TWI583689B - Compositions and methods for treating neoplasia, inflammatory disease and other disorders - Google Patents

Description

Composition and method for treating tumor formation, inflammatory diseases and other diseases [Related application]

The present application claims priority from the following US Provisional Patent Application: Serial No. 61/370,745 filed on August 4, 2010, and Serial No. 61/375,863, filed on August 22, 2010, filed on March 24, 2011 The serial number of the application is 61/467,376. The entire contents of each of their respective applications are incorporated herein by reference.

[United States Federal Funded Research Invention Rights Statement]

This research work is funded by the National Institutes of Health grant, grant number: K08CA128972; the US government has specific rights to the invention.

The present invention relates to compositions and methods for treating or preventing tumor formation. More specifically, the present invention provides compositions and methods for disrupting the interaction between a BET family polypeptide comprising a bromo-containing domain and chromatin.

The N-terminal tail of histones maintains chromatin stability and undergoes modifications associated with transcriptional regulation. The best features of their modification are acetylation, methylation and phosphorylation. For each modification, there is an appropriate mark or a mark In addition to the enzymes; they must then be interpreted by transcriptional mechanisms. Acetyl-isamino acid recognition is primarily facilitated by the bromine functional site of the general component of the transcription factor complex. The bromine functional site and the extra-terminal (BET)-family (eg, BRD2, BRD3, BRD4, and BRDT) share two N-terminal bromine functional sites that exhibit high sequence conservation and interact with protein-proteins. There is a general functional part structure that is implicated in the C-terminal functional part. Abnormal regulation of histone modifications affects gene activity and plays a role in tumor formation. In the action of non-histone proteins [including, but not limited to, Hsp90, p53, STAT transcription factors, cortactin, β-catenin, and α-tubulin], the amino acid side chain is acetylated to Important regulatory items. Therefore, regulation of the recognition of the side chain of the amino acid is expected to clearly exert important phenotypes and therapeutic effects in the formation of the disease. Despite the importance of ethionyl-isoaminic acid recognition for tumor formation, almost no modulators of acetyl-isogenic acid recognition were identified.

As described below, the invention features for the treatment or prevention of tumor formation, inflammatory diseases, obesity, fatty liver (NASH or other), diabetes, atherosclerosis, arterial stent obstruction, heart failure, cachexia, grafts Contaminant diseases related to host diseases and bromodomain-related infectious diseases, treatment of parasites, malaria, trypanosomes, and compositions and methods for reducing male fertility. In a particular embodiment, the compounds of the invention are used to overcome the resistance of tumor formation (eg, cancer and non-malignant diseases). Other uses of the compositions of the present invention include cell state adjustment for organ transplantation, regenerative medicine (i.e., by promoting or inhibiting cell differentiation) and promoting Pluripotency, but not limited to this. More specifically, the present invention provides an interaction for disrupting a BET-type polypeptide comprising a bromo-containing domain and an etidinyl-iso-acid and/or chromatin (eg, disrupting the bromine-containing domain and presenting Compositions and methods for inhibiting cancer production in the interaction of the thiol-ammonic acid modification at the N-terminal tail of histones. In another embodiment, the invention prevents or treats an inflammatory disease.

In one aspect, the invention provides a compound of Formula I, or a salt, solvate, or hydrate thereof: Wherein X is N or CR ₅ ; R ₅ is H, alkyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl, each of which is optionally substituted; R _B is H, alkyl, a hydroxyalkyl group, an aminoalkyl group, an alkoxyalkyl group, a haloalkyl group, a hydroxyl group, an alkoxy group, or a -COO-R ₃ group, each of which is optionally substituted; the ring A is an aryl group or a heteroaryl group; Each R _A is independently alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, each of which is optionally substituted; or any two R _A may be formed with the atoms to which they are attached a fused aryl or fused heteroaryl; R is alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, each of which is optionally substituted; R ₁ is -(CH ₂ ) _n -L, wherein 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 substituted aryl, or optionally substituted heteroaryl; R ₂ is H, D, halogen, or an alkyl group optionally substituted; each R ₃ is independently selected from the group consisting of: (i) H, aryl, substituted aryl, heteroaryl, or Substituted heteroaryl (ii) heterocycloalkyl or substituted heterocycloalkyl of; (iii) -C ₁ -C ₈ alkyl, -C ₂ -C ₈ alkenyl or -C ₂ -C ₈ alkynyl group, which each contain 0 1, 2 or 3 heteroatoms selected from O, S, or N; -C ₃ -C ₁₂ cycloalkyl, substituted -C ₃ -C ₁₂ cycloalkyl, -C ₃ -C ₁₂ cycloalkenene a substituted or substituted C ₃ -C ₁₂ cycloalkenyl group, each of which may be optionally substituted; and (iv) NH ₂ , N=CR ₄ R ₆ ; each R ₄ is independently H, alkyl, alkyl a cycloalkyl, heterocycloalkyl, aryl, or heteroaryl group, each of which is optionally substituted; or R ₃ and R ₄ and the nitrogen atom to which they are attached form a 4 to 10 membered ring; R ₆ Is an alkyl group, an alkenyl group, a cycloalkyl group, a cycloalkenyl group, a heterocycloalkyl group, an aryl group, or a heteroaryl group, each of which is optionally substituted; or R ₄ and R ₆ and the carbon atom to which it is attached Forming a 4 to 10 membered ring together; m is 0, 1, 2, or 3; the restriction is (a) if ring A is a thienyl group, X is N, R is a phenyl group or a substituted phenyl group, and R ₂ is H, R _B is a methyl group, and R ₁ is -(CH ₂ ) _n -L, wherein n is 1 and L is -CO-N(R ₃ R ₄ ), then R ₃ and R _{4 are} not attached thereto The nitrogen atoms together form an N-morpholine ring; (b) Ring A is thienyl, X is N, R is a substituted phenyl group of, R ₂ is H, R _B is methyl and R ₁ is - (CH _{2) n} -L, where, n is 1 and L is a -CO-N(R ₃ R ₄ ), and one of R ₃ and R ₄ is H, and the other of R ₃ and R ₄ is not methyl, hydroxyethyl, alkoxy, phenyl a substituted phenyl, pyridyl or substituted pyridyl; and (c) if ring A is a thienyl group, X is N, R is a substituted phenyl group, R ₂ is H, and R _B is a methyl group. And R ₁ is -(CH ₂ ) _n -L, wherein n is 1 and L is -COO-R ₃ , and R _{3 is} not a methyl group or an ethyl group.

In certain embodiments, R is aryl or heteroaryl, each of which is 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 an optionally substituted aryl group. In certain embodiments, each R ₃ is independently selected from the group consisting of H, 1, 2, or 3 -C ₁ -C ₈ alkane selected from heteroatoms of O, S, or N. Or; NH ₂ , 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 one of R ₃ and R ₄ is H, and the other of R ₃ and R ₄ is (CH ₂ ) _p -Y, wherein p is 1 to 3 (for example, p is 2) and Y is a nitrogen-containing ring (which may be aromatic or non- Aromatic).

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

In certain embodiments, R _B is alkyl, hydroxyalkyl, haloalkyl, or alkoxy; each of which is 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 group. In certain embodiments, Ring A is thiofuranyl, phenyl, naphthyl, biphenyl, tetrahydronaphthyl, hydroquinone, pyridyl, furyl, indolyl, pyrimidinyl, indole Base Base, imidazolyl, oxazolyl, thienyl, thiazolyl, triazolyl, isoxazolyl, quinolyl, 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, R _A is methyl and at least one occurrence.

In certain embodiments, each R _A is independently H, optionally substituted alkyl, or any two R _A may form an aryl group with the atoms to which they are attached, respectively.

In another aspect, the invention provides a compound of Formula II, a salt, solvate, or hydrate thereof: Wherein X is N or CR ₅ ; R ₅ is H, alkyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl, each of which is optionally substituted; R _B is H, alkyl, a hydroxyalkyl group, an aminoalkyl group, an alkoxyalkyl group, a haloalkyl group, a hydroxyl group, an alkoxy group, or a -COO-R ₃ group, each of which is optionally substituted; each R _A is independently an alkyl group or a ring An alkyl group, a heterocycloalkyl group, an aryl group, or a heteroaryl group, each of which is optionally substituted; or any two R _A may form a fused aryl group or a fused heteroaryl group together with the atoms to which they are respectively attached ; R is alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, each of which is optionally substituted; R' ₁ is H, -COO-R ₃ , -CO-R ₃ , a aryl group to be substituted, or a heteroaryl group optionally substituted; each R ₃ group is independently selected from the group consisting of: (i) H, an aryl group, a substituted aryl group, a heteroaryl group, a substituted group a heteroaryl group; (ii) a heterocycloalkyl group or a substituted heterocycloalkyl group; (iii) a -C ₁ -C ₈ alkyl group, a -C ₂ -C ₈ alkenyl group or a -C ₂ -C ₈ alkynyl group, It contains 0, 1, 2 or 3 heteroatoms selected from O, S, or N, respectively; -C ₃ -C ₁₂ cycloalkyl, substituted -C ₃ -C ₁₂ ring An alkyl group, a -C ₃ -C ₁₂ cycloalkenyl group, or a substituted -C ₃ -C ₁₂ cycloalkenyl group; each of which may be optionally substituted; m is 0, 1, 2, or 3; R' ₁ is -COO-R ₃ , X is N, R is a substituted phenyl group, and R _B is a methyl group, and R _{3 is} not a methyl group or an ethyl group.

In certain embodiments, R is aryl or heteroaryl, each of which is Replace as needed. In certain embodiments, R is phenyl or pyridyl, each of which is 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 pyridyl.

In certain embodiments, R' ₁ is -COO-R ₃ , optionally substituted aryl, or optionally substituted heteroaryl, and R ₃ is -C ₁ -C ₈ alkyl, which contains 0. 1, 2 or 3 heteroatoms selected from O, S, or N, and which may be substituted as needed. In certain embodiments, R' ₁ is -COO-R ₃ and R ₃ is methyl, ethyl, propyl, isopropyl, butyl, t-butyl, or t-butyl; R' ₁ is H or a phenyl group which is optionally substituted.

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 alkyl group optionally substituted, or any two R _A may form a fused aryl group with the atoms to which they are attached, respectively.

In certain embodiments, each R _A is a methyl group.

In another aspect, the invention provides a compound of Formula III, a salt, solvate, or hydrate thereof: Wherein X is N or CR ₅ ; R ₅ is H, alkyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl, each of which is optionally substituted; R _B is H, alkyl, a hydroxyalkyl group, an aminoalkyl group, an alkoxyalkyl group, a haloalkyl group, a hydroxyl group, an alkoxy group, or a -COO-R ₃ group, each of which is optionally substituted; the ring A is an aryl group or a heteroaryl group; Each R _A is independently alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, each of which is optionally substituted; or any two R _A may be formed with the atoms to which they are attached a fused aryl or a fused heteroaryl; R is an alkyl group, a cycloalkyl group, a heterocycloalkyl group, an aryl group, or a heteroaryl group, each of which is optionally substituted; each R ₃ group is independently selected from the following Group consisting of: (i) H, aryl, substituted aryl, heteroaryl, or substituted heteroaryl; (ii) heterocycloalkyl or substituted heterocycloalkyl; (iii) -C ₁ -C ₈ alkyl, -C ₂ -C ₈ alkenyl or -C ₂ -C ₈ alkynyl, each containing 0, 1, 2 or 3 heteroatoms selected from O, S, or N; -C ₃ -C ₁₂ cycloalkyl, substituted -C ₃ -C ₁₂ cycloalkyl the alkyl, -C ₃ -C ₁₂ cycloalkenyl group, or the substituted -C ₃ -C ₁₂ cycloalkenyl , Each of which are optionally substituted; and _{(iv) NH 2, N =} CR 4 R 6; R ₄ lines each independently H, alkyl, alkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, or Aryl groups, each of which is optionally substituted; or R ₃ and R ₄ together with the nitrogen atom to which they are attached form a 4 to 10 membered ring; R ₆ is alkyl, alkenyl, cycloalkyl, cycloalkenyl, hetero a cycloalkyl, aryl, or heteroaryl group, each of which is optionally substituted; or R ₄ and R ₆ together with the carbon atom to which they are attached form a 4 to 10 membered ring; m is 0, 1, 2, or 3; The limiting conditions are: (a) if ring A is a thienyl group, X is N, R is a phenyl group or a substituted phenyl group, and R _B is a methyl group, then R ₃ and R _{4 are} not attached to the nitrogen The atoms together form an N-morpholine ring; and (b) if ring A is a thienyl group, X is N, R is a substituted phenyl group, R ₂ is H, R _B is a methyl group, and R ₃ and R ₄ are In one case, H, the other of R ₃ and R ₄ is not methyl, hydroxyethyl, alkoxy, phenyl, substituted phenyl, pyridyl or substituted pyridyl.

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

In certain embodiments, R is p-Cl-phenyl, o-Cl-phenyl, m-Cl-phenyl, pF-phenyl, oF-phenyl, mF-phenyl, or pyridyl. 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 of which is optionally substituted.

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

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

In another aspect, the invention provides a compound of Formula IV, a salt, solvate, or hydrate thereof: Wherein X is N or CR ₅ ; R ₅ is H, alkyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl, each of which is optionally substituted; R _B is H, alkyl, a hydroxyalkyl group, an aminoalkyl group, an alkoxyalkyl group, a haloalkyl group, a hydroxyl group, an alkoxy group, or a -COO-R ₃ group, each of which is optionally substituted; the ring A is an aryl group or a heteroaryl group; Each R _A is independently alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, each of which is optionally substituted; or any two R _A may be formed with the atoms to which they are attached a fused aryl or a fused heteroaryl; R ₁ is -(CH ₂ ) _n -L, wherein 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 substituted aryl, or optionally substituted heteroaryl; R ₂ is H, D, halogen, or optionally substituted alkyl; each R ₃ is independently selected from the group consisting of : (i) H, aryl, substituted aryl, heteroaryl, or substituted heteroaryl; (ii) heterocycloalkyl or substituted heterocycloalkyl; (iii)-C ₁ - C ₈ alkyl, -C ₂ -C ₈ alkenyl or -C ₂ a -C ₈ alkynyl group containing 0, 1, 2 or 3 heteroatoms selected from O, S, or N, respectively; -C ₃ -C ₁₂ cycloalkyl, substituted -C ₃ -C ₁₂ naphthenic a group, a -C ₃ -C ₁₂ cycloalkenyl group, or a substituted -C ₃ -C ₁₂ cycloalkenyl group, each of which may be optionally substituted; and (iv) NH ₂ , N=CR ₄ R ₆ ; each R ₄ Is independently H, alkyl, alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, each of which is optionally substituted; or R ₃ and R ₄ together with the nitrogen atom to which it is attached Forming a 4 to 10 membered ring; R ₆ is alkyl, alkenyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, aryl, or heteroaryl, each of which is optionally substituted; or R ₄ and R ₆ forms a 4 to 10 membered ring together with the carbon atom to which it is attached; m is 0, 1, 2, or 3; the constraint is (a) if ring A is a thienyl group, X is N, and R ₂ is H, R _B is a methyl group, and R ₁ is -(CH ₂ ) _n -L, wherein n is 0 and L is -CO-N(R ₃ R ₄ ), then R ₃ and R _{4 are} not attached to the nitrogen The atoms together form an N-morpholine ring; (b) if ring A is a thienyl group, X is N, R ₂ is H, R _B is a methyl group, and R ₁ is -(CH ₂ ) _n -L, wherein n is 0 and L is _{-CO-N (R 3 R 4} ), R _3, and One of R ₄ are H, then the other of R ₃ and R ₄ in the not methyl, hydroxyethyl, alkoxy, phenyl, the substituted phenyl, pyridyl or substituted pyridyl group of And (c) if ring A is a thienyl group, X is N, R ₂ is H, R _B is a methyl group, and R ₁ is -(CH ₂ ) _n -L, wherein n is 0 and L is -COO -R ₃ , then R _{3 is} not methyl or ethyl.

In certain embodiments, R ₁ is —(CH ₂ ) _n —L, wherein n is 0 to 3, and L is —COO—R ₃ , optionally substituted aryl, or optionally substituted Aryl; and R ₃ is -C ₁ -C ₈ alkyl, which contains 0, 1, 2, or 3 heteroatoms selected from O, S, or N, and which may be substituted as desired. In certain embodiments, n is 1 or 2, and L is alkyl or -COO-R ₃ and R ₃ is methyl, ethyl, propyl, isopropyl, butyl, second butyl Or a tert-butyl group; or n is 1 or 2, and L is H or a phenyl group optionally substituted.

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

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, hydroquinone, pyridyl, furyl, indolyl, pyrimidinyl, indole Base Base, imidazolyl, oxazolyl, thienyl, thiazolyl, triazolyl, isoxazolyl, quinolyl, pyrrolyl, pyrazolyl, or 5,6,7,8-tetrahydroisoquinolinyl .

In certain embodiments, each R _A is independently an alkyl group optionally substituted, or any two R _A may form an aryl group with the atoms to which they are attached, respectively.

The invention also provides herein compounds of formula V to XXII, as well as any of the compounds described herein.

In another aspect, the invention provides a method for treating or preventing tumor formation in a subject, the method comprising administering an effective amount of any compound of Formulas I to XXII or any of the compounds described herein to The individual.

In certain embodiments, the compound is any compound of Formulas I through IV.

In another aspect, the invention provides a method for reducing the growth, proliferation, or survival of a neoplastic cell, the method comprising administering the cell and an effective amount of any compound of Formulas I to XXII or Any of the compounds described are contacted to reduce the growth, proliferation, or survival of tumor cells.

In another aspect, the invention provides a method for inducing differentiation of tumor cells The method comprises contacting the cell with any of the compounds of Formulas I to XXII or any of the compounds described herein to induce tumor cell differentiation.

In another aspect, the invention provides a method of inducing cell death in a tumor cell, the method comprising contacting the cell with a therapeutically effective amount of any compound of Formulas I to XXII or any of the compounds described herein, thereby inducing a tumor Cell death of cells.

In certain embodiments, the methods include selecting a compound for binding to a bromine-containing domain of the BET family.

In certain embodiments, the methods comprise selecting a compound for inhibiting binding of a bromo-containing domain domain to chromatin in a cellular environment.

In some embodiments, the methods include differential scanning fluorimetry (DSF), isothermal titration calorimetry, and/or fluorescence close-range phasing analysis. (luminescence proximity homogeneous assay; ALPHA-screen) to select compounds that bind to specificity. In certain embodiments, the compound increases the thermal stability of the bromine containing domain in the assay described.

In some embodiments of the methods, the BET family member is BRD2, BRD3, BRD4, or BRDT.

In certain embodiments of the methods, the cell line is located in an individual.

In another aspect, the invention provides a method for preventing or treating tumor formation in an individual, the method comprising administering an effective amount of any of the compounds of Formulas I to XXII or any of the compounds described herein to a desired individual, To prevent or treat tumor formation in an individual.

In certain embodiments, the individual is a mammal. In some embodiments, the individual is a human patient.

In certain embodiments, the method reduces the growth or proliferation of tumor formation in an individual.

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

In certain embodiments, the individual has tumor formation selected from the group consisting of: Burkitt's lymphoma, small cell lung cancer, breast cancer, colon cancer, neuroblastoma, polymorphism Glioblastoma, MLL-driven leukemia, chronic lymphocytic leukemia, NUT midline cancer, squamous cell carcinoma, or any other cancer associated with NUT rearrangement.

In another aspect, the invention provides a composition comprising a therapeutically effective amount of any of the compounds of Formulas I through XXII, or any of the compounds described herein, and a pharmaceutically acceptable excipient or carrier.

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

In another aspect, the invention provides a method for preventing or treating tumor formation in an individual, the method comprising administering to the individual a therapeutically effective amount of any of the compounds of Formulas I to XXII or any of the compounds described herein Wherein the compound interferes with binding of the bromo-containing domain to the acetamido-lysine or removes the BET family member from the chromatin, thereby preventing or treating the tumor form.

In certain embodiments, the compound inhibits binding of the 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 bromine containing domain.

In another aspect, the invention provides a method of identifying a compound for treating tumor formation, the method comprising contacting a test compound with a BET family member comprising a bromine-containing domain; and detecting the domain with a bromine-containing region Specificity is combined to identify test compounds suitable for the treatment of tumor formation.

In some embodiments, the combination of specificity uses differential scanning fluorimetry (DSF) analysis.

In certain embodiments, the binding system increases the thermal stability of the bromine containing domain.

In some embodiments, the binding system is detected using isothermal titration microcalorimetry.

In some embodiments, the binding is detected using a fluorescent close-range assay (ALPHA-screen).

In certain embodiments, the compound inhibits binding of a histone H4 Kac peptide to the bromo-containing domain.

In certain embodiments, the compound forms a hydrogen bond with the evolutionarily retained aspartic acid located in the bromine-containing domain domain.

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

In certain embodiments, the compound competitively binds to chromatin in a cellular environment.

In some embodiments, the competitive binding to chromatin is detected using fluorescence recovery after photobleaching (FRAP).

In certain embodiments, the method is performed in an in vitro tumor cell.

In certain embodiments, the method further comprises detecting a decrease in cell proliferation, an increase in cell death, or an increase in cell differentiation.

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

In certain embodiments, cell differentiation is confirmed by detecting an increase in keratin protein expression.

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

In another aspect, the invention provides a method for treating or preventing tumor formation in a subject, the method comprising administering to the individual an effective amount of any compound of Formulas I to XXII or any of the compounds described herein, Wherein the compound binds to the BET family bromine-containing domain and disrupts the interaction of the bromo-containing domain with chromatin, thereby preventing or treating the cancer.

In certain embodiments, the method induces cell death or differentiation of tumor cells of the individual.

In another aspect, the invention provides a composition for treating or preventing tumor formation, the composition comprising an effective amount of any compound selected from the group consisting of Formulas I to XXII or any of the compounds described herein. A compound and a pharmaceutically acceptable excipient, wherein the compound inhibits binding of the histone H4Kac peptide to the BET family bromine-containing domain.

In another aspect, the invention provides a method for reducing inflammation in an individual, the method comprising administering to the individual an effective amount of any of the compounds of Formulas I through XXII or any of the compounds described herein.

In another aspect, the invention provides a method for preventing or treating an inflammatory disease in a subject, the method comprising administering an effective amount of any of the compounds of Formulas I to XXII or any of the compounds described herein to a desired individual.

In certain embodiments, the individual is a mammal. In some embodiments, the individual is a human patient.

In certain embodiments, the method reduces cytokine concentration, histamine release, or biological activity of an immune response cell.

In another aspect, the invention provides a method of identifying a compound for treating inflammation, the method comprising contacting a test compound with a BET family member comprising a bromine-containing domain; and detecting a specificity with a bromine-containing domain Sexual combination to identify test compounds suitable for the treatment of inflammation.

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

definition

"Pharmaceutical" means any small molecule chemical compound, antibody, nucleic acid molecule, or polypeptide, or a fragment thereof.

The term "aromatic ring" or "aryl" as used herein means a monocyclic or polycyclic-aromatic ring or ring group including a carbon atom and a hydrogen atom. Examples of suitable aryl groups include, but are not limited to, phenyl, tolyl, fluorenyl, fluorenyl, fluorenyl, fluorenyl, and naphthyl, and benzo-fused ring moieties such as 5,6,7,8- Tetrahydronaphthyl. The aryl group may be unsubstituted or optionally substituted with one or more substituents such as the substituents of the alkyl groups described herein [including alkyl groups (preferably lower alkyl groups or via one or more a halogen-substituted alkyl group, but is not limited thereto, a hydroxyl group, an alkoxy group (preferably a lower alkoxy group), an alkylthio group, a cyano group, a halogen group, an amine group, or a boronic acid (-B ( OH) ₂ ), and nitro]. In certain embodiments, the aryl group is a single cyclic ring wherein the ring comprises 6 carbon atoms.

As used herein, the term "alkyl" means a saturated straight or branched chain acyclic hydrocarbon typically having from 1 to 10 carbon atoms. Representative saturated linear alkyl groups include methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, n-heptyl, n-octyl, n-decyl and n-decyl; Alkyl includes isopropyl, t-butyl, isobutyl, tert-butyl, isopentyl, 2-methylbutyl, 3-methylbutyl, 2-methylpentyl, 3-methyl Pentyl, 4-methylpentyl, 2-methylhexyl, 3-methylhexyl, 4-methylhexyl, 5-methylhexyl, 2,3-dimethylbutyl, 2,3-dimethyl Pentyl, 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 Base, 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-diethyl Pentyl, 3,3-diethylhexyl, 2,2-diethylhexyl, 3,3-diethylhexyl, and the like. The alkyl group contained in the compound of the present invention may be unsubstituted or optionally substituted with one or more substituents such as an amine group, an alkylamino group, an arylamino group, a heteroarylamino group, Alkoxy, alkylthio, pendant oxy, halo, fluorenyl, nitro, hydroxy, cyano, aryl, heteroaryl, alkylaryl, alkylheteroaryl, aryloxy, Heteroaryloxy, arylthio, heteroarylthio, arylamine, heteroarylamine, carbocyclyl, carbocyclyloxy, carbocyclylthio, carbocyclylamino, Heterocyclic group, heterocyclic oxy group, heterocyclic amino group, heterocyclic thio group and the like. Typical compounds are preferably lower alkyl groups for the compounds of the invention.

By "bromo-containing domain" is meant a portion of a multi-peptide that recognizes an acetamino acid lysine residue. In a specific embodiment, the bromo-containing region of the BET family member polypeptide comprises about 110 amino acids and shares a retained fold comprising a different loop region that interacts with chromatin. (loop region) The left-handed bundle of four alpha helices connected.

"BET family member polypeptide" means multiple wins including two bromodomains and an extraterminal (ET) domain or fragment thereof having transcriptional regulatory activity or acetamino acid lysate binding activity Peptide. Exemplary BET family members include BRD2, BRD3, BRD4, and BRDT.

"BRD2 polypeptide" means a protein or fragment thereof having 85% identity to NP_005095 which binds to chromatin or regulates transcription.

The sequence of the exemplary BRD2 polypeptide is as follows:

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

"BRD3 polypeptide" means a protein or a fragment thereof which is 85% identical to NP_031397.1 which binds to chromatin or regulates transcription.

The sequence of the exemplary BRD3 polypeptide is as follows:

"BRD3 nucleic acid molecule" means a polynucleotide encoding a BRD3 polypeptide or a fragment thereof.

"BRD4 polypeptide" means a protein or a fragment thereof which is 85% identical to NP_055114 which binds to chromatin or regulates transcription.

"BRD4 nucleic acid molecule" means a polynucleotide encoding a BRD4 polypeptide.

"BRDT polypeptide" means a protein or fragment thereof which is 85% identical to NP_001717 which binds to chromatin or regulates transcription.

"BRDT nucleic acid molecule" means a polynucleotide encoding a BRDT polypeptide.

"Improvement" means reducing, inhibiting, attenuating, reducing, preventing, or stabilizing the development or progression of a disease.

"Change" means the change (increase or decrease) in the amount or activity of a gene or polypeptide when detected by standard methods known in the art, such as those described herein. As used herein, the alterations include a 10% change in performance, preferably a 25% change, a better 40% change, and a 50% or higher change in performance.

"Analog" means a molecule that is not identical but has similar functional or structural characteristics. For example, a multi-peptide analog retains at least a portion of the biological activity of the corresponding naturally occurring multi-peptide, with certain biochemical modifications to enhance the function of the analog compared to the naturally occurring multi-peptide . Such biochemical modifications may increase the protease resistance, membrane permeability, or half-life of the analog, but do not alter, for example, ligand binding. Analogs can include non-natural amino acids.

"Compound" means any small molecule chemical compound, antibody, nucleic acid molecule, or polypeptide, or a fragment thereof.

The term "non-image isomer" means a stereoisomer having two or more asymmetric centers, and the molecules of the stereoisomers are not mirror images of each other.

The term "mirrible isomer" means two stereoisomers of a compound which are non-superimposable mirror images of each other. The molar mixture of the two mirror isomers is referred to as the "racemic mixture" or "racemate".

The term "halogen" means -F, -Cl, -Br, or -I.

The term "haloalkyl" is intended to include alkyl, as defined above, mono-, di- or polysubstituted by halogen, for example, fluoromethyl and trifluoromethyl.

The term "hydroxy" 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" means an aromatic 5 to 8 membered monocyclic ring, 8 to 12 membered bicyclic ring, or 11 to 14 membered tricyclic ring system, which ring system has 1 to 4 ring heteroatoms if it is a single ring. If it is a bicyclic ring, it has 1 to 6 ring hetero atoms, or if it is a tricyclic ring, it has 1 to 9 ring hetero atoms, the hetero atom is selected from O, N, or S, and the remaining ring atoms are carbon. The heteroaryl group may be substituted with a substituent of one or more aryl groups as desired. Examples of heteroaryl groups include pyridyl, furyl, benzodioxolyl, thienyl, pyrrolyl, oxazolyl, oxadiazolyl, imidazolyl, thiazolyl, isoxazolyl, Quinolinyl, pyrazolyl, isothiazolyl, anthracene Base, pyrimidinyl, pyridyl Base, three , triazolyl, thiadiazolyl, isoquinolyl, oxazolyl, benzoxazolyl, benzofuranyl, anthracene Base, imidazopyridyl, tetrazolyl, benzimidazolyl, benzothiazolyl, benzothiadiazolyl, benzooxadiazolyl, and fluorenyl, but are not limited thereto.

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

The term "isomer" or "stereoisomer" means having the same chemistry A compound that is composed of atoms or groups that are arranged differently in space.

The term "isotopic derivative" includes derivatives of compounds in which one or more atomic systems of a compound are replaced by a corresponding isotope of the atom. For example, an isotopic derivative of a compound containing a carbon atom (C ¹² ) may be one in which the carbon atom of the compound is substituted with a C ¹³ isotope.

The term "neoplastic" means a cell that has autonomous growth ability, that is, an abnormal state or condition characterized by rapid proliferative cell growth. Tumor disease states can be classified as pathological (ie, characterized by disease states or constituting disease states), or can be classified as non-pathological (ie, deviating from normal but not related to disease states). The term is intended to include all types of cancerous growth or oncogenic processes, metastatic tissues or cells, tissues or organs of malignant transformation, regardless of the histopathological type or stage of invasion. The "pathological hyperproliferation" cell line appears in a disease state characterized by malignant tumor growth. Examples of cells that are not pathologically hyperproliferating include cell proliferation associated with wound repair.

Exemplary tumors that can be treated using the compounds of the invention include, but are not limited to, leukemia (eg, acute leukemia, acute lymphocytic leukemia, acute myeloid leukemia, acute promyelocytic leukemia, acute myelomonocytic leukemia) , acute monocytic leukemia, acute erythrocytic leukemia, mixed-lineage leukemia, chronic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, multiple myeloma, polycythemia Vera), cutaneous T-cell lymphoma (CTCL), lymphoma (Hodgkin's) Disease), non-Hodgkin's disease, Waldenstrom's macroglobulinemia, heavy chain disease, and solid tumors such as sarcoma and malignant tumors (eg, fibrosarcoma, mucinous sarcoma, liposarcoma, chondrosarcoma, bone) Sarcoma, chordoma, angiosarcoma, endothelial sarcoma, lymphangiosarcoma, lymphatic endothelial sarcoma, synovial membranous tumor, mesothelioma, Ewing's sarcoma, uterine muscle tumor, rhabdomyosarcoma, colon cancer, colon Rectal cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland cancer, sebaceous gland cancer, mastoid carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medulla Carcinoma, bronchial cancer, renal cell carcinoma, liver cancer, cholangiocarcinoma, choriocarcinoma, seminoma, embryonal cancer, Wilm's 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, stellate cell tumor, neuroblastoma (medulloblastoma), cranial Pharyngeal tumor, ependymoma, pineal tumor, hemangioblastoma, acoustic neuroma, neuroendocrine tumor, oligodendroglioma, schwannomas, meningioma, melanoma, neuroblastoma and retinoblast tumor).

In a specific embodiment, tumor formation is driven by a primary transcriptional activator (eg, myc). Such cancers include Burkitt's lymphoma, small cell lung cancer, breast cancer, colon cancer, neuroblastoma, polymorphic glioblastoma, MLL-driven leukemia, chronic lymphocytic leukemia, and NUT rearrangement. Squamous cell carcinoma, and other cancers associated with proteins containing bromine-containing domain domains or NUT rearrangements, but are not limited thereto.

The term "inhibiting the growth of a tumor" includes slowing, interrupting, blocking, or Stop the growth and metastasis of the tumor, but do not have to completely eliminate the tumor growth.

"Computer simulation" means the use of a computer program to determine one or more of the following: the position of the ligand to the binding portion and the binding proximity, the occupied space of the ligand, the binding portion and the matching The amount of complementary contact surface between the ligands, the deformation energy of the specific ligand bound to the binding moiety, and the hydrogen bond strength between the ligand and the binding moiety, van der Waals interaction, hydrophobicity Several assessments of interaction and/or electrostatic interaction energy. Computer simulations can also provide a comparison between the model system and the characteristics of the candidate compounds. For example, a computer simulation test can compare a pharmacophore model of the present invention with a candidate compound to assess the fit of a candidate compound to a model.

"Computer system" means a hardware device, a software device, and a data storage device for analyzing atomic coordinates. The minimum hardware device of the computer system of the present invention includes a central processing unit (CPU), an input device, an output device, and a data storage device. It is desirable to provide a display to visualize the structure. The data storage device can be a random access memory (RAM) or a device for accessing the computer readable medium of the present invention. Examples of such systems are microcomputer workstations, Windows NT, or IBM OS/2 operating systems available from Silicon Graphics Incorporated and Sun Microsystems running Unix based.

"Computer readable media" means any medium that can be directly read and accessed by a computer, for example, by means of which the media is suitable for use in the aforementioned computer systems. Media includes, but is not limited to, magnetic storage media such as floppy disks, hard disk storage media and magnetic tape; optical storage media such as optical disks or CD-ROMs (CD-ROM); electrical storage media such as random access memory and read only memory (ROM); and mixtures of such types as magnetic/optical storage media.

In this disclosure, "including", "including" and "having" may have the meaning as defined in the US Patent Law and may mean "including", etc. "consisting essentially of or Consists essestially)) also has the meaning as defined in the U.S. Patent Law and the term is open-ended, allowing for the existence of those listed, as long as the existence of the listed person does not change the listed Basic or novel features, but prior art embodiments are excluded.

"Detection" means confirming the presence, absence or amount of an analyte to be detected.

"Detectable label" means a composition which, when attached to a molecule of interest, can be detected by spectroscopic, photochemical, biochemical, immunochemical, or chemical means. . For example, suitable labels include radioisotopes, magnetic beads, metal beads, colloidal particles, fluorescent dyes, electron-dense reagents, enzymes (eg, commonly used in ELISA), biotin, digoxigenin, or Hapten.

"Disease" means any condition or condition that would impair or interfere with the normal function of a cell, tissue or organ. Examples of diseases that can be treated using the compounds described herein include tumor formation, inflammatory diseases, obesity, fatty liver (NASH or other), diabetes, atherosclerosis, arterial stent obstruction, heart failure, cachexia, graft versus host Diseases, infectious diseases associated with bromine-containing domain domains, treatment of parasites, malaria, trypanosomes, and use to reduce male fertility. Other uses of the compositions of the invention include Cell state adjustment of organ transplantation, regenerative medicine (that is, by promoting or inhibiting cell differentiation) and promoting pluripotency, but is not limited thereto.

"Effective amount" means the dose required to ameliorate the symptoms of a disease compared to an untreated patient. The effective amount of the active compound which is used in the practice of the present invention to achieve the therapeutic treatment of the disease will vary depending upon the mode of administration, the age, weight, and general health of the subject. Finally, the appropriate amount and mode of administration will be determined by the attending physician or veterinarian. This amount is called the "effective" amount.

The term "mirrible isomer" means two stereoisomers of a compound which are non-superimposable mirror images of each other. The molar mixture of the two mirror isomers is referred to as the "racemic mixture" or "racemate".

The term "halogen" means -F, -Cl, -Br or -I.

The term "haloalkyl" is intended to include alkyl, as defined above, mono-, di- or polysubstituted by halogen, for example, fluoromethyl and trifluoromethyl.

The term "hydroxy" 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" means an aromatic 5 to 8 membered monocyclic ring, 8 to 12 membered bicyclic ring, or 11 to 14 membered tricyclic ring system, which ring system has 1 to 4 ring heteroatoms if it is a single ring. If it is a bicyclic ring, it has 1 to 6 ring hetero atoms, or if it is a tricyclic ring, it has 1 to 9 ring hetero atoms, the hetero atom is selected from O, N, or S, and the remaining ring atoms are carbon. The heteroaryl group can be substituted with one or more substituents (e.g., substituents for the aryl groups described herein) as desired. Examples of heteroaryl groups include pyridyl, furyl, benzodioxolyl, thienyl, pyrrolyl, oxazolyl, oxadiazolyl, imidazolyl, thiazolyl, isoxazolyl, Quinolinyl, pyrazolyl, isothiazolyl, anthracene Base, pyrimidinyl, pyridyl Base, three , triazolyl, thiadiazolyl, isoquinolyl, oxazolyl, benzoxazolyl, benzofuranyl, anthracene Base, imidazopyridyl, tetrazolyl, benzimidazolyl, benzothiazolyl, benzothiadiazolyl, benzooxadiazolyl, and fluorenyl, but are not limited thereto.

The term "heterocycle" as used herein, refers to an organic compound containing at least one atom other than carbon (eg, S, O, N) within the ring structure. The ring structure in such organic compounds may be aromatic or, in certain embodiments, non-aromatic. Some examples of the heterocyclic moiety include pyridine, pyrimidine, pyrrolidine, furan, tetrahydrofuran, tetrahydrothiophene, and dioxane, but are not limited thereto.

The term "isomer" or "stereoisomer" means a compound having the same chemical composition but differing in the arrangement of atoms or groups in space.

The term "isotopic derivative" includes derivatives of compounds in which one or more atomic systems of a compound are replaced by a corresponding isotope of the atom. For example, an isotopic derivative of a compound containing a carbon atom (C ¹² ) may be one in which the carbon atom of the compound is substituted with a C ¹³ isotope.

The present invention provides a number of targets suitable for the development of highly specific drugs to treat conditions characterized by the methods described herein. Moreover, the methods of the present invention provide an easy way to identify a therapy that can be safely used in an individual. Moreover, the methods of the present invention provide a pathway with high volume throughput, high sensitivity, and low complexity for actually analyzing any number of compounds that can act on the diseases described herein.

"Fitting" means determining one or more atoms or binding sites of one or more atoms of a drug molecule to a member of the BET family by an automated or semi-automated manner (eg, bromine-containing domains of BRD2, BRD3, BRD4, and BRDT) The interaction between them and determine the degree of stability of these interactions. Various computer-based methods for performing the fitting are further described herein.

"Fragment" means a portion of a multi-peptide or nucleic acid molecule. Preferably, the portion comprises 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 may 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 .

"Hybrid" means a hydrogen bond between complementary nucleobases which may be Watson-Crick, Hoogsteen or anti-Hodken hydrogen bonding. For example, adenine and thymine are complementary nucleobases paired by the formation of hydrogen bonds.

"Isolated polynucleotide" means a nucleic acid (eg, DNA) that does not contain a gene, which is located in the naturally occurring gene of the organism, and the nucleotide molecule of the present invention is derived from the naturally occurring gene of the organism And located on the side of the gene. Thus, the term includes, for example, a recombinant DNA that is incorporated into a vector; incorporated into an autonomously replicating plastid or virus; or incorporated into a chromosomal DNA of a prokaryote or eukaryote; or the recombinant DNA is not dependent Other sequences are present as separate molecules (e.g., cDNA or gene or cDNA fragments produced by PCR or restriction endonuclease cleavage). In addition, the term includes RNA transcribed from DNA molecules. Molecules and recombinant DNA (which are part of a hybrid gene encoding other multi-peptide sequences).

"Individually multi-peptide" means a multi-peptide of the present invention which has been isolated from a component naturally having the polypeptide. Typically, the multi-peptide is said to be isolated when it does not contain at least 60% by weight of the protein or naturally occurring organic molecule with which it is naturally associated. The formulation is preferably at least 75% by weight, more preferably at least 90% by weight, most preferably at least 99% by weight of the multipeptide of the invention. The isolated multi-peptide of the present invention can be obtained, for example, by extraction from a natural source; expression of a recombinant nucleic acid encoding the multi-peptide; or chemical synthesis of the protein. Purity can be measured by any suitable method, for example, column chromatography, polypropylene guanamine colloidal electrophoresis, or HPLC analysis.

"Sign" means any protein or polynucleotide that has an alteration in activity or activity that is associated with a disease or condition.

As used herein, "acquiring" in "acquiring a medicament" includes synthesizing, purchasing or otherwise obtaining the medicament.

The term "tumor" means a cell that has the ability to grow autonomously, that is, an abnormal state or condition characterized by rapid proliferating cell growth. Tumor disease states can be classified as pathological (ie, characterized by disease states or constituting disease states), or can be classified as non-pathological (ie, deviating from normal but not related to disease states). The term is intended to include all types of cancerous growth or carcinogenic processes, metastatic tissues or malignant transformation of cells, tissues or organs, regardless of the histopathological type or stage of invasion. The "pathologically over-proliferating" cell line is present in a disease state characterized by malignant tumor growth. Examples of non-pathologically hyperproliferating cells include fines associated with wound repair Cell proliferation.

The term "inhibiting the growth of a tumor" includes slowing, interrupting, stopping or stopping the growth and metastasis of the tumor, but does not necessarily eliminate the complete elimination of the tumor.

The general medical meaning of the term "tumor formation" refers to "new cell growth" caused by a loss of response to normal growth control, for example, the growth of tumor cells. "Excessive hyperplasia" means that the cells undergo abnormally high growth. However, as used herein, the term tumor formation generally refers to cells that experience abnormal cell growth rates. Tumor formation lines include "tumors," which can be benign, precancerous, or malignant.

The term "obtaining" in "obtaining a compound" is intended to include the purchase, synthesis or otherwise obtaining the compound.

The term "optical isomer" as used herein includes the following molecules (also known as palmitic molecules): they are mirror images of each other that are truly non-superimposable.

As used herein, the terms "parenteral administration" and "parenteral administration" mean a mode of administration that is different from enteral and topical administration, usually by injection, and includes intravenous, intramuscular, and intra-arterial administration. , intraspinal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, intraspinal and intrasternal injections and perfusion, but not Limited to this.

The term "polycyclic radical" or "polycyclic radical" means a radical of two or more cyclic rings (eg, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, and/or Or a heterocyclic group, wherein two adjacent ring systems share two or more carbon atoms, for example, the rings are "fused rings". A ring system that is bonded by non-adjacent atoms is called a "bridge" ring. Multiple rings of multiple rings Substituted by the substituents described above, such substituents are halogen, hydroxy, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate , alkylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylthiocarbonyl, alkoxy, phosphate, phosphonato, phosphinato, cyano, amine (including alkylamino, dialkylamino, arylamino, diarylamine, and alkylarylamine), mercaptoamine (including alkylcarbonylamino, arylcarbonylamino) , amidyl and ureido), carbaryl, imine, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfate, sulfonate Sulfonato, sulfonamide, sulfonylamino, nitro, trifluoromethyl, cyano, azido, heterocyclic, alkyl, alkylaryl, or aromatic or heteroaromatic section.

The term "polymorph" as used herein means 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 spectral properties. Different physical properties include stability (eg, heat or light), compressibility and density (very important in formulation and product manufacture), and dissolution rate (which can affect bioavailability), but are not limited thereto. . The difference in stability can be attributed to chemical reactivity (eg, differential oxidation causes a dosage form consisting of one polymorph to change color faster than a dosage form composed of another polymorph) or mechanical properties (eg , the tablet is broken during storage, because the kinetic tendency of the polymorph to be converted to a thermodynamically more stable polymorph) or both (for example, a polymorphic tablet is more susceptible to cracking at high humidity) . The different physical properties of the polymorph can affect its process.

The term "prodrug" includes a compound having a moiety that is metabolizable in vivo. In general, prodrugs are metabolized in vivo to the active drug by esterolytic enzymes or by other mechanisms. Examples of prodrugs and their use are well known in the art (see, for example, Berge et al . (1977) "Pharmaceutical Salts", J. Pharm. Sci. 66: 1-19). Prodrugs can be prepared in situ during the final isolation and purification of the compound, or by separately reacting the free acid form or hydroxyl group of the purified compound with a suitable esterifying agent. The hydroxyl group can be converted to an ester by treatment with a carboxylic acid. Examples of prodrug moieties include substituted and unsubstituted, branched or unbranched lower alkyl ester moieties (eg, propionates), lower alkyl alkenyl esters, di-lower alkyl groups - Amino-lower-alkyl ester (for example, dimethylaminoethyl ester), mercaptoamine-based lower alkyl ester (for example, ethoxymethyloxymethyl ester), low decyloxy group Alkyl alkyl ester (for example, trimethylacetoxymethyl methyl ester), aryl ester (phenyl ester), aryl-lower alkyl ester (for example, benzyl ester), substituted (for example, an aryl group having a methyl group, a halogen group, or a methoxy substituent) and an aryl-lower alkyl group, a decylamine, a lower carbon-alkylamine, a di-lower alkyl group Indoleamine, and hydroxyguanamine. Preferably, the prodrug moiety is a propionate and a decyl ester. It also includes a drug that is converted to the active form by other mechanisms in vivo.

Furthermore, the stereochemical representation across the carbon-carbon double bond from the general chemical field is also relative, where "Z" means often referred to as the "cis" (same) configuration, and "E" means the person often referred to as the "trans" (opposite) configuration. The compounds of the invention encompass both configurations: cis/trans and/or Z/E.

Regarding the nomenclature of the palm center, the terms "d" and "l" are used. As defined by the IUPAC Recommendation. As to the use of the terms non-image isomers, racemates, epimers, and mirror image isomers, these terms are used in their ordinary context to describe the stereochemistry of the formulation.

"Reduced" 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 that uses the basis of sequence alignment. A reference sequence can be a subset or all of a particular sequence; for example, a full-length cDNA or a fragment of a gene sequence, or a complete cDNA or gene sequence. In the case of a multi-peptide, the reference peptide sequence is typically at least about 16 amino acids in length, preferably at least about 20 amino acids, more preferably at least about 25 amino acids, even more preferably. It is about 35 amino acids, about 50 amino acids, or about 100 amino acids. In the case of nucleic acids, the nucleic acid sequence of reference is typically at least about 50 nucleotides in length, preferably at least about 60 nucleotides, more preferably at least about 75 nucleotides, even more preferably about 100 nucleotides. Nucleotide, or about 300 nucleotides, or any integer similar thereto or interposed therebetween.

"Specific binding" means a compound or antibody that recognizes and binds to the multi-peptide of the present invention, but the compound or antibody does not substantially recognize and bind to other molecules in the sample, for example, the sample naturally includes the present invention. Biological sample of multi-peptide.

Nucleic acid molecules suitable for use in the methods of the invention include any nucleic acid molecule encoding a multi-peptide of the invention or a fragment thereof. Such nucleic acid molecules need not be 100% identical to the endogenous nucleic acid sequence, but typically exhibit substantial identity. "substantial identity" with endogenous sequences A polynucleotide is typically hybridized to at least one strand of a double stranded nucleic acid molecule. Nucleic acid molecules suitable for use in the methods of the invention include any nucleic acid molecule encoding a multi-peptide of the invention or a fragment thereof. Such nucleic acid molecules need not be 100% identical to the endogenous nucleic acid sequence, but typically exhibit substantial identity. A polynucleotide having "substantial identity" to an endogenous sequence is typically hybridized to at least one strand of a double-stranded nucleic acid molecule. "Hybrid" means pairing a complementary polynucleotide sequence (eg, a gene described herein) or a portion thereof under various stringent conditions to form a double-stranded molecule. (See, for example, Wahl, G.M. and S.L. Berger (1987) Methods Enzymol. 152:399; Kimmel, A.R. (1987) Methods Enzymol. 153:507).

For example, harsh salt concentrations are typically less than about 750 mM NaCl and 75 mM trisodium citrate, preferably less than about 500 mM NaCl and 50 mM trisodium citrate, and more preferably less than about 250 mM NaCl and 25 mM trisodium citrate. Low stringency hybridization can be obtained in the absence of an organic solvent (e.g., formamide), while high stringency hybridization can occur in the presence of at least about 35% formamidine and more preferably at least about 50% formamidine. Obtained under. Severe 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. Altering additional parameters, such as hybridization time, concentration of detergent (such as sodium dodecyl sulfate (SDS)), and inclusion or exclusion of carrier DNA, is common in the art Well known to the knowledge. The various degrees of severity are achieved by combining the various conditions required. In a preferred embodiment, the hybrid system occurs at 30 ° C in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS. In a more preferred embodiment, the hybrid system is at 500 ° C at 37 ° C, 50 Occurred in mM trisodium citrate, 1% SDS, 35% formamide, and 100 μg/ml denatured salmon sperm DNA (ssDNA). In the most preferred embodiment, the hybrid system occurs at 42 ° C in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, and 200 μg/ml ssDNA. Applicable changes to these conditions are readily understood by those of ordinary skill in the art.

For most applications, the washing step after mixing is also different in terms of severity. Washing harsh conditions can be defined by salt concentration and by temperature. As noted above, the severity of the wash can be increased by lowering the salt concentration or by increasing the temperature. For example, the harsh salt concentration of the washing step is preferably less than about 30 mM NaCl and 3 mM trisodium citrate, and most preferably less than about 15 mM NaCl and 1.5 mM trisodium citrate. The stringent temperature conditions of the washing step typically comprise a temperature of at least about 25 ° C, more preferably a temperature of at least about 42 ° C, 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 1% SDS. In a more preferred embodiment, the washing step occurs at 42 ° C in 15 mM NaCl, 1.5 mM trisodium citrate, and 1% SDS. In a more preferred embodiment, the washing step occurs at 68 ° C in 15 mM NaCl, 1.5 mM trisodium citrate, and 1% SDS. Additional variations of these conditions are readily apparent to those of ordinary skill in the art.

Hybrid techniques are well known to those of ordinary skill in the art and are described, for example, in 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, Academic Press, New York); and Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York.

"Substantially identical" means at least 85 relative to a reference amino acid sequence (eg, any of the amino acid sequences described herein) or a nucleic acid sequence (eg, any of the nucleic acid sequences described herein). % identity peptide or nucleic acid molecule. Preferably, this sequence has at least 85%, 90%, 95%, 99%, or even 100% identity to the sequence used for alignment at the amino acid or nucleic acid level.

Sequence identity is typically measured using sequence analysis software (eg, Sequence Analysis Software Package of Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, BLAST, BESTFIT, GAP or PILEUP/PRETTYBOX program). Such soft systems align identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and/or other modifications. Reservative substitutions typically include substitutions in the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, aspartic acid , bran acid; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. In an exemplary method of determining the degree of identity, the BLAST software can be used, and when the probability score is between e.sup.-3 to e.sup.-100, it is shown as being close to the relevant sequence.

"lower" or "increase" means relative to the reference (reference) Has a negative or positive change of at least about 10%, 25%, 50%, 75%, or 100%.

"Rom-square root error" means the square root of the arithmetic mean from the square of the error of the mean.

"Reducing cell survival" means inhibiting cell viability or inducing cell death relative to a reference cell.

"Reference" means standard or control conditions.

A "reference sequence" is a defined sequence that uses the basis of sequence alignment. A reference sequence can be a subset or all of a particular sequence; for example, a full-length cDNA or a fragment of a gene sequence, or a complete cDNA or gene sequence. In the case of a multi-peptide, the reference peptide sequence is typically at least about 16 amino acids in length, preferably at least about 20 amino acids, more preferably at least about 25 amino acids, even more preferably. It is about 35 amino acids, about 50 amino acids, or about 100 amino acids. In the case of nucleic acids, the nucleic acid sequence of reference is typically at least about 50 nucleotides in length, preferably at least about 60 nucleotides, more preferably at least about 75 nucleotides, even more preferably about 100 nucleotides. Nucleotide, or about 300 nucleotides, or any integer similar thereto or interposed therebetween.

"Individual" means a mammal, including a human or a non-human mammal, such as, but not limited to, a cow, a horse, a dog, a sheep, or a cat.

"Specific binding" means a compound or antibody that recognizes and binds to the multi-peptide of the present invention, but the compound or antibody does not substantially recognize and bind to other molecules in the sample, for example, the sample naturally includes the present invention. Biological sample of multi-peptide.

The term "hydrogenthio" or "thiol" means -SH.

As used herein, the term "tautomer" means an isomer of an organic molecule that is readily interchangeable by tautomerism in which a hydrogen atom and a proton are tautomeric. Migration in the middle, and sometimes accompanied by the conversion of a single bond and a neighboring double bond.

As used herein, the term "treating" or the like means reducing or ameliorating a disease and/or a symptom associated with the disease. "Improvement" means reducing, inhibiting, attenuating, reducing, preventing, or stabilizing the development or progression of a disease. While not wishing to be limited, it is to be understood that treating a disease or condition does not require the complete elimination of the disease, condition, or symptoms associated therewith.

As used herein, the terms "prevention", "prophylactic treatment" and the like mean reducing the likelihood of an individual suffering from a disease or condition, wherein the individual does not have the disease or condition but is at risk of developing the disease or condition or possibility.

By "effective amount" is meant an amount of a compound that provides a therapeutic effect to the individual being treated. The therapeutic effect can be objective (i.e., can be measured by some test or marker) or subjective (i.e., the effect is indicated by the individual or perceived by the individual). An effective amount of a compound described herein can range from about 1 mg/Kg body weight to about 5000 mg/Kg body weight. The effective dose will also vary with the route of administration and the likelihood of co-administration with other agents.

It is to be understood that the scope of the invention is intended to be a For example, it should be understood that the range of 1 to 50 includes from 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, Any number, combination of numbers, or sub-range of groups of 42, 43, 44, 45, 46, 47, 48, 49, and 50.

As used herein, the term "treating" or the like means reducing or ameliorating a disease and/or a symptom associated with the disease. While not wishing to be limited, it is to be understood that treating a disease or condition does not require the complete elimination of the disease, condition, or symptoms associated therewith.

Unless specifically stated or apparent from the context, it is to be understood that the term "or" as used herein is inclusive. Unless specifically stated or apparent from the context, it is to be understood that the terms "a" and "the" are used in the singular or plural.

The term "about" as used herein is to be understood to be within the normal tolerances of the art, for example, within the standard deviation of the average value, unless specifically stated or apparent from the context. "About" is understood to mean 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05% of the stated value, Or within 0.01% range. All numerical values provided herein are modified by the term "about" unless the context dictates otherwise.

In any definition of a variable herein, the recitation of a listed chemical group includes the definition that the variable is any single group or combination of groups listed. The description of specific examples of the variables or aspects herein includes specific embodiments as any single embodiment or combination with any other specific embodiments or portions thereof.

Any of the compositions or methods provided herein can be combined with any one or more of the other compositions or methods provided herein.

Figure 1 shows the structure of two JQ1 stereoisomers. The C6 position indicated by an asterisk (*) is a stereocenter.

Figures 2A, 2B, and 2C are shown in the cells (-)-JQ1 without competition for binding to BRD4-NUT. Figure 2A shows that the fluorescence recovery after photobleaching (FRAP) of GFP-BRD4 is not affected by (-)-JQ1 (250 nM, 5 hours (h)) compared to the vector control group. influences. Data represent mean ± standard deviation (s.d.) (n = 5). Figure 2B is shown in a parallel comparative study showing that the GFP-BRD4 group demonstrated improved recovery in the presence of (+)-JQ1 (250 nM, 5 h). Data represent mean ± standard deviation (n = 5). Figure 2C provides a quantitative comparison of the mobility of GFP-BRD4 observed in the FRAP study (a, b). Data represent mean ± standard deviation (n = 5) and the p values obtained for the two-tailed t-test were compared against the ligand-treated sample and the vector control group.

Figures 3A to 3J show that JQ1 and chromatin and differentiated human NUT midline tumor cells competitively bind to BRD4. Figure 3A shows that fluorescence recovery (FRAP) after fading of GFP-BRD4 light confirmed an increase in recovery in the presence of JQ1. The nucleus displays a pseudo color in proportion to the fluorescence intensity. The white circle indicates the light faded area. Figures 3B to 3D show GFP-BRD4 transfection (Fig. 3B) and GFP-BRD4-NUT transfection (Fig. 3C) in FRAP experiments, JQ1 accelerates fluorescence recovery, but on nuclear GFP-NUT (p. The fluorescent response of 3D) has no effect. Figure 3E shows a quantitative comparison of the time to the half-maximal fluorescence recovery of the FRAP study (3B to 3D). Data represent mean ± standard deviation (n = 5) and annotate the p-value obtained from the two-tailed t-test. 3F JQ1 (500 nM, 48h) suggests a loss of focus for NUT (anti-NUT; 40x). Figure 3G shows cytological symptoms (H&E, 40x) of squamous differentiation confirmed by NQ cells treated with JQ1 (500 nM, 48 h). Figure 3H shows that differentiation of NMC cells treated with JQ1 (500 nM) is a time-dependent and significantly increased feature in cytokeratin expression (AE1/AE3; 40x). Figures 3I to 3J show rapid proliferation (Ki67; 40x) of JQ1 slowing 797 NMC cell line (Fig. 3I) and Per NMC cell line (Fig. 3J) in vitro.

Figures 4A, 4B and 4C are photomicrographs showing that JQ1 selectively induces squamous differentiation of NUT midline tumor cells. Figure 4A shows cytological symptoms confirmed by JQ1 (500 nM) treated NMC797 cells in a time-dependent squamous manner (as indicated by H&E, 40x and 100x), cell spinning, tiling and opening with chromatin Growth examples. Figure 4B shows that NMC Per403 cells treated with JQ1 (500 nM, 48 h) exhibited comparable squamous differentiation symptoms. Cell spinning and cell keratinization were visualized by H&E and keratin protein staining (40x), respectively. Figure 4C shows that there is no difference in response to JQ1 (500 nM) by plotting non-NMC squamous tumor cell line TE10 by H&E and keratin protein (AE1/AE3) staining (40x).

Figures 4D-a through 4D-b show that JQ1 attenuates NMC cell proliferation. Figure 4D-a shows two micrographs showing that JQ1 slows the rapid proliferation of NMC Per430 cell line in vitro (Ki67; 40x). The image is displayed under the same magnification. Figure 4D-b shows a graph showing the effect of JQ1 on cell proliferation (Ki67 staining and positive; %) as measured by IHC as performed in (4D-a) and 3J. The cell line was manually evaluated as Ki67 with a 5-fold high power field. Positive (dark nuclear staining) or negative (light blue nuclear staining). Data represent mean ± standard deviation (n = 5) and the p-value obtained with the two-tailed t-test is annotated.

Figures 4E-a, b, c, and d show that the induction of squamous differentiation of NUT midline tumor cells treated with JQ1 is stereospecific and time dependent. Figure 4E-a includes six micrographs showing that NMC797 cells treated with (-)-JQ1 (100 mM) on chamber culture slides exhibited comparable cell phenotypes compared to vehicle treated control groups. (+)-JQ1 (100 mM, 48 h) promotes squamous differentiation by cell spinning, tiling, and enhanced expression of keratin. Figure 4E-b shows the NMC797 cell line treated with palmar isomer or vehicle via JQ1 by centrifugation, fixation, sectioning and staining for keratin protein expression (left; AE1/AE3, 20x). At the same time, the image-based analysis of the keratin proteins in the prepared slides was performed using a fair image masking and quantitative calculation that can be used for the evaluation of the staining intensity. Figure 4E-c shows that (+)-JQ1 (250 nM) induces rapid expression of keratin protein in treated NMC797 cells compared to (-)-JQ1 (250 nM) and vector control groups, also by quantitative immunization Determined by histochemistry. Expressed as a percentage positive nuclear for each treatment condition. Data represent mean ± standard deviation (n = 3) and the p-value obtained with the two-tailed t-test is annotated. 4E-d shows that (+)-JQ1 (250 nM) caused a strong time-dependent induction of keratin protein staining (3+) in treated NMC797 cells compared to (-)-JQ1 (250 nM). Expressed as a percentage positive nuclear for each treatment condition. Data represent mean ± standard deviation (n = 3) and the p-value obtained with the two-tailed t-test is annotated. The images of each group are displayed in the same magnification.

Figures 4F-a to c show that squamous tumor cell lines that have not been translocated by BRD4-NUT are less susceptible to those who have been treated with JQ1. Gastrointestinal squamous tumor cell lines (TT and TE10) were cultured for 72 hours in the presence of (+)-JQ1 (250 nM, red nuclei) or (-)-JQ1 (250 nM, black nuclei). The minimal effect observed in cell proliferation with JQ1 is consistent with the rational role of mitotic cells ⁷ in the cell cycle. Data represent mean ± standard deviation (n = 3). IC ₅₀ values were calculated by logistic regression.

Figures 5A through 5K show that JQ1 treatment inhibits proliferation, rapid differentiation, and cell death in vitro and in vivo. Figure 5A shows the growth effect of BRD4 inhibition on BRD4-NUT-dependent cell lines. Cells were cultured with (+)-JQ1 (red nuclei) or (-)-JQ1 (black nuclei) and monitored after 72 hours of proliferation. Data represent mean ± standard deviation (n = 3). The IC ₅₀ value is calculated by Logis regression. Figure 5B shows that the progressive anti-proliferative effect of (+)-JQ1 on NMC797 cells can be confirmed by prolonged periods on days 1, 3, 7 and 10. Data points are mean ± standard deviation (n = 3). Figure 5C shows the flow cytometry results used to represent the DNA content in NMC797 cells. (+)-JQ1 (250 Nm, 48 h) induced G1 arrest compared to (-)-JQ1 (250 nM) and the vector control group. Figure 5D shows the results of flow cytometry analysis of NMC797 squamous tumor cells treated with vehicle, JQ1 or Staurosporine (STA), such as propidium iodide (PI) and annexin- V (annexin-V, AV) indication. Figure 5E shows the PET image results of NMC797 xenograft mice. The FDG uptake of the hind leg of xenografted mice treated with 50 mg (mg) / kg (kg) of JQ1 was reduced compared to the vehicle control group. Figure 5F shows that the mice with established disease (NMC797 xenograft) treated with 50 mg/kg daily JQ1 had a reduced tumor volume system compared to the vehicle control group. A significant response to treatment was observed on day 14 by a two-tailed t-test (p=0.039). Data represent mean ± standard deviation (n = 7), and Figure 5G shows that the histopathological analysis of NMC797 tumors excised from JQ1 treated animals showed an induced keratin protein expression (AE1) compared to vehicle treated animals. /AE) and weakened hyperplasia (Ki67, 40x). The expanded field of view is provided in Figures 11 and 12 below. Figure 5H shows the viability of artificial NMC11060 xenografts confirmed by PET images. Figure 5I shows the therapeutic response of primary 11060 NMC xenografts to (+)-JO1 (50 mg/kg daily for 4 days) by PET imaging. Figure 5J shows that the histopathological analysis of NMC11060 tumors excised from (+)-JQ1 treated animals showed an induction of keratin protein expression (AE1/AE, 20x; scale bar 20 μm) compared to vehicle-treated animals. And weakened hyperplasia (Ki67, 40x). Quantitative analysis of keratin protein induction was performed using image masking (Fig. 5J, right panel) and pixel positive analysis (Fig. 5K). A significant response to treatment was observed by a two-tailed t-test (p = 0.0001). Data represent the mean ± standard deviation of the field of view of three independent wide-angle microscopes. Figure 9 provides a comparative image of the stained resected tumor and the quantified mask.

The 5L-a and 5L-b plots are graphical representations of NJ797 xenograft mice enduring JQ1 treatment, which elicit an anti-tumor effect. Figure 5L-a shows treatment of reduced-load tumors in NMC797 xenograft mice treated with JQ1 (50 mg/kg daily) during 14 days. Data represent mean ± standard deviation (n = 7). Figure 5L-b shows that JQ1 did not produce adverse symptoms or weight loss. Data represent mean ± standard deviation (n = 7).

Figure 5M-a to d shows the NUT midline tumors treated by JQ1 The induction of squamous differentiation of cells is stereospecific and time dependent. Figure 5M-a is a photomicrograph showing that NMC797 cells treated with (-)-JQ1 (100 mM) on chamber culture slides exhibited comparable cell phenotypes compared to vehicle treated control groups. (+)-JQ1 (100 mM, 48 hours) promotes squamous differentiation by cell spinning, tiling, and enhanced expression of keratin. Figure 4M-b is a photomicrograph. NMC797 cell lines treated with palmar isomers or vehicle via JQ1 were centrifuged, fixed, sectioned and stained for keratin expression (left; AE1/AE3, 20x). At the same time, the image-based analysis of the keratin proteins in the prepared slides was performed using a fair image masking and quantitative calculation that can be used for the evaluation of the staining intensity. Figure 5M-c shows that (+)-JQ1 (250 nM) induces rapid expression of keratin protein in treated NMC797 cells compared to (-)-JQ1 (250 nM) and vector control groups, which is quantified by quantitative immunization Determined by histochemistry. Expressed as a percentage positive nuclear for each treatment condition. Data represent mean ± standard deviation (n = 3) and the p-value obtained with the two-tailed t-test is annotated. The 5M-d plot showed that (+)-JQ1 (250 nM) caused a strong time-dependent induction of keratin protein staining (3+) in treated NMC797 cells compared to (-)-JQ1 (250 nM). Expressed as a percentage positive nuclear for each treatment condition. Data represent mean ± standard deviation (n = 3) and the p-value obtained with the two-tailed t-test is annotated. The images of each group are displayed in the same magnification.

Figures 6A and 6B-a through 6B-e provide micrographs showing in vivo JQ1 initiation of squamous differentiation, growth arrest, and apoptosis as measured by IHC. Histopathological analysis of NMC tumors excised from animals treated with JQ1 (right panel) compared to vehicle-treated animals (left panel) Proteoglycan differentiation (H&E, 40x), nuclear NUT disappearance (NUT, 100x), attenuated hyperplasia (Ki67, 40x), induced keratin protein expression (AE1/AE3, 40x), and apoptotic response (TUNEL, 40x).

Figures 7A and 7B show that JQ1 selectively induces apoptosis of NMC in a human squamous cell cell line. Figure 7A provides the results of the FACS analysis. NMC Per403 cells treated with JQ1 (500 nM, 24 or 48 hours) exhibited induced apoptosis by flow cytometry compared to non-NMC squamous cell lines TE10 and TT. PI is propidium iodide and AV is annexin-V. Figure 7B shows the quantification and comparison of Annexin-V positive cells by flow cytometry as described in Figures 5B and A. Compared to NMC cell lines and non-NMC squamous cell lines, JQ1 (500 nM) was induced to induce apoptosis in a time-dependent manner. Data represent mean ± standard deviation (n = 3) and the p-value obtained with the two-tailed t-test is annotated.

Figures 8A through 8C are graphical representations showing that the NMC artificial tissue is sensitive to the anti-proliferative effect of JQ1 in vitro. Figure 8A shows the results from analysis derived from artificial NMC11060 cells. The cells were isolated from discarded clinical material and grown in short-term culture for in vitro studies. The anti-proliferative effect on BRD4 inhibition of NMC11060 cells was measured after 72 hours of culture with (+)-JQ1 (red nuclei) or (-)-JQ1 (black nuclei). NMC11060 cells are uniquely sensitive to (+)-JQ1 to palmitic isomers. Data represent mean ± standard deviation (n = 3). The IC ₅₀ value is calculated by Logis regression. Figure 8B shows that the progressive anti-proliferative effect of (+)-JQ1 on artificial NMC11060 cells can be confirmed by prolonging the time on days 1, 3, 7 and 10. Data points are mean ± standard deviation (n = 3). Figure 8C shows the flow cytometry results used to represent the DNA content in NMC11060 cells. (+)-JQ1 (250 Nm, 48 hours) induced G1 block compared to (-)-JQ1 (250 nM) and the vector control group.

Figures 9A and 9B are quantifications of extensive and intense keratin protein expression induced by JQ1 in artificial NMC11060 primary xenograft tumors. Figure 9A shows a low magnification (0.8x) image (AE1/AE3; left) taken from NMC11060 primary xenograft tumors excised from vehicle-treated animals, sectioned and stained for keratin protein expression. The overall staining of keratin proteins is light in all tumors of the section. Image-based analysis of keratin expression is accomplished using fair image occlusion and quantitative calculus that can be used to evaluate the intensity of individual pixels (right). Figure 9B shows NMC11060 primary xenograft tumors excised from (+)-JQ1 treated animals (50 mg/kg daily for 4 days), sectioned and stained for low magnification of keratin protein expression (0.8 x) Image (AE1/AE3; left). Extensive staining was consistent with the observed uniformity of keratin protein induction in JQ1-treated tumors. Image-based analysis of keratin expression is accomplished using fair image occlusion and quantitative calculus that can be used to evaluate the intensity of individual pixels (right). The pixels were quantitatively positive and visually described as blue (0), yellow (1+), orange (2+), and red (3+). All pairs of images are displayed under the same magnification

Figure 10 shows that JQ1 exhibits excellent oral bioavailability and exposure to appropriate drug motility in rodents. The pharmacokinetic study of (+)-JQ1 was performed by intravenous administration of CD1 mice (Fig. 10A, B) and oral administration (Fig. 10B, C). Figure 10A is a graph of mean plasma concentration-time profiles of (+)-JQ1 after intravenous administration (5 mg/kg). Data represent mean ± standard deviation (n = 3). Figure 10B shows the calculated pharmacokinetics of intravenous (+)-JQ1 demonstrating excellent drug exposure [area under the curve (AUC) = 2090 hr * ng/mL] and approximately one hour half-life (T _1/2 ) parameter. Figure 10C is a graph showing the mean plasma concentration-time profile of (+)-JQ1 after oral administration (10 mg/kg). Data represent mean ± standard deviation (n = 3). Figure 10D shows that oral (+)-JQ1 demonstrated excellent oral bioavailability (F = 49%), peak plasma concentration ( _Cmax = 1180 ng/mL), and drug exposure (AUC = 2090 hr * ng / mL). Calculated pharmacokinetic parameters. CL is the clearance rate; Vss is the volume in the steady state; MRT _INF is the average residence time pushed to infinity; _Tmax is the time to the maximum concentration.

Figure 11 is a graph showing the AlphaScreen binding data of JQ1 against palm isomer inhibition BRS4.1.

Figure 12 is a graph showing the AlphaScreen binding data of JQ1 against palm isomer inhibition BRS4.2.

Figure 13 is a graph showing the data representation of JQ1 against other BET family members (i.e., BET bromine-containing domain domains) (activity) and CBP (inactive).

Figure 14 is a graph showing the dose-range study of the JQ1 derivative for a pool of BRD4.1 and BRD4.2 inhibition.

Figures 15A and 15B are graphs. Figure 15A is shown in a xenograft mouse model of Nut midline tumors, and JQ1 reduces tumor burden. Figure 15B shows the average body weight of mice treated with JQ1.

Figures 16A to 16D are graphical representations showing inhibition of binding activity of BRD4 (1) and BRD4 (2) of JQ1 and its derivatives at 5 μM.

Figures 17A through 17D are graphical representations showing the effect of JQl and its derivatives on the % viability of Nut midline tumor cell (NMC) cells at 2 [mu]M.

Figures 18 to 55 show the dose response to viability of various cancer cell lines treated with JQ1 and its derivatives.

Figures 56A through 56D are graphical representations showing the results of the lead compound binding assay. Figure 56A is a graphical representation showing the results of a lead compound binding assay performed with BRD4.1. Figure 56B is a graphical representation showing the results of a lead compound binding assay performed with BRD4.2. Figure 56C shows a graphical representation of the results of a lead compound binding assay performed on a 797 cell line. Figure 56D shows a graphical representation of the results of a lead compound binding assay performed on a 10326 cell line.

The composition and method of the present invention are useful for treating or preventing tumor formation, inflammatory diseases, obesity, fatty liver (NASH or others), diabetes, atherosclerosis, arterial stent obstruction, heart failure, dysentery, graft It is resistant to host diseases, and infections associated with bromodomain-related infectious diseases, parasites, malaria, trypanosomes, and for reducing male fertility. Further uses of the compositions of the present invention include, but are not limited to, organ transplantation, regulation of cellular states for regenerative medicine (i.e., by promoting or inhibiting fine differentiation), and promoting differentiation pluripotency.

The present invention is based, at least in part, on the discovery of a BET-family with respect to a bromine-containing domain domain and the ability to modulate the bromine-containing domain domain family, which contains an ethionyl-isoamino acid residue that recognizes nuclear chromatin. More bromine-containing domains A related compound of the peptide family) is a biochemically selective cell-permeable, potent small molecule inhibitor (JQ1). Signal modification, which is widely associated with cell and disease biology, occurs with acetoacetate. The development of pharmaceutically active regions for many years has been the characterization of reversible enzymes that regulate side chain acetylation. To date, efforts have been made to limit the co-modification of "codons" and "eraser" (histone deacetylase) to the covalent modification of nuclear chromatin. However, there have been no descriptions of the acetyl-isamino acid recognition component or a protein inhibitor containing a bromine domain. A recent feature of the high resolution eutectic structure containing BRD4 reveals an excellent appearance that matches the acetamido- lysine-binding pocket. The JQ1 line competitively binds to ethionyl-isoamine to BRD4 which binds to the bromo-containing domain of BRD4 and replaces chromatin in human cells. Periodic translation of BRD4 was observed in subtypes of gene definitions of incurable human squamous tumors. The competitive binding of JQ1 to the BRD4 fusion oncogenic protein results in immediate squamous differentiation and specific anti-proliferation effects in BRD4-dependent tumors derived from artificial cell lines and murine models. These data confirm the feasibility of calibrating the epigenetic "epigenetic reader" protein-protein interactions and report general chemical scaffolds for the development of chemical probes that are broader in the bromine-containing domain family.

Protein containing bromine domain

Gene regulation is largely determined by reversible, non-covalent assembly of macromolecules. Signal transduction of RNA polymerase requires a higher array of protein complexes, by being able to interpret the regulatory state spatial regulation of chromatin translation. Epigenetic readers are structurally diverse proteins each having one or more evolutionarily conserved effector components that recognize histone proteins or DNA Covalent modification. The ε-N-acetylation (Kac) of the lysine residue at the caudal end of the histone is associated with open chromatin structure and transcriptional activation. The specific specific molecular regulation of ethionyl-isoamine is mainly regulated by the bromine-containing domain.

Proteins containing a bromo-containing domain are essentially biologically interesting, as components of transcription factor complexes (TAF1, PCAF, Gcn5, and CBP) and determine epigenetic memory. There are 41 human proteins containing a total of 57 multiple changes in the bromine-containing domain. In spite of the large sequence variation, all bromodomain-containing domain exclins include four left-handed α-helix clusters (α _Z , α _A , α _B , by multivariate cyclic regions (ZA and BC regions). Conservative folding of α _C ), which determines the specificity of the substrate. The co-crystal structure containing the peptide acceptor indicates that the ethionyl-isoamine acid is recognized by a central hydrophobic pocket and by residues of aspartic acid present in most of the bromine-containing domains The hydrogen bond of the base is anchored to the anchor ⁵ . The bromine-containing domain and the bromine-containing domain additional terminal (BET) family (BRD2, BRD3, BRD4, and BRDT) occupying a consensus domain architecture exhibit a high degree of sequence preservation and a more divergent C-terminally related domain ( Figure 1E) ⁶ .

BRD4

A strong and fundamental principle has been established in cancer research to calibrate BRD4. BRD4 is functional in promoting cell cycle and G1 arrest in cancer cells that have been knocked out of gene culture. BRD4 is an important regulator of transcriptional elongation and functions as a complement to the forward transcription factor complex (P-TEFb) ^7,8 . Cyclin dependent kinase -9 (P-TEFb central component) is a chronic lymphocytic haemophilia ⁹ valid target, and observation links to c-Myc-dependent transcription. The bromo-containing domain present in BRD4 complements the P-TEFb to the mitotic chromosome to increase the performance of the growth-promoting gene. BRD4 remains bound to the transcriptional start of the expression gene during the M/G1 cell cycle and does not occur at the onset of expression after the cell cycle. It has been shown that deletion of BRD4 in proliferating cells results in G1 arrest and apoptosis due to reduced levels of mitosis ¹³ and survival ¹⁴ important genes ¹² .

More importantly, BRD4 has recently been identified as human malignant squamous tumors period t (15; 19) translocation component chromatin fragments ^15,16. This translocation indicates a tandem N-terminal bromodomain containing domain of BRD4 as an in-frame chimera with a NUT (nuclear protein in testis) protein, genetically defined as a NUT midline tumor (NMC). Functional studies derived from NMC artificial cell lines have confirmed that BRD4-NUT oncoproteins play an important role in maintaining the dominant proliferative advantage and blocking the differentiation of this evenly fatal malignant tumor ¹⁷ . In particular, RNA silencing of the BRD4-NUT gene inhibits proliferation and promotes squamous differentiation and significantly increases cytokeratin expression. These observations highlight the broad availability and immediate therapeutic potential of direct action inhibitors of human bromodomain-containing proteins.

The characteristic compositions and methods of the present invention are useful for inhibiting human bromodomain-containing domain proteins.

Compound of the invention

The invention provides compounds (e.g., JQ1 and compounds of the formulae described herein) that bind to a binding pocket of the apo crystal structure of the first bromine-containing domain of a BET family member (e.g., BRD4). Without wishing to be bound by theory, these compounds are particularly effective in inhibiting the growth, proliferation or survival of proliferating retinal cells or inducing the differentiation of proliferating retinal cells. Chemical. In one method, the compound is useful for treating tumor formation, inflammatory diseases, obesity, fatty liver (NASH or others), diabetes, atherosclerosis, arterial stent obstruction, heart failure, dysentery, graft versus host disease And bromine-containing domain-associated infectious diseases, treatment of parasites, malaria, trypanosomes, and regulation of cell status for reducing male fertility; or for organ transplantation, for regenerative medicine (ie, by Promoting or inhibiting fine differentiation), and selectively using a molecular docking program to promote differentiation pluripotency to identify binding pockets for which the compound is expected to bind to the bromine-containing domain structure. In certain embodiments, a compound of the invention can bind to a BET family member and reduce the biological activity of the BET family member (eg, reduce prolongation) and/or disrupt subcellular localization of the BET family member (eg, reduce staining) Quality combination).

In certain embodiments, the compounds of the invention may prevent, inhibit, or disrupt or reduce at least 10%, 25%, 50%, 75%, or 100% of BET family members (eg, BRD2, BRD3, BRD4, BRDT) Biological activity and/or disruption of the protein subcellular localization, for example, by binding to the binding site of the apo binding pocket of the bromine containing domain.

In certain embodiments, the compounds of the invention have a molecular weight of less than about 1000 daltons, less than 800, less than 600, less than 500, less than 400, and less than about 300 ears. Small molecules swallowed. Examples of the compound of the present invention include JQ1 and other compounds of a binding pocket of apo crystal structure bound to the first bromine-containing domain of a BET family member (for example, BRD4) (hereinafter referred to as BRD4(1); PDB ID 2OSS ). JQ1 is a novel thieno-triazolo-1,4-diazepine. The invention further provides the compound A pharmaceutically acceptable salt.

In one aspect, the invention provides a compound of formula (I), or a salt, solvate, or hydrate thereof: Wherein X is N or CR ₅ ; R ₅ is H, alkyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl, each of which is optionally substituted; R _B is H, alkyl, hydroxy An alkyl group, an aminoalkyl group, an alkoxyalkyl group, a haloalkyl group, a hydroxyl group, an alkoxy group, or a -COO-R ₃ group, each of which is optionally substituted; the ring A is an aryl group or a heteroaryl group; A is independently alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, each of which is optionally substituted; or any two R _A together with their respective attached atoms may form a fused aryl group or a fused heteroaryl; R is alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, each of which is optionally substituted; R ₁ is -(CH ₂ ) _n -L, wherein n 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 substituted aryl, or optionally substituted heteroaryl; R ₂ is H, D (氘), halogen, or an alkyl group optionally substituted; each R _{3 is} independently selected from the group consisting of: (i) H, aryl, substituted aryl, heteroaryl, or substituted Aryl; (ii) The cycloalkyl group or substituted heterocycloalkyl; (iii) -C ₁ -C ₈ alkyl, -C ₂ -C ₈ alkenyl or -C ₂ -C ₈ alkynyl each containing 0, 1, 2 or 3 heteroatoms selected from O, S, or N, substituted -C ₃ -C ₁₂ cycloalkyl, -C ₃ -C ₁₂ cycloalkenyl, or substituted -C ₃ -C ₁₂ a cycloalkenyl group, each of which may be optionally substituted; and (iv) NH ₂ , N=CR ₄ R ₆ ; each R _{4 is} independently H, alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl , each of which is optionally substituted; or R ₃ and R ₄ together form a 4 to 10 membered ring with the nitrogen atom to which they are attached; R ₆ is alkyl, alkenyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, An aryl group or a heteroaryl group, each of which is optionally substituted; or R ₄ and R ₆ together form a 4 to 10 membered ring with the carbon atom to which they are attached; m is 0, 1, 2, or 3; : (a) if ring A is a thienyl group, X is N, R is a phenyl group or a substituted phenyl group, R ₂ is H, R _B is a methyl group, and R ₁ is -(CH ₂ ) _n -L, Wherein n is 1 and L is -CO-N(R ₃ R ₄ ), then R ₃ and R ₄ do not form a morpholine ring together with the nitrogen atom to which they are attached; (b) if ring A is a thienyl group, X is N, R is substituted phenyl R ₂ is H, R _B is methyl, and 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 are} In the case of H, the remainder of R ₃ and R ₄ are not methyl, hydroxyethyl, alkoxy, phenyl, substituted phenyl, pyridyl or substituted pyridyl; and (c) Ring A is a thienyl group, X is N, R is a substituted phenyl group, R ₂ is H, R _B is a methyl group, and R ₁ is -(CH ₂ ) _n -L, wherein n is 1 and L is - COO-R ₃ , then R _{3 is} not methyl or ethyl.

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

In certain embodiments, L is H, -COOR 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 an optionally substituted aryl group. In certain embodiments, each R _{3 is} independently selected from the group consisting of H, optionally substituted -C ₁ -C ₈ alkyl, which contains 0, 1, 2, or 3 a hetero atom selected from O, S, or N; or NH ₂ , N=CR ₄ R ₆ .

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

In certain embodiments, R _B is alkyl, hydroxyalkyl, haloalkyl, or alkoxy, each of which is 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 group. In certain embodiments, Ring A is thiofuranyl, phenyl, naphthyl, biphenyl, tetrahydronaphthyl, indanyl, pyridyl, furyl, indolyl, pyrimidinyl, indole Base Base, imidazolyl, oxazolyl, thienyl, thiazolyl, triazolyl, isoxazolyl, quinolyl, 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, R _A is methyl and at least one occurrence.

In certain embodiments, each R _{A is} independently H, optionally substituted aryl, or any two R _A, along with their respective attached atoms, can form an aryl group.

In another aspect, the invention provides a compound of formula (II), a salt, solvate thereof, or hydrate thereof: Wherein X is N or CR ₅ ; R ₅ is H, alkyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl, each of which is optionally substituted; R _B is H, alkyl, hydroxy An alkyl group, an aminoalkyl group, an alkoxyalkyl group, a haloalkyl group, a hydroxyl group, an alkoxy group, or a -COO-R ₃ group, each of which is optionally substituted; each ring A is independently an alkyl group, a cycloalkyl group, Heterocycloalkyl, aryl, or heteroaryl, each of which is optionally substituted; or any two R _A together with the atoms to which they are attached may form a fused aryl or fused heteroaryl; a cycloalkyl, heterocycloalkyl, aryl, or heteroaryl group, each of which is optionally substituted; R' ₁ is H, -COO-R ₃ , -CO-R ₃ , optionally substituted aryl, Or a heteroaryl group optionally substituted; each R _{3 is} independently selected from the group consisting of: (i) H, an aryl group, a substituted aryl group, a heteroaryl group, a substituted heteroaryl group; a heterocycloalkyl or substituted heterocycloalkyl; (iii) -C ₁ -C ₈ alkyl, -C ₂ -C ₈ alkenyl or -C ₂ -C ₈ alkynyl, each containing 0, 1 , 2, or 3 heteroatoms selected from O, S, or N of the hetero atom, -C ₃ -C ₁₂ cycloalkyl, substituted -C ₃ -C ₁₂ cycloalkyl of Group, -C ₃ -C ₁₂ cycloalkenyl group, or the substituted -C ₃ -C ₁₂ cycloalkenyl group, each optionally substituted; m is 1, 2, or 3; with the proviso that when R ' ₁ is -COO-R ₃ , X is N, R is a substituted phenyl group, and R _B is a methyl group, and R _{3 is} not a methyl group or an ethyl group.

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

In certain embodiments, R _'1 is -COO-R _3, the aryl group optionally substituted, or an optionally substituted aryl group the heteroaryl; and R ₃ is -C ₁ -C ₈ alkyl, containing 0 One, two, or three heteroatoms selected from O, S, or N, and which may be substituted as needed. In certain embodiments, R' ₁ is -COO-R ₃ and R ₃ is methyl, ethyl, propyl, isopropyl, butyl, t-butyl, or t-butyl; R' ₁ is H or a phenyl group which is optionally substituted.

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 alkyl group optionally substituted, or any two R _A together with their respective attached atoms may form a fused aryl group.

In certain embodiments, each R _A is a methyl group.

In another aspect, the invention provides a compound of formula (III), a salt, solvate, or hydrate thereof: Wherein X is N or CR ₅ ; R ₅ is H, alkyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl, each of which is optionally substituted; R _B is H, alkyl, hydroxy An alkyl group, an aminoalkyl group, an alkoxyalkyl group, a haloalkyl group, a hydroxyl group, an alkoxy group, or a -COO-R ₃ group, each of which is optionally substituted; the ring A is an aryl group or a heteroaryl group; A is independently alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, each of which is optionally substituted; or any two R _A together with their respective attached atoms may form a fused aryl group or a fused heteroaryl; R is alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, each of which is optionally substituted; each R _{3 is} independently selected from the group consisting of: ( i) H, aryl, substituted aryl, heteroaryl, or substituted heteroaryl; (ii) heterocycloalkyl or substituted heterocycloalkyl; (iii)-C ₁ -C ₈ An alkyl group, a -C ₂ -C ₈ alkenyl group or a -C ₂ -C ₈ alkynyl group, each containing 0, 1, 2 or 3 heteroatoms selected from O, S, or N, -C ₃ - C ₁₂ cycloalkyl group, the substituted -C ₃ -C ₁₂ -cycloalkyl, -C ₃ -C ₁₂ cycloalkenyl group, or the substituted -C ₃ -C ₁₂ cycloalkenyl group Each optionally substituted; and _{(iv) NH 2, N =} CR 4 R 6; each R ₄ is independently H, alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, each system Substituted as desired; or R ₃ and R ₄ together form a 4 to 10 membered ring with the nitrogen atom to which they are attached; R ₆ is alkyl, alkenyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, aryl, or a heteroaryl group, each of which is optionally substituted; or R ₄ and R ₆ together form a 4 to 10 membered ring with the carbon atom to which they are attached; m is 0, 1, 2, or 3; the limitation is: (a) If ring A is a thienyl group, X is N, R is a phenyl group or a substituted phenyl group, and R _B is a methyl group, then R ₃ and R ₄ do not form a morpholine ring together with the nitrogen atom to which they are attached; b) if ring A is a thienyl group, X is N, R is a substituted phenyl group, R ₂ is H, R _B is a methyl group, and one of R ₃ and R ₄ is H, then R ₃ and R ₄ The remainder are not methyl, hydroxyethyl, alkoxy, phenyl, substituted phenyl, pyridyl or substituted pyridyl.

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

In certain embodiments, R is p-Cl-phenyl, o-Cl-phenyl, m-Cl-phenyl, pF-phenyl, oF-phenyl, mF-phenyl or pyridyl. 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 of which is optionally substituted.

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

In another aspect, the invention provides a compound of formula (IV), a salt, solvate, or hydrate thereof: Wherein X is N or CR ₅ ; R ₅ is H, alkyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl, each of which is optionally substituted; R _B is H, alkyl, hydroxy An alkyl group, an aminoalkyl group, an alkoxyalkyl group, a haloalkyl group, a hydroxyl group, an alkoxy group, or a -COO-R ₃ group, each of which is optionally substituted; the ring A is an aryl group or a heteroaryl group; A is independently alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, each of which is optionally substituted; or any two R _A together with their respective attached atoms may form a fused aryl group or A fused heteroaryl group; R ₁ is -(CH ₂ ) _n -L, wherein 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 , as needed a substituted aryl group, or a heteroaryl group optionally substituted; R ₂ is H, D, halogen, or an alkyl group optionally substituted; each R _{3 is} independently selected from the group consisting of: (i) H, Aryl, substituted aryl, heteroaryl, or substituted heteroaryl; (ii) heterocycloalkyl or substituted heterocycloalkyl; (iii) -C ₁ -C ₈ alkyl, - C ₂ -C ₈ alkenyl or -C ₂ -C ₈ alkynyl group, Containing from 0, 1, 2, or 3 heteroatoms selected from O, S, or N heteroatom of, -C ₃ -C ₁₂ cycloalkyl, substituted -C ₃ -C ₁₂ cycloalkyl the alkyl, -C _3- C ₁₂ cycloalkenyl, or substituted -C ₃ -C ₁₂ cycloalkenyl, each of which may be optionally substituted; and (iv) NH ₂ , N=CR ₄ R ₆ ; each R _{4 is} independently H, alkane a group, a cycloalkyl group, a heterocycloalkyl group, an aryl group, or a heteroaryl group, each of which is optionally substituted; or R ₃ and R ₄ together with a nitrogen atom to which they are attached form a 4 to 10 membered ring; R ₆ is an alkane a base, alkenyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, aryl, or heteroaryl group, each of which is optionally substituted; or R ₄ and R ₆ together with the carbon atom to which they are attached form 4 to 10 Member ring; m is 0, 1, 2, or 3; the restrictions are: (a) if ring A is thienyl, X is N, R ₂ is H, R _B is methyl, and R ₁ is -( CH ₂ ) _n -L, wherein n is 0 and L is -CO-N(R ₃ R ₄ ), then R ₃ and R ₄ do not form a morpholine ring together with the nitrogen atom to which they are attached; (b) if ring A is a thienyl group, X is N, R ₂ is H, R _B is a methyl group, and R ₁ is -(CH ₂ ) _n -L, wherein n is 0 and L is -CO-N(R ₃ R ₄ ) and one of R ₃ and R ₄ are H, then R ₃ and R ₄ of which Are not methyl, hydroxyethyl, alkoxy, phenyl, the substituted phenyl, pyridyl or substituted pyridyl group of; and (c) if ring A is thienyl, X is N, R ₂ is H, R _B is methyl, and R ₁ is -(CH ₂ ) _n -L, wherein n is 0 and L is -COO-R ₃ , then R _{3 is} not methyl or ethyl.

In certain embodiments, R ₁ is —(CH ₂ ) _n —L, wherein n is 0 to 3 and L is —COO—R ₃ , optionally substituted aryl, or optionally substituted. And R ₃ is -C ₁ -C ₈ alkyl group containing 0, 1, 2 or 3 heteroatoms selected from O, S, or N, and which may be substituted as needed. In certain embodiments, n is 1 or 2 and L is alkyl or -COOR ₃ and R ₃ is methyl, ethyl, propyl, isopropyl, butyl, second butyl, or Tributyl; or n is 1 or 2 and L is H or a phenyl group optionally substituted.

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, furyl, indolyl, pyrimidinyl, indole Base Base, imidazolyl, oxazolyl, thienyl, thiazolyl, triazolyl, isoxazolyl, quinolyl, pyrrolyl, pyrazolyl, or 5,6,7,8-tetrahydroisoquinolinyl .

In certain embodiments, each ring A is independently an alkyl group that is optionally substituted, or any two R _A, along with their respective attached atoms, can form an aryl group.

The invention also provides compounds of formula (V-XXII) and compounds described herein.

In another aspect, the present invention provides a compound represented by the formula: a salt, a solvate thereof, or a hydrate thereof:

In certain embodiments, the compound is (+)-JQ1, a salt, solvate, or hydrate thereof:

In another aspect, the present invention provides a compound represented by the formula: a salt, a solvate thereof, or a hydrate thereof:

In another aspect, the present invention provides a compound represented by the formula: a salt, a solvate thereof, or a hydrate thereof:

In another aspect, the invention provides a compound, a salt, a solvate thereof, or a hydrate thereof, represented by any one of the following formulae:

In another aspect, the invention provides a compound, a salt, a solvate thereof, or a hydrate thereof, represented by any one of the following formulae:

In another aspect, the invention provides a compound, a salt, a solvate thereof, or a hydrate thereof, represented by any one of the following formulae: In certain embodiments, the compounds of the invention may be represented by any of the following structures:

In one embodiment, the compound is a lower structure, a salt, a solvate thereof, or a hydrate thereof: In another embodiment, the compound is a lower structure, a salt, a solvate thereof, or a hydrate thereof:

In another embodiment, the compound is a lower structure, a salt, a solvate thereof, or a hydrate thereof:

In certain embodiments, the compounds of the invention may have the relative palmarity of any of the compounds shown herein.

In certain embodiments, the invention provides a compound, salt, solvate, or hydrate thereof, of formula (V), (VI), or (VII): Wherein R, R ₁ , and R ₂ and R _{3 have} the same meanings of formula (I); Y is O, N, S, or CR ₅ , wherein R _{5 has} the same meaning as in formula (I); n is 0. Or 1; and the dotted circle in the formula (VII) represents an aromatic ring or a non-aromatic ring.

In certain embodiments of any of Formulas (I-IV) and (VI) (or any of the formulae herein), R ₆ represents a non-carbonyl moiety of the aldehyde shown in Table A below (ie, for Formula R ₆ In the case of an aldehyde of CHO, R ₆ is a non-carbonyl moiety of the aldehyde. In certain embodiments, R ₄ and R ₆ together represent a non-carbonyl moiety of the ketone shown in Table A below (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 a specific embodiment, the compound is represented by the following formula (III), a salt, a solvate thereof, or a hydrate thereof:

In certain embodiments, the compound is (racemic) JQ1; in certain embodiments, the compound is (+)JQ1. In certain embodiments, the compound is a compound selected from the group consisting of: or a salt, solvate, or hydrate thereof:

Additional examples of such compounds include compounds, salts, solvates, or hydrates thereof, according to any of the following formulae:

In the formula (IX-XXII), R and R' may be H, aryl, substituted aryl, 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 -C ₃ -C ₁₂ cycloalkenyl groups, each of which may be substituted as needed. In formula (XIV), X can be any substituent of the aryl groups described herein.

The compounds of the present invention can be prepared by a variety of methods, some of which are well known in the art. For example, the chemical examples provided below provide a synthetic scheme for the preparation of compound JQ1 (as a racemate) and stereoisomers (+)-JQ1 and (-)-JQ1 (see Schemes S1 and S2). A wide variety of compounds of formula (I) to (XXII) can be prepared in a similar manner using substitution of the appropriate starting materials.

For example, as shown in the following scheme 1, starting from JQ1, a similar preparation can be made. Amine.

As shown in Scheme 1, hydrolysis of the third butyl ester of IQ1 is provided to the carboxylic acid, which is treated with diphenylphosphorus azide (DDPA) and subjected to Curtius rearrangement to provide a Cbz protected amine, and then Protection produces amines. The amine is then refined, for example, by a reductive amination reaction to produce a secondary amine which can be further alkylated to provide a tertiary amine.

Scheme 2 shows the synthesis of a further embodiment of a compound of the invention (e.g., Formula I of the invention) wherein the center of the fused ring is modified (e.g., by a different aromatic ring (e.g., ring A in Formula I) Substitution). The use of an aminodiaryl ketone having an appropriate functionality (hereinafter, for example, instead of the aminodiaryl ketone S2 in Scheme S1) provides a novel compound having a plurality of fused ring centers and/or aryl groups (corresponding to Group I of R). The aminodiaryl ketones are commercially available or can be prepared by a variety of methods, some of which are well known in the art.

Scheme 3 provides an additional exemplary synthetic scheme for the preparation of further compounds of the invention.

As shown in Scheme 3, the fused bicyclic precursor (see Scheme S1, below, for the synthesis of this compound) is functionalized with a portion of R (DAM = dimethylaminomethyl) and then reacted with hydrazine. It is a tricyclic fused ring. The substituent R _x can be varied by selecting an appropriate hydrazine.

Additional examples of compounds of the invention which may be prepared by the methods described herein include: guanamine: guanamine may be prepared, for example, by the corresponding carboxylic acid or ester, followed by standard conditions and appropriate amines. Prepared by amidination. In certain embodiments, the guanamine provides a terminal nitrogen-containing terminal ring (eg, pyridyl, piperidinyl, piperidine) Two carbon "linkers" of the group, imidazolyl (including N-methyl-imidazolyl), morpholinyl, etc.). Exemplary guanamine structures include:

Preferably, two carbon linkers between the guanamine moiety and the terminal nitrogen-containing ring are used.

"Reverse guanamine":

Secondary guanamine:

Boric acid:

In certain embodiments, a compound having at least one chiral center is present in racemic form. In certain embodiments, a compound having at least one chiral center is enriched in a mirror stereoisomer, ie, having at least about 10%, 20%, 30%, 40%, 50%, 60%, 70 %, 80%, 85%, 90%, 90%, 95%, 99%, 99% or 100% of the excess of the enantiomeric excess (ee). In certain embodiments, the compound having the same absolute configuration is the compound (+)-JQ1 ((S)-2-(4-(4-chlorophenyl)-2,3,9- described herein). Trimethyl-6H-thienyl [3.2-f][1,2,4]triazolo[4,3-a][1,4]diazepine-6-yl)acetic acid tert-butyl ester) .

In certain embodiments of any of the formulae described herein, the compound is not represented by the following structures: Wherein R' ₁ is C ₁ -C ₄ alkyl; R' ₂ is hydrogen, halogen, or a C ₁ -C ₄ alkyl group optionally substituted by a halogen atom or a hydroxyl group; R' ₃ is a halogen atom, optionally a halogen atom, a C ₁ -C ₄ alkyl group, a C ₁ -C ₄ alkoxy group, or a cyano group-substituted phenyl group; -NR ₅ -(CH ₂ ) _m -R ₆ wherein 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 halogen atom; or -NR ₇ -CO--(CH ₂ ) _n -R ₈ wherein R ₇ is a hydrogen atom or a C ₁ -C ₄ alkyl group, n is an integer of 0 to 2, and R ₈ is a phenyl group or a pyridyl group optionally substituted by a halogen atom; and R' ₄ is -(CH ₂ ) _a -CO-NH-R ₉ wherein a is an integer from 1 to 4, and R ₉ is C ₁ -C ₄ alkyl; C ₁ -C ₄ hydroxymethyl; C ₁ -C ₄ alkoxy; a phenyl group or a pyridyl group or -(CH ₂ ) _b -COOR ₁₀ substituted by a C ₁ -C ₄ alkyl group, a C ₁ -C ₄ alkoxy group, an amine group or a hydroxyl group, wherein b is 1 to 4 An integer, and R ₁₀ is C ₁ -C ₄ alkyl.

In certain embodiments, the compound is not JQ1, (+)-JQ1, or (-)-JQ1. In certain embodiments, the compound is not a JQ1 inhibitor or JQ1-FITC conjugate.

The term "pharmaceutically acceptable salt" means a salt prepared from a compound disclosed herein (eg, JQ1, a compound of Formula I-XXII) or any other compound described herein, having an acidic functional group (eg, a carboxylic acid) Functional group) and a pharmaceutically acceptable inorganic or organic base. Suitable bases include, but are not limited to, alkali metal hydroxides such as sodium, potassium, and lithium; alkaline earth metal hydroxides such as calcium and magnesium; other metal hydroxides such as aluminum and zinc; aqueous ammonia, and organic amines Substituted - or hydroxy substituted - mono-, di-, or trialkylamine; dicyclohexylamine; tributylamine; pyridine; N-methyl, N-ethylamine; diethylamine Triethylamine; mono-, di-, or gin-(2-hydroxy-lower alkylamine), For example, mono-, di-, or cis-(2-hydroxy-hydroxyethyl)-amine, 2-hydroxy-tert-butylamine, or cis-(hydroxymethyl)methylamine; N,N,-di Lower alkyl-N-(hydroxyl lower alkyl)-amine, such as N,N,-dimethyl-N-(2-hydroxyethyl)-amine, or tris-(2-hydroxyethyl)amine; N - methyl-D-reduced glucosamine; and amino acids such as arginine, lysine, and the like. The term "pharmaceutically acceptable salt" means a salt prepared from a compound disclosed herein or any other compound described herein having a basic functional group (eg, an amine functional group) and a pharmaceutically acceptable mineral acid. Or organic acids. Suitable acids include, but are not limited to, sulfuric 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. , benzenesulfonic acid, fumaric acid, gluconic acid, glucuronic acid, glucose diacid, formic acid, benzoic acid, glutamic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid and p-toluenesulfonic acid.

Virtual screening method and system

In another aspect, the invention provides a machine for reading a storage medium comprising the structural coordinates of a binding site of a BET family member polypeptide (eg, a BRD4 domain) identified herein. This is the proposed combination of JQ1. A storage medium encoded with such data can display a three-dimensional graphical representation of a molecule or a molecular complex comprising such binding sites on a computer screen or similar viewing device.

The invention also provides methods for designing, evaluating and confirming compounds that bind to the aforementioned binding sites. Such compounds are expected to inhibit the biological activity of BET family members (eg, BRD2, BRD3, BRD4, BRDT) and/or disrupt the secondary cellular localization of BET family members (eg, BRD2, BRD3, BRD4, BRDT). The invention provides a computer for playing a) a molecular or molecular complex of three a dimensional representation, wherein the molecule or molecular complex comprises a binding site; or b) a three-dimensional graphical representation of a homologue of the molecule or molecular complex, wherein the homolog includes a binding site, which has The skeletal atom of the amino acid is a root mean square deviation of no more than about 2.0 (more preferably no more than 1.5) angstroms, wherein the computer comprises: (i) a machine that can read the data storage medium, including the machine The data encoded by the read data storage material, wherein the data includes structural coordinates of the amino acid residue based on the binding site of the bromine-containing domain structure binding pocket or other BET family members; (ii) working memory for performing a storage instruction of the readable data of the machine; (iii) a central processing unit coupled to the working memory and the readable data storage medium for processing the machine readable data into the three-dimensional graphic And (iv) a display coupled to the central processor for displaying the three-dimensional graphical representation.

Thus, the computer displays a three-dimensional graphical structure comprising molecular or molecular complexes with binding sites.

In another embodiment, the invention provides a computer to display a three-dimensional graphical representation of a molecular or molecular complex defined by structural coordinates of a BET family member (eg, BRD2, BRD3, BRD4, BRDT), an amino acid; Or a three-dimensional graphical representation of a homologue of the molecule or molecular complex, wherein the homolog includes a binding site having a backbone atom from the amino acid of no more than about 2.0 (more preferably no more than 1.5) ) RMS deviation of angstroms.

In an exemplary embodiment, the computer or computer system can include conventional components in the art, for example, as disclosed in U.S. Patent No. 5,978,740, the disclosure of which is incorporated herein by reference. For example, a computer system may include: a computer including a central processing unit ("CPU"), working memory (which may be like random access memory or "core" memory), mass storage memory (eg, one or more a disk drive 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 circuits, and one or more output circuits, All of them are interconnected with traditional system busses.

The machine readable data of the present invention can be input to a computer by using a data machine or a data machine connected to the data line. Alternatively or additionally, the input hardware can include a CD-ROM drive, a disk drive, or a flash memory. The keyboard can also be used as an input device when connecting the display terminal.

Output hardware coupled by an output circuit and a computer can also be implemented using conventional devices. By way of example, the output hardware can include a cathode ray tube or liquid crystal display terminal for displaying a graphical representation of the binding bag of the present invention using a program such as QUANTA or PYMOL. The output hardware can also include a printer, or a disk drive for subsequent use by the storage system.

In terms of operation, the central processing unit cooperates with various input and output devices to retrieve a large number of collaborative data paths for storing memory and accessing and extracting working memory, and determining a sequence of data processing steps. Some programs can be used to process the machine readable data of the present invention, including commercially available software.

Magnetic storage medium for storing machine readable data of the present invention For the sake of knowing. A magnetic data storage medium that can be encoded by machine readable data can be implemented by a system such as the computer system described above. The medium may be a conventional flexible magnetic disk or hard disk having a suitable substrate (which may be conventional) and a suitable coating (which may be conventional) on one or both sides, containing its polarity or direction to be magnetic Changed magnetic domain. The media may also contain openings (not shown) for receiving the spindle of a hard disk or other data storage device.

The magnetic domains of the medium are polarized or oriented to facilitate the encoding of machine readable data as described herein in a conventional manner to be performed by a system of computer systems as described herein.

The optically readable data storage medium can also be encoded by machine readable data or sets of instructions, which can be performed by a computer system. The medium can be a conventional closed disk read-only optical disc or an optically readable and magneto-optically readable rewritable medium such as a magneto-optical disc.

In the case of CD-ROM, it is clear that the hard-disk coating is reflective and imprints a plurality of recesses to encode machine-readable data. The recess is read and arranged by reflecting laser light that exits the surface of the coating. A substantially transparent protective coating is preferably provided on top of the reflective coating.

In the case of a magneto-optical disc, it is clear that the data-recording coating has no recessed holes, but has a magnetic domain whose polarity or direction can be magnetically changed by heating such as laser above a certain temperature. The direction of the domain is read by measuring the polarization of the reflected laser light from the coating. As described above, the arrangement of the fields encodes data.

When used to interface computer programs and software to translate three-dimensional structures that cooperate to form a molecular or molecular complex comprising a binding pocket, structural data can be used for various purposes, such as drug discovery.

For example, a structure encoded by data can be calculated to evaluate its ability to be associated with a chemical individual. Chemical individuals associated with binding sites of BET family members (eg, BRD2, BRD3, BRD4, BRDT) are expected to inhibit proliferation or induce differentiation of cancer cells to inhibit BET family members (eg, BRD2, BRD3, BRD4, BRDT) Biological activity and/or disruption of secondary cell localization. This compound is a potential drug candidate. Alternatively, the structure encoded by the data can be displayed on the computer screen in a graphical three-dimensional representation. This allows the structure to be visually inspected and the chemical entities associated with the structure to be visually inspected.

Thus, in accordance with another embodiment, the present invention is directed to a method for assessing a potential chemical entity associated with: a) a molecular or molecular complex comprising a binding site, as described herein by a BET family member (eg, BRD2, BRD3, BRD4, BRDT) as defined by the structural coordinates, or b) a homologue of the molecule or molecular complex, wherein the homologue comprises a binding pocket having a backbone from the amino acid The atom is a root mean square deviation of no more than about 2.0 (more preferably no more than 1.5) angstroms.

The method comprises the steps of: i) utilizing methods for performing appropriate manipulations between chemical individuals and binding sites of BET family members (eg, BRD2, BRD3, BRD4, BRDT) polypeptides or fragments or molecular complexes thereof; and ii) analysis The result of this suitable operation is to quantify the association between the chemical entity and the binding pocket. This particular embodiment relates to assessing the potential of association and binding of chemical individuals and BET family members (e.g., BRD2, BRD3, BRD4, BRDT) or fragments thereof.

The term "chemical entity" as used herein means a chemical compound, at least two A complex of a chemical compound, and a fragment of the compound or complex.

In certain embodiments, the method of assessing the association between the chemical entity and the molecular or molecular complex is by the structure of all of the amino acids of the BET family members (eg, BRD2, BRD3, BRD4, BRDT) as described herein. a coordinate, or a homologue of the molecule or molecular complex, wherein the homolog has a root mean square of no more than about 2.0 (more preferably no more than 1.5) angstroms from the backbone of the amino acid. deviation.

In further embodiments, the structural coordinates of a binding site described herein can be used to identify antagonists comprising molecules comprising a bromine-containing domain (eg, a bromine-containing domain-domain binding pocket). The method comprises the steps of: a) using molecular coordinates of a BET family member; and b) designing or selecting a potential agonist or antagonist using a three-dimensional structure. The compound can be obtained by any available method. "Acquiring" means obtaining the agonist or antagonist by, for example, synthesizing, purchasing, or otherwise. If desired, the method further involves contacting an agonist or antagonist comprising a BET family member polypeptide or a fragment thereof to determine the ability of the potential agonist or antagonist to interact with the molecule. If desired, the method further involves the steps of contacting a cancer cell containing a bromine-containing domain-binding compound, assessing cytotoxicity, assessing cancer cell proliferation, cell death, BET family member biological activity, or subcellular localization.

In another embodiment, the invention provides a method of identifying potential antagonists of BET family members (eg, BRD2, BRD3, BRD4, BRDT), the method comprising the steps of: a) using molecular coordinates of BET family members (eg, BRD2, BRD3, BRD4, BRDT) (eg, bromine-containing domain structure binding pockets); and b) designing or selecting potential agonists or antagonists using three-dimensional structures .

The inventors herein have not identified the binding sites for the bromo-containing domain to provide the necessary information to design novel chemical entities and compounds that interact with the bromine-containing domain (in whole or in part), and thus are regulatable (eg, Inhibition) Activity of BET family members (eg, BRD2, BRD3, BRD4, BRDT).

In accordance with the present invention, it is contemplated that a compound that binds to a BET family member (e.g., BRD2, BRD3, BRD4, BRDT) structural binding pocket sequence (which reduces the biological activity of the bromo-containing domain or disrupts the subcellular localization of the BET family member) is generally designed. There are some factors that are considered and so on. In a specific embodiment, at least a fragment of the compound and a member of the BET family (eg, BRD2, BRD3, BRD4, BRDT) is physically and/or structurally associated, for example, in a binding pocket sequence comprising a bromine-containing domain. This important important non-covalent molecular interaction includes hydrogen bonding, van der Waals interactions, hydrophobic interactions, and electrostatic interactions. Desirably, the compound is assumed to be in a configuration that is directly associated with the binding site of the bromo-containing domain. Although some parts of the compound may not directly participate in these associations, those parts of the individual may still affect the overall configuration of the molecule. That is, in order, the potency of the compound has a significant effect. The requirements of the configuration include the overall three-dimensional structure and orientation of the chemical compound associated with all or part of the binding site, or between the functional groups of some chemical compounds that directly interact with the binding site or its homolog. distance.

The effective inhibition or inhibition of the binding site of a chemical compound to a bromine-containing domain can be analyzed prior to its actual synthesis and testing using computer modeling techniques. The test is excluded if the theoretical structure of a given compound suggests that it is insufficiently interacting and associated with the binding site of the target. However, if computer modeling indicates a strong interaction, then the synthetic molecule is tested and tested for its ability to bind to the PEG family member (eg, BRD2, BRD3, BRD4, BRDT) to bind the pocket sequence, or to test its biological activity, for example, By determining the cytotoxicity in tumor cells, by determining the decrease in biological activity of a BET family member, or by determining the secondary cell location of a BET family member (eg, BRD2, BRD3, BRD4, BRDT). Candidate compounds can be calculated by a series of steps that screen chemical individual fragments and select their ability to associate with a bromine-containing domain structural binding pocket.

One of skill in the art can use one of several methods to screen for chemical compounds, or fragments of the ability to associate with a bromine-containing domain binding site. Based on the structural coordinates of the members of the BET family described herein or other definitions that are similar to those produced by machine readable storage media, this method can be initiated by visual inspection of binding sites such as on a computer screen. The selected fragment or chemical compound is then positioned or docked in multiple orientations within the binding sites as defined above. Using software such as Quanta and DOCK, docking can then be accomplished by using standard molecular mechanics energy minimization and molecular dynamics such as CHARMM and AMBER.

Specialized computer programs (e.g., those skilled in the art and/or commercially available and/or described herein) may also assist in the selection of fragments or chemistry. The process of the body.

Once selected suitable chemical entities or fragments are selected, they can be assembled into a single compound or complex. The assembly can be performed by visually checking on the computer screen the structural coordinates of the segment associated with the target binding site before displaying the relationship with each other in the three-dimensional image.

Binding of a binding agent to a fragment or chemical entity in a stepwise manner, as described herein, using an unoccupied binding site or, if desired, a whole or "re-design" inhibition of a portion of a known inhibitor or other Binding compound. A number of religand design methods are known in the art, and some are commercially available (e.g., Leap Frog, available from Tripos Associates, St. Louis, Mo.).

Other molecular modeling techniques can also be utilized in accordance with the present invention (see, for example, NC Cohen et al., "Molecular Modeling Software and 15 Methods for Medicinal Chemistry, J. Med. Chem., 33, pp. 883-894 (1990)) Or refer to MANavia and MA Murcko, "The Use of Structural Information in Drug Design", Current Opinions in Structural Biology, 2, pp. 202-210 (1992); LMBalbes et al., "A Perspective of Modern Methods in Computer-Aided Drug Design", in Reviews in Computational Chemistry, Vol. 5, KB Lipkowitz and DB Boyd, Eds., VCH, New 20 York, pp. 337-380 (1994); or reference WC Guida, "Software For Structure" -Based Drug Design", Curr. Opin. Struct. Biology,, 4, pp. 777-781 (1994)).

Once the compound has been designed or selected, it can be tested by calculation and evaluation. The effect of the individual being able to bind to the binding site is optimized.

A specific computer soft system is commercially available to evaluate the deformation energy and electrostatic interaction of a compound. Examples of programs designed for this use include: AMBER; QUANTA/CHARMM (Accelrys, Inc., Madison, WI), and the like. These programs can be executed, for example, using commercially available graphics work. Other hard disk systems and software packages are known to those skilled in the art.

Another technique involves screening a compound of a virtual library by computer simulation, for example, as described herein (see, for example, the examples). Thousands of compounds can be screened quickly and the virtual compound can be selected for further screening (eg, synthetic and in vitro or in vivo testing). For chemical entities or compounds that can bind, in whole or in part, to BET family members (eg, BRD2, BRD3, BRD4, BRDT), a small molecule library can be screened. In this screening, the quality of the binding site suitable for the individual can be judged or evaluated by the complementary or interacting energy.

A computer is used to generate a three-dimensional graphical representation of: a) a molecular or molecular complex, wherein the molecular or molecular complex comprises a structural binding pocket of a BET family member, which is based on a bromine-containing structure a three-dimensional graphical representation of a contiguous structural coordinate of a domain structure; or b) a three-dimensional graphical representation of a homologue of the molecule or molecular complex, wherein the homolog includes a binding site having a backbone from the amino acid The atom is a root mean square deviation of no more than about 2.0 (more preferably no more than 1.5) angstroms, wherein the computer comprises: (i) a machine readable data storage medium, including machine readable Data-encoded data storage material, wherein the data includes structural coordinates of the amino acid residue based on the binding pocket sequence of the BET family member; (ii) working memory for storing instructions for the machine readable data; (iii) a central processing unit coupled to the working memory and the machine readable data storage medium for processing the machine readable data into the three dimensional graphical representation; and (iv) a display The central processor is coupled for displaying the three-dimensional graphical representation. As described in the Examples, the compounds identified using computer simulation methods can be tested, if desired, in vitro or in vivo, for example, using the "additional screening methods" described below, or any other method known in the art.

Additional screening method

As described above, the present invention provides specific examples of chemical compounds, including JQ1, and other substituted compounds that bind to a bromine-containing domain binding pocket to reduce cell differentiation or reduce tumor cell proliferation. However, the invention is not limited thereto. The present invention provides a simple method for identifying reagents (including nucleic acids, peptides, small molecule inhibitors, and mimics) that bind to members of the BET family and are cytotoxic to tumor cells, which reduces the BET family The biological activity of the member, or disruption of the subcellular location of the BET family member. The compound is also expected to be useful for treating or preventing tumors, inflammatory diseases, obesity, fatty liver (NASH or others), diabetes, atherosclerosis, arterial stent obstruction, heart failure, dysentery, graft versus host disease, and Infectious diseases, parasites, and bromine-containing domains Malaria, treatment of trypanosomes, and use to reduce male fertility. Further uses of the compositions of the present invention include, but are not limited to, organ transplantation, regulation of cellular states for regenerative medicine (i.e., by promoting or inhibiting fine differentiation), and promoting differentiation pluripotency.

In particular, certain aspects of the invention are based on agents found to reduce, at least in part, the biological activity of a BET family member polypeptide, which may be suitable as a therapeutic agent for the treatment or prevention of tumors, inflammatory diseases, obesity, fatty liver ( NASH or others), diabetes, atherosclerosis, arterial stent obstruction, heart failure, dysentery, graft versus host disease, and bromine-containing domain-associated infectious diseases, parasites, malaria, trypanosomes And to reduce male fertility. Further uses of the compositions of the present invention include, but are not limited to, organ transplantation, regulation of cellular states for regenerative medicine (i.e., by promoting or inhibiting fine differentiation), and promoting differentiation pluripotency. In a particular embodiment, the effect of a compound or other agent of the invention is analyzed by examining cell proliferation, cell survival or cell death. In another approach, the agents and compounds of the invention are tested for their effect on transcriptional regulation or elongation. The agents and compounds of the invention that are found to reduce tumor growth, proliferation, or invasion are useful in the treatment or prevention of tumors. In another embodiment, the compounds of the invention are tested for their effect on the release of immune response cells, cytokines or histamine, or any other marker of inflammatory disease.

The methods of the invention may utilize any virtual agent that specifically binds to a BET family member or reduces the biological activity of a BET family member. The method of the present invention is useful for high throughput low cost screening of candidate agents (which reduce, slow, or It stabilizes the growth or proliferation of tumors, or is used to treat or prevent inflammatory diseases. Candidate agents that specifically bind to the bromodomain-containing domain of the BET family member are then isolated and used for in vitro or in vivo assays to reduce their ability to reduce tumor cell proliferation, reduce differentiation, and/or increase tumor cell death. test. Those skilled in the art will appreciate that the effect of a candidate agent on a cell is typically compared to a corresponding control cell that is not in contact with the candidate agent. Thus, screening methods include comparing proliferation of tumor cells contacted with a candidate agent to proliferation of untreated control cells.

In other specific embodiments, the expression or activity of a BET family member in a cell treated with a candidate agent reduces the BET family member in the contacted cell as compared to an untreated control sample identified as a candidate compound. Biological activity. It can be performed by conventional procedures in the art (for example, Western blotting, flow cytometry, immunocytochemistry, binding to magnetic and/or bromine-containing domain-specific antibodies). Coated beads, in situ hybridization, fluorescence in situ hybridization (FISH), ELISA, microarray analysis, RT-PCR, Northern blotting, or colorimetric assays such as Bradford analysis and Lowry analysis) to compare polypeptide performance or activity.

In one example of operation, one or more candidate agents are added to a culture dish containing tumor cells at various concentrations. Reagents that reduce the performance of BET family members in cells are suitable for use in the present invention; such agents can be used, for example, as therapeutic agents to prevent, delay, ameliorate, stabilize, or treat a tumor or inflammatory disease. Once identified, the agents of the invention (eg, agents that specifically bind to and/or oppose bromodomain-containing domains) can be used to treat tumors, inflammatory diseases, obesity, fat Liver (NASH or others), diabetes, atherosclerosis, arterial stent obstruction, heart failure, dysentery, graft versus host disease, and bromine-containing domain-associated infectious diseases, parasites, malaria, trypanosomes Treatment and use to reduce male fertility. Further uses of the compositions of the present invention include, but are not limited to, organ transplantation, regulation of cellular states for regenerative medicine (i.e., by promoting or inhibiting fine differentiation), and promoting differentiation pluripotency. The agent identified according to the method of the present invention is administered locally or systemically to treat tumors, inflammatory diseases, obesity, fatty liver (NASH or others), diabetes, atherosclerosis, arterial stent obstruction, heart failure, evil Diseases, grafts against host diseases, infections associated with bromine-containing domain domains, treatment of parasites, malaria, trypanosomes, and cells used to reduce male fertility, or for organ transplantation, for regenerative medicine Regulation of the state (ie, by promoting or inhibiting fine differentiation), or promoting in situ differentiation of pluripotency.

In one embodiment, the effect of the candidate agent can be measured in a BET family member by using the same general methods and standard immunological techniques (eg, Western blotting or immunoprecipitation with specific antibodies to BET family members). concentration. For example, immunoassays can be used to detect or monitor the performance of BET family members in tumor cells. In one embodiment, the invention identifies a multi- or monoclonal antibody (as described herein) that is capable of binding to a BET family member and blocking the biological activity of a BET family member or disrupting the subcellular location of a BET family member. Compounds that interrupt the secondary cell position of a BET family member, or that reduce the biological activity of a BET family member, are considered to be particularly useful. Also, the agent can be used as a therapeutic agent to prevent or treat a tumor or Inflammatory disease.

Alternatively, in addition, the candidate compounds are first specifically bound to and against the BET family members of the invention (eg, BRD2, BRD3, BRD4, BRDT) by analysis, and then tested for their effect on tumor cells (as described in the implementation) ) to be identified. In a particular embodiment, the efficacy of a candidate agent depends on its ability to interact with a BET family member. This interaction can be readily tested using any standard binding technique and functional analysis (e.g., as described by Ausubel et al., supra). For example, candidate compounds can be tested in vitro for their interaction and binding with the polypeptides of the invention and for testing the ability to modulate tumor cell proliferation by any standard assay (eg, those described herein). In one embodiment, flow cytometry analysis is used to determine tumor cell division by examining BrdU incorporation. In another specific embodiment, the performance of the BET family members is monitored immunohistochemically.

Potential bromine-containing domain antagonists include organic molecules, peptides, peptide mimetics, polypeptides, nucleic acid ligands, aptamers, and antibodies that bind to the bromine-containing domain of the BET family member and reduce Its activity. In a particular embodiment, candidate compounds that bind to members of the BET family can be identified using chromatographic-based techniques. For example, a recombinant BET family member polypeptide of the invention can be purified from a designed cell by standard techniques to express the polypeptide, or can be immobilized by chemical synthesis once the purified polypeptide is in a column. A solution of the candidate agent is then passed through the column, and an agent that binds specifically to the BET family member polypeptide or a fragment thereof according to its ability to bind to the BET family member polypeptide is immobilized to the column. In order to separate the reagent, the column is cleaned to remove non-specific binding molecules. The reagent of interest is then released from the column and collected. The reagents isolated by this method (or any other suitable method) can be further purified (e.g., by high performance liquid chromatography techniques) if desired. In addition, the ability of these candidate agents to reduce tumor cell proliferation or viability can be tested. Agents isolated by this method can also be used as, for example, therapeutic agents to treat or prevent tumors, inflammatory diseases, obesity, fatty liver (NASH or others), diabetes, atherosclerosis, arterial stent obstruction, heart Depletion, dyscrasia, graft versus host disease, and bromine-containing domain-associated infectious diseases, parasites, malaria, trypanosomes, and for reducing male fertility, or for organ transplantation, for regeneration Regulation of the cellular state of medicine (ie, by promoting or inhibiting fine differentiation), and promoting differentiation pluripotency. Compounds identified as binding to a member of the BET family have an affinity coefficient less than or equal to 1 nM, 5 nM, 10 nM, 100 nM, 1 μM, or 10 μM, in view of particular use in the present invention.

Test compounds and extracts

In certain embodiments, a BET family member antagonist (eg, an agent that specifically binds to and reduces the activity of a bromine-containing domain) is derived from a natural product or synthetic (or half) according to conventional methods in the art. A large database or chemical database of synthetic extracts, or identified from a polypeptide or nucleic acid database. Those skilled in the art of drug discovery and development should be able to understand that the exact source of the test extract or compound is not critical to the screening procedure of the present invention. Agents for screening may include those known to be used in the treatment of tumors or inflammatory diseases. Alternatively, virtually any number of unknown chemical extracts or compounds can be screened using the methods described herein. The extraction Examples of the substance or compound include, but are not limited to, plants, fungi, prokaryotes, or animal-based extracts, fermentation broths, and synthetic compounds, and modifications of existing polypeptides.

A database of natural polypeptides of the type of bacteria, fungi, plants, and animal extracts is commercially available from a number of sources, including Biotics (Sussex, UK), Xenova (Slough, UK), Harbor Branch Oceangraphics Institute ( Ft. Pierce, Fla.), and PharmaMar, USA (Cambridge, Mass.). The polypeptides can be modified to include those having a protein domain using methods well known in the art and as described herein. In addition, natural and synthetic homemade databases can be prepared, if desired, according to methods known in the art (e.g., by standard extraction and fractionation methods). Examples of methods for molecular library synthesis are available in the art, for example: DeWitt et al., Proc. Natl. Acad. Sci. USA 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. Furthermore, any library or compound can be easily modified using standard chemical, physical, or biochemical methods if desired.

Numerous methods can also be used for the random or direct synthesis (eg, semi-synthetic or total synthesis) of any number of polypeptides, chemical compounds including, but not limited to, compounds based on sugars, lipids, peptides, and nucleic acids. ). Commercially available from Brandon Associates (Merrimack, N.H.) and Aldrich Chemical (Milwaukee, Wis.) purchased a library of synthetic compounds. Alternatively, the chemical compound to be used as a candidate compound can be synthesized using starting materials which are readily available in the art by standard synthetic techniques and methods well known to those skilled in the art. Methods for the identification of synthetic chemical transformation and protecting groups (protection and deprotection) for the synthesis of compounds by methods described herein are well known in the art, for example, including those of the following: R. Larock, Comprehensive Organic Transformations, VCH Publishers (1989); TW Greene and PGM Wuts, Protective Groups in Organic Synthesis, 2nd ed., John Wiley and Sons (1991); L. Fieser and M. Fieser, Fieser and Fieser's Reagents For Organic Synthesis, John Wiley and Sons (1994); and L. Paquette, ed., Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons (1995).

The library of compounds may be present in solution (eg, Houghten, Biotechniques 13: 412-421, 1992), or small plants (Lam, Nature 354: 82-84, 1991), wafers (Fodor, Nature 364: 555-556, 1993), bacteria (Ladner, US Patent No. 5, 223, 409), spores (Ladner US Patent No. 5, 223, 409), plastids (Cull et al., Proc Natl Acad Sci USA 89: 1865-1869, 1992) or phage (Scott and Smith, Science 249: 386-390, 1990; Devlin, Science 249: 404-406, 1990; Cwirla et al. Proc. Natl. Acad. Sci. 87: 63786382, 1990; Felici, J. Mol. Biol. 301-310, 1991; Ladner supra.).

In addition, those skilled in the art of drug discovery and development should be well versed in the methods used to: copy (eg, classify deduplication, biological removal) Replica, and chemical de-duplication, or any combination thereof), elimination of replication, or repetitive materials known to be useful for their activity.

When a crude extract having a bromine domain domain binding activity is found, it is necessary to further fractionate the positive lead extract to separate the molecular component for observation. Therefore, the goal of the extraction, fractionation, and purification processes is to carefully characterize and identify chemical individuals within the crude extract that reduce tumor cell proliferation or viability. Methods of fractionation and purification of such heterogeneous extracts are known in the art. Compounds indicated as being suitable for use in therapeutic agents are chemically modified, if desired, according to methods known in the art.

The invention provides a method of treating a disease and/or a disorder or condition thereof, the method comprising administering to a subject (e.g., a mammal, such as a human) a therapeutically effective amount of a pharmaceutical composition comprising a compound of the formula described herein. Thus, a specific embodiment is a method of treating an individual suffering from or having a neoplastic disease or a condition or condition thereof. The method comprises the step of administering to a mammal a therapeutic amount of a compound of the invention sufficient to treat the disease, or a condition or condition thereof, under conditions of the disease or condition being treated.

The methods of the invention comprise administering to a subject (including identifying an individual in need of such treatment) an effective amount of a compound of the invention, or a composition of the invention, to produce the effects described above. The individual identifying the need for the treatment can be an individual's trial or medical professional and can be subjective (eg, judged) or objective (eg, measurable by a test or diagnostic method).

Therapeutic methods of the invention, which include the prevention of the treatment of a disease, generally comprise administering to a subject in need thereof (e.g., an animal, a human), including a mammal, particularly a human, a therapeutically effective amount of a compound of the invention, for example, as described herein. a compound of the formula. The treatment will suitably be administered to an individual suffering from, having, having or at risk of developing the disease or its condition or condition, particularly a human. The determination that their individuals are "at risk" may be subject to any subjective or diagnostic test or individual judgment or medical professional provider (eg, general test, enzyme or protein indicators, markers (as described herein), family history, etc.) Objectively decide to obtain it. The compounds of the invention may also be used in the treatment of any other condition that may be associated with a tumor or inflammation.

In one embodiment, the invention provides a method of monitoring the progress of a treatment. The method comprises, in an individual suffering from or suffering from a disease associated with a tumor or a condition thereof, wherein the system has administered a therapeutic amount of a compound of the present invention sufficient to treat the disease or its condition, determining a diagnostic marker (indicator) The concentration or diagnostic measurement method (eg, screening, assay) steps (eg, by any of the targets, proteins, or indicators thereof modified by the compounds herein). The concentration of the marker determined in the method can be compared to the known concentration of the marker in the healthy normal group or other diseased patient to establish the disease state of the individual. In a preferred embodiment, the second concentration of the marker in the individual is determined later than the point in time at which the first concentration is determined, and the effects of the two concentrations on monitoring the progression of the disease or the treatment are compared. In certain preferred embodiments, the desired therapeutic concentration of the marker in the individual is determined prior to initiating treatment of the invention; the pre-therapeutic concentration of the marker can then be correlated with the marker in the individual after initiation of treatment Concentration comparison to determine the effect of the treatment.

Medical therapeutics

In other specific embodiments, agents found to have medicinal properties using the methods of the invention (eg, JQ1 or a compound of the formula described herein) are suitable. Information for the modification of a drug or a structure that is an existing compound, for example, rationalizing drug design. This method is suitable for screening agents that are effective for: tumors, inflammatory diseases, obesity, fatty liver (NASH or others), diabetes, atherosclerosis, arterial stent obstruction, heart failure, dysentery, graft versus Host disease and treatment of infectious diseases associated with bromine-containing domains, parasites, malaria, trypanosomes, and regulation of cell status for reducing male fertility, or for organ transplantation, for regenerative medicine (ie, borrowing By promoting or inhibiting fine differentiation, and promoting in situ differentiation pluripotency.

For therapeutic use, the compositions or agents identified using the methods described herein can be administered systemically, for example, as a pharmaceutically acceptable buffer such as physiological saline. Preferred routes of administration include, for example, subcutaneous, intravenous, intraperitoneal, intramuscular, or intradermal injection to provide a continuous, sustained concentration of the patient's drug. The therapeutically effective amount of the treatment identified by the biologically acceptable carrier herein can be used to treat a human patient or other animal. Suitable carriers and other formulations are as described by Remington's Pharmaceutical Sciences by E. W. Martin. The amount of therapeutic agent may vary depending on the mode of administration, the age and weight of the patient, and the clinical signs of the following diseases: tumor, inflammatory disease, obesity, fatty liver (NASH or others), diabetes, atherosclerosis, arteries Stent obstruction, heart failure, dysentery, graft versus host disease, infection with bromine-containing domain-related infectious diseases, parasites, malaria, trypanosomes, and for reducing male fertility, or for organ transplantation, Regulatory of the cellular state of regenerative medicine (ie, by promoting or inhibiting fine differentiation), and promoting in situ differentiation pluripotency. In general, this amount can be used for their treatment and related to the above mentioned diseases or conditions. The range of other agents used in other diseases, but in some instances a lower amount is required due to the increased specificity of the compound. The administration of a compound at a dose that is cytotoxic to a tumor cell reduces the biological activity of the BET family member, as measured by methods known to those skilled in the art or using any assay to measure or have biological activity of a BET family member, or Reduce tumor cell proliferation, viability or aggression. In another embodiment, the compound is administered at a dose that reduces the symptoms of an inflammatory or inflammatory disease.

Inflammatory disease

Compositions of the invention, including compounds of the formulae described herein, are useful for reducing inflammatory effects and for treating inflammatory conditions. Inflammation involves a complex and continuous result of the molecular signals of the immune system, usually in response to infection or damage to cells or tissues. Inflammation usually refers to the onset of a healing of the body; however, if the inflammation is not properly regulated, the inflammatory effect can lead to chronic diseases such as arthritis.

"Inflammation reaction" or "immune response" means loss of function caused by living tissue to injury, infection, or irritation characterized by redness, heat, swelling, and pain, as well as increased blood flow and accumulation and secretion of immune cells. The reaction. The inflammatory effect is a response to the body invading the infectious microbial organism, thereby causing an increase in blood flow to the affected area, attracting white blood cells to release chemicals, increasing plasma flow, and causing the mononuclear ball to clear cell debris. Inflammation can be any one that stimulates an inflammatory response.

The non-specific response carried by the immune system is an innate cascade or innate immune response, and is characterized by cells at the site of injury or infection (eg, white blood cells, natural killer cells, mast cells, eosinophils) Infiltration of cells and basophils, and responses of phagocytic cells such as neutrophils, macrophages, and dendritic cells to chemical directional signals. Molecules secreted by the aforementioned cells, for example, histamine and various cytokines; and the complement system of circulating proteins all contribute to inflammatory effects. Diseases characterized by inflammatory effects are an important cause of human morbidity and mortality.

In certain embodiments, the inflammatory condition is a rheumatic disorder. As used herein, a rheumatic disorder refers to any of a variety of inflammatory conditions characterized by inflammatory effects, sometimes degeneration and/or metabolic degradation of connective tissue structures, particularly joints, ligaments, and tendons. Rheumatic conditions typically result in pain, stiffness, and/or limited movement. The particular tissue or tissue that is affected depends on the rheumatic disorder. Exemplary rheumatic conditions include, but are not limited to, rheumatoid arthritis, juvenile arthritis, bursitis, spondylitis, gout, scleroderma, Still's disease, and vasculature Inflammation (vasculitis).

In certain embodiments, the rheumatic condition is rheumatoid arthritis, and "treating" rheumatoid arthritis comprises reducing the severity, frequency, and/or occurrence of one or more symptoms of the rheumatoid arthritis. In other embodiments, the rheumatic disorder is juvenile arthritis, bursitis, spondylitis, gout, scleroderma, Styrian's disease, or vasculitis. The method of the invention reduces the severity, frequency and/or occurrence of any one or more of these conditions.

In various embodiments, the symptoms of arthritis or other inflammatory diseases include redness, swelling, inflammatory effects, fever, reduced range of motion, and pain. Examples of reducing the occurrence or severity of the condition include, but are not limited to, reducing the number of joint enlargements, reducing the amount of joint pain, reducing the dependence of analgesics, reducing the frequency or severity of self-assessment of the patient's pain, and increasing exercise. Freedom of freedom, increased mobility, reduced fever, and increased ability to perform daily work.

Neuroinflammatory effects (characterized by activated microglia and stellate cells and local manifestations of extensive inflammatory mediators) are essential responses to brain injury, whether traumatic, stroke, infection or neurodegeneration. This local tissue response is considered a partial repair and recovery process. Like many inflammatory conditions in peripheral diseases, neuroinflammatory effects contribute to the pathophysiology of CNS disorders.

In certain embodiments, the inflammatory condition is an inflammatory skin condition. Inflammatory skin conditions include, but are not limited to, rosacea, topical dermatitis, acne, seborrheic dermatitis, and cellulitis.

In another specific embodiment, the inflammatory disease is an ischemic or inflammatory cardiovascular disease. An inflammatory cardiovascular disease or condition can be, but is not limited to, a occlusive disease or condition, atherosclerosis, valvular heart disease, stenosis, restenosis after heart valve surgery, and in-stent restenosis In-stent-stenosis, myocardial infarction, coronary artery disease, acute coronary syndrome, septic heart failure, angina pectoris, myocardial hypoxia, or thrombosis. In other embodiments, the site is a secondary site of ischemic injury, such as the CNS or the kidney.

In other specific embodiments, the inflammatory disease is an ischemic or inflammatory bowel disease.

Inflammatory bowel disease (IBD) means chronic rectal inflammatory disease of unknown etiology of the small intestine and colon, including Crohn's disease (Crohn's disease, CD) and ulcerative colitis (UC). Crohn's disease may involve any part of the intestine and generally involves the terminal small intestine and/or colon. Ulcerative colitis involves only the colon and is usually confined to the rectum or terminal colon. Studies of mouse models of CD and UC strongly suggest that these two diseases are due to dysregulation of antibody mucosal immune responses in mucosal microbiota (Sartor, RB (1995). Gastroenterol Clin North Am 24, 475-507) (Strober W, et al. (2002) Annu. Rev. Immunol. 20:495-549).

Ulcerative colitis or undetermined colitis means a condition of the colon characterized by an inflammatory state in which one or more of the following histological features are detectable: in the presence of epithelial cell loss and spot ulceration, The massive depletion of mucinous goblet cells and the reduction in tubular gland density are characteristic of superficial inflammatory effects. In addition, in the lamina propia, lymphocytes and granulosa cells are involved in the exudation of cells entering the intestinal lumen (mostly neutrophils, and to a lesser extent, basophils) Composition) mixed inflammatory cells infiltrated. Similarly, when the artisan sees little or no indication of inflammatory effects in the outer muscle layer, the submucosa may indicate marked edema with a few inflammatory cells. For example, see Boirivant et al. Journal of Experimental Medicine 188: 1929-1939 (1998). Clinical signs may include, but are not limited to, diarrhea, rectal prolapse, weight loss, abdominal pain, and dehydration.

Crohn's disease involves inflammatory effects that affect any part of the digestive tract and usually affect the end of the small intestine and/or adjacent the descending colon. Frequently, the inflammatory effect is caused by alternating "normal mucosal areas and inflammatory areas" It is characterized by the fact that the diseased area of the intestine in Crohn's disease shows erythema, edema and increased fragility; the intestinal tract is often narrowed and connected to other abdominal organs or intestinal walls. Between the diseased intestines and other structures The fistula includes frequently occurring skin. Microscopic examination of the tissue in Crohn's disease shows erosion of the epithelial membrane, loss of mucin-producing goblet cells, and a large amount of lymphocytic infiltration involving all layers of the membrane; this infiltrate sometimes contains A giant cell that symbolizes the formation of granuloma tumors. When inflammation persists for a long period of time (chronic), scarring (fibrosis) sometimes occurs. Scar tissue is typically not as flexible as healthy tissue. Therefore, when fibers appear in the intestine When scarred, the scar narrows the width of the passage (inner lumen) involved in the segment of the intestine. These areas of the narrow 7 are called strictures. The stenosis may be slight or severe depending on how many obstructions there are. The amount of intestine contained in the stenotic region. The clinical symptoms/symptoms of Crohn's disease may include, but are not limited to, cachexia, weight loss, slow growth, and abdomen Pain, fistula drainage (draining fistulae), rectal prolapse and dehydration.

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

Preparation of pharmaceutical compositions

The compound for treating a neoplastic or inflammatory disease can be administered by any suitable means which effectively combines, reduces, or stabilizes a tumor formation or inflammatory disease in a concentration of a therapeutic agent in combination with other ingredients. The compound can be included in any suitable carrier material in any suitable amount and is generally present at from 1 to 95% by weight based on the total weight of the composition. Can be adapted for parenteral The composition is administered in a dosage form (e.g., subcutaneous, intravenous, intramuscular, or intraperitoneal) of the route of administration. The pharmaceutical composition can be based on traditional pharmaceutical practice (for example, see Remington: The Science and Practice of Pharmacy (20th ed.), ed. AR Gennaro, Lippincott Williams & Wilkins, 2000 and Encyclopedia of Pharmaceutical Technology, eds .J. Swarbrick and JC Boylan, 1988-1999, Marcel Dekker, New York).

The human dosage can be initially determined by extrapolation from the amount of compound used in the mouse, and it is well known to those skilled in the art that it is conventional in the art to compare humans to animal models to modify dosages. In certain embodiments, it is contemplated that the dosage can range from about 1 microgram (μg) compound per kilogram (kg) body weight to about 5000 milligrams (mg) compound per kg body weight; or from about 5 mg/kg body weight to about 4000 mg/kg. Weight; or from about 10 mg/kg body weight to about 3000 mg/kg body weight; or from about 50 mg/kg body weight to about 2000 mg/kg body weight; or from about 100 mg/kg body weight to about 1000 mg/kg body weight; or from about 150 mg/kg The body weight changes to about 500 mg/kg body weight. In other embodiments, the dose can be 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 mg/kg body weight. In other specific embodiments, the dosage can be about 8, 10, 12, 14, 16 or 18 mg/kg body weight. Of course, this dose can be adjusted up or down, as is conventional in the treatment protocol, depending on the initial The results of the bed test and the needs of specific patients.

The pharmaceutical compositions of the present invention can be formulated to provide sustained release of the active compound immediately upon administration or at any predetermined time or time period after administration. A late version of the composition is a generally known controlled release formulation comprising (i) a formulation that confers a substantially fixed concentration of the drug over a prolonged period of time in vivo; (ii) exceeds in vivo Prolonged for a period of time, after a predetermined delay time, the formulation is administered at a substantially fixed concentration; (iii) the formulation that continues to function during the predetermined time frame is varied by the plasma concentration of the active substance Relevant, undesired side effects are minimized to maintain relative, fixed, effective concentrations in the body (sawtooth dynamics); (iv) topical formulations are adjusted by the spatial configuration of the controlled release composition or Contact with the thymus; (v) a formulation that facilitates administration, for example, a dose administered once or twice a week; and (vi) a formulation that calibrates a tumor or inflammatory disease by using a carrier or chemical derivative To deliver a therapeutic agent to a particular cell type (eg, a tumor cell), for certain applications, the controlled release formulation eliminates the need for daily routine medication but continues blood. Concentration in therapeutic concentrations.

Any number of strategies can be performed to obtain a controlled release in which the release rate exceeds the metabolic challenge of the compound. In one example, controlled release is achieved by appropriate selection of a plurality of formulation parameters and components, including, for example, a plurality of controlled release compositions and types of coatings. Thus, the therapeutic agent is formulated with a suitable excipient to form a pharmaceutical composition which is released in a controlled manner in accordance with administration. Examples include single or complex unit tablets or capsule compositions, oily solutions, suspensions, emulsions, microcapsules, molecular complexes Compounds, nanoparticles, patches and liposomes.

Parenteral composition

The pharmaceutical composition can be prepared by injection, infusion or implantation (subcutaneous, intravenous, intramuscular, abdominal, etc.) in a dosage form, or by a conventional, non-toxic pharmaceutically acceptable carrier containing a suitable delivery device or implant. The agent or adjuvant is administered parenterally. Formulations or formulations of such compositions are well known in the art as pharmaceutical formulations well known to those skilled in the art. This formulation can be found in Remington: The Science and Practice of Pharmacy as described above.

Compositions for parenteral administration can be administered in unit dosage form (e.g., in a single dose ampule) or in vials containing several doses, and a suitable preservative (see below) can be added. The composition may be in the form of a solution, suspension, emulsion, infusion device, or delivery device for implantation, or in the form of a dry powder for recombination with water or other suitable vehicle prior to use. In addition to reducing or ameliorating tumor formation or inflammatory diseases, the compositions may also include carriers and/or excipients that are suitable for parenteral administration. The active therapeutic agent can be incorporated into microspheres, microcapsules, nanoparticles, liposomes, and the like for controlled release. Further, the composition may include a suspending agent, a solubilizing agent, a stabilizer, a pH-adjusting agent, an isotonic conditioning agent, and/or a dispersing agent.

As indicated above, the pharmaceutically acceptable compositions of the present invention may be in a form suitable for sterile injection. To prepare the composition, a suitable active anti-tumor therapeutic is dissolved or suspended in a parenterally acceptable liquid vehicle. The acceptable vehicle and solvent can be adjusted to a suitable pH value of water, 1,3-butanediol, Ringer's solution by using water, by adding an appropriate amount of hydrochloric acid, sodium hydroxide or a suitable buffer. (Ringer's solution), and isotonic Sodium chloride solution and glucose solution. Aqueous formulations may also include one or more preservatives (eg, methyl-, ethyl-, or n-propyl-p-hydroxybenzoate). When a compound is only slightly or slightly dissolved in water, a dissolution enhancer or solubilizer may be added, or the solvent may include 10 to 60 w/w% of propylene glycol or the like.

Controlled release of the parenteral composition

The controlled release parenteral composition can be in the form of an aqueous suspension, microspheres, microcapsules, magnetic microspheres, oily solution, oily suspension, or emulsion. Alternatively, the active drug can be incorporated into a biocompatible carrier, liposome, nanoparticle, implant, or infusion device.

Materials for preparing microspheres and/or microcapsules such as biodegradable/bioerodible polymers such as polygalactin, poly-(isobutylcyanoacrylate), poly(2- Hydroxyethyl-L-bromoamine), and poly(lactic acid). When a controlled release parenteral formulation is formulated as a saccharide (eg, dextrant), a protein (eg, albumin), a lipoprotein, or an antibody, a biocompatible carrier can be used. Materials for implants can be non-biodegradable (eg, polydimethyloxane) or biodegradable (eg, poly(caprolactone), poly(lactic acid), poly(glycolic acid) Or poly(orthoester) or a combination thereof).

Solid dosage form for oral use

Solid dosage forms for oral use include lozenges containing the active ingredient in a mixture of non-toxic pharmaceutically acceptable excipients. Such formulations are well known to those skilled in the art. The excipient can be, for example, an inert diluent or a filler (for example, sucrose, sorbitol, sugar, mannitol, microcrystalline cellulose, lake Powders include potato starch, calcium carbonate, calcium chloride, lactose, calcium phosphate, calcium sulfate, or sodium phosphate; granulating agents and disintegrating agents (for example, cellulose derivatives including microcrystalline cellulose, starch including potato starch, and Sodium carboxymethylcellulose, alginate or alginic acid; binders (eg, sucrose, glucose, sorbitol, acacia, alginic acid, sodium alginate, gelatin, starch, pregelatinized starch, microcrystalline fibers) , magnesium aluminum silicate, sodium carboxymethyl cellulose, methyl cellulose, hydroxypropyl methyl cellulose, ethyl cellulose, polyvinyl pyrrolidone, or polyethylene glycol); and lubricants; a flow agent; and an anti-adhesive agent (for example, magnesium stearate, zinc stearate, stearic acid, citric acid, hydrogenated vegetable oil or talc). Other pharmaceutically acceptable excipients can be colorants, sweeteners, plasticizers, wetting agents, buffers, and the like.

Tablets may be uncoated or may be coated by known techniques, optionally disintegrating and absorbed in the gastrointestinal tract, thereby providing a sustained action over a longer period of time. The coating may be adapted to release the active drug in a predetermined mode (eg, to achieve controlled release of the formulation) or it may be adapted to not release the active drug until after passage through the stomach (enteric coating). The coating may be a sugar coating or a film coating (for example, based on hydroxypropylmethylcellulose, methylcellulose, methylhydroxyethylcellulose, hydroxypropylcellulose, carboxymethylcellulose, acrylate copolymer) , polyethylene glycol and / or polyvinylpyrrolidone), or enteric coating (for example, methyl in methacrylic acid copolymer, cellulose acetate phthalate, hydroxypropyl methylcellulose phthalate Acid ester, hydroxypropyl methyl succinate acetate, polyvinyl acetate phthalate, shellac and/or ethyl cellulose). Further, a time delay material such as glyceryl monostearate or glyceryl distearate may be utilized.

The solid tablet composition can 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 can be applied in a similar manner to a solid dosage form, such as the Encyclopedia of Pharmaceutical Technology described above.

The at least two therapeutic agents can be mixed together into a lozenge or can be dispensed. In one example, the first active anti-tumor forming therapeutic agent is included in the interior of the tablet, and the second active therapeutic agent is external, such that a substantial portion of the second therapeutic agent is the first therapeutic agent Release before release.

Formulations for oral use can also be presented in the form of chewable lozenges or in hard gelatin capsules wherein the active ingredient is combined with an inert solid diluent (for example, potato starch, lactose, microcrystalline cellulose, calcium carbonate, phosphoric acid) Calcium or kaolin); or a soft gelatin capsule in which the active ingredient is mixed with water or an oily medium such as peanut oil, liquid paraffin, or olive oil. Powders and capsules can be prepared using the above ingredients and in conventional methods such as a mixer, a fluidized bed apparatus or a spray drying apparatus under the tablet and capsule.

Controlled release oral dosage form

Controlled release compositions for oral use can be constructed to release active anti-tumor forming or anti-inflammatory therapeutic agents by controlling the dissolution and/or diffusion of the active substance. Controlled release dissolution or diffusion can be achieved by suitably coating the lozenge, capsule, pill or granule formulation of the compound, or by incorporating the compound into a suitable matrix. The controlled release coating may comprise one or more of the above materials and/or the following: shellac, beeswax, sugar wax, ramie wax, carnauba wax, stearyl alcohol, monostearyl Acid glyceride, glyceryl distearate, glyceryl palmitate, ethyl cellulose, acrylic resin, dl-polylactose, cellulose acetate butyrate, polyvinyl chloride, polyethylene ethyl ester, polyethyl b Acetrolidone, polyethylene, polymethacrylate, methyl methacrylate, 2-hydroxy methacrylate, methacrylate hydrogel, 1,3 butanediol, hydroxyethyl methacrylate, And / or polyethylene glycol. In controlled release matrix formulations, the matrix material may also include, for example, hydrated methylcellulose, canobaba wax and stearyl alcohol, carbopol 934, polyfluorene oxide, glyceryl tristearate, methacrylic acid. Ester-methyl methacrylate, polyvinyl chloride, polyethylene and/or halogenated fluorocarbons.

The controlled release composition comprising one or more therapeutic compounds can also be in the form of a floating ingot or capsule (i.e., a lozenge or capsule that floats on top of the stomach volume over a period of time when administered orally). A floating ingot of a compound can be obtained by granulating a compound containing an excipient and a mixture of 20 to 75 w/w% of a hydrocolloid (for example, hydroxyethylcellulose, hydroxypropylcellulose or hydroxypropylmethylcellulose) And made. The resulting granules are then compression molded into tablets. When in contact with gastric fluid, the tablet forms a substantially water impermeable gel shield around its surface. This gel shield is involved in maintaining a density of less than one, thereby allowing the tablet to float in the gastric juice.

Combination therapy

Optionally, anti-tumor forming or anti-inflammatory therapeutic agents can be administered in combination with any of the other standard anti-tumor forming or anti-inflammatory therapeutic agents known in the art; such methods are well known to those skilled in the art. It is well known and described in Remington's Pharmaceutical Sciences by EW Martin. If desired, the agents of the invention (e.g., JQ1, a compound of the formulae described herein, and derivatives thereof) can be combined with any conventional anti-tumor treatment, including but not limited to surgery, radiation therapy, or chemotherapy. . Better concrete In the examples, the compounds of the invention are administered in combination with a genetic modulator or a transcriptional regulator (eg, a DNA methyltransferase inhibitor, a histone deacetylase inhibitor (HDAC inhibitor), an amine) Acid methyltransferase inhibitors), and anti-mitotic drugs (eg, taxanes, vinca alkaloids), hormone receptor modulators (eg, estrogen receptor modulators, androgen receptors) Regulators, cell signal pathway inhibitors (eg, tyrosine kinase inhibitors), protein stability regulators (proteasome inhibitors), hsp90 inhibitors, traditional chemotherapeutic agents, glucocorticoids, all-trans All-trans retinoic acid or other agent that promotes differentiation.

Set or medical system

The compositions of the invention may be assembled into kits or medical systems for use in ameliorating tumor formation or inflammatory diseases. In accordance with aspects of the present invention, a kit or medical system includes a carrier, i.e., a carton, carton, tube, etc., having a sealed confinement wherein one or more containers such as vials, tubes, ampoules, bottles, and the like. Kits or medical systems of the invention may also include instructions for using the reagents of the invention.

The practice of the present invention, unless otherwise indicated, utilizes the traditional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are within the skill of the art. This technique is fully illustrated in the following documents, for example: `: A Laboratory Manual", second edition (Sambrook, 1989); "Oligonucleotide Synthesis" (Gait, 1984); "Animal Cell Culture" (Freshney, 1987); "Methods in Enzymology""Handbook of Experimental Immunology" (Weir, 1996); "Gene Transfer Vectors for Mammalian Cells" (Miller and Calos, 1987): "Current Protocols in Molecular Biology" (Ausubel, 1987); "PCR: The Polymerase Chain Reaction", (Mullis, 1994) "Current Protocols in Immunology" (Coligan, 1991). These techniques can be applied to the preparation of polynucleotides and polypeptides of the invention, and thus can be used to prepare and practice the invention. Specific useful techniques for particular embodiments It will be discussed in the following sections.

The following examples are presented to enable those of ordinary skill in the art to fully disclose and describe the methods of <RTIgt; </ RTI> <RTIgt; </ RTI> <RTIgt; The inventors of the present invention are limited only to the scope of the present invention.

Example

The practice of the present invention, unless otherwise indicated, utilizes the traditional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are within the skill of the art. This technique is fully exemplified in the following documents, for example: Molecular Cloning: A Laboratory Manual", second edition (Sambrook, 1989); "Oligonucleotide Synthesis" (Gait, 1984); "Animal Cell Culture" (Freshney, 1987); "Methods In Enzymology ""Handbook of Experimental Immunology" (Weir, 1996); "Gene Transfer Vectors for Mammalian Cells" (Miller and Calos, 1987): "Current Protocols in Molecular Biology" (Ausubel, 1987); "PCR: The Polymerase Chain Reaction", (Mullis, 1994); "Current Protocols in Immunology" (Coligan, 1991). These techniques are applicable to the preparation of polynucleotides and polypeptides of the invention, and thus can be used to prepare and practice the invention. Specific useful techniques for particular embodiments are discussed in the following sections.

The following examples are presented to enable those of ordinary skill in the art to fully disclose and describe the methods of <RTIgt; </ RTI> <RTIgt; </ RTI> <RTIgt; The inventors of the present invention are limited only to the scope of the present invention.

I. Chemical Examples - Synthesis and Process

The compounds of the present invention can be synthesized according to the methods described herein, and/or by the methods described herein, in accordance with methods generally known to those skilled in the art.

(2-amino-4,5-dimethylthiophen-3-yl)(4-chlorophenyl)methanone ( S2 )

Compound JQ1 was prepared according to the reaction scheme shown above.

Add solid sulfur (220 mg, 6.9 mmol, 1.00 equiv) to 4-chlorobenzamide acetonitrile S1 (1.24 g, 6.9 mmol, 1 equiv), 2-butanone (0.62 ml, 6.9 mmol, 1.00) Equiv) and morpholine (0.60 ml, 6.9 mmol, 1.00 equiv) in ethanol (20 ml, 0.35 M); After 12 hours, the reaction mixture was cooled to 23 ° C ²¹ and poured into brine (100 mL). The aqueous layer was extracted with ethyl acetate (3×50 mL). The combined organic layers were washed with EtOAc EtOAc. The residue was purified by flash column chromatography eluting elut elut elut elut elut elut elut

3-({[(9H-茀-9-yl)methoxy)carbonyl}amino)-4-{[3-(4-chlorobenzylidenyl)-4,5-dimethylthiophene-2 -amino]amino}-4-ketobutyric acid ( S )-t-butyl ester ( S3 )

Add hexafluorophosphoric acid (2-(6-chloro-1H-benzotriazol-1-yl)-1,1,3,3-tetramethylammonium (HCTU) (827mg, 2.0mmol, 2.00equiv) and N , N -diisopropylethylamine (0.72ml, 4.0mmol, 4.00equiv) to 9-fluorenylmethoxycarbonyl-aspartic acid β- t -butyl ester [Fmoc-Asp(O t -Bu)-OH (864mg, 2.1mmol, 2.10equiv) in N , N -dimethylformamide (1.5ml, 1.0M). The mixture was then stirred for 5 minutes at 23 ° C. Then added S2 (266 mg, The reaction mixture was stirred at 23 ° C. After 16 hours, ethyl acetate (20 mL) and brine (20 mL) was evaporated. The organic layer was washed with EtOAc EtOAc (EtOAc m. Class) gave S3 (625 mg, 90%) as a brown oil.

3-amino-4-((3-(4-chlorobenzylidenyl)-4,5-dimethylthiophen-2-yl)amino)-4-ketobutyric acid ( S )-third Butyl ester ( S4 )

Compound S3 (560 mg, 0.85 mmol, 1 equiv) was dissolved in 20% piperidine in DMF (4.0 mL, 0.22 M). After 30 minutes, ethyl acetate (20 mL) and brine (20 mL) were evaporated. The two layers were separated and the aqueous extracted with EtOAc EtOAc. The combined organic layers were washed with EtOAcq. The residue was purified by flash column chromatography to (Combiflash RF system, 24 g silica gel, gradient 0 to 100% ethyl acetate - hexanes) to afford the free amine as a yellow solid of S4 (370mg, 90%). The image isomer purity was dropped to 75% (using an AS-H column, as determined by Berger Supercritical Fluid Chromatography (SFC)).

2-(5-(4-Chlorophenyl)-6,7-dimethyl-2-keto-2,3-dihydro-1H-thieno[2,3-e][1,4] (indolyl-3-yl)acetic acid ( S )-t-butyl ester ( S5 )

The amine ketone ( S4 ) (280 mg, 0.63 mmol) was dissolved in 10% aqueous acetic acid (21 mL, 0.03M); After 30 minutes, all solvents were removed under reduced pressure. The residue was purified by flash column chromatography (EtOAc EtOAc) The image isomer purity of S5 was 67% (determined by Berger Supercritical Fluid Chromatography (SFC) using an AS-H column).

2-(5-(4-chlorophenyl)-6,7-dimethyl-2-thioketo-2,3-dihydro-1H-thiophene [2,3-e][1,4]diazepin-3-yl)acetic acid tert-butyl ester (S6)

Add phosphorus pentasulfide (222 mg, 1.0 mmol, 2.00 equiv), sodium hydrogencarbonate (168 mg, 2.0 mmol, 4.00 equiv) to S5 (210 mg, 0.5 mmol, 1 equiv) of diethylene glycol dimethyl ether (1.25 ml, 0.4). M) in solution; heat the reaction mixture to 90 °C. After 16 hours, brine (20 ml) and ethyl acetate (35 ml) were added. The two layers were separated and the aqueous extracted with EtOAc EtOAc. The combined organic layers were washed with EtOAcq. The residue was purified by flash column chromatography (Combiflash EtOAc, EtOAc (EtOAc) 73mg, 34%).

2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a ][1,4]diazepine-6-yl)acetic acid tert-butyl ester [(±) JQ1 ]

A solution of hydrazine (0.015 ml, 0.45 mmol, 1.25 equiv) in THF (2.6 mL, 0.14 M) was obtained from EtOAc. The reaction mixture was warmed to 23 ° C and stirred at 23 ° C for 1 hour. All solvents were removed under reduced pressure. The obtained hydrazine was used without further purification. The hydrazine was then dissolved in a 2:3 mixture of trimethyl orthoacetate and toluene (6 ml, 0.06 M); the reaction mixture was heated to 120 °C. After 2 hours, all solvents were removed under reduced pressure. The residue was purified by flash column chromatography (EtOAc EtOAc EtOAc (EtOAc) The reaction conditions further heteroisomerize the stereogenic center to produce racemate JQ1 (as measured by Berger Supercritical Fluid Chromatography (SFC) using an AS-H column).

3-({[(9H-茀-9-yl)methoxy)carbonyl}amino)-4-{[3-(4-chlorobenzylidenyl)-4,5-dimethylthiophene-2 -amino]amino}-4-ketobutyric acid ( S )-t-butyl ester ( S3 )

Add (benzotriazol-1-yloxy)tripyrrolidinium oxime (PyBOP) (494 mg, 0.95 mmol, 0.95 equiv), N,N-diisopropylethylamine (0.50 ml, 2.8 mmol, 2.75 equiv) N-N-dimethyl group to 9-mercaptomethoxycarbonyl-aspartic acid β-t-butyl ester [Fmoc-Asp(Ot-Bu)-OH] (411 mg, 1.00 mmol, 1.0 equiv) This solution was taken up in decylamine (1.0 ml, 1.0 M); then the mixture was stirred at 23 ° C for 5 min. S2 (266 mg, 1.0 mmol, 1 equiv) was then added as a solid; the 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 extracted with EtOAc EtOAc. The combined organic layers were washed with EtOAc EtOAc m. The residue was purified by flash column chromatography eluting elut elut elut elut elut elut elut elut

3-amino-4-((3-(4-chlorobenzylidenyl)-4,5-dimethylthiophen-2-yl)amino)-4-ketobutyric acid ( S )-third Butyl ester ( S4 )

Compound S3 (310 mg, 0.47 mmol, 1 eq) was dissolved in 20% piperidine in DMF (2.2 mL, 0.22M). After 30 min, ethyl acetate (20 mL) Brine (20 ml). The two layers were separated and the aqueous extracted with EtOAc EtOAc. The combined organic layers were washed with EtOAcq. The residue was purified by flash column chromatography (EtOAc EtOAc (EtOAc) The purity was 91% (determined by Berger Supercritical Fluid Chromatography (SFC) using an AS-H column).

2-(5-(4-Chlorophenyl)-6,7-dimethyl-2-keto-2,3-dihydro-1H-thieno[2,3-e][1,4] (indolyl-3-yl)acetic acid ( S )-t-butyl ester ( S5 )

The amine ketone ( S4 ) (184 mg, 0.42 mmol) was dissolved in toluene (10 ml, 0.04M), EtOAc (300 mg) was added and the mixture was warmed to 90 °C. After 3 hours, the reaction mixture was cooled to 23 ° C, EtOAc was filtered, washed with ethyl acetate. The residue was purified by flash column chromatography to (Combiflash RF system, 12 g silica gel, gradient 0 to 100% ethyl acetate - hexanes) to afford the compound S5 as a white solid (168mg, 95%). The image isomer purity of S5 was 90% (determined by Berger Supercritical Fluid Chromatography (SFC) using an AS-H column).

2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a ][1,4]diazepine-6-5yl)acetic acid ( S ) -t- butyl ester [(+) JQ1 ]

Add a solution of potassium butoxide (1.0 M solution in THF, 0.3 ml, 0.30 mmol, 1.10 equiv) to S5 (114 mg, 0.27 mmol, 1 equiv) in THF (1.8 ml, 0.15 M) at -78 °C in. 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 to -10 °C over 45 min. Ethyl hydrazine (30 mg, 0.40 mmol, 1.50 equiv) was added to the reaction mixture, and the mixture was stirred at 23 °C. After 1 hour, 1-butanol (2.25 ml) was added to the reaction mixture, which was heated to 90 °C. After 1 h, all solvents were removed under reduced pressure and the residue was purified by flash column chromatography (Combiflash system, 4 g of silica gel, gradient 0 to 100% ethyl acetate-hexanes) to give a white solid (+) - JQ1 (114 mg, 92%), mirror image purity 90% (using AS-H column, determined by Berger supercritical fluid chromatography (SFC), 85% hexanes-methanol, 210 nm, t _R (R - enantiomer) = 1.59min, t _R (S- enantiomer) = 3.67min). This product was further purified by palm preparative HPLC (using an OD-H column, as determined by Agilent High Pressure Liquid Chromatography (SFC)) to afford an S -mirrion isomer of greater than 99% ee.

¹ H NMR (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.

C ₂₁ H ₂₄ ClN ₂ O ₃ S calculated values of HRMS (ESI) [M + H ] +: 457.1460, found 457.1451m / z.

TLC (EtOAc), Rf : 0.32 (UV)

[α] ²² _D = +75( c 0.5, CHCl ₃ )

Synthesis of (-)-JQ1 in a similar manner, using Fmoc-D-Asp(O t -Bu)-OH as starting material, further purified by palm preparative HPLC (using Agilent high pressure liquid layer of OD-H column) Analytical method), R -image isomers greater than 99% ee were obtained. [α] ²² _D =-72( c 0.5, CHCl ₃ )

Synthesis of other compounds

Other compounds of the invention were prepared as illustrated by Reaction Scheme S3.

As shown in the reaction scheme S3, the third butyl ester of (+)-JQ1 (1) is cleaved to obtain a free acid (2) which is coupled with hydrazine to obtain hydrazine (3); and then reacted with 4-hydroxybenzaldehyde. Get 腙(4).

醯肼(3) and 腙(4) exhibit activity in at least one biological assay.

Preparation of compounds by reaction of ruthenium (3) with various carbonyl containing compounds Library (see Table A above).

Other compounds are prepared for use, for example, as probes for analytical development. A synthetic example is shown in the reaction scheme S4 below.

Prepare other compounds as shown in Table B below:

The spectral data of each compound corresponds to the specified structural formula.

II. Biological activity Example 1: JQ1

Figure 1 shows the JQ1 mirror image isomer.

Example 2: JQ1 replaces BRD4 derived from nuclear chromatin in cells

To determine whether JQ1 competes with chromatin for binding to bromo functional sites in the nuclear environment, it is a fluorescence recovery (FRAP) experiment after photobleaching of BRD4. The previous studies have demonstrated that after the selective discrete area ²⁷ nuclear chromatin photobleaching, FRAP bromine-containing functional sites to assess the chimeric fluorescent lateral redistribution utility of progress. Human osteosarcoma cells (U2OS) transfected with GFP-BRD4 exhibited fluorescence intensity recovery over time (Figures 2A and 2B). In the presence of JQ1 (500 nM), the observed response was nearly identical to the replaced nuclear BRD4 (Figs. 3A and 3B). Cell FRAP studies confirmed that the effect of BRD4 migration differentiation was limited to biochemically active (+)-JQ1 stereoisomers (Fig. 2A-2C).

After potency and selective binding to BRD4 in homologous and cell line assays, the effect of JQ1 on disease-related, BRD4-dependent cell phenotypes was assessed. In NMC, the pathogenic BRD4-NUT fusion protein produced by the t(15;19) translocation is eager to bind to the concentration point of the acetylated chromatin discrete, conferring proliferation advantages and differentiation. The ability of JQ1 to directly target BRD4-NUT oncoprotein was assessed using FRAP. JQ1 (500 nM) significantly accelerated the fluorescence intensity recovery time in the photobleaching region of cells transfected with GFP-BRD4-NUT compared to the vehicle control group (Fig. 3C, 3E). Importantly, no effect on the redistribution of GFP-NUT was observed (Fig. 3D, 3E). Taken together, these data are consistent with the binding of JQ1 in cultured cells to BRD4.

Example 5: JQ1 induces squamous differentiation and growth arrest of BRD4-dependent carcinoma

A gene product that directly inhibits the expression of relapsing, oncogene translocation is a proven cancer treatment method ^28,29 . To investigate the phenotypic results of chemically inhibited BET family bromine functional sites on line cancer in BRD4-dependent NUT. The BRD4-NUT chromatin localization is mechanically linked to the conserved tandem bromine functional site of BRD4 in fusion protein 17. A typical feature of NMC is the appearance of discrete nuclear spots of BRD4-NUT oncoprotein when NUT-directed immunohistochemistry (IHC) ³⁰ is utilized. The 797 NMC cell line obtained from the patient was treated with JQ1 (500 nM) for 48 hours, and the nuclear lesion was erased to produce a diffused IHC-stained nuclear NUT (Fig. 3f).

In the NMC cell line ¹⁷ , a significant differentiation phenotype was observed when the BRD4-NUT was knocked down. JQ1 produces a heterogeneous, dose- and time-varying differentiation phenotype in NMC 797 and Per403 cells characterized by cell elongation and flattening, open chromatin and spindle morphology (3g and 4A, 4B, 4C) Figure). Rapid differentiation (<24 hours) and characterized by significant enhancement of cell f5c, a hallmark of squamous differentiation (Fig. 3H). Terminal differentiation was observed after 7 days of exposure to JQ1 at low doses below the micromolar concentration. Importantly, non-BRD4-dependent squamous cell lines (TE10 and TT) did not exhibit the differentiation effect of JQ1 (Figs. 4A, 4B, 4C). In BRD4-dependent NMC cells, differentiation was expected to be accompanied by growth arrest, as evidenced by a decrease in Ki67 staining (3i, j, and 4L-a, 4L-b map). Further support for the precise mechanism of action is that differentiation and growth stagnation phenotypes are only caused by (+)-JQ1, while (-)-JQ1 does not show observable effects (Fig. 4E-a-4E-d).

It is worth noting that the morphological changes in the JQ1 treatment phenotype and the BRD4-NUT using the RNA interference technique silently observed an increase in keratin expression (Fig. 4F-a, b). In order to confirm their morphological and IHC studies, the performance analysis of three typical squamous genes of RT-PCR was used to identify that keratin-14 was significantly (30-fold) induced by (+)-JQ1 in NMC 797 cells ( Figure 3i). And keratin-10 induced moderate epidermal transglutaminase enzyme Amides amine (TGM1) deficiency on the thorax with progressive differentiation of squamous epithelium consistent with the NMC 797 cells were obtained from primary tumors longitudinal septum ²⁸ coincide. The differentiation induction line stained with concentrated (3+) keratin using IHC was gradually performed during 72 hours as determined by quantitative IHC analysis (Fig. 4M). The support of the accurate mechanism of action is that the differentiated phenotype is only caused by (+)- JQ1 , while (-)- JQ1 does not show observable effects. Importantly, non-BRD4-dependent squamous cell lines (TE10) did not exhibit differentiation effects from JQ1 treatment (Fig. 4N-c). In BRD4-dependent NMC cells, differentiation was expected to be accompanied by growth arrest, as evidenced by a decrease in Ki67 staining (Fig. 4A-4G).

Next, the antiproliferative activity of the JQ1 mirror isomer in BRD4-dependent (797 and Per403) and BRD4-independent (TE10 and TT) human squamous carcinoma cell lines was evaluated. As shown in Figures 5a and 4F-a-c, (+)-JQ1 uniquely exhibited dose-dependent inhibition of cell growth in BRD4-dependent cell lines (797 IC50 = 140 nM; Per403 IC50 = 60 nM). The use of strong anti-proliferative effects and irreversible differentiation observed by IHC suggests induction of cell death. Therefore, early and late apoptosis was evaluated by flow cytometry with annexin-V and propidium iodide staining. As expected, JQ1 induced immediate and progressive apoptosis in BRD4-dependent human cancer cells, but in cells that did not carry BRD-NUT fusion, no significant amount of apoptosis was detected at the concentrations used. Cells (Fig. 5b and Fig. 7).

Example 6: In vivo efficacy and pharmacodynamics of JQ1 in NMC murine model effect

To determine whether JQ1 can attenuate the growth of BRD4-dependent cancer in a single preparation in vivo, a NMC 797 cell line was used to form a NMC xenograft pattern in mice. Short-term treatment studies were performed using PET imaging as the primary endpoint, followed by non-invasive imaging to investigate whether JQ1 anti-tumor activity can be demonstrated. An established and comparable burden of matched mouse populations with predictable disease was randomized to treatment with JQ1 (50 mg kg ^-1 ) or vehicle, administered daily via intraperitoneal injection. Mice were evaluated by PET imaging before randomization and after four days of treatment. It was observed that JQ1 treatment had a significant effect on FDG uptake, while animals treated with vehicle showed significant, progressive disease (Fig. 5e).

At the same time, the effect of JQ1 on tumor volume was studied by using calipers to measure and use the pharmacodynamic effects of quantitative IHC for matched mice with NMC 797 tumors. On the 14th day of treatment, the aggressive growth of NMC 797 xenografts prompted the study to be terminated, after which all vehicle-treated animals were close to the institutional tumor size limit. Animals receiving JQ1 exhibited statistically significant reductions in tumor growth (Fig. 5c, p=0.039; two-tailed t -test). To obtain comparative mechanical and pharmacodynamic data, all animals were sacrificed and removed from the tumor for histopathological analysis. It is worth noting that JQ1 is well tolerated at this dose and course of treatment, with no signs of toxicity or significant weight loss (5th, 5th).

In order to confirm that the anti-tumor effect observed by JQ1 treatment is associated with target engagement, it is to examine the BRD4-NUT oncoprotein of the sliced tumor tissue. As shown in Figures 5e and 6A, JQ1 treatment leads to the erasure of NUT nuclear spots, which is consistent with competitive binding to nuclear chromatin. Cell elongation and keratin expression increased, confirming the squamous differentiation of pharmacodynamics. The nuclear staining of the treated animal Ki67 was reduced, and TUNEL staining was increased, confirming that anti-proliferation and apoptosis effects were ongoing. In summary, their data provide a mechanical correlation between BRD4 inhibition in vivo and JQ1 treatment response.

In order to address the pharmacokinetic (PD) biomarkers of tumor keratin expression in an unbiased manner, an experimental procedure for quantitative IHC image acquisition and analysis was established. Paired samples from treated and untreated animals were prepared and analyzed using standardized experimental procedures and commercially available software (ImageScope; Aperio Technologies). JQ1 in the NMC 797 xenografts, uniformly in the excised tumor samples, caused strong (3+) keratin expression (Fig. 6Ba-e).

In parallel with their studies, a 29-year-old patient with BRD4-NUT-positive NMC who had extensive metastases from the mediastinum was identified. Under the goal of developing a more clinically relevant disease model, short-term cultures were established using clinical waste obtained from pleural effusions from patients with mild chest drainage. As shown in Figure 8, in vitro studies confirmed the robust effect of (+)-JQ1 on cell viability (IC50 = 4 nM), growth and cell cycle progression. Four animals transplanted from the patient's tumor material formed a foreseeable disease and were strongly PET positive (Fig. 5h). Animals were randomly assigned to vehicle or (+)-JQ1 treatment groups. Mice were evaluated by PET imaging before treatment assignment and after four days of treatment. Significant FDG uptake responses were observed with (+)-JQ1 treatment, while animals treated with vehicle showed significant, progressive disease (Fig. 5i). Again, tumor material was prepared for quantitative IHC analysis to demonstrate keratin expression after treatment with (+)-JQ1 (Fig. 5j, k, Fig. 9). In summary, their data provide a correlation between BRD4 inhibition in vivo and JQ1 response.

Under the emerging and complex variability of cancer genomes, repeated chromosomal rearrangements constitute a striking subset of clear, genetic targets in cancer. Successfully developed CML-targeted BCR-ABL kinase inhibitors, which can be used to fully characterize probe compounds ^31,32 , high-resolution crystallographic ^data33 , translation studies ³⁴ , and information-based rodent models ³⁵ will provide the best platform for ligand discovery and target validation. As described herein, novel BRD4-directed small molecule inhibitors are likely to be used to treat genetically defined human squamous cell carcinoma associated with NUT-BRD4 fusion.

In addition to NUT midline cancer, the BET family bromine functional site contributes to many other oncological and non-neoplastic diseases. BRD4 directs the P-TEFb complex to the mitotic chromosome, resulting in the expression of growth promoting genes such as c-Myc ^{11 9} and the identified cancer target Aurora B ¹³ . Further, BRD4 ³⁶ is amplified in breast cancer, the prediction of survival of breast cancer patients indicator ^37. In addition to their functions in cancer biology, members of the BET family have been identified for the replication of viruses ^{38, 39} and the genes necessary for the transmission of inflammatory responses ¹⁴ . Therefore, the availability of (+)-JQ1 and (-)-JQ1 for paired chemical probes will broadly stimulate research on information in transcription, development, and disease biology. It has also been found that JQ1 exhibits some off-target effects and excellent pharmacokinetic properties (including 49% oral bioavailability) for cell receptors (Fig. 10), establishing the credibility of drug-like derivatives for therapeutic applications.

The discovery and optimization of small molecule inhibitors of epigenetic targets is the main focus of current biomedical research. Methods success so far is limited to the identification of chromatin-modifying enzymes ligands of the body ^40. The most studied may be a regulator of deacetylation of the side chain of the amino acid, for which many pharmaceutical inhibitors have been developed clinically. The present invention provides compositions and methods for the formation of strong, selective inhibitors of epigenetic readers, including inhibitors of the first, fully characterized BET family bromine functional site. In view of this finding, the methods outlined herein can identify other candidates for identifying other inhibitors of selectivity within the BET family.

Example 7: JQ1 mirror image isomer binding and inhibition of BRD4.1 and BRD4.2

Figures 11 and 12 show the binding of JQ1 mirror image isomers and inhibition of BRD4.1 and BRD4.2. Figure 13 shows a comparison of JQ1S binding and inhibition of BET family members (activity) and CBP (inactive). Figure 14 shows the results of the dose range study of the JQ1 derivative focus library (see Table B for the compound structure).

Example 8: JQ1 is effective in treating BRD3-NUT cancer

The mice were implanted subcutaneously with BRD3-NUT cells. Tumor measurement data was collected once a week. When the tumor was palpable, the mice were divided into two treatment groups (n=10): the vehicle was administered intraperitoneally (IP) with 50 mg/kg JQ1 and 7 days per week IP for 7 days a week. On day 24 after the injection, five mice from each group were euthanized, and tumor samples were collected for laboratory analysis. Tumor samples were collected at the time of sacrifice at the time of the remainder of the study. Of all the samples, half of the tumors were fixed in 10% fumarin and the other half was rapidly frozen on dry ice. Treatment was terminated on day 32. Tumor volume and weight were collected weekly until all mice were dying or any size of the tumor reached 2 cm. Figure 15A shows that JQ1 exhibits in vivo efficacy against BRD3-NUT in a murine xenograft mode. Graphics. Treatment with JQ1 caused the tumor to resolve during treatment; after treatment discontinuation, the tumor resumed growth. Tumor volume differences were significant at all time points (p=7.65274E-09 on day 24); JQ1 dose was 50 mg/kg. Figure 15B shows that mice treated with JQ1 exhibited a mild weight loss, but responded promptly after treatment discontinuation.

Example 9: JQ1 and its analogs have efficacy in a variety of cancers

Figures 16A-16D show that JQ1 and its derivatives inhibit Brd4.1 and Brd4.2 binding at 5 μM JQ1 (see Table A for the structural formula of the compound). Figures 17A-17D show NUT midline cancer (NMC) cell viability after treatment with JQ1 and its derivatives at a compound concentration of 2 μM (see Table A for the structural formula of the compound). Figures 18-55 show the dose response efficacy for various cancer cell lines. Their data indicate that JQ1 and its derivatives have anti-cancer effects on a wide range of symptoms.

The results described herein were obtained using the methods and materials described below.

Example 10: Combining analysis results

The results of the combined analysis are shown in Table C below.

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

In the cell analysis, the activity of the lead compound was examined by the 797 cell line (derived from the patient) to determine the effect of BRD4 inhibition on the growth of the BRD4-NUT-dependent cell line. Cells and compounds were incubated together and proliferation was monitored after 72 hours. The curve fit was calculated using logistic regression (Fig. 56C). All lead compounds were tested in a cell-analysis using a 10326 cell line derived directly from the patient to determine the effect of BRD4 inhibition on the growth of BRD4-NUT-dependent cell lines (Fig. 56D). Cells and compounds were incubated together and proliferation was monitored after 72 hours. The curve fit was calculated using logistic regression.

Reagent

Endogenous BRD4-NUT exhibits midline cancer cell lines, 7971 and PER-4032, as previously described. Non-NMC human squamous carcinoma cell lines TE10 and TT were obtained from Drs. Anil Rustgi (University of Pennsylvania) and Adam Bass (Dana-Farber Cancer Institute). U2OS cells were obtained from ATCC. A large number of mammalian expression constructs for GFP-BRD4, GFP-NUT and GFP-BRD4-NUT have been previously described ³ . Tissue culture media, trypsin, and antibiotics were purchased from Mediatech. Antibodies and stains for immunohistochemistry and flow cytometry were obtained from Dako (cytokeratin AE1/AE3 antibody Cat# N1590), Millipore (TUNEL Cat# S7100), Vector (Ki67 Cat# VP-RM04), Cell Signaling Technologies (NUT Cat # 3625), BD Pharmingen (Annexin V-FITC Cat # 556547) and Invitrogen (Catidinium Catidine P#566).

Ethyl-histone binding assay

⁸ as previously described for analysis, but the manufacturer (PerkinElmer, USA) Protocol slight modifications. All reagents were diluted in 50 mM HEPES supplemented with 0.05% CHAPS, 100 mM NaCl, 0.1% BSA (pH 7.4) and allowed to equilibrate to room temperature before being added to the plates. Prepare 24 points 1:2 serial dilutions of ligands with a concentration range of 150-0 μM and transfer 4 μl into a low volume 384-well plate (ProxiPlateTM-384 Plus, PerkinElmer, USA) followed by 4 μl of HIS-labeled protein ( BRD4/1, 250nM, BRD4/2 and CREBBP, 2000nM). The wells were sealed and incubated at room temperature for 30 minutes, then 4 μl of biotin peptide with the molar concentration of the protein was added [BRD4(1) & BRD4(2) peptide: H4K5acK8acK12acK16ac, H-SGRGK(Ac)GGK(Ac GLGK (Ac) GGAK (Ac) RHRK (biotin) - OH; peptide of CREBBP: H3K36ac, biotin-KSAPATGGVK (Ac) KPHRYRPGT-OH (Cambridge Research Biochemicals, UK)]. The tray was sealed and incubated for another 30 minutes, then 4 μl of streptavidin-coated donor beads (25 μg/ml) and 4 μl of nickel chelate acceptor beads (25 μg/ml) were added under low light conditions. . The plates were sealed with aluminum foil to prevent light, incubated for 60 minutes at room temperature, and the data was read on a PHERAstar FS disk reader (BMG Labtech, Germany) using an AlphaScreen 680 excitation/570 emission filter set. After it is normalized to DMSO control corresponding to the calculated IC ₅₀ values in Prism 5 (GraphPad Software, USA), as the final concentration of the compound in a reaction volume of 20μl.

Sequence Alignment

The amino acid sequence of the full length human bromine functional site was obtained from NCBI (BRD2 Acession No. NP_005095, BRD3 Acession No. NP_031397.1, BRD4 Acession No. NP_055114.1, BRD-T Acession No. NP_001717.2). Multiple sequence alignments of individual bromo functional sites were performed using ClustalW ¹⁹ .

Fluorescence recovery after photobleaching (FRAP)

FRAP studies were performed on U2OS cells as previously described ^3,20 . Briefly, U2OS cells (Lipofectamine; Invitrogen) were transfected with a large number of mammalian expression constructs encoding GFP chimeras with BRD4, NUT and BRD4-NUT. The 5 μm ² nuclear region was bleached with high intensity laser in each field of view of the cells, and the recovery measurements were performed with low intensity laser and 150 μm pinhole. Images of the same field of view were acquired in 90 seconds using a Nikon C1 Plus confocal microscope equipped with a 37 ° C heating chamber and FRAP assembly. The time-averaged intensity of the bleached area was measured using MetaMorph v7 and normalized to an unrelated area of interest prior to bleaching; the data was then analyzed in Microsoft Excel Mac 12.2.4 to assess the time to recover half of the maximum fluorescence.

Differentiation and proliferation immunohistochemistry

Cultured cancer cell lines (797, Per403, at a concentration of 1.0×10 ⁴ cells per cell (4-chamber slide) or 5.0×10 ³ cells per cell (8-chamber slide) TT and TE10) were grown on chamber slides. Cells were treated with JQ1-racemate, (+)-JQ1, (-)-JQ1, or vehicle (DMSO) and incubated for different times. The medium was then removed and the chamber was washed with cold PBS. The cells were then fixed in 4% formaldehyde for 20 minutes at 4oC, washed with PBS, and transferred to Dana-Farber/Harvard Cancer Center (DF/HCC) Specialized Histopathology Services Core at Brigham and Women's Hospital for the following description. Dyeing. Immunoprecipitation of cell pellets The cancer cell lines (797 and Per403) were grown in T-75 flasks and treated with JQ1 or vehicle (DMSO) for 48 hours; then the cells were decomposed with trypsin at 2,000 Centrifuge at rpm for 10 minutes to form a cell pellet which was stabilized with a 1⁄2 volume HistoGel (Richard-Allen Scientific) and 10% bovine serum albumin. The cell pellet was washed with PBS and fixed in 4% formaldehyde for 20 minutes at 4 °C. The cells were then washed with PBS and transferred to DF/HCC Core Laboratory at Brigham and Women's Hospital for staining as described below. Quantitative analysis of Ki67 staining (cell scoring) was performed using an optical microscope, using five high magnification (40x) fields of view for each experiment. All fixed materials were embedded, sliced and stained by the DF/HCC Core Laboratory at Brigham and Women's Hospital using an established optimization protocol; Olympus BX40 microscope (Olympus Imaging America, Center Valley, PA) and Micropublisher 3.3 were used. Images were acquired by an RTV color camera (QImaging, Surrey, BC).

Cell proliferation analysis

The cells were seeded in a white, 384-well microtiter test plate (Nunc) in an amount of 500 cells per well in a total volume of 50 μL of medium. 797, TT and TE10 cells were grown in DMEM containing 1% penicillin/streptomycin and 10% FBS; Per403 cells were grown in DMEM containing 1% penicillin/streptomycin and 20% FBS. Compounds were delivered to microanalytical dishes using an automated needle transfer method (PerkinElmer JANUS, equipped with a V&P Scientific 100 nL needle instrument). After 48 hours of incubation at 37 ° C, the cells were lysed and the total ATP content of the troughs was assessed using a commercially available proliferation assay (CellTiterGlo; Promega). Dose for repeated measures Analysis calculated IC ₅₀ values estimated using logistic regression (GraphPad Prism).

Flow cytometry

In each groove starting concentration 5.0 × 10 ⁴ cells of the culture of human cancer cells (797, Per403, TT and the TE10) in the groove 6 tissue culture dish (BD Falcon) were grown. Cells were treated with JQ1 (500 nM), staurosporine (50 nM) or vehicle (DMSO 0.05%) for 24 or 48 hours. The trypsin-decomposed cells were 1:1 and annexin-V/iodine containing annexin V-FITC (BD Pharmingen, 1:500) and propidium iodide (Invitrogen, 1:1000) on ice. The propionate buffer (10 mM HEPES pH 7.4, 140 mM NaCl, 2.5 mM CaCl ₂ ) was mixed; the samples were immediately analyzed on a BD FACS Canto II. Visualization and analysis of apoptosis differentiation was generated using FlowJo flow cytometry analysis software (Tree Star, Inc.).

Xenotransplantation efficacy study

NMC xenografts were established by injection of 797 NMC cells (10 ⁷ ) in 30% Matrigel (BD Biosciences) into 6 weeks old female NCr nude mice (Charles River Laboratories). Twelve days after the injection, mice with measurable tumors were divided into groups treated with 50 mg/kg JQ1 IP or vehicle (5% DMSO in 5% dextrose solution). At the start of treatment, the average tumor size of the JQ1 treatment group (n=8) and the vehicle group (n=7) was similar (63.8 +/- 17.1 and 73.6 +/- 14.4 mm ^{3 , respectively} ). Pay close attention to the clinical symptoms of animals every day. The tumor size was evaluated using a caliper measurement method, and the volume was calculated by the formula Vol = 0.5 x LxW ² . After 2 weeks of treatment, all mice were humanely euthanized and tumors were fixed in 10% formalin for histopathological examination. Tumor volume was calculated to be statistically significant using the two-sided Student's t-test. All animal studies are accredited by the DFCI Laboratory Animal Management and Use Committee (IACUC).

Instrument use

Proton and carbon-13 nuclear magnetic resonance ( ¹ H NMR and ¹³ C NMR) spectra were recorded on a Varian reverse probe 600 INOVA spectrometer from Harvard Medical School East Quad NMR Facility. Chemical shifts are described in parts per million on the δ level, relating to ¹ H NMR, involving residual ruthenium in NMR solvents (CHCl ₃ : δ 7.24), and ¹³ C NMR, carbon resonance involving solvents (CDCl _{3 )} : δ 77.2). The data are described as follows: chemical shift [multimodality (s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, br = broad), unit is the coupling constant of Hertz, Integration]. High Resolution Mass Spectrometry (HRMS) was recorded on a Bruker APEX 4.7 Tesler FTMS spectrometer using an Electrical Spray Source (ESI) of the Instrumentation Facility of the Department of Chemistry, Massachusetts Institute of Technology. The intermediates and final products were purified using a Combiflash RF system (Teledyne Isco). The organic solution was concentrated on a Büchi R-205 rotary evaporator. The purity of the mirror image is tested using Berger Supercritical Fluid Chromatography (SFC) and AS-H column. The mirror image isomer was carried out using Agilent high pressure liquid chromatography and OD-H column (Broad Institute of Harvard and MIT).

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Other specific examples

It is apparent from the foregoing description that the invention described herein may be modified and modified to conform to various uses and circumstances; such specific examples are also within the scope of the following patent applications.

The recitation of a list of elements in any definition of the variables herein includes the definition of such a variable in any single element or combination (or sub-combination) of the listed elements. The detailed description of specific examples herein includes such specific examples in any single embodiment or in combination with any other specific examples or portions thereof.

This application is related to the following US provisional patent application: No. 61/334,991, filed on May 14, 2010, No. 61/370,745, filed on August 4, 2010; and application on August 22, 2010 The serial number is 61/375,863. The entire contents of each of their respective applications are incorporated herein by reference.

All patents and publications mentioned in this specification are hereby incorporated References are made as if each individual patent and publication were specifically and individually indicated as a reference.

<110> Dana-Farber Cancer Institute Ltd. DANA-FARBER CANCER INSTITUTE.INC.

<120> Composition and method for treating tumor formation, inflammatory diseases and other diseases

<130> 86363WO (70157)

<140> PCT/US2011/036701

<141> 2011-05-16

<150> 61/467,376

<151> 2011-03-24

<150> 61/375,863

<151> 2010-08-22

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<151> 2010-08-04

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Since the picture in this case is the result data, it is not the representative figure of this case.

Therefore, there is no designated representative map in this case.

Claims (11)

One selected from , , and The use of the group of compounds or a pharmaceutically acceptable salt thereof for the preparation of a medicament for treating a symptom selected from the group consisting of prostate cancer, renal cell carcinoma, liver cancer, lung cancer, breast cancer, colon cancer, and nerve A group consisting of a herma cell tumor, a polymorphic glioblastoma, a squamous cell carcinoma associated with NUT rearrangement, and a NUT midline cancer. The use according to claim 1, wherein the compound is represented by the following structural formula: Or a pharmaceutically acceptable salt thereof. The use according to claim 1, wherein the compound is represented by the following structural formula: Or a pharmaceutically acceptable salt thereof. The use of claim 2, wherein the compound is represented by the following structural formula: Or a pharmaceutically acceptable salt thereof. The use of claim 3, wherein the compound is represented by the following structural formula: Or a pharmaceutically acceptable salt thereof. The use of any one of claims 1 to 5 wherein the lung cancer is small cell lung cancer. The use according to any one of the preceding claims, wherein the lung cancer is non-small cell lung cancer. The use of any one of claims 1 to 5, wherein The symptom is NUT midline cancer. The use of any one of claims 1 to 5, wherein the symptom is prostate cancer. The use according to any one of claims 1 to 5, wherein the symptom is renal cell carcinoma. The use according to any one of claims 1 to 5, wherein the symptom is liver cancer.