WO2020093162A1

WO2020093162A1 - Treatment of bet inhibitor-resistant cancers and other diseases responsive to dual bet and cbp/ep300 inhibition therapy

Info

Publication number: WO2020093162A1
Application number: PCT/CA2019/051586
Authority: WO
Inventors: Francis J. Giles
Original assignee: Neomed Institute
Priority date: 2018-11-07
Filing date: 2019-11-07
Publication date: 2020-05-14

Abstract

There is provided a method for treating BET inhibitor-resistant cancers, comprising providing a subject having cancer cells that overexpress BET protein substrates of SPOP relative to corresponding non-cancerous cells; and administering a therapeutically effective amount of a BET inhibitor and a CBP/EP300 inhibitor to the subject having the BET protein-overexpressing cancer cells. In some embodiments, the BET inhibitor and the CBP/EP300 inhibitor can be a single compound, wherein the compound is a substituted benzimidazole or an aryl-substituted dihydroquinolinone having dual activity as a BET inhibitor and a CBP/EP300 inhibitor. In some embodiments, the method can further comprises assessing the presence or absence of a biomarker in the cancer cells indicative of BET inhibitor resistance, and/or the presence or absence of biomarker in the cancer cells indicative of responsiveness to BET inhibitor and CBP/EP300 inhibitor co-therapy.

Description

TREATMENT OF BET INHIBITOR-RESISTANT CANCERS AND OTHER DISEASES RESPONSIVE TO DUAL BET AND CBP/EP300 INHIBITION THERAPY

RELATED APPLICATIONS

This application claims priority to United States provisional application Nos. 62/756,812 filed on November 7, 2018 and 62/851,445 filed on May 22, 2019, the contents of which are incorporated herein by reference in their entirety for all purposes.

FIELD

The technical field relates to treatment of BET inhibitor-resistant cancers and other diseases responsive to dual BET and CBP/EP300 inhibition therapy, as well as compounds having dual BET and CBP/EP300 inhibition activity.

BACKGROUND

Bromodomains are found in a variety of mammalian DNA-binding proteins. The bromodomain, which is the conserved structural module in chromatin-associated proteins and histone acetyltransferases, is known to recognize acetyl-lysine residues on proteins. Bromodomain and extra-terminal domain (BET) inhibitors are a class of drugs presently being investigated for multiple therapeutic applications, including in anti-cancer therapies. However, some cancers and other diseases exhibit BET inhibitor resistance and are reported as being poorly responsive to BET inhibition therapy. There is therefore a need for compounds, compositions, and therapies for treating BET inhibitor-resistant diseases, such as BET inhibitor-resistant cancers.

SUMMARY

Using prostate cancer cell line, organoid, and patient-derived xenografts (PDX) as models, it is shown herein that cancer cells expressing E3 ubiquitin ligase substrate-binding adaptor speckle-type POZ protein (SPOP) variants linked to BET inhibitor resistance, exhibited increased sensitivity to simultaneous inhibition of BET protein and CREB binding protein (CBP)/histone acetyltransferase p300 (EP300), as compared to BET inhibitor-sensitive cancer cells expressing only wild type SPOP. Affinity binding studies revealed that members of the substituted benzimidazole family and aryl-substituted

dihydroquinolinone family of BRD4 inhibitors possess dual activity as BET and CBP/EP300 inhibitors. Evidence is shown herein that BET inhibitor-resistant cancers may be more sensitive to monotherapy with such dual inhibitors, as compared to BET inhibitor-sensitive cancers.

In one aspect, described herein is a method for treating BET inhibitor-resistant cancers, the method comprising providing a subject having cancer cells that overexpress BET protein substrates of SPOP relative to corresponding non-cancerous cells; and administering a therapeutically effective amount of a BET inhibitor and a CBP/EP300 inhibitor to the subject having the BET protein-overexpressing cancer cells. In some embodiments, the BET inhibitor and the CBP/EP300 inhibitor are a single compound, wherein the compound is a substituted benzimidazole or aryl-substituted dihydroquinolinone having dual activity as a BET inhibitor and a CBP/EP300 inhibitor. In some embodiments, the method further comprises assessing the presence or absence of a biomarker in the cancer cells indicative of BET inhibitor resistance, and/or the presence or absence of biomarker in the cancer cells indicative of responsiveness to BET inhibitor and CBP/EP300 inhibitor co-therapy.

In other aspects, described herein is a method of identifying a subject that may be suitable (or responsive) for anti-cancer co-therapy with a BET inhibitor and a CBP/EP300 inhibitor, the method comprising: (a) providing a biological sample comprising cancer cells from the subject; (b) measuring the presence or absence of biomarker in the cancer cells indicative of responsiveness to BET inhibitor and CBP/EP300 inhibitor co-therapy; and (c) identifying the subject as being suitable for anti-cancer co therapy with a BET inhibitor and a CBP/EP300 inhibitor when the cancer cells are positive for the biomarker. In some embodiments, the biomarker may be one or more of: overexpression of BET protein(s) (or other protein substrates whose degradation is mediated by SPOP) in the cancer cells relative to corresponding non-cancerous cells; and/or the expression of an SPOP variant in the cancer cells that impairs SPOP-mediated BET protein degradation.

In further aspects, described herein is a composition comprising: (i) a BET inhibitor and a CBP/EP300 inhibitor; or (ii) a compound which is a substituted benzimidazole or an aryl-substituted dihydroquinolinone dual inhibitor of BET and CBP/EP300, and a pharmaceutically acceptable excipient, for use in the treatment of BET inhibitor-resistant cancer cells in a subject. In some aspects, the present description relates to the use of such a composition or compound for treating BET inhibitor-resistant cancer cells in a subject, or for the manufacture of a medicament for same.

In some aspects, the present description relates to a compound which is a substituted benzimidazole or aryl-substituted dihydroquinolinone dual inhibitor of BET and CBP/EP300, for use as a BET inhibitor and a CBP/EP300 inhibitor in a BET and CBP/EP300 co-inhibition therapy.

In some embodiments, the methods, composition and compounds described herein are for the treatment of a solid cancer (e.g., a sarcoma, a carcinoma, a lymphoma, melanoma, glioma, prostate cancer, lung cancer, breast cancer, gastric cancer, liver cancer, colon cancer, tongue cancer, thyroid cancer, renal cancer, uterine cancer, cervical cancer, ovarian cancer, pancreatic cancer) and more particularly, BET inhibitor-resistant solid cancers. In other embodiments, the methods, composition and compounds described herein may be for the treatment of blood cancers (e.g., a leukemia, a lymphoma, or a myeloma), and more particularly, BET -inhibitor-resistant blood cancers.

Additional objects and features of the present compounds, compositions, methods and uses will become more apparent upon reading of the following non-restrictive description of exemplary embodiments, which should not be interpreted as limiting the scope of the invention.

BRIEF DESCRIPTION OF THE FIGURES

Fig. 1 is a graph showing the results of MTS cell proliferation studies in DU 145 prostate cancer cells transfected with either an empty vector (“EV”) or with an expression vector encoding an SPOP F133V variant (“F133V”), after treatment with vehicle alone (“DMSO”), the BET inhibitor JQ1 alone (“JQ1”), RNA interference-mediated knockdown of CREB binding protein expression (“iCBP”), or the combination of JQ1 and iCBP (“iCBP + JQ1”).

Fig. 2 is a graph showing the results of MTS cell proliferation studies in DU 145 prostate cancer cells transfected with either an empty vector (“EV”) or with an expression vector encoding an SPOP F133V variant (“F133V”), after treatment with vehicle alone (“DMSO”), the BET inhibitor JQ1 alone (“JQ1”), the CBP/EP300 inhibitor CPI-637 alone (“CPI-637”), the substituted benzimidazole Compound A1 having dual BET and CBP/EP300 inhibition activity (“Compound Al”), or co-treatment with JQ1 and CPI-637 (“CPI-637 + JQ1”).

Fig. 3 is a graph showing the results of dose/survival (kill curve) analyses of DU145 prostate cancer cells transfected with either an empty vector (“EV”) or with an expression vector encoding an SPOP F133V variant (“F133V”), exposed to increasing concentrations of Compound Al.

Fig. 4 is a scheme showing the main steps in the preparation of human prostate cancer patient- derived organoids.

Fig. 5 is a graph showing the effects of treatment with vehicle alone (“DMSO”), BET inhibitor alone (“JQ1”), CBP/EP300 inhibitor alone (“CPI-637”), co-treatment with BET inhibitor and CBP/EP300 inhibitor (“CPI-637 + JQ1”), or monotreatment with Compound Al (“Compound Al”), on the diameter of organoids prepared from human prostate cancers expressing either wild type (“SPOP^WT”) or a Q165P SPOP variant (“SPOP^Q165F”).

Fig. 6A-6J are representative microscopy images of prostate cancer organoids after culture for 10 days after treatment with vehicle alone (“DMSO”), BET inhibitor alone (“JQ1”), CBP/EP300 inhibitor alone (“CPI-637”), co-treatment with BET inhibitor and CBP/EP300 inhibitor (“CPI-637 + JQ1”), or monotreatment with Compound Al (“Compound Al”), wherein the organoids are prepared from human prostate cancers expressing either wild type (“SPOP^WT”) or a Q165P SPOP variant (“SPOP^Q165F”).

Fig. 7 and Fig. 8 show photographs of human prostate cancer tumors isolated from mouse xenografts models following treatment with vehicle alone (“DMSO”), BET inhibitor alone (“JQ1”), CBP/EP300 inhibitor alone (“CPI-637”), co-treatment with BET inhibitor and CBP/EP300 inhibitor (“CPI-637 + JQ1”), or monotreatment with Compound Al (“Compound Al”). Tumors expressing wild type (“SPOP^WT”) are shown in Fig. 7, while tumors expressing the Q165P SPOP variant (“SPOP^Q165F”) are shown in Fig. 8.

Fig. 9 and Fig. 10 are graphs showing corresponding tumor volume measurements of the tumors shown in Fig. 7 and Fig. 8, respectively.

Fig. 11 and Fig. 12 show the results of co-immunoprecipitation assays followed by immunoblot analysis for recombinantly expressed wild type or Q165P variant SPOP proteins tagged with either Flag or Myc epitopes.

Fig. 13 shows the chemical structures of exemplary substituted benzimidazole compounds described in WO 2017/024412.

Fig. 14 shows the chemical structures of exemplary aryl-substituted dihydroquinolinones described in WO 2017/024408. DESCRIPTION

In some aspects, described herein are methods for treating a cancer responsive to co-therapy with a bromodomain and extraterminal protein (BET) inhibitor and a CREB binding protein (CBP)/EP300 inhibitor (e.g., using two separate inhibitor molecules, or preferably a single molecule having dual activity as a BET inhibitor and a CBP/EP300 inhibitor). The method generally comprises providing, identifying, selecting, and/or screening for subjects having cancer cells that overexpress BET proteins (e.g., BRD2, BRD3, or BRD4), and more specifically BET proteins that are substrates of wild type E3 ubiquitin ligase substrate-binding adaptor speckle-type POZ protein (SPOP), relative to corresponding non-cancerous cells. For greater clarity, while the subject cancer cells overexpress or have elevated expression of BET proteins, the subject may be provided, identified, or selected based on the presence of one or more biomarkers indicative of BET inhibitor resistance, not necessarily elevated expression of BET proteins. In this regard, the results disclosed herein provide evidence that SPOP variants (e.g., SPOP variants identified from human prostate cancer tissue specimens) that impair SPOP -mediated BET protein degradation (e.g., SPOP variants defective or impaired for substrate binding, or SPOP variants that are defective or impaired for SPOP protein dimerization or oligomerization, thereby negatively affecting their ability to mediate BET protein degradation) may be more susceptible and/or responsive to BET inhibition and CBP/EP300 inhibition co-treatments, as compared to corresponding cells expressing wild type SPOP (e.g., see the dose-survival analysis shown in Fig. 3). The methods of treatment described herein generally comprise administering a therapeutically effective amount of a BET inhibitor and a CBP/EP300 inhibitor to the subject having the BET protein-overexpressing cancer cells. In some embodiments, BET protein overexpression in, for example, a cancer tissue specimen may be evaluated by comparison to the level of BET protein expression in neighbouring noncancerous, benign, or healthy tissue (e.g., via immunohistochemistry, immunocytochemistry, and/or other antibody-based detection assay) in a biological sample obtained from a cancer subject.

In some embodiments, the BET protein-overexpressing cancer cells described herein express an SPOP variant that impairs SPOP -mediated BET protein (e.g., BRD2, BRD3, BRD4) degradation. The human wild type SPOP protein has been purified and found to be composed of 374 amino acids and two domains: an N-terminal part containing residues 28-166 (MATH domain) and a C-terminal part containing residues 172-329 (BTB domain) (Zhuang et al., 2009), with the MATH domain mediating substrate binding and the BTB domain facilitating the formation of a 2:2 complex with the CUL3 N-terminal domain.

In some embodiments, the SPOP variants described herein may comprise a mutation in an amino acid residue of the MATH domain defined by amino acid positions 28-166 of human wild-type SPOP. Intriguingly, the vast majority of SPOP mutations found in prostate cancers were reported to be localized to the MATH domain (Barbieri et al., 2012). In some embodiments, the SPOP variant comprises a mutation in an amino acid residue of the substrate-binding cleft that impairs substrate interaction and ubiquitin transfer, such as mutations at positions R70, Y87, F102, Y123, K129, D130, W131, and/or F133 with respect to wild type SPOP (Zhuang et al., 2009; Janouskova et al., 2017).

In some embodiments, the methods described herein may comprise genotyping the cancer cells for the presence of a mutant SPOP gene encoding an SPOP variant as defined herein. In some embodiments, the methods described herein may comprise measuring one or more BET protein expression levels in the cancer cells (e.g., via immunocytochemistry, immunohistochemistry, immunoblot analysis, or other antibody -based BET protein detection assay), wherein the BET proteins measured comprise BET protein substrates of wild type SPOP (e.g., BRD2, BRD3, BRD4). In some embodiments, the BET protein-overexpressing cancer cells described herein are resistant to BET inhibitor mono treatment. As used herein, the expression“BET inhibitor resistant” or“resistant to BET inhibitor mono-treatment” refers to cancer cells that exhibit increased survival or tolerance to exposure to a BET inhibitor that lacks substantial CBP inhibiting activity, such as treatment with a pan-BET inhibitor such as JQ1, wherein the increased survival or tolerance is associated with overexpression of BET protein substrates of wild type SPOP, as compared to the level of survival or tolerance to the BET inhibitor in corresponding cells expressing lower or basal levels of SPOP. Such BET inhibitor resistant cancers have been previously widely reported to be poor candidates for BET inhibition anti-cancer therapy (Zhang et al., 2017; Dai et al., 2017; Janouskova et al., 2017).

Results disclosed herein using organoid models and human patient-derived mouse xenograft models show that solid cancers expressing SPOP mutant protein were more responsive to BET and CBP/EP300 dual inhibition treatment (e.g., using two separate inhibitor molecules, or a single molecule having dual activity as a BET inhibitor and a CBP/EP300 inhibitor), as compared to corresponding solid cancers expressing wild type SPOP protein (e.g. see Fig. 5 to Fig. 10). Accordingly, in some embodiments, the cancers or cancer cells described herein that may be particularly responsive or sensitive to BET and CBP/EP300 dual inhibition treatment comprise solid cancers (solid tumors) or cancer cells from solid cancers. In some embodiments, the solid cancer is a sarcoma, a carcinoma, a lymphoma, melanoma, glioma, prostate cancer, lung cancer, breast cancer, gastric cancer, liver cancer, colon cancer, tongue cancer, thyroid cancer, renal cancer, uterine cancer, cervical cancer, ovarian cancer, or pancreatic cancer. In other embodiments, the methods, composition and compounds described herein may be for the treatment of blood cancers (e.g., a leukemia, a lymphoma, or a myeloma), and more particularly, BET- inhibitor-resistant blood cancers.

In some embodiments, the BET and CBP/EP300 co-inhibition treatments described herein may comprise administering therapeutically effective amounts of a first molecule which is a BET inhibitor in combination with a second molecule which is a CBP/EP300 inhibitor. In some embodiments, the first and second molecules may be small molecule compounds. In some embodiments, the BET inhibitor administered may be a bromodomain-containing protein 2 (BRD2) inhibitor, bromodomain-containing protein 3 (BRD3) inhibitor, or bromodomain-containing protein 4 (BRD4) inhibitor. In particular embodiments, the BET inhibitor may be CPI-0610, DUAL946, GSK525762, 1-BET151, JQ1, OTX015, PFI-1, RVX-208, RVX2135, TEN-010, or a derivative thereof having BRD2, BRD3, or BRD4-inhibiting activity. In some embodiments, the CBP/EP300 inhibitor may be an agent that binds the CBP histone acetyltransferase (HAT) domain and/or the EP300 HAT domain and inhibits and/or reduces a biological activity of CBP and/or EP300 (e.g., CPI-637, A-485, C646, EML425, LB-A23, or functional derivatives thereof having CBP/EP300 inhibition activity).

Substituted benzimidazoles having BET and CBP/EP300 dual inhibition activity

In some embodiments, the BET and CBP/EP300 dual inhibition treatments described herein may comprise administration of a compound having BET and CBP/EP300 dual inhibition activity. Results disclosed herein demonstrate that members of the substituted benzimidazole family of BRD4 inhibitors, as described in WO 2017/024412, may have BET and CBP/EP300 dual inhibition activity (e.g., see

Example 6).

Accordingly, in some aspects, the compounds described herein may be a substituted

benzimidazole that binds BRD4 and CBP/EP300, as defined in WO 2017/024412. In some embodiments, the compound is as defined in one or more of the following embodiments 1 to 39:

1. A substituted benzimidazole of Formula A, or a pharmaceutically acceptable salt, solvate, ester, or prodrug thereof:

Formula A

wherein,

R¹ is:

(a) an imsubstituted Ci-Cealkyl;

(b) a Ci-Cealkyl substituted with one or more group(s) selected from halogen (such as

fluorine), CN, NO_¾ C(0)NHRⁿ, C(0)N(Rⁿ)₂, C0₂H, S0₂Rⁿ, S0₂NHRⁿ, and

S0₂N(Rⁿ)₂;

(c) a C₂-Cealkyl group substituted with a group selected from OR¹¹, halogenated OCi- Cealkyl, SH, SR¹¹, NH_¾ NHR¹¹, N(Rⁿ)_¾ NHC(0)Rⁿ, and N(Rⁿ)C(0)Rⁿ; or

(d) a group selected from C(0)Rⁿ, C(0)NHRⁿ, C(0)N(Rⁿ)₂, S0₂Rⁿ, S0₂NHRn, and

S0₂N(Rⁿ)₂;

R² is selected from hydrogen, NH₂ and a substituted or unsubstituted group selected from Ci- Cealkyl, C₂-C₆alkenyl, C₂-Cealkynyl, C3-Ciocycloalkyl, C3-Cioheterocycloalkyl, C(0)R¹², NHR¹², N(R¹²)_¾ C(0)NH_¾ C(0)NHR¹², C(0)N(R¹²)₂, NHC(0)R¹², S0₂R¹², S0₂NHR¹², S0₂N(R¹²)₂, NHSO_ZR¹², N(R¹²)S0₂R¹², NHSO_ZNHR¹², N(R¹²)S0₂NHR¹², NHS0₂N(R¹²)₂, and N(R¹²)S0₂N(R¹²)₂; R³ and R⁶ are each independently H, NH₂ or a substituted or unsubstituted group selected from Ci- Cealkyl, C(0)Rⁿ, NHR¹¹, N(Rⁿ)_¾ C(0)NH₂, C(0)NHRⁿ, C(0)N(Rⁿ)_¾ NHC(0)Rⁿ; and one of R⁴ and R⁵ is H, NH₂ or a substituted or unsubstituted group selected from Ci-Cealkyl, C(0)Rⁿ, NHR¹¹, N(Rⁿ)_¾ C(0)NH₂, C(0)NHRⁿ, C(0)N(Rⁿ)₂, and NHC(0)Rⁿ; the other of R⁴ and R⁵ is a group of Formula A.I:

Formula A.I

wherein,

R⁷, R⁸, and R¹⁰ are each independently H, halogen (such as F, Cl), CN, or a substituted or

unsubstituted Ci-Cealkyl or C3-C6cycloalkyl group, OR¹¹, SR¹¹, NHR¹¹, N(Rⁿ)_¾ NHC(0)Rⁿ, and N(Rⁿ)C(0)Rⁿ, provided that at least one of R⁷, R⁸, and R¹⁰ is other than H;

R⁹ is a substituted or unsubstituted Ci-C3alkyl or CYCscycloalkyl group;

R¹¹ is, independently in each occurrence, a substituted or unsubstituted C1-C6 alkyl group;

R¹² is, independently in each occurrence, a substituted or unsubstituted Ci-Cealkyl, C₂-C₆alkenyl, C₂-Cealkynyl, C3-Ciocycloalkyl, and C3-Cioheterocycloalkyl;

X¹, X², and X³ are each selected from N and C, wherein when X¹, X², or X³ is N, then the R⁷, R⁸, or R¹⁰ attached thereto is absent, provided that at least two of X¹, X², and X³ is C, wherein when any of the foregoing group contains an alkyl group, then said alkyl is a linear or branched acyclic alkyl group, and

optionally, wherein when any of R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹ and R¹² is substituted, it is substituted with one or more substituents selected from halogen, Ci-Cealkyl optionally substituted with an oxo group, Ci-Cealkoxy, C3-Cioheterocycloalkyl and Cearyl.

2. The compound of embodiment 1, wherein R⁴ is a group of Formula A.I.

3. The compound of embodiment 2, wherein R⁵ is a substituted or unsubstituted C₁-C₃ alkyl.

4. The compound of embodiment 2, wherein R⁵ is a hydrogen atom.

5. The compound of embodiment 1, wherein R⁵ is a group of Formula A.I.

6. The compound of embodiment 5, wherein R⁴is a substituted or unsubstituted C₁-C₃ alkyl.

7. The compound of embodiment 5, wherein R⁴ is a hydrogen atom.

8. The compound of any one of embodiments 1 to 7, wherein X¹, X², and X³ are all carbon atoms.

9. The compound of any one of embodiments 1 to 7, wherein X¹ is a nitrogen atom and R¹⁰ is

absent, and X² and X³ are carbon atoms.

10. The compound of any one of embodiments 1 to 9, wherein R⁹ is an unsubstituted C1-C3 alkyl or C3-C5 cycloalkyl group. 11 The compound of embodiment 10, wherein R⁹ is selected from methyl, ethyl, n-propyl, isopropyl, and cyclopropyl.

12 The compound of any one of embodiments 1 to 11, wherein R⁸ is halogen (such as F, Cl), CN, or a substituted or unsubstituted Ci-Cealkyl or C3-C6cycloalkyl group, OR¹¹, SR¹¹, NHR¹¹, N(Rⁿ)₂, NHC(0)Rⁿ, or N(Rⁿ)C(0)Rⁿ.

13. The compound of embodiment 8, wherein R⁷ and R¹⁰ are each hydrogen atoms and R⁸ is selected from Cl, CN, NHR¹¹ and a substituted or unsubstituted Ci-Cealkyl, or C3-C6cycloalkyl group.

14. The compound of any one of embodiments 1 to 13, wherein R³ is H or a substituted or

unsubstituted Ci-Cealkyl group.

15. The compound of embodiment 14, wherein R³ is H.

16. The compound of any one of embodiments 1 to 15, wherein R⁶ is H or a substituted or

unsubstituted Ci-Cealkyl group.

17. The compound of embodiment 16, wherein R⁶ is H.

18. The compound of embodiment 1, wherein the compound of Formula A is a compound of

Formula A.II:

Formula A.II

wherein R¹, R², R⁴ and R⁵ are as defined in embodiment 1, or a pharmaceutically acceptable salt, solvate, ester or prodrug thereof.

19. The compound of embodiment 18, wherein the compound of Formula A.II is a compound of

Formula A.III:

Formula A.III

wherein,

R¹, R², R⁷, R⁸, R⁹, and R¹⁰ are as defined in embodiment 1, or a pharmaceutically acceptable salt, solvate, ester or prodrug thereof.

20 The compound of embodiment 18, wherein the compound of Formula A.II is a compound of

Formula A.IV :

Formula A.IV

wherein,

The compound of embodiment 19 or 20, wherein R⁹ is an unsubstituted Ci-C3alkyl or C3- Cscycloalkyl group.

The compound of embodiment 19 or 20, wherein R⁹ is selected from methyl, trifluoromethyl, ethyl, n-propyl, isopropyl and cyclopropyl.

The compound of embodiment 19 or 20, wherein R⁷ and R¹⁰ are each hydrogen atoms and R⁸ is selected from Cl, CN, NHR¹¹ and a substituted or unsubstituted Ci-Cealkyl or C3-C6cycloalkyl group.

The compound of embodiment 19 or 20, wherein R⁸ and R⁹ are each independently a methyl, ethyl, isopropyl, fluoromethyl, difluoromethyl, trifluoromethyl, cyclopropyl, or

difluorocyclopropyl group.

The compound of any one of embodiments 1 to 24, wherein R² is hydrogen or a substituted or unsubstituted group selected from Ci-Cealkyl, C3-Ciocycloalkyl, or C3-Cioheterocycloalkyl group. The compound of embodiment 25, wherein R² is a substituted or unsubstituted Ci-C3alkyl, C3- Cecycloalkyl, or C3-C6heterocycloalkyl group.

The compound of embodiment 25, wherein R² is a substituted or unsubstituted group selected from methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, cyclopropyl, cyclobutyl, cyclopentyl, tetrahydrofuranyl, tetrahydropyranyl, dioxolanyl, piperidinyl, and pyrrolidinyl. The compound of any one of embodiments 1 to 27, wherein R¹ is a branched or linear unsubstituted Ci-Cealkyl.

The compound of any one of embodiments 1 to 27, wherein R¹ is a branched or linear Ci-Cealkyl substituted with one or more fluorine atom(s), or a branched or linear C2-Cealkyl substituted with a OCi-Cealkyl group or halogenated OCi-Cealkyl group.

The compound of embodiment 29, wherein R¹ is a branched or linear C2-C3alkyl substituted with a group selected from fluorine, OCi-Cealkyl, and halogenated OCi-Cealkyl.

The compound of any one of embodiments 1 to 30, wherein R¹ is branched.

The compound of embodiment 29, wherein R¹ is fluoromethyl, difluoromethyl, trifluoromethyl, 2,2,2-trifluoroethyl, 2-methoxyethyl, 2-ethoxyethyl, 2-(fluoromethoxy)ethyl, 2- (difluoromethoxy)ethyl, 2-(trifluoromethoxy)ethyl, 3,3,3-trifluoro-l-propyl, 3-methoxy-l-propyl, 3-ethoxy-l-propyl, 3 -(fluoromethoxy)-l -propyl, 3-(difluoromethoxy)-l-propyl, 3- (trifluoromethoxy)-l -propyl, 1-methoxy -2 -propyl, 1 -ethoxy -2-propyl, l-(fluoromethoxy)-2- propyl, l-(difluoromethoxy)-2-propyl, l-(trifluoromethoxy)-2-propyl, 2-methoxy-l-propyl, 2- ethoxy-l-propyl, 2-(fluoromethoxy)-l -propyl, 2-(difluoromethoxy)-l -propyl, or 2- (trifluoromethoxy) - 1 -propyl.

33. The compound of any one of embodiments 1 to 27, wherein R¹ is 2-methoxyethyl, 2- (trifluoromethoxy)ethyl, 1-methoxy -2-propyl, l-(trifluoromethoxy)-2 -propyl, 2-methoxy-l- propyl, or 2-(trifluoromethoxy)-l -propyl.

34. The compound of embodiment 1, wherein the compound of Formula A is selected from

Compounds A1 to A67 of Fig. 13, or a pharmaceutically acceptable salt, solvate, or prodrug thereof.

35. A substituted benzimidazole of Formula A.V

Formula A.V

wherein R² is selected from hydrogen, NEC and a substituted or unsubstituted group selected from Ci-Cealkyl, C2-C6alkenyl, C2-Cealkynyl, C3-Ciocycloalkyl, C3-Cioheterocycloalkyl, C(0)R¹², NHR¹², N(R¹²)₂, C(0)NH_¾ C(0)NHR¹², C(0)N(R¹²)_¾ NHC(0)R¹², S0₂R¹², SO2NHR¹², S0₂N(R¹²)₂, NHSO2R¹², N(R¹²)S0₂R¹², NHSO_ZNHR¹², N(R¹²)S0₂NHR¹², NHS0₂N(R¹²)₂, and N(R¹²)S0₂N(R¹²)₂;

R¹² is, independently in each occurrence, a substituted or unsubstituted Ci-Cealkyl, C2-C6alkenyl, C2-Cealkynyl, C3-Ciocycloalkyl, and C3-Cioheterocycloalkyl;

wherein when any of the foregoing group contains an alkyl group, then said alkyl is a linear or branched acyclic alkyl group; and

optionally, wherein when any of R² and R¹² is substituted, it is substituted with one or more substituents selected from halogen, Ci-Cealkyl optionally substituted with an oxo group, Ci-Cealkoxy, C3-Cioheterocycloalkyl and Cearyl,

or a pharmaceutically acceptable salt, solvate, ester or prodrug thereof.

36. The compound of embodiment 35, wherein R² is hydrogen or a substituted or unsubstituted group selected from Ci-Cealkyl, C3-Ciocycloalkyl, or C3-Cioheterocycloalkyl group.

37. The compound of embodiment 35, wherein R² is a substituted or unsubstituted Ci-C3alkyl, C3- Cecycloalkyl, or C3-C6heterocycloalkyl group.

38. The compound of embodiment 35, wherein R² is a substituted or unsubstituted group selected from methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, cyclopropyl, cyclobutyl, cyclopentyl, tetrahydrofuranyl, tetrahydropyranyl, dioxolanyl, piperidinyl, and pyrrolidinyl. 39. The compound of embodiment 35, wherein R² is cyclopropyl or tetrahydropyranyl.

Aryl-substituted dihydroquinolinones having BET and CBP/EP300 dual inhibition activity

In some embodiments, the BET and CBP/EP300 co-inhibition method, treatments and uses described herein may comprise administration of a compound having BET and CBP/EP300 dual inhibition activity. Results disclosed herein demonstrate that members of the aryl-substituted dihydroquinolinone family of BRD4 inhibitors, as defined in WO 2017/024408, may have BET and CBP/EP300 dual inhibition activity.

Accordingly, in some aspects, the compound described herein may be an aryl-substituted dihydroquinolinone that binds BRD4 and CBP/EP300, as defined in WO 2017/024408. In some embodiments, the compound is as defined in one or more of the following embodiments 40 to 48:

40. An aryl-substituted dihydroquinolinone of Formula B, or a pharmaceutically acceptable salt, solvate, ester, or prodrug thereof:

Formula B wherein

R¹’ is hydrogen or C -Ce alkyl;

R²’ is hydrogen, halogen, -CH₃, -C(R^a)(R^b)(R^c), -N(R^d)(R^e) or -0(R^r)

wherein

R^a is hydrogen or C -Ce alkyl;

R^b is hydrogen, hydroxyl or C -Ce alkyl;

R^c is C3-C6 cycloalkyl, C6-C10 aryl, 3-7 membered heterocycloalkyl, 5-10 membered heteroaryl, (C3-C6 cycloalkyl)Ci-C6 alkyl, (3-7 membered heterocycloalkyl)Ci-C6 alkyl, (C6-C10 aryl)Ci- Ce alkyl or (5-10 membered heteroaryl)Ci-C₆ alkyl, -0(R^g) or -N(R^g)(R^h);

R^d is hydrogen or C1-C6 alkyl;

R^e is C1-C6 alkyl, C3-C6 cycloalkyl, C6-C10 aryl, 3-7 membered heterocycloalkyl, 5-10 membered heteroaryl, (C3-C6 cycloalkyl)Ci-C₆ alkyl, (3-7 membered heterocycloalkyl)Ci-C₆ alkyl, ( - C10 aryl)Ci-C₆ alkyl, (5-10 membered heteroaryl)Ci-C₆ alkyl, keto(C₆-Cio aryl) or keto(5-10 membered heteroaryl);

R^f is C1-C6 alkyl, C3-C6 cycloalkyl, 3-7 membered heterocycloalkyl, C6-C10 aryl, 5-10 membered heteroaryl, (C3-C6 cycloalkyl)Ci-C6 alkyl, (3-7 membered heterocycloalkyl)Ci-C6 alkyl, (O- C10 aryl)Ci-C₆ alkyl or (5-10 membered heteroaryl)Ci-C₆ alkyl; R^g is Ci-Ce alkyl, C3-C6 cycloalkyl, C6-C10 aryl, 3-7 membered heterocycloalkyl, 5-10 membered heteroaryl, (C3-C6 cycloalkyl)Ci-C6 alkyl, (3-7 membered heterocycloalkyl)Ci-C6 alkyl, (Ce- C10 aryl)Ci-C₆ alkyl or (5-10 membered heteroaryl)Ci-C₆ alkyl;

R^h is hydrogen, C1-C4 alkyl, -C(0)(R^6’); -C(0)NH₂, -C(0)NH(R^6’) or -C(0)N(R^6’)(R⁶”) with R^6’ and R⁶”, which are the same or different, represent a C1-C6 alkyl group;

wherein in the definitions of R¹’ and R²’, each of cycloalkyl, heterocycloalkyl, aryl and heteroaryl is optionally substituted by 1 to 3 groups being halogen, CN, alkyl, haloalkyl, alkoxy or haloalkoxy; and

wherein when any of the foregoing group contains an alkyl group, then said alkyl is a linear or branched acyclic alkyl group.

The compound of embodiment 40, wherein R¹ is hydrogen or methyl.

The compound of embodiment 40, wherein R¹ is hydrogen.

The compound of any one of embodiments 40 to 42, wherein R²’ represents -C(R^a)(R^b)(R^c), - C(R^a)(R^b)0(R^g), -C(R^a)(R^b)N(R^g)(R^h), -N(R^d)(R^e) or -0(R^f)_, wherein:

R^a is H;

R^b is H or methyl;

R^c is a group benzyl, cyclobutyl, cyclopentyl, cyclohexyl, phenyl, thienyl, furanyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl, isothiazolyl, thiadiazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, indolizinyl, purinyl, naphthyridinyl, pteridinyl, [l,3]dioxolane, pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, tetrahydrofuryl or tetrahydropyranyl, each group R^c being optionally substituted by 1 to 3 groups being halogen, CN, alkyl, haloalkyl, alkoxy or haloalkoxy;

R^d is hydrogen or methyl;

R^e is a group cyclobutyl, cyclopentyl, cyclohexyl, phenyl, thienyl, furanyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl, isothiazolyl, thiadiazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, indolizinyl, purinyl, naphthyridinyl, pteridinyl, [l,3]dioxolane, pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl,

imidazolidinyl, piperidinyl, piperazinyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, tetrahydrofuryl, or tetrahydropyranyl, each of these groups being optionally substituted by 1 to 3 groups being halogen, CN, alkyl, haloalkyl, alkoxy or haloalkoxy, or R^e is a group -CH(R^h)(C3-C6 aryl) or -CH(R^h)(5-10 membered heteroaryl);

R^f is C3-C6 cycloalkyl, C6-C10 aryl, 3-10 membered heterocycloalkyl, 5-10 membered heteroaryl, -CH(R^h)(C3-C6 cycloalkyl), -CH(R^h)(C3-C6aryl), -CH(R^h)(3-7 membered heterocycloalkyl) or -CH(R^h)(5-10 membered heteroaryl);

R^g is a group methyl, ethyl, propyl, cyclobutyl, cyclopentyl, cyclohexyl, phenyl, thienyl, furanyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl, isothiazolyl, thiadiazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, indolizinyl, purinyl, naphthyridinyl, pteridinyl, [l,3]dioxolane, pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, tetrahydrofuryl or tetrahydropyranyl, each group R^g being optionally substituted by 1 to 3 groups being halogen, CN, alkyl, haloalkyl, alkoxy or haloalkoxy;

R^h is hydrogen, linear or branched C₁-C₄ alkyl or -(CO)R⁶ with R⁶ being a C₁-C₃ alkyl group; and wherein each of cycloalkyl, heterocycloalkyl, aryl and heteroaryl is optionally substituted by 1 to 3 groups being halogen, CN, alkyl, haloalkyl, alkoxy or haloalkoxy.

44. The compound of any one of embodiments 40 to 42, wherein R² represents -C(R^a)(R^b)(R^c), - C(R^a)(R^b)0(R^g), -C(R^a)(R^b)N(R^g)(R^h), -N(R^d)(R^e) or -0(R^f)_, wherein:

R^a is H;

R^b is H or methyl;

R^c is a group phenyl, benzyl, piperidinyl, tetrahydropyranyl or pyridyl;

R^d is hydrogen or methyl;

R^e is cyclohexyl, phenyl, benzyl, pyridyl, tetrahydropyranyl, -CH₂(pyridyl) or

CH(CH₃) (py ridy 1) ;

R^f is phenyl, cyclobutyl, cyclopentyl, cyclohexyl, benzyl, -CH₂(pyridyl), -CH(CH₃)(pyridyl), -CH₂(pyrazinyl), -CH₂(pyrimidinyl), -CH₂(tetrahydropyranyl), -CH₂(cyclopentyl), CH₂(cyclohexyl), -CH₂(oxazolyl) or -CH₂(thiazolyl);

R^g is ethyl, phenyl, cyclobutyl, cyclopentyl, cyclohexyl or tetrahydropyranyl;

R^h is hydrogen, methyl or acetyl;

wherein each of R^c, R^e, R^f and R^g is optionally substituted by a 1 to 3 groups being halogen, CN, C1-C6 alkyl or C1-C6 alkoxy.

45. The compound of any one of embodiments 40 to 42, wherein R² represents a group:

6. The compoimd of any one of embodiments 40 to 42, wherein R² represents a group:

47. The compound of any one of embodiments 40 to 42, wherein R² represents a group:

48. The compound of embodiment 40, wherein the compound of Formula B is selected from

Compounds B1 to B55 of Fig. 14, or a pharmaceutically acceptable salt, solvate, or prodrug thereof.

In some embodiments, the BET and CBP/EP300 co-inhibition method, treatments and uses described herein may comprise administration of a combination of compounds having BET and

CBP/EP300 dual inhibition activity. Hence, in some aspects, the BET and CBP/EP300 co-inhibition method, treatments and uses described herein may comprise administration of at least one substituted benzimidazole as defined in one or more of the above embodiments 1 to 39 in combination with at least one aryl-substituted dihydroquinolinone as defined in one or more of the above embodiments 40 to 48. Biomarkers indicative of responsiveness to BET inhibitor and CBP/EP300 inhibitor co-therapy

In some embodiments, described herein are methods of identifying a subject suitable for (or predicted to be responsive to, or predicted to be candidates of) anti-cancer co-therapy with a BET inhibitor and a CBP/EP300 inhibitor. The methods may comprise: (a) providing a biological sample comprising cancer cells from the subject (e.g., biological tissue previously obtained from surgery to remove, for example, part or all of an organ or tumor; or from a blood or tissue biopsy sample). The methods may further comprise (b) measuring the presence or absence of a biomarker in the cancer cells, in which the biomarker is indicative of the responsiveness of the cancer cells to BET inhibitor and CBP/EP300 inhibitor co-therapy. In some embodiments, biomarkers described herein may comprise overexpression of BET protein(s) (or other protein substrates (e.g., Activating Transcription Factor 2 (ATF2); Ma et al., 2018) that are ubiquitinated by wild type SPOP and targeted for degradation) in the cancer cells relative to corresponding non-cancerous cells, preferably one or more BET proteins that is/are substrates of, or is targeted for, degradation by SPOP (e.g., BRD2, BRD3, and/or BRD4). In some embodiments, biomarkers described herein may comprise expression of an SPOP variant in the cancer cells that impairs SPOP -mediated BET protein degradation (e.g., SPOP variants defective or impaired for substrate binding (e.g., BET protein binding), or SPOP variants that are defective or impaired for SPOP protein dimerization or oligomerization, thereby negatively affecting their ability to mediate BET protein degradation). In some embodiments, biomarkers described herein may comprise overexpression of a histone acetyltransferase (HAT) protein such as P300, as compared to non-cancerous cells, which have been reported to be elevated or upregulated in SPOP -mutated prostate cancer (Blattner et al., 2017). In some embodiments, biomarkers described herein may comprise a deletion or loss of function mutation of NCOR2 and/or overexpression of the deubiquitinase DUB3, which have been linked to BRD4 upregulation and BET inhibitor resistance (Jin et al., 2018). In some embodiments, biomarkers described herein may comprise the absence of a TMPRSS2 ETS gene fusion event, or the absence of alterations in EZH1 and ZNF148, which have been shown to be mutually exclusive in prostate tumors with SPOP mutations (Barbieri et al., 2012; Wei et al., 2018).

In some embodiments, the methods described herein may further comprise (c) identifying the subject as being suitable for (or predicted to be responsive to, or predicted to be candidates of) anti -cancer co-therapy with a BET inhibitor and a CBP/EP300 inhibitor when the cancer cells are positive for the biomarker. Conversely, in some embodiments, the methods described herein may further comprise (d) disqualifying the subject as being suitable for (or predicted to be responsive to, or predicted to be candidates of) anti-cancer co-therapy with a BET inhibitor and a CBP/EP300 inhibitor when the cancer cells are not positive for a biomarker as described herein.

In some embodiments, the biomarkers described herein may be measured by determining whether the subject’s cancer cells express the SPOP variant by genotyping the cancer cells for the presence of a mutant SPOP gene encoding the SPOP variant, and/or measuring BET protein expression levels in the cancer cells. In some embodiments, the biomarkers described herein may be detected or measured using an antibody specific for the SPOP variant as described herein. In some embodiments, the SPOP variants described herein may be evaluated in vitro for substrate binding activity and/or ubiquitin-transfer activity.

In some embodiments, the methods described herein relating to biomarker detection may further comprise treating the subjects which are positive for a biomarker as described herein. In some embodiments, the treatment comprises administering a therapeutically effective amount of a BET inhibitor and a CBP/EP300 inhibitor to the subject when the subject’s cancer cells are positive for the biomarker. In some embodiments, the BET inhibitor and the CBP/EP300 inhibitor are separate molecules as defined herein. In preferred embodiments, the BET inhibitor and the CBP/EP300 inhibitor are the same compound, and said compound is a compound as described herein.

Compositions and uses thereof

In some embodiments, described herein are compositions comprising, for example as separate molecules, a BET inhibitor as defined herein and a CBP/EP300 inhibitor as defined herein. In some embodiments, the compositions described herein may comprise a substituted benzimidazole as described herein, and/or an aryl-substituted dihydroquinolinone as described herein. In some embodiments, the compositions are pharmaceutical compositions comprising one or more pharmaceutically acceptable excipients.

In some embodiments, the compositions and compounds described herein are for use in the treatment of BET inhibitor resistant cancer cells in a subject. In some embodiments, described herein is the use of: (i) a BET inhibitor (e.g., as described herein) and a CBP/EP300 inhibitor (e.g., as described herein) as separate molecules; or (ii) a compound as described herein (e.g., a substituted benzimidazole as described herein, and/or an aryl-substituted dihydroquinolinone as described herein) for treating BET inhibitor-resistant cancer cells in a subject, or for the manufacture of a medicament for treating BET inhibitor-resistant cancer cells in a subject.

In some embodiments, the BET inhibitor-resistant cancer cells are positive for a biomarker as described herein, such as overexpressing BET protein substrates of SPOP relative to corresponding non- cancerous cells, and/or expressing an SPOP variant that impairs SPOP-mediated BET protein degradation as described herein.

In some embodiments, the BET inhibitor-resistant cancer cells treated may be positive for a biomarker as described herein, such as the cancer cells may overexpress BET protein substrates of SPOP relative to corresponding non-cancerous cells, and/or express an SPOP variant that impairs SPOP- mediated BET protein degradation (e.g., an SPOP variant as defined herein).

For greater clarity, as used herein unless otherwise indicated, the expressions“positive for a biomarker”,“having a biomarker”, or expressions relating to the fact that diseased cells overexpress or have elevated expression of a biomarker (e.g., overexpress BET proteins), relates to the presence of that particular biomarker in the diseased tissue, regardless of whether the biomarker is detected (e.g., in a diagnostic assay). In some embodiments, BET inhibitor resistant cancer cells treated may be from a type of cancer as described herein, such as solid cancers (solid tumors) or cancer cells from solid cancers, a sarcoma, a carcinoma, a lymphoma, melanoma, glioma, prostate cancer, lung cancer, breast cancer, gastric cancer, liver cancer, colon cancer, tongue cancer, thyroid cancer, renal cancer, uterine cancer, cervical cancer, ovarian cancer, or pancreatic cancer, or more particularly BET inhibitor-resistant solid cancers. In some embodiments, the subject’s cancer cells or cancer tissue is assessed or assayed for the presence of a biomarker as described herein, prior to, during, and/or after being treated with a compound or composition, or method of treatment, as described herein.

In some aspects, the present description relates to compounds as described herein (e.g., a substituted benzimidazole as described herein, and/or an aryl-substituted dihydroquinolinone as described herein) for use as a CBP/EP300 inhibitor in a CBP/EP300 inhibition therapy. In some aspects, described herein is the use of the compound as described herein (e.g., a substituted benzimidazole as described herein, and/or an aryl-substituted dihydroquinolinone as described herein) as a CBP/EP300 inhibitor in a CBP/EP300 inhibition therapy. In some embodiments, the CBP/EP300 inhibition therapy does not comprise an additional CBP/EP300 inhibitor (or an additional BET inhibitor) other than the compound described herein.

In some aspects, the present description relates to compounds as described herein for use as a BET inhibitor and a CBP/EP300 inhibitor in a BET and CBP/EP300 co-inhibition therapy. In some embodiments, the BET and CBP/EP300 co-inhibition therapy does not comprise an additional

CBP/EP300 inhibitor other than said compound.

In some embodiments, the compositions, compounds, treatment methods, and uses described herein may be employed for the treatment of a disease ameliorated by BET and CBP/EP300 co-inhibition therapy (e.g., chronic autoimmune or inflammatory conditions).

In some embodiments, the present description relates to the use of a compound as described herein as both a BET inhibitor and a CBP/EP300 inhibitor in a BET and CBP/EP300 co-inhibition therapy. In some embodiments, the BET and CBP/EP300 co-inhibition therapy does not comprise an additional CBP/EP300 inhibitor other than said compound, and/or does not comprise a BET inhibitor other than said compound.

In some aspects, the present description relates to the use of a compound as described herein for the manufacture of a medicament for CBP/EP300 inhibition therapy, wherein the CBP/EP300 inhibition therapy does not comprise an additional CBP/EP300 inhibitor other than said compound. In some aspects, the present description relates to the use of a compound as described herein for the manufacture of a medicament for BET and CBP/EP300 co-inhibition therapy, wherein the BET and CBP/EP300 co inhibition therapy does not comprise a BET inhibitor nor a CBP/EP300 inhibitor other than said compound.

In some embodiments, compositions, uses or treatment methods described herein may further comprise one or more drugs. In some embodiments, the compositions, uses or treatment methods described herein may be intended to treat prostate cancer and the one or more further drugs may be selected from drugs approved for prostate cancer, such as Abiraterone Acetate (e.g. Zytiga™),

Apalutamide (e.g. Erleada™), Bicalutamide (e.g. Casodex™), Cabazitaxel (e.g. Jevtana™), Degarelix (e.g. Firmagon™), Docetaxel (e.g. Taxotere™), Leuprolide Acetate (e.g. Eligard™, Lupron™, Lupron Depot™), Enzalutamide (e.g. Xtandi™), Flutamide, Goserelin Acetate (e.g. Zoladex™), Mitoxantrone Hydrochloride, Nilutamide (e.g. Nilandron™), Provenge (Sipuleucel-T™) and Radium 223 Dichloride (e.g. Xofigo™).

Definitions

All technical and scientific terms used herein have the same meaning as commonly understood by one ordinary skilled in the art to which the present technology pertains. For convenience, the meaning of certain terms and phrases used herein are provided below.

To the extent the definitions of terms in the publications, patents, and patent applications incorporated herein by reference are contrary to the definitions set forth in this specification, the definitions in this specification control. The section headings used herein are for organizational purposes only, and are not to be construed as limiting the subject matter disclosed.

The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. It should be noted that, the singular forms "a", "an", and "the" include plural forms as well, unless the content clearly dictates otherwise. Thus, for example, reference to a composition containing "a compound" also contemplates a mixture of two or more compounds. It should also be noted that the term "or" is generally employed in its sense including "and/or" unless the content clearly dictates otherwise. Furthermore, to the extent that the terms“including”, "includes", "having", "has", "with", or variants thereof are used in either the detailed description and/or the claims, such terms are intended to be inclusive in a manner similar to the term "comprising”.

The term "about" means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, "about" can mean within 1 or more than 1 standard deviation, per the practice in the art. Alternatively, "about" can mean a range of up to 20%, preferably up to 10%, more preferably up to 5%, and more preferably still up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5 -fold, and more preferably within 2-fold, of a value. Where particular values are described, unless otherwise stated the term "about" meaning within an acceptable error range for the particular value should be assumed.

As used herein, the terms "compounds herein described", "compounds of the present application" and equivalent expressions refer to compounds described in the present application, e.g., those encompassed by structural Formulae such as Formula A, A.II, A.III, A.IV, A.V and B, optionally with reference to any of the applicable embodiments, and also includes exemplary compounds, for example, Compounds A1-A67 of Fig. 13 and Compounds B1-B55 of Fig. 14, as well as their pharmaceutically acceptable salts, solvates, esters, and prodrugs when applicable. When a zwitterionic form is possible, the compound may be drawn as its neutral form for practical purposes, but the compound is understood to also include its zwitterionic form. Embodiments herein may also exclude one or more of the compounds. Compounds may be identified either by their chemical structure or their chemical name. In a case where the chemical structure and chemical name would conflict, the chemical structure will prevail.

Unless otherwise stated, structures depicted herein are also meant to include all isomeric (e.g., enantiomeric, diastereomeric, and geometric (or conformational)) forms of the structure; for example, the R and S configurations for each asymmetric center, Z and E double bond isomers, and Z and E conformational isomers. Therefore, single stereochemical isomers as well as enantiomeric,

diastereomeric, and geometric (or conformational) mixtures of the present compounds are within the scope of the present description. Unless otherwise stated, all tautomeric forms of the compounds are within the scope of the present description. Additionally, unless otherwise stated, structures depicted herein are also meant to include compounds that differ only in the presence of one or more isotopically enriched atoms. For example, compounds having the present structures including the replacement of hydrogen by deuterium or tritium, or the replacement of a carbon by a ¹³C- or ¹⁴C-enriched carbon are within the scope of the present description. Such compounds are useful, for example, as analytical tools, as probes in biological assays, or as therapeutic agents in accordance with the present description.

Where a particular enantiomer is preferred, it may, in some embodiments be provided substantially free of the corresponding enantiomer, and may also be referred to as "optically enriched." "Optically -emiched," as used herein, means that the compound is made up of a significantly greater proportion of one enantiomer. In certain embodiments the compound is made up of at least about 90% by weight of a preferred enantiomer. In other embodiments the compound is made up of at least about 95%, 98%, or 99% by weight of a preferred enantiomer. Preferred enantiomers may be isolated from racemic mixtures by any method known to those skilled in the art, including chiral high pressure liquid chromatography (HPLC) and the formation and crystallization of chiral salts or prepared by asymmetric syntheses. See, for example, Jacques et ak, Enantiomers, Racemates and Resolutions (Wiley Interscience, New York, 1981); Wilen, et ak, Tetrahedron 33:2725 (1977); Eliel, E.L. Stereochemistry of Carbon Compounds (McGraw-Hill, NY, 1962); Wilen, S.H. Tables of Resolving Agents and Optical Resolutions, p. 268 (E.L. Eliel, Ed., Univ. of Notre Dame Press, Notre Dame, IN 1972).

Definitions of specific functional groups and chemical terms are described in more detail below. For purposes of the present description, the chemical elements are identified in accordance with the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75^th, Ed., inside cover, and specific functional groups are generally defined as described therein. Additionally, general principles of organic chemistry, as well as specific functional moieties and reactivity, are described in Organic Chemistry, Thomas Sorrell, University Science Books, Sausalito, 1999; Smith and March March's Advanced Organic Chemistry, 5^th, Edition, John Wiley & Sons, Inc., New York, 2001; Larock, Comprehensive Organic Transformations, VCH Publishers, Inc., New York, 1989; Carruthers, Some Modem Methods of Organic Synthesis, 3^rd Edition, Cambridge University Press, Cambridge, 1987. The number of carbon atoms in a hydrocarbyl substituent can be indicated by the prefix "C_x-C_y," where x is the minimum and y is the maximum number of carbon atoms in the substituent. However, when the prefix“C_x-C_y” is associated with a group incorporating one or more heteroatom(s) by definition (e.g. heterocycloalkyl, heteroaryl, etc.), then x and y define respectively the minimum and maximum number of atoms in the cycle, including carbons as well as heteroatom(s).

The prefix "halo" indicates that the substituent to which the prefix is attached is substituted with one or more independently selected halogen radicals. More specifically, the terms "halo" and "halogen" as used herein refer to an atom selected from fluorine (fluoro, -F), chlorine (chloro, -Cl), bromine (bromo, - Br), and iodine (iodo, -I). For example, "haloalkyl" means an alkyl substituent wherein at least one hydrogen radical is replaced with a halogen radical.

The term "heteroatom" means one or more of oxygen, sulfur, nitrogen, phosphorus, or silicon (including, any oxidized form of nitrogen, sulfur, phosphorus, or silicon; the quatemized form of any basic nitrogen or; a substitutable nitrogen of a heterocyclic ring, for example N (as in 3,4-dihydro-2H- pyrrolyl), NH (as in pyrrolidinyl) or NR+ (as in N-substituted pyrrolidinyl).

Abbreviations may also be used throughout the application, unless otherwise noted, such abbreviations are intended to have the meaning generally understood by the field. Examples of such abbreviations include Me (methyl), Et (ethyl), Pr (propyl), i-Pr (isopropyl), Bu (butyl), t-Bu (tert-butyl), i- Bu (iso-butyl), s-Bu (sec-butyl), c-Bu (cyclobutyl), Ph (phenyl), Bn (benzyl), Bz (benzoyl), CBz or Cbz or Z (carbobenzyloxy), Boc or BOC (tert-butoxycarbonyl), and Su or Sue (succinimide). For greater certainty, examples of abbreviations used in the present application are listed in a table in the Examples section.

The chemical structures herein are drawn according to the conventional standards known in the art. Thus, where an atom, such as a carbon atom, as drawn appears to have an unsatisfied valency, then that valency is assumed to be satisfied by a hydrogen atom even though that hydrogen atom is not necessarily explicitly drawn. Hydrogen atoms should be inferred to be part of the compound.

The term "aliphatic" or "aliphatic group", as used herein, denotes a hydrocarbon moiety that may be straight-chain (i.e., unbranched), branched, or cyclic (including fused, bridging, and spiro-fused polycyclic) and may be completely saturated or may contain one or more units of unsaturation, but which is not aromatic. Unless otherwise specified, aliphatic groups contain 1-6 carbon atoms. In some embodiments, aliphatic groups contain 1-4 carbon atoms, and in yet other embodiments aliphatic groups contain 1-3 carbon atoms. Aliphatic groups include, but are not limited to, alkyl, alkenyl, alkynyl, carbocycle. Suitable aliphatic groups include, but are not limited to, linear or branched, alkyl, alkenyl, and alkynyl groups, and hybrids thereof such as (cycloalkyl)alkyl, (cycloalkenyl)alkyl or (cycloalkyl)alkenyl.

The term "alkyl" as used herein, refers to a saturated, straight- or branched-chain hydrocarbon radical typically containing from 1 to 20 carbon atoms. For example, "Ci-Cs alkyl" contains from one to eight carbon atoms. Examples of alkyl radicals include, but are not limited to, methyl, ethyl, propyl, isopropyl, w-butyl, tert- butyl, neopentyl, n-hexyl, heptyl, octyl radicals and the like.

The term "alkenyl" as used herein, denotes a straight- or branched-chain hydrocarbon radical containing one or more double bonds and typically from 2 to 20 carbon atoms. For example, "C2-C8 alkenyl" contains from two to eight carbon atoms. Alkenyl groups include, but are not limited to, for example, ethenyl, propenyl, butenyl, l-methyl-2-buten-l-yl, heptenyl, octenyl and the like.

The term "alkynyl" as used herein, denotes a straight- or branched-chain hydrocarbon radical containing one or more triple bonds and typically from 2 to 20 carbon atoms. For example, "C2-C8 alkynyl" contains from two to eight carbon atoms. Representative alkynyl groups include, but are not limited to, for example, ethynyl,l-propynyl, 1-butynyl, heptynyl, octynyl and the like.

The terms“cycloalkyl”,“alicyclic”,“carbocyclic” and equivalent expressions refer to a group comprising a saturated or partially unsaturated (non-aromatic) carbocyclic ring in a monocyclic or polycyclic ring system, including spiro (sharing one atom), fused (sharing at least one bond) or bridged (sharing two or more bonds) carbocyclic ring systems, having from three to fifteen ring members.

Examples of cycloalkyl groups include, without limitation, cyclopropyl, cyclobutyl, cyclopentyl, cyclopenten-l-yl, cyclopenten-2-yl, cyclopenten-3-yl, cyclohexyl, cyclohexen-l-yl, cyclohexen-2-yl, cyclohexen-3-yl, cycloheptyl, bicyclo[4,3,0]nonanyl, norbomyl, and the like. The term cycloalkyl includes both unsubstituted cycloalkyl groups and substituted cycloalkyl groups. The term“C3- C_ncycloalkyl” refers to a cycloalkyl group having from 3 to the indicated“n” number of carbon atoms in the ring structure. Unless the number of carbons is otherwise specified,“lower cycloalkyl” groups as herein used, have at least 3 and equal or less than 8 carbon atoms in their ring structure.

As used herein, the terms "heterocycle", "heterocycloalkyl", "heterocyclyl", "heterocyclic radical", and "heterocyclic ring" are used interchangeably and refer to a chemically stable 3- to 7- membered monocyclic or 7-10-membered bicyclic heterocyclic moiety that is either saturated or partially unsaturated, and having, in addition to carbon atoms, one or more, preferably one to four, heteroatoms, as defined above. When used in reference to a ring atom of a heterocycle, the term "nitrogen" includes a substituted nitrogen. As an example, in a saturated or partially unsaturated ring having 1-3 heteroatoms selected from oxygen, sulfur or nitrogen, the nitrogen may be N (as in 3,4-dihydro-2H-pyrrolyl), NH (as in pyrrolidinyl), or +NR (as in N-substituted pyrrolidinyl). A heterocyclic ring can be attached to its pendant group at any heteroatom or carbon atom that results in a chemically stable structure and any of the ring atoms can be optionally substituted. Examples of heterocycloalkyl groups include, but are not limited to, 1,3-dioxolanyl, pyrrolidinyl, pyrrolidonyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydrothiopyranyl, tetrahydrodithienyl, tetrahydrothienyl, thiomorpholino, thioxanyl, azetidinyl, oxetanyl, thietanyl, homopiperidinyl, oxepanyl, thiepanyl, oxazepinyl, diazepinyl, thiazepinyl, 1,2,3,6-tetrahydropyridinyl, 2-pyrrolinyl, 3-pyrrolinyl, 2H- pyranyl, 4H-pyranyl, dioxanyl, dithianyl, dithiolanyl, dihydropyranyl, dihydrothienyl, dihydrofuranyl, 3- azabicyclo[3,l,0]hexanyl, 3-azabicyclo[4,l,0]heptanyl, quinolizinyl, quinuclidinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl, and the like. Heterocyclic groups also include groups in which a heterocyclic ring is fused to one or more aryl, heteroaryl, or cycloaliphatic rings, such as indolinyl, 3H-indolyl, chromanyl, chromenyl, phenanthridinyl, 2-azabicyclo[2.2.1]heptanyl, octahydroindolyl, or tetrahydroquinolinyl, where the radical or point of attachment is on the heterocyclyl ring. A heterocyclyl group may be mono- or bicyclic. The term "heterocyclylalkyl" refers to an alkyl group substituted by a heterocyclyl, wherein the alkyl and heterocyclyl portions independently are optionally substituted.

As used herein, the term "partially unsaturated" refers to a ring moiety that includes at least one double or triple bond between ring atoms but is not aromatic. The term "partially unsaturated" is intended to encompass rings having multiple sites of unsaturation, but is not intended to include aryl or heteroaryl moieties, as herein defined.

The term "aryl" used alone or as part of a larger moiety as in "aralkyl", "aralkoxy", or

"aryloxyalkyl", refers to a monocyclic moiety or to a bicyclic or tricyclic fused ring system having a total of six to 15 ring members, wherein at least one ring in the system is aromatic and wherein each ring in the system contains three to seven ring members. The term "aryl" may be used interchangeably with the term "aryl ring". In certain embodiments of the present description, "aryl" refers to an aromatic ring system which includes, but not limited to, phenyl, biphenyl, naphthyl, azulenyl, anthracyl and the like, which may bear one or more substituents. The term "aralkyl" or "arylalkyl" refers to an alkyl residue attached to an aryl ring. Examples of aralkyl include, but are not limited to, benzyl, phenethyl, 1-phenylethyl, and the like. Also included within the scope of the term“aryl”, as it is used herein, is a group in which an aromatic ring is fused to one or more non-aromatic rings, such as indanyl, indenyl, phthalimidyl, naphthimidyl, fluorenyl, phenanthridinyl, or tetrahydronaphthyl, and the like.

The terms "heteroaryl" and "heteroar-", used alone or as part of a larger moiety, e.g.,

"heteroaralkyl", or "heteroaralkoxy", refer to groups having 5 to 18 ring atoms, preferably 5, 6, or 9 ring atoms; having 6, 10, or 14 p electrons shared in a cyclic array; and having, in addition to carbon atoms, from one to five heteroatoms. The term "heteroatom" includes but is not limited to nitrogen, oxygen, or sulfur, and includes any oxidized form of nitrogen or sulfur, and any quaternized form of a basic nitrogen. A heteroaryl may be a single ring, or two or more fused rings. Heteroaryl groups include, without limitation, thienyl, furanyl (furyl), thienyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl, isothiazolyl, thiadiazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, furopyridinyl, indolizinyl, purinyl, naphthyridinyl, and pteridinyl. The terms "heteroaryl" and "heteroar-", as used herein, also include groups in which a heteroaromatic ring is fused to one or more aryl, cycloaliphatic, or heterocyclyl rings, where the radical or point of attachment is on the

heteroaromatic ring. Nonlimiting examples include indolyl, 3H-indolyl, isoindolyl, benzothienyl (benzothiophenyl), benzofuranyl, dibenzofuranyl, indazolyl, benzimidazolyl, benzoxazolyl,

benzothiazolyl, quinolyl (quinolinyl), isoquinolyl (isoquinolinyl), quinolonyl, isoquinolonyl, cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, 4H-quinolizinyl, carbazolyl, acridinyl, phenanthridinyl, phenazinyl, phenothiazinyl, phenoxazinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, and pyrido[2,3- b]-l,4-oxazin-3(4H)-one. A heteroaryl group may be mono- or bicyclic. Heteroaryl groups include rings that are optionally substituted. The term "heteroaralkyl" refers to an alkyl group substituted by a heteroaryl, wherein the alkyl and heteroaryl portions independently are optionally substituted. Examples include, but are not limited to, pyridinylmethyl, pyrimidinylethyl and the like. The term "alkylene" refers to a divalent group derived from a straight or branched saturated hydrocarbyl chain typically containing from 1 to 20 carbon atoms, more typically from 1 to 8 carbon atoms. Examples of an "alkylene" include a polymethylene group, i.e., -(CEhV, wherein n is a positive integer, preferably from 1 to 6, from 1 to 4, from 1 to 3, from 1 to 2, or from 2 to 3; or -CEE-, -CH2CH2-, - CH2CH2CH2-, -CH2CH2CH2CH2-, and -CH₂CH(CH₃)CH2-. A substituted alkylene chain is a

polymethylene group in which one or more methylene hydrogen atoms are replaced with a substituent. Suitable substituents include those described below for a substituted aliphatic group.

The term "alkenylene" refers to a divalent unsaturated hydrocarbyl group which may be linear or branched and which has at least one carbon-carbon double bond. An alkenylene group typically contains 2 to 20 carbon atoms, more typically from 2 to 8 carbon atoms. Non-limiting examples of alkenylene groups include -C(H)=C(H)-, -C(H)=C(H)-CH₂-, -C(H)=C(H)-CH₂-CH₂-, -CH₂-C(H)=C(H)-CH₂-, - C(H)=C(H)-CH(CH₃)-, and -CH₂-C(H)=C(H)-CH(CH₂CH₃)-.

The term "alkynylene" refers to a divalent unsaturated hydrocarbon group which may be linear or branched and which has at least one carbon-carbon triple bond. Examples of alkynylene groups include, without limitation, -CºC-, -CºC-CH₂-, -CºC-CH₂-CH₂-, -CH₂-CºC-CH₂-, -CºC-CH(CH₃)-, and -CH₂- CºC-CH(CH₂CH₃)-.

As described herein, compounds of the present description may contain "optionally substituted" moieties. In general, the term "substituted", whether preceded by the term "optionally" or not, means that one or more hydrogens of the designated moiety are replaced with a suitable substituent. Unless otherwise indicated, an "optionally substituted" group may have a suitable substituent at each substitutable position of the group, and when more than one position in any given structure may be substituted with more than one substituent selected from a specified group, the substituent may be either the same or different at each position. Combinations of substituents envisioned under the present description are preferably those that result in the formation of chemically stable or chemically feasible compounds. The term "chemically stable", as used herein, refers to compounds that are not substantially altered when subjected to conditions to allow for their production, detection, and, in certain embodiments, their recovery, purification, and use for one or more of the purposes disclosed herein.

When an alkyl, alkenyl, alkynyl, carbocyclic, aryl, heteroaryl, heterocyclic, or any other group as used herein, is substituted or optionally substituted, this refers to a group that is substituted or unsubstituted by independent replacement of one, two, or three or more of the hydrogen atoms thereon with substituents including, but not limited to F, Cl, Br, I, OH, CO2H, alkoxy, oxo, thiooxo, NO2, CN, CF₃, N¾, protected amino, NHalkyl, NHalkenyl, NHalkynyl, NHcycloalkyl, NHaryl, NHheteroaryl, NHheterocyclic, dialkylamino, diarylamino, diheteroarylamino, O-alkyl, O-alkenyl, O-alkynyl, O- cycloalkyl, O-aryl, O-heteroaryl, O-haloalkyl, O-heterocyclic, C(0)alkyl, C(0)alkenyl, C(0)alkynyl, C(0)cycloalkyl, C(0)aryl, C(0)heteroaryl, C(0)heterocyeloalkyl, C0₂alkyl, C0₂alkenyl, C0₂alkynyl, C0₂cycloalkyl, C0₃aryl, C0₂heteroaryl, C0₂heterocycloalkyl, OC(0)alkyl, OC(0)alkenyl,

OC(0)alkynyl, OC(0)cycloalkyl, OC(0)aryl, OC(0)heteroaryl, OC(0)heterocycloalkyl, C(0)NH₂, C(0)NHalkyl, C(0)NHalkenyl, C(0)NHalkynyl, C(0)NHcycloalkyl, C(0)NHaryl, C(0)NHheteroaryl, C(0)NHheterocycloalkyl, 0C0₂alkyl, OCCkalkenyl, 0C0₂alkynyl, OC0₂cycloalkyl, 0C0₂aryl, OC0₂heteroaryl, OCCkheterocycloalkyl, OC(0)NH₂, OC(0)NHalkyl, OC(0)NHalkenyl,

OC(0)NHalkynyl, OC(0)NHcycloalkyl, OC(0)NHaryl, OC(0)NHheteroaryl,

OC(0)NHheterocycloalkyl, NHC(0)alkyl, NHC(0)alkenyl, NHC(0)alkynyl, NHC(0)cycloalkyl, NHC(0)aryl, NHC(0)heteroaryl, NHC(0)heterocycloalkyl, NHC0₂alkyl, NHC0₂alkenyl,

NHC0₂alkynyl, NHC0₂cycloalkyl, NHCCkaryl, NHC0₂heteroaryl, NHC0₂heterocycloalkyl,

NHC(0)NH₂, NHC(0)NHalkyl, NHC(0)NHalkenyl, NHC(0)NHalkenyl, NHC(0)NHcycloalkyl, NHC(0)NHaryl, NHC(0)NHheteroaryl, NHC(0)NHheterocycloalkyl, NHC(S)NH₂, NHC(S)NHalkyl, NHC(S)NHalkenyl, NHC(S)NHalkynyl, NHC(S)NHcycloalkyl, NHC(S)NHaryl, NHC(S)NHheteroaryl, NHC(S)NHheterocycloalkyl, NHC(NH)NH₂, NHC(NH)NHalkyl, NHC(NH)NHalkenyl,

NHC(NH)NHalkenyl, NHC(NH)NHcycloalkyl, NHC(NH)NHaryl, NHC(NH)NHheteroaryl,

NHC(NH)NHheterocycloalkyl, NHC(NH)alkyl, NHC(NH)alkenyl, NHC(NH)alkenyl,

NHC(NH)cycloalkyl, NHC(NH)aryl, NHC(NH)heteroaryl, NHC(NH)heterocycloalkyl, C(NH)NHalkyl, C(NH)NHalkenyl, C(NH)NHalkynyl, C(NH)NHcycloalkyl, C(NH)NHaryl, C(NH)NHheteroaryl, C(NH)NHheterocycloalkyl, S(0)alkyl, S(0)alkenyl, S(0)alkynyl, S(0)cycloalkyl, S(0)aryl, S(0)₂alkyl, S(0)₂alkenyl, S(0)₂alkynyl, S(0)₂cycloalkyl, S(0)₂aryl, S(0)heteroaryl, S(0)heterocycloalkyl, SO2NH2, S0₂NHalkyl, SCkNHalkenyl, S0₂NHalkynyl, S0₂NHcycloalkyl, S0₂NHaryl, S0₂NHheteroaryl, SCkNHheterocycloalkyl, NHS0₂alkyl, NHS0₂alkenyl, NHS0₂alkynyl, NHS0₂cycloalkyl, NHSC aryl, NHS0₂heteroaryl, NHS0₂heterocycloalkyl, CH₂NH₂, CH2SO2CH3, alkyl, alkenyl, alkynyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, heterocycloalkyl, cycloalkyl, carbocyclic, heterocyclic, polyalkoxyalkyl, polyalkoxy, methoxymethoxy, methoxyethoxy, SH, S-alkyl, S-alkenyl, S-alkynyl, S- cycloalkyl, S-aryl, S-heteroaryl, S-heterocycloalkyl, or methylthiomethyl.

In certain embodiments, suitable monovalent substituents on a substitutable carbon atom of a substituted or optionally substituted group are independently halogen; (CH₂)o-₄R°; (CH₂)o-₄0R°; 0(CH₂)o- ₄C(0)OR°; (CH₂)_O-4CH(OR⁰)₂; (CH₂)_O-4SR°; (CH₂)_0-4Ph, which may be substituted with R°; (CH₂)o- ₄0(CH₂)o-₄Ph which may be substituted with R°; -CH=CHPh, which may be substituted with R°; NO₂; CN; N₃; (CH₂) _O-4N(R⁰)₂; (CH₂)_O-4N(R⁰)C(0)R°; N(R°)C(S)R°; (CH₂)_O-4N(R⁰)C(0)NR⁰ ₂;

N(R°)C(S)NR°₂; (CH₂)O-₄N(R°)C(0)OR°; N(R°)N(R°)C(0)R°; N(R°)N(R⁰)C(0)NR⁰ ₂;

N (R°)N (R°)C(0)OR° ; (CH₂)o-₄C(0)R°; C(S)R°; (CH₂)_0-4C(O)OR°; (CH₂)_0-4C(O)SR°; (CH₂)o- ₄C(0)0SiR°₃; (CH₂)O-₄0C(0)R°; OC(0)(CH₂) _0-4SR-, SC(S)SR°; (CH₂)_0-4SC(O)R°; (CH₂)o-₄C(0)NR⁰ ₂; C(S)NR°₂; C(S)SR°; SC(S)SR°, (CH₂)O-₄0C(0)NR⁰ ₂; C(0)N(OR°)R°; C(0)C(0)R°; C(0)CH₂C(0)R°; C(NOR°)R°; (CH₂)O-₄SSR°; (CH₂)O-₄S(0)₂R⁰; (CH₂)O-₄(0)₂OR⁰; (CH₂)O-₄0S(0)₂R⁰; S(0)₂NR°₂; (CH₂)O- ₄S(0)R°; N(R⁰)S(0)₂NR⁰ ₂; N(R°)S(0)₂R°; N(OR°)R°; C(NH)NR°₂; P(0)₂R°; P(0)R°₂; OP(0)R°₂; OP(0)(OR°)₂; SiR°₃; (straight or branched Ci-₄alkylene)0-N(R°)₂; or (straight or branched Ci- ₄alkylene)C(0)0-N(R°)₂, wherein each R° may be substituted as defined below and is independently hydrogen, Ci ^aliphatic, CH₂Ph, 0(CH₂)o-iPh, or a 5-6-membered saturated, partially unsaturated, or aromatic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or, notwithstanding the definition above, two independent occurrences of R°, taken together with their intervening atom(s), may form a 3 to 12 membered saturated, partially unsaturated, or aryl mono- or bicyclic ring having 0 to 4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, which may be substituted as defined below.

Examples of monovalent substituents on R° (or the ring formed by taking two independent occurrences of R° together with their intervening atoms), are independently halogen, -(CH₂)o-2R*, - (haloR*), -(CH₂)_O-2OH, -(CH₂)_0-2OR*, -(CH₂y₂CH(OR*)₂, -O(haloR'), -CN, -N₃, -(CH₂)_0-2C(O)R*, - (CH₂y₂C(0)0H, -(CH₂y₂C(0)0R*, -(CH₂)_O-2SR*, -(CH₂)_O-2SH, -(CH₂)_O-2NH₂, -(CH₂)_0-2NHR*, -(CH₂) o- ₂NR*2, -N0₂, -SiR*₃, -OSiR*₃, -C(0)SR* -(Ci-4Straight or branched alkylene)C(0)OR*, or -SSR*, wherein each R* is unsubstituted or where preceded by "halo" is substituted only with one or more halogens, and is independently selected from C1 -4 aliphatic, -CH₂Ph, -O(CH₂)₀-iPh, or a 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. Suitable divalent substituents on a saturated carbon atom of R° include =0 and =S.

Examples of divalent substituents on a saturated carbon atom of a substituted or an optionally substituted group include the following: =0, =S, =NNR*_¾ =NNHC(0)R*, =NNHC(0)0R*,

=N HS(0)₂R*, =NR*, =NOR*, -0(C(R*₂))₂-_3O-, or -S(C(R*₂))₂-₃S-, wherein each independent occurrence of R* is selected from hydrogen, C1 -6 aliphatic which may be substituted as defined below, or an unsubstituted 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. Suitable divalent substituents that are bound to vicinal substitutable carbons of a substituted or an optionally substituted group include: -0(CR₂)_2-3o-, wherein each independent occurrence of R is selected from hydrogen, Ci -₆ aliphatic which may be substituted as defined below, or an unsubstituted 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

Exemplary substituents on the aliphatic group of R* include halogen, -R*, -(haloR*), -OH, -OR*, -O(haloR'), -CN, -C(0)OH, -C(0)OR*, -NH_¾ -NHR*, -NR*_¾ or -NO_¾ wherein each R* is unsubstituted or where preceded by "halo" is substituted only with one or more halogens, and is independently C1-4 aliphatic, -CH₂Ph, -0(CH₂)o-iPh, or a 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

The expression "pharmaceutically acceptable salt" refers to those salts of the compounds formed by the process of the present description which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit/risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, S. M. Berge, et al. describes pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences , 66: 1-19 (1977). The salts can be prepared in situ during the final isolation and purification of the compounds of the present description, or separately by reacting a free base function of the compound with a suitable organic or inorganic acid (acid addition salts) or by reacting an acidic function of the compound with a suitable organic or inorganic base (base- addition salts). Examples of pharmaceutically acceptable salts include, but are not limited to, nontoxic acid addition salts, or salts of an amino group formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid or with organic acids such as acetic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid or by using other methods used in the art such as ion exchange. Other pharmaceutically acceptable salts include, but are not limited to, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy -ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate salts, and the like. Representative base addition alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, or magnesium salts, and the like. Further pharmaceutically acceptable salts include, when appropriate, nontoxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, sulfonate and aryl sulfonate.

The term“solvate” refers to a physical association of one of the present compounds with one or more solvent molecules. This physical association includes hydrogen bonding. In certain instances, the solvate will be capable of isolation, for example when one or more solvent molecules are incorporated in the crystal lattice of a crystalline solid.“Solvate” encompasses both solution-phase and isolable solvates. Exemplary solvates include, without limitation, hydrates, hemihydrates, ethanolates, hemiethanolates, n- propanolates, iso-propanolates, 1-butanolates, 2-butanolate, and solvates of other physiologically acceptable solvents, such as the Clas 3 solvents described in the International Conference on

Harmonization (ICH), Guide for Industry, Q3C Impurities: Residual Solvents (1997). The compounds as herein described also include each of their solvates and mixtures thereof.

As used herein, the term "pharmaceutically acceptable ester" refers to esters of the compounds formed by the process of the present description which hydrolyze in vivo and include those that break down readily in the human body to leave the parent compound or a salt thereof. Suitable ester groups include, for example, those derived from pharmaceutically acceptable aliphatic carboxylic acids, particularly alkanoic, alkenoic, cycloalkanoic and alkanedioic acids, in which each alkyl or alkenyl moiety advantageously has not more than 6 carbon atoms. Examples of particular esters include, but are not limited to, formates, acetates, propionates, butyrates, acrylates and ethylsuccinates.

The expressions "prodrug" and "pharmaceutically acceptable prodrug" as used herein refers to those prodrugs of the compounds formed by the process of the present description which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals with undue toxicity, irritation, allergic response, and the like, commensurate with a reasonable benefit/risk ratio, and effective for their intended use. "Prodrug", as used herein means a compound which is convertible in vivo by metabolic means (e.g. by hydrolysis) to afford any compound delineated by the formulae of the instantdescription. Various forms of prodrugs are known in the art, for example, as discussed in Bundgaard, (ed.), Design of Prodrugs, Elsevier (1985); Widder, et al. (ed.), Methods in Enzymology, vol. 4, Academic Press (1985); Krogsgaard-Larsen, et al., (ed). "Design and Application of Prodrags, Textbook of Drag Design and Development", Chapter 5, 113-191 (1991); Bundgaard, et al., Journal of Drug Deliver Reviews, 8: 1-38(1992); Bundgaard, /. of Pharmaceutical Sciences, 77:285 et seq. (1988); Higuchi and Stella (eds.) Prodrags as Novel Drag Delivery Systems, American Chemical Society (1975); and Bernard Testa & Joachim Mayer, "Hydrolysis In Drag And Prodrug Metabolism: Chemistry, Biochemistry And Enzymology", John Wiley and Sons, Ltd. (2002).

Combinations of substituents and variables envisioned by the present description are only those that result in the formation of stable compounds. The term "stable", as used herein, refers to compounds which possess stability sufficient to allow manufacture and which maintains the integrity of the compound for a sufficient period of time to be useful for the purposes detailed herein (e.g., therapeutic or prophylactic administration to a subject).

As mentioned above, examples of the benzimidazole compounds described in the present application are disclosed in WO 2017/024412 and examples of the aryl-substituted dihydroquinolinone compounds described in the present application are disclosed WO 2017/024408. These compounds may be prepared by conventional chemical synthesis, such as exemplified in WO 2017/024412 and WO 2017/024408, which are herein incorporated by reference.

The compounds of the present description may be modified by appending various functionalities via any synthetic means delineated herein to enhance selective biological properties. Such modifications are known in the art and include those which increase biological penetration into a given biological system (e.g., blood, lymphatic system, central nervous system), increase oral availability, increase solubility to allow administration by injection, alter metabolism and alter rate of excretion.

As used herein, the term "effective amount" means that amount of a drag or pharmaceutical agent that will elicit the biological or medical response of a tissue, system, animal or human that is being sought, for instance, by a researcher or clinician. Furthermore, the term "therapeutically effective amount" means any amount which, as compared to a corresponding subject who has not received such amount, results in treatment, healing, prevention, or amelioration of a disease, disorder, or side effect, or a decrease in the rate of advancement of a disease or disorder. The term also includes within its scope amounts effective to enhance normal physiological function.

As used herein, the terms "treatment," "treat," and "treating" refer to reversing, alleviating, delaying the onset of, or inhibiting the progress of a disease or disorder, or one or more symptoms thereof, as described herein. In some embodiments, treatment may be administered after one or more symptoms have developed. In other embodiments, treatment may be administered in the absence of symptoms. For example, treatment may be administered to a susceptible individual prior to the onset of symptoms (e.g., in light of a history of symptoms and/or in light of genetic or other susceptibility factors). Treatment may also be continued after symptoms have resolved, for example to prevent or delay their recurrence.

As used herein, the expressions“bromodomain and extraterminal protein (BET) inhibitor” and “BET inhibitor”, which can be used interchangeably, denote a compound which inhibits the binding of BET family bromodomains to acetylated lysine residues (e.g., acetylated lysine residues on histones, particularly histones H3 and H4.). The BET family of bromodomain containing proteins comprises at least four proteins (BRD2, BRD3, BRD4, and BRD-t), which contain tandem bromodomains capable of binding to two acetylated lysine residues in close proximity, increasing the specificity of the interaction. Thus, as used herein, the expression "BET inhibitor" is defined as a compound that binds to and inhibits the target bromodomain-containing protein (such as a BET protein, e.g., BRD2, BRD3, BRD4, and/or BRDT). In some embodiments, the BET inhibitor substantially or completely inhibits the biological activity of the one or more target BET proteins. In some embodiments, the biological activity is binding of the one or more BET proteins to chromatin (e.g., histones associated with DNA) and/or another acetylated protein. In some embodiments, the biological activity is histone acetylation by the one or more BET proteins. In some embodiments, an inhibitor has an IC50 or binding constant of less about 50 mM, less than about 1 mM, less than about 500 nM, less than about 100 nM, or less than about 10 nM, to BRD2, BRD3, BRD4, and/or BRD-t (e.g., using the binding affinity methods described herein).

The terms“CBP” and“CREB binding protein,” as used herein, refers to any native CBP from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise indicated. The term encompasses“full-length,” unprocessed CBP as well as any form of CBP that results from processing in the cell. The term also encompasses naturally occurring variants of CBP, e.g., splice variants or allelic variants. In some embodiments, the amino acid sequence of an exemplary human CBP is UNIPROT Q92793-1. In some embodiments, the amino acid sequence of an exemplary human CBP is UNIPROT Q92793-2.

As used herein, the term“CBP/EP300 inhibitor” refers to a compound that binds to the CBP and/or EP300 and inhibits and/or reduces a biological activity of CBP and/or EP300. In some embodiments, CBP/EP300 inhibitor substantially or completely inhibits the biological activity of the CBP and/or EP300. In some embodiments, the biological activity is binding of the CBP and/or EP300 to chromatin (e.g., histones associated with DNA) and/or another acetylated protein. In some embodiments, the biological activity is histone acetylation by CBP and/or EP300. In some embodiments, an inhibitor has an IC50 or binding constant of less about 50 mM, less than about 1 mM, less than about 500 nM, less than about 100 nM, or less than about 10 nM (e.g., using the binding affinity methods described herein).

The terms“EP300”,“P300”, and“E1A binding protein p300” as used herein, refers to any native EP300 from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise indicated. The term encompasses“full-length,” unprocessed EP300 as well as any form of EP300 that results from processing in the cell. The term also encompasses naturally occurring variants of EP300, e.g., splice variants or allelic variants. In some embodiments, the amino acid sequence of an exemplary human EP300 is UNIPROT Q09472.

The term“patient” or“subject” as used herein refers to a mammal. A subject therefore refers to, for example, dogs, cats, horses, cows, pigs, guinea pigs, and the like. Preferably the subject is a human. When the subject is a human, the subject may be either a patient or a healthy human. In certain embodiments, a provided composition is formulated for administration to a subject or patient in need of such composition. In some embodiments, a provided composition is formulated for oral administration to a subject or patient.

In some embodiments, the therapeutically effective amount of a compound as defined herein can be administered to a subject or patient alone or admixed with a pharmaceutically acceptable carrier, adjuvant, or vehicle.

The expression "pharmaceutically acceptable excipient”,“pharmaceutically acceptable carrier”, “pharmaceutically acceptable adjuvant”, or“pharmaceutically acceptable vehicle" and equivalent expressions, refer to a non-toxic excipient, carrier, adjuvant, or vehicle that does not destroy the pharmacological activity of the compound with which it is formulated. Pharmaceutically acceptable excipient, carriers, adjuvants or vehicles that may be used in the compositions of this disclosure include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol and wool fat.

A "pharmaceutically acceptable derivative" means any non-toxic salt, ester, salt of an ester, prodrug, salt of a prodrug, or other derivative of a compound of the present description that, upon administration to a recipient, is capable of providing, either directly or indirectly, a compound of the present description or an inhibitory active metabolite or residue thereof.

Compositions and/or compounds described herein may be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir. The term "parenteral" as used herein includes subcutaneous, intravenous, intramuscular, intraarticular, intrasynovial, intrastemal, intrathecal, intrahepatic, intralesional and intracranial injection or infusion techniques. Other modes of administration also include intradermal or transdermal administration.

Liquid dosage forms for oral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active compounds, the liquid dosage forms may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3 -butylene glycol, dimethylformamide, oils (in particular, cottonseed, groundnut, com, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.

Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution, suspension or emulsion in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, U.S.P. and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil can be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid are used in the preparation of injectables.

Injectable formulations can be sterilized, for example, by filtration through a bacterial -retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.

In order to prolong the effect of a provided compound, it is often desirable to slow the absorption of the compound from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material with poor water solubility. The rate of absorption of the compound then depends upon its rate of dissolution that, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered compound form is accomplished by dissolving or suspending the compound in an oil vehicle. Injectable depot forms are made by forming microencapsule matrices of the compound in biodegradable polymers such as poly lactide -poly glycolide. Depending upon the ratio of compound to polymer and the nature of the particular polymer employed, the rate of compound release can be controlled.

Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by entrapping the compound in liposomes or microemulsions that are compatible with body tissues.

Compositions for rectal or vaginal administration are preferably suppositories which can be prepared by mixing the compounds of the present description with suitable non-irritating excipients or carriers such as cocoa butter, polyethylene glycol or a suppository wax which are solid at ambient temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the active compound.

Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active compound is mixed with at least one inert,

pharmaceutically acceptable excipient or carrier such as sodium citrate or dicalcium phosphate and/or a) fillers or extenders such as starches, lactose, sucrose, glucose, mannitol, and silicic acid, b) binders such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone (PVP), sucrose, and acacia, c) humectants such as glycerol, d) disintegrating agents such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate, e) solution retarding agents such as paraffin, f) absorption accelerators such as quaternary ammonium compounds, g) wetting agents such as, for example, cetyl alcohol and glycerol monostearate, h) absorbents such as kaolin and bentonite clay, and i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof. In the case of capsules, tablets and pills, the dosage form may also comprise buffering agents. Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like. The solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings and other coatings well known in the pharmaceutical formulating art. They may optionally contain opacifying agents and can also be of a composition that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes. Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like.

Provided compounds can also be in micro-encapsulated form with one or more excipients as noted above. The solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings, release controlling coatings and other coatings well known in the pharmaceutical formulating art. In such solid dosage forms the active compound may be admixed with at least one inert diluent such as sucrose, lactose or starch. Such dosage forms may also comprise, as is normal practice, additional substances other than inert diluents, e.g., tableting lubricants and other tableting aids such a magnesium stearate and microcrystalline cellulose. In the case of capsules, tablets and pills, the dosage forms may also comprise buffering agents. They may optionally contain opacifying agents and can also be of a composition that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner. Examples of embedding

compositions that can be used include polymeric substances and waxes.

Dosage forms for topical or transdermal administration of a compound of the present description include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants or patches. The active component is admixed under sterile conditions with a pharmaceutically acceptable carrier and any needed preservatives or buffers as may be required. Ophthalmic formulation, ear drops, and eye drops are also contemplated as being within the scope of the present description. Additionally, the description contemplates the use of transdermal patches, which have the added advantage of providing controlled delivery of a compound to the body. Such dosage forms can be made by dissolving or dispensing the compound in the proper medium. Absorption enhancers can also be used to increase the flux of the compound across the skin. The rate can be controlled by either providing a rate controlling membrane or by dispersing the compound in a polymer matrix or gel.

Pharmaceutically acceptable compositions provided herein may also be administered by nasal aerosol or inhalation. Such compositions are prepared according to techniques well-known in the art of pharmaceutical formulation and may be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promotors to enhance bioavailability, fluorocarbons, and/or other conventional solubilizing or dispersing agents.

Pharmaceutically acceptable compositions provided herein may be formulated for oral administration. Such formulations may be administered with or without food. In some embodiments, pharmaceutically acceptable compositions of this disclosure are administered without food. In other embodiments, pharmaceutically acceptable compositions of this disclosure are administered with food.

The amount of provided compounds that may be combined with carrier materials to produce a composition in a single dosage form will vary depending upon the subject or patient to be treated and the particular mode of administration. Provided compositions may be formulate such that a dosage of between 0.01 - 100 mg/kg body weight/day of the inhibitor can be administered to a subject or patient receiving these compositions.

It should also be understood that a specific dosage and treatment regimen for any particular subject or patient will depend upon a variety of factors, including age, body weight, general health, sex, diet, time of administration, rate of excretion, drug combination, the judgment of the treating physician, and the severity of the particular disease being treated. The amount of a provided compound in the composition will also depend upon the particular compound in the composition.

Compounds or compositions described herein may be administered using any amount and any route of administration effective for treating or lessening the severity of the disorders or diseases as contemplated herein. The exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the infection, the particular agent, its mode of administration, and the like. Provided compounds are preferably formulated in unit dosage form for ease of administration and uniformity of dosage. The expression "unit dosage form" as used herein refers to a physically discrete unit of agent appropriate for the subject or patient to be treated. It will be understood, however, that the total daily usage of the compounds and compositions of the present disclosure will be decided by the attending physician within the scope of sound medical judgment. The specific effective dose level for any particular subject or patient or organism will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the activity of the specific compound employed; the specific composition employed; the age, body weight, general health, sex and diet of the subject or patient; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed, and like factors well known in the medical arts.

Pharmaceutically acceptable compositions of this disclosure can be administered to humans and other animals orally, rectally, parenterally, intracisternally, intravaginally, intraperitoneally, topically (as by powders, ointments, or drops), buccally, as an oral or nasal spray, or the like, depending on the severity of the infection being treated. In certain embodiments, provided compounds may be administered orally or parenterally at dosage levels of about 0.01 mg/kg to about 50 mg/kg and preferably from about 1 mg/kg to about 25 mg/kg, of subject body weight per day, one or more times a day, to obtain the desired therapeutic effect.

The term“sample” or "biological sample", as used herein, includes, without limitation, cell cultures or extracts thereof, biopsied material obtained from a mammal or extracts thereof, and blood, saliva, mine, feces, semen, tears, or other body fluids or extracts thereof. Inhibition of activity of a protein, e.g., a bromodomain-containing protein such as a BET protein (e.g. BRD2, BRD3, BRD4 and/or BRDT), or a mutant thereof, in a biological sample is useful for a variety of purposes that are known to one of skill in the art. Examples of such purposes include, but are not limited to, blood transfusion, organ-transplantation, biological specimen storage, and biological assays.

Depending upon the particular condition, or disease, to be treated, additional therapeutic agents that are normally administered to treat that condition may also be used or administered separately as a part of a dosage regimen. As used herein, additional therapeutic agents that are normally administered to treat a particular disease, or condition, are known as "appropriate for the disease, or condition, being treated."

In some embodiments, the compositions, uses and methods of treatment described herein can comprise an additional therapeutic agent.

In some embodiments, the additional therapeutic agent can be an epigenetic drug. As used herein, the term "epigenetic drug" refers to a therapeutic agent that targets an epigenetic regulator.

Examples of epigenetic regulators include the histone lysine methyltransferases, histone arginine methyl transferases, histone demethylases, histone deacetylases, histone acetylases, and DNA methyltransferases. Histone deacetylase inhibitors include, but are not limited to, vorinostat.

Other therapies, chemotherapeutic agents, or other anti-proliferative agents may be combined with a provided compound to treat proliferative diseases and cancer. Examples of therapies or anticancer agents that may be used in combination with compounds herein described include surgery, radiotherapy (e.g., gamma-radiation, neutron beam radiotherapy, electron beam radiotherapy, proton therapy, brachy therapy, and systemic radioactive isotopes), endocrine therapy, a biologic response modifier (e.g., an interferon, an interleukin, tumor necrosis factor (TNF), hyperthermia and cryotherapy, an agent to attenuate any adverse effects (e.g., an antiemetic), and any other approved chemotherapeutic drug.

A provided compound may also be used to advantage in combination with one or more antiproliferative compounds. Such antiproliferative compounds include an aromatase inhibitor; an anti estrogen; an anti-androgen; a gonadorelin agonist; a topoisomerase I inhibitor; a topoisomerase II inhibitor; a microtubule active agent; an alkylating agent; a retinoid, a carotenoid, or a tocopherol; a cyclooxygenase inhibitor; an MMP inhibitor; an mTOR inhibitor; an antimetabolite; a platin compound; a methionine aminopeptidase inhibitor; a bisphosphonate; an antiproliferative antibody; a heparanase inhibitor; an inhibitor of Ras oncogenic isoforms; a telomerase inhibitor; a proteasome inhibitor; a compound used in the treatment of hematologic malignancies; a Flt-3 inhibitor; an Hsp90 inhibitor; a kinesin spindle protein inhibitor; a MEK inhibitor; an antitumor antibiotic; a nitrosourea; a compound targeting/decreasing protein or lipid kinase activity, a compound targeting/decreasing protein or lipid phosphatase activity, or any further anti-angiogenic compound.

Exemplary aromatase inhibitors include steroids, such as atamestane, exemestane and formestane, and non-steroids, such as aminoglutethimide, rogletimide, pyridoglutethimide, trilostane, testolactone, ketoconazole, vorozole, fadrozole, anastrozole and letrozole. Exemplary anti-estrogens include tamoxifen, fulvestrant, raloxifene and raloxifene

hydrochloride. Anti-androgens include, but are not limited to, bicalutamide. Gonadorelin agonists include, but are not limited to, abarelix, goserelin and goserelin acetate.

Exemplary topoisomerase I inhibitors include topotecan, gimatecan, irinotecan, camptothecin and its analogues, 9-nitrocamptothecin and the macromolecular camptothecin conjugate PNU-166148. Topoisomerase II inhibitors include, but are not limited to, the anthracyclines such as doxorubicin, daunorubicin, epirubicin, idarubicin and nemorubicin, the anthraquinones mitoxantrone and losoxantrone, and the podophillotoxins etoposide and teniposide.

Exemplary microtubule active agents include microtubule stabilizing, microtubule destabilizing compounds and microtubulin polymerization inhibitors including, but not limited to taxanes, such as paclitaxel and docetaxel; vinca alkaloids, such as vinblastine or vinblastine sulfate, vincristine or vincristine sulfate, and vinorelbine; discodermolides; colchicine and epothilones and derivatives thereof. Exemplary alkylating agents include cyclophosphamide, ifosfamide, melphalan or nitrosoureas such as cammstine and lomustine.

Exemplary cyclooxygenase inhibitors include Cox-2 inhibitors, 5-alkyl substituted 2- arylaminophenylacetic acid and derivatives, such as celecoxib, rofecoxib, etoricoxib, valdecoxib or a 5- alkyl-2-arylaminophenylacetic acid, such as lumiracoxib.

Exemplary matrix metalloproteinase inhibitors ("MMP inhibitors") include collagen peptidomimetic and non-peptidomimetic inhibitors, tetracycline derivatives, batimastat, marimastat, prinomastat, metastat, BMS-279251, BAY 12-9566, TAA211, MMI270B, and AAJ996.

Exemplary mTOR inhibitors include compounds that inhibit the mammalian target of rapamycin (mTOR) and possess antiproliferative activity such as sirolimus, everolimus, CCI-779, and ABT578.

Exemplary antimetabolites include 5-fluorouracil (5-FU), capecitabine, gemcitabine, DNA demethylating compounds, such as 5-azacytidine and decitabine, methotrexate and edatrexate, and folic acid antagonists such as pemetrexed.

Exemplary platin-containing compounds include carboplatin, cisplatin, nedaplatin, and oxaliplatin.

Exemplary methionine aminopeptidase inhibitors include bengamide or a derivative thereof and PPI-2458.

Exemplary bisphosphonates include etidronic acid, clodronic acid, tiludronic acid, pamidronic acid, alendronic acid, ibandronic acid, risedronic acid and zoledronic acid.

Exemplary antiproliferative antibodies include trastuzumab, trastuzumab-DMl, cetuximab, bevacizumab, rituximab, PR064553, and 2C4. The term "antibody" is meant to include intact monoclonal antibodies, polyclonal antibodies, multispecific antibodies formed from at least two intact antibodies, and antibody fragments, so long as they exhibit the desired biological activity.

Exemplary heparanase inhibitors include compounds that target, decrease or inhibit heparin sulfate degradation, such as PI- 88 and OGT2115. The term "an inhibitor of Ras oncogenic isoforms," such as H-Ras, K-Ras, or N-Ras, as used herein refers to a compound which targets, decreases, or inhibits the oncogenic activity of Ras; for example, a farnesyl transferase inhibitor such as L-744832, DK8G557, tipifarnib, and lonafarnib.

Exemplary telomerase inhibitors include compounds that target, decrease or inhibit the activity of telomerase, such as compounds which inhibit the telomerase receptor, such as telome statin.

Exemplary proteasome inhibitors include compounds that target, decrease or inhibit the activity of the proteasome including, but not limited to, bortezomib.

The phrase "compounds used in the treatment of hematologic malignancies" as used herein includes FMS-like tyrosine kinase inhibitors, which are compounds targeting, decreasing or inhibiting the activity of FMS-like tyrosine kinase receptors (Flt-3R); interferon, I-b-D-arabinofuransylcytosine (ara-c) and busulfan; and ALK inhibitors, which are compounds which target, decrease or inhibit anaplastic lymphoma kinase.

Exemplary Flt-3 inhibitors include PKC412, midostaurin, a staurosporine derivative, SU11248 and MLN518.

Exemplary HSP90 inhibitors include compounds targeting, decreasing or inhibiting the intrinsic ATPase activity of HSP90; degrading, targeting, decreasing or inhibiting the HSP90 client proteins via the ubiquitin proteosome pathway. Compounds targeting, decreasing or inhibiting the intrinsic ATPase activity of HSP90 are especially compounds, proteins or antibodies which inhibit the ATPase activity of HSP90, such as 17-allylamino,17-demethoxygeldanamycin (17AAG), a geldanamycin derivative; other geldanamycin related compounds; radicicol and HD AC inhibitors.

The phrase "a compound targeting/decreasing a protein or lipid kinase activity; or a protein or lipid phosphatase activity; or any further anti-angiogenic compound" as used herein includes a protein tyrosine kinase and/or serine and/or threonine kinase inhibitor or lipid kinase inhibitor, such as a) a compound targeting, decreasing or inhibiting the activity of the platelet-derived growth factor-receptors (PDGFR), such as a compound which targets, decreases, or inhibits the activity of PDGFR, such as an N- phenyl-2-pyrimidine-amine derivatives, such as imatinib, SU101, SU6668 and GFB-111; b) a compound targeting, decreasing or inhibiting the activity of the fibroblast growth factor-receptors (FGFR); c) a compound targeting, decreasing or inhibiting the activity of the insulin-like growth factor receptor I (IGF- IR), such as a compound which targets, decreases, or inhibits the activity of IGF-IR; d) a compound targeting, decreasing or inhibiting the activity of the Trk receptor tyrosine kinase family, or ephrin B4 inhibitors; e) a compound targeting, decreasing or inhibiting the activity of the Axl receptor tyrosine kinase family; f) a compound targeting, decreasing or inhibiting the activity of the Ret receptor tyrosine kinase; g) a compound targeting, decreasing or inhibiting the activity of the Kit/SCFR receptor tyrosine kinase, such as imatinib; h) a compound targeting, decreasing or inhibiting the activity of the c-Kit receptor tyrosine kinases, such as imatinib; i) a compound targeting, decreasing or inhibiting the activity of members of the c-Abl family, their gene-fusion products (e.g. Bcr-Abl kinase) and mutants, such as an N-phenyl-2 -pyrimidine-amine derivative, such as imatinib or nilotinib; PD180970; AG957; NSC 680410; PD 173955; or dasatinib; j) a compound targeting, decreasing or inhibiting the activity of members of the protein kinase C (PKC) and Raf family of serine/threonine kinases, members of the MEK, SRC, JAK, FAK, PDK1, PKB/Akt, and Ras/MAPK family members, and/or members of the cyclin-dependent kinase family (CDK), such as a staurosporine derivative disclosed in US 5,093,330, such as midostaurin;

examples of further compounds include UCN-01, safmgol, BAY 43-9006, bryostatin 1, perifosine;

ilmofosine; RO 318220 and RO 320432; GO 6976; ISIS 3521; LY333531/LY379196; a isochinoline compound; a famesyl transferase inhibitor; PD184352 or QAN697, or AT7519; k) a compound targeting, decreasing or inhibiting the activity of a protein-tyrosine kinase, such as imatinib mesylate or a tyrphostin such as Tyrphostin A23/RG-50810; AG 99; Tyrphostin AG 213; Tyrphostin AG 1748; Tyrphostin AG 490; Tyrphostin B44; Tyrphostin B44 (+) enantiomer; Tyrphostin AG 555; AG 494; Tyrphostin AG 556, AG957 and adaphostin (4-{ [(2,5- dihydroxyphenyl)methyl] amino} -benzoic acid adamantyl ester; NSC 680410, adaphostin); 1) a compound targeting, decreasing or inhibiting the activity of the epidermal growth factor family of receptor tyrosine kinases (EGFR, ErbB2, ErbB3, ErbB4 as homo- or

heterodimers) and their mutants, such as CP 358774, ZD 1839, ZM 105180; trastuzumab, cetuximab, gefitinib, erlotinib, OSI-774, Cl-1033, EKB-569, GW-2016, antibodies El l, E2.4, E2.5, E6.2, E6.4, E2.1 1, E6.3 and E7.6.3, and 7H-pyrrolo-[2,3-d]pyrimidine derivatives; and m) a compound targeting, decreasing or inhibiting the activity of the c-Met receptor.

Exemplary compounds that target, decrease or inhibit the activity of a protein or lipid phosphatase include inhibitors of phosphatase 1, phosphatase 2A, or CDC25, such as okadaic acid or a derivative thereof.

Further anti-angiogenic compounds include compounds having another mechanism for their activity unrelated to protein or lipid kinase inhibition, e.g. thalidomide and TNP-470.

Additional exemplary chemotherapeutic compounds, one or more of which may be used in combination with provided compounds, include: daunorubicin, adriamycin, Ara-C, VP- 16, teniposide, mitoxantrone, idarubicin, carboplatinum, PKC412, 6-mercaptopurine (6-MP), fludarabine phosphate, octreotide, SOM230, FTY720, 6-thioguanine, cladribine, 6-mercaptopurine, pentostatin, hydroxyurea, 2- hydroxy-lH-isoindole-l,3-dione derivatives,!- (4-chloroanilino)-4-(4-pyridylmethyl)phthalazine or a pharmaceutically acceptable salt thereof, l-(4-chloroanilino)-4-(4-pyridylmethyl)phthalazine succinate, angiostatin, endostatin, anthranilic acid amides, ZD4190, ZD6474, SU5416, SU6668, bevacizumab, rhuMAb, rhuFab, macugen; FLT-4 inhibitors, FLT-3 inhibitors, VEGFR-2 IgGI antibody, RPI 4610, bevacizumab, porfimer sodium, anecortave, triamcinolone, hydrocortisone, l la-epihydrocotisol, cortexolone, 17a-hydroxyprogesterone, corticosterone, desoxycorticosterone, testosterone, estrone, dexamethasone, fluocinolone, a plant alkaloid, a hormonal compound and/or antagonist, a biological response modifier, such as a lymphokine or interferon, an antisense oligonucleotide or oligonucleotide derivative, shRNA or siRNA, or a miscellaneous compound or compound with other or unknown mechanism of action.

In some embodiments, the compositions, uses or treatment methods described herein are intended to treat prostate cancer and the compositions, uses or treatment methods can comprise one or more further drugs selected from drugs approved for prostate cancer. The additional drug can be selected from Abiraterone Acetate (e.g. Zytiga™), Apalutamide (e.g. Erleada™), Bicalutamide (e.g. Casodex™), Cabazitaxel (e.g. Jevtana™), Degarelix (e.g. Firmagon™), Docetaxel (e.g. Taxotere™), Leuprolide Acetate (e.g. Eligard™, Lupron™, Lupron Depot™), Enzalutamide (e.g. Xtandi™), Flutamide, Goserelin Acetate (e.g. Zoladex™), Mitoxantrone Hydrochloride, Nilutamide (e.g. Nilandron™), Provenge (Sipuleucel-T™) and Radium 223 Dichloride (e.g. Xofigo™).

Provided compounds can be administered alone or in combination with one or more other therapeutic compounds as explained above. Possible combination therapy can take the form of fixed combinations or the administration of a provided compound and one or more other therapeutic compounds being staggered or given independently of one another, or the combined administration of fixed combinations and one or more other therapeutic compounds. Provided compounds can besides or in addition be administered especially for tumor therapy in combination with chemotherapy, radiotherapy, immunotherapy, phototherapy, surgical intervention, or a combination of these. Long-term therapy is equally possible as is adjuvant therapy in the context of other treatment strategies, as described above. Other possible treatments are therapy to maintain the subject’s or patient's status after tumor regression, or even chemopreventive therapy, for example in patients at risk.

Such additional agents may be administered separately from a composition containing a provided compound, as part of a multiple dosage regimen. Alternatively, those agents may be part of a single dosage form, mixed together with a provided compound in a single composition. If administered as part of a multiple dosage regimen, the two active agents may be submitted simultaneously, sequentially or within a period of time from one another normally within five hours from one another.

Upon improvement of a subject's or patient’s condition, a maintenance dose of a compound, composition or combination of the present description may be administered, if necessary. Subsequently, the dosage or frequency of administration, or both, may be reduced, as a function of the symptoms, to a level at which the improved condition is retained when the symptoms have been alleviated to the desired level, treatment should cease. The patient or subject may, however, require intermittent treatment on a long-term basis upon any recurrence of disease symptoms.

It will be understood, however, that the total daily usage of the compounds and compositions described herein will be decided by the attending physician within the scope of sound medical judgment. The specific inhibitory dose for any particular subject or patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the activity of the specific compound employed; the specific composition employed; the age, body weight, general health, sex and diet of the subject or patient; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed; and like factors well known in the medical arts.

The total daily inhibitory dose of the compounds described herein, administered to a patient or subject in single or in divided doses can be in amounts, for example, from 0.01 to 50 mg/kg body weight or more usually from 0.1 to 25 mg/kg body weight. Single dose compositions may contain such amounts or submultiples thereof to make up the daily dose. In one embodiment, treatment regimens according to the present description comprise administration to a subject or patient in need of such treatment from about 10 mg to about 1000 mg of the compound(s) of the present description per day in single or multiple doses.

As used herein, the term "combination," "combined," and related terms refers to the simultaneous or sequential administration of therapeutic agents in accordance with the present description. For example, a provided compound may be administered with another therapeutic agent simultaneously or sequentially in separate unit dosage forms or together in a single unit dosage form. Accordingly, an embodiment of the present description provides a single unit dosage form comprising a provided compound, an additional therapeutic agent, and a pharmaceutically acceptable carrier, adjuvant, or vehicle for use in the methods of the present description.

The amount of both, a provided compound and additional therapeutic agent (in those compositions which comprise an additional therapeutic agent as described above) that may be combined with the carrier materials to produce a single dosage form will vary depending upon the host treated and the particular mode of administration. Preferably, compositions should be formulated such that a dosage of between 0.01 - 100 mg/kg body weight/day of a provided compound can be administered.

In those compositions which comprise an additional therapeutic agent, that additional therapeutic agent and the provided compound may act synergistically. Therefore, the amount of additional therapeutic agent in such compositions will be less than that required in a monotherapy utilizing only that therapeutic agent. In such compositions a dosage of between 0.01 - 1,000 g/kg body weight/day of the additional therapeutic agent can be administered.

The amount of additional therapeutic agent present in the compositions of this disclosure will be no more than the amount that would normally be administered in a composition comprising that therapeutic agent as the only active agent. Preferably the amount of additional therapeutic agent in the presently disclosed compositions will range from about 50% to 100% of the amount normally present in a composition comprising that agent as the only therapeutically active agent.

Provided compounds, or pharmaceutical compositions thereof, may also be incorporated into compositions for coating an implantable medical device, such as prostheses, artificial valves, vascular grafts, stents and catheters. Vascular stents, for example, have been used to overcome restenosis (re narrowing of the vessel wall after injury). However, subjects or patients using stents or other implantable devices risk clot formation or platelet activation. These unwanted effects may be prevented or mitigated by pre-coating the device with a pharmaceutically acceptable composition comprising a provided compound. Implantable devices coated with a compound of the present description are another embodiment of the present description.

The recitation of an embodiment for a variable herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof. The recitation of an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof. EXAMPLES

Example 1 :

Materials and methods

Unless otherwise specified, the materials and methods employed herein are as described in Zhang et al., 2017 and/or Dai et al., 2017, or adapted therefrom with minor modifications.

Plasmids, chemicals and antibodies

Expression vectors for Myc-SPOP WT and F133V and Q165P mutants, Flag-SPOP WT and Q165P mutant, Myc-Cullin 3, Flag-BRD4 were generated or described previously (An et al., 2014; Zhang et al., 2017). SPOP mutant expression vectors were generated using KOD-Plus Mutagenesis Kit (Toyobo). CPI-637 and JQ1 were purchased from Sigma-Aldrich. Matrigel basement membrane Matrix (# 354248) was purchased from Corning Life Sciences. The antibodies used are as follows: anti-Myc tag from Santa Cruz Biotechnology; anti-HA from Covance; Flag-M2 (F-3165) from Cell Signaling Technology; BRD2 (abl39696) and BRD4 (abl28874) from Abeam; BRD3 (A302-368A) from Bethly Lab; SPOP (16750-1-AP) from Proteintech Group Inc.

Cell lines, cell culture, transfection and lentivirus infection

DU145 and 293T cells were obtained from the American Type Culture Collection (ATCC). 293T cells were maintained in DMEM medium with 10% FBS, and DU145 cells were maintained in RPMI medium with 10% FBS. Cells were transiently transfected using Lipofectamine 3000 (Thermo Fisher Scientific) according to the manufacturer’s instructions. pTsin-HA-SPOP mutant expression and virus packing constructs were transfected into 293T cells. Virus supernatant was collected 48 h after transfection. DU145 cells were infected with viral supernatant in the presence of polybrene (8 pg/mL) and were then selected in growth media containing 1.5 pg/mL puromycin. All the cell lines used were tested and authenticated by examining AR and PTEN expression. Plasmocin (InvivoGen) was added to cell culture media to prevent mycoplasma contamination. Mycoplasma contamination was tested regularly using Lookout Mycoplasma PCR Detection Kit from Sigma-Aldrich.

Subrenal capsule grafting and development of transplantable tumor lines from patients

The SPOP Q165P mutant PDX (LTL573R) was generated from the biopsy of a liver metastasis from a CRPC patient. SPOP WT PDX (LTL313 HR) is a CRPC model developed in castrated mice from LTL313H, which was derived from hormone naive primary PCa biopsy of a patient. Within 24 h of sample arrival, biopsy tissue was grafted into subrenal capsule of male NOD-SCID mice using the method as described previously (Lin et al., 2014; Wang et al., 2005). Growing tumors (transplantable tumor lines) were consistently maintained by serial subrenal capsule transplantation. Xenografts were harvested, measured and fixed for histopathological analysis. Animal care and experiments were carried out in accordance with the guidelines of the Canadian Council on Animal Care and were approved by the Institutional Animal Care and Use Committee. PDX maintenance and organoid culture

Both SPOP-WT and Q165P PDXs were transplanted subcutaneously and maintained in NOD- SCID IL-2-receptor gamma null (NSG) male mice. When the tumors reached the size of 1,000 mm³ about two months, they were passaged down to next generation.

PDX-derived organoid cultures were carried out generally using the protocol as described in Drost et al., 2016. Briefly, the tumor was cut into 1 mm³ pieces and digested in 5 mg/mL collagenase with 10 mM Y-27632 for 1 h at 37°C. The digested tumor tissue was washed with adDMEM/F12 medium and then treated with TrypLE with 10 mM Y-27632 for 10 min at 37°C, followed by another wash before embedded into Matrigel. Approximately 40 pL of Matrigel containing 50,000 cells was plated onto 24- well plate. The cultured medium for PDX was freshly prepared as described in Drost et al., 2016.

Drug treatment of PDX tumors

The PDX tumors including SPOP WT and Q165P mutant were established by passaging tumor pieces (~1 mm³) subcutaneously (s.c.) into 6 to 8 week-old NSG male mice. After tumors reach ~100 mm³ in size (approximately 4 weeks after transplantation), tumor positive animals in both SPOP WT and Q165P groups were randomly divided into different treatment groups (5 mice /group). JQ1, CPI-637 and Compound Al were dissolved in 40% polyethylene glycol (PEG400). Mice were treated via

intraperitoneal (IP) injection with the vehicle (40% PEG400), JQ1 (50 mg/kg), CPI-637 (30 mg/kg), JQ1 plus CPI-637 or Compound Al (30 mg/kg). Mice were treated with these drugs five days a week for 21 consecutive days. Tumor growth was measured by caliper twice a week.

Protein extraction and western blot analysis

For the cultured cells, the cells were washed with PBS and then lysed into cell lysis buffer (25 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% NP-40 and 5% glycerol). For the PDX tissues, frozen tissues were ground into powder on dry ice before adding the lysis buffer. Both cultured cells and ground tumor tissues were incubated for 30 mins on ice and then centrifuged at 13,000 rpm for 15 mins to remove the debris.

Hematoxylin and eosin staining (H&E), immunohistochemistry (IHC) and immunofluorescent cytochemistry (IFC)

The paraffin tissue sections for H&E, IHC and IFC were cut at 4-pm thickness. H&E and IHC were performed according to a previously published study (Blee et al., 2018). Specifically, the antigen retrieval was conducted via heat-induced epitope retrieval in 10 mM sodium citrate buffer (pH 6.0) for all antibodies used. Antibodies were diluted at appropriate concentrations as required and incubated in a humidified box overnight at 4°C. The secondary antibody for IHC was SignalStain®Boost IHC Detection Reagent (HRP, Rabbit) and the staining was developed with SignalStain®DAB Substrate Kit.

For IFC on PDX tissues, all the steps were the same as IHC paraffin tissues except that secondary florescence antibodies (Alexa Fluor 488 and Alexa Fluor 594) were used at 1 in 500 dilution. For IFC on the organoids, the organoid cultures were smeared onto slides and fixed with ice-cold Methanol/ Acetone (1: 1 dilution) at 4°C for 10 mins, followed by 3 washes of 0.2% Triton-100 in PBS. The organoids were blocked with 10% goat serum at room temperature for 1 h, and followed by primary antibody incubation at 4°C overnight. The following day, the organoids with incubated with secondary antibody at room temperature for 1 h. The nucleus was counterstained with DAPI.

MTS cell proliferation assay and clonogenic survival assay

For MTS cell proliferation assay, the cells were plated at a density of 3,000 cells/well in 96-well plates. At 4 h after plating, cells were treated with different concentrations of drugs and harvested at 72 h post-treatment. The OD value was read at a wavelength of 490 nm. The clonogenic survival assay was conducted as previously described (Yan et al., 2018). Briefly, an appropriate number of cells for different dosages of drugs were plated onto 6-well plate. At the following day, cells were treated with DMSO or Compound Al for 4 days and then cultured with fresh medium without drugs for another 8 days. 12 days later, colonies were fixed and stained with crystal violet 0.5% w/v) for 1 h. The colonies with more than 50 cells were counted, and the number of colonies in drug treated groups was normalized to the untreated group. The linear regression was applied to generate survival curves.

Statistical Analysis

Data are shown as mean values ± SD from experiments performed with at least three replicates. The difference among groups was analyzed using paired Student’s t-test unless otherwise specified. A P value less than 0.05 is considered statistically significant.

BROMOscan™ assay for measuring bromodomain binding affinities

Protocol Description of Bromodomain assays. T7 phage strains displaying bromodomains were grown in parallel in 24-well blocks in an E. coli host derived from the BL21 strain. E. coli were grown to log-phase and infected with T7 phage from a frozen stock (multiplicity of infection = 0.4) and incubated with shaking at 32°C until lysis (90-150 minutes). The lysates were centrifuged (5,000 x g) and filtered (0.2 pm) to remove cell debris. Streptavidin-coated magnetic beads were treated with biotinylated small molecule or acetylated peptide ligands for 30 minutes at room temperature to generate affinity resins for bromodomain assays. The liganded beads were blocked with excess biotin and washed with blocking buffer (SeaBlock (Pierce), 1% BSA, 0.05% Tween™ 20, 1 mM DTT) to remove unbound ligand and to reduce non-specific phage binding. Binding reactions were assembled by combining bromodomains, liganded affinity beads, and test compounds in lx binding buffer (17% SeaBlock, 0.33x PBS, 0.04% Tween 20, 0.02% BSA, 0.004% sodium azide, 7.4 mM DTT). Test compounds were prepared as lOOOx stocks in 100% DMSO and subsequently diluted 1: 10 in monoethylene glycol (MEG) to create stocks at lOOX the screening concentration (resulting stock solution is 10% DMSO/90% MEG). The compounds were then diluted directly into the assays such that the final concentration of DMSO and MEG were 0.1% and 0.9%, respectively. All reactions were performed in polystyrene 96-well plates in a final volume of 0.135 ml. The assay plates were incubated at room temperature with shaking for 1 hour and the affinity beads were washed with wash buffer (lx PBS, 0.05% Tween 20). The beads were then re-suspended in elution buffer (f x PBS, 0.05% Tween 20, 2 pM non-biotinylated affinity ligand) and incubated at room temperature with shaking for 30 minutes. The bromodomain concentration in the eluates was measured by qPCR. Compound Handling. An 11 -point 3 -fold serial dilution of each test compound was prepared in 100% DMSO at lOOOx final test concentration. This serial was then diluted to lOOx in ethylene glycol and subsequently diluted to lx in the assay (final DMSO concentration = 0.1%, Ethylene glycol

concentration^.9%). Most Kds were determined using a compound top concentration = 10,000 nM. If the initial Kd determined was < 0.169 nM (the lowest concentration tested), the measurement was repeated with a serial dilution starting at a lower top concentration. A Kd value reported as 40,000 nM indicates that the Kd was determined to be >10,000 nM.

Binding Constants (Kds). Binding constants (Kds) were calculated with a standard dose-response curve using the Hill equation: Response = Background + (Signal - Background)/(l + (Kd^{Hlll slop}7 Dose^{HlU slope})). The Hill Slope was set to -1. Curves were fitted using a non-linear least square fit with the Levenberg-Marquardt algorithm.

AlphaLISA™ BRD4 inhibitor testing protocol

Reagents and materials. AlphaLISA™ Glutathione Acceptor beads, PerkinElmer cat# AL109C; AlphaScreen Streptavidin Donor beads, PerkinElmer cat# 6760001. Bromodomain and biotinylated- peptide: GST-tagged BDR4 (BD1), EpiCypher cat#15-0012; Biotinylated H4:K5/K8/K12/K16(Ac), EpiCypher cat#12-0034. Other reagents: AlphaLISA™ 5x Epigenetics Buffer 1, 10 mL Kit Cat#

AL008C; DMSO, Fisher cat# D159-4; Protein LoBind™ tubes, 0.5 mL, Eppendorf cat# 13698793. All assays were performed in PerkinElmer OptiPlate™-384, Shallow Well, cat# 6007290. Assays were measured on a 2104 EnVision® multimode plate reader (PerkinElmer) using standard settings for Alpha detection.

Protocol. Assays were conducted in duplicate or triplicate in a total volume of 20 pL. Each test compound (including the positive control (+)-JQl) was tested at 9 concentrations (including zero (no compound)). The positive control was included in each plate. The following assay protocol was used: (1)

2 pL of 10X compound (1% DMSO final); (2) 4 pL of 5X BRD4 (BD1) 1 (5 nM final); (3) 10 min incubation at 23°C; (4) 4 pL of 5X Bio-H4 K(5,8,12,16)Ac (5 nM final); (5) 5 pL of 4X AlphaLISA™ GSH Acceptor beads (10 pg/mL final); (6) 60 min incubation at 23°C in the dark; (7) 5 pL of 4X SA-Donor beads (10 pg/mL final); (8) 30 min-incubation at 23°C in the dark; (9) Read with EnVision™. Data were fitted using the 4-parameter logistic curve to determine IC50 values (GraphPad Prism™ 5).

CREBBP (CBP) inhibitor testing protocol

Reagents and materials. AlphaLISA™ Glutathione Acceptor beads, PerkinElmer cat# AL109M; AlphaScreen™ Streptavidin Donor beads, PerkinElmer cat# 6760002. Proteins: Recombinant human CBP Bromodomain (1091-1190), containing an N-terminal GST tag, expressed in E. coli. EpiCypher cat# 15-0023; Recombinant CREBBP (1081-1197) protein, containing an N-terminal His-Tag and C-terminal FLAG-Tag, expressed in E. coli. Active Motif cat# 31373; Recombinant human CBP Bromodomain (1081-1197), containing an N-terminal GST tag, expressed in E. coli., Reaction Biology cat# RD-11-191; Recombinant human CBP Bromodomain (1081-1197), containing an N-terminal GST tag, expressed in E. coli. BPS Bioscience cat#31128; Biotinylated peptide: Biotinylated H4K5,8,12,16(Ac) (1-23) (Ac- SGRGK(Ac)GGK(Ac)GLGK(Ac)GGAK(Ac)RHRKVLR-Peg(Biot)), EpiCypher cat# 12-0034. Buffers: AlphaLISA™ 5X Epigenetics Buffer 1, 10 mL Kit Cat# AL008C; 50 mM Hepes pH 7.4, 50 mM NaCl, 0.1% BSA; 50 mM Tris-HCl pH 7.4, 50 mM NaCl, 0.1% BSA. Other reagents: DMSO, Fisher cat# D159-4; Protein LoBind tubes, 0.5 mL, Eppendorf cat# 13698793. All assays were performed in PerkinElmer OptiPlate™-384, Shallow Well, cat# 6007290. Assays were measured on a 2104 EnVision® multimode plate reader (PerkinElmer) using standard settings for Alpha detection.

Protocol. AlphaLISA™ assays were conducted in triplicate in a total volume of 20 pL. The following assay protocol was used: (1) 4 pL of CBP-CREBBP (50 ng per well); (2) 1 pL of inhibitor 5x (final 0.25% DMSO in 20 pL); (3) 5 pL of 4x Biotinylated-peptide (10 nM, final in 20 pL); (4) Incubate at 23°C for 30 min; (5) 5 pL of GSH conjugated Acceptor beads (10 pg/mL, final in 20 pL); (6) 30 min incubation at 23°C; (7) 5 pL of SA-Donor beads (10 pg/mL, final in 20 pL); (8) 30 min incubation at 23°C in the dark; (9) Read at RT with EnVision™ protocol AlphaLISA™ 384 OptiPlate™ BioAux. Data were fitted using the 4-parameter logistic curve to determine EC50 and IC50 values (GraphPad Prism 5). Compounds I-CBP112 (Cayman cat# 14468) and JQ1 (Tocris cat# 4499) were used as controls.

Examnle 2:

Prostate cancer cell lines expressing the SPOP FI 33V variant exhibit increased sensitivity to co-treatment with a BET inhibitor and a CBP/EP300 inhibitor

DU145 prostate cancer cells (Alimirah et al., 2006; ATCC^® HTB-81™) were transfected with either an empty vector (“EV”) or with an expression vector encoding an SPOP F133V variant (F133V”), one of the most frequent mutations found in prostate cancers. As shown in Fig. 1, cell proliferation studies revealed that DU145 cells expressing the SPOP F133V variant (“F133V”) exhibited resistance to treatment with a BET inhibitor (JQ1) alone (“JQ1”), and to RNA knockdown of CREB binding protein expression (“iCBP”), as compared to treatment with the vehicle alone (“DMSO”). Interestingly, DU145 cells expressing the SPOP F133V variant were particularly sensitive to co-treatment of DU145 cells with the BET inhibitor and knockdown of CREB binding protein expression (“iCBP + JQ1”).

Consistent with the above, further cell proliferation experiments shown in Fig. 2 revealed that DU145 cells expressing the SPOP F133V variant exhibited comparable resistance to treatment with the BET inhibitor JQ1 alone (“JQ1”), and to the CBP/EP300 inhibitor CPI-637 (“CPI-637”; Taylor et al., 2016), relative to control DU145 cells expressing their endogenous SPOP (“EV”). Both control (“EV”) and SPOP F133V-expressing DU145 cells showed greater sensitivity to co-treatment with the BET inhibitor and the CBP/EP300 inhibitor (“CPI-637 + JQ1”), as compared to the additive effects of monotreatment with either the BET inhibitor or the CBP/EP300 inhibitor. Interestingly, DU145 cells expressing the SPOP F133V variant showed particularly heightened sensitivity to co-treatment with the BET inhibitor and the CBP/EP300 inhibitor. Example 3:

Monotreatment with a substituted benzimidazole BRD4 inhibitor exhibited comparable efficacy to BET inhibitor and CBP/EP300 inhibitor co-treatment in DU145 cells expressing either wild-type or

FI 33V SPOP

Compound Al, which belongs to the substituted benzimidazole family of BRD4 inhibitors (e.g., as described in WO 2017/024412), was evaluated for efficacy as described in Example 2. Interestingly, compound Al showed comparable efficacy to co-treatment with the BET inhibitor and the CBP/EP300 inhibitor (see Fig. 2,“Compound Al”) in both control DU145 cells transfected with the empty vector (“EV”; expressing wild-type SPOP), and DU145 cells expressing the SPOP F133V variant (“SPOP- F133V”). A dose-survival analysis was then performed for compound Al in both control DU145 cells (“EV”) and SPOP F133V-expressing DU145 cells. This analysis revealed that the IC50 of compound Al dropped from 1.08 mM in the control cells to 0.69 pM in DU145 cells expressing the SPOP F133V variant, representing a 36% decrease (Fig. 3).

Examnie 4:

Efficacy of BET inhibitor and CBP/EP300 inhibitor co-treatment, and compound A1 monotreatment, evaluated in prostate cancer patient derived organoid and mice xenograft models

The results in Examples 2 and 3 were validated in an organoid model from a patient with prostate cancer expressing a different SPOP variant: Q165P (A > C), which was identified following Sanger sequencing of a tumor specimen (primary prostate cancer and liver metastasis biopsies) as generally described in Zhang et al., 2017 and Dai et al., 2017. Intriguingly, the primary tumor in this patient contained a heterozygous Q165P mutation, whereas the liver metastasis harbored a homozygous Q165P mutation.

Organoid cultures were established from a homozygous SPOP Q165P mutant PDX tumor, and a SPOP WT PDX-derived organoid was used as a control, using a protocol as generally described in Drost et al., 2016. The organoids were large at baseline, reflecting more aggressive biology. Organoids were prepared and tested as outlined in Fig. 4, and as previously described in Zhang et al., 2017 and Dai et al., 2017. Briefly, a fresh tumor was harvested and digested with collagenase and TrypLE to obtain the single live tumor cells, and cells were embedded into Matrigel™ allowed to grow into a three-dimension (3D) structure. Sanger sequencing further confirmed that the cultured organoids captured the same mutant of SPOP as their original Q165P tumors after 10-day culture in vitro. It was found that, after 10-day culture in vitro , WT organoids were easier to form a hollow and round-shaped structure and some of them were collapsed. Intriguingly, SPOP Q165P mutant organoids tended to form a solid and irregular sphere, and adhere to the bottom of culture dishes. Quantification of the diameter of organoids showed that SPOP Q165P organoids were bigger than their WT counterparts. These data suggest that the homozygous SPOP Q165P mutation confers a more aggressive cancer phenotype, which is consistent with the metastatic origin of this PDX in the patient.

The results in Figs. 5 and 6 show the effects of treatment with vehicle alone (“DMSO”), BET inhibitor alone (“JQ1”), CBP/EP300 inhibitor alone (“CPI-637”), co-treatment with BET inhibitor and CBP/EP300 inhibitor (“CPI-637 + JQ1”), or monotreatment with compound Al, on organoid diameter (Fig. 5) and morphology (Fig. 6A-6J).

Of note, Fig. 5 shows that - in the context of the organoid tumors expressing only the wild-type SPOP (“SPOP^WT”) - co-treatment with the BET inhibitor and the CBP/EP300 inhibitor (“CPI-637 +

JQ1”) did not result in significantly lower organoid diameter as compared to monotreatment with the BET inhibitor alone (“JQ1”) or the CBP/EP300 inhibitor alone (“CPI-637”). In contrast, in the context of the organoid tumors expressing the SPOP Q165P variant (“SPOP^Q165F”), co-treatment with the BET inhibitor and the CBP/EP300 inhibitor (“CPI-637 + JQ1”), as well as monotreatment with compound Al, resulted in smaller organoid tumors as compared to monotreatment with the BET inhibitor alone (“JQ1”) or the CBP/EP300 inhibitor alone (“CPI-637”).

Next, prostate cancer xenografts in mice were generated and utilized for efficacy studies, as previously described in Zhang et al., 2017 and Dai et al., 2017, except that the mice groups were treated with vehicle alone (“DMSO”), BET inhibitor alone (“JQ1”), CBP/EP300 inhibitor alone (“CPI-637”), co treatment with BET inhibitor and CBP/EP300 inhibitor (“CPI-637 + JQ1”), or monotreatment with compound Al. Sanger sequencing confirmed that PDX tumors harbored homozygous SPOP Q165P mutation and expression of BET proteins BRD2, BRD3 and BRD4 was confirmed by western blot and IHC analysis in PDX tumors prior to drug treatment. Fig. 7 and Fig. 8 respectively show representative images of tumors isolated from each group of mice for prostate cancer tumors expressing either wild type SPOP (“SPOP^WT”) or the SPOP variant Q165P (“SPOP^Q165F”). Corresponding tumor volume measurements are shown in Fig. 9 and Fig. 10, following 1 to 7 days of drug treatment for prostate cancer tumors expressing either wild type SPOP (“SPOP^WT”) or the SPOP variant Q165P (“SPOP^Q165F”), respectively.

Examnle 5:

Molecular characterization of the SPOP Q165P variant

The Q165P mutated residue is located at the edge of the MATH domain and almost in the junction between the MATH and BTB domains, prompting the determination of its effects on SPOP substrate binding, SPOP dimerization, and the expression of SPOP substrates. Computer simulation analysis revealed that the Q165P mutation causes larger conformational fluctuation, suggesting that the structure of the SPOP Q165P variant may less stable than that of wild type SPOP (data not shown). In parallel, co-immunoprecipitation assays suggested that the SPOP Q165P variant impairs the dimerization (or oligomerization) ability of SPOP, as shown in Fig. 11 and Fig. 12. SPOP Q165P mutation only partially (~50%) diminished the ability of SPOP to bind to BRD4 (data not shown).

Examnle 6:

Members of the substituted benzimidazole family of BRIM inhibitors exhibit binding affinity to both BET proteins and CBP/EP300

The BROMOscan™ platform (DiscoveRx Inc., Fremont, Calif.) was employed as described in Example 1 to measure the binding affinities of substituted benzimidazole BRD4 inhibitors to various bromodomain-containing biological targets. Briefly, the approach involves the use of DNA-tagged biological targets (e.g., DNA-tagged bromodomain polypeptides) and known ligands of the biological targets that are immobilized on a solid support. Test compounds that bind to the biological target prevents it from subsequently binding to the immobilized ligand, thus reducing the amount of the biological target that is ultimately captured on the solid support. Conversely, test compounds that do not bind to the biological target have no effect on the amount of the biological target captured on the solid support. The presence of the DNA tag on the biological targets enable sensitive and quantitative measurement of the biological targets captured, thereby enabling calculation of dissociation constants (Kds) for each test compound-biological target interaction.

Results are shown in Table A for the substituted benzimidazole compounds A1 and A2, whose syntheses and preparation were as previously described in WO 2017/024412.

Table A: Dissociation constants (Kds) of members of the substituted benzimidazole family of BRD4 inhibitors to multiple bromodomain-containing targets

Results shown represent averages of at least two replicates; Dissociation constants (Kds) less than

500 nM are bolded; Not determined; Numbers in parentheses, where present, distinguish between similar bromodomain binding sites on targets. Example 7:

Members of the aryl-substituted dihydroquinolinone family of BRD4 inhibitors exhibit binding affinity to both BET proteins and CBP/EP300

A competitive/displacement binding domain assay based on the homogeneous (no-wash) AlphaLISA™ technology was used to evaluate relative dual binding affinities of all the aryl-substituted dihydroquinolinone BRD4 inhibitors described in WO 2017/024408 to both BRD4 and CBP (CREBBP), using the methodologies as described in Example 1. Briefly, glutathione (GSH) AlphaLISA™ Acceptor beads are used to capture the GST-tagged BRD4 or GST-tagged CBP, while streptavidin-coated Donor beads are used to capture a biotinylated acetylated peptide, which is a known ligand of either BRD4 or CREBBP. Donor and Acceptor beads come into physical proximity through BRD4 or CBP binding to their respective acetylated peptide ligands. Excitation of the Donor beads at 680 nm results in the release of singlet oxygen, thereby triggering energy transfer to the Acceptor beads, if within 200 nm of each other, resulting in a sharp emission peak at 615 nm. This light emission can then be detected on an Alpha- enabled reader. As designed, a test compound which acts as an inhibitor of the BRD4/peptide ligand or CBP/peptide ligand interaction will thereby separate Donor and Acceptor beads, resulting in signal loss at 615 nm.

A plurality of different aryl-substituted dihydroquinolinones of WO 2017/024408 (synthesized as described therein) were tested. Results for test compounds exhibiting Kds for CBP of less than 500 nM are shown in Table B expressed as IC50 values. The other tested compounds showed Kds for CBP of between 570 nM and 100,000 nM.

Table B:

REFERENCES

Alimirah et al.,“DU-145 and PC-3 human prostate cancer cell lines express androgen receptor:

implications for the androgen receptor functions and regulation”, FEBS Lett. (2006),

580(9):2294-300.

An et al.,“Destruction of Full-Length Androgen Receptor by Wild-Type SPOP, but Not Prostate-Cancer- Associated Mutants”, Cell Rep (2014), 6(4): 657-69.

Barbieri et al.,“Exome sequencing identifies recurrent SPOP, FOXA1 and MED 12 mutations in prostate cancer”, Nat Genet. (2012), 44(6):685-9.

Blattner et al.,“SPOP Mutation Drives Prostate Tumorigenesis In Vivo through Coordinate Regulation of PI3K/mTOR and AR Signaling”, Cancer Cell. (2017), 31(3): 436-451.

Blee et al.,“TMPRSS2-ERG Controls Luminal Epithelial Lineage and Antiandrogen Sensitivity in PTEN and TP53-Mutated Prostate Cancer”, Clin Cancer Res (2018), 24(18): 4551-65.

Dai et al.,“Prostate cancer-associated SPOP mutations confer resistance to BET inhibitors through

stabilization of BRD4.” Nat Med. (2017), 23(9): 1063-1071.

Drost et al.,“Organoid culture systems for prostate epithelial and cancer tissue”, Nat Protoc (2016),

11(2): 347-58.

Janouskova et al.,“Opposing effects of cancer type-specific SPOP mutations on BET protein degradation and sensitivity to BET inhibitors”, Nat Med. (2017), 23(9): 1046-1054.

Jin et al.,“DUB3 Promotes BET Inhibitor Resistance and Cancer Progression by Deubiquitinating

BRD4”, Mol Cell. (2018), 71(4): 592-605.e4.

Lin et al.,“High fidelity patient-derived xenografts for accelerating prostate cancer discovery and drug development”, Cancer Res (2014), 74(4): 1272-83.

Ma et al.,“SPOP promotes ATF2 ubiquitination and degradation to suppress prostate cancer

progression”, Journal of Experimental & Clinical Cancer Research (2018) 37: 145. Taylor et al.,“Fragment-Based Discovery of a Selective and Cell-Active Benzodiazepinone CBP/EP300 Bromodomain Inhibitor (CPI-637)”, ACS Med Chem Lett. (2016), 7(5):531-6.

Wang et al.,“Development and characterization of efficient xenograft models for benign and malignant human prostate tissue”, Prostate (2005), 64(2): 149-59.

Wei et al.,“Functional roles of Speckle-Type Poz (SPOP) Protein in Genomic stability”, J Cancer (2018), 9(18): 3257-3262.

Yan et al.,“Dual inhibition of AKT-mTOR and AR signaling by targeting HDAC3 in PTEN- or SPOP- mutated prostate cancer”, EMBO Mol Med (2018), 10(4), pii: e8478.

Zhang et al.,“Intrinsic BET inhibitor resistance in prostate cancer caused by SPOP mutation-mediated BET protein stabilization”, Nat Med. (2017), 23(9): 1055-1062.

Zhuang et al.,“Structures of SPOP-Substrate Complexes: Insights into Molecular Architectures of BTB- Cul3 Ubiquitin Ligases”, Mol. Cell. (2009), 36(1): 39-50.

Claims

1. A method for treating a cancer responsive to co-therapy with a bromodomain and extraterminal protein (BET) inhibitor and a CREB binding protein (CBP)/EP300 inhibitor, the method comprising: providing a subject having cancer cells that overexpress BET protein substrates of wild-type E3 ubiquitin ligase substrate-binding adaptor speckle-type POZ protein (SPOP) relative to corresponding non-cancerous cells; and

administering a therapeutically effective amount of a BET inhibitor and a CBP/EP300 inhibitor to the subject having the BET protein-overexpressing cancer cells.

2. The method of claim 1, wherein the BET protein-overexpressing cancer cells express an SPOP variant that impairs SPOP -mediated BET protein degradation.

3. The method of claim 2, wherein the BET protein-overexpressing cancer cells express an SPOP variant comprising a mutation in an amino acid residue of the MATH domain defined by amino acid positions 28-166 of human wild-type SPOP.

4. The method of claim 2 or 3, the SPOP variant comprises a mutation in an amino acid residue of the substrate-binding cleft that impairs substrate interaction and ubiquitin transfer.

5. The method of claim 4, wherein the mutation in the substrate-binding cleft occurs at R70, Y87,

FI 02, Y123, K129, D130, W131, or F133 with respect to wild-type SPOP.

6. The method of any one of claims 1 to 5, further comprising genotyping the cancer cells for the presence of a mutant SPOP gene encoding the SPOP variant as defined in any one of claims 2 to 5, and/or measuring a BET protein expression levels in the cancer cells, wherein the BET proteins measured comprise BET protein substrates of wild-type SPOP.

7. The method of any one of claims 1 to 6, wherein the BET protein-overexpressing cancer cells are resistant to BET inhibitor mono-treatment.

8. The method of any one of claims 1 to 7, wherein the cancer is a solid cancer.

9. The method of claim 8, wherein the solid cancer is a sarcoma, a carcinoma, a lymphoma, melanoma, glioma, prostate cancer, lung cancer, breast cancer, gastric cancer, liver cancer, colon cancer, tongue cancer, thyroid cancer, renal cancer, uterine cancer, cervical cancer, ovarian cancer, or pancreatic cancer.

10. The method of any one of claims 1 to 9, wherein the BET inhibitor administered to the subject is a small molecule compound.

11. The method of any one of claims 1 to 10, wherein the BET inhibitor administered to the subject is a bromodomain-containing protein 2 (BRD2) inhibitor, bromodomain-containing protein 3 (BRD3) inhibitor, or bromodomain-containing protein 4 (BRD4) inhibitor.

12. The method of any one of claims 1 to 11, wherein the BET inhibitor administered to the subject is CPI-0610, DUAL946, GSK525762, 1-BET151, JQ1, OTX015, PFI-1, RVX-208, RVX2135, TEN-010, or a derivative thereof having BRD2, BRD3, or BRD4-inhibiting activity.

13. The method of any one of claims 1 to 12, wherein the CBP/EP300 inhibitor administered to the subject is a small molecule compound.

14. The method of any one of claims 1 to 13, wherein the CBP/EP300 inhibitor administered to the subject binds the CBP histone acetyltransferase (HAT) domain and/or the EP300 HAT domain and inhibits and/or reduces a biological activity of CBP and/or EP300.

15. The method of any one of claims 1 to 9, wherein the BET inhibitor and the CBP/EP300 inhibitor are the same compound, and said compound is a substituted benzimidazole that binds BRD4 and CBP/EP300.

16. The method of claim 15, wherein the compound is a substituted benzimidazole of Formula A, or a pharmaceutically acceptable salt, solvate, ester, or prodrug thereof:

Formula A

wherein,

R¹ is:

(a) an unsubstituted Ci-Cealkyl;

(b) a Ci-Cealkyl substituted with one or more group(s) selected from halogen (such as fluorine), CN, NOz, C(0)NHRⁿ, C(0)N(Rⁿ)₂, C0₂H, S0₂Rⁿ, S0₂NHRⁿ, and S0₂N(Rⁿ)₂;

(c) a C₂-Cealkyl group substituted with a group selected from OR¹¹, halogenated OCi-Cealkyl, SH, SR¹¹, NH₂, NHR¹¹, N(R")₂. NHC(0)Rⁿ, and N(Rⁿ)C(0)Rⁿ; or

(d) a group selected from C(0)Rⁿ, C(0)NHRⁿ, C(0)N(Rⁿ)₂, S0₂Rⁿ, SCENHRu, and S0₂N(Rⁿ)₂; R² is selected from hydrogen, N¾ and a substituted or unsubstituted group selected from Ci-Cealkyl, C2- Cealkenyl, C2-Cealkynyl, C3-Ciocycloalkyl, C3-Cioheterocycloalkyl, C(0)R¹², NHR¹², N(R¹²)₂, C(0)NH_¾ C(0)NHR¹², C(0)N(R¹²)_¾ NHC(0)R¹², SO2R¹², SO2NHR¹², S0₂N(R¹²)₂, NHSO2R¹², N(R¹²)S0₂R¹², NHSO2NHR¹², N(R¹²)S0₂NHR¹², NHS0₂N(R¹²)₂, and N(R¹²)S0₂N(R¹²)₂;

R³ and R⁶ are each independently H, NH₂ or a substituted or unsubstituted group selected from Ci-Cealkyl, C(0)Rⁿ, NHR¹¹, N(Rⁿ)₂, C(0)NH₂, C(0)NHRⁿ, C(0)N(Rⁿ)_¾ NHC(0)Rⁿ; and

one of R⁴ and R⁵ is H, N¾ or a substituted or unsubstituted group selected from Ci-Cealkyl, C(0)Rⁿ, NHR¹¹, N(Rⁿ)_¾ C(0)NH₂, C(0)NHRⁿ, C(0)N(Rⁿ)₂, and NHC(0)Rⁿ; the other of R⁴ and R⁵ is a group of Formula A.I:

Formula A.I

wherein,

R⁷, R⁸, and R^{1 0} are each independently H, halogen (such as F, Cl), CN, or a substituted or unsubstituted Ci-Cealkyl or C₃-C₆cycloalkyl group, OR¹¹, SR¹¹, NHR¹¹, N(Rⁿ)_¾ NHC(0)Rⁿ, and N(Rⁿ)C(0)Rⁿ, provided that at least one of R⁷, R⁸, and R^{1 0} is other than H;

R⁹ is a substituted or unsubstituted Ci-C3alkyl or CN-Cscycloalkyl group;

R¹² is, independently in each occurrence, a substituted or unsubstituted Ci-Cealkyl, C2-C6alkenyl, C2- Cealkynyl, C3-Ciocycloalkyl, and C3-Cioheterocycloalkyl;

X¹, X², and X³ are each selected from N and C, wherein when X¹, X², or X³ is N, then the R⁷, R⁸, or R¹⁰ attached thereto is absent, provided that at least two of X¹, X², and X³ is C,

wherein when any of the foregoing group contains an alkyl group, then said alkyl is a linear or branched acyclic alkyl group, and

optionally, wherein when any of R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹ and R¹² is substituted, it is

substituted with one or more substituents selected from halogen, Ci-Cealkyl optionally substituted with an oxo group, Ci-Cealkoxy, C3-Cioheterocycloalkyl and Cearyl.

17. The method of claim 16, wherein R⁴ is a group of Formula A.I.

18. The method of claim 17, wherein R⁵ is a substituted or unsubstituted C₁-C₃ alkyl.

19. The method of claim 17, wherein R⁵ is a hydrogen atom.

20. The method of claim 16, wherein R⁵ is a group of Formula A.I.

21. The method of claim 20, wherein R⁴is a substituted or unsubstituted C1-C3 alkyl.

22. The method of claim 20, wherein R⁴ is a hydrogen atom.

23. The method of any one of claims 16 to 22, wherein X¹, X², and X³ are all carbon atoms.

24. The method of any one of claims 16 to 22, wherein X¹ is a nitrogen atom and R¹⁰ is absent, and

X² and X³ are carbon atoms.

25. The method of any one of claims 16 to 24, wherein R⁹ is an unsubstituted C₁-C₃ alkyl or C₃- C₅ cycloalkyl group.

26. The method of claim 25, wherein R⁹ is selected from methyl, ethyl, n-propyl, isopropyl, and cyclopropyl.

27. The method of any one of claims 16 to 26, wherein R⁸ is halogen (such as F, Cl), CN, or a substituted or unsubstituted Ci-Cealkyl or C3-C6cycloalkyl group, OR¹¹, SR¹¹, NHR¹¹, N(Rⁿ)2,

NHC(0)R¹¹ , or N(R' 'jCTOjR^{1 1}.

28. The method of claim 23, wherein R⁷ and R¹⁰ are each hydrogen atoms and R⁸ is selected from Cl, CN, NHR¹¹ and a substituted or unsubstituted Ci-Cealkyl, or C3-C6cycloalkyl group.

29. The method of any one of claims 16 to 28, wherein R³ is H or a substituted or unsubstituted Ci- Cealkyl group.

30. The method of claim 29, wherein R³ is H.

31. The method of any one of claims 16 to 30, wherein R⁶ is H or a substituted or unsubstituted Ci-

Cealkyl group.

32. The method of claim 31, wherein R⁶ is H.

33. The method of claim 16, wherein the compound of Formula A is a compound of Formula A.II:

Formula A.II wherein R¹, R², R⁴ and R⁵ are as defined in claim 16, or a pharmaceutically acceptable salt, solvate, ester or prodrug thereof.

34. The method of claim 33, wherein the compound of Formula A.II is a compound of Formula A.III:

Formula A.III

wherein,

R¹, R², R⁷, R⁸, R⁹, and R¹⁰ are as defined in claim 16, or a pharmaceutically acceptable salt, solvate, ester or prodrug thereof.

35. The method of claim 33, wherein the compound of Formula A.II is a compound of Formula

A.IV:

Formula A.IV

wherein,

36. The method of claim 34 or 35, wherein R⁹ is an unsubstituted Ci-C3alkyl or CN-CTcycloalkyl group.

37. The method of claim 34 or 35, wherein R⁹ is selected from methyl, trifluoromethyl, ethyl, n- propyl, isopropyl and cyclopropyl.

38. The method of claim 34 or 35, wherein R⁷ and R¹⁰ are each hydrogen atoms and R⁸ is selected from Cl, CN, NHR¹¹ and a substituted or unsubstituted Ci-Cealkyl or C3-C6cycloalkyl group.

39. The method of claim 34 or 35, wherein R⁸ and R⁹ are each independently a methyl, ethyl, isopropyl, fluoromethyl, difluoromethyl, trifluoromethyl, cyclopropyl, or difluorocyclopropyl group.

40. The method of any one of claims 16 to 39, wherein R² is hydrogen or a substituted or unsubstituted group selected from Ci-Cealkyl, C3-Ciocycloalkyl, or C3-Cioheterocycloalkyl group.

41. The method of claim 40, wherein R² is a substituted or unsubstituted Ci-C3alkyl, C3-C6cycloalkyl, or C3-C6heterocycloalkyl group.

42. The method of claim 40, wherein R² is a substituted or unsubstituted group selected from methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, cyclopropyl, cyclobutyl, cyclopentyl,

tetrahydrofuranyl, tetrahydropyranyl, dioxolanyl, piperidinyl, and pyrrolidinyl.

43. The method of any one of claims 16 to 42, wherein R¹ is a branched or linear unsubstituted Ci- Cealkyl.

44. The method of any one of claims 16 to 42, wherein R¹ is a branched or linear Ci-Cealkyl substituted with one or more fluorine atom(s), or a branched or linear C2-Cealkyl substituted with a OCi- Cealkyl group or halogenated OCi-Cealkyl group.

45. The method of claim 44, wherein R¹ is a branched or linear C2-C3alkyl substituted with a group selected from fluorine, OCi-Cealkyl, and halogenated OCi-Cealkyl.

46. The method of any one of claims 16 to 45, wherein R¹ is branched.

47. The method of claim 44, wherein R¹ is fluoromethyl, difluoromethyl, trifluoromethyl, 2,2,2- trifluoroethyl, 2-methoxyethyl, 2-ethoxyethyl, 2-(fluoromethoxy)ethyl, 2-(difluoromethoxy)ethyl, 2- (trifluoromethoxy)ethyl, 3,3 ,3 -trifluoro-1 -propyl, 3-methoxy-l-propyl, 3 -ethoxy- 1 -propyl, 3- (fluoromethoxy) - 1 -propyl, 3 -(difluoromethoxy) - 1 -propyl, 3 -(trifluoromethoxy) - 1 -propyl, 1 -methoxy -2- propyl, 1 -ethoxy -2 -propyl, l-(fluoromethoxy)-2 -propyl, l-(difluoromethoxy)-2-propyl, 1- (trifluoromethoxy)-2-propyl, 2-methoxy-l-propyl, 2-ethoxy- 1 -propyl, 2-(fluoromethoxy)-l-propyl, 2- (difluoromethoxy)-l -propyl, or 2-(trifluoromethoxy)-l-propyl.

48. The method of any one of claims 16 to 42, wherein R¹ is 2-methoxyethyl, 2- (trifluoromethoxy)ethyl, 1 -methoxy -2 -propyl, l-(trifluoromethoxy)-2-propyl, 2-methoxy-l-propyl, or 2- (trifluoromethoxy)- 1 -propyl.

49. The method of claim 16, wherein the compound of Formula A is selected from Compounds A1 to A67 of Fig. 13, or a pharmaceutically acceptable salt, solvate, or prodrug thereof.

50. The method of claim 15, wherein the compound is a substituted benzimidazole of Formula A.V

Formula A.V

wherein R² is selected from hydrogen, NEC and a substituted or unsubstituted group selected from Ci- Cealkyl, C2-C6alkenyl, C2-Cealkynyl, C3-Ciocycloalkyl, C3-Cioheterocycloalkyl, C(0)R¹², NHR¹², N(R¹²)₂, C(0)NH_¾ C(0)NHR¹², C(0)N(R¹²)₂, NHC(0)R¹², SO_ZR¹², SO2NHR¹², S0₂N(R¹²)₂, NHSO2R¹², N(R¹²)S0₂R¹², NHSO2NHR¹², N(R¹²)S0₂NHR¹², NHS0₂N(R¹²)₂, and N(R¹²)S0₂N(R¹²)₂;

optionally, wherein when any of R² and R¹² is substituted, it is substituted with one or more substituents selected from halogen, Ci-Cealkyl optionally substituted with an oxo group, Ci-Cealkoxy, C3- Cioheterocycloalkyl and Cearyl.

or a pharmaceutically acceptable salt, solvate, ester or prodrug thereof.

51. The method of claim 50, wherein R² is hydrogen or a substituted or unsubstituted group selected from Ci-Cealkyl, C3-Ciocycloalkyl, or C3-Cioheterocycloalkyl group.

52. The method of claim 50, wherein R² is a substituted or unsubstituted Ci-C3alkyl, C3-C6cycloalkyl, or C3-C6heterocycloalkyl group.

53. The method of claim 50, wherein R² is a substituted or unsubstituted group selected from methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, cyclopropyl, cyclobutyl, cyclopentyl, tetrahydrofuranyl, tetrahydropyranyl, dioxolanyl, piperidinyl, and pyrrolidinyl.

54. The method of claim 50, wherein R² is cyclopropyl or tetrahydropyranyl.

55. The method of any one of claims 1 to 9, wherein the BET inhibitor and the CBP/EP300 inhibitor are the same compound, and said compound is an aryl-substituted dihydroquinolinone that binds BRD4 and CBP/EP300.

56. The method of claim 55, wherein the compound is an aryl-substituted dihydroquinolinone of Formula B, or a pharmaceutically acceptable salt, solvate, ester, or prodrug thereof:

Formula B wherein

R¹’ is hydrogen or C -Ce alkyl;

R²’ is hydrogen, halogen, -CH₃, -C(R^a)(R^b)(R^c), -N(R^d)(R^e) or -OCR*);

wherein

R^a is hydrogen or Ci-C’e alkyl;

R^b is hydrogen, hydroxyl or Ci-C’e alkyl;

R^c is C3-C6 cycloalkyl, Ce-Ciu aryl, 3-7 membered heterocycloalkyl, 5-10 membered heteroaryl, (C3-C6 cycloalkyl)Ci-C₆ alkyl, (3-7 membered heterocycloalkyl)Ci-C₆ alkyl, (Ce-Cm aryl)Ci-C₆ alkyl or (5-10 membered heteroaryl)Ci-C₆ alkyl, -0(R^g) or -N(R^g)(R^h);

R^d is hydrogen or Ci-Ce alkyl;

R^e is C1-C6 alkyl, C3-C6 cycloalkyl, Ce-Cm aryl, 3-7 membered heterocycloalkyl, 5-10 membered heteroaryl, (C₃-C₆ cycloalkyl)Ci-C6 alkyl, (3-7 membered heterocycloalky 1)Oi-Ob alkyl, (Ce-Ciu aryl)Ci-C6 alkyl, (5-10 membered heteroaryl)Ci-C₆ alkyl, keto(C₆-Ci₀ aryl) or keto(5-10 membered heteroaryl);

R^f is C1-C6 alkyl, C3-C6 cycloalkyl, 3-7 membered heterocycloalkyl, C6-C10 aryl, 5-10 membered heteroaryl, (C3-C6 cycloalkyl)Ci-C6 alkyl, (3-7 membered heterocycloalky l)Ci-C6 alkyl, (C6-C10 aryl)Ci-C₆ alkyl or (5-10 membered heteroaryl)Ci-C₆ alkyl;

R^g is C1-C6 alkyl, C3-C6 cycloalkyl, C6-C10 aryl, 3-7 membered heterocycloalkyl, 5-10 membered heteroaryl, (C3-C6 cycloalkyl)Ci-C6 alkyl, (3-7 membered heterocycloalky l)Ci-C6 alkyl, (C6-C10 aryl)Ci-C₆ alkyl or (5-10 membered heteroaryl)Ci-C₆ alkyl;

wherein in the definitions of R¹’ and R²’, each of cycloalkyl, heterocycloalkyl, aryl and heteroaryl is

optionally substituted by 1 to 3 groups being halogen, CN, alkyl, haloalkyl, alkoxy or haloalkoxy; and wherein when any of the foregoing group contains an alkyl group, then said alkyl is a linear or branched acyclic alkyl group.

57. The method of claim 56, wherein R¹ is hydrogen or methyl.

58. The method of claim 56, wherein R¹ is hydrogen.

59. The method of any one of claims 56 to 58, wherein R²’ represents -C(R^a)(R^b)(R^c), -C(R^a)(R^b)0(R^g),

-C(R^a)(R^b)N(R^g)(R^h), -N(R^d)(R^e) or -0(R^f)_, wherein:

- R^a is H;

- R^b is H or methyl;

- R^c is a group benzyl, cyclobutyl, cyclopentyl, cyclohexyl, phenyl, thienyl, furanyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl, isothiazolyl, thiadiazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, indolizinyl, purinyl, naphthyridinyl, pteridinyl, [l,3]dioxolane, pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, tetrahydrofuryl or tetrahydropyranyl, each group R^c being optionally substituted by 1 to 3 groups being halogen, CN, alkyl, haloalkyl, alkoxy or haloalkoxy;

- R^d is hydrogen or methyl;

- R^e is a group cyclobutyl, cyclopentyl, cyclohexyl, phenyl, thienyl, furanyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl, isothiazolyl, thiadiazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, indolizinyl, purinyl, naphthyridinyl, pteridinyl, [l,3]dioxolane, pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, tetrahydrofuryl, or tetrahydropyranyl, each of these groups being optionally substituted by 1 to 3 groups being halogen, CN, alkyl, haloalkyl, alkoxy or haloalkoxy, or R^e is a group -CH(R^h)(C₃-C₆ aryl) or -CH(R^h)(5-10 membered heteroaryl);

- R^f is C3-C6 cycloalkyl, C6-C10 aryl, 3-10 membered heterocycloalkyl, 5-10 membered heteroaryl, - CH(R^h)(C3-C6 cycloalkyl), -CH(R^h)(C3-C6 aryl), -CH(R^h)(3-7 membered heterocycloalkyl) or - CH(R^h)(5-10 membered heteroaryl);

- R^g is a group methyl, ethyl, propyl, cyclobutyl, cyclopentyl, cyclohexyl, phenyl, thienyl, furanyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl, isothiazolyl, thiadiazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, indolizinyl, purinyl, naphthyridinyl, pteridinyl, [l,3]dioxolane, pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, tetrahydrofuryl or tetrahydropyranyl, each group R^g being optionally substituted by 1 to 3 groups being halogen, CN, alkyl, haloalkyl, alkoxy or haloalkoxy; - R^h is hydrogen, linear or branched C₁-C₄ alkyl or -(CO)R⁶ with R⁶ being a C₁-C₃ alkyl group; and wherein each of cycloalkyl, heterocycloalkyl, aryl and heteroaryl is optionally substituted by 1 to 3 groups being halogen, CN, alkyl, haloalkyl, alkoxy or haloalkoxy.

60. The method of any one of claims 56 to 58, wherein R² represents -C(R^a)(R^b)(R^c), -C(R^a)(R^b)0(R^g), -C(R^a)(R^b)N(R^g)(R^h), -N(R^d)(R^e) or -0(R^f)_, wherein:

- R^a is H;

- R^b is H or methyl;

- R^c is a group phenyl, benzyl, piperidinyl, tetrahydropyranyl or pyridyl;

- R^d is hydrogen or methyl;

- R^e is cyclohexyl, phenyl, benzyl, pyridyl, tetrahydropyranyl, -CH₂(pyridyl) or -CH(CH₃) (pyridyl);

- R^f is phenyl, cyclobutyl, cyclopentyl, cyclohexyl, benzyl, -CH₂(pyridyl), -CH(CH₃)(pyridyl), -CH₂(pyrazinyl), -CH₂(pyrimidinyl), -CH₂(tetrahydropyranyl), -CH₂(cyclopentyl),

-CH₂(cyclohexyl), -CH₂(oxazolyl) or -CH₂(thiazolyl);

- R^g is ethyl, phenyl, cyclobutyl, cyclopentyl, cyclohexyl or tetrahydropyranyl;

- R^h is hydrogen, methyl or acetyl;

wherein each of R^c, R^e, R^f and R^g is optionally substituted by a 1 to 3 groups being halogen, CN, Ci-Ce alkyl or C1-C6 alkoxy.

61. The method of any one of claims 56 to 58, wherein R² represents a group:

62. The method of any one of claims 56 to 58, wherein R^2’ represents a group:

63. The method of any one of claims 56 to 58, wherein R² represents a group:

64. A method of identifying a subject suitable for anti-cancer co-therapy with a BET inhibitor and a CBP/EP300 inhibitor, the method comprising:

(a) providing a biological sample comprising cancer cells from the subject;

(b) measuring the presence of absence of biomarker in the cancer cells indicative of

responsiveness to BET inhibitor and CBP/EP300 inhibitor co-therapy, wherein the biomarker is: overexpression of BET protein(s) in the cancer cells relative to corresponding non-cancerous cells; and/or the expression of an SPOP variant in the cancer cells that impairs SPOP -mediated BET protein degradation; and

(c) identifying the subject as being suitable for anti -cancer co-therapy with a BET inhibitor and a CBP/EP300 inhibitor when the cancer cells are positive for the biomarker.

65. The method of claim 64, wherein the biomarker is measured by determining the expression of the SPOP variant by genotyping the cancer cells for the presence of a mutant SPOP gene encoding the SPOP variant, and/or measuring BET protein expression levels in the cancer cells.

66. The method of claim 65, wherein the SPOP variant is as defined in any one of claims 3 to 5.

67. The method of any one of claims 64 to 66, wherein the cancer cells or type of cancer is as defined in any one of claims 7 to 9.

68. The method of any one of claims 64 to 66, further comprising administering a therapeutically effective amount of a BET inhibitor and a CBP/EP300 inhibitor to the subject when the subject’s cancer cells are positive for the biomarker.

69. The method of claim 68, wherein:

(i) the BET inhibitor is as defined in any one of claims 10 to 12;

(ii) the CBP/EP300 inhibitor is as defined in claim 13 or 14; or

(iii) the BET inhibitor and the CBP/EP300 inhibitor are the same compound, and said compound is as defined in any one of claims 15 to 54 or 55 to 63.

70. A composition comprising:

(i) the BET inhibitor as defined in any one of claims 10 to 12, and the CBP/EP300 inhibitor as defined in claim 13 or 14; or

(ii) the compound as defined in any one of claims 15 to 54 or 55 to 63,

and a pharmaceutically acceptable excipient, for use in the treatment of BET inhibitor resistant cancer cells in a subject.

71. The composition for the use of claim 70, wherein the BET inhibitor resistant cancer cells overexpress BET protein substrates of SPOP relative to corresponding non-cancerous cells, and/or express an SPOP variant that impairs SPOP -mediated BET protein degradation.

72. The composition for the use of claim 71, wherein: the SPOP variant is as defined in any one of claims 3 to 5; and/or the cancer cells or type of cancer is as defined in any one of claims 7 to 9.

73. The composition for the use of any one of claims 70 to 72, wherein the subject is assessed for the presence of the biomarker as defined in claim 64 or 65.

74. Use of:

(ii) the compound as defined in any one of claims 15 to 54 or 55 to 63,

for treating BET inhibitor resistant cancer cells in a subject.

75. Use of:

(ii) the compound as defined in any one of claims 15 to 54 or 55 to 63,

for the manufacture of a medicament for treating BET inhibitor resistant cancer cells in a subject.

76. The use of claim 74 or 75, wherein the BET inhibitor resistant cancer cells overexpress BET protein substrates of SPOP relative to corresponding non-cancerous cells, and/or express an SPOP variant that impairs SPOP -mediated BET protein degradation.

77. The use of claim 76, wherein: the SPOP variant is as defined in any one of claims 3 to 5; and/or the cancer cells or type of cancer is as defined in any one of claims 7 to 9.

78. The use of any one of claims 74 to 77, wherein the subject is assessed for the presence of the biomarker as defined in claim 64 or 65.

79. The use of any one of claims 74 to 78, wherein the cancer cells or type of cancer is as defined in any one of claims 7 to 9.

80. A compound as defined in any one of claims 15 to 54 or 55 to 63 for use as a CBP/EP300 inhibitor in a CBP/EP300 inhibition therapy.

81. The compound for the use of claim 80, wherein the CBP/EP300 inhibition therapy does not comprise an additional CBP/EP300 inhibitor other than said compound.

82. The compound for the use of claim 80 or 81, wherein the CBP/EP300 inhibition therapy does not comprise a BET inhibitor other than said compound.

83. A compound as defined in any one of claims 15 to 54 or 56 to 63 for use as a BET inhibitor and a CBP/EP300 inhibitor in a BET and CBP/EP300 co-inhibition therapy.

84. The compound for the use of claim 83, wherein the BET and CBP/EP300 co-inhibition therapy does not comprise an additional CBP/EP300 inhibitor other than said compound.

85. The compound for the use of claim 83 or 84, wherein the BET and CBP/EP300 co-inhibition therapy does not comprise a BET inhibitor nor a CBP/EP300 inhibitor other than said compound.

86. The compound for the use of any one of claims 80 to 85, for use in the treatment of a disease ameliorated by BET and CBP/EP300 dual inhibition therapy.

87. Use of the compound as defined in any one of claims 15 to 54 or 55 to 63 as a CBP/EP300 inhibitor in a CBP/EP300 inhibition therapy.

88. The use of claim 87, wherein the CBP/EP300 inhibition therapy does not comprise an additional CBP/EP300 inhibitor other than said compound.

89. The use of claim 87 or 88, wherein the CBP/EP300 inhibition therapy does not comprise a BET inhibitor other than said compound.

90. Use of the compound as defined in any one of claims 15 to 54 or 55 to 63 as a BET inhibitor and a CBP/EP300 inhibitor in a BET and CBP/EP300 co-inhibition therapy.

91. The use of claim 90, wherein the BET and CBP/EP300 co-inhibition therapy does not comprise an additional CBP/EP300 inhibitor other than said compound.

92. The use of claim 90 or 91, wherein the BET and CBP/EP300 co-inhibition therapy does not comprise a BET inhibitor other than said compound.

93. The use of any one of claims 87 to 92, for the treatment of a disease ameliorated by BET and CBP/EP300 co-inhibition therapy.

94. Use of the compound as defined in any one of claims 15 to 54 or 55 to 63 for the manufacture of a medicament for CBP/EP300 inhibition therapy, wherein the CBP/EP300 inhibition therapy does not comprise an additional CBP/EP300 inhibitor other than said compound.

95. Use of the compound as defined in any one of claims 15 to 54 or 55 to 63 for the manufacture of a medicament for BET and CBP/EP300 co-inhibition therapy, wherein the BET and CBP/EP300 co- inhibition therapy does not comprise a BET inhibitor nor a CBP/EP300 inhibitor other than said compound.

96. The use of claim 94 or 95, in the treatment of a disease ameliorated by BET and CBP/EP300 co inhibition therapy.