[go: up one dir, main page]

WO2008008841A2 - Utilisation de dérivés de benzimidazoles pour le traitement et/ou la prévention de désordres autoimmunitaires - Google Patents

Utilisation de dérivés de benzimidazoles pour le traitement et/ou la prévention de désordres autoimmunitaires Download PDF

Info

Publication number
WO2008008841A2
WO2008008841A2 PCT/US2007/073266 US2007073266W WO2008008841A2 WO 2008008841 A2 WO2008008841 A2 WO 2008008841A2 US 2007073266 W US2007073266 W US 2007073266W WO 2008008841 A2 WO2008008841 A2 WO 2008008841A2
Authority
WO
WIPO (PCT)
Prior art keywords
substituted
avp
alkyl
group
cells
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/US2007/073266
Other languages
English (en)
Other versions
WO2008008841A3 (fr
Inventor
Jagadish C. Sircar
Mark L. Richards
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Avanir Pharmaceuticals Inc
Original Assignee
Avanir Pharmaceuticals Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Avanir Pharmaceuticals Inc filed Critical Avanir Pharmaceuticals Inc
Publication of WO2008008841A2 publication Critical patent/WO2008008841A2/fr
Publication of WO2008008841A3 publication Critical patent/WO2008008841A3/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
    • C07D471/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains two hetero rings
    • C07D471/04—Ortho-condensed systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D209/00—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom
    • C07D209/02—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom condensed with one carbocyclic ring
    • C07D209/04—Indoles; Hydrogenated indoles
    • C07D209/10—Indoles; Hydrogenated indoles with substituted hydrocarbon radicals attached to carbon atoms of the hetero ring
    • C07D209/14—Radicals substituted by nitrogen atoms, not forming part of a nitro radical
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
    • C07D401/12—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings linked by a chain containing hetero atoms as chain links

Definitions

  • Preferred aspects of the present invention relate to treating and/or preventing autoimmune disorders, such as systemic lupus erythematosus and multiple sclerosis, using compounds that target the underlying mechanisms of such autoimmune disorders.
  • autoimmune disorders are systemic diseases which are divided into organ- specific autoimmune diseases such as pernicious anemia, Goodpasture's syndrome, myasthenia gravis, insulin-resistant diabetes, atrophic nephritis, multiple sclerosis (MS) and the like, and organ-nonspecific autoimmune diseases such as rheumatoid arthritis, systemic lupus erythematosus, polymyositis and the like.
  • organ-specific autoimmune diseases such as pernicious anemia, Goodpasture's syndrome, myasthenia gravis, insulin-resistant diabetes, atrophic nephritis, multiple sclerosis (MS) and the like
  • organ-nonspecific autoimmune diseases such as rheumatoid arthritis, systemic lupus erythematosus, polymyositis and the like.
  • Systemic lupus erythematosus (also called SLE or lupus) is a systemic autoimmune disease that results from inflammatory reactions caused by the deposition of antinuclear antibody or anti-DNA antibody and their immune complexes in various organs and tissues. SLE causes a variety of problems. It may cause skin rashes, arthritis, anemia, seizures or psychiatric illness, and often affects internal organs including the kidneys, lungs and heart. ' Once a disease with high mortality, SLE is now considered a chronic disease. [0005] Prevalence of SLE is 40 to 50 per 100,000. It is more common in certain ethnic groups, particularly among blacks. More than 85 percent of lupus patients are women.
  • SLE SLE
  • Typical features of SLE include: (1) a butterfly shaped rash over the cheeks; (2) a skin rash appearing in areas exposed to the sun; (3) sores in the mouth and nose; (4) arthritis involving one or more joints; (5) kidney inflammation; (6) nervous system disorders including seizures, mental disorders, and strokes; (7) fever, weight loss, hair loss, poor circulation in the fingers and toes, chest pain when taking deep breaths (pleurisy) and abdominal pain are often seen; and (8) people with lupus are more likely to have clogged arteries that can lead to heart attack and stroke at a younger age, likely due to the inflammation from lupus.
  • NSAIDs nonsteroidal anti-inflammatory drugs
  • Plaquenil antimalarial drugs
  • Antimalarial medications for SLE include hydroxychloroquine sulfate (Plaquenil) and chloroquine hydrochloride (Aralen). These antimalarial medications are not labeled by the U.S. Food and Drug Administration (FDA) for the treatment of lupus but are often prescribed for people with lupus to reduce inflammation.
  • FDA U.S. Food and Drug Administration
  • Hormonal therapy is another form of treatment for symptoms of SLE.
  • Hormone therapies include DHEA (dehydroxyepiandrosterone).
  • DHEA is an androgenic dietary supplement that is derived from the wild yam; only pharmaceutical-grade (versus "natural") DHEA is considered effective. Research suggests that it can improve stamina and sense of well-being when used to treat people with autoimmune diseases. People with SLE have reportedly had a decrease in symptoms when using DHEA. The most common side effects are acne and facial hair growth in women and hair loss in men.
  • Corticosteroids the single most prescribed drugs to treat SLE, must be used judiciously. Bone protection is important when steroids are used. Common prescription corticosteroids include prednisone, dexamethasone, and hydrocortisone. Corticosteroids have many side effects, including weight gain, stomach ulcers, sleeping difficulties, increased blood pressure, increased blood sugar (glucose), delayed wound healing, and a reduced ability to fight infection. Other problems associated with corticosteroid use include cataract formation, decreased blood flow to the hip joint causing deterioration of the joint (aseptic necrosis), and osteoporosis. Pharmacologic treatment for SLE often involves reaching a balance between preventing severe, possibly life-threatening organ damage, maintaining an acceptable quality of life, and minimizing side effects.
  • the autoimmune disorder is preferably SLE or MS.
  • the method comprises administering an effective amount of a compound to a patient in need of such therapeutic and/or prophylactic treatment, wherein the compound is selected from the group consisting of:
  • X and Y are independently selected from the group consisting of H, alkyl, alkoxy, aryl, substituted aryl, hydroxy, halogen, amino, alkylamino, nitro, cyano, CF 3 , OCF 3 , CONH 2 , CONHR and NHCOR 1 ;
  • R is selected from the group consisting of H, CH 3 , C 2 H 5 , C 3 H 7 , C 4 H 9 , CH 2 Ph, and CH 2 C 6 H 4 -F(p ⁇ );
  • R 1 and R 2 are independently selected from the group consisting of H, aryl, substituted aryl, cycloaryl substituted cycloaryl, multi-ring cycloaryl, benzyl, substituted benzyl, alkyl, cycloalkyl substituted cycloalkyl, multi-ring cycloalkyl, fused-ring aliphatic, cyclopropyl, substituted cyclopropyl, cyclobutyl, substituted cyclobutyl, cyclopentyl, substituted cyclopentyl, cyclohexyl, substituted cyclohexyl, cycloheptyl, substituted cycloheptyl, bicycloheptyl, bicyclooctyl, bicyclononyl, substituted bicycloalknyl, adamantyl, and substituted adamantyl,
  • X and Y are selected independently from the group consisting of alkyl, alkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, hydroxy, halogen, NO 2 ,
  • Z is selected from the group consisting of O, S, NH, and N-R'; wherein R' is further selected from the group consisting of H, alkyl, aminoalkyl, and dialkylaminoalkyl;
  • R is selected from the group consisting of H, alkyl, halogen, alkoxy, CF3 and OCF3; and [0019] Rl and R2 are independently selected from the group consisting of H, alkyl, aminoalkyl, dialkylaminoalkyl, hydoxyalkyl, alkoxyalkyl, cycloalkyl, oxacycloalkyl and thiocycloalkyl,
  • X and Y are independently selected from the group consisting of mono, di, tri, and tetra substituted H, alkyl, alkoxy, aryl, substituted aryl, hydroxy, halogen, amino, alkylamino, nitro, cyano, CF3, OCF3, CONH2, CONHR, and NHCORl;
  • R is selected from the group consisting of H, CH3, C2H5, C3H7, C4H9, CH2Ph, and CH2C6H4-F(p-);
  • Rl and R2 are independently selected from the group consisting of alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, multi-ring cycloalkyl, fused-ring aliphatic, cyclopropyl, substituted cyclopropyl, cyclobutyl, substituted cyclobutyl, cyclopentyl, substituted cyclopentyl, cyclohexyl, substituted cyclohexyl, cycloheptyl, substituted cycloheptyl, bicycloheptyl, bicyclooctyl, bicyclononyl, substituted bicycloalkenyl, adamantyl, substituted adamantyl; and
  • substituents on said substituted alkyl, substituted cycloalkyl, substituted cyclopropyl, substituted cyclobutyl, substituted cyclopentyl, substituted cyclohexyl, substituted cycloheptyl, substituted bicycloalkenyl, and substituted adamantyl are selected from the group consisting of alkyl, aryl, CF3, CH3, OCH3, OH, CN, COOR5, and COOH,
  • X and Y are independently selected from the group consisting of mono, di, tri, and tetra substituted H, alkyl, alkoxy, aryl, substituted aryl, hydroxy, halogen, amino, alkylamino, nitro, cyano, CF3, OCF3, CONH2, CONHR, and NHCORl;
  • R is selected from the group consisting of H, CH3, C2H5, C3H7, C4H9, CH2Ph, and CH2C6H4-F(p-);
  • Rl and R2 are independently selected from the group consisting of alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, multi-ring cycloalkyl, fused-ring aliphatic, cyclopropyl, substituted cyclopropyl, cyclobutyl, substituted cyclobutyl, cyclopentyl, substituted cyclopentyl, cyclohexyl, substituted cyclohexyl, cycloheptyl, substituted cycloheptyl, bicycloheptyl, bicyclooctyl, bicyclononyl, substituted bicycloalkenyl, adamantyl, substituted adamantyl, heterocyclic rings, and substituted heterocyclic rings; and
  • substituted alkyl, substituted cycloalkyl, substituted cyclopropyl, substituted cyclobutyl, substituted cyclopentyl, substituted cyclohexyl, substituted cycloheptyl, substituted bicycloalkenyl, substituted adamantyl and substituted heterocyclic rings are selected from the group consisting of alkyl, acyl, aryl, CF3, CH3, OCH3, OH, CN, COOR5, COOH, COCF3, and heterocyclic rings,
  • X is selected from the group consisting of mono, di, tri, and tetra substituted H, alkyl, alkoxy, aryl, substituted aryl, hydroxy, halogen, amino, alkylamino, nitro, cyano, CF3, OCF3, CONH2, CONHR, and NHCORl;
  • R is selected from the group consisting of H, CH3, C2H5, C3H7, C4H9, CH2Ph, and CH2CH4-F(p-);
  • Y is selected from the group consisting of mono, di, tri, and tetra substituted H, alkyl, alkoxy, aryl, benzo, substituted aryl, hydroxy, halogen, amino, alkylamino, nitro, cyano, CF3, OCF3, COPh, COOCH3, CONH2, CONHR, NHCONHRl, and NHCORl;
  • Rl is selected from the group consisting of alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, multi-ring cycloalkyl, fused-ring aliphatic, cyclopropyl, substituted cyclopropyl, cyclobutyl, substituted cyclobutyl, cyclopentyl, substituted cyclopentyl, cyclohexyl, substituted cyclohexyl, cycloheptyl, substituted cycloheptyl, bicycloheptyl, bicyclooctyl, bicyclononyl, substituted bicycloalkenyl, adamantyl, substituted adamantyl, heterocyclic rings containing one or more heteroatoms, and substituted heterocyclic rings; and
  • substituted alkyl, substituted cycloalkyl, substituted cyclopropyl, substituted cyclobutyl, substituted cyclopentyl, substituted cyclohexyl, substituted cycloheptyl, substituted bicycloalkenyl, substituted adamantyl, and substituted heterocyclic rings are selected from the group consisting of alkyl, aryl, CF3, CH3, OCH3, OH, CN, COOR, COOH, and heterocyclic rings,
  • X and Y are independently selected from the group consisting of H, alkyl, alkoxy, aryl, substituted aryl, hydroxy, halogen, amino, alkylamino, nitro, cyano, CF3, OCF3. CONH2, CONHR and NHCORl;
  • R is selected from the group consisting of H, CH3, C2H5, C3H7, C4H9, CH2Ph, CH2C6H4-F(p-); and
  • Rl and R2 are independently selected from the group consisting of H, aryl, substituted aryl, cycloaryl substituted cycloaryl, multi-ring cycloaryl, benzyl, substituted benzyl, alkyl, cycloalkyl substituted cycloalkyl, multi-ring cycloalkyl, fused-ring aliphatic, cyclopropyl, substituted cyclopropyl, cyclobutyl, substituted cyclobutyl, cyclopentyl, substituted cyclopentyl, cyclohexyl, substituted cyclohexyl, cycloheptyl, substituted cycloheptyl, bicycloheptyl, bicyclooctyl, bicyclononyl, substituted bicycloalknyl, adamantyl, substituted adamantyl and the like,
  • X and Y are independently selected from the group consisting of H, alkyl, alkoxy, aryl, substituted aryl, hydroxy, halogen, amino, alkylamino, nitro, cyano, CF 3 , OCF 3 .
  • X and Y are independently selected from the group consisting of H, alkyl, alkoxy, aryl, substituted aryl, hydroxy, halogen, amino, alkylamino, nitro, cyano, CF3, OCF3. CONH2, CONHR and NHCORl; [0038] wherein R is selected from the group consisting of H, CH3, C2H5, C3H7, C4H9, CH2Ph, CH2C6H4-F(p-), COCH3, CO2CH2CH3, aminoalkyl and dialkylaminoalkyl; and
  • Rl and R2 are independently selected from the group consisting of H, aryl, heteroaryl, thiophene, pyridyl, thiazolyl, isoxazolyl, oxazolyl, pyrimidinyl, substituted aryl, substituted heteroaryl, substituted thiophene, substituted pyridyl, substituted thiazolyl, substituted isoxazolyl, substituted oxazolyl, cycloaryl, cycloheteroaryl, quinolinyl, isoquinolinyl, substituted cycloaryl, substituted cycloheteroaryl, substituted quinolinyl, substituted isoqunolinyl, multi-ring cycloaryl, multi-ring cycloheteroaryl, benzyl, heteroaryl- methyl, substituted benzyl, substituted heteroaryl-methyl alkyl, dialkylaminoalkyl, cycloal
  • R is selected from the group consisting of H, C1-C5 alkyl, benzyl, p-fluorobenzyl and di-alkylamino alkyl, wherein said C1-C5 alkyl is selected from the group consisting of a straight chain, branched or cyclic alkyl;
  • Rl and R2 are independently selected from the group consisting of H, alkyl, substituted alkyl, C3-C9 cycloalkyl, substituted C3-C9 cycloalkyl, polycyclic aliphatic groups, substituted polycyclic aliphatic groups, phenyl, substituted phenyl, naphthyl, substituted naphthyl, heteroaryl and substituted heteroaryl, wherein said heteroaryl and said substituted heteroaryl contain 1-3 heteroatoms, wherein said heteroatom is independently selected from the group consisting of nitrogen, oxygen and sulfur;
  • R3 and R4 are independently selected from the group consisting of H, alkyl, aryl, heteroaryl and COR';
  • R' is selected from the group consisting of H, alkyl, substituted alkyl, C3-C9 cycloalkyl, substituted C3-C9 cycloalkyl, polycyclic aliphatics, substituted polycyclic aliphatics, phenyl, substituted phenyl, naphthyl, substituted naphthyl, heteroaryl and substituted heteroaryl, wherein said heteroaryl and said substituted heteroaryl contain 1-3 heteroatoms, wherein said heteroatom is independently selected from the group consisting of nitrogen, oxygen and sulfur;
  • Rl, R2, and R' is selected from the group consisting of H, halogens, polyhalogens, alkoxy group, substituted alkoxy, alkyl, substituted alkyl, dialkylaminoalkyl, hydroxyalkyl, carbonyl, OH, OCH3, COOH, OCOR', COOR', COR', CN, CF3, OCF3, NO2, NR'R', NHCOR' and CONR'R';
  • X and Y are independently selected from the group consisting of H, halogens, alkoxy, substituted alkoxy, alkyl, substituted alkyl, dialkylaminoalkyl, hydroxyalkyl, OH, OCOR", OCH3, COOH, CN, CF3, OCF3, N02, COOR", CHO and COR"; and
  • R" is a C1-C8 alkyl, wherein said C1-C8 alkyl is selected from the group consisting of a straight chain, branched or cyclic alkyl,
  • X and Y may be different or the same and are independently selected from the group consisting of H, halogen, alkyl, alkoxy, aryl, substituted aryl, hydroxy, amino, alkylamino, cycloalkyl, morpholine, thiomorpholine, nitro, cyano, CF3, OCF3, CORl, COORl, CONH2, CONHRl, and NHCORl;
  • n is an integer from one to three;
  • m is an integer from one to four;
  • R is selected from the group consisting of H, CH3, C2H5, C3H7, C4H9, CH2Ph, CH2C6H4-F(p-), COCH3, COCH2CH3, CH2CH2N(CH3)2, and CH2CH2CH2N(CH3)2; and
  • Rl and R2 are independently selected from the group consisting of H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, polycycloalkyl, substituted polycycloalkyl, polycycloalkenyl, substituted polycycloalkenyl, arylalkyl, substituted arylalkyl, heteroarylalkyl, substituted heteroarylalkyl, arylcycloalkyl, substituted arylcycloalkyl, heteroarylcycloalkyl, substituted heteroarylcycloalkyl, heterocyclic ring, substituted heterocyclic ring, heteroatom, and substituted heteroatom,
  • R is selected from the group consisting of H, C1-C5 alkyl, benzyl, p-fluorobenzyl and di-alkylamino alkyl, wherein said C1-C5 alkyl is selected from the group consisting of a straight chain, branched or cyclic alkyl;
  • Rl and R2 are independently selected from the group consisting of H, alkyl, substituted alkyl, C3-C9 cycloalkyl, substituted C3-C9 cycloalkyl, polycyclic aliphatic groups, phenyl, substituted phenyl, naphthyl, substituted naphthyl, heteroaryl and substituted heteroaryl, wherein said heteroaryl and said substituted heteroaryl contain 1-3 heteroatoms, wherein said heteroatom is independently selected from the group consisting of nitrogen, oxygen and sulfur;
  • substituted phenyl, substituted naphthyl and substituted heteroaryl contain 1-3 substituents, wherein said substituent is selected from the group consisting of H, halogens, polyhalogens, alkoxy group, substituted alkoxy, alkyl, substituted alkyl, dialkylaminoalkyl, hydroxyalkyl, OH, OCH3, COOH, COOR' COR', CN, CF3, OCF3, NO2, NR 'R', NHCOR' and CONR 'R';
  • R' is selected from the group consisting of H, alkyl, substituted alkyl, C3-C9 cycloalkyl, substituted C3-C9 cycloalkyl, polycyclic aliphatics, phenyl, substituted phenyl, naphthyl, substituted naphthyl, heteroaryl and substituted heteroaryl, wherein said heteroaryl and said substituted heteroaryl contain 1-3 heteroatoms, wherein said heteroatom is independently selected from the group consisting of nitrogen, oxygen and sulfur; and [0056] wherein R" is a C1-C8 alkyl, wherein said C1-C8 alkyl is selected from the group consisting of a straight chain, branched or cyclic alkyl,
  • R is selected from the group consisting of H, C1-C5 alkyl, benzyl, p-fluorobenzyl and di-alkylamino alkyl, wherein said C1-C5 alkyl is selected from the group consisting of a straight chain, branched or cyclic alkyl;
  • Rl and R2 are independently selected from the group consisting of H, alkyl, substituted alkyl, C3-C9 cycloalkyl, substituted C3-C9 cycloalkyl, polycyclic aliphatic groups, phenyl, substituted phenyl, naphthyl, substituted naphthyl, heteroaryl and substituted heteroaryl, wherein said heteroaryl and said substituted heteroaryl contain 1-3 heteroatoms, wherein said heteroatom is independently selected from the group consisting of nitrogen, oxygen and sulfur; [0059] wherein said substituted phenyl, substituted naphthyl and substituted heteroaryl contain 1-3 substituents, wherein said substituent is selected from the group consisting of H, halogens, polyhalogens, alkoxy group, substituted alkoxy, alkyl, substituted alkyl, dialkylaminoalkyl, hydroxyalkyl, OH, OCH3, COOH, COOR' COR',
  • R' is selected from the group consisting of H, alkyl, substituted alkyl, C3-C9 cycloalkyl, substituted C3-C9 cycloalkyl, polycyclic aliphatics, phenyl, substituted phenyl, naphthyl, substituted naphthyl, heteroaryl and substituted heteroaryl, wherein said heteroaryl and said substituted heteroaryl contain 1-3 heteroatoms, wherein said heteroatom is independently selected from the group consisting of nitrogen, oxygen and sulfur; and
  • R" is a C1-C8 alkyl, wherein said C1-C8 alkyl is selected from the group consisting of a straight chain, branched or cyclic alkyl,
  • A, B, D, E, G, V, X, Y, and Z are independently selected from carbon and nitrogen, with the proviso that at least one of A, B, D, E, G is nitrogen;
  • R is selected from the group consisting of H, C1-C5 alkyl, benzyl, p-fluorobenzyl and di-alkylamino alkyl, wherein said C1-C5 alkyl is selected from the group consisting of a straight chain, branched or cyclic alkyl;
  • Rl and R2 are independently selected from the group consisting of H, alkyl, substituted alkyl, C3-C9 cycloalkyl, substituted C3-C9 cycloalkyl, polycyclic aliphatic groups, phenyl, substituted phenyl, naphthyl, substituted naphthyl, heteroaryl and substituted heteroaryl, wherein said heteroaryl and said substituted heteroaryl contain 1-3 heteroatoms, wherein said heteroatom is independently selected from the group consisting of nitrogen, oxygen and sulfur;
  • substituted phenyl, substituted naphthyl and substituted heteroaryl contain 1-3 substituents, wherein said substituent is selected from the group consisting of H, halogens, polyhalogens, alkoxy group, substituted alkoxy, alkyl, substituted alkyl, dialkylaminoalkyl, hydroxyalkyl, OH, OCH3, COOH, COOR' COR', CN, CF3, OCF3, NO2, NR'R', NHCOR' and CONR'R'; and
  • R' is selected from the group consisting of H, alkyl, substituted alkyl, C3-C9 cycloalkyl, substituted C3-C9 cycloalkyl, polycyclic aliphatics, phenyl, substituted phenyl, naphthyl, substituted naphthyl, heteroaryl and substituted heteroaryl, wherein said heteroaryl and said substituted heteroaryl contain 1-3 heteroatoms, wherein said heteroatom is independently selected from the group consisting of nitrogen, oxygen and sulfur,
  • R is selected from the group consisting of H, C1-C5 alkyl, benzyl, p-fluorobenzyl and di-alkylamino alkyl, wherein said C1-C5 alkyl is selected from the group consisting of a straight chain, branched or cyclic alkyl;
  • R3, X, and Y are independently selected from the group consisting of H, halogen, alkoxy, substituted alkoxy, alkyl, substituted alkyl, dialkylaminoalkyl, hydroxyalkyl, OH, OCH3, COOH, CN, CF3, OCF3, NO2, COOR", CHO, and COR";
  • Rl and R2 are independently selected from the group consisting of H, alkyl, substituted alkyl, C3-C9 cycloalkyl, substituted C3-C9 cycloalkyl, polycyclic aliphatic groups, phenyl, substituted phenyl, naphthyl, substituted naphthyl, heterocyclic, and substituted heterocyclic, wherein said heterocyclic and said substituted heterocyclic contain 1-3 heteroatoms, wherein said heteroatom is independently selected from the group consisting of nitrogen, oxygen and sulfur; [0070] wherein said substituents are selected from the group consisting of H, halogen, alkoxy, substituted alkoxy, alkyl, substituted alkyl, dialkylaminoalkyl, hydroxyalkyl, OH, OCH3, COOH, COOR' COR', CN, CF3, OCF3, N02, NR'R', NHCOR' and CONR'R';
  • R' is selected from the group consisting of H, alkyl, substituted alkyl, C3-C9 cycloalkyl, substituted C3-C9 cycloalkyl, polycyclic aliphatics, phenyl, substituted phenyl, naphthyl, substituted naphthyl, heteroaryl and substituted heteroaryl, wherein said heteroaryl and said substituted heteroaryl contain 1-3 heteroatoms, wherein said heteroatom is independently selected from the group consisting of nitrogen, oxygen and sulfur; and
  • R" is selected from the group consisting of C1-C9 alkyl, wherein said C1-C9 alkyl is selected from the group consisting of straight chain alkyl, branched alkyl, and cyclic alkyl.
  • a method for treating and/or preventing SLE comprises administering an effective amount of a compound to a patient in need of such therapeutic and/or prophylactic treatment, wherein the compound is:
  • the compound is:
  • AVP 27457 [0075] In another preferred embodiment of the method for treating and/or preventing SLE, the compound is:
  • the compound is:
  • a method for treating and/or preventing MS comprises administering an effective amount of a compound to a patient in need of such therapeutic and/or prophylactic treatment, wherein the compound is:
  • the compound is:
  • the compound is:
  • the compound is:
  • a method is also disclosed in accordance with another preferred embodiment of the present invention for treating and/or preventing an immune disorder, comprising administering an amount of a compound sufficient to suppress expression of at least one Golgi resident protein associated with protein trafficking and/or processing such that the immune disorder is treated and/or prevented.
  • the immune disorder is an allergy or an autoimmune disorder. More preferably, the autoimmune disorder is SLE or MS.
  • the Golgi resident protein is selected from the group consisting of GS15, GS28, mannosidase II, and GPP130.
  • the compound is selected from the group consisting of compounds 1-42.
  • Figure 1 is a schematic illustrating intracellular protein trafficking.
  • Figure 2 shows the IgE response to antigen ex vivo.
  • Figure 3 shows the IgE response to IL-4 + ⁇ CD40 Ab in human PBL in vitro.
  • Figure 4 illustrates murine spleen T cell cytokine responses in vitro.
  • Figure 5 shows human PBL T cell cytokine responses.
  • Figures 6 show CD23 on human monocytes.
  • Figure 7 shows spleen cell proliferation response to AVP 893.
  • Figure 8 shows proliferation of human PBL in response to stimulus and drug in vitro.
  • Figure 9 shows an NCI 60-cell panel.
  • Figure 10 is a schematic of a BAL protocol #1 and illustrates the cells in
  • Figure 11 shows the AHR response in vivo.
  • Figure 12 shows the effect of AVP 25752 on B 16-Fl mouse melanoma tumor growth.
  • Figure 13 shows the effect of AVP 893 on HS294t human melanoma tumor growth.
  • Figure 14 is a dose response of AVP 13358 on various biochemical assays.
  • Figure 15 is a kinase screen of AVP 13358.
  • Figure 16 shows the PowerBlot results of the effect of AVP 893 on protein expression.
  • Figure 17 shows the time course of AVP 893 action in B16 cells.
  • Figure 18 shows the effect of AVP 893 on nicastrin and GS28 expression in various cells at 16 hours.
  • Figure 19 shows the effect of AVP 893 on nicastrin, calnexin and GS28 expression in various cells overnight.
  • Figure 20 shows the effect of AVP 893 on nicastrin, n-gly, calnexin and GS28 expression in various cells overnight.
  • Figure 21 shows inhibition of stimulated protein expression in BALB/c spleen cells by AVP 893.
  • Figure 22 shows dose-responsive inhibition of PMA/ionomycin-stimulated nicastrin and GS28 expression in BALB/c spleen cells by various compounds.
  • Figure 23 shows the PMA effect on AVP 893 inhibition of PBL proliferation response to IL-4/ ⁇ CD40 Ab.
  • Figure 24 shows the selective dose-response of AVP 893 in down- regulating IL-4/ ⁇ CD40 Ab induced protein expression after 48 hours in the presence and absence of PMA.
  • Figure 25 shows GS28 mRNA response to AVP 893 in human PBL.
  • Figure 26 is a schematic showing involvement of various cellular proteins in protein trafficking pathways.
  • Figure 27 shows dose-responsive inhibition by AVP 893 of GS 15 and GS28 expression in 18-20 hour cultures of B16-F10 cells.
  • Figure 28 shows the time-dependent effect of AVP 893 on mannosidase II expression.
  • Figure 29 shows the time-dependent effect of AVP 893 on Rab ⁇ expression in Vero cells.
  • Figure 30 shows shows the effect of AVP 893 on Golgi morphology in MOLT4 cells.
  • Figure 31 shows the effect of AVP 893 on GPP130 distribution in NIH- 3T3 cells.
  • Figure 32 shows the Rab ⁇ distribution in B16 cells.
  • Figure 33 compares the NCI results with AVP 893 and Brefeldin A.
  • Figure 34 shows the effects of AVP 893 and Brefeldin A on GS28 and nicastrin expression.
  • Figure 35 shows the activity of AVP 893 on Golgi resident proteins compared to known pharmacological agents in 3T3 cells.
  • Figure 36 shows the differential effects of AVP 893, brefeldin A, and nocodozole on mannosidase II expression.
  • Figure 37 compares the effect of AVP compounds and COG deficient mutants on cell phenotype.
  • Figure 38 shows the effects of AVP 13358 on GS28 and ⁇ COP expression in HeLa Hl cells by confocal microscopy and Western blotting.
  • Figure 39 shows the protective effect of proteosome and lysosome inhibitors when co-treating with AVP 13358 in MOLT-4 and 3T3 cells.
  • Figure 40 shows the protective effect of proteosome and lysosome inhibitors when co-treating with AVP 13358 in B16-F10 cells.
  • Figure 41 compares GS28, GPP130 and TGN38 expression in H460 cells.
  • Figure 42 compares GS28, GPP 130 and GMl 30 expression in HT-29 cells.
  • Figure 43 shows the concentration-dependent effects of AVP 893 on Golgi proteins in sensitive and resistant cells.
  • Figure 44 shows the effect of AVP 13358 on CD23 transcription in vitro.
  • Figure 45 shows the expression of CD23 at the plasma membrane and at the total cell protein level in relation to AVP 13358 concentration.
  • Figure 46 shows the reversibility of the effect of AVP 13358 on CD23 glycosylation.
  • Figure 47 compares the effect of AVP 13358 with several inhibitors of protein glycosylation and trafficking in spleen cells and M 12.4.5 cells.
  • Figure 48 shows the susceptibility of CD23 sugar residues to the effects of endoglycosidases.
  • Figure 49 quantitates the effect of AVP 893 on HSV-2 propagation in Vero cells in vitro.
  • Figure 50 shows the effect of AVP 893 on HSV-2 propagation in Vero cells in vitro though the expression of glycoprotein E.
  • Figure 51 compares the effect of AVP-26296, Acyclovir, Docosanol, and vehicle in an in vivo model of HSV-2 infection.
  • Figure 52 is a schematic of the cellular effect of AVP compounds.
  • Figure 53 shows the effect of AVP 13358 on survival of NZBAV-Fl female mice (SLE model).
  • Figure 54 shows results of pilot MS study.
  • Figure 55 shows scoring results from a second MS model study.
  • Figure 56 shows the effect of drug treatment on the body weight of NZBW-Fl female mice.
  • Figure 57 shows periodic serum drug levels 1 hr after dosing.
  • Figure 58 shows the impact of drug treatment on proteinuria and survival in the second NZBAV SLE model.
  • Figure 59 shows effect of AVP 13358 on total serum IgG amd anti-histone Ig in the mouse SLE model.
  • Figure 60 shows effect of AVP 13358 on total anti-DNA antibody (single- stranded and double-stranded) in the mouse SLE model.
  • Figure 61 shows effect of AVP 13358 on anti-dsDNA IgM and IgG detected in mouse SLE model.
  • Figure 62 compares oral bioavailability of back-up compounds with AVP 13358.
  • Figure 63 compares the potency of lead 2-phenyl benzimidazole coumpounds for inhibition of IgE (mouse spleen cells) and cytokines (human PBL).
  • Figure 64 shows efficacy of back-up compounds in asthma models in vivo.
  • the inventors have synthesized and characterized a novel family of 2- phenyl benzimidazole derivatives that down-regulate IgE associated with allergic reactions. Besides their ability to suppress IgE responses ex vivo, in vitro, and in vivo, these compounds also inhibit cytokine production/release, suppress cell surface receptor expression, and inhibit cellular proliferation. See e.g., U.S. Patent Nos. 6,271,390, 6,451,829, 6,369,091, 6,911,462; 6,303,645, 6,919,366 and 6,759,425, and co-pending U.S. Patent Application Nos.
  • Applicants postulate that modulation of protein trafficking/processing may be a fundamental mechanism through which these benzimidazole derivatives exert their various biological actions.
  • X and Y are independently selected from the group consisting of H, alkyl, alkoxy, aryl, substituted aryl, hydroxy, halogen, amino, alkylamino, nitro, cyano, CF 3 , OCF 3 , CONH 2 , CONHR and NHCORi;
  • R is selected from the group consisting of H, CH 3 , C 2 H 5 , C 3 H 7 , C 4 H 9 , CH 2 Ph, and CH 2 C 6 H 4 -F(p ⁇ );
  • R 1 and R 2 are independently selected from the group consisting of H, aryl, substituted aryl, cycloaryl substituted cycloaryl, multi-ring cycloaryl, benzyl, substituted benzyl, alkyl, cycloalkyl substituted cycloalkyl, multi-ring cycloalkyl, fused-ring aliphatic, cyclopropyl, substituted cyclopropyl, cyclobutyl, substituted cyclobutyl, cyclopentyl, substituted cyclopentyl, cyclohexyl, substituted cyclohexyl, cycloheptyl, substituted cycloheptyl, bicycloheptyl, bicyclooctyl, bicyclononyl, substituted bicycloalknyl, adamantyl, and substituted adamantyl,
  • X and Y are selected independently from the group consisting of alkyl, alkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, hydroxy, halogen, NO 2 ,
  • Z is selected from the group consisting of O, S, NH, and N-R'; wherein R' is further selected from the group consisting of H, alkyl, aminoalkyl, and dialkylaminoalkyl;
  • R is selected from the group consisting of H, alkyl, halogen, alkoxy, CF3 and OCF3; and [0155] Rl and R2 are independently selected from the group consisting of H, alkyl, aminoalkyl, dialkylaminoalkyl, hydoxyalkyl, alkoxyalkyl, cycloalkyl, oxacycloalkyl and thiocycloalkyl,
  • X and Y are independently selected from the group consisting of mono, di, tri, and tetra substituted H, alkyl, alkoxy, aryl, substituted aryl, hydroxy, halogen, amino, alkylamino, nitro, cyano, CF3, OCF3, CONH2, CONHR, and NHCORl;
  • R is selected from the group consisting of H, CH3, C2H5, C3H7, C4H9, CH2Ph, and CH2C6H4-F(p-);
  • Rl and R2 are independently selected from the group consisting of alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, multi-ring cycloalkyl, fused-ring aliphatic, cyclopropyl, substituted cyclopropyl, cyclobutyl, substituted cyclobutyl, cyclopentyl, substituted cyclopentyl, cyclohexyl, substituted cyclohexyl, cycloheptyl, substituted cycloheptyl, bicycloheptyl, bicyclooctyl, bicyclononyl, substituted bicycloalkenyl, adamantyl, substituted adamantyl; and
  • substituents on said substituted alkyl, substituted cycloalkyl, substituted cyclopropyl, substituted cyclobutyl, substituted cyclopentyl, substituted cyclohexyl, substituted cycloheptyl, substituted bicycloalkenyl, and substituted adamantyl are selected from the group consisting of alkyl, aryl, CF3, CH3, OCH3, OH, CN, COOR5, and COOH,
  • X and Y are independently selected from the group consisting of mono, di, tri, and tetra substituted H, alkyl, alkoxy, aryl, substituted aryl, hydroxy, halogen, amino, alkylamino, nitro, cyano, CF3, OCF3, CONH2, CONHR, and NHCORl;
  • R is selected from the group consisting of H, CH3, C2H5, C3H7, C4H9, CH2Ph, and CH2C6H4-F(p-);
  • Rl and R2 are independently selected from the group consisting of alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, multi-ring cycloalkyl, fused-ring aliphatic, cyclopropyl, substituted cyclopropyl, cyclobutyl, substituted cyclobutyl, cyclopentyl, substituted cyclopentyl, cyclohexyl, substituted cyclohexyl, cycloheptyl, substituted cycloheptyl, bicycloheptyl, bicyclooctyl, bicyclononyl, substituted bicycloalkenyl, adamantyl, substituted adamantyl, heterocyclic rings, and substituted heterocyclic rings; and
  • substituted alkyl, substituted cycloalkyl, substituted cyclopropyl, substituted cyclobutyl, substituted cyclopentyl, substituted cyclohexyl, substituted cycloheptyl, substituted bicycloalkenyl, substituted adamantyl and substituted heterocyclic rings are selected from the group consisting of alkyl, acyl, aryl, CF3, CH3, OCH3, OH, CN, COOR5, COOH, COCF3, and heterocyclic rings,
  • X is selected from the group consisting of mono, di, tri, and tetra substituted H, alkyl, alkoxy, aryl, substituted aryl, hydroxy, halogen, amino, alkylamino, nitro, cyano, CF3, OCF3, CONH2, CONHR, and NHCORl;
  • R is selected from the group consisting of H, CH3, C2H5, C3H7, C4H9, CH2Ph, and CH2CH4-F(p-);
  • Y is selected from the group consisting of mono, di, tri, and tetra substituted H, alkyl, alkoxy, aryl, benzo, substituted aryl, hydroxy, halogen, amino, alkylamino, nitro, cyano, CF3, OCF3, COPh, COOCH3, CONH2, CONHR, NHCONHRl, and NHCORl;
  • Rl is selected from the group consisting of alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, multi-ring cycloalkyl, fused-ring aliphatic, cyclopropyl, substituted cyclopropyl, cyclobutyl, substituted cyclobutyl, cyclopentyl, substituted cyclopentyl, cyclohexyl, substituted cyclohexyl, cycloheptyl, substituted cycloheptyl, bicycloheptyl, bicyclooctyl, bicyclononyl, substituted bicycloalkenyl, adamantyl, substituted adamantyl, heterocyclic rings containing one or more heteroatoms, and substituted heterocyclic rings; and
  • substituted alkyl, substituted cycloalkyl, substituted cyclopropyl, substituted cyclobutyl, substituted cyclopentyl, substituted cyclohexyl, substituted cycloheptyl, substituted bicycloalkenyl, substituted adamantyl, and substituted heterocyclic rings are selected from the group consisting of alkyl, aryl, CF3, CH3, OCH3, OH, CN, COOR, COOH, and heterocyclic rings,
  • X and Y are independently selected from the group consisting of H, alkyl, alkoxy, aryl, substituted aryl, hydroxy, halogen, amino, alkylamino, nitro, cyano, CF3, OCF3. CONH2, CONHR and NHCORl;
  • R is selected from the group consisting of H, CH3, C2H5, C3H7, C4H9, CH2Ph, CH2C6H4-F(p-); and
  • Rl and R2 are independently selected from the group consisting of H, aryl, substituted aryl, cycloaryl substituted cycloaryl, multi-ring cycloaryl, benzyl, substituted benzyl, alkyl, cycloalkyl substituted cycloalkyl, multi-ring cycloalkyl, fused-ring aliphatic, cyclopropyl, substituted cyclopropyl, cyclobutyl, substituted cyclobutyl, cyclopentyl, substituted cyclopentyl, cyclohexyl, substituted cyclohexyl, cycloheptyl, substituted cycloheptyl, bicycloheptyl, bicyclooctyl, bicyclononyl, substituted bicycloalknyl, adamantyl, substituted adamantyl and the like,
  • X and Y are independently selected from the group consisting of H, alkyl, alkoxy, aryl, substituted aryl, hydroxy, halogen, amino, alkylamino, nitro, cyano, CF 3 , OCF 3 .
  • X and Y are independently selected from the group consisting of H, alkyl, alkoxy, aryl, substituted aryl, hydroxy, halogen, amino, alkylamino, nitro, cyano, CF3, OCF3. CONH2, CONHR and NHCORl;
  • R is selected from the group consisting of H, CH3, C2H5, C3H7, C4H9, CH2Ph, CH2C6H4-F(p-), COCH3, CO2CH2CH3, aminoalkyl and dialkylaminoalkyl; and [0175] wherein Rl and R2 are independently selected from the group consisting of H, aryl, heteroaryl, thiophene, pyridyl, thiazolyl, isoxazolyl, oxazolyl, pyrimidinyl, substituted aryl, substituted heteroaryl, substituted thiophene, substituted pyridyl, substituted thiazolyl, substituted isoxazolyl, substituted oxazolyl, cycloaryl, cycloheteroaryl, quinolinyl, isoquinolinyl, substituted cycloaryl, substituted cycloheteroaryl, substituted quinolinyl, isoquinolin
  • R is selected from the group consisting of H, C1-C5 alkyl, benzyl, p-fluorobenzyl and di-alkylamino alkyl, wherein said C1-C5 alkyl is selected from the group consisting of a straight chain, branched or cyclic alkyl;
  • Rl and R2 are independently selected from the group consisting of H, alkyl, substituted alkyl, C3-C9 cycloalkyl, substituted C3-C9 cycloalkyl, polycyclic aliphatic groups, substituted polycyclic aliphatic groups, phenyl, substituted phenyl, naphthyl, substituted naphthyl, heteroaryl and substituted heteroaryl, wherein said heteroaryl and said substituted heteroaryl contain 1-3 heteroatoms, wherein said heteroatom is independently selected from the group consisting of nitrogen, oxygen and sulfur;
  • R3 and R4 are independently selected from the group consisting of H, alkyl, aryl, heteroaryl and COR';
  • R' is selected from the group consisting of H, alkyl, substituted alkyl, C3-C9 cycloalkyl, substituted C3-C9 cycloalkyl, polycyclic aliphatics, substituted polycyclic aliphatics, phenyl, substituted phenyl, naphthyl, substituted naphthyl, heteroaryl and substituted heteroaryl, wherein said heteroaryl and said substituted heteroaryl contain 1-3 heteroatoms, wherein said heteroatom is independently selected from the group consisting of nitrogen, oxygen and sulfur;
  • Rl, R2, and R' is selected from the group consisting of H, halogens, polyhalogens, alkoxy group, substituted alkoxy, alkyl, substituted alkyl, dialkylaminoalkyl, hydroxyalkyl, carbonyl, OH, OCH3, COOH, OCOR', COOR', COR', CN, CF3, OCF3, NO2, NR'R', NHCOR' and CONR 'R';
  • X and Y are independently selected from the group consisting of H, halogens, alkoxy, substituted alkoxy, alkyl, substituted alkyl, dialkylaminoalkyl, hydroxyalkyl, OH, OCOR", OCH3, COOH, CN, CF3, 0CF3, NO2, COOR", CHO and COR"; and
  • R" is a C1-C8 alkyl, wherein said C1-C8 alkyl is selected from the group consisting of a straight chain, branched or cyclic alkyl,
  • X and Y may be different or the same and are independently selected from the group consisting of H, halogen, alkyl, alkoxy, aryl, substituted aryl, hydroxy, amino, alkylamino, cycloalkyl, morpholine, thiomorpholine, nitro, cyano, CF3, OCF3, CORl, COORl, CONH2, CONHRl, and NHCORl;
  • n is an integer from one to three;
  • m is an integer from one to four;
  • R is selected from the group consisting of H, CH3, C2H5, C3H7, C4H9, CH2Ph, CH2C6H4-F(p-), COCH3, COCH2CH3, CH2CH2N(CH3)2, and CH2CH2CH2N(CH3)2; and
  • Rl and R2 are independently selected from the group consisting of H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, polycycloalkyl, substituted polycycloalkyl, polycycloalkenyl, substituted polycycloalkenyl, arylalkyl, substituted arylalkyl, heteroarylalkyl, substituted heteroarylalkyl, arylcycloalkyl, substituted arylcycloalkyl, heteroarylcycloalkyl, substituted heteroarylcycloalkyl, heterocyclic ring, substituted heterocyclic ring, heteroatom, and substituted heteroatom,
  • R is selected from the group consisting of H, C1-C5 alkyl, benzyl, p-fluorobenzyl and di-alkylamino alkyl, wherein said C1-C5 alkyl is selected from the group consisting of a straight chain, branched or cyclic alkyl;
  • Rl and R2 are independently selected from the group consisting of H, alkyl, substituted alkyl, C3-C9 cycloalkyl, substituted C3-C9 cycloalkyl, polycyclic aliphatic groups, phenyl, substituted phenyl, naphthyl, substituted naphthyl, heteroaryl and substituted heteroaryl, wherein said heteroaryl and said substituted heteroaryl contain 1-3 heteroatoms, wherein said heteroatom is independently selected from the group consisting of nitrogen, oxygen and sulfur;
  • substituted phenyl, substituted naphthyl and substituted heteroaryl contain 1-3 substituents, wherein said substituent is selected from the group consisting of H, halogens, polyhalogens, alkoxy group, substituted alkoxy, alkyl, substituted alkyl, dialkylaminoalkyl, hydroxyalkyl, OH, OCH3, COOH, COOR' COR', CN, CF3, OCF3, NO2, NR 'R', NHCOR' and CONR 'R';
  • R' is selected from the group consisting of H, alkyl, substituted alkyl, C3-C9 cycloalkyl, substituted C3-C9 cycloalkyl, polycyclic aliphatics, phenyl, substituted phenyl, naphthyl, substituted naphthyl, heteroaryl and substituted heteroaryl, wherein said heteroaryl and said substituted heteroaryl contain 1-3 heteroatoms, wherein said heteroatom is independently selected from the group consisting of nitrogen, oxygen and sulfur; and [0192] wherein R" is a C1-C8 alkyl, wherein said C1-C8 alkyl is selected from the group consisting of a straight chain, branched or cyclic alkyl,
  • R is selected from the group consisting of H, C1-C5 alkyl, benzyl, p-fluorobenzyl and di-alkylamino alkyl, wherein said C1-C5 alkyl is selected from the group consisting of a straight chain, branched or cyclic alkyl;
  • Rl and R2 are independently selected from the group consisting of H, alkyl, substituted alkyl, C3-C9 cycloalkyl, substituted C3-C9 cycloalkyl, polycyclic aliphatic groups, phenyl, substituted phenyl, naphthyl, substituted naphthyl, heteroaryl and substituted heteroaryl, wherein said heteroaryl and said substituted heteroaryl contain 1-3 heteroatoms, wherein said heteroatom is independently selected from the group consisting of nitrogen, oxygen and sulfur; [0195] wherein said substituted phenyl, substituted naphthyl and substituted heteroaryl contain 1-3 substituents, wherein said substituent is selected from the group consisting of H, halogens, polyhalogens, alkoxy group, substituted alkoxy, alkyl, substituted alkyl, dialkylaminoalkyl, hydroxyalkyl, OH, OCH3, COOH, COOR' COR',
  • R' is selected from the group consisting of H, alkyl, substituted alkyl, C3-C9 cycloalkyl, substituted C3-C9 cycloalkyl, polycyclic aliphatics, phenyl, substituted phenyl, naphthyl, substituted naphthyl, heteroaryl and substituted heteroaryl, wherein said heteroaryl and said substituted heteroaryl contain 1-3 heteroatoms, wherein said heteroatom is independently selected from the group consisting of nitrogen, oxygen and sulfur; and
  • R" is a C1-C8 alkyl, wherein said C1-C8 alkyl is selected from the group consisting of a straight chain, branched or cyclic alkyl,
  • A, B, D, E, G, V, X, Y, and Z are independently selected from carbon and nitrogen, with the proviso that at least one of A, B, D, E, G is nitrogen;
  • R is selected from the group consisting of H, C1-C5 alkyl, benzyl, p-fluorobenzyl and di-alkylamino alkyl, wherein said C1-C5 alkyl is selected from the group consisting of a straight chain, branched or cyclic alkyl;
  • Rl and R2 are independently selected from the group consisting of H, alkyl, substituted alkyl, C3-C9 cycloalkyl, substituted C3-C9 cycloalkyl, polycyclic aliphatic groups, phenyl, substituted phenyl, naphthyl, substituted naphthyl, heteroaryl and substituted heteroaryl, wherein said heteroaryl and said substituted heteroaryl contain 1-3 heteroatoms, wherein said heteroatom is independently selected from the group consisting of nitrogen, oxygen and sulfur;
  • substituted phenyl, substituted naphthyl and substituted heteroaryl contain 1-3 substiruents, wherein said substituent is selected from the group consisting of H, halogens, polyhalogens, alkoxy group, substituted alkoxy, alkyl, substituted alkyl, dialkylaminoalkyl, hydroxyalkyl, OH, OCH3, COOH, COOR' COR', CN, CF3, OCF3, NO2, NR 'R', NHCOR' and CONR'R'; and
  • R' is selected from the group consisting of H, alkyl, substituted alkyl, C3-C9 cycloalkyl, substituted C3-C9 cycloalkyl, polycyclic aliphatics, phenyl, substituted phenyl, naphthyl, substituted naphthyl, heteroaryl and substituted heteroaryl, wherein said heteroaryl and said substituted heteroaryl contain 1-3 heteroatoms, wherein said heteroatom is independently selected from the group consisting of nitrogen, oxygen and sulfur,
  • R is selected from the group consisting of H, C1-C5 alkyl, benzyl, p-fluorobenzyl and di-alkylamino alkyl, wherein said C1-C5 alkyl is selected from the group consisting of a straight chain, branched or cyclic alkyl;
  • R3, X, and Y are independently selected from the group consisting of H, halogen, alkoxy, substituted alkoxy, alkyl, substituted alkyl, dialkylaminoalkyl, hydroxyalkyl, OH, OCH3, COOH, CN, CF3, OCF3, NO2, COOR", CHO, and COR";
  • Rl and R2 are independently selected from the group consisting of H, alkyl, substituted alkyl, C3-C9 cycloalkyl, substituted C3-C9 cycloalkyl, polycyclic aliphatic groups, phenyl, substituted phenyl, naphthyl, substituted naphthyl, heterocyclic, and substituted heterocyclic, wherein said heterocyclic and said substituted heterocyclic contain 1-3 heteroatoms, wherein said heteroatom is independently selected from the group consisting of nitrogen, oxygen and sulfur; [0206] wherein said substituents are selected from the group consisting of H, halogen, alkoxy, substituted alkoxy, alkyl, substituted alkyl, dialkylaminoalkyl, hydroxyalkyl, OH, OCH3, COOH, COOR' COR', CN, CF3, 0CF3, N02, NR'R', NHCOR' and CONR 'R';
  • R' is selected from the group consisting of H, alkyl, substituted alkyl, C3-C9 cycloalkyl, substituted C3-C9 cycloalkyl, polycyclic aliphatics, phenyl, substituted phenyl, naphthyl, substituted naphthyl, heteroaryl and substituted heteroaryl, wherein said heteroaryl and said substituted heteroaryl contain 1-3 heteroatoms, wherein said heteroatom is independently selected from the group consisting of nitrogen, oxygen and sulfur; and
  • R" is selected from the group consisting of C1-C9 alkyl, wherein said C1-C9 alkyl is selected from the group consisting of straight chain alkyl, branched alkyl, and cyclic alkyl.
  • the present invention is directed to small molecule inhibitors of IgE (synthesis and/or release) which are useful in the treatment of allergy and/or asthma or any diseases where IgE is pathogenic.
  • IgE synthesis and/or release
  • the particular compounds disclosed herein were identified by their ability to suppress IgE levels in both ex vivo and in vivo assays. Development and optimization of clinical treatment regimens can be monitored by those of skill in the art by reference to the ex vivo and in vivo assays described below.
  • mice were immunized i.p. with 10 ⁇ g DNP-KLH adsorbed onto 4 mg alum and sacrificed after 15 days.
  • Spleens were excised and homogenized in a tissue grinder, washed twice, and maintained in DMEM supplemented with 10% FBS, 100 U/ml penicillin, 100 ⁇ g/ml streptomycin and 0.0005% 2-mercaptoethanol.
  • Spleen cell cultures were established (2-3 million cells/ml, 0.2 ml/well in quadruplicate, 96-well plates) in the presence or absence of DNP-KLH (10 ng/ml).
  • Test compounds (2 ⁇ g/ml and 50 ng/ml) were added to the spleen cell cultures containing antigen and incubated at 37° C for 8 days in an atmosphere of 10% CO 2 .
  • Antigen-specific IgGl was measured similarly, except that culture supernatants were diluted 200-fold and biotinylated-goat antimouse IgGl (b-GAMGl) was substituted for b-GAME.
  • IgG2a was measured in ELISA plates that were coated with DNP- KLH following a 1 :20 dilution of culture supernatants and incubation with biotinylated-goat antimouse IgG2a (b-GAMG2a). Quantitation of each isotype was determined by comparison to a standard curve. The level of detectability of all antibodies was about 200-400 pg/ml and there was less than 0.001% cross-reactivity with any other Ig isotype in the ELISA for IgE.
  • mice Female BALB/cByj mice were irradiated with 250 rads 7 hours after initiation of the daily light cycle. Two hours later, the mice were immunized i.p. with 2 ⁇ g of KLH in 4 mg alum. Two to seven consecutive days of drug injections were initiated 6 days later on either a once or twice daily basis. Typically, i.p. injections and oral gavages were administered as suspensions (150 ⁇ l/injection) in saline with 10% ethanol and 0.25% methylcellulose. Each treatment group was composed of 5-6 mice. On the second day of drug administration, 2 ⁇ g of DNP-KLH was administered i.p. in 4 mg alum, immediately following the morning injection of drug. Mice were bled 7-21 days following DNP-KLH challenge.
  • Antigen-specific IgE, IgGl and IgG2a antibodies were measured by ELISA. Periorbital bleeds were centrifuged at 14,000 rpm for 10 min, the supernatants were diluted 5-fold in saline, and centrifuged again. Antibody concentrations of each bleed were determined by ELISA of four dilutions (in triplicate) and compared to a standard curve: anti- DNP IgE (1:100 to 1 :800), anti-DNP IgG2a (1 :100 to 1:800), and anti-DNP IgGl (1:1600 to 1 :12800). In Vitro Measures of Drug Action
  • T cells were isolated from murine spleen and cultured for 16 hours in the presence of stimulus +/- AVP 13358 ( Figure 4). T cells were also isolated from donor PBL and cultured for 16-36 hours in the presence of phytohemaglutin (PHA, 5 ⁇ g/ml) or ConA (5 ⁇ g/ml) +/- AVP 13358 ( Figure 5). Supernatants of both mouse and human cell cultures were quantified for cytokines using Luminex beads. All cytokines achieved levels of at least 200 pg/ml and these levels were at least 10-fold higher than background.
  • AVP 13358 potently suppressed the levels of most cytokines, including those important for the development of allergy, i.e., IL-4, IL-5, and IL-13.
  • a third group of activities discovered for these compounds is the suppression of membrane receptor expression.
  • CD23 the B cell IgE receptor; Figure 6
  • IL-4 receptor IL- 4R ⁇ , not shown
  • the data shown in Figure 9 represents the IC50s of AVP 893 and AVP 13358 for the suppression of a variety of tumor cell lines based on measures of total protein from 2-day cultures of tumor cell lines wherein the level of protein is roughly proportional to the level of proliferation.
  • Total protein was assessed by the SRB assay, as adopted for many of the proliferation experiments outline below.
  • SRB Sulforhodamine B
  • cells are inoculated into 96 well microtiter plates in 100 ⁇ l at plating densities ranging from 5,000 to 40,000 cells/well depending on the doubling time of individual cell lines. After cell seeding, the microtiter plates are incubated at 37°C, 5 % or 10% CO 2 — depending on the cell line and media — 95 % air and 100 % relative humidity for 24 h prior to addition of experimental drugs. After 24 h, two plates of each cell line are fixed in situ with trichloroacetic acid (TCA), to represent a measurement of the cell population for each cell line at the time of drug addition.
  • TCA trichloroacetic acid
  • the plates are incubated for an additional 48 h at 37°C, 5 %/10% CO2, and 100 % relative humidity.
  • the assay is terminated by the addition of cold TCA.
  • Cells are fixed in situ by the gentle addition of 50 ⁇ l of cold 50 % (w/v) TCA (final concentration, 10 % TCA) and incubated for 60 minutes at 4°C. The supernatant is discarded, and the plates are washed five times with tap water and air-dried.
  • Sulforhodamine B (SRB) solution (100 ⁇ l) at 0.4 % (w/v) in 1 % acetic acid is added to each well, and plates are incubated for 10 minutes at room temperature.
  • NCI National Cancer Institute
  • AVP drug effects on the variety of responses observed in vitro are also noted in vivo. This not only provides a level of confidence that the in vitro findings can be carried over to the intact animal, but also indicates that these agents may have utility in treating human diseases wherein these effects would be beneficial. Screening for Biological Targets
  • a second series of experiments tested the activity of AVP 893 on the expression of over 950 proteins by Western blotting in vitro (in triplicate) using methods detailed below.
  • B16 tumor cells were chosen for this screen and a 16-hour duration of AVP 893 treatment was selected to optimize the number of proteins that might be modified by drug.
  • B16-F10 cells were cultured for 16 hr in the presence or absence of 100 ng/ml AVP 893. Samples were harvested and lysates prepared according to instructions supplied by Becton-Dickinson. Samples were placed on dry ice and submitted to the same for expression analysis of 950 proteins. Only 6 proteins were found to be consistently and significantly modified in lysates derived from drug-treated cells (Figure 16).
  • the culture medium was removed by aspiration (for attached cells) or by low speed centrifugation (for suspension cells) for 5-7 minutes at room temperature.
  • the cells were washed with PBS, spun at 1200 rpm and the cell pellets were kept on ice.
  • Ice cold lysis buffer 300 ⁇ l/2.0xl0 7 cells
  • Cell pellets were gently resuspended and incubated on ice for at least 30 min while vortexing a few times during incubation.
  • Cell lysate was spun at 14,000 rpm for 2-5 min at 4 0 C, the supernatant transferred to a new microfuge tube, and the pellet was discarded.
  • An aliquot of sample was mixed with an equal volume of 2X sample buffer (InVitrogen), and stored at -80 0 C. Protein concentration was determined by using "BCA protein assay reagent kit" from Pierce.
  • Protein samples (in sample buffer) were boiled for 1-3 minutes and put on ice. Samples containing equivalent protein were loaded on the NuPage gel (InVitrogen). After the electrophoresis was complete, proteins were transferred from the gel to a PVDF membrane using the electro-blotting apparatus from InVitrogen. Non-specific binding was suppressed by incubating membranes with 5% milk (in PBS, 0.1% tween 20) for at least 30 min at room temperature or overnight at 4 0 C. The blocked membrane was incubated with primary antibody (See TABLE 3) diluted in 5% milk for 1 hour at room temperature. Optimal dilution depends on the antibody and the amount of protein. Dilutions of 1:1000 were generally used for the primary antibodies.
  • the membrane was washed with PBS/0.1% tween 3-4 times 5 mins.
  • the membrane was incubated for 30-60 minutes at room temperature with horseradish peroxidase (HRP) conjugated secondary antibody diluted in 5% milk. We usually used 1 :4000 dilution for the secondary antibody from Santa Cruz.
  • the membrane was washed 3-4 times with PBS/0.1% tween, each time 15 minutes.
  • the detection solutions A and B were mixed in a ratio 40:1 and pipetted onto the membrane, and incubated for 5 min at RT.
  • a sheet of Hyper film ECL was placed on the top of the membrane in the dark and exposed for 1 min, or adjust accordingly.
  • HCAM H300 sc-7946 rabbit polyclonal m,r,h Santa Cruz
  • NSF N-18 sc-15915 goat polyclonal m,r,h Santa Cruz
  • Notch1 (H-131 ) sc-9170 rabbit polyclonal m,r,h Santa Cruz
  • Presenilin 1 (N-19) sc-1245 goat polyclonal m,r,h Santa Cruz
  • Presenilin 2 (C-20) sc-1456 goat polyclonal m,r,n Santa Cruz
  • Rab5A (S-19) sc-309 rabbit polyclonal m,r,h Santa Cruz
  • Rab1A (C-19) sc-311 rabbit polyclonal m,r,h Santa Cruz
  • Rab1 B (G-20) sc-599 rabbit polyclonal m,r,h Santa Cruz
  • Rab2 (P-19) sc-307 rabbit polyclonal m,r,h Santa Cruz
  • Rab6 (C-19) sc-310 rabbit polyclonal m,r,h Santa Cruz
  • VAMP-1 (FL-118) sc-13992 rabbit polyclonal m,r,h Santa Cruz
  • VAMP-3 (N-12) sc-18208 goat polyclonal m,r,h Santa Cruz p115 (N-20) sc-16272 goat polyclonal m,r,h Santa Cruz secondary antibodies
  • GS 15 and GS28 are t-SNARE proteins that are involved in the docking and fusion of vesicles in the Golgi and the intermediate compartment (IC, located between the ER and Golgi). Thus, these GS proteins are intimately involved in the movement of proteins (via vesicles) both between the ER and Golgi and within the Golgi cisternae.
  • Nicastrin is a part of the ⁇ -secretase complex that is responsible for intramembrane cleavage of a number of proteins that subsequently translocate into the nucleus and act as transcription factors. Included amongst these proteins are amyloid precursor protein (APP), Notch, erbB4, E-cadherin, and others.
  • nicastrin normally passes through the ER where it is partially glycosylated and then to the Golgi where glycosylation and sialation is completed.
  • nicastrin is essentially acting as a cargo protein whose changes reflect of how the cell processes and transports it.
  • AVP 893 treatment appears to prevent the Golgi-associated processing changes of nicastrin, which is perhaps associated with its effect on GS28.
  • the normal fibroblast cell line, 3T3, showed a more profound response to drug as levels of GS28 and mature nicastrin were virtually eliminated by AVP 893 exposure.
  • An AVP 893 concentration/response evaluation for 3T3 cells suggests that the IC50 for GS28 and mature nicastrin expression is between 10 and 100 ng/ml ( Figure 20), which is consistent with the IC50 for AVP 893 inhibition of 3T3 cell proliferation.
  • AVP 893 also suppressed GS28 expression in mouse spleen cells that were stimulated with various agents (Figure 21).
  • BALB/c spleen cells were cultured for 20 hours in the presence of stimulus +/- AVP 893 (100 ng/ml) and harvested and prepared as described in Figure 17.
  • Stimulus conditions include: LPS (10 ⁇ g/ml), IL-4 (10 ng/ml) plus anti-CD40 Ab (100 ng/ml), PMA (10 ng/ml) plus ionomycin (100 nM), or Con A (5 ⁇ g/ml).
  • LPS (10 ⁇ g/ml)
  • IL-4 10 ng/ml
  • PMA 10 ng/ml
  • ionomycin 100 nM
  • Con A 5 ⁇ g/ml
  • Figure 22 compares the effects of 3 compounds that possess different potencies for inhibition of IL-4/anti-CD40 Ab-stimulated IgE production or proliferation by mouse spleen cells. This experiment was carried out as described for Figure 21, except that different AVP compounds with high (AVP 893, 5 nM), medium (AVP 26297, 50 nM), and low (AVP 25630, 500 nM) anti-proliferative potency were tested and compared. AVP 893 was tested at 1, 10, and 100 ng/ml; AVP 26297 was tested at 1, 10, 100, and 1000 ng/ml; AVP 25630 was tested at 10, 100, and 1000 ng/ml. For each compound, the effect on both GS28 and mature nicastrin expression paralleled their effect on proliferation in vitro suggesting that these effects at the cellular and protein level are linked.
  • AVP 893 was tested at 1, 10, and 100 ng/ml
  • AVP 26297 was tested at 1, 10, 100, and 1000 ng/ml
  • RNA purity was checked by spectrophotometer. RT-PCR (36 cycles) was performed following the RT-PCR One-Step protocol (Qiagen). Similar results were obtained when testing mRNA samples obtained from other cell sources (not shown). Primers
  • GS28 is but one member of a complicated pathway of interacting proteins that are responsible for the movement of vesicles through the cell.
  • SNARE proteins that are involved in vesicular docking and fusion
  • Rabs a group of small Ras-like GTPases known as Rabs are responsible for activating many of these proteins to permit their interaction.
  • Rab proteins known to play a prominent role in the ER-Golgi protein trafficking include Rabla, Rablb and Rab ⁇ ( Figure 26). Both Rabl proteins help COPII protein-coated vesicles to travel from the ER to the Golgi, while Rab ⁇ is involved in the retrograde movement of vesicles back to the ER via COPI vesicles.
  • AVP 893 was found to suppress the total cellular expression of resident Golgi proteins such as GS28 and GS 15 ( Figure 27) and GPPl 30 (data not shown), and the juxtanuclear expression of mannosidase II ( Figure 28) in a time-dependent manner.
  • GS 15 staining in B16-F10 cells was greatly diminished by AVP 893 beginning at 4 hrs of exposure, whereas GS28 levels started dropping off after 8 hrs of exposure, culminating in significantly reduced levels after 16 hrs of drug incubation.
  • GM- 130 a Golgi-structural protein, did not appear to be affected by AVP 893 (data not shown).
  • the juxtanuclear expression of the non-resident Golgi protein Rab ⁇ is unaffected in Vero cells, as illustrated in Figure 29. Similar results were obtained in other cells.
  • mannosidase II a resident Golgi enzyme involved in carbohydrate processing
  • Figure 28 the staining of the GTPase Rab ⁇ was neither diminished nor significantly altered by the presence of AVP 893, even after 18 hrs.
  • AVP 893 discriminately affects Golgi resident proteins while leaving ER proteins (e.g. calnexin), non-resident proteins (e.g. Rab6) or structural proteins (e.g. GM- 130) unaffected.
  • the mannosidase II data is yet another example of the time course of AVP 893 action on resident Golgi proteins, wherein a slow decrease in expression levels culminates in severely diminished levels after 16-20 hrs of drug incubation.
  • endoplasmic reticulum (calnexin) and Golgi ( ⁇ -adaptin) reference markers were previously shown to be neither quantitatively altered nor their distribution changed by overnight culture with AVP-893 (not shown).
  • Each gradient was adjusted in terms of protein loaded per gel and film exposure time to optimize visualization and indicate the qualitative and quantitative changes in GPP 130 within each culture condition.
  • an aliquot of the original lysates (containing 30 ⁇ g of protein) was removed prior to centrifugation, and directly compared by Western blot (left lane).
  • GPP 130 shifts into lighter layers of the gradient suggesting that AVP-893 causes a rapid redistribution of the protein out of the Golgi and into smaller vesicles.
  • the protein found in the lighter layers is the incompletely processed form of GPP 130 while the mature form remains in the same fractions as from control cultures. This suggests that the protein is gradually leaching out of the Golgi rather than exiting the organelle en masse.
  • GPP 130 Moreover as illustrated in the pre-gradient lysates (left lane), the redistribution of GPP 130 precedes its loss; an observation that can be explained by GPP 130 movement out of the Golgi cisternae into a compartment that ultimately targets the protein for proteolysis.
  • Figure 32 shows the redistribution of Rab ⁇ following a 16 hr culture with AVP 893 in B16 cells
  • the levels of Rab ⁇ present in each fraction are compared with the presence of marker proteins; calnexin for the endoplasmic reticulum (ER), ⁇ -adaptin for the Golgi (G), and Rab5a for vesicles/endosomes (V).
  • marker proteins calnexin for the endoplasmic reticulum (ER), ⁇ -adaptin for the Golgi (G), and Rab5a for vesicles/endosomes (V).
  • ER endoplasmic reticulum
  • G ⁇ -adaptin for the Golgi
  • V vesicles/endosomes
  • the distribution of these Golgi proteins may vary depending on the cell type, hi any event, the results illustrated in Figure 31 show that there is a shift in the distribution of GPP 130 to smaller vesicles in the presence of AVP- 893, and this shift precedes its loss.
  • the results support the concept that the primary event initiated by 2-PB compounds is the dissociation of these proteins from the Golgi. Bl 6 and 3T3 Density Gradient Protocol
  • B16-F10 and 3T3 cells were seeded into 175cm 2 flasks one day prior to drug application. On the subsequent day, fresh media +/- drug was applied to the cultures. After 16 hours, the cells were washed with cold Dulbecco's PBS, then harvested in ice-cold homogenization buffer: 13OmM KCl, 25mM NaCl, ImM EGTA, 25mM Tris pH7.4, plus 15 ⁇ l protease inhibitor per 5 ml buffer. 1 ml of buffer was used per flask, and the cells were scrapped off into 14ml round-bottom culture tubes and kept on ice.
  • the harvested cells were then homogenized with a tissue homogenizer (Polytron PTl 0/35), transferred into 2ml centrifuge tubes, and spun at 1,000 rpm for 8 min at 4°C. The supernatant was collected and placed on top of a 30% to 2.5% iodixanol (Optiprep) gradient, previously prepared with homogenization buffer and kept cold. 16x100mm ultracentrifuge tubes were used in a Sorval OTD50B Ultracentrifuge with an AH-627 rotor, spinning the samples at 27,000 rpm for 1 hr. 1 ml samples were carefully removed from the top of the gradient, then diluted with a 2X sample buffer for Western blot analysis (16 ⁇ l loaded per lane). Throughout this protocol, samples were kept on ice as much as possible. Comparison with Brefeldin A
  • Monensin is a sodium ionophore that shares some of the effects noted for the AVP compounds (e.g., cytokine inhibition). However, because it acts in a post-Golgi compartment, there are qualitative differences in their activity that clearly demonstrate that the compounds act on distinct targets. Brefeldin A, however, blocks movement of proteins from the ER to the Golgi and shares many of the effects observed for AVP 893, including cytokine production/release and tumor cell proliferation.
  • Brefeldin A was previously tested by the NCI for inhibition of tumor cell proliferation in their 60-cell screen.
  • the NCI 60-cell screen was performed essentially as described for Figure 9.
  • Data available from the NCI database for brefeldin A was compared with more recent AVP 893 data. Comparison of the results obtained for brefeldin A with that of AVP 893 show that while brefeldin A inhibits proliferation of virtually all cells at concentrations of 10 to 100 nM, AVP 893 showed considerable variation in potency ( ⁇ 10 nM to >10 ⁇ M) depending upon the cell line tested (Figure 33).
  • Several tumor cell lines were cultured in the presence of either AVP 893 or brefeldin A for about 48 hours before assessing proliferation response by measuring total protein (SRB), as described for Figure 9.
  • SRB total protein
  • AVP 893 inhibits GS28 (and mature nicastrin) expression in the 2 "sensitive" cell lines at concentrations that closely paralleled their activity on proliferation.
  • MOLT-4, Hs294T, and H460 cells were cultured overnight with either AVP 893 or brefeldin A and harvested and prepared for Western blotting as described for Figure 17.
  • AVP 893 had little effect on GS28 or nicastrin in the resistant line, H-460.
  • brefeldin A had variable effects on GS28 ranging from a small diminution (MOLT4, Hs578T) to a large increase in expression (H-460) at high concentrations.
  • AVP 893 has unique activity against Golgi resident proteins, as compared to pharmacological agents known to affect the Golgi. This comparison between the activity of AVP 893 and the known agents monensin, brefeldin A, and rapamycin, helps demonstrate that AVP 893 affects resident Golgi proteins in a unique fashion.
  • the first agent was added 1 hr before the second agent; 18 hour incubations followed.
  • the concentrations of agents were as follows: AVP 893, 200 ng/ml; brefeldin A, 10 ⁇ g/ml; monensin, 10 ⁇ g/ml; rapamycin, 10 nM.
  • AVP 893 decreased the expression of GS28 and GS 15 more markedly than the other three agents, and its effect on GPP 130 (causing expression of the lower, putative immature-form of the glycoprotein) was matched only by monensin.
  • brefeldin A and monensin when combined with AVP 893, dominated its activity, showing only a brefeldin A or monensin-induced 'phenotype' of expression. Only when AVP 893 was combined with rapamycin did the '893 phenotype' of protein expression occur.
  • the activity of AVP 893 against resident Golgi proteins is unique and distinct from the known pharmacological agents monenin, brefeldin A, and rapamycin.
  • COG conserved oligomeric Golgi
  • COG-Deficient and AVP Compound Treated Cells Express Similar Phenotypes
  • the cassette of resident Golgi proteins reported to be depleted in CHO cells that lack either COGl or COG2 are all type II membrane constituents and include GS 15, GS28, mannosidase II, GPP 130, Giantin, Golgin-84, and CASP ( Figure 37).
  • the first 4 are the proteins most affected by the AVP benzimidazoles, while we have not generated data on the latter 3 proteins.
  • Oka, et al tested 24 other proteins involved in the trafficking-secretory system and found them to be unaffected in COG mutant cells. We tested the expression of 15 of the latter proteins and found that AVP compounds had no consistent effects on any.
  • the proteins tested by both laboratories there is perfect agreement as to those that are or are not suppressed in either cells that lack one of the COGs or cells that are treated with AVP compounds.
  • COGl and COG2 mutant CHO cells and C0G3 knockdown HeLa cells express abnormal Golgi morphology. Instead they appear dilated or are dispersed into small vesicles.
  • MOLT-4, Vero, and 3T3 cells treated with AVP compounds also contain dilated or dispersed Golgi.
  • OVCAR-3 cells are resistant to the antiproliferative effects of AVP 893 but its Golgi proteins are inhibited by drug with reasonable potency.
  • maximal inhibition of Golgi proteins by AVP 893 in this cell line is only 70%.
  • the Golgi apparatus functions in the processing and sorting of proteins to distal cellular compartments such as the endosomes and plasma membrane.
  • COGs are important for maintaining the integrity of Golgi cisternae and many of the processing effects, but appear to have little effect on protein secretion. Indeed, the complete dismantling of Golgi cisternae by brefeldin A does not inhibit anterograde movement of newly synthesized or recycled proteins.
  • the primary function of COGs is in the retrograde movement of Golgi resident proteins. We have shown that AVP benzimidazoles inhibit secretion or cell surface expression of a number of proteins, including cytokines, immunoglobulins, CD23, and IL-4 receptor.
  • EndoH cleaves high mannose sugar i.e., the addition of complex carbohydrates, which normally occurs in the Golgi, prevents cleavage by the enzyme.
  • Other Golgi-specific functions such as addition of sialic acid residues and O-linked sugars are also blocked.
  • CD23 contains both N-linked and O-linked sugars, and in the absence of stimulus the production of this protein is minimal.
  • stimulation of the cell surface expression of CD23 is inhibited by AVP-13358 in approximately the same concentrations as observed for IgE and cytokine response inhibition.
  • AVP-13358 is inhibited by AVP-13358 in approximately the same concentrations as observed for IgE and cytokine response inhibition.
  • CD23 transcription is unaffected by AVP- 13358 except for a small induction of transcript levels after 20 hours ( Figure 44), a possible compensatory mechanism for the effects at the protein level.
  • the effects on CD23 protein (Western blot) are illustrated together with the effect on its cell surface expression (FACS analysis) in Figure 45.
  • PNGaseF reduces CD23 in lysates of control cells to a size that is about 2 kDa larger than lysates from drug-treated cells, a difference that is made up by the 2 kDa of O-linked sugar not found in the latter cells.
  • nicastrin reportedly contains no O-linked sugar and thus the size of the PNGase-treated protein is the same in both control and drug-treated cell lysates (not shown). EndoH removes all remaining sugar from CD23 of drug treated cell lysates (to the level of the PNGaseF-treated protein) while leaving control lysates unaffected.
  • AVP 893 has minimal effects on the expression of proteins involved in exocytosis ( Figure 26), particularly VAMP, SNAP23 (non-neuronal cells), and SNAP25 (neuronal cells). Accordingly, the compound does not affect the release of norepinephrine or the re-uptake of dopamine in PC 12 pheochromocytoma cells (not shown).
  • the AVP 893 analog, AVP 13358 does not inhibit degranulation of rat basophilic leukemia (RBL) cells when induced with PMA/ionomycin or IgE-antigen complexes (not shown).
  • plaque forming units PFU
  • AVP 893 suppressed plaque formation at all concentrations tested, with a total block occurring at 300 ng/ml.
  • the steep concentration-response curve suggests a non-competitive inhibition, as would be expected of a drag that acts on the host cell rather than the virus.
  • AVP 893 was demonstrated to exert antiviral activity against representatives of other viral families. As shown in TABLE 5, the spread of many other viral families that utilize the Golgi or the intermediate compartment were inhibited by AVP 893 in vitro. In contrast, viruses in the Papovaviridae and Rhabdoviridae families, which do not use the Golgi, are not inhibited by AVP compounds. Finally, a guinea pig topical HSV model has shown that AVP compounds inhibit viral infectivity in v/v ⁇ . The efficacy of topical AVP 26296 was tested in HSV-2 cutaneous and vaginal guinea pig disease models.
  • Adenoviridae D ne nucleus, lysis common cold, adeno Junin cytoplasmic assembly, buds measels, mumps, RSV,
  • R RNA virus
  • D DNA virus
  • e lipid enveloped Inhibitors of Golgi Resident Proteins
  • Preferred aspects of the described invention encompass chemical compounds of at least seventeen structural classes (disclosed in co-pending US Application No. 10/915,722; incorporated herein in its entirety by reference thereto). Compounds representing all of these series inhibit IgE response and cell proliferation in vitro at similar concentrations where Golgi resident proteins are inhibited. The latter is evidenced by inhibition of GS28 expression in non-transformed cells ( Figure 52).
  • Preferred aspects of the present invention relate to a novel mechanism for selectively modulating Golgi proteins, which impacts numerous biological processes, including allergy, cell proliferation, and viral replication. More particularly, aspects of the present invention relate to the identification and characterization of compounds that regulate this mechanism and thereby modulate the biological processes.
  • both the t-SNARE proteins, GS 15 and GS28 which are involved in the docking and fusion of vesicles in the Golgi and the intermediate compartment (IC, located between the ER and Golgi) and nicastrin, which participates in the intramembrane cleavage of proteins that translocate into the nucleus and act as transcription factors, were found to be affected by compounds that exhibit a wide range of biological activities.
  • Golgi protein targets besides GS 15, GS28, mannosidase II, and GPP 130 (shown herein to be suppressed by the AVP compounds), that influence protein trafficking/processing in disease states ⁇ inter alia allergy, cancer, viral infection), via the same or redundant pathways described.
  • pharmacologic suppression of GS28 levels has been identified by the inventors as one preferred means for selectively regulating Golgi proteins or lipids that are necessary for proliferative (or viral replicative) cellular responses
  • modulation of other Golgi-associated proteins that act in concert with GS28 or which supplement or enhance the effects of GS28 may represent other preferred means for treating proliferative/replicative disorders (as shown in schematic form in Figure 26).
  • combination therapies with other agents that target other Golgi proteins such that suppression of the pathologic trafficking/processing response is enhanced, represent another embodiment within the scope of the present invention.
  • a compelling aspect of the preferred embodiments of the present invention is that redundant protein trafficking and processing pathways, and the proteins involved therein, operate to allow cells to carry out their normal (or “good") processes, despite selectively suppressing the "bad" trafficking/processing associated with cells implicated in the disease condition (e.g., transformed, infected, etc.). Accordingly, the inventors have found that toxicity is minimized (in contrast to treatment regimens employing Brefeldin A) using the selective pharmacologic therapies disclosed herein. Other indications: SLE and MS
  • SLE is characterized immunologically by an exagerated response to self antigen. SLE involves B and T lymphocytes and dendritic cells. Desirable drug effects for treatment of SLE would include inhibition of IgG Ab response, inhibition of ThI cytokines (IFN- ⁇ , IFN- ⁇ , and IL-6), and inhibition of recruitment and proliferation of activated lymphoid cells.
  • the immune disorder immunological consequence
  • cells involved are summarized for SLE, allergy, and MS in TABLE 6.
  • MS is characterized immunologically by an exagerated response to self antigen. MS involves B and T lymphocytes. Desirable drug effects for treatment of MS would include inhibition of IgG Ab response, inhibition of ThI cytokines (IFN- ⁇ , IL-6, and TNF ⁇ ), and inhibition of recruitment and proliferation of activated lymphoid cells. TABLE 6
  • NZB/W mouse model The most widely accepted disease model for the study of SLE in animals is the NZB/W mouse model. , The survival of NZB/W Fl female mice treated with AVP 13358 was tested and the results are shown in Figure 53. There is a clear survival advantage for mice that received AVP 13358 versus those that received vehicle.
  • EAE experimental auotimmune encephalomyelitis
  • TABLE 7 shows the protocol for an ongoing NZB/W SLE model.
  • *-renal histology to include 3 specific areas of pathology (glomerulosclerosis, nephropathy, and interstitial lymphocytic infiltrates), as well as staining for IgG and IgM deposition in the glomeruli.
  • cytokines spleen cells will be cultured with ConA and CpG to assess their propensity to secrete IL-2, IL-4, IL-6, IL-10, IFN ⁇ and IFN ⁇ [0273]
  • the weight of NZB/W-F1 female mice treated with vehicle, AVP 27457, 13358, prednisolone and AVP 13358 + prednisolone was monitored over a period of several months. Body weight changes are shown in Figure 56 and serum drug levels are shown in Figure 57.
  • Mouse serum was obtained approximately every 2 months and tested for various anti-nuclear antibodies using commercial kits, as shown in Figures 59-61.
  • Total serum IgG and anti-histone antibody are shown in Figure 59.
  • Anti-DNA antibody in the serum of treated and untreated mice is shown in Figure 60, including graphs of anti-ssDNA Ig and anti-dsDNA Ig. Data has not been corrected for serum dilution (1 : 100).
  • IgM- and IgG- specific anti-dsDNA antibody responses are shown in Figure 61.
  • the preliminary results of the second SLE trial show that both AVP compounds potentially have inhibitory effects on the appearance of the inflammatory immune complexes, as well as delay the appearance of proteinuria.
  • mice treated with AVP 13358 appear to develop disease later than those receiving vehicle alone.
  • AVP 27591 and AVP 27457 provide good potency, oral bioavailability, and AVP 27591 has no active metabolites that achieve significant serum concentrations after oral administration. Both compounds attain higher serum levels than AVP 13358 at their respective maximum tolerated doses.
  • AVP 13358 provides a survival advantage in a NZB/W model of SLE, and has shown an early propensity for suppressing certain markers of the disease (urine protein, anti-DNA antibodies).
  • MS treatment experiments with a mouse model of MS (MBP immunized, Pertussis toxin treated) have shown a survival and/or morbidity advantage in AVP 13358-treated animals. Best results appear to be in the relapsing phase of the disease.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Communicable Diseases (AREA)
  • Oncology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

La présente invention concerne sous ses aspects préférés, le traitement et/ou la prévention de désordres autoimmunitaires, tels que le lupus érythémateux disséminé (SLE) et la sclérose en plaques, en utilisant une famille de dérivés de benzimidazoles.
PCT/US2007/073266 2006-07-14 2007-07-11 Utilisation de dérivés de benzimidazoles pour le traitement et/ou la prévention de désordres autoimmunitaires Ceased WO2008008841A2 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US83104106P 2006-07-14 2006-07-14
US60/831,041 2006-07-14

Publications (2)

Publication Number Publication Date
WO2008008841A2 true WO2008008841A2 (fr) 2008-01-17
WO2008008841A3 WO2008008841A3 (fr) 2008-05-29

Family

ID=38924149

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2007/073266 Ceased WO2008008841A2 (fr) 2006-07-14 2007-07-11 Utilisation de dérivés de benzimidazoles pour le traitement et/ou la prévention de désordres autoimmunitaires

Country Status (1)

Country Link
WO (1) WO2008008841A2 (fr)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20110118324A1 (en) * 2009-01-15 2011-05-19 Boris Vitalyevich Stradomskiy Method and composition for treating multiple sclerosis (variants)
RU2418582C1 (ru) * 2010-04-08 2011-05-20 Аверин Константин Михайлович 1,3-диалкилбензимидазолия галогениды - средства для лечения рассеянного склероза
US10974882B2 (en) 2018-03-28 2021-04-13 In-Tech Enterprise Ltd. Container security system
CN116114657A (zh) * 2022-10-11 2023-05-16 上海交通大学医学院附属瑞金医院 一种红斑狼疮样小鼠模型的构建方法

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040006117A1 (en) * 2002-02-15 2004-01-08 Schering Aktiengesellschaft Microglia inhibitors for interrupting interleukin 12 and IFN-gamma-mediated immune reactions
EP1651198A2 (fr) * 2003-08-08 2006-05-03 Avanir Pharmaceuticals Inhibition pharmacologique selective du trafic proteique et methodes associees de traitement de maladies humaines

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20110118324A1 (en) * 2009-01-15 2011-05-19 Boris Vitalyevich Stradomskiy Method and composition for treating multiple sclerosis (variants)
RU2418582C1 (ru) * 2010-04-08 2011-05-20 Аверин Константин Михайлович 1,3-диалкилбензимидазолия галогениды - средства для лечения рассеянного склероза
US10974882B2 (en) 2018-03-28 2021-04-13 In-Tech Enterprise Ltd. Container security system
CN116114657A (zh) * 2022-10-11 2023-05-16 上海交通大学医学院附属瑞金医院 一种红斑狼疮样小鼠模型的构建方法
CN116114657B (zh) * 2022-10-11 2024-06-11 上海交通大学医学院附属瑞金医院 一种红斑狼疮样小鼠模型的构建方法

Also Published As

Publication number Publication date
WO2008008841A3 (fr) 2008-05-29

Similar Documents

Publication Publication Date Title
US20050256179A1 (en) Selective pharmacologic inhibition of protein trafficking and related methods of treating human diseases
US8329656B2 (en) Use of a PDE5 inhibitor for treating and preventing hypopigmentary disorders
CA2304505A1 (fr) Methodes permettant d'augmenter les taux d'apolipoproteine dans le traitement de maladies neurodegeneratives
HUP0101543A2 (hu) Női szexuális zavarok kezelésére szolgáló gyógyszerkészítmények
CN108430475B (zh) 用于治疗慢性咳嗽的奥维匹坦
AU2024200537A1 (en) Compositions and methods for treating galactosemia
JP2020513005A (ja) Ｃｃｒ３阻害剤を用いて、加齢性機能障害を治療するための方法及び組成物
AU2016226006A1 (en) Method of treatment with tradipitant
TW201605443A (zh) 治療ｘ染色體脆折症及相關病症的方法
WO2008008841A2 (fr) Utilisation de dérivés de benzimidazoles pour le traitement et/ou la prévention de désordres autoimmunitaires
EP4395753A1 (fr) Fenfluramine pour le traitement de maladies et d'affections démyélinisantes
EP3197445B1 (fr) Proline ou dérivés de proline pour le traitement de la démence
Jin et al. Zeaxanthin attenuates OVA-induced allergic asthma in mice by regulating the p38 MAPK/β-catenin signaling pathway
US20230181572A1 (en) Carbamoyl cyclohexane derivatives for treating autism spectrum disorder
KR102845328B1 (ko) 인지 장애의 치료시 사용하기 위한 3β-(벤질옥시)-17α-메틸-프레근-5-엔-20-온
US10512637B2 (en) Use of inhibitor of cystine-glutamate transporter
JP2025000672A (ja) Ｌｏｕ０６４を使用した治療の方法
Zhang et al. E3 ubiquitination ligase XIAP lightens diabetes‐induced cognitive impairment by inactivating TXNIP‐ERS‐mediated neuronal injury
Hung et al. Regulation of stromal cell-derived factor-1 and exhaled nitric oxide in asthmatic children following montelukast and ketotifen treatment
JP2015532927A (ja) ホスファチジルイノシトール３−キナーゼ阻害剤およびアロマターゼ阻害剤を含む組合せ医薬
RU2277096C2 (ru) Средства против слабоумия, содержащие производное 2-арил-8-оксодигидропурина в качестве активного ингредиента
RU2796005C2 (ru) 3β-(БЕНЗИЛОКСИ)-17α-МЕТИЛ-ПРЕГН-5-ЕН-20-ОН ДЛЯ ПРИМЕНЕНИЯ ПРИ ЛЕЧЕНИИ КОГНИТИВНЫХ РАССТРОЙСТВ
CN118401242A (zh) 使用lou064的治疗方法
CN116322689A (zh) 用于治疗与冠状病毒病毒感染相关的呼吸窘迫和细胞因子风暴综合征的多功能配体的用途
Zohar A Congratulatory Note

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 07799493

Country of ref document: EP

Kind code of ref document: A2

NENP Non-entry into the national phase

Ref country code: DE

NENP Non-entry into the national phase

Ref country code: RU

32PN Ep: public notification in the ep bulletin as address of the adressee cannot be established

Free format text: NOTING OF LOSS OF RIGHTS PURSUANT TO RULE 112(1)EPC

122 Ep: pct application non-entry in european phase

Ref document number: 07799493

Country of ref document: EP

Kind code of ref document: A2