JP5715241B2 - Compositions and methods for treating neoplasms, inflammatory diseases, and other disorders - Google Patents

Description

This application is related to US Provisional Patent Application No. 61 / 334,991, filed May 14, 2010, US Provisional Patent Application No. 61 / filed on August 4, 2010, and No. 370,745, US Provisional Patent Application No. 61 / 375,863, filed Aug. 22, 2010, and US Provisional Patent Application No. 61/467, filed Mar. 24, 2011. , 376 claim priority. The contents of each of these patent applications are incorporated herein by reference in their entirety.

Declaration of Invention Rights Made under Federally Assisted Research and Development This study was supported by grant number K08CA128972 from the National Institute of Health. The US government has certain rights in the invention.

  The histone N-terminal tail preserves chromatin stability and is the subject of modifications associated with transcriptional regulation. Among these modifications, acetylation, methylation, and phosphorylation are the best characterized. For each modification, there is an enzyme that applies or removes the appropriate label. These modifications must then be interpreted by the transcription machinery. Acetyllysine recognition is mediated primarily by bromodomains, which are generally components of transcription factor complexes. Bromodomains and the extra terminal (BET) family (eg, BRD2, BRD3, BRD4, and BRDT) are common domain structures that comprise two N-terminal bromodomains that exhibit high levels of sequence conservation and protein-protein interactions. Share a wider variety of C-terminal domains that are believed to be related to Abnormal regulation of histone modifications can affect gene activity and contribute to carcinogenesis. Lysine side chain acetylation is a key regulatory event in the function of non-histone proteins including but not limited to Hsp90, p53, STAT transcription factor, cortactin, β-catenin, and α-tubulin. . Thus, it is expected that modulation of lysine side chain recognition will exert significant expression and therapeutic effects widely in development and disease. Despite the importance of acetyllysine recognition for carcinogenesis, few modulators of acetyllysine recognition have been identified.

  As described below, the present invention relates to neoplasms, inflammatory diseases, obesity, fatty liver (NASH or not), diabetes, atherosclerosis, arterial stent occlusion, heart failure, cachexia, graft pair It features compositions and methods for treating or preventing host diseases, infectious diseases associated with bromodomains, treating parasites, malaria, trypanosoma, and reducing male fertility. In certain embodiments, the compounds of the present invention are used to overcome drug resistance of neoplasms (eg, cancer and non-malignant diseases). Further uses of the compositions of the present invention include, but are not limited to, use in organ transplantation, cell condition regulation for regenerative medicine (ie by promoting or inhibiting cell differentiation), and promoting pluripotency. It is not a thing. More specifically, the present invention prevents the interaction of a BET family polypeptide comprising a bromodomain with acetyllysine and / or chromatin (eg, bromodomain and acetyl present on the histone N-terminal tail). Compositions and methods are provided that interfere with interaction with lysine modifications) and inhibit carcinogenesis. In another embodiment, the present invention prevents or treats inflammatory diseases.

In one aspect, the invention provides a compound of formula I,

Where
X is N or CR ₅ ;
R ₅ is H, alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, each optionally substituted;
R _B is H, alkyl, hydroxyaralkyl, aminoalkyl, alkoxyalkyl, haloalkyl, hydroxy, alkoxy, or —COO—R ₃ , each optionally substituted;
Ring A is aryl or heteroaryl;
Each R _A is independently alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, each optionally substituted; or any two R _A are atoms attached to each of them Together can form a fused aryl or heteroaryl group;
R is alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, each optionally substituted;
R ₁ is — (CH ₂ ) _n —L (where n is 0 to 3 and L is H), —COO—R ₃ , —CO—R ₃ , —CO—N (R ₃ R _{4), - S (O)} 2 -R 3, -S (O) 2 -N (R 3 R 4), N (R 3 R 4), N (R 4) C (O) R 3, optionally Optionally substituted aryl, or optionally substituted heteroaryl;
R ₂ is H, D, halogen, or optionally substituted alkyl;
Each R ₃ is independently
(I) H, aryl, substituted aryl, heteroaryl, or substituted heteroaryl;
(Ii) heterocycloalkyl or substituted heterocycloalkyl;
(Iii) O, S or is selected from N, containing 0, 1, 2, or 3 heteroatoms each, _-C 1 -C ₈ alkyl, _-C 2 -C ₈ alkenyl, or -C _2, -C ₈ alkynyl; each may be optionally _substituted, -C 3 _{-C 12} cycloalkyl, substituted _-C 3 _{-C 12 cycloalkyl,} -C 3 _{-C 12} cycloalkenyl or substituted -C, ₃ -C ₁₂ cycloalkenyl; and _{(iv) NH 2, N =} CR 4 R 6
Selected from the group consisting of;
Each R ₄ is independently H, alkyl, alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, each optionally substituted; or R ₃ and R ₄ are attached to them Together with the nitrogen atom to form a 4-10 membered ring;
R ₆ is alkyl, alkenyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, aryl, or heteroaryl, each optionally substituted; or R ₄ and R ₆ are the carbon atoms to which they are attached. Together with it to form a 4-10 membered ring;
m is 0, 1, 2, or 3;
However (a) Ring A is thienyl, X is N, R is phenyl or substituted phenyl, _{R 2} is H, _{R B} is methyl, _{R 1} is - _{(CH 2) n} - If L (wherein n is 1 and L is —CO—N (R ₃ R ₄ )) then R ₃ and R ₄ will not come with the nitrogen atom to which they are attached. Does not form a morpholino ring;
(B) Ring A is thienyl, X is N, R is a substituted phenyl, _{R 2} is H, _{R B} is methyl, _{R 1} is - _{(CH 2)} n -L _(wherein Wherein n is 1 and L is —CO—N (R ₃ R ₄ ), and if one of R ₃ and R ₄ is H, then the other of R ₃ and R ₄ is methyl , Not hydroxyethyl, alkoxy, phenyl, substituted phenyl, pyridyl or substituted pyridyl;
(C) Ring A is thienyl, X is N, R is a substituted phenyl, _{R 2} is H, _{R B} is methyl, _{R 1} is - _{(CH 2)} n -L _(wherein In which n is 1 and L is —COO—R ₃ ), then R ₃ is a compound that is neither methyl nor ethyl, or a salt, solvate or hydrate thereof.

  In certain embodiments, R is aryl or heteroaryl, each optionally substituted.

In certain _{embodiments, L} is _{H, -COO-R 3, -CO} -N (R 3 R 4), - S (O) 2 -R 3, -S (O) 2 -N (R 3 R 4 ), N (R ₃ R ₄ ), N (R ₄ ) C (O) R ₃ or optionally substituted aryl. In certain embodiments, each R ₃ is independently H; —C ₁ -C ₈ alkyl containing 0, 1, 2, or 3 heteroatoms selected from O, S, or N; or NH ₂ ; selected from the group consisting of N = CR ₄ R ₆ .

In certain embodiments, R ₁ is — (CH ₂ ) _n —L, wherein n is 1 and L is —CO—N (R ₃ R ₄ ), and R ₃ and R ₄ One is H, the other of R ₃ and R ₄ is (CH ₂ ) _p —Y, p is 1-3 (eg, p is 2) and Y is (aromatic or non-aromatic) Good) A nitrogen-containing ring.

In certain embodiments, R ₂ is H, D, halogen or methyl.

In certain embodiments, R _B is alkyl, hydroxyalkyl, haloalkyl, or alkoxy, each optionally substituted.

In certain embodiments, R _B is methyl, ethyl, hydroxymethyl, methoxymethyl, trifluoromethyl, COOH, COOMe, COOEt, or COOCH ₂ OC (O) CH ₃ .

  In certain embodiments, Ring A is a 5 or 6 membered aryl or heteroaryl. In certain embodiments, ring A is thiofuranyl, phenyl, naphthyl, biphenyl, tetrahydronaphthyl, indanyl, pyridyl, furanyl, indolyl, pyrimidinyl, pyridinyl, pyrazinyl, imidazolyl, oxazolyl, thienyl, thiazolyl, triazolyl, isoxazolyl, quinolinyl, pyrrolyl, Pyrazolyl or 5,6,7,8-tetrahydroisoquinolinyl.

  In certain embodiments, Ring A is phenyl or thienyl.

In certain embodiments, m is 1 or 2 and at least one occurrence of R _A is methyl.

In certain embodiments, each R _A is independently H, optionally substituted alkyl, or any two R _A can form an aryl together with the atoms to which each is attached.

In another aspect, the present invention provides compounds of formula II,

Where
X is N or CR ₅ ;
R ₅ is H, alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, each optionally substituted;
R _B is H, alkyl, hydroxyaralkyl, aminoalkyl, alkoxyalkyl, haloalkyl, hydroxy, alkoxy, or —COO—R ₃ , each optionally substituted;
Each R _A is independently alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, each optionally substituted; or any two R _A are atoms attached to each of them Together can form a fused aryl or heteroaryl group;
R is alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, each optionally substituted;
R ′ ₁ is H, —COO—R ₃ , —CO—R ₃ , optionally substituted aryl, or optionally substituted heteroaryl;
Each R ₃ is independently
(I) H, aryl, substituted aryl, heteroaryl, substituted heteroaryl;
(Ii) heterocycloalkyl or substituted heterocycloalkyl;
(Iii) O, S or is selected from N, containing 0, 1, 2, or 3 heteroatoms each, _-C 1 -C ₈ alkyl, _-C 2 -C ₈ alkenyl, or -C _2, -C ₈ alkynyl; each may be optionally _substituted, -C 3 _{-C 12} cycloalkyl, substituted _-C 3 _{-C 12 cycloalkyl,} -C 3 _{-C 12} cycloalkenyl or substituted -C, Selected from the group consisting of _{3 to} C ₁₂ cycloalkenyl;
m is 0, 1, 2, or 3;
Provided that when R ′ ₁ is —COO—R ₃ , X is N, R is substituted phenyl, and R _B is methyl, then R ₃ is not methyl or ethyl, or a salt thereof, Solvates or hydrates are provided.

  In certain embodiments, R is aryl or heteroaryl, each optionally substituted. In certain embodiments, R is phenyl or pyridyl, each optionally substituted. In certain embodiments, R is p-Cl-phenyl, o-Cl-phenyl, m-Cl-phenyl, p-F-phenyl, o-F-phenyl, m-F-phenyl or pyridinyl.

In certain embodiments, R ′ ₁ is —COO—R ₃ , optionally substituted aryl, or optionally substituted heteroaryl; R ₃ is selected from O, S, or N -C ₁ -C ₈ alkyl, which contains 0, 1, 2, or 3 heteroatoms and is optionally substituted. In certain embodiments, R ′ ₁ is —COO—R ₃ and R ₃ is methyl, ethyl, propyl, i-propyl, butyl, sec-butyl, or t-butyl; or R ′ ₁ is H or optionally substituted phenyl.

In certain embodiments, R _B is methyl, ethyl, hydroxymethyl, methoxymethyl, trifluoromethyl, COOH, COOMe, COOEt, COOCH ₂ OC (O) CH ₃ .

In certain embodiments, R _B is methyl, ethyl, hydroxymethyl, methoxymethyl, trifluoromethyl, COOH, COOMe, COOEt, or COOCH ₂ OC (O) CH ₃ .

In certain embodiments, each R _A is independently an optionally substituted alkyl, or any two R _A together with the atom to which each is attached can form a fused aryl.

In certain embodiments, each R _A is methyl.

In another aspect, the present invention provides compounds of formula III,

Where
X is N or CR ₅ ;
R ₅ is H, alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, each optionally substituted;
R _B is H, alkyl, hydroxyaralkyl, aminoalkyl, alkoxyalkyl, haloalkyl, hydroxy, alkoxy, or —COO—R ₃ , each optionally substituted;
Ring A is aryl or heteroaryl;
Each R _A is independently alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, each optionally substituted; or any two R _A are atoms attached to each of them Together can form a fused aryl or heteroaryl group;
R is alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, each optionally substituted;
Each R ₃ is independently
(I) H, aryl, substituted aryl, heteroaryl, or substituted heteroaryl;
(Ii) heterocycloalkyl or substituted heterocycloalkyl;
(Iii) O, S or is selected from N, containing 0, 1, 2, or 3 heteroatoms each, _-C 1 -C ₈ alkyl, _-C 2 -C ₈ alkenyl, or -C _2, -C ₈ alkynyl; each may be optionally _substituted, -C 3 _{-C 12} cycloalkyl, substituted _-C 3 _{-C 12 cycloalkyl,} -C 3 _{-C 12} cycloalkenyl or substituted -C, ₃ -C ₁₂ cycloalkenyl; and _{(iv) NH 2, N =} CR 4 R 6
Selected from the group consisting of;
Each R ₄ is independently H, alkyl, alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, each optionally substituted; or R ₃ and R ₄ are attached to them Together with the nitrogen atom to form a 4-10 membered ring;
R ₆ is alkyl, alkenyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, aryl, or heteroaryl, each optionally substituted; or R ₄ and R ₆ are the carbon atoms to which they are attached. Together with it to form a 4-10 membered ring;
m is 0, 1, 2, or 3;
However (a) Ring A is thienyl, X is N, R is phenyl or substituted phenyl, if methyl R _B, then R ₃ and R ₄ and the nitrogen atom to adhere them thereto Do not combine to form a morpholino ring;
(B) Ring A is thienyl, X is N, R is a substituted phenyl, _{R 2} is H, _{R B} is methyl, one of _{R 3} and _{R 4} if H, The other of R ₃ and R ₄ then provides a compound that is methyl, hydroxyethyl, alkoxy, phenyl, substituted phenyl, pyridyl or non-substituted pyridyl, or a salt, solvate or hydrate thereof.

  In certain embodiments, R is aryl or heteroaryl, each optionally substituted. In certain embodiments, R is phenyl or pyridyl, each optionally substituted.

In certain embodiments, R is p-Cl-phenyl, o-Cl-phenyl, m-Cl-phenyl, p-F-phenyl, o-F-phenyl, m-F-phenyl or pyridinyl. In certain embodiments, R ₃ is H, NH ₂ , or N = CR ₄ R ₆ .

In certain embodiments, each R ₄ is independently H, alkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, each optionally substituted.

In certain embodiments, R ₆ is alkyl, alkenyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, aryl, or heteroaryl, each optionally substituted.

In certain embodiments, one of R ₃ and R ₄ is H, the other of R ₃ and R ₄ is (CH ₂ ) _p —Y, p is 1-3 (eg, p is 2), Y is a nitrogen-containing ring (which may be aromatic or non-aromatic).

In another aspect, the present invention provides compounds of formula IV,

Where
X is N or CR ₅ ;
R ₅ is H, alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, each optionally substituted;
R _B is H, alkyl, hydroxyaralkyl, aminoalkyl, alkoxyalkyl, haloalkyl, hydroxy, alkoxy, or —COO—R ₃ , each optionally substituted;
Ring A is aryl or heteroaryl;
Each R _A is independently alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, each optionally substituted; or any two R _A are atoms attached to each of them Together can form a fused aryl or heteroaryl group;
R ₁ is — (CH ₂ ) _n —L (where n is 0 to 3 and L is H), —COO—R ₃ , —CO—R ₃ , —CO—N (R ₃ R _{4), - S (O)} 2 -R 3, -S (O) 2 -N (R 3 R 4), N (R 3 R 4), N (R 4) C (O) R 3, optionally Optionally substituted aryl, or optionally substituted heteroaryl;
R ₂ is H, D, halogen, or optionally substituted alkyl;
Each R ₃ is independently
(I) H, aryl, substituted aryl, heteroaryl, or substituted heteroaryl;
(Ii) heterocycloalkyl or substituted heterocycloalkyl;
(Iii) O, S or is selected from N, containing 0, 1, 2, or 3 heteroatoms each, _-C 1 -C ₈ alkyl, _-C 2 -C ₈ alkenyl, or -C _2, -C ₈ alkynyl; each may be optionally _substituted, -C 3 _{-C 12} cycloalkyl, substituted _-C 3 _{-C 12 cycloalkyl,} -C 3 _{-C 12} cycloalkenyl or substituted -C, ₃ -C ₁₂ cycloalkenyl; and _{(iv) NH 2, N =} CR 4 R 6
Selected from the group consisting of;
Each R ₄ is independently H, alkyl, alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, each optionally substituted; or R ₃ and R ₄ are attached to them Together with the nitrogen atom to form a 4-10 membered ring;
R ₆ is alkyl, alkenyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, aryl, or heteroaryl, each optionally substituted; or R ₄ and R ₆ are the carbon atoms to which they are attached. Together with it to form a 4-10 membered ring;
m is 0, 1, 2, or 3;
However (a) Ring A is thienyl, X is N, _{R 2} is H, _{R B} is methyl, _{R 1} is - _{(CH 2)} in n -L (wherein, n is 0 Yes, L is —CO—N (R ₃ R ₄ ), then R ₃ and R ₄ do not combine with the nitrogen atom to which they are attached to form a morpholino ring;
(B) Ring A is thienyl, X is N, _{R 2} is H, _{R B} is methyl, _{R 1} is - _{(CH 2)} n -L _(wherein, n is 0 , L is —CO—N (R ₃ R ₄ ), one of R ₃ and R ₄ is H, then the other of R ₃ and R ₄ is methyl, hydroxyethyl, alkoxy, phenyl , Not substituted phenyl, pyridyl or substituted pyridyl;
(C) Ring A is thienyl, X is N, _{R 2} is H, _{R B} is methyl, _{R 1} is - _{(CH 2)} n -L _(wherein, n is 0 , L is —COO—R ₃ ), then R ₃ provides a compound that is neither methyl nor ethyl, or a salt, solvate or hydrate thereof.

In certain embodiments, R ₁ is — (CH ₂ ) _n —L, wherein n is 0-3, L is —COO—R ₃ , optionally substituted aryl, or optionally R ₃ is optionally substituted containing 0, 1, 2, or 3 heteroatoms selected from O, S, or N it may also be a _-C 1 -C ₈ alkyl. In certain embodiments, n is 1 or 2, L is alkyl or —COO—R ₃ , and R ₃ is methyl, ethyl, propyl, i-propyl, butyl, sec-butyl, or t-butyl. Or n is 1 or 2, and L is H or optionally substituted phenyl.

In certain embodiments, R ₁ is — (CH ₂ ) _n —L, wherein n is 0 and L is —CO—N (R ₃ R ₄ ), and R ₃ and R ₄ One is H, the other of R ₃ and R ₄ is (CH ₂ ) _p —Y, p is 1-3 (eg, p is 2), and Y is (aromatic or non-aromatic) It may be a nitrogen-containing ring.

In certain embodiments, R ₂ is H or methyl.

In certain embodiments, R _B is methyl, ethyl, hydroxymethyl, methoxymethyl, trifluoromethyl, COOH, COOMe, COOEt, COOCH ₂ OC (O) CH ₃ .

  In certain embodiments, ring A is phenyl, naphthyl, biphenyl, tetrahydronaphthyl, indanyl, pyridyl, furanyl, indolyl, pyrimidinyl, pyridinyl, pyrazinyl, imidazolyl, oxazolyl, thienyl, thiazolyl, triazolyl, isoxazolyl, quinolinyl, pyrrolyl, pyrazolyl, Or 5,6,7,8-tetrahydroisoquinolinyl.

In certain embodiments, each R _A is independently an optionally substituted alkyl, or any two R _A together with the atom to which each is attached can form an aryl.

  The present invention also provides compounds of formulas V-XXII herein and any compound described herein.

  In another aspect, the invention treats a neoplasm in a subject comprising administering to the subject an effective amount of a compound of any of Formulas I-XXII, or any compound described herein. Or provide a way to prevent.

  In certain embodiments, the compound is a compound of any of formulas I-IV.

  In another aspect, the invention contacts a cell with an effective amount of a compound of Formulas I-XXII or any compound described herein, thereby reducing neoplastic cell growth, proliferation or survival. A method of reducing the growth, proliferation or survival of neoplastic cells comprising the step of:

  In another aspect, the invention comprises contacting a cell with a compound of any of Formulas I-XXII or any compound described herein, thereby inducing differentiation in a neoplastic cell. A method is provided for inducing differentiation in a neoplastic cell comprising.

  In another aspect, the invention provides contacting a cell with a therapeutically effective amount of any compound of Formulas I-XXII or any compound described herein, thereby causing the cell to be A method of inducing cell death in a neoplastic cell comprising the step of inducing death is provided.

  In certain embodiments, the method further comprises selecting a compound that binds to a bromodomain of the BET family.

  In certain embodiments, the method further comprises selecting a compound that inhibits binding of the bromodomain to chromatin in the cellular environment.

  In certain embodiments, the method uses differential scanning fluorimetry (DSF), isothermal titration calorimetry, and / or luminescent proximity homogeneous assay (ALPHA-screen) to select compounds for binding specificity. The method further comprises a step. In certain embodiments, the compound increases the thermal stability of the bromodomain in the assay.

  In particular embodiments of the method, the BET family member is BRD2, BRD3, BRD4 or BRDT.

  In certain embodiments of the method, the cell is in a subject.

  In another aspect, the invention administers to a subject in need thereof an effective amount of a compound of any of Formulas I-XXII or any compound described herein, thereby causing a neoplasm in the subject. A method of preventing or treating a neoplasm in a subject comprising the step of preventing or treating

  In certain embodiments, the subject is a mammal. In certain embodiments, the subject is a human patient.

  In certain embodiments, the method reduces neoplastic growth or proliferation in the subject.

  In certain embodiments, the neoplasm is driven by a transcriptional activator. In certain embodiments, the transcriptional activator is myc.

  In certain embodiments, the subject is Burkitt lymphoma, small cell lung cancer, breast cancer, colon cancer, neuroblastoma, glioblastoma multiforme, MLL-driven leukemia, chronic lymphocytic leukemia, NUT median cancer, Having a neoplasm selected from the group consisting of squamous cell carcinoma or any other carcinoma associated with NUT rearrangement.

  In another aspect, the invention provides a therapeutically effective amount of a compound of any of Formulas I-XXII or any compound described herein, and a pharmaceutically acceptable excipient or carrier therefor Is provided.

  In another aspect, the invention provides a packaged pharmaceutical comprising a therapeutically effective amount of any compound of Formulas I-XXII or any compound described herein, and written instructions for administering the compound. I will provide a.

  In another aspect, the invention provides a neoplasm in a subject comprising administering to the subject a therapeutically effective amount of a compound of any of Formulas I-XXII or any compound described herein. Wherein the compound interferes with the binding of the bromodomain to acetyllysine or otherwise replaces the BET family member from chromatin, thereby preventing or treating said neoplasm.

  In certain embodiments, the compound inhibits binding of a histone H4 Kac peptide to a BET family member.

  In certain embodiments, the BET family member is BRD2, BRD3, BRD4 or BRDT.

  In certain embodiments, the compound binds to the Kac binding site of the BET family bromodomain.

  In another aspect, the invention includes contacting a test compound with a BET family member comprising a bromodomain; detecting specific binding to the bromodomain, thereby providing a test compound useful for treating a neoplasia. A method of identifying a compound for neoplastic therapy comprising the step of identifying is provided.

  In certain embodiments, binding specificity is assayed using differential scanning fluorimetry (DSF).

  In certain embodiments, the binding increases the thermal stability of the bromodomain.

  In certain embodiments, binding is detected using an isothermal titration calorimeter.

  In certain embodiments, binding is detected using a luminescence proximity homogeneous assay (ALPHA-screen).

  In certain embodiments, the compound inhibits binding of the histone H4 Kac peptide to the bromodomain.

  In certain embodiments, the compound forms a hydrogen bond with an evolutionarily conserved asparagine in the bromodomain.

  In certain embodiments, the BET family member is BRD4 or BRD2, and the asparagine is Asn140 in BRD4 (1) and Asn429 in BRD2 (2).

  In certain embodiments, the compound binds competitively with chromatin within the cellular environment.

  In certain embodiments, competitive binding with chromatin is detected using post-photobleaching fluorescence recovery (FRAP).

  In certain embodiments, the method is performed in neoplastic cells in vitro.

  In certain embodiments, the method further comprises detecting decreased cell proliferation, increased cell death, or increased cell differentiation.

  In certain embodiments, the cell death is apoptotic cell death.

  In certain embodiments, cell differentiation is identified by detecting increased cytokeratin expression.

  In certain embodiments, the method further comprises detecting a decrease in transcription elongation.

  In another aspect, the invention provides a neoplasm in a subject comprising administering to said subject an effective amount of a compound of any of Formulas I-XXII or any compound described herein. A method of treating or preventing is provided, wherein the compound binds to a BET family bromodomain and interferes with the interaction of the bromodomain and chromatin, thereby preventing or treating the cancer.

  In certain embodiments, the method induces cell death or differentiation in neoplastic cells in the subject.

  In another aspect, the invention provides an effective amount of a compound selected from the group consisting of any compound of Formulas I-XXII or any compound described herein, and a pharmaceutically acceptable excipient. And a composition for treating or preventing a neoplasm comprising inhibiting the binding of a histone H4 Kac peptide to a BET family bromodomain.

  In another aspect, the invention reduces inflammation in a subject comprising administering to the subject an effective amount of a compound of any of Formulas I-XXII or any compound described herein. Provide a method.

  In another aspect, the invention provides the step of administering to a subject in need thereof an effective amount of any compound of Formulas I-XXII or any compound described herein, thereby in the subject. A method for preventing or treating an inflammatory disease in a subject comprising the step of preventing or treating the inflammatory disease.

  In certain embodiments, the subject is a mammal. In certain embodiments, the subject is a human patient.

  In certain embodiments, the method reduces cytokine levels, histamine release, or biological activity of immune responsive cells.

  In another aspect, the invention comprises contacting a test compound with a BET family member comprising a bromodomain; detecting specific binding to the bromodomain, thereby identifying a test compound useful for treating inflammation Providing a method for identifying a compound for treating inflammation.

  Other features and advantages of the invention will be apparent from the detailed description, and from the claims.

Definitions “Agent” means any small molecule compound, antibody, nucleic acid molecule, or polypeptide, or fragment thereof.

As used herein, the term “aromatic ring” or “aryl” means a mono- or polycyclic aromatic ring or ring radical comprising carbon and hydrogen atoms. Examples of suitable aryl groups include phenyl, tolyl, anthracenyl, fluorenyl, indenyl, azulenyl, and naphthyl, and benzo-fused carbocyclic moieties such as 5,6,7,8-tetrahydronaphthyl. It is not limited. Aryl groups can be unsubstituted or substituted, for example, as described herein for alkyl groups (without limitation alkyl (preferably lower alkyl or one or more halo-substituted alkyl)), hydroxy, alkoxy (preferably Can be optionally substituted with one or more substituents such as lower alkoxy), alkylthio, cyano, halo, amino, boronic acid (including —B (OH) ₂ and nitro). In form, the aryl group is monocyclic and the ring comprises 6 carbon atoms.

  As used herein, the term “alkyl” means a saturated straight chain or branched acyclic hydrocarbon, typically having from 1 to 10 carbon atoms. Exemplary saturated straight chain alkyls include methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, n-heptyl, n-octyl, n-nonyl, and n-decyl; On the other hand, as saturated branched alkyl, isopropyl, sec-butyl, isobutyl, tert-butyl, isopentyl, 2-methylbutyl, 3-methylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2-methylhexyl , 3-methylhexyl, 4-methylhexyl, 5-methylhexyl, 2,3-dimethylbutyl, 2,3-dimethylpentyl, 2,4-dimethylpentyl, 2,3-dimethylhexyl, 2,4-dimethylhexyl 2,5-dimethylhexyl, 2,2-dimethylpentyl, 2,2-dimethylhexyl, 3,3 Dimethylpentyl, 3,3-dimethylhexyl, 4,4-dimethylhexyl, 2-ethylpentyl, 3-ethylpentyl, 2-ethylhexyl, 3-ethylhexyl, 4-ethylhexyl, 2-methyl-2-ethylpentyl, 2- Methyl-3-ethylpentyl, 2-methyl-4-ethylpentyl, 2-methyl-2-ethylhexyl, 2-methyl-3-ethylhexyl, 2-methyl-4-ethylhexyl, 2,2-diethylpentyl, 3,3 -Diethylhexyl, 2,2-diethylhexyl, 3,3-diethylhexyl and the like. The alkyl group included in the compounds of the present invention may be unsubstituted or amino, alkylamino, arylamino, heteroarylamino, alkoxy, alkylthio, oxo, halo, acyl, nitro, hydroxyl, cyano, aryl, Heteroaryl, alkylaryl, alkylheteroaryl, aryloxy, heteroaryloxy, arylthio, heteroarylthio, arylamino, heteroarylamino, carbocyclyl, carbocyclyloxy, carbocyclylthio, carbocyclylamino, heterocyclyl, heterocyclyloxy Optionally substituted with one or more substituents such as, heterocyclylamino, heterocyclylthio and the like. In the compounds of the present invention, lower alkyl is typically preferred.

  “Bromodomain” means a polypeptide moiety that recognizes an acetylated lysine residue. In one embodiment, the bromodomain of a BET family member polypeptide comprises approximately 110 amino acids and comprises a left-handed bundle of four alpha helices that are bound by a diverse loop region that interacts with chromatin. Share saved folds.

  “BET family polypeptide” means a polypeptide comprising two bromodomains and an extraterminal (ET) domain or fragment thereof having transcriptional regulator activity or acetylated lysine binding activity. Exemplary BET family members include BRD2, BRD3, BRD4, and BRDT.

  “BRD2 polypeptide” means a protein or fragment thereof having at least 85% identity with NP_005095 and capable of chromatin binding or transcriptional regulation.

The sequence of an exemplary BRD2 polypeptide is shown below.
MLQNVTPHNKLPGEGNAGLLGLGPEAAAPGKRRIRKPSLLLYEGFSPTMASVPALQLPTPNPPPPEVSNPNPK
KPGVRTNQLQYLHKVVMKALWKHQFAWPFRQPMVDAVKLGLPDYHKIIKQPMDMTGTIKRRLENNYYWASE
CMQDFNTMFTTNCYIYNKPTDDIVLMAQTLEKIFLQKVASMPQEEQELVVTIPKNSHKKGAKLAALQGSVT
SAHQVPAVSSVSHTALYTPPPEIPTTVLNIPHPSVISLLPLSLSLSAGPPLLAVTAAPPAQPPLAKKKVGVK
RKADTTTPTPTAILAPPGSSPASPPGSLEPKAARLPPMRRESGPRPIKPPRKDLDPDSQQQQQSSKKGKLSEQL
KHCNGILKELLSKKKHAAYAPFPFYKPVDASALGLHDYHDIIKHPMDLSTVKRKMENRDYRDAQEFAADVRL
MFSNCYKYNPPDHDVVAMARKLQDVFEFRYAKMPDEPLEPGPLPVSTAMPPGLAKSSSESSESSESSESS
SEEEEEEEEDEEEEESSSDSEEEERAHRLAELQEQLRAVHEQLAALSQGPISKPKRKREKKKEKKKRKA
EKHRGRADADDKGPRAPRPQPQPKKSKASGSGGGGSAALGPSGFGPSGGSGTKLPKKATKTTAPLPTG
YDSEEEEESRPMSYDEKRRQLSLDNKLPGEKLGLVVHIIQAREPSLRDSNPEEIEIDFETLKPSTLRELLE
RYVLSCLRKKPRKPYTIKKPVGKTKEELALEKKRELEKRLQDVSGQLNSTKKPPKKANEKTESSSAQQVA
VSRLSSSSSSSSSSSSSSSSSDTSDSDSG

  “BRD2 nucleic acid molecule” means a polynucleotide encoding a BRD2 polypeptide or fragment thereof.

  "BRD3 polypeptide" means a protein or fragment thereof having at least 85% identity with NP_031397.1 capable of chromatin binding or transcriptional regulation.

The sequence of an exemplary BRD3 polypeptide is shown below.
1 mstatvapa gipatpgpvn ppppevsnps kpggrktnqlq ymqnvvvktl wkhqfawpfy
61 qpvdaiklnl pdyhkiknp mdmgtikkrl nynywase cmqdfntmft ncyynnkptd
121 divlmaqale kiflqkvaqm pqevevelpp apkgkgrkpagaqsagtqq vaavsvspa
181 tpfqsvppptv sqtpviaatp vptitanvts vpvpppaaapp pppatpivpvv pppppvvkk
241 gvkrkadttt pttsaitasr sesppplsdp kqakvvarre sggrpikppk kdlegeevpq
301 hagkgkgklse hrycdsilr emmlskkhaya awpfykpvda ealhlhdyhd iihpmddlst
361 vkrkmmdrey pdaqgfaadv llmfsncyky nppdhevvam arklkdvdvem rfampmpev
421 eapalpapa pmvskgaess rsseessds gssdseeera trlaqeql kavheqlaal
481 sqapvnkpkkk kkekkekk kkdkekeke hkvkaeeeek akvappakqa qqkakapakka
541
601 repslrdsn pdieididf lkpttlrel ryvksclqkk qrkpfsasgk kqakakskeel
661 akkekkelek rlqdvsgqls ssskparkek pgsapsggps rssssess gssssgsgss
721 dssdse

  “Brd3 nucleic acid molecule” means a polynucleotide encoding a BRD3 polypeptide.

“BRD4 polypeptide” means a protein or fragment thereof having at least 85% identity with NP — 055114 and capable of chromatin binding or transcriptional regulation.
1 msaesgpgtr lrnlpvmgdg letsqmstq aqaqpqpana astnpppppet snpnkpkkrqt
61 nqlqyllrvv lktlwkhqfa wpfqqpvdav klnlpdyyki iktpmdmgti kkrlennyyw
121 naqiqiqdfn tmftncyyyn kpgddivlma ealklflqk inelpettee imvqkgrg
181 rgrgketgtak pgvstvpntt qastppqtqt pqpnppppvqa tphppfavvtp drivqtpvmt
241 vvppqplqtp ppvppqpqpp papappqpvqs hppiaatpq pvktkkgvkr kadtttptti
301 dpiheppslp pepktklggq rressrpvkp pkkdvpdsqq hapeksskv seqlkccsgi
361 lkemfakha ayawpfykpv dvelglhldy cdiihppmdm stikclear eyrdaqefga
421 dvrlmfsncy kynppdhevv amarklqdvf emrfakmpde peepvvavss pavpptpkvv
481 appsdsssss dssssdsst ddsseeaaqr laelqeqlka vheqlaalsq pqqnkpkkk
541 kdkkkekkek hkrkeeven kkskepppp ktknkknsn snvskepapp mkskpppptie
601 seedkckpm syeekrqlsl dinklpggekl grvvhiiqsr epslknsnpd eiidfeltlk
661 pstlreary vtsclrkrkrk pqaekvdvia gsskmkgfss sessessess sssedsetg
721 pa

  “Brd4 nucleic acid molecule” means a polynucleotide encoding a BRD4 polypeptide.

“BRDT polypeptide” means a protein or fragment thereof having at least 85% identity with NP_001717 and capable of chromatin binding or transcriptional regulation.
1 mslpsrqtai ivnppppeii ntkknggrltn qlqylqkvvl kdlwkhsfsw pfqrpvdavk
61 lqlpdytyi knpmdnntik krlenkyyak authiednt mfsncylynk pgddivivlmaq
121 aleklfmqkl sqmpqeqvv gvkerikkgt qqniavsak kesspask vfkqqeipsv
181 fpktsisspln vvqgasvnss sqtaaqvtkg vkrkadttt atsavkasse fsptfteksv
241 alppikemp knvlpdsqqq ynvvktvkvt eqlrhcseil kemlakkhfs yawpfynpvd
301 vnalglhny dvvknpmmdlg tikekmdnq ykdaykfaad vrlfmmncyk ynppdhevvt
361 marmlqdvfe thfskitiep vesmplcyik tditettgre ntneasseg ssddsederv
421 krklklqeql kavhqqlqvl sqvpffrnklk kkekskkekk ekvnnsnen prkmseqmrl
481 kekskrnqpk krkqqfiglk sednakpm nydekrqlsl niklppgdkl grvvhiiqsr
541 epslsnsnpnd eiidfeltlk astreleleky vsacrlkrpr kppakkimmms keelhsqkkq
601 elekrlldvn nqlnsrkrqt ksdktqpska venvrssress sssssses essssssdlss
661 dssdssemf pkftevkpnd spskenvkmm knecipeggr tgvtgigyv qdttsanttl
721 vhqttpshvm ppnhhqlafn yqlehlqtv knisplqilp psgdseqlsn gtvhmhpsgd
781 sdtmlsec sec qapvqkdiki knadswkslg kpvkpsgvmk ssdelfnqfr kaaiekevka
841 rtqerirkhl eqntkelkas quqrdlgng ltvesfsnki qnkcsgeeqk ehqqsseaqd
901 ksklwllkdr dlarqkeqer rrramvgti dmtlqsdimt mfennfd

“BRDT nucleic acid molecule” means a polynucleotide encoding a BRDT polypeptide.

  “Improve” means to reduce, inhibit, attenuate, diminish, stop, or stabilize the onset or progression of a disease.

  “Alteration” means a change (increase or decrease) in the expression level or activity of a gene or polypeptide, as detected by standard methods known in the art as described herein. As used herein, alterations include a 10% change in expression level, preferably a 25% change, more preferably a 40% change, and most preferably a 50% or more change in expression level.

  “Analog” means molecules that are not identical but have similar functional or structural characteristics. For example, a polypeptide analog retains at least some biological activity of the corresponding natural polypeptide while having certain biochemical modifications that enhance the function of the analog compared to the natural polypeptide. Such biochemical modifications can increase the protease resistance, membrane permeability, or half-life of the analog without changing, for example, ligand binding. Analogs include unnatural amino acids.

  “Compound” means any small molecule compound, antibody, nucleic acid molecule, or polypeptide, or fragment thereof.

  The term “diastereomers” refers to stereoisomers with two or more centers of chirality and whose molecules are not mirror images of one another.

  The term “enantiomer” refers to two stereoisomers of a compound that are non-superimposable mirror images of each other. An equimolar mixture of two enantiomers is referred to as a “racemic mixture” or “racemate”.

  The term “halogen” refers to —F, —Cl, —Br or —I.

  The term “haloalkyl” is intended to include alkyl groups as defined above, mono-, di- or polysubstituted with halogens such as fluoromethyl and trifluoromethyl.

  The term “hydroxyl” means —OH.

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

  The term "heteroaryl" has 1 to 4 ring heteroatoms for monocyclic systems, 1 to 6 heteroatoms for bicyclic systems, and 1 to 9 heteroatoms for tricyclic systems. , Aromatic 5-8 membered monocyclic system, 8-12 membered bicyclic ring system, or 11-14 membered ring tricyclic system, wherein the heteroatom is selected from O, N, or S, and the rest The ring atom is carbon. A heteroaryl group may be optionally substituted with one or more substituents such as aryl groups. Examples of heteroaryl groups include pyridyl, furanyl, benzodioxolyl, thienyl, pyrrolyl, oxazolyl, oxadiazolyl, imidazolylthiazolyl, isoxazolyl, quinolinyl, pyrazolyl, isothiazolyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, triazolyl, thiadiazolyl, Examples include, but are not limited to, isoquinolinyl, indazolyl, benzoxazolyl, benzofuryl, indolizinyl, imidazopyridyl, tetrazolyl, benzimidazolyl, benzothiazolyl, benzothiadiazolyl, benzooxadiazolyl, and indolyl.

  The term “heterocyclic”, as used herein, refers to an organic compound that contains at least one atom other than carbon (eg, S, O, N) in the ring structure. The ring structure in these organic compounds can be either aromatic or non-aromatic. Some examples of heterocyclic moieties include, but are not limited to, pyridine, pyrimidine, pyrrolidine, furan, tetrahydrofuran, tetrahydrothiophene, and dioxane.

  The terms “isomer” or “stereoisomer” refer to compounds that have the same chemical configuration but differ in the arrangement of atoms or groups in space.

The term “isotopic derivative” includes a derivative of a compound in which one or more atoms in the compound are replaced with the corresponding isotopes of the atoms. For example, an isotope derivative of a compound containing a carbon atom (C ¹² ) is one in which the compound's carbon atom is replaced with a C ¹³ isotope.

  The term “neoplasm” refers to an abnormal situation or condition characterized by the growth of cells with autonomous proliferative capacity, ie rapidly proliferating cells. Neoplastic conditions may be classified as pathological, i.e. characterize or constitute the pathology, or may be classified as non-pathological, i.e. a deviation from normal but not associated with a pathological condition. The term is intended to include all types of cancerous growth or carcinogenic processes, metastatic or malignant transformed cells, tissues, or organs, regardless of histopathology type or stage of invasiveness. “Pathologic hyperproliferative” cells occur in diseases characterized by malignant tumor growth. Examples of non-pathological hyperproliferative cells include cell proliferation associated with wound repair.

  Illustrative neoplasms susceptible to treatment with compounds of the invention include leukemias (eg, acute leukemia, acute lymphocytic leukemia, acute myeloid leukemia, acute myeloblastic leukemia, acute promyelocytic leukemia, acute Bone marrow mononuclear leukemia, acute monocytic leukemia, acute red blood leukemia, mixed lineage myeloid leukemia, chronic leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia), multiple myeloma, polycythemia vera, cutaneous T cells Lymphoma (CTCL), lymphoma (Hodgkin's disease, non-Hodgkin's disease), Waldenstrom's macroglobulinemia, heavy chain disease, and (eg fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma , Angiosarcoma, endothelial sarcoma, lymphangiosarcoma, lymphatic endothelial sarcoma, synovial tumor, mesothelioma, Ewing tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, colorectal cancer, pancreas Breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell tumor, adenocarcinoma, sweat gland cancer, sebaceous gland cancer, papillary cancer, papillary adenocarcinoma, cystadenocarcinoma, medullary Cancer, bronchogenic lung cancer, renal cell carcinoma, hepatocellular carcinoma, cholangiocarcinoma, choriocarcinoma, seminoma, fetal cancer, Wilms tumor, cervical cancer, uterine cancer, testicular cancer, lung cancer, small Cell lung cancer, non-small cell lung cancer, bladder cancer, epithelial cancer, glioma, glioblastoma, glioblastoma multiforme, astrocytoma, medulloblastoma, craniopharyngioma, ependymal cell Sarcomas (such as tumors, pineal tumors, hemangioblastomas, auditory neuromas, neuroendocrine tumors, oligodendroma, Schwann celloma, meningiomas, melanoma, neuroblastoma, and retinoblastoma) Solid tumors such as cancer and carcinoma, but are not limited thereto.

  In one embodiment, the neoplasm is driven by a major transcriptional activator such as myc. Such cancers include Burkitt lymphoma, small cell lung cancer, breast cancer, colon cancer, neuroblastoma, glioblastoma multiforme, MLL-driven leukemia, chronic lymphocytic leukemia, squamous epithelium with NUT translocation. Examples include, but are not limited to, cancer, and other cancers with bromodomain-containing proteins or NUT rearrangements.

  The language “inhibiting growth” of a neoplasm includes delaying, interrupting, arresting or stopping its growth and metastasis, and it is not essential to indicate complete elimination of neoplastic growth.

  “Computer modeling” means the application of a computational program to determine one or more of the following: position and proximity of the ligand to the binding moiety, occupied space of the binding ligand, complementation between the binding moiety and the ligand. Contact area, deformation energy of binding of a given ligand to a binding moiety, and some estimation of hydrogen bond strength, van der Waals interaction, hydrophobic interaction, and / or electrostatic interaction between ligand and binding moiety Working energy. Computer modeling can also provide a comparison between the characteristics of the model system and the candidate compound. For example, a computer modeling experiment may compare the pharmacophore model of the present invention with a candidate compound to assess the fit of the candidate compound with the model.

  “Computer system” means hardware means, software means, and data storage means used for atomic coordinate data analysis. The minimum hardware means of the computer-based system of the present invention comprises a central processing unit (CPU), input means, output means, and data storage means. Desirably, a monitor for visualizing the structural data is provided. The data storage means may be RAM or means for accessing the computer readable medium of the present invention. Examples of such systems are microcomputer workstations available from Silicon Graphics Incorporated and Sun Microsystems that run Unix-based, Windows NT or IBM OS / 2 operating systems.

  “Computer-readable medium” means any medium that can be read and accessed directly by a computer, for example, such that the medium is suitable for use in the computer systems described above. Media include magnetic storage media such as floppy disks, hard disk storage media, and magnetic tapes; optical storage media such as optical discs or CD-ROMs; electrical storage media such as RAM and ROM; and magnetic / optical storage media These categories include, but are not limited to, hybrids.

  In this disclosure, “comprises”, “comprising”, “containing”, “having” and the like are attributed to them in US patent law. May have meaning and may mean “includes”, “including”, etc .; likewise “consisting essentially of” or “consists essentially” ) "Has the meaning ascribed to U.S. Patent Law, the term is unrestricted, and the enumeration is only required as long as the basic or novel characteristics of the enumeration remain unchanged due to the presence beyond the enumeration Is allowed, but prior art embodiments are excluded.

  “Detection” refers to identifying the presence, absence or amount of the analyte to be detected.

  “Detectable label” means a composition that, when bound to a molecule of interest, makes the molecule of interest detectable via spectroscopic, photochemical, biochemical, immunochemical, or chemical means. To do. For example, useful labels include radioisotopes, magnetic beads, metal beads, colloidal particles, fluorescent dyes, high electron density reagents, enzymes (such as those commonly used in ELISAs), biotin, digoxigenin, or haptens. .

  “Disease” means any medical condition or disorder that impairs or prevents the normal function of a cell, tissue, or organ. Examples of diseases susceptible to treatment with the compounds described herein include neoplasms, inflammatory diseases, obesity, fatty liver (NASH or not), diabetes, atherosclerosis, arterial stent occlusion, Heart failure, cachexia, graft-versus-host disease, infectious diseases associated with bromodomains, parasites, malaria, trypanosoma treatment, and male fertility reduction. Further uses of the compositions of the present invention include, but are not limited to, use in organ transplantation, cell condition regulation for regenerative medicine (ie by promoting or inhibiting cell differentiation), and promoting pluripotency. It is not a thing.

  “Effective amount” means the amount of an agent required to ameliorate disease symptoms compared to an untreated patient. The effective amount of active compound used to practice the invention for disease treatment will vary depending on the mode of administration, the subject's age, weight and overall health. Ultimately, the attending physician or veterinarian will decide the appropriate amount and dosage regimen. Such an amount is referred to as an “effective” amount.

  The term “enantiomer” refers to two stereoisomers of a compound that are non-superimposable mirror images of each other. An equimolar mixture of two enantiomers is referred to as a “racemic mixture” or “racemate”.

  The term “halogen” refers to —F, —Cl, —Br or —I.

  The term “haloalkyl” is intended to include alkyl groups as defined above, mono-, di- or polysubstituted with halogens such as fluoromethyl and trifluoromethyl.

  The term “hydroxyl” means —OH.

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

  The term "heteroaryl" has 1 to 4 ring heteroatoms for monocyclic systems, 1 to 6 heteroatoms for bicyclic systems, and 1 to 9 heteroatoms for tricyclic systems. , Aromatic 5-8 membered monocyclic system, 8-12 membered bicyclic ring system, or 11-14 membered ring tricyclic system, wherein the heteroatom is selected from O, N, or S, and the rest The ring atom is carbon. A heteroaryl group may be optionally substituted with one or more substituents such as, for example, those described herein for aryl groups. Examples of heteroaryl groups include pyridyl, furanyl, benzodioxolyl, thienyl, pyrrolyl, oxazolyl, oxadiazolyl, imidazolyl, thiazolyl, isoxazolyl, quinolinyl, pyrazolyl, isothiazolyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, triazolyl, thiadiazolyl, isoquinolinyl , Indazolyl, benzoxazolyl, benzofuryl, indolizinyl, imidazopyridyl, tetrazolyl, benzimidazolyl, benzothiazolyl, benzothiadiazolyl, benzooxadiazolyl, and indolyl.

  The term “heterocyclic”, as used herein, refers to an organic compound that contains at least one atom other than carbon (eg, S, O, N) in the ring structure. The ring structure in these organic compounds can be either aromatic or, in certain embodiments, non-aromatic. Some examples of heterocyclic moieties include, but are not limited to, pyridine, pyrimidine, pyrrolidine, furan, tetrahydrofuran, tetrahydrothiophene, and dioxane.

  The terms “isomer” or “stereoisomer” refer to compounds that have the same chemical configuration but differ in the arrangement of atoms or groups in space.

The term “isotopic derivative” includes a derivative of a compound in which one or more atoms in the compound are replaced with the corresponding isotopes of the atoms. For example, an isotope derivative of a compound containing a carbon atom (C ¹² ) is one in which the compound's carbon atom is replaced with a C ¹³ isotope.

  The present invention provides a number of targets that are useful in the development of highly specific drugs for treating disorders characterized by the methods described herein. In addition, the methods of the present invention provide an easy means to identify treatments that are safe for the subject. In addition, the methods of the present invention provide a means to analyze virtually any number of compounds for effects on the diseases described herein with high throughput, high sensitivity, and low complexity.

  “Compatible” refers to one or more atoms of an agent molecule and one or more atoms or binding sites of a BET family member (eg, BRD2, BRD3, BRD4, and BRDT bromodomains) by automated or semi-automated means. Means to measure the interaction between and to determine the degree to which such interaction is stable. Various computer-based methods for adaptation are further described herein.

  “Fragment” means a portion of a polypeptide or nucleic acid molecule. This portion preferably contains at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the total length of the reference nucleic acid molecule or polypeptide. Fragments contain 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nucleotides or amino acids May be.

  “Hybridization” means hydrogen bonding, which may be Watson-Crick, Hoogsteen or reverse Hoogsteen hydrogen bonding between complementary nucleobases. For example, adenine and thymine are complementary nucleobases that pair through the formation of hydrogen bonds.

  An “isolated polynucleotide” refers to a nucleic acid (eg, DNA) that does not include genes located on the gene side in the natural genome of the organism from which the nucleic acid molecule of the invention is derived. The term thus includes, for example, recombinant DNA incorporated in a vector, in a self-replicating plasmid or virus, or in prokaryotic or eukaryotic genomic DNA; or other molecules independent of other sequences ( For example, recombinant DNA present as cDNA or PCR or genomic or cDNA fragments produced by restriction endonuclease digestion. In addition, the term includes RNA molecules that are transcribed from DNA molecules, as well as recombinant DNA that is part of a hybrid gene that encodes additional polypeptide sequences.

  "Isolated polypeptide" means a polypeptide of the invention that has been separated from the components that naturally accompany it. Typically, a polypeptide is isolated if it is at least 60% by weight and there is no protein or natural organic molecule to which it naturally binds. Preferably, the preparation is at least 75% by weight, more preferably at least 90% by weight, most preferably at least 99% by weight of the polypeptides of the invention. Isolated polypeptides of the present invention may be obtained, for example, by extraction from natural sources; by expression of recombinant nucleic acids encoding such polypeptides; or by chemical synthesis of proteins. Purity can be measured by any suitable method such as, for example, column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.

  “Marker” means any protein or polynucleotide having a change in expression level or activity associated with a disease or disorder.

  As used herein, “obtaining” as found in “obtaining an agent” includes synthesizing, purchasing, or otherwise obtaining the agent.

  The term “neoplasm” refers to a cell that has an autonomous proliferative capacity, ie, an abnormal situation or condition characterized by rapidly proliferating cell growth. Neoplastic conditions may be classified as pathological, i.e. characterize or constitute the pathology, or may be classified as non-pathological, i.e. a deviation from normal but not associated with a pathological condition. The term is intended to include all types of cancerous growth or carcinogenic processes, metastatic or malignant transformed cells, tissues, or organs, regardless of histopathology type or stage of invasiveness. “Pathologic hyperproliferative” cells occur in diseases characterized by malignant tumor growth. Examples of non-pathological hyperproliferative cells include cell proliferation associated with wound repair.

  The language “inhibiting growth” of a neoplasm includes delaying, interrupting, arresting or stopping its growth and metastasis, and does not necessarily suggest complete elimination of neoplastic growth.

  The normal medical meaning of the term “neoplasm” refers to “new cell growth”, which is the result of, for example, a loss of responsiveness to normal growth control for neoplastic cell growth. “Hyperplasia” refers to cells that exhibit an abnormally high growth rate. However, as used herein, the term neoplasm generally refers to cells that have an abnormal rate of cell growth. Neoplasms include “tumors”, which may be benign, premalignant or malignant.

  The term “obtaining” as found in “obtaining a compound” is intended to include purchasing, synthesizing or otherwise obtaining the compound.

  As used herein, the term “optical isomer” includes molecules also known as chiral molecules, which are exact mirror images that cannot be superimposed on each other.

  The terms “parenteral administration” and “administered parenterally” are used herein without limitation, intravenously, intramuscularly, intraarterially, intrathecally, intraarticularly, intraorbitally, Intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, epidermal, intraarticular, subcapsular, subarachnoid, intraspinal, and intrasternal injections and infusions, usually by injection, other than enteral and local administration Means the mode of administration.

  The term “polycyclyl” or “polycyclic group” refers to bicyclic or higher radicals (eg, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl and / or heterocyclyl), in which two or more carbons are present. Shared by two adjacent rings, for example, a ring is a “fused ring”. Rings that are joined through non-adjacent atoms are termed “bridged” rings. Each of the polycyclic rings is halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonate, Phosphinato, cyano, amino (including alkylamino, dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl, and ureido), amidino, imino, sulfhydryl, Alkylthio, arylthio, thiocarboxylate, sulfate, sulfonate, sulfamoyl, Including Ruhon'amido, nitro, trifluoromethyl, cyano, azido, heterocyclyl, alkyl, alkylaryl, or an aromatic or heteroaromatic moiety.

  The term “polymorph” as used herein refers to a solid crystalline form of a compound of the invention or a complex thereof. Different polymorphs of the same compound may exhibit different physical, chemical and / or spectroscopic properties. Different physical properties include stability (eg against heat or light), compressibility and density (important in formulation and product manufacture), and dissolution rate (which can affect bioavailability). It is not limited to. Differences in stability may be due to changes in chemical reactivity (eg, a differential oxidation that changes color more rapidly when the dosage form consists of one polymorph than when it consists of another polymorph) or a change in mechanical properties (eg, power Tablets disintegrate during storage as the morphologically preferred polymorph transforms into a thermodynamically more stable polymorph) or both (eg, at high humidity, one polymorph tablet is more susceptible to breakage) Can result from Different physical properties of polymorphs can affect their processing.

  The term “prodrug” includes compounds with moieties that can be metabolized in vivo. In general, prodrugs are metabolized to the active drug in vivo by esterases or by other mechanisms. Examples of prodrugs and their use are well known in the art (see, eg, Berge et al. (1977) “Pharmaceutical Salts”, J. Pharm. Sci. 66: 1-19). Prodrugs can be prepared in situ during final isolation and purification of the compound, or can be prepared by reacting the free acid form or hydroxyl of the purified compound individually with a suitable esterifying agent. The hydroxyl group can be converted to an ester via treatment with a carboxylic acid. Examples of prodrug moieties include substituted and unsubstituted, branched or unbranched lower alkyl ester moieties (eg, propionic acid esters), lower alkenyl esters, di-lower alkylamino lower alkyl esters (eg, dimethylaminoethyl esters) Acylamino lower alkyl esters (eg acetyloxymethyl esters), acyloxy lower alkyl esters (eg pivaloyloxymethyl esters), aryl esters (phenyl esters), aryl lower alkyl esters (eg benzyl esters), substituted (eg Aryl and aryl lower alkyl esters, amides, lower alkyl amides, di-lower alkyl amides, and hydroxy amides (with methyl, halo, or methoxy substituents). Preferred prodrug moieties are propionic acid esters and acyl esters. Also included are prodrugs that have been converted to active forms through other mechanisms in vivo.

  Furthermore, the stereochemical indicators of the entire carbon-carbon double bond are also in conflict with the general chemical field, in which “Z” refers to what is often referred to as “cis” (same side) conformation. , “E” refers to what is often referred to as the “trans” (opposite) conformation. Both cis / trans and / or Z / E configurations are encompassed by the compounds of the present invention.

  With respect to chiral center nomenclature, the terms “d” and “l” configuration are as defined by the IUPAC recommendation. For the use of the terms diastereomers, racemates, epimers, and enantiomers, these are used in their usual context describing the stereochemistry of the preparation.

  “Reduce” means a negative change of at least 10%, 25%, 50%, 75%, or 100%.

  “Reference” means standard or control conditions.

  A “reference sequence” is a defined sequence used as a basis for sequence comparison. The reference sequence may be a subset or the entire specified sequence, for example, a full-length cDNA or a fragment of a gene sequence, or a complete cDNA or gene sequence. For polypeptides, the length of the reference polypeptide sequence is generally at least about 16 amino acids, preferably at least about 20 amino acids, more preferably at least about 25 amino acids, and even more preferably about 35 Amino acids, about 50 amino acids, or about 100 amino acids. For nucleic acids, the length of the reference nucleic acid sequence is generally at least about 50 nucleotides, preferably at least about 60 nucleotides, more preferably at least about 75 nucleotides, and even more preferably about 100 nucleotides or about 300 nucleotides; Or any integer before or after them or between them.

  “Specifically binds” recognizes and binds a polypeptide of the invention, but substantially recognizes other molecules in a sample, eg, a biological sample that naturally contains a polypeptide of the invention. Means a compound or antibody that does not bind.

  Nucleic acid molecules useful in the methods of the present invention include any nucleic acid molecule that encodes a polypeptide of the present invention or a fragment thereof. Such nucleic acid molecules may not be 100% identical to the endogenous nucleic acid sequence, but typically exhibit considerable identity. A polynucleotide having “substantial identity” with an endogenous sequence is typically capable of hybridizing to at least one strand of a double-stranded nucleic acid molecule. Nucleic acid molecules useful in the methods of the present invention include any nucleic acid molecule that encodes a polypeptide of the present invention or a fragment thereof. Such nucleic acid molecules may not be 100% identical to the endogenous nucleic acid sequence, but typically exhibit considerable identity. A polynucleotide having “substantial identity” with an endogenous sequence is typically capable of hybridizing to at least one strand of a double-stranded nucleic acid molecule. “Hybridize” means a pair of complementary polynucleotide sequences (eg, a gene described herein) or a pair that forms a double-stranded molecule between portions thereof under various stringency conditions. (See, eg, Wahl, GM and SL Berger (1987) Methods Enzymol. 152: 399; Kimmel, AR (1987) Methods Enzymol. 152: 507).

  For example, stringent salt concentrations are typically below about 750 mM NaCl and 75 mM trisodium citrate, preferably below about 500 mM NaCl and 50 mM trisodium citrate, more preferably about 250 mM NaCl and Below 25 mM trisodium citrate. Low stringency hybridization is obtained in the absence of an organic solvent such as formamide, whereas high stringency hybridization is obtained in the presence of at least about 35% formamide, more preferably at least about 50% formamide. can get. Stringent temperature conditions typically include a temperature of at least about 30 ° C, more preferably at least about 37 ° C, and most preferably at least about 42 ° C. It is well known to those skilled in the art to vary additional parameters such as hybridization time, eg, concentration of detergents such as sodium dodecyl sulfate (SDS), and inclusion or exclusion of carrier DNA. By combining these various conditions as needed, various stringency levels are achieved. In a preferred embodiment, hybridization occurs at 30 ° C. in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS. In a more preferred embodiment, hybridization occurs at 37 ° C. in 500 mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100 μg / ml denatured salmon sperm DNA (ssDNA). . In the most preferred embodiment, hybridization occurs at 42 ° C. in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, and 200 μg / ml ssDNA. Useful variations of these conditions are readily apparent to those skilled in the art.

  For most applications, the washing step following hybridization will also vary in stringency. Wash stringency conditions can be defined by salt concentration and temperature. As above, wash stringency can be increased by decreasing salt concentration or increasing temperature. For example, the stringent salt concentration for the wash step is preferably below about 30 mM NaCl and 3 mM trisodium citrate, and most preferably below about 15 mM NaCl and 1.5 mM trisodium citrate. Stringent temperature conditions for the washing step typically include a temperature of at least about 25 ° C, more preferably at least about 42 ° C, and even more preferably at least about 68 ° C. In a preferred embodiment, the washing step occurs at 25 ° C. in 30 mM NaCl, 3 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, the washing step occurs at 42 ° C. in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, the washing step occurs at 68 ° C. in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. Additional variations in these conditions will be readily apparent to those skilled in the art. Hybridization techniques are well known to those skilled in the art and are described, for example, Benton and Davis (Science 196: 180, 1977); Grunstein and Hogness (Proc. Natl. Acad. Sci., USA 72: 3961, 1975); Ausubel et al. (Current Protocols in Molecular Biology, Wiley Interscience, New York, 2001); Berger and Kimmel (Guide to Molecular Cloning Techniques, 1987, AcademicS); , Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York.

  “Substantially identical” refers to a reference amino acid sequence (eg, any one of the amino acid sequences described herein) or a nucleic acid sequence (eg, any one of the nucleic acid sequences described herein). Means a polypeptide or nucleic acid molecule exhibiting at least 85% identity. Preferably, such sequences are at least 85%, 90%, 95%, 99% or 100% identical at the amino acid or nucleic acid level to the sequences used for comparison.

  Sequence identity is typically measured using sequence analysis software. (For example, Sequence Analysis Software Package of the Genetics Computer Group, Universal of Wisconsin Biotechnology, ST, 1710 University. Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and / or other modifications. Conservative substitutions typically include glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. An exemplary approach for determining the degree of identity may use a BLAST program, e. sup. -3 and e. sup. A probability score between -100 suggests a closely related sequence.

  “Reduce” or “increase” means a negative or positive change of at least about 10%, 25%, 50%, 75%, or 100%, respectively, as compared to a reference.

  “Mean square deviation” means the square root of the arithmetic mean of the square of deviations from the mean.

  “Reducing cell survival” means inhibiting cell survival or inducing cell death as compared to a reference cell.

  “Reference” means standard or control conditions.

  A “reference sequence” is a defined sequence used as a basis for sequence comparison. The reference sequence may be a subset or the entire specified sequence, for example, a full-length cDNA or a fragment of a gene sequence, or a complete cDNA or gene sequence. For polypeptides, the length of the reference polypeptide sequence is generally at least about 16 amino acids, preferably at least about 20 amino acids, more preferably at least about 25 amino acids, and even more preferably about 35 Amino acids, about 50 amino acids, or about 100 amino acids. For nucleic acids, the length of the reference nucleic acid sequence is generally at least about 50 nucleotides, preferably at least about 60 nucleotides, more preferably at least about 75 nucleotides, and even more preferably about 100 nucleotides or about 300 nucleotides; Or any integer before or after them or between them.

  “Subject” means a mammal, including, but not limited to, humans or non-human mammals such as cows, horses, dogs, sheep, or cats.

  “Specifically binds” recognizes and binds a polypeptide of the invention, but substantially recognizes other molecules in a sample, eg, a biological sample that naturally contains a polypeptide of the invention. Means a compound or antibody that does not bind.

  The term “sulfhydryl” or “thiol” means —SH.

  As used herein, the term “tautomer” refers to an isomer of an organic molecule that is readily interconverted by tautomerization, in which a hydrogen atom or proton is transferred during the reaction. In some cases, replacement of a single bond with an adjacent double bond is accompanied.

  As used herein, the terms “treat”, “treating”, “treatment” and the like refer to reducing or ameliorating a disorder and / or associated symptoms. “Improve” means to reduce, inhibit, attenuate, diminish, stop, or stabilize the onset or progression of a disease. It will be appreciated that, although not excluded, treating a disorder or condition does not require complete elimination of the disorder and its associated symptoms or condition.

  As used herein, terms such as “prevent”, “preventing”, “prevention”, “prophylactic treatment” do not have a disorder or medical condition. Refers to reducing the probability that a disorder or condition will develop in a subject at risk or prone to develop.

  “Effective amount” refers to the amount of the compound that provides a therapeutic effect to the treated subject. The therapeutic effect may be objective (ie, measurable with some test or marker) or subjective (ie, subject has signs of effect or control feels effect). Effective amounts of the compounds described herein may range from about 1 mg / Kg to about 5000 mg / Kg body weight. Effective doses will also vary depending on route of administration, as well as the possibility of simultaneous use of other agents.

  It is understood that the ranges provided herein are shorthand for all values within the range. For example, the range of 1-50 is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46 , 47, 48, 49, or 50 is understood to include any number, combination of numbers, or subrange from the group consisting of.

  As used herein, the terms “treat”, “treating”, “treatment” and the like refer to reducing or ameliorating a disorder and / or associated symptoms. It will be appreciated that, although not excluded, treating a disorder or condition does not require complete elimination of the disorder and its associated symptoms or condition.

  Unless otherwise noted or except where apparent from the context, as used herein, the term “or” is understood to be inclusive. Unless otherwise indicated or otherwise apparent from context, as used herein, the terms “a”, “an”, and “the” are understood to be singular or plural.

  Unless otherwise noted or apparent from the context, as used herein, the term “about” means within the normal tolerance of the art, eg within 2 standard deviations of the mean. Understood. About 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05% of the stated value Or within 0.01%. Except where otherwise apparent from the context, all numerical values provided herein are modified by the term about.

  The recitation of a list of chemical groups in the definition of any variable herein includes the definition of the variable as any single group or combination of listed groups. The recitation of an embodiment for a variable or aspect herein includes that embodiment as any single embodiment, or that embodiment in combination with any other embodiments or portions thereof.

  Any composition or method provided herein may be combined with one or more of any of the other compositions and methods provided herein.

The structures of the two JQ1 stereoisomers are shown. The stereocenter in C6 is indicated by an asterisk (*). (-)-JQ1 enantiomer is a graph showing that it does not competitively bind to BRD4-NUT in cells. FIG. 2A shows that GFP-BRD4 post-photobleaching fluorescence recovery (FRAP) is not affected by the presence of (−)-JQ1 (250 nM, 5 h) compared to the vehicle control. Data are mean ± s. d. (N = 5). FIG. 2B shows that in a side-by-side comparative study, expressed GFP-BRD4 demonstrates improved recovery in the presence of (+)-JQ1 (250 nM, 5 h). Data are mean ± s. d. (N = 5). FIG. 2C provides a quantitative comparison of the transferred fraction of GFP-BRD4 observed in the FRAP test (a, b). Data are mean ± s. d. Represents (n = 5) and is annotated by the p-value obtained from a two-tailed t-test comparing the ligand-treated sample with the vehicle control. JQ1 binds to BRD4 competitively with chromatin and shows that human NUT median cancer cells are differentiated. FIG. 3A shows GFP-BRD4 post-photobleaching fluorescence recovery (FRAP) demonstrating improved recovery in the presence of JQ1. The nucleus is pseudo-colored in proportion to the fluorescence intensity. White circles indicate photo bleaching areas. 3B-D show that JQ1 accelerates fluorescence recovery in FRAP experiments performed with transfected GFP-BRD4 (FIG. 3B) and GFP-BRD4-NUT (FIG. 3C), but nuclear GFP-NUT (FIG. 3D) shows no effect on recovery. FIG. 3E shows a quantitative comparison of time vs half half fluorescence recovery of the FRAP test (FIGS. 3B-3D). Data are mean ± s. d. Represents (n = 5) and is annotated by the p-value obtained from the two-sided t test. FIG. 3F shows that JQ1 (500 nM, 48 h) promotes loss of focal nuclear staining of NUT (anti-NUT; 40 ×). FIG. 3G shows that NMC cells treated with JQ1 (500 nM, 48 h) demonstrate cytological signs of squamous epithelial differentiation (H &E; 40 ×). FIG. 3H shows that differentiation of NMC cells by JQ1 (500 nM) is immediate, time-dependent and characterized by a marked increase in cytokeratin expression (AE1 / AE3; 40 ×). FIGS. 3I-J show that JQ1 attenuates rapid proliferation of the 797 cell line (FIG. 3I) and the Per403NMC cell line (FIG. 3J) in vitro (Ki67; 40 ×). It is a microscope picture which shows that JQ1 selectively induces squamous epithelial differentiation in NUT median cancer. FIG. 4a demonstrates NMC 797 cells treated with JQ1 (500 nM) demonstrate time-dependent cytological signs of squamous differentiation, exemplified by cell spindle, flattening, and development of open chromatin. (H &E; illustrated at 40 × and 100 ×). FIG. 4B shows that NMC Per403 cells treated with JQ1 (500 nM, 48 h) show comparable signs of squamous differentiation. Cell spindle and cytosol keratinization are illustrated by H & E and keratin staining, respectively (40 ×). FIG. 4C shows that the non-NMC squamous cell carcinoma cell line TE10 cannot differentiate in response to JQ1 (500 nM) and is illustrated by H & E and keratin (AE1 / AE3) staining (40 ×). JQ1 impairs NMC cell proliferation. FIG. 4D-a includes two photomicrographs (Ki67; 40 ×) showing that JQ1 attenuates rapid growth of the NMC Per403 cell line in vitro. Images are shown at the same magnification. FIG. 4D-b is a graph showing the effect of JQ1 on cell proliferation as measured by (4D-a) and IHC performed in FIG. 3J (Ki67 staining and positive;%). Cells were manually scored as Ki67 positive (dark stained nuclei) or negative (light blue stained nuclei) in 5 high power fields. Data are mean ± s. d. Represents (n = 5) and is annotated by the p-value obtained from the two-sided t test. It shows that the induction of squamous epithelial differentiation in NUT midline cancer cells by JQ1 is stereospecific and time dependent. FIG. 4E-a shows 6 NMC 797 cells treated in the chamber slide with (−)-JQ1 (100 nM) exhibit an equivalent cytosolic phenotype compared to the vehicle treated control. Including micrographs. (+)-JQ1 (100 nM, 48 h) stimulated squamous epithelial differentiation exhibited by cell spindleing, flattening, and increased keratin expression. FIG. 4E-b shows that NMC 797 cells treated with JQ1 enantiomer or vehicle were centrifuged, fixed, sliced and stained for keratin expression (left; AE1 / AE3, 20 ×). Image-based keratin expression analysis was performed on slides prepared simultaneously using unbiased masking and quantification algorithms that could score nuclei for staining intensity (right; 20 ×). FIG. 4E-c shows that (+)-JQ1 (250 nM) is keratin in treated NMC 797 cells compared to (−)-JQ1 (250 nM) and vehicle control as measured by quantitative immunohistochemistry. It shows that the rapid expression of was induced. Present percent positive nuclei per treatment condition. Data are mean ± s. d. Represents (n = 3) and is annotated by the p-value obtained from a two-sided t test. FIG. 4E-d shows that (+)-JQ1 (250 nM) causes a time-dependent induction of potent (3+) keratin staining in treated NMC 797 cells compared to (−)-JQ1 (250 nM). . Percent positive nuclei per treatment condition are presented. Data are mean ± s. d. Represents (n = 3) and is annotated by the p-value obtained from a two-sided t test. The grouped images are shown at the same magnification. Squamous cell carcinoma cell lines that do not possess a BRD4-NUT translocation are less sensitive to treatment with JQ1. Gastrointestinal squamous cell carcinoma cell lines (TT and TE10) were cultured for 72 hours in the presence of (+)-JQ1 (250 nM, red circle) or (−)-JQ1 (250 nM, black circle). With JQ1, the minimal effect observed on cell proliferation is consistent with a plausible role in cell cycle progression in mitotic cells ⁷ . Data are mean ± s. d. Presented as (n = 3). IC ₅₀ values were calculated by logistic regression. Squamous cell carcinoma cell lines that do not possess a BRD4-NUT translocation are less sensitive to treatment with JQ1. Gastrointestinal squamous cell carcinoma cell lines (TT and TE10) were cultured for 72 hours in the presence of (+)-JQ1 (250 nM, red circle) or (−)-JQ1 (250 nM, black circle). With JQ1, the minimal effect observed on cell proliferation is consistent with a plausible role in cell cycle progression in mitotic cells ⁷ . Data are mean ± s. d. Presented as (n = 3). IC ₅₀ values were calculated by logistic regression. It shows that JQ1 treatment inhibits proliferation, promotes differentiation and cell death in vitro and in vivo. FIG. 5a shows the growth effect of BRD4 inhibition on BRD4-NUT dependent cell lines. Cells were cultured with (+)-JQ1 (red circles) or (−)-JQ1 (black circles) and growth was monitored after 72 hours. (+)-JQ1 uniquely attenuated proliferation by the NMC cell line. Data are mean ± s. d. Presented as (n = 3). IC ₅₀ values were calculated by logistic regression. FIG. 5B shows that the progressive anti-proliferative effect of (+)-JQ1 over time on NMC 797 cells was demonstrated on days 1, 3, 7, and 10. Data points are mean ± s. d. (N = 3). FIG. 5C shows flow cytometry results for DNA content in NMC 797 cells. (+)-JQ1 (250 nM, 48 h) induced G1 arrest compared to (−)-JQ1 (250 nM) and the vehicle control. FIG. 5D shows the results of flow cytometric analysis of NMC 797 squamous cell carcinoma cells treated with the indicated vehicle, JQ1 or staurosporine (STA). PI, propidium iodide. AV, Annexin V. FIG. 5E shows the results of PET imaging of a mouse NMC 797 xenograft. FDG uptake in hindlimb xenografts is reduced compared to vehicle control by 50 mg kg ⁻¹ JQ1 treatment. FIG. 5F shows that tumor volumes are reduced in mice with established disease (NMC 797 xenografts) treated with 50 mg kg ⁻¹ JQ1 daily compared to vehicle control. A significant response to treatment was observed by a two-sided t-test on day 14 (p = 0.039). Data are mean ± s. d. (N = 7). FIG. 5G shows that histopathological analysis on NMC 797 tumors removed from JQ1-treated animals showed keratin expression induction (AE1 / AE3, 40 ×) and decreased proliferation (Ki67, 40 ×) compared to vehicle-treated animals. It shows that it is clarified (bar scale is 20 μm). In supplemental FIGS. 11 and 12, an enlarged field of view is provided. FIG. 5H shows that the viability of patient-derived NMC 11060 xenografts was confirmed by PET imaging. FIG. 5I shows that the therapeutic response of primary 11060 NMC xenografts to (+)-JQ1 (50 mg kg ⁻¹ daily for 4 days) was demonstrated by PET imaging. FIG. 5J shows that histopathological analysis of primary NMC 11060 tumors excised from (+)-JQ1 treated animals reveals keratin expression induction compared to vehicle treated animals (AE1 / AE3, 20 ×: Bar scale is 20 μm). Quantitative analysis of keratin induction was performed using contrast masking (FIG. 5J, right panel) and pixel positive analysis (FIG. 5K). Significant response to treatment is observed by a two-sided t test (p = 0.0001). Data are the mean ± sd of three independent wide field microscope fields. d. Represents. A comparative image of the stained excised tumor and the quantitative mask is provided in FIG. FIG. 4 is a graph showing that mice bearing NMC 797 xenografts can tolerate JQ1 therapy, causing an anti-tumor effect. FIG. 5L-a shows that treatment with NQ 797 xenograft-bearing mice with JQ1 (50 mg kg ⁻¹ per day) reduced tumor burden during 14 days of treatment. Data are mean ± s. d. (N = 7). FIG. 5L-b shows that JQ1 did not cause adverse symptoms or weight loss. Data are mean ± s. d. (N = 7). It shows that the induction of squamous epithelial differentiation in NUT midline cancer cells by JQ1 is stereospecific and time dependent. FIG. 5M-a is a photomicrograph showing that NMC 797 cells treated in chamber slides with (−)-JQ1 (100 nM) exhibit an equivalent cytosolic phenotype compared to vehicle-treated controls. is there. (+)-JQ1 (100 nM, 48 h) stimulates squamous epithelial differentiation exhibited by cell spindleing, flattening, and increased keratin expression. FIG. 5M-b is a photomicrograph. NMC 797 cells treated with JQ1 enantiomer or vehicle were centrifuged, fixed, sliced, and stained for keratin expression (left; AE1 / AE3, 20 ×). Image-based keratin expression analysis was performed on slides prepared simultaneously using unbiased masking and quantification algorithms that could score nuclei for staining intensity (right; 20 ×). FIG. 5M-c shows that (+)-JQ1 (250 nM) was measured in treated NMC 797 cells compared to (−)-JQ1 (250 nM) and vehicle control as measured by quantitative immunohistochemistry. It is a graph which shows that the rapid expression of keratin is induced | guided | derived. Present percent positive nuclei per treatment condition. Data are mean ± s. d. Represents (n = 3) and is annotated by the p-value obtained from a two-sided t test. FIG. 5M-d shows that (+)-JQ1 (250 nM) causes a time-dependent induction of potent (3+) keratin staining in treated NMC 797 cells compared to (−)-JQ1 (250 nM). It is a graph which shows that. Present percent positive nuclei per treatment condition. Data are mean ± s. d. Represents (n = 3) and is annotated by the p-value obtained from a two-sided t test. The grouped images are shown at the same magnification. Provided are photomicrographs showing that JQ1 promotes squamous differentiation, growth arrest, and apoptosis in vivo as measured by IHC. Histopathological analysis of NMC tumors removed from JQ1-treated animals (right panel) was all compared to vehicle-treated animals (left panel), squamous differentiation (H & E, 40 ×), nuclear NUT lesion progression ( NUT, 100x), reduced proliferation (Ki67, 40x), keratin expression induction (AE1 / AE3, 40x), and apoptotic response (TUNEL, 40x) are revealed. Provided are photomicrographs showing that JQ1 promotes squamous differentiation, growth arrest, and apoptosis in vivo as measured by IHC. Histopathological analysis of NMC tumors removed from JQ1-treated animals (right panel) was all compared to vehicle-treated animals (left panel), squamous differentiation (H & E, 40 ×), nuclear NUT lesion progression ( NUT, 100x), reduced proliferation (Ki67, 40x), keratin expression induction (AE1 / AE3, 40x), and apoptotic response (TUNEL, 40x) are revealed. FIG. 4 shows that JQ1 selectively induces apoptosis in NMC in human squamous cell carcinoma cell lines. FIG. 7A provides the results of FACS analysis. NMC Per403 cells (500 nM, 24 or 48 h) treated with JQ1 exhibit apoptosis induction by flow cytometry as opposed to non-NMC squamous cell carcinoma cell lines TE10 and TT. PI, propidium iodide; AV, annexin V. FIG. 7B shows quantification and comparison of annexin V positive cells by flow cytometry performed in FIGS. 5b and a. JQ1 (500 nM) showed an immediate and time-dependent induction of apoptosis in the NMC cell line compared to the non-NMC squamous cell carcinoma cell line. Data are mean ± s. d. Represents (n = 3) and is annotated by the p-value obtained from a two-sided t test. It is a graph which shows that a tissue derived from a NMC patient is sensitive to the antiproliferative effect of (+)-JQ1 in vitro. FIG. 8A shows the results from analysis of patient-derived NMC 11060 cells. Cells were isolated from waste clinical material and cultured in short-term cultures for in vitro experiments. The antiproliferative effect of BRD4 inhibition on NMC 11060 cells was measured after 72 hours of culture with (+)-JQ1 (red circles) or (−)-JQ1 (black circles). NMC 11060 cells are uniquely sensitive to the (+)-JQ1 enantiomer. Data are mean ± s. d. Presented as (n = 3). IC ₅₀ values were calculated by logistic regression. FIG. 8B shows the progressive anti-proliferative effect of (+)-JQ1 over time on patient-derived NMC 11060 cells, demonstrated on days 1, 3, 7, and 10. Data points are mean ± s. d. (N = 3). FIG. 8C shows flow cytometry results for DNA content in NMC 11060 cells. (+)-JQ1 (250 nM, 48 h) induces G1 arrest compared to (−)-JQ1 (250 nM) and the vehicle control. Provide quantification of diffuse strong keratin expression induced by jq1 in patient-derived NMC 11060 primary xenograft tumors. FIG. 9a shows a low magnification (0.8 ×) image of an isolated NMC 11060 primary xenograft from a vehicle-treated animal, sliced and stained for keratin expression (AE1 / AE3; left). Throughout the sliced tumor, the overall staining for keratin is weak. Image-based keratin expression analysis was performed using unbiased masking and quantification algorithms that can score individual pixels for staining intensity (right). FIG. 9B shows low magnification (0.8 ×) of isolated NMC 11060 primary xenografts from (+)-JQ1-treated animals (50 mg kg ⁻¹ daily for 4 days) sliced and stained for keratin expression. Images are shown (AE1 / AE3; left). A diffuse staining is observed, consistent with the uniform effect of keratin induction in JQ1-treated tumors. Image-based keratin expression analysis was performed using unbiased masking and quantification algorithms that can score individual pixels for staining intensity (right). Pixel positives are scored quantitatively and reported visually in blue (0), yellow (1+), orange (2+) and red (3+). All image pairs are shown at the same magnification. Show that JQ1 exhibits excellent oral bioavailability and adequate pharmacokinetic exposure in rodents. The pharmacokinetic study of (+)-JQ1 was performed in CD1 mice following intravenous (FIG. 10A, B) and oral (FIG. 10B, C) administration. a, Mean plasma concentration-time profile after intravenous administration of (+)-JQ1 (5 mg kg ⁻¹ ). Data are mean and s. d. (N = 3). FIG. 10B shows calculated intravenous (+) − JQ1 demonstrating excellent drug exposure [area under the curve (AUC) = 2090 hr * ng / mL] and a half-life of approximately 1 hour (T _1/2 ). The pharmacokinetic parameters of are shown. FIG. 10C shows the mean plasma concentration-time profile after oral administration of (+)-JQ1 (10 mg kg ⁻¹ ). Data are mean and s. d. (N = 3). FIG. 10D shows calculated oral (+) demonstrating superior oral bioavailability (F = 49%), peak plasma concentration (Cmax = 1180 ng / mL) and drug exposure (AUC = 2090 hr * ng / mL). -Shows the pharmacokinetic parameters of JQ1. CL, clearance; Vss, volume at steady state; MRT _INF , average residence time extrapolated to infinite time; T _max , maximum concentration arrival time. FIG. 6 is a graph showing AlphaScreen binding data for JQ1 enantiomers that inhibit BRD4.1. FIG. 6 is a graph showing AlphaScreen binding data for JQ1 enantiomers that inhibit BRD4.2. FIG. 6 is a graph showing profiling of JQ1S for other BET family members (active) and CBP (inactive). It is a graph which shows the dosage determination test of the priority library of a JQ1 derivative. FIG. 15A is a graph showing that JQ1 reduces tumor burden in a mouse xenograft model of NUT median carcinoma. FIG. 15B is a graph showing the body weight of mice treated with JQ1. FIG. 15A is a graph showing that JQ1 reduces tumor burden in a mouse xenograft model of NUT median carcinoma. FIG. 15B is a graph showing the body weight of mice treated with JQ1. It is a graph which shows BRD4 (1) and BRD4 (2) binding activity of JQ1 and its derivative (s). It is a graph which shows BRD4 (1) and BRD4 (2) binding activity of JQ1 and its derivative (s). It is a graph which shows BRD4 (1) and BRD4 (2) binding activity of JQ1 and its derivative (s). It is a graph which shows BRD4 (1) and BRD4 (2) binding activity of JQ1 and its derivative (s). 2 is a graph showing the effect of JQ1 and its derivatives on NUT median carcinoma (NMC) cell viability. 2 is a graph showing the effect of JQ1 and its derivatives on NUT median carcinoma (NMC) cell viability. 2 is a graph showing the effect of JQ1 and its derivatives on NUT median carcinoma (NMC) cell viability. 2 is a graph showing the effect of JQ1 and its derivatives on NUT median carcinoma (NMC) cell viability. 2 shows the dose response viability of various cancer cell lines treated with JQ1 and its derivatives. 2 shows the dose response viability of various cancer cell lines treated with JQ1 and its derivatives. 2 shows the dose response viability of various cancer cell lines treated with JQ1 and its derivatives. 2 shows the dose response viability of various cancer cell lines treated with JQ1 and its derivatives. 2 shows the dose response viability of various cancer cell lines treated with JQ1 and its derivatives. 2 shows the dose response viability of various cancer cell lines treated with JQ1 and its derivatives. 2 shows the dose response viability of various cancer cell lines treated with JQ1 and its derivatives. 2 shows the dose response viability of various cancer cell lines treated with JQ1 and its derivatives. 2 shows the dose response viability of various cancer cell lines treated with JQ1 and its derivatives. 2 shows the dose response viability of various cancer cell lines treated with JQ1 and its derivatives. 2 shows the dose response viability of various cancer cell lines treated with JQ1 and its derivatives. 2 shows the dose response viability of various cancer cell lines treated with JQ1 and its derivatives. 2 shows the dose response viability of various cancer cell lines treated with JQ1 and its derivatives. 2 shows the dose response viability of various cancer cell lines treated with JQ1 and its derivatives. 2 shows the dose response viability of various cancer cell lines treated with JQ1 and its derivatives. 2 shows the dose response viability of various cancer cell lines treated with JQ1 and its derivatives. 2 shows the dose response viability of various cancer cell lines treated with JQ1 and its derivatives. 2 shows the dose response viability of various cancer cell lines treated with JQ1 and its derivatives. 2 shows the dose response viability of various cancer cell lines treated with JQ1 and its derivatives. 2 shows the dose response viability of various cancer cell lines treated with JQ1 and its derivatives. 2 shows the dose response viability of various cancer cell lines treated with JQ1 and its derivatives. 2 shows the dose response viability of various cancer cell lines treated with JQ1 and its derivatives. 2 shows the dose response viability of various cancer cell lines treated with JQ1 and its derivatives. 2 shows the dose response viability of various cancer cell lines treated with JQ1 and its derivatives. 2 shows the dose response viability of various cancer cell lines treated with JQ1 and its derivatives. 2 shows the dose response viability of various cancer cell lines treated with JQ1 and its derivatives. 2 shows the dose response viability of various cancer cell lines treated with JQ1 and its derivatives. 2 shows the dose response viability of various cancer cell lines treated with JQ1 and its derivatives. 2 shows the dose response viability of various cancer cell lines treated with JQ1 and its derivatives. 2 shows the dose response viability of various cancer cell lines treated with JQ1 and its derivatives. 2 shows the dose response viability of various cancer cell lines treated with JQ1 and its derivatives. 2 shows the dose response viability of various cancer cell lines treated with JQ1 and its derivatives. 2 shows the dose response viability of various cancer cell lines treated with JQ1 and its derivatives. 2 shows the dose response viability of various cancer cell lines treated with JQ1 and its derivatives. 2 shows the dose response viability of various cancer cell lines treated with JQ1 and its derivatives. 2 shows the dose response viability of various cancer cell lines treated with JQ1 and its derivatives. 2 shows the dose response viability of various cancer cell lines treated with JQ1 and its derivatives. 2 shows the dose response viability of various cancer cell lines treated with JQ1 and its derivatives. It is a graph which shows the result of a lead compound binding assay. It is a graph which shows the result of a lead compound binding assay. It is a graph which shows the result of a lead compound binding assay. It is a graph which shows the result of a lead compound binding assay.

  The present invention includes neoplasms, inflammatory diseases, obesity, fatty liver (NASH or not), diabetes, atherosclerosis, arterial stent occlusion, heart failure, cachexia, graft-versus-host disease, bromodomain and Features compositions and methods useful for treating or preventing associated infectious diseases, treating parasites, malaria, trypanosoma, and reducing male fertility. Further uses of the compositions of the present invention include, but are not limited to, use in organ transplantation, cell condition regulation for regenerative medicine (ie by promoting or inhibiting cell differentiation), and promoting pluripotency. It is not a thing.

  The present invention is based, at least in part, on the discovery of a cell-permeable potent small molecule inhibitor (JQ1) and related compounds that are biochemically selective for the BET family of bromodomains, which compounds on nuclear chromatin. The bromodomain family, which is a family of polypeptides containing bromodomains that recognize the acetyl lysine residues, can be regulated. Lysine acetylation has been shown to be a signaling modification that is widely associated with cellular and disease biology. Targeting enzymes that reversibly mediate side chain acetylation has been an active area of drug discovery research for many years. To date, successful efforts have been limited to covalently modified “lighters” (acetyltransferases) and “eraser” (histone deacetylases) that have emerged in the context of nuclear chromatin. An acetyllysine recognition module, or a potent inhibitor of the bromodomain, has not yet been described. Recent characterization of the high resolution co-crystal structure of BRD4 revealed excellent shape complementarity with the acetyl lysine binding fossa. The binding of JQ1 to the tandem bromodomain of BRD4 is acetyllysine competitive and displaces BRD4 from chromatin in human cells. A recurrent translocation of BRD4 is observed in an incurable genetically defined subtype of human squamous cell carcinoma. Competitive binding of JQ1 to the BRD4 fusion oncoprotein results in immediate squamous differentiation and specific antiproliferative effects in patient-derived cell lines and in mouse models of BRD4-dependent carcinoma. These data establish the feasibility of epigenetic “leader” targeted protein-protein interactions and provide a versatile chemical framework for developing chemical probes across the broader bromodomain family. Report.

Bromodomain-containing proteins Gene regulation is basically governed by a non-covalent assembly of reversible macromolecules. Information transfer to RNA polymerase requires higher order protein complexes that are spatially controlled by assembly elements that can interpret the post-translational modification state of chromatin. Epigenetic leaders are structurally diverse proteins, each carrying one or more evolutionarily conserved effector modules that recognize covalent modifications of histone proteins or DNA. Ε-N-acetylation of a lysine residue (Kac) on the histone tail is associated with open chromatin structure and transcriptional activation ³ . Context specific molecular recognition of acetyl lysine is mainly mediated by the bromodomain.

Bromodomain-containing proteins have very high biological interest as components of transcription factor complexes (TAF1, PCAF, Gcn5, and CBP), and epigenetic memory determinants ⁴ . There are 41 human proteins containing a total of 57 diverse bromodomains. Despite large sequence diversity, all bromodomains are bound by four α helices (α _Z , α _A , α _B , Share a conserved fold comprising a left-handed bundle of α _C ). A co-crystal structure with a peptidic substrate showed that acetyllysine was recognized by the central hydrophobic fovea and anchored by hydrogen bonding with asparagine residues present in most bromodomains ⁵ . The bromodomain and extra-terminal (BET) families (BRD2, BRD3, BRD4, and BRDT) have a general domain structure comprising two N-terminal bromodomains that exhibit a high level of sequence conservation and a more diverse C- Share the terminal mobilization domain (FIG. 1E) ⁶ .

BRD4
Recent studies have established a reluctant theoretical basis for targeting BRD4 in cancer. BRD4 functions to promote cell cycle progression, and knockdown in cultured cancer cell lines promotes G1 arrest. BRD4 is an important mediator of transcription elongation and functions to recruit a positive transcription elongation factor complex (P-TEFb) ^7,8 . Cyclin-dependent kinase-9, the core component of P-TEFb, is a validated target in chronic lymphocytic leukemia ⁹ and has recently been associated with c-Myc-dependent transcription ¹⁰ . The bromodomain present in BRD4 recruits P-TEFb to the mitotic chromosome, leading to increased expression of growth promoting genes ¹¹ . BRD4 remains bound to the transcription start site of the gene expressed in M / G1, but its presence in the start site expressed later in the cell cycle has not been discovered. Knockdown of BRD4 in proliferating cells has been shown to result in G1 arrest and apoptosis by reducing expression levels ¹³ and survival ¹⁴ of genes important for mitotic progression ¹² .

Most importantly, BRD4 has recently been identified as a component of a recurrent t (15; 19) chromosomal translocation in high-grade human squamous cell carcinoma ^15,16 . Such a translocation expresses the tandem N-terminal bromodomain of BRD4 as an in-frame chimera with the NUT (nuclear protein in testis) protein that genetically defines the so-called NUT midline carcinoma (NMC). Functional studies in patient-derived NMC cell lines have verified the essential role of the BRD4-NUT oncoprotein in maintaining the growth benefits and differentiation arrest characteristic of this uniformly fatal malignancy ¹⁷ . In particular, RNA silencing of BRD4-NUT gene expression halts proliferation and promotes squamous epithelial differentiation with a marked increase in cytokeratin expression. These observations highlight the broad utility and immediate therapeutic potential of human bromodomain protein direct-acting inhibitors.

  The present invention features compositions and methods useful for inhibiting human bromodomain proteins.

Compounds of the Invention The present invention provides compounds that bind in the binding pocket of the apo crystal structure of the first bromodomain (eg, BRD4) of a BET family member (eg, JQ1 and compounds of the formulas described herein) . While not wishing to be bound by theory, these compounds are particularly effective in inhibiting the growth, proliferation or survival of proliferative neoplastic cells or inducing the differentiation of such cells. Also good. One approach is neoplasia, inflammatory disease, obesity, fatty liver (NASH or not), diabetes, atherosclerosis, arterial stent occlusion, heart failure, cachexia, graft-versus-host disease, bromodomain Treatment of infectious diseases associated with, treatment of parasites, malaria, trypanosoma, and use in reducing male fertility or in organ transplantation, cell condition regulation for regenerative medicine (ie promoting cell differentiation) Compounds, or by inhibition), and compounds useful for promoting pluripotency are selected using a molecular docking program that identifies compounds that are expected to bind to the bromodomain structural binding pocket. In certain embodiments, a compound of the invention binds to a BET family member, decreases the biological activity of the BET family member (eg, prolongation), and / or subcellularly localizes the BET family member. Can interfere (eg, reduced chromatin binding).

  In certain embodiments, the compounds of the invention interfere with and inhibit the biological activity of BET family members (eg, BRD2, BRD3, BRD4, BRDT) by at least 10%, 25%, 50%, 75%, or 100%. Or may interfere with or reduce and / or interfere with the intracellular localization of such proteins, for example by binding to a binding site in the bromodomain apo binding pocket.

  In certain embodiments, the compounds of the invention are small molecules having a molecular weight of less than about 1000 daltons, less than 800, less than 600, less than 500, less than 400, less than about 300 daltons. Examples of compounds of the invention include JQ1 binding to the binding pocket of the apo crystal structure of the first bromodomain of a BET family member (eg BRD4 (hereinafter referred to as BRD4 (1); PDB ID 2OSS)) Other compounds include JQ1 is a novel thieno-triazolo-1,4-diazepine The present invention further provides pharmaceutically acceptable salts of such compounds.

In one aspect, the invention provides a compound of formula I,

Where
X is N or CR ₅ ;
R ₅ is H, alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, each optionally substituted;
R _B is H, alkyl, hydroxyaralkyl, aminoalkyl, alkoxyalkyl, haloalkyl, hydroxy, alkoxy, or —COO—R ₃ , each optionally substituted;
Ring A is aryl or heteroaryl;
Each R _A is independently alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, each optionally substituted; or any two R _A are atoms attached to each of them Together can form a fused aryl or heteroaryl group;
R is alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, each optionally substituted;
R ₁ is — (CH ₂ ) _n —L (where n is 0 to 3 and L is H), —COO—R ₃ , —CO—R ₃ , —CO—N (R ₃ R _{4), - S (O)} 2 -R 3, -S (O) 2 -N (R 3 R 4), N (R 3 R 4), N (R 4) C (O) R 3, optionally Optionally substituted aryl, or optionally substituted heteroaryl;
R ₂ is H, D (deuterium), halogen, or optionally substituted alkyl;
Each R ₃ is independently
(I) H, aryl, substituted aryl, heteroaryl, or substituted heteroaryl;
(Ii) heterocycloalkyl or substituted heterocycloalkyl;
(Iii) O, S or is selected from N, containing 0, 1, 2, or 3 heteroatoms each, _-C 1 -C ₈ alkyl, _-C 2 -C ₈ alkenyl, or -C _2, -C ₈ alkynyl; each may be optionally _substituted, -C 3 _{-C 12} cycloalkyl, substituted _-C 3 _{-C 12 cycloalkyl,} -C 3 _{-C 12} cycloalkenyl or substituted -C, ₃ -C ₁₂ cycloalkenyl; and _{(iv) NH 2, N =} CR 4 R 6
Selected from the group consisting of;
Each R ₄ is independently H, alkyl, alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, each optionally substituted; or R ₃ and R ₄ are attached to them Together with the nitrogen atom to form a 4-10 membered ring;
R ₆ is alkyl, alkenyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, aryl, or heteroaryl, each optionally substituted; or R ₄ and R ₆ are the carbon atoms to which they are attached. Together with it to form a 4-10 membered ring;
m is 0, 1, 2, or 3;
However (a) Ring A is thienyl, X is N, R is phenyl or substituted phenyl, _{R 2} is H, _{R B} is methyl, _{R 1} is - _{(CH 2) n} - If L (wherein n is 1 and L is —CO—N (R ₃ R ₄ )) then R ₃ and R ₄ will not come with the nitrogen atom to which they are attached. Does not form a morpholino ring;
(B) Ring A is thienyl, X is N, R is a substituted phenyl, _{R 2} is H, _{R B} is methyl, _{R 1} is - _{(CH 2)} n -L _(wherein Wherein n is 1 and L is —CO—N (R ₃ R ₄ ), and if one of R ₄ is R ₃ and R ₄ is H, then the other of R ₃ and R ₄ is methyl , Not hydroxyethyl, alkoxy, phenyl, substituted phenyl, pyridyl or substituted pyridyl;
(C) Ring A is thienyl, X is N, R is a substituted phenyl, _{R 2} is H, _{R B} is methyl, _{R 1} is - _{(CH 2)} n -L _(wherein In which n is 1 and L is —COO—R ₃ ), then R ₃ is a compound that is neither methyl nor ethyl, or a salt, solvate or hydrate thereof.

  In certain embodiments, R is aryl or heteroaryl, each optionally substituted.

In certain _{embodiments, L} is _{H, -COO-R 3, -CO} -N (R 3 R 4), - S (O) 2 -R 3, -S (O) 2 -N (R 3 R 4 ), N (R ₃ R ₄ ), N (R ₄ ) C (O) R ₃ or optionally substituted aryl. In certain embodiments, each R ₃ is independently H; optionally substituted —C containing 0, 1, 2, or 3 heteroatoms selected from O, S, or N. It is selected from or the group consisting of _{NH 2, N = CR 4 R} 6; 1 ~C 8 alkyl.

In certain embodiments, R ₂ is H, D, halogen or methyl.

In certain embodiments, R _B is alkyl, hydroxyalkyl, haloalkyl, or alkoxy, each optionally substituted.

In certain embodiments, R _B is methyl, ethyl, hydroxymethyl, methoxymethyl, trifluoromethyl, COOH, COOMe, COOEt, or COOCH ₂ OC (O) CH ₃ .

  In certain embodiments, Ring A is a 5 or 6 membered aryl or heteroaryl. In certain embodiments, ring A is thiofuranyl, phenyl, naphthyl, biphenyl, tetrahydronaphthyl, indanyl, pyridyl, furanyl, indolyl, pyrimidinyl, pyridinyl, pyrazinyl, imidazolyl, oxazolyl, thienyl, thiazolyl, triazolyl, isoxazolyl, quinolinyl, pyrrolyl, Pyrazolyl or 5,6,7,8-tetrahydroisoquinolinyl.

  In certain embodiments, Ring A is phenyl or thienyl.

In certain embodiments, m is 1 or 2 and at least one occurrence of R _A is methyl.

In certain embodiments, each R _A is independently H, optionally substituted alkyl, or any two R _A can form an aryl together with the atoms to which each is attached.

In another aspect, the present invention provides compounds of formula II,

Where
X is N or CR ₅ ;
R ₅ is H, alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, each optionally substituted;
R _B is H, alkyl, hydroxyaralkyl, aminoalkyl, alkoxyalkyl, haloalkyl, hydroxy, alkoxy, or —COO—R ₃ , each optionally substituted;
Each R _A is independently alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, each optionally substituted; or any two R _A are atoms attached to each of them Together can form a fused aryl or heteroaryl group;
R is alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, each optionally substituted;
R ′ ₁ is H, —COO—R ₃ , —CO—R ₃ , optionally substituted aryl, or optionally substituted heteroaryl;
Each R ₃ is independently
(I) H, aryl, substituted aryl, heteroaryl, substituted heteroaryl;
(Ii) heterocycloalkyl or substituted heterocycloalkyl;
(Iii) O, S or is selected from N, containing 0, 1, 2, or 3 heteroatoms each, _-C 1 -C ₈ alkyl, _-C 2 -C ₈ alkenyl, or -C _2, -C ₈ alkynyl; each may be optionally _substituted, -C 3 _{-C 12} cycloalkyl, substituted _-C 3 _{-C 12 cycloalkyl,} -C 3 _{-C 12} cycloalkenyl or substituted -C, Selected from the group consisting of _{3 to} C ₁₂ cycloalkenyl;
m is 0, 1, 2, or 3;
Provided that when R ′ ₁ is —COO—R ₃ , X is N, R is substituted phenyl, and R _B is methyl, then R ₃ is not methyl or ethyl, or a salt thereof, Solvates or hydrates are provided.

  In certain embodiments, R is aryl or heteroaryl, each optionally substituted. In certain embodiments, R is phenyl or pyridyl, each optionally substituted. In certain embodiments, R is p-Cl-phenyl, o-Cl-phenyl, m-Cl-phenyl, p-F-phenyl, o-F-phenyl, m-F-phenyl or pyridinyl.

In certain embodiments, R ′ ₁ is —COO—R ₃ , optionally substituted aryl, or optionally substituted heteroaryl; R ₃ is selected from O, S, or N -C ₁ -C ₈ alkyl, which contains 0, 1, 2, or 3 heteroatoms and is optionally substituted. In certain embodiments, R ′ ₁ is —COO—R ₃ and R ₃ is methyl, ethyl, propyl, i-propyl, butyl, sec-butyl, or t-butyl; or R ′ ₁ is H Or optionally substituted phenyl.

In certain embodiments, R _B is methyl, ethyl, hydroxymethyl, methoxymethyl, trifluoromethyl, COOH, COOMe, COOEt, COOCH ₂ OC (O) CH ₃ .

In certain embodiments, R _B is methyl, ethyl, hydroxymethyl, methoxymethyl, trifluoromethyl, COOH, COOMe, COOEt, or COOCH ₂ OC (O) CH ₃ .

In certain embodiments, each R _A is independently an optionally substituted alkyl, or any two R _A together with the atom to which each is attached can form a fused aryl.

In certain embodiments, each R _A is methyl.

In another aspect, the present invention provides compounds of formula III,

Where
X is N or CR ₅ ;
R ₅ is H, alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, each optionally substituted;
R _B is H, alkyl, hydroxyaralkyl, aminoalkyl, alkoxyalkyl, haloalkyl, hydroxy, alkoxy, or —COO—R ₃ , each optionally substituted;
Ring A is aryl or heteroaryl;
Each R _A is independently alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, each optionally substituted; or any two R _A are atoms attached to each of them Together can form a fused aryl or heteroaryl group;
R is alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, each optionally substituted;
Each R ₃ is independently
(I) H, aryl, substituted aryl, heteroaryl, or substituted heteroaryl;
(Ii) heterocycloalkyl or substituted heterocycloalkyl;
(Iii) O, S or is selected from N, containing 0, 1, 2, or 3 heteroatoms each, _-C 1 -C ₈ alkyl, _-C 2 -C ₈ alkenyl, or -C _2, -C ₈ alkynyl; each may be optionally _substituted, -C 3 _{-C 12} cycloalkyl, substituted _-C 3 _{-C 12 cycloalkyl,} -C 3 _{-C 12} cycloalkenyl or substituted -C, ₃ -C ₁₂ cycloalkenyl; and _{(iv) NH 2, N =} CR 4 R 6
Selected from the group consisting of;
Each R ₄ is independently H, alkyl, alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, each optionally substituted; or R ₃ and R ₄ are attached to them Together with the nitrogen atom to form a 4-10 membered ring;
R ₆ is alkyl, alkenyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, aryl, or heteroaryl, each optionally substituted; or R ₄ and R ₆ are the carbon atoms to which they are attached. Together with it to form a 4-10 membered ring;
m is 0, 1, 2, or 3;
However (a) Ring A is thienyl, X is N, R is phenyl or substituted phenyl, if methyl R _B, then R ₃ and R ₄ and the nitrogen atom to adhere them thereto Do not combine to form a morpholino ring;
(B) Ring A is thienyl, X is N, R is a substituted phenyl, _{R 2} is H, _{R B} is methyl, one of _{R 3} and _{R 4} if H, The other of R ₃ and R ₄ then provides a compound that is methyl, hydroxyethyl, alkoxy, phenyl, substituted phenyl, pyridyl or non-substituted pyridyl, or a salt, solvate or hydrate thereof.

  In certain embodiments, R is aryl or heteroaryl, each optionally substituted. In certain embodiments, R is phenyl or pyridyl, each optionally substituted.

In certain embodiments, R is p-Cl-phenyl, o-Cl-phenyl, m-Cl-phenyl, p-F-phenyl, o-F-phenyl, m-F-phenyl or pyridinyl. In certain embodiments, R ₃ is H, NH ₂ , or N = CR ₄ R ₆ .

In certain embodiments, each R ₄ is independently H, alkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, each optionally substituted.

In certain embodiments, R ₆ is alkyl, alkenyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, aryl, or heteroaryl, each optionally substituted.

In another aspect, the present invention provides compounds of formula IV,

Where
X is N or CR ₅ ;
R ₅ is H, alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, each optionally substituted;
R _B is H, alkyl, hydroxyaralkyl, aminoalkyl, alkoxyalkyl, haloalkyl, hydroxy, alkoxy, or —COO—R ₃ , each optionally substituted;
Ring A is aryl or heteroaryl;
Each R _A is independently alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, each optionally substituted; or any two R _A are atoms attached to each of them Together can form a fused aryl or heteroaryl group;
R ₁ is — (CH ₂ ) _n —L (where n is 0 to 3 and L is H), —COO—R ₃ , —CO—R ₃ , —CO—N (R ₃ R _{4), - S (O)} 2 -R 3, -S (O) 2 -N (R 3 R 4), N (R 3 R 4), N (R 4) C (O) R 3, optionally Optionally substituted aryl, or optionally substituted heteroaryl;
R ₂ is H, D, halogen, or optionally substituted alkyl;
Each R ₃ is independently
(I) H, aryl, substituted aryl, heteroaryl, or substituted heteroaryl;
(Ii) heterocycloalkyl or substituted heterocycloalkyl;
(Iii) O, S or is selected from N, containing 0, 1, 2, or 3 heteroatoms each, _-C 1 -C ₈ alkyl, _-C 2 -C ₈ alkenyl, or -C _2, -C ₈ alkynyl; each may be optionally _substituted, -C 3 _{-C 12} cycloalkyl, substituted _-C 3 _{-C 12 cycloalkyl,} -C 3 _{-C 12} cycloalkenyl or substituted -C, ₃ -C ₁₂ cycloalkenyl; and _{(iv) NH 2, N =} CR 4 R 6
Selected from the group consisting of;
Each R ₄ is independently H, alkyl, alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, each optionally substituted; or R ₃ and R ₄ are attached to them Together with the nitrogen atom to form a 4-10 membered ring;
R ₆ is alkyl, alkenyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, aryl, or heteroaryl, each optionally substituted; or R ₄ and R ₆ are the carbon atoms to which they are attached. Together with it to form a 4-10 membered ring;
m is 0, 1, 2, or 3;
However (a) Ring A is thienyl, X is N, _{R 2} is H, _{R B} is methyl, _{R 1} is - _{(CH 2)} in n -L (wherein, n is 0 Yes, L is —CO—N (R ₃ R ₄ ), then R ₃ and R ₄ do not combine with the nitrogen atom to which they are attached to form a morpholino ring;
(B) Ring A is thienyl, X is N, _{R 2} is H, _{R B} is methyl, _{R 1} is - _{(CH 2)} n -L _(wherein, n is 0 , L is —CO—N (R ₃ R ₄ ), one of R ₃ and R ₄ is H, then the other of R ₃ and R ₄ is methyl, hydroxyethyl, alkoxy, phenyl , Not substituted phenyl, pyridyl or substituted pyridyl;
(C) Ring A is thienyl, X is N, _{R 2} is H, _{R B} is methyl, _{R 1} is - _{(CH 2)} n -L _(wherein, n is 0 , L is —COO—R ₃ ), then R ₃ provides a compound that is neither methyl nor ethyl, or a salt, solvate or hydrate thereof.

In certain embodiments, R ₁ is — (CH ₂ ) _n —L, wherein n is 0-3, L is —COO—R ₃ , optionally substituted aryl, or optionally R ₃ is optionally substituted containing 0, 1, 2, or 3 heteroatoms selected from O, S, or N it may also be a _-C 1 -C ₈ alkyl. In certain embodiments, n is 1 or 2, L is alkyl or —COO—R ₃ , and R ₃ is methyl, ethyl, propyl, i-propyl, butyl, sec-butyl, or t-butyl. Yes; or n is 1 or 2, and L is H or optionally substituted phenyl.

In certain embodiments, R ₂ is H or methyl.

In certain embodiments, R _B is methyl, ethyl, hydroxymethyl, methoxymethyl, trifluoromethyl, COOH, COOMe, COOEt, COOCH ₂ OC (O) CH ₃ .

  In certain embodiments, ring A is phenyl, naphthyl, biphenyl, tetrahydronaphthyl, indanyl, pyridyl, furanyl, indolyl, pyrimidinyl, pyridinyl, pyrazinyl, imidazolyl, oxazolyl, thienyl, thiazolyl, triazolyl, isoxazolyl, quinolinyl, pyrrolyl, pyrazolyl, Or 5,6,7,8-tetrahydroisoquinolinyl.

In certain embodiments, each R _A is independently an optionally substituted alkyl, or any two R _A together with the atom to which each is attached can form an aryl.

  The present invention also provides compounds of Formula V-XXII and any compounds described herein.

In another aspect, the invention provides a compound of formula

Provide a compound represented by a salt, solvate or hydrate thereof

In certain embodiments, the compound is (+)-JQ1:

Its salt, solvate or hydrate.

In another aspect, the invention provides a compound of formula

Or

Provided is a compound represented by a salt, solvate or hydrate thereof.

In another aspect, the invention provides a compound of formula

Or

Or a salt, solvate or hydrate thereof.

In another aspect, the invention provides a compound of formula

Or a salt, solvate or hydrate thereof is provided.

In another aspect, the invention provides a compound of formula

Or a salt, solvate or hydrate thereof is provided.

In another aspect, the invention provides:






A compound represented by any one of the following structures, or a salt, solvate or hydrate thereof is provided.

In certain embodiments, the compounds of the present invention are





Or a salt, solvate or hydrate thereof.

In one embodiment, the compound has the structure

Or a salt, solvate or hydrate thereof.

In another embodiment, the compound has the structure

Or a salt, solvate or hydrate thereof.

In another embodiment, the compound has the structure

Or a salt, solvate or hydrate thereof.

  In certain embodiments, the compounds of the invention may have the reverse chirality of any compound shown herein.

In certain embodiments, the invention provides a compound represented by formula (V), (VI), or (VII):

Wherein R, R ₁ and R ₂ and R _B have the same meaning as in formula (I); Y is O, N, S or CR ₅ and R ₅ is in formula (I) N is 0 or 1; the dashed circle in formula (VII) represents an aromatic or non-aromatic ring), or a salt, solvate or hydrate thereof .

In certain embodiments of any of Formulas I-IV and VI (or any formulas herein), R ₆ corresponds to the non-carbonyl moiety of the aldehyde shown in Table A below (ie, Formula R ₆ In the CHO aldehyde, R ₆ is the non-carbonyl portion of the aldehyde). In certain embodiments, R ₄ and R ₆ in combination correspond to a non-carbonyl moiety of a ketone shown in Table A (ie, for a ketone of formula R ₆ C (O) R ₄ , R ₄ and R ₆ Is the non-carbonyl moiety of the ketone).

In one embodiment, the compound has the formula:

Or a salt, solvate, or hydrate thereof.

In certain embodiments, the compound is (racemic) JQ1; in certain embodiments, the compound is (+)-JQ1. In certain embodiments, the compound is

and

Or a salt, solvate or hydrate thereof selected from the group consisting of

Examples of additional compounds include the formula:


Or a salt, solvate or hydrate thereof.

In Formulas IX-XXII, R and R ′ can be, for example, each optionally substituted H, aryl, substituted aryl, heteroaryl, heteroaryl, heterocycloalkyl, —C ₁ -C ₈ alkyl, _-C 2 -C ₈ alkenyl, _-C 2 -C _{8 alkynyl,} -C 3 _{-C 12} cycloalkyl, substituted _-C 3 _{-C 12 cycloalkyl,} -C 3 _{-C 12} cycloalkenyl or substituted, - It may be C ₃ -C ₁₂ cycloalkenyl. In formula XIV, X can be any substituent for an aryl group as described herein.

  The compounds of the present invention can be prepared by a variety of methods, some of which are known in the art. For example, examples of chemicals provided below are for preparing compound JQ1 (as a racemate) and enantiomers (+)-JQ1 and (−)-JQ1 (see Schemes S1 and S2). A synthesis scheme is provided. The various compounds of formulas (I)-(XXII) can be prepared by analogous methods by substitution of the appropriate starting materials.

For example, starting from JQ1, similar amines can be prepared as shown in Scheme 1 below.

Scheme 1

As shown in Scheme 1, hydrolysis of the t-butyl ester of JQ1 gives a carboxylic acid that is treated with diphenylphosphoric acid azide (DPPA) to provide a Cbz protected amine upon exposure to Curtius rearrangement conditions. Then it is deprotected to give the amine. For example, subsequent synthesis of amine groups by reductive amination results in secondary amines that can be further alkylated to provide tertiary amines.

Scheme 2

  Scheme 2 shows the synthesis of a further example of a compound of the invention of formula I in which, for example, the fused ring core is modified (eg by replacing ring A of formula I with a different aromatic ring). . A variety of fused ring cores and / or attached aryl groups (corresponding to group R in formula I) can be used by using aminodiaryl ketones with the appropriate functionality (eg instead of aminodiaryl ketone S2 in Scheme S1 below). A new compound is provided. Such aminodiaryl ketones are commercially available or some can be prepared by a variety of methods known in the art.

Scheme 3 provides additional exemplary synthetic schemes for preparing additional compounds of the invention.

Scheme 3

As shown in Scheme 3, the fused bicyclic precursor (see Scheme S1 below for the synthesis of this compound) is functionalized with the moiety R (DAM = dimethylaminomethylene protecting group), followed by hydrazide and To form a tricyclic fusion core. The substituent R _x can be varied by selection of a suitable hydrazide.

  Additional examples of compounds of the present invention (which can be prepared by the methods described herein) include the following.

Amides Amides can be prepared, for example, by preparation of the corresponding carboxylic acid or ester followed by amidation with an appropriate amine using standard conditions. In certain embodiments, the amide provides a two-carbon “linker” with a terminal terminal nitrogen-containing ring (eg, pyridyl, piperidyl, piperazinyl, imidazolyl (including N-methyl-imidazolyl), morpholinyl, etc.) As a structure,

Is mentioned.

  The use of a two-carbon linker between the amide moiety and the terminal nitrogen-containing ring is preferred.

"Reverse amide"

Secondary amine

Boronic acid

  In certain embodiments, the compound having at least one chiral center exists in racemic form. In certain embodiments, compounds having at least one chiral center are enantiomerically enriched, ie, at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, Has an enantiomeric excess (ee) of 85%, 90%, 95%, 99%, 99% or 100%. In certain embodiments, the compound is a compound (+)-JQ1 ((S) -tert-butyl 2- (4- (4-chlorophenyl) -2,3,9-trimethyl-6H) described herein. -Thieno [3,2-f] [1,2,4] triazole [4,3-a] [1,4] diazepam-6-yl) acetate).

In certain embodiments of any of the formulas disclosed herein, the compound is not represented by the following structure:

Where
R ′ ₁ is C ₁ -C ₄ alkyl;
R ′ ₂ is hydrogen, halogen, or C ₁ -C ₄ alkyl optionally substituted with a halogen atom or hydroxyl group;
R ′ ₃ is a halogen atom, phenyl optionally substituted with a halogen atom, C ₁ -C ₄ alkyl, C ₁ -C ₄ alkoxy, or cyano; —NR ₅ — (CH ₂ ) _m —R ₆ (formula In which R ₅ is a hydrogen atom or C ₁ -C ₄ alkyl, m is an integer from 0 to 4 and R ₆ is phenyl or pyridyl optionally substituted by a halogen atom); or —NR ₇ —CO —— (CH ₂ ) _n —R ₈ , wherein R ₇ is a hydrogen atom or C ₁ -C ₄ alkyl, n is an integer from 0 to 2, and R ₈ is optionally halogenated. Phenyl or pyridyl substituted by an atom);
R ′ ₄ is — (CH ₂ ) _a —CO—NH—R ₉ (wherein a is an integer of ₁ to ₄ and R ₉ is C _{1 to} C ₄ alkyl); C _{1 to} C _{4 hydroxyalkyl;} C 1 -C ₄ alkoxy; or optionally _C 1 -C _{4 alkyl,} C 1 -C ₄ alkoxy, amino or hydroxyl group or _{- (CH 2) b -COOR 10} ( wherein, b is 1 Is an integer of ˜4, and R ₁₀ is C ₁ -C ₄ alkyl).

  The term “pharmaceutically acceptable salt” is prepared from a compound disclosed herein (eg, JQ1, a compound of Formulas I-XXII) or any other compound described herein, Refers to a salt having an acidic functional group, such as a carboxylic acid functional group, and a pharmaceutically acceptable inorganic or organic base. Suitable bases include alkali metal hydroxides such as sodium, potassium, and lithium; hydroxides of alkaline earth metals such as calcium and magnesium; hydroxides of other metals such as aluminum and zinc; ammonia, And organic amines such as unsubstituted or hydroxy substituted mono-, di-, or trialkylamine; dicyclohexylamine; tributylamine; pyridine; N-methyl, N-ethylamine; diethylamine; triethylamine; mono-, bis-, or tris- Mono-, bis-, or tris- (2-hydroxy-lower alkylamine, N, N-, such as (2-hydroxyethyl) -amine, 2-hydroxy-tert-butylamine, or tris- (hydroxymethyl) methylamine Dimethyl-N- (2 N, N, -di-lower alkyl-N- (hydroxy lower alkyl) -amine, or tri- (2-hydroxyethyl) amine) such as hydroxyethyl) -amine); N-methyl-D-glucamine; and arginine; Examples include amino acids such as lysine, but are not limited thereto. The term “pharmaceutically acceptable salt” is prepared from a compound disclosed herein or any other compound described herein, and a basic functional group, such as an amino functional group, and a pharmaceutical Also refers to salts having a chemically acceptable inorganic or organic acid. Suitable acids include hydrogen sulfate, citric acid, acetic acid, oxalic acid, hydrochloric acid, hydrogen bromide, hydrogen iodide, nitric acid, phosphoric acid, isonicotinic acid, lactic acid, salicylic acid, tartaric acid, ascorbic acid, succinic acid, maleic acid , Besylic acid, fumaric acid, gluconic acid, glucaronic acid, sugar acid, formic acid, benzoic acid, glutamic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, and p-toluenesulfonic acid. It is not something.

Computer Simulation Screening Methods and Systems In another aspect, the present invention provides a machine-readable storage medium comprising the structural coordinates of a BET family member polypeptide (eg, BRD4 domain) binding site identified herein. . This is the proposed JQ1 binding site. The storage medium encoding these data can show a three-dimensional graphical representation of a molecule or molecular complex comprising such a binding site on a computer screen or similar visual device.

The present invention also provides methods for designing, evaluating and identifying compounds that bind to the aforementioned binding sites. Such compounds inhibit the biological activity of BET family members (eg BRD2, BRD3, BRD4, BRDT) and / or subcellular localization of BET family members (eg BRD2, BRD3, BRD4, BRDT) Is expected to interfere. The present invention provides: a) a three-dimensional representation of a molecule or molecular complex comprising a binding site; or b) a mean square deviation from the main chain atom of said amino acid of about 2.0 (more preferably 1.5). Providing a computer that generates a three-dimensional representation of a homologue of the molecule or molecule complex comprising a binding site that is less than or equal to
(I) a machine readable data storage medium comprising a data storage material encoded by machine readable data;
(Ii) a working memory storing instructions for processing the machine readable data;
(Iii) a central processing unit connected to the working memory and the machine readable data storage medium for processing the machine readable data into the three dimensional display; and (iv) for displaying the three dimensional display. Comprising a display connected to the central processor, the data comprising structural coordinates of amino acid residues in a bromodomain structure binding pocket or other BET family member binding site.

  Thus, the computer generates a three-dimensional graphical structure of the molecule or molecular complex that comprises the binding site.

  In another embodiment, the present invention provides a three-dimensional representation of a molecule or molecular complex, as defined by the structural coordinates of the amino acids of a BET family member (eg, BRD2, BRD3, BRD4, BRDT), or the main chain atoms of said amino acids There is provided a computer for generating a three-dimensional representation of a homologue of said molecule or molecule complex comprising a binding site whose mean square deviation from is 2.0 (more preferably 1.5) Å or less.

  In an exemplary embodiment, the computer or computer system is, for example, in US Pat. No. 5,978,740 and / or US Pat. No. 6,183,121 (incorporated herein by reference). It may include components conventional in the art as disclosed. For example, a computer system may include a central processing unit (“CPU”), a working memory (eg, may be a random access memory (RAM) or “core” memory), a mass storage memory (one or more disk drives). Or a CD-ROM drive), one or more cathode ray tube (CRT) or liquid crystal display (LCD) display terminals, one or more keyboards, one or more input lines, and one or more output lines. It can include a computer, all of which are interconnected by a conventional system bus.

  The machine-readable data of the present invention may be input to a computer through the use of a modem or group of modems connected by a data line. Alternatively or in addition, the input hardware may include a CD-ROM drive, a disk drive or flash memory. A keyboard may also be used as an input device in conjunction with the display terminal.

  Similarly, output hardware connected to a computer via an output line may be implemented by a conventional apparatus. As an example, the output hardware may include a CRT or LCD display terminal that uses a program such as QUANTA or PYMOL to show a graphical display of the binding pockets of the present invention. Output hardware may also include printers or disk drives to store system output for later use.

  In operation, the CPU coordinates the use of various input and output devices, coordinates data access from mass storage and access to and from working memory, and determines the sequence of data processing steps. Several programs, including commercially available software, may be used to process the machine readable data.

  A magnetic storage medium for storing machine readable data according to the present invention may be conventional. A magnetic data storage medium may encode machine-readable data, which may be implemented by a system such as the computer system described above. The medium has a suitable substrate that can be conventional and a suitable coating on one or both sides that can also be conventional and contains magnetic domains whose polarity or orientation can be changed magnetically. Can be a conventional floppy diskette or hard disk. The medium may also have an opening (not shown) for receiving a spindle of a disk drive or other data storage device.

  The magnetic domains of the media are encoded in a manner that may be conventional, such as machine-readable data as described herein, for execution by a system such as the computer system described herein. Polarized or oriented.

  Optical readable data storage media may also be used to code machine readable data or a set of instructions that can be implemented by a computer system. The medium can be a conventional compact disk read only memory (CD-ROM) or a rewritable medium such as an optically readable and magneto-optically writable magneto-optical disk.

  In the case of a CD-ROM, as is well known, the disk coating is reflective and is imprinted with a plurality of pits to encode machine-readable data. The arrangement of the pits is read by reflecting the laser beam on the coating surface. A protective coating, preferably substantially transparent, is provided on the top surface of the reflective coating.

  In the case of a magneto-optical disk, as is well known, a data recording coating does not have pits, but has a plurality of magnetic domains that can change its polarity or orientation magnetically when heated above a specific temperature with a laser or the like. . The orientation of the magnetic domains can be read by measuring the polarization of the laser light reflected from the coating. The arrangement of magnetic domains encodes the above data.

  Structural data can be used for various purposes such as drug discovery when used in conjunction with a software-programmed computer that translates these coordinates into a three-dimensional structure of a molecule or molecular complex comprising a binding pocket. May be used for.

  For example, the structure encoded by the data may be evaluated computationally for its ability to bind to a chemical entity. A chemical entity that binds to the binding site of a BET family member (eg, BRD2, BRD3, BRD4, BRDT) inhibits neoplastic cell proliferation or induces differentiation, and a BET family member (eg, BRD2, BRD3, BRD4, BRDT) Is expected to inhibit the biological activity of and / or interfere with subcellular localization. Such compounds are potential drug candidates. As an alternative, the structure encoded by the data may be shown in a three-dimensional graphic display on the computer screen. This allows visual inspection of the structure as well as visual inspection of the bond with the chemical entity of the structure.

  Thus, according to another embodiment, the present invention provides that the chemical entity comprises a) a binding site defined by the structural coordinates of a BET family member (eg, BRD2, BRD3, BRD4, BRDT) as described herein. Or b) a binding pocket having a mean square deviation from the main chain atom of the amino acid of 2.0 (more preferably 1.5) ２．０ or less, or The present invention relates to a homolog of a molecular complex and a method for evaluating the possibility of binding.

This method
i) performing a matching operation between the chemical entity and the binding site of the BET family member (eg, BRD2, BRD3, BRD4, BRDT) polypeptide or fragment or molecular complex thereof using computational means;
ii) analyzing the result of the fitting operation and quantifying the binding between the chemical entity and the binding pocket. This embodiment relates to assessing the potential of chemical entities that associate or bind to the binding site of a BET family member (eg, BRD2, BRD3, BRD4, BRDT) or fragments thereof.

  The term “chemical entity” as used herein refers to a compound, a complex of at least two compounds, and a fragment of such a compound or complex.

  In certain embodiments, the method comprises a molecule or molecular complex wherein the chemical entity is defined by the structural coordinates of all amino acids of a BET family member (eg, BRD2, BRD3, BRD4, BRDT) as described herein. Or the average square deviation from the main chain atom of the amino acid is 2.0 (more preferably 1.5) Å or less, and the possibility of binding to the homologue of the molecule or molecular complex is evaluated.

In further embodiments, the structural coordinates of one binding site described herein may be utilized in a method for identifying an antagonist of a molecule comprising a bromodomain binding site (eg, a bromodomain structural binding pocket). This method
a) using atomic coordinates of BET family members;
b) using the three-dimensional structure to design or select a potential agonist or antagonist. The compound may be obtained by any available means. “Obtain” means, for example, synthesizing, purchasing, or otherwise procuring an agonist or antagonist. If desired, the method further involves contacting the agonist or antagonist with the BET family member polypeptide or fragment thereof to determine the ability of the potential agonist or antagonist to interact with the molecule. If desired, the method can also contact neoplastic cells with a bromodomain binding compound to assess cytotoxicity, assess neoplastic cell proliferation, cell death, BET family member biological activity, or subcellular localization. Is further accompanied by a step.

In another embodiment, the present invention provides:
a) using atomic coordinates of a BET family member (eg BRD2, BRD3, BRD4, BRDT) (eg bromodomain structural binding pocket);
b) A method for identifying potential antagonists of BET family members (eg, BRD2, BRD3, BRD4, BRDT) comprising the step of designing or selecting potential agonists or antagonists using the three-dimensional structure. provide.

  The elucidation of bromodomain binding sites by the present inventors by the present inventors may interact in whole or in part with the bromodomain, and thus the BET family members (eg BRD2, BRD3, BRD4, BRDT) It provides the information necessary to design new chemical entities and compounds that may modulate (eg, inhibit) activity.

  In accordance with the present invention, binding to structural binding pocket sequences of BET family members (eg, BRD2, BRD3, BRD4, and BRDT) reduces biological activity of the bromodomain or subcellular localization of BET family members The design of compounds that interfere with this generally involves consideration of several factors. In one embodiment, the compound is physically and / or structurally at least a fragment of a BET family member (eg, BRD2, BRD3, BRD4, and BRDT), such as a binding site in the structural binding pocket sequence of the bromodomain. Join. Non-covalent molecular interactions important in this binding include hydrogen bonding, van der Waals interactions, hydrophobic interactions, and electrostatic interactions. Desirably, the compound is in a conformation that allows it to bind directly to the bromodomain binding site. Certain parts of the compound may not be directly involved in these bonds, but these parts of the entity may still affect the overall conformation of the molecule. This in turn may have a significant impact on the potency of the compound. Such conformational requirements include the overall three-dimensional structure and orientation of the compound relative to all or part of the binding site, or between functional groups comprising several compounds that interact directly with the binding site or its homologue. An interval is mentioned.

  The potential inhibition or binding effect of a compound on the bromodomain binding site may be analyzed by use of computer modeling techniques prior to its actual synthesis and testing. If the theoretical structure of a given compound suggests insufficient interaction and binding between it and the target binding site, testing of the compound becomes unnecessary. However, if computer modeling suggests a strong interaction, the molecule can be synthesized and tested for the ability to bind to the structural binding pocket sequences of BET family members (eg, BRD2, BRD3, BRD4, and BRDT), or For example, by assaying for cytotoxicity in neoplastic cells, by assaying for decreased biological activity of BET family members, or for intracellular localization of BET family members (eg, BRD2, BRD3, BRD4, and BRDT) It is tested for its biological activity by assaying. Candidate compounds may be evaluated computationally by means of a series of steps in which chemical entities or fragments are screened and selected for their ability to bind to the structural binding pocket of the bromodomain.

  One skilled in the art may use one of several methods in which compounds or fragments are screened for their ability to bind to a bromodomain binding site. This process may include visual inspection of binding sites on a computer screen based on, for example, the structural coordinates of the BET family members described herein, or other coordinates that define similar shapes created from machine-readable storage media. You may start with. The selected fragments or compounds are then placed in a variety of orientations or docked within the binding sites defined above. Docking may be accomplished using software such as Quanta and DOCK, followed by energy minimization and molecular dynamics with standard molecular mechanics force fields such as CHARMM and AMBER.

  Specialty computer programs (eg, as known in the art and / or commercially available and / or described herein) may also assist in the process of selecting fragments or chemical entities.

  Once the appropriate chemicals or fragments are selected, they can be assembled into a single compound or complex. Visual inspection of the interrelationship of the fragments on the three-dimensional image shown on the computer screen relative to the structural coordinates of the target binding site may precede the assembly.

  As described above, instead of proceeding with the construction of a binding pocket inhibitor in a step-by-step manner one fragment at a time, using empty binding sites or optionally known inhibitors Inhibitory or other binding compounds may be designed entirely or “newly”, including several parts of There are a number of new ligand design methods known in the art, some of which are commercially available (eg, LeapFrog available from Tripos Associates, St. Louis, Mo.).

  Other molecular modeling techniques may also be used in accordance with the present invention (eg, NC Cohen et al., “Molecular Modeling Software and Methods for Medicinal Chemistry, J. Med. Chem., 33, pp. 883). 894 (1990); MA Navia and MA Murcko, “The Use of Structural Information in Drug Design”, Current Opinions in Structural 2, Biop. 202, Biop. M. Balbes et al., “A Perspective of Modern Methods in C” See also mputer-Aided Drug Design ", in Reviews in Computational Chemistry, Vol. 5, KB Lipkowitz and DB Boyd, Eds., VCH, New York, pp. 337-380. See also WC Guida, “Software for Structure-Based Drug Design”, Curr. Opin. Struct. Biology, 4, pp. 777-781 (1994)).

  Once the compound is designed or selected, the efficiency with which the entity may bind to the binding site may be tested and optimized by computational evaluation.

  Specific computer software for assessing the deformation energy and electrostatic interaction of compounds is available in the art. Examples of programs designed for such applications include AMBER; QUANTA / CHARMM (Accelrys, Inc., Madison, Wis.). These programs may be implemented using, for example, a commercially available graphics workstation. Other hardware systems and software packages are known to those skilled in the art.

  Another technique involves screening by computer simulation of a virtual library of compounds as described herein (see, eg, Examples). Thousands of compounds can be rapidly screened and the best virtual compounds can be selected for further screening (eg, by synthesis and in vitro or in vivo testing). Small molecule databases can be screened for chemical entities or compounds that can bind, in whole or in part, to BET family members (eg, BRD2, BRD3, BRD4, and BRDT) bromodomain binding sites. In this screening, the quality of matching between such entities and binding sites may be determined by shape complementarity and by estimated interaction energy.

a) a three-dimensional representation of a molecule or molecular complex comprising a structural binding pocket of a BET family member, defined by the structural coordinates of the amino acid residues in the structural binding pocket sequence of the bromodomain; or b) the amino acid Produces a three-dimensional representation of a homologue of said molecule or molecule complex comprising a binding site whose mean square deviation from the main chain atom is about 2.0 (more preferably 1.5) １．５ or less A computer for said computer,
(I) a machine readable data storage medium comprising a data storage material encoded by machine readable data;
(Ii) a working memory storing instructions for processing the machine readable data;
(Iii) a central processing unit connected to the working memory and the machine-readable data storage medium for processing the machine-readable data into the three-dimensional display; and (iv) indicating the three-dimensional display; Comprising a display connected to a central processor, the data comprising structural coordinates of the structural coordinates of amino acid residues in the structure binding pocket sequence of a BET family member. As identified in the examples, compounds identified using computer simulation methods can be used, for example, using the “additional screening methods” described below, or any other art-known method. Thus, it may optionally be tested in vitro or in vivo.

Additional Screening Methods As noted above, the present invention relates to compounds comprising JQ1, as well as other substituted compounds, that bind to the bromodomain binding pocket and induce cell differentiation or reduce cell proliferation in neoplastic cells. A specific example is provided. However, the present invention is not limited to this. The present invention is capable of binding to BET family members, is cytotoxic to neoplastic cells, reduces the biological activity of BET family members, or interferes with the intracellular localization of BET family members (nucleic acids, peptides It further provides a simple means of identifying agents (including small molecule inhibitors, and mimetics). Such compounds also include neoplasms, inflammatory diseases, obesity, fatty liver (NASH or not), diabetes, atherosclerosis, arterial stent occlusion, heart failure, cachexia, graft-versus-host disease, Expected to be useful in treating or preventing infectious diseases associated with bromodomains, treating parasites, malaria, trypanosoma, and reducing male fertility. Further uses of the compositions of the present invention include, but are not limited to, use in organ transplantation, cell condition regulation for regenerative medicine (ie by promoting or inhibiting cell differentiation), and promoting pluripotency. It is not a thing.

  In particular, certain aspects of the invention provide that the agent that decreases the biological activity of a BET family member polypeptide is a neoplasm, inflammatory disease, obesity, fatty liver (NASH or not), diabetes, atheroma Atherosclerosis, arterial stent occlusion, heart failure, cachexia, graft-versus-host disease, treatment or prevention of infectious diseases associated with bromodomains, treatment of parasites, malaria, trypanosoma, and male fertility Based at least in part on the discovery that it is probably useful as a therapeutic agent for reducing. Further uses of the compositions of the present invention include, but are not limited to, use in organ transplantation, cell condition regulation for regenerative medicine (ie by promoting or inhibiting cell differentiation), and promoting pluripotency. It is not a thing. In certain embodiments, the effect of a compound of the present invention or other agent is analyzed by assaying cell proliferation, cell survival or cell death. In another approach, the agents and compounds of the invention are assayed for effects on transcriptional regulation or elongation. Agents and compounds of the present invention that reduce neoplastic growth, proliferation, or invasiveness are identified as useful in the treatment or prevention of neoplasms. In yet another embodiment, the compounds of the invention are assayed for effects on immune responsive cells, cytokine or histamine release, or any other marker of inflammatory disease.

  Virtually any agent that specifically binds to or reduces the biological activity of a BET family member may be used in the methods of the invention. The methods of the invention are useful for high-throughput, low-cost screening of candidate agents that reduce, retard or stabilize neoplastic growth or proliferation, or treat or prevent inflammatory diseases. A candidate agent that specifically binds to the bromodomain of a BET family member is then isolated and its ability to reduce neoplastic cell proliferation, induce differentiation and / or increase neoplastic cell death. Test for activity in in vitro or in vivo assays. One skilled in the art understands that the effect of a candidate agent on a cell is typically compared to a corresponding control cell that has not been contacted with the candidate agent. Thus, the screening method involves comparing the growth of neoplastic cells contacted with the candidate agent to the growth of untreated control cells.

  In another embodiment, the expression or activity of a BET family member in a cell treated with a candidate agent is compared to an untreated control sample to determine the biological activity of the BET family member in the contacted cell. Identify candidate compounds to decrease. Polypeptide expression or activity is determined by Western blotting, flow cytometry, immunocytochemistry, binding to magnetic and / or bromodomain specific antibody-coated beads, in situ hybridization, fluorescence in situ hybridization (FISH), ELISA , Microarray analysis, RT-PCR, Northern blotting, or colorimetric assays such as Bradford assay or Lowry assay.

  In one example, one or more candidate agents are added at various concentrations to a culture containing neoplastic cells. Agents that reduce the expression of BET family members expressed in cells are considered useful in the present invention; such agents prevent, delay, improve, for example, neoplasms or inflammatory diseases. It may be used as a therapeutic agent to stabilize, treat or treat. Once identified, an agent of the invention (eg, an agent that specifically binds and / or antagonizes a bromodomain) is used to neoplasm, inflammatory disease, obesity, fatty liver (NASH or not) ), Diabetes, atherosclerosis, arterial stent occlusion, heart failure, cachexia, graft-versus-host disease, treatment of infectious diseases associated with bromodomains, treatment of parasites, malaria, trypanosoma, and Men's fertility may be reduced. Further uses of the compositions of the present invention include, but are not limited to, use in organ transplantation, cell condition regulation for regenerative medicine (ie by promoting or inhibiting cell differentiation), and promoting pluripotency. It is not a thing. Agents identified according to the methods of the present invention can be delivered locally or systemically to form neoplasms, inflammatory diseases, obesity, fatty liver (NASH or otherwise), diabetes, atherosclerosis, Treat arterial stent occlusion, heart failure, cachexia, graft-versus-host disease, infectious diseases associated with bromodomains, treat parasites, malaria, trypanosoma, and reduce male fertility or organ transplantation , For use in regulating cell conditions for regenerative medicine (ie by promoting or inhibiting cell differentiation) or in situ pluripotency.

  In one embodiment, as an alternative, the effect of a candidate agent is determined by an antibody specific for a BET family member using the same general methods and standard immunological techniques, such as Western blotting or immunoprecipitation. , Measured at the production level of BET family members. For example, immunoassays may be used to detect or monitor the expression of BET family members in neoplastic cells. In one embodiment, the present invention can bind to a BET family member to block biological activity or inhibit subcellular localization, polyclonal (generated as described herein) or Identify monoclonal antibodies. Compounds that interfere with the intracellular localization of BET family members or reduce biological activity are considered particularly useful. Again, such agents may be used as therapeutic agents to prevent or treat neoplasms or inflammatory diseases, for example.

  As an alternative or in addition, as described in the Examples, those that specifically bind and antagonize BET family members of the invention (eg, BRD2, BRD3, BRD4, and BRDT) are first assayed. Candidate compounds may then be identified by subsequently testing their effects on neoplastic cells. In one embodiment, the effectiveness of a candidate agent depends on its ability to interact with BET family members. Such interactions can be easily assayed using a number of standard binding techniques and functional assays such as those described in Ausubel et al., Supra. For example, a candidate compound can be tested in vitro for interaction and binding with a polypeptide of the present invention to modulate its ability to modulate neoplastic cell proliferation in any standard assay (eg, those described herein). May be assayed. In one embodiment, neoplastic cell division is determined by assaying BrdU incorporation using flow cytometric analysis. In another embodiment, the expression of BET family members is monitored immunohistochemically.

  Potential bromodomain antagonists include organic molecules, peptides, peptidomimetics, polypeptides, nucleic acid ligands, aptamers, and antibodies that bind to and reduce the activity of BET family member bromodomains. In one particular example, candidate compounds that bind to BET family members may be identified using chromatography-based techniques. For example, the recombinant BET family member polypeptides of the present invention may be purified from cells that have been genetically modified to express the polypeptide by standard techniques, or once the purified peptide is immobilized on a column, it may be chemically synthesized. Also good. Next, a solution of the candidate agent is passed through the column, binds to the BET family member polypeptide, and specifically binds to the BET family member polypeptide or fragment thereof based on its ability to be immobilized on the column. Identify the substance. To isolate the agent, the column is washed to remove non-specifically bound molecules, and then the agent of interest is released from the column and collected. Agents isolated by this method (or any other suitable method) may be further purified if desired (eg, by high performance liquid chromatography). In addition, these candidate agents may be tested for their ability to reduce neoplastic cell proliferation or viability. Agents isolated by this approach also include, for example, neoplasms, inflammatory diseases, obesity, fatty liver (NASH or not), diabetes, atherosclerosis, arterial stent occlusion, heart failure, cachexia, Graft-versus-host disease, treat or prevent infectious diseases associated with bromodomains, treat parasites, malaria, trypanosoma, and reduce male fertility or use in organ transplantation, for regenerative medicine It may be used as a therapeutic agent that promotes cell state regulation (ie, by promoting or inhibiting cell differentiation) and pluripotency. Compounds identified as binding to BET family members with affinity constants of 1 nM, 5 nM, 10 nM, 100 nM, 1 μM or 10 μM or less are considered particularly useful in the present invention.

Test Compounds and Extracts In certain embodiments, a BET family member antagonist (eg, an agent that specifically binds to a bromodomain and decreases activity) is a large live of a natural or synthetic (or semi-synthetic) extract. From a library or from a chemical library or polypeptide or nucleic acid library according to methods known in the art. Those skilled in the field of drug discovery research and development understand that the precise source of the test extract or compound is not critical to the screening procedure of the present invention. Agents used in screening may include those known as therapeutic agents for neoplasms or inflammatory diseases. Alternatively, the methods described herein can be used to screen virtually any number of unknown chemical extracts or compounds. Examples of such extracts or compounds include, but are not limited to, plant-based, fungal-based, prokaryotic-based or animal-based extracts, fermentation broths, and synthetic compounds, as well as modifications of existing polypeptides. It is not a thing.

  Libraries of natural polypeptides in the form of bacterial, fungal, plant, and animal extracts are Biotics (Sussex, UK), Xenova (Slough, UK), Harbor Branch Oceanographic Institute (Ft. Pierce, Fla.), And PharmaMar. , U. S. A. (Cambridge, Mass.) Are commercially available from several sources. Such polypeptides can be modified using methods described herein known in the art to include protein transduction domains. In addition, if desired, natural and synthetically generated libraries are generated according to methods known in the art, eg, by standard extraction and fractionation methods. Examples of molecular library synthesis methods are described in, for example, DeWitt et al. , Proc. Natl. Acad. Sci. U. S. A. 90: 6909, 1993; Erb et al. , Proc. Natl. Acad. Sci. USA 91: 11422, 1994; Zuckermann et al. , J .; Med. Chem. 37: 2678, 1994; Cho et al. , Science 261: 1303, 1993; Carrell et al. , Angew. Chem. Int. Ed. Engl. 33: 2059, 1994; Carell et al. , Angew. Chem. Int. Ed. Engl. 33: 2061, 1994; and Gallop et al. , J .; Med. Chem. 37: 1233, 1994, and the like. Furthermore, if desired, any library or compound is readily modified using standard chemical, physical, or biochemical methods.

  Generate random or directed synthesis (eg, semi-synthetic or total synthesis) of any number of polypeptides, compounds, including but not limited to saccharide-based, lipid-based, peptide-based, and nucleic acid-based compounds A number of methods are available. Synthetic compound libraries are commercially available from Brandon Associates (Merrimack, NH) and Aldrich Chemical (Milwaukee, Wis.). Alternatively, compounds used as candidate compounds can be synthesized from readily available starting materials using standard synthetic techniques and techniques known to those skilled in the art. Synthetic chemical transformations and protecting group techniques (protection and deprotection) useful for synthesizing compounds identified by the methods described herein are known in the art and are described, for example, in R.A. Larock, Comprehensive Organic Transformations, VCH Publishers (1989); W. Greene and P.M. G. M.M. Wuts, Protective Groups in Organic Synthesis, 2nd ed. John Wiley and Sons (1991); Fieser and M.M. Fieser, Fieser and Fieser's Reagents for Organic Synthesis, John Wiley and Sons (1994); Paquette, ed. , Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons (1995), and subsequent editions.

  Compound libraries can be in solution (eg Houghten, Biotechniques 13: 412-421, 1992) or on beads (Lam, Nature 354: 82-84, 1991), on chip (Fodor, Nature 364: 555-556, 1993). On bacteria (Ladner, US Pat. No. 5,223,409), on spores (Ladner US Pat. No. 5,223,409), on plasmids (Cull et al., Proc Natl Acad Sci USA 89 1865-1869, 1992) or on phage (Scott and Smith, Science 249: 386-390, 1990; Devlin, Science 249: 404-406, 1990; Cwirla et al. Proc. Natl. Acad. Sci. 87: 6378-6382, 1990; Felici, J. Mol. Biol. 222: 301-310, 1991; Ladner, supra).

  In addition to this, those skilled in drug discovery research and development will be able to replicate or repeat de-replication of materials of known activity (eg taxonomic de-replication, biological de-replication, and chemical de-replication, or any of its Easily understand that combination) or exclusion should be used whenever possible. If the crude extract is found to have BET family member bromodomain binding activity, further fractionation of the positive lead extract is required to isolate the molecular components responsible for the observed effects . Thus, the goal of the extraction, fractionation, and purification process is the careful characterization and identification of chemical entities that reduce neoplastic cell growth or viability in the crude extract. Methods for fractionating and purifying such heterogeneous extracts are known in the art. If desired, compounds shown to be useful as therapeutic agents are chemically modified according to methods known in the art.

  The present invention provides a disease and / or disorder or symptom thereof comprising administering to a subject (eg, a mammal such as a human) a therapeutically effective amount of a pharmaceutical composition comprising a compound of the formulas herein. Provide a method of treatment. Accordingly, one embodiment is a method of treating a subject suffering from or susceptible to a neoplastic disease or disorder or symptom thereof. The method includes administering to the mammal a therapeutic amount of an amount of a compound herein sufficient to treat the disease or disorder or symptom thereof under conditions such that the disease or disorder is treated.

  The methods herein provide an effective amount of a compound or composition described herein as described herein to produce such an effect (subjects identified as requiring such treatment). Administering to a subject. Identification of a subject in need of such treatment can be determined by the subject or health care professional and can be subjective (eg, opinion) or objective (eg, measurable by a test or diagnostic method).

  Therapeutic methods (including prophylactic treatments) of the present invention generally require a therapeutically effective amount of a compound herein, such as a compound of formula herein, including mammals, particularly humans. Administering to a subject (eg, animal, human). Such treatment is suitably administered to a subject, particularly a human, who is suffering from, having, susceptible to, or at risk of having a disease, disorder, or symptom thereof. The determination of a “risk” subject is by diagnostic test or by the opinion of the subject or health care provider (eg, genetic test, enzyme or protein marker, Marker (as defined herein), family history, etc.) It can be made by any objective or subjective judgment. The compounds herein may be used in the treatment of any other disorder where neoplasia or inflammation is implicated.

  In one embodiment, the present invention provides a method of monitoring treatment progress. The method comprises a diagnostic marker (such as any target, protein or indicator thereof described herein modulated by a compound herein), or a disorder or symptom thereof associated with a neoplasm Measuring a level of a diagnostic measurement (eg, screening, assay) in a subject suffering from or susceptible to, a therapeutic amount of a compound herein sufficient to treat the disease or symptom thereof, Administered to the subject. The level of Marker measured by the method can be compared to the known Marker level of either a healthy control or other patient to establish the disease state of the subject. In a preferred embodiment, at a time later than the first level measurement, a second level of Marker in the subject is measured and the two levels are compared to monitor the course of the disease or the effectiveness of the therapy. . In certain preferred embodiments, the pre-treatment level of the marker in the subject is measured prior to initiating treatment according to the present invention; then this pre-treatment level of Marker is compared to the Marker level in the subject after initiation of treatment. The effectiveness of the treatment can be determined.

In another embodiment, an agent (eg, JQ1 or a compound of the formula described herein) that has been discovered to have a pharmacological effect using the methods described herein is an agent. Or as information on the structural modification of existing compounds by rational drug design. Such methods are effective against neoplasms, inflammatory diseases, obesity, fatty liver (NASH or not), diabetes, atherosclerosis, arterial stent occlusion, heart failure, cachexia, graft-versus-host disease Treatment of infectious diseases, parasites, malaria, trypanosoma associated with bromodomain, and use in reducing male fertility or organ transplantation, cell conditioning for regenerative medicine (ie cell differentiation) Is useful for screening agents that promote pluripotency).

  For therapeutic use, a composition or agent identified using the methods disclosed herein is systemically administered in a pharmaceutically acceptable buffer such as, for example, physiological saline. May be. Preferred routes of administration include, for example, subcutaneous, intravenous, intraperitoneal, intramuscular, or intradermal injection that provides continuous sustained drug levels in the patient. Treatment of human patients or other animals is performed using a therapeutically effective amount of the therapeutic agent identified herein in a physiologically acceptable carrier. For suitable carriers and their formulations, see, for example, Remington's Pharmaceutical Sciences by E.M. W. Described in Martin. The amount of therapeutic agent administered is: mode of administration, patient age and weight, neoplasm, inflammatory disease, obesity, fatty liver (NASH or not), diabetes, atherosclerosis, arterial stent occlusion, heart failure , Cachexia, graft-versus-host disease, clinical manifestations of infectious diseases associated with bromodomains, treatment of parasites, malaria, trypanosoma, and use in male fertility or organ transplantation, regenerative medicine Fluctuate depending on the cellular state regulation for (ie, by promoting or inhibiting cell differentiation) and promoting pluripotency. In general, the amount is within the range used for other agents used in the treatment of other diseases associated with such diseases or conditions, but in some cases, increased compound specificity Less amount is needed for that. The compound is cytotoxic to neoplastic cells, as determined by methods known to those skilled in the art, or as determined using any assay that measures BET family member expression or biological properties, and the BET family member Is administered at a dosage that reduces the biological activity of or reduces the growth, survival, or invasiveness of neoplastic cells. In another embodiment, the compound is administered at a dosage that reduces inflammation or symptoms of an inflammatory disease.

Inflammatory Diseases Compositions of the invention comprising compounds of the formulas described herein are useful for reducing inflammation and for treating inflammatory diseases. Inflammation is usually the result of a complex series of molecular signals involved in the immune system in response to infection or cell or tissue damage. Inflammation normally constitutes the body's onset of healing; however, if not properly regulated, inflammation can lead to chronic diseases such as arthritis.

  An “inflammatory response” or “immune response” is against injury, infectivity or irritation characterized by redness, warmth, swelling, pain, and loss of function resulting from increased blood flow and immune cell influx and secretion It means the reaction of living tissue. Inflammation is the body's reaction to invading infectious microorganisms, resulting in increased blood flow to the affected area, release of chemicals that attract white blood cells, increased plasma flow, and arrival of monocytes that clear off necrotic tissue debris. Anything that stimulates an inflammatory response is considered inflammatory.

  The innate cascade, or innate immune response, is developed by the immune system in response to chemotactic signaling at the site of injury or infection, and leukocytes, natural killer cells, mast cells, eosinophils and basophils, and It is a nonspecific response characterized by the invasion of cells such as phagocytic cells such as neutrophils, macrophages, and dendritic cells. Molecules such as histamine and various cytokines secreted by the aforementioned cells; and the complement system of circulating proteins contribute to inflammation.

  The disease is characterized by inflammation and is a prominent etiology and cause of death in humans.

  In certain embodiments, the inflammatory disease is a rheumatoid disorder. Rheumatoid disorders, as used herein, refer to any of a variety of inflammatory diseases characterized by inflammation, sometimes degeneration and / or metabolic abnormalities of connective tissue structures, particularly joints, ligaments, and tendons. Rheumatoid disorders typically result in pain, stiffness, and / or limited mobility. The particular tissue or tissue that is affected depends on the rheumatoid disorder. Exemplary rheumatoid disorders include, but are not limited to, rheumatoid arthritis, juvenile arthritis, bursitis, spondylitis, gout, scleroderma, Still's disease, and vasculitis. .

  In certain embodiments, the rheumatoid disorder is rheumatoid arthritis and "treatment" of rheumatoid arthritis includes reducing the severity, frequency, and / or appearance of one or more rheumatoid arthritis symptoms. . In another embodiment, the rheumatoid disorder is juvenile arthritis, bursitis, spondylitis, gout, scleroderma, Still's disease, or vasculitis. The methods of the present invention reduce the severity, frequency, and / or appearance of any one or more of these pathologies.

  In various embodiments, symptoms of arthritis or other inflammatory diseases include redness, swelling, inflammation, fever, decreased range of motion, and pain. Examples of symptom appearance or reduced severity include: decreased number of swollen joints, reduced number of painful joints, reduced dependence on analgesics, reduced patient frequency of pain or self-assessment of severity, freedom of movement Include, but are not limited to, increased degrees, increased mobility, decreased fever, and increased daily performance.

  Neuroinflammation characterized by microglial and astrocyte activation and local expression of various inflammatory mediators is the basis for brain injury, whether due to trauma, stroke, infectivity, or neurodegeneration It is a reaction. This local tissue response is considered part of the repair and recovery process. Like many inflammatory conditions in peripheral diseases, neuroinflammation can contribute to the pathophysiology of CNS disorders.

  In certain embodiments, the inflammatory disease is an inflammatory skin disease. Inflammatory skin diseases include, but are not limited to, rosacea, atopic dermatitis, acne, seborrheic dermatitis, and cellulitis.

  In another embodiment, the inflammatory disease is an ischemic or inflammatory cardiovascular disease. Inflammatory cardiovascular disease or disorder is obstructive disease or disorder, atherosclerosis, heart valve disease, stenosis, restenosis, in-stent stenosis, myocardial infarction, coronary artery disease, acute coronary syndrome, congestive heart failure, angina May be, but is not limited to, myocardial ischemia or thrombosis. In another embodiment, the site is a secondary site of ischemic injury such as CNS or kidney.

  In another embodiment, the inflammatory disease is ischemic or inflammatory bowel disease.

  Inflammatory bowel disease (IBD) refers to a chronic recurrent inflammatory disease of unknown cause affecting the small and large intestine, and includes both Crohn's disease (CD) and ulcerative colitis (UC). Crohn's disease can affect any part of the intestinal tract, but usually affects the distal small and / or large intestine. Ulcerative colitis affects only the large intestine and is usually limited to the rectum or distal colon. Studies of CD and UC mouse models strongly suggest that both of these diseases are due to dysregulation of the mucosal immune response to antigens in the mucosal flora (Sartor, RB (1995). Gastroenterol. Clin North Am 24,475-507) (Strober W, et al. (2002) Annu. Rev. Immunol. 20: 495-549).

  Ulcerative colitis or unclassified colitis refers to the pathology of the large intestine, characterized by an inflammatory condition, in which one or more of the following histological features can be detected: epithelial cell loss and patches Superficial inflammation characterized by the presence of cervical ulceration, apparent depletion of mucin producing goblet cells, and a decrease in tubular gland density. In addition, within the lamina propria, a mixture consisting of lymphocytes and granulocytes (the latter mostly consisting of neutrophils and to a lesser extent eosinophils) associated with cell exudation into the intestinal lumen Inflammatory cell infiltrates are observed. Also, submucosal levels may show significant edema with few inflammatory cells, while within the outer muscle layer, those skilled in the art will see little or no evidence of inflammation. See, for example, Boirivant et al. See Journal of Experimental Medicine 188: 1929-1939 (1998). Clinical symptoms include, but are not limited to, diarrhea, rectal prolapse, weight loss, abdominal pain, and dehydration.

  Crohn's disease refers to inflammation that affects any part of the gastrointestinal tract, but most often affects the distal small intestine and / or the adjacent ascending colon. Inflammation is often characterized by “interlaced lesions” consisting of inflammatory areas alternating with normal mucosal areas. Crohn's disease intestinal lesions are characterized by erythema, edema, and increased friability; sometimes the intestine narrows and attaches to other abdominal organs or the intestinal wall. A fistula between the affected intestine and other structures including the skin is not uncommon. Microscopic examination of Crohn's disease tissue reveals epithelial erosion, loss of mucin-producing goblet cells, and extensive lymphocyte infiltration affecting the entire mucosa; this invasion is sometimes a giant cell that is a sign of granulomas Containing. If inflammation is present for a long time (chronically), it can sometimes cause scars (fibrosis). Scar tissue is typically not as flexible as healthy tissue. Thus, when fibrosis occurs in the intestine, the scar may narrow the width of the passage (lumen) in the lesioned intestinal area. These clamped areas are called stenosis. The stenosis may be mild or severe depending on how much it prevents the intestinal contents from passing through the narrowed area. Clinical signs / symptoms of Crohn's disease can include, but are not limited to, cachexia, weight loss, growth failure, abdominal pain, drainage fistulas, rectal prolapse, and dehydration.

  In certain embodiments, an inflammatory liver disease or disorder. For example, the inflammatory liver disease or disorder is selected from the group consisting of autoimmune hepatitis, cirrhosis, and biliary cirrhosis.

Formulation of a pharmaceutical composition Administration of a compound to treat a neoplasm or inflammatory disease, in combination with other components, is effective in improving, reducing or stabilizing a neoplasm or inflammatory disease May be by any suitable means that results in a therapeutic drug concentration. The compound may be contained in any suitable amount in any suitable carrier material and is usually present in an amount of 1 to 95% by weight of the total composition. The composition may be provided in a dosage form suitable for parenteral (eg, subcutaneous, intravenous, intramuscular, or intraperitoneal) route of administration. The pharmaceutical composition may be formulated according to conventional pharmaceutical practice (eg, Remington: The Science and Practice of Pharmacy (20th ed.), Ed. A. R. Gennaro, Lippincott Williams & Wilkins, 2000 and Penedhc. Technology, eds. J. Swarbrick and JC Boylan, 1988-1999, Marcel Dekker, New York).

  Human dosage can be initially determined by extrapolation of the amount of compound used in mice, and as those skilled in the art will recognize, this is a technique that alters dosage for humans compared to animal models. Routine in the field. In certain embodiments, the dosage is about 1 μg compound / Kg body weight to about 5000 mg compound / Kg body weight; or about 5 mg / Kg body weight to about 4000 mg / Kg body weight or about 10 mg / Kg body weight to about 3000 mg / Kg body weight; It is anticipated that it may vary between about 50 mg / Kg body weight to about 2000 mg / Kg body weight; or about 100 mg / Kg body weight to about 1000 mg / Kg body weight; or about 150 mg / Kg body weight to about 500 mg / Kg body weight. . In another embodiment, the dose is about 1, 5, 10, 25, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750. 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1600, 1700, 1800, 1900, 2000, 2500, 3000, 3500, 4000, 4500 Or 5000 mg / Kg body weight. In another embodiment, it is envisioned that the dose may be in the range of about 5 mg compound / Kg body weight to about 20 mg compound / Kg body weight. In another embodiment, the dose may be about 8, 10, 12, 14, 16, or 18 mg / Kg body weight. Of course, this dosage may be adjusted upward or downward depending on the results of the initial clinical trial and the needs of the particular patient, as is routinely done with such treatment protocols.

  A pharmaceutical composition according to the invention may be formulated to release the active compound substantially immediately upon administration or at any predetermined time or period after administration. The latter type of composition is commonly known as a controlled release formulation and includes the following: (i) a formulation that provides a substantially constant concentration of drug over time in the body; (ii) a long term in the body after a predetermined delay time; (Iii) a relatively constant and effective in the body while simultaneously minimizing undesirable side effects associated with plasma level fluctuations (sawtooth kinetic pattern) of the active substance A formulation that maintains its effect for a predetermined period of time by maintaining the level; (iv) a formulation that localizes the effect, for example, by spatial arrangement of a controlled release composition, eg, adjacent to or in contact with the thoracic line; A formulation that allows convenient administration, eg, every week or every two weeks; and (vi) delivering a therapeutic agent to a particular cell type (eg, neoplastic cell) , The use of carriers or chemical derivatives, formulations neoplastic or inflammatory disease targets. In some applications, controlled release formulations eliminate the need for frequent administration to keep plasma levels at therapeutic levels during the day.

  Several strategies can be pursued to obtain controlled release where the release rate more than compensates for the metabolic rate of the compound in question. In one example, controlled release is obtained by appropriate selection of various formulation parameters and ingredients, including, for example, various types of controlled release compositions and coatings. Accordingly, the therapeutic agent is formulated into a pharmaceutical composition with suitable excipients, which release the therapeutic agent in a controlled manner upon administration. Examples include single or multiple unit tablet or capsule compositions, oils, suspensions, emulsions, microcapsules, microspheres, molecular complexes, nanoparticles, patches, and liposomes.

Parenteral compositions Pharmaceutical compositions can be administered by injection, infusion or implantation (subcutaneous, intravenous, intramuscular, intraperitoneal, etc.) in dosage forms or formulations, or by conventional non-toxic pharmaceutically acceptable carriers and It may be administered parenterally via a suitable delivery device or implant containing the adjuvant. The formulation and preparation of such compositions is well known to those skilled in pharmaceutical formulation. The prescription is in Remington: The Science and Practice of Pharmacy, supra.

  The parenteral composition may be provided in a unit dose form (eg, a single-dose ampoule) or in a vial containing several doses into which an appropriate preservative may be added ( See below). The composition may be in the form of a solution, suspension, emulsion, infusion device, or implantable delivery device, or it is presented as a dry powder that is reconstituted with water or another suitable vehicle prior to use. May be. In addition to the active agent that alleviates or ameliorates the neoplasm or inflammatory disease, the composition may include suitable parenterally acceptable carriers and / or excipients. Active therapeutic agents may be incorporated into microspheres, microcapsules, nanoparticles, liposomes, and the like for controlled release. In addition, the composition may include suspending agents, solubilizing agents, stabilizing agents, pH adjusting agents, osmotic pressure adjusting agents, and / or dispersing agents.

  As mentioned above, the pharmaceutical composition according to the invention may be in a form suitable for sterile injection. To prepare such a composition, a suitable active anti-neoplastic therapeutic is dissolved or suspended in a parenterally acceptable liquid vehicle. Among the acceptable vehicles and solvents that may be employed are water, water adjusted to the appropriate pH by the addition of appropriate amount of hydrochloric acid or sodium hydroxide or appropriate buffer, 1,3-butanediol, Ringer's solution, and isotonic Sodium chloride solution and dextrose solution. Aqueous formulations may also contain one or more preservatives (eg, methyl, ethyl or n-propyl p-hydroxybenzoate). In some cases, if one of the compounds is poorly soluble or slightly water soluble, a dissolution promoter or solubilizer may be added, or the solvent may include 10-60% w / w propylene glycol, etc. it can.

Controlled release parenteral compositions Controlled release parenteral compositions may be in the form of aqueous suspensions, microspheres, microcapsules, magnetic microspheres, oils, oil suspensions, or emulsions. Alternatively, the active agent may be incorporated into a biocompatible carrier, liposome, nanoparticle, implant, or infusion device.

  Materials for use in microspheres and / or microcapsule preparations are biodegradable, such as polygalactin, poly- (isobutylcyanoacrylate), poly (2-hydroxyethyl-L-glutamine), and poly (lactic acid). / A biodegradable polymer. Biocompatible carriers that may be used in formulating a controlled release parenteral formulation are carbohydrates (eg, dextran), proteins (eg, albumin), lipoproteins, or antibodies. The material for use in the implant is non-biodegradable (eg polydimethylsiloxane), or biodegradable (eg poly (caprolactam), poly (lactic acid), poly (glycolic acid) or poly (orthoester)) or combinations thereof ).

Solid dosage forms for oral use Formulations for oral use include tablets containing the active ingredient in admixture with non-toxic pharmaceutically acceptable excipients. Such formulations are known to those skilled in the art. Excipients include, for example, inert diluents or bulking agents (eg sucrose, sorbitol, sugar, mannitol, microcrystalline cellulose, starches including potato starch, calcium carbonate, sodium chloride, lactose, calcium phosphate, calcium sulfate, or phosphate Sodium); granulating and disintegrating agents (eg cellulose derivatives including microcrystalline cellulose, starches including potato starch, croscarmellose sodium, alginate or alginic acid); binders (eg sucrose, glucose, sorbitol, acacia) Alginate, sodium alginate, gelatin, starch, pregelatinized starch, microcrystalline cellulose, magnesium aluminum silicate, sodium carboxymethylcellulose, methylcellulose, hydroxypropi Methylcellulose, ethylcellulose, polyvinylpyrrolidone, or polyethylene glycol); and smoothing agents, glidants, and anti-sticking agents (eg, magnesium stearate, zinc stearate, stearic acid, silica, hydrogenated vegetable oil, or talc) Good. Other pharmaceutically acceptable excipients can be colorants, flavoring agents, plasticizers, wetting agents, buffering agents, and the like.

  The tablets may be uncoated or they may be coated by known techniques to optionally delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. The coating may be adapted to release the active agent in a predetermined pattern (eg to achieve a controlled release formulation) or it may be adapted not to release the active agent until after passage through the stomach. (Enteric coating). The coating can be a sugar coating, a film coating (eg hydroxypropylmethylcellulose, methylcellulose, methylhydroxyethylcellulose, hydroxypropylcellulose, carboxymethylcellulose, acrylate copolymer, polyethylene glycol and / or polyvinylpyrrolidone base) or an enteric coating (eg methacrylic acid). Copolymer, cellulose acetate phthalate, hydroxypropylmethylcellulose phthalate, methylcellulose hydroxypropyl succinate acetate, polyvinyl acetate phthalate, shellac, and / or ethylcellulose base). In addition, a time delay material such as glyceryl monostearate or glyceryl distearate may also be employed.

  Solid tablet compositions may include a coating adapted to protect the composition from unwanted chemical changes (eg, chemical degradation prior to release of the active therapeutic agent). The coating may be applied onto the solid dosage form in a manner similar to that described in the above-mentioned Encyclopedia of Pharmaceutical Technology.

  At least two therapeutic agents may be mixed together in the tablet or divided. In one example, the first active anti-neoplastic therapeutic is within the tablet and the second active therapeutic is external such that a substantial portion of the second therapeutic is released prior to the release of the first therapeutic. Contained in

  Formulations for oral use are active as chewable tablets or as hard gelatin capsules where the active ingredient is mixed with an inert solid diluent (eg potato starch, lactose, microcrystalline cellulose, calcium carbonate, calcium phosphate or kaolin) or active Ingredients may be presented as soft gelatin capsules, for example, mixed with water or an oil medium such as peanut oil, liquid paraffin, or olive oil. Powders and granules may be prepared in a conventional manner using the ingredients described above for tablets and capsules, for example using a mixer, fluid bed apparatus or spray drying apparatus.

Controlled release oral dosage forms Controlled release compositions for oral use are constructed to release active antineoplastic or anti-inflammatory therapeutics, for example, by controlling the dissolution and / or diffusion of the active agent. Also good. Controlled dissolution or diffusion release may be achieved by appropriate coating of tablets, capsules, pellets, or by granule formulation of the compound, or by incorporating the compound into a suitable matrix. Controlled release coatings include one or more of the coating materials described above and / or, for example, shellac, beeswax, glycowax, hydrogenated castor oil, carnauba wax, stearyl alcohol, glyceryl monostearate, glyceryl distearate, palmito Glyceryl stearate, ethyl cellulose, acrylic resin, dl-polylactic acid, cellulose acetate butyrate, polyvinyl chloride, polyvinyl acetate, vinyl pyrrolidone, polyethylene, polymethacrylate, methyl methacrylate, 2-hydroxymethacrylate, methacrylate hydrogel, 1,3 Examples include butylene glycol, ethylene glycol methacrylate, and / or polyethylene glycol. In a controlled release matrix formulation, the matrix material can be, for example, hydrated methylcellulose, carnauba wax and stearyl alcohol, carbopol 934, silicone, glyceryl tristearate, methyl acrylate-methyl methacrylate, polyvinyl chloride, polyethylene, and / or Halogenated fluorocarbons may also be included.

  Controlled release compositions containing one or more therapeutic compounds may also be in the form of buoyant tablets or capsules (ie, tablets or capsules that float over the stomach contents for a period of time upon oral administration). . A buoyant tablet formulation of the compound can be prepared by granulating a mixture of compound and excipient with 20-75% w / w hydrocolloid, such as hydroxyethylcellulose, hydroxypropylcellulose, or hydroxypropylmethylcellulose. The resulting granules can then be compressed into tablets. Upon contact with gastric juice, the tablet forms a substantially water-impermeable gel barrier around its surface. This gel barrier is responsible for maintaining a density of less than 1, thereby allowing the tablets to remain buoyant in the gastric juice.

Combination therapy Optionally, the antineoplastic or anti-inflammatory therapeutic agent may be administered in combination with any other standard anti-neoplastic or anti-inflammatory therapy known in the art; Methods are known to those skilled in the art, and Remington's Pharmaceutical Sciences by E.M. W. Described in Martin. If desired, agents of the invention (eg, JQ1, compounds of formulas described herein, and derivatives thereof) include, but are not limited to surgery, radiation therapy, or chemotherapy. Not in combination with any conventional antineoplastic therapy. In preferred embodiments, the compounds of the present invention are combined with epigenetic or transcriptional regulators (eg, DNA methyltransferase inhibitors, histone deacetylase inhibitors (HDAC inhibitors), lysine methyltransferase inhibitors) Mitotic inhibitors (eg taxanes, vinca alkaloids), hormone receptor modulators (eg estrogen receptor modulators, androgen receptor modulators), cell signaling pathway inhibitors (eg tyrosine kinase inhibitors), regulation of protein stability It is administered in combination with factors (proteasome inhibitors), hsp90 inhibitors, conventional chemotherapeutic drugs, glucocorticoids, all-trans retinoic acid or other agents that promote differentiation.

Kits or pharmaceutical systems The compositions may be assembled into kits or pharmaceutical systems used to ameliorate neoplasms or inflammatory diseases. Kits or pharmaceutical systems according to this aspect of the invention carry boxes, cartons, cylinders, etc. in which one or more containment means such as vials, tubes, ampoules, bottles, etc. are tightly sealed. Means. The kit or pharmaceutical system of the present invention may also comprise accompanying instructions for using the agent of the present invention.

  The practice of the present invention, unless otherwise specified, is well within the skill of the artisan, and includes conventional (including recombinant techniques) molecular biology, microbiology, cell biology, biochemistry, and immunology. Use technology. Such techniques are described in the literature “Molecular Cloning: A Laboratory Manual”, second edition (Sambrook, 1989); “Oligonucleotide Synthesis” (Gait, 1984); “Animal Cell Culture” (E7, 1987); “Handbook of Experimental Immunology” (Weir, 1996); “Gene Transfer Vectors for Mammalian Cells” (Miller and Calos, 1987); “Current Protocols in Mols.” Ausubel, 1987); “PCR: The Polymerase Chain Reaction” (Mullis, 1994); “Current Protocols in Immunology” (Coligan, 1991). These techniques can be applied to the production of the polynucleotides and polypeptides of the invention and can therefore be considered in the creation and implementation of the invention. In the following sections, techniques that are particularly useful for specific embodiments are discussed.

  The following examples are presented to provide one of ordinary skill in the art with a complete disclosure and description of how to make and use the assays, screening, and treatment methods of the present invention, which the inventors regard as their invention. It is not intended to limit the scope.

  The practice of the present invention, unless otherwise specified, is well within the skill of the artisan, and includes conventional (including recombinant techniques) molecular biology, microbiology, cell biology, biochemistry, and immunology. Use technology. Such techniques are described in the literature “Molecular Cloning: A Laboratory Manual”, second edition (Sambrook, 1989); “Oligonucleotide Synthesis” (Gait, 1984); “Animal Cell Culture” (E7, 1987); “Handbook of Experimental Immunology” (Weir, 1996); “Gene Transfer Vectors for Mammalian Cells” (Miller and Calos, 1987); “Current Protocols in Mols.” Ausubel, 1987); “PCR: The Polymerase Chain Reaction”, (Mullis, 1994); “Current Protocols in Immunology” (Coligan, 1991). These techniques can be applied to the production of the polynucleotides and polypeptides of the invention and can therefore be considered in the creation and implementation of the invention. In the following sections, techniques that are particularly useful for specific embodiments are discussed.

  The following examples are presented to provide one of ordinary skill in the art with a complete disclosure and description of how to make and use the assays, screening, and treatment methods of the present invention, which the inventors regard as their invention. It is not intended to limit the scope.

I. Chemical Examples—Synthesis and Preparation Methods The compounds of the present invention can be synthesized by the methods described herein and / or according to methods known to those skilled in the art in view of the description herein. obtain.

Scheme S1. Synthesis of racemic bromodomain inhibitor (±) -JQ1

(2-Amino-4,5-dimethylthiophen-3-yl) (4-chlorophenyl) methanone (S2)
Compound JQ1 was prepared according to the scheme shown above.

4-chlorobenzoylacetonitrile S1 (1.24 g, 6.9 mmol, 1 eq), 2-butanone (0.62 ml, 6.9 mmol, 1.00 eq) in ethanol (20 ml, 0.35 M) at 23 ° C. ), And sulfur (220 mg, 6.9 mmol, 1.00 equiv) as a solid to a solution of morpholine (0.60 ml, 6.9 mmol, 1.00 equiv) ²¹ . The mixture was then heated to 70 ° C. After 12 hours, the reaction mixture was cooled to 23 ° C. and poured into brine (100 ml). The aqueous layer was extracted with ethyl acetate (3 × 50 ml). The combined organic layers were washed with brine (50 ml), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by flash column chromatography (Combiflash RF system, 40 grams of silica gel, gradient 0-100% ethyl acetate-hexane) to give S2 (1.28 g, 70%) as a yellow solid.

(S) -tert-butyl-3-({[(9H-fluoren-9-yl) methoxy] carbonyl} amino) -4-{[3- (4-chlorobenzoyl) -4,5-dimethylthiophene-2 -Yl] amino} -4-oxobutanoate (S3)
9-Fluorenylmethoxycarbonyl-aspartic acid β-tert-butyl ester [Fmoc-Asp (Ot-Bu) -OH] (864 mg, 2.M) in N, N-dimethylformamide (1.5 ml, 1.0 M). 1 mmol, 2.10 eq.) Was added to (2- (6-chloro-1H-benzotriazol-1-yl) -1,1,3,3-tetramethylaminium hexafluorophosphate (HCTU) ( 827 mg, 2.0 mmol, 2.00 equiv), and N, N-diisopropylethylamine (0.72 ml, 4.0 mmol, 4.00 equiv) were added sequentially, then the mixture was stirred at 23 ° C. for 5 min. S2 (266 mg, 1.0 mmol, 1 eq) was then added as a solid and the reaction mixture was stirred at 23 ° C. After 16 hours, ethyl acetate 20 ml) and brine (20 ml) were added, the two layers were separated and the aqueous layer was extracted with ethyl acetate (2 × 20 ml) The combined organic layers were washed with brine (30 ml) and dried over anhydrous sodium sulfate. Filtered and concentrated under reduced pressure The residue was purified by flash column chromatography (Combiflash RF system, 40 grams of silica gel, gradient 0-100% ethyl acetate-hexanes) and S3 (625 mg, 90%) was brown Obtained as an oil.

(S) -tert-butyl 3-amino-4-((3- (4-chlorobenzoyl) -4,5-dimethylthiophen-2-yl) amino) -4-oxobutanoate (S4)
Compound S3 (560 mg, 0.85 mmol, 1 equivalent) was dissolved in 20% piperidine in a 23 ° C. DMF solution (4.0 ml, 0.22 M). After 30 minutes, ethyl acetate (20 ml) and brine (20 ml) were added to the reaction mixture. The two layers were separated and the aqueous layer was extracted with ethyl acetate (2 × 20 ml). The combined organic layers were washed with brine (3 × 25 ml), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by flash column chromatography (Combiflash RF system, 24 grams of silica gel, gradient 0-100% ethyl acetate-hexanes) to give the free amine S4 (370 mg, 90%) as a yellow solid. The enantiomeric purity dropped to 75% (determined by Berger supercritical fluid chromatography (SFC) using AS-H column).

(S) -tert-butyl 2- (5- (4-chlorophenyl) -6,7-dimethyl-2-oxo-2,3-dihydro-1H-thieno [2,3-e] [1,4] diazepam -3-yl) acetate (S5)
Amino ketone (S4) (280 mg, 0.63 mmol) was dissolved in 10% acetic acid ethanol solution (21 ml, 0.03 M). The reaction mixture was heated to 85 ° C. After 30 minutes, all solvent was removed under reduced pressure. The residue was purified by flash column chromatography (Combiflash RF system, 12 grams of silica gel, gradient 0-100% ethyl acetate-hexanes) to give compound S5 (241 mg, 95%) as a white solid. The enantiomeric purity of S5 was 67% (determined by Berger supercritical fluid chromatography (SFC) using AS-H column).

tert-Butyl 2- (5- (4-chlorophenyl) -6,7-dimethyl-2-thioxo-2,3-dihydro-1H-thieno [2,3-e] [1,4] diazepam-3-yl Acetate (S6)
To a solution of S5 (210 mg, 0.5 mmol, 1 eq) in diglyme (1.25 ml, 0.4 M) was added phosphorus pentasulfide (222 mg, 1.0 mmol, 2.00 eq), sodium bicarbonate (168 mg, 2.0 mmol, 4.00 equivalents) was added sequentially. The reaction mixture was heated to 90 ° C. After 16 hours, brine (20 ml) and ethyl acetate (35 ml) were added. The two layers were separated and the aqueous layer was extracted with ethyl acetate (3 × 30 ml). The combined organic layers were washed with brine (2 × 15 ml), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by flash column chromatography (Combiflash RF system, 24 gram silica gel, gradient 0-100% ethyl acetate-hexanes) with S5 (73 mg, 34%) recovered and S6 (141 mg, 65%) brown Obtained as a solid.

tert-Butyl 2- (4- (4-chlorophenyl) -2,3,9-trimethyl-6H-thieno [3,2-f] [1,2,4] triazole [4,3-a] [1, 4] diazepam-6-yl) acetate [(±) JQ1]
To a solution of S6 (158 mg, 0.36 mmol, 1 equiv) in THF (2.6 ml, 0.14 M) at 0 ° C. was added hydrazine (0.015 ml, 0.45 mmol, 1.25 equiv). The reaction mixture was warmed to 23 ° C. and stirred at 23 ° C. for 1 hour. All solvents were removed under reduced pressure. The resulting hydrazine was used directly without purification. The hydrazine was then dissolved in a 2: 3 mixture of trimethyl orthoacetate and toluene (6 ml, 0.06M). The reaction mixture was heated to 120 ° C. After 2 hours, all solvent was removed under reduced pressure. The residue was purified by flash column chromatography (Combiflash RF system, 4 g silica gel, gradient 0-100% ethyl acetate-hexane) to give JQ1 (140 mg, 85% over 2 steps) as a white solid. Reaction conditions further epimerized the asymmetric center, resulting in racemic JQ1. (Measured by Berger Supercritical Fluid Chromatography (SFC) using AS-H column).

Scheme S2. Synthesis of enantiomerically enriched (+)-JQ1

(S) -tert-butyl-3-({[(9H-fluoren-9-yl) methoxy] carbonyl} amino) -4-{[3- (4-chlorobenzoyl) -4,5-dimethylthiophene-2 -Yl] amino} -4-oxobutanoate (S3)
9-Fluorenylmethoxycarbonyl-aspartic acid β-tert-butyl ester [Fmoc-Asp (Ot-Bu) —OH] (411 mg, 1.N, N-dimethylformamide (1.0 ml, 1.0 M)). (00 mmol, 1.0 eq) in solution, (benzotriazol-1-yloxyl) tripyrrolidinophosphonium (PyBOP) (494 mg, 0.95 mmol, 0.95 eq), N, N-diisopropylethylamine (0.50 ml) 2.8 mmol, 2.75 equivalents). The mixture was then stirred at 23 ° C. for 5 minutes. Then S2 (266 mg, 1.0 mmol, 1 eq) was added as a solid. The reaction mixture was stirred at 23 ° C. After 4 hours, ethyl acetate (20 ml) and brine (20 ml) were added. The two layers were separated and the aqueous layer was extracted with ethyl acetate (2 × 20 ml). The combined organic layers were washed with brine, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by flash column chromatography (Combiflash RF system, 40 grams of silica gel, gradient 0-100% ethyl acetate-hexanes) to give S3 (452 mg, 72%) as a brown oil.

(S) -tert-butyl 3-amino-4-((3- (4-chlorobenzoyl) -4,5-dimethylthiophen-2-yl) amino) -4-oxobutanoate (S4)
Compound S3 (310 mg, 0.47 mmol, 1 equivalent) was dissolved in 20% piperidine in DMF solution (2.2 ml, 0.22 M) at 23 ° C. After 30 minutes, ethyl acetate (20 ml) and brine (20 ml) were added to the reaction mixture. The two layers were separated and the aqueous layer was extracted with ethyl acetate (2 × 20 ml). The combined organic layers were washed with brine (3 × 25 ml), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by flash column chromatography (Combiflash RF system, 24 grams of silica gel, gradient 0-100% ethyl acetate-hexanes) to give the free amine S4 (184 mg, 90%) as a yellow solid. The enantiomeric purity was 91%. (Checked by Berger Supercritical Fluid Chromatography (SFC) using AS-H column).

(S) -tert-butyl 2- (5- (4-chlorophenyl) -6,7-dimethyl-2-oxo-2,3-dihydro-1H-thieno [2,3-e] [1,4] diazepam -3-yl) acetate (S5)
Aminoketone (S4) (184 mg, 0.42 mmol) was dissolved in toluene (10 ml, 0.04 M). Silica gel (300 mg) was added and the reaction mixture was heated to 90 ° C. After 3 hours, the reaction mixture was cooled to 23 ° C. The silica gel was filtered and washed with ethyl acetate. The combined filtrate was concentrated. The residue was purified by flash column chromatography (Combiflash RF system, 12 grams of silica gel, gradient 0-100% ethyl acetate-hexane) to give compound S5 (168 mg, 95%) as a white solid. The enantiomeric purity was 90% (determined by Berger supercritical fluid chromatography (SFC) using an AS-H column).

(S) -tert-butyl 2- (4- (4-chlorophenyl) -2,3,9-trimethyl-6H-thieno [3,2-f] [1,2,4] triazole [4,3-a ] [1,4] diazepam-6-yl) acetate [(+) JQ1]
To a solution of S5 (114 mg, 0.27 mmol, 1 eq) in THF (1.8 ml, 0.15 M) at −78 ° C. was added potassium tert-butoxide (1.0 M solution in THF, 0.3 ml,. 30 mmol, 1.10 eq) was added. The reaction mixture was warmed to −10 ° C. and stirred at 23 ° C. for 30 minutes. The reaction mixture was cooled to -78 ° C. Diethyl chlorophosphate (0.047 ml, 0.32 mmol, 1.20 equiv) was added to the reaction mixture ²² . The resulting mixture was warmed at −10 ° C. for 45 minutes. Acetic hydrazide (30 mg, 0.40 mmol, 1.50 equiv) was added to the reaction mixture. The reaction mixture was stirred at 23 ° C. After 1 hour, 1-butanol (2.25 ml) was added to the reaction mixture heated to 90 ° C. After 1 hour, all solvent was removed under reduced pressure. The residue was purified by flash column chromatography (Combiflash system, 4 g silica gel, gradient 0-100% ethyl acetate-hexane) to give (+)-JQ1 (114 mg, 92%) as a white solid with 90% enantiomeric purity. (AS-H column, 85% hexane-methanol, 210 nm, t _R (R-enantiomer) = 1.59 min, t _R (S-enantiomer) = 1.67 min, Berger super Measured by critical fluid chromatography (SFC)). The product was further purified by chiral preparative HPLC (Agilent high pressure liquid chromatography using an OD-H column) to give the S enantiomer above 99% ee.
¹ HNMR (600 MHz, CDCl ₃ , 25 ° C.) δ 7.39 (d, J = 8.4 Hz, 2H), 7.31 (d, J = 8.4 Hz, 2H), 4.54 (t, J = 6 .6 MHz, 1H), 3.54-3.52 (m, 2H), 2.66 (s, 3H), 2.39 (s, 3H), 1.67 (s, 3H), 1.48 ( s, 9H).
¹³ C NMR (150 MHz, CDCl ₃ , 25 ° C.) δ 171.0, 163.8, 155.7, 150.0, 136.9, 131.1, 130.9, 130.6, 130.3, 128.9 81.2, 54.1, 38.1, 28.4, 14.6, 13.5, 12.1.
HRMS (ESI) calcd _{C 21 H 24 ClN 2 O 3} S [M + H] +: 457.1460, Found 457.1451m / z.
TLC (EtOAc), Rf: 0.32 (UV)
[Α] ²² _D = + 75 (c0.5, CHCl ₃ )

(-)-JQ1 was similarly synthesized using Fmoc-D-Asp (Ot-Bu) -OH as a starting material, and by chiral preparative HPLC (Agilent high pressure liquid chromatography using OD-H column). Further purification gave R enantiomers in excess of 99% ee. [Α] ²² _D = −72 (c0.5, CHCl ₃ )

Synthesis of Additional Compounds Additional compounds of the invention were prepared as illustrated in Scheme S3.

Scheme S3. Synthesis of hydrazine derivatives

  As shown in Scheme S3, the t-butyl ester (1) of (+)-JQ1 was cleaved to give the free acid (2), which was combined with hydrazine to give hydrazide (3). Reaction with 4-hydroxybenzaldehyde yielded hydrazone (4).

  Both hydrazide (3) and hydrazone (4) showed activity in at least one biological assay.

  A library of compounds was prepared by reaction of hydrazide (3) with various carbonyl-containing compounds (see Table A above).

  For example, additional compounds were prepared for use as probes for assay development. An exemplary synthesis is shown in Scheme S4 below.

Scheme S4. Synthesis of derivatives useful as probes

  Additional compounds were prepared as shown in Table B below.

  The spectral data for each compound was consistent with the assigned structure.

II. Biological activity Example 1: JQ1
FIG. 1 shows the JQ1 enantiomer.

Example 2: JQ1 replaces BRD4 from nuclear chromatin in cells.
To establish whether JQ1 binds to the bromodomain competitively with chromatin in the cellular environment, a post-photobleaching fluorescence recovery (FRAP) experiment was performed on BRD4. Previous studies have demonstrated the utility of FRAP in assessing the lateral redistribution pace of bromodomain-containing fluorescent chimeras following selective photobleaching of discrete regions of nuclear chromatin ²⁷ . Human osteosarcoma cells (U2OS) transfected with GFP-BRD4 exhibit a time-dependent recovery of fluorescence intensity (FIGS. 2A and 2B). In the presence of JQ1 (500 nM), the observed recovery is in immediate agreement with the displaced nuclear BRD4 (FIGS. 3A and 3B). The cellular FRAP test confirmed that the effect of BRD4 on the migrating fraction was limited to the biochemical activity (+)-JQ1 stereoisomer (FIGS. 2A-2C).

  Having demonstrated strong selective binding to BRD4 in a homogeneous cell-based assay, the effect of JQ1 on disease-related BRD4-dependent cell phenotype was evaluated. The pathogenic BRD4-NUT fusion protein resulting from the t (15; 19) translocation in NMC binds to discrete lesions and greed of acetylated chromatin, giving it a growth advantage and differentiation barrier. FRAP was used to evaluate the ability of JQ1 to directly target the BRD4-NUT oncoprotein. Compared to the vehicle control, JQ1 (500 nM) significantly accelerated the time-versus-recovery of fluorescence intensity within the photobleached region of cells transfected with GFP-BRD4-NUT (FIG. 3C). 3E). Importantly, no effect on redistribution of GFP-NUT was observed (FIGS. 3D, 3E). In summary, these data are consistent with competitive binding of JQ1 to BRD4 in cultured cells.

Example 5: JQ1 induces squamous differentiation and growth arrest in BRD4-dependent carcinoma.
Direct inhibition of gene products expressed from recurrent carcinogenic translocations is a validated therapeutic approach in ^cancer28,29 . We searched for phenotypic consequences of chemical inhibition of BET family bromodomains on BRD4-dependent NUT median carcinoma. BRD4-NUT chromatin localization is mechanistically linked to the conserved tandem bromodomain of BRD4 in the fusion protein ¹⁷ . A characteristic of NMC is the emergence of discrete nuclear speckles of BRD4-NUT oncoprotein by NUT-induced immunohistochemistry (IHC) ³⁰ . Treatment of the patient-derived 797 NMC cell line with JQ1 (500 nM) for 48 hours spreads the nuclear foci and resulted in diffuse nuclear NUT staining with IHC (FIG. 3f).

A marked differentiation phenotype was observed by knockdown of BRD4-NUT in the NMC cell line ¹⁷ . JQ1 produced an equivalent dose and time-dependent differentiation phenotype characterized by cell spreading and flattening, open chromatin, and fusiform morphology in NMC 797 and Per403 cells (FIGS. 3g and 4A). 4B, 4C). Differentiation was rapid (<24 hours) and was characterized by a markedly increased cytokeratin expression that was evidence of squamous differentiation (FIG. 3h). Terminal differentiation was observed after 7 days in culture exposed to submicromolar JQ1. Importantly, non-BRD4-dependent squamous cell carcinoma cell lines (TE10 and TT) cannot exhibit the differentiation effects of JQ1 (FIGS. 4A, 4B, 4C). In BRD4-dependent NMC cells, differentiation is naturally accompanied by growth arrest, as evidenced by a decrease in Ki67 staining (FIGS. 3i, j, and 4L-a, 4L-b). Furthermore, in support of the targeted mechanism of action, the differentiation and growth arrest phenotype is driven only by (+)-JQ1, whereas (−)-JQ1 shows no observable effect (FIG. 4E-a ~ 4E-d).

In particular, JQ1 treatment phenotypically replicated morphological changes and increased keratin expression observed with BRD4-NUT silencing by RNA interference (FIGS. 4F-a, b). In support of these morphological and IHC studies, expression analysis of three canonical squamous tissue genes by RT-PCR revealed significant (30-fold) induction of keratin-14 by (+)-JQ1 in NMC 797 cells Was identified (FIG. 3i). The moderate induction of keratin-10 and the absence of an effect on epidermal transglutaminase (TGM1) is then consistent with progressive differentiation towards the thoracic squamous epithelium, consistent with the primary mediastinal tumor from which NMC 797 cells were derived ²⁸ . Induction of differentiation with strong (3+) keratin staining by IHC proceeds over 72 hours as measured by quantitative IHC analysis (FIG. 4M). In support of the targeted mechanism of action, the differentiation phenotype is only driven by (+)-JQ1, whereas (-)-JQ1 shows no observable effect. Importantly, the non-BRD4-dependent squamous cell carcinoma cell line (TE10) cannot exhibit differentiation effects from JQ1 treatment (FIG. 4N-c). In BRD4-dependent NMC cells, differentiation is naturally accompanied by growth arrest, as evidenced by a decrease in Ki67 staining (FIGS. 4A-4G).

The antiproliferative activity of JQ1 enantiomers was then evaluated in BRD4-dependent (797 and Per403) and BRD4-independent (TE10 and TT) human squamous cell carcinoma cell lines. As shown in FIGS. 5a and 4F-ac, (+)-JQ1 uniquely exhibited a dose-dependent inhibition of cell growth only in a BRD4-dependent cell line (797 IC ₅₀ = 140 nM; Per403 IC ₅₀ = 60 nM). The strong antiproliferative effect and irreversible differentiation observed by IHC suggests the induction of cell death. Therefore, early and late apoptosis was assessed by annexin V and propidium iodide staining by flow cytometry. As expected, JQ1 induces immediate progressive apoptosis in BRD4-dependent human cancer cells, but at the concentrations used, significant levels of apoptotic cells in cell lines that do not possess a BRD-NUT fusion Was not detected (FIGS. 5b and 7).

Example 6: In vivo efficacy and pharmacodynamic effects of JQ1 in a mouse model of NMC To establish whether JQ1 can attenuate BRD4-dependent carcinoma growth as a single agent in vivo A mouse xenograft model of NMC was developed in mice using the NMC 797 cell line. Short-term treatment studies were conducted using PET imaging as the primary endpoint to explore whether JQ1's anti-tumor activity could be demonstrated, followed by non-invasive imaging. A match cohort of mice with an established equivalent burden of measurable disease was randomized for treatment with JQ1 (50 mg kg ⁻¹ ) or vehicle administered by daily intraperitoneal injection. Mice were evaluated by PET imaging prior to randomization and after 4 days of treatment. A significant response to FDG uptake was observed with JQ1 treatment, whereas vehicle treated animals demonstrated overt progressive disease (FIG. 5e).

  In parallel, a match cohort of NMC 797 tumor-bearing mice was studied for the effect of JQ1 on tumor volume by caliper measurements and for pharmacodynamic effects by quantitative IHC. Aggressive growth of the NMC 797 xenograft prompted the conclusion of the study on day 14 of treatment, at which time all vehicle-treated animals approached institutional tumor size limits. Animals administered JQ1 exhibited a statistically significant reduction in tumor growth (FIG. 5c, p = 0.039; two-sided t-test). In order to capture relatively mechanistic and pharmacodynamic data, all animals were sacrificed and tumors were explanted for histopathological analysis. In particular, JQ1 was well tolerated at this dose and dosing regimen with no adverse signs of toxicity or obvious weight loss (FIGS. 5d, 5L).

  To confirm that the antineoplastic effect observed with JQ1 treatment was associated with target engagement, the sliced tumor tissue was examined for BRD4-NUT oncoprotein. As presented in FIGS. 5e and 6A, JQ1 treatment resulted in the spread of NUT nuclear speckle, consistent with competitive binding to nuclear chromatin. Cell proliferation and increased keratin expression demonstrated pharmacodynamic squamous differentiation. Reduction of Ki67 nuclear staining and increased Tanel staining in treated animals demonstrated ongoing antiproliferative apoptotic effects. Taken together, these data provide a mechanistic link between BRD4 inhibition and therapeutic response to JQ1 in vivo.

  As part of an effort to report pharmacodynamic (PD) biomarkers of tumor keratin expression in an unbiased manner, a protocol was established for quantitative IHC image collection and analysis. Paired samples from treated and untreated animals were prepared and analyzed using standardized protocols and commercially available software (ImageScope; Aperio Technologies). JQ1 caused strong (3+) keratin expression in NMC 797 xenografts uniformly in isolated tumor specimens (FIGS. 6Ba-e).

  In parallel with these studies, a 29 year old patient with a broadly metastatic BRD4-NUT positive NMC arising from the mediastinum was identified. With the goal of developing a more clinically relevant disease model, a short-term culture was established using discarded clinical material obtained from pleural fluid drained from the patient's symptomatic chest tube. As presented in FIG. 8, in vitro studies demonstrated a potent stereoselective effect of (+)-JQ1 on cell viability (IC50 = 4 nM), growth, and cell cycle progression. Four animals implanted with patient-derived tumor material developed measurable disease, which was strongly PET positive (FIG. 5h). Animals were randomly assigned to vehicle or (+)-JQ1 treatment cohorts. Mice were evaluated by PET imaging prior to treatment assignment and after 4 days of treatment. A significant response to FDG uptake was observed with JQ1 treatment, whereas vehicle treated animals demonstrated overt progressive disease (FIG. 5i). Again, tumor material was prepared for quantitative IHC analysis, which demonstrated induction of keratin expression following (+)-JQ1 treatment (FIGS. 5j, k, FIG. 9). Taken together, these data provide a mechanistic link between BRD4 inhibition and therapeutic response to JQ1 in vivo.

Throughout the complex mutation landscape of the emerging cancer genome, recurrent chromosomal rearrangements comprise a convincing subset of overt genetic targets in cancer. As demonstrated by successful development of kinase inhibitors targeting BCR-ABL in CML, well-characterized probe compounds ³¹ , ³² , high-resolution crystallographic data ³³ , translation research studies ³⁴ , The mouse model ^35, which provides information when available, provides an optimal platform for ligand discovery and target validation. As reported herein, novel BRD4-directed small molecule inhibitors are likely to be useful in the treatment of genetically defined human squamous cell carcinomas associated with NUT-BRD4 fusion.

Other than NUT midline carcinoma, BET family bromodomains contribute to a number of other neoplastic and non-neoplastic diseases. BRD4 is to targeting the P-TEFb complex mitotic chromosomes, expression of the growth-promoting gene ¹¹⁹ and established cancer target Aurora ^{B 13,} such as c-Myc is provided. In addition, BRD4 is amplified in breast cancer ³⁶ and is a survival predictive marker in breast cancer patients ³⁷ . In addition to these functions, BET family members are recognized in cancer biology as essential genes for viral replication ³⁸ , ³⁹ and in inflammatory response mediator ¹⁴ . Thus, the availability of (+)-JQ1 and (−)-JQ1 as paired chemical probes broadly facilitates informative research in transcription, development, and disease biology. JQ1 has also been shown to exhibit excellent pharmacokinetic properties, including 49% oral bioavailability (FIG. 10), with minimal off-target effects on cellular receptors, and a drug-like derivative for therapeutic use. Establish development validity.

The discovery and optimization of epigenetic targeted small molecule inhibitors is a major focus of current biomedical research. Previous successful approaches have been limited to ligand identification for chromatin modifying enzymes ⁴⁰ . Perhaps the best studied are modulators of lysine side chain deacetylation, for which a number of pharmaceutical inhibitors have been clinically developed. The present invention provides compositions and methods for developing potent selective inhibitors of epigenetic leaders, including the first fully characterized inhibitors of the BET family of bromodomains. In view of this discovery, the approaches outlined herein may be useful in identifying additional candidates for identifying additional inhibitors that have selectivity in the BET family.

Example 7: JQ1 enantiomer binds to and inhibits BRD4.1 and BRD4.2.
Figures 11 and 12 show that the JQ1 enantiomer binds to and inhibits BRD4.1 and BRD4.2. FIG. 13 shows a comparison of binding and inhibition of JQ1S to BET family members (active) and CBP (inactive). FIG. 14 shows the results of a dose range study of a centralized library of JQ1 derivatives (see Table B above for compound structure).

Example 8: JQ1 is effective in treating BRD3-NUT cancer BRD3-NUT cells were implanted subcutaneously into mice. Tumor measurement data was collected weekly. Once the tumor became palpable, mice were divided into two treatment groups (n = 10): JQ1 was administered at 50 mg / kg ip (IP) 7 days / week and vehicle was administered at IP 7 days / week. . On day 24 after injection, 5 mice from each group were euthanized and tumor samples were collected and sent for laboratory analysis. Tumor samples were collected at the time of mouse sacrifice throughout the remainder of the study. In all samples, half of the tumors were fixed in 10% formalin and the other half was snap frozen on dry ice. The process ended on the 32nd day. Tumor volume and weight were collected weekly until all mice were moribund or either of the tumor dimensions reached 2 cm. FIG. 15A is a graph showing that JQ1 shows in vivo efficacy against BRD3-NUT in a mouse xenograft model. Treatment with JQ1 resulted in tumor regression at the time of treatment. Upon cessation of treatment, tumor growth resumed. The difference in tumor volume was significant at any time point (p = 7.665274E-09 on day 24). JQ1 was administered at 50 mg / kg. FIG. 15B shows that mice treated with JQ1 show mild weight loss and rapid recovery after treatment cessation.

Example 9: JQ1 and its analogs are effective against various cancers FIGS. 16A-16D show that JQ1 and its derivatives inhibit Brd4.1 and Brd4.2 binding at 5 μM JQ1. Shown (see Table A above for compound structure). FIGS. 17A-17D show NUT median carcinoma (NMC) cell viability following treatment with 2 μM compound with JQ1 and its derivatives (see Table A above for compound structure). Figures 18-55 show the dose response viability of various cancer cell lines. These data suggest that JQ1 and its derivatives show anticancer efficacy against a wide range of cancers.

  The results reported herein were obtained using the following methods and materials.

Example 10: Binding assay results The results of the binding assay are shown in Table C below.

  The binding activity of lead compounds with BRD4 site 1 was determined by α-assay using a 12-point dose response curve (FIG. 56A). Compound (S) -JQ1 (JQS) was used as a positive control. (R) -JQ1 (JQR) was used as a negative control. Compounds JQ6, JQ8, JQ13, JQ33, and JQ35 exhibited excellent binding activity. The binding activity results of all lead compounds with BRD4 site 2 were also determined by α-assay using a 12-point dose response curve (FIG. 56B). Compounds JQ6, JQ8, JQ13, JQ33, and JQ35 exhibited excellent binding activity.

  In a 797 cell line cell assay (from a patient), the activity of lead compounds was examined to determine the growth effect of BRD4 inhibition on BRD4-NUT dependent cell lines. Cells were cultured with compounds and growth was monitored after 72 hours. Curve fitting was calculated by logistic regression (FIG. 56C). In a cell assay of the 10326 cell line directly derived from the patient, all lead compounds were examined to determine the growth effect of BRD4 inhibition on BRD4-NUT dependent cell lines (FIG. 56D). Cells were cultured with compounds and growth was monitored after 72 hours. Curve fitting was calculated by logistic regression.

The reagent endogenous BRD4-NUT expression midline carcinoma cell lines 797 ¹ and PER-403 ^2, already mentioned. Non-NMC human squamous cell carcinoma cell lines TE10 and TT were obtained from Dr. Anil Rustgi (University of Pennsylvania) and Dr. Adam Bass (Dana-Farber Cancer Institute). U2OS cells were obtained from ATCC. Mammalian overexpression constructs for GFP-BRD4, GFP-NUT, and GFP-BRD4-NUT have been previously described ³ . Medium for culture, trypsin, and antibiotics were purchased from Mediatech. Antibodies and strains for immunohistochemistry and flow cytometry are: Dako (cytokeratin AE1 / AE3 antibody catalog number N1590), Millipore (tanel catalog number S7100), Vector (Ki67 catalog number VP-RM04), Cell Signaling Technologies ( NUT catalog number 3625), BD Pharmingen (Annexin V-FITC catalog number 556547), and Invitrogen (propidium iodide catalog number P3566).

Acetyl-histone binding assay ^Eight assays were performed as previously described, with slight modifications to the manufacturer's protocol (PerkinElmer, USA). All reagents were diluted in 50 mM HEPES, 100 mM NaCl, 0.1% BSA, pH 7.4 supplemented with 0.05% CHAPS and allowed to equilibrate to room temperature prior to addition to the plate. Prepare 24 point 1: 2 serial dilutions of ligand ranging from 150-0 μM and transfer 4 μl to small volume 384 well plates (ProxiPlate ™ -384 Plus, PerkinElmer, USA) and transfer 4 μl of HIS labeled protein (BRD4 / Followed by 1,250 nM, BRD 4/2 and CREBBP, 2000 nM). The plate was sealed and incubated for 30 minutes at room temperature before addition of equimolar concentrations of 4 μl biotinylated peptide to the protein [peptides for BRD4 (1) & BRD4 (2): H4K5acK8acK12acK16ac, H-SRGRGK (Ac) GGK (Ac) GLGK (Ac) GGAK (Ac) RHRK (biotin) -OH; peptide for CREBBP: H3K36ac, biotin-KSAPATGGVK (Ac) KPHRYRPGT-OH (Cambridge Research Biochemicals, UK)]. The plate was sealed and incubated for another 30 minutes before adding 4 μl streptavidin coated donor beads (25 μg / ml) and 4 μl nickel chelate acceptor beads (25 μg / ml) under low light conditions. Plates were sealed with foil, protected from light, incubated at room temperature for 60 minutes, and read on a PHERAstar FS plate reader (BMG Labtech, Germany) using an AlphaScreen 680 excitation / 570 emission filter set. IC ₅₀ values are calculated in Prism 5 (GraphPad Software, USA) after normalization to the corresponding DMSO control and are presented as the final concentration of compound in a 20 μl reaction volume.

Sequence Alignment The amino acid sequence of the full length human bromodomain was obtained from NCBI (BRD2 accession number NP_005095, BRD3 accession number NP_031397.1, BRD4 accession number NP_055114.1, BRD-T accession number NP_001717.2). Use ClustalW ^19, it was performed multiple sequence comparison of the individual bromodomain.

Fluorescence recovery after photo bleaching (FRAP)
^3,20 FRAP tests were performed on U2OS cells as previously described. Briefly, U2OS cells were transfected (Lipfectamine; Invitrogen) with a mammalian overexpression construct encoding a GFP chimera containing BRD4, NUT, and BRD4-NUT. In one cell in each field of view, a 5 μm ² nucleus region was bleached with a high laser light intensity, and recovery was measured with a low laser light intensity and a 150 μm pinhole. Images of the same field of view were obtained for 90 seconds using a Nikon C1 plus confocal microscope equipped with a 37 ° C. heating chamber and FRAP module. The average intensity of the bleaching area was measured over time using MetaMorph v7 and normalized to the independent area of the subject prior to bleaching. The data was then analyzed in Microsoft Excel Mac 12.2.4 to assess half-maximal fluorescence recovery.

Differentiation and Proliferation Immunohistochemistry Cultured cancer cell lines (797, Per403, TT, and TE10) at 1.0 × 10 ⁴ cells per chamber (4 chamber slide) or 5.0 × per chamber (8 chamber slide) 10 ³ cells were cultured in chamber slides. Cells were treated with JQ1-racemic, (+)-JQ1, (−)-JQ1, or vehicle (DMSO) and cultured at various time intervals. The medium was then removed and the chamber was washed with cold PBS. Cells were then fixed in 4% formaldehyde at 4 ° C. for 20 minutes, washed with PBS, and stained for Dana-Farber / Harvard Cancer Center (DF / HCC) Specialized Histopathology Services, as described below. Transported to Britishman Women's Hospital. First, cancer cell lines (797 and Per403) were grown in T-75 flasks and treated with either JQ1 or vehicle (DMSO) for 48 hours to perform immunohistochemical studies of cell pellets. Cells were then trypsinized to form a cell pellet by centrifugation at 2,000 rpm for 10 minutes and stabilized with 1/2 volume HistoGel (Richard-Allen Scientific) and 10% bovine serum albumin. The cell pellet was washed with PBS and fixed in 4% formaldehyde at 4 ° C. for 20 minutes. Cells were then washed with PBS and transported to DF / HCC Core Laboratory at the Bridgeman's Hospital for staining as described below. Quantitative analysis (cell scoring) of Ki67 staining was performed by light microscopy using 5 high power (40 ×) fields per experiment. All fixation materials were embedded, sliced, and stained with DF / HCC Core Laboratory at Brighamman Women's Hospital using established optimization protocols. Images were obtained using an Olympus BX40 microscope (Olympus Imaging America, Center Valley, PA) and a Micropublisher 3.3 RTV color camera (QImaging, Surrey, BC).

Cell Proliferation Assay Cells were seeded in white 384 well microtiter plates (Nunc) at 500 cells in a total medium volume of 50 μL per well. 797, TT, and TE10 cells were cultured in DMEM containing 1% penicillin / streptomycin and 10% FBS. Per403 cells were cultured in DMEM containing 1% penicillin / streptomycin and 20% FBS. Compounds were delivered to microtiter assay plates by robotic pin transfer (Perkin Elmer JANUS equipped with a V & P Scientific 100 nL pin tool). Following incubation for 48 hours at 37 ° C., cells were lysed and wells were assessed for total ATP content using a commercial proliferation assay (Cell TiterGlo; Promega). Repeated measures were analyzed for dose and IC ₅₀ estimates calculated by logistic regression (GraphPad Prism).

Flow cytometry Cultured human cancer cells (797, Per403, TT, and TE10) were cultured in 6-well tissue culture plates (BD Falcon) at a starting concentration of 5.0 × 10 4 cells per well. Cells were treated with JQ1 (500 nM), staurosporine (50 nM) or vehicle (DMSO 0.05%) for 24 or 48 hours. Trypsinized cells and annexin V / propidium iodide buffer (10 mM HEPES pH 7.4, 140 mM NaCl, 2) containing annexin V-FITC (BD Pharmingen, 1: 500) and propidium iodide (Invitrogen, 1: 1000). .5 mM CaCl2) was mixed 1: 1 on ice. Samples were analyzed immediately on a BD FACS Canto II. FlowJo flow cytometry analysis software (Tree Star, Inc.) was used to create visualization and analysis of the apoptotic fraction.

Xenograft efficacy studies NMC xenografts were injected by injecting 797 NMC cells (10 ⁷ ) in 30% Matrigel (BD Biosciences) into the flank of 6 week old female NCr nude mice (Charles River Laboratories). Established. Twelve days after injection, mice with measurable tumors were divided into cohorts treated with 50 mg / kg IP of JQ1 or vehicle (5% DMSO in 5% dextrose). The mean tumor size in the JQ1 treatment group (n = 8) and vehicle group (n = 7) was similar at the start of treatment (63.8 +/− 17.1 and 73.6 +/− 14.4 mm3, respectively). Animals were followed daily for clinical symptoms. Tumor measurements were evaluated by caliper measurements and volumes were calculated using the formula, Vol = 0.5 × L × W ² . Two weeks after treatment, all mice were humanely euthanized and the statistical significance of tumor volume with tumors fixed in 10% formalin for histopathology was calculated by a two-sided student t test. All animal studies were approved by the DFCI IACUC.

Equipment Used Proton and carbon-13 nuclear magnetic resonance ( ¹ HNMR and ¹³ CNMR) spectra were recorded on a Varian Invertase Probe 600 INOVA spectrometer from Harvard Medical School East Quad NMR Facility. Chemical shifts are recorded in ppm on the δ scale, ¹ H NMR refers to residual protium in NMR solvent (CHCl ₃ : δ 7.24), ¹³ C NMR refers to solvent carbon resonance (CDCl ₃ : δ 77.2), respectively. To do. Data are reported as follows: chemical shift [multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, br = broad), cup Ring constant (Hz), integration]. High-resolution mass spectra (HRMS) were recorded using the Bruker APEX 4.7 T Spectra on the Instrument Facility of the Department of Chemistry, Massachusetts Institute of Technology. The intermediate and final product were purified using a CombiFlash RF system (Teledine Isco). The organic solution was concentrated on a Büchi R-205 rotary evaporator. The enantiomeric purity was checked using a Berger supercritical fluid chromatography (SFC) and an AS-H column. Enantiomeric preparative purification was performed using Agilent high pressure liquid chromatography and OD-H columns (Broad Institute of Harvard and MIT).

Reference 1 Ptashne, M.M. Binding reactions: epigenetic switches, signal transduction and cancer. Curr Biol 19, R234-241 (2009).
2 Schreiber, S .; L. & Bernstein, B.C. E. Signaling network model of chromatin. Cell 111, 771-778, (2002).
3 Marushige, K .; Activation of chromatin by acylation of histone side chains. Proc Natl Acad Sci USA 73, 3937-3941, (1976).
4 Dey, A.M. , Nishiyama, A .; Karpova, T .; McNally, J.A. & Ozato, K .; Brd4 marks select genes on mitochromatic chromatin and directions postmitotic transcription. Mol Biol Cell 20, 4899-4909, (2009).
5 Owen, D.W. J. et al. et al. The structural basis for the recognition of acetated histone H4 by the bromodomain of histone tranferase gcn5p. Embo J 19, 6141-6149, (2000).
6 Zeng, L.M. & Zhou, M .; M.M. Bromodomain: an acetyl-lysine binding domain. FEBS Lett 513, 124-128, (2002).
7 Yang, X. et al. J. et al. Multisite protein modification and intramolecular signaling. Oncogene 24, 1653-1662 (2005).
8 Yang, Z .; et al. Recruitment of P-TEFb for simulation of transcriptional elongation by the bromodomain protein Brd. Mol Cell 19, 535-545, (2005).
9 Phelps, M.M. A. et al. Clinical response and pharmacokinetics from a phase 1 study of an active dosing schedule of flavopiridol in relaxed chronic lymphatic. Blood 113, 2637-2645, (2009).
10 Rahl, P.M. B. et al. c-Myc Regulates Transactional Pause Release. Cell 141, 4323-4445, (2010).
11 Yang, Z .; , He, N .; & Zhou, Q. Brd4 replicates P-TEFb to chromosomes at plate mitosis to promote G1 gene expression and cell cycle progression. Mol Cell Biol 28, 967-976, (2008).
12 Mochizuki, K .; et al. The bromodomain protein Brd4 stimulates G1 gene transcription and promotes progression to phase. J Biol Chem 283, 9040-9048, (2008).
13 You, J .; et al. Regulation of aurora B expression by the bromodomain protein Brd4. Mol Cell Biol 29, 5094-5103, (2009).
14 Huang, B.H. Yang, X .; D. , Zhou, M .; M.M. Ozato, K .; & Chen, L.A. F. Brd4 coactivates transactional activation of NF-kappaB via specific binding to acetated RelA. Mol Cell Biol 29, 1375-1387, (2009).
15 French, C.I. A. , Miyoshi, I .; Aster, J .; C. & Et al. BRD4 bromodomain gene rearrangement in aggressive carcinoma with translocation (15; 19). Am J Pathol 159 (6), 1987-1992, (2001).
16 French, C.I. A. et al. BRD4-NUT fusion oncogene: a novel mechanism in aggressive carcinoma. Cancer Res 63, 304-307, (2003).
17 French, C.I. A. et al. BRD-NUT oncoproteins: a family of closed related proteins Oncogene 27, 2237-2242, (2008).
18 Miyoshi, S .; Ooike, S .; Iwata, K .; , Hikawa, H .; & Sugaraha, K .; ANTITUMOR AGENT. (2009).
19 Adachi, K .; et al. (2006).
20 Sueoka, H .; , Komatsu, H .; , Kobayashi, H .; & Ehara, S .; Thienotriazolodiazepine compounds and medicinal uses thereof. (1998).
21 VonVoiglander, P.M. F. & Straw, R.A. N. Alprazolam: Review of Pharmacological, Pharmacokinetic and Clinical Data. Drug Development Research 6, 1-12, (1985).
22 Niesen, F.M. H. Berglund, H .; & Vedadi, M.M. The use of differential scanning fluorimetry to detect ligand interactions that promote protein stability. Nat Protoc 2, 2212-2221, (2007).
23 Fedorov, O .; et al. A systematic interaction map of validated kinase inhibitors with Ser / Thr kinases. Proc Natl Acad Sci USA 104, 20523-20528, (2007).
24 Bullock, A.M. N. et al. Structural basis of inhibitor specificity of the human protocol cognitive produci tion site in molly mureine leukemia virus (PIM-1). J Med Chem 48, 7604-7614, (2005).
25 Quinn, A.M. M.M. et al. A homogeneous method for investing of methylation-dependent protein-protein interactions in epigenetics. Nucleic Acids Res 38, e11, (2010).
26 Volmuth, F.M. Blankenfeldt, W. & Geyer, M.M. Structures of the dual bromodomains of the P-TEFb-activating protein Brd4 atomic resolution. J Biol Chem 284, 36547-36556, (2009).
27 French, C.I. A. Molecular pathology of NUT midline carcinomas. J Clin Pathol, (2008).
28 Huang, M.M. E. et al. Use of all-trans retinoic acid in the treatment of acne proteolytic leukemia. Blood 72, 567-572 (1988).
29 Druker, B.D. J. et al. et al. Efficacy and safety of a specific inhibitor of the BCR-ABL tyrosine kinase in chronic myeloid leukemia. N Engl J Med 344, 1031-1037, (2001).
30 Haack, H.M. et al. Diagnostics of NUT Midline Carcinoma Using a NUT-specific Monoclonal Antibody. Am J Surg Pathol, (2009).
31 Buchdunger, E .; et al. Selective inhibition of the platelet-derived growth factor signal transduction pathway by a protein-tyrosine kinase inhibitor. Proc Natl Acad Sci USA 92, 2558-2562 (1995).
32 Buchdunger, E .; et al. Inhibition of the Abl protein-tyrosine kinase in vitro and in vivo by a 2-phenylaminopyridine derivative. Cancer Res 56, 100-104, (1996).
33 Schindler, T .; et al. Structural mechanism for STI-571 inhibition of abelson tyrosine kinase. Science 289, 1938-1942, (2000).
34 Druker, B.M. J. et al. et al. Effects of a selective inhibitor of the Abl tyrosine kinase on the growth of Bcr-Abl positive cells. Nat Med 2, 561-566, (1996).
35 le Coutre, P.M. et al. In vivo education of human BCR / ABL-positive leukemia cells with an ABL kinase inhibitor. J Natl Cancer Inst 91, 163-168, (1999).
36 Kadota, M .; et al. Identification of novel gene amplifications in breast cancer and coexistence of gene amplification with an activation of PIK3CA. Cancer Res 69, 7357-7365, (2009).
37 Crawford, N.M. P. et al. Bromodain 4 activation predicates breast cancer survival. Proc Natl Acad Sci USA 105, 6380-6385, (2008).
38 You, J .; et al. Kaposi's sarcoma-associated herpesviral lateness-associated nuclear antigen interacts with bromodomain protein Brd4 on host. J Virol 80, 8909-8919, (2006).
39 Abate, E.E. A. Voitenleitner, C.I. & Botchan, M.M. R. Structure of the papillomavirus DNA-thetering complex E2: Brd4 and a peptide that ablates HPV chromosomal association. Mol Cell 24, 877-889, (2006).
40 Cole, P.M. A. Chemical probes for histone-modifying enzymes. Nat Chem Biol 4, 590-597, (2008).

Other Embodiments From the foregoing description, it will be apparent that changes and modifications may be made to the invention described herein to adapt it to various uses and conditions. Such embodiments are also within the scope of the following claims.

  The recitation of a list of elements in the definition of any variable herein includes the definition of the variable as any single element or combination of listed elements. The recitation of an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.

  This application is based on US Provisional Patent Application No. 61 / 334,991 filed on May 14, 2010; US Provisional Patent Application No. 61 / 370,745 filed on August 4, 2010. And US Provisional Patent Application No. 61 / 375,863, filed Aug. 22, 2010. The contents of each of these applications are incorporated herein by reference.

  All patents and publications referred to herein are hereby incorporated by reference as if the patents and publications were each individually incorporated individually for any purpose. .

Claims (5)

A compound represented by a structural formula selected from the group consisting of: or a pharmaceutically acceptable salt thereof. Structural formula

The compound of Claim 1 represented by these, or its pharmaceutically acceptable salt. Structural formula

The compound of Claim 1 represented by these, or its pharmaceutically acceptable salt. Structural formula

The compound of Claim 2 represented by these, or its pharmaceutically acceptable salt. Structural formula

The compound of Claim 3 represented by these, or its pharmaceutically acceptable salt.