylic acid, ester, ether, halide, hydroxy, ketone, azide, nitro, silyl, sulfo-oxo, or thiol as described herein. The term “biaryl” is a specific type of aryl group and is included in the definition of “aryl.” As used herein, the term 5- or 6-membered substituted or unsubstituted aryl ring refers to any carbon-based aromatic group containing 5 or 6 carbons, and that can be substituted or unsubstituted.

[0223] As used herein, the term “heteroaryl” refers to an aromatic group that has at least one heteroatom incorporated within the ring of the aromatic group. Examples of heteroatoms include, but are not limited to, nitrogen, oxygen, sulfur, and phosphorus, where N-oxides, sulfur oxides, and dioxides are permissible heteroatom substitutions. The heteroaryl group can be substituted or unsubstituted. The heteroaryl group can be substituted with one or more groups including, but not limited to, alkyl, cycloalkyl, alkoxy, amino, ether, halide, hydroxy, nitro, silyl, sulfo-oxo, or thiol as described herein. Heteroaryl groups can be monocyclic, or alternatively fused ring systems.

[0224] Heteroaryl groups include, but are not limited to, furyl, imidazolyl, pyrimidinyl, tetrazolyl, thienyl, pyridinyl, pyrrolyl, N-methylpyrrolyl, quinolinyl, isoquinolinyl, pyrazolyl, triazolyl, thiazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiadiazolyl, isothiazolyl, pyridazinyl, pyrazinyl, benzofuranyl, benzodioxolyl, benzothiophenyl, indolyl, indazolyl, benzimidazolyl, imidazopyridinyl, pyrazolopyridinyl, and pyrazolopyrimidinyl. Further not limiting examples of heteroaryl groups include, but are not limited to, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl,thiophenyl, pyrazolyl, imidazolyl, benzo[d]oxazolyl, benzo[d]thiazolyl, quinolinyl, quinazolinyl, indazolyl, imidazo[1,2- b]pyridazinyl, imidazo[1,2-a]pyrazinyl, benzo[c][1,2,5]thiadiazolyl, benzo[c][1,2,5]oxadiazolyl, and pyrido[2,3-b]pyrazinyl. As used herein, the term 5- or 6- membered substituted or unsubstituted heteroaryl ring refers to any aromatic group containing 5 or 6 members, and that can be substituted or unsubstituted.

[0225] The term “aryloxy” as used herein, refers to an aryl group singly bonded to an oxygen. In addition, the aryl group can be a single ring structure or comprise multiple ring structures that are either fused ring structures or attached via one or more bridging groups such as a carbon-carbon bond. For example, in some embodiments of the compounds disclosed herein, the aryloxy can be substituted with 5- or 6-membered aryl or heteroaryl. For example, biaryl to two aryl groups that are bound together via a fused ring structure, as in naphthalene, or are attached via one or more carbon-carbon bonds, as in biphenyl. Benzyloxy is an example of an aryl single ring structure singly bonded to oxygen. In some embodiments of the compounds disclosed herein, the benzyloxy can be substituted with 5- or 6-membered aryl or heteroaryl. Fused aryl groups including, but not limited to, indene and naphthalene groups are also contemplated.

[0226] The term “aldehyde” as used herein is represented by the formula –C(O)H. Throughout this specification “C(O)” is a short hand notation for a carbonyl group, i.e., C=O. As an example, in some embodiments of the compounds disclosed herein, the term “aldehyde” is represented by the formula −C(O)− or −R12C(O)−, wherein R12is C1-C6 alkyl.

[0227] The terms “amine” or “amino” as used herein are represented by the formula – NA1A2, where A1and A2can be, independently, hydrogen or alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl group as described herein. A specific example of amino is –NH2.

[0228] The term “alkylamino” as used herein is represented by the formula –NH(-alkyl) and –N(-alkyl)2, where alkyl is a described herein. Representative examples include, but are not limited to, methylamino group, ethylamino group, propylamino group, isopropylamino group, butylamino group, isobutylamino group, (sec-butyl)amino group, (tert-butyl)amino group, pentylamino group, isopentylamino group, (tert-pentyl)amino group, hexylamino group, dimethylamino group, diethylamino group, dipropylamino group, diisopropylamino group,dibutylamino group, diisobutylamino group, di(sec-butyl)amino group, di(tert-butyl)amino group, dipentylamino group, diisopentylamino group, di(tert-pentyl)amino group, dihexylamino group, N-ethyl-N-methylamino group, N-methyl-N-propylamino group, N-ethyl-N-propylamino group and the like. As used herein, the term C1-C6alkylamino refers to an alkylamino group having from one to six carbon atoms. As an example, in some embodiments of the compounds disclosed herein, the term “amine” or “alkylamino” is represented by the formula NR112 or R12NR112, wherein R11is selected from hydrogen and substituted or unsubstituted C1-C6 alkyl, and R12is C1-C6alkyl.

[0229] As used herein, the terms “amide” and “carboxamide” refer to compounds represented by the formula –C(O)NA1A2, where A1and A2can independently be hydrogen, an alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl group as described herein. As an example, in some embodiments of the compounds disclosed herein, the term carboxamide is represented by the formula −C(O)NR11− or −R12C(O)NR11−, wherein R11is selected from hydrogen and substituted or unsubstituted C1-C6alkyl, and R12is C1-C6alkyl. The term carboxamide also is represented by the formula, wherein each R11is independently selected from hydrogen and substituted or unsubstituted C1-C6 alkyl, and each R12independently is C1-C6 alkyl.

[0230] The term “carboxylic acid” as used herein is represented by the formula –C(O)OH.

[0231] The term “ester” as used herein is represented by the formula –OC(O)A1or –C(O)OA1, where A1can be alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl group as described herein. The term “polyester” as used herein is represented by the formula2can be, independently, an alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl group described herein and “a” is an integer from 1 to 500. “Polyester” is as the term used to describe a group that is produced by the reaction between a compound having at least two carboxylic acid groups with a compound having at least two hydroxyl groups. As an example, in some embodiments of the compounds disclosed herein, the term “ester” is represented12, wherein each R independently is C1-C6 alkyl. In other embodiments, the term “ester” is represented by−C(O)OR11−, −R12C(O)OR11−, wherein R11is selected from hydrogen and substituted or unsubstituted C1-C6alkyl, and R12is C1-C6alkyl.

[0232] The term “ether” as used herein is represented by the formula A1OA2, where A1and A2can be, independently, an alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl group described herein. The term “polyether” as used herein is represented by the formula –(A1O-A2O)a–, where A1and A2can be, independently, an alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl group described herein and “a” is an integer of from 1 to 500. Examples of polyether groups include polyethylene oxide, polypropylene oxide, and polybutylene oxide.

[0233] The terms “halo,” “halogen” or “halide,” as used herein can be used interchangeably and refer to F, Cl, Br, or I.

[0234] The terms “pseudohalide,” “pseudohalogen” or “pseudohalo,” as used herein can be used interchangeably and refer to functional groups that behave substantially similar to halides. Such functional groups include, by way of example, cyano, thiocyanato, azido, trifluoromethyl, trifluoromethoxy, perfluoroalkyl, and perfluoroalkoxy groups.

[0235] The term “heteroalkyl” as used herein refers to an alkyl group containing at least one heteroatom. Suitable heteroatoms include, but are not limited to, O, N, Si, P and S, wherein the nitrogen, phosphorous and sulfur atoms are optionally oxidized, and the nitrogen heteroatom is optionally quaternized. Heteroalkyls can be substituted as defined above for alkyl groups.

[0236] The term “heteroaryl” as used herein refers to an aromatic group that has at least one heteroatom incorporated within the ring of the aromatic group. Examples of heteroatoms include, but are not limited to, nitrogen, oxygen, sulfur, and phosphorus, where N-oxides, sulfur oxides, and dioxides are permissible heteroatom substitutions. The heteroaryl group can be substituted or unsubstituted. The heteroaryl group can be substituted with one or more groups including, but not limited to, alkyl, cycloalkyl, alkoxy, amino, ether, halide, hydroxy, nitro, silyl, sulfo-oxo, or thiol as described herein. Heteroaryl groups can be monocyclic, or alternatively fused ring systems. Heteroaryl groups include, but are not limited to, furyl, imidazolyl, pyrimidinyl, tetrazolyl, thienyl, pyridinyl, pyrrolyl, N-methylpyrrolyl, quinolinyl, isoquinolinyl, pyrazolyl, triazolyl, thiazolyl, oxazolyl, oxadiazolonyl, isoxazolyl, oxadiazolyl, thiadiazolyl, isothiazolyl, pyridazinyl, pyrazinyl, benzofuranyl, benzodioxolyl, benzothiophenyl, indolyl, indazolyl,benzimidazolyl, imidazopyridinyl, pyrazolopyridinyl, and pyrazolopyrimidinyl. Further not limiting examples of heteroaryl groups include, but are not limited to, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, thiophenyl, pyrazolyl, imidazolyl, benzo[d]oxazolyl, benzo[d]thiazolyl, quinolinyl, quinazolinyl, indazolyl, 1,2,3-triazolyl, 1,2,4-triazolyl, imidazo[1,2- b]pyridazinyl, imidazo[1,2-a]pyrazinyl, benzo[c][1,2,5]thiadiazolyl, benzo[c][1,2,5]oxadiazolyl, and pyrido[2, 3-b ]pyrazinyl.

[0237] The terms “heterocycle” or “heterocyclyl,” as used herein can be used interchangeably and refer to single and multi-cyclic aromatic or non-aromatic ring systems in which at least one of the ring members is other than carbon. Thus, the term is inclusive of, but not limited to, “heterocycloalkyl,” “heteroaryl,” “bicyclic heterocycle,” and “polycyclic heterocycle.” Heterocycle includes pyridine, pyrimidine, furan, thiophene, pyrrole, isoxazole, isothiazole, pyrazole, oxazole, thiazole, imidazole, oxazole, including, 1,2,3-oxadiazole, 1,2,5- oxadiazole and 1,3,4-oxadiazole, thiadiazole, including, 1,2,3-thiadiazole, 1,2,5-thiadiazole, and 1,3,4-thiadiazole, triazole, including, 1,2,3-triazole, 1,3,4-triazole, tetrazole, including 1,2,3,4- tetrazole and 1,2,4,5-tetrazole, pyridazine, pyrazine, triazine, including 1,2,4-triazine and 1,3,5- triazine, tetrazine, including 1,2,4,5-tetrazine, pyrrolidine, piperidine, piperazine, morpholine, azetidine, tetrahydropyran, tetrahydrofuran, dioxane, and the like. The term heterocyclyl group can also be a C2heterocyclyl, C2-C3heterocyclyl, C2-C4heterocyclyl, C2-C5heterocyclyl, C2-C6heterocyclyl, C2-C7 heterocyclyl, C2-C8 heterocyclyl, C2-C9 heterocyclyl, C2-C10 heterocyclyl, C2-C11 heterocyclyl, and the like up to and including a C2-C18 heterocyclyl. For example, a C2 heterocyclyl comprises a group which has two carbon atoms and at least one heteroatom, including, but not limited to, aziridinyl, diazetidinyl, dihydrodiazetyl, oxiranyl, thiiranyl, and the like. Alternatively, for example, a C5 heterocyclyl comprises a group which has five carbon atoms and at least one heteroatom, including, but not limited to, piperidinyl, tetrahydropyranyl, tetrahydrothiopyranyl, diazepanyl, pyridinyl, and the like. It is understood that a heterocyclyl group may be bound either through a heteroatom in the ring, where chemically possible, or one of carbons comprising the heterocyclyl ring.

[0238] The term “bicyclic heterocycle” or “bicyclic heterocyclyl” as used herein refers to a ring system in which at least one of the ring members is other than carbon. Bicyclic heterocyclyl encompasses ring systems wherein an aromatic ring is fused with another aromatic ring, orwherein an aromatic ring is fused with a non-aromatic ring. Bicyclic heterocyclyl encompasses ring systems wherein a benzene ring is fused to a 5- or a 6-membered ring containing 1, 2 or 3 ring heteroatoms or wherein a pyridine ring is fused to a 5- or a 6-membered ring containing 1, 2 or 3 ring heteroatoms. Bicyclic heterocyclic groups include, but are not limited to, indolyl, indazolyl, pyrazolo[1,5-a]pyridinyl, benzofuranyl, quinolinyl, quinoxalinyl, 1,3-benzodioxolyl, 2,3-dihydro-1,4-benzodioxinyl, 3,4-dihydro-2H-chromenyl, 1H-pyrazolo[4,3-c]pyridin-3-yl; 1H- pyrrolo[3,2- b]pyridin-3-yl; and 1 H-pyrazolo[3,2-b]pyridin-3-yl.

[0239] The term “heterocycloalkyl” as used herein refers to an aliphatic, partially unsaturated or fully saturated, 3- to 14-membered ring system, including single rings of 3 to 8 atoms and bi- and tricyclic ring systems. The heterocycloalkyl ring-systems include one to four heteroatoms independently selected from oxygen, nitrogen, and sulfur, wherein a nitrogen and sulfur heteroatom optionally can be oxidized and a nitrogen heteroatom optionally can be substituted. Representative heterocycloalkyl groups include, but are not limited to, pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, tetrahydrofuryl, and −O-Bn- tetrazolyl.

[0240] The term “hydroxyl” or “hydroxy” as used herein is represented by the formula –OH. As an example, in some embodiments of the compounds disclosed herein, the term “hydroxyl” is represented by –R12OH, wherein R12is C1-C6 alkyl.

[0241] The term “ketone” as used herein is represented by the formula A1C(O)A2, where A1and A2can be, independently, an alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl group as described herein.

[0242] The term “azide” or “azido” as used herein is represented by the formula –N3.

[0243] The term “nitro” as used herein is represented by the formula –NO2. As an example, in some embodiments of the compounds disclosed herein, the term “nitro” is represented by −R12NO2, wherein R12is C1-C6 alkyl.

[0244] The term “nitrile” or “cyano” as used herein is represented by the formula –CN. As an example, in some embodiments of the compounds disclosed herein, the term “cyano” is represented by −R12CN, wherein R12is C1-C6 alkyl.

[0245] The term “silyl” as used herein is represented by the formula –SiA1A2A3, where A1, A2, and A3can be, independently, hydrogen or an alkyl, cycloalkyl, alkoxy, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl group as described herein.

[0246] The term “sulfo-oxo” as used herein is represented by the formulas –S(O)A1, – S(O)2A1, –OS(O)2A1, or –OS(O)2OA1, where A1can be hydrogen or an alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl group as described herein. Throughout this specification “S(O)” is a short hand notation for S=O. The term “sulfonyl” is used herein to refer to the sulfo-oxo group represented by the formula –S(O)2A1, where A1can be hydrogen or an alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl group as described herein. As an example, in some embodiments of the compounds disclosed herein, the term “sulfonyl” is represented by −S(O)2−. In other embodiments of the compounds disclosed herein, the sulfonyl is represented by a sulfonamide, such as −S(O)2NR11− or −R12S(O)2NR11−, wherein each R11is independently selected from hydrogen and substituted or unsubstituted C1-C6alkyl, and each R12independently is C1-C6alkyl. In other embodiments of the compounds disclosed herein, the sulfonamide is −S(O)2NR112or −R12S(O)2NR112, wherein each R11is independently selected from hydrogen and substituted or unsubstituted C1-C6 alkyl, and each R12independently is C1-C6 alkyl.

[0247] The term “sulfone” as used herein is represented by the formula A1S(O)2A2, where A1and A2can be, independently, an alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl group as described herein. The term “sulfoxide” as used herein is represented by the formula A1S(O)A2, where A1and A2can be, independently, an alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl group as described herein.

[0248] The term “thiol” as used herein is represented by the formula −SH.

[0249] The term “boronic ester” as used herein is represented by the formula −B(OR11)2or −R12B(OR11)2), The term “boronic acid” as used herein is represented by the formula −B(OH)2or −R12B(OH)2.

[0250] Compounds described herein can contain one or more double bonds and, thus, potentially give rise to cis / trans (E / Z) isomers, as well as other conformational isomers. Unless stated to the contrary, the invention includes all such possible isomers, as well as mixtures of such isomers.

[0251] Unless stated to the contrary, a formula with chemical bonds shown only as solid lines and not as wedges or dashed lines contemplates each possible isomer, e.g., each enantiomer and diastereomer, and a mixture of isomers, such as a racemic or scalemic mixture. Compounds described herein can contain one or more asymmetric centers and, thus, potentially give rise to diastereomers and optical isomers. Unless stated to the contrary, the present invention includes all such possible diastereomers as well as their racemic mixtures, their substantially pure resolved enantiomers, all possible geometric isomers, and pharmaceutically acceptable salts thereof. Mixtures of stereoisomers, as well as isolated specific stereoisomers, are also included. During the course of the synthetic procedures used to prepare such compounds, or in using racemization or epimerization procedures known to those skilled in the art, the products of such procedures can be a mixture of stereoisomers.

[0252] Many organic compounds exist in optically active forms having the ability to rotate the plane of plane-polarized light. In describing an optically active compound, the prefixes D and L or R and S are used to denote the absolute configuration of the molecule about its chiral center(s). The prefixes d and I or (+) and (-) are employed to designate the sign of rotation of plane-polarized light by the compound, with (-) or meaning that the compound is levorotatory. A compound prefixed with (+) or d is dextrorotatory. For a given chemical structure, these compounds, called stereoisomers, are identical except that they are non-superimposable mirror images of one another. A specific stereoisomer can also be referred to as an enantiomer, and a mixture of such isomers is often called an enantiomeric mixture. A 50:50 mixture of enantiomers is referred to as a racemic mixture. Many of the compounds described herein can have one or more chiral centers and therefore can exist in different enantiomeric forms. If desired, a chiral carbon can be designated with an asterisk (*). When bonds to the chiral carbon are depicted as straight lines in the disclosed formulas, it is understood that both the (R) and (S) configurations of the chiral carbon, and hence both enantiomers and mixtures thereof, are embraced within the formula. As is used in the art, when it is desired to specify the absolute configuration about a chiral carbon, one of the bonds to the chiral carbon can be depicted as a wedge (bonds to atoms above the plane) and the other can be depicted as a series or wedge of short parallel lines is (bonds to atoms below the plane). The Cahn-lngold-Prelog system can be used to assign the (R) or (S) configuration to a chiral carbon.

[0253] The compounds described in the invention can be present as a solvate. In some cases, the solvent used to prepare the solvate is an aqueous solution, and the solvate is then often referred to as a hydrate. The compounds can be present as a hydrate, which can be obtained, for example, by crystallization from a solvent or from aqueous solution. In this connection, one, two, three or any arbitrary number of solvent or water molecules can combine with the compounds according to the invention to form solvates and hydrates. Unless stated to the contrary, the invention includes all such possible solvates.

[0254] It is also appreciated that certain compounds described herein can be present as an equilibrium of tautomers. For example, ketones with an a-hydrogen can exist in an equilibrium of the keto form and the enol form. OH ~ H keto form enol form amide form imidic acid form Likewise, amides with an N-hydrogen can exist in an equilibrium of the amide form and the imidic acid form. Unless stated to the contrary, the invention includes all such possible tautomers.

[0255] It is known that chemical substances form solids which are present in different states of order which are termed polymorphic forms or modifications. The different modifications of a polymorphic substance can differ greatly in their physical properties. The compounds according to the invention can be present in different polymorphic forms, with it being possible for particular modifications to be metastable. Unless stated to the contrary, the invention includes all such possible polymorphic forms.

[0256] Certain materials, compounds, compositions, and components disclosed herein can be obtained commercially or readily synthesized using techniques generally known to those of skill in the art.

[0257] The invention provides a pharmaceutical composition comprising any of the described compounds (e.g., a compound of Formula (I) or a compound of Formula (II)), or a pharmaceutically acceptable salt thereof, and a pharmaceutical carrier. The compound of Formula (I) or compound of Formula (II) can be present in the pharmaceutical composition in any suitable amount. For example, the compound of Formula (I) or the compound of Formula (II) can present in the pharmaceutical composition in an amount of about 0.01 mg per milliliter (mL) or more (e.g., about 0.05 mg / mL or more, about 0.1 mg / mL or more, about 0.5 mg / mL or more, about 1 mg / mL or more, about 2 mg / mL or more, about 5 mg / mL or more, about 10mg / mL or more, about 20 mg / mL or more, or about 50 mg / mL or more. Typically, the pharmaceutical composition will comprise the compound of Formula (I) or compound of Formula (II) in an amount of about 100 mg / mL or less (e.g., about 75 mg / mL or less, about 50 mg / mL or less, about 40 mg / mL or less, about 30 mg / mL or less, about 20 mg / mL or less, about 15 mg / mL or less, about 10 mg / mL or less, or about 5 mg / mL or less). Any two of the foregoing endpoints can be used to define a close-ended range, or a single endpoint can be used to define an open-ended range. For example, the compound of Formula (I) or compound of Formula (II) can be present in the pharmaceutical composition in a concentration of from about 0.1 mg / mL to about 50 mg / mL, about 0.1 mg / mL to about 20 mg / mL, or about 1 mg / mL to about 20 mg / mL.

[0258] In some embodiments the pharmaceutical composition further comprises one or more pharmaceutically acceptable excipients. The pharmaceutically acceptable excipients described herein, for example, vehicles, adjuvants, carriers or diluents, are well-known to those who are skilled in the art and are readily available. Typically, the pharmaceutically acceptable carrier is one that is chemically inert to the active compounds and one that has no detrimental side effects or toxicity under the conditions of use.

[0259] The pharmaceutical compositions can be administered as oral, sublingual, transdermal, subcutaneous, topical, absorption through epithelial or mucocutaneous linings, intravenous, intranasal, intraarterial, intraperitoneal, intramuscular, intratumoral, peritumoral, intraperitoneal, intrathecal, rectal, vaginal, or aerosol formulations. In some aspects, the pharmaceutical composition is administered orally, intravenously, or intraperitoneally. In certain embodiments, the pharmaceutical composition is administered orally or intraperitoneally. In preferred embodiments, the pharmaceutical composition is administered intraperitoneally.

[0260] In accordance with any of the aspects, compounds of Formula (I), compounds of Formula (II), or pharmaceutically acceptable salts thereof, can be administered orally to a subject in need thereof. Formulations suitable for oral administration can consist of (a) liquid solutions, such as of the compound dissolved in diluents, such as water, saline, or orange juice and include an additive, such as cyclodextrin (e.g., D-, E-, or J-cyclodextrin, hydroxypropyl cyclodextrin) or polyethylene glycol (e.g., PEG400); (b) capsules, sachets, tablets, lozenges, and troches, each containing a predetermined amount of the active ingredient, as solids or granules; (c) powders; (d) suspensions in an appropriate liquid; and (e) suitable emulsions and gels. Liquid formulationsmay include diluents, such as water and alcohols, for example, ethanol, benzyl alcohol, and the polyethylene alcohols, either with or without the addition of a pharmaceutically acceptable surfactant, suspending agent, or emulsifying agent. Capsule forms can be of the ordinary hard- or soft-shelled gelatin type containing, for example, surfactants, lubricants, and inert fillers, such as lactose, sucrose, calcium phosphate, and cornstarch. Tablet forms can include one or more of lactose, sucrose, mannitol, corn starch, potato starch, alginic acid, microcrystalline cellulose, acacia, gelatin, guar gum, colloidal silicon dioxide, croscarmellose sodium, talc, magnesium stearate, calcium stearate, zinc stearate, stearic acid, and other excipients, colorants, diluents, buffering agents, disintegrating agents, moistening agents, preservatives, flavoring agents, and pharmacologically compatible carriers. Lozenge forms can comprise the active ingredient in a flavor, usually sucrose and acacia or tragacanth, as well as pastilles comprising the active ingredient in an inert base, such as gelatin and glycerin, or sucrose and acacia, emulsions, gels, and the like containing, in addition to the active ingredient, such carriers as are known in the art.

[0261] Formulations suitable for parenteral administration include aqueous and non-aqueous, isotonic sterile injection solutions, which can contain anti-oxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. The compounds of Formula (I), compounds of Formula (II), or pharmaceutically acceptable salts thereof, can be administered in a physiologically acceptable diluent in a pharmaceutical carrier, such as a sterile liquid or mixture of liquids, including water, saline, aqueous dextrose and related sugar solutions, an alcohol, such as ethanol, isopropanol, or hexadecyl alcohol, glycols, such as propylene glycol or polyethylene glycol, glycerol ketals, such as 2,2-dimethyl-1,3-dioxolane-4-methanol, ethers, such as poly(ethyleneglycol) 400, an oil, a fatty acid, a fatty acid ester or glyceride, or an acetylated fatty acid glyceride with or without the addition of a pharmaceutically acceptable surfactant, such as a soap or a detergent, suspending agent, such as pectin, carbomers, methylcellulose, hydroxypropyl methylcellulose, or carboxymethylcellulose, or emulsifying agents and other pharmaceutical adjuvants.

[0262] Oils, which can be used in parenteral formulations include petroleum, animal, vegetable, or synthetic oils. Specific examples of oils include peanut, soybean, sesame,cottonseed, corn, olive, petrolatum, and mineral. Suitable fatty acids for use in parenteral formulations include oleic acid, stearic acid, and isostearic acid. Ethyl oleate and isopropyl myristate are examples of suitable fatty acid esters. Suitable soaps for use in parenteral formulations include fatty alkali metal, ammonium, and triethanolamine salts, and suitable detergents include (a) cationic detergents such as, for example, dimethyl dialkyl ammonium halides, and alkyl pyridinium halides, (b) anionic detergents such as, for example, alkyl, aryl, and olefin sulfonates, alkyl, olefin, ether, and monoglyceride sulfates, and sulfosuccinates, (c) nonionic detergents such as, for example, fatty amine oxides, fatty acid alkanolamides, and polyoxyethylene-polypropylene copolymers, (d) amphoteric detergents such as, for example, alkyl-beta-aminopropionates, and 2-alkyl-imidazoline quaternary ammonium salts, and mixtures thereof.

[0263] The parenteral formulations will typically contain from about 0.5 to about 25% by weight of the compounds of Formula (I), compounds of Formula (II), and / or pharmaceutically acceptable salts thereof. Suitable preservatives and buffers can be used in such formulations. In order to minimize or eliminate irritation at the site of injection, such compositions may contain one or more nonionic surfactants having a hydrophile-lipophile balance (HLB) of from about 12 to about 17. The quantity of surfactant in such formulations ranges from about 5 to about 15% by weight. Suitable surfactants include polyethylene sorbitan fatty acid esters, such as sorbitan monooleate and the high molecular weight adducts of ethylene oxide with a hydrophobic base, formed by the condensation of propylene oxide with propylene glycol. The parenteral formulations can be presented in unit-dose or multi-dose sealed containers, such as ampoules and vials, and can be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example, water, for injections, immediately prior to use. Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules, and tablets of the kind previously described.

[0264] Compounds of Formula (I), compounds of Formula (II), and / or pharmaceutically acceptable salts thereof, can be made into injectable formulations. The requirements for effective pharmaceutical carriers for injectable compositions are well known to those of ordinary skill in the art (see Banker, Gilbert S., and Robert K. Chalmers, Pharmaceutics and Pharmacy Practice, Lippincott, Philadelphia, Pa, 1982, pp.238–250; and Trissel, Lawrence A., ASHP Handbook onInjectable Drugs, American Society of Hospital Pharmacists, Washington, D.C., 1986, pp.622– 630).

[0265] Topically applied compositions are generally in the form of liquids (e.g., mouthwash), creams, pastes, lotions and gels. Topical administration includes application to the oral mucosa, which includes the oral cavity, oral epithelium, palate, gingival, and the nasal mucosa. In some aspects, the composition contains at least one active component and a suitable vehicle or carrier. It may also contain other components, such as an anti-irritant. The carrier can be a liquid, solid or semi-solid. In aspects, the composition is an aqueous solution, such as a mouthwash. Alternatively, the composition can be a dispersion, emulsion, gel, lotion or cream vehicle for the various components. In one aspect, the primary vehicle is water or a biocompatible solvent that is substantially neutral or that has been rendered substantially neutral. The liquid vehicle can include other materials, such as buffers, alcohols, glycerin, and mineral oils with various emulsifiers or dispersing agents as known in the art to obtain the desired pH, consistency and viscosity. It is possible that the compositions can be produced as solids, such as powders or granules. The solids can be applied directly or dissolved in water or a biocompatible solvent prior to use to form a solution that is substantially neutral or that has been rendered substantially neutral and that can then be applied to the target site. In aspects of the invention, the vehicle for topical application to the skin can include water, buffered solutions, various alcohols, glycols such as glycerin, lipid materials such as fatty acids, mineral oils, phosphoglycerides, collagen, gelatin and silicone based materials.

[0266] The compounds of Formula (I), compounds of Formula (II), and / or pharmaceutically acceptable salts thereof, alone or in combination with other suitable components, can be made into aerosol formulations to be administered via inhalation. These aerosol formulations can be placed into pressurized acceptable propellants, such as dichlorodifluoromethane, propane, nitrogen, and the like. They also may be formulated as pharmaceuticals for non-pressured preparations, such as in a nebulizer or an atomizer.

[0267] The dose administered to the subject, particularly human and other mammals, in accordance with the present invention should be sufficient to affect the desired response. One skilled in the art will recognize that dosage will depend upon a variety of factors, including the age, condition or disease state, predisposition to disease, genetic defect or defects, and bodyweight of the mammal. The size of the dose will also be determined by the route, timing and frequency of administration as well as the existence, nature, and extent of any adverse side- effects that might accompany the administration of a particular inhibitor and the desired effect. It will be appreciated by one of skill in the art that various conditions or disease states may require prolonged treatment involving multiple administrations.

[0268] In some aspects, the inventive methods comprise administering an effective amount of a compound of Formula (I), a compound of Formula (II), and / or a pharmaceutically acceptable salt thereof. An “effective amount” means an amount sufficient to show a meaningful benefit in an individual, e.g., promoting at least one aspect of tumor cell cytotoxicity (e.g., inducing apoptosis, inhibition of growth, inhibiting survival of a cancer cell, reducing proliferation, reducing size and / or mass of a tumor (e.g., solid tumor)), or treatment, healing, prevention, delay of onset, halting, or amelioration of other relevant medical condition(s) associated with a particular cancer. The meaningful benefit observed in the subject can be to any suitable degree (10, 20, 30, 40, 50, 60, 70, 80, 90% or more) relative to the state of the subject / tissue / cell prior to the inventive method. In some aspects, one or more symptoms of the cancer are prevented, reduced, halted, or eliminated subsequent to administration of a compound of Formula (I), a compound of Formula (II), and / or a pharmaceutically acceptable salt thereof, thereby effectively treating the cancer to at least some degree.

[0269] Effective amounts may vary depending upon the biological effect desired in the individual, condition to be treated, and / or the specific characteristics of the compound of Formula (I) and / or the compound of Formula (II), and the individual. In this respect, any suitable dose of the compound of Formula (I) and / or the compound of Formula (II) can be administered to the subject (e.g., human), according to the type of cancer to be treated. Various general considerations taken into account in determining the “effective amount” are known to those of skill in the art and are described, e.g., in Gilman et al., eds., Goodman and Gilman’s: The Pharmacological Bases of Therapeutics, 8th ed., Pergamon Press, 1990; and Remington’s Pharmaceutical Sciences, 17th Ed., Mack Publishing Co., Easton, Pa., 1990, each of which is herein incorporated by reference.

[0270] The dose of the compound of Formula (I) and / or the compound of Formula (II) desirably comprises about 0.01 mg per kilogram (kg) of the body weight of the subject (mg / kg)or more (e.g., about 0.05 mg / kg or more, about 0.1 mg / kg or more, about 0.5 mg / kg or more, about 1 mg / kg or more, about 2 mg / kg or more, about 5 mg / kg or more, about 10 mg / kg or more, about 15 mg / kg or more, about 20 mg / kg or more, about 30 mg / kg or more, about 40 mg / kg or more, about 50 mg / kg or more, about 75 mg / kg or more, about 100 mg / kg or more, about 125 mg / kg or more, about 150 mg / kg or more, about 175 mg / kg or more, about 200 mg / kg or more, about 225 mg / kg or more, about 250 mg / kg or more, about 275 mg / kg or more, about 300 mg / kg or more, about 325 mg / kg or more, about 350 mg / kg or more, about 375 mg / kg or more, about 400 mg / kg or more, about 425 mg / kg or more, about 450 mg / kg or more, or about 475 mg / kg or more) per day. Typically, the dose will be about 500 mg / kg or less (e.g., about 475 mg / kg or less, about 450 mg / kg or less, about 425 mg / kg or less, about 400 mg / kg or less, about 375 mg / kg or less, about 350 mg / kg or less, about 325 mg / kg or less, about 300 mg / kg or less, about 275 mg / kg or less, about 250 mg / kg or less, about 225 mg / kg or less, about 200 mg / kg or less, about 175 mg / kg or less, about 150 mg / kg or less, about 125 mg / kg or less, about 100 mg / kg or less, about 75 mg / kg or less, about 50 mg / kg or less, about 40 mg / kg or less, about 30 mg / kg or less, about 20 mg / kg or less, about 15 mg / kg or less, about 10 mg / kg or less, about 5 mg / kg or less, about 2 mg / kg or less, about 1 mg / kg or less, about 0.5 mg / kg or less, or about 0.1 mg / kg or less). Any two of the foregoing endpoints can be used to define a close-ended range, or a single endpoint can be used to define an open-ended range. For example, the dose of the compound of Formula (I) and / or the compound of Formula (II) can be from about 0.01 mg / kg to about 500 mg / kg, about 1 mg / kg to about 200 mg / kg, or about 10 mg / kg to about 100 mg / kg.

[0271] In some embodiments, the pharmaceutical composition comprises (i) a compound of Formula (I), a compound of Formula (II), or a pharmaceutically acceptable salt thereof, (ii) a polymer selected from methylcellulose, carboxymethylcellulose, polyvinylpyrrolidone, and hydroxypropyl cellulose, and (iii) a pharmaceutical carrier (e.g., water). The polymer selected from methylcellulose, carboxymethylcellulose, polyvinylpyrrolidone, and hydroxypropyl cellulose can have any suitable weight average molecular weight and can be present in the pharmaceutical composition in any suitable amount. For example, the pharmaceutical composition can comprise about 0.01 wt.% to about 10 wt.%, about 0.01 wt.% to about 5 wt.%, about 0.01 to about 1 wt.%, about 0.1 wt.% to about 10 wt.%, about 0.1 wt.% to about 5 wt.%, or about 0.1 wt.% to about 1 wt.%. In some embodiments, the pharmaceutical compositioncomprises (i) a compound of Formula (I), a compound of Formula (II), or a pharmaceutically acceptable salt thereof, (ii) polyvinylpyrrolidone, and (iii) a pharmaceutical carrier (e.g., water).

[0100] In some embodiments, the pharmaceutical composition comprises (i) a compound of Formula (I), a compound of Formula (II), or a pharmaceutically acceptable salt thereof, (ii) a surfactant, and (iii) a pharmaceutical carrier (e.g., water). The surfactant can be any suitable surfactant, i.e., a nonionic surfactant, a cationic surfactant, or an anionic surfactant. In some embodiments the surfactant is a nonionic surfactant comprising a polyethylene glycol unit, a polypropylene glycol unit, or a combination thereof. propylene oxide, or ethylene oxide and propylene oxide. For example, the nonionic surfactant can be of the formula:wherein a, b, and c are integers ranging from about 2 to about 200, and a, b, and c can be the same or different. In a preferred embodiment, the nonionic surfactant is PLURONIC F-127, i.e., OH(C2H4O)101(C3H6O)56(C2H4O)101H, marketed by BASF Corporation (Florham Park, New Jersey). Alternatively, the nonionic surfactant can be of the formula:wherein m is an integer ranging from about 2 to about 50 and n is an integer ranging from about 2 to about 200. In a preferred embodiment, the nonionic surfactant is BRIJ® S20, i.e., a polyethylene glycol octadecyl ether of the formula C18H37(OC2H4)nOH, marketed by Croda International PLC (East Yorkshire, United Kingdom).). In yet another embodiment, the nonionic surfactant is of the formula:wherein w, x, y, and z are integers and w, x, y, and z can be the same or different. In a preferred embodiment, the nonionic surfactant is TWEEN 80, i.e., w+x+y+z=80, marketed by Croda International PLC (East Yorkshire, United Kingdom). The surfactant (e.g., nonionic surfactant) can have any suitable weight average molecular weight and can be present in the pharmaceutical composition in any suitable amount. For example, the pharmaceutical composition can comprise about 0.001 wt.% to about 5 wt.%, about 0.001 to about 1 wt.%, about 0.005 wt.% to about 5 wt.%, about 0.005 wt.% to about 1 wt.%, about 0.01 wt.% to about 5 wt.%, or about 0.01 wt.%to about 1 wt.%. In some embodiments, the pharmaceutical composition comprises (i) a compound of Formula (I), a compound of Formula (II), or a pharmaceutically acceptable salt thereof, (ii) a nonionic surfactant comprising a polyethylene glycol unit, a polypropylene glycol unit, or a combination thereof, and (iii) a pharmaceutical carrier (e.g., water).

[0272] In certain embodiments, the pharmaceutical composition comprises the pharmaceutical composition comprises (i) a compound of Formula (I), a compound of Formula (II), or a pharmaceutically acceptable salt thereof, (ii) polyvinylpyrrolidone, (iii) a nonionic surfactant comprising a polyethylene glycol unit, a polypropylene glycol unit, or a combination thereof, and (iv) a pharmaceutical carrier (e.g., water). In some embodiments, the foregoing pharmaceutical compositions are administered orally or intraperitoneally. In certain embodiments, the foregoing pharmaceutical compositions are administered intraperitoneally.

[0273] The invention provides a method for treating a disease in a subject, the method comprising administering to the subject any one of the described compounds, or a pharmaceutically acceptable salt thereof, or the described pharmaceutical compositions.

[0274] As used herein, the terms “treat,” “treatment,” and “treating” refer to any indicia of success in the treatment or amelioration of a condition (e.g., cancer), including any objective or subjective parameter such as abatement; diminishing of symptoms or making the symptom, injury, or condition more tolerable to the patient; reduction in the rate of symptom progression; decreasing the frequency or duration of the symptom or condition; or, in some situations, preventing the onset of the symptom. The treatment or amelioration of symptoms can be based on any objective or subjective parameter, including, for example, the result of a physical examination. In this respect, the described compounds, pharmaceutical compositions, and methods can provide any amount of any level of treatment. Furthermore, the treatment provided by the disclosed method can include the treatment of one or more conditions or symptoms of the disease or condition being treated.

[0275] As used herein, the terms “subject” refers to any subject for whom diagnosis, treatment, or therapy is desired (e.g., mammals, or humans). In some embodiments, the subject is a human.

[0276] As used herein, the term “administering” refers to parenteral, intravenous, intraperitoneal, intramuscular, intratumoral, intralesional, intranasal, or subcutaneousadministration, oral administration, administration as a suppository, topical contact, intrathecal administration, or the implantation of a slow release device, e.g., a mini osmotic pump, to the subject. In some embodiments the administration is intravenous.

[0277] In some embodiments, the disease is a cancer. In some embodiments, the cancer is multiple myeloma, lung cancer, breast cancer, ovarian cancer, neuroblastoma, lymphoma, mantle cell lymphoma, acute leukemia, cancers associated with human papillomavirus, colorectal cancer, gastric cancer, liver cancer, cervical cancer, pancreatic cancer, prostate cancer, or a combination thereof. In some embodiments the cancer is multiple myeloma. In some embodiments the subject is a human.

[0278] In some embodiments the method further comprises administering at least one other therapeutic agent. The at least one other therapeutic agent can be any suitable therapeutic agent. In some embodiments the at least one other therapeutic agent is selected from carfilzomib, bortezomib, ixazomib, disulfiram, marizomib, oprozomib, epoxomicin, MG132, KZR-616, KZR-504, PKS2279, PKS2252, lenalidomide, pomalidomide, daratumumab, elotuzumab, isatuximab, and combinations thereof.

[0279] In some embodiments, the method further comprises administering a DNA damaging agent. As used herein, a DNA damaging agent is an agent that interferes with DNA transcription and cell division by inducing damage to DNA. Any suitable DNA damaging agent may be administered as part of the claimed methods. In some embodiments, the DNA damaging agent is radiation or an alkylating agent. In an embodiment, the DNA damaging agent is radiation.

[0280] As used herein, “IC50,” is intended to refer to the concentration of a substance (e.g., a compound or a drug) that is required for 50% inhibition of a biological process, or component of a process. For example, IC50 refers to the half maximal (50%) inhibitory concentration (IC) of a substance as determined in a suitable assay. For example, an IC50for hRPN13 can be determined in an in vitro or cell-based assay system. Frequently, receptor assays make use of a suitable cell line, e.g. a cell line that either expresses endogenously a target of interest, or has been transfected with a suitable expression vector that directs expression of a recombinant form of the target. For example, IC50for a compound disclosed herein can be determined using mammalian cells transfected with hRPN13.

[0281] As used herein, “DC50,” is intended to refer to the concentration of a substance (e.g., a compound or a drug) that is required for 50% degradation of a protein. For example, DC50refers to the half maximal (50%) degradation concentration (DC) of a substance as determined in a suitable assay. For example, a DC50for hRPN13 can be determined in an in vitro or cell-based assay system. Frequently, receptor assays make use of a suitable cell line, e.g. a cell line that either expresses endogenously a target of interest, or has been transfected with a suitable expression vector that directs expression of a recombinant form of the target. For example, DC50 for a compound disclosed herein can be determined using mammalian cells transfected with hRPN13.

[0282] As used herein, “DepC50” refers to a half-maximal depletion concentration value, which is the concentration of a substance (e.g., a compound or a drug) that is required for 50% depletion.

[0283] As used herein, “d1 / 2” refers to differential protein abundance following treatment, which is measured as the amount of time for the protein abundance to reach half the original amount following treatment.

[0284] The invention also provides a method for modifying the activity of hRPN13, hRPN13Pru, or a variant thereof in a subject, the method comprising administering to the subject any one of the described compounds, or a pharmaceutically acceptable salt thereof, or the described pharmaceutical compositions. In some embodiments the subject is a human.

[0285] The invention also provides a method for modifying the activity of one of the binding partners for hRPN13, hRPN13Pru, or a variant thereof in a subject, the method comprising administering to the subject any one of the described compounds, or a pharmaceutically acceptable salt thereof, or the described pharmaceutical compositions. In some embodiments the subject is a human.

[0286] As used herein, the term “modifying the activity” could mean using any one of the compounds described herein to cause reduced protein levels of hRPN13, hRPN13Pruor a variant thereof by altering its interactions with other binding partners, such as the proteasome, proteasome subunit PSMD1 / hRpn2, UCHL5, HDAC8, or PADI4. “Modifying the activity” could also mean using any one of the compounds described herein to cause changes in theinteractions formed between hRPN13, hRPN13Pruor a variant thereof and its binding partners, such as the proteasome, proteasome subunit PSMD1 / hRpn2, UCHL5, HDAC8, or PADI4.

[0287] The invention also provides a method for providing a molecular glue, the method comprising administering to a subject any one of the described compounds, or a pharmaceutically acceptable salt thereof, or the described pharmaceutical compositions, wherein the molecular glue enhances interactions between hRPN13, hRPN13Pru, or a variant thereof, and one of the binding partners thereof in a subject. As used herein, the term “molecular glue” refers to a compound binding to hRPN13, hRPN13Pru, or a variant thereof in a manner that enhances its interactions with its binding partners or causes new interactions with other biomolecules. In some embodiments, the subject is a human.

[0288] The invention also provides a method for inducing degradation of KEN box proteins in a subject, the method comprising administering to the subject any one of the described compounds, or a pharmaceutically acceptable salt thereof, or the described pharmaceutical compositions. As used herein, the term “KEN box proteins” refers to proteins that possess the KEN box sequence motif targeted by the APC / C. In particular, KEN box proteins contain the degradation motif of K-E-N for the APC / CFZR1multi-subunit E3 ligase. In some embodiments the KEN box proteins are PCNA clamp associated factor (PCLAF), ribonucleoside-diphosphate reductase subunit M2 (RRM2), cell division cycle 25A (CDC25A), proline rich 11 (PRR11), or Pituitary tumor transforming gene 1 (PTTG1). In some embodiments, the subject is a human.

[0289] The invention also provides a method for inducing apoptosis of cells in a subject, the method comprising administering to the subject any one of the described compounds, or a pharmaceutically acceptable salt thereof, or the described pharmaceutical compositions. In some embodiments the subject is a human.

[0290] The invention also provides a method for disrupting cell cycle progression of cells in a subject, the method comprising administering to the subject any one of the described compounds, or a pharmaceutically acceptable salt thereof, or the described pharmaceutical compositions. In some embodiments the subject is a human.

[0291] The invention also provides a method for restricting cell viability by degrading KEN box proteins in a subject, the method comprising administering to the subject any one of thedescribed compounds, or a pharmaceutically acceptable salt thereof, or the described pharmaceutical compositions.

[0292] The invention also provides a method for restricting cell cycle progression by degrading KEN box proteins in a subject, the method comprising administering to the subject any one of the described compounds, or a pharmaceutically acceptable salt thereof, or the described pharmaceutical composition.

[0293] In some embodiments the KEN box proteins are PCLAF, RRM2, CDC25A, PRR11, or PTTG1. In some embodiments the subject is a human.

[0294] Aspects, including embodiments, of the invention described herein may be beneficial alone or in combination, with one or more other aspects or embodiments. Without limiting the foregoing description, certain non-limiting aspects of the disclosure numbered 1-60 are provided below. As will be apparent to those of skill in the art upon reading this disclosure, each of the individually numbered aspects may be used or combined with any of the preceding or following individually numbered aspects. This is intended to provide support for all such combinations of aspects and is not limited to combinations of aspects explicitly provided below:

[0295] (1) In aspect (1) is provided a compound of Formula (I):Formula (I) or a pharmaceutically acceptable salt thereof, wherein X1is N or CR1; X2is N or CR2; X3is N or CR3;X4is N or CR4; X5is N or CR5; each of R1, R2, R3, R4, and R5is independently selected from hydrogen, halogen, −OH, −CF3, C1-C6alkylamino, substituted or unsubstituted C1-C6alkyl, C1-C6alkoxy, perhaloalkyl, perhaloalkoxy, haloalkoxy, heteroarylalkoxy, −O-Bn-tetrazolyl, aryloxy substituted with 5- or 6- membered aryl or heteroaryl, and benzyloxy substituted with 5- or 6-membered aryl or heteroaryl; provided that no more than two of X1-X5are N, and at least one of X1-X5is not CH; W is selected from substituted or unsubstituted C1-C6 alkyl, C1-C6 alkoxy, −S(O)2−, −R12S(O)2−, −C(O)−, −R12C(O)−, −C(O)NR11−, −R12C(O)NR11−, −S(O)2NR11−, −R12S(O)2NR11−, −C(O)O−, −R12C(O)O−, −C(O)OR12−, −R12C(O)OR12−, pyrazolyl, 1,2,3-triazolyl, 1,2,4-triazolyl, tetrazolyl, imidazolyl, oxadiazolonyl and isoxazolyl; Y is substituted or unsubstituted aryl or heteroaryl ring having 5-6 members, or substituted or unsubstituted bicyclic ring or two-ring system, the bicyclic ring or two-ring system having 9-11 members; and Z is selected from Z1, Z2, Z3, Z4, and Z5,wherein X6is N or CR6; X7is N or CR7; X8is N or CR8; X9is N or CR9; X10is N or CR10; each of R6, R7, R8, R9, and R10is independently selected from hydrogen, halogen, cyano, perhaloalkyl, substituted or unsubstituted C1-C6 alkyl, perhaloalkoxy, 5- or 6-membered substituted or unsubstituted aryl ring, 5- or 6-membered substituted or unsubstituted heteroaryl rprovided that no more than two of X6-X10are N; wherein R9and R10, R8and R9, R7and R8, or R6and R7together with the atoms to which they are attached optionally form a 5- or 6-membered substituted or unsubstituted aryl ring, or a5- or 6-membered substituted or unsubstituted heteroaryl ring; each R11is independently selected from hydrogen and substituted or unsubstituted C1-C6alkyl; and each R12independently is C1-C6alkyl.

[0296] (2) In aspect (2) is provided the compound of aspect (1), wherein Y is selected from:

[0297] (3) In aspect (3) is provided the compound of aspect (1) or aspect (2), wherein the compound is of Formula (Ia):Formula (Ia) or a pharmaceutically acceptable salt thereof, wherein R3is selected from hydrogen, chlorine, methyl, methoxy and –O-Bn-tetrazolyl; R5is selected from hydrogen and halogen; and provided that at least one of R3and R5is not hydrogen.

[0298] (4) In aspect (4) is provided the compound of any one of aspects (1)-(3), wherein the compound is of Formula (Ib):Formula (Ib) or a pharmaceutically acceptable salt thereof, wherein R3is selected from hydrogen, chlorine, methyl, methoxy and –O-Bn-tetrazolyl; R5is selected from hydrogen and chlorine; provided that at least one of R3and R5is not hydrogen; R7is selected from hydrogen and cyano; R8is selected from hydrogen, fluorine, −OH, −C(O)NH2, −C(O)OH, −S(O)2NH2, and −B(OH)2; provided that at least one of R7and R8is not hydrogen; and each R11is independently selected from hydrogen and methyl.

[0299] (5) In aspect (5) is provided the compound of aspect (1), wherein the compound is selected from:or a pharmaceutically acceptable salt thereof.

[0300] (6) In aspect (6) is provided the compound of aspect (1), wherein the compound is:, or a pharmaceutically acceptable salt thereof.

[0301] (7) In aspect (7) is provided the compound of aspect (1) or aspect (2), wherein the compound is of Formula (Ic):Formula (Ic) or a pharmaceutically acceptable salt thereof, wherein R3is selected from hydrogen, chlorine, and methoxy; R5is selected from hydrogen and chlorine; and provided that at least one of R3and R5is not hydrogen.

[0302] (8) In aspect (8) is provided the compound of aspect (1), wherein the compound is selected from:, or a pharmaceutically acceptable salt thereof.

[0303] (9) In aspect (9) is provided the compound of aspect (1) or aspect (2), wherein the compound is of Formula (Id):Formula (Id) or a pharmaceutically acceptable salt thereof, wherein R8is selected from hydrogen, fluorine, methyl, and methoxy; and R9is selected from hydrogen, methyl, and methoxy;R10is selected from hydrogen, fluorine, methyl, and methoxy; provided that at least one of R8-R10is not hydrogen; and R11is selected from hydrogen and methyl.

[0304] (10) In aspect (10) is provided the compound of aspect (1), wherein the compound is selected from:, or a pharmaceutically acceptable salt thereof.

[0305] (11) In aspect (11) is provided the compound of aspect (1), wherein the compound is of Formula (Ie):Formula (Ie) or a pharmaceutically acceptable salt thereof.

[0306] (12) In aspect (12) is provided the compound of aspect (11), wherein the compound is of Formula (If):Formula (If) or a pharmaceutically acceptable salt thereof, whereinX2is N or CR2, and when X2is CR2, R2is hydrogen or fluorine; R3is selected from hydrogen, chlorine, methyl, methoxy, and –O-Bn-tetrazolyl; R4is selected from hydrogen and chlorine; R5is selected from hydrogen and chlorine; provided that at least one of R3-R5is not hydrogen; R7is selected from hydrogen, fluorine, and cyano; R8is selected from hydrogen, fluorine, and cyano; provided that at least one of R7and R8is not hydrogen; each R11is independently selected from hydrogen and methyl; and each R12independently is C1-C6 alkyl.

[0307] (13) In aspect (13) is provided the compound of any one of aspect (1), wherein the compound is selected from:, or a pharmaceutically acceptable salt thereof.

[0308] (14) In aspect (14) is provided the compound of aspect (1) or aspect (2), wherein the compound is of Formula (Ig):or a pharmaceutically acceptable salt thereof, wherein R3is selected from hydrogen and methoxy; R5is selected from hydrogen and chlorine; provided that at least one of R3and R5is not hydrogen; R7is selected from hydrogen, fluorine, and cyano; R8is selected from hydrogen, fluorine, and cyano; and provided that at least one of R7and R8is not hydrogen.

[0309] (15) In aspect (15) is provided the compound of aspect (14), wherein the compound is of Formula (Ih):or a pharmaceutically acceptable salt thereof, wherein W is selected from −C(O)NH−, −S(O)2NH−, pyrazolyl, and 1,2,3-triazolyl.

[0310] (16) In aspect (16) is provided the compound of aspect (1), wherein the compound is:or a pharmaceutically acceptable salt thereof.

[0311] (17) In aspect (17) is provided the compound of aspect (1), wherein the compound is :or a pharmaceutically acceptable salt thereof.

[0312] (18) In aspect (18) is provided the compound of aspect (1) or aspect (2), wherein the compound is of Formula (Ii):Formula (Ii) or a pharmaceutically acceptable salt thereof.

[0313] (19) In aspect (19) is provided the compound of aspect (1) or aspect (2), wherein the compound is of Formula (Ij):Formula (Ij) or a pharmaceutically acceptable salt thereof.

[0314] (20) In aspect (20) is provided the compound of aspect (1) or aspect (2), wherein the compound is of Formula (Ik):Formula (Ik) or a pharmaceutically acceptable salt thereof, wherein R3is selected from hydrogen, chlorine, and methoxy; R5is selected from hydrogen and chlorine; and provided that at least one of R3and R5is not hydrogen.

[0315] (21) In aspect (21) is provided the compound of aspect (20), wherein the compound is of Formula (Im):Formula (Im) or a pharmaceutically acceptable salt thereof, wherein R3is selected from hydrogen, chlorine, and methoxy; R5is selected from hydrogen and chlorine; and provided that at least one of R3and R5is not hydrogen.

[0316] (22) In aspect (22) is provided the compound of aspect (1), wherein the compound is selected from:, or a pharmaceutically acceptable salt thereof.

[0317] (23) In aspect (23) is provided the compound of aspect (1) or aspect (2), wherein the compound is of Formula (In):or a pharmaceutically acceptable salt thereof.

[0318] (24) In aspect (24) is provided the compound of aspect (23), wherein the compound is of Formula (Io):Formula (Io) or a pharmaceutically acceptable salt thereof.

[0319] (25) In aspect (25) is provided the compound of aspect (24), wherein the compound is of Formula (Ip):Formula (Ip) or a pharmaceutically acceptable salt thereof.

[0320] (26) In aspect (26) is provided the compound of any one of aspect (25), wherein the compound is of Formula (Iq):Formula (Iq) or a pharmaceutically acceptable salt thereof, wherein each of R8, R9, and R10is independently selected from hydrogen, fluorine, methyl, and methoxy; provided that at least one of R8-R10is not hydrogen; and R11is selected from hydrogen and methyl.

[0321] (27) In aspect (27) is provided the compound of aspect (1), wherein the compound is selected from:or a pharmaceutically acceptable salt thereof.

[0322] (28) In aspect (28) is provided the compound of aspect (1) or aspect (2), wherein the compound is of Formula (Ir):Formula (Ir) or a pharmaceutically acceptable salt thereof.

[0323] (29) In aspect (29) is provided the compound of aspect (1), wherein the compound is:, or a pharmaceutically acceptable salt thereof.

[0324] (30) In aspect (30) is provided the compound of aspect (1) or aspect (2), wherein the compound is of Formula (Is):or a pharmaceutically acceptable salt thereof.

[0325] (31) In aspect (31) is provided the compound of aspect (1), wherein the compound is:, or a pharmaceutically acceptable salt thereof.

[0326] (32) In aspect (32) is provided a compound of Formula (II):or a pharmaceutically acceptable salt thereof, wherein Y is an aryl or heteroaryl ring having 5-6 members, or substituted or unsubstituted bicyclic ring or two-ring system, the bicyclic ring or two-ring system having 9-11 members; X6is N or CR6; X7is N or CR7; X8is N or CR8; X9is N or CR9; each of R6, R7, R8, and R9is independently selected from hydrogen, halogen, cyano, perhaloalkyl, substituted or unsubstituted C1-C6 alkyl, perhaloalkoxy, 5- or 6-membered substituted or unsubstituted aryl ring, or 5- or 6-membered substituted or unsubstituted heteroaryl ring, −R12CN, −NO2, −R12NO2, −OH, −R12OH, −C(O)OR11, −R12C(O)OR11, −NR112, −R12NR112, −C(O)NR112, −R12C(O)NR112, −B(OR11)2, −R12B(OR11)2, −S(O)2NR112, and −R12S(O)2NR112; provided that no more than two of X6-X9are N, and at least one of X6-X9is not CH; wherein R8and R9, R7and R8, or R6and R7together with the atoms to which they are attached optionally form a 5- or 6-membered substituted or unsubstituted aryl ring, or a 5- or 6- membered substituted or unsubstituted heteroaryl ring; each R11is independently selected from hydrogen and substituted or unsubstituted C1-C6 alkyl; andeach R12independently is C1-C6alkyl.

[0327] (33) In aspect (33) is provided the compound of aspect (32), wherein the compound is of Formula (IIa):Formula (IIa) or a pharmaceutically acceptable salt thereof.

[0328] (34) In aspect (34) is provided the compound of aspect (32), wherein the compound is:XL78, or a pharmaceutically acceptable salt thereof.

[0329] (35) In aspect (35) is provided a pharmaceutical composition comprising a compound of any one of aspects (1)-(34), or a pharmaceutically acceptable salt thereof, and a pharmaceutical carrier.

[0330] (36) In aspect (36) is provided the pharmaceutical composition of aspect (35), further comprising one or more pharmaceutically acceptable excipients.

[0331] (37) In aspect (37) is provided the pharmaceutical composition of aspect (35) or aspect (36), wherein the pharmaceutical composition further comprises a polymer selected from methylcellulose, carboxymethylcellulose, polyvinylpyrrolidone, and hydroxypropyl cellulose.

[0332] (38) In aspect (38) is provided the pharmaceutical composition of any one of aspects (35)-(37), wherein the pharmaceutical composition comprises polyvinylpyrrolidone.

[0333] (39) In aspect (39) is provided the pharmaceutical composition of any one of aspects (35)-(38), wherein the pharmaceutical composition further comprises a surfactant.

[0334] (40) In aspect (40) is provided the pharmaceutical composition of aspect (39), wherein the surfactant is a nonionic surfactant comprising a polyethylene glycol unit, a polypropylene glycol unit, or a combination thereof.

[0335] (41) In aspect (41) is provided a method for treating a disease in a subject, the method comprising administering to the subject a compound of any one of aspects (1)-(34), or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition of any one of aspect (35)-(40).

[0336] (42) In aspect (42) is provided the method of aspect (41), wherein the disease is a cancer.

[0337] (43) In aspect (43) is provided the method of aspect (42), wherein the cancer is multiple myeloma, lung cancer, breast cancer, ovarian cancer, neuroblastoma, lymphoma, mantle cell lymphoma, acute leukemia, cancers associated with human papillomavirus, colorectal cancer, gastric cancer, liver cancer, cervical cancer, pancreatic cancer, prostate cancer, or a combination thereof.

[0338] (44) In aspect (44) is provided the method of aspect (43), wherein the cancer is multiple myeloma.

[0339] (45) In aspect (45) is provided the method of any one of aspects (41)-(44), further comprising administering at least one other therapeutic agent.

[0340] (46) In aspect (46) is provided the method of aspect (45), wherein the at least one other therapeutic agent is selected from carfilzomib, bortezomib, ixazomib, disulfiram, marizomib, oprozomib, epoxomicin, MG132, KZR-616, KZR-504, PKS2279, PKS2252, lenalidomide, pomalidomide, daratumumab, elotuzumab, isatuximab, and combinations thereof.

[0341] (47) In aspect (47) is provided the method of any one of aspects (41)-(44), further comprising administering a DNA damaging agent.

[0342] (48) In aspect (48) is provided the method of aspect (47), wherein the DNA damaging agent is radiation.

[0343] (49) In aspect (49) is provided the method of any one of aspects (41)-(48), wherein the administration is intravenous.

[0344] (50) In aspect (50) is provided a method for modifying the activity of hRPN13, hRPN13Pru, or a variant thereof in a subject, the method comprising administering a compound of any one of aspects (1)-(34), or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition of any one of aspect (35)-(40).

[0345] (51) In aspect (51) is provided a method for modifying the activity of one of the binding partners for hRPN13, hRPN13Pru, or a variant thereof in a subject, the method comprising a compound of any one of aspects (1)-(34), or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition of any one of aspect (35)-(40).

[0346] (52) In aspect (52) is provided a method for providing a molecular glue, the method comprising administering to a subject a compound of any one of aspects (1)-(34), or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition of any one of aspect (35)-(40), wherein the molecular glue enhances interactions between hRPN13, hRPN13Pru, or a variant thereof, and one of the binding partners thereof in a subject.

[0347] (53) In aspect (53) is provided a method for inducing degradation of KEN box proteins in a subject, the method comprising administering to the subject a compound of any one of aspects (1)-(34), or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition of any one of aspect (35)-(40).

[0348] (54) In aspect (54) is provided the method of aspect (53), wherein the KEN box proteins are PCNA clamp associated factor (PCLAF), ribonucleotide-diphosphate reductase subunit M2 (RRM2), cell division cycle 25A (CDC25A), proline rich 11 (PRR11), or Pituitary tumor transforming gene 1 (PTTG1).

[0349] (55) In aspect (55) is provided a method for inducing apoptosis of cells in a subject, the method comprising administering to the subject a compound of any one of aspects (1)-(34), or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition of any one of aspect (35)-(40).

[0350] (56) In aspect (56) is provided a method for disrupting cell cycle progression of cells in a subject, the method comprising administering to the subject a compound of any one of aspects 1-34, or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition of any one of aspect (35)-(40).

[0351] (57) In aspect (57) is provided a method for restricting cell viability by degrading KEN box proteins in a subject, the method comprising administering to the subject a compound of any one of aspects (1)-(34), or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition of any one of aspect (35)-(40).

[0352] (58) In aspect (58) is provided a method for restricting cell cycle progression by degrading KEN box proteins in a subject, the method comprising administering a compound of any one of aspects (1)-(34), or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition of any one of aspect (35)-(40).

[0353] (59) In aspect (59) is provided the method of aspect (57) or aspect (58), wherein the KEN box proteins are PCLAF, RRM2, CDC25A, PRR11, or PTTG1.

[0354] (60) In aspect (60) is provided the method of any one of aspects (41)-(59), wherein the subject is a human.

[0355] The following examples further illustrate the invention but, of course, should not be construed as in any way limiting its scope.EXAMPLES

[0356] The following examples demonstrate the unexpected discovery of small molecule compounds according to the claimed invention, e.g., XL44, that deplete hRpn13Pruwithout need of ligation to a known ligand for ubiquitination machinery.

[0357] The examples also demonstrate that PCLAF and RRM2 are depleted following treatment with a compound of the claimed invention, e.g., XL44, in a proteasome dependent pathway (FIG.7B, 7E), and that this pathway was found to require the PCLAF KEN box (FIG. 7F). XL44 also caused reduction of PTTG1, a downstream target of PCLAF (Liu et al., Cell Death Dis., 13, 178 (2022)). Without wishing to be bound by any particular theory, it is believed that, because PTTG1 also has a KEN box, compounds of the claimed invention, e.g., XL44, may target PTTG1 in a PCLAF-independent manner.

[0358] The following methods were carried out in connection with the examples. Covalent Docking Virtual Screen

[0359] Covalent docking screens were conducted based on the hRpn13 Pru:XL5 structure (FIG.1) with the Schrödinger software package by running on 8 CPUs of the National Institutes of Health Biowulf cluster supercomputer. The reactive residue and reaction type for covalent docking was set up with hRPN13 residue Cys88 and Michael addition for eighteen (18) compounds (Table 1). Docking was performed with Pose Prediction (Thorough). Table 1: Compounds and Corresponding Docking ScoresSample Preparation

[0360] The DNA encoding hRPN13 Pru (spanning amino acids 1-150), hRPN13 or hRPN2 (spanning amino acids 940-953) was cloned to pRSET vector with His or glutathione S- transferase (GST) tags at the N-terminus followed by a PreScissionTMprotease cleavage site.PreScissionTMprotease is a tradename that refers to a fusion protein of GST and human rhinovirus type 143C protease. Plasmid expressing human PCLAF was generated commercially (GenScript) by inserting synthesized coding sequence with codon optimization for full length PCLAF between the BamHI and Not I restriction sites of pGEX-6p-1 with a GST tag at the N- terminus followed by a PreScissionTMprotease cleavage site. hRPN13 Pru, hRpn13 and hRpn2 (940-953) or PCLAF were expressed in E. coli BL21(DE3) pLysS or BL21 (DE3) cells (Invitrogen). Cells were grown at 37°C to optical density at 600 nm of 0.6 and induced for protein expression by addition of isopropyl-β-D-thiogalactoside (0.4 mM) for 20 hours at 17°C or 4 hours at 37°C. The cells were harvested by centrifugation at 4,550 g for 40 min, lysed by sonication, and cellular debris removed by centrifugation at 31,000 g for 30 min. The supernatant for hRpn13 Pru (1-150), hRpn13, hRpn2 (940-953) or PCLAF was incubated with Talon Metal AffinityTMresin (Clontech) for one hour or Glutathione S-sepharose 4BTM(GE Healthcare Life Sciences) for 3 hours and the resin washed extensively with buffer A (20 mM sodium phosphate, 300 mM NaCl, 10 mM βME, pH 6.5). hRpn13 Pru, hRpn13 or PCLAF was eluted from the resin by overnight incubation with 50 units per mL of PreScissionTMprotease (GE Healthcare Life Sciences) in buffer B (20 mM sodium phosphate, 50 mM NaCl, 2 mM DTT, pH 6.5) whereas GST-hRpn2 (940-953) was eluted in buffer B containing 20 mM reduced L-glutathione. The eluent of GST-hRpn2 (940-953) was concentrated to 2 mL, buffer exchanged to buffer B, and then incubated with 50 units per mL of PreScissionTMprotease overnight to cleave the GST tag. Following affinity chromatography, hRpn13 Pru, hRpn2 (940-953), PCLAF or hRpn13 was subjected to size exclusion chromatography with a Superdex75 or Superdex200 column on an FPLC system for further purification.15N ammonium chloride and 13C glucose were used for isotopic labeling. Acquisition of Compounds

[0361] General procedure for the synthesis of compounds according to the invention: In a thick-walled vial, indolin-2-one (1 equivalence) and aldehyde (1 equivalence) were dissolved in EtOH, and then piperidine (0.1 equivalence) was added. The vial was sealed and heated at 70oC for 4 to 6 hours (the reaction was monitored by LC-MS). The reaction precipitate was collected and washed with cold EtOH and dried under vacuum to afford the pure product. Whenthe precipitate was not observed during the reaction, the solvent was removed and the product purified by combi-flash silica-gel column chromatography.

[0362] Reaction for XL44:

[0363] Synthesis of XL44: In a thick-walled vial, 5-fluoroindolin-2-one (207 mg, 1.4 mmol) and N-(3-formylphenyl)-4-methoxybenzamide (250 mg, 1.4 mmol) were dissolved in EtOH (10 mL) and then piperidine (14 μL, 0.14 mmol) was added at room temperature. The vial was sealed and heated at 70oC for 6 hours (monitored by LC-MS). The yellow solid product was collected, washed with cold EtOH (3 x 10 mL) and dried under vacuum to afford yellow solid XL44 (290 mg, 53%, single isomer).1H NMR (400 MHz, dmso) δ 10.59 (s, 1H), 10.22 (s, 1H), 8.49 (t, J = 1.9 Hz, 1H), 8.26 (dt, J = 8.0, 1.2 Hz, 1H), 8.02 – 7.92 (m, 2H), 7.86 (s, 1H), 7.80 – 7.66 (m, 2H), 7.43 (t, J = 8.0 Hz, 1H), 7.10 – 6.98 (m, 3H), 6.78 (dd, J = 8.4, 4.4 Hz, 1H), 3.83 (s, 3H).13C NMR (101 MHz, dmso) δ 167.42, 165.39, 162.42, 158.42 (d), 139.51, 138.96, 137.50, 134.46, 130.10, 128.78, 127.48, 127.22, 127.14 (d), 126.80 (d), 125.02, 123.69, 115.65 (d), 114.08, 110.51 (d), 107.97 (d), 55.90.19F NMR (376 MHz, dmso) δ -122.51 (td). LC-MS [M+H]+: 389.00 (m / z).

[0364] Reaction for XL45:

[0365] Synthesis of XL45: XL45 was synthesized using the same reaction procedure of XL44.2-Oxindole (27 mg, 0.2 mmol), N-(3-formylphenyl)-4-methoxybenzamide (50 mg, 0.2 mmol), and piperidine (2 μL, 0.02 mmol) were used to obtain the yellow precipitate of XL45 (48 mg, 67%, single isomer).1H NMR (400 MHz, dmso) δ 10.60 (s, 1H), 10.24 (s, 1H), 8.17 (t, J =1.9 Hz, 1H), 8.01 – 7.92 (m, 2H), 7.84 – 7.79 (m, 1H), 7.71 – 7.67 (m, 1H), 7.59 (s, 1H), 7.48 (t, J = 7.9 Hz, 1H), 7.38 (ddt, J = 7.6, 1.7, 0.9 Hz, 1H), 7.22 (td, J = 7.7, 1.2 Hz, 1H), 7.10 – 7.02 (m, 2H), 6.92 – 6.81 (m, 2H), 3.83 (s, 3H).13C NMR (101 MHz, dmso) δ 169.10, 165.59, 162.47, 143.42, 140.12, 136.09, 135.24, 130.64, 130.14, 129.59, 128.16, 127.20, 125.14, 123.52, 121.98, 121.69, 121.26, 120.95, 114.09, 110.48, 55.90. LC-MS [M+H]+: 371.00 (m / z).

[0366] Reaction for XL46:

[0367] Synthesis of XL46: XL46 was synthesized using the same reaction procedure of XL44.1-Methyl-2-indolinone (29 mg, 0.2 mmol), N-(3-formylphenyl)-4-methoxybenzamide (50 mg, 0.2 mmol), and piperidine (2 μL, 0.02 mmol) were used and the product was purified by combi-flash silica-gel column chromatography (hexanes / EtOAc = 1:1) to obtain the XL46 (36 mg, 48%, single isomer).1H NMR (400 MHz, cdcl3) δ 8.29 (s, 1H), 7.96 – 7.82 (m, 3H), 7.82 – 7.76 (m, 2H), 7.67 (ddt, J = 7.6, 1.1, 0.5 Hz, 1H), 7.44 (t, J = 7.9 Hz, 1H), 7.36 (ddt, J = 7.7, 1.8, 1.0 Hz, 1H), 7.21 (td, J = 7.7, 1.2 Hz, 1H), 7.00 – 6.91 (m, 2H), 6.88 (td, J = 7.6, 1.1 Hz, 1H), 6.79 – 6.69 (m, 1H), 3.85 (s, 3H), 3.20 (s, 3H).13C NMR (101 MHz, cdcl3) δ 168.50, 165.46, 162.57, 144.11, 138.77, 136.72, 135.64, 129.82, 129.37, 129.15, 127.58, 126.90, 125.01, 123.11, 121.96, 121.40, 120.95, 120.66, 113.92, 108.13, 55.45, 26.14. LC-MS [M+H]+: 385.00 (m / z).

[0368] Reaction scheme for XL66:

[0369] Synthesis of XL66: 5-fluoroindolin-2-one (300 mg, 2 mmol), 4- bromopicolinaldehyde (1.14 g, 6 mmol) and sodium carbonate (233 mg, 2.1 mmol) in DMF (6 mL) were stirred at room temperature for 1 hour. Water (30 mL) was added and the precipitate was collected to provide the 3-((4-bromopyridin-2-yl)methylene)-5-fluoroindolin-2-one (325 mg, 51%, LC-MS [M+H]+: 319.00 (m / z)). To the solution of 3-((4-bromopyridin-2-yl)methylene)-5-fluoroindolin-2-one (100 mg, 0.3 mmol) in DMF (6 mL), Xantphos (29 mg, 0.05 mmol), cesium carbonate (194 mg, 0.57 mmol), palladium(II) acetate (8 mg, 0.03 mmol) and 4- methylbenzamide (68 mg, 0.5 mmol) were added under argon atmosphere. The reaction mixture was degassed several times by refilling with argon. The mixture was heated at 120oC for 1 hour and at 100oC for another 1 hour. To the cooled reaction mixture water (15 mL) was added and extracted with EtOAc. The organic phase was washed with saturated aqueous sodium chloride, dried over anhydrous Na2SO4 and the filtrate was concentrated. The crude material was purified by ISCO combi flash column chromatography (EtOAC / MeOH = 9:1) and combined only the pure fractions to provide XL66 (10 mg, 9%).1H NMR (400 MHz, dmso) δ 10.66 (d, J = 6.7 Hz, 2H), 8.94 (dd, J = 10.2, 2.8 Hz, 1H), 8.80 (d, J = 5.5 Hz, 1H), 8.18 (d, J = 2.0 Hz, 1H), 7.94 – 7.84 (m, 3H), 7.47 (s, 1H), 7.37 (d, J = 8.0 Hz, 2H), 7.13 (td, J = 8.9, 2.8 Hz, 1H), 6.84 (dd, J = 8.5, 4.6 Hz, 1H), 2.39 (s, 3H).13C NMR (101 MHz, dmso) δ 169.68, 166.88, 158.94, 156.62, 153.97, 151.05, 147.60, 142.97, 140.39 (d), 135.68, 131.61, 129.55, 128.43, 122.98 (d), 119.32, 117.50 (d), 115.62 (d), 114.71, 110.64 (d), 21.53.19F NMR (376 MHz, dmso) δ -121.82 (td). LC- MS [M+H]+: 374.20 (m / z).

[0370] Reaction scheme for XL67:

[0371] Synthesis of XL67: XL67 was synthesized using the same reaction procedure of XL66.4-methoxybenzamide was used instead of 4-methylbenzamide to afford XL67 (12 mg, 10%, single isomer).1H NMR (400 MHz, dmso) δ 10.65 (s, 1H), 10.60 (s, 1H), 8.94 (dd, J = 10.3, 2.8 Hz, 1H), 8.79 (d, J = 5.5 Hz, 1H), 8.17 (d, J = 2.0 Hz, 1H), 8.02 – 7.95 (m, 2H), 7.87 (dd, J = 5.6, 2.0 Hz, 1H), 7.47 (s, 1H), 7.18 – 7.05 (m, 3H), 6.84 (dd, J = 8.5, 4.6 Hz, 1H), 3.84 (s, 3H).13C NMR (101 MHz, DMSO) δ 169.71, 166.40, 162.97, 153.96, 151.05, 147.74, 140.42, 135.76, 130.48, 129.56, 129.53, 126.47, 123.06, 119.33, 117.65, 115.52, 114.70, 114.31, 110.71, 56.02. LC-MS [M+H]+: 390.20 (m / z).

[0372] Reaction scheme for XL68:

[0373] Synthesis of XL68: 3-(3-azidobenzylidene)-5-fluoroindolin-2-one was synthesized using the same reaction procedure as XL44. Starting materials 5-fluoroindolin-2-one (113 mg, 0.75 mmol), 3-azidobenzaldehyde (100 mg, 0.68 mmol), and piperidine (7 μL, 0.07 mmol) were used to obtain yellow precipitate of 3-(3-azidobenzylidene)-5-fluoroindolin-2-one (130 mg,281.20 (m / z).

[0374] Aforementioned 3-(3-azidobenzylidene)-5-fluoroindolin-2-one (50 mg, 0.18 mmol) and 1-ethynyl-4-methoxybenzene (26 mg, 0.2 mmol) were dissolved in DMSO (2 mL) and then CuI (3 mg, 0.02 mmol) was added and stirred for overnight. Aqueous NH4Cl (8 mL) was added and stirred for 1 hour. The solid precipitate was collected and washed with aqueous NH4Cl (2 x 10 mL) followed by water (20 mL), and then dried to afford XL68 (55 mg, 74%, single isomer).1H NMR (400 MHz, DMSO) δ 10.72 (s, 1H), 9.27 (s, 1H), 8.28 (d, J = 2.2 Hz, 1H), 8.08 (dt, J = 7.2, 2.0 Hz, 1H), 7.92 – 7.84 (m, 2H), 7.82 (dd, J = 10.8, 3.3 Hz, 3H), 7.24 (dd, J = 9.3, 2.6 Hz, 1H), 7.19 – 7.04 (m, 3H), 6.90 (dd, J = 8.6, 4.6 Hz, 1H), 3.82 (s, 3H).13C NMR (101 MHz, DMSO) δ 168.82, 159.84, 156.50, 147.88, 140.08, 137.48, 136.29, 131.10, 129.48, 129.19, 127.20, 123.13, 122.04, 121.95, 121.43, 120.76, 119.12, 117.53, 117.30, 114.95, 111.48, 110.33, 55.69.19F NMR (376 MHz, DMSO) δ -121.90 (td). LC-MS [M+H]+: 413.00 (m / z).

[0375] Reaction for XL69:

[0376] Synthesis of XL69: To a stirred solution of bis(triphenylphosphine)palladium(II) chloride (796.6 mg, 1.1 mmol) in DMF (45 mL), 1-ethynyl-4-methoxybenzene (3.0 g, 22.7 mmol), 3-bromobenzaldehyde (2.9 mL, 24.9 mmol), copper(I) iodide (432 mg, 2.3 mmol) and triethylamine (9.5 mL, 68.1 mmol) were added under argon. The reaction mixture was heated at 90oC under argon for 6 hours. Water was added to the cooled reaction mixture and extracted with EtOAc (200 mL). The organic layer was washed with aqueous sodium chloride (2 x 100 mL), dried, concentrated and purified by combiflash silica gel column chromatography (Hexanes / EtOAc = 4:1) to afford 3-((4-methoxyphenyl)ethynyl)benzaldehyde (3.8 g, 71 %).1H NMR (400 MHz, cdcl3) δ 9.99 (s, 1H), 7.98 (s, 1H), 7.80 (dd, J = 7.7, 1.5 Hz, 1H), 7.72 (dt, J = 7.7, 1.5 Hz, 1H), 7.47 (d, J = 8.9 Hz, 3H), 6.88 (d, J = 8.8 Hz, 2H), 3.81 (s, 3H).13C NMR (101 MHz, cdcl3) δ 191.63, 159.97, 136.91, 136.46, 133.19, 132.78, 129.05, 128.54, 124.86, 114.70, 114.11, 91.07, 86.69, 55.30.

[0377] To the solution of 3-((4-methoxyphenyl)ethynyl)benzaldehyde (1.6 g, 6.8 mmol) in DMF (60 mL), sodium azide (20.0 g, 307 mmol) was added. The reaction was heated at 170oC for 60 hours. The cooled reaction mixture filtered through pad of celite with the aid of EtOAc. The collected organic layer was washed with aqueous ammonia chloride (50 mL) and saturated aqueous sodium chloride (2 x 50 mL). The collected organic phase was dried over anhydrous Na2SO4, concentrated and purified by combi flash silica gel column chromatography (Hexanes / EtOAc = 3:7) to afford 3-(5-(4-methoxyphenyl)-1H-1,2,3-triazol-4-yl)benzaldehyde(1.24 g, 65 %).1H NMR (400 MHz, dmso) δ 10.00 (s, 1H), 8.06 (s, 1H), 7.89 (s, 1H), 7.77 (d, J = 7.7 Hz, 1H), 7.62 (t, J= 7.6 Hz, 1H), 7.51 – 7.31 (m, 2H), 6.98 (s, 2H), 3.77 (s, 3H).13C NMR (101 MHz, dmso) δ 193.41, 159.99, 137.04, 133.89, 133.47, 132.81, 131.70, 130.09, 129.98, 129.80, 128.81, 115.32, 114.80, 55.65. LC-MS [M+H]+: 280.20 (m / z).

[0378] In thick walled flask, piperidine (44 μL, 0.4 mmol) was added to the dichloromethane (20 mL) solution of 3-(5-(4-methoxyphenyl)-1H-1,2,3-triazol-4-yl)benzaldehyde (1.23 g, 4.4 mmol) and 5-fluoroindolin-2-one (666 mg, 4.4 mmol). The flask was sealed and heated at 70 °C for 16 hours. The yellow precipitate was collected and washed with dichloromethane (2 x 20 mL) and dried to afford XL69 (1.35 g, 74 %, single isomer).1H NMR (400 MHz, DMSO) δ 15.30 (s, 1H), 10.66 (s, 1H), 7.89 (s, 1H), 7.74 – 7.66 (m, 2H), 7.60 – 7.55 (m, 2H), 7.47 – 7.40 (m, 2H), 7.19 (dd, J = 9.4, 2.6 Hz, 1H), 7.14 – 7.06 (m, 1H), 6.97 (d, J = 8.7 Hz, 2H), 6.87 (dd, J = 8.5, 4.6 Hz, 1H), 3.76 (s, 3H).13C NMR (101 MHz, DMSO) δ 168.94, 159.94, 158.78, 156.44, 139.86, 137.32, 135.07, 129.83, 129.32, 128.42, 128.39, 122.20, 122.12, 117.19, 116.96, 114.71, 111.40, 111.32, 110.16, 109.90, 55.62.19F NMR (376 MHz, DMSO) δ -121.91. LC-MS [M+H]+: 413.20 (m / z). [

[0380] Synthesis of XL70: Pyridine (10 μL, 0.12 mmol) was added to a stirred solution of 3- (3-aminobenzylidene)-5-fluoroindolin-2-one (15 mg, 0.06 mmol) and 4-methoxybenzenesulfonyl chloride (15 mg, 0.07 mmol) in dichloromethane (5 mL) and the mixture was stirred for 6 hours. Water (5) was added, organic layer was collected and then the aqueous layer was extracted with dichloromethane (10 mL). The combined organic layers were washed with aqueous sodium chloride (15 mL), dried, and concentrated. The concentrated material was crystalized in hot EtOH to afford XL70 (16 mg, 64%, 5:1 mixture of E / Z isomers). Mixture of isomers1H NMR (400 MHz, CDCl3) δ 8.81 (s, 1H), 8.27 (s, 1H), 7.72 (d, J = 8.8 Hz, 2H), 7.62 (s, 1H), 7.32 – 7.04 (m, 5H), 6.88 – 6.67 (m, 4H), 3.70 (s, 3H). Major isomer19F NMR (376 MHz, CDCl3) δ -120.52 (td). Minor isomer19F NMR (376 MHz, CDCl3) δ -121.10 (td, J = 8.6, 4.2 Hz). LC-MS [M+H]+: 425.00 (m / z).

[0381] Reaction for XL71:

[0382] Synthesis of XL71: In a thick-walled vial, 1-(azidomethyl)-4-methoxybenzene (200 mg, 1.23 mmol) was dissolved in DMSO (2 mL), and then 3,3-diethoxyprop-1-yne (189 mg, 1.47 mmol) and copper iodide (23 mg, 0.123 mmol) were added under argon atmosphere. The vial was sealed and stirred for overnight. Aqueous ammonium chloride (10 mL) was added and extracted with EtOAc (20 mL). The organic layer was washed with aqueous sodium chloride (10 mL) and dried over anhydrous Na2SO4. The concentrated crude product was dissolved in 6 mL of dioxane / aq HCl (1:1) and heated the mixture at 50oC for 6 hours. The cooled mixture was quenched with cold aqueous NaHCO3(10 mL) and extracted with EtOAc (2*15 mL). The combined organic layers were washed with aqueous sodium chloride (10 mL), dried over anhydrous Na2SO4and concentrated to provide 1-(4-methoxybenzyl)-1H-1,2,3-triazole-4- carbaldehyde (265 mg, 1.22 mmol, 99 %), which was used directly for the next step without further purification.

[0383] In a thick-walled vial, 5-fluoroindolin-2-one (60 mg, 0.40 mmol) and 1-(4- methoxybenzyl)-1H-1,2,3-triazole-4-carbaldehyde (86 mg, 0.40 mmol) were dissolved in Ethanol (4 mL) and then piperidine (39 μL, 0.40 mmol) was added under argon atmosphere at room temperature. The vial was sealed and heated at 50oC for 2 hours (formation of yellow precipitate was observed around 30 min). The reaction vial was cooled to room temperature and the yellow precipitate was collected, washed with cold ethanol (10 mL) and dried to afford XL71 (134 mg, 96 %, single isomer).1H NMR (400 MHz, DMSO) δ 10.69 (s, 1H), 9.38 (d, J = 0.5 Hz, 1H), 8.00 (s, 1H), 7.78 (dd, J = 8.9, 2.6 Hz, 1H), 7.40 – 7.32 (m, 2H), 7.05 (ddd, J = 9.6, 8.5, 2.6 Hz, 1H), 7.00 – 6.94 (m, 2H), 6.83 (dd, J = 8.5, 4.4 Hz, 1H), 5.67 (s, 2H), 3.75 (s, 3H).13C NMR (101 MHz, DMSO) δ 167.79, 159.71, 157.29, 142.38, 137.48, 130.26, 128.88, 128.08, 126.24, 125.73 (d), 125.40 (d), 115.80 (d), 114.70, 110.74 (d), 108.29 (d), 55.62, 52.99.19F NMR (376 MHz, DMSO) δ -122.15. LC-MS [M+H]+: 351.00 (m / z).

[0384] Reaction for XL72:

[0385] Synthesis of XL72: XL72 was synthesized using the similar procedure as XL71. Click reaction of 3,3-diethoxyprop-1-yne and 2-azido-1-(4-methoxyphenyl)ethan-1-one followed by acetal deprotection and then the condensation with 5-fluoroindolin-2-one afforded XL72 (16 mg, 18%, single isomer).1H NMR (400 MHz, dmso) δ 10.59 (s, 1H), 8.94 (dd, J = 10.0, 2.8 Hz, 1H), 8.70 (s, 1H), 8.07 (d, J = 8.9 Hz, 2H), 7.65 (s, 1H), 7.13 (d, J = 9.0 Hz, 2H), 6.90 – 6.78 (m, 1H), 6.28 (s, 2H), 3.87 (s, 3H).19F NMR (376 MHz, dmso) δ -122.17. LC-MS [M+H]+: 379.00 (m / z).

[0386] Reaction for XL73:

[0387] Synthesis of XL73: In a thick-walled vial, 5-(2-fluorophenyl)-1-(pyridin-3- ylsulfonyl)-1H-pyrrole-3-carbaldehyde (50 mg, 0.15 mmol) and 5-fluoroindolin-2-one (24 mg, 0.16 mmol) were dissolved in Ethanol (5 mL)and then piperidine ( 15 μL, 0.15 mmol) was added under argon atmosphere at room temperature. The vial was sealed and heated at 60oC for 5 hours. The reaction vial was cooled to room temperature and the yellow precipitate was collected, washed with cold ethanol (10 mL), and dried to provide 5-fluoro-3-((5-(2- fluorophenyl)-1-(pyridin-3-ylsulfonyl)-1H-pyrrol-3-yl)methylene)indolin-2-one XL73 (56 mg, 80 %, single isomer).1H NMR (400 MHz, DMSO) δ 10.59 (s, 1H), 8.92 (dd, J = 4.8, 1.5 Hz, 1H), 8.66 (d, J = 2.4 Hz, 1H), 8.33 (dd, J = 1.8, 0.9 Hz, 1H), 8.01 (ddd, J = 8.2, 2.6, 1.6 Hz, 1H), 7.66 (dd, J = 8.2, 4.8 Hz, 1H), 7.61 – 7.52 (m, 2H), 7.41 (dd, J = 9.3, 2.6 Hz, 1H), 7.32 – 7.21 (m, 3H), 7.09 (td, J = 9.0, 2.6 Hz, 1H), 6.91 (d, J = 1.8 Hz, 1H), 6.86 (dd, J = 8.5, 4.6 Hz, 1H).13C NMR (101 MHz, DMSO) δ 169.27, 160.76 (d), 157.78 (d), 155.82, 147.66, 139.38, 135.55, 134.32, 133.54, 132.52 (d), 129.63, 129.14, 128.44, 126.69 (d), 125.18, 124.44 (d), 122.47,122.38, 118.35 (d), 117.96, 116.48 (d), 115.80 (d), 111.08 (d), 110.53 (d).19F NMR (376 MHz, DMSO) δ -111.20 (m), -121.93 (td). LC-MS [M+H]+: 464.40 (m / z).

[0388] Reaction for XL74:

[0389] Synthesis of XL74: In a thick-walled vial, 1-tosyl-1H-indole-3-carbaldehyde (50 mg, 0.17 mmol) and 5-fluoroindolin-2-one (27 mg, 0.18 mmol) were dissolved in Ethanol (5 mL) and then piperidine (16 μL, 0.17 mmol) was added under argon atmosphere at room temperature. The vial was sealed and heated at 60oC for 5 hours. The reaction vial was cooled to room temperature and the orange precipitate was collected, washed with cold ethanol (10 mL), and dried to provide 5-fluoro-3-((1-tosyl-1H-indol-3-yl)methylene)indolin-2-one XL74 (55 mg, 76 %, single isomer).1H NMR (400 MHz, DMSO) δ 10.75 (s, 1H), 9.76 (s, 1H), 8.34 – 8.25 (m, 1H), 8.12 (s, 1H), 8.05 – 7.88 (m, 4H), 7.51 – 7.40 (m, 4H), 7.06 (ddd, J = 9.6, 8.5, 2.6 Hz, 1H), 6.87 (dd, J = 8.5, 4.4 Hz, 1H), 2.32 (s, 3H).13C NMR (101 MHz, DMSO) δ 168.17, 158.48 (d), 146.62, 134.13, 133.97, 132.02, 131.05, 130.73, 127.31, 126.39 (d), 126.14 (d), 126.06, 125.66, 124.51, 120.49, 116.19, 115.48 (d), 113.66, 110.68 (d), 108.10 (d), 21.54.19F NMR (376 MHz, DMSO) δ -122.64 (td). LC-MS [M+H]+: 433.40 (m / z).

[0390] Reaction for XL75:

[0391] Synthesis of XL75: In a thick-walled vial, 1-(4-chlorobenzyl)-1H-indole-3- carbaldehyde (50 mg, 0.19 mmol) and 5-fluoroindolin-2-one (29 mg, 0.19 mmol) were dissolved in ethanol (5 mL) and then piperidine (18 μL, 1 Eq, 0.19 mmol) was added under argon atmosphere at room temperature. The vial was sealed and heated at 60oC for 4 hours. The reaction vial was cooled to room temperature and the yellow precipitate was collected, washed with cold ethanol (10 mL), and dried to afford 3-((1-(4-chlorobenzyl)-1H-indol-3-yl)methylene)-5-fluoroindolin-2-one XL75 (32 mg, 43 %, single isomer).1H NMR (400 MHz, dmso) δ 10.50 (s, 1H), 9.53 (s, 1H), 8.30 – 8.24 (m, 1H), 8.20 (s, 1H), 7.85 (dd, J = 9.4, 2.6 Hz, 1H), 7.61 – 7.53 (m, 1H), 7.44 – 7.35 (m, 2H), 7.31 – 7.21 (m, 4H), 6.93 (ddd, J = 9.6, 8.3, 2.6 Hz, 1H), 6.79 (dd, J = 8.4, 4.5 Hz, 1H), 5.60 (s, 2H).13C NMR (101 MHz, dmso) δ 168.52, 158.40 (d), 137.07, 136.62, 136.32, 135.91, 132.77, 129.53, 129.46, 129.20, 128.54, 127.55 (d), 123.41, 121.81, 119.76 (d), 119.46, 113.39 (d), 111.54 (d), 109.99 (d), 106.83, 106.57, 49.59.19F NMR (376 MHz, dmso) δ -123.23 (td). LC-MS [M+H]+: 403.40 (m / z).

[0392] Reaction for XL76:

[0393] Synthesis of XL76: In a thick-walled vial, 1-(3-chloro-5-fluorobenzyl)-1H-indole-3- carbaldehyde (50 mg, 0.17 mmol) and 5-fluoroindolin-2-one (28 mg, 0.18 mmol) were dissolved in ethanol (5 mL)and then piperidine (15 mg, 17 μL, 1 Eq, 0.17 mmol) was added under argon atmosphere at room temperature. The vial was sealed and heated at 60oC for 4 hours. The reaction vial was cooled to room temperature and the yellow precipitate was collected, washed with cold ethanol (10 mL), and dried to provide 3-((1-(3-chloro-5-fluorobenzyl)-1H-indol-3- yl)methylene)-5-fluoroindolin-2-one XL76 (34 mg, 46 %, single isomer).1H NMR (400 MHz, DMSO) δ 10.49 (s, 1H), 9.49 (s, 1H), 8.36 – 8.25 (m, 1H), 8.22 (s, 1H), 7.87 (dd, J = 9.4, 2.6 Hz, 1H), 7.66 – 7.53 (m, 2H), 7.35 – 7.13 (m, 4H), 6.96 (ddd, J = 9.6, 8.4, 2.6 Hz, 1H), 6.81 (dd, J = 8.4, 4.5 Hz, 1H), 5.65 (s, 2H).13C NMR (101 MHz, DMSO) δ 168.53, 162.02 (d), 158.42 (d), 137.09, 136.47, 135.94, 133.92 (d), 131.64 (d), 131.04 (d), 129.35, 128.49, 127.54 (d), 123.53, 121.92, 119.91 (d), 119.53, 117.61 (d), 115.37 (d), 113.45 (d), 111.59, 111.42, 110.03 (d), 106.75 (d), 47.67.19F NMR (376 MHz, DMSO) δ -111.83 (td), -123.19 (td). LC-MS [M+H]+: 421.40 (m / z).

[0394] Reaction for XL77:

[0395] Synthesis of XL77: In a thick-walled vial, 1-(2-(2-methoxyphenoxy)ethyl)-1H- indole-3-carbaldehyde (50 mg, 0.17 mmol) and 5-fluoroindolin-2-one (27 mg, 0.18 mmol) were dissolved in ethanol (5 mL) and then piperidine (17 μL, 0.17 mmol) was added under argon atmosphere at room temperature. The vial was sealed and heated at 70oC for 4 hours. The reaction vial was cooled to room temperature and the yellow precipitate was collected, washed with cold ethanol (10 mL), and dried to afford 5-fluoro-3-((1-(2-(2-methoxyphenoxy)ethyl)-1H- indol-3-yl)methylene)indolin-2-one XL77 (33 mg, 45 %, single isomer).1H NMR (400 MHz, DMSO) δ 10.53 (s, 1H), 9.58 (s, 1H), 8.30 – 8.22 (m, 1H), 8.21 (s, 1H), 7.86 (dd, J = 9.5, 2.6 Hz, 1H), 7.80 – 7.70 (m, 1H), 7.31 (tt, J = 7.2, 5.6 Hz, 2H), 7.00 – 6.77 (m, 6H), 4.73 (t, J = 5.0 Hz, 2H), 4.35 (t, J = 5.0 Hz, 2H), 3.65 (s, 3H).13C NMR (101 MHz, DMSO) δ 168.55, 158.42 (d), 149.81, 148.11, 137.87, 136.62, 135.86, 129.38, 128.74, 127.69 (d), 123.11, 122.22, 121.68, 121.22, 119.31 (d), 119.20, 114.83, 113.36 (d), 113.19, 111.81, 111.18, 109.93 (d), 106.61 (d), 68.30, 56.06, 46.92.19F NMR (376 MHz, DMSO) δ -123.25. LC-MS [M+H]+: 429.40 (m / z).

[0396] Reaction for XL78:

[0397] Synthesis of XL78: In In a thick-walled vial, 5-fluoro-3-methyl-1H-indole-2- carbaldehyde (50 mg, 0.28 mmol) and 5-fluoroindolin-2-one (45 mg, 0.30 mmol) were dissolved in Ethanol (5 mL)and then piperidine (28 μL, 0.28 mmol) was added under argon atmosphere at room temperature. The vial was sealed and heated at 60oC for 2 hours. The reaction vial was cooled to room temperature and the orange precipitate was collected, washed with cold ethanol (10 mL), and dried to provide 5-fluoro-3-((5-fluoro-3-methyl-1H-indol-2-yl)methylene)indolin- 2-one XL78 (73 mg, 83 %, single isomer).1H NMR (400 MHz, DMSO) δ 13.08 (s, 1H), 11.06 (s, 1H), 7.96 (s, 1H), 7.90 (dd, J = 9.3, 2.6 Hz, 1H), 7.57 (dd, J = 8.9, 4.5 Hz, 1H), 7.40 (dd, J = 9.6, 2.5 Hz, 1H), 7.15 (td, J = 9.2, 2.6 Hz, 1H), 7.03 (ddd, J = 9.6, 8.4, 2.6 Hz, 1H), 6.88 (dd, J = 8.5, 4.5 Hz, 1H), 2.58 (s, 3H).13C NMR (101 MHz, DMSO) δ 169.67, 159.26 (d, J = 139.0 Hz), 156.93 (d, J = 137.9 Hz), 136.42, 134.14, 133.03, 128.35 (d, J = 9.9 Hz), 126.82 (d, J = 9.4 Hz), 125.41, 122.55 – 120.77 (m), 115.17, 114.92 (d, J = 4.3 Hz), 114.70, 113.94 (d, J = 9.5 Hz), 110.90 (d, J = 8.5 Hz), 107.83 (d, J = 25.7 Hz), 104.67 (d, J = 22.9 Hz), 9.55.19F NMR (376 MHz, DMSO) δ -122.08, -123.43. LC-MS [M+H]+: 311.40 (m / z).

[0398] Reaction for XL79:

[0399] Synthesis of XL79: 5-((5-fluoro-2-oxoindolin-3-ylidene)methyl)-2,4-dimethyl-1H- pyrrole-3-carboxylic acid (100 mg, 0.333 mmol), 4-methoxyaniline (49 mg, 0.4 mmol) were dissolved in DMF (3 mL) and then HATU (133 mg, 0.35 mmol) and DIPEA (100 μL, 0.574 mmol) were added at room temperature. The reaction mixture stirred for 20 hours and water (20 mL) was added. The solid precipitate was collected and washed with water (20 mL). The collected solid was further recrystallized in EtOH to provide the yellow solid of 5-((5-fluoro-2- oxoindolin-3-ylidene)methyl)-N-(4-methoxyphenyl)-2,4-dimethyl-1H-pyrrole-3-carboxamide XL79 (116 mg, 86 %, 3:1 mixture of E / Z isomers). Mixture of E / Z isomers1H NMR (400 MHz, DMSO) δ 13.76 (s, 1H), 10.92 (s, 1H), 9.60 (s, 1H), 8.06 – 7.45 (m, 4H), 7.16 – 6.61 (m, 4H), 3.74 (s, 3H), 2.48 (s, 3H), 2.47 (s, 3H). Mixture of E / Z isomers13C NMR (101 MHz, DMSO) δ 170.08, 163.67, 160.74, 159.89, 157.56, 155.71, 153.01, 143.98, 140.75, 136.94, 135.05, 134.95,133.47, 133.06, 130.81, 130.26, 127.74, 127.64, 127.55, 126.38, 125.34, 124.84, 122.12, 121.88, 121.60, 115.42, 115.39, 114.24, 113.08, 112.84, 110.57, 110.48, 107.48, 106.64, 106.38, 56.50, 55.67, 15.41, 13.67, 12.09, 10.92. Major isomer19F NMR (376 MHz, DMSO) δ -122.49. Minor isomer19F NMR (376 MHz, DMSO) δ -121.99. LC-MS [M+H]+: 406.40 (m / z).

[0400] Reaction for XL80:

[0401] Synthesis of XL80: To the DMF solution of 3-(4-methoxyphenyl)-1H-pyrazole (4.5 g, 25.83 mmol), NBS (4.6 g, 25.83 mmol) was added at room temperature. The reaction mixture stirred for about 4 hours (monitored by LCMS) and water was added. Extracted with EtOAc and the organic layer was washed with brine, dried over anhydrous Na2SO4 and concentrated. The crude material was purified by ISCO combi flash silica gel column chromatography (Hexanes / EtOAc = 6 : 4) to afford 4-bromo-3-(4-methoxyphenyl)-1H-pyrazole (6.4 g, 25 mmol, 98 %).1H NMR (400 MHz, cdcl3) δ 11.70 (s, 1H), 7.70 – 7.62 (m, 2H), 7.53 (s, 1H), 6.97 – 6.89 (m, 2H), 3.85 (s, 3H).13C NMR (101 MHz, cdcl3) δ 160.00, 143.80, 137.06, 128.94, 121.91, 114.07, 91.71, 55.29. LC-MS [M-H]+: 251.20 (m / z).

[0402] 4-bromo-3-(4-methoxyphenyl)-1H-pyrazole (1000 mg, 3.951 mmol) was dissolved in THF (4 mL) and water (0.4 mL). (3-formylphenyl)boronic acid (622.0 mg, 4.149 mmol) and potassium carbonate (1.092 g, 7.902 mmol) were added. The mixture was degassed, PdCl2(dppf) (29 mg, 0.04 mmol) was added, and the reaction mixture was heated in a microwave synthesizer at 160 °C for 10 min. The solvents were removed in vacuo to afford a solid residue, which was adsorbed onto silica gel and then purified by combi flash silica gel column chromatography EtOAc / hexane (1:1) to provide 3-(3-(4-methoxyphenyl)-1H-pyrazol-4-yl)benzaldehyde (405 mg, 37 %).1H NMR (400 MHz, cdcl3) δ 9.96 (s, 1H), 7.87 – 7.81 (m, 1H), 7.80 – 7.72 (m, 2H), 7.56 – 7.51 (m, 1H), 7.45 (t, J = 7.6 Hz, 1H), 7.38 – 7.31 (m, 2H), 7.26 (s, 0H), 6.92 – 6.83 (m, 2H), 3.82 (s, 3H). LC-MS [M+H]+: 279.40 (m / z).

[0403] In a thick-walled vial, 3-(4-(4-methoxyphenyl)-1H-pyrazol-3-yl)benzaldehyde (392 mg, 1.41 mmol) was dissolved in EtOH (10 mL), and then 5-fluoroindolin-2-one (213 mg, 1.41 mmol) and piperidine (139 μL, 1.41 mmol) were added at room temperature. The vial was sealed and heated at 70oC for 4 hours by monitoring the reaction by LCMS. After cooling the reaction vial, solvent was removed and then the crude mixture was purified by Combi flash silica-gel column chromatography (MeOH:CH2Cl2 = 1:9) to provide the 5-fluoro-3-(3-(4-(4- methoxyphenyl)-1H-pyrazol-3-yl)benzylidene)indolin-2-one XL80 (225 mg, 39 %, 3:1 mixture of E / Z isomers). Mixture of isomer1H NMR (400 MHz, DMSO) δ 13.10 (s, 1H), 10.62 (s, 1H), 8.65 – 6.68 (m, 13H), 3.75 (s, 3H). Major isomer19F NMR (376 MHz, DMSO) δ -122.05. Minor isomer19F NMR (376 MHz, DMSO) δ -122.53. LC-MS [M+H]+: 412.20 (m / z).

[0404] Reaction for XL92:

[0405] Synthesis of XL92: In a thick-walled vial, 5-hydroxyindolin-2-one (50 mg, 0.34 mmol) and N-(3-formylphenyl)-4-methoxybenzamide (86 mg, 0.34 mmol) were dissolved in EtOH (2 mL), and then and piperidine (33 μL, 0.34 mmol) was added at room temperature under argon atmosphere. The vial was sealed and heated at 75oC for 4 hours (monitoring by LCMS). After cooling the reaction vial to room temperature, solvent was removed and re- dissolved in 20 mL CH2Cl2 / MeOH (19.5:0.5), which was stored in freezer for 1 hour to precipitate the red colored product. The precipitate was collected, washed with cold CH2Cl2 and dried to afford 23 mg of product. The collected solution was then stored in freezer for overnight and the precipitate was collected, washed with CH2Cl2(10 mL) and dried to afford the pure product (55 mg). XL92 (88 mg, 68 %, 10:1 mixture of E / Z isomers).(400 MHz, dmso) δ 10.27 (s, 1H), 10.23 (s, 1H), 8.95 (s, 1H), 8.05 (t, J = 1.9 Hz, 1H), 8.00 – 7.89 (m, 3H), 7.52 (s, 1H), 7.48 (t, J = 7.9 Hz, 1H), 7.34 (ddd, J = 8.5, 1.8, 0.9 Hz, 1H), 7.08 – 7.02 (m, 3H), 6.76 – 6.55 (m, 2H), 3.82 (s, 3H).13C NMR (101 MHz, dmso) δ 169.08, 165.56, 162.45, 152.33, 140.28, 135.77, 135.69, 135.30, 130.13, 129.53, 129.08, 127.29, 124.50, 121.95, 121.53, 120.75, 117.20, 114.09, 110.87, 110.85, 55.90. LC-MS [M+H]+: 387.20 (m / z).

[0406] Reaction for XL93:Synthesis of XL93: In a thick-walled vial, 2-oxoindoline-5-carboxylic acid (50 mg, 0.28 mmol), and N-(3-formylphenyl)-4-methoxybenzamide (72 mg, 0.28 mmol) were dissolved in EtOH (2 mL), and then piperidine (28 μL, 0.28 mmol) was added at room temperature under argon atmosphere. The vial was sealed and heated at 75oC for about 3 hours (monitoring by LCMS). The reaction mixture cooled to room temperature and 1M HCl was added to adjust the pH = 2. The yellow precipitate was formed during the addition of 1M HCl. The solid was collected, washed with EtOH (8 mL) followed by water (8 mL), and then dried to afford 3-(3-(4- methoxybenzamido)benzylidene)-2-oxoindoline-5-carboxylic acid XL93 (106 mg, 91 %, single isomer).1H NMR (400 MHz, dmso) δ 12.55 (s, 1H), 10.99 (s, 1H), 10.25 (s, 1H), 8.23 (d, J = 1.6 Hz, 1H), 8.17 (d, J = 1.9 Hz, 1H), 7.97 – 7.88 (m, 3H), 7.85 (dd, J = 8.2, 1.6 Hz, 1H), 7.68 (s, 1H), 7.50 (t, J = 7.9 Hz, 1H), 7.41 (ddt, J = 7.6, 1.8, 0.9 Hz, 1H), 7.09 – 7.01 (m, 2H), 6.95 (d, J = 8.2 Hz, 1H), 3.82 (s, 3H).13C NMR (101 MHz, dmso) δ 169.32, 167.39, 165.61, 162.44, 147.11, 140.39, 137.52, 134.90, 132.50, 130.11, 129.59, 127.46, 127.31, 124.64, 124.40, 124.08, 121.89, 121.15, 120.85, 114.08, 110.22, 55.91. LC-MS [M+H]+: 415.20 (m / z).

[0407] Reaction for XL94:

[0408] Synthesis of XL94: In a thick-walled vial, 2-oxoindoline-5-sulfonamide (30 mg, 0.14 mmol) and N-(3-formylphenyl)-4-methoxybenzamide (36 mg, 0.14 mmol) were dissolved in EtOH (2 mL), and then piperidine (14 μL, 0.14 mmol) was added at room temperature under argon. The vial was sealed and heated at 75oC for 3 hours by monitoring the reaction by LCMS. The yellow precipitate was started to form in about 5 minutes. Precipitate was collected, washed with cold EtOH (8 mL) and dried to afford 4-methoxy-N-(3-((2-oxo-5-sulfamoylindolin-3-ylidene)methyl)phenyl)benzamide XL94 (56 mg, 88 %, 5:1 mixture of E / Z isomers).1H NMR (400 MHz, dmso) δ 11.01 (s, 1H), 10.38 (s, 1H), 8.31 (t, J = 1.7 Hz, 2H), 8.02 – 7.84 (m, 2H), 7.82 – 7.64 (m, 3H), 7.62 – 7.36 (m, 2H), 7.21 – 6.99 (m, 5H), 3.83 (s, 3H).13C NMR (101 MHz, dmso) δ 169.24, 166.27, 162.63, 146.10, 139.94, 138.27, 137.32, 134.67, 130.14, 129.84, 128.70, 126.92, 126.79, 126.00, 122.74, 121.32, 121.25, 121.13, 114.20, 110.24, 55.95. LC-MS [M+H]+: 450.20 (m / z).

[0409] Reaction for XL95:

[0410] Synthesis of XL95: In a thick-walled vial, (2-oxoindolin-5-yl)boronic acid (50 mg, 0.28 mmol) and N-(3-formylphenyl)-4-methoxybenzamide (72 mg, 0.28 mmol) were dissolved in EtOH (2 mL), and then piperidine (28 μL, 0.28 mmol) was added at room temperature under argon atmosphere. The vial was sealed and heated at 75oC for 4 hours by monitoring the reaction by LCMS. The reaction vial was cooled to room temperature and poured into water (20 mL) to precipitate. The yellow precipitate was collected, washed with CH2Cl2(3 x 10 mL) and dried to afford the (3-(3-(4-methoxybenzamido)benzylidene)-2-oxoindolin-5-yl)boronic acid XL95 (44 mg, 38 %, mostly single isomer).1H NMR (400 MHz, dmso) δ 10.68 (s, 1H), 10.41 (s, 1H), 8.32 (s, 1H), 8.30 (d, J = 1.9 Hz, 1H), 8.03 – 7.88 (m, 4H), 7.76 – 7.63 (m, 2H), 7.57 (s, 1H), 7.53 – 7.39 (m, 2H), 7.11 – 7.03 (m, 2H), 6.85 (d, J = 7.8 Hz, 1H), 3.83 (s, 3H).13C NMR (101 MHz, dmso) δ 169.50, 166.15, 162.67, 145.24, 139.63, 137.24, 135.73, 135.14, 130.22, 129.59, 129.30, 127.78, 126.73, 126.26, 122.42, 121.76, 120.71, 114.18, 109.72, 55.95. LC-MS [M+H]+: 415.20 (m / z).

[0411] Reaction for XL96:

[0412] Synthesis of XL96: 2-Oxoindoline-5-carbonitrile (1000 mg, 6.323 mmol) in H2SO4 (18 g, 10 mL, 85% Wt, 0.16 mol) was heated at 80 °C for 6 hours. The reaction mixture cooledto room temperature and left it stand for overnight. The reaction mixture poured into an ice-water and adjusted the pH = 5 using 50% aqueous NaOH solution. The precipitate was collected, washed with water and dried to provide 2-oxoindoline-5-carboxamide (558 mg, 53 %).1H NMR (400 MHz, dmso) δ 10.58 (s, 1H), 8.02 – 7.58 (m, 3H), 7.12 (s, 1H), 6.81 (dd, J = 7.9, 0.7 Hz, 1H), 3.50 (s, 2H).13C NMR (101 MHz, dmso) δ 177.11, 168.16, 146.90, 128.22, 127.70, 126.01, 124.20, 108.80, 36.03. LC-MS [M+H]+: 177.40 (m / z).

[0413] XL5B, XL44B, XL52-XL56, XL44-13C6-CB were synthesized under the following general conditions. Starting materials were used as received unless otherwise noted. All moisture sensitive reactions were performed in an inert atmosphere of argon with oven dried glassware. Reagent grade solvents were used for extractions and flash chromatography. Reaction progress was monitored by LC-MS analysis performed on an Agilent UPLC / MS instrument equipped with a RP-C18 column (Poroshell 120 SB-C18, 4.6 X 50 mm, 2.7 μm or Zorbax 300SB-C18, 4.6 X 50 mm, 3.5 μm), dual atmospheric pressure chemical ionization (APCI) / electrospray (ESI) mass spectrometry detector, and photodiode array detector. Flash chromatography was performed by using a RediSepRf NP-silica (40-63 μm 60 Å) or a Teledyne RediSepRf Gold RP- C18 column (20-40 μm 100 Å) in a Teledyne ISCO CombiFlash Rf 200 purification system unless otherwise specified.

[0414] XL5B was synthesized as follows:To a stirred solution of azide (Lu et al., Nat. Commun., 12, 7318 (2021)), S1 (30 mg, 0.06 mmol) in CH3CN (5 mL), biotin-PEG4-alkyne S2 (33 mg, 0.06 mmol), CuI (2.6 mg, 0.012 mmol), and Et3N (9 μL, 0.06 mmol) were added under argon atmosphere (see Lu et al., Nat. Commun., 12, 7318 (2021)). The reaction mixture was stirred for overnight at room temperature and then solvent was removed by rotary evaporator. Saturated aqueous NH4Cl (3 mL) and CH2Cl2 (5 mL) were added to the crude mixture and stirred for 5 min. Then organic layer was separated, and the aqueous layer was extracted with CH2Cl2(5 x 5 mL). The combined organic layers were dried over Na2SO4, filtered and concentrated to afford the crude material. The crude product was dissolved in 20 mol% TFA in CH2Cl2 (1 mL) and stirred for 3 hours at room temperature. Afterthe deprotection was completed, the solvent was removed and purified by a preparatory HPLC with a XBridge BEH C18 OBD Prep Column, 130Å, 5 μm, 30 mm X 150 mm reverse-phase column as the stationary phase. Water (buffered with 0.05% trifluoroacetic acid) and MeCN were used as the mobile phase and HPLC conditions: UV collection 254 nm, flow rate 30 mL / min, 20% MeCN as linear gradient for 5 min and 20% → 65% MeCN for 5 to 20 min. The HPLC fractions were combined and lyophilized to yield XL5B (28 mg, 52%, single isomer).1H NMR (400 MHz, dmso) δ 12.24 (s, 1H), 10.67 (s, 1H), 8.94 (s, 1H), 8.62 (dd, J = 8.4, 1.1 Hz, 1H), 8.49 (t, J = 2.0 Hz, 1H), 8.40 (s, 1H), 8.23 – 8.17 (m, 2H), 8.14 – 8.09 (m, 2H), 8.05 (dd, J = 7.9, 1.7 Hz, 1H), 7.97 (ddd, J = 8.2, 2.1, 1.0 Hz, 1H), 7.83 – 7.76 (m, 2H), 7.68 (ddd, J = 8.6, 7.3, 1.6 Hz, 1H), 7.60 (t, J = 8.0 Hz, 1H), 7.31 – 7.22 (m, 1H), 6.39 (s, 1H), 6.33 (s, 1H), 4.64 (s, 2H), 4.27 (dd, J = 7.8, 5.0 Hz, 1H), 4.10 (ddd, J = 7.8, 4.4, 1.8 Hz, 1H), 3.67 – 3.59 (m, 2H), 3.60 – 3.54 (m, 2H), 3.52 – 3.44 (m, 8H), 3.36 (t, J = 5.9 Hz, 2H), 3.15 (q, J = 5.8 Hz, 2H), 3.06 (ddd, J = 8.6, 6.2, 4.4 Hz, 1H), 2.79 (dd, J = 12.4, 5.1 Hz, 1H), 2.55 (d, J = 12.4 Hz, 1H), 2.04 (t, J = 7.4 Hz, 2H), 1.65 – 1.36 (m, 4H), 1.28 (dq, J = 14.1, 7.0 Hz, 2H).13C NMR (101 MHz, dmso) δ 172.56, 170.22, 165.13, 163.14, 159.45, 153.23, 145.91, 140.64, 140.25, 139.26, 134.78, 134.67, 132.60, 131.69, 130.17, 130.06, 126.34, 125.35, 124.29, 122.79, 122.48, 120.86, 120.07, 117.52, 115.98, 106.66, 70.27, 70.25, 70.17, 70.01, 69.66, 69.61, 63.87, 61.48, 59.63, 55.86, 40.29, 38.89, 35.54, 28.63, 28.48, 25.70. HRMS (m / z): [M+H]+calcd. for C45H52N9O10S, 910.3558; found, 910.3541.

[0415] XL44B was synthesized as follows:In a thick-walled vial, 5-fluoroindolin-2-one S3 (1 g, 6.6 mmol), and 3-nitrobenzaldehyde (1 g, 6.6 mmol) were dissolved in EtOH (15 mL) and then piperidine (0.2 mL, 2 mmol) was added at room temperature. The vial was sealed and heated at 70oC for 6 hours. The yellow solid precipitate was collected, washed with cold EtOH (2 x 20 mL) and dried under vacuum to afford yellow solid S4 (1.3 g, 70%).1H NMR (400 MHz, dmso) δ 10.75 (s, 1H), 9.38 (t, J = 2.0 Hz, 1H), 8.74 – 8.51 (m, 1H), 8.28 (dd, J = 7.9, 2.3 Hz, 1H), 8.02 (s, 1H), 7.75 (t, J = 8.0 Hz, 1H), 7.65 (dd, J = 8.9, 2.6 Hz, 1H), 7.07 (td, J = 9.1, 2.6 Hz, 1H), 6.81 (dd, J = 8.5, 4.4 Hz, 1H).13CNMR (101 MHz, dmso) δ 167.41, 158.42 (d), 148.13, 138.45, 137.97 (d), 136.01, 135.52, 130.24, 129.27 (d), 126.46, 126.23 (d), 125.22, 116.50 (d), 110.92 (d), 108.29 (d).19F NMR (376 MHz, dmso) δ -122.21. HRMS (m / z): [M+H]+calcd. for C15H10FN2O3, 285.0675; found, 285.0666.

[0416] Nitro compound S4 (500 mg, 1.76 mmol) and SnCl4(1 g, 5.28 mmol) were dissolved in EtOH (20 mL) and then refluxed for 4 hours. To the cooled reaction mixture, saturated aqueous potassium fluoride (10 mL) was added and stirred for 1 hour. The crude product was extracted with EtOAc (3 x 20 mL), and dried over anhydrous Na2SO4. After concentration, the crude product was purified by an ISCO combi flash silica gel column (EtOAc / hexanes) to provide brown colored aniline derivative S5 (295 mg, 66%, mixture of isomers E / Z = 5:1). Nitro compound S4 (500 mg, 1.76 mmol) and SnCl4(1 g, 5.28 mmol) were dissolved in EtOH (20 mL) and then refluxed for 4 hours. To the cooled reaction mixture, saturated aqueous potassium fluoride (10 mL) was added and stirred for 1 hour. The crude product was extracted with EtOAc (3 x 20 mL), and dried over anhydrous Na2SO4. After concentration, the crude product was purified by an ISCO combi flash silica gel column (EtOAc / hexanes) to provide brown colored aniline derivative S5 (295 mg, 66%, mixture of isomers E / Z = 5:1). Major isomer:1H NMR (400 MHz, DMSO) δ 10.60 (s, 1H), 7.58 (s, 1H), 7.34 (dd, J = 9.4, 2.7 Hz, 1H), 7.19 (t, J = 7.8 Hz, 1H), 7.07 (ddd, J = 9.4, 8.5, 2.7 Hz, 1H), 6.88 – 6.82 (m, 2H), 6.79 (dd, J = 7.5, 0.9 Hz, 1H), 6.72 – 6.67 (m, 1H), 5.37 (s, 2H).13C NMR (101 MHz, DMSO) δ 169.24, 157.62 (d), 149.55, 139.55 (d), 139.19, 134.98, 129.90, 127.16 (d), 122.55 (d), 117.21, 116.57 (d), 116.12, 114.13, 111.11 (d), 110.24 (d).19F NMR (376 MHz, DMSO) δ -122.09 (td). Selected for minor isomer:1H NMR (400 MHz, DMSO) δ 10.56 (s, 1H), 7.71 (s, 1H), 7.64 (dd, J = 9.1, 2.7 Hz, 1H), 7.61 (s, 1H), 7.13 (t, J = 7.8 Hz, 1H), 7.01 (ddd, J = 9.6, 8.4, 2.6 Hz, 1H), 5.18 (s, 2H).13C NMR (101 MHz, DMSO) δ 167.52, 157.41 (d), 148.90, 140.28, 137.22, 134.72, 129.11, 127.21, 126.05 (d), 120.97, 117.65, 117.34, 115.20 (d), 110.30, 107.59 (d).19F NMR (376 MHz, DMSO) δ -122.65 (td). HRMS (m / z): [M+H]+calcd. for C15H12FN2O, 255.0934; found, 255.0938.

[0417] To a stirred solution of boc-6-Ahx-OH S7 (210 mg, 0.91 mmol) in DMF (10 mL), benzyl 4-aminobenzoate hydrochloride S6 (240 mg, 0.91 mmol), N-(3-dimethylaminopropyl)- N′-ethylcarbodiimide hydrochloride (EDC-HCl) (280 mg, 1.46 mmol), 1-hydroxy-7- azabenzotriazole (HOAt) (178 mg, 1.46 mmol), and Et3N (0.46 mL, 3.2 mmol) were added at room temperature and stirred for overnight. Water (20 mL) was added and extracted with EtOAc (30 mL). The organic layer was washed with aqueous NaCl (2 x 20 mL) and dried over anhydrous Na2SO4. After concentration, the crude product was purified by an ISCO combi flash silica gel column (EtOAc / Hexanes) to afford S8 (165 mg, 44%).1H NMR (400 MHz, cdcl3) δ 8.24 (s, 1H), 8.05 – 7.96 (m, 2H), 7.69 – 7.61 (m, 2H), 7.46 – 7.28 (m, 5H), 5.33 (s, 2H), 4.69 (s, 1H), 3.08 (q, J = 6.7 Hz, 2H), 2.35 (t, J = 7.5 Hz, 2H), 1.71 (p, J = 7.6 Hz, 2H), 1.53 – 1.44 (m, 2H), 1.42 (s, 8H), 1.40 – 1.28 (m, 2H).13C NMR (101 MHz, cdcl3) δ 171.83, 166.10, 156.23, 142.72, 136.08, 130.88, 128.58, 128.22, 128.10, 125.13, 118.81, 79.26, 66.59, 40.22, 37.43, 29.70, 28.43, 26.21, 24.91. HRMS (m / z): [M+Na]+calcd. for C25H32N2O5Na, 463.2209; found, 463.2199.

[0418] To a methanolic solution of S8 (150 mg, 0.34 mmol), palladium on carbon (4 mg) was added under inert atmosphere. The resulting slurry was purged with argon (1 min) before H2gas was added via a double walled balloon. Upon completion as indicated by LC-MS analysis, the reaction mixture was purged with nitrogen (2 min) and filtered through a pad of celite with the aid of MeOH. The concentrated crude product S9 was dried and used for the next step without further purification.NMR (400 MHz, DMSO) δ 10.17 (s, 1H), 7.87 (d, J = 8.3 Hz, 2H), 7.69 (d, J = 8.3 Hz, 2H), 6.77 (t, J = 5.9 Hz, 1H), 2.91 (q, J = 6.6 Hz, 2H), 2.33 (t, J = 7.4 Hz, 2H), 1.59 (p, J = 7.5 Hz, 2H), 1.45 – 1.38 (m, 2H), 1.36 (s, 9H), 1.33 – 1.21 (m, 2H).13C NMR (101 MHz, DMSO) δ 172.18, 167.60, 156.05, 143.56, 130.74, 126.08, 118.64, 77.76,40.24, 36.92, 29.79, 28.74, 26.45, 25.21. HRMS (m / z): [M+Na]+calcd. for C18H26N2O5Na, 373.1739; found, 373.1726.

[0419] The benzoic acid S9 (62 mg, 0.18 mmol) was dissolved in DMF (3 mL), aniline S5 (49 mg, 0.19 mmol), 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3- oxid hexafluorophos-phate (HATU) (75 mg, 0.19 mmol), and DIPEA (83 μL, 0.45 mmol) were added at room temperature and the mixture was stirred for overnight. Water (5 mL) was added and extracted with EtOAc (20 mL). The organic layer was washed with aqueous NaCl (2 x 10 mL) and dried over anhydrous Na2SO4. After concentration, the crude product was purified by an ISCO combi flash silica gel column (CH2Cl2 / MeOH). The purified material was subjected to 20 mol% TFA in CH2Cl2 (1 mL) and stirred for 3 hours at room temperature. Upon completion of Boc deprotection as indicated by LC-MS analysis, the solvent was removed and lyophilized to afford TFA salt of S10 (70 mg, 68% over two steps), which was used for the next step without further purification. TFA salt of S10 (35 mg, 0.072 mmol) was dissolved in 3 mL of CH2Cl2 / DMF (1:1), and then biotin-NHS (21 mg, 0.079 mmol) and Et3N (52 μL, 0.36 mmol) were added at room temperature under argon atmosphere. The reaction mixture was stirred for overnight, water (10 mL) was added and then stirring was continued for 30 minutes. The yellow precipitate was collected and washed thoroughly with water (30 mL) and dried under vacuum to afford pure XL44B (48 mg, 94%, E / Z = 6:1). Major isomer:NMR (400 MHz, dmso) δ 10.63 (s, 1H), 10.30 (s, 1H), 10.14 (s, 1H), 8.20 (s, 1H), 7.94 – 7.90 (m, 2H), 7.82 – 7.77 (m, 1H), 7.74 – 7.70 (m, 3H), 7.67 (s, 1H), 7.51 (t, J = 7.9 Hz, 1H), 7.45 (dd, J = 9.4, 2.6 Hz, 1H), 7.38 (d, J = 7.6 Hz, 1H), 7.08 (td, J = 9.0, 2.6 Hz, 1H), 6.85 (dd, J = 8.5, 4.6 Hz, 1H), 6.40 (s, 1H), 6.34 (s, 1H), 4.27 (dd, J = 7.7, 5.1 Hz, 1H), 4.13 – 4.07 (m, 1H), 3.11 – 2.98 (m, 4H), 2.79 (dd, J = 12.5, 5.1 Hz, 1H), 2.55 (d, J = 12.4 Hz, 1H), 2.33 (t, J = 7.4 Hz, 3H), 2.02 (t, J = 7.4 Hz, 2H), 1.63 – 1.53 (m, 3H), 1.50 – 1.35 (m, 4H), 1.33 – 1.22 (m, 5H).13C NMR (101 MHz, dmso) δ 172.25, 172.14, 169.07, 165.63, 163.15, 157.72 (d, J = 235.2 Hz), 142.93, 140.12, 139.76, 137.78, 134.81, 129.78, 129.13, 129.07, 127.90 (d, J = 2.9 Hz), 125.35, 122.29, 122.19 (d, J = 8.9 Hz), 120.77, 118.63, 116.95 (d, J = 23.7 Hz), 111.14 (d, J = 8.0 Hz), 110.71= 25.9 Hz), 61.48, 59.63, 55.88, 40.29, 38.72, 36.86, 35.67, 29.47, 28.66, 28.48, 26.57, 25.79, 25.16.19F NMR (376 MHz, dmso) δ -121.90 (td). Selected for minor isomer:1H NMR (400 MHz, dmso) δ 10.59 (s, 1H), 10.24 (s, 1H), 8.50 (s, 1H), 8.25 (d, J = 7.8 Hz, 1H), 7.86 (s, 1H), 6.78 (dd, J = 8.5, 4.4 Hz,1H), 3.15 (d, J = 5.3 Hz, 1H).19F NMR (376 MHz, dmso) δ -122.50 (td, J = 9.2, 4.3 Hz). HRMS (m / z): [M+H]+calcd. for C38H42FN6O5S, 713.2921; found, 713.2897.

[0420] XL44-13C6-CB was synthesized as follows:In a thick-walled vial, 5-fluoroindolin-2-one S3 (74 mg, 0.49 mmol) and 3-nitro-benz-13C6- aldehyde (77 mg, 0.49 mmol) were dissolved in EtOH (3 mL) and then piperidine (48 μL, 0.49 mmol) was added at room temperature. The vial was sealed and heated at 70oC for 6 hours. The yellow solid product was collected, washed with cold EtOH (2 x 5 mL) and dried under vacuum to afford S4-13C6(95 mg, 67%). To a stirred solution of S4-13C6(85 mg, 0.29 mmol) in 30 mL EtOH / H2O (2:1), iron (82 mg, 1.45 mmol) and NH4Cl (8 mg, 0.15 mmol) were added at room temperature. The reaction mixture was refluxed for 4 days by monitoring the reaction by LCMS. The reaction mixture cooled to room temperature and filtered through a thick pad of celite with the aid of CH2Cl2 / MeOH (9:1). The filtrate was transferred to separating funnel, organic layer was separated and then aqueous layer was extracted with CH2Cl2(2 x 10 mL). The combined organic layers were dried over anhydrous Na2SO4, and concentrated to provide S5-13C6, which was used for the next step without further purification. The crude S5-13C6(46 mg, 0.17 mmol) was dissolved in CH2Cl2(3 mL), and then 4-methoxybenzoyl chloride (26 μL, 0.19 mmol) and Et3N (50 μL, 0.34 mmol) were added at room temperature. The reaction mixture stirred for overnight and water (3 mL) was added. The organic layer was separated and the aqueous layer was extracted with CH2Cl2 (2 x 5 mL). Combined organic layers were dried, concentrated and purified by ISCO combi flash column chromatography to afford XL44-13C6-CB (6 mg, 9%).1H NMR (400 MHz, dmso) δ 10.65 (s, 1H), 10.29 (s, 1H), 8.44 – 8.00 (m, 1H), 7.99 – 7.93 (m, 2H), 7.76 – 7.55 (m, 3H), 7.47 (dd, J= 9.5, 2.6 Hz, 1H), 7.38 – 7.05 (m, 4H), 6.87 (dd, J = 8.5, 4.6 Hz, 1H), 3.84 (s, 3H).1H Bilevel Dec NMR (400 MHz, dmso) δ 10.64 (s, 1H), 10.29 (s, 1H), 8.22 (s, 1H), 8.02 – 7.92 (m, 2H), 7.82 (d, J = 7.8 Hz, 1H), 7.69 (s, 1H), 7.59 – 7.43 (m, 2H), 7.39 (d, J = 7.5 Hz, 1H), 7.15 – 7.02 (m, 3H), 6.87 (dd, J = 8.5, 4.6 Hz, 1H), 3.84 (s, 3H).13C NMR (101 MHz, dmso) δ 168.64, 168.58, 165.18, 162.04, 158.44, 156.10, 139.69 (td), 139.30, 134.32 (ddd), 129.68, 129.30 (td), 124.77 (td), 121.78 (dddd), 120.28 (ddd), 116.59, 116.36,113.66, 110.67 , 110.38, 110.12, 55.45.19F NMR (376 MHz, dmso) δ -121.94 (td). HRMS (m / z): [M+Na]+calcd. for C1713C6H17FN2O3Na, 417.1322; found, 417.1318.

[0421] XL52 was synthesized as follows:To a stirred solution of 4-cyanobenzyl bromide S12 (1 g, 5.1 mmol) and methyl 4- hydroxybenzoate S11 (815 mg, 5.3 mmol) in DMF (15 mL), K2CO3 (2.11 g, 15.3 mmol) was added and stirred for 15 hours at room temperature. Water (30 mL) and EtOAc (50 mL) were added and then the organic layer was separated. The collected organic layer was washed with aqueous NaCl (2 x 30 mL) and dried over anhydrous Na2SO4. After concentration, the crude product was purified by an ISCO combi flash silica gel column (hexanes / EtOAc) to afford S13 (white solid, 1.2 g, 88%).1H NMR (400 MHz, CDCl3) δ 7.97 – 7.89 (m, 2H), 7.65 – 7.57 (m, 2H), 7.50 – 7.43 (m, 2H), 6.94 – 6.86 (m, 2H), 5.10 (s, 2H), 3.81 (s, 3H).13C NMR (101 MHz, CDCl3) δ 166.65, 161.81, 141.71, 132.50, 131.74, 127.59, 123.44, 118.59, 114.41, 111.99, 68.94, 51.97. HRMS (m / z): [M+H]+calcd. for C16H14NO3, 268.0974; found, 268.0968.

[0422] Nitrile S13 (1 g, 3.74 mmol) was dissolved in DMF (20 mL) and then NaN3(365 mg, 5.61 mmol), and NH4Cl (300 mg, 5.61 mmol) were added at room temperature under argon atmosphere. The reaction was heated at 125oC for 16 hours and cooled to room temperature. Water (30 mL) was added to a cooled reaction mixture and extracted with EtOAc (50 mL). The organic layer was washed with brine (2 x 30 mL), dried (anhydrous Na2SO4), and purified the concentrated material by an ISCO combi flash silica gel column (CH2Cl2 / MeOH) to provide S14 (white solid, 625 mg, 54%).1H NMR (400 MHz, DMSO) δ 8.08 (d, J = 8.3 Hz, 2H), 7.94 (d, J = 8.9 Hz, 2H), 7.69 (d, J = 8.0 Hz, 2H), 7.21 – 7.14 (m, 3H), 5.31 (s, 2H), 3.82 (s, 3H).13C NMR(101 MHz, DMSO) δ 166.31, 162.50, 140.16, 132.95, 131.74, 128.95, 127.60, 124.38, 122.70, 115.35, 69.38, 52.33. HRMS (m / z): [M+H]+calcd. for C16H15N4O3, 311.1144; found, 311.1140.

[0423] To a stirred solution of S14 (220 mg, 0.71 mmol) in 6 mL MeOH / THF (1:1), LiOH (85 mg, 3.55 mmol) in water (1 mL) was added at 0oC. The reaction mixture was warmed to room temperature and stirred for 6 hours (monitored by LC-MS). Ice cold aqueous HCl (1M) was added to the reaction and then stirred for 30 minutes. The white precipitate was collected and washed with water (10 mL) and dried under vacuum to afford S15 (196 mg, 93%).1H NMR (400 MHz, dmso) δ 12.63 (s, 1H), 8.05 (d, J = 8.0 Hz, 2H), 7.97 – 7.83 (m, 2H), 7.67 (d, J = 7.9 Hz, 2H), 7.11 (d, J = 8.8 Hz, 2H), 5.28 (s, 2H).13C NMR (101 MHz, dmso) δ 167.37, 162.18, 140.30, 132.90, 131.83, 128.92, 127.60, 123.82, 115.13, 69.30. HRMS (m / z): [M+H]+calcd. for C15H13N4O3, 297.0988; found, 297.0984.

[0424] Carboxylic acid S15 (30 mg, 0.10 mmol) and S5 (28 mg, 0.11 mmol) were dissolved in DMF (2 mL), HATU (42 mg, 0.11 mmol), and DIPEA (46 μL, 0.25 mmol) were then added at room temperature. The reaction stirred for overnight and purified by reverse-phase ISCO combi flash column chromatography. Water (buffered with 0.05% trifluoroacetic acid) and MeCN were used as the mobile phase, and the collected fractions were combined and lyophilized to provide XL52 (5 mg, 9%, E / Z = 3:1).1H NMR (400 MHz, DMSO) δ 10.75 (s, 1H), 10.40 (s, 1H), 8.28 (s, 1H), 8.17 – 8.00 (m, 5H), 7.89 (d, J = 8.1 Hz, 1H), 7.78 – 7.66 (m, 3H), 7.60 – 7.40 (m, 3H), 7.28 – 7.20 (m, 2H), 7.15 (td, J = 8.9, 2.6 Hz, 1H), 6.93 (dd, J = 8.5, 4.6 Hz, 1H), 5.34 (s, 2H).13C NMR (101 MHz, DMSO) δ 169.10, 165.67, 161.46, 158.92, 156.58, 140.21, 139.80, 139.00, 138.68, 137.82, 134.82, 130.20, 129.77, 128.74, 127.93, 127.49, 127.21, 125.31, 122.30, 122.17, 120.79, 116.95 (d), 115.03, 111.22, 110.73 (d), 69.53.19F NMR (376 MHz, DMSO) δ -121.89 (td). HRMS (m / z): [M+H]+calcd. for C30H22FN6O3, 533.1737; found, 533.1735. Selected for minor isomer:1H NMR (400 MHz, DMSO) δ 10.70 (s, 1H), 10.33 (s, 1H), 8.57 (s, 1H), 8.33 (d, J = 7.9 Hz, 1H), 7.93 (s, 1H), 7.11 – 7.04 (m, 1H), 6.86 (dd, J = 8.4, 4.4 Hz, 1H).19F NMR (376 MHz, DMSO) δ -122.47 (td). HRMS (m / z): [M+H]+calcd. for C30H22FN6O3, 533.1737; found, 533.1732.

[0425] XL53 was synthesized as follows:In a thick-walled vial, 5-fluoroindolin-2-one S3 (16 mg, 0.11 mmol) and aldehyde S16 (30 mg, 0.1 mmol) were dissolved in EtOH (5 mL) and then piperidine (10 μL, 0.1 mmol) was added at room temperature. The vial was sealed and heated at 70oC for 6 hours (monitored by LC-MS). The yellow solid product was collected, washed with cold EtOH (2 x 5 mL) and dried under vacuum to afford yellow solid XL53 (21 mg, 48%).1H NMR (400 MHz, dmso) δ 10.74 (s, 1H), 10.61 (s, 1H), 8.23 (d, J = 2.1 Hz, 1H), 8.09 (dd, J = 9.1, 2.1 Hz, 1H), 7.94 – 7.82 (m, 3H), 7.74 (d, J = 2.6 Hz, 1H), 7.34 (d, J = 8.0 Hz, 2H), 7.09 (td, J = 9.0, 2.6 Hz, 1H), 6.87 (dd, J = 8.6, 4.5 Hz, 1H), 6.77 (dd, J = 9.0, 2.6 Hz, 1H), 2.37 (s, 3H).13C NMR (101 MHz, dmso) δ 168.29, 166.45, 157.67 (d), 143.53, 142.74, 140.08 (d), 133.66 (d), 133.00, 131.70, 130.34, 129.48, 128.34, 128.11, 125.85, 123.14, 121.90 (d), 121.59 (d), 120.87 (d), 117.62 (d), 111.49 (d), 110.88 (d), 21.49.19F NMR (376 MHz, dmso) δ -58.46 (d), -121.77 (td). HRMS (m / z): [M+H]+calcd. for C24H17F4N2O2, 441.1226; found, 441.1225.

[0426] XL54 was synthesized as follows:Intermediate S18 was synthesized using the reported procedure (Khairnar, P. V., Su, Y-H., Chen, Y-C., Edukondalu, A., Chen, Y-R., and Lin, W., Organophosphane-Catalyzed Direct β- Acylation of 4-Arylidene Pyrazolones and 5-Arylidene Thiazolones with Acyl Chlorides, Org. Lett.22, 6868–6872 (2020)). To a stirred solution of S17 (1 g, 7.3 mmol) in CH2Cl2(40 mL), NaHCO3(1.23 g, 14.6 mmol) in 20 mL water was added. The reaction mixture cooled to 0oCand then chloroacetyl chloride (0.58 mL, 7.3 mmol) was added. The reaction warmed to room temperature and stirred for overnight. The organic phase was separated, and aqueous phase was extracted with CH2Cl2 (2 x 50 mL). The combined organic layers were washed with aqueous NaCl (50 mL) and dried over anhydrous Na2SO4. The concentrated yellow crude product S18 was used for the next step without further purification. In a thick-walled vial, aldehyde S19 (20 mg, 0.08 mmol) in AcOH, thiazole S18 (15 mg, 0.088 mmol), and NaOAc (3 mg, 0.024 mmol) were added. The vial was sealed and heated at 100oC for 6 hours. The vial was cooled to room temperature and then yellow precipitate was collected by washing with cold ethanol (10 mL). The collected solid was purified by ISCO combi flash silica gel column chromatography (CH2Cl2 / MeOH) to afford XL54 (21 mg, 63%).1H NMR (400 MHz, dmso) δ 10.34 (s, 1H), 8.30 (t, J = 1.7 Hz, 1H), 8.23 – 8.16 (m, 2H), 8.05 – 7.96 (m, 3H), 7.95 – 7.87 (m, 1H), 7.86 – 7.78 (m, 1H), 7.74 – 7.65 (m, 2H), 7.62 – 7.46 (m, 2H), 7.14 – 7.00 (m, 2H), 3.84 (d, J = 0.6 Hz, 3H).13C NMR (101 MHz, dmso) δ 187.32, 182.57, 165.74, 162.55, 140.81, 138.46, 136.12, 134.12, 131.63, 130.22, 130.18, 128.99, 127.14, 126.97, 126.80, 123.64, 121.35, 114.13, 55.93. HRMS (m / z): [M+H]+calcd. for C24H19N2O3S, 415.1116; found, 415.1114.

[0427] XL55 was synthesized as follows:Intermediate S22 was synthesized using the reported procedure starting from S20 (Worley et al., J. Org. Chem., 40, 1731–1734 (1975)). Sulfuryl chloride (162 μL, 2 mmol) was added to a stirred solution of 2,4-dihydro-1,4-benzothiazin-3-one S20 (300 mg, 1.82 mmol) in CH2Cl2(10 mL) at room temperature. After the disappearance of starting material (monitored by LC-MS), solvent was removed and the product was recrystallized in CH2Cl2to provide S21 (335 mg, 92%).1H NMR (400 MHz, dmso) δ 10.87 (s, 1H), 7.33 (dd, J = 7.7, 1.4 Hz, 1H), 7.19 (ddd, J = 8.0, 7.3, 1.4 Hz, 1H), 7.05 – 6.95 (m, 2H), 5.11 (d, J = 1.0 Hz, 1H).13C NMR (101 MHz, dmso) δ 161.24, 136.83, 128.88, 127.48, 123.63, 117.52, 115.87, 79.30. HRMS (m / z): [M-Cl]+calcd. for C8H6NOS, 164.0170; found, 164.0166.

[0428] In a sealed tube, 2-chloro-4H-1,4-benzothiazin-3-one (255 mg, 1.28 mmol) and triethyl phosphite (1.1 mL, 6.39 mmol) were heated at 120oC for 18 hours. The reaction was concentrated and dried to afford yellow solid S22 which was used directly for the next step.1H NMR (400 MHz, dmso) δ 10.78 (s, 1H), 7.30 (dd, J = 7.8, 1.4 Hz, 1H), 7.14 (td, J = 7.7, 1.5 Hz, 1H), 7.01 – 6.83 (m, 2H), 4.38 (d, J = 20.8 Hz, 1H), 4.02 – 3.67 (m, 4H), 1.12 (t, J = 7.0 Hz, 3H), 0.93 (t, J = 7.0 Hz, 3H).13C NMR (101 MHz, dmso) δ 161.65 (d), 137.50, 127.42, 127.41, 123.55, 117.12, 117.09, 63.28 (d), 63.04 (d), 37.60 (d), 16.46 (d), 16.25 (d).31P NMR (162 MHz, DMSO) δ 17.76. HRMS (m / z): [M-Cl]+calcd. for C12H17NO4PS, 302.0616; found, 302.0617.

[0429] Aldehyde S19 (20 mg, 0.08 mmol) and diethyl (3-oxo-3,4-dihydro-2H-1,4- benzothiazin-2-yl)phosphonate S22 (24 mg, 0.08 mmol) were dissolved in MeOH (5 mL), and then 3.6 M NaOMe (145 μL, 0.52 mmol) in MeOH was added at room temperature. The formation of yellow precipitate was observed immediately after the addition of NaOMe. The reaction was stirred for about 2 hours and then yellow precipitate was collected. The collected precipitate was washed with cold MeOH (3 x 5 mL) and dried to afford pure XL55 (31 mg, 97%).1H NMR (400 MHz, dmso) δ 11.02 (s, 1H), 10.24 (s, 1H), 8.08 (d, J = 2.2 Hz, 1H), 7.98 (d, J = 8.6 Hz, 2H), 7.83 – 7.78 (m, 1H), 7.75 (s, 1H), 7.45 (t, J = 7.9 Hz, 1H), 7.39 – 7.26 (m, 2H), 7.22 – 7.12 (m, 1H), 7.10 – 7.04 (m, 3H), 7.02 – 6.98 (m, 1H), 3.83 (s, 3H).13C NMR (101 MHz, dmso) δ 165.56, 162.45, 158.89, 140.08, 135.04, 134.62, 131.06, 130.16, 129.34, 127.43, 127.27, 125.60, 125.50, 123.68, 121.63, 121.26, 121.12, 117.23, 115.48, 114.09, 55.91. HRMS (m / z): [M+H]+calcd. for C23H19N2O3S, 403.1116; found, 403.1106.

[0430] XL56 was synthesized as follows:To a stirred solution of 3'-aminoacetophenone S23 (500 mg, 3.7 mmol) in CH2Cl2 (10 mL), 4- methoxybenzoyl chloride (530 μL, 3.9 mmol), and Et3N (800 μL, 5.5 mmol) were added at 0oC. The reaction was warmed to room temperature and then stirred for 15 hours. Water (10 mL) and CH2Cl2 (20 mL) were added to the reaction mixture. Aqueous layer was removed and the organic layer was washed with aqueous NaHCO3(20 mL) and aqueous NaCl (20 mL). The organic phase was dried (Na2SO4) and concentrated to afford N-(3-acetylphenyl)-4-methoxybenzamide S24,which was used for the next step without further purification.1H NMR (400 MHz, cdcl3) δ 8.26 (s, 1H), 8.16 (t, J = 1.9 Hz, 1H), 8.05 (ddd, J = 8.1, 2.3, 1.0 Hz, 1H), 7.92 – 7.83 (m, 2H), 7.73 – 7.66 (m, 1H), 7.44 (t, J = 7.9 Hz, 1H), 6.99 – 6.89 (m, 2H), 3.86 (s, 3H), 2.59 (s, 3H).13C NMR (101 MHz, cdcl3) δ 198.10, 165.53, 162.66, 138.81, 137.73, 129.34, 129.07, 126.67, 124.88, 124.12, 119.70, 113.98, 55.47, 26.68. HRMS (m / z): [M+H]+calcd. for C16H16NO3, 270.1130; found, 270.1131.

[0431] In a thick-walled microwave vial, N-(3-acetylphenyl)-4-methoxybenzamide S24 (100 mg, 0.37 mmol) and 5-fluoroindolin-2-one S3 (280 mg, 1.85 mmol) were dissolved in EtOH (10 mL), and then piperidine (73 μL, 0.73 mmol) was added. The vial was sealed and irradiated in a microwave at 150oC for 4 hours (~80% conversion by LC-MS). The cooled reaction mixture was dissolved in CH2Cl2(30 mL) and washed with aqueous NH4Cl (10 mL) followed by aqueous NaCl (20 mL). The organic layer was dried over anhydrous Na2SO4, concentrated, and purified by ISCO combi flash silica gel column (hexanes / EtOAc) to provide XL56 (48 mg, 32%).1H NMR (400 MHz, cdcl3) δ 8.11 (s, 1H), 8.00 (s, 1H), 7.91 – 7.82 (m, 2H), 7.73 (dt, J = 8.2, 1.5 Hz, 1H), 7.64 (t, J = 1.9 Hz, 1H), 7.48 (t, J = 7.9 Hz, 1H), 7.03 (dt, J = 7.6, 1.3 Hz, 1H), 7.00 – 6.93 (m, 2H), 6.80 – 6.64 (m, 2H), 5.93 (dd, J = 9.7, 2.5 Hz, 1H), 3.87 (s, 3H), 2.79 (s, 3H).13C NMR (101 MHz, cdcl3) δ 169.34, 165.20, 162.66, 156.58, 143.12, 139.24, 135.28, 130.18, 128.93, 126.78, 121.94, 120.03, 117.49, 114.68, 114.44, 114.05, 111.02, 110.75, 109.39, 109.31, 55.48, 22.86.19F NMR (376 MHz, cdcl3) δ -121.28 (td). HRMS (m / z): [M+H]+calcd. for C24H20FN2O3, 403.1458; found, 403.1445. NMR Experiments

[0432] For screening of binding by1H,15N HSQC experiments, each small molecule was dissolved in DMSO-d6and added to 20 mM15N-labeled hRpn13 Pru at a molar excess of 2- or 10-fold as indicated in NMR buffer (20 mM sodium phosphate, 50 mM NaCl, 2 mM DTT, 10% DMSO-d6, pH 6.5). NMR experiments were conducted at 10°C or 25qC and on Bruker Avance 600, 800 or 850 MHz spectrometers equipped with cryogenically cooled probes. The13C-half- filtered NOESY spectrum was acquired with a 100 ms mixing time on a mixture of 0.4 mM13C- labeled hRpn13 Pru and 0.4 mM unlabeled XL44 or another mixture of 0.4 mM hRpn13 Pru and 0.4 mM XL44 with the central benzene ring13C-labeled (XL44-13C6-CB) in NMR buffer.19Fspectra were acquired on samples of 50 PM XL44 alone or with equimolar hRpn2 (940-953) or equimolar hRpn13 Pru as well as of mixtures of preincubated 50 PM XL44 and equimolar hRpn13 Pru with 25 or 50 PM hRpn2 (940-953) added. All samples were dissolved in NMR buffer without or with DTT as indicated and at 10qC. The1H,15N heteronuclear NOE experiment was acquired on 440 μM15N-labeled hRpn13 Pru with a saturation time of 6 seconds. Data were processed by NMRPipe (Delaglio et al., J. Biomol. NMR, 6, 277-93 (1995)) and visualized with XEASY (Bartels et al., J. Biomol. NMR, 6, 1-10 (1995)), MestReNova software or TopSpin software. Differential Scanning Fluorimetry

[0433] DSF experiments were performed on a Prometheus NT.48 instrument (NanoTemper Technologies, Germany) at 20°C.2 μM hRpn13 Pru was added to equal volume of serially diluted XL44 or XL5 in buffer C (20 mM sodium phosphate, 50 mM NaCl, 2 mM DTT, 10% DMSO, pH 6.5). Each sample was loaded into three capillaries of High Sensitivity grade (NanoTemper, cat # PR-C006) and the emission of intrinsic tryptophan fluorescence at 350 nm was monitored. LC-MS Experiment

[0434] 2 μM hRpn13 Pru was incubated with 10-fold molar excess XL44 or DMSO (vehicle control) in buffer D (20 mM sodium phosphate, 50 mM NaCl, pH 6.5) containing 0.2% DMSO for 2 hours at 4°C and the samples were then loaded onto a 6520 Accurate-Mass Q-TOF LC / MS system equipped with a dual electro-spray source, operated in the positive-ion mode. Acetonitrile was added to all samples to a final concentration of 10%. Mass Hunter Qualitative Analysis software (version B.07.00) with Bioconfirm Workflow was used for data analysis and deconvolution of mass spectra. Cell Culture and Antibodies

[0435] The RPMI 8226 (ATCC® CCL-155™), HS5-BMSCs and HCT116 WT (ATCC®CCL-247™) cell line were purchased from the American Tissue Culture Collection; Human Fibroblast 1634 (HF-1634) cells were a generous gift from Dr. Douglas Lowy, RPMI8226 trRpn13-MM2 or HCT116 trRpn13 cells were generated and described in previous studies (Lu et al., Nat. Commun., 12, 7318 (2021) and Osei-Amponsa et al., Mol. Cell Biol., 40 (2020)). RPMI 8226 cell lines were grown in RPMI-1640 media (ATCC® 30-2001™). HS5 and HF- 1634 cells were cultured in DMEM media supplemented with 100 mg / mL penicillin and streptomycin 100 U / mL (Gibco, Life Technologies). HCT116 cells were grown in McCoy’s 5A modified media (Thermo Fisher Scientific 16600082). In all cases, the media was supplemented with 10% fetal bovine serum (Atlanta Biologicals) and growth occurred in a 37°C humidified atmosphere of 5% CO2. Antibodies (dilutions) used in this study include primary antibodies anti- hRpn13 (100-200) (Abcam ab157185, 1:5,000), anti-β-actin (Cell Signaling Technology 3700s, 1:6,000 or 1:10,000), anti-cleaved caspase-9 (Cell Signaling, 52873s, 1:500), anti-PCLAF (Santa Cruz, sc-390515 HRP, 1:1000 or 1:500), anti-PTTG1 (Cell Signaling, 13445s, 1:1000), anti- RRM2 (Abcam ab57653, 1:1,000), anti-RRM2 (Abcam ab172476, 1:3,000), secondary antibodies anti-mouse (Sigma-Aldrich, A9917, 1:3,000 or 1:4,000), anti-rabbit (Life Technologies, A16110, 1:5,000) antibodies. MTT Assay

[0436] RPMI 8226 WT or trRpn13-MM2 cells were seeded at 8,000 cells / well with RPMI 1640 medium (no phenol red, Thermo Fisher Scientific 11835030) containing 10% fetal bovine serum in 96-well plates. After 24 hours, cells were treated with 0.4% DMSO (as a control) and this concentration was maintained with XL44 or XL52 at 2 μM, 4 μM, 6 μM or 8 μM concentration. Each condition was performed in sextuplicate. After 48 hours, 0.35 mg / mL MTT was added for 4 hours of incubation. Stop solution (40% DMF, 10% SDS (W / V), 25 mM HCl, 2.5% acetic acid in H2O) was added to the cells and incubated overnight. Absorbance at 570 nm was measured by using CLARIOstar (BMG LABTECH). MTS Assay

[0437] Cell proliferation experiments were performed using Cell Titer 96 Aqueous One Solution Cell Proliferation Assay System (Promega, Madison, WI, USA). Briefly, cells were plated in quadruplicate on clear, flat-bottomed 96-well tissue culture plates (Corning Costar) and treated with 0.1 to 50 μM concentrations of XL44. After incubation for 48 hours, MTS reagentwas added directly to the wells and incubated for 1.5 hours. The absorbance of MTS formazan was read at 490 nm (Omega 640 spectrophotometer, BMG Labtech, Cary, NC, USA). A blank measurement was taken for the absorbance of the wells with media only and subtracted accordingly. LDH Assay

[0438] LDH activity in cell supernatants was assessed using CQUANT LDH Cytotoxicity Assay Kit (ThermoFisher). RPMI 8226 WT and RPMI 8226 trRpn13-MM2 cells were plated with increasing cell number to determine optimal cell density for the assay. Briefly, 25000 cells / well were plated in quadripulate in 96-well plates and treated with 2.5 to 20 μM concentrations of XL44 or solvent control (DMSO) for 24 hours. Equal number of wells were plated for Spontaneous LDH activity and Maximum LDH activity controls. The chemical compound-mediated cytotoxicity assay was performed. % cytotoxicity was measured according to the manufacturer’s protocol. siRNA Knockdown Assay

[0439] HCT116 WT or trRpn13 cells were seeded at 4,0000 cells / mL with RPMI 1640 medium (no phenol red, Thermo Fisher Scientific 11835030) containing 10% fetal bovine serum and transfected with 50 nM scramble (Horizon Discovery, D-001810-10-05) or PCLAF siRNA (Horizon Discovery, L-017672-01-0010) by using the LipofectomineTM 300 transfection reagent for 48 hours. After 48 hours, the cells were seeded in a 10 cm dishes and treated with 0.8% DMSO or 20 μM XL44 for 24 hours or in parallel, seeded for the MTT assay into 96-well plates and treated with 0.4% DMSO or XL44 at 10 μM or 20 μM for 24 hours. Plasmids for Transfection

[0440] Plasmids expressing HA-tagged PCLAF WT or PCLAF mutant AA (K78A, E79A) were generated commercially (GenScript) by inserting synthesized coding sequence for full length PCLAF WT (UniProt ID: Q15004) or mutant AA with residues lysine 78 and glutamic acid 79 mutated to alanine between the BamHI and NotI restriction sites of pcDNA3.1+N-HA (GenScript, SC1317). Unmodified pcDNA3.1+N-HA was used as empty vector (EV) control.Transfection

[0441] HCT116 WT cells (8 x 104) were reverse transfected with 1 μg empty vector (EV), HA-PCLAF, HA-PCLAF (AA)-expressing plasmid by using lipofectamine 3000 (Thermo Fisher Scientific, L3000015) according to the manufacturer’s instructions. After 48 hours of transfection, cells were treated with 20 μM XL44 or DMSO (a control) for 24 hours before harvesting cells. XL44 Treatment

[0442] Two or four million RPMI 8226 WT or trRpn13-MM2 cells were seeded separately in a T75 flask. After 48 hours, the cells were treated with 0.8% DMSO (as a control, 0 hour or 24 hours) or 1, 2.5, 5, 7.5, 10, 15, 20 μM XL44, 1 μM MLN7243 and 20 μM XL44 for 24 hours or 20 μM XL44 for 1, 2, 3, 4, 5, 7, 8, 12 or 24 hours, or cells were pretreated with 100 nM carfilzomib, 1 μM MLN7243 or 1 μM MLN4924 for 2 hours and then 20 μM XL44 added for another 22 hours of co-treatment, as indicated. Biotin Pulldown Assay

[0443] RPMI 8226 WT cell lysates (2 mg) were preincubated with 50 μL pre-washed Dyna beads MyOne streptavidin T1 magnetic beads (Invitrogen 65602) for 1 hour at 4°C to remove non-specific biotinylated proteins in the cell lysate and then pre-cleared cell lysate was incubated with 40 μM biotin, XL5B or XL44B overnight at 4°C. Next, the mixture was incubated for an additional 3 hours at 4°C with 50 μL pre-washed Dyna beads MyOne streptavidin T1 magnetic beads. Following three washes with 1% Triton-TBS lysis buffer, proteins bound to the Dyna beads MyOne streptavidin T1 magnetic beads were eluted by using 2x LDS with 100 mM DTT and analyzed by immunoblotting. Immunoblotting

[0444] RPMI 8226 WT or trRpn13-MM2 cells were collected and washed with PBS followed by flash freezing in liquid nitrogen and storage at -80ºC for future use. Cells were lysed in 1% Triton-TBS lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1mM PMSF)supplemented with protease inhibitor cocktail (Roche). Total protein concentration was determined by bicinchoninic acid (Pierce). Protein lysates were prepared in 1x LDS (ThermoFisher, NP0007) buffer with 100 mM DTT and heating at 70°C for 10 min, loaded onto 4-12% Bis-Tris polyacrylamide gels (Life Technologies), subjected to SDS-PAGE and transferred to Invitrolon polyvinylidene difluoride membranes (Life Technologies). The membranes were blocked in Tris-buffered saline with 0.1% Tween-20 (TBST) supplemented with 5% skim milk or 5% BSA, incubated with primary antibody, washed in TBST, incubated with secondary antibodies and washed extensively in TBST. PierceTM ECL Western Blotting Substrate (32106; Thermo Fisher Scientific) or AmershamTM ECLTM Primer Western Blotting Detection Reagent (cytiva) was used for antibody signal detection. hRPN13 Degradation Assay

[0445] 640 nM purified hRpn13 was incubated without or with 30 nM commercial 26S proteasome (Enzo, BML-PW9310) for 10 min at 37°C in a buffer containing 50 mM Tris-HCl (pH 7.5), 5 mM MgCl2 and 5 mM ATP. The reaction was stopped by adding LDS loading buffer with 100 mM DTT. TMT Proteomic Analysis

[0446] RPMI 8226 WT or trRpn13-MM2 cells were treated with 0.8% DMSO (as a control) or 20 μM XL44 for 8 hours. Cell pellets were lysed in EasyPrep Lysis buffer (Thermo Fisher Scientific) according to the manufacturer’s protocol. Lysates were clarified by centrifugation and protein concentration was quantified by using a bicinchoninic acid (BCA) protein estimation kit (Thermo Fisher Scientific).20 μg of lysate was reduced, alkylated and digested by addition of trypsin at a ratio of 1:50 (Promega) and incubating overnight at 37°C. For TMT labeling, 100 μg of TMTpro label (Thermo Fisher Scientific) in 100% acetonitrile was added to each sample. After incubating the mixture for 1 hour at room temperature with occasional mixing, the reaction was terminated by adding 50 μL of 5% hydroxylamine, 20% formic acid. The peptide samples for each condition were pooled and clean-up was performed using the proprietary peptide clean up columns from the EasyPEP Mini MS Sample Prep kit (Thermo Fisher Scientific). The first dimensional separation of the peptides was performed by a Waters Acquity UPLC systemcoupled with a fluorescence detector (Waters, Milford, MA) and a 150 mm x 3.0 mm Xbridge Peptide BEMTM 2.5 μm C18 column (Waters, MA) operating at 0.35 mL / min. The dried peptides were reconstituted in 100 μL of mobile phase A solvent (3 mM ammonium bicarbonate, pH 8.0); mobile phase B was 100% acetonitrile (Thermo Fisher Scientific). The column was washed with mobile phase A for 10 minutes followed by gradient elution 0–50% B (10–60 min) and 50–75% B (60–70 min). Fractions were collected every minute and the resulting 60 fractions pooled into 24 fractions that were vacuum centrifuged to dryness and stored at -80°C until analysis by mass spectrometry. The dried peptide fractions were reconstituted in 0.1% trifluoroacetic acid and subjected to nanoflow liquid chromatography (Thermo UltimateTM 3000RSLC nano LC system, Thermo Fisher Scientific) coupled to an Orbitrap Eclipse mass spectrometer (Thermo Fisher Scientific). Peptides were separated by a low pH gradient with a 5- 50% acetonitrile over 120 minutes in mobile phase containing 0.1% formic acid at 300 nL / min flow rate. For TMT analysis, the FAIMS-MS2 based approach was used. MS scans were performed in the Orbitrap analyser at a resolution of 120,000 with an ion accumulation target set at 4e5and max IT set at 50 ms over a mass range of 350-1600 m / z. The FAIMS source was operated under standard resolution and four different compensation voltages (CVs) of -45, -60, - 75 and -90 was used. Ions with determined charge states between 2 and 6 were selected for MS2 scans. A cycle time of 0.75 seconds was used for each CV and a quadrupole isolation window of 0.4 m / z was used for MS / MS analysis. An Orbitrap at 15,000 resolution with a normalized automatic gain control set at 250 followed by maximum injection time set as “Auto” with a normalized collision energy setting of 38 was used for MS / MS analysis. The node “Turbo TMT” was switched on for high resolution acquisition of TMT reporter ions. Acquired MS / MS spectra were searched against a human uniprot protein database using a SEQUEST HT and percolator validator algorithms in the Proteome Discoverer 2.4 software (Thermo Fisher Scientific). The precursor ion tolerance was set at 10 ppm and the fragment ions tolerance was set at 0.02 Da along with methionine oxidation included as dynamic modification. Carbamidomethylation of cysteine residues and TMT16 plex (304.2071Da) was set as a static modification of lysine and the N-termini of the peptide. Trypsin was specified as the proteolytic enzyme, with up to 2 missed cleavage sites allowed. Searches used a reverse sequence decoy strategy to control for the false peptide discovery and identifications were validated using percolator software. Onlyproteins with two or more unique peptides were selected and those with less than 50% co- isolation interference were used for quantitative analysis. Reporter ion intensities were adjusted to correct for the impurities according to the manufacturer’s specification and the abundances of the proteins were quantified using the summation of the reporter ions for all identified peptides. The reporter abundances were normalized across all the channels to account for equal peptide loading. Data analysis and visualization were performed by Microsoft Excel or R. Crystallization of the hRPN13 Pru-XL44 Complex

[0447] A mixture of 374 μM hRpn13 Pru and 763 μM ubiquitin dissolved in 20 mM HEPES (pH 7.5), 100 mM NaCl and 1 mM TCEP and 509 μM XL44 dissolved in DMSO was crystallized by microbatch-under-oil at 4°C with equivolume of 0.1 M citric acid pH 4.6 and 20% PEG4000. Before data collection crystals were cryo-protected in a reservoir solution containing 25% ethylene glycol. Data Collection, Processing, and Structure Determination

[0448] X-ray diffraction data was collected at the Southeast Regional Collective Access Team (SER-CAT) using beamline ID-22 (wavelength 1.0 Å) available at the Advanced Photon Source, Argonne National Laboratory equipped with an EIGER 16M detector. The X-ray data sets were processed and scaled by SER-CAT auto-processing software and the structure solved by molecular replacement by using the program PHENIX (Adams et al., Acta. Crystallogr. D Biol. Crystallogr., 66, 213-221 (2010)) and the crystal structure of the hRpn13 Pru: ubiquitin: Rpn2 complex (PDB ID 5V1Y) as the search model (VanderLinden et al., J. Biol. Chem., 292, 9493-9504 (2017)). All structural refinement was performed by using PHENIX and REFMAC5 (CCP4 package) with default parameters. The Matthews coefficient (Matthews, J. Mol. Biol., 33, 491-497 (1968)) indicated the presence of two molecules in the asymmetric unit. The model building was done manually in Coot (Emsley et al., Acta. Crystallogr. D Biol. Crystallogr., 66, 486-501 (2010)). The protein atoms were fit into the electron density contoured at 3.0σ and 1.0σ for the Fo-Fc and 2Fo-Fc maps, respectively, and then XL44 was fit into the electron density near the Cys88 using the difference map contoured at 3.0σ. Water molecules were then fit into the electron density and refinement was performed. Electron density for the Leu33, Val38 andLys42 side chain atoms was not defined. Further refinement of these residues as well as the methyl group of XL44 was done by using the intermolecular NOE information obtained from the13C half-filtered NOESY experiment as described above using the program XPLOR-NIH 3.4 (Kassambara et al., “Drawing Survival Curves Using 'ggplot2',” R package version 0.4.9. (2021)) followed by REFMAC from CCP4 package. All data collection, refinement and validation statistics of the refined model are detailed in Table 2. Table 2: X-Ray Crystallographic Data Collection and Refinement Statistics for the hRPN13:Ubiquitin:XL44 Complex

[0449] The values in parenthesis represent the statistics for the last resolution shell. †Rmerge = ∑i|Ii(hkl) - 〈I(hkl)〉| / ∑hkl∑iIi(hkl), where I(hkl) is the intensity of reflection hkl, ∑hkl is the sum of overall reflections and ∑i is the sum over i measurements of reflection hkl. Modeling of XL44-Ligated hRPN13 Pru with SS Stereochemistry

[0450] An initial set of topology and parameter files for XL44 were generated by ACPYPE software and corrected to require the angles in the planar 6-membered rings to sum to 360°. XL44 was covalently bonded to the hRpn13 Cys88 sulfur atom with S, S stereochemistry at XL44 C12 and C13. With all other atoms frozen, NOE-derived distance restraints between hRpn13 Met31, Leu33, Val38, Lys42, Val85, Cys88, Val93, Val95 and XL44 were used to calculate 100 model structures of XL44-ligated hRpn13 Pru with S, S chirality for XL44 C12 and C13 by using XPLOR-NIH 3.4. The lowest energy structure was selected for analysis and display. Mining MMRF Data

[0451] MMRF CoMMpaSS data (release IA18) was accessed via the MMRF Researcher Gateway. After excluding patients with missing metadata, baseline bone marrow gene-level TPM estimates and overall survival measurements for n=754 patients were used to fit Cox survival regression models for PCLAF and PTTG1. Model fitting was performed using the survival package in R (Lin et al., Technometrics, 44, 85-86 (2002)). Kaplan-Meier plots wereconstructed for the patients in the bottom and top quartiles of gene expression using the ggsurvplot function from the survminer package for R (Kassambara et al., “Drawing Survival Curves Using 'ggplot2',” R package version 0.4.9. (2021)). Using the same TPM-normalized gene expression data, co-expression was assessed between PCLAF and PTTG1 using Pearson correlation for all 754 patient baseline bone marrow samples. Statistics and Reproductivity

[0452] The values for n represent replicates of biochemical assays displayed in FIG.1C, FIG.5B-5C (bottom panel), FIG.5D-5G, FIG.6D-6F (bottom panel), FIG.7G and FIG.13. For each figure, the number of replicates is indicated in the figure legend. IC50 values in FIG.5D-5E were analyzed by using the equation [Inhibitor] vs. normalized response - Variable slope in GraphPad Prism9. DepC50and d1 / 2values in FIG.5B-5C and FIG.6D-6F were calculated by using the equation [Inhibitor] vs. normalized response - Variable slope (FIG.5B and FIG.6D) and One phase decay (FIG.5C and FIG.6E-6F) in GraphPad Prism9. Mass Hunter Qualitative Analysis software (version B.07.00) with Bioconfirm Workflow was used to deconvolute mass spectra in FIG.1D. TMT analysis was performed in Microsoft Excel or R. Biophysical experiments and vitro assay including 2D NMR, DSF, MST, LC-MS and hRpn13 degradation assay were repeated at least once. Experiments using mammalian cells were repeated at least once. All replications were consistent. Only experiments in FIG.1F, 6A, 6D, and 6F were performed once. Data Availability

[0453] The structural coordinate for XL44-ligated hRPN13 Pru in this study has been deposited in the Protein Data Bank (PDB) under accession code 8FTQ. EXAMPLE 1

[0454] This example demonstrates that a compound of the claimed invention, XL44, binds to hRPN13 Pru. More specifically, this example utilizes 2D NMR spectra (FIG.1B), tryptophan quenching (FIG.1C) and mass spectrometry (FIG.1D) to demonstrate that a compound of the claimed invention, XL44, binds to hRpn13 Pru.

[0455] Addition of equimolar XL44 caused signal shifting in1H,15N HSQC spectra recorded on 20 PM15N-labeled hRpn13 Pru (FIG.1B), including for the intended Michael receptor Cys88 (FIG.1A and 1B). W108 is included among the amino acids shifted by XL44 addition (FIG.1B) and tryptophan quenching measured by differential scanning fluorimetry (λ350) has been an effective indicator of hRpn13 Pru interaction (Husnjak et al., Nature, 453, 481-8 (2008) and Lu et al., Nat. Commun., 12, 7318 (2021)). Tryptophan quenching by serial dilution of XL44 or XL5 (for comparison) into 1 PM hRpn13 Pru, was observed to have greater effects caused by XL44 compared to XL5 (FIG.1C). Consistent with a covalent interaction, liquid chromatography-mass spectrometry (LC-MS) detected a product of the appropriate molecular weight for XL44-ligated hRpn13 Pru following hRpn13 Pru incubation with 10-fold molar excess XL44 (FIG.1D). An isomeric mixture of XL44 was used for these biophysical experiments; however, separation of the two stereoisomers (FIG.8A) by silica gel column Liquid Chromatography (HPLC) indicated no preference for the E or Z isomer, as assessed by 2D1H,15N HSQC experiments of 20 PM15N-labeled hRpn13 Pru with 2-fold molar excess of E or Z XL44 stereoisomer (FIG.1B and FIG.8B). EXAMPLE 2

[0456] This example demonstrates that hRpn2 prevents the interaction of a compound of the claimed invention, XL44, with the proteasome-binding surface of hRpn13.

[0457] The XL44 indoline ring includes fluorine at the para position (FIG.1E, left panel) that is observable by 1D19F NMR. Experiments recorded on 50 PM XL44 dissolved in NMR buffer (20 mM sodium phosphate, 50 mM NaCl, 10% DMSO-d6, pH 6.5) without or with 2 mM DTT indicates DTT-dependent signal shifting (FIG.1E), indicating that XL44 can react with DTT. These signals are lost and a broadened signal at -122.63 ppm present following XL44 incubation with equimolar hRpn13 Pru (FIG.1E, orange). By contrast, no effect is observed following the incubation of equimolar hRpn2 (940-953) with XL44 (FIG.1E), indicating that XL44 binds to hRpn13 but not hRpn2. Addition of 0.5 molar or equimolar equivalents of hRpn2 (940-953) to the pre-incubated mixture of XL44 with equimolar hRpn13 Pru causes respective reduction or loss of hRpn13-bound XL44 signal, with corresponding reappearance of the DTTand unbound XL44 signals (FIG.1E). This experiment indicates that XL44 does not bind to hRpn2-bound hRpn13 Pru and moreover, that hRpn2 can displace XL44 from hRpn13. EXAMPLE 3

[0458] This example demonstrates that a compound of the invention, XL44, isolates hRPN13 from the whole cell lysate of RPMI 8226 cells.

[0459] To test whether XL44 can isolate hRpn13 from the whole cell lysate of RPMI 8226 cells, XL44 was fused with biotin, replacing the methoxy group with an amide linker (XL44B, FIG.9A, top panel). This modification reduced the affinity of the compound for hRpn13 Pru, as evaluated by 2D NMR showing a reduction in XL44-bound signals (FIG.9B compared to FIG. 1B). Despite the weaker affinity, streptavidin-bound biotinylated XL44 isolated hRpn13 from RPMI 8226 whole cell lysate (FIG.1F and FIG.18). For this experiment, biotin was used as a negative control and biotinylated XL5 (XL5B, FIG.9A, bottom panel) as a positive control. Since hRpn13 is a ubiquitin receptor, ubiquitin was also immunoprobed for to find co-enriched ubiquitinated proteins (Fig.18). EXAMPLE 4

[0460] This example demonstrates how a compound of the claimed invention, XL44, binds to hRPN13 Pru. In particular, this examples uses integrated NMR and X-ray crystallography to illustrate the structure of hRPN13 Pru-XL44.

[0461] To further characterize the interaction of XL44 with hRpn13 Pru, X-ray crystallography was used. Attempts to obtain crystals of hRpn13 Pru with XL44 were successful when ubiquitin was also included in the mixture, yielding a structure of hRpn13 Pru:ubiquitin:XL44 solved to 2.01 Å resolution (Table 2). Each asymmetric unit of the crystal contained two molecules of the hRpn13 Pru:ubiquitin:XL44 complex (Table 2). XL44 was found to bind to a similar region as XL5 (FIG.1A and 2A), tethered at Cys88 by a covalent bond between this amino acid’s sulfur atom and carbon 13 (C13) of XL44 (FIG.2A, insert). The electron density for XL44 was well resolved for the methoxybenzamide, central benzene and indoline-2-one rings, as was the thioester bond between XL44 and hRpn13 Cys88 and the surrounding region, allowing stereo assignment of the two chiral centers at C12 and C13 to Rand S respectively (FIG.2A, insert). Analysis of XL44 binding indicated that the XL44 indoline- 2-one ring interacts with hRpn13 Met31, Val85, and Phe106; the XL44 central benzene ring interacts with hRpn13 Pro89; and the XL44 methoxybenzamide ring interacts at opposite sides with hRpn13 Trp108 and Pro40 (FIG.2B). Akin to the three aromatic rings of XL44, the electron density for these interacting residues is well resolved (FIG.10A-10B). Except for the backbone atoms of Gly35 and Thr36, the hRpn13 β1 - β2 hairpin spanning Leu33 - Val38 was poorly defined (FIG.2C). Moreover, the electron density for the methyl group of the XL44 methoxybenzamide was also poorly defined (FIG.2a, insert). Disorder in the β1 - β2 hairpin is intrinsic to the hRpn13 Pru fold, as NMR experiments probing backbone amide dynamics of apo hRpn13 Pru indicated greater high frequency motions in this region of apo hRpn13 Pru (FIG.2D and FIG.10C).

[0462] To complement the crystal structure by providing information where the electron density map was poorly defined, NMR data on XL44-bound hRpn13 Pru was acquired. Unambiguous intermolecular NOE interactions were recorded between hRpn13 Pru and XL44 by acquiring a 3- or 2-dimensional1,13C half-filtered NOESY experiment on samples of13C- labeled hRpn13 Pru mixed with equimolar unlabeled XL44 (FIG.3A and FIG.10D-F) or of unlabeled hRpn13 Pru mixed with equimolar XL44 with its central benzene ring13C-labeled (XL44-13C6-CB) (FIG.3B). These NOESY experiments are designed to reveal intermolecular interactions for atoms within 6 Å of each other (Chen et al., Methods Mol. Biol., 832, 279-303 (2012)). Consistent with the saturation of the XL44 alkene by Michael addition, a signal for a hydrogen atom (H10) attached to C13 (FIG.10D, left panel) was observed with intermolecular NOE interactions to hRpn13 Pru Met31, Leu33, Val38, Val85, Cys88, and Val93 (FIG.10D, right panel).

[0463] In addition, interactions were recorded for the XL44 methyl group (FIG.2E) and hRpn13 Lys42 sidechain (FIG.3A); these atoms were missing in the electron density map (FIG. 2A and FIG.10B). The intermolecular interactions between the XL44 methyl group and hRpn13 Pru Met31, Pro40, Lys42, and Trp108 (FIG.3A) were used as distance constraints for structure calculations in XPLOR-NIH to define the orientation of the XL44 methyl group, freezing all other atoms to the XL44-bound hRpn13 crystal coordinates (FIG.2A). Further optimization was done by manual fitting of NOE-based intermolecular interactions between XL44 and hRpn13Leu33, Val38 and Lys42 (FIG.3A-3B and FIG.10D-10F) to reorient the side chains of these three residues in accordance with the experimental data. Finally, the structure was refined in CCP4 to obtain an NMR-assisted crystal structure that fit both the electron density map and intermolecular NOE information (FIG.3C).

[0464] Overall, the hRpn13 Pru:XL44:ubiquitin structure before and after NMR-based refinement are similar with a backbone r.m.s.d. of 0.117 Å for Cα atoms (FIG.3C), however the XL44 methyl group and side chain atoms of hRpn13 Leu33, Val38 and Lys42 are reoriented to fit the NMR data. The refined structure has identical molecular interactions for the two molecules in the asymmetric unit (FIG.11A-11B and Table 3). Moreover, the binding interface between hRpn13 Pru and ubiquitin was unaltered compared to a previously determined crystal structure of hRpn13 Pru with ubiquitin and hRpn2 (PDB ID 5V1Y, FIG.11C) (VanderLinden et al., J. Biol. Chem., 292, 9493-9504 (2017)). Table 3: Interactions Between XL44 and hRPN13 Pru Residues in A-and B-Chain

[0465] Whereas the crystal structure indicated RS stereochemistry for the C12,C13 chiral centers respectively, the in silico, structure predicted RR (C12,C13) stereochemistry (FIG.4A). Only the RS stereoisomer of XL44 was observed in the crystal structure, the NMR data indicatedheterogeneity at the C12 chiral center. For example, H18 was recorded to have two slightly different chemical shift values (FIG.10D), with one signal from XL44-bound to hRpn13 with an S chiral center at C12, as indicated by the hRpn13 Val38 methyl groups exhibiting stronger NOE interactions to XL44 H10 than XL44 H18 (FIG.10D). By contrast, NOEs (FIG.10D) from XL44 indoline-2-one H18 and H10 fit to either the R or S stereoisomer at C12. Specifically, NOEs to Leu33 (FIG.4B) and Met31 (FIG.4C) fit well to the R stereoisomer observed in the crystal structure, whereas those involving Val38, Val85, and Cys88 (FIG.10D) fit best to the S stereoisomer at C12, as modeled in FIG.11D.

[0466] These data indicate that XL44 binds to hRpn13 Pru with either R or S stereochemistry at C12 in solution even though only the R C12 chiral center crystalized. Neither the X-ray diffraction nor the NMR data provided evidence for the R chiral center at C13. EXAMPLE 5

[0467] This example demonstrates that a compound of the claimed invention, XL44, extends along an hRPN13 Pru channel mimicking hRPN2 interactions. In particular, this example demonstrates that a compound of the claimed invention, XL44, binds at the location of hRPN2 interaction (FIGS.4A, 4D, 4E) (Lu et al., Nat. Commun., 8, 15540 (2017) and VanderLinden et al., J. Biol. Chem., 292, 9493-9504 (2017)).

[0468] The XL44 methoxybenzamide group buried into a hRpn13 hydrophobic pocket formed by hRpn13 Pro40, Lys42, Val93 and Trp108, similar to hRpn2 Pro944 and Pro945 (FIG. 4D and 4E). By contrast, the XL54-methylbenzamide is directed towards hRpn13 Thr39 (FIG. 4F) where hRpn2 Pro947 is positioned (FIG.4E) when hRpn2 binds hRpn13. This positioning causes the XL54-methylbenzamide group to be more solvent exposed compared to the XL44 methoxybenzamide (FIG.11E).

[0469] Superposition of the hRpn13 Pru complexed structures shows translational movements of 4.2, 5.9 and 8.4 Å for the β1 – β2 hairpin upon binding XL5, XL44 and hRpn2 relative to apo state, respectively (FIG.11F). The positioning of the hRpn13 β1 – β2 hairpin is more similar to the hRpn2-bound state for XL44 compared to XL5 and allows interaction with Trp108 as well as closer contacts with Leu33 and Phe106 (FIG.4D-4F). Moreover, it was found that this end of XL44 could be extended. hRpn13 Pru bound to XL52 in which the XL44 methylgroup is replaced with 5-(4-methylphenyl)-2H-tetrazole (FIG.12). Addition of XL52 to15N- labeled hRpn13 Pru caused signal shifting similar to XL44 (FIG.1B). However, without wishing to be bound by any particular theory, and as discussed below, it is believed that XL52 may be unable to permeate the cells.

[0470] The XL44 central benzene ring forms hydrophobic interactions with hRpn13 Val38 at one side and Pro89 at the other (FIG.4G). Addition of a trifluoromethyl group (XL53) at the ortho position of this benzene ring reduced binding to hRpn13 Pru (FIG.12). Without wishing to be bound by any particular theory, it is believed that this could be the result of steric clashes with the backbone oxygen of hRpn13 Gln87 (FIG.4H). The XL44 indolin-2-one ring is buried within a pocked formed by hRpn13 Leu33, Val38, Val85, Gln87, Cys88, Val93, Val95 and Phe106 (FIG.4H). A weak hydrogen bond exists between the XL44 indolin-2-one oxygen and the hRpn13 Gln87 side chain amide group (FIG.4H). Replacement of the XL44 indolin-2-one ring with 2-phenylthiazol-4-one (XL54) or 4H-1,4-benzothiazin-3-one (XL55) caused reduced binding affinity to hRpn13 Pru (FIG.12). Similarly, and without wishing to be bound by any particular theory, it is believed that this is due to steric clashes with hRpn13 Phe106 or Leu33, respectively (FIG.4H). Reduced binding affinity was also caused by the substitution of XL44 H10 with a methyl group (XL56, FIG.12), also believed to be the result of steric clashes with hRpn13 Gln87 and Cys88 (FIG.4H). EXAMPLE 6

[0471] This example demonstrates that a compound of the claimed invention, XL44, induces loss of hRpn13Pruand hRpn13-dependent apoptosis.

[0472] To evaluate whether XL44 is efficacious in cells, XL44 was tested to determine whether it induces cell death by immunoblotting for the apoptosis marker cleaved caspase-9. Whole cell lysates from RPMI 8226 cells treated for 24 hours with 20 μM XL44 or DMSO vehicle control were immunoprobed to find cleaved caspase-9 prominent in XL44-treated cells whereas no effect was observed in the control experiment (FIG.5A, lane 3 versus 1). To test whether hRpn13 Pru is required for XL44 induction of apoptosis, a parallel experiment was performed with RPMI 8226 cells in which Exon 2 of the hRpn13 Pru domain is deleted and the resulting truncated hRpn13 protein (trRpn13) is present at low abundance (trRpn13-MM2), aspreviously described (Lu et al., Nat. Commun., 12, 7318 (2021)). Apoptosis was not induced in trRpn13-MM2 cells treated with XL44, indicating that hRpn13 Pru is required for XL44-induced apoptosis (FIG.5A, lane 4 versus 2).

[0473] The induction of apoptosis by XL44 is lost in RPMI 8266 trRpn13-MM2 cells, as indicated by apoptosis maker cleaved caspase-3 (FIG.19, lanes 1-4), and rescued by the reintroduction of hRpn13 into trRpn13-MM2 cells by using a lentiviral cDNA-based approach. Immunoblotting indicated successful reintroduction of hRpn13 into trRpn13-MM2 cells and its proteolysis to hRpn13Pru(FIG.19, lower panels, lanes 5-6). XL44 induced the production of cleaved caspase-3 in these hRpn13-expressing trRpn13-MM2 cells (FIG.19, lane 6 versus 5). Altogether, these data indicate that hRpn13 is required and sufficient for XL44-induced apoptosis (FIG.5A and FIG.19).

[0474] RPMI 8226 cells produce an hRpn13 fragment (hRpn13Pru) that were previously found to be degraded by XL5-derived PROTACs (XL5-VHL-2) but not XL5 itself (Lu, X. et al., Structure-Guided Bifunctional Molecules Hit a DEUBAD-lacking hRpn13 Species Upregulated In Multiple Myeloma, Nat Commun 12, 7318 (2021)). Surprisingly, XL44 treatment was found to cause a reduction in hRPN13Prulevels (FIG.5A, lane 3 versus lane 1). A half-maximal depletion concentration (DepC50) value with corresponding concentration-dependent increase in cleaved caspase-9 (FIG.5B) of 19 μM, reduced compared to the 39 μM DC50 of the XL5-derived PROTAC XL5-VHL-2 (Lu et al., Nat. Commun., 12, 7318 (2021)). Moreover, following treatment of RPMI 8226 cells with 20 μM XL44, a d1 / 2(differential protein abundance following treatment) value of 12 hours was measured (FIG.5C), shorter than the 16 hour d1 / 2 of XL5-VHL- 2 (Lu et al., Nat. Commun., 12, 7318 (2021)).

[0475] Since XL52 also bound to hRpn13 Pru, the efficacy of XL52 was tested in RPMI 8226 cells but it was found that it did not restrict cell viability (FIG.13). Without wishing to be bound by any particular theory, it is believed that this lack of activity might be due to the inability of XL52 to permeate the cells. An artificial membrane permeability assay (Eurofin) illustrated the permeability of XL44 and XL52 (Table 4).Table 4. Parallel Artificial Membrane Permeability Assay (PAMPA) for XL44 and XL52EXAMPLE 7

[0476] This example demonstrates that a compound of the claimed invention, XL44, reduces cell viability in an hRPN13-independent manner.

[0477] Cell viability was evaluated in RPMI 8226 WT and trRpn13-MM2 cells following 48-hour treatment with XL44 by measuring metabolism with an MTT (3-(4,5-dimethylthiazol-2- yl)-2,5-diphenyltetrazolium bromide) assay. This experiment revealed reduced viability in both cell lines, with IC50 values of 6.2 + 0.5 mM and 4.5 + 0.2 for WT and trRpn13-MM2 cells, respectively (FIG.5D), indicating a restriction of viability that is hRpn13-independent. Further testing found that each XL44 stereoisomer restricted RPMI 8226 cells with similar efficacy (FIG. 13B) consistent with the aforementioned NMR data (FIG.1B and FIG.8B).

[0478] XL44 also restricted the viability of other cell types, including breast cancer cell line MCF7, multidrug-resistant ovarian cancer cell line OVCAR-4, and metastatic gastrointestinal carcinoma model cell line OVCAR-5, with IC50 values of 2.3 + 0.5 μM, 3.9 + 0.2 μM and 7.1 + 0.9 μM, respectively (FIG.5E). By contrast, no effect was observed in non-cancerous human fibroblast 1634 and HS5 bone marrow-derived stem cells (BMSCs) up to 15 μM, albeit 25 μM and 50 μM XL44 reduced the viability of HS5 cells (FIG.5F). XL44-induced cell death was also measured in WT or trRpn13-MM2 cells by lactate dehydrogenase production following 48-hour treatment to find an equivalent cell killing effect (FIG.5G).

[0479] These data indicate that XL44 restricts the viability of multiple cancer cell lines through an hRpn13-independent mechanism. EXAMPLE 8

[0480] This example demonstrates that a compound of the claimed invention, XL44, causes loss of PCLAF, its downstream target PTTG1, and RRM2.

[0481] Tandem mass tag mass spectrometry (TMT-MS) was performed on lysates from RPMI 8226 WT cells treated for 8 hours with 20 μM XL44 or DMSO (vehicle control). Five proteins were identified as reduced, including PCNA clamp associated factor (PCLAF), ribonucleotide reductase regulatory subunit M2 (RRM2), proline rich 11 (PRR11), ribosomal biogenesis factor (RBIS), and cell division cycle 25A (CDC25A) (FIG.6A). To interrogate these findings further, lysates from RPMI 8226 WT or trRpn13-MM2 cells following 24-hour treatment with 20 μM XL44 or DMSO (vehicle control) were immunoprobed for proteins with available antibodies, including PCLAF and RRM2. PCLAF was depleted in RPMI 8226 WT and trRpn13-MM2 cells following XL44 treatment and was also at reduced levels in trRpn13-MM2 compared to WT cells (FIG.6B).

[0482] These results were corroborated in an experiment with RPMI 8226 WT cells treated with varying concentrations of XL44 (FIG.6C). Pituitary tumor-transforming gene 1 (PTTG1) is a downstream target of PCLAF(Liu et al., Cell Death Dis., 13, 178 (2022)) and this protein was also reduced by XL44 treatment (FIGs.6B-6C) and similar to PCLAF, reduced in trRpn13-MM2 (FIG.6B).

[0483] Two bands for RRM2 were detected (FIGs.6B-6C) by mouse anti-RRM2 antibodies, with the upper band reduced in XL44-treated RPMI 8226 WT or trRpn13 cells (FIG.6B) with dose dependency (FIG.6C). HCT116 cells are more easily transfected than RPMI 8226 cells and transfection with 50 nM RRM2 or scrambled (as a control) siRNAs caused the upper band and not the lower band to be reduced (FIG.14), indicating the lower band to be non-specific. Moreover, only the upper band was detected by rabbit anti-RRM2 antibodies (FIG.6B), which also indicated XL44-induced loss of RRM2 in WT and trRpn13-MM2 cells.

[0484] Treatment of RPMI 8226 cells with varying XL44 concentration indicated a DepC50 value of 8.4 + 1.3 μM or 7.8 + 1.7 μM for PCLAF or RRM2 (FIG.6D), respectively. Moreover d1 / 2values for PCLAF and RRM2 following 20 μM XL44 treatment were measured to be 3.4 + 1.3 and 11.2 + 5.8 hours, respectively (FIG.6E-6F).

[0485] The correlation of PCLAF, PTTG1 and RRM2 expression levels with overall survival was evaluated by using a large-scale patient dataset (Lonial et al., Blood, 124, 722 (2014)). These analyses indicated strong correlation, with p-values of <0.0001 for each (FIG.6G and FIG.15A). PCLAF and PTTG1 expression was also correlated (FIG.15B). A weakercorrelation was observed for the hRpn13-expressing gene ADRM1, with a p-value of 0.07 (FIG. 15C). However, these data are for full length hRpn13. Without wishing to be bound by any particular theory, it is believed that a stronger correlation exists for hRpn13Pruabundance, which is not available in this dataset.

[0486] Altogether, these data indicate that in addition to inducing the loss of hRpn13Pru, XL44 causes PCLAF, its downstream target PTTG1, and RRM2 to be depleted from cells.

[0487] The mechanism of RRM2 and PCLAF reduction by XL44 was examined further by testing whether these two proteins can be isolated from RPMI 8226 lysates by XL44B-bound streptavidin, as was done for hRpn13 (FIG.1F and FIG.18). RRM2 but not PCLAF was enriched by streptavidin-bound XL44B and migrated at a molecular weight characteristic of the unmodified (45 kDa, not ubiquitinated) protein (FIG.20).

[0488] In addition, any PCLAF interaction with XL44 was tested for using nuclear magnetic resonance (NMR) spectroscopy and liquid chromatography–mass spectrometry (LC–MS). Comparison of 2D1H,15N HSQC spectra recorded on 20 mM15N-labeled PCLAF without and with 10-fold molar excess XL44 yielded no spectral changes (FIG.21), indicating that XL44 does not bind to PCLAF in this assay. Similarly, incubation of XL44 with PCLAF yielded <1% XL44-adducted PCLAF (FIG.22). Altogether, these data suggest that XL44 can interact directly with RRM2, but not PCLAF.

[0489] hRpn13 targeting molecules, or its depletion by RNAi, disrupt cell cycle progression. As a result, it is possible that the loss of PCLAF and RRM2 (and perhaps also CDC25A and PRR11) is caused by a cell cycle defect. However, examination of all APC / C substrates detected in XL44-treated RPMI 8226 cells by TMT-MS (FIG.6A) indicated that only PCLAF, RRM2, and CDC25A were reduced by XL44 (FIG.23), suggesting a more specific mechanism of action. EXAMPLE 9

[0490] This example demonstrates that hRpn13Pruis generated spontaneously by proteasomes with clearance induced by a compound of the claimed invention, XL44, following ubiquitination.

[0491] Experiments were performed to test the role of the proteasome and ubiquitination machinery in XL44-induced loss of hRpn13Pruand PCLAF. The presence of hRpn13Pruin cellswas previously found to require proteasome activity (Lu et al., Nat. Commun., 12, 7318 (2021)). To test whether the proteasome can spontaneously generate hRpn13Pruby partially proteolyzing hRpn13, full length hRpn13 produced from E. coli was incubated with commercially available 26S proteasomes for 10 minutes and the mixture was immunoprobed with anti-hRpn13 antibodies. This experiment revealed the lower molecular weight hRpn13 bands following exposure to proteasomes, with an hRpn13 proeolyzed product that is consistent with the molecular weight of hRpn13Pru(FIG.7A).

[0492] To further study hRpn13 proteolysis in cells the proteasome inhibitor carfilzomib, the UAE inhibitor MLN7243 (UAEi), and the NAE inhibitor MLN4924 (NAEi) were used to assess the role of the proteasome, ubiquitin E1-activating, and Nedd8 E1-activating enzymes, respectively. Treatment with 100 nM carfilzomib for 24 hours led to reduced levels of hRpn13Pru(FIG.7B, lane 3 versus lane 1), in support of proteasome activity being required to generate this hRpn13 cleavage product. Carfilzomib treatment resulted in higher molecular weight bands being detected between 51 and 64 kDa for hRpn13, which were also present to a lesser extent in XL44-treated cells (FIG.7B, lane 2). Without wishing to be bound by any particular theory, and based on previous findings with XL5-based PROTACs (Lu et al., Nat. Commun., 12, 7318 (2021)), it is believed that these hRpn13 species were ubiquitinated hRpn13. Their reduction was observed following co-treatment for 22 hours of XL44 with 1 μM UAEi (with 2 hours of UAEi pre-treatment), with no impact by co-treatment with NAEi (FIG.7B, lane 6). Correspondingly, lower molecular weight hRpn13 species were increased with UAE inhibition (FIG.7B, lane 5). Depletion of hRpn13Prulevels by XL44 was not affected by UAEi (FIG.7B, lane 5, and FIG. 16A), indicating ubiquitin-independent proteolysis of hRpn13Pruto the smaller hRpn13 species (FIG.7C).

[0493] Collectively, these findings indicate a stepwise process of spontaneous proteolysis of hRpn13 by the proteasome to generate hRpn13Pruand XL44-induced further proteolysis to fragments which are cleared in a ubiquitin-dependent manner that does not involve neddylation or cullins (FIG.7B-7C).EXAMPLE 10

[0494] This example demonstrates that proteasome activity is required for depletion of PCLAF and RRM2 induced by a compound of the claimed invention, XL44.

[0495] The levels of PCLAF were also immunoprobed and showed an increase with carfilzomib treated RPMI 8226 cells (FIG.7B, lane 3 versus lane 1). Moreover, 22-hour co- treatment of 100 nM carfilzomib (with 2 hour pre-treatment) with 20 μM XL44 led to increased PCLAF abundance compared to untreated cells (FIG.7B, lane 4 versus 1) whereas negligible effects were observed by co-treatment of XL44 with UAEi (FIG.7B, lanes 5 compared to 2 and FIG.16A). Without wishing to be bound by any particular theory, it is believed that these findings suggest that PCLAF is turned over by proteasomes with and without XL44 treatment by a ubiquitin-independent process (FIG.7D). This is consistent with a previous report of it being intrinsically disordered (De Biasio et al., Biophys. J., 106, 865-874 (2014)).

[0496] XL44 as a single treatment induced loss of RRM2 (FIG.7E, lane 2 compared to 1), but its co-treatment with carfilzomib (FIG.7E, lane 4) and to a lesser extent UAEi (FIG.7E, lane 6) prevented this reduction. Carfilzomib as a single treatment or co-treatment with XL44 and UAEi co-treatment with XL44 caused a lower molecular weight band to appear suggesting partial proteolysis of RRM2; no such effect was observed with NAEi (FIG.7E, lane 7).

[0497] Altogether, these data indicate that XL44-induced depletion of PCLAF and RRM2 requires proteasome activity (Fig.7D). EXAMPLE 11

[0498] This example demonstrates that a KEN box but not uniquitination is required for depletion of PCLAF induced by a compound of the claimed invention, XL44.

[0499] The E3 ligase complex APC / CFZR1is reported to promote the degradation of PCLAF (Emanuele et al., Proc. Natl. Acad. Sci. USA, 108, 9845-9850 (2011)) and RRM2 (Chabes et al., Proc. Natl. Acad. Sci. USA, 100, 3925-9 (2003)) by recognition of KEN boxes in their protein sequences.

[0500] Whether XL44 enhances ubiquitination of PCLAF by APC / CFZR1was tested.5 μM fluorescently labeled PCLAF was preincubated with equimolar FZR1 and 8-fold molar excess XL44 or DMSO (as a control) for 1 hour, followed by addition of ubiquitin and other APC / Csubunits to initiate ubiquitination of PCLAF. XL44 did not affect PCLAF ubiquitination by APC / CFZR1(FIG.16B), consistent with the ubiquitin-independent clearance of PCLAF by XL44 (FIG.7B and FIG.16A).

[0501] This example further demonstrates that loss of hRpn13Pruby a compound of the claimed invention, e.g., XL44, is ubiquitin-independent, as its partial proteolysis to lower molecular weight species does not require UAE activity (FIG.7B-7C). These products, however, were stabilized by E1 inhibition. Thus, without wishing to be bound by any particular theory, it is believed that complete depletion of hRpn13 by proteasomes requires ubiquitination (FIG.7B- 7C). In the case of hRpn13, however, ubiquitin is not needed for its recruitment to the proteasome, as it binds to hRpn2 (Lu et al., PLoS One, 10, e0140518 (2015), Lu et al., Nat. Commun., 8, 15540 (2017), and Osei-Amponsa et al., Trends Biochem. Sci., 47, 950-964 (2022)). The proteasome generates hRpn13Prueven in isolation of other cellular factors (FIG.7A).

[0502] Without wishing to be bound by any particular theory, it also is believed that the compounds of the claimed invention, e.g., XL44, act as a molecular glue to induce or enhance hRpn13Prubinding to an interaction partner. hRpn13 is present both on and off proteasomes (Lu et al., Nat. Commun., 8, 15540 (2017)), and it is believed that an enhanced interaction through a compound of the claimed invention, e.g., XL44, could involve extraproteasomal hRpn13 and / or compete with hRpn13 binding to the proteasome. Unlike carfilzomib (FIG.7B) or XL5-VHL-2 (Lu et al., Nat. Commun., 12, 7318 (2021)), XL44 does not cause the accumulation of ubiquitinated hRpn13 (FIG.7B). This finding supports the distinct clearance mechanism of XL44 compared to XL5-VHL-2.

[0503] To test whether the KEN box sequence is required for XL44-induced depletion of PCLAF, HCT116 WT cells were transfected with 1 μg empty vector (pcDNA3.1+N-HA), HA- PCLAF (WT) or HA-PCLAF with alanine substitutions at KEN box amino acids K78 and E79 (K78A and E79A) and after 48 hours, the cells were treated with DMSO (control) or 20 μM XL44. XL44 treatment caused reduction of endogenous PCLAF (FIG.7F, lane 2 versus lane 1) and overexpressed HA-PCLAF (FIG.7F, lane 4 versus lane 3), but not of HA-PCLAF (AA) (FIG.7F, lane 6 versus lane 5).

[0504] Altogether, these data indicate that XL44 depletion of PCLAF occurs by a mechanism involving its KEN box, without altering its ubiquitination by APC / CFZR1.EXAMPLE 12

[0505] This example demonstrates that a compound of the claimed invention, XL44, restricts cell viability by dually targeting hRpn13Pruand PCLAF.

[0506] To test whether XL44 restriction of cell viability requires PCLAF, HCT116 WT and trRpn13 cells, were used; trRpn13 HCT116 cells, like trRpn13-MM2 cells, produce a truncated form of hRpn13 that begins at M109, thereby deleting much of the Pru domain (Lu et al., Nat. Commun., 8, 15540 (2017)). PCLAF levels were knocked down by siRNA in HCT116 WT (FIG. 16C, lane 2 versus lane 1) and trRpn13 (FIG.16C, lane 6 versus lane 5) cells(with comparative scramble siRNA), which were treated with XL44. PCLAF was reduced by XL44 in WT (FIG. 15C, lane 3 versus 1) and trRpn13 cells (FIG.16C, lane 7 versus lane 5) cells, with greatest loss by XL44 and PCLAF siRNA co-treatment (FIG.16C, lane 4 versus lane 2 and lane 8 versus lane 6).

[0507] In parallel with the experiment shown in FIG.16C, cell metabolism was measured by the MTT assay shown in FIG.4D. XL44 restriction of cell viability was reduced in HCT116 trRpn13 compared to WT cells (FIG.24 and FIG.7G, top left panel), indicating greater efficacy when hRpn13 Pru domain is present. Moreover, knockdown of PCLAF reduced XL44 restriction of cell viability in WT cells compared to the scramble control (FIG.7G, top right) and to a lesser extent, in trRpn13 (FIG.24 and FIG.7G, bottom left) cells. XL44 was less potent in HCT116 trRpn13 cells treated with PCLAF siRNA compared to the single loss of either hRpn13 or PCLAF, with a clear difference compared to WT control cells (FIG.24 and FIG.7G, bottom right panel). Without wishing to be bound by any particular theory, in view of this data, it is believed that PCLAF contributes to XL44 restriction of cell viability.

[0508] This example demonstrates that knockdown of PCLAF in trRpn13 HCT116 cells impaired XL44-induced restriction of cell viability (FIG.7G). PCLAF is required for cell stemness and plasticity of breast cancer cells and intestinal stem cells (Wang et al., Nat. Commun., 7, 10633 (2016) and Kim et al., Dev. Cell, 44, 582-596, e4 (2018)), with its levels negatively correlated with survival for patients with multiple myeloma (FIG.6G). XL44-induced apoptosis in multiple myeloma cells therefore has the potential to increase the survival probability of multiple myeloma patients. Notably, XL44 is more drug-like, with its small size compared to existing PROTACs. It is also cell permeable, motivating further testing inpreclinical trials for use in hRpn13Pru-producing cells, with upregulated PCLAF and RRM2, such as multiple myeloma cells. EXAMPLE 13

[0509] This example demonstrates that hRpn13 and hRpn13Pruare both highly expressed in human xenografts of myeloma and prostate cancer grown in mice. Xenografts of human cancer cell lines were grown in the flanks of 6-8 week old NOD-scid-gamma (NSG) mice with myeloma cells or athymic nude mice with prostate cancer cells. Immunoblots showed highly expressed hRpn13 and hRpn13Prulevels in both myeloma (FIG.17A-17B) and prostate cancer (FIG.17B) cell lysates.

[0510] These results suggest that Abcam antibody 157218 for ADRM1 recognizes hRpn13, and Abcam antibody 157185 recognizes both hRpn13 and the cleavage product hRpn13Pru. EXAMPLE 14

[0511] This example demonstrates that XL69 and XL80 are more potent than XL44 in an MTT assay of RPMI 8226.

[0512] Cell viability was evaluated in RPMI 8226 cells following 48-hour treatment with XL44, XL69, and XL80 by measuring metabolism with an MTT (3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide) assay. This experiment revealed reduced viability in RPMI 8226 cells, with XL69 and XL80 exhibiting increased potency relative to XL44 (FIG.25).

[0513] As mentioned in Example 7, XL44 exhibited no effect in HS5 bone marrow-derived stem cells (BMSCs) up to 15 μM, albeit 25 μM and 50 μM XL44 reduced the viability of HS5 cells (FIG.5F). For XL69 and XL80, no effect was observed in HS5 bone marrow-derived stem cells (BMSCs) up to 10 μM; however, 25 μM and 50 μM of XL69 and XL80 began to reduce the viability of HS5 cells (FIG.25).

[0514] These data indicate that XL69 and XL80 exhibit a similar, albeit more potent, effect to XL44 on RPMI 8226 cancer cells.EXAMPLE 15

[0515] This example provides exemplary compositions and methods for formulating compounds of the claimed invention.

[0516] To perform in vivo experiments (e.g., mouse pharmacokinetics experiments) using XL44, the following nanosuspension formulations were developed. However, prior to formulating XL44, the physical form of XL44 was determined by polarized light microscopy (PLM). With a multifunction microscope, XL44 compound powder was immersed in paraffin oil and visualized by cross-polarization at 20x magnification (FIG.26). XL44 bulk powder has a distinct rod-like morphology and exhibited birefringence, indicating a crystalline form.

[0517] Different wet-milling conditions were screened for converting XL44 into nanocrystals. In particular, approximately 5 mg of XL44 was weighed out into an amber vial (4 mL), followed by the addition of 1 gram of milling beads (0.2 mm or 0.5 mm), polymer (0.33%), and surfactant (0.01%) to achieve a 40 mM concentration using the process and vehicles set forth in FIG.27A. The compound load for the formulations was at 1.5%, as each sample was prepared at 40 mM or 15.5 mg / mL compound. XL44 slurries were stirred and wet-milled for 24 hours at room temperature and then collected for analysis.

[0518] 24 formulation preparations were performed on XL44 at 40 mM to vary vehicle’s polymer stabilizer, surfactant stabilizer, or milling bead size. After recovery from wet-milling, XL44 was diluted to 34.6 mM in each corresponding vehicle and analyzed by dynamic light scattering (DLS) to determine average particle size diameter of 50% (D50) or 90% (D90) of the particles. Each formulation was prepared in singlet. The results (conditions and outcomes) of the 24 different formulations are set forth in Table 5. Table 5. Conditions and Outcome for Nanosuspension Screening of XL44

[0519] The 24 formulations were ranked by their particle size when the formulation was freshly prepared, when suspended in biorelevant simulated fluid, and when stored at 4 ºC for two weeks. For the initial assessment of particle size, each formulation was diluted in water approximately 10-fold and measured by DLS at 37 ºC. The results are set forth in FIG.27B. The distribution of D50 and D90 values are the average particle size diameter of 50% and 90% the particles, respectively, and show the overall range of particle sizes within a formulation. A relatively large difference between the D50 and D90 values, such as with the carboxymethylcellulose (CMC) formulations, indicates a wide range of particle sizes and less particle uniformity. However, certain formulations containing methylcellulose (MC), polyvinylpyrrolidone (PVP K29-32), and hydroxypropyl cellulose (HPC) were observed to have D90 values <200 nm and uniform particle dispersion.

[0520] To identify the top formulations, the formulations were evaluated in biorelevant media, such as FaSSGF (fasted state simulated gastric fluid), FaSSIF (fasted state simulated intestinal fluid), or SPF (simulated peritoneal fluid). Without wishing to be bound by any particular theory, it is believed that stable nanocrystals are expected to have little to no change in particle size when suspended in biorelevant media, whereas an increase in particle size denotesaggregation and therefore nanocrystal instability. The 24 screening formulations were diluted approximately 10-fold into water (control) or the simulated biorelevant media and measured by DLS at 37 ºC. The results are set forth in FIG.28A. Formulations, such as 0.33% PVP K29-32 with surfactants, 0.01% Tween 80 or sodium dodecyl sulfate (SDS), were observed to have high variability in simulated fluids. Particularly, the D90 values for Formulations #14-16 increased from 200 – 500 nm to >800 nm in FaSSIF. However, such a significant increase is not observed with PVP K29-32 supplemented with Pluronic F127 (see Formulations #17 and #18), signifying that Pluronic F127 aids XL44 nanocrystal stability. CMC Formulations #7-12 exhibited an opposite effect, such that particle size decreased within the biorelevant media, indicating that CMC may encapsulate the larger particles in the formulation and decrease the overall particle size distribution.

[0521] Longer-term in vivo studies, such as efficacy and pharmacokinetic / pharmacodynamic (PK / PD), require a formulation to be stable over time. An unstable formulation may require frequent preparation, and higher frequency of nanosuspension preparation may result particle size variability of the dose. Therefore, the screening formulations were stored at 4 ºC, and particle sizes were measured fresh and after 1 - 2 weeks of storage. The results are set forth in FIG.28B. As is apparent from the results set forth in FIG.28B, CMC Formulations #7-12 had a decreasing D90 value over time, which is similar to the effect seen with simulated fluid. With the potential solubilization effect that CMC has on XL44 particles in biorelevant media and over time, these formulations were not further considered for the in vivo studies due to environmental variability. Overall, MC Formulations #1-6, PVP Formulation #18, and HPC Formulations #19- 24 all exhibited stable particle size with 4 ºC storage over 2 weeks.

[0522] Within this small-scale formulation screen and based on the following criteria, (1) day 0 particle size distribution (D50 versus D90), (2) stability in simulated fluids for oral or intraperitoneal administration, and (3) storage stability at 4 ºC for two weeks, MCT (Formulations #1 and #2 with 0.33% methylcellulose, 0.01% Tween 80) and PPF (formulation #18 with 0.33% PVP K29-32, 0.01% Pluronic F127) were selected as potential options for further screening of XL44, XL69, and XL80 nanosuspensions (see Example 16). Although milling bead size did not create a major difference between MCT Formulations #1 and #2, 0.5 mm beads in the PPF formulation #17 had a wider range between the D50 and D90 values thanPPF formulation #18 (FIG.28B). Therefore, 0.2 mm beads were found to be best performing. For the preliminary screening of XL44, XL69, and XL80 nanosuspensions set forth in Example 16, PPF was chosen due to preliminary saturated solubility data in simulated fluids, FaSSGF and FaSSIF (data not shown). EXAMPLE 16

[0523] This example demonstrates the stability of compounds disclosed herein, by preparing XL44, XL69, and XL80, as small-scale nanosuspensions in polyvinylpyrrolidone and surfactant (i.e., Pluronic F127).

[0524] Compounds XL44, XL69, and XL80 were used to prepare small-scale nanosuspensions in PPF at 40 mM, i.e., using Formulation #18 from Example 15. The average particle diameter results for XL44, XL69, and XL80 in biorelevant media and with 2-week storage at 4 ºC are set forth in FIG.29A.

[0525] As is apparent from the results set forth in FIG.29A, XL69 and XL80 yielded stable nanoparticles (D90 < 400 nm) in PPF at 40 mM, i.e., using Formulation #18 from Example 15, like XL44.

[0526] In addition, as demonstrated by the results set forth in FIG.29B, all three nanosuspensions (i.e., XL44, XL69, and XL80) exhibited birefringence under cross-polarization. Even the XL80-PPF nanosuspension, which visually had low birefringent intensity due to having the smallest particle size and rapid Brownian motion, exhibited birefringence under cross- polarization. EXAMPLE 17

[0527] This example demonstrates the bioavailability of compounds of the claimed invention, XL44, XL69, and XL80, as measured by oral gavage (PO) and intraperitoneal (IP) administration to mice.

[0528] Compounds XL44, XL69, and XL80 were formulated in PPF vehicle and dosed at 48 mg / kg in CD-1 male mice. A total of 7 groups (n=3 per group) were dosed by oral gavage (PO) and intraperitoneally (IP) administration. Formulations, which were nanosuspension (NS) orsuspension (S), were diluted approximately 10-fold in water and measured by dynamic light scattering (DLS), and the results are set forth in Table 6. Table 6. Dosing Formulations for Pharmokinetic Mice Studies

[0529] After administration, blood was sampled at 0.083, 0.25, 0.5, 1, 2, 4, 8, and 24 hours, and the concentration of XL44, XL69, and XL80 in the blood is plotted in FIG.30.

[0530] As is apparent from the results set forth in FIG.30, bioavailability of XL44, XL69, and XL80 was superior when administered by IP, as compared to oral administration. In addition, XL44, XL69, and XL80 achieved similar maximum blood concentrations at the given dose by IP administration. No adverse effects were observed for IP and oral administration during this time frame.

[0531] These results for XL44, XL69, and XL80 illustrate that a common vehicle and formulation can be used for administration of compounds disclosed herein.

[0532] When XL44 nanosuspensions in PPF were administered intraperitoneally (IP) at a dose of 48 mg / kg (daily for 5 days) to tumor (subcutaneously growing RPMI-8226 myeloma cells)-bearing NSG (NOD-SCID gamma) mice, tumor volumes (Day5-Day0: FIG.31A, FIG. 31B) and final weights at day 5 (FIG.31C) were lower in XL44-treated mice relative to vehicle treated controls.

[0533] All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.

[0534] The use of the terms “a” and “an” and “the” and “at least one” and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The use of the term “at least one” followed by a list of one or more items (for example, “at least one of A and B”) is to be construed to mean one item selected from the listed items (A or B) or any combination of two or more of the listed items (A and B), unless otherwise indicated herein or clearly contradicted by context. The terms “comprising,” “having,” “including,” and “containing” are to be construed as open-ended terms (i.e., meaning “including, but not limited to,”) unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0535] Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variationsthereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

Claims

CLAIM(S):

1. A compound of Formula (I):Formula (I) or a pharmaceutically acceptable salt thereof, wherein X1is N or CR1; X2is N or CR2; X3is N or CR3; X4is N or CR4; X5is N or CR5; each of R1, R2, R3, R4, and R5is independently selected from hydrogen, halogen, −OH, −CF3, C1-C6 alkylamino, substituted or unsubstituted C1-C6 alkyl, C1-C6 alkoxy, perhaloalkyl, perhaloalkoxy, haloalkoxy, heteroarylalkoxy, −O-Bn-tetrazolyl, aryloxy substituted with 5- or 6- membered aryl or heteroaryl, and benzyloxy substituted with 5- or 6-membered aryl or heteroaryl; provided that no more than two of X1-X5are N, and at least one of X1-X5is not CH; W is selected from substituted or unsubstituted C1-C6alkyl, C1-C6alkoxy, −S(O)2−, (O)2NR11−,R12−, pyrazolyl, 1,2,3-triazolyl, 1,2,4-triazolyl, tetrazolyl, imidazolyl, oxadiazolonyl and isoxazolyl; Y is substituted or unsubstituted aryl or heteroaryl ring having 5-6 members, orsubstituted or unsubstituted bicyclic ring or two-ring system, the bicyclic ring or two-ring system having 9-11 members; and Z is selected from Z1, Z2, Z3, Z4, and Z5,wherein X6is N or CR6; X7is N or CR7; X8is N or CR8; X9is N or CR9; X10is N or CR10; each of R6, R7, R8, R9, and R10is independently selected from hydrogen, halogen, cyano, perhaloalkyl, substituted or unsubstituted C1-C6 alkyl, perhaloalkoxy, 5- or 6-membered substituted or unsubstituted aryl ring, 5- or 6-membered substituted or unsubstituted heteroaryl ring, −R12CN, −NO2, −R12NO2, −OH, −R12OH, −C(O)OR11, −R12C(O)OR11, −NR112, −R12NR112, −C(O)NR112, −R12C(O)NR112, −B(OR11)2, −R12B(OR11)2, −S(O)2NR112, and −R12S(O)2NR112;provided that no more than two of X6-X10are N; wherein R9and R10, R8and R9, R7and R8, or R6and R7together with the atoms to which they are attached optionally form a 5- or 6-membered substituted or unsubstituted aryl ring, or a 5- or 6-membered substituted or unsubstituted heteroaryl ring; each R11is independently selected from hydrogen and substituted or unsubstituted C1-C6alkyl; and each R12independently is C1-C6 alkyl.

2. The compound of claim 1, wherein Y is selected from:

3. The compound of claim 1 or claim 2, wherein the compound is of Formula (Ia):Formula (Ia) or a pharmaceutically acceptable salt thereof, wherein R3is selected from hydrogen, chlorine, methyl, methoxy and –O-Bn-tetrazolyl; R5is selected from hydrogen and halogen; and provided that at least one of R3and R5is not hydrogen.

4. The compound of any one of claims 1-3, wherein the compound is of Formula (Ib):Formula (Ib) or a pharmaceutically acceptable salt thereof, wherein R3is selected from hydrogen, chlorine, methyl, methoxy and –O-Bn-tetrazolyl; R5is selected from hydrogen and chlorine; provided that at least one of R3and R5is not hydrogen; R7is selected from hydrogen and cyano; R8is selected from hydrogen, fluorine, −OH, −C(O)NH2, −C(O)OH, −S(O)2NH2, and −B(OH)2; provided that at least one of R7and R8is not hydrogen; and each R11is independently selected from hydrogen and methyl.

5. The compound of claim 1, wherein the compound is selected from:, or a pharmaceutically acceptable salt thereof.

6. The compound of claim 1, wherein the compound is:or a pharmaceutically acceptable salt thereof.

7. The compound of claim 1 or claim 2, wherein the compound is of Formula (Ic):Formula (Ic) or a pharmaceutically acceptable salt thereof, wherein R3is selected from hydrogen, chlorine, and methoxy; R5is selected from hydrogen and chlorine; and provided that at least one of R3and R5is not hydrogen.

8. The compound of claim 1, wherein the compound is selected from:, or a pharmaceutically acceptable salt thereof.

9. The compound of claim 1 or claim 2, wherein the compound is of Formula (Id):Formula (Id)or a pharmaceutically acceptable salt thereof, wherein R8is selected from hydrogen, fluorine, methyl, and methoxy; and R9is selected from hydrogen, methyl, and methoxy; R10is selected from hydrogen, fluorine, methyl, and methoxy; provided that at least one of R8-R10is not hydrogen; and R11is selected from hydrogen and methyl.

10. The compound of claim 1, wherein the compound is selected from:, or a pharmaceutically acceptable salt thereof.

11. The compound of claim 1, wherein the compound is of Formula (Ie):Formula (Ie) or a pharmaceutically acceptable salt thereof.

12. The compound of claim 11, wherein the compound is of Formula (If):Formula (If) or a pharmaceutically acceptable salt thereof, wherein X2is N or CR2, and when X2is CR2, R2is hydrogen or fluorine;R3is selected from hydrogen, chlorine, methyl, methoxy, and –O-Bn-tetrazolyl; R4is selected from hydrogen and chlorine; R5is selected from hydrogen and chlorine; provided that at least one of R3-R5is not hydrogen; R7is selected from hydrogen, fluorine, and cyano; R8is selected from hydrogen, fluorine, and cyano; provided that at least one of R7and R8is not hydrogen; each R11is independently selected from hydrogen and methyl; and each R12independently is C1-C6 alkyl.

13. The compound of any one of claim 1, wherein the compound is selected from:, or a pharmaceutically acceptable salt thereof.

14. The compound of claim 1 or claim 2, wherein the compound is of Formula (Ig):Formula (Ig) or a pharmaceutically acceptable salt thereof, whereinR3is selected from hydrogen and methoxy; R5is selected from hydrogen and chlorine; provided that at least one of R3and R5is not hydrogen; R7is selected from hydrogen, fluorine, and cyano; R8is selected from hydrogen, fluorine, and cyano; and provided that at least one of R7and R8is not hydrogen.

15. The compound of claim 14, wherein the compound is of Formula (Ih):Formula (Ih) or a pharmaceutically acceptable salt thereof, wherein W is selected from −C(O)NH−, −S(O)2NH−, pyrazolyl, and 1,2,3-triazolyl.

16. The compound of claim 1, wherein the compound is:or a pharmaceutically acceptable salt thereof.

17. The compound of claim 1, wherein the compound is:or a pharmaceutically acceptable salt thereof.

18. The compound of claim 1 or claim 2, wherein the compound is of Formula (Ii):Formula (Ii) or a pharmaceutically acceptable salt thereof.

19. The compound of claim 18, wherein the compound is of Formula (Ij):Formula (Ij) or a pharmaceutically acceptable salt thereof.

20. The compound of claim 1 or claim 2, wherein the compound is of Formula (Ik):Formula (Ik) or a pharmaceutically acceptable salt thereof, wherein R3is selected from hydrogen, chlorine, and methoxy; R5is selected from hydrogen and chlorine; and provided that at least one of R3and R5is not hydrogen.

21. The compound of claim 20, wherein the compound is of Formula (Im):Formula (Im) or a pharmaceutically acceptable salt thereof, wherein R3is selected from hydrogen, chlorine, and methoxy; R5is selected from hydrogen and chlorine; and provided that at least one of R3and R5is not hydrogen.

22. The compound of claim 1, wherein the compound is selected from:, or a pharmaceutically acceptable salt thereof.

23. The compound of claim 1 or claim 2, wherein the compound is of Formula (In):or a pharmaceutically acceptable salt thereof.

24. The compound of claim 23, wherein the compound is of Formula (Io):or a pharmaceutically acceptable salt thereof.

25. The compound of claim 24, wherein the compound is of Formula (Ip):Formula (Ip) or a pharmaceutically acceptable salt thereof.

26. The compound of claim 25, wherein the compound is of Formula (Iq):Formula (Iq) or a pharmaceutically acceptable salt thereof, wherein each of R8, R9, and R10is independently selected from hydrogen, fluorine, methyl, and methoxy; provided that at least one of R8-R10is not hydrogen; and R11is selected from hydrogen and methyl.

27. The compound of claim 1, wherein the compound is selected from: ,28. The compound of claim 1 or claim 2, wherein the compound is of Formula (Ir):

29. The compound of claim 1, wherein the compound is:, or a pharmaceutically acceptable salt thereof.

30. The compound of claim 1 or claim 2, wherein the compound is of Formula (Is):Formula (Is) or a pharmaceutically acceptable salt thereof.

31. The compound of claim 1, wherein the compound is:, or a pharmaceutically acceptable salt thereof.

32. A compound of Formula (II):Formula (II) or a pharmaceutically acceptable salt thereof, wherein Y is substituted or unsubstituted aryl or heteroaryl ring having 5-6 members, or substituted or unsubstituted bicyclic ring or two-ring system, the bicyclic ring or two-ring system having 9-11 members; X6is N or CR6; X7is N or CR7; X8is N or CR8; X9is N or CR9; each of R6, R7, R8, and R9is independently selected from hydrogen, halogen, cyano, perhaloalkyl, substituted or unsubstituted C1-C6alkyl, perhaloalkoxy, 5- or 6-membered substituted or unsubstituted aryl ring, or 5- or 6-membered substituted or unsubstituted heteroaryl ring, −R12CN, −NO2, −R12NO2, −OH, −R12OH, −C(O)OR11, −R12C(O)OR11, −NR112, −R12NR112, −C(O)NR112, −R12C(O)NR112, −B(OR11)2, −R12B(OR11)2, −S(O)2NR112, and −R12S(O)2NR112; provided that no more than two of X6-X9are N, and at least one of X6-X9is not CH; wherein R8and R9, R7and R8, or R6and R7together with the atoms to which they are attached optionally form a 5- or 6-membered substituted or unsubstituted aryl ring, or a 5- or 6- membered substituted or unsubstituted heteroaryl ring;each R11is independently selected from hydrogen and substituted or unsubstituted C1-C6alkyl; and each R12independently is C1-C6 alkyl.

33. The compound of claim 32, wherein the compound is of Formula (IIa):Formula (IIa) or a pharmaceutically acceptable salt thereof.

34. The compound of claim 32, wherein the compound is:XL78, or a pharmaceutically acceptable salt thereof.

35. A pharmaceutical composition comprising a compound of any one of claims 1-34,or a pharmaceutically acceptable salt thereof, and a pharmaceutical carrier.

36. The pharmaceutical composition of claim 35, further comprising one or more pharmaceutically acceptable excipients.

37. The pharmaceutical composition of claim 35 or claim 36, wherein the pharmaceutical composition further comprises a polymer selected from methylcellulose, carboxymethylcellulose, polyvinylpyrrolidone, and hydroxypropyl cellulose.

38. The pharmaceutical composition of any one of claims 35-37, wherein the pharmaceutical composition comprises polyvinylpyrrolidone.

39. The pharmaceutical composition of any one of claims 35-38, wherein the pharmaceutical composition further comprises a surfactant.

40. The pharmaceutical composition of claim 39, wherein the surfactant is a nonionic surfactant comprising a polyethylene glycol unit, a polypropylene glycol unit, or a combination thereof.

41. A method for treating a disease in a subject, the method comprising administering to the subject a compound of any one of claims 1-34, or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition of any one of claims 35-40.

42. The method of claim 41, wherein the disease is a cancer.

43. The method of claim 42, wherein the cancer is multiple myeloma, lung cancer, breast cancer, ovarian cancer, neuroblastoma, lymphoma, mantle cell lymphoma, acute leukemia, cancers associated with human papillomavirus, colorectal cancer, gastric cancer, liver cancer, cervical cancer, pancreatic cancer, prostate cancer, or a combination thereof.

44. The method of claim 43, wherein the cancer is multiple myeloma.

45. The method of any one of claims 41-44, further comprising administering at least one other therapeutic agent.

46. The method of claim 45, wherein the at least one other therapeutic agent is selected from carfilzomib, bortezomib, ixazomib, disulfiram, marizomib, oprozomib, epoxomicin, MG132, KZR-616, KZR-504, PKS2279, PKS2252, lenalidomide, pomalidomide, daratumumab, elotuzumab, isatuximab, and combinations thereof.

47. The method of any one of claims 41-44, further comprising administering a DNA damaging agent.

48. The method of claim 47, wherein the DNA damaging agent is radiation.

49. The method of any one of claims 41-48, wherein the administration is intravenous.

50. A method for modifying the activity of hRPN13, hRPN13Pru, or a variant thereof in a subject, the method comprising administering to the subject a compound of any one of claims 1-34, or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition of any one of claims 35-40.

51. A method for modifying the activity of one of the binding partners for hRPN13, hRPN13Pru, or a variant thereof in a subject, the method comprising administering to the subject a compound of any one of claims 1-34, or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition of any one of claims 35-40.

52. A method for providing a molecular glue, the method comprising administering to a subject a compound of any one of claims 1-34, or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition of any one of claims 35-40, wherein the molecular glue enhances interactions between hRPN13, hRPN13Pru, or a variant thereof, and one of the binding partners thereof in the subject.

53. A method for inducing degradation of KEN box proteins in a subject, the method comprising administering to the subject a compound of any one of claims 1-34, or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition of any one of claims 35-40.

54. The method of claim 53, wherein the KEN box proteins are PCNA clamp associated factor (PCLAF), ribonucleotide-diphosphate reductase subunit M2 (RRM2), cell division cycle 25A (CDC25A), proline rich 11 (PRR11), or Pituitary tumor transforming gene 1 (PTTG1).

55. A method for inducing apoptosis of cells in a subject, the method comprising administering to the subject a compound of any one of claims 1-34, or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition of any one of claims 35-40.

56. A method for disrupting cell cycle progression of cells in a subject, the method comprising administering to the subject a compound of any one of claims 1-34, or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition of any one of claims 35-40.

57. A method for restricting cell viability by degrading KEN box proteins in a subject, the method comprising administering to the subject a compound of any one of claims 1-34, or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition of any one of claims 35-40.

58. A method for restricting cell cycle progression by degrading KEN box proteins in a subject, the method comprising administering to the subject a compound of any one of claims 1-34, or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition of any one of claims 35-40.

59. The method of claim 57 or claim 58, wherein the KEN box proteins are PCLAF, RRM2, CDC25A, PRR11, or PTTG1.

60. The method of any one of claims 41-59, wherein the subject is a human.

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