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WO2008115259A2 - Inhibiteurs d'ubiquitine ligase - Google Patents

Inhibiteurs d'ubiquitine ligase Download PDF

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WO2008115259A2
WO2008115259A2 PCT/US2007/075511 US2007075511W WO2008115259A2 WO 2008115259 A2 WO2008115259 A2 WO 2008115259A2 US 2007075511 W US2007075511 W US 2007075511W WO 2008115259 A2 WO2008115259 A2 WO 2008115259A2
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alkyl
benzo
ylsulfonyl
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amino
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WO2008115259A3 (fr
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Rajinder Singh
Usha Ramesh
Jianing Huang
Sarkiz D. Issakani
Lyuben Tsvetkov
Matthew David Petroski
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Rigel Pharmaceuticals Inc
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Rigel Pharmaceuticals Inc
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D413/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms
    • C07D413/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings
    • C07D413/12—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings linked by a chain containing hetero atoms as chain links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D271/00—Heterocyclic compounds containing five-membered rings having two nitrogen atoms and one oxygen atom as the only ring hetero atoms
    • C07D271/02—Heterocyclic compounds containing five-membered rings having two nitrogen atoms and one oxygen atom as the only ring hetero atoms not condensed with other rings
    • C07D271/08—1,2,5-Oxadiazoles; Hydrogenated 1,2,5-oxadiazoles
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D413/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms
    • C07D413/14—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing three or more hetero rings

Definitions

  • This invention relates to the inhibition of ubiquitination. More particularly, the invention relates to compounds and methods for inhibiting ubiquitin ligase activity.
  • Ubiquitin is a 76 amino acid protein present throughout the eukaryotic kingdom. It is a highly conserved protein and is essentially the identical protein in diverse organisms ranging from humans to yeasts to fruit flies. In eukaryotes, ubiquitination is the key component of the ATP-dependent pathway for protein degradation and cellular regulatory processes. Proteins slated for degradation or that act as regulatory agents are covalently linked to ubiquitin via an ATP-dependent process catalyzed by three separate enzymes.
  • Ubiquitin is first activated in an ATP-dependent manner by a ubiquitin activating agent, for example, an El.
  • a ubiquitin activating agent for example, an El.
  • the C-terminus of a ubiquitin forms a high energy thioester bond with the ubiquitin activating agent.
  • the ubiquitin is then transferred to a ubiquitin conjugating agent, for example, an E2 (also called ubiquitin moiety carrier protein), also linked to this second ubiquitin agent via a thioester bond.
  • E2 also called ubiquitin moiety carrier protein
  • a ubiquitin ligating agent for example, an E3.
  • monomers or oligomers of ubiquitin are attached to the target protein.
  • each ubiquitin is covalently ligated to the next ubiquitin through the activity of a ubiquitin ligating agent to form polymers of ubiquitin.
  • the ubiquitination of target proteins by E3 in cells results in the formation of poly-ubiquitin chains.
  • An isopeptide bond is formed between the carboxyl terminus of the ubiquitin and the ⁇ -amino group of Lys in the target protein.
  • ubiquitin chains results from the formation of additional isopeptide bonds with the Lys 48 (and sometimes Lys 63 ) of a previously conjugated ubiquitin and the carboxyl-terminal GIy of an additional ubiquitin.
  • the efficient recognition of a ubiquitinated target protein by a proteosome requires at least four ubiquitins linked in this configuration.
  • Lys 48 or Lys 63 is involved in the formation of poly-ubiquitin chains.
  • Recent studies show that human Mdm2 mediates multiple mono-ubiquitination of p53 by a mechanism requiring enzyme isomerization (Zhihong et al.
  • the members of the El ubiquitin activating agents and E2 ubiquitin conjugating agents are structurally related and well characterized enzymes. There are numerous species of E2 ubiquitin conjugating agents, some of which act in preferred pairs with specific E3 ubiquitin ligating agents to confer specificity for different target proteins.
  • the family of ubiquitin and ubiquitin-like modifiers includes ubiquitin, NEDD8, ISG 15, SUMOl, SUM02, SUM03, APG 12, and APG8.
  • genome mining efforts have identified at least 530 human genes that encode enzymes responsible for ubiquitin conjugation and deconjugation. Many of these genes encode multiple splice variants, thereby increasing the diversity of enzyme families regulating ubiquitin conjugation and deconjugation. There are a multitude of E3's, reflecting their role as specificity determinants, an intermediate number of E2's, and few El's, which are redundant to multiple pathways. Thus, the same E2 in conjunction with different E3's recognizes distinct substrates.
  • ubiquitin and ubiquitin-like enzymes are encodes by at least 11 genes that comprise at least 11 isoforms; El 's are encoded by at least 13 genes that include at least 15 isoforms; E2's, which include Ubc (ubiquitin carrier proteins) and Uev (ubiquitin enzyme variants), are encoded by at least 49 genes that comprise at least 77 isoforms; E3's, which include RING, PHD, HECT and U-box domain containing proteins, are encoded by at least 391 genes that comprise at least 631 isoforms; and DUB 's (de-ubiquitylating enzymes), which includes USP, ULP, JAMM and UCH proteases, are encoded by at least 86 genes that comprise at least 136 isoforms.
  • Ubiquitin conjugation and deconjugation pathways regulate diverse biological pathways.
  • ubiquitin conjugation and deconjugation pathways play important roles in cancers, inflammation, metabolism, viral diseases and central nervous system disorders.
  • compounds that can modulate ubiquitin conjugation and deconjugation processes would serve as important therapeutic agents.
  • ubiquitin regulatory pathways see Wong et al. (DDT 8 (16), 746-754 (2003)).
  • ubiquitin ligating agents contain two separate activities: a ubiquitin ligase activity to attach, via an isopeptide bond, monomers or oligomers of ubiquitin to a target protein, and a targeting activity to physically bring the ligase and substrate together.
  • the substrate specificity of different ubiquitin ligating agents is a major determinant in the selectivity of the ubiquitin-mediated protein degradation process.
  • some ubiquitin ligating agents contain multiple subunits that form a complex having ubiquitin ligating activity.
  • SCFs which play an important role in regulating Gl progression and consists of at least three subunits: SKPl, Cullins (having at least seven family members) and an F-box protein (of which hundreds of species are known) which bind directly to and recruit the substrate to the complex.
  • SKPl a recently discovered family of RING finger proteins
  • the ROC/ APC 11 proteins have been shown to be the key elements conferring ligase activity to ubiquitin ligating agents.
  • ROC/Cullin combinations can regulate specific cellular pathways, as exemplified by the function of APCl 1-APC2, involved in the proteolytic control of sister chromatid separation and exit from telophase into Gl in mitosis ⁇ see King et al, supra; Koepp et al, Cell 97:431-34 (1999)), and ROCl-Cullin 1, involved in the proteolytic degradation of 1KB in NF-KB/IKB mediated transcription regulation (Tan et al, Mol Cell 3(4):527-533 (1999); Laney et al, Cell 97:427-30 (1999)).
  • the best characterized ubiquitin ligating agent is the APC (anaphase promoting complex), which is multi-component complex that is required both for entry into anaphase as well as exit from mitosis ⁇ see King et al, Science 274:1652-59 (1996) for review).
  • the APC plays a crucial role in regulating the passage of cells through anaphase by promoting ubiquitin-mediated proteolysis of many proteins.
  • the APC is also required for degradation of other proteins for sister chromatid separation and spindle disassembly.
  • proteins known to be degraded by the APC contain a conserved nine amino acid motif known as the "destruction box" that targets them for ubiquitin ubiquitination and subsequent degradation.
  • proteins that are degraded during Gl including Gl cyclins, CDK inhibitors, transcription factors and signaling intermediates, do not contain this conserved amino acid motif. Instead, substrate phosphorylation appears to play an important role in targeting their interaction with a ubiquitin ligating agent for ubiquitination (see Hershko et ah, Ann. Rev. Biochem. 67:429-75 (1998)).
  • E3 ubiquitin ligating agents Two major classes of E3 ubiquitin ligating agents are known: the HECT (homologous to E6-AP carboxy terminus) domain E3 ligating agents; and the RING finger domain E3 ligating agents.
  • E6AP is the prototype for the HECT domain subclass of E3 ligating agents and is a multi-subunit complex that functions as a ubiquitin ligating agent for the tumor suppressor p53 which is activated by papillomavirus in cervical cancer (Huang et al. (1999) Science 286:1321-1326).
  • Examples of the RING domain class of E3 ligating agents are TRAF6, involved in IKK activation; CbI, which targets insulin and EGF; Sina/Siah, which targets DCC; Itchy, which is involved in haematopoesis (B, T and mast cells); IAP, involved with inhibitors of apoptosis; and Mdm2 which is involved in the regulation of p53.
  • the RING finger domain subclass of E3 ligating agents can be further grouped into two subclasses.
  • the RING finger domain and the substrate recognition domain are contained on different subunits of a complex forming the ubiquitin ligating agent ⁇ e.g., the RBxI and the F-box subunit of the SCF complex).
  • the ligating agents In the second subclass of ubiquitin ligating agents, the ligating agents have the RING finger domain and substrate recognition domain on a single subunit. (e.g., Mdm2 and cbl) (Tyers et al. (1999) Science 284:601, 603-604; Joazeiro et al. (2000) 102:549-552).
  • a further class of ligating agents are those having a "PHD" domain and are homo logs of the RING finger domain ligating agents (Coscoy et al. (2001) J. Cell Biol. 155(7): 1265-1273), e.g., MEKKl.
  • the PHD domain ligating agents are a novel class of membrane-bound E3 ligating agents.
  • TRAF6 tumor necrosis factor (TNF) receptor associated factors
  • TRAF 6 promotes poly-ubiquitin chains formation that activates TAKl which in turn activates a number of important kinases such as IkB kinase and MAP kinases (reviewed by Wu et al., BioEssays 25, 1096-1105).
  • TRAF6 The ubiquitination of TRAF6 leads to activation of TAKl which then activates IkB kinase. IkB kinase in turn activates the NF-kB pathway as well as phosphorylates MKK6 in the JNK-p38 kinase pathway.
  • the NF-kB pathway includes many important processes such as inflammation, LPS-induces septic shock, viral infection such as HIV, and cell survival among others.
  • the ubiquitin ligase activity of TRAF 6 plays important regulatory roles in many cellular processes. Stem cells are characterized by their ability to self-renew as well as generate differentiated cells within each organ.
  • Bmi-1 was first isolated as an oncogene that cooperates with c-Myc in generating lymphomas in a murine model (see Haupt, et al. Cell 65:753-63 (1991); and van Lohuizen, et al. Cell 65:737-52 (1991)). It is a transcriptional repressor belonging to the Polycomb-group (PcG) family of proteins involved in axial patterning, hematopoiesis, regulation of proliferation, and senescence (see van der Lugt, et al. Genes Dev 8:757-69 (1994); and Pirrotta, Cell93:333-6 (1998)).
  • PcG Polycomb-group
  • Bmi-1 -deficient mouse embryonic fibroblasts overexpress INK4a/ARF locus- encoded genes pl6INK4a and pi 9ARF (mouse homologue of human pl4ARF) and undergo premature senescence in culture (see Jacobs, et al. Nature 397:164-8 (1999)).
  • pl6INK4a and pi 9ARF mouse homologue of human pl4ARF
  • overexpression of Bmi-1 reduces expression of pl6 INK4a and pi 9ARF and immortalizes MEFs (see Jacobs, et al. Nature 397:164-8 (1999)).
  • Bmi-1 is overexpressed in a variety of human cancers, such as mantle cell lymphomas (see Bea, et al.
  • Bmi-1 overexpression alone was able to immortalize post-selection pi 6INK4a-deficient human mammary epithelial cells (HMEC) and induced telomerase activity in these cells (see Dimri, et al. Cancer Res 62: 4736-45 (2002)).
  • human fibroblasts which expresses pl6INK4a Bmi-1 overexpression results in extension of replicative life span but no immortalization (see Itahana, et al. MoI Cell Biol 23:389-401 (2003)).
  • Bmi-1 was also reported to immortalize bone marrow stromal cells and cementoblast progenitor cells, albeit in combination with other oncogenes (see Saito, et al. J Bone Miner Res 20:50-7 (2005); Mori, et al. MoI Cell Biol 25:5183-905 (2005)).
  • Polycombgroup (PcG) proteins exist in at least twobiochemically distinct protein complexes, the EED-EZH2 complex and the PRCl complex, that respectively possess [0018] H3-K27 methyltransferase and H2A-K119 ubiquitin E3 ligase activities.
  • Bmi-1 and Ring IA two components of the PRCl complex, play important roles in H2A ubiquitylation and Hox gene silencing X-chromosome inactivation, tumorigenesis, and stem cell self-renewal; both proteins positively regulate H2A ubiquitylation.
  • the RING finger protein Ring IB is an E3 ligase that participates in the ubiquitination of lysine 119 of histone H2A, and the binding of Bmi-1 stimulates the E3 ligase activity.
  • the PRCl complex has at least two biochemical functions. One of which is to bind chromatin and prevent it from being remodeled by ATP-dependent remodeling factors (see Shao, et al. Cell 98: 37-46, (1999)). Using an electron microscope, a Drosophila PRCl sub-complex has been seen to compact nucleosome arrays in vitro (see Francis, et al. Science 306: 1574-1577, (2004)).
  • the E3 ligase activity has been shown to be important for the involvement of PRCl in X-chromosome inactivation and the control of Hox gene expression (see Fang, et al. J. Biol. Chem. 279: 52812- 52815 (2004); de Napoles, et al. Dev. Cell 7: 663-676 (2004); Hernandez-Munoz, et al. Proc. Natl. Acad. Sci. U. S. A. 102: 7635-7640 (2005); and Cao, et al. MoI. Cell 20: 845-854, (2005)).
  • Chromatin immunoprecipitation (ChIP) assays demonstrate that Bmi-1 and other components of the two PcG complexes bind to the promoter of HoxC13. Knockout Bmi-1 results in significant loss of H2A ubiquitylation and upregulation of Hoxcl3 expression, whereas EZH2 -mediated H3-K27 methylation is not affected. Results suggest that EZH2- mediated H3- K27 methylation functions upstream of PRCl and establishes a critical role for Bmi-1 and RinglA in H2A ubiquitylation and Hox gene silencing.
  • PRR postreplication repair
  • Rad6 a ubiquitin-conjugating enzyme
  • Radl8 a ubiquitin ligase, E3
  • Yeast Radl8 was shown to bind to single-stranded DNA and also to form a tight complex with Rad6 (see Bailly, et al. Genes Dev., 8: 811-820, (1994); and Bailly,et al. J.
  • PCNA proliferating cell nuclear antigen
  • Radl8 forms a tight complex with Rad6 both in yeast and vertebrate cells (see Tateishi, et al. Proc. Natl. Acad. Sci. U. S. A. 97: 7927-7932 (2000); and Bailly, V. et al, J. Biol. Chem. Ill: 23360-23365 (1997)).
  • Rad6 is a member of the E2 and is also named UBC2.
  • Yeast Rad6 is involved in amino end rule protein degradation through physical interaction with the E3 enzyme, Ubrl (see Dohmen, et al., Proc. Natl.
  • Rad6 plays a role that regulates monoubiquitination of histone 2B with the help of Brel, an E3 protein containing one RING finger domain (see Joazeiro and Weissman, A. M. Cell 102: 549-552, (2000)).
  • E3 protein containing one RING finger domain see Joazeiro and Weissman, A. M. Cell 102: 549-552, (2000).
  • yeast and vertebrate Radl8 also contain it in the NH2 -terminal region, suggesting that Radl8 functions as an E3.
  • the human and mouse RAD 18 proteins can interact with two forms of homo logs of yeast Rad6, RAD6A and RAD6B, both in vitro and in vivo (see Tateishi, et al. Proc. Natl Acad. Sci. USA, 97: 7927-7932, (2000); Tateishi, et al. MoI. Cell. Biol, 23: 474-481, (2003); and Xin, et al. Nucleic Acids Res., 28: 2847-2854, (2000)).
  • p27 is ubiquitously expressed and binds cyclin-Cdk2 to inhibit kinase activity.
  • p27 null animals exhibit multiorgan hyperplasia (see Fero et al., Nature 396: 177-180 (1996); Kiyokawa et al., Cell 85: 721-732 (1996); and Nakayama et al., Cell 85: 707-720 (1996)), and p27+/_ heterozygous animals are tumor prone (see Fero et al., Nature 396: 177-180 (1998)). While normal quiescent epithelial tissues express high p27 (see Catzavelos et al., Nat. Med.
  • Activated EGFR family receptor tyrosine kinases recruit and activate cSrc, and cSrc in turn further activates RTKs, stimulating cell proliferation (see Ishizawar and Parsons, Cancer Cell 6: 209-214 (2004)).
  • Drug-mediated cSrc inhibition blocks the effects of EGFR and Her2 on cell proliferation (see Belsches- Jablonski et al., Oncogene 20: 1465-1475 (2001); and Biscardi et al., J. Biol. Chem. 274:8335-8343 (1999)).
  • cSrc is also activated by liganded estrogen receptor (ER) in human breast cancer cells.
  • Estrogen ER binding stimulates rapid, transient recruitment of cSrc, She activation, and MAPK signaling (see Migliaccio et al., EMBO J. 15: 1292-1300 (1996)). Estrogen:ER-stimulated Src further recruits RTKs, Her2, EGFR (see Chu et al., Cancer Res. 65: 18-25 (2005)), and IGF-IR (see Song et al., Proc. Natl. Acad. Sci. USA 101 : 2076-2081 (2004)), to promote cell cycle progression.
  • ER blockade by tamoxifen or estrogen deprivation by aromatase inhibitors is of therapeutic utility in a majority of breast cancers that express the ER.
  • p27 is required for Gl arrest by tamoxifen or estrogen deprivation (see Cariou et al., Proc. Natl. Acad. Sci. USA 97: 9042-9046 (2000)).
  • the development of antiestrogen resistance limits treatment efficacy.
  • EGFRand Her2/ErbB2 overexpression can confer tamoxifen resistance in cultured lines (see Benz et al., Breast Cancer Res. Treat.
  • AZD0530 inhibits cell motility and invasion in vitro (see Hiscox et al., Breast Cancer Res. Treat. 97: 263-274 (2005)) and inhibits metastasis in animal models in vivo (see T. Green et al., 2005, American Association for Cancer Research, abstract). In three different Src-activated ER-positive breast cancer lines, AZD0530 or ER-saturating doses of tamoxifen each, when given alone, caused partial cell cycle inhibition.
  • AZD0530 together with tamoxifen increased p27, inhibited cyclin E- Cdk2, and causedGl arrest. In contrast, in lines lacking Src activation, addition of AZD0530 did not enhance the antiproliferative effect of tamoxifen.
  • Ubiquitin agents such as the ubiquitin activating agents, ubiquitin conjugating agents, and ubiquitin ligating agents, are key determinants not only in ubiquitin-mediated proteolytic pathway that results in the degradation of targeted proteins, but also in regulation of cellular processes. Consequently, agents that modulate the activity of such ubiquitin agents may be used to up-regulate or down-regulate specific molecules involved in cellular signal transduction.
  • Ubiquitin agents are involved in cancer, inflammation, adaptive immunity, innate immunity, bone metabolism, LPS-induced angiogenesis, osteoporosis, osteopinneal diseases, protozoan infection, viral infections, lymph node development, mammary gland development, skin development, and central nervous system development.
  • Viral and protozoan infections involving ubiquitination include infections caused by variola viruses such as smallpox, HIV and related conditions, human papillomavirus, HSV, adenovirus, coxsackie virus, HCMV, KSHV, EBV, paramyxovirus, myxomavirus, ebola, retrovirus, rhabdovirus, and the malaria parasite.
  • Other conditions in which ubiquitin is involved include aberrant cell growth, cancers, restenosis, psoriasis, and neoplastic cell proliferation.
  • Ubiquitin is also involved in gene regulation, such as in bone metabolism, and in signal transduction pathways that involve IL-I, CD40, RANKL, LPS, IL-17, LMPl, NF-kB, AP-I and kinases, such as MAP kinases, JNK/SAPK, ERK, p38, IkB kinase and Src-family tyrosine kinases.
  • ubiquitin plays a critical role in the TNFR/IL-IR/TLR signal transduction pathways of inflammation, for example, in autoimmune diseases such as rheumatoid arthritis (RA), chronic obstructive pulmonary disease (COPD), inflammatory bowel disease (IBD) and graft rejection, bone-destructive diseases, such as osteoporosis and RA, allergies and infective disease such as bacterial sepsis and associated systemic inflammation.
  • ubiquitin plays a role in diseases and conditions that involve non-degradative ubiquitination, for example, in diseases and conditions that involve activation of K-63 linked, non-degradative ubiquitination.
  • Ubiquitin has also been implicated as key components in other biochemical processes. Ubiquitination of the Gag structural protein of Rous Sarcoma virus has been linked to the targeting of Gag to the cell membrane of the host cell where it can assemble into spherical particles and bud from the cell surface. Production of HIV particles has also been associated with ubiquitination and may constitute an important cellular pathway for producing infectious particles. Thus, the ubiquitin pathway may be an important target for treatment of HIV positive patients.
  • the invention comprises compounds and compositions comprising the compounds for inhibiting ubiquitin agents.
  • the compositions can further comprise a pharmaceutically acceptable carrier, diluent, and/or excipient and can be used in inhibiting and treating various conditions where ubiquitination is involved. They can also be used as research tools to study the role of ubiquitin in various natural and pathological processes.
  • the invention comprises compounds that inhibit ubiquitination of target proteins.
  • the invention comprises a pharmaceutical composition comprising an inhibitor of ubiquitination according to the invention and a pharmaceutically acceptable carrier, excipient, or diluent.
  • the invention comprises methods of inhibiting ubiquitination in a cell, comprising contacting a cell in which inhibition of ubiquitination is desired with a pharmaceutical composition comprising a ubiquitin agent inhibitor according to the invention.
  • the invention provides methods for treating cell proliferative diseases or conditions, comprising administering to a patient in need thereof a pharmaceutical composition comprising an effective amount of a ubiquitin agent inhibitor according to the invention.
  • the invention provides methods for inhibiting TRAF6 activity in a cell, comprising administering to the cell a compound of the invention or a pharmaceutical composition comprising an effective amount of a compound according to the invention.
  • Figure 1 describes the Protein Ubiquitination System
  • Figure 2 describes Phosphorylation- and ubiquitin-dependent degradation of p27
  • Figure 3 describes Ubiquitination of p27Kipl by SCF-Skp2
  • Figure 4 describes a Plate -based p27-SCF ubiquitination assay
  • the invention provides compounds, compositions and methods for inhibiting ubiquitin ligase activity.
  • the invention provides compounds, compositions and methods for inhibiting TRAF6, APC as well as other enzymes that exhibit E3-like activity.
  • the invention also provides methods and compositions for treating cell proliferative diseases and conditions in which TRAF6 is involved.
  • the compounds of the invention inhibit the ubiquitination of p27, a tumor suppressor, and biochemical and cell based assays.
  • the compounds are expected to show selectivity over ligase counter assays such as p53-MDM2, APC, TRAF 6 and other biological targets and biochemical and cell based assays.
  • the compounds of the invention are expected to have good oral bioavailability, good ADME properties and are readily amenable to oral or IV formulation.
  • the compounds of the invention are expected to show efficacy in vivo graft animal models in diseases mediated via pathways involving p27/SCF.
  • the invention provides compounds according Formula I: and pharmaceutically acceptable salts, hydrates, solvates, polymorphs, atrophisomers, N-oxides, and prodrugs thereof, wherein
  • L' is a covalent bond or -SO 2 -;
  • X is H or -OH
  • Ri and R 2 are independently -H, -NO 2 , -OH, -CN, halo, Ci-C 6 alkyl, Ci-C 6 alkoxy, mono- to per-halogenated Ci-C 6 alkyl, heterocyclyl, heterocyclyl-O-aryl, -NH 2 , mono- or (Ii-(C 1 -C 6 alkyl) amino, -C(O)-OR 9 , -N(R 8 )-Z, -S-Z, -SO 2 -Z, -N(R 8 )-Z, aryl, or heteroaryl, wherein each of the aryl and heteroaryl is optionally substituted with 1 to 3 groups selected from -H, Ci-C 6 alkyl, Ci-C 6 alkoxy, mono- to per-halogenated Ci-C 6 alkyl, -NH 2 , mono- or di-(Ci-C 6 alkyl) amino, -NO 2 , halo, or -CN
  • Ri and R 2 together with the carbon atoms to which they are attached form an aryl group optionally substituted with 1 to 3 groups selected from -OH, halo, Ci-C 6 -alkyl, Ci-C 6 -alkoxy, -NH 2 . and -NO 2 ;
  • Y is -H, -NO 2 , -OH, -CN, halo, Ci-C 6 alkyl, Ci-C 6 alkoxy, -NH 2 , mono- or (Ii-(C 1 -C 6 alkyl) amino, -C(O)-OR 9 , -C(O)R 9 , -N(R 8 )-Z, Ci-C 6 alkyl-aryl, Ci-C 6 alkyl-heterocyclyl, Ci-C 6 alkyl-heteroaryl, C 0 -C 6 alkyl-C(O)-aryl, C 0 -C 6 alkyl-C(O)-heterocyclyl, C 0 -C 6 alkyl-C(O)-heteroaryl, -O-aryl, aryl, heterocyclyl, heteroaryl, or -Zi-S(O) 2 -Z 2 where Zi and Z 2 are independently aryl or heteroaryl, where
  • R 8 is -H, Ci-C 6 alkyl, or Ci-C 6 alkoxy
  • R 9 is -H, Ci-C 6 alkyl or C 3 -C 6 cycloalkyl
  • Z is -Ci-C 6 alkyl-O-C(O)-Ci-C 6 alkyl, aryl or heteroaryl wherein each of the aryl and heteroaryl is optionally substituted with -H, -NO 2 , -CN, halo, Ci-C 6 alkyl, Ci-C 6 alkoxy, -NH 2 , mono- or di-(Ci-C 6 alkyl) amino, or -C(O)-OR 9 .
  • the compounds of formula I are compounds of formula Ia:
  • the compounds of formula II are compounds of formulae III(i)-III(vi)
  • I ⁇ I(iii) X is H and Y is optionally substituted aryl;
  • I ⁇ I(iv) X is OH and Y is optionally substituted aryl;
  • III(v) X is H and Y is aryl substituted with halo (preferably -Cl); or
  • IV(i) Ri is -S-Z
  • IV(ii) Ri is -SO 2 -Z;
  • IV(iii) Ri is -N(R 8 )-Z.
  • the compounds according to formula I, II, III(i)-III(vi), and IV(i)-IV(iii) are compounds of formula V, which are compounds in which Z is aryl or heteroaryl wherein each of the aryl and heteroaryl is optionally substituted with -H, -NO 2 , -CN, halo, Ci-C 6 alkyl, Ci-C 6 alkoxy, -NH 2 , mono- or di-(Ci-C 6 alkyl) amino, or -C(O)-ORg.
  • Z is phenyl, pyrimidinyl, quinolinyl, or piperidinyl each of which is unsubstituted or substituted as described in the definition of Z in the description of compounds of formula I.
  • Z is pyrimidinyl
  • preferred are those compounds in which Z is pyrimidin-4-yl.
  • quinolinyl preferred are those compounds in which Z is quinolinyl-2-yl or quinolinyl-8-yl.
  • the compounds according to formula I, II, III(i)-III(vi), IV(i)-IV(iii), and V are compounds of formula VI, which are compounds in which R 2 is H.
  • Preferred compounds according to the invention for those of Table 1 are compounds according to the invention for those of Table 1 :
  • the invention also comprises compounds of Table 1, but the compounds of Table 1 are specifically excluded from the scope of the compounds of formulae I-V.
  • the compounds according to the first aspect of the invention are also useful as general ubiquitin ligase inhibitors.
  • the compounds of the invention can be used as inhibitors of enzymes that exhibit ligase activity, including but not limited to TRAF6, APC and E3 enzymes.
  • the compounds of the invention are also useful for regulating or inhibiting pathways in diseases and conditions that involve ubiquitin conjugation and deconjugation such as cancers, inflammation, metabolism, viral diseases and central nervous system disorders.
  • the compounds of the invention can be used to regulate or inhibit the products of genes that encode ubiquitin or ubiquitin- like enzymes described in Wong et al. (DDT 8 (16), 746-754 (2003)), which is incorporated by reference in its entirety.
  • the invention provides for pharmaceutical compositions comprising, together with a pharmaceutically acceptable carrier, diluent, or excipient, a compound of according to the first aspect of the invention described above.
  • the invention provides methods of inhibiting ubiquitination in a cell comprising contacting the cell in which inhibition of ubiquitination is desired with a compound according to the first aspect of the invention or a pharmaceutical composition according to the second aspect of the invention.
  • the compounds and formulations of the invention can inhibit ubiquitination in cells derived from animals, particularly, mammalian cells.
  • the compounds and formulations of the invention can also be used to inhibit the ubiquitin ligase activity of TRAF6.
  • the invention provides for methods of treating cell proliferative diseases or conditions comprising administering to a patient an effective amount of a compound of the first aspect of the invention or a pharmaceutical composition according to the second aspect of the invention.
  • Cell proliferative diseases or conditions include, but are not limited to, psoriasis, keloid scarring, and cancers, such as cancers of the breast, immune system, bone, nervous system, brain, blood, lymphatic system, and skin.
  • the compounds and pharmaceutical compositions of the invention are useful for treating cell proliferative diseases or conditions that involve TRAF6.
  • the invention provides for methods of inhibiting TRAF6 comprising administering to a patient an effective amount of a compound according to the first aspect of the invention or pharmaceutical composition according to the second aspect of the invention.
  • the compounds and pharmaceutical compositions are useful for treating conditions or diseases that involve TRAF6 such as those related to cancer, inflammation, adaptive immunity, innate immunity, bone metabolism, LPS-induced angiogenesis, osteoporosis, osteopinneal diseases, lymph node development, mammary gland development, skin development, and central nervous system development.
  • the compounds for use in the method according to the fifth aspect of the invention are also useful as general ubiquitin ligase inhibitors.
  • the compounds of the invention can be used as inhibitors of E3 enzymes that contain HECT and RING finger domains, Mdm2 with RING fingers and variants, and U-box-containing proteins.
  • the compounds of the invention are useful as protein modulators, immunologic agents anti-inflammatory agents, anti-osteoporosis agents, anti-viral agents, for example, inhibitors of variola viruses such as smallpox, HIV and related conditions, human papillomavirus, HSV, adenovirus, coxsackie virus, HCMV, KSHV, EBV, paramyxovirus, myxomavirus, ebola, retrovirus, and rhabdovirus, anti-protozoan agents, for example, inhibitors of the malaria parasite.
  • the compounds of the invention are also useful as oncologic and anti-pro liferative agents that inhibit aberrant cell growth, cancers, restenosis, psoriasis, and neoplastic cell proliferation.
  • Inhibition of TRAF 6 activity by the compounds and pharmaceutical compositions of the invention provides ways to regulate the expression of genes involved many biological processes. Such processes include but are not limited to bone metabolism and signal transduction pathways that involve IL-I, CD40, RANKL, LPS, IL-17, and LMPl.
  • the compounds and pharmaceutical compositions of the invention can be used to regulate the activities of transcription factors that activate the expression of genes, such as NF -kB and AP-I.
  • the compounds and pharmaceutical compositions can also be used to regulate the activities of kinases, such as MAP kinases, JNK/SAPK, ERK, p38, IkB kinase and Src-family tyrosine kinases.
  • TRAF6 serves as a therapeutic target for inflammatory and autoimmune diseases.
  • TRAF6 plays a critical regulator role of the TNFR/IL-1R/TLR signal transduction pathways and can serve as a broad anti-inflammation target for inflammatory diseases such as RA, COPD, IBD.
  • TRAF6 can also be a useful therapeutic target for treating autoimmune diseases, such as graft rejection because of its regulator role in the CD40 signaling cascade.
  • TRAF6 is also a target for treating bone-destructive diseases, such as osteoporosis and rheumatoid arthritis because TRAF6 plays a critical regulator role of the RANK signal transduction that mediate osteoclast activation and function.
  • TRAF6 may also serve as a novel allergic and infective disease target for treating bacterial sepsis and associated systemic inflammation because TRAF6 plays critical mediator roles in the TLR signal transduction which is involved in the interaction between dentritic cells, T lymphocytes and mast cells.
  • TRAF 6 may also serve as a therapeutic target in diseases and conditions that involve non-degradative ubiquitination.
  • TRAF6 acts as an E3 ubiquitin ligase that mediates kinase activation by K-63 linked, non-degradative ubiquitination.
  • Inhibiting TRAF ⁇ 's E3 ligase activity may provide novel anti-inflammation therapeutics.
  • the invention provides compounds according Formula X:
  • N-oxides, and prodrugs thereof wherein n is 0, 1, 2, 3, or 4; L' is a covalent bond or -SO 2 -; A is -C(X)(Y)-, -N(Y)-, or -O-, wherein X is H, -C(O)R 8 , or -OH; and
  • Y is -H, -NO 2 , -OH, -CN, halo, -Z, Ci-C 6 alkyl, Ci-C 6 alkoxy, -NH 2 , mono- or (Ii-(C 1 -C 6 alkyl) amino, -C(O)-OR 9 , -C(O)R 9 , -N(R 8 )-Z, Ci-C 6 alkyl-aryl, Ci-C 6 alkyl-heterocyclyl, C 1 -C 6 alkyl-heteroaryl, C 0 -C 6 alkyl-C(O)-aryl, C 0 -C 6 alkyl-C(O)-heterocyclyl, C 0 -C 6 alkyl-C(O)-heteroaryl, -O-aryl, C 3 -C 8 -cycloalkyl, heterocyclyl, , or -Zi-S(O) 2 -Z 2 where
  • Zi and Z 2 are independently aryl or heteroaryl, wherein each of the aryl, heterocyclyl and heteroaryl is optionally substituted with 1 to 3 groups selected from -H, Ci-C 6 alkyl, Ci-C 6 alkoxy, mono- to per-halogenated Ci-C 6 alkyl, -C(O)-OR 9 , -N(R 9 )-C(O)R 9 , -O-(halo Ci-C 6 alkyl), -NH 2 , mono- or di-(Ci-C 6 alkyl) amino, -NO 2 , halo, or -CN; or X and Y together with the atom to which they are attached, are oxo, a C 3 -Cs cycloalkyl or heterocyclyl wherein the cycloalkyl or heterocyclyl is optionally substituted by one to four R 4 groups;
  • Ri and R 2 are independently -H, -NO 2 , -OH, -CN, halo, Ci-C 6 alkyl, Ci-C 6 alkoxy, mono- to per-halogenated Ci-C 6 alkyl, heterocyclyl, heterocyclyl-O-aryl, -NH 2 , mono- or (Ii-(C 1 -C 6 alkyl) amino, -C(O)-OR 9 , -N(R 8 )-Z, -0-Z, -S-Z, -SO 2 -Z, -SO 2 -NH-, -N(Rg)-Z, aryl, or heteroaryl, wherein each of the aryl and heteroaryl is optionally substituted with 1 to 3 groups selected from -H, Ci-C 6 alkyl, Ci-C 6 alkoxy, mono- to per-halogenated Ci-C 6 alkyl, -NH 2 , mono- or di-(Ci-C 6 alkyl) amino,
  • each R 3 is independently halo or Ci-C 6 alkyl, or two R 3 attached to adjacent carbon atoms, together with the atoms to which they are attached, form a fused aryl or heteroaryl wherein the aryl or heteroaryl is optionally substituted by one to four R 4 groups; or Y and R 3 attached to adjacent carbon atoms, together with the atoms to which they are attached, form a fused aryl or heteroaryl and X is absent, wherein the aryl or heteroaryl is optionally substituted by one to four R 4 groups; each R 4 is independently halo, Ci-C 6 alkyl, oxo, or Ci-C 6 alkoxy; Rs is -H, Ci-C 6 alkyl, or Ci-C 6 alkoxy; R 9 is -H, Ci-C 6 alkyl or C 3 -C 6 cycloalkyl; and each Z is independently -Ci-C 6 alkyl-O-C(O)-
  • L' is SO 2 .
  • n is 0, 1, or 2. More preferably, n is 0. In other more preferred embodiments, n is 2.
  • A is -C(X)(Y)- wherein X is H or -OH; and Y is aryl, or heteroaryl wherein the aryl and heteroaryl is optionally substituted with one to four R5 groups, wherein each R5 is independently -NO 2 , -CN, halo, Ci-C 6 alkyl, Ci-C 6 alkoxy, -NH 2 , mono- or di-(Ci-C 6 alkyl) amino, or -C(O)ORg.
  • Y is aryl, or heteroaryl wherein the aryl and heteroaryl is optionally substituted with one to four R5 groups, wherein each R 5 is independently -NO 2 , -CN, halo, Ci-C 6 alkyl, Ci-C 6 alkoxy, -NH 2 , mono- or di-(Ci-C 6 alkyl) amino, or -C(O)OR 9 .
  • A is -CH(Y)- wherein Y is aryl, optionally substituted with one or two R 5 groups, wherein each R 5 is independently -NO 2 , -CN, halo, Ci-C 6 alkyl, Ci-C 6 alkoxy, -NH 2 , mono- or di-(Ci-C 6 alkyl) amino, or -C(O)OR 9 .
  • A is -N(Y)-, wherein
  • Y is aryl, or heteroaryl wherein the aryl and heteroaryl is optionally substituted with one to four R5 groups, wherein each R 5 is independently -NO 2 , -CN, halo, Ci-C 6 alkyl, Ci-C 6 alkoxy, -NH 2 , mono- or di-(Ci-C 6 alkyl) amino, or -C(O)OR 9 .
  • Y is aryl, optionally substituted with one or two R 5 groups, wherein each R 5 is independently -NO 2 , -CN, halo, Ci-C 6 alkyl, Ci-C 6 alkoxy, -NH 2 , mono- or di-(Ci-C 6 alkyl) amino, or -C(O)OR 9 .
  • Ci-C 6 alkyl Ci-C 6 alkoxy, -NH 2 , mono- or di-(Ci-C 6 alkyl) amino, or
  • Ci-C 6 alkyl Ci-C 6 alkoxy, -NH 2 , mono- or di-(Ci-C 6 alkyl) amino, or
  • A is -N(Y)- wherein Y is pyridyl, optionally substituted with one or two R 5 groups, wherein each R 5 is independently -NO 2 , -CN, halo, Ci-C 6 alkyl, Ci-C 6 alkoxy, -NH 2 , mono- or di-(Ci-C 6 alkyl) amino, or -C(O)OR 9 .
  • Ri is -H, halo, -O-Z, -S-Z, -SO 2 -Z, -N(Rg)-Z, aryl, or heteroaryl, wherein each of the aryl and heteroaryl is optionally substituted with 1 to 3 groups selected from -H, Ci-C 6 alkyl, Ci-C 6 alkoxy, mono- to per-halogenated Ci-C 6 alkyl, -NH 2 , mono- or di-(Ci-C 6 alkyl) amino, -NO 2 , halo, or -CN.
  • Ri is -H, halo, -O-Z, -S-Z, or N(Rg)-Z. Even more preferred are compounds of formula X, wherein Ri is -O-Z, -S-Z, or N(Rg)-Z. Other more preferred are compounds of formula X, wherein Ri is -H, or halo. Other more preferred are compounds of formula X, wherein Ri is chloro.
  • Ri is -S-Z, wherein Z is aryl or heteroaryl (preferably, quinolinyl) wherein the aryl and heteroaryl is optionally substituted with one to four R5 groups, wherein each R5 is independently -NO 2 , -CN, halo, Ci-C 6 alkyl, Ci-C 6 alkoxy, -NH 2 , mono- or di-(Ci-C 6 alkyl) amino, or -C(O)OR 9 .
  • Z is aryl or heteroaryl (preferably, quinolinyl) wherein the aryl and heteroaryl is optionally substituted with one to four R5 groups, wherein each R5 is independently -NO 2 , -CN, halo, Ci-C 6 alkyl, Ci-C 6 alkoxy, -NH 2 , mono- or di-(Ci-C 6 alkyl) amino, or -C(O)OR 9 .
  • Ri is -S-Z, wherein Z is aryl optionally substituted with one or two R5 groups, wherein each R 5 is independently -NO 2 , -CN, halo, Ci-C 6 alkyl, Ci-C 6 alkoxy, -NH 2 , mono- or di-(Ci-C 6 alkyl) amino, or -C(O)OR 9 .
  • Ri is -S-Z, wherein Z is phenyl optionally substituted with one or two R 5 groups, wherein each R 5 is independently -NO 2 , -CN, halo, Ci-C 6 alkyl, Ci-C 6 alkoxy, -NH 2 , mono- or di-(Ci-C 6 alkyl) amino, or -C(O)OR 9 .
  • Ri is -S-Z, wherein Z is heteroaryl (preferably, quinolinyl) optionally substituted with one or two R 5 groups, wherein each R 5 is independently -NO 2 , -CN, halo, Ci-C 6 alkyl, Ci-C 6 alkoxy, -NH 2 , mono- or di-(Ci-C 6 alkyl) amino, or -C(O)OR 9 .
  • Z is heteroaryl (preferably, quinolinyl) optionally substituted with one or two R 5 groups, wherein each R 5 is independently -NO 2 , -CN, halo, Ci-C 6 alkyl, Ci-C 6 alkoxy, -NH 2 , mono- or di-(Ci-C 6 alkyl) amino, or -C(O)OR 9 .
  • Ri is -S-Z, wherein Z is pyridyl optionally substituted with one or two R 5 groups, wherein each R 5 is independently -NO 2 , -CN, halo, Ci-C 6 alkyl, Ci-C 6 alkoxy, -NH 2 , mono- or di-(Ci-C 6 alkyl) amino, or -C(O)OR 9 .
  • R 2 is -H, -NO 2 , -OH,
  • Ci-C 6 alkyl halo, Ci-C 6 alkyl, Ci-C 6 alkoxy, mono- to per-halogenated Ci-C 6 alkyl, -NH 2 , mono- or
  • R 3 is methyl. More preferred compounds of formula X are those where R3 is methyl and n is O, 1, or 2.
  • the compounds of formula X are compounds of formula
  • L' is SO 2 .
  • n is 0, 1, or 2. More preferably, n is 0. In other more preferred embodiments, n is 2.
  • A is -C(X)(Y)- wherein X is H or -OH; and Y is aryl, or heteroaryl wherein the aryl and heteroaryl is optionally substituted with one to four R 5 groups, wherein each R 5 is independently -NO 2 , -CN, halo, Ci-C 6 alkyl, Ci-C 6 alkoxy, -NH 2 , mono- or (Ii-(C 1 -C 6 alkyl) amino, or -C(O)OR 9 .
  • More preferred are compounds of formula XI, wherein A is -CH(Y)- wherein
  • Y is aryl, or heteroaryl wherein the aryl and heteroaryl is optionally substituted with one to four R 5 groups, wherein each R 5 is independently -NO 2 , -CN, halo, Ci-C 6 alkyl, Ci-C 6 alkoxy, -NH 2 , mono- or di-(Ci-C 6 alkyl) amino, or -C(O)OR 9 .
  • Y is aryl, optionally substituted with one or two R 5 groups, wherein each R 5 is independently -NO 2 , -CN, halo, Ci-C 6 alkyl, Ci-C 6 alkoxy, -NH 2 , mono- or di-(Ci-C 6 alkyl) amino, or -C(O)OR 9 .
  • A is -N(Y)-, wherein Y is aryl, or heteroaryl wherein the aryl and heteroaryl is optionally substituted with one to four R 5 groups, wherein each R 5 is independently -NO 2 , -CN, halo, Ci-C 6 alkyl, Ci-C 6 alkoxy, -NH 2 , mono- or di-(Ci-C 6 alkyl) amino, or -C(O)ORg.
  • Y is aryl, optionally substituted with one or two R 5 groups, wherein each R 5 is independently -NO 2 , -CN, halo, Ci-C 6 alkyl, Ci-C 6 alkoxy, -NH 2 , mono- or di-(Ci-C 6 alkyl) amino, or -C(O)OR 9 .
  • Y is heteroaryl, optionally substituted with one or two R 5 groups, wherein each R 5 is independently -NO 2 , -CN, halo, Ci-C 6 alkyl, Ci-C 6 alkoxy, -NH 2 , mono- or di-(Ci-C 6 alkyl) amino, or -C(O)OR 9 .
  • Y is phenyl, optionally substituted with one or two R 5 groups, wherein each R 5 is independently -NO 2 , -CN, halo, Ci-C 6 alkyl, Ci-C 6 alkoxy, -NH 2 , mono- or di-(Ci-C 6 alkyl) amino, or -C(O)OR 9 .
  • A is -N(Y)- wherein Y is pyridyl, optionally substituted with one or two R 5 groups, wherein each R 5 is independently -NO 2 , -CN, halo, Ci-C 6 alkyl, Ci-C 6 alkoxy, -NH 2 , mono- or di-(Ci-C 6 alkyl) amino, or -C(O)OR 9 .
  • Ri is -H, halo, -O-Z, -S-Z, -SO 2 -Z, -N(Rg)-Z, aryl, or heteroaryl, wherein each of the aryl and heteroaryl is optionally substituted with 1 to 3 groups selected from -H, Ci-C 6 alkyl, Ci-C 6 alkoxy, mono- to per-halogenated Ci-C 6 alkyl, -NH 2 , mono- or di-(Ci-C 6 alkyl) amino, -NO 2 , halo, or -CN.
  • Ri is -S-Z, wherein Z is aryl or heteroaryl (preferably, quinolinyl) wherein the aryl and heteroaryl is optionally substituted with one to four R 5 groups, wherein each R 5 is independently -NO 2 , -CN, halo, Ci-C 6 alkyl, Ci-C 6 alkoxy, -NH 2 , mono- or di-(Ci-C 6 alkyl) amino, or -C(O)OR 9 .
  • Z is aryl or heteroaryl (preferably, quinolinyl) wherein the aryl and heteroaryl is optionally substituted with one to four R 5 groups, wherein each R 5 is independently -NO 2 , -CN, halo, Ci-C 6 alkyl, Ci-C 6 alkoxy, -NH 2 , mono- or di-(Ci-C 6 alkyl) amino, or -C(O)OR 9 .
  • Ri is -S-Z, wherein Z is aryl optionally substituted with one or two R 5 groups, wherein each R 5 is independently -NO 2 , -CN, halo, Ci-C 6 alkyl, Ci-C 6 alkoxy, -NH 2 , mono- or di-(Ci-C 6 alkyl) amino, or -C(O)ORg.
  • Ri is -S-Z, wherein Z is phenyl optionally substituted with one or two R 5 groups, wherein each R 5 is independently -NO 2 , -CN, halo, Ci-C 6 alkyl, Ci-C 6 alkoxy, -NH 2 , mono- or di-(Ci-C 6 alkyl) amino, or -C(O)ORg.
  • Ri is -S-Z, wherein Z is heteroaryl (preferably, quinolinyl) optionally substituted with one or two R 5 groups, wherein each R 5 is independently -NO 2 , -CN, halo, Ci-C 6 alkyl, Ci-C 6 alkoxy, -NH 2 , mono- or di-(Ci-C 6 alkyl) amino, or -C(O)ORg.
  • Z is heteroaryl (preferably, quinolinyl) optionally substituted with one or two R 5 groups, wherein each R 5 is independently -NO 2 , -CN, halo, Ci-C 6 alkyl, Ci-C 6 alkoxy, -NH 2 , mono- or di-(Ci-C 6 alkyl) amino, or -C(O)ORg.
  • Ri is -S-Z, wherein Z is pyridyl optionally substituted with one or two R 5 groups, wherein each R 5 is independently -NO 2 , -CN, halo, Ci-C 6 alkyl, Ci-C 6 alkoxy, -NH 2 , mono- or di-(Ci-C 6 alkyl) amino, or -C(O)ORg.
  • Ri is -S-Z, wherein Z is quinolinyl, optionally substituted with one or two R 5 groups, wherein each R 5 is independently -NO 2 , -CN, halo, Ci-C 6 alkyl, Ci-C 6 alkoxy, -NH 2 , mono- or di-(Ci-C 6 alkyl) amino, or -C(O)ORg.
  • R 2 is -H, -NO 2 , -OH, -CN, halo, Ci-C 6 alkyl, Ci-C 6 alkoxy, mono- to per-halogenated Ci-C 6 alkyl, -NH 2 , mono- or di-(Ci-C 6 alkyl) amino, -C(O)-OR 9 , -N(Rg)-Z, -0-Z, -S-Z, -SO 2 -Z, or -N(Rg)-Z.
  • R 2 is -H, -NO 2 , -OH, -CN, halo, Ci-C 6 alkyl, Ci-C 6 alkoxy, mono- to per-halogenated Ci-C 6 alkyl, -NH 2 , mono- or di-(Ci-C 6 alkyl) amino, -C(O)-ORg.
  • R 3 is methyl. More preferred compounds of formula XI are those where R 3 is methyl and n is O, 1 , or 2.
  • the compounds of formula XI are compounds of formula XII:
  • n is 0, 1, or 2.
  • n is 0. In other more preferred embodiments, n is 2.
  • A is -C(X)(Y)- wherein X is H or -OH; and Y is aryl, or heteroaryl wherein the aryl and heteroaryl is optionally substituted with one to four R5 groups, wherein each R5 is independently -NO 2 , -CN, halo, Ci-C 6 alkyl, Ci-C 6 alkoxy, -NH 2 , mono- or di-(Ci-C 6 alkyl) amino, or -C(O)ORg.
  • Ci-C 6 alkyl Ci-C 6 alkoxy, -NH 2 , mono- or di-(Ci-C 6 alkyl) amino, or -C(O)OR 9 .
  • A is -N(Y)-, wherein
  • Y is aryl, or heteroaryl wherein the aryl and heteroaryl is optionally substituted with one to four
  • R5 groups wherein each R 5 is independently -NO 2 , -CN, halo, Ci-C 6 alkyl, Ci-C 6 alkoxy, -NH 2 , mono- or di-(Ci-C 6 alkyl) amino, or -C(O)OR 9 .
  • Ci-C 6 alkyl Ci-C 6 alkoxy, -NH 2 , mono- or di-(Ci-C 6 alkyl) amino, or -C(O)OR 9 .
  • Ci-C 6 alkyl Ci-C 6 alkoxy, -NH 2 , mono- or di-(Ci-C 6 alkyl) amino, or
  • Ci-C 6 alkyl Ci-C 6 alkoxy, -NH 2 , mono- or di-(Ci-C 6 alkyl) amino, or
  • Ri is -H, halo, -O-Z, -S-Z, -SO 2 -Z, -N(Rg)-Z, aryl, or heteroaryl, wherein each of the aryl and heteroaryl is optionally substituted with 1 to 3 groups selected from -H, Ci-C 6 alkyl, Ci-C 6 alkoxy, mono- to per-halogenated Ci-C 6 alkyl, -NH 2 , mono- or di-(Ci-C 6 alkyl) amino, -NO 2 , halo, or -CN.
  • Ri is -S-Z, wherein Z is aryl or heteroaryl (preferably, quinolinyl) wherein the aryl and heteroaryl is optionally substituted with one to four R5 groups, wherein each R5 is independently -NO 2 , -CN, halo, Ci-C 6 alkyl, Ci-C 6 alkoxy, -NH 2 , mono- or di-(Ci-C 6 alkyl) amino, or -C(O)OR 9 .
  • Z is aryl or heteroaryl (preferably, quinolinyl) wherein the aryl and heteroaryl is optionally substituted with one to four R5 groups, wherein each R5 is independently -NO 2 , -CN, halo, Ci-C 6 alkyl, Ci-C 6 alkoxy, -NH 2 , mono- or di-(Ci-C 6 alkyl) amino, or -C(O)OR 9 .
  • Ri is -S-Z, wherein Z is aryl optionally substituted with one or two R5 groups, wherein each R 5 is independently -NO 2 , -CN, halo, Ci-C 6 alkyl, Ci-C 6 alkoxy, -NH 2 , mono- or di-(Ci-C 6 alkyl) amino, or -C(O)OR 9 .
  • Ri is -S-Z, wherein Z is phenyl optionally substituted with one or two R 5 groups, wherein each R 5 is independently -NO 2 , -CN, halo, Ci-C 6 alkyl, Ci-C 6 alkoxy, -NH 2 , mono- or di-(Ci-C 6 alkyl) amino, or -C(O)OR 9 .
  • Ri is -S-Z, wherein Z is heteroaryl (preferably, quinolinyl) optionally substituted with one or two R 5 groups, wherein each R 5 is independently -NO 2 , -CN, halo, Ci-C 6 alkyl, Ci-C 6 alkoxy, -NH 2 , mono- or di-(Ci-C 6 alkyl) amino, or -C(O)OR 9 .
  • Z is heteroaryl (preferably, quinolinyl) optionally substituted with one or two R 5 groups, wherein each R 5 is independently -NO 2 , -CN, halo, Ci-C 6 alkyl, Ci-C 6 alkoxy, -NH 2 , mono- or di-(Ci-C 6 alkyl) amino, or -C(O)OR 9 .
  • Ri is -S-Z, wherein Z is pyridyl optionally substituted with one or two R 5 groups, wherein each R 5 is independently -NO 2 , -CN, halo, Ci-C 6 alkyl, Ci-C 6 alkoxy, -NH 2 , mono- or di-(Ci-C 6 alkyl) amino, or -C(O)OR 9 .
  • Ci-C 6 alkyl halo, Ci-C 6 alkyl, Ci-C 6 alkoxy, mono- to per-halogenated Ci-C 6 alkyl, -NH 2 , mono- or
  • R 3 is methyl. More preferred compounds of formula XII are those where R3 is methyl and n is O, 1 , or 2.
  • the compounds of formula XII are compounds of formula
  • n is 0, 1, or 2. More preferably, n is 0. In other more preferred embodiments, n is 2. [0118] In another preferred embodiment of compounds of formula XIII, A is -C(X)(Y)- wherein X is H or -OH; and Y is aryl, or heteroaryl wherein the aryl and heteroaryl is optionally substituted with one to four R 5 groups, wherein each R 5 is independently -NO 2 , -CN, halo, Ci-C 6 alkyl, Ci-C 6 alkoxy, -NH 2 , mono- or di-(Ci-C 6 alkyl) amino, or -C(O)OR 9 .
  • A is -CH(Y)- wherein [0120] Y is aryl, or heteroaryl wherein the aryl and heteroaryl is optionally substituted with one to four R 5 groups, wherein each R 5 is independently -NO 2 , -CN, halo, Ci-C 6 alkyl, Ci-C 6 alkoxy, -NH 2 , mono- or di-(Ci-C 6 alkyl) amino, or -C(O)OR 9 .
  • A is -CH(Y)- wherein Y is aryl, optionally substituted with one or two R 5 groups, wherein each R 5 is independently -NO 2 , -CN, halo, Ci-C 6 alkyl, Ci-C 6 alkoxy, -NH 2 , mono- or di-(Ci-C 6 alkyl) amino, or -C(O)OR 9 .
  • A is -N(Y)-, wherein Y is aryl, or heteroaryl wherein the aryl and heteroaryl is optionally substituted with one to four R 5 groups, wherein each R 5 is independently -NO 2 , -CN, halo, Ci-C 6 alkyl, Ci-C 6 alkoxy, -NH 2 , mono- or di-(Ci-C 6 alkyl) amino, or -C(O)OR 9 .
  • A is -N(Y)- wherein Y is aryl, optionally substituted with one or two R 5 groups, wherein each R 5 is independently -NO 2 , -CN, halo, Ci-C 6 alkyl, Ci-C 6 alkoxy, -NH 2 , mono- or (Ii-(C 1 -C 6 alkyl) amino, or -C(O)OR 9 .
  • Other preferred compounds of formula XIII are those wherein A is -N(Y)- wherein Y is heteroaryl, optionally substituted with one or two R 5 groups, wherein each R 5 is independently -NO 2 , -CN, halo, Ci-C 6 alkyl, Ci-C 6 alkoxy, -NH 2 , mono- or di-(Ci-C 6 alkyl) amino, or -C(O)OR 9 .
  • A is -N(Y)- wherein Y is phenyl, optionally substituted with one or two R 5 groups, wherein each R 5 is independently -NO 2 , -CN, halo, Ci-C 6 alkyl, Ci-C 6 alkoxy, -NH 2 , mono- or di-(Ci-C 6 alkyl) amino, or -C(O)OR 9 .
  • Z is aryl or heteroaryl (preferably, quinolinyl) wherein the aryl and heteroaryl is optionally substituted with one to four R 5 groups, wherein each R 5 is independently -NO 2 , -CN, halo, Ci-C 6 alkyl, Ci-C 6 alkoxy, -NH 2 , mono- or di-(Ci-C 6 alkyl) amino, or -C(O)OR 9 .
  • Z is heteroaryl (preferably, quinolinyl) optionally substituted with one or two R 5 groups, wherein each R 5 is independently -NO 2 , -CN, halo, Ci-C 6 alkyl, Ci-C 6 alkoxy, -NH 2 , mono- or di-(Ci-C 6 alkyl) amino, or -C(O)OR 9 .
  • Z is pyridyl optionally substituted with one or two R 5 groups, wherein each R 5 is independently -NO 2 , -CN, halo, Ci-C 6 alkyl, Ci-C 6 alkoxy, -NH 2 , mono- or di-(Ci-C 6 alkyl) amino, or -C(O)ORg.
  • Z is quinolinyl optionally substituted with one or two R5 groups, wherein each R5 is independently -NO 2 , -CN, halo, Ci-C 6 alkyl, Ci-C 6 alkoxy, -NH 2 , mono- or di-(Ci-C 6 alkyl) amino, or -C(O)ORg.
  • R 2 is -H, -NO 2 , -OH, -CN, halo, Ci-C 6 alkyl, Ci-C 6 alkoxy, mono- to per-halogenated Ci-C 6 alkyl, -NH 2 , mono- or di-(Ci-C 6 alkyl) amino, -C(O)-OR 9 , -N(R 8 )-Z, -0-Z, -S-Z, -SO 2 -Z, or -N(R 8 )-Z.
  • R 2 is -H, -NO 2 , -OH, -CN, halo, Ci-C 6 alkyl, Ci-C 6 alkoxy, mono- to per-halogenated Ci-C 6 alkyl, -NH 2 , mono- or di-(Ci-C 6 alkyl) amino, -C(O)-OR 9 .
  • R 3 is methyl. More preferred compounds of formula XIII are those where R3 is methyl and n is O, 1 , or 2.
  • the compounds of formula XIII are compounds of formula XIV, XV, XVI, XVII, or XVIII:
  • n is 0, 1, or 2. More preferably, n is 0. In other more preferred embodiments, n is 2.
  • m is 0, 1 , or 2. More preferably, m is 1.
  • Y is aryl or heteroaryl wherein the aryl and heteroaryl is optionally substituted with one to four R 5 groups, wherein each R 5 is independently -NO 2 , -CN, halo, Ci-C 6 alkyl, Ci-C 6 alkoxy, -NH 2 , mono- or di-(Ci-C 6 alkyl) amino, or -C(O)OR 9 .
  • Y is heteroaryl, optionally substituted with one or two R 5 groups, wherein each R 5 is independently -NO 2 , -CN, halo, Ci-C 6 alkyl, Ci-C 6 alkoxy, -NH 2 , mono- or di-(Ci-C 6 alkyl) amino, or -C(O)OR 9 .
  • Z is heteroaryl (preferably, quinolinyl) optionally substituted with one or two R 5 groups, wherein each R 5 is independently -NO 2 , -CN, halo, Ci-C 6 alkyl, Ci-C 6 alkoxy, -NH 2 , mono- or di-(Ci-C 6 alkyl) amino, or -C(O)ORg.
  • R 5 is independently -NO 2 , -CN, halo, Ci-C 6 alkyl, Ci-C 6 alkoxy, -NH 2 , mono- or di-(Ci-C 6 alkyl) amino, or -C(O)ORg.
  • XVI, XVII, or XVIII Z is pyridyl optionally substituted with one or two R5 groups, wherein each R 5 is independently -NO 2 , -CN, halo, Ci-C 6 alkyl, Ci-C 6 alkoxy, -NH 2 , mono- or di-(Ci-C 6 alkyl) amino, or -C(O)ORg. Even more preferred are compounds of formula XIV, XV, XVI,
  • Z is quinolinyl optionally substituted with one or two R 5 groups, wherein each R 5 is independently -NO 2 , -CN, halo, Ci-C 6 alkyl, Ci-C 6 alkoxy, -NH 2 , mono- or di-(Ci-C 6 alkyl) amino, or -C(O)OR 9 .
  • R 2 is -H, -NO 2 , -OH, -CN, halo, Ci-C 6 alkyl, Ci-C 6 alkoxy, mono- to per-halogenated Ci-C 6 alkyl, -NH 2 , mono- or (Ii-(C 1 -C 6 alkyl) amino, -C(O)-OR 9 , -N(Rg)-Z, -0-Z, -S-Z, -SO 2 -Z, or -N(Rs)-Z.
  • R3 is methyl. More preferred compounds of formula XIV, XV, or XVI are those where R 3 is methyl and n is O, 1, or 2.
  • the compounds of formula XV are compounds of formula XIX or XX:
  • each R 6 is independently -NO 2 , -CN, halo, Ci-C 6 alkyl, Ci-C 6 alkoxy, -NH 2 , mono- or di-(Ci-C 6 alkyl) amino, or -C(O)OR 9 .
  • n is 0, 1, or
  • n is 0. In other more preferred embodiments, n is 2.
  • p is 0 or 1. In other more preferred embodiments, p is 1.
  • each R 6 is independently -NO 2 , -CN, halo, Ci-C 6 alkyl, or Ci-C 6 alkoxy.
  • each R 6 is independently -NO 2 , -CN, halo, Ci-C 6 alkyl, or Ci-C 6 alkoxy, and p is 1 or 2.
  • R 6 is -NO 2 ,
  • R 5 is independently -NO 2 , -CN, halo, Ci-C 6 alkyl, Ci-C 6 alkoxy, -NH 2 , mono- or di-(Ci-C 6 alkyl) amino, or -C(O)OR 9 .
  • Z is heteroaryl (preferably, quinolinyl) optionally substituted with one or two R 5 groups, wherein each R 5 is independently -NO 2 , -CN, halo, Ci-C 6 alkyl, Ci-C 6 alkoxy, -NH 2 , mono- or di-(Ci-C 6 alkyl) amino, or -C(O)OR 9 .
  • R 5 is independently -NO 2 , -CN, halo, Ci-C 6 alkyl, Ci-C 6 alkoxy, -NH 2 , mono- or di-(Ci-C 6 alkyl) amino, or -C(O)OR 9 .
  • Z is pyridyl optionally substituted with one or two R 5 groups, wherein each R 5 is independently
  • Ci-C 6 alkyl Ci-C 6 alkoxy, -NH 2 , mono- or di-(Ci-C 6 alkyl) amino, or
  • R 3 is methyl
  • More preferred compounds of formula XIX or XX are those where R3 is methyl and n is O, 1, or
  • the compounds of formula XVI are compounds of formula XXI:
  • Y is aryl or heteroaryl wherein the aryl and heteroaryl is optionally substituted with one to four R 5 groups, wherein each R 5 is independently -NO 2 , -CN, halo, Ci-C 6 alkyl, Ci-C 6 alkoxy, -NH 2 , mono- or (Ii-(C 1 -C 6 alkyl) amino, or -C(O)OR 9 .
  • Y is aryl, optionally substituted with one or two R5 groups, wherein each R5 is independently -NO 2 , -CN, halo, Ci-C 6 alkyl, Ci-C 6 alkoxy, -NH 2 , mono- or CIi-(C 1 -C 6 alkyl) amino, or -C(O)OR 9 .
  • R 5 is independently -NO 2 , -CN, halo, Ci-C 6 alkyl, Ci-C 6 alkoxy, -NH 2 , mono- or (Ii-(C 1 -C 6 alkyl) amino, or -C(O)OR 9 .
  • Z is aryl or heteroaryl (preferably, quinolinyl) wherein the aryl and heteroaryl is optionally substituted with one to four R 5 groups, wherein each R 5 is independently -NO 2 , -CN, halo, Ci-C 6 alkyl, Ci-C 6 alkoxy, -NH 2 , mono- or di-(Ci-C 6 alkyl) amino, or -C(O)ORg.
  • Z is heteroaryl (preferably, quinolinyl)optionally substituted with one or two R 5 groups, wherein each R 5 is independently -NO 2 , -CN, halo, Ci-C 6 alkyl, Ci-C 6 alkoxy, -NH 2 , mono- or di-(Ci-C 6 alkyl) amino, or -C(O)ORg.
  • Z is pyridyl optionally substituted with one or two R 5 groups, wherein each R 5 is independently -NO 2 , -CN, halo, Ci-C 6 alkyl, Ci-C 6 alkoxy, -NH 2 , mono- or di-(Ci-C 6 alkyl) amino, or -C(O)OR 9 .
  • Z is quinolinyl optionally substituted with one or two R 5 groups, wherein each R 5 is independently -NO 2 , -CN, halo, Ci-C 6 alkyl, Ci-C 6 alkoxy, -NH 2 , mono- or di-(Ci-C 6 alkyl) amino, or -C(O)ORg.
  • R 2 is -H, -NO 2 , -OH, -CN, halo, Ci-C 6 alkyl, Ci-C 6 alkoxy, mono- to per-halogenated Ci-C 6 alkyl, -NH 2 , mono- or (Ii-(C 1 -C 6 alkyl) amino, -C(O)-OR 9 , -N(Rg)-Z, -0-Z, -S-Z, -SO 2 -Z, or -N(Rs)-Z.
  • each R 7 is independently -NO 2 , -CN, halo, Ci-C 6 alkyl, Ci-C 6 alkoxy, -NH 2 , mono- or di-(Ci-C 6 alkyl) amino, or -C(O)ORg.
  • q is 0, 1, or 2.
  • q is 0 or 1. In other more preferred embodiments,q is 1.
  • each R 7 is independently -NO 2 , -CN, halo, Ci-C 6 alkyl, or Ci-C 6 alkoxy.
  • each R 7 is independently -NO 2 , -CN, halo, Ci-C 6 alkyl, or Ci-C 6 alkoxy, and q is 1 or 2.
  • R 7 is -NO 2 , -CN, halo, Ci-C 6 alkyl, or Ci-C 6 alkoxy, and q is 1.
  • R 5 is independently -NO 2 , -CN, halo, Ci-C 6 alkyl, Ci-C 6 alkoxy, -NH 2 , mono- or di-(Ci-C 6 alkyl) amino, or -C(O)OR 9 .
  • R 5 is independently -NO 2 , -CN, halo, Ci-C 6 alkyl, Ci-C 6 alkoxy, -NH 2 , mono- or di-(Ci-C 6 alkyl) amino, or -C(O)OR 9 .
  • each R 5 is independently -NO 2 , -CN, halo, Ci-C 6 alkyl, Ci-C 6 alkoxy, -NH 2 , mono- or (Ii-(C 1 -C 6 alkyl) amino, or --C(O)OR 9 .
  • Z is quinolinyl optionally substituted with one or two R 5 groups, wherein each R 5 is independently -NO 2 , -CN, halo, Ci-C 6 alkyl, Ci-C 6 alkoxy, -NH 2 , mono- or di-(Ci-C 6 alkyl) amino, or -C(O)OR 9 .
  • R 5 is independently -NO 2 , -CN, halo, Ci-C 6 alkyl, Ci-C 6 alkoxy, -NH 2 , mono- or di-(Ci-C 6 alkyl) amino, or -C(O)OR 9 .
  • Z is pyridyl optionally substituted with one or two R5 groups, wherein each R 5 is independently -NO 2 , -CN, halo, Ci-C 6 alkyl, Ci-C 6 alkoxy,
  • R 2 is -H, -NO 2 ,
  • Ci-C 6 alkyl Ci-C 6 alkoxy, mono- to per-halogenated Ci-C 6 alkyl, -NH 2 , mono- or (Ii-(C 1 -C 6 alkyl) amino, -C(O)-OR 9 , -N(R 8 )-Z, -0-Z, -S-Z, -SO 2 -Z, or -N(R 8 )-Z. More preferred are compounds of formula XXII, wherein R 2 is -H, -NO 2 , -OH, -CN, halo, Ci-C 6 alkyl,
  • Ci-C 6 alkoxy mono- to per-halogenated Ci-C 6 alkyl, -NH 2 , mono- or di-(Ci-C 6 alkyl) amino,
  • the invention provides one of the compounds listed in the following table,
  • the invention provides a compound of formula (XL),
  • R is Ci-C 6 alkyl, Ci-C 6 haloalkyl, aryl, or heteroaryl, wherein R is optionally substituted with one to four R 3 groups;
  • Li is L 3 , -Ci-C 6 alkyl-, -Ci-C 6 alkyl-L 3 -, -L 3 -Ci-C 6 alkyl-, or -L 3 -Ci-C 6 alkyl-L 3 , wherein each L 3 is independently -C(O)N(R 4 )-, -N(R 4 )C(O)-, -OC(O)-, -C(O)O-, -N(R 4 )C(O)N(R 4 )-, -OC(O)N(R 4 )-, -N(R 4 )C(O)O-, -OC(O)O-, -C(O)-, -S(O)-, -S(O) 2 -, -S(O) 2 N(R 4 )-, or -N(R 4 )S(O) 2 -; Ri is aryl or heteroaryl, wherein Ri is optionally
  • R 2 is aryl or heteroaryl, wherein R 2 is optionally substituted with one to four R 3 groups;
  • R 3 is halo, -OR 6 , -N(Re) 2 , -S(R 6 ), -S(O) 2 R 6 , -S(O) 2 N(Re) 2 , -S(O) 2 OR 6 ,
  • the compound of formula (XL) ⁇ is of formula (XLI),
  • the invention provides the compound of formula (XL) or (XLI), wherein R is Ci-C 6 alkyl. In other preferred embodiments of the seventh aspect, the invention provides the compound of formula (XL) or (XLI), wherein R is phenyl optionally substituted with one to four R 3 groups.
  • the invention provides the compound of formula (XL) or (XLI), wherein Li is -Ci-C 6 alkyl-L 3 - or -L 3 -Ci-C 6 alkyl-, wherein each L 3 is independently -C(O)N(R 4 )-, -N(R 4 )C(O)-, -OC(O)-, -C(O)O-, -N(R 4 )C(O)N(R 4 )-, -OC(O)N(R 4 )-, -N(R 4 )C(O)O-, -OC(O)O-, -C(O)-, -S(O)-, -S(O) 2 -, -S(O) 2 N(R 4 )-, or -N(R 4 )S(O) 2 -.
  • the invention provides the compound of formula (XL) or (XLI), wherein Li is -Ci-C 6 alkyl-L 3 - or -L 3 -Ci-C 6 alkyl-, wherein each L 3 is independently -C(O)N(R 4 )-, -N(R 4 )C(O)-, -S(O) 2 N(R 4 )-, or -N(R 4 )S(O) 2 -.
  • the invention provides the compound of formula (XL) or (XLI), wherein Li is -Ci-C 6 alkyl-L 3 - or -L 3 -Ci-C 6 alkyl-, wherein each L 3 is independently -C(O)N(R 4 )- or -N(R 4 )C(O)-.
  • the invention provides the compound of formula (XL) or (XLI), wherein L 2 is -C(O)N(R 4 )-, -N(R 4 )C(O)-, -OC(O)-, -C(O)O-, -N(R 4 )C(O)N(R 4 )-, -OC(O)N(R 4 )-, -N(R 4 )C(O)O-, -OC(O)O-.
  • the invention provides the compound of formula (XL) or (XLI), wherein L 2 is -N(R 4 )C(O)N(R 4 )-, -OC(O)N(R 4 )-, -N(R 4 )C(O)O-, -OC(O)O-.
  • the invention provides the compound of formula (XL) or (XLI), wherein L 2 is -N(R 4 )C(O)N(R 4 )-.
  • the invention provides the compound of formula (XL) or (XLI), wherein each R 3 is independently halo, -OR 6 , -N(Re) 2 , -S(O) 2 R 6 , -S(O) 2 N(R ⁇ ) 2 , -C(O)R 6 , -C(O)OR 6 , -C(O)N(Re) 2 , -CN, -C 1 -C 6 alkyl, -C 1 -C 6 haloalkyl, wherein each R 6 is independently-H, Ci-C 6 alkyl or Ci-C 6 haloalkyl.
  • the invention provides a compound of formula (LX),
  • Ri and R 2 are independently -H, -Ci-C 6 alkyl, -aryl, -heteroaryl, -Ci-C 6 alkyl-aryl, or -Ci-C 6 alkyl-heteroaryl, wherein each is optionally substituted with one to four R 5 groups;
  • R 3 , R 4 , and R 5 are each independently halo, -OR 6 , -N(Re) 2 , -S(R 6 ), -S(O) 2 R 6 , -S(O) 2 N(R6) 2 , -S(O) 2 OR 6 , -N(R 6 )S(O) 2 R 6 , -OS(O) 2 R 6 , -C(O)R 6 , -C(O)OR 6 , -C(O)N(Re) 2 , -OC(O)R 6 , -OC(O)OR 6 , -OC(O)N(Rg) 2 , -N(R 6 )C(O)R 6 , -N(R 6 )C(O)OR 6 , -N(R 6 )C(O)N(Rg) 2 , -CN, -NO 2 , -Ci-C 6 alkyl, -C 1 -C 6
  • the invention provides the compound of formula (LX), wherein m and n are independently 0, 1, or 2. More preferably, m and n are each 1.
  • the invention provides the compound of formula (LX), wherein Ri and R 2 are independently -Ci-C 6 alkyl-aryl or -Ci-C 6 alkyl-heteroaryl, wherein each is optionally substituted with one to four R5 groups.
  • Ri and R 2 are independently -Ci-C 6 alkyl-aryl or -Ci-C 6 alkyl-heteroaryl, wherein each is optionally substituted with one to four groups which are independently halo, -ORs, -N(Rs) 2 , -S(Rs), -S(O) 2 Rs, -S(O) 2 N(Rs) 2 , -C(O)R 8 , -C(O)OR 8 , -C(O)N(Rs) 2 , -CN, -NO 2 , -Ci-C 6 alkyl, or - Ci-C 6 haloalkyl, wherein each R 8 is independently -H, Ci-C 6 alkyl.
  • the compounds according to the sixth, seventh, and eighth aspects of the invention are also useful as general ubiquitin ligase inhibitors.
  • the compounds of the invention can be used as inhibitors of enzymes that exhibit ligase activity, including but not limited to p27/SCF, TRAF6, Radl8, BMI-I, APC and E3 enzymes.
  • the compounds of the invention are also useful for regulating or inhibiting pathways in diseases and conditions that involve ubiquitin conjugation and deconjugation such as cancers, inflammation, metabolism, viral diseases and central nervous system disorders.
  • the compounds of the invention can be used to regulate or inhibit the products of genes that encode ubiquitin or ubiquitin-like enzymes described in Wong et al. (DDT 8 (16), 746-754 (2003)), which is incorporated by reference in its entirety.
  • the invention provides for pharmaceutical compositions comprising, together with a pharmaceutically acceptable carrier, diluent, or excipient, a compound of according to the sixth, seventh, or eighth aspect of the invention described above or a compound selected from (2-((4-hydroxybenzyl)(4-methoxybenzyl)amino)-4-phenylthiazol-5- yl)(phenyl)methanone;
  • the invention provides methods of inhibiting ubiquitination in a cell comprising contacting the cell in which inhibition of ubiquitination is desired with a compound according to the sixth, seventh, or eighth aspect of the invention or a pharmaceutical composition according to the ninth aspect of the invention.
  • the compounds and formulations of the invention can inhibit ubiquitination in cells derived from animals, particularly, mammalian cells.
  • the compounds and formulations of the invention can also be used to inhibit the ubiquitin ligase activity of p27/SCF, TRAF6, Radl8, or BMI-I.
  • the invention provides for methods of treating cell proliferative diseases or conditions comprising administering to a patient an effective amount of a compound of the sixth, seventh, or eighth aspect of the invention or a pharmaceutical composition according to the ninth aspect of the invention.
  • Cell proliferative diseases or conditions include, but are not limited to, psoriasis, keloid scarring, and cancers, such as cancers of the breast, immune system, bone, nervous system, brain, blood, lymphatic system, and skin.
  • the compounds and pharmaceutical compositions of the invention are useful for treating cell proliferative diseases or conditions that involve p27/SCF, TRAF6, Radl8, or BMI-I.
  • the invention provides for methods of inhibiting TRAF6 comprising administering to a patient an effective amount of a compound according to the sixth, seventh, or eighth aspect of the invention or pharmaceutical composition according to the ninth aspect of the invention.
  • the compounds and pharmaceutical compositions are useful for treating conditions or diseases that involve TRAF6 such as those related to cancer, inflammation, adaptive immunity, innate immunity, bone metabolism, LPS-induced angiogenesis, osteoporosis, osteopinneal diseases, lymph node development, mammary gland development, skin development, and central nervous system development.
  • the invention provides for methods of inhibiting p27/SCF comprising administering to a patient an effective amount of a compound according to the sixth, seventh, or eighth aspect of the invention or pharmaceutical composition according to the ninth aspect of the invention.
  • the compounds and pharmaceutical compositions are useful for treating conditions or diseases that involve p27/SCF such as those related to cancer, inflammation, adaptive immunity, innate immunity, bone metabolism, LPS-induced angiogenesis, osteoporosis, osteopinneal diseases, lymph node development, mammary gland development, skin development, and central nervous system development.
  • a variety of cellular proliferative disorders may be treated using the drug and prodrug compounds via the disclosed methods.
  • the drug or prodrug compounds are used to treat various cancers in afflicted subjects. Cancers are traditionally classified based on the tissue and cell type from which the cancer cells originate. Carcinomas are considered cancers arising from epithelial cells while sarcomas are considered cancers arising from connective tissues or muscle. Other cancer types include leukemias, which arise from hematopoietic cells, and cancers of nervous system cells, which arise from neural tissue. For non-invasive tumors, adenomas are considered benign epithelial tumors with glandular organization while chondomas are benign tumor arising from cartilage.
  • Solid tumor cancers include malignant neoplastic masses of tissue or cancerous neoplasms characterized by the progressive or uncontrolled proliferation of cells.
  • the cells involved in the neoplastic growth have an intrinsic heritable abnormality such that they are not regulated properly by normal methods.
  • Malignant or cancerous neoplasms tend to grow rapidly, spread throughout the body, and recur if removed.
  • the cells of malignant tumors may be well differentiated, but most have some degree of anaplasia.
  • Anaplastic cells tend to be larger than normal and are abnormal, even playful, in shape.
  • the nuclei tend to be very large, and irregular, and they often stain darkly.
  • Malignant tumors may be partially encapsulated, but the cells of the cancer can infiltrate and destroy surrounding tissue.
  • cells from the primary tumor can migrate (metastasize) from the original tumor site and colonize in other tissues.
  • Tumors formed from cells that have spread are referred to as "secondary tumors" and contain cells that are similar to those in the original "primary” tumor.
  • Metastatic tumors typically form by migration of tumor cells from the original tumor site through the blood and lymph system to other tissues.
  • the drugs or prodrugs are used to treat solid tumors arising from various tissue types, including, but not limited to, cancers of the bone, breast, respiratory tract (e.g., bladder), brain reproductive organs, digestive tract, urinary tract, eye, liver, skin, head, neck, thyroid, parathyroid, and metastatic forms thereof.
  • various tissue types including, but not limited to, cancers of the bone, breast, respiratory tract (e.g., bladder), brain reproductive organs, digestive tract, urinary tract, eye, liver, skin, head, neck, thyroid, parathyroid, and metastatic forms thereof.
  • proliferative disorders include the following: a) proliferative disorders of the breast include, but are not limited to, invasive ductal carcinoma, invasive lobular carcinoma, ductal carcinoma, lobular carcinoma in situ, and metastatic breast cancer; b) proliferative disorders of the skin include, but are not limited to, basal cell carcinoma, squamous cell carcinoma, malignant melanoma, and Karposi's sarcoma; c) proliferative disorders of the respiratory tract include, but are not limited to, small cell and non-small cell lung carcinoma, bronchial adema, pleuropulmonary blastoma, and malignant mesothelioma; d) proliferative disorders of the brain include, but are not limited to, brain stem and hyptothalamic glioma, cerebellar and cerebral astrocytoma, medullablastoma, ependymal tumors, oligodendroglial, meningiomas, and
  • proliferative disorders is not limited to the conditions described above, but encompasses other disorders characterized by uncontrolled growth and malignancy. It is further understood that proliferative disorders include various metastatic forms of the tumor and cancer types described herein. The drug and prodrug compounds of the described methods may be tested for effectiveness against these disorders, and a therapeutically effective regimen established. Effectiveness, as further described below, includes reduction or remission of the tumor, decreases in the rate of cell proliferation, or cytostatic or cytotoxic effect on cell growth.
  • the invention provides for methods of inhibiting Radl8 comprising administering to a patient an effective amount of a compound according to the sixth, seventh, or eighth aspect of the invention or pharmaceutical composition according to the ninth aspect of the invention.
  • the compounds and pharmaceutical compositions are useful for treating conditions or diseases that involve Radl8 such as those related to cancer, inflammation, adaptive immunity, innate immunity, bone metabolism, LPS-induced angiogenesis, osteoporosis, osteopinneal diseases, lymph node development, mammary gland development, skin development, and central nervous system development.
  • the invention provides for methods of inhibiting BMI-I comprising administering to a patient an effective amount of a compound according to the sixth, seventh, or eighth aspect of the invention or pharmaceutical composition according to the ninth aspect of the invention.
  • the compounds and pharmaceutical compositions are useful for treating conditions or diseases that involve BMI-I such as those related to cancer, inflammation, adaptive immunity, innate immunity, bone metabolism, LPS-induced angiogenesis, osteoporosis, osteopinneal diseases, lymph node development, mammary gland development, skin development, and central nervous system development.
  • the compounds for use in the method according to the twelfth aspect of the invention are also useful as general ubiquitin ligase inhibitors.
  • the compounds of the invention can be used as inhibitors of E3 enzymes that contain HECT and RING finger domains, Mdm2 with RING fingers and variants, and U-box-containing proteins.
  • the compounds of the invention are useful as protein modulators, immunologic agents anti-inflammatory agents, anti-osteoporosis agents, anti-viral agents, for example, inhibitors of variola viruses such as smallpox, HIV and related conditions, human papillomavirus, HSV, adenovirus, coxsackie virus, HCMV, KSHV, EBV, paramyxovirus, myxomavirus, ebola, retrovirus, and rhabdovirus, anti-protozoan agents, for example, inhibitors of the malaria parasite.
  • the compounds of the invention are also useful as oncologic and anti-pro liferative agents that inhibit aberrant cell growth, cancers, restenosis, psoriasis, and neoplastic cell proliferation.
  • the invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a pharmaceutically acceptable carrier, diluent, or excipient and a compound of formula (XXX),
  • n 0, 1, 2, or 3;
  • R 1 is -CN or -C(O)NH(R 5 ), wherein R 5 is -H, Ci-C 6 alkyl, or Ci-C 6 haloalkyl; R 2 is -H or -L-R 7 , wherein
  • L is -C(O)- or -S(O) 2 -;
  • R 7 is Ci-C 6 alkyl, Ci-C 6 haloalkyl, C 3 -C 8 cycloalkyl, heterocyclyl, aryl, or heteroaryl, wherein R 7 is optionally substituted with one to four R 4 groups;
  • R is -Ci-C 6 alkyl, -Ci-C 6 haloalkyl, aryl, -Ci-C 6 alkyl-aryl, wherein R is optionally substituted with one to four R 4 groups; and
  • R 4 is halo, -OR 6 , -N(R 6 ) 2 , -S(R 6 ), -S(O) 2 R 6 , -S(O) 2 N(R 6 ) 2 , -S(O) 2 OR 6 ,
  • the compound of formula (XXX) is of formula (XXXI),
  • the compound of formula (XXXI) is of formula (XXXII),
  • the compound of formula (XXXII) is of formula (XXXIII),
  • the invention provides the pharmaceutical composition wherein the compound is one of formulas (XXX) - (XXXIII), and R 7 is aryl or heteroaryl, wherein R 7 is optionally substituted with one to four R 4 groups. More preferred embodiments include those where the compound is one of formulas (XXX) - (XXXIII), and R 7 is phenyl optionally substituted with one to four R 4 groups. Even more preferred embodiments include those where the compound is one of formulas (XXX) - (XXXIII), and R 7 is phenyl optionally substituted with one or two R 4 groups.
  • R 7 is furanyl, thienyl, pyrrolyl, thiazolyl, oxazolyl, isoxazoyl, isothiazoyl, imidazoyl, pyrazoyl, or triazolyl, wherein each R 7 is optionally substituted with one to four R 4 groups.
  • R 7 is furanyl, thienyl, or pyrrolyl, wherein each R 7 is optionally substituted with one or two R 4 groups.
  • the invention provides the pharmaceutical composition wherein the compound is one of formulas (XXX) - (XXXIII), and [0202] R 3 is -Ci-C 6 alkyl, phenyl, or benzyl, wherein R 3 is optionally substituted with one t ⁇ four R 4 groups.
  • More preferred embodiments include those where the compound is one of formulas (XXX) - (XXXIII), and R 3 is -Ci-C 6 alkyl, phenyl, or benzyl, wherein R 3 is optionally substituted with one or two R 4 groups. Even more preferred embodiments include those where the compound is one of formulas (XXX) - (XXXIII), and R 3 is -Ci-C 6 alkyl optionally substituted with one R 4 group. Even more preferred embodiments include those where the compound is one of formulas (XXX) - (XXXIII), and R 3 is benzyl optionally substituted with one R 4 group.
  • the invention provides the pharmaceutical composition wherein the compound is one listed in the following table,
  • the invention provides methods of inhibiting ubiquitination in a cell comprising contacting the cell in which inhibition of ubiquitination is desired with a pharmaceutical composition according to the sixteenth aspect of the invention.
  • the compounds and formulations of the invention can inhibit ubiquitination in cells derived from animals, particularly, mammalian cells.
  • the compounds and formulations of the invention can also be used to inhibit the ubiquitin ligase activity of p27/SCF, TRAF6, Radl8, or BMI-I.
  • the invention provides for methods of treating cell proliferative diseases or conditions comprising administering to a patient an effective amount of a pharmaceutical composition according to the sixteenth aspect of the invention.
  • Cell proliferative diseases or conditions include, but are not limited to, psoriasis, keloid scarring, and cancers, such as cancers of the breast, immune system, bone, nervous system, brain, blood, lymphatic system, and skin.
  • cancers such as cancers of the breast, immune system, bone, nervous system, brain, blood, lymphatic system, and skin.
  • the compounds and pharmaceutical compositions of the invention are useful for treating cell proliferative diseases or conditions that involve p27/SCF, TRAF6, Radl8, or BMI-I.
  • the invention provides for methods of inhibiting TRAF6 comprising administering to a patient an effective amount of a pharmaceutical composition according to the sixteenth aspect of the invention.
  • the compounds and pharmaceutical compositions are useful for treating conditions or diseases that involve TRAF6 such as those related to cancer, inflammation, adaptive immunity, innate immunity, bone metabolism, LPS-induced angiogenesis, osteoporosis, osteopinneal diseases, lymph node development, mammary gland development, skin development, and central nervous system development.
  • the invention provides for methods of inhibiting p27/SCF comprising administering to a patient an effective amount of a pharmaceutical composition according to the sixteenth aspect of the invention.
  • the compounds and pharmaceutical compositions are useful for treating conditions or diseases that involve p27/SCF such as those related to cancer, inflammation, adaptive immunity, innate immunity, bone metabolism, LPS-induced angiogenesis, osteoporosis, osteopinneal diseases, lymph node development, mammary gland development, skin development, and central nervous system development.
  • the invention provides for methods of inhibiting Radl8 comprising administering to a patient an effective amount of a pharmaceutical composition according to the sixteenth aspect of the invention.
  • the compounds and pharmaceutical compositions are useful for treating conditions or diseases that involve Radl8 such as those related to cancer, inflammation, adaptive immunity, innate immunity, bone metabolism, LPS-induced angiogenesis, osteoporosis, osteopinneal diseases, lymph node development, mammary gland development, skin development, and central nervous system development.
  • the invention provides for methods of inhibiting BMI-I comprising administering to a patient an effective amount of a pharmaceutical composition according to the sixteenth aspect of the invention.
  • the compounds and pharmaceutical compositions are useful for treating conditions or diseases that involve BMI-I such as those related to cancer, inflammation, adaptive immunity, innate immunity, bone metabolism, LPS-induced angiogenesis, osteoporosis, osteopinneal diseases, lymph node development, mammary gland development, skin development, and central nervous system development.
  • compositions for use in the method according to the nineteenth aspect of the invention are also useful as general ubiquitin ligase inhibitors.
  • the compounds of the invention can be used as inhibitors of E3 enzymes that contain HECT and RING finger domains, Mdm2 with RING fingers and variants, and U-box-containing proteins.
  • the compounds of the invention are useful as protein modulators, immunologic agents anti-inflammatory agents, anti-osteoporosis agents, anti-viral agents, for example, inhibitors of variola viruses such as smallpox, HIV and related conditions, human papillomavirus, HSV, adenovirus, coxsackie virus, HCMV, KSHV, EBV, paramyxovirus, myxomavirus, ebola, retrovirus, and rhabdovirus, anti-protozoan agents, for example, inhibitors of the malaria parasite.
  • the compounds of the invention are also useful as oncologic and anti-proliferative agents that inhibit aberrant cell growth, cancers, restenosis, psoriasis, and neoplastic cell proliferation.
  • alkyl generally refers to a monovalent group (e.g. CH3-CH2-)
  • a bivalent linking moiety can be "alkyl,” in which case those skilled in the art will understand the alkyl to be a divalent group (e.g.
  • aryl refers to the corresponding divalent moiety, arylene.
  • All atoms are understood to have their normal number of valences for bond formation (i.e., 4 for carbon, 3 for N, 2 for O, and 2, 4, or 6 for S, depending on the oxidation state of the S).
  • a moiety may be defined, for example, as (A) a -B-, wherein a is 0 or 1. In such instances, when a is 0 the moiety is B- and when a is 1 the moiety is A-B-.
  • alkyl refers to straight and branched chain aliphatic groups having from 1 to 12 carbon atoms, preferably 1-8 carbon atoms, and more preferably 1-6 carbon atoms, which is optionally substituted with one, two or three substituents.
  • Preferred alkyl groups include, without limitation, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, and hexyl.
  • a "Co” alkyl (as in "Co-C 3 _alkyl”) is a covalent bond.
  • alkenyl as used herein means an unsaturated straight or branched chain aliphatic group with one or more carbon-carbon double bonds, having from 2 to 12 carbon atoms, preferably 2-8 carbon atoms, and more preferably 2-6 carbon atoms, which is optionally substituted with one, two or three substituents.
  • Preferred alkenyl groups include, without limitation, ethenyl, propenyl, butenyl, pentenyl, and hexenyl.
  • alkynyl as used herein means an unsaturated straight or branched chain aliphatic group with one or more carbon-carbon triple bonds, having from 2 to 12 carbon atoms, preferably 2-8 carbon atoms, and more preferably 2-6 carbon atoms, which is optionally substituted with one, two or three substituents.
  • Preferred alkynyl groups include, without limitation, ethynyl, propynyl, butynyl, pentynyl, and hexynyl.
  • alkylene is an alkyl, alkenyl, or alkynyl group, as defined hereinabove, that is positioned between and serves to connect two other chemical groups.
  • Preferred alkylene groups include, without limitation, methylene, ethylene, propylene, and butylene.
  • Preferred alkenylene groups include, without limitation, ethenylene, propenylene, and butenylene.
  • Preferred alkynylene groups include, without limitation, ethynylene, propynylene, and butynylene.
  • cycloalkyl as employed herein includes saturated and partially unsaturated cyclic hydrocarbon groups having 3 to 12 carbons, preferably 3 to 8 carbons, and more preferably 3 to 6 carbons, wherein the cycloalkyl group additionally is optionally substituted.
  • Preferred cycloalkyl groups include, without limitation, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, and cyclooctyl.
  • heteroalkyl refers to an alkyl group, as defined hereinabove, wherein one or more carbon atoms in the chain are replaced by a heteroatom selected from the group consisting of O, S, and N.
  • An "aryl” group is a Ce-Cu aromatic moiety comprising one to three aromatic rings, which is optionally substituted.
  • the aryl group is a C 6 -Ci O aryl group.
  • Preferred aryl groups include, without limitation, phenyl, naphthyl, anthracenyl, and fluorenyl.
  • An "aralkyl” or “arylalkyl” group comprises an aryl group covalently linked to an alkyl group, either of which may independently be optionally substituted or unsubstituted.
  • the aralkyl group is (Ci-C 6 )alkyl(C 6 -Cio)aryl, including, without limitation, benzyl, phenethyl, and naphthylmethyl.
  • bis-aryl comprises an aryl group covalently linked to an aryl or heteroaryl group, any one of which may independently be optionally substituted or unsubstituted.
  • the bis-aryl group is (C6-Cio)aryl(C6-Cio)aryl or (C6-C 10 )aryl(C6-C 10 )heteroaryl, including, without limitation, 2,1,3-benzoxadiazolyl substituted in the benzo portion with phenyl or quinolinyl.
  • a “heterocyclic” group is an optionally substituted non-aromatic mono-, bi-, or tricyclic structure having from about 3 to about 14 atoms, wherein one or more atoms are selected from the group consisting of N, O, and S.
  • One ring of a bicyclic heterocycle or two rings of a tricyclic heterocycle may be aromatic, as in indan and 9,10-dihydro anthracene.
  • the heterocyclic group is optionally substituted on carbon with oxo or with one of the substituents listed above.
  • heterocyclic group is also optionally independently be substituted on nitrogen with alkyl, aryl, aralkyl, alkylcarbonyl, alkylsulfonyl, arylcarbonyl, arylsulfonyl, alkoxycarbonyl, aralkoxycarbonyl, or on sulfur with oxo or lower alkyl.
  • Preferred heterocyclic groups include, without limitation, epoxy, aziridinyl, tetrahydrofuranyl, pyrrolidinyl, piperidinyl, piperazinyl, thiazolidinyl, oxazolidinyl, oxazolidinonyl, and morpholino.
  • the heterocyclic group is fused to an aryl, heteroaryl, or cycloalkyl group.
  • fused heterocycles include, without limitation, tetrahydroquinoline and dihydrobenzofuran.
  • tetrahydroquinoline and dihydrobenzofuran.
  • an annular O or S atom is adjacent to another O or S atom.
  • heteroaryl refers to optionally substituted groups having 5 to 14 ring atoms, preferably 5, 6, 9, or 10 ring atoms; having 6, 10, or 14 pi electrons shared in a cyclic array; and having, in addition to carbon atoms, between one or more heteroatoms selected from the group consisting of N, O, and S.
  • a heteroaryl group may be pyrimidinyl, pyridinyl, benzimidazolyl, thienyl, benzothiazolyl, benzofuranyl and indolinyl.
  • Preferred heteroaryl groups include, without limitation, thienyl, benzothienyl, furyl, benzofuryl, dibenzofuryl, pyrrolyl, imidazolyl, pyrazolyl, pyridyl, pyrazinyl, pyrimidinyl, indolyl, quinolyl, isoquinolyl, quinoxalinyl, tetrazolyl, oxazolyl, thiazolyl, and isoxazolyl.
  • a “heteroaralkyl” or “heteroarylalkyl” group comprises a heteroaryl group covalently linked to an alkyl group, either of which is independently optionally substituted or unsubstituted.
  • Preferred heteroalkyl groups comprise a Ci-C 6 alkyl group and a heteroaryl group having 5, 6, 9, or 10 ring atoms. Specifically excluded from the scope of this term are compounds having adjacent annular O and/or S atoms.
  • heteroaralkyl groups examples include pyridylmethyl, pyridylethyl, pyrrolylmethyl, pyrrolylethyl, imidazolylmethyl, imidazolylethyl, thiazolylmethyl, and thiazolylethyl.
  • arylene is an aryl, heteroaryl, or heterocyclyl group, as defined hereinabove, that is positioned between and serves to connect two other chemical groups.
  • Preferred heterocyclyls and heteroaryls include, but are not limited to, acridinyl, azocinyl, benzimidazolyl, benzofuranyl, benzothiofuranyl, benzothiophenyl, benzoxazolyl, benzthiazolyl, benztriazolyl, benztetrazolyl, benzisoxazolyl, benzisothiazolyl, benzimidazolinyl, carbazolyl, 4aH-carbazolyl, carbolinyl, chromanyl, chromenyl, cinnolinyl, decahydroquinolinyl, 2H,6H-l,5,2-dithiazinyl, dihydrofuro[2,3-b]tetrahydrofuran, furanyl, furazanyl, imidazolidinyl, imidazolinyl, imidazolyl, lH-indazolyl, in
  • Suitable substituents include, without limitation, halo, hydroxy, oxo (e.g., an annular -CH- substituted with oxo is -C(O)-) nitro, alkyl, alkenyl, alkynyl, cycloalkyl, haloalkyl, haloalkenyl, haloalkynyl, halocycloalkyl, aryl, aralkyl, alkoxy, aryloxy, amino, acylamino, alkylcarbamoyl, arylcarbamoyl, aminoalkyl, acyl, carboxy, hydroxyalkyl, alkanesulfonyl, arenesulfonyl, alkanesulfonamido, arenesulfonamido, aralkylsulfonamido, alkylcarbonyl, acyloxy, cyano, and ureido groups.
  • R 3 ° and R 31 are each independently hydrogen, cyano, oxo, carboxamido, amidino, Ci-Cg hydroxyalkyl, C1-C3 alkylaryl, aryl-Ci-C 3 alkyl, Ci-C 8 alkyl, Ci-C 8 alkenyl, Ci-C 8 alkoxy, Ci-C 8 alkoxycarbonyl, aryloxycarbonyl, aryl-Ci-C 3 alkoxycarbonyl, C 2 -C 8 acyl, Ci-C 8 alkylsulfonyl, arylalkylsulfonyl, arylsulfonyl, aroyl, aryl, cycloalkyl, heterocyclyl, or heteroaryl, wherein
  • R 3 ° and R 31 taken together with the N to which they are attached form a heterocyclyl or heteroaryl, each of which is optionally substituted with from 1 to 3 substituents from (a), above.
  • substituents on cyclic moieties include 5-6 membered mono- and 9-14 membered bi-cyclic moieties fused to the parent cyclic moiety to form a bi- or tri-cyclic fused ring system.
  • an optionally substituted phenyl includes, but not limited to, the following:
  • haloalkyl is an alkyl, alkenyl, alkynyl, or cycloalkyl moiety in which from one to all hydrogens have been replaced with one or more halo.
  • halogen refers to chlorine, bromine, fluorine, or iodine.
  • acyl refers to an alkylcarbonyl or arylcarbonyl substituent.
  • acylamino refers to an amide group attached at the nitrogen atom (i.e., R-CO-NH-).
  • carbamoyl refers to an amide group attached at the carbonyl carbon atom (i.e., NH 2 -CO-). The nitrogen atom of an acylamino or carbamoyl substituent is additionally substituted.
  • sulfonamido refers to a sulfonamide substituent attached by either the sulfur or the nitrogen atom.
  • amino is meant to include NH 2 , alkylamino, arylamino, and cyclic amino groups.
  • ureido refers to a substituted or unsubstituted urea moiety.
  • a moiety that is substituted is one in which one or more hydrogens have been independently replaced with another chemical substituent.
  • substituted phenyls include 2-flurophenyl, 3,4-dichlorophenyl, 3-chloro-4-fluoro-phenyl, 2-fluoro-3-propylphenyl.
  • substituted iV-octyls include 2,4 dimethyl-5-ethyl-octyl and 3-cyclopentyl-octyl. Included within this definition are methylenes (-CH 2 -) substituted with oxygen to form carbonyl -CO-).
  • an "unsubstituted" moiety as defined above e.g., unsubstituted cycloalkyl, unsubstituted heteroaryl, etc. means that moiety as defined above that does not have any of the optional substituents for which the definition of the moiety (above) otherwise provides.
  • an "aryl” includes phenyl and phenyl substituted with a halo
  • "unsubstituted aryl” does not include phenyl substituted with a halo.
  • Some compounds of the invention may have chiral centers and/or geometric isomeric centers (E- and Z- isomers), and it is to be understood that the invention encompasses all such optical, diastereoisomers and geometric isomers.
  • the invention also comprises all tautomeric forms of the compounds disclosed herein.
  • Atropisomers are stereoisomers resulting from hindered rotation about single bonds where the barrier to rotation is high enough to allow for the isolation of the conformers (Eliel, E. L.; Wilen, S. H. Stereochemistry of Organic Compounds; Wiley & Sons: New York, 1994; Chapter 14, including pages 1150-1153 and the short definition on page 1193). Atropisomerism is significant because it introduces an element of chirality in the absence of stereogenic atoms.
  • the invention is meant to encompass atropisomers, for example in cases of limited rotation around the single bonds emanating from the core 2,1,3-benzoxadiazole or 2,1,3-benzothiadiazole structure. Atropisomers are also possible and are also specifically included in the compounds and/or prodrugs of the invention.
  • Polymorphism in chemical substances is the ability of a single compound to exist in two or more solid phases, each having different arrangements and/or conformations of the individual molecules in the solid form (D. J. W. Grant, Theory and Origin of Polymorphism. In H. G. Brittain (ed.) Polymorphism in Pharmaceutical Solids. Marcel Dekker, Inc., New York, 1999, pp. 1-34).
  • polymorphic solid forms can be crystalline or amorphous.
  • Polymorphs of molecules or their solvates for example, hydrates
  • Distinct polymorphic forms generally have different chemical and physical properties such as melting point, chemical reactivity, apparent solubility, dissolution rate, optical and electrical properties, vapor pressure, and density.
  • the invention is meant to encompass in its scope, different polymorphic forms of the compounds of the invention.
  • the compounds of the invention may be administered in the form of an in vivo hydrolyzable ester or in vivo hydrolyzable amide.
  • An in vivo hydrolyzable ester of a compound of the invention containing carboxy or hydroxy group is, for example, a pharmaceutically acceptable ester which is hydrolyzed in the human or animal body to produce the parent acid or alcohol.
  • Suitable pharmaceutically acceptable esters for carboxy include Ci_6-alkoxymethyl esters ⁇ e.g., methoxymethyl), Ci_ 6 -alkanoyloxymethyl esters ⁇ e.g., for example pivaloyloxymethyl), phthalidyl esters, C 3 _ 8 -cycloalkoxycarbonyloxyCi_ 6 -alkyl esters ⁇ e.g., 1-cyclohexylcarbonyloxyethyl); l,3-dioxolen-2-onylmethyl esters ⁇ e.g., 5-methyl-l,3-dioxolen-2-onylmethyl; and Ci_ 6 -alkoxycarbonyloxyethyl esters ⁇ e.g., 1-methoxycarbonyloxy ethyl) and may be formed at any carboxy group in the compounds of this invention.
  • An in vivo hydrolyzable ester of a compound of the invention containing a hydroxy group includes inorganic esters such as phosphate esters and a-acyloxyalkyl ethers and related compounds which as a result of the in vivo hydrolysis of the ester breakdown to give the parent hydroxy group.
  • inorganic esters such as phosphate esters and a-acyloxyalkyl ethers and related compounds which as a result of the in vivo hydrolysis of the ester breakdown to give the parent hydroxy group.
  • ⁇ -acyloxyalkyl ethers include acetoxymethoxy and 2,2-dimethylpropionyloxy-methoxy.
  • a selection of in vivo hydrolyzable ester forming groups for hydroxy include alkanoyl, benzoyl, phenylacetyl and substituted benzoyl and phenylacetyl, alkoxycarbonyl (to give alkyl carbonate esters), dialkylcarbamoyl and
  • TV ⁇ TVTV-dialkylaminoethy ⁇ -TV-alkylcarbamoyl to give carbamates
  • TV,TV-dialkylaminoacetyl and carboxyacetyl examples include substituents on benzoyl include morpholino and piperazino linked from a ring nitrogen atom via a methylene group to the 3- or 4- position of the benzoyl ring.
  • a suitable value for an in vivo hydrolyzable amide of a compound of the invention containing a carboxy group is, for example, a TV-Ci_6-alkyl or N,N-di-Ci_6-alkyl amide such as TV-methyl, TV-ethyl, TV-propyl, TVTV-dimethyl, TV-ethyl-TV-methyl or TVTV-diethyl amide.
  • the invention provides pharmaceutical compositions comprising an inhibitor of histone deacetylase according to the invention and a pharmaceutically acceptable carrier, excipient, or diluent.
  • Compounds of the invention may be formulated by any method well known in the art and may be prepared for administration by any route, including, without limitation, parenteral, oral, sublingual, transdermal, topical, intranasal, intratracheal, or intrarectal.
  • compounds of the invention are administered intravenously in a hospital setting.
  • administration may preferably be by the oral route.
  • compositions according to the invention may contain, in addition to the inhibitor, diluents, fillers, salts, buffers, stabilizers, solubilizers, and other materials well known in the art.
  • diluents fillers, salts, buffers, stabilizers, solubilizers, and other materials well known in the art.
  • the preparation of pharmaceutically acceptable formulations is described in, e.g., Remington's The Science and Practice of Pharmacy, 20th Edition, 2000.
  • salts refers to salts that retain the desired biological activity of the above-identified compounds and exhibit minimal or no undesired toxicological effects.
  • examples of such salts include, but are not limited to acid addition salts formed with inorganic acids (for example, hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid, and the like), and salts formed with organic acids such as acetic acid, oxalic acid, tartaric acid, succinic acid, malic acid, ascorbic acid, benzoic acid, tannic acid, pamoic acid, alginic acid, polyglutamic acid, naphthalenesulfonic acid, naphthalenedisulfonic acid, and polygalacturonic acid.
  • inorganic acids for example, hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid, and the like
  • organic acids such as acetic acid, oxalic acid, tartaric acid, succinic
  • the compounds can also be administered as pharmaceutically acceptable quaternary salts known by those skilled in the art, which specifically include the quaternary ammonium salt of the formula -NR + W-, wherein R is R 3 -R 7 , and W is a counterion, including chloride, bromide, iodide, -O-alkyl, toluenesulfonate, methylsulfonate, sulfonate, phosphate, or carboxylate (such as benzoate, succinate, acetate, glycolate, maleate, malate, citrate, tartrate, ascorbate, benzoate, cinnamoate, mandeloate, benzyloate, and diphenylacetate).
  • salt is also meant to encompass complexes, such as with an alkaline metal or an alkaline earth metal.
  • the active compound is included in the pharmaceutically acceptable carrier or diluent in an amount sufficient to deliver to a patient a therapeutically effective amount without causing serious toxic effects in the patient treated.
  • a preferred dose of the active compound for all of the above-mentioned conditions is in the range from about 0.01 to 300 mg/kg, preferably 0.1 to 100 mg/kg per day, more preferably 0.1 to about 50 mg per kilogram body weight of the recipient per day, and in some applications about 0.1 to about 25 mg per kilogram body weight of the recipient per day.
  • a typical topical dosage will range from 0.01-3% wt/wt in a suitable carrier.
  • the effective dosage range of the pharmaceutically acceptable derivatives can be calculated based on the weight of the parent compound to be delivered. If the derivative exhibits activity in itself, the effective dosage can be estimated as above using the weight of the derivative, or by other means known to those skilled in the art.
  • the compounds of the invention may be synthesized according to the methods known to those of ordinary skill in the art. For example, methods that may be used to make the compounds of the invention are described in Mallory, F. B. (Organic Syntheses, Coll. Vol. IV: pp 74-75 (John Wiley & Sons, 1963)); Smith, P.A.S. and Boyer, J.H. (Organic Syntheses, Coll. Vol. IV: pp 75-78 (John Wiley & Sons, 1963)), and in Can. J. Chem., pp 2482-2484 (1969). These references are incorporated by references in their entirety.
  • Scheme I outlines the general approach taken to synthesize the 2,1,3-benzoxadiazole scaffold. There are also commercially available 2,1,3-benzoxadiazoles and 2,1,3-benzothiadiazoles that can be used as starting materials for compounds of the invention. Schemes H-V outline some more specific synthetic methods used to make particular compounds of the invention.
  • the 2,1,3-benzoxadiazole scaffold is prepared starting with a 1 ,2-dinitrogen substituted aryl system.
  • a 1,2-bis aniline is used, for example.
  • the 1,2-bis aniline is treated with NaOCl and NaOH in a ring closure reaction to give the corresponding benzofurazan oxide, which in turn is reduced to the corresponding 2,1,3-benzoxadiazole.
  • the 1,2-bis aniline can be treated with NaNO 2 ZHCl (Sandmeyer reaction chemistry) to make an intermediate diazonium salt, which when treated with sodium azide (NaN 3 ) gives the corresponding ortho-amino phenyl azide. Heating the aryl azide in toluene gives the corresponding benzofurazan oxide, which, as outlined above, is reduced to the corresponding 2,1,3-benzoxadiazole.
  • the final reaction path in Scheme I shows that an ortho-amino nitrobenzene can be converted to the corresponding 2,1,3-benzoxadiazole by first converting the amino group to a carbamate and then heating to high temperature.
  • Scheme II outlines how compounds of the invention having a 4-sulfonamide group are made generally.
  • a 4-amino-2,l,3-benzoxadiazole is treated with a desired sulfonyl chloride in the presence of an acid scavenger, preferably an amine base.
  • Scheme II shows two preferred methods, one using pyridine as the base and heating to 95 0 C to effect the reaction, and the other using a resin-bound morpholine as the base and heating to 45 0 C to effect the reaction. Specific examples are described below and provide detailed synthetic procedures.
  • Scheme IV outlines how compounds of the invention having a 7-nitrogen-4-sulfur substitution are made generally.
  • the appropriate 4-choro-7-nitro-2,l,3-benzoxadiazole is reacted with for example a sulfide to give the corresponding 7-nitrogen-4-sulfur containing analog.
  • the sulfur group can be further oxidized to the corresponding sulfoxide or sulfone to make additional compounds of the invention.
  • compounds of the invention having a substituted 7-chloro-4-sulfur substitution are made via reaction of the appropriate benzoxadiazole sulfonyl chloride with, for example, an amine partner to form sulfonamides and the like.
  • the 7-choro group on the product can further be exchanged via substitution reactions with thiols and amines for example (as shown in the specific examples) to make the corresponding 4-sulfur-7-sulfur-containing-2, 1 ,3-benzoxadiazoles and 4-sulfur-7-nitrogen-containing-2, 1 ,3-benzoxadiazoles, respectively.
  • Scheme V outlines how compounds of the invention having, for example, a bis-aryl substitution are made generally.
  • the appropriate choro -2,1,3-benzoxadiazole is reacted with, for example, an aryl boronic acid under palladium-catalysis conditions to give the corresponding bis-aryl analog.
  • Scheme VI outlines how compounds of the invention having sulfonyl and a halo, thioaryl, or thioheteroaryl susbstituents on the benzoxadiazole core.
  • 2,6-dichloroaniline can be oxidixzed with MCPBA to yield the corresponding nitroso compound.
  • Reaction of the nitroso compound with sodium azide gives the
  • Scheme VII outlines how compounds of the invention having sulfonyl and a Z group linked to the core through a 'Q' moiety.
  • Q represents -O-, -S-, or -N(R8)-, wherein each variable is as defined for formula (XX).
  • Scheme VIII illustrates the synthesis of compounds of the invention of general formula (XL).
  • First compounds 801 and 802 were reacted to give a Schiff base which is further reacted in the same pot with an isonitrile (803) to form compound 804 which contains the imidazo[l,2-a]pyridine core.
  • Deprotection with LiOH in aqueous THF gave the carboxylic acid, 805.
  • Compound 806 was formed by standard amide coupling chemistry, in this case, HBTU in DMF with DIPEA, to yield 806.
  • the nitro group was reduced with 10% Pd/C in methanol to give 807, and finally, reaction of the free amine in 807 with a isocyanate produced the desired compound of the invention, 808.
  • Scheme IX illustrates the synthesis of compounds of the invention of general formula (XXX).
  • each variable is as defined for formula (XXX).
  • Reaction of 2,3-dichloroquinoxaline, 901, with malononitrile in the presence of a base gives 902.
  • reaction of 902 with a primary amine, followed by an acidic workup gives compound of the form of 903 with a lH-pyrrolo[2,3-b]quinoxaline core.
  • reaction of 903 with a substituted acid chloride yields compounds of the invention, 904.
  • Scheme X illustrates the synthesis of compounds of the invention of general formula (LX) one a solid support. Similar methods for preparation of compounds of the invention can be found in Katritzky, J. Comb. Chem. 2003, 392, which is hereby incorporated by reference.
  • Schemes I - X in conjunction with the examples described below, will make it sufficiently clear to one of ordinary skill in the art how to make the compounds of the invention.
  • Compounds in Table 1 were made using the techniques described herein and were isolated and characterized by either 1 H-NMR, LC/MS or both. Commercially available starting materials, for example, amines, thiols, aryl halides, sulfonyl chlorides, boronic acids, were used in most cases along with the chemistry described to make the compounds of the invention.
  • Example 1
  • This compound was prepared according to the procedure for the preparation of Cpd. No. 285 and was purified by reverse phase liquid chromatography.
  • This compound was prepared according to the procedure for the preparation of Cpd. No. 285 and was purified by reverse phase liquid chromatography.
  • This compound was prepared according to the procedure for the preparation of Cpd. No. 285 and was purified by reverse phase liquid chromatography.
  • This compound was prepared according to the procedure for the preparation of Cpd. No. 285 and was purified by reverse phase liquid chromatography.
  • This compound was prepared according to the procedure for the preparation of Cpd. No. 285 and was purified by reverse phase liquid chromatography.
  • This compound was prepared according to the procedure for the preparation of Cpd. No. 285 and was purified by reverse phase liquid chromatography.
  • This compound was prepared according to the procedure for the preparation of Cpd. No. 285 and was purified by reverse phase liquid chromatography.
  • This compound was prepared according to the procedure for the preparation of Cpd. No. 285 and was purified by reverse phase liquid chromatography.
  • This compound was prepared according to the procedure for the preparation of Cpd. No. 285 and was purified by reverse phase liquid chromatography.
  • This compound was prepared according to the procedure for the preparation of Cpd No. 285 and was purified by reverse phase liquid chromatography.
  • This compound was prepared according to the procedure for the preparation of Cpd No. 285 and was purified by reverse phase liquid chromatography.
  • This compound was prepared according to the procedure for the preparation of Cpd No. 285 and was purified by reverse phase liquid chromatography.
  • This compound was prepared according to the procedure for the preparation of Cpd. No. 285 and was purified by reverse phase liquid chromatography.
  • This compound was prepared according to the procedure for the preparation of Cpd. No. 285 and was purified by reverse phase liquid chromatography.
  • This compound was prepared according to the procedure for the preparation of Cpd. No. 285 and was purified by reverse phase liquid chromatography.
  • the compounds of the invention were characterized by LC/MS using methods with various conditions. All the methods comprised a mobile phase that included 0.05% formic acid in water (component A) and 0.05% formic acid in acetonitrile (component B) for making the gradient. Table 3 below list the various conditions of the methods.
  • the TRAF6/UevlA /UBC 13 assay is the biochemical plate-based assay of TRAF 6 ligase activity using Ubcl3 as the E2 enzyme.
  • the gel-based assay is the biochemical solution-based TRAF6 ligase assay (by SDS-PAGE and Western blot) to confirm the result from the plate-based ELISA assay.
  • the APC assay is an assay which is another E3 ligase (APC2/APC11) biochemical assay.
  • a 96-well Ni-plate was blocked with 100 ⁇ l of 1% casein in PBS for 1 hour at room temperature. The plates were then washed three times with 200 ⁇ l of Ix PBS and 80 ⁇ l of a reaction buffer was added per well which contained 50 ng of Flag-ubiquitin, 50ng of His-ubiquitin, 62.5 mM Tris pH 7.5, 6.25 mM MgCl 2 , 1.0 mM DTT, and 2 ⁇ M ATP. To each well was added 10 ⁇ l of a solution of the compound in DMSO.
  • the reaction was started by adding 10 ⁇ l of a solution consisting of IOng human El, 25ng each of Uevla and Ubcl3, and lOOng TRAF6 in the reaction buffer. The plates were shaken for 10 minutes and incubate at room temperature for 1 hour. After the incubation, the plates were washed three times with 200 ⁇ l Ix PBS in 0.05% Tween and lOO ⁇ l of an antibody mix consisting of anti-Flag (1 :30,000 dilution; Sigma F-3165) and anti-Mouse IgG-HRP (1/150,000 dilution; Jackson Immunoresearch #115-035-146) in Ix PBS with 0.25% BSA.
  • ICM intercellular adhesion molecule
  • TNF/IL-1 induces the NF-kB pathway in which NF -kB acts as a transcription factor and activation of NF-kB induces ICAM expression.
  • IkB inhibits NF-kB by binding to it and retaining NF-kB in the cytoplasm.
  • TRAF6, IKKs, and TAKl are activated which lead to the phosphorylation of IkB.
  • IkB Phosphorylated IkB is then ubiquitinated by the SCF complex and degraded by proteasome, thereby releasing NF-kB to translocate into the nucleus.
  • NF-kB binds to DNA and activates transcription of various genes that are involved in inflammation, cell survival, and apoptosis.
  • the ICAM assay is a primary assay for HTS as well as a cell based assay for TRAF6 inhibitors.
  • 96-well white plate The seeded plate was incubate at 37 0 C incubator in 5% CO 2 overnight. After the overnight incubation, 4 ⁇ l of diluted test compound was added and incubated for 1 hour at 37 0 C incubator in 5% CO 2 overnight. After the overnight incubation, 4 ⁇ l of diluted test compound was added and incubated for 1 hour at 37 0 C incubator in 5% CO 2 overnight. After the overnight incubation, 4 ⁇ l of diluted test compound was added and incubated for 1 hour at
  • the cells were stained with CD54 (1 :1000 diluted with medium) and anti-mouse IgG (1 : 1100 diluted with medium) for 1 hour. Subsequently, the medium was removed and each well was washed with 200 ⁇ l of PBS three times. Detection of activity was performed by addition of lOO ⁇ l/well of lumino substrate.
  • E3 (TRAF6) auto-ubiquitination was measured as described below. Activity in the presence of compound was determined relative to a parallel control in which only DMSO is added. The IC50 values were typically determined using 6 or 8 different concentrations of compound, although as few as 2 concentrations may be used to approximate the IC 50 values.
  • E&K 96_well plates (E&K-20201) were used for the solution based biochemical assay. 80 ⁇ l of the reaction buffer were added to each well that contained 100 ng/well of Flag ubiquitin. To this, 10 ⁇ l of the test compound diluted in DMSO were added.
  • reaction buffer consisted of 62.5 mM Tris pH 7.6 (Trizma Base - Sigma T-8524),
  • the Protein Buffer consisted of 20 mM Tris pH 7.6, 10% glycerol (Sigma G-5516) and 1 mM DTT.
  • the antibody mix consisted of 0.25% BSA (Sigma A-7906) in IX PBS, 1/50,000 anti-Flag (Sigma F-3165), 1/100,000 of anti-Mouse IgG-HRP (Jackson Immunoresearch
  • E3 His APCl 1/APC2 - "APC” auto ubiquitination was measured as described in US Patent Application No. 09/826,312 (Publication No. US 2002 0042083 Al), which is incorporated by reference in its entirety. Details of the protocol are described below. Activity in the presence of the compound was determined relative to a parallel control in which only DMSO was added. Values of the IC50 were typically determined using 6 or 8 different concentrations of the compound, although as few as 2 concentrations may be used to approximate the IC50 value.
  • Nickel coated 96 well plates (Pierce 15242) were blocked for 1 hour with 100 ⁇ l of blocking buffer at room temperature. The plates were washed 4 times with 225 ⁇ l of 1 DPBS and 80 ⁇ l of the reaction buffer were added that contained 100 ng/well of Flag ubiquitin. To this, 10 ⁇ l of the test compound diluted in DMSO were added. After the test compound was added, 10 ⁇ l of El (human), E2 (Ubch5c), and APC in Protein Buffer was added to obtain a final concentration of 5 ng/well of El, 20 ng/well of E2 and 100 ng/well of APC. The plates were shaken for 10 minutes and incubated at room temperature for 1 hour.
  • the plates were washed 4 times with 225 ⁇ l of IxPBS and 100 ⁇ l/well of Antibody Mix were added to each well.
  • the plates were incubated at room temperature for another hour after which they were washed 4 times with 225 ⁇ l of IxPBS and 100 ⁇ l/well of Lumino substrate were added to each well.
  • the luminescence was measured by using a BMG luminescence microplate reader.
  • Blocking Buffer 1% Casein in IxPBS was stored at 4°C until use.
  • reaction buffer consisted of 62.5 mM Tris pH 7.6 (Trizma Base - Sigma T 8524),
  • the Protein Buffer consisted of 20 mM Tris pH 7.6, 10% glycerol (Sigma G 5516) and 1 mM DTT.
  • the antibody mix consisted of 0.25% BSA (Sigma A 7906) in IX PBS, 1/50,000 anti
  • the substrate mix consisted of SuperSignal Substrate from Pierce (catalog number
  • the buffer solution is brought to a final volume of80 ⁇ l with Milipore-filtered water, followed by the addition of 10 ⁇ l DMSO.
  • 10 ⁇ l DMSO DMSO
  • To the above solution is then added 10 ⁇ l of ubiquitination enzymes in 20 mM Tris buffer, pH 7.5, and 5% glycerol.
  • E2-Ubch5c and E3-His ROCl/Cull, ROC1/CUL2, and ROC2/CUL5 are made as described in WO 01/75145. El is obtained commercially (Aff ⁇ niti Research Products, Singer, U. K.).
  • the wells are washed with 200 ⁇ l of PBST 3 times.
  • 100 gel of Mouse anti-Flag (1 : 10,000) and anti-Mouse Ig-HRP (1 :15, 000) in PBST are added to each well and allowed to incubate at room temperature for 1 hour.
  • the wells are then washed with 200 ⁇ l of PBST 3 times, followed by the addition of 100 ⁇ l of luminol substrate (1/5 dilution). Luminescence for each well is then measured using a fluorimeter.
  • ATCC American Type Culture Collection
  • A549 ATCC# CCL-185
  • HeLa ATCC# CCL-2
  • HCTl 16 ATCC# CCL-247
  • H1299 ATCC# CRL-5803
  • Flasks reaching approximately 70% confluency were trypsinized and resuspended in RPMI media (Cell-Gro catalog number 10-040-CM) modified to contain 5% FBS, lOOug/mlPen/Strep (Cell-Gro catalog number 30-002-CL), and 0.3mg/ml L-Glutamine (Cell-Gro catalog number 25 -003 -CL).
  • RPMI media Cell-Gro catalog number 10-040-CM
  • lOOug/mlPen/Strep Cell-Gro catalog number 30-002-CL
  • 0.3mg/ml L-Glutamine Cell-Gro catalog number 25 -003 -CL.
  • a 20,000 cells/ml solution was made for plating.
  • Cells were plated in black Packard 96 well plates by placing 100 ⁇ l per well (2,000 cells per well). Table 3 below shows these and additional cell line data.
  • Photographic Image Analysis of Proliferation, Apoptosis and Death (PAD Assay) Cells to be analyzed by photography were fixed and stained. One hundred microliters of media were removed and 100 ⁇ l of 9.3% formamide was added to each well. Plates were left on the benchtop for 45 minutes. A staining solution containing 1.55 ⁇ l of lmg/ml DAPI added to 18.75ml PBS was warmed for 15 minutes at 37 0 C. The cells were aspirated prior to washing with 100 ⁇ l of PBS. Seventy microliters of PBS were aspirated and 170 ⁇ l of the DAPI solution were added to each well of fixed cells.
  • PBS Photographic Image Analysis of Proliferation, Apoptosis and Death
  • Ubiquitination of p27 by SCF was assayed in a ubiquitin ligase assay essentially as described in US Patent No. 6,919,184 for screening ubiquitination of a ligase substrate and for modulators thereof.
  • reaction buffer 50 ⁇ L of reaction buffer with 20ng GST-CyclinE/Cdk2 was added to each well, and this was incubated for 30 minutes. lOul of compound in diluted in DMSO was added to each well, followed by a 20ul volume of reaction buffer with 20ng El, 180ng Ubc3 and 40ng Skpl/Skp2, and a 20ul volume of reaction buffer with 60ng Rocl/Cull, 20ng his-Cksl and 200ng FLAG-Ubiquitin. This was incubated for 90 minutes.

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Abstract

L'invention concerne des composés et des compositions pharmaceutiques utiles comme inhibiteurs d'agent d'ubiquitine, en particulier des inhibiteurs d'ubiquitine ligase. Les composés et les compositions pharmaceutiques proposés par la présente invention sont utiles en tant qu'inhibiteurs des voies biochimiques d'organismes impliquant une ubiquitine, comme des voies de transduction de signal. L'invention comprend également l'utilisation des composés et des compositions pharmaceutiques de la présente invention dans le traitement d'infections requérant une inhibition d'ubiquitination. De plus, l'invention concerne des procédés d'inhibition d'ubiquitination dans une cellule, incluant la mise en contact d'une cellule dans laquelle une inhibition d'ubiquitination est souhaitée avec un composé ou une composition pharmaceutique proposés par l'invention, en particulier pour inhiber l'activité d'ubiquitine ligase de TRAF 6.
PCT/US2007/075511 2006-08-10 2007-08-08 Inhibiteurs d'ubiquitine ligase Ceased WO2008115259A2 (fr)

Applications Claiming Priority (4)

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US82205006P 2006-08-10 2006-08-10
US60/822,050 2006-08-10
US88758507P 2007-01-31 2007-01-31
US60/887,585 2007-01-31

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WO2008115259A2 true WO2008115259A2 (fr) 2008-09-25
WO2008115259A3 WO2008115259A3 (fr) 2008-12-18

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8431593B2 (en) 2006-11-27 2013-04-30 H. Lundbeck A/S Heteroaryl amide derivatives
CN108658962A (zh) * 2017-03-30 2018-10-16 复旦大学 3-取代香豆素呋咱衍生物及其在制备抗多药耐药肿瘤药物中的用途
WO2019180207A1 (fr) * 2018-03-22 2019-09-26 Helmholtz Zentrum München - Deutsches Forschungszentrum für Gesundheit und Umwelt (GmbH) Inhibiteurs de traf 6

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006002284A1 (fr) * 2004-06-22 2006-01-05 Rigel Pharmaceuticals, Inc. Inhibiteurs de l'ubiquitine ligase

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8431593B2 (en) 2006-11-27 2013-04-30 H. Lundbeck A/S Heteroaryl amide derivatives
CN108658962A (zh) * 2017-03-30 2018-10-16 复旦大学 3-取代香豆素呋咱衍生物及其在制备抗多药耐药肿瘤药物中的用途
WO2019180207A1 (fr) * 2018-03-22 2019-09-26 Helmholtz Zentrum München - Deutsches Forschungszentrum für Gesundheit und Umwelt (GmbH) Inhibiteurs de traf 6
US12240854B2 (en) 2018-03-22 2025-03-04 Helmholtz Zentrum Muenchen—Deutsches Forschungszentrum Fuergesundheit Und Umwelt (Gmbh) TRAF 6 inhibitors

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