EP4629984A1 - Development of potent dual hdac/brd4 inhibitors - Google Patents

Abstract

Disclosed herein are compounds of Formula I or II, or pharmaceutically acceptable salts thereof, and their use as dual BRD4 and HDAC inhibitors. Also disclosed are pharmaceutical compositions comprising the compounds of Formula I or II and methods of using the compounds and the pharmaceutical compositions for treating cancer or psoriasis. Also disclosed herein are methods for preparing compounds of Formula I or II.

Description

DEVELOPMENT OF POTENT DUAL HDAC/BRD4 INHIBITORS CROSS-REFERENCE TO RELATED APPLICATIONS This application claims benefit of U.S. Provisional Application Serial No.63/430,584, filed December 6, 2022, the entire contents of which is incorporated by reference herein. REFERENCE TO AN ELECTRONIC SEQUENCE LISTING The contents of the electronic sequence listing (2023-12- 05_169852.00121_WIPO_Sequence_Listing_XML.xml; Size: 13,420 bytes; and Date of Creation: December 05, 2023) are herein incorporated by reference in its entirety. BACKGROUND OF THE INVENTION The traditional method of regulating functions of disease-related proteins is to develop small molecules that can bind at the active sites wof target proteins to reduce or block the interaction between target and natural ligands or other proteins/cofactors. However, the rapid dissociation of inhibitor-protein complex makes it hard to achieve deep and long-lasting protein inhibition. Although covalent inhibitors can reach long-lasting inhibition of protein by forming covalent linkages with nucleophiles residues, potential off-target effects of these types of compounds have hampered their development (Concepción González-Bello, ChemMedChem, 11(1), 22-30, 2015). Moreover, mutation in targets results in the resistance to current inhibitors (Ivana Bozic, et al., Proc. Natl. Acad. Sci. USA, 111(45), 15964-15968, 2014). In addition, not all disease associated proteins are “druggable” due to a flat protein interface or lack of a defined pocket for ligand interactions (Gregory L. Verdine and Loren D. Walensky, Clin. Cancer Res., 13(24), 2007). As such, there remains a need for new compounds and methods that can regulate the functions of disease-related proteins. Molecular recognition events between proteins are at the heart of every biological process (Zuzanna Kozicka and Nicolas Holger Thomä, Cell Chem. Biol., 28(7), 1032-1047, 2021). In contrast with the interactions between proteins and small ligands that generally involve a few residues within a specific site for binding with small molecular ligand, interfaces between proteins involve a multitude of polar and hydrophobic interactions distributed across a larger interface. Proteins with appropriate conformations can form stable protein-protein complexes that can regulate the function of one or more proteins in the complex. Thus, it is reasonable to remodel the conformation of protein for protein-protein interactions to regulate protein functions. Virus-associated lymphomas caused by infections of Kaposi's sarcoma associated herpesvirus (KSHV) and Epstein-Barr virus (EBV) are two of major human oncogenic herpesviruses. Virus-associated lymphomas are a group of aggressive malignancies with poor prognosis due to lack of effective treatments. Although treatment options are available for virus- associated lymphomas, current treatment options primarily depend on chemotherapy, immunotherapy, and antiviral therapy, the treatments are often ineffective due to drug resistance and dose-limiting toxicities. As such, there remains a need for development of effective therapies and agents in the treatment of virus-associated lymphomas. BRIEF SUMMARY OF THE INVENTION Disclosed herein are dual BRD4/HDAC inhibitors and methods of making and using the same. A first aspect of the technology provides for compounds of Formula I or Formula II, or pharmaceutically acceptable salts thereof. Formula 1 has a structure of Formula II has a structure of For compounds of Formula I or II, A is O or S; B is selected from the group consisting of O, NR¹, and S; R¹ is H or alkyl; X is selected from CR²R³ or a chemical bond; R² and R³ are independently selected from H or alkyl; and Y is selected from -(CH₂)₅C(O)NHOH, -OH, and H. When the compounds comprise CR²R³, R² and R³ may be the same or different. When R² and R³ are different, the carbon bonded to R² and R³ may be characterized by an S or R configuration. Exemplary R² and R³ include, without limitation, H or methyl. In some embodiments, R² and R³ are each H. In other embodiments, one of R² and R³ is H and the other is methyl. For certain compounds, A and B are each be oxygen. For other compounds, A is oxygen and B is NR¹. The compounds disclosed herein may be used in the manufacture of a medicament or for use in any of the methods disclosed herein. For example, the compounds disclosed herein may be used in methods for treating the indicated disease, condition, or disorder. Pharmaceutical compositions comprising the compounds disclosed herein are also provided. The pharmaceutical compositions comprise one or more of the compounds of Formular I and/or Formula II or pharmaceutically acceptable salts thereof. The pharmaceutical composition may further comprise a pharmaceutically acceptable excipient, diluent, or carrier. The pharmaceutical compositions disclosed herein may be for use in any of the methods disclosed herein. For example, the pharmaceutical compositions disclosed herein may be used in methods for treating the indicated disease, condition, or disorder. Methods of treating cancer are provided. The method may comprise administering an effective amount of any of the disclosed compounds, or a pharmaceutically acceptable salt thereof, to a subject having the cancer. Also provided are methods for reducing or inhibiting cancer cell growth in a subject having cancer. The method may comprise administering an effective amount of any of the disclosed compounds, or a pharmaceutically acceptable salt thereof, to a subject having the cancer. The cancer may be selected from the group consisting of Kaposi sarcoma (KS), primary effusion lymphoma (PEL), cutaneous T-cell lymphoma (CTCL), KSHV-associated lymphomas, and EBV-associated lymphomas Methods for inhibiting expression of c-MYC in a cancer cell are provided. The method may comprise contacting the cell with an effective amount of any of the disclosed compounds. Suitably, methods may be performed in vitro or ex vivo. In other instances, the methods are performed in vivo. The cancer cell may be selected from the group consisting of a Kaposi sarcoma cancer cell, a primary effusion lymphoma cancer cell, a cutaneous T-cell lymphoma cancer cell, a KSHV- associated lymphoma cancer cell, and an EBV-associated lymphoma cancer cell. Also provided is a method for treating psoriasis. The method may comprise administering an effective amount of any of the compounds disclosed herein to a subject having psoriasis. Also provided are methods for preparing the compounds disclosed herein. BRIEF DESCRIPTION OF THE DRAWINGS Non-limiting embodiments of the present invention will be described by way of example with reference to the accompanying figures, which are schematic and are not intended to be drawn to scale. In the figures, each identical or nearly identical component illustrated is typically represented by a single numeral. For purposes of clarity, not every component is labeled in every figure, nor is every component of each embodiment of the invention shown where illustration is not necessary to allow those of ordinary skill in the art to understand the invention. Figure 1 illustrates the working principle of ConCTACs. ConCTACs work by binding and inducing conformational change in one or more target proteins and facilitate the formation of a stable protein-protein complex with the target proteins are in proximity. The complex may alter the function some or all of the target protein that can abrogate or activate the function of a target proteins. Figures 2A-2B illustrate that there is no synergistic inhibitory effects of BET bromodomain inhibition by (+)-JQ1 and HDACs inhibition by SAHA (Vorinostat) or Entinostat in KSHV- positive BCBL-1 PEL cells. Figure 2A shows no synergistic inhibitory effects of BET bromodomain inhibition by (+)- JQ1 and HDACs inhibition by SAHA (Vorinostat) in KSHV-positive BCBL-1 PEL cells. Figure 2B shows no synergistic inhibitory effects of BET bromodomain inhibition by (+)- JQ1 and HDACs inhibition by Entinostat in KSHV-positive BCBL-1 PEL cells. KSHV-positive BCBL-1 PEL cells were treated with indicated concentrations of (+)-JQ1, Entinostat, or the combination with a fixed ratio (1:1) of (+)-JQ1 and Entinostat for 72 hours. Figures 3A-3G show that Example 2 or Example 5 inhibits BET bromodomains and HDACs pathways in a balanced fashion and alters the expressions of several cell death regulatory proteins in PEL cells. Figure 3A shows that Example 2 or Example 5 inhibits BET bromodomains pathway (decreased c-Myc protein level) and HDACs pathway (increased H3K9Ac, H4K9Ac, and Acetyl- tubulin protein levels) in a balanced fashion in BCBL-1 PEL cells. Meanwhile, Example 2 or Example 5 treatment alters the expression of several cell death regulatory proteins, including decreased p-Rb and increased cleaved-caspase 3, cleaved-PARP, p-ATM and p21 in BCBL-1 cells. BCBL-1 cells were treated with indicated concentrations of examples for 48 h, then protein expression was measured by western blot. Figure 3B shows that structural modification only for blocking the key BRD4 binding on Example 2 or Example 5 results in Example 3 or Example 6, respectively, demonstrating reduced inhibitory activity on BET bromodomains (little or no changes on c-Myc protein level versus vehicle control) but also reduced inhibitory activity on HDACs (increased H3K9Ac protein levels versus vehicle control) in BCBL-1 cells. Alternative structural modification only for blocking the key HDAC binding on Example 2 or Example 5 results in Example 4 or Example 7, respectively, demonstrating reduced inhibitory activity on HDACs (little or no changes on H3K9Ac protein levels versus vehicle control) but retaining inhibitory activity on BET bromodomains (decreased c-Myc protein level versus vehicle control) in BCBL-1 cells. BCBL-1 cells were treated with indicated concentrations of examples for 48 h, then protein expression was measured by western blot. The balanced and significantly enhanced BRD4 and HDAC inhibitions of Examples 2 and 5 are resulted from the induced HDAC and BRD4 protein-protein binding. Figures 3C-3G shows Example 2 or Example 5 demonstrates an overlapping gene expression pattern of altered genes when compared with known BET bromodomains inhibitor (+)- JQ1 and HDACs inhibitor SAHA. Figure 3C-3E illustrate the intersection analysis of significantly altered genes (expression change ≥ 2-fold and p < 0.05) identified by RNA-Seq from Example 2- , Example 5-, (+)-JQ1-, or SAHA-treated BCBL-1 cells, respectively. Figure 3F and 3G illustrate the Heat map and enrichment analysis of genes commonly altered in Example 2-, Example 5-, (+)- JQ1-, and SAHA-treated BCBL-1 cells. Genes from top to bottom in Figure 3F: FOS, CSF1R, CHAC1, IRF7, SOCS3, ATF3, DDIT4, ARRDC3, ARC, DDIT3, TPT1-AS1, TMEM189- UBE2V1, PCDH9, ST7-AS1, NBPF1, LINC00052, LINC00680, AC024592.12, BBS1, MALAT1, KLF10, NKPD1, SCN9A, GADD45B, TCL6, RNF185-AS1, GPRIN1, RP11- 20B24.4, PARP9, GSN, RP11-93B14.9, STAG3, ANKRD24, RP11-1070N10.5, IL6ST, APLP1, SH3BP5, SESN2, RP11-588H23.3, DOCK3, C7orf31, RN7SK, ERCC6-PGBD3, TUBB3, CUEDC1, TRIM36, CDKN2B, PTMS, PDIA4, ACADVL, HSPA5, GRN, NEK9, HSP90B1, RPS28, ZNF664, LAMC1, H1FX, COX7A2L, LGMN, PEA15, RPS15, SEMA4B, COPA, HSP90AA1, TAF10, RPN2, SLC25A5, RUVBL2, CDC123, RBBP7, KLK2, IVNS1ABP, CDC25C, RANBP1, AURKA, DPH2, MTFR2, FBXL19, WHSC1, FAM72B, NEK2, SPOCK1, ALYREF, LINC01088, CDC42EP1, TMC4, SLC6A20, TROAP, CD70, TPX2, FZD2, MYBL2, SEPT9, RAB31, RP11-707M3.3, CFAP58, SPINT1, MCM5, CCDC169-SOHLH2, IFNG, CD44, PRKCB, CD28, and LGALS9B. Figures 4A-4G show that compounds with balanced inhibitory activity on BET bromodomains and HDACs strongly induce KSHV lytic reactivation in KSHV-positive cells. Figures 4A-4C shows that treatment of Example 2 or Example 5 with balanced inhibitory activity on BET bromodomains and HDACs produces stronger RFP signals than Example 3 or Example 6 with reduced inhibitory activity on BET bromodomains and Example 4 or Example 7 with reduced inhibitory activity on HDACs in KSHV-positive iSLK.219 PEL cells. The iSLK.219 cells were treated by indicated concentrations of examples in the addition to Dox (0.05 µg/mL) for 24 h, then the fluorescence intensity was detected via microscopy. Figure 4D shows treatment of Example 2 or Example 5 with balanced inhibitory activity on BET bromodomains and HDACs significantly increases expression of viral genes, such as latent gene, LANA and two lytic genes, RTA and ORF26, and viral DNA levels in PEL cells. PEL cells were treated with compounds for 48 hours then the transcripts of viral genes and viral DNA levels were measured by using RT-qPCR and qPCR, respectively. SAHA and (+)-JQ1 were used as positive control. Error bars represent S.D. for 3 independent experiments, ** = p<0.01 (versus vehicle control). Figure 4E shows treatment of Example 2 or Example 5 with balanced inhibitory activity on BET bromodomains and HDACs strongly elevates the expression of LANA, RTA, ORF54 (an early gene), ORF62 (a late gene) from PEL cells even at low concentrations. The protein levels were measured by using western blotting after treatment PEL cells with compounds at indicated concentrations for 48 hours. SAHA and (+)-JQ1 were used as positive control. Figures 4F-4H show compounds with BET bromodomains and HDACs dual inhibitory activities induce viral lytic reactivation (Figure 4F), which is accompanied by a lower mature virion production under the treatment of compounds when compared to SAHA and (+)-JQ1 (Figure 4G and 4H). This suggests the compounds with BET bromodomains and HDACs dual inhibitory activities induce an alternative lytic reactivation with an incomplete lytic phase. The iSLK.219 cells were treated by indicated concentrations of compounds in conjunction with Dox (0.05 μg/mL), then RFP expression was evaluated at 72 h post-treatment as a measure of lytic reactivation (Figure 4F). Cells were stained with DAPI at 48 h post-infection and the fluorescence signals were examined using fluorescence microscopy (Figure 4G). The viral DNA levels were quantified using qPCR (Figure 4H). Data was normalized as the fold change compared to the Dox control. Figures 5A-5B show that compounds with balanced inhibitory activity on BET bromodomains and HDACs demonstrate augmented in vitro anti-cancer effects. Figure 5A shows that Example 2 with balanced inhibitory activity on BET bromodomains and HDACs demonstrates drastically cytotoxicity in four KSHV-positive PEL cells, including BCBL-1, JSC-1, BCP-1, and BC-1 cells. Comparing with Example 2, Example 3 with reduced inhibitory activity on BET bromodomains and Example 4 with reduced inhibitory activity on HDACs demonstrate 18˗ to 567-fold decreased cytotoxicity in all tested KSHV-positive PEL cells (please see CC₅₀ values in Table 2). Meanwhile, Example 2 has little cytotoxicity in primary cells such as HUVEC, a human primary endothelial cell line. Four KSHV-positive PEL cell lines and HUVEC cells were treated with indicated concentrations of compounds for 72 h, then the cytotoxicity was determined using the WST-1 assay. Figure 5B shows that Example 5 with balanced inhibitory activity on BET bromodomains and HDACs demonstrates enhanced cytotoxicity in four KSHV-positive PEL cells, including BCBL-1, JSC-1, BCP-1, and BC-1 cells. Comparing with Example 5, Example 6 with reduced inhibitory activity on BET bromodomains and Example 7 with reduced inhibitory activity on HDACs demonstrate 20˗ to 472-fold decreased cytotoxicity in all tested KSHV-positive PEL cells (please see CC₅₀ values in Table 2). Meanwhile, Example 5 has little cytotoxicity in primary cells such as HUVEC cells. Four KSHV-positive PEL cell lines and HUVEC cells were treated with indicated concentrations of compounds for 72 h, then the cytotoxicity was determined using the WST-1 assay. Figures 6A-6E show that compounds with balanced inhibitory activity on BET bromodomains and HDACs cause a higher degree of PEL cell cycle arrest and cell apoptosis. Figures 6A-6C show that Example 2 or Example 5 with balanced inhibitory activity on BET bromodomains and HDACs demonstrates a higher degree of PEL cell cycle arrest than Example 3 or Example 6 with reduced inhibitory activity on BET bromodomains and Example 4 or Example 7 with reduced inhibitory activity on HDACs. BCBL-1 cells were treated with these examples at the respective 2 × CC₅₀ for 24 h, then cell cycle was measured by flow cytometry analysis. Error bars represent S.D. for 3 independent experiments, ** = p < 0.01 (versus vehicle control). Figures 6D-6E show that Example 2 or Example 5 with balanced inhibitory activity on BET bromodomains and HDACs demonstrates a higher degree of PEL cell apoptosis than Example 3 or Example 6 with reduced inhibitory activity on BET bromodomains and Example 4 or Example 7 with reduced inhibitory activity on HDACs. BCBL-1 cells were treated with these examples at the respective 2 × CC₅₀ for 24 h, then cell apoptosis was measured by flow cytometry analysis. Error bars represent S.D. for 3 independent experiments, ** = p < 0.01 (versus vehicle control). Figures 7A-7B show that Example 5 demonstrates adequate pharmacokinetic (PK) properties for in vivo efficacy evaluations. Figure 7A shows that Example 5 demonstrates adequate PK parameters with AUC_last of 159 h × μg/mL and terminal t1/2 of 1.66 h when compared to (+)-JQ1 (AUC_last of 78 h × μg/mL, terminal t1/2 of 2.27 h). In contrast, SAHA is cleared too fast (AUC_last of 12 h × μg/mL, terminal t_1/2 of 0.72 h) and therefore it is not used in the following antitumor efficacy studies. The PK profiles of compounds were determined in plasma following a single intraperitoneal (i.p.) injection (50 mg per kg body weight) to NOD/SCID mice, 6–8-week-old, male (Jackson Laboratory, Ellsworth, Maine, USA). Values are calculated from arithmetic mean plasma concentrations (n = 3 mice per condition). Figure 7B shows that Example 5 demonstrates efficient oral PK parameters with AUC_last of 39 h × μg/mL, terminal t_1/2 of 1.12 h, and bioavailability (F) of 24.5%. The PK profiles of Example 5 were determined in plasma following a single oral gavage (p.o.) administration (50 mg per kg body weight) to NOD/SCID mice, 6–8-week-old, male (Jackson Laboratory, Ellsworth, Maine, USA). Values are calculated from arithmetic mean plasma concentrations (n = 3 mice per condition). The bioavailability F (%) = AUC_last for p.o. / AUC_last for i.p. × 100. Figures 8A-8F show that compounds with balanced inhibitory activity on BET bromodomains and HDACs strongly suppress PEL progression in vivo. Figures 8A-8B show that 20 mg/kg of Example 2 or 5 with balanced inhibitory activity on BET bromodomains and HDACs dramatically suppresses PEL tumor progression including reducing ascites formation over study timeframe, comparable to similar effects with 50 mg/kg of (+)-JQ1. Of note, 50 mg/kg of (+)-JQ1 shows visible toxicity in mice (abnormal weight loss in the curve shown in Figure 8A), which are not observed in either Examples 2- or 5-treated mice. NOD/SCID mice were injected i.p. with BCBL-1 cells.72 h later, the compounds or vehicle was i.p. administered initially at 72 h after BCBL-1 injections, and continued once daily, 2 days per week for 3 weeks. Weights were recorded weekly as a surrogate measure of tumor progression. Figure 8C shows that substantial tumor infiltration into the spleen of vehicle-treated mice, whereas only diffuse small tumor nodules are apparent in the spleen of either Examples 2- or 5- treated mice. Meanwhile, a dramatic reduction in c-Myc expression and an increased expression of H3K9Ac is observed within spleen tissues from either Examples 2- or 5-treated mice, when compared with those from vehicle-treated mice with IHC staining, suggesting the in vivo BET bromodomains and HDACs inhibitions by treatment of Examples 2 or 5. At the end of the treatment period in Figure 8A-8B, the splenic tissues were collected from the vehicle or compound treated mice, compared and processed with the H&E and IHC staining. Figures 8D-8F show that oral administration of 100 mg/kg of Example 2 or 5 with balanced inhibitory activity on BET bromodomains and HDACs dramatically represses tumor burden in mice and reduces ascites formation (Figure 8D-8E) and spleen enlargement (Figure 8F). Of note, oral administration of 100 mg/kg of (+)-JQ1 showed severe toxicity in the mice and the experiment regarding oral administration of 100 mg/kg of (+)-JQ1 was terminated based on institute policies. NOD/SCID mice were injected i.p. with BCBL-1 cells.72 h later, the compounds or vehicle were administered orally at 72 h after BCBL-1 injections, and continued once daily, 2 days per week for 3 weeks. Weights were recorded weekly as a surrogate measure of tumor progression. At the end of the treatment period, the splenic tissues were collected from the vehicle or compound treated mice for size comparison. Figures 9A-9C show the effects of compunds with balanced inhibitory activity on BET bromodomains and HDACs on EBV reactivation from EBV+ lymphomas. Figure 9A shows the cytotoxicity of Example 2 or 5 with balanced inhibitory activity on BET bromodomains and HDACs on three EBV+ B-lymphoblastoid cell lines (Akata, VAL, RPMI 6666) and two EBV-transformed lymphoblasts cell lines (GM22671 and GM16113). The cells were treated with indicated concentrations of compounds for 72 h, then cell viability was determined using the WST-1 assay. Data was normalized as the fold change compared to the DMSO control. Figures 9B-9C show the effect of Example 2, 5, or 19 with balanced inhibitory activity on BET bromodomains and HDACs on EBV reactivation. EBV+ cell lines were treated with tested compounds at 250 nM for 72 h, then the transcripts of viral genes were measured using RT-qPCR. Data were normalized as the fold change compared to the DMSO control. Error bars represent S.D. for 3 independent experiments. Figures 10A-10C show dose-response curve for compounds with Myla (Figure 10A), HH (Figure 10B), or Hut78 CTCL (Figure 10C) cells lines. Indicated cells were seeded at the indicated concentration: Myla 2000 cells/well, HH 10,000 cells/well, Hut78 10,000 cells/well in 80 ul RP10F, in Nunc™ Edge™ 96-well non-treated flat-bottom microplates (ThermoFisher Scientific, Waltham, MA) in RPMI + 10% FBS. Duplicate wells with cells were treated at each compound concentration. Cells were cultured in the presence of compounds for 72 hours followed by viability assay. The CellTiter Glo Luminescent Cell Viability Assay (Promega, Madison, WI) was performed according to the manufacturer’s instructions. Cells were lysed for 10 minutes at room temperature, and then the lysates were transferred to black 96-well microplates (Greiner Bio-One, Monroe, NC). Luminescence was read on a Cytation 5 plate reader (Agilent, Santa Clara, CA). Compounds and DMSO in culture medium without cells produced very little to no background luminescence, across all concentrations used. Background-subtracted luminescence for duplicate wells of treated cells were averaged and compared as a percentage of the luminescence from cells treated with vehicle alone (0.25% DMSO). Figure 11 shows dose-response curve for compounds with balanced inhibitory activity on BET bromodomains and HDACs against patient-derived CTCL cells. Dose-response curves were generated using purified malignant cells obtained from patients with CTCL. Peripheral blood mononuclear cells (PBMCs) were immediately separated from whole blood by Ficoll density gradient. Total CD4+ T cells were purified via either MACS CD4+ negative selection kit (Miltenyi Biotec) or RosetteSep™ human CD4+ T cell enrichment cocktail kit (STEMCELL Technologies). Malignant CD4+ T cells were purified via MACS CD4+ negative selection kit supplemented with biotin-conjugated anti-CD26 and/or anti-CD7 plus anti-biotin microbeads, based on the phenotype of aberrant T cells identified by clinical flow cytometry. 6000 cells/well were dispensed into microwell plates (Multidrop Combi, ThermoFisher Scientific) and cultured for 72 hours following addition of various concentrations of HDAC inhibitors (SAHA and Entinostat) and Exemplary compounds. Positive and negative controls were 10% and 0.2% DMSO, respectively. Cell viability was measured using CellTiter-Glo (Promega) with luminescence measurements taken on a Synergy Neo2 plate reader (BioTek Instruments). Mean and standard deviation of positive and negative control wells were used to quantify signal-to-background and Z′ values for each screening plate to ensure assay robustness. Drug data were normalized to the mean values of negative control (set as 0% effect) and positive control (set as 100% effect) wells, and 50% inhibitory concentrations (IC₅₀s) were calculated using GraphPad Prism (version 8.4.3). Nonparametric Mann-Whitney U Test was used for statistical comparison among groups. DETAILED DESCRIPTION OF THE INVENTION Disclosed herein are compounds that can simultaneously bind with more than one protein. The disclosed compounds can be used as dual BRD4 and HDAC inhibitors. Additionally, the present technology provides for novel and promising therapeutic agents against virus-associated lymphomas, such as those caused by infections of Kaposi's sarcoma associated herpesvirus (KSHV) and Epstein-Barr virus (EBV). Both histone deacetylases (HDACs) and bromodomain- containing protein 4 (BRD4) have been validated as therapeutic targets for the aforementioned virus-associated lymphomas in vitro and in vivo. Synergetic effects of spontaneously targeting HDAC and BRD4 on KSHV reactivation, cell cycle arrest, and cell apoptosis have been observed by using the disclosed compounds with balanced inhibitory activity on BET bromodomains and HDACs. Compounds with balanced inhibitory activity on BET bromodomains and HDACs exhibit 18˗ to 567-fold increased cytotoxicity when compared to compounds with reduced inhibitory activity on BET bromodomains or HDACs over in all tested KSHV-positive PEL cells. This indicates that a dual HDACs/BRD4 blockage is a viable therapeutic approach for treatment of these viruses-associated malignancies. Compounds of the present disclosure include compounds of Formula I or Formula II, or a pharmaceutically acceptable salt thereof. Compounds of Formula I have the structure Compounds of Formula II have the structure In some instances, compounds have the structure of Formula I. In other instances, the compounds have the structure of Formula II. For some compounds of Formulas I or II, the confirmation of the compound at the carbon of the diazepinyl bonded to the methylene bridging C(=A) is in an S configuration, e.g., For other compounds of Formulas I or II, the confirmation of the compound at the carbon of the diazepinyl bonded to the methylene bridging the C(=A) is in an S configuration. For the compounds of Formula I or II, A is O or S, B is selected from the group consisting of O, NR¹, and S, R¹ is H or alkyl, X is selected from CR²R³ or a chemical bond, R² and R³ are independently selected from H or alkyl, and Y is selected from , , , , -(CH₂)₅C(O)NHOH, -OH, and H. It is believed that the compounds disclosed herein can bind two different proteins of interest and facilitate a protein-protein complex between the proteins. The protein-protein complex may be due to the compound facilitating a conformational change in one or both of the proteins of interest. Such compounds may be referred to as a Conformation Change Targeting Chimera. See, Figure 1. As demonstrated in Figures 2A-2B, there is no synergistic effect when (+)-JQ1 and SAHA (Vorinostat) or Entinostat separately target the BET bromodomain and HDAC in KSHV- positive BCBL-1 PEL cells. As further described in the Examples, combining BET and HDAC inhibitory activity on the same compound results in a synergistic effect. Several different advantages may be realized when compounds facilitate a protein-protein complex. The function of one or more proteins may be altered. For example, this may result in activation or inhibition of protein function. The protein-protein complex may be more stable than a protein-ligand complex that can lead to a longer alteration of protein function than when a ligand is utilized. Compounds that facilitate protein-protein complexes may be able to target “undruggable” proteins due to a lack of defined ligand-binding pocket. Compounds that facilitate protein-protein interactions may also synergistically target more than one pathway. This may result in the need for lower effective amounts of therapeutic agents, enhance safety windows, or lower toxicity or unwanted side-effects. As used herein, a squiggly line is used to designate the point of attachment for any radical group or substituent group. As used herein, the term “alkyl” includes a straight-chain or branched alkyl radical in all of its isomeric forms, such as a straight or branched group of 1-12, 1-10, or 1-6 carbon atoms, referred to herein as C₁-C₁₂ alkyl, C₁-C₁₀-alkyl, and C₁-C₆-alkyl, respectively. Representative alkyl groups include methyl, ethyl, tert-butyl and the like. In some embodiments, the compound is selected from the group consisting of

In some embodiments, the compound of is a dual BRD4/HDAC inhibitor. As used herein, the term “dual BRD4 and HDAC inhibitor’ ’ or “dual inhibitor” refers to a compound capable of inhibiting both a BRD4 protein and an HDAC protein. In some embodiments, the compound as described herein has less cytotoxicity in a normal cell than a BRD4 inhibitor that is (+)-JQ1 (CAS: 1268524-70-4) or an HDAC inhibitor selected from the group consisting of entinostat (CAS: 209783-80-2) and vorinostat (CAS: 149647-78-9). As used herein, the term “cytotoxicity” refers to the quality of being toxic to cells and is measured by the CC₅₀ value in µM, which denotes the concentration of a compound that reduces the cell viability by 50% when compared to untreated cells. As used herein, the term “normal cell” refers to a non-cancerous cell that undergoes a regular cell cycle without unchecked cell growth like cancerous cells. In some embodiments, the compounds of the disclosure may contain one or more chiral centers and, therefore, exist as stereoisomers, such as enantiomers or diastereomers. As used herein, the term “stereoisomers” refers to the enantiomers or diastereomers of a compound. These compounds may be designated by the symbol “R” or “S,” depending on the configuration of substituents around the stereogenic carbon atom. Mixtures of enantiomers or diastereomers may be designated “(±)” in nomenclature, but the skilled artisan will recognize that a structure may denote a chiral center implicitly. It is understood that the generic chemical structures (e.g., Formula I and II) encompass all stereoisomeric forms of the specified compounds, unless indicated otherwise. Pharmaceutically acceptable salts of the disclosed compounds also are contemplated herein and may be utilized in the disclosed methods. For example, a substituent group of the disclosed compounds may be protonated or deprotonated and may be present together with an anion or cation, respectively, as a pharmaceutically acceptable salt of the compound. The term “pharmaceutically acceptable salt” as used herein, refers to salts of the compounds which are non- toxic to living organisms. Typical pharmaceutically acceptable salts include those salts prepared by reaction of the compounds as disclosed herein with a pharmaceutically acceptable mineral or organic acid or an organic or inorganic base. Such salts are known as acid addition and base addition salts. It will be appreciated by the skilled reader that most or all of the compounds as disclosed herein are capable of forming salts and that the salt forms of pharmaceuticals are commonly used, often because they are more readily crystallized and purified than are the free acids or bases. Acids commonly employed to form acid addition salts may include inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid, and the like, and organic acids such as p-toluenesulfonic acid, methanesulfonic acid, oxalic acid, p- bromophenylsulfonic acid, carbonic acid, succinic acid, citric acid, benzoic acid, acetic acid, and the like. Examples of suitable pharmaceutically acceptable salts may include the sulfate, pyrosulfate, bisulfate, sulfite, bisulfate, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate, bromide, iodide, acetate, propionate, decanoate, caprylate, acrylate, formate, hydrochloride, dihydrochloride, isobutyrate, caproate, heptanoate, propiolate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleat-, butyne- 1,4-dioate, hexyne-l,6-dioate, benzoate, chlorobenzoate, methylbenzoate, hydroxybenzoate, methoxybenzoate, phthalate, xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, alpha-hydroxybutyrate, glycolate, tartrate, methanesulfonate, propanesulfonate, naphthalene- 1 -sulfonate, naphthalene-2-sulfonate, mandelate, and the like.

Base addition salts include those derived from inorganic bases, such as ammonium or alkali or alkaline earth metal hydroxides, carbonates, bicarbonates, and the like. Bases useful in preparing such salts include sodium hydroxide, potassium hydroxide, ammonium hydroxide, potassium carbonate, sodium carbonate, sodium bicarbonate, potassium bicarbonate, calcium hydroxide, calcium carbonate, and the like.

It should be recognized that the counter-ion forming a part of any salt of a compound disclosed herein is usually not of a critical nature, so long as the salt as a whole is pharmacologically acceptable and as long as the counterion does not contribute undesired qualities to the salt as a whole. Undesired qualities may include undesirably solubility or toxicity.

It will be further appreciated that the disclosed compounds can be in equilibrium with various inner salts. For example, inner salts include salts wherein the compound includes a deprotonated substituent group and a protonated substituent group.

The application also provides a pharmaceutical composition. The pharmaceutical composition comprises a therapeutically effective amount of the compound as described herein, or a pharmaceutically acceptable salt thereof, and further comprises a pharmaceutically acceptable excipient, diluent, or carrier.

As used herein, the term “pharmaceutically acceptable excipient, diluent, or carrier” refers to a material that can be used as a vehicle for administering a therapeutic or prophylactic agent, (e.g., the compounds as described herein), because the material is inert or otherwise medically acceptable, as well as compatible with the agent. Such pharmaceutical compositions may be prepared by any method known in the art of pharmacy, for example by bringing into association the active ingredient with the carriers or excipients. In some embodiments, the compounds of Formula I or II disclosed herein may be formulated as pharmaceutical compositions that include an effective amount of one or more compounds as disclosed herein and one or more pharmaceutically acceptable carriers, excipients, or diluents. In some embodiments, pharmaceutical compositions as described herein comprise purified diastereomers or enantiomers of the compounds as disclosed herein (e.g., a composition comprising at least about 90%, 95%, or 99% pure diastereomer or enantiomer). Disclosed herein also includes methods of using the compounds as described herein for treating cancers. The method comprises administering an effective amount of the compounds as described herein, or a pharmaceutically acceptable salt thereof, or the pharmaceutical compositions as described herein, to a subject having cancer. As used herein, the terms “treating” or “to treat” each mean to alleviate symptoms, eliminate the causation of resultant symptoms either on a temporary or permanent basis, and/or to prevent or slow the appearance or to reverse the progression or severity of resultant symptoms of the named disease or disorder. As used herein, a “subject” may be interchangeable with “patient” or “individual,” which may be a human or a non-human animal in need of treatment. In some embodiments, the subject in need of treatment may include a subject having a cell proliferative disease, disorder, or condition such as cancer or cancer-associated pain. In some embodiments, the cancer to be treated is selected from the group consisting of Kaposi sarcoma (KS), primary effusion lymphoma (PEL), cutaneous T-cell lymphoma (CTCL), KSHV-associated lymphomas, and EBV-associated lymphomas. As used herein, the term “effective amount” refers to the amount or dose of the compound that provides the desired effect, upon single or multiple dose administration to the subject. A skilled artisan would understand that an effective amount can be readily determined by the attending diagnostician using known techniques and by observing results obtained under analogous circumstances. In some embodiments, the compound or the pharmaceutical composition as described herein is administered intraperitoneally, topically, and/or orally. Examples of pharmaceutical compositions containing the compounds of Formula I or II for oral administration include capsules, syrups, concentrates, powders, and granules. Examples of pharmaceutical compositions containing the compounds of Formula I or II for intraperitoneal administration include suspensions, concentrates, or solutions. A suitable solvent system for preparing suspensions, concentrates, or solutions of the compounds of Formula I or II includes the combination of DMSO, PEG300, TweenSO, and saline or water in any equivalents and sequences.

Another aspect of the invention provides for a method for inhibiting the expression of c- MYC in a cancer cell. The method comprises contacting the cell with an effective amount of the as described herein, or a pharmaceutically acceptable salt thereof, or the pharmaceutical compositions as described herein.

In some embodiments, the cancer cell in the methods described herein is selected from the group consisting of a Kaposi sarcoma cancer cell, a primary effusion lymphoma cancer cell, a cutaneous T-cell lymphoma cancer cell, a KSHV-associated lymphoma cancer cell, and an EBV- associated lymphoma cancer cell.

Another aspect of the invention provides for a method for reducing or inhibiting cancer cell growth in a subject having cancer. The method comprises administering an effective amount of the compounds as described herein, or a pharmaceutically acceptable salt thereof, or the pharmaceutical compositions as described herein, to the subject having cancer.

Another aspect of the invention provides for a method of treating psoriasis. The method comprises administering the compounds as described herein, or a pharmaceutically acceptable salt thereof, or the pharmaceutical compositions as described herein, to a subject having psoriasis. In some such embodiments, the compound or the pharmaceutical composition is administered intraperitoneally, topically, and/or orally.

Another aspect of the invention provides for preparing the compounds described herein, or a pharmaceutically acceptable salt thereof. The method comprises converting a BRD4-inhibiting subunit to the compounds of Formula I or Formular II.

As used herein, the term “BRD4-inhibiting subunit" refers to a chemical entity capable of inhibiting the activity of a BRD4 protein. The BRD4-inhibiting subunit may bind the binding domain of a BRD4 protein in a cellular environment, inhibiting the BRD4 protein’s ability to interact with histones or resulting in dissociation of the BRD4 protein from chromatin.

In some embodiments, the BRD4-inhibiting subunit comprises a thieno-triazolo-1,4- diazepine scaffold or thieno-l,4-diazepine scaffold. In some embodiments, the BRD4-inhibiting subunit comprises a carboxylic acid that can participate in the formation of a linker, for example, by esterification or amidation. One example of BRD4 inhibitor that comprises such scaffold includes (+)-JQ1. In some embodiments, the BRD4-inhibiting subunit is . In some embodiments, the method comprises contacting the BRD4-inhibiting subunit as disclosed herein with an HDAC-inhibiting subunit under conditions sufficient for linking the BRD4-inhibiting subunit and the HDAC-inhibiting subunit via esterification or amidation. As used herein, the term “linking” refers to the formation of a chemical bond (i.e., the linker) that may be cleaved in vivo, allowing for the separation of the BRD4- and HDAC-inhibiting subunit. Cleavage of the linker can result in production of inactive forms, such as acids, alcohols, amines, and the like. Suitably the linker may be cleaved by hydrolysis or other suitable bond- breaking reaction either with or without the contribution of an enzyme. In some embodiments, the linker may be an ester moiety capable of being hydrolyzed with an esterase. In some embodiments, the linker may be an amide moiety. The use of a linker, such as an ester or an amide, allows for the rapid breakdown of the compounds in blood plasma and organs after exerting the anti-tumor effects. As used herein, “esterification” refers to the formation of an ester bond (i.e., ), typically from an alcohol and a carboxylic acid. As used herein, the term “carboxylic acid” refers to a compound having a group of the formula -C(O)OH. As used herein, the term “alcohol” refers to the substituent having the structure . As used herein, “amidation” refers to the formation of an amide bond typically from an amine and a carboxylic acid. As used herein, the term “amine” refers to both unsubstituted and substituted amines, wherein substituents typically include, for example, alkyl, cycloalkyl, heterocyclyl, alkenyl, and aryl. As used herein, the term “HDAC-inhibiting subunit” refers to a chemical entity capable of inhibiting the activity of an HDAC protein. Histone deacetylases (HDACs) are critical epigenetic erasers that remove acetyl groups from lysine on histones. Inhibitors designed for HDAC may be composed of a hydroxamic acid or 1,2-diaminobenzene moiety as the zinc binding group (ZBG), attached to a linker chain mimicking the lysine side chain and fitting the tubular access to the zinc atom. This chain is terminated by a functional “cap” group, mainly aromatic, interacting with the external surface. Exemplary HDAC inhibitors include vorinostat (SAHA) and entinostat. In some embodiments, the HDAC-inhibiting subunit is selected from the group consisting of In some embodiments, the method of preparing the compounds as described herein further comprises deprotecting the HDAC-inhibiting subunit to form an amine or a hydroxamic acid moiety. In some embodiments, the method of preparing the compounds as described herein comprises contacting the BRD4-inhibiting subunit with O-(tetrahydro-2H-pyran-2- yl)hydroxylamine (OTX) under conditions sufficient to form a tetrahydropyranyl ether protected hydroxamic acid moiety. In some such embodiments, the method further comprises deprotecting the tetrahydropyranyl ether protected hydroxamic acid moiety under conditions sufficient for producing the compounds as described herein. As used herein, the term “tetrahydropyranyl ether protected hydroxamic acid moiety” refers to a structural moiety of w ¹ herein R is as defined herein. As used herein, the “conditions sufficient to form a tetrahydropyranyl ether protected hydroxamic acid moiety” and the conditions sufficient for “deprotecting the tetrahydropyranyl ether protected hydroxamic acid moiety” are described in literature and can be optimized by a skilled artisan depending on specific reactants. For literature reviewing tetrahydropyranyl ether protection and deprotection, see Wuts Peter G. M and Theodora W Greene, Greene's Protective Groups in Organic Synthesis, 4th ed., Wiley-Interscience 2007. Miscellaneous Unless otherwise specified or indicated by context, the terms “a”, “an”, and “the” mean “one or more.” For example, “a molecule” should be interpreted to mean “one or more molecules.” As used herein, “about”, “approximately,” “substantially,” and “significantly” will be understood by persons of ordinary skill in the art and will vary to some extent on the context in which they are used. If there are uses of the term which are not clear to persons of ordinary skill in the art given the context in which it is used, “about” and “approximately” will mean plus or minus ≤10% of the particular term and “substantially” and “significantly” will mean plus or minus >10% of the particular term. As used herein, the terms “include” and “including” have the same meaning as the terms “comprise” and “comprising.” The terms “comprise” and “comprising” should be interpreted as being “open” transitional terms that permit the inclusion of additional components further to those components recited in the claims. The terms “consist of” and “consisting of” should be interpreted as being “closed” transitional terms that do not permit the inclusion additional components other than the components recited in the claims. The term “consisting essentially of” should be interpreted to be partially closed and allowing the inclusion only of additional components that do not fundamentally alter the nature of the claimed subject matter. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention. All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein. Preferred aspects of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred aspects may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect a person having ordinary skill in the art to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context. EXAMPLES The disclosure is further illustrated by the following synthesis schemes and examples, which are not to be constructed as limiting this disclosure in scope or spirit to the specific procedures herein described. It is to be understood that the examples are provided to illustrate certain embodiments and that no limitation to the scope of the disclosure is intended thereby. It is to be further understood that resort may be had to various other embodiments, modifications, and equivalents thereof which may suggest themselves to those skilled in the art without departing from the spirit of the present disclosure and/or scope of the appended claims. Compounds of the present disclosure may be prepared by methods known in the art of organic synthesis. In all the methods it is understood that protecting groups for sensitive or reactive groups may be employed where necessary in accordance with general principles of chemistry. Protecting groups are manipulated according to standard methods of organic synthesis (T. W. Greene and P. G. M. Wuts (1999) Protective Groups in Organic Synthesis, ^3rd Edition, John Wiley & Sons). These groups are removed at a convenient stage of the compound synthesis using methods that are readily apparent to those skilled in the art. Unless otherwise noted, reagents and solvents were used as received from commercial suppliers. Proton nuclear magnetic resonance (¹H-NMR) spectra were obtained on Varian Mercury 400 NMR spectrometer, and carbon-13 nuclear magnetic resonance (¹³C-NMR) spectra were recorded on Varian Mercury 126 NMR spectrometer at ambient temperature. Chemicals shifts (δ) were reported in ppm, coupling constants (J) were in hertz, and the splitting patterns were described as follows: s for singlet; d for doublet; t for triplet; q for quartet; and m for multiplet. A Kinetex® 5 µm XB-C₁₈100 Å LC column (150 x 4.6 mm) with an Agilent 1100A high performance liquid chromatography (HPLC) system was used for the determination of compound purity. The column was maintained at 37 ℃ for the duration of HPLC. Elution was performed with a flow rate of 0.8 mL/min using a solvent system of deionized water with 0.1% TFA (v/v) (solvent A) and methanol with 0.1% TFA (v/v) (solvent B). Mass spectrometry was conducted using a Thermo Fisher LCQ-DECA spectrometer (ESI-MS mode). The sample was dissolved in acquirable solvents such as MeCN, DMSO, or MeOH and was injected directly into the column using an automated sample hander. Abbreviations used in the following examples and elsewhere herein are: aq. Aqueous Bn Benzyl (Boc)₂O di-tert-butyl dicarbonate br broad d doublet DCM dichloromethane dd double of doublets ddd double of doublet of doublets DMA N,N-dimethylacetamide DMAP 4-dimethylaminopyridine DMF N,N-dimethylformamide DMSO dimethylsulfoxide dq doublet of quartets dt doublet of triplet of doublets EDCI N-ethyl-N’-(3-dimethylaminopropyl)carbodiimide hydrochloride Et ethyl EtOAc ethyl acetate EtOH ethanol or ethyl alcohol Et₃N or TEA triethylamine equiv equivalents g gram h or hr hour HATU 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate HCl hydrochloric acid hept heptet HOAt 1-hydroxy-7-azabenzotriazole HPLC high performance liquid chromatography HRMS high resonance mass spectrometry i-Pr isopropyl i-PrOH isopropanol or isopropyl alcohol i-Pr₂nEt or DIPEA N,N-diisopropylethylamine LCMS liquid chromatography mass spectrometry LiOH·H₂O lithium hydroxide monohydrate LiBH₄ lithium borohydride MeCN or ACN acetonitrile MeOH methanol m multiplet M molar Me methyl mg milligram MHz megahertz min minutes mL milliliter mmol millimole MS mass spectrometry NaOH sodium hydroxide NH₂OH·HCl hydroxylammonium chloride NMM N-methylmorpholine NMR nuclear magnetic resonance NMP N-methyl-2-pyrrolidone Pd(OAc)₂ palladium(II) acetate pH potential of hydrogen Ph phenyl group PPh₃ triphenylphosphine PhMe toluene PyBOP benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate q quartet quint quintet quintd quintet of doublet rt room temperature Rt retention time s singlet sat. Saturated SOCl₂ thionyl chloride t-Bu tert-butyl t triplet tdd triplet of doublet of doublets TEA triethylamine TFA trifluoroacetic acid THF tetrahydrofuran THP tetrahydropyranyl THPONH₂ O-(tetrahydro-2H-pyran-2-yl)hydroxylamine Ttd triplet of triplet of doublets wt weight Example 1: 1-(4-((2-aminophenyl)carbamoyl)phenyl)ethyl 2-((S)-4-(4-chlorophenyl)- 2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetate Reagents and conditions: (a) (Boc)₂O, NaOH, 1,4-dioxane, H₂O, overnight; (b) 4-(1- hydroxyethyl)benzoic acid, HATU, DIPEA, DMF, rt, overnight; (c) TFA, DCM, rt, overnight; (d) 1-3, PyBOP, DIPEA, DMF, rt, overnight; (e) TFA, DCM, rt, 2 h. Step 1: tert-butyl (2-aminophenyl)carbamate (1-2) To a mixture of o-phenylenediamine (1-1, 23.6 g, 220.0 mmol) and 1N sodium hydroxide aqueous solution (118.0 mL) in 1,4-dioxane (150.0 mL) was added a suspension of di-tert-butyl dicarbonate (52.5 g, 240.0 mmol) in 1,4-dioxane (100.0 mL) dropwise at 0 ℃. After addition, the reaction mixture was stirred at room temperature overnight. LC/MS analysis indicated the completed conversion. The reaction mixture was diluted with water (200.0 mL) and extracted with dichloromethane (200.0 mL × 3). The combined organic phases were dried over anhydrous sodium sulfate, filtered, concentrated under vacuum, absorbed onto silica gel, and purified via flash chromatography (ethyl acetate: hexanes = 1: 99 to 1: 9) to afford a light yellow solid as tert-butyl (2-aminophenyl)carbamate (1-2, 23.3 g, 51% yields). ESI-MS m/z: 209 [M+H]⁺. Step 2: tert-butyl (2-(4-(1-hydroxyethyl)benzamido)phenyl)carbamate (1-3) A mixture of 4-(1-hydroxyethyl)benzoic acid (0.2 g, 1.0 mmol) and N, N- diisopropylethylamine (0.6 g, 5.0 mmol) in anhydrous dimethylformamide (10.0 mL) was stirred at 0 ℃ for 5 minutes. tert-butyl (2-aminophenyl)carbamate (1-2, 0.2 g, 1.0 mmol) and 1- [bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (0.5 g, 1.2 mmol) were successively added to the mixture at 0 ℃. After addition, the reaction mixture was stirred at room temperature overnight. LC/MS analysis indicated the completed conversion. The reaction mixture was diluted with iced water (20.0 mL) and extracted with dichloromethane (20.0 mL × 3). The combined organic phases were washed with brine (20 mL), dried over anhydrous sodium sulfate, filtered, concentrated under vacuum, absorbed onto silica gel, and purified via a flash chromatography (ethyl acetate: hexanes = 1: 99 to 1: 1) to afford a light yellow foam as tert-butyl (2-(4-(1- hydroxyethyl)benzamido)phenyl)carbamate (1-3, 0.3 g, 71.5% yields). ESI-MS m/z: 357 [M+H]⁺. Step 3: (S)-2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3- a][1,4]diazepin-6-yl)acetic acid (Example 21) To a stirred solution of (+)-JQ1 (4, 1.0 g, 2.2 mmol) in dichloromethane (15.0 mL) was added trifluoroacetic acid (5.0 mL). The reaction mixture was stirred at room temperature overnight. LC/MS analysis indicated the completed conversion. The reaction mixture was concentrated under vacuum, absorbed onto Celite and purified via a C18-reversed flash chromatography (deionized water: methanol = 95: 5 to 100% methanol) to afford a white foam as (+)-JQ1 carboxylic acid (Example 21, 0.7 g, 84.3% yields). ESI-MS m/z: 401 [M+H]⁺. Step 4: 1-(4-((2-((tert-butoxycarbonyl)amino)phenyl)carbamoyl)phenyl)ethyl 2-((S)-4-(4- chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6- yl)acetate (1-5) To a solution of (+)-JQ1 carboxylic acid (Example 21, 0.1 g, 0.3 mmol) and N, N- diisopropylethylamine (0.1 g, 0.9 mmol) in anhydrous dimethylformamide (5.0 mL) was added benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (0.2 g, 0.3 mmol) at 0 ℃. The resulting light-yellow solution was allowed to stir at 0 ℃ for 5 minutes under nitrogen atmosphere. Then tert-butyl (2-(4-(1-hydroxyethyl)benzamido)phenyl)carbamate (1-3, 0.1 g, 0.3 mmol) was added and the reaction mixture was stirred at room temperature overnight under nitrogen atmosphere. LC/MS analysis indicated the completed conversion. The resulting mixture was diluted with iced water (20.0 mL) and extracted with ethyl acetate (20.0 mL × 3). The combined organic phases were washed with brine (20 mL), dried over anhydrous sodium, filtered, concentrated under vacuum, absorbed onto silica gel, and purified via a C18-reversed flash chromatography (methanol : water = 5 : 95 to 100% methanol) to afford a beige foam as 1-(4-((2- ((tert-butoxycarbonyl)amino)phenyl)carbamoyl)phenyl)ethyl 2-((S)-4-(4-chlorophenyl)-2,3,9- trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetate (1-5, 84.0 mg, 37.9% yields). ESI-MS m/z: 739 [M+H]⁺. Step 5: 1-(4-((2-aminophenyl)carbamoyl)phenyl)ethyl 2-((S)-4-(4-chlorophenyl)-2,3,9- trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetate (Example 1) To a stirred solution of 1-(4-((2-((tert- butoxycarbonyl)amino)phenyl)carbamoyl)phenyl)ethyl 2-((S)-4-(4-chlorophenyl)-2,3,9- trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetate (1-5, 10.0 mg, 0.014 mmol) in dichloromethane (2.0 mL) was added trifluoroacetic acid (0.5 mL). The reaction mixture was stirred at room temperature for 3 hours. LC/MS analysis indicated the completed conversion, and the resulting mixture was diluted with 5.0 mL of dichloromethane. The mixture was basified via the addition of saturated sodium carbonate aqueous solution to a pH around 10 and the resulting mixture was stirred at room temperature for 5 minutes. Then the mixture was extracted with dichloromethane (20.0 mL × 3). The combined organic phases were washed with brine, dried over anhydrous sodium sulfate, filtered, concentrated under vacuum, absorbed onto silica gel, and purified via a flash chromatography (dichloromethane: methanol = 99: 1 to 95: 5) to afford a light- yellow solid as 1-(4-((2-aminophenyl)carbamoyl)phenyl)ethyl 2-((S)-4-(4-chlorophenyl)-2,3,9- trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetate (Example 1, 7.4 mg, 82.8% yields). ESI-MS m/z: 639 [M+H]⁺. Example 2: (S)-1-(4-((2-aminophenyl)carbamoyl)phenyl)ethyl 2-((S)-4-(4-chlorophenyl)- 2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetate Reagents and conditions: (a) LiOH·H₂O, THF, MeOH, H₂O, rt, overnight; (b) 1-2, HATU, DIPEA, DMF, rt, overnight; (c) Example 21, PyBOP, DIPEA, DMF, rt, overnight; (d) TFA, DCM, rt, 2 h. Step 1: (S)-4-(1-hydroxyethyl)benzoic acid (2-2) To a solution of methyl (S)-4-(1-hydroxyethyl)benzoate (2-1, 2.0g, 11.1 mmol) in tetrahydrofuran (42.0 mL) and menthol (42.0 mL) was added a solution of lithium hydroxide monohydrate (1.4 g, 33.3 mmol) in water (14.0 mL) dropwise at 0 ℃. After addition, the reaction mixture was stirred at room temperature overnight. LC/MS analysis indicated the completed conversion. The reaction mixture was concentrated under vacuum to remove the organic solvents. The residue was diluted with water (20.0 mL) and adjusted to pH 4~5 via the addition of 1N hydrochloric acid aqueous solution dropwise at 0 ℃ to generate a white suspension. After filtration, the collected solid was washed with iced water (20.0 mL) and dried under vacuum to afford a white solid as (S)-4-(1-hydroxyethyl)benzoic acid (2-2, 1.6 g, 84.0% yields). ESI-MS m/z: 189 [M+Na]⁺. Step 2: tert-butyl (S)-(2-(4-(1-hydroxyethyl)benzamido)phenyl)carbamate (2-3) A mixture of (S)-4-(1-hydroxyethyl)benzoic acid (2-2, 0.7 g, 4.2 mmol) and N, N- diisopropylethylamine (3.7 mL, 21.0 mmol) in anhydrous dimethylformamide (20.0 mL) was stirred at 0 ℃ for 5 minutes. tert-butyl (2-aminophenyl)carbamate (1-2, 0.9 g, 4.2 mmol) and 1- [bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (1.9 g, 5.0 mmol) were successively added to the mixture at 0 ℃. After addition, the reaction mixture was stirred at room temperature overnight. LC/MS analysis indicated the completed conversion. The reaction mixture was diluted with iced water (50.0 mL) and extracted with dichloromethane (50.0 mL × 3). The combined organic phases were washed with brine, dried over anhydrous sodium sulfate, filtered, concentrated under vacuum, absorbed onto silica gel, and purified via flash chromatography (ethyl acetate: hexanes = 1: 99 to 1: 1) to afford a white foam as tert-butyl (S)-(2-(4-(1-hydroxyethyl)benzamido)phenyl)carbamate (2-3, 1.2 g, 80.0% yields). ESI-MS m/z: 357 [M+H]⁺. Step 3: (S)-1-(4-((2-((tert-butoxycarbonyl)amino)phenyl)carbamoyl)phenyl)ethyl 2-((S)-4-(4- chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6- yl)acetate (2-4) To a solution of (+)-JQ1 carboxylic acid (Example 21, 1.4 g, 3.6 mmol), benzotriazol-1- yl-oxytripyrrolidinophosphonium hexafluorophosphate (2.2 g, 4.3 mmol) and N, N- diisopropylethylamine (1.3 g, 10.7 mmol) in anhydrous dimethylformamide (35.0 mL) was added tert-butyl (S)-(2-(4-(1-hydroxyethyl)benzamido)phenyl)carbamate (2-3, 1.4 g, 3.9 mmol) at 0 ℃. After addition, the reaction mixture was stirred at room temperature overnight. LC/MS analysis indicated the completed conversion. The resulting mixture was diluted with water (50 mL) and extracted with dichloromethane (50 mL × 3). The combined organic phases were washed with brine, dried over anhydrous sodium sulfate, filtered, concentrated under vacuum, absorbed onto silica gel, and purified via flash chromatography (ethyl acetate: hexanes = 1: 99 to 100% ethyl acetate) to afford a white form as (S)-1-(4-((2-((tert- butoxycarbonyl)amino)phenyl)carbamoyl)phenyl)ethyl 2-((S)-4-(4-chlorophenyl)-2,3,9- trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetate (2-4, 1.4 g, 53.0% yields). ESI-MS m/z: 739 [M+H]⁺. Step 4: (S)-1-(4-((2-aminophenyl)carbamoyl)phenyl)ethyl 2-((S)-4-(4-chlorophenyl)-2,3,9- trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetate (Example 2) To a stirred solution of (S)-1-(4-((2-((tert- butoxycarbonyl)amino)phenyl)carbamoyl)phenyl)ethyl 2-((S)-4-(4-chlorophenyl)-2,3,9- trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetate (2-4, 0.8 g, 1.1 mmol) in dichloromethane (20.0 mL) was added trifluoroacetic acid (4.0 mL). The reaction mixture was stirred at 0 ºC for 3 hours. LC/MS analysis indicated the completed conversion, and the resulting mixture was concentrated under vacuum to afford a light-yellow oil, which was diluted with 5.0 mL of dichloromethane. The mixture was basified via the addition of saturated sodium carbonate aqueous solution to pH = 10 and the resulting mixture was stirred at room temperature for 5 minutes. Then the mixture was extracted with dichloromethane (20.0 mL × 3). The combined organic phases were washed with brine, dried over anhydrous sodium sulfate, filtered, concentrated under vacuum, absorbed onto silica gel, and purified via flash chromatography (ethyl acetate: methanol = 1: 99 to 3: 97) to afford a beige foam as (S)-1-(4-((2- aminophenyl)carbamoyl)phenyl)ethyl 2-((S)-4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2- f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetate (Example 2, 0.6 g, 92.9% yields). ESI-MS m/z: 639 [M+H]⁺. Example 3: (S)-1-(4-((2-aminophenyl)carbamoyl)phenyl)ethyl 2-((R)-4-(4-chlorophenyl)- 2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetate Reagents and conditions: (a) TFA, DCM, rt, overnight; (b) 2-3, PyBOP, DIPEA, DMF, rt, overnight; (c) TFA, DCM, rt, 2 h. Step 1: (R)-2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3- a][1,4]diazepin-6-yl)acetic acid (3-2) To a stirred solution of (-)-JQ1 (3-1, 90.0 mg, 0.2 mmol) in dichloromethane (4.0 mL) was added trifluoroacetic acid (0.5 mL). The reaction mixture was stirred at room temperature overnight. LC/MS analysis indicated the completed conversion. The reaction mixture was concentrated under vacuum, absorbed onto Celite and purified via a C18 reversed flash chromatography (deionized water: methanol = 95: 5 to 100% methanol) to afford a white foam as (-)-JQ1 carboxylic acid (3-2, 78.0 mg, 99% yields). ESI-MS m/z: 401 [M+H]⁺. Step 2: (S)-1-(4-((2-((tert-butoxycarbonyl)amino)phenyl)carbamoyl)phenyl)ethyl 2-((R)-4- (4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6- yl)acetate (3-3) To a solution of (-)-JQ1 carboxylic acid (3-2, 25.0 mg, 0.06 mmol), benzotriazol-1-yl- oxytripyrrolidinophosphonium hexafluorophosphate (0.1 g, 0.2 mmol) and N, N- diisopropylethylamine (31.1 mg, 0.2 mmol) in anhydrous dimethylformamide (2.0 mL) was added tert-butyl (S)-(2-(4-(1-hydroxyethyl)benzamido)phenyl)carbamate (2-3, 22.1 mg, 0.06 mmol) at 0 ℃. After addition, the reaction mixture was stirred at room temperature overnight. LC/MS analysis indicated the completed conversion. The resulting mixture was diluted with water (20.0 mL) and extracted with dichloromethane (20.0 mL × 3). The combined organic phases were washed with brine, dried over anhydrous sodium sulfate, filtered, concentrated under vacuum, absorbed onto silica gel, and purified via flash chromatography (ethyl acetate: hexanes = 1: 99 to 100% ethyl acetate) to afford a white form as (S)-1-(4-((2-((tert- butoxycarbonyl)amino)phenyl)carbamoyl)phenyl)ethyl 2-((R)-4-(4-chlorophenyl)-2,3,9- trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetate (3-3, 3.3 mg, 7% yields). ESI-MS m/z: 739 [M+H]⁺. Step 3: (S)-1-(4-((2-aminophenyl)carbamoyl)phenyl)ethyl 2-((R)-4-(4-chlorophenyl)-2,3,9- trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetate (Example 3) To a stirred solution of (S)-1-(4-((2-((tert- butoxycarbonyl)amino)phenyl)carbamoyl)phenyl)ethyl 2-((R)-4-(4-chlorophenyl)-2,3,9- trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetate (3-3, 6.0 mg, 0.008 mmol) in dichloromethane (2.0 mL) was added trifluoroacetic acid (0.5 mL). The reaction mixture was stirred at room temperature for 2 hours. LC/MS analysis indicated the completed conversion, and the resulting mixture was concentrated under vacuum to afford a light-yellow oil, which was diluted with 5.0 mL of dichloromethane. The mixture was basified via the addition of saturated sodium carbonate aqueous solution to pH = 10 and the resulting mixture was stirred at room temperature for 5 minutes. Then the mixture was extracted with dichloromethane (20.0 mL × 3). The combined organic phases were washed with brine, dried over anhydrous sodium sulfate, filtered, concentrated under vacuum, absorbed onto silica gel, and purified via flash chromatography (ethyl acetate: methanol = 1: 99 to 3: 97) to afford a beige foam as (S)-1-(4-((2- aminophenyl)carbamoyl)phenyl)ethyl 2-((R)-4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2- f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetate (Example 3, 4.0 mg, 78% yields). ESI-MS m/z: 639 [M+H]⁺. Example 4: methyl 4-((S)-1-(2-((S)-4-(4-chlorophenyl)-2,3,9-trimethyl-6H-8λ²,10λ²- thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetoxy)ethyl)benzoate Reagents and conditions: (a) 2-1, PyBOP, DIPEA, DMF, rt, overnight. To a stirred solution of (+)-JQ1 carboxylic acid (Example 21, 40.0 mg, 0.1 mmol) and N, N-diisopropylethylamine (37.3 mg, 0.3 mmol) in anhydrous dimethylformamide (5.0 mL) was added benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (62.4 mg, 0.1 mmol) at 0 ℃. After stirring for 5 minutes, methyl (S)-4-(1-hydroxyethyl)benzoate (2-1, 19.8 mg, 0.1 mmol) was added and the resulting mixture was stirred at room temperature overnight. LC/MS analysis indicated the completed conversion. The reaction mixture was concentrated under vacuum, absorbed onto Celite and purified via a C18-reversed flash chromatography (deionized water : methanol = 95 : 5 to 100% methanol) to afford a white foam as methyl 4-((S)-1-(2-((S)-4- (4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6- yl)acetoxy)ethyl)benzoate (Example 4, 27.0 mg, 48% yields). ESI-MS m/z: 563 [M+H]⁺. Example 5: (R)-1-(4-((2-aminophenyl)carbamoyl)phenyl)ethyl 2-((S)-4-(4-chlorophenyl)- 2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetate

Reagents and conditions: (a) LiOH·H₂O, THF, MeOH, H₂O, rt, overnight; (b) 1-2, HATU, DIPEA, DMF, rt, overnight; (c) Example 21, PyBOP, DIPEA, DMF, rt, overnight; (d) TFA, DCM, rt, 2 h. Step 1: (R)-4-(1-hydroxyethyl)benzoic acid (5-2) To a solution of methyl (R)-4-(1-hydroxyethyl)benzoate (5-1, 2.0 g, 11.1 mmol) in tetrahydrofuran (42.0 mL) and menthol (42.0 mL) was added a solution of lithium hydroxide monohydrate (1.4 g, 33.3 mmol) in water (14.0 mL) dropwise at 0 ℃. After addition, the reaction mixture was stirred at room temperature overnight. LC/MS analysis indicated the completed conversion. The reaction mixture was concentrated under vacuum to remove the organic solvents. The residue was diluted with water (10.0 mL) and adjusted to pH 4~5 via the addition of 1N hydrochloric acid aqueous solution dropwise at 0 ℃ to generate a white suspension. After filtration, the collected solid was washed with iced water (10.0 mL) and dried under vacuum to afford a white solid as (R)-4-(1-hydroxyethyl)benzoic acid (5-2, 1.7 g, 92.5% yields). ESI-MS m/z: 167 [M+H]⁺. Step 2: tert-butyl (R)-(2-(4-(1-hydroxyethyl)benzamido)phenyl)carbamate (5-3) A mixture of (R)-4-(1-hydroxyethyl)benzoic acid (5-2, 0.6 g, 3.6 mmol) and N, N- diisopropylethylamine (3.2 mL, 18.0 mmol) in anhydrous dimethylformamide (50.0 mL) was stirred at 0 ℃ for 5 minutes. tert-butyl (2-aminophenyl)carbamate (1-2, 0.8 g, 3.6 mmol) and 1- [bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (1.6 g, 4.3 mmol) were successively added to the mixture at 0 ℃. After addition, the reaction mixture was stirred at room temperature overnight. LC/MS analysis indicated the completed conversion. The reaction mixture was diluted with iced water (200.0 mL) and extracted with dichloromethane (200.0 mL × 3). The combined organic phases were washed with brine (200 mL), dried over anhydrous sodium sulfate, filtered, concentrated under vacuum, absorbed onto silica gel, and purified via a flash chromatography (ethyl acetate: hexanes = 1: 99 to 1: 1) to afford a light yellow foam as tert-butyl (R)-(2-(4-(1- hydroxyethyl)benzamido)phenyl)carbamate (5-3, 1.2 g, 96.4% yields). ESI-MS m/z: 379 [M+Na]⁺. Step 3: (R)-1-(4-((2-((tert-butoxycarbonyl)amino)phenyl)carbamoyl)phenyl)ethyl 2-((S)-4- (4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6- yl)acetate (5-4) To a solution of (+)-JQ1 carboxylic acid (Example 21, 2.9 g, 7.2 mmol), benzotriazol-1- yl-oxytripyrrolidinophosphonium hexafluorophosphate (4.5 g, 8.7 mmol) and N, N- diisopropylethylamine (3.6 mL, 21.7 mmol) in anhydrous dimethylformamide (20.0 mL) was added tert-butyl (R)-(2-(4-(1-hydroxyethyl)benzamido)phenyl)carbamate (5-3, 2.8 g, 8.0 mmol) at 0 ℃. After addition, the reaction mixture was stirred at room temperature overnight. LC/MS analysis indicated the completed conversion. The resulting mixture was diluted with water (200.0 mL) and extracted with dichloromethane (200.0 mL ^ 3). The combined organic phases were washed with brine, dried over anhydrous sodium sulfate, filtered, concentrated under vacuum, absorbed onto silica gel, and purified via a flash chromatography (ethyl acetate: hexanes = 1: 99 to 100% ethyl acetate) to afford a white form as (R)-1-(4-((2-((tert- butoxycarbonyl)amino)phenyl)carbamoyl)phenyl)ethyl 2-((S)-4-(4-chlorophenyl)-2,3,9- trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetate (5-4, 3.3 g, 61.7 yields). ESI-MS m/z: 739 [M+H]⁺. Step 4: (R)-1-(4-((2-aminophenyl)carbamoyl)phenyl)ethyl 2-((S)-4-(4-chlorophenyl)-2,3,9- trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetate (Example 5) To a stirred solution of (R)-1-(4-((2-((tert- butoxycarbonyl)amino)phenyl)carbamoyl)phenyl)ethyl 2-((S)-4-(4-chlorophenyl)-2,3,9- trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetate (5-4, 1.0 g, 1.4 mmol) in dichloromethane (20.0 mL) was added trifluoroacetic acid (5.2 mL). The reaction mixture was stirred at room temperature for 3 hours. LC/MS analysis indicated the completed conversion, and the resulting mixture was concentrated under vacuum to afford a light-yellow oil, which was diluted with 5.0 mL of dichloromethane. The mixture was basified via the addition of saturated sodium carbonate aqueous solution to pH = 10 and the resulting mixture was stirred at room temperature for 5 minutes. Then the mixture was extracted with dichloromethane (20.0 mL × 3). The combined organic phases were washed with brine, dried over anhydrous sodium sulfate, filtered, concentrated under vacuum, absorbed onto silica gel, and purified via a flash chromatography (ethyl acetate: methanol = 1: 99 to 3: 97) to afford a beige foam as (R)-1-(4-((2- aminophenyl)carbamoyl)phenyl)ethyl 2-((S)-4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2- f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetate (Example 5, 0.9 g, 98.5% yields). ESI-MS m/z: 639 [M+H]⁺. Example 6: (R)-1-(4-((2-aminophenyl)carbamoyl)phenyl)ethyl 2-((R)-4-(4-chlorophenyl)- 2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetate Reagents and conditions: (a) 5-3, PyBOP, DIPEA, DMF, rt, overnight; (b) TFA, DCM, rt, 2 h. Step 1: (R)-1-(4-((2-((tert-butoxycarbonyl)amino)phenyl)carbamoyl)phenyl)ethyl 2-((R)-4- (4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6- yl)acetate (6-1) To a solution of (-)-JQ1 carboxylic acid (3-2, 25.0 mg, 0.06 mmol), benzotriazol-1-yl- oxytripyrrolidinophosphonium hexafluorophosphate (0.1 g, 0.2 mmol) and N, N- diisopropylethylamine (31.1 mg, 0.2 mmol) in anhydrous dimethylformamide (2.0 mL) was added tert-butyl (R)-(2-(4-(1-hydroxyethyl)benzamido)phenyl)carbamate (5-3, 22.1 mg, 0.06 mmol) at 0 ℃. After addition, the reaction mixture was stirred at room temperature overnight. LC/MS analysis indicated the completed conversion. The resulting mixture was diluted with water (20.0 mL) and extracted with dichloromethane (20.0 mL × 3). The combined organic phases were washed with brine, dried over anhydrous sodium sulfate, filtered, concentrated under vacuum, absorbed onto silica gel, and purified via a flash chromatography (ethyl acetate: hexanes = 1: 99 to 100% ethyl acetate) to afford a white form as (R)-1-(4-((2-((tert- butoxycarbonyl)amino)phenyl)carbamoyl)phenyl)ethyl 2-((R)-4-(4-chlorophenyl)-2,3,9- trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetate (6-1, 3.1 mg, 7% yields). ESI-MS m/z: 739 [M+H]⁺. Step 2: (R)-1-(4-((2-aminophenyl)carbamoyl)phenyl)ethyl 2-((R)-4-(4-chlorophenyl)-2,3,9- trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetate (Example 6) To a stirred solution of (R)-1-(4-((2-((tert- butoxycarbonyl)amino)phenyl)carbamoyl)phenyl)ethyl 2-((R)-4-(4-chlorophenyl)-2,3,9- trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetate (6-1, 6.0 mg, 0.008 mmol) in dichloromethane (2.0 mL) was added trifluoroacetic acid (0.5 mL). The reaction mixture was stirred at room temperature for 2 hours. LC/MS analysis indicated the completed conversion, and the resulting mixture was concentrated under vacuum to afford a light-yellow oil, which was diluted with 5.0 mL of dichloromethane. The mixture was basified via the addition of saturated sodium carbonate aqueous solution to pH = 10 and the resulting mixture was stirred at room temperature for 5 minutes. Then the mixture was extracted with dichloromethane (10.0 mL × 3). The combined organic phases were washed with brine, dried over anhydrous sodium sulfate, filtered, concentrated under vacuum, absorbed onto silica gel, and purified via a flash chromatography (ethyl acetate: methanol = 1: 99 to 3: 97) to afford a beige foam as (R)-1-(4-((2- aminophenyl)carbamoyl)phenyl)ethyl 2-((R)-4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2- f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetate (Example 6, 4.1 mg, 80% yields). ESI-MS m/z: 639 [M+H]⁺. Example 7: methyl 4-((R)-1-(2-((S)-4-(4-chlorophenyl)-2,3,9-trimethyl-6H-8λ²,10λ⁴- thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetoxy)ethyl)benzoate Reagents and conditions: (a) 5-1, PyBOP, DIPEA, DMF, rt, overnight. To a stirred solution of (+)-JQ1 carboxylic acid (Example 21, 40.0 mg, 0.1 mmol) and N, N-diisopropylethylamine (37.3 mg, 0.3 mmol) in anhydrous dimethylformamide (5.0 mL) was added benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (62.4 mg, 0.1 mmol) at 0 ℃. After stirring for 5 minutes, methyl (R)-4-(1-hydroxyethyl)benzoate (5-1, 19.8 mg, 0.1 mmol) was added and the resulting mixture was stirred at room temperature overnight. LC/MS analysis indicated the completed conversion. The reaction mixture was concentrated under vacuum, absorbed onto Celite and purified via a C18-reversed flash chromatography (deionized water: methanol = 95: 5 to 100% methanol) to afford a white foam as methyl 4-((R)-1-(2-((S)-4- (4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6- yl)acetoxy)ethyl)benzoate (Example 7, 32.0 mg, 56% yields). ESI-MS m/z: 563 [M+H]⁺. Example 8: 4-((2-aminophenyl)carbamoyl)benzyl (S)-2-(4-(4-chlorophenyl)-2,3,9-trimethyl- 6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetate Reagents and conditions: (a) SOCl₂, DMF, DCM, reflux, 8 h; (b) 1-2, TEA, DCM, 0 ℃ to rt, overnight; (c) LiBH₄, THF, 0 ℃ to rt, overnight; (d) Example 21, PyBOP, DIPEA, DMF, rt, overnight; (e) TFA, DCM, rt, 2 h. Step 1: methyl 4-(chlorocarbonyl)benzoate (8-2) To a solution of 4-(methoxycarbonyl)benzoic acid (8-1, 4.3 g, 24.0 mmol) and SOCl₂ (8.7 mL, 120.0 mmol) in dichloromethane (50.0 mL) was added anhydrous dimethylformamide (0.2 mL). The reaction mixture was stirred at 55 ℃ overnight. Then the reaction mixture was concentrated as a light-yellow oil as the crude product of methyl 4-(chlorocarbonyl)benzoate (8- 2), which was used directly for next steps without further purifications. Step 2: methyl 4-((2-((tert-butoxycarbonyl)amino)phenyl)carbamoyl)benzoate (8-3) A solution of methyl 4-(chlorocarbonyl)benzoate (8-2, 2.0 g, 10.1 mmol) in dichloromethane (25.0 mL) was added to a solution of tert-butyl (2-aminophenyl)carbamate (1-2, 2.1 g, 10.1 mmol) in dichloromethane (25.0 mL) and triethylamine (45.0 mL) at 0 ℃. Then the reaction mixture was stirred at room temperature overnight. The reaction mixture was concentrated under vacuum, absorbed onto silica gel, and purified via a flash chromatography (ethyl acetate: hexanes = 1: 4) to afford a white solid as methyl 4-((2-((tert- butoxycarbonyl)amino)phenyl)carbamoyl)benzoate (8-3, 1.8 g, 48.3% yields). ESI-MS m/z: 371 [M+H]⁺. Step 3: tert-butyl (2-(4-(hydroxymethyl)benzamido)phenyl)carbamate (8-4) Lithium borohydride was added to a solution of methyl 4-((2-((tert- butoxycarbonyl)amino)phenyl)carbamoyl)benzoate (8-3, 1.5 g, 4.0 mmol) in tetrahydrofuran (50.0 mL). The reaction mixture was stirred at room temperature overnight. Then 50.0 mL water was added to the reaction mixture to quench the reaction and the mixture was neutralized by the addition of 2N citric acid aqueous solution to a pH around 6. The resulting mixture was extracted with ethyl acetate (50.0 mL × 3). The combined organic layers were washed with brine, dried over anhydrous sodium sulfate, filtered, concentrated under vacuum, absorbed onto silica gel, and purified via a flash chromatography (ethyl acetate: hexanes = 1: 1) to afford a yellow solid as tert- butyl (2-(4-(hydroxymethyl)benzamido)phenyl)carbamate (8-4, 1.0 g, 74.5% yields). ESI-MS m/z: 343 [M+H]⁺. Step 4: 4-((2-((tert-butoxycarbonyl)amino)phenyl)carbamoyl)benzyl (S)-2-(4-(4- chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6- yl)acetate (8-5) To a solution of (+)-JQ1 carboxylic acid (Example 21, 24.0 mg, 0.06 mmol), benzotriazol- 1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (31.2 mg, 0.06 mmol) and N, N- diisopropylethylamine (23.3 mg, 0.18 mmol) in anhydrous dimethylformamide (2.0 mL) was added tert-butyl (2-(4-(hydroxymethyl)benzamido)phenyl)carbamate (8-4, 24.0 mg, 0.07 mmol) at 0 ℃. After addition, the reaction mixture was stirred at room temperature overnight. LC/MS analysis indicated the completed conversion. The resulting mixture was diluted with water (20.0 mL) and extracted with dichloromethane (20.0 mL × 3). The combined organic phases were washed with brine, dried over anhydrous sodium sulfate, filtered, concentrated under vacuum, absorbed onto silica gel, and purified via a flash chromatography (ethyl acetate: hexanes = 1: 99 to 100% ethyl acetate) to afford a white form as 4-((2-((tert- butoxycarbonyl)amino)phenyl)carbamoyl)benzyl (S)-2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H- thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetate (8-5, 40.0 mg, 91.9% yields). ESI-MS m/z: 726 [M+H]⁺. Step 5: 4-((2-aminophenyl)carbamoyl)benzyl (S)-2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H- thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetate (Example 8) To a solution of 4-((2-((tert-butoxycarbonyl)amino)phenyl)carbamoyl)benzyl (S)-2-(4-(4- chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetate (8-5, 40.0 mg, 0.06 mmol) in dichloromethane (2.0 mL) was added trifluoroacetic acid (0.5 mL). LC/MS analysis indicated the completed conversion, and the resulting mixture was concentrated under vacuum to afford a light-yellow oil, which was diluted with 5.0 mL of dichloromethane. The mixture was basified via the addition of saturated sodium carbonate aqueous solution to a pH around 10 and the resulting mixture was stirred at room temperature for 5 minutes. Then the mixture was extracted with dichloromethane (10.0 mL × 3). The combined organic phases were washed with brine, dried over anhydrous sodium sulfate, filtered, concentrated under vacuum, absorbed onto silica gel, and purified via a flash chromatography (ethyl acetate: methanol = 1: 99 to 3: 97) to afford a beige foam as 4-((2-aminophenyl)carbamoyl)benzyl (S)-2-(4-(4- chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetate (Example 8, 12.0 mg, 32.0% yields). ESI-MS m/z: 625 [M+H]⁺. Example 9: 4-((2-aminophenyl)carbamoyl)benzyl (R)-2-(4-(4-chlorophenyl)-2,3,9- trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetate Reagents and conditions: (a) 8-4, PyBOP, DIPEA, DMF, rt, overnight; (b) TFA, DCM, rt, 2 h. Step 1: 4-((2-((tert-butoxycarbonyl)amino)phenyl)carbamoyl)benzyl (R)-2-(4-(4- chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6- yl)acetate (9-1) To a solution of (-)-JQ1 carboxylic acid (3-2, 50.0 mg, 0.12 mmol), benzotriazol-1-yl- oxytripyrrolidinophosphonium hexafluorophosphate (65.0 mg, 0.125 mmol) and N, N- diisopropylethylamine (50.0 mg, 0.39 mmol) in anhydrous dimethylformamide (2.0 mL) was added tert-butyl (2-(4-(hydroxymethyl)benzamido)phenyl)carbamate (8-4, 50.0 mg, 0.15 mmol) at 0 ℃. After addition, the reaction mixture was stirred at room temperature overnight. LC/MS analysis indicated the completed conversion. The resulting mixture was diluted with water (20.0 mL) and extracted with dichloromethane (20.0 mL × 3). The combined organic phases were washed with brine, dried over anhydrous sodium sulfate, filtered, concentrated under vacuum, absorbed onto silica gel, and purified via a flash chromatography (ethyl acetate: hexanes = 1: 99 to 100% ethyl acetate) to afford a white form as 4-((2-((tert- butoxycarbonyl)amino)phenyl)carbamoyl)benzyl (R)-2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H- thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetate (9-1, 32.5 mg, 37.3% yields). ESI-MS m/z: 726 [M+H]⁺. Step 2: 4-((2-aminophenyl)carbamoyl)benzyl (R)-2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H- thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetate (Example 9) To a solution of 4-((2-((tert-butoxycarbonyl)amino)phenyl)carbamoyl)benzyl (R)-2-(4-(4- chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetate (9-1, 15.0 mg, 0.02 mmol) in dichloromethane (2.0 mL) was added trifluoroacetic acid (0.5 mL). LC/MS analysis indicated the completed conversion, and the resulting mixture was concentrated under vacuum to afford a light-yellow oil, which was diluted with 5.0 mL of dichloromethane. The mixture was basified via the addition of saturated sodium carbonate aqueous solution to pH = 10 and the resulting mixture was stirred at room temperature for 5 minutes. Then the mixture was extracted with dichloromethane (10.0 mL × 3). The combined organic phases were washed with brine, dried over anhydrous sodium sulfate, filtered, concentrated under vacuum, absorbed onto silica gel, and purified via a flash chromatography (ethyl acetate: methanol = 1: 99 to 3: 97) to afford a beige foam as 4-((2-aminophenyl)carbamoyl)benzyl (R)-2-(4-(4-chlorophenyl)-2,3,9- trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetate (Example 9, 10.0 mg, 80.0% yields). ESI-MS m/z: 625 [M+H]⁺. Example 10: 4-((2-aminophenyl)carbamoyl)benzyl (S)-2-(5-(4-chlorophenyl)-6,7-dimethyl- 2-oxo-2,3-dihydro-1H-thieno[2,3-e][1,4]diazepin-3-yl)acetate Reagents and conditions: (a) TFA, DCM, rt, 6 h; (b) 8-4, PyBOP, DIPEA, DMF, rt, overnight; (c) TFA, DCM, rt, 2 h. Step 1: (S)-2-(5-(4-chlorophenyl)-6,7-dimethyl-2-oxo-2,3-dihydro-1H-thieno[2,3- e][1,4]diazepin-3-yl)acetic acid (10-2) tert-butyl (S)-2-(5-(4-chlorophenyl)-6,7-dimethyl-2-oxo-2,3-dihydro-1H-thieno[2,3- e][1,4]diazepin-3-yl)acetate (10-1) was generated following the procedure in publication Panagis Filippakopoulos et al., Nature, 468, 1067-1073, 2010. To a solution of tert-butyl (S)-2-(5-(4- chlorophenyl)-6,7-dimethyl-2-oxo-2,3-dihydro-1H-thieno[2,3-e][1,4]diazepin-3-yl)acetate (10-1, 0.1 g, 0.24 mmol) in dichloromethane (3.0 mL) was added trifluoroacetic acid (1.0 mL). LC/MS analysis indicated the completed conversion, and the resulting mixture was concentrated under vacuum to afford a yellow solid as the crude product of (S)-2-(5-(4-chlorophenyl)-6,7-dimethyl- 2-oxo-2,3-dihydro-1H-thieno[2,3-e][1,4]diazepin-3-yl)acetic acid (10-2), which was used directly for next steps without purifications. ESI-MS m/z: 363 [M+H]⁺. Step 2: 4-((2-((tert-butoxycarbonyl)amino)phenyl)carbamoyl)benzyl (S)-2-(5-(4- chlorophenyl)-6,7-dimethyl-2-oxo-2,3-dihydro-1H-thieno[2,3-e][1,4]diazepin-3-yl)acetate (10-3) To a solution of (S)-2-(5-(4-chlorophenyl)-6,7-dimethyl-2-oxo-2,3-dihydro-1H- thieno[2,3-e][1,4]diazepin-3-yl)acetic acid (10-2, 43.4 mg, 0.12 mmol), benzotriazol-1-yl- oxytripyrrolidinophosphonium hexafluorophosphate (62.4 mg, 0.12 mmol) and N, N- diisopropylethylamine (46.5 mg, 0.36 mmol) in anhydrous dimethylformamide (5.0 mL) was added tert-butyl (2-(4-(hydroxymethyl)benzamido)phenyl)carbamate (8-4, 49.3 mg, 0.14 mmol) at 0 ℃. After addition, the reaction mixture was stirred at room temperature overnight. LC/MS analysis indicated the completed conversion. The resulting mixture was diluted with water (20.0 mL) and extracted with dichloromethane (20.0 mL × 3). The combined organic phases were washed with brine, dried over anhydrous sodium sulfate, filtered, concentrated under vacuum, absorbed onto silica gel, and purified via a flash chromatography (ethyl acetate: hexanes = 1: 99 to 100% ethyl acetate) to afford a light-yellow form as 4-((2-((tert- butoxycarbonyl)amino)phenyl)carbamoyl)benzyl (S)-2-(5-(4-chlorophenyl)-6,7-dimethyl-2-oxo- 2,3-dihydro-1H-thieno[2,3-e][1,4]diazepin-3-yl)acetate (10-3, 70.0 mg, 84.9% yields). ESI-MS m/z: 687 [M+H]⁺. Step 3: 4-((2-aminophenyl)carbamoyl)benzyl (S)-2-(5-(4-chlorophenyl)-6,7-dimethyl-2-oxo- 2,3-dihydro-1H-thieno[2,3-e][1,4]diazepin-3-yl)acetate (Example 10) To a solution of 4-((2-((tert-butoxycarbonyl)amino)phenyl)carbamoyl)benzyl (S)-2-(5-(4- chlorophenyl)-6,7-dimethyl-2-oxo-2,3-dihydro-1H-thieno[2,3-e][1,4]diazepin-3-yl)acetate (10-3, 35.0 mg, 0.05 mmol) in dichloromethane (2.0 mL) was added trifluoroacetic acid (0.5 mL). LC/MS analysis indicated the completed conversion, and the resulting mixture was concentrated under vacuum to afford a light-yellow oil, which was diluted with 5.0 mL of dichloromethane. The mixture was basified via the addition of saturated sodium carbonate aqueous solution to pH = 10 and the resulting mixture was stirred at room temperature for 5 minutes. Then the mixture was extracted with dichloromethane (10.0 mL × 3). The combined organic phases were washed with brine, dried over anhydrous sodium sulfate, filtered, concentrated under vacuum, absorbed onto silica gel, and purified via a flash chromatography (ethyl acetate: methanol = 1: 99 to 3: 97) to afford a beige foam as 4-((2-aminophenyl)carbamoyl)benzyl (S)-2-(5-(4-chlorophenyl)-6,7- dimethyl-2-oxo-2,3-dihydro-1H-thieno[2,3-e][1,4]diazepin-3-yl)acetate (Example 10, 15.0 mg, 51.1% yields). ESI-MS m/z: 587 [M+H]⁺. Example 11: methyl (S)-4-((2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-8λ²,10λ⁴-thieno[3,2- f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetoxy)methyl)benzoate

Reagents and conditions: (a) methyl 4-(hydroxymethyl)benzoate, PyBOP, DIPEA, DMF, rt, overnight. To a solution of (+)-JQ1 carboxylic acid (Example 21, 10.0 mg, 0.025 mmol), benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (13.0 mg, 0.025 mmol) and N, N-diisopropylethylamine (9.3 mg, 0.075 mmol) in anhydrous dimethylformamide (3.0 mL) was added methyl 4-(hydroxymethyl)benzoate (4.2 mg, 0.025 mmol) at 0 ℃. After addition, the reaction mixture was stirred at room temperature overnight. LC/MS analysis indicated the completed conversion. The resulting mixture was diluted with water (10.0 mL) and extracted with dichloromethane (10.0 mL × 3). The combined organic phases were washed with brine, dried over anhydrous sodium sulfate, filtered, concentrated under vacuum, absorbed onto silica gel, and purified via a flash chromatography (ethyl acetate: hexanes = 1: 99 to 100% ethyl acetate) to afford a white form as methyl (S)-4-((2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-8λ²,10λ⁴-thieno[3,2- f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetoxy)methyl)benzoate (Example 11, 9.0 mg, 65.3% yields). ESI-MS m/z: 549 [M+H]⁺. Example 12: N-(2-aminophenyl)-4-(1-(2-((S)-4-(4-chlorophenyl)-2,3,9-trimethyl-6H- thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetamido)ethyl)benzamide

Reagents and conditions: (a) methyl 4-(1-aminoethyl)benzoate hydrochloride, PyBOP, DIPEA, DMF, rt, overnight, (b) LiOH·H₂O, MeOH, THF, H₂O, rt, overnight, (c) 1-2, PyBOP, DIPEA, DMF, rt, overnight, (d) TFA, DCM, rt, 3 h. Step 1: methyl 4-(1-(2-((S)-4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2- f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetamido)ethyl)benzoate (12-1) To a solution of (+)-JQ1 carboxylic acid (Example 21, 80.2 mg, 0.2 mmol), benzotriazol- 1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (43.0 mg, 0.2 mmol) and N, N- diisopropylethylamine (77.6 mg, 0.6 mmol) in anhydrous dimethylformamide (5.0 mL) was added methyl 4-(1-aminoethyl)benzoate hydrochloride (43.0 mg, 0.24 mmol) at 0 ℃. After addition, the reaction mixture was stirred at room temperature overnight. LC/MS analysis indicated the completed conversion. The resulting mixture was diluted with water (20.0 mL) and extracted with dichloromethane (20.0 mL × 3). The combined organic phases were washed with brine, dried over anhydrous sodium sulfate, filtered, concentrated under vacuum, absorbed onto silica gel, and purified via a flash chromatography (ethyl acetate: hexanes = 1: 99 to 100% ethyl acetate) to afford a white form as methyl 4-(1-(2-((S)-4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2- f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetamido)ethyl)benzoate (12-1, 0.1 g, 89.0% yields). ESI-MS m/z: 562 [M+H]⁺. Step 2: 4-(1-(2-((S)-4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3- a][1,4]diazepin-6-yl)acetamido)ethyl)benzoic acid (12-2) To a solution of methyl 4-(1-(2-((S)-4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2- f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetamido)ethyl)benzoate (12-1, 30.0 mg, 0.05 mmol) in methanol (2.1 mL) and tetrahydrofuran (2.1 mL) was added a solution of lithium hydroxide monohydrate (11.2 mg, 0.27 mmol) in water (0.7 mL) dropwise. The reaction mixture was stirred at room temperature overnight. Then the reaction mixture was concentrated under vacuum. The residue was diluted with water (5.0 mL), acidified by the addition of 2N HCl aqueous solution to a pH = 4 to afford a white suspension. After filtration, the white solid was dried under vacuum to afford a light-yellow solid as 4-(1-(2-((S)-4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2- f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetamido)ethyl)benzoic acid (12-2, 20.0 mg, 73.0% yields). ESI-MS m/z: 548 [M+H]⁺. Step 3: tert-butyl (2-(4-(1-(2-((S)-4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2- f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetamido)ethyl)benzamido)phenyl)carbamate (12- 3) To a solution of 4-(1-(2-((S)-4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2- f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetamido)ethyl)benzoic acid (12-2, 20.0 mg, 0.04 mmol), benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (20.8 mg, 0.04 mmol) and N, N-diisopropylethylamine (15.5 mg, 0.12 mmol) in anhydrous dimethylformamide (5.0 mL) was added tert-butyl (2-aminophenyl)carbamate (1-2, 7.6 mg, 0.048 mmol) at 0 ℃. After addition, the reaction mixture was stirred at room temperature overnight. LC/MS analysis indicated the completed conversion. The resulting mixture was diluted with water (20.0 mL) and extracted with dichloromethane (20.0 mL × 3). The combined organic phases were washed with brine, dried over anhydrous sodium sulfate, filtered, concentrated under vacuum, absorbed onto silica gel, and purified via a flash chromatography (ethyl acetate: methanol = 99: 1 to 97: 3) to afford a white form as tert-butyl (2-(4-(1-(2-((S)-4-(4-chlorophenyl)-2,3,9-trimethyl-6H- thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6- yl)acetamido)ethyl)benzamido)phenyl)carbamate (12-3, 13.0 mg, 44.0% yields). ESI-MS m/z: 738 [M+H]⁺. Step 4: N-(2-aminophenyl)-4-(1-(2-((S)-4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2- f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetamido)ethyl)benzamide (Example 12) To a solution of tert-butyl (2-(4-(1-(2-((S)-4-(4-chlorophenyl)-2,3,9-trimethyl-6H- thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6- yl)acetamido)ethyl)benzamido)phenyl)carbamate (12-3, 10.0 mg, 0.014 mmol) in dichloromethane (2.0 mL) was added trifluoroacetic acid (0.5 mL). The reaction mixture was stirred at room temperature for 3 hours. LC/MS analysis indicated the completed conversion, and the resulting mixture was concentrated under vacuum to afford a light-yellow oil, which was diluted with 5.0 mL of dichloromethane. The mixture was basified via the addition of saturated sodium carbonate aqueous solution to pH = 8 and the resulting mixture was stirred at room temperature for 5 minutes. Then the mixture was extracted with dichloromethane (10.0 mL × 3). The combined organic phases were washed with brine, dried over anhydrous sodium sulfate, filtered, concentrated under vacuum, absorbed onto silica gel, and purified via preparative TLC (dichloromethane: methanol = 15: 1) to afford a light-yellow foam as N-(2-aminophenyl)-4-(1-(2- ((S)-4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6- yl)acetamido)ethyl)benzamide (Example 12, 5.2 mg, 58.2% yields). ESI-MS m/z: 638 [M+H]⁺. Example 13: (S)-N-(2-aminophenyl)-4-((2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H- thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetamido)methyl)benzamide Reagents and conditions: (a) methyl 4-(aminomethyl)benzoate hydrochloride, PyBOP, DIPEA, DMF, rt, overnight, (b) LiOH·H₂O, MeOH, THF, H₂O, rt, overnight, (c) 1-2, PyBOP, DIPEA, DMF, rt, overnight, (d) TFA, DCM, rt, 2 h. Step 1: methyl (S)-4-((2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2- f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetamido)methyl)benzoate (13-1) To a solution of (+)-JQ1 carboxylic acid (Example 21, 80.0 mg, 0.2 mmol), benzotriazol- 1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (0.1 g, 0.2 mmol) and N, N- diisopropylethylamine (77.6 mg, 0.6 mmol) in anhydrous dimethylformamide (10.0 mL) was added methyl 4-(aminomethyl)benzoate hydrochloride (48.4 mg, 0.24 mmol) at 0 ℃. After addition, the reaction mixture was stirred at room temperature overnight. LC/MS analysis indicated the completed conversion. The resulting mixture was diluted with water (20.0 mL) and extracted with dichloromethane (20.0 mL × 3). The combined organic phases were washed with brine, dried over anhydrous sodium sulfate, filtered, concentrated under vacuum, absorbed onto silica gel, and purified via a flash chromatography (ethyl acetate: methanol = 99: 1 to 9: 1) to afford a white form as methyl (S)-4-((2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2- f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetamido)methyl)benzoate (13-1, 0.1 g, 91.2% yields). ESI-MS m/z: 548 [M+H]⁺. Step 2: (S)-4-((2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3- a][1,4]diazepin-6-yl)acetamido)methyl)benzoic acid (13-2) To a solution of methyl (S)-4-((2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2- f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetamido)methyl)benzoate (13-1, 80.0 mg, 0.15 mmol) in methanol (2.0 mL) and tetrahydrofuran (2.0 mL) was added a solution of lithium hydroxide monohydrate (18.4 mg, 0.44 mmol) in water (0.7 mL) dropwise. The reaction mixture was stirred at room temperature overnight. Then the reaction mixture was concentrated under vacuum. The residue was diluted with water (5.0 mL), acidified by the addition of 2N HCl aqueous solution to a pH = 4 to afford a light-yellow suspension. After filtration, the light-yellow solid was dried under vacuum to afford a beige solid as (S)-4-((2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2- f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetamido)methyl)benzoic acid (13-2, 58.0 mg, 72.2% yields). ESI-MS m/z: 534 [M+H]⁺. Step 3: tert-butyl (S)-(2-(4-((2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2- f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetamido)methyl)benzamido)phenyl)carbamate (13-3) To a solution of (S)-4-((2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2- f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetamido)methyl)benzoic acid (13-2, 50.0 mg, 0.09 mmol), benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (46.8 mg, 0.09 mmol) and N, N-diisopropylethylamine (34.9 mg, 0.27 mmol) in anhydrous dimethylformamide (5.0 mL) was added tert-butyl (2-aminophenyl)carbamate (1-2, 23.4 mg, 0.11 mmol) at 0 ℃. After addition, the reaction mixture was stirred at room temperature overnight. LC/MS analysis indicated the completed conversion. The resulting mixture was diluted with water (20.0 mL) and extracted with dichloromethane (20.0 mL × 3). The combined organic phases were washed with brine, dried over anhydrous sodium sulfate, filtered, concentrated under vacuum, absorbed onto silica gel, and purified via a flash chromatography (ethyl acetate: methanol = 99: 1 to 20: 1) to afford a white form as tert-butyl (S)-(2-(4-((2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2- f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetamido)methyl)benzamido)phenyl)carbamate (13-3, 40.0 mg, 61.4% yields). ESI-MS m/z: 724 [M+H]⁺. Step 4: (S)-N-(2-aminophenyl)-4-((2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2- f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetamido)methyl)benzamide (Example 13) To a solution of tert-butyl (S)-(2-(4-((2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H- thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6- yl)acetamido)methyl)benzamido)phenyl)carbamate (13-3, 15.0 mg, 0.02 mmol) in dichloromethane (3.0 mL) was added trifluoroacetic acid (0.5 mL). The reaction mixture was stirred at room temperature for 2 hours. LC/MS analysis indicated the completed conversion, and the resulting mixture was concentrated under vacuum to afford a light-yellow oil, which was diluted with 5.0 mL of dichloromethane. The mixture was basified via the addition of saturated sodium carbonate aqueous solution to pH = 10 and the resulting mixture was stirred at room temperature for 5 minutes. Then the mixture was extracted with dichloromethane (10.0 mL × 3). The combined organic phases were washed with brine, dried over anhydrous sodium sulfate, filtered, concentrated under vacuum, and purified via preparative TLC (dichloromethane: methanol = 20: 1) to afford a white foam as (S)-N-(2-aminophenyl)-4-((2-(4-(4-chlorophenyl)- 2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6- yl)acetamido)methyl)benzamide (Example 13, 7.0 mg, 56.1% yields). ESI-MS m/z: 624 [M+H]⁺. Example 14: (S,E)-3-(4-((2-(5-(4-chlorophenyl)-6,7-dimethyl-2-oxo-2,3-dihydro-1H- thieno[2,3-e][1,4]diazepin-3-yl)acetamido)methyl)phenyl)-N-hydroxyacrylamide

Reagents and conditions: (a) ethyl acrylate, Pd(OAc)₂, PPh₃, DIPEA, DMF, 90 ℃, overnight, (b) TFA, DCM, rt, overnight, (c) 10-2, PyBOP, DIPEA, DMF, rt, overnight, (d) LiOH·H₂O, EtOH, THF, H₂O, rt, overnight, (e) NH₂OH·HCl, PyBOP, DIPEA, DMF, rt, overnight. Step 1: ethyl (E)-3-(4-(((tert-butoxycarbonyl)amino)methyl)phenyl)acrylate (14-2) To a solution of tert-butyl (4-bromobenzyl)carbamate (14-1, 1.5 g, 5.2 mmol), ethyl acrylate (0.63 mL, 5.77 mmol), triphenylphosphine (0.27 g, 1.04 mmol), N, N- diisopropylethylamine (1.83 mL, 10.48 mmol) in anhydrous dimethylformamide (20.0 mL) was added palladium(II) acetate (0.12 g, 0.52 mmol). The reaction mixture was degassed with nitrogen for 15 minutes, sealed, and stirred at 90 ℃ overnight. After cooling, the reaction mixture was diluted with water (50.0 mL) and extracted with ethyl acetate (50.0 mL × 3). The combined organic layers were dried over anhydrous sodium sulfate, filtered, concentrated under vacuum, absorbed onto silica gel, and purified via a flash chromatography (ethyl acetate: hexanes = 1: 99 to 1: 3) to afford a yellow oil as ethyl (E)-3-(4-(((tert-butoxycarbonyl)amino)methyl)phenyl)acrylate (14-2, 1.2 g, 73.1% yields). ESI-MS m/z: 306 [M+H]⁺. Step 2: ethyl (E)-3-(4-(aminomethyl)phenyl)acrylate (14-3) To a solution of ethyl (E)-3-(4-(((tert-butoxycarbonyl)amino)methyl)phenyl)acrylate (14- 2, 0.5 g, 1.64 mmol) in dichloromethane (25.0 mL) was added trifluoroacetic acid (5.0 mL). The reaction mixture was stirred at room temperature overnight. LC/MS analysis indicated the completed conversion, and the resulting mixture was concentrated under vacuum to afford a yellow oil as the crude product of ethyl (E)-3-(4-(aminomethyl)phenyl)acrylate (14-3), which was used directly for next steps without further purifications. ESI-MS m/z: 206 [M+H]⁺. Step 3: ethyl (S,E)-3-(4-((2-(5-(4-chlorophenyl)-6,7-dimethyl-2-oxo-2,3-dihydro-1H- thieno[2,3-e][1,4]diazepin-3-yl)acetamido)methyl)phenyl)acrylate (14-4) To a solution of (S)-2-(5-(4-chlorophenyl)-6,7-dimethyl-2-oxo-2,3-dihydro-1H- thieno[2,3-e][1,4]diazepin-3-yl)acetic acid (10-2, 0.11 g, 0.30 mmol), benzotriazol-1-yl- oxytripyrrolidinophosphonium hexafluorophosphate (0.19 g, 0.36 mmol) and N, N- diisopropylethylamine (0.12 g, 0.9 mmol) in anhydrous dimethylformamide (5.0 mL) was added ethyl (E)-3-(4-(aminomethyl)phenyl)acrylate (14-3, 62.0 mg, 0.30 mmol) at 0 ℃. After addition, the reaction mixture was stirred at room temperature overnight. LC/MS analysis indicated the completed conversion. The resulting mixture was diluted with water (20.0 mL) and extracted with dichloromethane (20.0 mL × 3). The combined organic phases were washed with brine, dried over anhydrous sodium sulfate, filtered, concentrated under vacuum, absorbed onto silica gel, and purified via a flash chromatography (dichloromethane: methanol = 99: 1 to 97: 3) to afford a yellow form as ethyl (S,E)-3-(4-((2-(5-(4-chlorophenyl)-6,7-dimethyl-2-oxo-2,3-dihydro-1H-thieno[2,3- e][1,4]diazepin-3-yl)acetamido)methyl)phenyl)acrylate (14-4, 93.8 mg, 56.8% yields). ESI-MS m/z: 550 [M+H]⁺. Step 4: (S,E)-3-(4-((2-(5-(4-chlorophenyl)-6,7-dimethyl-2-oxo-2,3-dihydro-1H-thieno[2,3- e][1,4]diazepin-3-yl)acetamido)methyl)phenyl)acrylic acid (14-5) To a solution of ethyl (S,E)-3-(4-((2-(5-(4-chlorophenyl)-6,7-dimethyl-2-oxo-2,3-dihydro- 1H-thieno[2,3-e][1,4]diazepin-3-yl)acetamido)methyl)phenyl)acrylate (14-4, 55.0 mg, 0.1 mmol) in tetrahydrofuran (2.0 mL) and ethanol (2.0 mL) was added lithium hydroxide monohydrate (12.6 mg, 0.3 mmol) in water (1.0 mL) dropwise. The reaction mixture was stirred at room temperature overnight. Then the reaction mixture was concentrated under vacuum. The residue was diluted with water (5.0 mL), acidified by the addition of 2N HCl aqueous solution to a pH = 4 to afford a yellow suspension. After filtration, the yellow solid was dried under vacuum to afford a yellow solid as (S,E)-3-(4-((2-(5-(4-chlorophenyl)-6,7-dimethyl-2-oxo-2,3-dihydro-1H-thieno[2,3- e][1,4]diazepin-3-yl)acetamido)methyl)phenyl)acrylic acid (14-5, 50.4 mg, 96.6% yields). ESI- MS m/z: 522 [M+H]⁺ Step 5: (S,E)-3-(4-((2-(5-(4-chlorophenyl)-6,7-dimethyl-2-oxo-2,3-dihydro-1H-thieno[2,3- e][1,4]diazepin-3-yl)acetamido)methyl)phenyl)-N-hydroxyacrylamide (Example 14) To a solution of (S,E)-3-(4-((2-(5-(4-chlorophenyl)-6,7-dimethyl-2-oxo-2,3-dihydro-1H- thieno[2,3-e][1,4]diazepin-3-yl)acetamido)methyl)phenyl)acrylic acid (14-5, 50.4 mg, 0.1 mmol), benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (59.9 g, 0.12 mmol) and N, N-diisopropylethylamine (37.2 mg, 0.29 mmol) in anhydrous dimethylformamide (2.0 mL) was added hydroxylammonium chloride (10.0 mg, 0.14 mmol) at 0 ℃. After addition, the reaction mixture was stirred at room temperature overnight. LC/MS analysis indicated the completed conversion. The resulting mixture was diluted with water (20.0 mL) and extracted with dichloromethane (20.0 mL × 3). The combined organic phases were washed with brine, dried over anhydrous sodium sulfate, filtered, concentrated under vacuum, absorbed onto Celite, and purified via a C18 reversed flash chromatography (water: methanol = 5: 95 to 9: 1) to afford a white form as (S,E)-3-(4-((2-(5-(4-chlorophenyl)-6,7-dimethyl-2-oxo-2,3-dihydro-1H-thieno[2,3- e][1,4]diazepin-3-yl)acetamido)methyl)phenyl)-N-hydroxyacrylamide (Example 14, 31.2 mg, 60.5% yields). ESI-MS m/z: 537 [M+H]⁺. Example 15: (S)-4-((2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2- f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetamido)methyl)-N-hydroxybenzamide Reagents and conditions: (a) NH₂OH·HCl, PyBOP, DIPEA, DMF, rt, overnight. To a solution of (S)-4-((2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2- f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetamido)methyl)benzoic acid (13-2, 30.0 mg, 0.06 mmol), benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (37.5 mg, 0.072 mmol) and N, N-diisopropylethylamine (23.3 mg, 0.18 mmol) in anhydrous dimethylformamide (3.0 mL) was added hydroxylammonium chloride (6.1 mg, 0.09 mmol) at 0 ℃. After addition, the reaction mixture was stirred at room temperature overnight. LC/MS analysis indicated the completed conversion. The resulting mixture was diluted with water (20.0 mL) and extracted with dichloromethane (20.0 mL × 3). The combined organic phases were washed with brine, dried over anhydrous sodium sulfate, filtered, concentrated under vacuum, absorbed onto Celite, and purified via a C18 reversed flash chromatography (water: methanol = 1: 1) to afford a white form as (S)-4- ((2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6- yl)acetamido)methyl)-N-hydroxybenzamide (Example 15, 7.2 mg, 21.9% yields). ESI-MS m/z: 549 [M+H]⁺. Example 16: 4-(hydroxycarbamoyl)benzyl (S)-2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H- thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetate Reagents and conditions: (a) tert-butyl 4-(hydroxymethyl)benzoate, PyBOP, DIPEA, DMF, rt, overnight, (b) TFA, DCM, rt, 0.5 h; (c) NH₂OH·HCl, PyBOP, DIPEA, DMF, rt, overnight. Step 1: tert-butyl (S)-4-((2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2- f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetoxy)methyl)benzoate (16-1) To a solution of (+)-JQ1 carboxylic acid (Example 21, 0.12 g, 0.3 mmol), benzotriazol-1- yl-oxytripyrrolidinophosphonium hexafluorophosphate (0.16 g, 0.3 mmol) and N, N- diisopropylethylamine (0.11 g, 0.9 mmol) in anhydrous dimethylformamide (5.0 mL) was added tert-butyl 4-(hydroxymethyl)benzoate (62.5 mg, 0.3 mmol) at 0 ℃. After addition, the reaction mixture was stirred at room temperature overnight. LC/MS analysis indicated the completed conversion. The resulting mixture was diluted with water (20.0 mL) and extracted with dichloromethane (20.0 mL × 3). The combined organic phases were washed with brine, dried over anhydrous sodium sulfate, filtered, concentrated under vacuum, absorbed onto silica gel, and purified via a flash chromatography (dichloromethane: methanol = 99: 1 to 97: 3) to afford a white form as tert-butyl (S)-4-((2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2- f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetoxy)methyl)benzoate (16-1, 90.0 mg, 50.8% yields). ESI-MS m/z: 591 [M+H]⁺. Step 2: (S)-4-((2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3- a][1,4]diazepin-6-yl)acetoxy)methyl)benzoic acid (16-2) To a solution of tert-butyl (S)-4-((2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2- f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetoxy)methyl)benzoate (16-1, 80.0 mg, 0.14 mmol) in dichloromethane (5.0 mL) was added trifluoroacetic acid (0.5 mL). The reaction mixture was stirred at room temperature for 0.5 h. LC/MS analysis indicated the completed conversion, and the resulting mixture was concentrated under vacuum to afford a yellow oil as the crude product of (S)-4-((2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3- a][1,4]diazepin-6-yl)acetoxy)methyl)benzoic acid (16-2), which was used directly for next steps without further purifications. ESI-MS m/z: 535 [M+H]⁺. Step 3: 4-(hydroxycarbamoyl)benzyl (S)-2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H- thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetate (Example 16) To a solution of (S)-4-((2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2- f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetoxy)methyl)benzoic acid (16-2, 74.9 mg, 0.14 mmol), benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (87.4 mg, 0.17 mmol) and N, N-diisopropylethylamine (54.3 mg, 0.42 mmol) in anhydrous dimethylformamide (5.0 mL) was added hydroxylammonium chloride (14.6 mg, 0.21 mmol) at 0 ℃. After addition, the reaction mixture was stirred at room temperature overnight. LC/MS analysis indicated the completed conversion. The resulting mixture was diluted with water (20.0 mL) and extracted with dichloromethane (20.0 mL × 3). The combined organic phases were washed with brine, dried over anhydrous sodium sulfate, filtered, concentrated under vacuum, absorbed onto Celite, and purified via a C18 reversed flash chromatography (water: methanol = 5: 95 to 25: 75) to afford a white form as 4-(hydroxycarbamoyl)benzyl (S)-2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2- f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetate (Example 16, 23.0 mg, 29.9% yields). ESI-MS m/z: 550 [M+H]⁺. Example 17: (S)-4-(2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2- f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetamido)-N-hydroxybenzamide Reagents and conditions: (a) methyl 4-aminobenzoate, HATU, DIPEA, DMF, rt, overnight, (b) NH₂OH·HCl, MeOH, rt, 1 h. Step 1: methyl (S)-4-(2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2- f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetamido)benzoate (17-1) To a solution of (+)-JQ1 carboxylic acid (Example 21, 0.12 g, 0.3 mmol), 1- [bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (0.14 g, 0.36 mmol) and N, N-diisopropylethylamine (0.12 g, 0.9 mmol) in anhydrous dimethylformamide (5.0 mL) was added tert-butyl 4-aminobenzoate (54.4 mg, 0.36 mmol) at 0 ℃. After addition, the reaction mixture was stirred at room temperature overnight. LC/MS analysis indicated the completed conversion. The resulting mixture was diluted with water (20.0 mL) and extracted with dichloromethane (20.0 mL × 3). The combined organic phases were washed with brine, dried over anhydrous sodium sulfate, filtered, concentrated under vacuum, absorbed onto silica gel, and purified via a flash chromatography (ethyl acetate: methanol = 99: 1 to 97: 3) to afford a white form as methyl (S)-4-(2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H- thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetamido)benzoate (17-1, 60.0 mg, 37.5% yields). ESI-MS m/z: 534 [M+H]⁺. Step 2: (S)-4-(2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3- a][1,4]diazepin-6-yl)acetamido)-N-hydroxybenzamide (Example 17) Methyl (S)-4-(2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3- a][1,4]diazepin-6-yl)acetamido)benzoate (17-1, 10.0 mg, 0.02 mmol) was dissolved in the freshly prepared solution of 67N hydroxylammonium in methanol (3.0 mL). The mixture was stirred at room temperature for 1 hour. Then the reaction mixture was adjusted to a pH = 7 by the addition of 2N HCl in aqueous solution and concentrated under vacuum to afford a white suspension. The resulting suspension was extracted with dichloromethane (20.0 mL × 3). The combined organic layers were dried over anhydrous sodium sulfate, filtered, concentrated under vacuum, and purified via preparative TLC (dichloromethane: methanol = 10: 1) to afford a white solid as (S)-4-(2-(4-(4- chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6- yl)acetamido)-N-hydroxybenzamide (Example 17, 7.3 mg, 68.2% yields). ESI-MS m/z: 535 [M+H]⁺. Example 18: (S)-6-(2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2- f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetamido)-N-hydroxyhexanamide Reagents and conditions: (a) methyl 6-aminohexanoate, HATU, DIPEA, DMF, rt, overnight, (b) NaOH, MeOH, THF, H₂O, rt, overnight, (c) NH₂OH·HCl, PyBOP, TEA, DMF, rt, overnight. Step 1: methyl (S)-6-(2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2- f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetamido)hexanoate (18-1) To a solution of (+)-JQ1 carboxylic acid (Example 21, 80.0 mg, 0.2 mmol), 1- [bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (91.3 mg, 0.24 mmol) and N, N-diisopropylethylamine (77.6 g, 0.6 mmol) in anhydrous dimethylformamide (5.0 mL) was added methyl 6-aminohexanoate (29.0 mg, 0.20 mmol) at 0 ℃. After addition, the reaction mixture was stirred at room temperature overnight. LC/MS analysis indicated the completed conversion. The resulting mixture was diluted with water (20.0 mL) and extracted with dichloromethane (20.0 mL × 3). The combined organic phases were washed with brine, dried over anhydrous sodium sulfate, filtered, concentrated under vacuum, absorbed onto silica gel, and purified via a flash chromatography (dichloromethane: methanol = 20: 1) to afford a white form as methyl (S)-6-(2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H- thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetamido)hexanoate (18-1, 90.0 mg, 85.2% yields). ESI-MS m/z: 528 [M+H]⁺. Step 2: (S)-6-(2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3- a][1,4]diazepin-6-yl)acetamido)hexanoic acid (18-2) To a solution of methyl (S)-6-(2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2- f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetamido)hexanoate (18-1, 45.0 mg, 0.09 mmol) in tetrahydrofuran (2.0 mL) and methanol (2.0 mL) was added a solution of sodium hydroxide (10.2 mg, 0.27 mmol) in water (0.5 mL). The reaction mixture was stirred at room temperature overnight. Then the reaction mixture was concentrated under vacuum. The residue was diluted with water (5.0 mL), acidified by the addition of 2N HCl aqueous solution to a pH around 4 to afford a light- yellow suspension. After filtration, the light-yellow solid was dried under vacuum to afford a beige solid as (S)-6-(2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3- a][1,4]diazepin-6-yl)acetamido)hexanoic acid (18-2) which was used directly for the next step without further purifications. ESI-MS m/z: 514 [M+H]⁺. Step 3: (S)-6-(2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3- a][1,4]diazepin-6-yl)acetamido)-N-hydroxyhexanamide (Example 18) To a solution of (S)-6-(2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2- f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetamido)hexanoic acid (18-2, 46.3 mg, 0.09 mmol), benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (56.2 mg, 0.11 mmol) and N, N-diisopropylethylamine (69.8 mg, 0.5 mmol) in anhydrous dimethylformamide (5.0 mL) was added hydroxylammonium chloride (9.7 mg, 0.14 mmol) at 0 ℃. After addition, the reaction mixture was stirred at room temperature overnight. LC/MS analysis indicated the completed conversion. The resulting mixture was diluted with water (20.0 mL) and extracted with dichloromethane (20.0 mL × 3). The combined organic phases were washed with brine, dried over anhydrous sodium sulfate, filtered, concentrated under vacuum, absorbed onto silica gel, and purified via a preparative TLC (hexanes: dichloromethane: methanol = 45: 45: 10) to afford a white form as (S)-6-(2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3- a][1,4]diazepin-6-yl)acetamido)-N-hydroxyhexanamide (Example 18, 32.9 mg, 69.1% yields). ESI-MS m/z: 529 [M+H]⁺. Example 19: (S)-2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3- a][1,4]diazepin-6-yl)-N-hydroxyacetamide Reagents and conditions: (a) EDCI, HOAt, THPONH₂, NMM, DMF, rt, overnight; (o) 4N HCl in dioxane, rt, 0.5 h. Step 1: 2-((S)-4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3- a][1,4]diazepin-6-yl)-N-((tetrahydro-2H-pyran-2-yl)oxy)acetamide (19-1) To a solution of (+)-JQ1 carboxylic acid (Example 21, 0.75 g, 1.87 mmol) in anhydrous dimethylformamide (20.0 mL) were added 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (0.38 g, 2.47 mmol), 1-hydroxy-7-azabenzotriazole (0.34 g, 2.47 mmol), O- (tetrahydro-2H-pyran-2-yl)hydroxylamine (0.47 g, 4.04 mmol), and N-methyl morpholine (0.91 mL, 8.26 mmol) at 0 ℃. The reaction mixture was stirred at room temperature overnight. Then the reaction was quenched by the addition of iced-water (60 mL) and extracted with ethyl acetate (60 mL × 5). The combined organic layers were washed with brine, dried over anhydrous sodium sulfate, filtered, concentrated under vacuum, absorbed onto silica gel, and purified via a flash chromatography (ethyl acetate: methanol= 99: 1 to 9: 1) to afford a light-yellow foam as 2-((S)-4- (4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)-N- ((tetrahydro-2H-pyran-2-yl)oxy)acetamide (19-1, 0.25 g, 57% yield). ESI-MS m/z: 501 [M+H]⁺. Step 2: (S)-2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3- a][1,4]diazepin-6-yl)-N-hydroxyacetamide (Example 19) 2-((S)-4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3- a][1,4]diazepin-6-yl)-N-((tetrahydro-2H-pyran-2-yl)oxy)acetamide (19-1, 0.75 g, 1.5 mmol) was added to 4 N hydrogen chloride in 1,4-dioxane (50.0 mL). The resulting mixture was stirred at room temperature for 1 hour. LC/MS analysis indicated the completed conversion. The reaction mixture was concentrated under vacuum and purified via a C18-reversed flash chromatography (deionized water: methanol = 95: 5 to 100% methanol) to afford an off-white foam as (S)-2-(4-(4- chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)-N- hydroxyacetamide (Example 19, 0.56 g, 90% yield). ESI-MS m/z: 417 [M+H]⁺. Example 20: (R)-2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3- a][1,4]diazepin-6-yl)-N-hydroxyacetamide Reagents and conditions: (a) EDCI, HOAt, THPONH₂, NMM, DMF, rt, overnight; (o) 4N HCl in 1,4-dioxane, rt, 0.5 h. Step 1: 2-((R)-4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3- a][1,4]diazepin-6-yl)-N-((tetrahydro-2H-pyran-2-yl)oxy)acetamide (20-1) To a solution of (-)-JQ1 carboxylic acid (3-2, 25.0 mg, 0.062 mmol) in anhydrous dimethylformamide (2.0 mL) were added 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (12.7 mg, 0.082 mmol), 1-hydroxy-7-azabenzotriazole (11.2 mg, 2.082 mmol), O- (tetrahydro-2H-pyran-2-yl)hydroxylamine (15.8 mg, 0.14 mmol), and N-methyl morpholine (27.7 mg, 0.27 mmol) at 0 ℃. The reaction mixture was stirred at room temperature overnight. Then the reaction was quenched by the addition of iced-water (20 mL) and extracted with ethyl acetate (20 mL × 5). The combined organic layers were washed with brine, dried over anhydrous sodium sulfate, filtered, concentrated under vacuum, absorbed onto silica gel, and purified via a flash chromatography (ethyl acetate: methanol= 99: 1 to 9: 1) to afford a light-yellow foam as 2-((R)-4- (4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)-N- ((tetrahydro-2H-pyran-2-yl)oxy)acetamide (20-1, 17.0 mg, 54.8% yield). ESI-MS m/z: 501 [M+H]⁺. Step 2: (R)-2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3- a][1,4]diazepin-6-yl)-N-hydroxyacetamide (Example 20) 2-((R)-4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3- a][1,4]diazepin-6-yl)-N-((tetrahydro-2H-pyran-2-yl)oxy)acetamide (20-1, 10.0 mg, 0.02 mmol) was added to 4 N hydrogen chloride in 1,4-dioxane (0.7 mL). The resulting mixture was stirred at room temperature for 1 hour. LC/MS analysis indicated the completed conversion. The reaction mixture was concentrated under vacuum and purified via a C18-reversed flash chromatography (deionized water: methanol = 95: 5 to 100% methanol) to afford an off-white foam as (R)-2-(4-(4- chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)-N- hydroxyacetamide (Example 20, 7.0 mg, 84.2% yield). ESI-MS m/z: 417 [M+H]⁺. Table 1. Exemplary compounds Example 22: Cell culture and reagents BCBL-1 and iSLK.219 cells were kindly provided by Dr. Pinghui Feng (University of Southern California). BL-41, BJAB, BCP-1, BC-1, BC-3, JSC-1, HUVEC cells were purchased from American Type Culture Collection (ATCC) and cultured as recommended by the manufacturer. Example 23: Cytotoxicity assay The cell viability following treatment with compounds was assessed by the WST-1 assay (Roche, Indianapolis, Indiana, USA) according to the manufacturer’s protocol. The absorbance signal was measured using a microplate reader (Biotek Synergy 2). The 50% cytotoxic concentration (CC₅₀) for each compound was calculated from these dose-response curves using the software GraphPad Prism v5.0. Data were normalized as the fold change compared to the DMSO control. The representative cytotoxicity assay data is shown in Table 2. (*) represents ConCTAC with balanced inhibitory activity on BET bromodomains and HDACs, (-) represents ConCTAC with reduced inhibitory activity on BET bromodomains but retaining inhibitory activity on HDACs, and (|) represents ConCTAC with reduced inhibitory activity on HDACs but retaining inhibitory activity on BET bromodomains. Table 2. Screening of Anti-PEL activity Example 24: Infectivity assays and Fluorescence detection The iSLK.219 cells latently carry a recombinant rKSHV.219 virus and a doxycycline (Dox)-inducible gene expression system for expression of viral replication and transcription activator (RTA) protein, of which expression is essential and sufficient for triggering KSHV reactivation (Jinjong Myoung, et al., J. Virol. Methods, 174(1-2), 12-21, 2011). The rKSHV.219 contains two fluorescent protein genes, the green fluorescent protein (GFP) and red fluorescent protein (RFP), which are derived from the EF-1α promoter and KSHV lytic PAN promoter, respectively (Jinjong Myoung, et al., J. Virol. Methods, 174(1-2), 12-21, 2011). iSLK.219 cells were employed to evaluate viral reactivation and infectivity as described previously. The cells were treated by Dox (0.05µg/ml) in combination with tested compounds at concentrations and time-points as indicated, then RFP expression was detected by a fluorescent microscopy (Olympus DP80) and quantitatively analyzed by the imaging software CellSens Ver.2.2. The rest of the supernatants were used to infect HEK293T by spinoculation as previously reported by centrifugation at 1500 g for 60 min, then GFP expression was detected at 48 h post-infection by fluorescent microscopy. Example 25: Cell cycle and apoptosis analysis Flow cytometry was used for the quantitative assessment of cell cycle and apoptosis [33]. Briefly, to measure cell cycle response, PEL cell pellets were fixed in 70% ethanol, and incubated at 4°C overnight. Cell pellets were re-suspended in 0.5 mL of 0.05 mg/mL PI plus 0.2 mg/mL RNaseA and incubated at 37°C for 30 min. Cell cycle distribution was analyzed using a BD Accuri C6 flow cytometer. Apoptosis was assessed by the FITC-Annexin V/propidium iodide (PI) Apoptosis Detection Kit I (BD Pharmingen, San Jose, California, USA) on a flow cytometer. Data were normalized as the fold change compared to the DMSO control. Example 26: Quantitative Reverse Transcription-PCR (RT-qPCR) and quantitative PCR (qPCR) Transcripts of genes of interest were measured by RT-qPCR. Total cellular RNA was isolated and purified using the Qiagen RNeasy Kit (Qiagen, Germantown, Maryland, USA). The cDNA was synthesized using a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems). KSHV genomic DNA was isolated and purified using the QIAamp DNA mini-Kit (Qiagen, Germantown, Maryland, USA) and was also measured by QPCR. All RT-qPCR and QPCR assays were performed using a Real-Time PCR detection system (C1000 touch thermal cycler, Bio-Rad) using the iTaq™ Universal SYBR® Green Supermix (Bio-Rad) with specific primers (Table 3) and analyzed. Table 4 shows the top 10 common genes upregulated or downregulated from Example 2-, Example 5-, (+)-JQ1- and SAHA-treated BCBL-1 cells. Table 3. Primer sequences for qPCR and RT-qPCR

Table 4. The top 10 common genes upregulated or downregulated from Example 2-, Example 5-, (+)-JQ1- and SAHA-treated BCBL-1 cells Example 27: Western Blot The expression of the proteins of interest was detected by Western Blot using protein- specific antibodies. Immuno-reactive bands were identified using a Bio-rad Clarity Max Western ECL Substrate kit and visualized by Bio-rad Chemi Doc Imaging System. Anti-LANA antibody was purchased from Advanced Biotechnologies Inc (Eldersburg, MD, USA). Anti-ORF45 was purchased from Novus Biologicals (Centennial, CO, USA). Antibodies for KSHV RTA and ORF54 were purchased from Helmholtz-Munich, Germany and anti-ORF62 antibody was purchased from Novus Biologicals (Centennial, CO, USA). Antibodies for c-Myc, H3K9Ac, H4K9Ac, cleaved Caspase3, cleaved PARP, phospho-Rb, phospho-ATM, acetyl-tubulin and p21 were obtained from Cell Signaling Technology (Danvers, MA, USA). Example 28: Pharmacokinetic (PK) of compounds in mice The PK profiles of compounds were determined in plasma following a single intraperitoneal (IP) injection (50 mg per kg body weight) or oral gavage administration (50 mg per kg body weight) to NOD/SCID mice, 6–8-week-old, male (Jackson Laboratory, Ellsworth, Maine, USA). The IP and oral dose were formulated with DMSO/PEG300/Tween80/saline (5/30/10/55). Samples were obtained at 0.25, 0.5, 1, 2, 4, and 8 hours post dosing via tail snipping, transferred into plastic microcentrifuge tubes containing 4 μL of K2-EDTA (0.5 M) as anti-coagulant and placed on wet ice until centrifugation. Harvested blood samples were centrifuged shortly after collection at 4,000 g 4°C for 10 minutes. After centrifugation, the concentration of compounds in the plasma was determined using HPLC-coupled tandem mass spectrometry (LC-MS/MS). Values are calculated from arithmetic mean plasma concentrations (n = 3 mice per condition). AUC_last and t1/2 were determined using GraphPad Prism v5.0. The bioavailability F (%) = AUC_last for PO / AUC_last for IP × 100 (Jili Zhang, et al., BMC Vet. Res., 17(275), 2021). Table 5. Pharmacokinetic studies of (+)-JQ1, SAHA, and Example 5 in mice Example 29: PEL xenograft model In all NOD/SCID mice, 6–8-week-old, male (Jackson Laboratory, Ellsworth, Maine, USA), 1 × 10⁷ BCBL-1 cells in 200 µL RMPI-1640 without FBS were injected intraperitoneally and then mice were randomized into treatment groups of 6 mice. The tested compounds or vehicle was administered initially at 72 h after BCBL-1 injections, and continued once daily, 2 days per week for 3 weeks. Weights were recorded weekly as a surrogate measure of tumor progression. At the end of experiment, the spleens of mice were excised for immunoblots and immunohistochemical analyses. All protocols were approved by the University of Arkansas for Medical Sciences Animal Care and Use Committee in accordance with national guidelines. Example 30: RNA-Sequencing and enrichment analysis RNA-Sequencing (RNA-Seq) of triplicate samples was performed by BGI Americas Corporation using their unique DNBSEQ™ sequencing technology. The completed RNA- Sequencing data was submitted to NCBI Sequence Read Archive (SRA# PRJNA813422). Raw sequencing reads were analyzed using the RSEM software (version 1.3.0; human GRCh38 genome sequence and annotation) and gene expression was quantified as previously described (Fayez Kheir, et al., Cancers (Basel), 11(6), 759, 2019). The EBSeq software was utilized to call differentially expressed genes that were statistically significant using a false discovery rate (FDR) less than 0.05. Differentially expressed genes between compounds- and vehicle-treated PEL cells were used as input for the GO enrichment analyses. Example 31: Immunohistochemistry Formalin-fixed, paraffin-embedded tissues were microtome-sectioned to a thickness of 4 µm and placed on electromagnetically charged slides. Immunohistochemistry was performed, and the c-Myc and H3K9Ac antibodies were purchased from Abcam. Images were collected using an Olympus BX61 microscope equipped with a high resolution DP72 camera and CellSense image capture software. Example 32: Implications of the compounds as disclosed herein for the treatment of CTCL BET bromodomains or HDACs inhibition has been shown to be a major target for inducing cellular death of malignant cells isolated from patients with CTCL. Thus, the simultaneous and potent inhibition of both BET and HDAC using the Compound with balanced inhibitory activity on BET bromodomains and HDACs as described is predicted to have major therapeutic effects in the treatment of patients with CTCL. Example 33: Implications of the compounds as disclosed herein for the treatment of psoriasis. As psoriasis is mediate by activated T cells, the efficient targeting of CTCL cells, a malignancy of activated T cells, through the simultaneous and potent inhibition of both BET bromodomains and HDAC is predicted to translate to the treatment of psoriasis. This potential is further enhanced if such compounds are optimized through delivery to the skin by (1) increased absorption into the skin, and (2) inactivation upon systemic absorption.

Claims

CLAIMS 1. A compound of Formula I or Formula II, or a pharmaceutically acceptable salt thereof, having the structure wherein A is O or S; B is selected from the group consisting of O, NR¹, and S; R¹ is H or alkyl; X is selected from CR²R³ or a chemical bond; R² and R³ are independently selected from H or alkyl; and Y is selected from -(CH₂)₅C(O)NHOH, -OH, and H.

2. The compound of claim 1, wherein A and B are oxygen.

3. The compound of claim 1, wherein A is oxygen and B is NR¹.

4. The compound of claim 3, wherein R¹ is hydrogen.

5. The compound of any one of claims 1-4, wherein X is CR²R³.

6. The compound of claim 5, wherein Y is selected from

7. The compound of claim 6, wherein CR²R³ is CH₂ or CH(CH₃).

8. The compound of claim 7, wherein the compound has the formula

9. The compound of any one of claims 1-4, wherein X is a chemical bond.

10. The compound of claim 8, wherein Y is selected from -(CH₂)₅C(O)NHOH, -OH, and H.

11. The compound of claim 7, wherein the compound has the formula

12. The compound of any one of claims 1-11, wherein the compound has the formula

13. The compound of claim 1, wherein the compound is selected from the group consisting of 14. The compound of any one of claims 1-13, wherein the compound is a dual BRD4/HDAC inhibitor. 15. The compound of any one of claims 1-14, wherein the compound has less cytotoxicity in a normal cell than a BRD4 inhibitor that is (+)-JQ1 (CAS: 1268524-70-4) or an HDAC inhibitor selected from the group consisting of entinostat (CAS: 209783-80-2) and vorinostat (CAS: 149647-78-9). 16. A pharmaceutical composition comprising a therapeutically effective amount of the compound according to any one of claims 1-15, or a pharmaceutically acceptable salt thereof, and further comprising a pharmaceutically acceptable excipient, diluent, or carrier. 17. A method of treating cancer, the method comprising administering an effective amount of the compound of claim 1, or a pharmaceutically acceptable salt thereof, to a subject having the cancer. 18. The method of claim 17, wherein the cancer is selected from the group consisting of Kaposi sarcoma (KS), primary effusion lymphoma (PEL), cutaneous T-cell lymphoma (CTCL), KSHV-associated lymphomas, and EBV-associated lymphomas. 19. The method of any one of claims 17-18, wherein the compound comprises the compound according to any one of claims 2-15. 20. The method of any one of claims 17-18, wherein the compound comprises the compound according to claim 13. 21. The method of any one of claims 17-20, wherein the compound or the pharmaceutical composition is administered intraperitoneally, topically, and/or orally. 22. A method for inhibiting the expression of c-MYC in a cancer cell, the method comprising contacting the cell with an effective amount of the compound according to claim 1, or a pharmaceutically acceptable salt thereof. 23. The method of claim 22, wherein the cancer cell is selected from the group consisting of a Kaposi sarcoma cancer cell, a primary effusion lymphoma cancer cell, a cutaneous T- cell lymphoma cancer cell, a KSHV-associated lymphoma cancer cell, and an EBV- associated lymphoma cancer cell. 24. The method of any one of claims 22-23, wherein the compound comprises the compound according to any one of claims 2-15. 25. The method of any one of claims 22-23, wherein the compound comprises the compound according to claim 13. 26. A method for reducing or inhibiting cancer cell growth in a subject having cancer, the method comprising administering an effective amount of the compound according to claim 1, or a pharmaceutically acceptable salt thereof, to the subject having cancer. 27. The method of claim 23, wherein the cancer is selected from the group consisting of Kaposi sarcoma cancer, primary effusion lymphoma cancer, a cutaneous T-cell lymphoma cancer, a KSHV-associated lymphoma cancer, and an EBV-associated lymphoma cancer. 28. The method of any one of claims 26-27, wherein the compound comprises the compound according to any one of claims 2-15. 29. The method of any one of claims 26-27, wherein the compound comprises the compound according to claim 13. 30. A method of treating psoriasis, the method comprising administering an effective amount of the compound according to claim 1, or a pharmaceutically acceptable salt thereof, to a subject having psoriasis. 31. The method of claim 30, wherein the compound or the pharmaceutical composition is administered intraperitoneally, topically, and/or orally. 32. The method of any one of claims 30-31, wherein the compound comprises the compound according to any one of claims 2-15. 33. The method of any one of claims 30-31, wherein the compound comprises the compound according to claim 13. 34. A method for preparing the compound of any one of claims 1-15, or a pharmaceutically acceptable salt thereof, the method comprising converting a BRD4-inhibiting subunit to the compound. 35. The method of claim 34, wherein the BRD4-inhibiting subunit is . 36. The method of claim 34-35, wherein the method comprises contacting the BRD4- inhibiting subunit with an HDAC-inhibiting subunit under conditions sufficient for linking the BRD4-inhibiting subunit and the HDAC-inhibiting subunit via esterification or amidation. 37. The method of claim 36, wherein the HDAC-inhibiting subunit is selected from the group consisting of . 38. The method of claim 36 or 37, further comprising deprotecting the HDAC-inhibiting subunit to form an amine or a hydroxamic acid moiety. 39. The method of claim 38, comprising contacting the BRD4-inhibiting subunit with O- (tetrahydro-2H-pyran-2-yl)hydroxylamine (OTX) under conditions sufficient to form a tetrahydropyranyl ether protected hydroxamic acid moiety. 40. The method of claim 39, further comprising deprotecting the tetrahydropyranyl ether protected hydroxamic acid moiety under conditions sufficient for producing the compound.