WO2000032598A1 - Methodes et compositions destines au traitement de maladiesnflammatoires a base d'inhibiteurs de l'activite du facteur de necrose tumorale - Google Patents

Methodes et compositions destines au traitement de maladiesnflammatoires a base d'inhibiteurs de l'activite du facteur de necrose tumorale Download PDF

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WO2000032598A1
WO2000032598A1 PCT/US1999/028856 US9928856W WO0032598A1 WO 2000032598 A1 WO2000032598 A1 WO 2000032598A1 US 9928856 W US9928856 W US 9928856W WO 0032598 A1 WO0032598 A1 WO 0032598A1
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thioxo
thiazolidin
alkyl
methylene
ethyl
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PCT/US1999/028856
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WO2000032598A9 (fr
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Jing Wang
Kalyanaraman Ramnarayan
Darryl Rideout
Seymour Mong
Hengyi Zhu
Christina Niemeyer
Thomas P. Brady
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Structural Bioinformatics Inc.
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Publication of WO2000032598A9 publication Critical patent/WO2000032598A9/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D417/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00
    • C07D417/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings
    • C07D417/06—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings linked by a carbon chain containing only aliphatic carbon atoms
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/425—Thiazoles
    • A61K31/426—1,3-Thiazoles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/425—Thiazoles
    • A61K31/427—Thiazoles not condensed and containing further heterocyclic rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D277/00—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings
    • C07D277/02—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings not condensed with other rings
    • C07D277/20—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members
    • C07D277/32—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D277/36—Sulfur atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D417/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00
    • C07D417/14—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing three or more hetero rings

Definitions

  • the present invention relates to the prophylaxis and treatment of inflammatory diseases and, more particularly, to compounds that act as specific inhibitors of TNF-dependent NF- ⁇ B activation signaled by certain members of the TNF receptor superfamily, such as TNF-Rl , methods and means for making such inhibitors and then- use as pharmaceuticals.
  • TNFs Tumor necrosis factors
  • lymphotoxins formerly known as lymphotoxins
  • TNFs Tumor necrosis factors
  • TNFs are now known to elicit a wide range of biological effects, including playing an important role in endotoxic shock and in inflammatory, immunoregulatory, cardiovascular, proliferative, cytotoxic, and anti-viral activities .(reviewed by Goeddel et al. , CSH Symposia on Quantitative Biology 51:597-609 (1986)).
  • TNF- ⁇ has been said to have a central role in the immune response (Gamble et al. , Proc. Natl. Acad. Sci. USA 82:8667 (1985)), but the precise nature of that role remains clouded. It is known that TNF- ⁇ plays a multiple role as a mediator of inflammation and the immune response.
  • TNF- ⁇ The level of TNF- ⁇ is elevated in pathophysiological conditions, including sepsis syndrome, bacterial meningitis, CHF, cerebral malaria, AIDS, IBD, and RA (Eigler et al. , Immunol. Today 18:487-492 (1997)). Successful use of anti-TNF antibody therapy has recently been reported for patients with rheumatoid arthritis and Crohn's disease (Stack et ai , Lancet 349:521-524 (1997)).
  • Human TNF- ⁇ is synthesized as a precursor polypeptide consisting of 233 amino acids and is processed post-translationally to the secretory mature form consisting of the precursor's C-terminal 155 amino acids (Yamada et al. , Biotechnol. 3: 141-153 (1985)).
  • the three-dimensional structure and functional features of TNF- ⁇ and TNF- ⁇ have been well characterized by a combination of x-ray crystallography and site directed mutagenesis studies (Zhang et ai , J. Biol. Chem. 267:24069-24075 (1992); Van Ostade et al. , Protein Eng. 7:5-22 (1994); Banner et al.
  • TNF- ⁇ and TNF- ⁇ only share 32% identity in primary sequence, the crystal structures of both TNFs reveals that each monomer consists of two anti- parallel ⁇ -pleated sheets with a jelly roll topology and that monomers interact with each other in a head-to-tail fashion to form a homotrimeric structure (Eck et al. , J. Biol. Chem. 267:2119-2122 (1992)). In addition, both TNF- ⁇ and TNF- ⁇ bind to TNF receptors with similar affinities, suggesting that TNF- ⁇ and TNF- ⁇ bind to the same site on the TNF receptor.
  • TNF-Rl The induction of the various cellular responses mediated by TNF is initiated by its interaction with two distinct cell surface receptors, an approximately 55 kDa receptor termed TNF-Rl and an approximately 75 kDa receptor termed TNF-R2.
  • TNF-Rs share the typical structure of cell surface receptors including extracellular, transmembrane and intracellular regions. The extracellular portions of both receptors are found naturally also as soluble TNF-binding proteins (Nophar et al.
  • TNF receptors are independently active in signaling TNF responses. Direct signaling by TNF-R2 has been observed in lymphoid cells in which TNF-RJ stimulates the proliferation of thymocytes and a murine cytotoxic T cell line CT6
  • TNF-Rl and TNF-R2 along with other members of the TNF receptor superfamily, e.g. CD40, have been shown to independently mediate the activation of the transcription factor NF- ⁇ B (Lenardo & Baltimore, Cell 58:227-229 (1989); Legreid et al , J. Biol. Chem.
  • NF- ⁇ B is a member of the Rel family of transcriptional activators that control the expression of a variety of important cellular and viral genes (Lenardo & Baltimore, supra, and Thanos and Maniatis, Cell 80:529-532 (1995)).
  • TNF-R2 also mediates the transcriptional induction of the granulocyte-macrophage colony stimulating factor (GM-CSF) gene (Miyatake et al , EMBO J.
  • GM-CSF granulocyte-macrophage colony stimulating factor
  • TNF- ⁇ membrane bound TNF receptors TNF-Rl (p55) and TNF-R2 (p75) (Banner, et al , supra (1993)). While the TNF- ⁇ and TNF-Rl complex signals a large number of TNF activities, such as cytotoxicity, manganese superoxide dismutase induction, fibroblast proliferation, and NF- ⁇ B induction, the TNF- ⁇ and TNF-R2 complex is involved in the proliferation of primary thymocytes and T cells (Schalaby et al , J. Exp. Med. 172: 1517-1520 (1990); Tartaglia et al. , Proc. Natl Acad.
  • NF- ⁇ B activation of NF- ⁇ B is an essential step for TNF- ⁇ synthesis in synovial macrophages and demonstrated that design of a potent and long acting TNF-Rl antagonists to block NF- ⁇ B pathway should be sufficient and effective for the treatment of inflammatory diseases, such as rheumatoid arthritis (RA) and inflammatory bowel disease (IBD).
  • RA rheumatoid arthritis
  • IBD inflammatory bowel disease
  • compositions that are capable of precisely targeting acute immune inflammatory responses without producing significant undesirable side effects.
  • the present invention provides methods, compounds and compositions for treating inflammatory diseases by inhibiting tumor necrosis factor activity.
  • the invention provides a compound of the formula:
  • W1W2W3W4W5 is an alicyclic, heterocyclic, or heteroaromatic ring, with the provisos that the ring is not fused with any other ring, and the ring is not a pyrrazole, dihydropyrrazole, or tetrahydropyrrazole derivative; the ring containing W7, W8 and W9 is alicyclic, heterocyclic, aromatic or heteroaromatic; and the W10W11W12W13W14 ring is alicyclic, heterocyclic, or heteroaromatic.
  • the bonds between any two adjacent W atoms can be either single, double or aromatic bonds (valence permitting); W6 is not part of a ring; the W9-W10 bond is not part of a ring; W2, W7, W9, and W10 are each independently either N, C, or C with one substituent group (valence permitting); WI, W4, W5, and Wl l are each independently either SOx (where x is 1 or 2), N, C, or C with one substituent group (valence permitting); and W3, W6, W8, W12, W13, and W14 are each independently either O, S, SO, SO 2 , N, C, C with one substituent group (either single or double bonded), N one substituent group (single bonded), or PO(OR).
  • An additional aspect of the invention provides a related group of compounds which have proven particularly beneficial for treating inflammatory diseases by inhibiting tumor necrosis factor activity.
  • Such group comprises compounds of the formula: wherein
  • W1W2W3W4W5 is an aliphatic, heterocyclic, or heteroaromatic ring, with the provisos that:
  • R2 and R3 are independently any alicyclic, heterocyclic, aromatic, or heteroaromatic ring structure
  • W2 is O, S, NR11 , CR12R13, CR14, SO, or SO 2 , (valence permitting),
  • W3 is O, S, NR15, CR16R17, CR18, SO, or SO 2 , (valence permitting),
  • W4 is O, S, NR19, CR20R21, CR22, SO, or SO 2 , (valence permitting), W5 is N, C, or CR36 (valence permitting)
  • W2 is CR23 or NR24 where R23 or R24 is a five membered ring (alicyclic, heterocyclic, or heteroaromatic), then
  • WI is O, S, NR25, CR26R27, CR28, SO, or SO 2 , (valence permitting)
  • W3 is O, S, NR29, CR30R31 , CR32, SO, or SO 2 , (valence permitting) ,
  • W4 is O, S, NR33, CR34R35, CR36, SO, or SO 2 , (valence permitting),
  • W5 is N, C, or CR37 (valence permitting), If the ring W1W2W3W4W5 is neither 3-oxotetrahydrothiophene nor furan, or if the ring W1W2W3W4W5 is furan and Rl is not any of the groups in Exhibit A, then
  • W2 can be CR23 or NR24 where R23 or R24 is a six membered ring (alicyclic, heterocyclic, aromatic or heteroaromatic); in addition,
  • WI is O, S, NR25, CR26R27, CR28, SO, or SO 2 , (valence permitting) ,
  • W3 is O, S, NR29, CR30R31, CR32, SO, or SO 2 , (valence permitting)
  • W4 is O, S, NR33, CR34R35, CR36, SO, or SO 2 , (valence permitting) ,
  • W5 is N, C, or CR37 (valence permitting), Rl is independently H, heterocyclic, aromatic, heteroaromatic, small alkyl or cycloalkyl, optionally substimted with OH, O-alkyl, S-alkyl, hydroxy alkyl, CONH 2 , CONH-alkyl, OCF 3 , CON-dialkyl, halo, CF 3 , sulfonamide, phosphonamide, phosphonate ester, SO-alkyl, SO 2 -alkyl, O-aryl, S-aryl, SO-aryl, SO 2 -aryl, COO-alkyl, CONH-aryl, acyloxy, acylamino, alkylsulfonylamino, or arylsulfonylamino;
  • R3, R7 through Rl l, R15, R19, R24, R25, R29, R33 are each independently, H, heterocyclic, aromatic, heteroaromatic, small alkyl or cycloalkyl, optionally substimted with OH, O-alkyl, S-alkyl, CONH 2 , CONH-alkyl, CON-dialkyl, F, CF 3 , OCF 3 , sulfonamide, phosphonamide, or phosphonate ester; and
  • R2, R4 through R6, R12 through R14, R16 through R18, R20 through R23, R26 through R28, R30 through R32, and R34 through R37 are each independently H, halogen, OH, NH 2 , or O-alkyl, OCF 3 , O-cycloalkyl, heterocyclic, aromatic, heteroaromatic, small alkyl or cycloalkyl, optionally substimted with OH, O-alkyl, S-alkyl, SO-alkyl, SO 2 -alkyl, CONH 2 , CONH-alkyl, CON-dialkyl, F, CF 3 , sulfonamide, phosphonamide, phosphonate ester.
  • a further aspect of the invention provides a method for treating inflammatory diseases by inhibiting tumor necrosis factor activity comprising administering a compound of the formula:
  • W8 is CR16
  • W9 is CR17
  • W10 is N or CR18
  • Wl l is CH, N, CCH 3 , CF, CCH 2 CH 3 , or CC1
  • W12 is O, S, NR19 or CR20R21
  • O-cycloalkyl heterocyclic, aromatic, heteroaromatic, small alkyl or cycloalkyl, optionally substimted with OH, O-alkyl, S-alkyl, SO-alkyl, SO 2 -alkyl, CONH 2 , CONH-alkyl, CON-dialkyl, F, CF 3 , OCF 3 , sulfonamide, phosphonamide, or phosphonate ester; together with a pharmaceutically acceptable carrier to a patient in need of such treatment.
  • Such compounds and compositions will be found suitable for use as specific inhibitors of TNF-dependent NF- ⁇ B activation signaled by certain members of the TNF receptor superfamily for the prophylaxis and treatment of inflammatory diseases.
  • the present invention provides methods, compounds and compositions for treating inflammatory diseases by inhibiting tumor necrosis factor activity.
  • TNF shall include both tumor necrosis factor- ⁇ (TNF- ⁇ ) and tumor necrosis factor- ⁇ (TNF- ⁇ ), from animals or humans, together with naturally occurring alleles thereof.
  • TNF- ⁇ is described by Pennica et al. , Nature 312:721 (1984).
  • TNF- ⁇ is described by Gray et al , Nature 312:724 (1984).
  • TNF receptor antagonists are TNF receptor antagonists. These substances function by competing with native TNF for the cell surface receptor to which TNF binds and blocks the inflammatory events (hereinafter termed competitive antagonists). TNF receptor antagonists are useful, either alone or together with other therapeutic compositions, in the treatment of inflammatory responses.
  • the invention provides a compound of the formula:
  • W1W2W3W4W5 is an alicyclic, heterocyclic, or heteroaromatic ring, with the provisos that the ring is not fused with any other ring, and the ring is not a pyrrazole, dihydropyrrazole, or tetrahydropyrrazole derivative; the ring containing W7, W8 and W9 is alicyclic, heterocyclic, aromatic or heteroaromatic; and the
  • W10W11W12W13W14 ring is alicyclic, heterocyclic, or heteroaromatic.
  • the bonds between any two adjacent W atoms can be either single, double or aromatic bonds (valence permitting);
  • W6 is not part of a ring;
  • the W9-W10 bond is not part of a ring;
  • W2, W7, W9, and W 10 are each independently either N, C, or C with one substiment group (valence permitting);
  • WI , W4, W5, and Wl l are each independently either SOx (where x is 1 or 2), N, C, or C with one substiment group (valence permitting);
  • W3 , W6, W8, W12, W13 , and W14 are each independently either O, S, SO, S0 2 , N.
  • the invention provides a related group of compounds which have proven particularly beneficial for treating inflammatory diseases by inhibiting mmor necrosis factor activity.
  • Such group comprises compounds of the formula:
  • W1W2W3W4W5 is an aliphatic, heterocyclic, or heteroaromatic ring, with the provisos that:
  • R2 and R3 are independently any alicyclic, heterocyclic, aromatic, or heteroaromatic ring structure
  • W2 is O, S, NR11 , CR12R13, CR14, SO, or SO 2 , (valence permitting)
  • W3 is O, S, NR15, CR16R17, CR18, SO, or SO 2 , (valence permitting) ,
  • W4 is O, S, NR19, CR20R21 , CR22, SO, or SO 2 , (valence permitting),
  • W5 is N, C, or CR36 (valence permitting) If W2 is CR23 or NR24 where R23 or RJ4 is a five membered ring (alicyclic. heterocyclic, or heteroaromatic), then
  • WI is O, S, NR25, CR26R27, CR28, SO, or SO 2 , (valence permitting),
  • W3 is O, S, NR29, CR30R31 , CR32, SO, or SO 2 , (valence permitting),
  • W4 is O, S, NR33, CR34R35, CR36, SO, or SO 2 , (valence permitting),
  • W5 is N, C, or CR37 (valence permitting), If the ring W1W2W3W4W5 is neither 3-oxotetrahydrothiophene nor furan, or if the ring W1W2W3W4W5 is furan and Rl is not any of the groups in Exhibit A, then
  • W2 can be CR23 or NR24 where R23 or R24 is a six membered ring (alicyclic, heterocyclic, aromatic or heteroaromatic); in addition, WI is O, S, NR25, CR26R27, CR28, SO, or SO 2 , (valence permitting),
  • W3 is O, S, NR29, CR30R31, CR32, SO, or SO 2 , (valence permitting),
  • W4 is O, S, NR33, CR34R35, CR36, SO, or SO 2 , (valence permitting),
  • W5 is N, C, or CR37 (valence permitting), Rl is independently H, heterocyclic, aromatic, heteroaromatic, small alkyl or cycloalkyl, optionally substimted with OH, O-alkyl, S-alkyl, hydroxy alkyl, CONH 2 , CONH-alkyl, OCF 3 , CON-dialkyl, halo, CF 3 , sulfonamide, phosphonamide, ' phosphonate ester, SO-alkyl, SO 2 -alkyl, O-aryl, S-aryl, SO-aryl, SO 2 -aryl, COO-alkyl, CONH-aryl, acyloxy, acylamino, alkylsulfonylamino, or arylsulfonylamino;
  • R15. R19, RJ4, R25, R29, R33 are each independently, H, heterocyclic, aromatic, heteroaromatic, small alkyl or cycloalkyl, optionally substimted with OH, O-alkyl, S-alkyl, CONH 2 , CONH-alkyl, CON-dialkyl, F, CF 3 , OCF 3 , sulfonamide, phosphonamide, or phosphonate ester; and R2, R4 through R6, R12 thorugh R14, R16 through R18, R20 through R23, R26 through R28, R30 through R32, and R34 through R37 are each independently H, halogen, OH, NH 2 , or O-alkyl, OCF 3 , O-cycloalkyl, heterocyclic, aromatic, heteroaromatic, small alkyl or cycloalkyl, optionally substimted with OH, O-alkyl, S-alkyl
  • An additional aspect of the invention provides a method for treating inflammatory diseases by inhibiting mmor necrosis factor activity comprising administering a compound of the formula:
  • an alternative group of related compounds will also find use in the present method for treating inflammatory diseases by inhibiting mmor necrosis factor activity.
  • This group of inhibitors includes compounds of the formula:
  • R3 is H or short alkyl or cycloalkyl
  • Rl is H, or straight or branched alkyl (C1-C10) optionally substimted by:
  • COOR4 (where R4 is H, short alkyl, cycloalkyl, branched alkyl), an aromatic or heteroaromatic ring, or by aryloxy; or alkenyl, especially 2-propenyl; or aromatic or heteroaromatic ring-especially furan, optionally substimted by alkyl, hydroxy or alkoxy;
  • R6 is aromatic or heteroaromatic (especially phenyl, furyl, thiophenyl, pyrrolyl, thiazolyl) optionally substimted by short alkyl; nitro; alkoxy (including multiple alkoxy); aryloxy optionally substimted by alkoxy or alkyl; aromatic or heteroaromatic (especially thiophenyl) optionally substimted by halo, trifluoromethyl, trifluoromethoxy, alkoxy, alkyl, COORl l (where Rll is H, short alkyl, cycloalkyl, branched alkyl) or optionally fused to a 5 membered carboxyclic or heterocyclic ring; together with a pharmaceutically acceptable carrier to a patient in need of such treatment.
  • aromatic or heteroaromatic especially phenyl, furyl, thiophenyl, pyrrolyl, thiazolyl
  • R6 is aromatic or heteroaromatic (especially phenyl,
  • the present inhibitor compounds will be recognized as bearing a strucmral relationship to thiazolidmedione and 2-thiazolidinedione compounds, although a wide variation of the atomic components of the strucmres will be expected to preserve the TNF receptor antagonist activity.
  • the compounds of the present invention can be prepared in accordance with chemical synthetic protocols well known to those of skill in this art.
  • One desirable category of such techniques is know by the generic term "combinatorial chemistry.”
  • Such techniques are well know in the art, and can be generally summarized as follows:
  • preparation of libraries can be by the "split synthesis" method, as described in Gallop et al , J. Med. Chem. , 37: 1233-1251 (1994).
  • the split synthesis procedure involves dividing a resin support into n equal fractions, in a separate reaction carry out a single reaction to each aliquot, and then thoroughly mixing all the resin particles together. Repeating the protocol for a total of x cycles can produce a stochastic collection of up to n x different compounds.
  • An alternative format is by preparing sublibraries in the O 3 O 2 X, format, wherein two positions on the compounds, O 3 and O 2 are explicitly defined and a third position, X,, varies.
  • Such sublibraries can be conveniently prepared by the tea-bag technique, as is known in the art, and described, for example in U.S. Pat. No. 4,631,211 and Houghten et al, Proc. Natl. Acad. Sci. , 82:5131-5135 (1985).
  • the iterative selection and enhancement process of screening and sublibrary resynthesis can be employed.
  • a sublibrary of various Rl substituents can be screened to select the most active Rl substiment.
  • the compound having the most active Rl is then resynthesized and with the Rl position being defined, a new RJ position mixture library is prepared, screened, and the most active R2 selected.
  • the above process can then be repeated to identify R3 and the other most active R substituents on the W1W2W3W4W5 ring.
  • the positional scanning technique only a single position is defined in a given sublibrary and the most preferred substiment at each position of the compound is identified.
  • SCLs synthetic combinatorial libraries
  • Solution phase combinatorial chemistry methods can be used when the product can be separated from side products and starting materials through rapid techniques. Examples of these are: (1) selective precipitation of product and removal of byproducts and precursors by washing, (2) selective removal of side products and starting materials using chemically reactive polymers and/or ion exchange polymers ("scavenge"), (3) selective binding of product to a chemically reactive polymer, followed by removal of the product through a second chemical reaction (“capture”) (4) selective binding of product to an ion exchange polymer, followed by removal with acid, base, or high salt buffer (“capture”), and (5) selective solubilization of product.
  • Solution phase combinatorial chemistry approaches are covered in a recent set of reviews (Tetrahedron, 54:3955-4150 (1998)).
  • the synthetic approaches described in examples 1-20 can be optimally carried out using solution phase combinatorial chemistry.
  • Several reactions are carried out simultaneously using a multiple reaction vessel block such as, but not limited to, the Charybdis CalypsoTM temperature controlled blocks, with gas manifolds to maintain an argon or nitrogen atmosphere.
  • the reactions can be carried out simultaneously in multiple vials filled with argon or nitrogen and fitted with magnetic stirbars and polytetrafluoroethylene-lined, sealed caps, by heating and stirring them simultaneously in a magnetic stirrer/heater such as, but not limited to, the Pierce
  • ReactThermTM III Heating/Stirring Module The products are isolated by addition of water and filtration using a system such as, but not limited to, the Charybdis CalypsoTM filtration block or polypropylene syringes fitted with filter disks made from polyethylene, polytetrafluoroethylene, or glass and attached to a vacuum manifold.
  • a system such as, but not limited to, the Charybdis CalypsoTM filtration block or polypropylene syringes fitted with filter disks made from polyethylene, polytetrafluoroethylene, or glass and attached to a vacuum manifold.
  • reaction scheme I a heterocycle containing a chemically reactive methylene group in an inert solvent such as dimethylacetamide is treated with an appropriately-substimted aldehyde in the presence of a catalyst such as acetic acid plus sodium acetate, piper idine, or ammonium hydroxide.
  • a catalyst such as acetic acid plus sodium acetate, piper idine, or ammonium hydroxide.
  • Rl is any alkyl or aryl group
  • R2 and R3 are O or no group, (for W4 and W5, see "broadly defined claim for TNF-active compounds", valence permitting).
  • dppf is taken to mean l J'bis(diphenylphosphino)ferrocene, and W1-W5, is as previously defined.
  • a 5-membered ring aromatic halide (XI is Br or I) is converted to the corresponding boronic pinacolate ester in an inert solvent such as DMSO or DMF, and then coupled to a monoalkyamine or monoarylamine (R1NH 2 ) in toluene (dppf and W1-W5 are as previously defined).
  • Thee are numerous assays available to routinely identify compounds which display activity as TNF receptor antagonists.
  • One assay technique which has been found particularly useful is the Eu 3+ labeling of TNF- ⁇ using a Eu 3+ -chelate of DTTA, which has demonstrated specific binding to TNF-Rl .
  • Eu-labeling reagents are commercially available to link the Eu-chelate covalently to either a free amino group or a sulfhydryl group on the protein.
  • six lysines are exposed on the protein surface, whereas two cysteine residues form an intra-disulfide bond, which leaves no free-cysteine accessible for chemical modification.
  • TNF- ⁇ , TNF- ⁇ , and anti-human TNF-Rl antibodies are able to compete with Eu 3+ - labeled TNF- ⁇ for binding to the receptor.
  • highly sensitive, non- radioactive probes are found very useful for high throughput screening of potential small molecule TNF-Rl antagonists.
  • the screening assay is performed generally as follows: Eu 3+ -chelate of N 1 -(p-isothiocyanatobenzyl)-diethylenetriamine-N 1 , N 2 , N 3 , N 3 - tetraacetic acid (DTTA; Wallac, Gathersburg, MD) is used to prepare [Eu 3+ ]TNF- ⁇ . lOO ⁇ g lyophilized TNF- ⁇ is resuspended in lOO ⁇ L of labeling buffer (50mM NaHCO 3 , pH 8.5, containing 0.9% NaCl). [Eu 3+ ]-DTTA (50 ⁇ g) is then added to TNF- ⁇ in the labeling buffer. The reaction is carried out at 4°C for 48 hours.
  • DTTA Wallac, Gathersburg, MD
  • the sample is then diluted 2-fold into 50mM Tris buffer (pH 7.8) containing 0.9% NaCl and 0.05 % NaN 3 , and dialyzed against 1 liter 50mM Tris buffer to remove free Eu 3+ -DTTA label.
  • the protein concentration is determined by the Bradford method (Bradford, 1976) and the specific activity is calculated using a europium standard solution (Wallac).
  • the ligand binding assay is performed as follows: 96-well plates are coated with lOng of TNF receptor in 50mM NaHCO 3 (pH 9.6) overnight at 4°C. Plates are then blocked with 0.2% BSA in PBS buffer, washed once with binding buffer (0.2 BSA/PBS/0.1 % Tween-20), and incubated with Eu 3+ -labeled TNF- ⁇ and the test compound for one and a half-hours at room temperature. The plates are then washed three times with DELFIA Wash Solution (Wallac) and lOO ⁇ l of DELFIA Enhancement Solution (Wallace) is added. The plate is placed on a plate shaker for 10 min before reading using a Victor Flurometer 1420 (Wallac).
  • the europium counting protocol is used with a 320nm excitation pulse at a frequency of 1000 s '1 and detection at 615nm (emission wavelength). Fluorescence signal is measured after a delay of 400 ⁇ sec between each excitation pulse. Non-specific binding is defined using TNF- ⁇ with 500- fold excess of [Eu 3+ ]TNF- ⁇ . Each experimental point is carried out in duplicate.
  • the data ligand receptor interaction data is analyzed using Prizm (GraphPad Software).
  • Ligand binding data are analyzed by non-linear least-square regression. Saturation data are fitted to a rectangular hyperbola model and competition data are fitted to a sigmoidal curve with a variable slope.
  • Inhibition constants (Ki) are determined from IC 50 's using the Chang-Prusoff equation (Cheng, Y.C. and W.H. Prusoff, Biochem. Pharmacol. 22:3099-3108 (1973)).
  • [Eu 3+ ]TNF- ⁇ could be displaced by an anti-human TNF-Rl neutralizing monoclonal antibody, as well as unlabeled TNF- ⁇ and TNF- ⁇ , suggesting that this antibody neutralizes TNF-Rl -mediated bioactivities by blocking TNF- ⁇ or TNF- ⁇ binding to TNF-Rl.
  • TNF- ⁇ is know to cause rapid degradation of I ⁇ B ⁇ , with a concomitant translocation of NF- ⁇ B from the cytoplasm to the nucleus in MRC-5 cells (a human embryonal lung cell line (ATCC CCL-171)).
  • MRC-5 cells are incubated at a density of 5x10" cells per chamber in culture medium (Eagle's MEM with 2mM L-glutamine and Earle's BSS adjusted to contain 1.5g/L sodium bicarbonate, O.lmM non-essential amino acid, l.OmM sodium'pyruvate, 10% fetal bovine serum (FBS)) on Fisher culture slides overnight at 37°C and 5 % CO 2 .
  • culture medium Eagle's MEM with 2mM L-glutamine and Earle's BSS adjusted to contain 1.5g/L sodium bicarbonate, O.lmM non-essential amino acid, l.OmM sodium'pyruvate, 10% fetal bovine serum (FBS)
  • the chambers are washed twice with PBS.
  • the cells are fixed by incubating in ice cold methanol for five minutes and allowed to air dry.
  • the cells are then washed three times with PBS.
  • the specimen is incubated with 10% FBS in PBS for 20 minutes to suppress non-specific binding of IgG, then washed once with PBS.
  • the chambers are then incubated with goat anti-human NF- ⁇ B p65 (Santa Cruz
  • Inflammatory or immune-potentiated inflammatory events to be treated with the present TNF receptor antagonists are characterized by the presence of a humoral and/or cellular response directed against an undesired foreign or self target tissue or by uncertain etiology.
  • immune potentiating inflammatory events are characterized by antibodies directed against host tissue by way of an aberrant host response, host antibodies against grafted tissue, or antibodies of graft origin directed against host tissue.
  • Such events also are characterized by infiltration of polymorphonuclear neutrophils and mononuclear leukocytes into the target tissue, and subsequent induction of pain, localized edema, possible vascular endothelial injury and excessive production of cytokines by stimulated cells.
  • TNF receptor antagonist will be a function of many variables, including the affinity of the antagonist for the TNF receptor, any residual cytotoxic activity exhibited by competitive antagonists, the route of administration, the clinical condition of the patient (including the desirability of maintaining a non-toxic level of endogenous TNF activity), and whether the receptor antagonist is to be used for the prophylaxis or for the treatment of acute response episodes.
  • the molar dose of TNF receptor antagonist will be expected to vary about from 0.001 to 10 times the maximum tolerated molar dose of TNF- ⁇ , although as noted above this range will be subject to a great deal of therapeutic discretion. It is to be expected that concentrations of TNF localized at the sites of inflammation may exceed the whole body maximum therapeutic dose. Assay of the TNF concentration in inflammatory infiltrates will provide guidance as to the amount of TNF receptor antagonist to be employed, particularly if localized administration is practical, e.g. in Crohn's disease (suppositories) or arthritis (injections into synovial fluid).
  • TNF- ⁇ Similar dosages and considerations apply in the case of TNF- ⁇ .
  • the key factor in selecting an appropriate dose is the result obtained: If the patient's inflammatory response does not at least partially resolve within about 48 hours after administration, the dose should be gradually elevated until the desired effect is achieved. Also, relatively higher doses will be initially needed for the treatment for acute rejection or inflammatory episodes, i.e. , for patients in acute organ transplant rejection or undergoing arthritic flares.
  • the therapeutic preparation will be administered to a patient in need of anti-inflammatory treatment at a therapeutically effective dosage level.
  • the lowest effective dosage levels can be determined routinely by initiating treatment at higher dosage levels and reducing the dosage level until relief from inflammatory reaction is no longer obtained.
  • therapeutic dosage levels will range from about 0.01-100 g/kg of host body weight.
  • the present TNF receptor antagonist can also administered in conjunction with other anti-inflammatory agents used in or proposed for the treatment of individual immuno inflammatory conditions as appropriate, e.g. gold salts, cyclosporin antibiotics, salicylate and corticosteroids (such as methylprednisolone).
  • anti-inflammatory agents used in or proposed for the treatment of individual immuno inflammatory conditions as appropriate, e.g. gold salts, cyclosporin antibiotics, salicylate and corticosteroids (such as methylprednisolone).
  • these agents may be employed in lesser dosages than when used alone.
  • the present invention contemplates combinations as simple mixtures as well as chemical hybrids.
  • One example of the latter is where the present compound is covalently linked to a pharmaceutical such as a corticosteroid, or where two or more compounds are joined.
  • covalent binding of the distinct chemical moieties can be accomplished by any one of many commercially available cross-linking compounds.
  • the TNF receptor antagonist should be intravenously infused or introduced at the inflammatory lesion immediately upon the development of symptoms or serological evidence of TNF activity.
  • prophylaxis is suitably accomplished by intramuscular or subcutaneous administration.
  • the compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to injection may also be prepared.
  • These therapeutic preparations can be administered to mammals for veterinary use, such as with domestic animals, and clinical use in humans in a manner similar to other therapeutic agents.
  • the dosage required for therapeutic efficacy will vary according to the type of use and mode of administration, as well as the particularized requirements of individual hosts.
  • compositions can be provided together with physiologically tolerable liquid, gel or solid carriers, diluents, adjuvants and excipients.
  • Such compositions are typically prepared as sprays (e.g. intranasal aerosols) for topical use. However, they may also be prepared either as liquid solutions or suspensions, or in solid forms including respirable and nonrespirable dry powders.
  • Oral formulations e.g.
  • compositions usually include such normally employed additives such as binders, fillers, carriers, preservatives, stabilizing agents, emulsifiers, buffers and excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like.
  • binders such as binders, fillers, carriers, preservatives, stabilizing agents, emulsifiers, buffers and excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like.
  • binders such as binders, fillers, carriers, preservatives, stabilizing agents, emulsifiers, buffers and excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like.
  • the compounds of the present invention are often mixed with diluents or excipients which are physiologically tolerable and compatible.
  • Suitable diluents and excipients are, for example, water, saline, dextrose, glycerol, or the like, and combinations thereof.
  • the compositions may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, stabilizing or pH buffering agents.
  • Additional formulations which are suitable for other modes of administration, such as topical administration, include salves, tinctures, creams, lotions, and, in some cases, suppositories.
  • traditional binders, carriers and excipients may include, for example, polyalkylene glycols or triglycerides.
  • the mixmre is stirred under argon at 90 °C for 40 hours, cooled to ambient temperature, and treated with 20mL water to precipitate the product.
  • the mixmre is filtered and the residue washed with lOOmL water, air dried, and dried under high vacuum to yield 576 mg of the title product (82 % based on 3-(phenylmethoxy)benzaldehyde).
  • the resin is washed 3 times with 25mL Dioxane, 3 times with 25mL dioxone/H 2 O, 6 times with 25mL H 2 O, 3 times with 25mL EtOH, 3 times with 25mL MeOH, and dried under high vacuum.
  • the resin is then treated with a 1/1 mixmre of dioxane and IM HCl [concentrated] for 48 hours at 25 °C.
  • the resin is again washed with 3 times with 25mL Dioxane, 3 times with 25mL H 2 O, 3 times with 25mL Dioxane, 3 times with 25mL Acetone, 3 times with 25mL EtOH, 3 times with 25mL
  • the resin is washed 2 times with 25mL pyridine, 3 times with 25 mL pyridine/H 2 O, 6 times with 25mL H 2 O, 3 times with 25mL EtOH, 3 times with 25mL MeOH, and dried in vacuo to yield the title compound.
  • the resin mix is cooled, then filtered and washed 3 times with 25mL THF, 3 times with 25mL Dioxane, 3 times with 25mL MeOH, 3 times with 25mL Dioxane, 3 times with 25mL MeOH, and dried in vacuo.
  • the product resin (lg) is then treated with a solution of 8mL cone. HCl in 8mL Dioxane for 3 hours at 25°C. To this solution is added lOmL of dichloromethane and ImL H 2 O. The filtrate is collected and the resin is washed 3 times with 2mL of dichloromethane followed by 5mL of H 2 O. The resulting filtrates are all combined and the organic layers separated.
  • the aqueous layer is extracted with dichloromethane, the organic layers are combined, dried with sodium sulfate, and evaporated to give an oily residue which is dissolved in ether then extracted with saturated KHCO 3 .
  • the aqueous layer is acidified with HCl and extracted with DCM dried and evaporated to yield 41mg of the title product (30% based on the resin from Example 41).
  • the resin is washed 2 times with 15mL of trimethylorthoformate, 3 times with 15mL of pyridine, 3 times with 15mL of DMF, 3 times with 15mL of dichloromethane, 3 times with 15mL of MeOH, 2 times with 15mL of dichloromethane, 3 times with 15mL of
  • the first fraction is collected and evaporated to give 360mg of a yellow/green oil, which is used without further purification.
  • reaction mixmre is stirred for 18 hours while allowing it to warm to 25 °C.
  • the solvent is evaporated, the residue is dissolved in ether, filtered, and washed twice with aqueous potassium carbonate.
  • the ether layer is dried with magnesium sulfate, filtered, stripped of solvent and partially purified on silica gel using hexane/ethyl acetate to yield 230mg of crude product, containing 40% triphenylphosphine oxide.
  • the product is used for subsequent steps without further purification.
  • 2,5-thiophenedicarboxaldehyde (541mg, 3.86mmol) and hydroxylamine hydrochloride (402.34mg, 5.789mmol) are dissolved in 12mL of 1: 1 pyridine/ 1-butanol and heated to 90 °C for 28 hours. The solvent is removed by warming under a stream of argon followed by high vacuum. The crude product is diluted to a volume of 4mL with dimethylacetamide to produce a solution containing 0.48mol/liter of the title compound. This solution is used without purification in subsequent steps.
  • the resin is washed 2 times with 15mL of trimethylorthoformate, 3 times with 15mL of pyridine, 3 times with 15mL of D lF, 3 times with 15mL of dichloromethane, 3 times with 15mL of MEOH, 2 times with 15mL of dichloromethane, 3 times with 15mL of MeOH and dried in vacuo.
  • the resin is treated with Dioxane/IM HCl (cone.) (1/1) for 48 hours, then diluted with H 2 0, filtered and extracted with DCM (15mL x 3). The filtrates are combined and neutralized with potassium hydrogen carbonate.
  • the organic layer is isolated, dried with sodium sulfate, and evaporated in vacuo to give the title product
  • purification with reverse phase HPLC on octadecylsilica is carried out using a gradient from 5 to 95 % acetonitrile in water containing 0.1 % trifluoroacetic acid.
  • purification with reverse phase HPLC on octadecylsilica is carried out using a gradient from 5 to 95 % acetonitrile in water containing 0J % trifluoroacetic acid.
  • Methyl (3-(5-formylfuryl)thiophene-2-carboxylate and N-allylrhodanine are combined to yield the title compound using the procedure in Example 4, with the exception that, if necessary to dissolve the precursors, additional solvent is added (1 : 1 acetic acid/dimethylacetamide containing 0.5M sodium acetate).
  • purification with reverse phase HPLC on octadecylsilica is carried out using a gradient from 5 to 95 % acetonitrile in water containing 0.1 % trifluoroacetic acid.
  • purification with reverse phase HPLC on octadecylsilica is carried out using a gradient from 5 to 95 % acetonitrile in water containing 0.1 % trifluoroacetic acid.
  • purification with reverse phase HPLC on octadecylsilica is carried out using a gradient from 5 to 95 % acetonitrile in water containing OJ % trifluoroacetic acid.
  • purification with reverse phase HPLC on octadecylsilica is carried out using a gradient from 5 to 95 % acetonitrile in water containing 0.1 % trifluoroacetic acid.
  • Methyl (3-(5-formylfuryl)thiophene-2-carboxylate and rhodanine are combined to yield the title compound using the procedure in Example 4, with the exception that, if necessary to dissolve the precursors, additional solvent is added (1: 1 acetic acid/dimethylacetamide containing 0.5M sodium acetate).
  • purification with reverse phase HPLC on octadecylsilica is carried out using a gradient from 5 to 95 % acetonitrile in water containing OJ % trifluoroacetic acid.
  • purification with reverse phase HPLC on octadecylsilica is carried out using a gradient from 5 to 95 % acetonitrile in water containing 0.1 % trifluoroacetic acid.
  • purification with reverse phase HPLC on octadecylsilica is carried out using a gradient from 5 to 95 % acetonitrile in water containing OJ % trifluoroacetic acid.
  • purification with reverse phase HPLC on octadecylsilica is carried out using a gradient from 5 to 95% acetonitrile in water containing 0.1 % trifluoroacetic acid.
  • purification with reverse phase HPLC on octadecylsilica is carried out using a gradient from 5 to 95% acetonitrile in water containing 0.1 % trifluoroacetic acid.
  • purification with reverse phase HPLC on octadecylsilica is carried out using a gradient from 5 to 95% acetonitrile in water containing 0.1 % trifluoroacetic acid.
  • purification with reverse phase HPLC on octadecylsilica is carried out using a gradient from 5 to 95 % acetonitrile in water containing OJ % trifluoroacetic acid.
  • purification with reverse phase HPLC on octadecylsilica is carried out using a gradient from 5 to 95 % acetonitrile in water containing OJ % trifluoroacetic acid.
  • purification with reverse phase HPLC on octadecylsilica is carried out using a gradient from 5 to 95% acetonitrile in water containing OJ % trifluoroacetic acid.
  • purification with reverse phase HPLC on octadecylsilica is carried out using a gradient from 5 to 95% acetonitrile in water containing OJ % trifluoroacetic acid.
  • purification with reverse phase HPLC on octadecylsilica is carried out using a gradient from 5 to 95 % acetonitrile in water containing 0.1 % trifluoroacetic acid.
  • purification with reverse phase HPLC on octadecylsilica is carried out using a gradient from 5 to 95 % acetonitrile in water containing OJ % trifluoroacetic acid.
  • purification with reverse phase HPLC on octadecylsilica is carried out using a gradient from 5 to 95 % acetonitrile in water containing 0.1 % trifluoroacetic acid.
  • purification with reverse phase HPLC on octadecylsilica is carried out using a gradient from 5 to 95% acetonitrile in water containing 0J % trifluoroacetic acid.
  • Example 47 The product from Example 47 and the product from Example 49 are combined to yield the title compound using the procedure in Example 4, with the exception that, if necessary to dissolve the precursors, additional solvent is added (1: 1 acetic acid/dimethylacetamide containing 0.5M sodium acetate).
  • purification with reverse phase HPLC on octadecylsilica is carried out using a gradient from 5 to 95% acetonitrile in water containing OJ % trifluoroacetic acid.
  • Example 50 The product from Example 50 and 5-(3-nitrophenyl)furan-2-carbaldehyde are combined to yield the title compound using the procedure in Example 4, with the exception that, if necessary to dissolve the precursors, additional solvent is added (1: 1 acetic acid/dimethylacetamide containing 0.5M sodium acetate).
  • purification with reverse phase HPLC on octadecylsilica is carried out using a gradient from 5 to 95 % acetonitrile in water containing 0.1 % trifluoroacetic acid.
  • Example 47 The product from Example 47 and N-ethylrhodanine are combined to yield the title compound using the procedure in Example 4, with the exception that, if necessary to dissolve the precursors, additional solvent is added (1: 1 acetic acid/dimethylacetamide containing 0.5M sodium acetate).
  • purification with reverse phase HPLC on octadecylsilica is carried out using a gradient from 5 to 95 % acetonitrile in water containing 0.1 % trifluoroacetic acid.
  • Example 50 The product from Example 50 and the product from Example 52 are combined to yield the title compound using the procedure in Example 4, with the exception that, if necessary to dissolve the precursors, additional solvent is added (1 : 1 acetic acid/dimethylacetamide containing 0.5M sodium acetate).
  • purification with reverse phase HPLC on octadecylsilica is carried out using a gradient from 5 to 95% acetonitrile in water containing 0.1 % trifluoroacetic acid.
  • purification with reverse phase HPLC on octadecylsilica is carried out using a gradient from 5 to 95 % acetonitrile in water containing 0.1 % trifluoroacetic acid.
  • Example 55 (containing 0.64mmol of 5-Cyanothiophene-2-carboxaldehyde and 1- methylpiperazine (0.02mL, 0J8mmol) and heated at 75 degrees for 38. hours under argon while stirring. The product is precipitated with 50mL water, washed with 120mL water and 20mL diethyl ether, and dried in vacuo. Yield: 93 mg, 54% .
  • further purification is carried out with reverse phase HPLC on octadecylsilica is carried out using a gradient from 5 to 95 % acetonitrile in water containing 0.1 % trifluoroacetic acid.
  • Example 96 3-ethyl-5- ⁇ r5-(hydroxymethyl)(2-thienyl)1methylene ⁇ -2-thioxo-1.3-thiazolidin-4-one
  • Example 44 The product from Example 44 and N-ethylrhodanine are combined to yield the title compound using the procedure in Example 4, with the exception that, if necessary to dissolve the precursors, additional solvent is added (1: 1 acetic acid/dimethylacetamide containing 0.5M sodium acetate).
  • purification with reverse phase HPLC on octadecylsilica is carried out using a gradient from 5 to 95 % acetonitrile in water containing 0.1 % trifluoroacetic acid.
  • Example 99 2- ⁇ 5-[ ( 3-ethyl-4-oxo-2-thioxo-1.3-thiazolidin-5-ylidene ) methyl1-2-thienyl ⁇ acetic acid
  • Example 43 The product from Example 43 and N-ethylrhodanine are combined to yield the title compound using the procedure in ' Example 4, with the exception that, if necessary to dissolve the precursors, additional solvent is added (1 : 1 acetic acid/dimethylacetamide containing 0.5M sodium acetate).
  • Example 56 The product from Example 56 and N-ethylrhodanine are combined to yield the title compound using the procedure in Example 4, with the exception that, if necessary to dissolve the precursors, additional solvent is added (1 : 1 acetic acid/dimethylacetamide containing 0.5M sodium acetate).
  • purification with reverse phase HPLC on octadecylsilica is carried out using a gradient from 5 to 95 % acetonitrile in water containing 0.1 % trifluoroacetic acid.
  • Example 85 The product from Example 85 (58Jmg, OJlmmol) is combined with hydroxylamine hydrochloride (63Jmg, 0.91mmol) in pyridine (ImL) and stirred under argon at 25 °C for 48 hours. 125mL water and 75mL hexanes are added and filtered.
  • Example 105 3-ethyl-5-
  • purification with reverse phase HPLC on octadecylsilica is carried out using a gradient from 5 to 95 % acetonitrile in water containing 0.1 % trifluoroacetic acid.
  • purification with reverse phase HPLC on octadecylsilica is carried out using a gradient from 5 to 95 % acetonitrile in water containing 0J % trifluoroacetic acid.
  • Example 55 The product of Example 55 and N-methylrhodanine were combined to yield the title compound using the procedure in Example 4, with the exception that, if necessary to dissolve the precursors, additional solvent is added (1: 1 acetic acid/dimethylacetamide containing 0.5M sodium acetate).
  • purification with reverse phase HPLC on octadecylsilica is carried out using a gradient from 5 to 95 % acetonitrile in water containing 0.1 % trifluoroacetic acid.
  • N-ethylrhodanine (94 mg, 0.64mmol) is combined with 1.33mL of the product from Example 55 (contammg 0.64mmol of 5-Cyanothiophene-2-carboxaldehyde) and 1- methylpiperazine (0.02mL, 0J8mmol) and heated at 75 °C for 38 hours.
  • the product is precipitated with 50mL water, washed with 120mL water and 20mL diethyl ether, and dried in vacuo. Yield: 52 mg, 29% .
  • further purification is carried out with reverse phase HPLC on octadecylsilica is carried out using a gradient from 5 to 95 % acetonitrile in water containing 0.1 % trifluoroacetic acid.
  • N-allylrhodanine (l l lmg, O. ⁇ lmmol) is combined with 1.33mL of the product from Example 55 (containing 0.64mmol of 5-Cyanothiophene-2-carboxaldehyde) and
  • Example 47 The product from Example 47 and the product from Example 48 are combined to yield the title compound using the procedure in Example 4, with the exception that, if necessary to dissolve the precursors, additional solvent is added (1:1 acetic acid/dimethylacetamide containing 0.5M sodium acetate), and instead of precipitation, purification with reverse phase HPLC on octadecylsilica is carried out using a gradient from 5 to 95 % acetonifrile in water containing 0.1 % trifluoroacetic acid.
  • Example 111 The product from Example 47 and the product from Example 48 are combined to yield the title compound using the procedure in Example 4, with the exception that, if necessary to dissolve the precursors, additional solvent is added (1:1 acetic acid/dimethylacetamide containing 0.5M sodium acetate), and instead of precipitation, purification with reverse phase HPLC on octadecylsilica is carried out using a gradient from 5 to 95 % acetonifrile in water containing 0.1 % trifluor
  • Example 47 The product from Example 47 and the product from Example 51 are combined to yield the title compound using the procedure in Example 4, with the exception that, if necessary to dissolve the precursors, additional solvent is added (1 : 1 acetic acid/dimethylacetamide containing 0.5M sodium acetate), and instead of precipitation, purification with reverse phase HPLC on octadecylsilica is carried out using a gradient from 5 to 95 % acetonitrile in water containing 0.1 % trifluoroacetic acid.
  • Example 53 The product from Example 53 and the product from Example 48 are combined to yield the title compound using the procedure in Example 4, with the exception that, if necessary to dissolve the precursors, additional solvent is added (1:1 acetic acid/dimethylacetamide containing 0.5M sodium acetate), and instead of precipitation, purification with reverse phase HPLC on octadecylsilica is carried out using a gradient from 5 to 95% acetonitrile in water containing OJ % trifluoroacetic acid.
  • Example 4 with the exception that, if necessary to dissolve the precursors, additional solvent is added (1: 1 acetic acid/dimethylacetamide containing 0.5M sodium acetate), and instead of precipitation, purification with reverse phase HPLC on octadecylsilica is carried out using a gradient from 5 to 95 % acetonitrile in water containing 0.1 % trifluoroacetic acid.
  • Example 49 The product from Example 49 and 5-(l-naphthyl)furan-2-carbaldehyde are combined to yield the title compound using the procedure in Example 4, with the exception that, if necessary to dissolve the precursors, additional solvent is added (1: 1 acetic acid/dimethylacetamide containing 0.5M sodium acetate), and instead of precipitation, purification with reverse phase HPLC on octadecylsilica is carried out using a gradient from 5 to 95% acetonitrile in water containing OJ % trifluoroacetic acid.
  • Example 50 The product from Example 50 and 5-(l-naphthyl)furan-2-carbaldehyde are combined to yield the title compound using the procedure in Example 4, with the exception that, if necessary to dissolve the precursors, additional solvent is added (1 : 1 acetic acid/dimethylacetamide containing 0.5M sodium acetate), and instead of precipitation, purification with reverse phase HPLC on octadecylsilica is carried out using a gradient from 5 to 95% acetonitrile in water containing OJ % trifluoroacetic acid.
  • Example 118 to demonstrate their suitability for use as specific inhibitors of TNF-dependent NF- ⁇ B activation signaled by certain members of the TNF receptor superfamily for the prophylaxis and treatment of inflammatory diseases.
  • Example 118 to demonstrate their suitability for use as specific inhibitors of TNF-dependent NF- ⁇ B activation signaled by certain members of the TNF receptor superfamily for the prophylaxis and treatment of inflammatory diseases.
  • Example 117 The compounds synthesized in Examples 1-116, and those obtained as described in Example 117 are screened for their ability to bind to TNF receptor, in order to identify candidates with potential TNF inhibitory activity, generally according to the following protocol:
  • Eu 3+ -chelate of N'-(p-isothiocyanatobenzyl)-diethylenetriamine-N 1 , N 2 , N ⁇ N 3 - tetraacetic acid (DTTA; EG&G Wallac, Gathersburg, MD) is used to prepare [Eu 3+ ]TNF- ⁇ .
  • DTTA N'-(p-isothiocyanatobenzyl)-diethylenetriamine-N 1 , N 2 , N ⁇ N 3 - tetraacetic acid
  • the sample is then diluted 2-fold into 50mM Tris buffer (pH 7.8) containing 0.9% NaCl and 0.05% NaN 3 , and dialyzed against 1 liter 50mM Tris buffer to remove free Eu 3+ -DTTA label.
  • the protein concentration is determined by the Bradford method (Bradford, 1976) and the specific activity is calculated using a europium standard solution (Wallac).
  • the ligand binding assay is performed as follows: 96-well plates are coated with lOng of TNF receptor in 50mM NaHCO 3 (pH 9.6) overnight at 4°C. Plates are then blocked with 0.2% BSA in PBS buffer, washed once with binding buffer
  • the data ligand receptor interaction data is analyzed using Prizm (GraphPad Software). Ligand binding data are analyzed by non-linear least-square regression. Samration data are fitted to a rectangular hyperbola model and competition data are fitted to a sigmoidal curve with a variable slope. Inhibition constants (Ki) are determined from IC 50 values using the Chang-Prusoff equation (Cheng & Prusoff , 1973). In analyzing the data generated from testing the compounds of Examples 1-20, it is determined that each compound is capable of binding the TNF receptor Rl . Thus, each compound serves as a candidate for analysis in an assay of biological activity.
  • Example 116 and 117 The compounds synthesized ⁇ i Examples 1-20 and identified in Example 116 and 117 as being able to bind to TNF receptor are tested for their TNF inhibitory activity in a biological assay, generally according to the following protocol: MRC-5 cells (available as ATCC CCL-171 from the American Type Culture
  • the chambers are washed twice with PBS.
  • the cells are fixed by incubating in ice cold MeOH for five minutes and allowed to air dry.
  • the cells are then washed three times with PBS.
  • the specimen is incubated with 10% FBS in PBS for 20 minutes to suppress non-specific binding of IgG, then washed once with PBS.
  • the chambers are then incubated with goat anti-human NF- ⁇ B p65 (Santa Cruz Biotechnology, Inc.) at 1:500 dilution in PBS with 1.5 % FBS for 60 minutes.
  • the chambers are then washed three times with PBS for 5 minutes each, and incubated for
  • the following new compounds are proven to be useful compounds for preventing or treating inflammatory diseases by inhibiting tumor necrosis factor activity: 5-(5-methylfuran-2-ylmethylene)-2-thioxo-3-methyl-thiazolidin-4-one, 5-(5-methylfuran-2-ylmethylene)-2-thioxo-3-allyl-thiazolidin-4-one,

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Abstract

L'invention concerne des procédés et des compositions agissant en tant qu'inhibiteurs spécifiques de l'activation du NF-λB dépendante du TNF, dans laquelle certains membres de la superfamille de récepteurs du TNF jouent le rôle de signaux. Ces méthodes et compositions sont destinés à la prophylaxie et au traitement des maladies inflammatoires.
PCT/US1999/028856 1998-12-04 1999-12-06 Methodes et compositions destines au traitement de maladiesnflammatoires a base d'inhibiteurs de l'activite du facteur de necrose tumorale WO2000032598A1 (fr)

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Cited By (24)

* Cited by examiner, † Cited by third party
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WO2001030771A1 (fr) * 1999-10-28 2001-05-03 Kyowa Hakko Kogyo Co., Ltd. Derives de thiazolidinedione
WO2002032894A1 (fr) * 2000-10-18 2002-04-25 Ortho-Mcneil Pharmaceutical, Inc. Imidazoles substitues utilises dans le traitement de maladies inflammatoires
GB2387172A (en) * 2002-03-28 2003-10-08 Pantherix Ltd [(Aryl-/arylthio-)aryl]methylene substituted azole & azine derivatives and their therapeutic use as antibacterials
WO2004028441A2 (fr) * 2002-09-27 2004-04-08 L'oreal Compose heterocyclique pour stimuler ou induire la pousse des cheveux ou des cils et/ou freiner leur chute, composition le contenant, ses utilisations
WO2004043955A1 (fr) * 2002-11-13 2004-05-27 Rigel Pharmaceuticals, Inc. Derives de rhodanine et compositions pharmaceutiques les contenant
US6765013B2 (en) 1999-08-31 2004-07-20 Incyte San Diego Thiazolidinedione derivatives for the treatment of diabetes and other diseases
WO2005020990A1 (fr) * 2003-07-30 2005-03-10 Centre National De La Recherche Scientifique Thiazolidines antibiotiques
WO2005026127A1 (fr) * 2003-09-11 2005-03-24 Institute Of Medicinal Molecular Design. Inc. Inhibiteur d'inhibiteur-1 activateur de plasmogene
JP2005535593A (ja) * 2002-05-17 2005-11-24 キューエルティー インコーポレーティッド チアゾリジンジチオン誘導体の使用方法
WO2006024699A1 (fr) * 2004-08-30 2006-03-09 Karyon-Ctt Ltd Composes de thioxothiazolidinone utilises comme produits pharmaceutiques
US7071218B2 (en) 2001-11-15 2006-07-04 Incyte San Diego Incorporated N-substituted heterocycles for the treatment of hypercholesteremia, dyslipidemia and other metabolic disorders; cancer, and other diseases
US7102000B2 (en) 2002-03-08 2006-09-05 Incyte San Diego Inc. Heterocyclic amide derivatives for the treatment of diabetes and other diseases
US7153875B2 (en) 2001-03-07 2006-12-26 Incyte San Diego Heterocyclic derivatives for the treatment of cancer and other proliferative diseases
US7196108B2 (en) 2002-03-08 2007-03-27 Incyte San Diego Inc. Bicyclic heterocycles for the treatment of diabetes and other diseases
US7265139B2 (en) 2001-03-08 2007-09-04 Incyte San Diego Inc. RXR activating molecules
WO2008010601A1 (fr) * 2006-07-20 2008-01-24 Otsuka Pharmaceutical Co., Ltd. INHIBITEUR NF- ϰB
JP2008532999A (ja) * 2005-03-24 2008-08-21 コリア リサーチ インスティチュートオブ ケミカルテクノロジー 抗癌剤として有用な５−（１，３−ジアリール−１ｈ−ピラゾール−４−イルメチレン）−チアゾリジン−２，４−ジオン誘導体
KR101118768B1 (ko) * 2005-03-24 2012-03-20 한국화학연구원 항암제로 유용한5-(3-아릴-1-피리딜-1ｈ-피라졸-4-일메틸렌)-티아졸리딘-2,4-디온 유도체
KR101118827B1 (ko) * 2005-08-17 2012-03-20 한국화학연구원 항암제로 유용한5-(3-아릴-1-페닐-1ｈ-피라졸-4-일메틸렌)-3-알킬카복시-로다닌 유도체
CN103003271A (zh) * 2010-06-18 2013-03-27 爱维斯健有限公司 新型罗丹宁衍生物及其制备方法，含罗丹宁衍生物为活性成分的预防或治疗aids的药物组合物
WO2015070122A1 (fr) * 2013-11-08 2015-05-14 The Cleveland Clinic Foundation Agents anticancéreux inhibant la protéine disulfure isomérase
US9255088B2 (en) 2010-08-11 2016-02-09 The Regents Of The University Of California Premature-termination-codons readthrough compounds
US9598395B2 (en) 2012-03-23 2017-03-21 The Regents Of The University Of California Premature-termination-codons readthrough compounds
CN106588909A (zh) * 2017-01-06 2017-04-26 广东工业大学 一种喹啉类衍生物的制备及其在抗炎中的应用

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WO1998053790A2 (fr) * 1997-05-30 1998-12-03 Texas Biotechnology Corporation Composes pouvant inhiber le facteur de croissance de l'endothelium vasculaire pour l'empecher de se fixer a ses recepteurs

Cited By (35)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6765013B2 (en) 1999-08-31 2004-07-20 Incyte San Diego Thiazolidinedione derivatives for the treatment of diabetes and other diseases
US7226940B2 (en) 1999-08-31 2007-06-05 Incyte San Diego, Inc. Substituted heterocycles for the treatment of diabetes and other diseases
US6974826B2 (en) 1999-08-31 2005-12-13 Incyte San Diego Inc. Imidazolidinedione derivatives for the treatment of diabetes and other diseases
WO2001030771A1 (fr) * 1999-10-28 2001-05-03 Kyowa Hakko Kogyo Co., Ltd. Derives de thiazolidinedione
US7041686B2 (en) 2000-10-18 2006-05-09 Scott Beers Substituted imidazoles useful in the treatment of inflammatory diseases
WO2002032894A1 (fr) * 2000-10-18 2002-04-25 Ortho-Mcneil Pharmaceutical, Inc. Imidazoles substitues utilises dans le traitement de maladies inflammatoires
US7153875B2 (en) 2001-03-07 2006-12-26 Incyte San Diego Heterocyclic derivatives for the treatment of cancer and other proliferative diseases
US7265139B2 (en) 2001-03-08 2007-09-04 Incyte San Diego Inc. RXR activating molecules
US7071218B2 (en) 2001-11-15 2006-07-04 Incyte San Diego Incorporated N-substituted heterocycles for the treatment of hypercholesteremia, dyslipidemia and other metabolic disorders; cancer, and other diseases
US7102000B2 (en) 2002-03-08 2006-09-05 Incyte San Diego Inc. Heterocyclic amide derivatives for the treatment of diabetes and other diseases
US7196108B2 (en) 2002-03-08 2007-03-27 Incyte San Diego Inc. Bicyclic heterocycles for the treatment of diabetes and other diseases
GB2387172A (en) * 2002-03-28 2003-10-08 Pantherix Ltd [(Aryl-/arylthio-)aryl]methylene substituted azole & azine derivatives and their therapeutic use as antibacterials
JP2005535593A (ja) * 2002-05-17 2005-11-24 キューエルティー インコーポレーティッド チアゾリジンジチオン誘導体の使用方法
WO2004028441A3 (fr) * 2002-09-27 2004-05-06 Oreal Compose heterocyclique pour stimuler ou induire la pousse des cheveux ou des cils et/ou freiner leur chute, composition le contenant, ses utilisations
WO2004028441A2 (fr) * 2002-09-27 2004-04-08 L'oreal Compose heterocyclique pour stimuler ou induire la pousse des cheveux ou des cils et/ou freiner leur chute, composition le contenant, ses utilisations
WO2004043955A1 (fr) * 2002-11-13 2004-05-27 Rigel Pharmaceuticals, Inc. Derives de rhodanine et compositions pharmaceutiques les contenant
WO2005020990A1 (fr) * 2003-07-30 2005-03-10 Centre National De La Recherche Scientifique Thiazolidines antibiotiques
WO2005026127A1 (fr) * 2003-09-11 2005-03-24 Institute Of Medicinal Molecular Design. Inc. Inhibiteur d'inhibiteur-1 activateur de plasmogene
WO2006024699A1 (fr) * 2004-08-30 2006-03-09 Karyon-Ctt Ltd Composes de thioxothiazolidinone utilises comme produits pharmaceutiques
JP2008532999A (ja) * 2005-03-24 2008-08-21 コリア リサーチ インスティチュートオブ ケミカルテクノロジー 抗癌剤として有用な５−（１，３−ジアリール−１ｈ−ピラゾール−４−イルメチレン）−チアゾリジン−２，４−ジオン誘導体
KR101118842B1 (ko) * 2005-03-24 2012-03-16 한국화학연구원 항암제로 유용한5-(1,3-디아릴-1ｈ-피라졸-4-일메틸렌)-티아졸리딘-2,4-디온 유도체
KR101118768B1 (ko) * 2005-03-24 2012-03-20 한국화학연구원 항암제로 유용한5-(3-아릴-1-피리딜-1ｈ-피라졸-4-일메틸렌)-티아졸리딘-2,4-디온 유도체
KR101118827B1 (ko) * 2005-08-17 2012-03-20 한국화학연구원 항암제로 유용한5-(3-아릴-1-페닐-1ｈ-피라졸-4-일메틸렌)-3-알킬카복시-로다닌 유도체
WO2008010601A1 (fr) * 2006-07-20 2008-01-24 Otsuka Pharmaceutical Co., Ltd. INHIBITEUR NF- ϰB
AU2007276071B2 (en) * 2006-07-20 2010-12-09 Otsuka Pharmaceutical Co., Ltd. NF-kappaB inhibitor
EP2583969A2 (fr) * 2010-06-18 2013-04-24 Avixgen Inc. Nouveaux dérivés de rhodanine, procédé pour les préparer et composition pharmaceutique pour la prévention ou le traitement du sida contenant les dérivés de rhodanine comme principes actifs
CN103003271A (zh) * 2010-06-18 2013-03-27 爱维斯健有限公司 新型罗丹宁衍生物及其制备方法，含罗丹宁衍生物为活性成分的预防或治疗aids的药物组合物
EP2583969A4 (fr) * 2010-06-18 2013-10-23 Avixgen Inc Nouveaux dérivés de rhodanine, procédé pour les préparer et composition pharmaceutique pour la prévention ou le traitement du sida contenant les dérivés de rhodanine comme principes actifs
US8759536B2 (en) 2010-06-18 2014-06-24 Avixgen Inc. Rhodanine derivatives, method for preparing same, and pharmaceutical composition for the prevention or treatment of aids containing the rhodanine derivatives as active ingredients
US9255088B2 (en) 2010-08-11 2016-02-09 The Regents Of The University Of California Premature-termination-codons readthrough compounds
US9598395B2 (en) 2012-03-23 2017-03-21 The Regents Of The University Of California Premature-termination-codons readthrough compounds
WO2015070122A1 (fr) * 2013-11-08 2015-05-14 The Cleveland Clinic Foundation Agents anticancéreux inhibant la protéine disulfure isomérase
US9359342B2 (en) 2013-11-08 2016-06-07 The Cleveland Clinic Foundation Protein disulfide isomerase inhibiting anticancer agents
CN106588909A (zh) * 2017-01-06 2017-04-26 广东工业大学 一种喹啉类衍生物的制备及其在抗炎中的应用
CN106588909B (zh) * 2017-01-06 2019-08-27 广东工业大学 一种喹啉类衍生物的制备及其在抗炎中的应用

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