USE OF BENZIMIDAZOLE DERIVATIVES FOR THE TREATMENT AND/OR PREVENTION OF AUTOIMMUNE DISORDERS

Cross-Reference to Related Applications

[0001] The present application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Application No. 60/831,041 filed on July 14, 2006, the disclosure of which is incorporated by reference herein in its entirety.

Background of the Invention Field of the Invention

[0002] 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. Description of the Related Art

[0003] Humans and other mammals may exhibit undesirable immune reactions not only to foreign substances (allergies), but also to their own components (autoimmune disorders). Such 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.

[0004] 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. Because of its wide variety of symptoms, diagnosis is often difficult and requires a high degree of awareness among physicians. 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.

[0006] Current medications cannot cure SLE, but only control symptoms in an attempt to prevent or slow organ damage. Because most SLE symptoms are caused by inflammation, first-line medications are anti-inflammatory. A person having mild disease or symptoms that affect quality of life, but does not have life-threatening organ problems, may be treated with nonsteroidal anti-inflammatory drugs (NSAIDs) or antimalarial drugs (such as hydroxychloroquine; Plaquenil).

[0007] 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.

[0008] The most significant side effect of antimalarials is damage to the tissue that lines the eye (retina); this is rare when appropriate doses are used. Regular eye examinations are critical to preventing eye damage. An initial eye examination will be done if you are taking Plaquenil or Aralen for more than 3 months. Other side effects include occasional rash, nausea, or diarrhea.

[0009] 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.

[0010] 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.

[0011] Accordingly, there remains an important unmet need for development of therapeutic and/or prophylactic agents that target the mechanisms of inappropriate allergic reactions, both to foreign (allergy) and self (autoimmune) substances.

Summary of the Invention

[0012] Methods for treating and/or preventing an autoimmune disorder are disclosed in accordance with embodiments of the present invention. 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:

[0013] wherein 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 ;

[0014] wherein 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~); and

[0015] wherein 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,

(2) [0016] wherein X and Y are selected independently from the group consisting of alkyl, alkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, hydroxy, halogen, NO ₂ ,

CF ₃ , OCF ₃ , NH ₂ , NHR ₃ , NR ₃ R ₄ and CN;

[0017] wherein 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;

[0018] wherein 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,

[0020] wherein 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;

[0021] wherein R is selected from the group consisting of H, CH3, C2H5, C3H7, C4H9, CH2Ph, and CH2C6H4-F(p-);

[0022] wherein 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

[0023] wherein the 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,

[0024] wherein 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;

[0025] wherein R is selected from the group consisting of H, CH3, C2H5, C3H7, C4H9, CH2Ph, and CH2C6H4-F(p-);

[0026] wherein 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

[0027] wherein the substituents on said 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,

[0028] wherein 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;

[0029] wherein R is selected from the group consisting of H, CH3, C2H5, C3H7, C4H9, CH2Ph, and CH2CH4-F(p-);

[0030] wherein 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;

[0031] wherein 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

[0032] wherein the substituents on said 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,

(6)

[0033] wherein 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;

[0034] wherein R is selected from the group consisting of H, CH3, C2H5, C3H7, C4H9, CH2Ph, CH2C6H4-F(p-); and

[0035] wherein 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,

(9)

[0036] wherein 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 NHCORj;

[0037] wherein 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

[0039] 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, substituted isoqunolinyl, multi-ring cycloaryl, multi-ring cycloheteroaryl, benzyl, heteroaryl- methyl, substituted benzyl, substituted heteroaryl-methyl alkyl, dialkylaminoalkyl, cycloalkyl, cycloalkyl containing 1-3 heteroatoms, substituted cycloalkyl, substitute cycloalkyl containing 1-3 heteroatoms, multi-ring cycloalkyl, multiring cycloalkyl containing 1-3 heteroatoms, fused-ring aliphatic, fused-ring aliphatic containing 1-3 heteroatoms, cyclopropyl, substituted cyclopropyl, cyclobutyl, substituted cyclobutyl, cyclopentyl, pyrrole, piperidine, substituted cyclopentyl, cyclohexyl, substituted cyclohexyl, cycloheptyl, substituted cycloheptyl, bicycloheptyl, substituted pyrrole, substituted piperidine, bicyclooctyl, bicyclononyl, substituted bicycloalkenyl, adamantyl, and substituted adamantyl, heterocyclic ring, and substituted heterocyclic ring,

[0040] wherein 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;

[0041] wherein 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;

[0042] wherein R3 and R4 are independently selected from the group consisting of H, alkyl, aryl, heteroaryl and COR';

[0043] wherein 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;

[0044] wherein the substituent on 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';

[0045] wherein 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

[0046] 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,

[0047] 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;

[0048] n is an integer from one to three;

[0049] m is an integer from one to four;

[0050] 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

[0051] 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,

[0052] wherein 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;

[0053] wherein 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;

[0054] 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', CN, CF3, OCF3, NO2, NR 'R', NHCOR' and CONR 'R';

[0055] wherein 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,

[0057] wherein 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;

[0058] wherein 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', CN, CF3, OCF3, NO2, NR 'R', NHCOR' and CONR'R';

[0060] wherein 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

[0061] 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,

[0062] wherein 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;

[0063] wherein 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;

[0064] wherein 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;

[0065] 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', CN, CF3, OCF3, NO2, NR'R', NHCOR' and CONR'R'; and

[0066] wherein 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,

[0067] wherein 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;

[0068] wherein 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";

[0069] wherein 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';

[0071] wherein 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

[0072] wherein 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.

[0073] In preferred embodiments, a method is disclosed for treating and/or preventing SLE. 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:

AVP 13358

[0074] In another preferred embodiment of the method for treating and/or preventing SLE, the compound is:

AVP 27457

[0075] In another preferred embodiment of the method for treating and/or preventing SLE, the compound is:

AVP 27591

[0076] In another preferred embodiment of the method for treating and/or preventing SLE, the compound is:

AVP 26296

[0077] A method is also disclosed for treating and/or preventing 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:

AVP 13358

[0078] In another preferred embodiment of the method for treating and/or preventing MS, the compound is:

AVP 27457

[0079] In another preferred embodiment of the method for treating and/or preventing MS, the compound is:

AVP 27591

[0080] In another preferred embodiment of the method for treating and/or preventing MS, the compound is:

AVP 26296

[0081] 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. In preferred embodiments, the immune disorder is an allergy or an autoimmune disorder. More preferably, the autoimmune disorder is SLE or MS. In preferred variations, the Golgi resident protein is selected from the group consisting of GS15, GS28, mannosidase II, and GPP130. hi further preferred embodiments, the compound is selected from the group consisting of compounds 1-42.

Brief Description of the Drawings

[0082] Figure 1 is a schematic illustrating intracellular protein trafficking. [0083] Figure 2 shows the IgE response to antigen ex vivo. [0084] Figure 3 shows the IgE response to IL-4 + αCD40 Ab in human PBL in vitro.

[0085] Figure 4 illustrates murine spleen T cell cytokine responses in vitro. [0086] Figure 5 shows human PBL T cell cytokine responses. [0087] Figures 6 show CD23 on human monocytes. [0088] Figure 7 shows spleen cell proliferation response to AVP 893. [0089] Figure 8 shows proliferation of human PBL in response to stimulus and drug in vitro. [0090] Figure 9 shows an NCI 60-cell panel. [0091] Figure 10 is a schematic of a BAL protocol #1 and illustrates the cells in

BAL wash.

[0092] Figure 11 shows the AHR response in vivo. [0093] Figure 12 shows the effect of AVP 25752 on B 16-Fl mouse melanoma tumor growth. [0094] Figure 13 shows the effect of AVP 893 on HS294t human melanoma tumor growth. [0095] Figure 14 is a dose response of AVP 13358 on various biochemical assays. [0096] Figure 15 is a kinase screen of AVP 13358. [0097] Figure 16 shows the PowerBlot results of the effect of AVP 893 on protein expression.

[0098] Figure 17 shows the time course of AVP 893 action in B16 cells. [0099] Figure 18 shows the effect of AVP 893 on nicastrin and GS28 expression in various cells at 16 hours.

[0100] Figure 19 shows the effect of AVP 893 on nicastrin, calnexin and GS28 expression in various cells overnight.

[0101] Figure 20 shows the effect of AVP 893 on nicastrin, n-gly, calnexin and GS28 expression in various cells overnight.

[0102] Figure 21 shows inhibition of stimulated protein expression in BALB/c spleen cells by AVP 893.

[0103] Figure 22 shows dose-responsive inhibition of PMA/ionomycin-stimulated nicastrin and GS28 expression in BALB/c spleen cells by various compounds.

[0104] Figure 23 shows the PMA effect on AVP 893 inhibition of PBL proliferation response to IL-4/αCD40 Ab.

[0105] Figure 24 shows the selective dose-response of AVP 893 in down- regulating IL-4/αCD40 Ab induced protein expression after 48 hours in the presence and absence of PMA.

[0106] Figure 25 shows GS28 mRNA response to AVP 893 in human PBL.

[0107] Figure 26 is a schematic showing involvement of various cellular proteins in protein trafficking pathways.

[0108] Figure 27 shows dose-responsive inhibition by AVP 893 of GS 15 and GS28 expression in 18-20 hour cultures of B16-F10 cells.

[0109] Figure 28 shows the time-dependent effect of AVP 893 on mannosidase II expression.

[0110] Figure 29 shows the time-dependent effect of AVP 893 on Rabό expression in Vero cells.

[0111] Figure 30 shows shows the effect of AVP 893 on Golgi morphology in MOLT4 cells.

[0112] Figure 31 shows the effect of AVP 893 on GPP130 distribution in NIH- 3T3 cells.

[0113] Figure 32 shows the Rabό distribution in B16 cells.

[0114] Figure 33 compares the NCI results with AVP 893 and Brefeldin A.

[0115] Figure 34 shows the effects of AVP 893 and Brefeldin A on GS28 and nicastrin expression.

[0116] Figure 35 shows the activity of AVP 893 on Golgi resident proteins compared to known pharmacological agents in 3T3 cells.

[0117] Figure 36 shows the differential effects of AVP 893, brefeldin A, and nocodozole on mannosidase II expression.

[0118] Figure 37 compares the effect of AVP compounds and COG deficient mutants on cell phenotype.

[0119] Figure 38 shows the effects of AVP 13358 on GS28 and βCOP expression in HeLa Hl cells by confocal microscopy and Western blotting.

[0120] Figure 39 shows the protective effect of proteosome and lysosome inhibitors when co-treating with AVP 13358 in MOLT-4 and 3T3 cells.

[0121] Figure 40 shows the protective effect of proteosome and lysosome inhibitors when co-treating with AVP 13358 in B16-F10 cells.

[0122] Figure 41 compares GS28, GPP130 and TGN38 expression in H460 cells.

[0123] Figure 42 compares GS28, GPP 130 and GMl 30 expression in HT-29 cells.

[0124] Figure 43 shows the concentration-dependent effects of AVP 893 on Golgi proteins in sensitive and resistant cells.

[0125] Figure 44 shows the effect of AVP 13358 on CD23 transcription in vitro.

[0126] Figure 45 shows the expression of CD23 at the plasma membrane and at the total cell protein level in relation to AVP 13358 concentration.

[0127] Figure 46 shows the reversibility of the effect of AVP 13358 on CD23 glycosylation.

[0128] 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.

[0129] Figure 48 shows the susceptibility of CD23 sugar residues to the effects of endoglycosidases.

[0130] Figure 49 quantitates the effect of AVP 893 on HSV-2 propagation in Vero cells in vitro.

[0131] Figure 50 shows the effect of AVP 893 on HSV-2 propagation in Vero cells in vitro though the expression of glycoprotein E.

[0132] Figure 51 compares the effect of AVP-26296, Acyclovir, Docosanol, and vehicle in an in vivo model of HSV-2 infection.

[0133] Figure 52 is a schematic of the cellular effect of AVP compounds.

[0134] Figure 53 shows the effect of AVP 13358 on survival of NZBAV-Fl female mice (SLE model).

[0135] Figure 54 shows results of pilot MS study.

[0136] Figure 55 shows scoring results from a second MS model study.

[0137] Figure 56 shows the the effect of drug treatment on the body weight of NZBW-Fl female mice.

[0138] Figure 57 shows periodic serum drug levels 1 hr after dosing.

[0139] Figure 58 shows the impact of drug treatment on proteinuria and survival in the second NZBAV SLE model.

[0140] Figure 59 shows effect of AVP 13358 on total serum IgG amd anti-histone Ig in the mouse SLE model.

[0141] Figure 60 shows effect of AVP 13358 on total anti-DNA antibody (single- stranded and double-stranded) in the mouse SLE model.

[0142] Figure 61 shows effect of AVP 13358 on anti-dsDNA IgM and IgG detected in mouse SLE model.

[0143] Figure 62 compares oral bioavailability of back-up compounds with AVP 13358.

[0144] Figure 63 compares the potency of lead 2-phenyl benzimidazole coumpounds for inhibition of IgE (mouse spleen cells) and cytokines (human PBL).

[0145] Figure 64 shows efficacy of back-up compounds in asthma models in vivo.

Detailed Description of the Preferred Embodiment

[0146] 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. 90/006,945 (reexam), 10/951,515, 10/795,006, 10/661,139, 10/661,296, 10/821,667,

10/508,968, 11/168,711; all of which are incorporated herein in their entirety by reference thereto. Synthetic methods are detailed in the inventors earlier-filed applications (listed above). More recently, it was discovered that these same benzimidazole derivatives also modulate certain Golgi proteins that are involved in intracellular protein trafficking and/or processing. See e.g., U.S. Patent Application No. 10/915,722 (2005/0256179-Al ); incorporated herein in its entirety by reference thereto. Thus, Applicants postulate that modulation of protein trafficking/processing may be a fundamental mechanism through which these benzimidazole derivatives exert their various biological actions. Applicants now show in accordance with preferred embodiments of the present invention that in addition to suppressing inappropriate immune reactions to foreign substances (allergies), the benzimidazole derivatives also suppress inappropriate immune reactions associated with autoimmune disorders such as SLE and MS.

[0147] The compounds were not identified on the basis of a target-based assay but rather based on their cellular activity. Thus, the mechanism of action has until recently been a mystery. The activity profile of these compounds is highly unusual and suggests that their shared mechanism of action is novel. These agents do not affect the activity of more than 70 kinases and other enzymes. Moreover, a screen of drug activity on the expression of over 950 proteins revealed only a handful of modulated proteins in vitro. These results and the studies subsequent to this form the basis of the patent application described herein.

[0148] Several distinct series of chemical compounds are described that have in common a suppressive action on the expression of IgE, elicitation of cytokines, expression of membrane receptors, and cellular proliferation. These series include the following compounds:

[0149] wherein 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;

[0150] wherein 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~); and

[0151] wherein 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,

(2) [0152] wherein X and Y are selected independently from the group consisting of alkyl, alkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, hydroxy, halogen, NO ₂ ,

CF ₃ , OCF ₃ , NH ₂ , NHR ₃ , NR ₃ R ₄ and CN;

[0153] wherein 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;

[0154] wherein 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,

[0156] wherein 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;

[0157] wherein R is selected from the group consisting of H, CH3, C2H5, C3H7, C4H9, CH2Ph, and CH2C6H4-F(p-);

[0158] wherein 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

[0159] wherein the 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,

[0160] wherein 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;

[0161] wherein R is selected from the group consisting of H, CH3, C2H5, C3H7, C4H9, CH2Ph, and CH2C6H4-F(p-);

[0162] wherein 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

[0163] wherein the substituents on said 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,

[0164] wherein 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;

[0165] wherein R is selected from the group consisting of H, CH3, C2H5, C3H7, C4H9, CH2Ph, and CH2CH4-F(p-);

[0166] wherein 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;

[0167] wherein 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

[0168] wherein the substituents on said 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,

(6)

[0169] wherein 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;

[0170] wherein R is selected from the group consisting of H, CH3, C2H5, C3H7, C4H9, CH2Ph, CH2C6H4-F(p-); and

[0171] wherein 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,

(9)

[0172] wherein 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 ;

[0173] wherein 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;

[0174] wherein 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, substituted isoqunolinyl, multi-ring cycloaryl, multi-ring cycloheteroaryl, benzyl, heteroaryl- methyl, substituted benzyl, substituted heteroaryl-methyl alkyl, dialkylaminoalkyl, cycloalkyl, cycloalkyl containing 1-3 heteroatoms, substituted cycloalkyl, substitute cycloalkyl containing 1-3 heteroatoms, multi-ring cycloalkyl, multiring cycloalkyl containing 1-3 heteroatoms, fused-ring aliphatic, fused-ring aliphatic containing 1-3 heteroatoms, cyclopropyl, substituted cyclopropyl, cyclobutyl, substituted cyclobutyl, cyclopentyl, pyrrole, piperidine, substituted cyclopentyl, cyclohexyl, substituted cyclohexyl, cycloheptyl, substituted cycloheptyl, bicycloheptyl, substituted pyrrole, substituted piperidine, bicyclooctyl, bicyclononyl, substituted bicycloalkenyl, adamantyl, and substituted adamantyl, heterocyclic ring, and substituted heterocyclic ring,

[0176] wherein 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;

[0177] wherein 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;

[0178] wherein R3 and R4 are independently selected from the group consisting of H, alkyl, aryl, heteroaryl and COR';

[0179] wherein 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;

[0180] wherein the substituent on 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';

[0181] wherein 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

[0182] 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,

[0183] 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;

[0184] n is an integer from one to three;

[0185] m is an integer from one to four;

[0186] 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

[0187] 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,

[0188] wherein 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;

[0189] wherein 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;

[0190] 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', CN, CF3, OCF3, NO2, NR 'R', NHCOR' and CONR 'R';

[0191] wherein 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,

[0193] wherein 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;

[0194] wherein 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', CN, CF3, 0CF3, NO2, NR 'R', NHCOR' and CONR'R';

[0196] wherein 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

[0197] 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,

[0198] wherein 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;

[0199] wherein 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;

[0200] wherein 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;

[0201] wherein said 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

[0202] wherein 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,

[0203] wherein 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;

[0204] wherein 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";

[0205] wherein 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';

[0207] wherein 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

[0208] wherein 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.

[0209] Numerous specific compounds that exemplify the generic formulas 1-42 have been synthesized and tested in accordance with preferred aspects of the present invention. For the sake of clarity, hydrogen atoms have been left out of many of the structural diagrams. In particular, many of the nitrogen atoms are depicted without complete valance (3 bonds); it is understood, therefore that where an N is shown with only 2 bonds, the third bond is to an H (not shown). Likewise, the carbon rings are shown without H atoms. Some of the preferred compounds that Applicant has synthesized and tested are listed below in TABLE 1.

TABLE 1 AVP NUMBER STRUCTURE

[0210] Some preferred aza-benzimidazole derivatives that have been synthesized, tested and found to be active are listed below in TABLE 2. The aza-benzimidazoles listed in TABLE 2 and below were synthesized as detailed in co-pending US Application No. 10/661,296; which is incorporated in its entirety by reference thereto. As mentioned above with respect to TABLE 1, H atoms are not shown in many of the structures.

TABLE 2

MOL FORMULA MOL WEIGHT COMPOUND NUMBER

C26 H31 N5 O2 445.5639 26294

C34 H39 N5 O2 549.7151 26296

C25 H24 N6 O2 440.5046 26345

C28 H35 N5 02 473.6175 26375

C29 H28 N6 02 492.5802 26390

C34 H37 N5 03 563.6983 26425

C29 H26 N6 03 506.5634 26427

C34 H39 N5 03 565.7141 26436

C29 H28 N6 03 508.5792 26437

C28 H33 N5 03 487.6007 26624

C29 H28 N6 02 492.5802 26648

C24 H17 N7 O2 435.4453 26991

C25 H24 N6 O2 440.5046 26993

C26 H26 N602 454.5314 26994

C24H17N7O2 435.4453 26995

C26 H26 N602 454.5314 26996

C24H17N7O2 435.4453 26997

C26 H26 N602 454.5314 26998

C25H18N6O2 434.4572 26999

C25H17CIN6O2 468.9023 27000

C25H16CI2N6O2 503.3474 27001

C25H17FN6O2 452.4473 27002

C26H24N6O2 452.5156 27003

C29 H26 N603 506.5634 27005

C34H35N5O4 577.6815 27006

-αfiCG

C27 H33 N5 02 459.5907 27008

C25 H24 N6 02 440.5046 27009

C27 H33 N5 O2 459.5907 27010

C25 H24 N6 02 440.5046 27011

C27 H33 N5 02 459.5907 27012

C26 H25 N5 O2 439.5165 27013

C26 H24 Cl N5 O2 473.9616 27014

C26 H23 CI2 N5 02 508.4067 27015

C26 H24 F N5 O2 457.5066 27016

C27 H27 N5 03 469.5423 27017

C27 H31 N5 02 457.5749 27018

-P

C30 H33 N5 O3 511.6227 27020

C26 H26 N6 O2 454.5314 27021

C31 H37 N5 O2 511.6663 27022

C27 H33 N5 02 459.5907 27023

C26 H26 N6 02 454.5314 27024

C28 H35 N5 02 473.6175 27025

C26 H26 N6 02 454.5314 27026

C28 H35 N5 O2 473.6175 27027

C27 H27 N5 02 453.5433 27028

C27 H26 Cl N5 02 487.9884 27029

C27 H25 CI2 N5 02 522.4335 27030

C27 H26 F N5 O2 471.5334 27031

C28 H29 N5 03 483.5691 27032

C28 H33 N502 471.6017 27033

C24H17N7O2 435.4453 27034

C29 H28 N6 O2 492.5802 27035

C25 H24 N602 440.5046 27036

C26 H26 N6 O2 454.5314 27037

C24H17N7O2 435.4453 27038

C26 H26 N602 454.5314 27040

C26 H26 N6 O2 454.5314 27041

C25H18N6O2 434.4572 27042

C25H17CIN6O2 468.9023 27043

C25H16CI2N6O2 503.3474 27044

C25H17FN6O2 452.4473 27045

C28H35N5O2 473.6175 27046

C24H17N7O2 435.4453 27047

C26 H26 N602 454.5314 27049

C31 H37 N502 511.6663 27050

QTJ H33 N5 O2 459.5907 27051

C28H35N5O2 473.6175 27052

C26 H26 N6 O2 454.5314 27053

C26 H26 N602 454.5314 27054

C28 H35 N502 473.6175 27055

C27 H27 N502 453.5433 27056

C27 H26 Cl N502 487.9884 27057

C27 H25 CI2 N502 522.4335 27058

C27 H26 F N502 471.5334 27059

C28 H29 N503 483.5691 27060

C28 H33 N502 471.6017 27061

C31 H37 N503 527.6653 27062

C25H18N6O2 434.4572 27063

C30 H29 N502 491.5921 27064

C26 H25 N502 439.5165 27065

C27 H27 N502 453.5433 27066

C25H18N6O2 434.4572 27067

C25H18N6O2 434.4572 27068

C26H19N5O2 433.4691 27069

C26H18CIN5O2 467.9142 27070

C26H17CI2N5O2 502.3593 27071

C26H18FN5O2 451.4592 27072

C27 H21 N503 463.4949 27073

C27 H25 N502 451.5275 27074

C30 H29 N503 507.5911 27075

C25H17CIN6O2 468.9023 27076

C27 H20 Cl N503 497.94 27077

C30 H26 Cl N503 540.0204 27078

C26 H24 F N5 O2 457.5066 27079

C27H26FN5O2 471.5334 27080

C25H17FN6O2 452.4473 27112

C25H17FN6O2 452.4473 27113

C27 H26 F N502 471.5334 27114

C26H17CIFN5O2 485.9043 27115

C27 H20 F N5 O3 481.485 27116

C26 H24 N6 O2 452.5156 27117

C31 H35 N502 509.6505 27118

C27 H31 N5 O2 457.5749 27119

C28 H33 N502 471.6017 27120

C28H33N5O2 471.6017 27122

C26H24N6O2 452.5156 27123

C28 H33 N5 02 471.6017 27124

C27 H25 N5 02 451.5275 27125

C27 H24 Cl N5 02 485.9726 27126

C27 H23 CI2 N5 02 520.4177 27127

C27 H24 F N5 02 469.5176 27128

C28 H27 N5 03 481.5533 27129

C28 H31 N5 02 469.5859 27130

C31 H35 N5 O3 525.6495 27131

C31 H33 N5 03 523.6337 27132

C26 H24 N6 O2 452.5156 27121

C26 H24 Cl N5 02 473.9616 27133

C25 H17 Cl N6 02 468.9023 27134

C27 H26 Cl N502 487.9884 27135

C27 H24 Cl N502 485.9726 27136

C25H16CI2N6O2 503.3474 27137

C27 H25 CI2 N502 522.4335 27138

C27 H25 CI2 N502 522.4335 27139

C25H17FN6O2 452.4473 27140

C31 H35 N5 O3 525.6495 27141

C31 H35 N503 525.6495 27142

C30 H26 Cl N503 540.0204 27143

C31 H33 N503 523.6337 27144

C31 H28 F3 N5 03 575.5882 27238

C27 H24 F3 N5 03 523.5126 27237

C34 H37 N5 03 563.6983 27297

C34 H39 N5 03 565.7141 27298

C31 H28 F3 N5 03 575.5882 27326

C33 H39 N7 03 S 613.7831 27333

C34 H39 N5 04 581.7131 27433

C30 H28 F N5 O2 509.5822 27456

C26 H24 F N5 02 457.5066 27457

C27 H26 F N5 02 471.5334 27458

C30 H35 N5 02 497.6395 27007

C26 H24 F N5 02 457.5066 27565

C26 H23 F2 N5 02 475.4967 27591

C26 H24 F N5 02 457.5066 27590

C29 H28 N6 03 508.5792 27004

C30 H35 N5 03 513.6385 27019

C27 H26 F N5 03 487.5324 27937

[0211] Other aza-benzimidazoles within the disclosed genera include:

TABLE 2(b) (Additional aza-benzimidazoles within the disclosed genera)

MOL FORMULA MOL WEIGHT COMPOUND

NUMBER

C27 H26 F N5 03 487.5324 27937

C26 H23 F2 N5 02 475.4967 27969

C26 H23 F2 N5 02 475.4967 27978

C26 H17 F2 N5 02 469.4493 27987

C26 H15 F4 N5 O2 505.4295 27988

C26 H17 F2 N5 02 469.4493 27998

C26 H17 F2 N5 O2 469.4493 27999

C26 H18 F N5 O2 451.4592 28011

C26 H 16 F3 N5 02 487.4394 28019

C26 H18 F N5 02 451.4592 28023

C27 H18 F3 N5 02 501.4662 28024

1OK>* C27 H18 F3 N5 O2 501.4662 28025

[0212] Recently, the mechanism of action of these compounds was investigated in order to link the diverse actions of these compounds. These studies led to the revelation that the trafficking and processing of cellular proteins (Figure 1) is affected by drug treatment in vitro. This novel mechanism of action has no known duplication by drugs utilized in the treatment of human disease. Moreover, only a handful of chemicals used in the dissection of molecular mechanisms of cellular processes are known to inhibit intracellular protein trafficking. The compounds described herein affect the expression of particular proteins responsible for movement and processing of cellular proteins through the Golgi in all primary cells and many tumor cell lines. Moreover, studies designed to track intracellular protein movement show that the compounds block the Golgi movement of proteins through the Golgi in vitro by a mechanism that is distinct from that utilized by other known inhibitors such as Monensin and Brefeldin A. The described activity helps explain the known diverse actions of the AVP compounds and successfully predicts additional activity, particularly inhibition of viral propagation. Assays

[0213] In one preferred embodiment, 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. 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.

[0214] Ex Vivo Assay - This system begins with in vivo antigen priming and measures secondary antibody responses in vitro. The basic protocol was documented and optimized for a range of parameters including: antigen dose for priming and time span following priming, number of cells cultured in vitro, antigen concentrations for eliciting secondary IgE (and other Ig' s) response in vitro, fetal bovine serum (FBS) batch that will permit optimal IgE response in vitro, the importance of primed CD4+ T cells and hapten-specific B

cells, and specificity of the ELISA assay for IgE (Marcelletti and Katz, Cellular Immunology 135:471-489 (1991); incorporated herein by reference).

[0215] The actual protocol utilized for this project was adapted for a more high throughput analyses. BALB/cByj mice were immunized i.p. with 10 μg 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 μg/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 μg/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 .

[0216] Culture supernatants were collected after 8 days and Ig's were measured by a modification of the specific isotype-selective ELISA assay described by Marcelletti and Katz {Supra). The assay was modified to facilitate high throughput. ELISA plates were prepared by coating with DNP-KLH overnight. After blocking with bovine serum albumin (BSA), an aliquot of each culture supernatant was diluted (1:4 in phosphate buffered saline (PBS) with BSA, sodium azide and Tween 20), added to the ELISA plates, and incubated overnight in a humidified box at 4° C. IgE levels were quantitated following successive incubations with biotinylated-goat antimouse IgE (b-GAME), AP-streptavidin and substrate.

[0217] 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.

[0218] In Vivo Assay - Compounds found to be active in the ex vivo assay (above) were further tested for their activity in suppressing IgE responses in vivo. Mice receiving low- dose radiation prior to immunization with a carrier exhibited an enhanced IgE response to antigen sensitization 7 days later (Bargatze and Katz, J. Immunol. 125:2306-2310, (1980)).

Administration of the test compounds immediately prior to and after antigen sensitization, measured the ability of that drug to suppress the IgE response. The levels of IgE, IgGl and IgG2a in serum were compared.

[0219] 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 μg 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 μl/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 μg 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.

[0220] 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

[0221] These series of compounds were initially identified on the basis of their IgE-blocking activity in an ex vivo IgE response protocol (Figure 2), and the biological activity of all analogs are characterized on the basis of their activity in this assay. Activity in the ex vivo assay is corroborated in the in vitro assay of B cell response to IL-4 / anti-CD40 Ab-stimulated IgE in human PBL (Figure 3) using standard procedures, and mouse splenic B cells (not shown). Drug action on T cells was shown by testing T cell cytokine responses to various stimuli in vitro. The response of a cadre of cytokines and chemokines to several alternative stimuli was tested in T cells from both mouse spleen and human PBL. The data for cytokines that were enhanced at least 10-fold by stimulus are shown in Figures 4 and 5. 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 μg/ml) or ConA (5 μg/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. Using a similar approach for stimulating the expression of IgE, both CD23 (the B cell IgE receptor; Figure 6) and the IL-4 receptor (IL- 4Rα, not shown) were potently blocked by AVP 13358 on human monocytes and murine B cells in vitro. The fourth activity discovered for these compounds was the inhibition of cellular proliferation. This effect was noted first in the proliferation of primary cells in response to a variety of stimuli, including IL-4/anti-CD40 Ab, PMA/ionomycin, LPS, ConA, anti-CD3 antibody, or epidermal growth factor (EGF). Drug effects on the proliferation of mouse spleen cells and human PBL are shown in Figures 7 and 8, respectively. Compounds of these series also were shown to have anti-proliferative effects on tumor cell growth in vitro. AVP compounds were submitted to the NCI for testing in their 60-cell screening panel. 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. Sulforhodamine B (SRB) Assay Protocol (adapted from NCI protocol)

[0222] For a typical screening experiment, cells are inoculated into 96 well microtiter plates in 100 μl 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. Following drug addition, the plates are incubated for an additional 48 h at 37°C, 5 %/10% CO2, and 100 % relative humidity. For adherent cells, the assay is terminated by the addition of cold TCA.

Cells are fixed in situ by the gentle addition of 50 μl 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 μl) at 0.4 % (w/v) in 1 % acetic acid is added to each well, and plates are incubated for 10 minutes at room temperature. After staining, unbound dye is removed by washing five times with 1 % acetic acid and the plates are air-dried. Bound stain is subsequently solubilized with 10 mM trizma base, and the absorbance is read on an automated plate reader at a wavelength of 515 nm. For suspension cells, the methodology is the same except that the assay is terminated by fixing settled cells at the bottom of the wells by gently adding 50 μl of 80 % TCA (final concentration, 16 % TCA). Using the seven absorbance measurements [time zero, control growth, and test growth in the presence of drug at the five concentration levels], the percentage growth is calculated at each of the drug concentrations levels.

[0223] Testing performed at the National Cancer Institute (NCI) revealed the compounds to be novel both in structure and the profile of cells against which the compounds were active. The latter is indicative of a distinctly different mechanism of action for the AVP benzimidazoles compared to other compounds submitted to the NCI for this analysis. Corroboration of In Vitro Action by In Vivo Activity.

[0224] Several of the compounds have been tested in in vivo models of human disease that also reflect the results observed in vitro. Two models of allergic asthma were tested in mice, the broncho-alveolar lavage (BAL) and airway hyper-reactivity (AHR) models. Both models are initiated by a similar protocol to generate an "allergic" response to chicken ovalbumin (OVA). The BAL model measures cellular and cytokine infiltration into the lungs in response to nebulized OVA. AVP drug administration suppresses the eosinophil and lymphocyte infiltration in the standard protocol (Figure 10) as well as other similar BAL models. Where increases of BAL cytokines were noted, drug also suppressed these responses (not shown). Airway hyper-reactivity response to methacholine challenge also was inhibited by drug in these mice (Figure 11).

[0225] Compounds have been tested for activity in a number of in vivo tumor models. Subcutaneous inoculation of B16 melanoma tumor cells into syngeneic (C57BL/6)

mice results in the rapid tumor growth. Drug (AVP 25752) treatment of mice that had been inoculated with tumor cells experienced a significant decrease in the rate of tumor growth compared to vehicle-treated mice (Figure 12). Similar but more dramatic results were obtained when 8 million Hst294t human melanoma tumor cells were inoculated into 25 Nu/Nu mice in a xenographic model (Figure 13). Twelve days later mice were separated into two groups and treated with AVP 893 (10-40 mg/kg/day) or vehicle i.p. daily.

[0226] Thus, 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

[0227] In an effort to understand how these compounds might be acting at the cellular and molecular level, several screens of drug activity were initiated. The first 2 screens were designed to test the activity of drug on certain binding events and the activity of a variety of enzymes in vitro (Figures 14 and 15). The results of in vitro biochemical assays (as indicated on the Y-axis) are shown in Figure 14. AVP 13358 (1 μM) was also tested for activity in 60 kinase assays as a part of a screening protocol performed by Upstate Inc (Figure 15). However, no drug activity was observed at concentrations of less than 1 μg/ml, far above it's IC50 for the pharmacological activities described above.

[0228] 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. Thus, 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). Of these, only GS 15, GS28, and nicastrin were subsequently found to be consistently changed in the B16 and other cell lines. Although the GS proteins and nicastrin have entirely different functions in the cell, and

have not been linked (apparently) in the scientific literature, there is a rational explanation for the changes noted in each protein, as described below. Western Blotting and Sample Preparation

[0229] 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 μl/2.0xl0 ⁷ cells) was added with freshly added protease inhibitors. 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.

[0230] 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.

TABLE 3

primarv antibodies

Name Cat # Species company source

ARF (H-50) sc-9063 rabbit polyclonal m,r,h Santa Cruz

Y1-adaptin (M-300) sc-10763 rabbit polyclonal m,r,h Santa Cruz

Bet1 612038 mouse monoclonal m,r,h,d,chick BD bioscience

Copβ (T-14) sc-13335 goat polyclonal m,r,h Santa Cruz

Calnexin (C-20) sc-6465 goat polyclonal m,r,h Santa Cruz

EEA1 (N-19) sc-6415 goat polyclonal m,h Santa Cruz

E-Cadherin (H-108) sc-7870 rabbit polyclonal m,r,h Santa Cruz

Copε (E-20) sc-12104 goat polyclonal m,r,h Santa Cruz

ErbB-4 (C-18) sc-283 rabbit polyclonal m,r,h Santa Cruz

GS27 (G-20) sc-14157 goat polyclonal m,r,h Santa Cruz

GS15 610960 mouse monoclonal d,Hu,Ms,r,Bov, Frog BD Bioscience

GS28 611184 mouse monoclonal m,r,h BD Bioscience

GS28(N-16) sc-15270 rabbit polyclonal m,r,h Santa Cruz

HCAM (H300) sc-7946 rabbit polyclonal m,r,h Santa Cruz

HSV-1VP16(vA-19) sc-17547 goat polyclonal HSV-1 protein Santa Cruz

HSV-2glycoproteinD(vl-20) sc-17538 goat polyclonal HSV-2 protein Santa Cruz

HCAM (DF1485) sc-7297 mouse monoclonal h Santa Cruz

Histone H1(FL-219) sc-10806 rabbit polyclonal broad Santa Cruz

NSF (C-19) sc-15917 goat 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

Nicastrin (N-19) sc-14369 goat 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

Rab8 (P-16) sc-306 rabbit polyclonal h Santa Cruz

Rab6 (C-19) sc-310 rabbit polyclonal m,r,h Santa Cruz

SNAP25 (N-19) sc-7539 goat polyclonal m,r,h Santa Cruz

SYP (C-20) sc-7568 goat polyclonal m,r,h Santa Cruz

Syntaxin (FL-288) sc-13994 rabbit polyclonal m,r,h Santa Cruz

Syntaxin-1 (HPC-1) sc-12736 mouse monoclonal m,r,h Santa Cruz

Ykt6p (K-16) sc-10835 goat polyclonal m,r,h Santa Cruz α-SNAP (N-19) sc-7770 goat polyclonal m,r,h Santa Cruz

SNAP 23 111 202 rabbit polyclonal m,h SYSY.Germany

SNAP 23A VAP-SV013 rabbit polyclonal hu,ha,ca,bov stressgen β-Tubulin (D-IO) sc-5274 mouse monoclonal 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

Rabbit anti-goat IgG-HRP sc-2768 Santa Cruz

Goat anti-rabbit IgG-HRP sc-2004 Santa Cruz anti-mouse IgG-HRP sc-2005 Santa Cruz

Goat anti-mouse IgG-HRP sc-2005 Santa Cruz

Expression of Cellular Proteins

[0231] 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 γ-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. Drug treatment of B16 cells results in a block of nicastrin maturation such that the immature, partially glycosylated form of nicastrin accumulates at the expense of the fully glycosylated moiety. Nicastrin normally passes through the ER where it is partially glycosylated and then to the Golgi where glycosylation and sialation is completed. Thus nicastrin is essentially acting as a cargo protein whose changes reflect of how the cell processes and transports it. By suppressing the maturation of nicastrin, AVP 893 treatment appears to prevent the Golgi-associated processing changes of nicastrin, which is perhaps associated with its effect on GS28.

[0232] To further examine the putative effects of AVP 893, other proteins in the trafficking pathway were tested in vitro in B16 and other cell lines. The effect of AVP 893 on GS28 and nicastrin was corroborated in Bl 6 cells and extended to include a time-course (Figure 17). B16-F10 tumor cells were seeded in T75 flasks at 20% confluence and cultured overnight. AVP 893 (100 ng/ml) was added to several flasks and harvested at several different time points following addition of compound. Lysates were prepared, separated by electrophoresis, and probed with antibody as described above in the general Western blotting protocol. Drug effects on GS28 and nicastrin paralleled each other and were progressively stronger with longer drug incubations. Two days of culture with AVP 893 resulted in a complete loss of GS28 (not shown). Other cell lines were also tested for their expression of

GS28 and nicastrin and found to respond similarly to drug, although quantitative differences were evident. For the experiments shown in Figures 18, 19 and 20, all cell lines were treated as described for Figure 17. Tumor cell lines found to respond similarly to AVP 893 include 3T3, CAKI, SF295, PC3, MOLT4, Neuro2a, and RBL (Figures 18, 19, and 20). The effects on LOX cells were less evident. 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. Levels of calnexin, a resident ER protein used as a control, were unchanged in drug-treated cells. 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.

[0233] 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 μg/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 μg/ml). As with the 3T3 fibroblasts, spleen cell expression of GS28 was abrogated by drug while calnexin expression was minimally affected. 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.

[0234] A similar experiment performed on mouse spleen cells was repeated in human PBL except that some samples were also treated with the protein kinase C activator, PMA. The addition of PMA to IL-4/anti-CD40 Ab (ICD) in in vitro cultures does not affect the proliferation of human PBL or their IgE response but does enhance the potency of AVP

893 for inhibiting both measures (Figure 23). PBL were prepared and cultured in the presence of either IL-4/anti-CD40 Ab or the combination of PMA and IL-4/anti-CD40 Ab +/- AVP 893 for 4 days before pulsing with 3H-thymidine and harvesting. The following concentrations of human-specific reagents were used: PMA (100 ng/ml), IL-4 (100 ng/ml), and anti-CD40 Ab (300 ng/ml). Similarly, the addition of PMA to PBL does not increase the level of GS28 but enhances the potency of AVP 893 for inhibiting GS28 expression (Figure 24). PBL cultures were carried out as described for Figure 23 except that the cells were harvested after 48 hours and lysates prepared for Western blotting (as in Figure 17). These results provide additional evidence for the existence of a link between the cellular effects of AVP 893 and GS28 expression in primary (non-transformed) cells.

[0235] The specific mode by which AVP 893 diminishes expression of GS28 protein is not yet known but does not appear to involve transcription, as AVP 893 did not affect the level of GS28 mRNA when tested at 4 and 24 hours following addition of drug (Figure 25). Human buffy coats were purchased from the San Diego Blood Bank. Buffy Coat was purified of red blood cells using Histopaque-1077 following Sigma Diagnostic protocol. Lymphocytes (20 million) were then cultured in 75cm ² flasks in cDMEM (+/- stimulus & AVP 893) for either 4 or 24 hrs. Cells were harvested and reconstituted in a guanidine/phenol solution essentially as described by Maniatis. The aqueous layer was removed and washed with guanidine solution and finally 70% EtOH. 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 5'-GATCTCAGGAAACAGGCTCG-S', 5'-CCTGTAAGCCTTGCCAAAAG-S' ACTIN 5'-GTGGGCCGCTCTAGGCACCA-S' 5'-TGGCCTTAGGGTGCAGGGGG-S'

[0236] GS28 is but one member of a complicated pathway of interacting proteins that are responsible for the movement of vesicles through the cell. In addition to the SNARE proteins that are involved in vesicular docking and fusion, 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 Rabό (Figure 26). Both Rabl proteins help COPII protein-coated vesicles to travel from the ER to the Golgi, while Rabό is involved in the retrograde movement of vesicles back to the ER via COPI vesicles. Despite their role in ER to Golgi trafficking, however, the responses of these Rab proteins to AVP compounds were found to be variable depending on the cell, suggesting that the effects on these Rab proteins are secondary to a primary event. Numerous other proteins involved in intracellular trafficking were also tested and found to be largely unaffected by AVP benzimidazole compounds (Figure 26).

[0237] 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). Likewise, the juxtanuclear expression of the non-resident Golgi protein Rabό is unaffected in Vero cells, as illustrated in Figure 29. Similar results were obtained in other cells.

[0238] These results demonstrate that AVP 893 acts discriminately on the expression of Golgi resident proteins GS 15, GS28, GPP 130, and mannosidase II, sparing the Golgi structural protein GM-130 and having little effect on the Rab GTPase Rabό. Furthermore, these affects were most pronounced following overnight (16-20 hr) incubations with AVP 893, although some effects at early time points were seen. These conclusions were drawn from the Western blot analysis (Figure 27), as well as from the immunocytochemical studies (Figures 28-29). More particularly, mannosidase II, a resident Golgi enzyme involved in carbohydrate processing, was shown to move out of the Golgi beginning after 1 hr of AVP 893 application (Figure 28), with little to no discernible amount of the enzyme remaining after 4 hrs, and certainly none after 18 hrs. In contrast, as shown in Figure 29, the staining of the GTPase Rabό was neither diminished nor significantly altered by the presence of AVP 893, even after 18 hrs.

[0239] Accordingly, it can be concluded that 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. In addition, 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.

[0240] Experiments were conducted to examine the Golgi structure and morphology on the ultrastructural level following treatment with AVP 893. Electron microscopy analyses of untreated MOLT4 cells vs. MOLT4 cells treated with AVP 893 (200 ng/mL) for 2 hrs or 18 hrs demonstrated that AVP 893 disrupts Golgi structure (Figure 30). At 2 hrs of AVP 893 treatment, shadows of Golgi cisternae appeared while after 18 hrs treatment, no Golgi cisternae were found. This finding was repeated with Vero cells, where AVP 893 was applied for 1 hr, 4 hrs, and 18 hrs, with the later two exposures resulting in disruption of cisternal structure to a more vesicular pattern (data not shown). We therefore conclude that AVP 893 disturbs the structure of the Golgi cisternae within a brief timeframe (2 hrs) of treatment. Intracellular Protein Movement

[0241] The distribution of Golgi resident proteins was examined further by passing cell lysates through an iodixanol density gradient. For this experiment, GPP 130 was measured because it is partially retained following an overnight culture with AVP-893 in NIH-3T3 cells. Moreover, because GPP 130 is glycosylated, it can reveal changes in processing in concert with its distribution. NIH-3T3 cells were cultured with AVP-893, and cell lysates were layered on an iodixanol gradient before centrifugation and analysis of aliquots of the sequential 1 ml fractions by Western blotting (Figure 31). The distribution of endoplasmic reticulum (calnexin) and Golgi (γ-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. To illustrate the difference between each

condition an aliquot of the original lysates (containing 30 μg of protein) was removed prior to centrifugation, and directly compared by Western blot (left lane).

[0242] Following a 1 hr culture with drug, 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. Significantly, 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. 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.

[0243] hi a similar experiment, Figure 32 shows the redistribution of Rabό following a 16 hr culture with AVP 893 in B16 cells The levels of Rabό present in each fraction are compared with the presence of marker proteins; calnexin for the endoplasmic reticulum (ER), γ-adaptin for the Golgi (G), and Rab5a for vesicles/endosomes (V). Also shown are the unfractionated levels of Rabό that were obtained prior to density gradient centrifugation. As noted in Figure 31, no difference in the expression of these 3 marker proteins was observed between the control and drug-treated cells. Although Rabό was not quantitatively reduced in unfractionated lysates it appeared to redistribute to the ER at the expense of the Golgi compartment. Similar results were noted for Rabla (not shown).

[0244] Density gradient separations with other Golgi proteins and cell lines, however, often yielded results that diverge from that described in Figure 31 and Figure 32. In part, this might be explained by differences in the pathway by which these Golgi proteins follow in their return to early Golgi membranes. GPP 130 has been reported to recycle between the Golgi and distal compartments while Rabό, GS 15, and GS28 cycle back to the ER and early Golgi membranes. Indeed, it has been reported that disruption of Golgi protein recycling in yeast results in the redistribution of a Golgi mannosyltransferase to heavier membranes. Moreover, as described in detail below, 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

[0245] 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 μl 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 (16μl loaded per lane). Throughout this protocol, samples were kept on ice as much as possible. Comparison with Brefeldin A

[0246] Of the few chemical compounds known to affect the intracellular trafficking of proteins, the two most studied are monensin and brefeldin A (BFA). 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. The mechanism of brefeldin A is reasonably well mapped out and involves Golgi disruption through inhibition of GDP-GTP transfer on Arfl, a GTPase responsible for activating budding of anterograde COPII vesicles from the ER to the Golgi. However, although Arfl is primarily located in the ER-Golgi region, it is

also found in other compartments and appears to have more broad effects than just the ER- Golgi area.

[0247] 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 μM) 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. The results of the head-to-head comparisons performed in-house also show substantial differences in the relative proliferative responses of cells to brefeldin A and AVP 893 in vitro (not shown).

[0248] As shown in Figure 34, 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. In contrast, 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. Moreover, the changes observed for GS28 did not parallel the IC50 of brefeldin A for proliferation in these cell lines. Quantitative effects of brefeldin A on nicastrin were minimal, and no changes in the glycosylation of the protein were observed. These results clearly show that brefeldin A and AVP 893 act via different mechanisms to inhibit protein trafficking.

[0249] Further studies were conducted to show that 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. For combination treatments, 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 μg/ml; monensin, 10 μg/ml; rapamycin, 10 nM. As shown in Figure 35, 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. La addition, 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. Thus, the activity of AVP 893 against resident Golgi proteins is unique and distinct from the known pharmacological agents monenin, brefeldin A, and rapamycin.

[0250] Additional evidence that AVP 893 has unique activity against Golgi resident proteins (e.g. mannosidase II), was found using both shorter durations of drug exposure and immunocytochemistry instead of Western blot analysis (Figure 36). This experiment showed that lhr of treatment of brefeldin A or nocodozole disrupted the normal pattern of staining of mannosidase II. The crescent-shaped Golgi labeling was either completely dispersed, in the case of brefeldin A, or spread into a myriad of small, punctate fragments, in the case of nocodozole. However, 1 hr of AVP 893 exposure had no apparent effect in this experiment, and certainly not any perturbation of mannosidase II localization or expression levels. This contrasts with the result observed following longer incubations with AVP compounds wherein mannosidase II is depleted from the juxtanuclear region starting at about 4 hours (Figure 30). In conclusion, the results shown in Figure 36 provide further evidence that AVP 893 acts in a unique fashion against Golgi resident proteins.

[0251] The effect of AVP 893 on the expression of an array of other trafficking proteins was also tested but no other proteins appeared to be modulated quantitatively, including several of the putative interacting partners of GS28 (VAMPl, Gs 15, Yktό) and a variety of tethering proteins and GTPases (Figures 26 & 37). Most of these proteins function outside of the ER-Golgi region while the locations of many have not been defined. Figure 37 also illustrates an important comparison with published data of phenotypes of cells that have had one or more of an 8-member complex of proteins depleted. These proteins, which comprise the COG (conserved oligomeric Golgi) complex, are located on early Golgi

membranes and putatively are responsible for the transfer of the contents of COPI vesicles. The latter are responsible for the recycling of Golgi resident proteins to ensure their continued function. COG-Deficient and AVP Compound Treated Cells Express Similar Phenotypes

[0252] Much of the evidence that supports the involvement of COGs in the action of AVP benzimidazoles is circumstantial. Initially, it was based on a comparison with the phenotype of COGl and COG2 deletion mutants of CHO cells described by Oka, et al {Molecular Biology of the Cell, 15:2423-2435, (2004)). Subsequently, a paper by Zolov and Lupashin {Journal of Cell Biology, 168:747-759 (2005)) characterized the cellular phenotype of COG3-deficiency established by gene knock down. Work in yeast form the balance of the information available about the structure and function of COGs.

[0253] 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. In addition, 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. Thus, of 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.

[0254] There are other parallels. While total cellular βCOP expression is not suppressed, it moves out of the juxtanuclear Golgi region dispersing into small vesicles, either in the COG deletion mutants or in cells treated with AVP 13358 (Figure 38). As noted above, COPI proteins interact with COGs and the disruption of this interaction would plausibly cause dispersal of COPI vesicles from the perinuclear region. Indeed this appears to be the case as the COPI vesicle protein βCOP and the Golgi resident protein GS28 begin to disperse from the juxtanuclear region after 1.5 hr of exposure to AVP 893 and by 4.5 hr they no longer co-localize. Finally, 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. As noted earlier, MOLT-4, Vero, and 3T3 cells treated with AVP compounds also contain dilated or dispersed Golgi. The Fate of Golgi Resident Proteins

[0255] In COG-deficient cells, Golgi resident proteins undergo an accelerated degradation, rather than reduced synthesis. This process is likely carried out in either the lysosome or proteosome (the 2 important pathways of protein degradation in the cell) following transport from the Golgi to the ER in COPI vesicles. In CHO cell mutants, inhibition of proteosomal enzymes blocks the degradation of Golgi resident proteins caused by COG mutation, hi cells treated with AVP benzimidazoles, a similar pattern exists. Overnight cultures of MOLT-4 or 3T3 cells (Figure 39) or B16-F10 cells (Figure 40) with AVP 13358 with lysosomal (NH ₄ Cl) or proteosomal (MGl 32) inhibitors prevent the loss of Golgi resident proteins, although variability exists among different cell lines. Moreover, the combination of lysosome and proteosome inhibitors more effectively conserves GS 15 and GS28 than either agent alone (Figure 40). Thus, AVP compounds appear to accelerate the degradation of these proteins, while the individual cell determines the degradative pathway. The Role in Cell Proliferation

[0256] Much of what is known of COGs in cell growth has been learned from studies in yeast. The study of the effect of COG depletion on proliferation of mammalian cells is difficult because deletion mutants that block cell growth are naturally selected out. Thus, if this is the typical phenotype, as suggested by yeast studies for COGs 1-4, few COG deletion mutants would be identified. Nonetheless, CHO cells lacking COGl or COG2 have been identified, as with COG3 knockdowns in HeLa cells. As noted above, AVP compounds have variable effects of the growth of tumor cells, with IC50s ranging from sub-nM to > 10 μM. All primary cells stimulated with mitogens, however, are very sensitive to the growth inhibitory properties of AVP compounds. We have evidence to suggest that the difference between sensitive and resistant cells is in how they handle the flux of Golgi proteins. That is, although these proteins leave the juxtanuclear region in response to AVP compounds (Figures 41-42), they appear to be sequestered by resistant cells in a compartment that is impervious to proteolytic enzymes. Thus, while all sensitive cells are able to degrade resident Golgi enzymes in the presence of AVP compounds, the concentrations of AVP 893 that

displace GS 15, GS28, and GPP 130 from its juxtanuclear expression in H460 and SW480 cells affect neither the total cellular level of the protein nor the proliferation of these cells in culture (Figure 43, Table 4). Interestingly, OVCAR-3 cells are resistant to the antiproliferative effects of AVP 893 but its Golgi proteins are inhibited by drug with reasonable potency. However, maximal inhibition of Golgi proteins by AVP 893 in this cell line is only 70%.

Table 4

'expression determined by Western blotting.

*maximum suppression = 70%; all other sensitive proteins (IC50s of 100 nM or less) were maximally inhibited by AVP 893 greater than 95% nd = not determined

The Role in Protein Secretion

[0257] 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. Many other proteins are likely to be affected as well. However, we do not know how these compounds specifically block the expression/release of these proteins, and nothing is known of the effects of disrupting COG activity on their intracellular movement. What we

do know is that both COG mutant and AVP compound-treated cells have no apparent deficits in the secretion of pre-formed mediators (e.g., exocytosis). Moreover, effects on secretion or cell surface expression may be secondary to changes in glycosylation, as reported for the LDL receptor. There have also been reports suggesting that IgE oligosaccharides are important for its secretion. The difficulty in studying secretion of un-stored proteins such as cytokines and immunoglobulins however exaggerates the difficulties in establishing the role of COGs in these processes. Glvcosylation Defects

[0258] Deficits in glycosylation are easier to visualize and study than the movement of cytokines and immunoglobulins. Moreover, all cells perform these processing tasks and thus the deficits in protein glycosylation have been studied in both mutant CHO cells and HeLa cells. In cells where COG expression has been disrupted, broad effects on both N-linked and 0-linked glycosylation exist. As would be expected for a Golgi-specific effect, addition of high mannose CHO chains are not blocked (an ER-mediated event) but conversion of these oligosaccharides to an EndoH resistant form is prevented. 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.

[0259] CD23 contains both N-linked and O-linked sugars, and in the absence of stimulus the production of this protein is minimal. As noted earlier, 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. There is, however, a time-dependence of the effect on CD23 expression that may give some hint as to the mechanism of its interference by these compounds. 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. There appear to be both quantitative and qualitative effects on CD23 protein that ultimately influences its cell surface expression. Clearly, the immature form can be expressed at the cell surface, although the efficiency may be decreased. At 100

ng/ml of AVP 13358, high levels of the immature protein are produced at 48 hrs but the cell- surface expression barely exceeds that of non-stimulated cells. Interestingly, the effects on CD23 processing are mirrored by nicastrin, although the latter expresses some residual fully glycosylated protein (not shown). As observed for IgE production, the effects on glycosylation are fully reversible upon removal of AVP 13358 (Figure 46). The process is quite slow (Ty ₂ ~ 20 hr) however, which is a likely reflection of the inefficient synthetic machinery for replacing constitutive proteins.

[0260] To explore the nature of the glycosylation defects induced by AVP compounds, we compared their effects with that of selective glycosylation and trafficking inhibitors. In spleen cells, AVP 13358 more profoundly inhibits CD23 processing when compared with other agents (Figure 47). Minimal effects were noted following inhibition of mannosidase II (swainsonine) or glucosidases (castanospermine). Even tunicamycin, which inhibits addition of all N-linked sugars, yielded CD23 at a slightly larger size than allowed by AVP 13358. The latter difference is likely accounted by the 2 kDa of O-linked oligosaccharides that reportedly comprise CD23 sugars. The effects of monensin, brefeldin A, and nocodazole also were different from that induced by AVP 13358. The latter agrees with the phenotypic differences observed between cells treated with AVP compounds and these agents. Lysates of control and AVP 13358-treated cells were digested with EndoH and PNGaseF and then analyzed by Western blot to reveal the CHO residues that are exposed following drug treatment (Figure 48). As described above, EndoH removes all exposed N- linked mannose residues, i.e. those to which complex carbohydrates have not been added. PNGase F removes all N-linked sugars regardless of its level of complexity. In both spleen cells and the CD23-positive mouse B-lymphoma cell line M12.4.5, 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. In comparison 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. These results clearly

show that complexation of N-linked sugars and addition of O-linked sugar, two processes that take place exclusively in the Golgi, are inhibited by AVP 13358. Other Biological Effects Predicted by Inhibition of Golgi Proteins

[0261] Demonstration of a disruptive effect on the Golgi provides a clearer view of how AVP compounds work. The classical ER-Golgi pathway is the preferred transportation/maturation path of most intracellular proteins, including IgE, many membrane receptors, and many cytokines. Exceptions to the latter are IL-I and MIF, which by-pass the ER-Golgi via the "non-classical" secretion pathway. As predicted, despite inhibiting the secretion of many cytokines, AVP 13358 does not affect IL-I or MIF secretion in vitro (not shown).

[0262] The proposed mechanism of the AVP compounds on intracellular protein transit also allows certain predictions as to other effects and non-effects that these compounds might share. For example, inhibition of vesicle fusion or budding between the ER and Golgi should not affect exocytosis as would be expected of a post-Golgi active compound such as monensin. Indeed, 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). Moreover, 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).

[0263] An important potential consequence of blocking normal vesicle movement between the ER and Golgi is the inhibition of the propagation of certain viruses. Most viruses rely on the Golgi for assimilating its proteins, lipid coats and, ultimately, infectivity. The capacity of AVP 893 to inhibit viral propagation was tested in vitro by infecting Vero cells with HSV-2 and observing the effect of increasing concentrations of drug (Figure 49). Vero cells (1 million/ml) were cultured overnight and inoculated with about 150 PFU of live type 2 Herpes virus (HSV-2) about 1 hour after addition of AVP 893. After 48 hours, media was removed and the cells washed with saline and stained with Biological Plaque Stain for 20 min. One ml of water was added and the liquid removed before quantifying virus by

enumerating plaque forming units (PFU). AVP 893 suppressed plaque formation at all concentrations tested, with a total block occurring at 300 ng/ml. Moreover, 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.

[0264] We next determined whether viral particles (as visualized in Figure 50 by labeling HS V-2 glycoprotein E) spread beyond the initial site of infection in the presence of drug. HSV-2-infected cultures were treated with AVP 893. The results demonstrate that little to no labeling was found in cells surrounding beyond the initial site of infection. Other HSV proteins including gB, gD, and the capsid protein ICP5 were also examined with similar results (data not shown). In conclusion, AVP 893 blocks the spread of HSV-2 virus particles (or at least blocks the spread of infectious particles). Furthermore, AVP 893 didn't appear to stop the initial infection of the culture, only the subsequent spread of the virus. These results provide proof-of-concept that AVP 893, through its effect on resident Golgi proteins, may be inhibiting the spread of virus particles that utilize the Golgi in their lifecycle, as HSV-2 does.

[0265] In addition to affecting the expression of HSV proteins, 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/vø.The efficacy of topical AVP 26296 was tested in HSV-2 cutaneous and vaginal guinea pig disease models. Results for treatment of the cutaneous model with vehicle (Formulation W: 38% Gelucire 44/14, 38% polyethylene glycol 400, 10% tween 80, and 10% l-methyl-2-pyrrolidinone), Acyclovir (commercial ointment), Docosanal (Abreva), and AVP 26296 (2% in Formulation W) are shown in Figure 51.

TABLE 5

Family type assembly, budding, exit examples

Adenoviridae D, ne nucleus, lysis common cold, adeno Junin

cytoplasmic assembly, buds measels, mumps, RSV,

Paramyxovindae R, e apical PM parainfluenza, Sendai

ER (bypasses G), to apical PM _, tick fever, rota (infant Reoviridae R, ne lysis gastroenteritis) ass inner PM or cytoplasm _b uds HIV, HTLV-I & II, RSV Retrovindae R, e basoiaterai PM (rous) cytoplasmic assembly, buds

Rhabdoviridae R, e basoiaterai PM rabies, VSV cytoplasmic assembly, buds at

Togaviridae R, e ER (to G-?) rubella, Smbis, SFV

[0266] R= RNA virus; D = DNA virus; e = lipid enveloped

Inhibitors of Golgi Resident Proteins

[0267] 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).

[0268] 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. As described herein, 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. It was further elucidated that treatment with these compounds blocked nicastrin maturation such that the immature, partially glycosylated form of nicastrin accumulates at the expense of the fully glycosylated active moiety. Nicastrin normally passes through the ER where it is partially glycosylated and then to the Golgi where glycosylation and sialation is completed. Thus, changes in nicastrin state seem to correlate with its intracellular compartment as it moves through the cell.

[0269] The above description of preferred embodiments of the present invention is not intended to be limiting on the scope of the invention. Indeed, Jung et al. {Electrophoresis (2000) 21:3369-3377) indicate that there are 157 resident proteins (SWISS- PROT database; Table 1) associated with the ER and Golgi apparatus. Taylor et al. {Electrophoresis (1997) 18:643-654) reported 173 proteins in rat hepatocyte Golgi. Thus, there may be many other 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. Accordingly, whereas pharmacologic suppression of GS28 levels, for example, 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). Alternatively, 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.

[0270] 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

[0271] 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-γ, IFN-α, and IL-6), and inhibition of recruitment and proliferation of activated lymphoid cells. The immune disorder (immunological consequence), cells involved, and desired therapeutic and/or prophylactic drug effects are summarized for SLE, allergy, and MS in TABLE 6.

[0272] 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-γ, IL-6, and TNFα), and inhibition of recruitment and proliferation of activated lymphoid cells.

TABLE 6

* all desired drug effects (except for TNFα inhibition) are achieved by the 2-PB compounds disclosed herein

[0273] 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.

[0274] A pilot MS study (EAE, experimental auotimmune encephalomyelitis) was also conducted and the results are shown in Figure 54. EAE was induced in 6-8 week/old female PLSJL-Fl mice by s.c injection of 100 mg myelin basic protein emulsified in complete Freud's adjuvant (CFA) containing 1 mg/ml H37Ra Mycobacterium tuberculosis on day 0, followed by 400 ng Pertussis toxin i.p. on days 0 & 3. Animals were treated from days 3 to 44 with 20 or 40 mg/kg AVP 13358 in Formulation W 6 days/wk p.o.

[0275] A second MS model study was perfomed using the scoring methodology outlined below. Animals were immunized as above and then treated with Formulation W + AVP 13358 at a daily oral dose of 30 mg/kg. The results (scoring and animal weight) are illustrated in Figure 55 and show potential therapeutic benefit for AVP 13358.

Score Observation

0 normal

1 tail swelling

2 walking slowly

3 dragging feet

4 death

[0276] TABLE 7 shows the protocol for an ongoing NZB/W SLE model.

TABLE 7

Group Animal Drug Dose # per group End-point

1 NZBAV vehicle — 20 survival

2 NZBAV 13358 25 mpk 20 survival

3 NZBAV prednisolone 0.3 mg tiw 20 survival

4 NZBAV 27457 25 mpk 20 survival

5 NZBAV 13358/pred above 20 survival

6 BALB/c none — 10 control

Assessment Frequency morbidity, mortality daily body weight weekly urine protein 4 weeks serum antibodies 4-6 weeks serum BUN, creatinine 4-6 weeks spleen cell phenotype @7 months of age serum cytokines @5, 8 months of age serum drug/metabolite @3, 8 months of age

Assessment Frequency Comment renal histology at 7 months of age 5 kidneys per group; 20 total cytokine response at 7 months of age ex vivo, 5 spleens per group; 25 total (see below*)

*-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, IFNα and IFNγ

[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.

[0277] Results of urine protein changes and preliminary animal survival are shown in Figure 58. Urine was collected from NZBAV mice and tested using a dipstick and read colorimetrically by instrument (Bayer). Early survival results appear to favor treatment with AVP-13358.

[0278] 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. Thus, 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. Moreover, as shown in the first SLE protocol, mice treated with AVP 13358 appear to develop disease later than those receiving vehicle alone.

[0279] Methods of preventing MS and SLE are also contemplated within preferred embodiments of the present invention. Administration of any of compounds 1-42 prior to the onset of MS or SLE symptoms in a patient in need of prophylactic treatment are expected to provide similar beneficial effects as those illustrated in Figures 53-61. Development of Back-up Compounds

[0280] Oral bioavailablity of AVP 13358, AVP 27457, and AVP 27591 was assayed and the results compared in Figure 62. Groups of 5 BALB/c mice were dosed with 15 mg/kg of drug either via i.v. or p.o. Bleeds were obtained at approximately 0.5, 1, 1.5, 2, 3, 4, and 6 hr after dosing. No group of mice was bled more than twice. Drug and active metabolites were quantified by HPLC and the area under the curve (AUC) was determined using the Prism graph function based on integration of the time-concentration curve between 0.5 to 6 hr. The respective oral and intravenous curves were compared to determine oral

bioavailability. 6M = 6 min active metabolite of AVP 13358; 7.8M = 7.8 min active metabolite of AVP 27457

[0281] An experiment designed to compare the potency of the lead AVP compounds for inhibition of IgE (mouse spleen cells) and cytokines (human PBL) was performed, and the results shown in Figure 63. Compounds AVP 27591, AVP 27457, and AVP 893 were assayed for their effects on IFN-γ, IL-10, IL-2, IgE, IL-6 and TNF-α. Fresh human PBLs were prepared and cultured overnight with ConA (5 mg/ml) and drug. Supernatants were quantified using cytokine-specifϊc Luminex beads. Mouse IgE response was measured from supernatants of BALB/c spleen cells cultured for 5 days with IL-4 and anti-CD40 Ab. The parallel activity profiles of the 3 compounds suggest that they are acting via the same target.

[0282] Compounds were tested for therapeutic efficacy using the BAL and AHR asthma in vivo models. Both models were initiated in BALB/c mice by i.p. injection of OVA adsorbed to alum. After 2 weeks, mice were administered 2 consecutive daily exposures to OVA by nebulization and this was repeated at 4 weeks post-sensitization (3 daily nebulizations). Drug or vehicle was administered in the indicated doses orally on the days that OVA was administered by nebulization. 24 hours after the last nebulization, mice were either challenged with methylcholine in a whole body plethsmograph (AHR) or their BAL gavage fluid was obtained and characterized according to cell phenotype. Compounds AVP 13358 and AVP 27457 were used in the BAL model experiment. Compound AVP 27457 was used in the AHR experiment. The results shown in Figure 64 demonstrate that the backup compound has similar efficacy as AVP- 13358.

[0283] In summary, 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). With regard to 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.

[0284] Additional experimental results with other compounds are summarized in Table 8.

TABLE 8

MOL STRUCTURE MOL MOL FORMULA MOL WEIGHT Compound Number In Vitro IgE Inhibition IC50

STRUCTURE (nM) chiral

FALSE C27 H26 F N503 487.5324 AVP-0027937

FALSE C26 H23 F2 N502 475.4967 AVP-0027969 <1.0

FALSE C26 H23 F2 N502 475.4967 AVP-0027978

FALSE C26H15F4N5O2 505.4295 AVP-0027988 10

FALSE C26H17F2N5O2 469.4493 AVP-0027998 10

FALSE C26H17F2N5O2 469.4493 AVP-0027999 15

FALSE C26 H18 F N502 451.4592 AVP-0028011 20

FALSE C26H16F3N5O2 487.4394 AVP-0028019

FALSE C26H18FN5O2 451.4592 AVP-0028023 15

FALSE C27H18F3N5O2 501.4662 AVP-0028024 20

FALSE C27H18F3N5O2 501.4662 AVP-0028025

[0285] Those skilled in the art will recognize or be able to ascertain, using no more than routine experimentation, many equivalents of the specific embodiments of the invention described therein. Such equivalents are intended to be encompassed by the following claims. All figures, tables, and appendices, as well as patents, applications, and publications, referred to above, are hereby incorporated by reference.