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EP4629984A1 - Développement d'inhibiteurs puissants de hdac/brd4 - Google Patents

Développement d'inhibiteurs puissants de hdac/brd4

Info

Publication number
EP4629984A1
EP4629984A1 EP23901526.6A EP23901526A EP4629984A1 EP 4629984 A1 EP4629984 A1 EP 4629984A1 EP 23901526 A EP23901526 A EP 23901526A EP 4629984 A1 EP4629984 A1 EP 4629984A1
Authority
EP
European Patent Office
Prior art keywords
compound
mmol
thieno
diazepin
chlorophenyl
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP23901526.6A
Other languages
German (de)
English (en)
Inventor
Hong-Yu Li
Zhengyu Wang
Zhiqing QIN
Michael Girardi
Henry Wong
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Yale University
BioVentures LLC
Original Assignee
Yale University
BioVentures LLC
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Yale University, BioVentures LLC filed Critical Yale University
Publication of EP4629984A1 publication Critical patent/EP4629984A1/fr
Pending legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D495/00—Heterocyclic compounds containing in the condensed system at least one hetero ring having sulfur atoms as the only ring hetero atoms
    • C07D495/02—Heterocyclic compounds containing in the condensed system at least one hetero ring having sulfur atoms as the only ring hetero atoms in which the condensed system contains two hetero rings
    • C07D495/04—Ortho-condensed systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/06—Antipsoriatics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D495/00—Heterocyclic compounds containing in the condensed system at least one hetero ring having sulfur atoms as the only ring hetero atoms
    • C07D495/12—Heterocyclic compounds containing in the condensed system at least one hetero ring having sulfur atoms as the only ring hetero atoms in which the condensed system contains three hetero rings
    • C07D495/14—Ortho-condensed systems

Definitions

  • 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
  • A is O or S
  • B is selected from the group consisting of O, NR 1 , and S
  • R 1 is H or alkyl
  • X is selected from CR 2 R 3 or a chemical bond
  • R 2 and R 3 are independently selected from H or alkyl
  • Y is selected from -(CH 2 ) 5 C(O)NHOH, -OH, and H.
  • R 2 and R 3 may be the same or different.
  • the carbon bonded to R 2 and R 3 may be characterized by an S or R configuration.
  • Exemplary R 2 and R 3 include, without limitation, H or methyl.
  • R 2 and R 3 are each H. In other embodiments, one of R 2 and R 3 is H and the other is methyl.
  • a and B are each be oxygen.
  • A is oxygen and B is NR 1 .
  • 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.
  • compositions disclosed herein may be for use in any of the methods disclosed herein.
  • 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.
  • methods for reducing or inhibiting cancer cell growth in a subject having cancer 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
  • KS Kaposi sarcoma
  • PEL primary effusion lymphoma
  • CCL cutaneous T-cell lymphoma
  • KSHV-associated lymphomas KSHV-associated lymphomas
  • EBV-associated lymphomas EBV-associated lymphomas
  • 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.
  • a method for treating psoriasis may comprise administering an effective amount of any of the compounds disclosed herein to a subject having psoriasis.
  • methods for preparing the compounds disclosed herein are BRIEF DESCRIPTION OF THE DRAWINGS
  • FIG. 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.
  • 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.
  • Example 4 or Example 7 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.
  • 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 ⁇ g/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.
  • viral genes such as latent gene, LANA and two lytic genes, RTA and ORF26
  • 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.
  • 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).
  • the iSLK.219 cells were treated by indicated concentrations of compounds in conjunction with Dox (0.05 ⁇ g/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 CC 50 values in Table 2).
  • Example 2 has little cytotoxicity in primary cells such as HUVEC, a human primary endothelial cell line.
  • primary cells such as HUVEC, a human primary endothelial cell line.
  • 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.
  • 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 CC 50 values in Table 2). Meanwhile, Example 5 has little cytotoxicity in primary cells such as HUVEC cells.
  • 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.
  • 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.
  • Figures 7A-7B show that Example 5 demonstrates adequate pharmacokinetic (PK) properties for in vivo efficacy evaluations.
  • PK pharmacokinetic
  • Figure 7A shows that Example 5 demonstrates adequate PK parameters with AUC last of 159 h ⁇ ⁇ g/mL and terminal t1/2 of 1.66 h when compared to (+)-JQ1 (AUC last of 78 h ⁇ ⁇ g/mL, terminal t1/2 of 2.27 h).
  • SAHA is cleared too fast (AUC last of 12 h ⁇ ⁇ g/mL, terminal t 1/2 of 0.72 h) and therefore it is not used in the following antitumor efficacy studies.
  • Figure 7B shows that Example 5 demonstrates efficient oral PK parameters with AUC last of 39 h ⁇ ⁇ g/mL, terminal t 1/2 of 1.12 h, and bioavailability (F) of 24.5%.
  • 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.
  • 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.
  • FIGS 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).
  • 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.
  • FIGS. 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.
  • FIG. 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 NuncTM EdgeTM 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.
  • Dose-response curves were generated using purified malignant cells obtained from patients with CTCL.
  • Peripheral blood mononuclear cells PBMCs
  • Total CD4+ T cells were purified via either MACS CD4+ negative selection kit (Miltenyi Biotec) or RosetteSepTM 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 (IC 50 s) 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).
  • KSHV Kaposi's sarcoma associated herpesvirus
  • EBV Epstein-Barr virus
  • HDACs histone deacetylases
  • BRD4 bromodomain- containing protein 4
  • 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.
  • A is O or S
  • B is selected from the group consisting of O, NR 1 , and S
  • R 1 is H or alkyl
  • X is selected from CR 2 R 3 or a chemical bond
  • R 2 and R 3 are independently selected from H or alkyl
  • Y is selected from , , , , -(CH 2 ) 5 C(O)NHOH, -OH, and H.
  • 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.
  • a squiggly line is used to designate the point of attachment for any radical group or substituent group.
  • 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 C 1 -C 12 alkyl, C 1 -C 10 -alkyl, and C 1 -C 6 -alkyl, respectively.
  • Representative alkyl groups include methyl, ethyl, tert-butyl and the like.
  • the compound is selected from the group consisting of
  • the compound of is a dual BRD4/HDAC inhibitor.
  • 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.
  • 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).
  • cytotoxicity refers to the quality of being toxic to cells and is measured by the CC 50 value in ⁇ M, which denotes the concentration of a compound that reduces the cell viability by 50% when compared to untreated cells.
  • normal cell refers to a non-cancerous cell that undergoes a regular cell cycle without unchecked cell growth like cancerous cells.
  • the compounds of the disclosure may contain one or more chiral centers and, therefore, exist as stereoisomers, such as enantiomers or diastereomers.
  • stereoisomers refers to the enantiomers or diastereomers of a compound.
  • 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.
  • pharmaceutically acceptable salt 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.
  • 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.
  • inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid, and the like
  • 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.
  • 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, phenylbuty
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • a “subject” may be interchangeable with “patient” or “individual,” which may be a human or a non-human animal in need of treatment.
  • 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.
  • 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.
  • KS Kaposi sarcoma
  • PEL primary effusion lymphoma
  • CCL cutaneous T-cell lymphoma
  • KSHV-associated lymphomas KSHV-associated lymphomas
  • EBV-associated lymphomas EBV-associated lymphomas.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the BRD4-inhibiting subunit comprises a thieno-triazolo-1,4- diazepine scaffold or thieno-l,4-diazepine scaffold.
  • 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.
  • the BRD4-inhibiting subunit is .
  • 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.
  • 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.
  • the linker may be cleaved by hydrolysis or other suitable bond- breaking reaction either with or without the contribution of an enzyme.
  • the linker may be an ester moiety capable of being hydrolyzed with an esterase.
  • the linker may be an amide moiety.
  • esterification refers to the formation of an ester bond (i.e., ), typically from an alcohol and a carboxylic acid.
  • the term “carboxylic acid” refers to a compound having a group of the formula -C(O)OH.
  • the term “alcohol” refers to the substituent having the structure .
  • “amidation” refers to the formation of an amide bond typically from an amine and a carboxylic acid.
  • the term “amine” refers to both unsubstituted and substituted amines, wherein substituents typically include, for example, alkyl, cycloalkyl, heterocyclyl, alkenyl, and aryl.
  • the term “HDAC-inhibiting subunit” refers to a chemical entity capable of inhibiting the activity of an HDAC protein.
  • Histone deacetylases 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.
  • the HDAC-inhibiting subunit is selected from the group consisting of
  • 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.
  • 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.
  • O-(tetrahydro-2H-pyran-2- yl)hydroxylamine (OTX) under conditions sufficient to form a tetrahydropyranyl ether protected hydroxamic acid moiety.
  • the method further comprises deprotecting the tetrahydropyranyl ether protected hydroxamic acid moiety under conditions sufficient for producing the compounds as described herein.
  • tetrahydropyranyl ether protected hydroxamic acid moiety refers to a structural moiety of w 1 herein R is as defined 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.
  • a Kinetex® 5 ⁇ m XB-C 18 100 ⁇ 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 °C 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).
  • 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 °C. After addition, the reaction mixture was stirred at room temperature overnight. LC/MS analysis indicated the completed conversion.
  • 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 °C for 5 minutes.
  • 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)
  • (+)-JQ1 carboxylic acid Example 21, 0.1 g, 0.3 mmol
  • 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 °C.
  • 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).
  • 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 °C. 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.
  • Step 2 tert-butyl (S)-(2-(4-(1-hydroxyethyl)benzamido)phenyl)carbamate (2-3)
  • S tert-butyl
  • (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 °C for 5 minutes.
  • 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
  • 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.
  • 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)benza
  • Reagents and conditions (a) LiOH ⁇ H 2 O, THF, MeOH, H 2 O, 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 °C. 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.
  • Step 2 tert-butyl (R)-(2-(4-(1-hydroxyethyl)benzamido)phenyl)carbamate (5-3)
  • R tert-butyl
  • (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 °C for 5 minutes.
  • 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-hydroxyethy
  • 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)benza
  • Example 7 methyl 4-((R)-1-(2-((S)-4-(4-chlorophenyl)-2,3,9-trimethyl-6H-8 ⁇ 2 ,10 ⁇ 4 - 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.
  • 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) SOCl 2 , DMF, DCM, reflux, 8 h; (b) 1-2, TEA, DCM, 0 °C to rt, overnight; (c) LiBH 4 , THF, 0 °C 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 SOCl 2 (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 °C 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 °C. Then the reaction mixture was stirred at room temperature overnight.
  • 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.
  • 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).
  • 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)
  • (+)-JQ1 carboxylic acid Example 21, 24.0 mg, 0.06 mmol
  • benzotriazol- 1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (31.2 mg, 0.06 mmol)
  • 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 m
  • 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)
  • (-)-JQ1 carboxylic acid 3-2, 50.0 mg, 0.12 mmol
  • benzotriazol-1-yl- oxytripyrrolidinophosphonium hexafluorophosphate (65.0 mg, 0.125 mmol)
  • N, N- diisopropylethylamine 50.0 mg, 0.39 mmol
  • tert-butyl (2-(4-(hydroxymethyl)benzamido)phenyl)carbamate 8-4, 50.0 mg, 0.15 m
  • 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.
  • 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)
  • benzotriazol-1-yl- oxytripyrrolidinophosphonium hexafluorophosphate (62.4 mg, 0.12 mmol)
  • N, N- diisopropylethylamine (46.5 mg, 0.36 mmol)
  • Reagents and conditions (a) methyl 4-(1-aminoethyl)benzoate hydrochloride, PyBOP, DIPEA, DMF, rt, overnight, (b) LiOH ⁇ H 2 O, MeOH, THF, H 2 O, 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)
  • (+)-JQ1 carboxylic acid Example 21, 80.2 mg, 0.2 mmol
  • benzotriazol- 1-yl-oxytripyrrolidinophosphonium hexafluorophosphate 43.0 mg, 0.2 mmol
  • N, N- diisopropylethylamine 77.6 mg, 0.6 mmol
  • anhydrous dimethylformamide 5.0 mL
  • 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, 0.05 mmol) in methanol (2.1 mL) and t
  • 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)
  • (+)-JQ1 carboxylic acid Example 21, 80.0 mg, 0.2 mmol
  • benzotriazol- 1-yl-oxytripyrrolidinophosphonium hexafluorophosphate 0.1 g, 0.2 mmol
  • N, N- diisopropylethylamine 77.6 mg, 0.6 mmol
  • anhydrous dimethylformamide (10.0 mL) was added methyl 4-(aminomethyl)benzoate hydrochloride (48.4 mg, 0.24 mmol) at 0 °C.
  • Reagents and conditions (a) ethyl acrylate, Pd(OAc) 2 , PPh 3 , DIPEA, DMF, 90 °C, overnight, (b) TFA, DCM, rt, overnight, (c) 10-2, PyBOP, DIPEA, DMF, rt, overnight, (d) LiOH ⁇ H 2 O, EtOH, THF, H 2 O, rt, overnight, (e) NH 2 OH ⁇ HCl, PyBOP, DIPEA, DMF, rt, overnight.
  • Step 1 ethyl (E)-3-(4-(((tert-butoxycarbonyl)amino)methyl)phenyl)acrylate (14-2)
  • 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).
  • 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.
  • 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)
  • (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)
  • 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) NH 2 OH ⁇ 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)
  • (+)-JQ1 carboxylic acid Example 21, 0.12 g, 0.3 mmol
  • benzotriazol-1- yl-oxytripyrrolidinophosphonium hexafluorophosphate (0.16 g, 0.3 mmol)
  • N, N- diisopropylethylamine (0.11 g, 0.9 mmol
  • anhydrous dimethylformamide 5.0 mL
  • 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).
  • 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
  • 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)
  • (+)-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)
  • N, N-diisopropylethylamine (0.12 g, 0.9 mmol
  • tert-butyl 4-aminobenzoate 54.4 mg, 0.36 mmol) at 0 °C.
  • 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, H 2 O, rt, overnight, (c) NH 2 OH ⁇ 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)
  • (+)-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 °C.
  • 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)
  • (+)-JQ1 carboxylic acid Example 21, 0.75 g, 1.87 mmol
  • anhydrous dimethylformamide (20.0 mL) 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.
  • 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, THPONH 2 , 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
  • 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).
  • 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
  • ) represents ConCTAC with reduced inhibitory activity on HDACs but retaining inhibitory activity on BET bromodomains.
  • 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).
  • RTA viral replication and transcription activator
  • the rKSHV.219 contains two fluorescent protein genes, the green fluorescent protein (GFP) and red fluorescent protein (RFP), which are derived from the EF-1 ⁇ 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 (0.05 ⁇ g/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.
  • 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.
  • 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 iTaqTM 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
  • 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
  • IP intraperitoneal
  • oral gavage administration 50 mg per kg body weight
  • 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 ⁇ L of K2-EDTA (0.5 M) as anti-coagulant and placed on wet ice until centrifugation.
  • RNA-Sequencing and enrichment analysis RNA-Sequencing of triplicate samples was performed by BGI Americas Corporation using their unique DNBSEQTM 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).
  • Example 31 Immunohistochemistry Formalin-fixed, paraffin-embedded tissues were microtome-sectioned to a thickness of 4 ⁇ m 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.

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Abstract

Sont divulgués dans la présente invention des composés de formule I ou II, ou des sels pharmaceutiquement acceptables de ceux-ci, et leur utilisation en tant qu'inhibiteurs doubles BRD4 et HDAC. Sont également également divulguées des compositions pharmaceutiques comprenant les composés de formule I ou II et des méthodes d'utilisation des composés et des compositions pharmaceutiques pour le traitement du cancer ou du psoriasis. Sont en outre divulgués dans la présente invention des méthodes de préparation de composés de formule I ou II.
EP23901526.6A 2022-12-06 2023-12-06 Développement d'inhibiteurs puissants de hdac/brd4 Pending EP4629984A1 (fr)

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WO2025053994A1 (fr) * 2023-09-08 2025-03-13 The Regents Of The University Of California Amplificateurs à petites molécules de clusterine sécrétée (sclu)

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