WO2007084625A2 - Composes et procedes atypiques d’inhibition d’activite p53 - Google Patents

Composes et procedes atypiques d’inhibition d’activite p53 Download PDF

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WO2007084625A2
WO2007084625A2 PCT/US2007/001350 US2007001350W WO2007084625A2 WO 2007084625 A2 WO2007084625 A2 WO 2007084625A2 US 2007001350 W US2007001350 W US 2007001350W WO 2007084625 A2 WO2007084625 A2 WO 2007084625A2
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group
compound
aralkyl
heteroaryl
pharmaceutical composition
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Ming-Ming Zhou
D. Phil Sachchidanand
Lei Zeng
James J. Manfredi
Lois Resnick-Silverman
Stuart Aaronson
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Mount Sinai School Of Medicine
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    • C07C317/00—Sulfones; Sulfoxides
    • C07C317/26—Sulfones; Sulfoxides having sulfone or sulfoxide groups and nitrogen atoms, not being part of nitro or nitroso groups, bound to the same carbon skeleton
    • C07C317/32—Sulfones; Sulfoxides having sulfone or sulfoxide groups and nitrogen atoms, not being part of nitro or nitroso groups, bound to the same carbon skeleton with sulfone or sulfoxide groups bound to carbon atoms of six-membered aromatic rings of the carbon skeleton
    • C07C317/34—Sulfones; Sulfoxides having sulfone or sulfoxide groups and nitrogen atoms, not being part of nitro or nitroso groups, bound to the same carbon skeleton with sulfone or sulfoxide groups bound to carbon atoms of six-membered aromatic rings of the carbon skeleton having sulfone or sulfoxide groups and amino groups bound to carbon atoms of six-membered aromatic rings being part of the same non-condensed ring or of a condensed ring system containing that ring
    • C07C317/38—Sulfones; Sulfoxides having sulfone or sulfoxide groups and nitrogen atoms, not being part of nitro or nitroso groups, bound to the same carbon skeleton with sulfone or sulfoxide groups bound to carbon atoms of six-membered aromatic rings of the carbon skeleton having sulfone or sulfoxide groups and amino groups bound to carbon atoms of six-membered aromatic rings being part of the same non-condensed ring or of a condensed ring system containing that ring with the nitrogen atom of at least one amino group being part of any of the groups, X being a hetero atom, Y being any atom, e.g. N-acylaminosulfones
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    • C07C233/04—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having nitrogen atoms of carboxamide groups bound to hydrogen atoms or to carbon atoms of unsubstituted hydrocarbon radicals with carbon atoms of carboxamide groups bound to acyclic carbon atoms of an acyclic saturated carbon skeleton
    • C07C233/05—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having nitrogen atoms of carboxamide groups bound to hydrogen atoms or to carbon atoms of unsubstituted hydrocarbon radicals with carbon atoms of carboxamide groups bound to acyclic carbon atoms of an acyclic saturated carbon skeleton having the nitrogen atoms of the carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms
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    • C07C233/04—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having nitrogen atoms of carboxamide groups bound to hydrogen atoms or to carbon atoms of unsubstituted hydrocarbon radicals with carbon atoms of carboxamide groups bound to acyclic carbon atoms of an acyclic saturated carbon skeleton
    • C07C233/07—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having nitrogen atoms of carboxamide groups bound to hydrogen atoms or to carbon atoms of unsubstituted hydrocarbon radicals with carbon atoms of carboxamide groups bound to acyclic carbon atoms of an acyclic saturated carbon skeleton having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a six-membered aromatic ring
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    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C233/00—Carboxylic acid amides
    • C07C233/01—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms
    • C07C233/16—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by singly-bound oxygen atoms
    • C07C233/17—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by singly-bound oxygen atoms with the substituted hydrocarbon radical bound to the nitrogen atom of the carboxamide group by an acyclic carbon atom
    • C07C233/18—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by singly-bound oxygen atoms with the substituted hydrocarbon radical bound to the nitrogen atom of the carboxamide group by an acyclic carbon atom having the carbon atom of the carboxamide group bound to a hydrogen atom or to a carbon atom of an acyclic saturated carbon skeleton
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    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C233/00—Carboxylic acid amides
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    • C07C233/16—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by singly-bound oxygen atoms
    • C07C233/24—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by singly-bound oxygen atoms with the substituted hydrocarbon radical bound to the nitrogen atom of the carboxamide group by a carbon atom of a six-membered aromatic ring
    • C07C233/25—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by singly-bound oxygen atoms with the substituted hydrocarbon radical bound to the nitrogen atom of the carboxamide group by a carbon atom of a six-membered aromatic ring having the carbon atom of the carboxamide group bound to a hydrogen atom or to a carbon atom of an acyclic saturated carbon skeleton
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    • C07C233/45—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by carboxyl groups
    • C07C233/53—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by carboxyl groups with the substituted hydrocarbon radical bound to the nitrogen atom of the carboxamide group by a carbon atom of a six-membered aromatic ring
    • C07C233/54—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by carboxyl groups with the substituted hydrocarbon radical bound to the nitrogen atom of the carboxamide group by a carbon atom of a six-membered aromatic ring having the carbon atom of the carboxamide group bound to a hydrogen atom or to a carbon atom of a saturated carbon skeleton
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    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C323/00—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups
    • C07C323/50—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and carboxyl groups bound to the same carbon skeleton
    • C07C323/51—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and carboxyl groups bound to the same carbon skeleton having the sulfur atoms of the thio groups bound to acyclic carbon atoms of the carbon skeleton
    • C07C323/52—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and carboxyl groups bound to the same carbon skeleton having the sulfur atoms of the thio groups bound to acyclic carbon atoms of the carbon skeleton the carbon skeleton being acyclic and saturated
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    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D209/00—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom
    • C07D209/02—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom condensed with one carbocyclic ring
    • C07D209/04—Indoles; Hydrogenated indoles
    • C07D209/10—Indoles; Hydrogenated indoles with substituted hydrocarbon radicals attached to carbon atoms of the hetero ring
    • C07D209/12—Radicals substituted by oxygen atoms
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    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D209/00—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom
    • C07D209/02—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom condensed with one carbocyclic ring
    • C07D209/04—Indoles; Hydrogenated indoles
    • C07D209/30—Indoles; Hydrogenated indoles with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, directly attached to carbon atoms of the hetero ring
    • C07D209/32—Oxygen atoms
    • C07D209/38—Oxygen atoms in positions 2 and 3, e.g. isatin
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    • C07D209/00—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom
    • C07D209/56—Ring systems containing three or more rings
    • C07D209/80—[b, c]- or [b, d]-condensed
    • C07D209/82—Carbazoles; Hydrogenated carbazoles
    • C07D209/88—Carbazoles; Hydrogenated carbazoles with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to carbon atoms of the ring system
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    • C07D215/00—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems
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    • C07D215/04—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen atoms or carbon atoms directly attached to the ring nitrogen atom with only hydrogen atoms or radicals containing only hydrogen and carbon atoms, directly attached to the ring carbon atoms
    • C07D215/08—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen atoms or carbon atoms directly attached to the ring nitrogen atom with only hydrogen atoms or radicals containing only hydrogen and carbon atoms, directly attached to the ring carbon atoms with acylated ring nitrogen atom
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    • C07D221/02—Heterocyclic compounds containing six-membered rings having one nitrogen atom as the only ring hetero atom, not provided for by groups C07D211/00 - C07D219/00 condensed with carbocyclic rings or ring systems
    • C07D221/04—Ortho- or peri-condensed ring systems
    • C07D221/06—Ring systems of three rings
    • C07D221/08—Aza-anthracenes

Definitions

  • the present invention provides novel compounds that block p53 association with the co-activator CBP. This interaction is implicated in p53-induced transcription of the cell cycle inhibitor p21 in response to DNA damage.
  • the human tumor suppressor ⁇ 53 is a transcription factor that binds in a sequence- specific manner to particular sites in the genome and activates transcription of target genes Vousden, et al, (2002) Nat Rev Cancer 2, 594-604; Oren (2003) Cell Death Differ 10, 431-442; el-Deiry (1998) Semin Cancer Biol 8, 345-357). It plays a pivotal role in cellular response to stress signals in cell cycle arrest, senescence, DNA repair or apoptosis (Vogelstein, et al, (2000) Nature 408, 307-310; Levine (1997) Cell 88, 323-331 ; Prives et al. (1999) J. Pathol.
  • p53 The biological activity of p53 is tightly regulated by post-translational modifications in its N- and C-terminal regions (Alarcon- Vargas et al., (2002) Carcinogenesis 23, 541-547). Upon DNA damage, p53 is extensively phosphorylated within the N-terminal activation domain, which relieves it from association with the negative regulator Mdm2, resulting in p53 stabilization and activation (Haupt et al., (1997) Nature 387, 296-299; Kubbutat et al, (1997) Nature 387, 299-303; Momand et al, (1997) J. Cell. Biochem.
  • the transcriptional co-activator histone acetyltransferase p300/CBP (CREB-binding protein) has been shown to acetylate K373, K382 and to a lesser extent K372 and K381 , whereas another co- activator p300/CBP-asso dated factor (PCAF) acetylates K320.
  • PCAF co- activator p300/CBP-asso dated factor
  • Acetylated K382 in p53 may serve as a binding site for the CBP bromodomain and that this bromodomain/acetyl-lysine binding is responsible for p53 acetylation-dependent coactivator recruitment after DNA damage, a step that is essential for p53-induced transcriptional activation of the cyclin-dependent kinase inhibitor p21 in Gl cell cycle arrest (Mujtaba et al, (2004) MoI Cell 13, 251-263). [0004] Despite the fact that these post-translational modifications are known to play an important role in p53 function, specific effects of single or combinatorial modifications on p53 function remain elusive.
  • R and R' are independently selected from the group consisting of hydrogen, lower alkyl, aryl, aralkyl; substituted aralkyl, heteroaryl; substituted heteroaryl, phenyl, benzyl, SO 2 , NH 21 NO 2, SO 2 , CH 3 , CH 2 CH 3 , OCH 3 , OCOCH 3 , CH 2 COCH 3 , OH, CN 3 halogen, carboxy, and alkoxy and their pharmaceutically acceptable salts of acids or bases.
  • the general formula (I) includes every stereoisomer, epimer and diastereoisomer, as a mixture or in isolated form.
  • R may be selected from the group consisting of
  • R' may be lower alkyl or hydrogen.
  • each compound consists of one aromatic ring connected to an -NHCOCH 3 group either directly or via a two-three carbon chain.
  • each compound consists of one aromatic ring fused to an alicyclic ring.
  • each compound consists of two fused aromatic rings.
  • each compound consists of one aromatic ring substituted with -(OHb) 2 NHCOCH 3 .
  • the present invention features a pharmaceutical composition
  • a pharmaceutical composition comprising a compound of formula I wherein
  • R and R' are independently selected from the group consisting of hydrogen, lower alkyl, aryl, aralkyl; substituted aralkyl, heteroaryl; substituted heteroaryl, phenyl, benzyl, SO 2 , NH 21 NO 2 , SO 2 , CH 3 , CH 2 CH 3 , OCH 3 , OCOCH 3 , CH 2 COCH 3 , OH, CN, halogen, carboxy, and alkoxy, and their pharmaceutically acceptable salts of acids or bases, together with a pharmaceutically acceptable earner.
  • the general formula (I) includes every stereoisomer, epimer and diastereoisomer, as a mixture or in isolated form.
  • R may be selected from the group consisting of
  • R' may be lower alkyl or hydrogen.
  • each compound consists of one aromatic ring connected to an -NHCOCH 3 group either directly or via a two-three carbon chain.
  • each compound consists of one aromatic ring fused to an alicyclic ring.
  • each compound consists of two fused aromatic rings.
  • each compound consists of one aromatic ring substituted with -(CHa) 2 NHCOCHs.
  • the present invention features methods for inhibiting the biological activity of p53 comprising the step of contacting a biological sample with a therapeutically effective amount of a compound according to general formula (I) or a pharmaceutical composition comprising a compound of the following general formula(I) wherein:
  • R and R' are independently selected from the group consisting of hydrogen, lower alkyl, aryl, aralkyl; substituted aralkyl, heteroaryl; substituted heteroaryl, phenyl, benzyl, SO 2 , NH 21 NO 2, SO 2 , CH 3 , CH 2 CH 3 , OCH 3 , OCOCH 3 , CH 2 COCH 3 , OH 3 CN, halogen, carboxy, and alkoxy, and their pharmaceutically acceptable salts of acids or bases.
  • the general formula (I) includes every stereoisomer, epimer and diastereoisomer, as a mixture or in isolated form.
  • R may be selected from the group consisting of
  • R' may be lower alkyl or hydrogen.
  • each compound consists of one aromatic ring connected to an -NHCOCH 3 group cither directly or via a two-three carbon chain.
  • each compound consists of one aromatic ⁇ ng fused to an alicyclic ring.
  • each compound consists of two fused aromatic rings.
  • each compound consists of one aromatic ring substituted with -(CH 2 ) 2 NH COCH 3 .
  • the present invention features methods for treating a disease associated with p53 transcription comprising the step of contacting a biological sample with a therapeutically effective amount of a compound according to general formula (I) or a pharmaceutical composition comprising a compound of the following general formula(I) wherein:
  • R and R' are independently selected from the group consisting of hydrogen, lower alkyl, aryl, aralkyl; substituted aralkyl, heteroaryl; substituted heteroaryl, phenyl, benzyl, SO 2 , NH 2, NO 2, SO 2 , CH 3 , CH 2 CH 3 , OCH 3 , OCOCH 3 , CH 2 COCH 3 , OH, CN, halogen, carboxy, and alkoxy, and their pharmaceutically acceptable salts of acids or bases.
  • the general formula (I) includes every stereoisomer, epimer and diastereo isomer, as a mixture or in isolated form.
  • R may be selected from the group consisting of
  • R' may be lower alkyl or hydrogen.
  • each compound consists of one aromatic ring connected to an -NHCOCH 3 group either directly or via a two-three carbon chain.
  • each compound consists of one aromatic ring fused to an alicyclic ring.
  • each compound consists of two fused aromatic rings.
  • each compound consists of one aromatic ring substituted with -(CHa) 2 NHCOCH 3 .
  • the disease may be selected from the group consisting of cancer, cell death caused all or in part by cancer treatments such as chemotherapy or radiation thereapy, neuron cell death, stroke, Alzheimer's Disease and Parkinson's Disease.
  • the present invention features compounds of the following general formula(II) wherein:
  • R and R' are independently selected from the group consisting of hydrogen, lower alkyl, aryl, aralkyl; substituted aralkyl, heteroaryl; substituted heteroaryl, phenyl, benzyl, SO 2 , NH 21 NO 2, SO 2>.
  • X is selected from the group consisting of O, S, and N.
  • the general formula (II) includes every stereoisomer, epimer and diastereoisomer, as a mixture or in isolated form.
  • the compound of formula (II) may be selected from the group consisting of
  • the present invention features a pharmaceutical composition
  • a pharmaceutical composition comprising a compound of formula II wherein
  • R and R' are independently selected from the group consisting of hydrogen, lower alkyl, aryl, aralkyl; substituted aralkyl, heteroaryl; substituted heteroaryl, phenyl, benzyl, SO 2 , NH 2 , NO 2, SO 2 , CH 3 , CH 2 CH 3 , OCH 3 , OCOCH 3 , CH 2 COCH 3 , OH, CN, halogen, carboxy, and alkoxy, and their pharmaceutically acceptable salts of acids or bases, together with a pharmaceutically acceptable carrier.
  • X is selected from the group consisting of O, S, and N.
  • the general formula (II) includes every stereoisomer, epimer and diastereoisomer, as a mixture or in isolated form. 10016] In preferred embodiments, the compound of formula (II) may be selected from the group consisting of
  • the present invention features methods for inhibiting the biological activity of p53 comprising the step of contacting a biological sample with a therapeutically effective amount of a compound according to general formula (II) or a pharmaceutical composition comprising a compound of the following general formula(II) wherein: R-X-R' (H)
  • R and R' are independently selected from the group consisting of hydrogen, lower alkyl, aryl, aralkyl; substituted aralkyl, heteroaryl; substituted heteroaryl, phenyl, benzyl, SO2, NH 2 , NO 2 , SO 2, CH 3 , CH 2 CH 3 , OCH 3 , OCOCH 3 , CH 2 COCH 3 , OH, CN, halogen, carboxy, and alkoxy, and their pharmaceutically acceptable salts of acids or bases.
  • X is selected from the group consisting of O, S, and N.
  • the general formula (I) includes every stereoisomer, epimer and diastereoisomer, as a mixture or in isolated form.
  • the compound of general formula (II) may be selected from the group consisting of
  • the present invention features methods for treating a disease associated with p53 transcription comprising the step of contacting a biological sample with a therapeutically effective amount of a compound according to general formula (II) or a pharmaceutical composition comprising a compound of the following general formula ⁇ ll) wherein:
  • R and R" are independently selected from the group consisting of hydrogen, lower alkyl, aryl, aralkyl; substituted aralkyl, heteroaryl; substituted heteroaryl, phenyl, benzyl, SO 2 , NH 2, NO 2, SO 2 , CH 3 , CH 2 CH 3 , OCH 3 , OCOCH 3 , CH 2 COCH 3 , OH 3 CN, halogen, carboxy, and alkoxy, and their pharmaceutically acceptable salts of acids or bases.
  • X is selected from the group consisting of O, S, and N.
  • the general formula (I) includes every stereoisomer, epimer and diastereoisomer, as a mixture or in isolated form.
  • the compound of general formula (II) may be selected from the group consisting of
  • FIG. 1 Physicochemical properties of the acetyl-lysine binding sites in the CBP and PCAF bromodomains.
  • a Ribbon diagram of the 3D structure of the CBP bromodomain in complex with lysine 382- acetylated p53 peptide (PDB USP).
  • b. & c Surface electrostatic potential representation of the bromodomains of CBP and PCAF, respectively. The electrostatic potential of the protein molecular surface was calculated using Delphi [47] and figures were produced using GRASP [49] and rendered using Pov4Grasp fhttp://pov4grasp.free. fiV). d. & e.
  • FIG. 1 Discovery of initial lead compounds for the CBP bromodomain a. Small-molecule compounds that bind to the CBP bromodomain. b. Superposition of 2D 1 H- 15 N-HSQC spectra of the CBP bromodomain showing changes of the protein NMR resonances from the free form (black) to the complex form with a representative compound MS2126. c. Weighted 1 H and 15 N chemical shift changes ( ⁇ ) by the CBP bromodomain induced by binding to representative ligands from each group, i.e. MS2126, MS7972, MS9802 or MS0433. The AcK binding site lies between ZA and BC loops.
  • Amino acid residues exhibiting major chemical shift perturbations are color-coded on the protein surface: red for 0.05 ppm ⁇ ⁇ ⁇ 0.08 ppm and blue for ⁇ >0.08 ppm.
  • the orientation of the protein structure is similar to that in Figure ⁇ a & Ib. d.
  • FIG. 3 Structural analysis of CBP bromodomain interactions with small molecules ⁇ . Identification of binding locations of small molecules in the CBP bromodomain by Autodock 3.0 calculations (left panels) and j-surface calculations using NMR chemical shift perturbation data at 2 ⁇ (right panels). Ribbons diagrams depict best binding modes of ligands of each group, as determined by Autodock 3.0 calculations. The aromatic ring of each compound is color-coded according to the corresponding j-surface calculation.
  • b Three-dimensional structure of the CBP bromodomain bound to MS7972, as determined by NMR spectroscopy, illustrating the ligand binding site between the ZA and BC loops.
  • Figure 4 Small-molecule inhibition of CBP bromodomain and p53-AcK382 interaction ⁇ . Inhibition of CBP bromodomain and p53-AcK382 peptide binding by lead compounds in a competition assay detected by anti-GST Western blot. In this assay, a lead compound competes against the biotinylated p53-AcK382 peptide that was immobilized on strep tavidin agarose beads for binding to the GST CBP bromodomain.
  • Concentration of the biotinylated or the non- biotinylated p53 peptide used in the assay is 10 ⁇ M and 25 ⁇ M, respectively, whereas compound concentration ranges from 0 to 100 ⁇ M, as indicated.
  • b Fluorescence titration of CBP bromodomain binding to MS7972. Superimposition of fluorescence spectra of the CBP bromodomain ( ⁇ 5 ⁇ M) with increasing amount of MS7972 (0- 80 ⁇ M). Binding affinity was determined by monitoring fluorescence intensity change at 450 nm as a function of ligand concentration (inset).
  • FIG. 5 Modulation of p53 function in response to DNA damage by small molecules
  • a Increased p53 expression in response to DNA damage agent doxorubicin treatment, as illustrated with U2OS cells. Wild-type p53 expressing U2OS cells were incubated with 0.1 ⁇ g/ml doxorubicin for the indicated time (up to 24 hours) and then subjected to immunoblotting analysis with specific anti-bodies.
  • b Small-molecule inhibition of the increase in p53 levels in response to DNA damage. Wild- type p53 expressing U2OS cells were either incubated with DMSO or treated with 20, 200 ⁇ M of each small-molecule compound for 16 hours.
  • c & L Modulation of p53 function in response to DNA damage by MS2126 or MS7972, respectively. Wild-type p53 expressing U2OS cells were either incubated with DMSO or treated with 200 ⁇ M of MS2126 or MS7972 for 16 hours. Cells were then further incubated with 0.1 ⁇ g/ml doxorubicin for the indicated times and then subjected to immunoblotting analysis with specific antibodies. e. Compound MS2126 does not affect the increase in HIFl ⁇ level in response to hypoxia.
  • Wild-type p53 expressing U2OS cells were incubated in the absence or presence of 200 ⁇ M of MS2126 for 16 hours. Cells were then further incubated in either normoxic or hypoxic conditions for an additional 24 ' hours as shown. Cell lysates were then subjected to immunoblotting with the indicated antibodies. /. Compound MS5557 does not affect p53 function in response to DNA damage, as demonstrated in wild-type ⁇ 53 expressing U2OS cells with experimental conditions similar to those in c and d. The cells were treated with DMSO, 200 ⁇ M of MS7972 or MS5557 for 16 hours, and then further incubated with 0.1 ⁇ g/ml doxorubicin for 24 hours and then subjected to immunoblotting analysis with specific antibodies.
  • Figure 6 demonstrates protection of radiation induced cell death by two p53 inhibitors MS7972 and MS5557.
  • Figure 7 demonstrates the effect of the p53 inhibitors MS2126 and MS7972 on TNF ⁇ inhibition of myogenic differentiation in myoblast cell cline C2C12.
  • Figure 8 demonstrates the effects of the p53 inhibitors MS2126 and MS7972 on reducing TNF ⁇ inhibition of myogenic differentiation in a myoblast cell line.
  • Compounds of the present in invention and pharmaceutical compositions comprising the same are useful for modulating, preventing, retarding the progression and treating diseases associated with p53 transcription.
  • Lysine acetylation of human tumor suppressor p53 in response to cellular stress signals is required for its function as a transcription factor that regulates cell cycle arrest, senescence or apoptosis.
  • the present invention features a series of small-molecule chemical compounds that can block, for instance, K382-acetylated p53 association with the bromodomain of CBP.
  • These small molecules were indcntified in target structure-based nuclear magnetic resonance (NMR) spectroscopy screening of a focused library of chemical compounds that was constructed based on the structural knowledge of the CBP bromodomain/p53-AcK382 interaction.
  • NMR nuclear magnetic resonance
  • Cell-based functional assays further demonstrate that these compounds can modulate p53 stability, protein level, modification patterns as well as transcriptional activation of downstream target gene p21 in response to DNA damage.
  • the combined in-vitro and in-vivo data demonstrate the clinical efficacy of these small molecule compounds.
  • lower alkyl and lower alkoxy are understood as meaning straight or branched alkyl and alkoxy groups having from 1 to 8 carbon atoms;
  • aryl is understood as meaning an aromatic group selected from phenyl and naphthyl groups
  • heteroaryl is understood as meaning a mono- or bicyclic aromatic group, each cycle, or ring, comprising five or six atoms and said cycle, or ring, or both cycles, or rings, including in its carbon skeleton from one to three heteroatoms selected from nitrogen, oxygen and sulphur;
  • the terras "lower aralkyl” and “lower heteroaralkyl” are understood as meaning, in view of the definitions above, phenyl(Cj -Cs)alkyl or naphthyl(Ci -Cg)alkyl and heteroar(Ci -Cg)alkyl respectively;
  • substituted concerning the terms aryl, aralkyl, phenyl, radical (f ⁇ ve-membered, including Z), heteroaryl, heteroaralkyl, as defined above, signifies that the groups in question are substituted on the aromatic part with one or more identical or different groups selected from the groups: (Ci -C 8 )alkyl, trifluoromethyl, (Ci -Cg)alkoxy, hydroxy, nitro, amino, (Ci - C8)alkylamino, di(Ci -Cs)alkylamino, sulphoxyl, sulphonyl, sulphonamide, sulpho(Ci -Cg)alkyl, carboxyl, carbalkoxyl, carbamide (it being possible for said (C] -Cg)alkyl groups to be linear or branched) or substituted with one or more halogen atoms; the term aminoacyl, which concerns the glutathionyl, cysteinyl
  • a "bromodomain-acetyl-lysine binding complex” is a binding complex between a bromodomain or fragment thereof and either a peptide/polypeptide comprising an acetyl-lysine (or an analog of acetyl-lysine), or a free analog of acetyl-lysine, such as acetyl- histamine disclosed in the Example below.
  • the peptide comprises at least six amino acids in addition to the acetyl-lysine.
  • a fragment of a bromodomain preferably comprises a ZA loop as defined below.
  • the dissociation constant of a bromodomain-acetyl-lysine binding complex is dependent on whether the lysine residue or analog thereof is acetylated or not, such that the affinity for the bromodomain and the peptide comprising the lysine residue (for example) significantly decreases when that lysine residue is not acetylated.
  • a bromodomain-acetyl-lysine binding complex is that formed between P/CAF with Tat (the "Tat- P/CAF complex") as exemplified below.
  • acetyl -lysine analog is used interchangeably with the term “analog of acetyl-lysine” and is a compound that contains the acetyl-amine-like structure.
  • a "ZA loop" of a bromodomain is a key protion of a bromodomain that is involved in the binding of the bromodomain to the acetyl-lysine.
  • the structure of the actual ZA loop of the bromodon ⁇ ain of P/CAF is depicted in Fig. 2A.
  • a ZA loop has between about 20 and 40 amino acids and preferably comprises the amino acid sequence set forth in United States Patent Application 09/784,553 and United States Patent Application 10/209/201, now published as United States Patent Publication No. 2004/0009613, the disclosure of which is hereby incorporated by reference in its entirety. More preferably the ZA loop comprises between about 23 to 34 amino acids.
  • a "polypeptide” or “peptide” comprising a fragment of a bromodomain, such as the ZA loop, or a peptide or polypeptide comprising an acetyl- lysine, as used herein can be the “fragment” alone, or a larger chimeric or fusion peptide/protein which contains the "fragment”.
  • fusion protein and “fusion peptide” are used interchangeably and encompass “chimeric proteins and/or chimeric peptides” and fusion "intein proteins/peptides”.
  • a fusion protein comprises at least a portion of a protein or peptide of the present invention, e.g., a bromodomain, joined via a peptide bond to at least a portion of another protein or peptide including e.g., a second bromodomain in a chimeric fusion protein.
  • the portion of the bromodomain is antigenic.
  • Fusion proteins can comprise a marker protein or peptide, or a protein or peptide that aids in the isolation and/or purification of the protein, for example.
  • agents As used herein, and unless otherwise specified, the terms “agent”, “potential drug”, “compound”, “test compound” or “potential compound” are used interchangeably, and refer to chemicals which potentially have a use as an inhibitor or activator/stabilizer of bromodomain- acetyl-lysine binding. Therefore, such “agents”, “potential drugs”, “compounds” and “potential compounds” may be used, as described herein, in drug assays and drug screens and the like.
  • a "small organic molecule” or “small molecule” is an organic compound, including a peptide or organic compound complexed with an inorganic compound (e.g., metal) that has a molecular weight of less than 3 Kilodaltons. Such small organic molecules can be included as agents, etc. as defined above.
  • the term "binds to” is meant to include all such specific interactions that result in two or more molecules showing a preference for one another relative to some third molecule. This includes processes such as covalent, ionic, hydrophobic and hydrogen bonding but does not include non-specific associations such as solvent preferences.
  • homologous in all its grammatical forms refers to the relationship between proteins that possess a "common evolutionary origin,” including proteins from superfamilies ⁇ e.g., the immunoglobulin superfamily) and homologous proteins from different species ⁇ e.g., myosin light chain, etc.) (Reeck et al., Cell, 50:667 (1987)).- Such proteins have sequence homology as reflected by their high degree of sequence similarity.
  • sequence similarity in all its grammatical forms refers to the degree of identity or correspondence between nucleic acid or amino acid sequences of proteins that may or may not share a common evolutionary origin ⁇ see Reeck et al. , supra).
  • sequence similarity when modified with an adverb such as “highly,” may refer to sequence similarity and not a common evolutionary origin.
  • Two DNA sequences are "substantially homologous" when at least about 60% (preferably at least about 80%, and most preferably at least about 90 or 95%) of the nucleotides match over the defined length of the DNA sequences. Sequences that are substantially homologous can be identified by comparing the sequences using standard software available in sequence data banks, or in a Southern hybridization experiment under, for example, stringent conditions as defined for that particular system. Defining appropriate hybridization conditions is within the skill of the art ⁇ See, e.g., Sambrook et al, 1989 supra; DNA Cloning, VoIs. I & II, supra; Nucleic Acid Hybridization, supra., and Sambrook and Russell, 2001).
  • an amino acid sequence is 100% "homologous" to a second amino acid sequence if the two amino acid sequences are identical, and/or differ only by neutral or conservative substitutions as defined below. Accordingly, an amino acid sequence is 50% "homologous" to a second amino acid sequence if 50% of the two amino acid sequences are identical, and/or differ only by neutral or conservative substitutions.
  • DNA and protein sequence percent identity can be determined using Mac Vector 6.0.1 , Oxford Molecular Group PLC (1996) and the Clustal W algorithm with the alignment default parameters, and default parameters for identity. These commercially available programs can also be used to determine sequence similarity using the same or analogous default parameters.
  • biological activity of p53 refers not only to the binding affinity or activity of a single molecule but also to the pathological or clinical sequelae of the molecule as it functions in various interactions, pathways and cascades in vivo in an organism. Specifically, the term refers to p53 function as a transcriptional activator for its target genes. Similarly, the term “disease associated with p53 transcription” refers to any disease or pathology caused all or in part by any biological activity of p53 that is altered relative to its normal or homeostatic level.
  • NMR-based screening of chemical compounds for a given target protein is considered to be reliable and target site-specific, making it preferable over random high-throughput screening (Shuker et al., (1996) Science 274, 1531- 1534; Hajduk et al, (1997) Science 278, 498-499; Hajduk et al., (1999) Q Rev Biophys 32, 211- 240; Moore (1999) Curr. Opin. in Biotech. 10, 54-58).
  • NMR is generally not best suited for chemical screening in a high-throughout fashion.
  • the bromodomain (BRD) is found in a large number of chromatin associated proteins and nuclear histone lysine acetyltransferases (HATs), and has been recently shown to function as an acetyl-lysine (AcK) binding domain. Bromodomain/AcK. binding plays a pivotal role in regulation of chromatin remodeling and gene transcription (Zeng et al., (2002) FEBS Lett 513, 124-128; Winston et al., (1999) Nature Struct. Biol. 6, 601-604; Marmorstein et al., (2001) Gene 272, 1-9).
  • BRDs adopt a conserved structural fold of a left-handed four-helix bundle ( ⁇ Z, ⁇ A, ⁇ B and ⁇ C), as first shown in the PCAF BRD (Dhalluin et al., (1999) Nature 399, 491-496).
  • the ZA and BC loops at one end of the bundle form a hydrophobic pocket for AcK binding.
  • the structure of the CBP BRD bound to a p53-AcK382 peptide shows that AcK382 intercalates into the protein hydrophobic cavity and interacts with residues of the ZA and BC loop ( Figure Ia).
  • the AcK binding pocket is hydrophobic in nearly all BRDs, whereas electrostatics at the opening of the AcK binding pocket displays significant variations in different BRDs. For example, in CBP BRD, the opening is slightly positively charged, while it is more negatively charged in PCAF BRD ( Figure Ib vs. Ic and Id vs. Ie). These differences at the ligand binding site serve as the basis for selectivity of chemical ligands targeting a particular BRD. Given these structural feature differences at the AcK binding pocket and the fact that most of known drug molecules contain one aromatic ring, we constructed a knowledge-based library of about 200 compounds from a collection of about 14,000 small molecules (ChemBridge, Inc.).
  • each compound consists of one aromatic ring connected to an -NHCOCH 3 group either directly or via a two-three carbon chain; and (2) drug-like properties of compounds are evaluated according to the Lipinsky's Rule of Five (Lipinski et al., (1997) Advanced Drug Delivery Reviews 23, 3-25).
  • Ligand docking into a target protein can be performed using Autodock 3.0 that uses a genetic search algorithm as a global optimizer and energy minimization as a local search method (Morris et al., (1998) Journal of Computational Chemistry 19, 1639-1662). Although as a grid- based method, Autodock limits itself to a rigid model of a target protein, ligand flexibility is allowed. To predict the best docking mode for a given ligand, docking calculations generate a number of clusters (i.e. solutions with pair-wise RMSD of all atoms of 1.0 A) and rank of each docking mode (cluster rank). Docking mode is selected from the lowest-energy solution of a cluster corresponding to the minimum docking energy.
  • cluster rank rank of each docking mode
  • Calculation of electron current density surface using chemical shift perturbation data can also help localize a ligand when bound to a target protein.
  • This method calculates the center of electron current density for a ligand aromatic ring using point-dipole that is represented as dot density (j-surface), where the highest dot density correlates to the center of the ligand aromatic ring.
  • the surface can, therefore, guide to locate the binding site for the aromatic ring of the ligand.
  • the structure also shows that the ligand forms a network of inter-molecular hydrophobic and aromatic interactions with VaIl 1 15, Leul l20, Ilel l22, Tyrl l25 and Tyrl 167, and that the acetyl and ketone groups with VaIl 115 and Leul 120, and Glnl 113, respectively.
  • the residues are involved in interactions with the p53-AcK382 peptide, binding of MS7972 to CBP BRD likely block the protein interaction with an acetyl-lysine-containing binding partner such as p53 (see below).
  • p53 In response to DNA damage, p53 also becomes acetylated on its C-terminal lysine residues including lysine 382, promoting its recruitment of the transcriptional coactivator CBP/p300 via BRD/AcK binding, which leads to histone acetylation and transcriptional activation of target genes such as the cyclin-dependent kinase inhibitor p21 in cell cycle arrest.
  • target genes such as the cyclin-dependent kinase inhibitor p21 in cell cycle arrest.
  • treatment of U2OS cells with MS2126 or MS7972 at 200 ⁇ M, prior to the doxorubicin stimulation results in a dramatic decrease of the doxorubicin- induced p53 increase as compared to the DMSO control.
  • the lysine-acetyl atcd p53 in a free state is not stable in the cell, as it is subject to rapid de-acetylation by histone deacetylases and subsequent ⁇ biqutinalion and protein degradation by Mdm2. This effect is consistent with the corresponding decrease in p53-mediated p21 activation in response to doxorubicin-induced DNA damage.
  • Treatment of U2OS cells with compound MS9802 or MS0433 showed much less effects, if any, on p53 protein level and activation in cells, which is consistent with their inability to inhibit CBP BRD/p53-AcK382 association in vitro ( Figure 4a).
  • MS2126 does not seem to affect the up-regulation of HIFl ⁇ under hypoxic conditions (Figure 5e), and MS5557, which is a structural analog of MS7972 but does not bind to the CBP BRD ( Figure S3), does not modulate p53 function under DNA damage condition ( Figure 5f), thus further highlighting the specificity of MS2126 and MS7972 for p53 function.
  • Figure 5e shows that inhibition of CBP BRD/p53 at AcK.382 by small-molecule compounds causes a dramatic inactivation of p53 transcriptional activity through promoting its protein instability by changes of its post-translational modification states.
  • these cell-based assays provide a valuable assessment of cell permeability and in vivo efficacy of these small-molecule compounds on p53 function as a transcriptional activator for its target genes, which is essential for further lead optimization through SAR-guided chemical modifications.
  • Bromodomain The present invention utilizes detailed structural information regarding a bromodomain and a bromodomain complexed with its acetylated binding partner.
  • the present invention further utilizes knowledge of the three-dimensional structure of the bromodomain and a bromodomain acetylated binding partner complex. Since the interaction of the bromodomain with a ligand can play a significant role in remodeling/regulation/activation, the structural information can be employed in methods of identifying drugs that can modulate basic cell processes by modulating the transcription. In a particular, the three-dimensional structural information is used in the design of a small organic molecule for the treatment of disease.
  • the bromodomain and lysine-acetylated protein interaction can now be implicated to play a causal role in the development of a number of diseases.
  • the resulting fusion protein MLL-CBP contains the tandem bromodomain-PHD f ⁇ nger-HAT domain of CBP. It also has been shown that both the bromodomain and HAT domain of CBP are required for leukomogenesis, because deletion of either the bromodomain or the HAT domain results in loss of the MLL-CBP fusion protein's ability for cell transform.
  • the CBP bromodomain and more particularly, the ZA loop of the CBP bromodomain, is an excellent target for developing drugs that interfere with the bromodomain acetyl-lysine interaction that can be used in the treatment of disease.
  • an antibody e.g., a humanized antibody raised specifically against a peptide from the ZA loop of the CBP bromodomain could also be effective for treating these conditions.
  • Compounds may be active to bind to two nearby sites on the bromodomain.
  • a compound that binds a first site of the bromodomain does not bind a second nearby site. Binding to the second site can be determined by monitoring changes in a different set of amide chemical shifts in either the original screen or a second screen conducted in the presence of a ligand (or potential ligand) for the first site. From an analysis of the chemical shift changes the approximate location of a potential ligand for the second site is identified. Optimization of the second ligand for binding to the site is then carried out by screening structurally related compounds (e.g., analogs as described above).
  • ligands for the first site and the second site are identified, their location and orientation in the ternary complex can be determined experimentally either by NMR spectroscopy or X-ray crystallography.
  • a linked compound is synthesized in which the ligand for the first site and the ligand for the second site are linked.
  • the two ligands are covalently linked.
  • This linked compound is tested to determine if it has a higher binding affinity for the bromodomain than either of the two individual ligands.
  • a linked compound is selected as a ligand when it has a higher binding affinity for the bromodomain than either of the two ligands.
  • the affinity of the linked compound with the bromodomain is determined monitoring the 15 N- or ⁇ -amide chemical shift changes in two dimensional ' 5 N-heteronuclear single-quantum correlation ( 15 N-HSQC) spectra upon the addition of the linked compound to the l5 N-labeled bromodomain as described above.
  • a larger linked compound can be constructed in an analogous manner, e.g., linking three ligands which bind to three nearby sites on the bromodomain to form a multilinked compound that has an even higher affinity for the bromodomain than the linked compound.
  • compositions In yet another aspect of the present invention, pharmaceutical compositions of the compounds of formulae I and II are provided. Such pharmaceutical compositions maybe for administration for injection, or for oral, pulmonary, nasal or other forms of administration.
  • pharmaceutical compositions comprising effective amounts of a low molecular weight component or components, or derivative products, of the invention together with pharmaceutically acceptable diluents, preservatives, solubilizers, emulsifiers, adjuvants and/or carriers.
  • compositions include diluents of various buffer content (e.g., Tris-HCl, acetate, phosphate), pH and ionic strength; additives such as detergents and solubilizing agents (e.g., Tween 80, Polysorbate 80), anti-oxidants (e.g., ascorbic acid, sodium metabisulfite), preservatives (e.g., Thimersol, benzyl alcohol) and bulking substances (e.g., lactose, mannitol); incorporation of the material into particulate preparations of polymeric compounds such as polylactic acid, polyglycolic acid, etc. or into liposomes. Hylauronic acid may also be used.
  • buffer content e.g., Tris-HCl, acetate, phosphate
  • additives e.g., Tween 80, Polysorbate 80
  • anti-oxidants e.g., ascorbic acid, sodium metabisulfite
  • preservatives e.g.
  • compositions may influence the physical state, stability, rate of in vivo release, and rate of in vivo clearance of the present proteins and derivatives. See, e.g., Remington's Pharmaceutical Sciences, 18th Ed. (1990, Mack Publishing Co., Easton, PA 18042) pages 1435-1712 which are herein incorporated by reference.
  • the compositions may be prepared in liquid form, or may be in dried powder, such as lyophilized form.
  • Oral Delivery. Contemplated for use herein are oral solid dosage forms, which are described generally in Remington's Pharmaceutical Sciences, 18th Ed.1990 (Mack Publishing Co. Easton PA 18042) at Chapter 89, which is herein incorporated by reference.
  • Solid dosage forms include tablets, capsules, pills, troches or lozenges, cachets or pellets.
  • liposomal or proteinoid encapsulation may be used to formulate the present compositions (as, for example, proteinoid microspheres reported in U.S. Patent No. 4,925,673).
  • Liposomal encapsulation may be used and the liposomes may be derivatized with various polymers (e.g., U.S. Patent No. 5,013,556).
  • a description of possible solid dosage forms for the therapeutic is given by Marshall, K. In: Modern Pharmaceutics Edited by G.S. Banker and CT. Rhodes Chapter 10, 1979, herein incorporated by reference.
  • the formulation will include an agent of the present invention (or chemically modified forms thereof) and inert ingredients which allow for protection against the stomach environment, and release of the biologically active material in the intestine.
  • oral dosage forms of the above derivatized component or components are also specifically contemplated.
  • the component or components may be chemically modified so that oral delivery of the derivative is efficacious.
  • One skilled in the art has available formulations which will not dissolve in the stomach, yet will release the material in the duodenum or elsewhere in the intestine. Preferably, the release will avoid the deleterious effects of the stomach environment, either by protection of the protein (or derivative) or by release of the biologically active material beyond the stomach environment, such as in the intestine.
  • the therapeutic can be included in the formulation as fine multiparticulates in the form of granules or pellets of particle size about 1 mm.
  • the fo ⁇ nulation of the material for capsule administration could also be as a powder, lightly compressed plugs or even as tablets.
  • the therapeutic could be prepared by compression. One may dilute or increase the volume of the therapeutic with an inert material.
  • These diluents could include carbohydrates, especially mannitol, a-lactose, anhydrous lactose, cellulose, sucrose, modified dextrans and starch.
  • Certain inorganic salts may be also be used as fillers including calcium triphosphate, magnesium carbonate and sodium chloride.
  • Disintegrants may be included in the formulation of the therapeutic into a solid dosage form.
  • Materials used as disintegrates include but are not limited to starch, including the commercial disintegrant based on starch, Explotab. Binders also may be used to hold the therapeutic agent together to form a hard tablet and include materials from natural products such as acacia, tragacanth, starch and gelatin.
  • An anti-frictional agent may be included in the formulation of the therapeutic to prevent sticking during the formulation process. Lubricants may be used as a layer between the therapeutic and the die wall.
  • Glidants that might improve the flow properties of the drug during formulation and to aid rearrangement during compression also might be added.
  • the glidants may include starch, talc, pyrogenic silica and hydrated silicoaluminate.
  • a surfactant might be added as a wetting agent. Additives which potentially enhance uptake of the protein (or derivative) are for instance the fatty acids oleic acid, linoleic acid and linolenic acid.
  • Transdermal administration Various and numerous methods are known in the art for transdermal administration of a drug, e.g., via a transdermal patch. Transdermal patches are described in for example, U.S. Patent No. 5,407,713, issued April 18, 1995 to Rolando et al.; U.S. Patent No. 5,352,456, issued October 4, 1004 to Fallon et al.; U.S. Patent No. 5,332,213 issued August 9, 1994 to D'Angelo et al.; U.S. Patent No. 5,336,168, issued August 9, 1994 to Sibalis; U.S. Patent No. 5,290,561, issued March 1, 1994 to Farhadieh et al; U.S. Patent No.
  • a transdermal route of administration may be enhanced by use of a dermal penetration enhancer, e.g., such as enhancers described in U.S. Patent No. 5,164,189 ⁇ supra), U.S. Patent No. 5,008,110 ⁇ supra), and U.S. Patent No. 4,879,119, issued November 7, 1989 to Aruga et al, the disclosure of each of which is incorporated herein by reference in its entirety.
  • a dermal penetration enhancer e.g., such as enhancers described in U.S. Patent No. 5,164,189 ⁇ supra), U.S. Patent No. 5,008,110 ⁇ supra), and U.S. Patent No. 4,879,119, issued November 7, 1989 to Aruga et al, the disclosure of each of which is incorporated herein by reference in its entirety.
  • Pulmonary Delivery also contemplated herein is pulmonary delivery of the pharmaceutical compositions of the present invention.
  • a pharmaceutical composition of the present invention is delivered to the lungs of a mammal while inhaling and traverses across the lung epithelial lining to the blood stream.
  • Other reports of this include Adjei et al. [Pharmaceutical Research, 7:565-569 (1990); Adjei et al., InternationalJournal of Pharmaceutics, 63:135-144 (1990) (leuprolide acetate); Braquet et al., Journal of Cardiovascular Pharmacology, 13(suppl.
  • NMR samples contained the CBP BRD (0.5 mM) in complex with a chemical ligand MS7972 (—2 mM) in 100 mM phosphate buffer of pH 6.5, containing 5 mM perdeuterated DTT and 0.5 mM EDTA in H 2 O/ 2 H 2 O (9/1) or 2 H 2 O.
  • AU NMR spectra were acquired at 30 0 C on a Bruker 500 or 600 MHz NMR spectrometer.
  • the backbone 1 H, 13 C and 15 N resonances were assigned using 3D HNCACB and HN(CO)CACB spectra.
  • the side-chain atoms were assigned from 3D HCCH-TOCSY and (H)C(CO)NH- TOCSY data.
  • the NOE-derived distance restraints were obtained from 15 N- or l 3 C-edited 3D NOESY spectra.
  • the 3 J HN1Ha coupling constants measured from 3D HNHA data were used to determine cA-angle restraints.
  • Slowly exchanging amide protons were identified from a series of 2D 15 N-HSQC spectra recorded after H 2 O/ 2 H2O exchange.
  • the intermolecular NOEs used in defining the structure of the CBP bromodomain/ligand complex were detected in l3 C-edited (F / ), I3 C/ I5 N- filtered (Fj) 3D NOESY spectra (Clore et al., (1994) Meth. Enzymol. 239, 249-363). Protein structures were calculated with a distance geometry-simulated annealing protocol with X-PLOR (Brunger (1993) X-PLOR Version 3.1: A system for X-Ray crystallography and NMR, version 3.1 Edition (New Haven, CT: Yale University Press). Initial structure calculations were performed with manually assigned NOE-derived distance restraints.
  • Hydrogen-bond distance restraints generated from the H/D exchange data, were added at a later stage of structure calculations for residues with characteristic NOEs.
  • the converged structures were used for iterative automated NOE assignment by ARIA for refinement (Nilges et al., (1998) Prog. NMR Spectroscopy 32, 107-139). Structure quality was assessed by Procheck-NMR (Laskowski et al., J. Biomol. NMR 8, 477-486).
  • the structure of the protein/ligand complex was determined using intermolecular NOE-derived distance restraints.
  • each grid map consisted of a 126x126x126 grid with the centre of the map assigned to the geometric centre of the protein.
  • Each LGA job consists of 50 runs with 270,000 generations in each run and maximum number of energy evaluations set to 2.5x10 .
  • a 62x76x80 grid was generated with each LGA job that consisted of 200 runs with 270,000 generations in each run and maximum number of energy evaluations of 5.OxIO 6 .
  • Resulting docked orientations within 1.0 A RMSD tolerance of each other were clustered together. Docked conformations were analyzed using AutoDockTools (http://www.scripps.edu/ ⁇ sanner) and LIGPLOT (Wallace et al., (1995) Protein Eng ⁇ 5, 127-134).
  • the MD protocol used the LINCS method (Hess et al., (1997) Journal of Computational Chemistry 18, 1463-1472) to constrain covalent bond lengths. Temperature and pressure were kept constant separately by coupling the protein, ions and solvent to external temperature and pressure baths with respective coupling constant ( ⁇ ) of 0.1 ps and 0.5 ps. The reference temperature was adjusted to 300 K. To relax the solvent configuration, a steepest descent minimization was adopted. The following step was position-restrained dynamics, which restrains atom positions of the protein while letting the solvent move in the simulation box to reach equilibrium before a full molecular dynamics simulation starts.
  • rP l k is the distance between the carbonyl oxygen of residue /-1 to the heavy atom k
  • r" k is the distance between the amide proton and the heavy atom k.
  • the parameter b was set to -
  • Fluorescence Binding Experiment. Fluorescence measurements were performed on ISS PCl photon counting spectro-fluorometer at room temperature. The concentration of the protein (calculated using the theoretical absorption coefficient of 24,750 M " 'cm ' ' at 280 nm) was 5 ⁇ M in 100 mM phosphate buffer, pH 6.5, containing 5 mM DTT. Protein intrinsic fluorescence was measured at an excitation wavelength of 295 nm and emission was collected from 300-500 nm using 8nm band passes for both excitation and emission. The protein sample was titrated with a ligand MS7972 to a final concentration of 80 ⁇ M with 0.7 % final dilution.
  • the CBP BRD eluted from the beads was run on SDS-PAGE, and visualized in western blots by anti-GST antibody and horseradish-peroxidase-conjugated goat anti-rabbit IgG.
  • Small-molecule inhibition assay was performed by incubating the CBP BRD and the biotinylated p53 AcK382 peptide with increasing amount of small-molecule compound.
  • Wild-type p53 expressing U2OS cells were either incubated with DMSO or treated with 20 or 200 ⁇ M of various small-molecule compounds for about 16 hours. The cells were further incubated with 0.1 ⁇ g/ml doxorubicin for a specified time of 2 to 24 hours, and then cell lysates were subjected to immunoblotting analysis using specific antibodies for p53, phosphorylated Serl5 of p53, acetylated Lys382 of p53, ⁇ 21 or Ku70.
  • HIFl ⁇ For the up-regulation of HIFl ⁇ , U2OS cells were incubated in the absence or presence of 200 ⁇ M of MS2126 for 16 hours, and then further incubated in either normoxic or hypoxic conditions for an additional 24 hours. Cell lysates were then subjected to immunoblotting with the specific antibodies for HIFl ⁇ and Ku70.
  • Radiation therapy is a widely used clinical treatment of human cancers including locally advanced cervix, lung, head and neck, rectal, esophagus, anal and prostate cancers, and it compares favorably to radical prostatectomy (DeVita, et ai, Cancer J, 2001. 7 Suppl I: S2-13; D'Amico, et al, JAMA, 1998. 280(1 1):969-74).
  • IR ionizing radiation
  • a chemical agent that is capable of down-regulating p53's transcriptional activation could inhibit p53-induce programmed cell death.
  • This idea was tested with a DUl 45 prostate cancer cell line using two lead compounds MS7972 and NS5557, which have been shown to inhibit lysine 382- acetylated p53 interaction with the bromodomain of the transcriptional co-activator CBP (Mujtaba, S., et al., MoI Cell, 2004. 13(2):251-63 and Sachchidanand, et al., Chem Biol, 2006. 13(l): ⁇ l-90).
  • the percent of colonies surviving 2 Gy of ionizing radiation for DU145 cells is about 65%, whereas in the presence of MS7972 or MS5557 (either at 20 or 200 ⁇ M), the cell survival rate was significantly increased to about 85- 89%.
  • DU- 145 has a mutation in p53
  • published data demonstrate that DUl 45 derived mutants are capable of inducing p21 after UV-induced DNA damage, that DNA damage agents etoposide and doxorubicin can induce p53 -dependent apopotosis and that DU145 mutations do not have a dominant-negative effect on p53 function (Gurova, K. V., et al., Cancer Res, 2003.
  • p53 inhibitors are also effective in additional cell lines including a wild- type p53 cell line LnCAP and a p53 deleted cell line PC-3.
  • the p53 inhibitors capable of modulating p53 function in gene transcription activation are especially effective as therapeutic agents in combined chemotherapy or radiation therapy to minimize the deleterious effects of radiation damage to normal issues in cancer treatment.
  • a cell survival test by ionizing radiation treatment was performed using a procedure as described in a recently published study (Taneja, et al., J Biol Chem, 2004. 279(3):2273-80; Fernandez-Capetillo, et al., Nat Cell Biol, 2002. 4(12):993-7). Briefly, the DU145 cells were cultured in culture dishes and grown on glass coverslips in 24 well plates to about 50% confluence before irradiation. Ionizing radiation was performed with a 137 Cs ⁇ irradiator at 1.5 Gy/min to a dose ranging from 0-2 Gy. Cell death was measured by a clonogenic survival analysis using a colony formation assay that counts and compares cell colonies before and after IR treatment. Typically, at least assays were performed to assess relative radio-sensitivity of these cells.
  • Muscle wasting is a severe complication associated with chronic infection and cancers that leads to an overall poor prognosis for recovery.
  • TNF ⁇ tumor necrosis factor-alpha

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  • Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)

Abstract

La présente invention concerne des composés et des compositions pharmaceutiques les comprenant, qui sont utiles pour la modulation d’activité p53. L’invention fournit aussi des procédés d’inhibition de l’activité biologique du p53 et de traitement de maladies telles que, par exemple, le cancer, la mort de cellules causées entièrement ou en partie par les traitements du cancer tels qu’une chimiothérapie ou une thérapie par radiation, la mort de cellules neuronales, un infarctus, la maladie d’Alzheimer et la maladie de Parkinson.
PCT/US2007/001350 2006-01-19 2007-01-19 Composes et procedes atypiques d’inhibition d’activite p53 WO2007084625A2 (fr)

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WO2013033268A2 (fr) 2011-08-29 2013-03-07 Coferon, Inc. Ligands bromodomaines bivalents et procédés d'utilisation de ceux-ci
WO2013033269A1 (fr) 2011-08-29 2013-03-07 Coferon, Inc. Monomères bioorthogonaux capables de se dimériser et de cibler des bromodomaines et procédés d'utilisation correspondant
JP2013510125A (ja) * 2009-11-05 2013-03-21 グラクソスミスクライン エルエルシー ブロモドメイン阻害剤としてのテトラヒドロキノリン誘導体
JP2014526518A (ja) * 2011-09-15 2014-10-06 タイペイ メディカル ユニバーシティ 心不全またはニューロン損傷を治療するための化合物およびその方法
WO2016001452A1 (fr) 2014-07-04 2016-01-07 Universität Zürich Composés, destinés plus particulièrement à être utilisés dans le traitement d'une maladie ou d'une pathologie pour laquelle un inhibiteur du bromodomaine est indiqué
EP2955524A3 (fr) * 2009-11-05 2016-03-23 GlaxoSmithKline LLC Nouveau procédé
EP4051283A4 (fr) * 2019-11-01 2024-03-27 Univ California Modulateurs de p53 et utilisations de ceux-ci

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Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2013510125A (ja) * 2009-11-05 2013-03-21 グラクソスミスクライン エルエルシー ブロモドメイン阻害剤としてのテトラヒドロキノリン誘導体
EP2955524A3 (fr) * 2009-11-05 2016-03-23 GlaxoSmithKline LLC Nouveau procédé
US9360482B2 (en) 2009-11-05 2016-06-07 Glaxosmithkline Llc Process for the identification of a compound which inhibits the binding of the second bromodomain of each of human BRD-2, BRD-3, and BRD-4
US9753034B2 (en) 2009-11-05 2017-09-05 Glaxosmithkline Llc Process for the identification of a compound which inhibits the binding of the first bromodomain of each of human BRD-2, BRD-3, and BRD-4
WO2013033268A2 (fr) 2011-08-29 2013-03-07 Coferon, Inc. Ligands bromodomaines bivalents et procédés d'utilisation de ceux-ci
WO2013033269A1 (fr) 2011-08-29 2013-03-07 Coferon, Inc. Monomères bioorthogonaux capables de se dimériser et de cibler des bromodomaines et procédés d'utilisation correspondant
JP2014526518A (ja) * 2011-09-15 2014-10-06 タイペイ メディカル ユニバーシティ 心不全またはニューロン損傷を治療するための化合物およびその方法
WO2016001452A1 (fr) 2014-07-04 2016-01-07 Universität Zürich Composés, destinés plus particulièrement à être utilisés dans le traitement d'une maladie ou d'une pathologie pour laquelle un inhibiteur du bromodomaine est indiqué
EP4051283A4 (fr) * 2019-11-01 2024-03-27 Univ California Modulateurs de p53 et utilisations de ceux-ci

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