WO2022240641A1 - High-throughput assay for identification of myc inhibitors - Google Patents

Abstract

Provided, inter alia, is a high-throughput screening assay for identifying a MYC inhibitor. Further disclosed are pharmaceutical compositions, and methods for treating cancer using the MYC inhibitor identified via the high-throughput screening assay described herein.

Description

HIGH-THROUGHPUT ASSAY FOR IDENTIFICATION OF MYC INHIBITORS

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The subject patent application claims the benefit of priority to U.S. Provisional Patent Application No. 63/187,573 (filed May 12, 2021; now pending). The full disclosures of the priority application is incorporated herein by reference in its entirety and for all purposes.

[0002] Throughout this application various publications, patents, and/or patent applications are referenced. The disclosures of the publications, patents and/or patent applications are hereby incorporated by reference in their entireties into this application in order to more fully describe the state of the art to which this disclosure pertains.

TECHNICAL FIELD

[0003] The present disclosure relates to high-throughput screening assay for identifying MYC inhibitor scaffolds using a fluorescent small molecule probe. The present disclosure further relates to pharmaceutical compositions, and methods for treating cancer with the MYC inhibitors identified via the high-throughput screening assay described herein.

INTRODUCTION AND SUMMARY

[0004] The v-myc myelocytomatosis viral oncogene homolog (MYC) protein is an essential regulator of cell-cycle progression occupying and apical space in the transcriptome (Adhikary et al., Nat. Rev. Mol. Cell Biol. 2005, 6 (8), 635-645). The proto-oncogene c-myc encodes a transcription factor (MYC) that controls cell proliferation. MYC also plays a role in regulating cell cycle, cell growth, angiogenesis, apoptosis, and oncogenesis. MYC is involved in almost all cancers, and a gain of function in MYC is seen in nearly all human cancers. MYC’s activity can increase in tumors as a consequence of mutations, chromosomal rearrangements, increased expression, or gene amplification. Elevated or deregulated expression of c-MYC has been detected in a wide range of human cancers and is often associated with aggressive, poorly differentiated tumors. Such cancers include colon, breast, cervical, small cell lung carcinomas, osteosarcomas, glioblastomas, melanoma and myeloid leukemias.

[0005] Part of the difficulty in studying MYC is its frenetic mode of action: although having an ephemeral existence, it is able to seemingly affect transcription in both a local and global manner. Moreover, MYC exists as an intrinsically disordered protein (IDP), taking on structure only in the presence of other basic helix-loop-helix leucine zipper (bHLH-LZ) transcription factors of the MAX network. Its control of mitogenesis requires association with another bHLH-LZ protein, MAX, which exists in the cell stoichiometric excess to MYC (Adhikary et al., Nat. Rev. Mol. Cell Biol. 2005, 6 (8), 635-645; Blackwood et al., Science 1991, 251, 1211-1217; Conacci-Sorrell et al., Cold Spring Harb. Perspect Med. 2014, 4 (1), a014357-a014357; and McKeown and Bradner, Cold Spring Harb. Perspect Med, 2014, 4 (10), aO 14266).

[0006] MYC and MAX are both intrinsically disordered proteins (IDP) that heterodimerize and bind preferentially to the palindromic E-box motif in DNA to stimulate or promote transcriptional activity (Ji et al., PLoS One , 2011, 6, e26057; Nie et al., Cell , 2012, 151, 68-79; and Lin et al., Cell, 2012, 151, 56-67). Dysregulation of MYC family gene/proteins occurs in over half of all human tumors and is recognized as a general hallmark of cancer initiation and maintenance (Adhikary et al., Nat. Rev. Mol. Cell Biol. 2005, 6 (8), 635-645; Eilers et al., Genes Dev., 2008, 22, 2755-2766; Varlakhanova et al., Cancer Res., 2009, 69, 7487-7490; Gabay et al., Perspect. Med., 2014, 4, aO 14241; and Dang, Cell, 2012, 149, 22-35). Its gain of function in cancer is largely a result of overabundance (Kalkat et al., Genes {Basel), 2017, 8, 2-30), rather than mutation, stemming from changes in gene copy number, transcriptional overexpression, post-translational modification (Zhang et al., Proc. Natl. Acad. Sci., 2012, 109, 2790-2795; Malempati et al., Leukemia, 2006, 20, 1572-1581), and decreased degradation resulting from suppression of ubiquitin E3 ligases (Choi et al., Genes Dev., 2010, 24, 1236-1241). Beyond accelerating proliferation, MYC has been implicated in immune evasion by regulating immune checkpoint genes (Casey et al., Blood, 2018, 131, 2007-2015). Furthermore, MYC has been found to have unexpected roles in cancers that nominally have a non-MYC etiology (Soucek et al., Genes Dev., 2013, 27, 504- 513; Liu et al., Nat. Med., 2011, 17, 1116-1120).

[0007] MYC is a pleiotropic transcription factor, yet it possesses broad pathogenic prominence making it a coveted cancer target. This MYC dichotomy has produced a forbidding fear that inhibiting a gene that controls essential cellular activities would lead to unacceptable side effects in vivo. Surprisingly, studies involving the systemic inhibition of MYC through conditional expression of a dominant-negative Omomyc allele in tumorbearing mice have demonstrated dramatic losses of tumor phenotypes in lung adenocarcinomas (Soucek et al., Genes Dev., 2013, 27, 504-513; Beaulieu et al., Sci. Transl. Med., 2019, 11, eaar5012). Glioblastomas (Annibali et al., Nat. Commun., 2014, 5, 4632), skin papillomatosis (Soucek et al., Cell Death Differ., 2004, 11, 1038-1045), and pancreatic tumors (Sodir et al., Genes Dev., 2011, 25, 907-915). This occurs without intolerable side- effects and those which do occur are reversible when MYC inhibition is relieved. Furthermore, this absence of toxicity has to a large extent been mirrored by small molecules at therapeutic concentrations (Hart et al., Proc. Natl. Acad. Sci., 2014, 111, 12556-12561; Stellas et al., J. Natl. Cancer Inst., 2014, 106, dju320; Soodgupta et al ,,Mol. Cancer Ther., 2015, 14, 1286-1294; Han et al., Cancer Cell, 2019, 36, 483-497; and Bouvard et al., Proc. Natl. Acad. Sci., 2017, 114, 3497-3502). Thus, while MYC is a pleiotropic transcription factor, its inhibition has had only mild and reversible side effects to normal tissues in adult animals indicating that a therapeutic window is achievable despite global MYC inhibition. [0008] This lack of structure and instability greatly impairs the ability to structurally or biophysically characterize MYC interactions. In all, these attributes have worked to make MYC an attractive, but elusive target in drug discovery. Even as one of the most established oncoproteins, decades of drug development have failed to produce a clinically viable MYC inhibitor. As a result, indirect methods of modulating MYC dysregulation through epigenetic silencing or by manipulating post-translational regulators of MYC have been examined (Allen-Petersen et al., BioDrugs, 2019, 33, 539-553; Janghorban et al ,,Proc. Natl. Acad. Sci., 2014, 111, 9157-9162). Although these methods are promising, many of these targets are not MYC-specific, presenting potential off-target effects and resistance pathways (Allen-Petersen et al., BioDrugs, 2019, 33, 539-553). The longstanding strategy has been to directly disrupt the protein-protein interaction (PPI) between MYC and MAX to inhibit dimerization or DNA complexing, ultimately interrupting gene transcription. This strategy has the potential for high specificity with low mutational evasion (Allen-Petersen et al., BioDrugs, 2019, 33, 539- 553). The challenge of this approach is two-fold: 1. IDPs like MYC lack well-defined binding pockets, and small molecule interactions come at a high entropic cost (Jin et al., PLOS Comput. Biol., 2013, 9, el003249; Michel et al., PLoS One, 2012, 7, e41070; and Yu et al., Sci. Rep., 2016, 6, 22298). 2. PPI interfaces are highly dynamic and expansive areas (flat, featureless and relatively large) that are challenging to disrupt with low molecular weight entities (Mabonga et al., Biophys. Rev., 201911, 559-581). Even so, a number of inhibitors capable of interfering with the MYC-MAX dimer have been identified, demonstrating the feasibility of this strategy (Hart et al., Proc. Natl. Acad. Sci., 2014, 111, 12556-12561; Stellas et al., J. Natl. Cancer Inst., 2014, 106, dju320; Han et al., Cancer Cell, 2019, 36, 483-497; Wang et al., Mol. Cancer Ther., 2007, 6, 2399-2408; Yin et al.,

Oncogene, 2003, 22, 6151-6159; Berg et al., Proc. Natl. Acad. Sci., 2002, 99, 3830-3835; Kiessling et al., Chem. Biol., 2006, 13, 745-751; Shi et al., Bioorganic Med. Chem. Lett., 2009, 19, 6038-6041; Chauhan et al., ChemMedChem , 2014, 9, 2274-2285; Kiessling et al., ChemMedChem , 2007, 2, 627-630; Choi et al., ACS Chem. Biol., 2017, 12, 2715-2719; and Castell et al., Sci. Rep., 2018, 8, 10064).

[0009] To discover small molecule MYC inhibitors, screening platforms have utilized Forster resonance energy transfer (FRET) (Berg et al., Proc. Natl. Acad. Sci., 2002, 99, 3830- 3835), fluorescence polarization (Hart et al., Proc. Natl. Acad. Sci., 2014, 111, 12556-12561), bimolecular fluorescence/luciferase complementation (Kiessling et al., Chem. Biol., 2006, 13, 745-751; Choi et al., ACS Chem. Biol., 2017, 12, 2715-2719), electrophoretic mobility shift (EMSA) (Jeong et al., Mol. Biosyst., 2010, 6, 1503-1509), and yeast two-hybrid (Wang et al., Mol. Cancer Idler., 2007, 6, 2399-2408) methodologies. While these platforms have identified compounds, they exhibit a variety of limitations, including cost, complexity, intellectual property, and the potential to yield artificial hits by adulterating the structure of the IDP MYC.

[0010] Thus, there exists a need for a simple, reliable, and cost effective high-throughput screening (HTS) assay for identification of small molecule MYC inhibitors. The present disclosure provides a simple and cost effective high-throughput screening assay for identifying MYC inhibitor scaffolds using a fluorescent small molecule probe. This high- throughput screening assay does not require covalent modifications, immobilizations, or the binding elements MAX and the DNA E-box.

[0011] In an aspect, provided herein is a compound of formula I:

or a pharmaceutically acceptable salt thereof, wherein: each R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³, and R¹⁴ is independently hydrogen, halogen, haloalkyl, -OH, C₁-C₅ alkoxy, -NO2, -CN, thiazolyl, or C1-C5 alkyl. L¹ is a bond, -S(O)₂-, -N(H)-, -O-, -S-, -C(O)-, -C(O)N(H)-, -N(H)C(O)-, -NHC(O)NH-, -C(O)O-, -OC(O)-, substituted or unsubstituted alkylene, substituted or unsubstituted heteroalkylene, an amino acid, or a combination thereof, and Q is a fluorophore.

[0012] In embodiments, each R¹, R², R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³, and R¹⁴ is independently hydrogen, halogen, C₁-C₅ alkyl, or -OH. In embodiments, each R¹, R², R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³, and R¹⁴ is independently hydrogen.

[0013] In embodiments, R³ is hydrogen, halogen, thiazolyl, C₁-C₅ alkoxy, -NO₂, or -CN. In embodiments, R³ is -NO₂, thiazolyl, or -CN. In embodiments, R³ is -CN.

[0014] In embodiments, L¹ is -C(O)-, unsubstituted alkylene or unsubstituted heteroalkylene, or a combination thereof. In embodiments, L¹ is -NHCH₂CH₂NH- or - NHCH₂CH₂NHC(O)-.

[0015] In embodiments, Q is a coumarin derivative. In embodiments, Q is

[0016] In embodiments, the compound is 1, or a

pharmaceutically acceptable salt thereof.

[0017] In an aspect, provided herein is a method of identifying a test compound as a MYC inhibitor, the method comprising: incubating a fluorescent probe with MYC, thereby forming a probe-MYC complex. Incubating the test compound with the probe-MYC complex. Measuring emission from test compound incubated with probe-MYC complex, and identifying the test compound as MYC inhibitor when detected fluorescence signal decreased in the presence of the test compound relative to the detected fluorescence signal in the absence of the test compound.

[0018] In embodiments, the fluorescent probe is a compound of formula I:

Formula I or a pharmaceutically acceptable salt thereof, wherein: each R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³, and R¹⁴ is independently hydrogen, halogen, haloalkyl, -OH, C1-C5 alkoxy, -NO2, -CN, thiazolyl, or C1-C5 alkyl. L¹ is bond, -S(O)₂-, -N(H)-, -O-, -S-, -C(O)-, -C(O)N(H)-, -N(H)C(O)-, -NHC(O)NH-, -C(O)O-, -OC(O)-, substituted or unsubstituted alkylene, substituted or unsubstituted heteroalkylene, an amino acid, or a combination thereof, and Q is a fluorophore.

[0019] In embodiments, each R¹, R², R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³, and R¹⁴ is independently hydrogen, halogen, C₁-C₅ alkyl, or -OH. In embodiments, each R¹, R², R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³, and R¹⁴ is independently hydrogen.

[0020] In embodiments, R³ is hydrogen, halogen, thiazolyl, C₁-C₅ alkoxy, -NO₂, or -CN. In embodiments, R³ is -NO₂, thiazolyl, or -CN. In embodiments, R³ is -CN.

[0021] In embodiments, L¹ is -C(O)-, unsubstituted alkylene or unsubstituted heteroalkylene, or a combination thereof. In embodiments, L¹ is -NHCH₂CH₂NH- or - NHCH₂CH₂NHC(O)-.

[0022] In embodiments, Q is a coumarin derivative. In embodiments, Q is

[0023] In embodiments, the compound is 1, or a

pharmaceutically acceptable salt thereof.

[0024] In an aspect, provided herein is a method of inhibiting MYC-MAX dimerization, comprising contacting MYC with an effective amount of a MYC inhibitor, wherein the MYC inhibitor is identified by any one of the methods described herein.

[0025] In an aspect, provided herein is a method of inhibiting transcriptional activation by MYC, comprising contacting MYC with an effective amount of a MYC inhibitor, wherein the MYC inhibitor is identified by any one of the methods described herein.

[0026] In an aspect, provided herein is a method of inhibiting MYC-induced cellular proliferation, comprising contacting MYC with an effective amount of a MYC inhibitor, wherein the MYC inhibitor is identified by any one of the methods described herein.

[0027] In an aspect, provided herein is a MYC inhibitor which is identified by any one of the methods described herein.

[0028] In an aspect, provided herein is a pharmaceutical composition comprising a MYC inhibitor and a pharmaceutically acceptable carrier. In embodiments, the MYC inhibitor is identified by any one of the methods described herein.

[0029] In an aspect, provided herein is a method of treating a disease in a subject in need thereof, the method comprising administering to the subject an effective amount of a MYC inhibitor, wherein the MYC inhibitor is identified by any one of the methods described herein. In embodiments, the disease is cancer.

BRIEF DESCRIPTION OF THE DRAWINGS

[0030] FIG. 1 A depicts the process of the high-throughput screening assay for MYC inhibitor.

[0031] FIG. IB shows chemical structures of MYC inhibitor KJ-Pyr-9 and fluorescent probe 1 (derived from KJ-Pyr-9). [0032] FIG. 2 shows fluorescence enhancement of fluorescent probe 1 in the presence of MYC. Emission sweep of 1 (5 mM, excitation: 427 nm) after incubation (21 °C, 1 hr) with varying MYC concentrations (1:3 dilution, 6.7-0 pM) in buffer (50 mM MES, pH 6.0, 2% DMSO).

[0033] FIG. 3 shows fluorescence perturbation of 1, and two coumarin derivatives (which do not inhibit MYC - 7-diethylamino-4-methylcoumarin and 7-amino-4-methylcoumarin) in the presence of MYC. Dilution series of MYC (2 pM-0.9 nM, 1:3) in buffer (50 mM MES, pH 6.0, Tween20 0.01%, BSA 0.002%, DMSO 2%) were prepared with and without fluorophore (0.8 mM). Plates were incubated for 1 hr at room temperature before measuring emission at the maximum excitation: 1 (424 nm), 7-diethylamino-4-methylcoumarin (375 nm), and 7-amino-4-methylcoumarin (339 nm). Emission values from wells with fluorophore were subtracted by the respective wells without fluorophore.

[0034] FIG. 4 Chemical structures of the fluorescent probe 1 and three established MYC inhibitors are shown at the top, with results of a competition assay, where following incubation of 1 with MYC, known MYC inhibitors were added at different concentrations, shown below. Solutions of 1 (0.8 pM) in buffer (50 mM MES, pH 6.0, Tween20 0.01%, BSA 0.002%, DMSO 2%) were prepared with and without MYC (1 pM). Inhibitors were serially diluted across wells (1:3 dilution, 100 pM - 45.7 nM). The plate was incubated (1 hr, 21 °C, dark) before fluorescence was measured (excitation: 424 nm, emission: 470 nm). Fluorescence from wells with MYC were subtracted by the respective wells without MYC and the results were fit in GraphPad Prism. Data are presented as the average of three replicates ± SD.

[0035] FIG. 5A shows the binding of KJ-Pyr-9 against MYC-monomer and MYC-MAX- dimer proteins using SPR. KJ-Pyr-9 was flowed over MYC-monomer or MYC-MAX-dimer protein immobilized on the CM5 chip surface, and the k_a , k_d and K_D values were determined by Biacore Insight Evaluation software. The blank and reference subtracted and solvent- corrected sensorgrams (dotted lines) and fitted curves (continuous curves) are shown.

[0036] FIG. 5B shows the binding of 1 against MYC-monomer and MYC-MAX-dimer proteins using SPR. 1 was flowed over MYC-monomer or MYC-MAX-dimer protein immobilized on the CM5 chip surface, and the k_a , k_d and K_D values were determined by Biacore Insight Evaluation software. The blank and reference subtracted and solvent- corrected sensorgrams (dotted lines) and fitted curves (continuous curves) are shown.

[0037] FIG. 6 shows structural elucidation and HDX-MS binding data. Rendering of the X- ray crystal structure of the MYC (white)/MAX (dark grey)/DNA complex (PDB 1NKP). The binding region for 1 is depicted in the highlighted region (light grey). Enlarged area of a part of the MYC/MAX/DNA complex is shown in the box on the right side of the figure. The enlarged area shows the induced-fit docking of the best-ranked poses of 1 and 5 into the binding region.

[0038] FIG. 7 shows the results of LOPAC 1280 (library of pharmacologically active compounds; N=1280) screen for discovery of MYC inhibitors. The scatter plot of 1280 compounds screened (in triplicate) in a high throughput assay is shown. Each dot represents the activity result of a well containing test compound (black dots) or controls (grey dots). Overall screening statistics are described in the box below the graph. In this viewing format, the y-axis represents % inhibition (response), i.e., full enzymatic activity is defined as “0” and fluorescence signal of the substrate in the presence of the 10074-G5 as “100”, with a hit cut-off of 23.74%. An example of a dose-response curve for 10074-G5 (and chemical structure of 10074-G5) is also shown.

[0039] FIG. 8 shows select high affinity hits selected from the 1536-well (LOPAC 1280) and 384-well pilot screens.

[0040] FIG. 9 A shows the inhibition of MYC-MAX DNA binding by SPR for pilot screen hits (whose chemical structures shown in figure 8). 100% inhibition set as buffer without MYC-MAX dimer and 0% inhibition set as buffer without inhibitor. Inhibitors were evaluated at 10 mM. Compounds KJ-Pyr-9, 5b and 5d are reported inhibitors (Jacob et ak, 2018, Bioorg. Med. Chem., 26, 4234-4239). 1 is the fluorescent probe used for the high throughput assays.

[0041] FIG. 9B shows results of CEF assay. MYC specificity oncogenic transformation assay with KJ-Pyr-9 and 5 (see figure 8) against virally induced oncogenic transformation by MYC, Src, Jun, or H1047R. Efficiency of transformation (%) is relative to vehicle (DMSO) for the respective oncogene. DETAILED DESCRIPTION OF THE INVENTION Definitions:

[0042] Unless defined otherwise, technical and scientific terms used herein have meanings that are commonly understood by those of ordinary skill in the art unless defined otherwise. Generally, terminologies pertaining to techniques of cell and tissue culture, molecular biology, immunology, microbiology, genetics, transgenic cell production, protein chemistry and nucleic acid chemistry and hybridization described herein are well known and commonly used in the art. The methods and techniques provided herein are generally performed according to conventional procedures well known in the art and as described in various general and more specific references that are cited and discussed herein unless otherwise indicated. See, e.g., Sambrook et al. Molecular Cloning: A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989) and Ausubel et al.,

Current Protocols in Molecular Biology, Greene Publishing Associates (1992). All of the references cited herein are incorporated herein by reference in their entireties. The terminology used in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are well known and commonly used in the art. Standard techniques can be used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients.

[0043] The headings provided herein are not limitations of the various aspects of the disclosure, which aspects can be understood by reference to the specification as a whole. Unless otherwise required by context herein, singular terms shall include pluralities and plural terms shall include the singular. Singular forms “a”, “an” and “the”, and singular use of any word, include plural referents unless expressly and unequivocally limited on one referent. In addition, the phrase "substituted with a[n]," as used herein, means the specified group may be substituted with one or more of any or all of the named substituents. For example, where a group, such as an alkyl or heteroaryl group, is "substituted with an unsubstituted C₁-C₂₀ alkyl, or unsubstituted 2 to 20 membered heteroalkyl," the group may contain one or more unsubstituted C₁-C₂₀ alkyls, and/or one or more unsubstituted 2 to 20 membered heteroalkyls.

[0001] Moreover, where a moiety is substituted with an R substituent, the group may be referred to as “R-substituted.” Where a moiety is R-substituted, the moiety is substituted with at least one R substituent and each R substituent is optionally different. Where a particular R group is present in the description of a chemical genus (such as Formula (I)), a Roman alphabetic symbol may be used to distinguish each appearance of that particular R group. For example, where multiple R¹³ substituents are present, each R¹³ substituent may be distinguished as R^{13 A}, R^{13 B}, R^{13 c}, R^{13 D}, etc., wherein each of R^{13 A}, R^{13 B}, R^{13 c}, R^{13 D}, etc. is defined within the scope of the definition of R¹³ and optionally differently.

[0002] A “detectable agent” or “detectable moiety” is a composition, substance, element, or compound; or moiety thereof; detectable by appropriate means such as spectroscopic, photochemical, biochemical, immunochemical, chemical, magnetic resonance imaging, or other physical means. For example, useful detectable agents include ¹⁸F, ³²P, ³³P, ⁴⁵Ti, ⁴⁷Sc, ⁵²Fe, ⁵⁹Fe, ⁶²Cu, ⁶⁴Cu, ⁶⁷Cu, ⁶⁷Ga, ⁶⁸Ga, ⁷⁷ As, ⁸⁶Y, ⁹⁰Y. ⁸⁹Sr, ⁸⁹Zr, ⁹⁴Tc, ⁹⁴Tc, ^99mTc, ⁹⁹Mo, ¹⁰⁵Pd, ¹⁰⁵Rh, ^luAg, ^luIn, ¹²³I, ¹²⁴I, ¹²⁵I, ¹³¹I, ¹⁴²Pr, ¹⁴³Pr, ¹⁴⁹Pm, ¹⁵³Sm, ^154'1581Gd, ¹⁶¹Tb, ¹⁶⁶Dy, ¹⁶⁶HO, ¹⁶⁹Er, ¹⁷⁵LU, ¹⁷⁷LU, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁸⁹Re, ¹⁹⁴Ir, ¹⁹⁸ Au, ¹⁹⁹ Au, ²¹¹At, ²¹¹Pb, ²¹²Bi, ²¹²Pb, ²¹³Bi, ²²³Ra, ²²⁵ Ac, Cr, V, Mn, Fe, Co, Ni, Cu, La, Ce, Pr, Nd, Pm, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, Lu, ³²P, fluorophore (e.g. fluorescent dyes), electron-dense reagents, enzymes ( e.g ., as commonly used in an ELISA), biotin, digoxigenin, paramagnetic molecules, paramagnetic nanoparticles, ultrasmall superparamagnetic iron oxide ("USPIO") nanoparticles, USPIO nanoparticle aggregates, superparamagnetic iron oxide ("SPIO") nanoparticles, SPIO nanoparticle aggregates, monochrystalline iron oxide nanoparticles, monochrystalline iron oxide, nanoparticle contrast agents, liposomes or other delivery vehicles containing Gadolinium chelate ("Gd-chelate") molecules, Gadolinium, radioisotopes, radionuclides (e.g. carbon-11, nitrogen-13, oxygen-15, fluorine-18, rubidium- 82), fluorodeoxy glucose (e.g. fluorine- 18 labeled), any gamma ray emitting radionuclides, positron-emitting radionuclide, radiolabeled glucose, radiolabeled water, radiolabeled ammonia, biocolloids, microbubbles (e.g. including microbubble shells including albumin, galactose, lipid, and/or polymers; microbubble gas core including air, heavy gas(es), perfluorcarbon, nitrogen, octafluoropropane, perflexane lipid microsphere, perflutren, etc.), iodinated contrast agents (e.g. iohexol, iodixanol, ioversol, iopamidol, ioxilan, iopromide, diatrizoate, metrizoate, ioxaglate), barium sulfate, thorium dioxide, gold, gold nanoparticles, gold nanoparticle aggregates, fluorophores, two-photon fluorophores, or haptens and proteins or other entities which can be made detectable, e.g., by incorporating a radiolabel into a peptide or antibody specifically reactive with a target peptide. A detectable moiety is a monovalent detectable agent or a detectable agent capable of forming a bond with another composition.

[0003] Radioactive substances (e.g., radioisotopes) that may be used as imaging and/or labeling agents in accordance with the embodiments of the disclosure include, but are not limited to, ¹⁸F, ³²P, ³³P, ⁴⁵Ti, ⁴⁷Sc, ⁵²Fe, ⁵⁹Fe, ⁶²Cu, ⁶⁴Cu, ⁶⁷Cu, ⁶⁷Ga, ⁶⁸Ga, ⁷⁷ As, ⁸⁶Y, ⁹⁰Y.

⁸⁹ Sr, ⁸⁹Zr, ⁹⁴Tc, ⁹⁴Tc, ^99mTc, "Mo, ¹⁰⁵Pd, ¹⁰⁵Rh, ^mAg, ^U1ln, ¹²³I, ¹²⁴I, ¹²⁵I, ¹³¹I, ¹⁴²Pr, ¹⁴³Pr, ¹⁴⁹Pm, ¹⁵³Sm, ¹⁵⁴-¹⁵⁸¹Gd, ¹⁶¹Tb, ¹⁶⁶Dy, ¹⁶⁶Ho, ¹⁶⁹Er, ¹⁷⁵Lu, ¹⁷⁷Lu, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁸⁹Re, ¹⁹⁴Ir, ¹⁹⁸Au, ¹⁹⁹Au, ²¹¹At, ²¹¹Pb, ²¹²Bi, ²¹²Pb, ²¹³Bi, ²²³Ra and ²²⁵ Ac. Paramagnetic ions that may be used as additional imaging agents in accordance with the embodiments of the disclosure include, but are not limited to, ions of transition and lanthanide metals (e.g. metals having atomic numbers of 21-29, 42, 43, 44, or 57-71). These metals include ions of Cr, V, Mn, Fe, Co, Ni, Cu, La, Ce, Pr, Nd, Pm, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb and Lu.

[0022] It is understood the use of the alternative (e.g., “or”) herein is taken to mean either one or both or any combination thereof of the alternatives.

[0023] The term “and/or” used herein is to be taken mean specific disclosure of each of the specified features or components with or without the other. For example, the term “and/or” as used in a phrase such as “A and/or B” herein is intended to include “A and B,”

“A or B,” “A” (alone), and “B” (alone). Likewise, the term “and/or” as used in a phrase such as “A, B, and/or C” is intended to encompass each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0024] As used herein, the term “about” refers to a value or composition that is within an acceptable error range for the particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined, i.e., the limitations of the measurement system. For example, “about” or “approximately” can mean within one or more than one standard deviation per the practice in the art. Alternatively, “about” or “approximately” can mean a range of up to 10% (i.e., ±10%) or more depending on the limitations of the measurement system. For example, about 5 mg can include any number between 4.5 mg and 5.5 mg. Furthermore, particularly with respect to biological systems or processes, the terms can mean up to an order of magnitude or up to 5-fold of a value. When particular values or compositions are provided in the instant disclosure, unless otherwise stated, the meaning of “about” or “approximately” should be assumed to be within an acceptable error range for that particular value or composition. In embodiments, about includes the specified value.

[0025] In this disclosure, “comprises,” “comprising,” “containing” and “having” and the like can have the meaning ascribed to them in U.S. Patent law and can mean “includes,” “including,” and the like. “Consisting essentially of or “consists essentially” likewise has the meaning ascribed in U.S. Patent law and the term is open-ended, allowing for the presence of more than that which is recited so long as basic or novel characteristics of that which is recited is not changed by the presence of more than that which is recited, but excludes prior art embodiments.

[0026] The terms "polypeptide," "peptide" and "protein" and other related terms used herein are used interchangeably to refer to a polymer of amino acid residues, wherein the polymer may in embodiments be conjugated to a moiety that does not consist of amino acids. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers.

A "fusion protein" refers to a chimeric protein encoding two or more separate protein sequences that are recombinantly expressed as a single moiety. Polypeptides include mature molecules that have undergone cleavage. These terms encompass native and artificial proteins, protein fragments and polypeptide analogs (such as muteins, variants, chimeric proteins and fusion proteins) of a protein sequence as well as post-translationally, or otherwise covalently or non-covalently, modified proteins. Two or more polypeptides (e.g., 3 polypeptide chains) can associate with each other, via covalent and/or non-covalent association, to form a multimeric polypeptide complex (e.g., multi-specific antigen binding protein complex). Association of the polypeptide chains can also include peptide folding. Thus, a polypeptide complex can be dimeric, trimeric, tetrameric, or higher order complexes depending on the number of polypeptide chains that form the complex.

[0027] As used herein, the terms “cancer,” “neoplasm,” and “tumor” are used interchangeably and, in either the singular or plural form, refer to cells that have undergone a malignant transformation that makes them pathological to the host organism. Primary cancer cells can be readily distinguished from non-cancerous cells by well-established techniques, particularly histological examination. The definition of a cancer cell, as used herein, includes not only a primary cancer cell, but any ceil derived from a cancer cell ancestor. This includes metastasized cancer cells, and in vitro cultures and cell lines derived from cancer ceils. When referring to a type of cancer that normally manifests as a solid tumor, a “clinically detectable” tumor is one that is detectable on the basis of tumor mass: e.g., by procedures such as computed tomography (CT) scan, magnetic resonance imaging (MKI), X-ray, ultrasound or palpation on physical examination, and/or which is detectable because of the expression of one or more cancer-specific antigens in a sample obtainable from a patient. Tumors may be a hematopoietic (or hematologic or hematological or blood-related) cancer, for example, cancers derived from blood cells or immune cells, which may be referred to as “liquid tumors.” Specific examples of clinical conditions based on hematologic tumors include leukemias such as chronic myelocytic leukemia, acute myelocytic leukemia, chronic lymphocytic leukemia and acute lymphocytic leukemia; plasma cell malignancies such as multiple myeloma, MGUS and Waldenstrom's macroglobulinemia; lymphomas such as non- Hodgkin's lymphoma, Hodgkin's lymphoma, and the like,

[0028] The cancer may be any cancer in which an abnormal number of blast cells or unwanted ceil proliferation is present or that is diagnosed as a hematological cancer, including both lymphoid and myeloid malignancies. Myeloid malignancies include, but are not limited to, acute myeloid (or myelocytic or myelogenous or myelohlastic) leukemia (undifferentiated or differentiated), acute promyeioid (or promyelocytic or promyelogenous or promyeloblastic) leukemia, acute myelomonocytic (or myelomonoblastic) leukemia, acute monocytic (or monoblastic) leukemia, erythroleukemia and megakaryocytic (or megakaryoblastic) leukemia. These leukemias may be referred together as acute myeloid (or myelocytic or myelogenous) leukemia (AML). Myeloid malignancies also include myeloproliferative disorders (MPD) which include, but are not limited to, chronic myelogenous (or myeloid) leukemia (CML), chronic myelomonocytic leukemia (CMML), essential thrombocythemia (or thrombocytosis), and polcythemia vera (PCV). Myeloid malignancies also include myelodysplasia (or myelodysplastic syndrome or MOS), which may be referred to as refractor/ anemia (RA), refractory anemia with excess blasts (RAEB), and refractory anemia with excess blasts in transformation (RAEBT); as well as myelofibrosis (MFS) with or without agnogenic myeloid metaplasia.

[0029] Hematopoietic cancers also include lymphoid malignancies, which may affect the lymph nodes, spleens, bone marrow, peripheral blood, and/or extranodal sites. Lymphoid cancers include B-cell malignancies, which include, but are not limited to, B-cel! non- Hodgkin’s lymphomas (B-NHLs). B-NHLs may be indolent (or low-grade), intermediate- grade (or aggressive) or high-grade (very aggressive). Indolent Bcell lymphomas include follicular lymphoma (FL); small lymphocytic lymphoma (SLL); marginal zone lymphoma (MZL) including nodal MZL, extranodal MZL, splenic MZL and splenic MZL with villous lymphocytes; lymphopiasmacytic lymphoma (LPL); and mucosa-associated-lymphoid tissue (MALT or extranodal marginal zone) lymphoma. Intermediate-grade B~NHLs include mantle ceil lymphoma (MCL) with or without leukemic involvement, diffuse large cell lymphoma (DLBCL), follicular large cell (or grade 3 or grade 3B) lymphoma, and primary mediastinal lymphoma (PML), High-grade B-NHLs include Burkitt's lymphoma (BL), Burkitt-like lymphoma, small non-eleaved ceil lymphoma (SNCCL) and lymphoblastic lymphoma. Other B-NHLs include immunoblastic lymphoma (or immunocytoma), primary effusion lymphoma, HIV associated (or AIDS related) lymphomas, and post-transplant lymphoproliferative disorder (PTLD) or lymphoma. B-cell malignancies also include, but are not limited to, chronic lymphocytic leukemia (CLL), prolymphocytic leukemia (PLL), Waldenstrom’s rnacroglobulinemia (WM), hairy cell leukemia (HCL), large granular lymphocyte (LGL) leukemia, acute lymphoid (or lymphocytic or lymphoblastic) leukemia, and Castieman's disease. NHL may also include T-cell non-Hodgkin’s lymphoma s (T-NHLs), which include, but are not limited to T-ceil non-Hodgkin’s lymphoma not otherwise specified (NOS), peripheral T-cell lymphoma (PTCL), anaplastic large cell lymphoma (ALCL), angioimmunoblastic lymphoid disorder (A!LD), nasal natural killer (NK) cell/T-cell lymphoma, gamma/delta lymphoma, cutaneous T ceil lymphoma, mycosis fungoides, and Sezary syndrome,

[0030] Hematopoietic cancers also include Hodgkin’s lymphoma (or disease) including classical Hodgkin’s lymphoma, nodular sclerosing Hodgkin's lymphoma, mixed cellularity Hodgkin’s lymphoma, lymphocyte predominant (LP) Hodgkin's lymphoma, nodular LP Hodgkin's lymphoma, and lymphocy te depleted Hodgkin's lymphoma. Hematopoietic cancers also include plasma cell diseases or cancers such as multiple myeloma (MM) including smoldering MM, monoclonal gammopathy of undetermined (or unknown or unclear) significance (MGUS), plasmacytoma (bone, extramedullary), lymphoplasmacytic lymphoma (LPL), Waldenstrom's Macroglobulinemia, plasma cell leukemia, and primary amyloidosis (AL). Hematopoietic cancers may also include other cancers of additional hematopoietic cells, including polymorphonuclear leukocytes (or neutrophils), basophils, eosinophils, dendritic cells, platelets, erythrocytes and natural killer cells. Tissues which include hematopoietic ceils referred herein to as “hematopoietic cell tissues” include bone marrow; peripheral blood; thymus; and peripheral lymphoid tissues, such as spleen, lymph nodes, lymphoid tissues associated with mucosa (such as the gut-associated lymphoid tissues), tonsils, Peyer's patches and appendix, and lymphoid tissues associated with other mucosa, for example, the bronchial linings.

[0031] The term "isolated", means altered “by the hand of man” from its natural state, has been changed or removed from its original environment, or both. When the term “isolated” is applied to a nucleic acid or protein, denotes that the nucleic acid or protein is essentially free of other cellular components with which it is associated in the natural state. It can be, for example, in a homogeneous state and may be in either a dry or aqueous solution. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis, high-performance liquid chromatography or mass spectrophotometry. A protein that is the predominant species present in a preparation is substantially purified. For example, a polynucleotide or a polypeptide naturally present in a living organism is not “isolated,” but the same polynucleotide or polypeptide separated from the coexisting materials of its natural state is “isolated”, including but not limited to when such polynucleotide or polypeptide is introduced back into a cell, even if the cell is of the same species or type as that from which the polynucleotide or polypeptide was separated.

[0032] As used herein, the term “variant” polypeptides and “variants” of polypeptides refers to a polypeptide comprising an amino acid sequence with one or more amino acid residues inserted into, deleted from and/or substituted into the amino acid sequence relative to a reference polypeptide sequence. Polypeptide variants include fusion proteins. In the same manner, a variant polynucleotide comprises a nucleotide sequence with one or more nucleotides inserted into, deleted from and/or substituted into the nucleotide sequence relative to another polynucleotide sequence. Polynucleotide variants include fusion polynucleotides.

[0033] As used herein the term “domain” refers to a folded protein structure which has tertiary structure independent of the rest of the protein. Generally, domains are responsible for discrete functional properties of proteins and in many cases may be added, removed or transferred to other proteins without loss of function of the remainder of the protein and/or of the domain. An “antibody single variable domain” is a folded polypeptide domain comprising sequences characteristic of antibody variable domains. It therefore includes complete antibody variable domains and modified variable domains, for example, in which one or more loops have been replaced by sequences which are not characteristic of antibody variable domains, or antibody variable domains which have been truncated or comprise N- or C-terminal extensions, as well as folded fragments of variable domains which retain at least the binding activity and specificity of the full-length domain.

[0034] An “individual” “patient” or “subject” is a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In certain embodiments, the individual or subject is a human. In certain embodiments, the subject is an adult, an adolescent, a child, or an infant. In some embodiments, the terms “individual” or “patient” are used and are intended to be interchangeable with “subject”.

[0035] “Percent (%) amino acid sequence identity” with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, with the aid of the local homology algorithm by Smith and Waterman, 1981, Ads App. Math. 2, 482, with the aid of the local homology algorithm by Needleman and Wunsch, 1970, J. Mol. Biol. 48, 443, with the aid of the similarity search algorithm by Pearson and Lipman, 1988, Proc. Natl Acad. Sci. USA 88, 2444, or with the aid of computer programs using said algorithms (e.g., EMBOSS Needle or EMBOSS Water, available at www.ebi.ac.uk/Tools/psa/ ). Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. "Percentage of sequence identity" or "percent (%) [sequence] identity", as used herein, is determined by comparing two optimally locally aligned sequences over a comparison window defined by the length of the local alignment between the two sequences. (This may also be considered percentage of homology or "percent (%) homology".) The amino acid sequence in the comparison window may comprise additions or deletions (e.g., gaps or overhangs) as compared to the reference sequence for optimal alignment of the two sequences. Local alignment between two sequences only includes segments of each sequence that are deemed to be sufficiently similar according to a criterion that depends on the algorithm used to perform the alignment (e.g., EMBOSS Water), "identical" or percent "identity," refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (i.e., about 60% identity, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over a specified region, when compared and aligned for maximum correspondence over a comparison window or designated region). The percentage identity is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100. Optimal alignment of sequences for comparison may be conducted by the local homology algorithm of Smith and Waterman (Add. APL. Math.

2:482, 1981), by the global homology alignment algorithm of Needleman and Wunsch (J. Mol. Biol. 48:443, 1970), by the search for similarity method of Pearson and Lipman (Proc. Natl. Acad. Sci. USA 85: 2444, 1988), or by inspection. GAP and BESTFIT, as additional examples, can be employed to determine the optimal alignment of two sequences that have been identified for comparison. Typically, the default values of 5.00 for gap weight and 0.30 for gap weight length are used.

[0036] A comparison of the sequences and determination of the percent identity between two polypeptide sequences, or between two polynucleotide sequences, may be accomplished using a mathematical algorithm. For example, the "percent identity" or "percent homology" of two polypeptide or two polynucleotide sequences may be determined by comparing the sequences using the GAP computer program (a part of the GCG Wisconsin Package, version 10.3 (Accelrys, San Diego, Calif.)) using its default parameters. Expressions such as “comprises a sequence with at least X% identity to Y” with respect to a test sequence mean that, when aligned to sequence Y as described above, the test sequence comprises residues identical to at least X% of the residues of Y.

[0037] An amino acid or nucleotide base "position" is denoted by a number that sequentially identifies each amino acid (or nucleotide base) in the reference sequence based on its position relative to the N-terminus (or 5'-end). Due to deletions, insertions, truncations, fusions, and the like that must be taken into account when determining an optimal alignment, in general the amino acid residue number in a test sequence determined by simply counting from the N-terminus will not necessarily be the same as the number of its corresponding position in the reference sequence. For example, in a case where a variant has a deletion relative to an aligned reference sequence, there will be no amino acid in the variant that corresponds to a position in the reference sequence at the site of deletion. Where there is an insertion in an aligned reference sequence, that insertion will not correspond to a numbered amino acid position in the reference sequence. In the case of truncations or fusions there can be stretches of amino acids in either the reference or aligned sequence that do not correspond to any amino acid in the corresponding sequence.

[0038] The terms "numbered with reference to" or "corresponding to," when used in the context of the numbering of a given amino acid or polynucleotide sequence, refers to the numbering of the residues of a specified reference sequence when the given amino acid or polynucleotide sequence is compared to the reference sequence. An amino acid residue in a protein "corresponds" to a given residue when it occupies the same essential structural position within the protein as the given residue. For example, a selected residue in a selected protein corresponds to Lys397 of MYC when the selected residue occupies the same essential spatial or other structural relationship as Lys397 in MYC. Instead of a primary sequence alignment, a three-dimensional structural alignment can also be used. In this case, an amino acid that occupies the same essential position as Lys397 in the structural model is said to correspond to the Lys residue.

[0039] “Linker” refers to a chemical moiety comprising a covalent bond or a chain of atoms that covalently attaches an antibody to a drug moiety. In various embodiments, linkers include a divalent radical. In various embodiments, linkers can comprise one or more amino acid residues.

[0040] As used herein, the terms “bioconjugate” and “bioconjugate linker” refers to the resulting association between atoms or molecules of “bioconjugate reactive groups” or “bioconjugate reactive moieties”. The association can be direct or indirect. For example, a conjugate between a first bioconjugate reactive group (e.g., -NFL, -C(O)OH, -N- hydroxysuccinimide, or -maleimide) and a second bioconjugate reactive group (e.g., sulfhydryl, sulfur-containing amino acid, amine, amine sidechain containing amino acid, or carboxylate) provided herein can be direct, e.g., by covalent bond or linker (e.g. a first linker of second linker), or indirect, e.g., by non-covalent bond (e.g. electrostatic interactions (e.g. ionic bond, hydrogen bond, halogen bond), van der Waals interactions (e.g. dipole-dipole, dipole-induced dipole, London dispersion), ring stacking (pi effects), hydrophobic interactions and the like). In embodiments, bioconjugates or bioconjugate linkers are formed using bioconjugate chemistry (i.e. the association of two bioconjugate reactive groups) including, but are not limited to nucleophilic substitutions (e.g., reactions of amines and alcohols with acyl halides, active esters), electrophilic substitutions (e.g., enamine reactions) and additions to carbon-carbon and carbon-heteroatom multiple bonds (e.g., Michael reaction, Diels-Alder addition). These and other useful reactions are discussed in, for example, March, ADVANCED ORGANIC CHEMISTRY, 3rd Ed., John Wiley & Sons,

New York, 1985; Hermanson, BIOCONJUGATE TECHNIQUES, Academic Press, San Diego, 1996; and Feeney et al., MODIFICATION OF PROTEINS; Advances in Chemistry Series, Vol. 198, American Chemical Society, Washington, D.C., 1982. In embodiments, the first bioconjugate reactive group (e.g., maleimide moiety) is covalently attached to the second bioconjugate reactive group (e.g. a sulfhydryl). In embodiments, the first bioconjugate reactive group (e.g., haloacetyl moiety) is covalently attached to the second bioconjugate reactive group (e.g. a sulfhydryl). In embodiments, the first bioconjugate reactive group (e.g., pyridyl moiety) is covalently attached to the second bioconjugate reactive group (e.g. a sulfhydryl). In embodiments, the first bioconjugate reactive group (e.g., -N- hydroxysuccinimide moiety) is covalently attached to the second bioconjugate reactive group (e.g. an amine). In embodiments, the first bioconjugate reactive group (e.g., maleimide moiety) is covalently attached to the second bioconjugate reactive group (e.g. a sulfhydryl).

In embodiments, the first bioconjugate reactive group (e.g., -sulfo-N-hydroxysuccinimide moiety) is covalently attached to the second bioconjugate reactive group (e.g. an amine).

[0041] Useful bioconjugate reactive moieties used for bioconjugate chemistries herein include, for example:

(a) carboxyl groups and various derivatives thereof including, but not limited to, N-hydroxysuccinimide esters, N-hydroxybenztriazole esters, acid halides, acyl imidazoles, thioesters, p-nitrophenyl esters, alkyl, alkenyl, alkynyl and aromatic esters;

(b) hydroxyl groups which can be converted to esters, ethers, aldehydes, etc.

(c) haloalkyl groups wherein the halide can be later displaced with a nucleophilic group such as, for example, an amine, a carboxylate anion, thiol anion, carbanion, or an alkoxide ion, thereby resulting in the covalent attachment of a new group at the site of the halogen atom; (d) dienophile groups which are capable of participating in Diels-Alder reactions such as, for example, maleimido or maleimide groups;

(e) aldehyde or ketone groups such that subsequent derivatization is possible via formation of carbonyl derivatives such as, for example, imines, hydrazones, semicarbazones or oximes, or via such mechanisms as Grignard addition or alkyllithium addition;

(f) sulfonyl halide groups for subsequent reaction with amines, for example, to form sulfonamides;

(g) thiol groups, which can be converted to disulfides, reacted with acyl halides, or bonded to metals such as gold, or react with maleimides;

(h) amine or sulfhydryl groups (e.g., present in cysteine), which can be, for example, acylated, alkylated or oxidized;

(i) alkenes, which can undergo, for example, cycloadditions, acylation,

Michael addition, etc;

(j) epoxides, which can react with, for example, amines and hydroxyl compounds;

(k) phosphoramidites and other standard functional groups useful in nucleic acid synthesis;

(l) metal silicon oxide bonding; and

(m) metal bonding to reactive phosphorus groups (e.g. phosphines) to form, for example, phosphate diester bonds.

(n) azides coupled to alkynes using copper catalyzed cycloaddition click chemistry.

(o) biotin conjugate can react with avidin or strepavidin to form an avidin-biotin complex or streptavidin-biotin complex.

[0004] The bioconjugate reactive groups can be chosen such that they do not participate in, or interfere with, the chemical stability of the conjugate described herein. Alternatively, a reactive functional group can be protected from participating in the crosslinking reaction by the presence of a protecting group. In embodiments, the bioconjugate comprises a molecular entity derived from the reaction of an unsaturated bond, such as a maleimide, and a sulfhydryl group. The term “protecting group” is used in accordance with its ordinary meaning in organic chemistry and refers to a moiety covalently bound to a heteroatom, heterocycloalkyl, or heteroaryl to prevent reactivity of the heteroatom, heterocycloalkyl, or heteroaryl during one or more chemical reactions performed prior to removal of the protecting group. Typically a protecting group is bound to a heteroatom (e.g., O) during a part of a multipart synthesis wherein it is not desired to have the heteroatom react (e.g., a chemical reduction) with the reagent. Following protection the protecting group may be removed (e.g., by modulating the pH). In embodiments the protecting group is an alcohol protecting group. Non-limiting examples of alcohol protecting groups include acetyl, benzoyl, benzyl, methoxymethyl ether (MOM), tetrahydropyranyl (THP), and silyl ether (e.g., trimethyl silyl (TMS)). In embodiments the protecting group is an amine protecting group. Non-limiting examples of amine protecting groups include carbobenzyloxy (Cbz), tert-butyloxycarbonyl (BOC), 9-Fluorenylmethyloxycarbonyl (FMOC), acetyl, benzoyl, benzyl, carbamate, p-methoxybenzyl ether (PMB), and tosyl (Ts).

[0043] The term “pharmaceutically acceptable salts” is meant to include salts of the active compounds that are prepared with relatively nontoxic acids or bases, depending on the particular substituents found on the compounds described herein. When compounds of the present disclosure contain relatively acidic functionalities, base addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired base, either neat or in a suitable inert solvent. Examples of pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amino, or magnesium salt, or a similar salt. When compounds of the present disclosure contain relatively basic functionalities, acid addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired acid, either neat or in a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include those derived from inorganic acids like hydrochloric, hydrobromic, nitric, carbonic, monohydrogencarbonic, phosphoric, monohydrogenphosphoric, dihydrogenphosphoric, sulfuric, monohydrogensulfuric, hydriodic, or phosphorous acids and the like, as well as the salts derived from relatively nontoxic organic acids like acetic, propionic, isobutyric, maleic, malonic, benzoic, succinic, suberic, fumaric, lactic, mandelic, phthalic, benzenesulfonic, p- tolylsulfonic, citric, tartaric, oxalic, methanesulfonic, and the like. Also included are salts of amino acids such as arginate and the like, and salts of organic acids like glucuronic or galactunoric acids and the like (see, for example, Berge etal. , “Pharmaceutical Salts”, Journal of Pharmaceutical Science , 1977, 66, 1-19). Certain specific compounds of the present disclosure contain both basic and acidic functionalities that allow the compounds to be converted into either base or acid addition salts.

[0044] Thus, the compounds of the present disclosure may exist as salts, such as with pharmaceutically acceptable acids. The present disclosure includes such salts. Non-limiting examples of such salts include hydrochlorides, hydrobromides, phosphates, sulfates, methanesulfonates, nitrates, maleates, acetates, citrates, fumarates, proprionates, tartrates (e.g., (+)-tartrates, (-)-tartrates, or mixtures thereof including racemic mixtures), succinates, benzoates, and salts with amino acids such as glutamic acid, and quaternary ammonium salts (e.g. methyl iodide, ethyl iodide, and the like). These salts may be prepared by methods known to those skilled in the art.

[0045] The neutral forms of the compounds are preferably regenerated by contacting the salt with a base or acid and isolating the parent compound in the conventional manner. The parent form of the compound may differ from the various salt forms in certain physical properties, such as solubility in polar solvents.

[0046] In addition to salt forms, the present disclosure provides compounds, which are in a prodrug form. Prodrugs of the compounds described herein are those compounds that readily undergo chemical changes under physiological conditions to provide the compounds of the present disclosure. Prodrugs of the compounds described herein may be converted in vivo after administration. Additionally, prodrugs can be converted to the compounds of the present disclosure by chemical or biochemical methods in an ex vivo environment, such as, for example, when contacted with a suitable enzyme or chemical reagent.

[0047] Certain compounds of the present disclosure can exist in unsolvated forms as well as solvated forms, including hydrated forms. In general, the solvated forms are equivalent to unsolvated forms and are encompassed within the scope of the present disclosure. Certain compounds of the present disclosure may exist in multiple crystalline or amorphous forms. In general, all physical forms are equivalent for the uses contemplated by the present disclosure and are intended to be within the scope of the present disclosure.

[0048] “Pharmaceutically acceptable excipient” and “pharmaceutically acceptable carrier” refer to a substance that aids the administration of an active agent to and absorption by a subject and can be included in the compositions of the present disclosure without causing a significant adverse toxicological effect on the patient. Non-limiting examples of pharmaceutically acceptable excipients include water, NaCl, normal saline solutions, lactated Ringer’s, normal sucrose, normal glucose, binders, fillers, disintegrants, lubricants, coatings, sweeteners, flavors, salt solutions (such as Ringer's solution), alcohols, oils, gelatins, carbohydrates such as lactose, amylose or starch, fatty acid esters, hydroxymethycellulose, polyvinyl pyrrolidine, and colors, and the like. Such preparations can be sterilized and, if desired, mixed with auxiliary agents such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, and/or aromatic substances and the like that do not deleteriously react with the compounds of the disclosure. One of skill in the art will recognize that other pharmaceutical excipients are useful in the present disclosure.

[0049] The term “pharmaceutical formulation” refers to a preparation which is in such form as to permit the biological activity of an active ingredient contained therein to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered.

[0050] The term “administering”, “administered” and grammatical variants refers to the physical introduction of an agent to a subject, using any of the various methods and delivery systems known to those skilled in the art. Exemplary routes of administration for the formulations disclosed herein include intravenous, intramuscular, subcutaneous, intraperitoneal, spinal or other parenteral routes of administration, for example by injection or infusion. The phrase “parenteral administration” as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion, as well as in vivo electroporation. In some embodiments, the formulation is administered via a non-parenteral route, e.g., orally. Other non-parenteral routes include a topical, epidermal or mucosal route of administration, for example, intranasally, vaginally, rectally, sublingually or topically. Administering can also be performed, for example, once, a plurality of times, and/or over one or more extended periods.

[0051] "Treating" and "treatment" as used herein include prophylactic treatment.

Treatment methods include administering to a subject a therapeutically effective amount of an active agent. The administering step may consist of a single administration or may include a series of administrations. The length of the treatment period depends on a variety of factors, such as the severity of the condition, the age of the patient, the concentration of active agent, the activity of the compositions used in the treatment, or a combination thereof. It will also be appreciated that the effective dosage of an agent used for the treatment or prophylaxis may increase or decrease over the course of a particular treatment or prophylaxis regime. Changes in dosage may result and become apparent by standard diagnostic assays known in the art. In some instances, chronic administration may be required. For example, the compositions are administered to the subject in an amount and for a duration sufficient to treat the patient. In embodiments, the treating or treatment is no prophylactic treatment.

[0052] An “effective amount” is an amount sufficient for a compound to accomplish a stated purpose relative to the absence of the compound (e.g. achieve the effect for which it is administered, treat a disease, reduce enzyme activity, increase enzyme activity, reduce a signaling pathway, or reduce one or more symptoms of a disease or condition). 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.” A “reduction” of a symptom or symptoms (and grammatical equivalents of this phrase) means decreasing of the severity or frequency of the symptom(s), or elimination of the symptom(s). A “prophylactically effective amount” of a drug is an amount of a drug that, when administered to a subject, will have the intended prophylactic effect, e.g., preventing or delaying the onset (or reoccurrence) of an injury, disease, pathology or condition, or reducing the likelihood of the onset (or reoccurrence) of an injury, disease, pathology, or condition, or their symptoms. The full prophylactic effect does not necessarily occur by administration of one dose, and may occur only after administration of a series of doses. Thus, a prophylactically effective amount may be administered in one or more administrations. An “activity decreasing amount,” as used herein, refers to an amount of antagonist required to decrease the activity of an enzyme relative to the absence of the antagonist. A “function disrupting amount,” as used herein, refers to the amount of antagonist required to disrupt the function of an enzyme or protein relative to the absence of the antagonist. The exact amounts will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques (see, e.g., Lieberman, Pharmaceutical Dosage Forms (vols. 1-3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999); Pickar, Dosage Calculations (1999); and Remington: The Science and Practice of Pharmacy, 20th Edition, 2003, Gennaro, Ed., Lippincott, Williams & Wilkins).

[0053] For any compound described herein, the therapeutically effective amount can be initially determined from cell culture assays. Target concentrations will be those concentrations of active compound(s) that are capable of achieving the methods described herein, as measured using the methods described herein or known in the art.

[0054] As is well known in the art, therapeutically effective amounts for use in humans can also be determined from animal models. For example, a dose for humans can be formulated to achieve a concentration that has been found to be effective in animals. The dosage in humans can be adjusted by monitoring compounds effectiveness and adjusting the dosage upwards or downwards, as described above. Adjusting the dose to achieve maximal efficacy in humans based on the methods described above and other methods is well within the capabilities of the ordinarily skilled artisan.

[0055] The term “therapeutically effective amount,” as used herein, refers to that amount of the therapeutic agent sufficient to ameliorate the disorder, as described above. For example, for the given parameter, a therapeutically effective amount will show an increase or decrease of at least 5%, 10%, 15%, 20%, 25%, 40%, 50%, 60%, 75%, 80%, 90%, or at least 100%. Therapeutic efficacy can also be expressed as “-fold” increase or decrease. For example, a therapeutically effective amount can have at least a 1.2-fold, 1.5-fold, 2-fold, 5-fold, or more effect over a control.

[0056] Dosages may be varied depending upon the requirements of the patient and the compound being employed. The dose administered to a patient, in the context of the present disclosure, should be sufficient to effect a beneficial therapeutic response in the patient over time. The size of the dose also will be determined by the existence, nature, and extent of any adverse side-effects. Determination of the proper dosage for a particular situation is within the skill of the practitioner. Generally, treatment is initiated with smaller dosages which are less than the optimum dose of the compound. Thereafter, the dosage is increased by small increments until the optimum effect under circumstances is reached. Dosage amounts and intervals can be adjusted individually to provide levels of the administered compound effective for the particular clinical indication being treated. This will provide a therapeutic regimen that is commensurate with the severity of the individual's disease state. [0057] “Affinity” refers to the strength of the sum total of noncovalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless indicated otherwise, as used herein, “binding affinity” refers to intrinsic binding affinity which reflects a 1 : 1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (Kd). Affinity can be measured by common methods known in the art, including those described herein. Specific illustrative and exemplary embodiments for measuring binding affinity are described in the following.

[0058] In embodiments, a dissociation constant (K_D) can be measured using a BIACORE surface plasmon resonance (SPR) assay. Surface plasmon resonance refers to an optical phenomenon that allows for the analysis of real-time interactions by detection of alterations in protein concentrations within a biosensor matrix, for example using the BIACORE system (Biacore Life Sciences division of GE Healthcare, Piscataway, NJ).

[0059] An “inhibitor” refers to a compound (e.g. compounds described herein) that reduces activity when compared to a control, such as absence of the compound or a compound with known inactivity. The terms “inhibitor,” “repressor” or “antagonist” or “downregulator” interchangeably refer to a substance capable of detectably decreasing the expression or activity of a given gene or protein. The antagonist can decrease expression or activity 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more in comparison to a control in the absence of the antagonist. In certain instances, expression or activity is 1.5-fold, 2-fold, 3- fold, 4-fold, 5-fold, 10-fold or lower than the expression or activity in the absence of the antagonist.

[0060] 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 activity or function of the protein in the absence of the inhibitor. In embodiments inhibition means negatively affecting (e.g. decreasing) the concentration or levels of the protein relative to the concentration or level of the protein in the absence of the inhibitor. In embodiments inhibition refers to reduction of a disease or symptoms of disease. In embodiments, inhibition refers to a reduction in the activity of a particular protein target. 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 embodiments, inhibition refers to a reduction of activity of a target protein resulting from a direct interaction (e.g. an inhibitor binds to the target protein). In embodiments, inhibition refers to a reduction of activity of a target protein from an indirect interaction (e.g. an inhibitor binds to a protein that activates the target protein, thereby preventing target protein activation).

[0061] The term “MYC,” as used herein, refers to the protein product of the c-Myc gene. Dysregulation of MYC family gene/proteins occurs in over half of all human tumors and is recognized as a general hallmark of cancer initiation and maintenance. M YC is a transcriptional regulator that belongs to a family of basic helix-loop-helix leucine zipper (bHLH-LZ) proteins that dimerize with the bHLH-LZ protein MAX to become functional . The MYC-MAX heterodimer preferentially binds to the E-Box motif, a palindromic DNA sequence. MYC affects transcription at two molecular levels. As a transcription factor, it can bind to the promoters of target genes to stimulate or repress transcriptional activity. As an amplifier of transcription in cancer cells that show MYC gain of function, it enhances the activity of existing transcriptional programs. In both situations, MYC must dimerize with MAX to be effective.

[0062] The human genome contains three MYC genes and corresponding proteins, c-MYC N-MYC and L-MYC. Unless otherwise noted, MYC is used herein to indicate the e-MYC protein. c-Myc can also act as a transcriptional repressor. By binding Miz-1 transcription factor and displacing the p300 co-activator, it inhibits expression of Miz-1 target genes. In addition, Myc has a direct role in the control of DNA replication. Myc is activated upon various mitogenic signals such as Wnt, Shh and EGF (via the MAPK/ERK pathway). By modifying the expression of its target genes, Myc activation results in numerous biological effects. The first to be discovered was its capability to drive cell proliferation (upregulates 3ycling, downregulates p21), but it also plays a very important role in regulating cell growth (upregulates ribosomab RN.A and proteins), apoptosis (downregulates Bel-2), differentiation and stem cell self-renewal. Myc is a very strong proto-oncogene and it is very often found to be upregu!ated in many types of cancers. Myc overexpression stimulates gene amplification, presumably through DNA over-replication. As used herein, c-Myc signaling pathway or c- Myc mediated cellular activity refers to any biochemical effect or cellular response that will occur as a result of activation of Myc.

[0063] “Contacting” is used in accordance with its plain ordinary meaning and refers to the process of allowing at least two distinct species (e.g. chemical compounds including biomolecules or cells) to become sufficiently proximal to react, interact or physically touch.

It should be appreciated; however, the resulting reaction product can be produced directly from a reaction between the added reagents or from an intermediate from one or more of the added reagents that can be produced in the reaction mixture.

[0064] The term “contacting” may include allowing two species to react, interact, or physically touch, wherein the two species may be a compound as described herein and a protein or enzyme. In some embodiments contacting includes allowing a compound described herein to interact with a protein or enzyme that is involved in a signaling pathway.

[0065] The term “associated” or “associated with” in the context of a substance or substance activity or function associated with a disease (e.g. a protein associated disease, a cancer (e.g., cancer, inflammatory disease, autoimmune disease, or infectious disease)) means that the disease (e.g. cancer, inflammatory disease, autoimmune disease, or infectious disease) is caused by (in whole or in part), or a symptom of the disease is caused by (in whole or in part) the substance or substance activity or function. As used herein, what is described as being associated with a disease, if a causative agent, could be a target for treatment of the disease.

[0066] The terms “disease” or “condition” refer to a state of being or health status of a patient or subject capable of being treated with the compounds or methods provided herein. The disease may be a cancer. The disease may be an autoimmune disease. The disease may be an inflammatory disease. The disease may be an infectious disease. In some further instances, “cancer” refers to human cancers and carcinomas, sarcomas, adenocarcinomas, lymphomas, leukemias, etc., including solid and lymphoid cancers, kidney, breast, lung, bladder, colon, ovarian, prostate, pancreas, stomach, brain, head and neck, skin, uterine, testicular, glioma, esophagus, and liver cancer, including hepatocarcinoma, lymphoma, including B-acute lymphoblastic lymphoma, non-Hodgkin’s lymphomas (e.g., Burkitt’s,

Small Cell, and Large Cell lymphomas), Hodgkin’s lymphoma, leukemia (including AML, ALL, and CML), or multiple myeloma.

[0067] 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 known in the chemical arts. [0068] Descriptions of compounds of the present disclosure are limited by principles of chemical bonding known to those skilled in the art. Accordingly, where a group may be substituted by one or more of a number of substituents, such substitutions are selected so as to comply with principles of chemical bonding and to give compounds which are not inherently unstable and/or would be known to one of ordinary skill in the art as likely to be unstable under ambient conditions, such as aqueous, neutral, and several known physiological conditions. For example, a heterocycloalkyl or heteroaryl is attached to the remainder of the molecule via a ring heteroatom in compliance with principles of chemical bonding known to those skilled in the art thereby avoiding inherently unstable compounds.

[0069] Where substituent groups are specified by their conventional chemical formulae, written from left to right, they equally encompass the chemically identical substituents that would result from writing the structure from right to left, e.g., -CH₂O- is equivalent to - OCH₂-.

[0070] The term “alkyl,” by itself or as part of another substituent, means, unless otherwise stated, a straight (i.e., unbranched) or branched carbon chain (or carbon), or combination thereof, which may be fully saturated, mono- or polyunsaturated and can include mono-, di- and multivalent radicals. The alkyl may include a designated number of carbons (e.g., C₁-C₁₀ means one to ten carbons). Alkyl is an uncyclized chain. Examples of saturated hydrocarbon radicals include, but are not limited to, groups such as methyl, ethyl, n-propyl, isopropyl, n- butyl, t-butyl, isobutyl, sec-butyl, methyl, homologs and isomers of, for example, n-pentyl, n- hexyl, n-heptyl, n-octyl, and the like. An unsaturated alkyl group is one having one or more double bonds or triple bonds. Examples of unsaturated alkyl groups include, but are not limited to, vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl), 2,4-pentadienyl, 3-(l,4- pentadienyl), ethynyl, 1- and 3-propynyl, 3-butynyl, and the higher homologs and isomers.

An alkoxy is an alkyl attached to the remainder of the molecule via an oxygen linker (-O-).

An alkyl moiety may be an alkenyl moiety. An alkyl moiety may be an alkynyl moiety. An alkyl moiety may be fully saturated. An alkenyl may include more than one double bond and/or one or more triple bonds in addition to the one or more double bonds. An alkynyl may include more than one triple bond and/or one or more double bonds in addition to the one or more triple bonds.

[0071] The term “alkylene,” by itself or as part of another substituent, means, unless otherwise stated, a divalent radical derived from an alkyl, as exemplified, but not limited by, -CH₂CH₂CH₂CH₂-. Typically, an alkyl (or alkylene) group will have from 1 to 24 carbon atoms, with those groups having 10 or fewer carbon atoms being preferred herein. A “lower alkyl” or “lower alkylene” is a shorter chain alkyl or alkylene group, generally having eight or fewer carbon atoms. The term “alkenylene,” by itself or as part of another substituent, means, unless otherwise stated, a divalent radical derived from an alkene.

[0072] The term “heteroalkyl,” by itself or in combination with another term, means, unless otherwise stated, a stable straight or branched chain, or combinations thereof, including at least one carbon atom and at least one heteroatom (e.g., O, N, P, Si, or S), and wherein the nitrogen and sulfur atoms may optionally be oxidized, and the nitrogen heteroatom may optionally be quatemized. The heteroatom(s) (e.g., O, N, S, Si, or P) may be placed at any interior position of the heteroalkyl group or at the position at which the alkyl group is attached to the remainder of the molecule. Heteroalkyl is an uncyclized chain. Examples include, but are not limited to: -CH₂-CH₂-O-CH₃, -CH₂-CH₂-NH-CH₃, -CH₂-CH₂-N(CH₃)- CH3, -CH₂-S-CH₂-CH₃, -CH₂-S-CH₂, -S(O)-CH₃, -CH2-CH₂-S(O)₂-CH3, -CH=CH-O-CH₃, - Si(CH₃)₃, -CH₂-CH=N-OCH₃, -CH=CH-N(CH₃)-CH₃, -O-CH₃, -O-CH₂-CH₃, and -CN. Up to two or three heteroatoms may be consecutive, such as, for example, -CH₂-NH-OCH₃ and - CH₂-O-Si(CH₃)_3. A heteroalkyl moiety may include one heteroatom (e.g., O, N, S, Si, or P).

A heteroalkyl moiety may include two optionally different heteroatoms (e.g., O, N, S, Si, or P). A heteroalkyl moiety may include three optionally different heteroatoms (e.g., O, N, S,

Si, or P). A heteroalkyl moiety may include four optionally different heteroatoms (e.g., O, N, S, Si, or P). A heteroalkyl moiety may include five optionally different heteroatoms (e.g., O, N, S, Si, or P). A heteroalkyl moiety may include up to 8 optionally different heteroatoms (e.g., O, N, S, Si, or P). The term “heteroalkenyl,” by itself or in combination with another term, means, unless otherwise stated, a heteroalkyl including at least one double bond. A heteroalkenyl may optionally include more than one double bond and/or one or more triple bonds in addition to the one or more double bonds. The term “heteroalkynyl,” by itself or in combination with another term, means, unless otherwise stated, a heteroalkyl including at least one triple bond. A heteroalkynyl may optionally include more than one triple bond and/or one or more double bonds in addition to the one or more triple bonds.

[0073] Similarly, the term “heteroalkylene,” by itself or as part of another substituent, means, unless otherwise stated, a divalent radical derived from heteroalkyl, as exemplified, but not limited by, -CH₂-CH₂-S-CH₂-CH₂- and -CH₂-S-CH₂-CH₂-NH-CH₂-. For heteroalkylene groups, heteroatoms can also occupy either or both of the chain termini (e.g., alkyleneoxy, alkylenedioxy, alkyleneamino, alkylenediamino, and the like). Still further, for alkylene and heteroalkylene linking groups, no orientation of the linking group is implied by the direction in which the formula of the linking group is written. For example, the formula - C(O)₂R'- represents both -C(O)₂R'- and -R'C(O)₂-. As described above, heteroalkyl groups, as used herein, include those groups that are attached to the remainder of the molecule through a heteroatom, such as -C(O)R, -C(O)NR', -NR'R", -OR', -SR, and/or -SO2R'. Where “heteroalkyl” is recited, followed by recitations of specific heteroalkyl groups, such as - NR'R" or the like, it will be understood that the terms heteroalkyl and -NR'R" are not redundant or mutually exclusive. Rather, the specific heteroalkyl groups are recited to add clarity. Thus, the term “heteroalkyl” should not be interpreted herein as excluding specific heteroalkyl groups, such as -NR'R" or the like.

[0074] The terms “cycloalkyl” and “heterocycloalkyl,” by themselves or in combination with other terms, mean, unless otherwise stated, cyclic versions of “alkyl” and “heteroalkyl,” respectively. Cycloalkyl and heterocycloalkyl are not aromatic. Additionally, for heterocycloalkyl, a heteroatom can occupy the position at which the heterocycle is attached to the remainder of the molecule. Examples of cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, 1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, and the like. Examples of heterocycloalkyl include, but are not limited to, 1- (1,2,5,6-tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-morpholinyl, 3- morpholinyl, tetrahydrofuran-2-yl, tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl, 1-piperazinyl, 2-piperazinyl, and the like. A “cycloalkylene” and a “heterocycloalkylene,” alone or as part of another substituent, means a divalent radical derived from a cycloalkyl and heterocycloalkyl, respectively.

[0075] In embodiments, the term “cycloalkyl” means a monocyclic, bicyclic, or a multicyclic cycloalkyl ring system. In embodiments, monocyclic ring systems are cyclic hydrocarbon groups containing from 3 to 8 carbon atoms, where such groups can be saturated or unsaturated, but not aromatic. In embodiments, cycloalkyl groups are fully saturated. Examples of monocyclic cycloalkyls include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, and cyclooctyl. Bicyclic cycloalkyl ring systems are bridged monocyclic rings or fused bicyclic rings. In embodiments, bridged monocyclic rings contain a monocyclic cycloalkyl ring where two non adjacent carbon atoms of the monocyclic ring are linked by an alkylene bridge of between one and three additional carbon atoms (i.e., a bridging group of the form (CH₂)_w, where w is 1, 2, or 3). Representative examples of bicyclic ring systems include, but are not limited to, bicyclo[3.1.1]heptane, bicyclo[2.2.1]heptane, bicyclo[2.2.2]octane, bicyclo[3.2.2]nonane, bicyclo[3.3.1]nonane, and bicyclo[4.2.1]nonane. In embodiments, fused bicyclic cycloalkyl ring systems contain a monocyclic cycloalkyl ring fused to either a phenyl, a monocyclic cycloalkyl, a monocyclic cycloalkenyl, a monocyclic heterocyclyl, or a monocyclic heteroaryl. In embodiments, the bridged or fused bicyclic cycloalkyl is attached to the parent molecular moiety through any carbon atom contained within the monocyclic cycloalkyl ring. In embodiments, cycloalkyl groups are optionally substituted with one or two groups which are independently oxo or thia. In embodiments, the fused bicyclic cycloalkyl is a 5 or 6 membered monocyclic cycloalkyl ring fused to either a phenyl ring, a 5 or 6 membered monocyclic cycloalkyl, a 5 or 6 membered monocyclic cycloalkenyl, a 5 or 6 membered monocyclic heterocyclyl, or a 5 or 6 membered monocyclic heteroaryl, wherein the fused bicyclic cycloalkyl is optionally substituted by one or two groups which are independently oxo or thia. In embodiments, multicyclic cycloalkyl ring systems are a monocyclic cycloalkyl ring (base ring) fused to either (i) one ring system selected from the group consisting of a bicyclic aryl, a bicyclic heteroaryl, a bicyclic cycloalkyl, a bicyclic cycloalkenyl, and a bicyclic heterocyclyl; or (ii) two other ring systems independently selected from the group consisting of a phenyl, a bicyclic aryl, a monocyclic or bicyclic heteroaryl, a monocyclic or bicyclic cycloalkyl, a monocyclic or bicyclic cycloalkenyl, and a monocyclic or bicyclic heterocyclyl. In embodiments, the multicyclic cycloalkyl is attached to the parent molecular moiety through any carbon atom contained within the base ring. In embodiments, multicyclic cycloalkyl ring systems are a monocyclic cycloalkyl ring (base ring) fused to either (i) one ring system selected from the group consisting of a bicyclic aryl, a bicyclic heteroaryl, a bicyclic cycloalkyl, a bicyclic cycloalkenyl, and a bicyclic heterocyclyl; or (ii) two other ring systems independently selected from the group consisting of a phenyl, a monocyclic heteroaryl, a monocyclic cycloalkyl, a monocyclic cycloalkenyl, and a monocyclic heterocyclyl. Examples of multicyclic cycloalkyl groups include, but are not limited to tetradecahydrophenanthrenyl, perhydrophenothiazin-l-yl, and perhydrophenoxazin-l-yl.

[0076] In embodiments, a cycloalkyl is a cycloalkenyl. The term “cycloalkenyl” is used in accordance with its plain ordinary meaning. In embodiments, a cycloalkenyl is a monocyclic, bicyclic, or a multicyclic cycloalkenyl ring system. In embodiments, monocyclic cycloalkenyl ring systems are cyclic hydrocarbon groups containing from 3 to 8 carbon atoms, where such groups are unsaturated (i.e., containing at least one annular carbon carbon double bond), but not aromatic. Examples of monocyclic cycloalkenyl ring systems include cyclopentenyl and cyclohexenyl. In embodiments, bicyclic cycloalkenyl rings are bridged monocyclic rings or a fused bicyclic rings. In embodiments, bridged monocyclic rings contain a monocyclic cycloalkenyl ring where two non adjacent carbon atoms of the monocyclic ring are linked by an alkylene bridge of between one and three additional carbon atoms (i.e., a bridging group of the form (CH₂)_w, where w is 1, 2, or 3). Representative examples of bicyclic cycloalkenyls include, but are not limited to, norbomenyl and bicyclo[2.2.2]oct 2 enyl. In embodiments, fused bicyclic cycloalkenyl ring systems contain a monocyclic cycloalkenyl ring fused to either a phenyl, a monocyclic cycloalkyl, a monocyclic cycloalkenyl, a monocyclic heterocyclyl, or a monocyclic heteroaryl. In embodiments, the bridged or fused bicyclic cycloalkenyl is attached to the parent molecular moiety through any carbon atom contained within the monocyclic cycloalkenyl ring. In embodiments, cycloalkenyl groups are optionally substituted with one or two groups which are independently oxo or thia. In embodiments, multicyclic cycloalkenyl rings contain a monocyclic cycloalkenyl ring (base ring) fused to either (i) one ring system selected from the group consisting of a bicyclic aryl, a bicyclic heteroaryl, a bicyclic cycloalkyl, a bicyclic cycloalkenyl, and a bicyclic heterocyclyl; or (ii) two ring systems independently selected from the group consisting of a phenyl, a bicyclic aryl, a monocyclic or bicyclic heteroaryl, a monocyclic or bicyclic cycloalkyl, a monocyclic or bicyclic cycloalkenyl, and a monocyclic or bicyclic heterocyclyl. In embodiments, the multicyclic cycloalkenyl is attached to the parent molecular moiety through any carbon atom contained within the base ring. In embodiments, multicyclic cycloalkenyl rings contain a monocyclic cycloalkenyl ring (base ring) fused to either (i) one ring system selected from the group consisting of a bicyclic aryl, a bicyclic heteroaryl, a bicyclic cycloalkyl, a bicyclic cycloalkenyl, and a bicyclic heterocyclyl; or (ii) two ring systems independently selected from the group consisting of a phenyl, a monocyclic heteroaryl, a monocyclic cycloalkyl, a monocyclic cycloalkenyl, and a monocyclic heterocyclyl.

[0077] In embodiments, a heterocycloalkyl is a heterocyclyl. The term “heterocyclyl” as used herein, means a monocyclic, bicyclic, or multicyclic heterocycle. The heterocyclyl monocyclic heterocycle is a 3, 4, 5, 6 or 7 membered ring containing at least one heteroatom independently selected from the group consisting of O, N, and S where the ring is saturated or unsaturated, but not aromatic. The 3 or 4 membered ring contains 1 heteroatom selected from the group consisting of O, N and S. The 5 membered ring can contain zero or one double bond and one, two or three heteroatoms selected from the group consisting of O, N and S.

The 6 or 7 membered ring contains zero, one or two double bonds and one, two or three heteroatoms selected from the group consisting of O, N and S. The heterocyclyl monocyclic heterocycle is connected to the parent molecular moiety through any carbon atom or any nitrogen atom contained within the heterocyclyl monocyclic heterocycle. Representative examples of heterocyclyl monocyclic heterocycles include, but are not limited to, azetidinyl, azepanyl, aziridinyl, diazepanyl, 1,3-dioxanyl, 1,3-dioxolanyl, 1,3-dithiolanyl, 1,3-dithianyl, imidazolinyl, imidazolidinyl, isothiazolinyl, isothiazolidinyl, isoxazolinyl, isoxazolidinyl, morpholinyl, oxadiazolinyl, oxadiazolidinyl, oxazolinyl, oxazolidinyl, piperazinyl, piperidinyl, pyranyl, pyrazolinyl, pyrazolidinyl, pyrrolinyl, pyrrolidinyl, tetrahydrofuranyl, tetrahydrothienyl, thiadiazolinyl, thiadiazolidinyl, thiazolinyl, thiazolidinyl, thiomorpholinyl, 1,1-dioxidothiomorpholinyl (thiomorpholine sulfone), thiopyranyl, and trithianyl. The heterocyclyl bicyclic heterocycle is a monocyclic heterocycle fused to either a phenyl, a monocyclic cycloalkyl, a monocyclic cycloalkenyl, a monocyclic heterocycle, or a monocyclic heteroaryl. The heterocyclyl bicyclic heterocycle is connected to the parent molecular moiety through any carbon atom or any nitrogen atom contained within the monocyclic heterocycle portion of the bicyclic ring system. Representative examples of bicyclic heterocyclyls include, but are not limited to, 2,3-dihydrobenzofuran-2-yl, 2,3- dihydrobenzofuran-3-yl, indolin-l-yl, indolin-2-yl, indolin-3-yl, 2,3-dihydrobenzothien-2-yl, decahydroquinolinyl, decahydroisoquinolinyl, octahydro-lH-indolyl, and octahydrobenzofuranyl. In embodiments, heterocyclyl groups are optionally substituted with one or two groups which are independently oxo or thia. In certain embodiments, the bicyclic heterocyclyl is a 5 or 6 membered monocyclic heterocyclyl ring fused to a phenyl ring, a 5 or 6 membered monocyclic cycloalkyl, a 5 or 6 membered monocyclic cycloalkenyl, a 5 or 6 membered monocyclic heterocyclyl, or a 5 or 6 membered monocyclic heteroaryl, wherein the bicyclic heterocyclyl is optionally substituted by one or two groups which are independently oxo or thia. Multicyclic heterocyclyl ring systems are a monocyclic heterocyclyl ring (base ring) fused to either (i) one ring system selected from the group consisting of a bicyclic aryl, a bicyclic heteroaryl, a bicyclic cycloalkyl, a bicyclic cycloalkenyl, and a bicyclic heterocyclyl; or (ii) two other ring systems independently selected from the group consisting of a phenyl, a bicyclic aryl, a monocyclic or bicyclic heteroaryl, a monocyclic or bicyclic cycloalkyl, a monocyclic or bicyclic cycloalkenyl, and a monocyclic or bicyclic heterocyclyl. The multicyclic heterocyclyl is attached to the parent molecular moiety through any carbon atom or nitrogen atom contained within the base ring. In embodiments, multicyclic heterocyclyl ring systems are a monocyclic heterocyclyl ring (base ring) fused to either (i) one ring system selected from the group consisting of a bicyclic aryl, a bicyclic heteroaryl, a bicyclic cycloalkyl, a bicyclic cycloalkenyl, and a bicyclic heterocyclyl; or (ii) two other ring systems independently selected from the group consisting of a phenyl, a monocyclic heteroaryl, a monocyclic cycloalkyl, a monocyclic cycloalkenyl, and a monocyclic heterocyclyl. Examples of multicyclic heterocyclyl groups include, but are not limited to 1 OH-phenothiazin- 10-yl, 9, 10-dihydroacridin-9-yl, 9, 10-dihydroacridin- 10-yl, lOH-phenoxazin-10-yl, 10,1 l-dihydro-5H-dibenzo[b,f]azepin-5-yl, 1, 2,3,4- tetrahydropyrido[4,3-g]isoquinolin-2-yl, 12H-benzo[b]phenoxazin-12-yl, and dodecahydro- lH-carbazol-9-yl.

[0078] The terms “halo” or “halogen,” by themselves or as part of another substituent, mean, unless otherwise stated, a fluorine, chlorine, bromine, or iodine atom. Additionally, terms such as “haloalkyl” are meant to include monohaloalkyl and polyhaloalkyl. For example, the term “halo(C₁-C₄)alkyl” includes, but is not limited to, fluoromethyl, difluoromethyl, trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, and the like.

[0079] The term “acyl” means, unless otherwise stated, -C(O)R where R is a substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl.

[0080] The term “aryl” means, unless otherwise stated, a polyunsaturated, aromatic, hydrocarbon substituent, which can be a single ring or multiple rings (preferably from 1 to 3 rings) that are fused together (i.e., a fused ring aryl) or linked covalently. A fused ring aryl refers to multiple rings fused together wherein at least one of the fused rings is an aryl ring. The term “heteroaryl” refers to aryl groups (or rings) that contain at least one heteroatom such as N, O, or S, wherein the nitrogen and sulfur atoms are optionally oxidized, and the nitrogen atom(s) are optionally quatemized. Thus, the term “heteroaryl” includes fused ring heteroaryl groups (i.e., multiple rings fused together wherein at least one of the fused rings is a heteroaromatic ring). A 5,6-fused ring heteroarylene refers to two rings fused together, wherein one ring has 5 members and the other ring has 6 members, and wherein at least one ring is a heteroaryl ring. Likewise, a 6,6-fused ring heteroarylene refers to two rings fused together, wherein one ring has 6 members and the other ring has 6 members, and wherein at least one ring is a heteroaryl ring. And a 6,5-fused ring heteroarylene refers to two rings fused together, wherein one ring has 6 members and the other ring has 5 members, and wherein at least one ring is a heteroaryl ring. A heteroaryl group can be attached to the remainder of the molecule through a carbon or heteroatom. Non-limiting examples of aryl and heteroaryl groups include phenyl, naphthyl, pyrrolyl, pyrazolyl, pyridazinyl, triazinyl, pyrimidinyl, imidazolyl, pyrazinyl, purinyl, oxazolyl, isoxazolyl, thiazolyl, furyl, thienyl, pyridyl, pyrimidyl, benzothiazolyl, benzoxazoyl benzimidazolyl, benzofuran, isobenzofuranyl, indolyl, isoindolyl, benzothiophenyl, isoquinolyl, quinoxalinyl, quinolyl, 1 -naphthyl, 2- naphthyl, 4-biphenyl, 1 -pyrrolyl, 2-pyrrolyl, 3 -pyrrolyl, 3 -pyrazolyl, 2-imidazolyl, 4- imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl, 2-thienyl, 3- thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl, 4-pyrimidyl, 5 -benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl, 1 -isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl, 3- quinolyl, and 6-quinolyl. Substituents for each of the above noted aryl and heteroaryl ring systems are selected from the group of acceptable substituents described below. An “arylene” and a “heteroarylene,” alone or as part of another substituent, mean a divalent radical derived from an aryl and heteroaryl, respectively. A heteroaryl group substituent may be -O- bonded to a ring heteroatom nitrogen.

[0081] A fused ring heterocyloalkyl-aryl is an aryl fused to a heterocycloalkyl. A fused ring heterocycloalkyl-heteroaryl is a heteroaryl fused to a heterocycloalkyl. A fused ring heterocycloalkyl-cycloalkyl is a heterocycloalkyl fused to a cycloalkyl. A fused ring heterocycloalkyl-heterocycloalkyl is a heterocycloalkyl fused to another heterocycloalkyl. Fused ring heterocycloalkyl-aryl, fused ring heterocycloalkyl-heteroaryl, fused ring heterocycloalkyl-cycloalkyl, or fused ring heterocycloalkyl-heterocycloalkyl may each independently be unsubstituted or substituted with one or more of the substitutents described herein.

[0082] Spirocyclic rings are two or more rings wherein adjacent rings are attached through a single atom. The individual rings within spirocyclic rings may be identical or different. Individual rings in spirocyclic rings may be substituted or unsubstituted and may have different substituents from other individual rings within a set of spirocyclic rings. Possible substituents for individual rings within spirocyclic rings are the possible substituents for the same ring when not part of spirocyclic rings (e.g. substituents for cycloalkyl or heterocycloalkyl rings). Spirocylic rings may be substituted or unsubstituted cycloalkyl, substituted or unsubstituted cycloalkylene, substituted or unsubstituted heterocycloalkyl or substituted or unsubstituted heterocycloalkylene and individual rings within a spirocyclic ring group may be any of the immediately previous list, including having all rings of one type (e.g. all rings being substituted heterocycloalkylene wherein each ring may be the same or different substituted heterocycloalkylene). When referring to a spirocyclic ring system, heterocyclic spirocyclic rings means a spirocyclic rings wherein at least one ring is a heterocyclic ring and wherein each ring may be a different ring. When referring to a spirocyclic ring system, substituted spirocyclic rings means that at least one ring is substituted and each substituent may optionally be different.

[0083] The symbol - ” (a wavy line) denotes the point of attachment of a chemical moiety to the remainder of a molecule or chemical formula.

[0084] The term “oxo,” as used herein, means an oxygen that is double bonded to a carbon atom.

[0085] The term “alkylsulfonyl,” as used herein, means a moiety having the formula -S(O₂)-R', where R' is a substituted or unsubstituted alkyl group as defined above. R' may have a specified number of carbons (e.g., “ C₁-C₄ alkylsulfonyl”).

[0086] The term “alkylarylene” as an arylene moiety covalently bonded to an alkylene moiety (also referred to herein as an alkylene linker). In embodiments, the alkylarylene group has the formula:

[0087] An alkylarylene moiety may be substituted (e.g. with a substituent group) on the alkylene moiety or the arylene linker (e.g. at carbons 2, 3, 4, or 6) with halogen, oxo, -N3, - CF₃, -CCI₃, -CBr₃, -CI₃, -CN, -CHO, -OH, -NH₂, -COOH, -CONH₂, -NO2, -SH, -SO₂CH₃ - SO₃H, , -OSO₃H, -SO2NH₂, -NHNH₂, -ONH₂, -NHC(O)NHNH₂, substituted or unsubstituted C1-C5 alkyl or substituted or unsubstituted 2 to 5 membered heteroalkyl). In embodiments, the alkylarylene is unsubstituted.

[0088] Each of the above terms (e.g., “alkyl,” “heteroalkyl,” “cycloalkyl,” “heterocycloalkyl,” “aryl,” and “heteroaryl”) includes both substituted and unsubstituted forms of the indicated radical. Preferred substituents for each type of radical are provided below.

[0089] Substituents for the alkyl and heteroalkyl radicals (including those groups often referred to as alkylene, alkenyl, heteroalkylene, heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, and heterocycloalkenyl) can be one or more of a variety of groups selected from, but not limited to, -OR', =O, =NR', =N-OR, -NR'R", -SR', -halogen, - SiR'R"R", -OC(O)R', -C(O)R, -CO₂R, -CONR'R", -OC(O)NR'R", -NR"C(O)R', -NR- C(O)NR"R", -NR"C(O)₂R, -NR-C(NR'R"R"')=NR"", -NR-C(NR'R")=NR"', -S(O)R', - S(O)₂R, -S(O)₂NR'R", -NRSO₂R, -NR'NR'R'", -ONR'R", -NR'C(O)NR"NR"'R"", -CN, - NO₂, -NR'SO₂R", -NR'C(O)R", -NR'C(O)-OR", -NR'OR", in a number ranging from zero to (2m'+l), where rri is the total number of carbon atoms in such radical. R, R, R", R", and R"" each preferably independently refer to hydrogen, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl (e.g., aryl substituted with 1-3 halogens), substituted or unsubstituted heteroaryl, substituted or unsubstituted alkyl, alkoxy, or thioalkoxy groups, or arylalkyl groups. When a compound described herein includes more than one R group, for example, each of the R groups is independently selected as are each R, R", R", and R"" group when more than one of these groups is present. When R and R" are attached to the same nitrogen atom, they can be combined with the nitrogen atom to form a 4-, 5-, 6-, or 7- membered ring. For example, -NR'R" includes, but is not limited to, 1-pyrrolidinyl and 4- morpholinyl. From the above discussion of substituents, one of skill in the art will understand that the term “alkyl” is meant to include groups including carbon atoms bound to groups other than hydrogen groups, such as haloalkyl (e.g., -CF3 and -CH₂CF₃) and acyl (e.g., - C(O)CH₃, -C(O)CF₃, -C(O)CH₂0CH₃, and the like).

[0090] Similar to the substituents described for the alkyl radical, substituents for the aryl and heteroaryl groups are varied and are selected from, for example: -OR', -NR'R", -SR, - halogen, -SiR'R'R", -OC(O)R, -C(O)R, -CO₂R, -CONR'R", -OC(O)NR'R", -NR"C(O)R, - NR'-C(O)NR"R", -NR"C(O)₂R, -NR-C(NR'R"R"')=NR"", -NR-C(NRR")=NR", -S(O)R, - S(O)₂R, -S(O)₂NR'R", -NRSO_ZR, -NR'NR'R", -ONR'R", -NR'C(O)NR"NR'"R"", -CN, - NO₂, -R, -N₃, -CH(Ph)₂, fluoro( C₁-C₄)alkoxy, and fluoro(C₁-C₄)alkyl, -NRSO₂R", - NRC(O)R", -NR'C(O)-OR", -NR'OR", in a number ranging from zero to the total number of open valences on the aromatic ring system; and where R, R", R", and R"" are preferably independently selected from hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl. When a compound described herein includes more than one R group, for example, each of the R groups is independently selected as are each R, R", R", and R"" groups when more than one of these groups is present.

[0091] Substituents for rings (e.g. cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkylene, heterocycloalkylene, arylene, or heteroaryl ene) may be depicted as substituents on the ring rather than on a specific atom of a ring (commonly referred to as a floating substituent). In such a case, the substituent may be attached to any of the ring atoms (obeying the rules of chemical valency) and in the case of fused rings or spirocyclic rings, a substituent depicted as associated with one member of the fused rings or spirocyclic rings (a floating substituent on a single ring), may be a substituent on any of the fused rings or spirocyclic rings (a floating substituent on multiple rings). When a substituent is attached to a ring, but not a specific atom (a floating substituent), and a subscript for the substituent is an integer greater than one, the multiple substituents may be on the same atom, same ring, different atoms, different fused rings, different spirocyclic rings, and each substituent may optionally be different. Where a point of attachment of a ring to the remainder of a molecule is not limited to a single atom (a floating substituent), the attachment point may be any atom of the ring and in the case of a fused ring or spirocyclic ring, any atom of any of the fused rings or spirocyclic rings while obeying the rules of chemical valency. Where a ring, fused rings, or spirocyclic rings contain one or more ring heteroatoms and the ring, fused rings, or spirocyclic rings are shown with one more floating substituents (including, but not limited to, points of attachment to the remainder of the molecule), the floating substituents may be bonded to the heteroatoms. Where the ring heteroatoms are shown bound to one or more hydrogens (e.g. a ring nitrogen with two bonds to ring atoms and a third bond to a hydrogen) in the structure or formula with the floating substituent, when the heteroatom is bonded to the floating substituent, the substituent will be understood to replace the hydrogen, while obeying the rules of chemical valency.

[0092] Two or more substituents may optionally be joined to form aryl, heteroaryl, cycloalkyl, or heterocycloalkyl groups. Such so-called ring-forming substituents are typically, though not necessarily, found attached to a cyclic base structure. In one embodiment, the ring-forming substituents are attached to adjacent members of the base structure. For example, two ring-forming substituents attached to adjacent members of a cyclic base structure create a fused ring structure. In another embodiment, the ring-forming substituents are attached to a single member of the base structure. For example, two ring-forming substituents attached to a single member of a cyclic base structure create a spirocyclic structure. In yet another embodiment, the ring-forming substituents are attached to non- adjacent members of the base structure.

[0093] Two of the substituents on adjacent atoms of the aryl or heteroaryl ring may optionally form a ring of the formula -T-C(O)-(CRR')_p-U-, wherein T and U are independently -NR-, -O-, -CRR'-, or a single bond, and p is an integer of from 0 to 3. Alternatively, two of the substituents on adjacent atoms of the aryl or heteroaryl ring may optionally be replaced with a substituent of the formula -A-(CH₂)_r-B-, wherein A and B are independently -CRR'-, -O-, -NR.-, -S-, -S(O) -, -S(O)₂-, -S(O)₂NR'-, or a single bond, and r is an integer of from 1 to 4. One of the single bonds of the new ring so formed may optionally be replaced with a double bond. Alternatively, two of the substituents on adjacent atoms of the aryl or heteroaryl ring may optionally be replaced with a substituent of the formula - (CRR')_s-X'- (C"R"R"')_d-, where s and d are independently integers of from 0 to 3, and X' is - O-, -NR'-, -S-, -S(O)-, -S(O)₂-, or -S(O)₂NR-. The substituents R, R, R", and R" are preferably independently selected from hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl.

[0094] As used herein, the terms “heteroatom” or “ring heteroatom” are meant to include oxygen (O), nitrogen (N), sulfur (S), phosphorus (P), and silicon (Si).

[0095] A “substituent group,” as used herein, means a group selected from the following moieties:

(A) oxo, halogen, -CCI3, -CBr₃, -CF₃, -CI3, -CH₂CI, -CH₂Br, -CH₂F, -CH₂I, -CHCh, -CHBr₂, -CHF₂, -CHI₂, -CN, -OH, -NH₂, -COOH, -CONH₂, -NO₂, -SH, -SO₃H,

-SO4H, -SO₂NH₂, -NHNH₂, -ONH₂, -NHC(O)NHNH₂, -NHC(O)NH₂, -NHSO₂H, -NHC(O)H, -NHC(O)0H, -NHOH, -OCCI3, -OCF3, -OCBr₃, -OCI₃,-OCHCl₂,

-OCHBr₂, -OCHI₂, -0CHF₂, -N3, unsubstituted alkyl (e.g., C₁-C₈ alkyl, C1-C6 alkyl, or C₁-C₄ alkyl), unsubstituted heteroalkyl (e.g., 2 to 8 membered heteroalkyl, 2 to 6 membered heteroalkyl, or 2 to 4 membered heteroalkyl), unsubstituted cycloalkyl (e.g., C₃-C₈ cycloalkyl, C3-C6 cycloalkyl, or C5-C6 cycloalkyl), unsubstituted heterocycloalkyl (e.g., 3 to 8 membered heterocycloalkyl, 3 to 6 membered heterocycloalkyl, or 5 to 6 membered heterocycloalkyl), unsubstituted aryl (e.g., C6-C10 aryl, C10 aryl, or phenyl), or unsubstituted heteroaryl (e.g., 5 to 10 membered heteroaryl, 5 to 9 membered heteroaryl, or 5 to 6 membered heteroaryl), and

(B) alkyl (e.g., C₁-C₈ alkyl, C1-C6 alkyl, or C₁-C₄ alkyl), heteroalkyl (e.g., 2 to 8 membered heteroalkyl, 2 to 6 membered heteroalkyl, or 2 to 4 membered heteroalkyl), cycloalkyl (e.g., C₃-C₈ cycloalkyl, C3-C6 cycloalkyl, or C5-C6 cycloalkyl), heterocycloalkyl (e.g., 3 to 8 membered heterocycloalkyl, 3 to 6 membered heterocycloalkyl, or 5 to 6 membered heterocycloalkyl), aryl (e.g., C6-C10 aryl, C10 aryl, or phenyl), heteroaryl (e.g., 5 to 10 membered heteroaryl, 5 to 9 membered heteroaryl, or 5 to 6 membered heteroaryl), substituted with at least one substituent selected from:

(i) oxo, halogen, -CCI3, -CBr₃, -CF₃, -CI3, -CH₂CI, -CH₂Br, -CH₂F, -CH₂I, -CHCb, -CHBr₂, -CHF₂, -CHI₂, -CN, -OH, -NH₂, -COOH, -CONH₂, -NO₂, -SH, -SO3H,

-SO4H, -SO₂NH₂, -NHNH₂, -ONH₂, -NHC(O)NHNH₂, -NHC(O)NH₂, -NHSO₂H, -NHC(O)H, -NHC(O)0H, -NHOH, -OCCI3, -OCF3, -OCBr₃, -OCI₃,-OCHCl₂,

-OCHBr₂, -OCHI₂, -0CHF₂, -N3, unsubstituted alkyl (e.g., C₁-C₈ alkyl, C1-C6 alkyl, or C₁-C₄ alkyl), unsubstituted heteroalkyl (e.g., 2 to 8 membered heteroalkyl, 2 to 6 membered heteroalkyl, or 2 to 4 membered heteroalkyl), unsubstituted cycloalkyl (e.g., C₃-C₈ cycloalkyl, C3-C6 cycloalkyl, or C5-C6 cycloalkyl), unsubstituted heterocycloalkyl (e.g., 3 to 8 membered heterocycloalkyl, 3 to 6 membered heterocycloalkyl, or 5 to 6 membered heterocycloalkyl), unsubstituted aryl (e.g., C6-C10 aryl, C10 aryl, or phenyl), or unsubstituted heteroaryl (e.g., 5 to 10 membered heteroaryl, 5 to 9 membered heteroaryl, or 5 to 6 membered heteroaryl), and

(ii) alkyl (e.g., C₁-C₈ alkyl, C1-C6 alkyl, or C₁-C₄ alkyl), heteroalkyl (e.g., 2 to 8 membered heteroalkyl, 2 to 6 membered heteroalkyl, or 2 to 4 membered heteroalkyl), cycloalkyl (e.g., C₃-C₈ cycloalkyl, C3-C6 cycloalkyl, or C5-C6 cycloalkyl), heterocycloalkyl (e.g., 3 to 8 membered heterocycloalkyl, 3 to 6 membered heterocycloalkyl, or 5 to 6 membered heterocycloalkyl), aryl (e.g., C6-C10 aryl, C10 aryl, or phenyl), heteroaryl (e.g., 5 to 10 membered heteroaryl, 5 to 9 membered heteroaryl, or 5 to 6 membered heteroaryl), substituted with at least one substituent selected from: (a) oxo, halogen, -CCb, -CBr₃, -CF₃, -Cb, -CH₂C1, -CH₂Br, -CH₂F, -CH₂I,

-CHCb, -CHBr₂, -CHF₂, -CHI₂, -CN, -OH, -NH₂, -COOH, -CONH₂, -NO₂, -SH,

-S0₃H, -SO4H, -S0₂NH₂, -NHNH₂, -ONH₂, -NHC(O)NHNH₂, -NHC(O)NH₂,

-NHS0₂H, -NHC(O)H, -NHC(O)0H, -NHOH, -0CC1₃, -OCF₃, -OCBr₃, -OCI₃,

-OCHCb, -OCHBr₂, -OCHI₂, -OCHF₂, -N₃, unsubstituted alkyl (e.g., C₁-C₈ alkyl, C₁-C6 alkyl, or C₁-C₄ alkyl), unsubstituted heteroalkyl (e.g., 2 to 8 membered heteroalkyl, 2 to 6 membered heteroalkyl, or 2 to 4 membered heteroalkyl), unsubstituted cycloalkyl (e.g., C₃-C₈ cycloalkyl, C₃-C₆ cycloalkyl, or C5-C6 cycloalkyl), unsubstituted heterocycloalkyl (e.g., 3 to

8 membered heterocycloalkyl, 3 to 6 membered heterocycloalkyl, or 5 to 6 membered heterocycloalkyl), unsubstituted aryl (e.g., C6-C10 aryl, C10 aryl, or phenyl), or unsubstituted heteroaryl (e.g., 5 to 10 membered heteroaryl, 5 to 9 membered heteroaryl, or 5 to 6 membered heteroaryl), and

(b) alkyl (e.g., C₁-C₈ alkyl, C₁-C₆ alkyl, or C₁-C₄ alkyl), heteroalkyl (e.g., 2 to 8 membered heteroalkyl, 2 to 6 membered heteroalkyl, or 2 to 4 membered heteroalkyl), cycloalkyl (e.g., C₃-C₈ cycloalkyl, C₃-C₆ cycloalkyl, or C₅-C₆ cycloalkyl), heterocycloalkyl (e.g., 3 to 8 membered heterocycloalkyl, 3 to 6 membered heterocycloalkyl, or 5 to 6 membered heterocycloalkyl), aryl (e.g., C6-C10 aryl, C10 aryl, or phenyl), heteroaryl (e.g., 5 to 10 membered heteroaryl, 5 to 9 membered heteroaryl, or 5 to 6 membered heteroaryl), substituted with at least one substituent selected from: oxo, halogen, -CCb, -CBr₃, -CF₃, -Cb, -CH₂C1, -CH₂Br,

-CH₂F, -CH₂I, -CHCl₂, -CHBr₂, -CHF₂, -CHI₂, -CN, -OH, -NH₂, -COOH, -CONH₂, -NO₂, -S H, -SO₃H, -SO4H, -SO₂NH₂, -NHNH₂, -ONH₂, -NHC(O)NHNH₂,

-NHC(O)NH₂, -NHSO₂H, -NHC(O)H, -NHC(O)0H, -NHOH, -OCCb, -OCF₃,

-OCBr₃, -OCb, -OCHCb, -OCHBr₂, -OCHb, -OCHF₂, -N₃, unsubstituted alkyl (e.g., C₁-C₈ alkyl, C₁-C₆ alkyl, or C₁-C₄ alkyl), unsubstituted heteroalkyl (e.g., 2 to 8 membered heteroalkyl, 2 to 6 membered heteroalkyl, or 2 to 4 membered heteroalkyl), unsubstituted cycloalkyl (e.g., C₃-C₈ cycloalkyl, C₃-C₆ cycloalkyl, or C5-C6 cycloalkyl), unsubstituted heterocycloalkyl (e.g., 3 to 8 membered heterocycloalkyl, 3 to 6 membered heterocycloalkyl, or 5 to 6 membered heterocycloalkyl), unsubstituted aryl (e.g., C₆-C₁₀ aryl, C₁₀ aryl, or phenyl), or unsubstituted heteroaryl (e.g., 5 to 10 membered heteroaryl, 5 to 9 membered heteroaryl, or 5 to 6 membered heteroaryl).

[0096] A “size-limited substituent” or “ size-limited substituent group,” as used herein, means a group selected from all of the substituents described above for a “substituent group,” wherein each substituted or unsubstituted alkyl is a substituted or unsubstituted C₁-C₂₀ alkyl, each substituted or unsubstituted heteroalkyl is a substituted or unsubstituted 2 to 20 membered heteroalkyl, each substituted or unsubstituted cycloalkyl is a substituted or unsubstituted C₃-C₈ cycloalkyl, each substituted or unsubstituted heterocycloalkyl is a substituted or unsubstituted 3 to 8 membered heterocycloalkyl, each substituted or unsubstituted aryl is a substituted or unsubstituted C₆-C₁₀ aryl, and each substituted or unsubstituted heteroaryl is a substituted or unsubstituted 5 to 10 membered heteroaryl.

[0097] A “lower substituent” or “ lower substituent group,” as used herein, means a group selected from all of the substituents described above for a “substituent group,” wherein each substituted or unsubstituted alkyl is a substituted or unsubstituted C₁-C₈ alkyl, each substituted or unsubstituted heteroalkyl is a substituted or unsubstituted 2 to 8 membered heteroalkyl, each substituted or unsubstituted cycloalkyl is a substituted or unsubstituted C₃- C₇ cycloalkyl, each substituted or unsubstituted heterocycloalkyl is a substituted or unsubstituted 3 to 7 membered heterocycloalkyl, each substituted or unsubstituted aryl is a substituted or unsubstituted phenyl, and each substituted or unsubstituted heteroaryl is a substituted or unsubstituted 5 to 6 membered heteroaryl.

[0098] In some embodiments, each substituted group described in the compounds herein is substituted with at least one substituent group. More specifically, in some embodiments, each substituted alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl, substituted aryl, substituted heteroaryl, substituted alkylene, substituted heteroalkylene, substituted cycloalkylene, substituted heterocycloalkylene, substituted arylene, and/or substituted heteroarylene described in the compounds herein are substituted with at least one substituent group. In other embodiments, at least one or all of these groups are substituted with at least one size-limited substituent group. In other embodiments, at least one or all of these groups are substituted with at least one lower substituent group.

[0099] In other embodiments of the compounds herein, each substituted or unsubstituted alkyl may be a substituted or unsubstituted C₁-C₂₀ alkyl, each substituted or unsubstituted heteroalkyl is a substituted or unsubstituted 2 to 20 membered heteroalkyl, each substituted or unsubstituted cycloalkyl is a substituted or unsubstituted C₃-C₈ cycloalkyl, each substituted or unsubstituted heterocycloalkyl is a substituted or unsubstituted 3 to 8 membered heterocycloalkyl, each substituted or unsubstituted aryl is a substituted or unsubstituted C₆- C₁₀ aryl, and/or each substituted or unsubstituted heteroaryl is a substituted or unsubstituted 5 to 10 membered heteroaryl. In some embodiments of the compounds herein, each substituted or unsubstituted alkylene is a substituted or unsubstituted C₁-C₂₀ alkylene, each substituted or unsubstituted heteroalkylene is a substituted or unsubstituted 2 to 20 membered heteroalkylene, each substituted or unsubstituted cycloalkylene is a substituted or unsubstituted C₃-C₈ cycloalkylene, each substituted or unsubstituted heterocycloalkylene is a substituted or unsubstituted 3 to 8 membered heterocycloalkylene, each substituted or unsubstituted arylene is a substituted or unsubstituted C₆-C₁₀ arylene, and/or each substituted or unsubstituted heteroarylene is a substituted or unsubstituted 5 to 10 membered heteroaryl ene.

[00100] In some embodiments, each substituted or unsubstituted alkyl is a substituted or unsubstituted C₁-C₈ alkyl, each substituted or unsubstituted heteroalkyl is a substituted or unsubstituted 2 to 8 membered heteroalkyl, each substituted or unsubstituted cycloalkyl is a substituted or unsubstituted C₃-C₇ cycloalkyl, each substituted or unsubstituted heterocycloalkyl is a substituted or unsubstituted 3 to 7 membered heterocycloalkyl, each substituted or unsubstituted aryl is a substituted or unsubstituted C₆-C₁₀ aryl, and/or each substituted or unsubstituted heteroaryl is a substituted or unsubstituted 5 to 9 membered heteroaryl. In some embodiments, each substituted or unsubstituted alkylene is a substituted or unsubstituted C₁-C₈ alkylene, each substituted or unsubstituted heteroalkylene is a substituted or unsubstituted 2 to 8 membered heteroalkylene, each substituted or unsubstituted cycloalkylene is a substituted or unsubstituted C₃-C₇ cycloalkylene, each substituted or unsubstituted heterocycloalkylene is a substituted or unsubstituted 3 to 7 membered heterocycloalkylene, each substituted or unsubstituted arylene is a substituted or unsubstituted C₆-C₁₀ arylene, and/or each substituted or unsubstituted heteroarylene is a substituted or unsubstituted 5 to 9 membered heteroarylene. In some embodiments, the compound is a chemical species set forth in the Examples section, figures, or tables below.

[00101] In embodiments, a substituted or unsubstituted moiety (e.g., substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted alkylene, substituted or unsubstituted heteroalkylene, substituted or unsubstituted cycloalkylene, substituted or unsubstituted heterocycloalkylene, substituted or unsubstituted arylene, and/or substituted or unsubstituted heteroarylene) is unsubstituted (e.g., is an unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl, unsubstituted heteroaryl, unsubstituted alkylene, unsubstituted heteroalkylene, unsubstituted cycloalkylene, unsubstituted heterocycloalkylene, unsubstituted arylene, and/or unsubstituted heteroaryl ene, respectively). In embodiments, a substituted or unsubstituted moiety (e.g., substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted alkylene, substituted or unsubstituted heteroalkylene, substituted or unsubstituted cycloalkylene, substituted or unsubstituted heterocycloalkylene, substituted or unsubstituted arylene, and/or substituted or unsubstituted heteroarylene) is substituted (e.g., is a substituted alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl, substituted aryl, substituted heteroaryl, substituted alkylene, substituted heteroalkylene, substituted cycloalkylene, substituted heterocycloalkylene, substituted arylene, and/or substituted heteroarylene, respectively).

[00102] In embodiments, a substituted moiety (e.g., substituted alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl, substituted aryl, substituted heteroaryl, substituted alkylene, substituted heteroalkylene, substituted cycloalkylene, substituted heterocycloalkylene, substituted arylene, and/or substituted heteroarylene) is substituted with at least one substituent group, wherein if the substituted moiety is substituted with a plurality of substituent groups, each substituent group may optionally be different. In embodiments, if the substituted moiety is substituted with a plurality of substituent groups, each substituent group is different.

[00103] In embodiments, a substituted moiety (e.g., substituted alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl, substituted aryl, substituted heteroaryl, substituted alkylene, substituted heteroalkylene, substituted cycloalkylene, substituted heterocycloalkylene, substituted arylene, and/or substituted heteroarylene) is substituted with at least one size-limited substituent group, wherein if the substituted moiety is substituted with a plurality of size-limited substituent groups, each size-limited substituent group may optionally be different. In embodiments, if the substituted moiety is substituted with a plurality of size-limited substituent groups, each size-limited substituent group is different.

[00104] In embodiments, a substituted moiety (e.g., substituted alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl, substituted aryl, substituted heteroaryl, substituted alkylene, substituted heteroalkylene, substituted cycloalkylene, substituted heterocycloalkylene, substituted arylene, and/or substituted heteroarylene) is substituted with at least one lower substituent group, wherein if the substituted moiety is substituted with a plurality of lower substituent groups, each lower substituent group may optionally be different. In embodiments, if the substituted moiety is substituted with a plurality of lower substituent groups, each lower substituent group is different.

[00105] In embodiments, a substituted moiety (e.g., substituted alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl, substituted aryl, substituted heteroaryl, substituted alkylene, substituted heteroalkylene, substituted cycloalkylene, substituted heterocycloalkylene, substituted arylene, and/or substituted heteroarylene) is substituted with at least one substituent group, size-limited substituent group, or lower substituent group; wherein if the substituted moiety is substituted with a plurality of groups selected from substituent groups, size-limited substituent groups, and lower substituent groups; each substituent group, size-limited substituent group, and/or lower substituent group may optionally be different. In embodiments, if the substituted moiety is substituted with a plurality of groups selected from substituent groups, size-limited substituent groups, and lower substituent groups; each substituent group, size-limited substituent group, and/or lower substituent group is different.

[00106] Certain compounds of the present disclosure possess asymmetric carbon atoms (optical or chiral centers) or double bonds; the enantiomers, racemates, diastereomers, tautomers, geometric isomers, stereoisometric forms that may be defined, in terms of absolute stereochemistry, as (R)-or (S)- or, as (D)- or (L)- for amino acids, and individual isomers are encompassed within the scope of the present disclosure. The compounds of the present disclosure do not include those that are known in art to be too unstable to synthesize and/or isolate. The present disclosure is meant to include compounds in racemic and optically pure forms. Optically active (R)- and (S)-, or (D)- and (L)-isomers may be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques. When the compounds described herein contain olefmic bonds or other centers of geometric asymmetry, and unless specified otherwise, it is intended that the compounds include both E and Z geometric isomers. [00107] As used herein, the term “isomers” refers to compounds having the same number and kind of atoms, and hence the same molecular weight, but differing in respect to the structural arrangement or configuration of the atoms.

[00108] The term “tautomer,” as used herein, refers to one of two or more structural isomers which exist in equilibrium and which are readily converted from one isomeric form to another.

[00109] 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.

[00110] Unless otherwise stated, structures depicted herein are also meant to include all stereochemical forms of the structure; i.e., the R and S configurations for each asymmetric center. Therefore, single stereochemical isomers as well as enantiomeric and diastereomeric mixtures of the present compounds are within the scope of the disclosure.

[00111] It should be noted that throughout the application that alternatives are written in Markush groups, for example, each amino acid position that contains more than one possible amino acid. It is specifically contemplated that each member of the Markush group should be considered separately, thereby comprising another embodiment, and the Markush group is not to be read as a single unit.

[0005] “Analog,” or “analogue” is used in accordance with its plain ordinary meaning within Chemistry and Biology and refers to a chemical compound that is structurally similar to another compound (i.e., a so-called “reference” compound) but differs in composition, e.g., in the replacement of one atom by an atom of a different element, or in the presence of a particular functional group, or the replacement of one functional group by another functional group, or the absolute stereochemistry of one or more chiral centers of the reference compound. Accordingly, an analog is a compound that is similar or comparable in function and appearance but not in structure or origin to a reference compound.

[0006] As used herein, common organic abbreviations are defined as follows:

Ac Acetyl

ACN Acetonitrile

ADME Absorption, distribution, metabolism, and excretion aq. Aqueous BOC or Boc tert-Butoxy carbonyl

BSA Bovine serum Albumin

°C Temperature in degrees Centigrade

DCM dichloromethane

DIEA Diisopropylethylamine

DIPEA N,N -Dii sopropy 1 ethyl amine

DMF A^f,A^f'-Di methyl form amide

DMSO Dimethyl sulfoxide

EDC 1 -Ethyl-3 -(3 -dimethylaminopropyl)carbodiimide

EDTA Ethylenediaminetetraacetic acid

Et Ethyl

EtOAc Ethyl acetate

Eq Equivalents

Fmoc 9-Fluorenylmethoxy carbonyl g Gram(s) hr Hour (hours)

HEPES 2-[4-(2-hydroxyethyl)piperazin-l-yl]ethanesulfonic acid

HPLC High-performance liquid chromatography

HPLC/MS High-performance liquid chromatography-mass spectrometry

LC/MS Liquid chromatography-mass spectrometry

Me Methyl mg milligrams

MeOH Methanol mL Milliliter(s) mL / uL Microliter(s) mol moles mmol millimoles mmol/umol micromoles

MS mass spectrometry

NHS N-Hydroxysuccinimide

PBS Phosphate-buffered saline

Phe Phenylalanine

Pip piperidine

RP-HPLC Reverse phase HPLC

SPR Surface plasmon resonance rt Room temperature t-Bu tert-Butyl

TCEP Tris(2-carboxyethyl)phosphine TFA Trifluoracetic acid

THF Tetrahydrofuran

I. Overview

[00113] c-MYC drives a variety of pro-growth and anti-apoptotic genes and is frequently overexpressed in cancer. Accordingly, inhibition of oncogenic transformation caused by c- MYC would represent a potent chemotherapeutic strategy with wide ranging utility in treating both solid tumors and leukemia. A simple and cost effective high-throughput screening assay for identifying MYC inhibitor scaffolds using a fluorescent small molecule probe is described herein. The fluorescent probe is derived from the inhibitor chemical scaffold, KJ-Pyr-9, that has been shown to: 1. specifically bind MYC and MYC-MAX with excellent affinity; 2. inhibit MYC-driven oncogenic transformation and MYC-dependent transcriptional regulation; 3. interfere with tumor growth in a MYC-driven model; 4. be specific for MYC relative to the other oncogenic transformation factors Src, Jun, and PI3K H1047R; 5. revert the transcriptional signature of MYC to that of a tetracycline-induced knockdown in P493-6 cells (Hart et al., Proc. Natl. Acad. Sci., 2014, 111, 12556-12561). [00114] While an effective inhibitor, KJ-Pyr-9 lacks the drug-like qualities (ADME/Tox) required for clinical application and the scaffold is not amenable to extensive derivatization without compromising affinity and specificity (Jacob et al., 2018, Bioorg. Med. Chem., 26, 4234-4239).

II. Compositions

[00115] In one aspect, provided herein is a compound of formula I:

Formula I or a pharmaceutically acceptable salt thereof, wherein: each R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³, and R¹⁴ is independently hydrogen, halogen, haloalkyl, -OH, C1-C5 alkoxy, -NO2, -CN, thiazolyl, or C1-C5 alkyl;

L¹ is bond, -S(O)₂-, -N(H)-, -O-, -S-, -C(O)-, -C(O)N(H)-, -N(H)C(O)-, -NHC(O)NH-, -C(O)O-, -OC(O)-, substituted or unsubstituted alkylene, substituted or unsubstituted heteroalkylene, an amino acid, or a combination thereof; and Q is a fluorophore.

[00116] In embodiments, each R¹, R², R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³, and R¹⁴ is independently hydrogen, halogen, C1-C5 alkyl, or -OH.

[00117] In embodiments, each R¹, R², R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³, and R¹⁴ is independently halogen. In embodiments, each R¹, R², R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³, and R¹⁴ is independently C1-C5 alkyl. In embodiments, each R¹, R², R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³, and R¹⁴ is independently -OH.

[00118] In embodiments, each R¹, R², R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³, and R¹⁴ is independently methyl. In embodiments, each R¹, R², R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³, and R¹⁴ is independently ethyl. In embodiments, each R¹, R², R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³, and R¹⁴ is independently propyl.

[00119] In embodiments, each R¹, R², R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³, and R¹⁴ is independently hydrogen.

[00120] In embodiments, R³ is hydrogen, C1-C5 alkoxy, halogen, thiazolyl, -NO2, or -CN. In embodiments, R³ is -NO2, thiazolyl, or -CN.

[00121] In embodiments, R³ is hydrogen. In embodiments, R³ is halogen. In embodiments, R³ is -NO2_. In embodiments, R³ is C1-C5 alkoxy. In embodiments, R³ is -OMe. In embodiments, R³ is thiazolyl.

[00122] In embodiments, R³ is -CN.

[00123] In embodiments, L¹ is -C(O)-, unsubstituted alkylene or unsubstituted heteroalkylene, or a combination thereof. In embodiments, L¹ is -NHCH₂CH₂NH- or - NHCH₂CH₂NHC(O)-. In embodiments, L¹ is -NHCH₂CH₂NHC(O)-. [00124] In embodiments, Q is a coumarin derivative.

[00125] In embodiments, Q is:

[00126] In embodiments, the compound provided herein is:

1 or a pharmaceutically acceptable salt thereof.

III. Screening Methods

[00127] In an aspect, provided herein is a method of identifying a test compound as a MYC inhibitor, the method comprising: (a) incubating a fluorescent probe with MYC, thereby forming a probe-MYC complex; (b) incubating the test compound with the probe- MYC complex; (c) measuring emission from test compound incubated with probe-MYC complex (d) identifying the test compound as MYC inhibitor when detected fluorescence signal decreased in the presence of the test compound relative to the detected fluorescence signal in the absence of the test compound.

[00128] In embodiments, a probe-MYC complex includes a fluorescent probe and a MYC. In embodiments, the probe-MYC complex is formed by incubating or contacting a fluorescent probe with a MYC protein. In embodiments, the fluorescent probe is non- covalently bound to the MYC. In embodiments, the fluorescent probe is covalently bound to the MYC. [00129] In embodiments, fluorescence signal from the fluorescent probe increases when fluorescent probe is incubated or contacted with a MYC protein. In embodiments, the fluorescence of probe-MYC complex is higher than fluorescence of the fluorescent probe alone.

[00130] In embodiments, the fluorescent probe contacts a MYC protein amino acid corresponding to Lys 397, Lys 398, Ala 399, Thr 400, Ala 401, Tyr 402, He 403, and Leu 404, as shown in FIG. 6. In embodiments, the fluorescent probe contacts a MYC protein amino acid corresponding to Lys 397, Ala 401, and Leu 404.

[00131] In embodiments, the fluorescent probe contacts at least one amino acid residue of the MYC protein. In embodiments, the fluorescent probe contacts multiple amino acids of the MYC protein.

[00132] In embodiments, a MYC inhibitor is selected from a library by screening. In embodiments, fluorescence of probe-MYC complex is compared in the presence and in the absence of a test compound.

[00133] Some test compounds decrease fluorescence of the probe-MYC complex. Some test compounds do not affect the fluorescence of the probe-MYC complex (see figure 1 A). Test compounds decreasing fluorescence of the probe-MYC complex are competing MYC inhibitors and may be selected for further studies from the initial screen.

[00134] In embodiments, identified test compounds produce at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 99% decrease in fluorescence of the probe-MYC complex upon incubation. In embodiments, identified test compounds produce at least 25%, at least 35%, at least 45%, at least 55%, at least 65%, at least 75%, at least 85%, at least 95%, or at least 99% decrease in fluorescence of the probe-MYC complex upon incubation.

[00135] In embodiments, the fluorescent probe incubated or contacted with MYC protein is a compound of formula I:

Formula I or a pharmaceutically acceptable salt thereof, wherein: each R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³, and R¹⁴ is independently hydrogen, halogen, haloalkyl, -OH, C1-C5 alkoxy, -NO2, -CN, thiazolyl, or C1-C5 alkyl;

L¹ is bond, -S(O)₂-, -N(H)-, -O-, -S-, -C(O)-, -C(O)N(H)-, -N(H)C(O)-, -NHC(O)NH-, -C(O)O-, -OC(O)-, substituted or unsubstituted alkylene, substituted or unsubstituted heteroalkylene, an amino acid, or a combination thereof; and Q is a fluorophore.

[00136] In embodiments, each R¹, R², R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³, and R¹⁴ is independently hydrogen, halogen, C1-C5 alkyl, or -OH.

[00137] In embodiments, each R¹, R², R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³, and R¹⁴ is independently halogen. In embodiments, each R¹, R², R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³, and R¹⁴ is independently C1-C5 alkyl. In embodiments, each R¹, R², R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³, and R¹⁴ is independently -OH.

[00138] In embodiments, each R¹, R², R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³, and R¹⁴ is independently methyl. In embodiments, each R¹, R², R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³, and R¹⁴ is independently ethyl. In embodiments, each R¹, R², R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³, and R¹⁴ is independently propyl.

[00139] In embodiments, each R¹, R², R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³, and R¹⁴ is independently hydrogen. [00140] In embodiments, R³ is hydrogen, C₁-C₅ alkoxy, halogen, thiazolyl, -NO₂, or -CN. In embodiments, R³ is -NO₂, thiazolyl, or -CN.

[00141] In embodiments, R³ is hydrogen. In embodiments, R³ is halogen. In embodiments, R³ is -NO_2. In embodiments, R³ is C₁-C₅ alkoxy. In embodiments, R³ is -OMe. In embodiments, R³ is thiazolyl.

[00142] In embodiments, R³ is -CN.

[00143] In embodiments, L¹ is -C(O)-, unsubstituted alkylene or unsubstituted heteroalkylene, or a combination thereof. In embodiments, L¹ is -NHCH₂CH₂NH- or - NHCH₂CH₂NHC(O)-. In embodiments, L¹ is -NHCH₂CH₂NHC(O)-.

[00144] In embodiments, Q is a coumarin derivative.

[00145] In embodiments, Q is:

[00146] In embodiments, the fluorescent probe incubated or contacted with MYC protein is:

or a pharmaceutically acceptable salt thereof.

IV. Pharmaceutical compositions [00147] In an aspect, provided herein is a pharmaceutical composition including a MYC inhibitor and a pharmaceutically acceptable carrier. In embodiments, provided herein is a pharmaceutical composition including a MYC inhibitor as identified herein, including embodiments, and a pharmaceutically acceptable carrier.

[00148] In embodiments, the pharmaceutical composition is formulated as a tablet, a powder, a capsule, a pill, a cachet, or a lozenge as described herein. The pharmaceutical composition may be formulated as a tablet, capsule, pill, cachet, or lozenge for oral administration. The pharmaceutical composition may be formulated for dissolution into a solution for administration by such techniques as, for example, intravenous administration. The pharmaceutical composition may be formulated for oral administration, suppository administration, topical administration, intravenous administration, intraperitoneal administration, intramuscular administration, intralesional administration, intrathecal administration, intranasal administration, subcutaneous administration, implantation, transdermal administration, or transmucosal administration as described herein.

[00149] The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, etc.

[00150] In embodiments, the pharmaceutical composition may include optical isomers, diastereomers, enantiomers, isoforms, polymorphs, hydrates, solvates or products, or pharmaceutically acceptable salts of the compound described herein. The compound described herein (including pharmaceutically acceptable salts thereof) included in the pharmaceutical composition may be covalently attached to a carrier moiety, as described above. In embodiments, the compound described herein (including pharmaceutically acceptable salts thereof) included in the pharmaceutical composition is not covalently linked to a carrier moiety. A combination of covalently and not covalently linked compound described herein may be in a pharmaceutical composition herein.

[00151] In embodiments, the pharmaceutical composition includes a second agent. In embodiments, the pharmaceutical composition includes a second agent in a therapeutically effective amount.

[00152] The compound described herein (including pharmaceutically acceptable salts thereof) may be administered alone or co-administered to a subject in need thereof with a second agent. Co-administration is meant to include simultaneous or sequential administration as described herein of the compound described herein individually or in combination (e.g. more than one compound - e.g. second agent).

V. Methods of use

[00153] In an aspect, provided herein is a method of inhibiting MYC-MAX dimerization, comprising contacting MYC with a MYC inhibitor. In an aspect, provided herein is a method of inhibiting MYC-MAX dimerization, comprising contacting MYC with a MYC inhibitor as identified herein, including embodiments.

[00154] In an aspect, provided herein is a method of inhibiting MYC-MAX dimerization, comprising contacting MYC with an effective amount of MYC inhibitor.

[00155] In an aspect, provided herein is a method of inhibiting MYC-MAX dimerization, comprising contacting MYC with an effective amount of MYC inhibitor as identified herein, including embodiments.

[00156] In an aspect, provided herein is a method of inhibiting transcriptional activation by MYC, comprising contacting MYC with a MYC inhibitor. In an aspect, provided herein is a method of inhibiting transcriptional activation by MYC, comprising contacting MYC with a MYC inhibitor as identified herein, including embodiments.

[00157] In an aspect, provided herein is a method of inhibiting transcriptional activation by MYC, comprising contacting MYC with an effective amount of MYC inhibitor.

[00158] In an aspect, provided herein is a method of inhibiting transcriptional activation by MYC, comprising contacting MYC with an effective amount of MYC inhibitor as identified herein, including embodiments.

[00159] In an aspect, provided herein is a method of inhibiting MYC-induced cellular proliferation, comprising contacting MYC with a MYC inhibitor. In an aspect, provided herein is a method of inhibiting MYC-induced cellular proliferation, comprising contacting MYC with a MYC inhibitor as identified herein, including embodiments.

[00160] In an aspect, provided herein is a method of inhibiting MYC-induced cellular proliferation, comprising contacting MYC with an effective amount of MYC inhibitor. [00161] In an aspect, provided herein is a method of inhibiting MYC-induced cellular proliferation, comprising contacting MYC with an effective amount of MYC inhibitor as identified herein, including embodiments.

[00162] In an aspect, provided herein is a method of inhibiting MYC-induced cellular growth, comprising contacting MYC with a MYC inhibitor. In an aspect, provided herein is a method of inhibiting MYC-induced cellular growth, comprising contacting MYC with a MYC inhibitor as identified herein, including embodiments.

[00163] In an aspect, provided herein is a method of inhibiting MYC-induced cellular growth, comprising contacting MYC with an effective amount of MYC inhibitor.

[00164] In an aspect, provided herein is a method of inhibiting MYC-induced cellular growth, comprising contacting MYC with an effective amount of MYC inhibitor as identified herein, including embodiments.

[00165] In an aspect, provided herein is a method of treating a disease in a subject in need thereof, said method including administering to the subject a MYC inhibitor. In an aspect, provided herein is a method of treating a disease in a subject in need thereof, said method including administering to the subject an effective amount of a MYC inhibitor.

[00166] In an aspect, provided herein is a method of treating a disease in a subject in need thereof, said method including administering to the subject a MYC inhibitor as identified herein, including embodiments.

[00167] In an aspect, provided herein is a method of treating a disease in a subject in need thereof, said method including administering to the subject an effective amount of a MYC inhibitor as identified herein, including embodiments.

[00168] In another aspect, a MYC inhibitor for use as a medicament is provided. In further aspects, a MYC inhibitor for use in a method of treatment is provided. In another aspect, provided herein is a method of treating a disease in a subject in need thereof, said method including administering an effective amount of a pharmaceutical composition of the MYC inhibitor as identified herein.

[00169] In another aspect, a MYC inhibitor as identified herein, including embodiments, for use as a medicament is provided. In further aspects, a MYC inhibitor as identified herein, including embodiments, for use in a method of treatment is provided. In another aspect, provided herein is a method of treating a disease in a subject in need thereof, said method including administering an effective amount of a pharmaceutical composition of the MYC inhibitor as identified herein, including embodiments.

[00170] In embodiments, the disease or disorder (e.g., tumor growth) can be is associated with or mediated by abnormal c-Myc expression or biochemical activities. In embodiments, the disease is cancer. In embodiments, the cancer is colon, breast, cervical, small cell lung carcinomas, osteosarcomas, glioblastomas, melanoma or myeloid leukemia.

[00171] In embodiments, the cancers and tumors suitable for treatment with compositions and methods can be those present in a variety of tissues and organs. They also include cancer cells, tumor cells, which include malignant tumor cells, and the like that are found in the component cells of these tissues and/or organs. Examples include brain tumors (glioblastoma multiforme and the like), spinal tumors, maxillary sinus cancer, cancer of the pancreatic gland, gum cancer, tongue cancer, lip cancer, nasopharyngeal cancer, mesopharyngeal cancer, hypopharyngeal cancer, laryngeal cancer, thyroid cancer, parathyroid cancer, lung cancer, pleural tumors, cancerous peritonitis, cancerous pleuritis, esophageal cancer, stomach cancer, colon cancer, bile duct cancer, gallbladder cancer, pancreatic cancer, hepatic cancer, kidney cancer, bladder cancer, prostate cancer, penile cancer, testicular tumors, cancer of the adrenal glands, uterocervical cancer, endometrial cancer, vaginal cancer, vulvar cancer, ovarian cancer, ciliated epithelial cancer, malignant bone tumors, soft-tissue sarcomas, breast cancer, skin cancer, malignant melanomas, basal cell tumors, leukemia, myelofibrosis with myeloid metaplasia, malignant lymphoma tumors, Hodgkin's disease, plasmacytomas, and gliomas.

EXAMPLES

[00172] The following examples are meant to be illustrative and can be used to further understand embodiments of the present disclosure and should not be construed as limiting the scope of the present teachings in any way.

[00173] The chemical reactions described in the Examples can be readily adapted to prepare a number of other compounds of the present disclosure, and alternative methods for preparing the compounds of this disclosure are deemed to be within the scope of this disclosure. For example, the synthesis of non-exemplified compounds according to the present disclosure can be successfully performed by modifications apparent to those skilled in the art, e.g., by utilizing other suitable reagents known in the art other than those described, or by making routing modifications of reaction conditions, reagents, and starting materials. Alternatively, other reactions disclosed herein or known in the art will be recognized as having applicability for preparing other compounds of the present disclosure.

[00174] Reactions were carried out under atmospheric conditions and all reagents were obtained from commercial sources and used without further purification. Reactions were monitored using thin-layer chromatography (TLC) or high-performance liquid chromatography-MS (HPLC-MS). TLC was performed using Merck precoated analytical plates (0.25 mm thick, silica gel 60 F254) and visualized under UV light. HPLC-MS analysis was performed on an Agilentl260 Infinity II instrument coupled to a single quadrupole InfmityLab LC/MSD instrument running a linear gradient of eluent A (0.1% formic acid in H₂0) and eluent B (0.1% formic acid in MeCN) from 0 to 95% of A during t = 0-6 min and then with eluent B from t = 6-10 min, at a flow rate of 0.5 mL/min on a Zorbax300SB-C8 column at 35°.

Synthetic Examples

Example SI: Synthesis of the fluorescent probe 1.

1

[00175] 4-(2-(4-cyanophenyl)-6-(furan-2-yl)pyridin-4-yl)benzoic acid 10 was synthesized as previously described in Jacob et al., 2018, Bioorg. Med. Chem., 26, 4234-4239, incorporated herein in its entirety.

[00176] 4-(2-(4-cyanophenyl)-6-(furan-2-yl)pyridin-4-yl)benzoic acid 10 (82.9 μmol) was dissolved in DMF (1 mL) followed by the addition of /V-hydroxysuccinimide (248.7 μmol) and /VpY-diisopropylcarbodiimide (182.3 μmol). The mixture was stirred overnight at room temperature. Reaction completion was determined by HPLC-MS. The solution was extracted with DCM and washed with water, dried over sodium sulfate, filtered, and concentrated under reduced pressure.

[00177] The crude (not isolated) was dissolved in DMF (1 mL) with /V-Boc- ethylenediamine (91.2 μmol) and stirred at room temperature for 5 hr. The solution was extracted with DCM and washed with water, dried over sodium sulfate, filtered, and concentrated under reduced pressure.

[00178] The crude (not isolated) was dissolved in anhydrous DCM (7 mL), and trifluoroacetic acid (0.4 mL) was added while stirring at room temperature. Deprotection of the hoc group was monitored by HPLC-MS. Once complete, the mixture was poured into diethyl ether (15 mL) to precipitate a white solid which was rinsed diethyl ether and dried on the frit.

[00179] The solid and 7-(diethylamino)coumarin-3-carboxylic acid A-succinimidyl ester 11 (48.3 μmol) were dissolved in a solution of N,N-diisopropylethylamine (96.6 μmol) and DMF (1 mL). The mixture was stirred overnight at room temperature. The solvent was removed under reduced pressure and the oil was dissolved in minimal MeOH before purification by preparative TLC (1:10 MeOH:DCM) to yield a yellow solid (24.9 μmol, 30.1%).

[00180] ¹H NMR (600 MHz, CDCl₃) δ 9.29 (br.t, J= 6.0 Hz, 1H), 8.71 (s, 1H), 8.25 (d, J = 8.0 Hz, 2H), 8.07 (br.t, J= 4.1 Hz, 1H), 8.04 (d, J= 8.0 Hz, 2H), 7.92 (s, 1H), 7.82-7.76 (m, 5H), 7.59 (s, 1H), 7.42 (d, J= 8.9 Hz, 1H), 7.25 (d, J= 3.4 Hz, 1H), 6.64 (dd, J= 9.0, 2.5 Hz, 1H), 6.59 (dd, J= 3.4, 1.7 Hz, 1H), 6.48 (d, J= 2.4 Hz, 1H), 3.79-3.73 (m, 2H), 3.73- 3.67 (m, 2H), 3.44 (q, J= 7.1 Hz, 4H), 1.23 (t, J= 7.0 Hz, 3H). ¹³C NMR (151 MHz, CDCh) d 166.9, 165.8, 162.8, 157.9, 155.6, 153.6, 153.0, 150.3, 149.6, 148.5, 143.8, 143.4, 140.9, 135.2, 132.7, 131.4, 128.2, 127.8, 127.3, 117.3, 116.2, 112.7, 112.4, 110.3, 109.8, 109.5, 108.4, 96.7, 45.3, 42.9, 39.4, 29.8, 12.5. HPLC, t_R 7.74 min (>98%, UV₂₈₀). HRMS (ES⁺) m/z calcd for [C₃₉H₃₄N₅O₅] ⁺ : 652.2554; found 652.2568.

Biological Examples

Example Bl: Fluorescence enhancement of fluorescent probe 1 in the presence of MYC.

[00181] Fluorescent probe 1 (5 pM) was incubated with varying concentrations of MYC protein (1:3 dilution in buffer - 50 mM MES, pH 6.0, 2% DMSO - 6.68pM-1.6nM) for 1 hr. at 21°C. 1 Figure 2 shows increase in fluorescence of 1 with increasing concentrations of MYC. Under these conditions 1 exhibited a dose-dependent emission increase.

Example B2: Fluorescence perturbation of coumarin derivatives compared to fluorescent probe 1 in the presence of MYC.

[00182] Dilution series of MYC (2 pM-0.9 nM, 1 :3) in buffer (50 mM MES, pH 6.0, Tween20 0.01%, BSA 0.002%, DMSO 2%) were prepared with and without fluorophore (0.8 mM). Plates were incubated for 1 hr. at room temperature before measuring emission at the maximum excitation: 1 (424 nm), 7-diethylamino-4-methylcoumarin (375 nm), or 7-amino-4- methylcoumarin (339 nm). Emission values from wells with fluorophore were subtracted by the respective wells without fluorophore. Emission was measured using SpectraMax i3x (Molecular Devices)

[00183] Figure 3 (top panel) shows a dose-dependent fluorescence increase upon incubation of MYC with 1. Coumarin derivatives evaluated under the same conditions exhibited the opposite effect (Figure 3, middle and bottom panels), an emission decrease, suggesting the KJ-Pyr-9 scaffold (i.e. inhibition of MYC) is required for the emission increase of 1.

Example B3: Competition Assays.

[00184] The dose-dependent fluorescence increase of 1 upon incubation with MYC was further evaluated by the addition of established MYC inhibitors KJ-Pyr-9, 10058-F4, and 10074-G5 to displace 1 and attenuate the emission increase. Solutions of 1 (0.8 mM) in buffer (50 mM MES, pH 6.0, Tween200.01%, BSA 0.002%, DMSO 2%) were prepared with and without MYC (1 mM). These solutions were plated in 96-well black half-area plates, and known MYC inhibitors KJ-Pyr-9, 10058-F4 and 10074-G5, from DMSO stocks were added and serially diluted across wells (1 :3 dilution, 100 pM - 45.7 nM) with a final volume of 90 μL. The plates were incubated (1 hr, 21 °C, dark) before fluorescence was measured (excitation: 424 nm, emission: 470 nm), as shown in figure 4. To limit the influence of independent fluorescence attenuation, control wells without MYC were measured, and their fluorescence values were subtracted from wells with MYC. IC₅₀ values were determined by fitting the points to a log(inhibitor) vs. response equation in GraphPad Prism: Y =

where X is the log(inhibitor, pM), Y is the emission, and Top and

Bottom are the Y Plateaus. Data are presented as the average of three replicates ± SD.

Example B4: KJ-Pyr-9 Scaffold Specificity and Affinity.

[00185] A pulldown study was performed to investigate the ability of the KJ-Pyr-9 scaffold to sequester MYC within a complex cell lysate and to determine if manipulation of the amide would hinder engagement in this context. Both active (2) and inactive (3) biotinylation probes were prepared based on previously reported inhibitors (Jacob et ak,

2018, Bioorg. Med. Chem., 26, 4234-4239), and binding rates mirrored those of their progenitors by bio-layer interferometry (BLI).

2 3

[00186] Bio-layer Interferometry (BLI): Streptavidin sensors (ForteBio) were equilibrated in buffer consisting of 50 mM MES, pH 6.0, 0.1% BSA, 0.02% Tween-20 and 10 mM NaCl (note: NaCl concentration optimized for individual sensor trays) for five minutes, and this buffer was used throughout the experiment. Sensors were then subjected to real-time kinetic analysis in the following steps: baseline (60 s), ligand loading (2/3-250 nM, 1% DMSO, 180 s), quenching (biocytin-10 ug/mL, 60 s), baseline (60 s), association (MYC-150-100 nM, 90 s), dissociation (240 s). Kinetic analysis was carried out using ForteBio Analysis 9.0 software provided with the instrument and the data fitted with a 2: 1 (heterogenous ligand) model (R² = 0.99).

[00187] Pulldown experiment: P493-6 cells were cultured as previously described (Gossen et al., 1992, Proc. Natl. Acad. Sci. U.S.A., 89, 5547-5551; Schuhmacher et al., 1999, Curr. Biol., 9, 1255-12580). After reaching a density of 1 x 10⁶ cells/mL, cultures were transferred to tubes (1 mL/tube) and pelleted by centrifugation (2500 x g, 10 min). The pellets were rinsed with HEPES buffer (30 mM HEPES, 50 mM NaCl, 0.005% Tween20, 3 mM EDTA, pH 7.4) and centrifuged to remove supernatant (2500 x g, 10 min). Proteins were extracted from pellets (10 μL) according to the protocol in the M-PER instructions (Thermo Fisher, #78501). Pierce high capacity streptavidin agarose (200 μL) was rinsed with MES buffer (600 μL, 50 mM MES, pH 6.0) three times before incubation (12 hr, rt) with the biotinylated compound (60 nmol, 2 or 3) in 5% DMSO MES buffer (600 μL). The resins were then rinsed with MES as described above to remove unbound compound. [00188] Extracted P493-6 cell lysates (100 mE) and purified MYC (100 μL, 1.5 mM) were added to the ligand-bound resins and gently mixed (24 hr, rt). The mixtures were centrifuged (500 x g, 2 min, rt) to remove supernatant and the resin was washed three times with MES buffer (600 μL, 15 min, rt) and once with 4% DMSO MES buffer (600 μL, 15 min, rt). The respective competitor (90 nmol, unlabeled 2 or 3) was added in MES buffer (600 μL, 3% DMSO) and gently mixed (3 hr, rt). The supernatant was isolated by centrifugation (500 x g,

2 min, rt). All washes and elutions were evaluated by polyacrylamide gel electrophoresis with Bolt LDS sample buffer, Bolt premade 17-well gel (4-12% Bis-Tris Plus), and MES SDS running buffer. Gels were visualized by silver stain (SilverSNAP, #24602, Pierce) or western blot (c-Myc 9E10, Horseradish peroxidase, Super Signal West Dura, iBlot 2 NC mini). MYC enrichment observed by western blot and was quantified relative to lysate by ImageJ to determine the percent retained by the resin. The immobilized inhibitor 2 was able to demonstrate high specificity by enriching MYC in P493-6 cell lysates and eluting it when incubated with the original inhibitor (data not shown).

[00189] Binding affinity and ability to inhibit MYC-MAX DNA complexation were evaluated by previously described SPR methods (Jacob et al., 2018, Bioorg. Med. Chem., 26, 4234-4239): Briefly, the binding of KJ-Pyr-9 and 1 against MYC-monomer and MYC-MAX- dimer proteins were evaluated using SPR technique.

[00190] MYC monomer protein and MYC-MAX dimer proteins were immobilized onto the CM5 sensor chip surface (GE Healthcare Life Sciences, # 29149603) via amine-coupling methodology aimed at a ligand density around 1,000 and 1,400 RUs, respectively. A Biacore 8K (GE Healthcare Life Sciences) built-in LMW single-cycle kinetics protocol was used for kinetics determination. The contact time was set as 120 s for each concentration, and the entire dissociation time was set as 3600 s. There were 5 concentrations per compound per cycle, each tested compound had its own blank buffer injection as reference. Compound KJ- Pyr-9 was made into stock solution of 30 mM using 100% DMSO, while 1 was made into stock solution of 10 mM using 100% DMSO. Each of the compounds were diluted using lx PBS-P + buffer with 1% DMSO to get working concentrations of KJ-Pyr-9 compound as 0.625, 1.250, 2.500, 5.000 and 10.000 μM and that of 1 as 0.123, 0.370, 1.111, 3.333, and 10.000 mM. The association rate constant (k_a), dissociation rate constant (k_d) and equilibrium dissociation constant (K_D) values were calculated using Biacore Insight Evaluation software (ver. 3.0.11.15423) following the two-state reaction model with global fit parameters for reference, blank and solvent-corrected sensorgrams. [00191] The blank and reference subtracted and solvent-corrected sensorgrams (dotted lines) and fitted curves (continuous lines) were presented. Surprisingly, 1 exhibited a lower equilibrium dissociation constant for MYC than KJ-Pyr-9 (0.38 mM vs. 3.5 mM, Figures 5A and 5B) and a significantly greater capacity to disrupt DNA complexation (figure 9A). Accordingly, affinity and specificity are retained when the amide of the KJ-Pyr-9 scaffold is manipulated.

Example B5: Inhibitor Binding Site.

[00192] Molecular Modeling: Calculations were performed with modules from the Schrodinger Small Molecule Drug Discovery Suite (Maestro), release 2018-3, using the OPLS3 force field for parameterization (Harder et al., 2016, J. Chem. Theory Comput., 12, 281-296). The X-ray co-crystal structure of the MYC/MAX/DNA complex (PDB 1NKP) was imported from the protein data bank and prepared with the Protein Preparation Wizard using default settings (Nair et al., 2003, Cell, 112, 193-205; Madhavi et al., 2013, J. Comput. Aided. Mol. Des., 27, 221-234). Ligands were imported into and prepared using LigPrep (Madhavi et al., 2013, J. Comput. Aided. Mol. Des., 27, 221-234). Docking was carried out using the Induced Fit Docking module in Glide (Sherman et al., 2006, J. Med. Chem., 49, 534-553). A receptor box grid center was built 20 A around the KKATAYIL peptide sequence of MYC (the binding residues derived from HDX-MS) and compounds were docked with Extra Precision Glide redocking and otherwise default settings, with the lowest energy binding mode represented (Friesner et al., 2006, J. Med. Chem., 49, 6177-6196). The binding pocket surrounding the binding region was predicted using SiteMap, and predicted chemical properties were calculated in QikProp (Halgren T., 2007, Chem. Biol. Drug Des., 69, 146- 148; Halgren T., 2009, J. Chem. Ihf. Model., 49, 377-389). Figures were generated using PyMol Molecular Graphics System (Schrodinger, LLC).

[00193] HDX-MS (hydrogen-deuterium exchange detected by mass spectrometry) experiments were performed to determine the inhibitor binding site. Differential HDX-MS experiments were conducted as previously described with a few modifications detailed below (Chalmers et al., 2006, Anal. Chem., 78, 1005-1014). Peptides were identified using tandem MS (MS/MS) with an Orbitrap mass spectrometer (Q Exactive Thermo Fisher). Product ion spectra were acquired in data-dependent mode with the top five most abundant ions selected for the product ion analysis per scan event. The MS/MS data files were submitted to Mascot (Matrix Science) for peptide identification. Peptides included in the HDX analysis peptide set had a MASCOT score greater than 20. The MASCOT search was repeated against a decoy (reverse) sequence, and ambiguous identifications were ruled out and not included in the HDX peptide set.

[00194] MYC-MAX dimer (10 mM) was incubated (lh, rt) with the respective ligands at a 1:10 protein-to-ligand molar ratio. Next, 5 μL of sample was diluted into 20 μL D2O buffer (50 mM MES, pH 7.4; 28 mM NaCl) and incubated for various time points (0, 10, 60, 300, 900, and 3600 s) at 4 °C. The deuterium exchange was then slowed by mixing with 25 μL of cold (4 °C) 0.1 M Sodium Phosphate, 50 mM TCEP and 1% trifluoroacetic acid. Quenched samples were immediately injected into the HDX platform. Upon injection, samples were passed through an immobilized pepsin column (2 mm x 2 cm) at 200 μL min^-1, and the digested peptides were captured on a 2 mm ^c 1 cm C₈ trap column (Agilent) and desalted. Peptides were separated across a 2.1 mm x 5 cm C₁₈ column (1.9 μL Hypersil Gold, Thermo Fisher) with a linear gradient of 4-40% MeCN and 0.3% formic acid, over 5 min. Sample handling, protein digestion and peptide separation were conducted at 4 °C. Mass spectrometric data were acquired using an Orbitrap mass spectrometer (Exactive, Thermo Fisher). The intensity weighted mean m/z centroid value of each peptide envelope was calculated and subsequently converted into a percentage of deuterium incorporation. This is accomplished determining the observed averages of the non-deuterated and fully deuterated spectra and using the conventional formula. Corrections for back-exchange were made based on an estimated 70% deuterium recovery and accounting for the known 80% deuterium content of the deuterium exchange buffer. HDX analyses were performed in triplicate, with single preparations of each purified protein/complex. Statistical significance for the differential HDX-MS data is determined by t-test for each time point and is integrated into the HDX-MS Workbench software.

[00195] The MYC-MAX dimer, in the presence of 1, exhibited reduced exchange in residues KKATAYIL (C-MYC397-404) of the MYC sequence relative to DMSO as demonstrated via deuterium exchange experiments (data not shown). This binding site, a depiction of which (based on molecular modeling) is shown in figure 6, is within the bHLH- LZ motif (C-MYC_351-439), a critical interface for MYC-MAX dimerization. Based on these experiments, it is proposed that the accommodation of the cavity below the ion pair of E373 and K398 (figure 6) by an inhibitor distorts the positioning of the linked helix and hence disrupt the DNA binding properties of the dimeric MYC/MAX protein. Example B6: Small Molecule High-Throughput Screening.

[00196] To assess the high-throughput screening potential of the competitive assay, a diversity library (N = 1408) was evaluated in triplicate in a 384-well format (data not shown). The competitive assay, described above, was scaled to 20 μL. Buffer was transferred to plates with an auto-dispenser, before compounds were added using a 100 nL pintool head affixed to a PerkinElmer FX instrument. Positive (10074-G5) and negative (DMSO) control wells were run on each plate with 16 replicates. Under these conditions, a high average Z’ factor was observed (0.71), hit rate (0.57%) and high plate-to-plate replicate fidelity (r² > 0.78). These values were calculated using previously described equations. For the data normalization, activity of each compound was calculated on Avg + 3SD cutoff basis using the following equation:

Percent response of compound

where the “High Control” represents wells containing MYC+1+10074-G5 and the “Data Wells” represent wells containing both test compounds and the “Low Control” which contains MYC+l+DMSO.

[00197] A mathematical algorithm was used to determine active compounds. Two values were calculated: 1) the average percent response of all compounds tested for the screen, and 2) three times their standard deviation. The sum of these two values was used as a cutoff parameter, i.e. any compound that exhibited greater percent inhibition than the cutoff parameter (35.5% in the case here) was declared active. Using this cutoff, the diversity assay yielded 6 active compounds (“hits”). Six molecules were selected for evaluation, see figure 8, compounds 12, 13, 14, 15, 16, and 17.

[00198] This assay was further validated using the dose-response curve of the reference 10074-G5, as shown in figure 7, and then the assay was miniaturized to a 1536-well format. The conditions were optimized, and a LOP AC (Library of Pharmacologically Active Compounds) pilot screen was run (N = 1280) in triplicate (Figure 7).

[00199] The protocol for the 1536-well plate format: 5 μL/well of MYC and fluorescent probe 1 were dispensed as follows: 50 mM MES pH 6 and 22.1 mM NaCl+ 1 mM MYC enzyme and 0.8 mM final 1. Then, 50 nL/well test compounds were pinned at 10 pM final concentration in 2% DMSO. The plates were centrifuged for 4 minutes at 1,000 rpm at room temperature and incubated in the dark for 60 minutes at room temperature. Fluorescence was read using TECAN M200.

[00200] Raw assay data was imported into the Scripps’ corporate database and analyzed using Symyx software. The normalization of data and determination of cutoff parameter (23.74%) were performed as described above. The assay had excellent reproducibility, Z’ factor, and hit rate (1.25%) and identified 16 candidate inhibitors using the cutoff parameter 23.74%. In this HTS assay, 10074-G5 exhibited an IC50 of 1.2 mM and showed no evidence of creating artifactual fluorescence. Plate-to-plate reproducibility was excellent with a correlation of r² > 0.9 and a slope very close to 1 (data not shown). A counter- screen assay was implemented to identify compounds acting as non-specific chromophores, enhancers of fluorescence, or optically interfere with fluorescence. The counter-screen titration assay is similar in format to the MYC inhibitor assay but employs a method wherein fluorescence in the presence of MYC, the probe 1 and the hit compound is subtracted by the fluorescence in the absence of MYC. Specifically, the titration assay employed the same reagents, protocols, and detection systems as the confirmation, but tested each of the selected/available compounds as 10-point dose-response titrations (3 -fold dilutions) in triplicate. The titration assay performance for inhibitors were consistent with an average Z’ of 0.87 ± 0.01 and a S:B of 8.59 ± 0.12. A four-parameter equation describing a sigmoidal dose-response curve was then fitted with adjustable baseline using Assay Explorer software (Symyx Technologies Inc.). The reported IC50 values were generated from fitted curves by solving for the X- intercept value at the 50% inhibition/activation level of the Y-intercept value. The following rule was used to declare a compound as “active” or “inactive”: Compounds with an IC50 greater than 10 mM were considered inactive. Compounds with an IC50 less than 10 pM were considered active. Of those, 7 compounds demonstrated nominal potency (IC50 < 10 pM) selective to MYC inhibitors.

Example B7: Evaluation of Screening Hits: Inhibition of MYC-MAX DNA Binding by SPR.

[00201] Select compounds from both the 384-well and 1536-well screens (N = 14) were evaluated by several orthogonal assays. The ability of the selected inhibitors (10 pM) to disrupt MYC-MAX DNA binding was assessed utilizing previously described SPR assays (see Example B4 above). As shown in figure 9A, while all compounds exhibited some level of inhibition, three molecules demonstrated greater inhibition than KJ-Pyr-9. Compounds KJ-Pyr-9, 5b and 5d are reported inhibitors (Jacob et al., 2018, Bioorg. Med. Chem., 26, 4234-4239). Furthermore, predicted ADME properties display higher cell permeable, oral absorption, and solubility for the selected compounds over KJ-Pyr-9 (Table 1).

[00202] Table 1. Selected ADME properties calculated in QikProp. MW, molecular weight (g/mol); HBD, hydrogen bond donors; HBA, hydrogen bond acceptors; cLogP, predicted octanol/water partition coefficient; cLogS, predicted aqueous solubility (mol/L); predicted apparent non-active transport Caco-2 cell permeability (nm/sec); %Abs, predicted human oral absorption; Van der Waals polar surface area (PSA) of nitrogen and oxygen atoms (A²); Ro5, violations of Lipinski’s rule of five; Ro3, violations of Jorgensen’s rule of three: (LogS > -5.7, Caco2 > 22 nm/s, # primary metabolites <

7).

Example B8: Evaluation of Screening Hits: CEF (Chicken Embryo Fibroblast) Oncogenic Transformation Assay. [00203] MYC-induced oncogenic transformation in chicken embryo fibroblasts (CEF) was used as a secondary screen to determine inhibition of MYC in a biological setting. Cellular efficacy and specificity were evaluated by previously described CEF oncogenic transformation assays (Hart et al., Proc. Natl. Acad. Sci., 2014, 111, 12556-12561; Jacob et al., 2018, Bioorg. Med. Chem., 26, 4234-4239). Six of the fourteen compounds demonstrated the ability to reduce foci formation at concentrations 10 mM or lower (data not shown). These inhibitors were evaluated in the CEF assay with other oncoproteins (Src, Jun and PI3K). One lead, Compound 5, exhibited significant specificity. MYC specificity oncogenic transformation assay with KJ-Pyr-9 and 5 against virally induced oncogenic transformation by MYC, Src, Jun, or PI3K H1047R. The oncogenic activity of ATG-MYC was strongly inhibited by KJ-Pyr-9 and new lead compound 5, whereas the unrelated oncoproteins (Src, v- Jun, PI3K H1047R) were inhibited only at significantly higher concentrations, as shown in figure 9B.

[00204] Identified hits were structurally distinct from 1 and possessed favorable predicted drug-like properties. Furthermore, these molecules disrupted MYC-MAX DNA complexation and several inhibited oncogenic transformation in CEFs.

[00205] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, the descriptions and examples should not be construed as limiting the scope of the invention. The disclosures of all patent and scientific literature cited herein are expressly incorporated herein in their entirety by reference.

Claims

WHAT IS CLAIMED IS:

1. A compound of formula I:

or a pharmaceutically acceptable salt thereof; wherein: each R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³, and R¹⁴ is independently hydrogen, halogen, haloalkyl, -OH, C1-C5 alkoxy, -NO2, -CN, thiazolyl, or C1-C5 alkyl; L¹ is bond, -S(O)₂-, -N(H)-, -O-, -S-, -C(O)-, -C(O)N(H)-, -N(H)C(O)-,

-NHC(O)NH-, -C(O)O-, -OC(O)-, substituted or unsubstituted alkylene, substituted or unsubstituted heteroalkylene, an amino acid, or a combination thereof; and Q is a fluorophore.

2. The compound of claim 1, wherein each R¹, R², R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³, and R¹⁴ is independently hydrogen, halogen, C₁-C₅ alkyl, or -OH.

3. The compound of claim 1, wherein each R¹, R², R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³, and R¹⁴ is independently hydrogen.

4. The compound of claim 1, wherein R³ is hydrogen, halogen, thiazolyl, C₁-C₅ alkoxy, -NO2, or -CN.

5. The compound of claim 1, wherein R³ is -NO2, thiazolyl, or -CN.

6. The compound of claim 1, wherein R³ is -CN.

7. The compound of claim 1, wherein L¹ is -C(O)-, unsubstituted alkylene or unsubstituted heteroalkylene, or a combination thereof.

8. The compound of claim 1, wherein L¹ is -NHCH₂CH₂NH- or -NHCH₂CH₂NHC(O)-. The compound of claim 1, wherein Q is a coumarin derivative. The compound of claim 1, wherein Q is:

The compound of claim 1, wherein the compound is:

or a pharmaceutically acceptable salt thereof. A method of identifying a test compound as a MYC inhibitor, the method comprising:

(a) incubating a fluorescent probe with MYC, thereby forming a probe-MYC complex;

(b) incubating the test compound with the probe-MYC complex;

(c) measuring emission from test compound incubated with probe-MYC complex.

(d) identifying the test compound as MYC inhibitor when detected fluorescence signal decreased in the presence of the test compound relative to the detected fluorescence signal in the absence of the test compound. The method of claim 12, wherein the fluorescent probe is a compound of formula I:

or a pharmaceutically acceptable salt thereof, wherein: each R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³, and R¹⁴ is independently hydrogen, halogen, haloalkyl, -OH, C1-C5 alkoxy, -NO2, -CN, thiazolyl, or C1-C5 alkyl; L¹ is bond, -S(O)₂-, -N(H)-, -O-, -S-, -C(O)-, -C(O)N(H)-, -N(H)C(O)-,

-NHC(O)NH-, -C(O)O-, -OC(O)-, substituted or unsubstituted alkylene, substituted or unsubstituted heteroalkylene, an amino acid, or a combination thereof; and Q is a fluorophore. The method of claim 13, wherein each R¹, R², R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³, and R¹⁴ is independently hydrogen, halogen, C₁-C₅ alkyl, or -OH. The method of claim 13, wherein each R¹, R², R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³, and R¹⁴ is independently hydrogen. The method of claim 13, wherein R³ is hydrogen, halogen, thiazolyl, C₁-C₅ alkoxy, -NO2, or -CN. The method of claim 13, wherein R³ is -NO2, thiazolyl, or -CN. The method of claim 13, wherein R³ is -CN. The method of claim 13, wherein L¹ is -C(O)-, unsubstituted alkylene or unsubstituted heteroalkylene, or a combination thereof. The method of claim 13, wherein L¹ is -NHCH₂CH₂NH- or -NHCH₂CH₂NHC(O)-. The method of claim 13, wherein Q is a coumarin derivative. The method of claim 13, wherein Q is:

The method of claim 13, wherein the fluorescent probe is:

or a pharmaceutically acceptable salt thereof. A method of inhibiting MYC-MAX dimerization, comprising contacting MYC with an effective amount of a MYC inhibitor, wherein the MYC inhibitor is identified by any one of the methods of claims 12-23. A method of inhibiting transcriptional activation by MYC, comprising contacting MYC with an effective amount of a MYC inhibitor, wherein the MYC inhibitor is identified by any one of the methods of claims 12-23. A method of inhibiting MYC-induced cellular proliferation, comprising contacting MYC with an effective amount of a MYC inhibitor, wherein the MYC inhibitor is identified by any one of the methods of claims 12-23. A MYC inhibitor, wherein the MYC inhibitor is identified by any one of the methods of claims 12-23. A pharmaceutical composition comprising a MYC inhibitor and a pharmaceutically acceptable carrier. The pharmaceutical composition of claim 28, wherein the MYC inhibitor is identified by any one of the methods of claims 12-23. A method of treating a disease in a subject in need thereof, the method comprising administering to the subject an effective amount of a MYC inhibitor, wherein the MYC inhibitor is identified by any one of the methods of claims 12-23. The method of claim 30, wherein the disease is cancer.