SOX10 inhibitors and uses thereof

Abstract

This disclosure provides compounds of Formula (I) and (II) and (III), and pharmaceutically acceptable salts of any of the foregoing, that inhibit SOX10 function. These compounds are useful, e.g., for treating a disease in which increased SOX10 activity contributes to the pathology and / or symptoms and / or progression of the disease (e.g., cancer) in a subject (e.g., a human).

Description

SOX10 INHIBITORS AND USES THEREOFSTATEMENT OF GOVERNMENT SUPPORT

[0001] This invention was made with government support under NIH / NCI (1R21CA226082-01, R37CA260062) DF / HCC SPORE in Gastrointestinal Cancer, NIH / NCI (P50CA127003). The government has certain rights in the invention.PRIORITY CLAIM

[0002] This application claims the benefit of priority to U.S. Provisional Application No. 63 / 542,729, filed on October 5, 2023, which is incorporated by reference in its entirety, including any drawings and exhibits.FIELD

[0003] The present disclosure relates to compounds of Formula (I) or (II) or (HI), and pharmaceutically acceptable salts of any of the foregoing, that inhibit SOX10 function. These compounds are useful, e.g., for treating a disease in which increased SOX10 activity contributes to the pathology and / or symptoms and / or progression of the disease (e.g., cancer) in a subject (e.g.. a human).BACKGROUND

[0004] SOX10 is a member of the SOX family of high mobility group box (HMG-box) transcription factors that is essential for the development of the neural crest a lineage that ultimately gives rise to melanocytes. Transcription factors like SOX10 are hijacked during tumorigenesis and underly cancer progression, spread, and resistance to standard of care therapies. Oncogenic transcription factors are generally considered difficult to target with small-molecule inhibitors, limiting the development of transformative cancer therapies.SUMMARY

[0005] Some embodiments provide compounds of Formula (I):or a pharmaceutically acceptable salt thereof, whereinLis a bond or -LA-LB-, wherein LBis atached to Ring A;LAis -(C1-C3 alkyl)(CO)n-* or phenylene optionally substituted with 1-2 independently selected R5, whereinc'*’’ indicates the point of atachment to LB; orLAand R3together with the nitrogen to which they are atached join to form a 4-10 membered heterocyclyl optionally substituted with 1-2 independently selected R4;LBis a bond, -C(O)-, -C(0)NHNHC(0)-, -NH-, -C(O)NR6-, -C(O)(C1-C3 alkyl)-*, - C(O)NH(C 1-C3 alkyl)-*, -NH(C1-C3 alkyl)-*, -NH(C1-C3 alkyl)C(O)-*. -NHNHC(O)-*, -C(O)NHNH(CS)NH-*, -C(O)(C1-C3 alkyl optionally substituted with 1-3 independently selected R7)-*, -C(O)O(C1-C3 alkyl optionally substituted with 1-3 independently selected R7)-*, or -C(O)N=*, wherein “*’" indicates the point of atachment to Ring A;Ring A is phenyl, 4-13 membered heterocyclyl, or C3-C6 cycloalkyl;R1is selected from:(i) halogen,(ii) C1-C3 alkoxyl,(iii) C1-C3 alkyl optionally substituted with 1-4 independently selected RA, phenyl or 5-6 membered heteroaryl each optionally substituted with 1-2 RB,(iv) -NRCC(O)(CI-C3haloalkyl),(v) -NRCC(O)(CI-C3alkyl),(vi) -C(O)NRDRE,(vu) -SO2NR10R11,(viii) 5-10 membered heteroaryl optionally substituted with 1-2 R12,(ix) phenyl optionally substituted with 1-2 R13,(x) 4-6 membered heterocyclyl optionally substituted with 1-2 R14,(xi) -C(O)R15,(xii) -NR6(CO)R15,(xiii) cyano,(xiv) -SO2(C1-C3 alkyl),(xv) -N=S(O)(C1-C3 alkyl)N(Cl-C3 alkyl)2,(xvi) -NHSO2(C1-C3 alkyl),(xvii) -NO2,(xviii) C3-C7 cycloalkyl,(xix) -NRDRE,(xx) C2-C4 alkynyl optionally substituted with -C(O)C1-C3 alkyl, and(xxi) hydrogen;R2is cyano, -CO2CI-C3 alkyl. C1-C6 alkyl. C1-C3 alkoxyl, halogen. -OH, or -SH;R3is hydrogen or C1-C3 alkyl optionally substituted with phenyl optionally substituted with 1-2 halogen;R4is C1-C3 alkyl or phenyl;R5is -OH, halo, or phenyl;R6is hydrogen or C1-C3 alkoxyl;R7is halogen, -OH, C2-C3 alkenyl, C2-C3 alkynyl, or 5-6 membered heteroaryl optionally substituted with 1-2 independently selected halogen;R8is halogen or C1-C3 alkyl;R9is hydrogen or C1-C3 alkyl;R10and R11are each independently hydrogen or C1-C3 alkyl; orR10and R11, together with the nitrogen to which they are attached, join together to form a 5-6 membered heterocyclyl;R12is halogen, -OH, C1-C3 alkyl, cyano, -NO2, or -NH2;R13is halogen -OH, C1-C3 alkyl. C1-C3 haloalkyl, C1-C3 alkoxyl, cyano, or C1-C3 haloalkoxyl;R14is halogen or C1-C3 alkyl;R15is C1-C3 alkyl, C1-C4 alkoxyl optionally substituted with phenyl, C1-C3 haloalkyl, C3-C7 cycloalkyl, phenoxyl. or phenyl optionally substituted with 1-2 independently selected R16;R16is halogen, -OH, -NH(phenyl optionally substituted with halogen), -NH(C1-C4 alkyl), or -NH(C3-C7 cycloalkyl);RAis halogen, -OH, phenyl, -CO2CI-C3 alkyl, C2-C4 alkenyl, and C2-C4 alkynyl;RBis halogen or cyano;Rcis hydrogen or C1-C3 alkyl;RDand REare each independently hydrogen, C1-C3 alkyl, C3-C7 cycloalkyl; orRDand RE, together with the nitrogen to which they are attached, join together to form a 5-6 membered heterocyclyl; n is 0 or 1; and m is 0, 1, 2, 3, or 4;wherein the compound of Formula (I) is not

[0006] Some embodiments provide a method of treating a SOXIO-associated disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a compound of Formula (I), or a pharmaceutically acceptable salt thereof.

[0007] Some embodiments provide a method of treating a SOXIO-associated disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a compound of Formula (II):or a pharmaceutically acceptable salt thereof, wherein:Lis a bond or -LA-LB-, wherein LBis attached to Ring A;LAis -(C1-C3 alkyl)(CO)n-* or phenylene optionally substituted with 1-2 independently selected R5. whereinL‘*” indicates the point of attachment to LB: orLAand R3together with the nitrogen to which they are attached join to form a 4-10 membered heterocyclyl optionally substituted with 1-2 independently selected R4;LBIS a bond, -C(O)-, -C(O)NHNHC(O)-, -NH-, -C(O)NR6, -C(O)(C1-C3 alkyl)-*, - C(O)NH(C1-C3 alkyl)-* -NH(C1-C3 alkyl)-*, -NH(C1-C3 alkyl)C(O)-*, -NHNHC(O)-*, or -C(O)NHNH(CS)NH-*, -C(O)(C1-C3 alkyl optionally substituted with 1-3 independently selected R7)-*, or -C(O)O(C 1 -C3 alkyl optionally substituted with 1 -3 independently selected R7)-*-C(O)N=*, wherein “*” indicates the point of attachment to Ring A;Ring A is phenyl, 4-13 membered heterocyclyl, or C3-C6 cycloalkyl;R1is selected from:(i) halogen,(ii) C1-C3 alkoxy 1,(iii) C1-C3 alky l optionally substituted with 1-4 independently selected RA, phenyl or 5-6 membered heteroaryl each optionally substituted with 1-2 RB,(iv) -NRcC(O)(Ci-C?, haloalkyl),(v) -NRCC(O)(CI-C3alkyl),(vi) -C(O)NRDRE,(vii) -SO2NR10R11,(viii) 5-10 membered heteroaryl optionally substituted with 1-2 R12,(ix) phenyl optionally substituted with 1-2 R13,(x) 4-6 membered heterocyclyl optionally substituted with 1-2 R14,(xi) -C(O)R15,(xii) -NR6(CO)R15.(xiii) cyano,(xiv) -SO2(C1-C3 alkyl),(xv) -N=S(O)(C1-C3 alkyl)N(Cl-C3 alkyl)2,(xvi) -NHSO2(C1-C3 alkyl),(xvii) -NO2,(xviii) C3-C7 cycloalkyl,(xix) -NRDRE,(xx) C2-C4 alkynyl optionally substituted with -C(O)C1-C3 alkyl, and(xxi) hydrogen;R2is cyano, -CO2C1 -C3 alkyl, C1 -C6 alkyl, C1 -C3 alkoxyl, halogen, -OH, or -SH;R3is hydrogen or C1-C3 alkyl optionally substituted with phenyl optionally substituted with 1-2 halogen;R4is C1-C3 alkyl or phenyl;R5is -OH, halo, or phenyl;R6is hydrogen or C1-C3 alkoxyl;R7is halogen, -OH, C2-C3 alkenyl, C2-C3 alkynyl, or 5-6 membered heteroaryl optionally substituted with 1-2 independently selected halogen;R8is halogen or C1-C3 alkyl;R9is hydrogen or C1-C3 alkyl;R10and R11are each independently hydrogen or C1-C3 alkyd; orR10and R11, together with the nitrogen to which they are attached, join together to form a 5-6 membered heterocyclyl;R12is halogen, -OH, C1-C3 alkyl, cyano, -NO2, or -NH2;R1?is halogen -OH, C1-C3 alkyl, C1-C3 haloalkyl, C1-C3 alkoxyl, cyano, or C1-C3 haloalkoxyl;R14is halogen or C1-C3 alkyl;R15is C1-C3 alkyl, C1-C4 alkoxyl optionally substituted with phenyl, C1-C3 haloalkyl, C3-C7 cycloalkyl, phenoxyl, or phenyl optionally substituted with 1-2 independently selected R16;R16is halogen. -OH, -NH(phenyl optionally substituted with halogen). -NH(C1-C4 alkyl), or -NH(C3-C7 cycloalkyl);RAis halogen, -OH, phenyl, -CO2CI-C3 alkyl, C2-C4 alkenyl, and C2-C4 alkynyl;RBis halogen or cyano;Rcis hydrogen or C1-C3 alkyl;RDand REare each independently hydrogen, C1-C3 alkyl, C3-C7 cycloalkyl; orRDand RE, together with the nitrogen to which they are attached, join together to form a 5-6 membered heterocyclyl; n is 0 or 1; and m is 0, 1. 2, 3, or 4.

[0008] Some embodiments provide a compound of Formula (III):or a pharmaceutically acceptable salt thereof, whereinL is -(C=O)NRL1(CH2)q-* , -NRL1SO2(CH2)q-*. wherein indicates the attachment point to Rx;Rxis phenyl or 5-6 membered heteroaryl, each optionally substituted with 1-2 independently selected RX1;RL1is hydrogen or C1-C3 alkyl;RX1is halo, -NR RZ, or 5-6 membered heteroaryl;RYand Rzare independently H or phenyl; and q is 0 or 1.

[0009] Some embodiments provide a method of treating a SOXIO-associated disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a compound of Formula (III), or a pharmaceutically acceptable salt thereof.

[0010] The details of one or more embodiments of the disclosure are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the disclosure will be apparent from the description and drawings, and from the claims.BRIEF DESCRIPTION OF DRAWINGS

[0011] FIG. 1 shows breakdown of 25 cancer lineages with their representative cell lines and overview of isotopic Tandem Orthogonal Proteolysis-Activity-Based Protein Profiling (isoTOP-ABPP) platform used to develop the Pan-Cancer DrugMap in Example 1.

[0012] FIG. 2 shows representative cysteine reactive electrophilic fragments and three key scout fragments identified as covalently binding to cysteine by the isoTOP-ABPP platform.

[0013] FIG. 3 shows number and abundance of cysteines engaged with fragment KB03.

[0014] FIG. 4 shows overlap of cysteine binding by scout fragments KB02, KB03, andKB05 relative to the other covalently binding fragments.

[0015] FIG. 5 shows substantial overlap in cysteine reactivity changes in K562 cells between 180 covalent and the three scout fragments.

[0016] FIG. 6 shows a connectivity map identifying differences in cysteine ligandability, domain ligandability, protein ligandability, and pathway ligandability across 400+ cancer cell lines.

[0017] FIG. 7 shows representative breakdown of ligandable cysteines identified in UDP -glucose dehydrogenase (UGDH) in non-small cell lung cancer (NSCLC).

[0018] FIG. 8 shows representative ligandable protein classes and pathways identified through the isoTOP-ABPP platform.

[0019] FIG. 9 A and FIG. 9B show amino acids enriched within a 10A sphere of ligandable cysteines in kinases, and ligandable cysteines localized near protein features, respectively.

[0020] FIG. 10 shows lineage-restricted ligandable dependencies, with SOX10 highlighted.

[0021] FIG. 11 shows SOX10 depletion blocks the growth of a majority of melanomas (inset is crystal violet staining of SK-MEL5 cell line following SOX10 depletion). Column order for each cell line from left to right is sgControl. and lentivirus vectors SOX10 1 and SOX 10 4.

[0022] FIG. 12 shows alpha-fold predicted structure for SOX10 (encompassing amino acids 62-172) suggesting that Cys71 is localized near a small molecule binding groove (black regions) within the dimerization domain. HMG-box (dotted box) is the protein domain involved in DNA binding.

[0023] FIG. 13 shows SOX10*C71 is localized to the SOXE dimerization domain, with ligandability of SOX10»C71 evidenced by interactions with KB03 across 33 melanoma cell lines.

[0024] FIG. 14A and FIG. 14B show validation of SOXIO transcriptional reporter following SOXIO depletion, and over-expression, respectively.

[0025] FIG. 15 shows SOXIO transcriptional reported screen with a library of 1,000 cysteine-reactive compounds in U257 cells in which hit 2-A01 was identified.

[0026] FIG. 16 shows exemplary compounds and SAR undertaken on hit 2-A01 to generate SH29.

[0027] FIG 17 shows the activity of exemplary SAR compounds in SOXIO transcriptional assay.

[0028] FIG. 18 shows dose response of SOXIO transcriptional activity with SH29 IC50.

[0029] FIG. 19 shows SH29 disrupts SOXIO activity in multiple melanoma cell lines. Bars from left to right represent 0, 2.5, 5, and 10 pM SH29 treatment in each cell line.

[0030] FIG. 20 shows mutation of SOX10*C71 “ * W, which disrupts protein complex formation as assessed with bismaleimidoethane (BMOE) crosslinking.

[0031] FIG. 21 shows over-expressed SOX10»C71W mutant in HEK-293T cells with dimerization via with crosslinker bis-maleimidoethane (BMOE) results in decrease SOX10»C71W mobility compared to wildtype SOXIO.

[0032] FIG. 22 shows SH29 blocks SOXIO protein in U257 cells.

[0033] FIG. 23 shows SH29 blocks WT-SOX10 dimerization but not SOX10«C71A.

[0034] FIG. 24 shows immunoblot analysis of HEK293T cells expressing FLAG-SOX10 or FLAG-SOX10»C71A following enrichment with biotinylated SH29 analog (SH-0029- DTB).

[0035] FIG. 25 shows SH-0029 (SH29) increases SOXIO-SOXIO interactions in vitro in a cys71 -dependent manner.

[0036] FIG. 26A and FIG. 26B show SH-0029 increases SOXIO-SOXIO interactions in cells, with immunoblot analysis of SOXIO interactions following treatment with SH-0029 or SH-0105, respectively.

[0037] FIG. 27 A and FIG. 27B show size-exclusion chromatography (SEC) fraction of SOXIO treated with DMSO control and SH-0029, and immunoblot analysis of the SEC fractions, respectively.

[0038] FIG. 28 shows identification of SOXIO-interacting proteins in melanoma and verification of SOXIO-MITF interaction (inset).

[0039] FIG. 29A and FIG. 29B show MITF depletion inhibits SOXIO transcriptional activity, and SH29 disrupts MITF protein complex, respectively.

[0040] FIG. 30 shows schematic for nuclear fractionation.

[0041] FIG. 31 shows SOXIO-MITF interaction is compartment restricted.

[0042] FIG. 32 shows schematic for oligonucleotide-enrichment and SOXIO binding motifs.

[0043] FIG. 33 shows SOXIO can be specifically enriched with oligonucleotides.

[0044] FIG. 34 shows SOX10«C71W mutant cannot interact with oligonucleotide.

[0045] FIG. 35 shows differential enrichment of SOXIO and MITF in nuclear compartments.

[0046] FIG. 36 shows identification of SOXIO by proteomics following oligonucleotide enrichment.

[0047] FIG. 37 shows deciphering SOXIO transcriptional complex. Analytical ultracentrifugation reveals SOXIO exists in multiple fractions.

[0048] FIG. 38A and 38B show purity of active recombinant SOXIO and analysis of recombinant SOXIO produced in bacteria, respectively.

[0049] FIG. 39 shows an exemplary scheme to improve a SOXIO probe.

[0050] FIG. 40 shows profiling SOXIO activity using SH29 analogs identifies SH73 as a more potent hit.

[0051] FIG. 41A and FIG. 41B show SH73 disrupts SOXIO protein complex, and comparison of SOX10«C71 engagement and SOXIO transcriptional activity following treatment with SH29 analogs, respectively.

[0052] FIG. 42 shows SH73 dose response curve.

[0053] FIG. 43 shows heatmap depicting global changes in cysteine reactivity following SH29 analog treatment in U257 cells.

[0054] FIG. 44 shows SH29-alkyne probe for SOXIO enrichment.

[0055] FIG. 45 shows gene expression changes following treatment with siSOX 10 in melanoma cell lines. Range bars at the bottom refer to fold change (-LOG, siCTRL / siSOXlO).

[0056] FIG. 46 shows pathways downregulated following SOXIO depletion.

[0057] FIG. 47 shows overlap between downregulated genes following SOXIO and MITF depletion.

[0058] FIG. 48 shows comparison of H3K27ac and SOXIO genomic locations in SK- MEL5 cells.

[0059] FIG. 49A and FIG. 49B shows genome browser tracks for SOXIO target genes ERBB3 and IL16, respectively.

[0060] FIG. 50 shows comparison of gene expression changes following genetic or pharmacological inhibition of SOXIO in SK-MEL5 cells.

[0061] FIG. 51 shows SH29 blocks proliferation of SOX 10-positive melanoma cell lines. Bars from left to right represent DMSO control and 4 pM SH29 treatment in each cell line.

[0062] FIG. 52 shows comparison between SH29 and sgSOXlO sensitivity.

[0063] FIG. 53 shows the negative control compound SHI 05 does not block proliferation of melanoma cell lines. Bars from left to right represent DMSO control, and 5 pM SHI 05, and 5 pM SH29 treatment in each cell line.

[0064] FIG. 54 shows schematic for PDOTS system.

[0065] FIG. 55 shows characterization of emerging therapy TBKli with immune checkpoint blockade using PDOTS system.

[0066] FIG. 56 shows SH29 proliferation effects correlate with depletion of SOXIO in melanomas.

[0067] FIG. 57A and FIG. 57B show relative proliferation following SH29 treatment in U257 cells expressing sgSOXIO l and PAM-resistant FLAG-SOX10 or FLAG-SOX10-C71, and immunoblot analysis of SOXIO levels, respectively.

[0068] FIG. 58 shows proliferation assay in 51 cancer cell lines treated with 4 pM SH29 or SH105, with SH29 IC50 values for the indicated cell lines.

[0069] FIG. 59 shows SH29 IC50 values correlate with SOXIO expression.

[0070] FIG. 60 shows relative expression levels of SOXIO-target genes in seven melanoma cell lines by RNA sequencing (RNA-seq) after SH29 treatment.

[0071] FIG. 61 shows SH29 disrupts SOXIO transcriptional signaling in melanomas.GSEA in melanoma cell lines following treatment with SH29.

[0072] FIG. 62 shows results of quantitative polymerase chain reaction (qPCR) analysis of 7 gene products in melanoma cell lines following treatment with SH29.

[0073] FIG. 63 shows siRNA levels of SOX10 target genes following treatment with 2.5 pM of SH29 or SHI 05 relative to DMSO control in SKMEL5 cells along with gene overlap.

[0074] FIG. 64 shows volcano plots highlighting SOXIO-regulated genes following treatment with SH29.

[0075] FIG. 65 shows SOXIO-regulated genes are differentially impacted in melanoma cell lines following SH29 treatment. Data are represented as mean ± SD. *p < 0.05. Student’s t test (two-tailed, unpaired) was used to determine statistical significanceDETAILED DESCRIPTION

[0076] The present application is based, in part, on the discovery and exploitation of a ligandable cysteine (Cys71) of SOXIO within a conserved domain of the protein. This cysteine is required for proliferation in >85% of melanoma models examined (n=22 / 25). Without being bound by any theory7, blocking this residue (S0X1OC71) disrupts its proteinprotein interactions corresponding transcriptional complex, interrupting a critical node required for melanoma proliferation.Definitions

[0077] To facilitate understanding of the disclosure set forth herein, a number of additional terms are defined below. Generally, the nomenclature used herein and the laboratory procedures in organic chemistry, medicinal chemistry, and pharmacology described herein are those well-known and commonly employed in the art. Unless defined otherwise, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Each of the patents, applications, published applications, and other publications that are mentioned throughout the specification and the attached appendices are incorporated herein by reference in their entireties.

[0078] Definitions of specific functional groups and chemical terms are described in more detail below. The chemical elements are identified in accordance with the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75th Ed., inside cover, and specific functional groups are generally defined as described therein. Additionally, general principles of organic chemistry, as well as specific functional moieties and reactivity7, are described in Thomas Sorrell, Organic Chemistry, University Science Books, Sausalito, 1999; Smith and March, March's Advanced Organic Chemistry, 5th Edition. John Wiley7& Sons, Inc., New York, 2001; Larock, Comprehensive Organic Transformations, VCH Publishers, Inc., New York, 1989; and Carruthers, Some Modem Methods of Organic Synthesis, 3rd Edition, Cambridge University Press, Cambridge, 1987.

[0079] The abbreviations used herein have their conventional meaning within the chemical and biological arts. The chemical structures and formulae set forth herein are constructed according to the standard rules of chemical valency know n in the chemical arts.

[0080] Compounds described herein can comprise one or more asymmetric centers or double bonds, and thus can exist in various isomeric forms, e.g., enantiomers, diastereomers, racemates, geometric isomers, stereoisomeric forms that may be defined, in terms of absolute stereochemistry, as (R)-or (S)-. For example, the compounds described herein can be in the form of an individual enantiomer, diastereomer or geometric isomer, or can be in the form of a mixture of stereoisomers, including racemic mixtures and mixtures enriched in one or more stereoisomer. Isomers can be isolated from mixtures by methods known to those skilled in the art. including chiral high pressure liquid chromatography (HPLC) and the formation and crystallization of chiral salts; or preferred isomers can be prepared by asymmetric syntheses. See, for example, Jacques et al., Enantiomers, Racemates and Resolutions (Wiley Interscience, New' York, 1981); Wilen et al., Tetrahedron 33:2725 (1977); Eliel, Stereochemistry of Carbon Compounds (McGraw-Hill, NY, 1962); and Wilen, Tables of Resolving Agents and Optical Resolutions p. 268 (E.L. Eliel. Ed.. Univ, of Notre Dame Press. Notre Dame, IN 1972). The present disclosure includes compounds in racemic and optically pure forms. When the compounds described herein contain olefinic bonds or other centers of geometric asymmetry, and unless specified otherwise, it is intended that the compounds include both E and Z geometric isomers. The disclosure additionally encompasses compounds described herein as individual isomers substantially free of other isomers, and alternatively, as mixtures of various isomers.

[0081] When a range of values is listed, it is intended to encompass each value and subrange within the range. For example “C1-C6 alkyl” is intended to encompass, Cl, C2, C3, C4, C5, C6, C 1-C6, C1-C5, C1-C4, C1-C3, C1-C2, C2-C6, C2-C5, C2-C4, C2-C3, C3-C6, C3-C5, C3-C4, C4-C6, C4-C5, and C5-C6 alkyl.

[0082] The following terms are intended to have the meanings presented therewith below and are useful in understanding the description and intended scope of the present disclosure.

[0083] "Alkyl” refers to a radical of a straight-chain or branched saturated hydrocarbon group having from 1 to 50 carbon atoms (‘"C1-C50 alkyl”). In some embodiments, an alkyl group has, for example, 1 to 20 carbon atoms (“C1-C20 alkyd”), 1 to 10 carbon atoms (“Cl- C10 alkyl”), or 1 to 6 carbom atoms (“C1-C6 alkyl”). Examples of C1-C6 alky l groups include methyl (Cl), ethyl (C2). n-propyl (C3). isopropyl (C3). n-butyl (C4), tert-butyl (C4), sec-butyl (C4), iso-butyl (C4), n-pentyl (C5), 3-pentanyl (C5), amyl (C5), neopentyl (C5),3-methyl-2-butanyl (C5), tertiary amyl (C5), and n-hexyl (C6). Additional examples of alkyl groups include n-heptyl (C7). n-octyl (C8) and the like. Common alkyl abbreviations include Me (-CH ,). Et (-CH2CH3), iPr (-CH(CH3)2), nPr (-CH2CH2CH3), nBu (- CH2CH2CH2CH3), or i-Bu (-CH2CH(CH3)2).

[0084] “Halo’' or “halogen,” independently or as part of another substituent, means a fluorine (F), chlorine (Cl), bromine (Br), or iodine (I) atom. The term “halide” by itself or as part of another substituent, refers to a fluoride, chloride, bromide, or iodide atom. In certain embodiments, the halo group is fluorine.

[0085] “Haloalkyl” refers to an alkyl group as described herein (e.g., a C1-C6 alkyd group) in which one or more of the hydrogen atoms are replaced by a halogen (e.g., mono- haloalkyl, di-haloalkyl and tri-haloalkyl). Such groups include but are not limited to, chloromethyl, fluoromethyl, difluoromethyl, trifluoromethyl, chloro-fluoroalkyl, chlorodifluoroalkyl, and 2-fluoroisobutyl.

[0086] “Alkoxy” or “alkoxy!” refers to an alkyl group as described herein (e.g., a C1-C6 alkyl group), which is attached to a molecule via oxygen atom. This includes moieties where the alkyl part may be linear or branched, such as methoxy, ethoxy, n-propoxy, iso-propoxy, n- butoxy, sec-butoxy, tert-butoxy, n-pentoxy and n-hexoxy.

[0087] “Haloalkoxy” refers to an alkoxy group as described herein (e.g., a C1-C6 alkoxy group), in which one or more of the hydrogen atoms are replaced by a halogen (e.g., monohaloalkoxy, di-haloalkoxy and tri-haloalkoxy). Such groups include but are not limited to, chloromethoxy, fluoromethoxy, difluoromethoxy, trifluoromethoxy, chloro-fluoroalkoxy, chloro-difluoroalkoxy, and 2-fluoroisobutoxy.

[0088] “Ary l” refers to a radical of a monocyclic or polycyclic (e.g., bicyclic or tricyclic) 4n+2 aromatic ring system (e.g., having 6, 10, or 14 71 electrons shared in a cyclic array) having 6-14 ring carbon atoms and zero heteroatoms provided in the aromatic ring system (“C6-C14 aryl”). In some embodiments, an ary l group has six ring carbon atoms (“C6 aryl”; e.g., phenyl). In some embodiments, an aryl group has ten ring carbon atoms (“CIO ary l”; e.g., naphthyl such as 1-naphthyl and 2-naphthyl). In some embodiments, an aryl group has fourteen ring carbon atoms (“C14 aryl”; e.g.. anthracyl). An aryl group may be described as. e.g., a C6-C10 aryl. Aryl groups include, but are not limited to, phenyl, naphthyl, indenyl, and tetrahydronaphthyl. As used herein, the term “arylene”, employed alone or in combination with other terms, refers to a divalent aryl linking group having 6 to 14 ring carbon atoms. Examples of arylene group include phenylene and naphthylene.

[0089] “Heteroaryl” refers to a radical of a 5-14 membered monocyclic, bicyclic or tricyclic 4n+2 aromatic ring system (e.g., having 6, 10 or 14 n electrons shared in a cyclic array) having ring carbon atoms and 1-4 ring heteroatoms provided in the aromatic ring system, wherein each heteroatom is independently selected from nitrogen, oxygen and sulfur (“6-10 membered heteroa ’). In heteroaryl groups that contain one or more nitrogen atoms, the point of attachment can be a carbon or nitrogen atom, as valency permits. Heteroaryl groups can also contain one or more oxidized atoms such as carbon (oxo), sulfur (sulfone, sulfoxide), and nitrogen (N-oxide). Heteroaryl bicyclic ring systems can include one or more heteroatoms in one, two or three rings. “Heteroaryl” also includes ring systems wherein the heteroaryl ring, as defined above, is fused with one or more aryl groups wherein the point of attachment is either on the aryl or heteroaryl ring, and in such instances, the number of ring members designates the number of ring members in the fused (aryl / heteroaryl) ring system. Bicyclic or tricylic heteroaryl groups wherein one ring does not contain a heteroatom (e.g., indolyl, quinolinyl, carbazolyl, and the like) the point of attachment can be on either ring, i.e., either the ring bearing a heteroatom (e.g., 2-indolyl) or the ring that does not contain a heteroatom (e.g., 5-indolyl). A heteroar l group may be described as, e.g., a 6-10-membered heteroaryl, wherein the term “membered” refers to the non-hydrogen ring atoms within the moiety. As used herein, the term “heteroarylene”, employed alone or in combination with other terms, refers to a divalent heteroaryl linking group having 5 to 14 ring atoms. Examples of heteroarylene group include indolylene, pyridinylene, and quinolinylene.

[0090] In some embodiments, a heteroaryl group is a 6-10 membered aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms provided in the aromatic ring system, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“6-10 membered heteroaryl”). In some embodiments, a heteroaryl group is a 5-8 membered aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms provided in the aromatic ring system, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5-8 membered heteroaryl”). In some embodiments, a heteroaryl group is a 5-6 membered aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms provided in the aromatic ring system, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5-6 membered heteroaryl”). In some embodiments, the 5-6 membered heteroaryl has 1-3 ring heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, the 5-6 membered heteroaryl has 1-2 ring heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, the 5-6 memberedheteroaryl has 1 ring heteroatom selected from nitrogen, oxygen, and sulfur. In certain embodiments, the heteroaryl group is unsubstituted 5-14 membered heteroaryl. In certain embodiments, the heteroaryl group is substituted 5-14 membered heteroaryl.

[0091] Exemplary 5-membered heteroaryl groups containing one heteroatom include, without limitation, pyrrolyl, furanyl and thiophenyl. Exemplary' 5-membered heteroaryl groups containing two heteroatoms include, without limitation, imidazolyl, pyrazolyl, oxazolyl. isoxazolyl. thiazolyl, and isothiazolyl. Exemplary 5-membered heteroaryl groups containing three heteroatoms include, without limitation, triazolyl, oxadiazolyl, and thiadiazolyl. Exemplary' 5-membered heteroaryl groups containing four heteroatoms include, without limitation, tetrazolyl. Exemplary 6-membered heteroaryl groups containing one heteroatom include, without limitation, pyridinyl. Exemplary 6-membered heteroaryl groups containing two heteroatoms include, without limitation, pyridazinyl, pyrimidinyl, and pyrazinyl. Exemplary 6-membered heteroary l groups containing three or four heteroatoms include, without limitation, triazinyl and tetrazinyl, respectively. Exemplary' 7-membered heteroaryl groups containing one heteroatom include, without limitation, azepinyl, oxepinyl, and thiepinyl. Exemplary 5.6-bicychc heteroaryl groups include, without limitation, indolyl. isoindolyl, indazolyl, benzotriazolyl, benzothiophenyl, isobenzothiophenyl, benzofuranyl, benzoisofuranyl, benzimidazolyl, benzoxazolyl, benzisoxazolyl, benzoxadiazolyl, benzthiazolyl, benzisothiazolyl, benzthiadiazolyl, indolizinyl, and purinyl. Exemplary 6,6- bicyclic heteroaryl groups include, without limitation, naphthyridinyl. pteridinyl, quinolinyl, isoquinolinyl, cinnolinyl, quinoxalinyl, phthalazinyl, and quinazolinyl.

[0092] “Cycloalkyl” refers to a radical of a saturated or partially unsaturated cyclic hydrocarbon group having from 3 to 12 ring carbon atoms (“C3-C6 cycloalkyl'’) and zero heteroatoms in the non-aromatic ring system. In some embodiments, a cycloalkyl group has, for example, 3 to 6 ring carbon atoms (“C3-C6 cycloalkyl”). Exemplary C3-C6 cycloalkyl groups include, without limitation, cyclopropyl (C3), cyclopropenyl (C3), cyclobutyl (C4), cyclobutenyl (C4), cyclopentyl (C5), cyclopentenyl (C5), cyclohexyl (C6), cyclohexenyl (C6), cyclohexadienyl (C6), and the like. "‘Cycloalkyl” also includes ring systems wherein the cycloalky l ring, as defined above, is fused with one or more aryl groups wherein the point of attachment is on the cycloalkyl ring, and in such instances, the number of carbons continue to designate the number of carbons in the cycloalkyl ring system. As used herein, the term “cycloalkylene”, employed alone or in combination with other terms, refers to a divalent cycloalkyl linking group having 3 to 10 ring carbon carbons. Examples of cycloalkylene groups include cyclopropylene and cyclohexylene.

[0093] “Heterocyclyl” refers to a radical of a 4-12 membered saturated or partially unsaturated ring system having ring carbon atoms and 1 to 4 ring heteroatomic groups, wherein each heteroatomic group is independently selected from nitrogen, oxygen, sulfur and oxidized forms of sulfur (for example, S, S(O) and S(O)2), boron, phosphorus, and silicon (“3-12 membered heterocyclyl”). Heterocyclyl groups can also contain one or more oxidized carbon atoms (oxo groups). In heterocyclyl groups that contain one or more nitrogen atoms, the point of attachment can be a carbon, nitrogen, phosphorus, or silicon atom, as valency permits. A heterocyclyl group can either be monocyclic (“monocyclic heterocyclyl”) or a fused, bridged, or spiro ring system such as a bicyclic system (“bicyclic heterocyclyl”). Heterocyclyl bicyclic ring systems can include one or more heteroatoms in one or both rings. “Heterocyclyl” also includes ring systems wherein the heterocyclyl ring, as defined above, is fused with one or more cycloalkyl groups wherein the point of attachment is either on the cycloalkyl or heterocyclyl ring, or ring systems wherein the heterocyclyl ring, as defined above, is fused with one or more ary l or heteroary l groups, wherein the point of attachment is on the heterocyclyl ring, and in such instances, the number of ring members continue to designate the number of ring members in the heterocyclyl ring system. A heterocyclyl group may be described as, e.g., a 4-7-membered heterocyclyl, wherein the term “membered” refers to the non-hydrogen ring atoms, i.e., carbon, nitrogen, oxygen, and sulfur and oxidized forms of sulfur (for example, S, S(O) and S(O)2), within the moiety. As used herein, the term “heterocyclylene”. employed alone or in combination with other terms, refers to a divalent heterocyclyl linking group having 4 to 12 ring atoms. Examples of heterocyclylene groups include piperazinylene, tetrahydrofuranylene, and pyrrolidinylene.

[0094] Exemplary 4-membered heterocyclyl groups containing one heteroatom include, without limitation, azetidinyl, oxetanyl and thietanyl. Exemplary 5-membered heterocyclyl groups containing one heteroatom include, without limitation, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothiophenyl, dihydrothiophenyl, pyrrolidinyl, dihydropyrrolyl and pyrrolyl-2, 5-dione. Exemplary75-membered heterocyclyl groups containing two heteroatoms include, without limitation, dioxolanyl, oxasulfuranyl, disulfuranyl, and oxazolidin-2-one. Exemplary 5-membered heterocyclyl groups containing three heteroatoms include, without limitation, triazolinyl, oxadiazolinyl, and thiadiazolinyl. Exemplary 6- membered heterocyclyl groups containing one heteroatom include, without limitation, piperidinyl, tetrahydropyranyl, dihydropyridinyl, and thianyl. Exemplary 6-membered heterocyclyl groups containing two heteroatoms include, without limitation, piperazinyl, morpholinyl, dithianyl, dioxanyl. Exemplary76-membered heterocyclyl groups containingtwo heteroatoms include, without limitation, triazinanyl. Exemplary 7-membered heterocyclyl groups containing one heteroatom include, without limitation, azepanyl, oxepanyl and thiepanyl. Exemplary 8-membered heterocyclyl groups containing one heteroatom include, without limitation, azocanyl, oxecanyl and thiocanyl. Exemplary 5- membered heterocyclyl groups fused to a C6 aryl ring (also referred to herein as a 5,6- bicyclic heterocyclic ring) include, without limitation, indolinyl, isoindolinyl, dihydrobenzofuranyl, dihydrobenzothienyl, benzoxazolinonyl. and the like. Exemplary 6- membered heterocyclyl groups fused to an aryl ring (also referred to herein as a 6,6-bicyclic heterocyclic ring) include, without limitation, tetrahydroquinolinyl, tetrahydroisoquinolinyl, and the like.

[0095] "Hydroxy'’ or "hydroxyl” refers to the radical -OH.

[0096] Whenever a group is described as being “optionally substituted”, that group may be unsubstituted or substituted with one or more of the indicated substituents. Likew ise, w hen a group is described as being “substituted” the substituent(s) may be selected from one or more the indicated substituents. If no substituents are indicated, it is meant that the indicated “optionally substituted” or “substituted” group may be substituted with one or more individually and independently selected group(s) that are stable and chemically acceptable for the group being substituted. Non-limiting examples of optional substituents are halogen, cyano, hydroxyl, nitro, sulfhydryl, amino, acyl, alkyl, hydroxyalkyl, aminoalkyl, haloalkyl, alkenyl, alkynyl, alkoxy, alkenoxy. alkynoxy, haloalkoxy, haloalkenoxy, haloalkynoxy, cycloalkyl, halocycloalkyl, cycloalkoxy, aryl, aryloxy, arylalkoxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclyloxy, aralkyl, cycloalkylalkyl, cycloalkyl alkoxy, heteroaralkyl, alkoxyalkyl, heterocyclylalkyl, O-carbamyl, N-carbamyl, alkoxycarbonyl, C-amido, N- amido, alkyd phosphine oxide, SFs, S-sulfonamido, N-sulfonamido. C-carboxy, sulfoxide, and sulfone.

[0097] In some embodiments one or more of the nitrogen atoms of a disclosed compound if present are oxidized to the corresponding N-oxide.

[0098] The term "pharmaceutically acceptable salts" is meant to include salts that are prepared with relatively nontoxic acids or bases, depending on the particular substituents found on the compounds described herein. In some instances, pharmaceutically acceptable salts are obtained by reacting a compound having acidic group described herein with a base to form a salt such as an ammonium salt, an alkali metal salt, such as a sodium or a potassium salt, an alkaline earth metal salt, such as a calcium or a magnesium salt, a salt of organic bases such as dicyclohexylamine, N-methyl-D-glucamine, tris(hydroxymethyl)methylamine,and salts with amino acids such as arginine, lysine, and the like, or by other methods previously determined. The pharmacologically acceptable salts not specifically limited as far as it can be used in medicaments. Examples of a salt that the compounds described herein form with a base include the following: salts thereof with inorganic bases such as sodium, potassium, magnesium, calcium, and aluminum; salts thereof with organic bases such as methylamine, ethylamine and ethanolamine; salts thereof with basic amino acids such as lysine and ornithine; and ammonium salt.

[0099] The term "pharmaceutically acceptable excipients" refers to a carrier or an adjuvant that may be administered to a patient, together with a compound of the present disclosure, or a pharmaceutically acceptable salt, solvate, salt of the solvate or prodrug thereof, and which does not destroy the pharmacological activity thereof and is nontoxic when administered in doses sufficient to deliver a therapeutic amount of the compound.

[0100] The term “pharmaceutical composition” refers to a mixture of a compound described herein with other chemical components (referred to collectively herein as “pharmaceutically acceptable carriers”), such as stabilizers, diluents, dispersing agents, suspending agents, thickening agents, and / or other excipients. The pharmaceutical composition facilitates administration of the compound to an organism.

[0101] The term “tautomer” as used herein refers to compounds whose structures differ markedly in arrangement of atoms, but which exist in easy and rapid equilibrium, and it is to be understood that compounds provided herein may be depicted as different tautomers, and when compounds have tautomeric forms, all tautomeric forms are intended to be within the scope of the disclosure, and the naming of the compounds does not exclude any tautomer. An example of a tautomeric forms includes the following example:

[0102] It will be apparent to one skilled in the art that certain compounds of this disclosure may exist in tautomeric forms, all such tautomeric forms of the compounds being within the scope of the disclosure.

[0103] Compounds provided herein may also contain unnatural proportions of atomic isotopes at one or more of the atoms that constitute such compounds. That is, an atom, in particular when mentioned in relation to a compound according to Formula (I), comprises all isotopes and isotopic mixtures of that atom, either naturally occurring or synthetically produced, either with natural abundance or in an isotopically enriched form. For example,when hydrogen is mentioned, it is understood to refer to 'l l.2H, H or mixtures thereof; when carbon is mentioned, it is understood to refer tonC,12C,13C,14C or mixtures thereof; when nitrogen is mentioned, it is understood to refer to13N,14N,15N or mixtures thereof; when oxygen is mentioned, it is understood to refer to14O,15O,16O,17O,18O or mixtures thereof; and when fluoro is mentioned, it is understood to refer to18F,19F or mixtures thereof; unless expressly noted otherwise. For example, in deuteroalkyl and deuteroalkoxy groups, where one or more hydrogen atoms are specifically replaced with deuterium (2H). As some of the aforementioned isotopes are radioactive, the compounds provided herein therefore also comprise compounds with one or more isotopes of one or more atoms, and mixtures thereof, including radioactive compounds, wherein one or more non-radioactive atoms has been replaced by one of its radioactive enriched isotopes. Radiolabeled compounds are useful as additional agents, e.g., therapeutic agents, research reagents, e.g., assay reagents, and diagnostic agents, e.g., in vivo imaging agents. All isotopic variations of the compounds provided herein, whether radioactive or not, are intended to be encompassed within the scope of the present disclosure. In some embodiments, the compounds described herein are labeled with18F.

[0104] “Treating” or “treatment” refers to reducing the symptoms or arresting or inhibiting further development of the disease (in whole or in part). “Treating” or “treatment” includes any effect, e.g., lessening, reducing, modulating, or eliminating, that results in the improvement of the disease and the like. For example, certain methods herein treat a SOX10- associated disease by decreasing or reducing the progression, re-occurrence, or symptoms of the a SOXIO-associated disease.

[0105] An “effective amount” is an amount sufficient to accomplish a stated purpose (e.g. achieve the effect for which it is administered, treat a disease, reduce protein activity, reduce or increase protein levels, detect the presence and / or amount of a protein, or reduce one or more symptoms of a disease). An example of an “effective amount” is an amount sufficient to contribute to the treatment, prevention, or reduction of a symptom or symptoms of a disease, which could also be referred to as a “therapeutically effective amount.”

[0106] As defined herein, the term “inhibition”, “inhibit”, “inhibiting” and the like in reference to a protein-inhibitor interaction means negatively affecting (e.g., decreasing) the activity or function of the protein relative to the activity7or function of the protein in the absence of the inhibitor. In some embodiments, inhibition refers to reduction in the progression of a disease and / or symptoms of disease. In some embodiments, inhibition refers to a reduction in the activity of a signal transduction pathway or signaling pathway. Thus,inhibition includes, at least in part, partially or totally blocking stimulation, decreasing, preventing, or delaying activation, or inactivating, desensitizing, or down-regulating signal transduction or enzymatic activity or the amount of a protein. In some embodiments, inhibition of SOXIO disrupts its protein-protein interactions corresponding transcriptional complex.

[0107] A "subject." as used herein, refers to a living organism suffering from or prone to a disease that can be treated by administration of a compound or pharmaceutical composition, as provided herein. Non-limiting examples include mammals such as humans. In some embodiments, a subject is human. In some embodiments, a subject is a newborn human. In some embodiments, a subject is an elderly human. In some embodiments, the subject is a pediatric subject (e.g., a subject 21 years of age or less).

[0108] Some embodiments provide compounds of Formula (I)or a pharmaceutically acceptable salt thereof, whereinLis a bond or -LA-LB-, wherein LBis attached to Ring A;LAis -(C1-C3 alkyl)(CO)n-* or phenylene optionally substituted with 1-2 independently selected R5. wherein indicates the point of attachment to LB; orLAand R3together with the nitrogen to which they are attached join to form a 4-10 membered heterocyclyl optionally substituted with 1-2 independently selected R4;LBIS a bond, -C(O)-, -C(O)NHNHC(O)-, -NH-, -C(O)NR6-, -C(O)(C1-C3 alkyl)-*, - C(O)NH(C1-C3 alkyl)-*, -NH(C1-C3 alkyl)-*, -NH(C1-C3 alkyl)C(O)-*, -NHNHC(O)-*, -C(O)NHNH(CS)NH-*, -C(O)(C1-C3 alkyl optionally substituted with 1-3 independently selected R7)-*, -C(O)O(C1-C3 alky l optionally substituted with 1-3 independently selected R7)-*, or -C(O)N=*, wherein indicates the point of attachment to Ring A;Ring A is phenyl, 4-13 membered heterocyclyl, or C3-C6 cycloalkyl;R1is selected from:(i) halogen,(ii) C1-C3 alkoxyl,(iii) C1-C3 alkyl optionally substituted with 1-4 independently selected RA, phenyl or 5-6 membered heteroaryl each optionally substituted with 1-2 RB,(iv) -NRCC(O)(CI-C3haloalkyl),(v) -NRCC(O)(CI-C3alkyl),(vi) -C(O)NRDRE,(vii) -SO2NR10R11,(viii) 5-10 membered heteroaryl optionally substituted with 1-2 R12,(ix) phenyl optionally substituted with 1-2 R13,(x) 4-6 membered heterocyclyl optionally substituted with 1-2 R14,(xi) -C(O)R15,(xii) -NR6(CO)R15,(xiii) cyano,(xiv) -SO2(C1-C3 alkyl).(xv) -N=S(O)(C1-C3 alkyl)N(Cl-C3 alkyl)2,(xvi) -NHSO2(C1-C3 alkyl),(xvii) -NO2,(xviii) C3-C7 cycloalkyl.(xix) -NRDRE,(xx) C2-C4 alkynyl optionally substituted with -C(O)C1-C3 alkyl, and(xxi) hydrogen;R2is cyano, -CO2C1-C3 alkyl, C1-C6 alkyl, C1-C3 alkoxyl, halogen, -OH, or -SH;R3is hydrogen or C1-C3 alkyl optionally substituted with phenyl optionally substituted with 1-2 halogen;R4is C1-C3 alkyl or phenyl;R5is -OH, halo, or phenyl;R6is hydrogen or C1-C3 alkoxyl;R7is halogen, -OH, C2-C3 alkenyl, C2-C3 alkynyl, or 5-6 membered heteroaryl optionally substituted with 1 -2 independently selected halogen;R8is halogen or C1-C3 alky l;R9is hydrogen or C1-C3 alkyl;R10and R11are each independently hydrogen or C1-C3 alkyl; orR10and R11, together with the nitrogen to which they are attached, join together to form a 5-6 membered heterocyclyl;R12is halogen, -OH, C1-C3 alkyl, cyano, -NO2, or -NH2;R13is halogen -OH, C1-C3 alkyl, C1-C3 haloalkyl, C1-C3 alkoxyl, cyano, or C1-C3 haloalkoxyl;R14is halogen or C1-C3 alkyl;R15is C1-C3 alkyl, C1-C4 alkoxyl optionally substituted with phenyl, C1-C3 haloalkyl, C3-C7 cycloalkyl, phenoxyl, or phenyl optionally substituted with 1-2 independently selected R16;R16is halogen, -OH, -NH(phenyl optionally substituted with halogen), -NH(C1-C4 alkyl), or -NH(C3-C7 cycloalkyl);RAis halogen, -OH, phenyl, -CO2CI-C3 alkyl, C2-C4 alkenyl, and C2-C4 alkynyl;RBis halogen or cyano;Rcis hydrogen or C1-C3 alkyl;RDand REare each independently hydrogen. C1-C3 alkyl, C3-C7 cycloalkyl; orRDand RE, together with the nitrogen to which they are attached, join together to form a 5-6 membered heterocyclyl; n is 0 or 1; and m is 0, 1, 2, 3, or 4; wherein the compound of Formula (I) is not

[0109] Some embodiments provide a method of treating a SOXIO-associated disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a compound of Formula (I), or a pharmaceutically acceptable salt thereof.

[0110] Some embodiments provide a method of treating a SOXIO-associated disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a compound of Formula (II):or a pharmaceutically acceptable salt thereof, wherein:Lis a bond or -LA-LB-, wherein LBis atached to Ring A;LAis -(C1-C3 alkyl)(CO)n-* or phenylene optionally substituted with 1-2 independently selected R5, whereinc'*’’ indicates the point of atachment to LB; orLAand R3together with the nitrogen to which they are atached join to form a 4-10 membered heterocyclyl optionally substituted with 1-2 independently selected R4;LBis a bond, -C(O)-, -C(0)NHNHC(0)-, -NH-, -C(O)NR6, -C(O)(C1-C3 alkyl)-*, - C(O)NH(C 1-C3 alkyl)-* -NH(C 1-C3 alkyl)-*, -NH(C1-C3 alkyl)C(O)-*, -NHNHC(O)-*, or -C(O)NHNH(CS)NH-*, -C(O)(C1-C3 alkyl optionally substituted with 1-3 independently selected R7)-*, or -C(O)O(C1-C3 alkyl optionally substituted with 1-3 independently selected R7)-*-C(O)N=*, wherein indicates the point of atachment to Ring A;Ring A is phenyl, 4-13 membered heterocyclyl, or C3-C6 cycloalkyl;R1is selected from:(i) halogen,(ii) C1-C3 alkoxyl,(iii) C1-C3 alkyl optionally substituted with 1-4 independently selected RA, phenyl or 5-6 membered heteroaryl each optionally substituted with 1-2 RB,(iv) -NRCC(O)(CI-C3haloalkyl),(v) -NRCC(O)(CI-C3alkyl),(vi) -C(O)NRDRE,(vii) -SO2NR10R11.(viii) 5-10 membered heteroaryl optionally substituted with 1 -2 R12,(ix) phenyl optionally substituted with 1-2 R13,(x) 4-6 membered heterocyclyl optionally substituted with 1-2 R14,(xi) -C(O)R15,(xn) -NR6(CO)R15,(xiii) cyano,(xiv) -SO2(C1-C3 alkyl),(xv) -N=S(O)(C1-C3 alkyl)N(Cl-C3 alkyl)2,(xvi) -NHSO2(C1-C3 alkyl).(xvii) -NO2,(xviii) C3-C7 cycloalkyd,(xix) -NRDRB,(xx) C2-C4 alkynyl optionally substituted with -C(O)C1-C3 alkyl, and(xxi) hydrogen;R2is cyano, -CO2CI-C3 alkyl, C1-C6 alkyl, C1-C3 alkoxyl, halogen, -OH, or -SH;R3is hydrogen or C1-C3 alkyl optionally substituted with phenyl optionally substituted with 1-2 halogen;R4is C1-C3 alkyl or phenyl;R5is -OH, halo, or phenyl;R6is hydrogen or C1-C3 alkoxyl;R7is halogen. -OH. C2-C3 alkenyl. C2-C3 alkynyl. or 5-6 membered heteroaryl optionally substituted with 1 -2 independently selected halogen;R8is halogen or C1-C3 alkyl;R9is hydrogen or C1-C3 alky l;R10and R11are each independently hydrogen or C1-C3 alkyl; orR10and R11, together with the nitrogen to which they are attached, join together to form a 5-6 membered heterocyclyl;R12is halogen, -OH, C1-C3 alkyl, cyano, -NO2, or -NH2;R13is halogen -OH, C1-C3 alkyl, C1-C3 haloalkyl, C1-C3 alkoxyl, cyano, or C1-C3 haloalkoxyl;R14is halogen or C1-C3 alkyl;R15is C1-C3 alkyd, C1-C4 alkoxyl optionally substituted with phenyl, C1-C3 haloalkyl, C3-C7 cycloalkyl, phenoxyl, or phenyl optionally substituted with 1-2 independently selected R16;R16is halogen, -OH, -NH(phenyl optionally substituted with halogen), -NH(C1 -C4 alkyl), or -NH(C3-C7 cycloalkyd);RAis halogen, -OH, phenyl, -CO2CI-C3 alkyd, C2-C4 alkenyl, and C2-C4 alkynyl;RBis halogen or cyano;Rcis hydrogen or C1-C3 alkyl;RDand REare each independently hydrogen, C1-C3 alkyl, C3-C7 cycloalkyd; orRDand RE, together with the nitrogen to which they are attached, join together to form a 5-6 membered heterocyclyl; n is 0 or 1 ; and m is 0, 1, 2, 3, or 4.

[0111] In some embodiments, L is or -LA-LB-; wherein LBis attached to Ring A.In some embodiments, LAis -(C1-C3 alkyl)(CO)n-*; wherein indicates the point of attachment to LB.

[0112] In some embodiments, LAis phenylene optionally substituted with 1-2 independently selected R5.

[0113] In some embodiments, LAand R3together with the nitrogen to which they are attached join to form a 4-10 membered heterocyclyl optionally substituted with 1-2 independently selected R4.

[0114] In some embodiments, LBis a bond.

[0115] In some embodiments, LBis -C(O)-.

[0116] In some embodiments, LBis -C(O)NHNHC(O)-.

[0117] In some embodiments, LBis -NH-.

[0118] In some embodiments, LBis -C(O)NR6-.

[0119] In some embodiments, LBis -C(O)(C1-C3 alkyl)-*; wherein "‘*?' indicates the point of attachment to Ring A.

[0120] In some embodiments, LBis -C(O)NH(C1-C3 alkyl)-*; wherein “*” indicates the point of attachment to Ring A.

[0121] In some embodiments, LBis -NH(C1-C3 alkyl)-*; wherein “*” indicates the point of attachment to Ring A.

[0122] In some embodiments, LBis -NH(C1-C3 alkyl)C(O)-*; wherein "‘*” indicates the point of attachment to Ring A.

[0123] In some embodiments. LBis -NHNHC(O)-*; wherein indicates the point of attachment to Ring A.

[0124] In some embodiments, LBis -C(O)NHNH(CS)NH-*; whereinc'*” indicates the point of attachment to Ring A.

[0125] In some embodiments. LBis -C(O)(C1-C3 alky l optionally substituted with 1-3 independently selected R7)-*; wherein indicates the point of attachment to Ring A.

[0126] In some embodiments, LBis -C(O)O(C1-C3 alkyl optionally substituted with 1-3 independently selected R7)-*; wherein “*” indicates the point of attachment to Ring A.

[0127] In some embodiments, LBis -C(O)N=*; wherein “*’" indicates the point of attachment to Ring A.

[0128] In some embodiments. L is a bond.

[0129] In some embodiments, Ring A is phenyl.

[0130] In some embodiments, Ring A is 5-13 membered heterocyclyl.

[0131] In some embodiments, Ring A is C3-C6 cycloalkyl.

[0132] In some embodiments, R1is halogen.

[0133] In some embodiments, R1is C1-C3 alkoxy 1.

[0134] In some embodiments, R1is C1-C3 alkyl optionally substituted with 1-4 independently selected R7.

[0135] In some embodiments, R1is C1-C3 alkyl substituted with phenyl optionally substituted with 1-2 R8.

[0136] In some embodiments, R1is C1-C3 alkyl substituted with 5-6 membered heteroaryl optionally substituted with 1-2 R8.

[0137] In some embodiments, haloalkyl).

[0138] In some embodiments, alky 1).

[0139] In some embodiments,

[0140] In some embodiments,

[0141] In some embodiments, R1is 5-10 membered heteroaryl optionally substituted with 1-2 R12

[0142] In some embodiments, R1is phenyl optionally substituted with 1-2 R13.

[0143] In some embodiments, R1is 4-6 membered heterocyclyl optionally substituted with 1 -2 R14.

[0144] In some embodiments. R1is -C(O)R15.

[0145] In some embodiments, R1is -NR6(CO)R15.

[0146] In some embodiments, R1is cyano.

[0147] In some embodiments, alkyl).

[0148] In some embodiments, -C3 alkyl)N(Cl-C3 alkyl)2.

[0149] In some embodiments, -C3 alkyl).

[0150] In some embodiments,

[0151] In some embodiments, R1is C3-C7 cycloalkyl.

[0152] In some embodiments, R1is hydrogen.

[0153] In some embodiments, R2is C1-C6 alkyl.

[0154] In some embodiments, R2is C1-C3 alkoxyl.

[0155] In some embodiments, R2is halogen.

[0156] In some embodiments, R2is -OH.

[0157] In some embodiments, R2is -SH.

[0158] In some embodiments, RJis hydrogen.

[0159] In some embodiments, R3is C1-C3 alkyl optionally substituted with phenyl optionally substituted with 1-2 halogen.

[0160] In some embodiments, R4is C1-C3 alkyl.

[0161] In some embodiments, R5is -OH.

[0162] In some embodiments, R5is halogen.

[0163] In some embodiments, R5is phenyl.

[0164] In some embodiments, R6is hydrogen.

[0165] In some embodiments, R6is C1-C3 alkoxyl.

[0166] In some embodiments, R7is halogen.

[0167] In some embodiments, R7is OH.

[0168] In some embodiments, R7is C2-C3 alkenyl.

[0169] In some embodiments, R7is C2-C3 alkynyl.

[0170] In some embodiments, R7is 5-6 membered heteroaryl optionally substituted with 1-2 independently selected halogen.

[0171] In some embodiments, R8is halogen.

[0172] In some embodiments, R8is C1-C3 alkyl.

[0173] In some embodiments, R9is hydrogen.

[0174] In some embodiments, R9is C1-C3 alky l.

[0175] In some embodiments, R10and R11are each hydrogen.

[0176] In some embodiments. R10and R11are each independently selected C1-C3 alkyl.

[0177] In some embodiments, one of R10and R11is C1-C3 alkyl and the other one of R10and R11is hydrogen.

[0178] In some embodiments, R10and R11, together with the nitrogen to which they are attached, join together to form a 5-6 membered heterocyclyl.

[0179] In some embodiments, R12is halogen.

[0180] In some embodiments, R12is C1-C3 alkyl’

[0181] In some embodiments, R12is cyano.

[0182] In some embodiments, R12is -NH2.

[0183] In some embodiments, R13is halogen.

[0184] In some embodiments, R13is -OH.

[0185] In some embodiments, R13is C1-C3 alkyl.

[0186] In some embodiments, R13C1-C3 haloalkyl.

[0187] In some embodiments, R13is CI-C3 alkoxyl.

[0188] In some embodiments, R14is halogen.

[0189] In some embodiments, R14is C1-C3 alkyl.

[0190] In some embodiments, R15is C1-C3 alkyl.

[0191] In some embodiments, R15is C1-C4 alkoxyl optionally substituted with phenyl.

[0192] In some embodiments, R15is C1-C3 haloalkyl.

[0193] In some embodiments, R15is C3-C7 cycloalkyl.

[0194] In some embodiments, R15is phenyl optionally substituted with 1-2 independently selected R16.

[0195] In some embodiments, R16is halogen.

[0196] In some embodiments, R16is -OH.

[0197] In some embodiments, R16is -NH(phenyl optionally substituted with halogen).

[0198] In some embodiments, R16is -NH(C1-C4 alkyl).

[0199] In some embodiments, R16is -NH(C3-C7 cycloalkyl).

[0200] In some embodiments, Rcis hydrogen.

[0201] In some embodiments, Rcis C1-C3 alkyl.

[0202] In some embodiments, RDand REare each hydrogen.

[0203] In some embodiments, RDand REare each independently selected C1-C3 alkyl.

[0204] In some embodiments, one of RDand REis C1-C3 alkyl and the other one of RDand REis hydrogen.

[0205] In some embodiments. RDand RE, together with the nitrogen to which they are attached, join together to form a 5-6 membered heterocyclyl.

[0206] In some embodiments, n is 1 .

[0207] In some embodiments, n is 0.

[0208] In some embodiments, m is 1.

[0209] In some embodiments, m is 2.

[0210] In some embodiments, m is 3.

[0211] In some embodiments, m is 4.

[0212] In some embodiments, m is 0.Table 1. Exemplary compounds of Formula (I) and Formula (II)Table 2. Additional exemplar}' compounds of Formula (I) and Formula (II)

[0213] Some embodiments provide a compound of Formula (III):or a pharmaceutically acceptable salt thereof, whereinL is -(C=O)NRL1(CH2)q-* , -NRL1SO2(CH2)q-*. wherein indicates the attachment point to Rx;Rxis phenyl or 5-6 membered heteroaryl, each optionally substituted with 1-2 independently selected RX1;RL1is hydrogen or C1-C3 alkyl;RX1is halo, -NRARZ, or 5-6 membered heteroaryl;RYand Rzare independently H or phenyl; and q is 0 or 1.Table 3. Exemplary compounds of Formula (III)Pharmaceutical Compositions

[0214] Some embodiments provide a pharmaceutical composition comprising the compound of Formula (I), or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable excipients.

[0215] Some embodiments provide a pharmaceutical composition comprising the compound of Formula (II), or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable excipients.

[0216] Some embodiments provide a pharmaceutical composition comprising the compound of Formula (III), or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable excipients.Methods of Treatment

[0217] Provided herein are methods for inhibiting SOX10 (encoded by SOX10 gene) function. For example, provided herein are compounds that inhibit SOX10 function that are useful for treating or preventing diseases associated with dysregulation of SOX10 function, such as cancer (e.g., SOXIO-associated cancer).

[0218] The ability of test compounds to act as a SOX 10 inhibitor may be demonstrated by assays known in the art. The activity of the compounds and compositions provided herein as SOX10 inhibitors can be assayed in vitro, in vivo, or in a cell line. Potency of a SOX10 inhibitor as provided herein can be determined by ECso and / or IC50 value. A compound with a lower EC50 value or a lower IC50 value, as determined under substantially similar conditions, is a more potent SOX10 inhibitor relative to a compound with a higher EC50 or IC50 value.

[0219] Some embodiments provide a method of treating or preventing diseases associated with dysregulation of SOX10 function, such as cancer (e.g.. SOXIO-associated cancer) in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a compound of Formula (I), or a pharmaceutically acceptable salt thereof.

[0220] Some embodiments provide a method of treating or preventing diseases associated with dysregulation of SOX10 function, such as cancer (e.g.. SOXIO-associated cancer) in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a compound of Formula (II), or a pharmaceutically acceptable salt thereof.

[0221] Some embodiments provide a method of treating or preventing diseases associated with dysregulation of SOX10 function, such as cancer (e.g.. SOXIO-associated cancer) in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a compound of Formula (III), or a pharmaceutically acceptable salt thereof. Indications

[0222] Compounds of Formula (I) or (II) or (III), or pharmaceutically acceptable salts of any of the foregoing, are useful for treating diseases which can be treated with a SOX10inhibitor, such as SOXIO-associated diseases, e.g., proliferative disorders such as cancers, including hematological cancers and solid tumors (e.g., advanced or metastatic solid tumors).

[0223] Some embodiments provide a method of treating cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a compound of Formula (I) or (II) or (III), or pharmaceutically acceptable salts of any of the foregoing. In some embodiments, the cancer is a SOXIO-associated cancer.

[0224] Some embodiments provide a method of treating cancer in a subject that has been identified or diagnosed as having a SOXIO-associated cancer, comprising administering to the subject a therapeutically effective amount of a compound of Formula (I) or (II) or (III), or pharmaceutically acceptable salts of any of the foregoing.

[0225] Some embodiments provide a method of treating cancer in a subject in need thereof, comprising (a) determining that the subject has a SOXIO-associated cancer, and (b) administering to the subject a therapeutically effective amount of a compound of Formula (I) or (II) or (III), or pharmaceutically acceptable salts of any of the foregoing.

[0226] The term “SOXIO-associated disease" as used herein refers to diseases associated with or having a dysregulation of a SOX10 gene, a SOX10 protein, or the activity of any (e g., one or more) of the same (e.g., any of the types of dysregulation of a SOX10 gene, or a SOX10 protein, or the activity of any of the same described herein). Non-limiting examples of a SOXIO-associated disease include, for example, cancer (e.g.. SOXIO-associated cancer).

[0227] The term “SOX-associated cancer” as used herein refers to cancers associated with or having a dysregulation of SOX10 activity, e.g., a SOX10 gene, a SOX10 protein, or activity of any of the same. Non-limiting examples of SOXIO-associated cancers are described herein.

[0228] Also provided herein are methods for treating a subject identified or diagnosed as having a SOXIO-associated cancer that include administering to the subject a therapeutically effective amount of a compound of Formula (I) or (II) or (III), or pharmaceutically acceptable salts of any of the foregoing, or a pharmaceutical composition thereof. In some embodiments, the subject that has been identified or diagnosed as having a SOXIO-associated cancer through the use of a regulatory agency-approved, e.g., FDA-approved test or assay for identifying dysregulation of a TSOX10 gene, a SOX10 protein, or activity of any of the same, in a subject or a biopsy sample from the subject or by performing any of the non-limiting examples of assays described herein. In some embodiments, the test or assay is provided as a kit. In some embodiments, the cancer is an SOXIO-associated cancer.

[0229] In some embodiments of any of the methods or uses described herein, the subject has a clinical record indicating that the subject has a tumor that has a dysregulation of a SOXIO gene, a SOXIO protein, or activity of any of the same. Also provided are methods of treating a subject that include administering a therapeutically effective amount of a compound of Formula (I) or (II) or (III), or pharmaceutically acceptable salts of any of the foregoing, to a subject having a clinical record that indicates that the subject has a dysregulation of a SOXIO gene, a SOXIO protein, or activity of any of the same.

[0230] In some embodiments of any of the methods or uses described herein, the cancer (e.g., SOXIO-associated cancer) is selected from a hematological cancer and a solid tumor.

[0231] In some embodiments of any of the methods or uses described herein, the cancer (e.g., SOXIO-associated cancer) is selected from melanoma, prostate cancer, breast cancer, bladder cancer, hepatocellular carcinoma, schwannoma, ovarian cancer, oral cancer, sweat ductal / glandular tumors, adenoid cystic carcinoma, mammary' analog secretory carcinoma, sialoblastoma, basal cell adenocarcinoma, basal cell adenoma, pleomorphic adenoma, neurofibromas, adenocarcinoma, mesenchymal melanoma, lung cancer, colorectal cancer, esophageal cancer, gastric cancer, and gliomas.

[0232] In some embodiments of any of the methods or uses described herein, the cancer (e.g., SOXIO-associated cancer) is selected from melanoma, prostate cancer, and breast cancer.

[0233] In some embodiments of any of the methods or uses described herein, the cancer (e.g., SOXI O-associated cancer) is selected from bladder cancer, hepatocellular carcinoma, skin cancer, schwannoma, ovarian cancer, oral cancer, sweat ductal / glandular tumors, adenoid cystic carcinoma, mammary analog secretory carcinoma, sialoblastoma, basal cell adenocarcinoma, basal cell adenoma, pleomorphic adenoma, neurofibromas, adenocarcinoma, mesenchymal melanoma, lung cancer, colorectal cancer, esophageal cancer, gastric cancer, and gliomas.

[0234] In some embodiments of any of the methods or uses described herein, the cancer is melanoma.

[0235] In some embodiments of any of the methods or uses described herein, the cancer is prostate cancer.

[0236] In some embodiments of any of the methods or uses described herein, the cancer is breast cancer.Inhibition of SOXIO Activity

[0237] Also provided is a method for restoring SOXIO function in a cell, comprising contacting the cell with a compound of Formula (I) or (II) or (III), or pharmaceutically acceptable salts of any of the foregoing. In some embodiments, the contacting is in vitro. In some embodiments, the contacting is in vivo. In some embodiments, the contacting is in vivo, wherein the method comprises administering an effective amount of a compound of Formula (I) or (II) or (III), or pharmaceutically acceptable salts of any of the foregoing, to a subject having a cell having aberrant SOXIO function. In some embodiments, the cell is a cancer cell. In some embodiments, the cancer cell is any cancer as described herein. In some embodiments, the cancer cell is a SOXIO-associated cancer cell. As used herein, the term “contacting” refers to the bringing together of indicated moieties in an in vitro system or an in vivo system. For example, “contacting” a SOXIO protein with a compound provided herein includes the administration of a compound provided herein to an individual or subject, such as a human, having a SOXIO protein, as well as, for example, introducing a compound provided herein into a sample containing a cellular or purified preparation containing the SOXIO protein.

[0238] Also provided herein is a method of inhibiting cell proliferation, in vitro or in vivo, the method comprising contacting a cell with an effective amount of a compound of Formula (I) or (II) or (III), or pharmaceutically acceptable salts of any of the foregoing, or a pharmaceutical composition thereof as defined herein.

[0239] A number of embodiments of the disclosure have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the disclosure.EXAMPLESCell culture:

[0240] Cell lines. A375, SKMEL2, RPM17951, MeWo, IGR39, WM266-4, SKMELS, HMCB, HS944T, SKMEL3, SKMEL30, HCT116, and HEK-293T were grown in DMEM (Coming) supplemented with 10% fetal bovine serum (FBS, Coming), Penicillin- Streptomycin (100 mg / ml. Thermo Fisher Scientific) and L-Glutamine (2 mM, Coming). K562, G361, U62, COLO 679, IPC298, COLO 792, RVH421, MELJUSO, WM793B, M234, M189, M194, M270, M258, M283, M187, M307, M321, M272, M148, M249, M171, M144, M308, M176, M175, 10164, CALU6. HARA, HOP62, OVISE. EF0 27. OVTOKO, and EF021 were grown in RPM1-1640 (Coming) supplemented with 10% fetal bovine serum(FBS, Coming), Penicillin- Streptomycin (100 mg / ml, Thermo Fisher Scieitific), and 1% GlutaMax (Thermo Fisher Scientific). SW620, SKMEL28, IGR1, A2058, C32, IGR37, LOX- IMVI, 10150, 10170, OVK18, TOV21G, and OAW28 were grown in DMEM / F12 (Coming) supplemented with 10% fetal bovine serum (FBS, Coming), Penicillin-Streptomycin (100 mg / ml, Thermo Fisher Scientific), and 1% GlutaMax (Thermo Fisher Scientifc). SKOV3 cells were grown in McCoy's 5A (Coming) supplemented with 10% fetal bovine serum (FBS, Coming), Penicillin-Streptomycin (100 mg / ml. Thermo Fisher Scientific) and L-Glutamine (2 mM, Coming). PC9 cells were grown in RPMI (Coming) supplemented with 10% fetal bovine serum (FBS, Coming), 1% Antibiotic-Antimycotic (Thermo Fisher Scientific), and L- Glutamine (Thermo Fisher Scientific). All the cell lines were routinely tested for Mycoplasma and if not noted elsewhere were obtained from American Tissue Type Collection (ATCC).Transient transfection:

[0241] HEK-293T cells were plated at a confluence of 3 x 106cells / 10 cm dish. 24 hrs post seeding, cells were transfected with indicated plasmid in polyethyleneimine (PEI, Polysciences) with a ratio of 1 :3 (DNA:PEI). Cells were harvested 48 hrs post transfection, washed once in ice-cold PBS buffer, and snap-frozen in liquid nitrogen. In experiments where cells w ere treated with compounds prior to harvest, tissue culture dishes w ere coated with poly-L-ly sine (Sigma Aldrich, 1 : 100 diluted) for 20 min at 37°C prior to cell seeding. 48 hrs after transfection, cells were treated with SH-0029 or SH-0105 for 3h in serum-free media and then harvested.Lenti virus generation:

[0242] Lentiviral plasmids for either sgRNA expression or over-expression w ere cloned into the indicated lentiviral expression plasmids. Lentiviral expression plasmids were co-transfected with psPAX2 (Addgene #12260) and pMD2.G (Addgene #12259) envelope packaging plasmids into 1.8 x 106HEK293T cells using X-tremeGENE™ 360 Transfection Reagent (Sigma- Aldrich). Virus-containing supernatants were collected 48 hrs after transfection. siRNA transfection:

[0243] siRNAs were obtained from Dharmacon. siRNA oligonucleotide pools of 4 different siRNAs were transfected into melanoma cells using Lipofectamine RNAimax (Invitrogen) at a final concentration of 20 nM following the manufacturer's instructions. Briefly, melanoma cells were trypsinized and seeded at a confluency of 3 x 105cells / w-ell in 6-well plates with pre-plated transfection complexes incubated in Opti-MEM (LifeTechnologies). Culture media were replaced 24 hrs post transfection. 48 hrs post siRNA transfection, adherent cells were trypsinized and replated at a confluency of 1 x 104cells / well in 96-well plates for luciferase assays. Cells were harvested 72 hrs post-transfection to examine protein levels by immunoblotting.Quantitative reverse-transcriptase (qRT)-PCR:

[0244] U62, SK-MEL28, COLO-792, IPC-298, SK-MEL3 cells were replated at a confluency of 1.5 x 105cells / well in 6-well plates. After 24 hrs, cells were treated with vehicle control (DMSO), 3 pM SH-0029, or SH-0105 for 48 hrs. MM1S, AM01, RPM18226, KMS11, and KMS27 were replated at a confluency of 5 x 105cells / well in 12-well plates and treated with vehicle (DMSO), SH-9791, or SH-1696 at the concentrations described for 3 hrs. Total RNA was extracted with Quick-RNA extraction kits (Zymo Research) and cDNA synthesized using qScript cDNA synthesis kit (Quanta) according to manufacturer's instructions. qRT-PCR was performed using LightCycler® 480 System (Roche) using the FastStart SYBR Green master mix (Roche). The relative expression of each gene was normalized to UBC (Ubiquitin C).Cloning and mutagenesis:

[0245] cDNAs were amplified using Q5 High-Fidelity 2X master mix (NEB), Phusion (NEB), or Pfu DNA polymerase (Promega) and subcloned into the pRK5 (Addgene #32004), pLJMl (Addgene #100510) or pLX304 (Addgene #25890) by T4 ligation or Gibson cloning (NEB). Site directed mutants were constructed using primers containing the desired mutations. All constructs were verified by DNA sequencing.SOX 10 transcriptional reporter assays:

[0246] To generate U257, SK-MEL5, HMCB, U62, and WM-2664 SOX10 reporter cell lines, cells were transduced with lentiviral particles harboring pEZX vector (Genecopoeia) with a secreted luciferase under the control of a SOX10 target gene promoter. PAX8, YAP1, and SOX2 reporter cells were established in a similar manner in OVTOKO, MDA-MB-231, and H3122 cells, respectively. To identify small molecule inhibitors of SOX10 transcriptional activity, U257 SOX10 reporter cells were replated at a confluency of 5,000 cells / well in 96- well plates. 24 hrs post seeding, cells were treated with vehicle (DMSO) or the indicated small molecule library (Sourced from Enamine) for 48 hrs. To determine SOX10 transcriptional activity7, culture supernatants were collected and secreted luciferase (GLUC) and secreted alkaline phosphatase (SEAP, expression control) levels were determined according to manufacturer's instructions (Genecopoeia and Invivogen, respectively).Cell Ivsis and Immunoprecipitations:

[0247] Unless noted otherwise, all lysis buffers were supplemented with protease inhibitors (Roche), 10 mM Sodium Fluoride (Sigma Aldrich), and 1 mM Sodium Orthovanadate (Sigma Aldrich). To determine melanoma protein expression, pellets were lysed with 1% Triton-XlOO lysis buffer (40 mM HEPES pH 7.4, 10 mM KCI, 5 mM MgCL, 1% Triton-XlOO) supplemented with benzonase (Santacruz Biotechnology) using a chilled bath sonicator (Q700, QSonica). Lysates were rotated for 30 min at 4°C and centrifuged at 21.000 x g for 10 min. and soluble fractions were collected. For co-transfection experiments. HEK-293T cells co-transfected with indicated plasmids were washed with PBS once and treated with SH-0029 or SH-0105 for 3 hrs in serum-free media. Plates were washed once with ice-cold PBS. snap-frozen for storage, then lysed using a chilled bath sonicator (Q700, QSonica) in CHAPS lysis buffer (40 mM HEPES pH 7.4, 10 mM KCI, 5 mM MgCh, 0.3 % CHAPS). Lysates were clarified by centrifugation at 21,000 x g for 10 min. Anti-FLAG M2 resin (Sigma Aldrich) was added to the HEK-293T lysates generated as described above, resin was incubated for 2 hat 4°C with end-over-end rotation. Following immunoprecipitation, beads were washed three times with CHAPS lysis buffer supplemented with 150 mM NaCl. Loading buffer was subsequently added and samples were denatured by boiling at 95°C for 5 min. Proteins were resolved by SOS-PAGE and analyzed by immunoblotting.Flow cytometric analysis of cellular ROS levels:

[0248] Cellular ROS levels were determined using CM-H2DCFDA (Thermo Fisher Scientific). For CM-H2DCFDA staining in EFO27, SKOV3, EFO21, OVISE, OVTOKO, TOV21G, OVK18, and OAW28, cells were washed with prewarmed PBS, trypsin digested and harvested by centrifugation at 1200 x g at room temperature for 2 mins. The cell pellet was resuspended in PBS with 1 pM of CM-H2DCFDA and incubated for 30 min in a 37°C incubator with 5% CO2. For CM-H2DCFDA staining in K562 cells, cells were pretreated with 1 mM TMCEP (Sigma Aldrich) for 30 min or 1 pM K1696 (MedChemExpress) for 48 hrs and harvested by centrifugation at 1200 x g at room temperature for 2 mins. The cell pellet was resuspended in PBS with 1 pM of CM-H2DCFDA and 1 mM TMCEP or 1 pM K1696. then incubated for 30 min in a 37°C incubator with 5% CO2. Cells were subsequently washed with PBS and changes in CM-H2DCFDA fluorescence were determined via flow cytometry using Aurora (Cytek). Data was analyzed using Flowjo vl0.6 for FITC intensity. SQX10 interaction with SH-0029-DTB:

[0249] U257 or U62 melanoma cells were plated at a confluency of 4 x 106cells per 15 cm dishes. After 48 hrs, cells were treated with DMSO or SH-0029 (30, 60, or 90 pM) for 3hin serum-free RPMI, followed by SH-0029-DTB (30 pM) treatment for 1 hr. Cells were washed once with ice-cold PBS, snap-frozen for storage and then lysed in Buffer A (15 mM Tns-HCI pH 8.0, 15 mM NaCl, 60 mM KCI, 1 mM EDTA, 0.5 mM EGTA, 0.05% NP-40) for 5 min on ice. Lysates were clarified by centrifugation at 400 x g for 5 min. For streptavidin enrichment, streptavidin agarose beads (Thermo Fisher Scientific) were added to lysates and incubated for 3 hrs at 4°C with end- over-end rotation. Following enrichment, beads were washed three times with Buffer A supplemented with 150 mM NaCl. Loading buffer was added to the immunoprecipitated proteins which were subsequently denatured by boiling at 95°C for 5 min. Proteins were resolved by SOS- PAGE and analyzed by immunoblotting.SOXIO in vitro binding assays:

[0250] HEK-293T cells expressing the indicated proteins were washed once with ice-cold PBS, snap-frozen for storage, then lysed using a chilled bath sonicator (Q700, QSonica) in CHAPS lysis buffer (40 mM HEPES pH 7.4, 10 mM KCI, 5 mM MgCb, 0.3 % CHAPS). Lysates were clarified by centrifugation at 21,000 x g for 10 mins. For FLAG immunoprecipitations. anti-FLAG M2 resin (Sigma Aldrich) was added to lysate from cells expressing SOX10-FLAG, SOX10»C71A-FLAG or FLAG-MET AP2 and incubated for 2 hrs at 4°C with end-over-end rotation. Lysates isolated from cells expressing SOX10-HA or SOX10«C71A-HA, were treated with DMSO or the indicated compounds for 3 hrs at 4°C and then incubated with SOXIO-FLAG-bound resin for 1 hr at 4°C with end-over-end rotation. Following immunoprecipitation, beads were washed three times with CHAPS lysis buffer supplemented with 150 mM NaCl. Loading buffer was added to the immunoprecipitated proteins which were subsequently denatured by boiling at 95 °C for 5 min. Proteins were resolved by SOS-PAGE and analyzed by immunoblotting.Thermal Shift Assays:

[0251] The HEK-293T pellets transiently expressing each protein were lysed with DPBS supplemented with protease inhibitors (Roche), Sodium Fluoride (Sigma Aldrich), and Sodium Orthovanadate (Sigma Aldrich), using a chilled bath sonicator (Q700, QSonica). Lysates were clarified by centrifugation at 300 x g for 3 min. Supernatants were diluted to 1.25 mg / ml using DPBS, aliquoted at 40-50p Uwell in a PCR plate and allowed to warm to RT. Vehicle (DMSO) or the indicated compound were subsequently added, and the samples were allowed to incubate at room temperature for 1 hr in a PCR plate. Samples were heated to the indicated temperature for 3 min in a BioRad T100 Thermal Cycler (BioRad). Samples were allowed to cool at room temperature for 5 min, incubated for 3 min on ice, transferredinto 1.5 ml tubes, and centrifuged at 21,000 x g for 1 hr. Soluble fractions were collected for immunoblot analysis as described above. Band intensity was assessed with Image J analysis and normalized to the value at the lowest temperature (RACK1: 36°C, UGDH: 48°C) used in each experiment. The melting curve for each protein was generated with Prism v9 (GraphPad).CRISPR-mediated knockdown in melanoma models:

[0252] 3 x 105melanoma cell lines were transduced with lentiviral particles containing non-targeting sgRNAs or sgRNAs targeting Exon 1 of SOX10 in the presence of 10 pg / ml polybrene (Millipore Sigma). 24 hrs post infection, cell culture media were replaced, and cells were cultured for an additional 24 hrs. Cells were subsequently transferred into 10 cm dishes in fresh media to culture for 3 days. Afterwards, cells were replated into 96-well plates in the presence of puromycin (0.25-1 pg / mL) for proliferation assays 6 days post-seeding (see below) or were replated in a 6 cm dish or a 10 cm dish for immunoblot analysis blot analysis following 6 days of puromycin selection.Cell proliferation assay:

[0253] To examine the impact of SOX10 depletion on melanoma cell lines. 1,000-7.000 melanoma cells expressing the indicated sgRNAs (see above) were seeded into a 96-well plate, and proliferation was determined 6 days post-seeding by measuring relative ATP levels. 50 pL Cell Titer Gio™ (Promega) was added to each sample well, and the luminescence was read on the SpectraMax M5 plate reader (Molecular Devices). To examine the impact of SH-0029 or SH-0105 on cell proliferation, 2,000-7,000 cells were seeded in 96- well plates / well in 100 pL media. The following day, cells were treated with the indicated compounds for 96 hrs. At which point culture media were removed, and cells were stained with Crystal Violet staining solution (0.5 % in 20 % methanol) for 30 min at room temperature. Viability was assessed with Image J analysis. To calculate half maximal inhibitory concentrations (IC50), cells w ere treated with 0.0156-10 pM compounds for 96 hrs, cell proliferation was analyzed with crystal violet images, and IC50 values were calculated using log(inhibitor) vs % normalized response formula in Prism v9 (GraphPad). For proliferation In U257 stably co-expressing sgSOXIO-l and PAM- resistant FLAG-SOX10 or FLAG-SOX10«C71A, cells were replated in 96 well plates at 5,000 cells / well in 100 pl medium after six days of puromycin selection. The following day, cells w ere treated with 0.5- 10 pM SH-0105 or SH-0029 for 96 hrs. Cell proliferation was determined by measuring relative ATP levels as described above.RNAseq analysis of melanomas treated with SH-0029 or SH-0105:

[0254] U257, COLO679, and IGR-1 were replated at 1.5 x 105cells / well into 6-well plates. The following day cells were treated with 3 pM SH-0029 or SH-0105 for 48 hrs. For SK-MEL5 treatment, 3 x 105 cells were first transfected with the indicated siRNAs in 6-well plates. 24 hrs later, cells were treated with vehicle (DMSO) or 2.5 pM SH-0029 or SH-0105 for 48 hrs. Total RNA was extracted with NucleoSpin RNA extraction kit (MACHEREY- NAGEL) according to manufacturer's instructions, and 1500 ng / 30 pL RNA was sent to Novogene for sequencing.RNA-seq analysis following SOXIO depletion in melanoma using modified Smart-seq2 protocol:

[0255] Seven melanoma cells (HMCB, MeWO, U257. U62, SKMEL5,SKMEL28, and SKMEL2) transfected with 20 nM of each indicated siRNA were seeded in 96-well plates (5,000 cells / well) 48 hrs post transfection. Following 24 hrs proliferation in 96-well plates, cells were lysed with 25 pL of Buffer TCL (QIAGEN) supplemented with 1% mercaptoethanol (Sigma Aldrich) and stored at -80°C until use. Libraries from each cell line were generated based on the Smart-seq2 protocol 131 1 3 2 with the following modifications. RNA from 10 pL lysates was first purified with Agencourt RNAClean XP beads (Beckman Coulter) before oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Scientific) and locked TSO oligonucleotide, which was followed by 10 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (Roche) with subsequent Agencourt AMPure XP bead purification (Beckman Coulter). Libraries were prepared with tagmentation using the Nextera XT Library' Prep kit (Illumina) with custom barcode adapters (sequences available upon request). Libraries from the seven melanoma cell lines transfected with different siRNAs with unique barcodes were combined and sequenced with paired-end, 38- base reads, using aNextSeq 500 sequencer (Illumina).RNAseq Data Processing:

[0256] Melanoma RNA Samples were processed in Novogene. Reads were aligned using STAR v.2.4.0h. FeatureCounts was used to quantify the aligned fragments and FPKM expression values were calculated by removing duplicates and using Cufflinks v.2.2.1 and hgl9 RefSeq gene definitions. DEseq2 v3.10 was used to perform the differential expression analysis for the datasets in this study. Genes with 2-fold changes and P value < 0.05 were defined as differentially expressed genes. GO analysis was performed using datasets downloaded from the GSEA website. After integrating the SOXIO target genes, ahypergeometric test was performed to calculate p-values for the given gene sets. FDR was calculated by p.adjust(method = "fdr") using R.ChlP-seq:

[0257] ChIP assays were performed using 5 million cells per sample. Chromatin from 1% formaldehyde-fixed cells was fragmented to a size range of 200-700 bases with a Branson 250 Sonifier. Solubilized chromatin was immunoprecipitated with 5 pg antibodies against SOX10 (Abeam) or H3K27ac (Active Motif) at 4 °C overnight. Antibody -chromatin complexes were pulled down with protein G Dynabeads (Life Technologies), washed, and then eluted. After cross-link reversal and RNAseg (Roche) and proteinase K (Thermo Fisher Scientific) treatment, immunoprecipitated DNA was extracted with AMP Pure beads (Beckman Coulter). DNA was quantified with Qubit dsDNA HS Assay kit (Invitrogen). ChIP DNA samples were used to prepare sequencing libraries with Ultralow V2 DNA-Seq Library Preparation Kit (NuGEN). A Nextseq 1000 Illumina genome analyzer was used for sequencing.ChlP-seq Data Processing:

[0258] ChlP-seq sequencing reads were aligned to the hg!9 genome using bwa v.0.7.12 with default settings. After the removal of duplicate reads using picard-tools v.1.95, aligned reads w ere extended to 200 bp to approximate fragment sizes. Density maps were normalized to 10 M reads. IGV was used to visualize ChlP-seq coverage at specified loci. ChlP-seq peaks were identified using MACS2 v.2.2.7. 1 with a q-value of 10‘5The narrow peak setting was used for TFs while broad peaks were used for histone marks. Peaks within 2 kb of a TSS were considered promoter sites and the remaining sites were considered distal sites. Chromatin and TF signals associated with peaks w ere quantified using pyBigWig v0.3.18. iso-TMT sample preparation:

[0259] Adherent or suspension cells were cultured until reaching -80% confluency. Cells were washed once with ice-cold PBS, snap-frozen in liquid nitrogen, and stored at -80°C until use. To measure the impact of intracellular redox on cysteine ligandability, K562 cells were seeded at 2 x 105cells / ml and treated with 1 mM Tris(2- methoxycarbonylethyl)phosphine (TMCEP) (Sigma-Aldrich) for 1 hr or 1 pM K1696 (MedChem Express) for 48 hrs and subsequently harvested as described above. Frozen cell pellets were lysed in DPBS supplemented with Benzonase (Santacruz) and protease inhibitors (Roche) using a chilled bath sonicator (QSONICA) and centrifuged for 3 min at 300 x g. Proteins were quantified by BCA assay (Thermo Fisher Scientific) and a total of 50 pg of total protein extracts were used per compound treatment. Lysates were treatedwith vehicle (DMSO) or 500 pM of KB02, KB03, or KB05 (Sigma-Aldirch) for 1 hr, followed by 1 mM DBIA treatment for 1 hr. For NFkBl hit finding, lysates were treated with 200 pM compound for 90 minutes and subsequently treated with 1 mM nBIA for 1 hr. For experiments that investigated UGDH complex formation, K562 cell lysates were treated with 20, 100, or 1000 pM UDP-xylose (UGA Complex Carbohydrate Resource Center) for 1 hr before KB05 treatment. For experiments that investigated EGFR or XPO1 liganding, PC9 or K562 cell lysates were treated with 1 pM Osimertinib (Selleck Chem) or Selinexor (Selleck Chem), respectively, for 1 hr before KB05 treatment.

[0260] Following DBIA incubation, lysates were reduced with 5 mM 5- tris (2-carboxyethyl)phosphine hydrochloride (TCEP) (Sigma-Aldrich) for 2 min at room temperature, followed by alkylation using 20 mM iodoacetamide (Sigma- Aldrich) for 30 min in the dark at room temperature. Proteins were precipitated using SP3 magnetic beads. In brief, SP3 magnetic beads (Cytiva) were prewashed with LC-MS grade water (Sigma Aldrich), and 250 pg combined SP3 beads (1: 1, hydrophobic:hydrophilic) and LC-MS grade ethanol (Sigma Aldrich)were added to each sample to reach a final concentration of 50% ethanol. SP3 incubation was performed for 30 min at room temperature, and beads were subsequently washed 3 times with 80% HPLC grade-ethanol (Sigma Aldrich) and then resuspended with 175 pL of Trypsin / Lys-C (1 pg, Thermo Fisher Scientific) in 200 mM EPPS (Sigma Aldrich) pH 8.4, 5 mM CaCh. Proteins were digested overnight (16 h) at 37°C and digested peptides were enriched with streptavidin magnetic beads (Cytiva) for 1 hr at room temperature. Beads were subsequently washed three times with DPBS, twice with HPLC grade-water (Sigma Aldrich). Peptides were eluted with 50 % acetonitrile (Sigma Aldrich), 0.1% formic acid (Thermo Fisher Scientific), and dried using a Speedvac (Thermo Fisher Scientific).

[0261] Cysteine-enriched peptides were reconstituted with 30 % acetonitrile, 70 % 200 mM EPPS pH 8.4 and labeled with 25 pg of TMT reagent (Thermo Fisher Scientific) per channel for 75 min at room temperature with rotation. Labeling was terminated by the addition of 5% hydroxylamine (Acros Organics) for 15 min followed by addition of 10% formic acid. Samples were pooled and dried using a Speedvac (Thermo Fisher Scientific). Peptides were desalted with stage tips using the following procedure: peptides were reconstituted with 5% acetonitrile / 0.1 % formic acid and loaded onto Cl 8 Micro Spin columns (Nest Group) pre-equilibrated with LC-MS grade methanol (Fisher Chemical) and LC / MS-grade water containing 0.1% formic acid. C18 spin columns were washed 10 timeswith LC / MS grade water containing 0.1 % formic acid and subsequently eluted with 80% acetonitrile, 0. 1% formic acid and dried using a Speedvac (Thermo Fisher Scientific).Mass spectrometry Data Acquisition:

[0262] All mass spectrometry data were acquired using an Orbitrap Eclipse™ Tribrid™ Mass Spectrometer in-line with an Easy NanoLC-1200 system (Thermo Fisher Scientific). Peptides were separated using 75pm capillary column packed with 50 cm of Cl 8 resin (2pm, 100 A; Thermo Fisher Scientific) using 180, 205, or 210 min gradients of 10-35% acetonitrile in 0.1% FA per run, unless otherwise noted. Eluted peptides were acquired by data-dependent acquisition and quantified using the synchronous precursor selection (DDA-SPS-MS3) method for TMT quantification. Briefly, MSI spectra were acquired at 120-K resolving power with a maximum of 50-ms ion injection in the Orbitrap with high-field asymmetric- waveform ion-mobility spectrometry (FAIMS) values at -40, -50, and -70 compensate voltage (CV). MS2 spectra were acquired by selection of the top twenty most abundant features via collisional induced dissociation in the ion trap using an automatic gain control (AGC) setting of 10 K, quadrupole isolation width of 0.7 m / z, and a maximum ion accumulation time of 50 ms. These spectra were passed in real time to the external computer for online database searching. Intelligent data acquisition using Comet real-time searching (RTS) was performed with a database including cell line mutations (DepMap) and human protein databases (release_20210506). The same forward- and reversed-sequence human protein databases were used for both the RTS search and the final search (Uniprot). Next, peptides were filtered using simple, initial parameters that included the following: not a match to a reversed-sequence, containing TMTProl6 isobaric tags, maximum PPM error <50, minimum PPM error >5, minimum Corr 0.10. If peptide spectra matched those above, an SPS- MS3 scan was performed using up to 20 b- and y-type fragment ions as precursors with an AGC of 250 K for a maximum of 250 ms, with a normalized HCD collision energy setting of 55 (TMTProl6).Mass spectrometry Data Analysis:

[0263] The mass spectrometry data were searched by MSFragger (v3.4), Tandem (v2013.06.15.1), Comet (v2021.01.rev0). MSGF+ (v20220107) or Proteome Discover (2.5, Thermo Fisher Scientific) using the following parameters : 6 amino acids for minimum peptide length, 20 ppm precursor tolerance and 700 ppm fragment ion tolerance, allow tryptic peptides only, up to two missed cleavages, oxidation of methionine (+15.9949 Da) and DBIA on cysteine residues (+239.1634) as variable modifications while TMTProl6 (+304.2071 Da) on lysine and peptide N-termini, and cysteine carbamidomethylation (+57.0214 Da) werestatic modifications. For each mass spectrometry run, the searches were performed against a custom cell-line specific FASTA database that included the canonical UniProt human (v2021) protein sequences, common contaminants, cell-line-specific mutated peptide sequences, and reversed versions of UniProt sequences. The reversed sequences were added as decoys for target-decoy analysis. The initial search result from each search engine was then analyzed by PeptideProphet. Then the PeptideProphet results from the multiple search engines were merged by iProphet analysis. Peptide ion FDRs were estimated by target-decoy approach at cell-line level based on iProphet probability. For the collective DrugMap dataset, peptides were filtered to obtain a 1% global FDR. PeptideProphet and iProphet analyses were performed via Trans-Proteomic Pipelinel48 (TPP) (v6.0.0). TMT reporter-ion quantification from the MS3 scans of all identified PSMs was extracted by an in-house program. The raw reporter ion intensities were adjusted for impurity correction according to the manufacturer's specifications. For quantification of each MS3 spectrum, a total sum signal-to-noise of all reporter ions of 100 (TMT16-plex) was used.Calculation of Cysteine Engagement:

[0264] To study cysteine ligandability, "engagement" (e) was calculated by cysteinereactive compounds (e.g. KB05, KB03, KB02) (Equation 1). Cysteine engagement is measured in units of percent using equation 1 :

[0265] Engagement is formally equivalent to a probability7of liganding by a cysteinereactive compound, whereas the commonly calculated ratio (Equation 2) is like a mathematical odds.DMS* 05,3,2 (2)

[0266] These similar constructions have different emphases. For example, ratios exhibit more prominent fluctuations at positive extremes, whereas engagement equalizes the width of the number line that is allotted to different levels of cysteine ligandability7. To allow direct comparison, a conversion from ratios to engagement is provided in Equation 3.£ “ 200 0.5 j(3)

[0267] To compare these two metrics, consider a cysteine whose ratio is initially 10 and doubles to 20 in a second measurement. While this equals a 100% absolute increase inreactivity as understood with ratios, this difference only spans roughly 10 percentage points in the engagement space. Thus, engagement gives equal attention to cysteines which fluctuate from "reactive" to "highly reactive" and those which fluctuate from "not reactive" to "reactive." To complete the data processing workflow, the data was partitioned by scout (KB05, KB03, KB02) and quantile-normalized the engagement values for all cell lines profiled, effectively creating three datasets. Per scout, this operation equalized both the centers and the widths of the distributions of cysteine engagement values across all cell lines. CSEA:

[0268] In order to systematically enumerate features that are unusually prevalent among a set of cysteines, such as biological pathways or protein structural elements, a Cysteine Set Enrichment Analysis (CSEA) was developed. CSEA requires two user-defined lists of cysteines: one should contain cysteines of interest, and the other background set should contain cysteines not expected to share meaningful sources of variation with cysteines in the set of interest. CSEA uses the background set as a reference against which to detect unexpectedly high enrichment of a feature among the cysteines of interest. >6000 sets of cysteines were compiled, conceptually binned into four kinds of set, based on: molecular functions, biological pathways, experimental literature, and protein structures. This repository constitutes a library against which users can check for features enriched in their cysteines of interest. CSEA can be run against the entire library, or any subset of it- even a single cysteine set.

[0269] CSEA uses the common permutation test to establish the significant enrichment of a particular feature in a user-defined set of interest. Provided an appropriately large background set of cysteines, CSEA will tabulate the overlap between random draws of cysteines from the background set and a set of cysteines from the library, e.g. the set of all cysteines which belong to myosin motor domains. This process is formally equivalent to evaluating the intersection in a two-way Venn diagram. Each overlap calculation represents one permutation. Users can choose how many permutations CSEA performs, but the results typically stabilize after -200 permutations. Collectively, these permutations form an empirical null distribution against which the "true" overlap between the cysteines of interest and the feature in question is compared. This comparison is made mathematically formal by fitting a Gaussian to the null overlap distribution(Equation 4)where a, [3, and y are fitting constants, and % is the probability density of the null distribution. After fitting, a one-sided tail integration is computed from the "true overlap" to infinity. This tail area equals the p value for the feature of interest. In a case where the null set is completely devoid of any cysteines having the feature studied, CSEA interpolates a Gaussian with minimal density near 0 in order to facilitate p-value calculation. P-values generated in this case are denoted in the output; however, this use illustrates one setting where the user- defined background may be suboptimal and warrant further consideration by the user. This procedure is repeated for each feature or library set the user wishes to evaluate. The Benjamini -Hochberg method is then applied to control the false discovery’ rate in a set of p- values generated over a single run. Users are free to substitute more stringent, family-wise error rate correction procedures, such as the Bonferroni correction.Identification of Lineage-restricted Dependencies:

[0270] In order to delineate targets for chemical probe development, genes that exhibit lineage-restricted genetic dependency and also contain ligandable cysteines were identified. Cell lines profiled in this study were intersected with those that underwent corresponding CRISPR-based knockout and RNA-Seq profiling in DepMap. Then the data was row- normalized and reported for both 'omic features (i.e. CRISPR scores and RNA-Seq log TPM) and summarized these two tables into their constituent lineage subtypes as reported in CCLE. Specifically, columns were collapsed by taking medians across all available cell lines belonging to a single lineage subtype (e.g. melanoma). At the level of lineage subtypes and not individual cell lines, the relative importance of a particular gene within a lineage subty pe was estimated by subtracting the row-normalized values of all other lineage subtypes from the one in question, forming a distribution of differences. For example, in this study cysteine ligandability in >20 tissue types spanning ~70 lineage subtypes was profiled, meaning that ~70 subtractions were performed for each gene, where one lineage subtype at a time was held constant. Each distribution was summarized by taking its median. In principle, any gene showing persistent, positive differences, as encapsulated in the median difference, across tens of lineage subtypes, should be uniquely important for cell lines of that lineage subtype, but not other lineages. Finally, the inner product of the different matrices w as calculated for each 'omic feature, obtaining a final "score" for all genes jointly assayed in both datasets, across all lineages. Higher scores indicate mutual elevations in both transcriptional exclusivity andCRISPR-based essentiality for a particular gene within a given lineage subty pe. The angular coordinate of the plot is used to summarize scores for each subtype within a particular lineage, and the radial coordinate indicates the magnitude of the score. Negative scores were filtered out. Given that transcription factors constituted the class of proteins most enriched at the periphery of the plot (as evidenced by simple counting), subsetting to transcription factors was performed. The maximum ligandability, per lineage, of cysteines belonging to multiple transcription factors detected in this study are shown.The SOXIO signature calculation:

[0271] To estimate SOXIO activity in cell lines, row-normalized z-scores were calculated on the log TPM data in the CCLE. Specifically, for each transcript, z-scores were calculated across all available cell lines, irrespective of tissue of origin. Then the median of these row-normalized transcript levels were calculated across canonical SOXIO target genes. This step defined a single number, i.e. a SOXIO signature level, for each cell line. Visualization of protein structures:

[0272] Representations of protein structures were generated in Chimera 1. 17.3.Quantification and statistical analysis:

[0273] Representations Statistical analyses were performed with Excel (Microsoft), Prism (GraphPad v9.5.1), and MATLAB (R2022b). Error bars represent mean± s.d. Statistical significance was assessed using unpaired, two-tailed Student's t-tests. P values are indicated in figure legends and source data. < 0.05 is indicated with single asterisks, P < 0.001 with double asterisks, and P < 0.0001 with triple asterisks.Example 1. Development of a Pan-Cancer DrugMap

[0274] Herein is described chemical a proteomics technology comprising cysteine druggability mapping (CDM) that enables the global identification of ligandable proteins. Chemical proteomics combines the specificity of cysteine-reactive probes with the comprehensive analytical scale of next generation proteomics, enabling one to globally assess the landscape of protein ligandability in native biological settings where the physiological states of proteins are preserved. CDM is powered by the isotopic Tandem Orthogonal Proteolysis-Activity-Based Protein Profiling (isoTOP-ABPP) platform which maps global changes in cysteine reactivity following treatment with cysteine reactive electrophiles. The focus was on cysteines given their critical roles in protein function: nucleophiles in enzyme active sites, metal coordination and sites of post-translational modification (e.g. palmitoylation). Because of their importance and unique reactivity, cysteines are the targets of multiple clinically approved inhibitors supporting the premise that targeting cysteines is aI l lviable strategy for cancer treatment. Examples include ibrutinib and Osimertinib, used for the treatment of B cell lymphomas and Non Small Cell Lung Cancers (NSCLCs), respectively. By mapping cysteine engagement by covalent fragments with drug-like scaffolds, it was discovered that this approach is predictive in identifying ligandable cysteines that are readily engaged in downstream small molecule screens with advanced drug-like inhibitors. By employing covalent compound libraries that target cysteines the scope of ligandable proteins expands by 6-fold, including many unconventionally druggable protein classes such as transcription factors, scaffolds and protein-protein interactions.

[0275] To date, a significant body of knowledge about cysteine ligandability in cancer has been limited to chemical proteomic analysis of a handful of cancer models, restricting the understanding of how ligandability is influenced by a cancer's tissue of origin, mutational status or oncogenic addiction. Importantly, whether cysteine ligandability is restricted to a handful of models or is consistent throughout cancer remains unknown. To address these questions, CDM in 400+ cancer cell lines spanning 25 cancer ty pes was done to generate a pan-cancer DrugMap.

[0276] In this process. 6000+ ligandable cysteines corresponding to 6106 proteins were identified, including sites that were commonly ligandable as well as sites that were unique to a given lineage. By comparing cysteine ligandability with gene essentiality, a number of lineage restricted transcription factors were uncovered a that contain ligandable cysteines. The focus was on SOX10, because it had been discovered to contain a highly ligandable cysteine (Cys71) within a conserved domain and demonstrate its requirement for proliferation in >85% of melanoma models examined (n=22 / 25). By developing a SOX10 transcriptional reporter assay, it was found that Cys71 is critical for SOXIO activity and this information was leveraged to screen a library of 1000+ advanced covalent inhibitors. In this way, multiple compounds that target SOXIO were identified and through a series of medicinal chemistry optimizations a more potent analog was developed and used to demonstrate that liganding SOXIO disrupts its transcriptional program in multiple melanoma cell lines. Importantly , this chemical probe selectively blocks the proliferation of melanomas addicted to SOXIO signaling. Accordingly, it was hypothesized that liganding of SOX10»C71 disrupts its protein-protein interactions corresponding transcriptional complex, blocking a critical node required for melanoma proliferation. Studies suggested that the development of a potent and selective SOXIO inhibitor may be useful as a frontline therapy in the treatment of skin cancer, in particular melanomas. Collectively, these discoveries provide a roadmap for thedevelopment of new-generation of anti-cancer drugs targeting an oncogene traditionally considered beyond the realm of small molecule inhibition.

[0277] To develop a comprehensive portrait of cysteine ligandability across multiple cancers, 400+ cell lines were profiled representative of 25 cancers ('‘lineages”, See Table 4) via isoTOP-ABBP platform coupled to 16-plex tandem mass tag (TMT)-based quantification (FIG. 1). On average, each cancer was represented by ~5 cell lines, with rarer cancers (e.g. rhabdomyosarcoma) covered by a minimum of two. For melanoma, non-small cell lung cancer (NSCLC) and bile duct, it was elected to profile 30+ cell lines, leveraging the larger number of cell lines to determine if there was heterogeneity in ligandability (see below). In this approach, cell lysates are first treated with cysteine-reactive "scout" compounds or vehicle control, allowing reactive cysteines a chance to form covalent adducts, and then this is followed by a chase with a pan-cysteine-reactive probe (iodoacetamide-desthiobiotin, DBIA) which reacts with all remaining free cysteine thiolate groups. Cysteines that reacted with the scout compound will escape being tagged by DBIA.Table 4. Cell lines and corresponding lineage probed with isoTOP-ABBP platform coupled to 16-plex tandem mass tag (TMT)-based quantification*CNS = central nervous system; PNS = peripheral nervous system

[0278] To broadly identify ligandable cysteines, three scout fragments were relied upon, which accounted for >50% cysteine reactivity of 180 covalent fragments (FIG. 2). On average 10,000+ cysteines were quantified per cell line (profiled in technical triplicates) accounting for 1000+ TMT 16-plex runs. Ligandable cysteines were those engaged (□- value) >60% by cysteine-reactive compounds (FIG. 3). To select these scouts, cysteines targeted by a larger set of 152 covalent fragments were mapped. The three covalent scout probes were identified (FIG. 2), to engage -74% of cysteines covered by the larger fragment library (FIG. 4). More than 78 thousand cysteines were identified of which 6000+ were ligandable (defined as a competition ratio-value (R-Value) DMSO / Scout >2.5 which corresponds to 60% occupancy at a cysteine (FIG. 5). Within each cancer subtype, both common and uniquely ligandable cysteines were identified, including a core set of -800 ligandable cysteines defining a ligandability signature across the catalog of cell lines. Cell line specific ligandability was largely driven by differential gene expression between lineages, however for cancers represented by >20 cell lines it was uncovered that a subset of cysteines (-84) were differential ligandability, despite having comparable protein expression (FIG. 6). While no corresponding protein class was enriched among this class of heterogeneous ligandable cysteines, about 70% (59 of 84) of them were found localized to proteins involved in protein-protein interactions as defined by Bioplex.

[0279] Ligandability was systematically assessed across different protein classes and domains, reassuringly finding that cysteines near or in kinase active sites were disproportionately ligandable. Curiously, zinc-finger transcription factors and proteins with intrinsically disordered domains which are classically considered ‘undruggable’ had many members with ligandable cysteines. Also identified were multiple ligandable cysteines withinkey oncogenic pathways including those involved in non-small cell lung cancer (NSCLC) (FIG. 7) and the Rho cell migration pathway (FIG. 8).

[0280] To begin a more detailed analysis of the structural contributors to cysteine ligandability, 55,000+ human protein structures corresponding to 35% of cysteines identified in this study were downloaded. The nearest amino acid neighbors within a 10A sphere of ligandable cysteines were quantified, finding an enrichment of serine and aspartate associated with ligandable cysteines in protein kinases (FIG. 9A). A cntical determinant of whether a protein is druggable is the presence of pockets which can accommodate small molecule binding near a critical portion of a protein. Using P2Rank, a machine learning-based pocket prediction algorithm, -41000 pockets across 55,500 structures were identified, and it was determined that -15% predicted ligand-binding pockets neighbor a liganded cysteine. Furthermore, liganded cysteines near diverse functional sites in proteins were identified, such as active sites, allosteric sites, and DNA- and cofactor-binding sites (FIG. 9B). The presence of ligandable cysteines near pockets suggests a path forward to developing more advanced ligands that take advantage of a binding site rather than pure cysteine reactivity.Example 2. Development of a chemical probe for SOX 10.

[0281] Herein describes leveraging the DrugMap developed in Example 1 to guide the development of a chemical probe for lineage-restricted transcription factors that do not have literature precedent. To this end cysteine ligandability was compared with corresponding gene essentiality identified in cancer dependency screens (DEPMAP) for each cancer type (FIG. 10). Among the most differentially essential and ligandable proteins was SOX 10, a member of the SOX family of high mobility group box (HMG-box) transcription factors that is essential for the proliferation of melanoma cell lines. SOX10 is critical for the development of the neural crest a lineage that ultimately gives rise to melanocytes. Targeting SOX 10 should have a high therapeutic index, because its essentiality stems from its role in developmental rather than function in adult mice. Accordingly, SOX10 specificity to melanoma is used as a histological marker for high-grade melanomas in the clinic. It was established that within 22 melanoma cell lines (out of 25 examined) genetic depletion of SOX10 blocks their proliferation (FIG. 11). Within SOX10, Cys71 was identified as consistently ligandable across melanomas with Cys71 localized to the conserved SOXE dimerization domain and predicted to be adjacent to a small molecule binding groove (FIGS. 12 and 13). In light of this, it was reasoned that S0X1OC71 represents a highly compelling ligandable dependency to prioritize for inhibitor development. To readout SOX10 activity, a SOX10 reporter was developed that places promoter elements from a SOXIO-target geneupstream of luciferase, finding the reporter highly responsive to over-expression of SOXIO as well as its depletion in the melanoma cell line U257 (FIG. 14A and 14B). Mutation of SOX10»C71 a hydrophobic tryptophan was found, which results in a significant decrease in SOXIO transcriptional activity. Because of the importance of Cys71 for SOXIO activity and its ligandability', it was elected to identity potential ligands for SOX10*C71, screening a library of cysteine reactive compounds (1000+ members of chemically diverse acrylamides, average molecular weight= -370 Da) in U257 melanoma cells harboring the SOXIO reporter. This screen was not biased for a particular mechanism of inhibition. More than 10 compounds were found that scored as hits having a z-score <-2. Because there are no inhibitors for SOXIO, it was not possible to calculate a Z-prime for this screen, however other metrics for screening including the coefficient of variation (CV=0.0706) and the signal to noise ratio (S / Navg= 132.6) were in accordance with a technically successful screen. Validation of the hits directed attention to 2-A01, 3-acrylamido-N-(2-bromo-5- hydroxyphenyl)benzamide (FIG. 15). To determine if there was tractable structure activity relationship (SAR) for this hit, a limited exploratory medicinal chemistry campaign was initiated, and generated 55 additional analogs functionalizing the two cores of 2-A01 (e.g.. Core A and B) with sulfones, amines, lactones (FIG. 16). These efforts yielded a more potent analog, SH29 (3-acty lamido-N-(l,4-dimethyl-2-oxo-l,2,3,4-tetrahydroquinolin-6- yl)benzamide), and revealed the general parameters for what broad alterations are tolerated and their impact on SOXIO inhibition. For example, inhibition was increased with aniline- ty pe motifs in core A whereas the addition of hydrogen bond donating (HBD) groups in core B reduced activity' (FIG. 17). It was found that SH29 had an ICstr-llpM (FIG. 18), and decreased SOXIO activity in five melanoma cell lines in a dose-dependent manner (FIG. 19). SH29 treatment further decreased the expression of established SOXIO target genes including MIA, RXRG and GAS7 involved in signaling and proliferation (FIG. 20). Cys71 has been suggested to take part in SOXIO dimerization through the conserved SOXE domain, which is thought to be important for the transcription of some SOXIO target genes. To confirm the importance of Cys71 for SOXIO dimerization, the SOX10»C71W mutant was over-expressed in HEK-293T cells and assessed dimerization with the crosslinker bis-maleimidoethane (BMOE), finding a decrease SOX10«C71W mobility compared to wildtype SOXIO (FIG. 21). Because HEK-293T cells do not express endogenous express SOXIO or its potential interacting proteins, it was elected to monitor SOXIO complex formation in U257 cells, finding that SH29 disrupted presumed SOXIO, which migrate at a molecular weight largerthan the predicted dimer (FIG. 22). SH29 only influenced WT SOXIO in this way, but not S0X1OC71 A. providing a complimentary validation of these cell results (FIG. 23).

[0282] Using a biotinylated version of the SOXIO ligand (SH-0029-DTB), it was verified that SH-0029 specifically engages SOX10»C71 but not a SOX10»C71A mutant, in HEK- 293T cells (FIG. 24). Without wishing to be bound by theory', this result suggests an alternative mechanism of inhibition other than one that requires high-occupancy binding.

[0283] The localization of S0X1OC71 to the SOXE domain, which has been implicated in SOXIO dimerization, led to the question whether SH-0029 might modulate SOXIO multimerization. A SOXIO in vitro binding assay revealed that SH-0029, rather than disrupting SOXIO-SOXIO dimerization, instead increased the interaction (FIG. 25). The increase in SOXIO-SOXIO interaction was recapitulated in cells (FIG. 26A and 26B), and it was additionally found that SH-0029 treatment resulted in SOXIO migrating at a greater molecular weight (FIG 27 A and 27B). This ‘glue-like’ effect of SH-0029 was completely dependent on Cys71, and SH-0029 did not increase the binding in a SOX10«C71A mutant, either in vitro or in cells. This result was echoed by the negative control probe, SH-0105 (FIG. 26B).

[0284] By leveraging a chemical proteomic drug discovery engine, a highly ligandable oncogenic driver was identified and developed a corresponding chemical probe that disrupts a protein-protein interaction in a target traditionally considered undruggable.Example 3. Determination of how SH29 blocks SOXIO transcriptional complex formation

[0285] Transcription factors rarely function in isolation but as larger multiprotein signaling complexes. To identify the SOXIO transcriptional complex in melanoma FLAG- SOX10 and a control protein (METAP2) from SK-MEL5 cells were immunoprecipitated and identified over 100 proteins that specifically interact with SOXIO including MITF and SOX8 (FIG. 28). It was additionally confirmed that MITF interacts with SOXIO and found that the depletion of MITF led to a substantial decrease in SOXIO transcriptional activity (FIGs. 28 and 29). Previous studies have established that MITF and SOXIO form a melanocytic transcriptional program required for differentiation and proliferation within melanomas.

[0286] A question that emerges from these studies is whether SOXIO interacts with its complex members as a monomer or dimer, information that is required to interpret downstream cellular signaling experiments. To probe this question, the established in cell cross-linking and in vitro binding assay will be used to monitor SOXIO interaction with MITF as dimer or as a monomer based on its migration in a panel of cell lines. Preliminary data indicated that MITF runs at higher molecular weight when treated with a crosslinker andthat this migration is blocked by SH29 treatment suggesting the formation of higher-order transcriptional complexes that are sensitive to the SOXIO chemical probe (FIG. 29B). In addition to these directed approaches to probe the role of SOXIO dimerization in seeding transcriptional complexes, an unbiased approach will be taken to monitor by IP-MS how SOXIO interacting proteins are altered when cells are treated with SH29. Preliminary proteomics analysis suggested that SOXIO interacts with CHD4, a member of the nucleosome remodeling complex and previously unappreciated SOXIO interactor. This approach highlights the utility of using proteomics in defining new members of SOXIO transcriptional complex. Complimenting these studies, changes in protein interactions will be monitored using a SOX10*C71W mutant expressed in U257 cells. Although an imperfect mimetic for small molecule adduction, proteins that are disrupted by both mutation and treatment with SH29 will provide a complementary basis for binding to SOXIO as a dimer. It is important to note that these protein interactions were conducted from whole cell lysate a combination of both nuclear and cytosolic extracts, which may represent a mixture of SOXIO existing as both a monomer and dimer. Even within the nucleus, it is now appreciated that transcription factors have differential activity within multiple nuclear compartments. Thus, it was elected to generate a series of cellular fractions encompassing the cytosol (C), nucleoplasm (Nl) and euchromatin (N2 and N3) compartments, with the latter nuclear fractions prepared by isolating the nucleus and eluting proteins from chromatin with increasing NaCl concentrations (FIG. 30). Curiously, it was found that SOXIO interaction with MITF only occurred in the euchromatin compartment, despite the presence of both factors in multiple compartments (FIG. 31). Determining how SOX10»C71 covalent adduction alters its transcriptional complex formation will be informative.Does regulating SOXIO dimerization alter its DNA binding, capacity?

[0287] One measure of transcription factor activity can be distilled down to its interaction with DNA. To this end, a panel of biotinylated oligonucleotides encompassing a diverse array of SOXIO binding sites was generated, which combined with streptavidin enrichment, can isolate SOXIO from cellular lysate (FIG. 32). Specificity of interaction is generated because mutation of the SOXIO binding site in the oligonucleotide disrupts SOXIO interaction (FIG. 33). Moreover, the SOX10»C71W dimerization mutant was not enriched by a SOX10- directed oligonucleotide compared to WT SOXIO (FIG. 34). By combining oligonucleotide enrichment with nuclear fractionation, it was found that SOXIO interacted with the MITF promoter (MITF) oligonucleotide in the N2 and N3 nuclear fractions, whereas a DCT promoter (DCT) oligonucleotide only bound SOXIO in the N3 fraction. This is the samefraction where the MITF is found, suggesting SOXIO must be in a complex with MITF to interact with this sequence (FIG. 35). Using the array of SOXIO binding oligonucleotides, determine how disruption of SOXIO dimerization with SH29 shapes its complex formation on DNA will be determined. In addition to monitoring SOXIO and MITF by immunoblot, which proteins interact with these oligonucleotides by proteomics will be unbiasedly assess. In preliminary experiments, strong enrichment for SOXIO with a wildtype but not mutant MITF oligonucleotide was found using proteomics (FIG. 36). Using this platform, proteins which are enriched by oligonucleotides across the different nuclear fractions in the presence or absence of SH29 will sequentially be monitored, providing a comprehensive portrait of SOXIO transcriptional complexes that interact with DNA. These experiments will be done in a panel of six cutaneous melanoma cell lines, to determine if these results are broadly applicable to the role of blocking SOXIO protein interactions and DNA binding.Expected results, potential pitfalls and alternatives

[0288] Herein, is provided a rationale experimental approach to test how liganding of SOX I 0«C7 l disrupts the SOXIO protein complexes and corresponding DNA interactions. While SOXIO interactions with DNA have been previously explored, these experiments have been conducted in the context of purified recombinant protein, eschewing the native transcriptional complexes that form within the cell. The approach taken herein to investigate protein-protein and protein complex-DNA interactions, preserves native SOXIO protein complex interactions allowing dissection of how liganding can disrupt transcriptional activity at the biochemical level. This experimental plan is a modem take on classical biophysical experiments using the resolving power of mass spectrometry to characterize how adduction of SOX10«C71 alters its active state in an unbiased format. The proposed studies to interrogate how SOXIO-MITF interactions are regulated by liganding will provide a potential mechanistic explanation for the cellular phenotypes of SOXIO covalent modifications. While the initial biochemical characterization of SOXIO dimerization involves immunoblot and mass spectrometry analysis, it is not yet known whether disrupting higher-order SOXIO complexes composed of multiple copies of MITF or other proteins is ocurring. If the current approaches suggest the formation of higher order complexes, analytical ultracentrifugation will be used to determine the stoichiometry of SOXIO-MITF higher order complexes following treatment with SH29. Excitingly, the preliminary' results indicate that SOXIO is localized to multiple fractions in a glycerol gradient potentially corresponding to different macromolecular complexes (FIG. 36). Pairing analytical ultracentrifugation with oligonucleotide enrichment, will provide a greater depth of analysis to determine theoligomeric state of SOXIO as interacts with DNA. Finally, as a complement to the cellular oligonucleotide enrichment assays, electric mobility shift assays (EMSA) using highly purified recombinant and active SOXIO will be employed (FIG. 38) to provide an in vitro model of how SOXIO liganding alters its interactions with DNA.Scientific Rigor and Biological Variables

[0289] In vitro assays that test the effect of disrupting SOXIO protein interactions will serve as a complimentary readout and validation of IP-MS and oligonucleotide interactions that occur in the cell. Cellular assays will be conducted in six SOXIO positive melanoma cell lines, isolated from patients of various ages, half of which are male and half of which are female. The role of SOXIO in cutaneous melanoma will be studied, which disproportionally impacts Caucasians, and as a result, these models are derived from Caucasian patients. Proteomics experiments will employ > 3 biological replicates. Statistical significance of changes in protein interactions as determined by mass spectrometry is determined by using student’s t-test followed by multiple hypothesis testing using the Benjamini -Hochberg correction.Example 4. Determining the optimization potential of SOXIO probes

[0290] The goal of this example study is to improve upon the initial SOXIO chemical probe (SH29) and develop a more potent and selective analog. A deep structure activity relationship (SAR) exploration of the binding element and warhead portions of SH29 will be undertaken. The on-target and off-target engagement of new SH29-inspired analogs will be assessed using chemical proteomics. Determination whether high level occupancy of SOXIO is required to disrupt its activity will be done using a combination of synthetic and chemical proteomic approaches.Additional Data

[0291] Additional data demonstrates that SH29 can disrupt SOXIO transcriptional activity' by disrupting SOXIO dimer formation. Unexpectedly, analogs with the greatest inhibition of SOXIO transcriptional activity were rooted in minor changes in core B (FIG. 39 and FIG. 40). Also identified was a structurally related SH29 analog. SHI 05 which did not disrupt SOXIO transcriptional activity (FIG. 39 and FIG. 40), which can serve as a negative control compound. To better understand SOXIO engagement, cells were treated with a series of SH29 analogs of vary ing activity' and monitored SOX10*C71 engagement using isoTOP- ABPP, finding a positive correlation between engagement and inhibition (FIGs. 41A and 41D). In general. SOX10»C71 was engaged at -30% by SH29, which while encouraging also indicates a need for further development of these probes. Importantly, this work also suggeststhat low-level engagement of this TF may be sufficient to disrupt its activity, an unexpected result stemming from these preliminary studies.Exploring, SOX 10 Probe SAR

[0292] Over 150 compounds were designed and tested based on the positive and negative correlations to SOXIO transcriptional activity' observed in preliminary studies (FIG. 16 and FIG. 40). Moderate modifications to core B revealing that varying the arylamide substitution pattern: 1,3 (meta = SH29)1,4 (para = SH73, see FIG. 42) had the greatest impact. The heatmap depicting global changes in cysteine reactivity following SH29 analog treatment in U257 cells supports this (FIG. 43). Additionally, it was found that hydrogen bond donors (such as the hydroxy group) in core B had little impact on SOXIO activity whereas preserving the tetrahydroisoquinlines scaffolds in both cores correlated with a blockade of SOXIO. Importantly, the preliminary results suggests that the l,4-dimethyl-3,4- dihydroquinolin-2(lH)-one core offers an advanced binding element, and indicates an endstage for large, extensive modifications. To test the importance of chirality, four constituents of SH29 and SH73 will be separated via chiral HPLC to determine if one enantiomer has a more dramatic impact on SOXI O inhibition. Importantly, chirality may also afford us a unique negative control. With regards to the modification of core B a greater degree of rigidity will be introduced to the warhead fused ring systems encouraged by the positive SAR observed with the tetrahydroisoquinline scaffold. Then an array of analogs with warheads of varying geometries and reactivities will be generated (e.g., 2-fluoroacrylamide, butyramide). Finally, a combinatory approach will be taken when derivatizing cores A and B by combining these different cores via amide coupling (e.g., HATU or COMU) to access greater chemical space. Throughout this medicinal chemistry campaign, analogs be triaged based on their IC50 values made across five melanoma cell lines expressing the SOXIO transcriptional reporter (FIG. 19), ensuring the selection of the most potent analog is active in multiple models. The most potent analogs will be brought forth to SOXIO in vitro binding assay and in cell SOXIO complex formation assay (FIG. 20, FIGS. 21-22). As an additional specificity control, a SOX10»C71A mutant will be employed that does not disrupt SOXIO dimerization in the presence of SH29 can be used to demonstrate cysteine-specific liganding (FIG. 23).Example 5. Chemical proteomic determination of 13-F10 analogs on-target and off-target engagement

[0293] Compound activity will depend both on its engagement of SOX10»C71 and minimal interaction with other off-targets. Specificity will be assessed using the isoTOP-ABPP platform. A panel of melanoma cell lines expressing SOXIO (e.g. U257, SK-MEL2, SK-MEL5. MELJUSO, U62) and lung cancer cell lines that do not-express SOXIO (e.g. CALU6, H1975, H441, H562, H3122) will be treated with analogs that disrupt SOXIO activity and those that do not. Analogs will be prioritized if they engage SOXIO at >30% but engage less than 40 additional cysteines, which from previous experience, meet the required specificity for functional assays. To account for common reactive cysteines that will be non- specifically bound by analogs that are active against SOXIO, analogs will be leveraged which do not disrupt SOXIO activity and will subtract common targets as non-specific hits. Collectively, this chemical proteomic focused interrogation of on-target and off-target engagement will provide another window into the specificity' of these compounds, allowing optimization both on potency and proteome-wide selectivity.

[0294] Alpha fold modeling of SOXIO, hints that C71 is within a structured domain adjacent to a substantial groove (FIG. 12). The presence of a groove is further supported by SAR studies as described herein. To demonstrate SOX10«C71 is localized near a bona-fide pocket will require solving the structure of SOXIO engaged by SH29. Highly purified recombinant SOXIO that is active and can bind DNA has been generated (FIG. 38A and FIG. 38B). As an alternative to cysteine-focused competition assays to assess engagement, SH29 labeling of SOXIO will be examined. To this end, an alkyne derivative of SH29, (SH29- alkyne) was generated, and found by immunoblot that it both binds SOXIO and is competed by parental SH29 (FIG. 44). While the immunoblot analysis allows for rapid determination of SOXIO engagement, bio-orthogonal conjugation and streptavidin enrichment can be coupled to proteomics to globally profile on-target and off-target engagement, as previously described. Collectively, these chemical proteomic approaches when combined with robust biochemical analysis, provide a comprehensive method to analyze the efficacy of the SAR campaign targeting SOXIO.

[0295] A strength of the current approach is the integration of multiple orthogonal platforms (including biochemical and chemical proteomic) to monitor progress in medicinal chemistry’ efforts to improve SOXIO targeting with covalent inhibitors. Furthermore, functional rescues of SOXIO inhibitors using SOXIO mutants to further validate on target engagement of SOXIO will be examined.

[0296] Statistical comparisons for disruption of SOXIO transcriptional activity with different SH29 analogs will also be based on ANOVA with Tukey's correction for multiple comparisons. ANOVA models will be appropriately nested to allow for replication in the design. Proteomics experiments will employ > 3 biological replicates. Statistical significanceof changes in protein interactions as determined by mass spectrometry is determined by using student’s t-test followed by multiple hypothesis testing using the Benjamini -Hochberg correction.Example 6. Deciphering the cellular impact of SOXIO inhibition in melanoma

[0297] The overall goal of this example is to examine the cellular impact of SOXIO covalent inhibitors on melanoma transcriptional networks and proliferation. Preliminary data indicates that SOXIO liganding phenocopies depletion of SOXIO for select target genes (FIG. 20). By using transcriptional approaches (e.g. RNAseq) and probing the genomic location of SOXIO (e.g. ChlP-Seq) it will be elucidated how pharmacological disruption of SOXIO impacts its oncogenic transcriptional program in comparison to its genetic loss. The near complete proliferation arrest of many melanoma cell lines following SOXIO depletion (FIG. 11) suggest a very specific oncogenic dependency that is anticipated to be amplified with pharmacological agents. To test this hypothesis, multiple melanoma models will be employed including advanced patient derived models where the immune system is preserved, allowing insights into how checkpoint blockade synergizes with SOXIO inhibition.

[0298] To decipher how liganding SOXIO disrupts its oncogenic gene expression program, the loss of SOXIO using RNAseq was examined in seven melanoma cell lines (FIG. 45). It was elected to deplete SOXIO with siRNA because these models could not tolerate SOXIO deletion. Across all cell lines it was identified 400+ genes downregulated including well established targets such as IL-16 and ERBB3 (FIGs 45 and FIGS. 49A and 49B). Geneset enrichment analysis revealed a significant downregulation in genes involved in melanin biosynthesis and neuronal development, consistent with the established functions of SOXIO in development (FIG. 50). Interestingly, also identified were several clusters related to immune signaling, including inflammatory and interferon signaling that were upregulated following SOXIO depletion (FIG. 45). When MITF was depleted in these lines, it was found a strong overlap in downregulated genes between the two factors, consistent with their formation of a transcriptional complex (FIG. 47). To further refine which genes are specifically regulated by SOXIO, the genomic location in SK-MEL5 cells was mapped using ChlP-Seq finding a strong overlap with H3K27Ac. an epigenetic modification associated with active transcription, and genes in siSOXlO treated cells that were downregulated in (FIG. 48 and FIGS. 49A and 49B).

[0299] Because pharmacological and genetic regulation of SOXIO may differ with regards to kinetics of inhibition and downstream transcriptional regulation, it was decided to investigate the effects of SH29 on SOXIO transcriptional output and genomic location atmultiple time points (e.g. 6. 12, 24, 48 and 72 hrs.). In parallel, cells will be treated with SH105 a structurally related molecule that does not disrupt SOXIO transcriptional activity (FIG. 40) as a control compound in RNAseq and ChlP-seq experiments. Preliminary profiling of genes regulated by SH29 in SK-MEL5 cells indicates a strong overlap (-70%) with downregulated genes following expression of siSOXlO but not the control probe SHI 05 (FIG. 50). Upon profiling five additional melanoma cell lines, including two models derived from therapy -resistant patients, a gene regulon specific to pharmacological inhibition of SOXIO will be identified. It will then be asked whether this set of genes is enriched in sequenced melanoma tumors, to identify particular subtypes that may benefit from SOXIO inhibition. Collectively, completion of these genomic studies will help reach the goals of this example study, and establishing a strong foundation for how pharmacological manipulation of SOXIO alters transcriptional programs in melanomas.Example 6. Pharmacological blockade of melanoma proliferation with a SOXIO inhibitor.

[0300] To determine the initial feasibility of disrupting melanoma proliferation using SOXIO inhibitors, A panel of cell lines were treated (nine melanoma; three non-small cell lung cancer (NSCLC)) with varying levels of SOXIO expression with SH29 and monitored proliferation for 4 days by crystal violet staining (FIG. 51 and FIG. 52). A good correlation between SH29 cytotoxicity and SOXIO dependence was observed in melanoma cell lines with U62 demonstrating the greatest sensitivity to both SH29 treatment and SOXIO depletion (FIG. 52). Cell lines which were not dependent on SOXIO (e.g. RPMI-7951) or from different lineages (e.g. NSCLC lines) showed attenuated sensitivity to SH29 (FIG. 51 and FIG. 52), suggesting that the expression of SOXIO may serve as a biomarker for advanced agents targeting this factor. There was no observed cytotoxicity with the control compound SH105 in comparison to SH29 (FIG. 53). These preliminary’ data, suggest good specificity for SH29 in accordance with the selective dependency elicited by SOXIO expression. Given the importance of immune checkpoint blockade (ICB) in the management of melanoma, it is important to determine if SOXIO inhibition synergizes with ICB. To this end, the effect of SOXIO inhibition alone or in combination using 3D microfluidic cultures of patient derived organotypic tumor spheroids (PDOTS) from melanoma patients will be examined. PDOTS are multicellular spheroids (40-100 pm in size) comprised of cancer cells and autologous immune and stromal cells which faithfully recapitulate the in vivo response to treatment with anti-PD-1 antibodies and synergies between emerging therapies and ICB (FIG. 54). The efficacy of SOXIO inhibition with SH29 in the presence of anti-PD-1 antibody (pembrolizumab) or isotype control using a panel of melanoma PDOTS (n=35) will also beexamined. To determine therapeutic benefit, proliferation as measured by dual labeling deconvolution fluorescence microscopy using Hoechst (all cells), acridine orange (live cells) and propidium iodide (dead cells), as previously described will be monitored. Image acquisition will be performed using a fully motorized Nikon Eclipse NiE upright microscope. Raw cell area will be determined using NIS Elements software and raw7cell area and percent cell area will be determined to assess absolute and relative amount of cell killing, as previously described.

[0301] Given the expected heterogeneity of PDOTS responses, waterfall plots ordering specimens by response to PD-1 blockade relative to IgG control both to define responders and non-responders will be generated. PDOTS response parameters are defined as follows: responder (reduction >30% compared to control), stable / partial responder (<30% reduction and <20% growth compared to control), and non-responder (>20% growth compared to control). Additionally, it will be determined if the population distribution of ex vivo PDOTS response characteristics is representative of melanoma patients treated with ICB clinically, including analysis of difference by age (<60 yrs. or >60yrs) and gender. Preliminary data indicates that the response rates to ex vivo PD-1 blockade in melanoma PDOTS approximates the clinical response rate expected in the clinical population without measurable difference in PDOTS viability7responses based on age, gender, or tumor mutational profile. Statistical analysis will be performed using 1-way ANOVA with matching between conditions from individual PDOTS specimens with Dunnetf s multiple comparisons test. Violin plots wall be generated for 2-way ANOVA with Sidak’s multiple comparisons to examine the effect between the 4 treatment arms averaged over the entire PDOTS cohort. The completion of these studies will help support determination of the efficacy of targeting SOX10 in advanced melanoma models.

[0302] The use of PDOTS and established melanoma cell lines to test the impact of SOX 10 inhibition will establish the generalizability of the findings. All randomization, treatments and analysis will be performed in a blinded fashion. Quantification of staining for proliferation and cell death will be performed automatically using ImageJ software (NIH).

[0303] Bioinformatic analysis pipelines for RNAseq will use STAR for mapping, featureCounts for gene expression measurement and DESeq2 for differential expression assessment and statistical evaluation (log2 fold change and FDR values). ChlP-seq peaks signals will be quantified with bwtools. Duplicate experiments will be performed for statistical analysis. T-tests will be used to determine statistical significance of changes in log2 signal intensities and p-values will be converted to FDR q-values to correct for multiplehypothesis testing as previously described. Statistical comparisons of PDOTS live / dead analysis after 5-6 days will also be based on ANOVA with Tukey’s correction for multiple comparisons. ANOVA models will be appropriately nested to allow for replication in the design. For PDOTS studies, assuming an overall comparison-wide type-I error of 0.1 (2- sided) and sample of size 35, each pairwise comparison would have at least 85% power to detect a difference in proliferation that is approximately 0.8 times the common standard deviation (effect size=0.8). Additional investigations assess the proportion of patient samples within each of the treatments with response (i.e., at least 50% dead cells). Based on prior data, it is expected a null rate of 25%. A treatment would be considered promising if at least 50% of patient samples achieved response. Statistical assessments are based on one-sample, exact binomial tests, with an overall, two-sided alpha=0. 1 divided equally among the three treatment comparisons. With 35 patient samples, there would be approximately 85% power to detect the difference between response rates of 25% and 50%. Using these approaches, emerging therapy with TBKli with immune checkpoint blockade using PDOTS sy stem can be evaluated (FIG. 55).

[0304] Example 7. A covalent SOX10 ligand disrupts melanoma transcriptional signaling and proliferation

[0305] To examine the effect of SH29 on melanoma proliferation, SH29 sensitivity was compared with SOX10 dependency following genomic depletion. Sensitivity to SOX10 depletion correlated with sensitivity to SH29 and with an established SOX 10 transcriptional signature (FIG. 56). Expression of a SOX10»C71 A mutant in U257 melanoma cells partially rescued the proliferation block of SH29 in comparison with cells expressing WT SOX10 (FIG. 57A and FIG. 57B). Given the consistent proliferation defect induced by depletion of SOX10 in melanoma cell lines, the activity of SH29 in 40+ melanoma cell lines of diverse origins was examined. This analysis revealed a wide range of inhibition of proliferation (FIG. 58). In contrast, treatment with SH29 or depletion of SOX10 showed only modest activity in SOX10-negative melanomas (e.g., IGR39, LOX-IMVI, and RPMI7951) and in lung (HOP62, HARA, and CALU6) or colon (SW620 and HCT116) models deficient in SOX10 expression.

[0306] To investigate how liganding of SOX10 impacts melanoma transcriptional networks, an extended SOX10 transcriptional signature was defined by depleting SOX10 in seven melanoma cell lines and analyzing the resulting transcriptional changes by RNA sequencing (RNA-seq) (FIG. 60). SH29 treatment in three melanoma cell lines revealed a significant enrichment for the SOX10 target genes as determined by gene set enrichment analysis (GSEA) (FIG. 61), which was confirmed in other melanomas by qPCR (FIG. 62).These results were further corroborated examining siRNA levels of SOXIO target genes following treatment with 2.5 pM of SH29 or SH105 relative to DMSO control in SKMEL5 cells (FIG. 63). Representative lava plots are shown in FIG. 64, and summary of SOX 10- regulated genes differentially impacted in melanoma cell lines following SH29 treatment is shown in FIG. 65. As noted previously, in SKMEL5 cells there was consistent overlap between cellular pathways perturbed following SH29 treatment and SOXIO depletion, and it was found that many of the genes downregulated by SH29 in this model were bound by SOXIO near active enhancers, including those of the genes ERBB3 and interleukin- 16 (IL- 16) (FIG. 49A and FIG. 49B). ERBB3 is a known melanoma-specific dependency and IL- 16, an immunomodulatory' regulator, is preferentially expressed in SOXIO-high melanocytic cells, suggesting that the SOXIO ligand causes downregulation of the SOXIO transcriptional network. Collectively, these findings demonstrate the ligandability of SOXIO and show that its covalent adduction can disrupt oncogenic transcriptional signaling.Example 8. Biological activity7of covalent inhibitors in U257 melanoma cells harboring the SOXIO luciferse reporter.

[0307] The U257 cells were transduced with lentiviral particles harboring pEZX vector (Genecopoeia) with a secreted luciferase under the control of a SOXIO target gene promoter. The U257 SOXIO reporter cells were replated at a confluency of 5,000 cells / well in 96-well plates. 24 hrs post seeding, cells were treated with vehicle (DMSO) or the indicated small molecule for 48 hrs. To determine SOXIO transcriptional activity, culture supernatants were collected and secreted luciferase (GLUC) and secreted alkaline phosphatase (SEAP, expression control) levels were determined according to manufacturer's instructions (Genecopoeia and Invivogen, respectively). In brief, SOXIO transcriptional activity was monitored by Gaussia luciferase (Glue) activity, and cell viability was monitored by secreted alkaline phosphatase (SEAP) activity. The Gluc / SEAP ratio was determined and then normalized to DMSO control so the percentage of DMSO-sample was 100%. Accordingly, the Gluc / SEAP ratio is expressed as a percentage. The results are shown in Table 5.Table 5. Biological activity ofGeneral synthetic methods

[0308] Reagents and solvents were obtained from commercial suppliers and used without further purification, unless otherwise stated. Reactions were carried out under a positive atmosphere of nitrogen and monitored by thin layer chromatography (TLC) using shortwave UV light or by SHIMADZU LC / MS. Normal phase flash column chromatography was performed using silica gel 60 N (spherical, 75-150 pM). ' f l NMR spectra were recorded on Bruker Avance 400 MHz spectrometer and were calibrated using residual non-deuterated solvent as the internal references (CDC13: 7.26 ppm; MeOH-D4: 3.31 ppm, acetone-D6: 2.05 ppm; DMSO-D6: 2.50 ppm). The following abbreviations were used to explain NMR peakmultiplicities: s = singlet, d= doublet, t = triplet, q = quartet, p = pentet, m = multiplet, br = broad.LCMSInstrument specifications:Agilent 1100 Series LC / MSD system with DADVELSD Alltech 2000ES and Agilent LC\MSD VL (G1956B), SL (G1956B) mass-spectrometer. Agilent 1200 Series LC / MSD system with DADVELSD Alltech 3300 and Agilent LC\MSD G6130A. G6120B mass- spectrometer. Agilent Technologies 1260 Infinity LC / MSD system with DAD\ELSD Alltech 3300 and Agilent LC\MSD G6120B mass- spectrometer. Agilent Technologies 1260 Infinity II LC / MSD system with DAD\ELSD G7102A 1290 Infinity II and Agilent LC\MSD G6120B mass-spectrometer.General Parameters:Detection: DAD - DAD1A 215 nm, DAD1B 254 nm MSD - single quadrupole, AP-ESI (positive / negative mode switching)Temperature: 25 °CColumn: InfinityLab Poroshell 120 SB-C 18 4.6x30mm 2.7 Micron with Guard: UHPLC Guard 3PK InfinityLab Poroshell 120 SB-C18 4.6x5mm 2.7 MicronMobile phases: A - Deionized water: Formic acid (99.9:0.1%). B - HPLC-grade MeCN: (Deionized water: Formic acid (99.9:0.1%)) (95:5%)Gradient: from A - 99%, B - 1 % to A - 1 %, B - 99%HPLC procedures:Instrument specifications:Agilent 1260 Infinity' systems equipped with DAD and mass-detector General Parameters: Temperature: 25 °C HPLC procedure 1 :Column: Chromatorex 18 SMB 100-5T 100 A, 5 pm, 19 mm x 100mm with SiliaSphere C18 100A 5pm 100 A, 19mm x 10 mm. Detection: DAD - DAD1A 215 nm, DAD IB 254 nm. MSD - single quadrupole. AP-ESIMobile phases: A - Deionized water (100%). B - HPLC-grade CH3CN (100%)HPLC procedure 2:Column: Chromatorex 18 SMB 100-5T 100 A, 5 pm, 19 mm x 100mm with SiliaSphere C18 100A 5pm 100 A, 19mm x 10 mm Detection: DAD - DAD1A 215 nm, DAD IB 254 nm. MSD - single quadrupole. AP-ESIMobile phases: A - Deionized water (100%). B - HPLC-grade MeOH (100%)HPLC procedure 3:Column: Chromatorex 18 SMB 100-5T 100 A, 5 pm, 19 mm x 100mm with SiliaSphere C18 100A 5pm 100 A, 19mm x 10 mm. Detection: DAD - DAD1A 200 nm, DAD IB 215 nm. MSD - single quadrupole, AP-ESIMobile phases: A - Deionized water (100%). B - HPLC-grade CH3CN (100%)HPLC procedure 4:Column: Chromatorex 18 SMB 100-5T 100 A, 5 pm, 19 mm x 100mm with SiliaSphere C18 100 A 5 pm 100 A, 19mm x 10 mmDetection: DAD - DAD1A 200 nm, DAD1B 254 nm. MSD - single quadrupole, AP-ESIMobile phases: A - Deionized water (100%). B - HPLC-grade MeOH (100%)HPLC procedure 5:Column: XBridge Shield RP18 OBD Column, 30*150 mm, 5pm Detection: UV - wavelength: 254 and 220 nm.Mobile Phases: A: Water (10 mmol / L NH4HCO3). B: ACN; Flow rate: 60 mL / min.HPLC procedure 6:Column: conditions (Column: YMC-Actus Triart Cl 8, 30*150 mm, 5pm Detection: UV - wavelength: 254 and 220 nmMobile Phases: A: Water (10 mmol / L NH4HCO3). B: ACN; Flow rate: 60 mL / min.Intermediates:Intermediate 1. Preparation of 3-acrylamido-5-(prop-2-yn-l-yloxy)benzoic acid

[0309] Step 1 : Preparation of methyl 3-nitro-5-(prop-2-yn-l-yloxy)benzoate

[0310] To a flame-dried round bottom flask, methyl 3-hydroxy-5-nitrobenzoate (5.00 g, 25.4 mmol), potassium carbonate (4.20 g, 30.5 mmol), and propargyl bromide (1.68 g, 30.5 mmol) in DMF (20 ml) stirred at RT for 16 h. The mixture was diluted with diethyl ether (100 ml) and water (100 ml) and then transferred to a separatory funnel. Theorganic layer was separated, dried with magnesium sulfate, concentrated, and used in the next step without further purification.

[0311] Step 2: Preparation of methyl 3-amino-5-(prop-2-yn-l-yloxy)benzoate

[0312] The isolated alkyne intermediate (1.00 g, 4.25 mmol) was suspended in MeOH (30 ml) and stirred atRT, then aqueous sat. NH4CI (10ml) and Zn metal (1.00 g, 15.4mmol) were added portion-wise. The mixture was raised to reflux for 1 h, cooled to RT, and filtered through celite. The filtrate w as concentrated to remove methanol then redissolved in EtOAc. The mixture was transferred to a separatory funnel and w ashed with sat. NaHCCh, dried with magnesium sulfate, concentrated, and purified via column chromatography to afford methyl 3-amino-5-(prop-2-yn-l- yloxy)benzoate.

[0313] Step 3: Preparation of methyl 3-acrylamido-5-(prop-2-yn-l-yloxy)benzoate

[0314] In a flame-dried round bottom flask, the amine intermediate (1.00 g, 4.88 mmol) was dissolved in THF and cooled to 0° C. Acryloyl chloride (0.530 g. 5.86 mmol) and NEts (0.591 g, 5.86 mmol) were added dropwise. The reaction mixture was stirred for 3 h, quenched with 10 ml of DI water, and then transferred to a separatory funnel. The aqueous layer was extracted three times with EtOAc, dried with magnesium sulfate, concentrated, and purified via column chromatography to afford methyl 3-acrylamido-5-(prop-2-yn- l - yloxy)benzoate.

[0315] Step 4: Preparation of 3-acrylamido-5-(prop-2-yn- l-yloxy)benzoic acid

[0316] Methyl 3-acrylamido-5-(prop-2-yn-l-yloxy)benzoate (1.00 g, 3.86 mmol) was dissolved in THF / H2O (10: 1, 11 mL), then LiOH monohydrate (0.195 g, 4.63 mmol) was added portion wise. The reaction mixture was stirred for 12 hat RT, then quenched with 20 ml of 1MHC1. The solution was transferred to a separatory funnel, extracted 3xEtOAc, dried with magnesium sulfate, concentrated, and purified via column chromatography to afford the title compound.Intermediate 2. Preparation of 6-amino-l,4-dimethyl-3,4-dihydroquinolin-2(lH)-one

[0317] Step 1: Preparation of l,4-dimethyl-3,4-dihydroquinolin-2(lH)-one

[0318] l,4-dimethylquinolin-2(H)-one (2.00 g, 11.7 mmol) was dissolved in 50.0 ml of AcOH, then Pd / C (cat.) was added, and the atmosphere was replaced with hydrogen gas. The reaction mixture was raised to 75° C, and then stirred for 16 h. The crude mixture was cooledto RT and filtered through celite. The filtrate was then poured into ice-water, and the product was collected via filtration and used in the next step without further purification.

[0319] Step 2: Synthesis of l,4-dimethyl-6-nitro-3,4-dihydroquinolin-2(lH)-one

[0320] l,4-dimethyl-3,4-dihydroquinolin-2(lH)-one (1.00 g, 5.71 mmol) was dissolved in H2SO4 (10 ml) and cooled to 0° C. Fuming nitric acid (5.71 mmol) was added dropwise, then the mixture was allowed to warm to RT and stirred for 3 h. The crude mixture was poured into a 2: 1 ice-water mixture and extracted with EtOAc, dried with magnesium sulfate, concentrated, and purified via column chromatography to afford 1,4- dimethyl-6-nitro-3,4-dihydroquinolin-2(lH)-one.

[0321] Step 3: Preparation of 6-amino-l,4-dimethyl-3.4-dihydroquinolin-2(lH)-one

[0322] l,4-dimethyl-6-nitro-3,4-dihydroquinolin-2(lH)-one (1.00 g. 4.59 mmol) was suspended in MeOH (70 ml}. Then, aqueous sat. NH4CI (20 ml) and Zn metal (1.00 g, 15.4 mmol) were added portion-wise. The mixture was raised to reflux for 1 h, cooled to RT, and filtered through celite. The filtrate was concentrated to remove methanol and then redissolved in EtOAc. The mixture was transferred to a separatory funnel, washed with sat. NaHCOs, dried with magnesium sulfate, concentrated, and purified via column chromatography .Intermediate 3. Preparation of (N-(14-azido-3,6,9,12-tetraoxatetradecyl)-6-((4 5S)-5- meth yl-2-oxoimidazolidin-4-yl)hexanamide)

[0323] Step 1 : Preparation of 2,5-dioxopyrrolidin-l-yl-6-((4R,5S)-5-methyl-2- oxoimidazolidin-4-yl)hexanoate

[0324] Desthiobiotin (200 mg, 0.93 mmol), N-hydroxysuccinimide (241 mg, 1.11 mmol), and EDC-HCI (213 mg, 1.11 mmol) were stirred in DMF under a nitrogen atmosphere for 12 hours. The crude mixture was diluted with EtOAc, washed with water, dried with magnesium sulfate, and concentrated to afford crude product subsequently used in the next step.

[0325] Step 2: Preparation of (N-(14-azido-3,6,9,12-tetraoxatetradecyl)-6-((47?,5S)-5- meth yl-2-oxoimidazolidin-4-yl)hexanamide)

[0326] To a solution of crude 2,5-dioxopyrrolidin-l-yl-6-((4R,5S)-5-methyl-2- oxoimidazolidin-4-yl)hexanoate (100 mg, 0.32 mmol) in dichloromethane, N,N- diisopropylethylamine (42 mg, 0.32 mmol) and 14-azido-3,6,9,12-tetraoxatetradecan-l-amine (83 mg, 0.32 mmol) were added. The reaction mixture was stirred overnight at room temperature under a nitrogen atmosphere. The solvent was then removed in vacuo, and the resulting oil was subjected to flash chromatography to obtain N-(14-azido- 3,6,9, 12- tetraoxatetradecyl)-6-((47?, 5S)-5-meth yl-2-oxoimidazolidin-4-yl)hexanamide).Compounds:General amide coupling procedure:

[0327] Amide coupling procedure: The -NH2 substrate, -COOH substrate, EDC, HOAt, NEts, and DMF were added to a flame dried round bottom flask under nitrogen atmosphere and stirred for 12 h. The reaction mixture is then diluted with EtOAc, transferred to a separatory funnel, washed with H2O, dried over magnesium sulfate, and concentrated.Example 9. Preparation of A-(4-(4-methyl-3-oxo-6-(trifluoromethyl)-l, 2,3,4- tetrahydroquinoxaline-1 -carbonyljbenzy Ijacrylamide

[0328] The general amide coupling procedure performed using 51 mg (0.22 mmol) of 1- methyl-7-(trifluoromethyl)-3,4-dihydroquinoxalin-2(177)-one hydrochloride, 54 mg (0.263 mmol) of 4-[(prop-2-enamido)methyl]benzoic acid, 54 mg (0.348 mmol) of EDC, 33 mg (0.326 mmol) of EtsN, and 31.4 mg (0.231 mmol) of HOAt. Purified by HPLC procedure 4 (gradient: from A-85%: B-15% to A-35%: B-65%; Rf = 0.81; run time = 6.5 min). Yield: 40.0 mg (40 %). Beige solid. ' H NMR (400 MHz, Methanol-D4) 5 7.53 (s, 1H), 7.42 (d, J = 8.3 Hz. 2H), 7.32 (d. J = 8.5 Hz. 2H), 7. 14 (d. J = 8.6 Hz, 1H). 6.98-6.96 (m, 1H). 5.69 (dd. J = 8.3, 3.7 Hz, 1H), 4.57 (s, 2H), 4.48 (s, 2H), 3.48 (s, 3H). El MS m / z: pos. 418.0 (M+H)+.

[0329] Example 10. Preparation of 4-(N-methylprop-2-enamido)-N-(2-oxo-2,3,4,5- tetrahydro-lH-l-benzazepin-7-yl)benzamide

[0330] The general amide coupling procedure performed using 53 mg (0.301 mmol) of 7- amino-2,3,4,5-tetrahydro-lH-l- benzazepin-2-one, 57 mg (0.278 mmol) of 4-(N-methylprop-2- enamido)benzoic acid, 49 mg (0.316 mmol) of EDC, and 43 mg (0.316 mmol) of HO At. Purified by HPLC procedure 1 (gradient: from A- 70%: B-30% to A-45%: B-55%). Yield: 56.9 mg (62 %). White powder. LCMS purity: 95.4 % (LCMS procedure 1, Rf = 0.51, run time = 2 min). ' H NMR (400 MHz, Methanol-D4) 5 8.03 (d, J = 8.6 Hz, 2H), 7.62-7.60 (m, 2H), 7.44-7.42 (m, 2H), 7.04 (d, J = 8.9 Hz, 1H), 6.31 (dd, J = 16.8, 2.1 Hz, 1H), 6.22-6.15 (m, 1H), 5.63 (dd, J = 10.2, 2.2 Hz, 1H), 3.39 (s, 3H), 2.81 (t, J = 6.9 Hz, 2H), 2.34-2.21 (m, 4H). El MS m / z: pos. 364.1 (M+H)+.Example 11. Preparation of N-(2-methyl-l-oxo-l,2-dihydroisoquinolin-7-yl)-4-(N- methylprop-2-enamido)benzamide

[0331] The general amide coupling procedure performed using 51 mg (0.293 mmol) of 7- amino-2-methyl-1.2-dihydroisoquinolin- 1-one. 62 mg (0.302 mmol) of 4-(N-methylprop-2- enamido)benzoic acid, 59 mg (0.38 mmol) of EDC, and 41.9 mg (0.308 mmol) of HO At. Purified by HPLC procedure 1 (gradient: from A-75%: B- 25% to A-50%: B-50%). Yield: 43.8 mg (44.1 %). Beige powder. LCMS purity : 98.5 % (LCMS procedure 1, Rf = 0.43, run time = 2 min). ' H NMR (400 MHz, Methanol-D4) 5 8.63 (d, J = 2.4 Hz, 1H), 8.14 (dd, J = 8.7, 2.3 Hz, 1H), 8.08 (d, J = 8.8 Hz, 2H), 7.68 (d, J = 8.6 Hz, 1H), 7.45 (d, J = 8.6 Hz, 2H), 7.33 (d, J = 7.3 Hz, 1H), 6.70 (d, J = 7.3 Hz, 1H), 6.31 (dd, J = 16.8, 2.2 Hz, 1H), 6.23-6.16 (m, 1H), 5.64 (dd, J = 10.1, 2.1 Hz, 1H), 3.63 (s, 3H), 3.40 (s, 3H). El MS m / z: pos. 362.2 (M+H)+.Example 12. Preparation of N-(7-fluoro-2-oxo-l,2,3,4-tetrahydroquinolin-6-yl)-4-(prop-2- enamido)benzamide

[0332] The general amide coupling procedure performed using 44 mg (0.244 mmol) of 6- amino-7-fluoro-l,2,3,4-tetrahydroquinolin-2-one, 46 mg (0.241 mmol) of 4-(prop-2- enamido)benzoic acid. 40 mg (0.258 mmol) of EDC, and 34.9 mg (0.256 mmol) of HOAt. Purified by HPLC procedure 1 (gradient: from A-85%: B-15% to A-60%: B- 40%). Yield: 24 mg (31.1 %). Beige powder. LCMS purity: 96.9 % (LCMS procedure 1, Rf = 0.51, run time= 2 min). ’ l l NMR (400 MHz, Methanol-D4) 5 7.95 (d, J = 8.9 Hz, 1H), 7.81 (d, J = 9.0 Hz, 1H), 7.47 (d, J = 7.9 Hz. 1H), 6.75 (d. J = 11.0 Hz, 1H), 6.50-6.38 (m, 1H), 5.82 (dd, J = 9.3, 2.6 Hz, 1H), 2.96 (t, J = 7.6 Hz, 1H), 2.60 (dd, J = 8.5, 6.6 Hz, 1H). El MS m / z: pos. 354.0 (M+H)Example 13. Preparation of 2-acrylamido-N-(l,4-dimethyl-2-oxo-l,2,3,4-tetrahydroquinolin- 6-yl)benzamide

[0333] The general amide coupling procedure performed using 56 mg (0.298 mmol) of 6- amino-1,4- dimethyl-3,4-dihydroquinolin-2(lH)-one, 63 mg (0.33 mmol) of 3-(prop-2- enamido)benzoic acid, 61 mg (0.393 mmol) of EDC, and 42.5 mg (0.312 mmol) of HOAt. Purified by HPLC procedure 2 (gradient: from A-60%: B-40% to A-10%: B-90%). Yield: 20.0 mg (19.0 %). Beige solid. 'H NMR (400 MHz, CDCh) 5 8.21 (s, 1H), 8.17 (s, 1H), 7.94 (s, 1H), 7.72 (d, J = 8.0 Hz, 1H), 7.62 (d, J = 8.3 Hz, 1H), 7.54-7.44 (m, 2H), 7.41 (t, J = 7.9 Hz, 1H), 6.96 (d, J = 8.7 Hz, 1H), 6.45 (d, J = 16.8 Hz, 1H), 6.28 (dd, J = 16.9, 10.2 Hz, 1H), 5.79 (d, J = 11.5 Hz, 1H), 3.35 (s, 3H), 3.08- 2.98 (m, 1H), 2.72 (dd, J = 15.8, 5.4 Hz, 1H), 2.44 (dd, J = 15.8, 7.5 Hz, 1H), 1.27 (d, J = 7.0 Hz, 3H). El MS m / z: pos. 364.2 (M+H)+.Example 14. Preparation of N-(1.3-dimethyl-2-oxo-l,2-dihydroquinolin-6-yl)-3-(prop-2- enamido)benzamide

[0334] The general amide coupling procedure performed using 56 mg (0.298 mmol) of 6- amino-1.3-dimethyl-1.2-dihydroquinolin-2-one. 63 mg (0.33 mmol) of 3-(prop-2- enamido)benzoic acid, 61 mg (0.393 mmol) of EDC, and 42.5 mg (0.312 mmol) of HOAt. Purified by HPLC procedure 2 (gradient: from A-60%: B-40% to A-10%: B-90%). Yield: 9.6 mg (8.9 %). Beige powder. LCMS purity: 100 % (LCMS procedure 1, Rf = 0.6, run time = 2 min). ’H NMR (400 MHz, Methanol-D4) 5 8.24 (s, 1H), 8.06 (d. J = 2.4 Hz, 1H), 7.88 (dd, J = 9.1, 2.4 Hz, 1H), 7.82 (d, J = 8.1 Hz, 1H), 7.77 (s, 1H), 7.70 (d, J = 8.1 Hz, 1H), 7.57 (d, J= 9.1 Hz, 1H), 7.50 (t, J = 7.9 Hz, 1H), 6.50-6.38 (m, 2H), 5.82 (dd, J = 9.2, 2.7 Hz, 1H), 3.79 (s, 4H), 2.24 (s, 3H). El MS m / z: pos. 362.2 (M+H)+.Example 15. Preparation of 3-acrylamido-N-(l,4-dimethyl-2-oxo-l, 2,3,4- tetrahydroquinolin-6-yl)benzamide

[0335] The general amide coupling procedure performed using 56 mg (0.298 mmol) of 6- amino-1,4- dimethyl-3,4-dihydroquinolin-2(lH)-one, 63 mg (0.33 mmol) of 3-(prop-2- enamido)benzoic acid. 61 mg (0.393 mmol) of EDC, and 42.5 mg (0.312 mmol) of HOAt. Purified by HPLC procedure 2 (gradient: from A-60%: B-40% to A- 10%: B-90%). Yield: 48.0 mg (45.0 %). Beige solid. ’H NMR (400 MHz, Methanol-D4) 5 8.17 (s, 1H), 7.78 (dm, J = 8.1 Hz. 1H), 7.64-7.59 (m. 3H), 7.46 (t, J = 7.9 Hz, 1H), 7.12 (d, J = 9.4 Hz, 1H), 6.47-6.35 (m. 2H), 5.78 (dd, J = 9.2, 2.7 Hz, 1H), 3.35 (s, 3H), 3.35 (s, 3H), 3.06 (h, J = 7.0 Hz. 1H), 2.72 (dd, J = 15.9, 5.5 Hz, 1H), 2.42 (dd, J = 15.9, 7.0 Hz, 1H). El MS m / z: pos. 364.2 (M+H)+.Example 16. Preparation of N-(l-methyl-2-oxo-l,2,5,6,7,8-hexahydroquinolin-5-yl)-4-(prop- 2-enamido)benzamide

[0336] The general amide coupling procedure performed using 49 mg (0.275 mmol) of 5- amino-l-methyl-1,2,5,6,7,8- hexahydroquinolin-2-one. 57 mg (0.298mmol) of 4-(prop-2- enamido)benzoic acid. 57 mg (0.367 mmol) of EDC, and 39.3 mg (0.289 mmol) of HOAt. Purified by HPLC procedure 2 (gradient: from A- 70%: B-30% to A-20%: B-80%; Rf = 0.56; run time = 6.5 min). Yield: 52.9 mg (55.5 %). Yellow powder. LCMS purity: 100 % (LCMS procedure 1, Rf = 0.39, run time = 2 min). 'l l NMR (400 MHz, Methanol-D4) 5 7.83 (d, J = 8.9 Hz. 2H), 7.74 (d. J = 9.0 Hz. 2H), 7.42 (d. J = 9.3 Hz, 1H), 6.49-6.36 (m, 3H). 5.80 (dd. J = 9.3, 2.6 Hz, 1H), 5.16-5.15 (m, 1H), 3.56 (s, 3H), 2.87-2.73 (m, 2H), 2.1-1.91 (m, 4H). El MS m / z: pos. 352.2 (M+H)+.Example 17. Preparation of N-(l-methyl-2-oxo-l,2,3,4-tetrahydroquinolin-6-yl)-4-(N- methylprop-2-enamido)benzamide

[0337] The general amide coupling procedure performed using 59 mg (0.335 mmol) of 6-amino-l -methyl- 1.2.3.4- tetrahydroquinolin-2-one, 69 mg (0.336 mmol) of 4-(N- methylprop-2-enamido)benzoic acid, 52 mg (0.335 mmol) of EDC, and 47.9 mg (0.352 mmol) of HO At. Purified by HPLC procedure 1 (gradient: from A-75%: B-25% to A-50%: B-50%). Yield: 69.4 mg (62.5 %). Beige powder. LCMS purity': 100 % (LCMS procedure 1, Rf = 0.58, run time = 2 mm). ' H NMR (400 MHz, Methanol-D4) 5 8.04-8.02 (m, 2H), 7.64- 7.60 (m, 2H), 7.44-7.41 (m. 2H), 7.14 (d, J = 8.5 Hz, 1H), 6.31 (dd, J = 16.9, 2.1 Hz. 1H). 6.21-6.15 (m, 1H), 5.63 (dd, J = 10.2, 2.2 Hz, 1H), 3.38 (d, J = 8.6 Hz, 6H), 2.96-2.93 (m, 2H), 2.66-2.63 (m, 2H). El MS m / z: pos. 364.2 (M+H)+.Example 18. Preparation of N-(2-chloro-l,3-benzothiazol-6-yl)-4-(prop-2- enamido)benzamide

[0338] The general amide coupling procedure performed using 52 mg (0.283 mmol) of 2- chloro-l,3-benzothiazol-6-amine, 59 mg (0.309 mmol) of 4-(prop-2-enamido)benzoic acid, 52 mg (0.335 mmol) of EDC, and 40.4 mg (0.297mmol) of HOAt. Purified by HPLC procedurel (gradient: from A-60%: B-40% to A-35%: B-65%). Yield: 13.1 mg (13 %). Yellow powder. LCMS purity: 100 % (LCMS procedure 1, Rf = 0.65. run time = 2 min). 'H NMR (400 MHz, Methanol-D4) 8 8.50 (d, J = 2.1 Hz, 1H), 7.97 (dm, J = 8.9 Hz, 2H), 7.89 (d, J = 8.8 Hz, 1H), 7.83 (dm, J = 8.9 Hz, 2H), 7.75 (dd, J = 8.9, 2.1 Hz, 1H), 6.51-6.38 (m, 2H), 5.82 (dd, J = 9.3, 2.6 Hz, 1H). El MS m / z: pos. 358.0 (M+H)+.Example 19. Preparation of N-[(4-{6-oxa-2-thia-9-azaspiro[4.6]undecane-9- carbonyl}phenyl)methyl]prop-2-enamide

[0339] The general amide coupling procedure performed using 46 mg (0.22 mmol) of 6-oxa-2-thia-9-azaspiro[4.6]undecane hydrochloride, 54 mg (0.263 mmol) of 4 [(prop-2- enamido)methyl]benzoic acid. 54 mg (0.348 mmol) of EDC, 33 mg (0.326 mmol) of EtsN, and 31.4 mg (0.231 mmol) of HOAt. Purified by HPLC procedure 4 (gradient: from A-85%: B-15% to A-35%: B-65%; Rf = 0.81; run time = 6.5 min). Yield: 50 mg (58 %). Pink powder. LCMS purity: 100 % (LCMS procedure 1, Rf = 0.52, run time = 2 min).JH NMR (400 MHz, Methanol-D4) 5 7.40-7.39 (m. 4H), 6.33-6.23 (m, 2H), 5.69 (dd, J = 8.5, 3.5 Hz, 1H), 4.48 (s, 2H), 3.94-3.54 (m, 6H), 3.00-2.90 (m. 2H), 2.83-2.63 (d, J = 57.7 Hz. 2H), 2.27- 2.08 (m, 3H), 1.84-1.68 (m, 1H). El MS m / z: pos. 361.2 (M+H)+.Example 20. Preparation of N-(l,l-dimethyl-3-oxo-l,3-dihydro-2-benzofuran-5-yl)-4-(N- methylprop-2-enamido)benzamide

[0340] The general amide coupling procedure performed using 36 mg (0.203 mmol) of 6- amino 3,3-dimethyl-l,3-dihydro-2- benzofuran- 1 -one, 47 mg (0.229 mmol) of 4-(N- methylprop-2-enamido)benzoic acid, 43 mg (0.277 mmol) of EDC, and 29.1 mg (0.214 mmol) of HOAt. Purified by HPLC procedure 3 (gradient: from A- 65%: B-35% to A-40%: B-60%). Yield: 35.8 mg (47.2 %). Beige powder. LCMS purity: 100 % (LCMS procedure 1, Rf = 0.55, run time = 2 min). 'H NMR (400 MHz, MeOD-D4) 5 8.26 (d, J = 2.0 Hz, 1H), 8.07-8.04 (m, 3H), 7.62 (d, J = 8.4 Hz, 1H), 7.45 (d, J = 8.6 Hz, 2H), 6.31 (dd, J = 16.8, 2.1 Hz. 1H), 6.22-6.15 (m. 1H), 5.64 (dd, J = 10.2, 2.2 Hz, 1H), 3.40 (s, 3H), 1.68 (s, 6H). El MS m / z: pos. 365.0 (M+H)+.Example 21. Preparation of 4-acrylamido-N-(5-bromo-2-hydroxyphenyl)benzamide

[0341] The general amide coupling procedure performed using 38 mg (0.203 mmol) of 4- acrylamidobenzoic acid. 43 mg (0.229 mmol) of 2-amino-4-bromophenol, 43 mg (0.277 mmol) of EDC, and 29 mg (0.214 mmol) of HOAt. Purified by preparatory HPLC. Beige powder. 'H NMR (400 MHz, DMSO-D6) 5 10.44 (s, 1H), 10.21 (bs, 1H), 9.39 (s, 1H), 7.97- 7.93 (m, 2H), 7.82-7.80 (m, 2H), 7.18 (dd, J = 8.6, 2.5 Hz, 1H), 6.88 (d, J = 8.6 Hz, 1H), 6.47(dd, J = 17.0, 10.1 Hz. 1H), 6.31 (dd, J = 17.0, 2.0 Hz, 1H), 5.81 (dd, J = 10.0, 2.0 Hz, 1H). El MS m / z: pos. 361.0 (M+H)+.Example 22. Preparation of 4-acrylamido-N-(3-oxo-3,4-dihydro-2H-benzo[b][l,4]thiazin-7- yl)benzamide

[0342] The general amide coupling procedure performed using 38 mg (0.203 mmol) of 4- acrylamidobenzoic acid. 41 mg (0.229 mmol) of 7-amino-2H benzo[b][l,4]thiazin-3(4H)- one, 43 mg (0.277 mmol) of EDC, and 29 mg (0.214 mmol) of HO At. Purified by preparatory HPLC. Beige powder. El MS m / z: pos. 354.1 (M+H)+.Example 23. Preparation of (R)-N-benzyl-l-cyanopyrrolidine-3-carboxamide

[0343] Step 1 : Preparation of tert-butyl (3R)-3-(benzylcarbamoyl)pyrrolidine-l- carboxylateA solution of (3R)-l-(tert-butoxycarbonyl)pyrrolidine-3-carboxylic acid (400 mg, 1.86 mmol, 1 equiv) in DMF (5 mL) was treated with HATU (1060 mg, 2.79 mmol, 1.5 equiv) and DIEA (721 mg, 5.57 mmol, 3 equiv) at 0°C under nitrogen atmosphere followed by the addition of benzylamine (219 mg, 2.04 mmol, 1.1 equiv) in portions at room temperature 159. The resulting mixture was stirred for 3 hrs at room temperature under nitrogen atmosphere. The resulting mixture was diluted with water (20 mL). The aqueous layer was extracted with CH2CI2 (3x10 mL). The organic layers were concentrated under vacuum. The residue was purified by Prep-TLC (PE / EA 1:3) to afford terl-butyl (3R)-3-(benzylcarbamoyl)pyrrolidine- 1-carboxylate (520 mg, 92%) as ayellow solid. LC-MS: (M-H)+ found 303.15.

[0344] Step 2: Preparation of (3R)-N-benzylpyrrolidine-3-carboxamideA solution of tert-butyl (3R)-3-(benzylcarbamoyl)pyrrolidine- 1 -carboxylate (200 mg, 0.66 mmol, 1 equiv) in DCM (3 mL) was stirred at room temperature under nitrogen atmosphere, then the addition of TFA (1 mL) was administered dropwise 160. The resulting mixture was stirred for 2 hrs under nitrogen atmosphere. The resulting mixture was concentrated under vacuum to afford (3R)-N-benzylpyrrolidine-3-carboxamide (200 mg, crude) and was used in the next step directly without further purification. LC-MS: (M+H)+ found 205.10.

[0345] Step 3: Preparation of (3R)-N-benzyl-l-cyanopyrrolidine-3-carboxamideA solution of (3R)-N-benzylpyrrolidine-3-carboxamide (200 mg, 0.98 mmol. 1 equiv) in DCM (3 mL) was treated with Et3N (495 mg, 4.89 mmol, 5 equiv) at 0°C under nitrogen atmosphere followed by the addition of BrCN (114.08 mg, 1.077 mmol, 1.1 equiv) in portions at 0°C. The resulting mixture was stirred for 2 hrs at room temperature under nitrogen atmosphere. The mixture was diluted with CH2CI2 saturated NaHCOs and the aqueous layer was extracted with CH2CI2 (3x10 mL). The organic layers were concentrated under vacuum. The crude product (150 mg) was purified by HPLC procedure 5 (Gradient: 10% B to 34% B) in 9 min to afford (3R)-N-benzyl-l-cyanopyrrolidine-3-carboxamide (40 mg, 18%) as a white solid. 'H NMR (400 MHz, Chloroform-d) 5 7.42 -7.31(m, 3H). 7.31 - 7.29 (m, 1H), 7.28 - 7.25 (m, 1H). 5.91 (s, 1H), 4.47 (d, J = 5.6 Hz, 2H), 3.71 - 3.57 (m. 3H), 3.45 - 3.40 (m, 1H), 2.92 - 2.85 (m, 1H), 2.25 - 2.10 (m, 2H). LC-MS: 228.00 (M+H)+.Example 24. Preparation of (S)-l-(2-bromo-4-chlorophenyl)-N-(l-cyanopyrrolidin-3- yl)methanesulfonamide

[0346] A solution of (3S)-3-aminopyrrolidine-l-carbonitrile (100 mg, 0.900 mmol, 1 equiv) in DCM (0.7 mL) was treated with ELN (455 mg, 4.50 mmol. 5 equiv) for 5 min at room temperature under nitrogen atmosphere followed by the addition of (2-bromo-4- chlorophenyl) methanesulfonyl chloride (273.49 mg, 0.900 mmol, 1 equiv) in DCM (0.3 mL) dropwise at 0°C. The resulting mixture was stirred for 2 hrs at room temperature under nitrogen atmosphere. The reaction was quenched with water at 0°C. The resulting mixture was extracted with CH2CI2 (2 x 15 mL). The combined organic layers were washed with brine (1x10 mL) and dried over anhydrous Na2SC>4. After filtration, the filtrate was concentrated under reduced pressure. The residue (100 mg) was purified by HPLC procedure 6, gradient: 33% B to 55% B in 9 min to afford l-(2-bromo-4-chlorophenyl)-N-[(3S)-l- cyanopyrrolidin-3-yl] methanesulfonamide (39 mg, 11%) as a yellow solid. LCMS: (M+H)+found 377.75. ' H NMR (300 MHz, Chloroform-d) 5 7.67 (d, J = 2.0 Hz, 1H), 7.53 (d, J = 8.3 Hz, 1H), 7.38 (d, J = 8.3, 1H), 4.52 (s, 3H), 3.84 (t, J = 5.7 Hz, 1H), 3.58 - 3.37 (m, 3H), 3.22 (d, J = 10.2, 1H), 2.15 - 2.01 (m, 1H), 1.87 - 1.75 (m, 1H).Example 25. Preparation of (S)-N-(l-cyanopyrrolidin-3-yl)-l-phenylmethanesulfonamide

[0347] Step 1 : Preparation of tert-butyl (S)-3-((phenylmethyl)sulfonamido)pyrrolidine-l- carboxylate tert-butyl (S)-3-aminopyrrolidine-l-carboxylate (130 mg, 0.70 mmol, 1.00 equiv.) and phenylmethanesulfonyl chloride (130 mg, 1.05 mmol. 1.50 equiv.) were dissolved in ACN (5.00 mL), and then, TEA (0.31 mL, 2.10 mmol, 3.00 equiv.) was added to the solutionl60. The mixture was heated at 30 °C for 2 hrs. The mixture was concentrated in vacuo and the crude product was used into next step without further purification.

[0348] Step 2: Preparation of (S)-l-phenyl-N-(pyrrolidin-3-yl)methanesulfonamideTo a solution of tert-butyl (S)-3-((phenylmethyl)sulfonamido)pyrrolidine-l -carboxylate (crude) in DCM (0.50 mL), HCl-Dioxane (3.50 mL, 4.00 M) was added dropwise, then mixture was heated at 30 °C for 2 hrs 162. The mixture was concentrated in vacuo and the crude product was used without further purification.

[0349] Step 3: Preparation of (S)-N-(l-cyanopyrrolidin-3-yl)-l- phenylmethanesulfonamide(S)-l-phenyl-N-(pyrrolidin-3-yl)methanesulfonamide (crude) was dissolved in DMSO (3.00 mL), and then DIEA (0.37 mL, 2.10 mmol, 3.00 equiv.) and cyanic bromide (147 mg, 1.40 mmol, 2.00 equiv.) were added to the solutionl61. The mixture was heated at 30 °C for 2 hrs. The mixture was purified by prep HPLC to give (S)-N-(l- cyanopyrrolidin-3-yl)-l- phenylmethanesulfonamide (40 mg, 0.15 mmol, 99%).XH NMR (400 MHz, DMSO-d6) 5 ppm 1.70 - 1.82 (m, 1 H) 1.98 - 2.10 (m, 1 H) 3.13 (dd, J=9.69, 4.82 Hz, 1 H) 3.35 - 3.39 (m, 1 H) 3.40 - 3.54 (m, 2 H) 3.88 (sxL J=5.88 Hz, 1 H) 4.39 (s, 2 H) 7.31 - 7.43 (m, 5 H) 7.55 (br d. J=6.63 Hz. 1 H). MS-ESI. 266.1 (M+H)+. HRMS-TOF. (M+H)+, 266.0945.Example 26. Preparation of (S)-N-(l -cyanopyrrolidin-3-yl)-N-methyl-2- (phenylamino)pyridine-3-sulfonamide

[0350] Step 1 : Preparation of tert-butyl (3S)-3-[N-methyl2-(phenylamino)pyridine-3- sulfonamido]pyrrolidine-l -carboxylate

[0351] To a stirred solution of NaH (38 mg. 1.60 mmol, 2 equiv) and aniline (82 mg, 0.878 mmol, 1.1 equiv) in THF (5 mL) at 0°C. tert-butyl (3S)-3-(N-methyl2-chloropyridine- 3- sulfonamido)pyrrolidine-l -carboxylate (300 mg, 0.798 mmol, 1 equiv) was added. The resulting mixture was stirred for 12 hrs at 50°C then concentrated under reduced pressure. The residue was purified by reverse-phase flash chromatography with the following conditions: column, Cl 8 silica gel; mobile phase, ACN in water. 0% to 100% gradient in 20 min; detector, UV 254 nm to afford tert-butyl (3S)-3-[N-methyl2-(phenylamino)pyridine-3- sulfonamido]pyrrolidine-l-carboxylate (80 mg, 23%) as yellow^ oil. LC-MS: (M+H)+, 433.

[0352] Step 2: preparation of N-methyl-2-(phenylamino)-N-[(3S)-pyrrolidin-3- yl]pyridine-3-sulfonamide

[0353] To a stirred solution of tert-butyl (3S)-3-[N-methyl2-(phenylamino)pyridine-3- sulfonamido]pyrrolidine-l-carboxylate (80 mg, 0.185 mmol, 1 equiv) in DCM (2 mL), HC1 in 1,4-dioxane (0.5 mL, 16.456 mmol, 88.98 equiv) was added drop wise at room temperature. The resulting mixture was stirred for Ihr and then concentrated under reduced pressure to afford crude N-methyl-2-(phenylamino)-N-[(3S)-pyrrolidin-3-yl]pyridine-3-sulfonamide (90 mg, crude) as a yellow solid. LC-MS: (M+H)+, found 333.

[0354] Step 3: Preparation of N-[(3S)-l-cyanopyrrolidin-3-yl]-N-methyl-2- (phenylamino)pyridine-3-sulfonamide

[0355] To a stirred solution of N-methyl-2-(phenylamino)-N-[(3S)-pyrrolidin-3- yl]pyridine-3-sulfonamide (90 mg, 0.27 mmol. 1 equiv) and BrCN (32 mg, 0.30 mmol, 1.1 equiv) in DMF (3 mL), K2CO3 (75 mg, 0.54 mmol, 2 equiv) was added at room temperaturel61. The resulting mixture was stirred for 1 hr then concentrated under reduced pressure and purified by HPLC procedure 5, gradient: 35% B to 57% B in 10 min to afford N-[(3S)-l-cyanopyrrolidin-3-yl]-N-methyl-2-(phenylamino)pyridine-3-sulfonamide (12 mg, 12%) as a yellow' oil. 'H NMR (400 MHz, DMSO-d6) 6 8.54 (s, 1H), 8.41 (dd, 1H), 8.09 (dd, 1H), 7.63 - 7.57 (m, 2H), 7.39 - 7.30 (m, 2H), 7.12 - 7.04 (m, 1H), 7.03 - 6.96 (m, 1H), 4.79 - 4.67 (m, 1H), 3.46 - 3.40 (m, 2H), 3.30 - 3.27 (m, 1H), 3.27 - 3.23 (m, 1H), 2.77 (s, 3H), 1.95 - 1.77 (m. 2H). LC-MS: 358.0 (M+H)+.Example 27. Preparation of (S)-N-(l-cyanopyrrolidin-3-yl)-N-methyl-l- phenylmethanesulfonamide

[0356] Step 1 : Preparation of tert-butyl (3S)-3-(N-methylphenylmethanesulfonamido) pyrrolidine- 1 -carboxylate

[0357] A round bottomed flask was charged with tert-butyl (3S)-3-(methylamino) pyrrolidine- 1 -carboxylate (500 mg, 2.50 mmol, 1 equiv), TEA (758 mg, 7.49 mmol, 3 equiv), and a stir bar. THF (10 m ) was added, the mixture was cooled to 0°C, then phenylmethanesulfonyl chloride (523 mg, 2.75 mmol, 1.1 equiv) was addedl60. The solution was stirred for 1 hr at room temperature before quenching the reaction by the addition of MeOH. The resulting mixture was concentrated in vacuo, and the residue was purified by silica gel column chromatography using PE / EA (2: 1) to afford tert-butyl (3S)-3-(N- methylphenylmethanesulfonamido) pyrrolidine-l-carboxylate (480 mg, 54%) as off-white oil. LC-MS: (M+H)+, found: 355.20.

[0358] Step 2: Preparation of N-methyl-1 -phenyl -N-[(3S)-pyrrolidin-3-yl] methanesulfonamide

[0359] A round bottomed flask was charged with tert-buty l (3S)-3 (N- methylphenylmethanesulfonamido) pyrrolidine-1 -carboxylate (480 mg, 1.35 mmol, 1 equiv) and a stir bar. A solution of HC1 in 1.4-di oxane (10 mL) was added, and the solution was stirred for 1 h at room temperature. The solution was concentrated in vacuo to afford crude N-methyl-1 -phenyl-N-[(3S)-pyrrolidin-3-yl] methanesulfonamide (300 mg, 87%) as an off- white amorphous solid that was used without further purification. LC-MS: (M+H)+, found: 255.05.

[0360] Step 3: Preparation of (S)-N-(l-cyanopyrrolidin-3-yl)-N-methyl-l - phenylmethanesulfonamide

[0361] A round bottomed flask was charged with N-methyl-1 -phenyl-N-[(3S)-pyrrolidin- 3-yl] methanesulfonamide (100 mg, 0.393 mmol. 1 equiv), TEA (119 mg, 1.18 mmol, 3 equiv), DCM (3 mL), and a stir bar. The mixture was cooled to 0°C, cyanogen bromide (46 mg, 0.43 mmol, 1.1 equiv) was added, and the mixture stirred for 1 hr at room temperature. The mixture was diluted with water (30 mL), and the aqueous phase was extracted with DCM (30 mL) three times. The combined organic layers were washed with brine, dried over sodium sulfate, filtered, and concentrated in vacuo. The resulting crude material was punfied by Prep-HPLC to give (S)-N-(l-cyanopyrrolidin-3-yl)-N-methyl-l- phenylmethanesulfonamide (54 mg, 49%) as an off-white amorphous solid.JH NMR (400 MHz, DMSO-d6) 5 7.45 - 7.33 (m, 5H), 4.46 (s, 2H), 4.34 (p, J = 7.8 Hz, 1H), 3.46 (ddd, J = 9.2, 6.8, 5.5 Hz, 1H), 3.34 - 3.25 (m, 2H), 3.22 (dd, J = 9.7, 7.3 Hz, 1H), 2.68 (s, 3H), 1.98 - 1.84 (m, 2H). LC-MS: 280.05 (M+H)+.Example 28. Preparation of (S)-N-(l-cyanopyrrolidin-3-yl)-N-methyl-2-(lH-pyrazol-4- yl)benzenesulfonamide

[0362] Step 1 : Preparation of tert-butyl (3S)-3-[N-methyl2-(lH-pyrazol-4-yl) benzenesulfonamido] pyrrolidine- 1 -carboxylate

[0363] To a stirred solution of tert-butyl (3S)-3-(N-methyl-2-bromobenzenesulfonamido) pyrrolidine- 1 -carboxylate (200 mg, 0.477 mmol, 1 equiv) and K2CO3 (198 mg, 1.43 mmol, 3 equiv) in DMF (2 mL) and H2O (0.4 mL) were added Pd(dppf)Ch. CH2CI2 (38.9 mg, 0.048 mmol, 0.1 equiv) and 4-(4,4,5,5-tetramethyl-l,3,2-dioxaborolan-2-yl)-lH-pyrazole (111 mg, 0.572 mmol. 1.2 equiv) in portions at room temperature under nitrogen atmosphere. The resulting mixture was stirred for 2hrs at 80°C. The resulting mixture was extracted with EtOAc (3x3 mL) and concentrated under reduced pressure. The residue was purified by Prep- TLC (CH2CI2 / MeOH 10: 1) to afford tert-butyl (3S)-3-[N-methyl2-(lH-pyrazol-4-yl) benzenesulfonamido] pyrrolidine- 1 -carboxylate (110 mg, 56 %) as a yellow solid.

[0364] Step 2: preparation of N-methyl-2-(lH-pyrazol-4-yl)-N-[(3S)-pyrrolidin-3- yl] benzenesulfonamide

[0365] To a stirred solution of tert-butyl (3S)-3-[N-methyl-2-(lH-pyrazol-4-yl) benzenesulfonamido] pyrrolidine-1 -carboxylate (110 mg, 0.271 mmol, 1 equiv) in 4 N HC1 in 1,4-di oxane (2 mL) at room temperature. The resulting mixture was stirred for 1 hr at room temperature and concentrated under reduced pressure to afford crude N-methyl-2-(lH- pyrazol-4-yl)-N-[(3S)-pyrrolidin-3-yl] benzenesulfonamide (100 mg) as a yellow solid.

[0366] Step 3: Preparation of N-[(3S)-l-cyanopyrrolidin-3-yl]-N-methyl-2-(lH-pyrazol- 4-yl)benzenesulfonamide

[0367] To a stirred solution of N-methyl-2-(lH-pyrazol-4-yl)-N-[(3S)-pyrrolidin-3- yl] benzenesulfonamide (70 mg, 0.228 mmol, 1 equiv) and K2CO3 (94.73 mg, 0.684 mmol, 3 equiv) in DMF (0.7 mL) was added BrCN (26.6 mg, 0.251 mmol, 1.1 equiv) in portions at 0°C under nitrogen atmosphere. The resulting mixture was stirred for 1 hr at room temperature then concentrated under reduced pressure. The crude material was purified by Prep-HPLC (Column: XBridge Prep OBD C18 Column, 30*150 mm, 5pm; Mobile Phase A: Water (10 mmol / L NFLHCCL), Mobile Phase B: ACN; Flow rate: 60 mL / min; Gradient: 26% B to 45% B in 9 min) to afford N-[(3S)-l-cyanopyrrolidin-3-yl]-N-methyl-2-(lH-pyrazol-4-yl)benzenesulfonamide (13.9 mg, 18.30%) as a white solid. 1H NMR (400 MHz, Chloroform-d) 5 8.16 - 8.10(m, 1H), 7.85 (s, 2H), 7.61 (td. J = 7.6, 1.4 Hz, 1H). 7.48 (td, J = 7.7, 1.4 Hz, 1H), 7.42 - 7.36(m, 1H), 4.16 (p, J = 7.6 Hz, 1H), 3.43 - 3.37 (m, 1H), 3.27 - 3.22 (m, 1H), 3.12 - 3.08 (m, 1H), 2.99 - 2.95 (m, 1H), 2.40 (s, 3H), 1.80 - 1.75 (m, 2H). LC / MS: 332.00 (M+H)+.Example 29. Preparation of 3-acrylamido-N-(l,4-dimethyl-2-oxo-l,2,3,4-tetrahydroquinolin- 6-yl)-5-((l-(21-((4S.5R)-5-methyl-2-oxoimidazolidin-4-yl)-16-oxo-3,6,9,12-tetraoxa-15- azahenicosyl)-lH-l,2,3-triazol-5-yl)methoxy)benzamide (SH29-DTB)

[0368] Step 1 : Preparation of 3-acrylarnido-N-(l,4-dimethyl-2-oxo-l,2,3,4- tetrahydroquinolin-6-yl)-5-(prop-2-yn-l-yloxy)benzamide

[0369] Obtained by using the aforementioned standard coupling conditions with 49 mg (0.203 mmol} of Intermediate 1, 45 mg (0.229 mmol) of Intermediate 2, 43 mg (0.277 mmol) of EDC, and 29 mg (0.214 mmol) of HO At. The crude reaction mixture was purified by preparatory HPLC.

[0370] Step 2: Preparation of SH29-DTB

[0371] In a round bottom flask, were added 3-acrylamido-N-(l,4-dimethyl-2-oxo- l,2,3,4-tetrahydroquinolin-6-yl)-5-(prop-2-yn-l-yloxy)benzamide (0.100 g, 0.240 mmol), (+)-sodium L-ascorbate (8.5 mg, 0.048 mmol), copper(II) sulfate powder (7.6 mg, 0.048 mmol), and Intermediate 3 (110 mg, 0.240 mmol) were added. The atmosphere was purged with argon gas, then DMF (3 ml) was added. The reaction mixture was stirred at RT for 16 h. The solvent was removed in vacuo and directly purified via column chromatography to isolate SH-0029-DTB.NMR (400 MHz, Methanol-D4) 6 8.16 (s, 1H), 7.73 (s, 1H), 7.63-7.61 (m, 3H), 7.32 (s, 1H), 7.12 (d, J = 9.5 Hz, 1H), 6.47-6.36 (m, J = 42.9 Hz, 2H), 5.79 (dd, J = 8.8, 3.0 Hz, 1H), 5.25 (s, 2H), 4.59 (t. J = 4.9 Hz, 2H), 3.87 (t, J =5.0 Hz, 2H), 3.78-3.52 (m, 12H), 3.45 (t, J = 5.5 Hz, 2H), 3.36-3.32 (m, 6H), 3.11- 3.04 (m, 1H), 2.72 (dd, J = 1 6.0, 5.5 Hz, 1H), 2.42 (dd, J = 1 5.8, 7.0 Hz, 1H), 2.13 (t, J =7.4 Hz, 2H), 1.59-1.53(m, J = 7.5 Hz, 2H), 1.51-1.26 (m, 12H), 1.04 (d, J = 6.5 Hz, 3H). El MS m / z: pos 876.4 (M+H)+.Example 30. Preparation of N-(2-(2-iodoacetamido)ethyl)-6-((4R,5S)-5-methyl-2- oxoimidazolidin-4-yl)hexanamide (DBIA)

[0372] Step 1 : Preparation of 2,5-dioxopyrrolidin-l-yl 6-((4R,5S)-5-methyl-2- oxoimidazolidin-4-yl)hexanoate

[0373] Desthiobiotin (200 mg, 0.93 mmol), N-hydroxysuccinimide (241 mg, 1.11 mmol), and EDC-HCI (213 mg, 1.11 mmol) were stirred in DMF under a nitrogen atmosphere for 12 hrs. The crude mixture was diluted with EtOAc. washed with water, dried with magnesium sulfate, and concentrated (used without further purification).

[0374] Step 2: Preparation of N-(2-aminoethyl)-6-((4R,5S)-5-methyl-2-oxoimidazolidin- 4-yl)hexanamide

[0375] To a solution of 2,5-dioxopyrrolidin-l-yl 6-((4R,5S)-5-methyl-2- oxoimidazolidin-4-yl)hexanoate (100 mg, 0.32 mmol) in dichloromethane, N,N-diisopropylethylamine (0.32 mmol) and N-boc ethylenediamine (0.32 mmol) were added. The reaction mixture was stirred overnight at room temperature under a nitrogen atmosphere. The solvent was then removed in vacuo, and the resulting oil was dispersed in 20% TFA in dichloromethane (DCM) and stirred for 3 hrs at room temperaturee. The TFA and DCM were removed in vacuo to yield a yellow oil, which was washed repeatedly with diethyl ether to remove impurities.

[0376] Step 3: Preparation of N-(2-(2-iodoacetamido)ethyl)-6-((4R,5S)-5-methyl-2- oxoimidazolidin-4-yl)hexanamide

[0377] N-(2-aminoethyl)-6-((4R5S)-5-methyl-2-oxoimidazolidin-4-yl)hexanamide (0.182 g, 0.71 mmol) dispersed in 10 ml of methanol / DCM (1:9 v / v), to which was added N.N- diisopropylethylamine (0.71 mmol) and iodoacetic anhydride (378 mg, 1.07 mmol) [note: this was freshly prepared by combining lodoacetic acid (8.0 g,43.0 mmol) and DCC (4.2 g, 20.5 mmol) in EtOAc (120 ml) for 2 hrs under a nitrogen atmosphere. The crude mixture was filtered and concentrated for direct use in the next reaction]. The reaction was stirred in the dark for 1 h at room temperature. The reaction mixture was concentrated, then triturated with acetone to isolated DBIA as a white solid.Note: the compound is light sensitive. Thus, work was done in low light conditions and the flask was wrapped in foil. 'H NMR (400 MHz, DMSO-D6) 5 8.23 (s, 1H), 7.76 (s, 1H), 6.27 (s. 1H), 6.08 (s. 1H). 3.57-3.53 (m. 3H), 3.46-3.39(m. 1H). 3.03 (bs. 4H). 2.00 (t, J = 7.4 Hz, 2H), 1.46-1.40 (m, 2H), 1.29-1.17 (m 6H), 0.91 (d, J =6.4 Hz, 3H). El MS m / z: 425.1 (M+H)+.

Claims

WHAT IS CLAIMED IS:

1. A compound of Formula (I)or a pharmaceutically acceptable salt thereof, whereinLis a bond or -LA-LB-, wherein LBis attached to Ring A;LAis -(C1-C3 alkyl)(CO)n-* or phenylene optionally substituted with 1-2 independently selected R5, wherein “*” indicates the point of attachment to LB; orLAand R3together with the nitrogen to which they are attached join to form a 4-10 membered heterocyclyl optionally substituted with 1-2 independently selected R4;LBIS a bond. -C(O)-, -C(O)NHNHC(O)-. -NH-, -C(O)NR6-. -C(O)(C1-C3 alkyl)-*. - C(O)NH(C1-C3 alkyl)-*, -NH(C1-C3 alkyl)-*, -NH(C1-C3 alkyl)C(O)-*, -NHNHC(O)-*, - C(O)NHNH(CS)NH-*, -C(O)(C1-C3 alkyl optionally substituted with 1-3 independently selected R7)-*, -C(O)O(C1-C3 alkyl optionally substituted with 1-3 independently selected R7)-*, or -C(O)N=*, wherein "*” indicates the point of attachment to Ring A;Ring A is phenyl, 4-13 membered heterocyclyl, or C3-C6 cycloalkyl;R1is selected from:(i) halogen,(ii) C1-C3 alkoxyl,(iii) C1-C3 alkyl optionally substituted with 1-4 independently selected RA, phenyl or 5-6 membered heteroaryl each optionally substituted with 1-2 RB,(iv) -NRCC(O)(CI-C3haloalkyl),(v) -NRCC(O)(CI-C3alkyl),(vi) -C(O)NRDRE,(vn) -SO2NR10R11,(viii) 5-10 membered heteroaryl optionally substituted with 1-2 R12,(ix) phenyl optionally substituted with 1-2 R13,(x) 4-6 membered heterocyclyl optionally substituted with 1-2 R14,(xi) -C(O)R15,(xii) -NR6(CO)R15,(xiii) cyano,(xiv) -SO2(C1-C3 alkyl),(xv) -N=S(O)(C1-C3 alkyl)N(Cl-C3 alkyl)2,(xvi) -NHSO2(C1-C3 alkyl),(xvii) -NO2,(xviii) C3-C7 cycloalkyl.(xix) -NRDRE,(xx) C2-C4 alkynyl optionally substituted with -C(O)C1-C3 alky l, and(xxi) hydrogen;R2is cyano, -CO2C1-C3 alkyl. C1-C6 alkyl. C1-C3 alkoxyl, halogen. -OH. or -SH;R3is hydrogen or C1-C3 alkyl optionally substituted with phenyl optionally substituted with 1-2 halogen;R4is C1-C3 alkyl or phenyl;R5is -OH, halo, or phenyl;R6is hydrogen or C1-C3 alkoxyl;R7is halogen, -OH, C2-C3 alkenyl, C2-C3 alkynyl, or 5-6 membered heteroaryl optionally substituted with 1-2 independently selected halogen;R8is halogen or C1-C3 alkyl;R9is hydrogen or C1-C3 alkyl;R10and R11are each independently hydrogen or C1 -C3 alkyl; orR10and R11, together with the nitrogen to which they are attached, join together to form a 5-6 membered heterocyclyl;R12is halogen, -OH, C1-C3 alkyl, cyano, -NO2, or -NH2;R13is halogen -OH, C1-C3 alkyl, C1-C3 haloalkyl, C1-C3 alkoxyl, cyano, or C1-C3 haloalkoxyl;R14is halogen or C1-C3 alkyl;R15is C1-C3 alkyl, C1-C4 alkoxyl optionally substituted with phenyl, C1-C3 haloalkyl, C3-C7 cycloalkyl, phenoxyl. or phenyl optionally substituted with 1-2 independently selected R16;R16is halogen, -OH, -NH(phenyl optionally substituted with halogen), -NH(C1-C4 alkyl), or -NH(C3-C7 cycloalkyl);RAis halogen, -OH, phenyl, -CO2C1-C3 alkyl, C2-C4 alkenyl, and C2-C4 alkynyl;RBis halogen or cyano;Rcis hydrogen or C1-C3 alkyl;RDand REare each independently hydrogen. C1-C3 alkyl, C3-C7 cycloalkyl; orRDand RE, together with the nitrogen to which they are attached, join together to form a 5-6 membered heterocyclyl; n is 0 or 1; and m is 0, 1, 2, 3, or 4; wherein the compound of Formula (I) is not2. The compound of Claim 1. wherein L is or -LA-LB-; wherein LBis attached toRing A.

3. The compound of any one of Claims 1-2, wherein LAis -(C1-C3 alkyl)(CO)n- *; wherein indicates the point of attachment to LB.

4. The compound of any one of Claims 1 -3, wherein LAis phenylene optionally substituted with 1-2 independently selected R5.

5. The compound of any one of Claims 1-4, wherein LAand R3together with the nitrogen to which they are attached join to form a 4-10 membered heterocyclyl optionally substituted with 1-2 independently selected R4.

6. The compound of any one of Claims 1-5, wherein LBis a bond.

7. The compound of any one of Claims 1-5, wherein LBis -C(O)-.

8. The compound of any one of Claims 1-5, wherein LBis -C(O)NHNHC(O)-.

9. The compound of any one of Claims 1-5, wherein LBis -NH-.

10. The compound of any one of Claims 1-5, wherein LBis -C(O)NR6-.

11. The compound of any one of Claims 1-5, wherein LBis -C(O)(C1-C3 alkyl)-*; wherein “*” indicates the point of attachment to Ring A.

12. The compound of any one of Claims 1-5, wherein LBis -C(O)NH(C1-C3 alkyl)-*; wherein “*” indicates the point of attachment to Ring A.

13. The compound of any one of Claims 1-5, wherein LBis -NH(C1-C3 alkyl)-*; whereinc'*’’ indicates the point of attachment to Ring A.

14. The compound of any one of Claims 1-5, wherein LBis -NH(C1-C3 alkyl)C(O)-*; wherein indicates the point of attachment to Ring A.

15. The compound of any one of Claims 1-5, wherein LBis -NHNHC(O)-*; wherein “*” indicates the point of attachment to Ring A.

16. The compound of any one of Claims 1-5, wherein LBis -C(O)NHNH(CS)NH- *; wherein indicates the point of attachment to Ring A.

17. The compound of any one of Claims 1-5, wherein LBis -C(O)(C1-C3 alkyl optionally substituted with 1-3 independently selected R7)-*; wherein indicates the point of attachment to Ring A.

18. The compound of any one of Claims 1-5, wherein LBis -C(O)O(C1-C3 alkyl optionally substituted with 1-3 independently selected R7)-*; wherein “*” indicates the point of attachment to Ring A.

19. The compound of any one of Claims 1-5, wherein LBis -C(O)N=*; wherein “*’■ indicates the point of attachment to Ring A.

20. The compound of Claim 1, wherein L is a bond.

21. The compound of any one of Claims 1-20, wherein Ring A is phenyl.

22. The compound of any one of Claims 1-20, wherein Ring A is 4-13 membered heterocyclyl.

23. The compound of any one of Claims 1-20. wherein Ring A is C3-C6 cycloalkyl.

24. The compound of any one of Claims 1-20, wherein R1is halogen.

25. The compound of any one of Claims 1-20, wherein R1is C1-C3 alkoxyl.

26. The compound of any one of Claims 1-20, wherein R1is C1-C3 alkyl optionally substituted with 1-4 independently selected RA.

27. The compound of any one of Claims 1-20, wherein R1is C 1-C3 alkyl substituted with phenyl optionally substituted with 1-2 RB.

28. The compound of any one of Claims 1-20, wherein R1is C1-C3 alkyl substituted with 5-6 membered heteroaryl optionally substituted with 1 -2 RB.

29. The compound of any one of Claims 1-20, wherein R1is -NRCC(O)(CI-C3 haloalkyl).

30. The compound of any one of Claims 1-20, wherein R1is -NRCC(O)(CI-C3 alkyl).

31. The compound of any one of Claims 1-20. wherein R1is -C(O)NRDRE.

32. The compound of any one of Claims 1-20, wherein R1is -SChNR^R11.

33. The compound of any one of Claims 1-20, wherein R1is 5-10 membered heteroaryl optionally substituted with 1-2 R12.

34. The compound of any one of Claims 1-20, wherein R1is phenyl optionally substituted with 1-2 R13.

35. The compound of any one of Claims 1-20, wherein R1is 4-6 membered heterocyclyl optionally substituted with 1-2 R14.

36. The compound of any one of Claims 1-20, wherein R1is -C(O)R15.

37. The compound of any one of Claims 1-20, wherein R1is -NR6(CO)R15.

38. The compound of any one of Claims 1-20, wherein R1is cyano.

39. The compound of any one of Claims 1-20, wherein R1is -SC>2(C1-C3 alky l).

40. The compound of any one of Claims 1-20. wherein R1is -N=S(O)(C1-C3 alkyl)N(Cl-C3 alkyl)2.

41. The compound of any one of Claims 1-20, wherein R1is -NHSO2(C1-C3 alkyl).

42. The compound of any one of Claims 1-20, wherein R1is -NO2.

43. The compound of any one of Claims 1-20, wherein R1is C3-C7 cycloalkyl.

44. The compound of any one of Claims 1-20, wherein R1is NRDRE.

45. The compound of any one of Claims 1-20. wherein R1is C2-C4 alkynyl optionally substituted with -C(O)C1-C3 alkyl.

46. The compound of any one of Claims 1-20, wherein R1is hydrogen.

47. The compound of any one of Claims 1-46. wherein R2is C1-C6 alkyl.

48. The compound of any one of Claims 1-46, wherein R2is C1-C3 alkoxyl.

49. The compound of any one of Claims 1-46, wherein R2is halogen.

50. The compound of any one of Claims 1-46, wherein R2is -OH.

51. The compound of any one of Claims 1-46. wherein R2is -SH.

52. The compound of any one of Claims 1-51, wherein R3is hydrogen.

53. The compound of any one of Claims 1-49, wherein R3is C1-C3 alkyl optionally substituted with phenyl optionally substituted with 1-2 halogen.

54. The compound of any one of Claims 1-51, wherein R4is C1-C3 alkyl.The compound of any one of Claims 1-52. wherein R5is -OH.

56. The compound of any one of Claims 1-52, wherein R5is halogen.

57. The compound of any one of Claims 1-52. wherein R5is phenyl.

58. The compound of any one of Claims 1-55, wherein R6is hydrogen.

59. The compound of any one of Claims 1-55, wherein R6is C1-C3 alkoxyl.

60. The compound of any one of Claims 1-57, wherein R7is halogen.

61. The compound of any one of Claims 1-57, wherein R7is OH.

62. The compound of any one of Claims 1-57, wherein R7is C2-C3 alkenyl.

63. The compound of any one of Claims 1-57, wherein R7is C2-C3 alkynyl.

64. The compound of any one of Claims 1-57. wherein R7is 5-6 membered heteroaryl optionally substituted with 1-2 independently selected halogen.

65. The compound of any one of Claims 1-62, wherein R8is halogen.

66. The compound of any one of Claims 1-62, wherein R8is C1-C3 alkyl.

67. The compound of any one of Claims 1-64, wherein R9is hydrogen.

68. The compound of any one of Claims 1-64, wherein R9is C1-C3 alkyl.

69. The compound of any one of Claims 1-66, wherein R10and R11are each hydrogen.

70. The compound of any one of Claims 1-66, wherein R10and R11are each independently selected C1-C3 alkyl.

71. The compound of any one of Claims 1-66, wherein one of R10and R11is Cl- C3 alkyl and the other one of R10and R11is hydrogen.

72. The compound of any one of Claims 1 -66, wherein R10and R11, together with the nitrogen to which they are attached, join together to form a 5-6 membered heterocyclyl.

73. The compound of any one of Claims 1-70, wherein R12is halogen.

74. The compound of any one of Claims 1-70, wherein R12is C 1-C3 alkyl’75. The compound of any one of Claims 1-70, wherein R12is cyano.

76. The compound of any one of Claims 1-70, wherein R12is -NH2.

77. The compound of any one of Claims 1-74, wherein R13is halogen.

78. The compound of any one of Claims 1-74, wherein R13is -OH.

79. The compound of any one of Claims 1-74, wherein R13is C1-C3 alkyl.

80. The compound of any one of Claims 1-74, wherein R13C1-C3 haloalkyl.

81. The compound of any one of Claims 1-74, wherein R13is C1-C3 alkoxyl.

82. The compound of any one of Claims 1-79, wherein R14is halogen.

83. The compound of any one of Claims 1-79, wherein R14is C1-C3 alkyl.

84. The compound of any one of Claims 1-81, wherein R15is C1-C3 alkyl.

85. The compound of any one of Claims 1-81, wherein R15is C1-C4 alkoxyl optionally substituted with phenyl.

86. The compound of any one of Claims 1-81, wherein R15is C 1-C3 haloalkyl.

87. The compound of any one of Claims 1-81, wherein R15is C3-C7 cycloalkyl.

88. The compound of any one of Claims 1 -81, wherein R15is phenyl optionally substituted with 1-2 independently selected R16.

89. The compound of any one of Claims 1-86, wherein R16is halogen.

90. The compound of any one of Claims 1-86, wherein R16is -OH.

91. The compound of any one of Claims 1-86, wherein R16is -NH(phenyl optionally substituted with halogen).

92. The compound of any one of Claims 1-86, wherein R16is -NH(C1-C4 alkyl).

93. The compound of any one of Claims 1-86. wherein R16is -NH(C3-C7 cycloalkyl).

94. The compound of any one of Claims 1-91, wherein Rcis hydrogen.

95. The compound of any one of Claims 1-91, wherein Rcis C1-C3 alkyd.

96. The compound of any one of Claims 1-93, wherein RDand REare each hydrogen.

97. The compound of any one of Claims 1-93, wherein RDand REare each independently selected C1-C3 alky l.

98. The compound of any one of Claims 1 -93, wherein one of RDand REis C1-C3 alkyl and the other one of RDand REis hydrogen.

99. The compound of any one of Claims 1-93, wherein RDand RE, together with the nitrogen to which they are attached, join together to form a 5-6 membered heterocyclyl.

100. The compound of any one of Claims 1-97, wherein n is 1.

101. The compound of any one of Claims 1-97. wherein n is 0.

102. The compound of any one of Claims 1-99, wherein m is 1.

103. The compound of any one of Claims 1-99, wherein m is 2.

104. The compound of any one of Claims 1-99, wherein m is 3.

105. The compound of any one of Claims 1-99, wherein m is 4.

106. The compound of any one of Claims 1-99, wherein m is 0.

107. A pharmaceutical composition comprising the compound of Formula (I), or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable excipients.

108. A compound of Formula (III):or a pharmaceutically acceptable salt thereof, whereinL is -(C=O)NRL1(CH2)q-* , -NRL1SO2(CH2)q-*, wherein indicates the attachment point to Rx;Rxis phenyl or 5-6 membered heteroaryl, each optionally substituted with 1-2 independently selected RX1;RL1is hydrogen or C1-C3 alkyl;RX1is halo. -NR Rz. or 5-6 membered heteroaryl;RYand Rzare independently H or phenyl; and q is 0 or 1.

109. A pharmaceutical composition comprising the compound of Formula (III), or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable excipients.

110. A method of treating a SOXIO-associated disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a compound of Formula (11):or a pharmaceutically acceptable salt thereof, wherein:Lis a bond or -LA-LB-, wherein LBis attached to Ring A;LAis -(C1-C3 alkyl)(CO)n-* or phenylene optionally substituted with 1-2 independently selected R5, wherein indicates the point of attachment to LB; orLAand R3together with the nitrogen to which they are attached join to form a 4-10 membered heterocyclyl optionally substituted with 1-2 independently selected R4;LBIS a bond, -C(O)-, -C(O)NHNHC(O)-, -NH-, -C(O)NR6, -C(O)(C1-C3 alkyl)-*, - C(O)NH(C1-C3 alkyl)-* -NH(C1-C3 alkyl)-*, -NH(C1-C3 alkyl)C(O)-*, -NHNHC(O)-*, or -C(O)NHNH(CS)NH-*, -C(O)(C1-C3 alkyl optionally substituted with 1-3 independentlyselected R7)-*, or -C(O)O(C1-C3 alkyl optionally substituted with 1-3 independently selected R7)-*-C(O)N=*, wherein indicates the point of atachment to Ring A;Ring A is phenyl, 4-13 membered heterocyclyl, or C3-C6 cycloalkyl;R1is selected from:(i) halogen,(ii) C1-C3 alkoxyl,(iii) C1-C3 alkyl optionally substituted with 1-4 independently selected RA, phenyl or 5-6 membered heteroaryl each optionally substituted with 1-2 RB,(iv) -NRCC(O)(CI-C3haloalkyl),(v) -NRCC(O)(CI-C3alkyl),(vi) -C(O)NRDRE,(vii) -SO2NR10R11,(viii) 5-10 membered heteroaryl optionally substituted with 1-2 R12,(ix) phenyl optionally substituted with 1-2 R13,(x) 4-6 membered heterocyclyl optionally substituted with 1-2 R14).(xi) -C(O)R15,(xii) -NR6(CO)R15,(xiii) cyano,(xiv) -SO2(C1-C3 alkyl),(xv) -N=S(O)(C1-C3 alkyl)N(Cl-C3 alkyl)2,(xvi) -NHSO2(C1 -C3 alkyl);(xvii) -NO2,(xviii) C3-C7 cycloalky l.(xix) -NRDRE,(xx) C2-C4 alkynyl optionally substituted with -C(O)C1-C3 alkyl, and(xxi) hydrogen;R2is cyano, -CO2C1-C3 alkyl, C1-C6 alkyl, C1-C3 alkoxyl, halogen, -OH, or -SH;R3is hydrogen or C1-C3 alkyl optionally substituted with phenyl optionally substituted with 1-2 halo;R4is C1-C3 alkyl or phenyl;R5is -OH, halo, or phenyl;R6is hydrogen or C1-C3 alkoxyl;R7is halogen. -OH, C2-C3 alkenyl. C2-C3 alkynyl, or 5-6 membered heteroaryl optionally substituted with 1-2 independently selected halogen;R8is halogen or C1-C3 alkyl;R9is hydrogen or C1-C3 alkyl;R10and R11are each independently hydrogen or C1-C3 alkyl; orR10and R11, together with the nitrogen to which they are attached, join together to form a 5-6 membered heterocyclyl;R12is halo, -OH, C1-C3 alkyl, cyano, -NO2, or -NH2;R13is halogen -OH, C1-C3 alkyl. C1-C3 haloalkyl, cyano, or C1-C3 haloalkoxyl, or C1-C3 alkoxyl;R14is halogen or C1-C3 alkyl;R15is C1-C3 alkyl, C1-C4 alkoxyl optionally substituted with phenyl, C1-C3 haloalkyl, C3-C7 cycloalkyl, phenoxyl. or phenyl optionally substituted with 1-2 independently selected R16;R16is halogen, -OH, -NH(phenyl optionally substituted with halogen), -NH(C1-C4 alkyl), or -NH(C3-C7 cycloalkyl);RAis halogen, -OH, phenyl, -CO2CI-C3 alkyl, C2-C4 alkenyl, and C2-C4 alkynyl;RBis halogen or cyano;Rcis hydrogen or C1-C3 alkyl;RDand REare each independently hydrogen, C1-C3 alkyl, C3-C7 cycloalkyl; orRDand RE, together with the nitrogen to which they are attached, join together to form a 5-6 membered heterocyclyl; n is 0 or 1 ; and m is 0, 1, 2, 3, or 4.

111. A method of treating an SOXIO-associated disease or disorder, in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a compound of any one of Claims 1-106, or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition of Claim 107.

112. A method of treating an SOXIO-associated disease or disorder, in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a compound of Claim 108, or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition of Claim 109.

113. The method of any one of Claims 110-112, wherein the SOXIO-associated disease or disorder is cancer.

114. The method of any one of Claims 110-113, wherein the SOXIO-associated disease or disorder is skin cancer115. The method of Claim 114, wherein skin cancer is melanoma.

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