WO2012061754A2 - Composés et procédés pour traiter des maladies auto-immunes - Google Patents

Composés et procédés pour traiter des maladies auto-immunes Download PDF

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WO2012061754A2
WO2012061754A2 PCT/US2011/059405 US2011059405W WO2012061754A2 WO 2012061754 A2 WO2012061754 A2 WO 2012061754A2 US 2011059405 W US2011059405 W US 2011059405W WO 2012061754 A2 WO2012061754 A2 WO 2012061754A2
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predicted
protein
similar
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domain
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PCT/US2011/059405
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WO2012061754A3 (fr
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Bridget Wagner
Jeremy Duvall
Danny Hung-Chieh Chou
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The Broad Institute, Inc.
President And Fellows Of Harvard College
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Publication of WO2012061754A2 publication Critical patent/WO2012061754A2/fr
Publication of WO2012061754A3 publication Critical patent/WO2012061754A3/fr
Priority to US13/887,466 priority Critical patent/US8741905B2/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D267/00—Heterocyclic compounds containing rings of more than six members having one nitrogen atom and one oxygen atom as the only ring hetero atoms
    • C07D267/22—Eight-membered rings
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D498/00—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and oxygen atoms as the only ring hetero atoms
    • C07D498/02—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and oxygen atoms as the only ring hetero atoms in which the condensed system contains two hetero rings
    • C07D498/04—Ortho-condensed systems

Definitions

  • the invention was supported, in whole or in part, by a grant DP2-DK083048 from NIH- NIDDK. The Government has certain rights in the invention.
  • This invention relates to the treatment autoimmune diseases including type-1 diabetes, lupus and rheumatoid arthritis.
  • Type-1 diabetes is caused by the autoimmune destruction of insulin producing ⁇ -cells in the pancreas, ⁇ -cell apoptosis involves a set of signaling cascades initiated by interleukin-1 ⁇ (IL- 1 ⁇ ), interferon- ⁇ (IFN- ⁇ ), and tumor necrosis factor-a (TNF- a).
  • IL-1 ⁇ and TNF- a induce NFKB expression, and downstream activation of gene expression is thought to occur through nitric oxide (NO) signaling, which both increases endoplasmic reticulum stress-response pathways and decreases ⁇ cell-specific functions.
  • NO nitric oxide
  • NO is a highly reactive molecule that inhibits the electron-transport chain, leading to decreases in glucose oxidation rates, ATP generation, and insulin production; cellular nitrite is more stable and serves as a surrogate marker for NO.
  • NFKB activation and IFN-a-induced STAT-1 signaling work together to effect ⁇ -cell apoptosis, mainly involving the intrinsic apoptotic pathway in both rodents and humans.
  • the downstream effector of this cascade, caspase-3 results in apoptosis and the loss of the ability to secrete insulin in response to glucose stimulation.
  • the invention relates to compounds of Formula I or a prodrug or metabolite thereof:
  • and X3 is independently -O- or -S-;
  • Each X 4 , X 5 and X 6 is independently -CH or -N-;
  • Each X 2i Yi and Y 2 is independently -NR
  • Cyi is an optionally substituted aryl group
  • Cy 2 is an optionally substituted cycloalkyl, optionally substituted cycloalkenyl, optionally substituted heterocyclyl or optionally substituted aryl containing one, two or three rings;
  • R is hydrogen, halogen, aliphatic, substituted aliphatic, aryl or substituted aryl;
  • Each R 2 , R3, R4 and R5 is independently selected from absent, hydrogen, halogen, -OR10, -SR10, - NR10R1 1 , -C(0)R,o, -C(O)OR )0 , -C(O)NR 10 Rn, -N(R, 0 )C(O)Rn, -CF 3 , -CN, -N0 2 , -N 3 , acyl, alkoxy, substituted alkoxy, alk lamino, substituted alkylamino, dialkylamino, substituted dialkylamino, substituted or unsubstituted alkylthio, substituted or unsubstituted alkylsulfonyl, optionally substituted aliphatic, optionally substituted aryl or optionally substituted heterocyclyl; G, is -N(R,o)C(0)-, -N(R,o)C(S)-, -N(R 10 )
  • n and m is independently selected from 0, 1 , 2 or 3;
  • Each v and w is independently selected from 0, 1 , 2, 3, 4, 5 or 6;
  • Each Rio, Ri i, Ri 2 , Ri3 and RH is independently absent, hydrogen, halogen, -OH, -SH, -NH 2 , - CF 3 , -CN, -N0 2 , -N 3, -C(0)OH, -C(0)NH 2 , acyl, alkoxy, substituted alkoxy, alkylamino, substituted alkylamino, dialkylamino, substituted dialkylamino, substituted or unsubstituted alkylthio, substituted or unsubstituted alkylsulfonyl, aliphatic, substituted aliphatic, aryl or substituted aryl; alternatively two of Rio, Ri ⁇ , R12, R13 and R14 together with the atoms to which they are attached and any intervening atoms may form an additional optionally substituted, 3, 4, 5, 6 or 7 membered ring.
  • the invention further relates to the treatment of autoimmune diseases by the
  • Compounds of Formula I can be useful for the treatment of autoimmune diseases including multiple sclerosis, Crohn's disease, lupus erythematosus, rheumatoid arthritis, osteoarthritis, psoriasis, ulcerative colitis, type-1 diabetes, pancreatitis, asthma, idiopathic thrombocytopenia purpura, uveitis, Guillain-Barre syndrome or myasthenia gravis.
  • autoimmune diseases including multiple sclerosis, Crohn's disease, lupus erythematosus, rheumatoid arthritis, osteoarthritis, psoriasis, ulcerative colitis, type-1 diabetes, pancreatitis, asthma, idiopathic thrombocytopenia purpura, uveitis, Guillain-Barre syndrome or myasthenia gravis.
  • FIG. 1 Cellular ATP Levels after incubation with Compound- 1 A.
  • FIG. 2 Caspase activation after incubation with Compound- 1 A.
  • FIG. 3 Glucose-stimulated insulin secretion with Compound-21A.
  • FIG. 4 The effect of Compound-21 A (10 ⁇ ) on beta cell apoptosis indicated by relative levels of ATP in the presence of common modes of beta-cell apoptosis: (A) ER stress induced by thapsigargin, (B) ER stress induced by tunicamycin, and (C) glucose toxicity. All three graphs show a DMSO control ( ⁇ ) and Compound-21 A ( ⁇ ).
  • FIG. 5 Gene-set enrichment analysis after 6-hr treatment with Compound-21 A: (A) Enrichment plot showing SANA response to interferon- ⁇ , (B) Enrichment plot showing Browne response to interferon- ⁇ .
  • FIG. 6 Schematic showing role of interferon- ⁇ in STAT1 -dependent signaling pathway.
  • FIG. 7 Compound-21 A inhibits cytokine-induced STAT1 reporter gene activity.
  • FIG. 8 Suppression of STAT1 phosphorylation by Compound-21 A.
  • FIG. 9 Inhibition of nuclear STAT1 translocation by Compound-21 A.
  • FIG. 10 The effect of putative JA 2 inhibitors, SD-1029 and Z3, on cytokine-induced beta cell apoptosis.
  • FIG. 1 1 Schematic of candidate protein binder by Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC).
  • FIG. 12 Candidate proteins identified by SILAC and mass spectrometry analysis.
  • FIG. 13 Usp9x isolated by low-throughput Compound-21 A pull-down and Western blot.
  • FIG. 14 Usp9x purification and thermal shift assay and comparison of Compound-21 A with non-specific debiquitinase inhibitor WP1 130, suggests binding.
  • FIG. 15 Compound-21 enhances effect of ABT-737 on DLD-1 cell death.
  • FIG. 16 STAT1 levels and phosphorylation after Usp9x knock-down.
  • Each i and X3 is independently -O- or -S-;
  • Each X 4 , X 5 and Xe is independently -CH or -N-;
  • Each X? , Yi and Y 2 is independently -NR10, -S- or -0-;
  • Cyi is an optionally substituted aryl group
  • Cy 2 is an optionally substituted cycloalkyl, optionally substituted cycloalkenyl, optionally substituted heterocyclyl or optionally substituted aryl containing one, two or three rings;
  • Ri is hydrogen, halogen, aliphatic, substituted aliphatic, aryl or substituted aryl;
  • Each R 2 , R3, R4 and R 5 is independently selected from absent, hydrogen, halogen, -OR10, -SR10, - NR10R1 1 , -C(0)R,o, -C(0)OR,o, -C(O)NR 10 Rn, -N(R, 0 )C(O)Ru, -CF 3 , -CN, -N0 2 , -N 3 , acyl, alkoxy, substituted alkoxy, alkylamino, substituted alkylamino, dialkylamino, substituted dialkylamino, substituted or unsubstituted alkylthio, substituted or unsubstituted alkylsulfonyi, optionally substituted aliphatic, optionally substituted aryl or optionally substituted heterocyclyl;
  • G is -N(R,o)C(0)-, -N(R,o)C(S)-, -N(R )0 )S(O) 2 -, -N(R 1 o)S(0) 2 -[C(R,o)(Rn)]w-, -N(R 10 )C(O)- [C(R 10 )(Rn)]w-, -NCR ⁇ CS ⁇ CCCR.oXR.
  • Each v and w is independently selected from 0, 1 , 2, 3, 4, 5 or 6;
  • Each Rio, R1 1 , Ri 2 , Rn and R14 is independently absent, hydrogen, halogen, -OH, -SH, -NH 2 , - CF 3 , -CN, -N0 2 , -N 3, -C(0)OH, -C(0)NH 2 , acyl, alkoxy, substituted alkoxy, alkylamino, substituted alkylamino, dialkylamino, substituted dialkylamino, substituted or unsubstituted alkylthio, substituted or unsubstituted alkylsulfonyi, aliphatic, substituted aliphatic, aryl or substituted aryl; alternatively two of R ]0 , R1 1 , Ri 2 , R13 and RH together with the atoms to which they are attached and any intervening atoms may form an additional optionally substituted, 3, 4, 5, 6 or 7 membered ring.
  • each R100, Rioi, Rio2 and R103 is independently absent, hydrogen, halogen, -ORio, -SRio, - NR10R1 1 , -C(0)R,o, -C(O)OR 10 , -C(O)NR 10 Ri i, -N(R, 0 )C(O)R, ,, -CF 3 , -CN, -N0 2 , -N 3 , acyl, alkoxy, substituted alkoxy, alkylamino, substituted alkylamino, dialkylamino, substituted dialkylamino, substituted or unsubstituted alkylthio, substituted or unsubstituted alkylsulfonyl, aliphatic, substituted aliphatic, aryl or substituted aryl.
  • Cy 2 is selected from;
  • Xi is -0-.
  • X 2 is -0-.
  • 3 is -0-.
  • X is -CH.
  • X5 is -CH.
  • X(, is -CH.
  • n 1
  • R5 is -CH3.
  • n 0.
  • Yi is -NH.
  • Y 2 is -NH.
  • R ⁇ is hydroxyl substituted alkyl.
  • R 2 and R3 are hydrogen and v is 1.
  • Cyi is naphthyl or substituted naphthyl.
  • Cy 2 is an optionally substituted phenyl.
  • Gi is -N(H)S(0) 2 -.
  • the invention relates to a compound of Formula II:
  • the invention further relates to the treatment of autoimmune diseases by the
  • Compounds of Formula I can be useful for the treatment of autoimmune diseases including multiple sclerosis, Crohn's disease, lupus erythematosus, rheumatoid arthritis, osteoarthritis, psoriasis, ulcerative colitis, type- 1 diabetes, pancreatitis, asthma, idiopathic thrombocytopenia purpura, uveitis, Guillain-Barre syndrome or myasthenia gravis.
  • a patient suffering from type-1 diabetes is treated with a compound of Formula I.
  • Human beta-cell apoptosis involves a complex set of signaling cascades initiated by IL-1 ⁇ , IFN- ⁇ , and TNF-a.
  • IL-1 ⁇ and TNF-a induce NF-KB expression, while downstream activation of gene expression is thought to occur through nitric oxide (NO) signaling, which both increases the endoplasmic reticulum stress-response pathway and decreases beta-cell function; this pathway works together with IFN-y-induced STAT-1 signaling to effect beta-cell death.
  • NO nitric oxide
  • cytokines are beta cell-specific, and small- molecule suppressors should have little to no effect on other cell types in the pancreas.
  • Small molecules that increase beta-cell survival in the presence of cytokines could be of potential clinical benefit to early-stage type 1 diabetic patients.
  • a number of studies have described small molecules with protective effects in the presence of cytokines; most of these were discovered because of their nonspecific antioxidant or anti-inflammatory effects.
  • small-molecule inhibition of histone deacetylases (HDAC) with suberoylanilide hydroxamic acid (SAHA) or trichostatin A (TSA) can prevent cytokine-induced beta-cell death, presumably by decreasing NF-KB transactivation.
  • HDAC histone deacetylases
  • SAHA suberoylanilide hydroxamic acid
  • TSA trichostatin A
  • the invention further relates to a method of treating a BCL-2 related disease or disorder by administering a compound of Formula I to a subject in need thereof.
  • the BCL-2 mediated disease or disorder is a cell proliferative disorder.
  • the cell proliferative disorder is selected from breast cancer, prostate cancer, lymphoma, skin cancer, pancreatic cancer, colon cancer, melanoma, malignant melanoma, ovarian cancer, brain cancer, primary brain carcinoma, head-neck cancer, glioma, glioblastoma, liver cancer, bladder cancer, non-small cell lung cancer, head or neck carcinoma, breast carcinoma, ovarian carcinoma, lung carcinoma, small-cell lung carcinoma, Wilms' tumor, cervical carcinoma, testicular carcinoma, bladder carcinoma, pancreatic carcinoma, stomach carcinoma, colon carcinoma, prostatic carcinoma, genitourinary carcinoma, thyroid carcinoma, esophageal carcinoma, myeloma, multiple myeloma, adrenal carcinoma, renal cell carcinoma, endometrial
  • the cell proliferative disorder is selected from the group consisting of papilloma, blastoglioma, Kaposi's sarcoma, melanoma, lung cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, astrocytoma, head cancer, neck cancer, bladder cancer, breast cancer, lung cancer, colorectal cancer, thyroid cancer, pancreatic cancer, gastric cancer, hepatocellular carcinoma, leukemia, lymphoma, Hodgkin's disease and Burkitt's disease.
  • the invention relates to a composition
  • a composition comprising a compound of
  • the composition comprises a compound of Formula I and a second compound selected from ABT-263, ABT-737, gossypol, chelerythrine chloride, apogossypolone, antimycin A, TW-37, HA14- 1 , obatoclax (GX 15-070), ApoG2, NMB and TM 12-06.
  • the second compound is ABT- 737.
  • the invention relates to a method of treating a deubiquitinase related disease or disorder comprising administering a compound of Formula I to a subject in need thereof.
  • the deubiquitinase related disease or disorder is selected from inflammation, cell proliferative disorder, macular degeneration and diabetic retinopathy, Alzheimer's, atherosclerosis, psoriasis, rheumatoid arthritis and endometriosis.
  • the disease or disorder is a cell proliferative disorder, preferably cancer.
  • the invention relates to the treatment of a disease or disorder mediated by the abnormal or aberrant expression of one or more gene or gene product selected from Table 1 .
  • cytochrome P450 family 2, subfamily j, polypeptide 10 (predicted)
  • transporter 1 ATP-binding cassette, sub-family B (MDR/TAP) /// transporter 2
  • ATP-binding cassette, sub-family B (MDR/TAP) /// proteosome (prosome, macropain) subunit, beta type 9 /// similar to butyrophilin-like 8 (predicted) /// RT1 class II, locus Da /// RT1 class II, locus Dbl /// major histocompatibility complex, class II, DM beta /// major histocompatibility complex, class II, DM alpha /// RT1 class II, locus Ba /// RT1 class II, locus Bb /// RT1 class II, locus
  • vacuolar protein sorting 54 yeast
  • Vamp5 vesicle-associated membrane protein 5
  • RTl class lb locus Aw2 /// RTl class la, locus A l /// RTl class la, locus A2 /// RTl class lb, locus CI /// RTl class II, locus DOa /// RTl class II, locus Ha /// TAP binding protein /// discoidin domain receptor family, member 1 /// leucocyte specific transcript 1 /// Fas death domain-associated protein /// UDP- Gal:betaGlcNAc beta 1,3-galactosyltransferase, polypeptide 4 /// synaptic Ras GTPase activating protein 1 homolog (rat) /// general transcription factor II H, polypeptide 4 /// procollagen, type XI, alpha 2 /// RTl class I, locus Ke4 /// RAB2, member RAS oncogene family-like /// PHD finger protein 1 /// zinc finger and BTB domain containing 9 /// POU domain, class 5, transcription factor 1 /// valyl-t
  • NADP+ dependent methylenetetrahydrofolate dehydrogenase
  • methenyltetrahydrofolate cyclohydrolase methenyltetrahydrofolate cyclohydrolase
  • formylterrahydrofolate synthase methylenetetrahydrofolate dehydrogenase
  • Ig immunoglobulin domain
  • TM transmembrane domain
  • RNA binding protein 2 Cugbp2
  • ring finger protein 128 /// hypothetical protein LOC680663, LOC680663 ///
  • proteasome prosome, macropain 28 subunit, beta, Psme2
  • proteasome prosome, macropain 28 subunit, alpha, Psme l
  • CD47 antigen Rh-related antigen, integrin-associated signal transducer
  • serine (or cysteine) peptidase inhibitor serine (or cysteine) peptidase inhibitor, clade A (alpha- 1 antiproteinase,
  • GTP binding protein (gene overexpressed in skeletal muscle) (predicted)
  • Vldlr very low density lipoprotein receptor
  • NK2 transcription factor related locus 2 (Drosophila) (predicted), Nkx2-
  • Vldlr very low density lipoprotein receptor
  • pantothenate kinase 1 predicted
  • G protein-coupled receptor family C, group 5, member B (predicted),
  • T transmembrane domain
  • Epb4.114a predicted
  • Solute carrier family 35 (UDP-glucuronic acid/UDP-N-acetylgalactosamine dual
  • solute carrier family 16 (monocarboxylic acid transporters), member 10
  • GABA-A gamma-am inobutyric acid
  • solute carrier family 12 sodium/potassium/chloride transporters
  • solute carrier family 24 sodium/potassium/calcium exchanger
  • the invention further relates to a compound of Formula II:
  • e is 0, 1 , 2, 3 or 4;
  • R 50 is -C(0)R 54 , -C(0)N(H)Rs4, -C(S)R 54 , -C(S)N(H)R 54 ;
  • R 54 is alkyl or substituted alky], -(CH 2 ) 2 0(CH 2 CH 2 0)fCH 2 CH 2 NH 2 , - (CH 2 ) 2 0(CH 2 CH 2 ) g NH 2 , -(CH 2 CH 2 ) g NH 2, -(CH 2 ) 2 0(CH 2 CH 2 0)fCH 2 CH 2 SH, - (CH 2 ) 2 0(CH 2 CH 2 ) g SH, -(CH 2 CH 2 ) g OH, -(CH 2 ) 2 0(CH 2 CH 2 0)(CH 2 CH 2 OH, - (CH 2 ) 2 0(CH 2 CH 2 ) g OH, -(CH 2 CH 2 ) g OH;
  • each f and g is independently, an integer between 0 and 500, preferably, 0, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15 orl 7.
  • Each R51 , R52 and R53 is independently hydrogen, halogen, -OR10, -SR10, -NR10R11, - C(0)R,o, -C(0)OR,o, -C(0)NR,oRi i, -N(R 10 )C(O)Rn, -CF 3 , -CN, -N0 2 , -N 3 , acyl, alkoxy, substituted alkoxy, alkylamino, substituted alkylamino, dialkylamino, substituted dialkylamino, substituted or unsubstituted alkylthio, substituted or unsubstituted alkylsulfonyl, optionally substituted aliphatic, optionally substituted aryl or optionally substituted heterocyclyl.
  • the compounds of Formula II can be immobilized onto a surface or a bead through the R50 group.
  • the immobilized compounds can be used to identify proteins, genes or DNA that can interact with the immobilized compound.
  • the invention relates to a compound of Formula II having the structure:
  • aliphatic group refers to a non-aromatic moiety that may be saturated (e.g. single bond) or contain one or more units of unsaturation, e.g., double and/or triple bonds.
  • An aliphatic group may be straight chained, branched or cyclic, contain carbon, hydrogen or, optionally, one or more heteroatoms and may be substituted or unsubstituted.
  • aliphatic groups include, for example, polyalkoxyalkyls, such as polyalkylene glycols, polyamines, and polyimines, for example. Such aliphatic groups may be further substituted.
  • aliphatic groups may include alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, and substituted or unsubstituted cycloalkyl groups as described herein.
  • acyl refers to a carbonyl substituted with hydrogen, alkyl, partially saturated or fully saturated cycloalkyl, partially saturated or fully saturated heterocycle, aryl, or heteroaryl.
  • acyl includes groups such as (C ⁇ -Ce) alkanoyl (e.g., formyl, acetyl, propionyl, butyryl, valeryl, caproyl, t-butylacetyl, etc.), (C3-C6)cycloalkylcarbonyl (e.g.,
  • heterocyclic carbonyl e.g., pyrrol idinylcarbony
  • alkyl, cycloalkyl, heterocycle, aryl and heteroaryl portion of the acyl group may be any one of the groups described in the respective definitions.
  • the acyl group may be unsubstituted or optionally substituted with one or more substituents (typically, one to three substituents) independently selected from the group of substituents listed below in the definition for "substituted” or the alkyl, cycloalkyl, heterocycle, aryl and heteroaryl portion of the acyl group may be substituted as described above in the preferred and more preferred list of substituents, respectively.
  • alkyl is intended to include branched, straight chain and cyclic, substituted or unsubstituted saturated aliphatic hydrocarbon radicals/groups having the specified number of carbons.
  • Preferred alkyl groups comprise about 1 to about 24 carbon atoms (“C1-C24”)
  • C7-C24 preferably about 7 to about 24 carbon atoms
  • C8-C24 preferably about 8 to about 24 carbon atoms
  • C9-C24 preferably about 9 to about 24 carbon atoms
  • Other preferred alkyl groups comprise at about 1 to about 8 carbon atoms (“Ci-Cs”) such as about 1 to about 6 carbon atoms (“C1-C6”), or such as about 1 to about 3 carbon atoms (“C1-C3").
  • Ci-C 6 alkyl radicals include, but are not limited to, methyl, ethyl, propyl, isopropyl, «-butyl, tert-buty ⁇ , n- pentyl, neopentyl and n-hexyl radicals.
  • alkenyl refers to linear or branched radicals having at least one carbon-carbon double bond. Such radicals preferably contain from about two to about twenty-four carbon atoms (“C2-C24”) preferably about 7 to about 24 carbon atoms (“C7-C24”), preferably about 8 to about 24 carbon atoms (“C8-C24”), and preferably about 9 to about 24 carbon atoms (“C9-C2 ”)- Other preferred alkenyl radicals are "lower alkenyl” radicals having two to about ten carbon atoms (“C2-C10”) such as ethenyl, allyl, propenyl, butenyl and 4-methylbutenyl.
  • Preferred lower alkenyl radicals include 2 to about 6 carbon atoms ("C 2 -C6").
  • alkenyl and “lower alkenyl”, embrace radicals having “cis” and “trans” orientations, or alternatively, "E” and “Z” orientations.
  • alkynyl refers to linear or branched radicals having at least one carbon-carbon triple bond. Such radicals preferably contain from about two to about twenty-four carbon atoms ("C2-C24") preferably about 7 to about 24 carbon atoms ("C 7 -C 2 4"), preferably about 8 to about 24 carbon atoms ("C 8 -C 2 4"), and preferably about 9 to about 24 carbon atoms ("C9-C24").
  • C2-C24 carbon atoms
  • Other preferred alkynyl radicals are "lower alkynyl” radicals having two to about ten carbon atoms such as propargyl, 1-propynyl, 2-propynyl, 1-butyne, 2-butynyl and 1 -pentynyl.
  • Preferred lower alkynyl radicals include 2 to about 6 carbon atoms ("C 2 -C6").
  • cycloalkyl refers to saturated carbocyclic radicals having three to about twelve carbon atoms ("Ci-Cn").
  • cycloalkyl embraces saturated carbocyclic radicals having three to about twelve carbon atoms. Examples of such radicals include cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl.
  • cycloalkenyl refers to partially unsaturated carbocyclic radicals having three to twelve carbon atoms. Cycloalkenyl radicals that are partially unsaturated carbocyclic radicals that contain two double bonds (that may or may not be conjugated) can be called
  • cycloalkyldienyl More preferred cycloalkenyl radicals are “lower cycloalkenyl” radicals having four to about eight carbon atoms. Examples of such radicals include cyclobutenyl, cyclopentenyl and cyclohexenyl.
  • alkylene refers to a divalent group derived from a straight chain or branched saturated hydrocarbon chain having the specified number of carbons atoms.
  • alkylene groups include, but are not limited to, ethylene, propylene, butylene, 3- methyl-pentylene, and 5-ethyl-hexylene.
  • alkenylene denotes a divalent group derived from a straight chain or branched hydrocarbon moiety containing the specified number of carbon atoms having at least one carbon-carbon double bond.
  • Alkenylene groups include, but are not limited to, for example, ethenylene, 2-propenylene, 2-butenylene, l -methyl-2-buten- l -ylene, and the like.
  • alkynylene denotes a divalent group derived from a straight chain or branched hydrocarbon moiety containing the specified number of carbon atoms having at least one carbon-carbon triple bond.
  • Representative alkynylene groups include, but are not limited to, for example, propynylene, 1 -butynylene, 2-methyl-3-hexynylene, and the like.
  • alkoxy refers to linear or branched oxy-containing radicals each having alkyl portions of one to about twenty-four carbon atoms or, preferably, one to about twelve carbon atoms. More preferred alkoxy radicals are "lower alkoxy" radicals having one to about ten carbon atoms and more preferably having one to about eight carbon atoms. Examples of such radicals include methoxy, ethoxy, propoxy, butoxy and tert-butoxy.
  • alkoxyalkyl refers to alkyl radicals having one or more alkoxy radicals attached to the alkyl radical, that is, to form monoalkoxyalkyl and dialkoxyalkyl radicals.
  • aryl alone or in combination, means a carbocyclic aromatic system containing one, two or three rings wherein such rings may be attached together in a pendent manner or may be fused.
  • aryl embraces aromatic radicals such as phenyl, naphthyl, tetrahydronaphthyl, indane and biphenyl.
  • heterocyclyl refers to saturated, partially unsaturated and unsaturated heteroatom-containing ring-shaped radicals, which can also be called “heterocyclyl”, “heterocycloalkenyl” and “heteroaryl” correspondingly, where the heteroatoms may be selected from nitrogen, sulfur and oxygen.
  • saturated heterocyclyl radicals include saturated 3 to 6-membered heteromonocyclic group containing 1 to 4 nitrogen atoms (e.g.
  • pyrrolidinyl imidazolidinyl, piperidino, piperazinyl, etc.
  • saturated 3 to 6- membered heteromonocyclic group containing 1 to 2 oxygen atoms and 1 to 3 nitrogen atoms e.g. morpholinyl, etc.
  • saturated 3 to 6-membered heteromonocyclic group containing 1 to 2 sulfur atoms and 1 to 3 nitrogen atoms e.g., thiazolidinyl, etc.
  • partially unsaturated heterocyclyl radicals include dihydrothiophene, dihydropyran, dihydrofuran and dihydrothiazole.
  • Heterocyclyl radicals may include a pentavalent nitrogen, such as in tetrazolium and pyridinium radicals.
  • the term "heterocycle” also embraces radicals where heterocyclyl radicals are fused with aryl or cycloalkyl radicals. Examples of such fused bicyclic radicals include benzofuran, benzothiophene, and the like.
  • heteroaryl refers to unsaturated aromatic heterocyclyl radicals.
  • heteroaryl radicals include unsaturated 3 to 6 membered heteromonocyclic group containing 1 to 4 nitrogen atoms, for example, pyrrolyl, pyrrolinyl, imidazolyl, pyrazolyl, pyridyl, pyrimidyl, pyrazinyl, pyridazinyl, triazolyl (e.g., 4H- l ,2,4-triazolyl, l H- l ,2,3-triazolyl, 2H- l ,2,3-triazolyl, etc.) tetrazolyl (e.g.
  • benzimidazolyl quinolyl, isoquinolyl, indazolyl, benzotriazolyl, tetrazolopyridazinyl (e.g., tetrazolo[l ,5-b]pyridazinyl, etc.), etc.; unsaturated 3 to 6-membered heteromonocyclic group containing an oxygen atom, for example, pyranyl, furyl, etc.; unsaturated 3 to 6-membered heteromonocyclic group containing a sulfur atom, for example, thienyl, etc.; unsaturated 3- to 6- membered heteromonocyclic group containing 1 to 2 oxygen atoms and 1 to 3 nitrogen atoms, for example, oxazolyl, isoxazolyl, oxadiazolyl (e.g., 1 ,2,4-oxadiazolyl, 1 ,3,4-oxadiazolyl, 1 ,2,5- oxadiazolyl, etc.
  • benzoxazolyl, benzoxadiazolyl, etc. unsaturated 3 to 6- membered heteromonocyclic group containing 1 to 2 sulfur atoms and 1 to 3 nitrogen atoms, for example, thiazolyl, thiadiazolyl (e.g., 1 ,2,4- thiadiazolyl, 1 ,3,4-thiadiazolyl, 1 ,2,5-thiadiazolyl, etc.) etc.; unsaturated condensed heterocyclyl group containing 1 to 2 sulfur atoms and 1 to 3 nitrogen atoms (e.g., benzothiazolyl, benzothiadiazolyl, etc.) and the like.
  • thiazolyl, thiadiazolyl e.g., 1 ,2,4- thiadiazolyl, 1 ,3,4-thiadiazolyl, 1 ,2,5-thiadiazolyl, etc.
  • heterocycloalkyl refers to heterocyclo-substituted alkyl radicals. More preferred heterocycloalkyl radicals are "lower heterocycloalkyl” radicals having one to six carbon atoms in the heterocyclo radical.
  • alkylthio refers to radicals containing a linear or branched alkyl radical, of one to about ten carbon atoms attached to a divalent sulfur atom.
  • Preferred alkylthio radicals have alkyl radicals of one to about twenty-four carbon atoms or, preferably, one to about twelve carbon atoms. More preferred alkylthio radicals have alkyl radicals which are "lower alkylthio" radicals having one to about ten carbon atoms. Most preferred are alkylthio radicals having lower alkyl radicals of one to about eight carbon atoms. Examples of such lower alkylthio radicals include methylthio, ethylthio, propylthio, butylthio and hexylthio.
  • aralkyl or "arylalkyl” refer to aryl-substituted alkyl radicals such as benzyl, diphenylmethyl, triphenylmethyl, phenylethyl, and diphenylethyl.
  • aryloxy refers to aryl radicals attached through an oxygen atom to other radicals.
  • aralkoxy or "arylalkoxy” refer to aralkyl radicals attached through an oxygen atom to other radicals.
  • aminoalkyl refers to alkyl radicals substituted with amino radicals.
  • Preferred aminoalkyl radicals have alkyl radicals having about one to about twenty-four carbon atoms or, preferably, one to about twelve carbon atoms. More preferred aminoalkyl radicals are "lower aminoalkyl” that have alkyl radicals having one to about ten carbon atoms. Most preferred are aminoalkyl radicals having lower alkyl radicals having one to eight carbon atoms. Examples of such radicals include aminomethyl, aminoethyl, and the like.
  • alkylamino denotes amino groups which are substituted with one or two alkyl radicals.
  • Preferred alkylamino radicals have alkyl radicals having about one to about twenty carbon atoms or, preferably, one to about twelve carbon atoms. More preferred alkylamino radicals are "lower alkylamino” that have alkyl radicals having one to about ten carbon atoms. Most preferred are alkylamino radicals having lower alkyl radicals having one to about eight carbon atoms.
  • Suitable lower alkylamino may be monosubstituted N-alkylamino or disubstituted ⁇ , ⁇ -alkylamino, such as N-methylamino, N-ethylamino, ⁇ , ⁇ -dimethylamino, N,N-diethylamino or the like.
  • substituted refers to the replacement of one or more hydrogen radicals in a given structure with the radical of a specified substituent including, but not limited to: halo, alkyl, alkenyl, alkynyl, aryl, heterocyclyl, thiol, alkylthio, arylthio, alkylthioalkyl, arylthioatkyl, alkylsulfonyl, alkylsulfonylalkyl, arylsulfonylalkyl, alkoxy, aryloxy, aralkoxy, aminocarbonyl, alkylaminocarbonyl, arylaminocarbonyl, alkoxycarbonyl, aryloxycarbonyl, haloalkyl, amino, trifluoromethyl, cyano, nitro, alkylamino, arylamino, alkylaminoalkyl, arylaminoalkyl, aminoalkylamino, hydroxy
  • chemical moieties that are defined and referred to throughout can be univalent chemical moieties (e.g., alkyl, aryl, etc.) or multivalent moieties under the appropriate structural circumstances clear to those skilled in the art.
  • an "alkyl” moiety can be referred to a monovalent radical (e.g. CH3-CH2-), or in other instances, a bivalent linking moiety can be "alkyl,” in which case those skilled in the art will understand the alkyl to be a divalent radical (e.g., -CH2-CH2-), which is equivalent to the term "alkylene.”
  • divalent moieties are required and are stated as being "alkoxy", "alkylamino”
  • alkoxy alkoxy
  • alkylamino alkyloxy
  • alkylthio alkyl
  • alkylthio alkyl
  • alkyl alkyl
  • alkyl alkenyl
  • alkynyl alkynyl
  • aliphatic or cycloalkyl
  • halogen refers to an atom selected from fluorine, chlorine, bromine and iodine.
  • carbocyclic biaryl refers to fused bicyclic moieties, typically containing 4-20 carbon atoms.
  • An example is naphthalene.
  • the biaryl groups may contain 1 -4 heteroatoms. Examples include indoles, isoindoles, quinolines, isoquinolines, benzofurans, isobenzofurans, benzothiophenes, benzo[c]thiophenes, benzimidazoles, purines, indazoles, benzoxazole, benzisoxazole, benzothiazole, quinoxalines, quinazolines, cinnolines, and the like.
  • compound and “drug” as used herein all include pharmaceutically acceptable salts, co-crystals, solvates, hydrates, polymorphs, enantiomers, diastereoisomers, racemates and the like of the compounds and drugs having the formulas as set forth herein.
  • the term "effective amount of the subject compounds,” with respect to the subject method of treatment, refers to an amount of the subject compound which, when delivered as part of desired dose regimen, brings about management of the disease or disorder to clinically acceptable standards.
  • Treatment refers to an approach for obtaining beneficial or desired clinical results in a patient.
  • beneficial or desired clinical results include, but are not limited to, one or more of the following: alleviation of symptoms, diminishment of extent of a disease, stabilization (i.e., not worsening) of a state of disease, preventing spread (i.e., metastasis) of disease, preventing occurrence or recurrence of disease, delay or slowing of disease progression, amelioration of the disease state, and remission (whether partial or total).
  • compositions of the present invention comprise a therapeutically effective amount of a compound of the present invention formulated together with one or more pharmaceutically acceptable carriers or excipients.
  • the term "pharmaceutically acceptable carrier or excipient” means a nontoxic, inert solid, semi-solid, gel or liquid filler, diluent, encapsulating material or formulation auxiliary of any type.
  • materials which can serve as pharmaceutically acceptable carriers are sugars such as lactose, glucose and sucrose; cyclodextrins such as alpha- (a), beta- ( ⁇ ) and gamma- ( ⁇ ) cyclodextrins; starches such as corn starch and potato starch;
  • cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols such as propylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol, and phosphate buffer solutions, as well as other non-toxic compatible lubricants such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, releasing agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and
  • compositions of this invention may be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir.
  • the pharmaceutical compositions of this invention may contain any conventional nontoxic pharmaceutically-acceptable carriers, adjuvants or vehicles.
  • the pH of the formulation may be adjusted with pharmaceutically acceptable acids, bases or buffers to enhance the stability of the formulated compound or its delivery form.
  • parenteral as used herein includes subcutaneous, intracutaneous, intravenous, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal, intralesional and intracranial injection or infusion techniques.
  • Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs.
  • the liquid dosage forms may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.
  • the oral compositions can also include adjuvants such as wetting agents, e
  • Injectable preparations for example, sterile injectable aqueous or oleaginous suspensions, may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents.
  • the sterile injectable preparation may also be a sterile injectable suspension or emulsion, such as INTRALIPID®, LIPOSYN® or OMEGAVEN®, or solution, in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1 ,3-butanediol.
  • INTRALIPID® is an intravenous fat emulsion containing 10-30% soybean oil, 1-10% egg yolk phospholipids, 1 - 10% glycerin and water.
  • LIPOSYN® is also an intravenous fat emulsion containing 2- 15% safflower oil, 2-15% soybean oil, 0.5-5% egg phosphatides 1- 10% glycerin and water.
  • OMEGAVEN® is an emulsion for infusion containing about 5-25% fish oil, 0.5-10% egg phosphatides, 1-10% glycerin and water.
  • acceptable vehicles and solvents that may be employed are water, Ringer's solution, USP and isotonic sodium chloride solution.
  • sterile, fixed oils are conventionally employed as a solvent or suspending medium.
  • any bland fixed oil can be employed including synthetic mono- or diglycerides.
  • fatty acids such as oleic acid are used in the preparation of injectables.
  • the injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.
  • compositions for rectal or vaginal administration are preferably suppositories which can be prepared by mixing the compounds of this invention with suitable non-irritating excipients or carriers such as cocoa butter, polyethylene glycol or a suppository wax which are solid at ambient temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the active compound.
  • suitable non-irritating excipients or carriers such as cocoa butter, polyethylene glycol or a suppository wax which are solid at ambient temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the active compound.
  • Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules.
  • the active compound is mixed with at least one inert, pharmaceutically acceptable excipient or carrier such as sodium citrate or dicalcium phosphate and/or: a) fillers or extenders such as starches, lactose, sucrose, glucose, mannitol, and silicic acid, b) binders such as, for example, carboxymethylcellulose, alginates, gelatin,
  • the dosage form may also comprise buffering agents.
  • compositions of a similar type may also be employed as fillers in soft and hard- filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like.
  • the solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings and other coatings well known in the pharmaceutical formulating art. They may optionally contain opacifying agents and can also be of a composition that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes.
  • Dosage forms for topical or transdermal administration of a compound of this invention include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants or patches.
  • the active component is admixed under sterile conditions with a pharmaceutically acceptable carrier and any needed preservatives or buffers as may be required.
  • Ophthalmic formulation, ear drops, eye ointments, powders and solutions are also contemplated as being within the scope of this invention.
  • the ointments, pastes, creams and gels may contain, in addition to an active compound of this invention, excipients such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.
  • excipients such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.
  • Powders and sprays can contain, in addition to the compounds of this invention, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances.
  • Sprays can additionally contain customary propellants such as chlorofluorohydrocarbons.
  • Transdermal patches have the added advantage of providing controlled delivery of a compound to the body.
  • dosage forms can be made by dissolving or dispensing the compound in the proper medium.
  • Absorption enhancers can also be used to increase the flux of the compound across the skin.
  • the rate can be controlled by either providing a rate controlling membrane or by dispersing the compound in a polymer matrix or gel.
  • a therapeutic composition of the invention is formulated and administered to the patient in solid or liquid particulate form by direct administration e.g., inhalation into the respiratory system.
  • Solid or liquid particulate forms of the active compound prepared for practicing the present invention include particles of respirable size: that is, particles of a size sufficiently small to pass through the mouth and larynx upon inhalation and into the bronchi and alveoli of the lungs. Delivery of aerosolized therapeutics, particularly aerosolized antibiotics, is known in the art (see, for example U.S. Pat. No. 5,767,068 to VanDevanter et al., U.S. Pat. No.
  • a “therapeutically effective amount” of a compound of the invention is meant an amount of the compound which confers a therapeutic effect on the treated subject, at a reasonable benefit/risk ratio applicable to any medical treatment.
  • the therapeutic effect may be objective (i.e., measurable by some test or marker) or subjective (i.e., subject gives an indication of or feels an effect).
  • one or more therapeutic agents of the dosage unit may exist in an extended or control release formulation and additional therapeutic agents may not exist in extended release formulation.
  • the extended release contemplates the substantially continuous delivery of drug over an extended period of time, such as greater than one, two, three, four or more weeks.
  • an agent described herein may exist in a controlled release formulation or extended release formulation in the same dosage unit with another agent that may or may not be in either a controlled release or extended release formulation.
  • the rate of delivery may be altered, for example, by varying the lactide/glycolide ratio in a poly(D,L-lactide-co-glycolide) encapsulation.
  • Other polymers that may be used include polyacetal polymers, polyorthoesters, polyesteramides, polycaprolactone and copolymers thereof, polycarbonates, polyhydroxybuterate and copolymers thereof, polymaleamides, copolyaxalates and polysaccharides.
  • a compound of formula I is formulated with an extended release component, such as a coated extended release matrix, an extended release matrix, or an extended release bead matrix.
  • a compound of Formula I is used in combination with a polymer matrix (e.g., Eudragit), Hydroxypropyl methyl cellulose (HPMC) and/or a polymer coating (e.g., Eudragit).
  • a polymer matrix e.g., Eudragit
  • HPMC Hydroxypropyl methyl cellulose
  • a polymer coating e.g., Eudragit
  • Such formulations can, for example, be compressed into solid tablets or granules or formed into pellets for capsules or tablets.
  • Extended release oral formulation can be prepared using additional methods known in the art.
  • a suitable extended release form of a compound of Formula I may be a matrix tablet composition.
  • Suitable matrix forming materials include, for example, waxes (e.g., carnauba, bees wax, paraffin wax, ceresine, shellac wax, fatty acids, and fatty alcohols), oils, hardened oils or fats (e.g., hardened rapeseed oil, castor oil, beef tallow, palm dil, and soya bean oil), and polymers (e.g., hydroxypropyl cellulose, polyvinylpyrrolidone, hydroxypropyl methyl cellulose, and
  • Extended release formulations can be made by spray drying polymer-drug mixtures, emulsion-based technologies, coacervation based technologies, film casting, extrusion based technologies and other processes to manufacture polymer-drug micropartic!es possessing an extended release profile.
  • Examples of suitable extended release technologies that can be used to incorporate the compounds of formula I described herein include, without limitation, the MEDISORB ® technology, as described in, for example, U.S. Pat. Nos. 6,264,987 to Wright, 5,654,008 and/or 5,792,477, for example; the PROLEASE ® technology, as described, for example in U.S. Pat. No. 6,358,443 to Herbert; the technologies described by Southern Research Institute, as described for example in U.S. Pat. No. 6,306,425; and "Method of Preparing Sustained Release Microparticles," U.S. Application No. 60/441 ,946, filed Jan. 23, 2003, and the technologies described by Alza Corp., including the ALZAMER Depot injection technology. The contents of these patents are incorporated herein by reference in their entirety.
  • EMF-4 To a stirred solution of EMF-2 (21 g, 40.0 mmol, 1 equiv) and 2-fluoro-3-nitrobenzoyl chloride (20.36 g, 100 mmol, 2.5 equiv) in CH 2 C1 2 (120 mL) at 0 °C was added triethyl amine NEt 3 ) (27.7 mL, 200 mmol, 5 equiv). The reaction was allowed to warm to room temperature as it stirred, and no starting material remained after 1.5 h. H 2 0 (50 mL) was added to the reaction and it was extracted with CH 2 C1 2 (2 x 100 mL). The combined organic portion was dried over MgS0 4 , filtered, and concentrated.
  • SynPhase L-series alkyl tethered diisopropylarylsilane lanterns (32 Lanterns, ⁇ 15 ⁇ / lantern) were prepared for loading by washing with CH 2 CI 2 (3 x 20 min) and dried overnight under reduced pressure. The lanterns were then activated in an oven-dried vial by addition of 3% TfOH in CH 2 CI 2 . The vial was shaken for 10 minutes, and the lanterns turned bright red. The liquid was removed, 2,6-lutidine was added, and the lanterns were shaken until the red color disappeared. A small amount of CH 2 CI 2 was added to the lanterns followed by core IB (353 mg, 0.576 mmol, 1.2 equiv) in CH 2 CI 2 .
  • the lanterns were washed with CH 2 C1 2 (1 x 8 mL), DMF (2 x 8 mL), THF H 2 0 (3: 1 , 1 x 8 mL), THF/isopropanol (3: 1, 1 x 8 mL), THF (1 x 8 mL), and CH 2 C1 2 (2 x 8 mL).
  • General Protocol A was used to release the compound from the lantern for subsequent for characterization to ascertain that the reaction was complete.
  • the remaining lanterns were then cleaved according to General Protocol A to give the product as a white solid.
  • INS-I E cells (generously provided by C. Wollheim and P. Maechler, University of Geneva) were maintained in RPMI 1640 containing 1 1 mM glucose, 10% fetal bovine serum, 10 mM HEPES, 50 ⁇ 2-mercaptoethanol, 1 mM sodium pyruvate, cultivated at 37 °C with 5% CCb in a humidified atmosphere, and split every week.
  • Recombinant rat IL- ⁇ ⁇ and recombinant mouse TNF-a were purchased from R&D Systems.
  • Recombinant mouse IFN- ⁇ , Griess reagent, and dexamethasone were purchased from Sigma.
  • CellTiter-Glo and Caspase- Glo 3/7 reagents were purchased from Promega. Alsterpaullone and Ro 31 -8220 were purchased from EMD Biosciences. The pyrazole derivatives were purchased from Maybridge. CHIR99021 was synthesized as described in Wang et al. (Proc. Natl. Acad. Sci. U.S.A. 106, 1427-1432, 2009).
  • INS- I E cells were seeded at 10,000 cells per well using a Multidrop Combi (Thermo Labsystems) in white optical 384- well plates (Corning Life Sciences). After overnight incubation, medium was removed and 50 ⁇ L ⁇ of RPMI containing 1 % FBS and a combination of cytokines ( 10 ng mL- 1 IL- ⁇ ⁇ , 50 ng mL IFN- ⁇ , 25 ngmL " 1 TNF-a) was added to every well. Using libraries of compounds dissolved in DMSO and a CyBi-Well pin-transfer robot (CyBio Corp.), 0.1 uL of each compound was added. After 48 h, medium was removed and 20 uL of CellTiter-Glo reagent was added.
  • cytokines 10 ng mL- 1 IL- ⁇ ⁇ , 50 ng mL IFN- ⁇ , 25 ngmL " 1 TNF-a
  • Luminescence was measured after 10 min of incubation using an EnVision plate reader
  • the uncertainty in a single background-subtracted measurement was estimated using the number of mock-treated wells on the plate and, as a measure of the assay noise, the standard deviation of the per-day mock-treatment distribution.
  • the signal was scaled by the noise, the standard deviation of the mock treatment distribution.
  • INS- I E cells were seeded and treated as described for high-throughput screening. After treatment with cytokine and compounds for 48 h, 10 ⁇ . of modified Griess reagent (1 : 1 mixture of 1 % sulfanilamide in 30% acetic acid and 0.1 % N-(l - naphthyl) ethylenediamine dihydrochloride in 60% acetic acid) was added to each well. After 5 min of incubation at RT, the absorbance at 540 nm was measured using an EnVision (Perkin Elmer) plate reader.
  • modified Griess reagent 1 : 1 mixture of 1 % sulfanilamide in 30% acetic acid and 0.1 % N-(l - naphthyl) ethylenediamine dihydrochloride in 60% acetic acid
  • Caspase-3 Activity Assay INS- I E cells were seeded at 5,000 cells per well in white optical 384- well plates and treated as described for highthroughput screening. After treatment with cytokines and compounds for 48 h, medium was removed and 20 uL Caspase-Glo 3/7 reagent was added. Luminescence was measured after 2 h of incubation using an EnVision plate reader. The data from the screening is tabulated in Table 3 below.
  • INS- I E cells were seeded in 96-well plates at 20,000 cells per well and incubated for 48 h in 100 of fresh RPMI containing 1 % FBS and the cytokine cocktail, in the presence or absence of compounds. Cells were washed and incubated for 2 h in KRBH ( 135 mM NaCl, 3.6 mM KC1, 5 mM NaHC0 3 , 0.5 mM NaH 2 P0 4 , 0.5 mM MgCl 2 , 1.5 mM CaCl 2 , 10 mM HEPES, pH 7.4, 0.1 % BSA) without glucose.
  • KRBH 135 mM NaCl, 3.6 mM KC1, 5 mM NaHC0 3 , 0.5 mM NaH 2 P0 4 , 0.5 mM MgCl 2 , 1.5 mM CaCl 2 , 10 mM HEPES, pH 7.4, 0.1 % BSA
  • INS- I E cells were seeded at 10,000 cells/well using a Multidrop Combi (Thermo Labsystems) in white optical 384-well plates (Corning Life Sciences). After overnight incubation, medium was removed and 50 ⁇ . RPMI containing the treated compound, 1 % FBS and a combination of cytokines (10 ng mL IL- ⁇ ⁇ , 50 ng mL IFN- ⁇ , 25 ng/mL TNF- ⁇ ) was added to every well. After incubation for 48 hr, medium was removed and 20 ⁇ . CellTiter-Glo reagent was added. Luminescence was measured after 10-min incubation using an EnVision plate reader (PerkinElmer).
  • GSEA Gene-expression profiling and Gene-Set Enrichment Analysis
  • GAS reporter assay INS- I E cells were transfected with plasmids (GAS reporter assay kit) using DharmaFECT reagent according to the manufacturer's protocol (SABiosciences) in white optical 384-well plates (Corning Life Sciences). After overnight incubation, medium was removed and 50 RPMI containing the treated compound, 1 % FBS and a combination of cytokines (10 ng/mL IL- ⁇ ⁇ , 50 ng/mL IFN- ⁇ , 25 ng/mL TNF-a) was added to every well. After treatment with cytokines and compounds for 18 hr, luminescence was measure using Dual-Glo according to the manufacturer's protocol (Promega).
  • RNA interference and Western blotting siRNAs (100 nM) were transfected into INS- I E cells (5,000 cells/well in a 384-well plate) using DharmaFECT reagent. Transfected cells were cultured for 72hr, then collected for Western blot analysis and cell-based assays. For Western blotting, cells were lysed in RIPA buffer. Total protein was separated by 4-12% SDS-PAGE and transferred to a PVDF membrane. Blots were developed using the chemiluminescence detection system SuperSignal (Thermo Fishe Scientific) and light emission was captured using an Imaging Station 4000MM (Carestream).
  • Kinase profiling was performed by Miilipore (KinaseProfiler) according to the manufacturer's protocols.
  • ATP concentrations were within 1 5 ⁇ of the apparent KM for each enzyme.
  • SILAC and mass spectrometry We performed stable isotope labeling of amino acids in cell culture (SILAC) on INS-I E cells, followed by mass spectrometry to identify proteins enriched in the absence of soluble BRD0476 competitor, as described in Ong SE et al. (2009) Proc. Natl. Acad. Sci. USA 106:4617-4622.
  • the IgG-agarose was washed extensively with lysis buffer containing 0.15 M NaCl, then with several washes in buffer B prior to incubation with 0.250 ml of buffer B containing 0.1 mg of TEV protease (Invitrogen). After 3 h at 4 °C -70-90% of the TAP-tagged protein had been cleaved from the IgG-agarose and the eluted protein was incubated with 0.1 ml of rabbit calmodulin-Sepharose (Roche) equilibrated in buffer C. After 1 h at 4 °C, the calmodulin- Sepharose was washed with buffer C.
  • the calmodulin-Sepharose was then incubated with 0.1 ml of buffer D for 10 min at 4 °C.
  • the eluate was removed from the beads and the elution repeated two or three times.
  • the eluates were centrifuged at 1500 g for 1 min at 4 °C in protein desalting spin columns.
  • Lysis buffer contained 50 mM Tris HCl (pH 7.5), 1 mM EGTA, 1 mM EDTA, 1 % (w/v) Nonidet P40, 1 mM sodium orthovanadate, 10 mM sodium b-glycerophosphate, 50 mM sodium fluoride, 5 mM sodium pyrophosphate, 0.27 M sucrose, 1 mM DTT (dithiothreitol) and complete proteinase inhibitor cocktail (one tablet/50 ml).
  • Buffer B contained 50 mM Tris/HCl (pH 7.5), 0.15 M NaCl, 0.27 M sucrose, 1 % (w/v) Nonidet P40 and 1 mM DTT.
  • Buffer C contained 50mM Tris/HCl (pH 7.5), 0.15 M NaCl, 1 mM MgCl 2 , 1 mM imidazole, 2 mM CaCI 2 , 0.27 M sucrose and 1 mM DTT.
  • Buffer D contained 50 mM Tris/HCl (pH 7.5), 20 mM EGTA, 150 mM NaCl and 5 mM DTT.
  • TBS-Tween buffer contained 50 mM Tris/HCl (pH 7.5), 0.15 M NaCl and 0.2% (v/v) Tween 20.

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Abstract

L'invention concerne un composé de Formule I :
PCT/US2011/059405 2010-11-05 2011-11-04 Composés et procédés pour traiter des maladies auto-immunes WO2012061754A2 (fr)

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US9802904B2 (en) 2012-12-28 2017-10-31 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Inhibitors of the USP1/UAF1 deubiquitinase complex and uses thereof
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WO2022065965A1 (fr) * 2020-09-28 2022-03-31 경상국립대학교산학협력단 Composition comprenant la protéine arl6ip5 en tant que principe actif pour la prévention, le soulagement ou le traitement d'un cancer
CN114272378A (zh) * 2020-09-27 2022-04-05 四川大学华西医院 一种使cttnbp2nl功能缺失的试剂在制备治疗疾病的药物中的用途
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