Inhibitors of RNA binding protein IGF2BP3

Abstract

The present disclosure relates compounds, pharmaceutical compositions thereof, and methods of using said compounds and compositions for inhibiting the activity of RNA binding protein IGF2BP3 and for the treatment of cancer.

Description

[0001] INHIBITORS OF RNA BINDING PROTEIN IGF2BP3

[0002] RELATED APPLICATIONS

[0003] This application claims the benefit of U.S. Provisional Application No. 63 / 528,250, filed July 21, 2023, which is hereby incorporated by reference in its entirety.

[0004] BACKGROUND

[0005] Leukemias are the most common childhood cancers, accounting for 28% of all cancers in children. The most common types are acute lymphocytic leukemia (ALL) and acute myeloid leukemia (AML). In the United States, there are almost 7000 new cases of ALL each year, with a fatality rate of over 20%. Further, AML is one of the most common forms of leukemia in adults, with about 20,000 new cases diagnosed each year. AML remains a highly fatal disease with a five-year survival rate of only 28.3%.

[0006] Recent work has established that RNA-binding proteins, via diverse mechanisms, can be important regulators of oncogenesis. The RNA-binding protein IGF2BP3 is an oncofetal protein that was discovered to be specifically overexpressed in B-acute lymphoblastic leukemia (B-ALL). Not normally expressed in adult tissues, Igf2bp3 is dispensable for normal development in a mouse genetic model. However, functional studies demonstrated that IGF2BP3 was requisite for leukemogenesis in vitro and in vivo, in both human and murine model systems. In B-ALL cell lines, knockdown of IGF2BP3 led to a dramatic reduction in growth and an increase in apoptosis. In vivo, overexpression of IGF2BP3 in the bone marrow led to hyperproliferation in hematopoietic stem and progenitor cells, and a pre- leukemic disease with expansion of B- and myeloid cells. Deletion of Igf2bp3 in a murine bone marrow transplantation assay significantly limited leukemia development and increased leukemia-free survival. Collectively, the literature demonstrates the functional role of IGF2BP3 as an oncogene and establishes it as a target in acute leukemia. Further, it is estimated that up to 15% of all human cancers overexpress IGF2BP3. In view of the foregoing, there is a clear and unmet need for cancer therapies targeting IGF2BP3.

[0007] SUMMARY OF THE INVENTION

[0008] The present disclosure relates to inhibitors of IGF2BP3 activity and pharmaceutically acceptable salts thereof. In certain aspects, the present disclosure provides a compound having a structure represented by Formula I or a pharmaceutically acceptable salt thereof: wherein

[0009] A is aryl, heteroaryl, or heterocyclyl; each Ri is independently alkyl, alkoxy, alkenyl, alkynyl, aralkyl, heteroaralkyl, aryl, heteroaryl, halo, haloalkyl, hydroxyl, carboxyl, acyl, ester, thioester, phosphoryl, amino, amido, cyano, nitro, azido, cycloalkyl, heterocyclyl, alkylsulfoxidyl, alkylsulfonyl, or sulfonamido;

[0010] Xi and X2 are each independently N or CH;

[0011] X3 is N, CH, or absent;

[0012] X4is S, O, C(H)(RA), or NRA;

[0013] X5 is O or NRB;

[0014] X6is N(Rx)(Ry) or ORX;

[0015] X7 is O, S, C(H)(Rc), NRc, or is absent;

[0016] Xs is O, S, C(H)(RD), NRD, or is absent;

[0017] RA, RB, RC, RD, RX, and Ryare each independently selected from H, alkyl, alkoxy, aryl, aralkyl, heteroaryl, heteroaralkyl, and cycloalkyl; nl is 0, 1, 2, 3, or 4; and n2 is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12.

[0018] In certain aspects, the present disclosure provides a method of treating cancer in a subject in need thereof comprising administering the compound of formula I or a pharmaceutically acceptable salt thereof. In certain embodiments, the cancer is characterized by overexpression of RNA-binding protein IGF2BP3. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] FIG. 1A shows a schematic representation of IGF2BP3-GFP-Flag (I3-GFP-F) and IGF2BP3 -Flag construct.

[0020] FIG. IB shows immunofluorescence microscopy of 293T cells transduced with 13- GFP-F.

[0021] FIG. 1C shows western blot analysis for the Flag-IGF2BP3 and I3-GFP-F protein.

[0022] FIG. ID shows a western blot of elutions of purified protein that were obtained after purification on M2 anti-Flag resin.

[0023] FIG. IE shows Coomassie stained gel showing quantification of purified protein compared with BSA standards.

[0024] FIG. IF shows western blot of input, control and anti-Flag IP used for RNA immunoprecipitation .

[0025] FIG. 1G shows RT-qPCR for CDK6 and MYC, a known target of IGF2BP3 in immunoprecipitate (p<0.05 for IgG vs. Flag IP).

[0026] FIG. 2A shows a schematic of the assay. TR-FRET readings (Fluorescence Intensity at 520nm) plotted as fold over “no RNA / maximum protein / maximum Strep-Tb control” in these assays.

[0027] FIG. 2B shows fluorescence intensity as a function of protein concentration with lOnM RNA concentration.

[0028] FIG. 2C shows fluorescence intensity as a function of RNA concentration (25 nM protein).

[0029] FIG. 2D shows a demonstration of assay validation; GFP fluorescence intensity with various combinations of assay components is shown; Tb= Terbium; M6A RNA= m6A modified, biotinylated RNA oligonucleotide; Biotin RNA= biotinylated RNA without m6A modification; 13= I3-GFP-F fusion protein.

[0030] FIG. 3A shows GFP fluorescence in three different experiments performed on three different days (E1-E3; n=110 (replicates); assay volume= 10 pL) in control versus a test samples.

[0031] FIG. 3B shows the FRET Ratio, calculated as GFP fluorescence / Terbium fluoresce performed on three different days (E1-E3; n=110 (replicates); assay vol urn e= 10 pL) in control versus a test samples. FIG. 3C shows a comparison of GFP fluorescence in the control versus a test sample in 100 pL and 10 pL assay volume.

[0032] FIG. 3D shows the GFP / Terbium FRET ratio comparison in the control versus a test sample in 100 pL and 10 pL assay volumes.

[0033] FIG. 3E shows Z’ values for plates with different assay volumes, demonstrating minimal inter-day variation of assay system. Each bar represents Z’ value for one plate.

[0034] FIG. 4A shows a bar graph indicating number of compounds screened and number of hits at specified statistical cutoffs.

[0035] FIG. 4B shows the hit rate percentage based on different statistical cutoffs.

[0036] FIG. 5A shows a scatter plot of plate standard deviations of positive control across 617 plates utilized in the assay. Briefly, this involved measurement of fluorescence ratios for control samples and deriving mean and standard deviation. Standard deviations(s) are represented as percent of mean.

[0037] FIG. 5B shows a scatter plot of plate Z’ -scores for controls.

[0038] FIG. 5C shows the percent of standard deviations less than the indicated % standard deviation (s).

[0039] FIG. 5D shows the percent of Z-scores greater than indicated value for controls.

[0040] FIG. 6A shows the raw and refined hit rates in the preliminary and full-scale screens as a percentage of total compounds screened in each.

[0041] FIG. 6B shows the total number of hits in the library compared to hits amongst compounds containing Core Structure #1 (“‘Core’ structure); Fisher’s exact test, p<0.0001.

[0042] FIG. 6C shows the total number of hits in the library compared to hits amongst compounds containing Core Structure #2 (“2ndcore structure”); Fisher’s exact test, p<0.0001.

[0043] FIG. 7A shows the fluorescence ratios of Tier 1, Tier 2, and Tier 3 compounds in validation assays (data graphed as mean + / - 95% CI). To confirm hits identified in the full- scale screen, several replicate experiments were carried out to assess changes in fluorescence ratios. 415 high-probability hits were re -pinned into two 384-well plates and were subjected to the same TR-FRET assay as before. An individual compound was defined as being a “hit” in the validation screen when the fluorescence ratio deviated from negative controls by greater than three standard deviations. Next, Tier 1 hit compounds were defined as those that were positive in both experimental replicates; Tier 2 hit compound were defined as those that were positive in both experimental replicates, and Tier 3 compounds were defined as those that were “non-hits” in the validation experiment based on their not being positive in either replicate.

[0044] FIG. 7B shows the relative frequency of Tier 1, Tier 2 and Tier 3 compounds amongst all “primary hits” versus amongst compounds with Core structure #1 (“ ‘Core’ structure”).

[0045] FIG. 8A shows a schematic demonstrating the strategy for the counterscreening assay. Briefly, the assay measured cell growth as assessed by CTG in WT and KO cells in the presence of the compound. Cell growth was expressed as fold-change from control wells where no compound was applied. 415 compounds that were subjected to the validation screen with TR-FRET were also assessed here. The assay was performed in triplicate and repeated twice.

[0046] FIG. 8B shows cell growth in I3KO plotted against cell growth in NT; the dashed- line box indicates the area where inhibition of cell growth in the NT cells exceeds inhibition in the KO cells.

[0047] FIG. 8C shows the cell growth ratio (NT / KO) for each individual compound. The outliers correspond to the compounds represented in the dashed- line box in FIG. 8B.

[0048] FIG. 9A shows the compound I3IN-001 identified in the counter-screen (left) and the growth curves of cells exposed to said compound (right). Cell growth curves, based off of a CTG assay, of SEM NT and SEM-I3K0 cells are expressed as fold-change from no-drug treatment controls.

[0049] FIG. 9B shows the compound I3IN-002 identified in the counter- screen (left) and the growth curves of cells exposed to said compound (right). Cell growth curves, based off of a CTG assay, of SEM NT and SEM-I3K0 cells are expressed as fold-change from no-drug treatment controls. I3IN-002 shows a clear-cut increased inhibition of cell growth in SEM- NT cells as opposed to SEM-I3K0 cells.

[0050] FIG. 9C shows the compound I3IN-003 identified in the counter-screen (left) and the growth curves of cells exposed to said compound (right). Cell growth curves, based off of a CTG assay, of SEM NT and SEM-I3K0 cells are expressed as fold-change from no-drug treatment controls.

[0051] FIG. 9D shows the structure of I3IN-115, along with the calculated IC50 in the cellbased IC50 assays performed in SEM-NT (“NT IC50”) and SEM-I3K0 cells (“I3KO IC50”), and IC50 experiments showing primary data of cell growth inhibition, represented as “fold change” for SEM-NT cells (“NT 115”) and SEM-I3K0 cells (“KO 115”) treated with I3IN- 115.

[0052] FIG. 10A shows cell growth assays on SEM WT cells treated with I3IN-002 or DMSO.

[0053] FIG. 10B shows cell growth assays on RS4;11 cells treated with I3IN-002 or DMSO.

[0054] FIG. IOC shows cell growth assays on OCI-AML 8227 cells treated with I3IN-002 or DMSO.

[0055] FIG. 10D shows cell growth assays on Lin-MLL-Af4 cells treated with I3IN-002 or DMSO.

[0056] FIG. 10E shows cell cycle analysis using propidium iodide.

[0057] FIG. 10F shows cell cycle analysis using propidium iodide.

[0058] FIG. 10G shows caspase 3 / 7 assay on I3IN-002 treated RS4;11 cells.

[0059] FIG. 10H shows caspase 3 / 7 assay on I3IN-002 treated SEM cells.

[0060] FIG. 11A shows qRT-PCR data on CDK6, MYC, HoxAO, and IGFlr (IGF2BP3 targets), demonstrating target specificity of the I3IN-002. qRT-PCR was conducted after RNA immunoprecipitation on cells treated with DMSO and / or I3IN-002.

[0061] FIG. 11B shows cellular thermal shift assays on SEM cells treated with DMSO / I3IN- 002, 13IN-115, and I3IN-157 and probed for IGF2BP3 and Actin-beta.

[0062] FIG. 11C shows quantification of CDK6, BCL2, MYC, and HOXA9 RNA recovered from RNA immunoprecipitation experiments with or without treatment with I3IN-002. The bottom panel shows Western blot analysis of RNA immunoprecipitation samples following treatment of SEM cells with 5 pM I3IN-002. Statistical significance was evaluated by student’s T-test, *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.

[0063] FIG. 12A shows differentially expressed genes between SEM-NT and SEM-I3KO cells. SEM-13KO is a SEM strain with a knockout of IGF2BP3. Common genes are highlighted.

[0064] FIG. 12B shows differentially expressed genes between SEM-NT cells treated with DMSO versus I3IN-002. Common genes are highlighted. Treatment of SEM cells with KIN- 002 leads to similar gene expression changes as the knockout of IGF2BP3, as seen in FIG. 12A. FIG. 12C shows differentially expressed genes between SEM-NT and SEM-I3K0 cells. SEM-13K0 is a SEM strain with a knockout of IGF2BP3. IGF2BP3-CLIP targets are highlighted.

[0065] FIG. 12D shows differentially expressed genes between SEM-NT cells treated with DMSO versus I3IN-002. IGF2BP3-CLIP targets are highlighted.

[0066] FIG. 12E shows the overlap between differentially expressed genes upon IGF2BP3 knockout (“KO”) or treatment with I3IN-002 (“I3IN-002 Rx”).

[0067] FIG. 12F shows pathway enrichment analysis of the genes that were differently expressed upon IGF2BP3 knockout or treatment with I3IN-002.

[0068] FIG. 12G shows RT-qPCR based confirmation of genes showing common differential regulation between I3IN-002 treatment and genetic knockout of IGF2BPe (I3KO). Data are shown as fold change, following normalization to DMSO, with 3 replicate measurements per sample. Statistical significance was evaluated by student’s T-test, *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.

[0069] FIG. 12H shows Western blot measurements of CDK6 and BCL2, which are known targets of IGFBP3.

[0070] FIG. 121 shows RT-qPCR based confirmation of genes showing common differential regulation between I3IN-002 treatment and genetic knockout of IGF2BPe (13 KO) in spleen samples. Data are shown as fold change, following normalization to DMSO. Statistical significance was evaluated by student’s T-test, *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.

[0071] FIG. 13A shows a schematic of assay design. Lin- cells extracted from mouse bone marrow are transduced with MLL-AF4, selected on G418 and then are subjected to treatment with 13 inhibitor compounds. Alternately, they are genetically engineered with a deletion for the Igf2bp3 gene. The cells were then subjected to a colony formation assay in methylcellulose.

[0072] FIG. 13B shows the results of a colony formation assay following treatment with I3IN-002 or with knockout of IGF2BP3 (“I3KO”).

[0073] FIG. 13C shows the results of a colony formation assay following treatment with I3IN-115 or with knockout of IGF2BP3 (“I3KO”).

[0074] FIG. 13D shows flow cytometry analysis of CD34 and c-kit, two markers that have previously been associated with leukemic stem cells, following treatment of cells with I3IN- 002 and I3IN-115. (+ represents 5mM treatment; ++ represents lOmM treatment). Statistical test utilized here was 1-way ANOVA followed by Bonferroni’s multiple comparison test (* p<0.05; **p<0.01).

[0075] FIG. 14A shows fold change in cell viability of diffuse large B-cell lymphoma cell lines (SUDHL-4, OCI-Ly3) tested against I3IN-002.

[0076] FIG. 14B shows fold change in cell viability of diffuse large B-cell lymphoma cell lines (SUDHL-4, OCI-Ly3) tested against I3IN-115.

[0077] FIG. 14C shows fold change in cell viability of Glioblastoma multiforme (GBM) cell lines LN18, U87, and U251 treated with I3IN-002.

[0078] FIG. 14D shows fold change in cell viability of Glioblastoma multiforme (GBM) cell lines LN18, U87, and U251 treated with I3IN-115.

[0079] FIG. 15A shows the measurement of colony formation in methylcellulose following treatment of a murine model of MLL-AF4 driven leukemia with I3IN-002. Data represents the mean number of total colonies for each condition, with 3 replicates per condition. I3KO represents genetic knockout of IGF2BP3, which results in reduced colony formation.

[0080] FIG. 15B shows a FACS histogram of a leukemic stem-cell enriched population by assaying the CD34+ c-kit+ population following in vitro treatment of Lin- / MLL-Af4 cells in culture.

[0081] FIG. 15C shows a FACS histogram of a leukemic stem-cell enriched population by assaying the CD34+ CD38- population following in vitro treatment of OCI- AML-8227 cells in culture.

[0082] FIG. 15D shows quantitation of the FACS data from FIG. 15C. Statistical significance was evaluated by student’s T-test. ***p<0.001; ****p<0.0001.

[0083] FIG. 16A shows a schematic of the mouse experiment that generated the data for FIG. 16B-G. Mice syngeneically transplanted with the Lin-MLL-Af4 cells (either WT or 13 KO) were injected intraperitoneally with I3IN-002 or vehicle control using the schedule indicated.

[0084] FIG. 16B shows gross pathology photographs off murine spleens from mice syngeneically transplanted with Lin- / MLL-Af4 cells. 13 KO are genetic knockouts of IGF2BP3, which markedly reduced leukemic engraftment. FIG. 16C shows splenic weights from mice, treated as indicated. Statistical significance was evaluated by student’s T-test, *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.

[0085] FIG. 16D shows histology of liver, spleen, lung, and kidney from treated mice, as indicated. Arrow indicates a leukemic infiltrate in the liver in the portal area. Dotted lines indicate splenic white pulp, with leukemic infiltrates in the red pulp (area outside the dotted lines). Hematoxylin and eosin staining, original magnification, 200X.

[0086] FIG. 16E shows FACS staining for CD34 and c-kit in the spleen of treated mice, as indicated. Data represents one experiment composed of the following number of animals: vehicle, n=8; 13 IN-002, n=7; I3KO, n=5.

[0087] FIG. 16F shows quantification of overall leukemia engraftment and various leukemia subsets in the spleen. Data represents one experiment composed of the following number of animals: vehicle, n=8; 13 IN-002, n=7; I3KO, n=5. Statistical significance was evaluated by student’s T-test, *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.

[0088] FIG. 16G shows quantification of overall leukemia engraftment and various leukemia subsets in the bone marrow. Data represents one experiment composed of the following number of animals: vehicle, n=8; 13 IN-002, n=7; I3KO, n=5. Statistical significance was evaluated by student’s T-test, *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.

[0089] DETAILED DESCRIPTION OF THE INVENTION

[0090] Defined here as regulation that occurs following the production of an mRNA transcript, post-transcriptional gene regulation involves a number of players, including RNA binding proteins. Recent work has established that RNA-binding proteins, via diverse mechanisms, can be important regulators of oncogenesis. It was found that a set of 36 RNA binding proteins were specifically upregulated in MLL-translocated B-ALL, of which the most significantly dysregulated protein was IGF2BP3. In line with these findings, MLL-AF4 directly interacts with the IGF2BP3 promoter region, based on a recent study, which was confirmed. In B-ALL cell lines, knockdown of IGF2BP3 led to a dramatic reduction in growth and an increase in apoptosis. In vivo, overexpression of IGF2BP3 in the bone marrow led to hyperproliferation in hematopoietic stem and progenitor cells, and a pre-leukemic disease with expansion of B- and myeloid cells. More recently, a deletion allele for murine Igf2bp3 was engineered, and a bone marrow transplantation assay was utilized to induce MLL-AF4-driven leukemia . Remarkably, the deletion of Igf2bp3 significantly limited leukemia development and increased leukemia- free survival. At baseline, the Igf2bp3 knockout mouse did not show any overt phenotypes, strongly suggesting that it is dispensable for normal development and homeostasis in the adult animal. This work, which was recently published , clearly demonstrates the functional role of IGF2BP3 as an oncogene and establishes it as a target in acute leukemia.

[0091] To analyze the mechanistic basis of IGF2BP3 action, iCLIP-seq, a technique designed to globally detect RNA molecules bound by IGF2BP3 in conjunction with differential expression analyses in cells that were depleted for IGF2BP3, was undertaken. These analyses revealed that IGF2BP3 binds to the 3’UTR of mRNA transcripts. The regulated mRNA transcripts included numerous downregulated oncogenes such as HOXA9, CDK6 and MYC . Importantly, deletion of the RNA-binding RRM domains of IGF2BP3 abrogated stabilization of these transcripts, and reversed IGF2BP3-driven pre-leukemic disease in vivo. Together these data suggest that IGF2BP3’s binding to specific oncogenic mRNAs is central to its role in promoting oncogenesis in the hematopoietic system. In addition, data generated by others has now revealed that epitranscriptomic modification of target mRNAs with N6-methyladenosine (m6A) is a determinant of IGF2BP3 binding and function in several different model systems. These specific determinants of mRNA binding (i.e., sequence, m6A modifications) by IGF2BP3 formed the basis of mechanistic understanding of the activity of this protein. Interference with IGF2BP3 RNA-binding function phenocopies IGF2BP3 loss-of-function, based on published data.

[0092] This provided the impetus to inhibit IGF2BP3 as a method to inhibit cancer cell growth, proliferation and to develop a potential therapeutic strategy in cancer that overexpresses IGF2BP3. To this end, a time-resolved Forester resonance energy transfer (TR-FRET) assay was developed and validated, using purified IGF2BP3 protein and a synthetic m6A-modified target RNA molecule to measure binding. A library of small molecule compounds was applied to this assay, with resultant hit compounds being confirmed in TR-FRET and cell-based assays. Confirmatory work to demonstrate activity in a number of leukemia cell lines with high-level IGF2BP3 expression has been completed. The first of these compounds, christened I3IN-002, shows on-target activity based on RNA immunoprecipitation assays from leukemic cells treated with the compound, cellular thermal shift assays, and global gene expression analyses. Together, this data shows a valuable method to screen for and identify small molecule compounds using a biochemical screen and a phenotypic counter-screen, and the first in a series of compounds that appear to inhibit IGF2BP3 and may prove to pave the way for therapeutic development.

[0093] In certain aspects, the present disclosure provides a compound having a structure represented by Formula I or a pharmaceutically acceptable salt thereof: wherein

[0094] A is aryl, heteroaryl, or heterocyclyl; each Ri is independently alkyl, alkoxy, alkenyl, alkynyl, aralkyl, heteroaralkyl, aryl, heteroaryl, halo, haloalkyl, hydroxyl, carboxyl, acyl, ester, thioester, phosphoryl, amino, amido, cyano, nitro, azido, cycloalkyl, heterocyclyl, alkylsulfoxidyl, alkylsulfonyl, or sulfonamido;

[0095] Xi and X2 are each independently N or CH;

[0096] X3 is N, CH, or absent;

[0097] X4is S, O, C(H)(RA), or NRA;

[0098] X5 is O or NRB;

[0099] X6is N(Rx)(Ry) or ORX;

[0100] X7 is O, S, C(H)(Rc), NRc, or is absent;

[0101] Xs is O, S, C(H)(RD), NRD, or is absent;

[0102] RA, RB, RC, RD, RX, and Ryare each independently selected from H, alkyl, alkoxy, aryl, aralkyl, heteroaryl, heteroaralkyl, and cycloalkyl; nl is 0, 1, 2, 3, or 4; and n2 is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12.

[0103] In certain embodiments, RA, RB, RC, RD, RX, and Ryare each independently selected from alkyl, alkoxy, aryl, aralkyl, heteroaryl, heteroaralkyl, or cycloalkyl, each of which is optionally substituted with trifluoromethane or isopropyl.

[0104] In some embodiments, the compound has a structure represented by Formula II or a pharmaceutically acceptable salt thereof: wherein X9 is NH or CH2 and X10 is N or CH.

[0105] In some embodiments, the compound has a structure represented by Formula III or a pharmaceutically acceptable salt thereof: wherein n3 is 0, 1, or 2.

[0106] In some embodiments, A is aryl.

[0107] In some embodiments, the compound has a structure represented by Formula IV or a pharmaceutically acceptable salt thereof:

[0108] IV.

[0109] In some embodiments, Xs is absent.

[0110] In some embodiments, the compound has a structure represented by Formula Va or a pharmaceutically acceptable salt thereof: wherein X9 is N or CH. In some embodiments, the compound has a structure represented by Formula Vb or a pharmaceutically acceptable salt thereof: wherein X9 is N or CH.

[0111] In some embodiments, the compound has a structure represented by Formula Vc or a pharmaceutically acceptable salt thereof: wherein X9 is N or CH.

[0112] In some embodiments, the compound has a structure represented by Formula Vd or a pharmaceutically acceptable salt thereof:

[0113] Vd wherein X9 is N or CH.

[0114] In some embodiments, X9 is CH.

[0115] In some embodiments, the compound has a structure represented by Formula VI or a pharmaceutically acceptable salt thereof:

[0116]

[0117] In some embodiments, Xi is N. In some embodiments, X2 is N. In some embodiments, X3 is N. In some embodiments, X7 is NRc, wherein Rc is selected from H, alkyl, alkoxy, aryl, aralkyl, heteroaryl, heteroaralkyl, and cycloalkyl.

[0118] In some embodiments, the compound has a structure represented by Formula VII or a pharmaceutically acceptable salt thereof:

[0119] VII wherein Rc is selected from H, alkyl, alkoxy, aryl, aralkyl, heteroaryl, heteroaralkyl, and cycloalkyl.

[0120] In some embodiments, X4 is S. In some embodiments, X5 is O. In some embodiments, Xe is N(Rx)(Ry). In some embodiments, nl is 0 or 1. In some embodiments, n2 is 1.

[0121] In some embodiments, the compound has a structure represented by Formula VIII or a pharmaceutically acceptable salt thereof:

[0122] VIII.

[0123] In some embodiments, Rc is H or alkyl (e.g., methyl). In some preferred embodiments, Rc is H. In some preferred embodiments, Rc is methyl. In some embodiments, Rxis H or alkyl (e.g., methyl). In some preferred embodiments, Rxis H. In some preferred embodiments, Rxis methyl.

[0124] In some embodiments, Ryis aryl (e.g., phenyl), aralkyl (e.g., benzyl), heteroaryl (e.g., pyridyl), or cycloalkyl (e.g., cyclopropyl or cyclohexane). In some embodiments, Ryis aryl (e.g., phenyl), or cycloalkyl (e.g., cyclopropyl). In some preferred embodiments, Ryis phenyl.

[0125] In some embodiments, the compound has a structure represented by Formula IX or a pharmaceutically acceptable salt thereof: wherein, each Rzis independently alkyl, alkoxy, alkenyl, alkynyl, aralkyl, heteroaralkyl, aryl, heteroaryl, halo, haloalkyl, hydroxyl, carboxyl, acyl, ester, thioester, phosphoryl, amino, amido, cyano, nitro, azido, cycloalkyl, heterocyclyl, alkylsulfoxidyl, alkylsulfonyl, or sulfonamido; and n3 is 0, 1, 2, 3, 4, or 5.

[0126] In some embodiments, each Rzis independently alkyl (e.g., methyl, difluoromethyl, trifluoromethyl, or isopropyl), alkoxy (e.g., methoxyl), hydroxyl, nitro, or azido. In some embodiments, Rzis alkyl (e.g., methyl, trifluoromethyl, or isopropyl) or alkoxy (e.g., methoxyl). In some embodiments, each Rzis independently methyl, trifluoromethyl, or chlorine.

[0127] In some embodiments, n3 is 0, 1, 2, or 3. In some embodiments, n3 is 0, 1, or 2. In some preferred embodiments, n3 is 1 or 2. In some embodiments, n3 is 1.

[0128] In some embodiments, Rzis ortho to the N-H. In some embodiments, each Rzis para or ortho to the N-H. In some embodiments, each Rzis meta or ortho to the N-H. In some embodiments, each Rzis ortho to the N-H.

[0129] In some embodiments, Ri is alkyl (e.g., methyl, trifluoromethyl, ethyl, or isopropyl), alkoxy (e.g., methoxyl), or halo (e.g., fluorine or chlorine). In some preferred embodiments, Ri is methyl. In some preferred embodiments, Ri is ethyl. In some embodiments, the compound is selected from

[0130] ; or a pharmaceutically acceptable salt thereof.

[0131] In some preferred embodiments, the compound is pharmaceutically acceptable salt thereof. In some preferred embodiments, the compound pharmaceutically acceptable salt thereof.

[0132] In some preferred embodiments, the compound i or a pharmaceutically acceptable salt thereof. In some preferred embodiments, the compound pharmaceutically acceptable salt thereof. In some preferred embodiments, the compound pharmaceutically acceptable salt thereof. In some preferred embodiments, the compound is pharmaceutically acceptable salt thereof. In some preferred embodiments, the compound pharmaceutically acceptable salt thereof. In some preferred embodiments, the compound is pharmaceutically acceptable salt thereof. In some preferred embodiments, the compound pharmaceutically acceptable salt thereof. In some preferred embodiments, the compound is pharmaceutically acceptable salt thereof. In some pharmaceutically acceptable salt thereof. In some preferred embodiments, the compound is pharmaceutically acceptable salt thereof.

[0133] In another aspect, the present disclosure provides compounds having a structure pharmaceutically acceptable salt thereof. In yet another aspect, the present disclosure provides compounds having a structure selected from

[0134] Table 1 or Table 2.

[0135] In another aspect, the present disclosure provides a pharmaceutical composition comprising the compounds recited herein and a pharmaceutically acceptable excipient.

[0136] In another aspect, the present disclosure provides a method of treating cancer in a subject in need thereof, comprising administering a compound recited herein or a pharmaceutically acceptable salt thereof. In certain preferred embodiments, the cancer is characterized by overexpression of RNA-binding protein IGF2BP3.

[0137] In certain embodiments, the cancer is a hematological malignancy, such as a leukemia or lymphoma, for example a non-Hodgkin’s lymphoma. In certain embodiments, the hematological malignancy is myelogenous leukemia, myeloid leukemia (e.g., acute myeloid leukemia), myelodysplastic syndrome, lymphoblastic leukemia (e.g., acute lymphoblastic leukemia), chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), high risk CLL, follicular lymphoma, diffuse large B-cell lymphoma (DLBCL) (e.g., DLBCL or ABC-DLBLC), mantle cell lymphoma (MCL), Waldenstrom’s macroglobulinemia (WM), multiple myeloma, marginal zone lymphoma (MZL), Burkitt’s lymphoma, non-Burkitt high grade B cell lymphoma, extranodal marginal zone B cell lymphoma, transformed high grade B-cell lymphoma (HGBL), lymphoplasmacytic lymphoma (LPL), central nervous system lymphoma (CNSL), or MALT lymphoma. In certain embodiments, the hematological malignancy is myelogenous leukemia. In other embodiments, the hematological malignancy is myeloid leukemia (e.g., acute myeloid leukemia). In certain embodiments, the hematological malignancy is acute myeloid leukemia (e.g., AML). In certain embodiments, the AML is primary AML. In other embodiments, the AML is secondary AML. In yet other embodiments, the hematological malignancy is myelodysplastic syndrome. In certain embodiments, the myelodysplastic syndrome is high grade. In other embodiments, the myelodysplastic syndrome is low grade. In certain embodiments, the myelodysplastic syndrome is high risk. In yet other embodiments, the hematological malignancy is lymphoblastic leukemia (e.g., acute lymphoblastic leukemia). In yet other embodiments, the hematological malignancy is chronic lymphocytic leukemia (CLL). In certain embodiments, the CLL is high risk CLL. In yet other embodiments, the hematological malignancy is small lymphocytic lymphoma (SLL). In yet other embodiments, the hematological malignancy is follicular lymphoma. In yet other embodiments, the hematological malignancy is diffuse large B-cell lymphoma (DLBCL). In yet other embodiments, the hematological malignancy is activated B cell-like (ABC) DLBCL. In yet other embodiments, the hematological malignancy is germinal center B cell-like (GCB) DLBCL. In certain embodiments, the DLBCL is extranodal. In certain embodiments, the DLBCL is extranodal leg lymphoma, extranodal testicle lymphoma, or extra nodal not otherwise specified (NOS) type lymphoma. In yet other embodiments, the hematological malignancy is mantle cell lymphoma. In further embodiments, the hematological malignancy is Waldenstrom’s macroglobulinemia. In yet other embodiments, the hematological malignancy is multiple myeloma. In still other embodiments, the hematological malignancy is marginal zone lymphoma. In yet other embodiments, the hematological malignancy is Burkitt’s lymphoma. In yet other embodiments, the hematological malignancy is non-Burkitt high grade B cell lymphoma. In still other embodiments, the hematological malignancy is extranodal marginal zone B cell lymphoma. In yet other embodiments, the hematological malignancy is transformed high grade B-cell lymphoma (HGBL). In yet other embodiments, the hematological malignancy is lymphoplasmacytic lymphoma (LPL). In yet other embodiments, the hematological malignancy is CNS lymphoma. In yet other embodiments, the CNS lymphoma is primary CNS lymphoma (PCNSL). In yet other embodiments, the hematological malignancy is MALT lymphoma. In certain embodiments, the hematological malignancies described above may be relapsed or refractory.

[0138] In certain embodiments, the cancer is leukemia. In certain preferred embodiments, the leukemia is acute lymphoblastic leukemia (B-ALL). In certain preferred embodiments, the ALL is B -acute lymphoblastic leukemia with translocations involving the MLL (KMT2A) gene. In certain embodiments, the leukemia is acute myeloid leukemia (AML).

[0139] In some embodiments, the hematological malignancy is acute lymphoblastic leukemia (ALL). In some embodiments, the ALL is B-cell acute lymphoblastic leukemia with translocations involving the MLL (KMT2A) gene. In some embodiments, the hematological malignancy is myelogenous leukemia. In some embodiments, the hematological malignancy is myeloid leukemia. In some embodiments, the myeloid leukemia is acute myeloid leukemia (AML). In some embodiments, the AML is primary AML. In some embodiments, the AML is secondary AML.

[0140] Pharmaceutical Compositions

[0141] The compositions and methods of the present disclosure may be utilized to treat an individual in need thereof. In certain embodiments, the individual is a mammal such as a human, or a non-human mammal. When administered to an animal, such as a human, the composition or the compound is preferably administered as a pharmaceutical composition comprising, for example, a compound of the invention and a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are well known in the art and include, for example, aqueous solutions such as water or physiologically buffered saline or other solvents or vehicles such as glycols, glycerol, oils such as olive oil, or injectable organic esters. In preferred embodiments, when such pharmaceutical compositions are for human administration, particularly for invasive routes of administration (i.e., routes, such as injection or implantation, that circumvent transport or diffusion through an epithelial barrier), the aqueous solution is pyrogen-free, or substantially pyrogen-free. The excipients can be chosen, for example, to effect delayed release of an agent or to selectively target one or more cells, tissues or organs. The pharmaceutical composition can be in dosage unit form such as tablet, capsule (including sprinkle capsule and gelatin capsule), granule, lyophile for reconstitution, powder, solution, syrup, suppository, injection or the like. The composition can also be present in a transdermal delivery system, e.g., a skin patch. The composition can also be present in a solution suitable for topical administration, such as a lotion, cream, or ointment.

[0142] A pharmaceutically acceptable carrier can contain physiologically acceptable agents that act, for example, to stabilize, increase solubility or to increase the absorption of a compound such as a compound of the invention. Such physiologically acceptable agents include, for example, carbohydrates, such as glucose, sucrose or dextrans, antioxidants, such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins or other stabilizers or excipients. The choice of a pharmaceutically acceptable carrier, including a physiologically acceptable agent, depends, for example, on the route of administration of the composition. The preparation or pharmaceutical composition can be a self-emulsifying drug delivery system or a self-microemulsifying drug delivery system. The pharmaceutical composition (preparation) also can be a liposome or other polymer matrix, which can have incorporated therein, for example, a compound of the invention. Liposomes, for example, which comprise phospholipids or other lipids, are nontoxic, physiologically acceptable and metabolizable carriers that are relatively simple to make and administer.

[0143] The phrase "pharmaceutically acceptable" is employed herein to refer to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0144] The phrase "pharmaceutically acceptable carrier" as used herein means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials which can serve as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, com oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21) other non-toxic compatible substances employed in pharmaceutical formulations.

[0145] A pharmaceutical composition (preparation) can be administered to a subject by any of a number of routes of administration including, for example, orally (for example, drenches as in aqueous or non-aqueous solutions or suspensions, tablets, capsules (including sprinkle capsules and gelatin capsules), boluses, powders, granules, pastes for application to the tongue); absorption through the oral mucosa (e.g., sublingually); subcutaneously; transdermally (for example as a patch applied to the skin); and topically (for example, as a cream, ointment or spray applied to the skin). The compound may also be formulated for inhalation. In certain embodiments, a compound may be simply dissolved or suspended in sterile water. The formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the host being treated and the particular mode of administration. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound which produces a therapeutic effect. Generally, out of one hundred percent, this amount will range from about 1 percent to about ninety-nine percent of active ingredient, preferably from about 5 percent to about 70 percent, most preferably from about 10 percent to about 30 percent.

[0146] Methods of preparing these formulations or compositions include the step of bringing into association an active compound, such as a compound of the invention, with the carrier and, optionally, one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association a compound of the present disclosure with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product.

[0147] Formulations of the disclosure suitable for oral administration may be in the form of capsules (including sprinkle capsules and gelatin capsules), cachets, pills, tablets, lozenges (using a flavored basis, usually sucrose and acacia or tragacanth), lyophile, powders, granules, or as a solution or a suspension in an aqueous or non-aqueous liquid, or as an oil-in- water or water-in-oil liquid emulsion, or as an elixir or syrup, or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and acacia) and / or as mouth washes and the like, each containing a predetermined amount of a compound of the present disclosure as an active ingredient. Compositions or compounds may also be administered as a bolus, electuary or paste.

[0148] To prepare solid dosage forms for oral administration (capsules (including sprinkle capsules and gelatin capsules), tablets, pills, dragees, powders, granules and the like), the active ingredient is mixed with one or more pharmaceutically acceptable carriers, such as sodium citrate or dicalcium phosphate, and / or any of the following: (1) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and / or silicic acid; (2) binders, such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and / or acacia; (3) humectants, such as glycerol; (4) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) solution retarding agents, such as paraffin; (6) absorption accelerators, such as quaternary ammonium compounds; (7) wetting agents, such as, for example, cetyl alcohol and glycerol monostearate; (8) absorbents, such as kaolin and bentonite clay; (9) lubricants, such a talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof; (10) complexing agents, such as, modified and unmodified cyclodextrins; and (11) coloring agents. In the case of capsules (including sprinkle capsules and gelatin capsules), tablets and pills, the pharmaceutical compositions may also comprise buffering agents. Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugars, as well as high molecular weight polyethylene glycols and the like.

[0149] A tablet may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared using binder (for example, gelatin or hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (for example, sodium starch glycolate or cross-linked sodium carboxymethyl cellulose), surface-active or dispersing agent. Molded tablets may be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.

[0150] The tablets, and other solid dosage forms of the pharmaceutical compositions, such as dragees, capsules (including sprinkle capsules and gelatin capsules), pills and granules, may optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical-formulating art. They may also be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile, other polymer matrices, liposomes and / or microspheres. They may be sterilized by, for example, filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions that can be dissolved in sterile water, or some other sterile injectable medium immediately before use. These compositions may also optionally contain opacifying agents and may be of a composition that they release the active ingredient(s) only, or preferentially, in a certain portion of the gastrointestinal tract, optionally, in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes. The active ingredient can also be in micro- encapsulated form, if appropriate, with one or more of the above-described excipients. Liquid dosage forms useful for oral administration include pharmaceutically acceptable emulsions, lyophiles for reconstitution, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active ingredient, the liquid dosage forms may contain inert diluents commonly used in the art, such as, for example, water or other solvents, cyclodextrins and derivatives thereof, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.

[0151] Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents.

[0152] Suspensions, in addition to the active compounds, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.

[0153] Dosage forms for the topical or transdermal administration include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants. The active compound may be mixed under sterile conditions with a pharmaceutically acceptable carrier, and with any preservatives, buffers, or propellants that may be required.

[0154] The ointments, pastes, creams and gels may contain, in addition to an active compound, excipients, such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.

[0155] Powders and sprays can contain, in addition to an active compound, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances. Sprays can additionally contain customary propellants, such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane.

[0156] Transdermal patches have the added advantage of providing controlled delivery of a compound of the present disclosure to the body. Such dosage forms can be made by dissolving or dispersing the active compound in the proper medium. Absorption enhancers can also be used to increase the flux of the compound across the skin. The rate of such flux can be controlled by either providing a rate controlling membrane or dispersing the compound in a polymer matrix or gel.

[0157] The phrases "parenteral administration" and "administered parenterally" as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal and intrastemal injection and infusion. Pharmaceutical compositions suitable for parenteral administration comprise one or more active compounds in combination with one or more pharmaceutically acceptable sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents.

[0158] Examples of suitable aqueous and nonaqueous carriers that may be employed in the pharmaceutical compositions of the invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

[0159] These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents that delay absorption such as aluminum monostearate and gelatin.

[0160] In some cases, in order to prolong the effect of a drug, it is desirable to slow the absorption of the drug from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material having poor water solubility. The rate of absorption of the drug then depends upon its rate of dissolution, which, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered drug form is accomplished by dissolving or suspending the drug in an oil vehicle.

[0161] Injectable depot forms are made by forming microencapsulated matrices of the subject compounds in biodegradable polymers such as polylactide-polyglycolide. Depending on the ratio of drug to polymer, and the nature of the particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions that are compatible with body tissue.

[0162] For use in the methods of this invention, active compounds can be given per se or as a pharmaceutical composition containing, for example, 0.1 to 99.5% (more preferably, 0.5 to 90%) of active ingredient in combination with a pharmaceutically acceptable carrier.

[0163] Methods of introduction may also be provided by rechargeable or biodegradable devices. Various slow release polymeric devices have been developed and tested in vivo in recent years for the controlled delivery of drugs, including proteinaceous biopharmaceuticals. A variety of biocompatible polymers (including hydrogels), including both biodegradable and non-degradable polymers, can be used to form an implant for the sustained release of a compound at a particular target site.

[0164] Actual dosage levels of the active ingredients in the pharmaceutical compositions may be varied so as to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.

[0165] The selected dosage level will depend upon a variety of factors including the activity of the particular compound or combination of compounds employed, or the ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion of the particular compound(s) being employed, the duration of the treatment, other drugs, compounds and / or materials used in combination with the particular compound(s) employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.

[0166] A physician or veterinarian having ordinary skill in the art can readily determine and prescribe therapeutically effective amount of the pharmaceutical composition required. For example, the physician or veterinarian could start doses of the pharmaceutical composition or compound at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. By “therapeutically effective amount” it is meant the concentration of a compound that is sufficient to elicit the desired therapeutic effect. It is generally understood that the effective amount of the compound will vary according to the weight, sex, age, and medical history of the subject. Other factors which influence the effective amount may include, but are not limited to, the severity of the patient's condition, the disorder being treated, the stability of the compound, and, if desired, another type of therapeutic agent being administered with the compound of the invention. A larger total dose can be delivered by multiple administrations of the agent. Methods to determine efficacy and dosage are known to those skilled in the art (Isselbacher et al. (1996) Harrison’s Principles of Internal Medicine 13 ed., 1814-1882, herein incorporated by reference).

[0167] In general, a suitable daily dose of an active compound used in the compositions and methods of the invention will be that amount of the compound that is the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally depend upon the factors described above.

[0168] If desired, the effective daily dose of the active compound may be administered as one, two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms. In certain embodiments of the present disclosure, the active compound may be administered two or three times daily. In preferred embodiments, the active compound will be administered once daily.

[0169] The patient receiving this treatment is any animal in need, including primates, in particular humans; and other mammals such as equines, cattle, swine, sheep, cats, and dogs; poultry; and pets in general.

[0170] In certain embodiments, compounds of the invention may be used alone or conjointly administered with another type of therapeutic agent.

[0171] The present disclosure includes the use of pharmaceutically acceptable salts of compounds of the invention in the compositions and methods of the present disclosure. In certain embodiments, contemplated salts of the invention include, but are not limited to, alkyl, dialkyl, trialkyl or tetra-alkyl ammonium salts. In certain embodiments, contemplated salts of the invention include, but are not limited to, L-arginine, benethamine, benzathine, betaine, calcium hydroxide, choline, deanol, diethanolamine, diethylamine, 2- (diethylamino)ethanol, ethanolamine, ethylenediamine, N-methylglucamine, hydrabamine, IH-imidazole, lithium, L-lysine, magnesium, 4-(2-hydroxyethyl)morpholine, piperazine, potassium, l-(2-hydroxyethyl)pyrrolidine, sodium, triethanolamine, tromethamine, and zinc salts. In certain embodiments, contemplated salts of the invention include, but are not limited to, Na, Ca, K, Mg, Zn or other metal salts. In certain embodiments, contemplated salts of the invention include, but are not limited to, l-hydroxy-2-naphthoic acid, 2,2-dichloroacetic acid, 2-hydroxyethanesulfonic acid, 2-oxoglutaric acid, 4-acetamidobenzoic acid, 4-aminosalicylic acid, acetic acid, adipic acid, 1-ascorbic acid, 1-aspartic acid, benzenesulfonic acid, benzoic acid, (+)-camphoric acid, (+)-camphor-10-sulfonic acid, capric acid (decanoic acid), caproic acid (hexanoic acid), caprylic acid (octanoic acid), carbonic acid, cinnamic acid, citric acid, cyclamic acid, dodecylsulfuric acid, ethane- 1,2-disulfonic acid, ethanesulfonic acid, formic acid, fumaric acid, galactaric acid, gentisic acid, d-glucoheptonic acid, d-gluconic acid, d-glucuronic acid, glutamic acid, glutaric acid, glycerophosphoric acid, glycolic acid, hippuric acid, hydrobromic acid, hydrochloric acid, isobutyric acid, lactic acid, lactobionic acid, lauric acid, maleic acid, 1-malic acid, malonic acid, mandelic acid, methanesulfonic acid , naphthalene- 1,5-disulfonic acid, naphthalene-2- sulfonic acid, nicotinic acid, nitric acid, oleic acid, oxalic acid, palmitic acid, pamoic acid, phosphoric acid, propionic acid, 1- pyroglutamic acid, salicylic acid, sebacic acid, stearic acid, succinic acid, sulfuric acid, 1-tartaric acid, thiocyanic acid, p-toluenesulfonic acid, trifluoroacetic acid, and undecylenic acid salts.

[0172] The pharmaceutically acceptable acid addition salts can also exist as various solvates, such as with water, methanol, ethanol, dimethylformamide, and the like. Mixtures of such solvates can also be prepared. The source of such solvate can be from the solvent of crystallization, inherent in the solvent of preparation or crystallization, or adventitious to such solvent.

[0173] Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions.

[0174] Examples of pharmaceutically acceptable antioxidants include: (1) water-soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal-chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

[0175] Definitions

[0176] Unless otherwise defined herein, scientific and technical terms used in this application shall have the meanings that are commonly understood by those of ordinary skill in the art. Generally, nomenclature used in connection with, and techniques of, chemistry, cell and tissue culture, molecular biology, cell and cancer biology, neurobiology, neurochemistry, virology, immunology, microbiology, pharmacology, genetics and protein and nucleic acid chemistry, described herein, are those well-known and commonly used in the art.

[0177] The methods and techniques of the present disclosure are generally performed, unless otherwise indicated, according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout this specification. See, e.g. “Principles of Neural Science”, McGraw-Hill Medical, New York, N.Y. (2000); Motulsky, “Intuitive Biostatistics”, Oxford University Press, Inc. (1995); Lodish et al., “Molecular Cell Biology, 4th ed ”, W. H. Freeman & Co., New York (2000); Griffiths et al., “Introduction to Genetic Analysis, 7th ed.”, W. H. Freeman & Co., N.Y. (1999); and Gilbert et al., “Developmental Biology, 6th ed.”, Sinauer Associates, Inc., Sunderland, MA (2000).

[0178] Chemistry terms used herein, unless otherwise defined herein, are used according to conventional usage in the art, as exemplified by “The McGraw-Hill Dictionary of Chemical Terms”, Parker S., Ed., McGraw-Hill, San Francisco, C.A. (1985).

[0179] All of the above, and any other publications, patents and published patent applications referred to in this application are specifically incorporated by reference herein. In case of conflict, the present specification, including its specific definitions, will control.

[0180] The term “agent” is used herein to denote a chemical compound (such as an organic or inorganic compound, a mixture of chemical compounds), a biological macromolecule (such as a nucleic acid, an antibody, including parts thereof as well as humanized, chimeric and human antibodies and monoclonal antibodies, a protein or portion thereof, e.g., a peptide, a lipid, a carbohydrate), or an extract made from biological materials such as bacteria, plants, fungi, or animal (particularly mammalian) cells or tissues. Agents include, for example, agents whose structure is known, and those whose structure is not known. The ability of such agents to inhibit AR or promote AR degradation may render them suitable as “therapeutic agents” in the methods and compositions of this disclosure.

[0181] A “patient,” “subject,” or “individual” are used interchangeably and refer to either a human or a non-human animal. These terms include mammals, such as humans, primates, livestock animals (including bovines, porcines, etc.), companion animals (e.g., canines, felines, etc.) and rodents (e.g., mice and rats).

[0182] “Treating” a condition or patient refers to taking steps to obtain beneficial or desired results, including clinical results. Beneficial or desired clinical results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, preventing spread of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. “Treatment” can also mean prolonging survival as compared to expected survival if not receiving treatment.

[0183] The term “preventing” is art-recognized, and when used in relation to a condition, such as a local recurrence (e.g., pain), a disease such as cancer, a syndrome complex such as heart failure or any other medical condition, is well understood in the art, and includes administration of a composition which reduces the frequency of, or delays the onset of, symptoms of a medical condition in a subject relative to a subject which does not receive the composition. Thus, prevention of cancer includes, for example, reducing the number of detectable cancerous growths in a population of patients receiving a prophylactic treatment relative to an untreated control population, and / or delaying the appearance of detectable cancerous growths in a treated population versus an untreated control population, e.g., by a statistically and / or clinically significant amount.

[0184] “Administering” or “administration of’ a substance, a compound or an agent to a subject can be carried out using one of a variety of methods known to those skilled in the art. For example, a compound or an agent can be administered, intravenously, arterially, intradermally, intramuscularly, intraperitoneally, subcutaneously, ocularly, sublingually, orally (by ingestion), intranasally (by inhalation), intraspinally, intracerebrally, and transdermally (by absorption, e.g., through a skin duct). A compound or agent can also appropriately be introduced by rechargeable or biodegradable polymeric devices or other devices, e.g., patches and pumps, or formulations, which provide for the extended, slow or controlled release of the compound or agent. Administering can also be performed, for example, once, a plurality of times, and / or over one or more extended periods.

[0185] Appropriate methods of administering a substance, a compound or an agent to a subject will also depend, for example, on the age and / or the physical condition of the subject and the chemical and biological properties of the compound or agent (e.g., solubility, digestibility, bioavailability, stability and toxicity). In certain embodiments, a compound or an agent is administered orally, e.g., to a subject by ingestion. In certain embodiments, the orally administered compound or agent is in an extended release or slow release formulation, or administered using a device for such slow or extended release.

[0186] A “therapeutically effective amount” or a “therapeutically effective dose” of a drug or agent is an amount of a drug or an agent that, when administered to a subject will have the intended therapeutic effect. The full therapeutic effect does not necessarily occur by administration of one dose, and may occur only after administration of a series of doses. Thus, a therapeutically effective amount may be administered in one or more administrations. The precise effective amount needed for a subject will depend upon, for example, the subject’s size, health and age, and the nature and extent of the condition being treated, such as cancer or MDS. The skilled worker can readily determine the effective amount for a given situation by routine experimentation.

[0187] As used herein, the terms “optional” or “optionally” mean that the subsequently described event or circumstance may occur or may not occur, and that the description includes instances where the event or circumstance occurs as well as instances in which it does not. For example, “optionally substituted alkyl” refers to the alkyl may be substituted as well as where the alkyl is not substituted.

[0188] It is understood that substituents and substitution patterns on the compounds of the present disclosure can be selected by one of ordinary skilled person in the art to result chemically stable compounds which can be readily synthesized by techniques known in the art, as well as those methods set forth below, from readily available starting materials. If a substituent is itself substituted with more than one group, it is understood that these multiple groups may be on the same carbon or on different carbons, so long as a stable structure results. As used herein, the term “optionally substituted” refers to the replacement of one to six hydrogen radicals in a given structure with the radical of a specified substituent including, but not limited to: hydroxyl, hydroxyalkyl, alkoxy (e.g. methoxy), halogen (e.g. fluorine or chlorine), alkyl (e.g. methyl, trifluoromethyl, ethyl, or isopropyl), alkenyl, alkynyl, aralkyl, heteroaralkyl, nitro, silyl, acyl, acyloxy, aryl, heteroaryl, cycloalkyl, heterocyclyl, amino, aminoalkyl, cyano, haloalkyl, haloalkoxy, -OCO-Ctk-O-alkyl, -OP(O)(O-alkyl)2 or -CH2- OP(O)(O-alkyl)2. Preferably, “optionally substituted” refers to the replacement of one to five hydrogen radicals in a given structure with the substituents mentioned above. More preferably, one to two hydrogen radicals are replaced by the substituents as mentioned above. It is understood that the substituent can be further substituted.

[0189] As used herein, "alkyl" refers to a straight-chain or branched-chain aliphatic saturated hydrocarbon group, and may be preferably an alkyl having 1 to 6 carbon atoms, and more preferably an alkyl having 1 to 4 carbon atoms. Examples of such alkyls include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl, 1- ethylpropyl, hexyl, isohexyl, 1,1-dimethyl butyl, 2,2-dimethylbutyl, 3,3-dimethylbutyl, and 2- ethylbutyl. The “alkyl” group may be optionally substituted. Moreover, the term “alkyl” as used throughout the specification, examples, and claims is intended to include both unsubstituted and substituted alkyl groups, the latter of which refers to alkyl moieties having substituents replacing a hydrogen on one or more carbons of the hydrocarbon backbone, including haloalkyl groups such as trifluoromethyl and 2,2,2-trifluoroethyl, etc.

[0190] The term “acyl” is art-recognized and refers to a group represented by the general formula hydrocarbylC(O)-, preferably alkylC(O)-.

[0191] The term “acylamino” is art-recognized and refers to an amino group substituted with an acyl group and may be represented, for example, by the formula hydrocarbylC(O)NH-.

[0192] The term “acyloxy” is art-recognized and refers to a group represented by the general formula hydrocarbylC(O)O-, preferably alkylC(O)O-.

[0193] The term “alkoxy” refers to an alkyl group having an oxygen attached thereto. Representative alkoxy groups include methoxy, ethoxy, propoxy, tert-butoxy and the like.

[0194] The term “alkoxyalkyl” refers to an alkyl group substituted with an alkoxy group and may be represented by the general formula alkyl-O-alkyl.

[0195] The term “Cx.y” or “Cx-Cy”, when used in conjunction with a chemical moiety, such as, acyl, acyloxy, alkyl, alkenyl, alkynyl, or alkoxy is meant to include groups that contain from x to y carbons in the chain. Coalkyl indicates a hydrogen where the group is in a terminal position, a bond if internal. A Ci -ealkyl group, for example, contains from one to six carbon atoms in the chain.

[0196] The term “alkylamino”, as used herein, refers to an amino group substituted with at least one alkyl group.

[0197] The term “alkylthio”, as used herein, refers to a thiol group substituted with an alkyl group and may be represented by the general formula alkylS-.

[0198] The term “amide”, as used herein, refers to a group wherein R9and R10each independently represent a hydrogen or hydrocarbyl group, or R9and R10taken together with the N atom to which they are attached complete a heterocycle having from 4 to 8 atoms in the ring structure.

[0199] The terms “amine” and “amino” are art-recognized and refer to both unsubstituted and substituted amines and salts thereof, e.g., a moiety that can be represented by wherein R9, R10, and R10’ each independently represent a hydrogen or a hydrocarbyl group, or R9and R10taken together with the N atom to which they are attached complete a heterocycle having from 4 to 8 atoms in the ring structure.

[0200] The term “aminoalkyl”, as used herein, refers to an alkyl group substituted with an amino group.

[0201] As used herein, "aryl" refers to a carbocyclic aromatic group that may be further fused with a second 5- or 6-membered carbocyclic group that may be aromatic, saturated or unsaturated, and examples of aryl may include, but are not limited to, phenyl, indanyl, 1- naphthyl, 2-naphthyl, tetrahydronapthyl, and the like. Aryl may be linked to other groups at appropriate positions on the aromatic ring. Preferably the ring is a 5- to 7-membered ring, more preferably a 6-membered ring. Aryl groups include benzene, naphthalene, phenanthrene, phenol, aniline, and the like.

[0202] The term “carbamate” is art-recognized and refers to a group wherein R9and R10each independently represent hydrogen or a hydrocarbyl group.

[0203] The term “carbocyclylalkyl”, as used herein, refers to an alkyl group substituted with a carbocycle group.

[0204] The term “carbocycle” includes 5-7 membered monocyclic and 8-12 membered bicyclic rings. Each ring of a bicyclic carbocycle may be selected from saturated, unsaturated and aromatic rings. Carbocycle includes bicyclic molecules in which one, two or three or more atoms are shared between the two rings. The term “fused carbocycle” refers to a bicyclic carbocycle in which each of the rings shares two adjacent atoms with the other ring. Each ring of a fused carbocycle may be selected from saturated, unsaturated and aromatic rings. In an exemplary embodiment, an aromatic ring, e.g., phenyl, may be fused to a saturated or unsaturated ring, e.g., cyclohexane, cyclopentane, or cyclohexene. Any combination of saturated, unsaturated and aromatic bicyclic rings, as valence permits, is included in the definition of carbocyclic. Exemplary “carbocycles” include cyclopentane, cyclohexane, bicyclo[2.2.1]heptane, 1,5 -cyclooctadiene, 1,2,3,4-tetrahydronaphthalene, bicyclo[4.2.0]oct-3-ene, naphthalene and adamantane. Exemplary fused carbocycles include decalin, naphthalene, 1,2,3,4-tetrahydronaphthalene, bicyclo[4.2.0]octane, 4,5,6,7-tetrahydro- IH-indene and bicyclo[4.1.0]hept-3-ene. “Carbocycles” may be substituted at any one or more positions capable of bearing a hydrogen atom.

[0205] The term “carbocyclylalkyl”, as used herein, refers to an alkyl group substituted with a carbocycle group.

[0206] The term “carbonate” is art-recognized and refers to a group -OCO2-. The term “carboxy”, as used herein, refers to a group represented by the formula -CO2H.

[0207] The term “cycloalkyl” includes substituted or unsubstituted non-aromatic single ring structures, preferably 4- to 8-membered rings, more preferably 4- to 6-membered rings. The term “cycloalkyl” also includes polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjoining rings wherein at least one of the rings is cycloalkyl and the substituent (e.g., R100) is attached to the cycloalkyl ring, e.g., the other cyclic rings can be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and / or heterocyclyls. Heteroaryl groups include, for example, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, pyrazole, pyridine, pyrazine, pyridazine, pyrimidine, denzodioxane, tetrahydroquinoline, and the like.

[0208] The term “ester”, as used herein, refers to a group -C(O)OR9wherein R9represents a hydrocarbyl group.

[0209] The term “ether”, as used herein, refers to a hydrocarbyl group linked through an oxygen to another hydrocarbyl group. Accordingly, an ether substituent of a hydrocarbyl group may be hydrocarbyl-O-. Ethers may be either symmetrical or unsymmetrical. Examples of ethers include, but are not limited to, heterocycle-O-heterocycle and aryl-O- heterocycle. Ethers include “alkoxyalkyl” groups, which may be represented by the general formula alkyl-O-alkyl.

[0210] The terms “halo” and “halogen” as used herein means halogen and includes chloro, fluoro, bromo, and iodo.

[0211] The terms “hetaralkyl” and “heteroaralkyl”, as used herein, refers to an alkyl group substituted with a hetaryl group.

[0212] As used herein, "heteroaryl" refers to a heteroaromatic compound containing at least one heteroatom selected from the group consisting of N, O and S, unless otherwise specified, and preferably the heteroaryl group may include, but are not limited to, a pyridine group, a pyrazine group, a pyrimidine group, a pyridazine group, a pyrazole group, an imidazole group, a triazole group, an indole group, an oxadiazole group, a thiadiazole group, a quinoline group, an isoquinoline group, an isoxazole group, an oxazole group, a thiazole group, and pyrrole group. The terms “heteroaryl” and “hetaryl” also include polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjoining rings wherein at least one of the rings is heteroaromatic, e.g., the other cyclic rings can be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and / or heterocyclyls. Heteroaryl groups include, for example, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, pyrazole, pyridine, pyrazine, pyridazine, and pyrimidine, and the like.

[0213] The term “heteroatom” as used herein means an atom of any element other than carbon or hydrogen. Preferred heteroatoms are nitrogen, oxygen, and sulfur.

[0214] The term “heterocyclylalkyl”, as used herein, refers to an alkyl group substituted with a heterocycle group. As used herein, "heterocycle" refers to an aromatic or non-aromatic ring containing a heteroatom selected from a nitrogen atom, a sulfur atom and an oxygen atom other than a carbon atom as a ring member atom, and preferably includes a 4- to 10-membered and more preferably a 5- to 9-membered aromatic or non-aromatic ring containing 1 to 4 of the above heteroatoms. Examples of such aromatic rings include thienyl, furyl, pyrrolyl, imidazolyl, pyrazolyl, thiazolyl, isothiazolyl, oxazolyl, isoxazolyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, 1,2,4-oxadiazolyl, 1,3,4-oxadiazolyl, 1,2,4-thiadiazolyl, 1,3,4-thiadiazolyl, triazolyl, tetrazolyl, triazinyl, and benzo thiazolyl. In addition, examples of such non-aromatic rings include tetrahydrothienyl, tetrahydrofuranyl, pyrrolinyl, pyrrolidinyl, imidazolinyl, imidazolidinyl, oxazolinyl, oxazolidinyl, pyrazolinyl, pyrazolidinyl, thiazolinyl, thiazolidinyl, tetrahydroisothiazolyl, tetrahydrooxazolyl, tetrahydroisoxazolyl, piperidinyl, piperazinyl, tetrahydropyridinyl, dihydropyridinyl, dihydrothiopyranyl, tetrahydropyrimidinyl, tetrahydropyridazinyl, dihydropyranyl, tetrahydropyranyl, tetrahydrothiopyranyl, morpholinyl, thiomorpholinyl, azepanyl, diazepanyl, and azepinyl. The terms “heterocyclyl” and “heterocyclic” also include polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjoining rings wherein at least one of the rings is heterocyclic, e.g., the other cyclic rings can be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and / or heterocyclyls.

[0215] As used herein, "arylene" and “heteroarylene” refer to divalent radicals of an aromatic ring and a heteroaromatic ring.

[0216] The term “hydrocarbyl”, as used herein, refers to a group that is bonded through a carbon atom that does not have a =0 or =S substituent, and typically has at least one carbonhydrogen bond and a primarily carbon backbone, but may optionally include heteroatoms. Thus, groups like methyl, ethoxyethyl, 2-pyridyl, and even trifluoromethyl are considered to be hydrocarbyl for the purposes of this application, but substituents such as acetyl (which has a =0 substituent on the linking carbon) and ethoxy (which is linked through oxygen, not carbon) are not. Hydrocarbyl groups include, but are not limited to aryl, heteroaryl, carbocycle, heterocycle, alkyl, alkenyl, alkynyl, and combinations thereof.

[0217] The term “hydroxy alkyl”, as used herein, refers to an alkyl group substituted with a hydroxy group.

[0218] The term “lower” when used in conjunction with a chemical moiety, such as, acyl, acyloxy, alkyl, alkenyl, alkynyl, or alkoxy is meant to include groups where there are ten or fewer atoms in the substituent, preferably six or fewer. A “lower alkyl”, for example, refers to an alkyl group that contains ten or fewer carbon atoms, preferably six or fewer. In certain embodiments, acyl, acyloxy, alkyl, alkenyl, alkynyl, or alkoxy substituents defined herein are respectively lower acyl, lower acyloxy, lower alkyl, lower alkenyl, lower alkynyl, or lower alkoxy, whether they appear alone or in combination with other substituents, such as in the recitations hydroxyalkyl and aralkyl (in which case, for example, the atoms within the aryl group are not counted when counting the carbon atoms in the alkyl substituent).

[0219] The term “phosphoryl” is art-recognized and refers to the group represented by the general formula wherein R9and R10each independently represent a negative charge, hydrogen, or hydrocarbyl.

[0220] The term “phosphoramidityl” is art-recognized and refers to the group represented by the general formula wherein R9represents a negative charge, hydrogen, or hydrocarbyl; and each R10independently represents hydrogen or hydrocarbyl.

[0221] The term “phosphoramidatyl” is art-recognized and refers to the group represented by the general formula wherein each R9and R10independently represent hydrogen or hydrocarbyl.

[0222] The terms “polycyclyl”, “polycycle”, and “polycyclic” refer to two or more rings (e.g., cycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and / or heterocyclyls) in which two or more atoms are common to two adjoining rings, e.g., the rings are “fused rings”. Each of the rings of the polycycle can be substituted or unsubstituted. In certain embodiments, each ring of the polycycle contains from 3 to 10 atoms in the ring, preferably from 5 to 7.

[0223] The term “sulfate” is art-recognized and refers to the group -OSO3H, or a pharmaceutically acceptable salt thereof.

[0224] The term “sulfonamide” is art-recognized and refers to the group represented by the general formulae wherein each R9and R10independently represents hydrogen or hydrocarbyl.

[0225] The term “sulfoxide” is art-recognized and refers to the group-S(O)-.

[0226] The term “sulfonate” is art-recognized and refers to the group SO3H, or a pharmaceutically acceptable salt thereof.

[0227] The term “sulfone” is art-recognized and refers to the group -S(O)2-.

[0228] The term “iminosulfanonyl” is art-recognized and refers to the group represented by the general formula wherein R9and R10each independently represents hydrogen or hydrocarbyl.

[0229] In certain embodiments, as used herein, "substituent" includes a halogen group, a cyano group, a nitro group, a substituted or unsubstituted amino group including a substituted or unsubstituted alkyl group and a substituted or unsubstituted carboxyl group, a substituted or unsubstituted hydrocarbon group, substituted or unsubstituted heterocyclic group, acyl group, substituted or unsubstituted amino group, substituted or unsubstituted carbamoyl group, substituted or unsubstituted thiocarbamoyl group, substituted or unsubstituted sulfamoyl group, substituted or unsubstituted hydroxy group, a substituted or unsubstituted sulfonyl(SH) group, and a substituted or unsubstituted silyl group.

[0230] The term “substituted” refers to moieties having substituents replacing a hydrogen on one or more carbons of the backbone. It will be understood that “substitution” or “substituted with” includes the implicit proviso that such substitution is in accordance with permitted valence of the substituted atom and the substituent, and that the substitution results in a stable compound, e.g., which does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, etc. As used herein, the term “substituted” is contemplated to include all permissible substituents of organic compounds. In a broad aspect, the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and non-aromatic substituents of organic compounds. The permissible substituents can be one or more and the same or different for appropriate organic compounds. For purposes of this invention, the heteroatoms such as nitrogen may have hydrogen substituents and / or any permissible substituents of organic compounds described herein which satisfy the valences of the heteroatoms. Substituents can include any substituents described herein, for example, a halogen, a hydroxyl, a carbonyl (such as a carboxyl, an alkoxycarbonyl, a formyl, or an acyl), a thiocarbonyl (such as a thioester, a thioacetate, or a thioformate), an alkoxy, a phosphoryl, a phosphate, a phosphonate, a phosphinate, an amino, an amido, an amidine, an imine, a cyano, a nitro, an azido, a sulfhydryl, an alkylthio, a sulfate, a sulfonate, a sulfamoyl, a sulfonamido, a sulfonyl, a heterocyclyl, an aralkyl, or an aromatic or heteroaromatic moiety. It will be understood by those skilled in the art that the moieties substituted on the hydrocarbon chain can themselves be substituted, if appropriate.

[0231] The term “thioalkyl”, as used herein, refers to an alkyl group substituted with a thiol group.

[0232] The term “thioester”, as used herein, refers to a group -C(O)SR9or -SC(O)R9wherein R9represents a hydrocarbyl.

[0233] The term “thioether”, as used herein, is equivalent to an ether, wherein the oxygen is replaced with a sulfur.

[0234] The term “urea” is art-recognized and may be represented by the general formula wherein R9and R10each independently represent hydrogen or a hydrocarbyl.

[0235] The term “modulate” as used herein includes the inhibition or suppression of a function or activity (such as cell proliferation) as well as the enhancement of a function or activity. As used herein, the term "pharmaceutically acceptable salt" according to the present disclosure equally retains the biological effectiveness and properties of a compound disclosed herein according to an embodiment, and may collectively refer to any preferred salt in terms of pharmaceutical, biological or other properties. Non-limiting examples of these salts may be salts in which an inorganic base or an organic base is added to the compound disclosed herein, or acid addition salts. Examples of organic acids capable of forming such acid addition salts may be acetic acid, glycolic acid, lactic acid, pyruvic acid, malonic acid, succinic acid, glutaric acid, fumaric acid, malic acid, mandelic acid, tartaric acid, citric acid, ascorbic acid, palmitic acid, maleic acid, hydroxymaleic acid, benzoic acid, hydroxybenzoic acid, phenylacetic acid, cinnamic acid, salicylic acid, methanesulfonic acid, benzenesulfonic acid, toluenesulfonic acid, and the like, and examples of inorganic acids may be hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like.

[0236] The term “pharmaceutically acceptable acid addition salt” as used herein means any non-toxic organic or inorganic salt of any base compounds represented by Formula I. Illustrative inorganic acids which form suitable salts include hydrochloric, hydrobromic, sulfuric and phosphoric acids, as well as metal salts such as sodium monohydrogen orthophosphate and potassium hydrogen sulfate. Illustrative organic acids that form suitable salts include mono-, di-, and tricarboxylic acids such as glycolic, lactic, pyruvic, malonic, succinic, glutaric, fumaric, malic, tartaric, citric, ascorbic, maleic, benzoic, phenylacetic, cinnamic and salicylic acids, as well as sulfonic acids such as p-toluene sulfonic and methanesulfonic acids. Either the mono or di-acid salts can be formed, and such salts may exist in either a hydrated, solvated or substantially anhydrous form. In general, the acid addition salts of compounds of Formula I are more soluble in water and various hydrophilic organic solvents, and generally demonstrate higher melting points in comparison to their free base forms. The selection of the appropriate salt will be known to one skilled in the art. Other non-pharmaceutically acceptable salts, e.g., oxalates, may be used, for example, in the isolation of compounds of Formula I for laboratory use, or for subsequent conversion to a pharmaceutically acceptable acid addition salt.

[0237] The term “pharmaceutically acceptable basic addition salt” as used herein means any non-toxic organic or inorganic base addition salt of any acid compounds represented by Formula I or any of their intermediates. Illustrative inorganic bases which form suitable salts include lithium, sodium, potassium, calcium, magnesium, or barium hydroxide. Illustrative organic bases which form suitable salts include aliphatic, alicyclic, or aromatic organic amines such as methylamine, trimethylamine and picoline or ammonia. The selection of the appropriate salt will be known to a person skilled in the art.

[0238] “Prodrug” or “pharmaceutically acceptable prodrug” refers to a compound that is metabolized, for example hydrolyzed or oxidized, in the host after administration to form the compound of the present disclosure (e.g., compounds of formula I). Typical examples of prodrugs include compounds that have biologically labile or cleavable (protecting) groups on a functional moiety of the active compound. Prodrugs include compounds that can be oxidized, reduced, aminated, deaminated, hydroxylated, dehydroxylated, hydrolyzed, dehydrolyzed, alkylated, dealkylated, acylated, deacylated, phosphorylated, or dephosphorylated to produce the active compound. Examples of prodrugs using ester or phosphoramidate as biologically labile or cleavable (protecting) groups are disclosed in U.S. Patents 6,875,751, 7,585,851, and 7,964,580, the disclosures of which are incorporated herein by reference. The prodrugs of this disclosure are metabolized to produce a compound of Formula I. The present disclosure includes within its scope prodrugs of the compounds described herein. Conventional procedures for the selection and preparation of suitable prodrugs are described, for example, in “Design of Prodrugs” Ed. H. Bundgaard, Elsevier, 1985.

[0239] The phrase “pharmaceutically acceptable carrier” as used herein means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filter, diluent, excipient, solvent or encapsulating material useful for formulating a drug for medicinal or therapeutic use.

[0240] The term “Log of solubility”, “LogS” or “logS” as used herein is used in the art to quantify the aqueous solubility of a compound. The aqueous solubility of a compound significantly affects its absorption and distribution characteristics. A low solubility often goes along with a poor absorption. LogS value is a unit stripped logarithm (base 10) of the solubility measured in mol / liter.

[0241] EXAMPLES

[0242] The invention now being generally described, it will be more readily understood by reference to the following examples which are included merely for purposes of illustration of certain aspects and embodiments of the present disclosure and are not intended to limit the invention. Example 1: Identification of a small molecule inhibitor of RNA-binding protein

[0243] IGF2BP3

[0244] Summary

[0245] The RNA-binding protein IGF2BP3 is an oncofetal protein that was discovered to be specifically overexpressed in B-acute lymphoblastic leukemia with translocations involving the MLL (KMT2A) gene. Not normally expressed in adult tissues, Igf2bp3 was found to be dispensable for normal development in a mouse genetic model. However, functional studies demonstrated that IGF2BP3 was requisite for leukemogenesis in vitro and in vivo, in human and murine model systems. Based on these findings and widespread evidence from others of a pro-oncogenic role, a biochemical screen was undertaken to identify small molecule inhibitors of IGF2BP3-RNA interactions. Approximately 190,000 compounds were screened with a high-throughput screening (HTS)-optimized time-resolved Forester resonance energy transfer assay. From this screen, a list of 415 high-probability hits was identified and was further pared down with a cell-based counter- screen. The cell-based counter screen leveraged differential sensitivity of IGF2BP3-suffcient and deficient cell lines to compounds and identified compounds with potential IGF2BP3- specific activity. One of these compounds was confirmed, its synthesis was developed and described, and its on-target activity was determined through a combination of RNA immunoprecipitation experiments, RNA- sequencing, and cellular thermal shift assay. This compound, designated I3IN-002, shows cell growth inhibitory activity in multiple leukemia cell lines tested, with an IC50 of approximately 2 pM. Further, the compound results in a partial cell cycle arrest in G0 / G1 and induces apoptosis. Together, these findings identify the first small molecule targeting the IGF2BP3 protein to be identified by a biochemical screen, paving the way for clinical development of this and related small molecules.

[0246] Results

[0247] Cloning and production of a GFP -tagged IGF2BP 3 fusion protein.

[0248] A GFP- and Flag-tagged IGF2BP3 protein-encoding mammalian expression vector was generated, allowing for the quick and efficient production of assay-scale quantities of protein in human cells. The purified protein was validated in terms of its RNA-binding properties, and reactivity with anti-IGF2BP3 antibodies. The GFP- and Flag tags were added to the carboxy-terminus of the protein (3’ end of the DNA construct) and cloned into pcDNA3.1 (FIG. 1A). Transfection of this plasmid into 293T cells resulted in bright fluorescence (FIG. IB), indicating a successful construction of the fusion protein. Moreover, the fusion protein was recognized by anti-Flag and anti-IGF2BP3 antibodies, indicating that IGF2BP3, Flag and GFP moieties were successfully expressed as a fusion protein (FIG. 1C). The protein was then purified using an M2 anti-Flag resin, with successful elution as per methods (FIG. ID). Quantification of the protein on a Coomassie stained gel shows the relative quantitation of the protein (FIG. IE), with purified fractions showing between 125 and 250 pg / mL of purified protein. Further characterization of the protein for RNA binding activity was carried out, showing that the fusion protein could be immunoprecipitated with anti-Flag antibodies, and the protein bound to mRNAs encoding CDK6 and MYC, known targets of IGF2BP3 (FIG. IF and FIG. 1G). Together, these data indicate the successful cloning, expression, and purification of a functional IGF2BP3-Flag-GFP fusion protein (13- GFP-F).

[0249] Time resolved Forester Resonance energy transfer (TR-FRET) assay can successfully measure IGF2BP3 binding to m6A modified oligonucleotide.

[0250] Next, a time-resolved Forester resonance energy transfer assay, based on a biotinylated RNA oligonucleotide carrying a m6A modification, was designed and validated. Briefly, the principle of the assay is that if I3-GFP-F and the oligonucleotide / terbium complex are in proximity a signal (GFP fluorescence from I3-GFP-F) is generated (FIG. 2A). A small molecule that interferes with this interaction will lead to a loss of signal transfer, and decreased GFP fluorescence. The assay was tested over a range of protein and RNA concentrations, finding that the assay behaved as expected (FIG. 2B and FIG. 2C), with saturation of the signal at approximately 50 nM of I3-GFP-F and 25 nM RNA oligonucleotide. In addition, the assay’s dependence on the presence of all components of the assay was validated, and I3-GFP-F, biotinylated mRNA, and Terbium had to be present for GFP fluorescent signal to be generated (FIG. 2D). In addition, the addition of a m6A modification of the oligonucleotide results in an approximately two-fold increase in signal intensity, in line with prior reports that this modification increases the affinity of IGF2BP3 for its target mRNAs and the function of IGF2BP3 as a m6A-reader protein.

[0251] Miniaturized assay retains adequate performance characteristics for a high throughput screen (HTS). To adapt the assay to HTS, equipment located within the UCLA MSSR was utilized. The main factors to consider are plate homogeneity, intra-time point and inter-time point heterogeneity: mean values and standard deviation of the positive and negative controls were calculated. From these measurements a parameter was derived, Z’, which assesses the likelihood of obtaining a successful high-throughput screening approach. The basic necessity for an automated assay is a G+ (standard deviation of positive control) <20% and Z’>0.5 . Using IpL microfluidic chip, volumes from 0.5 pL-20 pL can be accurately dispensed. Briefly, the GFP fluorescence was highly reproducible in the assay performed with all components as in FIG. 2A - FIG. 2D, across three independent experiments performed on three different days (FIG. 3A). Similarly, the main assay parameter that was measured, the FRET ratio, was also highly reproducible across these three pilot experiment performed with 100 pL (FIG. 3B). Next, the assay was miniaturized down to assay volume ranging from 100 pL to 10 pL and 5 pL. In a set of pilot experiments, the system was successfully miniaturized to 10 pL, while maintaining G+ <20% and Z’>0.5. The GFP fluorescence was highly reproducible (FIG. 3C), as was the TR-FRET ratio (Fig 3d). Furthermore, as stated, the Z’ for the miniaturized experiment was maintained at Z’>0.5 (FIG. 3E), with minimal variation between three separate plates performed on 3 different days. Together, this data shows that the assay was highly reproducible and that it demonstrated maintained performance during miniaturized to a volume amenable to HTS.

[0252] Pre-screen Assay Performance characteristics indicated a successful path to full scale screening.

[0253] For plate layout, 32 control wells per plate with DMSO in columns 1 and 2 were utilized, and no RNA in columns 23 and 24 as internal control. After incubation, plates were read on a Perkin Elmer EnVision plate reader using 340 nm pulse, 100 psec delay and 200 psec window for signal. Emission filters were 520 ± 10 nm and 486 ± 5 nm. Data collected during the assay was transferred to the Collaborative Drug Discovery database (CDD) for mining. To determine real-world performance of the assay, an initial “pre-screen” study was performed using the miniaturized validated assay on -6909 compounds. Briefly, plate heatmaps were used to visualize wells with alterations in the fluorescence ratio, demonstrating a “hit” (middle of plate), with signal closer to control (left two lanes of plate). Using Z-score criteria of 1, 2, and 3, there were -940, -180, and 92 hits in the initial prescreen assay (FIG. 4A). This corresponded with a “raw” hit rate of -13.6%, 2.60% and 1.33% respectively (FIG. 4B). To refine this result, hits were restricted to those where Terbium fluorescence (“Wl”) was close to plate control values for this parameter, in this case within 1 standard deviation of the mean for the plate (i.e., IW1 ZI<1). This “refined hit rate” further reduced the numbers to 746 (10.8%), 135 (1.95%), and 65 (0.94%) (FIG. 4A and 4B). Together, these results demonstrated that the assay was sufficiently validated and performed well in “real-world” HTS, allowing for the procession to a full-scale screen with the entire deck of compounds available at MSSR.

[0254] Full-scale screen of -190,000 compounds.

[0255] The HTS screen of 196,567 compounds was completed recently. Success criteria were to achieve standard deviation of <20% and Z’>0.5 in a given plate, in the majority of the plates that were screened. Across 617 plates that were analyzed for this assay, >99% of plates passed the first criterion (i.e., showed a standard deviation for controls less than 20% of the mean for the controls). In fact, only a single plate did not meet this criterion, and the vast majority of control samples showed standard deviations <10% of the control mean, with a significant majority with standard deviations <5% (FIG. 5A, FIG. 5C). Similarly, the Z’- score criterion showed that 95% of the plates met the primary criterion, which was to have a Z ’score greater than 0.5 (FIG. 5B, FIG. 5D). Based on the completion of the full-scale screen, hit rates for the compounds were calculated. Briefly, the raw hit rate for the preliminary screen and the full-scale screen were 1.33 and 1.73% respectively, which are quite similar (FIG. 6A). As before, a secondary criterion was employed to decrease nonspecific hits by limiting the Z-score of terbium fluorescence (so-called W 1) in the pool of hits. These criteria successfully reduced the hit rate to 0.94% and 0.191% in the preliminary and full-scale screens, respectively (FIG. 6A). Out of 186,978 evaluable compounds that were screened, this translated to approximately 358 hits in the full-scale screen, which were further curated by examination of the individual data points. Using a different Wl Z-score definition, based on the deviation of Wl from the mean of the overall assay, 547 “refined” hits were obtained. These lists were then combined and narrowed by manual curation and visual assessment of plate performance, etc. This led to an initial, high- stringency hit selection of 415 compounds that were assayed further, as described below. Structure-function analyses of hits reveals enrichment of structurally related compounds.

[0256] Initial analyses on the structures of the compounds that comprised the hits in the full- scale screen were performed. These analyses consisted of manual inspection of the chemical structures by an expert organic chemist (N.K.G.), an expert in HTS (R.D.) and an expert in RNA biology and medicine (D.R.). These analyses resulted in the identification of “core structures” that were common amongst many of the hits. The first core structure contains an indolyltriazine motif and is depicted below:

[0257] Core Structure #1

[0258] As a reminder, 358-547 hits were found (depending on the definition of the W1 Z) in the full- scale screen out of 186,978 evaluable compounds, whereas there were 29 indolyltriazine hits out of 174 total compounds with that core structure in the screen (FIG. 6B). This represents an indolyltriazine hit rate of nearly 16.7%, which is ~57-87-fold enrichment for these compounds, a highly statistically significant result (p<0.0001; Fisher’s exact test). Similarly, a second core structure was identified, depicted below:

[0259] Core Structure #2 where 16 hits out of 183 compounds in the screen (hit rate of -8.74%; -31-46-fold enrichment) were found, again, a highly statistically significant amount (p<0.0001; Fisher’s exact test). Together, these data indicated a successful completion of the full-scale screening effort and point to a series of structures with potential activity against IGF2BP3. Confirmatory TR-FRET analyses reveal high-probability hits.

[0260] To confirm hits identified in the full-scale screen, several replicate experiments were carried out to assess changes in fluorescence ratios. 415 high-probability hits were re-pinned into two 384-well plates and were subjected to the same TR-FRET assay as before. An individual compound was defined as being a “hit” in the validation screen when the fluorescence ratio deviated from negative controls by greater than three standard deviations. Next, a Tier 1 hit compound was defined as positive in both experimental replicates; Tier 2 hit compound as positive in both experimental replicates, and Tier 3 compounds were “nonhits” in the validation experiment based on their not being positive in either replicate. Based on this definition, 371 compounds or 89.3% of all the 415 compounds were Tier 1 or Tier 2 hits (FIG. 7A). Of the indolyltriazine compounds, 27 or 93.1% of 29 compounds were Tier 1 or Tier 2 hits (FIG. 7B). Of note, mapping these compounds back to the primary data from the full-scale screen did not show any obvious trends in terms of the original single measured parameters in terms of compounds that were Tier 1, Tier 2, or Tier 3 (non-hits). Together, these data indicate that compound lists were able to be generated with reliable performance on the TR-FRET assays.

[0261] Counter-screen strategy reveals compounds with specific activity in IGF2BP3- overexpressing cell lines.

[0262] Next, hit compounds were tested against cell lines to assay for an effect on cancer cell growth. For this purpose, a strategy of comparing the effectiveness of the compound in cell lines sufficient and deficient for IGF2BP3 was utilized. Prior work generated extensive data that IGF2BP3 deficient cells showed decreased growth compared to parental or control cell lines. SEM cells are a B-ALL cell line that carry the MLL-AF4 translocation and show high levels of IGF2BP3 expression. Here, it was reasoned that a compound that was specific for IGF2BP3 would only show activity in cell lines that contained intact, increased expression of the protein (SEM-NT) and should show reduced activity in cell lines where the protein was experimentally deleted via CRISPR-Cas9 (SEM-I3K0) (FIG. 8A). To identify compounds with cellular activity and potentially with IGF2BP3-specfic activity, the 415 compounds from the validation experiments were screened against SEM-NT and SEM-I3K0 cells. Cells were treated with compound and grown for 4 days under normal growth conditions, described previously . Cell growth was then assayed with Cell Titer Gio, a luminescence-based reagent.

[0263] Fold change from control (i.e., no drug treated wells) was plotted for SEM-NT versus SEM- I3KO. Remarkably, there were many compounds (red box) that showed enhanced reduction of cell growth in SEM-NT, as opposed to SEM-I3K0 (FIG. 8B). With the data represented in a different manner, it was found that these represented a decreased cellular growth ratio in NT versus KO (FIG. 8C). Compounds with a significant effect on growth in NTs were also included, even if the change in the cellular growth ratio was more marginal. Using the cutoff (Growth Ratio<0.7 OR NT Growth<0.7), 16 compounds (shown in Table 1) were identified and moved into further testing.

[0264] Table 1. Compounds confirmed on counter-screen

[0265]

[0266] Powder based testing of compounds with activity in lGF2BP3-overexpressing cell lines

[0267] To confirm the findings, synthesized compounds were obtained from commercial vendors to test the activity and to obtain preliminary data regarding the inhibitory concentration- 50 (IC50) for the 16 compounds that were identified. The results for representative compounds are shown in FIG. 9 and below.

[0268] ICIN-115

[0269] These compounds were designated as I3IN-001, 13IN-002, 13IN-003, and I3IN-115 (for 13- inhibitor-#). I3IN-002 showed IGF2BP3-dependent activity in SEM-NT cell lines; the growth inhibition was blunted in SEM-I3K0 cells (FIG. 9B). I3IN-001 and I3IN-003 showed activity in both cell lines, with I3IN-003 showing some IGF2BP3 -dependent activity (FIG. 9A, FIG. 9C). I3IN-115 had a calculated IC50 of 0.917 pM in SEM-NT cell lines (FIG. 9D). The structure of I3IN-002 contains the indolyltriazine core structure that was previously identified as being enriched in hits from the full-scale screen. Based on these and other experiments, the IC50 determined for inhibition of cell growth in I3IN-002 cells is estimated to be -2 pM. Together, these data demonstrate the feasibility of the approach in identifying compounds that have activity in cell lines, and moreover, in an IGF2BP3- dependent manner.

[0270] The performance of several dozen compounds in IC50 assays was also tested. Table 2 shows structures and calculated IC50 values for derivative compounds tested. The IC50 shown for I3IN-002 in Table 2 is based on one experiment; the value for I3IN-002 IC50 in I3KO ranges from 7.1-25 pM.

[0271]

[0272] Successful synthesis ofI3IN-002

[0273] To enable further biological evaluation, I3IN-002 was prepared in high purity and in larger quantities compared to what was available commercially. Moreover, chemical characterization data for I3IN-002 was not identified in the literature. To access I3IN-002, the synthetic sequence shown below was performed.

[0274] First isatin 1 was treated with thiosemicarbazide (2) and potassium carbonate in methanol at ambient temperature to give hydrazone formation. The hydrazone intermediate was subjected to potassium carbonate in water at reflux to give triazinoindolothione 3 . In the next step, intermediate 3 was treated with alkylchloride 4 in the presence of potassium carbonate in DMSO . This delivered I3IN-002, which was isolated as a powder. The purity of I3IN-002 was deemed to be >97% by both quantitative NMR and HPLC analysis.

[0275] I3IN-002 shows activity against a number of cell lines that overexpress IGF2BP3.

[0276] To further characterize the activity of I3IN-002, it was tested in a number of cell lines that were available. RS4;11 cells are similar to SEM cells in that they contain the MLL-AF4 translocation protein that induces IGF2BP3. OCI-AME8227 are an AME cell line that overexpressed IGF2BP3 and is a model for AME leukemia stem cell function. Lastly, the Lin-MLL Af4 cell line is a cell line that was developed from murine hematopoietic stem and progenitor cells that were transduced with the fusion oncoprotein. This system is amenable to experiments of syngeneic transplantation into mouse recipients, which in the future will allow for detailed in vivo testing of the compound’s activity. Remarkably, treatment with a single dose of I3IN-002 results in a dose-dependent growth inhibition of SEM, RS4; 11, OCI- AML8227 and Lin-MLL Af4 cells at the concentrations tested (FIG. 10A, FIG. 10B, FIG. IOC, FIG. 10D). Moreover, cell cycle profding using propidium iodide in RS4;11 cells showed a decrease in S-phase and G2-M, indicating partial cell cycle arrest in G0 / G1 following treatment with the compound (FIG. 10E, FIG 11G). Lastly, apoptosis induction on cells treated with I3IN-002 was analyzed. These studies revealed a dose-dependent increase in apoptosis, as detected by Caspase 3 / 7 activity in both RS4;11 and SEM cells (FIG. 10G and FIG. 10G, respectively). Together, these studies indicate that I3IN-002 impacts cell growth, cell cycle and apoptosis in a variety of target cell lines, including three different models of MLL-AF4-driven leukemia, and one AML cell line. Moreover, the effect is conserved between species, in both human and murine cell lines.

[0277] I3IN-002 inhibits the molecular function of IGF2BP3 in post-transcriptional gene regulation

[0278] IGF2BP3 functions in post-transcriptional gene regulation by binding to mRNA molecules and regulating their expression, as described in Bell et al. “Insulin-like growth factor 2 mRNA-binding proteins (IGF2BPs): post-transcriptional drivers of cancer progression?” Cell Mol Life Sci. 2013;70(15):2657-75. To assess target specificity, three sets of experiments to characterize the effects of I3IN-002 were undertaken.

[0279] First, as an RNA binding protein, it would be expected that the treatment of cells with I3IN-002 would result in reduced binding of IGF2BP3 to its target mRNAs. To assess this, RNA-immunoprecipitation and RT-qPCR to assess the levels of targets bound by IGF2BP3 was undertaken, as previously described. Following 2 hours of treatment with I3IN-002, the levels of four IGF2BP3 targets, CDK6, MYC, H0XA9, and IGF1R, were significantly reduced in RNA-immunoprecipitated with anti-IGF2BP3 antibody (FIG. 11A).

[0280] Next, a cellular thermal shift assay (CETSA) was utilized to confirm binding of I3IN-002, 13IN-115, and I3IN-157 to their target. In all three cases, Western blotting for IGF2BP3 demonstrates stabilization of the protein with increasing temperature in the presence of the compound (FIG. 11B). To test whether I3IN-002, a compound discovered via biochemical screening and confirmed to have cellular activity, acts through IGF2BP3, RNA immunoprecipitation (RIP) following treatment with I3IN-002 was performed. It was found that four mRNAs (CDK6, MYC, BCL2, and H0XA9) that were previously found to be associated with IGF2BP3 were decreased in the immunoprecipitate following IGF2BP3 pulldown (FIG. 11C).

[0281] To understand the downstream consequences of IGF2BP3 inhibition, RNA-seq of DMSO-treated, I3IN-002-treated and I3KO SEM cells was performed. Briefly, RNA was isolated from the SEM-NT, SEM-NT-I3-IN-002 treated, and SEM-I3K0 cells at the same time in triplicate and subjected to library construction and RNA sequencing as described in the methods section. These studies resulted in the identification of differentially expressed genes between SEM-NT and SEM-I3K0 (n=319 genes) as well as between SEM-NT and SEM-NT-I3IN-002 treated cells (n=226 genes). Volcano plots demonstrated that the overall changes in gene expression were more pronounced in genetic knockout of IGF2BP3 compared to I3IN-002 treated cells (FIG. 12A, FIG. 12C). However, the relative proportion of downregulated and upregulated genes appeared to be somewhat similar. Remarkably, 63 genes were common between the two datasets, which represents a highly significant overlap (Hypergeometric test, p-value< IO6; FIG. 12E). Overlap of the datasets with known targets of IGF2BP3 based on cross link immunoprecipitation and RNA sequencing (iCLIP-seq and eCLIP-seq) assays demonstrated many overlapping targets (FIG. 12C, FIG. 12D). Lastly, gene set enrichment analyses of the overlap genes between 13 KO and I3IN-002 treated cells showed many pathways that were previously associated with IGF2BP3 function, including response to oxidative stress, cell activation, cellular response to cytokine stimulus, neutrophil degranulation, etc. (FIG. 12F). Together these high-throughput, unbiased analyses provide further evidence that I3IN-002 acts directly on IGF2BP3, given the highly significant overlap and function of I3IN-002-regulated genes and IGF2BP3 genetic knockout-regulated genes.

[0282] A selected panel of differentially regulated genes were confirmed by RT-qPCR (FIG. 12G), and Western blotting confirmed the downregulation of CDK6 and BCL2 proteins that we have previously observed with I3KO (FIG. 12H). Given these findings, the expression of various IGF2BP3 targeted mRNAs in spleen specimens from mice treated in vivo, as described in FIG. 16A, was measured. Several mRNAs, including Myc, Cdk6, and Hoxa9 were downregulated in spleen samples from mice treated with I3IN-002 based on RT-qPCR (FIG. 121). Together, these findings strongly support on-target activity for 13 IN-002 in situ and provide insight into the mechanism of the anti-leukemic impact observed in vitro and in vivo.

[0283] Reduction of leukemia initiating cells and leukemia engraftment in vivo by I3IN-002

[0284] Given that genetic ablation of Igf2bp3 leads to a reduction in leukemia initiating cells, it was next assayed whether 13 IN-002 caused an effect on plating efficiency in methylcellulose colony formation assays. Like I3KO, 13 IN-002 led to a reduction in total colony counts in a dose-dependent manner and a reduction in the fraction of cells that showed expression of CD34 and c-kit (FIG. 13A-B). Similarly, treatment of OCL AML-8227 cells led to a dose-dependent reduction in CD34+CD38- cells, which are thought to correspond to the leukemic stem cell fraction (FIG. 13C-D).

[0285] With these findings, the compound was next tested in vivo. Following preliminary testing to ensure lack of toxicity, mice syngeneically transplanted with the Lin-MLL-Af4 cells (either WT or I3KO) were injected intraperitoneally with I3IN-002 or vehicle control using the schedule indicated (FIG. 16A). Upon harvest at 4 weeks post-transplant, WT- transplanted mice that received I3IN-002 showed significantly reduced spleen size and weight compared to vehicle treated control mice, but larger than I3KO-transplanted mice (FIG. 16B-C). Histologically, we observed lower levels of leukemic engraftment in the liver (compare cellular area next to arrow) and reduced architectural distortion and lower leukemic engraftment in the spleen (compare size of dotted areas and intervening cellularity) in I3IN- 002 treated mice, and as expected in 13 KO (FIG. 16D). Similar reductions in engraftment were also noted in lung and kidney (FIG. 16D). Similar to what was observed in vitro, FACS analysis revealed that in vivo treatment reduced the number of CD34+ c-kit+ cells in both the spleen and the bone marrow (FIG. 16E, F, and G). In the spleen, leukemic engraftment, the proportion of CDllb+ cells, CD34+ Seal- cells were all reduced (FIG. 16D, E, and G). In the bone marrow, overall leukemic engraftment was not reduced following 13 IN-002 treatment, but reductions in Cllb+ and CD34+ Seal- cells were noted (FIG. 16G). Together, these data indicate a potent and significant effect by intraperitoneal injection of I3IN-002 leukemic burden, by gross and microscopic histology and flow cytometry, corresponding with a reduction in the leukemic initiating cell fraction.

[0286] Materials and. Methods

[0287] Cell lines and cell culture

[0288] All cell lines were maintained in standard conditions in incubator at 37 °C and 5% CO2. Human B-ALL cell lines, RS4;11 (ATCC CRL-1873), NALM6 (ATCC CRL-3273), and SEM (DMZ-ACC 546) were cultured as previously described. Immortalized MLL-Af4 transformed hematopoietic stem and progenitor cells derived from mouse bone marrow (MLL-Af4 Lin- cells) were cultured in IMDM with 15% FBS, supplemented with recombinant mouse stem cell factor (SCF, 100 ng / mL, Thermo Fisher), recombinant mouse Interleukin-6 (IL-6, 4 ng / mL, Thermo Fisher), recombinant human FMS like tyrosine kinase 3 ligand (FLT3-L, 50 ng / mL, Thermo Fisher) and mouse thrombopoietin (TPO, 50 ng / mL, Thermo Fisher). OCI-AML8227 cells were a kind gift from Dr. John Dick and Dr. Eric Lechman . OCLAML 8227 cells were cultured in a specialized media AMEM (Wisent Bio), BITS 9000 (Stem Cell Technologies), stem cell factor (SCF, 200 ng / mL, R&D systems), thrombopoietin (TPO, 25 ng / mL, R&D systems), FMS like tyrosine kinase 3 ligand (FLT3-L, 50 ng / mL, R&D systems), Interleukin-6 (IL-6, 20 ng / mL, R&D systems), Interleukin-3 (IL-3, 10 ng / mL, R&D systems), Granulocyte Colony- Stimulating Factor (G-CSF, 10 ng / mL, R&D systems). OCLAML8227 cells were cultured by plating 400,000 cells / well of non-tissue culture treated 24-well plate (Eppendorf co.) and were passaged every 7 days.

[0289] Plasmids and viral transduction

[0290] IGF2BP3-GFP-Flag over-expression plasmid was constructed by fusing full length IGF2BP3 CDS with GFP CDS and cloned in pCDNA 3.0 vector. pLenti-Cas9.GFP vector (Addgene) was used to stably express Cas9 in B-ALL cells. Guide RNAs (sgRNA) to target the igf2bp3 gene were cloned in pLKO5. sgRNA. EFS.tRFP vector, protocol described previously. CRlSPR / Cas9-mediated deletion of 1GF2BP 3 in cell lines

[0291] Human B-ALL cell lines, SEM, RS4;11, and NALM6 were depleted for IGF2BP3 using lentiviral delivery of CRISPR / Cas9 components in a two-vector system previously described. Immortalized MLL-Af4 Lin- cells were initially isolated from bone marrow of Cas9-GFP mice and then transformed using retroviral transduction with MLL-Af4 retroviral supernatant, with four rounds of transduction with MLL-Af4 retroviral supernatant, followed by selection in G418 supplemented media at 400 ug / mL for 7 days, as previously described. Cells were then stably transduced with lentiviral supernatant containing sgRNA against igf2bp3 (I3Cr2) or non-targeting (NT-1) and sorted on GFP and mCherry positivity .

[0292] Protein over-expression and purification pCDNA 3.IGF2BP3.GFP.Flag construct was transfected in 293T cells plated in 150 mm tissue culture dish. After 48 h of transfection, cells were lysed in non-denaturing lysis buffer (50 mM Tris- HC1, pH 7.4, 300 mM NaCl, 5% glycerol, 0.1% NP40, IX DNase, 10 units RNase / mL, 7.0 mg / 50 mL of DTT, lx Protease inhibitor) and incubated on ice for 1 h followed by 25 cycles of sonication with 10 sec ‘on’ and 20 sec ‘off’ pulse. Lysate was cleared by centrifugation at 12,000 x g for 15 mins. Cleared lysates were loaded in the column filled with Anti-flag M2 resin (Sigma Aldrich) and passed multiple times to assist the binding. The bound protein fraction was eluted by adding 0.5 M Glycine HC1, pH 3.5 buffer. The eluent was concentrated using Vivaspin MwCO 50 column (Greiner Bio One). The concentrated protein was stored in 100 mM Tris-HCl buffer at pH 7.2. The purity and integrity of the protein was determined by mass spectroscopy and SDS-PAGE Commassie staining. The specificity of the purified protein was determined by western blotting and probing for IGF2BP3 using rabbit Anti-human IGF2BP3 antibody.

[0293] Protein extraction and Western blot

[0294] Cell lysates were made in non-denaturing cell lysis buffer and RIPA lysis buffer. Lysates were electrophoresed using SDS-PAGE using standard conditions . Antibodies used were IGF2BP3 anti-rabbit polyclonal (catalog #RNP009, MBL) and P-actin anti-mouse monoclonal (catalog A5441, Sigma- Aldrich).

[0295] TR-FRET assay

[0296] TR-FRET assay was performed using the purified IGF2BP3-GFP fusion protein. Briefly, FRET assay was performed in assay buffer (50mM Tris-HCl, pH 7.4, 150 mM NaCl, DTT, 5% Glycerol, protease inhibitor (lx), RNase Inhibitor (10 units / mL)). First, the purified protein was diluted at 50 nM concentration (2x) in the assay buffer and added to the plate. In the second step, 20 nM (2x) concentration of biotinylated M6A labelled RNA oligo was incubated with Streptavidin-Terbium in the assay buffer for 30 min on ice. After the incubation was over, the Streptavidin-Tb + RNA complex was added to the wells with purified proteins. The plate was incubated for 1 h at RT for binding and read on EnVision microplate reader (Perkin Elmer) at a wavelength of 488 nM (Terbium fluorescence) and 525 nM (GFP fluorescence).

[0297] Drug treatment and. RNA Immunoprecipitation assay

[0298] SEM cells plated at a density of 0.5xl06 / mL treated with DMSO and I3IN-002 compound, and incubated for 24 h. After incubation is over, cells were pelleted down and washed with PBS to remove the trace of compound. Cells were lysed in 1 mL of nondenaturing lysis buffer containing protease inhibitor and RNase inhibitor, and incubated on ice for 45 min. Post incubation, lysate was cleared by centrifugation and immunoprecipitation was performed using IgG and anti-IGF2BP3 antibody. cDNA was prepared from the RNA and qPCR was performed to test the enrichment of known IGF2BP3 targets such as CDK6, Myc, HoxA9 and IGFlr.

[0299] In vitro assays for cell viability, apoptosis, and proliferation, with drug treatment

[0300] Cell viability assays were performed using a luminescent assay based on ATP quantitation (CellTiterGlo, Promega, catalog G7571), per manufacturer’s protocol. 1,500 cells / well (for 384 well format) and 5,000 cells / well (for 96 well format) were plated and incubated with different concentrations of drug for 4 days at 37 °C in a 5% CO2 incubator, followed by endpoint assay. Apoptosis study was performed on the compound treated cells using Annexin V staining described previously.

[0301] Cell Cycle using Propidium Iodide method

[0302] SEM and RS4; 11 cells were treated with 5 pM concentration of I3IN-002 and DMSO for 48 h. After 48 h cells were harvested, washed with PBS, and fixed in pre-chilled 70% ethanol overnight. Fixed cells were washed with PBS and 350 pL Propidium Iodide staining solution (20 pg / mL of propidium Iodide and 200 pg / mL of DNase free RNase) was added to the cells and incubated for 1 h at RT. Stained cells were run on the flow cytometer and analyzed using FlowJo software V10. Apoptosis

[0303] Apoptosis assay was performed using luminescence based Caspase Gio assay from Promega Corp. Briefly, SEM and RS4;11 cells were treated with 5 pM concentration of I3IN-002 and DMSO for 48 h. Post treatment, 100 pL of Caspase Gio reagent was added to each well (1:1) and incubated for 1 h at 37 °C. Plate was read on Varioscan lux microplate reader from Thermo Scientific. The increase in luminescence in I3IN-002 treated cells compared to DMSO corresponds to an increase in Caspase 3 / 7 activity.

[0304] RNA immunoprecipitation and qPCR assays.

[0305] RNA immunoprecipitation assay was performed on SEM cells treated with 5 pM concentration of I3IN-002 and DMSO for 48 h. After incubation, cells were lysed in a nondenaturing lysis buffer supplemented with Protease inhibitor and RNase inhibitor. Lysate was cleared at 12,000 rpm for 15 min and RIP was performed on the cleared lysate by adding 5 pg of IGF2BP3 antibody / IgG and 50 pL of Protein G agarose beads and incubated overnight at 4 °C. RIP and qRT-PCR were performed using protocol described previously (15).

[0306] Cellular thermal shift assay ( CETSA)

[0307] A cellular thermal shift assay was performed on SEM cells treated with the test compound, based on the protocol published in Jafari el al. “The cellular thermal shift assay for evaluating drug target interactions in cells.” Nature Protocols. 2014; 9(9):2100-22. Briefly, 20 xlO6cells were treated with 25 pM concentration of test compound in 40 mL of complete growth media, and incubated for 24 hr at 37 °C in COi incubator, DMSO was used as vehicle control. After incubation, cells were spun down and washed with PBS to remove traces of test compounds. Cells were further re-suspended in 1 mL of PBS added with lx protease inhibitor cocktail and 100 pL of cell suspension was distributed in individual PCR tubes. Cells were subjected thermal denaturation at different temperature ranging from 38 °C - 52 °C on a thermal cycler for 3 min. Thermal treated cells were lysed by flash freeze and thaw cycle in liquid Nitrogen and centrifuged at 20,000 x g for 20 min at 4 °C. Lysates recovered were boiled in the sample buffer and loaded on the gel for western blotting.

[0308] RNA isolation and qPCR

[0309] Previous protocols were adapted for RT-qPCR as standard procedures. For normalization to housekeeping genes, RT-qPCR primers for human 18S (or actin) and mouse L32 were used. RNA Sequencing, Bioinformatics Methods & Data Analysis

[0310] Total RNA was extracted from cell pellets using Qiazol (Qiagen) per manufacturer’s protocol with the following modification to include an additional RNA ethanol wash step: after the total RNA was solubilized in ddH2O, one overnight ethanol precipitation step was included for further purification. Libraries for RNA-Seq were prepared with KAPA mRNA- Seq Hyper Prep Kit. The workflow consists of mRNA enrichment and fragmentation, first strand cDNA synthesis using random priming followed by second strand synthesis converting cDNA:RNA hybrid to double-stranded cDNA (dscDNA), and incorporates dUTP into the second cDNA strand. cDNA generation is followed by end repair to generate blunt ends, A- tailing, adaptor ligation and PCR amplification. Different adaptors were used for multiplexing samples in one lane. Sequencing was performed on Illumina HiSeq3000 for a SE 1 x 50 run. Data quality check was done on Illumina SAV. Demultiplexing was performed with Illumina Bcl2fastq v2.19.1.403 software. The reads were mapped by STAR 2.7.9a and read counts per gene were quantified using the human genome GRCh38. In Partek Flow, read counts were normalized by CPM +1.0E-4. All results of differential gene expression analysis utilized the statistical analysis tool, DESeq2. P-value (p < 0.05), FDR (< 0.05), and fold change (FC > 2-fold) filters were applied for differentially expressed gene lists prior to downstream analysis. Multiple testing correction was performed using the Benjamini -Hochberg method. Significant differentially expressed genes have adjusted P value =< 0.05 and absolute log2FC >= I . Enrichment analyses were completed with Metascape .

[0311] Synthesis of I3IN-002

[0312] A round-bottom flask containing a magnetic stir bar was charged with K2CO3 (1.52 g, 11.0 mmol, 1.1 equiv), isatin 1 (1.75 g, 10.0 mmol, 1.0 equiv), thiosemicarbazide 2 (911 mg, 10.0 mmol, 1.0 equiv,) and MeOH (17 mF, 0.60 M). The reaction mixture was allowed to stir at 23 °C for 18 h. At this point, the reaction mixture was acidified by dropwise addition of acetic acid over roughly 5 min (5 mF). The resulting mixture was stirred for an additional hour. The mixture was then vacuum filtered over a fritted funnel, and the filter cake containing the product was washed with H2O (100 mF), followed by Et2O (50 mF) to afford (E)-2-(5-ethyl-2-oxoindolin-3-ylidene)hydrazine-l-carbothioamide (2.10 g, crude mass) as a yellow-orange solid. This was used directly in the subsequent reaction without further purification. To a round-bottom flask containing a magnetic stir bar, (E)-2-(5-ethyl-2-oxoindolin- 3-ylidene)hydrazine-l -carbothioamide (2.10 g, 8.46 mmol, 1.0 equiv), K2CO3 (1.17 g, 8.46 mmol, 1.0 equiv) were added, and dissolved in H2O (100 mL, 0.08 M). The round-bottom flask was fitted with a reflux condenser and the reaction mixture was stirred at 100 °C for 16 h. At this point, the crude mixture was allowed to cool to 23 °C. It was acidified by the drop wise addition of acetic acid over roughly 5 min (10 mL) and then stirred for an additional hour. The mixture was vacuum filtered over a fritted funnel, and the filter cake containing the product was washed with H2O (100 mL), followed by Et2O (50 mL) to afford triazinoindolothione 3 (1.47 g, 65% yield over two steps, >95% purity) as a yellow-orange solid.

[0313] To a 1-dram vial charged with a magnetic stir bar was added triazinoindolothione 3 (50 mg, 0.22 mmol, 1.0 equiv), chloroacetamide 4 (52 mg, 0.22 mmol, 1.0 equiv), K2CO3 (60 mg, 0.43 mmol, 2.0 equiv), and DMSO (1.1 mL, 0.20 molar). The reaction mixture was allowed to stir at 23 °C for 16 h. The reaction mixture was transferred to a vial containing H2O (5 mL), which led to the formation of a precipitate. The mixture was sonicated for 5 minutes to afford the crude product as a suspension in water. This mixture was then heated to reflux for - 5 seconds to effect partial dissolution and was then allowed to cool to 23 °C under ambient conditions. The product precipitated and was collected by vacuum filtration over a paper filter. The filter cake was successively washed with H2O (5 x 1 mL), MeOH (l x 0.5 mL), and Et2O (2 x 1 mL) to give I3IN-002 (42 mg, 45% yield, >95% purity) as a tan solid. I3IN-002: Mp: 253 °C;JH NMR (500 MHz, (CD3)2SO): d 12.53 (s, 1H), 9.89 (s, 1H), 8.14 (s, 1H), 7.72 (d, J = 7.8 Hz, 1H), 7.68 (t, 7= 7.8 Hz, 1H), 7.60 (d, J= 8.0 Hz, 1H), 7.56 (dd, J= 8.3, 1.4 Hz, 1H), 7.51 (d, J= 8.3 Hz, 1H), 7.44 (t, J= 7.6 Hz, 1H), 4.27 (s, 2H), 2.80 (q, 7= 7.5 Hz, 2H), 1.28 (t, 7= 7.6 Hz, 3H);13C NMR (125 MHz, (CD3)2SO): d 167.3, 165.9, 146.6, 141.3, 138.7, 138.4, 135.2, 133.1, 131.2, 129.3, 126.6, 126.3 (q, 7= 5.0 Hz), 124.0 (q, 7= 29.6 Hz), 123.5 (q, 7 = 273.4 Hz), 120.1, 117.6, 112.6, 34.5, 28.0, 16.2; IR (Film): 3263, 2961, 1662, 1536, 1095 cm1; HRMS-APCI (m / z) [M+H]+calcd for C2oHi7F3N5OS+, 432.11004; found 432.11002; HPLC purity 97 %; Rt= 4.91 min.

[0314] Animal Experiments

[0315] For in vivo studies, C57BE / 6J , B6.SJE-PtprcaPepc^ / BoyJ (B6 CD45.1), and B6J.129(Cg)-Gt(ROSA)26Sortml l(CAG'cas9*’'EGFP)Fezh / J(Cas9-GFP, BE / 6J) were procured from

[0316] The Jackson Laboratory. The primary murine leukemia cells WT and Igf2bp3 KO previously described in Lin et al. “Targeting IGF2BP3 enhances antileukemic effects of menin-MLL inhibition in MLL-AF4 leukemia.” Blood Adv. 2024;8(2):261-75 were transplanted into busulfan conditioned recipients. One week following transplantation, mice were injected with vehicle or I3IN-002, three times a week at a dose of 25 mg / kg, for three weeks, with an end point at four weeks post-transplantation. The peripheral blood engraftment of the leukemic cells was checked by FACS at week 2 and week 4. Once the peripheral blood engraftment reached >20% at 4 weeks, the experiment was terminated and tissues were harvested to be analyzed by FACS, histology and RT-qPCR.

[0317] Statistical analysis

[0318] Data shown represents mean ± SD for continuous numerical data. Two-tailed student’s t tests or one-way ANOVA followed by Bonferroni’s multiple comparisons test were performed using GraphPad Prism software and conducted as described in the figure legends. Survival analyses were performed using Kaplan-Meier method with comparisons made using log-rank tests, followed by Bonferroni’s correction for multiple comparisons. A P value less than 0.05 was considered significant.

[0319] Discussion

[0320] The RNA-binding protein IGF2BP3 is a potent oncofetal protein. IGF2BP3 was chosen as a target in acute leukemia following over a decade of work in, showing that is overexpressed in patients with MLL-AF4 translocated acute leukemia, genetic gain and loss of function experiments in cell lines and mouse models, and detailed mechanistic work. In this manuscript, the successful completion of a high throughput biochemical screening assay that utilizes the RNA-binding function of IGF2BP3 as the discriminant for small molecule activity was described. Combining the biochemical screen with a cell-based assay to detect IGF2BP3 -dependent impacts on leukemia cell growth and survival, 16 compounds were identified that are likely to have activity in an IGF2BP3 specific manner. The first of these compounds to be confirmed in I3IN-002, which shows an IC50 of approximately 2 pM in initial assays. Specifically, I3IN-002 inhibits cell growth, which was measured at an endpoint four days after seeding the cells with compound. In addition, I3IN-002 impacted cell cycle progression of treated cells, and increased apoptosis. Furthermore, I3IN-002 impacted global gene expression in a manner similar to genetic deletion of IGF2BP3, and RNA immunoprecipitation experiments similarly supported an on-target activity against IGF2BP3. Together, these results provide compelling evidence that I3IN-002 and potentially its derivatives will be specific inhibitors of this important oncofetal protein.

[0321] Considerations related to the targeting of RNA binding proteins

[0322] RNA binding proteins have recently been appreciated as biologically and clinically significant regulators of gene expression. This recognition has led to a growing effort to target RNA binding proteins with small molecule or oligonucleotide-based strategies . However, a large number of cancer-associated RNA binding proteins are also required for normal homeostasis, and hence, targeting these proteins might engender significant toxicity. Given this concern, discerning RBPs that have cancer- specific expression is a major effort in the field, and the IGF2BP family of proteins have emerged as cancer- specific or cancer- enriched proteins. Second, while oligonucleotide-based therapies hold great promise, and a few approaches to deliver antisense oligonucleotides or siRNAs have even achieved regulatory approvals for use in patients, targeting of many tissues, including cancer, remains a major challenge in the field. Hence, small molecule targeting holds many advantages, including delivery, with development of reproducible and straightforward synthesis methods allowing for future large-scale manufacturing and distribution. In recent years, several small molecules have been reported to inhibit the IGF2BP family of proteins. Perhaps the earliest inhibitor reported was an isocorydine derivative, 8-amino-ICD, which was reported to target hepatocellular carcinoma and decrease IGF2BP3 at EC50 of 20 pM or greater . Another inhibitor, BTYNB, was reported to target IGF2BP1 in the 5-10 pM range. In the last two years, several inhibitors have been reported to target IGF2BP2 at a range of concentrations including in acute T-lymphoblastic leukemia and acute myeloid leukemia, including CWI1-2 (sub-micromolar IC50 in AML cell lines) and JX5(10-20 pM IC50). Hence the targeting of IGF2BP family proteins is of great interest in the field, and the discoveries here add to the repertoire of molecules that have been recently reported, with the first specific inhibitor of IGF2BP3.

[0323] Structures identified by this assay

[0324] This manuscript represents the first set of efforts to develop inhibitors of IGF2BP3 based on a biochemical assay. Thus far, the indolyltriazine moiety -based compounds were confirmed as having activity against IGF2BP3. Early work confirms a second compound with a very similar structure to I3IN-002 as also having activity in the cell-based assay, providing some reassurance of the basis of activity (data not shown). In addition, there is a second core structure that was already identified by this assay, and further work is ongoing to confirm compounds containing the second core structure.

[0325] Efforts around I3IN-002 analogs

[0326] These efforts indicate that I3IN-002 can be readily synthesized using widely commercially available component molecules. Based on the fact that twenty-nine of these compounds showed activity in the TR-FRET assay, but only two of them showed activity in the cell-based assay (Fig. 8), there were some early indications of structural constraints on compounds that might be viable biologically. As noted, early analoging efforts have yielded one additional compound that shows activity in an IGF2BP3-dependent manner, as with I3IN-002. Current chemistry efforts are focused on additional side chain alterations and modifications in an effort to generate compounds with improved IC50 characteristics. Future work with I3IN-002 and improved derivatives will focus on assessing and improving safety / toxicity profiles, generating formulations for injection into animals, and mouse-based experimentation to determine pharmacokinetics and in vivo efficacy.

[0327] IGF2BP3 is a key target in multiple types of cancer

[0328] While this Example focuses on the role of IGF2BP3 in acute leukemia, it is estimated that up to 15% of all human cancers overexpress IGF2BP3, based on the COSMIC database, and there are several excellent reviews on this topic. This includes a number of distinct epithelial malignances and subtypes of hematolymphoid cancer. Knockdown of IGF2BP3 inhibits cancer cell growth, motility and features of aggressive cancer in a variety of cancer subtypes. The IGF2BP3 shows a pattern of expression consistent with an “oncofetal protein”, with very low to absent expression in adult tissues. Consistent with this notion, IGF2BP3 appears to be largely dispensable in homeostatic development, providing a significant therapeutic window for targeting. It should be noted that IGF2BP3 expression is quite variable within different cancer types, and hence future indications would need to be more carefully defined. IGF2BP3 expression could be evaluated in different cancer types by widely available techniques such as immunohistochemistry; and compounds such as I3IN-002 could be administered as a precision — medicine intervention in carefully selected patients. Hence, the application of this compound in cancer therapy might be profound and will move the field of RBP-targeting small molecule development forward. INCORPORATION BY REFERENCE

[0329] All publications and patents mentioned herein are hereby incorporated by reference in their entirety as if each individual publication or patent was specifically and individually indicated to be incorporated by reference. In case of conflict, the present application, including any definitions herein, will control.

[0330] EQUIVALENTS

[0331] While specific embodiments of the subject invention have been discussed, the above specification is illustrative and not restrictive. Many variations of the invention will become apparent to those skilled in the art upon review of this specification and the claims below. The full scope of the invention should be determined by reference to the claims, along with their full scope of equivalents, and the specification, along with such variations.

Claims

We claim:

1. A compound having a structure represented by Formula I or a pharmaceutically acceptable salt thereof:I whereinA is aryl, heteroaryl, or heterocyclyl; each Ri is independently alkyl, alkoxy, alkenyl, alkynyl, aralkyl, heteroaralkyl, aryl, heteroaryl, halo, haloalkyl, hydroxyl, carboxyl, acyl, ester, thioester, phosphoryl, amino, amido, cyano, nitro, azido, cycloalkyl, heterocyclyl, alkylsulfoxidyl, alkylsulfonyl, or sulfonamido;Xi and X2 are each independently N or CH;X3 is N, CH, or absent;X4is S, O, C(H)(RA), or NRA;X5 is O or NRB;X6is N(Rx)(Ry) or ORX;X7 is O, S, C(H)(Rc), NRc, or is absent;Xs is O, S, C(H)(RD), NRD, or is absent;RA, RB, RC, RD, RX, and Ryare each independently selected from H, alkyl, alkoxy, aryl, aralkyl, heteroaryl, heteroaralkyl, and cycloalkyl; nl is 0, 1, 2, 3, or 4; and n2 is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12.

2. The compound of claim 1, wherein the compound has a structure represented by Formula II or a pharmaceutically acceptable salt thereof:wherein X9 is NH or CH2 and X10 is N or CH.

3. The compound of claim 1, wherein the compound has a structure represented byFormula III or a pharmaceutically acceptable salt thereof:wherein n3 is 0, 1, or 2.

4. The compound of claim 1, wherein A is aryl.

5. The compound of claim 1 or 4, wherein the compound has a structure represented by Formula IV or a pharmaceutically acceptable salt thereof:

6. The compound of any one of claims 1, 4, and 5, wherein Xs is absent.

7. The compound of any one of claims 1 and 4-6, wherein the compound has a structure represented by Formula Va or a pharmaceutically acceptable salt thereof:Va wherein X9 is N or CH.

8. The compound of any one of claims 1 and 4-6, wherein the compound has a structure represented by Formula Vb or a pharmaceutically acceptable salt thereof:wherein X9 is N or CH.

9. The compound of any one of claims 1 and 4-6, wherein the compound has a structure represented by Formula Vc or a pharmaceutically acceptable salt thereof:wherein X9 is N or CH.

10. The compound of any one of claims 1 and 4-6, wherein the compound has a structure represented by Formula Vd or a pharmaceutically acceptable salt thereof:wherein X9 is N or CH.

11. The compound of any one of claims 7-10, wherein X9 is CH.

12. The compound of any one of claims 1 and 4-11, wherein the compound has a structure represented by Formula VI or a pharmaceutically acceptable salt thereof:

13. The compound of any one of claims 1 and 4-12, wherein Xi is N.

14. The compound of any one of claims 1 and 4-12, wherein X2 is N.

15. The compound of any one of claims 1, 4-11, 13, and 14, wherein X3 is N.

16. The compound of any one of claims 1 and 4-15, wherein X7 is NRc, wherein Rc is selected from H, alkyl, alkoxy, aryl, aralkyl, heteroaryl, heteroaralkyl, and cycloalkyl.

17. The compound of any one of claims 1, 4-11, and 13-16, wherein the compound has a structure represented by Formula VII or a pharmaceutically acceptable salt thereof:VII wherein Rc is selected from H, alkyl, alkoxy, aryl, aralkyl, heteroaryl, heteroaralkyl, and cycloalkyl.

18. The compound of any one of claims 1 and 4-17, wherein X4 is S.

19. The compound of any one of claims 1 and 4-18, wherein X5 is O.

20. The compound of any one of claims 1 and 4-19, wherein X<> is N(Rx)(Ry).

21. The compound of any one of claims 1 and 4-20, wherein nl is 0 or 1.

22. The compound of any one of claims 1 and 4-21, wherein n2 is 1.

23. The compound of any one of claims 1, 4-11, and 13-22, wherein the compound has a structure represented by Formula VIII or a pharmaceutically acceptable salt thereof:VIII.

24. The compound of any one of claims 1, 4-11, and 13-23, wherein Rc is H or alkyl (e.g., methyl).

25. The compound of claim 24, wherein Rc is H.

26. The compound of any one of claims 1, 4-11, and 13-25, wherein Rxis H or alkyl (e.g., methyl).

27. The compound of claim 26, wherein Rxis H.

28. The compound of any one of claims 1, 4-11, and 13-27, wherein Ryis aryl (e.g., phenyl), aralkyl (e.g., benzyl), heteroaryl (e.g., pyridyl), or cycloalkyl (e.g., cyclopropyl or cyclohexane).

29. The compound of any one of claims 1, 4-11, and 13-27, wherein Ryis aryl (e.g., phenyl), or cycloalkyl (e.g., cyclopropyl).

30. The compound of claim 29, wherein Ryis phenyl.

31. The compound of any one of claims 1, 4-11, and 13-30, wherein the compound has a structure represented by Formula IX or a pharmaceutically acceptable salt thereof:IX wherein, each Rzis independently alkyl, alkoxy, alkenyl, alkynyl, aralkyl, heteroaralkyl, aryl, heteroaryl, halo, haloalkyl, hydroxyl, carboxyl, acyl, ester, thioester, phosphoryl, amino, amido, cyano, nitro, azido, cycloalkyl, heterocyclyl, alkylsulfoxidyl, alkylsulfonyl, or sulfonamido; andn3 is 0, 1, 2, 3, 4, or 5.

32. The compound of claim 31, wherein each Rzis independently alkyl (e.g., methyl, difluoromethyl, trifluoromethyl, or isopropyl), alkoxy (e.g., methoxy), hydroxyl, nitro, or azido.

33. The compound of claim 31, wherein Rzis alkyl (e.g., methyl, trifluoromethyl, or isopropyl) or alkoxy (e.g., methoxy).

34. The compound of claim 34, wherein each Rzis independently methyl, trifluoromethyl, or chloro.

35. The compound of any one of claims 31-35, wherein n3 is 0, 1, 2, or 3.

36. The compound of any one of claims 31-35, wherein n3 is 0, 1, or 2.

37. The compound of claim 36, wherein n3 is 1.

38. The compound of any one of claims 31-37, wherein Rzis ortho to the N-H.

39. The compound of any one of claims 31-35, wherein each Rzis para or ortho to theN-H.

40. The compound of any one of claims 31-35, wherein each Rzis meta or ortho to the N-H.

41. The compound of any one of claims 31-35, wherein each Rzis ortho to the N-H.

42. The compound of any one of claims 1-41, wherein Ri is alkyl (e.g., methyl, trifluoromethyl, ethyl, or isopropyl), alkoxy (e.g., methoxy), or halo (e.g., fluorine or chlorine).

43. The compound of claim 42, wherein Ri is methyl.

44. The compound of claim 42, wherein Ri is ethyl.or a pharmaceutically acceptable salt thereof.

46. The compound of claim 45, wherein the compound is47. The compound of claim 45, wherein the compound ispharmaceutically acceptable salt thereof.

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

49. The compound of claim 48, wherein the compound ispharmaceutically acceptable salt thereof.

50. The compound of claim 48, wherein the compound is51. The compound of claim 48, wherein the compound ispharmaceutically acceptable salt thereof.

52. The compound of claim 48, wherein the compound ispharmaceutically acceptable salt thereof.

53. The compound of claim 48, wherein the compound ispharmaceutically acceptable salt thereof.

54. The compound of claim 48, wherein the compound ispharmaceutically acceptable salt thereof.

55. The compound of claim 48, wherein the compound is56. The compound of claim 48, wherein the compound ispharmaceutically acceptable salt thereof.

57. The compound of claim 48, wherein the compound ispharmaceutically acceptable salt thereof.

58. The compound of claim 48, wherein the compound is59. A compound having a structure selected from60. A pharmaceutical composition comprising the compound of any one of claims 1-59 and a pharmaceutically acceptable excipient.

61. A method of treating cancer in a subject in need thereof, comprising administering the compound of any one of claims 1-59, a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of claim 60, to the subject.

62. The method of claim 61, wherein the cancer is characterized by overexpression of RNA-binding protein IGF2BP3.

63. The method of claim 61 or 62, wherein the cancer is a hematological malignancy.

64. The method of claim 63, wherein the is hematological malignancy is myelogenous leukemia, myeloid leukemia (e.g., acute myeloid leukemia), myelodysplastic syndrome (MDS), lymphoblastic leukemia (e.g., acute lymphoblastic leukemia), chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), high risk CLL, follicular lymphoma, diffuse large B-cell lymphoma (DLBCL) (e.g., DLBCL or ABC-DLBLC), mantle celllymphoma (MCL), Waldenstrom’s macroglobulinemia (WM), multiple myeloma, marginal zone lymphoma (MZL), Burkitt’s lymphoma, non-Burkitt high grade B cell lymphoma, extranodal marginal zone B cell lymphoma, transformed high grade B-cell lymphoma (HGBL), lymphoplasmacytic lymphoma (LPL), central nervous system lymphoma (CNSL), or MALT lymphoma.

65. The method of claim 64, wherein the hematological malignancy is acute lymphoblastic leukemia (ALL).

66. The method of claim 65, wherein the ALL is B-cell acute lymphoblastic leukemia with translocations involving the MLL (KMT2A) gene.

67. The method of claim 64, wherein the hematological malignancy is myelogenous leukemia.

68. The method of claim 64, wherein the hematological malignancy is myeloid leukemia.

69. The method of claim 68, wherein the myeloid leukemia is acute myeloid leukemia(AML).

70. The method of claim 69, wherein the AML is primary AML.

71. The method of claim 69, wherein the AML is secondary AML.

Images