Chemical matters for targeting histone acetyltransferase p300 (EP300) and CREB-binding protein (CBP)

Abstract

The present disclosure relates to compounds, compositions, and methods for treating cancers mediated by aberrant Histone Acetyltransferase p300 (EP300) and / or CREB-Binding Protein (CBP) activity.

Description

VIA EFS Attorney Docket No.046094-784001WO Date of Deposit: December 17, 2024 CHEMICAL MATTERS FOR TARGETING HISTONE ACETYLTRANSFERASE P300 (EP300) AND CREB-BINDING PROTEIN (CBP) RELATED APPLICATIONS

[0001] This application claims the benefit of priority under 35 U.S.C. § 119(e) to U.S. Provisional Application No: 63 / 611,562, filed December 18, 2023, which is incorporated herein by reference in its entirety. SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on December 16, 2024 is named “046094-784001WO.xml” and is 4 KB bytes in size. BACKGROUND OF THE DISCLOSURE

[0003] The multidomain and paralogous histone acetyltransferases (HATs), histone acetyltransferase p300 (EP300) and CREB-binding protein (CBP) are two key proteins that function to maintain and reinforce malignant gene expression programs through regulation of transcription(Arany et al., Cell, 1994, 77:799-800; Welti et al., Cancer Discov., 2021, 11:1118- 1137; Bose et al., Cell, 2017, 168:135-149; Ramos et al., Nucleic Acids Res., 2010, 38:5396- 408). EP300 and CBP broadly regulate the activity of other proteins through their protein acetyltransferase catalytic activity (Weinert et al., Cell, 2018, 174:231-244; Boija et al., Mol. Cell, 2017, 68:491-503; Dancy et al., Chem. Rev., 2015, 115:2419-52). Further, these proteins contain several highly homologous domains, including KIX, bromodomains (BRDs) and HAT domains, through which they interact with, dock to, and acetylate target proteins, respectively (Welti et al., Cancer Discov., 2021, 11:1118-1137; Dancy et al., Chem. Rev., 2015, 115:2419- 52; Zucconi et al., Biochemistry, 2016, 55:3727-34). Evidence from knockout studies in the mouse (Kasper et al., Mol. Cell. Biol., 2006, 26:789-809; Rebel et al., Proc. Natl. Acad. Sci. USA, 2002, 99:14789-94; Oike et al., Blood, 1999, 93:2771-9; Yao et al., Cell, 1998, 93:361- 72), and studies in cancer cells (Welti et al., Cancer Discov., 2021, 11:1118-1137; Durbin et al., Cancer Discov., 2022, 12:730-751; Jin et al., Cancer Res., 2017, 77:5564-5575; Ogiwara et al., Cancer Discov., 2016, 6:430-45) have implicated these proteins as critical to the 1 AFSDOCS:300782609.1VIA EFS Attorney Docket No.046094-784001WO Date of Deposit: December 17, 2024 development of normal tissues and as potential targets for therapeutic development in disease states.

[0004] Much like many other epigenetic regulatory proteins (Wimalasena et al., Mol. Cell, 2020, 78:1086-1095), significant medicinal chemistry efforts have led to the production of a variety of compounds targeting distinct domains within EP300 and CBP proteins, in addition to newer proteolysis-targeted chimaera molecules which induce the degradation of one or the other proteins (Zucconi et al., Biochemistry, 2016, 55:3727-34; Michaelides et al., ACS Med. Chem. Lett., 2018, 9:28-33; Joy et al., J. Am. Chem. Soc., 2021, 143:15056-15062). Currently available conventional small molecule inhibitors are nonselective between EP300 and CBP, resulting in toxicity due to cross-reactivity when targeting these critical proteins. Accordingly, there is a need for more selective inhibitors that would minimize toxicity. SUMMARY OF THE INVENTION

[0005] A first aspect of the present disclosure is directed to a compound having a structure represented by formula I: a pharmaceutically acceptable salt or stereoisomer thereof,R1, R2, and R3are each independently H, Br, CHF2, CF3, or t-Bu, provided that when at least one of R1, R2, and R3 is Br, CHF2, CF3, or t-Bu, at least one of R1, R2, and R3 is H, or R1and R2, together with the carbon atoms to which they are attached, form 1,4-dioxane.

[0006] Another aspect of the present disclosure is directed to a pharmaceutical composition containing the compound of formula I, or a pharmaceutically acceptable salt or stereoisomer thereof, and a pharmaceutically acceptable carrier.

[0007] A further aspect of the present disclosure is directed to a method of treating a neurologic, endometrial, or hematologic cancer, that includes administering the compound of formula I, or a pharmaceutically acceptable salt or stereoisomer thereof, to a subject in need thereof. 2 AFSDOCS:300782609.1VIA EFS Attorney Docket No.046094-784001WO Date of Deposit: December 17, 2024

[0008] As shown in the working examples, the difluorophenyl group of CCS1477, a known EP300 / CBP inhibitor is critical for facilitating potent interaction with the bromodomain of EP300 / CBP and for efficacy (shown in vitro). Compounds of the present disclosure, which contain different modifications on the phenyl group, may offer advantages over CCS1477 such as, for example, increased selectivity for EP300 / CBP compared with other proteins, enhanced pharmacokinetic stability and other properties such as plasma protein binding and tissue penetration, which increase efficacy. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] FIG.1A-FIG.1H depicts the chemical targeting of the bromodomains of EP300 / CBP causes reduced cell growth in medulloblastoma cells, compared with HAT domain targeting. FIG.1A is a schematic of compound targeting of EP300 and CBP proteins by either CCS1477 (bromodomain) or A485 (HAT domain)-targeted compounds. FIG.1B-FIG.1D are graphs for Kelly cells (FIG.1B), RhJT cells (FIG.1C) and 143B cells (FIG.1D) that were tested for dose- response effects of CCS1477 and A485 after six days. n = 3 independent biological replicates for each dose. Error bars represent standard error of the mean (SEM). FIG. 1E is a pie graph showing the distribution of 454 cancer cell lines subjected to PRISM screening with either CCS1477 or A485. FIG. 1F is a plot of normalized AUC values for 454 cell lines tested for CCS1477 or A485 effects in dose response after 5 days. Dots reflect individual cell lines. Dotted line indicates normalized AUC value of 1, where normalized effect of CCS1477 is equal to A485. Arrows denote higher or lower AUC values, representing increased relative effects of A485 (higher) or CCS1477 (lower). Red bars indicate median value of lineages. n = 454 cell lines, 31 tumor types. FIG. 1G and FIG. 1H are graphs for HDMBO3 (FIG. 1G) and MB002 (FIG. 1H) group 3 medulloblastoma cell lines that were tested for dose-response effects of CCS1477 and A485 after six days. n = 3 independent biological replicates for each dose. Error bars represent SEM.

[0010] FIG. 2A-FIG. 2G depict that CCS1477 preferentially targets the bromodomains of EP300 and CBP. FIG. 2A is a profiling chart of CCS1477 (1 µM) against human bromodomains showing preferential interaction of CCS1477 with CBP and EP300, and additional interactions with BD1 of the BET proteins BRD2, BRD3, BRD4 and BRDT. FIG. 2B is a series of representative melting curves from DSF studies of the bromodomains of EP300, CBP and BRD4-BD1 in the presence of CCS1477 or SGC-CBP30 along with a summary graph. FIG. 2C shows an isothermal titration calorimetry (ITC) analysis of the interaction of CCS1477 with the bromodomains of EP300, CBP and BRD4-BD1; the Kdvalues 3 AFSDOCS:300782609.1VIA EFS Attorney Docket No.046094-784001WO Date of Deposit: December 17, 2024 were 25.0, 4.0, and 403 nM, respectively. FIG. 2D is a series of cocrystal structures of CCS1477 (yellow) bound to CBP (beige, PDB code 8FV2), EP300 (grey, PDB code 8FVF), or BRD4-BD1 (green, PDB code 8FVK). Black dotted lines indicate hydrogen bonding interactions, the critical asparagine residue is highlighted in magenta, water molecules are shown as pink spheres. FIG.2E shows the mixed H-bonding and Pi-cation interactions (green dotted lines) between the side chain of R1137and the difluorophenyl-piperidone moiety of CCS1477 in EP300. The same interaction pattern is seen in CBP with R1173, while BRD4-1 lacks an equivalent arginine residue in this region of the binding site. FIG. 2F shows that CCS1477 adopts significantly different conformational states in CBP (beige) and BRD4-BD1 (green), reflecting differences in shape complementarity with the respective KAc sites. FIG. 2G is an immunoblot of biotinylated-CCS1477 pulldowns in HDMB03 cell lysates showing pulldown of EP300 and CBP, but not BRD4 at low concentrations, and interaction with EP300, CBP and BRD4 at higher concentrations of compound. Data is representative of n = 3 independent lysates and reactions.

[0011] FIG. 3A-FIG. 3K shows that the structure-activity relationship (SAR) studies of CCS1477 analogues revealed the importance of compound engagement with R1173(CBP) or R1137(EP300) for inhibitory activity. FIG. 3A is a graphical representation of the binding affinity of CCS1477 and analogues thereof for CBP, EP300 and BRD4-BD1 as assessed by DSF. FIG. 3B is a plot showing the correlation of ΔTmvalues for the bromodomains of CBP and EP300. The Pearson’s r and statistical significance P values are indicated. FIG. 3C is a plot showing the correlation of ΔTmand Kdvalues from MST experiments for compound interaction with CBP. FIG.3D-FIG.3H is a series of co-crystal structures of CBP with different compounds. Compound structures are shown below the co-crystal structures. FIG. 3I is an immunoblot of biotinylated-CCS1477 pulldowns in HDMBO3 medulloblastoma cell lysates showing pulldown of EP300 but not BRD4 at low concentrations of CCS1477, and interaction with EP300 and BRD4 at higher concentrations of CCS1477. CCS1477-int1 fails to interact with either EP300 or BRD4 at either concentration. Data is representative of n = 3 independent lysates and reactions. FIG.3J and FIG.3K are graphs of HDMBO3 (FIG.3J) and MB002 (FIG. 3K) cells that were tested for dose-response effects of CCS1477 and CCS1477-int1 after six days. n = 3 independent biological replicates for each dose. Error bars represent SEM.

[0012] FIG. 4A-FIG. 4G show that CCS1477 selectively disrupts an interlinked transcriptional genetic dependency network in group 3 medulloblastoma cells including MYC. FIG. 4A is a heatmap showing HDMBO3 and MB002 cells that were treated with CCS1477 (HDMBO3 = 39nM, MB002 = 1280nM), A485 (HDMBO3 = 400nM, MB002 = 1970nM), 4 AFSDOCS:300782609.1VIA EFS Attorney Docket No.046094-784001WO Date of Deposit: December 17, 2024 JQ1 (HDMBO3 = 100nM, MB002 = 107nM) or matched concentration of DMSO control for 6 h, followed by ERCC-controlled spike-in RNAseq analysis. DESEQ2 analysis was performed to detect significantly changed transcripts (adjusted p<0.05 comparing treatment and DMSO). Transcripts that are significant in any treatment or cell line are shown (n=5271). Heatmap displays k-means ranked clusters of significantly different genes found in HDMBO3 and MBOO2 cells (k=4). FIG. 4B is a bar graph showing the union of statistically significant up and downregulated transcripts, relative to DMSO controls from FIG. 4A found in both HDMB03 and MB002 cells. n = 3 independent biological replicates per treatment. FIG. 4C is a gene set enrichment analysis comparing ERCC spike-in normalized transcriptomes of A485 or CCS1477 treated HDMBO3 samples, using the Hallmarks dataset identified the top differentially regulated gene set to be “Hallmarks_MYC_Targets_V2” with an NES=3.67 and FDR q-value of 0. FIG. 4D is an immunoblot of HDMBO3 cells that were treated with A485 (400 nM), CCS1477 (39 nM) or JQ1 (100 nM) for 6 h followed by lysis with GAPDH as a loading control. Data is representative of >3 independent treatments and lysates. FIG. 4E is a bar graph showing a metascape analysis using the MSigDB gene set “Oncogenic Signatures” database, demonstrating the log10 q-value of oncogenic signatures lost in genes downregulated by either CCS1477, A485 or JQ1 treatment. FIG. 4F is a bar grap showing gene annotations from Protein Analysis THrough Evolutionary Relationships (PANTHER) analysis of genetic dependencies in 7 medulloblastoma cell lines that are downregulated by treatment with CCS1477, A485 or JQ1. FIG. 4G shows a string-database analysis of high-stringency CCS1477-downregulated gene dependencies in medulloblastoma cell lines demonstrates a highly interlinked dependency network centered on transcriptional regulation. Line width indicates strength of known protein-protein interactions. Red indicates proteins involved in control of mRNA transcription / cell cycle, blue indicates proteins involved in RNA metabolism.

[0013] FIG. 5A-FIG. 5F show PRISM screening validation for CCS1477 and A485 across cancer cell lines. FIG.5A is a graph of raw AUC values of all cell lines (n = 460) treated with either CCS1477 or A485 for 5 days, resolved by PRISM barcode sequencing. Median shown by dashed central line, S.D. dotted lines. FIG.5B is a graph of median normalized AUCs of all cell lines (n = 460) treated with either CCS1477 or A485 for 5 days, resolved by PRISM barcode sequencing. Median shown by dashed central line, S.D. dotted lines. FIG. 5C is a dot plot of individual cell lines plotted by median normalized AUC ratio (CCS1477 / A485). FIG. 5D is a graph of CCLE mutational data that was extracted and compared with median normalized AUC ratio. The relationship between median normalized AUC ratio and cell line mutational status of EP300, CBP was determined. N = 328 (non-mutated), 97 (either EP300 or 5 AFSDOCS:300782609.1VIA EFS Attorney Docket No.046094-784001WO Date of Deposit: December 17, 2024 CBP mutated), 30 (both EP300 and CBP mutated), p=NS. FIG. 5E and FIG. 5F are dot plots showing genetic dependency on CBP (FIG. 5E) or EP300 (FIG. 5F) demonstrated compared with median normalized AUC ratio. Data extracted from depmap.org, 22Q4 dataset.

[0014] FIG. 6 shows a plasma pharmacokinetic analysis of CCS1477 following intraperitoneal injection in CD1 nude mice. Female CD1 nude mice were injected with CCS1477 in 10% DMSO, 90% sterile water containing 20% hydroxy-propyl-cyclodextran, and plasma recovered for analysis following retro-orbital puncture at timepoints following injection. n = 3 mice per timepoint. IV and oral dosing data retrieved from Welti et al., Cancer Discov., 2021, 11:1118-1137.

[0015] FIG. 7A-FIG. 7G shows the differential binding affinity of CCS1477 and analogues thereof for BRD4-BD1. FIG.7A shows the sructural alignment of the bromodomains of EP300, CBP and BD1 of BRD4 demonstrate high amino acid sequence identity between EP300 and CBP, with lowered identity with BRD4. FIG.7B shows the overlay of the WPF shelf of BRD4- BD1 liganded with a fragment-like dimethylisoxazole (PDB 6FT3, blue) and CCS1477 (PDB 8FVK, green). W81 undergoes a conformational change as a result of steric hindrance upon binding of CCS1477. FIG.7C is an immunoblot of biotinylated-CCS1477 pulldowns in Kelly neuroblastoma cell lysates demonstrates pulldown of EP300 but not BRD4 at low concentrations, and interaction with EP300 and BRD4 at higher concentrations of compound. Data is representative of n=3 independent lysates and reactions. FIG.7D is a dot plot showing the correlation of ΔTm values determined by DSF for CBP and BRD4-BD1 interaction with the analogues. FIG. 7E is a bar graph showing the ratio of ΔTm values (CBP / BRD4) indicating changes in target specificity relative to CCS1477. FIG. 7F shows ITC thermograms of the interaction of CCS1477int (compound 1) with the bromodomains of CBP, EP300 and BRD4. FIG.7G is a graph showing binding affinity of compound 1 for indicated proteins.

[0016] FIG.8 is a series of electron density maps of ligands bound to CBP, EP300 or BRD4- BD1. The left panel shows the 2Fo-Fc density map upon refinement with ligand (blue mesh, contoured at 1σ). The right panel shows the Fo-Fc density map upon refinement omitting the ligand (red mesh, contoured at 3σ).

[0017] FIG.9 is a series of 2D diagrams of the binding interactions of compounds in cocrystal structures with CBP, EP300 or BRD4 as computed by Poseview (proteins.plus / ).

[0018] FIG. 10A-FIG. 10G show that CCS1477, but not A485 or JQ1, disrupts a densely interacting network of genes implicated in transcriptional control in group 3 medulloblastoma. FIG. 10A is a series of Venn diagrams of intersection of ERCC-spike in normalized downregulated (left, blue) and upregulated (right, red) genes in HDMBO3 and MB002 cells 6 AFSDOCS:300782609.1VIA EFS Attorney Docket No.046094-784001WO Date of Deposit: December 17, 2024 after treatment with CCS1477, A485 or JQ1 for 6 h, compared with DMSO controls. n=3 independent biological replicates. Intersection genes represent “high-confidence” down and upregulated targets. FIG. 10B shows a gene set enrichment analysis comparing ERCC spike- in normalized transcriptomes of A485 or CCS1477 treated samples in MB002 cells, using the Hallmarks dataset identified the top differentially regulated gene set to be “Hallmarks_MYC_Targets_V2” with an NES=1.82 and FDR q-value of 9.45x10-4. FIG. 10C is a bar graph showing normalized RNAseq gene expression of c-MYC in HDMBO3 and MB002 cells treated for 6h with DMSO, A485, CCS1477 or JQ1. *-p<0.05 by two-way ANOVA. FIG.10D is a series of bar graphs for a METASCAPE analysis of transcription factor binding sites in high-confidence genes downregulated after 6 h treatment with CCS1477, A485 or JQ1 in HDMBO3 and MB002 cells. Highlighted in red are c-MYC relevant binding sites. FIG. 10E is a series of pie charts of relative ratios of dependent and non-dependent genes in high-confidence downregulated genes in HDMBO3 and MB002 cells after treatment with CCS1477, A485 or JQ1 for 6h. Dependency was determined using the 22Q4 release of Depmap, profiling exome-wide CRISPR-cas9 dropout screening data in seven medulloblastoma cell lines. FIG. 10F and FIG. 10G show a string-database analysis of high- stringency A485 (FIG. 10F) and JQ1 (FIG. 10G) downregulated gene dependencies in medulloblastoma cell lines. Line width indicates strength of known protein-protein interactions. Red indicates gene ontologies associated with transcription and cell cycle, blue indicates nucleic acid metabolism, grey indicates other.

[0019] FIG.11 shows the mean plasma and brain concentration-time data of CCS1477.

[0020] FIG.12 shows the mean plasma and brain concentration-time data of iCBP-1.

[0021] FIG.13 shows the mean plasma and brain concentration-time data of iCBP-2.

[0022] FIG.14 shows the mean plasma and brain concentration-time data of iCBP-4.

[0023] FIG.15 shows the mean plasma and brain concentration-time data of iCBP-6.

[0024] FIG. 16 shows the brain-plasma ratio of CCS1477 and iCBP4 after 50 mg / kg IP injection of compounds over 8 h in normal CD1 mice.

[0025] FIG. 17 shows the brain concentration of CCS1477 and iCBP4 after 50 mg / kg IP injection of compounds over 24 h in normal CD1 mice.

[0026] FIG.18 Plasma concentration of CCS1477 and iCBP4 after 50 mg / kg IP injection of compounds over 24 h in normal CD1 mice. 7 AFSDOCS:300782609.1VIA EFS Attorney Docket No.046094-784001WO Date of Deposit: December 17, 2024 DETAILED DESCRIPTION

[0027] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which the subject matter herein belongs. As used in the specification and the appended claims, unless specified to the contrary, the following terms have the meaning indicated in order to facilitate the understanding of the present disclosure.

[0028] As used in the description and the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise. Therefore, for example, reference to “a composition” includes mixtures of two or more such compositions, reference to “an inhibitor” includes mixtures of two or more such inhibitors, and the like.

[0029] Unless stated otherwise, the term “about” means within 10% (e.g., within 5%, 2%, or 1%) of the particular value modified by the term “about.”

[0030] The transitional term “comprising,” which is synonymous with “including,” “containing,” or “characterized by,” is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. When used in the context of the number of heteroatoms in a heterocyclic structure, it means that the heterocyclic group that that minimum number of heteroatoms. By contrast, the transitional phrase “consisting of” excludes any element, step, or ingredient not specified in the claim. The transitional phrase “consisting essentially of” limits the scope of a claim to the specified materials or steps “and those that do not materially affect the basic and novel characteristic(s)” of the disclosure.

[0031] In one aspect, compounds of the disclosure are represented by formula I: a pharmaceutically acceptable salt or stereoisomer thereof,R1, R2, and R3 are each independently H, Br, CHF2, CF3, or t-Bu, provided that when at least one of R1, R2, and R3is Br, CHF2, CF3, or t-Bu, at least one of R1, R2, and R3is H, or R1 and R2, together with the carbon atoms to which they are attached, form 1,4-dioxane. 8 AFSDOCS:300782609.1VIA EFS Attorney Docket No.046094-784001WO Date of Deposit: December 17, 2024

[0032] In some embodiments, R1 and R2 are each independently H, Br, CHF2, CF3, or t-Bu, and R3is H.

[0033] In some embodiments, R1 and R3 are each independently H, Br, CHF2, CF3, or t-Bu, and R2is H.

[0034] In some embodiments, two of R1, R2, and R3 are Br or CF3.

[0035] In some embodiments, R1and R3are both CHF2or t-Bu.

[0036] In some embodiments, R1 and R2 are both H.

[0037] In some embodiments, R1 and R3 are both H.

[0038] In some embodiments, R1is Br, CHF2, CF3, or t-Bu and R2and R3are both H.

[0039] In some embodiments, R2 is Br, CHF2, CF3, or t-Bu and R1 and R3 are both H.

[0040] In some embodiments, compounds of formula I are represented by any one of the following structures: 2), 5),9 AFSDOCS:300782609.1VIA EFS Attorney Docket No.046094-784001WO Date of Deposit: December 17, 2024 aor a pharmaceutically acceptable salt. A pharmaceutically acceptable salt of the compounds of this disclosure can be formed, for example, by reaction of an appropriate free base of a compound of the invention and an appropriate pharmaceutically acceptable acid in a suitable solvent under standard conditions well known in the art. See, for example, Gould, P. L., “Salt selection for basic drugs,” International Journal of Pharmaceutics, 33:201-217 (1986); Bastin, R. J., et al., “Salt Selection and Optimization Procedures for Pharmaceutical New Chemical Entities,” Organic Process Research and Development, 4:427-435 (2000); and Berge, S. M., et al., “Pharmaceutical Salts,” Journal of Pharmaceutical Sciences, 66:1-19 (1977).

[0042] Compounds of the present disclosure may have at least one chiral center and thus may be in the form of a stereoisomer, which as used herein, embraces all isomers of individual compounds that differ only in the orientation of their atoms in space. The term stereoisomer includes mirror image isomers (enantiomers which include the (R-) or (S-) configurations of the compounds), mixtures of mirror image isomers (physical mixtures of the enantiomers, and racemates or racemic mixtures) of compounds, geometric (cis / trans or E / Z, R / S) isomers of compounds and isomers of compounds with more than one chiral center that are not mirror images of one another (diastereoisomers). The chiral centers of the compounds may undergo epimerization in vivo; thus, for these compounds, administration of the compound in its (R-) form is considered equivalent to administration of the compound in its (S-) form. Accordingly, the compounds of the present disclosure may be made and used in the form of individual isomers and substantially free of other isomers, or in the form of a mixture of various isomers, e.g., racemic mixtures of stereoisomers.

[0043] In some embodiments, the compound of formula I is an isotopic derivative in that it has at least one desired isotopic substitution of an atom, at an amount above the natural abundance of the isotope, i.e., enriched. In one embodiment, the compound includes deuterium 10 AFSDOCS:300782609.1VIA EFS Attorney Docket No.046094-784001WO Date of Deposit: December 17, 2024 or multiple deuterium atoms. As used herein, the term “hydrogen”, i.e., H, refers to all isotopes of hydrogen, including protium (1H) and deuterium (2H). As used herein, the term “compound” embraces isotopic derivatives.

[0044] Compounds of the present disclosure may also be in the form of N-oxides, crystalline forms (also known as polymorphs), co-crystals, active metabolites of the compounds having the same type of activity, prodrugs, tautomers, and unsolvated as well as solvated (e.g., hydrated) forms with pharmaceutically acceptable solvents such as water, ethanol, and the like, of the compounds. As used herein, the term “compound” embraces all these forms. Methods of Synthesis

[0045] Broadly, the compounds or pharmaceutically acceptable salts or stereoisomers thereof, may be prepared by any process known to be applicable to the preparation of chemically related compounds. The compounds of the present disclosure will be better understood in connection with the synthetic schemes that described in various working examples that illustrate non-limiting methods by which the compounds of the disclosure may be prepared. Pharmaceutical Compositions

[0046] Another aspect of the present disclosure is directed to a pharmaceutical composition that includes the compound of formula I, or a pharmaceutically acceptable salt or stereoisomer thereof, and a pharmaceutically acceptable carrier. The phrase "pharmaceutically acceptable" indicates that the substance or composition must be compatible chemically and / or toxicologically with the other ingredients comprising a formulation, and / or the patient being treated therewith. Suitable carriers may include, for example, liquids (both aqueous and non- aqueous alike, and combinations thereof), solids, encapsulating materials, gases, and combinations thereof (e.g., semi-solids), and gases, that function to carry or transport the compound from one organ, or portion of the body, to another organ, or portion of the body. A carrier is “acceptable” in the sense of being physiologically inert to and compatible with the other ingredients of the formulation and not injurious to the subject or patient. Depending on the type of formulation, the carrier may be a pharmaceutically acceptable excipient, a pharmaceutically acceptable diluent, a pharmaceutically acceptable vehicle, and / or a pharmaceutically acceptable solvent.

[0047] Broadly, compounds of formula I, and their pharmaceutically acceptable salts and stereoisomers may be formulated into a given type of composition in accordance with conventional pharmaceutical practice such as conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping and compression processes 11 AFSDOCS:300782609.1VIA EFS Attorney Docket No.046094-784001WO Date of Deposit: December 17, 2024 (see, e.g., Remington: The Science and Practice of Pharmacy (20thed.), ed. A. R. Gennaro, Lippincott Williams & Wilkins, 2000 and Encyclopedia of Pharmaceutical Technology, eds. J. Swarbrick and J. C. Boylan, 1988-1999, Marcel Dekker, New York), each of which is incorporated herein by reference in its entirety. The type of formulation depends on the mode of administration which may include enteral (e.g., oral, buccal, sublingual and rectal), parenteral (e.g., subcutaneous (s.c.), intravenous (i.v.), intramuscular (i.m.), and intrasternal injection, or infusion techniques, intra-ocular, intra-arterial, intramedullary, intrathecal, intraventricular, transdermal, interdermal, intravaginal, intraperitoneal, mucosal, nasal, intratracheal instillation, bronchial instillation, and inhalation) and topical (e.g., transdermal).

[0048] Representative types of compositions suitable for oral administration includetablets, coated tablets, dragees, troches, lozenges, aqueous or oily suspensions, liquid solutions, dispersible powders or granules, emulsions, hard or soft capsules, or syrups or elixirs. Compositions intended for oral administration use may be prepared according to any method known in the art for the manufacture of pharmaceutical compositions. They may further contain one or more of sweetening agents, flavoring agents, coloring agents and preserving agents.

[0049] Tablets contain the compound in admixture with a non-toxic pharmaceutically acceptable excipient. Representative examples of excipients include inert diluents, such as calcium carbonate, sodium carbonate, lactose, dextrose, saccharose, cellulose, corn starch, potato starch, calcium phosphate or sodium phosphate; granulating and disintegrating agents, for example, maize starch, alginic acid, alginates or sodium starch glycolate; binding agents, for example starch, gelatin or acacia; lubricating agents, for example silica, magnesium or calcium stearate, stearic acid or talc; effervescing mixtures; dyestuffs, sweeteners, wetting agents such as lecithin, polysorbates or lauryl sulphate. The tablets may be uncoated, or they may be coated by known techniques to delay disintegration and adsorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as glyceryl monostearate or glyceryl distearate may be employed. Such preparations may be manufactured in a known manner, for example by means of mixing, granulating, tableting, sugar coating or film coating processes.

[0050] Compositions intended for oral administration use may be formulated as hard gelatin capsules wherein the compound is in admixture with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules wherein the compound is in admixture with water or an oily medium, such as peanut oil, liquid paraffin, or olive oil. 12 AFSDOCS:300782609.1VIA EFS Attorney Docket No.046094-784001WO Date of Deposit: December 17, 2024

[0051] Aqueous suspensions may contain the compound in admixture with an excipient. Representative examples of excipients includesuspending agents, e.g., sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethyl-cellulose, sodium alginate, polyvinylpyrrolidone gum tragacanth and gum acacia; and dispersing or wetting agents, e.g., naturally-occurring phosphatides such as lecithin, condensation products of an alkylene oxide with fatty acids such as example polyoxy ethylene stearate, condensation products of ethylene oxide with long chain aliphatic alcohols such as heptadecaethyleneoxycetanol, condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate, and condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides such as polyoxyethylene sorbitan monooleate.

[0052] Aqueous suspensions may also contain a preservative, such as for example, ethyl or n-propyl p-hydroxybenzoate, and / or a coloring agent such as sucrose or saccharin.

[0053] Oily suspensions may be formulated by suspending the active ingredient in a vegetable oil, representative examples of which include arachis oil, olive oil, sesame oil, coconut oil, and a mineral oil such as liquid paraffin. Oily suspensions may contain a thickening agent such as, for example beeswax, hard paraffin or cetyl alcohol.

[0054] Sweetening agents, such as those set forth above, and flavoring agents may be added. These compositions may be preserved by this addition of an antioxidant such as ascorbic acid. Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the compound in admixture with a dispersing or wetting agent, a suspending agent and a preservative. Suitable dispersing or wetting agents and suspending agents are exemplified by those already mentioned above.

[0055] The pharmaceutical compositions may be formulated as an oil-in-water emulsion. The oily phase may be a vegetable oil, for example olive oil or arachis oils, or a mineral oil, for example liquid paraffin or mixtures of these. Suitable emulsifying agents include naturally occurring gums, for example gum acacia or gum tragacanth, naturally occuring phosphatides, for example soybean lecithin, and esters or partial esters derived from fatty acids an hexitol anhydrides, for example sorbitan mono-oleate, and condensation products of the said partial esters with ethylene oxide, for example polyoxyethylene sorbitan monooleate. The emulsion may also contain sweetening and flavoring agents. Syrups and elixirs may be formulated with sweetening agents, for example glycerol, sorbitol and sucrose. In particular a syrup for diabetic patients can contain as carriers only products, for example sorbitol, which do not metabolize to glucose or which only metabolize a very small amount to glucose. 13 AFSDOCS:300782609.1VIA EFS Attorney Docket No.046094-784001WO Date of Deposit: December 17, 2024

[0056] Such formulations may also contain a demulcent, a preservative and flavoring and coloring agents.

[0057] Representative types of compositions suitable for parenteral administration (e.g., subcutaneous, intravenous, intramuscular, intrasternal, or by infusion techniques) includesterile injectable aqueous and oleaginous suspensions. The suspensions may be formulated according to the known art using those suitable dispersing of wetting agents and suspending agents as described above. The injectable compositions may also be a sterile injectable solution or suspension in a non-toxic paternally acceptable diluent or solvent, for example as a solution in 1,3-butane diol.

[0058] Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables.

[0059] Representative types of compositions suitable for inhalationaerosols and solutions for nebulizers.

[0060] Representative types of compositions suitable for rectal administration includesuppositories. They may be prepared by mixing the compound with an excipient which is solid at ordinary temperature but liquid at the rectal temperature and will therefore melt in the rectum to release the drug. Representative examples of such excipients include cocoa butter and poly-ethylene glycols.

[0061] Representative types of compositions suitable for topical administration includecreams, ointments, jellies, collyriums, solutions and suspensions. Dosage Amounts

[0062] The amount of the compound of formula I administered to a brain or hematologic cancer patient may be effective in producing the desired therapeutic response. Therefore, the amount of the compound of formula I, or a pharmaceutically acceptable salt or a stereoisomer thereof, may for example, achieve remission of the cancer, prevent or delay progression of the cancer, alleviate one or more of the symptoms of the cancer, or kill or inhibit the growth of cancer cells.

[0063] The total daily dosage of the compounds and usage thereof may be decided in accordance with standard medical practice, e.g., by the attending physician using sound medical judgment. The specific dose for any particular subject may depend upon a variety of factors including the disease or disorder being treated and the severity thereof (e.g., its present 14 AFSDOCS:300782609.1VIA EFS Attorney Docket No.046094-784001WO Date of Deposit: December 17, 2024 status); the age, body weight, general health, sex and diet of the subject; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the compound; and like factors well known in the medical arts (see, for example, Goodman and Gilman’’s The Pharmacological Basis of Therapeutics, 10thEdition, A. Gilman, J. Hardman and L. Limbird, eds., McGraw-Hill Press, 155-173, 2001), which is incorporated herein by reference in its entirety.

[0064] The daily dosages can vary within wide limits and will be adjusted to the individual requirements in each particular case. Typically, however, the dosage adopted for each route of administration when a compound is administered alone to adult humans is 0.0001 to 50 mg / kg, most commonly in the range of 0.001 to 10 mg / kg, body weight, for instance 0.01 to 1 mg / kg. Such a dosage may be given, for example, from 1 to 5 times daily. For intravenous injection a suitable daily dose is from 0.0001 to 1 mg / kg body weight, preferably from 0.0001 to 0.1 mg / kg body weight. A daily dosage can be administered as a single dosage or according to a divided dose schedule. Methods of Use

[0065] In some aspects, the present disclosure is directed to methods of treating a neurologic (brain or peripheral nervous system), endometrial, or hematologic cancer. The methods entail administration of a compound formula I, or a pharmaceutically acceptable salt or stereoisomer thereof, to a subject in need thereof.

[0066] These cancers may involve (e.g., are characterized or mediated by) aberrant EP300 and / or CBP activity (e.g., elevated levels of EP300 / CBP or otherwise functionally abnormal EP300 / CBP, e.g., mutant EP300 / CBP activity) relative to a non-pathological state.

[0067] The term “subject” (or “patient”) as used herein includes all members of the animal kingdom prone to or suffering from the indicated cancer. In some embodiments, the subject is a mammal, e.g., a human or a non-human mammal. A subject “in need of” treatment according to the present disclosure may be “suffering from or suspected of suffering from” cancer may have been positively diagnosed or otherwise presents with a sufficient number of risk factors or a sufficient number or combination of signs or symptoms such that a medical professional could diagnose or suspect that the subject was suffering from the cancer. Thus, subjects suffering from, and suspected of suffering from, the cancer are not necessarily two distinct groups. 15 AFSDOCS:300782609.1VIA EFS Attorney Docket No.046094-784001WO Date of Deposit: December 17, 2024

[0068] In some embodiments, the neurologic (brain or peripheral nervous system), endometrial, or hematologic cancer is a solid tumor or sarcoma. In some embodiments, the sarcoma is osteosarcoma.

[0069] In some embodiments, the neurologic cancer is medulloblastoma or neuroblastoma.

[0070] In some embodiments, the endometrial cancer is endometrial carcinoma or uterine adenosquamous carcinoma.

[0071] In some embodiments, the hematologic cancer is multiple myeloma or leukemia (e.g. acute myeloid leukemia).

[0072] These and other aspects of the present disclosure will be further appreciated upon consideration of the following Examples, which are intended to illustrate certain particular embodiments of the disclosure but are not intended to limit its scope, as defined by the claims. EXAMPLES

[0073] Example 1: Cancer cell lines are broadly sensitive to EP300 / CBP inhibitors

[0074] Several small molecule inhibitors of distinct protein domains of EP300 / CBP are available. However, the majority of them targets the histone acetyltransferase domain (HAT) or bromodomain (BRD) (Welti et al., Cancer Discov., 2021, 11:1118-1137; Dancy et al., Chem. Rev., 2015, 115:2419-52; Michaelides et al., ACS Med. Chem. Lett., 2018, 9:28-33; Lasko et al., Nature, 2017, 550:128-132). Nearly equipotent inhibitors of each of the HAT and BRD have been developed: the spirooxazolidinedione A485 (Michaelides et al., ACS Med. Chem. Lett., 2018, 9:28-33; Lasko et al., Nature, 2017, 550:128-132) and the dimethylisoxazol-benzimidazole CCS1477 (Welti et al., Cancer Discov., 2021, 11:1118-1137). These compounds bind the HAT or BRD of EP300 / CBP respectively, with low nanomolar potency, are cell permeant, and are largely specific for EP300 / CBP over related HAT- and BRD-containing proteins. These compounds provide a unique opportunity to compare the effects of domain-specific inhibition of EP300 and CBP. These compounds were used as tools to investigate the relative contribution of the EP300 / CBP BRD or HAT domain to tumor cell growth (FIG.1A). A 10-point, dose-response growth assays was performed using mixed pools of 460 barcoded cancer cell lines, over five days of treatment in vitro (Corsello et al., Nat. Cancer, 2020, 1:235-248; Yu et al., Nat. Biotechnol., 2016, 34:419-23). Growth was measured using an area-under-the-curve (AUC) approach (Table S1). These results demonstrated that several cancers, including medulloblastoma, endometrial carcinoma, uterine adenosquamous carcinoma, multiple myeloma, osteosarcoma and acute myeloid leukemia were cancer types 16 AFSDOCS:300782609.1VIA EFS Attorney Docket No.046094-784001WO Date of Deposit: December 17, 2024 most preferentially affected by CCS1477, compared with A485. These tumor types are also commonly affected by genetic knockout of EP300 and CBP, in addition to BRD4. All of these targets (EP300, CBP, BRD4) are affected by CCS1477.

[0075] Comparison across all cancer cell lines indicated that the HAT inhibitor A485 had a greater effect on growth suppression than the BRD inhibitor CCS1477 (FIG.5A), and that the individual effects of CCS1477 and A485 across all cell lines were not well correlated (R2=0.13). Given potential differences in compound penetration and solubility, and to compare the relative cell line and tumor-specific effects of these two compounds in assays performed non-simultaneously, a median normalization of each dataset was performed (Table S2). This had no effect on the correlation of individual compound activity (R2remained 0.13) but provided more comparable AUC ranges between A485 and CCS1477 treatments (FIG. 5B). To examine the relative cell line-specific response to either A485 or CCS1477 each individual cell line was directly compared by analyzing the median normalized ratio (CCS1477 / A485) on a per-cell-line basis. The median-normalized AUC ratio of CCS1477 / A485 demonstrated that the majority of cell lines were nearly equivalently affected by HAT or BRD inhibition, with a fraction of cell lines displaying median normalized ratios of >1.2 (13.7%, 63 / 460) or <0.8 (10.6%, 49 / 460) (FIG. 5C). To orthogonally validate these findings, the effects of A485 and CCS1477 on the growth of three cell lines from distinct tumor types were examined, each of which was differentially affected by these two compounds. Low-throughput testing in 143B osteosarcoma, RHJT rhabdomyosarcoma and Kelly neuroblastoma cell lines (FIG. 1B-FIG. 1D) demonstrated that 143B and RHJT cells were more sensitive to CCS1477 than A485 (ratio 0.67 143B, 0.76 RHJT) while in contrast, Kelly cells were more sensitive to A485 than CCS1477 (ratio 1.43).

[0076] Specific characteristics of cell lines were examined for enhanced effects of either CCS1477 or A485. To do so, expression, mutation and dependency data from the Cancer Cell Line Encyclopedia (CCLE) and the Cancer Dependency Map (DepMap) was integrated to investigate whether mutational status of EP300 or CBP predicted differential response to CCS1477 or A485. Univariate analysis of gene expression, mutation and exome-wide DepMap CRISPR-cas9 dependency did not reveal clear associations between these variables and the median normalized AUC value. Since prior data had demonstrated that cells with mutations in CBP were more sensitive to loss of EP300 (Ogiwara et al., Cancer Discov., 2016, 6:430-45), CCLE data was examined for the mutational status of EP300 and CBP. These findings demonstrated that mutational status had no relationship with median-normalized AUC, indicating that the differential response to CCS1477 or A485 was not associated with individual 17 AFSDOCS:300782609.1VIA EFS Attorney Docket No.046094-784001WO Date of Deposit: December 17, 2024 or combined mutational status of the target (FIG. 5D). DepMap CRISPR-cas9 knockout data (Dharia et al., Nat. Genet., 2021, 53:529-538; Meyers et al., Nat. Genet., 2017, 49:1779-1784) was used to identify whether there was a correlation between the median normalized AUC ratio and genetic dependency on either EP300 or CBP. There was no association between the individual CRISPR-cas9 knockout of EP300 or CBP and the relative effect of BRD or HAT domain inhibition (FIG. 5E and FIG. 5F). These data indicated that the relative susceptibility to EP300 / CBP BRD vs HAT domain inhibition, as determined by the ratio of median normalized AUC is not determined by a single driver and is likely multifactorial in nature.

[0077] Specific tumor types were examined for enhanced sensitivity to A485 or CCS1477. Cell lines were collapsed into 31 distinct tumor types, excluding those with n<3 cell lines (FIG. 1E), and sorted by median-normalized AUC ratio. This yielded 454 cell lines for analysis. By this metric, most tumors were similarly inhibited by A485 or CCS1477, with a median normalized AUC ratio of ~1 (FIG. 1F). Few tumor types, including medulloblastoma, endometrial / uterine cancer and multiple myeloma displayed a reduced median normalized AUC ratio, indicating an increased relative effect of CCS1477, compared with A485 (FIG.1F). In contrast, others such as B-cell leukemia, kidney cancer and thyroid carcinoma displayed enhanced relative effects of A485, compared with CCS1477 (FIG. 1F). These data indicated that some tumor types display differential sensitivity to EP300 / CBP BRD or HAT domain inhibition.

[0078] Example 2: Medulloblastoma shows enhanced sensitivity to bromodomain inhibition of EP300 / CBP

[0079] CCS1477 is an EP300 / CBP-specific BRD inhibitor (Welti et al., Cancer Discov., 2021, 11:1118-1137), and has entered clinical trials for adult patients with metastatic carcinomas and advanced hematologic malignancies (NCT04068597, NCT03568656). Examination of tumor-specific responses, however, revealed that CCS1477 demonstrated greatest activity, relative to A485, in cell lines derived from the high-risk, pediatric brain tumor, medulloblastoma (FIG.1F). Medulloblastoma (MB) is an aggressive pediatric malignant brain tumor composed of distinct disease subtypes regulated by different driver oncogenes (Hovestadt et al., Nat. Rev. Cancer, 2020, 20:42-56; Roussel et al., Cerebellum, 2018, 17:28- 36; Northcott et al., Nature, 2017, 547:311-317; Lin et al., Nature, 2016, 530:57-62). Classically, MB tumors are characterized by four subgroups, Sonic hedgehog (SHH)-activated, WNT-activated, Group 3 and Group 4 (Northcott et al., Nature, 2017, 547:311-317; Taylor et al., Acta Neuropathol, 2012, 123:465-72; Northcott et al., Nature, 2012, 488:49-56). Two of 18 AFSDOCS:300782609.1VIA EFS Attorney Docket No.046094-784001WO Date of Deposit: December 17, 2024 three MB cell lines, ONS76 and UW228 cells were preferentially sensitive to CCS1477, as compared with A485 (FIG.1F). These cell lines are reported to be SHH-activated MB cell lines with high expression of c-MYC (Ivanov et al., J. Biotechnol., 2016, 236:10-25.

[0080] One of the most aggressive MB tumor types are Group 3 medulloblastoma (G3MB), which are commonly characterized by overexpression by amplification of the driver oncogene c-MYC in 17% of cases 4 (Northcott et al., Nature, 2017, 547:311-317; Taylor et al., Acta Neuropathol, 2012, 123:465-72; Northcott et al., Nature, 2012, 488:49-56), and poor patient survival (Hovestadt et al., Nat. Rev. Cancer, 2020, 20:42-56). Given prior reports linking CCS1477 to disruption of c-MYC expression (Welti et al., Cancer Discov., 2021, 11:1118- 1137), higher risk subtypes of MB were explored in models that more closely pattern in vivo gene expression states. A comparative analysis of CCS1477 and A485 effects was performed by CellTiter-Glo® analysis using two G3MB cell lines, HDMB03 and MB002, growing in neurosphere cultures (Kawauchi et al., Cancer Cell, 2012, 21:168-80). Both HDMB03 and MB002 cells displayed dramatically enhanced sensitivity to CCS1477, as compared with A485 (FIG. 1G and FIG. 1H). Given these findings, preclinical pharmacokinetic (PK) analysis of CCS1477 was performed after either 25 or 50 mg / kg i.p. dosing.

[0081] These results demonstrated similar plasma PK properties as previously (FIG. 6) (Welti et al., Cancer Discov., 2021, 11:1118-1137). Since medulloblastoma is a primary brain tumor, treated mice were sacrificed at three timepoints after dosing (8, 16, 24 h) and perfused the murine vasculature with PBS to eliminate contaminating blood from the brain. Following equilibrium dialysis, LC / MS / MS was performed to identify concentrations of CCS1477 in the murine brain. At these timepoints and doses, CCS1477 was not detected in brain tissues (below lower limit of detection of 6 ng / mL). These data indicated that MB cells, and G3MB cell lines in particular, may display enhanced sensitivity to BRD-based inhibition of EP300 / CBP, as compared with HAT domain inhibition, though the ability of CCS1477 to target EP300 / CBP in vivo may be limited by poor blood-brain barrier penetration.

[0082] Example 3: CCS1477 preferentially targets EP300 and CBP

[0083] G3MB cells were exceptionally sensitive to CCS1477, as compared to A485, though this compound appeared to be non-CNS penetrant. The BRDs of EP300 and CBP are highly structurally related to each other (Dancy et al., Chem. Rev., 2015, 115:2419-52). Further, the BRDs of EP300 and CBP have structural similarity with minor sequence homology to those found in other proteins, including the BD1 N-terminal bromodomains of the BET proteins BRD2,3,4 and BRDT (FIG. 7A). Prior studies have demonstrated strong efficacy of BRD4 19 AFSDOCS:300782609.1VIA EFS Attorney Docket No.046094-784001WO Date of Deposit: December 17, 2024 inhibitors, such as JQ1, in G3MB cells in vitro (Bandopadhayay et al., Nat. Commun., 2019, 10:2400; Bandopadhayay et al., Clin. Cancer Res., 2014, 20:912-25; Pribnow et al., Mol. Cancer Ther., 2022, 21:1306-1317; Vo et al., Sci. Rep., 2018, 8:8733). To ensure that the effects that were observed on suppression of G3MB cell growth were specific to inhibition of the EP300 / CBP BRD and not due to cross-reactivity with other BRD-containing proteins, the effects of CCS1477 by Bromoscan profiling across 32 human BRDs were tested. Strong binding of CCS1477 to the BRD of CBP and EP300, but also with the BD1 BRDs of BRD2, 3 and 4 was observed (FIG.2A and Table 1). Table 1. Bromoscan profiling of CCS1477 (1 µM) DiscoveRx Gene Entrez Gene Percent Compound Symbol Symbol Control CCS1477ATAD2A ATAD2 100CCS1477ATAD2B ATAD2B 79CCS1477BAZ2A BAZ2A 79CCS1477BAZ2B BAZ2B 82CCS1477BRD1 BRD1 88CCS1477BRD2(1) BRD2 3.5CCS1477 BRD2(2) BRD2 57 CCS1477 BRD3(1) BRD3 1.7 CCS1477 BRD3(2) BRD3 66 CCS1477 BRD4(1) BRD4 3.3 CCS1477BRD4(2) BRD4 75CCS1477BRD7 BRD7 64CCS1477BRD9 BRD9 39CCS1477BRDT(1) BRDT 14CCS1477BRDT(2) BRDT 76CCS1477 BRPF1 BRPF1 31 CCS1477 BRPF3 BRPF3 75 CCS1477 CECR2 CECR2 78 CCS1477 CREBBP CREBBP 0.7 CCS1477EP300 EP300 1.6CCS1477FALZ BPTF 63CCS1477GCN5L2 KAT2A 100CCS1477PBRM1(2) PBRM1 78CCS1477 PBRM1(5) PBRM1 71 CCS1477 PCAF KAT2B 88 CCS1477 SMARCA2 SMARCA2 50 CCS1477 SMARCA4 SMARCA4 61 CCS1477TAF1(2) TAF1 69CCS1477TAF1L(2) TAF1L 80CCS1477(PHD,Bromo.) TRIM24 54CCS1477TRIM33(PHD,Bromo.) TRIM33 90CCS1477WDR9(2) BRWD1 1420 AFSDOCS:300782609.1VIA EFS Attorney Docket No.046094-784001WO Date of Deposit: December 17, 2024 These data contrasted with published Kdvalues derived from surface plasmon resonance measurements of 1.3, 1.7 and 222 nmol / L for EP300, CBP and BRD4, respectively. To resolve these findings, binding studies were performed using differential scanning fluorimetry (DSF), to assess the binding of CCS1477 with recombinant BRDs from EP300, CBP or the first BRD (BD1) of BRD4 (FIG. 2B). These data demonstrated a greater thermal shift induced by CCS1477 with EP300 or CBP, as compared with BRD4. Similar observations were seen using the parental compound for CCS1477, SGC-CBP30 (FIG.2B). Further, analysis of the binding energies by isothermal titration calorimetry (ITC) demonstrated significantly stronger interaction of CCS1477 with the bromodomains of CBP and EP300 (Kd = 4.0 and 26 nM, respectively) than with BD1 of BRD4 (Kd= 403 nM) (FIG. 2C and Table 2). As shown, CCS1477 displays preferential binding to the bromodomains of EP300 and CBP, compared with BRD4. 21 AFSDOCS:300782609.1VIA EFS Attorney Docket No.046094-784001WO Date of Deposit: December 17, 2024 Table 2. Activity and interactions of compoundsb Single ITC experiment (data fit ± SEM) c Three independent MST experiments (average ± SD) 22 AFSDOCS:300782609.1VIA EFS Attorney Docket No.046094-784001WO Date of Deposit: December 17, 2024

[0084] To understand the structural basis for the observed differential binding affinities between CCS1477 and EP300 / CBP and BRD4, co-crystal structures of the respective bromodomains with CCS1477 were obtained (FIG. 2D). CCS1477 binds through canonical hydrogen-bonding interactions of its dimethylisoxazole moiety with a conserved asparagine residue in the acetyl-lysine (Kac) binding site (CBPN1168, EP300N1132, BRD4N140). A major difference, however, between the inhibitor binding pattern in EP300 / CBP and BRD4 is the involvement of an arginine side chain (EP300R1137, CBPR1173) that interacts with the difluorophenyl ring of CCS1477 through Pi-cation interactions, while establishing an additional hydrogen-bond with the piperidinone oxygen (FIG. 2E). BRD4 is devoid of an arginine in this region of the Kac site, which explains the reduced binding affinity for CCS1477. Additionally, the BRD4 binding pocket contains a WPF shelf that imposes steric hindrance on CCS1477 such that the side chain of BRD4W81undergoes a conformational change to accommodate the difluorophenyl moiety (FIG. 7B). In EP300 / CBP, an analogous tryptophan residue is not present, and instead the equivalent LPF shelf is less bulky and facilitates interaction with CCS1477. Superposition of CCS1477 as bound in the respective Kac sites reveals an altered binding pose, reflecting the reduced shape complementarity between CCS1477 and BRD4, as compared with EP300 / CBP (FIG.2F).

[0085] The interactions of CCS1477 with EP300, CBP and / or BRD4 were investigated with in vitro biochemical assays. Biotinylated pulldown assays were performed in cell extracts derived from HDMB03 G3MB and Kelly neuroblastoma cells (FIG.2G and FIG.7C). Lysates were treated with biotinylated-CCS1477 at 1 or 10 ^M, prior to western blotting. These data demonstrated that at low concentrations, biotinylated-CCS1477 interacted with EP300 but not BRD4 (FIG.2G and FIG.7C). As a control, this interaction could be ablated by co-incubation with a 10-fold excess of unlabeled CCS1477. At supraphysiologic high doses (10^M), biotin- CCS1477 interacted with EP300 and BRD4.

[0086] Together, these data demonstrate that CCS1477 is a potent inhibitor of EP300 / CBP and a moderate inhibitor of the first bromodomain of BRD4 and other BET proteins. At concentrations relevant to inhibition in cells (≤1µM) CCS1477 likely predominantly interacts with EP300 and CBP. 23 AFSDOCS:300782609.1VIA EFS Attorney Docket No.046094-784001WO Date of Deposit: December 17, 2024

[0087] Example 4: Structure-activity relationship (SAR) studies of the CCS1477 pharmacophore

[0088] To further understand the selectivity of CCS1477 for EP300 and CBP over BRD4, a set of CCS1477 derivatives was generated. To see if the difluorophenyl moiety of CCS1477 ensures efficient interactions with the BRDs of EP300 or CBP (FIG. 2E), analogues of CCS1477 were synthesized and characterized for binding affinity by DSF, microscale thermophoresis (MST) and ITC (Table 2). The ΔTm values from DSF studies strongly correlated for compound interactions with EP300 and CBP (FIG.3A and FIG.3B), reflecting the high similarity of the Kac site between these paralogs. The correlation of DSF values for CBP and BRD4 was less significant, indicating differences for compound interactions with the respective Kac sites (FIG. 7D). This is also evident from the ratio of ΔTmvalues for CBP / BRD4, which indicates changes in selectivity of certain compounds for CBP over BRD4 (FIG. 7E). Since recombinantly expressed CBP BRD was more robust in biochemical assays and crystallization studies, Kd values were determined for all compound-CBP interactions by MST, and co-crystal structures of the CBP BRD with five of the seven analogues were determined. The ΔTm values from DSF strongly correlated with the Kd values from MST, which confirmed the robustness of binding affinity assessment by the chosen orthogonal assays (FIG.3C).

[0089] To directly test the contribution of the difluorophenyl moiety, derivatives lacking the difluorophenyl portion were generated (compounds 1 and 2, structures for all derivative compounds detailed in Table 2). The resultant compounds displayed a >1000-fold loss of affinity for EP300 and CBP (Table 2). A co-crystal structure of the CBP bromodomain with compound 2 demonstrated that the side chain R1173is more flexible than in co-crystal structures with CCS1477, and assumes a conformation incompatible for hydrogen-bonding with the piperidinone oxygen, while weakly interacting with a nitrogen of the benzimidazole core (FIG. 3D). Therefore, loss of Pi-cation as well as hydrogen-bonding interactions render compounds 1 and 2 less efficient binders to EP300 and CBP. Notably, activity against BRD4 was reduced by ~ 10-fold, indicating that the hydrophobic van-der-Waals (VDW) interactions observed between the difluorophenyl moiety of CCS1477 and the WPF shelf of BRD4 contribute to binding affinity, but relatively less so in the EP300 / CBP bromodomain. Substitution of difluorophenyl with phenyl (compound 5) resulted in a ~ 10-fold loss of binding activity, and the cocrystal revealed that R1173is now hydrogen-bonded to the piperidinone oxygen but not quite close enough to the phenyl ring to establish a Pi-cation interaction (FIG.3E). Introduction of a butyl group in meta position of the phenyl (compound 6) did not change the binding affinity 24 AFSDOCS:300782609.1VIA EFS Attorney Docket No.046094-784001WO Date of Deposit: December 17, 2024 relative to 5, and the cocrystal structure with CBP showed that R1173establishes the same Pi- cation and hydrogen-bonding interactions as seen with CCS1477 (FIG. 3F). Introducing bis- trifluoromethyl in meta position of the phenyl ring (compound 4) reduced the binding affinity by ~ 40-fold; while the co-crystal structure revealed Pi-cation and hydrogen-bonding interactions with R1173, steric hindrance imposed on the LPF shelf leads to conformational changes that negatively impact overall binding affinity (FIG. 3G). Notably, replacement with the less bulky dihydrobenzodioxine moiety (compound 7) maintained high binding affinity (Kd = 17 nM), and the cocrystal structure confirmed unimpeded positioning and interaction with R1173(FIG. 3H). Illustrating these SAR findings, electron density maps of ligand binding and binding poses of ligand-protein pairs are found in FIG.8 and FIG.9.

[0090] While none of the analogues was superior to CCS1477 in terms of binding affinity, substitutions were well tolerated if productive interactions with R1173were maintained. Notably, among the analogues tested only compound 6 maintained high activity against CBP but was considerably less potent against BRD4, indicating an increase in target selectivity. Combined, these SAR studies suggest that modifications of the CCS1477 parent compound to increase efficacy for certain in vivo applications, such as improved target selectivity or facilitating brain penetrance, are feasible.

[0091] These biochemical data were concordant with in vitro pulldown binding assays using biotinylated CCS1477-int1, where, in contrast to CCS1477, even high doses failed to interact with EP300, CBP or BRD4 (FIG. 3I). These structural studies were further emphasized by in vitro testing in G3MB cells, where loss of the difluorophenyl moiety, resulting in ablated binding to EP300 / CBP (compounds 1, 2) and caused a drastic decrease in medulloblastoma cell growth inhibition (Table 2). In contrast, milder changes to compound structure that retain the ability of compound to form Pi-cation as well as hydrogen-bonding interactions with the bromodomain resulted in only minor changes in IC50(Table 2). To further evaluate the specific effects of CCS1477-int1, CellTiter-Glo® dose-response growth assays were performed in HDMB03 and MB002 G3MB cells, demonstrating a blunting of anti-growth effect in cells treated with CCS1477-int1, as compared with CCS1477 (FIG.3J and FIG.3K).

[0092] Example 5: The bromodomain of EP300 / CBP is required to maintain transcriptional dependency networks in Group 3 medulloblastoma

[0093] HDMB03 or MB002 cells were treated with the day 3 IC50dose of CCS1477 (HDMB03 = 39 nM, MB002 = 1280 nM) for a short time-period of 6 hours, followed by extraction of total cellular RNA for ERCC-controlled spike-in RNAseq analysis. As a 25 AFSDOCS:300782609.1VIA EFS Attorney Docket No.046094-784001WO Date of Deposit: December 17, 2024 comparison, cells were treated in parallel with the day 3 IC50 dose of A485 (HDMB03 = 400 nM, MB002 = 1970 nM) or the BRD4 inhibitor, JQ1 (HDMB03 = 100 nM, MB002 = 107 nM) for the same timepoints. First, global changes in gene expression induced by drug treatment were examined. Independently, for each cell line and condition, significantly differentially expressed genes were determined using DEseq2, relative to DMSO treated controls (FIG.10A).

[0094] By examining the expression of any significantly altered gene across all treatments and cell lines, relative to DMSO controls, a distinct modular pattern of gene expression changes induced by CCS1477, A485 or JQ1 was observed (FIG. 4A). Next, only high-confidence changes in gene expression were identified, by examining the genes that were significantly and coordinately changed by treatment with each compound, in both cell lines. Each treatment predominantly caused loss of high-confidence gene expression, with A485 demonstrating the fewest effects, followed by CCS1477, and then JQ1 (FIG.4B and FIG.10A).

[0095] Since CCS1477 and A485 demonstrated dramatically different effects on G3MB cell growth (FIG. 1G and FIG. 1H), and different patterns of gene dysregulation (FIG. 4B), pathways that were dysregulated by domain-specific compound treatment in HDMB03 and MB002 G3MB cells were identified. The relative effects of A485 and CCS1477 treatment on HDMB03 and MB002 cells were compared by gene set enrichment analysis (GSEA) using the Hallmarks genesets from the Molecular Signatures Database (MSigDB). The most consistently altered Hallmark geneset between CCS1477 and A485-treated cells was the MYC_Targets_V2 geneset, which was downregulated in both cell lines when treated with CCS1477, as compared with A485 (FIG. 4C and FIG. 10B). Concordant with these findings, loss of MYC gene expression and protein levels was observed in HDMB03 cells treated with CCS1477, and to a lesser extent also with A485 and JQ1, at this timepoint (FIG. 4D and FIG. 10C). Despite the observation of moderate loss of c-MYC expression with A485, this remains enhanced with CCS1477 treatment (FIG.4C, FIG.4D, FIG.10B, and FIG.10C).

[0096] A Metascape analysis of the significantly downregulated genes driven by CCS1477, as compared with DMSO, focusing on the oncogenic signatures dataset from MSigDB was performed. Filtering this for significant enrichment (log10Q-value<-3), an enrichment of MYC-regulated genesets was identified, in addition to several others (FIG. 4E). Similar analysis comparing A485 or JQ1 against DMSO demonstrated enrichment for alternative oncogenic signatures, including TP53 and E2F3 signatures for JQ1-treated cells, and MEK signatures for A485-treated cells (FIG. 4E). Since MYC-regulated genesets appeared to be critically important to the CCS1477 effect, the promoters of genes downregulated by CCS1477, A485 and JQ1 were examined and investigated for MYC consensus binding sequences. 26 AFSDOCS:300782609.1VIA EFS Attorney Docket No.046094-784001WO Date of Deposit: December 17, 2024 Concordant with the effects on Hallmarks and oncogenic signatures, enrichment of MYC binding motifs for CCS1477-downregulated genes were identified, which was less prominent in A485- or JQ1-downregulated genesets (FIG.10D). These findings showed a key enhanced, but not sole role for c-MYC in driving G3MB cell responses to EP300 / CBP BRD inhibitor treatment, compared with HAT inhibitor treatment.

[0097] To identify the early gene networks that are disrupted by each individual inhibitor treatment that were functionally responsible for reduced HDMB03 cell growth, genes that were significantly downregulated by each compound and intersected these data with orthogonal exome-wide CRISPR-cas9 dropout screening data from seven MB cell lines (Dharia et al., Nat. Genet., 2021, 53:529-538)) were examined. 19.4% of genes significantly downregulated by CCS1477 were required for growth of MB cells, which was higher than that found after treatment with JQ1 (12.5%) or A485 (8.6%) (p=0.0027 (A485 vs. CCS1477) and p=0.0034 (JQ1 vs. CCS1477) by two-sided Fisher’s exact test (FIG.10E). To identify the ontologies of these targets, a PANTHER analysis (Mi et al., Nat. Protoc., 2013, 8:1551-66) was performed, which demonstrated that >50% of genes downregulated by CCS1477 and required for MB growth were nucleic-acid-binding proteins, transcription factors or chromatin-binding proteins (FIG.4F). In contrast, these ontologies were distinct for A485 and JQ1 treatment (Table 3). Table 3. PANTHER annotations of high confidence downregulated gene targets # MB cell Compound lines with e it or it e27 AFSDOCS:300782609.1VIA EFS Attorney Docket No.046094-784001WO Date of Deposit: December 17, 2024 CCS1477 RRP9 7 RNA binding RNA metabolism CCS1477 TBCC 7 Chaperone cytoskeletal e e or28 AFSDOCS:300782609.1VIA EFS Attorney Docket No.046094-784001WO Date of Deposit: December 17, 2024 JQ1 RAD51D 6 Enzyme DNA metabolism JQ1 MVK 5 Enzyme g or or n eAFSDOCS:300782609.1VIA EFS Attorney Docket No.046094-784001WO Date of Deposit: December 17, 2024 A485 ABHD15 1 Enzyme A485 CCND3 1 Cell Cyclegenes implying dysregulation of a central process, the STRING database (Szklarczyk et al., Nucleic Acids Res., 2015, 43:D447-52) was used to determine whether the functionally relevant genes downregulated by CCS1477, A485 or JQ1 formed a candidate protein-protein interaction network. This analysis demonstrated that the majority of genes required for MB cell growth and disrupted by CCS1477 was involved in a highly interconnected, integrated protein- protein interaction network involved in coordination of mRNA transcription / cell cycle regulation (red) or RNA metabolism (blue) (FIG. 4G, terms determined by Gene Ontology analysis). In contrast, the dependency genes disrupted by A485 produced proteins that were largely unlinked from each other, and not enriched for a specific functional category by GO analysis (FIG. 10F denoted by lack of interconnections between proteins, Table 1). As a control, JQ1 disrupted several small networks of proteins whose genes are required for growth of MB cells, though these networks were far less interconnected than those targeted by CCS1477 (FIG.10G and Table 3).

[0099] These findings indicate that the primary effect of CCS1477 on transcription in HDMB03 cells is through dysregulation of a network of genes involved in RNA metabolism and coordinated by MYC proteins, that is critically required for MB cell growth.

[0100] Example 6: Discussion of Examples 1-5

[0101] To understand the modes of binding and specificity of CCS1477 for EP300, CBP and BRD4, crystallographic experiments were performed, combined with modifications of the CCS1477 molecule, to develop a structure-activity relationship of the catalytic core of CCS1477. The findings demonstrated key residue interactions in the bromodomain of EP300 / CBP and CCS1477 (FIG. 2A-FIG. 3K). Chemical substitution of the difluorophenyl 30 AFSDOCS:300782609.1VIA EFS Attorney Docket No.046094-784001WO Date of Deposit: December 17, 2024 ring of CCS1477 yielded a compound with drastically reduced inhibition and poor binding to either EP300 or CBP, highlighting a critical functional role for this moiety within the catalytic core of the CCS1477 molecule. These studies illuminating the pharmacophore of CCS1477 are crucial to the development of derivatives that maintain on-target activity, with evolved characteristics, such as improved CNS penetration, for use in high-risk medulloblastoma.

[0102] Prior evidence has implicated a key role for BRD4 inhibition in multiple tumor states, including G3MB (Bandopadhayay et al., Nat. Commun., 2019, 10:2400; Bandopadhayay et al., Clin. Cancer Res., 2014, 20:912-25) among many others, where dominant phenotypic outcomes on transcription were related to disruption of MYC transcription. Of note, these studies typically have used longer treatments (>24 h) of JQ1 or related molecules to elicit effects on MYC transcription. Two key observations were made: 1) using biochemical, structural and medicinal chemistry approaches, CCS1477 has enhanced specificity for the bromodomains of EP300 and CBP relative to BD1 of BRD4; and 2) the early effects of CCS1477, but not A485 or JQ1 treatment, in G3MB cell lines involves selective disruption of networks of genes required for survival of these cells, including c-MYC as previously reported (Welti et al., Cancer Discov., 2021, 11:1118-1137), in addition to networks of genes beyond this as well. The observations highlight that inhibition of EP300 / CBP bromodomains functions predominantly to disrupt transcriptional networks involved in regulation of transcription itself, in addition to coordinators of transcriptional amplification, such as c-MYC (Lin et al., Cell, 2012, 151:56-67; Nie et al., Cell, 2012, 151:68-79). Critically, these findings were not observed with HAT domain inhibitors, which demonstrated striking differences with BRD inhibition. Despite having similar outcomes in cell phenotype at day 3, the transcriptional effects of JQ1 and A485 were strikingly different from those of CCS1477, indicating a role for consideration of domain-specific inhibitory effects.

[0103] These findings implicate specific inhibition of EP300 / CBP bromodomains or HAT domains as having exceptional relative effects on cell growth in a restricted range of tumors. This is particularly true for the use of EP300 / CBP bromodomain inhibitors in high-risk tumors such as Group 3 medulloblastoma, due to rapid and selective effects on disrupting networks of transcriptional regulators that are crucial to disease progression.

[0104] Example 7: Methods

[0105] Cell Lines and Reagents

[0106] Cell lines used in PRISM screening have been previously described (Corsello et al., Nat. Cancer, 2020, 1:235-248; Yu et al., Nat. Biotechnol., 2016, 34:419-23).143B cells were 31 AFSDOCS:300782609.1VIA EFS Attorney Docket No.046094-784001WO Date of Deposit: December 17, 2024 obtained from ATCC® and cultured in Eagle’s minimum essential medium (MEM) with 0.015 mg / mL 5-bromo-2’-deoxyuridine and 90% FBS. Kelly cells were obtained from DSMZ and cultured in RPMI with 10% FBS. RhJT cells were provided by the Broad Institute Pediatric Dependencies Project and cultured in RPMI with 10% FBS. HDMB03 and MB002 cells were provided by Till Milde (KiTZ, Heidelberg) and Yoon-Jae Cho (OHSU), and cultured in neurosphere medium as previously described (Pribnow et al., Mol. Cancer Ther., 2022, 21:1306-1317). All cells were validated to be free of mycoplasma spp. with routine testing for identity by short-tandem repeat testing.

[0107] PRISM and Bromoscan analysis

[0108] Dose-response sequencing data was processed to determine area-under-the-curve (AUC) values, as previously described (Corsello et al., Nat. Cancer, 2020, 1:235-248; Yu et al., Nat. Biotechnol., 2016, 34:419-23). Tumor type assignments for cell lines were determined using the Cancer Cell Line Encyclopedia annotations (Ghandi et al., Nature, 2019, 569:503- 508). Data was filtered on a per-cell line basis to determine AUC values for both CCS1477 and A485, prior to comparison. Tumor grouping analyses were restricted to those with >3 representative cell lines. AUC data for each compound was performed across all cell lines treated by calculating the median AUC and then median normalizing the data. Bromoscan assay was performed by Eurofins DiscoverX (San Francisco, CA), using 1 ^M concentrations of CCS1477.

[0109] CellTiter-Glo® Assay

[0110] CellTiter-Glo® assay was performed as per the manufacturer’s instructions, at the noted timepoint after compound treatment or plating. For Kelly, RhJT and 143B, 500 cells were plated in normal growth media per well in a 384-well plate (Corning®). HDMB03 and MB002 cells were plated in growth media at 500 cells / well in a 384-well plate. Compounds were dispensed in a dose range from 1nM to 10^^M using a Tecan D300e compound dispenser (Tecan). Cells were incubated for the noted timepoints prior to performing CellTiter-Glo® assay as per the manufacturer’s instructions, and reading plates on an EnVision® 2104 microplate reader.

[0111] Western blotting and Immunoprecipitation

[0112] Western blotting protocols were as previously described (Durbin et al., Cancer Discov., 2022, 12:730-751; Durbin et al., Nat. Genet., 2018, 50:1240-1246). Cells growing in culture were lysed for whole-cell lysates using RIPA buffer, or for nuclear lysates and co- immunoprecipitation using the NE-PER™ nuclear lysate kit (Thermo Fisher Scientific™) 32 AFSDOCS:300782609.1VIA EFS Attorney Docket No.046094-784001WO Date of Deposit: December 17, 2024 according to the manufacturer’s protocol. Equal amounts of nuclear protein were immunoprecipitated using biotinylated CCS1477 with or without other compounds (CCS1477, CCS1477-int1) overnight, prior to recovery using streptavidin-coated dynabeads (Thermo Fisher Scientific™) as per the manufacturer’s instructions. Equal amounts of protein were resolved by Western blotting using 4% to 12% Bis-Tris NuPAGE™ gels (Thermo Fisher Scientific™) prior to transfer and immunoblotting using primary antibodies to EP300 (Abcam®), BRD4 (Abcam®), MYCN (Cell Signaling Technology®) or GAPDH (Cell Signaling Technology®). The secondary antibodies horseradish peroxidase–conjugated anti- rabbit and anti-mouse (Santa Cruz Biotechnology), were incubated prior to exposure to enhanced chemiluminescence reagents (GE Amersham).

[0113] Protein expression and purification

[0114] Expression plasmids (pNIC28-Bsa4) for the bromodomain of EP300 (residues 1048- 1161), CBP (residues 1081-1197), and BRD4-BD1 (residues 44-168) were obtained from Addgene, transformed into E. coli BL21 (DE3) RIL expression cells and grown at 37°C in LB medium (Fisher Scientific™) containing carbenicillin (0.1 mg / mL) and chloramphenicol. At OD600 of 0.6, the culture was cooled to 18°C and induced with 0.1 mM IPTG. After 18 h growth, the culture was harvested by centrifugation at 6,000 × g for 25 min and stored at -80°C. Harvested cell pellets were re-suspended in 50 mM Na / K phosphate buffer (pH 7.4) containing 100 mM NaCl, 40 mM imidazole, 0.01% w / v lysozyme and 0.01% v / v Triton™ X-100 at 4°C for 1 h, subjected to sonication. The lysate was clarified by centrifugation (30,000 × g for 45 min at 4°C). Proteins were purified by FPLC at 4°C using columns and chromatography materials obtained from GE Healthcare. The lysate was subjected to an immobilized Ni2+affinity chromatography column equilibrated with 50 mM Na / K phosphate buffer (pH 7.4) containing 100 mM NaCl and 40 mM imidazole using a gradient from 40 to 500 mM of imidazole. Fractions containing the target protein were combined and incubated overnight with TEV protease at 4°C, and the cleaved His6-tag was removed by a second Ni2+affinity column. Proteins were purified to homogeneity by size exclusion chromatography using Superdex™ 75 equilibrated with elution buffer (50 mM HEPES pH 7.5, 100 mM NaCl, 2 mM DTT). All BRDs eluted as monomeric protein and were of crystallization grade quality (> 95% purity as judged by SDS-PAGE). Pool fractions were combined, concentrated to 10-12 mg / mL, and aliquots were flash-frozen in liquid N2 and stored at -80°C.

[0115] Crystallization and X-ray crystallography

[0116] All crystallization experiments were performed at 18°C. Aliquots of purified EP300, CBP or BRD4-BD1 were set up for crystallization screening using a Mosquito liquid handler 33 AFSDOCS:300782609.1VIA EFS Attorney Docket No.046094-784001WO Date of Deposit: December 17, 2024 (TP Labtech) in 200 nL sitting droplets. For co-crystallization, the compound was pre-mixed with protein on ice and then diluted in a 1:1 ratio with precipitant to achieve a final concentration of 1 mM in 10% DMSO. Crystals were cryoprotected using the well solution supplemented with 25% ethylene glycol and flash frozen in liquid nitrogen. X-ray diffraction data were collected at −180°C in house (CuKα X-rays generated by a Rigaku Micro-Max 007- HF X-ray generator), and at the Synchrotron beamlines 22-ID / BM (SER-CAT) and 23-ID (GM / CA) of the Advanced Photon Source, Argonne National Laboratory. Data were reduced and scaled with XDS (Kabsch W., Acta Crystallogr. D. Biol. Crystallogr., 2010, 66:133-44) or DIALS (Winter et al., Acta Crystallogr. D. Biol. Crystallogr., 2018, 74:85-97) and Aimless (Evans et al., Acta Crystallogr. D. Biol. Crystallogr., 2013, 69:1204-14). The structures were solved by molecular replacement; structure refinement was carried out with PHENIX (Afonine et al., J. Appl. Crystallogr., 2010, 43:669-676); and model building was carried out with Crystallographic Object-Oriented Toolkit (COOT) (Emsley et al., Acta Crystallogr. D. Biol. Crystallogr., 2010, 66:486-501). Initial models for the small molecule ligands were generated with ligand restraints from eLBOW of the PHENIX suite. Figures were prepared using PyMOL (Schrödinger, LLC). The coordinate sets and structure factors were deposited in the Protein Data Bank (PDB) (Tables 4 and 5). 34 AFSDOCS:300782609.1VIA EFS Attorney Docket No.046094-784001WO Date of Deposit: December 17, 2024 Table 4. Crystallographic data collection and refinement statistics35 AFSDOCS:300782609.1VIA EFS Attorney Docket No.046094-784001WO Date of Deposit: December 17, 2024 Table 5. Crystallographic data collection and refinement statistics

[0117] Differential scanning fluorimetry (DSF)

[0118] DSF experiments were performed with an Applied Biosystem StepOnePlus™ real- time PCR system (Thermo Fisher Scientific™) using 96-well format plates, assayed in quadruplicate. For thermal shift determination, 5 μM protein was mixed with 100 μM compound in 50 mM HEPES, pH 7.5, 150 mM NaCl, 2 mM DTT, 2% DMSO, 5X SYPRO™ Orange (Thermo Fisher Scientific™) in 20 μL reaction volumes. Reaction mixtures were heated from 25°C to 95°C at 1°C / min with fluorescence readings every 0.5°C at 610 nm. The observed thermal shift (ΔTm) was recorded as the difference between the Tm of sample and DMSO reference wells.

[0119] Isothermal calorimetry (ITC)

[0120] All experiments were conducted using an PEAQ-ITC calorimeter (Malvern Scientific). BRDs were buffer exchanged using PD10 columns (GE life sciences) into 50 mM 36 AFSDOCS:300782609.1VIA EFS Attorney Docket No.046094-784001WO Date of Deposit: December 17, 2024 HEPES pH 7.5 and 100 mM NaCl (ITC buffer) before the experiment and concentrated to ∼10 mg / mL. Experiments were carried out in ITC buffer while stirring at 750 rpm through reverse titration. The microsyringe (40 μL load volume) was loaded with the protein sample (250–600 μM protein in ITC buffer) and inserted into the calorimetric cell (0.2 mL cell volume) consisting of compound (20–60 μM in ITC buffer). All titrations were conducted using an initial control injection of 0.2 μL followed by 13 identical injections (~3 μL per injection) with a duration of 2 s (per injection) and a spacing of 150 s between injections. The ratio of protein / compound was optimized to ensure complete saturation of the titrant before the final injection to ensure proper baseline determination. Data were corrected for dilution and analyzed using the MicroCal Origin software to determine the enthalpies of binding (ΔH) and binding constants as described (Wiseman et al., Analytical Biochemistry, 1989, 179:131-137). Thermodynamic parameters were calculated using the basic equation of thermodynamics (ΔG = ΔH - TΔS = -RTlnKB) where ΔG, ΔH and ΔS are the changes in free energy, enthalpy and entropy of binding, respectively. A single binding site model was used for all ligand-protein interactions.

[0121] Microscale thermophoresis (MST)

[0122] MST experiments were performed in a Monolith® NT.115 Pico instrument (NanoTemper Technologies) following published procedures (Huang et al., Methods Mol. Biol., 2021, 2213:187-193). Measurements were performed at medium MST power and 20 % excitation power (auto-detect) at 25°C with standard capillaries using a constant concentration of GFP-tagged CBP (50 nM) and increasing concentrations of inhibitor using a 16-point 2x serial dilution (1.5 nM to 100 μM) in 50 mM HEPES pH 7.5, 150 mM NaCl, 0.05% Tween and 2% DMSO. MST traces were recorded using standard parameters: 5 s MST power off, 30 s MST power on and 5 s MST power off. Measurements were taken at -1-0 s (cold region) and 10 s (hot region) and data were analyzed using the NTAnalysis software. The macroscopic dissociation constant (KD) was determined using the equation: [BL] / [B0] = (([L0]+[B0] + KD) − √(([L0]+[B0] + KD)2 − 4 × [L0]×[B0])) / 2 × [B0], where [B0] corresponds to the total concentration of target binding sites, [L0] to the concentration of titrated ligand, and [BL] to the concentration of formed complex between ligand and target binding sites (Wienken et al., Nat. Commun., 2010, 1:100).

[0123] RNA-sequencing

[0124] HDMB03 and MB002 cells growing in culture were treated with CCS1477, JQ1, A485 or matched concentrations of DMSO as a vehicle control, at the noted doses for a total 37 AFSDOCS:300782609.1VIA EFS Attorney Docket No.046094-784001WO Date of Deposit: December 17, 2024 of 6 h, prior to cell counting, isolation and lysis in TRIzol™ (Thermo Fisher Scientific™). External RNA consortium control RNAs (ERCC, Ambion®) were added directly to TRIzol™, based on cell number prior to RNA extraction, DNAse I treatment (Invitrogen™) and recovery. Samples were obtained in biological triplicate. Total RNA was subjected to ribosomal RNA depletion prior to library preparation and sequencing on a Nextseq-500 (paired end, 75bp reads). Reads were aligned to a reference index (hg19 revision of the human reference genome), quantified comparing to ERCC spike-in probes, and converted to transcripts per million (TPM). ERCC-normalized expression of each gene after 6 h of CCS1477, A485 or JQ1 was compared against its expression in DMSO-treated samples to create expression tables, which were processed using DESEQ2. Separate analyses were performed comparing CCS1477-treated and A485-treated samples. Genes were annotated as significantly different based on an adjusted p- value of <0.05 by DESEQ2 analysis, and data in both cell lines were integrated to identify high-confidence genes for each treatment subset. These data served as input for analysis in METASCAPE (Zhou et al., Nat. Commun., 2019, 10:1523). Subsetted gene lists were used for analysis by GSEA using the Gene Ontology Hallmarks collection in MSigDB (Subramanian et al., Proc. Natl. Acad. Sci. USA., 2005, 102:15545-50), PANTHER (Mi et al., Nat. Protoc., 2012, 8:1551-66) or the STRING database (Szklarczyk et al., Nucleic Acids Res., 2015, 43:D447-52).

[0125] Dependency-STRING database Analysis

[0126] Dependency analysis was performed using the 22Q4 release of the DepMap dataset, comprising seven medulloblastoma cell lines: UW228, DAOY, ONS76, D458, D425, D283MED, D341MED. Dependency was defined as a Chronos gene effect score of <-0.5, and gene lists were filtered for dependency in >2 / 7 medulloblastoma cell lines. Interaction networks were determined using the STRING database (Szklarczyk et al., Nucleic Acids Res., 2015, 43:D447-52), using medium confidence settings, network edges = confidence, no interactors, and hiding disconnected nodes (for JQ1, CCS1477) or showing disconnected nodes (A485). PANTHER was used to define gene annotations (Mi et al., Nat. Protoc., 2012, 8:1551-66), with unknown annotations assigned using individual assessment using the Genecards.org resource.

[0127] In vivo pharmacokinetic (PK) studies

[0128] The plasma pharmacokinetic (PK) profile of CCS1477 was evaluated in female CD- 1 nude mice (Charles River) at approximately 8-12 weeks in age. CCS1477 was dissolved in 10% DMSO / 90% (20% hydroxypropyl beta cyclodextrin (HP-β-CD) in sterile water) at 2.5 mg / mL for a 25 mg / kg dose and 5 mg / mL for a 50 mg / kg dose, each using a 10 mL / kg IP injection. Three survival blood samples were obtained from each mouse via retro-orbital plexus 38 AFSDOCS:300782609.1VIA EFS Attorney Docket No.046094-784001WO Date of Deposit: December 17, 2024 using 70 μL glass microhematocrit capillary tubes (Fisher Scientific™), and a fourth final sample by cardiac puncture immediately following the third survival sample, all using K- EDTA as anticoagulant. Samples were obtained at various times up to 24 hours post-dose, immediately processed to plasma, and stored at -80°C until analysis. At terminal time points of 8, 16, and 24 h, carcasses were perfused with PBS, brains extracted and rinsed. Samples were immediately stored on dry ice and transferred to - 80°C until analysis.

[0129] Brain samples were homogenized using a FastPrep-24 system (MP Biomedicals, Santa Ana, CA), and stored at -80°C until analysis by qualified liquid chromatography – tandem mass spectrometry (LC- MS / MS) assay. Plasma (CD-1 mouse, KEDTA, BioIVT) and brain homogenate (NSG mouse) calibrators and quality controls were spiked with solutions, corrected for salt content and purity as necessary, prepared in methanol. Plasma and brain homogenate samples were precipitated, and extracted supernatant was analyzed on an AB Sciex ExionLC high performance liquid chromatography system via an AB Sciex ExionLC autosampler (FIG.11-FIG.15).

[0130] Example 8: Compound Synthesis

[0131] tert-Butyl ((1r,4r)-4-((4-(3,5-dimethylisoxazol-4-yl)-2- nitrophenyl)amino)cyclohexyl)carbamate

[0132] In a 1003-nitrophenyl)-3,5- dimethylisoxazole (0.9442 g, 1.0 eq.) was dissolved in dry THF (30 mL) and TEA (1.68 mL). trans-N-Boc-1,4-cyclohexanediamine (1.029 g, 1.2 eq.) was added and the resulting mixture was heated at 60°C for 25 h. After cooling to room temperature (rt), the reaction mixture was diluted with water (100 mL), then extracted with ethyl acetate (2 x 100 mL). Combined organic layers were concentrated in vacuo to give the title compound, which was used in the next step without further purification. MS (ESI) calculated. For C22H31N4O5[M+1]+: 431.23, Found: 431.48.

[0133] tert-Butyl ((1r,4r)-4-((2-amino-4-(3,5-dimethylisoxazol-4- yl)phenyl)amino)cyclohexyl)carbamate 39 AFSDOCS:300782609.1VIA EFS Attorney Docket No.046094-784001WO Date of Deposit: December 17, 2024 NHBoc NHBoc Na2S2O4

[0134] In a butyl ((1r,4r)-4-((4- (3,5-- g, 1.0 eq.) was dissolved in THF (75 mL) and water (75 mL). Ammonia solution 7.0 M in methanol (10.87 mL, 20 eq.) and sodium dithionite (6.634 g, 10 eq) were added and the reaction stirred at rt for 6 h. The resulting mixture was diluted with ethyl acetate (200 mL), washed with 1M NaOH (100 mL) and brine (100 mL), the organic phase was concentrated in vacuo to give the title compound (2.0393 g), which was used in next step without further purification. MS (ESI) calculated. For C22H33N4O3 [M+1]+: 401.25, Found: 401.49.

[0135] tert-butyl ((1S,4r)-4-((4-(3,5-dimethylisoxazol-4-yl)-2-((S)-6-oxopiperidine-2- carboxamido)phenyl)amino)cyclohexyl)carbamate

[0136] In a((1r,4r)-4-((2- amino-4-(3,5-dimethylisoxazol-4-yl)phenyl)amino)cyclohexyl)carbamate (2.0393 g, 1.0 eq.), (S)-6-oxopiperidine-2-carboxylic acid (0.8015 g, 1.1 eq.), and DIEA (1.067 mL, 1.2 eq.) were dissolved in 10 mL DMF. HATU (2.1297 g, 1.1 eq.) was added and stirred at rt overnight. The resulting mixture was diluted with water, then extracted with ethyl acetate, dried over Na2SO4, filtered and concentrated in vacuo to give the title compound, which was used in next step without further purification. MS (ESI) calculated. For C28H40N5O5 [M+1]+: 526.30, Found: 526.59.

[0137] tert-butyl ((1S,4r)-4-(5-(3,5-dimethylisoxazol-4-yl)-2-((S)-6-oxopiperidin-2-yl)-1H- benzo[d]imidazol-1-yl)cyclohexyl)carbamate (1, CCS1477int) 40 AFSDOCS:300782609.1VIA EFS Attorney Docket No.046094-784001WO Date of Deposit: December 17, 2024

[0138] In a butyl ((1S,4r)-4-((4- (3,5-- - carboxamido)phenyl)amino)cyclohexyl)carbamate (4 g, 1.0 eq.) was dissolved in acetic acid (9 mL) then heated at 80°C for 6 h. The resulting mixture was concentrated then purified by silica gel chromatography (eluent: dichloromethane / methanol) to give the title compound as a white powder (511.2 mg). MS (ESI) calculated. For C28H38N5O4 [M+1]+: 508.29, Found: 508.39.

[0139] N1-((1S,4r)-4-(5-(3,5-dimethylisoxazol-4-yl)-2-((S)-6-oxopiperidin-2-yl)-1H- benzo[d]imidazol-1-yl)cyclohexyl)-N5-(15-oxo-19-((3aS,4S,6aR)-2-oxohexahydro-1H- thieno[3,4-d]imidazol-4-yl)-4,7,10-trioxa-14-azanonadecyl)glutaramide4-yl)-2-((S)-6-oxopiperidin-2-yl)-1H-benzo[d]imidazol-1-yl)cyclohexyl)carbamate (9.75 mg, 1.0 eq.) was dissolved in DCM (800 µL) followed by dropwise addition of trifluoroacetic acid (TFA, 200 µL). The reaction was stirred at 25°C for 1 h, and the solvent was removed. The residue was subjected to the next step reaction without further purification.

[0141] In a 4 mL vial fitted with a stir bar, the residue from the last step and N-Biotinyl- NH-(PEG)2-COOH DIPEA (20 atoms) (13.3 mg, 1 eq.) were dissolved in N,N- 41 AFSDOCS:300782609.1VIA EFS Attorney Docket No.046094-784001WO Date of Deposit: December 17, 2024 Dimethylformamide (DMF, 100 µL), followed by addition of triethylamine (TEA, 13 µL, 5 eq.) and HATU (7.3 mg, 1 eq.). Then the resulting mixture was stirred at 25°C for 2 h. After the reaction was completed, the mixture was directly purified by silica gel chromatography (eluent: dichloromethane / methanol) to give the title compound as a white powder (12.5 mg, 68%). MS (ESI) calculated. For C48H72N9O9S [M+1]+: 950.52, Found: 950.92.

[0143] (S)-1-(3-(difluoromethyl)phenyl)-6-(5-(3,5-dimethylisoxazol-4-yl)-1-((1r,4S)-4- methoxycyclohexyl)-1H-benzo[d]imidazol-2-yl)piperidin-2-one (3, iCBP-1)

[0144] In4-yl)-1-((1r,4S)- 4-methoxycyclohexyl)-1H-benzo[d]imidazol-2-yl)piperidin-2-one (8.5 mg, 1.0 eq.), (3- (difluoromethyl)phenyl)boronic acid (5.2 mg, 1.5 eq.), and Cu-TMEDA catalyst (1.4 mg, 0.15 eq.) were dissolved in acetonitrile (100 µL), followed by addition of 1,8- diazabicyclo[5.4.0]undec-7-ene (DBU, 1 µL, 0.1 eq.). The reaction mixture was stirred at 60°C for 24 h. The mixture was purified directly by silica gel chromatography (eluent: dichloromethane / methanol) to give the title compound as a white powder (7.9 mg, 71%).1H NMR (500 MHz, CDCl3) δ 7.61 (d, J = 1.3 Hz, 1 H), 7.37 (d, J = 8.6 Hz, 1 H), 7.29 (s, 1 H), 7.26-7.20 (m, 3 H), 7.00 (dd, J1 = 8.5 Hz, J2 = 1.5 Hz, 1 H), 6.45 (t, J = 56.5 Hz, 1 H), 5.25 (t, J = 5.3 Hz, 1 H), 3.99-3.89 (m, 1 H), 3.32 (s, 3 H), 3.24-3.12 (m, 1 H), 2.85-2.74 (m, 1 H), 2.68-2.57 (m, 1 H), 2.36 (s, 3 H), 2.23 (s, 3 H), 2.41-1.98, 1.95-1.83, 1.80-1.69, 1.39-1.05 (m, 12 H). MS (ESI) calculated. For C31H35F2N4O3[M+1]+: 549.27, Found: 549.44.

[0145] (S)-1-(3,5-bis(trifluoromethyl)phenyl)-6-(5-(3,5-dimethylisoxazol-4-yl)-1-((1r,4S)- 4-methoxycyclohexyl)-1H-benzo[d]imidazol-2-yl)piperidin-2-one (4, iCBP-4) 42 AFSDOCS:300782609.1VIA EFS Attorney Docket No.046094-784001WO Date of Deposit: December 17, 2024

[0146] -1-((1r,4S)-4- - eq.), (3,5- bis(trifluoromethyl)phenyl)boronic acid (7.7 mg, 1.5 eq.), and Cu-TMEDA catalyst (1.4 mg, 0.15 eq.) were dissolved in acetonitrile (100 uL), followed by addition of 1,8- diazabicyclo[5.4.0]undec-7-ene (DBU, 1 µL, 0.1 eq.). The reaction mixture was stirred at 60°C for 24 h. The mixture was purified directly by silica gel chromatography (eluent: dichloromethane / methanol) to give the title compound as a white powder (4.0 mg, 31%).1H NMR (500 MHz, CDCl3) δ 7.67 (s, 2 H), 7.59 (d, J = 1.3 Hz, 1 H), 7.56 (s, 1 H), 7.37 (d, J = 8.2 Hz, 1 H), 7.00 (dd, J1= 8.3 Hz, J2= 1.6 Hz, 1 H), 5.28 (t, J = 5.1 Hz, 1 H), 4.04-3.92 (m, 1 H), 3.33 (s, 3 H), 3.26-3.15 (m, 1 H), 2.88-2.75 (m, 1 H), 2.69-2.58 (m, 1 H), 2.34 (s, 3 H), 2.20 (s, 3 H), 2.41-1.98, 1.95-1.83, 1.80-1.69, 1.39-1.05 (m, 12 H). MS (ESI) calculated. For C32H33F6N4O3 [M+1]+: 635.25, Found: 635.42.

[0147] (S)-6-(5-(3,5-dimethylisoxazol-4-yl)-1-((1r,4S)-4-methoxycyclohexyl)-1H- benzo[d]imidazol-2-yl)-1-phenylpiperidin-2-one (5, iCBP-5)((1r,4S)- 4-methoxycyclohexyl)-1H-benzo[d]imidazol-2-yl)piperidin-2-one (8.5 mg, 1.0 eq.), phenylboronic acid (3.7 mg, 1.5 eq.), and Cu-TMEDA catalyst (1.4 mg, 0.15 eq.) were dissolved in acetonitrile (100 µL), followed by addition of 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU, 1 µL, 0.1 eq.). The reaction mixture was stirred at 60°C for 24 h. The mixture was purified directly by silica gel chromatography (eluent: dichloromethane / methanol) to give the title compound as a white powder (7.7, 77%).1H NMR (500 MHz, CDCl3) δ 7.62 (s, 1 H), 7.36 (d, J = 8.6 Hz, 1 H), 7.18 (t, J = 7.6 Hz, 2 H), 7.10 (t, J = 7.5 Hz, 1 H), 7.06 (d, J = 7.7 Hz, 2 43 AFSDOCS:300782609.1VIA EFS Attorney Docket No.046094-784001WO Date of Deposit: December 17, 2024 H), 6.99 (d, J = 8.6 Hz, 1 H), 5.21 (t, J = 5.2 Hz, 1 H), 3.95-3.83 (m, 1 H), 3.31 (s, 3 H), 3.23- 3.11 (m, 1 H), 2.85-2.75 (m, 1 H), 2.67-2.57 (m, 1 H), 2.36 (s, 3 H), 2.23 (s, 3 H), 2.41-1.98, 1.95-1.83, 1.80-1.69, 1.39-1.05 (m, 12 H). MS (ESI) calculated. For C30H35N4O3 [M+1]+: 499.27, Found: 499.44.

[0149] (S)-1-(3-(tert-butyl)phenyl)-6-(5-(3,5-dimethylisoxazol-4-yl)-1-((1r,4S)-4- methoxycyclohexyl)-1H-benzo[d]imidazol-2-yl)piperidin-2-one (6, iCBP-6) OB(OH)2OO ((1r,4S)-4-methoxycyclohexyl)-1H-benzo[d]imidazol-2-yl)piperidin-2-one (8.5 mg, 1.0 eq.), (3-(tert- butyl)phenyl)boronic acid (5.3 mg, 1.5 eq.), and Cu-TMEDA catalyst (1.4 mg, 0.15 eq.) were dissolved in acetonitrile (100 µL), followed by addition of 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU, 1 µL, 0.1 eq.). The reaction mixture was stirred at 60°C for 24 h. The mixture was purified directly by silica gel chromatography (eluent: dichloromethane / methanol) to give the title compound as a white powder (5.0 mg, 45%).1H NMR (500 MHz, CDCl3) δ 7.63 (d, J = 1.3 Hz, 1 H), 7.32 (d, J = 8.5 Hz, 1 H), 7.14 (t, J = 7.7 Hz, 1 H), 7.08 (d, J = 7.9 Hz, 1 H), 6.96 (dd, J1 = 8.6 Hz, J2 = 1.6 Hz, 1 H), 6.91 (d, J = 7.7 Hz, 1 H), 6.78 (t, J = 1.5 Hz, 1 H), 5.12 (dd, J1= 7.0 Hz, J2= 5.2 Hz, 1 H), 3.90-3.75 (m, 1 H), 3.30 (s, 3 H), 3.18-3.07 (m, 1 H), 2.84-2.73 (m, 1 H), 2.68-2.59 (m, 1 H), 2.33 (s, 3 H), 2.20 (s, 3 H), 2.41-1.98, 1.95-1.83, 1.80-1.69, 1.39- 1.05 (m, 12 H), 0.86 (s,9 H). MS (ESI) calculated. For C34H43N4O3[M+1]+: 555.33, Found: 555.48.

[0151] (S)-1-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-6-(5-(3,5-dimethylisoxazol-4-yl)-1- ((1r,4S)-4-methoxycyclohexyl)-1H-benzo[d]imidazol-2-yl)piperidin-2-one (7, iCBP-8)VIA EFS Attorney Docket No.046094-784001WO Date of Deposit: December 17, 2024

[0152] In a 4 mL vial fitted with a stir bar, (S)-6-(5-(3,5-dimethylisoxazol-4-yl)-1-((1r,4S)- 4-methoxycyclohexyl)-1H-benzo[d]imidazol-2-yl)piperidin-2-one (8.5 mg, 1.0 eq.), (2,3- dihydrobenzo[b][1,4]dioxin-6-yl)boronic acid (5.4 mg, 1.5 eq.), and Cu-TMEDA catalyst (1.4 mg, 0.15 eq.) were dissolved in acetonitrile (100 µL), followed by addition of 1,8- diazabicyclo[5.4.0]undec-7-ene (DBU, 1 µL, 0.1 eq.). The reaction mixture was stirred at 60°C for 24 h. The mixture was purified directly by silica gel chromatography (eluent: dichloromethane / methanol) to give the title compound as a white powder (6.3 mg, 56%).1H NMR (500 MHz, CDCl3) δ 7.62 (d, J = 0.9 Hz, 1 H), 7.38 (d, J = 8.6 Hz, 1 H), 6.99 (dd, J1 = 8.5 Hz, J2= 1.5 Hz, 1 H), 6.66 (d, J = 2.5 Hz, 1 H), 6.61 (d, J = 8.6 Hz, 1 H), 6.48 (dd, J1= 8.7 Hz, J2 = 2.3 Hz, 1 H), 5.13 (t, J = 5.1 Hz, 1 H), 4.08 (s, 4 H), 3.99-3.88 (m, 1 H), 3.33 (s, 3 H), 3.25-3.14 (m, 1 H), 2.84-2.71 (m, 1 H), 2.63-2.54 (m, 1 H), 2.37 (s, 3 H), 2.24 (s, 3 H), 2.41- 1.98, 1.95-1.83, 1.80-1.69, 1.39-1.05 (m, 12 H). MS (ESI) calculated. For C32H37N4O3 [M+1]+: 557.28, Found: 557.42.

[0153] Example 9: Brain:Plasma Ratios

[0154] 21 normal CD1 mice per compound (CCS1477 or iCBP4) were injected IP with a dose of 50 mg / kg specific compound (10% DMSO, 10% Solutol HS15, 80% (10% HP-β-CD in water), and animals were tested at 0.25, 0.5, 1, 2, 4, 8, and 24 h after injection. Three animals per timepoint were sacrificed with a terminal bleed for plasma concentrations of compound. The same mice sacrificed with terminal bleeds for plasma concentrations were perfused via direct cardiac puncture with 20 mL cold saline to perfuse the brain and remove contaminating blood. Brains were removed from these animals and homogenized for 2 min with 3 volumes of PBS by Mini-bead-beater before sample extraction.

[0155] On both samples (plasma and brain), detection of compound was performed by LC- MS, using defined concentrations of glipizide in acetonitrile as a standard curve, yielding a range of detection of 1-1000 ng / mL. Data for brain samples (ng compound / g brain tissue) are plotted as FIG. 17, and plasma samples (ng compound / g plasma) are plotted as FIG. 18. The ratio of these values is plotted as FIG.16 (brain / plasma).

[0156] iCBP4 sustained a higher presence in the brain as compared to CCS1477 (FIG.17). Also, iCBP4 has lower levels in circulation as compared to CCS1477 (FIG. 18). Together, these data suggest that iCBP4 is more brain penetrant than CCS1477, and has lower plasma circulation, and less toxicity. 45 AFSDOCS:300782609.1VIA EFS Attorney Docket No.046094-784001WO Date of Deposit: December 17, 2024

[0157] LCMS

[0158] All patent publications and non-patent publications are indicative of the level of skill of those skilled in the art to which this disclosure pertains. All these publications (including any specific portions thereof that are referenced) are herein incorporated by reference to the same extent as if each individual publication were specifically and individually indicated as being incorporated by reference. 46 AFSDOCS:300782609.1VIA EFS Attorney Docket No.046094-784001WO Date of Deposit: December 17, 2024

[0159] Although the disclosure herein has been described with reference to particular embodiments, it is to be understood that these embodiments are merely illustrative of the principles and applications of the present disclosure. It is therefore to be understood that numerous modifications may be made to the illustrative embodiments and that other arrangements may be devised without departing from the spirit and scope of the present disclosure as defined by the appended claims. 47 AFSDOCS:300782609.1VIA EFS Attorney Docket No.046094-784001WO Date of Deposit: December 17, 2024 APPENDIX Table S1. Area under the curve measurements for CCS1477 and A485 treated cell lines CCS1477  A485  Ratio  Cell Line   Lineage Association (AUC)  (AUC)   CCS1477 / A485 AFSDOCS:300782609.1VIA EFS Attorney Docket No.046094-784001WO Date of Deposit: December 17, 2024 G401  Rhabdoid  0.887  1.000  0.887 NCIH441  Lung Cancer  0.783  0.882  0.888 49 AFSDOCS:300782609.1VIA EFS Attorney Docket No.046094-784001WO Date of Deposit: December 17, 2024 MKN1  Gastric Cancer  0.961  0.967  0.993 SKMEL30  Melanoma  0.798  0.803  0.994 50 AFSDOCS:300782609.1VIA EFS Attorney Docket No.046094-784001WO Date of Deposit: December 17, 2024 T3M10  Lung Cancer  0.928  0.904  1.027 EN  Endometrial / Uterine Cancer  1.000  0.974  1.027 51 AFSDOCS:300782609.1VIA EFS Attorney Docket No.046094-784001WO Date of Deposit: December 17, 2024 CW2  Colon / Colorectal Cancer  0.994  0.932  1.066 SQ1  Lung Cancer  0.890  0.835  1.067 52 AFSDOCS:300782609.1VIA EFS Attorney Docket No.046094-784001WO Date of Deposit: December 17, 2024 MDAMB453  Breast Cancer  0.576  0.518  1.111 SNU878  Liver Cancer  0.797  0.716  1.112 53 AFSDOCS:300782609.1VIA EFS Attorney Docket No.046094-784001WO Date of Deposit: December 17, 2024 HH  Lymphoma  0.895  0.776  1.153 CORL105  Lung Cancer  0.878  0.762  1.153 54 AFSDOCS:300782609.1VIA EFS Attorney Docket No.046094-784001WO Date of Deposit: December 17, 2024 HCC38  Breast Cancer  0.887  0.743  1.193 CI1  B‐cell Lymphoma  0.928  0.778  1.193 55 AFSDOCS:300782609.1VIA EFS Attorney Docket No.046094-784001WO Date of Deposit: December 17, 2024 RL  B‐cell Lymphoma  0.890  0.706  1.262 CMLT1  CML  0.834  0.661  1.263 56 AFSDOCS:300782609.1VIA EFS Attorney Docket No.046094-784001WO Date of Deposit: December 17, 2024 769P  Kidney Cancer  0.866  0.633  1.368 OAW42  Ovarian Cancer  0.997  0.727  1.372 57 AFSDOCS:300782609.1VIA EFS Attorney Docket No.046094-784001WO Date of Deposit: December 17, 2024 TF1  AML  0.667  0.457  1.458 IGR37  Melanoma  1.000  0.684  1.461 58 AFSDOCS:300782609.1VIA EFS Attorney Docket No.046094-784001WO Date of Deposit: December 17, 2024 Table S2. Median normalized area under the curve measurements for CCS1477 and A485 treated cell lines CCS1477  A485  Ratio  Cell Line  Lineage Association (AUC)  (AUC)   CCS1477 / A485  h59 AFSDOCS:300782609.1VIA EFS Attorney Docket No.046094-784001WO Date of Deposit: December 17, 2024 NCIH441  Lung Cancer  0.853  1.111  0.768 RH28  Rhabdomyosarcoma  0.968  1.260  0.768 60 AFSDOCS:300782609.1VIA EFS Attorney Docket No.046094-784001WO Date of Deposit: December 17, 2024 SKMEL30  Melanoma  0.871  1.012  0.860 HEYA8  Ovarian Cancer  1.061  1.233  0.860 61 AFSDOCS:300782609.1VIA EFS Attorney Docket No.046094-784001WO Date of Deposit: December 17, 2024 EN  Endometrial / Uterine Cancer  1.091  1.227  0.889 CHLA15  Neuroblastoma  0.807  0.906  0.891 62 AFSDOCS:300782609.1VIA EFS Attorney Docket No.046094-784001WO Date of Deposit: December 17, 2024 SQ1  Lung Cancer  0.971  1.052  0.923 BICR16  Head and Neck Cancer  0.793  0.857  0.925 63 AFSDOCS:300782609.1VIA EFS Attorney Docket No.046094-784001WO Date of Deposit: December 17, 2024 SNU878  Liver Cancer  0.869  0.903  0.962 TE4  Esophageal Cancer  1.091  1.132  0.963 64 AFSDOCS:300782609.1VIA EFS Attorney Docket No.046094-784001WO Date of Deposit: December 17, 2024 CORL105  Lung Cancer  0.958  0.960  0.998 PANC0813  Pancreatic Cancer  1.091  1.091  1.000 65 AFSDOCS:300782609.1VIA EFS Attorney Docket No.046094-784001WO Date of Deposit: December 17, 2024 CI1  B‐cell Lymphoma  1.012  0.980  1.032 RERFLCKJ  Lung Cancer  0.963  0.932  1.033 66 AFSDOCS:300782609.1VIA EFS Attorney Docket No.046094-784001WO Date of Deposit: December 17, 2024 CMLT1  CML  0.910  0.833  1.093 KNS60  Glioblastoma  0.975  0.891  1.094 67 AFSDOCS:300782609.1VIA EFS Attorney Docket No.046094-784001WO Date of Deposit: December 17, 2024 OAW42  Ovarian Cancer  1.087  0.916  1.187 LMSU  Gastric Cancer  1.082  0.910  1.188 68 AFSDOCS:300782609.1VIA EFS Attorney Docket No.046094-784001WO Date of Deposit: December 17, 2024 IGR37  Melanoma  1.091  0.862  1.265 NCIH1437  Lung Cancer  1.083  0.847  1.278 69 AFSDOCS:300782609.1

Claims

VIA EFS Attorney Docket No.046094-784001WO Date of Deposit: December 17, 2024 What is claimed is:

1. A compound having a structure represented by formula I: a pharmaceutically acceptable salt or stereoisomer thereof,H, Br, CHF2, CF3, or t-Bu, provided that when at least one of R1, R2, and R3 is Br, CHF2, CF3, or t-Bu, at least one of R1, R2, and R3 is H, or R1and R2, together with the carbon atoms to which they are attached, form 1,4-dioxane.

2. The compound of claim 1, wherein R1and R2are each independently H, Br, CHF2, CF3, or t-Bu, and R3 is H.

3. The compound of claim 1, wherein R1 and R3 are each independently H, Br, CHF2, CF3, or t-Bu, and R2is H.

4. The compound of claim 1, wherein two of R1, R2, and R3are Br or CF3.

5. The compound of claim 1, wherein R1and R2are both H.

6. The compound of claim 1, wherein R1and R3are both H.

7. The compound of claim 1, which is: 70 AFSDOCS:300782609.1VIA EFS Attorney Docket No.046094-784001WO Date of Deposit: December 17, 2024 2), 5), a8. The compound of claim 7, a pharmaceutically acceptable salt or71 AFSDOCS:300782609.1VIA EFS Attorney Docket No.046094-784001WO Date of Deposit: December 17, 2024 9. The compound of claim 7, a pharmaceutically acceptable salt or10. The compound of claim 7, a pharmaceutically acceptable salt or11. The compound of claim 7, a pharmaceutically acceptable salt or72 AFSDOCS:300782609.1VIA EFS Attorney Docket No.046094-784001WO Date of Deposit: December 17, 2024 12. The compound of claim 7, a pharmaceutically acceptable salt or13. The compound of claim 7, a pharmaceutically acceptable salt or14. A pharmaceutical composition, comprising the compound of any one of claims 1-13, or pharmaceutically acceptable salt or stereoisomer thereof, and a pharmaceutically acceptable carrier.

15. A method of treating a neurologic, endometrial, or hematologic cancer, comprising administering to a subject in need thereof, the compound of any one of claims 1-13, or a pharmaceutically acceptable salt or stereoisomer thereof, or the pharmaceutical composition of claim 14.

16. The method of claim 15, wherein the neurologic, endometrial, or hematologic cancer is neuroblastoma, endometrial carcinoma, uterine adenosquamous carcinoma, multiple myeloma, acute myeloid leukemia, or medulloblastoma. 73 AFSDOCS:300782609.1

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